Gene therapy prolongs survival and restores function in murine and canine models of myotubular myopathy

Abstract

Loss-of-function mutations in the myotubularin gene (MTM1) cause X-linked myotubular myopathy (XLMTM), a fatal, congenital pediatric disease that affects the entire skeletal musculature. Systemic administration of a single dose of a recombinant serotype 8 adeno-associated virus (AAV8) vector expressing murine myotubularin to Mtm1-deficient knockout mice at the onset or at late stages of the disease resulted in robust improvement in motor activity and contractile force, corrected muscle pathology, and prolonged survival throughout a 6-month study. Similarly, single-dose intravascular delivery of a canine AAV8-MTM1 vector in XLMTM dogs markedly improved severe muscle weakness and respiratory impairment, and prolonged life span to more than 1 year in the absence of toxicity or a humoral or cell-mediated immune response. These results demonstrate the therapeutic efficacy of AAV-mediated gene therapy for myotubular myopathy in small- and large-animal models, and provide proof of concept for future clinical trials in XLMTM patients.

Figure 1

Intravascular delivery of AAV8-Mtm1 in myotubularin-deficient mice improves lifespan and body growth. (A) Experimental design. (B) Survival and (C) body mass of wild-type mice (WT) and constitutive KO-Mtm1 mice injected at 3 weeks of age with saline (WT + saline, KO + saline, n=10 per genotype). Myotubularin-deficient mice were injected with AAV8-Mtm1 at 3×10¹³ viral genomes per kg (vg/kg) at 3 (KO + AAV Early, n=8) and 5 weeks of age (KO Late + AAV, n=11) during a 6 months follow-up study. (D) Mass of representative skeletal muscles of KO-Mtm1 mice 2 weeks after injection of saline (KO + saline, n=4) and 6 months (n=10 after injection of AAV8-Mtm1 (KO Early + AAV, n=8, and KO Late + AAV, n=8). Values were normalized to muscle mass of age-matched, saline-injected WT mice (n=10), taken as 100%. (E) Myotubularin protein quantification by immunoblot, GAPDH immunodetection was used an internal control. The number of animals was as in (C). Muscles: TA = tibialis anterior; EDL = extensor digitorum longus; SOL = soleus; GA = gastrocnemius; QUA = quadriceps; TRI = triceps; BI = biceps brachii; DIA = diaphragm. Statistical significance: P < 0.05 (one symbol); P < 0.01 (two symbols); P < 0.001 (three symbols); each condition versus WT + saline values.

Figure 2

Mtm1 gene replacement therapy corrects the internal architecture and hypotrophy of skeletal muscle fibers in myotubularin-knockout mice. Treatment groups were as described in Fig. 1. Mice were injected with either saline (+ saline) or AAV8-Mtm1 vector (+ AAV). Sections were obtained after 2 weeks (5 weeks of age) and after 6 months of treatment. (A) Cross-sections from tibialis anterior (TA) muscle stained with hematoxylin and eosin (HE) and NADH-TR, and by immunofluorescence with antibodies against DHPR1α and dysferlin. Scale bars = 10 µm. (B) Mean diameter of muscle fibers from TA and biceps brachii muscles from mice injected with either saline or AAV8-Mtm1 after 2 weeks (left graph, WT + saline, n=10; KO + saline, n=4) and after 6 months of treatment (right graph, WT + saline, n=10; KO Early + AAV, n=7, and KO Late + AAV, n=8). Statistical significance: P < 0.05 (one symbol); P < 0.01 (two symbols); each condition versus WT + saline values.

Figure 3

Gene replacement therapy with AAV8-Mtm1 improves strength, activity and long-term survival in myotubularin deficient mice. (A) Whole-body spontaneous mobility of normal (WT + saline), mutant (KO + saline) and AAV-treated mutant (KO Early and Late + AAV) mice, 2 weeks (5 weeks of age) and 6 months after PBS or vector injection. The distance covered over the 90-min test was assessed using an open field actimeter. (B) Escape test measurements in the 5 groups of mice. (C) Specific tetanic force of isolated EDL muscles from KO mice injected at an early and late stage of the disease 6 months after vector delivery compared to saline-injected KO and WT littermates. Drawings of the tests are shown on the left side of each figure section. WT + saline, n=6 and KO + saline, n=4 at 5 weeks; WT + saline, n=10, KO Early + AAV, n=8, and KO Late + AAV, n=8 at 6 months. Symbols for statistical significance: same as in Fig. 1.

