Precision genome surgery in autologous stem cell transplant

自体干细胞移植中的精准基因组手术

• 批准号: 9811117
• 负责人: Janet Ruthe Sparrow
• 金额: $ 40.5万
• 依托单位: COLUMBIA UNIVERSITY HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-07-01 至 2021-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9811117
• 关键词: Address; Affect; Age related macular degeneration; Aging; Alleles; American; Animals; Autologous; Autologous Stem Cell Transplantation; Benchmarking; Biological Models; Cell Transplantation; Cell Transplants; Cells; Clinical; Clustered Regularly Interspaced Short Palindromic Repeats; Data; Development; Disease; Dominant-Negative Mutation; Electrophysiology (science); Epithelial; Excision; Eye; FDA approved; Functional disorder; Genes; Genome; Genomics; Goals; Human; Immune; Immunocompromised Host; In Vitro; Laboratories; Measurement; Methods; Monitor; Morphology; Mus; Mutation; Operative Surgical Procedures; Optics; Organ; Outcome; Patients; Phase I Clinical Trials; Phenotype; Population; Preclinical Testing; Proteins; Protocols documentation; Public Health; RPE65 protein; Regenerative Medicine; Research; Retina; Retinal; Retinal Pigments; Retinitis Pigmentosa; Retinoids; Safety; Signal Transduction; Source; Stem cell transplant; Stem cells; Structure of retinal pigment epithelium; Supplementation; Testing; Therapeutic; Therapeutic immunosuppression; Tissues; Transplantation; Tumorigenicity; Work; gain of function; gain of function mutation; gene repair; gene therapy; gene transplantation for gene therapy; genome integrity; genome sequencing; healing; human subject; individual patient; induced pluripotent stem cell; innovation; loss of function; novel; novel strategies; precision medicine; repaired; safety testing; stem; success; tool; transplantation medicine; whole genome

Retinal pigmented epithelial (RPE) disorders, including autosomal dominant retinitis pigmentosa (RP) and age- related macular degeneration, are an enormous burden and a growing public health concern, given the aging U.S. population. Autosomal dominant (ad) conditions emerge from mutations that allow expression of a defective protein and are generally not amenable to current “gene addition” therapies in humans, as they require precise repair to remove the gain-of-function mutation. The long-term goal is to identify ways to use patient stem cells as a tissue source for regenerative medicine (RM). The objective of this application is to determine whether autologous induced pluripotent stem (iPS) cell transplantation is a RM approach or treatment of dominant disorders. The eye provides an ideal "proving ground" for RM therapy, as it is an organ with relative immune privilege that is both accessible and isolated, therapies for the eye are generally neither invasive nor systemic, and its optically transparency allows treatment to be monitored easily and noninvasively in living animals. The central hypothesis is that a gene-editing strategy using a novel state-of-the-art therapeutic editing approach can suppress manifestation of a dominant RPE65D477G/+ mutation in human RP. This hypothesis will be tested by pursuing three specific aims: 1) Obtain functionally recovered RPE after CRISPR repair of corresponding RPE65D477G/+ iPS cells. Gene-editing tools and methods will be optimized for CRISPR repair of the RPE65D477G/+ mutation in iPS cells. The cells will be assessed to determine whether they are able to differentiate into RPE and to confirm their genomic integrity. 2) Determine whether in vitro- repaired patient iRPE functionally integrates into the retinae of live mice by assessing inner retinal electrophysical signals and retinoid signaling. RPE grafts will be derived from either RPE65 gene-repaired or unrepaired patient iPS cells. 3) Determine whether transplanted gene-repaired iRPE is safe and nontumorigenic. Immunocompromised Rpe65rd12/Rpe65rd12; Prkdcscid/Prkdcscid mice will be subretinally injected with iRPE grafts and tested for safety, tumorigenicity and survival. The proposed research is significant, as it will advance treatment guided by the precise pathophysiology of patient-specific mutations. The proposed research is innovative because it uses a new approach to generate repaired RPE cells for transplantation, should not require immunosuppressive therapy, and make use of innovative outcome measurements. The research is expected to have an important positive impact as it represents the initial steps of a precision medicine approach directed toward developing treatments targeting dominant mutations unique to individual patients with RP, AMD, and other dominant disorders, while introducing new methods and model systems. .

视网膜色素上皮（RPE）疾病，包括常染色体显性遗传性视网膜色素变性（RP）和年龄- 考虑到老龄化，相关的黄斑变性是一个巨大的负担，也是一个日益严重的公共卫生问题 美国人口。常染色体显性 (ad) 病症是由允许表达某种基因的突变引起的 有缺陷的蛋白质，通常不适用于人类目前的“基因添加”疗法，因为它们 需要精确修复以消除功能获得性突变。长期目标是确定使用方法 患者干细胞作为再生医学（RM）的组织来源。该应用程序的目的是 确定自体诱导多能干 (iPS) 细胞移植是否是 RM 方法或 治疗显性疾病。眼睛为 RM 疗法提供了理想的“试验场”，因为它是一个器官 由于相对免疫特权既可接近又可隔离，眼部治疗通常既不方便又不隔离。 非侵入性或全身性，其光学透明性使治疗能够轻松且非侵入性地进行监测 在活体动物中。中心假设是使用一种新颖的最先进的基因编辑策略 治疗性编辑方法可以抑制人类 RP 中显性 RPE65D477G/+ 突变的表现。 该假设将通过追求三个具体目标来检验：1) 获得功能恢复的 RPE 对相应的 RPE65D477G/+ iPS 细胞进行 CRISPR 修复后。基因编辑工具和方法将得到优化 用于 CRISPR 修复 iPS 细胞中的 RPE65D477G/+ 突变。将评估细胞以确定是否 它们能够分化为 RPE 并确认其基因组完整性。 2) 确定是否在体外- 通过评估视网膜内部，修复后的患者 iRPE 功能性地整合到活小鼠的视网膜中 电物理信号和类维生素A信号传导。 RPE 移植物将源自 RPE65 基因修复或 未修复的患者 iPS 细胞。 3) 确定移植的基因修复iRPE是否安全 非致瘤性。免疫功能低下的 Rpe65rd12/Rpe65rd12； Prkdcscid/Prkdcscid 小鼠将被视网膜下注射 使用 iRPE 移植物进行安全性、致瘤性和存活率测试。拟议的研究意义重大，因为它 将在患者特异性突变的精确病理生理学指导下推进治疗。拟议的 研究具有创新性，因为它使用了一种新方法来生成用于移植的修复 RPE 细胞， 不应需要免疫抑制治疗，并利用创新的结果测量。这 研究预计将产生重要的积极影响，因为它代表了精确化的初始步骤 旨在开发针对个体独特显性突变的治疗方法的医学方法 患有 RP、AMD 和其他显性疾病的患者，同时引入新的方法和模型系统。 。