Calbindin-D28k protection against glucocorticoid induced cell death

Abstract

The present invention provides novel compositions containing a calbindin-D 28k therapeutic element, which is involved in the regulation of apoptosis, and may be administered for the prevention of an abnormal apoptosis response in cells. In particular the compositions and methods of the present invention may be used for the prevention or induction of apoptosis in such cells types as osteoblasts and osteocytes. Specifically, the compositions and methods of the present invention are useful for the prevention of diseases associated with glucocorticoid induced cell death. Specifically, the compositions and methods of the present invention may be useful in the prevention of glucocorticoid induced cell death in osteoblasts and the treatment of such conditions as glucocorticoid induced osteoporosis.

What is claimed is:  
     
         1 . A vector suitable for use in a human comprising a polynucleotide, wherein said polynucleotide encodes a calbindin-D 28k  polypeptide.  
     
     
         2 . The vector of  claim 1 , wherein said polynucleotide sequence encoding the CALBINDIN-D 28k  polypeptide is SEQ ID NO:1.  
     
     
         3 . The vector of  claim 1 , wherein the polynucleotide sequence has at least 70% identity to SEQ ID NO:1, said identity being calculated over the entire length of SEQ ID NO:1.  
     
     
         4 . The vector of  claim 1 , wherein the polynucleotide sequence is identical to SEQ ID NO:1.  
     
     
         5 . The vector of  claim 1 , wherein the CALBINDIN-D 28k  polypeptide comprises an amino acid sequence of SEQ ID NO:2.  
     
     
         6 . The vector of  claim 5 , wherein the CALBINDIN-D 28k  polypeptide comprises an amino acid sequence that has at least 70% identity to SEQ ID NO:2, said identity being calculated over the entire length of SEQ ID NO:2.  
     
     
         7 . The vector of  claim 1 , wherein said polynucleotide encodes a calbindin-D 28k  antisense sequence to a nucleotide sequence encoding a CALBINDIN-D 28k  polypeptide.  
     
     
         8 . The vector of  claim 7 , wherein the polynucleotide sequence has at least 70% identity to the antisense polynucleotide sequence of  claim 7 , said identity being calculated over the entire length of the sequence.  
     
     
         9 . A vector comprising the polynucleotide sequence of  claim 7 , wherein said vector is capable of inhibiting the expression of a CALBINDIN-D 28k  polypeptide when said vector is present in a compatible host cell.  
     
     
         10 . A pharmaceutical composition suitable for use in a human comprising a biologically effective amount of a CALBINDIN-D 28k  polynucleotide and an acceptable carrier.  
     
     
         11 . The composition of  claim 10 , wherein the CALBINDIN-D 28k  polynucleotide sequence is substantially similar to SEQ ID NO: 1.  
     
     
         12 . The composition of  claim 10 , wherein the CALBINDIN-D 28k  polynucleotide sequence is an antisense sequence to SEQ ID NO: 1  
     
     
         13 . A pharmaceutical composition suitable for use in a human comprising a biologically effective amount of a CALBINDIN-D 28k  polypeptide and an acceptable carrier.  
     
     
         14 . The composition of  claim 13 , wherein the CALBINDIN-D 28k  polypeptide sequence is substantially similar to SEQ ID NO: 2.  
     
     
         15 . A method of treating a disease associated with abnormal glucocorticoid induced cell death comprising the administration of a pharmaceutical composition comprising a biologically effective amount of a CALBINDIN-D 28k  polynucleotide and an acceptable carrier.  
     
     
         16 . The method of  claim 15 , wherein the disease comprises glucocorticoid induced osteoporosis.  
     
     
         17 . A method of treating a disease associated with abnormal glucocorticoid induced cell death comprising the administration of a pharmaceutical composition comprising a biologically effective amount of a CALBINDIN-D 28k  polypeptide and an acceptable carrier.  
     
     
         18 . The method of  claim 17 , wherein the disease comprises glucocorticoid induced osteoporosis.  
     
     
         19 . A method of treating a disease associated with a lack of normal cell death comprising the administration of a pharmaceutical composition comprising a biologically effective amount of a polynucleotide coding for the antisense sequence to SEQ. ID. No. 1, and an acceptable carrier.  
     
