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New Studies toward Understanding Alzheimer Disease

Posted in Alzheimer's Disease, Biological Networks, Biomarkers & Medical Diagnostics, Disease Biology, Etiology, Kinase, Metabolism, Metabolomics, Neurodegenerative Diseases, Neurological Diseases, Neuroscience, Pharmaceutical Drug Discovery, Phosphorylase, tagged , , , , , , on June 4, 2016| Leave a Comment »

New Studies toward Understanding Alzheimer Disease

Curators: Larry H. Bernstein, MD, FCAP and Aviva Lev-Ari, PhD, RN

 

There is no unifying concept of Alzheimer Disease beyond the Tau and beta amyloid roles.  Recently, Ingenbleek and Bernstein (journal AD) made the connection between the age related decline of liver synthesis of plasma transthyretin and the more dramatic decline of transthyretin at the blood brain barrier, and the relationship to inability to transfer vitamin A via retinol binding protein to the brain.  Related metabolic events are reported by several groups.

 

What else is New?

 

Amyloid-β peptide protects against microbial infection in mouse and worm models of Alzheimer’s disease.

Kumar DK, Choi SH, Washicosky KJ, Eimer WA, Tucker S, Ghofrani J, Lefkowitz A, McColl G, Goldstein LE, Tanzi RE, Moir RD.

Sci Transl Med. 2016 May 25;8(340):340ra72.  http://dx.doi.org:/10.1126/scitranslmed.aaf1059

They show that Aβ oligomerization, a behavior traditionally viewed as intrinsically pathological, may be necessary for the antimicrobial activities of the peptide. Collectively, our data are consistent with a model in which soluble Aβ oligomers first bind to microbial cell wall carbohydrates via a heparin-binding domain. Developing protofibrils inhibited pathogen adhesion to host cells. Propagating β-amyloid fibrils mediate agglutination and eventual entrapment of unatttached microbes….Salmonella Typhimurium bacterial infection of the brains of transgenic 5XFAD mice resulted in rapid seeding and accelerated β-amyloid deposition, which closely colocalized with the invading bacteria.

This is quite interesting in that infection drives the production of acute phase reactants resulting in decreased production of transthyretin.  Whether this also has ties to chronic disease in the elderly and risk of AD is not known.

Gain-of-function mutations in protein kinase Cα (PKCα) may promote synaptic defects in Alzheimer’s disease.

Alfonso SI, Callender JA, Hooli B, Antal CE, Mullin K, Sherman MA, Lesné SE, Leitges M, Newton AC, Tanzi RE, Malinow R.

Sci Signal. 2016 May 10;9(427):ra47.  http://dx.doi.org:/10.1126/scisignal.aaf6209.

Through whole-genome sequencing of 1345 individuals from 410 families with late-onset AD (LOAD), they identified three highly penetrant variants in PRKCA, the gene that encodes protein kinase Cα (PKCα), in five of the families. All three variants linked with LOAD displayed increased catalytic activity relative to wild-type PKCα as assessed in live-cell imaging experiments using a genetically encoded PKC activity reporter. Deleting PRKCA in mice or adding PKC antagonists to mouse hippocampal slices infected with a virus expressing the Aβ precursor CT100 revealed that PKCα was required for the reduced synaptic activity caused by Aβ. In PRKCA(-/-) neurons expressing CT100, introduction of PKCα, but not PKCα lacking a PDZ interaction moiety, rescued synaptic depression, suggesting that a scaffolding interaction bringing PKCα to the synapse is required for its mediation of the effects of Aβ. Thus, enhanced PKCα activity may contribute to AD, possibly by mediating the actions of Aβ on synapses.

 

Science Signaling Podcast for 10 May 2016: PKCα in Alzheimer’s disease.

Newton AC, Tanzi RE, VanHook AM.

Sci Signal. 2016 May 10;9(427):pc11. doi: 10.1126/scisignal.aaf9436.

Relevance of the COPI complex for Alzheimer’s disease progression in vivo.

Bettayeb K, Hooli BV, Parrado AR, Randolph L, Varotsis D, Aryal S, Gresack J,Tanzi RE, Greengard P, Flajolet M.

Proc Natl Acad Sci U S A. 2016 May 10;113(19):5418-23. http://dx.doi.org:/10.1073/pnas.1604176113

Inhibition of death-associated protein kinase 1 attenuates the phosphorylation and amyloidogenic processing of amyloid precursor protein.

Kim BM, You MH, Chen CH, Suh J, Tanzi RE, Ho Lee T.

Hum Mol Genet. 2016 Apr 19. pii: ddw114.

Extracellular deposition of amyloid-beta (Aβ) peptide, a metabolite of sequential cleavage of amyloid precursor protein (APP), is a critical step in the pathogenesis of Alzheimer’s disease (AD). While death-associated protein kinase 1 (DAPK1) is highly expressed in AD brains and its genetic variants are linked to AD risk, little is known about the impact of DAPK1 on APP metabolism and Aβ generation. This study demonstrated a novel effect of DAPK1 in the regulation of APP processing using cell culture and mouse models. DAPK1, but not its kinase deficient mutant (K42A), significantly increased human Aβ secretion in neuronal cell culture models. Moreover, knockdown of DAPK1 expression or inhibition of DAPK1 catalytic activity significantly decreased Aβ secretion. Furthermore, DAPK1, but not K42A, triggered Thr668 phosphorylation of APP, which may initiate and facilitate amyloidogenic APP processing leading to the generation of Aβ. In Tg2576 APPswe-overexpressing mice, knockout of DAPK1 shifted APP processing toward non-amyloidogenic pathway and decreased Aβ generation. Finally, in AD brains, elevated DAPK1 levels showed co-relation with the increase of APP phosphorylation. Combined together, these results suggest that DAPK1 promotes the phosphorylation and amyloidogenic processing of APP, and that may serve a potential therapeutic target for AD.

Recapitulating amyloid β and tau pathology in human neural cell culture models: clinical implications.

Choi SH, Kim YH, D’Avanzo C, Aronson J, Tanzi RE, Kim DY.

US Neurol. 2015 Fall;11(2):102-105.    Free PMC Article

The “amyloid β hypothesis” of Alzheimer’s disease (AD) has been the reigning hypothesis explaining pathogenic mechanisms of AD over the last two decades. However, this hypothesis has not been fully validated in animal models, and several major unresolved issues remain. Our 3D human neural cell culture model system provides a premise for a new generation of cellular AD models that can serve as a novel platform for studying pathogenic mechanisms and for high-throughput drug screening in a human brain-like environment.

The two key pathological hallmarks of AD are senile plaques (amyloid plaques) and neurofibrillary tangles (NFTs), which develop in brain regions responsible for memory and cognitive functions (i.e. cerebral cortex and limbic system) 3. Senile plaques are extracellular deposits of amyloid-β (Aβ) peptides, while NFTs are intracellular, filamentous aggregates of hyperphosphorylated tau protein 4.

The identification of Aβ as the main component of senile plaques by Drs. Glenner and Wong in 1984 5 resulted in the original formation of the “amyloid hypothesis.” According to this hypothesis, which was later renamed the “amyloid-β cascade hypothesis” by Drs. Hardy and Higgins 6, the accumulation of Aβ is the initial pathological trigger in the disease, subsequently leading to hyperphosphorylation of tau, causing NFTs, and ultimately, neuronal death and dementia 4,7–10. Although the details have been modified to reflect new findings, the core elements of this hypothesis remain unchanged: excess accumulation of the pathogenic forms of Aβ, by altered Aβ production and/or clearance, triggers the vicious pathogenic cascades that eventually lead to NFTs and neuronal death.

Over the last two decades, the Aβ hypothesis of AD has reigned, providing the foundation for numerous basic studies and clinical trials 4,7,10,11. According to this hypothesis, the accumulation of Aβ, either by altered Aβ production and/or clearance, is the initial pathological trigger in the disease. The excess accumulation of Aβ then elicits a pathogenic cascade including synaptic deficits, altered neuronal activity, inflammation, oxidative stress, neuronal injury, hyperphosphorylation of tau causing NFTs and ultimately, neuronal death and dementia 4,7–10.

One of the major unresolved issues of the Aβ hypothesis is to show a direct causal link between Aβ and NFTs 12–14. Studies have demonstrated that treatments with various forms of soluble Aβ oligomers induced synaptic deficits and neuronal injury, as well as hyperphosphorylation of tau proteins, in mouse and rat neurons, which could lead to NFTs and neurodegeneration in vivo 18–21. However, transgenic AD mouse models carrying single or multiple human familial AD (FAD) mutations in amyloid precursor protein (APP) and/or presenilin 1 (PS1) do not develop NFTs or robust neurodegeneration as observed in human patients, despite robust Aβ deposition 13,22,23. Double and triple transgenic mouse models, harboring both FAD and tau mutations linked with frontotemporal dementia (FTD), are the only rodent models to date displaying both amyloid plaques and NFTs. However, the NFT pathology in these models stems mainly from the overexpression of human tau as a result of the FTD, rather than the FAD mutations24,25.

Human neurons carrying FAD mutations are an optimal model to test whether elevated levels of pathogenic Aβ trigger pathogenic cascades including NFTs, since those cells truly share the same genetic background that induces FAD in humans. Indeed, Israel et al., observed elevated tau phosphorylation in neurons with an APP duplication FAD mutation 33. Blocking Aβ generation by β-secretase inhibitors significantly decreased tau phosphorylation in the same model, but γ-secretase inhibitor, another Aβ blocker, did not affect tau phosphorylation 33. Neurons with the APP V717I FAD mutation also showed an increase in levels of phospho tau and total tau levels 28. More importantly, Muratore and colleagues showed that treatments with Aβ-neutralizing antibodies in those cells significantly reduced the elevated total and phospho tau levels at the early stages of differentiation, suggesting that blocking pathogenic Aβ can reverse the abnormal tau accumulation in APP V717I neurons 28.

Recently, Moore et al. also reported that neurons harboring the APP V717I or the APP duplication FAD mutation showed increases in both total and phospho tau levels 27. Interestingly, altered tau levels were not detected in human neurons carrying PS1 FAD mutations, which significantly increased pathogenic Aβ42 species in the same cells 27. These data suggest that elevated tau levels in these models were not due to extracellular Aβ accumulation but may possibly represent a very early stage of tauopathy. It may also be due to developmental alterations induced by the APP FAD mutations.

As summarized, most human FAD neurons showed significant increases in pathogenic Aβ species, while only APP FAD neurons showed altered tau metabolism that may represent very early stages of tauopathy. However, all of these human FAD neurons failed to recapitulate robust extracellular amyloid plaques, NFTs, or any signs of neuronal death, as predicted in the amyloid hypothesis.

In our recent study, we moved one step closer to proving the amyloid hypothesis. By generating human neural stem cell lines carrying multiple mutations in APP together with PS1, we achieved high levels of pathogenic Aβ42 comparable to those in brains of AD patients 44–46.

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Platform for AD drug screening in human neural progenitor cells with FAD mutations in a 3D culture system, which successfully reproduce human AD pathogenesis (amyloid plaques-driven tauopathy).

In addition to the impact on toxic Aβ species, our 3D culture model can test if these antibodies can block tau pathologies in 3D human neural cell culture systems 44–46. Human cellular AD models can also be used to determine optimal doses of candidate AD drugs to block Aβ and/or tau pathology without affecting neuronal survival (Fig. 1).

While much progress has been made, many challenges still lie on the path to creating human neural cell culture models that comprehensively recapitulate pathogenic cascades of AD. A major difficulty lies in reconstituting the brain regions most affected in AD: the hippocampus and specific cortical layers. Recent progress in 3D culture technology, such as “cerebral organoids,” may also be helpful in rebuilding the brain structures that are affected by AD in a dish 52,53. These “cerebral organoids” were able to model various discrete brain regions including human cortical areas 52, which enabled them to reproduce microcephaly, a brain developmental disorder. Similarly, pathogenic cascades of AD may be recapitulated in cortex-like structures using this model. Adding neuroinflammatory components, such as microglial cells, which are critical in AD pathogenesis, will illuminate the validity of the amyloid β hypothesis. Reconstitution of robust neuronal death stemming from Aβ and tau pathologies will be the next major step in comprehensively recapitulating AD in a cellular model.

 

Family-based association analyses of imputed genotypes reveal genome-wide significant association of Alzheimer’s disease with OSBPL6, PTPRG, and PDCL3.

Herold C, Hooli BV, Mullin K, Liu T, Roehr JT, Mattheisen M, Parrado AR, Bertram L, Lange C, Tanzi RE.

Mol Psychiatry. 2016 Feb 2. http://dx.doi.org:/10.1038/mp.2015.218.

Relationship between ubiquilin-1 and BACE1 in human Alzheimer’s disease and APdE9 transgenic mouse brain and cell-based models.

Natunen T, Takalo M, Kemppainen S, Leskelä S, Marttinen M, Kurkinen KM, Pursiheimo JP, Sarajärvi T, Viswanathan J, Gabbouj S, Solje E, Tahvanainen E, Pirttimäki T, Kurki M, Paananen J, Rauramaa T, Miettinen P, Mäkinen P, Leinonen V, Soininen H, Airenne K, Tanzi RE, Tanila H, Haapasalo A, Hiltunen M.

Neurobiol Dis. 2016 Jan;85:187-205. http://dx.doi.org:/10.1016/j.nbd.2015.11.005.

Accumulation of β-amyloid (Aβ) and phosphorylated tau in the brain are central events underlying Alzheimer’s disease (AD) pathogenesis. Aβ is generated from amyloid precursor protein (APP) by β-site APP-cleaving enzyme 1 (BACE1) and γ-secretase-mediated cleavages. Ubiquilin-1, a ubiquitin-like protein, genetically associates with AD and affects APP trafficking, processing and degradation. Here, we have investigated ubiquilin-1 expression in human brain in relation to AD-related neurofibrillary pathology and the effects of ubiquilin-1 overexpression on BACE1, tau, neuroinflammation, and neuronal viability in vitro in co-cultures of mouse embryonic primary cortical neurons and microglial cells under acute neuroinflammation as well as neuronal cell lines, and in vivo in the brain of APdE9 transgenic mice at the early phase of the development of Aβ pathology. Ubiquilin-1 expression was decreased in human temporal cortex in relation to the early stages of AD-related neurofibrillary pathology (Braak stages 0-II vs. III-IV). There was a trend towards a positive correlation between ubiquilin-1 and BACE1 protein levels. Consistent with this, ubiquilin-1 overexpression in the neuron-microglia co-cultures with or without the induction of neuroinflammation resulted in a significant increase in endogenously expressed BACE1 levels. Sustained ubiquilin-1 overexpression in the brain of APdE9 mice resulted in a moderate, but insignificant increase in endogenous BACE1 levels and activity, coinciding with increased levels of soluble Aβ40 and Aβ42. BACE1 levels were also significantly increased in neuronal cells co-overexpressing ubiquilin-1 and BACE1. Ubiquilin-1 overexpression led to the stabilization of BACE1 protein levels, potentially through a mechanism involving decreased degradation in the lysosomal compartment. Ubiquilin-1 overexpression did not significantly affect the neuroinflammation response, but decreased neuronal viability in the neuron-microglia co-cultures under neuroinflammation. Taken together, these results suggest that ubiquilin-1 may mechanistically participate in AD molecular pathogenesis by affecting BACE1 and thereby APP processing and Aβ accumulation.

