Muteins of the pyrroline-5-carboxylate reductase 1

Abstract

The invention relates to muteins of the pyrroline-5-carboxylate reductase 1 (PYCR1), to nucleic acid molecules comprising a nucleotide sequence encoding such muteins, to methods of determining in a subject a predisposition of having an age related disorder associated with PYCR1, to methods of identifying a compound capable of modifying the expression of PYCR1 and methods of treating a subject having an age-related disorder associated with PYCR1. The invention further relates to a genetically modified animal and a method of modifying the expression of the PYCR1 gene in an animal.

1 . A mutein of the pyrroline-5-carboxylate reductase 1 (PYCR1), wherein at least one of the amino acid residues at sequence positions 4 to 220 and 222 to 266 of the wild type amino acid sequence of PYCR1 as set forth in Swiss-Prot Accession No. P32322 is mutated; or wherein the mutein comprises a mutation of at least one of the amino acid residues at sequence positions 4 to 266 of the wild type amino acid sequence of PYCR1 as set forth in Swiss-Prot Accession No. P32322, wherein the mutation leads to a reduced function or loss of function of the mutein compared to the wild-type protein. 
     
     
         2 . (canceled) 
     
     
         3 . The mutein according to  claim 1 , wherein at least one of the amino acid residues at sequence positions 4, 119, 179, 189, 206, 251, 257 and 266 is mutated. 
     
     
         4 . The mutein according to  claim 3 , wherein the mutation of the amino acid residue at sequence position 4 is a frameshift mutation leading to a translation stop codon occurring at the codon that encodes for sequence position 50 of the wiid type amino acid sequence of PYCR1. 
     
     
         5 . The mutein according to  claim 3 , wherein the amino acid residue at sequence position 119 is replaced by glycine or histidine. 
     
     
         6 . The mutein according to  claim 3 , wherein the amino acid residue at sequence position 179 is replaced by a hydrophilic amine acid, or wherein the hydrophilic amino acid is a hydroxyl-containing amino acid. 
     
     
         7 . (canceled) 
     
     
         8 . (canceled) 
     
     
         9 . The mutein according to  claim 3 , wherein the amino acid residue at sequence position 189 is replaced by a hydrophobic amino acid, or wherein the hydrophobic amino acid is an aliphatic amino acid. 
     
     
         10 . (canceled) 
     
     
         11 . (canceled) 
     
     
         12 . The mutein according to  claim 3 , wherein the amino acid residue at sequence position 206 is replaced by an aromatic amino acid. 
     
     
         13 . (canceled) 
     
     
         14 . The mutein according to  claim 3 , wherein the amino acid residue at sequence position 206 is replaced by a positively charged amino acid, or wherein the tositively charged amino acid is arginine or lysine. 
     
     
         15 . (canceled) 
     
     
         16 . The mutein according to  claim 3 , wherein the amino acid residue at sequence position 251 is replaced by histidine. 
     
     
         17 . The mutein according to  claim 3 , wherein the amino acid residue at sequence position 257 is replaced by a hydrophilic amino acid, or wherein the hydrophilic amino acid is a hydroxyl-containing amino acid. 
     
     
         18 . (canceled) 
     
     
         19 . (canceled) 
     
     
         20 . The mutein according to  claim 3 , wherein the amino acid residue at sequence position 266 is replaced by glutamine or asparagine. 
     
     
         21 . (canceled) 
     
     
         22 . A nucleic acid molecule comprising a nucleotide sequence encoding a mutein according to  claim 1 . 
     
     
         23 . (canceled) 
     
     
         24 . (canceled) 
     
     
         25 . A method of determining in a subject a predisposition of having an age-related disorder associated with pyrroline-5-carboxylate reductase 1 (PYCR1), the method comprising analyzing a nucleic acid sample obtained from a subject for the presence of a nucleotide sequence encoding a mutein according to  claim 1 , wherein the presence of the nucleotide sequence indicates a predisposition of the subject having or being at risk for having the age-related disorder wherein the age-related disorder is selected from the group consisting of cutis laxa (wrinkly skin syndrome), de Barsy syndrome and gerodermia osteodysplasia. 
     
