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Familial transthyretin amyloid polyneuropathy

Curator: Larry H. Bernstein, MD, FCAP

 

UPDATED on 6/3/2020

Treatment of Cardiac Transthyretin Amyloidosis

Authors:
Emdin M, Aimo A, Rapezzi C, et al.
Citation:
Treatment of Cardiac Transthyretin Amyloidosis: An Update. Eur Heart J 2019;40:3699-3706.

The following are key points to remember from this update on the treatment of cardiac transthyretin amyloidosis:

  1. Transthyretin (TTR) is a highly conserved protein involved in transportation of thyroxine (T4) and retinol-binding protein. TTR is synthesized mostly by the liver and is rich in beta strands with an intrinsic propensity to aggregate into insoluble amyloid fibers, which deposit within tissue leading to the development of TTR-related amyloidosis (ATTR). ATTR can follow the deposition of either variant TTR (ATTRv, previously known as mutant ATTR) or wild type TTR (ATTRwt).
  2. Cardiac ATTR has a favorable survival rate compared to light chain (AL) amyloidosis, with a median survival of 75 versus 11 months. However, ATTR cardiomyopathy is a progressive disorder but newer therapeutic options include tafamidis (positive phase 3 clinical trial), and possibly patisiran and inotersen.

Inhibition of the Synthesis of Mutated Transthyretin

  1. Liver transplantation removes the source of mutated TTR molecules and prolongs survival, with a 20-year survival of 55.3%. However, tissue accumulation of TTR can continue after liver transplantation because TTR amyloid fibers promote subsequent deposition of ATTRwt. Combined liver–heart transplantation is feasible in younger patients with ATTRv cardiomyopathy and a small series suggests better prognosis than cardiac transplantation.
  2. Inhibition of TTR gene expression: Patisiran is a small interfering RNA blocking the expression of both variant and wt TTR. On the basis of the APOLLO trial, it was approved for therapy of adults with ATTRv-related polyneuropathy both in the United States and European Union. In this trial, patisiran promoted favorable myocardial remodeling based on echocardiographic and N-terminal B-type natriuretic peptide (NT-BNP) changes (this effect was not demonstrated for inotersen) and is still under investigation for tafamidis.
  3. Antisense oligonucleotides inotersen inhibits the production of both variant and wt TTR. Based on the findings of the NEURO-TTR trial, the Food and Drug Administration (FDA) approved this agent for patients with ATTRv-related polyneuropathy. In the NEURO-TTR trial, cardiomyopathy was present in 63%, but the study was not powered to measure effects of inotersen on heart disease. Inotersen can cause thrombocytopenia and must be used cautiously with bleeding risk.

Tetramer Stabilization

  1. Selective stabilizers include tafamidis and AG10. Tafamidis is a benzoxazole and a small molecule that inhibits the dissociation of TTR tetramers by binding the T4-binding sites. The phase ATTR-ACT study showed that when comparing the pooled tafamidis arms (80 and 20 mg) with the placebo arm, tafamidis was associated with lower all-cause mortality than placebo (78 of 264 [29.5%] vs. 76 of 177 [42.9%]; hazard ratio, 0.70; 95% confidence interval, 0.51-0.96) and a lower rate of cardiovascular hospitalizations. Based on the results of the ATTR-ACT trial, it has received Breakthrough Therapy designation from the FDA for treatment of ATTR cardiomyopathy.
  2. Nonselective agents: Diflunisal, a nonsteroidal anti-inflammatory drug, is reported to stabilize TTR tetramers. More studies are needed to confirm its clinical efficacy.

Inhibition of Oligomer Aggregation and Oligomer Disruption

  1. Epigallocatechin gallate is the most abundant catechin in green tea. One single-center open-label 12-month study did not show survival benefits or any change in echocardiographic parameters or NT-BNP compared to baseline.

Degradation and Reabsorption of Amyloid Fibers

  1. Doxycycline-taurosodeoxycholic acid (TUDCA) has been evaluated in two small studies and the results appear to be modest. More data are needed to confirm its efficacy.
  2. Antibodies targeting serum amyloid P protein or amyloid fibrils: Patient enrollment for miridesap followed by anti-SAP antibodies was suspended, and this approach is not being evaluated currently. However, a monoclonal antibody designed to specifically target TTR amyloid deposits (PRX004) has entered clinical evaluation, with an ongoing phase 1 study on ATTRv.

Supportive Treatment of Cardiac Involvement

  1. Drug therapies: Although angiotensin-converting enzyme (ACE) inhibitors/angiotensin-receptor blockers (ARBs) and beta-blockers may have been poorly tolerated in the ATTR-ACT trial, 30% of the patients were on ACE inhibitors/ARBs. There are no data with digoxin in TTR amyloid, and non-dihydropyridine calcium channel blockers are contraindicated due to negative inotropy.
  2. Implantable cardioverter-defibrillators (ICDs): In one study, which included 53 patients with amyloid, ICD shocks occurred exclusively in the AL amyloid group and none in the TTR amyloid patients. Higher defibrillation thresholds and complication rates are of concern.
  3. Cardiac pacing: In a large series of ATTRv-related polyneuropathy (n = 262), a pacemaker was implanted in 110 patients with His ventricular interval >700 ms. The authors recommend that any conduction disturbance on 12-lead electrocardiogram (ECG) warrants further investigation with Holter monitoring to determine candidacy for a pacemaker.
  4. Left ventricular assist device (LVAD): Although an LVAD is technically feasible, it is associated with high short-term mortality and worse outcomes than in dilated cardiomyopathy.
  5. Cardiac transplantation: This is a valuable option for patients with end-stage heart failure when significant extracardiac disease is excluded. In one study with 10 patients, only episodes of amyloid recurrence occurred.

This is an outstanding overview of this topic and recommended reading for anyone who cares for patients with cardiac transthyretin amyloid.

 

First-Ever Evidence that Patisiran Reduces Pathogenic, Misfolded TTR Monomers and Oligomers in FAP Patients

We reported data from our ongoing Phase 2 open-label extension (OLE) study of patisiran, an investigational RNAi therapeutic targeting transthyretin (TTR) for the treatment of TTR-mediated amyloidosis (ATTR amyloidosis) patients with familial amyloidotic polyneuropathy (FAP). Alnylam scientists and collaborators from The Scripps Research Institute and Misfolding Diagnostics, Inc. were able to measure the effects of patisiran on pathogenic, misfolded TTR monomers and oligomers in FAP patients. Results showed a rapid and sustained reduction in serum non-native conformations of TTR (NNTTR) of approximately 90%. Since NNTTR is pathogenic in ATTR amyloidosis and the level of NNTTR reduction correlated with total TTR knockdown, these results provide direct mechanistic evidence supporting the therapeutic hypothesis that TTR knockdown has the potential to result in clinical benefit. Furthermore, complete 12-month data from all 27 patients that enrolled in the patisiran Phase 2 OLE study showed sustained mean maximum reductions in total serum TTR of 91% for over 18 months and a mean 3.1-point decrease in mNIS+7 at 12 months, which compares favorably to an estimated increase in mNIS+7 of 13 to 18 points at 12 months based upon analysis of historical data sets in untreated FAP patients with similar baseline characteristics. Importantly, patisiran administration continues to be generally well tolerated out to 21 months of treatment.

Read our press release

View the non-native TTR poster (480 KB PDF)

View the complete 12-month patisiran Phase 2 OLE data presentation (620 KB PDF)

We are encouraged by these new data that provide continued support for our hypothesis that patisiran has the potential to halt neuropathy progression in patients with FAP. If these results are replicated in a randomized, double-blind, placebo-controlled study, we believe that patisiran could emerge as an important treatment option for patients suffering from this debilitating, progressive and life-threatening disease.

 

Hereditary ATTR Amyloidosis with Polyneuropathy (hATTR-PN)

ATTR amyloidosis is a progressive, life-threatening disease caused by misfolded transthyretin (TTR) proteins that accumulate as amyloid fibrils in multiple organs, but primarily in the peripheral nerves and heart. ATTR amyloidosis can lead to significant morbidity, disability, and mortality. The TTR protein is produced primarily in the liver and is normally a carrier for retinol binding protein – one of the vehicles used to transport vitamin A around the body.  Mutations in the TTR gene cause misfolding of the protein and the formation of amyloid fibrils that typically contain both mutant and wild-type TTR that deposit in tissues such as the peripheral nerves and heart, resulting in intractable peripheral sensory neuropathy, autonomic neuropathy, and/or cardiomyopathy.

Click to Enlarge

 

ATTR represents a major unmet medical need with significant morbidity and mortality. There are over 100 reported TTR mutations; the particular TTR mutation and the site of amyloid deposition determine the clinical manifestations of the disease whether it is predominantly symptoms of neuropathy or cardiomyopathy.

Specifically, hereditary ATTR amyloidosis with polyneuropathy (hATTR-PN), also known as familial amyloidotic polyneuropathy (FAP), is an inherited, progressive disease leading to death within 5 to 15 years. It is due to a mutation in the transthyretin (TTR) gene, which causes misfolded TTR proteins to accumulate as amyloid fibrils predominantly in peripheral nerves and other organs. hATTR-PN can cause sensory, motor, and autonomic dysfunction, resulting in significant disability and death.

It is estimated that hATTR-PN, also known as FAP, affects approximately 10,000 people worldwide.  Patients have a life expectancy of 5 to 15 years from symptom onset, and the only treatment options for early stage disease are liver transplantation and TTR stabilizers such as tafamidis (approved in Europe) and diflunisal.  Unfortunately liver transplantation has limitations, including limited organ availability as well as substantial morbidity and mortality. Furthermore, transplantation eliminates the production of mutant TTR but does not affect wild-type TTR, which can further deposit after transplantation, leading to cardiomyopathy and worsening of neuropathy. There is a significant need for novel therapeutics to treat patients who have inherited mutations in the TTR gene.

Our ATTR program is the lead effort in our Genetic Medicine Strategic Therapeutic Area (STAr) product development and commercialization strategy, which is focused on advancing innovative RNAi therapeutics toward genetically defined targets for the treatment of rare diseases with high unmet medical need.  We are developing patisiran (ALN-TTR02), an intravenously administered RNAi therapeutic, to treat the hATTR-PN form of the disease.

Patisiran for the Treatment hATTR-PN

APOLLO Phase 3 Trial

In 2012, Alnylam entered into an exclusive alliance with Genzyme, a Sanofi company, to develop and commercialize RNAi therapeutics, including patisiran and revusiran, for the treatment of ATTR amyloidosis in Japan and the broader Asian-Pacific region. In early 2014, this relationship was extended as a significantly broader alliance to advance RNAi therapeutics as genetic medicines. Under this new agreement, Alnylam will lead development and commercialization of patisiran in North America and Europe while Genzyme will develop and commercialize the product in the rest of world.

 

Hereditary ATTR Amyloidosis with Cardiomyopathy (hATTR-CM)

ATTR amyloidosis is a progressive, life-threatening disease caused by misfolded transthyretin (TTR) proteins that accumulate as amyloid fibrils in multiple organs, but primarily in the peripheral nerves and heart. ATTR amyloidosis can lead to significant morbidity, disability, and mortality. The TTR protein is produced primarily in the liver and is normally a carrier for retinol binding protein – one of the vehicles used to transport vitamin A around the body.  Mutations in the TTR gene cause misfolding of the protein and the formation of amyloid fibrils that typically contain both mutant and wild-type TTR that deposit in tissues such as the peripheral nerves and heart, resulting in intractable peripheral sensory neuropathy, autonomic neuropathy, and/or cardiomyopathy.

Click to Enlarge                            http://www.alnylam.com/web/assets/tetramer.jpg

ATTR represents a major unmet medical need with significant morbidity and mortality. There are over 100 reported TTR mutations; the particular TTR mutation and the site of amyloid deposition determine the clinical manifestations of the disease, whether it is predominantly symptoms of neuropathy or cardiomyopathy.

Specifically, hereditary ATTR amyloidosis with cardiomyopathy (hATTR-CM), also known as familial amyloidotic cardiomyopathy (FAC), is an inherited, progressive disease leading to death within 2 to 5 years. It is due to a mutation in the transthyretin (TTR) gene, which causes misfolded TTR proteins to accumulate as amyloid fibrils primarily in the heart. Hereditary ATTR amyloidosis with cardiomyopathy can result in heart failure and death.

While the exact numbers are not known, it is estimated hATTR-CM, also known as FAC affects at least 40,000 people worldwide.  hATTR-CM is fatal within 2 to 5 years of diagnosis and treatment is currently limited to supportive care.  Wild-type ATTR amyloidosis (wtATTR amyloidosis), also known as senile systemic amyloidosis, is a nonhereditary, progressive disease leading to death within 2 to 5 years. It is caused by misfolded transthyretin (TTR) proteins that accumulate as amyloid fibrils in the heart. Wild-type ATTR amyloidosis can cause cardiomyopathy and result in heart failure and death. There are no approved therapies for the treatment of hATTR-CM or SSA; hence there is a significant unmet need for novel therapeutics to treat these patients.

Our ATTR program is the lead effort in our Genetic Medicine Strategic Therapeutic Area (STAr) product development and commercialization strategy, which is focused on advancing innovative RNAi therapeutics toward genetically defined targets for the treatment of rare diseases with high unmet medical need.  We are developing revusiran (ALN-TTRsc), a subcutaneously administered RNAi therapeutic for the treatment of hATTR-CM.

Revusiran for the Treatment of hATTR-CM

ENDEAVOUR Phase 3 Trial

In 2012, Alnylam entered into an exclusive alliance with Genzyme, a Sanofi company, to develop and commercialize RNAi therapeutics, including patisiran and revusiran, for the treatment of ATTR amyloidosis in Japan and the broader Asian-Pacific region. In early 2014, this relationship was extended as a broader alliance to advance RNAi therapeutics as genetic medicines. Under this new agreement, Alnylam and Genzyme have agreed to co-develop and co-commercialize revusiran in North America and Europe, with Genzyme developing and commercializing the product in the rest of world. This broadened relationship on revusiran is aimed at expanding and accelerating the product’s global value.

Pre-Clinical Data and Advancement of ALN-TTRsc02 for Transthyretin-Mediated Amyloidosis

We presented pre-clinical data with ALN-TTRsc02, an investigational RNAi therapeutic targeting transthyretin (TTR) for the treatment of TTR-mediated amyloidosis (ATTR amyloidosis).  In pre-clinical studies, including those in non-human primates (NHPs), ALN-TTRsc02 achieved potent and highly durable knockdown of serum TTR of up to 99% with multi-month durability achieved after just a single dose, supportive of a potentially once quarterly dose regimen. Results from studies comparing TTR knockdown activity of ALN-TTRsc02 to that of revusiran showed that ALN-TTRsc02 has a markedly superior TTR knockdown profile.  Further, in initial rat toxicology studies, ALN-TTRsc02 was found to be generally well tolerated with no significant adverse events at doses as high as 100 mg/kg.

Read our press release

View the presentation

http://www.alnylam.com/product-pipeline/hereditary-attr-amyloidosis-with-cardiomyopathy/

 

Emerging Therapies for Transthyretin Cardiac Amyloidosis Could Herald a New Era for the Treatment of HFPEF

Oct 14, 2015   |  Adam Castano, MDDavid Narotsky, MDMathew S. Maurer, MD, FACC

http://www.acc.org/latest-in-cardiology/articles/2015/10/13/08/35/emerging-therapies-for-transthyretin-cardiac-amyloidosis#sthash.9xzc0rIe.dpuf

Heart failure with a preserved ejection fraction (HFPEF) is a clinical syndrome that has no pharmacologic therapies approved for this use to date. In light of failed medicines, cardiologists have refocused treatment strategies based on the theory that HFPEF is a heterogeneous clinical syndrome with different etiologies. Classification of HFPEF according to etiologic subtype may, therefore, identify cohorts with treatable pathophysiologic mechanisms and may ultimately pave the way forward for developing meaningful HFPEF therapies.1

A wealth of data now indicates that amyloid infiltration is an important mechanism underlying HFPEF. Inherited mutations in transthyretin cardiac amyloidosis (ATTRm) or the aging process in wild-type disease (ATTRwt) cause destabilization of the transthyretin (TTR) protein into monomers or oligomers, which aggregate into amyloid fibrils. These insoluble fibrils accumulate in the myocardium and result in diastolic dysfunction, restrictive cardiomyopathy, and eventual congestive heart failure (Figure 1). In an autopsy study of HFPEF patients, almost 20% without antemortem suspicion of amyloid had left ventricular (LV) TTR amyloid deposition.2 Even more resounding evidence for the contribution of TTR amyloid to HFPEF was a study in which 120 hospitalized HFPEF patients with LV wall thickness ≥12 mm underwent technetium-99m 3,3-diphosphono-1,2-propranodicarboxylic acid (99mTc-DPD) cardiac imaging,3,4 a bone isotope known to have high sensitivity and specificity for diagnosing TTR cardiac amyloidosis.5,6 Moderate-to-severe myocardial uptake indicative of TTR cardiac amyloid deposition was detected in 13.3% of HFPEF patients who did not have TTR gene mutations. Therefore, TTR cardiac amyloid deposition, especially in older adults, is not rare, can be easily identified, and may contribute to the underlying pathophysiology of HFPEF.

Figure 1

As no U.S. Food and Drug Administration-approved drugs are currently available for the treatment of HFPEF or TTR cardiac amyloidosis, the development of medications that attenuate or prevent TTR-mediated organ toxicity has emerged as an important therapeutic goal. Over the past decade, a host of therapies and therapeutic drug classes have emerged in clinical trials (Table 1), and these may herald a new direction for treating HFPEF secondary to TTR amyloid.

Table 1

TTR Silencers (siRNA and Antisense Oligonucleotides)

siRNA

Ribonucleic acid interference (RNAi) has surfaced as an endogenous cellular mechanism for controlling gene expression. Small interfering RNAs (siRNAs) delivered into cells can disrupt the production of target proteins.7,8 A formulation of lipid nanoparticle and triantennary N-acetylgalactosamine (GalNAc) conjugate that delivers siRNAs to hepatocytes is currently in clinical trials.9 Prior research demonstrated these GalNAc-siRNA conjugates result in robust and durable knockdown of a variety of hepatocyte targets across multiple species and appear to be well suited for suppression of TTR gene expression and subsequent TTR protein production.

The TTR siRNA conjugated to GalNAc, ALN-TTRSc, is now under active investigation as a subcutaneous injection in phase 3 clinical trials in patients with TTR cardiac amyloidosis.10 Prior phase 2 results demonstrated that ALN-TTRSc was generally well tolerated in patients with significant TTR disease burden and that it reduced both wild-type and mutant TTR gene expression by a mean of 87%. Harnessing RNAi technology appears to hold great promise for treating patients with TTR cardiac amyloidosis. The ability of ALN-TTRSc to lower both wild-type and mutant proteins may provide a major advantage over liver transplantation, which affects the production of only mutant protein and is further limited by donor shortage, cost, and need for immunosuppression.

Antisense Oligonucleotides

Antisense oligonucleotides (ASOs) are under clinical investigation for their ability to inhibit hepatic expression of amyloidogenic TTR protein. Currently, the ASO compound, ISIS-TTRRx, is under investigation in a phase 3 multicenter, randomized, double-blind, placebo-controlled clinical trial in patients with familial amyloid polyneuropathy (FAP).11 The primary objective is to evaluate its efficacy as measured by change in neuropathy from baseline relative to placebo. Secondary measures will evaluate quality of life (QOL), modified body mass index (mBMI) by albumin, and pharmacodynamic effects on retinol binding protein. Exploratory objectives in a subset of patients with LV wall thickness ≥13 mm without a history of persistent hypertension will examine echocardiographic parameters, N-terminal pro–B-type natriuretic peptide (NT-proBNP), and polyneuropathy disability score relative to placebo. These data will facilitate analysis of the effect of antisense oligonucleotide-mediated TTR suppression on the TTR cardiac phenotype with a phase 3 trial anticipated to begin enrollment in 2016.

TTR Stabilizers (Diflunisal, Tafamidis)

Diflunisal

Several TTR-stabilizing agents are in various stages of clinical trials. Diflunisal, a traditionally used and generically available nonsteroidal anti-inflammatory drug (NSAID), binds and stabilizes familial TTR variants against acid-mediated fibril formation in vitro and is now in human clinical trials.12,13 The use of diflunisal in patients with TTR cardiac amyloidosis is controversial given complication of chronic inhibition of cyclooxygenase (COX) enzymes, including gastrointestinal bleeding, renal dysfunction, fluid retention, and hypertension that may precipitate or exacerbate heart failure in vulnerable individuals.14-17 In TTR cardiac amyloidosis, an open-label cohort study suggested that low-dose diflunisal with careful monitoring along with a prophylactic proton pump inhibitor could be safely administered to compensated patients.18 An association was observed, however, between chronic diflunisal use and adverse changes in renal function suggesting that advanced kidney disease may be prohibitive in diflunisal therapy.In FAP patients with peripheral or autonomic neuropathy randomized to diflunisal or placebo, diflunisal slowed progression of neurologic impairment and preserved QOL over two years of follow-up.19 Echocardiography demonstrated cardiac involvement in approximately 50% of patients.20 Longer-term safety and efficacy data over an average 38 ± 31 months in 40 Japanese patients with hereditary ATTR amyloidosis who were not candidates for liver transplantation showed that diflunisal was mostly well tolerated.12 The authors cautioned the need for attentive monitoring of renal function and blood cell counts. Larger multicenter collaborations are needed to determine diflunisal’s true efficacy in HFPEF patients with TTR cardiac amyloidosis.

Tafamidis

Tafamidis is under active investigation as a novel compound that binds to the thyroxine-binding sites of the TTR tetramer, inhibiting its dissociation into monomers and blocking the rate-limiting step in the TTR amyloidogenesis cascade.21 The TTR compound was shown in an 18-month double-blind, placebo-controlled trial to slow progression of neurologic symptoms in patients with early-stage ATTRm due to the V30M mutation.22 When focusing on cardiomyopathy in a phase 2, open-label trial, tafamidis also appeared to effectively stabilize TTR tetramers in non-V30M variants, wild-type and V122I, as well as biochemical and echocardiographic parameters.23,24 Preliminary data suggests that clinically stabilized patients had shorter disease duration, lower cardiac biomarkers, less myocardial thickening, and higher EF than those who were not stabilized, suggesting early institution of therapy may be beneficial. A phase 3 trial has completed enrollment and will evaluate the efficacy, safety, and tolerability of tafamidis 20 or 80 mg orally vs. placebo.25 This will contribute to long-term safety and efficacy data needed to determine the therapeutic effects of tafamidis among ATTRm variants.

Amyloid Degraders (Doxycycline/TUDCA and Anti-SAP Antibodies)

Doxycycline/TUDCA

While silencer and stabilizer drugs are aimed at lowering amyloidogenic precursor protein production, they cannot remove already deposited fibrils in an infiltrated heart. Removal of already deposited fibrils by amyloid degraders would be an important therapeutic strategy, particularly in older adults with heavily infiltrated hearts reflected by thick walls, HFPEF, systolic heart failure, and restrictive cardiomyopathy. Combined doxycycline and tauroursodeoxycholic acid (TUDCA) disrupt TTR amyloid fibrils and appeared to have an acceptable safety profile in a small phase 2 open-label study among 20 TTR patients. No serious adverse reactions or clinical progression of cardiac or neuropathic involvement was observed over one year.26 An active phase 2, single-center, open-label, 12-month study will assess primary outcome measures including mBMI, neurologic impairment score, and NT-proBNP.27 Another phase 2 study is examining the tolerability and efficacy of doxycycline/TUDCA over an 18-month period in patients with TTR amyloid cardiomyopathy.28 Additionally, a study in patients with TTR amyloidosis is ongoing to determine the effect of doxycycline alone on neurologic function, cardiac biomarkers, echocardiographic parameters, modified body mass index, and autonomic neuropathy.29

Anti-SAP Antibodies

In order to safely clear established amyloid deposits, the role of the normal, nonfibrillar plasma glycoprotein present in all human amyloid deposits, serum amyloid P component (SAP), needs to be more clearly understood.30 In mice with amyloid AA type deposits, administration of antihuman SAP antibody triggered a potent giant cell reaction that removed massive visceral amyloid deposits without adverse effects.31 In humans with TTR cardiac amyloidosis, anti-SAP antibody treatments could be feasible because the bis-D proline compound, CPHPC, is capable of clearing circulating human SAP, which allow anti-SAP antibodies to reach residual deposited SAP. In a small, open-label, single-dose-escalation, phase 1 trial involving 15 patients with systemic amyloidosis, none of whom had clinical evidence of cardiac amyloidosis, were treated with CPHPC followed by human monoclonal IgG1 anti-SAP antibody.32 No serious adverse events were reported and amyloid deposits were cleared from the liver, kidney, and lymph node. Anti-SAP antibodies hold promise as a potential amyloid therapy because of their potential to target all forms of amyloid deposits across multiple tissue types.

Mutant or wild-type TTR cardiac amyloidoses are increasingly recognized as a cause of HFPEF. Clinicians need to be aware of this important HFPEF etiology because the diverse array of emerging disease-modifying agents for TTR cardiac amyloidosis in human clinical trials has the potential to herald a new era for the treatment of HFPEF.

References

  1. Maurer MS, Mancini D. HFpEF: is splitting into distinct phenotypes by comorbidities the pathway forward? J Am Coll Cardiol 2014;64:550-2.
  2. Mohammed SF, Mirzoyev SA, Edwards WD, et al. Left ventricular amyloid deposition in patients with heart failure and preserved ejection fraction. JACC Heart Fail 2014;2:113-22.
  3. González-López E, Gallego-Delgado M, Guzzo-Merello G, et al. Wild-type transthyretin amyloidosis as a cause of heart failure with preserved ejection fraction. Eur Heart J 2015.
  4. Castano A, Bokhari S, Maurer MS. Unveiling wild-type transthyretin cardiac amyloidosis as a significant and potentially modifiable cause of heart failure with preserved ejection fraction. Eur Heart J 2015 Jul 28. [Epub ahead of print]
  5. Rapezzi C, Merlini G, Quarta CC, et al. Systemic cardiac amyloidoses: disease profiles and clinical courses of the 3 main types. Circulation 2009;120:1203-12.
  6. Bokhari S, Castano A, Pozniakoff T, Deslisle S, Latif F, Maurer MS. (99m)Tc-pyrophosphate scintigraphy for differentiating light-chain cardiac amyloidosis from the transthyretin-related familial and senile cardiac amyloidoses. Circ Cardiovasc Imaging 2013;6:195-201.
  7. Fire A, Xu S, Montgomery MK, Kostas SA, Driver SE, Mello CC. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 1998;391:806-11.
  8. Elbashir SM, Harborth J, Lendeckel W, Yalcin A, Weber K, Tuschl T. Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature 2001;411:494-8.
  9. Kanasty R, Dorkin JR, Vegas A, Anderson D. Delivery materials for siRNA therapeutics. Nature Mater 2013;12:967-77.
  10. U.S. National Institutes of Health. Phase 2 Study to Evaluate ALN-TTRSC in Patients With Transthyretin (TTR) Cardiac Amyloidosis (ClinicalTrials.gov website). 2014. Available at: https://www.clinicaltrials.gov/ct2/show/NCT01981837. Accessed 8/19/2015.
  11. U.S. National Institutes of Health. Efficacy and Safety of ISIS-TTRRx in Familial Amyloid Polyneuropathy (Clinical Trials.gov Website. 2013. Available at: http://www.clinicaltrials.gov/ct2/show/NCT01737398. Accessed 8/19/2015.
  12. Sekijima Y, Dendle MA, Kelly JW. Orally administered diflunisal stabilizes transthyretin against dissociation required for amyloidogenesis. Amyloid 2006;13:236-49.
  13. Tojo K, Sekijima Y, Kelly JW, Ikeda S. Diflunisal stabilizes familial amyloid polyneuropathy-associated transthyretin variant tetramers in serum against dissociation required for amyloidogenesis. Neurosci Res 2006;56:441-9.
  14. Epstein M. Non-steroidal anti-inflammatory drugs and the continuum of renal dysfunction. J Hypertens Suppl 2002;20:S17-23.
  15. Wallace JL. Pathogenesis of NSAID-induced gastroduodenal mucosal injury. Best Pract Res Clin Gastroenterol 2001;15:691-703.
  16. Mukherjee D, Nissen SE, Topol EJ. Risk of cardiovascular events associated with selective COX-2 inhibitors. JAMA 2001;286:954-9.
  17. Page J, Henry D. Consumption of NSAIDs and the development of congestive heart failure in elderly patients: an underrecognized public health problem. Arch Intern Med 2000;160:777-84.
  18. Castano A, Helmke S, Alvarez J, Delisle S, Maurer MS. Diflunisal for ATTR cardiac amyloidosis. Congest Heart Fail 2012;18:315-9.
  19. Berk JL, Suhr OB, Obici L, et al. Repurposing diflunisal for familial amyloid polyneuropathy: a randomized clinical trial. JAMA 2013;310:2658-67.
  20. Quarta CCF, Solomon RH Suhr SD, et al. The prevalence of cardiac amyloidosis in familial amyloidotic polyneuropathy with predominant neuropathy: The Diflunisal Trial. International Symposium on Amyloidosis 2014:88-9.
  21. Hammarstrom P, Jiang X, Hurshman AR, Powers ET, Kelly JW. Sequence-dependent denaturation energetics: A major determinant in amyloid disease diversity. Proc Natl Acad Sci U S A 2002;99 Suppl 4:16427-32.
  22. Coelho T, Maia LF, Martins da Silva A, et al. Tafamidis for transthyretin familial amyloid polyneuropathy: a randomized, controlled trial. Neurology 2012;79:785-92.
  23. Merlini G, Plante-Bordeneuve V, Judge DP, et al. Effects of tafamidis on transthyretin stabilization and clinical outcomes in patients with non-Val30Met transthyretin amyloidosis. J Cardiovasc Transl Res 2013;6:1011-20.
  24. Maurer MS, Grogan DR, Judge DP, et al. Tafamidis in transthyretin amyloid cardiomyopathy: effects on transthyretin stabilization and clinical outcomes. Circ Heart Fail 2015;8:519-26.
  25. U.S. National Institutes of Health. Safety and Efficacy of Tafamidis in Patients With Transthyretin Cardiomyopathy (ATTR-ACT) (ClinicalTrials.gov website). 2014. Available at: http://www.clinicaltrials.gov/show/NCT01994889. Accessed 8/19/2015.
  26. Obici L, Cortese A, Lozza A, et al. Doxycycline plus tauroursodeoxycholic acid for transthyretin amyloidosis: a phase II study. Amyloid 2012;19 Suppl 1:34-6.
  27. U.S. National Institutes of Health. Safety, Efficacy and Pharmacokinetics of Doxycycline Plus Tauroursodeoxycholic Acid in Transthyretin Amyloidosis (ClinicalTrials.gov website). 2011. Available at: http://www.clinicaltrials.gov/ct2/show/NCT01171859. Accessed 8/19/2015.
  28. U.S. National Institutes of Health. Tolerability and Efficacy of a Combination of Doxycycline and TUDCA in Patients With Transthyretin Amyloid Cardiomyopathy (ClinicalTrials.gov website). 2013. Available at: http://www.clinicaltrials.gov/ct2/show/NCT01855360. Accessed 8/19/2015.
  29. U.S. National Institutes of Health. Safety and Effect of Doxycycline in Patients With Amyloidosis (ClinicalTrials.gov website).2015. Available at: https://clinicaltrials.gov/ct2/show/NCT01677286. Accessed 8/19/2015.
  30. Pepys MB, Dash AC. Isolation of amyloid P component (protein AP) from normal serum as a calcium-dependent binding protein. Lancet 1977;1:1029-31.
  31. Bodin K, Ellmerich S, Kahan MC, et al. Antibodies to human serum amyloid P component eliminate visceral amyloid deposits. Nature 2010;468:93-7.
  32. Richards DB, Cookson LM, Berges AC, et al. Therapeutic Clearance of Amyloid by Antibodies to Serum Amyloid P Component. N Engl J Med 2015;373:1106-14.

 

The Acid-Mediated Denaturation Pathway of Transthyretin Yields a Conformational Intermediate That Can Self-Assemble into Amyloid

Zhihong Lai , Wilfredo Colón , and Jeffery W. Kelly *
Department of Chemistry, Texas A&M University, College Station, Texas 77843-3255
Biochemistry199635 (20), pp 6470–6482   http://dx.doi.org:/10.1021/bi952501g
Publication Date (Web): May 21, 1996  Copyright © 1996 American Chemical Society

Transthyretin (TTR) amyloid fibril formation is observed during partial acid denaturation and while refolding acid-denatured TTR, implying that amyloid fibril formation results from the self-assembly of a conformational intermediate. The acid denaturation pathway of TTR has been studied in detail herein employing a variety of biophysical methods to characterize the intermediate(s) capable of amyloid fibril formation. At physiological concentrations, tetrameric TTR remains associated from pH 7 to pH 5 and is incapable of amyloid fibril formation. Tetrameric TTR dissociates to a monomer in a process that is dependent on both pH and protein concentration below pH 5. The extent of amyloid fibril formation correlates with the concentration of the TTR monomer having an altered, but defined, tertiary structure over the pH range of 5.0−3.9. The inherent Trp fluorescence-monitored denaturation curve of TTR exhibits a plateau over the pH range where amyloid fibril formation is observed (albeit at a higher concentration), implying that a steady-state concentration of the amyloidogenic intermediate with an altered tertiary structure is being detected. Interestingly, 1-anilino-8-naphthalenesulfonate fluorescence is at a minimum at the pH associated with maximal amyloid fibril formation (pH 4.4), implying that the amyloidogenic intermediate does not have a high extent of hydrophobic surface area exposed, consistent with a defined tertiary structure. Transthyretin has two Trp residues in its primary structure, Trp-41 and Trp-79, which are conveniently located far apart in the tertiary structure of TTR. Replacement of each Trp with Phe affords two single Trp containing variants which were used to probe local pH-dependent tertiary structural changes proximal to these chromophores. The pH-dependent fluorescence behavior of the Trp-79-Phe mutant strongly suggests that Trp-41 is located near the site of the tertiary structural rearrangement that occurs in the formation of the monomeric amyloidogenic intermediate, likely involving the C-strand−loop−D-strand region. Upon further acidification of TTR (below pH 4.4), the structurally defined monomeric amyloidogenic intermediate begins to adopt alternative conformations that are not amyloidogenic, ultimately forming an A-state conformation below pH 3 which is also not amyloidogenic. In summary, analytical equilibrium ultracentrifugation, SDS−PAGE, far- and near-UV CD, fluorescence, and light scattering studies suggest that the amyloidogenic intermediate is a monomeric predominantly β-sheet structure having a well-defined tertiary structure.

 

Prevention of Transthyretin Amyloid Disease by Changing Protein Misfolding Energetics

Per Hammarström*, R. Luke Wiseman*, Evan T. Powers, Jeffery W. Kelly   + Author Affiliations

Science  31 Jan 2003; 299(5607):713-716   http://dx.doi.org:/10.1126/science.1079589

Genetic evidence suggests that inhibition of amyloid fibril formation by small molecules should be effective against amyloid diseases. Known amyloid inhibitors appear to function by shifting the aggregation equilibrium away from the amyloid state. Here, we describe a series of transthyretin amyloidosis inhibitors that functioned by increasing the kinetic barrier associated with misfolding, preventing amyloidogenesis by stabilizing the native state. The trans-suppressor mutation, threonine 119 → methionine 119, which is known to ameliorate familial amyloid disease, also functioned through kinetic stabilization, implying that this small-molecule strategy should be effective in treating amyloid diseases.

 

Rational design of potent human transthyretin amyloid disease inhibitors

Thomas Klabunde1,2, H. Michael Petrassi3, Vibha B. Oza3, Prakash Raman3, Jeffery W. Kelly3 & James C. Sacchettini1

Nature Structural & Molecular Biology 2000; 7: 312 – 321.                http://dx.doi.org:/10.1038/74082

The human amyloid disorders, familial amyloid polyneuropathy, familial amyloid cardiomyopathy and senile systemic amyloidosis, are caused by insoluble transthyretin (TTR) fibrils, which deposit in the peripheral nerves and heart tissue. Several nonsteroidal anti-inflammatory drugs and structurally similar compounds have been found to strongly inhibit the formation of TTR amyloid fibrils in vitro. These include flufenamic acid, diclofenac, flurbiprofen, and resveratrol. Crystal structures of the protein–drug complexes have been determined to allow detailed analyses of the protein–drug interactions that stabilize the native tetrameric conformation of TTR and inhibit the formation of amyloidogenic TTR. Using a structure-based drug design approach ortho-trifluormethylphenyl anthranilic acid and N-(meta-trifluoromethylphenyl) phenoxazine 4,6-dicarboxylic acid have been discovered to be very potent and specific TTR fibril formation inhibitors. This research provides a rationale for a chemotherapeutic approach for the treatment of TTR-associated amyloid diseases.

 

First European consensus for diagnosis, management, and treatment of transthyretin familial amyloid polyneuropathy

Adams, Davida; Suhr, Ole B.b; Hund, Ernstc; Obici, Laurad; Tournev, Ivailoe,f; Campistol, Josep M.g; Slama, Michel S.h; Hazenberg, Bouke P.i; Coelho, Teresaj; from the European Network for TTR-FAP (ATTReuNET)

Current Opin Neurol: Feb 2016; 29 – Issue – p S14–S26      http://dx.doi.org:/10.1097/WCO.0000000000000289

Purpose of review: Early and accurate diagnosis of transthyretin familial amyloid polyneuropathy (TTR-FAP) represents one of the major challenges faced by physicians when caring for patients with idiopathic progressive neuropathy. There is little consensus in diagnostic and management approaches across Europe.

Recent findings: The low prevalence of TTR-FAP across Europe and the high variation in both genotype and phenotypic expression of the disease means that recognizing symptoms can be difficult outside of a specialized diagnostic environment. The resulting delay in diagnosis and the possibility of misdiagnosis can misguide clinical decision-making and negatively impact subsequent treatment approaches and outcomes.

Summary: This review summarizes the findings from two meetings of the European Network for TTR-FAP (ATTReuNET). This is an emerging group comprising representatives from 10 European countries with expertise in the diagnosis and management of TTR-FAP, including nine National Reference Centres. The current review presents management strategies and a consensus on the gold standard for diagnosis of TTR-FAP as well as a structured approach to ongoing multidisciplinary care for the patient. Greater communication, not just between members of an individual patient’s treatment team, but also between regional and national centres of expertise, is the key to the effective management of TTR-FAP.

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Transthyretin familial amyloid polyneuropathy (TTR-FAP) is a highly debilitating and irreversible neurological disorder presenting symptoms of progressive sensorimotor and autonomic neuropathy [1▪,2▪,3]. TTR-FAP is caused by misfolding of the transthyretin (TTR) protein leading to protein aggregation and the formation of amyloid fibrils and, ultimately, to amyloidosis (commonly in the peripheral and autonomic nervous system and the heart) [4,5]. TTR-FAP usually proves fatal within 7–12 years from the onset of symptoms, most often due to cardiac dysfunction, infection, or cachexia [6,7▪▪].

The prevalence and disease presentation of TTR-FAP vary widely within Europe. In endemic regions (northern Portugal, Sweden, Cyprus, and Majorca), patients tend to present with a distinct genotype in large concentrations, predominantly a Val30Met substitution in the TTR gene [8–10]. In other areas of Europe, the genetic footprint of TTR-FAP is more varied, with less typical phenotypic expression [6,11]. For these sporadic or scattered cases, a lack of awareness among physicians of variable clinical features and limited access to diagnostic tools (i.e., pathological studies and genetic screening) can contribute to high rates of misdiagnosis and poorer patient outcomes [1▪,11]. In general, early and late-onset variants of TTR-FAP, found within endemic and nonendemic regions, present several additional diagnostic challenges [11,12,13▪,14].

Delay in the time to diagnosis is a major obstacle to the optimal management of TTR-FAP. With the exception of those with a clearly diagnosed familial history of FAP, patients still invariably wait several years between the emergence of first clinical signs and accurate diagnosis [6,11,14]. The timely initiation of appropriate treatment is particularly pertinent, given the rapidity and irreversibility with which TTR-FAP can progress if left unchecked, as well as the limited effectiveness of available treatments during the later stages of the disease [14]. This review aims to consolidate the existing literature and present an update of the best practices in the management of TTR-FAP in Europe. A summary of the methods used to achieve a TTR-FAP diagnosis is presented, as well as a review of available treatments and recommendations for treatment according to disease status.

