Zhong Li, Zhou Xiaohuai, Li Yanjun, Qing Keyun, Xiao Xiao, Samulski Richard Jude, Srivastava Arun
Department of Pediatrics, Division of Cellular and Molecular Therapy, Powell Gene Therapy Center, University of Florida College of Medicine, Gainesville, Florida 32610-3633, USA.
Mol Ther. 2008 Feb;16(2):290-5. doi: 10.1038/sj.mt.6300376. Epub 2007 Dec 18.
Recombinant adeno-associated virus 2 (AAV) vectors encapsidate single-stranded genomes of either polarity equally frequently in separate mature virions. Because viral genomes of either polarity are transcriptionally inactive, both the failure to undergo viral second-strand DNA synthesis and the failure to undergo DNA strand annealing have been proposed as possible reasons to account for the observed low efficiency of transgene expression. We compared the transduction efficiencies of conventional AAV vectors containing both [-] and [+] polarity genomes with those containing either the [-] or the [+] polarity genomes, in vitro as well as in vivo. We document that the transduction efficiency of single-polarity AAV vectors is significantly enhanced by (i) co-infection with adenovirus; (ii) small interfering RNA (siRNA)-mediated down-modulation of a cellular protein, FKBP52, tyrosine-phosphorylated forms of which inhibit AAV second-strand DNA synthesis; (iii) over-expression of a cellular protein tyrosine phosphatase, T cell protein tyrosine phosphatase (TC-PTP), which catalyzes tyrosine-dephosphorylation of FKBP52; and (iv) deliberate over-expression of TC-PTP, or the absence of FKBP52, respectively, in TC-PTP-transgenic mice and in FKBP52-knockout mice. These data confirm that viral second-strand DNA synthesis, rather than DNA strand annealing, is the rate-limiting step in efficient transduction by AAV vectors. This finding has implications in the use of these vectors in human gene therapy.
重组腺相关病毒2型（AAV）载体在单独的成熟病毒粒子中，以相同的频率等量包装正负两种极性的单链基因组。由于两种极性的病毒基因组均无转录活性，因此有人提出，病毒第二链DNA合成失败以及DNA链退火失败，可能是导致观察到的转基因表达效率低下的原因。我们在体外和体内比较了含有正负两种极性基因组的传统AAV载体与仅含负或正极性基因组的载体的转导效率。我们证明，单极性AAV载体的转导效率可通过以下方式显著提高：（i）与腺病毒共感染；（ii）小干扰RNA（siRNA）介导下调一种细胞蛋白FKBP52，其酪氨酸磷酸化形式会抑制AAV第二链DNA合成；（iii）过表达一种细胞蛋白酪氨酸磷酸酶，即T细胞蛋白酪氨酸磷酸酶（TC-PTP），它可催化FKBP52的酪氨酸去磷酸化；以及（iv）分别在TC-PTP转基因小鼠和FKBP52基因敲除小鼠中刻意过表达TC-PTP或缺失FKBP52。这些数据证实，病毒第二链DNA合成而非DNA链退火，是AAV载体高效转导的限速步骤。这一发现对这些载体在人类基因治疗中的应用具有重要意义。
