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

描述(申请人提供)：在美国，每年有超过13,000名儿童被诊断为I型糖尿病(T1D)，即胰岛自身免疫性破坏导致内源性胰岛素分泌丧失。目前对这些患者的治疗标准是胰岛素疗法，这种疗法可以恢复血糖稳态，旨在预防与高血糖相关的并发症。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，如果在诊断出高血糖时重新建立免疫耐受，受损的胰岛可以再生。我建议研究利用基因治疗载体制造治疗性细胞来治疗T1 D的方法。我测试了表达几种不同转基因的腺病毒载体(包括FasL、IDO、IL-10以及一种可溶性和膜结合形式的IL-4(SLL-4和mblL-4))感染的DC在静脉过继转移一百万细胞后延缓10-12周龄雌性NOD小鼠糖尿病发病的能力。我同样筛选了1微克的外切体，这是由这些修饰的DC产生的小的膜结合囊泡。我的初步结果表明，表达sIL-4的DC能够完全预防疾病的发生，而单独治疗表达mbll-4的外切体可将糖尿病的发病延迟约5周。本研究的目的是利用表达sIL-4或mblL-4的腺病毒载体优化DC和DC来源的外切体的治疗潜力，并了解这些DC和DC来源的外切体发挥治疗作用的机制。为此，我建议优化这些初步结果，通过过继转移DC来源的外切体或亲本DC来治疗12周大的NOD/ShiLTJ小鼠，这些DC来源的外切体或亲本DC已被修饰为表达sIL-4或mbll-4。我会先确定最有效的治疗方案，然后再确定最有效的剂量。在优化DC和DC来源的外切体预防糖尿病发病的剂量后，我将确定这个治疗方案在逆转已确立的T1 D方面是否有效。I假设过继转移IL-4转导的DC或其外切体通过调节内源性T细胞亚群的功能来保护T1D免受T1D的影响。由于IL-4似乎是有效的，我将首先检查我们的DC和Exosome疗法对NOD小鼠Th1/Th2平衡的影响，然后将进行过继转移研究到NOD/SCID小鼠以及FACS和ELISPOT，看看这些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.