US2018312571A1PendingUtilityA1
CpG REDUCED FACTOR VIII VARIANTS, COMPOSITIONS AND METHODS AND USES FOR TREATMENT OF HEMOSTASIS DISORDERS
Est. expiryOct 30, 2035(~9.3 yrs left)· nominal 20-yr term from priority
A61P 37/06A61P 43/00A61P 7/04A61K 45/06C12N 2710/10343C12P 21/00C07K 14/755A61K 38/37A61K 48/00A61K 48/0066C12N 15/8645C12N 15/86C12N 2750/14145C12N 7/00C12N 2750/14122C12N 2710/10342C12N 2750/14141C12N 2750/14143
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Claims
Abstract
CpG reduced nucleic acid variants encoding FVIII protein and methods of use thereof are disclosed. In particular embodiments, CpG reduced nucleic acid variants encoding FVIII are expressed more efficiently by cells, are secreted at increased levels by cells over wild-type Factor VIII proteins, exhibit enhanced expression and/or activity over wild-type Factor VIII proteins or are packaged more efficiently into viral vectors.
Claims
exact text as granted — not AI-modifiedWhat is claimed is:
1 . A nucleic acid variant encoding Factor VIII (FVIII) having a B domain deletion, wherein the nucleic acid variant has 92% or greater identity to SEQ ID NO:7.
2 . The nucleic acid variant of claim 1 , wherein the nucleic acid variant has 93% or greater sequence identity to SEQ ID NO:7.
3 . The nucleic acid variant of claim 1 , wherein the nucleic acid variant has 94% or greater sequence identity to SEQ ID NO:7.
4 . The nucleic acid variant of claim 1 , wherein the nucleic acid variant has 95% or greater sequence identity to SEQ ID NO:7.
5 . The nucleic acid variant of claim 1 , wherein the nucleic acid variant has 95%-100% sequence identity to SEQ ID NO:7.
6 . The nucleic acid variant of claim 1 , wherein the nucleic acid variant has 20 or fewer, 15 or fewer, or 10 or fewer cytosine-guanine dinucleotides (CpGs).
7 . The nucleic acid variant of claim 1 , wherein the nucleic acid variant has no more than 5 cytosine-guanine dinucleotides (CpGs).
9 . The nucleic acid variant of claim 1 , wherein the nucleic acid variant has 4, 3, 2, 1 or 0 cytosine-guanine dinucleotides (CpGs).
10 . The nucleic acid variant of claim 1 , wherein the nucleic acid variant encodes SEQ ID NO:25 having a deletion of one or more amino acids of the sequence SFSQNPPVLKRHQR (SEQ ID NO:29), or a deletion of the entire sequence SFSQNPPVLKRHQR.
11 . The nucleic acid variant of claim 1 , wherein the nucleic acid variant encodes SEQ ID NO:25.
12 . A nucleic acid variant encoding Factor VIII (FVIII) having a B domain deletion (FVIII-BDD), wherein the nucleic acid variant has fewer cytosine-guanine dinucleotides (CpG) than SEQ ID NO: 19.
13 . The nucleic acid variant of claim 12 , wherein said FVIII-BDD is mammalian.
14 . A nucleic acid variant encoding human Factor VIII having a B domain deletion (hFVIII-BDD), wherein the nucleic acid variant has no more than 2 cytosine-guanine dinucleotides (CpGs).
15 . The nucleic acid variant of claim 14 , wherein the nucleic acid variant has 1 cytosine-guanine dinucleotide (CpG).
16 . The nucleic acid variant of claim 14 , wherein the nucleic acid variant has no cytosine-guanine dinucleotides (CpGs).
17 . The nucleic acid variant of any of claims 1 - 16 , wherein the encoded FVIII-BDD or hFVIII-BDD is identical to hFVIII-BDD encoded by SEQ ID NO: 19.
18 . The nucleic acid variant of any of claims 1 - 17 , wherein the nucleic acid variant is distinct from FVIII-V3 (SEQ ID NO:20) and CO3 (SEQ ID NO:21).
