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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.

项目总结/文摘