Self Complementary Recombinant AAV Vectors

描述（由申请人提供）：重组腺相关病毒（rAAV）载体受限于宿主细胞DNA合成的要求，以从单链病毒体DNA模板生成互补链。这在不同的细胞类型中以不同的效率发生，并且可以通过用共感染的腺病毒、UV照射或其他DNA损伤或细胞应激手段进行处理来诱导。我们已经利用AAV在基因组是野生型基因组长度的一半时包装二聚体反向重复DNA分子的倾向来规避这种限制。这些基因组可以在从衣壳释放后重新退火以形成ds-DNA，而不需要宿主细胞DNA合成。自身互补rAAV（scAAV）比同源单链rAAV转导更有效，并且不受DNA合成抑制剂的影响。在小鼠肌肉和肝脏中，scAAV转基因表达开始更快，达到更高的水平，并且在比常规单链rAAV载体更低的剂量下观察到。在小鼠肝脏中，rAAV的转导以前仅限于少于5%的肝细胞。使用scAAV，我们可以在使用相同剂量的单次尾静脉注射后使大约50%的小鼠肝细胞存活。这说明了scAAV载体的定量和定性优势。在大脑中，当消除DNA合成的需要时，注射区域的表达水平和饱和度就会提高。scAAV载体具有足够的遗传能力用于基因治疗应用，包括递送小蛋白编码基因和核酶以及反义RNA策略。除了作为载体的效用之外，scAAV还代表rAAV转导途径中的独特中间体，这可以进一步我们对rAAV转导障碍的理解。在目的1中，将使用肝脏缺血-再灌注模型来询问scAAV基因表达的快速开始是否将允许其用于递送抗氧化剂基因以保护移植的组织和器官。在目标2中，将关注scAAV载体是否可以比单链rAAV载体更有效地在中枢神经系统中表达治疗基因。将在小鼠脑中测试包装成不同AAV血清型的scAAV的转导效率，并通过单独包装转基因和调节基因并在单次注射中共同递送它们来测试使泰特调节适应scAAV载体的可行性。该系统将适用于递送编码艾杜糖醛酸硫酸酯酶的基因，以校正Hunters病小鼠模型中的MPS II。在目的3中，将测试通过使用固有AAV末端重复（TR）启动子活性来扩展scAAV编码能力的可行性。scAAV载体的生产和纯化已经通过在一个TR中产生具有突变的构建体来简化，从而迫使二聚体基因组复制。将测试这些突变对末端重复序列转录起始的影响。

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.