The mouse dystrophin muscle promoter/enhancer drives expression of mini-dystrophin in transgenic mdx mice and rescues the dystrophy in these mice.

Successful gene therapy for Duchenne muscular dystrophy (DMD) requires the restoration of dystrophin protein in skeletal muscles. To achieve this goal, appropriate regulatory elements that impart tissue-specific transgene expression need to be identified. Currently, most muscle-directed gene therapy studies utilize the muscle creatine kinase promoter.

The mouse dystrophin muscle enhancer-1 imparts skeletal muscle, but not cardiac muscle, expression onto the dystrophin Purkinje promoter in transgenic mice.

A subset of patients harboring mutations in the dystrophin gene suffer from X-linked dilated cardiomyopathy (XLCM), a familial heart disease that is not accompanied by any clinical signs of skeletal muscle myopathy. As the muscle (M) isoform of dystrophin is not expressed in these patients, the absence of skeletal muscle symptoms has been attributed to expression of the brain (B) and cerebellar Purkinje (CP) isoforms of dystrophin in skeletal, but not cardiac, muscles of XLCM patients. The compensatory mechanism of dystrophin B and CP promoter upregulation is not known but it has been suggested that the dystrophin muscle enhancer from intron 1, DME-1, may be important in this activity.

Mouse dystrophin enhancer preferentially targets lacZ expression in skeletal and cardiac muscle.

Duchenne muscular dystrophy is a muscle wasting disease that results from a dystrophin deficiency in skeletal and cardiac muscle. Studies concerning the regulatory elements that govern dystrophin gene expression in skeletal and/or cardiac muscle in both mouse and human have identified a promoter and an enhancer located in intron 1. In transgenic mice, the muscle promoter alone targets the expression of a lacZ reporter gene only to the right ventricle of the heart, suggesting the need for other regulatory elements to target skeletal muscle and the rest of the heart.

The mouse dystrophin enhancer is regulated by MyoD, E-box-binding factors, and by the serum response factor.

In vivo studies in the mouse have revealed that the muscle promoter of the mouse dystrophin gene can target the right ventricle of the heart only, suggesting the need for other regulatory elements to target the skeletal muscle as well as other compartments of the heart. In this study we report the identification of the mouse dystrophin gene enhancer that is located approximately 8.5 kilobases downstream from the mouse dystrophin gene muscle promoter.

Transfection of cultured myoblasts in high serum concentration with DODAC:DOPE liposomes.

The inhibitory effect of serum is one of the main obstacles to the in vivo use of cationic liposomes as a DNA delivery system. We have found that a novel liposome formulation, DODAC:DOPE (1:1) is totally resistant to the inhibitory effects of serum for transfection of cultured myoblasts and myotubes. Transfection with a lacZ reporter gene in the presence of 95% fetal bovine serum gave up to 25% beta-gal-positive cells in C2C12 myoblasts and about six-fold less in primary human myoblasts.

Dystrophin isoforms DP71 and DP427 have distinct roles in myogenic cells.

Duchenne muscular dystrophy is caused by mutations in the dystrophin gene, a complex gene that generates a family of distinct isoforms. In immature muscle cells, two dystrophin isoforms are expressed, Dp427 and Dp71. To characterize the function of Dp71 in myogenesis, we have examined the expression of Dp71 in myogenic cells.

A muscle-specific enhancer within intron 1 of the human dystrophin gene is functionally dependent on single MEF-1/E box and MEF-2/AT-rich sequence motifs.

In previous studies we have described a 5.0 kb Hin dIII fragment downstream of muscle exon 1 that exhibits properties consistent with a muscle-specific transcriptional enhancer. The goal of this study has been to identify the sequence elements responsible for muscle-specific enhancer activity.

Modulation of the specificity and activity of a cellular promoter in an adenoviral vector.

Most gene therapy studies with recombinant adenoviruses employ viral promoters and lack tissue specificity. To determine whether a tissue-specific cellular promoter inserted into the adenoviral genome can direct the expression of a reporter gene in a tissue-specific manner, recombinant adenoviruses containing a nuclear lacZ gene driven by a human ventricular/slow muscle myosin light chain 1 promoter with and without a muscle creatine kinase enhancer were constructed. The ability of these viruses to express the reporter genes in infected myogenic and nonmyogenic cell lines was studied.

Identification of a transcriptional enhancer within muscle intron 1 of the human dystrophin gene.

