MCK Promoter Research Articles

Expression and function of Four AAV-based constructs for dystrophin restoration in the mdx mouse model of duchenne muscular dystrophy.

Robust expression of shortened, functional dystrophin provided impetus to develop adeno-associated virus (AAV)-based constructs for clinical application. Because several cassettes are being tested in clinical trials, this study compared efficacy of the following four shortened dystrophin-promoter combinations with implications for outcomes in clinical trials: MHCK7 or MCK promoter with a shortened dystrophin transgene containing the N-terminus and spectrin repeats R1, R2, R3 and R24 (rAAVrh74.MHCK7.micro-dystrophin and rAAVrh74.MCK.micro-dystrophin, respectively); shortened dystrophin construct containing the neuronal nitric oxide (nNOS) binding site (rAAVrh74.MHCK7.DV.mini-dystrophin); and shortened dystrophin containing the C-terminus (rAAVrh74.MHCK7.micro-dystrophin.Cterm). Functional and histological benefit were examined at 4 weeks following intramuscular delivery in mdx mice. rAAVrh74.MHCK7.micro-dystrophin provided the most robust transgene expression and significantly increased specific force output in the tibialis anterior muscle. Muscle environment was normalized (i.e., reductions in central nucleation), indicating functional and histological advantages of rAAVrh74.MHCK7.micro-dystrophin. Thus, promoter choice and transgene design are critical for optimal dystrophin expression/distribution for maximal functional improvement.

Pre Clinical Assessment of AAVrh74.MCK.GNE Viral Vector Therapeutic Potential: Robust Activity Despite Lack of Consistent Animal Model for GNE Myopathy

Background:GNE myopathy is a unique adult onset rare neuromuscular disease caused by recessive mutations in the GNE gene. The pathophysiological mechanism of this disorder is not well understood and to date, there is no available therapy for this debilitating disease. We have previously established proof of concept that AAV based gene therapy can effectively deliver the wild type human GNE into cultured muscle cells from human patients and in mice, using a CMV promoter driven human wild type GNE plasmid delivered through an adeno associated virus (AAV8) based platform.Objective:In the present study we have generated a muscle specific GNE construct, driven by the MCK promoter and packaged with the AAVrh74 serotype for efficacy evaluation in an animal model of GNE Myopathy.Methods:The viral vector was systemically delivered at 2 doses to two age groups of a Gne–/– hGNED207V Tg mouse described as a preclinical model of GNE Myopathy, and treatment was monitored for long term efficacy.Results:In spite of the fact that the full described characteristics of the preclinical model could not be reproduced, the systemic injection of the rAAVrh74.MCK.GNE viral vector resulted in a long term presence and expression of human wt GNE in the murine muscles and in some improvements of their mild phenotype. The Gne–/– hGNED207V Tg mice are smaller from birth, but cannot be differentiated from littermates by muscle function (grip strength and Rotarod) and their muscle histology is normal, even at advanced age.Conclusions:The rAAVrh74.MCK.GNE vector is a robust tool for the development of GNE Myopathy therapies that supply the intact GNE. However, there is still no reliable animal model to fully assess its efficacy since the previously developed Gne–/– hGNED207V Tg mice do not present disease characteristics.

Sema3a-Nrp1 Signaling Mediates Fast-Twitch Myofiber Specificity of Tw2+ Cells.

We previously identified a unique population of interstitial muscle progenitors, marked by expression of the Twist2 transcription factor, which fuses specifically to type IIb/x fast-twitch myofibers. Tw2+ progenitors are distinct from satellite cells, a muscle progenitor that expresses Pax7 and contributes to all myofiber types. Through RNA sequencing and immunofluorescence, we identify the membrane receptor, Nrp1, as a marker of Tw2+ cells but not Pax7+ cells. We also found that Sema3a, a chemorepellent ligand for Nrp1, is expressed by type I and IIa myofibers but not IIb myofibers. Using stripe migration assays, chimeric cell-cell fusion assays, and a Sema3a transgenic mouse model, we identify Sema3a-Nrp1 signaling as a major mechanism for Tw2+ cell fiber-type specificity. Our findings reveal an extracellular signaling mechanism whereby a cell-surface receptor for a chemorepellent confers specificity of intercellular fusion of a specific muscle progenitor with its target tissue.

