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

摘要 我们最近的临床试验使用腺相关病毒载体（AAV），以提供迷你肌营养不良蛋白， 杜氏肌营养不良症（DMD）患者肌肉出现了意想不到的 结果：治疗后，在两名患者中发现了抗肌萎缩蛋白特异性T细胞反应， 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 有可能根除所有导致无效治疗的遗传修饰肌肉， 以及对基因治疗团体的普遍关注。当前没有任何 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- 诱导和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， 合成仅在转导细胞中抑制抗原呈递的小肽。在 初步实验，我们证明，这些病毒抑制剂的细胞合成， 肽（称为VIPRs）阻止明确定义的抗原的表面呈递，因此 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VIPRs逃避肌营养不良蛋白的能力， 特异性CTL介导的AAV转导肌肉的消除。首先，我们将测试 VIPRs的利用可以阻断AAV-1后肌营养不良蛋白特异性CTL应答的诱导。 肌肉注射（Aim 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/或增强载体盒的功能， 针对VIPR的免疫逃避能力，我们将优化VIPR域并测试免疫逃避能力， 突变体的能力（目标3）。通过在相同的细胞中递送VIPRs和迷你肌养蛋白， 载体，这种方法确保抗原呈递将仅在AAV-1中减弱。 没有对免疫系统的全身性副作用的转导细胞（如将是这种情况 使用免疫抑制药物或通过应用调节性T细胞）。总的来说，这 该提案概述了一项有希望的策略，以克服我们的临床DMD观察和关注 对于一般的基因治疗研究，通过创建能够避免宿主的AAV载体， 对外来蛋白的免疫反应

Abstract 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.