Gene therapy for tuberous sclerosis

我们的研究小组将评估基因治疗减少结节性硬化症危及生命症状的潜力 TSC综合征（TSC），影响全球200万人。TSC是一种常染色体显性肿瘤抑制基因， 由TSC 1（编码错构瘤蛋白）或TSC 2（编码块茎蛋白）中的遗传突变引起的疾病， 相应正常等位基因的体细胞丢失导致错构瘤（局灶性细胞过度生长）， 机关神经系统受累包括皮质结节、室管膜下结节和室管膜下巨细胞 星形细胞瘤伴脑积水癫痫、自闭症、认知障碍和心理健康问题。我们有 建立了一个随机的中枢神经系统（CNS）小鼠模型的TSC 2，表现出室管膜下 过度生长，脑积水和早期死亡，反映了TSC患者的情况。现行标准 脑室管膜下病变的护理包括神经外科开颅术和/或长期治疗， 雷帕霉素，两者都对大脑发育有潜在的破坏作用。我们的论点是，一个威胁生命的 TSC -脑积水的并发症可以通过侵入性较小且持续时间较长的手术来预防，即 静脉内（IV）递送编码替代蛋白的腺相关病毒（AAV）载体，而不 如果需要，会影响其他护理标准的使用。第四，交付应实现“额外的副本”的 外周组织中的替代基因，降低了其他危及生命的错构瘤形成的可能性， 身体我们将使用我们的CNS Tsc 2小鼠模型来评估编码AAV载体的能力。 浓缩版的块茎素（ceparin），以减少室管膜异常的大小和延长寿命。 R61目的1 -评估培养物中的ceptin蛋白，包括使用来自 患者分化成神经祖细胞，细胞毒性和功能测定，去无去：ceparin 必须显示至少50%的tuberin抑制mTOR活性的活性，以及细胞大小的正常化， 细胞增殖率。目的2 -利用CNS Tsc 2小鼠中的AAV-cycloin优化AAV血清型和启动子 建模并评估治疗效果。行为、存活时间、神经病理学和全身病理学 将被监测，以及载体分布和载体靶向脑细胞的能力， 外周组织Go-no-go：在该CNS Tsc 2模型中，AAV-cavin必须至少使平均寿命加倍 并显示出与雷帕霉素（阳性对照）相当的有效性。在R33目标3中-评价剂量递增 和Tsc 2 +/-和CNS Tsc 2小鼠模型中AAV-cavin的潜在毒性，以及获得 通过磁共振定量测量治疗和未治疗小鼠的心室容积 显像我们的TSC专家团队包括Breakefield博士（神经系统疾病的临床前基因治疗）， Maguire（AAV载体），Ramesh（生物化学和细胞生物学），Stemmer-Rachamimov（神经解剖学）和 Thiele（临床护理）。这些活动旨在开展临床前研究，以支持临床试验， 通过我们的机构保护知识产权，并促进向生物技术公司发放许可证。

Our team will evaluate the potential for gene therapy to reduce life threatening symptoms in tuberous sclerosis complex (TSC), which affects 2 million people world-wide. TSC is an autosomal dominant, tumor suppressor disorder caused by an inherited mutation in either TSC1 (encoding hamartin) or TSC2 (encoding tuberin) with somatic loss of the corresponding normal allele leading to hamartomas (focal cell overgrowths) in different organs. Neurological involvement includes cortical tubers, subependymal nodules and subependymal giant cell astrocytomas with hydrocephalus epilepsy, autism, cognitive impairment and mental health issues. We have created a stochastic central nervous system (CNS) mouse model of TSC2 which manifests subependymal overgrowths, hydrocephalus and early death, reflecting what happens in TSC patients. Current standards-of- care for subependymal lesions in the brain involve neurosurgical craniotomy and/or long term treatment with rapamycin, both having potentially damaging effects on brain development. Our thesis is that a life-threatening complication of TSC – hydrocephalus can be prevented with a less invasive and longer lasting procedure, i.e. intravenous (IV) delivery of an adeno-associated virus (AAV) vector encoding a replacement protein, without compromising use of other standards-of-care if needed. IV delivery should achieve “extra copies” of the replacement gene in peripheral tissues reducing the likelihood of other life threatening hamartomas forming in the body. We will use our CNS Tsc2 mouse model to evaluate the ability of an AAV vector encoding a condensed version of tuberin (cTuberin) to reduce the size of ependymal abnormalities and extend lifespan. R61 Aim 1 - Evaluation of cTuberin protein in culture, including use of induced pluripotent stem cells from patients differentiated into neural progenitor cells, with cytoxicity and functional assays, Go-no-go: cTuberin must show at least 50% of tuberin activity in suppression of mTOR activity, and normalization of cell size and rate of cell proliferation. Aim 2 - Optimize AAV serotype and promoter with AAV-cTuberin in CNS Tsc2 mouse model and evaluate therapeutic efficacy. Behavior, survival times, neuropathology and whole body pathology will be monitored, as well as vector distribution and the ability of the vector to target cells in the brain and peripheral tissues. Go-no-go: AAV-cTuberin must at least double the average lifespan in this CNS Tsc2 model and show comparable effectiveness to rapamycin (positive control). In R33 Aim 3 - Evaluate dose escalation and potential toxicity of AAV-cTuberin in Tsc2+/- and CNS Tsc2 mouse models, as well as obtaining quantitative measurements of ventricular volumes in treated and non-treated mice by magnetic resonance imaging. Our team of TSC experts includes Drs. Breakefield (preclinical gene therapy for neurologic diseases), Maguire (AAV vectors), Ramesh (biochemistry and cell biology), Stemmer-Rachamimov (neuroanatomy) and Thiele (clinical care). These activities are aimed to carry out preclinical research in support of clinical trials, to secure intellectual property rights through our institution, and to facilitate licensing to biotechnology companies.