IMPROVED VIRAL VECTORS FOR GENE TRANSFER TO MUSCLE

该项目的目标是开发和测试用于将基因转移到横纹肌的改进的病毒载体系统。特别关注的是将基因转移到成年和老年小鼠肌肉的能力，重点是Duchenne肌营养不良症(DMD)模型。DMD是人类最常见的遗传病之一，也是影响骨骼肌的最常见的遗传病。将探索两种载体系统：空洞腺病毒(Ad)和腺相关病毒(AAV)载体。这两个系统都有巨大的潜力。 用于基因转移，但每一种都需要额外的开发，以便能够以安全有效的方式进行临床使用。因此，对这些媒介系统的技术改进将构成项目目标的一个主要组成部分。该项目的第二部分将探索使用这些载体技术来修改成年和老年动物的肌肉，目标是纠正正常和营养不良肌肉随着年龄的增长而逐渐增加的功能缺陷。我们将开发改进的包装细胞系、载体和辅助病毒，以提高去内脏的Ad载体的产量、纯度和易制备性。我们还将探索使用AAV 6型将基因转移到肌肉中。AAV血清型6显示出高效的肌肉基因转移，我们建议优化方法以获得高滴度的表达肌肉特异性表达的高纯度AAV6载体。这两种类型的载体都将被用来表征通过传递全长、微型或微肌营养不良蛋白来预防、阻止或纠正DMD的MDX小鼠模型中的肌营养不良的能力。研究将在小鼠、成年小鼠和老年小鼠身上进行，并将探索将这些载体输送到横纹肌的潜在免疫学后果。最后，我们将使用这些系统来探索衰老小鼠营养不良的其他表型特征，目标是 通过基因转导抗肌营养不良蛋白和胰岛素样生长因子-1来阻止或至少部分逆转横纹肌的功能缺陷。改进基因传递方法和改善成年和老年动物营养不良肌肉异常的研究不仅将有助于开发DMD的治疗方法，而且还可以作为开发其他遗传病和与衰老相关的疾病的遗传疗法的模型。

The goals of this project are to develop and test improved viral vector systems for gene transfer to striated muscle. A particular focus is on the ability to transfer genes to muscles of adult and old mice, with an emphasis on models for Duchenne muscular dystrophy (DMD). DMD is among the most common human genetic diseases, and represents the most common genetic disease affecting skeletal muscle. Two vector systems will be explored: gutted adenoviral (Ad) and adeno-associated viral (AAV) vectors. Both of these systems have significant potential for use in gene transfer, but they each require additional development to enable clinical use in a safe and efficacious manner. Technological improvements to these vectors systems will therefore constitute a major component of the project goals. A second component of the project will explore the use of these vector technologies to modify muscles of adult and old animals, with a goal of correcting functional deficits that progressively increase with aging in both normal and dystrophic muscles. We will develop improved packaging cell lines, vectors and helper viruses to increase the yield, purity and ease of preparation of gutted Ad vectors. We will also explore the use of AAV serotype 6 for gene transfer to muscle. AAV serotype 6 displays highly efficient gene transfer to muscle, and we propose to optimize methods to obtain high titer stocks of highly purified AAV6 vectors displaying muscle-specific expression. Both types of vectors will be used to characterize the ability to prevent, halt or correct features of muscular dystrophy in the mdx mouse model of DMD by delivery of full-length, mini or micro-dystrophins. Studies will be conducted in young, adult and old mice, and will explore potential immunological consequences of delivery of these vectors to striated muscle. Finally, we will use these systems to explore additional phenotypic features of dystrophy in aging mice, with a goal of developing methods to arrest, and at least partially reverse, functional deficits of striated muscles by gene delivery of dystrophin and Igf-1. Studies that lead to improved methods for gene delivery and the amelioration of abnormalities of dystrophic muscles in adult and old animals will not only facilitate development of a treatment for DMD, but can also serve as a model for the development of genetic therapies for other genetic diseases and conditions associated with aging.