Engineered B Cells as a Universal Platform for the Treatment of Enzymopathies

工程 B 细胞作为治疗酶病的通用平台

• 批准号: 10582595
• 负责人: Branden S Moriarity
• 金额: $ 38.75万
• 依托单位: UNIVERSITY OF MINNESOTA
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-03-01 至 2025-02-28
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10582595
• 关键词: Affect; Animals; Antibodies; Antibody-Producing Cells; Antigens; Automobile Driving; B cell therapy; B-Lymphocytes; Back; Blood Cells; Bone Marrow Transplantation; CD34 gene; CRISPR/Cas technology; Cardiopulmonary; Cell Line; Cell Therapy; Cell Transplantation; Cell model; Cells; Cessation of life; Complementary DNA; Cornea; DNA cassette; Defect; Development; Disease; Disease model; Effectiveness; Emu species; Engineering; Engraftment; Enhancers; Enzymes; Excretory function; Future; GAG Gene; Gene Delivery; Gene Expression; Genes; Genetic; Genetic Diseases; Genetic Transcription; Genome engineering; Glycosaminoglycans; Goals; Hematopoietic Stem Cell Transplantation; Hematopoietic stem cells; Hepatosplenomegaly; Human; Human Engineering; Immunization; Immunologic Deficiency Syndromes; Immunosuppression; Impairment; In Vitro; Individual; Infection; Knock-in; Knock-out; L-Iduronidase; Life; Link; Lymphocyte; Lysosomal Storage Diseases; Medical; Memory B-Lymphocyte; Metabolic; Methods; Modeling; Morbidity - disease rate; Mucopolysaccharidosis I; Mucopolysaccharidosis I H; Mus; National Institute of Allergy and Infectious Disease; Neurologic; Obstruction; Pathology; Patients; Phycoerythrin; Physicians; Plasma Cells; Pre-Clinical Model; Preclinical Testing; Process; Production; Proteins; Reagent; Recombinant adeno-associated virus (rAAV); Regimen; Research Personnel; Risk; Serotyping; Signal Transduction; Site; Specificity; System; Systemic disease; T-Lymphocyte; T-Lymphocyte Subsets; Technology; Therapeutic; Transplantation; Urine; Work; autosome; biological research; cancer immunotherapy; cell type; cellular engineering; cost; enzyme deficiency; enzyme replacement therapy; experimental study; expression vector; gene correction; gene therapy; genome editing; graft vs host disease; improved; in vivo; in vivo evaluation; insight; integration site; model organism; mouse model; novel therapeutics; overexpression; preconditioning; promoter; public health relevance; skeletal dysplasia; success; therapeutic transgene; transgene expression; treatment group; Affect; Animals; Antibodies; Antibody-Producing Cells; Antigens; Automobile Driving; B cell therapy; B-Lymphocytes; Back; Blood Cells; Bone Marrow Transplantation; CD34 gene; CRISPR/Cas technology; Cardiopulmonary; Cell Line; Cell Therapy; Cell Transplantation; Cell model; Cells; Cessation of life; Complementary DNA; Cornea; DNA cassette; Defect; Development; Disease; Disease model; Effectiveness; Emu species; Engineering; Engraftment; Enhancers; Enzymes; Excretory function; Future; GAG Gene; Gene Delivery; Gene Expression; Genes; Genetic; Genetic Diseases; Genetic Transcription; Genome engineering; Glycosaminoglycans; Goals; Hematopoietic Stem Cell Transplantation; Hematopoietic stem cells; Hepatosplenomegaly; Human; Human Engineering; Immunization; Immunologic Deficiency Syndromes; Immunosuppression; Impairment; In Vitro; Individual; Infection; Knock-in; Knock-out; L-Iduronidase; Life; Link; Lymphocyte; Lysosomal Storage Diseases; Medical; Memory B-Lymphocyte; Metabolic; Methods; Modeling; Morbidity - disease rate; Mucopolysaccharidosis I; Mucopolysaccharidosis I H; Mus; National Institute of Allergy and Infectious Disease; Neurologic; Obstruction; Pathology; Patients; Phycoerythrin; Physicians; Plasma Cells; Pre-Clinical Model; Preclinical Testing; Process; Production; Proteins; Reagent; Recombinant adeno-associated virus (rAAV); Regimen; Research Personnel; Risk; Serotyping; Signal Transduction; Site; Specificity; System; Systemic disease; T-Lymphocyte; T-Lymphocyte Subsets; Technology; Therapeutic; Transplantation; Urine; Work; autosome; biological research; cancer immunotherapy; cell type; cellular engineering; cost; enzyme deficiency; enzyme replacement therapy; experimental study; expression vector; gene correction; gene therapy; genome editing; graft vs host disease; improved; in vivo; in vivo evaluation; insight; integration site; model organism; mouse model; novel therapeutics; overexpression; preconditioning; promoter; public health relevance; skeletal dysplasia; success; therapeutic transgene; transgene expression; treatment group

