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:
- 金额:$ 182.94万
- 依托单位:
- 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:
- 关键词:AgingAlzheimer&aposs disease riskAmyloidAnimal ModelAnimalsBar CodesBiologyBloodBlood CellsBlood PlateletsBrainCD34 geneCOVID-19COVID-19 severityCRISPR/Cas technologyCell CountCellsClinicClinicalClonal ExpansionClustered Regularly Interspaced Short Palindromic RepeatsControl AnimalDNA Double Strand BreakDiseaseDisease modelEngineeringEngraftmentFailureGenerationsGenesGeneticGenetic DiseasesHematopoiesisHematopoietic stem cellsHumanInflammatoryInheritedKnock-outLaboratoriesLentivirus VectorLinkLungMacacaMacaca mulattaMarrowMediatingMicrogliaModelingModificationMutateMutationMyeloid CellsNonhomologous DNA End JoiningNormal CellOther GeneticsOutcomePhenotypePilot ProjectsPre-Clinical ModelPredispositionPrognosisRUNX1 geneReportingResearchRhesusRodent ModelSARS-CoV-2 infectionSafetySyndromeSystemTimeTissuesToxic effectVirusWorkagedbase editingcardiovascular disorder riskcohortfunctional lossgene correctiongene therapygenome editinggenotoxicityhuman diseasehuman modelimprovedinsertion/deletion mutationlentiviral integrationleukemialoss of functionloss of function mutationmouse modelmutantpatient prognosispredictive testpreventrepairedsevere COVID-19targeted sequencingtargeted treatmenttau Proteinstherapeutic 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32年以上,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。在恒河猴模型中,被证明是人类临床结果的唯一预测试验,我们专注于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,以及了解和提高已建立的和新的基因治疗系统的安全性。
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:
- 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:
- 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:
- 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:
- 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.
- DOI:10.1007/s11684-011-0159-1
- 发表时间:2011-12
- 期刊:
- 影响因子:8.1
- 作者:Wu, Chuanfeng;Dunbar, Cynthia E
- 通讯作者:Dunbar, Cynthia E
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CYNTHIA E DUNBAR其他文献
CYNTHIA E DUNBAR的其他文献
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{{ truncateString('CYNTHIA E DUNBAR', 18)}}的其他基金
GENE TRANSFER AND EX VIVO MANIPULATION OF HEMATOPOIETIC CELLS
造血细胞的基因转移和离体操作
- 批准号:
6290425 - 财政年份:
- 资助金额:
$ 182.94万 - 项目类别:
Gene Transfer And Ex Vivo Manipulation Of Hematopoietic
基因转移和造血的离体操作
- 批准号:
6809652 - 财政年份:
- 资助金额:
$ 182.94万 - 项目类别:
The rhesus macaque as a preclinical model for induced pluripotent stem cells
恒河猴作为诱导多能干细胞的临床前模型
- 批准号:
8344862 - 财政年份:
- 资助金额:
$ 182.94万 - 项目类别:
Clonal and imaging analyses of in vivo hematopoiesis, immune cell ontogeny and adoptive cell therapies
体内造血、免疫细胞个体发育和过继细胞疗法的克隆和成像分析
- 批准号:
10929124 - 财政年份:
- 资助金额:
$ 182.94万 - 项目类别:
Novel therapies for bone marrow failure and Diamond-Blackfan Anemia
骨髓衰竭和戴蒙德-布莱克范贫血的新疗法
- 批准号:
10929163 - 财政年份:
- 资助金额:
$ 182.94万 - 项目类别:
Gene Transfer And Ex Vivo Manipulation Of Hematopoietic
基因转移和造血的离体操作
- 批准号:
6690539 - 财政年份:
- 资助金额:
$ 182.94万 - 项目类别:
Developing Efficient and Safe Gene Transfer to Primate Hematopoietic Stem Cells
开发高效、安全的灵长类造血干细胞基因转移方法
- 批准号:
8557916 - 财政年份:
- 资助金额:
$ 182.94万 - 项目类别: