Gnatenko D, Arnold T E, Zolotukhin S, Nuovo G J, Muzyczka N, Bahou W F
Department of Medicine, State University of New York at Stony Brook 11794-8151, USA.
J Investig Med. 1997 Feb;45(2):87-98.
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.
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 x 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 approximately 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).
Based on these initial data, in vivo studies were completed using a rat carotid artery model. Both wild-type AAV (titers -1X10(9) IU/mL) and rAAV (pTRCol alpha 1(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 microU/mg protein vs 40.7 +/- 17 microU/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.
我们已使用野生型和重组腺相关病毒2型（AAV）来研究体外和体内的转导、复制效率、功能性蛋白表达以及向血管细胞的基因递送。
由β-半乳糖苷酶盒上游6个不同启动子驱动的重组腺相关病毒2型（rAAV）质粒（大小范围为野生型AAV的110%）被有效地用于产生复制缺陷型病毒，滴度始终在2.5×10⁵IU/mL范围内。AAV在人脐静脉内皮细胞（HUVEC）中的感染性和复制与细胞增殖指数无关，这确立了该病毒用于转导静止血管细胞的潜在效用。AAV感染的HUVEC的长期培养显示在18天时存在游离形式，尽管未观察到19号染色体特异性整合。使用鼠胶原蛋白α1（I）启动子（pTRColα1（I）β）或巨细胞病毒启动子（pTRCMVβ）时，HUVEC中功能性β-半乳糖苷酶活性比对照高约400%。
基于这些初步数据，使用大鼠颈动脉模型完成了体内研究。野生型AAV（滴度-1×10⁹IU/mL）和rAAV（pTRColα1（I）β或pTRCMVβ）在体内均能有效感染血管细胞，通过DNA原位聚合酶链反应判断，内皮细胞和血管平滑肌细胞的转导频率接近90%，且无血管结构破坏的证据。感染后48小时使用总血管提取物进行的蛋白表达显示，与注射生理盐水的对照血管相比，使用pTRColα1（I）β时功能性β-半乳糖苷酶活性增加了20倍（799±236微单位/毫克蛋白对40.7±17微单位/毫克蛋白）。
这些数据首次证明rAAV可适用于在体外和体内将转基因定向高水平递送至正常静止的血管内皮细胞和平滑肌细胞并实现表达。