Correction to Cathepsin L Mediates the Degradation of Novel APP C-Terminal Fragments.

Wang H, Sang N, Zhang C, Raghupathi R, Tanzi RE, Saunders A.

Biochemistry. 2015 Sep 22;54(37):5781.  http://dx.doi.org:/10.1021/acs.biochem.5b00968. Epub 2015 Sep 8. No abstract available.

Massachusetts Alzheimer’s Disease Research Center: progress and challenges.

Hyman BT, Growdon JH, Albers MW, Buckner RL, Chhatwal J, Gomez-Isla MT, Haass C, Hudry E, Jack CR Jr, Johnson KA, Khachaturian ZS, Kim DY, Martin JB, Nitsch RM, Rosen BR, Selkoe DJ, Sperling RA, St George-Hyslop P, Tanzi RE, Yap L, Young AB, Phelps CH, McCaffrey PG.

Alzheimers Dement. 2015 Oct;11(10):1241-5. http://dx.doi.org:/10.1016/j.jalz.2015.06.1887. Epub 2015 Aug 19. No abstract available.

Alzheimer’s in 3D culture: challenges and perspectives.

D’Avanzo C, Aronson J, Kim YH, Choi SH, Tanzi RE, Kim DY.

Bioessays. 2015 Oct;37(10):1139-48. doi: 10.1002/bies.201500063. Epub 2015 Aug 7. Review.

Free PMC Article

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Synaptotagmins interact with APP and promote Aβ generation.

Gautam V, D’Avanzo C, Berezovska O, Tanzi RE, Kovacs DM.

Mol Neurodegener. 2015 Jul 23;10:31. doi: 10.1186/s13024-015-0028-5.

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Near-infrared fluorescence molecular imaging of amyloid beta species and monitoring therapy in animal models of Alzheimer’s disease.

Zhang X, Tian Y, Zhang C, Tian X, Ross AW, Moir RD, Sun H, Tanzi RE, Moore A, Ran C.

Proc Natl Acad Sci U S A. 2015 Aug 4;112(31):9734-9. doi: 10.1073/pnas.1505420112. Epub 2015 Jul 21.

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A 3D human neural cell culture system for modeling Alzheimer’s disease.

Kim YH, Choi SH, D’Avanzo C, Hebisch M, Sliwinski C, Bylykbashi E, Washicosky KJ, Klee JB, Brüstle O, Tanzi RE, Kim DY.

Nat Protoc. 2015 Jul;10(7):985-1006. doi: 10.1038/nprot.2015.065. Epub 2015 Jun 11.

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Cathepsin L Mediates the Degradation of Novel APP C-Terminal Fragments.

Wang H, Sang N, Zhang C, Raghupathi R, Tanzi RE, Saunders A.

Biochemistry. 2015 May 12;54(18):2806-16. doi: 10.1021/acs.biochem.5b00329. Epub 2015 Apr 28. Erratum in: Biochemistry. 2015 Sep 22;54(37):5781.

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γ-Secretase modulators reduce endogenous amyloid β42 levels in human neural progenitor cells without altering neuronal differentiation.

D’Avanzo C, Sliwinski C, Wagner SL, Tanzi RE, Kim DY, Kovacs DM.

FASEB J. 2015 Aug;29(8):3335-41. doi: 10.1096/fj.15-271015. Epub 2015 Apr 22.

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PLD3 gene variants and Alzheimer’s disease.

Hooli BV, Lill CM, Mullin K, Qiao D, Lange C, Bertram L, Tanzi RE.

Nature. 2015 Apr 2;520(7545):E7-8. doi: 10.1038/nature14040. No abstract available.

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Breakup of amyloid plaques

Posted in Alzheimer's Disease, Etiology, Neurological Diseases, Neuroscience, Pharmacotherapy and Cell Activity, tagged , , , , on February 15, 2016| Leave a Comment »

Breakup of amyloid plaques

Larry H. Bernstein, MD, FCAP, Curator

LPBI

 

 

Small Molecule EPPS Breaks Up Amyloid Plaques

Alzheimers Plaque Therapy, Alzheimers small molecule, amyloid plaque treatment

One of the hallmarks of Alzheimer’s disease has been the generation of Amyloid-β (Aβ) oligomers, fibrils, and ultimately plaques. It is currently contended whether these plaques are a cause of Alzheimer’s disease and related mental deficits, or merely an effect. Researchers at the Korea Institute of Science and Technology have demonstrated in vivo formation and disaggregation of Aβ plaques. They previously reported small ionic molecules which could accelerate the formation of Aβ plaques. Six small molecules which inhibited aggregate formation were discovered at the same time. One of these molecules, 4-(2-hydroxyethyl)-1-piperazinepropanesulphonic acid (EPPS), works as a therapeutic in a Alzheimer’s mouse model. EPPS was found to be both orally available and cross the blood brain barrier where it directly binds to Aβ plaques. Double transgenic mice , APPswe/PS1-dE9 (amyloid precursor protein/presenilin protein 1) mice were administered EPPS in their drinking water for 3.5 months and compared to non-treated transgenic controls. EPPS treated mice both improved from their baseline and out-performed transgenic controls in both the Morris water maze and contextual fear response tests. Immunofluorescent staining of matched brain regions demonstrated elimination of Aβ plaques in the hippocampus of EPPS treated mice. Further study is required to completely understand the mechanism by which EPPS disaggregates the Aβ plaques. This study demonstrates the cause and effects Aβ plaque generation, and subsequent removal, has on Alzheimer’s disease related cognitive function. Should the effect transfer to humans, this could prove a significant discovery for the treatment of Alzheimer’s disease.

 

Kim, et al. (October, 2015) EPPS rescues hippocampus-dependent cognitive deficits in APP/PS1 ice by disaggregation of amyloid-b oligomers and plaques Nature Communications

 

EPPS  rescues hippocampus-dependent cognitive deficits in APP/PS1 mice by disaggregation of amyloid-β oligomers and plaques

Hye Yun Kim, Hyunjin Vincent Kim, Seonmi Jo, C. Justin Lee, Seon Young Choi, Dong Jin Kim & YoungSoo Kim

Nature Communications 2016; 6(8997)     http://dx.doi.org:/10.1038/ncomms9997

Alzheimer’s disease (AD) is characterized by the transition of amyloid-β (Aβ) monomers into toxic oligomers and plaques. Given that Aβ abnormality typically precedes the development of clinical symptoms, an agent capable of disaggregating existing Aβ aggregates may be advantageous. Here we report that a small molecule, 4-(2-hydroxyethyl)-1-piperazinepropanesulphonic acid (EPPS), binds to Aβ aggregates and converts them into monomers. The oral administration of EPPS substantially reduces hippocampus-dependent behavioural deficits, brain Aβ oligomer and plaque deposits, glial γ-aminobutyric acid (GABA) release and brain inflammation in an Aβ-overexpressing, APP/PS1 transgenic mouse model when initiated after the development of severe AD-like phenotypes. The ability of EPPS to rescue Aβ aggregation and behavioural deficits provides strong support for the view that the accumulation of Aβ is an important mechanism underlying AD.

 

During Alzheimer’s disease (AD) pathogenesis, amyloid-β (Aβ) monomers aberrantly aggregate into toxic oligomers, fibrils and eventually plaques. The concentration of misfolded Aβ species highly correlates with the severity of neurotoxicity and inflammation that leads to neurodegeneration in AD1, 2, 3. Accordingly, substantial efforts have been devoted to reducing Aβ levels, including methods to prevent the production and aggregation of Aβ4, 5, 6, 7. Although these approaches effectively prevent the de novo formation of Aβ aggregates, existing Aβ oligomers and plaques will still remain in the patient’s brain8, 9, 10. Thus, the desirable effects of Aβ inhibitors may be expected when administered before a patient develops toxic Aβ deposits5, 6, 7. However, in AD patients with mild-to-moderate symptoms, anti-amyloidogenic agents have not yielded expected outcomes, which may be due to the incomplete removal of pre-existing Aβ aggregates11. As Aβ typically begins to aggregate long before the onset of AD symptoms, interventions specifically aimed at disaggregating existing plaques and oligomers may constitute a useful approach to AD treatment, perhaps in parallel with agents aimed at inhibiting aggregate formation8, 9, 10, 11, 12.

 

Result highlights  

EPPS reduces Aβ-aggregate-induced memory deficits in mice

Figure 1: EPPS ameliorates Aβ-induced memory deficits in mice.

 

EPPS ameliorates A[beta]-induced memory deficits in mice.

(a) Time course of the experiments. (b) Intracerebroventricular (i.c.v.) injection site brain schematic diagram. (c) Pretreated effects of EPPS on Aβ-aggregate-induced memory deficits observed by the % alternation on the Y-maze. EPPS, 0 (n=10), 30 (n=9) or 100 mg kg−1 per day (n=10), was orally given to 8.5-week-old ICR male mice for 1 week; then, vehicle (10% DMSO in PBS, n=10) or Aβ aggregates (50 pmol per 10% DMSO in PBS; Supplementary Fig. 1A) were injected into the intracerebroventricular region (P=0.022). (d) Co-treated effects of EPPS on Aβ-aggregate-induced memory deficits observed by the % alternation on the Y-maze. Male, 8.5-week-old ICR mice received an injection of vehicle (n=9) or Aβ aggregates into the intracerebroventricular region, and then EPPS, 0 (n=10), 30 (n=10) or 100 mg kg−1 per day (n=10), was orally given to these mice for 5 days. From the top, P=0.003, 0.006, 0.015. The error bars represent the s.e.m. One-way analysis of variance followed by Bonferroni’s post-hoc comparisons tests were performed in all statistical analyses. (*P<0.05, **P<0.01, ***P<0.001; other comparisons were not significant).

 

EPPS is orally safe and penetrates the blood–brain barrier

Orally administered EPPS rescues cognitive deficits in APP/PS1 mice

 

Figure 2: EPPS rescues hippocampus-dependent cognitive deficits.

http://www.nature.com/ncomms/2015/151208/ncomms9997/images_article/ncomms9997-f2.jpg

 

Figure 3: EPPS does not affect synaptic plasticity in mice.

http://www.nature.com/ncomms/2015/151208/ncomms9997/images_article/ncomms9997-f3.jpg

 

Figure 4: EPPS disaggregates Aβ plaques and oligomers in APP/PS1 mice.

EPPS disaggregates A[beta] plaques and oligomers in APP/PS1 mice.

APP/PS1 mice and WTs from the aforementioned behavioural tests were killed and subjected to brain analyses. EPPS, 0 (TG(−), male, n=15), 10 (TG(+), male, n=11) or 30 mg kg-1 per day (TG(++), male,n=8), was orally given to 10.5-month-old APP/PS1 for 3.5 months and their brains were compared with age-matched WT brains (WT(−), male, n=16). (a) ThS-stained Aβ plaques in whole brains (scale bars, 1 mm) and the hippocampal region (scale bars, 200 μm) of each group. The mouse brain schematic diagram was created by authors (green and red boxes: regions of brain images, a and f, respectively). (b) Number or area of plaques normalized (%) to the level in 10.5-month-old TG mice. Plaque number: P-values compared with TG (male, 10.5-month-old) are all <0.0001 (#). P-values compared with TG(−) (male, 14-month-old) are all <0.0001 (*). Plaque area: P-values compared with TG (male, 10.5-month-old) are all <0.0001 (#). P-values compared with TG(−) (male, 14-month-old) are all <0.0001 (*). (c–e) Aβ-insoluble and -soluble fractions analyses from brain lysates. (c) Sandwich ELISA of Aβ-insoluble fractions. Hippocampus: all P<0.0001; cortex: P=0.004, 0.046. (d) Sandwich ELISA of Aβ-soluble fractions. (e) Dot blotting of the total Aβ (anti-Aβ: 6E10, also recognizes APP) and oligomers (anti-amyloidogenic protein oligomer: A11). (f) Histochemical analyses of Aβ deposition. Aβs were stained with the 6E10 antibody and ThS. Aβ plaques (first row): green; all Aβs (second row): red; 4,6-diamidino-2-phenylindole (DAPI): blue (as a location indicator). The third and bottom rows show merged images of plaques and Aβs, and plaques and Aβs with DAPI staining. Scale bars, 50 μm. (g) Western blotting analyses of APP expression in hippocampal and cortical lysates (detected at ~100 kDa by 6E10 antibody). Densitometry (see Supplementary Fig. 3A). Full version (see Supplementary Fig. 7). The error bars represent the s.e.m. One-way analysis of variance followed by Bonferroni’s post-hoc comparisons tests were performed in all statistical analyses (*P<0.05, **P<0.01, ***P<0.001, #P<0.05, ##P<0.01,###P<0.001; other comparisons were not significant).

 

EPPS removes Aβ plaques and oligomers in APP/PS1 mice

Collectively, these results indicate that EPPS rescues hippocampus-dependent cognitive deficits when orally administered to aged, symptomatic APP/PS1 TG mice.

Collectively, these results indicate that orally administered EPPS effectively decreases Aβ plaques and oligomers in APP/PS1 model mouse brains.

 

EPPS lowers Aβ-dependent inflammation and glial GABA release

Figure 5: EPPS lowers inflammation and glial GABA release.

EPPS disaggregates Aβ oligomers and fibrils by direct interaction and reduces cytotoxicity

Figure 6: EPPS disaggregates Aβ aggregates by selective binding.

 

(1) a small molecule, EPPS, converts neurotoxic oligomers and plaques into non-toxic monomers by directly binding to Aβ aggregates;

(2) orally administered EPPS produces a dose-dependent reduction of Aβ plaque deposits and behavioural deficits in APP/PS1 TG mice, even when administration was delayed until after the pathology was well established;

(3) the beneficial effect of EPPS probably operates through an Aβ-related mechanism rather by facilitating cognitive processes; and

(4) large doses of EPPS appeared to be well tolerated in initial toxicity studies6, 7, 33.

Dr. T. Ronald Theodore
Email rtheodore@integratedbiologics.com
URL http://www.integratedbiologics.com
In Response To Breakup of amyloid plaques
Submitted on 2016/05/18 at 3:33 am
Comment Re: “EPPS rescues hippocampus-dependent cognitive deficits in APP/PS1 mice by disaggregation of amyloid-β oligomers and plaques” Kim et al, Nature Communications 8 December 2015
HEPES, Zwitterions, and the “Good” Buffers as Biological Response Modifiers

In reference to the article “EPPS rescues hippocampus-dependent cognitive deficits in APP/PS1 mice by disaggregation of amyloid-β oligomers and plaques” Kim et al, Nature Communications 8 December 2015, we note some important omissions.

