Alpha cells conversion to beta cells in non-human primates

项目概要和相关性 治疗或治愈1型糖尿病的理想解决方案是形成新的功能性β细胞 从患者自身的未被自身免疫攻击的组织，从而避免了对任何免疫的需要。 免疫抑制最近大量的数据表明，α-细胞是内源性的一个可行的潜在来源， 转分化为β细胞。在此，我们描述了小鼠中的胰腺导管内病毒递送系统，其中， 将携带pdx 1/mafA表达载体的腺相关病毒（AAV）单次输注给予毒素诱导的 （四氧嘧啶）糖尿病小鼠。这种AAV基因治疗诱导了稳健和持久的α细胞转分化为β细胞样细胞， 细胞通过新生，在4周内恢复超过60%的β细胞群，并且具有持久的，持久的 真的。偶然地，当这种β细胞样细胞新生在自身免疫NOD小鼠中被类似地诱导时， 在模型中，小鼠在4个月或更长时间内血糖正常，没有任何额外的治疗或免疫抑制。对我们 根据现有知识，在NOD小鼠中没有临床上适用的β细胞替代疗法是成功的， 免疫抑制我们怀疑新生β细胞样细胞可能不受自身免疫的攻击，因为它们 通过RNA-seq分析是“不完美的”β细胞。由于胰管注射是作为一种常规方法在人体中进行的， 相对简单，非手术程序，因为许多病毒基因治疗试验目前正在进行的几个 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。在这一提议中， 我们将在非人类灵长类动物中进行重要的原理验证研究，作为人类基因工程的最后准备步骤。 治疗临床试验灵长类动物的胰腺与小鼠胰腺相比具有非常不同的质地和稠度（并且 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。在 此外，胰高血糖素启动子比CMV启动子更适合表达pdx 1和mafA，因此我们将努力 开发和优化在灵长类动物中有效的胰高血糖素启动子载体。进一步的研究将对此进行调查 在AAV中和抗体的情况下，使用胰管输注方法。我们还将进行详细的分析 新的β-细胞样细胞，包括生理学，基因表达表型和解剖学。总之，我们认为， 如果研究成功，我们将为1型糖尿病患者的临床试验做好准备 糖尿病。

PROJECT SUMMARY AND RELEVANCE 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 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. In addition, a glucagon promoter is preferable to the CMV promoter for expression of pdx1 and mafA, so we will strive to develop and optimize a glucagon promoter vector that is effective in primates. 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.