Systemic Delivery of Dysferlin Overlap Vectors Provides Long-Term Gene Expression and Functional Improvement for Dysferlinopathy

Rachael A Potter, Danielle A Griffin, Patricia C Sondergaard, Ryan W Johnson, Eric R Pozsgai, Kristin N Heller, Ellyn L Peterson, Kimmo K Lehtimäki, Hillarie P Windish, Plavi J Mittal, Douglas E Albrecht, Jerry R Mendell, Louise R Rodino-Klapac, Rachael A Potter, Danielle A Griffin, Patricia C Sondergaard, Ryan W Johnson, Eric R Pozsgai, Kristin N Heller, Ellyn L Peterson, Kimmo K Lehtimäki, Hillarie P Windish, Plavi J Mittal, Douglas E Albrecht, Jerry R Mendell, Louise R Rodino-Klapac

Abstract

Dysferlinopathies comprise a family of disorders caused by mutations in the dysferlin (DYSF) gene, leading to a progressive dystrophy characterized by chronic muscle fiber loss, fat replacement, and fibrosis. To correct the underlying histopathology and function, expression of full-length DYSF is required. Dual adeno-associated virus vectors have been developed, defined by a region of homology, to serve as a substrate for reconstitution of the full 6.5 kb dysferlin cDNA. Previous work studied the efficacy of this treatment through intramuscular and regional delivery routes. To maximize clinical efficacy, dysferlin-deficient mice were treated systemically to target all muscles through the vasculature for efficacy and safety studies. Mice were evaluated at multiple time points between 4 and 13 months post treatment for dysferlin expression and functional improvement using magnetic resonance imaging and magnetic resonance spectroscopy and membrane repair. A systemic dose of 6 × 1012 vector genomes resulted in widespread gene expression in the muscles. Treated muscles showed a significant decrease in central nucleation, collagen deposition, and improvement of membrane repair to wild-type levels. Treated gluteus muscles were significantly improved compared to placebo-treated muscles and were equivalent to wild type in volume, intra- and extramyocellular lipid accumulation, and fat percentage using magnetic resonance imaging and magnetic resonance spectroscopy. Dual-vector treatment allows for production of full-length functional dysferlin with no toxicity. This confirms previous safety data and validates translation of systemic gene delivery for dysferlinopathy patients.

Keywords: AAV; LGMD2B; dysferlin; gene therapy; systemic delivery.

Conflict of interest statement

Dr. Rodino-Klapac is the inventor of the AAV.dysferlin dual vector technology. This technology has been licensed exclusively to Myonexus Therapeutics, Inc. Dr. Rodino-Klapac is a co-founder and Chief Scientific Officer for Myonexus Therapeutics, Inc. No competing financial interests exist for the remaining authors.

