Gene editing for hemophilia A with improved factor VIII expression
View Patent ↗Provided herein, in some embodiments, are materials and methods for treating hemophilia A in a subject ex vivo or in vivo. Also provided herein, in some embodiments, are materials and methods for knocking in a coding sequence encoding a synthetic FVIII having a B domain substitute into a genome.
1. A system for altering a host cell DNA sequence, comprising:
a deoxyribonucleic acid (DNA) endonuclease or a nucleic acid encoding the DNA endonuclease, wherein the DNA endonuclease is Cas9;
a guide RNA (gRNA) comprising a spacer sequence complementary to a host cell locus or a nucleic acid encoding the gRNA; and
a donor template comprising a nucleic acid sequence encoding a synthetic FVIII protein, wherein the synthetic FVIII protein comprises a B domain substitute, wherein the B domain substitute comprises the amino acid sequence of any one of SEQ ID NOs: 362-364, 366-369, 371, and 373.
2. The system of claim 1 , wherein the B domain substitute comprises the amino acid sequence of SEQ ID NO: 364.
3. The system of claim 1 , wherein the host cell locus is the locus of a gene expressed in the liver, the locus of a gene encoding an acute phase protein, or a safe harbor locus.
4. The system of claim 3 , wherein the acute phase protein is an albumin, a transferrin, or a fibrinogen.
5. The system of claim 1 , wherein the nucleic acid encoding the DNA endonuclease is codon-optimized for expression in the host cell.
6. The system of claim 1 , wherein the nucleic acid encoding the DNA endonuclease is a deoxyribonucleic acid (DNA) or a ribonucleic acid (RNA).
7. The system of claim 6 , wherein the RNA encoding the DNA endonuclease is an mRNA.
8. The system of claim 1 , wherein the donor template nucleic acid sequence is codon optimized for expression in the host cell.
9. The system of claim 1 , wherein the donor template nucleic acid sequence comprises a reduced content of CpG di-nucleotides as compared to a wild type nucleic acid sequence encoding a FVIII protein, or wherein the donor template nucleic acid sequence does not comprise CpG di-nucleotides.
10. The system of claim 1 , wherein the donor template is encoded in an Adeno Associated Virus (AAV) vector.
11. The system of claim 1 , wherein the donor template comprises a donor cassette comprising the nucleic acid sequence encoding a synthetic FVIII protein, and wherein the donor cassette is flanked on one or both sides by a gRNA target site.
12. The system of claim 11 , wherein the donor cassette is flanked on both sides by a gRNA target site, or wherein the donor cassette is flanked on its 5′ side by a gRNA target site.
13. The system of claim 11 , wherein the gRNA target site is a target site for a gRNA in the system.
14. The system of claim 13 , wherein the gRNA target site of the donor template is the reverse complement of a genomic gRNA target site for a gRNA in the system.
15. The system of claim 1 , wherein the DNA endonuclease or nucleic acid encoding the DNA endonuclease is contained in a liposome or lipid nanoparticle.
16. The system of claim 15 , wherein the liposome or lipid nanoparticle also comprises the gRNA.
17. The system of claim 1 , wherein the DNA endonuclease is complexed with the gRNA, thereby providing a Ribonucleoprotein (RNP) complex.
18. A method of editing a genome in a host cell, the method comprising providing to the cell:
(a) a gRNA comprising a spacer sequence complementary to a host cell locus or a nucleic acid encoding the gRNA;
(b) a DNA endonuclease or a nucleic acid encoding the DNA endonuclease, wherein the DNA endonuclease is Cas9; and
(c) a donor template comprising a nucleic acid sequence encoding a synthetic FVIII protein, wherein the synthetic FVIII protein comprises a B domain substitute, wherein the B domain substitute comprises the amino acid sequence of any one of SEQ ID NOs: 362-364, 366-369, 371, and 373.
19. A method of treating hemophilia A in a subject, the method comprising:
providing the following to a cell in the subject:
(a) a gRNA comprising a spacer sequence complementary to a host cell locus or a nucleic acid encoding the gRNA;
(b) a DNA endonuclease or a nucleic acid encoding the DNA endonuclease, wherein the DNA endonuclease is Cas9; and
(c) a donor template comprising a nucleic acid sequence encoding a synthetic FVIII protein, wherein the synthetic FVIII protein comprises a B domain substitute, wherein the B domain substitute comprises the amino acid sequence of any one of SEQ ID NOs: 362-364, 366-369, 371, and 373.
20. A method of increasing the amount of FVIII in a subject, the method comprising:
providing the following to a cell in the subject, wherein the subject has a first serum level of FVIII:
(a) a gRNA comprising a spacer sequence complementary to a host cell locus or a nucleic acid encoding the gRNA;
(b) a DNA endonuclease or a nucleic acid encoding the DNA endonuclease, wherein the DNA endonuclease is Cas9; and
(c) a donor template comprising a nucleic acid sequence encoding a synthetic FVIII protein, wherein the synthetic FVIII protein comprises a B domain substitute, wherein the B domain substitute comprises the amino acid sequence of any one of SEQ ID NOs: 362-364, 366-369, 371, and 373.