瞬态可逆性Rb1敲除对毛细胞再生和存活的体内研究

Severe hearing loss affects tens of millions of people in China with no effective treatment. One of the major causes of hearing loss is the permanent loss of inner ear hair cells. Unlike lower vertebrate animals such as birds and fish, hair cells in mammalian inner ear cannot be regenerated intrinsically. One of the major approaches to regenerate hair cells is through cell cycle re-entry of hair cells that leads to the production of new hair cells. Retinoblastoma gene (Rb1) is known to be required for cell cycle exit and maintenance of postmitotic state of hair cells. In a mouse model in which Rb1 is deleted in the inner ear sensory progenitor cells, the progenitor cells continue proliferation while differentiating into both hair cells and supporting cells. As the result supernumerary hair cells are produced. Significantly the new hair cells differentiate properly and are functional. However in postnatal inner ear, hair cells with permanent Rb1 deletion ultimately die due to the function of Rb1 is required for survival, which prevents the use to generate new surviving hair cells in postnatal animals. Ideally the function of Rb1 should be blocked transiently to induce proliferation of hair cells, with Rb1 function subsequently resumes so that new hair cells can survive. As an alternative approach to evaluate restoration of Rb1 function for survival of new hair cells, we propose to use gene therapy approach to delivery Rb1 gene into supernumerary hair cells in which Rb1 is deleted. In particular, we will microinject adeno-associate virus serotype 2 (AAV2) that carries Rb1 into cochlea of a transgenic mouse, in which Rb1 is deleted specifically in hair cells due to Gfi1-cre activity (Gfi1-cre/Rb1flox/flox). This study is based on our data that in Gfi1-cre/Rb1flox/flox mice, supernumerary functional hair cells are produced but die by p7. Further we have shown that AAV2 can infect young (p0-p3) cochlear hair cells. Thus by delivering Rb1 gene into Rb1-null hair cells, we hypothesize that Rb1 function will be restored and supernumerary hair cells will survive. We will perform injection using p0/ p1 mouse and study the survive of hair cells one month later. We will further study differentiation and synapse formation of survival hair cells with different markers, and study function of hair cells by FM1-43 uptake and transduction current recording. Finally we will perform acoustic brainstem recording (ABR) of mice to evaluate hearing of injected. No hearing can be measured in Gfi1-cre/Rb1flox/flox mice without injection, whereas we expect some hearing in the injected Gfi1-cre/Rb1flox/flox mice. Success of the project is the proof of principle that transient blockade of Rb1 function can be used to achieve hair cell regeneration for functional recovery of hearing. It will further demonstrate gene therapy is a valid tool in hair cell regeneration.

调控视网膜母细胞瘤基因（Rb1）的表达为获得耳蜗毛细胞再生的一条重要途径。在胚胎早期敲除Rb1，可再生有功能的毛细胞；在分化后的毛细胞中敲除Rb1，其重新进入细胞周期，但逐渐死亡，认为Rb1对于分化后毛细胞的存活和功能起重要作用。前期工作比较了12种不同血清型的腺相关病毒（AAV），作为内耳基因治疗的载体，第2型AAV成功转染新生鼠毛细胞，并且对听力无明显影响。本课题拟构建Gfi1-cre-Rb1 flox/flox小鼠模型，特异性敲除毛细胞Rb1，使得毛细胞增殖；在新生期以纳升级显微手术操作系统行耳蜗显微注射，转染携带Rb1的2型AAV，使得毛细胞增殖后重新表达Rb1，在不同时间点行听力及内耳形态学检测，并行增殖后存活的新生毛细胞的功能检测。为以瞬态可逆性阻断Rb1途径获得毛细胞再生提供理论依据，也证实以AAV为载体进行内耳基因治疗的可行性。

本课题比较了12种不同血清型的腺相关病毒（AAV），作为内耳基因治疗的载体，第2型AAV成功转染新生鼠毛细胞，并且对听力无影响。课题拟构建Gfi1-cre-Rb1 flox/flox小鼠模型，特异性敲除毛细胞Rb1，使得毛细胞增殖；在新生期以纳升级显微手术操作系统行耳蜗显微注射，转染携带Rb1的2型AAV，使得毛细胞增殖后重新表达Rb1，在3、6周后行听力检查，发现未能纠正听力，也未能使得增殖后细胞的存活。我们构建Cas9蛋白质，以gRNA（针对GFP）和脂质结合，注射入Math1-GFP新生鼠内耳。以脂质体和Cas9蛋白注射为对照，在内耳注射10天后，发现CRISPR/Cas9蛋白系统可以精准的对毛细胞进行基因编辑，效率达到20%，通过测序，证实了对GFP进行了编辑。为基因治疗的进展提供了强有力的依据。

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