Method of modulating cell survival and reagents useful for same

Abstract

The present invention relates generally to a method for modulating cell survival. Modulation of cell survival includes inducing, enhancing or otherwise promoting cell survival such as the survival of neural cells as well as facilitating cell death such as the death of targeted cancer cells. The modulation of cell survival is mediated by a region identified on the p75 neurotrophin receptor (p75 NTR ) required for death signalling. The present invention further provides genetic molecules which encode the death signalling region of p75 NTR which are useful in antagonizing death signal function as well as promoting cell death when expressed in targeted cells. The present invention also contemplates recombinant peptides, polypeptides and proteins s well as chemical equivalents, derivatives and homologues thereof which comprise the death signalling portion of p75 NTR . Particularly useful molecules of the present invention comprise peptides corresponding to soluble forms of the death signalling portion of p75 NTR . These molecules antagonize p75 NTR -mediated cell death.

1 . An isolated nucleic acid molecule comprising a sequence of nucleotides or complementary sequence of nucleotides which encodes an amino acid sequence which is capable of signalling, inducing or otherwise facilitating the death of a cell in which said amino acid sequence is adjacent, proximal or otherwise juxtaposed to the membrane of said cell or when said amino acid is in multimeric form.  
     
     
         2 . An isolated nucleic acid molecule according to  claim 1  wherein the amino acid sequence comprises a membrane-associating portion and/or multimer-forming portion and a portion which corresponds to all or part of the cytoplasmic region of p75 NTR  or a functional equivalent, derivative or homologue thereof.  
     
     
         3 . An isolated nucleic acid molecule according to  claim 2  wherein the membrane-associated portion is derived from p75 NTR  or a functional equivalent, derivative or homologue thereof.  
     
     
         4 . An isolated nucleic acid molecule according to  claim 2  wherein the membrane-associating portion is from a receptor or other ligand-binding molecule.  
     
     
         5 . An isolated nucleic acid molecule according to  claim 4  wherein the receptor or other ligand-binding molecule is a cytokine receptor, immunoglobulin or T-cell receptor.  
     
     
         6 . A homologue, analogue or derivative of the nucleic acid molecule of any one of  claims 1  to  5 .  
     
     
         7 . An isolated nucleic acid molecule comprising a sequence of nucleotides which encodes an amino acid sequence which inhibits or reduces p75 NTR -mediated cell death wherein said amino acid sequence is a soluble form of the p75 NTR  receptor corresponding to an intracellular region adjacent, proximal or otherwise juxtaposed to the membrane of said cell.  
     
     
         8 . An isolated nucleic acid molecule according to  claim 7  wherein the p75 NTR  is of human, primate or murine origin.  
     
     
         9 . An isolated nucleic acid molecule according to any one of the proceeding claims comprising a nucleotide sequence substantially capable of hybridizing to <400>1 or its complementary form under low stringency conditions.  
     
     
         10 . An isolated nucleic acid molecule according to  claim 9  comprising a nucleotide sequence substantially as set forth in <400>7 or a nucleotide sequence capable of hybridizing to <400>7 or its complementary form under low stringency conditions or a nucleotide sequence having at least 60% identity to <400>7.  
     
     
         11 . An isolated nucleic acid molecule according to  claim 9  comprising a nucleotide sequence encoding an amino acid sequence set forth in <400>8 or an amino acid sequence having at least 60% identity thereto.  
     
     
         12 . A nucleic acid molecule comprising the nucleotide sequence: 
       {n 1  - - - n x } b a{n′ l  - - - n′ y } c a{n″ l  - - - n″ z } d   
       wherein 
 {n 1  - - - n x } is a sequence of x nucleotides encoding an extracellular portion of a receptor or ligand-binding molecule;  
 {n′ l  - - - n′ y } is a sequence of y nucleotides encoding a transmembrane peptide, polypeptide or protein or a molecule capable of inducing multimerisation;  
 {n″ l  - - - n″ z } is a sequence of z nucleotides comprising a nucleotide sequence substantially as set forth in <400>7 or a nucleotide sequence encoding an amino acid sequence substantially as set forth in <400>8 or a nucleotide sequence capable of hybridising to <400>7 or a complementary form thereof under low stringency conditions such as at 42° C. or a nucleotide sequence having at least 60% identity to <400>7;  
 b, c and d may be the same or difference and each is 0, 1 or >1;  
 x, y and z may be the same or different and each is 0, 1 or >1;  
 a is a nucleotide bond;  
 wherein when c is 1 or >1 and d is 1 or >1 and wherein when the molecule is expressed in a neural cell, the expression product signals, induces or otherwise facilitates cell death.  
 
