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），这是一种影响体内几乎所有肌肉的严重肌肉疾病。治疗DMD需要全身治疗。腺相关病毒（Adeno-associated virus， AAV）是目前唯一能有效转导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。尽管前景广阔，但AAV基因治疗受到病毒包装能力小（最大5 kb）的挑战。全长11.5 kb的肌营养不良蛋白编码序列不能由单个AAV载体传递。为了克服这一障碍，研究人员开发了缩短的微/迷你肌营养不良蛋白基因。微基因（< 4 kb）可以装入单个AAV中，但它们只能部分提高肌肉力量。6~ 8kb的小基因可以使肌肉力量正常化，但对于AAV包装来说太大了。此外，它们不能恢复全长蛋白的神经元一氧化氮合酶（nNOS）募集功能。亲本资助的目的是：(1)确定肌营养不良蛋白基因中的nNOS定位域，并开发能够恢复肌层nNOS的新型合成微基因；(2)探索双AAV载体介导的全身微肌营养不良蛋白基因治疗小鼠模型；(3)建立犬DMD模型的AAV系统基因转移。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是肌营养不良蛋白的nNOS锚定结构域。我们还生成了一个新的？H2-R15基因可以恢复肌上皮nNOS并提高运动表现（发表在2009年3月24日的《临床研究杂志》上）。肌营养不良蛋白的缺失导致收缩相关的肌层损伤和组织病理学。然而，肌肉力量减少并不总是与组织病理学相关。导致DMD患者肌肉力量丧失的分子途径目前尚不清楚。在亲本R01基金的支持下，我们最近获得了初步数据，表明细胞质nNOS错定位和随后的亚硝化/氧化应激可能是DMD力降低的基础。在这篇文章中，我们建议进一步探讨nNOS错定位在DMD发病和治疗中的分子机制/后果。具体来说，我们将检验以下两个假设：(1)R16/17介导的nNOS募集具有序列特异性和期特异性；(2) nNOS错定位导致亚硝化/氧化应激，收缩蛋白的s -亚硝基化导致DMD肌肉力降低。这些研究将我们的项目扩展到新的方向，超出了父母资助的范围。这些发现将为我们对疾病过程的理解提供新的亮点，并为DMD治疗开辟新的治疗途径。

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.