Mechanisms of AAV transduction in glaucoma-associated human trabecular meshwork cells

被引:45
作者
Borrás, T
Xue, W
Choi, VW
Bartlett, JS
Li, GR
Samulski, RJ
Chisolm, SS
机构
[1] Univ N Carolina, Sch Med, Dept Ophthalmol, Chapel Hill, NC 27515 USA
[2] Univ N Carolina, Gene Therapy Ctr, Chapel Hill, NC USA
[3] Ohio State Univ, Dept Pediat, Columbus, OH 43210 USA
关键词
human trabecular meshwork; adeno-associated viruses; recombinant replication-deficient adenoviruses; self-complementary adeno-associated viruses; perfused human anterior; segment cultures; microarrays;
D O I
10.1002/jgm.886
中图分类号
Q81 [生物工程学(生物技术)]; Q93 [微生物学];
学科分类号
071005 ; 0836 ; 090102 ; 100705 ;
摘要
Background: Glaucoma is a chronic eye disease which leads to irreversible blindness. The trabecular meshwork tissue controls intraocular pressure (IOP), which is the major risk factor for glaucoma. Gene therapy treatment of chronic diseases requires the use of long-term expression, low toxicity and lack of immune response vectors. Adeno-associated viruses (AAV) possess these characteristics but have been unable to transduce the trabecular meshwork. Because of the importance of regulating elevated IOP by long-term gene therapy, we investigated mechanisms of AAV transduction to the human trabecular meshwork (TM). Methods: Primary human trabecular meshwork cells (HTM) and perfused organ cultures were infected with rAAV2-GFP, RGD-pseudotyped rAAV2-GFP alone, or combined with recombinant Delta E1/E3 adenoviruses. Intracellular rAAV2 DNA and RNA were measured by relative quantitative and real-time TaqMan polymerase chain reaction (PCR). Host transcriptome was analyzed using high-density oligonucleotide microarrays. One transduction mechanism was tested using self-complementary AAV (scAAV). Results: The dramatic transduction enhancement obtained upon coinfection of rAAV2 with Delta E1/E3 adenoviruses provides insights into transduction mechanisms in the HTM. Even if not transduced, rAAV2 enters TM cells. GeneChip analysis showed significant changes in host genes involved in cell cycle and DNA replication. Consequently, scAAV-GFP transduction was highly efficient. Other transduction-enhancement genes included coxsackie adenovirus receptor (CAR) and genes relevant to trabecular meshwork function. Conclusions: The rate-limiting step of AAV transduction was not viral entry failure but, at least in part, host downregulation of DNA replication. Additional specific host genes might be involved. Our study revealed genes and mechanisms which led for the first time to efficient AAV transduction of the HTM. Copyright (c) 2006 John Wiley & Sons, Ltd.
引用
收藏
页码:589 / 602
页数:14
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