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Meet the 2016 MacArthur Fellows

September 23, 2016 by David Orchard-Webb, PhD

Meet the 2016 MacArthur Fellows

Reporter: David Orchard-Webb

Article ID #214: Meet the 2016 MacArthur Fellows. Published on 9/23/2016

WordCloud Image Produced by Adam Tubman

“While our communities, our nation, and our world face both historic and emerging challenges, these 23 extraordinary individuals give us ample reason for hope. They are breaking new ground in areas of public concern, in the arts, and in the sciences, often in unexpected ways. Their creativity, dedication, and impact inspire us all.”


—MacArthur President Julia Stasch

Meet the 2016 fellows here: https://www.macfound.org/fellows/class/class-2016/

Posted in Interviews with Scientific Leaders | Leave a Comment »

UPDATED Postmarketing Safety or Effectiveness Data Needed: The 2013 paper was funded by the firm Sarepta Therapeutics, sellers of eteplirsen, a surge in its shares seen after the approval. Eteplirsen will cost patients around $300,000 a year.

September 22, 2016 by 2012pharmaceutical

UPDATED Postmarketing Safety or Effectiveness Data Needed: The 2013 paper was funded by the firm Sarepta Therapeutics, sellers of eteplirsen, a surge in its shares seen after the approval. Eteplirsen will cost patients around $300,000 a year.

 

Curator: Aviva Lev-Ari, PhD, RN

 

UPDATED on 5/15/2023

FDA advisers narrowly vote in favor of Sarepta’s DMD gene therapy

By Max Bayer May 12, 2023 07:09pm
FDA advisers have narrowly voted in favor of Sarepta Therapeutics’ gene therapy for patients with Duchenne muscular dystrophy (DMD), a stunning decision that runs counter to regulators who expressed skepticism heading into the meeting.

Experts from the Cellular, Tissue, and Gene Therapies Advisory Committee narrowly voted 8 to 6 that the benefit-risk profile of SRP-9001 was strong enough to support accelerated approval even as questions about the gene therapy’s efficacy linger. No members of the committee abstained. SRP-9001 was developed to treat ambulatory patients with a DMD gene mutation.

Advisers were tasked with considering four discussion topics about SRP-9001 that centered on the available data on the treatment, the benefit-risk profile of administering it and the implications of allowing the company to use an ongoing phase 3 trial as a confirmatory study. There was little alignment between Sarepta and the FDA’s interpretation of the data, forcing advisers to break the tie.

“I think we owe it to the patients to help them intervene,” said committee member and consumer representative Kathleen O’Sullivan-Fortin, who voted in favor. Others expressed confidence in the safety profile of the gene therapy even amid concerns about the efficacy.

FDA shocks Sarepta, ordering AdComm for DMD gene therapy in sudden change of heart

Sarepta argued that a surrogate endpoint quantifying the expression of micro-dystrophin protein in patients’ muscles was an adequate biomarker to predict clinical benefit and grant accelerated approval. The company cited a natural history study as evidence that correcting the expression addresses the root cause of DMD. The FDA has for years been wary of the proposed biomarker, suggesting as far back as 2018 that Sarepta reconsider the endpoint.

Some of the concerns raised by both committee members and the FDA were aimed at Sarepta’s manufacturing of SRP-9001, which changed between the second and third clinical trials. The new process, which uses a lower percentage of full capsid, is what Sarepta is using in its accelerated approval application. The agency cautioned this could reduce efficacy and increase the risk of side effects. It also wasn’t the process used in Sarepta’s most detailed clinical trial, part of Study 102.

That trial was the only randomized, placebo-controlled study of the gene therapy conducted to date. The trial found that there was not a statistically significant change in functional motor ability between treated patients and placebo as assessed by a common functional rating scale for DMD. Sarepta argued that treated patients had a numerically greater score at all time points, but the FDA concluded those figures were within the bounds of uncertainty, “which is also demonstrated by the lack of even a trend toward statistical significance.”

Sarepta pointed to a post hoc analysis to show that the results varied by age, however, with treated patients ages 4 to 5 showing improvement compared to placebo while 6- to 7-year-old patients had worse function scores than placebo. Sarepta used external control groups from natural history studies to prove a larger benefit between treated and untreated patients across the company’s studies to date. But the FDA said Friday that the natural history data are “challenging to interpret.”

https://www.fiercebiotech.com/biotech/fda-sarepta-advisors-meeting-dmd-gene-therapy-vote-against

UPDATED on 8/18/2019

Sarepta Duchenne drug rejected by FDA in surprise setback

 

Dive Brief:

  • In an unexpected decision, the Food and Drug Administration rejected Sarepta Therapeutics’ experimental drug for Duchenne muscular dystrophy, issuing on Monday a Complete Response Letter to the rare disease biotech.
  • According to Sarepta, the agency cited in its refusal infection risk tied to the drug’s delivery as well as preclinical signs of kidney toxicity. Called Vyondys 53, the medicine is designed for roughly 8% of Duchenne patients with a specific genetic mutation.
  • Shares in Cambridge, Mass.-based Sarepta fell sharply in post-market trading. Approval of the drug was widely anticipated, making the rejection a setback in Sarepta’s ambitions to treat a wider pool of Duchenne patients.

SOURCE

https://www.biopharmadive.com/news/sarepta-surprise-fda-rejection-duchenne-vyondys-53/561200/

 

On September 19, the FDA okayed eteplirsen to treat Duchenne muscular dystrophy (DMD), a rare genetic disorder that results in muscle degeneration and premature death. Several of its top officials disagreed with the drug’s approval, questioning how beneficial it will be for patients, as Forbes, MedPage Today and others reported.

http://retractionwatch.com/2016/09/21/amid-controversial-sarepta-approval-decision-fda-head-calls-for-key-study-retraction/

Factors at play for FDA Approval of eteplirsen

  1. the help of the families of young boys with Duchenne muscular dystrophy, emotional scenes from these families who have campaigned for so long
  2. an executive team from Sarepta who wouldn’t give up,

Ed Kaye, Sarepta, CEO – EK: It’s all about resilience. One of the things we’ve had is a group of people of like minds and anytime one of us gets down, somebody else is there to pick you up. One of the things we’ve always done is: Every time we’ve felt sorry for ourselves, we just need to think about those patients and what they go through. Our struggles in comparison very quickly become meaningless. You end up saying to yourself: What am I complaining about? Quit whining; get up and do your job.

and

3. an emerging new philosophy from some within the FDA, eteplirsen, now Exondys 51, was approved in patients with a confirmed mutation of the dystrophin gene amenable to exon 51 skipping.

http://www.fiercebiotech.com/biotech/sarepta-ceo-ed-kaye-fda-courage-nice-and-resilience?utm_medium=nl&utm_source=internal&mrkid=993697&mkt_tok=eyJpIjoiTXpBeU56aGpNREV3T1RZMiIsInQiOiJIM2poTkVOQ0N6YmxaenVHZDM1RlVvbTFmRkdwZGdxQ0pmYXNVOG5PKzRyenFXTkRMV0dcL3l0bVBPNkJ2NFV3Rnc3bWVFVnUwMCs3YVhWeVhvRkkrUU5FMFJ1RndSQTlHWFRnQmFTbUo3ODg9In0%3D

9/19/2016

FDA grants accelerated approval to first drug for Duchenne muscular dystrophy

The accelerated approval of Exondys 51 is based on the surrogate endpoint of dystrophin increase in skeletal muscle observed in some Exondys 51-treated patients. The FDA has concluded that the data submitted by the applicant demonstrated an increase in dystrophin production that is reasonably likely to predict clinical benefit in some patients with DMD who have a confirmed mutation of the dystrophin gene amenable to exon 51 skipping. A clinical benefit of Exondys 51, including improved motor function, has not been established. In making this decision, the FDA considered the potential risks associated with the drug, the life-threatening and debilitating nature of the disease for these children and the lack of available therapy.

The FDA granted Exondys 51 fast track designation, which is a designation to facilitate the development and expedite the review of drugs that are intended to treat serious conditions and that demonstrate the potential to address an unmet medical need. It was also granted priority review and orphan drug designation. Priority review status is granted to applications for drugs that, if approved, would be a significant improvement in safety or effectiveness in the treatment of a serious condition. Orphan drug designation provides incentives such as clinical trial tax credits, user fee waiver and eligibility for orphan drug exclusivity to assist and encourage the development of drugs for rare diseases.

SOURCE

http://www.fda.gov/NewsEvents/Newsroom/PressAnnouncements/ucm521263.htm

The viability of this drug approval depends  on “to be gathered” Postmarketing safety or effectiveness data, aka follow-up confirmatory trials.

Sarepta CEO Ed Kaye on FDA courage, NICE and resilience

BA: When it comes to flexibility, however, the FDA will likely not be flexible if your drug doesn’t prove the desired efficacy in your longer term postmarketing studies. If at the end of this period your drug doesn’t come through, how easy will it be for you to take this off the market? I don’t think anyone, including the FDA, wants a repeat of what happened in 2011 when Roche saw its breast cancer license for Avastin, which had been approved under an accelerated review, pulled after not being safe or effective enough in the follow-up confirmatory trials. But you face this as a possible scenario.

EK: That’s true, but one of the things we’re trying to do to mitigate that is to obviously, with our ongoing studies, prove the efficacy that the FDA wants to see. And you know, if there is a problem with one study then we’d hope to have other data that are supportive. The other thing we’re doing of course is developing that next-generation chemistry in DMD that could prove more effective, so we could certainly consider using that next-gen chemistry to take our work forward and try and make it better.

We have a lot of shots on goal to make sure we can continue to supply a product for these boys, but there is always a risk. If we can’t show efficacy in the way the FDA wants, then yes they have the option to take it off the market.

http://www.fiercebiotech.com/biotech/sarepta-ceo-ed-kaye-fda-courage-nice-and-resilience?utm_medium=nl&utm_source=internal&mrkid=993697&mkt_tok=eyJpIjoiTXpBeU56aGpNREV3T1RZMiIsInQiOiJIM2poTkVOQ0N6YmxaenVHZDM1RlVvbTFmRkdwZGdxQ0pmYXNVOG5PKzRyenFXTkRMV0dcL3l0bVBPNkJ2NFV3Rnc3bWVFVnUwMCs3YVhWeVhvRkkrUU5FMFJ1RndSQTlHWFRnQmFTbUo3ODg9In0%3D

Need for follow-up confirmatory trials remains outstanding

FDA’s Postmarketing Surveillance Programs

http://www.fda.gov/Drugs/GuidanceComplianceRegulatoryInformation/Surveillance/ucm090385.htm

FDA’s Regulations and Policies and Procedures for Postmarketing Surveillance Programs

http://www.fda.gov/Drugs/GuidanceComplianceRegulatoryInformation/Surveillance/ucm090394.htm

 

Positions on Sarepta’s eteplirsen Scientific Approach

Gene Editing for Exon 51: Why CRISPR Snipping might be better than Exon Skipping for DMD

Reporter: Aviva Lev-Ari, PhD, RN

https://pharmaceuticalintelligence.com/2016/01/23/gene-editing-for-exon-51-why-crispr-snipping-might-be-better-than-exon-skipping-for-dmd/

 

QUOTE START

Retraction Watch

Tracking retractions as a window into the scientific process

Amid controversial Sarepta approval decision, FDA head calls for key study retraction

with one comment

FDAThe head of the U.S. Food and Drug Administration (FDA) has called for the retraction of a study about a drug that the agency itself approved earlier this week, despite senior staff opposing the approval.

On September 19, the FDA okayed eteplirsen to treat Duchenne muscular dystrophy (DMD), a rare genetic disorder that results in muscle degeneration and premature death. Several of its top officials disagreed with the drug’s approval, questioning how beneficial it will be for patients, as Forbes, MedPage Today and others reported.

In a lengthy report Commissioner Robert Califf sent to senior FDA officials on September 16 — that was made public on September 19 — he called for the retraction of a 2013 study published in Annals of Neurologyfunded by the seller of eteplirsen, which showed beneficial effects of the drug in DMD patients. Califf writes inthe report:

The publication, now known to be misleading, should probably be retracted by its authors.

In a footnote in the report, Califf adds:

In view of the scientific deficiencies identified in this analysis, I believe it would be appropriate to initiate a dialogue that would lead to a formal correction or retraction (as appropriate) of the published report.

The study was not the key factor in the agency’s decision to approve the drug, according to Steve Usdin, Washington editor of the publication BioCentury; still, Usdin told Retraction Watch he is “really surprised” at the call for retraction from top FDA staff, the first he has come across in the last two decades.

The 2013 paper was funded by the firm Sarepta Therapeutics, sellers of eteplirsen, which has seen a surge in its shares after the approval. Eteplirsen will cost patients around $300,000 a year.

DMD affects around 1 in 3,600 boys due to a mutation in the gene that codes for the protein dystrophin, which is important for structural stability of muscles. Eteplirsen is the first drug to treat DMD, and was initially given a green light by Janet Woodcock, director of Center for Drug Evaluation and Research, after a split vote from the FDA’s advisory committee. Despite Califf’s issues with the literature supporting the drug’s use in DMD, he did not overturn Woodcock’s decision, and the agency approved the drug this week.

In 2014, an inspection team visited the Nationwide Children’s Hospital in Columbus, Ohio, where the research was conducted, according to the report. In the report, Ellis Unger, director of the Office of Drug Evaluation I in FDA’s Center for Drug Evaluation, notes:

We found the analytical procedures to be typical of an academic research center, seemingly appropriate for what was simply an exploratory phase 1/2 study, but not suitable for an adequate and well controlled study aimed to serve as the basis for a regulatory action. The procedures and controls that one would expect to see in support of a phase 3 registrational trial were not in evidence.

Specifically, Unger describes concerns about blinding during the experiments, and notes:

The immunohistochemistry images were only faintly stained, and had been read by a single technician using an older liquid crystal display (LCD) computer monitor in a windowed room where lighting was not controlled. (The technician had to suspend reading around mid-day, when brighter light began to fill the room and reading became impossible.)

Unger adds:

Having uncovered numerous technical and operational shortcomings in Columbus, our team worked collaboratively with the applicant to develop improved methods for a reassessment of the stored images…This re-analysis, along with the study published in 2013, provides an instructive example of an investigation with extraordinary results that could not be verified.

Luciana Borio, acting chief scientist at the FDA, is cited in the report saying:

I would be remiss if I did not note that the sponsor has exhibited serious irresponsibility by playing a role in publishing and promoting selective data during the development of this product. Not only was there a misleading published article with respect to the results of Study–which has never been retracted—but Sarepta also issued a press release relying on the misleading article and its findings…As determined by the review team, and as acknowledged by Dr. Woodcock, the article’s scientific findings—with respect to the demonstrated effect of eteplirsen on both surrogate and clinical endpoints—do not withstand proper and objective analyses of the data. Sarepta’s misleading communications led to unrealistic expectations and hope for DMD patients and their families.

