A gene and prgenitor cell therapy in Huntington disease mice

描述(申请人提供)：细胞替代疗法是一种可行的长期治疗亨廷顿病(HD)的选择，亨廷顿病(HD)中CAG重复序列的扩张会导致大多数纹状体神经元退化。受损的神经元可以被新的功能神经元取代，新的功能神经元可以从诱导的多能干细胞(IPSCs)中分化出来。然而，患者细胞中的IPSCs含有相同的突变HTT(MHTT)基因，最终会导致有毒的HTT蛋白积聚和细胞退化。因此，在细胞置换之前，应进行基因修复，以减少mHTT蛋白的过度生产。此外，将被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。这项研究是在我们最近成功开发出转基因HD猴子模型的基础上发展起来的。HD猴子出现了与人类患者相似的细胞变化和神经病理，这在啮齿动物模型中很少观察到。除了mHTT对猴子的神经毒性外，HD猴子还会出现类似HD患者的不自主运动、身体运动协调困难、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猴的IPSCs可以在体外分化为神经前体细胞和GABA能神经元，这两种细胞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)小鼠体内的神经干细胞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，来源于HD猴/患者自身的神经前体细胞可能被用来补充丢失的纹状体神经元的数量。然而，目前尚不清楚这些NPC是否能够恢复HD纹状体的功能。本建议的总体目标是确定神经干细胞替代在挽救HD小鼠模型表型异常方面的有效性。这项拟议的研究将被用作一项原则证明，它将通过对来自HD猴子的iPSC来源的NPC(RHD-NPC)进行基因纠正，为未来的HD猴子细胞替代疗法奠定基础。为了达到我们的目标，我们的方法是用针对HTT基因的小发夹RNA(shRNA-HTT)抑制mHTT在RHD-NPC中的表达，然后将得到的RHD-NPC移植到HD小鼠体内。ShRNA的基因调控可以减少毒性mHTT的表达和随后的蓄积，而细胞置换可以改善HD模型小鼠的异常行为和纹状体的萎缩。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；(2)确定RHD-NPC-siHD2移植对拯救HD小鼠异常表型的效果。

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.