Restoring Vision with High-Fidelity Nonsense Codon Correction

项目概要/摘要 无义突变导致大约15%的遗传性视网膜病和遗传性人类视网膜病变。 一般来说，美国有250万到300万患者患有这种疾病。 突变发生率可高达40%。因为无义突变会导致早产（PTC） 由于蛋白质翻译的限制，疾病表型通常是严重的。目前，只有有限数量的 在人类临床试验中测试的无义突变疗法，包括基因疗法，小分子 通读药物或基因组编辑。相关的挑战等于这些治疗方法的承诺 选项.展望未来，更新的技术可能会解决这些障碍，并提供更安全有效的 为患者治疗。在蛋白质翻译过程中，tRNA在核糖体位点起作用，将特定的 将氨基酸插入多肽序列中。我们的目标是开发下一代的核酸治疗， 反密码子编码转移RNA（ace-tRNA），其在以下位点掺入正确的野生型氨基酸： 一种致病的无意义突变由于眼睛提供的许多解剖学优势，我们 试图测试ace-tRNA疗法对导致视网膜病变的无义突变的广泛适用性 以及由于包括编码离子通道蛋白的基因在内的多种基因的缺陷而导致的相关失明。 具体来说，我们将集中在光感受器（PR）中表达的离子通道中的无义突变， 视网膜光输入和视网膜色素上皮（RPE），为PR提供支持。这两种细胞类型是 主要是失明发病的部位。 在这个项目中，我们将： 1)开发针对几种PR和RPE离子的特异性无义突变的ace-tRNA疗法 渠道 2)设计用于长期编辑的病毒和非病毒ace-tRNA递送系统。利用这些，我们将 使用培养的细胞和人iPSC衍生的细胞来确定ace-tRNA治疗的功能结果。 RPE和iPSC-PR视网膜类器官。 3)使用携带遗传缺陷的小鼠在体内测试我们的病毒和非病毒ace-tRNA， 人类失明;以及 4)在我们的临床前NHP模型系统中评估ace-tRNA治疗剂的安全性和生物利用度。 由于复杂性，没有FDA批准的针对通道病的治疗药物 与精确的翻译后修饰、仔细调控的表达和组装相关。我们 团队在ace-tRNA开发、纳米材料合成、人类多能干细胞 生物学、离子通道生理学和病理生理学模型系统是独特的，非常适合于发展 ace-tRNA技术用于临床试验，用于广泛的导致失明的遗传疾病。

PROJECT SUMMARY/ABSTRACT Nonsense mutations cause approximately 15% of genetically inherited retinopathies and inherited human diseases in general, accounting for 2.5 to 3 million patients in the U.S. For certain specific genes, nonsense mutation incidences can be as high as 40%. Because nonsense mutations cause premature termination (PTC) of protein translation, the disease phenotype is often severe. Currently, there are only a limited number of therapies for nonsense mutations being tested in human clinical trials, including gene therapy, small molecule read-through drugs, or genome editing. Associated challenges equal the promises of each of these therapeutic options. Looking forward, newer technologies may address these hurdles and provide more safe and efficacious treatments for patients. During protein translation, tRNA functions at the ribosomal site to incorporate a specific amino acid into the polypeptide sequence. We aim to develop the next generation of nucleic acid therapy based on anticodon encoding transfer RNA (ace-tRNA) that incorporates the correct wild type amino acid at the site of a disease-causing nonsense mutation. Because of the many anatomical advantages afforded by the eye, we seek to test the broad applicability of ace-tRNA therapeutics for nonsense mutations that cause retinopathies and related blindness due to defects in a variety of genes, including those encoding ion channel proteins. Specifically we will focus on nonsense mutation in ion channels expressed in photoreceptors (PR) which convert retinal light inputs and retinal pigment epithelium (RPE), which provide support for PR. These two cell types are primarily the site of blindness pathogenesis. In this project, we will: 1) Develop ace-tRNA therapeutics that target specific nonsense mutations across several PR and RPE ion channels. 2) Engineer both viral and non-viral ace-tRNA delivery systems for long-term editing. Using these we will determine the functional outcome of ace-tRNA treatment using cultured cells and human iPSC-derived RPE and iPSC-PR retinal organoids. 3) Test both our viral and non-viral ace-tRNA in vivo using mice harboring genetic defects that cause blindness in humans; and 4) Assess the safety and bioavailability of ace-tRNA therapeutics in our preclinical NHP model systems. There are no FDA-approved therapeutic drugs that target channelopathies because of the complexities associated with precise post-translational modifications, carefully regulated expression, and assembly. Our team’s combined expertise in ace-tRNA development, nanomaterial synthesis, human pluripotent stem cell biology, ion-channel physiology, and pathophysiological model systems is unique and ideally suited to advance ace-tRNA technology toward clinical trials for a wide range of genetic diseases that cause blindness.