Gene therapy for tuberous sclerosis

我们的团队将评估基因疗法减少结节性硬化症危及生命的症状的可能性。 复杂(TSC)，它影响着全球200万人。TSC是一种常染色体显性肿瘤抑制基因 由TSC1(编码Hamartin)或TSC2(编码tuberin)的遗传突变引起的疾病 相应的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)在不同的 器官。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。 星形细胞瘤有脑积水、癫痫、自闭症、认知障碍和精神健康问题。我们有 建立了TSC2的随机中枢神经系统(CNS)小鼠模型，该模型表现为室管膜下 过度生长、脑积水和过早死亡，反映了TSC患者发生的情况。当前的标准- 脑室管膜下病变的护理包括神经外科开颅手术和/或长期治疗 雷帕霉素，两者都对大脑发育有潜在的破坏性影响。我们的论点是，一个危及生命的 TSC-脑积水的并发症可以通过一种侵入性更小和持续时间更长的手术来预防。 静脉(IV)递送编码替换蛋白的腺相关病毒(AAV)载体，而不 如果需要，折衷使用其他护理标准。静脉输液应获得“额外的副本” 外周组织中的替换基因降低其他威胁生命的错构瘤形成的可能性 身体。我们将使用我们的CNS TSC2小鼠模型来评估AAV载体编码 凝集型结节蛋白(CTuberin)，以减少室管膜异常的大小，延长寿命。 R61目标1-评估培养中的cTuberin蛋白，包括使用来自 患者分化为神经前体细胞，进行细胞毒性和功能分析，Go-no-Go：cTuberin 必须显示至少50%的结节蛋白活性，以抑制mTOR活性，并使细胞大小和 细胞增殖率。目的用AAV-cTuberin优化CNS-TSC2小鼠AAV血清型和启动子 造模并评价治疗效果。行为、存活时间、神经病理学和全身病理学 将被监测，以及载体的分布和载体靶向大脑和 周围组织。Go-no-Go：AAV-cTuberin必须至少是CNS TSC2模型的平均寿命的两倍 并显示出与雷帕霉素(阳性对照)相当的疗效。在R33目标3--评估剂量递增 以及AAV-cTuberin对TSC2+/-和CNS TSC2小鼠模型的潜在毒性，以及获得 磁共振定量测量治疗和非治疗小鼠的脑室容量 成像。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。

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.