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: 10358566
• 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/10358566
• 关键词: Address; Affect; Animal Model; Animals; Antibodies; Antibody-Producing Cells; Antigens; Automobile Driving; B cell therapy; B-Cell Development; 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; 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; Immunosuppression; Impairment; In Vitro; Individual; Infection; Internet; 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; T-Lymphocyte; T-Lymphocyte Subsets; Technology; Testing; Therapeutic; Transplantation; Urine; Work; base; biological research; cancer cell; cancer immunotherapy; cell type; cellular engineering; cost; enzyme deficiency; enzyme replacement therapy; experimental study; expression vector; gene therapy; genome editing; graft vs host disease; in vivo; in vivo evaluation; insight; integration site; 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：酶病是酶功能的紊乱，包括遗传缺陷或酶缺陷。 特定的酶。目前治疗方法不足，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 造血干细胞移植非常不稳定，其中一部分会因移植物抗宿主病或感染而死亡 长期免疫抑制所致。另一种方法是对患者进行更多修改 可延展且可接近的细胞，例如淋巴细胞，表达大量活性酶并重新输注 这些细胞进入患者体内以产生缺乏的酶。这种多余的酶可以从工程化的产物中排出。 体内细胞并被内源细胞吸收，这一过程称为交叉校正。最近，有一个 人类 T 细胞基因组工程方面的大量工作，通常用于癌症免疫疗法。然而， 长寿的 T 细胞亚群代谢不活跃，不适合持续生产蛋白质。 相反，B 细胞可以产生大量的保护性抗体，并在感染后持续数年。 转化为长寿命浆细胞。已经证明这些浆细胞不仅仅是重新播种 是由记忆 B 细胞产生的，而是成为长寿命抗体产生细胞的结果，而这些细胞不会 增生。事实上，B 细胞可以长寿并且天生具有代谢活动来产生 大量的蛋白质（即抗体）使我们推测这些细胞可能是基因的理想平台 酶病的治疗。为了能够使用工程 B 细胞进行治疗，我们最近建立了使用 CRISPR/Cas9 在原代人 B 细胞中进行基因敲入和敲除（Johnson 等人，Sci Rep. 2018 年 8 月） 14；8(1)：12144)。现在，我们将应用这些方法来改造 B 细胞，以治疗酶病和 进行临床前测试。在这里，我们建议：1）优化表达载体和整合位点以获得最佳效果 治疗性转基因在人类 B 细胞中的表达，以及 2) 进行概念验证研究以使用 改造人类 B 细胞来治疗酶病。具体来说，我们将处理小鼠模型 NOD/SCID/Il2rγ 背景下的 I 型粘多糖贮积症 (MPS I)，通过移植工程人类 B细胞。 MPS I 是一种由 α-L-艾杜糖醛酸酶 (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物质的积累和多系统疾病。做作的 个体患有肝脾肿大、角膜混浊、骨骼发育不良、心肺阻塞、 以及严重的（Hurler 综合征）进行性神经功能障碍。 B细胞将被改造为 表达与 IDUA 转录连接的已知抗原特异性的 BCR，并随后进行免疫接种 在体内产生长寿的浆细胞。因此，本 R01 申请中提出的研究构成了 综合分析使用工程化 B 细胞治疗酶病，最终目标是治疗 人类的酶病。