Muscle Creatine Kinase Enhancer Research Articles

TH.I.9 - Development of microdystrophins for gene therapy of DMD

Gene therapy represents a promising approach to treat DMD as it has the potential to circumvent the primary cause of the disorder, a mutant gene. Gene replacement therapies have focused on methods to deliver expression cassettes encoding the dystrophin protein. Challenges to this approach have included the enormous size of the dystrophin gene (2.2 MB), its muscle mRNA (14 kb) and difficulties in safely delivering vectors to muscles bodywide. We and others have shown that vectors derived from specific serotypes of adeno-associated virus (AAV) can systemically deliver genes to muscles after intravascular infusion. However, AAV vectors have a limited carrying capacity of approximately 5 kb, necessitating the design of miniaturized cDNAs and gene regulatory elements that enable expression of “microdystrophins”. Early generation microdystrophins displayed functional limitations due to their small size (<4 kb) and the absence of some protein interaction domains. To develop improved vectors we tested a variety of novel microdystrophins lacking the C-terminal domain but carrying 4–6 spectrin-like repeats, including the nitric oxide synthase localization domain. We also tested a set of powerful gene regulatory elements based on the muscle creatine kinase (MCK) enhancer plus promoter. These novel microdystrophins were evaluated by AAV-mediated delivery to dystrophic mdx4cv mice followed by extensive analysis of muscle pathophysiology. We identified a 5 repeat vector that when driven by the CK8 regulatory cassette leads to stable expression and profound reductions in muscle pathophysiology. This AAV/CK8-µDys5 vector performs better than previously tested microdystrophin cassettes and represents a promising clinical candidate. We have also been testing AAV vectors to deliver components of the CRISPR/Cas9 system as a method to potentially repair, rather than replace, the mutant dystrophin gene. While this gene editing approach shows promise, its efficiency remains low and as currently applied is less effective than direct delivery of microd

Chromatin regulated interchange between polycomb repressive complex 2 (PRC2)-Ezh2 and PRC2-Ezh1 complexes controls myogenin activation in skeletal muscle cells

BackgroundPolycomb group (PcG) genes code for chromatin multiprotein complexes that are responsible for maintaining gene silencing of transcriptional programs during differentiation and in adult tissues. Despite the large amount of information on PcG function during development and cell identity homeostasis, little is known regarding the dynamics of PcG complexes and their role during terminal differentiation.ResultsWe show that two distinct polycomb repressive complex (PRC)2 complexes contribute to skeletal muscle cell differentiation: the PRC2-Ezh2 complex, which is bound to the myogenin (MyoG) promoter and muscle creatine kinase (mCK) enhancer in proliferating myoblasts, and the PRC2-Ezh1 complex, which replaces PRC2-Ezh2 on MyoG promoter in post-mitotic myotubes. Interestingly, the opposing dynamics of PRC2-Ezh2 and PRC2-Ezh1 at these muscle regulatory regions is differentially regulated at the chromatin level by Msk1 dependent methyl/phospho switch mechanism involving phosphorylation of serine 28 of the H3 histone (H3S28ph). While Msk1/H3S28ph is critical for the displacement of the PRC2-Ezh2 complex, this pathway does not influence the binding of PRC2-Ezh1 on the chromatin. Importantly, depletion of Ezh1 impairs muscle differentiation and the chromatin recruitment of MyoD to the MyoG promoter in differentiating myotubes. We propose that PRC2-Ezh1 is necessary for controlling the proper timing of MyoG transcriptional activation and thus, in contrast to PRC2-Ezh2, is required for myogenic differentiation.ConclusionsOur data reveal another important layer of epigenetic control orchestrating skeletal muscle cell terminal differentiation, and introduce a novel function of the PRC2-Ezh1 complex in promoter setting.

