The Vector

Volume 6, Issue 5: September 2017

Editorial Team

Guohua Yi, PhD - Editor, The Vector
Phillip Doerfler, PhD - Associate Editor, The Vector
Melvin Rincon, MD, PhD - Junior Editor, The Vector

Inside This Issue

President's Message
Breaking Through
Society News
Public Policy
Industry News

President's Message

Dear Colleagues,

Gene and cell therapy is entering a new and exciting era. Last week, the FDA approved the first in class CAR T-cell therapy CTL019 (tisagenlecleucel, marketed under the name Kymriah). The approach is the result of more than 20 years of research and the collective efforts of many ASGCT members. As patients benefit from the approval and commercialization of this therapy, it will undoubtedly have a lasting impact on our field driving growth and innovation.

Along with these rapid advances and the new therapeutics that will become available in the near term, ASGCT recognizes that there is a clear need for public education and patient outreach. To that end, the Society, in conjunction with the Hemophilia Federation of America, conducted two webinars for patients and patient advocates on gene therapy for hemophilia B. ASGCT is grateful to Dr. Mark Kay of Stanford University for his efforts in developing the content and presenting the webinars, both of which are available on ASGTC’s website here.

After many months of effort on the part of the Board of Directors, Outreach & Communications Committee, and staff, I am pleased to report that we are in the final stretch of our web site redesign process and are just one month away from the launch of our new site. It will provide more member-friendly experience, including vast improvements to the navigation, content, and aesthetic appeal.

This year has seen several milestones for our Society. Membership has crested at 2,400 individuals – matching the Society’s peak years in 2002 and 2003. Thank you to each and every one of you for joining ASGCT and renewing your membership in our Society. In the coming months, please do not forget to renew your membership for 2018. There are many exciting opportunities to get involved in ASGCT and we look forward to the 21st Annual Meeting in Chicago where there will be an accomplished lineup of plenary speakers for this year’s Annual Meeting. The George Stamatoyannopoulos Lecture will be given by Dr. Kathy High, the Co-Founder, President, and Chief Scientific Officer of Spark Therapeutics. The Presidential Symposium lecture will be given by Dr. Nick Restifo, a Senior Investigator at the National Cancer Institute Center for Cancer Research, Surgery Branch.

To support us through this profound growth, our Society also welcomes two new staff members, Betsy Foss-Campbell as Strategic Alliance Manager and Alex Wendland as Digital Communications Manager, both reporting directly to our Executive Director, David Barrett. In her newly created position, Betsy will manage ASGCT’s myriad and complex relationships with other external entities, supporting components of our Strategic Plan related influencing policy-makers. Betsy has deep experience working with professional research societies and holds master’s degrees in political science and communications. Alex will be driving our Society’s communications efforts through our web and social media presence as well as traditional media. Alex holds a master’s degree in journalism. Welcome Betsy and Alex!

Breaking Through

Long-term microdystrophin gene therapy is effective in a canine model of Duchenne muscular dystrophy

Summary written by: Alberto Malerba, George Dickson, and Caroline Le Guiner

Duchenne muscular dystrophy (DMD) is the most common genetic muscular disease in children affecting approximately 1 in 5,000 live newborn males. DMD is due to mutations in the DMD gene that deplete all muscles of dystrophin protein. Dystrophin has a crucial role in connecting the cytoplasmic actin of the myofibre to the dystrophin associated protein complex at the sarcolemma providing muscle stability. If dystrophin is missing, myofibres are extremely fragile and susceptible to injury and the progressive muscle deterioration leads to muscle weakness, respiratory insufficiency and cardiac failure eventually leading to premature death.

Gene addition strategy for DMD is based on replacing the faulty dystrophin gene with a new healthy copy delivered using the Adeno Associated viral vector (AAV), currently considered one of the most effective and safe viral vector for gene therapy applications. The caveat is that AAV have a limited packaging capacity of 4.7Kb while the dystrophin exonic DNA is about 11Kb and only a short version of the gene (called microdystrophin, MD), containing domains crucial for the protein functionality, can be delivered into muscle cells. The rationale is that patients affected by the allelic form of DMD, called Becker muscular dystrophy, exhibit natural in-frame deletions in their dystrophin gene that is associated to a milder dystrophinopathy.