ABSTRACT: Enzymopathies are a disturbance of enzyme function, including genetic deficiency or a defect in specific enzymes. Current treatment methods are insufficient and rely on hematopoietic stem cell transplant (HSCT) or lifelong enzyme replacement therapy (ERT). ERT can cost hundreds of thousands of dollars per year and HSCTs are highly precarious, with a subset resulting in death from graft versus host disease or infection brought on by prolonged immunosuppression. An alternative approach would be to modify a patients more malleable and accessible cells, such as lymphocytes, to express large quantities of active enzyme and re-infuse these cells into the patient to produce the lacking enzyme. This excess enzyme can be excreted from engineered cells in vivo and taken up by endogenous cells, a process termed cross correction. Recently, there has been a large amount of work on genome engineering of human T cells, typically for cancer immunotherapies. However, the subsets of T cells that are long-lived are 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 after conversion to 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 of enzymopathies. To enable the use of engineered B cells for therapy we recently established the use of CRISPR/Cas9 for gene knock-in and knockout in primary human B cells (Johnson et. al., Sci Rep. 2018 Aug 14;8(1):12144). Now, we will apply these approaches to engineer B cells for the treatment of enzymopathies and perform preclinical testing. Here, we propose to: 1) optimize expression vectors and integration sites for optimal expression of therapeutic transgenes in human B cells and 2) perform proof-of-concept studies to use engineered human B cells to treat enzymopathies. Specifically, we will treat a mouse model of mucopolysaccharidosis type I (MPS I) on a NOD/SCID/Il2rγ background by transplantation of engineered human B cells. MPS I is an autosomal recessive lysosomal disease caused by deficiency of alpha-L-iduronidase (IDUA) enzyme resulting in accumulation of glycosaminoglycan storage material and multi-systemic disease. Affected individuals suffer from hepatosplenomegaly, corneal clouding, skeletal dysplasias, cardiopulmonary obstruction, and in the severe form (Hurler syndrome) progressive neurologic impairment. B cells will be engineered to express a BCR of known antigen specificity transcriptionally linked to IDUA with subsequent immunization to generate long lived plasma cells in vivo. The studies proposed in this R01 application thus constitute a comprehensive analysis of the use of engineered B cells to treat enzymopathies with the ultimate goal of treating enzymopathies in humans.

摘要：酶病是酶功能的灾难，包括遗传缺陷或缺陷 特定酶。当前的治疗方法不足，依赖造血干细胞移植 （HSCT）或终身酶替代疗法（ERT）。 ERT每年可能花费数十万美元 HSCTS高度不稳定，子集导致移植物与宿主疾病或感染导致死亡 由长时间的免疫抑制购买。另一种方法是更多地修改患者 可延展且可及的细胞，例如淋巴细胞，以表达大量活性酶并重新输入 这些细胞进入患者以产生缺乏酶。可以超过工程的多余酶 细胞在体内并被内源性细胞收集，该过程称为交叉校正。最近，有一个 人类T细胞的基因组工程大量工作，通常用于癌症免疫疗法。然而， 长期寿命的T细胞的子集在代谢上是非活性的，对于持续的蛋白质产生而言并不理想。 相反，B细胞可以生成大量受保护的抗体，并在多年后继续这样做 转换为长寿命的浆细胞。已经证明这些浆细胞不仅是重新种子 通过记忆B细胞，而是成为长寿命抗体产生的细胞的结果 增生。 B细胞可以长期存在并固有地具有代谢活性以生成的事实 大量蛋白质（即抗体）使我们假设这些细胞可能是基因的理想平台 酶病的治疗。为了使工程B细胞用于治疗，我们最近建立了使用 crispr/cas9用于原代人B细胞中的基因敲入和敲除（Johnson等，SciRep。2018） 14; 8（1）：12144）。现在，我们将将这些方法应用于工程师B细胞，以治疗酶病和 执行临床前测试。在这里，我们建议：1）优化表达式向量和集成位点以达到最佳 人类B细胞中热转基因的表达和2）进行概念验证研究以使用 设计的人类B细胞以治疗酶病。具体来说，我们将处理鼠标模型 通过移植工程人的人类，在点头/scID/IL2Rγ背景上，I型粘二糖（MPS I） B细胞。 MPS I是由α-L-二维罗替酶（IDUA）缺乏引起的常染色体隐性溶酶体疾病 酶导致糖胺聚糖储存材料和多系统疾病的积累。做作的 患者患有肝肾上腺肿，角膜蒙阴影，骨骼发育不良，心肺阻塞， 并以严重的形式（Hurler综合征）进行性神经系统障碍。 B单元将设计为 表达与IDUA相关的已知抗原特异性的BCR，随后免疫到 在体内产生长期活的浆细胞。因此，在此R01应用中提出的研究构成了 全面分析工程B细胞治疗酶病的使用的最终目的 人类的酶病。