Phosphorylase re-expression, increase in the force of contraction and decreased fatigue following notexin-induced muscle damage and regeneration in the ovine model of McArdle disease.

McArdle disease is caused by a deficiency of myophosphorylase and currently a satisfactory treatment is not available. The injection of notexin into, or the layering of notexin onto, the muscles of affected sheep resulted in necrosis followed by regeneration of muscle fibres with the expression of both non-muscle isoforms of phosphorylase within the fibres and a reduction of the amount of glycogen in the muscle with an increase in the strength of contraction and a decrease in fatiguability in the muscle fibres. The sustained re-expression of both the brain and liver isoforms of phosphorylase within the muscle fibres provides further emphasis that strategies to enhance the re-expression of these isoforms should be investigated as a possible treatment for McArdle disease.

Challenges for the genetic screening in dysferlin deficiency--report of an instructive case and review of the literature.

The homozygous or compound heterozygous mutation of the alleles of DYSF gene causes dysferlinopathy resulting in limb girdle muscular dystrophy Type 2B (LGMD 2B) or Miyoshi myopathy. However, patients with only 1 (heterozygous) mutation on 1 allele are increasingly recognized. Based on the Leiden database (www.dmd.nl) among 257 different mutations resulting in dysferlin-deficient muscular dystrophy, pathogenic mutations were detected only on 1 allele in 45 cases, while the exons of the other allele did not show any pathological alterations. The relatively high number of these so-called heterozygous cases raises the question if present routine molecular techniques are sufficient alone for confirming the diagnosis of dysferlin deficiency. In fact, the heterogenous genetic background of the disease makes it impossible to make the correct diagnosis without Western blot of the muscle dysferlin. This paper presents the clinical, myopathological and molecular genetic results of a 30-year-old male with dysferlinopathy as an instructive case. The cDNA sequencing of the dysferlin gene revealed a single C5302T heterozygous mutation resulting in Arg1768Trp exchange. The paper highlights the importance of the protein analysis in the diagnosis of dysferlin deficiency, discusses the difficulties of the complete genomic analysis of the dysferlin gene alterations and the possible etiopathogenetic role of the noncoding DNA sequence of the dysferlin gene in dysferlin deficiencies.

Adenovirus and adeno-associated virus-mediated delivery of human myophosphorylase cDNA and LacZ cDNA to muscle in the ovine model of McArdle's disease: expression and re-expression of glycogen phosphorylase.

At present there is no satisfactory treatment for McArdle's disease, deficiency of myophosphorylase. Injection of modified adenovirus 5 (AdV5) and adeno-associated virus 2 (AAV2) vectors containing myophosphorylase expression cassettes, into semitendinosus muscle of sheep with McArdle's disease, produced expression of functional myophosphorylase and some re-expression of the non-muscle glycogen phosphorylase isoforms (both liver and brain) in regenerating fibres. Expression of both non-muscle isoforms was also seen after control injections of AdV5LacZ vectors. There was up to an order of magnitude greater expression of phosphorylase after myophosphorylase vector injection than after LacZ controls (62% of sections with over 1000 positive muscle fibres, versus 7%). The results presented here suggest that the use of viral vector-mediated phosphorylase gene transfer may be applicable to the treatment of McArdle's disease and that sustained re-expression of the brain and liver isoforms should also be investigated as a possible treatment.

Localization of coxsackie virus and adenovirus receptor (CAR) in normal and regenerating human muscle.

The primary receptor for Adenovirus and Coxsackie virus (CAR) serves as main port of entry of the adenovirus vector mediating gene transfer into skeletal muscle. Information about CAR expression in normal and diseased human skeletal muscle is lacking. C'- or N'-terminally directed polyclonal antibodies against CAR were generated and immunohistochemical analysis of CAR on morphologically normal and regenerating human skeletal muscle of children and adults was performed. In morphologically normal human muscle fibers, CAR immunoreactivity was limited to the neuromuscular junction. In regenerating muscle fibers, CAR was abundantly co-expressed with markers of regeneration. The function of CAR at the neuromuscular junction is currently unknown. Co-expression of CAR with markers of regeneration suggests that CAR is developmentally regulated, and may serve as a marker of skeletal muscle fiber regeneration.

Dystrophic phenotype of canine X-linked muscular dystrophy is mitigated by adenovirus-mediated utrophin gene transfer.