T.I.2: Therapeutic efforts in GNE myopathy

GNE myopathy (also known as HIBM or DMRV) is an autosomal recessive disease due to biallelic mutations in GNE , a gene encoding for a key enzyme in sialic acid biosynthesis. It is assumed that the presence of the mutated protein (or lack of its normal activity) and muscle hyposialylation contribute to the pathophysiology of the disease. Given the putative pathophysiology several therapy approaches are being tried: 1. Metabolic bypass with- a. oral supplementation of high dose sialic acid (both as extended release and regular capsules). 48weeks results of an ongoing trial in 46 patients show clinically meaningful effect in stabilizing upper extremities muscle strength associated with increased serum levels of sialic acid (H. Lau – this meeting). b. oral ManNAc (a precursor of sialic acid). Phase 1 trial has been concluded. c. IVG which contains high sialic acid showed modest effect and was abandoned. 2. Gene therapy- a. compassionate trial in a single patient used IV liposomal delivery of GNE coupled to CMV promoter. Proof of concept for mild increased sialylation was observed. b. viral-mediated gene therapy. A platform for human therapy trial has been developed based on AAV8 harboring the wild type human GNE and driven by MCK promoter. Single IV injection of this construct proved safe and resulted in human GNE expression in normal mice (Mitrani Rosenbaum – this meeting). Now that therapies are under investigation, it is critical that patients with GNE myopathy should be identified at the early stages of the disease. Disclosure: ZA is a consultant for Ultragenyx and Bioblast pharma.

G.P.55: GNE myopathy: Assessment of an adeno associated virus AAV2/8 based GNE gene delivery system

GNE Myopathy is a unique neuromuscular disorder characterized by adult onset and slowly progressive distal and proximal muscle and a typical histology including rimmed vacuoles and filamentous inclusions. The disease is caused by recessive mutations in the UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase gene (GNE), encoding the key enzyme in the biosynthetic pathway of sialic acid. (In an attempt to develop a possible treatment for GNE Myopathy patients.) We have established a platform for gene therapy trials based on adeno associated virus (AAV 8) vectors harboring the wild type human GNE gene driven by an MCK promoter. These viral constructs proved to be safe when injected systemically (tail vein) in healthy C57Bl6 mice. No mice presented any physical or behavioral change. All tissues analyzed for histology 35 days post infection showed no pathological findings. Real Time PCR analysis of the expression of human GNE showed it is expressed in all tissues analyzed, including skeletal muscles of the upper and lower limbs. This was confirmed by the expression of a control virus, where GNE was replaced by the luciferase gene, which showed luciferase expression disseminated in all organs. To assess the efficacy of the system, this AAV8/MCKGNE viral vectors are being assayed in the GNE Myopathy mouse model generated by Malicdan et al. (2007) We believe that the assessment of the effects of GNE viral delivery will be of great value by setting the stage for potential human trials.

Upregulation of triglyceride synthesis in skeletal muscle overexpressing DGAT1

The gene encoding diacylglycerol acyltransferase (DGAT1) is a functional and positional candidate gene for milk and intramuscular fat content. A bovine DGAT1 overexpression vector was constructed containing mouse MCK promoter and bovine DGAT1 cDNA. MCK-DGAT1 transgene in FVB mice was researched in present study. The transgene DGAT1 had a high level of expression in contrast to the endogenous DGAT1 in posterior tibial muscle of the transgenic mice, but a low expression level in the cardiac muscle. Compared with wild-type mice, triglyceride and DGAT1 content were approximately fourfold and 50% increased in posterior tibial muscle of the transgenic mice, respectively, while a little increase in cardiac muscle.

