Assessment of systemic AAV-microdystrophin gene therapy in the GRMD model of Duchenne muscular dystrophy

Abstract

Duchenne muscular dystrophy (DMD) is a progressive muscle wasting disease caused by the absence of dystrophin, a membrane-stabilizing protein encoded by the DMD gene. Although mouse models of DMD provide insight into the potential of a corrective therapy, data from genetically homologous large animals, such as the dystrophin-deficient golden retriever muscular dystrophy (GRMD) model, may more readily translate to humans. To evaluate the clinical translatability of an adeno-associated virus serotype 9 vector (AAV9)-microdystrophin (μDys5) construct, we performed a blinded, placebo-controlled study in which 12 GRMD dogs were divided among four dose groups [control, 1 × 10¹³ vector genomes per kilogram (vg/kg), 1 × 10¹⁴ vg/kg, and 2 × 10¹⁴ vg/kg; n = 3 each], treated intravenously at 3 months of age with a canine codon-optimized microdystrophin construct, rAAV9-CK8e-c-μDys5, and followed for 90 days after dosing. All dogs received prednisone (1 milligram/kilogram) for a total of 5 weeks from day -7 through day 28. We observed dose-dependent increases in tissue vector genome copy numbers; μDys5 protein in multiple appendicular muscles, the diaphragm, and heart; limb and respiratory muscle functional improvement; and reduction of histopathologic lesions. As expected, given that a truncated dystrophin protein was generated, phenotypic test results and histopathologic lesions did not fully normalize. All administrations were well tolerated, and adverse events were not seen. These data suggest that systemically administered AAV-microdystrophin may be dosed safely and could provide therapeutic benefit for patients with DMD.

Fig. 1.. Experimental design and timeline for AAV9-microdystrophin injection in GRMD dogs.

(A) An AAV9 construct containing a microdystrophin (μDys5) consisting of the N-terminal actin binding domain (ABD1), two hinges (H1 and H4), five spectrin-like repeats (R1, R16, R17, R23, and R24), and the cysteine-rich (CR) domain with the muscle-specific CK8e expression cassette was injected. (B) Dogs were randomly assigned to one of four groups (n = 3 for each) that received a single intravenous dose of the AAV9-μDys5 or vehicle based on the dose of AAV-9 received: Group 1: 1E13 vg/kg, Low Dose; Group 2: 1E14 vg/kg, Mid Dose; Group 3: 2E14 vg/kg, High Dose; and Group 4: 0 vg/kg, Vehicle (Control). (C) AAV9-μDys5 was administered on Day 0 (baseline) when dogs were about 90±10 days (3 months) of age. Prednisone (1 mg/kg orally) was begun 7 days prior to AAV9-μDys5 injection and continued without tapering the dose for a total of 35 days. Anti-AAV9 circulating antibodies were measured at baseline and on several subsequent days until termination at about study Day 90. Phenotypic testing was done within 5 days of the AAV9-μDys5 injection and then repeated on Days 45 and 90 (about 4½ and 6 months of age), each ± 5 days to accommodate scheduling the various tests.

Fig. 2.. Biodistribution of vector genomes (vg).

qPCR analysis of vector genome copies per microgram of genomic DNA in muscles (left) and non-muscle tissues (right) at study day 90. Means and standard deviation for all study subjects within a dose group and time point are plotted. LV = left ventricle.

Fig. 3.. Protein expression quantified by immunofluorescence (IF) and western blot.

(A) Imaging by IF of biceps femoris muscle sections at Day 90 (top) and a histogram depicting group means of the percent cross sectional area containing positive fibers (bottom). Antibody-mediated IF stains include microdystrophin (top row, red), the dystrophin glycoprotein complex (DGC) members β-sarcoglycan and nNOS (rows 2 and 3; both green), MHCd (row 4, green), and utrophin (row 5; green) (bar = 100μm). (B). Western blot quantification of microdystrophin protein in tissues collected from dogs at necropsy as a % of normal dystrophin (top). Each blot (middle: diaphragm and bottom: liver) contains 13 lanes in the following order: Lanes 1–5 – Dystrophin standard curve composed of 100%, 50%, 25%, 10%, and 0% of normal dystrophin; Lanes 6–13 – Vehicle, Mid, High, Low, Low, Mid, Vehicle, High. GAPDH used to confirm sample loading.

Fig. 4.. AAV9-μDys5-treated dogs demonstrate reduced histopathologic lesions and fibrosis.

(A) Images of cranial sartorius muscle at baseline and Day 90 from GRMD dog with H&E staining (upper two panels). Immunofluorescence of microdystrophin (red) and dystrophin (green) at Day 90 (lower two panels). (B) Average lesion scores varied across skeletal muscles but were generally lower in the mid and high dose groups. (C) A heat map demonstrating lesion severity in skeletal muscle and cardiac samples from all dogs obtained at Day 90. (D) Representative images from each dose group of muscle collected at necropsy and stained with Masson’s trichrome (left), together with a histogram of mean collagen per group quantified as a percent of area (right). (E) Data presented as a histogram of relative frequency of fiber size using a bin of 5 μm and a maximum bin of 50 μm and mean fiber size for each dose group (left). Fiber size evaluation of biceps femoris muscles collected at necropsy using minimal Feret’s diameter measurements (right). Bar = 100μm in A and D.

Fig. 5.. Absolute tibiotarsal joint extension and flexion torque percent change for combined dose groups from baseline to Days 45 (interim) and 90 (final).

(A) Dose-related increases in the percent change for both absolute extension and flexion torque from baseline to Day 45 (left) and to Day 90 (right). Means and standard deviation for all study subjects within a dose group and time point are plotted. (B and C) Percent changes for extension (B) and flexion (C) torque plotted for individual dogs show evidence of a dose effect. Each dose group n = 3; combined dose groups n = 6. Combined ‘Control & Low dose’ versus ‘Mid & High dose’ groups were compared using the Mann-Whitney-Wilcoxon test. * P<0.05; ** P<0.01; *** P<0.001.

Fig. 6.. Respiratory peak expiratory flow: peak inspiratory flow (PEF:PIF) ratios from baseline to end of study.

(A) Adult GRMD dog showing the instrumentation for respiratory inductance plethysmography (RIP). The solid black arrows point to the elastic inductance bands that circle the chest and the abdomen. The bands are looped through an inner lycra undershirt. Leads from the bands pass through the loose, outer mesh shirt and directly into the telemetry unit within the outer pouch (open arrow). (B) Graphs showing the absolute (left) PEF:PIF ratios at Day 90 and the percent change from Day 0 to Days 45 and 90 (right). Each dose group n = 3; combined dose groups n = 6. Combined ‘Control & Low dose’ versus ‘Mid & High dose’ groups were compared using the Mann-Whitney-Wilcoxon test. *** P<0.001. (C) RIP peak expiratory flow:peak inspiratory flow (PEF:PIF) ratios for individual dogs show a dose effect, with a disease-associated increase in the control/low dose dogs compared to relative stability of values for the mid/high dose groups.