Utrophin is highly homologous and structurally similar to dystrophin, and in gene delivery experiments in mdx mice was able to functionally replace dystrophin. We performed mini-utrophin gene transfer in Golden Retriever dogs with canine muscular dystrophy (CXMD). Unlike the mouse model, the clinicopathological phenotype of CXMD is similar to that of Duchenne muscular dystrophy (DMD). We injected an adenoviral vector expressing a synthetic utrophin into tibialis anterior muscles of newborn dogs affected with CXMD and examined transgene expression by RNA and protein analysis at 10, 30 and 60 days postinjection in cyclosporin-treated and -untreated animals. Immunosuppression by cyclosporin was required to mitigate the immune response to viral and transgene antigens. RT-PCR analysis showed the presence of the exogenous transcript in the muscle of cyclosporin-treated and -untreated animals. The transgenic utrophin was efficiently expressed at the extrajunctional membrane in immunosuppressed dogs and this expression was stable for at least 60 days. We found reduced fibrosis and increased expression of dystrophin-associated proteins (DAPs) in association with muscle areas expressing the utrophin minigene, indicating that mini-utrophin can functionally compensate for lack of dystrophin in injected muscles. For this reason, utrophin transfer to dystrophin-deficient muscle appears as a promising therapeutic approach to DMD.

Forced myofiber regeneration promotes dystrophin gene transfer and improved muscle function despite advanced disease in old dystrophic mice.

Duchenne muscular dystrophy (DMD) is caused by defects in the dystrophin gene. In young dystrophic mdx mice, immature regenerating myofibers represent the principal substrate for adenovirus vector (AdV)-mediated dystrophin gene transfer. However, in DMD patients immature regenerating myofibers are generally sparse. Such a situation also exists in old mdx mice, which may represent a more realistic model. Therefore, here we have used old mdx mice (of 14- to 17 months of age) to test the hypothesis that one-time administration of a myonecrotic agent can transiently re-establish a population of immature myofibers susceptible to AdV-mediated dystrophin gene transfer. This strategy led to upregulation of the coxsackie/adenovirus attachment receptor by means of induction of regenerating myofibers, significantly augmented AdV-mediated dystrophin gene expression, and enhanced force-generating capacity. In addition, it led to an increased resistance to contraction-induced injury compared with untreated controls. The latter protective effect was positively correlated with the number of dystrophin-expressing myofibers (r=0.83, P<0.05). Accordingly, the risk:benefit ratio associated with the sequential use of forced myofiber regeneration and AdV-mediated dystrophin gene transfer was favorable in old mdx mice despite advanced disease. These findings have implications for the potential applicability of AdV-mediated gene therapy to DMD and other muscle diseases in which immature regenerating myofibers are lacking.

Dystrophin expression in muscle following gene transfer with a fully deleted ("gutted") adenovirus is markedly improved by trans-acting adenoviral gene products.

Helper-dependent adenoviruses (HDAd) are Ad vectors lacking all or most viral genes. They hold great promise for gene therapy of diseases such as Duchenne muscular dystrophy (DMD), because they are less immunogenic than E1/E3-deleted Ad (first-generation Ad or FGAd) and can carry the full-length (Fl) dystrophin (dys) cDNA (12 kb). We have compared the transgene expression of a HDAd (HDAdCMVDysFl) and a FGAd (FGAdCMV-dys) in cell culture (HeLa, C2C12 myotubes) and in the muscle of mdx mice (the mouse model for DMD). Both vectors encoded dystrophin regulated by the same cytomegalovirus (CMV) promoter. We demonstrate that the amount of dystrophin expressed was significantly higher after gene transfer with FGAdCMV-dys compared to HDAdCMVDysFl both in vitro and in vivo. However, gene transfer with HDAdCMVDysFl in the presence of a FGAd resulted in a significant increase of dystrophin expression indicating that gene products synthesized by the FGAd increase, in trans, the amount of dystrophin produced. This enhancement occurred in cell culture and after gene transfer in the muscle of mdx mice and dystrophic golden retriever (GRMD) dogs, another animal model for DMD. The E4 region of Ad is required for the enhancement, because no increase of dystrophin expression from HDAdCMVDysFl was observed in the presence of an E1/E4-deleted Ad in vitro and in vivo. The characterization of these enhancing gene products followed by their inclusion into an HDAd may be required to produce sufficient dystrophin to mitigate the pathology of DMD by HDAd-mediated gene transfer.

The UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase gene is mutated in recessive hereditary inclusion body myopathy.

Hereditary inclusion body myopathy (HIBM; OMIM 600737) is a unique group of neuromuscular disorders characterized by adult onset, slowly progressive distal and proximal weakness and a typical muscle pathology including rimmed vacuoles and filamentous inclusions. The autosomal recessive form described in Jews of Persian descent is the HIBM prototype. This myopathy affects mainly leg muscles, but with an unusual distribution that spares the quadriceps. This particular pattern of weakness distribution, termed quadriceps-sparing myopathy (QSM), was later found in Jews originating from other Middle Eastern countries as well as in non-Jews. We previously localized the gene causing HIBM in Middle Eastern Jews on chromosome 9p12-13 (ref. 5) within a genomic interval of about 700 kb (ref. 6). Haplotype analysis around the HIBM gene region of 104 affected people from 47 Middle Eastern families indicates one unique ancestral founder chromosome in this community. By contrast, single non-Jewish families from India, Georgia (USA) and the Bahamas, with QSM and linkage to the same 9p12-13 region, show three distinct haplotypes. After excluding other potential candidate genes, we eventually identified mutations in the UDP-N-acetylglucosamine-2-epimerase/N-acetylmannosamine kinase (GNE) gene in the HIBM families: all patients from Middle Eastern descent shared a single homozygous missense mutation, whereas distinct compound heterozygotes were identified in affected individuals of families of other ethnic origins. Our findings indicate that GNE is the gene responsible for recessive HIBM.

Muscle-specific overexpression of the adenovirus primary receptor CAR overcomes low efficiency of gene transfer to mature skeletal muscle.

Significant levels of adenovirus (Ad)-mediated gene transfer occur only in immature muscle or in regenerating muscle, indicating that a developmentally regulated event plays a major role in limiting transgene expression in mature skeletal muscle. We have previously shown that in developing mouse muscle, expression of the primary Ad receptor CAR is severely downregulated during muscle maturation. To evaluate how global expression of CAR throughout muscle affects Ad vector (AdV)-mediated gene transfer into mature skeletal muscle, we produced transgenic mice that express the CAR cDNA under the control of the muscle-specific creatine kinase promoter. Five-month-old transgenic mice were compared to their nontransgenic littermates for their susceptibility to AdV transduction. In CAR transgenics that had been injected in the tibialis anterior muscle with AdVCMVlacZ, increased gene transfer was demonstrated by the increase in the number of transduced muscle fibers (433 +/- 121 in transgenic mice versus 8 +/- 4 in nontransgenic littermates) as well as the 25-fold increase in overall beta-galactosidase activity. Even when the reporter gene was driven by a more efficient promoter (the cytomegalovirus enhancer-chicken beta-actin gene promoter), differential transducibility was still evident (893 +/- 149 versus 153 +/- 30 fibers; P < 0.001). Furthermore, a fivefold decrease in the titer of injected AdV still resulted in significant transduction of muscle (253 +/- 130 versus 14 +/- 4 fibers). The dramatic enhancement in AdV-mediated gene transfer to mature skeletal muscle that is observed in the CAR transgenics indicates that prior modulation of the level of CAR expression can overcome the poor AdV transducibility of mature skeletal muscle and significant transduction can be obtained at low titers of AdV.

The spread of transgene expression at the site of gene construct injection.

Seven 2-day-old golden retriever pups were given focal intramuscular injections of a first generation adenovirus-dystrophin minigene construct and adenovirus-beta-galactosidase construct as a 2:1 mixture into the left anterior tibial muscle. The spread of transgene expression within the anterior tibial muscle was compared with the spread of methylene blue dye after identical injection into the contralateral muscle. Transgene expression 5-7 days after intramuscular injection was shown to extend between 5.8 and 11.6 mm along the biopsied muscle length (range of biopsy lengths 11.1-12.2 mm). The level of transgene expression at 2-2.5-mm intervals from the site of injection was significantly related to the distance from the site of injection (dystrophin, P = 0.009; beta-galactosidase, P = 0.015). The spread of methylene blue dye within the anterior tibial muscle < or =24 h after identical intramuscular injection demonstrated a similar pattern to the transgene expression, with dye staining measured between 5.5 and 8.5 mm along the muscle sample length (range of biopsy lengths 5.6-15.6 mm). The greatest transgene expression and dye staining was measured 2-2.5 mm proximal to the site of injection with a maximum of 23% of muscle fibers expressing the dystrophin transgene, 95.2% expressing the beta-galactosidase transgene, and 98% of the tissue section stained with methylene blue dye. These results suggest transgene expression after focal intramuscular injection is relatively localized around the site of injection. Further research is required to develop techniques that will provide transgene expression throughout the length and breadth of a muscle.

