Methods of expressing LIM mineralization protein in non-osseous cells
View Patent ↗Methods of expressing LIM mineralization protein in non-osseous mammalian cells, such as stem cells or intervertebral disc cells (e.g., cells of the annulus fibrosus, or cells of the nucleus pulposus) are described. The methods involve transfecting the cells with an isolated nucleic acid comprising a nucleotide sequence encoding a LIM mineralization protein operably linked to a promoter. Transfection may be accomplished ex vivo or in vivo by direct injection of virus or naked DNA, or by a nonviral vector such as a plasmid. Expression of the LIM mineralization protein can stimulate proteoglycan and/or collagen production in cells capable of producing proteoglycyan and/or collagen. Methods for treating disc disease associated with trauma or disc degeneration are also described.
1. A method of increasing an amount of a proteoglycan produced by a cell in a mammal comprising transfecting an isolated nucleic acid sequence encoding a Human LIM Protein-1 (HLMP-1) protein into said cell in vivo by delivering the isolated nucleic acid sequence directly into an intervertebral disc of the mammal; wherein the isolated nucleic acid sequence is operably linked to a promoter and is included within a viral vector and whereupon expression of the isolated nucleic acid sequence for the HLMP-1 protein results in the proteoglycan synthesis in the cell, and wherein said cell is selected from nucleus pulposus cells, annulus fibrosis cells and any combination thereof.
2. The method according to claim 1 , wherein the cell is an intervertebral disc cell and wherein the cell is transfected in vivo by direct injection of the isolated nucleic acid into an intervertebral disc of the mammal.
3. A method of increasing an amount of a proteoglycan produced by a cell in a mammal comprising transfecting an isolated nucleic acid sequence encoding a HLMP-1 protein into said cell ex vivo and implanting said transfected cell into an intervertebral disc of the mammal, wherein the isolated nucleic acid sequence is operably linked to a promoter, wherein said cell is selected from nucleus pulposus cells, annulus fibrosis cells and any combination thereof, and
whereupon expression of the isolated nucleic acid sequence for the HLMP-1 protein results in increasing the proteoglycan synthesis in the cell.
4. The method of claim 1 , wherein the isolated nucleic acid is in an adenoviral vector.
5. The method of claim 1 , wherein the isolated nucleic acid is in a retroviral vector.
6. The method of claim 4 , wherein the adenoviral vector is Recombinant Type 5 Adenovirus comprising nucleotide sequence encoding HLMP-1 (AdHLMP-1).
7. The method of claim 1 , wherein the promoter is a cytomegalovirus promoter.
8. The method of claim 3 , further comprising: combining the transfected cell with a carrier prior to implantation into the mammal.
9. The method of claim 8 , wherein the carrier comprises a porous matrix.
10. The method of claim 9 , wherein the porous matrix comprises a synthetic polymer or collagen matrix.
11. The method of claim 3 , wherein the isolated nucleic acid is in an expression vector.
12. The method of claim 11 , wherein the expression vector is a viral vector.
13. The method of claim 12 , wherein the viral vector is an adenoviral vector.
14. The method of claim 11 , wherein the promoter is a cytomegalovirus promoter.