Utrophin gene expression

Details Utrophin gene expression Utrophin gene expression

1998-07-14

2003-02-11

Carlson, Karen Cochrane (Department: 1653)

Organic compounds -- part of the class 532-570 series

Organic compounds

Carbohydrates or derivatives

C536S024100, C536S023500, C435S069100, C435S069700, C435S006120, C435S325000, C435S369000, C435S320100, C435S252300, C435S458000, C435S071100, C530S350000, C530S300000, C514S012200, C514S04400A, C424S450000, C424S093200, C264S004100

Reexamination Certificate

active

06518413

ABSTRACT:

The present invention generally relates to the provision of nucleic acid from which a polypeptide with utrophin function can be expressed, especially mini-genes and chimaeric constructs. Expression of a utrophin transgene significantly decreases the severity of the dystrophic muscle phenotype in an animal model.
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. Dystrophin is a large cytoskeletal protein which in muscle is located at the cytoplasmic surface of the sarcolemma, the neuromuscular junction (NMJ) and myotendinous junction (MTJ). The protein is composed of four domains: an actin-binding domain (shown in vitro to bind actin), a rod domain containing triple helical repeats, a cysteine rich (CR) domain and a carboxy-terminal (CT) domain. The majority of the CRCT binds to a complex of proteins and glycoproteins (called the dystrophin protein complex, DPC) spanning the sarcolemma. This complex consists of cytoskeletal syntrophins and dystrobrevin, transmembrane, &bgr;-dystroglycan, &agr;-, &bgr;-&dgr;-, &ggr;-sarcoglycans and extracellular &agr;-dystroglycan. The DPC links to laminin-&agr;2 (merosin) in the extracellular matrix and to the actin cytoskeleton via dystrophin within the cell. The breakdown of the integrity of the DPC due to the loss of, or impairment of dystrophin function, leads to muscle degeneration and the DMD phenotype. The structure of dystrophin and protein interactions within the DPC have been recently reviewed [1,2,3].
There are various approaches which can be adopted for the gene therapy of DMD. These include myoblast transfer, retroviral infection, adenoviral infection and direct injection of plasmid DNA. In most cases the dystrophin gene used in the experiments generates a truncated protein approximately half the size of the full size protein. This dystrophin minigene was modelled on a natural mutation identified in a very mild Becker patient [4]. The cloned version of this truncated minigene is able to reverse the pathological phenotype in the dystrophin deficient mdx mouse [5,6,7] and has had limited success when delivered to mdx muscle by viral vectors [8,9,10]. Although some progress is being made in each of these areas using the mdx mouse as a model system, there are 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 [11]. Another problem to be addressed is the rejection of cells expressing dystrophin because of immunological intolerance i.e. dystrophin within these cells will appear foreign to the host immune system given that most DMD patients will never have expressed dystrophin [12,13].
In order to circumvent some of these problems, possibilities of compensating for dystrophin loss using a related protein, utrophin, are being explored.
Utrophin is a 395 kDa protein encoded by a gene located on chromosome 6q24 and shown to have strong sequence similarity to dystrophin [14]. The actin binding domain of dystrophin and utrophin has 85% similarity and the DPC binding region has 88% similarity. Both of these domains have been shown to function as predicted in vitro. The structure and potential protein interactions are described in detail in reviews [1,2,3].
There is a substantial body of evidence demonstrating that utrophin is capable of localising to the sarcolemma. During normal fetal muscle development there is increased utrophin expression, localised to the sarcolemma up until 18 weeks and 20 days gestation in human and mouse respectively. 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 and MTJ [15,16,17]. The decrease in utrophin expression coincides with increased expression of dystrophin [17]. Many studies have shown that utrophin is bound to the sarcolemma in DMD and BMD patients. However the levels of utrophin localised at the sarcolemma vary from report to report [18,19,20,21]. In some other non Xp21 myopathies, utrophin and dystrophin are simultaneously bound to the sarcolemma of adult skeletal muscle [22].
High levels of utrophin may protect muscle from the consequences of dystrophin loss. Matsumara et al. [23] demonstrated that purified membranes from the mdx mouse contained complexes of utrophin and the DPC. When quadricep muscles (which show necrosis) from these mice were analysed by immunoblotting, the level of utrophin remained approximately the same, however the level of the &agr;-dystroglycan was drastically reduced. In cardiac muscle (which shows no pathology) the level of utrophin was elevated four fold with no loss of the &agr;-dystroglycan. Immunocytochemical analysis of other mdx small calibre skeletal muscles (extraocular and toe) which also have no pathology shows increased utrophin expression and normal levels of &agr;-sarcoglycan. This result suggests that the increased levels of utrophin interacts with the DPC (or an antigenically related complex) at the sarcolemma and prevents loss of the complex thus the structure of these cells remains normal. In the mdx mouse, utrophin levels in muscle remain elevated soon after birth compared with normal mice; however once the utrophin levels have decreased to the adult levels (about 1 week after birth), the first signs of muscle fibre necrosis are detected [15,16].
Thus, in certain circumstances utrophin can localise to the sarcolemma probably at the same binding sites as dystrophin, namely actin and the DPC. If the expression of utrophin is high enough, it may maintain the DPC and thus alleviate the DMD phenotype. It is unlikely that such external upregulation could be tightly controlled giving rise to potentially high levels of utrophin within the cell. However, this may not be a problem as Cox et al. [24] have demonstrated that gross over expression of dystrophin in the muscle of transgenic mdx mice reverts the muscle pathology to normal with no obvious detrimental side effects.
The present invention has arisen from cloning of nucleic acid encoding utrophin and fragments of utrophin from various species. The original aim was to clone nucleic acid encoding human utrophin, but major problems were encountered. A previous paper (14) reported the amino acid sequence of utrophin (so-called “dystrophin-related protein”), obtained by cloning of overlapping cDNAs. However, two regions around the amino terminal actin binding domain were not represented in these clones. These regions could be amplified by PCR and sequenced, but it has proved not to be possible to clone them. Either clones which should have included these regions were rearranged (as determined by restriction mapping) or simply no clones were isolated even if highly recombination deficient
E. coli
host strains (SURE and STBL2) were used. The gaps in the sequence were identified by comparing the sequence generated from the utrophin cDNAs to the published human dystrophin sequence. It became apparent as further utrophin clones were isolated, none spanned these two gaps.
Sequence information obtained from the amino terminus of the human cDNA was used to design probes and rat and mouse cDNA libraries were screened. Rat cDNAs were also unstable or rearranged in the region corresponding to the unclonable regions in the human sequence. Some large rat clones covering these regions were obtained, but all attempts to generate subclones failed due to rearrangements of the inserts as determined by restriction mapping. Surprisingly, in view of the difficulties with the human and rat sequences, cDNA from the mouse library, covering the regions in question, was found to be stable and amenable to further manipulation including the. generation of smaller subclones.
FIG. 1
shows a comparison between human, rat and mouse utrophi

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