Kim et al state specific effects of EPPS affecting Alzheimer’s disease. We would point out that EPPS is also referenced as HEPPS.1 HEPPS has been accepted as a “Good” buffer and a zwitterion. The authors attribute the effects of EPPS to anti-inflammatory action. The authors omit reference that EPPS (HEPPS) is a listed “Good” buffer and a zwitterion.1 The anti-inflammatory effects of zwitterions and “Good” buffers have been previously described.3,4 The effects of these zwitterions as biological response modifiers with effects on neurological diseases including Alzheimer’s have been previously noted.4,5 ( HEPES has been used preferentially based on Good’s original data showing HEPES has the highest ability to increase the rate of mitochondrial oxidative phosphorylation). Kim et al attribute the effects of EPPS to anti-inflammatory actions. The anti-inflammatory effects of the buffers are well known.3,4 We would suggest that anti-inflammatory effects of the buffers may be singular, synergistic or combined effects of other biological responses that have been noted including mitochondrial and other actions.4,5,6,7 Prior literature and data would certainly anticipate the findings of Kim et al. It is noted that all these zwitterionic buffers have effects on the neurological system.

What is important is that further research to determine the effects of these zwitterionic buffers as biological response modifiers on neurological diseases including Alzheimer’s is continued. The ability of the zwitterionic buffers on brain and other organ injury are currently under review.

T. Ronald Theodore
Integrated Biologics, LLC
rtheodore@integratedbiologics.com

1. Merck Index, 15th Edition, Feb 2015.
2. Norman E. Good et al., Hydrogen Ion Buffers for Biological Research, Biochemistry vol.5, No. 2, Feb. 1966.
3. “Effects of In-vivo Administration of Taurine and HEPES on the Inflammatory Response in Rats” Pharmacy and Pharmacology, vol. 46, No. 9, Sept. 1994.
4. Theodore et al., Zwitterionic Compositions and Methods as Biological Response Modifiers, US Patent No. 6,071,919.
5. Garvey et al., Phosphate and HEPES buffers potently affect the fibrillation and oligomerization mechanism of Alzheimer’s Aβ peptide, Biochemical and Biophysical Research Communications, 06/2011; 409(3):385-8. DOI: 10.1016/j.bbrc.2011.04.141.
6. Theodore et al., Pilot Ascending Dose Tolerance Study of Parenterally Administered 4-(2 Hydroxyethyl)-l-piperazine Ethane Sulfonic Acid (TVZ-7) in Dogs, Cancer Biotherapy & Radiopharmaceuticals, Volume 12, Number 5, 1997.
7. Theodore et al., Preliminary Evaluation of a Fixed Dose of Zwitterionic Piperazine (TVZ-7) in Clinical Cancer, Cancer Biotherapy and Radiopharmaceuticals, Volume 12, Number 5, 1997.

 

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Tumor Models

Posted in Genome Biology, tagged , , on October 22, 2015| Leave a Comment »

Tumor-Models

Larry H. Bernstein, MD, FCAP, Curator

LPBI

 

 

Tumor Models Bridge Mouse-Human Gap

http://www.genengnews.com/gen-articles/tumor-models-bridge-mouse-human-gap/5610/

 

Revamped Allograft and Innovative Xenograft Models Can Reduce the Risks of Late-Stage Clinical Trials and Increase the Odds of Translational Success

http://www.genengnews.com/Media/images/Article/HorizonDiscovery_CRISPR1417421418.jpg

 

CRISPR-Cas9 sgRNA in vitro screens can be used to look for genes that when lost induce resistance to a drug (positive screen) or increase sensitivity to a drug (negative screen). The technology might also be a powerful tool for target ID screens in vivo, and looks set to aid in our understanding of tumor development and progression in animal models. [Horizon Discovery]

 

 

 

Precision medicine is all about the two T’s—targets and treatments—targets that emerge from analyses of patient-specific information, and treatments that hit these targets. Both T’s, however, pose difficulties. All too often, insights and drug candidates originating in precision medicine fail to translate into clinical practice.

This reality prompted sober comments at the most recent event in the Tumor Models series organized by Hanson Wade. “One of the current problems with identifying new targets for precision medicine is identifying biologically meaningful targets likely to translate to the clinic,” complained Nicola McCarthy, Ph.D., oncology program manager, Horizon Discovery.

That pretty much sums up the difficulty with the first of the T’s. Now, what’s so hard about the second one? “It’s estimated that about 94–96% of drugs making it through preclinical testing stages to clinical phases ultimately fail in the clinic as a result of poor efficacy or poor safety,” lamented Leon Hall, Ph.D., senior director, global scientific development, Taconic Biosciences.

  • Drs. McCarthy and Hall were among presenters at Tumor Models Summit Boston 2015. Meeting highlights, discussed herein, included in vitro approaches, such as the powerful gene-editing technology known as CRISPR, as well as in vivo approaches, such as mouse models for immuno-oncology strategies.

    Dr. Hall spoke to the common goal: “We believe, as others do, that by improving the ability of mouse models to mimic human biology, the models will better predict drug responses in the human patient.”

     

    CRISPR-Modified Screens

    HumanizedMice

    HumanizedMice

     

     

    Crown Bioscience is developing research platforms for preclinical evaluation of drugs that harness the human immune system to fight human tumors. For example, the company provides HuPrime® 3.0, a patient-derived xenograft model. It is based on humanized mice, HuMice™, immunocompromised mice that have been inoculated with human hematopoietic cells (from cord blood stem cells).

    http://www.genengnews.com/Media/images/Article/thumb_CrownBioscience_HumanizedMice3669342311.jpg

     

    “Cell lines are such fantastic tools when you have very targeted mechanistic questions,” exclaimed Tommy Broudy, Ph.D., general manager, Crown Bioscience. In collaboration with Horizon Discovery, Crown Bioscience has developed isogenic cancer models for in vivo compound screening. For example, using Horizon’s rAAV-based Genesis™ gene-editing platform, the companies introduced mutations to genes such as KRAS, PIK3CA, PTEN, IDH1 and IDH2, and p53.

    Expanding its technological capabilities, Horizon Discovery recently added CRISPR-Cas9 single-guide RNA (sgRNA) screening capabilities to a tool collection that already included siRNA cell-line screens.

    Directed by sgRNAs, Cas9 nucleases cut at specific locations in the genome. “Unlike siRNA screens, CRISPR-Cas9 sgRNA screens enable complete loss of a particular protein,” said Dr. McCarthy. “This can be informative in terms of identifying and validating targets for which a phenotype of interest is evident only on complete loss of protein expression.” Such targets, Dr. McCarthy emphasized, can be missed by siRNA screens because full depletion of a target often does not occur consistently between siRNAs.

    Horizon employs CRISPR-Cas9 screens to identify targets amenable to a synthetic lethality approach. Synthetic lethality arises if two conditions are met: 1) mutation of either of two genes is compatible with viability, and 2) mutations in both genes result  in cell death. Potential “synthetic lethal” targets identified by Horizon include specific common mutations occurring in colorectal cancer, such as p53 and KRAS.

    The screens involve putting an sgRNA library targeting thousands of genes into a cancer cell population, aiming for one sgRNA infection per cell so multiple genes aren’t cut in any one cell.
    The sgRNA population is exposed to a target drug of interest, perhaps one being considered for combination drug therapy. Next-generation sequencing identifies baseline and survivor sgRNAs.

    The sgRNAs lost during treatment are potential synthetic lethal targets, suggesting that the corresponding knocked out gene is essential for the viability of a cancer cell line.

    Identifying targets in this way can be “like looking for a needle in a haystack,” asserted Dr. McCarthy. “Difficult cellular changes that once took a long time to engineer, however, are now generally much more straightforward with CRISPR-Cas9 technology.”

  • From Cell Lines to Mouse Models

    “We certainly run many cell-line studies, but we have slightly more translational questions when trying to understand or predict what may happen in the clinic, in the patient population,” informed Dr. Broudy. “Cell lines are not the best tools for that. For translational questions, you want to have models that are as clinically relevant as possible, models that maintain the heterogeneity of cancer and genomic equivalency to a patient tumor.”

    Mouse models fit the bill. Today’s models are able to accommodate ever-evolving scientific and precision/personalized medicine missions, which include precisely editing the genome and harnessing the immune system to fight cancer.

    Case in point: Dr. McCarthy said that potential targets identified from siRNA or CRISPR-Cas9 sgRNA cell-line gene-editing screens are validated through more complex in vitro models such as 3D culture. Targets potentially involved in modulating tumor immune responses might advance to in vivo models, such as syngeneic mouse models, for further validation.

  • Transplantation Model Options

    In general, transplantation mouse models come in two types: allograft (mouse on mouse) and xenograft (human on mouse). An allograft mouse tumor system, which typically consists of a mouse cell line on a mouse immune system, is known as a syngeneic model. Xenograft systems include cell-line xenograft models and patient-derived xenograft (PDX) models. Yet another xenograft system is the humanized hybrid.

    “In a humanized hyrid,” explained Dr. Broudy, “a human tumor or a tumor cell line is engrafted into a mouse in which a human immune system has been reconstituted.” Models of this sort are also called humanized mice. Such models, Dr. Hall added, are meant to better emulate human biology.

    Crown, the premier PDX company, has almost 1,600 PDX models. Their expanding collection represents a global population including models established from American, Asian, and European patients. “And that accounts for the incredible diversity of models our clients, pharma drug developers, can select from,” declared Dr. Broudy.

    “Syngeneic” means genetically similar such as putting a tumor that originated in a C57 black mouse into a different C57 black mouse, meaning the animal has an intact immune system. With increasing efforts to harness the immune system to treat cancer, syngeneics have a big role in immuno-oncology drug development.

    “The tool set just keeps getting bigger and bigger,” Dr. Broudy insisted. “It is allowing us to ask lots of different questions.” A complementary sentiment is expressed by Walter Ausserer, Ph.D., associate general manager of clinical and in vivo services at The Jackson Laboratory (JAX): “I think the science is still young enough that you need to be using every tool you can.”

  • Immuno-Oncology Modeling

    humanized mouse

    Immune-inhibitory pathways (immune checkpoints) are essential for maintaining self-tolerance and modulating immune responses. Immune T cells have a natural ability to destroy cancer cells. This capability of T cells can be inhibited by tumors, which develop immune suppressor mechanisms and thereby escape immune surveillance, gaining the capacity for uncontrolled growth. For example, cells in the tumor microenvironment or a tumor itself may express high levels of immune checkpoint proteins such as CTLA4 or the programmed cell death PD-1 ligand, respectively, which negatively regulate T-cell activation.

    “In immuno-oncology, we are trying to activate a patient’s immune system to recognize and attack that patient’s tumor,” said Dr. Ausserer. Immune checkpoint inhibitor drugs that counteract tumor immune suppression mechanisms and promote immune system activation represent a key focus of immuno-oncology drug development.

    The co-engrafted NOD scid gamma (NSG) humanized mouse developed by JAX is “grafted with both the human immune system and human tumors,” Dr. Ausserer continued. “It is one of the first in vivo model for studying interactions between human immune system cells and human tumors.

    “This area of immuno-oncology is uniquely personal, akin to personalized medicine. You’re trying to coax an individual patient’s immune system to recognize and attack that same patient’s tumor.”

    http://www.genengnews.com/Media/images/Article/thumb_CharlesRiver_Roden2t2972422088.jpg

     

    According to Charles River Laboratories, immuno-oncology development may be ill-served by con- ventional xenograft models. Such models may lack relevance because they rely on immuno-compromised animals. Syngeneic mouse models, however, represent an attractive alternative. They can show how cancer therapies perform in the presence of a functional immune system.

     

    Dr. Broudy concurred. “We’ve been putting significant effort into extending the utility of PDX and cell-line xenograft mouse models for immuno-oncology drug development, including utilizing humanized mice. The truth is, if you don’t have a human immune system in the mix and are using only typical human patient-derived tumor xenograft models, there’s not a whole lot of value for immuno-oncology.”

    Another angle was emphasized by Aidan Synnott, Ph.D., site director at Charles River Laboratories. “With the advent of immunotherapies,” he said, “we’ve taken a look at the very old syngeneic mouse models and basically revitalized them.”

    Syngeneic models have been used since the 1960s. In the 1990s and 2000s, however,  people started moving toward xenograft models. According to Dr. Synnott, this trend gained momentum when immunodeficient mouse technology became available.

    The real impetus to immunotherapy came with the 2011 approval of Bristol-Myers Squibb’s Yervoy (ipilimumab), the first checkpoint inhibitor. This therapeutic, which blocks CTLA4, led to widespread recognition that syngeneics offer advantages over conventional human-on-mouse PDX models for immuno-oncology studies because of their functional immune systems.

    Indeed, Merck, Amgen, and other pharmaceutical companies used syngenic mouse models in developing their approved therapies, recalled Dr. Synnott. Syngeneic mouse tumor models of B16-F10 melanoma and M38 colon adenocarcinoma were both used in developing Amgen’s leukemia drug Blinatumomab and Merck’s melanoma drug Keytruda.

  • Mechanistic Insights

    However, mechanisms underlying drug efficacy and efficacy endpoints still remain to be fully elucidated and defined. So Charles River is running studies to do exactly that. For example, the company employs flow cytometry to assess drug effects on the balance of different types of immune cells with antibodies.

    “In some of our syngeneic models following checkpoint inhibitor treatment, the number of CD4-positive T cells will go down,” explained Dr. Synnott. “However, the number of CD8 effector T cells that actually attack the cancer will go up.

    “We help our clients understand how their drug is actually having an effect. You don’t just tell them, ‘Your tumor shrank, so your therapy works.’ You tell them the tumor shrank because it enhanced a certain subset of immune cells which then presumably attacked the tumor and shrank it.”

  • Mighty Human Mouse Models

    Taconic Biosciences’ portfolio of precision research models consists of genetically engineered humanized mice carrying human genes, and mice engrafted with human cells and tissues. “We work very closely with Japan’s Central Institute for Experimental Animals,” said Dr. Hall. “They focus on the genetic development of what we call super-immunodeficient mice that can be readily engrafted with foreign cells and tissues. These become the basis of our cell- and tissue-engraftment portfolio.”

    huNOG, Taconic’s primary immuno-oncology model, is a NOG (NOD/Shi-scid/IL-2Rγnull) mouse engrafted with a human immune system. It expresses a variety of human immune cell lineages including T and B cells.

    Much of today’s immuno-oncology work focuses on “enhancing T-cell functionality within cancer patients to enable their T cells to activate, target the tumor, and destroy tumor cells,” stated Dr. Hall. Human huNOG T cells are functional and mimic responses seen in human patients treated with immune checkpoint inhibitory drugs. A huNOG mouse can successfully engraft a human primary tumor.

