Precision genome surgery in autologous stem cell transplant

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- 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，因为 美国人口。常染色体显性遗传(AD)条件出现于允许A基因表达的突变 有缺陷的蛋白质，通常不适用于目前人类的“基因添加”疗法，因为它们 需要精确的修复来移除功能获得突变。长期目标是找出使用 患者干细胞作为再生医学(RM)的组织来源。此应用程序的目标是 确定自体诱导多能干细胞(IPS)移植是一种RM方法还是 治疗显性疾病。眼睛是一个器官，它为rm治疗提供了一个理想的“试验场”。 由于相对免疫特权既是可获得的，也是孤立的，眼睛的治疗通常既不是 非侵入性和全身性，其光学透明性使治疗可以轻松和非侵入性地进行监测 在活着的动物身上。中心假设是使用一种新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。 这一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 相应的RPE65D477G/+iPS细胞CRISPR修复后。基因编辑工具和方法将得到优化 用于iPS细胞RPE65D477G/+突变的CRISPR修复。将对细胞进行评估，以确定 它们能够分化为RPE并确认其基因组的完整性。2)确定在体外是否- 通过评估视网膜内部，修复的患者虹膜PE功能整合到活体小鼠的视网膜中 电物理信号和视黄醇信号。RPE移植物将来自RPE65基因修复的或 未修复的患者iPS细胞。3)确定移植的基因修复的虹膜是否安全和 不会致癌的。免疫受损的Rpe65rd12/Rpe65rd12；Prkdcscd/Prkdcscd小鼠将被注射到视网膜下 并对其安全性、致瘤性和存活率进行测试。拟议的研究具有重要意义，因为它 将在患者特定突变的精确病理生理学指导下推进治疗。建议数 这项研究具有创新性，因为它使用了一种新的方法来产生修复的RPE细胞用于移植， 不应要求免疫抑制治疗，并使用创新的结果测量方法。这个 预计研究将产生重要的积极影响，因为它代表着精确度的初始步骤 针对个体特有的显性突变开发治疗方法的医学方法 RP、AMD等显性疾病患者，同时引入新的方法和模型系统。 。

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. .