19 . The nucleic acid variant of any of claims 1 - 18 , wherein the nucleic acid variant encodes SEQ ID NO:25 having a deletion of one or more amino acids of the sequence SFSQNPPVLKRHQR (SEQ ID NO:29), or a deletion of the entire sequence SFSQNPPVLKRHQR.
20 . The nucleic acid variant of any of claims 1 - 18 , wherein the nucleic acid variant encodes SEQ ID NO:25.
21 . A vector comprising the nucleic acid variant of any of claims 1 - 20 .
22 . An expression vector comprising the nucleic acid variant of any of claims 1 - 20 .
23 . The expression vector of claim 22 , selected from the group consisting of an adenovirus-associated virus (AAV) vector, a retroviral vector, an adenoviral vector, a plasmid, or a lentiviral vector.
24 . The expression vector of claim 23 , wherein said AAV vector comprises an AAV serotype or an AAV pseudotype, wherein said AAV pseudotype comprise an AAV capsid serotype different from an ITR serotype.
25 . The expression vector of 23 or 24 , further comprising an intron, an expression control element, one or more adeno-associated virus (AAV) inverted terminal repeats (ITRs) and/or a filler polynucleotide sequence.
26 . The expression vector of claim 25 , wherein the intron is within or flanks the nucleic acid variant.
27 . The expression vector of claim 25 , wherein the expression control element is operably linked to the nucleic acid variant.
28 . The expression vector of claim 25 , wherein the AAV ITR(s) flanks the 5′ or 3′ terminus of the nucleic acid variant.
29 . The expression vector of claim 25 , wherein the filler polynucleotide sequence flanks the 5′ or 3′terminus of the nucleic acid variant.
30 . The expression vector of any of claims claim 25 - 29 , wherein the intron, expression control element, one or more adeno-associated virus (AAV) inverted terminal repeats (ITRs) and/or a filler polynucleotide sequence has been modified to have reduced cytosine-guanine dinucleotides (CpGs).
31 . The expression vector of any of claims claim 25 - 29 , wherein the intron, expression control element, one or more adeno-associated virus (AAV) inverted terminal repeats (ITRs) and/or a filler polynucleotide sequence has been modified to have 20 or fewer, 15 or fewer, 10 or fewer, 5 or fewer or 0 cytosine-guanine dinucleotides (CpGs).
32 . The expression vector of any of claims 25 , 27 , 30 and 31 , wherein the expression control element comprises a constitutive or regulatable control element, or a tissue-specific expression control element or promoter.
33 . The expression vector of any of claims 25 , 27 , 30 and 31 , wherein the expression control element comprises an element that confers expression in liver.
34 . The expression vector of any of claims 25 , 27 , 30 and 31 , wherein the expression control element comprises a TTR promoter or mutant TTR promoter.
35 . The expression vector of claim 34 , wherein the mutant TTR promoter comprises SEQ ID NO:22.
36 . The expression vector of any of claims claim 25 - 35 , 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.
37 . The expression vector of any of claims claim 24 - 30 , wherein the vector comprises an ITR, a promoter, a polyA signal and/or intron sequence set forth in SEQ ID NO:23.
38 . An AAV vector comprising the nucleic acid variant of any of claims 1 - 20 or the expression vector of any of claims 25 - 37 .
39 . The AAV vector of claim 38 , wherein the AAV vector comprises a modified or variant AAV VP1, VP2 and/or VP3 capsid sequence, or wild-type AAV VP1, VP2 and/or VP3 capsid sequence.
40 . The AAV vector of claim 38 , wherein the AAV vector comprises a modified or variant AAV VP1, VP2 and/or VP3 capsid sequence having 90% or more identity to AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, Rh10, Rh74 or AAV-2i8 VP1, VP2 and/or VP3 sequences.
