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

描述（由申请人提供）：我们最近使用腺相关病毒载体（AAV）向杜氏肌营养不良症（DMD）患者的肌肉输送Mini-Dystrophin的临床试验取得了意想不到的结果；治疗后，两名患者出现抗肌营养不良蛋白特异性t细胞反应，这与治疗前纤维的逆转发育有关。这些抗肌萎缩蛋白特异性T细胞具有根除所有基因修饰肌肉的潜力，导致治疗无效，同时也引起了基因治疗界的普遍关注。目前，没有策略可以避免转基因特异性ctl，无论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进化出一种潜在的解决方案，通过合成仅在转导细胞中抑制抗原呈递的小肽来解决这一困境。在初步实验中，我们证明了这些病毒抑制肽（称为vipr）的细胞合成阻止了明确定义的抗原的表面呈现，从而保护转导细胞免受宿主免疫反应的影响。在目前的提案中，我们将使用犬DMD模型（GRMD）来评估vipr在抗肌营养不良蛋白特异性ctl介导的AAV转导肌肉消除中的逃避能力。首先，我们将测试使用vipr是否可以阻断AAV肌肉注射后抗肌萎缩蛋白特异性CTL反应的诱导（Aim 1）。接下来，我们将研究vipr的应用是否会帮助AAV转导的肌纤维逃脱预先存在的肌营养不良蛋白特异性ctl介导的消除（Aim 2）。为了减小载体盒大小以有效包装病毒粒子和/或提高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）。通过在同一载体中递送vipr和mini-dystrophin，该方法确保抗原呈递仅在AAV转导细胞中减弱，而不会对免疫系统产生全身性副作用（如使用免疫抑制药物或应用调节性t细胞）。总的来说，该建议概述了一种有希望的策略，通过创建能够避免宿主对外来蛋白质的免疫反应的AAV载体，来克服我们的临床DMD观察和一般基因治疗研究的担忧。

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.