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NZ729107B2 - An improved expression cassette for packaging and expression of variant factor viii for the treatment of hemostasis disorders - Google Patents

An improved expression cassette for packaging and expression of variant factor viii for the treatment of hemostasis disorders Download PDF

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Publication number
NZ729107B2
NZ729107B2 NZ729107A NZ72910715A NZ729107B2 NZ 729107 B2 NZ729107 B2 NZ 729107B2 NZ 729107 A NZ729107 A NZ 729107A NZ 72910715 A NZ72910715 A NZ 72910715A NZ 729107 B2 NZ729107 B2 NZ 729107B2
Authority
NZ
New Zealand
Prior art keywords
fviii
nucleic acid
variant
aav
vector
Prior art date
Application number
NZ729107A
Other versions
NZ729107A (en
Inventor
Liron Elkouby
Katherine A High
Denise Sabatino
Original Assignee
Liron Elkouby
Katherine A High
Denise Sabatino
The Children's Hospital Of Philadelphia
Filing date
Publication date
Application filed by Liron Elkouby, Katherine A High, Denise Sabatino, The Children's Hospital Of Philadelphia filed Critical Liron Elkouby
Priority claimed from PCT/US2015/045142 external-priority patent/WO2016025764A2/en
Publication of NZ729107A publication Critical patent/NZ729107A/en
Publication of NZ729107B2 publication Critical patent/NZ729107B2/en

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    • 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
    • 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
    • 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
    • A61K48/0066—Manipulation of the nucleic acid to modify its expression pattern, e.g. enhance its duration of expression, achieved by the presence of particular introns in the delivered nucleic acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
    • 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
    • A61P7/02—Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • 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
    • 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
    • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
    • C12N2750/00011—Details
    • C12N2750/14011—Parvoviridae
    • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
    • C12N2750/14141—Use of virus, viral particle or viral elements as a vector
    • C12N2750/14143—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
    • C12N2840/00—Vectors comprising a special translation-regulating system
    • C12N2840/007—Vectors comprising a special translation-regulating system cell or tissue specific

Abstract

Factor VIII variants and methods of use thereof are disclosed. In particular, the disclosure provides a Factor VIII (FVIII) encoding nucleic acid variant, wherein said nucleic acid variant has one or more leucine codons changed to CTG compared to TTA, TTG, CTT, CTC or CTA in wild type FVIII, wherein said nucleic acid variant has at least 50% GC content, wherein said nucleic acid variant is at least 94% identical to the sequence of SEQ ID NO:1, and wherein the FVIII encoded by said nucleic acid variant exhibits greater expression when compared to wild type FVIII expression. The disclosure also provides a Factor VIII (FVIII) encoding nucleic acid variant, wherein said nucleic acid variant has one or more leucine codons changed to CTG compared to TTA, TTG, CTT, CTC or CTA in wild type FVIII, wherein said nucleic acid variant has at least 50% GC content, wherein said nucleic acid variant is at least 94% identical to the sequence of SEQ ID NO:1, and the FVIII encoded by said nucleic acid variant comprises a B domain deletion and exhibits greater expression when compared to wild type FVIII comprising a B domain deletion expression.

Claims (51)