Patients with TTR-FAP can present with a range of symptoms [11], and care should be taken to acquire a thorough clinical history of the patient as well as a family history of genetic disease. Delay in diagnosis is most pronounced in areas where TTR-FAP is not endemic or when there is no positive family history [1▪]. TTR-FAP and TTR-familial amyloid cardiomyopathy (TTR-FAC) are the two prototypic clinical disease manifestations of a broader disease spectrum caused by an underlying hereditary ATTR amyloidosis [19]. In TTR-FAP, the disease manifestation of neuropathy is most prominent and definitive for diagnosis, whereas cardiomyopathy often suggests TTR-FAC. However, this distinction is often superficial because cardiomyopathy, autonomic neuropathy, vitreous opacities, kidney disease, and meningeal involvement all may be present with varying severity for each patient with TTR-FAP.

Among early onset TTR-FAP with usually positive family history, symptoms of polyneuropathy present early in the disease process and usually predominate throughout the progression of the disease, making neurological testing an important diagnostic aid [14]. Careful clinical examination (e.g., electromyography with nerve conduction studies and sympathetic skin response, quantitative sensation test, quantitative autonomic test) can be used to detect, characterize, and scale the severity of neuropathic abnormalities involving small and large nerve fibres [10]. Although a patient cannot be diagnosed definitively with TTR-FAP on the basis of clinical presentation alone, symptoms suggesting the early signs of peripheral neuropathy, autonomic dysfunction, and cardiac conduction disorders or infiltrative cardiomyopathy are all indicators that further TTR-FAP diagnostic investigation is warranted. Late-onset TTR-FAP often presents as sporadic cases with distinct clinical features (e.g., milder autonomic dysfunction) and can be more difficult to diagnose than early-onset TTR-FAP (Table 2) [1▪,11,12,13▪,14,20].

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Genetic testing is carried out to allow detection of specific amyloidogenic TTR mutations (Table 1), using varied techniques depending on the expertise and facilities available in each country (Table S2, http://links.lww.com/CONR/A39). A targeted approach to detect a specific mutation can be used for cases belonging to families with previous diagnosis. In index cases of either endemic and nonendemic regions that do not have a family history of disease, are difficult to confirm, and have atypical symptoms, TTR gene sequencing is required for the detection of both predicted and new amyloidogenic mutations [26,27].

Following diagnosis, the neuropathy stage and systemic extension of the disease should be determined in order to guide the next course of treatment (Table 4) [3,30,31]. The three stages of TTR-FAP severity are graded according to a patient’s walking disability and degree of assistance required [30]. Systemic assessment, especially of the heart, eyes, and kidney, is also essential to ensure all aspects of potential impact of the disease can be detected [10].

Table 4

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Image Tools

The goals of cardiac investigations are to detect serious conduction disorders with the risk of sudden death and infiltrative cardiomyopathy. Electrocardiograms (ECG), Holter-ECG, and intracardiac electrophysiology study are helpful to detect conduction disorders. Echocardiograms, cardiac magnetic resonance imaging, scintigraphy with bone tracers, and biomarkers (e.g., brain natriuretic peptide, troponin) can all help to diagnose infiltrative cardiomyopathy[10]. An early detection of cardiac abnormalities has obvious benefits to the patient, given that the prophylactic implantation of pacemakers was found to prevent 25% of major cardiac events in TTR-FAP patients followed up over an average of 4 years [32▪▪]. Assessment of cardiac denervation with 123-iodine meta-iodobenzylguanidine is a powerful prognostic marker in patients diagnosed with FAP [33].

…..

Tafamidis

Tafamidis is a first-in-class therapy that slows the progression of TTR amyloidogenesis by stabilizing the mutant TTR tetramer, thereby preventing its dissociation into monomers and amyloidogenic and toxic intermediates [55,56]. Tafamidis is currently indicated in Europe for the treatment of TTR amyloidosis in adult patients with stage I symptomatic polyneuropathy to delay peripheral neurological impairment [57].

In an 18-month, double-blind, placebo-controlled study of patients with early-onset Val30Met TTR-FAP, tafamidis was associated with a 52% lower reduction in neurological deterioration (P = 0.027), a preservation of nerve function, and TTR stabilization versus placebo [58▪▪]. However, only numerical differences were found for the coprimary endpoints of neuropathy impairment [neuropathy impairment score in the lower limb (NIS-LL) responder rates of 45.3% tafamidis vs 29.5% placebo; P = 0.068] and quality of life scores [58▪▪]. A 12-month, open-label extension study showed that the reduced rates of neurological deterioration associated with tafamidis were sustained over 30 months, with earlier initiation of tafamidis linking to better patient outcomes (P = 0.0435) [59▪]. The disease-slowing effects of tafamidis may be dependent on the early initiation of treatment. In an open-label study with Val30Met TTR-FAP patients with late-onset and advanced disease (NIS-LL score >10, mean age 56.4 years), NIS-LL and disability scores showed disease progression despite 12 months of treatment with tafamidis, marked by a worsening of neuropathy stage in 20% and the onset of orthostatic hypotension in 22% of patients at follow-up [60▪].

Tafamidis is not only effective in patients exhibiting the Val30Met mutation; it also has proven efficacy, in terms of TTR stabilization, in non-Val30Met patients over 12 months [61]. Although tafamidis has demonstrated safe use in patients with TTR-FAP, care should be exercised when prescribing to those with existing digestive problems (e.g., diarrhoea, faecal incontinence) [60▪].

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Diflunisal

Diflunisal is a nonsteroidal anti-inflammatory drug (NSAID) that, similar to tafamidis, slows the rate of amyloidogenesis by preventing the dissociation, misfolding, and misassembly of the mutated TTR tetramer [62,63]. Off-label use has been reported for patients with stage I and II disease, although diflunisal is not currently licensed for the treatment of TTR-FAP.

Evidence for the clinical effectiveness of diflunisal in TTR-FAP derives from a placebo-controlled, double-blind, 24-month study in 130 patients with clinically detectable peripheral or autonomic neuropathy[64▪]. The deterioration in NIS scores was significantly more pronounced in patients receiving placebo compared with those taking diflunisal (P = 0.001), and physical quality of life measures showed significant improvement among diflunisal-treated patients (P = 0.001). Notable during this study was the high rate of attrition in the placebo group, with 50% more placebo-treated patients dropping out of this 2-year study as a result of disease progression, advanced stage of the disease, and varied mutations.

One retrospective analysis of off-label use of diflunisal in patients with TTR-FAP reported treatment discontinuation in 57% of patients because of adverse events that were largely gastrointestinal [65]. Conclusions on the safety of diflunisal in TTR-FAP will depend on further investigations on the impact of known cardiovascular and renal side-effects associated with the NSAID drug class [66,67].

 

 

 

 

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CRISPR/Cas9, Familial Amyloid Polyneuropathy ( FAP) and Neurodegenerative Disease


CRISPR/Cas9, Familial Amyloid Polyneuropathy ( FAP) and Neurodegenerative Disease

Curator: Larry H. Bernstein, MD, FCAP

 

CRISPR/Cas9 and Targeted Genome Editing: A New Era in Molecular Biology

https://www.neb.com/tools-and-resources/feature-articles/crispr-cas9-and-targeted-genome-editing-a-new-era-in-molecular-biology

The development of efficient and reliable ways to make precise, targeted changes to the genome of living cells is a long-standing goal for biomedical researchers. Recently, a new tool based on a bacterial CRISPR-associated protein-9 nuclease (Cas9) from Streptococcus pyogenes has generated considerable excitement (1). This follows several attempts over the years to manipulate gene function, including homologous recombination (2) and RNA interference (RNAi) (3). RNAi, in particular, became a laboratory staple enabling inexpensive and high-throughput interrogation of gene function (4, 5), but it is hampered by providing only temporary inhibition of gene function and unpredictable off-target effects (6). Other recent approaches to targeted genome modification – zinc-finger nucleases [ZFNs, (7)] and transcription-activator like effector nucleases [TALENs (8)]– enable researchers to generate permanent mutations by introducing doublestranded breaks to activate repair pathways. These approaches are costly and time-consuming to engineer, limiting their widespread use, particularly for large scale, high-throughput studies.

The Biology of Cas9

The functions of CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) and CRISPR-associated (Cas) genes are essential in adaptive immunity in select bacteria and archaea, enabling the organisms to respond to and eliminate invading genetic material. These repeats were initially discovered in the 1980s in E. coli (9), but their function wasn’t confirmed until 2007 by Barrangou and colleagues, who demonstrated that S. thermophilus can acquire resistance against a bacteriophage by integrating a genome fragment of an infectious virus into its CRISPR locus (10).

Three types of CRISPR mechanisms have been identified, of which type II is the most studied. In this case, invading DNA from viruses or plasmids is cut into small fragments and incorporated into a CRISPR locus amidst a series of short repeats (around 20 bps). The loci are transcribed, and transcripts are then processed to generate small RNAs (crRNA – CRISPR RNA), which are used to guide effector endonucleases that target invading DNA based on sequence complementarity (Figure 1) (11).

Figure 1. Cas9 in vivo: Bacterial Adaptive Immunity

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In the acquisition phase, foreign DNA is incorporated into the bacterial genome at the CRISPR loci. CRISPR loci is then transcribed and processed into crRNA during crRNA biogenesis. During interference, Cas9 endonuclease complexed with a crRNA and separate tracrRNA cleaves foreign DNA containing a 20-nucleotide crRNA complementary sequence adjacent to the PAM sequence. (Figure not drawn to scale.)

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One Cas protein, Cas9 (also known as Csn1), has been shown, through knockdown and rescue experiments to be a key player in certain CRISPR mechanisms (specifically type II CRISPR systems). The type II CRISPR mechanism is unique compared to other CRISPR systems, as only one Cas protein (Cas9) is required for gene silencing (12). In type II systems, Cas9 participates in the processing of crRNAs (12), and is responsible for the destruction of the target DNA (11). Cas9’s function in both of these steps relies on the presence of two nuclease domains, a RuvC-like nuclease domain located at the amino terminus and a HNH-like nuclease domain that resides in the mid-region of the protein (13).

To achieve site-specific DNA recognition and cleavage, Cas9 must be complexed with both a crRNA and a separate trans-activating crRNA (tracrRNA or trRNA), that is partially complementary to the crRNA (11). The tracrRNA is required for crRNA maturation from a primary transcript encoding multiple pre-crRNAs. This occurs in the presence of RNase III and Cas9 (12).

During the destruction of target DNA, the HNH and RuvC-like nuclease domains cut both DNA strands, generating double-stranded breaks (DSBs) at sites defined by a 20-nucleotide target sequence within an associated crRNA transcript (11, 14). The HNH domain cleaves the complementary strand, while the RuvC domain cleaves the noncomplementary strand.

The double-stranded endonuclease activity of Cas9 also requires that a short conserved sequence, (2–5 nts) known as protospacer-associated motif (PAM), follows immediately 3´- of the crRNA complementary sequence (15). In fact, even fully complementary sequences are ignored by Cas9-RNA in the absence of a PAM sequence (16).

Cas9 and CRISPR as a New Tool in Molecular Biology

The simplicity of the type II CRISPR nuclease, with only three required components (Cas9 along with the crRNA and trRNA) makes this system amenable to adaptation for genome editing. This potential was realized in 2012 by the Doudna and Charpentier labs (11). Based on the type II CRISPR system described previously, the authors developed a simplified two-component system by combining trRNA and crRNA into a single synthetic single guide RNA (sgRNA). sgRNAprogrammed Cas9 was shown to be as effective as Cas9 programmed with separate trRNA and crRNA in guiding targeted gene alterations (Figure 2A).

To date, three different variants of the Cas9 nuclease have been adopted in genome-editing protocols. The first is wild-type Cas9, which can site-specifically cleave double-stranded DNA, resulting in the activation of the doublestrand break (DSB) repair machinery. DSBs can be repaired by the cellular Non-Homologous End Joining (NHEJ) pathway (17), resulting in insertions and/or deletions (indels) which disrupt the targeted locus. Alternatively, if a donor template with homology to the targeted locus is supplied, the DSB may be repaired by the homology-directed repair (HDR) pathway allowing for precise replacement mutations to be made (Figure 2A) (17, 18).

Cong and colleagues (1) took the Cas9 system a step further towards increased precision by developing a mutant form, known as Cas9D10A, with only nickase activity. This means it cleaves only one DNA strand, and does not activate NHEJ. Instead, when provided with a homologous repair template, DNA repairs are conducted via the high-fidelity HDR pathway only, resulting in reduced indel mutations (1, 11, 19). Cas9D10A is even more appealing in terms of target specificity when loci are targeted by paired Cas9 complexes designed to generate adjacent DNA nicks (20) (see further details about “paired nickases” in Figure 2B).

The third variant is a nuclease-deficient Cas9 (dCas9, Figure 2C) (21). Mutations H840A in the HNH domain and D10A in the RuvC domain inactivate cleavage activity, but do not prevent DNA binding (11, 22). Therefore, this variant can be used to sequence-specifically target any region of the genome without cleavage. Instead, by fusing with various effector domains, dCas9 can be used either as a gene silencing or activation tool (21, 23–26). Furthermore, it can be used as a visualization tool. For instance, Chen and colleagues used dCas9 fused to Enhanced Green Fluorescent Protein (EGFP) to visualize repetitive DNA sequences with a single sgRNA or nonrepetitive loci using multiple sgRNAs (27).

Figure 2. CRISPR/Cas9 System Applications

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  1. Wild-type Cas9 nuclease site specifically cleaves double-stranded DNA activating double-strand break repair machinery. In the absence of a homologous repair template non-homologous end joining can result in indels disrupting the target sequence. Alternatively, precise mutations and knock-ins can be made by providing a homologous repair template and exploiting the homology directed repair pathway.
    B. Mutated Cas9 makes a site specific single-strand nick. Two sgRNA can be used to introduce a staggered double-stranded break which can then undergo homology directed repair.
    C. Nuclease-deficient Cas9 can be fused with various effector domains allowing specific localization. For example, transcriptional activators, repressors, and fluorescent proteins.

Targeting Efficiency and Off-target Mutations

Targeting efficiency, or the percentage of desired mutation achieved, is one of the most important parameters by which to assess a genome-editing tool. The targeting efficiency of Cas9 compares favorably with more established methods, such as TALENs or ZFNs (8). For example, in human cells, custom-designed ZFNs and TALENs could only achieve efficiencies ranging from 1% to 50% (29–31). In contrast, the Cas9 system has been reported to have efficiencies up to >70% in zebrafish (32) and plants (33), and ranging from 2–5% in induced pluripotent stem cells (34). In addition, Zhou and colleagues were able to improve genome targeting up to 78% in one-cell mouse embryos, and achieved effective germline transmission through the use of dual sgRNAs to simultaneously target an individual gene (35).

A widely used method to identify mutations is the T7 Endonuclease I mutation detection assay (36, 37) (Figure 3). This assay detects heteroduplex DNA that results from the annealing of a DNA strand, including desired mutations, with a wildtype DNA strand (37).

Figure 3. T7 Endonuclease I Targeting Efficiency Assay

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Genomic DNA is amplified with primers bracketing the modified locus. PCR products are then denatured and re-annealed yielding 3 possible structures. Duplexes containing a mismatch are digested by T7 Endonuclease I. The DNA is then electrophoretically separated and fragment analysis is used to calculate targeting efficiency.

Another important parameter is the incidence of off-target mutations. Such mutations are likely to appear in sites that have differences of only a few nucleotides compared to the original sequence, as long as they are adjacent to a PAM sequence. This occurs as Cas9 can tolerate up to 5 base mismatches within the protospacer region (36) or a single base difference in the PAM sequence (38). Off-target mutations are generally more difficult to detect, requiring whole-genome sequencing to rule them out completely.

Recent improvements to the CRISPR system for reducing off-target mutations have been made through the use of truncated gRNA (truncated within the crRNA-derived sequence) or by adding two extra guanine (G) nucleotides to the 5´ end (28, 37). Another way researchers have attempted to minimize off-target effects is with the use of “paired nickases” (20). This strategy uses D10A Cas9 and two sgRNAs complementary to the adjacent area on opposite strands of the target site (Figure 2B). While this induces DSBs in the target DNA, it is expected to create only single nicks in off-target locations and, therefore, result in minimal off-target mutations.

By leveraging computation to reduce off-target mutations, several groups have developed webbased tools to facilitate the identification of potential CRISPR target sites and assess their potential for off-target cleavage. Examples include the CRISPR Design Tool (38) and the ZiFiT Targeter, Version 4.2 (39, 40).

Applications as a Genome-editing and Genome Targeting Tool

Following its initial demonstration in 2012 (9), the CRISPR/Cas9 system has been widely adopted. This has already been successfully used to target important genes in many cell lines and organisms, including human (34), bacteria (41), zebrafish (32), C. elegans (42), plants (34), Xenopus tropicalis (43), yeast (44), Drosophila (45), monkeys (46), rabbits (47), pigs (42), rats (48) and mice (49). Several groups have now taken advantage of this method to introduce single point mutations (deletions or insertions) in a particular target gene, via a single gRNA (14, 21, 29). Using a pair of gRNA-directed Cas9 nucleases instead, it is also possible to induce large deletions or genomic rearrangements, such as inversions or translocations (50). A recent exciting development is the use of the dCas9 version of the CRISPR/Cas9 system to target protein domains for transcriptional regulation (26, 51, 52), epigenetic modification (25), and microscopic visualization of specific genome loci (27).

The CRISPR/Cas9 system requires only the redesign of the crRNA to change target specificity. This contrasts with other genome editing tools, including zinc finger and TALENs, where redesign of the protein-DNA interface is required. Furthermore, CRISPR/Cas9 enables rapid genome-wide interrogation of gene function by generating large gRNA libraries (51, 53) for genomic screening.

The Future of CRISPR/Cas9

The rapid progress in developing Cas9 into a set of tools for cell and molecular biology research has been remarkable, likely due to the simplicity, high efficiency and versatility of the system. Of the designer nuclease systems currently available for precision genome engineering, the CRISPR/Cas system is by far the most user friendly. It is now also clear that Cas9’s potential reaches beyond DNA cleavage, and its usefulness for genome locus-specific recruitment of proteins will likely only be limited by our imagination.

 

Scientists urge caution in using new CRISPR technology to treat human genetic disease

By Robert Sanders, Media relations | MARCH 19, 2015
http://news.berkeley.edu/2015/03/19/scientists-urge-caution-in-using-new-crispr-technology-to-treat-human-genetic-disease/

http://news.berkeley.edu/wp-content/uploads/2015/03/crispr350.jpg

The bacterial enzyme Cas9 is the engine of RNA-programmed genome engineering in human cells. (Graphic by Jennifer Doudna/UC Berkeley)

A group of 18 scientists and ethicists today warned that a revolutionary new tool to cut and splice DNA should be used cautiously when attempting to fix human genetic disease, and strongly discouraged any attempts at making changes to the human genome that could be passed on to offspring.

Among the authors of this warning is Jennifer Doudna, the co-inventor of the technology, called CRISPR-Cas9, which is driving a new interest in gene therapy, or “genome engineering.” She and colleagues co-authored a perspective piece that appears in the March 20 issue of Science, based on discussions at a meeting that took place in Napa on Jan. 24. The same issue of Science features a collection of recent research papers, commentary and news articles on CRISPR and its implications.    …..

A prudent path forward for genomic engineering and germline gene modification

David Baltimore1,  Paul Berg2, …., Jennifer A. Doudna4,10,*, et al.
http://science.sciencemag.org/content/early/2015/03/18/science.aab1028.full
Science  19 Mar 2015.  http://dx.doi.org:/10.1126/science.aab1028

 

Correcting genetic defects

Scientists today are changing DNA sequences to correct genetic defects in animals as well as cultured tissues generated from stem cells, strategies that could eventually be used to treat human disease. The technology can also be used to engineer animals with genetic diseases mimicking human disease, which could lead to new insights into previously enigmatic disorders.

The CRISPR-Cas9 tool is still being refined to ensure that genetic changes are precisely targeted, Doudna said. Nevertheless, the authors met “… to initiate an informed discussion of the uses of genome engineering technology, and to identify proactively those areas where current action is essential to prepare for future developments. We recommend taking immediate steps toward ensuring that the application of genome engineering technology is performed safely and ethically.”

 

Amyloid CRISPR Plasmids and si/shRNA Gene Silencers

http://www.scbt.com/crispr/table-amyloid.html

Santa Cruz Biotechnology, Inc. offers a broad range of gene silencers in the form of siRNAs, shRNA Plasmids and shRNA Lentiviral Particles as well as CRISPR/Cas9 Knockout and CRISPR Double Nickase plasmids. Amyloid gene silencers are available as Amyloid siRNA, Amyloid shRNA Plasmid, Amyloid shRNA Lentiviral Particles and Amyloid CRISPR/Cas9 Knockout plasmids. Amyloid CRISPR/dCas9 Activation Plasmids and CRISPR Lenti Activation Systems for gene activation are also available. Gene silencers and activators are useful for gene studies in combination with antibodies used for protein detection.    Amyloid CRISPR Knockout, HDR and Nickase Knockout Plasmids

 

CRISPR-Cas9-Based Knockout of the Prion Protein and Its Effect on the Proteome


Mehrabian M, Brethour D, MacIsaac S, Kim JK, Gunawardana C.G, Wang H, et al.
PLoS ONE 2014; 9(12): e114594. http://dx.doi.org/10.1371/journal.pone.0114594

The molecular function of the cellular prion protein (PrPC) and the mechanism by which it may contribute to neurotoxicity in prion diseases and Alzheimer’s disease are only partially understood. Mouse neuroblastoma Neuro2a cells and, more recently, C2C12 myocytes and myotubes have emerged as popular models for investigating the cellular biology of PrP. Mouse epithelial NMuMG cells might become attractive models for studying the possible involvement of PrP in a morphogenetic program underlying epithelial-to-mesenchymal transitions. Here we describe the generation of PrP knockout clones from these cell lines using CRISPR-Cas9 knockout technology. More specifically, knockout clones were generated with two separate guide RNAs targeting recognition sites on opposite strands within the first hundred nucleotides of the Prnp coding sequence. Several PrP knockout clones were isolated and genomic insertions and deletions near the CRISPR-target sites were characterized. Subsequently, deep quantitative global proteome analyses that recorded the relative abundance of>3000 proteins (data deposited to ProteomeXchange Consortium) were undertaken to begin to characterize the molecular consequences of PrP deficiency. The levels of ∼120 proteins were shown to reproducibly correlate with the presence or absence of PrP, with most of these proteins belonging to extracellular components, cell junctions or the cytoskeleton.

http://journals.plos.org/plosone/article/figure/image?size=inline&id=info:doi/10.1371/journal.pone.0114594.g001

http://journals.plos.org/plosone/article/figure/image?size=inline&id=info:doi/10.1371/journal.pone.0114594.g003

 

Development and Applications of CRISPR-Cas9 for Genome Engineering

Patrick D. Hsu,1,2,3 Eric S. Lander,1 and Feng Zhang1,2,*
Cell. 2014 Jun 5; 157(6): 1262–1278.   doi:  10.1016/j.cell.2014.05.010

Recent advances in genome engineering technologies based on the CRISPR-associated RNA-guided endonuclease Cas9 are enabling the systematic interrogation of mammalian genome function. Analogous to the search function in modern word processors, Cas9 can be guided to specific locations within complex genomes by a short RNA search string. Using this system, DNA sequences within the endogenous genome and their functional outputs are now easily edited or modulated in virtually any organism of choice. Cas9-mediated genetic perturbation is simple and scalable, empowering researchers to elucidate the functional organization of the genome at the systems level and establish causal linkages between genetic variations and biological phenotypes. In this Review, we describe the development and applications of Cas9 for a variety of research or translational applications while highlighting challenges as well as future directions. Derived from a remarkable microbial defense system, Cas9 is driving innovative applications from basic biology to biotechnology and medicine.

The development of recombinant DNA technology in the 1970s marked the beginning of a new era for biology. For the first time, molecular biologists gained the ability to manipulate DNA molecules, making it possible to study genes and harness them to develop novel medicine and biotechnology. Recent advances in genome engineering technologies are sparking a new revolution in biological research. Rather than studying DNA taken out of the context of the genome, researchers can now directly edit or modulate the function of DNA sequences in their endogenous context in virtually any organism of choice, enabling them to elucidate the functional organization of the genome at the systems level, as well as identify causal genetic variations.

Broadly speaking, genome engineering refers to the process of making targeted modifications to the genome, its contexts (e.g., epigenetic marks), or its outputs (e.g., transcripts). The ability to do so easily and efficiently in eukaryotic and especially mammalian cells holds immense promise to transform basic science, biotechnology, and medicine (Figure 1).

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4343198/bin/nihms659174f1.jpg

For life sciences research, technologies that can delete, insert, and modify the DNA sequences of cells or organisms enable dissecting the function of specific genes and regulatory elements. Multiplexed editing could further allow the interrogation of gene or protein networks at a larger scale. Similarly, manipulating transcriptional regulation or chromatin states at particular loci can reveal how genetic material is organized and utilized within a cell, illuminating relationships between the architecture of the genome and its functions. In biotechnology, precise manipulation of genetic building blocks and regulatory machinery also facilitates the reverse engineering or reconstruction of useful biological systems, for example, by enhancing biofuel production pathways in industrially relevant organisms or by creating infection-resistant crops. Additionally, genome engineering is stimulating a new generation of drug development processes and medical therapeutics. Perturbation of multiple genes simultaneously could model the additive effects that underlie complex polygenic disorders, leading to new drug targets, while genome editing could directly correct harmful mutations in the context of human gene therapy (Tebas et al., 2014).

Eukaryotic genomes contain billions of DNA bases and are difficult to manipulate. One of the breakthroughs in genome manipulation has been the development of gene targeting by homologous recombination (HR), which integrates exogenous repair templates that contain sequence homology to the donor site (Figure 2A) (Capecchi, 1989). HR-mediated targeting has facilitated the generation of knockin and knockout animal models via manipulation of germline competent stem cells, dramatically advancing many areas of biological research. However, although HR-mediated gene targeting produces highly precise alterations, the desired recombination events occur extremely infrequently (1 in 106–109 cells) (Capecchi, 1989), presenting enormous challenges for large-scale applications of gene-targeting experiments.

Genome Editing Technologies Exploit Endogenous DNA Repair Machinery

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4343198/bin/nihms659174f2.gif

To overcome these challenges, a series of programmable nuclease-based genome editing technologies have been developed in recent years, enabling targeted and efficient modification of a variety of eukaryotic and particularly mammalian species. Of the current generation of genome editing technologies, the most rapidly developing is the class of RNA-guided endonucleases known as Cas9 from the microbial adaptive immune system CRISPR (clustered regularly interspaced short palindromic repeats), which can be easily targeted to virtually any genomic location of choice by a short RNA guide. Here, we review the development and applications of the CRISPR-associated endonuclease Cas9 as a platform technology for achieving targeted perturbation of endogenous genomic elements and also discuss challenges and future avenues for innovation.   ……

Figure 4   Natural Mechanisms of Microbial CRISPR Systems in Adaptive Immunity

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4343198/bin/nihms659174f4.gif

……  A key turning point came in 2005, when systematic analysis of the spacer sequences separating the individual direct repeats suggested their extrachromosomal and phage-associated origins (Mojica et al., 2005Pourcel et al., 2005Bolotin et al., 2005). This insight was tremendously exciting, especially given previous studies showing that CRISPR loci are transcribed (Tang et al., 2002) and that viruses are unable to infect archaeal cells carrying spacers corresponding to their own genomes (Mojica et al., 2005). Together, these findings led to the speculation that CRISPR arrays serve as an immune memory and defense mechanism, and individual spacers facilitate defense against bacteriophage infection by exploiting Watson-Crick base-pairing between nucleic acids (Mojica et al., 2005Pourcel et al., 2005). Despite these compelling realizations that CRISPR loci might be involved in microbial immunity, the specific mechanism of how the spacers act to mediate viral defense remained a challenging puzzle. Several hypotheses were raised, including thoughts that CRISPR spacers act as small RNA guides to degrade viral transcripts in a RNAi-like mechanism (Makarova et al., 2006) or that CRISPR spacers direct Cas enzymes to cleave viral DNA at spacer-matching regions (Bolotin et al., 2005).   …..

As the pace of CRISPR research accelerated, researchers quickly unraveled many details of each type of CRISPR system (Figure 4). Building on an earlier speculation that protospacer adjacent motifs (PAMs) may direct the type II Cas9 nuclease to cleave DNA (Bolotin et al., 2005), Moineau and colleagues highlighted the importance of PAM sequences by demonstrating that PAM mutations in phage genomes circumvented CRISPR interference (Deveau et al., 2008). Additionally, for types I and II, the lack of PAM within the direct repeat sequence within the CRISPR array prevents self-targeting by the CRISPR system. In type III systems, however, mismatches between the 5′ end of the crRNA and the DNA target are required for plasmid interference (Marraffini and Sontheimer, 2010).  …..

In 2013, a pair of studies simultaneously showed how to successfully engineer type II CRISPR systems from Streptococcus thermophilus (Cong et al., 2013) andStreptococcus pyogenes (Cong et al., 2013Mali et al., 2013a) to accomplish genome editing in mammalian cells. Heterologous expression of mature crRNA-tracrRNA hybrids (Cong et al., 2013) as well as sgRNAs (Cong et al., 2013Mali et al., 2013a) directs Cas9 cleavage within the mammalian cellular genome to stimulate NHEJ or HDR-mediated genome editing. Multiple guide RNAs can also be used to target several genes at once. Since these initial studies, Cas9 has been used by thousands of laboratories for genome editing applications in a variety of experimental model systems (Sander and Joung, 2014). ……

The majority of CRISPR-based technology development has focused on the signature Cas9 nuclease from type II CRISPR systems. However, there remains a wide diversity of CRISPR types and functions. Cas RAMP module (Cmr) proteins identified in Pyrococcus furiosus and Sulfolobus solfataricus (Hale et al., 2012) constitute an RNA-targeting CRISPR immune system, forming a complex guided by small CRISPR RNAs that target and cleave complementary RNA instead of DNA. Cmr protein homologs can be found throughout bacteria and archaea, typically relying on a 5 site tag sequence on the target-matching crRNA for Cmr-directed cleavage.

Unlike RNAi, which is targeted largely by a 6 nt seed region and to a lesser extent 13 other bases, Cmr crRNAs contain 30–40 nt of target complementarity. Cmr-CRISPR technologies for RNA targeting are thus a promising target for orthogonal engineering and minimal off-target modification. Although the modularity of Cmr systems for RNA-targeting in mammalian cells remains to be investigated, Cmr complexes native to P. furiosus have already been engineered to target novel RNA substrates (Hale et al., 20092012).   ……

Although Cas9 has already been widely used as a research tool, a particularly exciting future direction is the development of Cas9 as a therapeutic technology for treating genetic disorders. For a monogenic recessive disorder due to loss-of-function mutations (such as cystic fibrosis, sickle-cell anemia, or Duchenne muscular dystrophy), Cas9 may be used to correct the causative mutation. This has many advantages over traditional methods of gene augmentation that deliver functional genetic copies via viral vector-mediated overexpression—particularly that the newly functional gene is expressed in its natural context. For dominant-negative disorders in which the affected gene is haplosufficient (such as transthyretin-related hereditary amyloidosis or dominant forms of retinitis pigmentosum), it may also be possible to use NHEJ to inactivate the mutated allele to achieve therapeutic benefit. For allele-specific targeting, one could design guide RNAs capable of distinguishing between single-nucleotide polymorphism (SNP) variations in the target gene, such as when the SNP falls within the PAM sequence.

 

 

CRISPR/Cas9: a powerful genetic engineering tool for establishing large animal models of neurodegenerative diseases

Zhuchi Tu, Weili Yang, Sen Yan, Xiangyu Guo and Xiao-Jiang Li

Molecular Neurodegeneration 2015; 10:35  http://dx.doi.org:/10.1186/s13024-015-0031-x

Animal models are extremely valuable to help us understand the pathogenesis of neurodegenerative disorders and to find treatments for them. Since large animals are more like humans than rodents, they make good models to identify the important pathological events that may be seen in humans but not in small animals; large animals are also very important for validating effective treatments or confirming therapeutic targets. Due to the lack of embryonic stem cell lines from large animals, it has been difficult to use traditional gene targeting technology to establish large animal models of neurodegenerative diseases. Recently, CRISPR/Cas9 was used successfully to genetically modify genomes in various species. Here we discuss the use of CRISPR/Cas9 technology to establish large animal models that can more faithfully mimic human neurodegenerative diseases.

Neurodegenerative diseases — Alzheimer’s disease(AD),Parkinson’s disease(PD), amyotrophic lateral sclerosis (ALS), Huntington’s disease (HD), and frontotemporal dementia (FTD) — are characterized by age-dependent and selective neurodegeneration. As the life expectancy of humans lengthens, there is a greater prevalence of these neurodegenerative diseases; however, the pathogenesis of most of these neurodegenerative diseases remain unclear, and we lack effective treatments for these important brain disorders.

CRISPR/Cas9,  Non-human primates,  Neurodegenerative diseases,  Animal model

There are a number of excellent reviews covering different types of neurodegenerative diseases and their genetic mouse models [812]. Investigations of different mouse models of neurodegenerative diseases have revealed a common pathology shared by these diseases. First, the development of neuropathology and neurological symptoms in genetic mouse models of neurodegenerative diseases is age dependent and progressive. Second, all the mouse models show an accumulation of misfolded or aggregated proteins resulting from the expression of mutant genes. Third, despite the widespread expression of mutant proteins throughout the body and brain, neuronal function appears to be selectively or preferentially affected. All these facts indicate that mouse models of neurodegenerative diseases recapitulate important pathologic features also seen in patients with neurodegenerative diseases.

However, it seems that mouse models can not recapitulate the full range of neuropathology seen in patients with neurodegenerative diseases. Overt neurodegeneration, which is the most important pathological feature in patient brains, is absent in genetic rodent models of AD, PD, and HD. Many rodent models that express transgenic mutant proteins under the control of different promoters do not replicate overt neurodegeneration, which is likely due to their short life spans and the different aging processes of small animals. Also important are the remarkable differences in brain development between rodents and primates. For example, the mouse brain takes 21 days to fully develop, whereas the formation of primate brains requires more than 150 days [13]. The rapid development of the brain in rodents may render neuronal cells resistant to misfolded protein-mediated neurodegeneration. Another difficulty in using rodent models is how to analyze cognitive and emotional abnormalities, which are the early symptoms of most neurodegenerative diseases in humans. Differences in neuronal circuitry, anatomy, and physiology between rodent and primate brains may also account for the behavioral differences between rodent and primate models.

 

Mitochondrial dynamics–fusion, fission, movement, and mitophagy–in neurodegenerative diseases

Hsiuchen Chen and David C. Chan
Human Molec Gen 2009; 18, Review Issue 2 R169–R176
http://dx.doi.org:/10.1093/hmg/ddp326

Neurons are metabolically active cells with high energy demands at locations distant from the cell body. As a result, these cells are particularly dependent on mitochondrial function, as reflected by the observation that diseases of mitochondrial dysfunction often have a neurodegenerative component. Recent discoveries have highlighted that neurons are reliant particularly on the dynamic properties of mitochondria. Mitochondria are dynamic organelles by several criteria. They engage in repeated cycles of fusion and fission, which serve to intermix the lipids and contents of a population of mitochondria. In addition, mitochondria are actively recruited to subcellular sites, such as the axonal and dendritic processes of neurons. Finally, the quality of a mitochondrial population is maintained through mitophagy, a form of autophagy in which defective mitochondria are selectively degraded. We review the general features of mitochondrial dynamics, incorporating recent findings on mitochondrial fusion, fission, transport and mitophagy. Defects in these key features are associated with neurodegenerative disease. Charcot-Marie-Tooth type 2A, a peripheral neuropathy, and dominant optic atrophy, an inherited optic neuropathy, result from a primary deficiency of mitochondrial fusion. Moreover, several major neurodegenerative diseases—including Parkinson’s, Alzheimer’s and Huntington’s disease—involve disruption of mitochondrial dynamics. Remarkably, in several disease models, the manipulation of mitochondrial fusion or fission can partially rescue disease phenotypes. We review how mitochondrial dynamics is altered in these neurodegenerative diseases and discuss the reciprocal interactions between mitochondrial fusion, fission, transport and mitophagy.

 

Applications of CRISPR–Cas systems in Neuroscience

Matthias Heidenreich  & Feng Zhang
Nature Rev Neurosci 2016; 17:36–44   http://dx.doi.org:/10.1038/nrn.2015.2

Genome-editing tools, and in particular those based on CRISPR–Cas (clustered regularly interspaced short palindromic repeat (CRISPR)–CRISPR-associated protein) systems, are accelerating the pace of biological research and enabling targeted genetic interrogation in almost any organism and cell type. These tools have opened the door to the development of new model systems for studying the complexity of the nervous system, including animal models and stem cell-derived in vitro models. Precise and efficient gene editing using CRISPR–Cas systems has the potential to advance both basic and translational neuroscience research.
Cellular neuroscience
, DNA recombination, Genetic engineering, Molecular neuroscience

Figure 3: In vitro applications of Cas9 in human iPSCs.close

http://www.nature.com/nrn/journal/v17/n1/carousel/nrn.2015.2-f3.jpg

a | Evaluation of disease candidate genes from large-population genome-wide association studies (GWASs). Human primary cells, such as neurons, are not easily available and are difficult to expand in culture. By contrast, induced pluripo…

  1. Genome-editing Technologies for Gene and Cell Therapy

Molecular Therapy 12 Jan 2016

  1. Systematic quantification of HDR and NHEJ reveals effects of locus, nuclease, and cell type on genome-editing

Scientific Reports 31 Mar 2016

  1. Controlled delivery of β-globin-targeting TALENs and CRISPR/Cas9 into mammalian cells for genome editing using microinjection

Scientific Reports 12 Nov 2015

 

Alzheimer’s Disease: Medicine’s Greatest Challenge in the 21st Century

https://www.physicsforums.com/insights/can-gene-editing-eliminate-alzheimers-disease/

The development of the CRISPR/Cas9 system has made gene editing a relatively simple task.  While CRISPR and other gene editing technologies stand to revolutionize biomedical research and offers many promising therapeutic avenues (such as in the treatment of HIV), a great deal of debate exists over whether CRISPR should be used to modify human embryos. As I discussed in my previous Insight article, we lack enough fundamental biological knowledge to enhance many traits like height or intelligence, so we are not near a future with genetically-enhanced super babies. However, scientists have identified a few rare genetic variants that protect against disease.  One such protective variant is a mutation in the APP gene that protects against Alzheimer’s disease and cognitive decline in old age. If we can perfect gene editing technologies, is this mutation one that we should be regularly introducing into embryos? In this article, I explore the potential for using gene editing as a way to prevent Alzheimer’s disease in future generations. Alzheimer’s Disease: Medicine’s Greatest Challenge in the 21st Century Can gene editing be the missing piece in the battle against Alzheimer’s? (Source: bostonbiotech.org) I chose to assess the benefit of germline gene editing in the context of Alzheimer’s disease because this disease is one of the biggest challenges medicine faces in the 21st century. Alzheimer’s disease is a chronic neurodegenerative disease responsible for the majority of the cases of dementia in the elderly. The disease symptoms begins with short term memory loss and causes more severe symptoms – problems with language, disorientation, mood swings, behavioral issues – as it progresses, eventually leading to the loss of bodily functions and death. Because of the dementia the disease causes, Alzheimer’s patients require a great deal of care, and the world spends ~1% of its total GDP on caring for those with Alzheimer’s and related disorders. Because the prevalence of the disease increases with age, the situation will worsen as life expectancies around the globe increase: worldwide cases of Alzheimer’s are expected to grow from 35 million today to over 115 million by 2050.

Despite much research, the exact causes of Alzheimer’s disease remains poorly understood. The disease seems to be related to the accumulation of plaques made of amyloid-β peptides that form on the outside of neurons, as well as the formation of tangles of the protein tau inside of neurons. Although many efforts have been made to target amyloid-β or the enzymes involved in its formation, we have so far been unsuccessful at finding any treatment that stops the disease or reverses its progress. Some researchers believe that most attempts at treating Alzheimer’s have failed because, by the time a patient shows symptoms, the disease has already progressed past the point of no return.