41 . The AAV vector of claim 38 , wherein the AAV vector comprises a VP1, VP2 or VP3 capsid sequence selected from any of: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, Rh10, Rh74 or AAV-2i8 AAV serotypes.
42 . The AAV vector of claim 38 , wherein the AAV vector comprises a capsid having 90% or more sequence identity to LK03 capsid (SEQ ID NO:27).
43 . The AAV vector of claim 38 , wherein the AAV vector comprises a capsid having 90% or more sequence identity to SPK capsid (SEQ ID NO:28).
44 . The AAV vector of claim 38 , wherein the AAV vector comprises LK03 capsid (SEQ ID NO:27).
45 . The AAV vector of claim 38 , wherein the AAV vector comprises SPK capsid (SEQ ID NO:28).
46 . The AAV vector of claim 38 , wherein the AAV vector comprises the nucleic acid variant SEQ ID NO:7 and LK03 capsid sequence (SEQ ID NO:27).
47 . The AAV vector of claim 38 , wherein the AAV vector comprises the nucleic acid variant SEQ ID NO:7 and SPK capsid (SEQ ID NO:28).
48 . The AAV vector of claim 38 , wherein the AAV vector comprises the nucleic acid variant and one or more of a mutated TTR promoter (TTRmut), synthetic intron, poly A and ITR in SEQ ID NO:23.
49 . The AAV vector of claim 38 , wherein the AAV vector comprises the nucleic acid variant and one or more of a mutated TTR promoter (TTRmut), synthetic intron, poly A and ITR in SEQ ID NO:23 and LK03 capsid sequence (SEQ ID NO:27) or SPK capsid (SEQ ID NO:28).
50 . A host cell comprising the nucleic acid variant of any of claims 1 - 20 , or the vector or expression vector of any of claims 21 - 37 .
51 . The host cell of claim 50 , said host cells expressing the FVIII encoded by said nucleic acid variant.
52 . A host cell comprising the AAV vector of any of claims 38 - 49 .
53 . The host cell of claim 52 , said host cells producing the AAV vector of any of claims 38 - 49 .
54 . A pharmaceutical composition comprising the nucleic acid variant of any of claims 1 - 20 , the vector or expression vector of any of claims 21 - 37 , or the AAV vector of any of claims 38 - 49 in a biologically compatible carrier or excipient.
55 . The nucleic acid variant of any claims 1 - 20 , the vector or expression vector of any of claims 21 - 37 , or the AAV vector of any of claims 38 - 49 encapsulated in a liposome or mixed with phospholipids or micelles.
56 . The pharmaceutical composition of claim 54 or 55 , further comprising empty capsid AAV.
57 . The pharmaceutical composition of claim 54 or 55 , further comprising empty capsid of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11 and/or AAV-Rh74 serotype.
58 . The pharmaceutical composition of claim 54 or 55 , further comprising empty capsid AAV of the same serotype as the AAV vector administered.
59 . The pharmaceutical composition of claim 54 or 55 , wherein the empty capsid is LK03 capsid (SEQ ID NO:27) or SPK capsid (SEQ ID NO:28).
60 . The pharmaceutical composition of any of claims 56 - 59 , wherein the ratio of said empty capsids to said AAV vector is about 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, or 10:1.
61 . A method for delivering or transferring a nucleic acid sequence into a mammal or a mammalian cell, comprising administering or contacting the nucleic acid variant of any of claims 1 - 20 , vector or expression vector of any of claims 21 - 37 , or the AAV vector of any of claims 38 - 49 to said mammal or mammalian cell, thereby delivering or transferring the nucleic acid sequence into the mammal or mammalian cell.
62 . A method of treating a mammal in need of Factor VIII, comprising:
(a) providing the nucleic acid variant of any of claims 1 - 20 , vector or expression vector of any of claims 21 - 37 , or the AAV vector of any of claims 38 - 49 ; and (b) administering an amount of the nucleic acid variant of any of claims 1 - 20 , vector or expression vector of any of claims 21 - 37 , or the AAV vector of any of claims 38 - 49 to the mammal wherein said Factor VIII is expressed in the mammal.
