No abstract

Objectives Alpha-sarcoglycan deficiency is a severe form of muscular dystrophy (LGMD2D) without treatment. Gene replacement represents a strategy for correcting the underlying defect. Questions related to this approach were addressed in this clinical trial, particularly the need for immunotherapy, and persistence of gene expression. Methods A double-blind, randomized controlled trial using rAAV1.tMCK.hSGCA injected into the extensor digitorum brevis (EDB) muscle was conducted. Control sides received saline. A three-day course of methylprednisolone accompanied gene transfer without further immune suppression. Results No adverse events were encountered. SGCA gene expression increased 4-5 fold over control sides when examined at 6 weeks (two subjects) and 3 months (one subject). The full sarcoglycan complex was restored in all subjects and muscle fiber size was increased in the 3-month subject. AAV1 neutralizing antibodies were seen as early as 2 weeks. Neither CD4+ nor CD8+ cells were increased over contralateral sides. Scattered foci of inflammation could be found but showed features of programmed cell death. ELISpot showed no IFN-γ response to α-SG or AAV1 capsid peptide pools with the exception of a minimal capsid response in one subject. Restimulation to detect low frequency capsid specific T cells by ELISpot assays was negative. Results of the first three subjects successfully achieved study aims precluding the need for additional enrollment. Interpretation The finding of this gene replacement study in LGMD2D has important implications for muscular dystrophy. Sustained gene expression was seen, but studies over longer time periods without immunotherapy will be required for design of vascular delivery gene therapy trials

Objective The aim of this study was to attain long-lasting alpha-sarcoglycan gene expression in LGMD2D subjects mediated by adeno-associated virus (AAV) gene transfer under control of a muscle specific promoter (tMCK) Methods rAAV1.tMCK.hSGCA (3.25 × 1011 vg) was delivered to the extensor digitorum brevis (EDB) muscle of three subjects with documented SGCA mutations via a double-blind, randomized, placebo controlled trial. Control sides received saline. The blind was not broken until the study was completed at 6 months and all results reported to the oversight committee. Results Persistent alpha-sarcoglycan gene expression was achieved for six months in two of three LGMD2D subjects. Markers for muscle fiber transduction other than alpha-sarcoglycan included expression of major histocompatability complex I (MHC I), increase in muscle fiber size, and restoration of the full sarcoglycan complex. Mononuclear inflammatory cells recruited to the site of gene transfer appeared to undergo programmed cell death demonstrated by TUNEL and caspase-3 staining. A patient failing gene transfer demonstrated an early rise in neutralizing antibody titers and T cell immunity to AAV validated by enzyme-linked immunospot (ELISpot) on the second day post gene injection. This was in clear distinction to other participants with satisfactory gene expression. Interpretation The findings of this gene replacement study in LGMD2D subjects have important implications not previously demonstrated in muscular dystrophy. Long-term, sustainable gene expression of alpha-sarcoglycan was observed following gene transfer mediated by AAV. The merit of a muscle specific tMCK promoter, not previously used in clinical trial was evident, and the potential for reversal of disease was displayed.

Charcot–Marie–Tooth (CMT) neuropathies represent a heterogeneous group of peripheral nerve disorders affecting 1 in 2,500 persons. One variant, CMT1A, is a primary Schwann cell (SC) disorder, and represents the single most common variant. In previous studies, we showed that neurotrophin-3 (NT-3) improved the tremblerJ (TrJ) mouse and also showed efficacy in CMT1A patients. Long-term treatment with NT-3 was not possible related to its short half-life and lack of availability. This led to considerations of NT-3 gene therapy via adenoassociated virus (AAV) delivery to muscle, acting as secretory organ for widespread distribution of this neurotrophic agent. In the TrJ model of demyelinating CMT, rAAV1.NT-3 therapy resulted in measurable NT-3 secretion levels in blood sufficient to provide improvement in motor function, histopathology, and electrophysiology of peripheral nerves. Furthermore, we showed that the compound muscle action potential amplitude can be used as surrogate for functional improvement and established the therapeutic dose and a preferential muscle-specific promoter to achieve sustained NT-3 levels. These studies of intramuscular (i.m.) delivery of rAAV1.NT-3 serve as a template for future CMT1A clinical trials with a potential to extend treatment to other nerve diseases with impaired nerve regeneration.