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Mutant factor viii compositions and methods

a technology of mutant factor viii and composition, which is applied in the field of recombinant human factor viii mutants, can solve the problems of insufficient expression level of fviii, excessive bleeding when hemophiliac is injured, and inefficient secretion of human fviii

Inactive Publication Date: 2016-04-14
XIAO WEIDONG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The present invention provides modified factor VIII proteins, as well as methods for using them to treat factor VIII deficiencies such as hemophilia A. The modified factor VIII proteins have increased expression or secretion compared to wild-type factor VIII. The mutations can include amino acid substitutions in various parts of the factor VIII protein, such as I86, Y105, A108, D115, Q117, F129, G132, H134, M147, L152, and combinations thereof. The mutant human factor VIII can also include a deletion in the B domain or a2 and / or a3 domain(s) of human factor VIII. The invention also provides isolated polynucleotide sequences and expression vectors containing the modified factor VIII genes, as well as a method for treating patients with factor VIII deficiencies by administering the pharmaceutical composition containing the expression vector.

Problems solved by technology

In patients with Hemophilia A, the blood does not clot properly resulting in excessive bleeding when the hemophiliac is injured.
Difficulties in implementation of gene therapy techniques include problems encountered in the use of viruses as gene vectors and insufficient expression levels of FVIII.
For example, human FVIII secretes very inefficiently and the yield is logs lower comparing to similar proteins such as factor V. Further, while viruses are effective as gene vectors because they can be used to transduce cells leading to protein expression in vivo, the proteins coating the virus particle may activate the body's immune system.

Method used

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  • Mutant factor viii compositions and methods
  • Mutant factor viii compositions and methods
  • Mutant factor viii compositions and methods

Examples

Experimental program
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Effect test

example 1

Construction, Expression and Purification of B-Domainless Factor VIII Mutants

[0164]Plasmid pAAV-CB-F8 carries a B-domainless human factor VIII (hF8) cDNA under the control of a CB promoter (beta-actin promoter with a CMV enhancer). This plasmid, consistent with the construction shown in FIG. 10, was used as template for making various hF8 mutants. A hF8 cDNA fragment encoding the substitution mutations I86V, Y105F, A108S, D115E, Q117H, F129L, G132K, H134Q, M147T and L152P was synthesized chemically and used to replace the corresponding region of pAAV-CB-F8. The resulting plasmid (pAAV-CB-F8-X10) expresses a mutant factor VIII protein with the above 10 mutations (F8-X10; SEQ ID NO: 3).

[0165]A factor VIII cDNA fragment encoding the substitution mutations I86V, A108S, G132K, M147T, L152P was synthesized chemically and used to replace the corresponding region of pAAV-CB-F8. The resulting plasmid (pAAV-CB-F8-X5) expresses a mutant factor VIII protein with the above 5 mutations (F8-X5; SE...

example 2

Comparison of Different Factor VIII Mutants for Secretion in Tissue Culture Cells

[0171]Plasmids pAAV-CB-hBDD-F8 (wt), pAAV-CB-hBDD-F8-X10, pAAV-CB-hBDD-F8-X5 and pAAV-CB-hBDD-F8-G312K were separately transfected in BHK cells (panel A) or 293 cells (panel B). Secreted F8 in the media was harvested and assayed by aPTT assay at 48 hours post transfection. The expression / secretion by wild type human BDD-F8(hBDD) was set as 100%. As shown in FIG. 7, the hF8 mutants were secreted at about 2-8.5 fold higher expression levels than the wild type hF8.

example 3

Comparison of Human Factor VIII Mutant Secretion In Vivo

[0172]Plasmids pAAV-CB-hBDDF8 (B-domain deleted (BDD) wt hF8), pAAV-CB-hBDDF8-X10 (hF8-BDD with 10 substitutions; SEQ ID NO:3), pAAV-CB-hBDD-F8-X5(hF8 with 5 substitutions; SEQ ID NO:4) and pAAV-CB-hBDD-F8-G312K (hF8 with G132K substitution) were separately injected to Blab / c and F8 double knock-out mice. Secreted F8 in the blood was collected and assayed by aPTT assay at 48 hours post injection. The expression / secretion by wild type human F8 (hBDD-F8) was set as 100%. All mutants described here outperform the wild type factor VIII. As shown in FIG. 8, the hF8 mutants were secreted at about 1.2-5.2 fold higher expression levels than the wild type hF8.

EXAMPLE 4

Comparison of Different Factor VIII Mutants in Secretion

[0173]Plasmids encoding amino acids in hBDD-F8-X10 were modified to revert back the mutant substitutions to their corresponding wild type amino acids as indicated in the table. For example, hBDD-F8-X10-V86I means that...

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Abstract

In one aspect, present invention provides a recombinant mutant human factor VIII having increased expression and / or secretion as compared to wild-type factor VIII. In certain embodiments, the recombinant factor VIII includes one or more amino acid substitution(s) selected from the group consisting of I86, Y105, A108, D115, Q117, F129, G132, H134, M147 and L152. In other aspects, the present invention provides FVIII encoding nucleic acids, FVIII-expression vectors, as well as methods of using the modified FVIII genes in the treatment of FVIII deficiencies, such as hemophilia A.

Description

[0001]This application claims priority to U.S. Provisional Patent Application Ser. No. 61 / 838,867, filed on Jun. 24, 2013. The entirety of the aforementioned application is incorporated herein by reference.GOVERNMENT LICENSE RIGHTS[0002]This invention was made with U.S. Government support under grant HL084381 from the National Institutes of Health. The U.S. Government thus may have certain license rights in this invention.FIELD[0003]The present invention relates to recombinant human factor VIII mutants exhibiting higher expression levels than the corresponding wild-type human factor VIII. The present invention also relates to methods of making and using the recombinant human factor VIII mutants.BACKGROUND[0004]Hemophilia A, the most common of the severe, inherited bleeding disorders, results from a deficiency or defect in the plasma protein factor VIII. In patients with Hemophilia A, the blood does not clot properly resulting in excessive bleeding when the hemophiliac is injured. Tr...

Claims

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Application Information

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IPC IPC(8): C07K14/755
CPCA61K38/00C07K14/755
Inventor XIAO, WEIDONGCAO, WENJINGDONG, BIAO
Owner XIAO WEIDONG
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