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

摘要：酶病是一种酶功能紊乱，包括遗传缺陷或基因缺陷。 特定的酶。目前的治疗方法是不够的，依赖于造血干细胞移植 (HSCT)或终生酶替代疗法(ERT)。ERT每年可能花费数十万美元 而造血干细胞移植是高度不稳定的，有一种亚型会导致移植物抗宿主疾病或感染死亡。 是由长期的免疫抑制引起的。另一种方法是对患者进行更多修改 可延展性和可获得性的细胞，如淋巴细胞，以表达大量活性酶并重新注入 这些细胞进入患者体内，产生缺乏的酶。这种多余的酶可以从基因工程中排泄出来 细胞在体内并被内源性细胞摄取，这一过程被称为交叉校正。最近，有一个 在人类T细胞基因组工程方面的大量工作，通常用于癌症免疫治疗。然而， 长寿的T细胞亚群在新陈代谢方面不活跃，不适合持续的蛋白质生产。 相反，B细胞可以产生大量的保护性抗体，并在几年后继续这样做 转化为长寿的浆细胞。已经证明，这些浆细胞不仅仅是重新播种 通过记忆B细胞，而不是成为长寿抗体产生细胞的结果 扩散。B细胞可以活得很长并且天生就有代谢活动来产生 大量的蛋白质(即抗体)使我们假设这些细胞可能是基因的理想平台 内脏疾病的治疗。为了能够使用工程B细胞进行治疗，我们最近建立了一种使用 CRISPR/Cas9在原代人类B细胞中的基因敲入和敲除(Johnson et.美国科学代表2018年8月 14；8(1)：12144)。现在，我们将把这些方法应用于工程B细胞来治疗酶病和 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，以获得最优 治疗性转基因在人类B细胞中的表达和2)执行概念验证研究以使用 改造了人类B细胞来治疗酶病。具体地说，我们将处理一个小鼠模型 以NOD/SCID/IL2Rγ为背景的工程人移植的I型粘多糖病 B细胞。MPS I是由α-L艾杜糖醛酸酶缺乏引起的常染色体隐性遗传性溶酶体病 酶导致糖胺多聚糖储藏物质积聚和多系统疾病。受影响 个人患有肝脾肿大、角膜混浊、骨骼发育不良、心肺阻塞、 严重形式(赫勒综合征)的进行性神经损害。B细胞将被改造成 表达已知抗原特异性的BCR，转录上与IDUA连锁，随后免疫 在体内产生长寿的浆细胞。因此，在本R01申请中提议的研究构成了 利用工程B细胞治疗酶病的综合分析最终目的是治疗 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.