    “You can treat those mice with a drug such as ipilimumab, and the human immune system will respond showing typical markers of activation seen in human patients, such as increases in T cells,” Dr. Hall continued. “Immune cells such as cytotoxic T cells infiltrate the tumor, resulting in tumor regression.”

    After putting a human patient tumor on board, immune responses generated by the tumor’s presence are assessed by flow cytometry and other techniques. Both immune responses and interactions between the human immune system and human tumor are evaluated following therapeutic intervention.

    Historically, pharmaceutical and biotechnology companies developed drugs for preclinical studies in mouse models that were, well, mouse specific. Due to species specificity, these drugs would not be exactly the same as the large molecule drug that would be utilized in humans.

    Dr. Hall wrapped up as follows: “Being able to place a human immune system within the mouse allows investigators to utilize the same therapeutic agent in the mouse model that will move to the clinic.” As a result, more drugs should be successful once they reach clinical testing.

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CRISPR/Cas9 Finds Its Way As an Important Tool For Drug Discovery & Development

Posted in Cancer and Current Therapeutics, CANCER BIOLOGY & Innovations in Cancer Therapy, Cell Biology, Cell Biology, Signaling & Cell Circuits, Conference Coverage with Social Media, CRISPR/Cas9 & Gene Editing, Disease Biology, Disease Biology, Small Molecules in Development of Therapeutic Drugs, Drug Delivery Platform Technology, Drug Toxicity, Gene Regulation, Gene Regulation and Evolution, Genetics & Innovations in Treatment, Genetics & Pharmaceutical, Metabolism, Metabolomics, Molecular Genetics & Pharmaceutical, Personalized and Precision Medicine & Genomic Research, Pharmaceutical Drug Discovery, Pharmacodynamics and Pharmacokinetics, Pharmacogenomics, Signaling & Cell Circuits, Small Molecules in Development of Therapeutic Drugs, tagged , , , , , , , , , , , , , , , on September 2, 2015| Leave a Comment »

CRISPR/Cas9 Finds Its Way As an Important Tool For Drug Discovery & Development

  UPDATED 6/11/2021

CRISPR Diagnostics: CRISPR-dx Comes of Age: Tool in Drug Development

The past five years has seen a rapid expansion of the ability of CRISPR based tools toward diagnostic testing. Recently, CRISPR has been used to detect SARS-CoV-2 in patients. An article in the journal Science describes the different classes of CRISPR diagnostics in use today .

Update near end of post

UPDATED 8/08/2020

Association to Causation: Using GWAS to Identify Druggable Targets

A Gen Webinar Thursday, August 6, 2020; 11:00am – 12:30pm EST

See at end of post

Curator: Stephen J. Williams, Ph.D.

Article 21.4.8-  CRISPRCas9 Finds Its Way As an Important Tool For Drug Discovery & Development

 

2.1.2.1

CRISPR/Cas9 Finds Its Way As an Important Tool For Drug Discovery & Development, Volume 2 (Volume Two: Latest in Genomics Methodologies for Therapeutics: Gene Editing, NGS and BioInformatics, Simulations and the Genome Ontology), Part 2: CRISPR for Gene Editing and DNA Repair

The RNA-guided Cas9 nuclease from the microbial clustered regularly interspaced short palindromic repeats (CRISPR) adaptive immune system can be used to facilitate efficient genome engineering in eukaryotic cells by simply specifying a 20-nt targeting sequence within its guide RNA.

CRISPR/Cas systems are part of the adaptive immune system of bacteria and archaea, protecting them against invading nucleic acids such as viruses by cleaving the foreign DNA in a sequence-dependent manner. Although CRISPR arrays were first identified in the Escherichia coli genome in 1987 (Ishino et al., 1987), their biological function was not understood until 2005, when it was shown that the spacers were homologous to viral and plasmid sequences suggesting a role in adaptive immunity (Bolotin et al., 2005; Mojica et al., 2005; Pourcel et al., 2005). Two years later, CRISPR arrays were confirmed to provide protection against invading viruses when combined with Cas genes (Barrangou et al., 2007). The mechanism of this immune system based on RNA-mediated DNA targeting was demonstrated shortly thereafter (Brouns et al., 2008; Deltcheva et al., 2011; Garneau et al., 2010; Marraffini and Sontheimer, 2008).

Jennifer Doudna, PhD Professor of Molecular and Cell Biology and Chemistry, University of California, Berkeley Investigator, Howard Hughes Medical Institute has recently received numerous awards and accolades for the discovery of CRISPR/Cas9 as a tool for mammalian genetic manipulation as well as her primary intended research target to understand bacterial resistance to viral infection.

A good post on the matter and Dr. Doudna can be seen below:

http://pharmaceuticalintelligence.com/2014/06/13/215-245-6132014-jennifer-doudna-the-biology-of-crisprs-from-genome-defense-to-genetic-engineering/

In Delineating a Role for CRISPR-Cas9 in Pharmaceutical Targeting inheritable metabolic disorders in which may benefit from a CRISPR-Cas9 mediated therapy is discussed. However this curation is meant to focus on CRISPR/CAS9 AS A TOOL IN PRECLINICAL DRUG DEVELOPMENT.

 

Three Areas of Importance of CRISPR/Cas9 as a TOOL in Preclinical Drug Discovery Include:

  1. Gene-Function Studies: CRISPR/CAS9 ability to DEFINE GENETIC LESION and INSERTION SITE
  2. CRISPR/CAS9 Use in Developing Models of Disease
  3. CRISPR/CAS9 Use as a Diagnostic Tool
  • Using CRISPR/Cas9 in PRECLINICAL TOXICOLOGY STUDIES

I.     Gene-Function Studies: CRISPR/CAS9 ability to DEFINE GENETIC LESION and INSERTION SITE

The advent of the first tools for manipulating genetic material (cloning, PCR, transgenic technology, and before microarray and other’omic methods) allowed scientists to probe novel, individual gene functions as well as their variants and mutants in a “one-gene-at-a time” process. In essence, a gene (or mutant gene) was sequenced, cloned into expression vectors and transfected into recipient cells where function was evaluated.

However, some of the experimental issues with this methodology involved

  • Most transfections experiments result in NON ISOGENIC cell lines – by definition the insertion of a transgene alters the genetic makeup of a cell line. Simple transfection experiments with one transgene compared to a “null” transfectant compares non-isogenic lines, possibly confusing the interpretation of gene-function studies. Therefore a common technique is to develop cell lines with inducible gene expression, thereby allowing the investigator to compare a gene’s effect in ISOGENIC cell lines.
  1. Use of CRSPR in Highthrough-put Screening of Genetic Function

A very nice presentation and summary of CRSPR’s use in determining gene function in a high-throughput manner can be found below

www.rna.uzh.ch/events/journalclub/20140429JCCaihong.pdf

  1. Determining Off-target Effects of Gene Therapy Simplified with CRSPR

In GUIDE-seq: First genome-wide method of detecting off-target DNA breaks induced by CRISPR-Cas nucleases (from This Journal’s series on Live Meeting Coverage) at a 2014 Koch lecture

Shengdar Q Tsai and J Keith Joung describe

“

an approach for global detection of DNA double-stranded breaks (DSBs) introduced by RGNs and potentially other nucleases. This method, called genome-wide, unbiased identification of DSBs enabled by sequencing (GUIDE-seq), relies on capture of double-stranded oligodeoxynucleotides into DSBs. Application of GUIDE-seq to 13 RGNs in two human cell lines revealed wide variability in RGN off-target activities and unappreciated characteristics of off-target sequences. The majority of identified sites were not detected by existing computational methods or chromatin immunoprecipitation sequencing (ChIP-seq). GUIDE-seq also identified RGN-independent genomic breakpoint ‘hotspots’.

SOURCE http://www.nature.com/nbt/journal/vaop/ncurrent/full/nbt.3117.html

“

II. CRISPR/Cas9 Use in Developing Models of Disease

 

  1. Developing Animal Tumor Models

In a post this year I discussed a talk at the recent 2015 AACR National Meeting on a laboratories ability to use CRISPR gene editing in-vivo to produce a hepatocarcinoma using viral delivery. The post can be seen here: Notes from Opening Plenary Session – The Genome and Beyond from the 2015 AACR Meeting in Philadelphia PA; Sunday April 19, 2015

1) In this talk Dr. Tyler Jacks discussed his use of CRSPR to generate a mouse model of liver tumor in an immunocompetent mouse. Some notes from this talk are given below

  1. B) Engineering Cancer Genomes: Tyler Jacks, Ph.D.; Director, Koch Institute for Integrative Cancer Research
  • Cancer GEM’s (genetically engineered mouse models of cancer) had moved from transgenics to defined oncogenes
  • Observation that p53 -/- mice develop spontaneous tumors (lymphomas)
  • then GEMs moved to Cre/Lox systems to generate mice with deletions however these tumor models require lots of animals, much time to create, expensive to keep;
  • figured can use CRSPR/Cas9 as rapid, inexpensive way to generate engineered mice and tumor models
  • he used CRSPR/Cas9 vectors targeting PTEN to introduce PTEN mutations in-vivo to hepatocytes; when they also introduced p53 mutations produced hemangiosarcomas; took ONLY THREE months to produce detectable tumors
  • also produced liver tumors by using CRSPR/Cas9 to introduce gain of function mutation in β-catenin

See an article describing this study by MIT News “A New Way To Model Cancer: New gene-editing technique allows scientists to more rapidly study the role of mutations in tumor development.”

The original research article can be found in the August 6, 2014 issue of Nature[1]

And see also on the Jacks Lab site under Research

2)     In the Upcoming Meeting New Frontiers in Gene Editing multiple uses of CRISPR technology is discussed in relation to gene knockout/function studies, tumor model development and

New Frontiers in Gene Editing

Session Spotlight:
BUILDING IN VIVO MODELS FOR DRUG DISCOVERY

Genome Editing Animal Models in Drug Discovery
Myung Shin, Ph.D., Senior Principal Scientist, Biology-Discovery, Genetics and Pharmacogenomics, Merck Research Laboratories

Recent advances in genome editing have greatly accelerated and expanded the ability to generate animal models. These tools allow generating mouse models in condensed timeline compared to that of conventional gene-targeting knock-out/knock-in strategies. Moreover, the genome editing methods have expanded the ability to generate animal models beyond mice. In this talk, we will discuss the application of ZFN and CRISPR to generate various animal models for drug discovery programs.

In vivo Cancer Modeling and Genetic Screening Using CRISPR/Cas9
Sidi Chen, Ph.D., Postdoctoral Fellow, Laboratories of Dr. Phillip A. Sharp and Dr. Feng Zhang, Koch Institute for Integrative Cancer Research at MIT and Broad Institute of Harvard and MIT

Here we describe a genome-wide CRISPR-Cas9-mediated loss-of-function screen in tumor growth and metastasis. We mutagenized a non-metastatic mouse cancer cell line using a genome-scale library. The mutant cell pool rapidly generates metastases when transplanted into immunocompromised mice. Enriched sgRNAs in lung metastases and late stage primary tumors were found to target a small set of genes, suggesting specific loss-of-function mutations drive tumor growth and metastasis.

FEATURED PRESENTATION: In vivo Chromosome Engineering Using CRISPR-Cas9
Andrea Ventura, M.D., Ph.D., Assistant Member, Cancer Biology and Genetics Program, Memorial Sloan Kettering Cancer Center

We will discuss our experience using somatic genome editing to engineer oncogenic chromosomal rearrangements in vivo. More specifically, we will present the results of our ongoing efforts aimed at modeling cancers driven by chromosomal rearrangements using viral mediated delivery of Crispr-Cas9 to adult animals.

RNAi and CRISPR/Cas9-Based in vivo Models for Drug Discovery
Christof Fellmann, Ph.D., Postdoctoral Fellow, Laboratory of Dr. Jennifer Doudna, Department of Molecular and Cell Biology, The University of California, Berkeley

Genetically engineered mouse models (GEMMs) are a powerful tool to study disease initiation, treatment response and relapse. By combining CRISPR/Cas9 and “Sensor” validated, tetracycline-regulated “miR-E” shRNA technology, we have developed a fast and scalable platform to generate RNAi GEMMs with reversible gene silencing capability. The synergy of CRISPR/Cas9 and RNAi enabled us to not only model disease pathogenesis, but also mimic drug therapy in mice, providing us capability to perform preclinical studies in vivo.

In vivo Genome Editing Using Staphylococcus aureus Cas9
Fei Ann Ran, Ph.D., Post-doctoral Fellow, Laboratory of Dr. Feng Zhang, Broad Institute and Junior Fellow, Harvard Society of Fellows

The RNA-guided Cas9 nuclease from the bacterial CRISPR/Cas system has been adapted as a powerful tool for facilitating targeted genome editing in eukaryotes. Recently, we have identified an additional small Cas9 nuclease from Staphylococcus aureus that can be packaged with its guide RNA into a single adeno-associated virus (AAV) vector for in vivo applications. We demonstrate the use of this system for effective gene modification in adult animals and further expand the Cas9 toolbox for in vivo genome editing.

OriGene, Making the Right Tools for CRISPR Research
Xuan Liu, Ph.D., Senior Director, Marketing, OriGene

CRISPR technology has quickly revolutionized the scientific community. Its simplicity has democratized the genome editing technology and enabled every lab to consider its utility in gene function research. As the largest tool box for gene functional research, OriGene created a large collection of CRISPR-related tools, including various all-in-one vectors for gRNA cloning, donor vector backbones, genome-wide knockout kits, AAVS1 insertion vectors, etc. OriGene’s high quality products will accelerate CRISPR research.

  1. Transgenic Animals : Custom Mouse and Rat Model Generation Service Using CRISPR/Cas9 by AppliedStem Cell Inc. (http://www.appliedstemcell.com/)

A critical component of producing transgenic animals is the ability of each successive generations to pass on the transgene. In her post on this site, A NEW ERA OF GENETIC MANIPULATION  Dr. Demet Sag discusses the molecular biology of Cas9 systems and their efficiency to cause point mutations which can be passed on to subsequent generations

“

This group developed a new technology for editing genes that can be transferable change to the next generation by combining microbial immune defense mechanism, CRISPR/Cas9 that is the latest ground breaking technology for translational genomics with gene therapy-like approach.

  • In short, this so-called “mutagenic chain reaction” (MCR) introduces a recessive mutation defined by CRISPR/Cas9 that lead into a high rate of transferable information to the next generation. They reported that when they crossed the female MCR offspring to wild type flies, the yellow phenotype observed more than 95 percent efficiency.

“

The advantage of CRISPR/Cas9 over ZFNs or TALENs is its scalability and multiplexibility in that multiple sites within the mammalian genome can be simultaneously modified, providing a robust, high-throughput approach for gene editing in mammalian cells.

Applied StemCell, Inc. offers various services related to animal models including conventional transgenic rats, and phenotype analysis using knock-in, knock-out strategies.

