Optimization of gene transfer and gene editing safety and efficacy focusing on the NHP model

以NHP模型为重点的基因转移和基因编辑安全性和有效性的优化

• 批准号: 10253804
• 负责人: CYNTHIA E DUNBAR
• 金额: $ 173.15万
• 依托单位: NATIONAL HEART, LUNG, AND BLOOD INSTITUTE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10253804
• 关键词: Abnormal Cell; Acute Myelocytic Leukemia; Algorithms; Animal Model; Animals; Bar Codes; Behavior; Biology; Blood; Bone Marrow Purging; CAR T cell therapy; CD34 gene; COVID-19; CRISPR/Cas technology; Cells; Clinic; Clinical; Clinical Trials; Clonal Expansion; Clone Cells; Clustered Regularly Interspaced Short Palindromic Repeats; Development; Diamond-Blackfan anemia; Disease; Disease model; Engraftment; Enhancers; Eosinophilia; Erythroblasts; Erythroid; Gene Transfer; Generations; Genes; Genetic; Genetic Diseases; Genome engineering; Hematopoiesis; Hematopoietic stem cells; Human; In Vitro; Individual; Inflammatory; International; Knock-out; Laboratories; Lentivirus Vector; Macaca; Macaca mulatta; Maps; Marrow; Measures; Mediating; Modeling; Molecular; Mutate; Myeloid Cells; Myeloid Leukemia; Nonhomologous DNA End Joining; Pancytopenia; Patients; Phenotype; Population; Pre-Clinical Model; Primates; Publishing; RNA Splicing; RUNX1 gene; Reporting; Research; Resistance; Retrieval; Rhesus; Risk; Safety; Site; Stem Cell Factor; Syndrome; System; Technology; Thrombocytopenia; Time; Transplantation; Validation; Work; cardiovascular disorder risk; chimeric antigen receptor T cells; comparative; cytokine; design; gene correction; gene therapy; genome editing; genotoxicity; human model; in silico; in vitro Assay; in vivo; insertion/deletion mutation; interest; lentiviral integration; lentivirally transduced; leukemia; leukemogenesis; loss of function mutation; meetings; mouse model; mutant; myeloid cell development; neoplastic; neutrophil; novel therapeutics; off-target site; outcome forecast; peripheral blood; predictive test; promoter; rat Piga protein; repaired; stem cells; targeted treatment; therapeutic genome editing; transcription factor; vector

My research group has worked for over 30 years in the laboratory and in the clinic to develop safe and effective gene addition and gene correction therapies directed at hematopoietic stem and progenitor cells (HSPC). In the rhesus model, shown to be the only predictive assay for human clinical results, we have focused on optimizing gene transfer to primitive stem and progenitor cells, and on understanding and enhancing safety of established and new vector systems. We retrieve and analyze clonal contributions to peripheral blood populations following transplantation of CD34+ transduced progenitor cells. We have applied our genetic barcoding technology to map contributions of thousands of individual hematopoietic stem and progenitor cell clones, and investigated whether clonal expansion as an early measure of genotoxicity can be assessed in a high throughput manner using this approach. Relevant preclinical model for assessing genotoxicity prior to clinical trials are an unmet need, since in vitro assays and murine models have not been predictive. The quantitative assessment of oligoclonality in vivo, via our highly sensitive and quantitative barcoding approach, allows relevant comparisons between vectors. In animals followed for 1-7.5 years, we have now clonally tracked the behavior of almost 200,000 lentivirally-transduced HSPC over time, and whether the vector contains strong (SFFV), medium (MSCV) or weak promoter/enhancers, with a single exception we have not seen clonal expansion or other evidence of genotoxicity. This direct comparative work was published recently (Yabe et al MTCMD, 2019) However recently we have now reported the first clear evidence of genotoxicity utilizing a lentiviral vector to transduce HSPC in a human or primate. A rhesus macaque receiving lentivirally-barcoded cells, with a vector containing a moderately strong enhancer, developed markedly abnormal neoplastic hematopoiesis, with profound thrombocytopenia, eosinophilia, and