In the Le Guiner et al study, microdystrophin 1 (MD1) driven by a synthetic muscle and heart specific promoter (Spc5.12) and sequence optimized to enhance its expression in canine cells was packaged into recombinant AAV2/8 vector (rAAV2/8-Spc5.12-cMD1) and delivered systemically in young (2-4 month old) Golden retriever muscular dystrophy (GRMD) dogs, a large animal model considered to be valuable platform to test preclinical gene therapy strategies for DMD. A similar vector codon-optimized for MD expression in mouse previously showed great body wide transgene expression after single intravascular administration in mdx mice, the most used murine model of DMD.

The importance of Le Guiner et al study is that for the first time the functional effects of a gene therapy treatment based on rAAV-MD delivery by systemic administration and without immunosuppression have been studied in a large animal model of DMD for a significantly long period after vector injection. This allowed to investigate relevant clinical outcomes, similarly to what would be applied in a phase I/II clinical trial in DMD boys. In particular this study demonstrates that delivering rAAV2/8-Spc5.12-cMD1 by either locoregional (LR) or systemic intravascular (IV) administration is safe and is associated to a significant expression of cMD1 body wide that leads to the stabilization of the clinical parameters in the treated dogs and to a clinical benefit sustained for at least 24 months after vector administration. Notably no toxicity or deleterious humoral or cellular -mediated immune responses were observed against the transgene product although no immune suppression was performed before or after vector delivery.

The first part of the study is based on the single LR delivery of 1e13 vector genome (vg)/kg of the therapeutic vector rAAV2/8-Spc5.12-cMD1 in a forelimb of 4, 3.5 - 4 month old GRMD dogs with the main aims of testing the safety of the procedure, the level of expression of cMD1 and the related functional efficacy. Three months after vector delivery, dogs were euthanized and multiple muscles were harvested from treated and untreated forelimbs. All treated muscles exhibited significant cMD1 expression ranging from 40 to 60% of cMD1+ fibres. The beneficial effect of cMD1 expression on the histopathology was assessed by analyzing Collagen I deposition and developmental myosin heavy chain expression: both markers were reduced suggesting decrease in fibrosis and reduction of myofibre turnover in muscles of injected limbs compared with contralateral limbs. The improvement in functionality was assessed by testing the muscle extension strength in the wrist of the forelimbs in both injected and non-injected legs before euthanasia. cMD1 expression significantly improved the muscle extension strength in the injected forelimbs compared to the controls. Furthermore Proton 1H-NMR imaging and phosphorous 31P-NMR spectroscopy analyses of muscles of treated forelimbs suggested an improved sarcolemmal membrane stability.

The second part of the study was based on a single intravascular injection of rAAV2/8-Spc5.12-cMD1 in two cohorts of 2 month old GRMD dogs: the first (n=5) received a higher dose of 1e14 vg/kg and the second (n=3) received a lower dose of 2e13 vg/kg (5 times less). WT and untreated GRMD dogs were used as controls. Again, no immunosuppression was performed during the study. Dogs were followed for at least 24 months (2 dogs treated with higher dose) or 7-8 months (remaining 6 dogs). As expected the administration of the higher dose induced markedly more cMD1 expression (20-80% of myofibres depending on the muscles analysed) compared to the lower dose (3-13%) showing a sustained transgene expression for at least 14 months after vector administration. As expected, a correlation between the number of the vector genomes and the % of cMD1+ fibres was observed for both groups of treated dogs. Analysis of fibrosis and muscle regeneration (as performed for the locoregional infusion) in multiple biopsies obtained at different time points confirmed that the administration of the higher dose of rAAV2/8-Spc5.12-cMD1 was accompanied by a clear and sustained reduction of the dystrophic pathology. Furthermore a global clinical score designed to quantify the clinical improvement demonstrated that while untreated dogs and dogs treated with low dose of the vector deteriorated rapidly and had to be euthanized at about 1 year of age, the clinical score of dogs treated with the higher dose were much improved with a stabilization to about 50% and 80% of clinical score  that was associated to an increased lifespan of over2 years after vector administration. Other functional analyses including those assessing dysphagia, respiratory activities and gait quality of the dogs demonstrated a substantial improvement in the high-dose group compared to untreated or low dose treated dogs.

