Novel cell therapy for sustained therapeutic protein delivery in vivo

能够在体内持续递送治疗性蛋白质的细胞疗法具有有益于肾脏和肾脏的潜力。 疾病及其并发症。在上一个资助周期中，我们使用 用于使用促红细胞生成素（EPO）体内递送治疗性蛋白质的转座子修饰的抗原特异性T细胞 作为一个模型系统。我们证明了小鼠EPO的递送和对慢性肾病贫血的治疗 在小鼠中作为模型系统，并且我们证明了从抗原特异性的人促红细胞生成素表达系统中诱导人促红细胞生成素表达。 体外T细胞。我们建议通过将我们的研究扩展到 体内环境中的人T细胞和测试增强长期治疗酶的创新方法 α-半乳糖苷酶A（α-gal A）缺失导致的法布里病。α-gal A -/-小鼠代表一种 人类法布里病的动物模型，其起因于缺乏代谢脂肪所需的酶， 包括肾脏疾病在内的全身性疾病。在目标1中，我们将测试转座子修饰的抗原特异性T细胞 用于在法布里病模型中表达α-gal A。我们将把EPO的小鼠研究扩展到α-gal A， 使用抗原特异性小鼠T细胞和疫苗接种的法布里病模型。我们将对人类T细胞进行基因改造 表达嵌合抗原受体（CAR）与荧光素酶或人α-galA一起沿着。细胞将被注入 NOD/SCID/Fabry小鼠以评估工程化的抗原特异性人T细胞植入、应答和免疫应答的能力。 接种，并在体内模型中短期表达α-gal A。虽然穿孔素在T细胞中很重要， 对于恶性细胞的清除，穿孔素途径也有助于抗原表达细胞的清除 接种疫苗后。在目标2中，我们将测试T细胞中的穿孔素敲除作为增强长期治疗的一种方式。 从抗原特异性T细胞的蛋白质递送。我们建议在抗原- 特异性T细胞，以确定它是否会促进长期持久的细胞提供治疗性蛋白质。在aim中 3，我们将在小鼠模型中测试转座子修饰的抗原特异性T细胞长期表达α-gal A Fabry病我们建议以一种新的方式递送表达人α-gal A的优化的人CAR-T细胞。 NOD/SCID/Fabry小鼠模型。我们将评估植入、疫苗接种应答、α-gal A活性和 组织中的神经酰胺三己糖苷水平。拟议的研究将导致新的细胞疗法的发展 治疗肾脏疾病及其并发症，并有可能产生远远超出肾脏的治疗影响。

A cell therapy capable of sustained therapeutic protein delivery in vivo has the potential to benefit both kidney disease and its complications. During our previous grant cycle, we developed and validated technology using transposon-modified antigen specific T cells for therapeutic protein delivery in vivo using erythropoietin (EPO) as a model system. We demonstrated delivery of murine EPO and therapy for anemia of chronic kidney disease in mice as a model system, and we demonstrated inducible human EPO expression from antigen-specific human T cells in vitro. We propose to significantly advance beyond our previous grant by extending our studies to human T cells in an in vivo context and testing an innovative approach to enhance long-term therapeutic enzyme delivery for Fabry disease that results from loss of α-galactosidase A (α-gal A). α-gal A -/- mice represent an animal model of human Fabry disease, which results from lack of an enzyme needed to metabolize fats leading systemic disease including kidney disease. In aim 1, we will test transposon-modified antigen-specific T cells for expression of α-gal A in a Fabry disease model. We will extend our mouse studies with EPO to α-gal A in a Fabry disease model using antigen-specific mouse T cells and vaccination. We will gene modify human T cells to express a chimeric antigen receptor (CAR) along with luciferase or human α-gal A. Cells will be infused into NOD/SCID/Fabry mice to evaluate the ability of engineered antigen-specific human T cells to engraft, respond to vaccination, and short-term expression α-gal A in an in vivo model. Although perforin is important in T cells for clearance of malignant cells, the perforin pathway also contributes to clearance of antigen expressing cells post vaccination. In aim 2, we will test perforin knockout in T cells as a way of enhancing long-term therapeutic protein delivery from antigen-specific T cells. We propose to test perforin knockout in the setting of antigen- specific T cells to determine if it will boost long-term persistence of cells delivering therapeutic proteins. In aim 3, we will test transposon-modified antigen-specific T cells for long-term expression of α-gal A in a mouse model of Fabry disease. We propose to deliver optimized human CAR-T cells expressing human α-gal A in a NOD/SCID/Fabry mouse model. We will evaluate for engraftment, vaccination response, α-gal A activity, and globotriaosylceramide levels in tissues. The proposed studies will lead to the development of new cell therapies for kidney disease and its complications and have the potential for therapeutic impact well beyond the kidney.