Aldrich W A, Ren C, White A F, Zhou S-Z, Kumar S, Jenkins C B, Shaw D R, Strong T V, Triozzi P L, Ponnazhagan S
Department of Medicine, The University of Alabama at Birmingham, Birmingham, AL, USA.
Gene Ther. 2006 Jan;13(1):29-39. doi: 10.1038/sj.gt.3302601.
The potential of adeno-associated virus (AAV)-based vectors in human gene therapy is being explored for several diseases. Although sustained transgene expression and low vector-associated cellular immunity are attractive features of recombinant (r) AAV, the wider application of rAAV vectors encapsidated in serotype 2 capsid is hampered by poor transduction efficiency in many target tissues. These include ex vivo-generated dendritic cells (DC), which have demonstrated promising immunotherapeutic activity. We report here that efficient transduction of mouse bone marrow-derived DC can be achieved with self-complementary (sc) rAAV encapsidated in serotype 6 capsid. Sequential exposure of DC precursor cultures to IL-4 and GM-CSF with sc rAAV6 encoding the human tumor antigen, carcinoembryonic antigen (CEA), for 7 days followed by activation with CpG oligodeoxynucleotides (ODN) and anti-mouse CD40 antibody resulted in highly efficient transduction of DC. DC surface markers as determined by flow cytometry analysis of sc rAAV6-transduced DC were comparable to nontransduced DC. Efficiency of vector transduction and transgene expression were confirmed by immunostaining and real-time PCR. Microarray analysis of RNA from CpG ODN and CD40 antibody stimulated sc AAV6-transduced DC revealed upregulation of transcription factors and cytokines involved in immune activation and downregulation of inhibitory factors, suggesting a possible role of transcriptional activation in the observed effect. The adoptive transfer into syngeneic mice of the ex vivo-transduced and activated DC resulted in the development of CEA-specific antibody and T-helper 1-associated immune responses. Immunized mice also developed antibody to AAV6 capsid protein, which did not crossreact with AAV2 capsid protein. These studies demonstrate the potential utility of sc rAAV serotype 6-based vectors in transduction of DC for genetic vaccination approaches.
腺相关病毒（AAV）载体在人类基因治疗中对多种疾病的应用潜力正在被探索。尽管重组（r）AAV具有持续的转基因表达和较低的载体相关细胞免疫等吸引人的特性，但衣壳为2型的rAAV载体在许多靶组织中的转导效率较低，这阻碍了其更广泛的应用。这些靶组织包括体外生成的树突状细胞（DC），它们已显示出有前景的免疫治疗活性。我们在此报告，衣壳为6型的自我互补（sc）rAAV能够高效转导小鼠骨髓来源的DC。将DC前体细胞培养物先后暴露于白细胞介素-4和粒细胞-巨噬细胞集落刺激因子，并加入编码人类肿瘤抗原癌胚抗原（CEA）的sc rAAV6，持续7天，随后用CpG寡脱氧核苷酸（ODN）和抗小鼠CD40抗体激活，可实现DC的高效转导。通过对sc rAAV6转导的DC进行流式细胞术分析确定的DC表面标志物与未转导的DC相当。通过免疫染色和实时PCR证实了载体转导效率和转基因表达。对来自CpG ODN和CD40抗体刺激的sc AAV6转导的DC的RNA进行微阵列分析，发现参与免疫激活的转录因子和细胞因子上调，抑制因子下调，这表明转录激活在观察到的效应中可能起作用。将体外转导并激活的DC过继转移到同基因小鼠中，导致了CEA特异性抗体和辅助性T细胞1相关免疫反应的产生。免疫小鼠还产生了针对AAV6衣壳蛋白的抗体，该抗体与AAV2衣壳蛋白无交叉反应。这些研究证明了基于sc rAAV 6型载体在转导DC用于基因疫苗接种方法方面的潜在效用。
