Alpha cell conversion to beta cells in non-human primates

非人灵长类动物中α细胞转化为β细胞

• 批准号: 10451657
• 负责人: GEORGE K. GITTES
• 金额: $ 64.15万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-09-30 至 2024-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10451657
• 关键词: Address; Alloxan; Alpha Cell; Anatomy; Animals; Autoimmune; Autoimmunity; Beta Cell; Biology; Blood Glucose; Cell physiology; Cells; Clinical; Clinical Trials; D Cells; Data; Dependovirus; Development; Diabetes Mellitus; Diabetic mouse; Disease; Duct (organ) structure; Electrons; Endocrine; Endoscopic Retrograde Cholangiopancreatography; Gene Delivery; Gene Expression; Gene Expression Profile; Gene therapy trial; Generations; Genes; Genetic; Glare; Glucagon; Histology; Human; Hyperplasia; Hypoglycemia; Immunosuppression; In Situ; In Vitro; Inbred NOD Mice; Individual; Infusion procedures; Injections; Insulin-Dependent Diabetes Mellitus; Intervention; Islet Cell; Islets of Langerhans Transplantation; Kidney; Location; Mechanics; Methods; Microscopic; Modeling; Monitor; Mus; Oral cavity; Pancreas; Pancreatic duct; Patients; Phenotype; Physiology; Population; Positioning Attribute; Preparation; Primates; Problem Solving; Procedures; Quality Control; Recovery; Recurrence; Replacement Therapy; Reporting; Serotyping; Signal Transduction; Single-Stranded DNA; Source; System; Texture; Therapeutic; Tissues; Translating; Viral; Viral Genes; Viral Vector; Virus; alpha Toxin; autoimmune pathogenesis; beta cell replacement; blood glucose regulation; capsule; cell replacement therapy; clinical application; diabetic; endoplasmic reticulum stress; euglycemia; experimental study; expression vector; gene therapy; gene therapy clinical trial; glucose metabolism; immunogenic; islet; mouse model; neutralizing antibody; non-diabetic; nonhuman primate; prevent; single-cell RNA sequencing; success; transcriptome sequencing; transdifferentiation; viral gene delivery

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.

治疗或治愈1型糖尿病的一种理想方法是从患者自身组织中形成新的功能β细胞，这些细胞不受自身免疫系统的攻击，从而避免了任何免疫抑制的需要。最近大量的研究表明，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。AAV基因治疗通过新生诱导α-细胞向β-细胞样细胞的转变，使α-细胞在4周内恢复60%以上的β-细胞质量，并产生持久的血糖。偶然的是，当这种β细胞样细胞新生在自身免疫性NOD小鼠模型中同样被诱导时，小鼠在没有任何额外治疗或免疫抑制的情况下，在4个月或更长时间内血糖正常。据我们所知，临床上适用于NOD小鼠的β细胞替代疗法在没有免疫抑制的情况下是成功的。通过RNA-seq分析，我们怀疑新生β细胞样细胞可能不会受到自身免疫的攻击，因为它们是“不完美的”β细胞。由于胰管注射是一种相对简单的非手术治疗方法，而且目前正在进行多种疾病的病毒基因治疗试验，我们认为我们的方法可能很快适用于1型糖尿病患者。在本提案中，我们将在非人类灵长类动物（NHP’s）中进行重要的原理验证研究，作为为人类基因治疗临床试验做准备的最后一步。灵长类动物的胰腺与老鼠的胰腺有着非常不同的质地和稠度（与人类的胰腺非常相似）。因此，病毒传递的机制可能需要进行实质性的改变。NHP患者最初出现严重低血糖的问题。我们现在已经解决了这个问题，并在糖尿病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的分析，包括生理学、基因表达表型和解剖学。总之，我们认为这些研究如果成功，将使我们为1型糖尿病患者的临床试验做好准备。