GENE TRANSFER INTO NON DIVIDING CELLS BY MEANS OF HIV

我们已经开发了利用HIV载体将基因转移到非分裂细胞中的系统。CD4+HeLa细胞在G2期通过γ照射和细胞术分析被捕获。这些不分裂的细胞与含有β -gal基因的HIV载体孵育，两天后用X-gal染色。在G2和G1/S阻滞的CD4+HeLa细胞中检测到的蓝色细胞数量大致相同。HIV载体在这些非分裂细胞上的转导效率与在分裂细胞上的转导效率相当。此外，我们利用链接物介导的PCR （LMPCR）技术分析了HIV载体在非分裂细胞中的整合位点。在一些克隆中检测到逆转录病毒整合特征的连接序列，表明HIV载体能够稳定地整合到非分裂细胞的染色体中。下一步，我们开发了包含CD4基因的腺病毒载体和HIV载体的两步基因转移系统，以扩大HIV载体的宿主范围。腺病毒介导的CD4的瞬时表达有效地使各种非t细胞对HIV介导的稳定基因转移敏感。由于腺病毒和艾滋病毒载体都可以转导非分裂细胞，因此这两种载体的结合可以作为基因转移到非分裂细胞的一般策略。因此，HIV载体不仅可用于艾滋病的基因治疗，而且可用于各种针对非分裂细胞的基因治疗方案。

We have developed the gene transfer system into non-dividing cells using HIV vectors. CD4+HeLa cells were arrested at the G2 phase by gamma irradiation and at the cytometry analysis. These non-dividing cells were incubated with the HIV based vector containing the beta-gal gene and stained with X-gal after two days. Approximately the same numbers of blue cells were detected in both G2 and G1/S arrested CD4+HeLa cells. The transduction efficiency of HIV vectors on these non-dividing cells was comparable to that on dividing cells. Moreover, we analyzed the integration sites of HIV vectors in non-dividing cells by the linker mediated PCR (LMPCR) technique. The junction sequences chracteristic for retrovirus integration was detected in some clones, indicating that HIV vectors are able to integrate stably into the chromosome of non-dividing cells.The next step, we have developed the two step gene transfer system using an adenoviral vector containing the CD4 gene and HIV vectors to expand the host range of HIV vectors. Adenoviral mediated transient expression of CD4 was efficiently render various non-T cells susceptible to HIV mediated stable gene transfer. Since both adenovirus and HIV vectors can transduce non-dividing cells, the combination of these two vectors may be used as a general strategy for gene transfer into non-dividing cells. Therefor, HIV vector may be useful not only for gene therapy of AIDS but also for a variety of gene therapy protocols targeting non-dividing cells.