Insulin stimulates fusion, but not tethering, of GLUT4 vesicles in skeletal muscle of HA-GLUT4-GFP transgenic mice

Insulin regulates glucose uptake into fat and muscle by modulating the subcellular distribution of GLUT4 between the cell surface and intracellular compartments. However, quantification of these translocation processes in muscle by classical subcellular fractionation techniques is confounded by contaminating microfibrillar protein; dynamic studies at the molecular level are almost impossible. In this study, we introduce a muscle-specific transgenic mouse model in which HA-GLUT4-GFP is expressed under the control of the MCK promoter. HA-GLUT4-GFP was found to translocate to the plasma membrane and T-tubules after insulin stimulation, thus mimicking endogenous GLUT4. To investigate the dynamics of GLUT4 trafficking in skeletal muscle, we quantified vesicles containing HA-GLUT4-GFP near the sarcolemma and T-tubules and analyzed insulin-stimulated exocytosis at the single vesicle level by total internal reflection fluorescence and confocal microscopy. We found that only 10% of the intracellular GLUT4 pool comprised mobile vesicles, whereas most of the GLUT4 structures remained stationary or tethered at the sarcolemma or T-tubules. In fact, most of the insulin-stimulated exocytosis emanated from pretethered vesicles, whereas the small pool of mobile GLUT4 vesicles was not significantly affected by insulin. Our data strongly suggest that the mobile pool of GLUT4 vesicles is not a major site of insulin action but rather locally distributed. Most likely, pretethered GLUT4 structures are responsible for the initial phase of insulin-stimulated exocytosis.

Transcription Factor Rational Design Improves Directed Differentiation of Human Mesenchymal Stem Cells Into Skeletal Myocytes

There is great interest in transdifferentiating cells from one lineage into those of another and in dedifferentiating mature cells back into a stem/progenitor cell state by deploying naturally occurring transcription factors (TFs). Often, however, steering cellular differentiation pathways in a predictable and efficient manner remains challenging. Here, we investigated the principle of combining domains from different lineage-specific TFs to improve directed cellular differentiation. As proof-of-concept, we engineered the whole-human TF MyoDCD, which has the NH(2)-terminal transcription activation domain (TAD) and adjacent DNA-binding motif of MyoD COOH-terminally fused to the TAD of myocardin (MyoCD). We found via reporter gene and marker protein assays as well as by a cell fusion readout system that, targeting the TAD of MyoCD to genes normally responsive to the skeletal muscle-specific TF MyoD enforces more robust myogenic reprogramming of nonmuscle cells than that achieved by the parental, prototypic master TF, MyoD. Human mesenchymal stem cells (hMSCs) transduced with a codon-optimized microdystrophin gene linked to a synthetic striated muscle-specific promoter and/or with MyoD or MyoDCD were evaluated for complementing the genetic defect in Duchenne muscular dystrophy (DMD) myocytes through heterotypic cell fusion. Cotransduction of hMSCs with MyoDCD and microdystrophin led to chimeric myotubes containing the highest dystrophin levels.

Nfix Regulates Fetal-Specific Transcription in Developing Skeletal Muscle

Skeletal myogenesis, like hematopoiesis, occurs in successive developmental stages that involve different cell populations and expression of different genes. We show here that the transcription factor nuclear factor one X (Nfix), whose expression is activated by Pax7 in fetal muscle, in turn activates the transcription of fetal specific genes such as MCK and beta-enolase while repressing embryonic genes such as slow myosin. In the case of the MCK promoter, Nfix forms a complex with PKC theta that binds, phosphorylates, and activates MEF2A. Premature expression of Nfix activates fetal and suppresses embryonic genes in embryonic muscle, whereas muscle-specific ablation of Nfix prevents fetal and maintains embryonic gene expression in the fetus. Therefore, Nfix acts as a transcriptional switch from embryonic to fetal myogenesis.