63 . The method of claim 61 or 62 , wherein said Factor VIII encoded by the nucleic acid variant is expressed in a cell, tissue or organ of said mammal.
64 . The method of claim 63 , wherein, the cell comprises a secretory cell.
65 . The method of claim 63 , wherein the cell comprises an endocrine cell or an endothelial cell.
66 . The method of claim 63 , wherein the cell comprises a hepatocyte, a sinusoidal endothelial cell, a megakaryocyte, a platelet or hematopoetic stem cell.
67 . The method of claim 63 , wherein the tissue or organ of said mammal comprises liver.
68 . The method of any of claims 61 - 67 , wherein the mammal produces an insufficient amount of Factor VIII protein, or a defective or aberrant Factor VIII protein.
69 . The method of any of claims 61 - 67 , wherein the mammal has hemophilia A.
70 . The method of any of claims 61 - 67 , wherein the Factor VIII encoded by the nucleic acid variant is expressed at levels having a beneficial or therapeutic effect on the mammal.
71 . A method for treatment of a hemostasis related disorder in a patient in need thereof comprising administration of a therapeutically effective amount of the nucleic acid variant of any of claims 1 - 20 , vector or expression vector of any of claims 21 - 37 , or the AAV vector of any of claims 38 - 49 in a biologically acceptable carrier to the patient.
72 . The method of claims 61 , 62 or 71 , wherein said mammal or said patient has a disorder selected from the group consisting of hemophilia A, von Willebrand diseases and bleeding associated with trauma, injury, thrombosis, thrombocytopenia, stroke, coagulopathy, disseminated intravascular coagulation (DIC) and over-anticoagulation treatment disorders.
73 . The method of any of claims 61 - 72 , wherein the nucleic acid variant of any of claims 1 - 20 , vector or expression vector of any of claims 21 - 37 , or the AAV vector of any of claims 38 - 49 is delivered to said mammal or said patient intravenously, intraarterially, intramuscularly, subcutaneously, intra-cavity, or by intubation, or via catheter.
74 . The method of any of claims 61 - 72 , wherein FVIII is expressed at levels without substantially increasing risk of thrombosis.
75 . The method of claim 74 , wherein said thrombosis risk is determined by measuring fibrin degradation products.
76 . The method of any of claims 61 - 72 , wherein FVIII is expressed at levels greater than 1% of the levels of FVIII found in a subject that does not have hemophilia A.
77 . The method of any of claims 61 - 72 , wherein FVIII is expressed at levels greater than 3% of the levels of FVIII found in a subject that does not have hemophilia A.
78 . The method of any of claims 61 - 72 , wherein activity of FVIII is detectable for at least 1, 2, 3 or 4 weeks, or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 months, or at least 1 year.
79 . The method of any of claims 61 - 72 , wherein FVIII is expressed at levels greater than 1% or 3% of the levels of FVIII found in a subject that does not have hemophilia A for at least 1, 2, 3 or 4 weeks, or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 months, or at least 1 year.
80 . The method of any of claims 61 - 72 , wherein FVIII is expressed at levels having a therapeutic effect for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 days, weeks or months.
81 . The method of any of claims 61 - 72 , wherein said FVIII is present in the mammal or patient at levels of about 20% FVIII activity or greater than 20% activity for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 continuous days, weeks or months.
82 . The method of any of claims 61 - 72 , wherein said FVIII is expressed at levels at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36 % , 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, or 50% of normal FVIII levels.
83 . The method of any of claims 61 - 72 , wherein the AAV vector is administered at a dose of less than 1×10 12 vector genomes per kilogram (vg/kg) of the mammal or patient, and said FVIII is produced in the mammal or patient at levels of about 20% activity or greater than 20% activity for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 continuous days, weeks or months
84 . The method of any of claims 61 - 72 , wherein the AAV vector is administered at a dose of about 5×10 11 vector genomes per kilogram (vg/kg) of the mammal or patient, and said FVIII is produced in the mammal or patient at levels of about 20% activity or greater than 20% activity for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 continuous days, weeks or months.
