Adenylosuccinic acid therapy ameliorates murine Duchenne Muscular Dystrophy.

Arising from the ablation of the cytoskeletal protein dystrophin, Duchenne Muscular Dystrophy (DMD) is a debilitating and fatal skeletal muscle wasting disease underpinned by metabolic insufficiency. The inability to facilitate adequate energy production may impede calcium (Ca2+) buffering within, and the regenerative capacity of, dystrophic muscle. Therefore, increasing the metabogenic potential could represent an effective treatment avenue. The aim of our study was to determine the efficacy of adenylosuccinic acid (ASA), a purine nucleotide cycle metabolite, to stimulate metabolism and buffer skeletal muscle damage in the mdx mouse model of DMD. Dystrophin-positive control (C57BL/10) and dystrophin-deficient mdx mice were treated with ASA (3000 µg.mL-1) in drinking water. Following the 8-week treatment period, metabolism, mitochondrial density, viability and superoxide (O2-) production, as well as skeletal muscle histopathology, were assessed. ASA treatment significantly improved the histopathological features of murine DMD by reducing damage area, the number of centronucleated fibres, lipid accumulation, connective tissue infiltration and Ca2+ content of mdx tibialis anterior. These effects were independent of upregulated utrophin expression in the tibialis anterior. ASA treatment also increased mitochondrial viability in mdx flexor digitorum brevis fibres and concomitantly reduced O2- production, an effect that was also observed in cultured immortalised human DMD myoblasts. Our data indicates that ASA has a protective effect on mdx skeletal muscles.

A Simple and Low-cost Assay for Measuring Ambulation in Mouse Models of Muscular Dystrophy.

Measuring functional outcomes in the treatment of muscular dystrophy is an essential aspect of preclinical testing. The assessment of voluntary ambulation in mouse models is a non-invasive and reproducible activity assay that is directly analogous to measures of patient ambulation such as the 6-minute walk test and related mobility scores. Many common methods for testing mouse ambulation speed and distance are based on the open field test, where an animal's free movement within an arena is measured over time. One major downside to this approach is that commercial software and equipment for high-resolution motion tracking is expensive and may require transferring mice to specialized facilities for testing. Here, we describe a low-cost, video-based system for measuring mouse ambulation that utilizes free and open-source software. Using this protocol, we demonstrate that voluntary ambulation in the dystrophin-null mdx mouse model for Duchenne muscular dystrophy (DMD) is decreased relative to wild-type mouse activity. In mdx mice expressing the utrophin transgene, these activity deficits are not observed and the total distance traveled is indistinguishable from wild-type mice. This method is effective for measuring changes in voluntary ambulation associated with dystrophic pathology, and provides a versatile platform that can be readily adapted to diverse research settings.

Combinatorial therapeutic activation with heparin and AICAR stimulates additive effects on utrophin A expression in dystrophic muscles.

Upregulation of utrophin A is an attractive therapeutic strategy for treating Duchenne muscular dystrophy (DMD). Over the years, several studies revealed that utrophin A is regulated by multiple transcriptional and post-transcriptional mechanisms, and that pharmacological modulation of these pathways stimulates utrophin A expression in dystrophic muscle. In particular, we recently showed that activation of p38 signaling causes an increase in the levels of utrophin A mRNAs and protein by decreasing the functional availability of the destabilizing RNA-binding protein called K-homology splicing regulatory protein, thereby resulting in increases in the stability of existing mRNAs. Here, we treated 6-week-old mdx mice for 4 weeks with the clinically used anticoagulant drug heparin known to activate p38 mitogen-activated protein kinase, and determined the impact of this pharmacological intervention on the dystrophic phenotype. Our results show that heparin treatment of mdx mice caused a significant ∼1.5- to 3-fold increase in utrophin A expression in diaphragm, extensor digitorum longus and tibialis anterior (TA) muscles. In agreement with these findings, heparin-treated diaphragm and TA muscle fibers showed an accumulation of utrophin A and ß-dystroglycan along their sarcolemma and displayed improved morphology and structural integrity. Moreover, combinatorial drug treatment using both heparin and 5-amino-4-imidazolecarboxamide riboside (AICAR), the latter targeting 5' adenosine monophosphate-activated protein kinase and the transcriptional activation of utrophin A, caused an additive effect on utrophin A expression in dystrophic muscle. These findings establish that heparin is a relevant therapeutic agent for treating DMD, and illustrate that combinatorial treatment of heparin with AICAR may serve as an effective strategy to further increase utrophin A expression in dystrophic muscle via activation of distinct signaling pathways.