most strikingly an erythroid expansion with very high levels of circulating nucleated red blood cells. These abnormal cell populations were shown to be clonal by barcode retrieval, and the clone contains 9 independent barcoded insertions. We have retrieved the insertion sites, and analysis of which site or sites is most likely responsible for the syndrome has identified at least two genes over expressed and aberrantly spliced that likely contributed to the phenotype, specifically the transcription factor PLAG1 and the cytokine Stem Cell Factor. This important information has been presented a several international meetings, generated interest and concern at the FDA, and was recently published (Espinoza et al, Molecular Therapy, 2019). Given the potential for genotoxicity with random integration of lentiviral vectors, and other drawbacks of gene addition as compared to targeted gene correction approaches, we have utilized the rhesus macaque to explore CRISPR/Cas9 genome editing to create disease models and to develop gene editing therapies targeting HSPC. We have optimized CRISPR/Cas9 gene editing of rhesus CD34+ HSPC, initially knocking out loci and creating indels via non-homologous end joining repair. We have successfully engrafted 9 animals with gene-edited cells, with long-term engraftment at levels of up to 90% for cells that have targeted indels. We created a model to investigate whether clonal expansion in paroxysmal nocturnal hemoglobinuria is intrinsic to HSPC via targeting of the PIG-A locus (Shin et al, Blood, 2019). We knocked out CD33 in neutrophils produced from edited HSPC as an approach to make marrow resistant to CAR-T cells targeting CD33 in acute myeloid leukemia, demonstrating no change in any aspect of myeloid cell development or function following CD33 knockout, and demonstrating the utility of this approach to safely treat myeloid leukemias with CAR-T cells (Kim, Yu et al Cell, 2018). This work was published last FY, but this FY we have set up a rhesus macaque CAR-T model and will move forward to deliver CD33 CAR-T to macaques with and without engrafted CD33KO HSPC created by CRISPR/Cas gene editing to evaluate whether the CD33 KO HSPC protect the animals from CAR-T-associated myeloablation. We have created a robust macaque model of clonal hematopoiesis by targeting DNMT3, TET2 and ASXL1 with CRISPR/Cas9 mediated editing to create loss of function mutations. We have shown marked clonal expansion of TET2 mutated clones in three animals, and less marked expansion of DNMT2 or ASXL1 edited clones, and we have documented a highly inflammatory phenotype for TET2 mutant myeloid cells, relevant to the increased risk of cardiovascular disease in CHIP patients. We have multiple ongoing studies to investigate the biology of clonal expansion in these animals, adn this year we have shown that treatment with tociluzumab reverses clonal expansion due to TET2 deficiency in this model. We hypothesize that clonal hematopoiesis accompanied by an inflammatory phenotype could be associated with severe COVID-19 disease, and we are now investigating this using our macaque clonal hematopoiesis mode. Recently we have developed a macaque gene editing model for RUNX1 deficiency and this is being used to understand the biology of the condition and the feasbility of gene therapies. A model is being developed for Diamond-Blackfan anemia as well to be used to identify new therapies and better understand this bone marrow failure disorder. We are studying the most predictive approaches to identify and detect off-target effects of CRISPR/Cas9 in HSPC and their progeny in our engrafted rhesus macaques. We have the first comprehensive analysis of on target versus off target editing of HSPC, at sites identified by in silico algorithms versus In vitro site ID via CircleSeq, in a relevant macaque animal model. We have identified persistent CD33 off-target sites in vivo and are now mapping these clones over time and in various lineages. This work was presented in the Plenary Session at ASGCT 2020.

