Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Patent
1997-10-22
1999-10-26
Brusca, John S.
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
435 691, 435325, 536 231, 536 241, C12Q 168, C12P 2100, C12N 510, C12N 1511
Patent
active
059726091
DESCRIPTION:
BRIEF SUMMARY
The present invention is based on cloning of a genomic promoter region of the human utrophin gene and of the mouse utrophin gene.
The severe muscle wasting disorders Duchenne muscular dystrophy (DMD) and the less debilitating Becker muscular dystrophy (BMD) are due to mutations in the dystrophin gene resulting in a lack of dystrophin or abnormal expression of truncated forms of dystrophin, respectively. Dystrophin is a large cytoskeletal protein (427 kDa with a length of 125 nm) which in muscle is located at the cytoplasmic surface of the sarcolemma, the neuromuscular junction (NMJ) and myotendinous junction (MTJ). It binds to a complex of proteins and glycoproteins spanning the sarcolemma called the dystrophin associated glycoprotein complex (DGC). The breakdown of the integrity of this complex due to loss of, or impairment of dystrophin function, leads to muscle degeneration and the DMD phenotype.
The dystrophin gene is the largest gene so far identified in man, covering over 2.7 megabases and containing 79 exons. The corresponding 14 kb dystrophin mRNA is expressed predominantly in skeletal, cardiac and smooth muscle with lower levels in brain. Transcription of dystrophin in different tissues is regulated from either the brain promoter (predominantly active in neuronal cells) or muscle promoter (differentiated myogenic cells, and primary glial cells) giving rise to differing first exons. A third promoter between the muscle promoter and the second exon of dystrophin regulates expression in cerebellar Purkinje neurons. Recently reviewed in [1,2,3].
There are various approaches which have been adopted for the gene therapy of DMD, using the mdx mouse as a model system. However, there are considerable problems related to the number of muscle cells that can be made dystrophin positive, the levels of expression of the gene and the duration of expression [4]. It has also become apparent that simply re-introducing genes expressing the dystrophin carboxy-terminus has no effect on the dystrophic phenotype although the DGC appears to be re-established at the sarcolemma [5,6].
In order to circumvent some of these problems, possibilities of compensating for dystrophin loss using a related protein, utrophin, are being explored as an alternative route to dystrophin gene therapy. A similar strategy is currently being evaluated in clinical trials to upregulate foetal haemoglobin to compensate for the affected adult-globin chains in patients with sickle cell anaemia [7,8].
Utrophin is a 395 kDa protein encoded by multiexonic 1Mb UTRN gene located on chromosome 6q24 [9]. At present the tissue regulation of utrophin is not fully understood. Unlike dystrophin, only one promoter has so far been detected. In the dystrophin deficient mdx mouse, utrophin levels in muscle remain elevated soon after birth compared with normal mice; once the utrophin levels have decreased to the adult levels (about 1 week after birth), the first signs of muscle fibre necrosis are detected. However there is evidence to suggest that in the small calibre muscles, continual increased levels of utrophin can interact with the DGC complex (or an antigenically related complex) at the sarcolemma thus preventing loss of the complex with the result that these muscles appear normal. There is also a substantial body of evidence demonstrating that utrophin is capable of localising to the sarcolemma in normal muscle. During fetal muscle development there is increased utrophin expression, localised to the sarcolemma, up until 18 weeks in the human and 20 days gestation in the mouse. After this time the utrophin sarcolemmal staining steadily decreases to the significantly lower adult levels shortly before birth where utrophin is localised almost exclusively to the NMJ. The decrease in utrophin expression coincides with increased expression of dystrophin. See reviews [1,2,3].
Thus, in certain circumstances utrophin can localise to the sarcolemma probably at the same binding sites as dystrophin, through interactions with actin and the DGC. Accordingly, if expression
REFERENCES:
Hum. Mol. Genet., vol. 2, no. 11, 1993, pp. 1765-1772, Pearce et al.: "The utrophin and dystrophin genes share similarities in genomic structure".
Nature Genetics vol. 6, Mar. 1994, pp. 236-244, Cross et al.: "Purification of CpG islands using a methylated DNA binding column".
Database Strand Embl Empri AN: HS54B8R, Oct. 22, 1995 Mac Donald et al.
Dev Dynamics vol. 198, Dec. 1993, pp. 254-264, Schofield et al.: "Expression of the dystrophin-related protein (utrophine) gene during mouse embryogenesis".
Biotech Business News, vol. 3, No. 72, Jan. 28, 1994, p. 15, Oncogene Sci. Corpo. et al.: "Muscular dystrophy therapy sought".
Nature, vol. 360-593, XP002010740 Tinsley et al.: "Primary structure of dystrophin-related protein".
Davies Kay Elizabeth
Dennis Carina
Tinsley Jonathon Mark
Brusca John S.
Medical Research Council
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