Characterization of recombinant adeno-associated virus-2 as a vehicle for gene delivery and expression into vascular cells
Gnatenko, D.; Arnold, T.E.; Zolotukhin, S.; Nuovo, G.J.; Muzyczka, N.; Bahou, W.F.
Journal of Investigative Medicine the Official Publication of the American Federation for Clinical Research 45(2): 87-98
1997
ISSN/ISBN: 1081-5589 PMID: 9084579 Document Number: 478077
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 beta-galactosidase cassette were effectively used for generation of replication-deficient virus, with titers consistently ranging from 2-5 times 10-5 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 beta-galactosidase activity -400% above control was evident in HUVEC using either a murine collagen alpha-1(I) promoter (pTRCol-alpha-1(I)beta) or CMV promoter (pTRCMV-beta). Results: Based on these initial data, in vivo studies were completed using a rat carotid artery model. Both wild-type AAV (titers sbd 1 times 10-9 IU/mL) and rAAV (pTRCol-alpha(I)beta or pTRCMV-beta) 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 beta-galactosidase activity using pTRCol-alpha-1(I)beta compared to saline-injected controls vessels (799 +- 236 mu-U/mg protein vs 40.7 +- 17 mu-U/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.