Nonenzymatic Gene Editing in Treatment of Heredity Spherocytosis

非酶基因编辑治疗遗传性球形红细胞增多症

基本信息

批准号：
10305603
负责人：
PATRICK G GALLAGHER
金额：
$ 62.02万
依托单位：
YALE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2019
资助国家：
美国
起止时间：
2019-02-01 至 2023-11-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10305603
关键词：
Address Anemia Base Pairing Biological Assay Bone Marrow Bone Marrow Cells Cell Line Cells Chemicals Chemistry Clustered Regularly Interspaced Short Palindromic Repeats Communication Complex DNA DNA Modification Process Development Disease Effectiveness Encapsulated Engineered Gene Engraftment Ensure Erythrocyte Membrane Erythrocytes Erythroid Progenitor Cells Fetal Liver Formulation Frequencies Genes Genome engineering Genomics Goals Hematology Hematopoietic Hematopoietic Stem Cell Transplantation Hematopoietic stem cells Hemolytic Anemia Hereditary Disease Hereditary Spherocytosis Heredity Immune Inflammatory Inflammatory Response Inherited Injections Intravenous Laboratories Life Link Mediating Methods Modification Monitor Morphology Mus Mutation Nature Nucleotides Patients Penetration Peptide Nucleic Acids Phenotype Point Mutation Polymers Procedures Protocols documentation Publishing Reagent Reporting Sickle Cell Anemia Site Splenectomy Splenomegaly Techniques Testing Thalassemia Toxic effect Transfusion Transplantation Work alpha Spectrin base beta Globin beta Thalassemia biomaterial compatibility clinically relevant deep sequencing design disease phenotype gene correction gene therapy genotoxicity human model improved in vivo in vivo Model infancy innovation mouse model nanoparticle nanoparticle delivery next generation novel nuclease palliative peptide G success targeted delivery tool transcription activator-like effector nucleases treatment strategy zinc finger nuclease

项目摘要

Project Summary/Abstract Approximately a quarter of patients with hereditary spherocytosis, a common inherited anemia, suffer from alpha- spectrin linked recessive HS (rHS), the most severe form of the disease. rHS patients present in infancy with life-threatening hemolytic anemia, many are transfusion-dependent. In these patients, splenectomy is only palliative; the only cure is hematopoietic stem cell (HSC) transplant. Development of genome engineering has expanded the repertoire of potential strategies for treatment of inherited erythrocyte disorders. We have developed a non-nuclease based gene editing technique using biocompatible and biodegradable nanoparticles (NP) encapsulating chemically modified peptide nucleic acids (PNAs) and donor DNAs. Using this strategy, we have cured a murine model of beta-thalassemia using IV injections of NP-PNAs, achieving gene correction of 6% after a single treatment with unmodified PNA, establishing proof-of-principle and demonstrating feasibility of our approach. This technique avoids ex vivo manipulation and its associated challenges, as well as obviates most of the genotoxicity associated with nuclease-based methods. This project leverages a multi-PI collaborative effort to develop the NP-PNA approach for gene editing in sph mice, a spontaneous murine model of rHS due to a point mutation in the alpha-spectrin gene. It addresses the hypothesis that NP-PNAs and donor DNAs can be used to correct the alpha-spectrin mutation in an in vivo model of HS at clinically relevant frequencies sufficient to ameliorate the HS disease phenotype with minimal toxicity and extremely low genomic off-target effects. We will establish robust protocols for in vivo DNA modification in hematopoietic cells after simple IV administration of NP-PNAs, providing a facile, non-toxic strategy for treatment of HS without the need for complex transplantation procedures or ex vivo manipulation. The goal of aim one is to optimize triplex-forming PNAs for site-specific gene editing of the alpha-spectrin gene. Relevant studies include assays of gene editing, off target effects, and genotoxicity. The goal of aim two is to identify and develop nanoparticle formulations for systemic in vivo editing of the alpha-spectrin gene in hematopoietic stem and progenitor cells (HSPCs). Relevant studies include development and characterization of NPs with novel size and polymer composition to improve target delivery of PNAs after systemic administration to HSPCs as well as improving penetration of the bone marrow compartment. The goal of aim three is the establishment of robust gene editing protocols for in vivo modification of the alpha-spectrin gene in HSPCs in alpha-spectrin deficient sph/sph mice to ameliorate or cure the HS phenotype. Results will be monitored by detailed laboratory analyses of the HS phenotype, including functional analyses of the erythrocyte membrane. Gene editing efficiency in hematopoietic stem cells and genotoxicity will also be analyzed. These approaches are widely applicable not only to other inherited disorders of the erythrocyte, but also to many disorders arising in the hematopoietic stem cell. Many of the observations from this work will likely extend beyond the field of hematology.

项目摘要/摘要大约四分之一的遗传性球细胞增多症患者是一种常见的遗传性贫血，患有α- Spectrin连接的隐性HS（RHS），这是该疾病中最严重的形式。 RHS患者婴儿期威胁生命的溶血性贫血，许多是输血依赖的。在这些患者中，脾切除术仅是姑息唯一的疗法是造血干细胞（HSC）移植。基因组工程的发展具有扩大了遗传性红细胞疾病的潜在策略的曲目。我们有使用生物相容性和可生物降解的纳米颗粒开发了一种基于非核酶的基因编辑技术（NP）封装化学修饰的肽核酸（PNAS）和供体DNA。使用此策略，我们已经使用NP-PNA注射IV静脉注射β-丘脑的鼠模型，以实现基因校正单次治疗未修饰的PNA后6％，建立原则证明并证明了可行性我们的方法。该技术避免了离体操纵及其相关的挑战，并避免了大多数与基于核酸酶的方法相关的遗传毒性。该项目利用多PI合作努力开发用于SPH小鼠基因编辑的NP-PNA方法，SPH小鼠是由RHS的自发鼠模型 α-光谱基因中的点突变。它解决了以下假设：NP-PNA和供体DNA可以是用于在临床相关频率下校正HS体内模型中α-谱蛋白突变以最小的毒性和极低的基因组脱靶效应来改善HS疾病表型。我们将在简单静脉内给予造血细胞中的体内DNA修饰的强大方案 NP-PNA，为HS的治疗提供了一种易感的无毒策略，而无需复杂移植程序或离体操纵。 AIM ONE的目的是优化三型PNA α-谱蛋白基因的位点特异性基因编辑。相关研究包括基因编辑的测定，关闭目标作用和遗传毒性。目标二的目的是识别和开发用于系统性的纳米颗粒制剂造血茎和祖细胞（HSPC）中α-谱蛋白基因的体内编辑。相关研究包括具有新颖尺寸和聚合物组成的NP的开发和表征以改善目标全身给药后向HSPCs进行PNA并改善骨髓的渗透率车厢。目标三的目的是建立用于体内修饰的强大基因编辑方案 α-谱蛋白缺乏SPH/SPH小鼠中HSPC中α-谱蛋白基因的改善或治愈HS 表型。结果将通过对HS表型的详细实验室分析来监视结果，包括功能红细胞膜的分析。造血干细胞和遗传毒性的基因编辑效率将也可以分析。这些方法不仅适用于红细胞的其他遗传疾病而且还出现了许多在造血干细胞中引起的疾病。这项工作的许多观察结果将可能延伸到血液学领域。