The Vector

Volume 7, Issue 5: June 2018

Editorial Team

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

Inside This Issue

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

President's Message

Dear Colleagues,

Thank you to each and every one of the 3,474 people who traveled to Chicago, Illinois to attend our 21st Annual Meeting last month! Congratulations to Dr. Helen Heslop, Dr. Dave Schaffer, the Program Committee, abstract reviewers, and each of 30 standing and scientific committees who contributed to the scientific and educational content at the meeting. Though I am not surprised that this was a record-setting year for ASGCT, I am thrilled by just how much we have grown! In one year alone, on the heels of three FDA-approved gene therapies in the U.S., our attendance increased by 31%, abstract submissions by 28%, exhibitors by 40%, and Society membership by 20%. The Annual Meeting continues to be the best place to find out about the latest breakthroughs in the field, meet and connect with new people, and discuss technological developments and clinical successes.

As I assume the role of president, I am looking forward to serving our members by completing the goals established in the 2016 strategic plan and setting a course for the future of our Society. ASGCT is continuing to make trainee advancement a priority by investing in the creation of career development and travel awards. The Society has announced the availability of seven career development awards of $25,000 each and seven career travel awards of $10,000 each. The application period is open through August 1, 2018. Please visit the Grants & Awards page for more information.

The Government Relations as well as the Clinical Trials & Regulatory Affairs Committees, have been hard at work! On May 22-23, ASGCT traveled to Washington, DC and met with officials from fifteen different legislative offices. We advocated for enhanced research funding, continued investment in regulatory innovation, and adequate access to gene and cell therapies, in addition to educating lawmakers about our field. On July 16, we return to Washington to brief the House Medical Technology Caucus on gene therapy, while later this summer we will hold the first-ever ASGCT-FDA liaison meeting, providing a forum to educate and inform the Agency on advances in our field.

Lastly, I wish to remind you all to join us again April 29-May 2, 2019 in Washington, DC for the Society’s 22nd Annual Meeting!


Michele Calos, PhD

Breaking Through

Counteracting CRISPR/Cas9 toxicity in human hematopoietic stem cells by anti-CRISPR peptides

HDAd5/35++ Adenovirus Vector Expressing Anti-CRISPR Peptides Decreases CRISPR/Cas9 Toxicity in Human Hematopoietic Stem Cells
Li, Chang et al. Molecular Therapy - Methods & Clinical Development , Volume 9 , 390 - 401

Summary by: Chang Li1*, André Lieber1
1University of Washington, Department of Medicine, Division of Medical Genetics, Box 357720, Seattle, WA 98195; *corresponding author, Email:

The prokaryote-derived CRISPR/Cas9 nuclease has contributed to revolutionary advances of our ability to edit the mammalian genome. Among its versatile applications, genome editing in hematopoietic stem cells (HSCs) represents a promising cure for genetic disorders. It is thought that the CRISPR/Cas9 need to be expressed only for a short time to achieve permanent genomic modification. Extended expression of nucleases is likely to cause cytotoxic effects on HSCs, as indicated by our previous report using ZFNs1. Here we utilized anti-CRISPR (Acr) peptides naturally existing in phages2 to control the duration of CRISPR/Cas9 activity, and consequently counteract its cytotoxicity in human CD34+ cells.

To deliver the CRISPR machinery into HSCs, we employed a previously developed non-integrating adenoviral vector HDAd5/35++ which is devoid of all viral genes and with enhanced tropism for non-dividing primitive HSCs3. On-target cleavage analyses showed that the designed guide RNAs were capable of modifying the two genomic target sites, an erythroid specific enhancer of BCL11A and a g-globin promoter, resulting in the reactivation of fetal g-globin for potential gene therapy of b-thalassemia and sickle cell disease. After subjecting the transduced CD34+ cells to in vitro erythroid differentiation, cells infected with HDAd-globin-CRISPR showed no significant difference in expansion kinetics and enucleation, compared to untransduced control or cells transduced with HDAd expressing a scrambled non-targeting CRISPR (HDAd-scr-CRISPR). However, CRISPR vector transduced cells formed significantly fewer progenitor colonies than untransduced control, while HDAd-globin-CRISPR caused a more dramatic reduction than HDAd-scr-CRISPR. HDAd expressing a GFP reporter (HDAd-GFP) did not affect the progenitor colony numbers. These data indicated HDAd-CRISPR transduction negatively affected a primitive subset with colony forming capability in CD34+ cells. Further flow cytometry analysis for primitive CD34+ CD38- CD45RA- CD90+ confirmed this observation.

