Restoring Vision with High-Fidelity Nonsense Codon Correction

通过高保真无义密码子校正恢复视力

• 批准号: 10627046
• 负责人: Christopher A Ahern
• 金额: $ 4.25万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-02-01 至 2025-11-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10627046
• 关键词: Accounting; Address; Amino Acids; Anatomy; Anticodon; Biochemical; Biological Assay; Biological Availability; Biological Models; Blindness; Cell Differentiation process; Cell membrane; Cells; Childhood; Clinical Trials; Communication; Communities; Complex; Cone dystrophy; Cultured Cells; DNA; DNA Sequence; Data; Defect; Development; Disease; Drug Delivery Systems; Drug Targeting; Electrophysiology (science); Engineering; Evaluation; Eye; FDA approved; Gene Targeting; Genes; Genetic Diseases; Glutamine; Human; In Vitro; Incidence; Inherited; Ion Channel; Ion Channel Protein; Iowa; Libraries; Light; Location; Membrane Proteins; Methods; Missense Mutation; Molecular; Monitor; Mus; Mutation; Neural Retina; Night Blindness; Nonsense Codon; Nonsense Mutation; Nucleotides; Ophthalmologist; Organoids; Pathogenesis; Patients; Pharmaceutical Preparations; Pharmacology; Photoreceptors; Phototransduction; Physiology; Point Mutation; Post-Translational Protein Processing; Production; Protein Biosynthesis; Protein translocation; Proteins; Proteomics; Research; Research Personnel; Resources; Retina; Retinal Diseases; Retinal Photoreceptors; Ribosomes; Safety; Scientist; Silicon Dioxide; Site; Structure; Structure of retinal pigment epithelium; System; TRPM1 gene; Technology; Terminator Codon; Testing; Therapeutic; Therapeutic Intervention; Tissues; Toxic effect; Transfer RNA; Translations; Universities; Viral; Vitelliform macular dystrophy; base; cell type; disease phenotype; efficacious treatment; functional outcomes; gene therapy; genome editing; human disease; human pluripotent stem cell; in vivo; induced pluripotent stem cell; intravitreal injection; mouse model; nanomaterials; nanomedicine; nanoparticle; new technology; next generation; nonhuman primate; novel; nucleic acid-based therapeutics; polypeptide; pre-clinical; pre-clinical therapy; preclinical safety; premature; repaired; retinal progenitor cell; safety assessment; safety testing; sight restoration; small molecule; small molecule therapeutics; stem cell biology; tRNA Precursor; targeted delivery; therapeutic gene; therapeutic target; transcriptomics; virtual

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.

项目摘要/摘要 废话突变导致大约15％的遗传遗传性视网膜病和遗传的人 一般疾病，在美国的某些特定基因，胡说八道，在美国占2.5至300万患者 突变发动机可能高达40％。因为废话突变会导致过早终止（PTC） 在蛋白质翻译中，疾病表型通常很严重。目前，只有有限的 在人类临床试验中测试的废话突变的疗法，包括基因疗法，小分子 读取药物或基因组编辑。相关的挑战等于每种治疗的承诺 选项。展望未来，较新的技术可能会解决这些障碍，并提供更安全和高效的 患者的治疗。在蛋白质翻译过程中，tRNA在核糖体位点起作用以结合特定 氨基酸进入多肽序列。我们旨在发展基于核酸疗法的下一代 在编码转移RNA（ACE-TRNA）的反密码子上，该转移RNA（ACE-tRNA）在位置上掺入正确的野生型氨基酸 引起疾病的胡说八道突变。由于眼睛带来的许多解剖学优势，我们 寻求测试Ace-tRNA疗法在引起视网膜病变的胡说八道突变中的广泛适用性 以及由于多种基因的缺陷而引起的相关失明，包括编码离子通道蛋白的那些基因。 具体而言，我们将专注于在转换的光感受器（PR）中表达的离子通道中的胡说八道突变 视网膜光输入和视网膜色素上皮（RPE），可为PR提供支持。这两种细胞类型是 主要是失明发病机理的部位。 在这个项目中，我们将： 1）开发ACE-tRNA疗法，该疗法靶向几个PR和RPE离子的特定废话突变 频道。 2）工程师的病毒和非病毒ACE-tRNA输送系统用于长期编辑。使用这些我们会 使用培养的细胞和人IPSC来确定ACE-TRNA处理的功能结果 RPE和IPSC-PR视网膜器官。 3）使用带有遗传缺陷的小鼠在体内测试我们的病毒和非病毒ACE-tRNA 人类的失明；和 4）评估ACE-TRNA疗法在我们的临床前NHP模型系统中的安全性和生物利用度。 由于复杂性，没有FDA批准的治疗药物靶向通道病 与精确的翻译后修饰，仔细调节表达和组装相关。我们的 团队在ACE-tRNA开发，纳米材料合成，人类多能干细胞方面的综合专业知识 生物学，离子通道生理学和病理生理模型系统是独一无二的，非常适合促进 ACE-TRNA技术针对引起失明的广泛遗传疾病的临床试验。