Base editing and prime editing for sickle cell disease
镰状细胞病的碱基编辑和引物编辑
基本信息
- 批准号:10579903
- 负责人:
- 金额:$ 77.71万
- 依托单位:
- 依托单位国家:美国
- 项目类别:
- 财政年份:2021
- 资助国家:美国
- 起止时间:2021-01-01 至 2024-12-31
- 项目状态:已结题
- 来源:
- 关键词:Acute PainAdenosineAdultAffectAffinityAlanineAllelesAllogenicAmericanAmino AcidsAntisickling AgentsArchitectureAutologousBase PairingBenignBiochemicalBiological AssayBloodCD34 geneCaringCell DeathCell LineCell TherapyCell physiologyCellsCessation of lifeChromosomal RearrangementClinical ResearchCodon NucleotidesComplexDNADNA Double Strand BreakDeteriorationDevelopmentDiseaseElementsEngineeringEngraftmentEnzymesErythrocytesErythroid CellsErythroid Progenitor CellsEscherichia coliEvolutionFetal HemoglobinFrequenciesFunctional disorderFutureGene ExpressionGene SilencingGenesGeneticGenetic DiseasesGenetic EngineeringGenetic TemplateGenomeGenomic DNAGenomicsGlobinGuide RNAHLA AntigensHematological DiseaseHematopoieticHematopoietic Stem Cell TransplantationHematopoietic stem cellsHemoglobinHemoglobin F DiseaseHemolytic AnemiaHumanHypoxiaIn VitroIndividualInheritedMaintenanceMalignant - descriptorMediatingMedicalMessenger RNAMethodsMissense MutationModificationMorbidity - disease rateMultiple Organ FailureMusMutationNucleotidesOrganOutcomePainPatientsPolymersProteinsQuality of lifeRNAReagentRecombinantsRegulationResearchSafetySickle CellSickle Cell AnemiaSickle Cell TraitSickle HemoglobinSiteTechnologyTestingTherapeuticToxic effectTranscriptional Silencer ElementsTransplantationTreatment EfficacyValineVariantXenograft procedurebase editingbase editorbeta Globinchronic paincurative treatmentsexperiencegene therapygenetic approachgenetic informationgenetic manipulationgenome editingimprovedin vivoinsightmanufacturemortalitymouse modelmutantnew technologynovelnovel strategiesoff-target mutationpolymerizationprecision geneticsprematurepreventprime editingprime editorpromoterrepairedsafety testingsickle erythroidsicklingtargeted treatmenttherapeutic targettool
项目摘要
PROJECT SUMMARY
Despite advances in the medical care of sickle cell disease (SCD), most patients continue to experience severe
pain, poor quality of life, progressive organ deterioration and premature death. Allogeneic hematopoietic stem
cell transplantation (HSCT) can cure SCD but is associated with numerous toxicities and only 20% of patients
have Human Leukocyte Antigen (HLA)-matched donors. Therefore, improved and more widely accessible
curative therapies are needed. Genetic modification of autologous HSCs is a promising experimental approach
for treating SCD that circumvents some of the problems associated with allogeneic HSCT, although the optimal
technical strategies are not yet established. This proposal explores the use of adenosine base editors (ABEs)
and prime editors (PEs) for genetic correction of SCD. In contrast to conventional genome editing, these novel
approaches create precise nucleotide alterations independent of double-stranded DNA breaks (DSBs), which
can cause structural DNA abnormalities, cell death or malignant transformation. Adenosine base editors convert
targeted A·T base pairs to G·C pairs. Prime editors copy edited sequence information from a guide RNA template
into a targeted DNA locus. We will test these potentially transformative tools in 3 different strategies for SCD
therapy. Aim 1 employs ABEs to create HSC alterations that recapitulate hereditary persistence of fetal
hemoglobin (HPFH), a benign genetic condition that alleviates the pathophysiology of co-inherited SCD by
inducing the expression of red blood cell (RBC) fetal hemoglobin (HbF), a potent anti-sickling agent. We have
used protein evolution strategies to create new high-efficiency ABEs that generate HPFH mutations at
frequencies of up to 60% in CD34+ hematopoietic stem and progenitor cells (HSPCs), with HbF being induced
to levels that inhibit hypoxic sickling of erythroid progeny. Aim 2 uses ABEs to convert the mutant SCD codon
from valine to alanine, thereby generating “Hemoglobin Makassar (HbG)”, a naturally occurring benign non-
sickling variant. We have developed an altered PAM-specific ABE that converts HbS alleles to HbG in SCD
donor HSPCs at frequencies of up to 80%, with inhibition of RBC sickling. Aim 3 employs prime editing to revert
the mutant SCD codon to normal (Val→Glu), which we have shown to occur efficiently in the HEK293T cell line
and now aim to optimize in HSPCs from affected individuals. Overall, our preliminary studies have shown proof
of principle for three novel, independent editing approaches to treating SCD without the need to enrich for edited
cells or to create DSBs. Through the proposed research, we seek to optimize the efficiency of these approaches
in primary HSPCs and to further determine their safety and efficacy by using mouse models, in vitro culture
methods and biochemical assays. Developing three approaches simultaneously will enable us to compare their
outcomes directly and to determine the best therapeutic strategy to pursue in future clinical studies. More
generally, our planned studies have the potential to generate new paradigms for using base editors and PEs to
treat numerous genetic blood disorders via precise genetic manipulation of HSCs.
