Establishing a novel gene editing strategy for BBS7 using human retinal organoids

使用人类视网膜类器官建立 BBS7 的新型基因编辑策略

• 批准号: 10427680
• 负责人: Kathleen R Chirco
• 金额: $ 8.95万
• 依托单位: OREGON HEALTH & SCIENCE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-30 至 2023-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10427680
• 关键词: Accounting; Affect; Bardet-Biedl Syndrome; Biological Models; CRISPR/Cas technology; Cell physiology; Cells; Cilia; Clinic; Complex; Defect; Development; Disease; Disease Progression; Disease model; Dose; Evaluation; Formulation; Functional disorder; Future; Genes; Goals; Growth; Human; Immunologics; In Vitro; Individual; Inherited; Investigation; Knowledge; Lead; Life; Link; Measures; Mediating; Membrane; Mentors; Methods; Mitotic; Modeling; Modification; Morphogenesis; Mutation; Organoids; Persons; Phenotype; Photoreceptors; Retina; Retinal Degeneration; Retinal Diseases; Retinitis Pigmentosa; Safety; Specificity; Syndrome; System; Testing; Therapeutic; TimeLine; Training; Translational Research; Validation; Variant; Viral Vector; Vision research; Work; base; career; ciliopathy; cytotoxic; disease phenotype; disease-causing mutation; effective therapy; experience; experimental study; genetic variant; genome editing; immunogenicity; improved; in vivo; induced pluripotent stem cell; inherited retinal degeneration; lipid nanoparticle; nanoparticle delivery; nonhuman primate; novel; optimal treatments; photoreceptor degeneration; prevent; prime editing; prime editor; protein transport; recessive genetic trait; side effect; therapeutic gene; tool; translational model; treatment optimization

PROJECT SUMMARY/ABSTRACT As the leading cause of inherited retinal degeneration, retinitis pigmentosa (RP) affects about 1.5 million people worldwide. Bardet-Biedl syndrome (BBS) is the second most common causes of syndromic RP, and is characterized as an autosomal recessive ciliopathy with severe photoreceptor degeneration occurring by the first or second decade of life. BBS has been linked to variants in 21 genes, with those in BBS7 accounting for roughly 2% of all BBS cases. The overall goal of this proposal is to overcome two major hurdles in vision research: (1) the ability to accurately recapitulate disease mechanisms and progression for BBS7 in a translatable model system, and (2) the ability to permanently correct disease-causing variants and restore photoreceptor cell function with high efficiency and specificity. In Aim 1, disease mechanisms responsible for the onset of BBS7 will be examined by generating a retinal organoid model system from human induced pluripotent stem cells harboring disease-causing mutations in the BBS7 gene. In Aims 2 and 3, BBS7 variants will be corrected using prime editing tools and a lipid nanoparticle (LNP)-based delivery strategy within the human retinal organoid model to study timing and efficiency of disease rescue. Furthermore, the therapies found to be most effective in vitro will be tested in nonhuman primates to determine dose, immunogenicity, and efficiency of Cas9 delivery into photoreceptor cells in vivo. Successful completion of these aim will 1) contribute to our basic understanding of the pathophysiological mechanisms underlying photoreceptor dysfunction in BBS7, 2) provide the field with a thorough evaluation of a targeted gene editing strategy to treat BBS7 in the retina, and 3) establish LNPs as an ideal delivery system to limit cytotoxic and immunologic side effects commonly observed with AAV-based delivery methods. Taken together, this work will establish a pipeline for testing and optimization of therapies to treat BBS7 and other inherited retinal diseases, including additional forms of BBS as well as RP.

项目摘要/摘要 作为遗传性视网膜变性的主要原因，视网膜炎色素（RP）影响约150万人 全世界。 Bardet-Biedl综合征（BBS）是综合征RP的第二大最常见原因，IS 以第一次出现的严重感受器变性为特征是常染色体隐性纤毛病 或生活的第二个十年。 BBS已与21个基因的变体相关 所有BBS案件中有2％。该提案的总体目标是克服视力研究中的两个主要障碍：（1） 在可翻译模型中准确概括BBS7的疾病机制和进展的能力 系统，以及（2）永久纠正引起疾病变体和恢复感光细胞的能力 具有高效率和特异性的功能。在AIM 1中，疾病机制负责BBS 7 将通过从人类诱导的多能干细胞中生成视网膜器官模型系统来检查 在BBS7基因中含有引起疾病的突变。在AIMS 2和3中，将使用BBS7变体纠正 人类视网膜器官中的主要编辑工具和脂质纳米颗粒（LNP）的输送策略 研究疾病救援的时间和效率的模型。此外，发现最有效的疗法 体外将在非人类灵长类动物中进行测试，以确定CAS9递送的剂量，免疫原性和效率 在体内进入感光细胞。这些目标的成功完成1）有助于我们的基本理解 BBS7中的感光下功能障碍的病理生理机制，2）为该领域提供了A 对靶向基因编辑策略的彻底评估，以治疗视网膜中的BBS7，3）将LNPS作为一种 理想的输送系统，以限制基于AAV的细胞毒性和免疫副作用通常观察到 递送方法。综上所述，这项工作将建立一条用于测试和优化疗法的管道 治疗BBS7和其他遗传性视网膜疾病，包括其他形式的BB和RP。