In another functional assay for primitive HSCs involving the transplantation of the transduced cells into irradiated NOD/Shi-scid/interleukin-2 receptor g (IL-2Rg) null (NSG) mice, the engraftment potency of HDAd-CRISPR-transduced cells was severely compromised. HDAd-globin-CRISPR showed stronger effect than HDAd-scr-CRISPR, while HDAd-GFP transduction did not significantly affect engraftment. Notably, cellular composition analyses in the bone marrow revealed that CD34+ HSC population was mostly affected by the CRISPR vector.

To address CRISPR toxicity in primitive HSCs by controlled inhibition of its activity, we constructed an HDAd35++ vector expressing anti-CRISPR peptides, AcrIIA4 and AcrIIA2 (HDAd-Acr)2. The ability of HDAd-Acr to block CRISPR/Cas9 cleavage was first tested in Human Umbilical cord blood-Derived Erythroid Progenitor (HUDEP-2) cells, which faithfully model the suppression of g-globin expression in adult erythroid cells. Simultaneous co-infection with CRISPR and Acr vectors (equal MOI) inhibited on-target cleavage and g-globin reactivation after erythroid differentiation. When HUDEP-2 cells were infected with the HDAd-Acr two days after HDAd-CRISPR transduction, target cleavage was not blocked, leading to g-globin reactivation. These data collectively indicated that HDAd-Acr is capable of blocking HDAd-CRISPR activity after cleaving the target site. While we observed ~90% inhibition of CRISPR activity by delivering equal MOI of HDAd-Acr, complete inhibition is likely achievable by fine-tuning the ratio of CRISPR and Acr vectors.

The HDAd-Acr vector was then tested in CD34+ cells using the same regimen of sequential transduction, followed by transplantation into NSG mice. The resulting engraftment was significantly improved, compared to cells transduced with HDAd-CRISPR alone. Moreover, comparable numbers of human CD34+ HSCs were found in the untransduced setting and the group received sequential transduction with two vectors. Importantly, target site editing in bone marrow human CD45+ cells was similar to pre-transplantation levels, indicating the edited cells survived. After subjecting bone marrow human CD45+ cells to erythroid differentiation, significantly higher percentages of g-globin+ cells and a partial switch from adult globin to fetal g-globin were detected in HDAd-globin-CRISPR + HDAd-Acr samples compared with samples from mice that were transplanted with untransduced cells.

HDAd5/35++ vectors are an attractive vehicle mediating transgene delivery. In addition to its aforementioned efficient transduction rate in non-dividing HSCs and absence of vector/transduction-associated toxicity, other properties include their episomal existence in the nucleus, up to 32-kb large packaging capacity, and the relatively low cost of vector production at high yields. Our recent study employed HDAd5/35++ vectors for in vivo transduction of mouse HSCs4. When combined with selection, we efficiently reactivated g-globin expression in a transgenic b-YAC mouse model via ex vivo and in vivo transduction5. However, while mouse HSCs are less sensitive to DNA double strand breaks (DSB)6, quiescent human HSCs are sensitized to apoptosis after DSB-inducing irradiation7.

Solving the problem of CRISPR/Cas9 toxicity in HSCs is not only relevant for HDAd5/35++ vector mediated CRISPR delivery. A diminished potential to engraft in NSG mice due to prolonged expression of CRISPR/Cas9 has also been reported after CRISPR plasmid8 or mRNA transfection9. Consistent with these reports and our findings in primitive HSCs, recently published studies have also highlighted concerns on CRISPR/Cas9 toxicity in human pluripotent stem cells after CRISPR delivery with Dox-inducible lentivirus vectors or in immortalized human retinal pigment epithelial cells10, 11. Notably, we are not aware of reports about CRISPR-related cytotoxicity after nucleofection of CRISPR ribonucleoproteins (RNPs), consisting of purified Cas9 protein in complex with a synthetic gRNA. In this approach, in contrast to Cas9 (and sgRNA) expression from plasmids, lentivirus, or HDAd5/35++ vectors, the duration of CRISPR activity is restricted by the intracellular turnover of the two components, which most likely reduces potential side effects on the cell.