The 14 kb muscle isoform of the Duchenne muscular dystrophy (DMD) gene is expressed primarily in skeletal and cardiac muscle. Transcription of the muscle isoform is induced as myoblasts differentiate into multinucleated myotubes and transcript levels are increased a further 10-fold in mature skeletal muscle. In previous studies we have demonstrated that the core muscle promoter of the human DMD gene contains sequences that regulate the induction of DMD gene expression with myoblast differentiation.

Expression of the dystrophin isoform Dp71 in differentiating human fetal myogenic cultures.

The dystrophin gene defective in Duchenne muscular dystrophy (DMD) is extreme in size and complexity with several promoters which direct expression of different isoforms in different tissues. In contrast with adult skeletal muscle which expresses 427 kDa dystrophin, fetal muscle tissue expresses the 71 kDa ubiquitous isoform Dp71 as well as 427 kDa muscle dystrophin. To examine Dp71 expression in fetal muscle further, we have monitored its expression pattern in differentiating myogenic cultures of human fetal muscle origin.

Stability of the human dystrophin transcript in muscle.

The human dystrophin gene has 79 exons spanning >2300 kb making it the largest known gene. In previous studies we showed that approximately 16 h are required to transcribe the gene in myogenic cultures [Tennyson, C.N.

Condensation of plasmid DNA with polylysine improves liposome-mediated gene transfer into established and primary muscle cells.

Cationic liposomes provide a means to introduce genes into cells both ex vivo and in vivo. In the past few years their use has been described in several tissues, e.g.

The human dystrophin gene requires 16 hours to be transcribed and is cotranscriptionally spliced.

The largest known gene is the human dystrophin gene, which has 79 exons spanning at least 2,300 kilobases (kb). Transcript accumulation was monitored from four regions of the gene following induction of expression in muscle cell cultures. Quantitative reverse transcription-polymerase chain reaction (RT-PCR) results indicate that approximately 12 h are required for transcription of 1,770 kb (at an average elongation rate of 2.

Mapping of the human NMDA receptor subunit (NMDAR1) and the proposed NMDA receptor glutamate-binding subunit (NMDARA1) to chromosomes 9q34.3 and chromosome 8, respectively.

A role for the N-methyl-D-aspartate (NMDA) receptor in the molecular pathology underlying Huntington disease (HD) has been proposed on the basis of neurochemical studies in HD and the ability of the NMDA receptor to mediate neuronal cell death. The molecular cloning of the human NMDA receptor subunit (NMDAR1) and a proposed glutamate-binding subunit of the NMDA receptor (NMDARA1) have provided an opportunity to test the hypothesis that either of these genes might be directly involved in the causation of HD. We have mapped NMDAR1 to 9q34.

Dystrophin in frameshift deletion patients with Becker muscular dystrophy.

In a previous study we identified 14 cases with Duchenne muscular dystrophy (DMD) or its milder variant, Becker muscular dystrophy (BMD), with a deletion of exons 3-7, a deletion that would be expected to shift the translational reading frame of the mRNA and give a severe phenotype. We have examined dystrophin and its mRNA from muscle biopsies of seven cases with either mild or intermediate phenotypes. In all cases we detected slightly lower-molecular-weight dystrophin in 12%-15% abudance relative to the normal.

Polymorphisms and deduced amino acid substitutions in the coding sequence of the ryanodine receptor (RYR1) gene in individuals with malignant hyperthermia.

Twenty-one polymorphic sequence variants of the RYR1 gene, including 13 restriction fragment length polymorphisms (RFLPs), were identified by sequence analysis of human ryanodine receptor (RYR1) cDNAs from three individuals predisposed to malignant hyperthermia (MH). All RFLPs were detectable in PCR-amplified products, and their segregation was consistent with our initial finding of linkage to MH in the nine families previously informative for one or more intragenic markers (MacLennan et al., 1990, Nature 343:559-561).

Duchenne muscular dystrophy: gene and gene product; mechanism of mutation in the gene.

The X-linked gene responsible for Duchenne muscular dystrophy encodes dystrophin, a high-molecular-weight cytoskeletal protein. Studies in several laboratories have revealed deletion of one or more exons in 60% of affected boys; quantitative analysis in our laboratory has detected duplication of exons in another 6%. The severe Duchenne phenotype is associated with deletions or duplications that shift the reading frame of the message, whereas the milder Becker muscular dystrophy is associated with deletions or duplications that maintain the reading frame.