Transduction Efficiency of Adeno-Associated Virus Serotypes After Local Injection in Mouse and Human Skeletal Muscle.

The adeno-associated virus (AAV) vector is an efficient tool for gene delivery in skeletal muscle. AAV-based therapies show promising results for treatment of various genetic disorders, including muscular dystrophy. These dystrophies represent a heterogeneous group of diseases affecting muscles and typically characterized by progressive skeletal muscle wasting and weakness and the development of fibrosis. The tropism of each AAV serotype has been extensively studied using systemic delivery routes, but very few studies have compared their transduction efficiency through direct intramuscular injection. Yet, in some muscular dystrophies, where only a few muscles are primarily affected, a local intramuscular injection to target these muscles would be the most appropriate route. A comprehensive comparison between different recombinant AAV (rAAV) serotypes is therefore needed. In this study, we investigated the transduction efficiency of rAAV serotypes 1-10 by local injection in skeletal muscle of control C57BL/6 mice. We used a CMV-nls-LacZ reporter cassette allowing nuclear expression of LacZ to easily localize targeted cells. Detection of ß-galactosidase activity on muscle cryosections demonstrated that rAAV serotypes 1, 7, 8, 9, and 10 were more efficient than the others, with rAAV9 being the most efficient in mice. Furthermore, using a model of human muscle xenograft in immunodeficient mice, we observed that in human muscle, rAAV8 and rAAV9 had similar transduction efficiency. These findings demonstrate for the first time that the human muscle xenograft can be used to evaluate AAV-based therapeutical approaches in a human context.

A novel technique for diaphragm biopsies in human patients.

BACKGROUND: The diaphragm is difficult to biopsy because of its anatomic location. We describe a new laparoscopic diaphragm biopsy technique. MATERIAL AND METHODS: Fifty one patients with amyotrophic lateral sclerosis gave their consent to diaphragm biopsy in the context of an implanted phrenic nerve stimulation protocol (NCT01583088). The biopsy was taken from the costal diaphragm, after opening the parietal peritoneum with scissors, and by grasping the diaphragmatic muscle over the rib with toothed laparoscopy forceps. RESULTS: The first four electrocoagulation biopsies were unsuitable for morphologic examination. The following 47 biopsies were therefore performed without electrocoagulation. The mean size of the biopsy fragments obtained after preparation was 3 ± 1 × 2 ± 1 × 1 ± 1 mm (maximum: 4 × 3 × 2 mm). A diaphragmatic injury occurred during the section in three cases requiring immediate suture without causing pneumothorax. A small pleural effusion was observed on the postoperative chest x-ray in one patient with a spontaneously favorable outcome. Numerous stains were able to be performed on the fragments obtained. CONCLUSIONS: Diaphragm biopsy can be safely performed by laparoscopy and yields tissue suitable for our future histologic evaluation.