Figures

Figure 1.
Figure 1.
Sustained long-term expression following intramuscular delivery in dysferlin null mice. Four-week-old Dysf–/– mice were injected with 2 × 1011 vg total dose of AAVrh74.MHCK7.DYSF.DV into the left tibialis anterior (TA) muscle and followed for 3–24 months to monitor expression and histology (n = 3 per time point). (A) Hematoxylin and eosin (H&E) staining demonstrates a significant reduction in centrally located nuclei in the treated TA compared to (B) contralateral control TA muscles injected with saline. (C) Anti-dysferlin immunofluorescence staining demonstrates dysferlin transgene expression was sustained throughout all time points (3–24 months). (D) At 6, 9, 12, 18, and 24 months post delivery, there was a significant decrease in central nucleation to <5%. Excluding the 3 month time point, all values were significant (p ≤ 0.01) for central nucleation quantification. (E) There was no significant decrease in the percentage of dysferlin-positive fivers over time, and contralateral (right side) muscles demonstrated no dysferlin expression. Two-way analyses of variance (ANOVAs) were utilized to identify differences in the means between treated and untreated cohorts. Error bars are represented as mean ± standard error of the mean (SEM; p < 0.05).
Figure 2.
Figure 2.
Intravenous delivery of AAVrh74.MHCK7.DYSF.DV improves histological parameters in BlaJ mice. (A) H&E staining of the psoas muscle 28 weeks post treatment demonstrated improvement in fiber size distribution and reduced central nuclei following intravenous delivery of 6 × 1012 vg total dose of AAVrh74.MHCK7.DYSF.DV in treated BlaJ mice versus saline-treated mice. (B) Picosirius red staining for collagen demonstrated reduction in fibrosis in AAVrh74.MHCK7.DYSF.DV-treated psoas muscles. (C) Following AAVrh74.MHCK7.DYSF.DV delivery, there was a trend toward normal fiber diameters, with a significant increase in mean fiber diameter in treated muscle (27.8 ± 0.17 μm) versus control (18.4 ± 0.099 μm), and reduced central nucleation in the TA, quadriceps, triceps, and psoas muscles. (E) Additionally, there was a significant decrease from 37.0% to 9.7% collagen deposition in treated mice compared to saline-treated mice. Two-way ANOVAs were utilized with post hoc analysis to locate differences in central nucleation and fiber diameters. Error bars are represented as mean ± SEM (p < 0.05).
Figure 3.
Figure 3.
Long-term improvement in collagen deposition and immune staining with AAVrh74.MHCK7.DYSF.DV treatment. (A) At 15 months of age, H&E staining demonstrated normalization of histology in treated psoas muscles (BlaJ-AAV). (B) Picosirius staining in the gluteus muscle demonstrated a reduction in collagen deposition. (C) Following long-term treatment in systemically treated mice, there was a trend toward normalization of fiber diameters and (D) reduced centrally located nuclei in the quadriceps muscle, as shown with H&E staining. (E) Finally, there was a 9.0% reduction in collagen deposition using picosirius staining in the gluteus muscle. One-way ANOVAs were utilized with post hoc analysis to locate differences in the means. Error bars are represented as mean ± SEM (p < 0.05).
Figure 4.
Figure 4.
Long-term expression and normalization of centrally located nuclei in AAVrh74.MHCK7.DYSF.DV-treated mice. (A) Robust dysferlin expression was seen following systemic delivery (6 × 1012 dose) using anti-dysferlin immune staining with an N-terminal antibody. Gastrocnemius (GAS) and TA muscles are shown for representation. (B) Sections were co-stained with DAPI to quantify dysferlin positive fibers co-localized with centrally located nuclei. (C) There was widespread dysferlin transgene expression at 15 months of age >5% in all tissues, with the diaphragm expressing the highest at 53%. (D) It is demonstrated that the majority of fibers with centrally located nuclei are absent of dysferlin expression (<0.5% are positive for dysferlin and centrally located nuclei). (E) Full-length dysferlin in treated muscles at 15 months of age was confirmed by Western blot analysis (TA and GAS) compared to wild-type and BlaJ-untreated muscle.
Figure 5.
Figure 5.
Long-term improvements using magnetic resonance imaging (MRI) in systemically treated mice with AAVrh74.MHCK7.DYSF.DV. (A) Animals underwent MRI after 7, 9, 12, and 15 months of age and demonstrated improvements in gluteus volume (p < 0.001), (B) decreased gluteus fat (p < 0.0001), (C) increased water content to wild-type levels (p = 0.223; p < 0.01), and (D) decreased fat percentage significantly at the 15-month time points (p < 0.0001). (E) MR-spectroscopy was used to evaluate metabolite accumulation and decreased extramyocellular lipid accumulation was demonstrated in the gluteus muscle (p < 0.0001), as well as decreased creatine levels in the muscle to wild-type levels (p = 0.021) (F). Fifteen-month-old BLAJ animals were unable to undergo magnetic resonance spectroscopy evaluation due to excessive fat infiltration in the saline-treated cohort. Two-way ANOVAs with Tukey post hoc analysis were utilized to locate differences in the means. Error bars are represented as mean ± SEM (p < 0.05).
Figure 6.
Figure 6.
Systemic expression and histological improvements in an aged cohort treated with AAVrh74.MHCK7.DYSF.DV. (A) Animals treated at 6 months of age and sacrificed at 9 months of age demonstrated reductions in centrally located nuclei in psoas muscle using H&E staining. (B) There was slightly reduced collagen deposition between 6 and 9 months of age with treatment. (C) Dysferlin transgene expression was noted across all muscles following AAV treatment in this aged cohort, which led to significant improvement in membrane repair capacity in flexor digitorum brevis muscles (p < 0.05 at 190 s).
Figure 7.
Figure 7.
Systemic delivery in rhesus macaque with AAVrh74.MHCK7.DYSF.DV. (A) Anti-dysferlin immunofluorescence staining in untreated naïve rhesus macaque diaphragm and quadriceps muscles (left panel) and systemically treated diaphragm, quadriceps, and hamstring demonstrates robust dysferlin expression. (B) Enzyme-linked immunosorbent spot assay analysis of T-cell response to dysferlin and AAV peptide pools demonstrates no adverse T-cell response in the presence of (C) widespread vector genome (vg/μg of genomic DNA) biodistribution quantified using quantitative polymerase chain reaction (qPCR). Vastus lateralis control and heart control in qPCR biodistribution refers to an uninjected male rhesus macaque.

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