Down-regulation of MyoD by Calpain 3 Promotes Generation of Reserve Cells in C2C12 Myoblasts

Calpain 3 is a calcium-dependent cysteine protease that is primarily expressed in skeletal muscle and is implicated in limb girdle muscular dystrophy type 2A. To date, its best characterized function is located within the sarcomere, but this protease is found in other cellular compartments, which suggests that it exerts multiple roles. Here, we present evidence that calpain 3 is involved in the myogenic differentiation process. In the course of in vitro culture of myoblasts to fully differentiated myotubes, a population of quiescent undifferentiated "reserve cells" are maintained. These reserve cells are closely related to satellite cells responsible for adult muscle regeneration. In the present work, we observe that reserve cells express higher levels of endogenous Capn3 mRNA than proliferating myoblasts. We show that calpain 3 participates in the establishment of the pool of reserve cells by decreasing the transcriptional activity of the key myogenic regulator MyoD via proteolysis independently of the ubiquitin-proteasome degradation pathway. Our results identify calpain 3 as a potential new player in the muscular regeneration process by promoting renewal of the satellite cell compartment.

Construction and analysis of compact muscle-specific promoters for AAV vectors

Adeno-associated viral (AAV) vectors have been broadly used for gene transfer in vivo for various applications. However, AAV precludes the use of most of the original large-sized tissue-specific promoters for expression of transgenes. Efforts are made to develop highly compact, active and yet tissue-specific promoters for use in AAV vectors. In this study, we further abbreviated the muscle creatine kinase (MCK) promoter by ligating a double or triple tandem of MCK enhancer (206-bp) to its 87-bp basal promoter, generating the dMCK (509-bp) and tMCK (720-bp) promoters. The dMCK promoter is shorter but stronger than some previously developed MCK-based promoters such as the enh358MCK (584-bp) and CK6 (589-bp) in vitro in C2C12 myotubes and in vivo in skeletal muscles. The tMCK promoter is the strongest that we tested here, more active than the promiscuous cytomegalovirus (CMV) promoter. Furthermore, both the dMCK and tMCK promoters are essentially inactive in nonmuscle cell lines as well as in the mouse liver (>200-fold weaker than the CMV promoter). The dMCK promoter was further tested in a few lines of transgenic mice. Expression of LacZ or minidystrophin gene was detected in skeletal muscles throughout the body, but was weak in the diaphragm, and undetectable in the heart and other tissues. Similar to other miniature MCK promoters, the dMCK promoter also shows preference for fast-twitch myofibers. As a result, we further examined a short, synthetic muscle promoter C5-12 (312-bp). It is active in both skeletal and cardiac muscles but lacks apparent preference on myofiber types. Combination of a MCK enhancer to promoter C5-12 has increased its strength in muscle by two- to threefold. The above-mentioned compact muscle-specific promoters are well suited for AAV vectors in muscle-directed gene therapy studies.

Muscle creatine kinase/SV40 hybrid promoter for muscle-targeted long-term transgene expression

Gene therapy for congenital protein deficiencies requires lifelong expression of a deficient protein. Current gene therapy approaches preferentially employ the strong cytomegalovirus (CMV) promoter/enhancer or its derivative CAG promoter; however, these promoters provide only temporary transgene expression. To create a promoter that enables long-lasting expression in muscle, hybrid promoters were constructed by coupling the muscle creatine kinase (MCK) enhancer to various strong promoters for enhancement of tissue specificity and improved transcriptional activity. A hybrid promoter containing the MCK enhancer and the simian virus 40 promoter (MCK/SV40 promoter) yielded long-term (>6 months) expression of a human secretory alkaline phosphatase (huSEAP) reporter gene following electrotransfer of the plasmid into mice, whereas expression using a conventional CMV or CAG promoter faded away within a few weeks. To explore the mechanism behind the sustained expression obtained with the MCK/SV40 promoter, mice were immunized with a LacZ expression plasmid driven by MCK/SV40 or a conventional promoter. Minimal cellular and humoral responses to LacZ were observed in MCK/SV40 promoter-treated animals, and mouse SEAP gene expression in vivo was successfully maintained by both the MCK/SV40 and conventional promoters. These results suggest that the lower immunogenicity of the MCK/SV40 promoter contributed to long-lasting gene expression in mice. Therefore, the MCK/SV40 promoter may provide the basis for development of an effective transgene expression cassette for treatment of congenital protein deficiencies in which therapeutic proteins are recognized as foreign by the host immune system.