     
         20 . The method of  claim 19 , wherein the disease is selected from the group consisting of osteoblastic cancer, osteocytic cancer, prostrate cancer, lymphocytic cancer, leukemia and lymphoma.  
     
     
         21 . A vector for the delivery of a calbindin-D 28k  therapeutic element to a human for the treatment of diseases associated with glucocorticoid induced cell death or an abnormal decrease in cell death, wherein the vector comprises an expression cassette encoding the calbindin-D 28k  therapeutic.  
     
     
         22 . The vector of  claim 21 , wherein the calbindin-D 28k  therapeutic is selected from the group consisting of an CALBINDIN-D 28k  polynucleotide, a CALBINDIN-D 28k  polynucleotide antisense sequence, a CALBINDIN-D 28k  protein, and a CALBINDIN-D 28k  protein fragment.  
     
     
         23 . The vector of  claim 21 , wherein the expression cassette comprises one or more elements selected from the group consisting of a host cell origin of replication, suitable promoter operably linked to a heterologous genetic element, internal ribosome entry site, splice donor site, splice acceptor site, suitable enhancer, PPT track, heterologous genetic element, a reporter gene, and an appropriate termination sequence.  
     
     
         24 . The vector of  claim 34  wherein the vector is selected from the group consisting of: retrovirus, lentivirus, adenovirus, herpes simplex viruses (HSV), cytomegalovirus (CMV), and adeno-associated virus (AAV).  
     
     
         25 . A method for introducing a CALBINDIN-D 28k  therapeutic into a human for the treatment of a disease associated with glucocorticoid induced cell death, comprising transducing the cell with the vector of  claim 21 .  
     
     
         26 . The method of  claim 25 , wherein the transduction occurs in vivo.  
     
     
         27 . The method of  claim 25 , wherein the transduction occurs ex vivo.  
     
     
         28 . The method of  claim 25 , wherein the cell is selected from the group consisting of osteoblasts and osteocytes.  
     
     
         29 . The cell of  claim 28 , wherein the cell comprises a neural cell.  
     
     
         30 . The method of  claim 25 , wherein the disease associated with glucocorticoid induced cell death is osteoporosis.  
     
     
         31 . A method for introducing a CALBINDIN-D 28k  therapeutic into a human for the treatment of a disease associated with glucocorticoid induced cell death, comprising transfecting the cell with a plasmid comprising an expression cassette encoding the CALBINDIN-D 28k  therapeutic.  
     
     
         32 . The method of  claim 31 , wherein the CALBINDIN-D 28k  therapeutic is selected from the group consisting of a CALBINDIN-D 28k  polynucleotide, a CALBINDIN-D 28k  polynucleotide antisense sequence, a CALBINDIN-D 28k  protein and a CALBINDIN-D 28k  protein fragment.  
     
     
         33 . The methods of  claim 31 , wherein said transfection is carried out by a procedure selected from the group consisting of calcium phosphate transfection, DEAE-dextran mediated transfection, transvection, microinjection, cationic lipid-mediated transfection, electroporation, scrape loading, ballistic introduction or infection, use of a gene gun, lyposome and lipofectamine transfection  
     
     
         34 . The method of  claim 31 , wherein the transfection occurs in vivo.  
     
     
         35 . The method of  claim 31 , wherein the transfection occurs in vitro.  
     
     
         36 . The method of  claim 31 , wherein the cell is selected from the group consisting of osteoblasts and osteocytes.  
     
     
         37 . The method of  claim 31 , wherein the disease associated with glucocorticoid induced cell death is osteoporosis.  
     
     
         38 . The cell of  claim 31 , wherein the cell comprises a neural cell.  
     
     
         39 . The method of  claim 31 , wherein the transfection takes place as part of an ex vivo procedure.  
     
     
         40 . The method of  claim 17 , wherein the disease comprises glucocorticoid induced neuronal cell death.  
     
     
         41 . The method of  claim 40 , wherein the neuronal cell comprises a hippocampal cell.