XI-006 induces potent p53-independent apoptosis in Ewing sarcoma.

There is an imperious need for the development of novel therapeutics for the treatment of Ewing sarcoma, the second most prevalent solid bone tumour observed in children and young adolescents. Recently, a 4-nitrobenzofuroxan derivative, XI-006 (NSC207895) was shown to diminish MDM4 promoter activity in breast cancer cell lines. As amplification of MDM4 is frequently observed in sarcomas, this study examined the therapeutic potential of XI-006 for the treatment of Ewing and osteosarcoma. XI-006 treatment of Ewing and osteosarcoma cell lines (n = 11) resulted in rapid and potent apoptosis at low micro-molar concentrations specifically in Ewing sarcoma cell lines (48 hr IC50 0.099-1.61 µM). Unexpectedly, apoptotic response was not dependent on MDM4 mRNA/protein levels or TP53 status. Alkaline/neutral comet and γH2AX immunofluorescence assays revealed that the cytotoxic effects of XI-006 could not be attributed to the induction of DNA damage. RNA expression analysis revealed that the mechanism of action of XI-006 could be accredited to the inhibition of cell division and cycle regulators such as KIF20A and GPSM2. Finally, potent synergy between XI-006 and olaparib (PARP inhibitor) were observed due to the down-regulation of Mre11. Our findings suggest that XI-006 represents a novel therapeutic intervention for the treatment of Ewing sarcoma.

Second-generation compound for the modulation of utrophin in the therapy of DMD.

Duchenne muscular dystrophy (DMD) is a lethal, X-linked muscle-wasting disease caused by lack of the cytoskeletal protein dystrophin. There is currently no cure for DMD although various promising approaches are progressing through human clinical trials. By pharmacologically modulating the expression of the dystrophin-related protein utrophin, we have previously demonstrated in dystrophin-deficient mdx studies, daily SMT C1100 treatment significantly reduced muscle degeneration leading to improved muscle function. This manuscript describes the significant disease modifying benefits associated with daily dosing of SMT022357, a second-generation compound in this drug series with improved physicochemical properties and a more robust metabolism profile. These studies in the mdx mouse demonstrate that oral administration of SMT022357 leads to increased utrophin expression in skeletal, respiratory and cardiac muscles. Significantly, utrophin expression is localized along the length of the muscle fibre, not just at the synapse, and is fibre-type independent, suggesting that drug treatment is modulating utrophin transcription in extra-synaptic myonuclei. This results in improved sarcolemmal stability and prevents dystrophic pathology through a significant reduction of regeneration, necrosis and fibrosis. All these improvements combine to protect the mdx muscle from contraction induced damage and enhance physiological function. This detailed evaluation of the SMT C1100 drug series strongly endorses the therapeutic potential of utrophin modulation as a disease modifying therapeutic strategy for all DMD patients irrespective of their dystrophin mutation.

Safety, tolerability, and pharmacokinetics of SMT C1100, a 2-arylbenzoxazole utrophin modulator, following single- and multiple-dose administration to healthy male adult volunteers.

