Résumé :
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From The Gene Therapy Center, The Center for Vaccines and Immunity, and the Department of Radiology at Nationwide Children's Hospital and the Powell Gene Therapy Center at the University of FloridaGene replacement is a therapeutic strategy suitable for monogenic disorders caused by mutations of genes <4kb that permit packaging of the full cDNA in adeno-associated virus (AAV). LGMD2D, caused by mutations of the alpha-sarcoglycan gene (SGCA), is a target for this approach. The disease is without treatment and severity parallels the clinical spectrum of dystrophinopathies. Pre-clinical studies of SGCA transfer under control of the tMCK promoter in aSG KO mice prepared the way for the first successful clinical gene transfer trial in muscular dystrophy. Six LGMD2D subjects (ages 11 - 43) participated in a double-blind, controlled trial of rAAV1.tMCK.hSGCA injected into the extensor digitorum brevis (EDB) muscle. All subjects received 3.25 X 1011 vg delivered in 1.5 ml. Four hours preand at 24, and 48 hours post-gene transfer, intravenous methylprednisolone was given to reduce inflammation. EDB muscles were removed at 6 weeks (n =2), 12 weeks (n =1), and 6 months post gene transfer (n = 3). Robust gene expression in the first three patients negated a need to increase the dose in subsequent enrollees. No serious adverse events have been encountered in over 2 years of follow up. Gene transfer of rAAV1.tMCK.hSGCA demonstrated a favorable outcome in 5 of 6 patients with persistent gene expression for as long as 6 months with increase in muscle fiber size at 3 months and 6 months. Immunological findings differed from scenarios encountered in our DMD gene transfer trial where we observed an immune response related to transgene expression (in an area of an endogenous DMD gene deletion and related to novel epitopes expressed on revertant fibers). On the other hand, we did find that robust pre-existing immunity to AAV precluded gene expression in one patient in the LGMD2D trial in whom we observed a rapid amnestic response to AAV. A powerful predictor of pre-existing immunity in this case was the AAV binding Elisa assay, a potential biomarker for patient selection for future gene therapy trials. Inflammatory cells encountered in post gene transfer muscle biopsies in LGMD2D gene transfer were focal, perivascular, without inclination to invade transduced muscle fibers, and were TUNEL and caspase-3 positive favoring an apoptotic fate. These findings using a muscle specific promoter and sustained gene expression for 6 months pave the way for a vascular delivery trial to the isolated limb through the femoral artery (ILPfa) where we can target specific muscle groups to potentially improve clinical function. Using ILPfa we can transduce >80% of quadriceps muscle fibers in a paradigm we plan to take to clinical trial. Supported by National Institute of Arthritis, Musculoskeletal, and Skin Diseases, National Institutes of Health 1U54NS055958 and the Muscular Dystrophy Association, and performed under FDA IND# BB-IND 13434
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