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

项目总结 造血干细胞/祖细胞(HSPC)基因治疗在几个患者中提供了临床益处 受到多种遗传病的影响，其中一些已经达到市场授权的选定 有迹象表明。然而，使用半随机整合载体会带来插入突变和 异位/不受调控的转基因表达。当受影响的基因受到影响时，这些问题变得更加相关 需要高度表达才能发挥其功能，以及当其活性直接影响基因组稳定性时，例如 重组激活基因1(RAG1)的情况。RAG1以高度但严格调控方式表达 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严重... 联合免疫缺陷(SCID)。而由于不受调控的RAG1导致的基因组损伤的高风险 表达到目前为止阻碍了利用病毒载体治疗RAG1缺陷，有必要开发 新的有效的治疗方法，特别是对于缺乏相合的HSPC捐赠者或 没有资格进行同种异体移植。我们建议的长期目标是解决这一未得到满足的医疗需求 并开发了一种有效的新型治疗方法，旨在恢复细胞的功能和表达控制 自体患者的RAG1基因来源于HSC。我们的中心假设是基因修复策略 保存生理表达调控是治疗RAG1缺陷的一种安全有效的方法。 我们报告说，通过调整培养条件和基因输送载体，有可能部分克服 在最原始和临床相关的HSPC亚群中限制基因编辑的生物障碍 (Genovese，自然，2014；Schioloi，科学-转化-医学，2017)。在这个项目中，我们将利用 我们以前的成果是：1)直接修复RAG1突变；2)提高当前HSPC基因编辑的效率 协议和iii)在合适的小鼠模型上研究非遗传毒性的条件反射。功能矫正术 通过利用体外最先进的技术，工程RAG1基因将在患者来源的细胞上进行严格评估 T细胞分化实验和体内异种移植实验。我们将利用我们最近的 优化的基因编辑程序和条形码技术(BAR-SEQ，Ferrari等人，NAT。生物技术。2020)至 在最大限度地提高编辑效率的同时，降低了处理后的HSPC的细胞毒性，从而提高了长 定期移植淋巴样细胞。为了支持临床试验的合理性，我们将评估疾病的纠正情况 限制功能性HSPC在两种RAG1小鼠模型中的表型和新出现的疗效 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一种治疗RAG1缺陷的创新方法， 将基于同源性的基因编辑定位为精确的HSC工程的标准，提供更安全和更多 有效的治疗策略，在血液学中具有广泛的适用性。

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.