MEK1 plays contrary stage-specific roles in skeletal myogenic differentiation

The role of extracellular signal-regulated kinase (ERK) signaling in skeletal myogenesis has been reported to be both stimulatory and inhibitory. We propose that this discrepancy may arise from the stage-specific, different roles of mitogen-activated protein kinase kinase 1 (MEK1). We found that the phosphorylated MEK1 level of differentiating C2C12 cells was low on day 1 (early-stage) and reached a maximum on days 2–3 (mid-stage). Cells treated at early stage with the MEK-specific inhibitors, PD184352 and U0126, reduced both the MHC protein level and MCK promoter activity, demonstrating that high MEK1 activity at the mid-stage is required for myogenic differentiation. In contrast, treatment with the ERK-specific inhibitors, FR180204 and ERK inhibitor I, had no effect. However, because the sustained overexpression of constitutively active MEK1 inhibits myogenic differentiation, we further analyzed the stage-specific role of MEK1 using the Tet-Off expression system. The results demonstrated that myogenic differentiation was inhibited if active MEK1 expression was induced earlier than day 1 in differentiation condition, but stimulated if induced after that, demonstrating that activated MEK1 plays differential roles depending on activation time. In addition, the induction of active MEK1 at 12h enhanced the Id2 protein level, while the induction at 36h resulted in reduction. Thus, MEK1 plays stage-specific and contrary roles in myogenesis, and MEK1 activated at the mid-stage promotes muscle differentiation independent of ERK.

2008 Robert Levy Memorial Lecture—Tissue-Specific Regulation of Lipoprotein Lipase and Energy Balance: The Story Gets Even More Interesting

Lipoprotein lipase (LPL) is a multifunctional enzyme produced by and studied in many tissues, including adipose tissue, cardiac and skeletal muscle, islets, and macrophages. After synthesis by parenchymal cells, the lipase is transported to the capillary endothelium, where it is rate-limiting for the hydrolysis of the triglyceride (TG) core of the circulating TG-rich lipoproteins, chylomicrons, and very low density lipoproteins (VLDL). The reaction products, fatty acids and monoacylglycerol, are in part taken up by the tissues locally, where they are processed in a tissue-specific manner, e.g., stored as neutral lipids (TG > cholesteryl esters[CE]) in adipose tissue, oxidized or stored in muscle, or as CE/TG in foam cells in macrophages. LPL is regulated in a tissue-specific manner. In adipose tissue, LPL is increased by insulin and meals but decreased by fasting, whereas muscle LPL is decreased by insulin and increased by fasting. In obesity, adipose tissue LPL is increased; however, the insulin dose-response curve is shifted to the right. After weight reduction and stabilization of the reduced obese state, adipose tissue LPL is increased, as is the response of the enzyme to insulin and meals. In skeletal muscle, insulin does not stimulate LPL nor is the enzyme activity changed in obesity; however, after weight reduction, LPL in skeletal muscle is decreased by 70%. These tissue-specific changes in LPL set the stage for lipid partitioning to help explain the recidivism of obesity. To examine this divergent regulation further, transgenic and knockout murine models of tissue-specific LPL expression have been developed. Mice with overexpression of LPL in skeletal muscle develop TG accumulation in muscle, develop insulin resistance, are protected from excessive weight gain, and increase their metabolic rate in the cold. When placed onto the LPL knockout and leptin deficient background, overexpression of LPL using an MCK promoter reduces obesity. Alternatively, a deletion of LPL in skeletal muscle reduces TG accumulation and increases insulin-mediated glucose transport into muscle but leads to lipid partitioning to other tissues, insulin resistance, and obesity. In the heart, loss of LPL is associated with hypertriglyceridemia and a greater utilization of glucose, implying that free fatty acids are not a sufficient fuel for optimal cardiac function. LPL is also produced in the brain, and that’s where the “story gets even more interesting.” We have just created mice with a neuron-specific deletion of LPL (NEXLPL−/−) using cre recombinase driven by the helix-loop-helix nuclear transcription factor NEX promoter. By 6 months of age, NEXLPL−/− mice weigh 50% more than their litter mates. This phenotype provides convincing evidence that lipoprotein sensing occurs in the brain and is important to energy balance and body weight regulation. Overall, LPL is a fascinating enzyme that contributes in a pronounced way to normal lipoprotein metabolism, tissue-specific substrate delivery and utilization, and to the many aspects of metabolism that relate to cardiovascular disease, including energy metabolism, insulin action, body weight regulation, and atherosclerosis.

