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：
10323054
负责人：
DAVID R LIU
金额：
$ 71.57万
依托单位：
ST. JUDE CHILDREN'S RESEARCH HOSPITAL
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：
https://reporter.nih.gov/project-details/10323054
关键词：
Abnormal Cell Acute Pain Adenosine Adult Affect Affinity Alanine Alleles Allogenic American Amino Acids Antisickling Agents Architecture Autologous Base Pairing Benign Biochemical Biological Assay Blood CD34 gene Caring Cell Death Cell Line Cell Therapy Cell physiology Cells Cessation of life Chromosomal Rearrangement Clinical Research Codon Nucleotides Complex DNA DNA Double Strand Break Deterioration Development Disease Elements Engineering Engraftment Enzymes Erythrocytes Erythroid Cells Erythroid Progenitor Cells Escherichia coli Evolution Fetal Hemoglobin Frequencies Functional disorder Future Gene Expression Gene Silencing Genes Genetic Genetic Diseases Genetic Engineering Genetic Template Genome Genomic DNA Genomics Globin Guide RNA HLA Antigens Hematological Disease Hematopoietic Hematopoietic Stem Cell Transplantation Hematopoietic stem cells Hemoglobin Hemoglobin F Disease Hemolytic Anemia Human Hypoxia In Vitro Individual Inherited Maintenance Malignant - descriptor Mediating Medical Messenger RNA Methods Missense Mutation Modification Morbidity - disease rate Multiple Organ Failure Mus Mutation Nucleotides Organ Outcome Pain Patients Proteins Quality of life RNA Reagent Recombinants Regulation Research Safety Sickle Cell Sickle Cell Anemia Sickle Cell Trait Sickle Hemoglobin Site Technology Testing Therapeutic Toxic effect Transcriptional Silencer Elements Transplantation Treatment Efficacy Valine Variant Xenograft procedure base base editing base editor beta Globin chronic pain curative treatments experience gamma Globin gene therapy genetic approach genetic information genetic manipulation genome editing hemoglobin polymer improved in vivo insight mortality mouse model mutant new technology novel novel strategies off-target mutation polymerization precision genetics premature prevent prime editing prime editor promoter repaired safety testing sickle erythroid sickling targeted treatment therapeutic target tool

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疾病（SCD）的医疗护理进展，大多数患者仍在经历严重疼痛，生活质量差，进行性器官劣化和过早死亡。同种异体造血茎细胞移植（HSCT）可以治愈SCD，但与许多毒性有关，只有20％的患者有人类白细胞抗原（HLA）匹配的供体。因此，改进并更广泛地访问需要治疗疗法。自体HSC的遗传修饰是一种有希望的实验方法为了治疗SCD，绕过一些与同种异体HSCT相关的问题，尽管最佳技术策略尚未建立。该建议探讨了腺苷基础编辑器（ABES）的使用和主要编辑（PES）用于SCD的遗传校正。与传统的基因组编辑相反，这些小说方法创造了与双链DNA断裂（DSB）无关的精确核苷酸改变，该核苷酸（DSB）会导致结构性DNA异常，细胞死亡或恶性转化。腺苷基础编辑器转换靶向A·T碱基对g·c对。 Prime Editor副本从指南RNA模板中编辑了序列信息进入靶向DNA基因座。我们将在SCD的3种不同策略中测试这些潜在的变革性工具治疗。目标1员工会创建HSC改变，以概括胎儿的遗传持久性血红蛋白（HPFH），一种良性的遗传疾病，可以通过诱导潜在的抗助剂剂的红细胞（RBC）胎儿血红蛋白（HBF）的表达。我们有使用的蛋白质进化策略来创建新的高效ABE，以产生HPFH突变 CD34+造血茎和祖细胞（HSPC）中高达60％的频率，HBF诱导到抑制红细胞后代缺氧性疾病的水平。 AIM 2使用ABS转换突变体SCD密码子从缬氨酸到丙氨酸，从而产生了“血红蛋白Makassar（HBG）”，这是一种天然存在的非良性非 - 令人讨厌的变体。我们已经开发了一个改变的PAM特异性ABE，将HBS等位基因转换为SCD中的HBG 供体HSPC的频率高达80％，并抑制了RBC的疾病。 AIM 3员工的主要编辑以恢复突变的SCD密码子至正常（Val→GLU），我们已证明在HEK293T细胞系中有效地发生现在旨在优化受影响个体的HSPC。总体而言，我们的初步研究显示了证据三种新颖的独立编辑方法来处理SCD的原理，而无需丰富编辑单元格或创建DSB。通过拟议的研究，我们试图优化这些方法的效率在初级HSPC中，并通过使用鼠标进一步确定其安全性和效率方法和生化测定。开发三种方法只会使我们能够比较他们的直接结局，并确定未来临床研究中追求的最佳治疗策略。更多的通常，我们计划的研究有可能生成新的范式，用于使用基本编辑和PES 通过精确的HSC遗传操纵来治疗众多遗传疾病。