Strategies to overcome immunity in gene therapy of DMD

杜氏肌营养不良症（DMD）的特征是由于肌营养不良蛋白（已知最大的人类基因）突变导致的进行性肌肉变性。在许多情况下，肌营养不良蛋白质没有功能，因为 突变缺失将翻译转移到不正确的阅读框。使用重组腺相关病毒载体（rAAV）的基因置换是治疗DMD的一种方法。功能性小型化肌营养不良蛋白基因已被开发用于通过重组AAV载体递送。宿主免疫反应 AAV衣壳和微小肌萎缩蛋白转基因产物代表了基因治疗成功的潜在重要风险。一方面，针对衣壳的中和抗体和T细胞可能阻止或减少持续的转基因表达。也有一种可能性是针对小型化的T细胞 肌营养不良蛋白将消除转导的肌细胞/TES。事实上，我们最近在用重组AAV载体转导肌肉后的四或六名受试者中检测到了对微型肌营养不良蛋白的CD 4+辅助和CD 8-I-细胞毒性T细胞应答。在至少一个人类受试者中，转基因编码的表位位于从人类受试者缺失的序列中。 dystrophin基因 我们已经开发了一种外源转基因表达的非人灵长类动物模型，以更好地了解rAAV载体转导肌肉后宿主免疫对结果的影响。恒河猴与人类相似之处在于，它们经常携带中和captain特异性抗体和T细胞，这些抗体和T细胞可以干扰 成功的治疗此外，骨骼肌中非自身转基因的表达被细胞免疫应答快速终止，所述细胞免疫应答可以针对编码的外源蛋白或rAAV衣壳蛋白。 在项目2中，我们建议使用AAV免疫恒河猴（i）通过瞬时消耗针对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细胞应答的回忆的影响。此外，将评估针对AAV衣壳或转基因产物的记忆T细胞是否在转导的肌细胞的破坏中起主导作用。 将评价在猴中缓和这些反应的方法。

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.