Figure 4

Myotubularin is expressed and increases muscle mass and volume after local gene therapy in XLMTM dogs. (A) Cranial tibialis canine muscles 6 weeks post AAV8-MTM1 intramuscular injection. Immunoblot of myotubularin (MTM1, green) and GAPDH (red) from cranial tibialis muscle lysates at proximal (Prox), middle or distal section of the muscle. (B) MTM1 gene replacement therapy increases hind limb strength in XLMTM dogs. (Drawing) Method used to measure hind limb flexion strength in dogs. A nerve stimulator delivers electrical frequencies from 1-110 Hz to muscles that pull the paw toward the stifle (knee). A transducer captures the torque generated when the paw pulls on the foot pedal. (Upper graph) Baseline prior to injection, 10 weeks of age (WT, n=3; XLMTM, n=3); (Middle graph) 4 weeks post-injection, 14 weeks of age (WT, n=3; XLMTM, n=3); (Bottom graph) 6 weeks post-injection, 16 weeks of age (WT, n=2; XLMTM, n=2). Symbols for statistical significance: same as in Fig. 1. (C) Local myotubularin gene replacement therapy improves muscle fiber architecture in XLMTM dogs. Cryosections of the cranial tibialis muscle (middle part) were assessed microscopically: boxes in NADH-TR staining show areas magnified below; immunofluorescence staining for DHPRα1 and dysferlin show correction of abnormal organelles with AAV8-MTM1. Size bars = 25 µm. Electron microscopy (EM) shows normal T-tubules (black arrows) and abnormal L-tubules (white arrow). Bar = 500 nm. Bottom panel: Close-up of WT muscle and schematic showing normal relationship of sarcomere ends (Z-line), and triads of T-tubles and sarcoplasmic reticulum (SR).

Figure 5

(A-F) Hind limb strength of XLMTM dogs after intravascular administration of AAV8-MTM1. Data presented as mean ± SD combined values of both limbs. (A) Peak hind limb torque at various times up to 1 year after infusion. (B) Baseline prior to infusion, 9 weeks-of-age (WT, n=2; XLMTM + AAV8, n=3; XLMTM + Saline, n=1); (C) 6 weeks post-infusion, 15 weeks of age (WT, n=2; XLMTM + AAV8, n=2; XLMTM + Saline, n=2); (D) 8 weeks post-infusion, 17 weeks of age (WT, n=3; XLMTM + AAV8, n=3; XLMTM + Saline (n=3)); (E) 14 weeks post-infusion, 23 weeks of age (WT, n=3; XLMTM + AAV8, n=3). Note: XLMTM dogs infused only with saline did not survive beyond 18 weeks of age. (F). One year post-infusion (WT, n=1, carrier, n=3, XLMTM + AAV8, n=3). (G) Peak inspiratory flow (PIF), a respiratory functional measure reflecting diaphragm muscle strength, taken in anesthetized dogs at baseline and at 8, 14 weeks and 1 year post infusion with AAV8. Number of animals/group: same as in b–f. Symbols for statistical significance: same as in Fig. 1.

Figure 6

MTM1 gene replacement therapy corrects the internal architecture and hypotrophy of skeletal muscle fibers in myotubularin-mutant dogs. Muscle cryosections from age-matched wild-type (WT) or rAAV8-MTM1 infused XLMTM dogs were assessed microscopically. Comparison is shown between the left (infused) hindlimb and the right (contralateral non-infused) limb. (A) Representative micrographs of HE and NADH-TR staining of quadriceps muscle cross-sections from muscle biopsies taken 4 weeks-post infusion from dog, “D4”. (B) HE stained cranial tibialis cross-sections taken 1 year post AAV infusion. (Lower graph) myofiber diameter frequency distribution of images shown above. Size bars = 25 µm.

Figure 7

Biodistribution of AAV8 vector and myotubularin transgene expression in XLMTM dogs infused with AAV8-MTM1. Comparison of vector distribution (A) and MTM1 transgene expression (B) among upper and lower limb muscle biopsies collected 4 weeks post-infusion in three XLMTM dogs, Dog 4, Dog 5 and Dog 6. Legend: Upper limb muscles: triceps, biceps brachii (TRI, BI bra, respectively). Lower limb muscles: biceps femoris, quadriceps (BI fem, QUA). R=right, L=left, inf = infused limb. (B) Expression of canine MTM1 protein relative to the housekeeping gene, GAPDH in whole muscle lysates probed with an anti-myotubularin antibody. Comparison of vector distribution (C) and MTM1 transgene expression (D) among upper and lower limb muscle necropsy samples collected 1 year post-infusion in D4, an XLMTM dog. Legend: muscles of the infused leg, VL=vastus lateralis; VM=vastus medialis; RF=rectus femoris; Ad=adductor magnus; Pec=pectineus; Sar=cranial Sartorius; Gra=gracilis; BI fem=biceps femoris; ST=semitendinosis; SM=semimembranosis; CT=cranial tibialis; Gast=gastrocnemius; per=peroneus longus. Muscles distal to the infused leg, Dia=diaphragm, Interc=intercostals, He=heart.