Methods and means for treating dna repeat instability associated genetic disorders

Abstract

The current invention provides for methods and medicaments that apply oligonucleotide molecules complementary only to a repetitive sequence in a human gene transcript, for the manufacture of a medicament for the diagnosis, treatment or prevention of a cis-element repeat instability associated genetic disorders in humans. The invention hence provides a method of treatment for cis-element repeat instability associated genetic disorders. The invention also pertains to modified oligonucleotides which can be applied in method of the invention to prevent the accumulation and/or translation of repeat expanded transcripts in cells.

1 . A method for preventing or treating a genetic disorder in a subject, comprising administering to a subject with a genetic disorder that is associated with human cis-element repeat instability a single stranded oligonucleotide comprising or consisting of a sequence that is complementary only to a repetitive element sequence in a gene transcript of a gene with said repeat instability. 
     
     
         2 . The method according to  claim 1  wherein the repetitive element is present in a coding sequence of the gene transcript. 
     
     
         3 . The method according to  claim 1  wherein the repetitive element is present in a non-coding sequence of the gene transcript. 
     
     
         4 . The method according to  claim 1  wherein the sequence of the repetitive element is selected from the group consisting of CAG; GCG; CUG; CGG; CCG; GAA; GCC; and CCUG. 
     
     
         5 . The method according to  claim 2 , wherein the oligonucleotide comprises or consists of a sequence that is complementary to a CAG repeat and wherein the disorder is Huntington's disease, spino-cerebellar ataxias, Haw River syndrome, X-linked spinal and bulbar muscular atrophy or dentatorubral-pallidoluysian atrophy. 
     
     
         6 . The method according to  claim 2 , wherein the oligonucleotide comprises or consists of a sequence that is complementary to a GCG repeat and wherein the disorder is infantile spasm syndrome, deidocranial dysplasia, blepharophimosis, hand-foot-genital disease, synpolydactyl, oculopharyngeal muscular dystrophy or holoprosencephaly. 
     
     
         7 . The method according to  claim 3 , wherein the oligonucleotide comprises or consists of a sequence that is complementary to a CUG repeat and wherein the disorder is myotonic dystrophy type 1, spino-cerebellar ataxia 8 or Huntington's disease-like 2. 
     
     
         8 . The method according to  claim 3  wherein the oligonucleotide comprises or consists of a sequence that is complementary to a CCUG repeat and wherein the disorder is myotonic dystrophy type 2. 
     
     
         9 . The method according to  claim 3 , wherein the oligonucleotide comprises or consists of a sequence that is complementary to a CGG repeat and wherein the disorder is fragile X syndrome. 
     
     
         10 . The method according to  claim 3 , wherein the oligonucleotide comprises or consists of a sequence that is complementary to a GAA repeat and wherein the disorder is Friedreich's ataxia. 
     
     
         11 . The method according to  claim 1  wherein the oligonucleotide has a length of about 10 to about 50 nucleotides. 
     
     
         12 . The method according to  claim 1  wherein the single stranded oligonucleotide comprises ribonucleotides, deoxyribonucleotides, nucleotides of a locked nucleic acid (LNA), nucleotides of a peptide nucleic acid (PNA), morpholino phosphorodiamidates, nucleotides of an ethylene-bridged nucleic acid or a mixture thereof. 
     
     
         13 . The method according to  claim 12 , wherein the oligonucleotide comprises 2′-O-substituted RNA phosphorothioate nucleotides. 
     
     
         14 . The method according to  claim 1  wherein the administered oligonucleotide is in the form of an expressible nucleic acid vector. 
     
     
         15 . The method according to  claim 1 , wherein the oligonucleotide is in a pharmaceutical composition which further comprises an excipient and/or targeting ligand that delivers the oligonucleotide to cells and/or enhances intracellular delivery of the oligonucleotide. 
     
     
         16 . A single stranded oligonucleotide comprising or consisting of a sequence of about 10 to about 50 nucleotides that is complementary to a repetitive sequence of a tri- or tetranucleotide selected from the group consisting of
 (a) CAG;   (b) GCG;   (c) CUG;   (c) CGG;   (d) GAA;   (e) GCC;   (f) CCUG.   
     
     
         17 . The oligonucleotide according to  claim 16 , comprising 2′-O-substituted phosphorothioate ribonucleotides, phosphorothioate deoxyribonucleotides, LNA nucleotides, morpholino nucleotides, and/or combinations thereof. 
     
     
         18 . The oligonucleotide according to  claim 26  wherein the label is a radioactive label or a fluorescent label. 
     
     
         19 . A pharmaceutical composition comprising an oligonucleotide according to  claim 16 , and a pharmaceutically acceptable excipient. 
     
     
         20 . The pharmaceutical composition of  claim 19 , further comprising a targeting ligand that delivers the oligonucleotide to a cell and/or enhances intracellular delivery of the oligonucleotide. 
     
     
         21 . A nucleic acid vector, that expresses the oligonucleotide according to  claim 16  in human cells. 
     
     
         22 . A method for reducing the number of repeat-containing gene transcripts in a cell comprising providing to said cell the oligonucleotide according to  claim 16 . 
     
     
         23 . (canceled) 
     
     
         24 . The method according to  claim 11  wherein the oligonucleotide has a length of about 12 to about 30 nucleotides. 
     
     
         25 . The method of  claim 12  wherein the single stranded oligonucleotide has a phosphorothioate-containing backbone. 
     
     
         26 . The oligonucleotide of  claim 16  that further comprises a detectable label. 
     
     
         27 . A method for detecting the presence of nucleic acid repetitive elements in cells, comprising:
 (a) contacting the cells or a lysate or extract thereof with the oligonucleotide according to  claim 18 , under conditions wherein said oligonucleotide hybridizes with cellular DNA and/or RNA;   (b) detecting hybridization of said oligonucleotide,   
       wherein the hybridization of said oligonucleotide is indicative of the presence of said repeat elements in said cells. 
     
     
         28 . A method for diagnosing a genetic disorder associated with human cis-element repeat instability in a subject, comprising performing the method of  claim 27  on cells, or obtained from a subject with, suspected of having, or at risk for said disorder, or on a lysate or extract of said cells,
 wherein detection of the presence of said repeat elements in said cells, lysate or extract is diagnostic of said genetic disorder.