Hyaluronic Acid Binding Domain-Growth Factor Fusion Protein cDNAs and Fusion Proteins for Cartilage Matrix Preservation and Report

Abstract

The present invention provides isolated nucleic acid compounds, amino acids compounds and related materials, along with methods to make and use the compounds. In particular, there are provided gene therapy materials useful for inducing positive physiological responses in tissues, and fusion proteins encoded by the gene therapy materials.

We claim: 
     
         1 . A fusion protein, comprising:
 a.) a hyaluronic acid-binding domain (HAB) of a cartilage matrix protein linked to   b.) a conserved region of a growth factor (GF) protein,   wherein said fusion protein is capable of upregulating glycosaminoglycan (GAG) expression in chondrocytes.   
     
     
         2 . The fusion protein of  claim 1 , which further comprises at least one additional element selected from the group consisting of: signal peptide (SP); linker peptide (linker); protease cleavage site; E peptide; and additional functional peptide. 
     
     
         3 . The fusion protein of  claim 2 , wherein the order of elements, N-terminus to C-terminus is: SP-HAB-GF-E peptide. 
     
     
         4 . The fusion protein of  claim 2 , wherein the order of elements, N-terminus to C-terminus is selected from the group consisting of: SP-HAB-GF; SP-HAB-GF-E peptide; SP-HAB-GF-additional peptide sequence; SP-HAB-linker-GF-additional peptide sequence; SP-HAB-linker-GF-E peptide; SP-HAB-linker-protease cleavage sequence-GF-additional peptide sequence; and SP-HAB-linker-protease cleavage sequence-GF-E peptide. 
     
     
         5 . The fusion protein of  claim 2 , wherein the SP is selected from the group consisting of: aggrecan signal peptide, CD44 signal peptide; link protein signal peptide; TSG-6 signal peptide, versican signal peptide; and other HA-binding protein signal peptide. 
     
     
         6 . The fusion protein of  claim 1 , wherein the HAB is selected from the group consisting of: aggrecan; CD44; link protein; TSG-6; versican; and other HA-binding proteins. 
     
     
         7 . The fusion protein of  claim 2 , wherein the linker is selected from the group consisting of: Linker 1: GGSG (SEQ ID NO: 1); Linker 2: GGSGGGSG (SEQ ID NO: 2); Linker 3: GGSGGGSGGGSG (SEQ ID NO: 3); Linker 4: GGGGS (SEQ ID NO: 4); Linker 5: GGGGSGGGGS (SEQ ID NO: 5); Linker 6: GGGGSGGGGSGGGGS (SEQ ID NO: 6); Linker 7: GGSGGS (SEQ ID NO: 7); and Linker 8: VIGHPIDSE (SEQ ID NO: 8). 
     
     
         8 . The fusion protein of  claim 2 , wherein the protease cleavage site is selected from the group consisting of: SEQ ID NO: 9; SEQ ID NO: 10; SEQ ID NO: 11; SEQ ID NO: 12; and SEQ ID NO: 13. 
     
     
         9 . The fusion protein of  claim 1 , wherein the GF is selected from the group consisting of: IGF-1; BMP2; BMP4; BMP7; FGF2; FGF18; GDF5; TGF-β1 and TGF-β3 
     
     
         10 . The fusion protein of  claim 1 , wherein the CD44 HAB element is a polypeptide having at least 90 percent identity to a polypeptide selected from the group consisting of: amino acids 1-132 of SEQ ID NO: 64; amino acids 1-156 of SEQ ID NO: 68; amino acids 1-178 of SEQ ID NO: 72; and amino acids 1-222 of SEQ ID NO: 76. 
     
     
         11 . The fusion protein of  claim 1 , further comprising: flanking amino acids to the HAB, wherein the number of flanking amino acids is selected from the group consisting of: 5 amino acids; 10 amino acids; 15 amino acids; 20 amino acids; 25 amino acids; 30 amino acids; 35 amino acids; 40 amino acids; 45 amino acids; 50 amino acids; 55 amino acids; and 60 amino acids. 
     
