Novel cell therapy for sustained therapeutic protein delivery in vivo

一种能够在体内持续传递治疗性蛋白质的细胞疗法有可能对两个肾脏都有利 疾病及其并发症。在我们之前的资助周期中，我们使用以下工具开发和验证技术 转座子修饰的抗原特异性T细胞利用促红细胞生成素(EPO)体内传递治疗蛋白 作为一个模范系统。我们展示了小鼠促红细胞生成素的传递和慢性肾脏病贫血的治疗。 在小鼠中作为模型系统，我们展示了抗原特异性的人EPO的诱导性表达 T细胞的体外培养。我们建议将我们的学习扩展到以下几个方面，从而大大提高我们以前的拨款 体内人类T细胞和测试一种增强长期治疗酶的创新方法 由于α-半乳糖苷酶A(α-GAL A)缺失而导致的法布里病的分娩。α-GAL A-/-小鼠代表一种 人类Fabry病的动物模型，这种疾病是由于缺乏代谢脂肪所需的酶而引起的 包括肾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 α-Gal A在法布里病模型中的表达。我们将用促红细胞生成素将我们的小鼠研究扩展到α-GalA Fabry病模型采用抗原特异的小鼠T细胞和疫苗接种。我们将对人类T细胞进行基因改造 为了表达嵌合抗原受体(CAR)和荧光素酶或人α-Gal A，细胞将被注入 NOD/SCID/Fabry小鼠评估工程化抗原特异性T细胞移植、应答的能力 给疫苗接种，并在体内模型中短期表达α-GalA。尽管穿孔素在T细胞中很重要 为了清除恶性细胞，穿孔素途径也有助于清除表达抗原的细胞。 接种疫苗后。在目标2中，我们将测试T细胞中穿孔素的敲除，以此作为加强长期治疗的一种方式 抗原特异性T细胞的蛋白质传递。我们建议在抗原的背景下测试穿孔素的敲除- 以确定它是否会提高提供治疗性蛋白质的细胞的长期持久性。在AIM 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-GalA 法布里病。我们建议将表达人α-Gal A的优化的人CAR-T细胞 NOD/SCID/Fabry小鼠模型。我们将评估植入率、疫苗接种反应、α-GAL A活性和 组织中球状三糖神经酰胺的水平。拟议的研究将导致新的细胞疗法的发展。 对肾脏疾病及其并发症具有潜在的治疗作用，远远超出肾脏的范围。

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.