SMT C1100 is a small molecule utrophin modulator in development to treat Duchenne muscular dystrophy. This study evaluated the safety, tolerability, and pharmacokinetics of SMT C1100 in healthy volunteers. This double-blind, placebo-controlled Phase 1 study comprised: Part 1, an escalating, single-dose with/without fasting involving 50 mg/kg, 100 mg/kg, 200 mg/kg, and 400 mg/kg doses; and Part 2, a multiple 10 day dose evaluation involving 100 mg/kg bid and 200 mg/kg bid doses. Adverse events were recorded. SMT C1100 was absorbed rapidly following single and multiple oral doses, with median tmax attained within 2-3.5 hour across all doses. Considerable variability of pharmacokinetic parameters was noted among subjects. Following single doses, systemic exposure increased in a sub-proportional manner, with the 8.0-fold dose increment resulting in 2.7- and 2.4-fold increases in AUC0-∞ and Cmax , respectively. AUC0-∞ and Cmax were estimated as 4.2- and 4.8-fold greater, respectively, following food. Systemic exposure reduced upon repeat dosing with steady-state concentrations achieved within 3-5 days of multiple bid dosing. No serious or severe adverse events were reported. SMT C1100 was safe and well tolerated with plasma concentrations achieved sufficient to cause a 50% increase in concentrations of utrophin in cells in vitro.

The pros and cons of common actin labeling tools for visualizing actin dynamics during Drosophila oogenesis.

Dynamic remodeling of the actin cytoskeleton is required for both development and tissue homeostasis. While fixed image analysis has provided significant insight into such events, a complete understanding of cytoskeletal dynamics requires live imaging. Numerous tools for the live imaging of actin have been generated by fusing the actin-binding domain from an actin-interacting protein to a fluorescent protein. Here we comparatively assess the utility of three such tools--Utrophin, Lifeact, and F-tractin--for characterizing the actin remodeling events occurring within the germline-derived nurse cells during Drosophila mid-oogenesis or follicle development. Specifically, we used the UAS/GAL4 system to express these tools at different levels and in different cells, and analyzed these tools for effects on fertility, alterations in the actin cytoskeleton, and ability to label filamentous actin (F-actin) structures by both fixed and live imaging. While both Utrophin and Lifeact robustly label F-actin structures within the Drosophila germline, when strongly expressed they cause sterility and severe actin defects including cortical actin breakdown resulting in multi-nucleate nurse cells, early F-actin filament and aggregate formation during stage 9 (S9), and disorganized parallel actin filament bundles during stage 10B (S10B). However, by using a weaker germline GAL4 driver in combination with a higher temperature, Utrophin can label F-actin with minimal defects. Additionally, strong Utrophin expression within the germline causes F-actin formation in the nurse cell nuclei and germinal vesicle during mid-oogenesis. Similarly, Lifeact expression results in nuclear F-actin only within the germinal vesicle. F-tractin expresses at a lower level than the other two labeling tools, but labels cytoplasmic F-actin structures well without causing sterility or striking actin defects. Together these studies reveal how critical it is to evaluate the utility of each actin labeling tool within the tissue and cell type of interest in order to identify the tool that represents the best compromise between acceptable labeling and minimal disruption of the phenomenon being observed. In this case, we find that F-tractin, and perhaps Utrophin, when Utrophin expression levels are optimized to label efficiently without causing actin defects, can be used to study F-actin dynamics within the Drosophila nurse cells.

Muscle structure influences utrophin expression in mdx mice.

