Self Complementary Recombinant AAV Vectors

描述(申请人提供)：重组腺相关病毒(RAAV)载体受限于宿主细胞DNA合成的要求，以从单链病毒DNA模板产生互补链。这在不同类型的细胞中发生的效率不同，可以通过联合感染腺病毒、紫外线照射或其他方式的DNA损伤或细胞应激来诱导。我们绕过了这一限制，利用AAV在基因组长度为野生型基因组长度的一半时包装二聚体反向重复DNA分子的趋势。当这些基因组从衣壳中释放出来时，可以重新退火形成ds-DNA，而不需要宿主细胞DNA合成。自互补rAAV(ScAAV)的转导效率高于同源单链rAAV，且不受DNA合成抑制剂的影响。在小鼠肌肉和肝脏中，与传统的单链rAAV载体相比，scAAV转基因表达开始得更早，表达水平更高，并且可以在更低的剂量下观察到。以前，rAAV在小鼠肝脏中的转导仅限于不到5%的肝细胞。单次尾静脉注射相同剂量的ScAAV后，我们可以转导大约50%的小鼠肝细胞。这说明了scAAV载体的数量和质量优势。在大脑中，当不需要DNA合成时，注射区域获得了更高的表达水平和饱和度。单链AAV载体具有足够的遗传容量用于基因治疗，包括运送小蛋白编码基因、核酶和反义RNA策略。除了作为载体的作用外，scAAV还是rAAV转导途径中一种独特的中间体，它可以加深我们对rAAV转导障碍的理解。在目标1中，将使用肝脏缺血-再灌注模型来研究scAAV基因的快速开始表达是否会使其能够传递抗氧化基因来保护移植的组织和器官。在目标2中，将重点研究scAAV载体是否能比单链rAAV载体更有效地在中枢神经系统表达治疗基因。包装成不同AAV血清型的单链AAV的转导效率将在小鼠脑内进行测试，并将通过单独包装转基因和调控基因并在一次注射中共同传递的方式来测试将Tet调节适应于单链AAV载体的可行性。该系统将适用于在亨特斯病小鼠模型中传递编码艾杜糖酸硫酸酯酶的基因，以纠正MPS II。在目的3中，将测试利用固有的AAV末端重复(TR)启动子活性来扩大scAAV编码量的可行性。ScAAV载体的生产和纯化已经通过创建在一个tR中具有突变的结构来简化，从而迫使二聚体基因组的复制。这些都将被测试突变对末端重复序列转录启动的影响。

DESCRIPTION (provided by applicant): Recombinant adeno-associated virus (rAAV) vectors are limited by the requirement for host-cell DNA synthesis to generate a complementary strand from the single-stranded virion DNA template. This occurs with varying efficiency in different cell types and can be induced through treatment with co-infecting adenovirus, UV irradiation, or other means of DNA damage or cell stress. We have circumvented this limitation using the tendency of AAV to package dimeric inverted repeat DNA molecules when the genome is half the wild-type genome length. These genomes can re-anneal to form ds-DNA upon release from the capsid with no host-cell DNA synthesis required. The self-complementary rAAV (scAAV) transduces more efficiently than the homologous single-strand rAAV and is unaffected by inhibitors of DNA synthesis. In mouse muscle and liver, scAAV transgene expression began sooner, reached higher levels, and was observable at lower doses than conventional single-strand rAAV vectors. Transduction in mouse liver with rAAV had previously been limited to less than 5% of hepatocytes. Using the scAAV, we can transduce approximately 50% of mouse hepatocytes after a single tail-vein injection using the same dose. This illustrates both the quantitative and qualitative advantage of the scAAV vectors. In brain, greater expression levels and saturation of the injected area were achieved when the need for DNA synthesis was eliminated. The scAAV vectors have sufficient genetic capacity for gene therapy applications including delivery of small protein coding genes and ribozyme and anti-sense RNA strategies. Apart from its utility as a vector, scAAV represents a unique intermediate in the rAAV transduction pathway, which can further our understanding of barriers to rAAV transduction. In Aim 1, a liver ischemia-reperfusion model will be used to ask whether the rapid onset of scAAV gene expression will allow its use to deliver an anti-oxidant gene for protection of transplanted tissue and organs. In Aim 2, will focus on whether scAAV vectors can express therapeutic genes in the central nervous system more effectively than single-strand rAAV vectors. The transducing efficiency of scAAV, packaged into different AAV serotypes, will be tested in mouse brain and the feasibility of adapting Tet regulation to scAAV vectors will be tested by separately packaging the transgene and regulatory gene and co-delivering them in a single-injection. This system will be adapted to the delivery of the gene coding iduronate sulfatase for correction of MPS II in a Hunters disease mouse model. In Aim 3, the feasibility of expanding the scAAV coding capacity by using the intrinsic AAV terminal repeat (TR) promoter activity will be tested. The production and purification of scAAV vectors has been streamlined by creating constructs with mutations in one TR, forcing the replication of dimeric genomes. These will be tested for the effects of the mutations on transcription initiation from the terminal repeats.