Further explanation of their use of CRSPR can be found at the site below:

http://pharmaceuticalintelligence.com/2014/10/29/gene-editing-at-crispr-speed-services-and-tools/

In addition, ReproCELL Inc., a Tokyo based stem cell company, uses CRSPR to develop

· Tailored disease model cells (hiPSC-Disease Model Cells)

  • 2 types of services
  • ReproUNUS™-g：human iPS cell derived functional cells involving gene editing by CRISPR/Cas9 system
  • eproUNUS™-p：patient derived iPS cell derived functional cells

III. Using CRISPR/Cas9 in PRECLINICAL TOXICOLOGY STUDIES

As of now it is unclear as to the strategy of pharma in how to use this technology for toxicology testing however a few companies have licensed the technology to use across their R&D platforms including

A recent paper used a sister technique TALEN to generate knock-in pigs which suggest that it would be possible to generate pigs with human transgenes, especially in human liver isozymes in orer to study hepatotoxicity of drugs.

Efficient bi-allelic gene knockout and site-specific knock-in mediated by TALENs in pigs

Jing Yao, Jiaojiao Huang, Tang Hai, Xianlong Wang, Guosong Qin, Hongyong Zhang, Rong Wu, Chunwei Cao, Jianzhong Jeff Xi, Zengqiang Yuan, Jianguo Zhao

Sci Rep. 2014; 4: 6926. Published online 2014 November 5. doi: 10.1038/srep06926

UPDATED 8/08/2020

Association to Causation: Using GWAS to Identify Druggable Targets

A Gen Webinar Thursday, August 6⋅11:00am – 12:30pm

This webinar is available at https://www.genengnews.com/resources/webinars/association-to-causation-using-gwas-to-identify-druggable-target/

Speakers:

Martin Kampmann, PhD

matinkampmann ucsf

Associate Professor
UCSF
Investigator
Chan Zuckerberg Biohub

Kevin Holden, PhD

kevinholdn sythego

Head of Science
Synthego

Abhi Saharia, PhD

abhisharia sythego

VP, Commercial Development
Synthego

Human genetics provides perhaps the single best opportunity to innovate and improve clinical success rates, through the identification of novel drug targets for complex disease. Even as correlation identifies multiple genetic variants associated with disease, it is challenging to conduct requisite functional studies to identify the causal variants, especially since most association signals map to non-coding regions of the genome.

Genetic editing technologies, such as CRISPR, have enabled the modeling of associated variants at their native loci, including non-coding loci, empowering the identification of underlying biological mechanisms of disease with potential causal genes. However, genome editing is largely manual today severely limiting scale, and forcing the use of rational filters to prioritize which variants to investigate functionally.

In this GEN webinar, we will discuss several strategies enabling large-scale functional investigation of disease-associated variants in a cost- and time-effective manner, including different types of pooled CRISPR-based screens and the development of a fully automated genome engineering platform. We will also review how optimization of genome engineering on this platform enables the engineering of disease-associated variants at scale in pluripotent cells.

  • They will be presenting on use of wide scale CRSPR screens to validate druggable targets
  • The presenters will also discuss new platforms for these wide scale screens

Martin Kampmann, PhD UCSF

  • Multiple genetic variants associated with disease
  • Big gap between accumulation of genetic variant information and functions of these variants
  • CRSPRi or CRSPa (siRNA coupled or enhancer coupled CRSPR guides)
  • Arrayed screens: multiplate guide RNAs and phenotype measured (phenotype can be morphology, complex biological systems like organoids or non autonomous functions
  • Using pooled screens and use of suitable cell model critical for this strategy
  • For example in iPSC vs. neurons has different expression patterns upon same CRSPR of UBA1
  • Advantage is using CRSPR to take iPSC from diseased variant patient to make a corrected isogenic control then introduce gRNAs and use modifier screens to determine phenotypes
  • Generated a platform called CRISPRbrain.org to do bioinformatics on various experiments with different guide RNAs (CRSPRs)

Abhi Saharia, PhD Syntheco

  • Target identification with CSRSPR at Scale
  • Nature medicine paper did GWAS and found 27 SNV associated with high risk disease and a rational filter focused on 1 SNV in noncoding region but why study a single variant and if studied all 27 would they have been able to identify a more representative druggable set?
  • Goal is to reduce or eliminate these rational filters
  • HALO (scalable RNA guide), ECLIPSE platform (automated generation of modified cell lines, BIOINFORMATIC platform (integrated informatics)
  • Syntheco uses an electroporation with ribonucleic proteins (RNP) to give highest efficiency and minimizes off target as complex is only in cells for a short period of time
  • They confirm they are doing single cell cloning by using automated microscopy to confirm single cell growth in each cloning well

Kevin Holden, Head of Science at Syntheco

  • Engineering iPSc genetically modified cells at scale
  • The closer you get to your target site the more efficient your CRSPR so a big factor when making guides, especially for knock-in CRSPR
  • Adding a small molecule non homologous end joining inhibitor increases efficiency to 95%
  • Cold shocking the cells also assists in homologous repair
  • Use cleavage resistant templates

III. CRISPR/CAS9 AS A DIAGNOSTIC TOOL

     In the journal Science, Omar Abudayyeh and Jonathan Gootenberg discuss how CRISPR-based diagnostic (CRISPR-dx) tools offer a solution, and multiple CRISPR-dx products for detection of the SARS-CoV-2 RNA genome have been authorized by the US Food and Drug Administration (FDA).  In addition they discuss the work by Jiao et al. in combining this technique to develop a rapid and sensitive SARS-CoV2 diagnostic test.

Omar O. Abudayyeh, Jonathan S. Gootenberg. Science  28 May 2021: CRISPR Diagnostics
Vol. 372, Issue 6545, pp. 914-915; DOI: 10.1126/science.abi9335

Summary

Although clinical diagnostics take many forms, nucleic acid–based testing has become the gold standard for sensitive detection of many diseases, including pathogenic infections. Quantitative polymerase chain reaction (qPCR) has been widely adopted for its ability to detect only a few DNA or RNA molecules that can unambiguously specify a particular disease. However, the complexity of this technique restricts application to laboratory settings. The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has underscored the need for the development and deployment of nucleic acid tests that are economical, easily scaled, and capable of being run in low-resource settings, without sacrifices in speed, sensitivity or specificity. CRISPR-based diagnostic (CRISPR-dx) tools offer a solution, and multiple CRISPR-dx products for detection of the SARS-CoV-2 RNA genome have been authorized by the US Food and Drug Administration (FDA). On page 941 of this issue, Jiao et al. (1) describe a new CRISPR-based tool to distinguish several SARS-CoV-2 variants in a single reaction.

Although clinical diagnostics take many forms, nucleic acid–based testing has become the gold standard for sensitive detection of many diseases, including pathogenic infections. Quantitative polymerase chain reaction (qPCR) has been widely adopted for its ability to detect only a few DNA or RNA molecules that can unambiguously specify a particular disease. However, the complexity of this technique restricts application to laboratory settings. The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has underscored the need for the development and deployment of nucleic acid tests that are economical, easily scaled, and capable of being run in low-resource settings, without sacrifices in speed, sensitivity or specificity. CRISPR-based diagnostic (CRISPR-dx) tools offer a solution, and multiple CRISPR-dx products for detection of the SARS-CoV-2 RNA genome have been authorized by the US Food and Drug Administration (FDA). On page 941 of this issue, Jiao et al. (1) describe a new CRISPR-based tool to distinguish several SARS-CoV-2 variants in a single reaction.

There are multiple types of CRISPR systems comprising basic components of a single protein or protein complex, which cuts a specific DNA or RNA target programmed by a complementary guide sequence in a CRISPR-associated RNA (crRNA). The type V and VI systems and the CRISPR-associated endonucleases Cas12 (2, 3) and Cas13 (4, 5) bind and cut DNA or RNA, respectively. Furthermore, upon recognizing a target DNA or RNA sequence, Cas12 and Cas13 proteins exhibit “collateral activity” whereby any DNA or RNA, respectively, in the sample is cleaved regardless of its nucleic acid sequence (4, 6). Thus, reporter DNAs or RNAs, which allow for visual or fluorescent detection upon cleavage, can be added to a sample to infer the presence or absence of specific DNA or RNA species (4–8).

Initial versions of CRISPR-dx utilizing Cas13 alone were sensitive to the low picomolar range, corresponding to a limit of detection of millions of molecules in a microliter sample. To improve sensitivity, preamplification methods, such as recombinase polymerase amplification (RPA), PCR, loop-mediated isothermal amplification (LAMP), or nucleic acid sequence–based amplification (NASBA), can be used with Cas12 or Cas13 to enable a limit of detection down to a single molecule (8). This preamplification approach, applicable to both Cas12 and Cas13 (6, 7), enabled a suite of detection methods and multiplexing up to four orthogonal targets (7). Additional developments expanded CRISPR-dx readouts beyond fluorescence, including lateral flow (7), colorimetric (9), and electronic or material responsive readouts (10), allowing for instrument-free approaches. In addition, post–collateral-cleavage amplification methods, such as the use of the CRISPR-associated enzyme Csm6, have been combined with Cas13 to further increase the speed of CRISPR-dx tests (7). As an alternative to collateral-cleavage–based detection, type III CRISPR systems, which involve large multiprotein complexes capable of targeting both DNA and RNA, have been used for SARS-CoV-2 detection through production of colorimetric or fluorometric readouts (11).

FDA-authorized CRISPR-dx tests are currently only for use in centralized labs, because the most common CRISPR detection protocols require fluid handling steps and two different incubations, precluding their immediate use at the point of care. Single-step formulations have been developed to overcome this limitation, and these “one-pot” versions of CRISPR-dx are simple to run, operate at a single temperature, and run without complex equipment, producing either fluorescence or lateral flow readouts. The programmability of CRISPR makes new diagnostic tests easier to develop, and within months of the release of the SARS-CoV-2 genome, many COVID-19–specific CRISPR tests were reported and distributed around the world.

The broader capability for Cas enzyme–enhanced nucleic acid binding or cleavage has led to several other detection modalities. Cas9-based methods for cleaving nucleic acids in solution for diagnostic purposes have been combined with other detection platforms, such as destruction of undesired amplicons for preparation of next-generation sequencing libraries (12), or selective removal of alleles for nucleotide-specific detection (13). Alternatively, the programmable cleavage event from the Cas nuclease can be used to initiate an amplification reaction (14). Cas9-based DNA targeting has also been used for nucleotide detection in combination with solid-state electronics, promising an amplification-free platform for detection. In this platform, called CRISPR-Chip, the Cas9 protein binds nucleotide targets of interest (often in the context of the native genome) to graphene transistors, where the presence of these targets alters either current or voltage (15). By utilizing additional Cas9 orthologs and specific guide designs, CRISPR-Chip approaches have been tuned for single–base-pair sensitivity (15). Because they are integrated with electronic readers, CRISPR-Chip platforms may allow facile point-of-care detection with handheld devices.

 

Different classes of CRISPR diagnostics. GRAPHIC: ERIN DANIEL

Jiao et al. use a distinct characteristic of type II CRISPR systems, which involve Cas9, to develop a new type of noncollateral based CRISPR detection. Unlike Cas12s and Cas13, Cas9-crRNA complex formation requires an additional RNA known as the trans-activating CRISPR RNA (tracrRNA). By sequencing RNAs bound to Cas9 from Campylobacter jejuni in its natural host, the authors identified unexpected crRNAs, called noncanonical crRNA (ncrRNA), that corresponded to endogenous transcripts. Upon investigation of this surprising observation, it became clear that the tracrRNA was capable of hybridizing to semi-complementary sequences from a variety of RNA sources, leading to biogenesis of ncrRNAs of various sizes. Recognizing that they could program tracrRNAs to target a transcript of interest, the authors generated a reprogrammed tracrRNA (Rptr) that could bind and cleave a desired transcript, converting a piece of that transcript into a functional guide RNA. By then creating fluorescent DNA sensors that would be cleaved by the Rptr and ncrRNAs, the sensing of RNA by Cas9 could be linked to a detectable readout. This platform, called LEOPARD (leveraging engineered tracrRNAs and on-target DNAs for parallel RNA detection), can be combined with gel-based readouts and enables multiplexed detection of several different sequences in a single reaction (see the figure).

Jiao et al. also combined LEOPARD with PCR in a multistep workflow to detect SARS-CoV-2 genomes from patients with COVID-19. Although more work is needed to integrate this Cas9-based detection modality into a single step with RPA or LAMP to create a portable and sensitive isothermal test, an advantage of this approach is the higher-order multiplexing that can be achieved, allowing multiple pathogens, diseases, or variants to be detected simultaneously. More work is also needed to combine this technology with extraction-free methods for better ease of use; alternative readouts to gel-based readouts, such as lateral flow and colorimetric readouts, would be beneficial for point-of-care detection.

In just 5 years, the CRISPR-dx field has rapidly expanded, growing from a set of peculiar molecular biology discoveries to multiple FDA-authorized COVID-19 tests and spanning four of the six major subtypes of CRISPR systems. Despite the tremendous promise of CRISPR-dx, substantial challenges remain to adapting these technologies for point-of-care and at-home settings. Simplification of the chemistries to operate as a single reaction in a matter of minutes would be revolutionary, especially if the reaction could be run at room temperature without any complex or expensive equipment. These improvements to CRISPR-dx assays can be achieved by identification or engineering of additional Cas enzymes with lower-temperature requirements, higher sensitivity, or faster kinetics, enabling rapid and simple amplification-free detection with single-molecule sensitivity.

Often overlooked is the necessity for a sample DNA or RNA preparation step that is simple enough to be added directly to the CRISPR reaction to maintain a simple workflow for point-of-care testing. In addition, higher-order multiplexing developments would allow for expansive testing menus and approach the possibility of testing for all known diseases. As these advancements are realized, innovative uses of CRISPR-dx will continue in areas such as surveillance, integration with biomaterials, and environmental monitoring. In future years, CRISPR-dx assays may become universal in the clinic and at home, reshaping how diseases are diagnosed.

References and Notes

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Other related articles on CRISPR/Cas9 were published in this Open Access Online Scientific Journal, include the following:

Search Results for ‘CRISPR’

Where is the most promising avenue to success in Pharmaceuticals with CRISPR-Cas9?

