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NZ745831B2 - Optimized factor viii genes - Google Patents
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NZ745831B2 - Optimized factor viii genes - Google Patents

Optimized factor viii genes

Info

Publication number
NZ745831B2
NZ745831B2 NZ745831A NZ74583117A NZ745831B2 NZ 745831 B2 NZ745831 B2 NZ 745831B2 NZ 745831 A NZ745831 A NZ 745831A NZ 74583117 A NZ74583117 A NZ 74583117A NZ 745831 B2 NZ745831 B2 NZ 745831B2
Authority
NZ
New Zealand
Prior art keywords
seq
nucleic acid
nucleotides
acid molecule
nucleotide sequence
Prior art date
Application number
NZ745831A
Other versions
NZ745831A (en
Inventor
Tongyao Liu
Siyuan Tan
Original Assignee
Bioverativ Therapeutics Inc
Filing date
Publication date
Application filed by Bioverativ Therapeutics Inc filed Critical Bioverativ Therapeutics Inc
Priority to NZ784737A priority Critical patent/NZ784737B2/en
Priority claimed from PCT/US2017/015879 external-priority patent/WO2017136358A1/en
Publication of NZ745831A publication Critical patent/NZ745831A/en
Publication of NZ745831B2 publication Critical patent/NZ745831B2/en

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/0008—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition
    • A61K48/0016—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition wherein the nucleic acid is delivered as a 'naked' nucleic acid, i.e. not combined with an entity such as a cationic lipid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/04—Antihaemorrhagics; Procoagulants; Haemostatic agents; Antifibrinolytic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/745—Blood coagulation or fibrinolysis factors
    • C07K14/755—Factors VIII, e.g. factor VIII C (AHF), factor VIII Ag (VWF)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/31—Fusion polypeptide fusions, other than Fc, for prolonged plasma life, e.g. albumin
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/15011—Lentivirus, not HIV, e.g. FIV, SIV
    • C12N2740/15041—Use of virus, viral particle or viral elements as a vector
    • C12N2740/15043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16041—Use of virus, viral particle or viral elements as a vector
    • C12N2740/16043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2800/00—Nucleic acids vectors
    • C12N2800/22—Vectors comprising a coding region that has been codon optimised for expression in a respective host
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof

Abstract

The present disclosure 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 disclosure 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.

Claims (31)