While research towards a cure continues, researchers have sought effective ways to prevent Alzheimer’s disease. Although some studies show that mental and physical exercise may lower ones risk of Alzheimer’s disease, approximately 60-80% of the risk for Alzheimer’s disease appears to be genetic. Thus, if we’re serious about prevention, we may have to act at the genetic level. And because the brain is difficult to access surgically for gene therapy in adults, this means using gene editing on embryos.

Reference https://www.physicsforums.com/insights/can-gene-editing-eliminate-alzheimers-disease/

 

Utilising CRISPR to Generate Predictive Disease Models: a Case Study in Neurodegenerative Disorders


Dr. Bhuvaneish.T. Selvaraj  – Scottish Centre for Regenerative Medicine

http://www.crisprsummit.com/utilising-crispr-to-generate-predictive-disease-models-a-case-study-in-neurodegenerative-disorders

  • Introducing the latest developments in predictive model generation
  • Discover how CRISPR is being used to develop disease models to study and treat neurodegenerative disorders
  • In depth Q&A session to answer your most pressing questions

 

Turning On Genes, Systematically, with CRISPR/Cas9

http://www.genengnews.com/gen-news-highlights/turning-on-genes-systematically-with-crispr-cas9/81250697/

 

Scientists based at MIT assert that they can reliably turn on any gene of their choosing in living cells. [Feng Zhang and Steve Dixon]  http://www.genengnews.com/media/images/GENHighlight/Dec12_2014_CRISPRCas9GeneActivationSystem7838101231.jpg

With the latest CRISPR/Cas9 advance, the exhortation “turn on, tune in, drop out” comes to mind. The CRISPR/Cas9 gene-editing system was already a well-known means of “tuning in” (inserting new genes) and “dropping out” (knocking out genes). But when it came to “turning on” genes, CRISPR/Cas9 had little potency. That is, it had demonstrated only limited success as a way to activate specific genes.

A new CRISPR/Cas9 approach, however, appears capable of activating genes more effectively than older approaches. The new approach may allow scientists to more easily determine the function of individual genes, according to Feng Zhang, Ph.D., a researcher at MIT and the Broad Institute. Dr. Zhang and colleagues report that the new approach permits multiplexed gene activation and rapid, large-scale studies of gene function.

The new technique was introduced in the December 10 online edition of Nature, in an article entitled, “Genome-scale transcriptional activation by an engineered CRISPR-Cas9 complex.” The article describes how Dr. Zhang, along with the University of Tokyo’s Osamu Nureki, Ph.D., and Hiroshi Nishimasu, Ph.D., overhauled the CRISPR/Cas9 system. The research team based their work on their analysis (published earlier this year) of the structure formed when Cas9 binds to the guide RNA and its target DNA. Specifically, the team used the structure’s 3D shape to rationally improve the system.

In previous efforts to revamp CRISPR/Cas9 for gene activation purposes, scientists had tried to attach the activation domains to either end of the Cas9 protein, with limited success. From their structural studies, the MIT team realized that two small loops of the RNA guide poke out from the Cas9 complex and could be better points of attachment because they allow the activation domains to have more flexibility in recruiting transcription machinery.

Using their revamped system, the researchers activated about a dozen genes that had proven difficult or impossible to turn on using the previous generation of Cas9 activators. Each gene showed at least a twofold boost in transcription, and for many genes, the researchers found multiple orders of magnitude increase in activation.

After investigating single-guide RNA targeting rules for effective transcriptional activation, demonstrating multiplexed activation of 10 genes simultaneously, and upregulating long intergenic noncoding RNA transcripts, the research team decided to undertake a large-scale screen. This screen was designed to identify genes that confer resistance to a melanoma drug called PLX-4720.

“We … synthesized a library consisting of 70,290 guides targeting all human RefSeq coding isoforms to screen for genes that, upon activation, confer resistance to a BRAF inhibitor,” wrote the authors of the Nature paper. “The top hits included genes previously shown to be able to confer resistance, and novel candidates were validated using individual [single-guide RNA] and complementary DNA overexpression.”

A gene signature based on the top screening hits, the authors added, correlated with a gene expression signature of BRAF inhibitor resistance in cell lines and patient-derived samples. It was also suggested that large-scale screens such as the one demonstrated in the current study could help researchers discover new cancer drugs that prevent tumors from becoming resistant.

More at –  http://www.genengnews.com/gen-news-highlights/turning-on-genes-systematically-with-crispr-cas9/81250697/

 

Susceptibility and modifier genes in Portuguese transthyretin V30M amyloid polyneuropathy: complexity in a single-gene disease
Miguel L. Soares1,2, Teresa Coelho3,6, Alda Sousa4,5, …, Maria Joa˜o Saraiva2,5 and Joel N. Buxbaum1
Human Molec Gen 2005; 14(4): 543–553   http://dx.doi.org:/10.1093/hmg/ddi051
https://www.researchgate.net/profile/Isabel_Conceicao/publication/8081351_Susceptibility_and_modifier_genes_in_Portuguese_transthyretin_V30M_amyloid_polyneuropathy_complexity_in_a_single-gene_disease/links/53e123d70cf2235f352733b3.pdf

Familial amyloid polyneuropathy type I is an autosomal dominant disorder caused by mutations in the transthyretin (TTR ) gene; however, carriers of the same mutation exhibit variability in penetrance and clinical expression. We analyzed alleles of candidate genes encoding non-fibrillar components of TTR amyloid deposits and a molecule metabolically interacting with TTR [retinol-binding protein (RBP)], for possible associations with age of disease onset and/or susceptibility in a Portuguese population sample with the TTR V30M mutation and unrelated controls. We show that the V30M carriers represent a distinct subset of the Portuguese population. Estimates of genetic distance indicated that the controls and the classical onset group were furthest apart, whereas the late-onset group appeared to differ from both. Importantly, the data also indicate that genetic interactions among the multiple loci evaluated, rather than single-locus effects, are more likely to determine differences in the age of disease onset. Multifactor dimensionality reduction indicated that the best genetic model for classical onset group versus controls involved the APCS gene, whereas for late-onset cases, one APCS variant (APCSv1) and two RBP variants (RBPv1 and RBPv2) are involved. Thus, although the TTR V30M mutation is required for the disease in Portuguese patients, different genetic factors may govern the age of onset, as well as the occurrence of anticipation.

Autosomal dominant disorders may vary in expression even within a given kindred. The basis of this variability is uncertain and can be attributed to epigenetic factors, environment or epistasis. We have studied familial amyloid polyneuropathy (FAP), an autosomal dominant disorder characterized by peripheral sensorimotor and autonomic neuropathy. It exhibits variation in cardiac, renal, gastrointestinal and ocular involvement, as well as age of onset. Over 80 missense mutations in the transthyretin gene (TTR ) result in autosomal dominant disease http://www.ibmc.up.pt/~mjsaraiv/ttrmut.html). The presence of deposits consisting entirely of wild-type TTR molecules in the hearts of 10– 25% of individuals over age 80 reveals its inherent in vivo amyloidogenic potential (1).

FAP was initially described in Portuguese (2) where, until recently, the TTR V30M has been the only pathogenic mutation associated with the disease (3,4). Later reports identified the same mutation in Swedish and Japanese families (5,6). The disorder has since been recognized in other European countries and in North American kindreds in association with V30M, as well as other mutations (7).

TTR V30M produces disease in only 5–10% of Swedish carriers of the allele (8), a much lower degree of penetrance than that seen in Portuguese (80%) (9) or in Japanese with the same mutation. The actual penetrance in Japanese carriers has not been formally established, but appears to resemble that seen in Portuguese. Portuguese and Japanese carriers show considerable variation in the age of clinical onset (10,11). In both populations, the first symptoms had originally been described as typically occurring before age 40 (so-called ‘classical’ or early-onset); however, in recent years, more individuals developing symptoms late in life have been identified (11,12). Hence, present data indicate that the distribution of the age of onset in Portuguese is continuous, but asymmetric with a mean around age 35 and a long tail into the older age group (Fig. 1) (9,13). Further, DNA testing in Portugal has identified asymptomatic carriers over age 70 belonging to a subset of very late-onset kindreds in whose descendants genetic anticipation is frequent. The molecular basis of anticipation in FAP, which is not mediated by trinucleotide repeat expansions in the TTR or any other gene (14), remains elusive.

Variation in penetrance, age of onset and clinical features are hallmarks of many autosomal dominant disorders including the human TTR amyloidoses (7). Some of these clearly reflect specific biological effects of a particular mutation or a class of mutants. However, when such phenotypic variability is seen with a single mutation in the gene encoding the same protein, it suggests an effect of modifying genetic loci and/or environmental factors contributing differentially to the course of disease. We have chosen to examine age of onset as an example of a discrete phenotypic variation in the presence of the particular autosomal dominant disease-associated mutation TTR V30M. Although the role of environmental factors cannot be excluded, the existence of modifier genes involved in TTR amyloidogenesis is an attractive hypothesis to explain the phenotypic variability in FAP. ….

ATTR (TTR amyloid), like all amyloid deposits, contains several molecular components, in addition to the quantitatively dominant fibril-forming amyloid protein, including heparan sulfate proteoglycan 2 (HSPG2 or perlecan), SAP, a plasma glycoprotein of the pentraxin family (encoded by the APCS gene) that undergoes specific calcium-dependent binding to all types of amyloid fibrils, and apolipoprotein E (ApoE), also found in all amyloid deposits (15). The ApoE4 isoform is associated with an increased frequency and earlier onset of Alzheimer’s disease (Ab), the most common form of brain amyloid, whereas the ApoE2 isoform appears to be protective (16). ApoE variants could exert a similar modulatory effect in the onset of FAP, although early studies on a limited number of patients suggested this was not the case (17).

In at least one instance of senile systemic amyloidosis, small amounts of AA-related material were found in TTR deposits (18). These could reflect either a passive co-aggregation or a contributory involvement of protein AA, encoded by the serum amyloid A (SAA ) genes and the main component of secondary (reactive) amyloid fibrils, in the formation of ATTR.

Retinol-binding protein (RBP), the serum carrier of vitamin A, circulates in plasma bound to TTR. Vitamin A-loaded RBP and L-thyroxine, the two natural ligands of TTR, can act alone or synergistically to inhibit the rate and extent of TTR fibrillogenesis in vitro, suggesting that RBP may influence the course of FAP pathology in vivo (19). We have analyzed coding and non-coding sequence polymorphisms in the RBP4 (serum RBP, 10q24), HSPG2 (1p36.1), APCS (1q22), APOE (19q13.2), SAA1 and SAA2 (11p15.1) genes with the goal of identifying chromosomes carrying common and functionally significant variants. At the time these studies were performed, the full human genome sequence was not completed and systematic singlenucleotide polymorphism (SNP) analyses were not available for any of the suspected candidate genes. We identified new SNPs in APCS and RBP4 and utilized polymorphisms in SAA, HSPG2 and APOE that had already been characterized and shown to have potential pathophysiologic significance in other disorders (16,20–22). The genotyping data were analyzed for association with the presence of the V30M amyloidogenic allele (FAP patients versus controls) and with the age of onset (classical- versus late-onset patients). Multilocus analyses were also performed to examine the effects of simultaneous contributions of the six loci for determining the onset of the first symptoms.  …..

The potential for different underlying models for classical and late onset is supported by the MDR analysis, which produces two distinct models when comparing each class with the controls. One could view the two onset classes as unique diseases. If this is the case, then the failure to detect a single predictive genetic model is consistent with two related, but different, diseases. This is exactly what would be expected in such a case of genetic heterogeneity (28). Using this approach, a major gene effect can be viewed as a necessary, but not sufficient, condition to explain the course of the disease. Analyzing the cases but omitting from the analysis of phenotype the necessary allele, in this case TTR V30M, can then reveal a variety of important modifiers that are distinct between the phenotypes.

The significant comparisons obtained in our study cohort indicate that the combined effects mainly result from two and three-locus interactions involving all loci except SAA1 and SAA2 for susceptibility to disease. A considerable number of four-site combinations modulate the age of onset with SAA1 appearing in a majority of significant combinations in late-onset disease, perhaps indicating a greater role of the SAA variants in the age of onset of FAP.

The correlation between genotype and phenotype in socalled simple Mendelian disorders is often incomplete, as only a subset of all mutations can reliably predict specific phenotypes (34). This is because non-allelic genetic variations and/or environmental influences underlie these disorders whose phenotypes behave as complex traits. A few examples include the identification of the role of homozygozity for the SAA1.1 allele in conferring the genetic susceptibility to renal amyloidosis in FMF (20) and the association of an insertion/deletion polymorphism in the ACE gene with disease severity in familial hypertrophic cardiomyopathy (35). In these disorders, the phenotypes arise from mutations in MEFV and b-MHC, but are modulated by independently inherited genetic variation. In this report, we show that interactions among multiple genes, whose products are confirmed or putative constituents of ATTR deposits, or metabolically interact with TTR, modulate the onset of the first symptoms and predispose individuals to disease in the presence of the V30M mutation in TTR. The exact nature of the effects identified here requires further study with potential application in the development of genetic screening with prognostic value pertaining to the onset of disease in the TTR V30M carriers.

If the effects of additional single or interacting genes dictate the heterogeneity of phenotype, as reflected in variability of onset and clinical expression (with the same TTR mutation), the products encoded by alleles at such loci could contribute to the process of wild-type TTR deposition in elderly individuals without a mutation (senile systemic amyloidosis), a phenomenon not readily recognized as having a genetic basis because of the insensitivity of family history in the elderly.

 

Safety and Efficacy of RNAi Therapy for Transthyretin Amyloidosis

Coelho T, Adams D, Silva A, et al.
N Engl J Med 2013;369:819-29.    http://dx.doi.org:/10.1056/NEJMoa1208760

Transthyretin amyloidosis is caused by the deposition of hepatocyte-derived transthyretin amyloid in peripheral nerves and the heart. A therapeutic approach mediated by RNA interference (RNAi) could reduce the production of transthyretin.

Methods We identified a potent antitransthyretin small interfering RNA, which was encapsulated in two distinct first- and second-generation formulations of lipid nanoparticles, generating ALN-TTR01 and ALN-TTR02, respectively. Each formulation was studied in a single-dose, placebo-controlled phase 1 trial to assess safety and effect on transthyretin levels. We first evaluated ALN-TTR01 (at doses of 0.01 to 1.0 mg per kilogram of body weight) in 32 patients with transthyretin amyloidosis and then evaluated ALN-TTR02 (at doses of 0.01 to 0.5 mg per kilogram) in 17 healthy volunteers.

Results Rapid, dose-dependent, and durable lowering of transthyretin levels was observed in the two trials. At a dose of 1.0 mg per kilogram, ALN-TTR01 suppressed transthyretin, with a mean reduction at day 7 of 38%, as compared with placebo (P=0.01); levels of mutant and nonmutant forms of transthyretin were lowered to a similar extent. For ALN-TTR02, the mean reductions in transthyretin levels at doses of 0.15 to 0.3 mg per kilogram ranged from 82.3 to 86.8%, with reductions of 56.6 to 67.1% at 28 days (P<0.001 for all comparisons). These reductions were shown to be RNAi mediated. Mild-to-moderate infusion-related reactions occurred in 20.8% and 7.7% of participants receiving ALN-TTR01 and ALN-TTR02, respectively.

ALN-TTR01 and ALN-TTR02 suppressed the production of both mutant and nonmutant forms of transthyretin, establishing proof of concept for RNAi therapy targeting messenger RNA transcribed from a disease-causing gene.

 

Alnylam May Seek Approval for TTR Amyloidosis Rx in 2017 as Other Programs Advance


https://www.genomeweb.com/rnai/alnylam-may-seek-approval-ttr-amyloidosis-rx-2017-other-programs-advance

Officials from Alnylam Pharmaceuticals last week provided updates on the two drug candidates from the company’s flagship transthyretin-mediated amyloidosis program, stating that the intravenously delivered agent patisiran is proceeding toward a possible market approval in three years, while a subcutaneously administered version called ALN-TTRsc is poised to enter Phase III testing before the end of the year.

Meanwhile, Alnylam is set to advance a handful of preclinical therapies into human studies in short order, including ones for complement-mediated diseases, hypercholesterolemia, and porphyria.

The officials made their comments during a conference call held to discuss Alnylam’s second-quarter financial results.

ATTR is caused by a mutation in the TTR gene, which normally produces a protein that acts as a carrier for retinol binding protein and is characterized by the accumulation of amyloid deposits in various tissues. Alnylam’s drugs are designed to silence both the mutant and wild-type forms of TTR.

Patisiran, which is delivered using lipid nanoparticles developed by Tekmira Pharmaceuticals, is currently in a Phase III study in patients with a form of ATTR called familial amyloid polyneuropathy (FAP) affecting the peripheral nervous system. Running at over 20 sites in nine countries, that study is set to enroll up to 200 patients and compare treatment to placebo based on improvements in neuropathy symptoms.

According to Alnylam Chief Medical Officer Akshay Vaishnaw, Alnylam expects to have final data from the study in two to three years, which would put patisiran on track for a new drug application filing in 2017.

Meanwhile, ALN-TTRsc, which is under development for a version of ATTR that affects cardiac tissue called familial amyloidotic cardiomyopathy (FAC) and uses Alnylam’s proprietary GalNAc conjugate delivery technology, is set to enter Phase III by year-end as Alnylam holds “active discussions” with US and European regulators on the design of that study, CEO John Maraganore noted during the call.

In the interim, Alnylam continues to enroll patients in a pilot Phase II study of ALN-TTRsc, which is designed to test the drug’s efficacy for FAC or senile systemic amyloidosis (SSA), a condition caused by the idiopathic accumulation of wild-type TTR protein in the heart.

Based on “encouraging” data thus far, Vaishnaw said that Alnylam has upped the expected enrollment in this study to 25 patients from 15. Available data from the trial is slated for release in November, he noted, stressing that “any clinical endpoint result needs to be considered exploratory given the small sample size and the very limited duration of treatment of only six weeks” in the trial.

Vaishnaw added that an open-label extension (OLE) study for patients in the ALN-TTRsc study will kick off in the coming weeks, allowing the company to gather long-term dosing tolerability and clinical activity data on the drug.

Enrollment in an OLE study of patisiran has been completed with 27 patients, he said, and, “as of today, with up to nine months of therapy … there have been no study drug discontinuations.” Clinical endpoint data from approximately 20 patients in this study will be presented at the American Neurological Association meeting in October.

As part of its ATTR efforts, Alnylam has also been conducting natural history of disease studies in both FAP and FAC patients. Data from the 283-patient FAP study was presented earlier this year and showed a rapid progression in neuropathy impairment scores and a high correlation of this measurement with disease severity.

During last week’s conference call, Vaishnaw said that clinical endpoint and biomarker data on about 400 patients with either FAC or SSA have already been collected in a nature history study on cardiac ATTR. Maraganore said that these findings would likely be released sometime next year.

Alnylam Presents New Phase II, Preclinical Data from TTR Amyloidosis Programs
https://www.genomeweb.com/rnai/alnylam-presents-new-phase-ii-preclinical-data-ttr-amyloidosis-programs

 

Amyloid disease drug approved

Nature Biotechnology 2012; (3http://dx.doi.org:/10.1038/nbt0212-121b

The first medication for a rare and often fatal protein misfolding disorder has been approved in Europe. On November 16, the E gave a green light to Pfizer’s Vyndaqel (tafamidis) for treating transthyretin amyloidosis in adult patients with stage 1 polyneuropathy symptoms. [Jeffery Kelly, La Jolla]

 

Safety and Efficacy of RNAi Therapy for Transthyretin …

http://www.nejm.org/…/NEJMoa1208760?&#8230;

The New England Journal of Medicine

Aug 29, 2013 – Transthyretin amyloidosis is caused by the deposition of hepatocyte-derived transthyretin amyloid in peripheral nerves and the heart.

 

Alnylam’s RNAi therapy targets amyloid disease

Ken Garber
Nature Biotechnology 2015; 33(577)    http://dx.doi.org:/10.1038/nbt0615-577a

RNA interference’s silencing of target genes could result in potent therapeutics.

http://www.nature.com/nbt/journal/v33/n6/images/nbt0615-577a-I1.jpg

The most clinically advanced RNA interference (RNAi) therapeutic achieved a milestone in April when Alnylam Pharmaceuticals in Cambridge, Massachusetts, reported positive results for patisiran, a small interfering RNA (siRNA) oligonucleotide targeting transthyretin for treating familial amyloidotic polyneuropathy (FAP).  …

  1. Analysis of 589,306 genomes identifies individuals resilient to severe Mendelian childhood diseases

Nature Biotechnology 11 April 2016

  1. CRISPR-Cas systems for editing, regulating and targeting genomes

Nature Biotechnology 02 March 2014

  1. Near-optimal probabilistic RNA-seq quantification

Nature Biotechnology 04 April 2016

 

Translational Neuroscience: Toward New Therapies

https://books.google.com/books?isbn=0262029863

Karoly Nikolich, ‎Steven E. Hyman – 2015 – ‎Medical

Tafamidis for Transthyretin Familial Amyloid Polyneuropathy: A Randomized, Controlled Trial. … Multiplex Genome Engineering Using CRISPR/Cas Systems.

 

Is CRISPR a Solution to Familial Amyloid Polyneuropathy?

Author and Curator: Larry H. Bernstein, MD, FCAP

Originally published as

https://pharmaceuticalintelligence.com/2016/04/13/is-crispr-a-solution-to-familial-amyloid-polyneuropathy/

 

http://scholar.aci.info/view/1492518a054469f0388/15411079e5a00014c3d

FAP is characterized by the systemic deposition of amyloidogenic variants of the transthyretin protein, especially in the peripheral nervous system, causing a progressive sensory and motor polyneuropathy.

FAP is caused by a mutation of the TTR gene, located on human chromosome 18q12.1-11.2.[5] A replacement of valine by methionine at position 30 (TTR V30M) is the mutation most commonly found in FAP.[1] The variant TTR is mostly produced by the liver.[citation needed] The transthyretin protein is a tetramer.    ….

 

 

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Translational Gene Editing – June 16-17, 2016 in Boston, MA

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Learn More | Sponsorship & Exhibit Details | Register by April 29 & SAVE up to $200!

IMPROVING CRISPR FOR BETTER FUNCTIONAL SCREENING

Optimized sgRNA Libraries for Genetic Screens with CRISPR-Cas9
John Doench, Ph.D., Associate Director, Genetic Perturbation Platform, Broad Institute of Harvard and MIT

Optimizing CRISPR for Pooled Genome-Wide Functional Genetic Screens
Paul Diehl, Ph.D., Director, Business Development, Cellecta, Inc.

CRISPR-Cas9 Whole Genome Screening: Going Where No Screen Has Gone Before
Ralph Garippa, Ph.D., Director, RNAi Core Facility, Sloan-Kettering Institute, Memorial Sloan-Kettering Cancer Center

Cross-Species Synthetic Lethal Screens and Applications to Drug Discovery
Norbert Perrimon, Ph.D., Professor, Department of Genetics, Harvard Medical School and Investigator, Howard Hughes Medical Institute

Interactive Breakout Discussion Groups with Continental Breakfast
This session features various discussion groups that are led by a moderator/s who ensures focused conversations around the key issues listed. Attendees choose to join a specific group and the small, informal setting facilitates sharing of ideas and active networking. Continental breakfast is available for all participants.

Topic: CRISPR/Cas9 System for In vivo Drug Discovery
Moderator: Danilo Maddalo, Ph.D., Lab Head, ONC Pharmacology, Novartis Institutes for BioMedical Research

  • Impact of CRISPR/Cas9 system on in vivo mouse models
  • Application of the CRISPR/Cas9 system in in vivo screens
  • Technical limitations/safety issues

Topic: Getting Past CRISPR Pain Points
Moderators: John Doench, Ph.D., Associate Director, Genetic Perturbation Platform, Broad Institute of Harvard and MITStephanie Mohr, Ph.D., Lecturer, Genetics & Director of the Drosophila RNAi Screening Center, Harvard Medical School

  • Challenges and solutions for CRISPR gRNA design
  • Methods for detecting engineered changes

Topic: Cellular Delivery of CRISPR/Cas9
Moderator: Daniel E Bauer M.D., Ph.D., Assistant Professor of Pediatrics, Harvard Medical School and Staff Physician in Pediatric Hematology/Oncology, Boston Children’s Hospital and Dana-Farber Cancer Institute, Principal Faculty, Harvard Stem Cell Institute

GENE EDITING FOR SCREENING DISEASE PATHWAYS AND DRUG TARGETS

Scouring the Non-Coding Genome by Saturating Edits
Daniel E. Bauer, M.D., Ph.D., Assistant Professor of Pediatrics, Harvard Medical School and Staff Physician in Pediatric Hematology/Oncology, Boston Children’s Hospital and Dana-Farber Cancer Institute, Principal Faculty, Harvard Stem Cell Institute

Parallel shRNA and CRISPR/Cas9 Screens Reveal Biology of Stress Pathways and Identify Novel Drug Targets
Michael Bassik, Ph.D., Assistant Professor, Department of Genetics, Stanford University

BUILDING THE CRISPR TOOLBOX

Beyond Cas9: Discovering Single Effector CRISPR Tools
Jonathan Gootenberg, Member, Laboratories of Dr. Aviv Regev and Dr. Feng Zhang, Department of Systems Biology, Harvard Medical School, and Broad Institute of Harvard and MIT

CRISPR-Cas9 Genome Editing Improves Sub-Cellular Localization Studies
Netanya Y. Spencer, M.D., Ph.D., Research Fellow in Medicine, Joslin Diabetes Center, Harvard Medical School

TECHNOLOGY PANEL: Trends in CRISPR Technologies
Panelists to be Announced

This panel will bring together 2-3 technical experts from leading technology and service companies to discuss trends and improvements in CRISPR libraries, reagents and platforms that users can expect to see in the near future. (Opportunities Available for Sponsoring Panelists)

APPLICATIONS OF CRISPR FOR DRUG DISCOVERY

Use of CRISPR and Other Genomic Technologies to Advance Drug Discovery
Namjin Chung, Ph.D., Head, Functional Genomics Platform, Discovery Research, AbbVie, Inc.

Application of Genome Editing Tools to Model Human Genetics Findings in Drug Discovery
Myung Shin, Ph.D., Senior Principal Scientist, Genetics and Pharmacogenomics, Merck & Co. Inc.

In vivo Application of the CRISPR/Cas9 Technology for Translational Research
Danilo Maddalo, Ph.D., Lab Head, ONC Pharmacology, Novartis Institutes for BioMedical Research

DEVELOPING TOOLS FOR BETTER TRANSLATION

Improving CRISPR-Cas9 Precision through Tethered DNA-Binding Domains
Scot A. Wolfe, Ph.D., Associate Professor, Department of Molecular, Cell and Cancer Biology, University of Massachusetts Medical School

Nucleic Acid Delivery Systems for RNA Therapy and Gene Editing
Daniel G. Anderson, Ph.D., Professor, Department of Chemical Engineering, Institute for Medical Engineering & Science, Harvard-MIT Division of Health Sciences & Technology and David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology

Translating CRISPR/Cas9 into Novel Medicines
Alexandra Glucksmann, Ph.D., COO, Editas Medicine

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RNAi Discovery

Larry H. Bernstein, MD, FCAP, Curator

LPBI

 

RETURN TO THE RNAi WORLD: RETHINKING GENE EXPRESSION AND EVOLUTION

Nobel Lecture, December 8, 2006 by Craig C. Mello

Abstracted

 

It’s wonderful to be here today, I would like to start with the most important part, by saying thank you. First of all, I want to thank Andy Fire for being such a tremendous colleague, friend and collaborator going back over the years. Without Andy I definitely wouldn’t be here today. I need to thank the University of Massachusetts for providing for my laboratory, for believing in me and for giving me not only a place and money, but great colleagues with whom to pursue my research.

I want to get down to the theme of my talk today, which really is, in part, about how we continually underestimate the complexity of life. It’s the correction of these underestimations that is quite often what this prize is really recognizing. As science progresses, our knowledge expands, we think we understand, and too often we become overconfident. The fact is, I think we almost always underestimate the complexity of life and of nature. Today has been a true celebration of that beauty and complexity.

If one looks carefully, the complexity of living things becomes strikingly clear. Consider for example the natural environment of C. elegans. Figure 3 is an electron micrograph taken by George Barron, who works on nematophagous fungi. The unfortunate worm shown here has become ensnared in a trap set by a fungus that preys on nematodes….These fungi can sense the motion or contact of a worm and, after the worm has entered its lariat, the fungus inflates it to constrict the snare around the animal, trapping it.

In Roger Kornberg’s talk, we heard about an RNA polymerase that can transcribe the DNA to produce RNA copies of the genetic information. These copies provide templates for the polymerization of the proteins through another elaborate and really beautiful process, called translation.

Does the DNA sequence information control all of the events in the cell? Cells are constantly responding to their environment and to surrounding cells, and these external influences can alter the cell in heritable ways that do not require changes in the primary sequence information in the DNA. Consider the early C. elegans embryo. During these early divisions, maternal mRNA and protein products that are stored in the egg direct numerous cellcell signaling and differentiation events that give rise to the multicellular organism. These are exemplified by the distribution of the PIE-1 protein (Figure 4). PIE-1 tracks with, and is essential for, germline specification…..two daughter cells differ with respect to their content of maternally provided products, like PIE-1. These products, in turn, can direct the subsequent development of these cells such that, once differentiated in this way, these cells remain committed to their specific tasks in the animal through numerous rounds of cell division. These remarkably stable differentation events can be maintained for the entire life of an organism without any underlying changes in the DNA sequence.

How do developing cells, all with the same DNA content, lock in different programs of gene expression that are stable through so many rounds of cell division? One possibility, as I will discuss below, is that mechanisms related to those that mediate RNA interference have a role in this process. It has been suggested that the origin of life on Earth may have begun with selfreplica­ting nucleic acid polymers that were more similar chemically to RNA than to DNA, a classic hypothesis referred to as the “RNA World” hypothesis.

….primordial germ cells in the common metazoan (probably worm-like) ancestor of worms and humans, and going even farther back are direct descendants of the hypothetical self-replicating RNA molecules that gave rise to all life on Earth some 3.5 billion years ago….. RNAi itself is at least one billion years old. Biological mechanisms are far more constant than the positions of continents on our planet. That fact and the implicit concept of deep time are among the most profound discoveries of science.

1) there is a particle, containing siRNAs, for some traits; 2) these siRNAs can grow and multiply independent of cell division; 3) both the nucleus and the cytoplasm can contain the siRNAs; 4) a given siRNA may be represented by many replicas; and 5) that during cell division the daughter cells may receive different kinds and numbers of siRNAs through unequal cell division.

…here’s what CBS Evening News came up with (Figure 5). In the movie, the double stranded RNA flies onto the scene then opens at one end and begins to open and close as though it’s chewing. Defective genes, shaped like colored cheese puffs, then begin to fly into the mouth of the RNA from the left. The RNA is literally eating the DNA for lunch. Now, Andy and I knew that RNA interference was something incredible when we started working on it, but we really didn’t have any idea that it was this incredible.

Public broadcasting has the luxury of an audience that tends to have a bit more patience, and they came up with a 15 minute segment and another strategy, “the cop”, to explain RNAi (Figure 6). They describe a little policeman who looks out for viruses and other misbehavior in the cell. When he sees double-stranded RNA he realizes something is not right. He then goes on to use “enzymatic kung fu” to destroy not only the dsRNA with that sequence, but all RNAs with that sequence that he encounters in the cell.

I like both of these movies because they illustrate a really important concept; that is, that RNAi is an active process, that there is an organismal response to the dsRNA [4]. We realized this at an early stage, because, first of all, as Andy mentioned, the silencing was heritable. RNA injected into an animal resulted in silencing that was transmitted to progeny and even transmitted through crosses for multiple generations via the egg or the sperm. So, the interference mechanism can be initiated in one generation and then transmitted in the germline.

The inheritance properties and systemic nature of RNAi, along with its remarkable potency in C. elegans, all pointed toward an active organismal response to the double-stranded RNA. What we wanted to do immediately, upon realizing that there was an active response in the organism, was to find the genes in the animal that encode that response. Therefore, we set out to use the powerful genetics of C. elegans to look for mutant strains defective in RNAi. We imagined that these mutants would define genes required for the recognition of the foreign double-stranded RNA, genes required for the transport of the silencing signal from cell to cell, genes required for the amplification of silencing, and genes required for the silencing apparatus itself.

Hiroaki was able to identify numerous mutants. Some of these lacked the RNAi response and had no other obvious phenotypes, like rde-1 and rde-4. However, some of his mutants had additional defects, including a very striking phenotype in which the transposons, which are selfreplicating DNA elements present in the genomes of all organisms, became hyperactive, causing mutations by jumping from place to place in the genome. In addition, these same mutants had a reduced tendency to silence transgenes in the germline (transgenes are genes that are experimentally introduced into the organism). In normal worms, transgenes have the vexing property of becoming silent after introduction into the animal experimentally. The same mutants with activated transposons also exhibited activation of transgenes in the germline. These observations suggested that the normal physiological function of RNAi might be to defend cells against the potentially damaging effects of transposons and other foreign genetic elements (perhaps including viruses)…..The rde- 1 and rde-4 mutants, as I indicated earlier, had no other phenotypes. They Figure 7. Hiroaki Tabara 249 were strongly deficient in RNAi in response to double-stranded RNA, but the transposon silencing and the transgene silencing mechanisms were still functioning in these mutant strains. These observations indicated to us, even at that very early stage of our analysis, the existence of some additional, very interesting complexity.

Hiroaki cloned the rde-1 gene and showed that it encodes a highly conserved protein that we now refer to as an Argonaute protein [6]. RDE-1 was an interesting protein for a couple of reasons. It had highly conserved domains found in related genes in organisms as diverse as plants and humans, and yet nothing was known about the enzymatic activities or the biological functions of these domains. This was a very exciting time in the laboratory. We at last had a gene that we knew was involved in the mechanism. Furthermore, previous work on one gene closely related to RDE-1 from Drosophila had linked this gene family to an epigenetic silencing pathway in the fruit fly [7, 8], and work in plants had linked a member of the family to the control of development [9]. Very shortly after our paper was published, Carlo Cogoni and Giuseppe Macino [10] published a very nice paper implicating an RDE-1 family member in silencing triggered by the introduction of a transgene in the fungus Neurospora. So from these findings in other organisms, and from Hiroaki’s genetics, we hypothesized that there may be other types of triggers that initiate related silencing pathways either through natural developmental mechanisms or in response to transposons and transgenes.

…, the lin-4 gene appeared to encode two RNA products: an ~70 nucleotide-long RNA capable of forming a double-stranded RNA molecule with a hairpin-like structure, and a single-stranded 22 nucleotide RNA that appeared to be derived from this longer RNA (Figure 10). This short RNA was capable of binding directly to sites in the transcript of the lin-14 gene, a gene that is negatively regulated by lin-4 during the normal course of worm development.

Even before we identified RDE-1, we were interested in the possibility of a relationship between the RNAi pathway and the lin-4 pathway. Indeed, Hiroaki had raised the concern that RNAi-defective mutants could be hard to recover as viable strains since they might also cause disruption of the lin-4 pathway. Making all of these possibilities even more exciting – while we were conducting genetic screens for RNAi deficient strains, beautiful work was published by Hamilton and Baulcombe [12], linking small RNAs of ~21 nucleotides to viral gene silencing in plants, and by Gary Ruvkun’s lab, identifying a second lin-4- like worm gene, let-7 [13]. Whereas lin-4 was a worm-specific gene, it turned out that the let-7 gene had homologs in every animal, including humans. Remarkably, e­ve­ry single nucleotide in the twenty-one nucleotide mature let-7 RNA products from the worm and human were identical to each other. The conservation of let-7 initiated a gold rush to find small RNA encoding genes, now referred to as micro-RNA genes, in the genomes of numerous organisms.

….activities present in Drosophila cells could process double-stranded RNA into tiny RNAs approximately 21 nucleotides long. Tom Tuschl and colleagues were the first to show that these small RNAs could silence gene expression in vertebrate cells [16]. Thus genetic studies in worms had identified small RNAs as silencing agents beginning in 1993, experimental studies of virus infections in plants identified small RNAs accumulating in infected plants, biochemical studies in fly extracts identified small RNAs in extracts, and finally experimental studies identified silencing activity in cellular assays with vertebrate cells. But were these small RNA molecules only similar in size, or did their similarity extend to mechanism?

Alla’s work provided an answer. When Alla knocked out alg-1 and alg-2, she observed a phenotype that was very similar to that observed when you knock out let-7. To confirm this connection we collaborated with Gary Ruvkun and Amy Pasquinelli, who had recently developed probes for following the processing of the lin-4 and let-7 precursor RNAs into their mature 21 nucleotide RNAs. In wild-type animals, the precursor forms are barely detectable. However, we found that, after inactivation of alg-1 and -2, this precursor accumulates to high levels while the product, the mature twenty-one nucleotide RNA, is greatly diminished [17] (Figure 11).

We also looked at the involvement of Dicer in this process. Dicer was identified by Greg Hannon’s lab as a nuclease required for processing long do­uble-stranded RNA into approximately 21-nucleotide fragments in Drosophila cells. We were able to show, as did several other groups [18, 19], that when you knock out Dicer you also see defects in the processing of these micro­RNAs (Figure 11). With these findings, the first link was established between RNA interference and a natural developmental mechanism for regulating gene expression. This was extremely exciting, and we envisioned a model (Figure 12), in which the RNAi and microRNA pathways utilized different members of the RDE-1 family and converged on Dicer. Downstream of Dicer these pathways appeared to diverge again, through the action of unknown effectors that direct different types of silencing, including mRNA destruction, transcriptional silencing and inhibition of translation. And yet, we still had not identified the RDE-1 family member involved in transposon and transgene silencing.

At that time we thought of the RDE-1 family members (also known as Argonaute proteins) as initiators of the silencing pathways. Genetic stu­dies had placed RDE-1 at an upstream step in the pathway and, as I showed you, ALG-1 and -2 are required for processing the microRNA precursors. However, there was mounting evidence that these proteins might also function downstream in the silencing step. Definitive support for this idea came from Greg Hannon’s group through a collaboration with Ji-Joon Song and Leemor Joshua-Tor at Cold Spring Harbor [20]. They showed that Argonaute proteins have structural similarity to an enzyme domain that can cut RNA, and they presented a model for how Argonaute proteins can bind the ends of the short RNAs and utilize the sequence information to find and destroy target mRNAs in the cell. These studies demonstrated that Argonaute proteins represent the long sought “slicer” activity (or the cop) that lies at the heart of the RNA-induced silencing pathway. We were surprised to learn that RDE-1 was probably the slicer enzyme because our genetic studies had placed RDE-1 activity at an upstream step in the pathway. However, we realized that this observation could be explained if Argonautes function more than once during RNAi in C. elegans. For example (Figure 13), we imagined that RDE-1 could function along with small RNAs derived from processing of the trigger dsRNA in an initial round of target mRNA cleavage. The cleaved target mRNA could then serve as a template for an RNA-dependent RNA polymerase that produces new siRNAs that could, in turn, interact with other Argonautes to mediate efficient silencing of the gene.

The last concept I want to discuss relates to the question of how RNAi can interact with chromatin to silence genes, and the potential importance of this mechanism for gene regulation during both development and evolution. As indicated earlier in my talk, many of the genes involved in RNAi are also required for the silencing of transgenes in the germline.

….Beautiful direct evidence for a link between RNAi and chromatin silencing has more recently come from work in the fission yeast S. pombe, where a complex containing an Argonaute protein and known chromatin interacting factors has been shown to interact directly with silenced genes in the nucleus [24]. To explain how RNAi could regulate DNA directly, I have to tell you a little bit about the physiological nature of DNA inside cells. Your DNA isn’t just lying around by itself. The unit of packaging for DNA is a protein structure called the nucleosome. The DNA wraps around the nucleosome twice, and the nucleosomes are in turn wrapped up and packaged into even thicker fibers. Chromosomes are composed of these protein/DNA fibers, also referred to as chromatin. Partly, what’s achieved by packaging the DNA into chromatin is a silencing effect. Structural stu­dies of the nucleosome core suggest that short protein tails stick out past the DNA in such a way that they are readily accessible for modification [25].

 

 

 

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Gene-Silencing and Gene-Disabling in Pharmaceutical Development

Larry H. Bernstein, MD, FCAP, Curator

LPBI

 

Down and Out with RNAi and CRISPR

http://www.genengnews.com/gen-articles/down-and-out-with-rnai-and-crispr/5619/

 

RNA interference (RNAi) silences, or knocks down, the translation of a gene by inducing degradation of a gene target’s transcript. To advance RNAi applications, Thermo Fisher Scientific has developed two types of small RNA molecules: short interfering RNAs and microRNAs. The company also offers products for RNAi analysis in vitro and in vivo, including libraries for high-throughput applications.

 

Genes can be knocked down with RNA interference (RNAi) or knocked out with CRISPR-Cas9. RNAi, the screening workhorse, knocks down the translation of genes by inducing rapid degradation of a gene target’s transcript.

CRISPR-Cas9, the new but already celebrated genome-editing technology, cleaves specific DNA sequences to render genes inoperative. Although mechanistically different, the two techniques complement one another, and when used together facilitate discovery and validation of scientific findings.

RNAi technologies along with other developments in functional genomics screening were discussed by industry leaders at the recent Discovery on Target conference. The conference, which emphasized the identification and validation of novel drug targets and the exploration of unknown cellular pathways, included a symposium on the development of CRISPR-based therapies.

RNAi screening can be performed in either pooled or arrayed formats. Pooled screening provides an affordable benchtop option, but requires back-end deconvolution and deep sequencing to identify the shRNA causing the specific phenotype. Targets are much easier to identify using the arrayed format since each shRNA clone is in an individual well.

“CRISPR complements RNAi screens,” commented Ryan Raver, Ph.D., global product manager of functional genomics at Sigma-Aldrich. “You can do a whole genome screen with either small interfering RNA (siRNA) or small hairpin RNA (shRNA), then validate with individual CRISPRs to ensure it is a true result.”

A powerful and useful validation method for knockdown or knockout studies is to use lentiviral open reading frames (ORFs) for gene re-expression, for rescue experiments, or to detect whether the wild-type phenotype is restored. However, the ORF randomly integrates into the genome. Also, with this validation technique, gene expression is not acting under the endogenous promoter. Accordingly, physiologically relevant levels of the gene may not be expressed unless controlled for via an inducible system.

In the future, CRISPR activators may provide more efficient ways to express not only wild-type but also mutant forms of genes under the endogenous promoter.

Choice of screening technique depends on the researcher and the research question. Whole gene knockout may be necessary to observe a phenotype, while partial knockdown might be required to investigate functions of essential or lethal genes. Use of both techniques is recommended to identify all potential targets.

For example, recently, a whole genome siRNA screen on a human glioblastoma cell line identified a gene, known as FAT1, as a negative regulator of apoptosis. A CRISPR-mediated knockout of the gene also conferred sensitivity to death receptor–induced apoptosis with an even stronger phenotype, thereby validating FAT1’s new role and link to extrinsic apoptosis, a new model system.

Dr. Raver indicated that next-generation RNAi libraries that are microRNA-adapted might have a more robust knockdown of gene expression, up to 90–95% in some cases. Ultracomplex shRNA libraries help to minimize both false-negative and false-positive rates by targeting each gene with ~25 independent shRNAs and by including thousands of negative-control shRNAs.

Recently, a relevant paper emerged from the laboratory of Jonathan Weissman, Ph.D., a professor of cellular and molecular pharmacology at the University of California, San Francisco. The paper described how a new ultracomplex pooled shRNA library was optimized by means of a microRNA-adapted system. This system, which was able to achieve high specificity in the detection of hit genes, produced robust results. In fact, they were comparable to results obtained with a CRISPR pooled screen. Members of the Weissman group systematically optimized the promoter and microRNA contexts for shRNA expression along with a selection of guide strands.

Using a sublibrary of proteostasis genes (targeting 2,933 genes), the investigators compared CRISPR and RNAi pooled screens. Data showed 48 hits unique to RNAi, 40 unique to CRISPR, and an overlap of 21 hits (with a 5% false discovery rate cut-off). Together, the technologies provided a more complete research story.

 

 

“RNA screens are well accepted and will continue to be used, but it is important biologically that researchers step away from the RNA mechanism to further study and validate their hits to eliminate potential bias,” explained Louise Baskin, senior product manager, Dharmacon, part of GE Healthcare. “The natural progression is to adopt CRISPR in the later stages.”

RNAi uses the cell’s endogenous mechanism. All of the components required for gene knockdown are already within the cell, and the delivery of the siRNA starts the process. With the CRISPR gene-editing system, which is derived from a bacterial immune defense system, delivery of both the guide RNA and the Cas9 nuclease, often the rate limiter in terms of knockout efficiency, are required.

 

Arrayed CRISPR Screens

Synthetic crRNA:tracrRNA reagents can be used in a similar manner to siRNA reagents for assessment of phenotypes in a cell population. Top row: A reporter cell line stably expressing Cas9 nuclease was transfected with GE Dharmacon’s Edit-R synthetic crRNA:tracrRNA system, which was used to target three positive control genes (PSMD7, PSMD14, and VCP) and a negative control gene (PPIB). Green cells indicate EGFP signaling occuring as a result of proteasome pathway disruption. Bottom row: A siGENOME siRNA pool targeting the same genes was used in the same reporter cell line.

 

In pooled approaches, the cell has to either drop out or overexpress so that it is sortable, limiting the types of addressable biological questions. A CRISPR-arrayed approach opens up the door for use of other analytical tools, such as high-content imaging, to identify hits of interest.

To facilitate use of CRISPR, GE recently introduced Dharmacon Edit-R synthetic CRISPR RNA (crRNA) libraries that can be used to carry out high-throughput arrayed analysis of multiple genes. Rather than a vector- or plasmid-based gRNA to guide the targeting of the Cas9 cleavage, a synthetic crRNA and tracrRNA are used. These algorithm-designed crRNA reagents can be delivered into the cells very much like siRNA, opening up the capability to screen multiple target regions for many different genes simultaneously.

GE showed a very strong overlap between CRISPR and RNAi using this arrayed approach to validate RNAi screen hits with synthetic crRNA. The data concluded that CRISPR can be used for medium- or high-throughput validation of knockdown studies.

“We will continue to see a lot of cooperation between RNAi and gene editing,” declared Baskin. “Using the CRISPR mechanism to knockin could introduce mutations to help understand gene function at a much deeper level, including a more thorough functional analysis of noncoding genes.

“These regulatory RNAs often act in the nucleus to control translation and transcription, so to knockdown these genes with RNAi would require export to the cytoplasm. Precision gene editing, which takes place in the nucleus, will help us understand the noncoding transcriptome and dive deeper into how those genes regulate disease progression, cellular development and other aspects of human health and biology.”

 

Tool Selection

The functional genomics tool should fit the specific biology; the biology should not be forced to fit the tool. Points to consider include the regulation of expression, the cell line or model system, as well as assay scale and design. For example, there may be a need for regulatable expression. There may be limitations around the cell line or model system. And assay scale and design may include delivery conditions and timing to optimally complete perturbation and reporting.

“Both RNAi- and CRISPR-based gene modulation strategies have pros and cons that should be considered based on the biology of the genes being studied,” commented Gwen Fewell, Ph.D., chief commercial officer, Transomic Technologies.

RNAi reagents, which can produce hypomorphic or transient gene-suppression states, are well known for their use in probing drug targets. In addition, these reagents are enriching studies of gene function. CRISPR-Cas9 reagents, which produce clean heterozygous and null mutations, are important for studying tumor suppressors and other genes where complete loss of function is desired.

 

Schematic of a pooled shRNA screening workflow developed by Transomic Technologies. Cells are transduced, and positive or negative selection screens are performed. PCR amplification and sequencing of the shRNA integrated into the target cell genome allows the determination of shRNA representation in the population.

 

Timing to readout the effects of gene perturbation must be considered to ensure that the biological assay is feasible. RNAi gene knockdown effects can be seen in as little as 24–72 hours, and inducible and reversible gene knockdown can be realized. CRISPR-based gene knockout effects may become complete and permanent only after 10 days.

Both RNAi and CRISPR reagents work well for pooled positive selection screens; however, for negative selection screens, RNAi is the more mature tool. Current versions of CRISPR pooled reagents can produce mixed populations containing a fraction of non-null mutations, which can reduce the overall accuracy of the readout.

To meet the needs of varied and complex biological questions, Transomic Technologies has developed both RNAi and CRISPR tools with options for optimal expression, selection, and assay scale. For example, the company’s shERWOOD-UltramiR shRNA reagents incorporate advances in design and small RNA processing to produce increased potency and specificity of knockdown, particularly important for pooled screens.

Sequence-verified pooled shRNA screening libraries provide flexibility in promoter choice, in vitro formats, in vivo formats, and a choice of viral vectors for optimal delivery and expression in biologically relevant cell lines, primary cells or in vivo.

The company’s line of lentiviral-based CRISPR-Cas9 reagents has variable selectable markers such that guide RNA- and Cas9-expressing vectors, including inducible Cas9, can be co-delivered and selected for in the same cell to increase editing efficiency. Promoter options are available to ensure expression across a range of cell types.

“Researchers are using RNAi and CRISPR reagents individually and in combination as cross-validation tools, or to engineer CRISPR-based models to perform RNAi-based assays,” informs Dr. Fewell. “Most exciting are parallel CRISPR and RNAi screens that have tremendous potential to uncover novel biology.”

 

Converging Technologies

The convergence of RNAi technology with genome-editing tools, such as CRISPR-Cas9 and transcription activator-like effector nucleases, combined with next-generation sequencing will allow researchers to dissect biological systems in a way not previously possible.

“From a purely technical standpoint, the challenges for traditional RNAi screens come down to efficient delivery of the RNAi reagents and having those reagents provide significant, consistent, and lasting knockdown of the target mRNAs,” states Ross Whittaker, Ph.D., a product manager for genome editing products at Thermo Fisher Scientific. “We have approached these challenges with a series of reagents and siRNA libraries designed to increase the success of RNAi screens.”

Thermo Fisher’ provides lipid-transfection RNAiMax reagents, which effectively deliver siRNA. In addition, the company’s Silencer and Silencer Select siRNA libraries provide consistent and significant knockdown of the target mRNAs. These siRNA libraries utilize highly stringent bioinformatic designs that ensure accurate and potent targeting for gene-silencing studies. The Silencer Select technology adds a higher level of efficacy and specificity due to chemical modifications with locked nucleic acid (LNA) chemistry.

The libraries alleviate concerns for false-positive or false-negative data. The high potency allows less reagent use; thus, more screens or validations can be conducted per library.

Dr. Whittaker believes that researchers will migrate regularly between RNAi and CRISPR-Cas9 technology in the future. CRISPR-Cas9 will be used to create engineered cell lines not only to validate RNAi hits but also to follow up on the underlying mechanisms. Cell lines engineered with CRISPR-Cas9 will be utilized in RNAi screens. In the long term, CRISPR-Cas9 screening will likely replace RNAi screening in many cases, especially with the introduction of arrayed CRISPR libraries.

 

Validating Antibodies with RNAi

Unreliable antibody specificity is a widespread problem for researchers, but RNAi is assuaging scientists’ concerns as a validation method.

The procedure introduces short hairpin RNAs (shRNAs) to reduce expression levels of a targeted protein. The associated antibody follows. With its protein knocked down, a truly specific antibody shows dramatically reduced or no signal on a Western blot. Short of knockout animal models, RNAi is arguably the most effective method of validating research antibodies.

The method is not common among antibody suppliers—time and cost being the chief barriers to its adoption, although some companies are beginning to embrace RNAi validation.

“In the interest of fostering better science, Proteintech felt it was necessary to implement this practice,” said Jason Li, Ph.D., founder and CEO of Proteintech Group, which made RNAi standard protocol in February 2015. “When researchers can depend on reproducibility, they execute more thorough experiments and advance the treatment of human diseases and conditions.”

 

Down and Out with RNAi and CRISPR

Genes can be knocked down with RNA interference (RNAi) or knocked out with CRISPR-Cas9. RNAi, the screening workhorse, knocks down the translation of genes by inducing rapid degradation of a gene target’s transcript.

RNA-Based Therapeutics and Vaccines

RNA-based biopharmaceuticals, which includes therapeutics and vaccines, is a relatively new class of treatment and prophylactic for a number of chronic and rare diseases, including cancer, diabetes, tuberculosis, and certain cardiovascular conditions. The field holds great promise in the prevention and treatment of these diseases as demonstrated by early-phase clinical trials as well as significant investment by the drug development community.

Ready, Aim, CRISPR (or RNAi)

Recent progress in probing gene function via the RNAi and CRISPR methods were a strong theme of the Discovery On Target conference. Both methods enable researchers to impair the function of a targeted gene.

Masked RNAi Drug Slips through Membrane, Sheds Guise within Cell

For small interfering RNA, approaching a cell is like walking up to the door of an old speakeasy. Such doors were heavily reinforced and had a narrow, built-in sliding panel at eye level, and if the eyes peering out though the open panel didn’t like the look of you, well, you were not getting inside. Failing to gain entry is something that happens all too frequently to small interfering RNAs, which admittedly are anything but “life of the party” types.

Read Full Post »


RNAi, CRISPR and Gene Expression

Larry H. Bernstein, MD, FCAP, Curator

LPBI

 

Down and Out with RNAi and CRISPR

Gene-Silencing and Gene-Disabling Techniques Are Moving To the Heart of Drug Discovery

  • Click Image To Enlarge +
    RNA interference (RNAi) silences, or knocks down, the translation of a gene by inducing degradation of a gene target’s transcript. To advance RNAi applications, Thermo Fisher Scientific has developed two types of small RNA molecules: short interfering RNAs and microRNAs. The company also offers products for RNAi analysis in vitro and in vivo, including libraries for high-throughput applications.

     

     

     

     

     

     

     

     

    Genes can be knocked down with RNA interference (RNAi) or knocked out with CRISPR-Cas9. RNAi, the screening workhorse, knocks down the translation of genes by inducing rapid degradation of a gene target’s transcript.

    CRISPR-Cas9, the new but already celebrated genome-editing technology, cleaves specific DNA sequences to render genes inoperative. Although mechanistically different, the two techniques complement one another, and when used together facilitate discovery and validation of scientific findings.

    RNAi technologies along with other developments in functional genomics screening were discussed by industry leaders at the recent Discovery on Target conference. The conference, which emphasized the identification and validation of novel drug targets and the exploration of unknown cellular pathways, included a symposium on the development of CRISPR-based therapies.

    RNAi screening can be performed in either pooled or arrayed formats. Pooled screening provides an affordable benchtop option, but requires back-end deconvolution and deep sequencing to identify the shRNA causing the specific phenotype. Targets are much easier to identify using the arrayed format since each shRNA clone is in an individual well.

    “CRISPR complements RNAi screens,” commented Ryan Raver, Ph.D., global product manager of functional genomics at Sigma-Aldrich. “You can do a whole genome screen with either small interfering RNA (siRNA) or small hairpin RNA (shRNA), then validate with individual CRISPRs to ensure it is a true result.”

    A powerful and useful validation method for knockdown or knockout studies is to use lentiviral open reading frames (ORFs) for gene re-expression, for rescue experiments, or to detect whether the wild-type phenotype is restored. However, the ORF randomly integrates into the genome. Also, with this validation technique, gene expression is not acting under the endogenous promoter. Accordingly, physiologically relevant levels of the gene may not be expressed unless controlled for via an inducible system.

    In the future, CRISPR activators may provide more efficient ways to express not only wild-type but also mutant forms of genes under the endogenous promoter.

    Choice of screening technique depends on the researcher and the research question. Whole gene knockout may be necessary to observe a phenotype, while partial knockdown might be required to investigate functions of essential or lethal genes. Use of both techniques is recommended to identify all potential targets.

    For example, recently, a whole genome siRNA screen on a human glioblastoma cell line identified a gene, known as FAT1, as a negative regulator of apoptosis. A CRISPR-mediated knockout of the gene also conferred sensitivity to death receptor–induced apoptosis with an even stronger phenotype, thereby validating FAT1’s new role and link to extrinsic apoptosis, a new model system.

    Dr. Raver indicated that next-generation RNAi libraries that are microRNA-adapted might have a more robust knockdown of gene expression, up to 90–95% in some cases. Ultracomplex shRNA libraries help to minimize both false-negative and false-positive rates by targeting each gene with ~25 independent shRNAs and by including thousands of negative-control shRNAs.

    Recently, a relevant paper emerged from the laboratory of Jonathan Weissman, Ph.D., a professor of cellular and molecular pharmacology at the University of California, San Francisco. The paper described how a new ultracomplex pooled shRNA library was optimized by means of a microRNA-adapted system. This system, which was able to achieve high specificity in the detection of hit genes, produced robust results. In fact, they were comparable to results obtained with a CRISPR pooled screen. Members of the Weissman group systematically optimized the promoter and microRNA contexts for shRNA expression along with a selection of guide strands.

    Using a sublibrary of proteostasis genes (targeting 2,933 genes), the investigators compared CRISPR and RNAi pooled screens. Data showed 48 hits unique to RNAi, 40 unique to CRISPR, and an overlap of 21 hits (with a 5% false discovery rate cut-off). Together, the technologies provided a more complete research story.

    Arrayed CRISPR Screens

  • Click Image To Enlarge +
    Synthetic crRNA:tracrRNA reagents can be used in a similar manner to siRNA reagents for assessment of phenotypes in a cell population. Top row: A reporter cell line stably expressing Cas9 nuclease was transfected with GE Dharmacon’s Edit-R synthetic crRNA:tracrRNA system, which was used to target three positive control genes (PSMD7, PSMD14, and VCP) and a negative control gene (PPIB). Green cells indicate EGFP signaling occuring as a result of proteasome pathway disruption. Bottom row: A siGENOME siRNA pool targeting the same genes was used in the same reporter cell line.

     

     

     

     

     

     

     

     

    “RNA screens are well accepted and will continue to be used, but it is important biologically that researchers step away from the RNA mechanism to further study and validate their hits to eliminate potential bias,” explained Louise Baskin, senior product manager, Dharmacon, part of GE Healthcare. “The natural progression is to adopt CRISPR in the later stages.”

    RNAi uses the cell’s endogenous mechanism. All of the components required for gene knockdown are already within the cell, and the delivery of the siRNA starts the process. With the CRISPR gene-editing system, which is derived from a bacterial immune defense system, delivery of both the guide RNA and the Cas9 nuclease, often the rate limiter in terms of knockout efficiency, are required.

    In pooled approaches, the cell has to either drop out or overexpress so that it is sortable, limiting the types of addressable biological questions. A CRISPR-arrayed approach opens up the door for use of other analytical tools, such as high-content imaging, to identify hits of interest.

    To facilitate use of CRISPR, GE recently introduced Dharmacon Edit-R synthetic CRISPR RNA (crRNA) libraries that can be used to carry out high-throughput arrayed analysis of multiple genes. Rather than a vector- or plasmid-based gRNA to guide the targeting of the Cas9 cleavage, a synthetic crRNA and tracrRNA are used. These algorithm-designed crRNA reagents can be delivered into the cells very much like siRNA, opening up the capability to screen multiple target regions for many different genes simultaneously.

    GE showed a very strong overlap between CRISPR and RNAi using this arrayed approach to validate RNAi screen hits with synthetic crRNA. The data concluded that CRISPR can be used for medium- or high-throughput validation of knockdown studies.

    “We will continue to see a lot of cooperation between RNAi and gene editing,” declared Baskin. “Using the CRISPR mechanism to knockin could introduce mutations to help understand gene function at a much deeper level, including a more thorough functional analysis of noncoding genes.

    “These regulatory RNAs often act in the nucleus to control translation and transcription, so to knockdown these genes with RNAi would require export to the cytoplasm. Precision gene editing, which takes place in the nucleus, will help us understand the noncoding transcriptome and dive deeper into how those genes regulate disease progression, cellular development and other aspects of human health and biology.”

    Tool Selection

    Click Image To Enlarge +
    Schematic of a pooled shRNA screening workflow developed by Transomic Technologies. Cells are transduced, and positive or negative selection screens are performed. PCR amplification and sequencing of the shRNA integrated into the target cell genome allows the determination of shRNA representation in the population.

    The functional genomics tool should fit the specific biology; the biology should not be forced to fit the tool. Points to consider include the regulation of expression, the cell line or model system, as well as assay scale and design. For example, there may be a need for regulatable expression. There may be limitations around the cell line or model system. And assay scale and design may include delivery conditions and timing to optimally complete perturbation and reporting.

    “Both RNAi- and CRISPR-based gene modulation strategies have pros and cons that should be considered based on the biology of the genes being studied,” commented Gwen Fewell, Ph.D., chief commercial officer, Transomic Technologies.

    RNAi reagents, which can produce hypomorphic or transient gene-suppression states, are well known for their use in probing drug targets. In addition, these reagents are enriching studies of gene function. CRISPR-Cas9 reagents, which produce clean heterozygous and null mutations, are important for studying tumor suppressors and other genes where complete loss of function is desired.

    Timing to readout the effects of gene perturbation must be considered to ensure that the biological assay is feasible. RNAi gene knockdown effects can be seen in as little as 24–72 hours, and inducible and reversible gene knockdown can be realized. CRISPR-based gene knockout effects may become complete and permanent only after 10 days.

    Both RNAi and CRISPR reagents work well for pooled positive selection screens; however, for negative selection screens, RNAi is the more mature tool. Current versions of CRISPR pooled reagents can produce mixed populations containing a fraction of non-null mutations, which can reduce the overall accuracy of the readout.

    To meet the needs of varied and complex biological questions, Transomic Technologies has developed both RNAi and CRISPR tools with options for optimal expression, selection, and assay scale. For example, the company’s shERWOOD-UltramiR shRNA reagents incorporate advances in design and small RNA processing to produce increased potency and specificity of knockdown, particularly important for pooled screens.

    Sequence-verified pooled shRNA screening libraries provide flexibility in promoter choice, in vitro formats, in vivo formats, and a choice of viral vectors for optimal delivery and expression in biologically relevant cell lines, primary cells or in vivo.

    The company’s line of lentiviral-based CRISPR-Cas9 reagents has variable selectable markers such that guide RNA- and Cas9-expressing vectors, including inducible Cas9, can be co-delivered and selected for in the same cell to increase editing efficiency. Promoter options are available to ensure expression across a range of cell types.

    “Researchers are using RNAi and CRISPR reagents individually and in combination as cross-validation tools, or to engineer CRISPR-based models to perform RNAi-based assays,” informs Dr. Fewell. “Most exciting are parallel CRISPR and RNAi screens that have tremendous potential to uncover novel biology.”

    Converging Technologies

    The convergence of RNAi technology with genome-editing tools, such as CRISPR-Cas9 and transcription activator-like effector nucleases, combined with next-generation sequencing will allow researchers to dissect biological systems in a way not previously possible.

    “From a purely technical standpoint, the challenges for traditional RNAi screens come down to efficient delivery of the RNAi reagents and having those reagents provide significant, consistent, and lasting knockdown of the target mRNAs,” states Ross Whittaker, Ph.D., a product manager for genome editing products at Thermo Fisher Scientific. “We have approached these challenges with a series of reagents and siRNA libraries designed to increase the success of RNAi screens.”

    Thermo Fisher’ provides lipid-transfection RNAiMax reagents, which effectively deliver siRNA. In addition, the company’s Silencer and Silencer Select siRNA libraries provide consistent and significant knockdown of the target mRNAs. These siRNA libraries utilize highly stringent bioinformatic designs that ensure accurate and potent targeting for gene-silencing studies. The Silencer Select technology adds a higher level of efficacy and specificity due to chemical modifications with locked nucleic acid (LNA) chemistry.

    The libraries alleviate concerns for false-positive or false-negative data. The high potency allows less reagent use; thus, more screens or validations can be conducted per library.

    Dr. Whittaker believes that researchers will migrate regularly between RNAi and CRISPR-Cas9 technology in the future. CRISPR-Cas9 will be used to create engineered cell lines not only to validate RNAi hits but also to follow up on the underlying mechanisms. Cell lines engineered with CRISPR-Cas9 will be utilized in RNAi screens. In the long term, CRISPR-Cas9 screening will likely replace RNAi screening in many cases, especially with the introduction of arrayed CRISPR libraries.

     

    Validating Antibodies with RNAi

    Unreliable antibody specificity is a widespread problem for researchers, but RNAi is assuaging scientists’ concerns as a validation method.

    The procedure introduces short hairpin RNAs (shRNAs) to reduce expression levels of a targeted protein. The associated antibody follows. With its protein knocked down, a truly specific antibody shows dramatically reduced or no signal on a Western blot. Short of knockout animal models, RNAi is arguably the most effective method of validating research antibodies.

    The method is not common among antibody suppliers—time and cost being the chief barriers to its adoption, although some companies are beginning to embrace RNAi validation.

    “In the interest of fostering better science, Proteintech felt it was necessary to implement this practice,” said Jason Li, Ph.D., founder and CEO of Proteintech Group, which made RNAi standard protocol in February 2015. “When researchers can depend on reproducibility, they execute more thorough experiments and advance the treatment of human diseases and conditions.”

     

Junk DNA Kept in Good Repair by Nuclear Membrane  

Heterochromatin has the dubious distinction of being called the “dark matter” of DNA, and it has even suffered the indignity of being dismissed as “junk DNA.” But it seems to get more respectful treatment inside the nucleus, where it has the benefit of a special repair mechanism. This mechanism, discovered by scientists based at the University of Southern California (USC), transports broken heterochromatin sequences from the hurly-burly of the heterochromatin domain so that they can be repaired in the relative peace and quiet of the nuclear periphery.

This finding suggests that the nuclear membrane is more versatile than is generally appreciated. Yes, it serves as a protective container for nuclear material, and it uses its pores to manage the transport of molecules in and out of the nucleus. But it may also play a special role in maintaining the integrity of heterochromatin, which tends to be overlooked because it consists largely of noncoding DNA, including repetitive stretches of no apparent function.

“Scientists are now starting to pay a lot of attention to this mysterious component of the genome,” said Irene E. Chiolo, Ph.D., an assistant professor at USC. “Heterochromatin is not only essential for chromosome maintenance during cell division; it also poses specific threats to genome stability. Heterochromatin is potentially one of the most powerful driving forces for cancer formation, but it is the ‘dark matter’ of the genome. We are just beginning to unravel how repair works here.”

Dr. Chilo led an effort to understand how heterochromatin stays in good repair, even though it is particularly vulnerable to a kind of repair error called ectopic recombination. This kind of error is apt to occur when flaws in repeated sequences undergo homologous recombination (HR) by means of double-strand break (DSB) repair. Specifically, repeated sequences tend to recombine with each other during DNA repair.

Working with the fruit fly Drosophila melanogaster, Dr. Chilo’s team observed that breaks in heterochromatin are repaired after damaged sequences move away from the rest of the chromosome to the inner wall of the nuclear membrane. There, a trio of proteins mends the break in a safe environment, where it cannot accidentally get tangled up with incorrect chromosomes.

The details appeared October 26 in Nature Cell Biology, in an article entitled, “Heterochromatic breaks move to the nuclear periphery to continue recombinational repair.”

“[Heterochromatic] DSBs move to the nuclear periphery to continue HR repair,” the authors wrote. “Relocalization depends on nuclear pores and inner nuclear membrane proteins (INMPs) that anchor repair sites to the nuclear periphery through the Smc5/6-interacting proteins STUbL/RENi. Both the initial block to HR progression inside the heterochromatin domain, and the targeting of repair sites to the nuclear periphery, rely on SUMO and SUMO E3 ligases.”

“We knew that nuclear membrane dysfunctions are common in cancer cells,” Dr. Chiolo said. “Our studies now suggest how these dysfunctions can affect heterochromatin repair and have a causative role in cancer progression.”

This study may help reveal how and why organisms become more predisposed to cancer as they age—the nuclear membrane progressively deteriorates as an organism ages, removing this bulwark against genome instability.

Next, Dr. Chiolo and her team will explore how the movement of broken sequences is accomplished and regulated, and what happens in cells and organisms when this membrane-based repair mechanism fails. Their ultimate goal is to understand how this mechanism functions in human cells and identify new strategies to prevent their catastrophic failure and cancer formation.

 

 

Gene Found that Regulates Stem Cell Number Production

Gene Found that Regulates Stem Cell Number Production

The gene Prkci promotes the generation of differentiated cells (red). However if Prkci activity is reduced or absent, neural stem cells (green) are promoted. [In Kyoung Mah]

A scientific team from the University of Southern California (USC) and the University of California, San Diego have described an important gene that maintains a critical balance between producing too many
and too few stem cells. Called Prkci, the gene influences whether stem cells self-renew to produce more stem cells, or differentiate into more specialized cell types, such as blood or nerves.

When it comes to stem cells, too much of a good thing isn’t necessarily a benefit: producing too many new stem cells may lead to cancer; making too few inhibits the repair and maintenance of the body.

In their experiments, the researchers grew mouse embryonic stem cells, which lacked Prkci, into embryo-like structures in the laboratory. Without Prkci, the stem cells favored self-renewal, generating large numbers of stem cells and, subsequently, an abundance of secondary structures.

Upon closer inspection, the stem cells lacking Prkci had many activated genes typical of stem cells, and some activated genes typical of neural, cardiac, and blood-forming cells. Therefore, the loss of Prkci can also encourage stem cells to differentiate into the progenitor cells that form neurons, heart muscle, and blood.

Prkci achieves these effects by activating or deactivating a well-known group of interacting genes that are part of the Notch signaling pathway. In the absence of Prkci, the Notch pathway produces a protein that signals to stem cells to make more stem cells. In the presence of Prkci, the Notch pathway remains silent, and stem cells differentiate into specific cell types.

These findings have implications for developing patient therapies. Even though Prkci can be active in certain skin cancers, inhibiting it might lead to unintended consequences, such as tumor overgrowth. However, for patients with certain injuries or diseases, it could be therapeutic to use small molecule inhibitors to block the activity of Prkci, thus boosting stem cell production.

“We expect that our findings will be applicable in diverse contexts and make it possible to easily generate stem cells that have typically been difficult to generate,” said Francesca Mariani, Ph.D., principal investigator at the Eli and Edythe Broad Center for Regenerative Medicine and Stem Cell Research at USC.

Their study (“Atypical PKC-iota Controls Stem Cell Expansion via Regulation of the Notch Pathway”) was published in a Stem Cell Reports.

Atypical PKC-iota Controls Stem Cell Expansion via Regulation of the Notch Pathway

In Kyoung Mah,1 Rachel Soloff,2,3 Stephen M. Hedrick,2 and Francesca V. Mariani1, *

Stem Cell Reports (2015),     http://dx.doi.org/10.1016/j.stemcr.2015.09.021

The number of stem/progenitor cells available can profoundly impact tissue homeostasis and the response to injury or disease. Here, we propose that an atypical PKC, Prkci, is a key player in regulating the switch from an expansion to a differentiation/maintenance phase via regulation of Notch, thus linking the polarity pathway with the control of stem cell self-renewal. Prkci is known to influence symmetric cell division in invertebrates; however a definitive role in mammals has not yet emerged. Using a genetic approach, we find that loss of Prkci results in a marked increase in the number of various stem/progenitor cells. The mechanism used likely involves inactivation and symmetric localization of NUMB, leading to the activation of NOTCH1 and its downstream effectors. Inhibition of atypical PKCs may be useful for boosting the production of pluripotent stem cells, multipotent stem cells, or possibly even primordial germ cells by promoting the stem cell/progenitor fate.

 

The control of asymmetric versus symmetric cell division in stem and progenitor cells balances self-renewal and differentiation to mediate tissue homeostasis and repair and involves key proteins that control cell polarity. In the case of excess symmetric division, too many stem-cell-like daughter cells are generated that can lead to tumor initiation and growth. Conversely, excess asymmetric cell division can severely limit the number of cells available for homeostasis and repair (Go´mez-Lo´pez et al., 2014; Inaba and Yamashita, 2012). The Notch pathway has been implicated in controlling stem cell self-renewal in a number of different contexts (Hori et al., 2013). However, how cell polarity, asymmetric cell division, and the activation of determinants ultimately impinges upon the control of stem cell expansion and maintenance is not fully understood. In this study, we examine the role of an atypical protein kinase C (aPKC), PRKCi, in stem cell self-renewal and, in particular, determine whether PRKCi acts via the Notch pathway. PKCs are serine-threonine kinases that control many basic cellular processes and are typically classified into three subgroups—conventional, novel, and the aPKCs iota and zeta, which, in contrast to the others, are not activated by diacylglyceride or calcium. The aPKC proteins are best known for being central components of an evolutionarily conserved Par3-Par6-aPKC trimeric complex that controls cell polarity in C. elegans, Drosophila, Xenopus, zebrafish, and mammalian cells (Suzuki and Ohno, 2006).

Before Notch influences stem cell self-renewal, the regulation of cell polarity, asymmetric versus symmetric cell division, and the segregation of cell fate determinants such as NUMB may first be required (Knoblich, 2008). For example, mutational analysis in Drosophila has demonstrated that the aPKC-containing trimeric complex is required for maintaining polarity and for mediating asymmetric cell division during neurogenesis via activation and segregation of NUMB (Wirtz-Peitz et al., 2008). NUMB then functions as a cell fate determinant by inhibiting Notch signaling and preventing self-renewal (Wang et al., 2006). In mammals, the PAR3-PAR6-aPKC complex also can bind and phosphorylate NUMB in epithelial cells and can regulate the unequal distribution of Numb during asymmetric cell division (Smith et al., 2007). During mammalian neurogenesis, asymmetric division is also thought to involve the PAR3-PAR6-aPKC complex, NUMB segregation, and NOTCH activation (Bultje et al., 2009).

Mice deficient in Prkcz are grossly normal, with mild defects in secondary lymphoid organs (Leitges et al., 2001). In contrast, deficiency of the Prkci isozyme results in early embryonic lethality at embryonic day (E)9.5 (Seidl et al., 2013; Soloff et al., 2004). A few studies have investigated the conditional inactivation of Prkci; however, no dramatic changes in progenitor generation were detected in hematopoietic stem cells (HSCs) or the brain (Imai et al., 2006; Sengupta et al., 2011), although one study found evidence of a role for Prkci in controlling asymmetric cell division in the skin (Niessen et al., 2013). Analysis may be complicated by functional redundancy between the iota and zeta isoforms and/or because further studies perturbing aPKCs in specific cell lineages and/or at specific developmental stages are needed.

Here, we investigate the requirement of Prkci in mouse cells using an in vitro system that bypasses early embryonic lethality. Embryonic stem (ES) cells are used to make embryoid bodies (EBs) that develop like the early post-implantation embryo in terms of lineage specification and morphology and can also be maintained in culture long enough to observe advanced stages of cellular differentiation (Desbaillets et al., 2000). Using this approach, we provide genetic evidence that inactivation of Prkci signaling leads to enhanced generation of pluripotent cells and some types of multipotent stem cells, including cells with primordial germ cell (PGC) characteristics. In addition, we provide evidence that aPKCs ultimately regulate stem cell fate via the Notch pathway.

Figure 1. Prkci/ EBs Contain Cells with Pluripotency Characteristics (A and A0 ) Day (d) 12 heterozygous EBs have few OCT4/E-CAD+ cells, while null EBs contain many in clusters at the EB periphery. Inset: OCT4 (nucleus)/E-CAD (cytoplasm) double-positive cells. (B and B0 ) Adjacent sections in a null EB show that OCT4+ cells are likely also SSEA1+. (C) Dissociated day-12 Prkci/ EBs contain five to six times more OCT4+ and approximately three times more SSEA1+ cells than heterozygous EBs (three independent experiments). (D and D0 ) After 2 days in ES cell culture, no colonies are visible in null SSEA1 cultures while present in null SSEA1+ cultures (red arrows). (E–E00) SSEA1+ sorted cells can be maintained for many passages, 27+. (E) Prkci+/ sorted cells make colonies with differentiated cells at the outer edges (n = 27/35). (E0 ) Null cells form colonies with distinct edges (n = 39/45). (E00) The percentage of undifferentiated colonies is shown. ***p < 0.001. (F) Sorted null cells express stem cell and differentiation markers at similar levels to normal ES cells (versus heterozygous EBs) (three independent experiments). (G) EBs made from null SSEA1+ sorted cells express germ layer marker genes at the indicated days. Error bars indicate mean ± SEM, three independent experiments. Scale bars, 100 mm in (A, D, and E); 25 mm in (B). See also Figure S1.

RESULTS

Prkci/ Cultures Have More Pluripotent Cells Even under Differentiation Conditions First, we compared Prkci null EB development to that of Prkci/ embryos. Consistent with another null allele (Seidl et al., 2013), both null embryos and EBs fail to properly cavitate (Figures S1A and S1B). The failure to cavitate is unlikely to be due to the inability to form one of the three germ layers, as null EBs express germ-layer-specific genes (Figure S1E). A failure of cavitation could alternatively be caused by an accumulation of pluripotent cells. For example, EBs generated from Timeless knockdown cells do not cavitate and contain large numbers of OCT4-expressing cells (O’Reilly et al., 2011). In addition, EBs generated with Prkcz isoform knockdown cells contain OCT4+ cells under differentiation conditions (Dutta et al., 2011; Rajendran et al., 2013). Thus, we first evaluated ES colony differentiation by alkaline phosphatase (AP) staining. After 4 days without leukemia inhibitory factor (LIF), Prkci/ ES cell colonies retained crisp boundaries and strong AP staining. In contrast, Prkci+/ colonies had uneven colony boundaries with diffuse AP staining (Figures S1F–S1F00). To definitively detect pluripotent cells, day-12 EBs were assayed for OCT4 and E-CADHERIN (E-CAD) protein expression. Prkci+/ EBs had very few OCT4/E-CAD double-positive cells (Figure 1A); however, null EBs contained large clusters of OCT4/E-CAD double-positive cells, concentrated in a peripheral zone (Figure 1A0 ). By examining adjacent sections, we found that OCT4+ cells could also be positive for stage-specific embryonic antigen 1 (SSEA1) (Figures 1B and 1B0 ). Quantification by fluorescence-activated cell sorting (FACS) analysis showed that day-12 Prkci/ EBs had more OCT4+ and SSEA1+ cells than Prkci+/ EBs (Figure 1C). We did not find any difference between heterozygous and wild-type cells with respect to the number of OCT4+ or SSEA1+ cells or in their levels of expression for Oct4, Nanog, and Sox2 (Figures S1I, S1I0 and S1J). However, we did find that Oct4, Nanog, and Sox2 were highly upregulated in OCT4+ null cells (Figure S1G). Thus, together, these data indicate that Prkci/ EBs contain large numbers of pluripotent stem cells, despite being cultured under differentiation conditions.

Functional Pluripotency Tests If primary EBs have a pluripotent population with the capacity to undergo self-renewal, they can easily form secondary EBs (O’Reilly et al., 2011). Using this assay, we found that more secondary EBs could be generated from Prkci/ versus Prkci+/ EBs, especially at days 6, 10, and 16; even when plated at a low density to control for aggregation (Figure S1H). To test whether SSEA1+ cells could maintain pluripotency long term, FACS-sorted Prkci/ SSEA1+ and SSEA1 cells were plated at a low density and maintained under ES cell culture conditions. SSEA1 cells were never able to form identifiable colonies and could not be maintained in culture (Figure 1D). SSEA1+ cells, however, formed many distinct colonies after 2 days of culture, and these cells could be maintained for over 27 passages (Figures 1D0 , 1E0 , and 1E00). Prkci+/ SSEA1+ cells formed colonies that easily differentiated at the outer edge, even in the presence of LIF (Figure 1E). In contrast Prkci/ SSEA1+ cells maintained distinct round colonies (Figure 1E0 ). Next, we determined whether null SSEA1+ cells expressed pluripotency and differentiation markers similarly to normal ES cells. Indeed, we found that Oct4, Nanog, and Sox2 were upregulated in both null SSEA1+ EB cells and heterozygous ES cells. In addition, differentiated markers (Fgf5, T, Wnt3, and Afp) and tissue stem/progenitor cell markers (neural: Nestin, Sox1, and NeuroD; cardiac: Nkx2-5 and Isl1; and hematopoietic: Gata1 and Hba-x) were downregulated in both SSEA1+ cells and heterozygous ES cells (Figure 1F). SSEA1+ cells likely have a wide range of potential, since EBs generated from these cells expressed markers for all three germ layers (Figure 1G).

