Factor IX polypeptide mutant, its uses and method for its production
Disclosed are a modified FIX (factor IX) polypeptide comprising a leucine, cysteine, aspartic acid, glutamic acid, histidine, lysine, asparagine, glutamine or tyrosine in position 338; pharmaceutical preparations containing said modified FIX polypeptide; a nucleotide sequence coding for the modified FIX polypeptide; and a method for producing the modified FIX polypeptide.
1. A method of treating a coagulopathy in a human patient, comprising administering a vector to the human patient, wherein:
a. the vector is an adeno-associated virus;
b. the vector comprises a nucleic acid encoding a modified FIX polypeptide, the modified FIX polypeptide comprising at least 70% identity to SEQ ID NO: 2 and a leucine in position 338 of SEQ ID NO: 2; and
c. the vector comprises promoter sequences and transcription termination and control elements;
thereby treating the coagulopathy.
2. The method of claim 1 , wherein the modified FIX polypeptide is expressed in the human patient at a level at least five fold less than the level of wild-type FIX of SEQ ID NO: 2 in a healthy human lacking the coagulopathy.
3. The method of claim 1 , wherein the nucleic acid encodes a modified FIX polypeptide identical to SEQ ID NO: 2 except for the leucine at position 338.
4. The method of claim 1 , wherein the nucleic acid integrates stably into the human patient's genome.
5. The method of claim 1 , wherein the adeno-associated virus vector is administered at a dose of 1 to 10 14 particles per kilogram of patient weight.
6. The method of claim 1 , wherein the adeno-associated virus vector is administered at a dose of 10 6 to 10 12 particles per kilogram of patient weight.
7. The method of claim 1 , wherein the coagulopathy is a deficiency in FIX.
8. The method of claim 7 , wherein the deficiency in FIX is a genetic deficiency in FIX.
9. The method of claim 7 , wherein the coagulopathy is hemophilia B.
10. The method of claim 8 , wherein the coagulopathy is hemophilia B.
11. The method of claim 2 , wherein the healthy human expresses 5 μg/ml of wild-type FIX.
12. The method of claim 11 , wherein the 5 μg/ml of wild-type FIX is measured in plasma from the healthy human.
13. The method of claim 1 , wherein the modified FIX polypeptide is expressed at a daily dosage between 0.1 μg/kg and 400 μg/kg body weight.
14. The method of claim 1 , wherein the modified FIX polypeptide is expressed at a level that does not cause thrombotic complications.
15. The method of claim 1 , wherein the modified FIX polypeptide is expressed at a level that does not lead to neutralizing antibody generation.
16. The method of claim 1 , wherein the modified FIX polypeptide is expressed at a level that reduces and/or prevents hemorrhages.
17. The method of claim 2 , wherein the modified FIX polypeptide is expressed in the human patient at a level of seven to nine fold less than the level of wild-type FIX of SEQ ID NO: 2 in the healthy human.
18. The method of claim 2 , wherein the modified FIX polypeptide is expressed in the human patient at a level of eight to nine fold less than the level of wild-type FIX of SEQ ID NO: 2 in the healthy human.
19. The method of claim 1 , wherein the nucleic acid encodes for a FIX polypeptide precursor comprising at least 70% identity to SEQ ID NO: 1, said FIX polypeptide precursor comprising the modified FIX polypeptide.
20. The method of claim 1 , wherein the nucleic acid comprises an overall sequence having at least 70% homology with the overall sequence of exon regions in the sequence having accession number K02402 (GenBank).