As mentioned, no immunosuppression was performed at any time during these studies so the humoral and T-cell response to cMD1 and the AAV8 capsid were assessed to verify any immunological response. In all treated dogs anti-cMD1 IgG were transiently detected only between 1 and 2 months after vector administration but their level decreased to undetectable levels at the following time points. Furthermore none of the dogs injected with the vector showed detectable T-cell responses directed against the cMD1 protein (measured using IFN-g enzyme-linked immunospot) at any time point. As expected only IgG and neutralizing antibodies were raised against the AAV8 capsid, while no T-cell mediated response was detected.

In conclusion the study shows that both the locoregional and systemic infusion of rAAV2/8-Spc512-cMD1 are safe and clinically relevant in GRMD dogs and suggests that a single transvenous infusion of a similar viral vector optimized for the expression of a human MD may be potentially therapeutic in DMD patients.

Long-term efficacy and safety of insulin and glucokinase gene therapy for diabetes: 8-year follow-up in dogs

Jaén et al., Mol. Ther. Methods Clin. Dev., 2017

Summary by Phillip A. Doerfler, Ph.D.

The progression of gene therapy applications has accelerated in recent years. The successes observed in the many clinical trials and the encouraging approval of gene therapy products in the U.S. and Europe suggests more widespread applications are immanent. Although gene therapy was foremost concerned with monogenic disorders, potential treatments for more complex disorders are now being pursued. Work performed by Maria Luisa Jaén and colleagues illustrates AAV-based gene therapy may be effective in improving the lives of millions affected by diabetes.

Fatima Bosch’s group has been pursuing an effective therapy for diabetes by engineering an in vivo glucose sensor. Through intramuscular injection of AAV1 encoding insulin and glucokinase, the transduced muscle is able to observe hyperglycemia and import glucose. The system is naturally shut down when glucose returns to physiologic concentrations. Having validated this method in experimentally induced diabetic mice and dogs, here the long-term follow-up in dogs was presented 8 years post-treatment.

In a previous study, two diabetic dogs were treated with AAV1 encoding insulin and glucokinase and were followed for long-term safety and efficacy. Through the lives of the dogs, both displayed normal fasting glucose levels compared to age-matched healthy dogs. Insulin levels were monitored and found to be within the healthy range in both dogs. As in other studies, such as hemophilia B, the ~8 years of monitoring shows the maintained, prolonged expression of a transgene product after a single injection and demonstrates the therapy maintains efficacy as the dogs aged.

The authors next monitored the dogs’ response to a glucose load. Not surprisingly, the diabetic dogs displayed initially higher blood glucose levels than normal dogs, but the difference between AAV-treated and untreated diabetic dogs was exceptionally different. The AAV-treated dogs at 1 hour post-glucose load had a blood glucose level 2-fold lower than untreated dogs. Additionally, the AAV-treated dogs returned to within normal blood glucose levels within 2 hours post-glucose load whereas the diabetic dogs maintained very high values. Importantly, glucose levels did not continue to decline in AAV-treated dogs to levels indicative of hypoglycemia. This demonstrates the quick response the in vivo glucose sensor has for hyperglycemia and its ability to normalize glucose levels.

Lastly, vector biodistribution and expression were evaluated. Muscles from the upper and lower leg as well as the liver were analyzed for vector genomes and transgene expression. Vector genomes were detected in both regions of the leg but no vector genomes were detected in the liver. Concomitantly, insulin and glucokinase gene expression was exclusive to the leg.

