Limb-girdle muscular dystrophy type 2D gene therapy restores alpha-sarcoglycan and associated proteins

Abstract

Objective: alpha-Sarcoglycan deficiency results in a severe form of muscular dystrophy (limb-girdle muscular dystrophy type 2D [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 muscle was conducted. Control sides received saline. A 3-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 (2 subjects) and 3 months (1 subject). The full sarcoglycan complex was restored in all subjects, and muscle fiber size was increased in the 3-month subject. Adeno-associated virus serotype 1 (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. Enzyme-linked immunospot (ELISpot) showed no interferon-gamma response to alpha-SG or AAV1 capsid peptide pools, with the exception of a minimal capsid response in 1 subject. Restimulation to detect low-frequency capsid-specific T cells by ELISpot assays was negative. Results of the first 3 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.

Trial registration: ClinicalTrials.gov NCT00494195.

Conflict of interest statement

Conflicts of Interest

There are no contributors to this manuscript who have any conflict of interest

Figures

Figure 1

A) Extensor digitorum brevis (EDB) muscle shown with dotted lines indicating plane of injection from apex to base (long axis) and across muscle from medial to lateral. B) The ultrasound picture shows the injection needle inserted through the long axis of the muscle (arrow). The arrowheads (two above and two below) define the margins of the muscle.

Figure 2

A) Post gene transfer tissue sections from EDB muscles for subjects 1, 2, and 3. The left panel shows α-SG gene expression on the side of vector injection (T) compared to muscle from opposite side (C)(scale bar = 100 μm). B) Western blots from all three cases show increased α-SG gene expression on the side of gene transfer compared to the contralateral side (treated on left, control on right) showing residual gene expression from mutant protein. α-SG is normalized to actin (lower band). C) β-Sarcoglycan staining demonstrates restoration on the side of gene transfer. Other sarcoglycans (γ and δ) were also restored (not shown). Muscle from subject (#2) is shown (scale bar = 100μm). D) Densitometry measurements show α-SG gene expression increased 4 to 5 fold on the side of vector injection.

Figure 3

A) Compares the number of CD4+ mononuclear cells/ mm2 area between control and the side of gene transfer; B) Compares the number of CD8+ mononuclear cells/ mm2 area between control and the side of gene transfer; C) MHC I staining of muscle sections shows lack of staining of muscle fibers on control (left) side, while the side of gene transfer shows distinct staining of the sarcolemmal membrane in this subject (#3). Microvascular circulation in MHC I stained on both sides (scale bar = 100 μm). All three cases showed this same staining pattern on control and gene transfer sides; D) TUNEL positive mononuclear cells (red) seen in a perivascular location in a muscle section from subject 2. Other nuclei appear blue in DAPI stain (scale bar = 100 μm).

Figure 4

IFN-γ ELISpot assays for all three subjects. α-SG and control enhanced Green fluorescent protein (eGFP) peptide stimulation showed no increase in spot forming colonies (SFC) per million peripheral blood mononuclear cells (PBMCs) in any subject. In Subject 1 there was a very minimal IFN-γ response specific to Pool 2 of the AAV1 capsid exceeding our confidence limits for a negative response (>50 spot forming cells /million PBMCs) at days 14 and 43. ELISpot assays were negative for Subjects 2 and 3. (dpi = days post injection; red = α–SG; black = eGFP; blue = AAV1 capsid pool 1; green = AAV1 capsid pool 2; purple = AAV1 capsid pool 3).