Modulation of Immune responses to gene therapy by Creation of AAV-specific Chimeric antigen receptor regulatory T cells (CAR-Tregs)

项目4-摘要 在项目4中，我们将使用我们的新型腺相关病毒(AAV)导向的嵌合抗原 受体(CAR)T细胞和T调节细胞(Tregs)调节和减轻免疫 AAV基因治疗α-1抗胰蛋白酶(AAT)病的反应。我们已经证明了我们的 AAV CARS可以清除AAV转导细胞，类似于早期观察到的情况 血友病试验和我们的AAV-CAR Tregs能够调节抗衣壳和抗- 转基因免疫反应，允许稳定和持久的转基因表达。我们会 优化我们目前的AAV汽车Treg结构和协议，以使最具抑制性和 实用的汽车Tregs。然后我们将进一步表征我们的AAV的抑制功能 免疫原性重组人32.33衣壳蛋白对小鼠模型的免疫应答。我们将进一步 确定他们是否能够克服先前存在的对衣壳病毒的免疫力，这是一个大问题 临床基因治疗由于患者中有大量既往存在的抗病毒免疫 AAV。而且，我们将研究AAV CAR T-regs是否能够克服先前存在的对转基因的免疫 就像那些参与CRISPR基因组编辑的人一样，同样，大多数患者都有免疫反应 使编辑细胞存活的可能性受到限制的CRISPR蛋白。我们还将 确定AAV CAR T-Regs是否允许重复注射AAV载体，允许多次 AAV基因治疗的剂量，以达到最佳的治疗表达。此外, 我们将进一步描述免疫系统对免疫原性转基因的调节。 通过AAV CAR Tregs允许稳定的免疫原性转基因表达。最后，我们会 AAV领域的一种新模型--雪貂体内对AAV传播的免疫应答特征 关于免疫反应。我们将研究雪貂模型如何很好地概括 临床观察以及衣壳特异性细胞毒和/或T调节细胞是否 全身或肌肉注射雪貂后产生的。总体而言，这个项目将使我们能够 深入了解AAV基因治疗后产生的免疫反应，并允许 美国将开发强大的工具来调节和缓解与以下疾病相关的免疫反应 临床基因治疗的发展。

Project 4 - Abstract In Project 4, we will use our novel adeno-associated virus (AAV) directed chimeric antigen receptor (CAR) T-cells and T regulatory cells (Tregs) to modulate and mitigate immune responses to AAV gene therapy for alpha-1-antitrypsin (AAT) disease. We have shown that our AAV CARs can clear AAV transduced cells similar to what was observed in the early Hemophilia trials and our AAV-CAR Tregs are able to allow modulate anti- capsid and anti- transgene immune responses allowing for stable and persistent transgene expression. We will optimize our current AAV CAR Treg construct and protocols to make the most suppressive and functional CAR Tregs. Then we will further characterize the suppressive function of our AAV CAR T-regs in response to the immunogenic rh32.33 capsid in mouse models. We will further determine if they are able to overcome pre-existing immunity to capsid which is a large issue for clinical gene therapy due to the large number of patients who have pre-existing immunity to AAV. And, we will study if AAV CAR T-regs can overcome pre-existing immunity to transgenes like those involved in CRISPR genome editing, as again most patients have immune response to CRISPR proteins causing the likelihood of survival of edited cells to be limited. We will also determine if AAV CAR T-regs can allow for re-dosing of AAV vectors, allowing for multiple doses of AAV gene therapy to be delivered to reach optimal therapeutic expression. In addition, we will further characterize the modulation of the immune system to immunogenic transgenes by AAV CAR Tregs allowing for stable immunogenic transgene expression. Finally, we will characterize the immune responses to AAV delivery by in ferrets, a novel model in the AAV field regarding immune responses. We will investigate how well the ferret model recapitulates the observations in the clinic and whether capsid specific cytotoxic and/or T-regulatory cells are generated after systemic or intramuscular injection of ferrets. Overall this project will allow us to learn considerably about the immune responses generated after AAV gene therapy and allow us to develop powerful tools to modulate and mitigate immune responses that are relevant for clinical gene therapy development.