Production of recombinant AAV vectors encoding insulin-like growth factor I is enhanced by interaction among AAV rep regulatory sequences.
Production of recombinant AAV vectors encoding insulin-like growth factor I is enhanced by interaction among AAV rep regulatory sequences.
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DOI：
10.1186/1743-422x-6-3
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发表时间：
2009-01-07
期刊：
影响因子：
4.8
通讯作者：
Trippel SB
中科院分区：
文献类型：
--
作者：
Shi S;Mercer SA;Dilley R;Trippel SB
腺相关病毒（AAV）载体是基因治疗的有前途的工具。目前，它们的潜力受到难以产生高载体产量以产生转基因蛋白产物的限制。AAV载体生产部分取决于病毒复制所需的复制（Rep）蛋白。我们测试了这样的假设，即在AAV辅助质粒中Rep 78/68的起始密码子和上游调控元件中的突变可以调控重组AAV（rAAV）载体的产生。我们进一步测试了所得rAAV载体制剂是否增加了潜在治疗性转基因胰岛素样生长因子I（IGF-I）的产生。我们构建了一系列含有不同Rep 78/68起始密码子和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，并将载体制备物用于转染HT 1080靶细胞。我们发现，在AAV辅助质粒（pAAV-RC）中，Rep 78/68起始密码子中的ATG被ACG取代消除了Rep 78/68翻译、rAAV和IGF-I的产生。用AAV 2内源性p5启动子替换pAAV-RC中Rep 78/68上游的异源序列恢复了ACG突变体的翻译活性，并恢复了rAAV和IGF-I的产生。将AAV 2 p19启动子序列插入到pAAV-RC中的异源序列之前也使得ACG能够充当Rep 78/68翻译的起始密码子。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）的功能可以通过用天然AAV 2 p5启动子替换其AAV 2不相关的异源序列来改善。综上所述，这些数据证明了在Rep 78/68的调节中起始密码子和上游调节序列之间的相互作用，并表明Rep 78/68调节元件中的选择性突变可用于增强rAAV载体的治疗价值。
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.