1. Saydaminova, K, Ye, X, Wang, H, Richter, M, Ho, M, Chen, H, et al. (2015). Efficient genome editing in hematopoietic stem cells with helper-dependent Ad5/35 vectors expressing site-specific endonucleases under microRNA regulation. Molecular therapy Methods & clinical development 1: 14057.
2. Rauch, BJ, Silvis, MR, Hultquist, JF, Waters, CS, McGregor, MJ, Krogan, NJ, et al. (2017). Inhibition of CRISPR-Cas9 with Bacteriophage Proteins. Cell 168: 150-158 e110.
3. Shayakhmetov, DM, Papayannopoulou, T, Stamatoyannopoulos, G, and Lieber, A (2000). Efficient gene transfer into human CD34(+) cells by a retargeted adenovirus vector. Journal of virology 74: 2567-2583.
4. Richter, M, Saydaminova, K, Yumul, R, Krishnan, R, Liu, J, Nagy, EE, et al. (2016). In vivo transduction of primitive mobilized hematopoietic stem cells after intravenous injection of integrating adenovirus vectors. Blood 128: 2206-2217.
5. Li, C, Psatha, N, Sova, P, Gil, S, Wang, H, Kim, J, et al. (2018). Reactivation of gamma-globin in adult beta-YAC mice after ex vivo and in vivo hematopoietic stem cell genome editing. Blood.
6. Doulatov, S, Notta, F, Laurenti, E, and Dick, JE (2012). Hematopoiesis: a human perspective. Cell stem cell 10: 120-136.
7. Mohrin, M, Bourke, E, Alexander, D, Warr, MR, Barry-Holson, K, Le Beau, MM, et al. (2010). Hematopoietic stem cell quiescence promotes error-prone DNA repair and mutagenesis. Cell stem cell 7: 174-185.
8. Bjurstrom, CF, Mojadidi, M, Phillips, J, Kuo, C, Lai, S, Lill, GR, et al. (2016). Reactivating Fetal Hemoglobin Expression in Human Adult Erythroblasts Through BCL11A Knockdown Using Targeted Endonucleases. Molecular therapy Nucleic acids 5: e351.
9. Dever, DP, Bak, RO, Reinisch, A, Camarena, J, Washington, G, Nicolas, CE, et al. (2016). CRISPR/Cas9 beta-globin gene targeting in human haematopoietic stem cells. Nature 539: 384-389.
10. Haapaniemi, E, Botla, S, Persson, J, Schmierer, B, and Taipale, J (2018). CRISPR–Cas9 genome editing induces a p53-mediated DNA damage response. Nature Medicine.
11. Ihry, RJ, Worringer, KA, Salick, MR, Frias, E, Ho, D, Theriault, K, et al. (2018). p53 inhibits CRISPR–Cas9 engineering in human pluripotent stem cells. Nature Medicine.

Society News

ASGCT to Award Up To $245,000 in Support of Students and Trainees

ASGCT recently created 14 new grant opportunities to support members developing their careers. The Society will award up to $175,000 in Career Development Awards and up to $70,000 in Career Travel Awards—applications will be accepted through August 1. For more information on eligibility, applications, deadlines, and required documents, please visit Grants & Awards.

Scientific Symposia Summaries

Novel Treatment Approaches for Respiratory and Gastrointestinal Diseases
Organized by the Respiratory & GI Tract Committee Symposium

Effective gene and cell therapies for respiratory and gastrointestinal diseases are currently lacking due to the complexity of these organs. Recent advances, however, have shown great promise at overcoming several delivery challenges.

Dr. Joseph Zabner from the University of Iowa presented on AAV mediated gene transfer to airway stem cells for the treatment of cystic fibrosis (CF) lung disease. Lung disease accounts for 95% of the morbidity and mortality associated with CF. Thus, targeting stem cells is central to the development of an impactful therapy. To study this, his group relied on the use of a CFTR-null pig model. Dr. Zabner and his colleagues demonstrated that they could generate porcine-specific AAV vectors with specific mutations on the AAV capsid that enhanced tropism for pig airway epithelium. When tested invivo, these porcine-specific AAV vectors were effective at transducing the surface airway cells and more impressive were effective at correcting the CFTR-associated ion transport defect. He also presented on strategies to transduce stem cells which could extend the longevity of gene expression.

Dr. Frank McKeon from the University of Houston discussed strategies for regenerating the lung in patients with chronic lung conditions. As part of his presentation he demonstrated that p63/Krt5 lung stem cells play an essential role in lung regeneration. Dr. McKeon discussed future prospects for treating acute respiratory distress syndrome, necrotizing pneumonia, chronic obstructive pulmonary disease, and CF. His presentation provided an invaluable insight into the stem cell development path for treatments of diseases for which there are currently no cures.

