Optimization of genetic modification of HSCs in the NHP model and creation of relevant preclinical models of human disease and therapies

NHP模型中HSC基因修饰的优化以及人类疾病和治疗相关临床前模型的创建

基本信息

批准号：
10929089
负责人：
CYNTHIA E DUNBAR
金额：
$ 182.94万
依托单位：
NATIONAL HEART, LUNG, AND BLOOD INSTITUTE
依托单位国家：
美国
项目类别：
财政年份：
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/10929089
关键词：
Aging Alzheimer&apos s disease risk Amyloid Animal Model Animals Bar Codes Biology Blood Blood Cells Blood Platelets Brain CD34 gene COVID-19 COVID-19 severity CRISPR/Cas technology Cell Count Cells Clinic Clinical Clonal Expansion Clustered Regularly Interspaced Short Palindromic Repeats Control Animal DNA Double Strand Break Disease Disease model Engineering Engraftment Failure Generations Genes Genetic Genetic Diseases Hematopoiesis Hematopoietic stem cells Human Inflammatory Inherited Knock-out Laboratories Lentivirus Vector Link Lung Macaca Macaca mulatta Marrow Mediating Microglia Modeling Modification Mutate Mutation Myeloid Cells Nonhomologous DNA End Joining Normal Cell Other Genetics Outcome Phenotype Pilot Projects Pre-Clinical Model Predisposition Prognosis RUNX1 gene Reporting Research Rhesus Rodent Model SARS-CoV-2 infection Safety Syndrome System Time Tissues Toxic effect Virus Work aged base editing cardiovascular disorder risk cohort functional loss gene correction gene therapy genome editing genotoxicity human disease human model improved insertion/deletion mutation lentiviral integration leukemia loss of function loss of function mutation mouse model mutant patient prognosis predictive test prevent repaired severe COVID-19 targeted sequencing targeted treatment tau Proteins therapeutic genome editing

项目摘要

My research group has worked for over 32 years in the laboratory and in the clinic to develop safe and effective gene therapies directed at hematopoietic stem and progenitor cells (HSPC). In the rhesus macaque model, shown to be the only predictive assay for human clinical results, we have focused on optimizing both lentiviral gene addition and gene editing therapies targeting hematopoietic stem and progenitor cells, and on understanding and enhancing the safety of established and new gene therapy systems. Given the potential for genotoxicity with random integration of lentiviral vectors, and other drawbacks of semi-random gene addition as compared to targeted gene correction approaches, we have utilized the rhesus macaque to explore CRISPR/Cas9 genome editing and more recently base editing to create disease models and to develop gene editing therapies targeting HSPC. We have optimized CRISPR/Cas9 gene editing and base editing of rhesus CD34+ HSPC, initially knocking out loci via CRISPR/CAs-induced non-homologous end joining repair, creating loss-of function indels, and now focusing on improving the safety and efficacy of HDR-mediated gene correction and of single mutation-directed base editing. We have successfully engrafted 22 animals with gene-edited cells, with long-term levels of up to 70-90% for blood cells with targeted NHEJ indels. We have focused on investigating the quantitative adverse impact of gene editing on the engraftment and long-term function of HSPCs in the macaque model. Using quantitative barcoding together with gene editing, we have demonstrated marked loss of functional HSPC numbers with both NHEJ but even more markedly HDR editing, and thus far less adverse impact on HSPCs with base editing, which does not result in double stranded DNA breaks. 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, and have shown that treatment with tociluzumab reverses or slows clonal expansion due to TET2 deficiency in this model (Shin et al, Blood, 2022). We hypothesized that clonal hematopoiesis accompanied by an inflammatory phenotype could be associated with severe COVID-19 disease, and carried out pilot studies investigating this using our macaque clonal hematopoiesis model, comparing outcomes of SARS-CoV-2 infection in clonal hematopoiesis versus control animals, documenting higher levels of virus in tissue and shed in the lungs (Shin et al, 2023). We have also carried out a large scale targeted sequencing study of rhesus macaque blood cells from cohorts of aged animals, use deep error-corrected sequencing to look at 56 clonal hematopoiesis genes initially identified in aging humans. We have uncovered for the first time a natural animal model of clonal hematopoiesis, showing exactly the same genes mutated as in humans, in contrast to lack of such mutations in rodent models (Shin et al, Blood, 2022). We extended these studies to human cohorts in terms of analyzing the relationship between COVID-19 severity and the presence of clonal hematopoiesis, and in the largest and most definitive study to date, did not demonstrate an impact on COVID-19 severity (Zhou et al, Blood, 2022). We have also developed a gene editing macaque model for RUNX1 deficiency in order to better understand the biology of the inherited marrow failure/leukemia predisposition syndrome and to assess the feasibility of gene therapies in correcting the phenotype, asking whether mutant vs normal cells predominate over time in a chimeric state. Mutant cells predominate, a concerning finding for gene therapies of this condition (Lee et al, Blood, 2023). This model also recapitulates the platelet and HSPC phenotype of human RUNX1 deficiency, in contrast to murine models. A recent report linked clonal hematopoiesis to surprisingly a decreased risk of Alzheimer's disease, and postulated that CH myeloid cells were more potent in entering or functioning in the brain to prevent accumulation of amyloid or tau plaques. We have utilized our macaque CH model and control barcoded non-CH animals to investigate TET2 or other CH mutations results in higher replacement of microglial cells in the brain by analyzing purified macaque microglial cells for CH mutations compared to levels in blood myeloid cells. We have not found enhancement of microglial replacement by HSPC-derived cells in the setting of CH. Mechanistic studies are ongoing.