With the persistence of the genomes, improved response to glucose challenge, and transgene expression, the overall effect of a single injection of AAV1 encoding insulin and glucokinase is potent. The report by Jaén et al. demonstrates the safety and efficacy of gene therapy to treat diabetes, paving the way for great potential change in diabetes treatment options.

Society News

Check out the 20th Annual Meeting Session Summaries

Cardiovascular Symposium

Organized by the Cardiovascular Gene & Cell Therapy Committee

The Symposium was conducted on May 10, 2017 and was Co-Chaired by Michael A. Laflamme, MD, PhD and Charles R. Bridges, MD, ScD.  The symposium was entitled “Targeting Non-coding RNA in Cardiovascular Disease”.  The symposium was very well-attended.

The symposium was kicked off by a presentation entitled; “The Role of Non-coding RNAs in Vascular Pathology and Therapy” given by Andrew Baker, PhD.  Dr. Baker discussed the central role of certain microRNAs – including the roles of mir21 and mir143 in neointimal formation in stents and in pulmonary arterial hypertension.  He also provided new insights into the role of long non-coding RNAs in cardiovascular pathogenesis.  Susmita Sahoo, PhD, gave an outstanding lecture on “RNA Modifications in Cardiac Remodeling and Regeneration”.  Her talk highlighted the emerging evidence that post-translational modifications of RNAs are vital to their stability and translation to proteins.  In particular, she highlighted the role of N6-methyladenosine (mdA) which is upregulated in humans and in preclinical models of myocardial ischemia.  The fat mass and obesity-associated protein (FTO) directly regulates m6A and SERCA2a expression and its overexpression improves cardiac function significantly in murine models of cardiac ischemia.  Mark Feinberg, MD, who’s talk was entitled, “Targeting MicroRNAs in Atherosclerosis” identified critical microRNAs that are implicated in atherogenesis.  As both a scientist and a clinical cardiologist, he provided novel insights into how to translate these discoveries into new therapies for cardiovascular disease.

Traslational Science Symposium

Technology Roadmap for Cell and Gene Therapy – Achieving Large Scale, Cost-effective, Reproducible Manufacturing

A subset of gene based cell therapies for treatment of genetic disorders, regenerative medicine, and immunotherapy, have reached clinical proof of concept so that several of these products are now on the brink of licensure and a few have recently become medicines.  As a new modality, the infrastructure to support cell therapy manufacture and distribution is sparse, and cost of supply remains high. These issues dampen commercial potential and thus may impede broad delivery to patients.  

In 2016, the National Cell Manufacturing Consortium (NCMC) was established to bring together the various organizations involved in cell therapy standards and development, to help enhance the speed and success of cell therapy commercialization.  Krishnendu Roy, who helped to build and now leads the Consortium, presented an overview of the Technology Roadmap for cell therapy, developed by the NCMC.  Dr. Roy highlighted initiatives to support the building of an engineering feedback loop, to support continuous improvements to cell therapy which are informed by clinical evidence; The Marcus Center for Therapeutic Cell Characterization at Georgia Tech is leading an effort in this.  Also, standardization of cell therapy standards are important efforts that are being driven by groups such as the Standards Coordinating Body (SCB) of the Alliance for Regenerative Medicine (ARM).  Finally as a growing industry, the work force is not yet in place.  The development of training programs for cell therapy technicians, for example as an Associates degree, would be beneficial for the U.S. workforce to ensure leadership in the cell therapy industry.  Given the importance of these issues and the role of the ASGCT in the cell therapy industry, the Society is considering building a Committee focused on these topics.

Following this overview, two case studies of cell therapies, ADA modified stem cells (Strimvelis) which is approved in Europe, and Kite’s CD19 CAR which is on the cusp of regulatory approval in the U.S., were presented by Michelle Myers and Marc Better, respectively, with touch points relating to unique challenges in cell therapy for clinical and commercial development.  Each talk highlighted the challenges in building CMC teams, and detailed issues relating to the definition and establishment of critical quality attributes (CQAs) and comparability for this new class of products.