TGF-beta-activated Smad3 represses MEF2-dependent transcription in myogenic differentiation.

Transforming growth factor beta (TGF-beta) inhibits myogenesis and associated gene expression. We previously reported that the TGF-beta signaling effector Smad3 mediates this inhibition, by interfering with the assembly of myogenic bHLH transcription factor heterodimers on E-box sequences in the regulatory regions of muscle-specific genes. We now show that TGF-beta-activated Smad3 suppresses the function of MEF2, a second class of essential myogenic factors. TGF-beta signaling through Smad3 represses myogenin expression independently of E-boxes, and prevents a tethered MyoD-E47 dimer to activate transcription indirectly through MEF2-binding sites. In addition, Smad3 interacts with MEF2C, which requires its MADS domain, and disrupts its association with the SRC-family coactivator GRIP-1, thus diminishing the transcription activity of MEF2C. Consistent with this physical displacement, TGF-beta signaling blocks the GRIP-1-induced redistribution of MEF2C to discrete nuclear subdomains in 10T1/2 cells, and the recruitment of GRIP-1 to the myogenin promoter in differentiating myoblasts. These findings indicate that the TGF-beta/Smad3 pathway targets two critical components of the myogenic transcription machinery to inhibit terminal differentiation.

Quantitative proteomic identification of six4 as the trex-binding factor in the muscle creatine kinase enhancer.

Transcriptional regulatory element X (Trex) is a positive control site within the Muscle creatine kinase (MCK) enhancer. Cell culture and transgenic studies indicate that the Trex site is important for MCK expression in skeletal and cardiac muscle. After selectively enriching for the Trex-binding factor (TrexBF) using magnetic beads coupled to oligonucleotides containing either wild-type or mutant Trex sites, quantitative proteomics was used to identify TrexBF as Six4, a homeodomain transcription factor of the Six/sine oculis family, from a background of approximately 900 copurifying proteins. Using gel shift assays and Six-specific antisera, we demonstrated that Six4 is TrexBF in mouse skeletal myocytes and embryonic day 10 chick skeletal and cardiac muscle, while Six5 is the major TrexBF in adult mouse heart. In cotransfection studies, Six4 transactivates the MCK enhancer as well as muscle-specific regulatory regions of Aldolase A and Cardiac troponin C via Trex/MEF3 sites. Our results are consistent with Six4 being a key regulator of muscle gene expression in adult skeletal muscle and in developing striated muscle. The Trex/MEF3 composite sequence ([C/A]ACC[C/T]GA) allowed us to identify novel putative Six-binding sites in six other muscle genes. Our proteomics strategy will be useful for identifying transcription factors from complex mixtures using only defined DNA fragments for purification.

U7 snRNAs induce correction of mutated dystrophin pre-mRNA by exon skipping.

Most cases of Duchenne muscular dystrophy are caused by dystrophin gene mutations that disrupt the mRNA reading frame. Artificial exclusion (skipping) of a single exon would often restore the reading frame, giving rise to a shorter, but still functional dystrophin protein. Here, we analyzed the ability of antisense U7 small nuclear (sn)RNA derivatives to alter dystrophin pre-mRNA splicing. As a proof of principle, we first targeted the splice sites flanking exon 23 of dystrophin pre-mRNA in the wild-type muscle cell line C2C12 and showed precise exon 23 skipping. The same strategy was then successfully adapted to dystrophic immortalized mdx muscle cells where exon-23-skipped dystrophin mRNA rescued dystrophin protein synthesis. Moreover, we observed a stimulation of antisense U7 snRNA expression by the murine muscle creatine kinase enhancer. These results demonstrate that alteration of dystrophin pre-mRNA splicing could correct dystrophin gene mutations by expression of specific U7 snRNA constructs.

