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）基因治疗已在一些患者中提供临床益处 受各种遗传疾病的影响，其中一些已经达到了选定的市场授权， 迹象。然而，半随机整合载体的使用造成插入诱变的风险， 异位/不受调节的转基因表达。当受影响的基因 当其活性直接影响基因组稳定性时，如 RAG 1基因（RAG 1）RAG 1以高但严格调控的方式表达 在分化淋巴细胞前体中，它指导组装所需的VDJ重组过程 T细胞和B细胞受体及其失活突变是导致严重- 联合免疫缺陷（SCID）。尽管由于RAG 1不受调节而导致的基因组损伤的高风险 表达迄今为止阻碍了使用病毒载体治疗RAG 1缺陷，因此需要开发 新的和有效的治疗方法，特别是对于缺乏相容的HSPC供体或 不适合同种异体移植。我们提案的长期目标是解决这一未满足的医疗需求 并开发一种有效的新治疗方法， RAG 1基因在自体患者来源HSC上表达。我们的中心假设是，基因修复策略， 保持生理表达控制代表了治疗RAG 1缺陷的安全有效的方法。 我们报道了通过调整培养条件和基因传递载体， 在最原始和临床相关的HSPC亚群中限制基因编辑的生物屏障 （Genovese，Nature 2014; Schiroli，Science-Translational-Medicine 2017）。在这个项目中，我们将利用 我们之前的成就是i）直接修复RAG 1突变，ii）提高当前HSPC基因编辑的效率 方案和iii）在合适的小鼠模型上研究非遗传毒性调节。功能矫正 工程RAG 1基因将严格评估患者来源的细胞，通过利用最先进的体外 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 优化的基因编辑程序和条形码技术（BAR-seq，Ferrari等人，Nat.Biotech. 2020年）至 最大化编辑效率，同时降低对经处理的HSPC的细胞毒性，从而增加长- 术语移植淋巴细胞。为了支持临床试验的合理性，我们将评估疾病的纠正情况 通过在两个RAG 1鼠模型中限制功能性HSPC的量来检测表型，并测试新出现的HSPC的功效。 免疫毒素调节方案，以减少移植毒性和增加淋巴重建。总的来说， 该项目将有助于开发RAG 1缺陷的创新治疗方法， 将基于同源性的基因编辑定位为精确HSC工程的标准，提供更安全、更高效的 在血液学中具有广泛适用性的有效治疗策略。