项目摘要
尽管镰状细胞病(SCD)的医疗护理取得了进展,但大多数患者仍继续经历严重的贫血。
疼痛、生活质量差、进行性器官退化和过早死亡。异基因造血干
细胞移植(HSCT)可以治愈SCD,但与许多毒性相关,只有20%的患者
人类白细胞抗原(HLA)匹配的捐赠者。因此,改善和更广泛地获得
需要治愈性疗法。自体造血干细胞的基因修饰是一种很有前途的实验方法
用于治疗SCD,其避免了与同种异体HSCT相关的一些问题,尽管最佳的
技术战略尚未确立。该提案探讨了腺苷碱基编辑器(ABE)的使用
和用于SCD的遗传校正的初级编辑器(PE)。与传统的基因组编辑相比,
这些方法产生了不依赖于双链DNA断裂(DSB)的精确核苷酸改变,
可导致DNA结构异常、细胞死亡或恶性转化。腺苷碱基编辑器转换
靶向A·T碱基对到G·C碱基对。引物编辑器从指导RNA模板复制编辑的序列信息
插入到目标DNA位点我们将在3种不同的SCD策略中测试这些潜在的变革工具
疗法目的1利用ABE产生HSC改变,重现胎儿遗传性持续性
血红蛋白(HPFH)是一种良性遗传性疾病,通过以下方式阐明共同遗传性SCD的病理生理学:
诱导红细胞(RBC)胎儿血红蛋白(HbF)(一种有效的抗镰状化剂)的表达。我们有
使用蛋白质进化策略来创建新的高效ABE,
在CD 34+造血干细胞和祖细胞(HSPC)中的频率高达60%,HbF被诱导
达到抑制红细胞后代缺氧镰状化的水平。目的2使用ABE转换突变的SCD密码子
从缬氨酸到丙氨酸,从而产生“马卡萨尔血红蛋白(HbG)",一种天然存在的良性非-
镰状变种我们已经开发了一种改变的PAM特异性ABE,其在SCD中将HbS等位基因转化为HbG。
供体HSPC的频率高达80%,抑制RBC镰状化。Aim 3采用Prime编辑来还原
突变的SCD密码子变为正常密码子(瓦尔→Glu),我们已经证明这在HEK 293 T细胞系中有效发生
现在的目标是优化来自受影响个体的HSPC。总的来说,我们的初步研究表明
三个新的原则,独立的编辑方法来治疗SCD,而不需要丰富的编辑
细胞或创建DSB。通过所提出的研究,我们寻求优化这些方法的效率
在原代HSPC中,并通过使用小鼠模型、体外培养
方法和生化测定。同时开发三种方法将使我们能够比较它们的
结果直接,并确定最佳的治疗策略,在未来的临床研究中追求。更
一般来说,我们计划的研究有可能产生新的范例,使用基础编辑器和PE,
通过对HSC的精确遗传操作治疗多种遗传性血液疾病。
项目成果
期刊论文数量(0)
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{{ truncateString('DAVID R LIU', 18)}}的其他基金
Project 3: Therapeutic Gene Editing for Huntington's Disease
项目3:亨廷顿病的治疗性基因编辑
- 批准号:
10668769 - 财政年份:2023
- 资助金额:
$ 77.71万 - 项目类别:
Project 2: Therapeutic Gene Editing for Friedreich's Ataxia
项目 2:弗里德赖希共济失调的治疗性基因编辑
- 批准号:
10668768 - 财政年份:2023
- 资助金额:
$ 77.71万 - 项目类别:
Base editing and prime editing for sickle cell disease
镰状细胞病的碱基编辑和引物编辑
- 批准号:
10157511 - 财政年份:2021
- 资助金额:
$ 77.71万 - 项目类别:
Continuous Evolution of Proteins with Novel Therapeutic Potential
具有新治疗潜力的蛋白质的不断进化
- 批准号:
10181559 - 财政年份:2021
- 资助金额:
$ 77.71万 - 项目类别:
Base editing and prime editing for sickle cell disease
镰状细胞病的碱基编辑和引物编辑
- 批准号:
10323054 - 财政年份:2021
- 资助金额:
$ 77.71万 - 项目类别:
Continuous Evolution of Proteins with Novel Therapeutic Potential
具有新治疗潜力的蛋白质的不断进化
- 批准号:
10588186 - 财政年份:2021
- 资助金额:
$ 77.71万 - 项目类别:
Continuous Evolution of Proteins with Novel Therapeutic Potential
具有新治疗潜力的蛋白质的不断进化
- 批准号:
10393666 - 财政年份:2021
- 资助金额:
$ 77.71万 - 项目类别:
PedGeneRx - Admin Supplement to Base Editing and Prime Editing for Sickle Cell Disease R01
PedGeneRx - 镰状细胞病 R01 碱基编辑和 Prime 编辑的管理补充
- 批准号:
10594247 - 财政年份:2021
- 资助金额:
$ 77.71万 - 项目类别:
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