Figure 2. Prkci and Pluripotency Pathways (A) ERK1/2 phosphorylation (Y202/Y204) is reduced in null ES cells and early day (d)-6 null EBs compared to heterozygous EBs and strongly increased at later stages. The first lane shows ES cells activated (A) by serum treatment 1 day after serum depletion. (B) Quantification of pERK1/2 normalized to non-phosphorylated ERK1/2 (three independent experiments; mean ± SEM; **p < 0.01). (C) pERK1/2 Y202/Y204 is strongly expressed in the columnar epithelium of heterozygous EBs that have just cavitated. Null EBs have lower expression. OCT4 and pERK1/2 expression do not co-localize. Scale bar, 100 mm. (D) pERK1/2Y202/Y204 levels are lower in null SSEA1+ sorted cells than in heterozygous or in null day-12 EBs that have undergone further differentiation. pSTAT3 and STAT levels are unchanged. See also Figure S2.

ERK1/2 Signaling during EB Development Stem cell self-renewal has been shown to require the activation of the JAK/STAT3 and PI3K/AKT pathways and the inhibition of ERK1/2 and GSK3 pathways (Kunath et al., 2007; Niwa et al., 1998; Sato et al., 2004; Watanabe et al., 2006). We found that both STAT3 and phosphorylated STAT3 levels were not grossly altered and that the p-STAT3/STAT3 ratio was similar between heterozygous and null ES cells and EBs (Figures S2A and S2B). In addition we did not see any difference in AKT, pAKT, or b-CATENIN levels when comparing heterozygous to null ES cells or EBs (Figures S2A and S2C). Thus, the effects observed by the loss of Prkci are unlikely to be due to a significant alteration in the JAK/STAT3, PI3K/AKT, or GSK3 pathways.

Next, we investigated ERK1/2 expression and activation. Consistent with other studies showing ERK1/2 activation to be downstream of Prkci in some mammalian cell types (Boeckeler et al., 2010; Litherland et al., 2010), pERK1/2 was markedly inactivated in Prkci null versus heterozygous ES cells. In addition, during differentiation, null EBs displayed strong pERK1/2 inhibition early (until day 6). Later, pERK1/2 was activated strongly, as the EB began differentiating (Figures 2A and 2B). By immunofluorescence, pERK1/2 was strongly enriched in the columnar epithelium of control EBs, while overall levels were much lower in Prkci/ EBs (Figure 2C). In addition, high OCT4 expression correlated with a marked inactivation of pERK1/2 (Figure 2C). Next, we examined Prkci/ SSEA1+ cells by western blot. We found that SSEA1+ cells isolated from day-12 null EBs had pSTAT3 expression levels similar to whole EBs, while pERK1/2 levels were low (Figure 2D). Thus, these experiments indicate that the higher numbers of pluripotent cells in null EBs correlate with a strong inactivation of ERK1/2.

Neural Stem Cell Fate Is Favored in Prkci/ EBs It is well known that ERK/MEK inhibition is not sufficient for pluripotent stem cell maintenance (Ying et al., 2008); thus, other pathways are likely involved. Therefore, we used a TaqMan Mouse Stem Cell Pluripotency Panel (#4385363) on an OpenArray platform to investigate the mechanism of Prkci action. Day 13 and day 20 Prkci/ EBs expressed high levels of pluripotency and stemness markers versus heterozygous EBs, including Oct4, Utf1, Nodal, Xist, Fgf4, Gal, Lefty1, and Lefty2. However, interestingly, EBs also expressed markers for differentiated cell types and tissue stem cells, including Sst, Syp, and Sycp3 (neural-related genes), Isl1 (cardiac progenitor marker), Hba-x, and Cd34 (hematopoietic markers). Based on this first-pass test, we sought to determine whether loss of Prkci might favor the generation of neural, cardiac, and hematopoietic cell types and/or their progenitors.

Figure 3. Neural Stem Cell Populations Are Increased in Null EBs (A–C0 ) Prkci/ EBs (B) have more NESTINpositive cells than Prkci+/ EBs (A). (C and C0 ) MAP2 and TUJ1 are expressed in null EBs, similarly to heterozygous EBs (data not shown). (D) EBs were assessed for PAX6 expression, and the images were used for quantification (Figures S3A and S3B). The pixel count ratio of PAX6+ cells in null EBs (green) is substantially higher than that found in heterozygous EBs (black) (three independent experiments; mean ± SEM; *p < 0.05). (E–F000) Day 4 after RA treatment, Prkci/ EBs have more NESTIN- than TUJ1-positive neurons (E and F). However, null cells can still terminally differentiate into NEUROD-, NEUN-, and MAP2-positive cells (F0 –F000). Scale bars, 25 mm in (A and C) and 50 mm in (E). See also Figure S3. Ste

The Generation of Cardiomyocyte and Erythrocyte Progenitors Is Also Favored Next, we examined ISL1 expression (a cardiac stem cell marker) by immunofluorescence and found that Prkci/ EBs contained larger ISL1 clusters compared with Prkci+/ EBs; this was confirmed using an image quantification assay (Figures 4A, 4A0 , and 4C). Differentiated cardiac cells and ventral spinal neurons can also express ISL1 (Ericson et al., 1992); therefore, we also examined Nkx2-5 expression, a better stem cell marker and regulator of cardiac progenitor determination (Brown et al., 2004), by RT-PCR and immunofluorescence. In null EBs, Nkx2-5 was upregulated (Figure 4D). In addition, in response to RA, which can promote cardiac fates in vitro (Niebruegge et al., 2008), cells expressing NKX2-5 were more prevalent in null versus heterozygous EBs (Figures 4B and 4B0 ).The abundant cardiac progenitors found in null EBs were still capable of undergoing differentiation (Figures 4E–4F0 ).

 

Figure 4. Cardiomyocyte and Erythrocyte Progenitors Are Increased in Prkci/ EBs (A–F0 ) In (A, A0 , E, and E0 ), Prkci/ EBs cultured without LIF have more ISL1 (cardiac progenitor marker) and a-ACTININ-positive cells compared to heterozygous EBs. (C) At day (d) 9, the pixel count ratio for ISL1 expression indicates that null EBs (green) have larger ISL1 populations than heterozygous EBs (black) (three independent experiments, n = 20 heterozygous EBs, 21 null EBs total; mean ± SEM; *p < 0.05). In (B, B0 , D, F, and F0 ), RA treatment induces more NKX2-5 (both nuclear and cytoplasmic) and a-ACTININ expression in null EBs. Arrows point to fibers in (F0 ). (G) Null EBs (green) generate more beating EBs with RA treatment compared to heterozygous EBs (black) (four independent experiments; mean ± SEM; *p < 0.05, ***p < 0.001). (H) Dissociated null EBs of different stages (green) generate more erythrocytes in a colony-forming assay (CFU-E) (four independent experiments; mean ± SEM; **p < 0.01). (I) Examples of red colonies. (J) Gene expression for primitive HSC markers is upregulated in null EBs (relative to heterozygous EBs) (three independent experiments; mean ± SEM). Scale bars, 50 mm in (A, B, and E); 100 mm in (F), and 25 mm in (I). See also Figure S4. 6

Hba-x expression is restricted to yolk sac blood islands and primitive erythrocyte populations (Lux et al., 2008; Trimborn et al., 1999). Cd34 is also a primitive HSC marker (Sutherland et al., 1992). Next, we determined whether the elevated expression of these markers observed with OpenArray might represent higher numbers of primitive hematopoietic progenitors. Using a colony-forming assay (Baum et al., 1992), we found that red colonies (indicative of erythrocyte differentiation; examples in Figure 4I) were produced significantly earlier and more readily from cells isolated from null versus heterozygous EBs (Figure 4H). By quantitative real-time PCR, upregulation of Hba-x and Cd34 genes confirmed the OpenArray results (Figure 4J). In addition, we found Gata1, an erythropoiesis-specific factor, and Epor, an erythropoietin receptor that mediates erythroid cell proliferation and differentiation (Chiba et al., 1991), to be highly upregulated in null versus heterozygous EBs (Figure 4J). These data suggest that the loss of Prkci promotes the generation of primitive erythroid progenitors that can differentiate into erythrocytes.

To determine whether the aforementioned tissue stem cells identified were represented in the OCT4+ population that we described earlier, we examined the expression of PAX6, ISL1, and OCT4 in adjacent EB sections. We found that cells expressing OCT4 appeared to represent a distinct population from those expressing PAX6 and ISL1 (although some cells were PAX6 and ISL1 double-positive) (Figures S4A–S4C).

Prkci/ Cells Are More Likely to Inherit NUMB/aNOTCH1 Symmetrically The enhanced production of both pluripotent and tissue stem cells suggests that the mechanism underlying the action of Prkci in these different contexts is fundamentally similar. Because the Notch pathway controls stem cell self-renewal in many contexts (Hori et al., 2013), and because previous studies implicated a connection between PRKCi function and the Notch pathway (Bultje et al., 2009; Smith et al., 2007), we examined the localization and activation of a key player in the Notch pathway, NUMB, (Inaba and Yamashita, 2012). Differences in NUMB expression were first evident in whole EBs, where polarized expression was evident in the ectodermal and endodermal epithelia of heterozygous EBs, while Prkci/ EBs exhibited a more even distribution (Figures 5A–5B0 ). To more definitively determine the inheritance of NUMB during cell division, doublets undergoing telophase or cytokinesis were scored for symmetric (evenly distributed in both cells) or asymmetric (unequally distributed) NUMB localization (examples: Figures 5C and 5C0 ).

Because NUMB can be directly phosphorylated by aPKCs (both PRKCi and PRKCz) (Smith et al., 2007; Zhou et al., 2011), loss of Prkci might be expected to lead to decreased NUMB phosphorylation. Three NUMB phosphorylation sites—Ser7, Ser276, and Ser295—could be aPKC mediated (Smith et al., 2007). By immunofluorescence, we found that one of the most well-characterized sites (Ser276), was strongly inactivated in null versus heterozygous EBs, especially in the core (Figures 5F and 5G). Western analysis also confirmed that the levels of pNUMB (Ser276) were decreased in null versus heterozygous EBs (Figure S5F). Thus, genetic inactivation of Prkci leads to a marked decrease in the phosphorylation status of NUMB.

Notch pathway inhibition by NUMB has been observed in flies and mammals (Berdnik et al., 2002; French et al., 2002). Therefore, we investigated whether reduced Numb activity in Prkci/ EBs might lead to enhanced NOTCH1 activity and the upregulation of the downstream transcriptional readouts (Meier-Stiegen et al., 2010). An overall increase in NOTCH1 activation was supported by western blot analysis showing that the level of activated NOTCH1 (aNOTCH1) was strongly increased in day 6 and day 10 null versus heterozygous EBs (Figure S5G). This was supported by immunofluorescence in EBs, where widespread strong expression of aNOTCH1 was seen in most null cells (Figures 5I and 5I0 ), while in heterozygous EBs, this pattern was observed only in the OCT4+ cells (Figures 5H and 5H0 ).

Figure 5. Prkci/ Cells Preferentially Inherit Symmetric Localization of NUMB and aNOTCH1 and Notch Signaling Is Required for Stem Cell Self-Renewal in Null Cells (A–B0 ) In (A and B), day (d)-7 heterozygous EBs have polarized NUMB localization within epithelia and strong expression in the endoderm, while null EBs have a more even distribution. (A0 and B0 ) Enlarged views. (C and C0 ) Asymmetric and symmetric NUMB expression examples. (D) Doublets from day-10 null EBs have more symmetric inheritance when compared to day-10 heterozygous doublets (three independent experiments; mean ± SEM; **p < 0.01). A red line indicates a ratio of 1 (equal percent symmetric and asymmetric). (E) CD24high null doublets exhibited more symmetric NUMB inheritance than CD24high heterozygous doublets (three independent experiments; mean ± SEM; *p < 0.05). A red line indicates where the ratio is 1. (F and G) Decreased pNUMB (Ser276) is evident in the core of null versus heterozygous EBs (n = 10 of each genotype). (H–I0 ) In (H and I), aNOTCH1 is strongly expressed in heterozygous EBs, including both OCT4+ and OCT4 cells, while strong aNOTCH1 expression is predominant in OCT4+ cells of null EBs (n = 10 of each genotype)). (H0 and I0 ) Enlarged views of boxed regions. OCT4+ cells are demarcated with dotted lines. (J and J0 ) OCT4+ cells express HES5 strongly in the nucleus (three independent experiments). (K) Null doublets from dissociated EBs have more symmetric aNOTCH1 inheritance compared to heterozygous doublets (three independent experiments; mean ± SEM; **p < 0.01). A red line indicates where the ratio is 1. (L) CD24high Prkci/ doublets exhibit more symmetric aNOTCH1 than CD24high heterozygous doublets (three independent experiments; mean ± SEM; *p < 0.05). A red line indicates where the ratio is 1. (M and M0 ) Examples of asymmetric and symmetric aNOTCH1 localization. (N and O) Day-3 DMSO-treated null ES colonies show strong AP staining all the way to the colony edge in (N). Treatment with 3 mM DAPT led to more differentiation in (O). (P–R) OCT4 is strongly expressed in day-4 DMSO-treated null ES cultures (P). With DAPT (Q,R), OCT4 expression is decreased. (S) Working model: In daughter cells that undergo differentiation, PRKCi can associate with PAR3 and PAR6. NUMB is recruited and directly phosphorylated. The activation of NUMB then leads to an inhibition in NOTCH1 activation and stimulation of a differentiation/maintenance program. In the absence of Prkci, the PAR3/PAR6 complex cannot assemble (although it may do so minimally with Prkcz). NUMB asymmetric localization and phosphorylation is reduced. Low levels of pNUMB are not sufficient to block NOTCH1 activation, and activated NOTCH1 preserves the stem cell self-renewal program. We suggest that PRKCi functions to drive differentiation by pushing the switch from an expansion phase that is symmetric to a differentiation and/or maintenance phase that is predominantly asymmetric. In situations of low or absent PRKCi, we propose that the expansion phase is prolonged. Scale bars, 50 mm in (A, B, F, G, H, I, J, J0 , P–R); 200 mm in (A0 and B0 ); 25 mm in (C, C0 , M, and M0 ); and 100 mm in (H0 , I0 , N, and O). See also Figure S5.

 

Figure 6. Additional Inhibition of PRKCz Results in an Even Higher Percentage of OCT4-, SSEA1-, and STELLA-Positive Cells (A and A0 ) After day 4 without LIF, heterozygous ES cells undergo differentiation in the presence of Go¨6983, while null ES cells stay as distinct colonies in (A0 ). (B and B0 ) Go¨6983 stimulates an increase in OCT4+ populations in heterozygous EBs and an even larger OCT4+ population in null EBs in (B0 , insets: green and red channels separately). (C–D0 ) An even higher percentage of cells are OCT4+ (C and C0 ) and SSEA1+ (D and D0 ) with Go¨6983 treatment (day 12, three independent experiments). (E and F) More STELLA+ clusters containing a larger number of cells are present in drugtreated heterozygous EBs. (G and H) Null EBs also have more STELLA+ clusters and cells. Drug-treated null EBs exhibit a dramatic increase in the number of STELLA+ cells. (I–K) Some cells are double positive for STELLA and VASA in drug-treated null EBs (yellow arrows). There are also VASAonly (green arrows) and STELLA-only cells (red arrows) (three independent experiments). (L–P) Treatment with ZIP results in an increase in OCT4+ and STELLA+ cells. ZIP treatment also results in more cells that are VASA+ (three independent experiments); n = 11 for Prkci+/, and n = 13 for Prkci+/ + ZIP; n = 14 for Prkci/, and n = 20 for Prkci/ + ZIP; eight EBs assayed for both STELLA and VASA expression). Scale bars, 100 mm in (A and A0 ); 50 mm in (B and B0 ); and 25 mm in (E, I, and L).

 

DISCUSSION In this report, we suggest that Prkci controls the balance between stem cell expansion and differentiation/maintenance by regulating the activation of NUMB, NOTCH1, and Hes /Hey downstream effector genes. In the absence of Prkci, the pluripotent cell fate is favored, even without LIF, yet cells still retain a broad capacity to differentiate. In addition, loss of Prkci results in enhanced generation of tissue progenitors such as neural stem cells and cardiomyocyte and erythrocyte progenitors. In contrast to recent findings on Prkcz (Dutta et al., 2011), loss of Prkci does not appear to influence STAT3, AKT, or GSK3 signaling but results in decreased ERK1/2 activation. We hypothesize that, in the absence of Prkci, although ERK1/2 inhibition may be involved, it is the decreased NUMB phosphorylation and increased NOTCH1 activation that promotes stem and progenitor cell fate. Thus, we conclude that PRKCi, a protein known to be required for cell polarity, also plays an essential role in controlling stem cell fate and generation via regulating NOTCH1 activation.

Notch Activation Drives the Decision to Self-Renew versus Differentiate Notch plays an important role in balancing stem cell selfrenewal and differentiation in a variety of stem cell types and may be one of the key downstream effectors of Prkci signaling. Sustained Notch1 activity in embryonic neural progenitors has been shown to maintain their undifferentiated state (Jadhav et al., 2006). Similarly, sustained constitutive activation of NOTCH1 stimulates the proliferation of immature cardiomyocytes in the rat myocardium (Collesi et al., 2008). In HSCs, overexpression of constitutively active NOTCH1 in hematopoietic progenitors and stem cells supports both primitive and definitive HSC selfrenewal (Stier et al., 2002). Together, these studies suggest that activation and/or sustained Notch signaling can lead to an increase in certain tissue stem cell populations. Thus, a working model for how tissue stem cell populations are favored in the absence of Prkci involves a sequence of events that ultimately leads to Notch activation. Recent studies have shown that aPKCs can be found in a complex with NUMB in both Drosophila and mammalian cells (Smith et al., 2007; Zhou et al., 2011); hence, in our working model (Figure 5S), we propose that the localization and phosphorylation of NUMB is highly dependent on the activity of PRKCi. When Prkci is downregulated or absent (as shown here), cell polarity is not promoted, leading to diffuse distribution and decreased phosphorylation of NUMB. Without active NUMB, NOTCH1 activation is enhanced, Hes/Hey genes are upregulated, and stem/progenitor fate generation is favored. To initiate differentiation, polarization could be stochastically determined but could also be dependent on external cues such as the presentation of certain ligands or extracellular matrix (ECM) proteins (Habib et al., 2013). When PRKCi is active and the cell becomes polarized, a trimeric complex is formed with PRKCi, PAR3, and PAR6. Numb is then recruited and phosphorylated, leading to Notch inactivation, the repression of downstream Hes/Hey genes, and differentiation is favored (see Figure 5S). Support for this working model comes from studies in Drosophila showing that the aPKC complex is essential for Numb activation and asymmetric localization (Knoblich, 2008; Smith et al., 2007; Wang et al., 2006). Additional studies on mouse neural progenitors show that regulating Numb localization and Notch activation is critical for maintaining the proper number of stem/progenitor cells in balance with differentiation (Bultje et al., 2009). Thus, an important function for PRKCi may be to regulate the switch between symmetric expansion of stem/progenitor cells to an asymmetric differentiation/maintenance phase. In situations of low or absent PRKCi, we propose that the expansion phase is favored. Thus, temporarily blocking either, or both, of the aPKC isozymes may be a powerful approach for expanding specific stem/progenitor populations for use in basic research or for therapeutic applications.

Although we do not see changes in the activation status of the STAT3, AKT, or GSK3 pathway, loss of Prkci results in an inhibition of ERK1/2 (Figures 2A and 2B). This result is consistent with the findings that ERK1/2 inhibition is both correlated with and directly increases ES cell selfrenewal (Burdon et al., 1999). Modulation of ERK1/2 activity by Prkci has been observed in cancer cells and chondrocytes (Litherland et al., 2010; Murray et al., 2011). Although it is not clear whether a direct interaction exists between Prkci and ERK1/2, Prkcz directly interacts with ERK1/2 in the mouse liver and in hypoxia-exposed cells (Das et al., 2008; Peng et al., 2008). The Prkcz isozyme is still expressed in Prkci null cells but evidently cannot suf- ficiently compensate and activate the pathway normally. Furthermore, knocking down Prkcz function in ES cells does not result in ERK1/2 inhibition, suggesting that this isozyme does not impact ERK1/2 signaling in ES cells (Dutta et al., 2011). Therefore, although PRKCi may interact with ERK1/2 and be directly required for its activation, ERK1/2 inhibition could also be a readout for cells that are more stem-like. Further studies will be needed to address this question.

Utility of Inhibiting aPKC Function Loss of Prkci resulted in EBs that contained slightly more STELLA+ cells than EBs made from +/ cells. Furthermore, inhibition of both aPKC isozymes by treating Prkci null cells with the PKC inhibitor Go¨6983 or the more specific inhibitor, ZIP, strongly promoted the generation of large clusters of STELLA+ and VASA+ cells, suggesting that inhibition of both isozymes is important for PGC progenitor expansion (Figure 6). It is unclear what the mechanism for this might be; however, one possibility is that blocking both aPKCs is necessary to promote NOTCH1 activation in PGCs or in PGC progenitor cells that may ordinarily have strong inhibitions to expansion (Feng et al., 2014). Regardless of mechanism, the ability to generate PGC-like cells in culture is notoriously challenging, and our results provide a method for future studies on PGC specification and differentiation. Expansion of stem/progenitor pools may not be desirable in the context of cancer. Prkci has been characterized as a human oncogene, a useful prognostic cancer marker, and a therapeutic target for cancer treatment. Overexpression of Prkci is found in epithelial cancers (Fields and Regala, 2007), and Prkci inhibitors are being evaluated as candidate cancer therapies (Atwood et al., 2013; Mansfield et al., 2013). However, because our results show that Prkci inhibition leads to enhanced stem cell production in vitro, Prkci inhibitor treatment as a cancer therapy might lead to unintended consequences (tumor overgrowth), depending on the context and treatment regimen. Thus, extending our findings to human stem and cancer stem cells is needed.

In summary, here, we demonstrate that loss of Prkci leads to the generation of abundant pluripotent cells, even under differentiation conditions. In addition, we show that tissue stem cells such as neural stem cells, primitive erythrocytes, and cardiomyocyte progenitors can also be abundantly produced in the absence of Prkci. These increases in stem cell production correlate with decreased NUMB activation and symmetric NUMB localization and require Notch signaling. Further inhibition of Prkcz may have an additive effect and can enhance the production of PGC-like cells. Thus, Prkci (along with Prkcz) may play key roles in stem cell self-renewal and differentiation by regulating the Notch pathway. Furthermore, inhibition of Prkci and or Prkcz activity with specific small-molecule inhibitors might be a powerful method to boost stem cell production in the context of injury or disease.

 

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3D mapping of genome in combine FISH and RNAi

Larry H. Bernstein, MD, FCAP, Curator

LPBI

Cellular factors that shape the 3D landscape of the genome identified

http://www.nih.gov/news/health/aug2015/nci-13.htm

Researchers, using novel large-scale imaging technology, have mapped the spatial location of individual genes in the nucleus of human cells and identified 50 cellular factors required for the proper three-dimensional (3D) positioning of genes. These spatial locations play important roles in gene expression, DNA repair, genome stability, and other cellular activities. The study, by scientists at the National Cancer Institute (NCI), part of the National Institutes of Health, appeared August 13, 2015, in Cell.

One of the fundamental properties of the genomes of higher organisms is the non-random arrangement of DNA in the cell nucleus. Researchers have long known that most genes occupy preferred 3D positions in the nucleus and that the location of genes matters for their function, but it has been difficult to determine the molecular players and mechanisms that determine the positions. Although genes can be visualized routinely and their positions determined using fluorescence in situ hybridization, or FISH, this mapping method has traditionally been limited to the analysis of a few samples at a time and cannot be used for large-scale genome mapping.

NCI researchers, in close collaboration with NCI’s High-Throughput Imaging Facility, which was established earlier this decade, have developed a method called HIPMap (High-throughput Imaging Position Mapping) that makes the large-scale determination of 3D gene positions possible. This method uses an optimized FISH detection protocol, fully automated microscopy, and combines it with sophisticated computational image analysis that delivers high-precision gene mapping information for thousands of samples in a single experiment.

In the study, NCI researchers, led by Tom Misteli, Ph.D., associate director, NCI Center for Cancer Research, used HIPMap and a method known as RNA interference (RNAi) knockdown to screen nearly 700 proteins in the nucleus to identify those that are involved in the 3D positioning of several human genes. RNAi knockdown uses RNA molecules to block the production of specific proteins in cells.

By collecting data continuously from automated microscopes for 27 days and then analyzing more than three million data points, the scientists were able to identify 50 cellular factors that determine the location of genes in the cell nucleus. This list provides the basis for further investigation of the molecular mechanisms of genome organization.

“The importance of HIPMap is that it is a starting point for numerous applications, including cancer biology,” said Misteli. “In addition to addressing basic questions about the mechanisms of how genomes are organized in intact cells, the ability to map gene positions in a large number of samples and cells has already been used to detect very rare chromosome translocation events in cancer and to ask what cellular factors determine where chromosomes break.”  During translocations, chromosomes break and reattach, which can cause the fusion of otherwise unconnected genes, resulting in hybrid genes whose protein products may contribute to the development of cancer.

As an example of the implications of HIPMap, Misteli pointed to a study from his lab published last month (Burman et al., Genes and Development. July 1, 2015).  In that study researchers used a method derived from HIPMap to probe mechanisms that contribute to the susceptibility of chromosomes to break and form a cancer-causing translocation between the NPM1 gene and the ALK gene in a cancer known as anaplastic large cell lymphoma. Another possible application of HIPMap is in cancer diagnostics. The researchers have previously shown that some genes assume distinct positions in cancer. As a result, the 3D positions of genes could be used as diagnostic markers in diseases such as breast cancer and prostate cancer.

“HIPMap will be a powerful tool in many ongoing efforts to map the genome in 3D space and to translate the findings from these studies to cancer biology,” Misteli concluded.

About the National Institutes of Health (NIH): NIH, the nation’s medical research agency, includes 27 Institutes and Centers and is a component of the U.S. Department of Health and Human Services. NIH is the primary federal agency conducting and supporting basic, clinical, and translational medical research, and is investigating the causes, treatments, and cures for both common and rare diseases. For more information about NIH and its programs, visit www.nih.gov.

NIH…Turning Discovery Into Health®

Reference

Shachar S, Voss TC, Pegoraro G, Sciascia N, Misteli T. Identification of Gene Positioning Factors Using High-throughput Imaging Mapping. Cell. August 13, 2015. DOI: 10.1016/j.cell.2015.07.035.

Stephen J. Williams, PhD

It is interesting they were able to complete this work and develop this technology which I see has other applications than which they suggested. It has been shown how certain factors (HIRAII) accumulate in certain areas of the chromatin during earliest stages of transformation and facilitate massive chromatin remodeling. That genetic information is spatially regulated, particular specific genes, and their technology can map it in a way I feel which is more accurate than probe methodologies or common sequencing methodologies is exciting news. however it may be difficult to use this as an early detection platform, unless it is done post-biopsy. A proceedure, possibly sensor based would need to be developed as well an an invitral imaging methodology.
The particular interesting application I would find would be the detection of insertion sites of genetic therapy. Currently it requires a long procedure involving knowledge of flanking sequences but this mapping procedure would help greatly, especially as I see it in determining insertion sites in development of personalized CART therapy. I would be interested if this group have been able to transfect in a gene and use their HIMAP to determine the spatial insertion site. Will be nice to see this work evolve..

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Upregulate Tumor Suppressor Pathways

Writer and Curator: Larry H Bernstein, MD, FCAP

 

7.5  Upregulate Tumor Suppressor Pathways

7.5.1 NR4A nuclear receptors are orphans but not lonesome

7.5.2 The interplay of NR4A receptors and the oncogene–tumor suppressor networks in cancer

7.5.3 NLRX1 acts as tumor suppressor by regulating TNF-α induced apoptosis

7.5.4 The Mre11 Complex Suppresses Oncogene-Driven Breast Tumorigenesis and Metastasis

7.5.5 Expression of Stromal Cell-derived Factor 1 and CXCR4 Ligand Receptor System in Pancreatic Cancer

7.5.6 DLC1- a significant GAP in the cancer genome

7.5.7 DLC1 is a chromosome 8p tumor suppressor whose loss promotes hepatocellular carcinoma.

7.5.8 Smad7 regulates compensatory hepatocyte proliferation in damaged mouse liver and positively relates to better clinical outcome in human hepatocellular carcinoma

 

 

7.5.1 NR4A nuclear receptors are orphans but not lonesome

Kurakula K, Koenis DS, van Tiel CM, de Vries CJ.
Biochim Biophys Acta. 2014 Nov; 1843(11):2543-2555
http://dx.doi.org/10.1016/j.bbamcr.2014.06.010

Highlights

  • Nuclear receptors Nur77, Nurr1 and NOR-1 are ‘orphan’ receptors of the NR4A subfamily.
  • The NR4A receptors have no ligands.
  • The known protein–protein interactions of all three NR4A receptors are summarized.
  • Interacting proteins are transcription factors, coregulators or protein kinases.
  • Protein–protein interactions modulate NR4A receptor activity and function.

 

The NR4A subfamily of nuclear receptors consists of three mammalian members: Nur77, Nurr1, and NOR-1. The NR4A receptors are involved in essential physiological processes such as adaptive and innate immune cell differentiation, metabolism and brain function. They act as transcription factors that directly modulate gene expression, but can also form trans-repressive complexes with other transcription factors. In contrast to steroid hormone nuclear receptors such as the estrogen receptor or the glucocorticoid receptor, no ligands have been described for the NR4A receptors. This lack of known ligands might be explained by the structure of the ligand-binding domain of NR4A receptors, which shows an active conformation and a ligand-binding pocket that is filled with bulky amino acid side-chains. Other mechanisms, such as transcriptional control, post-translational modifications and protein–protein interactions therefore seem to be more important in regulating the activity of the NR4A receptors. For Nur77, over 80 interacting proteins (the interactome) have been identified so far, and roughly half of these interactions has been studied in more detail. Although the NR4As show some overlap in interacting proteins, less information is available on the interactome of Nurr1 and NOR-1. Therefore, the present review will describe the current knowledge on the interactomes of all three NR4A nuclear receptors with emphasis on Nur77.
Nur77 in the regulation of endocrine signals and steroid hormone synthesis

Nur77 is expressed in endocrine tissues and in organs that are crucial for steroid hormone synthesis such as the adrenal glands, the pituitary gland and the testes. The first functional NurRE was identified in the promoter of the pro-opiomelanocortin (POMC) gene of pituitary derived AtT-20 cells [2]. Nur77 can bind this NurRE either as a homodimer or as a heterodimer with either one of the other two NR4A receptors Nurr1 and NOR-1. Interestingly, it was shown that these heterodimers enhance POMC gene transcription more potently than homodimers of Nur77 do, suggesting that there is interdependency between the NR4A receptors in activating their target genes [3]. The NurRE sequence in the POMC promoter also partially overlaps with a STAT1-3 response element. Philips et al. showed that Nur77 and STAT1-3 bind simultaneously to this so called NurRE-STAT composite site and synergistically enhance transcription of the POMC gene. However, Nur77 and STAT1-3 do not interact directly, which suggests that oneor more facilitatingfactors are involved in NurRE-STAT driven transcription. Mynard et al. showed that this third factor is cAMP response element binding protein (CREB), which binds both STAT1-3 and Nur77 and indirectly enhances transcription of the POMC gene by facilitating the synergistic activation of the NurRE-STAT composite site by STAT1-3 and Nur77 [4]. Nur77also plays animportant role in the steroidogenic acute regulatory protein (StAR)-mediated testosterone production by Leydig cells. StAR is required for the transport of cholesterol through the mitochondrial membrane to initiate steroid hormone synthesis. Nur77 binds to an NBRE in the StAR promoter, which is in close proximity to an AP-1 response element. In response to cAMP stimulation c-Jun and Nur77 synergistically increase StAR gene expression [5], presumably through a direct interaction between c-Jun and the LBD of Nur77 [6]. On the other hand, c-Jun has also been shown to suppress expression of the hydroxylase P450 c17 gene by blocking the DNA-binding activity o fNur77 in response to stimulation of Leydig cells with reactive oxygen species [7].The effect of c-Jun on the transcriptional activity of Nur77 therefore seems to depend on other factors as well. One of these factors could be the atypical nuclear receptor DAX1 (NR0B1), which lacks a DBD and associates with multiple coregulatory proteins. DAX1 binds Nur77 directly and represses its ability to enhance transcription of the previously mentioned P450 c17 gene.

Fig.1.Schematic representation of the domain structure of nuclear receptors. Nuclear receptors are composed of an N-terminal domain (N-term), a central DNA-binding domain (DBD) and a ligand-binding domain (LBD). The amino acid similarity between the individual domains of Nur77 with Nurr1 and NOR-1 is given in percentages below the domains.

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The interactome of NOR-1

NOR-1 is less well studied than Nur77 and Nurr1 and most of the data on interacting proteins of NOR-1 are presented in studies that are mainly focused on its homologues. As a consequence, NOR-1 protein– protein interactions are described with limited detail, for example the HATp300/CBPacetylatesNOR-1similarlyasNur77,however,theeffect on NOR-1 activity has not been described [79]. Likewise, NOR-1 interacts with the co-regulator TIF1β resulting in enhanced NOR-1 activity, but the domain involved in the interaction is unknown [48]. Similar to Nur77, PKC and RSK1/2 were shown to induce NOR-1 mitochondrial translocation [73,79] and DNA-PK binds the DBD of NOR-1. Even though Nurr1 and Nur77 are both essential for optimal DSB repair the function of NOR-1 in this process remains to be studied [68]. Both FHL2 and the peptidyl-prolyl isomerase Pin1 bind the N-terminal domain and DBD of NOR-1, resulting in reduced or enhanced transcriptional activity of NOR-1, respectively [59,64]. Muscat and co-workers performed detailed studies to identify coregulatorsofNOR-1andwerethefirsttorevealtheabsenceofaconventional ligand-binding pocket in the LBD of NOR-1, through molecular modeling and hydrophobicity analysis of the LBD [104]. Based on these analyses, the relative importance of the N-terminal domain of NOR-1 in regulation of the transcriptional activity of NOR-1 became apparent and direct interaction of a number of crucial co-regulators to this domain was shown;SRC-2 (GRIP-1), SRC-1, SRC-3, p300, DRIP250/ TRAP220 and PCAF [104]. The interaction between the N-terminal domain of NOR-1 and TRAP220 is independent of PKA- and PKC phosphorylation sites in TRAP220. Most interestingly, the purine derivative 6-mercaptopurine, which enhances the activity of NR4As without directly binding these nuclear receptors promotes the interaction between NOR-1 and TRAP220 [105]. Both Nur77 and NOR-1 are involved in T-cell receptor mediated apoptosis of developing T cells [106]. During activation of T cells the expressionofNOR-1isinducedandproteinkinaseC(PKC)becomesactive.NOR-1is aPKCsubstratethat isphosphorylatedand subsequently translocatesfromthenucleustothemitochondriawhereitbindsBcl-2. Most interestingly, as already indicated above the interaction between NOR-1/Nur77 and Bcl-2 causes a conformational change in Bcl-2 allowing its BH3 domain to be exposed, resulting in the conversion of Bcl-2 from an anti-apoptotic into a pro-apoptotic protein. For Nur77 it is exactly known which amino acids are involved to provoke the functional switchin Bcl-2, whichis not thecasefor NOR-1 [57,79]. Initially, the homeobox domain containing protein Six3 was identified in a yeast-two-hybrid study as a protein that interacts uniquely withtheDBDandLBDofNOR-1withoutbindingorinhibitingtheactivity of Nur77 or Nurr1. Of interest, NOR-1 and Six3 show overlap in expression in the rat fetal forebrain on embryonic day 18 [107]. In a later study this specificity of Six3 forNOR-1 was not found, rather interaction with all three NR4As was observed [108]. NOR-1 is part of the EWS/NOR-1 fusion protein that is expressed in human extraskeletal myxoid chondrosarcoma tumors. Six3 enhances the activity of NOR-1 (and Nur77 and Nurr1), whereas the activity of EWS/NOR-1 is inhibited and the interaction only requires the DBD of NOR-1. The opposing data in these two studies may be explained by the use of different cell types for the activity assays, as well as the use of Gal4-fusion proteins in the latter study. PARP-1 specifically and effectively interacts with theDBD of NOR-1 independent of the enzymatic activity of PARP-1 [69]. Nurr1 interacts with lower affinity, whereas EWS/NOR-1 and Nur77 do not bind PARP-1, unless the N-terminal domain of Nur77 is deleted. The latter experiment nicely illustrates that the N-terminal domains of Nur77 and EWS/NOR-1 disturb PARP-1 interaction with the DBD. This may be the underlying mechanism of differential function of NOR-1 and the EWS/NOR-1 fusion protein. In line with the binding characteristics, PARP-1 only inhibits the activity of NOR-1 effectively, again independently of the ribose polymerase activity of PARP-1.

Table 5 NOR-1 interacting proteins.

Fig.2. Nur77 and its interacting proteins. Schematic overview of the protein–protein interactions with Nur77 for which the domains of interaction have been elucidated. Details are described in the text and in Tables 1–3, which also contain the full names of the indicated proteins. N-term, N-terminal domain; DBD, DNA-binding domain; LBD, ligand-binding domain.

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Fig.3. Nur77 and kinases modulating its activity and localization. A, Overview of the amino-acid sequence of Nur77 with known phosphorylation sites and associated kinases indicated (T= threonine,S= serine). B,Schematic illustration of effects of different kinases on Nur77 transcriptional activity and subcellular localization. See Table3 for definitions of the abbreviations of the kinases shown.

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Discussion and concluding remarks
This review summarizes the currently available knowledge on the protein–protein interactions of the NR4A nuclear receptor family and their downstream effects. When looking at the information gathered in this review three main observations can be made. First, there are a large number of protein–protein interactions that regulate the activity of Nur77 and there is a large variation in the effects of these interactions on the ‘target’ protein, be it Nur77 or the interacting protein itself. These effects include modulation of transcriptional activity, protein stability, post-translational modification and cellular localization: all processes that are tightly regulated by ligand binding in other nuclear receptors. In light of the many interactions it undergoes with other proteins, Nur77 could also be considered to be a molecular ‘chameleon’: a protein that selectively adopts the responsiveness of other proteins by directly interacting with them. Secondly, the protein–protein interactions with Nur77 described in this review have been studied in a wide range of cell types, such as immune cells (T-cells, thymocytes, monocytes and macrophages); somatic cells(neurons,smooth muscle cells,endothelial
cells and hepatocytes) and cancer cells from diverse origins.We reason that a stimulus- and cell type-specific expression pattern of interacting proteins may be decisive in determining both the interactions of NR4 As with other proteins and their activity in general.The well-studied interaction between Nur77 and RXRα, which has unique outcomes depending on both the cell type studied and the stimulus used, is one such interaction that is modulated by stimulus- or cell type- specific auxiliary proteins. Lastly, there is a large amount of overlap in interacting proteins between the three NR4A nuclear receptors. All three domains of the NR4As are involved in interactions with other proteins (Tables 1–5, Fig. 2), and we think that the unstructured N-terminal domains are of special interest as they have the lowest overall amino acid similarity (Fig. 1). Based on this dissimilarity, it could be hypothesized that the N-terminal domain of each NR4A receptor interacts with a unique set of proteins that specifically regulates each of their activities, if it were not for the fact that this review has shown that the interacting partners of the NR4As strongly overlap. However, a closer look at the N-terminal domains of Nur77, Nurr1 and NOR-1 reveals small stretches of relatively high similarity within the amino acid sequences (Fig. 4). The possible importance of these small stretches of high similarity is most readily apparent when looking at phosphorylation sites of the NR4As.

Fig. 4. Amino-acid sequence similarity between the N-terminal domains of the NR4A receptors. The amino-acid sequence of the N-terminal domains of Nur77, Nurr1 and NOR-1 was aligned and the extent of sequence similarity is indicated with colors; e.g. blue indicates the regions where the sequence of the three NR4As is identical. In the Nur77 sequence, the CHEK2 target Thr88, the JNK1 target Ser95, the ERK2 target Thr143, the CK2 target Ser152, and the DNA-PK target Ser164 are indicated with arrows. In the Nurr1 sequence, the ERK2 targets Ser126 and Thr132, and the ERK5 targets Thr168 and Ser177 are indicated with arrows.

 

 

7.5.2 The interplay of NR4A receptors and the oncogene–tumor suppressor networks in cancer

Beard JA, Tenga A, Chen T
Cell Signal. 2015 Feb; 27(2):257-66
http://dx.doi.org/10.1016/j.cellsig.2014.11.009

Highlights

  • The expression and function of NR4As are dysregulated in multiple cancer types.
  • NR4As are positively regulated by oncogenic signaling pathways.
  • NR4As are capable of inhibiting tumor suppressor signaling.
  • The connectedness of NR4As with these pathways mediate their functions in cancer.
  • NR4A agonists and antagonists offer therapeutic strategies for cancer treatment.

