In this regard, low-immunogenic, large-capacity, and human muscle-specific capsids will be particularly appealing. capsid engineering technologies to overcome hurdles in AAV-based DMD gene therapy. Introduction Duchenne muscular dystrophy (DMD) is the most common childhood muscle disease. It is characterized by progressive muscle weakness, loss of ambulation, and premature death caused by respiratory muscle and/or heart failure. DMD results from the loss of dystrophin, an essential cytoskeletal protein that protects muscle from contraction-induced injury (Fig. 1A). Soon after the discovery of the gene,1,2 it was postulated that expression of a functional gene in muscle may provide a cure for this relentless disease.3 Over the years, a number of nonviral and viral vectors have been explored to deliver the gene. Currently, adeno-associated computer virus (AAV) stands out as the leading candidate vector.4,5 Open in a separate window Determine 1. Adeno-associated viral vector (AAV) for Duchenne muscular dystrophy (DMD) gene therapy. The ultimate goal of AAV-mediated DMD gene therapy is usually to deliver a therapeutic gene using the gutless recombinant AAV (rAAV) A 922500 to achieve bodywide, robust, and persistent gene transfer in highly inflammatory and degenerative dystrophic muscle and heart to improve muscle and heart function, life quality, and lifespan. (A) Representative histology images from normal (left panel) and dystrophic (right panel) muscle. Normal muscle has well-organized, uniform myofibers with peripherally placed nuclei. Dystrophic muscle shows myofiber disorganization, centrally localized nuclei, and abundant infiltration of inflammatory cells. (B) Schematic outline of the wild-type AAV genome. The wild-type AAV has an 4.6C4.8?kb genome. It contains a gene for viral replication and a gene to generate viral capsid. Two inverted terminal repeats (ITRs) are positioned at the ends of the viral genome. ITR serves as the replication origin and packaging signal. (C) Schematic outline of the rAAV genome. The AAV vector is essentially gutted. A therapeutic expression cassette replaces the wild-type and genes. The only viral component in the rAAV genome is usually ITR. (D) The most commonly used AAV vector production method is usually triple-plasmid transfection in 293 cells. A rAAV plasmid carries the rAAV genome. A Rep/Cap plasmid expresses the replication and capsid proteins. Because the reproductive life cycle of AAV requires the help of adenovirus, an adenovirus helper plasmid is included in the transfection cocktail. AAV vector is usually purified using isopycnic ultracentrifugation and/or chromatography. Color images available online at www.liebertpub.com/hum AAV is a dependent parvovirus with an 5?kb single-stranded linear DNA genome.6 Wild-type AAV has two major open reading frames (ORFs) flanked by two inverted terminal repeats (ITRs). The 5 and 3 ORFs encode replication and capsid proteins, respectively.7 The ITR contains A 922500 145 nucleotides and serves as the AAV genome replication origin and packaging signal (Fig. 1B). In recombinant AAV, viral ORFs are replaced by the exogenous gene expression cassette, while the replication and capsid proteins are provided (Figs. 1C and D). AAV has many appealing features as DDR1 a gene therapy vector. For example, it has broad tissue tropism and high transduction efficiency. AAV can result in long-term persistent episomal expression.8,9 In addition, wild-type AAV is not associated with any known human diseases, and recombinant AAV vectors have shown an excellent safety profile in many clinical trials. Despite many appealing features, recombinant AAV vectors face several challenges for DMD gene therapy. First, AAV has a fairly limited packaging capacity. Two-thirds of the dystrophin coding sequence has to be removed in order to fit into an AAV particle. Second, the dystrophic muscle is an extremely hostile microenvironment for gene transfer (Fig. 1A). Myofiber degeneration and necrosis may result in the loss of the vector genome.10,11 Inflammation in the muscle further intensifies the immune response. Third, muscle consists of 40C50% the body mass and is spread throughout the body. An effective A 922500 gene therapy will require efficient systemic delivery to a variety of muscle groups, including the heart and diaphragm. Bodywide gene transfer brings in.