Bcl-2-modifying factor (bmf) sequences and their use in modulating apoptosis

Abstract

The present invention relates generally to novel molecules capable of, inter alia, modulating apoptosis in mammalian cells and to genetic sequences encoding same. More particularly, the present invention relates to a novel member of the Bcl-2 family of proteins, referred to herein as “Bmf”, and to genetic sequences encoding same and to regulatory sequences such as a promoter sequence directing expression of Bmf. Bmf comprises a BH3 domain which facilitates interaction to pro-survival Bcl-2 family members thereby triggering apoptosis. Bmf is regarded, therefore, as a BH3-only molecule. The molecules of the present invention are useful, for example, in therapy, diagnosis, antibody generation and as a screening tool for therapeutic agents capable of modulating physiological cell death or survival and/or modulating cell cycle entry. The present invention further contemplates genetically modified animals in which one or both alleles of Bmf are mutated or partially or wholly deleted alone or in combination with a mutation in one or both alleles of another Bcl-2-type molecule such as but not limited to Bim. The genetically modified animals are useful inter alia in screening for agents which ameliorate the symptoms of diseases caused by defects in apoptosis or which specifically promote apoptosis of target cells.

1 . An isolated nucleic acid molecule comprising a nucleotide sequence encoding or complementary to a sequence encoding an amino acid sequence substantially as set forth in one of SEQ ID NO:2 or SEQ ID NO:6 or SEQ ID NO:8 or a derivative or homolog thereof or having at least about 45% or greater similarity to one or more of SEQ ID NO:2 or SEQ ID NO:6 or SEQ ID NO:8 or a derivative or homolog thereof, wherein the derivative comprises a BH3 domain and binds a member of the pro-survival Bcl-2. 
     
     
         2 - 5 . (canceled) 
     
     
         6 . The nucleic acid molecule of  claim 1  comprising a nucleotide sequence set forth in SEQ ID NO:1 or a nucleotide sequence having at least about 45% similarity thereto or a nucleotide sequence capable of hybridizing to SEQ ID NO:1 or its complementary form under low stringency conditions. 
     
     
         7 . (canceled) 
     
     
         8 . The nucleic acid molecule of  claim 1  comprising a nucleotide sequence set forth in SEQ ID NO:5 or a nucleotide sequence having at least about 45% similarity thereto or a nucleotide sequence capable of hybridizing to SEQ ID NO:5 or its complementary form under low stringency conditions. 
     
     
         9 . The nucleic acid molecule of  claim 1  comprising a nucleotide sequence set forth in SEQ ID NO:7 or a nucleotide sequence having at least about 45% similarity thereto or a nucleotide sequence capable of hybridizing to SEQ ID NO:7 or its complementary form under low stringency conditions. 
     
     
         10 - 13 . (canceled) 
     
     
         14 . An isolated protein comprising an amino acid sequence encoded by a nucleotide sequence set forth in SEQ ID NO:1 or SEQ ID NO:5 or SEQ ID NO:7 or a nucleotide sequence having at least about 45% similarity to the nucleotide sequence set forth in SEQ ID NO: 1 or SEQ ID NO:5 or SEQ ID NO:7 or a nucleotide sequence capable of hybridizing to SEQ ID NO:1 or SEQ ID NO:5 or SEQ ID NO:7 or a complement thereof under low stringency conditions. 
     
     
         15 . The isolated protein of  claim 14  comprising an amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO:11. 
     
     
         16 - 18 . (canceled) 
     
     
         19 . The isolated protein of  claim 14  comprising an amino acid sequence as set forth in SEQ ID NO:2 or having at least 45% similarity thereto. 
     
     
         20 . (canceled) 
     
     
         21 . The isolated protein of  claim 14  comprising an amino acid sequence as set forth in SEQ ID NO:6 or having at least 45% similarity thereto. 
     
     
         22 . The isolated protein of  claim 14  comprising an amino acid sequence as set forth in SEQ ID NO:8 or having at least 45% similarity thereto. 
     
     
         23 - 26 . (canceled) 
     
     
         27 . A variant of an isolated bmf nucleic acid molecule comprising at least one nucleotide mutation in said nucleic acid molecule resulting in at least one amino acid addition, substitution and/or deletion to the polypeptide encoded by said variant wherein said polypeptide cannot bind, couple or otherwise associate with a dynein light chain, such as DLC2. 
     
     
         28 . The variant of  claim 27  wherein the mutation results in an altered amino acid sequence in the region which binds to the dynein light chain. 
     
     
         29 . A variant of an isolated Bmf polypeptide comprising at least one amino acid addition, substitution and/or deletion wherein said polypeptide cannot bind, couple or otherwise associate with the dynein light chain. 
     
     
         30 . A method of modulating activity of Bmf in a mammal, said method comprising administering to said mammal a modulating effective amount of an agent for a time and under conditions sufficient to increase or decrease Bmf activity. 
     
     
         31 . A method of modulating apoptosis in a mammal, said method comprising administering to said mammal an effective amount of an agent for a time and under conditions sufficient to modulate the expression of a nucleotide sequence encoding bmf. 
     
     
         32 . A method of modulating apoptosis in a mammal, said method comprising administering to said mammal an effective amount of an agent for a time and under conditions sufficient to modulate the activity of Bmf. 
     
     
         33 - 34 . (canceled) 
     
     
         35 . The method of  claim 30  or  31  or  32  wherein the mammal is a human. 
     
     
         36 . A pharmaceutical composition comprising bmf, Bmf or derivative thereof or an agent capable of modulating bmf expression or Bmf activity together with one or more pharmaceutically acceptable carriers and/or diluents, bmf, Bmf or said agent are referred to as the active ingredients. 
     
     
         37 . A monoclonal antibody having specificity for Bmf or bmf or derivative thereof. 
     
     
         38 - 39 . (canceled) 
     
     
         40 . A genetically modified animal in which one or both alleles of bmf are mutated alone or in combination with another mutation in one or both alleles for another Bcl-2 molecule such as but not limited to genes encoding Blk, Bad, Bik, Hrk, Bid, Bim, Noxa and/or Puma. 
     
     
         41 . The genetically modified animal of  claim 40  wherein said animal is a mouse, rat or pig. 
     
     
         42 - 47 . (canceled)