FKBP52在重组腺相关病毒载体胞内转运中的作用研究

Role of cellular FKBP52 protein in hydroxyurea treatment-mediated increase in transduction efficiency of recombinant adeno-associated virus 2 vectors

目的探讨细胞质中FKBP52对重组腺相关病毒(AAV)介导的基因转导效率的作用。方法测定AAV在FKBP52-野生型(WT)、FKBP52-杂合型(HE)及FKBP52-基因敲除(KO)鼠胚纤维母细胞(MEFs)中的基因转导效率,观察羟基脲(HU)对不同基因型MEFs中AAV转导效率的影响。采用流式细胞术、EMSA、Southernblot、免疫沉淀、West-ernblot等方法分析FKBP52对AAV在细胞内转运的作用。结果传统的单链AAV不能有效地转导FKBP52-WTMEFs,在FKBP52-HE及FKBP52-KOMEFs中的转导效率也未见明显增加。在这些细胞中,AAV不能有效地转运至细胞核。HU处理后能使AAV在WTMEFs中的转导效率提高25倍,但是在KOMEFs中AAV转导效率仅提高4倍。为避免AAV第二链合成的影响,进一步采用自身互补的AAV(scAAV)载体进行研究,结果发现,HU能使WTMEFsscAAV基因转导效率增强23倍,但在KOMEFs中转导效率仅增加4倍。提示,HU处理后KOMEFs中转导效率未增加的原因并非由于AAV第二链的合成障碍所致。HU处理后,59%的AAV基因组DNA出现在WTMEFs细胞核,KOMEFs细胞核中为28%,提示HU介导的AAV转运通路在KOMEFs中存在缺陷。转染FKBP52表达质粒的KOMEFs经HU处理后,AAV所介导的基因转导效率恢复至WTMEFs的水平,且直接与其转运至细胞核的转运功能改善相吻合。结论作为一种细胞分子伴侣蛋白,FKBP52能促进AAV在细胞内转运,有益于重组AAV载体在人类基因治疗中的应用。

Aim To explore the role of cytoplasmic FKBP52 in AAV-mediated transduction. Methods Murine embryo fibroblasts （MEFs）cultures from FKBP52 wild-type （WT）, heterozygous （HE）, and knockout （KO） mice were established. The role of FKBP52 in intracellular trafficking of AAV was analyzed by fluorescence-activated cell sorting （FACS） analyses, electrophoretic mobility shift assays （EMSA）, southern blot, immunoprecipitations and western blot analyses. Results Conventional AAV vectors failed to transduce WT MEFs efficiently, and the transduction efficiency was not significantly increased in HE or KO MEFs. AAV vectors failed to traffick efficiently to the nucleus in these cells. Treatment with hydroxyur-ea （HU） increased the transduction efficiency of conventional AAV vectors by- 25-fold in WT MEFs, but only by -4-fold in KO MEFs. The use of self-complementary AAV （scAAV） vectors, which bypass the requirement of viral second-strand DNA synthesis, revealed that HU treatment increased the transduction efficiency -23-fold in WT MEFs, but only -4- fold in KO MEFs, indicating that the lack of HU treatment-mediated increase in KO MEFs was not due to failure of AAV to undergo viral second-strand DNA synthesis. Following HU treatment, -59% of AAV genomes were present in the nuclear fraction from WT MEFs, but only -28% in KO MEFs, indicating that the pathway by which HU treatment mediates nuclear transport of AAV was impaired in KO MEFs. When KO MEFs were stably transfected with an FKBP52 expression plasmid, HU treatment-mediated increased in the transduction efficiency was restored in these cells, which correlated directly with improved intracellular trafficking. Intact AAV particles were also shown to interact with FKBP52 as well as with dynein, a known cellular protein involved in AAV trafficking. Conclusion These studies suggest that FKBP52, being a cellular chaperone protein, facilitates intracellular trafficking of AAV, which has implications in the optimal use of recombinant AAV vectors in human gene therapy.