IL-4 Overexpressing Dendritic Cells and Exosomes for Treatment of Type 1 Diabetes

描述（由申请人提供）：每年，美国有超过13，000名儿童被诊断患有I型糖尿病（T1 D），其中胰岛的自身免疫破坏导致内源性胰岛素产生的损失。目前对这些患者的标准治疗是胰岛素治疗，其恢复葡萄糖稳态并旨在预防与高血糖症相关的并发症。然而，阻止胰岛自身免疫破坏的新疗法可能是有用的，因为已经证明，如果在诊断高血糖症时重建免疫耐受，受损的胰岛可以再生。我建议研究使用基因治疗载体制造治疗细胞以治疗T1 D的方法。我已经测试了用表达几种不同转基因（包括FasL、IDO、IL-10以及可溶性和膜结合形式的IL-4（sIL-4和mblL-4））的腺病毒载体感染的DC在静脉内过继转移一百万个细胞后延迟10-12周龄雌性NOD小鼠中糖尿病发作的能力。我同样筛选了1 μ g外泌体，它们是由这些修饰的DC产生的小的膜结合囊泡。我的初步结果表明，施用表达mIL-4的DC能够完全预防疾病发作，而表达mIL-4的外来体的单次治疗将糖尿病发作延迟约5周。本研究的目标是优化DC和DC衍生的外泌体的治疗潜力，所述外泌体已经使用表达sIL-4或mblL-4的腺病毒载体被工程化为致耐受性的，用于治疗T1 D，并理解这些DC和DC衍生的外泌体发挥其治疗效果的机制。为此，我建议优化这些初步结果，用DC衍生的外泌体或已被修饰以表达sIL-4或mbIL-4的亲本DC的过继转移治疗12周龄NOD/ShiLTJ小鼠。我将首先确定最有效的治疗方案，然后确定最有效的剂量。在优化DC和DC衍生的外泌体的剂量以预防糖尿病发作后，我将确定该治疗方案是否有效逆转已建立的TlD。我推测IL-4转导的DC或其外泌体的过继转移通过调节内源性T细胞亚群的功能来保护T1 D。由于IL-4似乎是有效的，我将首先检查我们的DC和外泌体疗法对NOD小鼠中Th 1/Th 2平衡的影响，然后将对NOD/Scid小鼠以及FACS和ELISPOT进行过继转移研究，以观察治疗是否能够产生调节性T细胞群。

DESCRIPTION (provided by applicant): Each year, more than 13,000 children in the United Stated are diagnosed with type I diabetes (T1D), in which autoimmune destruction of the pancreatic islets results in loss of endogenous insulin production. The current standard of care for these patients is insulin therapy, which restores glucose homeostasis and aims to prevent complications associated with hyperglycemia. However, new therapies to halt the autoimmune destruction of the islets could be useful because it has been demonstrated that the damaged islets can regenerate if immune tolerance is reestablished at the time of diagnosis of hyperglycemia. I propose to study methods using gene therapy vectors to make therapeutic cells in order to treat T1 D. I have tested DC infected with adenoviral vectors expressing several different transgenes, including FasL, IDO, IL-10, as well as a soluble and membrane-bound form of IL-4 (slL-4 and mblL-4), for their ability to delay onset of diabetes in 10-12 week old female NOD mice after intravenous adoptive transfer of a million cells. I have similarly screened 1 ug of exosomes, which are small membrane-bound vesicles, produced by these modified DC. My preliminary results indicate that administration of DC expressing slL-4 are capable of completely preventing disease onset, whereas a single treatment of exosomes expressing mbll_-4 delays diabetes onset by approximately 5 weeks. The goal of this research is to optimize the therapeutic potential of DC and DC-derived exosomes which have been engineered to be tolerogenic using adenoviral vectors expressing either slL-4 or mblL-4 for the treatment of T1 D, and to understand the mechanisms by which these DC and DC-derived exosomes exert their therapeutic effects. To this end, I propose to optimize these preliminary results, treating 12-week-old NOD/ShiLTJ mice with adoptive transfer of either DC-derived exosomes or parental DC that have been modified to express either slL-4 or mbll_-4. I will first determine the most effective treatment schedule, and then I will determine the most efficacious dose. After optimizing the dosing of DC and DC-derived exosomes for the prevention of diabetes onset, I will determine if this treatment schedule is effective at reversing established T1 D. I hypothesize that adoptive transfer of IL-4 transduced DC or their exosomes protects against T1D by modulating the function of endogenous T cell subsets. Since IL-4 seems to be effective I will first examine the effect of our DC and exosome therapies of Th1/Th2 balance in the NOD mouse, and then will conduct adoptive transfer studies into NOD/Scid mice as well as FACS and ELISPOT to see if the treatments are capable of generating a regulatory T cell population.