Here’s how Sarepta describes the study’s findings in the press release Borio refers to:

Published study results showed that once-weekly treatment with eteplirsen resulted in a statistically significant increase from baseline in novel dystrophin, the protein that is lacking in patients with DMD. In addition, eteplirsen-treated patients evaluable on the 6-minute walk test (6MWT) demonstrated stabilization in walking ability compared to a placebo/delayed-treatment cohort. Eteplirsen was well tolerated in the study with no clinically significant treatment-related adverse events. These data will form the basis of a New Drug Application (NDA) to the U.S. Food and Drug Administration (FDA) for eteplirsen planned for the first half of 2014.

However, Usdin noted that the drug’s approval and the study are two independent events, adding that the 2013 study just “got the ball rolling” for eteplirsen, and the FDA conducted many of its own experiments analyses, as detailed in the newly released report.

Jerry Mendell, the corresponding author of the study (which has so far been cited 118 times, according to Thomson Reuters Web of Science) from Ohio State University in Columbus, told us the allegations were “unfounded” and said the data are “valid.” Therefore, he added, he will not be approaching the journal for a retraction, noting that the FDA asked him hundreds of questions about the paper and audited the trials.

Clifford Saper, the editor-in-chief of Annals of Neurology from the Beth Israel Deaconess Medical Center (which is part of Harvard Medical School), said in an email:

It takes more than a call by a politician for retraction of a paper. It takes actual evidence.

He added:

If the FDA commissioner has, or knows of someone who has, evidence for an error in a paper published in Annals of Neurology, I encourage him to send that evidence to me and a copy to the authors of the article, for their reply. At that point we will engage in a scientific review of the evidence and make appropriate responses.

Linda Lowes, sixth author of the present study, is the last author of a 2016 study in Physical Therapy that was retracted months after publication. Its notice reads:

This article has been retracted by the author due to unintentional deviations in the use of the described modified technique to assess plagiocephaly in the study participants, such that the use of the modified technique cannot be defended for the stated purpose in this population at this time.

Califf was a cardiologist at Duke University during the high-profile scandal of researcher Anil Potti at Duke, which led to more than 10 retractions, settled lawsuits, and medical board reprimands. In 2015, he told TheTriangle Business Journal:

I wish I had gotten myself more involved earlier…There were systems that were not adequate, as we stated. … That was a tough one, I think, for the whole institution.

We’ve contacted the FDA for comment, and will update the post with anything else we learn.

END QUOTE

Correction 9/21/16 10:44 p.m. eastern: When originally published, this post incorrectly reported that Califf was part of an inspection team that visited the Nationwide Children’s Hospital in Ohio, and attributed quotes from Ellis Unger to Califf. We have made appropriate corrections, and apologize for the error.

Like Retraction Watch? Consider making a tax-deductible contribution to support our growth. You can also follow us on Twitter, like us on Facebook, add us to your RSS reader, sign up on our homepage for an email every time there’s a new post, or subscribe to our daily digest. Click here to review our Comments Policy. For a sneak peek at what we’re working on, click here.

SOURCE

http://retractionwatch.com/2016/09/21/amid-controversial-sarepta-approval-decision-fda-head-calls-for-key-study-retraction/

Related Resources on FDA’s Policies on Drugs:

Posted in Exosomes, FDA, FDA Regulatory Affairs, Genome Biology, Genomics Pharmacy, Innovations in Neurophysiology & Neuropsychology, Pharmaceutical Discovery, Pharmacogenomics | Leave a Comment »

What AI Researchers Say About When Superintelligence will Arrive

September 22, 2016 by 2012pharmaceutical

Posted in Uncategorized | Leave a Comment »

LIVE 9/21 3:20PM to 6:40PM KINASE INHIBITORS FOR CANCER IMMUNOTHERAPY COMBINATIONS & KINASE INHIBITORS FOR AUTOIMMUNE AND INFLAMMATORY DISEASES at CHI’s 14th Discovery On Target, 9/19 – 9/22/2016, Westin Boston Waterfront, Boston

September 21, 2016 by 2012pharmaceutical

LIVE 9/21 3:20PM to 6:40PM KINASE INHIBITORS FOR CANCER IMMUNOTHERAPY COMBINATIONS & KINASE INHIBITORS FOR AUTOIMMUNE AND INFLAMMATORY DISEASES at CHI’s 14th  Discovery On Target, 9/19 – 9/22/2016, Westin Boston Waterfront, Boston

http://www.discoveryontarget.com/

http://www.discoveryontarget.com/crispr-therapies/

Leaders in Pharmaceutical Business Intelligence (LPBI) Group is a

Media Partner of CHI for CHI’s 14th Annual Discovery on Target taking place September 19 – 22, 2016 in Boston.

In Attendance, streaming LIVE using Social Media

Aviva Lev-Ari, PhD, RN

Editor-in-Chief

http://pharmaceuticalintelligence.com

#BostonDOT16

@BostonDOT

 

KINASE INHIBITORS FOR CANCER IMMUNOTHERAPY COMBINATIONS

3:20 Chairperson’s Opening Remarks

Guido J.R. Zaman, Ph.D., Managing Director & Head of Biology, Netherlands Translational Research Center B.V. (NTRC)

3:25 FEATURED PRESENTATION: Inhibition of PI3K and Tubulin

Doriano_Fabbro

Doriano Fabbro, Ph.D., CSO, PIQUR Therapeutics

The PI3K signaling pathway is frequently activated in tumors. PQR309 is a selective dual inhibitor of PI3K and mTOR (currently in Phase I) in cancer patients. The preclinical pharmacology and toxicology of PQR309 is presented, including its activity in lymphoma preclinical models. In addition, we elucidate structural factors defining the PI3K inhibitory activity and tubulin-binding of PQR309 derivatives.

  • PQR309 & GDC0941 arrest cells i G1/S (typical for PI3K/mTOR Inhibitor)
  • What drives Antiproliferative Activity of BKM120: PI3K or MT or both?
  • BKM120 Binds to beta-Tubulin/alpha -Tubulin Interfere
  • T2R-TTL complex
  • Orientation of BKM120 in PI3K
  • PQR309 – is a brain penetrating, PK and BAV by PO, good metabolic stability
  • PQR309 ANti-proliferative in Lymphoma
  • Clinical efficacy – Now in Phase II

4:05 Design and Development of a Novel PI3K-p110β/δ Inhibitor, KA2237 with Combined Tumor Immunotherapeutic, Growth Inhibition and Anti-Metastatic Activity

Stephen_Shuttleworth

Stephen Shuttleworth, Ph.D., FRSC, CChem, CSO, Karus Therapeutics Ltd.

The design and development of KA2237, a novel and selective inhibitor of PI3K-p110β/δ, will be described. This molecule has clinical potential in the treatment of solid and hematological malignancies, through its direct inhibition of tumor growth and metastatic spread, and through immunotherapeutic mechanisms. Phase I studies for KA2237 are scheduled to commence in Q2 2016 at the MD Anderson Cancer Center.

  • Design & Development of Novel, Oral, selective PI3K enzyme family: CLass I,II, III, IV based upon:
  • Class I IA IB
  • KA2237: DUal PI3K – p110beta/delta-selective inhibitor: CTL, Treg, p1 106 T sell response
  • Molecular signature in the tumor
  • WT p110delta, WT 1 10beta+, Mutant p1 10Beta+, PTEN-null, Ibrutinib-resistance, Growth inhibition; suppression of metastesis (p110beta
  • small molecule combination agents: potential aided by selectivity over p110
  • KA2237: clinical Pi3K-p110beta/delta Inhibitor- ATP -comtetitive
  • Doxorubicin -cytotoxic control
  • KA2237 superior activity to Idelasib
  • KA2237 – suppression of micro-metastasis in 4T1 synergenic model
  • Tumor Growth inhibition Pre-Surgery
  • Tumor Re-Growth Inhibition Post-Surgery
  • metastasis post surgery
  • Tumor-free mice post-surgery
  • CHemistry: IHC -pAKT; IHC – FOxp3+
  • KA2237 inhibits HGF-stimulated 4T1 tumor
  • 2004 – Preclinical develpemnt PI3K is reported
  • 2006 First PI#K is enter Clinical Trials
  • Targeting p1110Beta (PIKeCB) mutations in cancer with KA2237
  • DIscovery of the mutations lead drug discovery
  • KA@@#&: Potential in treatment of B-Cell Lymphom AS IN TARGETING IBRUTINIB RESISTENCE
  • GROWTH INHIBITION IN HEMATOLOGICAL CANCERS TUMOE CELL LINE PANEL
  • KA2237 – differentiated from competing Pi3K is Superior efficacy cf. p110delta
  • Combination: Not histone deacetylase but a tubulin deacetylase – Hsp90 ans Hsp70
  • T cell exhausion: Tumor growth inhibition vs Suppression of lung metastasis
  • Tumor BiologyRationale vs Clinical Agents
  • Oncogenic mutants, solid tumor supression magrophage, combination PD-1, CTLA$
  • FDA -approved kinase inhibitors

Summary

  1. phase I clinical study commenced in pathients with B cell Lymphoma
  2. Potential for treatment of solid and hematological malignancies

4:35 InCELL Pulse: A Novel Cellular Target Engagement Assay Platform for Drug Discovery

Treiber_Daniel

Daniel Treiber, Ph.D., Vice President, KINOMEscan, DiscoverX Corporation

InCELL Pulse is a quantitative and rapid method for measuring cellular target engagement potencies for small molecule inhibitors. InCELL Pulse capitalizes on two novel DiscoverX technologies, Enzyme Fragment Complementation (EFC) and Pulse Denaturation, which overcome the limitations of related target engagement methods. Examples across multiple target classes will be described.

  • InCELL Pulse – cellular Target ENgagement Assays
  • cellular thermal stabilization-based approach
  • simple, rapid and generig cellular alternative to CETSa
  • Thermal melting Curves vs Isothermal Inhibitor EC50 curves
  • Pulse Denaturation compound binding, or not binding
  • ABL1 Tyrosine Kinase – dose response curve – allosteric Inhibitor
  • MTH1 Hydrolase: InCELL Pulseassay validated for multiple substrate-competitive inhibitors
  • Validated InCELL Pulse Assays for Diverse Kinases
  • Kinase targets; BRAF, MEC1

Summary

  1. validation across proteins

TTP Labtech4:50 Potential Application of Fluorescence Lifetime Assays to Enable Robust, Rapid Protein Binding Assays

Wylie_Paul

Paul Wylie, Ph.D., Head, Applications, TTP Labtech

Current methods to screen protein binding interactions often have limitations due to the reliance on antibodies, but also interference from fluorescent molecules. Fluorescence lifetime has the potential to overcome these problems through directly labelled proteins and lifetime measurements that are independent of total fluorescence intensity.

  • Protein binding as a target class
  • protein-protein interactions (PPIs)
  1. FRET/HTRF
  2. FP
  3. AlphaScreen

What new in FLT?

  • long lifetime fluorophores, economical reagent platform
  • directly labelled reagents – no antibodies
  • independent of total intensity – reduced interference
  • robustness screen vs nuisance screen – caspase-3
  • productive; reduction false positives: FRET
  • protein-binding assays & FLT formats:
  1. protein – small molecule binding – CECR2
  2. protein – peptide binding: long and sholt lifetime
  3. Site-specific labelling vs Non-selective labelling
  4. Toolbox for PoC
  5. Detection reagents
  6. Further develop technology

5:05 Refreshment Break in the Exhibit Hall with Poster Viewing

 

6:40 End of Day

 

Posted in Autoimmune Inflammatory DIseases, CANCER BIOLOGY & Innovations in Cancer Therapy, Conference Coverage with Social Media, Immuno-Oncology & Genomics, Immunodiagnostics, Immunotherapy, Inflammasome, Innovation in Immunology Diagnostics, Monoclonal Immunotherapy | Leave a Comment »

LIVE 9/21 12:50 pm Plenary Keynote Program at CHI’s 14th Discovery On Target, 9/19 – 9/22/2016, Westin Boston Waterfront, Boston

September 21, 2016 by 2012pharmaceutical

LIVE 9/21 12:50 pm Plenary Keynote Program at CHI’s 14th Discovery On Target, 9/19 – 9/22/2016, Westin Boston Waterfront, Boston

http://www.discoveryontarget.com/

http://www.discoveryontarget.com/crispr-therapies/

#BostonDOT16

@BostonDOT

Leaders in Pharmaceutical Business Intelligence (LPBI) Group is a

Media Partner of CHI for CHI’s 14th AnnualDiscovery on Target taking place September 19 – 22, 2016 in Boston.

In Attendance, streaming LIVE using Social Media

Aviva Lev-Ari, PhD, RN

Editor-in-Chief

http://pharmaceuticalintelligence.com

Wednesday, September 21

12:50 pm Plenary Keynote Program

Wednesday, September 21, 2016 | 12:55 – 2:40 pm

12:55 Event Chairperson’s Opening Remarks

Cindy Crowninshield, RDN, LDN, Conference Director, Cambridge Healthtech Institute

1:00 Plenary Keynote Introduction – Enabling Epigenetic Drug Discovery through Quality Proteins and Assay Services

Ekaterina_Kuznetsova

Ekaterina Kuznetsova, Ph.D., Research Scientist, Reaction Biology Corporation

Epigenetic proteins have recently gained traction as a class of drug targets in both academia and industry. RBC is continuously expanding its epigenetic product and service line with a focus on providing (1) validated epigenetic biochemical assays, (2) epigenetic cell-based assays and (3) characterized epigenetic proteins to the drug discovery community.