我的研究小组在实验室和临床工作了超过 32 年，致力于开发针对造血干细胞和祖细胞 (HSPC) 的安全有效的基因疗法。恒河猴模型被证明是人类临床结果的唯一预测分析，我们重点优化针对造血干细胞和祖细胞的慢病毒基因添加和基因编辑疗法，并了解和增强已建立基因和新基因的安全性治疗系统。考虑到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半随机基因添加的其他缺点，我们利用恒河猴探索 CRISPR/Cas9 基因组编辑和最近的碱基编辑来创造疾病模型并开发针对 HSPC 的基因编辑疗法。我们优化了恒河猴 CD34+ HSPC 的 CRISPR/Cas9 基因编辑和碱基编辑，最初通过 CRISPR/CA 诱导的非同源末端连接修复敲除位点，产生功能缺失插入缺失，现在专注于提高治疗的安全性和有效性。 HDR 介导的基因校正和单突变定向碱基编辑。我们已成功将基因编辑细胞移植到 22 只动物中，具有靶向 NHEJ 插入/缺失的血细胞的长期水平高达 70-90%。我们重点研究基因编辑对猕猴模型中 HSPC 的植入和长期功能的定量不利影响。将定量条形码与基因编辑结合使用，我们证明了 NHEJ 功能性 HSPC 数量的显着损失，但 HDR 编辑的情况更为明显，因此碱基编辑对 HSPC 的不利影响要小得多，不会导致双链 DNA 断裂。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，并表明托珠单抗治疗可逆转或减缓该模型中由于 TET2 缺陷而导致的克隆扩增（Shin 等人，Blood，2022）。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，并使用我们的猕猴克隆造血模型进行了初步研究，比较了克隆造血与对照动物中 SARS-CoV-2 感染的结果，记录了组织和肺部排出的病毒水平较高（Shin 等人，2023）。我们还对老年动物群中的恒河猴血细胞进行了大规模靶向测序研究，使用深度纠错测序来研究最初在老年人中发现的 56 个克隆造血基因。我们首次发现了克隆造血的自然动物模型，显示出与人类完全相同的基因突变，而啮齿动物模型中则缺乏此类突变（Shin 等人，Blood，2022）。我们将这些研究扩展到人类队列，分析了 COVID-19 严重程度与克隆造血存在之间的关系，并且在迄今为止规模最大、最明确的研究中，并未证明对 COVID-19 严重程度的影响（Zhou 等人），血，2022）。我们还开发了一种针对 RUNX1 缺陷的基因编辑猕猴模型，以便更好地了解遗传性骨髓衰竭/白血病易感综合症的生物学，并评估基因疗法纠正表型的可行性，询问随着时间的推移突变细胞与正常细胞是否占主导地位处于嵌合状态。突变细胞占主导地位，这是这种情况的基因疗法的一个令人担忧的发现（Lee 等人，Blood，2023）。与小鼠模型相比，该模型还重现了人类 RUNX1 缺陷的血小板和 HSPC 表型。最近的一份报告将克隆造血与阿尔茨海默氏病风险降低联系起来，并推测 CH 骨髓细胞更有效地进入大脑或在大脑中发挥作用，以防止淀粉样蛋白或 tau 斑块的积累。我们利用我们的猕猴 CH 模型和对照条形码非 CH 动物来研究 TET2 或其他 CH 突变导致大脑中小胶质细胞的更高替换，通过分析纯化的猕猴小胶质细胞的 CH 突变与血液骨髓细胞中的水平相比。我们尚未发现 CH 环境中 HSPC 衍生细胞对小胶质细胞替代的增强作用。机理研究正在进行中。

项目成果

期刊论文数量（22）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Two Decades of ASGCT: Dreams Become Reality.

ASGCT 的两个十年：梦想变成现实。

DOI：
10.1016/j.ymthe.2017.04.011
发表时间：
2017
期刊：
Molecular therapy : the journal of the American Society of Gene Therapy
影响因子：
0
作者：
Dunbar,CynthiaE
通讯作者：
Dunbar,CynthiaE

A plethora of gene therapies for hemoglobinopathies.

大量针对血红蛋白病的基因疗法。

DOI：
10.1038/s41591-021-01235-7
发表时间：
2021
期刊：
Nature medicine
影响因子：
82.9
作者：
Dunbar,CynthiaE
通讯作者：
Dunbar,CynthiaE

Thrombopoietic status of patients on haemodialysis.

血液透析患者的血小板生成状态。

DOI：
10.1111/bjh.13918
发表时间：
2016
期刊：
British journal of haematology
影响因子：
6.5
作者：
Bat,Taha;Bat,BetulE;El-Moghraby,Ahmed;Patel,Samir;Feng,Xingmin;Dunbar,CynthiaE;Sarac,Erdal
通讯作者：
Sarac,Erdal

No evidence for clonal selection due to lentiviral integration sites in human induced pluripotent stem cells.

DOI：
10.1002/stem.322
发表时间：
2010-04
期刊：
STEM CELLS
影响因子：
5.2
作者：
Winkler, Thomas;Cantilena, Amy;Metais, Jean-Yves;Xu, Xiuli;Nguyen, Anh-Dao;Borate, Bhavesh;Antosiewicz-Bourget, Jessica E.;Wolfsberg, Tyra G.;Thomson, James A.;Dunbar, Cynthia E.
通讯作者：
Dunbar, Cynthia E.

Stem cell gene therapy: the risks of insertional mutagenesis and approaches to minimize genotoxicity.