Abstract:
This invention relates to a method of treating an inherited disease due to a point mutation producing a stop codon by administering an effective dose of an aminoglycoside antibiotic or a derivative thereof. Mdx mouse, which is an animal model for Duchenne muscular dystrophy, has been successfully treated with intramuscularly administered 1 and 5 mg gentamicin, which had for effect to suppress the premature stop mutation by inserting an amino acid at the stop codon. Dystrophin positive muscle fibers not different in number from those of normal mouse were detected at the dose of 5 mg gentamicin.

Description:
BACKGROUND OF THE INVENTION 
     Duchenne Muscular Dystrophy (DMD) is due to the mutation of a gene in the X chromosome coding for a protein called dystrophin (Koenig et al 1987; Hoffman et al. 1987; Bodrug et al. 1987, Arahata et al. 1988, Sugita et al. al 1988). The mutations of the dystrophin vary from one family of patients to another but always lead to the absence of a functional dystrophin protein under the membrane on the muscle fiber (Hoffman et al. 1987; Chelly et al. 1990; Chamberlain et al. 1991; Anderson et al 1992; Kilimann et al. 1992; Roberts et al 1992). The absence of dystrophin leads to an increase vulnerability of the muscle fibers during contraction (Menke 1995). Repeated cycles of damages and repairs produce a progressive reduction of the number of muscle fibers and to loss of strength which confine the patients to a wheel chair by the age of ten and to premature death in their early twenties. 
     Roughly 70% of the mutations of the dystrophin gene are large deletion of one of several exons (Anderson et al 1992; Kilimann et al. 1992). The other mutations are small point mutations due either to a small deletion of a few base pairs leading to a shift of the reading frame or changes of only one base pair producing a missense or a stop codon (Bullman et al 1991. Corrado et al. 1994; Roberts et al. 1992; Clemens et al 1992; Nicholson et al. 1993). Around 5% of all DMD mutations may be due to stop codons. 
     Cystic fibrosis (CF) is due to a mutation of a gene coding for the CF transmembrane conductance regulator (CFTR) protein. Howard et al. (1996) made experiments with a bronchial epithelial cell line obtained from a CF patient having a premature stop mutation in the CFTR gene. This mutation resulted in a premature end of the synthesis of the CFTR protein and thus in a non-functional protein. They incubated this cell line with aminoglycoside antibiotics G418 (100 mg/mL) or with a gentamicin (200 mg/mL) during 18 to 24 hours. This incubation with gentamicin permitted to suppress the premature stop mutation by inserting an amino acid at the stop codon. A full-length CFTR protein was thus obtained The suppression of the premature stop codon by gentamicin is mediated by mis-pairing between the stop codon and a near-cognate aminoacyl tRNA. Bedwell et al. (1997) recently demonstrated that this full length CFTR protein resulting from the incubation with the aminoglycoside antibiotics was present in the cell membrane and functional. 
     The mdx mouse is an animal model for DMD. It has a point mutation in the dystrophin gene resulting in a truncated protein which is not incorporated in the muscle fiber membrane (Hoffman et al 1987). 
     Therefore, this model is proper for testing the effect of aminoglycosides. 
     SUMMARY OF THE INVENTION 
     In accordance with the present invention is provided the first method for the in vivo treatment of a disease which is clue to the presence of a premature stop codon in a nucleic acid encoding a protein involved in the etiology of the disease, the method comprising the step of: 
     administering to the subject an effective dose of an aminoglycoside antibiotic, a derivative thereof or an aminoglycoside-like molecule to suppress the expression of said stop codon. 
     In a particular embodiment, each mention of the term “aminoglycoside” is intended to mean “gentamicin”. Other related molecules or derivatives are within the scope of this invention. 
     In a preferred embodiment, the aminoglycoside antibiotic is gentamicin sulfate. 
     The disease that has been treated in practice is Duchenne Muscular Dystrophy. Other diseases caused by a stop mutation would benefit from this invention. 
     The effective dose of aminoglycoside can be administered intra-muscularly, intravenously or subcutaneously, preferably intramuscularly. 