Abstract

Nuclear receptor (NR) subfamily 4 group A (NR4A) is a family of three highly homologous orphan nuclear receptors that have multiple physiological and pathological roles, including some in cancer. These NRs are reportedly dysregulated in multiple cancer types, with many studies demonstrating pro-oncogenic roles for NR4A1 (Nur77) and NR4A2 (Nurr1). Additionally, NR4A1 and NR4A3 (Nor-1) are described as tumor suppressors in leukemia. The dysregulation and functions of the NR4A members are due to many factors, including transcriptional regulation, protein-protein interactions, and post-translational modifications. These various levels of intracellular regulation result from the signaling cross-talk of the NR4A members with various signaling pathways, many of which are relevant to cancer and likely explain the family members’ functions in oncogenesis and tumor suppression. In this review, we discuss the multiple functions of the NR4A receptors in cancer and summarize a growing body of scientific literature that describes the interconnectedness of the NR4A receptors with various oncogene and tumor suppressor pathways.

NR4As are positively regulated by oncogenic signaling pathways

NR4A subfamily of nuclear receptors

NR4A subfamily of nuclear receptors

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intracellular regulation result from the signaling cross-talk of the NR4A members

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7.5.3 NLRX1 acts as tumor suppressor by regulating TNF-α induced apoptosis

Singh K, Poteryakhina A, Zheltukhin A, …Chumakov PM, Singh R.
Biochim Biophys Acta. 2015 May; 1853(5):1073-86
http://dx.doi.org/10.1016/j.bbamcr.2015.01.016

Highlights

  • NLRX1 sensitizes cancer cells to TNF induced cell death by regulating Caspase-8.
  • NLRX1 localizes to mitochondria (mt) and regulates TNF induced mt-ROS generation.
  • Mitochondrial association of Caspase-8 with NLRX1 may regulate mt-ETC function.
  • NLRX1 expression in cancer cells suppresses tumorigenicity in nude mice.

Chronic inflammation in tumor microenvironment plays an important role at different stages of tumor development. The specific mechanisms of the association and its role in providing a survival advantage to the tumor cells are not well understood. Mitochondria are emerging as a central platform for the assembly of signaling complexes regulating inflammatory pathways, including the activation of type-I IFN and NF-κB. These complexes in turn may affect metabolic functions of mitochondria and promote tumorigenesis. NLRX1, a mitochondrial NOD-like receptor protein, regulate inflammatory pathways, however its role in regulation of cross talk of cell death and metabolism and its implication in tumorigenesis is not well understood. Here we demonstrate that NLRX1 sensitizes cells to TNF-α induced cell death by activating Caspase-8. In the presence of TNF-α, NLRX1 and active subunits of Caspase-8 are preferentially localized to mitochondria and regulate the mitochondrial ROS generation. NLRX1 regulates mitochondrial Complex I and Complex III activities to maintain ATP levels in the presence of TNF-α. The expression of NLRX1 compromises clonogenicity, anchorage-independent growth, migration of cancer cells in vitro and suppresses tumorigenicity in vivo in nude mice. We conclude that NLRX1 acts as a potential tumor suppressor by regulating the TNF-α induced cell death and metabolism.

 

7.5.4 The Mre11 Complex Suppresses Oncogene-Driven Breast Tumorigenesis and Metastasis

Gupta GPVanness KBarlas AManova-Todorova KOWen YHPetrini JH
Mol Cell. 2013 Nov 7;52(3):353-65
http://dx.doi.org/10.1016%2Fj.molcel.2013.09.001

The DNA damage response (DDR) is activated by oncogenic stress, but the mechanisms by which this occurs, and the particular DDR functions that constitute barriers to tumorigenesis, remain unclear. We established a mouse model of sporadic onco-gene-driven breast tumorigenesis in a series of mutant mouse strains with specific DDR deficiencies to reveal a role for the Mre11 complex in the response to oncogene activation. We demonstrate that an Mre11-mediated DDR restrains mammary hyperplasia by effecting an oncogene-induced G2 arrest. Impairment of Mre11 complex functions promotes the progression of mammary hyperplasias into invasive and metastatic breast cancers, which are often associated with secondary inactivation of the Ink4a-Arf (CDKN2a) locus. These findings provide insight into the mechanism of DDR engagement by activated oncogenes and highlight genetic interactions between the DDR and Ink4a-Arf pathways in suppression of oncogene-driven tumorigenesis and metastasis.

The DNA damage response (DDR) network comprises DNA repair, DNA damage signaling, apoptosis, and cell-cycle checkpoint functions (Ciccia and Elledge, 2010). Two lines of evidence support the view that the DDR is a barrier to tumorigenesis. Mutations affecting components of the DDR are frequently associated with predisposition to cancer (Ciccia and Elledge, 2010). Also, indices of DDR activation are evident in preneoplastic lesions or in cultured cells harboring activated oncogenes (Bart-kova et al., 2005Gorgoulis et al., 2005). Despite supportive genetic data from in vitro and tumor inoculation studies (Bartkova et al., 2006;Di Micco et al., 2006), causal demonstration that the oncogene-induced DDR suppresses tumorigenesis within a tissue context remains limited (Gorrini et al., 2007Squatrito et al., 2010Takacova et al., 2012). In certain contexts, the role for ataxia telangiectasia mutated (ATM) in suppressing onco-gene-driven tumorigenesis was relatively minor, although these mouse models were limited by the fact that ATM−/− mice are prone to early spontaneous lymphomagenesis (Efeyan et al., 2009).

The mechanism for DDR activation in response to oncogene expression remains incompletely understood, but the prevailing view posits that oncogene activation leads to replication stress in the form of stalled, and subsequently collapsed, DNA replication forks (Halazonetis et al., 2008). Analysis of the ATRSeckel mouse has indicated that ATR may be required for cell viability upon oncogene activation, suggesting that DNA replication stress may indeed underlie these effects of oncogene activation (López-Contreras et al., 2012;Murga et al., 2011Schoppy et al., 2012). However, since ATR promotes viability, rather than elimination of the oncogene-expressing cells, this outcome is not consistent with a barrier function for that component of the DDR. The purpose of this study was to delineate the particular aspects of the DDR network that constitute barriers to oncogenesis using a mouse model of sporadic, oncogene-driven breast cancer.

The Mre11 complex is a sensor of DNA double-strand breaks (Stracker and Petrini, 2011). Hypomorphic mutations in this complex, modeled in the mouse after alleles inherited in ataxiatelangiectasia-like disorder (A-TLD) and Nijmegen breakage syndrome (NBS), have facilitated the elucidation of the Mre11 complex’s role in the ATM-dependent DDR. Here, we utilize these and other mutant mouse strains, individually and in combination, to define the tumor-suppressive functions of the DDR in mammary epithelium.

A Mouse Model of Sporadic, Oncogene-Induced Mammary Neoplasia

Expression of activated NeuT (Bargmann and Weinberg, 1988), the rodent ortholog of the ERBB2/HER2oncogene, in the mammary epithelium of adult mice via the RCAS/MMTVTVA system (Du et al., 2006) results in early DDR activation, and oligoclonal tumors with an average latency of 5 months (Reddy et al., 2010). To delineate the aspects of the DDR primarily relevant for tumor suppression in the face of oncogene activation, we interbred MMTV-TVA mice with a variety of mutant mouse strains with established DDR deficiencies. Age-matched cohorts of female animals (12–18 weeks old) were injected with either RCAS-HA-NeuT or control virus via mammary intraductal injection. The genotypes analyzed wereMre11ATLD1/ATLD1Nbs1ΔBBChk2−/−Nbs1ΔCChk2−/−p53515C/515Cp53−/−, and 53BP1−/−, each of which exhibits defects in DNA-damage-induced cell-cycle checkpoint activation, apoptosis, and/or DNA repair (Figures S1A and S1B available online; Liu et al., 2004Shibata et al., 2010Stracker et al., 20072008Stracker and Petrini, 2011Theunissen et al., 2003Williams et al., 2002). These mouse strains did not exhibit any histopathological deficits in mammary gland development (data not shown), circumventing the potential problem of differences in mammary tissue among the various genetic backgrounds confounding the analyses.

We performed digital quantification of glandular structures relative to total cellular content in the oncogene-expressing mammary glands and normalized this value to the glandular content observed in the matched control mammary glands (Figure 1C). These variations in mammary ductal enlargement, luminal filling, cellular turnover, and glandular density across the different genotypes are summarized in Figure 1D.

NeuT expression in Chk2−/− and Nbs1ΔCChk2−/− mammary epithelium produced hyperplasias that were only modestly dissimilar from WT (Figures 1B–1D; data not shown), suggesting that apoptosis and the intra-S phase checkpoint—diminished in both mutants (Stracker et al., 2008)—do not mediate the early response to oncogene activation. Consistent with that interpretation, p53515C/515C mutants, in which p53-dependent apoptosis is lost (Liu et al., 2004), also exhibited relatively modest hyper-plasia, although some morphological changes were noted (Figures 1B–1D). In contrast, p53−/− mammary glands resembled p53515C/515C morphologically, but exhibited more extensive NeuT-induced hyperplasia (Figures 1B–1D), consistent with additional deficiencies of the null mutant—including, but not limited to, induction of the G1/S checkpoint and senescence pathways.

In contrast to the aforementioned genotypes, oncogene-induced hyperplasia was markedly distinct in Mre11ATLD1/ATLD1 and Nbs1ΔBB mammary glands relative to WT mammary glands (Figures 1B–1D). The Mre11 complex mutant genotypes exhibited florid hyperplasia in response to oncogene expression that frequently filled the lumen of the enlarged mammary ducts. Quantification of hyperplasia across the entire mammary gland revealed that Mre11ATLD1/ATLD1 was associated with the most significant degree of oncogene-induced proliferative change (Figure 1C).

We examined oncogene-dependent activation of the DDR in WT and Mre11ATLD1/ATLD1 mammary hyperplasias. Consistent with prior reports (Reddy et al., 2010), we observed the formation of γH2AX foci and accumulation of 53BP1 nuclear staining in WT hyperplasias after the introduction of NeuT (Figures 2A and 2B). We observed a highly significant, >2-fold reduction in both NeuT-induced γH2AX foci formation and 53BP1 accumulation within Mre11ATLD1/ATLD1 lesions relative to WT (p < 0.0001; Figures 2A and 2B). In contrast to the effects of Mre11 complex hypomorphism, oncogene-dependent DDR activation was unperturbed in p53−/− mammary glands (Figure 2A; data not shown). These data demonstrate that the Mre11 complex is required for DDR activation upon NeuT expression.

The oncogene-driven, Mre11 complex-dependent DDR exhibited dissimilarities from that induced by ionizing radiation (IR). First, oncogene expression in the WT mammary gland resulted in finely punctate 53BP1 staining and did not induce the large foci that develop after irradiation of the mammary gland (Figure S4). In addition, phosphorylation of the ATM target KAP1 at Ser824 was not observed in the oncogene-expressing mammary gland, but was readily detected in IR-treated mammary tissue (Figure 2C). Similarly, we observed significantly less p53 stabilization in mammary epithelial cells after oncogene expression in comparison to irradiated tissue (Figure S4). Hence, the Mre11 complex-mediated response to oncogene activation appears to be qualitatively distinct from the response to clastogen-induced DNA damage.

We examined apoptosis and growth arrest—functional outcomes of DDR activation—in hyperplastic lesions. While NeuT expression was associated with increased proliferation and apoptosis rates relative to control mammary glands, we did not observe a statistically significant difference in TUNEL or Ki67 positivity between WT and Mre11ATLD1/ATLD1 oncogene-induced hyperplasias (Figures 3A and 3B). We observed a 4-fold increase in pHH3-S10 staining in WT versus Mre11ATLD1/ATLD1 hyperplasias (p < 0.001; Figure 3C), which was unexpected given the significantly increased cellularity of Mre11ATLD1/ATLD1 hyperplasias. The pHH3-S10 staining pattern that we observed was punctate, and pHH3-S10-positive nuclei did not exhibit morphological features of mitosis (Figure 3C, inset), suggesting that the pHH3-S10 signal represented pericentromeric staining characteristic of late G2 cells rather than mitotic cells.

Centriole duplication was evident in 84% of pHH3-S10-positive cells, compared to only 16% of pHH3-S10-negative cells (p < 0.0001; Figure 4B), indicating a cell-cycle state that is beyond the G1/S transition. These observations collectively suggest that NeuT expression in mammary epithelium activates a Mre11 complex-dependent G2 arrest or accumulation. Notably, this G2 arrest is distinct from the canonical IR-induced G2/M checkpoint, which is also Mre11 dependent (Theunissen et al., 2003). In that context, pHH3-S10 is not induced, suggesting that the heterochromatin-associated accumulation of this marker is oncogene specific.

The variable and prolonged latency of tumor onset in Mre11ATLD1/ATLD1 animals suggests that additional genetic alterations may be required for NeuT-mediated transformation of mammary epithelial cells. We examined p19Arf expression—a well-established oncogene-induced tumor-suppressive pathway (Sherr, 2001)—in the 3-week-old NeuT-expressing mammary hyperplasias from WT and Mre11ATLD1/ATLD1animals. We observed >10-fold induction of p19Arf after oncogene expression in Mre11ATLD1/ATLD1relative to control-injected mammary glands (Figure 6A). The extent of p19Arf induction in NeuT-expressingWT mammary glands was <50% of that observed in Mre11ATLD1/ATLD1 (p < 0.007, Figure 6A). Notably, there was no difference in HA-NeuT expression levels between the WT and Mre11ATLD1/ATLD1 mice that could account for the elevated levels of p19Arf (Figure S6A). As expected, p53 levels were modestly elevated in Mre11ATLD1/ATLD1 hyperplasias relative to WT (Figure S6B).

Collectively, the findings presented here indicate that the Mre11 complex constitutes an inducible barrier to oncogene-driven neoplasia. In response to oncogene activation, the Mre11 complex mediates a G2 arrest that appears to be qualitatively distinct from that revealed in previous analyses of Mre11 complex-dependent DDR functions (Figure 7EStracker et al., 2004). The arrest is associated with heterochromatin changes, including the appearance of macroH2A and histone H3 (Ser10) phosphorylation. Histone H3 phosphorylation at pericentric heterochromatin begins early in G2 phase and expands as cells enter mitosis (Crosio et al., 2002). That fact, along with the finding that H3 phosphorylation arises in cells that have undergone centriole duplication, indicates that cells in oncogene-expressing hyperplasias accumulate in G2. We cannot exclude the possibility that other NeuT-expressing cells also arrest in G1 without the observed heterochromatic changes. In Mre11ATLD1/ATLD1 mammary epithelium, the NeuT-induced arrest is lost, and macroH2A and histone H3 phosphorylation are not detected in hyperplastic tissue, demonstrating that the G2 accumulation depends on the Mre11 complex.

The Mre11 complex-dependent G2 arrest does not appear permanent, as WT cells are capable at low frequency of progressing to tumors. When the arrest is attenuated, as in Mre11ATLD1/ATLD1, we observe more extensive oncogene-induced mammary hyperplasia, and a significantly greater likelihood of progression to invasive breast cancer. Although previous studies show that the Mre11 complex suppresses genome instability, and thus the risk of spontaneous DNA-damage-associated tumorigenesis (Stracker et al., 2008Theunissen et al., 2003), this study demonstrates that the Mre11 complex also suppresses oncogene-driven neoplasia and tumorigenesis.

An important question concerns the underlying basis of the response to oncogene activation. Given the importance of the Mre11 complex in sensing DNA double-strand breaks and initiating an ATM-dependent DDR, a parsimonious interpretation is that oncogene activation results in DNA damage. Indeed, there are compelling genetic data supporting the induction of DNA replication stress upon oncogene activation (Bartkova et al., 2006Campaner and Amati, 2012Di Micco et al., 2006Dominguez-Sola et al., 2007;López-Contreras and Fernandez-Capetillo, 2010). DNA replication stress is a common precursor of frank DNA damage when forks collapse (Allen et al., 2011), which would readily account for the induction of DNA damage upon oncogene induction.

Potential crosstalk between the oncogene-induced DDR and the Arf tumor suppressor pathways has recently been described (Evangelou et al., 2013Monasor et al., 2013Velimezi et al., 2013). Our data provide direct evidence for a genetic interaction between these pathways during oncogene-driven tumorigenesis. We demonstrate that when Mre11 complex function is impaired, oncogene expression induces Arf expression, and Ink4a-Arf inactivation is commonly observed in the mammary tumors that ensue. The mechanism for how Mre11 hypomorphism promotes oncogene-induced Arf expression remains unclear.  We observe that 40% of the NeuT-induced mammary tumors that developed in Mre11ATLD1/ATLD1 mice had genetic inactivation of the Ink4a-Arf locus, and the remaining tumors exhibited reduced p19Arf expression, suggesting alternative modes of pathway suppression. These findings provide compelling genetic evidence for the cooperative roles of the Mre11 complex and Ink4a-Arf pathways in the suppression of oncogene-driven tumorigenesis and metastasis.

The behavior of the emergent tumors in Mre11ATLD1/ATLD1mice suggests a link between increased chromosomal instability and an elevated rate of metastatic dissemination from the primary tumor. The observation that all of the Ink4a-Arf mutated mammary tumors were lung metastatic also raises the possibility that Arf loss promotes metastatic progression in the context of Mre11 complex impairment.

Our genetic data suggest that functional hypomorphism of this pathway may be a driver of breast tumorigenesis, genomic instability, and metastasis. Given the profound DDR defects associated with Mre11 complex hypomorphism (Stracker and Petrini, 2011), this subset of human breast cancer may exhibit exquisite DNA damage sensitivities that could be therapeutically exploited to improve clinical outcomes.

 

 

7.5.5 Expression of Stromal Cell-derived Factor 1 and CXCR4 Ligand Receptor System in Pancreatic Cancer

Koshiba T, Hosotani R, Miyamoto Y, Ida J, …, Fujii N, Imamura M
Clin Cancer Res Sep; 6(9):3530-5
NR4A subfamily of nuclear receptors
http://clincancerres.aacrjournals.org/content/6/9/3530.long

To examine the expression of the stromal cell-derived factor 1 (SDF-1)/CXCR4 receptor ligand system in pancreatic cancer cells and endothelial cells, we performed immunohistochemical analysis for 52 pancreatic cancer tissue samples with anti-CXCR4 antibody and reverse transcription-PCR analysis for CXCR4 and SDF-1 in five pancreatic cancer cell lines (AsPC-1, BxPC-3, CFPAC-1, HPAC, and PANC-1), an endothelial cell line (HUVEC), and eight pancreatic cancer tissues. We then performed cell migration assay on AsPC-1 cells, HUVECs, and CFPAC-1 cells in the presence of SDF-1 or MRC-9 fibroblast cells. Immunoreactive CXCR4 was found mainly in pancreatic cancer cells and endothelial cells of relatively large vessels around a tumorous lesion. The immunopositive ratio in the pancreatic cancer was 71.2%. There was no statistically significant correlation with clinicopathological features. SDF-1 mRNA expressions were detected in all pancreatic cancer tissues but not in pancreatic cancer cell lines and HUVECs; meanwhile, CXCR4 mRNA was detected in all pancreatic cancer tissues, cancer cell lines, and HUVECs. The results indicate that the paracrine mechanism is involved in the SDF-1/CXCR4 receptor ligand system in pancreatic cancer. In vitro studies demonstrated that SDF-1 significantly increased the migration ability of AsPC-1 and HUVECs, and these effects were inhibited by CXCR4 antagonist T22, and that the coculture system with MRC-9 also increased the migration ability of CFPAC-1 cells, and this effect was significantly inhibited by T22. Our results suggested that the SDF-1/CXCR4 receptor ligand system may have a possible role in the pancreatic cancer progression through tumor cell migration and angiogenesis.

Chemokines belong to the small molecule chemoattractive cytokine family and are grouped into CXC chemokines and CC chemokines, on the basis of the characteristic presence of four conserved cysteine residues (123) . Chemokines mediate the chemical effect on target cells through G-protein-coupled receptors, which are characterized structurally by seven transmembrane spanning domains and are involved in the attraction and activation of mononuclear and polymorphonuclear leukocytes. The effects of CXC chemokines on cancer cells have been investigated in the case of IL3 -8. Several studies have demonstrated the presence of IL-8 and its receptor in tumor tissues, which were involved in vascular endothelial cell proliferation and tumor neovascularization ,(4567) . It was also reported that IL-8 inhibited non-small cell lung cancer proliferation via the autocrine and paracrine pathway (8) . IL-8 produced by malignant melanoma was found to induce cell proliferation via the autocrine pathway in vitro (9) . These studies indicate that IL-8 is involved in the regulation of tumor progression through tumor angiogenesis and/or direct cancer cell growth.

SDF-1 was initially cloned by Tashiro et al. (10) and later identified as a growth factor for B cell progenitors, a chemotactic factor for T cells and monocytes, and in B-cell lymphopoiesis and bone marrow myelopoiesis (111213) . SDF-1 is a member of the CXC subfamily of chemokines, and its chemotactic effect is mediated by the chemokine receptor CXCR4 (12 , 14) . Most of the chemokine receptors interact with pleural ligands, and vice versa, but the SDF-1/CXCR4 receptor ligand system has been shown to involve a one-on-one interaction (15 , 16) . Furthermore, CXCR4 has been shown to function as a coreceptor for T lymphocytotrophic HIV-1 isolates (17) . Recent studies have demonstrated that endothelial cells express CXCR4 and are strongly chemoattracted by SDF-1 (1819,20) . Tachibana et al. (15) reported that in the embryo of CXCR4 or SDF-1 knockout mice larger branches of the superior mesenteric artery were missing and that the resultant abnormal circulatory system led to gastrointestinal hemorrhage and intestinal obstruction. These findings suggest that SDF-1 and CXCR4 are involved in organ vascularization, as well as in the immune and hematopoietic system.

To clarify the role of the SDF-1/CXCR4 receptor ligand system in pancreatic cancer, we have investigated the expression of CXCR4 and SDF-1 with the aid of immunohistochemical analysis and RT-PCR in pancreatic cancer tissue and experimental chemotactic activity of SDF-1 in pancreatic cancer cells and vascular endothelial cells in vitro.

The distribution of CXCR4 protein expression in pancreatic cancer tissue was examined by means of immunohistochemical analysis of pancreatic cancer tissue samples obtained at surgical operation. Fig. 1<$REFLINK> shows representative immunostainings of cancerous and noncancerous regions in pancreatic cancer tissues. Staining of the CXCR4 protein was identified in the cytoplasm and/or cell membrane of cancer cells, but was not detected in the normal acinar cells and ductal cells of noncancerous region in pancreatic cancer tissue. Negative or weak staining for the CXCR4 protein was observed in a majority of the infiltrating inflammatory cells in the specimens. The immunopositive ratio of cancer cells in the pancreatic cancer tissue specimens was 71.2% (37 of 52). Table 1<$REFLINK>summarizes the relationship between CXCR4 expression and clinicopathological features of 52 pancreatic cancers. There was no significant correlation between the expression of CXCR4 protein and the clinicopathological variables examined (i.e., tumor extension, lymph node metastasis, liver metastasis, and Union International Contre Cancer stage). CXCR4 immunoreactivities were observed in endothelial cells of relatively large vessels around the tumorous lesions, but were scarcely found in the endothelial cells of microvessels inside tumorous lesions (Fig. 2, A and B)<$REFLINK> .

We performed RT-PCR using specific primers, as described in“ Materials and Methods,” to confirm CXCR4 and SDF-1 mRNA expression in pancreatic cancer cells, endothelial cells (HUVECs), and pancreatic cancer tissues. CXCR4 mRNA expressions were clearly detected in five pancreatic cancer cell lines, HUVECs, and eight pancreatic cancer tissue samples (Fig. 3a)<$REFLINK> . On the other hand, SDF-1 mRNA expression was not detected in five pancreatic cancer cell lines and HUVECs, but was identified in eight pancreatic cancer tissue samples (Fig. 3b)<$REFLINK> .

Transwell migration assays were performed to examine the effects of SDF-1 on motility of pancreatic cancer cells (AsPC-1) and endothelial cells (HUVEC). At a concentration of 100 ng/ml, SDF-1 induced chemotaxis of AsPC-1 cells, which was approximately double that of the control. One micromolar of T22 (CXCR4 antagonist) and 10 μg/ml of IVR7 (neutralizing CXCR4 antibody) completely blocked the chemotaxis of AsPC-1 induced by 100 ng/ml SDF-1 (Fig. 4a)<$REFLINK> . At a concentration of 100 g/ml SDF-1 induced an approximately quadruple chemotaxis of HUVECs. One micromolar of T22 caused a 33% reduction of the chemotaxis of HUVECs in the presence of containing 100 ng/ml SDF-1 (Fig. 4b)<$REFLINK> .

SDF-1 belongs to the CXC chemokine family and is a ligand for CXCR4. The role of the SDF-1/CXCR4 receptor ligand system has been investigated mainly in the field of immunology, especially in the mechanism of infection of T lymphocytotrophic HIV-1 and for the prevention of HIV-1 infection. Investigators have also paid attention to the role of the SDF-1/CXCR4 receptor ligand system in cancer tissues.

In this study, we first used immunohistochemical methods to examine CXCR4 expression in pancreatic cancer tissues. Immunoreactive CXCR4 was found in the cytoplasm and/or cell membrane of pancreatic cancer cells. Although CXCR4 staining in pancreatic cancer tissue was heterogeneous and showed differences between specimens, it was found mainly in cancer cells: the immunopositive ratio for the pancreatic cancer tissue specimens was 71.2% (37 of 52). There was a tendency for the immunopositive ratio of CXCR4 in tumors with lymph node metastasis or liver metastasis to be higher than in tumors without these features, but no statistically significant correlation with clinicopathological features were found. There is a diversity of views on the role of the SDF-1/CXCR4 receptor ligand system in malignant tissues. In the current study, SDF-1 mRNA expressions were detected in all pancreatic cancer tissues (eight of eight) but were not detected in pancreatic cancer cell lines (zero of five), whereas CXCR4 mRNA was detected in both pancreatic cancer tissues (eight of eight) and cancer cell lines (five of five). The results indicate that the paracrine mechanism may be involved in the SDF-1/CXCR4 receptor ligand system in pancreatic cancer.

Our results suggest that the SDF-1/CXCR4 receptor ligand system may have a possible role in the pancreatic cancer progression through tumor cell migration and angiogenesis. Because T22 suppressed the migration of both pancreatic cancer cells and endothelial cells in vitro, additional in vivo studies are warranted to examine whether T22 suppresses the tumor spread and tumor angiogenesis to clarify the role of the SDF-1/CXCR4 receptor ligand system in pancreatic cancer.

 

7.5.6 DLC1- a significant GAP in the cancer genome

Aurelia Lahoz and Alan Hall
Genes Dev. 2008 Jul 1; 22(13): 1724–1730
http://dx.doi.org/10.1101.2Fgad.1691408

Rho GTPases are believed to make important contributions to the development and progression of human cancer, but direct evidence in the form of somatic mutations analogous to those affecting Ras has been lacking. A recent study in Genes & Development by Xue and colleagues (1439–1444) now provides in vivo evidence that DLC1, a negative regulator of Rho, is a tumor suppressor gene deleted almost as frequently as p53 in common cancers such as breast, colon, and lung.

Cancer is a complex set of diseases arising from combinations of genetic and epigenetic events, including base mutations, chromosomal rearrangements, DNA methylation, and chromatin modification. Genetic changes were first seen cytologically and revealed gross chromosomal abnormalities, such as translocations, deletions, amplifications (of entire chromosomes or parts of chromosomes), and inversions. Subsequently, DNA sequencing of candidate genes and then whole genomes has uncovered large numbers of more subtle genetic alterations. The recent and continuing successes of sequencing and other nonfunctional based genomic approaches have raised new problems in how to determine which changes have significance for tumor development. This is not a trivial problem and will require combinations of cell-based assays, in vivo animal models, and ultimately clinical intervention.

The identification of the Ras oncogene was the first major triumph of the early application of molecular biology to the cancer problem (Malumbres and Barbacid 2003). Although originally identified as a viral oncogene in a rodent sarcoma-inducing retrovirus, it was the seminal work of the Weinberg and Cooper laboratories in 1981 (Krontiris and Cooper 1981Shih et al. 1981), using DNA transfection assays of human tumor DNA into immortalized mouse fibroblasts, that led to the identification of Ras as a true human oncogene. Several groups went on to show that any one of the three Ras genes (HRASKRAS, and NRAS) could be converted into a human oncogene by a single base mutation leading to a single amino acid substitution in the encoded Ras protein. Ras mutations are found in ∼30% of most, though not all, cancer types and it remains the most frequently mutated dominant oncogene so far identified (Bos 1989). We now know much about the consequences of those amino acid substitutions and the cellular and physiological importance of Ras in controlling proliferation and differentiation. Ras is an example of a regulatory GTPase that cycles between active (GTP-bound) and inactive (GDP-bound) conformations to control biochemical pathways and processes. These molecular switches are activated by guanine nucleotide exchange factors (GEFs), which catalyze exchange of GDP for GTP, and are inactivated by GTPase-activating proteins (GAPs), which promote the otherwise slow, intrinsic GTPase activity of the proteins (Fig. 1). The amino acid substitutions identified in Ras in human cancers are found at codons 12, 61, and to a lesser extent 13, and the common consequence of these changes is to prevent GAP-mediated stimulation of GTP hydrolysis leading to permanent activation of the switch (Trahey and McCormick 1987). Inspection of Figure 1 suggests possible alternative ways in which this molecular switch could be inappropriately activated. For example, activating mutations in one of the nine RasGEF genes or inactivation of one of the eight RasGAP genes could lead to hyperactivation of the switch. To date, no such mutations have been reported in GEF genes in human cancers, but one of the GAPs, neurofibromin, is encoded by the NF1 tumor suppressor gene. Patients with neurofibromatosis type I inherit only one functional NF1 gene and are then predisposed to cancer through complete loss of NF1. In addition, mutational activation of components of downstream signaling pathways (Fig. 1) could bypass the need for Ras and this is clearly the case with somatic mutations in BRAF (which encodes a Ras effector), found most frequently in malignant melanomas (>50%), but also in thyroid, colorectal, and ovarian cancer (Davies et al. 2002Wellbrock et al. 2004).

The Ras GTP.GDP cycle

The Ras GTP.GDP cycle

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2732422/bin/1724fig1.jpg

Figure 1. The Ras GTP/GDP cycle. Ras GTPases are molecular switches and the GDP/GTP cycle is controlled by GEFs and GAPs. The output of the switch is through the interaction of Ras.GTP with effector proteins.

Rho GTPases can trigger numerous downstream signaling pathways by interacting with distinct effectors—to date, ∼20 such target proteins have been reported that specifically interact with Rho (Etienne-Manneville and Hall 2002). One of the best-characterized is Rho kinase (ROCK), which regulates myosin II and actin filament contractility, through its ability to phosphorylate and inactivate myosin light chain phosphatase (Fukata et al. 2001). Rho kinase is involved in many aspects of normal cell biology, such as cell cycle, morphogenesis, and migration, and in addition has been shown to participate in the proliferation, invasion, and metastasis of cancer cells (Etienne-Manneville and Hall 2002Sahai and Marshall 2002Narumiya and Yasuda 2006). In the final part of their study, Xue et al. (2008) show that two small molecule Rho kinase inhibitors, Y-27632 and to a lesser extent Fasudil, inhibit in vitro colony formation of p53−/− liver progenitor cells expressing c-Myc and DCL1 shRNA. It should be noted, however, that both Y-27632 and Fasudil inhibit PRK/PKN and citron kinase, two other kinases activated by Rho, so the result is not entirely conclusive (Ishizaki et al. 2000).

Embryonic fibroblasts can be obtained from DLC1−/− mice and these display alterations in the organization of actin filaments and focal adhesion (Durkin et al. 2005). Confusingly, however, these knockout cells have fewer stress fibers and focal adhesions—the opposite of what would have been predicted for the loss of a GAP that regulates Rho. In fact the cytoskeletal and adhesion complex changes seen in DLC−/− fibroblasts appear to be more in keeping with Rac activation. Unfortunately the authors did not examine the levels of either Rho.GTP or Rac.GTP in these cells, which might have provided some insight into this unexpected result. In the absence of tissue-specific mouse knockouts, we must look to work in Drosophila on RhoGAP88C, the fly ortholog of DCL1, to provide some in vivo physiological data. Mutations in RhoGAP88C were first identified as crossveinless-c and result in defects in tissue morphogenesis during development (Denholm et al. 2005). Closer examination suggests that this GAP regulates tubulogenesis and convergent extension, two processes driven by reorganization of the actin cytoskeleton. An additional and provocative observation to emerge from this study is that RhoGAP88C acts through Rho in some tissues, but it acts through Rac and not Rho in others. The in vitro biochemical activity of this GAP has not been determined and so it is possible that it shows a different specificity from its mammalian counterpart. Otherwise, tissue-specific modification of its catalytic activity would need to be invoked, rendering the in vitro assays essentially useless for predicting specificity. Two subsequent studies have concluded that RhoGAP88C is localized basolaterally in epithelial cells and serves to restrict Rho activity to the apical surface and thereby generate morphogenetic tissue remodeling through polarized activation of myosin II (Brodu and Casanova 2006Simoes et al. 2006).

Taken together, a picture emerges of spatially localized DLC1 acting to control Rho activity so as to promote changes in the actin cytoskeleton during cell morphogenesis. The disruption of this pathway might be expected to lead to tissue disorganization during differentiation programs, which could promote inappropriate cell proliferation (Fig. 2).

DLC1 is a tumor suppressor.

DLC1 is a tumor suppressor.

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2732422/bin/1724fig2.jpg

Figure 2.  DLC1 is a tumor suppressor. Loss of DLC1 leads to deregulated and/or delocalized activation of Rho. This may disrupt tissue morphogenesis leading to inappropriate proliferation. (PM) Plasma membrane.

Directed therapeutic intervention depends on a deep understanding of the relevant signaling pathways through which DLC1 loss is manifest. It is a sobering thought that the signaling pathways downstream from Ras responsible for human cancer are still debated some 25 years after its discovery as a human oncogene and it would be optimistic to believe that identifying Rho pathways will be any easier. Inhibiting the GTPase itself, whether Ras or Rho, is challenging. One of the most promising potential targets for Ras inactivation has been farnesyltransferase (FT), the enzyme required for carboxy-terminal, post-translational modification by a farnesyl lipid (Wright and Philips 2006). FT inhibitors are currently in clinical trials, though the data reported so far are not encouraging. Inhibiting Rho using a similar strategy seems less attractive, since it uses a geranylgeranyltransferase to add a geranylgeranyl group; a much more widespread modification than farnesyl addition. Two other processing enzymes that act on both Ras and Rho, a carboxyl-protease and an isoprenylcysteine carboxyl methyltransferase, are being considered as Ras targets, but in tissue culture at least these seem not to be essential for Rho function (Michaelson et al. 2005). Another possibility that is distinctive to DLC1 might be to attack the epigenetic mechanisms that appear to be commonly used to silence this gene in human cancers. Inhibitors of DNA methyltransferase and histone deacetylase (HDAC) have already been shown to induce the restoration of DLC1 expression in cancer cells, making Zebularine, a new and highly effective DNA demethylating agent, as well as HDAC inhibitors attractive therapeutic approaches (Guan et al. 2006Neureiter et al. 2007Seng et al. 2007Xu et al. 2007). Finally, if it turns out that Rho kinase mediates the key signaling pathway downstream from DLC1 loss, then there is already a huge effort underway to develop small molecule inhibitors of this protein. Rho kinase has been implicated in various forms of cardiovascular disease—such as pulmonary hypertension, myocardial hypertrophy, and atherosclerosis—and in fact one compound, Fasudil, is already being used clinically in Japan for cerebral ischemia (Rikitake and Liao 2005Tawara and Shimokawa 2007). With over a dozen pharmaceutical companies reportedly working on this problem, and if the work from Xue et al. (2008) implicating Rho kinase downstream from DLC1 turns out to be correct, those companies may end up with a blockbuster!

 

7.5.7 DLC1 is a chromosome 8p tumor suppressor whose loss promotes hepatocellular carcinoma.

Xue W, Krasnitz A, Lucito R, Sordella R, … , Zender L, Lowe SW.
Genes Dev. 2008 Jun 1;22(11):1439-44
http://dx.doi.org/10.1101.2Fgad.1672608

Deletions on chromosome 8p are common in human tumors, suggesting that one or more tumor suppressor genes reside in this region. Deleted in Liver Cancer 1 (DLC1) encodes a Rho-GTPase activating protein and is a candidate 8p tumor suppressor. We show that DLC1 knockdown cooperates with Myc to promote hepatocellular carcinoma in mice, and that reintroduction of wild-type DLC1 into hepatoma cells with low DLC1 levels suppresses tumor growth in situ. Cells with reduced DLC1 protein contain increased GTP-bound RhoA, and enforced expression a constitutively activated RhoA allele mimics DLC1 loss in promoting hepatocellular carcinogenesis. Conversely, down-regulation of RhoA selectively inhibits tumor growth of hepatoma cells with disabled DLC1. Our data validate DLC1 as a potent tumor suppressor gene and suggest that its loss creates a dependence on the RhoA pathway that may be targeted therapeutically.

Tumor suppressor genes act in signaling networks that protect against tumor initiation and progression, and can be inactivated by deletions, point mutations, or promoter hypermethylation. Although tumor suppressors are rarely considered direct drug targets, they can negatively regulate pro-oncogenic signaling proteins that are amenable to small molecule inhibition. For instance, NF1 inhibits the Ras signaling pathway, which is deregulated in many cancers and has been pursued for its therapeutic potential (Downward 2003). Similarly, PTEN inhibits the PI3–kinase pathway, and inhibitors of PI3K pathway components such as PI3K, AKT, and mTORs have entered clinic trials (Luo et al. 2003).

Recurrent chromosomal deletions found in sporadic cancers often contain tumor suppressor genes. For example, PTEN loss on chromosome 10q23 frequently occurs in various cancers and promotes tumorigenesis by deregulating the PI3 kinase pathway (Maser et al. 2007). Similarly, heterozygous deletions on chromosome 8p22 in many hepatocellular carcinomas (HCC) (Jou et al. 2004) and other cancer types, including carcinomas of the breast, prostate, colon, and lung (Matsuyama et al. 2001Durkin et al. 2007). Several genes, including DLC1MTUS1FGL1 and TUSC3, have been identified as candidate tumor suppressors in this region (Yan et al. 2004). Deleted in Liver Cancer 1 (DLC1) is a particularly attractive candidate owing to its genomic deletion, promoter methylation, and underexpressed mRNA in cancer (Yuan et al. 19982003aNg et al. 2000Wong et al. 2003Guan et al. 2006Seng et al. 2007Ying et al. 2007;Zhang et al. 2007Pike et al. 2008; for review, see Durkin et al. 2007).

Despite its potential importance, functional data implicating DLC1 loss in tumorigenesis are lacking. DLC1encodes a RhoGAP protein that catalyzes the conversion of active GTP-bound RhoGTPase (Rho) to the inactive GDP-bound form and thus suppresses Rho activity (Yuan et al. 1998). DLC1 has potent GAP activity for RhoA and limited activity for CDC42 (Wong et al. 2003Healy et al. 2008). When overexpressed, DLC1 inhibits the growth of tumor cells and xenografts (Yuan et al. 2003b2004Zhou et al. 2004Wong et al. 2005Kim et al. 2007), but whether this requires its Rho-GAP activity or other functions remains unresolved (Qian et al. 2007Liao et al. 2007). Most functional studies to date have relied on DLC1 overexpression and, as yet, none have documented that loss of DLC1 promotes transformation in vitro or tumorigenesis in vivo. Indeed, homozygous dlc1 knockout mice die around embryonic day 10.5 (E10.5), and there is no overt phenotype in dlc1 heterozygous mice (Durkin et al. 2005).

Our laboratory recently developed a “mosaic” mouse model whereby liver carcinomas can be rapidly produced with different genetic alterations by manipulation of cultured embryonic liver progenitor cells (hepatoblasts) followed by transplantation into the livers of recipient mice (Zender et al. 20052006). We previously used this model to identify new oncogenes in HCC, which could be characterized in an appropriate biological and genetic context (Zender et al. 2006). Furthermore, using this system, we showed that shRNAs capable of suppressing gene function by RNAi could recapitulate the consequences of tumor suppressor gene loss on liver carcinogenesis (Zender et al. 2005Xue et al. 2007). Here we combine this mosaic model and RNAi to validate DLC1 as a potent tumor suppressor gene and study its action in vivo.