     
         26 . (canceled) 
     
     
         27 . (canceled) 
     
     
         28 . (canceled) 
     
     
         29 . A method of identifying a compound capable of modifying the expression of a PYCR1 gene, the method comprising:
 a. contacting a compound of interest with a nucleic acid molecule comprising a nucleotide sequence encoding pyrroline-5-carboxylate reductase 1 (PYCR1) or a functional fragment or mutant thereof, and   b. measuring the expression of the nucleotide sequence encoding pyrroline-5-carboxylate reductase 1 (PYCR1) or a functional fragment or functional mutant thereof, further comprising:   comparing the result of the measurement obtained from step (b) with that of a control measurement without the addition of the compound of interest.   
     
     
         30 . (canceled) 
     
     
         31 . (canceled) 
     
     
         32 . A method of modifying the expression of a PYCR1 gene in a cell, the method comprising introducing a nucleic acid molecule comprising a nucleotide sequence encoding pyrroline-b-carboxylate reductase 1 (PYCR1) or a functional fragment or functional mutant thereof into the cell. 
     
     
         33 . The method according to  claim 32 , wherein the expression of the PYCR1 gene is increased. 
     
     
         34 . (canceled) 
     
     
         35 . (canceled) 
     
     
         36 . (canceled) 
     
     
         37 . (canceled) 
     
     
         38 . (canceled) 
     
     
         39 . A method of treating a subject having an age-related disorder associated with PYCR1 or being at risk to develop an age-related disorder associated with PYCR1, the method comprising introducing a nucleic acid molecule comprising a nucleotide sequence encoding pyrroline-5-carboxylate reductase 1 (PYCR1) or a functional fragment or functional mutant thereof into the subject, or administering to the subject a compound capable of modifying the expression of a PYCR1 gene. 
     
     
         40 . (canceled) 
     
     
         41 . (canceled) 
     
     
         42 . A genetically modified animal, wherein the genetically modified animal comprises a nucleic acid encoding pyrroline-5-carboxylate reductase 1 (PYCR1), and wherein the nucleic acid is inactive, or wherein the inactive nucleic acid leads to a loss of function of the PYCR1. 
     
     
         43 . (canceled) 
     
     
         44 . (canceled) 
     
     
         45 . (canceled) 
     
     
         46 . (canceled) 
     
     
         47 . A method of modifying the expression of the PYCR1 gene in an animal, the method comprising administering to the animal a compound capable of modifying the expression of the PYCR1 gene, wherein the compound comprises a nucleic acid molecule that is capable of modifying the expression of the PYCR1 gene. 
     
     
         48 . (canceled) 
     
     
         49 . (canceled) 
     
     
         50 . A method of identifying a compound capable of modifying the expression of a PYCR1 gene, the method comprising:
 c. administering the compound of interest to an animal according to  claim 42 ; and   d. determining whether the compound is capable of modifying the expression of the PYCR1 gene.   
     
     
         51 . The method according to  claim 50 , wherein the compound increases the expression of the PYCR1 gene. 
     
     
         52 . The method according to  claim 50 , wherein the compound is formulated in a form of a cosmetic composition. 
     
     
         53 . A method of identifying a compound capable of modifying the expression of a PYCR1 gene, the method comprising:
 e, providing an isolated skin flap comprising at least one layer of living animal skin, said flap being attached to a test animal; and   f. applying the compound of interest to the living animal skin, wherein the skin flap includes cells that express the mutein as defined in  claim 1 .   
     
     
         54 . (canceled) 
     
     
         55 . The method according to  claim 53 , wherein the cells are fibroblasts. 
     
     
         56 . (canceled) 
     
     
         57 . (canceled) 
     
     
         58 . (canceled) 
     
     
         59 . (canceled) 
     
     
         60 . (canceled) 
     
     
         61 . The method according to  claim 53 , further comprising obtaining a sample of DNA from the skin flap to measure the expression of the PYCR1 gene. 
     
     
         62 . (canceled) 
     
     
         63 . (canceled) 
     
     
         64 . (canceled) 
     
     
         65 . (canceled) 
     
     
         66 . (canceled) 
     
     
         67 . (canceled) 
     
     
         68 . (canceled)