T-cell Response After Administration of AVVrh74.MHCK7 For Duchenne Muscular Dystrophy: Result from Open Labeled Control Trial

AAV or transgene associated T-cell Response may increase cytotoxicity and loss of transgene expression. In the earliest AAV2 factor IX clinical trials, transient transgene expression was seen for up to eight weeks before loss in a subset of patients. Subsequently, it was determined to be an AAV capsid-specific CD8+ cytotoxic T cell response that likely led to the decline of factor IX expression. Therefore, regulatory agency require sponsor to monitor the T-cell response against AAV viral vectors and transgene and their impact on safety and efficacy in clinical subjects.

Vector design and Administration to Clinical Subject of Phase 1a (nonrandomized control trial) for treatment of Duchenne Muscular Dystrophy

The gene construct containing the MHCK7 promoter and micro-dystrophin transgene packaged into the AAVrh74 capsid were administered a single dose of 2.0 × 1014 vg/kg rAAVrh74.MHCK7.micro-dystrophin in approximately 10 mL/kg. This dose was infused through peripheral limb vein over 1.25 to 1.5 h. 

Brief Protocol for ELISpot Assay for T cell Response:

Separation of Peripheral blood mononuclear cells (PBMCs) from whole blood using density gradient centrifugation
Plating of PBMCs in IP filter plates (Millipore) at the optimum cell density of approximately 200,000 PBMCs/wells. IP plate wells were pre-coated with monoclonal antibody provided in human interferon-γ ELISpot kit (U-Cytech Biosciences).
Stimulation of PBMCs with Viral vector (AAVrh74) capsid protein & Transgene (micro-dystrophin), positive controls (Concanavalin A), negative cotnrol ( 0.25% dimethylsulfoxide )
  • Three peptide pools for the AAVrh74 capsid protein (Genemed Synthesis, San Antonio, TX)        containing 34-36 peptides, each 18 amino acids long and overlapping by 11 residues.
  • Three peptide pools encompassing the micro-dystrophin transgene (Genemed Synthesis) containing 56 peptides, each 18 amino acids long and overlapping by 11 residues.
  • Concanavalin A (Sigma, 1 µg/mL) served as a positive control and 0.25% dimethylsulfoxide as a negative control.
  • Peptides were added directly to the wells at a final concentration of 1 µg/mL.
  • The plate was incubated at 37°C in 5-7% CO2 and 100% humidity for 36-48 hours.
  • Plate was developmed using biolynated detection reagent    
  • Interferon-γ spot formation was counted using Immunospot software (Series 3B Analyzer, Cellular Technology, Ltd, Cleveland, OH).
The study reported no adverse immune responses occurred. The study reported that  a transient increase in T-cell response toward AAV peptide pools 1 to 3 as early as 14 days after gene delivery. Increases in AAVrh74 antibodies were observed in all 4 patients, increasing by 14 days (range, 1:800-1:12 000) with post–gene therapy titers peaking around day 30 (range, 1:13-1:26 million) and remaining stable through 1 year.
No remarkable T-cell responses were observed toward 3 distinct peptide pools of micro-dystrophin transgene. 

Outcome: The safety of systemic delivery using the rAAVrh74.MHCK7.micro-dystrophin cassette at did not generate the clinically significant T-cell response in four study subject. The study also signifies the minimal risk for T-cell mediated cytotoxicity after administration of the rAAVrh74.MHCK7 microdystrophin

Sponsors: Sarepta Therapeutics

Sarepta Therapeutics Pipeline
Mendell et al 2020, Lancet
Rabinowitz et al 2019, Viruses
Ucytech Biosceinces