Gutted adenoviral vectors for gene transfer to muscle.

Adenoviral vectors are a popular choice for gene transfer protocols because they are well characterized, have a relatively large cloning capacity (up to 36 kB), and can be grown to high titers (1013 particles/mL) (). Despite these attributes, first-generation adenoviral vectors retain many viral genes that can elicit a strong immune response, severely limiting their utility for studies in vivo (). Our laboratory and others have been developing “gutted” or helper-dependent adenoviruses, which lack all viral coding sequences and therefore should greatly enhance the persistence of the vector in vivo (,). We have used this technology to deliver to muscle full-length cDNAs of the largest known gene, dystrophin, under control of the mouse muscle creatine kinase enhancer plus promoter (, , ).

Transgenic and tissue culture analyses of the muscle creatine kinase enhancer Trex control element in skeletal and cardiac muscle indicate differences in gene expression between muscle types.

The muscle creatine kinase (MCK) gene is expressed at high levels only in differentiated skeletal and cardiac muscle. The activity of the cloned enhancer-promoter has previously been shown to be dependent on the Trex element which is specifically bound by a yet unidentified nuclear factor, TrexBF. We have further characterized the function of the Trex site by comparing wild-type and Trex-mutated MCK transgenes in five mouse skeletal muscles: quadriceps, extensor digitorum longus (EDL), soleus, diaphragm, and distal tongue, as well as in heart ventricular muscle. Several types of statistical analysis including analysis of variance (ANOVA) and rank sum tests were used to compare expression between muscle types and between constructs. Upon mutation of the Trex site, median transgene expression levels decreased 3- to 120-fold in the muscles examined, with statistically significant differences in all muscles except the EDL. Expression in the largely slow soleus muscle was more affected than in the EDL, and expression in the distal tongue and diaphragm muscles was affected more than in soleus. Median expression of the transgene in ventricle decreased about 18-fold upon Trex mutation. Transfections into neonatal rat myocardiocytes confirmed the importance of the Trex site for MCK enhancer activity in heart muscle, but the effect is larger in transgenic mice than in cultured cells.

Analysis of the DNA-binding properties of MyoD, myogenin, and E12 by fluorescence anisotropy.

MyoD and Myogenin are dominant myogenic regulatory factors (MRFs), which are involved in control of muscle-specific gene expression. The ubiquitously expressed E12 dimerizes with MyoD and Myogenin and has been shown to enhance their DNA-binding and transcriptional activities. In this study, fluorescence anisotropy assays have been used to determine the Gibb's free energy of dissociation (DeltaG) for MyoD, Myogenin, and E12 as homo- and heterodimers to the well-characterized myosin light chain enhancer (MLC), muscle creatine kinase (MCK) enhancer, and mutant thereof. The heterodimers of MyoD or Myogenin with E12 bound the MCK enhancer equally well (DeltaG = 21 kcal/mol). The homodimers varied dramatically in both MLC and MCK enhancer binding affinity. MyoD homodimer bound the MCK enhancer with the highest affinity (DeltaG = 19.6 kcal/mol) in comparison with the Myogenin homodimer-MCK interaction (DeltaG = 16.6 kcal/mol) and E12 homodimer-MCK interaction (DeltaG = 18.0 kcal/mol). The slope and shape of the binding isotherms revealed that with the exception of the E12 homodimer-MCK enhancer interaction, the other proteins bound with high levels of positive cooperativity. In contrast, the E12 homodimer-MCK enhancer interaction actually occurs with significant negative cooperativity. The binding of these proteins to MLC enhancer mimicked binding to the MCK enhancer, but with much lower affinities. These data support the hypothesis that DNA acts as an allosteric ligand facilitating the dimerization of these proteins. The combination of differential affinity and cooperativity explains why the heterodimers are the active species in transcriptional regulation.

Analysis of muscle creatine kinase regulatory elements in recombinant adenoviral vectors.

