Optimized factor VIII gene
The present invention provides codon optimized Factor VIII sequences, vectors and host cells comprising codon optimized Factor VIII sequences, polypeptides encoded by codon optimized Factor VIII sequences, and methods of producing such polypeptides. The present invention also provides methods of treating bleeding disorders such as hemophilia comprising administering to the subject a codon optimized Factor VIII nucleic acid sequence or the polypeptide encoded thereby.
1 . A method of increasing expression of a polypeptide with Factor VIII activity in a subject comprising administering an isolated nucleic acid molecule to a subject in need thereof,
wherein the isolated nucleic acid molecule comprises a nucleotide sequence encoding a polypeptide with Factor VIII activity and has at least 96% sequence identity to SEQ ID NO: 2,
wherein the isolated nucleic acid molecule is comprised in a vector, and
wherein the expression of the polypeptide is increased relative to a reference nucleic acid molecule comprising SEQ ID NO: 3 or the vector comprising the reference nucleic acid molecule.
2 . The method of claim 1 , wherein plasma Factor VIII activity at 24 hours post administration is increased relative to a subject administered a reference nucleic acid molecule comprising SEQ ID NO: 3, a vector comprising the reference nucleic acid molecule, or a polypeptide encoded by the reference nucleic acid molecule.
3 . The method of claim 1 , wherein the nucleotide sequence comprises SEQ ID NO: 2.
4 . The method of claim 1 , wherein the nucleotide sequence has one or more of the following characteristics:
(a) the nucleotide sequence contains a higher percentage of G/C nucleotides compared to SEQ ID NO:3;
(b) the nucleotide sequence contains fewer MARS/ARS sequences compared to SEQ ID NO: 3;
(c) the nucleotide sequence does not contain the splice site GGTGAT (SEQ ID NO: 7);
(d) the nucleotide sequence contains fewer destabilizing elements (SEQ ID NO:8 and SEQ ID NO:9) relative to SEQ ID NO:3;
(e) the nucleotide sequence does not contain a poly-T sequence (SEQ ID NO:10);
(f) the nucleotide sequence does not contain a poly-A sequence (SEQ ID NO:11); or
(g) the human codon adaptation index is increased relative to SEQ ID NO:3.
5 . The method of claim 4 , wherein
(a) the human codon adaptation index is at least about 0.75;
(b) the percentage of G/C nucleotides is at least about 45%;
(c) the nucleotide sequence:
(i) contains at most one MARS/ARS sequence; or
(ii) does not contain a MARS/ARS sequence; and/or
(d) the nucleotide sequence:
(i) contains at most 4 destabilizing elements;
(ii) contains at most 2 destabilizing elements; or
(iii) does not contain a destabilizing element.
6 . The method of claim 1 , wherein the isolated nucleic acid molecule further comprises a heterologous nucleotide sequence.
7 . The method of claim 6 , wherein the heterologous nucleotide sequence encodes a heterologous amino acid sequence that is a half-life extender.
8 . The method of claim 6 , wherein the heterologous amino acid sequence is an immunoglobulin constant region or a portion thereof, transferrin, albumin, or a PAS sequence; optionally wherein the heterologous amino acid sequence is an Fc region.
9 . The method of claim 8 , wherein the heterologous amino acid sequence is linked to the N-terminus or the C-terminus of the amino acid sequence encoded by the nucleotide sequence or inserted between two amino acids in the amino acid sequence encoded by the nucleotide sequence.
10 . The method of claim 1 , wherein the vector is selected from a group consisting of an adenoviral vector, a lentiviral vector, a baculoviral vector, an Epstein Barr viral vector, a papovaviral vector, a vaccinia viral vector, a herpes simplex viral vector, and an adeno associated virus (AAV) vector.
11 . The method of claim 1 , wherein the subject has a bleeding disorder characterized by a deficiency in Factor VIII.
12 . The method of claim 1 , wherein the bleeding disorder is hemophilia.
13 . The method of claim 1 , wherein the bleeding disorder is hemophilia A.
14 . A method of treating a bleeding disorder characterized by a deficiency in Factor VIII, comprising administering to a subject in need thereof an isolated nucleic acid molecule,
wherein the isolated nucleic acid molecule is comprised in a vector, and
wherein the isolated nucleic acid molecule comprises a nucleotide sequence encoding a polypeptide with Factor VIII activity and has at least 96% sequence identity to SEQ ID NO: 2.
15 . The method of claim 14 , wherein the bleeding disorder is hemophilia.
16 . The method of claim 14 , wherein the bleeding disorder is hemophilia A.
17 . The method of claim 14 , wherein the vector is selected from a group consisting of an adenoviral vector, a lentiviral vector, a baculoviral vector, an Epstein Barr viral vector, a papovaviral vector, a vaccinia viral vector, a herpes simplex viral vector, and an adeno associated virus (AAV) vector.