Self Complementary Recombinant AAV Vectors

描述（由申请人提供）：重组腺相关病毒（rAAV）载体受到宿主细胞DNA合成的限制，需要从单链病毒粒子DNA模板生成互补链。这种情况在不同细胞类型中以不同的效率发生，可通过共感染腺病毒、紫外线照射或其他DNA损伤或细胞应激手段诱导。当基因组长度为野生型基因组长度的一半时，我们利用AAV包装二聚体反向重复DNA分子的倾向绕过了这一限制。这些基因组从衣壳释放后可以重新退火形成ds-DNA，而不需要宿主细胞DNA合成。自互补rAAV （scAAV）比同源单链rAAV更有效地转导，并且不受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，在较低剂量下，scAAV转基因表达开始得更快，表达水平更高。rAAV在小鼠肝脏中的转导以前仅限于不到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转导大约50%的小鼠肝细胞。这说明了scAAV载体的定量和定性优势。在大脑中，当对DNA合成的需要被消除时，更高的表达水平和注射区域的饱和被实现。scAAV载体具有足够的遗传能力，可用于小蛋白编码基因、核酶和反义RNA策略的基因治疗应用。除了作为载体的作用外，scAAV还代表了rAAV转导途径中一个独特的中间体，这可以进一步加深我们对rAAV转导障碍的理解。在Aim 1中，将使用肝脏缺血-再灌注模型来询问caav基因表达的快速发作是否允许其用于传递抗氧化基因以保护移植组织和器官。在Aim 2中，我们将重点关注scAAV载体是否比单链rAAV载体更有效地表达中枢神经系统的治疗基因。将在小鼠脑中测试包装成不同AAV血清型的scAAV的转导效率，并通过将转基因和调节基因分别包装并在单次注射中共递送，测试Tet调节对scAAV载体的可行性。该系统将适用于在猎人病小鼠模型中递送基因编码的杜醛酸酯硫酸酯酶以纠正MPS II。在Aim 3中，我们将测试利用固有的AAV末端重复序列（TR）启动子活性来扩展scAAV编码能力的可行性。通过在一个TR中创建具有突变的构建体，迫使二聚体基因组复制，简化了scAAV载体的生产和纯化。这些将被测试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.