WHAT IS CLAIMED IS:
1. A Factor VIII ) ng nucleic acid variant, wherein said nucleic acid variant has one or more leucine codons changed to CTG ed to TTA, TTG, CTT, CTC or CTA in wild type FVIII, wherein said nucleic acid variant has at least 50% GC t, wherein said nucleic acid variant is at least 94% identical to the sequence of SEQ ID NO:1, and n the FVIII encoded by said nucleic acid variant exhibits greater expression when compared to wild type FVIII expression.
2. A Factor VIII (FVIII) encoding nucleic acid variant, wherein said nucleic acid variant has one or more leucine codons changed to CTG compared to TTA, TTG, CTT, CTC or CTA in wild type FVIII, wherein said nucleic acid variant has at least 50% GC content, wherein said nucleic acid t is at least 94% identical to the sequence of SEQ ID NO:1, and the FVIII encoded by said nucleic acid variant comprises a B domain deletion and exhibits greater expression when compared to wild type FVIII comprising a B domain deletion expression.
3. The variant FVIII as claimed in claim 1 or 2, wherein said nucleic acid variant is more efficiently packaged into an adenovirus-associated virus (AAV) vector.
4. The variant FVIII as claimed in any of claims 1-3, wherein said FVIII encoded by said c acid variant exhibits greater biological activity when compared to wild type FVIII or when compared to wild type FVIII comprising a B domain deletion.
5. The variant FVIII as claimed in claim 4, wherein said biological activity is determined by a clotting assay or reduced bleeding in a FVIII assay or FVIII deficiency model.
6. The variant FVIII as claimed in any of claims 1-4, n said nucleic acid variant has 2-5, 5-10, 10-20, 20-50, 50-100, 0, 250-500, 500-750 or 750-850 CTG e codons modified from TTA, TTG, CTT, CTC or CTA leucine codons in wild type FVIII.
7. The variant FVIII as claimed in any of claims 1-4, wherein said nucleic acid variant has greater than 85% CTG leucine codons modified from TTA, TTG, CTT, CTC or CTA leucine codons in wild type FVIII.
8. The t FVIII as claimed in any of claims 1-4, wherein said nucleic acid variant has all CTG leucine codons modified from TTA, TTG, CTT, CTC or CTA leucine codons in wild type FVIII.
9. The variant FVIII as d in any of claims 1-4, wherein said nucleic acid variant has one or more AAG lysine codons compared to AAA lysine codons in wild type FVIII.
10. The variant FVIII as claimed in claim 1, wherein said nucleic acid variant has between about 50-59%, or 50-56%, or 50-53% GC content.
11. The t FVIII as claimed in any of claims 1-4, wherein said nucleic acid variant is at least 75% identical to wild type human FVIII nucleic acid or wild type human FVIII nucleic acid comprising a B domain deletion.
12. The variant FVIII as claimed in any of claims 1-4, wherein said nucleic acid variant is human FVIII, and/or wherein said wild type FVIII or wild type FVIII comprising a B domain on is human FVIII.
13. The variant FVIII as claimed in claim 1, wherein said nucleic acid variant comprises any of SEQ ID NOs:1-7 and 9.
14. The variant FVIII as d in any of claims 1-13, wherein 1, 2, 3 or all 4 of the codons encoding the PACE/furin cleavage site is/are deleted.
15. The variant FVIII as claimed in any of claims 1-13, wherein 1, 2, 3 or all 4 of the codons encoding the PACE/furin cleavage site set forth as HHQR or RHQR from positions 1645-1648 is/are deleted.
16. An expression vector comprising the variant FVIII of any of claims 1-15.
17. The expression vector of claim 16, selected from the group consisting of an adenovirusassociated virus (AAV) , a retroviral vector, an adenoviral vector, a plasmid, or a lentiviral vector.
18. The sion vector of claim 17, wherein said AAV vector comprises an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, Rh10, Rh74 or AAV-2i8 AAV serotype.
19. The expression vector of claim 16, further comprising an , an sion control element, one or more adeno-associated virus (AAV) inverted terminal s (ITRs) and/or a filler polynucleotide sequence.
20. The expression vector of claim 19, wherein the intron is within or flanks the variant FVIII, or wherein the expression control element is operably linked to the variant FVIII, or wherein the AAV ITR(s) flanks the 5’ or 3’ terminus of the t FVIII, or wherein the filler cleotide ce flanks the 5’ or 3’terminus of the variant FVIII.
21. The expression vector of claim 19, wherein the expression control element comprises a constitutive or regulatable control t, or a tissue-specific expression l element or promoter.
22. The expression vector of claim 19, wherein the expression control element comprises an element that confers expression in liver.
23. The expression vector of claim 19, wherein the expression control element comprises a TTR promoter or mutant TTR promoter.
24. The expression vector of claim 23, wherein the mutant TTR promoter comprises SEQ ID NO:8.
25. The expression vector of claim 19, wherein the ITR comprises one or more ITRs of any of: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, Rh10, Rh74 or AAV-2i8 AAV serotypes, or a combination thereof.
26. An in vitro host cell expressing the FVIII encoded by said nucleic acid variant of any of claims 1-15.
