Factor VIII mutation repair and tolerance induction
Methods of treating hemophilia A in a subject with an F8 gene mutation, wherein the F8 gene is repaired and the resultant repaired gene, upon expression, confers improved coagulation functionality to the encoded FVIII protein of the subject compared to the non-repaired F8 gene. The invention also includes methods of inducing immune tolerance to a FVIII replacement product ((r)FVIII) in a subject having a FVIII deficiency, wherein the F8 gene mutation is repaired and the repaired gene, upon expression, provides for the induction of immune tolerance to an administered replacement FVIII protein product. The invention also includes isolated nucleic acids, vectors, recombinant viruses, cells, and pharmaceutical compositions to repair the F8 gene.
1. An in vitro or ex vivo method of repairing a mutated F8 gene, the method comprising:
(i) providing an endothelial cell comprising an inversion mutation in the F8 gene;
(ii) introducing into the cell an isolated nucleic acid encoding a nuclease that targets an inversion mutation of the F8 gene and creates a double stranded break in the mutated F8 gene; and
a donor sequence comprising (a) a nucleic acid encoding a truncated FVIII polypeptide or (b) a native F8 3′ splice acceptor site operably linked to a nucleic acid encoding a truncated FVIII polypeptide, wherein the donor sequence is flanked by nucleic acid sequences homologous to the nucleic acid sequences upstream and downstream of the double stranded break in the F8 gene and
(iii) obtaining an endothelial cell that comprises a repaired inversion mutation in the F8 gene.
2. The method of claim 1 wherein the endothelial cell that comprises a repaired inversion mutation in the F8 gene is administered to a subject.
3. The method of claim 1 , wherein the nuclease is a zinc finger nuclease (ZFN), Transcription Activator-Like Effector Nuclease (TALEN), or a CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-associated (Cas) nuclease.
4. The method of claim 1 , wherein the nuclease targets intron 22 or the exon 22/intron 22 junction of the F8 gene.
5. The method of claim 1 , wherein the nuclease targets intron 1 or the exon 1/intron 1 junction of the F8 gene.
6. The method of claim 1 , wherein the mutation in the F8 gene is an intron 22 inversion.
7. The method of claim 1 , wherein the cells are blood outgrowth endothelial cells (BOECs) or liver sinusoidal endothelial cells (LSECs).
8. The method of claim 7 , wherein the blood outgrowth endothelial cells (BOECs) have been co-cultured with hepatocytes or liver sinusoidal endothelial cell (LSECs), or both.
9. The method of claim 7 , wherein the blood outgrowth endothelial cells (BOECs) have been co-cultured with induced pluripotent stem cells (iPSCs).
10. The method of claim 2 , wherein the endothelial cell that comprises a repaired inversion mutation in the F8 gene confers an improved coagulation functionality of the encoded FVIII protein of the subject compared to the coagulation functionality of the FVIII protein encoded by the mutated F8 gene of the subject.
11. The method of claim 2 , wherein the subject has hemophilia A.
12. The method of claim 2 , wherein prior to administering the endothelial cell that comprises a repaired inversion mutation in the F8 gene to the subject, the repaired endothelial cell is isolated out of a population of endothelial cells that do not have a repaired F8 gene.
13. The method of claim 12 , wherein prior to administering the endothelial cell that comprises a repaired inversion mutation in the F8 gene to the subject the endothelial cell that comprises a repaired inversion mutation in the F8 gene is expanded in vitro to produce a population of endothelial cells that comprise a repaired inversion mutation in the F8 gene.
14. The method of claim 13 , wherein the endothelial cells that comprise a repaired inversion mutation in the F8 gene are blood outgrowth endothelial cells (BOECs) or liver sinusoidal endothelial cells (LSECs).
15. The method of claim 14 , wherein the blood outgrowth endothelial cells (BOECs) (i) have been co-cultured with hepatocytes or liver sinusoidal endothelial cell (LSECs), or both; (ii) have been cultured in growth medium supplemented with conditioned medium from hepatocytes; or (iii) have been co-cultured with induced pluripotent stem cells (iPSCs).
16. The method of claim 15 , wherein the subject has hemophilia A.
17. The method of claim 11 , wherein the administration of the endothelial cell that comprises a repaired inversion mutation in the F8 gene induces a tolerogenic immune response to a replacement FVIII protein.
18. The method of claim 16 , wherein the administration of the endothelial cells that comprise a repaired inversion mutation in the F8 gene induces a tolerogenic immune response to a replacement FVIII protein.
19. The method of claim 7 , wherein the blood outgrowth endothelial cells (BOECs) have been cultured in growth medium supplemented with conditioned medium from hepatocytes.