Delivery of target specific nucleases
Described herein are lipid nanoparticles comprising cationic lipids and other lipids and also comprising engineered nucleases facilitate transfer of nucleic acids to cells.
1. A cationic lipid nanoparticle (LNP) comprising one or more polynucleotides encoding one or more zinc finger protein (ZFP) transcription factors (ZFP-TFs) or one or more zinc finger nucleases (ZFNs), wherein the ZFP-TFs or ZFNs comprise a ZFP designated 56691 or 56690, wherein
(i) the ZFP designated 56691 comprises 6 zinc finger domains ordered F1 to F6, the zinc finger domains comprising the following recognition helix regions:
F1: DRSHLTR (SEQ ID NO: 6);
F2: QSGDLTR (SEQ ID NO: 7);
F3: DRSNRTT (SEQ ID NO: 8);
F4: RSDALAR (SEQ ID NO: 9);
F5: RSDNLSE (SEQ ID NO: 10); and
F6: ERANRNS (SEQ ID NO: 11); and
(ii) the ZFN designated 56690 comprises 6 zinc finger domains ordered F1 to F6, the zinc finger domains comprising the following recognition helix regions:
F1: DRSALSR (SEQ ID NO: 12);
F2: TSGSLTR (SEQ ID NO: 13);
F3: QSSDLSR (SEQ ID NO: 14);
F4: WRKSLKV (SEQ ID NO: 15);
F5: DRSHLTR (SEQ ID NO: 6); and
F6: RLDWLPM (SEQ ID NO: 16).
2. The cationic LNP of claim 1 , wherein the one or more polynucleotides encode the one or more ZFNs and wherein the LNP further comprises one or more donor polynucleotides.
3. The cationic LNP of claim 2 , wherein the one or more polynucleotides and/or donor polynucleotide comprise mRNA.
4. The cationic LNP of claim 2 , wherein the one or more donor polynucleotides comprise an oligonucleotide that corrects a mutation in the endogenous CFTR gene.
5. The cationic LNP of claim 4 , wherein the mutation is a Δ508 mutation.
6. An isolated cell comprising one or more cationic LNPs according to claim 5 .
7. The cationic LNP of claim 4 , wherein the one or more donor polynucleotides comprise a transgene that expresses a functional CFTR protein.
8. An isolated cell comprising one or more cationic LNPs according to claim 7 .
9. An isolated cell comprising one or more cationic LNPs according to claim 2 .
10. The isolated cell of claim 9 , wherein the cell is a lung cell.
11. A composition comprising the cationic LNP of claim 2 , wherein the one or more donor polynucleotides comprise an oligonucleotide that corrects a mutation in the endogenous CFTR gene.
12. The composition of claim 11 , wherein the mutation is a Δ508 mutation.
13. The composition of claim 11 , wherein the one or more donor polynucleotides comprise a transgene that expresses a functional CFTR protein.
14. The composition of claim 11 , wherein the one or more donor polynucleotides are carried on a viral vector.
15. The composition of claim 14 , wherein the viral vector is an AAV vector.
16. A method of integrating a donor polynucleotide into an endogenous gene, the method comprising the composition of claim 11 to the cell such that the endogenous gene is cleaved by the one or more ZFNs and the donor polynucleotide is integrated into the cleaved endogenous gene.
17. A method of integrating a donor polynucleotide into an endogenous gene in a cell, the method comprising administering the cationic LNP of claim 2 to the cell such that the endogenous gene is cleaved by the one or more ZFNs and the donor polynucleotide is integrated into the cleaved endogenous gene.
18. The cationic LNP of claim 1 comprising:
(i) a first cationic lipid at a compositional molar ratio from about 0.18 to about 0.32 and of formula:
wherein R 1 and R 2 are independently substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl; R 3 and R 4 are independently hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl; m is an integer from 1 to 6; X a − is an anion;
(ii) a second cationic lipid at a compositional molar ratio from about 0.24 to about 0.51 and of formula:
wherein R 5 and R 8 are independently hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl; R 6 and R 7 are independently substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl; n is an integer from 1 to 6; and X b − is an anion;
(iii) a first helper lipid at a compositional molar ratio from about 0.20 to about 0.32;
(iv) a second helper lipid at a compositional molar ratio from about 0.01 to about 0.14; and
(v) a biostability enhancing agent at a compositional molar ratio from about 0.01 to about 0.02.
19. An isolated cell comprising one or more cationic LNPs according to claim 1 .
20. A composition comprising the cationic LNP of claim 1 .
21. A method of modulating endogenous gene expression in a cell, the method comprising administering the cationic LNP of claim 1 to the cell.
22. A method of treating and/or preventing cystic fibrosis in a subject, the method comprising administering the cationic LNP of claim 1 to the subject.
23. The method of claim 22 , wherein the cationic LNP of claim 1 is administered 1, 2, 3 or more times to the subject.
24. A kit comprising the cationic LNP of claim 1 .