SOCS-3 Induces Myoblast Differentiation

Myoblast differentiation is characterized by a sequence of events that includes an increase in insulin-like growth factor (IGF)-I and contractile gene expression. The increase in IGF-I expression activates cell signaling mechanisms that participate in the differentiation process. One potential contributor is the SOCS-3 (suppressor of cytokine signaling-3) gene, which regulates signaling mechanisms and may be sensitive to changes in IGF-I concentrations. For the first time, the role of SOCS-3 is investigated in myoblast differentiation. SOCS-3 mRNA levels and SOCS-3 transcriptional activity increase during myoblast differentiation. SOCS-3 gene expression is induced, at least in part, by activation of the IGF-I receptor during myoblast differentiation. Overexpression of SOCS-3 cDNA significantly increased transcriptional activation of the 2.0-kb skeletal alpha-actin promoter in differentiating C2C12 myoblasts. In addition, overexpression of SOCS-3 specifically increased serum response factor-driven transcriptional activity but had no effect on nuclear-factor of activated T cell-driven transcriptional activity. SOCS-3 overexpression induced skeletal alpha-actin transcription in a myoblast cell line that cannot respond to endogenous IGF-I, indicating that SOCS-3 can contribute to the myoblast differentiation process in the absence of IGF-I. These data suggest that IGF-I induces myoblast differentiation, in part, by increasing SOCS-3 expression.

AAV vector-mediated microdystrophin expression in a relatively small percentage of mdx myofibers improved the mdx phenotype

Duchenne muscular dystrophy (DMD) is a lethal disorder of skeletal muscle caused by mutations in the dystrophin gene. Adeno-associated virus (AAV) vector-mediated gene therapy is a promising approach to the disease. Although a rod-truncated microdystrophin gene has been proven to ameliorate dystrophic phenotypes, the level of microdystrophin expression required for effective gene therapy by an AAV vector has not been determined yet. Here, we constructed a recombinant AAV type 2 vector, AAV2-MCKΔCS1, expressing microdystrophin (ΔCS1) under the control of a muscle-specific MCK promoter and injected it into TA muscles of 10-day-old and 5-week-old mdx mice. AAV2-MCKΔCS1-mediated gene transfer into 5-week-old mdx muscle resulted in extensive and long-term expression of microdystrophin and significantly improved force generation. Interestingly, 10-day-old injected muscle expressed microdystrophin in a limited number of myofibers but showed hypertrophy of microdystrophin-positive muscle fibers and considerable recovery of contractile force. Thus, we concluded that AAV2-MCKΔCS1 could be a powerful tool for gene therapy of DMD.

