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

摘要：酶病是一种酶功能紊乱，包括遗传缺陷或 特定的酶。目前的治疗方法不足，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 和HSCT是高度不稳定的，其中一个亚组导致移植物抗宿主病或感染死亡 长期免疫抑制导致的另一种方法是对患者进行更多的修改 可塑性和可接近的细胞，如淋巴细胞，表达大量的活性酶并重新注入 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 细胞在体内并被内源性细胞吸收，这一过程称为交叉校正。最近，有一个 人类T细胞基因组工程的大量工作，通常用于癌症免疫治疗。然而，在这方面， 长寿命的T细胞亚群在代谢上是无活性的，并且对于恒定的蛋白质产生不是理想的。 相反，B细胞可以产生大量的保护性抗体，并在感染后持续多年。 转化为长寿的浆细胞。已经证明，这些浆细胞不仅仅是重新接种 而是成为长寿的抗体B细胞的结果， 增殖。事实上，B细胞可以变得长寿，并且固有地具有代谢活性， 大量的蛋白质（即抗体）使我们假设这些细胞可能是基因表达的理想平台。 酶病的治疗。为了使工程改造的B细胞能够用于治疗，我们最近建立了使用 用于在原代人B细胞中基因敲入和敲除的CRISPR/Cas9（约翰逊（Johnson）et.例如，科学报告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，用于治疗酶病， 进行临床前试验。在这里，我们建议：1）优化表达载体和整合位点，以获得最佳的 治疗性转基因在人B细胞中的表达和2）进行概念验证研究以使用 工程改造的人类B细胞来治疗酶病。具体来说，我们将治疗一个小鼠模型， 在NOD/SCID/IL 2 r γ背景下通过移植工程化人 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。影响 个体患有肝脾肿大、角膜混浊、骨骼发育不良，心肺阻塞， 和严重形式（Hurler综合征）的进行性神经功能缺损。B细胞将被改造成 表达与IDUA转录连接的已知抗原特异性的BCR，随后免疫， 在体内产生长寿的浆细胞。因此，本R 01申请中提出的研究构成了 全面分析使用工程化B细胞治疗酶病，最终目标是治疗 人类的酶病

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.