CRISPR/Cas9 genome editing tool for Staphylococcus aureus Cas9 complex (SaCas9) @ MIT’s Broad Institute

Delineating a Role for CRISPR-Cas9 in Pharmaceutical Targeting

Using CRISPR to investigate pancreatic cancer

Simple technology makes CRISPR gene editing cheaper

RNAi, CRISPR, and Gene Editing: Discussions on How To’s and Best Practices @14th Annual World Preclinical Congress June 10-12, 2015 | Westin Boston Waterfront | Boston, MA

CRISPR/Cas9: Contributions on Endoribonuclease Structure and Function, Role in Immunity and Applications in Genome Engineering

CRISPR-CAS editing brings cloning of woolly mammoth one step closer to reality

GUIDE-seq: First genome-wide method of detecting off-target DNA breaks induced by CRISPR-Cas nucleases

The Patents for CRISPR, the DNA editing technology as the Biggest Biotech Discovery of the Century

CRISPR: Applications for Autoimmune Diseases @UCSF

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Notes from Opening Plenary Session – The Genome and Beyond from the 2015 AACR Meeting in Philadelphia PA; Sunday April 19, 2015

Posted in Cancer and Current Therapeutics, CANCER BIOLOGY & Innovations in Cancer Therapy, Clinical Genomics, Conference Coverage with Social Media, CRISPR/Cas9 & Gene Editing, Health Economics and Outcomes Research, tagged , , , , , , , , , , , , , , , , , , , on April 30, 2015| Leave a Comment »

Notes from Opening Plenary Session – The Genome and Beyond from the 2015 AACR Meeting in Philadelphia PA; Sunday April 19, 2015

 

Reporter: Stephen J. Williams, Ph.D.

The following contain notes from the Sunday April 19, 2015 AACR Meeting (Pennsylvania Convention Center, Philadelphia PA) 9:30 AM Opening Plenary Session

The Genome and Beyond

Session Chairperson: Lewis C. Cantley, Ph.D.

Speakers: Michael R. Stratton, Tyler Jacks, Stephen B. Baylin, Robert D. Schreiber, Williams R. Sellers

  1. A) Insights From Cancer Genomes: Michael R. Stratton, Ph.D.; Director of the Wellcome Trust Sanger Institute
  • How do we correlate mutations with causative factors of carcinogenesis and exposure?
  • Cancer was thought as a disease of somatic mutations
  • UV skin exposure – see C>T transversion in TP53 while tobacco exposure and lung cancer see more C>A transversion; Is it possible to determine EXPOSURE SIGNATURES?
  • Use a method called non negative matrix factorization (like face pattern recognition but a mutation pattern recognition)
  • Performed sequence analysis producing 12,000 mutation catalogs with 8,000 somatic mutation signatures
  • Found more mutations than expected; some mutation signatures found in all cancers, while some signatures in half of cancers, and some signatures not found in cancer
  • For example found 3 mutation signatures in ovarian cancer but 13 for breast cancers (80,000 mutations); his signatures are actually spectrums of mutations
  • kataegis: defined as localized hypermutation; an example is a signature he found related to AID/APOBEC family (involved in IgG variability); kataegis is more prone in hematologic cancers than solid cancers
  • recurrent tumors show a difference in mutation signatures than primary tumor before drug treatment

 

  1. B) Engineering Cancer Genomes: Tyler Jacks, Ph.D.; Director, Koch Institute for Integrative Cancer Research
  • Cancer GEM’s (genetically engineered mouse models of cancer) had moved from transgenics to defined oncogenes
  • Observation that p53 -/- mice develop spontaneous tumors (lymphomas)
  • then GEMs moved to Cre/Lox systems to generate mice with deletions however these tumor models require lots of animals, much time to create, expensive to keep;
  • figured can use CRSPR/Cas9 as rapid, inexpensive way to generate engineered mice and tumor models
  • he used CRSPR/Cas9 vectors targeting PTEN to introduce PTEN mutations in-vivo to hepatocytes; when they also introduced p53 mutations produced hemangiosarcomas; took ONLY THREE months to produce detectable tumors
  • also produced liver tumors by using CRSPR/Cas9 to introduce gain of function mutation in β-catenin

 

See an article describing this study by MIT News “A New Way To Model Cancer: New gene-editing technique allows scientists to more rapidly study the role of mutations in tumor development.”

The original research article can be found in the August 6, 2014 issue of Nature[1]

And see also on the Jacks Lab site under Research

  1. C) Above the Genome; The Epigenome and its Biology: Stephen B. Baylin
  • Baylin feels epigenetic therapy could be used for cancer cell reprogramming
  • Interplay between Histone (Movers) and epigenetic marks (Writers, Readers) important for developing epigenetic therapy
  • Difference between stem cells and cancer: cancer keeps multiple methylation marks whereas stem cells either keep one on or turn off marks in lineage
  • Corepressor drugs are a new exciting class in chemotherapeutic development
  • (Histone Demythylase {LSD1} inhibitors in clinical trials)
  • Bromodomain (Brd4) enhancers in clinical trials
  1. D) Using Genomes to Personalize Immunotherapy: Robert D. Schreiber, Ph.D.,
  • The three “E’s” of cancer immunoediting: Elimination, Equilibrium, and Escape
  • First evidence for immunoediting came from mice that were immunocompetent resistant to 3 methylcholanthrene (3mca)-induced tumorigenesis but RAG2 -/- form 3mca-induced tumors
  • RAG2-/- unedited (retain immunogenicity); tumors rejected by wild type mice
  • Edited tumors (aren’t immunogenic) led to tolerization of tumors
  • Can use genomic studies to identify mutant proteins which could be cancer specific immunoepitopes
  • MHC (major histocompatibility complex) tetramers: can develop vaccines against epitope and personalize therapy but only good as checkpoint block (anti-PD1 and anti CTLA4) but personalized vaccines can increase therapeutic window so don’t need to start PD1 therapy right away
  • For more details see references Schreiker 2011 Science and Shankaran 2001 in Nature
  1. E) Report on the Melanoma Keynote 006 Trial comparing pembrolizumab and ipilimumab (PD1 inhibitors)

Results of this trial were published the morning of the meeting in the New England Journal of Medicine and can be found here.

A few notes:

From the paper: The anti–PD-1 antibody pembrolizumab prolonged progression-free survival and overall survival and had less high-grade toxicity than did ipilimumab in patients with advanced melanoma. (Funded by Merck Sharp & Dohme; KEYNOTE-006 ClinicalTrials.gov number, NCT01866319.)

And from Twitter:

Robert Cade, PharmD @VTOncologyPharm

KEYNOTE-006 was presented at this week’s #AACR15 conference. Pembrolizumab blew away ipilimumab as 1st-line therapy for metastatic melanoma.

2:02 PM – 21 Apr 2015

Jeb Keiper @JebKeiper

KEYNOTE-006 at #AACR15 has pembro HR 0.63 in OS over ipi. Issue is ipi is dosed only 4 times over 2 years (per label) vs Q2W for pembro. Hmm

11:55 AM – 19 Apr 2015

OncLive.com @OncLive

Dr Antoni Ribas presenting data from KEYNOTE-006 at #AACR15 – Read more about the findings, at http://ow.ly/LMG6T 

11:25 AM – 19 Apr 2015

Joe @GantosJ

$MRK on 03/24 KEYNOTE-006 vs Yervoy Ph3 stopped early for meeting goals of PFS, OS & full data @ #AACR15 now back to weekend & family

9:05 AM – 19 Apr 2015

Kristen Slangerup @medwritekristen

Keytruda OS benefit over Yervoy in frontline #melanoma $MRK stops Ph3 early & data to come @ #AACR15 #immunotherapy http://yhoo.it/1EYwwq8 

2:40 PM – 26 Mar 2015

Yahoo Finance @YahooFinance

Merck’s Pivotal KEYNOTE-006 Study in First-Line Treatment for…

Merck , known as MSD outside the United States and Canada, today announced that the randomized, pivotal Phase 3 study investigating KEYTRUDA® compared to ipilimumab in the first-line treatment of…

View on web

 

Stephen J Williams @StephenJWillia2

Progression free survival better for pembrolzumab over ipilimumab by 2.5 months #AACR15 @Pharma_BI #Cancer #Immunotherapy

11:56 AM – 19 Apr 2015

 

Stephen J Williams @StephenJWillia2

Melanoma Keynote 006 trial PD1 inhibitor #Immunotherapy 80% responders after 1 year @Pharma_BI #AACR15

 

References

  1. Xue W, Chen S, Yin H, Tammela T, Papagiannakopoulos T, Joshi NS, Cai W, Yang G, Bronson R, Crowley DG et al: CRISPR-mediated direct mutation of cancer genes in the mouse liver. Nature 2014, 514(7522):380-384.

 

Other related articles on Cancer Genomics and Social Media Coverage were published in this Open Access Online Scientific Journal, include the following:

Cancer Biology and Genomics for Disease Diagnosis

Introduction – The Evolution of Cancer Therapy and Cancer Research: How We Got Here?

Methodology for Conference Coverage using Social Media: 2014 MassBio Annual Meeting 4/3 – 4/4 2014, Royal Sonesta Hotel, Cambridge, MA

List of Breakthroughs in Cancer Research and Oncology Drug Development by Awardees of The Israel Cancer Research Fund

2013 American Cancer Research Association Award for Outstanding Achievement in Chemistry in Cancer Research: Professor Alexander Levitzki

Genomics and Epigenetics: Genetic Errors and Methodologies – Cancer and Other Diseases

Cancer Genomics – Leading the Way by Cancer Genomics Program at UC Santa Cruz

Genomics and Metabolomics Advances in Cancer

Pancreatic Cancer: Genetics, Genomics and Immunotherapy

Multiple Lung Cancer Genomic Projects Suggest New Targets, Research Directions for Non-Small Cell Lung Cancer

 

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Heroes in Medical Research: Developing Models for Cancer Research

Posted in CANCER BIOLOGY & Innovations in Cancer Therapy, Genome Biology, Health Economics and Outcomes Research, Human Immune System in Health and in Disease, Scientist: Career considerations, Translational Research, tagged , , , , , , , , , , , , , , , , on April 4, 2014| Leave a Comment »

Heroes in Medical Research: Developing Models for Cancer Research

Author, Curator: Stephen J. Williams, Ph.D.

 

The current rapid progress in cancer research would have never come about if not for the dedication of past researchers who had developed many of the scientific tools we use today. In this issue of Heroes in Medical Research I would like to give tribute to the researchers who had developed the some of the in-vivo and in-vitro models which are critical for cancer research.

 

The Animal Modelers in Cancer Research

Helen Dean King, Ph.D. (1869-1955)

Helen Dean King

Helen Dean King, Ph.D. from www.ExplorePAhistory.com; photo Courtesy of the Wistar Institute Archive Collection, Philadelphia, PA

 

 

The work of Dr. Helen Dean King on rat inbreeding led to development of strains of laboratory animals. Dr. King taught at Bryn Mawr College, then worked at University of Pennsylvania and the Wistar Institute under famed geneticist Thomas Hunt Morgan, researching if inbreeding would produce harmful genetic traits.   At University of Pennsylvania she examined environmental and genetic factors on gender determination.

 

 

 

 

Important papers include [1-6]as well as the following contributions:

“Studies in Inbreeding”, “Life Processes in Gray Norway Rats During Fourteen Years in Captivity”, doctoral thesis on embryologic development in toads (1899)

 

Milestones include:

 

1909    started albino rat breeding and bred 20 female and male from same litter (King colony) to 25

successive generations (inbreeding did not cause harmful traits)

 

1919     started to domesticate the wild Norwegian rats that ran thru Philadelphia (six pairs Norway rats

thru 28 generations)

A good reference for definitions of rat inbreeding versus line generation including a history of Dr. King’s work can be found at the site: Munificent Mischief Rattery and a brief history here.[7] In addition, Dr. King had investigated using rat strains as a possible recipient for tumor cells. The work was an important advent to the use of immunodeficient models for cancer research.

 

As shown below Philadelphia became a hotbed for research into embryology, development, genetics, and animal model development.

 

Beatrice Mintz, Ph.D.

(Beatrice Minz, Ph.D.; photo credit Fox Chase Cancer Center, www.pubweb.fccc.edu) Mintz

Dr. Mintz, an embryologist and cancer researcher from Fox Chase Cancer Center in Philadelphia, PA, contributed some of the most seminal discoveries leading to our current understanding of genetics, embryo development, cellular differentiation, and oncogenesis, especially melanoma, while pioneering techniques which allowed the development of genetically modified mice.

If you get the privilege of hearing her talk, take advantage of it. Dr. Mintz is one of those brilliant scientists who have the ability to look at a clinical problem from the viewpoint of a basic biological question and, at the same time, has the ability to approach the well-thought out questions with equally well thought out experimental design. For example, Dr. Mintz asked if a cell’s developmental fate was affected by location in the embryo. This led to her work by showing teratocarcinoma tumor cells in the developing embryo could revert to a more normal phenotype, essentially proving two important concepts in development and tumor biology:

  1. The existence of pluripotent stem cells
  2. That tumor cells are affected by their environment (which led to future concepts of the importance of tumor microenvironment on tumor growth

Other seminal discoveries included:

  • Development of the first mouse chimeras using novel cell fusion techniques
  • With Rudolf Jaenisch in 1974, showed integration of viral DNA from SV40, could be integrated into the DNA of developing mice and persist into adulthood somatic cells, the first transgenesis in mice which led ultimately to:
  • Development of the first genetically modified mouse model of human melanoma in 1993

Her current work, seen on the faculty webpage here, is developing mice with predisposition to melanoma to uncover risk factors associated with the early development of melanoma.

In keeping with the Philadelphia tradition another major mouse model which became seminal to cancer drug discovery was co-developed in the same city, same institute and described in the next section.

It is interesting to note that the first cloning of an animal, a frog, had taken place at the Institute for Cancer Research, later becoming Fox Chase Cancer Center, which was performed by Drs. Robert Briggs and Thomas J. King and reported in the 152 PNAS paper Transplantation of Living Nuclei From Blastula Cells into Enucleated Frogs’ Eggs.[8]

 

 The Immunodeficient Animal as a Model System for Cancer Research – Dr. Mel Bosma, Ph.D.

 

Bosma

Melvin J. Bosma, Ph.D.; photo credit Fox Chase Cancer Center

In the summer of 1980 at Fox Chase Cancer Center, Dr. Melvin J. Bosma and his co-researcher wife Gayle discovered mice with deficiencies in common circulating antibodies and since, these mice were littermates, realized they had found a genetic defect which rendered the mice immunodeficient (upon further investigation these mice were unable to produce mature B and T cells). These mice were the first scid (severe combined immunodeficiency) colony. The scid phenotype was later found to be a result of a spontaneous mutation in the enzyme Prkdc {protein kinase, DNA activated, catalytic polypeptide} involved in DNA repair, and ultimately led to a defect in V(D)J recombination of immunoglobulins.

The emergence of this scid mouse was not only crucial for AIDS research but was another turning point in cancer research , as researchers now had a robust in-vivo recipient for human tumor cells. The orthotopic xenograft of human tumor cells now allowed for studies on genetic and microenvironmental factors affecting tumorigenicity, as well as providing a model for chemotherapeutic drug development (see Suggitt for review and references)[9]. A discussion of the pros and cons of the xenograft system for cancer drug discovery would be too voluminous for this post and would warrant a full review by itself. But before the advent of such scid mouse systems researchers relied on spontaneous and syngeneic mouse tumor models such as the B16 mouse melanoma and Lewis lung tumor model.