  •  Epigenetics Drug Discovery – Reagents
  • Recombinant protein products
  • Bromodomains -family-wide coverage, BromoMELT -selectivity profiling – compound Profile
  • HMT substrate – histone tail peptidesnative nucleosomes
  • HIT – Assay Serives: in vitro Screening –
  • SBIR Funded Bromodomain Chimical probe Discovery projects
  • In-house libraries: BRD4-1 Screening statistics: Selectivity profiling of HIT
  • Radioisotope
  • Fluorescence, HTRF
  • Binding Interactions” Protein: Peptide; Protein: Compound
  • Custom assays – New tatgets, MOA studies
  • Profiilng: specificity
  • Assays and Proteins

1:15 Open Innovation Partnerships to Bridge the Gap from GWAS to Drug Targets

Jeffrey_Barrett

Jeffrey Barrett, D.Phil., Founding Director, OpenTargets.org; Group Leader, Wellcome Trust Sanger Institute

Public-Private Association: Publish all results for Global access

  1. Biogen/GSK
  2. European Bioinformatics Institute (EBI),
  3. Wellcome Trust Sanger Institute – Cellular, Infection, Cancer & Aging, Human genetics
  • GWAS for complex diseases
  • IBD – 200 genetic variations and risks for IBD – what genes
  • Oncology, Immunology, Neurodegeneration: Enabling resources, Genetics as tools Human cellualr experiments

Aaron_Day-Williams

Aaron Day-Williams, Ph.D., Biogen Scientific Lead, Open Targets; Associate Director and Head, Statistical Genetics, Biogen

Nearly 85% of candidate drugs that enter clinical trials fail, and many of these are found not to work only after progression to expensive late phase trials. Fewer new medicines are coming to market, and those that do are more expensive because their success must pay for themselves as well as the development costs of the failures. It is therefore essential to find analyses and experiments that can test whether modulating a particular target will achieve therapeutic benefit in a particular disease. The field of complex disease genetics has been transformed in the last ten years by genome-wide association studies, low cost genome sequencing and rapid advances in our understanding of cellular phenotypes. We will describe how human genetics and large-scale genomics can change how we approach therapeutic target validation, and how open innovation partnerships involving pharma industry scientists working closely with academics can best bring these cutting edge datasets to bear on the problem. For example, TargetValidation.org, which is open to users worldwide, in industry or academia, integrates a dozen databases for prioritizing targets in a single analysis framework enabled by new statistical techniques and disease ontologies. We will also describe our improved analysis pipeline to connect GWAS hits to causal genes.

  • Alzheimer’s Disease (AD) –>>>  antibody aducanumab reducd ABCA7  –  
  • Propagation of Inference
  • Ontology is a graph connecting information with evidence
  • AD GWAS – convert GWAS Locus to Therapeutical/Hypothesis Proposed MOA Target
  1. Biomarker association with disease
  2. SNPs
  3. Causal Variants/Causal Gene Identification: Aggregate SKAT – Posterior Probability: Neuronal integrity, Amyloid plaques, Neural brilliant

2:00 Cell-Penetrating Mini-Proteins

Gregory Verdine

Gregory L. Verdine, Ph.D., Erving Professor, Chemistry, Departments of Stem Cell and Regenerative Biology, Chemistry and Chemical Biology, and Molecular and Cellular Biology, Harvard University and Harvard Medical School

It has been estimated that as few as 10-15% of all potential targets are targetable in vivo by either biological or small molecules. To address this deficiency, we and FOG Pharmaceuticals are developing cell-penetrating mini-proteins, molecules that combine the ability of proteins to target large flat surfaces, with the ability of small molecules to penetrate cells. Progress on the development of cell-penetrating mini-proteins will be reviewed in this talk.

  • Cell-Penetrating Miniproteins – Biologics vs small molecules
  • 10% – Biologics – limited to targets outside the cell
  • 10% – Small molecules – limited targets with hydrophobic pockets – contact surface areas
  • Undrugable targets >80% and Drugable 20%
  • 80%-90% of human proteins cannot be targeted by established modalities
  • Biotech $250Bil and
  • Big Pharma $750Bil
  • Emerging of TARGETING SCIENCE – FILING the GAP enabling advancement:
  1. Gene Therapy
  2. pharmacogenomics
  3. CRISPR-directed
  4. Adeno Viruses – anti vescicule trafficing
  5. ADC
  6. antisense
  7. Intracellular
  8. Cell-Penetrating Mini-Proteins combine the synthetic
  9. Stripping DOwn a Protein to its Business End: Interaction domain come to contact with bioactive: ligand and interaction unfolding  – low binding affinity
  10. The all-hydrocarbon alpha helix stapling system – enhancement of alpha-helicity, proteolytic stability, serum half life
  11. Cellular uptake of a stapled peptide: FITC-labled Peptide, FITC-Labeled SAHBa,
  12. SAHB Suppresses Leukemia In Vivo: Vehicle vs SAHBs(g-E) – Tumor burden
  13. A dual antagonist of hDM2 and hDMx/4 [negative regulator: 253 tumor]
  14. alpha helic peptide drug development: for p53
  15. SAHM1 bridges the ICL/CSL interface: ICN and CSL and dnMAML

Gary Gilliland

Melanie Comejo

Andrew Kung

  • Discovery of direct-acting beta-catenin antagonists: Staple peptide  –
  • Transcription factors: DIscovery of direct-acting beta-catenin antagonists
  1. multiple staple configurations ->> lead optimization
  2. Amino-staple Target: Cell penetration by second-generation
  3. If no known helical interactor? Raf and K-Ras
  4. Sub-nM  antagonists, effector domain binder,
  5. Screening system = yeast Surface Display app – strip staple Yeast Surface expression and add Staple
  6. Ras-peptide interaction in vivo
  7. Ras double the peptide Complex at 2.15 A

Cell-penetrating all Ras isophorme

  • selection mutation inhibitors – Amide ptoton management in cyclosporine A – Hydrogen bonded –
  • Amide proton management in Pro-lock system
  • Pro-lock systme: N3
  • 3rd – Generation systems with Pfizer
  • Challenges:
  1. extensive screening campaign
  2. Endocytic uptake and release apthways
  3. blood is not reliable indicator of vs tissue concentration of drug

Fogpharma.com

 

 

 

Speaker Biographies:

Jeffrey Barrett, D.Phil., Founding Director, Open Targets; Group Leader, Wellcome Trust Sanger Institute

Jeffrey Barrett has been involved in Open Targets since its inception in 2014 and began his role as Head on 1 May 2015. Jeff is a statistical geneticist who has led several of the largest genome-wide association studies of complex disease. He first became interested in human genetics in Mark Daly’s lab at the Whitehead Institute before moving to Lon Cardon’s group in Oxford, where he received a D.Phil. in statistical genetics. Jeff was an analyst in the Wellcome Trust Case Control Consortium, and led early GWAS meta-analyses in inflammatory bowel disease and type 1 diabetes while a postdoc with David Clayton in Cambridge. Since joining the Sanger Institute as a group leader in 2008, he has used next-generation sequencing in thousands of individuals to find variation associated with both rare and common human diseases.

Aaron Day-Williams, Ph.D., Biogen Scientific Lead, Open Targets; Associate Director and Head, Statistical Genetics, Biogen

Aaron Day-Williams is a statistical geneticist and bioinformatician interested in utilizing human genetics to elucidate new targets, identify biomarkers and stratify patients. He joined Biogen in May 2012. Before joining Biogen he was a postdoctoral fellow at the Wellcome Trust Sanger Institute in Hinxton, UK, where he studied the genetics of osteoarthritis, type II diabetes and body mass index using 1000 genomes imputation-based GWAS, whole-exome sequencing and whole-genome sequencing. In addition to the gene-mapping projects, he was involved in developing methods for the analysis of rare variants. Aaron’s graduate work was performed in the labs of Dr. Eric Sobel and Dr. Kenneth Lange at UCLA, where he developed a set of methods to estimate kinship coefficients for use in gene mapping methods from GWAS chip genotyping without prior information on relationships.

Gregory L. Verdine, Ph.D., Erving Professor, Chemistry, Departments of Stem Cell and Regenerative Biology, Chemistry and Chemical Biology, and Molecular and Cellular Biology, Harvard University and Harvard Medical School

Gregory Verdine is a pioneer in the field of chemical biology, a serial biotech entrepreneur and a life science venture capitalist. In an independent academic career at Harvard University and Harvard Medical School spanning nearly three decades, he has elucidated the molecular mechanism of epigenetic DNA methylation and the pathways by which certain genotoxic forms of DNA damage are surveilled in and eradicated from the genome. He is a leading figure in the field of new therapeutic modalities, and has developed a new class of therapeutics termed stapled peptides, which are currently in clinical development and have received much attention for their ability to drug targets previously considered “undruggable”. Dr. Verdine has made important contributions in the translation of bench science to the bedside. He has founded or co-founded a number of successful biotechnology companies, including Enanta Pharmaceuticals (Nasdaq ENTA), Gloucester Pharmaceuticals (acquired by Celgene), Tokai Pharmaceuticals (Nasdaq TKAI), WaVe Life Sciences (Nasdaq WVE), Eleven Biotherapeutics (Nasdaq EBIO), Warp Drive Bio, Aileron Therapeutics, and FogPharma. To date, these companies have succeeded in gaining FDA approval for two drugs, romidepsin (Gloucester/Celgene) and paritaprevir (Enanta/AbbVie). He has also worked in the venture capital industry as a Venture Partner with Apple Tree Partners, Third Rock Ventures, and WuXi Healthcare Ventures, and as a Special Advisor to Texas Pacific Group. He co-founded and served as the founding President of the non-profit Gloucester Marine Genomics Institute and Gloucester Biotechnology Academy, for which he continues to serve as Director. Dr. Verdine is Chairman of the Board of Directors of WaVe Life Sciences, and is a Director of Warp Drive Bio, for which he also Chairs the company’s Scientific Advisory Board. He is currently President and CEO of FogPharma, a biotechnology company focused on a transformative drug discovery platform based on Cell-Penetrating Mini-Proteins. Dr. Verdine serves on the Board of Scientific Consultants of the Memorial Sloan Kettering Research Institute and on the Board of Scientific Advisors of the National Cancer Institute. Dr. Verdine holds a Ph.D. in chemistry from Columbia University, a BS in chemistry from St. Joseph’s University, and served as an NIH postdoctoral fellow in molecular biology at MIT and Harvard Medical School. He has won numerous prestigious awards, including most recently the AACR Award for Excellence in Chemistry in Cancer Research, and the Nobel Laureate Signature Award.

Posted in BioTechnology - Venture Creation, Venture Capital, Conference Coverage with Social Media | Leave a Comment »

LIVE 9/21 8AM to 2:40PM Targeting Cardio-Metabolic Diseases: A focus on Liver Fibrosis and NASH Targets at CHI’s 14th Discovery On Target, 9/19 – 9/22/2016, Westin Boston Waterfront, Boston

September 21, 2016 by 2012pharmaceutical

LIVE 9/21 8AM to 2:40PM Targeting Cardio-Metabolic Diseases: A focus on Liver Fibrosis and NASH Targets at CHI’s 14th Discovery On Target, 9/19 – 9/22/2016, Westin Boston Waterfront, Boston

http://www.discoveryontarget.com/

http://www.discoveryontarget.com/crispr-therapies/

#BostonDOT16

@BostonDOT

 

Nonalcoholic Steatohepatitis (NASH)

 

Leaders in Pharmaceutical Business Intelligence (LPBI) Group is a

Media Partner of CHI for CHI’s 14th Annual Discovery on Target taking place September 19 – 22, 2016 in Boston.

In Attendance, streaming LIVE using Social Media

Aviva Lev-Ari, PhD, RN

Editor-in-Chief

http://pharmaceuticalintelligence.com

 

Wednesday, September 21

7:30 am Registration Open and Morning Coffee

8:00 Chairperson’s Opening Remarks

Rebecca Taub, M.D., Ph.D., CEO, Madrigal Pharmaceuticals

  • Epidemic of NASH,
  • approaches to treating NASH – Fibrosis
  • NASH is a metabolic Disease of the Liver
  • Treating the HCV will treat the Fibrosis

8:10 FEATURED PRESENTATION: The Epidemic of Fatty Liver Disease: Silent, Serious and Still Growing?

Lee Kaplan, M.D., Ph.D., Director, Obesity, Metabolism and Nutrition Institute, Massachusetts General Hospital, Harvard Medical School

  • Silent, Serious and Growing
  • Obesity the Disease = BMI>30: Medical Complicastions for BMI >%) – On ANti-Obisity and Bariatric SUrgery, Type 2 Diabetis .. NAFLD .. NASH .. Cirrhosis .. HCC
  • Parkinson’s Disease
  • HIV/AIDS
  1. Medical Complications of Obisity =197 :
  2. NAFLD – Nonalcoholic Fatty Liver Disease >>> Liver transplantation replacing HCV
  3. Associated with obesity and type 2 diabetes
  4. NAFLD is UP 90% wiht Severe Obesity
  5. Viral hepatitis and Hemochromatosis
  6. NAFLD: Steatosis, Inflamamtion, Hepatocellular Necrosis, Fibrosis, Cirrhosis
  7. NASH: insulin resistence .. metabolic syndrom .. interaction
  8. Alternative Model: Metabolis Syndrom.. Steatosis .. NASH … FIbrosis
  9. Genetics of Liver DIsease
  10. PNPLA3 Associated with NAFLD – Not Weight Gain
  11. Other genes: A Partial List:
  12. Diagnosis of NASH: Liver biopsy macrovescicular fatty change: InflammationMollery bodies
  13. 75% Patients with Cirrhousis have obisity
  14. Alcoholoc hepatisis >> Progression to Cirrhousis
  15. Macrovesicular Steatosis
  16. NASH – inflammation
  17. Sinusoidal Pericellular Fibrosis –
  18. LAB Features of NAFLD
  • Transaminase elevation
  • Akaline phosphate
  1. Biomarkers – NASH – associated cirrhousis with lower rate 30% of elevation
  2. Fibrosure
  • Clinical Features of NASH: none presentation, Bright, Echo Fibroscan FibroscanScreen for HCC, Varices if Gray zone: Biopsy
  • Treatment of NASH
  • Treat liver disease: Treat steatosis then Inflamamtion and fibrosis
  1. NAFLD Treatment Strategy: Stepwise Approach
  • Treat the steatosis Piodlitazone
  • PPARalpha, delta,
  • Treat Inflammation: ANtioxidant
  • CCR2/CCR% inhibitors
  • Metabolic SUrgery
  • Weigh-independent for bariatric
  • Bariatic: improvrment of steatosis,effect on inflammationless clear
  • dramatic on weigh loss
  • NO clear is surgery improved cirhousis
  • If NASH developed >>>> progression s the rule
  • No great treatment of NASH

Medication-assciated NASH: Glucocorti

 

8:40 Non-Alcoholic Steatohepatitis and Cardiovascular Disease: Modulation by Novel PPAR Agonists

Bart Staels, Ph.D., Professor, INSERM, University of Lille, Pasteur Institute

Peroxisome proliferator-activated receptors (PPARs) are ligand-activated nuclear receptors which regulate lipid and glucose metabolism as well as inflammation. In this presentation, we will review recent findings on the pathophysiological role of PPARs in the different stages of non-alcoholic fatty liver disease (NAFLD), from steatosis development to steatohepatitis and fibrosis, as well as the preclinical and clinical evidences for potential therapeutical use of PPAR agonists in the treatment of NAFLD. PPARs play a role in modulating hepatic triglyceride accumulation, a hallmark of the development of NAFLD. Moreover, PPARs may also influence the evolution of reversible steatosis towards irreversible, more advanced lesions. Large controlled trials of long duration to assess the long-term clinical benefits of PPAR agonists in humans are ongoing.

