Sustained alpha-sarcoglycan gene expression after gene transfer in limb-girdle muscular dystrophy, type 2D

Abstract

Objective: The aim of this study was to attain long-lasting alpha-sarcoglycan gene expression in limb-girdle muscular dystrophy, type 2D (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 vector genomes) was delivered to the extensor digitorum brevis muscle of 3 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 were reported to the oversight committee.

Results: Persistent alpha-sarcoglycan gene expression was achieved for 6 months in 2 of 3 LGMD2D subjects. Markers for muscle fiber transduction other than alpha-sarcoglycan included expression of major histocompatibility complex 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 terminal deoxynucleotide transferase-mediated deoxyuridine triphosphate nick-end labeling 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 on the second day after 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 a clinical trial, was evident, and the potential for reversal of disease was displayed.

Figures

Figure 1

a). Subjects 4–6: post gene transfer tissue sections from EDB muscles were stained with antibody to α-SG. Subjects 4 and 5 showed increased staining on treated side (T) compared to control side (C). Subject 6 showed no difference in α-SG staining intensity pre- and post-gene transfer (findings verified by Bioquant Image Analysis®) (scale bar = 150 μm). b). Subjects 4–6: western blots show increased α-SG gene expression on the side of gene transfer compared to the contralateral side (treated on left, control on right) for Subjects 4 and 5; residual gene expression from mutant protein prominently exhibited in Subject 4. Subject 6 showed no increase in α-SG expression comparing pre- and post-gene transfer gels. WBs normalized to actin (lower band) and each is compared with normal (N) muscle for comparison. c). Subject 5: β-sarcoglycan staining (Subject 5) demonstrates restoration on the side of gene transfer with absent staining on control side (scale bar = 100 μm). Other sarcoglycans (delta and gamma) were also restored (not shown).

Figure 2

MHCI staining of sarcolemmal membrane of muscle sections on treated (T) and control (C) sides. Subjects 4 and 5 show staining on the treated (a, c) but not the control (b, d) sides. Subject 6 shows no MHCI staining on treated side (e) or control side (f). (scale bar = 100 μm). Microvascular circulation is positive for MHC I on both sides in all subjects.

Figure 3

a). Subject 4 (EO2-004): α-SG stimulated IFN-γ ELISpot assays showed no increase in spot forming colonies (SFC) per million peripheral blood mononuclear cells (PBMCs)at any time point (prior to gene transfer −14 days up to day 365). AAV1 capsid peptide pool stimulation (CP2 blue and CP3 red) were positive for T cell response (exceeded confidence limits of >50 spot forming cells /million PBMCs) at days 14 and 28. The response to AAV1 capsid pool 1 (CP1 green) was minimally positive at day 14. b). Subject 5 (E02–005):): α-SG and AAV1 capsid INF-γ ELISpot assays were negative preceding and throughout the course of gene transfer up to day 365 with exception of a slight reaction at day 194 to AAV1 capsid pool 2. c). Subject 6 (E02–006): the α-SG stimulated IFN-γ ELISpot assays (black) were consistently negative pre- and post-gene transfer. However, this patient showed a prolific AAV1 capsid response as early as day 2 and also present on day 7. This was distinctly earlier than other patients in Cohort 2 or in Cohort 1(previously published7).

Figure 4

Neutralizing antibody titers to AAV1 on Subjects 4–6 starting pre (day −14) through day 182 (D182) post-gene transfer. In all three subjects serum neutralizing antibody titers to AAV1 were very low (see Table 2 for pre-treatment titers). Follow up studies showed a slow rise in serum AAV1 titers in Subjects 4 and 5. This contrasted with the rapid rise and peak elevation by week 1 in Subject 6. Plateau in elevation for Subject 6 (red) represents limit of assay.

Figure 5

a) Subject 4. Focal collection of CD8+ T cells shown in perimysial connective tissue of muscle biopsy post gene transfer. b). Subject 4. TUNEL staining demonstrates inflammatory cells undergoing apoptosis confirmed by anti-caspase 3 antibody (inset).