Dystrophin Expression in Muscles of mdx Mice After Adenovirus-MediatedIn VivoGene Transfer

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Abstract
We have generated high-titer adenoviral recombinants (AVR) expressing a 6.3-kb partial dystrophin cDNA insert under the control of either the Rous sarcoma virus (RSV) or cytomegalovirus (CMV) promoter. These AVR preparations were free of both E1-containing AVR and AVR with a nonfunctional dystrophin expression cassette. With these optimal AVR preparations, we have obtained a high degree of short-term (10 days) expression of a truncated (approximately 200 kD) dystrophin in dystrophin-deficient mdx muscles injected in the neonatal period; a lesser degree of expression of dystrophin was found in muscles injected in the young adult age and in old animals. Microscopic indices of muscle damage revealed that the truncated dystrophin provided a significant protection of the transduced muscle fibers. However, by 60 days post-injection, a substantial reduction of the number of dystrophin-positive fibers was noted, even in the neonatally injected muscles, and near-total elimination of dystrophin-positive fibers occurred in muscles injected in the adult age. These effects appeared to be brought about by the activity of CD8+ cytotoxic lymphocytes directed against the transduced cells, leading to their eventual elimination. In severe combined immunodeficiency (SCID) mice, lacking both humoral and cellular immune competence, muscles transduced (either in the neonatal or adult age) by AVR containing a CMV-LacZ expression cassette maintained the early (10 day) transduction level up to 30 days post-injection. Systemic administration of AVR (i.e., into the left ventricle of the heart) led in 5 days to a high number of dystrophin-positive fibers in heart, diaphragm, and intercostal muscles but not in limb muscles. Injection of pure, high-titer suspension of adenoviral recombinants (AVR) containing a cytomegalovirus (CMV) or Rous sarcoma virus (RSV) promoter-driven 6.3-kb dystrophin cDNA (minigene) into mdx muscles produced a large number of dystrophin-positive muscle fibers by 10 days post-injection. The transduction efficiency 10 days post-injection was significantly greater for injections made during the first week of life compared to those animals injected in adult age. Microscopic indices suggested that even the truncated dystrophin (about 200 kD) was able to minimize damage to and loss of mdx muscle fibers. However, by 60 days post-injection, the prevalence of dystrophin-positive fibers had substantially declined. This appeared to be due largely to an elimination of the transduced muscle fibers by an immune response of the host, mediated mainly by cytotoxic, CD8+ lymphocytes. By contrast, the prevalence of AVR-transduced muscle fibers in severe combined immunodeficient (SCID) mice (which lack both cellular and humoral immune competence) did not significantly decline by 30 days post-injection. Therefore, a significant down-regulation of the host immune response is required (by vector modification and/or suppression of host immune system) if long-term AVR-mediated gene transfer is to become a practical modality for gene replacement therapy.