Non-Alcoholic Steatohepatisis and CVD – Meta inflammatory disease

  • NAFL — abnormal Lipid accumulation
  • NASH >> Balooning, FIbrosis inflamation
  • Resolution of NASH is associated with reduction of Fibrosis (Golden – 505 trial)

CVD is linked to NAFLD: Lipids elevated and therosclerosis

  • TG – elevated, APO B elevated, VLDL – elevated HDL decrease
  • PPAR Alpha
  • Gamma
  • PPAR Beta/Delta agonist: GENFIT – Elafibranor
  • SPPARM

Trans-activation: Lipid and Glucose homeostasis: Trans-repression – anti-inflammatory properties

  • Hapatic mitochondrial activity deseases upon progression from NAFL to NASH: Obese NAFL and NASH
  1. Upregulated hepatic respiratory in obese humans with or without NAFL
  2. Impaired
  3. Hepatic PPARalpha Expression Decreases upon Progression of Nash and Fibrosis
  4. hepatic PPARalpha expression – target genes increase in patients with improved NASH histology after 1 year
  5. Metabolic Regualtion by thehepatic JNK Signaling Pathway
  6. Target gene transcription – miR-21 expression increases in human
  7. PPAR Delta: Elafibbranor: – effect on plasma lipids: A Dual PPAR alpha/Delts (GFT505): 80mg vs placebo and 120mg vs placebo, improves plasma apolipolipids and glucose HbA1C – insulin sensitivity
  8. efficacy in NASH acting on: Steatosis, fibrosis and cirrhosis
  9. inflammatory markers: RESOLVE-IT Phase 3 Study Desing: NASH ressolution without adverse on FIbrosis and Cirrhosis

GOLDEN505 Trial: Improves plasma lipid levels: Triglycerides

Inclusion Criteria:

ALT, AST, GGT, ALP

Improve atherogenic dyslipidemia

  • APOC3 – associated with CVD

9:10 PANEL DISCUSSION: Liver Fibrosis and NASH Targets

Moderator: H. James Harwood, Ph.D., Delphi BioMedical Consultants, LLC

Panelists:

Lee Kaplan, M.D., Ph.D., Director, Obesity, Metabolism and Nutrition Institute, Massachusetts General Hospital, Harvard Medical School

Bart Staels, Ph.D., Professor, INSERM, University of Lille, Pasteur Institute

Rebecca Taub, M.D., Ph.D., CEO, Madrigal Pharmaceuticals

Weilin Xie, Ph.D., Senior Principal Scientist, Biotherapeutics, Celgene Corp.

  • FDA’s view on surrogate endpoints
  • Biomarkers of NASH
  • Regulatory challenges
  1. Liver biopsy: gold standard, invasive direct measure of endpoints pros/cons
  2. non-invasive functional tests – plasma bioamrkers
  3. non-invasisve liver imaging techniques: MRI to assess hepatic fat content MRE to assess hepatic fibrosis, Fibroscan,
  4. Endpoints acceptable by FDA: Current vs Future
  • Pre clinical Translational animal models

Discussion by Panel members

Progression from NAFLD to NASH: Oxidative stress and toxic lipids

NASH and Steatosis are different populations

Alcoholoc Steatosis vs Non-Alcoholic Steatosis

  • Obesity cause of Fatty liver
  • NASH in Diabetes
  • NASH progresses
  • Steatosis is associated with NASH
  • Different types of NASH: HTN, Dislipedemia,
  • GENETICS underlining factors, more genes are discovered
  • Limitations of Animal Studies for inference on Humans – careful in over generalizing results
  • Metabolic SYndrom -not all progresses to NASH
  • Nonalcoholic Steatohepatitis (NASH) depend on Steatosis

 

9:40 Coffee Break in the Exhibit Hall with Poster Viewing

10:25 Targeting Fibroblast Activation Protein (FAP) and FGF21 to Treat Fatty Liver Disease

Diana Ronai Dunshee, Ph.D., Department of Molecular Biology, Senior Scientific Researcher, Genentech, Inc.

FGF21 is a hormone with anti-obesity and hepatoprotective properties. However, the beneficial effects of FGF21 are limited by a relatively short half-life in circulation. We discovered that fibroblast activation protein (FAP), an endopeptidase overexpressed in liver with cirrhosis, cleaves and inactivates FGF21. Pharmacological inhibition of FAP increases endogenous levels of active FGF21, thus making FAP a promising target for the treatment of non-alcoholic-steatohepatitis (NASH).

  • Medical complications of obisity: NASH and DM-2
  • energy consumption
  • white adipose tissue – energy storage
  • brown adipose tissue matochondia’s energy
  • FGF21 – Human activation of protein cleavage: A Homone beneficial on metabolic health circulation, weigh loss
  • it suppreses hepatic Steatohepatitis
  • One singleinjection in mice — leads to energy expenditure induced weigh loss and metabolic improvement in Obese Humans
  • Negative FGF21 is Rapidly Eliminated from the body – renal degradation and Inactivation of FGF21 Endopeptidase Cleavage Site – Fibroblast Activation Protein Matched FAP Endopeptidease Specificity
  • Closest relative of DPP4 upregulted during tissue injury in NASH
  • FAP is SUfficient to Cleave FGF21
  • Recombinant FGF21 with Recombinant FAP in Serum or Plasma
  • FAP Protease – Serum Immunodepleted Ablates FGF21 Cleavage Activity: Peptide IgG vs anti-FAP
  • FAP Cleavage Inactivates Human FGF21 dependent on KLB-FGFR1c placed on the site
  • hFGF21 in Not Cleaved in FAP KO Mice
  • Fc-hFGF21 is more stable in FAP KO mice
  • FAR cleaves Endogenously Produced FGF21 In Vivo in monkeys and in dogs
  • The FAP Cleavage Consensus GLY-Pro is COnserved in most mammalian FGF21
  • FAP Does not Cleave the C-Terminal Residues of Mouse FGF21
  • Human: FAP, DPPIV
  • Mouse: FAP, DPP4
  • FAP INhibition
  • FAP is Overexpressed in Liver with Steatohepatitis: Early NASH vs Late NASH
  • Proposal: FAP Inhibition for FGF21 Stabilization in NASH
  1. Fatty hepatocytes – e.g. NASH
  2. Activated stellate cells, e.g. NASH

 

10:55 Thyroid Hormone Receptor Beta (THR-ß) Agonist for NASH: Correcting a Primary Deficiency in NASH Livers

Rebecca Taub, M.D., Ph.D., CEO, Madrigal Pharmaceuticals

NASH patients typically have metabolic syndrome including diabetes, dyslipidemia, obesity, and primarily die of cardiovascular disease. Hypothyroidism at the level of the thyroid gland and liver-specific hypothyroidism are common in NASH. Based on clinical and preclinical data, Thyroid receptor beta agonists decrease insulin resistance, reduce LDL-C, triglycerides fatty liver, inflammation and fibrosis in NASH. The target will also provide CV benefit to patients with NASH. MGL-3196 is a highly THR-ß selective liver-directed once daily oral medication that has shown excellent safety and lipid-lowering efficacy in humans; unlike prior thyroid receptor agonist(s), no cartilage findings in chronic toxicology or ALT increases in human studies. MGL-3196 is being advanced in Phase II studies in patients with genetic dyslipidemia or NASH.

Madrigal Portfolio of drugs:

  • MGL-3196: First-in-Class THR-Beta Agonist – discovered first at ROCHE – THR-beta selective targeted to the Liver – regulated by THR-Alpha  – in Phase II – no side effects on bone
  • Large & underserved Markets in NASH
  • Phase 2 HeFH Patients
  1. Hypothyroidism common in NASH patients
  2. Liver-specific Hypothyroidism present in human NASH degradation of thyroid hormone increases deiodised 9DIO) 3 produced by Stelllate cells in NASH liver
  3. Treating NASHrather than fibrosis is key in addressing the disease – approvable endpoint
  4. THR – Thyroid hormone reduces Cholesterol
  5. Thyroid hormone T3 thyroxine – treatment amy cause osteoporosis
  6. MGL 0 3196: Liver size, Live Triglycerides, Improve Insulin tolerance, decrease ALT
  7. Reduction of key NASH, Fibrosis Pathway Genes at Human Comparable Drug levels
  8. THR-beta: Decreased Liver Fibrosis, Apoptosis in mice:

HUMAN DATA

  • Single ascending dose study
  • Multiple – ascending studies: LDL and TG decrease
  • decrease Non-HDL CHolesterol
  • Decrease Apolipoprotein B
  • Pleiotropic Pioglitazone Effect in NASH at 6 month treatment and biopsy of liver – dramatic effect in NASH – ten years ago study
  • PPAR gamma agonist – NEGATIVE SIDE EFFECTS: weight gain, CHF, Bone osteoporosis
  • Anti-inflammatory: well tolerated

No Single NASH Therapeutics – Conbination agents

MGL – 3196 Phase 2 – Study: Proposed Phase 2 Proof of COncepts NASH Protocol

  • Unmet needs in FH, a severeGenetic Dyslipedemia
  • Weight loss in 6 weeksreduction in cholesterol and TG
  • Likelihood of Success
  • second study after 9 months
  • is different on NASH Patients in 12 weeks using MRI on Liver
  • prevalence
  • HeFH, PCSK9 inhibitors plus standard care
  • Unique and Complementary Lipid Lowering Profile
  1. Lowers Lp(a) and severely atherogenic practice
  2. Proposed Phase 2 HeFH Patients

 

11:25 Enjoy Lunch on Your Own

 

Posted in Fatty acids, Frontiers in Cardiology and Cardiovascular Disorders, Lipid metabolism, Lipidomics, Lipids, Liver & Digestive Diseases Research, PCSK9 Inhibitor Therapy | Leave a Comment »

LIVE 9/21 8AM to 10:55 AM Expoloring the Versatility of CRISPR/Cas9 at CHI’s 14th Discovery On Target, 9/19 – 9/22/2016, Westin Boston Waterfront, Boston

September 21, 2016 by sjwilliamspa

LIVE 9/21 8AM to 10:55 AM Expoloring the Versatility of CRISPR/Cas9 at CHI’s 14th Discovery On Target, 9/19 – 9/22/2016, Westin Boston Waterfront, Boston

http://www.discoveryontarget.com/

http://www.discoveryontarget.com/crispr-therapies/

Leaders in Pharmaceutical Business Intelligence (LPBI) Group is a

Media Partner of CHI for CHI’s 14th Annual Discovery on Targettaking place September 19 – 22, 2016 in Boston.

In Attendance, streaming LIVE using Social Media

Aviva Lev-Ari, PhD, RN

Editor-in-Chief

http://pharmaceuticalintelligence.com

#BostonDOT16

@BostonDOT

 

COMMENTS BY Stephen J Williams, PhD

EXPLORING THE VERSATILITY OF CRISPR/Cas9

 

8:00 Chairperson’s Opening Remarks

TJ Cradick , Ph.D., Head of Genome Editing, CRISPR Therapeutics

 

@CRISPRTX

 

8:10 Functional Genomics Using CRISPR-Cas9: Technology and Applications

Neville Sanjana, Ph.D., Core Faculty Member, New York Genome Center and Assistant Professor, Department of Biology & Center for Genomics and Systems Biology, New York University

 

CRISPR Cas9 is easier to target to multiple genomic loci; RNA specifies DNA targeting; with zinc finger nucleases or TALEEN in the protein specifies DNA targeting

 

  • This feature of crisper allows you to make a quick big and cheap array of a GENOME SCALE Crisper Knock out (GeCKO) screening library
  • How do you scale up the sgRNA for whole genome?; for all genes in RefSeq, identify consitutive exons using RNA-sequencing data from 16 primary human tissue (alot of genes end with ‘gg’) changing the bases on 3’ side negates crisper system but changing on 5’ then crisper works fine
  • Rank sequences to be specific for target
  • Cloned array into lentiviral and put in selectable markers
  • GeCKO displays high consistency betweens reagents for the same gene versus siRNA; GeCKO has high screening sensitivity
  • 98% of genome is noncoding so what about making a library for intronic regions (miRNA, promoter regions?)
  • So you design the sgRNA library by taking 100kb of gene-adjacent regions
  • They looked at CUL3; (data will soon be published in Science)
  • Do a transcription CHIP to verify the lack of binding of transcription factor of interest
  • Can also target histone marks on promoter and enhancer elements
  • NYU wants to explore this noncoding screens
  • sanjanalab.org

 

@nyuniversity

 

8:40 Therapeutic Gene Editing With CRISPR/Cas9

TJ Cradick , Ph.D., Head of Genome Editing, CRISPR Therapeutics

 

NEHJ is down and dirty repair of single nonhomologous end but when have two breaks the NEHJ repair can introduce the inversions or deletions

 

    • High-throughput screens are fine but can limit your view of genomic context; genome searches pick unique sites so use bioinformatic programs  to design specific guide Rna
    • Bioinformatic directed, genome wide, functional screens
    • Compared COSMID and CCTOP; 320 COSMID off-target sites, 333 CCtop off target
    • Young lab GUIDESeq program genome wide assay useful to design guides
    • If shorten guide may improve specificity; also sometime better sensitivity if lengthen guide

 

  • Manufacturing of autologous gene corrected product ex vivo gene correction (Vertex, Bayer, are partners in this)

 

 

They need to use a clones from multiple microarrays before using the GUidESeq but GUIDEseq is better for REMOVING the off targets than actually producing the sgRNA library you want (seems the methods for library development are not fully advanced to do this)

 

The score sometimes for the sgRNA design programs do not always give the best result because some sgRNAs are genome context dependent

9:10 Towards Combinatorial Drug Discovery: Mining Heterogeneous Phenotypes from Large Scale RNAi/Drug Perturbations

Arvind Rao, Ph.D., Assistant Professor, Department of Bioinformatics and Computational Biology, The University of Texas MD Anderson Cancer Center

 

Bioinformatics in CRISPR screens:  they looked at image analysis of light microscopy of breast cancer cells and looked for phenotypic changes

 

  • Then they modeled in a small pilot and then used the algorithm for 20,000 images (made morphometric measurements)
  • Can formulate training statistical algorithms to make a decision tree how you classify data points
  • Although their algorithms worked well there was also human input from scientists

Aggregate ranking of hits programs available on web like LINKS

 

@MDAndersonNews

 

10:25 CRISPR in Stem Cell Models of Eye Disease

Alexander Bassuk, M.D., Ph.D., Associate Professor of Pediatrics, Department of Molecular and Cellular Biology, University of Iowa

 

Blind athlete Michael Stone, biathlete, had eye disease since teenager helped fund and start the clinical trial for Starbardt disease; had one bad copy of ABCA4, heterozygous (inheritable in Ahkenazi Jewish) – a recessive inheritable mutation with juvenile macular degeneration

