Optimizing Gene Editing in Primary Human B Cells for Therapy and Research

优化人类原代 B 细胞中的基因编辑以用于治疗和研究

• 批准号: 9224508
• 负责人: Branden S Moriarity
• 金额: $ 22.88万
• 依托单位: UNIVERSITY OF MINNESOTA
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-01-01 至 2018-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9224508
• 关键词: Animal Model; Antibodies; Antibody Formation; Antibody-Producing Cells; Antigens; B cell differentiation; B-Cell Activation; B-Cell Development; B-Lymphocytes; BCL6 gene; Back; Biological Assay; Blood Cells; Bone Marrow Transplantation; CD19 gene; CD34 gene; CRISPR/Cas technology; Cell Line; Cell model; Cells; Cessation of life; Chemicals; Communicable Diseases; Complementary DNA; Complex; DNA; Defect; Development; Disease; Electroporation; Engineering; Engraftment; Enzymes; Formulation; Future; Gene Delivery; Gene Targeting; Genes; Genetic; Genetic Transcription; Genome engineering; Guide RNA; Health; Hematopoietic Stem Cell Transplantation; Hematopoietic stem cells; Hereditary Disease; Human; Human Biology; Human Engineering; Immunization; Immunize; Immunotherapy; In Situ; Knock-out; L-Iduronidase; Lead; Length; Lentivirus Vector; Life; Ligands; Ligation; Light; Link; Lymphocyte; Medical; Memory B-Lymphocyte; Messenger RNA; Metabolic; Methodology; Methods; Morbidity - disease rate; Mucopolysaccharidosis I; Mus; NUP214 gene; Nonhomologous DNA End Joining; Oligonucleotides; PRDM1 gene; Pathology; Patients; Phycoerythrin; Physiologic pulse; Plasma Cells; Plasmid Cloning Vector; Production; Proteins; Publications; RNA; RPS27 gene; Reagent; Regimen; Review Literature; Risk; Site; Source; Specificity; System; T-Lymphocyte; T-Lymphocyte Subsets; Technology; Testing; Therapeutic; Therapeutic Studies; Toxic effect; Transformed Cell Line; Transgenes; Transplantation; Width; Work; base; betacell therapy; biological research; cancer immunotherapy; cell type; cost; cost effective; cytokine; differentiated B cell; enzyme deficiency; enzyme replacement therapy; exhaustion; gene therapy; genome editing; graft vs host disease; homologous recombination; human disease; immunogenic; insight; mouse model; novel therapeutics; nuclease; preconditioning; public health relevance; small molecule; stem; success

ABSTRACT Enzymopathies are a disturbance of enzyme function, including genetic deficiency or defect in specific enzymes. Current methods for the treatment of enzymopathies are insufficient and rely on bone marrow transplant or life long enzyme replacement therapy. Enzyme replacement therapies can cost hundreds of thousands of dollars per year and bone marrow transplant are highly precarious, with a subset resulting in death form graft versus host disease. An alternative approach would be to modify a patients more malleable and accessible cells, such as lymphocytes, to express a wild type version of the corrupted enzyme and re-infuse these cells into the patient to produce the lacking enzyme. Recently, there has been a great amount of work on genome engineering of human T cells, largely for cancer immunotherapies. However, the subsets of T cells that are long-lived are largely metabolically inactive and not ideal for constant protein production. Conversely, B cells can generate large amounts of protective antibodies and continue to do so for years, largely due to the activity of long-lived plasma cells. It has been demonstrated that these plasma cells are not merely re-seeded by memory B cells but instead are the result of becoming long-lived antibody producing cells that do not proliferate. The fact that B cells can become long lived and inherently have the metabolic activity to generate large quantities of protein (i.e. antibody) led us to hypothesize that these cells might be an ideal platform for gene therapy for enzymopathies. This led us to investigate if others had attempted to modify B cells using targeted nucleases and to our surprise we found zero publications on the use of any targeted nuclease in primary human B cells. Thus, we performed preliminary studies using the CRISPR/Cas9 system to induce double strand breaks (DSBs) in B cells and found that we can gene edit primary human B with reasonable efficiencies, up to 43% by Surveyor nuclease assay. We have also qualitatively demonstrated that we can deliver genes to B cells using homologous recombination enhanced by DSB induction. Here, we propose to: 1) Optimize gene editing and delivery to primary human B cells using the CRISPR/Cas9 system, and 2) Perform proof- of-concept studies to treat the enzymopathies using gene edited B cells. Specifically, we will attempt to treat a mouse model of Mucopolysaccharidosis type I on a NOD/SCID/Il2rγ background by transplantation of engineered human B cells expressing a BCR of known antigen specificity transcriptionally linked to Alpha-L-iduronidase (IDUA) with subsequent immunization specific to the transgene BCR to generate long lived plasma cells.

摘要