Compositions and methods for modulating factor viii function
A FVIII and factor technology, applied in the fields of medicine and hematology, can solve problems such as immunogenicity
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[0047] Preparation of Nucleic Acid Molecules and Polypeptides Encoding the Variants
[0048] Nucleic acid molecules encoding variants of the invention can be prepared by methods using recombinant DNA technology. The availability of nucleotide sequence information enables the preparation of isolated nucleic acid molecules of the invention by a variety of means. For example, nucleic acid sequences encoding variants can be isolated from suitable biological sources using standard protocols well known in the art.
[0049] Nucleic acids of the invention may be maintained as RNA or DNA in any convenient cloning vector. In a specific embodiment, clones are maintained in plasmid cloning / expression vectors such as pBluescript (Stratagene, La Jolla, CA), which are propagated in suitable E. coli host cells. Alternatively, the nucleic acid can be maintained in a vector suitable for expression in mammalian cells. In cases where post-translational modifications affect the function of th...
Embodiment 1
[0088] Non-viral vectors (naked DNA, 5 μg / mouse) expressing various human FVIII B domain variants and FVIII-SQ under the control of liver-specific promoters were injected via the tail vein route under hydrodynamic conditions (5 mice mice / variants). The variant B domains tested were: B1: SFSQNSRHPS (SEQ ID NO: 13); B2: SFSQNSRHPSTRQKQ (SEQ ID NO: 14); B3: SFSQNSRHPSTRQKQFNATT (SEQ ID NO: 15); B4: SFSQN (SEQ ID NO : 16); B5: SFSQNSRH (SEQ ID NO: 17); and B6: SFSQNSRHPSTRQKQFNATTIPENDIEKTD (SEQ ID NO: 18). After 24 hours, blood was collected and FVIII antigen levels were measured by ELISA using Affinity Biologicals Matched Pair Antibody Set Product#F8C-EIA. Such as figure 2 As shown, all human FVIII B domain variants of the present invention were expressed at higher levels than FVIII-SQ in hemophilia A mice.
Embodiment 2
[0090] Wild-type FVIII (659K) and its amino acid substitution variants were transiently expressed in BHK cells. The specific activity of expressed FVIII was determined by a one-stage aPTT assay in conditioned expression medium. Such as image 3 As shown, most FVIII variants showed increased specific activities compared to wild-type FVIII.
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