Studies using low-resolution genome scanning methods have identified chromosome 8p deletions as common lesions in liver carcinoma and other tumor types. To confirm and extend these observations, we examined a series of data sets of copy number alterations in HCC obtained using representational oligonucleotide microarray analysis (ROMA), a variation of array-based CGH that enables genome scanning at high resolution (Lucito et al. 2003). In a panel of 86 liver cancers, heterozygous deletions encompassing theDLC1 were observed in 59 tumors (Fig. 1A,B; data not shown). Consistent with previous reports, these deletions were large (>5 Mb), encompassing >20 annotated genes but invariably included the DLC1 locus. Indeed, heterozygous deletions of DLC1 occurred more frequently than those observed for the well-established tumor suppressors such as INK4a/ARFPTEN, and TP53 (Fig. 1C). Furthermore, DLC1deletions were nearly as common as those for TP53 in other major tumor types such as lung, colon, and breast (Fig. 1C). Again, most 8p deletions were large, although in breast cancer DLC1 resided at a local deletion epicenter reminiscent of that surrounding the INK4a/ARF locus on chromosome 9p21 (Fig. 1D,E). Although we did not examine the status of the remaining allele in this tumor cohort, studies suggest that it can be silenced by promoter methylation (Yuan et al. 2003a; for review, see Durkin et al. 2007). Together, these data suggest that DLC1 loss plays an important role in human cancer but, in the absence of functional validation, are not conclusive.

Genetically modified liver progenitors were seeded into the livers of syngeneic recipients to assess their ability to form tumors in situ. In contrast to the modest impact of DLC1 loss in vitro, DLC1 shRNAs significantly accelerated tumor onset in vivo (P value < 0.0001 for shDLC1-1 and P < 0.0005 for shDLC1-2) (Fig. 2D,E). In fact, at 57 d post-transplantation, GFP-positive tumor nodules were observed in the livers of most animals receiving cells harboring DLC1 shRNAs, whereas the control animals showed no macroscopically detectable tumor burden (Fig. 2E). Furthermore, the pathology of tumors derived from DLC1 knockdown resembled aggressive human HCC and displayed a high proliferative index as assessed by Ki67 immunohistochemistry (Fig. 2F). Tumors also expressed the HCC markers α-fetoprotein (AFP) and albumin (Supplemental Fig. S3B). These data demonstrate that loss of DLC1 can efficiently promote the development of HCC.

We also ectopically expressed the murine dlc1 gene in mouse hepatoma cells and tested their ability to form tumors orthotopically. To this end, we cloned a Myc-tagged murine dlc1 cDNA and confirmed its ability to produce a protein of the correct molecular weight (Fig. 3A). A mouse hepatoma cell line harboring a luciferase reporter and expressing oncogenic Ras and undetectable DLC1 (see Fig. 1F, lane 8) was infected with the DLC1-expressing retrovirus or an empty vector. Consistent with the literature (Ng et al. 2000), reintroduction of DLC1 produced a modest effect on proliferation in colony formation assays (Supplemental Fig. S4A,B).

Although RhoA has been identified as a DLC1 effector, overexpression studies suggest that other DLC1 functions can contribute to its anti-proliferative activities (Liao et al. 2007Qian et al. 2007). To determine whether RhoA is required for maintaining tumorigenesis stimulated by DLC1 loss, we tested whether suppression of RhoA in DLC1-suppressed hepatoma lines would impact their expansion as subcutaneous tumors in immunocompromised mice. shRNAs capable of down-regulating RhoA to varying degrees (Fig. 5A) decreased the in vivo growth of two independent murine hepatoma lines with undetectable DLC1 (Fig. 5B, cell lines 1,2; Supplemental Fig. S6A,B). Of note, none of the shRNAs completely suppressed RhoA expression, and their ability to limit tumor expansion was proportional to their knockdown efficiency (Supplemental Fig. S6A). The impact of these shRNAs was less pronounced in hepatoma cell lines with higher DLC1 levels (Fig. 5B, cell lines 3,4; Supplemental Fig. S6C,D). Although complete inhibition of RhoA activity might be generally cytostatic (see Piekny et al. 2005), these data suggest that RhoA is required for maintaining the growth of tumors with attenuated DLC1 activity.

In this study, we combined in vivo RNAi and a mosaic mouse model of HCC to study the impact of DLC1 loss on liver carcinogenesis in mice, which to date has not been possible owing to the embryonic lethality of DLC1 knockout animals. We show that DLC1 loss, when combined with other oncogenic lesions, promotes HCC in vivo and that RhoA activation is both necessary and sufficient for its effects. In our survey of copy number alterations in human tumors, 8p22 deletions encompassing DLC1 occurred in >60% of heptocellular carcinomas as well as a large portion of human lung, breast, and colon carcinomas (see also Durkin et al. 2007). Similarly, RhoA is up-regulated in HCC and many other tumor types (Sahai and Marshall 2002;Fukui et al. 2006). Although other tumor suppressor genes may also reside in the 8p region, our results demonstrate that DLC1 is functionally important and highlight the potential importance of the RhoA signaling network in epithelial cancers.

Molecularly targeted therapies have been devised for inhibiting several oncogenic pathways, including those affected by BCR-ABL, activated Ras and PI3kinase (Downward 2003Luo et al. 2003). Although tumor suppressors are generally not amenable to direct therapeutic targeting, their mutation may confer a cellular dependency on downstream oncogenic proteins that can be inhibited with small molecule drugs. In this regard, the impact of DLC1 loss may parallel that produced by loss of PTEN, which deregulates the PI3K pathway and can sensitize cells to pharmacological inhibitors of downstream effectors such as mTOR (Maser et al. 2007). Our data indicate that RhoA is required for maintaining at least some tumors driven by DLC1 loss, and that cells with disabled DLC1 are particularly sensitive to inhibitors that target at least one RhoA effector. Clearly, more studies will be required to confirm and extend these observations; nevertheless, the high frequency of DLC1 loss in human cancer implies that pharmacologic intervention of the signaling pathways modulated by DLC1 may have broad therapeutic utility.

 

7.5.8 Smad7 regulates compensatory hepatocyte proliferation in damaged mouse liver and positively relates to better clinical outcome in human hepatocellular carcinoma

Feng T, Dzieran J, Gu X, Marhenke S, Vogel A, …, Dooley S, Meindl-Beinker NM.
Clin Sci (Lond). 2015 Jun 1; 128(11):761-74
http://dx.doi.org:/10.1042/CS20140606

Transforming growth factor β (TGF-β) is cytostatic towards damage-induced compensatory hepatocyte proliferation. This function is frequently lost during hepatocarcinogenesis, thereby switching the TGF-β role from tumour suppressor to tumour promoter. In the present study, we investigate Smad7 overexpression as a pathophysiological mechanism for cytostatic TGF-β inhibition in liver damage and hepatocellular carcinoma (HCC). Transgenic hepatocyte-specific Smad7 overexpression in damaged liver of fumarylacetoacetate hydrolase (FAH)-deficient mice increased compensatory proliferation of hepatocytes. Similarly, modulation of Smad7 expression changed the sensitivity of Huh7, FLC-4, HLE and HLF HCC cell lines for cytostatic TGF-β effects. In our cohort of 140 HCC patients, Smad7 transcripts were elevated in 41.4% of HCC samples as compared with adjacent tissue, with significant positive correlation to tumour size, whereas low Smad7 expression levels were significantly associated with worse clinical outcome. Univariate and multivariate analyses indicate Smad7 levels as an independent predictor for overall (P<0.001) and disease-free survival (P=0.0123). Delineating a mechanism for Smad7 transcriptional regulation in HCC, we identified cold-shock Y-box protein-1 (YB-1), a multifunctional transcription factor. YB-1 RNAi reduced TGF-β-induced and endogenous Smad7 expression in Huh7 and FLC-4 cells respectively. YB-1 and Smad7 mRNA expression levels correlated positively (P<0.0001). Furthermore, nuclear co-localization of Smad7 and YB-1 proteins was present in cancer cells of those patients. In summary, the present study provides a YB-1/Smad7-mediated mechanism that interferes with anti-proliferative/tumour-suppressive TGF-β actions in a subgroup of HCC cells that may facilitate aspects of tumour progression.

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RNAi – On Transcription and Metabolic Control

Writer and Curator: Larry H Bernstein, MD, FCAP

 

RNAi

This is the third contribution to a series on transcription and metabolic control. It reveals the enormous complexity in this emerging research.

 

mRNA, small RNAs, long RNAs, RNAi and DicAR

Aberrant mRNA translation in cancer pathogenesis
Pier Paolo Pandolfi
Oncogene (2004) 23, 3134–3137
http://dx.doi.org:/10.1038/sj.onc.1207618

As the molecular processes that control mRNA translation and ribosome biogenesis in the eukaryotic cell are extremely complex and multilayered, their deregulation can in principle occur at multiple levels, leading to both disease and cancer pathogenesis. For a long time, it was speculated that disruption of these processes may participate in tumorigenesis, but this notion was, until recently, solely supported by correlative studies. Strong genetic support is now being accrued, while new molecular links between tumor-suppressive and oncogenic pathways and the control of protein synthetic machinery are being unraveled. The importance of aberrant protein synthesis in tumorigenesis is further underscored by the discovery that compounds such as Rapamycin, known to modulate signaling pathways regulatory of this process, are effective anticancer drugs. A number of fundamental questions remain to be addressed and a number of novel ones emerge as this exciting field evolves.

 

mRNA Translation and Energy Metabolism in Cancer
I. Topisirovic and N. Sonenberg
Cold Spring Harbor Symposia on Quantitative Biology, Volume LXXVI
http://dx.doi.org:/10.1101/sqb.2011.76.010785

A prominent feature of cancer cells is the use of aerobic glycolysis under conditions in which oxygen levels are sufficient to support energy production in the mitochondria (Jones and Thompson 2009; Cairns et al. 2010). This phenomenon, named the “Warburg effect,” after its discoverer Otto Warburg, is thought to fuel the biosynthetic requirements of the neoplastic growth (Warburg 1956; Koppenol et al. 2011) and has recently been acknowledged as one of the hallmarks of cancer (Hanahan and Weinberg 2011). mRNA translation is the most energy-demanding process in the cell (Buttgereit and Brand 1995).In mammalian cells it consumes >20% of cellular ATP, not considering the energy that is required for the biosynthesis of the components of the translational machinery (e.g., ribosome biogenesis; Buttgereit and Brand 1995). Control of mRNA translation plays a pivotal role in the regulation of gene expression (Sonenberg and Hinnebusch 2009). In fact, a recent study demonstrated that mammalian proteome is mostly governed at the mRNA translation level (Schwanhausser et al. 2011). Malfunction of mRNA translation critically contributes to human disease, including diabetes, heart disease, blood disorders, and, most notably, cancer (Fig. 1; Crozier et al. 2006; Narla and Ebert 2010; Silvera et al. 2010; Spriggs et al. 2010). The first account of changes in the translational apparatus in cancer dates back to 1896, showing enlarged and irregularly shaped nucleoli that are the site of ribosome biogenesis (Pianese 1896). Rapidly proliferating cancer cells have more ribosomes than normal cells.

Figure 1. Dysregulated mRNA translation plays a pivotal role in cancer. Malignant cells are characterized by enlarged nucleoli and a larger number of ribosomes than their normal counterparts. Mutations and/or altered expression of ribosomal proteins (e.g., RPS19, RPS 24), rRNA-modifying enzymes (e.g., dyskerin), translation initiation factors (e.g., eIF4E), or the initiator tRNA (tRNAiMet) result in malignant transformation. Signaling pathways whose dysfunction is frequent in cancer (e.g., MAPK, PI3K/AKT) affect mRNA translation. Perturbations in the translatome result in aberrant cellular growth, proliferation, and survival characteristic of tumorigenesis.

 

In stark contrast to normal cells, in cancer cells ribosomal biogenesis is uncoupled from cell proliferation (Stanners et al. 1979). Accordingly, cancer cells exhibit abnormally high rates of protein synthesis (Silvera et al. 2010). That ribosomal dysfunction plays a central role in cancer is further corroborated by the findings that genetic alterations, which encompass the components of the ribosome machinery (i.e., “ribosomopathies”), are characterized by elevated cancer risk (Narla and Ebert 2010).

mRNA translation is the most energy-consuming process in the cell and strongly correlates with cellular metabolic activity. Translation and energy metabolism play important roles in homeostatic cell growth and proliferation, and when dysregulated lead to cancer. eIF4E is a key regulator of translation, which promotes oncogenesis by selectively enhancing translation of a subset of tumor-promoting mRNAs (e.g., cyclins and c-myc). PI3K/AKT and mitogen-activated protein kinase (MAPK) pathways, which are strongly implicated in cancer etiology, exert a number of their biological effects by modulating translation. The PI3K/AKT pathway regulates eIF4E function by inactivating the inhibitory 4E-BPs via mTORC1, whereas MAPKs activate MAP kinase signal-integrating kinases 1 and 2, which phosphorylate eIF4E. In addition, AMP-activated protein kinase, which is a central sensor of the cellular energy balance, impairs translation by inhibiting mTORC1. Thus, eIF4E plays a major role in mediating the effects of PI3K/AKT, MAPK, and cellular energetics on mRNA translation.Figure 2. eIF4E is regulated by multiple mechanisms. The expression of eIF4E is regulated by several transcription factors (e.g., c-myc, hnRNPK, p53) and adenine-uracil-rich element binding proteins (i.e., HuR and AUF1). eIF4E is suppressed by 4E-BPs, which are regulated by mTORC1. MAP kinase signal integrating kinases 1 and 2 (MNKs) phosphorylate eIF4E.

 

Figure 3. Ras/MAPK and PI3K/AKT/mTORC1 regulate the activity of eIF4E. Various stimuli activate phosphoinositide-3-kinase (PI3K) through the receptor tyrosine kinases (RTKs). Upon activation, PI3K converts phosphatidylinositol 4,5-bisphosphate (PIP2) into phosphatidylinositol-3,4,5-triphosphate (PIP3). This reaction is reversed by PTEN. Phosphoinositide-dependent protein kinase 1 (PDK1) and AKT bind to PIP3 via their pleckstrin homology domains, which allows for the phosphorylation and activation of AKT by PDK1. In addition, the mammalian target of rapamycin complex 2 (mTORC2) modulates the activity of AKT by phosphorylating its hydrophobic motif. AKT phosphorylates tuberous sclerosis complex 2 (TSC2) at multiple sites, which results in its inhibition and consequent activation of Ras homolog enriched in brain (Rheb), which is a small GTPase that activates mTORC1. mTORC1 phosphorylates 4E-BPs leading to their dissociation from eIF4E. In addition to the PI3K/AKT pathway, the activity of mTORC1 is regulated by the serine/threonine kinase 11/LKB1/AMP-kinase (LKB1/AMPK) pathway, regulated in development and DNA damage response 1 (REDD1) and Rag GTPases in response to the changes in cellular energy balance, oxygen and amino acid availability, respectively. Ras and the MAPK pathways are activated by various stimuli through receptor tyrosine kinases (RTKs). In addition the MAPK pathway isactivatedthrough theGprotein–coupled receptors(GPCRs) and byproteinkinaseC (PKC;notshown).TheMAPK pathways encompass an initial GTPase-regulated kinase (MAPKKK), which activates an effector kinase (MAPK) via an intermediate kinase (MAPKK). In response to stimuli such as growth factors, hormones, and phorbol-esters, Ras GTPase stimulates Raf kinase (MAPKKK), which activates extracellular signal-regulated kinases 1 and 2 (ERK 1 and 2) via extracellular signal-regulated kinase activator kinases MEK1 and 2 (MAPKK). Cellular stresses, including osmotic shock, inflammatory cytokines, and UV light, activate p38 MAPKs via multiple mechanisms including Rac kinase (MAPKKK) and MKK3 and 6 (MAPKK). p38 MAPK and ERK activate the MAPK signal–integrating kinases 1 and 2 (MNK1/2), which phosphorylate eIF4E. Additional abbreviations are provided in the text.

 

Cancer Exosomes Perform Cell-Independent MicroRNA Biogenesis and Promote Tumorigenesis
Cancer Cell Nov, 2014; 26: 707–721.
http://dx.doi.org/10.1016/j.ccell.2014.09.005

Breast cancer cells secrete exosomes with specific capacity for cell-independent miRNA biogenesis, while normal cellderivedexosomes lack thisability. Exosomes derivedfrom cancer cellsand serum frompatients withbreast cancer contain the RISC loading complex proteins, Dicer, TRBP, and AGO2, which process pre-miRNAs into mature miRNAs. Cancer exosomes alter the transcriptome of target cells in a Dicer-dependent manner, which stimulate nontumorigenic epithelial cells to form tumors.This study identifies a mechanism whereby cancer cells impart an oncogenic field effect by manipulating the surrounding cells via exosomes. Presence of Dicer in exosomes may serve as biomarker for detection of cancer.


Dicers at RISC. The Mechanism of RNAi

Marcel Tijsterman and Ronald H.A. Plasterk
Cell, Apr 2014; 117:1–4

Figure 1. Model for RNA Silencing in Drosophila In an ordered biochemical pathway, miRNAs (left panel) and siRNAs (right panel) are processed from double-stranded precursor molecules by Dcr-1and Dcr-2, respectively, and stay attached to Dicer-containing complexes, which assemble into RISC. The degree of complementarity between the RNA silencing molecule (in red) and its cognate target determines the fate of the mRNA: blocked translation or immediate destruction.

Argonaute2 Cleaves the Anti-Guide Strand of siRNA during RISC Activation
Cell 2005; 123:621-629
http://www.cell.com/cgi/content/full/123/4/621/DC1/
Dicing and slicing- The core machinery of the RNA interference pathway
Scott C Hammond
FEBS Letters 579 (2005) 5822–5829
http://dx.doi.org:/10.1016/j.febslet.2005.08.079

Fig. 1. Domain organization of RNaseIII gene family. Three classes of RNaseIII genes are shown. The PAZ domain in Dm-Dicer-2 contains mutations in several residues required for RNA binding and may not be functional.

Fig. 2. Model for Dicer catalysis. The PAZ domain binds the 2 nt 30 overhang of a dsRNA terminus. The RNaseIII domains form a pseudo-dimer. Each domain hydrolyzes one strand of the substrate. The binding site of the dsRBD is not defined. The function of the helicase domain is not known.

Fig. 3. Biogenesis pathway of microRNAs. MicroRNA genes are transcribed by RNA polymerase II. The primary transcript is referred to as ‘‘primicroRNA’’. Drosha processing occurs in the nucleus. The resulting precursor, ‘‘pre-microRNA’’, is exported to the cytoplasm for Dicer processing. In a coordinated manner, the mature microRNA is transferred to RISC and unwound by a helicase. mRNA targets that duplex in the Slicer scissile site are cleaved and degraded, if the microRNA is loaded into an Ago2 RISC. Mismatched targets are translationally suppressed. All Ago family members are believed to function in translational suppression.

Fig. 4. Model for Slicer catalysis. The siRNA guide strand is bound at the 50 end by the PIWI domain and at the 30 end by the PAZ domain. The 50 phosphate is coordinated by conserved basic residues. mRNA targets are initially bound by the seed region of the siRNA and pairing is extended to the 30 end. The RNaseH fold hydrolyzes the target in a cation dependent manner. Slicer cleavage is measured from the 50 end of the siRNA. Product is released by an unknown mechanism and the enzyme recycles.

 

 

RNA interference (RNAi) is a biological process in which RNA molecules inhibit gene expression, typically by causing the destruction of specific mRNA molecules. Historically, it was known by other names, including co-suppression, post transcriptional gene silencing (PTGS), and quelling. Only after these apparently unrelated processes were fully understood did it become clear that they all described the RNAi phenomenon. Andrew Fire and Craig C. Mello shared the 2006 Nobel Prize in Physiology or Medicine for their work on RNA interference in the nematode worm Caenorhabditis elegans, which they published in 1998.

 

Two types of small ribonucleic acid (RNA) molecules – microRNA (miRNA) and small interfering RNA (siRNA) – are central to RNA interference. RNAs are the direct products of genes, and these small RNAs can bind to other specific messenger RNA (mRNA) molecules and either increase or decrease their activity, for example by preventing an mRNA from producing a protein. RNA interference has an important role in defending cells against parasitic nucleotide sequences – viruses and transposons. It also influences development.

 

The RNAi pathway is found in many eukaryotes, including animals, and is initiated by the enzyme Dicer, which cleaves long double-stranded RNA (dsRNA) molecules into short double stranded fragments of ~20 nucleotide siRNAs. Each siRNA is unwound into two single-stranded RNAs (ssRNAs), the passenger strand and the guide strand. The passenger strand is degraded and the guide strand is incorporated into the RNA-induced silencing complex (RISC). The most well-studied outcome is post-transcriptional gene silencing, which occurs when the guide strand pairs with a complementary sequence in a messenger RNA molecule and induces cleavage by Argonaute, the catalytic component of the RISC complex. In some organisms, this process spreads systemically, despite the initially limited molar concentrations of siRNA.
http://en.wikipedia.org/wiki/RNA_interference

 

http://upload.wikimedia.org/wikipedia/commons/thumb/e/e4/ShRNA_Lentivirus.svg/481px-ShRNA_Lentivirus.svg.png

 

http://www.frontiersin.org/files/Articles/66078/fnmol-06-00040-HTML/image_m/fnmol-06-00040-g001.jpg
http://dx.doi.org:/10.3389/fnmol.2013.00040

The enzyme dicer trims double stranded RNA, to form small interfering RNA or microRNA. These processed RNAs are incorporated into the RNA-induced silencing.
MiRNA biogenesis and function. (A) The canonical miRNA biogenesis pathway is Drosha- and Dicer-dependent. It begins with RNA Pol II-mediated transcription..

 

Dicer Promotes Transcription Termination

Dicer Promotes Transcription Termination

Dicer Promotes Transcription Termination at Sites of Replication Stress to Maintain Genome Stability
Cell Oct 2014; 159(3): 572–583
http://dx.doi.org/10.1016/j.cell.2014.09.031

http://www.cell.com/cms/attachment/2019646604/2039684570/fx1.jpg

 

18-13 miRNA- protein complex ap-chap-18-pp-42-728

18-13 miRNA- protein complex ap-chap-18-pp-42-728

18-13 miRNA- protein complex (a) Primary miRNA transcript Translation blocked Hydrogen bond (b) Generation and function of miRNAs Hairpin miRNA miRNA Dicer …

http://image.slidesharecdn.com/ap-chap-18-pp-1229097198123780-1/95/ap-chap-18-pp-42-728.jpg?cb=1229090143

 

 

Identification and characterization of small RNAs involved in RNA silencing
FEBS Letters 579 (2005) 5830–5840
http://dx.doi.org:/10.1016/j.febslet.2005.08.009

Fig. 1. Small RNA cloning procedure. Outline of the small RNA cloning procedure. RNA is dephosphorylated (step 1) for joining the 30 adapter by T4 RNA ligase 1 in the presence of ATP (step 2). The use of a chemically adenylated adapter and truncated form of T4 RNA ligase 2 (Rnl2) allows eliminating the dephosphorylation step (step 4). If the RNA was dephosphorylated, it is re-phosphorylated (step 3) prior to 50 adapter ligation with T4 RNA ligase 1 and ATP (step 5). After 50 adapter ligation, a standard reverse transcription is performed (step 6). Alternatively, after 30 adapter ligation, the RNA is used directly for reverse transcription simultaneously with 50 adaptor joining (step 7). In this case, the property of reverse transcriptase to add non-templated cytidine residues at the 50 end of synthesized DNA is used to facilitate template switch of the reverse transcriptase to the 30 guanosine residues of the 50 adapter (SMART technology, Invitrogen). Abbreviations: P and OH indicate phosphate and hydroxyl ends of the RNA; App indicates 50 chemically adenylated adapter; L, 30 blocking group; CIP, calf alkaline phosphatase and PNK, polynucleotide kinase.

 

Transcriptional regulatory functions of nuclear long noncoding RNAs
Trends in Genetics, Aug 2014; 30(8):348-356
http://dx.doi.org/10.1016/j.tig.2014.06.001

Cis-acting lncRNAEnhancer-associated lncRNAIntergenic lncRNA

lncRNA

Promoter-associated lncRNA

Proximity transfer

Trans-acting lncRNA

 

Functional interactions among microRNAs and long noncoding RNAs
Sem Cell Dev Biol 2014; 34:9-14
http://dx.doi.org/10.1016/j.semcdb.2014.05.015
Genome-wide application of RNAi to the discovery of potential drug targets
FEBS Letters 579 (2005) 5988–599
http://dx.doi.org://10.1016/j.febslet.2005.08.015

Fig. 1. Schematic representation of gene silencing by an shRNA-expression vector. The shRNA is processed by Dicer. The processed siRNA enters the RNA-induced silencing complex (RISC), where it targets mRNA for degradation.

Fig. 2. Schematic representation of a transcription system for production of siRNA

Fig. 3. (A) Schematic representation of the proposed siRNA-expression system. Three or four C to U or A to G mutations are introduced into the sense strand. (B) Schematic representation of the discovery of a novel gene using an siRNA library.

 

Imperfect centered miRNA binding sites are common and can mediate repression of target mRNAs
Martin et al. Genome Biology 2014, 15:R51 http://genomebiology.com/2014/15/3/R51

 

 

 

 

Table 1 Number of inferred targets for each miRNA tested

miRNA Probes Transcripts Genes
miR-10a 2,206 5,963 1,887
miR-10a-iso 1,648 1,468 4,211
miR-10b 1,588 3,940 1,365
miR-10b-iso 963 2,235 889
miR-17-5p 1,223 2,862 1,137
miR-17-5p-iso 1,656 3,731 1,461
miR-182 2,261 6,423 2,008
miR-182-iso 1,569 4,316 1,444
miR-23b 2,248 5,383 1,990
miR-27a 2,334 5,310 2,069

Probes: number of probes significantly enriched in pull-downs compared to controls (5% FDR). Transcripts: number of transcripts to which those probes map exactly. Genes: number of genes from which those transcripts originate

Figure 2 Biotin pull-downs identify bone fide miRNA targets. (A) Volcano plot showing the significance of the difference in expression between the miR-17-5p pull-down and the mock-transfected control, for all transcripts expressed in HEK293T cells. Both targets predicted by TargetScan or validated previously via luciferase assay were significantly enriched in the pull-down compared to the controls. (B) Results from luciferase assays on previously untested targets predicted using TargetScan and uncovered using the biotin pull-down. The plot indicates mean luciferase activity from either the empty plasmid or from pMIR containing a miRNA binding site in the 3′ UTR, relative to a negative control. Asterisks indicate a significant reduction in luciferase activity (one-sided t-test; P<0.05) and error bars the standard error of the mean over three replicates. (C-E) Targets identified through PAR-CLIP or through miRNA over-expression studies show greater enrichment in the pull-down. Cumulative distribution of log fold-change in the pull-down for transcripts identified as targets by the indicated miRNA over-expression study or not. Red, canonical transcripts found to be miR-17-5p targets in the indicated study (Table S5 in Additional file 1); black, all other canonical transcripts; p, one-sided P-value from Kolmogorov-Smirnov test for a difference in distributions. (F) To confirm that our results were dependent on RISC association, cells were transfected with either single or double-stranded synthetic miRNAs, then subjected to AGO2 immunoprecipitation. The biotin pull-down was performed in the AGO2-enriched and AGO2-depleted fractions. (G-H) Quantitative RT-PCR revealed that, with double-stranded (ds) miRNA (G), four out of five known targets were enriched relative to input mRNA (*P≤0.05, **P<0.01, ***P<0.001) in the AGO2-enriched but not in the AGO2-depleted fractions, but this enrichment was not seen for the cells transfected with a single-stranded (ss) miRNA (H). The numbers on the x-axis correspond to those in Figure 2F. Error bars represent the standard error of mean (sem).

Figure 5 IsomiRs and canonical miRNAs target many of the same transcripts.

Hammerhead ribozymes in therapeutic target discovery and validation
Drug Disc Today 2009; 14(15/16): 776-783
http://dx.doi.org/10.1016/j.drudis.2009.05.003

Figure 1. Features of hammerhead ribozymes. A generic diagram of a hammerhead ribozyme bound to its target substrate: NUH is the cleavage triplet on target sequence, stems I and III are sites of the specific interactions between ribozyme and target, stem II is the structural element connecting separate parts of the catalytic core. Arrows represent the cleavage site, numbering system according to Hertel et al. [60].

hammerhead ribozyme

hammerhead ribozyme

https://www-ssrl.slac.stanford.edu/research/highlights_archive/ribozyme_fig1.jpg

 

Figure 1  Schematic (A) and ribbon (B) diagrams depicting the crystal structure of the full-length hammerhead ribozyme. The sequence and secondary structure

 

TABLE 1 Typical examples of successful applications of hammerhead ribozymes. Most of the data are derived from [10] and [11], the others are expressly specified.

  • Growth factors, receptors, transduction elements
  • Oncogenes, protoncogenes, fusion genes
  • Apoptosis, survival factors, drug resistance
  • Transcription factors
  • Extracellular matrix, matrix modulating factors
  • Circulating factors
  • Viral genome, viral genes

Figure 2.Target–ribozyme interactions. (a) As cheme of ribozyme binding to full substrate. The calculated energy of this binding ensures the formation of a stable complex. At the denaturating temperature, Tm, will allow this complex to survive to biological conditions. Conversely, after cleavage, binding energies calculated on single, (b) and (c), ribozyme arms are very low and no longer stable. These properties will ensure both the efficient release of cleavage fragments and the prevention of binding to unrelated targets. RNAs complementary to one binding arm only will not be bound or cleaved by the hammerhead catalytic sequence.

Figure 3. ‘Chemical omics’ approach. According to this target discovery strategy: (1) a first round of ‘omic’ study (proteomic, genomic, metabolomic, …) will enable the discovery of a set of (2) putative markers. A series of hammerhead ribozymes will then be prepared in order to target each marker. (4) A second ‘omic’ study round will be performed on (3) knocked down samples obtained after ribozymes administration. (5) A new series of markers will then be produced. An expanding analytical process of this type may be further repeated. Finally, a robust bioinformatic algorithm will make it possible to connect the different markers and draw new hypothetical links and pathways.

 

miRNA

ADAR Enzyme and miRNA Story
Sara Tomaselli, Barbara Bonamassa, Anna Alisi, et al.
Int. J. Mol. Sci. 2013, 14, 22796-22816;
http://dx.doi.org:/10.3390/ijms141122796

Adenosine deaminase acting on RNA (ADAR) enzymes convert adenosine (A) to inosine (I) in double-stranded (ds) RNAs. Since Inosine is read as Guanosine, the biological consequence of ADAR enzyme activity is an A/G conversion within RNA molecules. A-to-I editing events can occur on both coding and non-coding RNAs, including microRNAs (miRNAs), which are small regulatory RNAs of ~20–23 nucleotides that regulate several cell processes by annealing to target mRNAs and inhibiting their translation. Both miRNA precursors and mature miRNAs undergo A-to-I RNA editing, affecting the miRNA maturation process and activity. ADARs can also edit 3′ UTR of mRNAs, further increasing the interplay between mRNA targets and miRNAs. In this review, we provide a general overview of the ADAR enzymes and their mechanisms of action as well as miRNA processing and function. We then review the more recent findings about the impact of ADAR-mediated activity on the miRNA pathway in terms of biogenesis, target recognition, and gene expression regulation.

Figure 1. Structure of ADAR family proteins: ADAR1, ADAR2, and ADAR3. The ADAR enzymes contain a C-terminal conserved catalytic deaminase domain (DM), two or three dsRBDs in the N-terminal portion. ADAR1 full-length protein also contains a N-terminal Zα domain with a nuclear export signal (NES) and a Zβ domain, while ADAR3 has a  R-domain. A nuclear localization signal is also indicated.

 

Comprehensive modeling of microRNA targets predicts functional non-conserved and non-canonical sites
Doron Betel, Anjali Koppal, Phaedra Agius, Chris Sander, Christina Leslie
Genome Biology 2010, 11:R90 http://genomebiology.com/2010/11/8/R90

microRNAs are a class of small regulatory RNAs that are involved in post-transcriptional gene silencing. These small (approximately 22 nucleotide) single-strand RNAs guide a gene silencing complex to an mRNA by complementary base pairing, mostly at the 3′ untranslated region (3′ UTR). The association of the RNAinduced silencing complex (RISC) to the conjugate mRNA results in silencing the gene either by translational repression or by degradation of the mRNA. Reliable microRNA target prediction is an important and still unsolved computational challenge, hampered both by insufficient knowledge of microRNA biology as well as the limited number of experimentally validated targets.

mirSVR is a new machine learning method for ranking microRNA target sites by a down-regulation score. The algorithm trains a regression model on sequence and contextual features extracted from miRanda-predicted target sites. In a large-scale evaluation, miRanda-mirSVR is competitive with other target prediction methods in identifying target genes and predicting the extent of their downregulation at the mRNA or protein levels. Importantly, the method identifies a significant number of experimentally determined non-canonical and non-conserved sites.
Human RISC – MicroRNA Biogenesis and Posttranscriptional Gene Silencing
Cell 2005; 123:631-640
http://dx.doi.org:/10.1016/j.cell.2005.10.022
Development of microRNA therapeutics
Eva van Rooij & Sakari Kauppinen
EMBO Mol Med (2014) 6: 851–864
http://dx.doi.org:/10.15252/emmm.20110089

MicroRNAs (miRNAs) play key regulatory roles in diverse biological processes and are frequently dysregulated in human diseases. Thus, miRNAs have emerged as a class of promising targets for therapeutic intervention. Here, we describe the current strategies for therapeutic modulation of miRNAs and provide an update on the development of miRNA-based therapeutics for the treatment of cancer, cardiovascular disease and hepatitis C virus (HCV) infection.

Figure 1. miRNA biogenesis and modulation of miRNA activity by miRNA mimics and antimiR oligonucleotides. MiRNA genes are transcribed by RNA polymerase II from intergenic, intronic or polycistronic loci to long primary miRNA transcripts (pri-miRNAs) and processed in the nucleus by the Drosha–DGCR8 complex to approximately 70 nt pre-miRNA hairpin structures. The most common alternative miRNA biogenesis pathway involves short intronic hairpins, termed mirtrons, that are spliced and debranched to form pre-miRNA hairpins. Pre-miRNAs are exported into the cytoplasm and then cleaved by the Dicer–TRBP complex to imperfect miRNA: miRNA* duplexes about 22 nucleotides in length. In the cytoplasm, miRNA duplexes are incorporated into Argonaute-containing miRNA induced silencing complex (miRISC), followed by unwinding of the duplex and retention of the mature miRNA strand in miRISC, while the complementary strand is released and degraded. The mature miRNA functions as a guide molecule for miRISC by directing it to partially complementary sites in the target mRNAs, resulting in translational repression and/or mRNA degradation. Currently, two strategies are employed to modulate miRNA activity: restoring the function of a miRNA using double-stranded miRNA mimics, and inhibition of miRNA function using single-stranded anti-miR oligonucleotides.

Figure 2. Design of chemically modified miRNA modulators. (A) Structures of chemical modifications used in miRNA modulators. A number of different sugar modifications are used to increase the duplex melting temperature (Tm) of anti-miR oligonucleotides. The20-O-methyl(20-O-Me), 20-O-methoxyethyl(20-MOE )and 20-fluoro(20-F) nucleotides are modified at the 20 position of the sugar moiety, whereas locked nucleic acid (LNA) is a bicyclic RNA analogue in which the ribose is locked in a C30-endo conformation by introduction of a 20-O,40-C methylene bridge. To increase nuclease resistance and enhance the pharmacokinetic properties, most anti-miR oligonucleotides harbor phosphorothioate (PS) backbone linkages, in which sulfur replaces one of the non-bridging oxygen atoms in the phosphate group. In morpholino oligomers, a six-membered morpholine ring replaces the sugar moiety. Morpholinos are uncharged and exhibit a slight increase in binding affinity to their cognate miRNAs. PNA oligomers are uncharged oligonucleotide analogues, in which the sugar–phosphate backbone has been replaced by a peptide-like backbone consisting of N-(2-aminoethyl)-glycine units. (B) An example of a synthetic double-stranded miRNA mimic described in this review. One way to therapeutically mimic a miRNA is by using synthetic RNA duplexes that harbor chemical modifications for improved stability and cellular uptake. In such constructs, the antisense (guide) strand is identical to the miRNA of interest, while the sense (passenger) strand is modified and can be linked to a molecule, such as cholesterol, for enhanced cellular uptake. The sense strand contains chemical modifications to prevent mi-RISC loading. Several mismatches can be introduced to prevent this strand from functioning as an anti-miR, while it is further left unmodified to ensure rapid degradation.The20-F modification helps to protect the antisense strand against exonucleases, hence making the guide strand more stable, while it does not interfere with mi-RISC loading. (C) Design of chemically modified anti-miR oligonucleotides described in this review. Antagomirs are30 cholesterol-conjugated,20-O-Me oligonucleotides fully complementary to the mature miRNA sequence with several PS moieties to increase their in vivo stability. The use of unconjugated 20-F/MOE-, 20-MOE- or LNA-modified anti-miR oligonucleotides harboring a complete PS backbone represents another approach for inhibition of miRNA function in vivo. The high duplex melting temperature of LNA-modified oligonucleotides allows efficient miRNA inhibition using truncated, high-affinity 15–16-nucleotide LNA/DNA anti-miR oligonucleotides targeting the 50 region of the mature miRNA. Furthermore, the high binding affinity of fully LNA-modified 8-mer PS oligonucleotides, designated as tiny LNAs, facilitates simultaneous inhibition of entire miRNA seed families by targeting the shared seed sequence.

Human MicroRNA Targets
Bino John, Anton J. Enright, Alexei Aravin, Thomas Tuschl,.., Debora S. Mark
PLoS Biol 2004; 2(11): e363  http://www.plosbiology.org

More than ten years after the discovery of the first miRNA gene, lin-4 (Chalfie et al. 1981; Lee et al. 1993), we know that miRNA genes constitute about 1%–2% of the known genes in eukaryotes. Investigation of miRNA expression combined with genetic and molecular studies in Caenorhabditis elegans, Drosophila melanogaster, and Arabidopsis thaliana have identified the biological functions of several miRNAs (recent review, Bartel 2004). In C. elegans, lin-4 and let-7 were first discovered as key regulators of developmental timing in early larval developmental transitions (Ambros 2000; Abrahante et al. 2003; Lin et al. 2003; Vella et al. 2004). More recently lsy-6 was shown to determine the left–right asymmetry of chemoreceptor expression (Johnston and Hobert 2003). In D. melanogaster, miR-14 has a role in apoptosis and fat metabolism (Xu et al. 2003) and the bantam miRNA targets the gene hid involved in apoptosis and growth control (Brennecke et al. 2003).

MicroRNAs (miRNAs) interact with target mRNAs at specific sites to induce cleavage of the message or inhibit translation. The specific function of most mammalian miRNAs is unknown. We have predicted target sites on the 39 untranslated regions of human gene transcripts for all currently known 218 mammalian miRNAs to facilitate focused experiments. We report about 2,000 human genes with miRNA target sites conserved in mammals and about 250 human genes conserved as targets between mammals and fish. The prediction algorithm optimizes sequence complementarity using position-specific rules and relies on strict requirements of interspecies conservation. Experimental support for the validity of the method comes from known targets and from strong enrichment of predicted targets in mRNAs associated with the fragile X mental retardation protein in mammals. This is consistent with the hypothesis that miRNAs act as sequence-specific adaptors in the interaction of ribonuclear particles with translationally regulated messages. Overrepresented groups of targets include mRNAs coding for transcription factors, components of the miRNA machinery, and other proteins involved in translational regulation, as well as components of the ubiquitin machinery, representing novel feedback loops in gene regulation. Detailed information about target genes, target processes, and open-source software for target prediction (miRanda) is available at http://www.microrna.org. Our analysis suggests that miRNA genes, which are about 1% of all human genes, regulate protein production for 10% or more of all human genes.