     
     
         13 . A nucleic acid molecule according to  claim 12  wherein {n 1  - - - n x } comprises the nucleotide sequence substantially as set forth in <400>3 or is a nucleotide sequence having at least about 60% identity thereto or is capable of hybridising thereto under low stringency conditions at 42° C.  
     
     
         14 . A nucleic acid molecule according to  claim 12  wherein {n′ l  - - - n′ y } comprises the nucleotide sequence substantially as set forth in <400>5 or is a nucleotide sequence having at least about 60% identity thereto or is capable of hybridising thereto under low stringency conditions at 42° C.  
     
     
         15 . A genetic construct comprising an isolated nucleic acid molecule which comprises a sequence of nucleotides which corresponds or is complementary to a death signal region from p75 NTR  or a homologue, analogue or derivative thereof.  
     
     
         16 . A genetic construct according to  claim 15  wherein the coding region of the death signal from p75 NTR  is placed in operable connection with a promoter sequence such that a gene product is capable of being expressed under the control of said promoter sequence.  
     
     
         17 . A genetic construct according to  claim 15  or  16  wherein said genetic construct further comprises a terminator sequence.  
     
     
         18 . A method for inhibiting, reducing or otherwise antagonising p75 NTR -mediated death signal in a neural cell, said method comprising administering a peptide, polypeptide or protein or analogues or mimetics thereof which correspond to a non-membrane associated form of the p75 NTR  death signal region or a derivative, functional equivalent or homologue thereof.  
     
     
         19 . A peptide antagonist of the p75 NTR  death signal or functional analogues or mimetics thereof.  
     
     
         20 . A recombinant peptide, polypeptide or protein produced by expressing the isolated nucleic acid molecule according to any one of  claims 1  to  14  in a suitable host cell or a derivative, homologue or analogue of said peptide, polypeptide or protein.  
     
     
         21 . An isolated peptide, polypeptide or protein according to  claim 20  comprising the cytoplasmic region of p75 NTR  which signals, induces or otherwise facilitates cell death when said peptide, polypeptide or protein is adjacent, proximal or otherwise juxtaposed to a membrane-associating region such as from p75 NTR  or other membrane molecule and/or is in multimeric form or a derivative, homologue, chemical equivalent or analogue of said peptide, polypeptide or protein.  
     
     
         22 . A peptide, polypeptide or protein according to  claim 21  comprising an amino acid sequence substantially as set forth in <400>8 or an amino acid sequence having at least 60% identity thereto or a chemical equivalent, derivative, homologue or analogue of said peptide, polypeptide or protein.  
     
     
         23 . A method for inhibiting, reducing or otherwise antagonising a p75 NTR -mediated death signal in a neural cell, said method comprising introducing a nucleic acid molecule capable of being expressed to an expression product which corresponds to a non-membrane associated form of the p75 NTR  death signal region or a derivative, functional equivalent or homologue thereof.  
     
     
         24 . A method for inhibiting, reducing or otherwise antagonising a p75 NTR -mediated death signal in a neural cell, said method comprising contacting a cell carrying a p75 NTR  with a death signal-inhibiting effective amount of a molecule capable of antagonising the death signal of p75 NTR  or a component of the death signalling pathway.  
     
     
         25 . A method according to  claim 23  or  24  for the treatment of a range of neurodegenerative diseases such as cerebral palsy, trauma induced paralysis, vascular ischaemia associated with stroke, neural tumours, motoneurone disease, Parkinson's disease, Huntington's disease, Alzheimer's disease, multiple sclerosis and peripheral neuropathies associated with diabetes, heavy metal or alcohol toxicity, renal failure and/or infectious diseases such as herpes, rubella, measles, chicken pox, HIV and/or HTLV-1.  
     
     
         26 . A method according to  claim 23  or  24  for treating neurons and neural cells damaged by trauma or disease.  
     
     
         27 . A biological composition comprising a genetic molecule capable of being expressed into a p75 NTR  death signal antagonist or a p75 NTR  death signal said composition further comprising one or more pharmaceutically acceptable carriers and/or diluents.  
     
     
         28 . A method for modulating p75 NTR -mediated death signal in a neural cell, said method comprising administering an agent which antagonises or agonises cleavage of the extracellular domain of p75 NTR .  
     
     
         29 . Use of an antagonist of the death signal of p75 NTR  in the manufacture of a medicament for the treatment of neurodegenerative diseases in animals.  
     
     
         30 . Use according to  claim 29  wherein the animal is a human, primate, livestock animal, laboratory test animal, companion animal and/or captive wild animal.