Targeted Gene Therapy for Neurologic disease

描述(由申请人提供)： 遗传性神经系统疾病的有效基因治疗需要开发安全的载体，使转基因能够稳定地在神经元上表达。作为模型疾病，我们选择了共济失调-毛细血管扩张(A-T)，这是一种常染色体隐性遗传病，其特征是神经变性、免疫缺陷、癌症易感性和辐射敏感性。这些研究将集中在发展基于质粒的HSV扩增载体，以实现转基因到细胞基因组的定点整合或通过同源重组(HR)纠正内源基因的突变。替换载体将整合AAV的元件，包括ITRs和Rep，以定向插入ATM基因敲除小鼠基因组中的AAVS1位点。HR的载体将包括正常小鼠基因的8和16 kb区域，跨越敲入小鼠ATM基因9bp的缺失，以及应该增加HR频率的元件的序列和表达盒，如RAD51和HSV ICP8。通过转基因表达(免疫细胞化学和RT-PCR)和重组事件分析(PCR、Southern blotts和测序)，将在这些小鼠的成纤维细胞和神经前体细胞培养中以及在这些小鼠的原代神经元培养和小脑中的神经元中评估成功的整合。在新生小鼠小脑内注射载体后，将通过恢复神经元对电离辐射的正常凋亡反应，以及通过长期改善运动功能、减少氧化损伤和ATM的表达来评估功能恢复。这些治疗性载体设计应用于广泛的神经系统疾病，允许传递和稳定表达大的转基因盒，并有可能永久纠正神经元中的基因缺陷。

DESCRIPTION (provided by applicant): Effective gene therapy for hereditary neurologic disease will require the development of safe vectors, which can confer stable transgene expression on neurons. As a model disease we have chosen ataxia-telangiectasia (A-T), an autosomal recessive disorder characterized by neurodegeneration, immunodeficiency, cancer predisposition and radiation sensitivity. These studies will focus on the development of plasmid-based HSV amplicon vectors to achieve site-directed integration of transgenes into the cell genome or to correct mutations in endogenous genes by homologous recombination (HR). Replacement vectors will incorporate elements of AAV, including ITRs and Rep, to target insertion of the ATM cDNA into an AAVS1 site engineered into the genome of Atm knock-out mice. Vectors for HR will include 8 and16 kb regions of the normal mouse gene spanning a 9 bp deletion in the Atm gene in knock-in mice, as well as sequences and expression cassettes for elements that should increase the frequency of HR, such as Rad51 and HSV ICP8. Successful integrations will be evaluated in cultured fibroblasts and neuroprecursor cells from these mice, as well as in primary neuronal cultures and in neurons in the cerebella of these mice by expression of the transgene (immunocytochemistry and RT-PCR) and analysis of recombinant events (PCR, Southern blots and sequencing). Functional recovery will be assessed following injection of vectors in the newborn mouse cerebellum by restoration of the normal apoptotic response of neurons to ionizing radiation, as well as by long-term improvement in motor function, reduction of oxidative damage and expression of ATM. These therapeutic vector designs have applications for a broad range of neurologic diseases allowing delivery and stable expression of large transgene cassettes and the potential for permanent correction of gene defects in neurons.