Gene Delivery for Limb-Girdle Muscular Dystrophy Type 2D by Isolated Limb Infusion

Abstract

In a previous limb-girdle muscular dystrophy type 2D (LGMD2D) clinical trial, robust alpha-sarcoglycan gene expression was confirmed following intramuscular gene (SGCA) transfer. This paved the way for first-in-human isolated limb infusion (ILI) gene transfer trial to the lower limbs. Delivery of scAAVrh74.tMCK.hSGCA via an intravascular route through the femoral artery predicted improved ambulation. This method was initially chosen to avoid safety concerns required for large systemic vascular delivery viral loads. ILI methods were adopted from the extensive chemotherapy experience for treatment of malignancies confined to the extremities. Six LGMD2D subjects were enrolled in a dose-ascending open-label clinical trial. Safety of the procedure was initially assessed in the single limb of a non-ambulant affected adult at a dose of 1 × 1012 vg/kg. Subsequently, ambulatory children (aged 8-13 years) were enrolled and dosed bilaterally with either 1 × 1012 vg/kg/limb or 3 × 1012 vg/kg/limb. The six-minute walk test (6MWT) served as the primary clinical outcome; secondary outcomes included muscle strength (maximum voluntary isometric force testing) and SGCA expression at 6 months. All ambulatory participants except one had pre- and post-treatment muscle biopsies. All four subjects biopsied had confirmed SGCA gene delivery by immunofluorescence, Western blot analysis (14-25% of normal), and vector genome copies (5.4 × 103-7.7 × 104 vg/μg). Muscle strength in the knee extensors (assessed by force generation in kilograms) showed improvement in two subjects that correlated with an increase in fiber diameter post gene delivery. Six-minute walk times decreased or remained the same. Vascular delivery of AAVrh74.tMCK.hSGCA was effective at producing SGCA protein at low doses that correlated with vector copies and local functional improvement restricted to targeted muscles. Future trials will focus on systemic administration to enable targeting of proximal muscles to maximize clinical benefit.

Keywords: alpha-sarcoglycan; gene delivery; isolated limb infusion; limb-girdle muscular dystrophy.

Conflict of interest statement

J.R.M. is a Principal Investigator in the Center for Gene Therapy at Nationwide Children's Hospital. L.R.R.-K. is Vice-President for Gene Therapy, Sarepta Therapeutics. No competing financial interests exist for these contributors or for the remaining authors.

Figures

Figure 1.

SGCA expression following isolated limb infusion (ILI). SGCA gene expression demonstrated by immunofluorescence following alpha-sarcoglycan (α-SG) antibody staining of muscle biopsies taken post gene transfer for subject 1 (tibialis anterior) and pre and post gene transfer for subjects 3–6 (quadriceps). Except for subject 1 where it could not be compared, membrane staining intensity post gene transfer showed increased intensity. Confirmation of gene expression by quantitative Western blots revealed an increase in α-SG protein compared to baseline as follows: subject 3: 38%; subject 4: 12.5%; subject 5: 22%; and subject 6: 172%. Comparisons as a percent of normal for each subject were as follows: subject 1: 35%; subject 3: 14%; subject 4: 16%; subject 5: 15%; and subject 6: 25%. Western blots were quantified using a four-point standard curve (10%, 25%, 50%, and 100%) generated from non-dystrophic normal control muscle samples and normalized for muscle content using muscle actin as a loading control (lower band).

Figure 2.

Vector genome copy number following ILI. Delivery of scAAVrh74.tMCK.SGCA vector genomes was confirmed by quantitative polymerase chain reaction using the tMCK as the target amplicon. All baseline biopsy values were below the limit of detection. All subjects demonstrated vector genome copy numbers in the range of 5.4e3 and 7.76e4 vg/μg DNA (0.03–0.54 copies/nuclei): subject 1: 3.9e4 vg/μg; subject 3: 5.4e3 vg/μg, subject 4: 5.02e3 vg/μg; subject 5: 6.48e3 vg/μg; and subject 6: 7.76e4 vg/μg.