CpG REDUCED FACTOR VIII VARIANTS, COMPOSITIONS AND METHODS AND USES FOR TREATMENT OF HEMOSTASIS DISORDERS
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.
1 - 61 . (canceled)
62 . A method of providing Factor VIII activity to a human in need thereof, comprising administering to said human a recombinant adeno-associated viral (rAAV) vector comprising:
(a) a vector genome comprising a nucleic acid sequence encoding a variant Factor VIII having a B domain deletion, wherein (i) said variant Factor VIII having said B domain deletion comprises a sequence having a sequence identity of at least 99% to SEQ ID NO:25 and (ii) said nucleic acid sequence encoding said Factor VIII having said B domain deletion has 98% or greater identity to SEQ ID NO: 7 and has 10 or fewer cytosine-guanine dinucleotides (CpGs) and is operably linked to an expression control element providing for expression; and
(b) an rAAV capsid encapsidating said vector genome.
63 - 106 . (canceled)
107 . The method of claim 62 , wherein said human has hemophilia A.
108 . The method of claim 62 , wherein said human has a hemostasis related disorder.
109 . The method claim 107 , wherein said vector genome further comprises a polyA signal sequence 3′ of said nucleic acid sequence encoding said variant Factor VIII having said B domain deletion, and said expression control element is a promoter.
110 . The method of claim 109 , wherein said nucleic acid sequence encoding said Factor VIII having said B domain deletion has 99.5% or greater identity to SEQ ID NO:7 and has no CpGs.
111 . The method of claim 110 , wherein said promoter provides for expression in the liver.
112 . The method of claim 111 , wherein said capsid is an LK03 capsid comprising the sequence of SEQ ID NO:27.
113 . The method of claim 112 , wherein said vector genome further comprises an intron, a 3′ and 5′ adeno-associated virus inverted terminal repeat (ITR) flanking the terminus of the vector genome, and a filler polynucleotide.
114 . The method of claim 113 , wherein said promoter comprises the sequence of SEQ ID NO: 22.
115 . The method of claim 112 , wherein said vector genome comprises 5′ to 3′: a 5′ AAV2 inverted terminal repeat (ITR), said promoter, said nucleic acid sequence encoding said variant Factor VIII having said B domain deletion, an intron derived from human elongation factor EF-1 alpha, said polyA signal sequence, and a 3′ AAV2 ITR; and wherein said polyA signal sequence is a rabbit polyA signal sequence.
116 . The method of claim of claim 115 , wherein said promoter comprises the sequence of SEQ ID NO: 22.
117 . The method of claim 116 , wherein said intron is the intron sequence provided in SEQ ID NO: 23; and said rabbit polyA signal sequence is the polyA signal sequence provided in SEQ ID NO: 23.
118 . The method of claim 117 , wherein said ITRs are those present in SEQ ID NO: 23.
119 . The method of claim 115 , wherein said vector genome further comprises a Kozak consensus sequence.
120 . The method of claim 117 , wherein said vector genome further comprises a Kozak consensus sequence.
121 . The method of claim 118 , wherein said vector genome further comprises a Kozak consensus sequence.
122 . The method claim 108 , wherein said vector genome further comprises a polyA signal sequence 3′ of said nucleic acid sequence encoding said Factor VIII having said B domain deletion, and said expression control sequence is a promoter providing for liver expression.
123 . The method of claim 122 , wherein said nucleic acid sequence encoding said Factor VIII having said B domain deletion has 99.5% or greater identity to SEQ ID NO:7 and has no CpGs; wherein said vector genome comprises 5′ to 3′: a 5′ AAV2 ITR, said promoter, said nucleic acid sequence encoding said variant Factor VIII having said B domain deletion, an intron derived from human elongation factor EF-1 alpha, said polyA signal sequence, and a 3′ AAV2 ITR; and said capsid is an LK03 capsid comprising the sequence of SEQ ID NO:27.
124 . The method of claim of claim 123 , wherein said promoter comprises the sequence of SEQ ID NO: 22, said intron is the intron sequence provided in SEQ ID NO: 23, and said polyadenylation signal is the polyA signal sequence provided in SEQ ID NO: 23.
125 . The method of claim 124 , wherein said vector genome further comprises a Kozak consensus sequence.