Efficient and Stable Adeno-Associated Virus-Mediated Transduction in the Skeletal Muscle of Adult Immunocompetent Mice

Abstract

Recombinant adeno-associated virus (rAAV) vectors were evaluated for gene transfer into the skeletal muscle of adult immunocompetent mice. A study using a vector encoding nuclear localized beta-galactosidase (rAAV-nls-lacZ) examined: (i) the efficiency and duration of transgene expression; (ii) the status of the AAV genome in the transduced fibers; and (iii) the possibility of improving gene transfer by inducing muscle regeneration. In the absence of regeneration, the injection of 1.7 x 10(7) particles in the quadriceps resulted in gene transfer to 10-70% of myofibers. Histological analysis indicated that the vector was able to reach myofiber nuclei distant from the injection point. Cellular infiltrates were absent at early time points but became conspicuous in the vicinity of some positive fibers at 4-8 weeks and subsided by 26 weeks. Southern analysis indicated that one to three copies of the vector genome were present per cell genome equivalent. They were associated with high-molecular-weight DNA in the form of tandem oligomers or interlocked circles. Gene transfer was not facilitated in the regenerating muscle. Rather, an early inflammatory response resulted in the elimination of most positive fibers after 8 weeks. The presence of regenerated fibers with beta-galactosidase-positive nuclei suggested that myoblasts had been transduced and were able to fuse to form new fibers. Gene transfer in the absence of immune reactions against the transgene product was studied by injecting mice with a rAAV carrying the murine erythropoietin (mEpo) cDNA. Dose-dependent elevation in the hematocrit was measured for over 200 days and corresponded to 5- to 20-fold increases in plasma Epo levels. We conclude that AAV vectors efficiently and stably transduce post-mitotic muscle fibers and myoblasts in vivo.

Longevity of rAAV vector and plasmid DNA in blood after intramuscular injection in nonhuman primates: implications for gene doping

“…12 In contrast to plasmid, previous in vivo studies have shown that after a single injection, rAAV genomes can be detected in peripheral blood mononuclear cells (PBMCs) for several months. 8,19 Our goal in this study is to determine whether under stringent conditions (very low doses of rAAV), rAAV genomes can be detected in WBCs, which were targeted because they are easily accessible and have the potential of carrying vector sequences in the long term.…”

“…[5][6][7][8][9] Intramuscular (IM) injection is a convenient method owing to physical accessibility, mass of the tissue and access to the vasculature. [10][11][12] Moreover, recombinant adeno-associated viral (rAAV) vectors and naked plasmid are two different gene transfer systems used for IM delivery in animal models [13][14][15][16][17] and in humans. 18,19 Recently, regional vascular infusion of a vector to achieve skeletal muscle transduction has been reported for plasmid DNA (pDNA) 20,21 and for rAAV vectors.…”

Recombinant AAV vector encoding human VEGF165 enhances wound healing

“…Among the novel strategies that hold promise for therapeutic gene therapy applications in the skin is the use of vectors based on the adeno-associated virus (AAV). These vectors are derived from a non-pathogenic and widespread defective parvovirus, and are able to transduce both dividing and non-dividing cells, including skeletal and cardiac muscle, 23,24 brain 25 and liver. 26,27 Since recombinant AAV (rAAV) vectors are devoid of any viral genes -which are expressed in trans for the packaging process -they elicit virtually no inflammatory or immune response in the sites of injection.…”

Delivery of FGF-2 but not VEGF by encapsulated genetically engineered myoblasts improves survival and vascularization in a model of acute skin flap ischemia

“…Long-term expression of therapeutic transgenes is possible by direct gene transfer in vivo either through infection by viruses, including adenoviral 11 and adeno-associated vectors 12,13 or by direct injection of plasmid DNA. 14 Adenoviruses have been employed to continually deliver the angiogenic factors VEGF 165 , 15 FGF-2 (basic FGF), 16 FGF-1 (acidic FGF) 17 and FGF-5 18 in vivo, with each of these molecules stimulating the outgrowth of new vessels at the site of administration.…”