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新的具有功能的β细胞，这些细胞不受自身免疫的攻击，从而避免任何免疫抑制的需要。大量的新近研究表明，α-细胞是内源性转分化为β-细胞的潜在来源。在这里，我们描述了一种小鼠胰腺导管内病毒传递系统，其中携带PDX1/MafA表达载体的腺相关病毒(AAV)单次输注给毒素诱导的(四氧嘧啶)糖尿病小鼠。这种基因疗法通过新生诱导α细胞向β细胞样细胞转化，在4周内恢复了60%以上的β细胞质量，并伴有持续、持久的正常血糖。巧合的是，当在自身免疫性NOD小鼠模型中类似地诱导这种β细胞样细胞新生时，小鼠在没有任何额外治疗或免疫抑制的情况下，在4个月或更长时间内变得正常血糖。据我们所知，在NOD小鼠中，没有一种临床适用的β细胞替代疗法在没有免疫抑制的情况下是成功的。我们推测，新生的β细胞样细胞可能不会受到自身免疫的攻击，因为通过RNAseq分析，它们是“不完善的”β细胞。由于胰管注射在人类中是一种相对简单的非手术程序，而且目前许多针对几种疾病的病毒基因治疗试验正在进行，我们认为我们的方法可能很快就可以移植到患有1型糖尿病的人类身上。在这项提案中，我们将在非人类灵长类动物(NHP)中进行重要的原则证明研究，作为准备人类基因治疗临床试验的最后一步。灵长类胰腺的质地和一致性与小鼠胰腺非常不同(与人类胰腺非常相似)。因此，病毒传播的机制可能需要进行重大改变。我们在NHP的最初有严重低血糖的问题。我们现在已经在糖尿病NHP模型中解决了这个问题，并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的分析，包括生理、基因表达表型和解剖学。总而言之，我们认为，如果这些拟议的研究取得成功，我们应该为1型糖尿病患者的临床试验做好准备。

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.