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

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.

Direct intramuscular injection with recombinant AAV vectors results in sustained expression in a dog model of hemophilia

“…Cytotoxic lymphocyte (CTL) response to adenovirus vectors delivered to muscle is a predictable phenomenon, felt to be mediated by class I MHC-restricted CD8 + and CD4 + lymphocytes. 31,32 Cellular immune response following AAV vector delivery to muscle is absent 12 or limited to the period immediately following infection. 3 We have observed a striking histologic difference when recombinant adenovirus-␤-galactosidase was compared with rAAV-␤-galactosidase injection in mouse muscle.…”

Long-term expression of full-length human dystrophin in transgenic mdx mice expressing internally deleted human dystrophins

“…21 Immune responses to dystrophin have been reported in early myoblast transfer experiments, where only transient expression of dystrophin was achieved. 22 The delivery of dystrophin with viral vectors to animal models has also shown short-term expression of dystrophin in muscle fibres, 23 unless an immunosuppressive regime was used. 24 In most cases, it was not clear if the immune response was directed to the transgene itself or if this immune response was enhanced due to the vector system used, that is, cellular components or viral coat proteins.…”

Adeno-associated virus vector-mediated gene transfer into dystrophin-deficient skeletal muscles evokes enhanced immune response against the transgene product

“…These include the adenoviral vector-mediated introduction of functional dystrophin, [2][3][4][5][6] naked DNA, 7 and primary myoblast 8 or stem cell 9 transplantation. Alternatively, correction of the endogenous dystrophin gene was attempted using chimeric RNA/DNA oligonucleotides 10,11 or antisense 2'-O-methyl oligoribonucleotides.…”