Correction of Mucopolysaccharidosis type 1: Targeting safe harbor loci using genome editing

纠正 1 型粘多糖贮积症：使用基因组编辑瞄准安全港位点

• 批准号: 10213146
• 负责人: Natalia Gomez-Ospina
• 金额: $ 19.62万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-07-15 至 2023-02-15
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10213146
• 关键词: 2 year old; Advisory Committees; Affect; Allogenic; Area; Astrocytes; Basic Science; Biochemical; Biochemistry; Bioinformatics; Biological Assay; Blood; Blood - brain barrier anatomy; Brain; CCR5 gene; CRISPR/Cas technology; Cell Lineage; Cell Transplantation; Cell physiology; Cells; Child; Childhood; Chromosomal Rearrangement; Clinical; Cognition; Data; Densitometry; Deterioration; Development; Disease; Disease Progression; Engraftment; Enzymes; Failure; Flow Cytometry; Generations; Genetic; Genetic Diseases; Genetic Engineering; Glycosaminoglycans; Goals; Hematopoietic; Hematopoietic Stem Cell Transplantation; Hematopoietic System; Hematopoietic stem cells; Homologous Transplantation; Human; Immunocompromised Host; Immunohistochemistry; In Vitro; Intervention; L-Iduronidase; Lead; Lysosomal Storage Diseases; Measurement; Measures; Mediating; Medical; Methodology; Methods; Modeling; Modification; Molecular Neurobiology; Morbidity - disease rate; Mucopolysaccharidoses; Mucopolysaccharidosis I; Mus; Musculoskeletal; Musculoskeletal System; Mutation; Neuraxis; Neurologic; Neurologic Symptoms; Organ; Patients; Phenotype; Physicians; Reagent; Risk; Roentgen Rays; Safety; Scientist; Screening procedure; Symptoms; System; Technology; Testing; Therapeutic; Training; Translational Research; Transplantation; Tumorigenicity; Validation; behavioral phenotyping; body system; bone; career; clinically relevant; design; effective therapy; enzyme activity; enzyme replacement therapy; genome editing; genome-wide; improved; in vivo; innovation; man; model design; mortality; mouse model; nervous system disorder; neurobehavioral; neuropathology; novel strategies; off-target site; post-transplant; predictive tools; preservation; progenitor; skeletal; stem; stem cell biology; stem cells; therapeutic protein

Abstract Mucopolysaccharidosis type 1 (MPSI) is one of approximately 50 genetic disorders collectively known as lysosomal storage diseases (LSDs). Like many LSDs, MPSI presents with progressive neurologic and systemic manifestations, and like most LSDs it lacks effective treatments. Current interventions for MPSI include allogeneic hematopoietic stem cell transplantation (allo-HSCT) and enzyme replacement therapy (ERT). Both can slow the progression of the disease, but their therapeutic impact is limited, particularly on the neurologic and musculoskeletal systems. I propose a novel approach that overcomes the limitations of ERT and allo-HSCT by genetically engineering the patient’s own hematopoietic system to express high levels of the missing enzyme (IDUA). Specifically, CRISPR/Cas9 will be used to introduce IDUA into the CCR5 safe harbor locus in human hematopoietic stem and progenitor cells (HSCPs). A safe harbor approach can achieve high and sustained levels of enzyme expression, and is an adaptable platform for other lysosomal enzymes. The feasibility of this approach is supported by the preliminary data, describing an efficient method to target IDUA to the CCR5 locus in HSPCs. I show that the targeted cells secrete high levels of enzyme, differentiate into multiple hematopoietic lineages, and are capable of short-term engraftment in immunocompromised mice. The specific aims are designed to ascertain the potential of these cells to safely and effectively alleviate the MPSI symptoms. This will be tested in our newly established MPSI mouse model, designed for human cell engraftment by 1) measuring biochemical and multi-systemic phenotype improvement after transplantation, 2) establishing the stem cell potential and multi-lineage differentiation capacity of the modified cells, and 3) unbiasedly searching for off-target sites of our CRISPR/Cas9 methodology. Successful completion of the proposed studies will have strong impact on the management of MPSI, and establish a new paradigm for delivering therapeutic proteins for the treatment of non-hematological and neurological diseases, including other LSDs. My ultimate goal is to become an independent physician-scientist who helps advance our treatments for diseases, like the LSDs, with unmet clinical needs. My clinical training as a medical geneticist and my basic science background in genetics, biochemistry, and molecular neurobiology, make me ideally suited to contribute in the area. The training plan outlined in this application will permit me to develop additional needed expertise, including: 1) genome-editing technologies, 2) hematopoietic stem cell biology and transplantation, 3) bioinformatics, 4) neuropathology, and 5) the practice of translational research. With this additional training, and career and scientific guidance from my advisory committee, I believe I can accomplish my goal.

抽象的 1型粘二糖（MPSI）是大约50种遗传疾病之一，共同被称为 溶酶体储存疾病（LSD）。像许多LSD一样，MPSI呈现具有渐进性神经系统的 系统性表现，像大多数LSD一样，它缺乏有效的治疗方法。 MPSI的当前干预措施 包括同种异体造血干细胞移植（Allo-HSCT）和酶替代疗法 （ERT）。两者都可以减慢疾病的进展，但是它们的治疗影响受到限制，尤其是在 神经和肌肉骨骼系统。我提出了一种克服ERT局限性的新颖方法 和Allo-HSCT通过基因工程进行患者自己的造血系统，以表达高水平 缺少酶（IDUA）。特别是，CRISPR/CAS9将用于将IDUA引入CCR5安全港 人造血干和祖细胞（HSCP）中的基因座。安全港的方法可以达到高度 酶表达的持续水平，并且是其他溶酶体酶的适应性平台。 这种方法的可行性由初步数据支持，描述了一种有效的靶向IDUA的方法 到HSPC中的CCR5基因座。我表明，靶向细胞秘密高水平的酶分化为 多个造血谱系，能够在免疫功能低下的小鼠中短期植入。 具体目的旨在确定这些细胞安全有效地减轻MPSI的潜力 症状。这将在我们新建立的MPSI鼠标模型中进行测试，该模型是为人类细胞设计的 1）植入移植后测量生化和多系统的表型改善，2） 建立修饰细胞的干细胞电势和多条形分化能力，3） 公正地寻找我们CRISPR/CAS9方法的脱离目标。成功完成 拟议的研究将对MPSI的管理产生强大的影响，并为 提供治疗蛋白，用于治疗非血液学和神经系统疾病，包括 其他LSD。 我的最终目标是成为一个独立的身体科学家，以帮助我们促进我们的治疗 像LSD一样，有未满足的临床需求的疾病。我作为医学遗传学家和基本的临床培训 遗传学，生物化学和分子神经生物学的科学背景，使我非常适合 在该地区贡献。本申请中概述的培训计划将使我能够开发其他需要的 专业知识，包括：1）基因组编辑技术，2）造血干细胞生物学和移植，3） 生物信息学，4）神经病理学和5）转化研究实践。在此额外的培训中 以及我的咨询委员会的职业和科学指导，我相信我可以实现自己的目标。