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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.

我的研究小组已经在实验室和临床上工作了32年，开发了针对造血干细胞和祖细胞（HSPC）的安全有效的基因疗法。在恒河猴模型中，被证明是人类临床结果的唯一预测性试验，我们专注于优化靶向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和新的基因治疗系统的安全性。考虑到慢病毒载体随机整合的遗传毒性潜力，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，我们已经利用恒河猴来探索CRISPR/Cas9基因组编辑和最近的碱基编辑以创建疾病模型并开发靶向HSPC的基因编辑疗法。我们优化了恒河猴CD 34 + HSPC的CRISPR/Cas9基因编辑和碱基编辑，最初通过CRISPR/CAs诱导的非同源末端连接修复敲除基因座，创建功能缺失indel，现在专注于提高HDR介导的基因校正和单突变指导的碱基编辑的安全性和有效性。我们已经成功地用基因编辑的细胞移植了22只动物，具有靶向NHEJ indel的血细胞的长期水平高达70-90%。我们专注于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。使用定量条形码与基因编辑一起，我们已经证明了使用NHEJ但甚至更明显的HDR编辑的功能性HSPC数量的显著损失，并且因此使用碱基编辑对HSPC的不利影响小得多，这不会导致双链DNA断裂。我们已经通过用CRISPR/Cas9介导的编辑靶向DNMT 3、TET 2和ASXL 1以产生功能缺失突变来创建克隆造血的稳健猕猴模型。我们已经在三只动物中显示了TET 2突变克隆的显著克隆扩增，以及DNMT 2或ASXL 1编辑克隆的不太显著的扩增，并且我们已经记录了TET 2突变骨髓细胞的高度炎症表型，与CHIP患者心血管疾病风险增加相关。我们有多项正在进行的研究来研究这些动物中克隆扩增的生物学，并且已经表明，在该模型中，托西珠单抗治疗逆转或减缓了由于TET 2缺乏引起的克隆扩增（Shin等人，Blood，2022）。我们假设伴有炎性表型的克隆造血可能与严重的COVID-19疾病相关，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，记录了组织中更高水平的病毒和肺中的病毒脱落（Shin等人，2023）。我们还对来自老龄动物队列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龄人类中鉴定的56个克隆造血基因。我们首次发现了克隆造血的天然动物模型，显示出与人类完全相同的基因突变，与啮齿动物模型中缺乏此类突变相反（Shin等人，Blood，2022）。我们在分析COVID-19严重程度与克隆造血存在之间的关系方面将这些研究扩展到人类队列，并且在迄今为止最大和最确定的研究中，没有证明对COVID-19严重程度的影响（Zhou et al，Blood，2022）。我们还开发了一种RUNX 1缺陷的基因编辑猕猴模型，以更好地了解遗传性骨髓衰竭/白血病易感综合征的生物学，并评估基因疗法在纠正表型方面的可行性，询问突变细胞与正常细胞是否随时间推移在嵌合状态中占主导地位。突变细胞占优势，这是该病症的基因疗法的一个令人关注的发现（Lee等人，Blood，2023）。与鼠模型相比，该模型还概括了人RUNX 1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病的风险降低联系起来，并假设CH髓样细胞在进入大脑或在大脑中发挥作用以防止淀粉样蛋白或tau斑块的积累方面更有效。我们已经利用我们的猕猴CH模型和对照条形码化的非CH动物，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，研究TET 2或其他CH突变导致脑中小胶质细胞的更高替换。我们还没有发现增强HSPC衍生细胞的小胶质细胞替代设置CH。机制研究正在进行中。

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

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

由于人类诱导的多能干细胞中的慢病毒整合位点而导致克隆选择的证据。

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.

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

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