Dr. Liudmila Cebotaru from the Johns Hopkins University presented results from preclinical AAV-mediated gene transfer studies in rhesus macaques. AAV1 vector was evaluated for both efficiency of gene transfer and safety of repeat vector dosing. The goal of her program was to overcome one of the major hurdles of effective gene therapy for CF; namely, the development of neutralizing antibodies in response to repeat delivery. Dr. Cebotaru instilled into the airways of rhesus macaques two AAV1 vectors, one expressing the truncated CFTR (Δ27-264-CFTR) and one expressing GFP. No adverse events related to repeat AAV1 vector dosing were observed, suggesting that AAV1 gene therapy is safe. While, the titers of AAV-1 specific neutralizing antibody increased after each AAV vector dosing, there appeared to be minimal impact on effective CFTR (and GFP) expression as detected by both confocal microscopy and western blot analysis.

Dr. Mark Donowitz from Johns Hopkins University presented his work on the development of human enteroids and organoids. The former are self-perpetuating primary cultured mini-intestines generated from biopsies or surgical resections harvested from normal subjects or diseased patients. These self-perpetuate when provided appropriate growth factors. The latter are derived from induced pluripotent stem cells. Dr. Donowitz showed that enteroids can recapitulate the physiological function of several segments of the GI tract. He also indicated that enteroids, can be used to study human diseases and as such, are useful tools to evaluate promising molecular therapies including gene therapy. Since enteroids are derived from an individual subject, they are ideal for personalized medical approaches. Thus, Dr. Donowitz’s presentation provided invaluable insight and strategies for overcoming barriers for effective gene and cell-based therapies designed for lung and GI tract diseases.

New Directions and Clinical Trials for Muscle and Skeletal Disorders
Organized by the Musculo-Skeletal Gene & Cell Therapy Committee

The first two talks in the symposium focused on cell therapy approaches for muscular dystrophy and osteoarthritis. April Pyle, Ph.D. of UCLA spoke on “A CRISPR-Cas9 deletion strategy that targets the majority of DMD patients in hiPSC-derived muscle cells.” Dr. Pyle described her lab’s results with using the CRISPR-Cas9 system to cut out deleterious mutations in the dystrophin gene and restore partial protein function. Repaired iPSC were subjected to differentiation, but did not fuse well, and engraftment was poor compared to fetal satellite cells. The lab found compounds that seemed to create more fetal-like cells that fused better and were more mature. The lab is also pursuing in vivo delivery of gene editing components to muscle with nanoparticle-mediated delivery. Frank Barry, Ph.D. of the University of Ireland, Galway, spoke about “Adipose stromal cell therapy for osteoarthritis of the knee: Clinical progress and challenges.” Dr. Barry described a clinical trial involving injecting different amounts of adipose stromal cells in which an improvement in pain symptoms occurred. Dr. Barry stressed that lack of consistency of cells makes comparison between studies difficult and has adopted uniform conditions in an ongoing large clinical trial for osteoarthritis.

The second two talks in the symposium focused on AAV-mediated gene therapy. John Gray, Ph.D. of Audentes Therapeutics, Inc., San Francisco, spoke on “The ASPIRO trial for X-linked myotubular myopathy: Carefully taking systemic AAV treatments to the next level.” Dr. Gray described the development of systemic AAV delivery in his previous studies and development of AAV vectors for the present study, involving an AAV serotype 8 vector expressing MTM1 cDNA from a muscle-specific promoter. The vector was shown to be safe and effective in mouse and dog models of X-linked myotubular myopathy. Early results from the Phase 1-2 first-in-human clinical trial of the vector were summarized and indicated signs of clinical efficacy. Manuela Corti, Ph.D. of the University of Florida spoke on “Readministration of AAV vector in Pompe Disease.” Dr. Corti explained that exposure to AAV through gene therapy leads to development of anti-capsid antibodies. Dr. Corti presented a pharmacological regimen to modulate the humoral immune response based on transient B-cell ablation with rituximab and T cell modulation with sirolimus prior to vector exposure. Ongoing clinical trials in Pompe disease appeared to show the safety and efficacy of this approach to prevent antibody formation.

