Mice carrying mutations in both the dystrophin and utrophin genes die prematurely as a consequence of severe muscular dystrophy. Here, we demonstrate that intravascular administration of recombinant adeno-associated viral (rAAV) vectors carrying a microdystrophin gene restores dystrophin expression in the striated musculature of these animals, considerably reducing skeletal muscle pathology and extending lifespan. These findings suggest rAAV vectormediated systemic gene transfer may be useful for treatment of serious neuromuscular disorders such as Duchenne muscular dystrophy (DMD).Miniaturized dystrophin expression cassettes that restore sarcolemmal organization of the dystrophin-glycoprotein complex can be highly functional in transgenic mice 1 . However, delivering potentially therapeutic constructs throughout the musculature of animals with preexisting muscular dystrophy using traditional methods has proven inefficient. Recently, we established that intravascular administration of recombinant adeno-associated viral vectors pseudotyped with the serotype-6 capsid (rAAV6) can transduce the striated musculature of adult mice 2 . This advance enables assessment of systemic microdystrophin delivery in animal models of disease. Historically the dystrophin-deficient mdx mouse 3 has been employed as the primary model of DMD, although this animal does not experience the severe, body-wide dystrophy that shortens lifespan by 75% in patients [4][5][6] . The robustness of mdx mice is attributed to compensatory over-expression of the dystrophin-related protein utrophin, as knockout of both dystrophin and utrophin in mice causes progressive muscle wasting, impaired mobility and premature death 5,6 . In this study, we tested the hypothesis that systemic administration of rAAV6-microdystrophin can ameliorate the pathology associated with severe muscular dystrophy in dystrophin/utrophin double-knockout (dko) mice.A single intravenous administration of ~3×10 12 vector genomes of rAAV6-microdystrophin was administered to one month-old dko mice as described previously 2 , resulting in uniform, body-wide expression of dystrophin for at least one year ( Supplementary Fig. 1 online). Because muscle deterioration leading to respiratory failure is the primary cause of death in patients with DMD 7 , we examined the effects of treatment upon mouse diaphragm (DIA) muscles 4 . Dystrophin expression in DIA muscles examined 18 weeks after treatment was widespread (Fig. 1a,b) and was associated with a significant reduction in the prevalence of smaller regenerating muscle cells compared with untreated muscles (Fig. 1c). Treatment also reduced the frequency of centrally-nucleated myofibers -a feature of muscle regeneration -by ~85% compared with the DIA muscles of untreated mice (Fig. 1d). Importantly, the DIA muscles of treated animals demonstrated more than two-fold increased normalized force-producing capacity compared with the muscles of untreated mice (Fig. 1e).Using a protocol we developed for subjecting muscles to progressively ...

Systemic delivery of recombinant adeno-associated virus (rAAV) 6 vectors mediates efficient transduction of the entire striated musculature, making this an attractive strategy for muscle gene therapy. However, owing to widespread transduction of non-muscle tissues, optimization of this method would benefit from the use of muscle-specific promoters. Most such promoters either lack high-level expression in certain muscle types or are too large for inclusion in rAAV vectors encoding microdystrophin. Here, we describe novel regulatory cassettes based on enhancer/promoter regions of murine muscle creatine kinase (CK) and alpha-myosin heavy-chain genes. The strongest cassette, MHCK7 (770 bp), directs high-level expression comparable to cytomegalovirus and Rous sarcoma virus promoters in fast and slow skeletal and cardiac muscle, and low expression in the liver, lung, and spleen following systemic rAAV6 delivery in mice. Compared with CK6, our previous best cassette, MHCK7 activity is approximately 400-, approximately 50-, and approximately 10-fold higher in cardiac, diaphragm, and soleus muscles, respectively. MHCK7 also directs strong microdystrophin expression in mdx muscles. While further study of immune responses to MHCK7-regulated microdystrophin expression is needed, this cassette is not active in dendritic cell lines. MHCK7 is thus a highly improved regulatory cassette for experimental studies of rAAV-mediated transduction of striated muscle.

Duchenne muscular dystrophy (DMD), the most prevalent lethal genetic disorder in children, is caused by mutations in the 2.2-MB dystrophin gene. Absence of dystrophin and the dystrophin-glycoprotein complex (DGC) from the sarcolemma leads to severe muscle wasting and eventual respiratory and/or cardiac failure. There is presently no effective therapy for DMD. Several lines of evidence have suggested that methods to increase expression of utrophin, a dystrophin paralog, show promise as a treatment for DMD. Adeno-associated viral (AAV) vectors are a promising vehicle for gene transfer to muscle, but microutrophin transgenes small enough to be carried by AAV have not been tested for function. In this study, we intravenously administered recombinant AAV (rAAV2/6) harboring a murine codon-optimized microutrophin (DeltaR4-R21/DeltaCT) transgene to adult dystrophin(-/-)/utrophin(-/-) (mdx:utrn(-/-)) double-knockout mice. Five-month-old mice demonstrated localization of microutrophin to the sarcolemma in all the muscles tested. These muscles displayed restoration of the DGC, increased myofiber size, and a considerable improvement in physiological performance when compared with untreated mdx:utrn(-/-) mice. Overall, microutrophin delivery alleviated most of the pathophysiological abnormalities associated with muscular dystrophy in the mdx:utrn(-/-) mouse model. This approach may hold promise as a treatment option for DMD because it avoids the potential immune responses that are associated with the delivery of exogenous dystrophin.

The baculovirus-insect cell system is used routinely for foreign glycoprotein production, but the precise nature of the N-glycosylation pathway in this system remains unclear. Some studies indicate that these cells cannot process N-linked oligosaccharides to complex forms containing outer-chain galactose and sialic acid, while others indicate that they can. In this study, we used the major virion envelope glycoprotein of the baculovirus Autographa california multicapsid nuclear polyhedrosis virus (AcMNPV) to probe the N-glycosylation pathway in baculovirus-infected lepidopteran insect cells. The results showed that gp64 contained mannose, fucose, and probably N-acetylglucosamine, but no detectable galactose or sialic acid. These same results were observed with gp64 produced in any one of three different lepidopteran insect cell lines derived from Spodoptera frugiperda, Trichoplusia ni, or Estigmene acrea, whether it was produced at relatively earlier or later times after infection. These results indicated that the gp64 produced in AcMNPV-infected lepidopteran insect cells lacks complex N-linked oligosaccharides containing outer-chain galactose and sialic acid. By contrast, gp64 produced in mammalian cells contained both galactose and sialic acid, and endoglycosidase digestions revealed that these sugars were constituents of N-linked, not O-linked, oligosaccharides. This showed that at least one N-linked side chain on gp64 has the potential to be processed to a complex form. Together, these results suggest either that AcMNPV-infected lepidopteran insect cells are unable to convert any of the N-linked side chains on gp64 to complex structures or that outer-chain galactose and sialic acid residues are added to gp64 and then removed by cellular or viral exoglycosidases.