Zhang Nan, Clément Nathalie, Chen Dongmei, Fu Shuang, Zhang Haojiang, Rebollo Patricia, Linden R Michael, Bromberg Jonathan S
Recanati-Miller Transplantation Institute, Mount Sinai School of Medicine, New York, NY 10029, USA.
Transplantation. 2005 Sep 15;80(5):683-90. doi: 10.1097/01.tp.0000173381.97556.0d.
Recombinant adeno-associated viral (rAAV) vectors currently show promise for islet gene therapy. In the presence of complementing AAV2 Rep proteins, AAV2 genomes can be packaged with other serotype capsids to assemble infectious virions. During transduction, the ssDNA to dsDNA conversion is one of the major rate-limiting steps that contribute to the slow onset of transgene expression.
Using pseudotyping strategy, we produced double-stranded (dsAAV) and single-stranded (ssAAV) rAAV2 genomes carrying the GFP reporter gene packaged into AAV1, AAV2, and AAV5 capsids. The ability of cross-packaged AAV1, AAV2, and AAV5 at the same genome containing particle (gcp) concentration to transduce murine and human pancreatic islets was evaluated by GFP positive cell percentage. Transgenic expression was also determined by transplant transduced human islet into SCID mice.
Pseudotyped rAAV2/1 based vectors transduced murine islets at greater efficiency than either rAAV2/2 or rAAV2/5 vectors. For human islets transduction, the rAAV2/2 vector was more efficient than rAAV2/1 or rAAV2/5 vectors. rAAV2/2 transduced human islets more efficiently than murine islets, while rAAV2/1 transducted murine islets more efficiently than human islets. dsAAV, which do not require second strand synthesis and thus are potentially more efficient, evidenced 5 fold higher transduction ability than ssAAV vectors. Pseudotyped rAAV transduced islet grafts maintained normal function, expressed transgenic product persistently in vivo, and reversed diabetes.
The transduction efficiency of rAAV vectors was dependent on the cross-packaged capsid. The vector capsids permit species-specific transduction. For human islets, dsAAV2/2 vectors may be the most efficient vector for clinical development.
重组腺相关病毒（rAAV）载体目前在胰岛基因治疗方面显示出前景。在存在互补性AAV2 Rep蛋白的情况下，AAV2基因组可与其他血清型衣壳包装在一起以组装感染性病毒粒子。在转导过程中，单链DNA向双链DNA的转化是导致转基因表达起效缓慢的主要限速步骤之一。
采用假型化策略，我们制备了携带绿色荧光蛋白（GFP）报告基因的双链（dsAAV）和单链（ssAAV）rAAV2基因组，其被包装到AAV1、AAV2和AAV5衣壳中。通过GFP阳性细胞百分比评估相同基因组包含颗粒（gcp）浓度下交叉包装的AAV1、AAV2和AAV5转导小鼠和人胰岛的能力。还通过将转导的人胰岛移植到SCID小鼠中来确定转基因表达情况。
基于假型化rAAV2/1的载体转导小鼠胰岛的效率高于rAAV2/2或rAAV2/5载体。对于人胰岛转导，rAAV2/2载体比rAAV2/1或rAAV2/5载体更有效。rAAV2/2转导人胰岛比转导小鼠胰岛更有效，而rAAV2/1转导小鼠胰岛比转导人胰岛更有效。不需要第二链合成因而可能更有效的dsAAV，其转导能力比ssAAV载体高5倍。假型化rAAV转导的胰岛移植物保持正常功能，在体内持续表达转基因产物，并逆转糖尿病。
rAAV载体转导效率取决于交叉包装的衣壳。载体衣壳允许物种特异性转导。对于人胰岛，dsAAV2/2载体可能是临床开发中最有效的载体。
