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）长期治疗的可行选择，其中亨廷顿（HTT）基因中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性神经元取代，新的功能性神经元可以来自诱导多能干细胞（iPSC）。然而，来自患者细胞的iPSC具有相同的突变HTT（mHTT）基因，这最终将导致毒性HTT蛋白积累和细胞变性。因此，在细胞替换之前，应进行基因修复以减少mHTT蛋白的过度产生。此外，将替换的细胞整合到神经元网络中对于重建神经功能是重要的。本研究是基于我们最近成功开发转基因HD猴模型而进行的。HD猴出现与人类患者相似的细胞变化和神经病理学，这在啮齿动物模型中很少观察到。除了mHTT对猴的神经毒性外，HD猴还出现与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，来自我们的HD猴的iPSC可以在体外分化为神经祖细胞（NPC）和GABA能神经元，其形成大多数纹状体神经元。我们还表明，移植到严重联合免疫缺陷（SCID）小鼠的NPC继续分化成GABA表达神经元植入后，不形成肿瘤。这些结果表明，来源于HD猴/患者本身的NPC可用于补充丢失的纹状体神经元的群体。然而，目前尚不清楚这些NPC是否能够恢复HD纹状体的功能。本提案的总体目标是确定NPC替代在挽救HD小鼠模型的异常表型中的有效性。该研究将作为一种原理验证，通过遗传校正来自HD猴的iPSC衍生的NPC（rHD-NPC），为HD猴的未来细胞替代疗法奠定基础。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rHD-NPCs中mHTT的表达，然后将所得的rHD-NPCs移植到HD小鼠体内。在HD小鼠模型中，用shRNA进行遗传调节可以减少毒性mHTT的表达和随后的积累，而细胞替代可以改善异常行为和纹状体萎缩。为了实现我们的目标，我们有两个具体的目标：（1）确定基因调控的rHD-NPC的神经元分化特性和（2）确定rHD-NPC-siHD 2移植物在拯救HD小鼠异常表型中的功效。

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.