85 . The method of any of claims 61 - 84 , wherein said mammal or said patient is human.
86 . The method of any of claims 61 - 85 , wherein said mammal, said patient or said human is sero-positive or sero-negative for AAV.
87 . The method of any of claims 61 - 86 , further comprising administering AAV empty capsid to said mammal or said patient.
88 . The method of any of claims 61 - 86 , further comprising administering empty capsid of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11 and/or AAV-Rh74 serotype.
89 . The method of any of claims 61 - 86 , further comprising administering empty capsid AAV of the same serotype as the AAV vector administered.
90 . The method of claim 89 , wherein the empty capsid is LK03 capsid (SEQ ID NO:27) or SPK capsid (SEQ ID NO:28).
91 . The method of any of claims 87 - 90 , wherein the ratio of said empty capsids to said AAV vector is about 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, or 10:1.
92 . The method of any of claims 61 - 91 , further comprising administering an immunosuppressive agent.
93 . The method of any of claims 61 - 91 , further comprising administering an immunosuppressive agent after the AAV vector is administered.
94 . The method of any of claims 61 - 91 , further comprising administering an immunosuppressive agent from a time period within 1 hour to up to 45 days after the AAV vector is administered.
95 . The method of any of claims 92 - 94 , wherein the immunosuppressive agent comprises a steroid, cyclosporine (e.g., cyclosporine A), mycophenolate, Rituximab or a derivative thereof.
96 . The method of any of claims 61 - 95 , wherein the AAV vector is administered in a range from about 1×10 8 to about 1×10 14 vector genomes per kilogram (vg/kg) of the weight of the mammal or patient.
97 . The method of any of claims 61 - 95 , wherein the AAV vector is administered in a range from about 1×10 9 to about 1×10 13 vector genomes per kilogram (vg/kg) of the weight of the mammal or patient.
98 . The method of any of claims 61 - 95 , wherein the AAV vector is administered in a range from about 1×10 10 to about 1×10 12 vector genomes per kilogram (vg/kg) of the weight of the mammal or patient.
99 . The method of any of claims 61 - 95 , wherein the AAV vector is administered in a range from about 1×10 11 to about 1×10 12 vector genomes per kilogram (vg/kg) of the weight of the mammal or patient.
100 . The method of any of claims 61 - 95 , wherein the AAV vector is administered in a range from about 1×10 12 to about 1×10 13 vector genomes per kilogram (vg/kg) of the weight of the mammal or patient.
101 . The method of any of claims 61 - 95 , wherein the AAV vector is administered in a range from about 1×10 13 to about 1×10 14 vector genomes per kilogram (vg/kg) of the weight of the mammal or patient.
102 . The method of any of claims 61 - 95 , wherein the AAV vector is administered in a range from about 5×10 11 to about 1×10 12 vector genomes per kilogram (vg/kg) of the weight of the mammal or patient.
103 . The method of any of claims 61 - 95 , wherein the AAV vector is administered at a dose of about 5×10 11 vector genomes per kilogram (vg/kg) of the weight of the mammal or patient.
104 . The method of any of claims 61 - 103 , further comprising analyzing or monitoring the mammal for the presence or amount of AAV antibodies, an immune response against AAV, FVIII antibodies, an immune response against FVIII, FVIII amounts, FVIII activity level, amounts or levels of one or more liver enzymes or frequency, and/or severity or duration of bleeding episodes.
105 . A method of producing FVIII protein comprising expressing in a cell the nucleic acid variant as claimed in any of claims 1 - 20 , or the vector or expression vector of any of claims 21 - 37 , and recovering said FVIII protein produced by the cells.
106 . The method of claim 105 , further comprising purifying or isolating said FVIII protein produced by the cells.Cited by (0)
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