Overcoming our clinical complications: AAV vector design for the treatment of DMD

描述（由申请人提供）：我们最近的临床试验使用腺相关病毒载体（AAV）向杜氏肌营养不良症（DMD）患者的肌肉递送迷你肌营养不良蛋白，结果出乎意料;治疗后，在两名患者中发现肌营养不良蛋白特异性T细胞应答，这与治疗前的回复突变纤维发育有关。这些抗肌萎缩蛋白特异性T细胞有可能根除所有基因修饰的肌肉，导致无效的治疗，并引起基因治疗界的普遍关注。目前，不存在避免转基因特异性CTL的策略，无论它们是预先存在的还是治疗诱导的，并且全身性长期免疫抑制被认为是不可行的选择。然而，在自然界中发现的特定病毒已经通过合成仅在转导细胞中抑制抗原呈递的小肽而进化出了解决这一困境的潜在解决方案。在初步的实验中，我们证明，这些病毒抑制肽（称为VIPRs）的细胞合成，防止表面介绍一个明确的抗原，从而保护转导细胞从宿主的免疫反应。在当前的提议中，我们将使用犬DMD模型（GRMD）来评估VIPRs从肌营养不良蛋白特异性CTL介导的AAV转导肌肉消除的逃避能力。首先，我们将测试VIPRs的利用是否可以阻断AAV肌肉注射后肌营养不良蛋白特异性CTL应答的诱导（目的1）。接下来，我们将研究VIPRs的应用是否有助于AAV转导的肌纤维逃避预先存在的肌营养不良蛋白特异性CTL介导的消除（Aim 2）。为了减少载体盒的大小以有效包装病毒体和/或增强VIPR的逃避能力，我们将优化VIPR结构域并测试突变变体的免疫逃避能力（目的3）。通过在相同载体中递送VIPRs和微型肌养蛋白，该方法确保抗原呈递将仅在AAV转导的细胞中减弱，而对免疫系统没有全身性副作用（如使用免疫抑制药物或通过应用调节性T细胞的情况）。总的来说，这个建议概述了一个有前途的策略，以克服我们的临床DMD的意见和关注的基因治疗研究一般，通过创建一个AAV载体，能够避免宿主的免疫反应的外源蛋白。

DESCRIPTION (provided by applicant): Our recent clinical trial using adeno-associated viral vectors (AAV) to deliver Mini-Dystrophin to the muscle of patients with Duchenne muscular dystrophy (DMD) was met with an unexpected result; after treatment, a Dystrophin-specific T-cell response was found in two patients, which was related to revertant fiber development prior to therapy. These Dystrophin-specific T cells have the potential to eradicate all genetically modified muscle resulting in an ineffective therapy, as well as presenting a general concern for gene therapy communities in general. Currently, no strategy exists to avoid transgene-specific CTLs, whether they are pre-existing or therapy- induced, and systemic long-term immunosuppression is considered a non-viable option. However, particular viruses found in nature have evolved a potential solution to this dilemma by synthesizing small peptides that inhibit antigen presentation only in transduced cells. In preliminary experiments, we demonstrate that the cellular synthesis of these viral inhibitory peptides (termed VIPRs) prevents the surface presentation of a well-defined antigen, thus protecting transduced cells from the host's immune response. In the current proposal, we will use a canine DMD model (GRMD) to evaluate the evasion ability of VIPRs from Dystrophin- specific CTL-mediated elimination of AAV transduced muscles. First, we will test whether the utilization of VIPRs can block the induction of a Dystrophin-specific CTL response after AAV muscle injection (Aim 1). Next, we will study whether the application of VIPRs will help AAV transduced muscle fibers escape pre-existing Dystrophin-specific CTL-mediated elimination (Aim 2). To decrease the vector cassette size for efficient virion package and/or to enhance the evasion ability of VIPRs, we will optimize the VIPR domains and test the immune evasion capacity of mutant variants (Aim 3). By delivering VIPRs and mini-dystrophin in the same vector, this approach ensures that antigen presentation will be attenuated only in AAV- transduced cells without systemic side effects on the immune system (as would be the case using immunosuppressive drugs or by the application of regulatory T-cells). Collectively, this proposal outlines a promising strategy to overcome our clinical DMD observations and concerns for gene therapy studies in general, by creating an AAV vector capable of avoiding the host's immune response to a foreign protein.