Duchenne muscular dystrophy (DMD) is a severe muscle wasting disorder caused by mutations in the dystrophin gene. To examine the influence of muscle structure on the pathogenesis of DMD we generated mdx4cv:desmin double knockout (dko) mice. The dko male mice died of apparent cardiorespiratory failure at a median age of 76 days compared to 609 days for the desmin-/- mice. An ∼ 2.5 fold increase in utrophin expression in the dko skeletal muscles prevented necrosis in ∼ 91% of 1a, 2a and 2d/x fiber-types. In contrast, utrophin expression was reduced in the extrasynaptic sarcolemma of the dko fast 2b fibers leading to increased membrane fragility and dystrophic pathology. Despite lacking extrasynaptic utrophin, the dko fast 2b fibers were less dystrophic than the mdx4cv fast 2b fibers suggesting utrophin-independent mechanisms were also contributing to the reduced dystrophic pathology. We found no overt change in the regenerative capacity of muscle stem cells when comparing the wild-type, desmin-/-, mdx4cv and dko gastrocnemius muscles injured with notexin. Utrophin could form costameric striations with α-sarcomeric actin in the dko to maintain the integrity of the membrane, but the lack of restoration of the NODS (nNOS, α-dystrobrevin 1 and 2, α1-syntrophin) complex and desmin coincided with profound changes to the sarcomere alignment in the diaphragm, deposition of collagen between the myofibers, and impaired diaphragm function. We conclude that the dko mice may provide new insights into the structural mechanisms that influence endogenous utrophin expression that are pertinent for developing a therapy for DMD.

Nitric oxide signalling pathway in Duchenne muscular dystrophy mice: up-regulation of L-arginine transporters.

DMD (Duchenne muscular dystrophy) is an incurable rapidly worsening neuromuscular degenerative disease caused by the absence of dystrophin. In skeletal muscle a lack of dystrophin disrupts the recruitment of neuronal NOS (nitric oxide synthase) to the sarcolemma thus affecting NO (nitric oxide) production. Utrophin is a dystrophin homologue, the expression of which is greatly up-regulated in the sarcolemma of dystrophin-negative fibres from mdx mice, a mouse model of DMD. Although cardiomyopathy is an important cause of death, little is known about the NO signalling pathway in the cardiac muscle of DMD patients. Thus we used cardiomyocytes and hearts from two month-old mdx and mdx:utrophin-/- (double knockout) mice (mdx:utr) to study key steps in NO signalling: L-arginine transporters, NOS and sGC (soluble guanylyl cyclase). nNOS did not co-localize with dystrophin or utrophin to the cardiomyocyte membrane. Despite this nNOS activity was markedly decreased in both mdx and mdx:utr mice, whereas nNOS expression was only decreased in mdx:utr mouse hearts, suggesting that utrophin up-regulation in cardiomyocytes maintains nNOS levels, but not function. sGC protein levels and activity remained at control levels. Unexpectedly, L-arginine transporter expression and function were significantly increased, suggesting a novel biochemical compensatory mechanism of the NO pathway and a potential entry site for therapeutics.

Resveratrol decreases inflammation and increases utrophin gene expression in the mdx mouse model of Duchenne muscular dystrophy.

BACKGROUND & AIMS: Duchenne muscular dystrophy (DMD) is a lethal genetic disease with no cure. Reducing inflammation or increasing utrophin expression can alleviate DMD pathology. Resveratrol can reduce inflammation and activate the utrophin promoter. The aims of this study were to identify an active dose of resveratrol in mdx mice and examine if this dose decreased inflammation and increased utrophin expression. METHODS: 5-week old mdx mice were given 0, 10, 100, or 500 mg/kg of resveratrol everyday for 10 days. Sirt1 was measured by qRT-PCR and used to determine the most active dose. Muscle inflammation was measured by H&E staining, CD45 and F4/80 immunohistochemistry. IL-6, TNFα, PGC-1α, and utrophin gene expression were measured by qRT-PCR. Utrophin, Sirt1, and PGC-1α protein were quantified by western blot. RESULTS: The 100 mg/kg dose of resveratrol, the most active dose, increased Sirt1 mRNA 60 ± 10% (p < 0.01), reduced immune cell infiltration 21 ± 6% (H&E) and 42 ± 8% (CD45 immunohistochemistry (p < 0.05)), reduced macrophage infiltration 48 ± 10% (F4/80 immunohistochemistry (p < 0.05)), and increased IL-6, PGC-1α, and utrophin mRNA 247 ± 77%, 27 ± 17%, and 43 ± 23% respectively (p ≤ 0.05). Utrophin, Sirt1, and PGC-1α protein expression did not change. CONCLUSIONS: Resveratrol may be a therapy for DMD by reducing inflammation.

