利用CRISPR/Cas9基因组编辑技术治疗亨廷顿舞蹈症的深入研究

Huntington’s Disease (HD) is an autosomal dominantly inherited neurodegenerative disease, which is caused by CAG repeat expansion in the Huntingtin (HTT) gene. To date, there is no effective treatment for HD. Using the genome editing tool CRISPR/Cas9 to remove the expression of mutant HTT holds great promise and opens up a new avenue for treating HD. In our previous studies, we found that CRISPR/Cas9 treatment could ameliorate pathological phenotypes in a HD mouse model. However, we also found that Cas9, as a large bacterial protein, could induce neurotoxicity in the mouse brain to some extent. We therefore created a modified version of Cas9 (GCas9) with a lower toxicity to mouse neurons. In this proposal, we will further test the potential application of GCas9 in treating HD. We will compare the efficacy of wild type Cas9 and GCas9 in treating the HD mouse model, via behavioral tests and neuropathology. We will also examine the function of GCas9 in the brain of nonhuman primates. This proposal could provide important theoretical insights and innovative tools to the development of CRISPR/Cas9 based therapeutic approach for HD, as well as other hereditary neurological diseases.

亨廷顿舞蹈症（Huntington’s Disease，HD）是一种单基因常染色体显性遗传的神经退行性疾病，其致病基因为亨廷顿基因（Huntingtin，HTT）。目前尚未有针对HD的有效治疗手段。利用新型基因组编辑技术CRISPR/Cas9敲除变异HTT为治疗HD提供了崭新的思路。我们的前期工作利用CRISRP/Cas9技术能够有效缓解HD小鼠模型的病理表现。然而, 我们也发现Cas9作为－个大的细菌蛋白对小鼠神经元具有毒性作用。为此, 我们构建了对小鼠神经元毒性更小的新型Cas9蛋白（GCas9）。在本项目中，我们将进一步尝试利用GCas9治疗HD。我们将通过行为学和神经病理学实验在HD小鼠模型中比较野生型Cas9与GCas9的治疗效果，并且利用RNA测序在非人灵长类动物脑内验证GCas9的功能。本项目将为最终实现利用CRISPR/Cas9基因组编辑技术临床治疗HD提供重要技术手段。

亨廷顿舞蹈症（Huntington’s Disease，HD）是一种单基因常染色体显性遗传的神经退行性疾病，其致病基因为亨廷顿基因（Huntingtin，HTT）。目前尚未有针对HD的有效治疗手段。利用新型基因组编辑技术CRISPR/Cas9敲除变异HTT为治疗HD提供了崭新的思路。但敲除过程中也会降低正常HTT的表达，对神经元的功能和存活造成影响。本项目利用小鼠模型和猴模型深入验证了CRISPR/Cas9敲除HTT的安全性。我们发现在小鼠的原代神经元和胶质细胞中敲除HTT均不会影响细胞的存活和重要蛋白的表达，在猴脑中敲低HTT也不会影响神经元的形态和存活。上述结果提示HTT蛋白在成年动物的脑内发挥的功能不具有必要性，因此同时敲除正常和变异的HTT可以作为HD的一种潜在治疗手段，这为进一步推动CRISPR/Cas9基因编辑技术治疗HD提供了理论基础。

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