We have used wild-type and recombinant adenoassociated virus-2 (AAV) to study transduction, replication efficiencies, functional protein expression, and gene delivery to vascular cells in vitro and in vivo.
Methods
Recombinant adenoassociated 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 α1(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.
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