Expression and folding in the manufacturing process of cd-rap by using a cd-rap precursor protein

Abstract

The present invention relates to a CD-RAP precursor protein comprising a pre-sequence and CD-RAP, and its use in manufacturing of native CD-RAP. The present invention further relates to a composition comprising CD-RAP and at least one positively charged amino acid and a buffer, pharmaceuticals comprising said composition, their use in methods of treating and/or preventing cartilage disease or injury in patients suffering from aggrecan degradation, and/or increased influx of water into the cartilage, and/or decreased CD-RAP expression, as well as methods of producing said composition and methods of storing CD-RAP in said composition. The present invention further relates to a composition comprising liposomes comprising encapsulated CD-RAP or a variant thereof, its use in methods of treating joint disease or injury, and methods of producing such liposomal composition as well as storing CD-RAP therein.

What is claimed 
     
         1 . A CD-RAP precursor fusion protein comprising:
 a) a pre-sequence, which comprises at its C-terminus an enzymatic cleavage site and at its N-terminus, an amino acid sequence selected from the group consisting of MATTST (SEQ ID NO: 2), MATTLT (SEQ ID NO: 3), MATTSTG (SEQ ID NO: 4), MATLTG (SEQ ID NO: 5), MATTSTGN (SEQ ID NO: 6), MATTLTGN (SEQ ID NO: 7), MATTSTGNS (SEQ ID NO: 8), MATTLTGNS (SEQ ID NO: 9), MATTSTGNSA (SEQ ID NO: 10), and MATTLTGNSA (SEQ ID NO: 11; and,   b) CD-RAP having an amino acid sequence of SEQ ID NO: 1 or a polypeptide that has a sequence identity of at least 95% to the sequence of SEQ ID NO: 1.   
     
     
         2 . A nucleic add encoding the CD-RAP precursor fusion protein of  claim 1 . 
     
     
         3 . A vector comprising the nucleic acid of  claim 2 . 
     
     
         4 . A host cell comprising the CD-RAP precursor fusion protein of claim 
     
     
         5 . A host cell comprising the nucleic acid of 2. 
     
     
         6 . A host cell comprising the vector of  claim 3 . 
     
     
         7 . A method of produdng native CD-RAP comprising:
 a) cleaving the pre-sequence from the CD-RAP precursor fusion protein of  claim 1  at the C-terminus cleavage site of the pre-sequence by enzymatic cleavage; and   b) removing the cleaved pre-sequence to produce the native CD-RAP.   
     
     
         8 . A CD-RAP protein preparation comprising a CD-RAP protein having the mature CD-RAP sequence of SEQ ID NO: 1 with a length of 107 amino acids, wherein the ratio of said CD-RAP with a length of 107 amino acids to any other CD-RAP protein present in the CD-RAP protein preparation is ≥99 wt. %. 
     
     
         9 . A composition comprising CD-RAP or a variant thereof, and at least positively charged amino add and a buffer. 
     
     
         10 . A method of producing the composition of  claim 9 , comprising adding CD-RAP or a variant thereof, to a buffer comprising at least one positively charged amino acid. 
     
     
         11 . A composition comprising liposomes comprising encapsulated CD-RAP or a variant thereof, wherein the size of the liposomes is below 200 nm, below 150 nm, below 120 nm or below 100 nm. 
     
     
         12 . A method of producing a liposomal formulation comprising the steps of:
 a) dissolving at least one lipid in an organic solvent;   b) injecting the mixture of step a) into a circulating aqueous phase; and,   c) eliminating the solvent.   
     
     
         13 . A liposomal formulation produced by the method of  claim 12 . 
     
     
         14 . A method of reducing the size of liposomes in a liposomal formulation comprising the steps of:
 a) dissolving at least one lipid in an organic solvent;   b) injecting the mixture of step a) into a circulating aqueous phase; and,   c) eliminating the solvent.   
     
     
         15 . A method of reducing the polydispersity of a liposomal formulation comprising the steps of:
 a) dissolving at least one lipid in an organic solvent;   b) injecting the mixture of step a) into a circulating aqueous phase; and,   c) eliminating the solvent.   
     
     
         16 . A pharmaceutical comprising the composition of  claim 8 . 
     
     
         17 . A pharmaceutical comprising the composition of claim 
     
     
         18 . A pharmaceutical comprising the composition of  claim 11 . 
     
     
         19 . A method of treating and/or preventing cartilage disease or injury in a patient suffering from aggrecan degradation, and/or increased influx of water into the cartilage. and/or decreased CD-RAP expression comprising administering an effective amount of the composition of  claim 8  to the patient. 
     
     
         20 . A method of treating and/or preventing cartilage disease or injury in a patient suffering from aggrecan degradation, and/or increased influx of water into the cartilage, and/or decreased CD-RAP expression comprising administering an effective amount of the composition of  claim 9  to the patient. 
     
     
         21 . A method of treating and/or preventing cartilage disease or injury in a patient suffering from aggrecan degradation, and/or increased influx of water into the cartilage. and/or decreased CD-RAP expression comprising administering an effective amount of the composition of  claim 11  to the patient. 
     
     
         22 . A method of storing CD-RAP, comprising keeping CD-RAP in the composition of  claim 9 . 
     
     
         23 . A method of storing CD-RAP, comprising keeping CD-RAP in the composition of  claim 11 . 
     
     
         24 . The CD-RAP precursor fusion protein of  claim 1  wherein the polypeptide has a sequence identity of at least 98% to the sequence of SEQ ID NO: 1. 
     
     
         25 . The CD-RAP precursor fusion protein of  claim 1  wherein the polypeptide has a sequence identity of at least 99% to the sequence of SEQ ID NO: 1.