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）定向嵌合抗原 受体（CAR）T细胞和调节性T细胞（T细胞）来调节和减轻免疫应答。 AAV基因治疗对α-1-抗胰蛋白酶（AAT）疾病的反应。我们已经证明， AAV汽车可以清除AAV转导的细胞，类似于在早期实验中观察到的。 血友病试验和我们的AAV-CAR T细胞能够允许调节抗衣壳和抗 转基因免疫应答允许稳定和持续的转基因表达。我们将 优化我们目前的AAV CAR Treg构建体和方案，以使最具抑制性和 功能性CAR T细胞。然后，我们将进一步表征我们的AAV的抑制功能 在小鼠模型中响应免疫原性rh32.33衣壳的CAR T-T细胞。我们将进一步 确定他们是否能够克服预先存在的对衣壳的免疫力，这是一个大问题， 临床基因治疗由于大量的患者谁有预先存在的免疫力， AAV。而且，我们将研究AAV CAR T-B1是否可以克服对转基因的预先存在的免疫力。 就像那些参与CRISPR基因组编辑的人一样，因为大多数患者都有免疫反应， CRISPR蛋白导致编辑细胞存活的可能性受到限制。我们还将 确定AAV CAR T-T细胞是否可以允许AAV载体的再给药，允许多个 剂量的AAV基因治疗以达到最佳治疗表达。此外，本发明还提供了一种方法， 我们将进一步描述免疫系统对免疫原性转基因的调节 通过允许稳定的免疫原性转基因表达的AAV CAR T细胞。最后我们将 通过在雪貂中表征对AAV递送的免疫应答，雪貂是AAV领域中的一种新模型 关于免疫反应。我们将研究如何以及雪貂模型概括 在临床上的观察，以及是否衣壳特异性细胞毒性和/或T-调节细胞， 雪貂全身或肌肉注射后产生的。总的来说，这个项目将使我们能够 了解大量关于AAV基因治疗后产生的免疫反应，并允许 我们将开发强大的工具来调节和减轻与以下相关的免疫反应 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.