244. Successful AAV Vector-Mediated Gene Transfer into Canine Skeletal Muscle Required Suppression of Excess Immune Responses

Duchenne muscular dystrophy (DMD) is an X-linked, lethal muscle disorder caused by a mutation in the dystrophin gene (14 kb cDNA). An adeno-associated virus (AAV) vector-mediated gene transfer is one of attractive approaches to the treatment of DMD, but it has a limitation in insertion size up to 4.9 kb. To find a short but functional dystrophin cDNA, we have previously constructed three micro-dystrophin cDNAs, and generated transgenic (Tg) dystrophin-deficient mdx mice expressing micro-dystrophin. Among them, CS1-Tg mdx mice showed lowest levels of serum creatine kinase, complete amelioration of muscle pathology, and nearly full restoration of contractile force (BBRC. 293:1265, 2002). We also showed that muscle-specific MCK promoter in AAV vector could drive longer expression of the LacZ gene than the CMV promoter in skeletal muscle (Gene Ther. 23:1576, 2002). Furthermore, we constructed the AAV2 vector expressing ΔCS1 micro-dystrophin driven by MCK promoter, and demonstrated that AAV vector-mediated ΔCS1 transfer could ameliorate dystrophic phenotypes in mdx muscles (7th ASGT Annual Meeting 2004, in submission). For the application of this strategy to DMD patients, however, it is necessary to examine therapeutic effects and the safety issue in larger animal models, such as dystrophic dogs. We recently established a colony of beagle-based canine X-linked muscular dystrophy in Japan (Exp. Anim. 52: 93, 2003). When the AAV vector encoding the LacZ gene driven by a CMV promoter was introduced into skeletal muscles of dogs, β-galactosidase (β-gal) was expressed only in few fibers of injected muscle after 2 weeks of injection. No β-gal-positive fiver was detected in canine muscle at 4 and 8 weeks post-injection. Instead, large numbers of mononuclear cells appeared around β-gal-expressing fibers in injected muscle. To clarify mechanisms of low transduction and cellular infiltration in canine muscle after transfer of AAV vector, we examined viral infectivity, cytotoxicity and immune responses. First, we infected AAV vector into canine primary myotubes. This in vitro study showed that AAV vector could allow higher transgene expression in canine myotubes than in murine ones. Second, we tested whether injection of AAV particle elicit cytotoxicity or not. When the AAV vector expressing no transgene was injected into canine muscle, almost no infiltrating cells was observed in injected muscle. Third, we investigated immune responses. A lot of CD4- or CD8-positive cells were detected in clusters of infiltrating cells, together with elevated serum level of anti-β-gal IgG. To confirm low transduction depending on immune response, dogs received daily oral administration of cyclosporine (20 mg/kg/day) from -5 day of the introduction of the AAV vector. Immunosuppression considerably improved transduction efficiency by an AAV vector introduction in canine muscle. These results suggested that AAV vector-mediated gene transfer elicited stronger immune responses in canine muscle, and it was necessary to know the molecular background of excess immune responses and to find the way to minimize and suppress immune responses.

378. An AAV Vector-Mediated Micro-Dystrophin Expression in Relatively Small Percentage of Dystrophin-Deficient mdx Myofibers Still Improved the mdx Phenotype through Compensatory Hypertrophy

