Dual AAV Vectors for Duchenne Muscular Dystrophy Therapy

描述（由申请人提供）：肌营养不良蛋白基因突变导致杜氏肌营养不良症（DMD），这是一种严重的肌肉疾病，几乎影响身体的所有肌肉。治疗DMD需要全身治疗。腺相关病毒（Adeno-associated virus，AAV）是目前唯一能有效感染全身肌肉的病毒载体。因此，AAV被认为是DMD基因治疗的首选载体。尽管有很大的前景，但AAV基因治疗受到小病毒包装能力（最大5 kb）的挑战。11.5kb全长肌营养不良蛋白编码序列不能通过单个AAV载体递送。为了克服这一障碍，研究人员已经开发了简化的微小肌营养不良蛋白基因。微基因（<4kb）可以装入单个AAV，但它们只能部分改善肌肉力量。6~8 kb的小基因可以使肌肉力量正常化，但它们太大而不适合AAV包装。此外，它们不能恢复全长蛋白质的神经元型一氧化氮合酶（nNOS）募集功能。亲本资助的目标是（1）鉴定肌营养不良蛋白基因中的nNOS定位结构域，并开发可恢复肌膜nNOS的新型合成小基因;（2）在小鼠模型中探索双AAV载体介导的系统性小肌营养不良蛋白基因治疗;（3）在犬DMD模型中开发系统性AAV基因转移。 自从我们开始这个项目以来，已经取得了巨大的进展。最值得注意的是，我们已经确定R16/17作为nNOS锚定结构域的肌营养不良蛋白。我们还创造了一个新的？H2-R15小基因，恢复肌膜nNOS和增强运动能力（发表在2009年3月24日的临床研究杂志上）。肌营养不良蛋白的丢失导致收缩相关的肌膜损伤和组织病理学。然而，肌力下降并不总是与组织病理学相关。导致DMD中肌肉力量丧失的分子途径目前尚不清楚。在父母R 01资助的支持下，我们最近产生的初步数据表明，胞质nNOS错误定位和随后的亚硝化/氧化应激可能是DMD中力降低的基础。在本修订中，我们建议进一步探讨nNOS错误定位在DMD发病机制和治疗中的分子机制/后果。具体而言，我们将检验以下两个假设：（1）R16/17介导的nNOS募集是序列特异性和阶段特异性的;（2）nNOS错误定位导致亚硝化/氧化应激，收缩蛋白的S-亚硝基化有助于DMD中的肌力降低。这些研究将我们的项目扩展到父母补助金范围之外的新方向。这些发现将为我们对疾病过程的理解提供新的线索，并为DMD治疗开辟新的治疗途径。 公共卫生相关性：杜氏肌营养不良症（DMD）是一种威胁生命的疾病，影响相当大的人口。它是由肌营养不良蛋白基因突变引起的。有趣的是，肌营养不良蛋白的丢失破坏了肌细胞中神经元型一氧化氮合酶（nNOS）的亚细胞定位。在DMD发病机制中，nNOS稳态的改变被认为是一个关键因素。为了更好地开发DMD基因治疗，我们最近确定了肌营养不良蛋白基因中的nNOS招募结构域。在这里，我们建议进一步研究nNOS和dystrophin之间的分子相互作用。此外，我们将评估nNOS错误定位产生的亚硝化/氧化应激是否是DMD中力损失的原因。这些研究将加深我们对DMD发病机制的认识，为DMD的治疗打开新的大门。

DESCRIPTION (provided by applicant): Dystrophin gene mutations lead to Duchenne muscular dystrophy (DMD), a severe muscle disease that affected nearly all muscles in the body. A cure for DMD requires body-wide therapy. Adeno-associated virus (AAV) is currently the only viral vector that can efficiently transduce whole body muscle. For this reason, AAV has been considered as the vector-of-choice for DMD gene therapy. Despite great promise, AAV gene therapy is challenged by the small viral packaging capacity (5 kb maximal). The 11.5 kb full-length dystrophin coding sequence cannot be delivered by a single AAV vector. To overcome this obstacle, investigators have developed abbreviated micro/mini-dystrophin genes. Microgenes (< 4 kb) can fit into a single AAV but they only partially improve muscle force. The 6~8 kb minigenes can normalize muscle force but they are too big for AAV packaging. Furthermore, they cannot restore the neuronal nitric oxide synthase (nNOS) recruiting function of the full-length protein. The goals of the parental grant are (1) to identify the nNOS localization domain in the dystrophin gene and to develop novel synthetic minigenes that can restore sarcolemmal nNOS; (2) to explore dual AAV vector-mediated systemic mini-dystrophin gene therapy in the mouse model; (3) to develop systemic AAV gene transfer in a canine DMD model. Tremendous progress has been achieved since we started this project. Most remarkably, we have identified R16/17 as the nNOS anchoring domain in dystrophin. We have also generated a new ?H2-R15 minigene that restores sarcolemmal nNOS and enhances exercise performance (published in the Journal of Clinical Investigation on March 24, 2009). The loss of dystrophin leads to contraction-associated sarcolemmal injury and histopathology. However, muscle force reduction does not always correlate with histopathology. The molecular pathway(s) leading to the loss of muscle strength in DMD is currently unclear. With the support of the parental R01 grant, we recently generated preliminary data suggesting that cytosolic nNOS mislocalization and subsequent nitrosative/oxidative stress may underlie force reduction in DMD. In this revision, we propose to further explore the molecular mechanisms/consequencies of nNOS mislocalization in DMD pathogenesis and therapy. Specifically, we will test the following two hypotheses: (1) R16/17- mediated nNOS recruiting is sequence specific and phase specific; (2) nNOS mislocalization results in nitrosative/oxidative stress and S-nitrosylation of contractile proteins contributes to muscle force reduction in DMD. These studies expand our project to new directions beyond the scope of the parental grant. The findings will shed new light on our understanding of the disease process and uncover novel therapeutic avenues for DMD treatment. PUBLIC HEALTH RELEVANCE: Duchenne muscular dystrophy (DMD) is a life threatening diseases affecting a fairly large population. It is caused by dystrophin gene mutation. Interestingly, the loss of dystrophin disrupts subcellular localization of neuronal nitric oxide synthase (nNOS) in muscle cells. in the alteration of nNOS homeostasis has been suggested as a critical factor in DMD pathogenesis. In an effort to better develop DMD gene therapy, we recently identified the nNOS recruiting domain in the dystrophin gene. Here, we propose to further investigate the molecular interactions between nNOS and dystrophin. In addition, we will evaluate whether the nitrosative/oxidative stress generated by nNOS mislocalization is responsible for the force loss in DMD. These studies will advance our understanding on DMD pathogenesis and open new doors for DMD therapy.