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

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：酶病是一种酶功能的紊乱，包括基因缺陷或酶的缺陷