Pharmacological therapies for muscular dystrophies.

PURPOSE OF REVIEW: The study reviews recent advances in pharmacological management of muscular dystrophies. Similarities and differences among the pathophysiology of different forms of muscular dystrophy lead to a broad array of approaches to provide new treatments. RECENT FINDINGS: In this review, we include only those muscular dystrophies for which advances have been published in the past year. This represents the 'advancing edge' of a large body of research over more than 20 years. This runs the gamut of new discoveries in symptomatic management to mutation-specific strategies that attempt to correct the root cause of the disorder. SUMMARY: The field of pharmacological therapies for the muscular dystrophies continues to steadily advance. It is encouraging that research into new therapies is increasingly exploring pharmacological strategies with the potential to ameliorate disease pathology to a clinically significant degree.

Discovery of 2-arylbenzoxazoles as upregulators of utrophin production for the treatment of Duchenne muscular dystrophy.

A series of novel 2-arylbenzoxazoles that upregulate the production of utrophin in murine H2K cells, as assessed using a luciferase reporter linked assay, have been identified. This compound class appears to hold considerable promise as a potential treatment for Duchenne muscular dystrophy. Following the delineation of structure-activity relationships in the series, a number of potent upregulators were identified, and preliminary ADME evaluation is described. These studies have resulted in the identification of 1, a compound that has been progressed to clinical trials.

Dystrophins and DAPs are expressed in adipose tissue and are regulated by adipogenesis and extracellular matrix.

The dystrophin-associated protein complex (DAPC), consisting of dystrophin, dystroglycans, sarcoglycans, dystrobrevins and syntrophins, provides a linkage between the cytoskeleton and the extracellular matrix. The disruption of DAPC leads to Duchenne/Becker muscular dystrophy and other neuromuscular diseases. Although adipose-derived stem cells had been used for the experimental treatment of Duchenne/Becker disease with promising results, little is known on the expression and function of DAPC in adipose tissue. Here we show that visceral and subcutaneous rat adipose depots express mRNAs for all known dystrophin isoforms, utrophin, α- and ß-dystrobrevins, and α-, ßI-, ßII-, and γII-syntrophins. Visceral and subcutaneous rat preadipocytes express Dp116 and Dp71 mRNAs and proteins, and this expression is differentially regulated during adipogenesis. Rat preadipocytes also express ß-dystrobrevin, α-, ßI-, ßII- and γII-syntrophins, ß-dystroglycan and ß-, δ-, and ε-sarcoglycans with no changes during adipogenesis. We also show that α-dystrobrevin increases their expression during adipose differentiation and extracellular matrix differentially regulates the expression of dystrophin isoforms mRNAs during adipogenesis. Our results show that DAPC components are expressed in adipose tissues and suggest that this complex has a role on the adipose biology.

Marginal level dystrophin expression improves clinical outcome in a strain of dystrophin/utrophin double knockout mice.

Inactivation of all utrophin isoforms in dystrophin-deficient mdx mice results in a strain of utrophin knockout mdx (uko/mdx) mice. Uko/mdx mice display severe clinical symptoms and die prematurely as in Duchenne muscular dystrophy (DMD) patients. Here we tested the hypothesis that marginal level dystrophin expression may improve the clinical outcome of uko/mdx mice. It is well established that mdx3cv (3cv) mice express a near-full length dystrophin protein at ∼5% of the normal level. We crossed utrophin-null mutation to the 3cv background. The resulting uko/3cv mice expressed the same level of dystrophin as 3cv mice but utrophin expression was completely eliminated. Surprisingly, uko/3cv mice showed a much milder phenotype. Compared to uko/mdx mice, uko/3cv mice had significantly higher body weight and stronger specific muscle force. Most importantly, uko/3cv outlived uko/mdx mice by several folds. Our results suggest that a threshold level dystrophin expression may provide vital clinical support in a severely affected DMD mouse model. This finding may hold clinical implications in developing novel DMD therapies.