WE CLAIM:
1. An isolated nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide with FVIII activity, wherein the nucleotide sequence comprises a nucleic acid sequence comprising at least 90%, at least 91%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to nucleotides 58-2277 and 2320-4374 of SEQ ID NO:
2. The isolated nucleic acid molecule of claim 1, wherein the nucleotide sequence comprises at least 95% sequence identity to nucleotides 58-2277 and 2320-4374 of SEQ ID NO:
3. The isolated nucleic acid molecule of claim 1, wherein the nucleotide sequence comprises at least 99% sequence identity to nucleotides 58-2277 and 2320-4374 of SEQ ID NO:
4. The isolated nucleic acid molecule of any one of claims 1-3, wherein the nucleotide sequence further comprises a nucleic acid sequence encoding a signal peptide, wherein the nucleic acid sequence encoding a signal peptide has at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to: (i) nucleotides 1 to 57 of SEQ ID NO: 1; (ii) nucleotides 1 to 57 of SEQ ID NO: 2; (iii) nucleotides 1 to 57 of SEQ ID NO: 3; (iv) nucleotides 1 to 57 of SEQ ID NO: 4; (v) nucleotides 1 to 57 of SEQ ID NO: 5; (vi) nucleotides 1 to 57 of SEQ ID NO: 6; (vii) nucleotides 1 to 57 of SEQ ID NO: 70; (viii) nucleotides 1 to 57 of SEQ ID NO: 71; or (ix) nucleotides 1 to 57 of SEQ ID NO: 68.
5. The isolated nucleic acid molecule of any one of claims 1-4, wherein the nucleic acid molecule comprises one or more properties selected from the group consisting of: (a) the human codon adaptation index of the nucleic acid molecule or a portion thereof is increased relative to SEQ ID NO: 16; (b) the frequency of optimal codons of the nucleotide sequence or a portion thereof is increased relative to SEQ ID NO:16; (c) the nucleotide sequence or a portion thereof contains a higher percentage of G/C nucleotides compared to the percentage of G/C nucleotides in SEQ ID NO: 16; (d) the relative synonymous codon usage of the nucleotide sequence or a portion thereof is increased relative to SEQ ID NO: 16; (e) the effective number of codons of the nucleotide sequence or a portion thereof is reduced relative SEQ ID NO: 16; (f) the nucleotide sequence contains fewer MARS/ARS sequences (SEQ ID NOs: 21 and 22) relative to SEQ ID NO: 16; (g) the nucleotide sequence contains fewer destabilizing elements (SEQ ID NOs: 23 and 24) relative to SEQ ID NO: 16; and (h) any combination thereof.
6. The isolated nucleic acid molecule of any one of claims 1-5 further comprising a heterologous nucleotide sequence encoding a heterologous amino acid sequence.
7. The isolated nucleic acid molecule of claim 6, wherein the heterologous amino acid sequence is an immunoglobulin constant region or a portion thereof, XTEN, transferrin, albumin, or a PAS sequence.
8. The isolated nucleic acid molecule of claim 6, wherein the nucleotide sequence comprises SEQ ID NO: 72.
9. The isolated nucleic acid molecule of claim 6 or 7, 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 at one or more insertion sites.
10. The isolated nucleic acid molecule of claim 9, wherein the insertion site is immediately downstream of amino acid residue 3, 18, 22, 26, 40, 60, 65, 81, 116, 119, 130, 188, 211, 216, 220, 224, 230, 333, 336, 339, 375, 378, 399, 403, 409, 416, 442, 487, 490, 494, 500, 518, 599, 603, 713, 745, 1656, 1711, 1720, or 1725 corresponding to mature human FVIII (SEQ ID NO: 15).
11. The isolated nucleic acid molecule of any one of claims 1 to 10, wherein the FVIII polypeptide is a full length FVIII or a B domain deleted FVIII.
12. A vector comprising the nucleic acid molecule of any one of claims 1 to 11.
13. The vector of claim 12, wherein the vector is a lentiviral vector.
14. An isolated host cell comprising the nucleic acid molecule of any one of claims 1 to 11 or the vector of claim 12 or 13.
15. A method of producing a polypeptide with FVIII activity, comprising: culturing the host cell of claim 14 under conditions whereby a polypeptide with FVIII activity is produced, and recovering the polypeptide with FVIII activity.
16. Use of the isolated nucleic acid molecule of any one of claims 1 to 11, the vector of claim 12 or 13, or the polypeptide produced by the method of claim 15 in the manufacture of a medicament for the treatment of a bleeding disorder in a subject in need thereof.
17. An isolated nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide with FVIII activity, wherein the nucleotide sequence has at least 95% sequence identity to nucleotides 58 to 4374 of SEQ ID NO: 71.