Differential effects of dystrophin and utrophin gene transfer in immunocompetent muscular dystrophy (mdx) mice.

Duchenne muscular dystrophy (DMD) is a fatal disease caused by defects in the gene encoding dystrophin. Dystrophin is a cytoskeletal protein, which together with its associated protein complex, helps to protect the sarcolemma from mechanical stresses associated with muscle contraction. Gene therapy efforts aimed at supplying a normal dystrophin gene to DMD muscles could be hampered by host immune system recognition of dystrophin as a "foreign" protein. In contrast, a closely related protein called utrophin is not foreign to DMD patients and is able to compensate for dystrophin deficiency when overexpressed throughout development in transgenic mice. However, the issue of which of the two candidate molecules is superior for DMD therapy has remained an open question. In this study, dystrophin and utrophin gene transfer effects on dystrophic muscle function were directly compared in the murine (mdx) model of DMD using E1/E3-deleted adenovirus vectors containing either a dystrophin (AdV-Dys) or a utrophin (AdV-Utr) transgene. In immunologically immature neonatal animals, AdV-Dys and AdV-Utr improved tibialis anterior muscle histopathology, force-generating capacity, and the ability to resist injury caused by high-stress contractions to an equivalent degree. By contrast, only AdV-Utr was able to achieve significant improvement in force generation and the ability to resist stress-induced injury in the soleus muscle of immunocompetent mature mdx animals. In addition, in mature mdx mice, there was significantly greater transgene persistence and reduced inflammation with utrophin compared to dystrophin gene transfer. We conclude that dystrophin and utrophin are largely equivalent in their intrinsic abilities to prevent the development of muscle necrosis and weakness when expressed in neonatal mdx animals with an immature immune system. However, because immunity against dystrophin places an important limitation on the efficacy of dystrophin gene replacement in an immunocompetent mature host, the use of utrophin as an alternative to dystrophin gene transfer in this setting appears to offer a significant therapeutic advantage.

The mutation of Pro789 to Leu reduces the activity of the fast-twitch skeletal muscle sarco(endo)plasmic reticulum Ca2+ ATPase (SERCA1) and is associated with Brody disease.

Brody disease is a rare inherited disorder of fast-twitch skeletal muscle function and is characterized by a lifelong history of exercise-induced impairment of skeletal muscle relaxation, stiffness, and cramps. The autosomal recessive inheritance of mutations in ATP2A1, the gene encoding SERCA1, which is the fast-twitch skeletal muscle sarcoplasmic reticulum Ca2+ ATPase, has been associated with Brody disease in three of six Brody families in which ATP2A1 has been sequenced. In the present analysis of the ATP2A1 gene in four unrelated families with autosomal recessive inheritance of Brody disease, three mutations were found in two families, leading to premature stop codons and truncated SERCA1. In a third family, the homozygous substitution of T for C2366 led to the missense mutation of Pro789 to Leu. The Pro789 to Leu mutant was readily expressed in HEK-293 cells, but it demonstrated an almost complete loss of Ca2+ transport activity because of reduced Ca2+ affinity. In a fourth family, the heterozygous substitution of T for C2455, mutating Arg819 to Cys, was identified. This mutation was also readily expressed in HEK-293 cells and shown to have near normal Ca2+ transport activity, indicating that it is not causal for Brody disease. These results confirm the genetic heterogeneity of Brody disease and emphasize the importance of a functional test for mutant SERCA1; immunostaining of skeletal muscle to detect the loss of SERCA1a protein is not adequate for the diagnosis of ATP2A1-linked Brody disease.

Skeletal muscle gene transfer: regeneration-associated deregulation of fast troponin I fiber type specificity.