Effects of inhibitors of the arachidonic acid cascade on primary muscle culture from a Duchenne muscular dystrophy patient.

The aim of this study was to elucidate the mechanisms of action for potential targets of therapeutic intervention related to the arachidonic acid cascade in muscular dystrophy. Primary cultures from a Duchenne patient were used to study the expression of dystrophin-1, utrophin, desmin, neonatal myosin heavy chain (MHCn) and Bcl-2 during inhibition of phospholipase A2 (PLA2), cyclooxygenase (COX) and lipoxygenase (LOX). Hypo-osmotic treatment was applied in order to trigger Ca2+ influx and PLA2 activity. Inhibition of PLA2 and LOX with prednisolone and nordihydroguaiaretic acid (NDGA) caused a semi-quantitative increase of utrophin and Bcl-2-, and a dose-dependent, quantitative increase of desmin expression, an effect that was augmented by hypo-osmotic treatment. Our results indicate that LOX inhibitors, similarly to corticosteroids, can be beneficial in the treatment of muscular dystrophies.

UTRN on chromosome 6q24 is mutated in multiple tumors.

Though deletion of the long arm of chromosome 6 is one of the most common aberrations in tumors, its targeted gene(s) has not been convincingly identified. Using a functional screening approach, we found that UTRN (which encodes utrophin, a dystrophin-related protein) at 6q24, when expressed in an antisense orientation, induced cellular transformation, consistent with a tumor suppressor role. Northern blot analysis, semiquantitative reverse transcription-polymerase chain reaction (RT-PCR), and gene expression arrays all showed that UTRN expression was downregulated in primary tumors compared with matched normal tissues. Several UTRN neighbor genes were not affected in some tumors with UTRN downregulation, suggesting that UTRN was specifically targeted. RT-PCR, coupled with an in vitro transcription and translation assay, revealed inactivation mutations in 21/62 breast cancers, 4/20 neuroblastomas and 4/15 malignant melanomas. Most of the mutations were deletions involving one or more exons that led to the truncation of utrophin. Splicing errors were found in two cases, and nonsense mutation in one case. Overexpression of a wild-type UTRN in breast cancer cells inhibited tumor cell growth in vitro and reduced their tumor potential in nude mice. Our studies suggest that UTRN is a candidate tumor suppressor gene.

Increased expression of cSHMT, Tbx3 and utrophin in plasma of ovarian and breast cancer patients.

Plasma samples of ovarian and breast cancer patients were used to search for markers of cancer using 2-dimensional gel electrophoresis and matrix-assisted laser desorption/ionization time of flight mass spectrometry. Truncated forms of cytosolic serine hydroxymethyl transferase (cSHMT), T-box transcription factor 3 (Tbx3) and utrophin were aberrantly expressed in samples from cancer patients as compared to samples from noncancerous cases. Aberrant expression of proteins was validated by immunoblotting of plasma samples with specific antibodies to cSHMT, Tbx3 and utrophin. A cohort of 79 breast and 39 ovarian cancer patients and 31 individuals with noncancerous conditions was studied. We observed increased expression of truncated cSHMT, Tbx3 and utrophin in plasma samples obtained from patients at early stages of disease. Our data suggest that cSHMT, Tbx3 and utrophin can be used as components of multiparameter monitoring of ovarian and breast cancer (supplementary material for this article can be found on the International Journal of Cancer website at http://www.interscience.wiley.com/jpages/0020-7136/suppmat/index.html).

Calcineurin-NFAT signaling, together with GABP and peroxisome PGC-1{alpha}, drives utrophin gene expression at the neuromuscular junction.

