Lipid-encapsulated dual-cleaving endonuclease for DNA and gene editing
Disclosed herein are methods to edit genes, for example by administering a chimeric nuclease to a cell or organism without the use of a viral vector. Some methods herein include editing DNA of a cell by contacting the cell with a nuclease comprising a Cas9 sequence, where the nuclease edits the DNA of the cell.
1 . A method for editing DNA of a cell, comprising:
contacting the cell with a nuclease comprising a Cas9 sequence that is at least 99% identical to SEQ ID NO: 13 and comprises a glutamate at an amino acid corresponding to position 10 of SEQ ID NO: 13, wherein the nuclease edits the DNA of the cell.
2 . The method of claim 1 , wherein the cell is a mammalian cell.
3 . The method of claim 1 , wherein the DNA comprises genomic DNA.
4 . The method of claim 1 , wherein the cell comprises a cancer or airway cell.
5 . The method of claim 1 , wherein the editing comprises nicking or double-stranded cutting.
6 . The method of claim 1 , further comprising contacting the cell with a donor DNA.
7 . The method of claim 6 , wherein the editing comprises replacing a target site of the DNA of the cell with a sequence of the donor DNA.
8 . The method of claim 1 , wherein the nuclease cleaves phosphodiester bonds upstream and downstream of a target site of the DNA to remove the target site.
9 . The method of claim 1 , wherein the editing of the DNA comprises editing a gene of the DNA, the gene comprising a CFTR or EGFR gene.
10 . The method of claim 1 , wherein contacting the cell with the nuclease comprises administering a composition comprising the nuclease to a subject, wherein the subject comprises the cell.
11 . The method of claim 1 , wherein contacting the cell with the nuclease comprises administering a composition comprising a polynucleotide encoding the nuclease to a subject, wherein the subject comprises the cell.
12 . The method of claim 1 , wherein the method treats a disease of a subject.
13 . The method of claim 12 , wherein the disease comprises cancer or cystic fibrosis.
14 . The method of claim 12 , wherein the disease comprises non-small cell lung cancer.
15 . The method of claim 13 , wherein the nuclease is a dual nuclease that targets and cleaves 10 to 20 nucleotides on either side of a CFTR delta F508 mutation or an EGFR exon 19 mutation.
16 . The method of claim 1 , further comprising contacting the cell with a guide RNA.
17 . The method of claim 1 , wherein the nuclease is a chimeric nuclease that further comprises an I-TevI sequence coupled with the Cas9 sequence.
18 . The method of claim 17 , wherein the I-TevI sequence comprises the amino acid sequence of SEQ ID NO: 6, or a sequence thereof comprising 1, 2 or 3 mutations each selected from the group consisting of: an amino acid substitution, deletion, and insertion.
19 . The method of claim 1 , wherein contacting the cell with the nuclease comprises administering a pharmaceutical composition to a subject comprising the cell, the composition comprising a guide RNA or a nucleic acid encoding the guide RNA, and the nuclease or a nucleic acid encoding the nuclease, wherein the nuclease is a chimeric nuclease that further comprises an I-TevI sequence linked to the Cas9 sequence.
20 . The method of claim 19 , wherein the administration comprises intravenous administration, injection, or inhalation.
21 . The method of claim 20 , wherein the administration comprises inhalation.
22 . The method of claim 20 , wherein the administration comprises nebulization.
23 . The method of claim 19 , wherein the composition further comprises a lipid nanoparticle.
24 . The method of claim 23 , wherein the lipid nanoparticle is about 100 nM in size.
25 . The method of claim 23 , wherein the lipid nanoparticle comprises a cationic lipid.
26 . The method of claim 23 , wherein the lipid nanoparticle comprises DOPE, MPEG-5000-DMPE, DPPC, cholesterol, DOBA, or a combination thereof.
27 . The method of claim 1 , wherein the DNA comprises genomic DNA, the cell is a mammalian cell, the nuclease is a chimeric nuclease that further comprises an I-TevI nuclease domain connected via a peptide linker to the Cas9 sequence, and wherein the method further comprises contacting the cell with a guide RNA that binds the genomic DNA and the Cas9 sequence.
28 . The method of claim 23 , wherein the lipid nanoparticle comprises (a), (b), or (c):
(a) DOPE and MPEG-5000-DMPE at a molar ratio of about 2:0.05;
(b) DPPC, cholesterol and DOBA at a molar ratio of about 7:2:1; or
(c) DPPC, cholesterol and MPEG-5000-DMPE at a molar ratio of about 4:1:0.125.
29 . The method of claim 19 , wherein the composition further comprises N4-cholesteryl-spermine.
30 . The method of claim 19 , wherein the composition further comprises an excipient selected from the group consisting of: polysorbate, polyphosphate, calcium chloride, sodium chloride, sodium citrate, sodium hydroxide, sodium phosphate, sodium ethylenediaminetetraacetic acid, potassium chloride, potassium phosphate, and starch.