  • Also had another male in family with disease but he had another mutation in the RPGR gene
  • December 2015 paper Precision Medicine: Genetic Repair of retinitis pigmentosa in patient derived stem cells
  • They were able to correct the iPSCs in the RPGR gene derived from patient however low efficiency of repair, scarless repair, leaves changes in DNA, need clinical grade iPSCs, and need a humanized model of RPGR

@uiowa

10:55 CRISPR in Mouse Models of Eye Disease

Vinit Mahajan, M.D., Ph.D., Assistant Professor of Ophthalmology and Visual Sciences, University of Iowa College of Medicine

  • degeneration of the retina will see brown spots, the macula will often be preserved but retinal cells damaged but with RPGR have problems with peripheral vision, retinitis pigmentosa get tunnel vision with no peripheral vision (a mouse model of PDE6 Knockout recapitulates this phenotype)
  • the PDE6 is linked to the rhodopsin GTP pathway
  • rd1 -/- mouse has something that looks like retinal pigmentosa; has mutant PDE6; is actually a nonsense mutation in rd1 so they tried a crisper to fix in mice
  • with crisper fix of rd1 nonsense mutation the optic nerve looked comparible to normal and the retina structure restored
  • photoreceptors layers- some recovery but not complete
  • sequence results show the DNA is a mosaic so not correcting 100% but only 35% but stil leads to a phenotypic recovery; NHEJ was about 12% to 25% with large deletions
  • histology is restored in crspr repaired mice
  • CRSPR off target effects: WGS and analyze for variants SNV/indels, also looked at on target and off target regions; there were no off target SNVs indels while variants that did not pass quality control screening not a single SNV
  • Rhodopsin mutation accounts for a large % of patients (RhoD190N)
  • injection of gene therapy vectors: AAV vector carrying CRSPR and cas9 repair templates

CAPN mouse models

  • family in Iowa have dominant mutation in CAPN5; retinal degenerates
  • used CRSPR to generate mouse model with mutation in CAPN5 similar to family mutation
  • compared to other transgenic methods CRSPR is faster to produce a mouse model

To Follow LIVE CONFERENCE COVERAGE PLEASE FOLLOW ON TWITTER USING

Meeting #: #BostonDOT16

Meeting @: @BostonDOT

 

Overall good meeting #s:

#personalizedmedicine

#innovation

#cancer

#immunology

#immunooncology

#pharmanews

#CRSPR

#geneediting

#crisper

#biotech

 

AND FOLLOW these @

@pharma_BI

@AVIVA_1950

@BiotechNews

@CHI

@FierceBiotech

Posted in Big Data, Biological Networks, Cell Biology, Cell Biology, Signaling & Cell Circuits, Clinical Genomics, Conference Coverage with Social Media, CRISPR/Cas9 & Gene Editing, Disease Biology, Genome Biology, Mutagenesis, RNA Biology, RNA Biology, Cancer and Therapeutics, Scientific & Biotech Conferences: Press Coverage, Uncategorized, Variation in human protein-coding regions | Tagged , , , , , , , | Leave a Comment »

LIVE 9/20 2PM to 5:30PM New Viruses for Therapeutic Gene Delivery at CHI’s 14th Discovery On Target, 9/19 – 9/22/2016, Westin Boston Waterfront, Boston

September 20, 2016 by 2012pharmaceutical

LIVE 9/20 2PM to 5:30PM New Viruses for Therapeutic Gene Delivery at CHI’s 14th Discovery On Target, 9/19 – 9/22/2016, Westin Boston Waterfront, Boston

http://www.discoveryontarget.com/

http://www.discoveryontarget.com/crispr-therapies/

Leaders in Pharmaceutical Business Intelligence (LPBI) Group is a

Media Partner of CHI for CHI’s 14th Annual Discovery on Target taking place September 19 – 22, 2016 in Boston.

In Attendance, streaming LIVE using Social Media

Aviva Lev-Ari, PhD, RN

Editor-in-Chief

http://pharmaceuticalintelligence.com

#BostonDOT16

@BostonDOT

 

COMMENTS BY Stephen J Williams, PhD

Gene Therapy Breakthroughs

New Strategies for Better Specificity and Delivery

 

2:05  Chairman’s Remarks

Joseph Gold, Ph.D. Director Manufacturing Center for Biomedicine and Genetics, Beckman Research Institute City of Hope

 

  • CBG (center for biomedicine and Genetics) 20000 sq feet
  • CTPC (center therapy production) mainly CART
  • CBG 16 years operation do all stem cells and >400 products
  • New stem cell Beta cell progenitor
  • Do oncolytic VSV
  • CTPC is investigator driven CART islet cells,
  • Like to do novel work so work with CIRM
  • Banking of modified stem cells
  • Adherent scale out limitations: cost,inefficient; solution can be suspension
  • Establish hESC; plate on CELLstart > Accutase>StemPRO SFM>differentiation process; defined reagents — they use this for cardiomyocyte differentiation: they are functional (inotropy, chronotropy response to isoproterenol) can freeze back cells
  • Create a bank of intermediate cells and when you need it for surgery they will put on their matrix, enrich, expand and ship out
  • Allogeneic cells: project where take allogeneic neural stem cells to deliver a chemotherapy payload as they like to migrate to brain tumors
  • Allogeneic cells: for ALS modified to express GDNF
  • HIV resistance with engineered CCR5 negative blood stem cells
  • Release assay considerations: viability, sterility, if cryopreserved then can determine identity, viral insertions, VSV-G copy number, endotoxin and potency (FDA is wanting phase I potency assays) for CART potency is % transduced
  • Good in vivo activity of the neural stem cells loaded with chemotherapeutic

 

ALS  

  • If deliver GDNF to muscle  using genetically modified myoblasts
  • Best to use fetal stem cells – less issues

 

Canavan disease: progressive fatal neurologic disorder that begins in infancy and don’t make it past teenage years

  • Rossbach is taking autologous cells reprogramming generating iPS cells and then modifying by CRISPR but the CRISPR issues of off target effects persist as well the time required for process and verification; also don’t want to use a selectable marker and put in patients; so you can differentiate the cells and hit them with a lentiviral vector system

 

They have been named a PACT Center Production Assistance for Cell Therapy where you can apply for a project grant.  Applicable for startups up to larger mature companies

www.pactgroup.net

 

They do a standard panel of tests for viral infections.

They work with investigators or companies at all stages of manufacturing processes.

 

@BeckmanInst

@cityofhope

 

2:15 Large-Scale Production of Cell Therapies for Regenerative Medicine

Joseph Gold, Ph.D. Director Manufacturing Center for Biomedicine and Genetics, Beckman Research Institute

 

2:45  Directed Evolution of New Viruses for Therapeutic Gene Delivery

David Schaffer, Ph.D.  Professor of Chemical and Biomolecular Engineering, BioEngineering, Molecular and Cell Biology and Neuroscience;

 

AAV is very safe as many people already infected with it

 

  • Spark (Leber’s cogenital anaurosis
  • Hemophilia B
  • Lipoprotein lipase deficiency
  • Spinal muscular atrophy
  • Challenges: are we just getting the ‘low hanging fruit’ eg Spark therapy must be injected after retinal therapy, hemo B needs to be given at high doses
  • Their theory was AAV had been evolving for its own purposes so hence the limitations of AAV;
  • Utilized 25 different techniques to generate variants of AAV in a library then packaged (each will have its own barcode)
  • Broad platform technology: retina, lung, brain and spinal cord

Retinal:  AAV may be too large to get through layers of the eye, problems; subretinal injections and damage or retinal detachment.  Then they used their whole library in an in-vivo screen (as hard to recapitulate the multi cell layers of the retina).  

 

Cystic Fibrosis

  • AAV2H22 variant worked very well to supply the CFTR gene in pig model of cystic fibrosis and increases chloride transport and reduce bacterial load
  • Then found pig variant AAV did not work well on human tissue so designed a human variant and worked well in human tissues
  • The variant AAV2.5T surrounds sialic acid binding pockets and increases binding and endocytosis

 

Brain and Spinal Cord:  Sanfilippo B trial  8 holes drilled into skull followed by 16 AAV injections

 

  • Injected a generated AAV variant (by evolution process) : engineered AAV2 is 100 fold better getting through blood brain barrier… novel variant undergoes retrograde transport to cortex ; made a cas9 to remove a tdTomato gene overexpressed in mouse and found 90% knockdown
  • Also interesting point: the porcine variant did not work in human and the human variant did not work in porcine.  Implication for FDA safety and efficacy testing must do in monkeys

They started a spinout 4D Molecular Therapeutics

 

4:25 Lentiviral Vectors for Gene Therapy

 

Munapaty Swani, Ph.D. Texas Tech Health Science Center

 

  • Can express multiple shRNA under a separate promoter but toxic so if expressed in miRNA backbone could be safer under a pol II
  • How much of flanking sequence is needed?
  • 30 nt flanking sequence is enough for Drosha processing
  • Constructed 1 to 7 shRNA-miR targeting CCR5 and 6 viral genes; all constructs were functional
  • Problem with pol ii promoter
  • These 7 shRNA miRNA protect against HIV entry if against CCR5 and the 7 viral elements
  • Used the non-integrating lentivirus for transient to see if infect T cells or not versus integrating lentivirus ; results non-integrating lentivirus did not infect t cells so safer to use
  • CCR5 disruption reduced HIV infection in T cells in vitro;
  • ZFN treatment of HIV+ PBMC prevents activation of HIV
  • Encapsulted CAS9 within LV; cas9 protein is incorporated within LV and is functional
  • First transduce then come in with the Cas9 so made all in one lentivirus with Cas9 and an sgRNA expression vector *******
  • This shows that it is possible to put all in a nanoparticle based lentivirus and an all in one may make it easier and safer (supposedly)

 

4:55 AAV Capsid Design

Miguel Seria Esteves, PhD Associate Professor Neurology, Gene Therapy Center University of Massachusetts Medical School

 

-AAV replication dependent no known human disease with native AAV

  •  Multiple barriers to get across blood brain barrier
  • AAV9 preferentially target neonatal neurons and adult astrocytes
  • Multiple capsids can be used for AAV9 infection in brain but not complete
  • Can we design better capsids to give it better tropic properties and better penetration to blood brain barrier
  • Using a polyalanine in the 5’ end of the caspid was most efficeint
  • Increases gene transfer efficiency especially IN SELECT CELL TYPES; Glial transduction and increased in striatum: increase is structure specific so little in thalamus but good in cerebellum and spinal cord
  • AAV9 tranduces also in peripheral tissues with or without modified capsid

 

Huntington’s Disease

  • Polyglutamate disease polyy glu on huntingtin protein
  • They get a 40 to 50% reduction of huntingtin but not significant between capsid design
  • They did a directed evolution of AAV capsid and generated capsid gene delivery diversity: DNA shuffling and in vivo selection
  • AAV-B1 is a new tropic capsid showing transduction of different structures
  • Five fold reduction in tropism to the liver but massive increases in muscle and beta exocrine cells and lung
  • Presence of neutralizing antibodies is a problem with AAV therapy
  • In conclusion unknown mechanisms by whivh a highly hydrophobic string of 19 alanines modifies the CHS tropism of AAV9 kvariants
  • Chimeric capsids identified from in vivo screen can reveal interesting patterns of tropism

12:45 PM Screening with shRNA and CRISPR

Ryan Raver, PhD Global Product Manager, Functional Genomics, MilliporeSigma

  • KO – Knock Out
  • KD – Knock Down
  1. RNAi -KD
  2. CRISPR-Cas9 – KO

NEW STRATEGIES FOR BETTER SPECIFICITY AND DELIVERY

2:05 Chairperson’s Remarks

Joseph Gold, Center for Biomedicine and Genetics Beckman Research Institute, City of Hope

2:15 Large Scale Production of cell Therapies for Regenerative Medicine: COmbination Cell and Gene Therapy products

Joseph Gold, Center for Biomedicine and Genetics Beckman Research Institute, City of Hope

  • Biological & Cellular GMP manufacturing Core at COH
  • Establishing scalable hESC suspension Culture
  • Optimized small molecule concentration, induction timing, stirring rates
  1. Almost Xeno free
  2. defined
  3. very good reproducibility
  4. high purity and yield:
  • Immuno-staining,
  • FACS – cTnT, sMHC, Alpha-actinin
  • Cryopreservation, Multi-electrode Array (MEA)
  • hESC-RPE monolayer on synthetic substrate

Combination cell/gene therapy products at COH

  1. CAR T CCR5-inactivated CD34+ HSPC – Target: AIDS
  • adoptive immunotherapy using CAR-Engineering T cells – glioblastoma

2. HIV resistance with engineering CCR5-negative blood stem cells: gene KO by ZFNs

  • assay considerations 0 If cryopreservation : Identify, viral insertion, endotoxin, residual beads Potency: CAR T- % transduced cells, CCR5-?-CD34 cells: HIV resistance

3. Glioblastoma

4.  In vivo activity of transduced NSCs: Assay consideration – viability, sterility, mycobatom,

5.  ALS: degeneration of neurones

  • Embryonic stem cells
  • Fetal neural stem cells
  • Adult stem cells – human Proginetor neural cells

6.  Canavan Disease – Y.Shi – ASPA gene mutations cause Canavan disease

Strategy: autologous iPSC-derived, modified neural progenitors: DIferentiate to neural progenitors

Gene therapy: Correction (Off-taregt effects, correct individual cell lines)  or Over expression (Copy number, selection, transduce iPSC)

  • release assay consideration
  • identity – markers and HLA, contaminants, Potency: in vivo efficacy modified autologous
  • production Assistance for Cell therapy
  • cell therapy manufacturing development
  • roles of Biobanks

 

2:45 Directed Evolution of New Viruses for Therapeutic Gene Delivery

David Schaffer, Ph.D., Professor of Chemical and Biomolecular Engineering, Bioengineering, Molecular and Cell Biology, and Neuroscience; Director, Berkeley Stem Cell Center, University of California, Berkeley

Adeno-associated viral (AAV) vectors have been increasingly successful in clinical trials; however, viruses face many delivery barriers that limit their efficacy for most disease targets. We have developed directed vector evolution – the iterative genetic diversification of a viral genome and functional selection for desired properties – to engineer novel, optimized AAV vectors for efficient, selective delivery for a range of tissue and disease targets.