     
         12 . The fusion protein of  claim 11 , wherein the flanking amino acids are at the N terminus of HAB. 
     
     
         13 . The fusion protein of  claim 11 , wherein the flanking amino acids are at the C terminus of HAB. 
     
     
         14 . A nucleic acid molecule comprising a nucleic acid sequence that encodes a fusion protein comprising:
 a.) a hyaluronic acid-binding domain of a cartilage matrix protein (HAB) operably linked to   b.) a conserved region of a growth factor protein (GF),   wherein said fusion protein is capable of up-regulating glycosaminoglycan (GAG) expression in chondrocytes.   
     
     
         15 . The nucleic acid molecule of  claim 14 , wherein the nucleic acid molecule further comprises at least one additional nucleic acid sequence, wherein the additional nucleic acid sequence is selected from the group consisting of: a nucleic acid sequence that encodes at least one signal peptide (SP); a nucleic acid sequence that encodes at least one linker sequence (linker); a nucleic acid sequence that encodes at least one protease cleavage sequence; a nucleic acid sequence that encodes at least one E peptide; and a nucleic acid sequence that encodes at least one additional functional peptide; wherein the nucleic acids are operably linked so as to express a functional fusion protein. 
     
     
         16 . The nucleic acid molecule of  claim 15 , wherein the order of operably-linked elements, 5′ to 3′ is selected from the group consisting of: SP-HAB-GF; SP-HAB-GF-additional functional peptide; SP-HAB-GF-E peptide; SP-HAB-linker-GF-additional functional peptide; SP-HAB-linker-GF-E peptide; SP-HAB-linker-protease cleavage sequence-GF-E peptide; and SP-HAB-linker-protease cleavage sequence-GF-additional functional peptide. 
     
     
         17 . The nucleic acid molecule of  claim 15 , wherein the SP is selected from the group consisting of: aggrecan signal peptide; CD44 signal peptide; link protein signal peptide; TSG-6 signal peptide; versican signal peptide; and other HA-binding protein signal peptide. 
     
     
         18 . The nucleic acid molecule of  claim 14 , wherein the HAB comprises a polynucleotide fragment of a nucleotide sequence encoding a protein selected from the group consisting of: aggrecan; CD44; link protein; TSG-6; versican; and other HA-binding proteins. 
     
     
         19 . The nucleic acid molecule of  claim 15 , wherein the linker is selected from the group consisting of: Linker 1: GGSG (SEQ ID NO: 1); Linker 2: GGSGGGSG (SEQ ID NO: 2); Linker 3: GGSGGGSGGGSG (SEQ ID NO: 3); Linker 4: GGGGS (SEQ ID NO: 4); Linker 5: GGGGSGGGGS (SEQ ID NO: 5); Linker 6: GGGGSGGGGSGGGGS (SEQ ID NO: 6); Linker 7: GGSGGS (SEQ ID NO: 7); and Linker 8: VIGHPIDSE (SEQ ID NO: 8). 
     
     
         20 . The nucleic acid molecule of  claim 15 , comprising a cleavage site for a protease selected from the group consisting of: enterokinase (EK); Furin; Factor Xa; Matrix metalloproteinase (MMP); and Aggrecanase. 
     
     
         21 . The nucleic acid molecule of  claim 14 , wherein the GF is selected from the group consisting of: IGF-1; BMP2; BMP4; BMP7; FGF2; FGF18; GDF5; TGF-β1; and TGF-β3. 
     
     
         22 . The nucleic acid molecule of  claim 14 , wherein the HAB element comprises a sequence that is at least 80% identical to at least one sequence selected from the group consisting of: nucleotides 1-222; 1-178; 1-156; and 1-132 of SEQ ID NO: 54. 
     