We examined whether calcineurin-NFAT (nuclear factors of activated T cells) signaling plays a role in specifically directing the expression of utrophin in the synaptic compartment of muscle fibers. Immunofluorescence experiments revealed the accumulation of components of the calcineurin-NFAT signaling cascade within the postsynaptic membrane domain of the neuromuscular junction. RT-PCR analysis using synaptic vs. extrasynaptic regions of muscle fibers confirmed these findings by showing an accumulation of calcineurin transcripts within the synaptic compartment. We also examined the effect of calcineurin on utrophin gene expression. Pharmacological inhibition of calcineurin in mice with either cyclosporin A or FK506 resulted in a marked decrease in utrophin A expression at synaptic sites, whereas constitutive activation of calcineurin had the opposite effect. Mutation of the previously identified NFAT binding site in the utrophin A promoter region, followed by direct gene transfer studies in mouse muscle, led to an inhibition in the synaptic expression of a lacZ reporter gene construct. Transfection assays performed with cultured myogenic cells indicated that calcineurin acted additively with GA binding protein (GABP) to transactivate utrophin A gene expression. Because both GABP- and calcineurin-mediated pathways are targeted by peroxisome proliferator-activated receptor-gamma coactivator-1alpha (PGC-1alpha), we examined whether this coactivator contributes to utrophin gene expression. In vitro and in vivo transfection experiments showed that PGC-1alpha alone induces transcription from the utrophin A promoter. Interestingly, this induction is largely potentiated by coexpression of PGC-1alpha with GABP. Together, these studies indicate that the synaptic expression of utrophin is also driven by calcineurin-NFAT signaling and occurs in conjunction with signaling events that involve GABP and PGC-1alpha.

Evolution of pathological changes in the gastrocnemius of the mdx mice correlate with utrophin and beta-dystroglycan expression.

Utrophin can function in muscle as a substitute for dystrophin and its over-expression has been used successfully to ameliorate mdx muscle pathology. Despite of this fact, there are no detailed studies on the expression of endogenous skeletal muscle utrophin- and dystrophin-associated glycoproteins throughout the life span of mdx mice. We have monitored, sequentially, the expression of matrix metalloproteinase-9 (MMP-9), myosin heavy chain, utrophin and beta-dystroglycan, as well as the mRNA expression of utrophin and of structurally related proteins, in mdx and control mice. We found an inverse relationship between concentration of muscle utrophin and abundance of groups of degenerative-regenerative fibers and of MMP-9 expression. There was also temporal correlation between the decline of utrophin at 15 days of age and the onset of muscle necrosis. Conversely, reappearance of utrophin, with a peak around 2 months of age, was followed by a progressive decline of necrosis. A lineal correlation between utrophin and beta-dystroglycan levels, not seen in controls, indicates that improvement of mdx is due to utrophin binding to dystrophin-associated glycoproteins. Utrophin and other structurally related protein transcripts were not up-regulated, suggesting a post-transcriptional regulation for utrophin in skeletal muscle.

Factors influencing the efficacy, longevity, and safety of electroporation-assisted plasmid-based gene transfer into mouse muscles.

Intramuscular injection of plasmid is a potential alternative to viral vectors for the transfer of therapeutic genes into skeletal muscle fibers. The low efficiency of plasmid-based gene transfer can be enhanced by electroporation (EP) coupled with the intramuscular application of hyaluronidase. We have investigated several factors that can influence the efficiency of plasmid-based gene transfer. These factors include electrical parameters of EP, optimal use of hyaluronidase, age and strain of the host, and plasmid size. Muscles of very young and mature normal, mdx, and immunodeficient mice were injected with plasmids expressing beta-galactosidase, microdystrophin, full-length dystrophin, or full-length utrophin. Transfection efficiency, muscle fiber damage, and duration of transgene expression were analyzed. The best transfection level with the least collateral damage was attained at 175-200 V/cm. Pretreatment with hyaluronidase markedly increased transfection, which was also influenced by the plasmid size and the strain and the age of the mice. Even in immunodeficient mice, there was a significant late decline in transgene expression and plasmid DNA copies, although both still remained relatively high after 1 year. Thus, properly optimized EP-assisted plasmid-based gene transfer is a feasible, efficient, and safe method of gene replacement therapy for dystrophin deficiency of muscle but readministration may be necessary.