  • DNA (gene therapy and editing) >> RNAi (antisense) >> Protein: Small molecules and monoclonal antibodies
  • Dlivery
  • Adeno Associated Viral Vectors Adenoviral helper genes
  • AAV2, efficacy in Leber’s Congenital samaurosis (AAV)
  • Spinal muscular, lipolrotein lipase deficiency (AAV)

Gene Delivery

  1. neutralization of pre-esisting antibodies
  2. target tissue – deep penetration
  3. Inefficient transduction to target cells
  4. target specific cells

Fitness as Therapy – virus evolutionary: Tropism and immunity

  • AAV directed Vector evolution : Input /sequence >> Diversity/generation >> Packaging
  • Retinoschisis Model – Eye therapeutic gene to protect vision – AAV – transduction of retinal cell, vitreas in Human different scale — eventually Macualr degeneration Therapy for: Photo receptor is the target for therapy
  • Dog: Fundus Imaging of Engineered AAV: Variant Expression GFP Pool Carrying

Lung- Cystic Fibrosis (mucus production amplifies (enhanced transduction) due to MAC3 translation error) – Gene Therapy – cilia function to restore ability to clear mucos: Variant evolved on Human airway epithelia – AA  particles

  1. AAV2>>> AAV%>> T mutation
  • efficacy must be improved

Brain and Spinal Cord

  1. Scull – 8 drills followed by 16 AAV injections
  2. Spinal cord: injuction in muscle

Synthetic version of AAV — Engineered AAV for enhanced Retrograde Transport

  • AAV2-retrograded transport – Noval variant Undergoes Transport along multiple Projections
  • Cas9 – Retrograde Delivery of Cas9 to cortex of mouse – KD –

Summary

  • Virus as gene delivery mechanism
  • designer AAV variants

 

3:15 Sponsored Presentation (Opportunity Available)

3:45 Refreshment Break in the Exhibit Hall with Poster Viewing and Poster Competition Winner Announced

4:25 Lentiviral Vectors for Gene Therapy

Manjunath N. Swamy, MD, Professor of Biomedical Sciences and Co-DIrector of the Center of Emphasis in Infectious Diseases. Paul L Foster School of Medicine, Texas Tech University Health Science Center

  • RNAi targets for HIV
  • Expression of multiple shRNAs
  • COnstruction od 7 shRNA-miR targeting CCR5 and 6 viral genes – protect against both 5 ans x4 tropic HIV-1
  • shRNA expression does not decrease with distance from promoter
  • Lentiviral vector to express ZFNs: HIV envelop – ZFN-mediated CCR5 gene editing in Primary T cells
  • ZFN treatment of HIV+PBMC prevents activation of HIV
  • Strategy to encapsulate Cas9 Protein wihtin LV : AIm to deliver Cas9 protein deliver sgRNA expression vector. A Lentiviral
  • Gene editing by all-in-one Lentivirus = to prepack

 

4:55 AAV Caspid Engineering

Miguel Sena Esteves, Ph.D., Associate Professor, Department of Neurology, Gene Therapy Center, University of Massachusetts Medical School

Adeno-associated virus vectors have become the leading platform for development of in vivo gene therapies for neurological diseases. We have developed new AAV vectors for widespread gene delivery to the CNS through vascular infusion in adult animals through peptide grafting and in vivo library selection. These new neurotropic AAVs have achieved CNS-wide silencing of gene expression using gene-specific microRNAs.

  • Adeno-associated virus – Paravovirus family
  • Recombinant AAV vectors carry gene expression cassette of choice flanked by two
  • CNS – route of gene delivery
  • Crossing BBB
  • Systemic delivery of AAV9 vectors: IV
  • Peptide grafting – in vivo selection of novel CNS: DIstribution of GFP transduced cells – robust neuronal transducture with transduction AAV-AS
  • motor cortex, Straiatum, thalamus, motor cortex, ventral horn of spinal cord, cerebelum, liver, muscle, oculomotor nerve, nucleus of oculomotor nerve

Huntington’s Disease > 40 CAG vs <26 CAG in normal – Peptide grafting of AAV vectors for CNS

  • DNA shuffling and in vivo selection: Brain vs Liver

Next generation of AAV vectors for CNS 

  • Liver, pancreas, lung — same pattern

neural transduction after vascular delivery

  • AAV-B1 caspid vs AAV8 – 19 amino acids

Conclusion

New capsids with improved CNS tropism

19 alanines modifies the CNS tropism of AAV9 variants

Chimeric caspids identified from in vivo screens

 

5:25 Welcome Reception in the Exhibit Hall with Poster Viewing

 

Posted in Conference Coverage with Social Media, CRISPR/Cas9 & Gene Editing, Genetics & Innovations in Treatment, Genetics & Pharmaceutical, Oncolytic virus & OncoViro-Therapy, Personalized and Precision Medicine & Genomic Research, Regenerative Biology and Medicine | Leave a Comment »

LIVE 9/20 8AM to noon GENE THERAPIES BREAKTHROUGHS at CHI’s 14th Discovery On Target, 9/19 – 9/22/2016, Westin Boston Waterfront, Boston

September 20, 2016 by 2012pharmaceutical

LIVE 9/20 8AM to noon GENE THERAPIES BREAKTHROUGHS at CHI’s 14th Discovery On Target, 9/19 – 9/22/2016, Westin Boston Waterfront, Boston

http://www.discoveryontarget.com/

http://www.discoveryontarget.com/crispr-therapies/

#BostonDOT16

@BostonDOT

 

Leaders in Pharmaceutical Business Intelligence (LPBI) Group is a

Media Partner of CHI for CHI’s 14th Annual Discovery on Target taking place September 19 – 22, 2016 in Boston.

In Attendance, streaming LIVE using Social Media

Aviva Lev-Ari, PhD, RN

Editor-in-Chief

http://pharmaceuticalintelligence.com

COMMENTS BY Stephen J Williams, PhD

Gene Therapy Breakthroughs

New Strategies for Better Specificity and Delivery

 

2:05  Chairman’s Remarks

Joseph Gold, Ph.D. Director Manufacturing Center for Biomedicine and Genetics, Beckman Research Institute City of Hope

 

  • CBG (center for biomedicine and Genetics) 20000 sq feet
  • CTPC (center therapy production) mainly CART
  • CBG 16 years operation do all stem cells and >400 products
  • New stem cell Beta cell progenitor
  • Do oncolytic VSV
  • CTPC is investigator driven CART islet cells,
  • Like to do novel work so work with CIRM
  • Banking of modified stem cells
  • Adherent scale out limitations: cost,inefficient; solution can be suspension
  • Establish hESC; plate on CELLstart > Accutase>StemPRO SFM>differentiation process; defined reagents — they use this for cardiomyocyte differentiation: they are functional (inotropy, chronotropy response to isoproterenol) can freeze back cells
  • Create a bank of intermediate cells and when you need it for surgery they will put on their matrix, enrich, expand and ship out
  • Allogeneic cells: project where take allogeneic neural stem cells to deliver a chemotherapy payload as they like to migrate to brain tumors
  • Allogeneic cells: for ALS modified to express GDNF
  • HIV resistance with engineered CCR5 negative blood stem cells
  • Release assay considerations: viability, sterility, if cryopreserved then can determine identity, viral insertions, VSV-G copy number, endotoxin and potency (FDA is wanting phase I potency assays) for CART potency is % transduced
  • Good in vivo activity of the neural stem cells loaded with chemotherapeutic

 

ALS  

  • If deliver GDNF to muscle  using genetically modified myoblasts
  • Best to use fetal stem cells – less issues

 

Canavan disease: progressive fatal neurologic disorder that begins in infancy and don’t make it past teenage years

  • Rossbach is taking autologous cells reprogramming generating iPS cells and then modifying by CRISPR but the CRISPR issues of off target effects persist as well the time required for process and verification; also don’t want to use a selectable marker and put in patients; so you can differentiate the cells and hit them with a lentiviral vector system

 

They have been named a PACT Center Production Assistance for Cell Therapy where you can apply for a project grant.  Applicable for startups up to larger mature companies

www.pactgroup.net

 

They do a standard panel of tests for viral infections.

They work with investigators or companies at all stages of manufacturing processes.

 

@BeckmanInst

@cityofhope

 

2:15 Large-Scale Production of Cell Therapies for Regenerative Medicine

Joseph Gold, Ph.D. Director Manufacturing Center for Biomedicine and Genetics, Beckman Research Institute

 

2:45  Directed Evolution of New Viruses for Therapeutic Gene Delivery

David Schaffer, Ph.D.  Professor of Chemical and Biomolecular Engineering, BioEngineering, Molecular and Cell Biology and Neuroscience;

 

AAV is very safe as many people already infected with it

 

  • Spark (Leber’s cogenital anaurosis
  • Hemophilia B
  • Lipoprotein lipase deficiency
  • Spinal muscular atrophy
  • Challenges: are we just getting the ‘low hanging fruit’ eg Spark therapy must be injected after retinal therapy, hemo B needs to be given at high doses
  • Their theory was AAV had been evolving for its own purposes so hence the limitations of AAV;
  • Utilized 25 different techniques to generate variants of AAV in a library then packaged (each will have its own barcode)
  • Broad platform technology: retina, lung, brain and spinal cord

Retinal:  AAV may be too large to get through layers of the eye, problems; subretinal injections and damage or retinal detachment.  Then they used their whole library in an in-vivo screen (as hard to recapitulate the multi cell layers of the retina).  

 

Cystic Fibrosis

  • AAV2H22 variant worked very well to supply the CFTR gene in pig model of cystic fibrosis and increases chloride transport and reduce bacterial load
  • Then found pig variant AAV did not work well on human tissue so designed a human variant and worked well in human tissues
  • The variant AAV2.5T surrounds sialic acid binding pockets and increases binding and endocytosis

 

Brain and Spinal Cord:  Sanfilippo B trial  8 holes drilled into skull followed by 16 AAV injections

 

  • Injected a generated AAV variant (by evolution process) : engineered AAV2 is 100 fold better getting through blood brain barrier… novel variant undergoes retrograde transport to cortex ; made a cas9 to remove a tdTomato gene overexpressed in mouse and found 90% knockdown
  • Also interesting point: the porcine variant did not work in human and the human variant did not work in porcine.  Implication for FDA safety and efficacy testing must do in monkeys

They started a spinout 4D Molecular Therapeutics

 

4:25 Lentiviral Vectors for Gene Therapy

 

Munapaty Swani, Ph.D. Texas Tech Health Science Center

 

  • Can express multiple shRNA under a separate promoter but toxic so if expressed in miRNA backbone could be safer under a pol II
  • How much of flanking sequence is needed?
  • 30 nt flanking sequence is enough for Drosha processing
  • Constructed 1 to 7 shRNA-miR targeting CCR5 and 6 viral genes; all constructs were functional
  • Problem with pol ii promoter
  • These 7 shRNA miRNA protect against HIV entry if against CCR5 and the 7 viral elements
  • Used the non-integrating lentivirus for transient to see if infect T cells or not versus integrating lentivirus ; results non-integrating lentivirus did not infect t cells so safer to use
  • CCR5 disruption reduced HIV infection in T cells in vitro;
  • ZFN treatment of HIV+ PBMC prevents activation of HIV
  • Encapsulted CAS9 within LV; cas9 protein is incorporated within LV and is functional
  • First transduce then come in with the Cas9 so made all in one lentivirus with Cas9 and an sgRNA expression vector *******
  • This shows that it is possible to put all in a nanoparticle based lentivirus and an all in one may make it easier and safer (supposedly)

 

4:55 AAV Capsid Design

Miguel Seria Esteves, PhD Associate Professor Neurology, Gene Therapy Center University of Massachusetts Medical School

 

-AAV replication dependent no known human disease with native AAV

  •  Multiple barriers to get across blood brain barrier
  • AAV9 preferentially target neonatal neurons and adult astrocytes
  • Multiple capsids can be used for AAV9 infection in brain but not complete
  • Can we design better capsids to give it better tropic properties and better penetration to blood brain barrier
  • Using a polyalanine in the 5’ end of the caspid was most efficeint
  • Increases gene transfer efficiency especially IN SELECT CELL TYPES; Glial transduction and increased in striatum: increase is structure specific so little in thalamus but good in cerebellum and spinal cord
  • AAV9 tranduces also in peripheral tissues with or without modified capsid

 

Huntington’s Disease

  • Polyglutamate disease polyy glu on huntingtin protein
  • They get a 40 to 50% reduction of huntingtin but not significant between capsid design
  • They did a directed evolution of AAV capsid and generated capsid gene delivery diversity: DNA shuffling and in vivo selection
  • AAV-B1 is a new tropic capsid showing transduction of different structures
  • Five fold reduction in tropism to the liver but massive increases in muscle and beta exocrine cells and lung
  • Presence of neutralizing antibodies is a problem with AAV therapy
  • In conclusion unknown mechanisms by whivh a highly hydrophobic string of 19 alanines modifies the CHS tropism of AAV9 kvariants
  • Chimeric capsids identified from in vivo screen can reveal interesting patterns of tropism

8:20 AAV for Gene Therapy and Genome Editing

James Wilson, M.D., Ph.D., Professor, Department of Pathology and Laboratory Medicine, Perelman School of Medicine; Director, Orphan Disease Center and Director, Gene Therapy Program, University of Pennsylvania

AAV delivery for CNS can be direct into brain or into CSF but AAV are big and vectors usually don’t cross BBB

Mucopolysaccharidoses a lysosomal storage disease including Hunter, Morquio

  • There are canine models for these diseases
  • Data show that intrathecal delivery has good distribution to the CSF not the serum
  • With IV you only get spinal distribution but IT you get good distribution to cerebellum and frontal cortex
  • Intrathecal superior for clearing lesions in cortex of dogs
  • In the dog the disease is more skeletal and less neurologic so with IT dogs were better than control but still some problems
  • Avaxis: AAV9 gene therapy for SMA and ALS but trials are very small but seems to be dose dependent effect ; phase I/II done; Pfizer, Esteves and REGENEXBIO have trials
  • Liver transduction of AAV has always been a success; early vectors were not efficient for uptake but AAV9 hemophelia B (factor 9) was
  • OCTD disease of urea acid cycle (neonatal versus late onset); proteins altered metabolism
  • Have to create a metabolic sink to detoxify metabolites; not lie a gene replacement therapy
  • They got stable expression in mouse adult  model of OCTD with AAV and had rapid onset of expression; but when done in newborn mice they saw transient expression
  • Newborn liver is proliferating so gene vector may be diluted out versus the adult liver
  • So turned to gene editing (ZFN:NEHJ, TALEN:HR gene correction, CRSPR:transgene addition by HR
  • Staph aureus cas9 is smaller than most and can fit in a 4.79kb vector*****
  • Put in 2.6 kb doner OCTD gRNA
  • With a CRSPR-Cas9 mediated deliverycould maintain the expression of OCTD for over a week in newborn mice
  • BUT in adults the gene corrected animals started to die; they were losing their ability to break down protein
  • In newborns you got 10% gene editing with 30% indels but with adults 30% resulted only in 1% gene editing
  • There is a propensity to create large (>50bp) indels in the adult
  • NGS was needed to fully detect the target transgene integration, PCR is not good enough
  • Says that large animal models are needed for safety/efficacy studies
  • Problem with Rhesus monkey: started with a humanized mouse in Rag mice because did not want to do monkey studies; but did not get good expression in the monkeys (it was not the vector which was the problem)
  • AAV may be good enough of a donor to cause the HR recombination

 

Summary:  AAV vectors combined with a CRSPR CAS9 system is effective in neonatal delivery however 1) AAV by itself may be a good delivery system by itself but need the crsipir guide RNA to make the break to promote HR and get the best efficiency of integration 2) use of CRSPR CAS9 may direct the proper integration you want to deliver the OCTD exons to correct gene loci

 

Talk specific @ and #

#genetherapy

#virology

@PennMedicine

9:20 Using CRISPR/Cas9 to Target and Destroy Viral DNA Genomes; Inactivating HBV

 

Bryan R. Cullen, Ph.D., James B. Duke Professor of Molecular Genetics and Microbiology and Director, Center for Virology, Duke University

 

The CRISPR array is an evolutionary record of the bacteriophage that the bacteria have encountered.

