Genome Editing Therapy for Usher Syndrome Type 3

项目摘要/摘要 Usher综合征3A型(USH3A)是一种常染色体隐性遗传病，以进行性为特征 由于Clarin-1(CLRN1)基因突变而导致的听力和视力丧失。患有3型亚瑟综合征的人会 通常需要在成年中后期植入耳蜗，并被归类为法律上的盲人。这个 USH3A的慢性、进行性、严重视力丧失(视网膜色素变性，RP)的治疗仍然是一个 挑战。CLRN1在视网膜中的表达受到精细调控。AAV传递的基因增强疗法已经 对USH3A小鼠内耳有效，但对野生型小鼠视网膜功能有害 小鼠，表明正确的AAV载体剂量、启动子和递送方法需要良好的组合 优化开发一种安全的USH3A RP基因疗法。然而，AAV的表达水平 基因表达很难控制，随着时间的推移，基因表达会受到基因沉默的影响。基因的另类途径 编辑疗法可以直接修复基因组中的缺陷基因，导致内源性表达 调控元件的调控，可能为USH3A RP和USH3A RP的治疗提供一种有前景的治疗策略。 其他遗传性眼病。目前的基因编辑工具存在脱靶安全问题和低修复率问题 效率。2020年，我们的团队报告了一种新的Cas9变体，称为精细整合Cas9(MiCas9)， 提高了敲入(KI)效率，并显著减少了不必要的目标内外偏差 插入/删除事件(Indels)。我们相信，使用miCas9策略将提供安全和有效的 在视网膜中启用CLRN1基因校正。 缺乏临床相关的动物模型是发展有效的治疗方法的瓶颈。 USH3A中的RP。兔子是研究眼病的经典模型。我们最近开发了兔子模型 携带CLRN1移码(CLRN1−/−)和CLRN1N48K/N48K点突变。初步表征 研究表明，CLRN1突变会导致严重的进行性视力和听力退化。 据我们所知，这是第一个USH3A翻译动物模型，令人信服地概括了 进行性视力和听力退化表型。 在这个STTR快速通道应用中，利用了新的miCas9工具和新的 我们已经建立了兔模型，我们建议开发一种基因编辑疗法来治疗失明 USH3A。在第一阶段，我们将对CLRN1N48K/N48K兔(AIM1)的眼部表型进行表征，并开发出一种 MiCas9介导的hCLRN1 cDNA靶向整合策略适用于所有CLRN1突变(AIM2)。穿过 这项第一阶段的工作，我们将建立在USH3A兔视网膜上进行基因编辑的可行性。在第二阶段工作中， 我们将在我们的USH3A兔模型中测试这种USH3A基因编辑疗法的安全性和有效性 视网膜下注射，为miCas9介导的USH3A基因编辑治疗提供临床前证据。这 新的知识有可能推进3A型亚瑟综合征的一流临床治疗。

PROJECT SUMMARY/ABSTRACT Usher syndrome type 3A (USH3A), an autosomal recessive disorder, is characterized by progressive loss of hearing and vision due to a clarin-1 (CLRN1) gene mutation. A person with type 3 Usher syndrome will usually require cochlea implants by mid- to late adulthood and classified as being legally blind. The management of the chronic, progressive, severe vision loss (retinitis pigmentosa, RP) in USH3A remains a challenge. CLRN1 expression in retina is finely regulated. AAV delivered gene augmentation therapy has shown efficacy in the inner ear of USH3A mice but shown detrimental effects on retinal function in wildtype mice, indicating the right combination of AAV vector dose, promoter, and delivery method needs to be well optimized to develop a safe gene therapy for USH3A RP. However, the expression level of AAV delivered gene expression is hard to control and is subject to gene silencing over time. The alternative approach of gene editing therapy can directly repair a defective gene in the genome, result in expression under endogenous regulatory element control, and may provide a promising therapeutic strategy for treatment of USH3A RP and other genetic eye disorders. Current gene editing tools suffer from off-target safety concerns and low repair efficiency. In 2020, our group reported a novel Cas9 variant, known as meticulous integration Cas9 (miCas9), with improved knock-in (KI) efficiency and dramatically reduced undesirable on- and off-target insertion/deletion events (indels). We believe the use of miCas9 strategy will provide the safety and efficacy to enable the CLRN1 gene correction in the retina. The lack of clinically relevant animal models is the bottleneck to developing effective therapeutics for RP in USH3A. The rabbit is a classic model to study eye diseases. We recently developed rabbit models carrying the CLRN1 frameshift (CLRN1−/−) and the CLRN1N48K/N48K point mutation. Preliminary characterization of these rabbits has shown that CLRN1 mutations lead to severe progressive vision and hearing degeneration. To our knowledge these are the first USH3A translational animal models that convincingly recapitulates the progressive vision and hearing degeneration phenotype. In this STTR FAST TRACK application, taking advantage of the novel miCas9 tools and the novel rabbit models we have established, we propose to develop a gene editing therapy to treat vision loss in USH3A. In Phase1, we will characterize the eye phenotypes of the CLRN1N48K/N48K rabbits(aim1) and develop a miCas9 mediated hCLRN1 cDNA targeted integration strategy that fit for all CLRN1 mutations(aim2). Through this Phase I work, we will establish the feasibility of gene editing in the USH3A rabbit retina. In Phase II work, we will test the safety and efficacy of this USH3A gene editing therapy in our USH3A rabbit model through subretinal injection, to provide the preclinical evidence of miCas9 mediated USH3A gene editing therapy. This new knowledge has the potential to advance a first-in-class clinical treatment for Usher Syndrome Type 3A.