CHARACTERIZATION OF RECOMBINANT ADENOASSOCIATED VIRUS-2 AS A VEHICLE FOR GENE DELIVERY AND EXPRESSION INTO VASCULAR CELLS

Background: We have used wild-type and recombinant adeno-associated vi rus-2 (AAV) to study transduction, replication efficiencies, functiona l protein expression, and gene delivery to vascular cells in vitro and in vivo. Methods: Recombinant adeno-associated virus-2 (rAAV) plasmid s (ranging in size to 110% of wild-type AAV) driven by 6 distinct prom oters upstream of a beta-galactosidase cassette were effectively used for generation of replication-deficient virus, with titers consistentl y ranging from 2-5 x 10(5) IU/mL. AAV infectivity and replication in h uman umbilical vein endothelial cells (HUVEC) were unrelated to cellul ar proliferative index establishing the potential utility of the virus for transduction of quiescent vascular cells, Long-term cultures of A AV-infected HUVEC established the presence of episomal forms at 18 day s, although chromosome 19-specific integration was not evident, Functi onal beta-galactosidase activity similar to 400% above control was evi dent in HUVEC using either a murine collagen alpha 1(I) promoter (pTRC ol alpha 1(I)beta) or CMV promoter (pTRCMV beta). Results: Based on th ese initial data, in vivo studies were completed using a rat carotid a rtery model, Both wild-type AAV (titers similar to 1 x 10(9) IU/mL) an d rAAV (pTRCol alpha 1(I)beta or pTRCMV beta) efficiently infected vas cular cells in vivo with endothelial and vascular smooth muscle cell t ransduction frequencies approaching 90% as judged by DNA in situ polym erase chain reaction, with no evidence for disrupted vessel architectu re, Protein expression using total vessel extracts at 48 hours postinf ection demonstrated 20-fold increase in functional beta-galactosidase activity using pTRCol alpha 1(I)beta compared to saline-injected contr ols vessels (799 +/- 236 mu U/mg protein vs 40.7 +/- 17 mu U/mg protei n). Conclusions: These data provide the first evidence that rAAV may b e adapted for directed high-level transgene delivery and expression in to normally quiescent vascular endothelial and smooth muscle cells bot h in vitro and in vivo.