我的研究小组在实验室和临床工作了 30 多年，致​​力于开发针对造血干细胞和祖细胞 (HSPC) 的安全有效的基因添加和基因校正疗法。在被证明是人类临床结果的唯一预测分析的恒河猴模型中，我们专注于优化原始干细胞和祖细胞的基因转移，以及了解和增强已建立的和新的载体系统的安全性。 我们检索并分析了 CD34+ 转导祖细胞移植后对外周血群体的克隆贡献。 我们应用遗传条形码技术绘制了数千个造血干细胞和祖细胞克隆的贡献图，并研究了是否可以使用这种方法以高通量方式评估克隆扩增作为遗传毒性的早期测量。由于体外测定和小鼠模型尚无法预测，因此在临床试验之前评估遗传毒性的相关临床前模型的需求尚未得到满足。通过我们高度灵敏的定量条形码方法，对体内寡克隆性进行定量评估，从而可以在载体之间进行相关比较。在跟踪 1-7.5 年的动物中，我们现在已经克隆追踪了近 200,000 个慢病毒转导的 HSPC 随着时间的推移的行为，以及载体是否包含强 (SFFV)、中 (MSCV) 或弱启动子/增强子，只有一个例外我们还没有看到克隆扩张或其他遗传毒性的证据。这项直接比较工作最近发表（Yabe 等人 MTCMD，2019）然而，最近我们报告了利用慢病毒载体在人类或灵长类动物中转导 HSPC 的遗传毒性的第一个明确证据。一只恒河猴接受慢病毒条形码细胞（含有中等强度增强子的载体）后，出现明显异常的肿瘤性造血，伴有严重的血小板减少、嗜酸性粒细胞增多，最引人注目的是红细胞扩张，循环有核红细胞水平非常高。通过条形码检索显示这些异常细胞群是克隆的，并且该克隆包含 9 个独立的条形码插入。我们检索了插入位点，并分析了最有可能导致该综合征的一个或多个位点，确定了至少两个可能导致表型的基因过度表达和异常剪接，特别是转录因子 PLAG1 和细胞因子干细胞因子。这一重要信息已在多次国际会议上提出，引起了 FDA 的兴趣和关注，并于最近发表（Espinoza 等人，分子治疗，2019）。 鉴于慢病毒载体随机整合可能产生基因毒性，以及与靶向基因校正方法相比基因添加的其他缺点，我们利用恒河猴探索 CRISPR/Cas9 基因组编辑，以创建疾病模型并开发针对目标的基因编辑疗法HSPC。我们优化了恒河猴 CD34+ HSPC 的 CRISPR/Cas9 基因编辑，最初敲除基因座并通过非同源末端连接修复创建插入缺失。 我们已成功将基因编辑细胞移植到 9 只动物中，针对插入缺失的细胞的长期移植水平高达 90%。我们创建了一个模型，通过靶向 PIG-A 基因座来研究阵发性睡眠性血红蛋白尿症中的克隆扩张是否是 HSPC 所固有的（Shin 等人，Blood，2019）。我们敲除编辑过的 HSPC 产生的中性粒细胞中的 CD33，作为一种方法，使骨髓对急性髓性白血病中靶向 CD33 的 CAR-T 细胞产生耐药性，证明 CD33 敲除后骨髓细胞发育或功能的任何方面都没有变化，并证明了这种利用 CAR-T 细胞安全治疗骨髓性白血病的方法（Kim, Yu et al Cell, 2018）。这项工作于上个财年发表，但本财年我们已经建立了恒河猴 CAR-T 模型，并将继续将 CD33 CAR-T 传递给通过 CRISPR/Cas 基因编辑创建的植入和未植入 CD33KO HSPC 的猕猴，以评估是否CD33 KO HSPC 可保护动物免受 CAR-T 相关清髓作用的影响。我们通过 CRISPR/Cas9 介导的编辑以 DNMT3、TET2 和 ASXL1 为目标，创建功能丧失突变，从而创建了强大的猕猴克隆造血模型。我们已经在三只动物中展示了 TET2 突变克隆的显着克隆扩增，以及 DNMT2 或 ASXL1 编辑克隆的不显着扩增，并且我们已经记录了 TET2 突变骨髓细胞的高度炎症表型，这与 CHIP 患者心血管疾病风险增加有关。 我们正在进行多项研究来调查这些动物中克隆扩增的生物学特性，今年我们已经表明，托珠单抗治疗可逆转该模型中由于 TET2 缺陷而导致的克隆扩增。我们假设伴随炎症表型的克隆造血可能与严重的 COVID-19 疾病有关，我们现在正在使用猕猴克隆造血模式对此进行研究。最近我们开发了猕猴基因编辑 RUNX1 缺陷模型，该模型被用来了解该病症的生物学和基因疗法的可行性。正在开发一种针对 Diamond-Blackfan 贫血的模型，用于识别新疗法并更好地了解这种骨髓衰竭性疾病。 我们正在研究最具预测性的方法，以识别和检测 CRISPR/Cas9 对我们移植的恒河猴的 HSPC 及其后代的脱靶效应。我们在相关的猕猴动物模型中，首次对 HSPC 的在靶与脱靶编辑进行了全面分析，分别是通过计算机算法识别的位点与通过 CircleSeq 进行的体外位点 ID 识别。我们已经确定了体内持久的 CD33 脱靶位点，现在正在绘制这些克隆随时间的变化和不同谱系的图谱。这项工作已在 ASGCT 2020 的全体会议上提出。