Other scid systems have been developed such as in the dog, horse, and pig. Please see the following post on this site The SCID Pig: How Pigs are becoming a Great Alternate Model for Cancer Research. The athymic (nude) mouse (nu/nu) also is a popular immunodeficient mouse model used for cancer research

Two other in-vivo tumor models: Patient Derived Xenografts (PDX) and Genetically Engineered Mouse models (GEM) deserve their own separate discussion however the success of these new models can be attributed to the hard work of the aforementioned investigators. Therefore I will post separately and curate PDX and GEM models of cancer and highlight some new models which are having great impact on cancer drug development.

 

References

1.         Loeb L, King HD: Transplantation and Individuality Differential in Strains of Inbred Rats. The American journal of pathology 1927, 3(2):143-167.

2.         Lewis MR, Aptekman PM, King HD: Retarding action of adrenal gland on growth of sarcoma grafts in rats. J Immunol 1949, 61(4):315-319.

3.         Aptekman PM, Lewis MR, King HD: Tumor-immunity induced in rats by subcutaneous injection of tumor extract. J Immunol 1949, 63(4):435-440.

4.         Lewis MR, Aptekman PM, King HD: Inactivation of malignant tissue in tumor-immune rats. J Immunol 1949, 61(4):321-326.

5.         Lewis MR, King HD, et al.: Further studies on oncolysis and tumor immunity in rats. J Immunol 1948, 60(4):517-528.

6.         Aptekman PM, Lewis MR, King HD: A method of producing in inbred albino rats a high percentage of immunity from tumors native in their strain. J Immunol 1946, 52:77-86.

7.         Ogilvie MB: Inbreeding, eugenics, and Helen Dean King (1869-1955). Journal of the history of biology 2007, 40(3):467-507.

8.         Briggs R, King TJ: Transplantation of Living Nuclei From Blastula Cells into Enucleated Frogs’ Eggs. Proceedings of the National Academy of Sciences of the United States of America 1952, 38(5):455-463.

9.         Suggitt M, Bibby MC: 50 years of preclinical anticancer drug screening: empirical to target-driven approaches. Clinical cancer research : an official journal of the American Association for Cancer Research 2005, 11(3):971-981.

 

Other posts on this site about Cancer, Animal Models of Disease, and other articles in this series include:

The SCID Pig: How Pigs are becoming a Great Alternate Model for Cancer Research

A Synthesis of the Beauty and Complexity of How We View Cancer

Guidelines for the welfare and use of animals in cancer research

Importance of Funding Replication Studies: NIH on Credibility of Basic Biomedical Studies

FDA Guidelines For Developmental and Reproductive Toxicology (DART) Studies for Small Molecules

Report on the Fall Mid-Atlantic Society of Toxicology Meeting “Reproductive Toxicology of Biologics: Challenges and Considerations:

What`s new in pancreatic cancer research and treatment?

Heroes in Medical Research: Dr. Carmine Paul Bianchi Pharmacologist, Leader, and Mentor

Heroes in Medical Research: Dr. Robert Ting, Ph.D. and Retrovirus in AIDS and Cancer

Heroes in Medical Research: Barnett Rosenberg and the Discovery of Cisplatin

Richard Lifton, MD, PhD of Yale University and Howard Hughes Medical Institute: Recipient of 2014 Breakthrough Prizes Awarded in Life Sciences for the Discovery of Genes and Biochemical Mechanisms that cause Hypertension

Reuben Shaw, Ph.D., a geneticist and researcher at the Salk Institute: Metabolism Influences Cancer

 

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Jmjd3 and Cardiovascular Differentiation of Embryonic Stem Cells

Posted in Biological Networks, Gene Regulation and Evolution, Stem Cells for Regenerative Medicine, tagged , , , , , , , , , , , on October 26, 2013| Leave a Comment »

Jmjd3 and Cardiovascular Differentiation of Embryonic Stem Cells

Author and Curator: Larry H Bernstein, MD, FCAP

and

Curator: Aviva Lev-Ari, PhD, RN

This article is a presentation of recently published work on the basis for control of mesodermal and cardiovascular differentiation of embryonic stem cells, which has taken on increasing importance in the treatment of cardiovascular disease, with particular application to heart failure due to any cause, but with particular relevance to significant loss of myocardium, as may occur with acute myocardial infarction with more than 60% occlusion of the left anterior descending artery, near the osteum, with or without adjacent artery involvement, resulting in major loss of cardiac contractile force and insufficient ejection fraction. The article is of high interest and makes the following points:

  1. Ablation of Jmjd3 in mouse embryonic stem cells does not affect the maintenance of pluripotency and self-renewal
  2. Ablation of Jmjd3 in mouse embryonic stem cells compromised mesoderm and subsequent endothelial and cardiac differentiation 
  3. Jmjd3 reduces H3K27me3 marks at the Brachyury promoter and facilitates the recruitment of β-catenin
  4. β-catenin s critical for Wnt signal–induced mesoderm differentiation. 

Jmjd3 Controls Mesodermal and Cardiovascular Differentiation of Embryonic Stem Cells

K Ohtani, C Zhao, G Dobreva, Y Manavski, B Kluge, T Braun, MA Rieger, AM Zeiher and S Dimmeler

I The Institute of Cardiovascular Regeneration, Centre for Molecular Medicine, University of Frankfurt, Frankfurt, Germany (K.O., C.Z., Y.M., B.K., S.D.); Max-Planck-Institute for Heart and Lung Research, Bad Nauheim, Germany (G.D., T.B.); Department of Hematology/Oncology, Internal Medicine
II LOEWE Center for Cell and Gene Therapy, University of Frankfurt, Frankfurt, Germany (M.A.R.); and Department of Cardiology, Internal Medicine
III University of Frankfurt, Frankfurt, Germany (A.M.Z.).
This manuscript was sent to Benoit Bruneau, Consulting Editor, for review by expert referees, editorial decision, and final disposition.
Correspondence to Stefanie Dimmeler,  Institute of Cardiovascular Regeneration, Centre for Molecular Medicine, University of Frankfurt, Frankfurt, Germany. E-mail dimmeler@em.uni-frankfurt.de
 Circ Res. 2013;113:856-862;  http://dx.doi.org/10.1161/CIRCRESAHA.113.302035   http://circres.ahajournals.org/content/113/7/856  

Abstract

Rationale: The developmental role of the H3K27 demethylases Jmjd3, especially its epigenetic regulation at target genes in response to upstream developmental signaling, is unclear.

Objective: To determine the role of Jmjd3 during mesoderm and cardiovascular lineage commitment.

Methods and Results: Ablation of Jmjd3 in mouse embryonic stem cells does not affect the maintenance of pluripotency and self-renewal but compromised mesoderm and subsequent endothelial and cardiac differentiation. Jmjd3 reduces H3K27me3 marks at the Brachyury promoter and facilitates the recruitment of β-catenin, which is critical for Wnt signal–induced mesoderm differentiation.

Conclusions: These data demonstrate that Jmjd3 is required for mesoderm differentiation and cardiovascular lineage commitment. (Circ Res. 2013;113:856-862.)

  • Key Words: Brachyury protein ■ embryonic stem cells ■ epigenomics ■ Jmjd3 protein, mouse ■ mesodermn  –  Wnt signaling pathway

Introduction

Post-translational modifications of histone proteins represent essential epigenetic control mechanisms that can either allow or repress gene expression.1 Trimethylation of H3K27 is mediated by Polycomb group proteins and represses gene expression.2 The JmjC domain–containing proteins, UTX (ubiquitously transcribed tetratricopeptide repeat, X chromosome) and Jmjd3 (jumonji domain–containing protein 3, Kdm6b), not only act as demethylases to remove the repressive H3K27me3 marks, but also exhibit additional demethylase-independent functions.3–6 Jmjd3 is induced and participates in Hox gene expression during development,7 neuronal differentiation,8,9 and inflammation,5,10–12 and recent data suggest that Jmjd3 inhibits reprogramming by inducing cellular senescence.13 Because previous studies suggest that H3K27me3 regulates endothelial gene expression in adult proangiogenic cells,14 we addressed the function of Jmjd3 in cardiovascular lineage differentiation of embryonic stem cells (ESCs).

Methods

A detailed description of the experimental procedure is provided in the Online Data Supplement.  The online-only Data Supplement is available with this article at   http://circres.ahajournals.org/lookup/suppl/doi:10.1161/CIRCRESAHA. 113.302035/-/DC1.

Cell Culture

Plasmid Construction and Stable Transfection

The full-length Jmjd3, the mutants, and Brachyury were cloned into pEF1 vector (Invitrogen). The linearized plasmids were transfected in Jmjd3−/− ESCs using the Amaxa nucleofection system (Lonza).

Chromatin Immunoprecipitation

Nonstandard Abbreviations and Acronyms

EB                       embryoid body

ESC                    embryonic stem cell

WI                        wild type

Results

Jmjd3 knockout ESCs were generated by 2 rounds of gene targeting (Online Figure IA and IB). We obtained 7 Jmjd3−/− ESC clones, which lacked Jmjd3 mRNA and protein expression. All of the clones showed slightly increased global H3K27me3, but the expression of pluripotency genes, the morphology, the growth kinetic, and survival was indistinguishable from wild-type (WT) ESCs (Figure 1A–1C; Online Figure IC–IF). No significant changes of repressive H3K27me3 marks at the promoters of pluripotency genes were detected in Jmjd3−/− compared with WT ESCs (Online Figure IH). When

  • spontaneous differentiation was induced by leukemia inhibitory factor withdrawal,
  • Jmjd3 expression increased in WT ESCs (Figure 1D).
  • EBs derived from Jmjd3−/− ESCs were slightly smaller in size compared with WT EBs (Figure 1E).

mRNA expression profiling of Jmjd3−/− and WT ESC clones at day 4 after induction of differentiation showed

  • a distinct expression pattern of lineage-specific genes (Online Figure IIA).

Gene ontology functional analyses revealed a significant repression of genes that are involved in mesoderm development (Figure 1F; Online Figure IIB). Moreover,

  • repressed gene sets in Jmjd3−/− EBs were shown to be related to cardiac and vascular development,
  • consistent with impaired mesoderm differentiation (Figure 1F; Online Figure IIB).

Validation of the microarray results showed a similar reduction of pluripotency gene expression after leukemia inhibitory factor withdrawal in Jmjd3−/− compared with WT ESCs (Figure 1G). However, depletion of Jmjd3 substantially compromised the induction of mesodermal genes (Figure 1G). Especially, the pan-mesoderm marker, Brachyury, and the early mesoendoderm marker, Mixl1, were profoundly increased at day 4 of differentiation in WT ESCs, but not in Jmjd3−/− ESCs (Figure 1G). Moreover, the mesoendodermal marker, Eomes, and endodermal markers, such as Sox17 and FoxA2, were significantly suppressed, which is consistent with a very recent study showing that Jmjd3 is required for endoderm differentiation.19 Ectodermal markers were not significantly changed in Jmjd3−/− ESCs when using the spontaneous differentiation protocol (Figure 1G).

Because Jmjd3−/− ESCs showed a prominent inhibition of mesodermal markers after leukemia inhibitory factor withdrawal, we next questioned whether this phenotype can also be observed when directing differentiation of mesoderm using 2 different protocols. Consistent with our findings,

  • Jmjd3−/− ESCs showed a reduced expression of mesodermal marker genes when using the protocol for mesoderm differentiation described by Gadue et al20 (data not shown). Moreover,
  • mesoderm differentiation was significantly suppressed when Jmjd3−/− ESCs were cultured on OP9 stromal cells, which support mesodermal differentiation21 (Figure 2A).

Whereas WT ESCs showed the typical time-dependent increase in Brachyury+ cells, Jmjd3−/− ESCs generated significantly less Brachyury+ mesodermal cells (Figure 2B). Moreover, fluorescence activated cell sorting analysis revealed that fetal liver kinase (Flk)1+ vascular endothelial-cadherin−mesodermal cells were generated in WT ESCs but were reduced when Jmjd3−/− ESCs were used (Figure 2C). Interestingly, the formation of vascular endothelial-cadherin+ Flk+ cells was also significantly reduced by 96±1% and 88±3% in the 2 Jmjd3−/− ESC clones compared with WT ESCs (P<0.01), prompting us to explore the role of Jmjd3 in vascular differentiation further.
Endothelial differentiation was induced by a cytokine cocktail18 and was associated with a significant upregulation of Jmjd3 expression (Online Figure IIIA). Jmjd3−/− ESCs showed a marked reduction of endothelial differentiation as evidenced by

  • significantly reduced mRNA levels of the endothelial marker vascular endothelial-cadherin and endothelial-specific receptor tyrosine kinase Tie2 (Figure 3A).
  • The formation of endothelial marker expressing vascular structures after induction of endothelial differentiation was abolished in Jmjd3−/− ESCs (Figure 3B; Online Figure IIIB).
  • The impaired endothelial differentiation of Jmjd3−/− cells was partially rescued by the overexpression of Brachyury (Online Figure IIIC and IIID), suggesting that the inhibition of mesoderm formation, at least in part, contributes to the impaired endothelial commitment.

Because genes involved in heart development and morphogenesis were significantly downregulated in Jmjd3−/− ESCs on differentiation (Figure 1F; Online Figure II), we additionally determined the capacity of Jmjd3−/− ESCs to generate cardio-myocytes by inducing cardiac differentiation.17

  • Expression of cardiac progenitor cell markers, Mesp1 and Pdgfra, was inhibited in Jmjd3−/− ESCs compared with WT ESCs (Figure 3C).

Moreover, after plating on gelatin-coated dishes,

  • the Jmjd3−/− ESCs showed an impaired formation of EBs and
  • only 20% of EBs were contracting (Figure 3D).

Consistently, expression of the cardiac transcription factor Mef2c, the marker of working myocardium Nppa, and cardiac structural proteins TnT2 and α-myosin heavy chain were downregulated in Jmjd3−/− ESCs (Figure 3E and 3F; Online Figure IIIE).

  • Next, we addressed whether the impaired mesoderm differentiation observed in Jmjd3−/− ESCs might be mediated by an increase of repressive H3K27me3 marks at the promoters of developmental regulators. Of the various promoters studied, only Brachyury and Mixl1 showed a significant augmentation of H3K27me3 marks in Jmjd3−/− ESCs on differentiation (Figure 4A; Online Figure IVA). Consistently, the recruitment of RNA polymerase II to the transcription start sites of the promoters of Brachyury and Mixl1 was also significantly reduced (Online Figure IVC). In addition, Jmjd3 deficiency repressed polymerase II recruitment to the Flk1 and Mesp1 promoter but the inactivation of these promoters was not associated with changes in H3K27me3 marks (Figure IVA and IVC). These data were confirmed using protocols
  • that induce mesoderm differentiation by addition of Wnt3a (data not shown).20 Under these conditions,
  • Jmjd3−/− ESCs showed a 1.81±0.23-fold (P<0.05) enrichment of H3K27me3 marks at the Brachyury promoter compared with WT ESCs.