     
         23 . The nucleic acid molecule of  claim 14 , wherein the nucleic acid molecule encodes a fusion protein comprising HAB and IGF-1 linked by a number of amino acids selected from the group consisting of: at least about 60 amino acids, at least about 50 amino acids, at least about 40 amino acids, at least about 30 amino acids, at least about 20 amino acids, fewer than 20 amino acids; fewer than 15 amino acids; fewer than 10 amino acids; fewer than 5 amino acids; no amino acids. 
     
     
         24 . A composition comprising the nucleic acid molecule of  claim 14  and a pharmaceutically-acceptable carrier. 
     
     
         25 . A composition comprising the fusion protein of  claim 1  and a pharmaceutically-acceptable carrier. 
     
     
         26 . An expression vector encoding the fusion protein of  claim 1 , wherein the expression vector is a plasmid or a virus. 
     
     
         27 . The expression vector of  claim 26 , which is an adeno-associated virus plasmid (pAAV). 
     
     
         28 . A cell transformed, transfected, or transduced by a vector of  claim 26 . 
     
     
         29 . The cell of  claim 28 , wherein the cell is a chondrocyte. 
     
     
         30 . An animal model comprising the cell of  claim 28 . 
     
     
         31 . A method of producing a fusion protein comprising, expressing a nucleic acid molecule in the cell of  claim 28 . 
     
     
         32 . A method to upregulate glycosaminoglycan expression in at least one chondrocyte, comprising: expressing a nucleic acid molecule in at least one chondrocyte, wherein the nucleic acid molecule comprises a HAB element and a GF element. 
     
     
         33 . The method of  claim 32  wherein the nucleic acid molecule comprises at least one segment having at least 95% identity to a sequence selected from the group consisting of: SEQ ID NO: 28; SEQ ID NO: 35; SEQ ID NO: 37; SEQ ID NO: 42; SEQ ID NO: 46; SEQ ID NO: 50; SEQ ID NO: 62; SEQ ID NO: 66; SEQ ID NO: 70; and SEQ ID NO: 74. 
     
     
         34 . A method to treat a cartilage matrix protein-related pathology or injury in a subject, comprising: administering a fusion protein, wherein the fusion protein comprises:
 a.) a hyaluronic acid-binding domain (HAB) of a cartilage matrix protein linked to   b.) a conserved region of a growth factor (GF) protein,   wherein the fusion protein is capable of upregulating glycosaminoglycan (GAG) expression in chondrocytes;   
       upregulating GAG expression in chondrocytes in the subject; and 
       treating a cartilage matrix protein-related pathology or injury. 
     
     
         35 . The method of  claim 34  wherein the GF is IGF-I. 
     
     
         36 . The method of  claim 34  wherein the administering is performed by transforming at least one chondrocyte in vivo to express the fusion protein. 
     
     
         37 . The method of  claim 34  wherein the administering is performed by injection of a pharmaceutical composition comprising the fusion protein, wherein the fusion protein is produced in vitro. 
     
     
         38 . The method of  claim 34 , wherein the cartilage matrix protein-related pathology or injury is selected from the group consisting of: joint stiffness; joint pain; intervertebral disc pain; degenerative disease; facet disease; traumatic cartilage injury; surgical cartilage injury; osteoarthritis; and rheumatoid arthritis. 
     
     
         39 . The method of  claim 34  further comprising: increasing sports performance in the subject, wherein the sports performance is selected from the group consisting of: increased speed; increased endurance; increased weight-lifting ability; increased flexibility; increased strength; increased resistance to impact; increased concentration; and increased career length. 
     
     
         40 . The method of  claim 34  further comprising: decreasing GF removal rate from the joint synovium or intervertebral disc synovium of a subject, by introducing the fusion protein to at least one joint or intevertebral disc of a subject and decreasing GF removal rate from the joint synovium or intervertebral disc of the subject. 
     
     
         41 . The method of  claim 34  further comprising: increasing responsiveness rates of chondrocytes to GF in a subject, by introducing the fusion protein to at least one chondrocyte of a subject and increasing responsiveness rates of chondrocytes to GF in the subject.