Gene Therapy for Diabetes

项目摘要 存在两种替代1型糖尿病（T1 D）中丢失的β细胞的潜在方法。一是 移植来自同种异体胰腺供体或干细胞的新β细胞。第二种方法是 在T1 D患者中原位产生新的β细胞，而无需任何细胞移植。可以实现这一点 通过转录因子介导的与β细胞相关的内胚层细胞类型的重编程。基因治疗 载体用于递送重编程因子。 我们和其他人最近发现，有可能通过逆行导管治疗来纠正小鼠的糖尿病。 注射重编程载体。导管内递送具有递送高剂量基因的优点， 治疗载体局部，最大限度地减少全身副作用，并实现高局部浓度的 重编程因素此外，这种给药途径在人类中是容易可行的，如ERCP。 内窥镜逆行胰胆管造影术（endoscopic retrograde cholangiopancreatography）是临床胃肠病学中的常规程序。 啮齿动物的临床前研究表明，α细胞是重编程的主要目标，而 胰管也可转化为功能性β样细胞。 在这项提议中，我们将开发AAV载体，这些载体被优化用于重编程α细胞， 人和非人灵长类动物在导管内递送后的β-细胞命运。我们正在取得进展， 在我们目前的HIRN UC 4资助中，我们开发了能够转导 高效的人内分泌细胞。我们还进化出了顺式调节元件（克雷斯）， 将转基因表达限制在仅β细胞。 在目标1中，我们将产生新的AAV衣壳（变体），其在转导胰腺癌细胞中是高效的。 在非人灵长类动物体内逆行注射后的α细胞和导管细胞。高度创新的衣壳 将使用进化方法。在目标2中，我们将产生特异性指导转基因表达的克雷斯， 重编程目标，即α细胞。细胞类型特异性启动子将与microRNA组合 识别要素，以实现这一目标。最后，在Aim 3中，Aim 1和克雷斯产生的AAV衣壳 Aim 2中开发的两种病毒将结合起来，产生能够将重编程因子递送到 将在非人灵长类动物中评估α细胞及其重编程能力。 这项工作的成功执行将产生所需的临床前数据， 该方法具有在人类中临床应用的潜力。

PROJECT SUMMARY Two potential approaches exist for the replacement of the β-cells lost in type 1 diabetes (T1D). The first is to transplant new β-cells derived from either allogeneic pancreas donors or stem cells. The second approach is to generate new β-cells in situ in the T1D patient without any need for cell transplantation. This can be achieved by transcription factor mediated reprogramming of endodermal cell types related to β-cells. Gene therapy vectors are used to deliver the reprogramming factors. We and others have recently found that it is possible to correct diabetes in mice by retrograde ductal injection of reprogramming vectors. Intraductal delivery has the advantage of delivering a high dose of gene therapy vector locally, minimizing systemic side effects and achieving a high local concentration of reprogramming factors. Furthermore, this route of administration is readily feasible in humans, as ERCP (endoscopic retrograde cholangio-pancreatography) is a routine procedure in clinical gastroenterology. Preclinical work in rodents indicates that α-cells are the prime target for reprogramming, while pancreatic ducts may also be converted to functional β-like cells. In this proposal, we will develop AAV vectors that are optimized for reprogramming the α-cells of humans and non-human primates to the β-cell fate after intraductal delivery. We are building on the progress made in our current HIRN UC4 grant, in which we developed novel AAV capsids capable of transducing human endocrine cells with high efficiency. We also evolved cis-regulatory elements (CREs) capable of restricting transgene expression to only β-cells. In Aim 1, we will produce novel AAV capsids (variants) that are highly efficient in transducing pancreatic α-cells and duct cells after retrograde injection in non-human primates in vivo. Highly innovative capsid evolution methods will be used. In Aim 2, we will generate CREs that direct transgene expression specifically to the reprogramming target, i.e. α-cells. Cell-type specific promoters will be combined with microRNA recognition elements to achieve this goal. Finally, in Aim 3, AAV capsids generated by Aim 1 and CREs developed in Aim 2 will be combined to produce optimized AAV capable of delivering reprogramming factors to α-cells and its capability of reprogramming will be assessed in non-human primates. Successful execution of this work will generate the preclinical data needed to determine whether this approach has potential for clinical application in humans.