  • Incredible that a bacteria that never encountered a chromosomal structure would scan the genome with these gRNAs and then initiate HR and NEHJ (error prone in mammalian cells usually 3 nt
  • HBV is a very confused retrovirus because it first goes into the nucleus then becomes a template for RNA synthesis to make the particles that reinfect the cell and invisible to immune response
  • Although they do not produce infectious virus it still remains in genome
  • sgRNA screening: use a luciferase based assay to correct or knock out luc so looking for decrease of luciferase activity
  • In a model of HBV infection where they have an inducible HBV cccDNA in a single integrated copy the cas9 reduced the DNA but protein was not decreased that much (if you hit episomal DNA you see loss and the disintegration of cut out DNA but if you hit integrated DNA you see repair
  • In their case the integrated DNA was just mutated (A or AA insertion) so in essence a frameshift mutation
  • Future strategies: use two guide RNAs to permit deletions or allow use of nickase. Currently these gRNA use polIII which is very large
  • They are editing the VEGF loci using Sau Cas9 and two sgRNAs
  • In mice with HBV integrated in their genome get some cutting but they need to go to higher doses of Cas9 system
  • Potential future success if reduce viral load as HBV continually release antigen which results in T cell anergy and if reduce the viral load may help to reduce anergy and wake up the immune system

 

Meeting specific # and @

 

#genetherapy

#virology

#adenovirus

 @Duke

10:35 Targeted Endonucleases as Antiviral Agents: Promises and Pitfalls

Keith R. Jerome, M.D., Ph.D., Member, Vaccine and Infectious Disease Division, Fred Hutchinson Cancer Research Center; Professor and Head, Virology Division, Department of Laboratory Medicine, University of Washington

 

  • Can cure Hepatititis C because can stop replication
  • HSV in neurons and live for life: long lived form like HBV
  • Herpes simplex (HSV) establishes in dorsal root ganglion: acyclovir might be useful but came off patent
  • He reached out to advocacy groups to get data on need for HSV cure to convince funding agencies this is important
  • They found it is important to people; 90% want a cure if 5 years away
  • Homing endonucleases: small 800bp high specificity easy to put in vectors more difficult retargeting to other DNA targets
  • www.ltk.uzh.ch/de/dyn
  • HSV homing endonuclease from Cellectis AG targets a 24bp sequence in Ul19; introduces a DSB at target site with 4 bp 3’ overhang;
  • AAV targeted endonuclease delivery; nonimmunogenic; persists in episomal state
  • Exposure to HSV specific HE decreases virus production from neuronal cultures at all stages of replication cycle; if infect and let go for a month but the HE disrupts HSV in acute late-acute and late cycle
  • Developed a mouse model of HSV infection and AAV delivery in vivo system; so the AAV was accessing the nerve endings and going down to the trigenital dorsal ganglia; transport is independent of AAV serotype but transgene expression is HIGHLY dependent on AAV serotype
  • The in vivo HE treatment appears well tolerated however just a casual observation
  • Used NGS with bioinformatic approach to determine off target sites for the most likely mouse loci
  • HE suppresses viral reactivation (used PCR based reactivation assay)

 

Talk Specific @ and #

#drugdelivery

#genedelivery

#AIDS

#genetherapy

#HIV

@fredhutch

@defeatHIV

12:05 pm CRISPR/Cas9 for the Screening of the Human Kinome – A Pilot Study in an Aggressive Pediatric Cancer Cell Line

Simone T. Sredni, M.D., Ph.D., Research Assistant Professor, Neurological Surgery, Northwestern University Feinberg School of Medicine, Ann and Robert H. Lurie Children’s Hospital of Chicago

 

LentiArray CRISPR Kinase Array

  • Malignant rhabdoid tumors (MRT); among most aggressive of pediatric tumors rare but lethal
  • Inactivating mutations in SMARCB1 (INI1 gene)
  • Component of swi/snf chromatin remodeling complex
  • Can originate anywhere in kidney brain and spine
  • Kinase inhibitors may be effective (PLK1, ERBB2, AURAKA) : AURKA inhibitor in phase 2 and giving promising response
  • Used LentiArray viral vector system and stable expressed Cas9
  • Tested 160 kinases in screen; high transfection efficiency
  • PIMs; protooncogenes (proviral common integrations sites in Maloney leukemia; overexpressed in prostate  and hematologic; effects cell cycle cdc25a is a target and cell survival targets as well; PIM3 high copy number in MRT as well as PIM2
  • KO limits proliferation increases senescence and confirmed by MTT with pan PIM inhibitor CS6258 (Cyclene Pharmaceuticals)
  • PLK4 overexpression in peds haploinsufficient mice do make tumors; is it mutated? Yes inactivating mutations
  • PLK4 expression higher through cell cycle – dependent?
  • KI67 was down with Knockdown
  • Decrease in clonogenic assay on plastic not soft agar
  • CFI-400945 is PLK4 inhibitor is ovarian trials
  • Is effective in MRT and comparable to their cas9 knockdown

ssreni@luriechildrens.org

 

@NorthwesternMed

#cancer

#kinome

#brain

#ChildhoodCancerAwareness

 

GENE THERAPIES BREAKTHROUGHS

Tuesday, September 20

7:00 am Registration Open and Morning Coffee

 

KEYNOTE SESSION: GENOME EDITING FOR IN VIVO APPLICATIONS

8:05 Chairperson’s Opening Remarks

Bryan R. Cullen, Ph.D., James B. Duke Professor of Molecular Genetics and Microbiology and Director, Center for Virology, Duke University

8:20 AAV for Gene Therapy and Genome Editing

James Wilson, M.D., Ph.D., Professor, Department of Pathology and Laboratory Medicine, Perelman School of Medicine; Director, Orphan Disease Center and Director, Gene Therapy Program, University of Pennsylvania

In vivo delivery of nucleic acid therapeutics remains the primary barrier to success. My lab has focused on the use of vectors based on adeno-associated virus (AAV) for achieving success in pre-clinical and clinical applications of gene replacement therapy. Most of the current academic and commercial applications of in vivo gene replacement therapy are based on endogenous AAVs we discovered as latent viral genomes in primates. These vectors are reasonably safe and efficient for application of gene replacement therapy. The emergence of genome editing methods has suggested more precise and effective methods to treat inherited diseases in which genes are silenced or mutations are corrected. AAV vectors have been the most efficient platform for achieving genome editing in vivo. We will review our attempts to achieve therapeutic genome editing in animal models of liver disease using AAV.

  • AAV for delivery of vector to CNS
  • Novel AAV Platform – capsid platform – distributed by PENN Vector
  • direct
  • IV
  • into CNS: Head and Spinal MPS – Mucopolysaccharidosis – Class of lyposomal Storage Disorder
  1. AAV9: Binds Glycans with Terminal Gal – BBB

CNS gene transfer in canine model of MPS VII following IV and intrathecal AA( administration

  • Serum
  • CSF: Frontal cortex, Cerebelum, Spinal Cord
  • Intravenous (IV) and Intracisternal (IC) – AA9

Gene Therapy for Motor Neuron Disease: SMA and ALS: PhaseI/II clinical trial of AAV9-SMN IV in infants with SMA1 – Nationwide Children’s

  • High dose +23.3 Month survival

Summary

  • Avexis
  • Abeona
  • Pfizer
  • Nationwide
  • Esteves
  • REGENXBIO

Liver Transduction Following IV Adm of AAV8 Vectors

  • Mouth liver Day 3 vs Day 90
  • Hemophilia B: Therapeutic protein

Urea Cycle Disorder

  • newborn OTCD infant in HA crisis

Goals: efficient but Transient vorrection following AAV* Gene Therapy in Newborn

transfer of caspid – promoter vector  to correct diffect

  • Survival: neonatal gene therapy – two doses of vector, three injections – immunogenic
  • Liver transplantation of neonatal before 1 year of age

Gene Editing

  • gene targeting by ZFNs, TALENs or CRISPR/Cas9
  • WT donor DNA
  • Transgene donor DNA
  • Gene disruptions

In Vivo correction – liver mouse by AAV. CRISPR-SaCas9

  • OTC donor template
  • efficient restoration of OTC Expression in the liver  – mice treated at Neonatal Stage by AAV8.CRISPR-SaCas9 – Vector administration
  • Cas9 – Kinetics of Cas9 – Week 1,3,8 – dilutes with time
  • Should work in Adult mice vs Neonatal mice: Low dose vs High dose
  • Ureogensis increased
  • On-target Deep Sequensing and their Distribution in Neonatal-treated and Adult-treated Animals

In vivo gene editing: mixed results: Site-directed Insertion of hOTCco Gene Cassette in the OTC gene : Controls: WT and spf(ash)

Western Blot: NGS analysis demonstrates on target transgene integration 20 to 32%

High Protein DIet to Evaluate the Efficacy

  • NEW BORN – Gene Targeting in FIX-KO Mouse by CRISPR/Cas9: Infron1, Exon 2 Infron2 Exon 3
  • ADULTS – Gene Targeting in FIX-KO Mouse by CRISPR/Cas9:

Gene therapy must accumulate experience in Animal models: Safety and Efficacy

NEGATIVE RESULTS IN MONKEYS: Analysis of Liver Tissue for Editing and SaCas9

  • CRISPR/Cas9 -mediated Gene knock down of rhPCSK9 in Monkeys (Rhesus Macaque) in LIVER
  • In vitro sgRNAs in monkeys and human cells
  • EGFP sgRNA vs hrPCSK
  • In vivo does not infer In vitro
  • Gene Editing in Human — we are not yet there

9:20 Using CRISPR/Cas to Target and Destroy Viral DNA Genomes

Bryan R. Cullen, Ph.D., James B. Duke Professor of Molecular Genetics and Microbiology and Director, Center for Virology, Duke University

A number of pathogenic human DNA viruses, including HBV, HIV-1 and HSV1, cause chronic diseases in humans that remain refractory to cure, though these diseases can be controlled by antivirals. In addition the DNA virus HPV causes tumors that depend on the continued expression of viral genes. Here, I will present data demonstrating that several of these viruses can be efficiently cleaved and destroyed using viral vectors that express Cas9 and virus-specific guide RNAs, thus providing a potential novel approach to treatment.

  • HBV – as target intervention
  • CRISPR/Cas9 RNA Guided Nuclease System (RGN)
  • NGG- PAM >> dsDNA cleavage >> NHEJ Repair >> Prfect repair >> Completion Repair Cycle
  • vs Mutagenesis – cycle exit
  1. HBV – Inactivation with CRISPR/Cas9: HBV lifecycle of the virus — reverse transcripatse (RT) of caspid and envelope – release antigens in blood invisible to Immune response, SUrfacce Antigen,COre Antigen, X-protein,
  2. Inhibitors of HBV  – cccDNA is stable for decades – virus in blood do not create new virus
  3. tetracycline represses HBV expression
  4. HBsAG va HBeAG: on core, surface, RT and N.S. sgRNA

Vector Delivery Strategies to the Liver

  • Strep pyogenes Cas9 – a diffrence PAM (5′ – NNGRRT-3′) – too large – ~4.8kb, including th ITRs.
  • Can we identify smaller Promoters
  • Packaging Sau Cas9 and two sgRNAs into AAV

Summary

  • using HBV-infected hepatocytes of a humanized liver or transgenic mouse – HBV Dual Target

Using HBV with RT and CRISPR Cas9 — viral load reduced to awake the immune response – allergy stage  – potential for future therapeutics

 

 

9:50 Grand Opening Coffee Break in the Exhibit Hall with Poster Viewing

10:35 Targeted Endonucleases as Antiviral Agents: Promises and Pitfalls

Keith R. Jerome, M.D., Ph.D., Member, Vaccine and Infectious Disease Division, Fred Hutchinson Cancer Research Center; Professor and Head, Virology Division, Department of Laboratory Medicine, University of Washington

Genome editing offers the prospect of cure for infections such as HIV, hepatitis B virus, herpes simplex, and human papillomavirus, by disruption of essential viral nucleic acids or the human genes encoding receptors needed for viral entry. This talk will highlight the most recent laboratory data and the challenges still ahead in bringing this technology to the clinic.

  • Anti HSV – episonal DNA in Neuron, Acylovar – willingness to Participate in studies
  • Anti-HBV –
  • cART – HIV –

Viral replication returns if medication is taken away in two weeks

Herpes Simplex Virus HSV -1 – 50% HSV-2 16%

  1. CRISPR/Cas – difficult vectorization
  2. Targeted endonucleases
  3. Rare-cutting endonucleases in Gene Therapy – target distruction
  4. Derived from Crel enzyme by CELLECTIS AG (Paris) – co-expressed with Trex2 to remove 3″ hangover
  5. AAV as a targeted endonuclease delivery vector – mediated delivery to primary neuronal cultures
  6. Exposure to HSV-specific HE decreases virus production from neuronal cultures – virus production in treated cells
  • HSV-specific HE can disrupt HSV at all stages of the replication cycle – established in vivo
  1. cell planting
  2. AAV-mediated transgene delivery to the mouse TG in vivo – injection whiskerpad – eye scarified vs Eye not scarified
  3. AAV serotype: Transgene expression in TG is highly dependent on AAV serotype
  4. Dose dependence – Trigeminal ganglion (TG) – AAV-mediated transgene delivery to all branches ot TG
  5. In vivo mutugenesis of latent HSV
  6. Specificity – NGS analysis of Off target activity of NV1: Insertion vs Deletion vs NGS analysis of On and Off target activity of HSV1m8
  7. Endonuclease therapy suppresses viral reactivation
  8. Viral eradication: critical determinant: 3 of doses before cure occurs

Conclusion

Delivery system is the most important factor

 

11:05 Nucleic Acid Delivery Systems for RNA Therapy and Gene Editing

Daniel Anderson, Ph.D., Professor, Department of Chemical Engineering, Institute for Medical Engineering & Science, Harvard-MIT Division of Health Sciences & Technology and David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology

High throughput, combinatorial approaches have revolutionized small molecule drug discovery. Here we describe our high throughput methods for developing and characterizing RNA delivery and gene editing systems. Libraries of degradable polymers and lipid-like materials have been synthesized, formulated and screened for their ability to deliver RNA, both in vitro and in vivo. A number of delivery formulations have been developed with in vivo efficacy, and show potential applications for the treatment of genetic diseases, viral infections and cancers.