Figure 1. Target Prediction Pipeline for miRNA Targets in Vertebrates The mammalian (human, mouse, and rat) and fish (zebra and fugu) 39 UTRs were first scanned for miRNA target sites using position specific rules of sequence complementarity. Next, aligned UTRs of orthologous genes were used to check for conservation of miRNA– target relationships (‘‘target conservation’’) between mammalian genomes and, separately, between fish genomes. The main results (bottom) are the conserved mammalian and conserved fish targets, for each miRNA,as well as a smaller set of super-conserved vertebrate targets.   http://dx.doi.org:/10.1371/journal.pbio.0020363.g00
Figure 2. Distribution of Transcripts with Cooperativity of Target Sites and Estimated Number of False Positives Each bar reflects the number of human transcripts with a given number of target sites on their UTR. Estimated rate of false positives(e.g., 39%for2 targets) is given by the number of target sites predicted using shuffled miRNAs processed in a way identical to real miRNAs, including the use of interspecies conservation filter. http://dx.doi.org:/10.1371/journal.pbio.0020363.g002

Conserved Seed Pairing, Often improved an-Flanked by Adenosines, Indicates Thousands of Human Genes are MicroRNA Targets
Cell, Jan 2005; 120: 15–20
http://dx.doi.org:/10.1016/j.cell.2004.12.035

Integrated analysis of microRNA and mRNA expression. adding biological significance to microRNA target predictions.
Maarten van Iterson, Sander Bervoets, Emile J. de Meijer, et al.
Nucleic Acids Research, 2013; 41(15), e146
http://dx.doi.org:/10.1093/nar/gkt525

Current microRNA target predictions are based on sequence information and empirically derived rules but do not make use of the expression of microRNAs and their targets. This study aimed to improve microRNA target predictions in a given biological context, using in silico predictions, microRNA and mRNA expression. We used target prediction tools to produce lists of predicted targets and used a gene set test designed to detect consistent effects of microRNAs on the joint expression of multiple targets. In a single test, association between microRNA expression and target gene set expression as well as the contribution of the individual target genes on the association are determined. The strongest negatively associated mRNAs as measured by the test were prioritized. We applied our integration method to a well-defined muscle differentiation model. Validation of our predictions in C2C12 cells confirmed predicted targets of known as well as novel muscle-related microRNAs. We further studied associations between microRNA–mRNA pairs in human prostate cancer, finding some pairs that have been recently experimentally validated by others. Using the same study, we showed the advantages of the global test over Pearson correlation and lasso. We conclude that our integrated approach successfully identifies regulated microRNAs and their targets.

Long non-coding RNA and microRNAs might act in regulating the expression of BARD1 mRNAs
Int J Biol & Cell Biol 2014; 54:356-367
http://dx.doi.org/10.1016/j.biocel.2014.06.018

 

Passenger-Strand Cleavage Facilitates Assembly of siRNA into Ago2-Containing RNAi Enzyme Complexes
Cell 2006; 123:607-620
http://dx.doi.org:/10.1016/j.cell.2006.08.044

 

RNAi- RISC Gets Loaded
Cell 2005; 123:543-553
http://dx.doi.org:/10.1016/j.cell.2005.11.006
RNAi- The Nuts and Bolts of the RISC Machine
Cell 2005; 122:17-20
http://dx.doi.org:/10.1016/j.cell.2005.06.023
Structural domains in RNAi
FEBS Letters 579 (2005) 5841–5849
http://dx.doi.org:/10.1016/j.febslet.2005.07.072

Fig. 1. A ‘‘Domain-centric’’ view of RNAi. (A) The conserved pathways of RNA silencing. The domain structure of each protein in (hypothetical) interaction with its RNA is shown. For clarity, the second column lists domains in order N- to C-terminal. Figures are not to scale. In brief, Drosha, an RNase III enzyme, and its obligate binding partner, Pasha recognize pri-mRNA loops, and cut these into 70 nt hairpin pre-miRNAs. Dicer utilizes a PAZ domain to sense the 30 2-nt overhang created, and further processes these, and dsRNAs into miRNAs and siRNAs. Argonaute binds the 50 end of guide RNAs via its PIWI domain, and the 30 end via a PAZ domain, yielding RISCs that effect RNA silencing through several mechanisms. A Viral protein, VP19 can suppress RNA silencing by sequestering siRNAs. (B) A summary of known siRNA structural biology. Listed by domain are solved structures, their protein/organism of origin, and ligands, where applicable. Also shown are PDB codes.

Fig. 2. Novel modes of RNA recognition. (A) A typical dsRBD: Xenopus binding protein A (1DI2). A RNA helix is modeled pink, and the protein is rendered in transparent electrostatic contours (blue is basic, red acidic). Note the interaction of helices along the major groove, and the position of helix 1. A second dsRBD protein is visible, in the lower right. (B) A dsRBD, Saccharomyces Rnt1P (1T4L), recognizes hairpin loops. A novel third helix (top) pushes helix one into the loop of a hairpin RNA. (C) 30-OH recognition by PAZ. Human Eif2c1 (1SI3) bound to RNA (pink) is shown. PAZ is green, with transparent electrostatic surface plot. The OB-fold (nucleotide binding fold) and the insertion domain are labeled. Note the glove-and-thumb like cleft they form, that the 30-OH is inserted into. A basic groove (blue) the RNA binds along outside the cleft is visible. (D) A close-up view of PAZ, as in C (surface not-transparent, slightly rotated). See white arrows for orientation, and location of 30-OH binding site. RNA is shown red in sticks. The terminal –OH is barely visible, buried in a cleft. It and the carbon it bonds have been colored yellow for clarity. (E) The PIWI domain (2BGG). Note the insertion of the 50P red (labeled) into the binding site. Its complimentary strand (pink) is not annealed to it, and the 30 overhang and first complimentary bases sit on the protein surface. (F) An enlarged view of (E), with protein in slate and RNA modeled as red sticks. The coordinated magnesium is a grey sphere, which is coordinated by the terminal carboxylate of the protein, protein side chains, and RNA phosphate oxygens. The 50 base stacks against a conserved Tyr. Several other sidechain contacts are shown.

Fig. 3. Argonaute/RISC. (A) P. furiosus Argonaute (PDB 1Z26). A color-guided key to the domains is presented. PAZ sits over the PIWI/N/MID bowl and active site. The liganding atoms for the catalytic metal are depicted as yellow balls for clarity. The tungstate binding site (50P surrogate) is shown as tan spheres. (B) A guide strand channel. Looking down from the PAZ domain towards the active site, Z-sections are clipped off. Colors of domains are as in the key in (A). Wrapping down along a basic cleft from the PAZ 30OH binding site (approximate position labeled), a RNA binding groove passes the active site (yellow), and runs down to the 50P binding site (tan balls). A second cleft running perpendicular to this one at its entry may accommodate target strand RNA. For more detail, and models of siRNA placed into the grooves, see [27,29].

Fig. 4. VP19 sequestration of siRNA. (A) CIRV VP19 (1RPU, RNA removed). Two monomers (blue and cyan) form an 8 strand, concave b-sheet with bracketing helices at the ends. (B) Tombus viral VP19 bound to siRNA (1 monomer shown). RNA strands are modeled as sticks, with one strand pink and one red. The bracketing helix places two tryptophans in position to stack over the terminal RNA bases. On the b-sheet surface, and Arg and a Lys interact with the phosphate backbone, and at the center of the RNA binding surface, a number of Ser and Thr mediate an extensive hydrogen bond network. Both the Trp brackets and RNA binding by an extended b-sheet are unique.

 

Small RNA asymmetry in RNAi- Function in RISC assembly and gene regulation
FEBS Letters 579 (2005) 5850–5857
http://dx.doi.org:/10.1016/j.febslet.2005.08.071

 

The role of the oncofetal IGF2 mRNA-binding protein 3 (IGF2BP3) in cancer
Seminars in Cancer Biol 2014; 29:3-12
http://dx.doi.org/10.1016/j.semcancer.2014.07.006

Table 1 – Target mRNAs of IGF2BP3.

Target cis-Element Regulation
CD44 3’ -utr Control of mRNA stability
IGF2 5’ -utr Translational control
H19 ncRNA Unknown
ACTB 3’ -utr Unknown
MYC CRD Unknown
CD164 Unknown Control of mRNA stability
MMP9 Unknown Control of mRNA stability
ABCG2 Unknown Unknown
PDPN 3’ -utr Control of mRNA stability
HMGA2 3’ -utr Protection from miR directed degradation
CCND1 3’ -utr translational control
CCND3 3’ -utr translational control
CCNG1 3’ -utr translationalcontrol

 

Targeting glucose uptake with siRNA-based nanomedicine for cancer therapy
Biomaterials 2015; 51:1-11
http://dx.doi.org/10.1016/j.biomaterials.2015.01.068
The therapeutic potential of RNA interference
FEBS Letters 579 (2005) 5996–6007
http://dx.doi.og:/10.1016/j.febslet.2005.08.004

Table 1 Companies developing RNAi therapeutics that includes cancer

Company name Primary areas of interest
Atugen AG Metabolic disease; cancer ocular disease; skin disease
Benitec Australia Limited Hepatitis C virus; HIV/AIDS; cancer; diabetes/obesity
Calando Pharmaceuticals Nanoparticle technology
Genta Incorporated Cancer
Intradigm Corporation Cancer; SARS; arthritis
Sirna Therapeutics, Inc. AMD; Hepatitis C virus; asthma; diabetes; cancer; Huntington s disease; hearing loss

 

The Noncoding RNA Revolution—Trashing Old Rules to Forge New Ones
Cell 2014; 157:77-94
http://dx.doi.org/10.1016/j.cell.2014.03.008

Figure 1. Noncoding RNAs Function in Diverse Contexts Noncoding RNAs function in all domains of life, regulating gene expression from transcription to splicing to translation and contributing to genome organization and stability. Self-splicing RNAs, ribosomes, and riboswitches function in both eukaryotes and bacteria. Archaea (not shown) also utilize ncRNA systems including ribosomes, riboswitches, snoRNPs, and CRISPR. Orange strands, ncRNA performing the action indicated; red strands, the RNA acted upon by the ncRNA. Blue strands, DNA. Triangle, small-molecule metabolite bound by a riboswitch. Ovals indicate protein components of an RNP, such as the spliceosome (white oval), ribosome (two purple subunits), or other RNPs (yellow ovals). Because of the importance of RNA structure in these ncRNAs, some structures are shown but they are not meant to be realistic.

 

miRNAs and cancer targeting

Table 1 of targets

miRNA Cancer type reference
NA GI cancer Current status of miRNA-targeting therapeutics and preclinical studies against gastroenterological carcinoma
NA Renal cell Differential expression profiling of microRNAs and their potential involvement in renal cell carcinoma pathogenesis
NA urothelial
cancer
A microRNA expression ratio defining the invasive phenotype in bladder tumors
miR-31 breast A Pleiotropically Acting MicroRNA, miR-31, inhibits breast cancer growth
miR-512-3p NSCLC Inhibition of RAC1-GEF DOCK3 by miR-512-3p contributes to suppression of metastasis in non-small cell lung cancer
miR-495 gastric Methylation-associated silencing of miR-495 inhibit the migration and invasion of human gastric cancer cells
microRNA-218 prostate microRNA-218 inhibits prostate cancer cell growth and promotes apoptosis by repressing TPD52 expression
MicroRNA-373 cervical cancer MicroRNA-373 functions as an oncogene and targets YOD1 gene in cervical cancer
miR-25 NSCLC miR-25 modulates NSCLC cell radio-sensitivity – inhibiting BTG2 expression
miR-92a cervical cancer miR-92a. upregulated in cervical cancer & promotes cell proliferation and invasion by targeting FBXW7
MiR-153 NSCLC MiR-153 inhibits migration and invasion of human non-small-cell lung cancer by targeting ADAM19
miR-203 melanoma miR-203 inhibits melanoma invasive and proliferative abilities by targeting the polycomb group gene BMI1
miR-204-5p Papillary thyroid miR-204-5p suppresses cell proliferation by inhibiting IGFBP5 in papillary thyroid carcinoma
miR-342-3p Hepato-cellular miR-342-3p affects hepatocellular carcinoma cell proliferation via regulating NF-κB pathway
miR-1271 NSCLC miR-1271 promotes non-small-cell lung cancer cell proliferation and invasion via targeting HOXA5
miR-203 pancreas Pancreatic cancer derived exosomes regulate the expression of TLR4 in dendritic cells via miR-203
miR-203 metastatic SCC Rewiring of an Epithelial Differentiation Factor, miR-203, to Inhibit Human SCC Metastasis
miR-204 RCC TRPM3 and miR-204 Establish a Regulatory Circuit that Controls Oncogenic Autophagy in Clear Cell Renal Cell Carcinoma
NA urologic MicroRNAs and cancer. Current and future perspectives in urologic oncology
NA RCC MicroRNAs and their target gene networks in renal cell carcinoma
NA osteoSA MicroRNAs in osteosarcoma
NA urologic MicroRNA in Prostate, Bladder, and Kidney Cancer
NA urologic Micro-RNA profiling in kidney and bladder cancers

 

Current status of miRNA-targeting therapeutics and preclinical studies against gastroenterological carcinoma
Shibata et al. Molecular and Cellular Therapies 2013, 1:5 http://www.molcelltherapies.com/content/1/1/5

Differential expression profiling of microRNAs and their potential involvement in renal cell carcinoma pathogenesis
Clinical Biochemistry 43 (2010) 150–158
http://dx.doi.org:/10.1016/j.clinbiochem.2009.07.020

A microRNA expression ratio defining the invasive phenotype in bladder tumors
Urologic Oncology: Seminars and Original Investigations 28 (2010) 39–48
http://dx.doi.org:/10.1016/j.urolonc.2008.06.006

A Pleiotropically Acting MicroRNA, miR-31, inhibits breast cancer growth
Cell 137, 1032–1046, June 12, 2009
http://dx.doi.org:/10.1016/j.cell.2009.03.047

Inhibition of RAC1-GEF DOCK3 by miR-512-3p contributes to suppression of metastasis in non-small cell lung cancer
Intl JBiochem & Cell Biol 2015; 61:103-114
http://dx.doi.org/10.1016/j.biocel.2015.02.005

Methylation-associated silencing of miR-495 inhibit the migration and invasion of human gastric cancer cells by directly targeting PRL-3
Biochem Biochem Res Commun 2014; 456:344-350
http://dx.doi.org/10.1016/j.bbrc.2014.11.083

microRNA-218 inhibits prostate cancer cell growth and promotes apoptosis by repressing TPD52 expression
Biochem Biophys Res Commun 2015; 456:804-809
http://dx.doi.org/10.1016/j.bbrc.2014.12.026

MicroRNA-373 functions as an oncogene and targets YOD1 gene in cervical cancer
BBRC 2015; xx:1-6
http://dx.doi.org/10.1016/j.bbrc.2015.02.138

miR-25 modulates NSCLC cell radio-sensitivity – inhibiting BTG2 expression
BBRC 2015; 457:235-241
http://dx.doi.org/10.1016/j.bbrc.2014.12.094

miR-92a. upregulated in cervical cancer & promotes cell proliferation and invasion by targeting FBXW7
BBRC 2015; 458:63-69
http://dx.doi.org/10.1016/j.bbrc.2015.01.066

MiR-153 inhibits migration and invasion of human non-small-cell lung cancer by targeting ADAM19
BBRC 2015; 456:381-385
http://dx.doi.org/10.1016/j.bbrc.2014.11.093

miR-203 inhibits melanoma invasive and proliferative abilities by targeting the polycomb group gene BMI1
BBMC 2015; 456: 361-366
http://dx.doi.org/10.1016/j.bbrc.2014.11.087

miR-204-5p suppresses cell proliferation by inhibiting IGFBP5 in papillary thyroid carcinoma
BBRC 2015; 457:621-627
http://dx.doi.org/10.1016/j.bbrc.2015.01.037

miR-342-3p affects hepatocellular carcinoma cell proliferation via regulating NF-κB pathway
BBRC 2015; 457:370-377
http://dx.doi.org/10.1016/j.bbrc.2014.12.119

miR-1271 promotes non-small-cell lung cancer cell proliferation and invasion via targeting HOXA5
BBRC 2015; 458:714-719
http://dx.doi.org/10.1016/j.bbrc.2015.02.033

Pancreatic cancer derived exosomes regulate the expression of TLR4 in dendritic cells via miR-203
Cell Immunol 2014; 292:65-69
http://dx.doi.org/10.1016/j.cellimm.2014.09.004

Rewiring of an Epithelial Differentiation Factor, miR-203, to Inhibit Human Squamous Cell Carcinoma Metastasis
Cell Reports 2014; 9:104-117
http://dx.doi.org/10.1016/j.celrep.2014.08.062

TRPM3 and miR-204 Establish a Regulatory Circuit that Controls Oncogenic Autophagy in Clear Cell Renal Cell Carcinoma
Cancer Cell Nov 10, 2014; 26: 738–753
http://dx.doi.org/10.1016/j.ccell.2014.09.015

MicroRNA in Prostate, Bladder, and Kidney Cancer
Eur Urol 2011; 59:671-681
http://dx.doi.org/10.1016/j.eururo.2011.01.044

Micro-RNA profiling in kidney and bladder cancers
Urologic Oncology: Seminars and Original Investigations 2007; 25:387–392
http://dx.doi.org:/10.1016/j.urolonc.2007.01.019

MicroRNAs and cancer. Current and future perspectives in urologic oncology
Urologic Oncology: Seminars and Original Investigations 2010; 28:4–13
http://dx.doi.org:/10.1016/j.urolonc.2008.10.021

MicroRNAs and their target gene networks in renal cell carcinoma
BBRC 2011; 405:153-156
http://dx.doi.org/10.1016/j.bbrc.2011.01.019

MicroRNAs in osteosarcoma
Clin Chim Acta 2015; 444:9-17
http://dx.doi.org/10.1016/j.cca.2015.01.025

 

Table 2. miRNA cancer therapeutics

 

 

  • miRNA and mRNA cancer signatures determined by analysis of expression levels in large cohorts of patients
    | PNAS | Nov 19, 2013; 110(47): 19160–19165
    http://www.pnas.org/cgi/doi/10.1073/pnas.1316991110The study of mRNA and microRNA (miRNA) expression profiles of cells and tissue has become a major tool for therapeutic development. The results of such experiments are expected to change the methods used in the diagnosis and prognosis of disease. We introduce surprisal analysis, an information-theoretic approach grounded in thermodynamics, to compactly transform the information acquired from microarray studies into applicable knowledge about the cancer phenotypic state. The analysis of mRNA and miRNA expression data from ovarian serous carcinoma, prostate adenocarcinoma, breast invasive carcinoma, and lung adenocarcinoma cancer patients and organ specific control patients identifies cancer-specific signatures. We experimentally examine these signatures and their respective networks as possible therapeutic targets for cancer in single cell experiments.

 

 

RNA editing is vital to provide the RNA and protein complexity to regulate the gene expression. Correct RNA editing maintains the cell function and organism development. Imbalance of the RNA editing machinery may lead to diseases and cancers. Recently,RNA editing has been recognized as a target for drug discovery although few studies targeting RNA editing for disease and cancer therapy were reported in the field of natural products. Therefore, RNA  editing may be a potential target for therapeutic natural products

 

Aberrant microRNA (miRNA) expression is implicated in tumorigenesis. The underlying mechanisms are unclear because the regulations of each miRNA on potentially hundreds of mRNAs are sample specific.

 

We describe a novel approach to infer Probabilistic Mi RNA–mRNA  Interaction Signature (‘ProMISe’) from a single pair of miRNA–mRNA expression profile. Our model considers mRNA and miRNA competition as a probabilistic function of the expressed seeds (matches). To demonstrate ProMISe, we extensively exploited The Cancer Genome Atlas data. As a target predictor, ProMISe identifies more confidence/validated targets than other methods. Importantly, ProMISe confers higher cancer diagnostic power than using expression profiles alone.

Gene set enrichment analysis on averaged ProMISe uniquely revealed respective target enrichments of oncomirs miR-21 and 145 in glioblastoma and ovarian cancers. Moreover, comparing matched breast (BRCA) and thyroid (THCA) tumor/normal samples uncovered thousands of tumor-related interactions. For example, ProMISe– BRCA network involves miR-155/183/21, which exhibits higher ProMISe coupled with coherently higher miRNA expression and lower target expression; oncomirs miR-221/222 in the ProMISe–THCA network engage with many downregulated target genes. Together, our probabilistic approach of integrating expression and sequence scores establishes a functional link between the aberrant miRNA and mRNA expression, which was previously under-appreciated due to the methodological differences.

 

 

 

 

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Nanotechnology and HIV/AIDS Treatment

Author: Tilda Barliya, PhD

 

AIDS was first reported in 1981 followed by the identification of HIV as the cause of the disease in 1983 and is now a global pandemic that has become the leading infectious killer of adults worldwide. By 2006, more than 65 million people had been infected with the HIV virus worldwide and 25 million had died of AIDS (Merson MH. The HIV-AIDS pandemic at 25 – the global response. (1, 2). This has caused tremendous social and economic damage worldwide, with developing countries, particularly Sub-Saharan Africa, heavily affected.

A cure for HIV/AIDS has been elusive in almost 30 years of research. Early treatments focused on antiretroviral drugs that were effective only to a certain degree. The first drug, zidovudine, was approved by the US FDA in 1987, leading to the approval of a total of 25 drugs to date, many of which are also available in fixed-dose combinations and generic formulations for use in resource-limited settings (to date, only zidovudine and didanosine are available as true generics in the USA).

However, it was the advent of a class of drugs known as protease inhibitors and the introduction of triple-drug therapy in the mid-1990s that revolutionized HIV/AIDS treatment (3,4). This launched the era of highly active antiretroviral therapy (HAART), where a combination of three or more different classes of drugs are administered simultaneously.

Challenges of HIV/AIDS treatment

  • HIV resides in latent cellular and anatomical reservoirs where current drugs are unable to completely eradicate the virus.
  • Macrophages are major cellular reservoirs, which also contribute to the generation of elusive mutant viral genotypes by serving as the host for viral genetic recombination.
  • Anatomical latent reservoirs include secondary lymphoid tissue, testes, liver, kidney, lungs, the gut and the brain.
  • The major challenge facing current drug regimens is that they do not fully eramacrdicate the virus from these reservoirs; requiring patients take medications for life. Under current treatment, pills are taken daily, resulting in problems of patient adherence. The drugs also have side effects and in some people the virus develops resistance against certain drugs.

Current treatment in HIV/AIDS

The use of the HAART regimen, particularly in the developed world, has resulted in tremendous success in improving the expectancy and quality of lives for patients. However, some HAART regimens have serious side effects and, in all cases, HAART has to be taken for a lifetime, with daily dosing of one or more pills. Due to the need to take the medication daily for a lifetime, patients fail to adhere to the treatment schedule, leading to ineffective drug levels in the body and rebound of viral replication.Some patients also develop resistance to certain combinations of drugs, resulting in failure of the treatment. The absence of complete cure under current treatment underscores the great need for continued efforts in seeking innovative approaches for treatment of HIV/AIDS.

Drug resistance is mainly caused by the high genetic diversity of HIV-1 and the continuous mutation it undergoes. This problem is being addressed with individualized therapy, whereby resistance testing is performed to select a combination of drugs that is most effective for each patient (5). In addition, side effects due to toxicities of the drugs are also a concern. There are reports that patients taking HAART experience increased rates of heart disease, diabetes, liver disease, cancer and accelerated aging. Most experts agree that these effects could be due to the HIV infection itself or co-infection with another virus, such as co-infection with hepatitis C virus resulting in liver disease. However, the toxicities resulting from the drugs used in HAART could also contribute to these effects.

Under current treatment, complete eradication of the virus from the body has not been possible. The major cause for this is that the virus resides in ‘latent reservoirs’ within memory CD4+ T cells and cells of the macrophage–monocyte lineage. A major study recently found that, in addition to acting as latent reservoirs, macrophages significantly contribute to the generation of elusive mutant viral genotypes by serving as the host for viral genetic recombination (6).  The cells that harbor latent HIV are typically concentrated in specific anatomic sites, such as secondary lymphoid tissue, testes, liver, kidney, lungs, gut and the CNS. The eradication of the virus from such reservoirs is critical to the effective long-term treatment of HIV/AIDS patients.

Therefore, there is a great need to explore new approaches for developing nontoxic, lower-dosage treatment modalities that provide more sustained dosing coverage and effectively eradicate the virus from the reservoirs, avoiding the need for lifetime treatments.

Nanotechnology for HIV/AIDS treatment

The use of nanotechnology platforms for delivery of drugs is revolutionizing medicine in many areas of disease treatment.

Nanotechnology-based platforms for systemic delivery of antiretroviral drugs could have similar advantages.

  • Controlled-release delivery systems can enhance their half-lives, keeping them in circulation at therapeutic concentrations for longer periods of time. This could have major implications in improving adherence to the drugs.
  • Nanoscale delivery systems also enhance and modulate the distribution of hydrophobic and hydrophilic drugs into and within different tissues due to their small size. This particular feature of nanoscale delivery systems appears to hold the most promise for their use in clinical treatment and prevention of HIV. Specifically, targeted delivery of antiretroviral drugs to CD4+ T cells and macrophages as well as delivery to the brain and other organ systems could ensure that drugs reach latent reservoirs
  • Moreover, by controlling the release profiles of the delivery systems, drugs could be released over a longer time and at higher effective doses to the specific targets. Figure 1. Various nanoscale drug delivery systems.

Optional treatments:

  •    Antiretroviral drugs
  •    Gene Therapy
  •    Immune Therapy
  •    Prevention

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The use of nanotechnology systems for delivery of antiretroviral drugs has been extensively reviewed by Nowacek et al. and Amiji et al. (7,8).

In a recent study based on polymeric systems, nanosuspensions (200 nm) of the drug rilpivirine (TMC278) stabilized by polyethylene. A series of experiments by Dou et al. showed that nanosuspension of the drug indinavir can be stabilized by a surfactant system comprised of Lipoid E80 for effective delivery to various tissues. The indinavir nanosuspensions were loaded into macrophages and their uptake was investigated. Macrophages loaded with indinavir nanosuspensions were then injected intravenously into mice, resulting in a high distribution in the lungs, liver and spleen. More significantly, the intravenous administration of a single dose of the nanoparticle-loaded macrophages in a rodent mouse model of HIV brain infection resulted in significant antiviral activity in the brain and produced measureable drug levels in the blood up to 14 days post-treatment.polypropylene glycol (poloxamer 338) and PEGylated tocopheryl succinate ester (TPGS 1000) were studied in dogs and mice. A single-dose administration of the drug in nanosuspensions resulted in sustained release over 3 months in dogs and 3 weeks in mice, compared with a half-life of 38 h for free drug. These results serve as a proof-of-concept that nanoscale drug delivery may potentially lower dosing frequency and improve adherence.

Active targeting strategies have also been employed for antiretroviral drug delivery. Macrophages, which are the major HIV reservoir cells, have various receptors on their surface such as formyl peptide, mannose, galactose and Fc receptors, which could be utilized for receptor-mediated internalization. The drug stavudine was encapsulated using various liposomes (120–200 nm) conjugated with mannose and galactose, resulting in increased cellular uptake compared with free drug or plain liposomes, and generating significant level of the drug in liver, spleen and lungs. Stavudine is a water-soluble drug with a very short serum half-life (1 h). Hence, the increased cellular uptake and sustained release in the tissues afforded by targeted liposomes is a major improvement compared with free drug. The drug zidovudine, with half-life of 1 h and low solubility, was also encapsulated in a mannose-targeted liposome made from stearylamine, showing increased localization in lymph node and spleen. An important factor to consider here is that although most of the nucleoside drugs such as stavudine and zidovudine have short serum half-lives, the clinically relevant half-life is that of the intracellular triphosphate form of the drug. For example, despite zidovudine’s 1 h half-life in plasma, it is dosed twice daily based on intracellular pharmacokinetic and clinical efficacy data. Therefore, future nanotechnology-based delivery systems will have to focus in showing significant increase of the half-lives of the encapsulated drugs to achieve a less frequent dosing such as once weekly, once-monthly or even less.

Gene Therapy for HIV/AIDS

In addition to improving existing antiretroviral therapy, there are ongoing efforts to discover alternative approaches for treatment of HIV/AIDS. One promising alternative approach is gene therapy, in which a gene is inserted into a cell to interfere with viral infection or replication. Other nucleic acid-based compounds, such as DNA, siRNA, RNA decoys, ribozymes and aptamers or protein-based agents such as fusion inhibitors and zinc-finger nucleases can also be used to interfere with viral replication.

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RNAi is also considered to have therapeutic potential for HIV/AIDS. Gene silencing is induced by double stranded siRNA, which targets for destruction

he mRNA of the gene of interest. For HIV/AIDS, RNAi can either target the various stages of the viral replication cycle or various cellular targets involved in viral infection such as CD4, CCR5, and/or CXCR4, the major cell surface co-receptors responsible for viral entry. HIV replicates by reverse transcription to form DNA and uses the DNA to produce copies of its mRNA for protein synthesis; siRNA therapy could be used to knock down this viral mRNA. As with other gene therapy techniques, delivery of siRNA to specific cells and tissues has been the major challenge in realizing the potential of RNAi.

New nanotechnology platforms are tackling this problem by providing nonviral alternatives for effective and safe delivery. The first nontargeted delivery of siRNA in humans via self-assembling, cyclodextrin polymer-based nanoparticles for cancer treatment have recently entered Phase I clinical trials.

Although at an early stage, nonviral delivery of siRNA for treatment of HIV infection is also gaining ground. A fusion protein, with a peptide transduction domain and a double stranded RNA-binding domain, was used to encapsulate and deliver siRNA to T cells in vivo. CD4- and CD8-specific siRNA delivery caused RNAi responses with no adverse effects such as cyto-toxicity or immune stimulation. Similarly, a protamine-antibody fusion protein-based siRNA delivery demonstrated that siRNA knockdown of the gag gene can inhibit HIV replication in primary T cells

Single-walled nanotubes were shown to deliver CXCR4 and CD4 specific siRNA to human T cells and peripheral blood mononuclear cells. Up to 90% knockdown of CXCR4 receptors and up to 60% knockdown of CD4 expression on T cells was observed while the knockdown of CXCR4 receptors on peripheral blood mononuclear cells was as high as 60%. In a separate study, amino-terminated carbosilane dendrimers (with interior carbon-silicon bonds) were used for delivery of siRNA to HIV-infected lymphocytes.

These pioneering studies demonstrate that nonviral siRNA delivery is possible for HIV/AIDS treatment. However, more work needs to be done in optimizing the delivery systems and utilizing designs for efficient targeting and intracellular delivery. The recent developments in polymer- and liposome-based siRNA delivery systems could be optimized for targeting cells that are infected with HIV, such as T cells and macrophages. Moreover, since HIV mutates and has multiple strains with different genetic sequences, combination siRNA therapy targeting multiple genes should be pursued. For these applications, nanotechnology platforms with capability for co-delivery and targeting need to be developed specifically for HIV-susceptible cells. A macrophage and T-cell-targeted and nanotechnology-based combination gene therapy may be a promising platform for efficient HIV/AIDS treatment.

Immunotherapy for HIV/AIDS

The various treatment approaches described above focus on treating HIV/AIDS by directly targeting HIV at the level of the host cell or the virus itself. An alternative approach is immunotherapy aimed at modulating the immune response against HIV. CD8+ cytotoxic T-cell responses to acute HIV infection appear to be relatively normal, while neutralizing antibody production by B cells is delayed or even absent.

Immunotherapy is a treatment approach involving the use of immunomodulatory agents to modulate the immune response against a disease. Similar to vaccines, it is based on immunization of individuals with various immunologic formulations; however, the purpose is to treat HIV-infected patients as opposed to protect healthy individuals (preventive vaccines will be discussed in an upcoming section). The various immunotherapy approaches for HIV/AIDS could be based on delivering cytokines (such as IL-2, IL-7 and IL-15) or antigens. The development of cellular immunity, and to a large degree humoral immunity, requires antigen-presenting cells (APCs) to process and present antigens to CD4+and CD8+ T cells. Dendritic cells (DCs) are the quintessential professional APCs responsible for initiating and orchestrating the development of cellular and humoral (antibody) immunity.

Various polymeric systems have been explored for in vivo targeting of DCs and delivery of small molecules, proteins or DNAs showing potential for immunotherapy. Poly(ethylene glycol) (PEG) stabilized poly(propylene sulfide) polymer nanoparticles accumulated in DCs in lymph nodes. Following nanoparticle injection, DCs containing nanoparticles accumulated in lymph nodes, peaking at 4 days with 40–50% of DCs and other APCs having internalized nanoparticles.

In another study, nanoparticles of the copolymer poly(D,L-lacticide-co-glycolide) (PLGA) showed efficient delivery of antigens to murine bone marrow-derived DCs in vitro, suggesting their potential use in immunotherapy. More recently, a very interesting work showed that HIV p24 protein adsorbed on the surface of surfactant-free anionic poly(D,L-lactide) (PLA) nanoparticles were efficiently taken-up by mouse DCs, inducing DC maturation. he p24-nanoparticles induced enhanced cellular and mucosal immune responses in mice. Although this targeting is seen in ex vivo-generated DCs and not in vivo DCs, the efficient delivery of the antigen to DCs through the nanoparticles is an important demonstration that may eventually be applied to in vivo DC targeting.

Clinical Trial

he most clinically advanced application of nanotechnology for immunotherapy of HIV/AIDS is the DermaVir patch that has reached Phase II clinical trials (9). DermaVir is a targeted nanoparticle system based on polyethyleimine mannose (PEIm), glucose and HIV antigen coding DNA plasmid formulated into nanoparticles (~100 nm) and administered under a patch after a skin preparation. The nanoparticles are delivered to epidermal Langerhans cells that trap the nanoparticles and mature to become highly immunogenic on their way to the lymph nodes. Mature DCs containing the nanoparticles present antigens to T cells inducing cellular immunity. Preclinical studies and Phase I clinical trials showed safety and tolerability of the DermaVir patch, which led the progression to Phase II trials. This is the first nanotechnology-based immunotherapy for HIV/AIDS that has reached the clinic and encourages further work in this area.

Table 1

Summary of nanotechnology-based treatment approaches for HIV/AIDS.

Type of therapy Therapeutic agent (drug or gene) Nanotechnology delivery platform Development stage Refs.
Antiretroviral therapy Rilpivirine (TMC278) Poloxamer 338/TPGS 1000 Preclinical [35]
Indinavir Liposome-laden macrophages Preclinical [3638]
Stavudine Mannose- and galactose-targeted liposome Preclinical [3941]
Zidovudine Mannose-targeted liposome Preclinical [42]
Efavirenz Mannose-targeted dendrimer Preclinical [43,45]
Lamivudine Mannose-targeted dendrimer Preclinical [46]
Nanomaterials Fullerene derivatives Preclinical [4955]
Dendrimers Preclinical [56,57]
Silver nanoparticles Preclinical [58,59]
SDC-1721/gold nanoparticles Gold nanoparticles Preclinical [60]
Gene therapy siRNA Peptide fusion proteins, protamine–antibody fusion proteins, dendrimers, single walled carbon nanotubes, peptide–antibody conjugates Preclinical [7781]
Immunotherapy P24 protein Poly (D,L-lactide) nanoparticles/dendritic cells Preclinical [98]
Plasmid DNA Mannose-targeted polyethyleimine polymers Phase II clinical trials [99]

Note:  to open the references in the table 1, please go to ref 1 in this post to see full ref info.

Nanotechnology for HIV/AIDS prevention

The search for a safe and effective HIV/AIDS vaccine has been challenging in the almost three decades since the discovery of the disease. Recently, high-profile clinical trial failures have prompted great debate over the vaccine research, with some suggesting the need for a major focus on fundamental research, with fewer efforts on clinical trials.

The major challenges in the development of a preventive HIV/AIDS vaccine have been the extensive viral strain and sequence diversity, viral evasion of humoral and cellular immune responses, coupled with the lack of methods to elicit broadly reactive neutralizing antibodies and cytotoxic T cells. The challenge associated with delivery of any exogenous antigen (such as nanoparticles) to APCs, is that exogenous antigens require specialized ‘cross-presentation’ in order to be presented by MHC class I and activate CD8+cytotoxic T cells.

his requirement for cytosolic delivery of antigens and cross-presentation represents yet another hurdle for HIV intracellular antigen vaccine, but potentially an advantage of nanodelivery. Humoral responses (neutralizing antibodies produced by B cells) are generated to intact antigen presented on the surface for the virus, or nanoparticles, but these humoral responses typically require ‘help’ from CD4+ T cells, but rather both. Nanoparticles have potential as adjuvants and delivery systems for vaccines. Table 2 present the different approaches.

Table 2

Summary of nanotechnology developments for prevention of HIV/AIDS.

Type of preventive agent Antigen/adjuvant or drug Nanotechnology platform Development stage Refs.
Protein or peptide vaccine gp41, gp120, gp160, p24, Env, Gag, Tat Liposomes, nanoemulsion, MF59, PLA nanoparticles, poly(γ-glutamic acid) nanoparticles Preclinical [108111]
[119120]
[122125]
[128130]
DNA vaccine env, rev, gag, tat, CpG ODN Liposomes, nanoemulsion, PLA nanoparticles Preclinical [115,121]
Inactivated viral particle Inactivated HIV viral particle Polystyrene nanospheres Preclinical [126127]
Microbicides L-lysine dendrimer L-lysine dendrimer Phase I/II [136138]
PLGA nanoparticles
PSC-RANTES PLGA Preclinical [139]
siRNA Nanoparticles, lipids, cholesterol conjugation Preclinical [141144]

ODN: Oligonucleotides; PLA: Poly(D,L-lactide); PLGA: Poly(D,L-lacticide-co-glycolide).

Note:  to open the references in the table 2, please go to ref 1 in this post to see full ref info.

 

Summary

Nanotechnology can impact the treatment and prevention of HIV/AIDS with various innovative approaches. Treatment options may be improved using nanotechnology platforms for delivery of antiretroviral drugs. Controlled and sustained release of the drugs could improve patient adherence to drug regimens, increasing treatment effectiveness.

While there is exciting potential for nanomedicine in the treatment of HIV/AIDS, challenges remain to be overcome before the potential is realized. These include toxicity of nanomaterials, stability of nanoparticles in physiological conditions and their scalability for large-scale production. These are challenges general to all areas of nanomedicine and various works are underway to tackle them.

Another important consideration in investigating nanotechnology-based systems for HIV/AIDS is the economic aspect, as the hardest hit and most vulnerable populations reside in underdeveloped and economically poor countries. In the case of antiretroviral therapy, nanotherapeutics may increase the overall cost of treatment, reducing the overall value. However, if the nanotherapeutics could improve patient adherence by reducing dosing frequency as expected, and furthermore, if they can eradicate viral reservoirs leading to a sterile immunity, these advantages may effectively offset the added cost.

 

Ref:

1. Mamo T, Moseman EA., Kolishetti N., Salvadoe-Morales C., Shi J., Kuritzkes DR., Langer R., von-Adrian U and Farokhzad OF.   Emerging nanotechnology approaches for HIV/AIDS treatment and prevention. Nanomedicine (Lond) 2010; 5(2): 269-295.

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2861897/

2. Merson MH. The HIV-AIDS pandemic at 25 – the global response. N Engl J Med.2006;354(23):2414–2417

3. Walensky RP, Paltiel AD, Losina E, et al. The survival benefits of AIDS treatment in the United States. J Infect Dis. 2006;194(1):11–19

4. Richman DD, Margolis DM, Delaney M, Greene WC, Hazuda D, Pomerantz RJ. The challenge of finding a cure for HIV infection. Science. 2009;323(5919):1304–1307)

5.Sax PE, Cohen CJ, Kuritzkes DR. HIV Essentials. Physicians’ Press; Royal Oak, MI, USA: 2007.

6. Lamers SL, Salemi M, Galligan DC, et al. Extensive HIV-1 intra-host recombination is common in tissues with abnormal histopathology. PLoS One. 2009;4(3):E5065.

7. Vyas TK, Shah L, Amiji MM. Nanoparticulate drug carriers for delivery of HIV/AIDS therapy to viral reservoir sites. Expert Opin Drug Deliv. 2006;3(5):613–628.

8. Amiji MM, Vyas TK, Shah LK. Role of nanotechnology in HIV/AIDS treatment: Potential to overcome the viral reservoir challenge. Discov Med. 2006;6(34):157–162

9. Lori F, Calarota SA, Lisziewicz J. Nanochemistry-based immunotherapy for HIV-1. Curr Med Chem. 2007;14(18):1911–1919

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