Adenoviral gene transfer holds promise for gene therapy, but effective transduction of a large and distributed tissue such as muscle will almost certainly require systemic delivery. In this context, the use of muscle-specific regulatory elements such as the muscle creatine kinase (MCK) promoter and enhancer will avoid potentially harmful ectopic expression of transgenes. We describe here the development and testing of adenoviral vectors containing small, striated muscle-specific, highly active MCK expression cassettes. One of these regulatory elements (CK6) is less than 600 bp in length and is 12% as active as the CMV promoter/enhancer in muscle. A recombinant adenoviral vector containing this regulatory element retains very high muscle specificity, expressing 600-fold higher levels of transgene in muscle than in liver. Muscle-specific regulatory elements may also increase persistence of transduced muscle cells. Adenoviral transduction of dendritic cells has been shown to stimulate cytotoxic T-lymphocyte (CTL) responses directed against transgene epitopes. We show that human dendritic cells infected in vitro with MCK-containing adenoviruses do not express significant levels of transgene. Furthermore, while adenoviral vectors containing nonspecific promoters are normally cleared from muscle tissue within 1 month, we show that MCK-containing vectors express significant levels of transgene 4 months after intramuscular injection.

Muscle Differentiation Is Antagonized by SOX15, a New Member of the SOX Protein Family

SOX proteins belong to a multigenic family characterized by a unique DNA binding domain, known as the high mobility group box, that is related to that of the testis determining gene SRY. cDNA sequences for more than 30 SOX genes have been identified, and some are known to have diverse roles in vertebrate differentiation and development. Here, we report the isolation and characterization of mouse Sox15 that was uncovered during a screen for high mobility group box containing transcription factors that are expressed at different levels during skeletal muscle differentiation. Sox15 cDNAs were found at a much higher frequency in myoblasts prior to their differentiation into myotubes. Electrophoretic mobility shift assays indicated that recombinant SOX15 protein was capable of binding to a consensus DNA binding site for SOX proteins. When overexpressed in C2C12 myoblasts, wild type SOX15, but not a C-terminal truncated form or the related protein SOX11, specifically inhibited activation of muscle-specific genes and expression of the basic helix-loop-helix myogenic factors myogenin and MyoD, resulting in a failure of the cells to differentiate into myotubes. These results suggest a specific and repressive role for SOX15, requiring the C-terminal domain, during myogenesis.

Stimulation of Slow Skeletal Muscle Fiber Gene Expression by Calcineurin in Vivo

Adult skeletal muscle fibers can be categorized into fast and slow twitch subtypes based on specialized contractile and metabolic properties and on distinctive patterns of muscle gene expression. Muscle fiber-type characteristics are dependent on the frequency of motor nerve stimulation and are thought to be controlled by calcium-dependent signaling. The calcium, calmodulin-dependent protein phosphatase, calcineurin, stimulates slow fiber-specific gene promoters in cultured skeletal muscle cells, and the calcineurin inhibitor, cyclosporin A, inhibits slow fiber gene expression in vivo, suggesting a key role of calcineurin in activation of the slow muscle fiber phenotype. Calcineurin has also been shown to induce hypertrophy of cardiac muscle and to mediate the hypertrophic effects of insulin-like growth factor-1 on skeletal myocytes in vitro. To determine whether activated calcineurin was sufficient to induce slow fiber gene expression and hypertrophy in adult skeletal muscle in vivo, we created transgenic mice that expressed activated calcineurin under control of the muscle creatine kinase enhancer. These mice exhibited an increase in slow muscle fibers, but no evidence for skeletal muscle hypertrophy. These results demonstrate that calcineurin activation is sufficient to induce the slow fiber gene regulatory program in vivo and suggest that additional signals are required for skeletal muscle hypertrophy.