To determine whether the demethylase activity of Jmjd3 controls Brachyury expression by reducing repressive H3K27me3 marks during differentiation, we overexpressed full-length Jmjd3, the carboxyl-terminal part, including the JmjC-domain (cJmjd3: amino acids, 1141–1641), and a carboxyl-terminal mutant construct, which includes a point mutation (cJmjd3H1388A) to inactivate demethylase activity. Overexpression of full-length Jmjd3 and the carboxyl-terminal part of Jmjd3 in Jmjd3−/− ESCs partially rescued the expression of Brachyury on differentiation (Figure 4B and 4C). Howver, the inactive carboxyl-terminal part of Jmjd3 failed to rescue the impaired Brachyury expression in Jmjd3−/− ESCs (Figure 4C), suggesting that

  • the demethylase activity of Jmjd3 is required for the activation of the Brachyury promoter.

Because canonical Wnt signaling regulates the expression of Brachyury during development22,23 and Wnt/B-catenin–dependent genes were suppressed in Jmjd3−/− EBs compared with WT EBs (Online Figure V), we further explored whether Jmjd3 might interact with B-catenin signaling. Indeed,

  • B-catenin recruitment to the Brachyury promoter was significantly suppressed in Jmjd3−/− ESCs (Figure 4D) and
  • was rescued by Jmjd3 overexpression (Figure 4E).

Similar results were obtained when using the protocol for direct mesoderm differentiation described by Gadue et al20 (data not shown). To determine whether Jmjd3 might interact with B-catenin, we performed coimmunoprecipitation studies and showed that

  • Jmjd3 interacts with B-catenin in human embryonic kidney 293 cell and differentiated EBs (Figure 4F; Online Figure VI).

To assess a direct effect of Jmjd3 on B-catenin responsive promoter activity, we used a luciferase reporter assay. Coexpression of lymphoid enhancer binding factor 1 and the constitutive active form of B-catenin harboring a nuclear localization signal resulted in the activation of lymphoid enhancer binding factor 1 luciferase reporter activity in WT ESCs, but

  • this transcriptional activation was markedly impaired in Jmjd3−/− ESCs (Figure 4G).

Discussion

The data of the present study demonstrate that

  • deletion of Jmjd3 in ESCs does not affect self-renewal but
  • significantly impairs the formation of mesoderm on induction of differentiation.

The findings that Jmjd3 is not required for ESC maintenance are consistent with the dispensability of the Polycomb complex and the related demethylase UTX for self-renewal.1

  • The requirement of Jmjd3 for mesoderm differentiation was shown in spontaneous differentiation, as well as
  • when more specifically inducing mesoderm differentiation by the OP9 coculture system or under serum-free conditions followed by Wnt3a stimulation.
  • Jmjd3 deficiency profoundly suppressed the expression of Brachury, which is essential for mesoderm differentiation.

In the absence of Jmjd3,

  • repressive H3K27me3 marks at the Brachyury promoter are significantly increased, and
  • the recruitment of B-catenin, which is a prerequisite for Wnt-induced mesoderm differentiation, is impaired.
  • In addition, Jmjd3 is interacting with B-catenin and is contributing to B-catenin– dependent promoter activation.

This is consistent with the recent findings that cofactors can form a complex with B-catenin/ lymphoid enhancer binding factor 1

  • at Tcf/lymphoid enhancer binding factor 1 binding sites
  • at B-catenin–dependent promoters and
  • synergize with canonical Wnt signaling.24

Interestingly, a demethylase-independent regulation of B-catenin–dependent gene expression was recently described for UTX.25 However, our data provide evidence that

  • Brachyury expression in Jmjd3−/− ESCs is only rescued by catalytically active Jmjd3, which has maintained the demethylase activity.

On the basis of these findings, we propose a model in which Jmjd3 is recruited to the Brachury promoter to remove repressive H3K27me3 marks and on Wnt stimulation additionally promotes B-catenin–dependent promoter activation (Figure 4H). Such a model is similar to the recently described function of Jmjd3 in endoderm differentiation, whereby Jmjd3 associates with Tbx3 and is recruited to the poised promoter of Eomes, to mediate chromatin remodeling allowing subsequent induction of endoderm differentiation induced by activin A.19 The present study additionally demonstrates that

  • Jmjd3 contributes to endothelial and cardiac differentiation.
  • endothelial differentiation was profoundly impaired,

a finding that is consistent with previous findings in adult progenitor cells, showing a high H3K27me3 at endothelial genes.14 The relatively modest inhibition of cardiomyocyte differentiation in Jmjd3−/−  ESCs may be, in part, explained by a compensatory effect of UTX which was shown to regulate cardiac development.26 Together, our study provides first evidence for the regulation of B-catenin–dependent Wnt target genes by Jmjd3 during differentiation of ESCs.  However, the in vivo relevance of the findings is still unclear. The Jmjd3−/− mice that we have generated out of the ESCs, used in the present study, showed embryonic lethality before E6.5, suggesting a crucial role of Jmjd3 in early embryonic development.

Conclusions

Novelty and Significance

What Is Known?

•            Cell fate decisions require well-controlled changes in gene expression that are tightly controlled by epigenetic modulators.

•            The post-transcriptional modifications of histone proteins epigeneti-cally regulate gene expression.

•            Trimethylation of lysine 27 at histone K3 (H3K27me3) silences gene expression and can be reversed by the demethylase Jmjd3.

What New Information Does This Article Contribute?

•            The histone demethylase Jmjd3 is required for mesoderm differentiation and cardiovascular lineage commitment of mouse embryonic stem cells.

•            This effect is partially mediated by a silencing of the mesodermal regulator Brachyury.

•            Ablation of Jmjd3 further reduces β-catenin recruitment to the Brachyury promoter, which interferes with Wnt signaling that is required for proper mesoderm differentiation.

The differentiation of stem cells to specific lineages requires a well-defined modulation of gene expression programs, which is often controlled by epigenetic mechanisms. Although several epi-genetically active enzymes and complexes have been described, the function of the histone demethylase Jmjd3 for cardiovascu¬lar lineage commitment was unknown. Using mouse embryonic stem cells as a model, we now show that the demethylase Jmjd3 is required for mesoderm differentiation and for the differentia¬tion of embryonic stem cells to the vascular and cardiac lineage. We further identified the mechanism and showed that ablation of Jmjd3 resulted in a silencing of the Brachyury promoter that is associated with an increase in H3K27me3 marks. In addition, Jmjd3 was shown to facilitate the recruitment of β-catenin to the Brachyury promoter, which contributes to the Wnt-dependent ac-tivation of mesoderm differentiation. Together these data describe a novel epigenetic mechanism that controls cell fate decision.

Supplemental Methods

Generation of Jmjd3 knockout ES cell lines

Mouse genomic DNA encompassing the murine Jmjd3 gene region were isolated by PCR amplification and used to generate short (1.6kb) and long (6.2kb) arms of homology. The targeting vectors were constructed by inserting a loxP site together with an FRT flanked neomycin selection cassette within the intron 5 and a single distal loxP within the intron 3. This targeting strategy results in the deletion of 600bp coding sequences encoding for the ATG methionine codon and produces a frame shift of JmjC domain existing exon 19-21 required for Jmjd3 demethylase activity. The targeting vector was electroporated in 129Sv ES cells. G418 resistant ES cell clones were screened for homologous recombination by PCR analysis and targeting was verified by Southern blot analysis. Homozygous Jmjd3lox/lox ES cells were generated by electroporation of heterozygous Jmjd3lox/+ ES cells with the same targeting vector as above except that the neomycin resistance gene was replaced by puromycin gene using the Nucleofector (Lonza). Double-allele-recombined ES cells were selected for puromycin (1.3µg/mL, Invitrogen). Correct targeting of homozygous Jmjd3lox/lox ES clones were determined by PCR. To obtain Jmjd3-/- ES cells, Jmjd3lox/lox ES cells were electroporated with Cre-recombinase plasmid vector and loss of targeting cassettes was evaluated by loss of resistance of G418 and puromycin. Correct targeting of homozygous Jmjd3-/- ES cells was determined by PCR.

Reporter gene assays

3xLEF1 reporter plasmid, LEF1 expression construct and NLS-13-catenin were kind gifts from Rudolf Grosschedl. Mouse ES cells were seeded (5×104) on gelatin coated 24-well. After 24 hours of plating, 3xLEF1 reporter plasmid, LEF1, and NLS-13-catenin plasmids were transiently transfected with FugeneHD (Promega). 13-galactosidase plasmid was co-transfected for normalization of transfection efficiency. Each group was transfected in triplicates. 48 hours after transfection, cells were harvested. Cell lysis and luciferase assay were performed following the protocol of Luciferase Reporter Assay System (Promega). 13-galactosidase assays were performed using CPRG (Sigma) as substrate and the absorbance at 600nm was measured. Luciferase activity was normalized to 13-galactosidase activity.

Manuscript References

  1. Shen, X. et al. EZH1 mediates methylation on histone H3 lysine 27 and complements EZH2 in maintaining stem cell identity and executing pluripotency. Mol Cell 32, 491-502 (2008).
  2. Sargent, C.Y., Berguig, G.Y. & McDevitt, T.C. Cardiomyogenic differentiation of embryoid bodies is promoted by rotary orbital suspension culture. Tissue engineering. Part A 15, 331­342 (2009).
  3. Gadue, P., Huber, T.L., Paddison, P.J. & Keller, G.M. Wnt and TGF-beta signaling are required for the induction of an in vitro model of primitive streak formation using embryonic stem cells. Proc Natl Acad Sci U S A 103, 16806-16811 (2006).
  4. Ohtani, K. et al. Epigenetic regulation of endothelial lineage committed genes in pro-angiogenic hematopoietic and endothelial progenitor cells. Circ Res 109, 1219-1229 (2011).
  5. Yamaguchi, T.P., Takada, S., Yoshikawa, Y., Wu, N. & McMahon, A.P. T (Brachyury) is a direct target of Wnt3a during paraxial mesoderm specification. Genes Dev 13, 3185-3190 (1999).

Selected Figures   (Figure may not be shown)

Figure 1. Aberrant differentiation of Jmjd3−/− embryonic stem cells (ESCs). A, Quantitative polymerase chain reaction analysis of Jmjd3 in wild-type (WT) and Jmjd3−/− ESCs. B, Western blot analysis of Jmjd3 and Histone marks in WT and Jmjd3−/− ESCs. Histone H3 is used as a loading control. Quantification is shown in the right (n=3–5). C, Top, Morphology of WT and Jmjd3−/− ESCs on feeder cells. Bottom, Alkaline phosphatase staining of undifferentiated WT and Jmjd3−/− ESCs. D, Western blot analysis of Jmjd3 and Oct4 in WT ESCs during differentiation. α-Tubulin is used as a loading control. E, Bright field image of embryoid bodies at day 5. Scale bar, 200 μm. F, Gene ontology analysis for >2-fold repressed genes in Jmjd3−/− ESCs compared with WT ESCs 4 days after differentiation. The most highly represented categories are presented with ontology terms on the y-axis and P values for the significance of enrichment are shown on the x-axis. G, Gene expression changes of pluripotency and lineage-specific markers in WT and Jmjd3−/− ESCs after spontaneous differentiation by leukemia inhibitory factor withdrawal (n=4). Flk indicates fetal liver kinase.

Figure 2. Jmjd3−/− embryonic stem cells (ESCs) show an impaired ability to differentiate into mesoderm. A, Schematic illustration of the experimental protocol. Differentiation of ESCs (wild-type [WT] and 2 Jmjd3−/− ESCs clones) on OP9 feeder cells was analyzed. B, Left, Representative fluorescence activated cell sorting (FACS) plots showing Brachyury expression of ESC-derived cells. Right, Quantification of FACS analyses (n=3). C, Left, Representative FACS plots showing fetal liver kinase 1 (Flk1) and vascular endothelial-cadherin expression on ESC-derived cells. Right, Quantification of FACS analyses in Flk1+ cells (n=3).

Figure 3. Jmjd3 is required for embryonic stem cells (ESCs) differentiation to the endothelial and cardiac lineage. A, mRNA expression of endothelial markers at day 7 of endothelial differentiation (n=3). B, Platelet endothelial cell adhesion molecule (Pecam)-1 staining of wild-type (WT) and Jmjd3−/− ESCs at day 8 of endothelial differentiation. Phalloidin is used to stain F-actin. Nuclei are stained with 4′,6-diamidino-2-phenylindole (blue). Scale bar, 20 pm. C, Gene expression of cardiac progenitor markers at day 3 of cardiac differentiation. D, Number of beating embryoid bodies (EBs) at day 10 of cardiac differentiation (n=8). E, Gene expression of cardiac markers at day 7 of cardiac differentiation (n=6). F, α-Myosin heavy chain staining of WT and Jmjd3−/− ESCs at day 9 of cardiac differentiation. Nuclei are stained with Hoechst (blue). Scale bar, 20 pm. *P<0.05, **P<0.01, and ***P<0.001

Online Figure I. Generation and characterization of Jmjd3–‘– ESCs  (A) Targeting strategy to generate Jmjd3 mutant ESCs by homologous recombination. Primers used for PCR are shown. (B) Genotyping of Jmjd3–‘– ESCs by using 2 different primers. (C) Oct4 and Nanog staining in WT and Jmjd3−’− ESCs. Scale bar indicates 10µm. (D) Expression of Oct4 and Nanog in WT and Jmjd3–‘– ESCs. Data are presented as fold changes compared with day 0 WT ESCs. N=6. (E) Tunel staining (green) of WT and Jmjd3–‘– ESCs. Nuclei are stained with Hoechst (blue). Scale bar indicates 20µm. (F) Growth curves of WT and Jmjd3–‘– ESCs. N=6-8. (G) H3K27me3 staining in WT and Jmjd3–‘– ESCs. Nuclei are stained with Hoechst (blue). Scale bar indicates 20µm. (H) ChIP assay of undifferentiated WT and Jmjd3–‘– ESCs for H3K27me3. ChIP enrichments are normalized to Histone H3 density and represented as fold change relative to WT. N=3. Data represent mean ± SEM

Online Figure II. Jmjd3–‘– ESCs show an impaired mesoderm differentiation. (A) Microarray gene expression heat map depicting expression of representative pluripotency and lineage markers 4 days after differentiation in Jmjd3–‘– ESCs versus WI ESCs. Coloring illustrates log2 fold changes between Jmjd3–‘– ESCs and WI ESCs. Green and red colors represent down-regulation and up-regulation, respectively. (B) Gene ontology analysis for more than 2-fold altered genes in Jmjd3–‘– ESCs compared to WI ESCs 4 days after differentiation. Red and green colors represent down-regulation and up-regulation, respectively.

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