A gene and prgenitor cell therapy in Huntington disease mice

描述（由申请人提供）：细胞替代疗法是亨廷顿病（HD）的一种可行的长期治疗选择，亨廷顿病（HD）中亨廷顿蛋白（HTT）基因CAG重复序列的扩增导致大多数纹状体神经元变性。受损的神经元可以被新的功能神经元所取代，这些功能神经元可以从诱导多能干细胞（iPSCs）中获得。然而，来自患者细胞的iPSCs含有相同的突变HTT （mHTT）基因，最终会导致有毒的HTT蛋白积累和细胞变性。因此，在细胞替换之前，应该进行基因修复以减少mHTT蛋白的过量产生。此外，将被替换的细胞整合到神经网络中对于神经功能的重建也很重要。这项研究是基于我们最近成功开发的转基因HD猴模型。HD猴子的细胞变化和神经病变与人类患者相似，这在啮齿类动物模型中很少观察到。除了mHTT对猴子的神经毒性外，HD猴子还出现与HD患者相似的不自主运动、协调身体运动困难、认知能力下降和纹状体萎缩。我们的初步结果表明，我们的HD猴iPSCs可以在体外分化为神经祖细胞（neural progenitor cells, NPCs）和gaba能神经元，它们构成了纹状体神经元的大部分。我们还发现，移植到严重联合免疫缺陷（SCID）小鼠体内的npc在植入后继续分化为表达GABA的神经元，并且不会形成肿瘤。这些结果表明，来自HD猴/患者自身的npc可以用来补充丢失的纹状体神经元。然而，目前尚不清楚这些npc是否能够恢复HD的纹状体功能。本提案的总体目标是确定npc置换在挽救HD小鼠模型异常表型中的有效性。这项拟议的研究将被用作原理验证，这将为未来通过基因纠正来自HD猴子的ipsc衍生的npc （rhd - npc）对HD猴子进行细胞替代治疗奠定基础。为了实现我们的目标，我们的方法是用靶向HTT基因的小发夹RNA （shRNA-htt）抑制hhd - npc中的mHTT表达，然后将产生的hhd - npc移植到HD小鼠中。shRNA基因调控可以减少毒性mHTT的表达和随后的积累，而细胞替代可能改善HD小鼠模型纹状体的异常行为和萎缩。为了实现我们的目标，我们有两个具体的目标：(1)确定基因调节的rhd - npc的神经元分化特性；(2)确定rHD-NPC-siHD2移植物在挽救HD小鼠异常表型中的作用。

DESCRIPTION (provided by applicant): Cell replacement therapy is a viable option as a long-term treatment for Huntington's disease (HD), where expansion of CAG repeats in Huntingtin (HTT) gene results in the degeneration of the majority of striatal neurons. The damaged neurons may be replaced by new functional neurons, which can be derived from induced pluripotent stem cells (iPSCs). However, iPSCs from the patients' cells harbor the same mutant HTT (mHTT) gene, which eventually will lead to toxic HTT protein accumulation and cell degeneration. Therefore, genetic repair should be done to reduce overproduction of mHTT proteins before cell replacement. Moreover, the integration of the replaced cells into the neuronal network is important for reconstituting neural functions. This study is evolved based on our recent success in developing a transgenic HD monkey model. HD monkeys develop cellular changes and neuropathologies similar to that of human patients, which are rarely observed in rodent models. Besides the neurotoxicity of mHTT in monkeys, HD monkeys also develop involuntary movement, difficulties in coordinating body movement, cognitive decline and striatal atrophy similar to that of HD patients. Our preliminary results show that iPSCs from our HD monkey can be in vitro differentiated into neural progenitor cells (NPCs) and GABAergic neurons, which form the majority of striatal neurons. We also show that NPCs grafted into severe combined immunodeficient (SCID) mice continue to differentiate into GABA expressing neurons after implantation and do not form tumors. These results suggest that NPCs derived from HD monkey/patients themselves may be utilized to replenish the population of the lost striatal neurons. However, it is unclear whether these NPCs will be able to restore the function of the striatum of HD. The overall objective of this proposal is to determine the effectiveness of NPCs replacement in rescuing the abnormal phenotype of HD mouse model. The proposed study will be used as a proof-of-principle which will lay the groundwork for future cell replacement therapy in HD monkeys by genetically correcting iPSC-derived NPCs from HD monkeys (rHD-NPCs). To achieve our goals, our approach is to suppress mHTT expression in rHD-NPCs with small hairpin RNA targeting the HTT gene (shRNA-htt) and then grafts the resulted rHD-NPCs into HD mice. Genetic modulation with shRNA can reduce the expression and subsequent accumulation of toxic mHTT, whereas cell replacement may improve the abnormal behavior and the atrophy of the striatum in HD mice model. To achieve our goals, we have two specific aims: (1) Determine neuronal differentiation properties of genetically modulated rHD-NPCs and (2) Determine the efficacy of rHD-NPC-siHD2 graft in rescuing the abnormal phenotype of HD mice.