Skeletal muscle-specific expression of human blood coagulation factor IX rescues factor IX deficiency mouse by AAV-mediated gene transfer

The efficacy of recombinant adeno-associated virus (AAV) vector to deliver and express human blood clotting factor IX (hFIX) gene in skeletal muscle of coagulation factor IX deficiency mouse strain (FactorIX-knockout) is evaluated. The muscle creatine kinase enhancer (MCK) and beta-actin promoter (betaA) were used to drive the hFIX minigene (hFIXml), which was flanked by AAV inverted terminal repeats (ITRs). Following intramuscular injection of high titer (2.5 x 10(11) vector genomes/mL) of rAAV, increased hFIX expression (256 ng/mL of plasma) was achieved. The time course of hFIX expression demonstrated that the expression level gradually increased over a period of two weeks before anti-hFIX antibodies developed in mouse circulating plasma. Those results provided a promising evidence that rAAV-mediated gene transfer and skeletal muscle-specific expression of hFIX is a feasible strategy for treating patients for hemophilia B.

Regulation of N-acetylgalactosamine 4-sulfatase expression in retrovirus-transduced feline mucopolysaccharidosis type VI muscle cells.

As a preliminary step toward muscle-mediated gene therapy in the mucopolysaccharidosis (MPS) type VI cat, we have analyzed the transcriptional regulation of feline N-acetylgalactosamine 4-sulfatase (f4S) gene expression from various retroviral constructs in primary cultures of muscle cells. Two retroviral constructs were made containing the f4S cDNA under the transcriptional control of the human polypeptide chain-elongation factor 1alpha (EF1alpha) gene promoter or the cytomegalovirus (CMV) immediate-early promoter. Two further retroviral constructs were made with the murine muscle creatine kinase (mck) enhancer sequence upstream of the internal promoter. Virus made from each construct was used to transduce feline MPS VI myoblasts. The mck enhancer significantly upregulated f4S gene expression from both the EF1alpha promoter and the CMV promoter in transduced myoblasts and in differentiated myofibers. The highest level of 4S activity was observed in myoblasts and myofibers transduced with the retroviral construct Lmckcmv4S, in which the f4S gene is under the transcriptional regulation of the mck enhancer and CMV immediate-early promoter. Lmckcmv4S-transduced myofibers demonstrated correction of glycosaminoglycan storage and contained a 58-fold elevated level of 4S activity compared with normal myofibers. Recombinant f4S secreted from Lmckcmv4S-transduced myofibers was endocytosed by feline MPS VI myofibers, leading to correction of the biochemical storage phenotype.

Differential Biological Activities of Mammalian Id Proteins in Muscle Cells

Id proteins are helix-loop-helix (HLH) transcription factors that lack DNA-binding domains. These proteins form inactive heterodimers with basic HLH (bHLH) factors, inhibiting their DNA-binding and transcriptional activities. Consistent with a proposed role for Id proteins as inhibitors of terminal differentiation, Id1 and Id3 have been shown to negatively regulate myogenesis in cultured muscle cells. Here we have investigated the possibility that Id2 and/or Id4 can act in a similar manner. Surprisingly, while overexpression of Id2 resulted in inhibition of differentiation of Sol 8 myoblast cells, overexpression of Id4 did not. Sol 8 cells stably transfected with Id4 showed no apparent changes in expression of muscle-specific genes upon differentiation. DNA-binding activities present at the muscle creatine kinase (MCK) enhancer E-box and transcription of the MCK enhancer were not altered in Id4-overexpressing cells, compared with vector-transfected cells. Id2 is also a more potent inhibitor of protein/DNA complex formation at the MCK-R enhancer E-box than Identifiedin vitro.Therefore, our data support the notion that members of the Id family might be involved in the regulation of distinct developmental pathways.

Insertion of two independent enhancers in the long terminal repeat of a self-inactivating vector results in high-titer retroviral vectors with tissue-specific expression.

