HUMAN FACTOR IX VARIANTS WITH AN EXTENDED HALF LIFE
Factor IX variants are described with an increase in the number of glycosylation sites. The Factor IX variants have an increased half life and/or recovery.
1 . An isolated Factor IX (FIX) protein variant comprising one or more than one additional glycosylation site as compared to wild type Factor IX.
2 . The FIX variant of claim 1 , wherein at least one of the one or more than one additional glycosylation sites is in the activation peptide.
3 . The FIX variant of claim 1 , comprising a peptide segment inserted between position N157 and N167 of the human FIX amino acid sequence of SEQ ID NO:33.
4 . The FIX variant of claim 3 , wherein the peptide segment comprises 3-100 amino acid residues.
5 . The FIX variant of claim 4 , wherein the peptide segment comprises at least part of a mouse Factor IX activation peptide.
6 . The FIX variant of claim 5 , wherein the mouse activation peptide is modified to increase the number of glycosylation sites.
7 . The FIX variant of claim 1 , wherein the one or more than one additional glycosylation sites are selected from N-linked glycosylation site(s), O-linked glycosylation site(s) and a combination of N-linked glycosylation site(s) and O-linked glycosylation site(s).
8 . The FIX variant of claim 7 , wherein the glycosylation site(s) comprise N-linked glycosylation site(s) comprising a consensus sequence NXT/S, with the proviso that X is not proline.
9 . The FIX variant of claim 7 , wherein the glycosylation site(s) comprise O-linked glycosylation site(s) comprising a consensus sequence selected from the group consisting of CXXGGT/S-C (SEQ ID NO:9), NSTE/DA (SEQ ID NO:10), NITQS (SEQ ID NO:11), QSTQS (SEQ ID NO:12), D/E-FT-R/K-V (SEQ ID NO:13), C-E/D-SN (SEQ ID NO:14), GGSC-K/R (SEQ ID NO:15) and any combination thereof.
10 . The FIX variant of claim 1 , comprising 1-5 additional glycosylation sites.
11 . A vector comprising a nucleotide sequence encoding the FIX variant of claim 1 .
12 . A transformed cell comprising the vector of claim 11 .
13 . A transgenic animal comprising the FIX variant of claim 1 .
14 . The FIX variant of claim 1 , wherein at least one additional glycosylation site is outside of the activation peptide.
15 . The FIX variant of claim 14 , wherein the at least one additional glycosylation site corresponds to a site that is glycosylated in the native form of a non-human homolog of FIX.
16 . The FIX variant of claim 15 , wherein the non-human homolog is selected from the group consisting of dog, pig, cow, and mouse.
17 . A method of increasing the number of glycosylation sites in a Factor IX protein comprising:
a) aligning a first FIX amino acid sequence and a second FIX amino acid sequence;
b) identifying a glycosylation site in the first FIX amino acid sequence that is not present in the second FIX amino acid sequence; and
c) modifying the second FIX amino acid sequence to introduce a glycosylation site corresponding to the glycosylation site identified in the first FIX amino acid sequence of step (b),
wherein modifying the second FIX amino acid sequence increases the number of glycosylation sites in the FIX protein.
18 . The method of claim 17 , wherein the first FIX amino acid sequence is from a non-human species and the second FIX amino acid sequence is human FIX.
19 . The method of claim 17 , wherein the glycosylation site in the first FIX amino acid sequence is in the activation peptide.
20 . The method of claim 17 , wherein the glycosylation site in the first FIX amino acid sequence is outside of the activation peptide.
21 . The FIX variant of claim 1 , which is a human FIX protein.
22 . The FIX variant of claim 1 , wherein the one or more additional glycosylation sites are introduced by insertion of additional amino acids, deletion of amino acids, substitution of amino acids and/or rearrangement of amino acids, in any combination.
23 . The FIX variant of claim 1 , wherein the one or more additional glycosylation sites are introduced by site-directed mutagenesis.
24 . The FIX variant of claim 1 , wherein the one or more additional glycosylation sites are introduced by chemical synthesis of the FIX variant.
25 . An isolated FIX variant comprising one or more additional sugar chains as compared to wild type FIX.
26 . The isolated FIX variant of claim 25 , wherein said one or more additional sugar chains are added to the FIX protein by chemical and/or enzymatic methods.