     The effective dose is equivalent to about 8 to 40 mg gentamicin sulfate per kg of body weight per day in mice administered intra-muscularly, or to about 1.5 to 6 mg gentamicin sulfate per kg of body weight per day in humans administered intra-muscularly. 
     Is therefore contemplated a new use of an aminoglycoside antibiotic, a derivative thereof or an aminoglycoside-like molecule in the making of a medication to treat a subject affected by a disease due to the presence of a premature stop codon in a nucleic acid encoding a protein involved in the etiology of the disease, whereby the expression of said stop codon is suppressed and said nucleic acid is correctly translated in to a functional protein. 
    
    
     DESCRIPTION OF THE INVENTION 
     In the present series of experiments, 4 mdx mice were treated with I.M. injections of gentamicin, two of them received 1 mg/day and the other two 5 mg/day during 7 days. Control normal mice and mdx mice did not receive any gentamicin injections. All mice were then sacrificed, their skeletal muscle and their heart were frozen and cryostat sectioned. The presence of dystrophin in these sections was investigated by immunohistochemistry. Strong dystrophin immunostaining was observed in the normal skeletal and heart muscles. In the untreated mdx muscles, dystrophin immunostaining was observed only in a few revertant fibers (Hoffman et al. 1990). Dystrophin was detected by immunohistochemistry on all muscle fibers and all heart muscle cells of the mdx mice treated with gentamicin. In some muscles of mice treated with 5 mg/day of gentamicin the staining appeared as intense as that observed in the normal mouse muscles. The dystrophin staining was less intense in the muscles of mice treated with 1 mg/kg. This is the first demonstration that an aminoglycoside suppressed in vivo a premature stop codon. Therefore a dose of 8 to 40 mg per Kg of body weight per day successfully suppressed the premature stop codon by inserting an amino acid, and continuing translation of gentamicin-encoding nucleic acids Subcutaneous and intravenous administration would provide the same results. The duration of treatment may vary. For the purpose of the present demonstration, the i.m. treatment lasted for 7 days. Sub-cutaneous or intra-muscular daily treatments of 7 to 14 days or longer have been equivalently successful. 
     In humans, the usual antibiotic dosage rate of gentamicin is of about 3-5 mg/Kg/day, non-divided dose, for intramuscular route of administration. To avoid toxicity, it is recommended that gentamicin dosage rates be adjusted to avoid blood concentrations higher than 12 μg/mL for prolonged periods of time. For the purpose of using an optimally effective gentamicin dose, it may be necessary to administer maximal gentamicin doses achieving 12 μg/mL or higher blood concentration levels In mdx mice, a blood concentration which is about 65 μg/mL was achieved with a dose of about 35-40 gentamicin mg/Kg/day). No toxicity signs appeared in mdx mice. The blood circulation levels of gentamicin that would be effective in humans have not been calculated, but allometric calculation could bring a dose effective in mice close to an equivalent of about 1.5 to about 6 mg per Kg per day in humans. It is possible to co-administer agents to counteract the oto- or nephrotoxicity of high doses of gentamicin, such as 2,3 dihydroxybenzoate (DHB) as disclosed by Song and Schacht (1996) and by Pearce et al. (1985). Alternatively, aminoglycoside derivatives having an activity towards inserting an amino acid instead of stopping translation may be designed, taking gentamicin as a reference compound. Such gentamicin-like or derived molecules may be more potent than gentamicin and provide fewer toxic side effects. Further, any mean by which gentamicin or gentamicin-like or derived molecules can be driven more directly to the site of action is within the scope of this invention. This would lead to more selective treatment with lower side effects Liposomes or immunoliposomes may be envisageatle, for example, or else, conjugating a gentamicin-related molecule to a ligand specific to muscle tissue (targeting muscle tissue as opposed to systemic dispersion). 
     Any other disease caused by a stop mutation in nucleic acids of interest, such as certain types of hemophilia, are under the scope of this invention 
     Although the present invention has been described hereinabove by way of a preferred embodiment thereof, this embodiment can be modified at will, within the scope of the appended claims, without departing from the spirit and nature of the subject invention. 
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