New Directions in Viral Gene Delivery
Organized by the Viral Gene Transfer Vectors Committee

This session had four speakers, two devoted to AAV vector-based gene transfer, one to lentiviral vector-based gene transfer and one to non-invasive imaging of genetically modified cells. Luk Vandenberghe and Hildegard Buning provided updates on discovery platforms for the identification of novel AAV vectors with desired properties, such as various tissue tropisms. Dr. Vandenberghe focused on the use of ancestral sequence reconstruction and high throughput screening to select AAV variants and Dr. Buning focused on various rational design approaches. These approaches focused on creating novel AAV vectors to overcome the major barriers of AAV gene transfer, such as dealing with neutralizing antibodies, minimizing induction of innate and adaptive immune responses, optimizing vector production, episomal transgene persistence, and developing animal models that can better guide human trials. Dr. Bobby Gaspar provided an update on the use of lentiviral vector gene transfer, specifically for diseases of immunodeficiency. An excellent review of the past 17 years of clinical experience was presented. Importantly, 51 of 53 treated ADA-Scid subjects are off enzyme replacement therapy, which has resulted in one approved lentiviral vector-based product and the recent submission of documents for approval of a second product. Dr. Kah-Whye Peng presented exciting data showing how gene transfer of sodium/iodide symporters (NIS) in combination with PET imaging can be used for tracking transgene expression as well as genetically engineered cells in intact animals.

AFM-Téléthon Launches International Call for Projects focused on Bioproduction Breakthroughs in Gene Therapy Technologies

AFM-Téléthon, the French Association fighting neuromuscular disorders and other rare diseases, is pleased to announce the launching of its first international call for proposals for Research Projects on Bioproduction Breakthroughs in Gene Therapy Technologies. This call for proposals opened on June 4, 2018 to any project aimed at initiating, optimizing or translating disruptive research toward large-scale production of gene therapy viral vectors (notably AAV, lentivirus…), with emphasis on vectors that can accommodate large transgenes. Projects will be evaluated in two steps. The first step will be for the applicant to declare its intention to apply (see “How to apply” below) before the evaluation of a detailed application. The deadline for such declaration is July 6th more

Public Policy

ASGCT Advocates for Robust Funding to NIH

ASGCT emphasized the importance of continued generous appropriations to the National Institutes of Health (NIH) in fiscal year 2019 in its outside witness testimony submitted last month to the Senate Committee on Appropriations, Subcommittee on Labor, Health and Human Services, Education, and Related Agencies. These comments focus on the significance of prior NIH contributions to the development of life-altering gene and cell therapies, and the need for continued strong funding to the NIH to support further gene and cell therapy research. Such research has the potential to address immense unmet need and the resulting human and economic costs of diseases such as sickle cell disease, hemophilia, and muscular dystrophy that collectively impact the lives of 10 percent of the US population.

Patient Access to New Gene Therapies Integral to ASGCT Mission

The white paper ASGCT issued last month, Addressing the Value of Gene Therapy: Enhancing Patient Access to Transformative Treatment, asserts, “The most crucial issues to address are identifying and considering the unique value of gene therapy and its potential for transformative and durable improvements in human lives, and the importance of maximizing the ability of patients to access that value.” As part of ASGCT’s mission to advance the clinical application of genetic and cellular therapies to alleviate human disease, the 21st Annual Meeting continued to cover these crucial issues surrounding patient access to approved therapies.

The standing-room-only attendance level at the Ethics and Government Relations Committees’ symposium on value, pricing, and patient access to gene-based therapies reflects the importance of patient access to those in the field. That symposium, as well as sessions at pre-meeting workshops, focused on such topics as value frameworks; development and pricing considerations; the comparative costs of gene therapy relative to other cutting-edge treatments; barriers to patient access; coverage, coding, and reimbursement issues; and considerations for value-based payment. ASGCT will continue the conversation by convening key stakeholders to more specifically address current and proposed solutions to barriers to patient access at a September 24 event in Washington, DC— ASGCT Value Summit: Advancing Patient Access to the Benefits of Gene Therapy.

ASGCT Board Member Named to Second International Summit on Human Genome Editing Program Committee

ASGCT Board Member Matthew Porteus, MD, PhD, was appointed to the international, multidisciplinary program committee to develop the agenda for the Second International Summit on Human Genome Editing, which will take place Nov. 27-29 in Hong Kong. The summit, co-hosted by the Academy of Sciences of Hong Kong, the Royal Society of London, the U.S. National Academy of Sciences, and the U.S. National Academy of Medicine, will bring together a broad range of stakeholders to examine issues including the scientific advances in genome editing since the first international summit on human genome editing in 2015; prospects for developing international regulatory frameworks; and ethical and societal issues surrounding the pursuit of human genome-editing applications. ASGCT commends Dr. Porteus for his appointment to this prestigious group and looks forward to his contribution to addressing these significant issues related to genome editing in a global context.

Industry News

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22nd Annual Meeting
April 29 – May 2 | Washington D.C.