20th Annual Meeting video recordings are now available online!

Sessions include:

  • The ABC's of AAV
  • Getting Started in Genome Editing
  • Designing the Next Generation of Viral Vectors
  • Clinical Advancement of Gene Editing – Moving Science to the Clinic
  • Plus 25 more sessions and more than 34 hours of recordings

These recorded sessions are only available to ASGCT Members and through the Members Only Section. You must log in as a Member to view videos of the Education, Scientific, and Plenary Sessions.

ASCGT Call for Award Nominations!

The Outstanding Achievement Award (OAA) recognizes an ASGCT Member who has achieved a pioneering research success, a specific high impact accomplishment, or a lifetime of significant scientific contributions to the field of gene and cell therapy.

The Outstanding New Investigator Award (ONI) recognizes ASGCT Members who are newly independent researchers, 10 years or fewer from their first independent research position, based on their contributions to the field.

The Sonia Skarlatos Public Service Award (PSA) will recognize a person or group that has consistently fostered and enhanced the field of gene and cell therapy through governmental agencies, public policy groups, public education, or non-governmental charitable organizations.

Nominations are due to the ASGCT Executive Office by December 15, 2017 and may be submitted directly to:

Public Policy and Government Relations

ASGCT Signs On to Oppose Restrictions on Fetal Tissue and Embryonic Stem Cell Research

During the summer Congressional recess, HR 3358—the House Labor, Health and Human Services (HHS), and Education Appropriations Act, 2018—was introduced in the House. This bill contains a provision that would prohibit federal funds from being used to conduct or support research using human fetal tissue if such tissue is obtained pursuant to an induced abortion. Such restrictions, if enacted, would obstruct research on legally obtained tissues, which may affect the development of new treatments, including gene and cell therapies, for a wide range of serious diseases. Last week, ASGCT joined 62 other organizations in signing on to a letter opposing restrictions on the use of federal funding for fetal tissue or embryonic stem cell research. The recipients of the letter were Senator Thad Cochran (Miss.), Chairman of the U. S. Senate Committee on Appropriations; Senator Patrick Leahy (Vt.), Ranking Member of the U.S. Senate Committee on Appropriations; Senator Roy Blunt (Mo.), Chairman of the U.S. Senate Appropriations Subcommittee on Labor, HHS, Education, and Related Agencies; and Patty Murray (Wash.), Ranking Member of the U.S. Senate Appropriations Subcommittee on Labor, HHS, Education, and Related Agencies. The letter discourages the Senate from adopting restrictions in appropriations, such as those found in HR 3358.

HR 3358 and Senate Subcommittee Propose Increased NIH Funding

HR 3358, the 2018 House Appropriations bill for Labor, Health and Human Services, and Education proposes adding $1.1 billion in funding for the National Institutes of Health (NIH) over last year. The Senate version of the bill, after markup by the subcommittee yesterday, contains a proposed increase of $2 billion in funding for the NIH, which includes funding awarded for gene therapy research. The NIH estimates providing funding of $277 million for gene therapy research and $34 million for gene therapy clinical trials in fiscal year 2017. The Senate Subcommittee voted to report the bill to the full Appropriations Committee for markup, scheduled for today, September 7. However, the total proposed expenditures through all fiscal year 2018 appropriations bills exceed the spending cap that Congress established in 2011. Yesterday the President and Congressional leadership agreed to extend the debt ceiling and to create a stopgap three-month spending bill to fund government spending, both until December 15. These actions will give Congress and the White House additional time to reach consensus on the fiscal 2018 spending level, a long-term fix for the debt limit and an omnibus spending bill. 

