Shi Shuiliang, Mercer Scott A, Dilley Robert, Trippel Stephen B
Department of Orthopaedic Surgery, Indiana University School of Medicine, Indianapolis, IN, USA.
Virol J. 2009 Jan 7;6:3. doi: 10.1186/1743-422X-6-3.
Adeno-associated virus (AAV) vectors are promising tools for gene therapy. Currently, their potential is limited by difficulties in producing high vector yields with which to generate transgene protein product. AAV vector production depends in part upon the replication (Rep) proteins required for viral replication. We tested the hypothesis that mutations in the start codon and upstream regulatory elements of Rep78/68 in AAV helper plasmids can regulate recombinant AAV (rAAV) vector production. We further tested whether the resulting rAAV vector preparation augments the production of the potentially therapeutic transgene, insulin-like growth factor I (IGF-I).
We constructed a series of AAV helper plasmids containing different Rep78/68 start codon in combination with different gene regulatory sequences. rAAV vectors carrying the human IGF-I gene were prepared with these vectors and the vector preparations used to transduce HT1080 target cells. We found that the substitution of ATG by ACG in the Rep78/68 start codon in an AAV helper plasmid (pAAV-RC) eliminated Rep78/68 translation, rAAV and IGF-I production. Replacement of the heterologous sequence upstream of Rep78/68 in pAAV-RC with the AAV2 endogenous p5 promoter restored translational activity to the ACG mutant, and restored rAAV and IGF-I production. Insertion of the AAV2 p19 promoter sequence into pAAV-RC in front of the heterologous sequence also enabled ACG to function as a start codon for Rep78/68 translation. The data further indicate that the function of the AAV helper construct (pAAV-RC), that is in current widespread use for rAAV production, may be improved by replacement of its AAV2 unrelated heterologous sequence with the native AAV2 p5 promoter.
Taken together, the data demonstrate an interplay between the start codon and upstream regulatory sequences in the regulation of Rep78/68 and indicate that selective mutations in Rep78/68 regulatory elements may serve to augment the therapeutic value of rAAV vectors.
腺相关病毒（AAV）载体是基因治疗中很有前景的工具。目前，其潜力因难以产生高载体产量以生成转基因蛋白产物而受到限制。AAV载体的生产部分取决于病毒复制所需的复制（Rep）蛋白。我们测试了一个假设，即AAV辅助质粒中Rep78/68起始密码子和上游调控元件的突变可调节重组AAV（rAAV）载体的生产。我们进一步测试了所得的rAAV载体制剂是否能增加潜在治疗性转基因胰岛素样生长因子I（IGF-I）的产量。
我们构建了一系列包含不同Rep78/68起始密码子并结合不同基因调控序列的AAV辅助质粒。用这些载体制备携带人IGF-I基因的rAAV载体，并将载体制剂用于转导HT1080靶细胞。我们发现，在AAV辅助质粒（pAAV-RC）中，将Rep78/68起始密码子中的ATG替换为ACG会消除Rep78/68的翻译、rAAV和IGF-I的产生。用AAV2内源性p5启动子替换pAAV-RC中Rep78/68上游的异源序列，可使ACG突变体恢复翻译活性，并恢复rAAV和IGF-I的产生。将AAV2 p19启动子序列插入pAAV-RC中异源序列之前，也能使ACG作为Rep78/68翻译的起始密码子发挥作用。数据进一步表明，目前广泛用于rAAV生产的AAV辅助构建体（pAAV-RC）的功能，可通过用天然AAV2 p5启动子替换其AAV2无关的异源序列来改善。
综上所述，数据证明了起始密码子和上游调控序列在Rep78/68调控中的相互作用，并表明Rep78/68调控元件中的选择性突变可能有助于提高rAAV载体的治疗价值。
