Engineering pancreatic islets with TGβ protein to overcome rejection

描述(申请人提供)：1型糖尿病(T1D)仍然是超过0.9%的美国人口发病率和死亡率的主要来源，造成数十亿美元的经济负担。目前用于治疗T1D的两种方法是胰岛素治疗和同种异体胰岛/胰腺移植，这两种方法都有很大的局限性。胰岛素治疗并不能完全预防与糖尿病相关的并发症。同种异体胰岛/胰腺TX患有排斥反应，以及与长期使用免疫抑制药物相关的肾脏和胰岛毒性。因此，这项赠款提案的主要目标是建立一种新的免疫调节方案，以授予对同种异体胰岛移植的耐受性。 没有持续的免疫抑制。这将通过体外工程同种异体胰岛来实现，在其表面瞬时展示一种新型的转化生长因子1，SA-转化生长因子β蛋白，并使用工程的胰岛通过啮齿动物模型诱导耐受和治疗T1D。T1D和同种异体胰岛细胞的破坏主要是由T细胞针对独特的β细胞和Tx抗原介导的。因此，控制T致病细胞的功能对于诱导自身/同种异体抗原耐受和利用同种异体胰岛治疗T1D至关重要。在此，我们建议使用SA-转化生长因子作为免疫调节分子来实现这一目标。转化生长因子1具有多效性免疫功能，对自身抗原的耐受至关重要。通过各种免疫调节途径，转化生长因子在自身和同种异体抗原获得性耐受中也起着重要作用。最重要的是，利用转基因系统或病毒载体在胰岛异位表达转化生长因子？在人T1D NOD小鼠模型中有效地诱导了对自身抗原的耐受。然而，这种分子的持续表达与大量纤维化和长期的β细胞衰竭有关。重要的是，使用可诱导的转基因系统在β细胞中瞬时表达转化生长因子，克服了其有害影响，而不损害其耐受功能。由于这种转基因方法不适用于临床胰岛TX，我们设想以临床适用的方式在胰岛表面以蛋白水平瞬时显示转化生长因子β可能达到耐受，而不会因持续表达而出现报道的并发症。在有限的初步研究中，我们证明了在化学诱导的胰岛TX模型中，在胰岛上瞬时显示SA-转化生长因子ç在克服排斥反应方面是有效的。在这些强有力的初步数据的基础上，我们在此建议使用SA-转化生长因子ü工程的同种异体胰岛作为一种免疫调节手段，在化学和自发性糖尿病小鼠模型中防止移植物排斥反应和治疗糖尿病。将进行一系列研究来描述耐受的相关机制。在胰岛上快速和瞬时显示SA-转化生长因子蛋白为自身免疫和TX领域的干预提供了一种全新的手段。这种方法具有所需的简单性、安全性和有效性，使其成为一种临床相关的替代方案，在包括T1D在内的广泛免疫基础疾病的治疗中，可能实现与基因疗法相同的目标。

DESCRIPTION (provided by applicant): Type 1 diabetes (T1D) continues to be a major source of morbidity and mortality in over 0.9% US population with an economic burden in billions of dollars. The two current methods used to treat T1D are insulin therapy and allogeneic islet/pancreas transplantation (Tx), both of which have major limitations. Insulin administration does not completely prevent the complications associated with diabetes. Allogeneic islet/pancreas Tx suffers from rejection as well as renal and islet toxicities associated with chronic use of immunosuppressive drugs. Therefore, the primary objective of this grant proposal is to establish a novel immunomodulatory protocol to confer tolerance to allogeneic islet grafts in the absence of continuous immunosuppression. This will be achieved by engineering allogeneic islets ex vivo to transiently display on their surface a novel form of TGFß1, SA-TGFß, protein and use the engineered islets to induce tolerance and treat T1D using rodent models. T1D and allogeneic islet cell destruction is primarily mediated by T cells directed at unique beta cell and Tx antigens. Controlling the function of T pathogenic cells is, therefore, critical to tolerance induction to auto/alloantigens and treatment of T1D using allogeneic islets. We herein propose to use SA-TGFß as an immunomodulatory molecule to achieve this goal. TGFß1 has pleiotropic immune functions and is critical for tolerance to self-antigen. TGFß also plays an important role in acquired tolerance to auto and alloantigens using various immunomodulatory approaches. Most importantly, ectopic expression of TGFß in pancreatic islets using transgenic systems or viral vectors proved effective in inducing tolerance to autoantigens in NOD mouse model of human T1D. However, continuous expression of this molecule is associated with massive fibrosis and long-term beta cell failure. Importantly, transient expression of TGFß in beta cells using inducible transgenic systems overcame its deleterious effects without compromising its tolerogenic function. Inasmuch as this transgenic approach is not applicable to clinical islet Tx, we envisioned that the transient display of TGFβ at protein level on the surface of islets in a clinically applicable manner may achieve tolerance without its reported complications arising from continuous expression. In limited preliminary studies, we demonstrated that the transient display of SA-TGFß on pancreatic islets was effective in overcoming rejection in a chemically induced islet Tx model. Building on these strong preliminary data, we herein propose to use SA-TGFß-engineered allogeneic islets as a means of immunomodulation to prevent graft rejection and treat diabetes in chemically and spontaneously diabetic mouse models. A series of studies will be conducted to delineate the implicated mechanisms of the tolerance. Rapid and transient display of SA-TGFß protein on pancreatic islets offers a whole new means of intervention in the areas of autoimmunity and Tx. This approach possesses the simplicity, safety, and efficacy required to make it a clinically relevant alternative that may accomplish the same goals as gene therapy in the treatment of a broad spectrum of immune-based diseases, including T1D.