Top of pageAbstract [Background] Duchenne muscular dystrophy (DMD) is an X-linked, lethal muscle disorder caused by mutations in the dystrophin gene. An adeno-associated virus (AAV) vector-mediated gene transfer is one of attractive approaches to the treatment of DMD, though it has a limitation in insertion size up to 4.9 kb. Therefore, a full-length dystrophin cDNA (14 kb) cannot be incorporated into an AAV vector. We previously generated micro-dystrophin transgenic mdx mice. Micro-dystrophin CS1 transgenic mdx mice showed almost complete amelioration of dystrophic phenotypes (BBRC. 293: 1265, 2002). [Objective] We constructed an AAV vector expressing micro-dystrophin ΔCS1, and introduced it into skeletal muscles of mdx mice and examined whether the dystrophic process had been ameliorated or not. [Method] To incorporate micro-dystrophin CS1 cDNA (4.9 kb) into an AAV vector, we deleted 5′- and 3′-UTRs and exons 71-78 (alternative splicing regions), resulting 3.8 kb ΔCS1 cDNA. We produced type 2 AAV vector expressing ΔCS1 under the control of muscle specific MCK promoter to avoid immune response against transgene product (Gene Ther. 9: 1576, 2002), designated AAV2-MCKΔCS1. The vector was injected into anterior tibial (TA) muscles of 10-day-old and 5-week-old mdx mice. Mdx muscles show no obvious changes of degeneration at 10-day, whereas 5-week-old mdx muscles exhibit active cycles of muscle degeneration/regeneration. [Result] When the AAV2-MCKΔCS1 was injected at 5 weeks of age, dystrophin-positive fibers were 51.5 ± 17.3% at 24 weeks after the injection. The ratio of centrally nucleated fibers in ΔCS1-positive fibers was significantly reduced compared with that of ΔCS1-negative fibers, indicating protective function of ΔCS1 against muscle degeneration. Furthermore, AAV-injected muscles revealed complete recovery of the specific tetanic force. When injected at 10-day-old, ΔCS1-positive fibers was 16.5 ± 7.0% at 24 weeks. Most of ΔCS1-positive fibers were peripherally nucleated. Surprisingly, there was no statistical difference in specific tetanic force between AAV-injected mdx muscles and B10 muscles. To clarify the mechanism of physiological recovery due to small numbers of ΔCS1-positive fibers, we examined the relationship between myofiber hypertrophy and force generation. We found positive correlation between the muscle weight and the force generation when injected at 10-day-old. To confirm whether increased muscle weight reflected myofiber hypertrophy, we measured cross section areas (CSAs) of individual fibers. Fiber CSAs were remarkably larger in ΔCS1-positive mdx fibers compared with ΔCS1-nagative, non-treated mdx fibers, and even B10 fibers. Thus, selective hypertrophy of ΔCS1-positive fibers seemed to greatly assist contractile force generation. [Conclusion] The AAV2-MCKΔCS1 could be a powerful tool for the gene therapy of DMD. For clinical application of this strategy to DMD patients, experiments using a bigger animal model, e.g. canine X-linked muscular dystrophy will be very important.

Expression of dystrophin driven by the 1.35-kb MCK promoter ameliorates muscular dystrophy in fast, but not in slow muscles of transgenic mdx mice

Successful gene therapy of Duchenne muscular dystrophy may require the lifelong expression of a therapeutic gene in all affected muscles. The most promising gene delivery vehicles, viral vectors, suffer from several limitations, including immunogenicity, loss of therapeutic gene expression, and a limited packaging capacity. Therefore, various efforts were previously undertaken to use small therapeutic genes and to place them under the control of a strong and muscle-specific promoter. Here we report the effects of a minidystrophin (6.3 kb) under the control of a short muscle-specific promoter (MCK 1.35 kb) over most of the lifetime (4–20 months) of a transgenic mouse model. Dystrophin expression remained stable and muscle-specific at all ages. The dystrophic phenotype was greatly ameliorated and, most importantly, muscle function in limb muscles was significantly improved not only in young adult but also in aged mice compared to nontransgenic littermates. Dystrophin expression was strong in fast-twitch skeletal muscles such as tibialis anterior and extensor digitorum longus, but weak or absent in heart, diaphragm, and slow-twitch muscles. Additionally, expression was strong in glycolytic but weak in oxidative fibers of fast-twitch muscles. This study may have important implications for the design of future gene therapy trials for muscular dystrophy.

Adeno-associated virus vector-mediated gene transfer into dystrophin-deficient skeletal muscles evokes enhanced immune response against the transgene product.

