Dual AAV Vectors for Duchenne Muscular Dystrophy Therapy

描述(申请人提供)：dystrophin基因突变导致Duchenne肌营养不良症(DMD)，这是一种严重的肌肉疾病，几乎影响到身体的所有肌肉。要治愈DMD，需要进行全身治疗。腺相关病毒(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。尽管前景光明，但AAV基因治疗面临着病毒包装容量较小(最大为5kb)的挑战。11.5kb的全长抗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，研究人员开发了缩写的微型/微型肌营养不良蛋白基因。微基因可以适应单个AAV，但它不能将肌肉力量恢复到正常水平。6kb的H2-R19迷你GENE可以完全恢复肌肉力量。我的第一个也是唯一一个R01是开发双AAV载体，在MDX小鼠的单个肌肉中表达6kb的微型基因，这是一种轻度DMD小鼠模型。我们成功实现并超过了这一目标。在这次更新中，我们将进一步推进DMD的双重AAV基因治疗。尽管6kb的微型基因可以完全恢复肌力，但这种微型基因不能恢复肌膜上的神经元型一氧化氮合酶(NNOS)。在mdx小鼠和DMD患者中，肌膜nNOS的丢失导致功能性缺血。我们最近开发了一种新的7kb迷你基因，它可以将nNOS招募到肌膜上。在这次更新中，我们将剖析出负责在dystrophin基因中招募nNOS的结构基序(S)。此外，我们将通过测量肌力和血液灌注量来确定7kb微型基因在转基因小鼠中的治疗优势(S)。最重要的是，我们将开发新型的双AAV载体来表达7kb的微型基因。我们之前已经展示了一种有效的微型Ddystrophin基因治疗方法，即一对反式剪接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小鼠身上进行的。在这次更新中，我们将检验以下假设：(1)在有症状的dystrophin/utroin双基因敲除(DKO)小鼠DMD模型中，可以实现全身性双AAV载体的基因转移；(2)双AAV载体的系统微基因治疗可以改善dKO小鼠的肌肉病理，恢复肌力和血液灌注量。我们的长期目标是为DMD患者开发一种有效的微基因疗法。在大型动物模型中建立系统性基因转移是合乎逻辑的下一步。我们最近鉴定了一种用于DMD的柯基犬模型。然而，众所周知，狗的肌肉很难用任何载体进行转导。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。这是第一次在大型动物模型中进行全身基因转移的演示。在这次更新中，我们将检验以下假设：(1)新生犬可以实现全身双重AAV载体转导；(2)全身双重AAV微基因治疗在新生儿营养不良犬中是可行的。综上所述，我们的研究将为未来双AAV基因治疗DMD患者奠定基础。与公共卫生相关。杜氏肌营养不良症是一种威胁生命的疾病，影响着相当大的人口(每3500名新生儿中就有一人以上)。它是由营养不良蛋白基因突变引起的。双重AAV介导的微基因疗法有望治愈这种疾病。我们的工作将通过提供功能上优越的微型基因，通过在有症状的小鼠模型中靶向所有身体肌肉，通过在狗模型中通过全身治疗来推进当前的双载体治疗。我们的发现将为将来最终将双重AAV基因疗法应用于人体试验铺平道路。

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.