The use of retroviral vectors (RVs) derived from the murine oncoretroviruses for gene therapy is associated with the risk of malignant transformation of infected cells and ectopic expression of the proteins of interest. Targeting retroviral vectors to specific tissues would increase their safety and clinical applicability. To explore the potential of targeting vector expression to skeletal muscle, the murine leukemia virus broad transcriptional tropism was modified by substituting the viral promoter and/or enhancer with a transcriptional cassette containing the human T cell leukemia virus type I Tax-responsive element and the minimal muscle creatine kinase enhancer and promoter. The resulting retroviral vectors could be transcriptionally trans-activated by tax. In the absence of Tax, however, the viruses showed muscle-specific expression. Trans-complementing packaging and indicator cells stably expressing Tax were used to isolate high-titer producer cell clones (10(6) CFU/ml). In vitro, the levels of expression of these RVs in Tax-expressing fibroblasts were 10,000-fold higher than in normal fibroblasts and 1000-fold higher in C2C12 myotubes than in C2C12 myoblasts. Expression of the vectors and the endogenous muscle creatine kinase gene was similarly dependent on the maturity of the muscle cultures. One vector with modified LTRs was also tested in vivo in regenerating muscle and showed a delayed pattern of expression in myofibers compared with the vector containing the wild-type LTRs. These vectors can be easily modified to contain different tissue-specific enhancer and promoter elements and the availability of complementing packaging and indicator cells expressing Tax should allow their application in a variety of gene therapy settings.

High expression of human clotting factor IX cDNA in myoblasts C2C12 cells and C3H mice

Mouse myoblast C2C12 cell was used as target cell for gene transfer study of human clotting factor IX (hFIX) cDNA. In addition to the previously constructed retroviral vectors XLIX, LNCIX and G1NaCIX, G1NaMCIX with hFIX driven by muscle creatine kinase (MCK) enhancer and human cytomegalovirus (CMV) was constructed, based on the retroviral vector G1Na. These four retroviral vectors were used to transduce mouse myoblasts C2C12. With ELISA assays, it has been found that the expression levels of human clotting factor IX detected in those transduced C2C12 cells are G1NaMCIX> G1NaCIX> LNCIX> XLIX. Mixed colonal cells transduced with GlNaMCIX expressed hFIX protein at the level of 640 ng/10(6) cell every 24 h. The modified C2C12 cells transduced with G1NaMCIX were implanted into skeletal muscle of the hindlegs of C3H mice; a stable expression of hFIX was detected and lasted for 35 d, with a maximum level of 206 ng/mL plasma. The regulation of hFIX cDNA expression in myoblasts was discussed and it was strongly suggested that a myoblast-mediated gene delivery system had the potential to be optimized as a safe and effective therapeutic modality for hemophilia B.

HLH transcription factor activity in osteogenic cells

To examine possible mechanisms underlying osteoblast differentiation from mesenchymal stem cells, we investigated bHLH functional activity in cell lines representing different stages of osteoblast maturation. Interaction of nuclear proteins with oligonucleotides corresponding to various bHLH binding sequences (known as E-boxes) was determined in mobility shift assays. Both ADD-1 oligonucleotide, a binding site for transcription factor ADD-1, and OCE-1, an E-box from osteocalcin promoter, produced retarded bands after incubation with nuclear extracts from osteogenic cells. Cells at different stages of osteogenic maturation demonstrated similar patterns and intensity of binding, as did cells treated with different osteogenic inducers. Binding to ADD-1 and OCE-1 was not tissue-specific as it was also observed in fibroblastic 10T1/2 cells. MEF-1 oligonucleotide, the E-box sequence from the muscle creatine kinase enhancer, demonstrated no changes in binding with nuclear extracts from moderately differentiated (W-20) or relatively mature (ROS 17/2.8) cells under any conditions tested. However, in poorly differentiated RI-2J cells, which do not express osteogenic markers unless treated with dexamethasone, induction of differentiation was reflected in transient inhibition of binding to MEF-1. Inhibition of binding was not seen under differentiation-restrictive conditions. Promoter-reporter studies also demonstrated inhibition of MEF-1 driven CAT expression by dexamethasone under differentiation-permissive conditions in RI-2J cells. These data suggest that bHLH gene expression is not required for the early steps of osteogenesis; moreover, inhibition of bHLH protein binding to a MEF1-type E box might be an integral part of osteogenic commitment.