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: 10621348
• 负责人: Pietro Genovese
• 金额: --
• 依托单位: DANA-FARBER CANCER INST
• 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 2023-06-02
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10621348
• 关键词: Acceleration; Achievement; Address; Adverse effects; Affect; Allogenic; Authorization documentation; Autoimmune; Autoimmunity; Autologous; Automobile Driving; B-Cell Antigen Receptor; B-Cell Development; Bar Codes; Biological; Biological Assay; Biotechnology; CD34 gene; CRISPR/Cas technology; Cell Cycle Arrest; Cell Cycle Progression; Cell Transplantation; Cells; Chromosomal translocation; Clinical; Code; Collaborations; Complementary DNA; Cytotoxic agent; Defect; Development; Disease; Dose; Drug usage; Electroporation; Engineering; Engraftment; Exons; Gene Delivery; Genes; Genetic Diseases; Genetic Recombination; Genome Stability; Genomics; Goals; Hematology; Hematopoietic Stem Cell Transplantation; Hematopoietic Stem Cell subsets; Hematopoietic stem cells; Heterozygote; Homologous Transplantation; Human; IL2RG gene; Immunologic Deficiency Syndromes; Immunologics; Immunotoxins; In Vitro; Insertional Mutagenesis; Lentivirus; Lymphocyte; Lymphoid; Lymphoid Cell; Marketing; Mediating; Medical; Modeling; Morbidity - disease rate; Mus; Mutate; Mutation; Nature; Omenn syndrome; Opportunistic Infections; Organoids; Output; PTPRC gene; Patients; Physiological; Positioning Attribute; Pre-Clinical Model; Procedures; Process; Proteins; Protocols documentation; RAG1 gene; Rag1 Mouse; Reaction; Regimen; Reporting; Risk; Safety; Severe Combined Immunodeficiency; Site; Specificity; T cell differentiation; T-Lymphocyte; TP53 gene; Technology; Testing; Therapeutic; Thymus Gland; Toxic effect; Translational Research; Translations; Transplantation; Transplantation Conditioning; United States National Institutes of Health; V(D)J Recombination; Validation; Viral Vector; Xenograft procedure; authority; base editing; cell type; clinical development; clinically relevant; conditioning; congenital immunodeficiency; curative treatments; delivery vehicle; design; disease phenotype; disease-causing mutation; donor stem cell; efficacy evaluation; efficacy testing; engineered stem cells; experimental study; gene correction; gene repair; gene replacement; gene therapy; genotoxicity; high risk; homologous recombination; immune reconstitution; improved; in vivo; innovation; mortality; mouse model; new technology; novel; nuclease; preclinical study; preservation; promoter; reconstitution; repair strategy; research clinical testing; response; safety and feasibility; screening; single-cell RNA sequencing; stem cell gene therapy; stem cell genes; stem cell therapy; therapeutically effective; tool; transgene expression; translational medicine; treatment strategy; vector

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.

项目概要 造血干/祖细胞 (HSPC) 基因治疗已为数名患者带来临床益处 受多种遗传病影响，其中一些已获得选定的市场许可 迹象。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，这些问题变得更加重要 需要高度表达才能发挥其功能，并且当其活性直接影响基因组稳定性时，例如 以重组激活 1 (RAG1) 基因为例。 RAG1 以高但严格调控的方式表达 分化淋巴细胞前体，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）。虽然 RAG1 不受调控导致基因组损伤的风险很高 到目前为止，表达已经阻碍了使用病毒载体来治疗 RAG1 缺陷，因此需要开发 新颖有效的治疗方法，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型治疗方法，旨在恢复功能和表达控制 患者自体 HSC 上的 RAG1 基因。我们的中心假设是基因修复策略 保持生理表达控制是治疗 RAG1 缺陷的安全有效的方法。 我们报告说，通过定制培养条件和基因传递载体，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 （吉诺维斯，《自然》，2014 年；斯基罗利，《科学-转化-医学》，2017 年）。在这个项目中，我们将利用 我们之前的成就是 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) 在合适的小鼠模型上研究非基因毒性调节。功能校正 通过利用最先进的体外技术，将在患者来源的细胞上严格评估工程化的 RAG1 基因 T细胞分化测定和体内异种移植实验。我们将利用最近的 优化的基因编辑程序和条形码技术（BAR-seq，Ferrari 等人，Nat. Biotech. 2020） 最大限度地提高编辑效率，同时减少对处理过的 HSPC 的细胞毒性，从而提高长效基因的产量 术语“移植淋巴细胞”。为了支持临床检测的合理性，我们将评估疾病的纠正情况 通过限制两个 RAG1 小鼠模型中功能性 HSPC 的量来确定表型，并测试新兴的功效 免疫毒素预处理方案可减少移植毒性并增加淋巴重建。全面的， 该项目将有助于开发针对 RAG1 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、更高效的技术 有效的治疗策略在血液学中具有广泛的适用性。