Duchenne muscular dystrophy (DMD) is an X-linked, lethal muscular disorder caused by a defect in the DMD gene. AAV vector-mediated micro-dystrophin cDNA transfer is an attractive approach to treatment of DMD. To establish effective gene transfer into skeletal muscle, we examined the transduction efficiency of an AAV vector in skeletal muscles of dystrophin-deficient mdx mice. When an AAV vector encoding the LacZ gene driven by a CMV promoter (AAV-CMVLacZ) was introduced, beta-galactosidase expression markedly decreased in mdx muscle 4 weeks after injection due to immune responses against the transgene product. We also injected AAV-CMVLacZ into skeletal muscles of mini-dystrophin-transgenic mdx mice (CVBA3'), which show ameliorated phenotypes without overt signs of muscle degeneration. AAV vector administration, however, evoked substantial immune responses in CVBA3' muscle. Importantly, AAV vector using muscle-specific MCK promoter also elicited responses in mdx muscle, but at a considerably later period. These results suggested that neo-antigens introduced by AAV vectors could evoke immune reactions in mdx muscle, since increased permeability allowed a leakage of neo-antigens from the dystrophin-deficient sarcolemma of muscle fibers. However, resident antigen-presenting cells, such as myoblasts, myotubes and regenerating immature myofibers, might also play a role in the immune response.

Distinct distal and proximal p53-binding sites in the MCK promoter govern the transcriptional response to p53

We have investigated the functional importance of nucleotide sequence differences between proximal (P-) and distal (D-) p53-binding elements in the MCK promoter. P- and D-elements normally co-operate to permit synergistic promoter activation by p53. Interestingly, we find that P-elements cannot co-operate with each other. In contrast, co-operation between D-binding sites results in levels of p53-induced transcription far higher than those obtained by co-operation between P- and D-elements. These studies imply that distinct D- and P-p53-binding sites in the MCK promoter may dictate the promoter response to p53.

Different Regulatory Sequences Control Creatine Kinase-M Gene Expression in Directly Injected Skeletal and Cardiac Muscle

Regulatory sequences of the M isozyme of the creatine kinase (MCK) gene have been extensively mapped in skeletal muscle, but little is known about the sequences that control cardiac-specific expression. The promoter and enhancer sequences required for MCK gene expression were assayed by the direct injection of plasmid DNA constructs into adult rat cardiac and skeletal muscle. A 700-nucleotide fragment containing the enhancer and promoter of the rabbit MCK gene activated the expression of a downstream reporter gene in both muscle tissues. Deletion of the enhancer significantly decreased expression in skeletal muscle but had no detectable effect on expression in cardiac muscle. Further deletions revealed a CArG sequence motif at position -179 within the promoter that was essential for cardiac-specific expression. The CArG element of the MCK promoter bound to the recombinant serum response factor and YY1, transcription factors which control expression from structurally similar elements in the skeletal actin and c-fos promoters. MCK-CArG-binding activities that were similar or identical to serum response factor and YY1 were also detected in extracts from adult cardiac muscle. These data suggest that the MCK gene is controlled by different regulatory programs in adult cardiac and skeletal muscle.

Different regulatory sequences control creatine kinase-M gene expression in directly injected skeletal and cardiac muscle.

Regulatory sequences of the M isozyme of the creatine kinase (MCK) gene have been extensively mapped in skeletal muscle, but little is known about the sequences that control cardiac-specific expression. The promoter and enhancer sequences required for MCK gene expression were assayed by the direct injection of plasmid DNA constructs into adult rat cardiac and skeletal muscle. A 700-nucleotide fragment containing the enhancer and promoter of the rabbit MCK gene activated the expression of a downstream reporter gene in both muscle tissues. Deletion of the enhancer significantly decreased expression in skeletal muscle but had no detectable effect on expression in cardiac muscle. Further deletions revealed a CArG sequence motif at position -179 within the promoter that was essential for cardiac-specific expression. The CArG element of the MCK promoter bound to the recombinant serum response factor and YY1, transcription factors which control expression from structurally similar elements in the skeletal actin and c-fos promoters. MCK-CArG-binding activities that were similar or identical to serum response factor and YY1 were also detected in extracts from adult cardiac muscle. These data suggest that the MCK gene is controlled by different regulatory programs in adult cardiac and skeletal muscle.