AAV-Mediated Gene Transfer Restores a Normal Muscle Transcriptome in a Canine Model of X-Linked Myotubular Myopathy.

Multiple clinical trials employing recombinant adeno-associated viral (rAAV) vectors have been initiated for neuromuscular disorders, including Duchenne and limb-girdle muscular dystrophies, spinal muscular atrophy, and recently X-linked myotubular myopathy (XLMTM). Our previous work on a canine model of XLMTM showed that a single rAAV8-cMTM1 systemic infusion corrected structural abnormalities within the muscle and restored contractile function, with affected dogs surviving more than 4 years post injection. This remarkable therapeutic efficacy presents a unique opportunity to identify the downstream molecular drivers of XLMTM pathology and to what extent the whole muscle transcriptome is restored to normal after gene transfer.

Genome Editing of Expanded CTG Repeats within the Human DMPK Gene Reduces Nuclear RNA Foci in the Muscle of DM1 Mice.

Myotonic dystrophy type 1 (DM1) is caused by a CTG repeat expansion located in the 3' UTR of the DMPK gene. Expanded DMPK transcripts aggregate into nuclear foci and alter the function of RNA-binding proteins, leading to defects in the alternative splicing of numerous pre-mRNAs. To date, there is no curative treatment for DM1.

Long-term effects of systemic gene therapy in a canine model of myotubular myopathy.

Introduction: X-linked myotubular myopathy (XLMTM), a devastating pediatric disease caused by the absence of the protein myotubularin, results from mutations in the MTM1 gene. While there is no cure for XLMTM, we previously reported effects of MTM1 gene therapy using adeno-associated virus (AAV) vector on muscle weakness and pathology in MTM1-mutant dogs. Here, we followed 2 AAV-infused dogs over 4 years.

Systemic AAV8-Mediated Gene Therapy Drives Whole-Body Correction of Myotubular Myopathy in Dogs.

X-linked myotubular myopathy (XLMTM) results from MTM1 gene mutations and myotubularin deficiency. Most XLMTM patients develop severe muscle weakness leading to respiratory failure and death, typically within 2 years of age. Our objective was to evaluate the efficacy and safety of systemic gene therapy in the p.

Phosphatidylinositol 3-kinase inhibition restores Ca2+ release defects and prolongs survival in myotubularin-deficient mice.

Mutations in the gene encoding the phosphoinositide 3-phosphatase myotubularin (MTM1) are responsible for a pediatric disease of skeletal muscle named myotubular myopathy (XLMTM). Muscle fibers from MTM1-deficient mice present defects in excitation-contraction (EC) coupling likely responsible for the disease-associated fatal muscle weakness. However, the mechanism leading to EC coupling failure remains unclear.

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

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.

Phosphoinositide substrates of myotubularin affect voltage-activated Ca²⁺ release in skeletal muscle.

Skeletal muscle excitation–contraction (E–C) coupling is altered in several models of phosphatidylinositol phosphate (PtdInsP) phosphatase deficiency and ryanodine receptor activity measured in vitro was reported to be affected by certain PtdInsPs, thus prompting investigation of the physiological role of PtdInsPs in E–C coupling. We measured intracellular Ca2+ transients in voltage-clamped mouse muscle fibres microinjected with a solution containing a PtdInsP substrate (PtdIns(3,5)P2 or PtdIns(3)P) or product (PtdIns(5)P or PtdIns) of the myotubularin phosphatase MTM1. No significant change was observed in the presence of either PtdIns(5)P or PtdIns but peak SR Ca2+ release was depressed by ~30% and 50% in fibres injected with PtdIns(3,5)P2 and PtdIns(3)P, respectively, with no concurrent alteration in the membrane current signals associated with the DHPR function as well as in the voltage dependence of Ca2+ release inactivation.

Characterization of a recombinant adeno-associated virus type 2 Reference Standard Material.

A recombinant adeno-associated virus serotype 2 Reference Standard Material (rAAV2 RSM) has been produced and characterized with the purpose of providing a reference standard for particle titer, vector genome titer, and infectious titer for AAV2 gene transfer vectors. Production and purification of the reference material were carried out by helper virus-free transient transfection and chromatographic purification. The purified bulk material was vialed, confirmed negative for microbial contamination, and then distributed for characterization along with standard assay protocols and assay reagents to 16 laboratories worldwide.

Deleterious effect of peptide insertions in a permissive site of the AAV2 capsid.

A permissive site for insertion of heterologous peptide sequences has been identified in the capsid proteins of AAV2. While attempting to use this site for insertion of a nuclear localization sequence, we have observed a drastic reduction in the yield of DNA-containing particles. ELISA analysis showed that capsid assembly was modestly affected, whereas genome encapsidation was more profoundly altered, a phenomenon we did not observed when a RGD peptide was inserted at the same location.