18. The isolated nucleic acid molecule of claim 17, wherein the nucleotide sequence further comprises a nucleic acid sequence encoding a signal peptide, wherein the nucleic acid sequence encoding a signal peptide has at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to: (i) nucleotides 1 to 57 of SEQ ID NO: 1; (ii) nucleotides 1 to 57 of SEQ ID NO: 2; (iii) nucleotides 1 to 57 of SEQ ID NO: 3; (iv) nucleotides 1 to 57 of SEQ ID NO: 4; (v) nucleotides 1 to 57 of SEQ ID NO: 5; (vi) nucleotides 1 to 57 of SEQ ID NO: 6; (vii) nucleotides 1 to 57 of SEQ ID NO: 70; (viii) nucleotides 1 to 57 of SEQ ID NO: 71; or (ix) nucleotides 1 to 57 of SEQ ID NO: 68.
19. The isolated nucleic acid molecule of claim 17 or 18, wherein the nucleic acid molecule comprises one or more properties selected from the group consisting of: (a) the human codon adaptation index of the nucleic acid molecule or a portion thereof is increased relative to SEQ ID NO: 16; (b) the frequency of optimal codons of the nucleotide sequence or a portion thereof is increased relative to SEQ ID NO:16; (c) the nucleotide sequence or a portion thereof contains a higher percentage of G/C nucleotides compared to the percentage of G/C nucleotides in SEQ ID NO: 16; (d) the relative synonymous codon usage of the nucleotide sequence or a portion thereof is increased relative to SEQ ID NO: 16; (e) the effective number of codons of the nucleotide sequence or a portion thereof is reduced relative SEQ ID NO: 16; (f) the nucleotide sequence contains fewer MARS/ARS sequences (SEQ ID NOs: 21 and 22) relative to SEQ ID NO: 16; (g) the nucleotide sequence contains fewer destabilizing elements (SEQ ID NOs: 23 and 24) relative to SEQ ID NO: 16; and (h) any combination thereof.
20. The isolated nucleic acid molecule of any one of claims 17-19, further comprising a heterologous nucleotide sequence encoding a heterologous amino acid sequence.
21. The isolated nucleic acid molecule of claim 20, wherein the heterologous amino acid sequence is an immunoglobulin constant region or a portion thereof, XTEN, transferrin, albumin, or a PAS sequence.
22. The isolated nucleic acid molecule of claim 21, wherein the nucleotide sequence comprises SEQ ID NO: 72.
23. The isolated nucleic acid molecule of claim 21 or 22, 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 at one or more insertion sites.
24. The isolated nucleic acid molecule of claim 23, wherein the insertion site is immediately downstream of amino acid residue 3, 18, 22, 26, 40, 60, 65, 81, 116, 119, 130, 188, 211, 216, 220, 224, 230, 333, 336, 339, 375, 378, 399, 403, 409, 416, 442, 487, 490, 494, 500, 518, 599, 603, 713, 745, 1656, 1711, 1720, or 1725 corresponding to mature human FVIII (SEQ ID NO: 15).
25. The isolated nucleic acid molecule of any one of claims 17 to 24, wherein the FVIII polypeptide is a full length FVIII or a B domain deleted FVIII.
26. A vector comprising the nucleic acid molecule of any one of claims 17 to 25.
27. The vector of claim 26, wherein the vector is a lentiviral vector.
28. An isolated host cell comprising the nucleic acid molecule of any one of claims 17 to 25 or the vector of claim 26 or 27.
29. A method of producing a polypeptide with FVIII activity, comprising: culturing the host cell of claim 28 under conditions whereby a polypeptide with FVIII activity is produced, and recovering the polypeptide with FVIII activity.
30. Use of the isolated nucleic acid molecule of any one of claims 17 to 25, the vector of claim 27 or 28, or the polypeptide produced by the method of claim 29 in the manufacture of a medicament for the treatment of a bleeding disorder in a subject in need thereof.
31. An isolated nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide with FVIII activity, wherein the nucleotide sequence has: (i) at least 91%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to nucleotides 58-2277 and 2320-4374 of SEQ ID NO: 1; (ii) at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to nucleotides 58-2277 and 2320-4374 of SEQ ID NO: 2; (iii) at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to nucleotides 58- 2277 and 2320-4374 of SEQ ID NO: 3; (iv) at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to nucleotides 58-2277 and 2320-4374 of SEQ ID NO: 4; (v) at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to nucleotides 58- 2277 and 2320-4374 of SEQ ID NO: 5; (vi) at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to nucleotides 58- 2277 and 2320-4374 of SEQ ID NO: 6. Bioverativ Therapeutics Inc. By the Attorneys for the Applicant SPRUSON & FERGUSON
NZ745831A 2017-01-31 Optimized factor viii genes NZ745831B2 (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
NZ784737A NZ784737B2 (en) 2017-01-31 Optimized factor VIII genes

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
US201662289696P 2016-02-01 2016-02-01
US201662409739P 2016-10-18 2016-10-18
PCT/US2017/015879 WO2017136358A1 (en) 2016-02-01 2017-01-31 Optimized factor viii genes

Publications (2)

Publication Number Publication Date
NZ745831A NZ745831A (en) 2025-07-25
NZ745831B2 true NZ745831B2 (en) 2025-10-29

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