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.

抽象的 酶病是酶功能的灾难，包括遗传缺陷或缺陷 特定酶。当前治疗酶病的方法不足，并且依赖 骨髓移植或寿命长酶替代疗法。酶替代疗法可以 每年成千上万美元，骨髓移植非常不稳定， 子集导致死亡形式移植与宿主疾病。另一种方法是 修改患者更可延展和可及的细胞，例如淋巴细胞，以表达野生型 损坏的酶的版本，并将这些细胞重新输入到患者中以产生缺乏 酶。最近，在人类T细胞的基因组工程上进行了大量工作， 主要用于癌症免疫疗法。但是，长寿命的T细胞的子集很大程度上是 代谢不活跃，不适合持续的蛋白质产生。相反，B细胞可以生成 大量受保护的抗体，并继续这样做多年，主要是由于活性 长寿命的浆细胞。已经证明这些浆细胞不仅是重新种子 通过记忆B细胞，但取而代之的是产生长寿抗体的结果 不增殖。 B细胞可以长期存在并固有地具有代谢活性的事实 为了产生大量蛋白质（即抗体），使我们假设这些细胞可能是 用于酶病的基因疗法的理想平台。这使我们调查了其他人是否有 试图使用靶向核能修改B细胞，令我们惊讶的是，我们发现零出版物 关于在原代人B细胞中使用任何靶向核酸酶。那我们进行了初步 使用CRISPR/CAS9系统诱导双链断裂（DSB）的研究，发现 我们可以以合理的效率进行基因编辑原代人B，而测量师核酸酶高达43％ 测定。我们还定性地证明，我们可以使用 DSB诱导增强了同源重组。在这里，我们建议：1）优化基因 使用CRISPR/CAS9系统编辑和传递到原代人B细胞，2）进行证明 - 使用基因编辑的B细胞来治疗酶病的概念研究。具体来说，我们将尝试 通过点头/scID/scID/IL2Rγ背景来处理I型粘多糖的小鼠模型 表达已知抗原特异性BCR的工程人B细胞的移植 转录与α-L-二维罗苷酶（IDUA）相关，随后特有的免疫接种 转基因BCR产生长浆细胞。