Direct gene transfer into skeletal muscle in vivo presents a convenient experimental approach for studies of adult muscle gene regulatory mechanisms, including fast vs. slow fiber type specificity. Previous studies have reported preferential expression of fast myosin heavy chain and slow myosin light chain and troponin I (TnIslow) gene constructs in muscles enriched in the appropriate fiber type. We now report a troponin I fast (TnIfast) direct gene transfer study. We injected into the mouse soleus muscle plasmid DNA or recombinant adenovirus carrying a TnIfast/ beta-galactosidase (beta-gal) reporter construct that had previously been shown to be expressed specifically in fast fibers in transgenic mice. Surprisingly, microscopic histochemical analysis 1 and 4 wk postinjection showed similar TnIfast/beta-gal expression in fast and slow fibers. A low but significant level of muscle fiber segmental regeneration was evident in muscles 1 wk postinjection, and TnIfast/beta-gal expression was preferentially targeted to regenerating fiber segments. This finding can explain why TnIfast constructs are deregulated with regard to fiber type specificity, whereas the myosin constructs previously studied are not. The involvement of regenerating fiber segments in transduction by plasmid DNA and recombinant adenoviruses injected into intact normal adult muscle is an unanticipated factor that should be taken into account in the planning and interpretation of direct gene transfer experiments.

Modulation of Starling forces and muscle fiber maturity permits adenovirus-mediated gene transfer to adult dystrophic (mdx) mice by the intravascular route.

Duchenne muscular dystrophy (DMD) and other inherited myopathies lead to progressive destruction of most skeletal muscles in the body, including those responsible for maintaining respiration. DMD is a fatal disorder caused by defects in the dystrophin gene. Recombinant adenovirus vectors (AdV) are considered a promising means for therapeutic delivery of a functional dystrophin gene to DMD muscles. If AdV-mediated dystrophin gene replacement in DMD is to be successful, development of a systemic delivery method for targeting the large number of diseased muscles will be required. In this study we investigated two major factors preventing efficient AdV-mediated gene transfer to skeletal muscles of adult animals after intravascular AdV administration: (1) an inability of AdV particles to breach the endothelial barrier and enter into contact with myofibers, and (2) a relatively nonpermissive myofiber population for AdV infection due at least in part to insufficient levels of the coxsackie/adenovirus attachment receptor (CAR). On the basis of established principles governing the transendothelial flux of macromolecules, we further hypothesized that an alteration in Starling forces (increased hydrostatic and decreased osmotic pressures) within the intravascular compartment would facilitate AdV transendothelial flux via convective transport. In addition, experimental muscle regeneration was employed to increase the prevalence of immature myofibers in which CAR expression is upregulated. Here we report that by employing the above-described strategy, high-level heterologous reporter gene expression was achievable in hindlimb muscles of normal rats as well as dystrophic (mdx) mice (genetic homolog of DMD) after a single intraarterial injection of AdV. Microsphere studies confirmed enhanced transport into muscle of fluorescent tracer particles in the size range of AdV, and there was a high concordance between CAR upregulation and myofiber transduction after intraarterial AdV delivery. Furthermore, in mdx mice examined 10 days after intraarterial AdV delivery, the aforementioned procedures had no adverse effects on the force-generating capacity of targeted muscles. These findings have implications for eventual AdV-mediated gene therapy of generalized skeletal muscle diseases such as DMD using a systemic intraarterial delivery approach.

Prevention of the dystrophic phenotype in dystrophin/utrophin-deficient muscle following adenovirus-mediated transfer of a utrophin minigene.

Duchenne muscular dystrophy (DMD) is a progressive muscle wasting disorder caused by the lack of a subsarcolemmal protein, dystrophin. We have previously shown that the dystrophin-related protein, utrophin is able to compensate for the lack of dystrophin in the mdx mouse, the mouse model for DMD. Here, we explore whether utrophin delivered to the limb muscle of dystrophin/utrophin-deficient double knockout (dko) neonatal mice can protect the muscle from subsequent dystrophic damage. Utrophin delivery may avoid the potential problems of an immune response associated with the delivery of dystrophin to a previously dystrophin-deficient host. Dko muscle (tibialis anterior) was injected with a first generation recombinant adenovirus containing a utrophin minigene. Up to 95% of the fibres continued expressing the minigene 30 days after injection. Expression of utrophin caused a marked reduction from 80% centrally nucleated fibres (CNFs) in the uninjected dko TA to 12% in the injected dko TA. Within the region of the TA expressing the utrophin minigene, a significant decrease in the prevelance of necrosis was noted. These results demonstrate that the utrophin minigene delivered using an adenoviral vector is able to afford protection to the dystrophin/utrophin-deficient muscle of the dko mouse. Gene Therapy (2000) 7, 201-204.