Characterization of Recombinant Adeno-Associated Virus-2 as a Vehicle for Gene Delivery and Expression into Vascular Cells

Background: We have used wild-type and recombinant adeno-associated virus-2 (AAV) to study transduction, replication efficiencies, functional protein expression, and gene delivery to vascular cells in vitro and in vivo. Methods: Recombinant adeno-associated virus-2 (rAAV) plasmids (ranging in size to 110% of wild-type AAV) driven by 6 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 IU/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 AAV-infected HUVEC established the presence of episomal forms at 18 days, although chromosome 19-specific integration was not evident. Functional β-galactosidase activity -400% above control was evident in HUVEC using either a murine collagen αl(I) promoter (pTRColα1(I)β) or CMV promoter (pTRCMVβ). Results: Based on these initial data, in vivo studies were completed using a rat carotid artery model. Both wild-type AAV (titers -1X10⁹ IU/mL) and rAAV (pTRColα1(I)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 polymerase chain reaction, with no evidence for disrupted vessel architecture. Protein expression using total vessel extracts at 48 hours postinfection demonstrated 20-fold increase in functional β-galactosidase activity using pTRColα1(I)β compared to saline-injected controls vessels (799 +/- 236 μU/mg protein vs 40.7 +/-17 μU/mg protein). Conclusions: 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.