Alpha cell conversion to beta cells in non-human primates

治疗或治愈1型糖尿病的理想解决方案是从患者自身组织中形成不受自身免疫攻击的新的功能性β细胞，从而避免对任何免疫抑制的需要。最近大量的数据表明，α细胞是内源性转分化为β细胞的可行的潜在来源。在这里，我们描述了一种胰腺导管内病毒递送系统的小鼠，其中一个单一的输注腺相关病毒（AAV），携带pdx 1/mafA表达载体，给予毒素诱导的（四氧嘧啶）糖尿病小鼠。这种AAV基因疗法通过新生诱导了稳健和持久的α细胞转分化为β细胞样细胞，在4周内恢复了超过60%的β细胞质量，并且具有持续的、持久的真核细胞。偶然地，当在自身免疫NOD小鼠模型中类似地诱导这种β细胞样细胞新生时，小鼠变得血糖正常持续4个月或更长时间，而没有任何额外的治疗或免疫抑制。据我们所知，在没有免疫抑制的情况下，NOD小鼠中没有临床上适用的β细胞替代疗法是成功的。我们怀疑新生β细胞样细胞可能不受自身免疫攻击，因为它们通过RNA-seq分析是“不完美的”β细胞。由于胰管注射作为一种相对简单的非手术程序在人类中常规进行，并且由于许多病毒基因治疗试验目前正在进行中，因此我们认为我们的方法可以快速转化为1型糖尿病患者。在这项提案中，我们将在非人类灵长类动物（NHP）中进行重要的原理验证研究，作为人类基因治疗临床试验准备的最后一步。灵长类动物胰腺的质地和稠度与小鼠胰腺非常不同（与人类胰腺非常相似）。因此，病毒递送的机制可能需要实质性的改变。我们在NHP中最初遇到了严重低血糖的问题。我们现在已经解决了这个问题，在糖尿病NHP模型中取得了一些初步成功。进一步的研究将在AAV中和抗体的背景下研究这种胰腺导管输注方法。我们还将对新的β细胞样细胞进行详细分析，包括生理学，基因表达表型和解剖学。总之，我们认为，拟议的研究，如果成功的话，我们应该在1型糖尿病患者的临床试验准备。

An ideal solution to the treatment or cure of type 1 diabetes mellitus would be the formation of new functioning β-cells from the patient’s own tissues that are not attacked by the autoimmunity, thereby avoiding the need for any immunosuppression. Abundant recent data have suggested that α-cells are a viable potential source for endogenous transdifferentiation into β-cells. Here, we describe a pancreatic intraductal viral delivery system in the mouse, wherein a single infusion of an adeno-associated virus (AAV), carrying a pdx1/mafA expression vector, is given to a toxin-induced (alloxan) diabetic mouse. This AAV gene therapy induced robust and durable α-cell transdifferentiation into β-cell-like cells through neogenesis, with recovery of over 60% of the β-cell mass within 4 weeks, and with persistent, durable euglycemia. Serendipitously, when this β-cell-like cell neogenesis was similarly induced in the autoimmune NOD mouse model, the mice became euglycemic for 4 months or more, without any additional therapy or immunosuppression. To our knowledge, no clinically applicable β-cell replacement therapy in NOD mice has been successful without immunosuppression. We suspect that the neogenic β-cell-like cells may not be attacked by the autoimmunity because they are “imperfect” β-cells by RNA-seq analysis. Since pancreatic duct injection is routinely performed in humans as a relatively simple, non-surgical procedure, and since numerous viral gene therapy trials are currently ongoing for several diseases, we feel that our approach may be rapidly translatable to humans with type 1 diabetes mellitus. In this proposal, we will perform important proof-of-principle studies in non-human primates (NHP’s) as last steps in preparation for human gene therapy clinical trials. The primate pancreas has a very different texture and consistency than the mouse pancreas (and is very similar to the human pancreas). Thus, the mechanics of the viral delivery will likely require substantial alterations. We had initial problems in the NHP’s with severe hypoglycemia. We have now solved that problem with some preliminary success in a diabetic NHP model. Further studies will investigate this pancreatic ductal infusion approach in the context of AAV neutralizing antibodies. We will also perform detailed analyses of the new β-cell-like cells, including physiology, gene expression phenotype, and anatomy. In summary, we feel that the proposed studies, if successful, should position us well in preparation for clinical trials in humans with type 1 diabetes mellitus.