Method for gene editing
The present disclosure relates to compositions and methods for modifying a gene sequence, and for systems for delivering such compositions. For example, the disclosure relates to modifying a gene sequence using a CRISPR-Cas9 or other nucleic acid editing system, and methods and delivery systems for achieving such gene modification, such as viral or non-viral delivery systems.
1. A method for inducing repair of a gene in a subject in need thereof, the method comprising administering to the subject:
(i) one or more guide RNA (gRNA);
(ii) a repair template; and
(iii) a CRISPR-Cas system comprising a Cas mRNA,
wherein (i) and (ii) are provided in a same or different viral vector, and (iii) is provided in a lipid nanoparticle.
2. The method of claim 1 , wherein the viral vector is selected from the group consisting of adeno-associated virus (AAV), adenovirus, retrovirus, and lentivirus vectors.
3. The method of claim 1 , wherein the repair template is selected from the group consisting of a DNA repair template, an mRNA repair template, a sRNA repair template, an miRNA repair template, and an antisense oligonucleotide repair template.
4. The method of claim 1 , wherein the gRNA hybridizes to a target sequence associated with a genetic disease or disorder or a cancer in a cell in the subject.
5. The method of claim 4 , wherein the genetic disease or disorder is an inborn error of metabolism selected from disorders of amino acid transport and metabolism, lipid or fatty acid transport and metabolism, carbohydrate transport and metabolism, and metal transport and metabolism.
6. The method of claim 4 , wherein the genetic disease or disorder is hemophilia, cystic fibrosis, or sickle cell disease.
7. The method of claim 1 , wherein the subject is an animal.
8. The method of claim 1 , wherein the gRNA is administered prior to the CRISPR-Cas system.
9. The method of claim 7 , wherein the method achieves a modification rate of about 1% to about 10% of a population of cells in a target tissue of the subject.
10. The method of claim 1 , wherein the lipid nanoparticle comprises cKK-E12.
11. The method of claim 10 , wherein the lipid nanoparticle further comprises 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) and cholesterol C14-PEG2000.
12. A method for modifying a target nucleotide sequence in a target tissue of a subject in need thereof, comprising administering to the subject:
(i) a viral vector comprising a guide RNA (gRNA); and
(ii) a lipid nanoparticle comprising a CRISP R-Cas system comprising a Cas mRNA.
13. The method of claim 12 , wherein the viral vector is selected from the group consisting of adeno-associated virus (AAV), adenovirus, retrovirus, and lentivirus vectors.
14. The method of claim 12 , wherein the lipid nanoparticle comprises one or more of a phospholipid, cholesterol, polyethylene glycol (PEG)-lipid, and a lipophilic compound.
15. The method of claim 12 , wherein the lipid nanoparticle comprises cKK-E12.
16. The method of claim 15 , wherein the lipid nanoparticle further comprises 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) and cholesterol C14-PEG2000.
17. The method of claim 12 , further comprising administering to the subject a repair template selected from the group consisting of a DNA repair template, an mRNA repair template, a siRNA repair template, an miRNA repair template, and an antisense oligonucleotide repair template.
18. The method of claim 12 , wherein the gRNA is administered prior to the CRISPR-Cas system.
19. The method of claim 12 , wherein the CRISPR-Cas system is expressed in the target tissue for no more than about two months per administration.
20. The method of claim 19 , wherein the gRNA is expressed in the target tissue for at least 2 weeks.
21. The method of claim 20 , wherein about 1% to about 10% of cells within the target tissue are modified.
22. The method of claim 1 , wherein the lipid nanoparticle comprises one or more of a phospholipid, cholesterol, and polyethylene glycol (PEG)-lipid.
23. The method of claim 1 , wherein the gRNA and the repair template are provided in an adeno-associated virus (AAV) vector.
24. The method of claim 23 , wherein the CRISPR-Cas system is a CRISPR-Cas9 system.
25. The method of claim 1 , wherein the gRNA and the repair template are provided in adeno-associated virus 2/8 (AAV 2/8) vector.
26. The method of claim 12 , wherein the viral vector is an adeno-associated virus (AAV).
27. The method of claim 26 , wherein the CRISPR-Cas system is a CRISPR-Cas9 system.
28. The method of claim 12 , wherein the viral vector is an adeno-associated virus 2/8 (AAV 2/8).