Expert Input Requested on Gene Therapy Trials for Rare Diseases

The National Center for Advancing Translational Sciences (NCATS) has issued a Request for Information (RFI) on opportunities to increase the efficiency of human gene therapy trials in rare diseases. NCATS is particularly interested in input regarding the use of viral vectors as platforms for therapeutic gene delivery to specific organs or cell types, and how to most efficiently develop such platforms in clinical trials for the treatment of multiple diseases. The potential impact of a platform approach is great for rare genetic diseases, given the large number of rare diseases that may be impacted by gene therapy and the limited financial resources available for gene therapy trials for each rare disease. NCATS encourages feedback from all stakeholders, including basic, translational and clinical scientists; rare disease patients and health advocates; health care providers; biotechnology, venture capital and pharmaceutical industry members; and representatives from other governmental entities. Please submit responses to by September 19, 2017.

Industry News

Viewpoint: Editorial Commentary on the First CAR-T Cell Therapy Approved in the US

Written by Guohua Yi, PhD - Editor, The Vector

On August 30, the Food and Drug Administration approved the first ever CAR-T cell therapy, tisagenlecleucel (Kymriah®), developed by Novartis in partnership with the University of Pennsylvania, for the treatment of relapsed or refractory B-cell acute lymphoblastic leukemia (ALL) in children and young adults. This historic approval opens a new era of gene and cell therapy, encouraging more CD19-directed CAR-T cell therapies in the pipeline to be approved, and more importantly, energizing other gene therapy strategies to be tested against a variety of diseases.

Tisagenlecleucel (also known as CTL019) is modified T cells expressing a chimeric antigen receptor (CAR) that combines the CD19-targeting murine single-chain fragment antibody, spacer and transmembrane domains derived from human CD8-α, a human intracellular 4-1BB (CD137) co-stimulatory domain and a human intracellular CD3ζ T-cell-activation domain. After genetically modifying the patients’ own T cells with lentivirus carrying the above chimeric antigen receptor, the engineered T cells are infused back into the patients to target and kill leukemia-causing B cells that express CD19 molecules on the surface. In a clinical study of 63 patients with highly refractory leukemia who received treatment from April 2015 to August 2016, CTL019 showed dramatic efficacy— 82.5% (52 out of 63 patients) went into remission and 79% (50 patients) survived over 12 months. Given that these patients had received conventional therapy, but had been incurable, such high remission rates and survival probabilities signify an applaudable advancement in leukemia treatment.

As this “living-drug” contains re-engineered live cells, it is far more difficult to manufacture than traditional drugs. Therefore, how to shorten the turnaround time and make treatments more consistent are the main concerns following its approval. Moreover, frequently-occurring side effects, such as cytokine release syndrome (CRS), necessitate expert care after infusion of engineered T cells into the patients. Nevertheless, Novartis plans to limit initial treatment utilizing CTL019 to 30–35 accredited treatment centers in the US, providing training to transplant teams on the administration of the therapy and the management of CRS, as well as methodologies to monitor performance and compliance by Norvatis-authorized personnel to ensure the safety of patients. It is worth noting that another anti-CRS drug, tocilizumab (Actemra®, Genentech Inc.), has also been approved by the FDA as an accompanying drug for CTL019.

Beyond the approval of CTL019, there are some questions that remain to be answered, such as, 1) CTL019 can not only kill leukemic B cells, but also kill normal B cells, how does treatment influence normal B cell responses and how to solve or ameliorate this side effect? 2) Why do some patients not respond to treatment? 3) Are there any potential secondary malignancies associated with the treatment due to lentivirus-caused insertional mutagenesis? 4) Why did some patients display neurotoxic side effects?  5) Can CTL019 be expanded to treat other B cell malignancies?

Regardless, as the developer of CTL019, Dr. Carl June said, “"This therapy is a significant step forward in individualized cancer treatment that may have a tremendous impact on patients' lives." With the approval of the first gene therapy, we have enough reasons to believe that a new era of gene and cell therapy has begun, and that more and more gene therapy strategies will be applied to the bedside in the foreseeable future.


Register for the ASGCT Policy Summit

September 23-24, 2024 | Washington, D.C.

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