Jacqueline N. Robinson-Hamm1, Christopher E. Nelson1, Ruth M. Castellanos Rivera2, Annemieke Aartsma-Rus3, Aravind Asokan2,. Charles A. Gersbach1.
Cancer-Immunotherapy, Cancer Vaccines II 504. Restoration of Dystrophin Expression by Gene Editing with S. aureus Cas9 in Models of Duchenne Muscular Dystrophy
Jacqueline N. Robinson-Hamm1, Christopher E. Nelson1, Ruth M. Castellanos Rivera2, Annemieke Aartsma-Rus3, Aravind Asokan2, Charles A. Gersbach1 1 Biomedical Engineering, Duke University, Durham, NC, 2Genetics and Biochemistry & Biophysics, University of North Carolina at Chapel Hill, Chapel Hill, NC, 3Human Genetics, Leiden University Medical Center, Leiden, Netherlands Duchenne muscular dystrophy (DMD) is the most common fatal genetic disease characterized by progressive muscle wasting, loss of ambulation, and typically death in the third decade of life due to respiratory and cardiac complications. DMD results from deleterious mutations in the dystrophin gene that disrupts the translational reading frame and causes a complete lack of dystrophin protein. Becker muscular dystrophy (BMD) is similar to DMD in that it is the result of deletions in the dystrophin gene. However these deletions maintain the translational reading frame and result in the production of an internally truncated but partially functional dystrophin protein. The BMD phenotype is typically less severe than DMD, and thus converting DMD to a BMD-like phenotype by restoring the dystrophin reading frame is a widely explored therapeutic strategy. CRISPR/Cas9 can target precise loci to make specific DNA sequence changes in the genome. We have previously used S. pyogenes Cas9 (SpCas9) to restore dystrophin expression in immortalized myoblasts from DMD patients by deleting dystrophin exon 51 to repair the disrupted reading frame. A promising therapeutic application of this strategy involves in vivo viral delivery of the CRISPR/Cas9 system by AAV, however AAV cannot efficiently package the large SpCas9 gene along with full size promoters. Therefore we have made use of AAV delivery of the smaller S. aureus Cas9 (SaCas9) to delete exon 23 in the mouse dystrophin gene in vivo in the mdx mouse model of DMD and showed restored dystrophin expression and functional recovery. Here we are continuing this work by preparing a SaCas9 system targeted to the human dystrophin gene. gRNAs were designed to target the intronic regions flanking exon 51 and tested in vitro in HEK293T cells as well as immortalized DMD patient myoblasts that are correctable by removal of exon 51. The expected deletion was confirmed by PCR and sequencing of the genomic DNA and dystrophin cDNA. Western blot of lysates from differentiated cells confirmed restoration of dystrophin protein expression. We have also demonstrated exon 51 deletion in vivo following AAV delivery of this CRISPR/Cas9 system to the muscles of a mouse model containing the full length human dystrophin gene. Ongoing work involves testing this approach in a novel dystrophic mouse model carrying a mutated version of the human dystrophin gene that is correctable by exon 51 deletion. This work is important to the continued development of a translational strategy for gene editing as a potentially curative treatment for DMD.
Cancer-Immunotherapy, Cancer Vaccines II 505. VGX-3100 Drives Regression of HPV16/18 CIN2/3 and Robust Cellular Immune Responses in Blood and Cervical Tissue in a Blinded, Randomized, Placebo-Controlled Phase 2B Study
Matthew Morrow1, Connie Trimble2, Xuefei Shen3, Michael Dallas1, David Weiner4, Jean Boyer3, Jian Yan1, Kimberly Kraynyak1, Albert Sylester1, Mary Giffear1, Kathleen MarcozziPierce1, Divya Shah1, Kate Broderick3, Amir Khan1, Jessica Lee1, Laurent Humeau3, Niranjan Sardesai1, Mark Bagarazzi1 1 Inovio Pharmaceuticals, Plymouth Meeting, PA, 2Johns Hopkins University, Baltimore, MD, 3Inovio Pharmaceuticals, San Diego, CA, 4University of Pennsylvania, Philadelphia, PA
Objectives: Assessment of the safety and efficacy and immunogenicity of VGX-3100 in women with biopsy-proven CIN2/3 with concurrent HPV16 and/or HPV18 infection. Methods: The randomized, placebo-controlled, double-blind study, which was stratified by age and severity of CIN, evaluated cervical tissue changes after three 6 mg intramuscular doses of VGX-3100 followed by electroporation with Inovio’s CELLECTRA(r)2000 device at weeks 0, 4, and 12. Results: Among 167 vaccinated women, the study met its primary efficacy endpoint; the percentage of patients who had regression of CIN2/3 to CIN1 or no disease at 6 months post third dose was significantly higher in the VGX-3100 group compared to placebo (p=0.034). In addition, the trial demonstrated the ability of VGX-3100 to clear HPV infection concurrent with regression of CIN2/3 (p=0.003). Post-hoc immune analysis also revealed significantly elevated immune responses in treated patients who had CIN2/3 regression concurrent with HPV clearance when compared to those who did not. This included the presence of CD8+ T cells in the blood exhibiting CD137 expression concurrent with perforin (p=0.032) as well as perforin in addition to granzyme A (p=0.036) as well as an influx of CD8+ T cells into cervical tissue (p=0.008). Conclusion: The successful phase 2b results represent a significant milestone in the development of active immunotherapies to treat HPV-related dysplasia and cancer. The data generated from the trial reveal a significant clinical benefit afforded by treatment with VGX-3100 and underscore the mechanism of action of HPV specific T cells. Thus VGX-3100 has the potential to provide an important alternative or adjunct to surgery in treating CIN 2/3.
506. In Vivo Transduction of T Cells: The Future of Immunotherapy?
Inan Edes1, Irene Schneider2, Qi Zhou2, Veit R. Buchholz3, Christoph Kemna1, Dirk H. Busch3, Christian J. Buchholz2, Wolfgang Uckert1 1 Molecular Cell Biology and Gene Therapy, Humboldt University Berlin, Berlin, Germany, 2Molecular Biotechnology and Gene Therapy, Paul-Ehrlich-Institute, Langen, Germany, 3Institute for Medical Microbiology, Immunology and Hygiene, Technical University Munich, Munich, Germany
Genetically engineered T cells carrying antigen-specific chimeric antigen receptor (CAR) or T cell receptor (TCR) genes are highly effective in immunotherapy of leukemia and other solid cancers. At present, the modification of T cells requires an ex vivo gene transfer using viral or non-viral vector systems and the subsequent expansion of the engineered T cells to yield therapeutic numbers. However, extended periods of cell culture can lead to an alteration of the T cell phenotype due to antibody and cytokine-driven activation and may negatively influence T cell functionality. To counteract these drawbacks, efforts were undertaken to engineer naive or memory T cells in vitro. However, these T cell subsets are already Molecular Therapy Volume 24, Supplement 1, May 2016 Copyright © The American Society of Gene & Cell Therapy
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