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。微基因可以容纳在单个AAV中，但它不能将肌肉力量恢复到正常水平。一个6 kb的H2-R19基因可以完全恢复肌肉力量。我的第一个也是唯一的R01是在mdx小鼠（轻度DMD小鼠模型）的单个肌肉中开发双AAV载体来表达6kb的miniigene。我们已经成功地完成并超越了这一目标。在此次更新中，我们将进一步推进双AAV基因治疗DMD。尽管6kb的minigene可以完全恢复肌肉力量，但该minigene不能将神经元一氧化氮合酶（nNOS）恢复到肌膜。mdx小鼠和DMD患者肌层nNOS的缺失导致功能性缺血。我们最近开发了一种新的7 kb的迷你基因，可以将nNOS招募到肌膜上。在这次更新中，我们将剖析在肌营养不良蛋白基因中负责nNOS募集的结构基序。此外，我们将通过测量肌肉力量和血液灌注来确定7 kb minigene在转基因小鼠中的治疗优势。最重要的是，我们将开发新的双AAV载体来表达7 kb的迷你基因。我们之前已经证明了一种有效的微型肌营养不良蛋白基因治疗与一对反式剪接AAV （tsAAV，一种双载体方法）。这项原理验证研究是通过局部注射在mdx小鼠中进行的。在本次更新中，我们将验证以下假设：(1)在症状性肌营养不良蛋白/肌营养不良蛋白双敲除（dko）小鼠DMD模型中可以实现全身双AAV载体基因转移；(2)此外，双AAV载体的全身小基因治疗可以改善dko小鼠的肌肉病理，恢复肌肉力量和血液灌注。我们的长期目标是为DMD患者开发一种有效的迷你基因疗法。在大型动物模型中建立系统基因转移是顺理成章的下一步。我们最近描述了一种柯基犬的DMD模型。然而，犬的肌肉是出了名的难以用任何载体转导。我们现在已经克服了这一障碍，并在野生型新生犬中实现了系统的单一AAV转导。这是首次在大型动物模型中进行全身基因转移。在本次更新中，我们将验证以下假设：(1)在新生犬中可以实现系统性双AAV载体转导；(2)系统双AAV基因治疗对新生儿营养不良犬是可行的。综上所述，我们的研究将为今后双AAV迷你基因治疗DMD患者奠定基础。公共卫生相关性。杜氏肌营养不良症是一种危及生命的疾病，影响着相当多的人口（每3,500个新生儿中就有1个以上）。它是由肌营养不良蛋白基因突变引起的。双aav介导的迷你基因治疗有望治愈该病。我们的工作将通过提供功能优越的迷你基因，在有症状的小鼠模型中靶向所有身体肌肉，以及在狗模型中采用全身方法来推进当前的双载体治疗。我们的发现将为将来将双重AAV基因疗法最终推向人体试验铺平道路。

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. Thus AAV is 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 can fit into a single AAV but it cannot restore muscle force to the normal level. A 6 kb H2-R19 minigene fully recovers muscle force. My first and only R01 was to develop dual AAV vectors to express the 6 kb minigene in a single muscle in mdx mice, a mild mouse model for DMD. We have successfully accomplished and surpassed this goal. In this renewal, we will further advance dual AAV gene therapy for DMD. Despite the fact that the 6 kb minigene can fully restore muscle force, this minigene cannot restore neuronal nitric oxide synthase (nNOS) to the sarcolemma. The loss of sarcolemmal nNOS leads to functional ischemia in mdx mice and DMD patients. We recently developed a novel 7 kb minigene that recruits nNOS to the sarcolemma. In this renewal, we will dissect out the structure motif(s) responsible for nNOS recruiting in the dystrophin gene. Furthermore, we will establish the therapeutic advantage(s) of the 7 kb minigene in transgenic mice by measuring muscle force and blood perfusion. Most importantly, we will develop novel dual AAV vectors to express the 7 kb minigene. We have previously demonstrated an efficient mini-dystrophin gene therapy with a pair of the trans-splicing AAV (tsAAV, a dual vector approach). This proof-of-principle study is performed in mdx mice by local injection. In this renewal, we will test the hypotheses that (1) systemic whole body dual AAV vector gene transfer can be achieved in a symptomatic dystrophin/utrophin double knockout (dko) mouse DMD model; (2) furthermore, systemic minigene therapy with dual AAV vectors can ameliorate muscle pathology, restore muscle force and blood perfusion in dko mice. Our long-term goal is to develop an effective minigene therapy for DMD patients. Establishing systemic gene transfer in a large animal model is a logical next step. We have recently characterized a Corgi dog model for DMD. However canine muscle has been notoriously difficult to transduce with any vector. We have now overcome this hurdle and achieved systemic single AAV transduction in wild type newborn dogs. This is the first demonstration of a whole body gene transfer in a large animal model. In this renewal, we will test the hypotheses that (1) systemic dual AAV vector transduction can be achieved in newborn dogs; (2) systemic dual AAV minigene therapy is feasible in neonatal dystrophic dogs. In summary, our study will establish the foundation for dual AAV minigene gene therapy in DMD patients in the future. PUBLIC HEALTH RELEVANCE. Duchenne muscular dystrophy is a life threatening disease affecting a fairly large population (more than one in 3,500 newborns). It is caused by dystrophin gene mutations. Dual AAV-mediated minigene therapy hold great promise to cure the disease. Our work will advance current dual vector therapy by providing a functionally superior minigene, by targeting all body muscles in a symptomatic mouse model, and by systemic approach in a dog model. Our findings will pave the way to eventually move dual AAV gene therapy to human trials in the future.