Gene Therapy for Diabetes

项目总结 有两种潜在的方法可以替代1型糖尿病(T1D)中丢失的β细胞。第一个是 移植来自异基因胰腺捐赠者或干细胞的新β细胞。第二种方法是 在T1D患者体内原位生成新的β-细胞，而不需要任何细胞移植。这是可以实现的 通过转录因子介导的与β-细胞相关的内皮细胞类型的重编程。基因治疗 向量被用来传递重新编程因子。 我们和其他人最近发现，通过逆行导管可以纠正小鼠的糖尿病 重新编程向量的注入。导管内给药的优点是可以输送大剂量的基因。 局部治疗载体，最大限度地减少全身副作用，并实现较高的局部浓度 重新编程因素。此外，这种给药途径在人类身上是容易实现的，如ercp。 内窥镜逆行胰胆管造影术是临床胃肠病学的常规检查方法。 对啮齿动物的临床前研究表明，α细胞是重新编程的主要目标，而 胰管也可能转化为功能性的β样细胞。 在这项建议中，我们将开发针对α细胞的重新编程而优化的aav载体。 人类和非人类灵长类动物在导管内分娩后β细胞的命运。我们正在取得更大的进展 在我们目前的Hirn UC4拨款中，我们开发了能够传感的新型AAV衣壳 人的内分泌细胞效率高。我们还进化出顺式调节元件(CRE)，能够 将转基因表达仅限于β-细胞。 在目标1中，我们将生产高效转导胰腺的新型AAV衣壳(变体)。 非人灵长类体内逆行注射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，我们将生成指导特定转基因表达的Cres 至重编程目标，即α单元。细胞类型特异性启动子将与microRNA结合 实现这一目标的认可要素。最后，在AIM 3中，由AIM 1和Cres生成的AAV衣壳 在AIM 2中开发的AAV将组合在一起，以产生能够将重新编程因素传递到 α细胞及其重新编程的能力将在非人类灵长类动物中进行评估。 这项工作的成功执行将产生所需的临床前数据，以确定这是否 该方法具有在人类临床应用的潜力。

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.