Intracavitary Injection

Several studies have demonstrated that the diaphragm can be successfully targeted for gene transfer during the fetal period, by injecting gene transfer vectors directly into the uterine cavity. In a murine model of Pompe disease (Glycogen storage disease type II) which is defined by a lack of the enzyme acid a-glucosidase (GAA), Rucker et al (2004) injected adeno-associated virus vectors (AAV1 and AAV2) into the fetal peritoneal cavity on day 15 of gestation. These authors found that several weeks after birth, GAA levels in the diaphragm were significantly increased, and this was associated with improvements in histopathology and the contractile function of the diaphragm up to 6 months of age. Using fetal mice of the same gestational age, Gregory et al (2004) showed high-level LacZ gene transfer to the diaphragm by equine infectious anemia virus (EIAV) lentivirus vectors pseudo-typed with VSV-G. By combining intrapleural and intraperitoneal injections of EIAV in the fetus, not only the diaphragm but also the intercostal muscles and abdominal musculature were effectively transduced over a large proportion of their surface area. It should be noted that in the above studies there was little or no transgene expression in the distant muscles or organs, suggesting that the vectors were accessing the tissues locally rather than through their absorption into the systemic circulation (Rucker et al. 2004; Gregory et al. 2004). Moreover, an added advantage of performing gene transfer during the fetal period was that host immune responses against the involved vectors or transgenes were either mitigated or completely eliminated in mice (Rucker et al. 2004; Gregory et al. 2004). Jimenez et al (2005) administered HIV-1 derived lentiviral vectors into the peritoneum of early gestation macaque monkeys under ultrasound guidance, and also found consistent expression within the diaphragm. Remarkably, transgene expression in the diaphragm was reportedly sustained for up to 3 years (Tarantal et al. 2006). Finally, it has been reported that somite-derived cells expressing LacZ and injected into the uterine circulation of pregnant mdx mice can lead to significant transgene expression within the diaphragm at 2 months after birth, although the level of engraftment is low (Torrente et al. 2000).

During the postnatal period, the abilities of intraperitoneal or intrapleural administration of different vectors to transduce the diaphragm have also been evaluated. Howell et al. (1997) reported that after intraperitoneal injection of plasmid DNA containing a LacZ transgene together with lipofectin in Golden Retriever Muscular Dystrophy (GRMD) dog pups, positive staining could be found in the diaphragm, intercostal and abdominal muscles, albeit at extremely low levels. Huard et al (1995) performed intraperitoneal injection of first-generation adenoviral vectors in 2-day old rats, and showed significant transgene expression after 5 days in the diaphragm. In this study, a substantial adenovirus level in the blood was also demonstrated in the first few hours after intraperitoneal injection of the vector, raising a question as to whether transduction of the diaphragm may have been mediated through the systemic circulation. In adult mice, Mah et al (2004) compared the efficiencies of gene transfer to the diaphragm after direct local application ("painting") of AAV serotypes 1, 2 or 5 onto the peritoneal surface of the muscle. Interestingly, they also compared transduction efficiencies when the vectors were applied as free virus or mixed with a water-soluble, glycerine-based gel. These investigators found that at 6 weeks after vector delivery, the highest transduction of the diaphragm was achieved with gel-based application of vectors (AAV1 > AAV2 > AAV5), and diaphragmatic transduction was demonstrated not only for a reporter gene but also for the GAA transgene in the murine model of

Pompe disease. On the other hand, another group reported that in adult mice which received intrapleural instillation of AAV vectors containing a luciferase transgene, higher levels of diaphragmatic transduction were attained with AAV5 as compared to AAV2 (De et al. 2004). Blankinship et al (2004) showed high-level transduction of the diaphragm and intercostal muscles of adult mice after a single injection of AAV6 into the thoracic cavity. In addition, in the same study intraperitoneal injection of AAV6 in newborn mice achieved widespread transduction throughout the musculature, including the diaphragm.

Overall, the literature to date indicates that intracavitary injection of certain gene transfer vectors can achieve substantial local transduction of the respiratory muscles in animal models. These approaches are generally more effective in fetal or newborn animals, presumably due to incomplete formation of physical barriers to vector dispersion in the diaphragm and other respiratory muscles at this stage of development, as well as potentially higher levels of viral receptors and coreceptors within immature skeletal muscle tissues.

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