Development, Optimization and Preclinical Modeling of Hematopoietic Stem Cell Gene Editing for the Treatment of RAG1 Immunodeficiency

用于治疗 RAG1 免疫缺陷的造血干细胞基因编辑的开发、优化和临床前建模

• 批准号: 10949441
• 负责人: Pietro Genovese
• 金额: $ 44.25万
• 依托单位: BOSTON CHILDREN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-06-08 至 2026-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10949441
• 关键词: Development; Optimization; Preclinical; Modeling; Hematopoietic

PROJECT SUMMARY Hematopoietic Stem/Progenitor Cells (HSPC) gene therapy has provided clinical benefits in several patients affected by a variety of genetic diseases, some of which already reached market authorization for selected indications. However, the use of semi-randomly integrating vectors poses the risk of insertional mutagenesis and ectopic/unregulated transgene expression. These issues become even more relevant when the affected gene needs to be highly express to exert its function and when its activity directly impacts genome stability, such as the case for Recombination-Activating 1 (RAG1) gene. RAG1 is express in a high but tightly regulated manner in differentiating lymphocyte precursors, where it directs the VDJ recombination process required for assembly the T- and B-cell receptors, and its inactivating mutations are one of the most frequent causes of severe- combined immunodeficiency (SCID). While the high risk of genomic damage due to unregulated RAG1 expression has so far hampered the use of viral vectors to treat RAG1 deficiencies, there is a need to develop novel and effective therapeutic approaches, especially for patients who lacks a compatible HSPC donor or are not eligible for allogeneic transplant. The long-term goal of our proposal is to address this unmet medical need and develop an effective novel treatment directed at restoring both function and expression control of the RAG1 gene on autologous patient derived HSC. Our central hypothesis is that gene repair strategies that preserve physiologic expression control represent a safe and effective approach for treating RAG1 deficiencies. We reported that by tailoring culture conditions and gene delivery vehicles, it is possible to partially overcome the biologic barriers that constrain gene editing in the most primitive and clinically relevant HSPC subsets (Genovese, Nature 2014; Schiroli, Science-Translational-Medicine 2017). Within this project we will capitalize our previous achievements to i) directly fix RAG1 mutations, ii) improve efficiency of current HSPC gene editing protocols and iii) investigate non-genotoxic conditioning on suitable mouse models. Functional correction of the engineered RAG1 gene will be stringently assessed on patient derived cells, by exploiting state-of-the-art in vitro T cell differentiation assay and in vivo xenotransplantation experiments. We will take advantage of our recently optimized gene editing procedure and barcoding technology (BAR-seq, Ferrari et al, Nat. Biotech. 2020) to maximize editing efficiency while reducing cellular toxicity on the treated HSPC, thus increasing the yield of long- term engrafting lymphoid cells. To support the rational for clinical testing, we will assess correction of the disease phenotype by limiting amounts of functional HSPC in two RAG1 murine models and test efficacy of emerging immunotoxin conditioning regimens to reduce transplant toxicity and increase lymphoid reconstitution. Overall, this project will contribute to the development of an innovative treatment approach for RAG1 deficiencies and position homology-based gene editing as a standard for precise HSC engineering, providing for safer and more efficacious therapeutic strategies with broad applicability in hematology.

项目摘要 造血干/祖细胞（HSPC）基因疗法为几名患者提供了临床益处 受各种遗传疾病的影响，其中一些已经获得了选定的市场授权 适应症。但是，半随机整合向量的使用会带来插入诱变和 异位/不调节的转基因表达。当受影响的基因时，这些问题变得更加相关 需要高度表达以发挥其功能，并且当其活性直接影响基因组稳定性时，例如 重组激活1（rag1）基因的情况。 rag1以高但严格的方式表达 在区分淋巴细胞前体中，它指导组装所需的VDJ重组过程 T和B细胞受体及其灭活突变是严重的最常见原因之一 联合免疫缺陷（SCID）。而不受管制的RAG1引起的基因组损伤的高风险 到目前为止，表达阻碍了使用病毒载体来治疗RAG1缺陷的使用，需要发展 新颖有效的治疗方法，尤其是对于缺乏兼容HSPC供体的患者或 不符合同种异体移植的条件。我们建议的长期目标是解决这种未满足的医疗需求 并开发一种有效的新颖治疗方法，旨在恢复功能和表达控制 自体患者衍生的HSC的RAG1基因。我们的中心假设是基因修复策略 保存生理表达控制代表了治疗RAG1缺陷的安全有效方法。 我们报告说，通过调整培养条件和基因输送车，可以部分克服 在最原始和临床上相关的HSPC子集中限制基因编辑的生物屏障 （Genovese，Nature 2014; Schiroli，科学翻译 - 美食2017）。在这个项目中，我们将资本化 我们以前的成就i）直接修复RAG1突变，ii）提高当前HSPC基因编辑的效率 方案和iii）研究合适的小鼠模型上的非生物毒性调节。功能校正 通过利用最新的体外，将对患者衍生细胞进行严格评估工程的RAG1基因 T细胞分化测定和体内异种移植实验。我们将利用我们最近的优势 优化的基因编辑程序和条形码技术（Bar-Seq，Ferrari等，Nat。Biotech。2020） 最大化编辑效率，同时降低了处理过的HSPC的细胞毒性，从而增加了长期的产量 术语植入淋巴样细胞。为了支持临床测试的理性，我们将评估疾病的纠正 通过限制两个rag1鼠模型中功能性HSPC的量和新出现的测试功效 免疫毒素调节方案可降低移植毒性并增加淋巴样重构。全面的， 该项目将有助于开发Rag1缺陷的创新治疗方法和 位置基于同源的基因编辑是精确HSC工程的标准 有效的治疗策略在血液学中具有广泛适用性。