Strategies to overcome immunity in gene therapy of DMD

Duchenne肌营养不良症(DMD)的特征是由于已知的人类最大基因dystrophin的突变而导致肌肉进行性退化。在许多情况下，营养不良蛋白是不起作用的，因为 突变缺失会将翻译转移到错误的阅读框架。用重组腺相关病毒载体(RAAV)进行基因置换是治疗DMD的方法之一。功能小型化的抗肌营养不良蛋白基因已经被开发出来，可以通过重组AAV载体进行传递。宿主对该病毒的免疫应答 AAV衣壳蛋白和转小磷脂基因产物对基因治疗的成功具有潜在的重要风险。一方面，针对衣壳的中和抗体和T细胞可能会阻止或减少持续的转基因表达。还有一种可能性是T细胞针对小型化 Dystrophin将消除转导的肌痉挛/TES。事实上，我们最近在四到六名受试者中检测到，在用重组AAV载体转导肌肉后，CD4+辅助细胞和CD8-I-细胞毒T细胞对微型肌营养不良蛋白的反应。在至少一个人类受试者中，转基因编码的表位位于从 抗肌营养不良蛋白基因。 我们已经建立了一个外源转基因表达的非人类灵长类动物模型，以更好地了解宿主免疫对rAAV载体转导肌肉后结果的影响。恒河猴与人类的相似之处在于，它们经常藏有中和衣壳特异性抗体和T细胞，这些抗体和T细胞可能会干扰 治疗成功了。此外，非自身转基因在骨骼肌中的表达会被针对编码的外源蛋白或rAAV衣壳蛋白的细胞免疫反应迅速终止。 在项目2中，我们建议使用AAV免疫猕猴来(I)通过瞬时耗尽抗rAAV衣壳的血清中和抗体来增强肌肉转导，以及(Ii)评估转导对针对衣壳的记忆T细胞反应的回忆的影响。此外，是否针对AAV衣壳的记忆T细胞或转基因产物在破坏转导的心肌细胞中起主导作用将被评估。 在猴子身上缓和这些反应的方法将被评估。

Duchenne muscular dystrophy (DMD) is characterized by progressive muscle degeneration due to mutations in dystrophin, the largest known human gene. In many cases the dystrophin protein is not functional because mutational deletions shift translation to an incorrect reading frame. Gene replacement using recombinant adeno-associated virus vectors (rAAV) is one approach to treatment of DMD. Functional miniaturized dystrophin genes have been developed for delivery by recombinant AAV vectors. Host immune responses to the AAV capsid and the minidystrophin transgene product represent potentially important risks to the success of gene therapy. On one hand neutralizing antibodies and T cells directed against the capsid might prevent or reduce sustained transgene expression. There is also a possibility that T cells directed against miniaturized dystrophin will eliminate transduced myocj/tes. Indeed, we recently detected CD4+ helper and CD8-I- cytotoxic T cell responses to mini-dystrophin in four or six subjects after transduction of muscle with a recombinat AAV vector. In at least one human subject the transgene-encoded epitopes were located in sequences deleted from dystrophin gene. We have developed a non-human primate model of foreign transgene expression to better understand the influence of host immunity on outcomes after rAAV vector transduction of muscle. Rhesus macaques resemble humans in that they often harbor neutralizing capsid-specific antibodies and T cells that could interfere with successful therapy. Moreover expression of a non-self transgene in skeletal muscle is quickly terminated by a cellular immune response that could be directed against the encoded foreign protein or the rAAV capsid protein. In Project 2 we propose to use AAV-immune rhesus macaques to (i) enhance muscle transduction by transient depletion of serum neutralizing antibodies against the rAAV capsid and (ii) assess the impact of transduction on recall of memory T cell responses directed against the capsid. In addition, whether memory T cells against the AAV capsid or transgene product play a dominant role in destruction of transduced myocytes will be assessed. Methods to temper these responses in monkeys will be evaluated.