  1. Nanoparticulate approaches – repair your DNA while you still use it
  2. Barriers to Intracellular Delivery – What organs are most amenable to Targeting
  3. Liver, spleen, bone marrow, kidney
  4. Intracellular Drug Delivery:
  • Modular Pharmaci=ology with siRNA siRNA silences mRNA
  • Turning Nuclaic Acids into Drugs: Sequence Selection,  Mechanical modification (ligand conjugation), Encapsulation

Materials used for RNA Delivery – increase diversity of materials

  • Liquid light material: Combinatorial synthesis of lipid-like materials
  • RNA NAnoparticles – Lipid -siRNA-Nanoformulations targeting TTR in the liver of Primates
  • Mechanism of ApoE mediated iLNP Delivery [Phil Sharp/Alnylam]
  • si delivery to ENdothelium
  • Lipid modified Polymers: Short amino polymers – Total Dose 5 siRNA
  • Nanoformulation Chemistry: Endothelium in many organs(preferred) vs Hepatocytes
  • Immune cells as a target: CD45 or control
  • In vivo mediated Homologous Recombination Gene repair:  Nanoformulation deliver sgRNA
  1. In vivo delivery of sgRNA to endothelium wiht nanoparticle: mediated guide RNA delivery to endothilium: DNA Repair and Protein delivery
  2. Can CRISPR be used to repair a disease gene in vivo – PLASMID encoding Cas9 and GuideRNA + 199nt ss DNA repair template
  3. in vivo CRISPR rescue repairs defect, restores body weight and stops
  4. mRNA with nanoparticles: In vivo delivery- EPO Protein: Mean Human EPO
  • AAV Only
  • AAV +CAs9 – nano – 6% repain is therapeutic
  • In vivo mediated Gene Knockout without the Virus
  1. PCSK( Blood Cholesterol Liver PCSK9 Analysis – 60% gene mutated 65% reduction in CHolesterol — Synthetic system to do gene knockout

 

11:35 PANEL DISCUSSION: CRISPR/Cas: A Realistic and Practical Look at What the Future Could Hold

Moderator: Bryan R. Cullen, Ph.D., James B. Duke Professor of Molecular Genetics and Microbiology and Director, Center for Virology, Duke University

Participants: Session Speakers

Each speaker will spend a few minutes sharing their viewpoints and experiences on where things stand with using the CRISPR/Cas system for in vivo applications. Attendees will have an opportunity to ask questions and share their opinions.

Discussion

  • Ex vivo delivery to Immune cell rather than to the tumor itself
  • solid cells therapy needs to reach each cell alternatives are needed

12:05 pm CRISPR/Cas9 for the Screening of the Human Kinome – A Pilot Study in an Aggressive Pediatric Cancer Cell Line

Simone T. Sredni, M.D., Ph.D., Research Associate Professor, Neurological Surgery, Northwestern University Feinberg School of Medicine, Ann and Robert H. Lurie Children’s Hospital of Chicago

The CRISPR-Cas9 system for genome editing is a powerful tool to identify genes involved in vital biological processes. A systematic functional screening of the human kinome has the potential to reveal molecules that are essential for tumor survival, growth, and migration. We will describe our experience using the Invitrogen LentiArray™CRISPR library to mutate 160kinases in a highly malignant pediatric tumor cell line. We will discuss our approach for screening, monitoring of cells lines, and validation.

  • LentiArray CRISPR Kinase Library – Bet Test 160 Kinase inhibitors
  • MRT – Malignant Rhabdoid Tumors – Children lexx 3 Years old
  • Genetic landmark, histology anatomical location: Kidney, Brain, Spine
  • Kinase Inhibitors and MRT
  • Finding Novel Targets
  1. Invitrogen – lentiArray CRISPR Library – edit 160 kinase genes – Viral Vector design
  • Cas9
  • gRNA-Kinase
  • Positive Control
  • Negative Control

2.  Kinome Screening – Impact on Proliferation 160 – only EIGHT were tested – significal=nly impaired cell profiferation

Retransaction and Confirmation of the identified  targets

PIM – Leukemia virus induce lymphomas: Proviral – PIM-1,2,3 KO

  • Verification of Genome Editing
  • PIMs – Proliferation
  • PIMs – Senescence – Adult hematological diseases and refractory solid tumors
  • PLK4 – Direct Mitosis Regulator – Activated Protein (mRNA) – expression in cytoplasm
  • PLK-4 and Cancer: Over-expression vs Deregulation – Colony Formation – colonygenic
  • Gene expression – Frozen Tumors – abnormality in children and in adults
  • Verification of Gene Editing: Cleavage and deletion
  • PLK-4 as a Cancer Drug Target – inhibitor enzymatic  – PLK-4 Inhibitor Xenografs

 

 

 

 

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LIVE 9/19 4PM – 5:30PM NK CELL-BASED CANCER IMMUNOTHERAPY @CHI’s 14th Discovery On Target, 9/19 – 9/22/2016, Westin Boston Waterfront, Boston

September 19, 2016 by 2012pharmaceutical

LIVE 9/19 4PM – 5:30PM NK CELL-BASED CANCER IMMUNOTHERAPY @CHI’s 14th Discovery On Target, 9/19 – 9/22/2016, Westin Boston Waterfront, Boston

 

 

CHI’s 14th Discovery On Target, 9/19 – 9/22/2016, Westin Boston Waterfront, Boston

http://www.discoveryontarget.com/

http://www.discoveryontarget.com/crispr-therapies/

#BostonDOT16

@BostonDOT

Leaders in Pharmaceutical Business Intelligence (LPBI) Group is a Media Partner of CHI for CHI’s 14th Annual Discovery on Target taking place September 19 – 22, 2016 in Boston.

In Attendance, streaming LIVE using Social Media

Aviva Lev-Ari, PhD, RN

Editor-in-Chief

http://pharmaceuticalintelligence.com

COMMENTS BY Stephen J Williams, PhD

Technological Innovations enabling NK Cell based Cancer immunotherapies

 

4 PM Autologous ex-vivo expanded NK cells for Solid Tumor Immunotherapies

Ali Ashkar DVM PhD McMaster university

 

NK cells

  1. Healer reg NK cells
  2. Inflammatory NK like TAMS
  3. Cytotoxic 90% in blood but in healthy tissue not that much

 

CD56+ CD16- healer NK near fetal maternal interface

 

Ex vivo expansion from breastCA donor most wind up cd56+ they produce IFN gamma

Their model is using ascites and testing in a PDX model but that is immunosuppressed, what about immunocompetent

 

Her2+ cell lines are extremely sensitive what about her2- then? Or triple negative?

 

Do NK cells from lung kill lung cancer cells? Yes both stimulated and unstimulated NK cells specifically

 

Ovarian ascites NK cells can purify and expand and produce cytokine

However they used survival as endpoint so possibly not reflective of NK effect on tumor

 

Other problem with model have to give them IL2 or IL15 or human NK cells would not survive in mice; however humanized mice might work better as don’t need exogenous IL2 or IL15 (mice produce the humanized version of these

 

4:30 Novel CARs introduced into NK cells facilitate Potent Tumor Cell Killing

 

NK-92 based CAR-tumor targeting (TNK) CEA CARTNK

 

CD19 CARTNK  CD123 CARTNK CD38 CAR-TNK

 

The CEA CAR work on colon cancer cell lines and all CEA positive lines however there is a NK resistant LS174T cell line

 

Early killing using a CAR NK don’t see with just NK and introducing a CAR provides killing for resiistant tumors

So the CD18 CARTNK worked well on Raji cells

 

Used the CD123 CARTNK on RPMI 8226 cell line so it would appear that you have to personalize the CAR NK cell model with the markers on each tumor – what about tumor heterogeneity?

In Raji they get about a 50% improvement in tumor killing using a CAR

There is significantly higher CD38 expression in the tumor setting

 

5) Understanding of NK Cell Effector Functions: A Single Cell Lab on a Chip Perspective

 

Tania Konry Northeastern University

 

Dynamics of cancer cell signaling responses to immune cell signals

 

High throughput single cell characterization of the effects of immune cell heterogeneity

 

Phenotypic drug profiling for better targeting of drug-resistant tumors

 

So with a microfluidic can encapsulate a cell with an assay/reagent

Could you encapsulte with nanoparticle?

 

They hold IP for a bead based multiplex assay.  The microfluidic system is a platform to detect heterogeneity among tumor cells.

 

Looking at interaction of T-cell tumor cell and NK cell using multiplex bead assays in a microfluidic system

 

Can detect cell killing within the droplet in the microfluidic system and test different drugs as well as cell death kinetics

For Example they had looked at the interaction of PD-L1 with the NK cell and tumor cell by coencapsulating the tumor and effector cells

 

With breast cancer cell spheroids you can use alginate as a polymer to generate those spheres and can perfuse with drug of your choice

 

TECHNOLOGICAL INNOVATIONS ENABLING NK CELL-BASED CANCER IMMUNOTHERAPY

4:00 Autologous ex vivo Expanded NK Cells for Solid Tumor Immunotherapy

Ali_Ashkar

Ali Ashkar, D.V.M., Ph.D., Professor, Pathology and Molecular Medicine, McMaster Immunology Research Centre, McMaster University

In healthy individuals, the innate immune system, particularly natural killer (NK) cells, are crucial for immune surveillance. In patients with advanced tumors number and activity of NK cells decline significantly. Recent advances in NK cell expansion and activation have generated renewed interest in adoptive NK cell therapy for cancers. We have expanded NK cells from blood of breast, lung and ovarian cancer patients and have investigated their activities against autologous primary tumor cells. In addition, we have established xenograft models with the primary tumors to study the antitumor activities of autologous NK cells against primary tumor cells in vivo. Ex vivo expanded NK cells survive and proliferate in vivo in the presence of autologous PBMCs.

  • CD56 and CD16 – phynotype in Human
  • Healer NK cells (regNK)
  • Inflammatory NK cells (Similar to TAM)
  • In Malignant Ascites – NK cells CD16 negative – changed phynotype from positive
  • Model of autologous NK Cell CAncer Immunotherapy for OC, BC and LC
  • Cytokine Activated Cancer patients vs Health donors
  • Expanded NK cells IV control group and NK injected
  • Stimulated vs unstimulated NK cells: All cells, non-Epithelial, Epithelial cells
  • NK treated and IL-2 + NK Treated vs COntrol (IL-12): Liver vs Lung
  • Ex vivo expanded autologous PB-NK vs allogenous PB-NK – NK from ovarian cancer patients: Peripheral blood vs Ascites
  • Expand NK to produce IFN-gamma
  • Can expanded-NK cells from ascites kill aotologous primary ovarian cancer cells in vivo? Untreated vs NK treated
  • CD3 vs CD56 – in vivo expansion of NK cells in the peritoneal cavity?

4:30 Novel CARs Introduced into NK Cells Facilitate Potent Tumor Cell Killing that Results in Tumor Regression

Rohit_Duggal

Rohit Duggal, Ph.D., Director, Experimental Cellular Therapy, Sorrento Therapeutics

This presentation features an introduction of chimeric antigen receptors (CARs), which provide homing and specificity to cytotoxic cells of the immune system. Novel CARs isolated from Sorrento’s G-MAB library targeting various tumor antigens will be described. The characterization of the NK cells modified to express these CARs will also be described.

  • NK Cell therapy
  1. mmodification od existing NK tumor cells engineered
  2. stem-cells
  • Proof of Concept:
  1. NK-92 antiCEA – CD28z 4inf  – specificity – colon cancer carcinoma : NK-92 vs Anti CEA
  2. improve potency of CAN upon expression of the anti CEA CAR in NK-resistant
  3. STI-NK 0n targeting NK (TNK)
  4. tumor control in orthotopic colon cancer model
  5. Tumor regression in Colon Cancer upon treatment with anti CEA
  6. NK-resistant LS74T colon carcinoma cells
  • GBM patient segmentation by TCGA
  1. STI NK line cytotoxicity against K562 LEUKEMIA LINE
  2. CAR-TNK
  3. Anti CD38 CAR-TNK killing specific to CD38 expressing cells – low killing

Conclution

  • NK Products in Tumor models
  • Tumorigenic capabilities of NK and of STI NK

 

5:00 Understanding of NK Cell Effector Functions: A Single-Cell Lab-on-a-Chip Perspective

Tania_Konry

Tania Konry, Ph.D., Assistant Professor, Department of Pharmaceutical Sciences, Northeastern University

Natural Killer (NK) cells are an essential component of innate immunity that actively inhibit tumor development. Here we present a novel single-cell method of analyzing the mechanisms underlying the cellular interactions of NK cells with multiple myeloma cells. The integrated droplet microfluidics device developed by our group permits compartmentalization of cell pairs and secreted products within sub-nanoliter volumes and thereby controls cell-to-cell communication by limiting it to interactions between the co-encapsulated cells. It allows monitoring of both contact-dependent (immune synapse formation, delivery of lytic hits) and contact-independent cellular interactions (release of cytokines, chemokines) simultaneously. This dynamic single-cell experimental model is expected to provide preclinical information particularly relevant to the scenario of NK cell-cancer cell interactions.

  • DC- T cell
  • Antigen on DC-T interaction
  • DC-T-Tumor cell interaction
  • cytotoxic molecule – secretion
  • Beads
  • modeling Myeloma Cell
  • Dynamic analysis of NK-tumor cell interaction: cell motility >> conjugation >> detachment >> lytic hit >> cell death
  • Brefeldin inhibits cytokine secretion – in presence of NK cells: FAst vs slow kill
  • Cancer cell lysis: EGTA blocks calcium-dependent perforin polymerization
  • Duration od contact, killing time: contact dependent and Independent and Target cell death: Response to PD-L1 blockage
  • Immunotherapy regulation
  • conjugate duration, detection of lytic hit and cell death

Conclusion

  1. Novel 3D Tumor mimicking microenvironment to differentiate aggressive breast cancer from Indolent Cancers and to evaluate cancer Drugs
  2. LAB-on-a CHIP: 3D Tumor cell model

DFCI/NEW Joint Program in Cancer Drug Development

 

5:30 Close of Symposium

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