Recombinant adeno-associated virus-2 (raav) as a vector for gene delivery into vascular cells

We have utilized wild-type and recombinant AAV to study transduction, replication efficiencies, functional protein expression, and gene delivery to vascular cells in vitro and in vivo. rAAV plasmids (ranging in size to 110% of wild-type AAV) driven by six distinct promoters upstream of a β-galactosidase cassette were effectively used for generation of replication-deficient virus, with titers consistently ranging from 2-5 × 10⁵ i.u./ml. AAV infectivity and replication in human umbilical vein endothelial cells (HUVEC) were unrelated to cellular proliferative index establishing the potential utility of the virus for transduction of quiescent vascular cells. Long-term cultures of AAVinfected HUVEC established the presence of both episomal and integrative forms at 18 days, although chromosome 19-specific integration was not evident. Functional βgalactosidase activity ∼400% above control was evident in HUVEC using either a murine collagenal(l) promoter (pTRColal(l)β) or CMV promoter (pTRCMVβ). Based on these initial data, in vivo studies were completed using a rat carotid artery model. Both wildtype AAV (titers ∼1×10⁹ i.u./ml) and rAAV (pTRColod(l)β or pTRCMVβ) efficiently infected vascular cells in vivo with endothelial and vascular smooth muscle cell transduction frequencies approaching 90% as judged by DNA in situ PCR, with no evidence for disrupted vessel architecture. Protein expression using total vessel extracts at 24 hours post-infection demonstrated 20-fold increase in functional βgalaclosidase activity using pTRCola1(l)β compared to saline-injected controls vessels (799 +/- 236 μU/mg protein vs. 40.7 +/-17 μU/mg protein). These data provide the first evidence that rAAV may be adapted for directed high-level transgene delivery and expression into normally quiescent vascular endothelial and smooth muscle cells both in vitro and in vivo.