Enhancing immune regulation in gene therapy for hemophilia

项目摘要/摘要 首次显示，腺相关病毒(AAV)基因转移是一种很有前途的血友病治疗方法。 长期全身凝血FIX表达的I/II期临床试验取得成功 血友病B。然而，CD8+T细胞对输入病毒衣壳抗原的反应已经成为一种重要的 障碍。此外，血友病的治疗通常有抑制抗体(抑制物)形成的风险。 对抗治疗性凝血因子。而我们的临床前数据表明，肝脏AAV基因能够 转移诱导免疫耐受修复，向更具免疫原性的FVIII基因迈进 血友病A的治疗仍然是一个挑战。载体免疫排斥反应的复杂问题 转基因产品需要多管齐下的协同战略，旨在减少 载体的免疫原性和免疫调节通路的激活 抑制仍可能发生的获得性免疫反应，最终实现长期 免疫耐受性，从而确保持续治疗。这项提议的重点是发展 抑制调节性T细胞(Treg)不需要的免疫反应的策略和方案，以及 创造增强免疫调节的条件，包括Treg的诱导、扩增和迁移。 在此之前，我们已经证实，肝细胞来源的转基因可以诱导CD4+CD25+FoxP3+Treg 在肝脏导向的基因转移中，表达对于转基因产物的耐受性至关重要。在这里，我们将 继续利用定义Treg和树突状细胞之间相互作用的机制来诱导和 展开FoxP3+Treg和Alternative Treg(CD4+CD25-LAP+和TR1细胞)。通过改进归纳和 Treg的扩增及其迁移能力，我们使这些细胞能够以最佳方式控制不需要的免疫 对载体和转基因产物的反应。拟议的实验分为以下几个具体目标： 目的1：剖析FoxP3+Treg、TR1或LAP+的扩张和迁移到肝脏的机制 受体GITR、CX3CR1和VEGFR的配体可以触发Treg细胞。 目的2：验证血浆细胞样细胞和CX3CR1+树突状细胞增强功能的假说 血友病肝脏AAV基因转移过程中Treg细胞在肝脏中的扩增和迁移。 目标3：基于不同子集的替代和协同使用来开发免疫耐受协议 Treg细胞：Foxp3+Treg、TR1和/或LAP+Treg细胞。 目的4：建立体内系统，研究免疫反应和对肝脏基因的调节 与人肝细胞和免疫细胞转移。

Project Summary / Abstract Adeno-associated viral (AAV) gene transfer represents a promising treatment for hemophilia, showing first success in Phase I/II clinical trials with long-term systemic coagulation FIX expression in patients with hemophilia B. However, CD8+ T cell responses to input viral capsid antigen have emerged as a substantial hurdle. In addition, treatment of hemophilia generally bears a risk for inhibitory antibody (inhibitor) formation against the therapeutic clotting factor. While our pre-clinical data have shown the ability of hepatic AAV gene transfer to induce immune tolerance to FIX, moving forward toward the more immunogenic FVIII in gene therapy for hemophilia A remains a challenge. The complex problems of immune rejection of vector or transgene product require a multi-pronged approach of synergistic strategies aimed at reducing immunogenicity of the vector and at activation of immune regulatory pathways that serve as a “backup mechanism” to suppress adaptive immune responses that may still occur, ultimately accomplishing long-term immune tolerance and thereby assuring sustained therapy. The focus of this proposal is the development of strategies and protocols for suppression of unwanted immune responses by regulatory T cells (Treg), and to create conditions that enhance immune regulation, including induction, expansion, and migration of Treg. Previously, we established that induction of CD4+CD25+FoxP3+ Treg by hepatocyte-derived transgene expression is crucial for tolerance to the transgene product upon liver-directed gene transfer. Here, we will continue to exploit the mechanisms that define the interplay between Treg and dendritic cells to induce and expand FoxP3+ Treg and alternative Treg (CD4+CD25-LAP+ and Tr1 cells). By improving induction and expansion of Treg, and their ability to migrate, we empower these cells to optimally control unwanted immune responses to vector and transgene product. Proposed experiments are grouped in the following specific aims: Aim 1: Dissect the mechanisms by which expansion and migration to the liver of FoxP3+ Treg, Tr1, or LAP+ Treg cells can be triggered by ligands of the receptors GITR, CX3CR1 and VEGFR. Aim 2: Test the hypothesis that empowering by plasmacytoid and CX3CR1+ dendritic cells promotes expansion and migration of Treg cells in the liver in hepatic AAV gene transfer for hemophilia. Aim 3: Develop immune tolerance protocols based on the alternative and synergistic use of distinct subsets of Treg cells: FoxP3+ Treg, Tr1 and/or LAP+ Treg cells. Aim 4: Develop an in vivo system that enables studies of immune responses and regulation to liver gene transfer with human hepatocytes and immune cells.