27. An in vitro host cell comprising the nucleic acid variant of any of claims 1-15 or the expression vector of any of claims 16-25.
28. A virus vector comprising the nucleic acid t of any of claims 1-15 or the expression vector of any of claims 16-25.
29. An AAV vector comprising the nucleic acid variant of any of claims 1-15 or the expression vector of any of claims 16-25.
30. The AAV vector of claim 29, wherein the AAV vector comprises a VP1, VP2 and/or VP3 capsid ce having 75% or more sequence identity to AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, Rh10, Rh74 or AAV-2i8 VP1, VP2 and/or VP3 sequences.
31. The AAV vector of claim 29, n the AAV vector comprises a VP1, VP2 and/or VP3 capsid sequence selected from any of: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, Rh10, Rh74 and AAV-2i8 AAV serotypes.
32. A pharmaceutical composition comprising the nucleic acid variant of any of claims 1-15, the expression vector of any of claims 16-25, or the virus or AAV vector of any of claims 29-31 in a biologically compatible carrier or excipient, ally ing empty capsid AAV.
33. The nucleic acid variant of any of claims 1-15, the expression vector of any of claims 16- 25, or the virus or AAV vector of any of claims 29-31 encapsulated in a liposome or mixed with phospholipids or micelles.
34. Use of a nucleic acid sequence for the manufacture of a medicament for the treatment of a hemostasis disorder in a human subject, wherein the nucleic acid sequence comprises the nucleic acid t of any of claims 1-15, the expression vector of any of claims 16-25, or the virus or AAV vector of any of claims 29-31.
35. A c acid variant of any of claims 1-15, the expression vector of any of claims 16- 25, or the virus or AAV vector of any of claims 29-31 for use in the ent to a mammal in need of Factor VIII.
36. The nucleic acid variant of any of claims 1-15, the sion vector of any of claims 16- 25, or the virus or AAV vector of any of claims 29-31 wherein said Factor VIII is for expression in a mammal.
37. The use of claim 34, wherein said Factor VIII is for expression at levels having a beneficial or therapeutic effect on a cell.
38. The use of claim 34, wherein said medicament is formulated for delivery to a cell, tissue or organ.
39. The use of claim 34, wherein the medicament is ated for delivery to a secretory cell.
40. The use of claim 34, wherein the medicament is formulated for delivery to an endocrine cell or an endothelial cell.
41. The use of claim 34, n the medicament is formulated for delivery to a hepatocyte, a sinusoidal endothelial cell, a megakaryocyte, a platelet or hematopoetic stem cell.
42. The use of claim 38, wherein the tissue or organ comprises liver.
43. The use of claim 34, wherein the human subject produces an insufficient amount of Factor VIII protein, or a defective or aberrant Factor VIII protein.
44. The use of claim 34, wherein hemostasis disorder is hemophilia A.
45. A nucleic acid variant of any of claims 1-15, the expression vector of any of claims 16- 25, or the virus or AAV vector of any of claims 29-31 in a biologically acceptable carrier for use in the treatment of a hemostasis related disorder.
46. The c acid variant of any of claims 1-15, the expression vector of any of claims 16-25, or the virus or AAV vector of any of claims 29-31 in a ically acceptable carrier as claimed in claim 45, wherein said hemostasis related disorder selected from the group consisting of hemophilia A, von Willebrand diseases and ng associated with trauma, injury, thrombosis, ocytopenia, stroke, coagulopathy, disseminated intravascular coagulation (DIC) and overanticoagulation treatment disorders.
47. The use of claim 43 or claim 44, wherein the medicament is formulated to be delivered intravenously, intraarterially, intramuscularly, subcutaneously, intra-cavity, or by intubation, or via catheter.
48. The use of claim 34, wherein the medicament further comprises empty capsid AAV.
49. The use of claim 34, wherein the ment further comprises empty capsid of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV-12, AAVRh10 and/or AAV-Rh74 serotype.
50. An in vitro method of producing FVIII protein comprising expressing in a cell the variant FVIII as claimed in any of claims 1-15, or the sion vector of claim 16 and recovering said FVIII protein produced by the cells.
51. The in vitro method of claim 50, r comprising purifying or isolating said FVIII protein produced by the cells. CHOP0440902_ST25
NZ729107A 2015-08-13 An improved expression cassette for packaging and expression of variant factor viii for the treatment of hemostasis disorders NZ729107B2 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US201462036936P 2014-08-13 2014-08-13
PCT/US2015/045142 WO2016025764A2 (en) 2014-08-13 2015-08-13 An improved expression cassette for packaging and expression of variant factor viii for the treatment of hemostasis disorders

Publications (2)

Publication Number Publication Date
NZ729107A NZ729107A (en) 2024-07-26
NZ729107B2 true NZ729107B2 (en) 2024-10-30

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