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Posts Tagged ‘RNAi’


Familial transthyretin amyloid polyneuropathy

Curator: Larry H. Bernstein, MD, FCAP

LPBI

 

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/

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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).

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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

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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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