Compositions and methods for editing nucleic acids in cells utilizing oligonucleotides
The present invention includes compositions and methods for the treatment of a medical condition or disease utilizing editing oligonucleotides. The editing oligonucleotides contain an oligonucleotide strand of about 10 to about 50 nucleotides on each side of the editing moiety which may contain a sugar or linker that positions the active editing moiety in the proper location for hybridization to the target nucleic acid. The editing oligonucleotides may also contain at least one nucleotide sequence change from the targeted sequence in the genome. The method includes modifying a genomic sequence within a cell utilizing an editing oligonucleotide without additional proteins or nucleic acids to assist in the editing process. The editing oligonucleotide may comprise backbone modifications that increase the nuclease stability of the oligonucleotide as compared to unmodified oligonucleotides or oligonucleotides having three phosphorothioates on each terminus.
1. A method of modifying a human genomic sequence within a human cell or a human individual comprising the steps of:
introducing into said human cell or said human individual a single-stranded chemically modified oligonucleotide without additional exogenous proteins or nucleic acids to assist in editing said human genomic sequence, wherein said chemically modified oligonucleotide comprises one or more 2′ sugar modification(s), one or more backbone modification(s), and one or more nucleobase modification(s), wherein the one or more 2′ sugar modification(s) is selected from 2′-O-methyl, 2′-fluoro, or a combination of 2′-O-methyl and 2′-fluoro, wherein said oligonucleotide comprises, in 5′ to 3′ order, a 5′ arm, an editing site, and a 3′ arm, wherein said oligonucleotide is substantially complementary to a target sequence in said human genomic sequence with the exception of one or more mismatches in the editing site that modify the target sequence, wherein the chemically modified oligonucleotide is conjugated to Gal-NAc or a lipophilic group, wherein the human genomic sequence comprises a disease-causing mutation, and wherein:
a) one of the 3′ arm or the 5′ arm does not comprise a 2′-sugar modification, and/or
b) the chemically modified oligonucleotide comprises a methylphosphonate end-block.
2. The method of claim 1 , wherein the 3′ arm comprises one or more 2′-fluoro sugar modifications and the 5′ arm does not comprise a 2′-sugar modification.
3. The method of claim 1 , wherein the 5′ arm comprises one or more 2′-O-methyl sugar modifications and the 3′ arm does not comprise a 2′-sugar modification.
4. The method of claim 1 , wherein the 5′ arm comprises one or more 2′-O-methyl sugar modifications or one or more 2′-fluoro sugar modifications and the 3′ arm comprises one or more 2′-fluoro sugar modifications.
5. The method of claim 1 , wherein the one or more backbone modifications is selected from phosphorothioate, methyl phosphonate or a combination of phosphorothioate and methyl phosphonate.
6. The method of claim 5 , wherein the chemically modified oligonucleotide comprises one or more phosphorothioates at a 5′ terminus of the 5′ arm and one or more phosphorothioates at a 3′ terminus of the 3′ arm.
7. The method of claim 5 , wherein the chemically modified oligonucleotide comprises one or more methyl phosphonates at a 5′ terminus of the 5′ arm and one or more phosphorothioates at a 3′ terminus of the 3′ arm.
8. The method of claim 1 , wherein the nucleobase modification is one or more 5′-methyl-cytosine.
9. The method of claim 1 , wherein the chemically modified oligonucleotide is formulated into a pharmaceutically acceptable composition additionally comprising a pharmaceutically acceptable carrier.
10. The method of claim 1 wherein the modification to the human genomic sequence reverts a mutated nucleotide to a wild-type nucleotide.
11. The method of claim 1 wherein the modification to the human genomic sequence modifies a codon in a mutated-disease-causing protein to produce a non-wild-type protein that is a non-disease-causing protein.
12. The method of claim 1 , wherein the modification to the human genomic sequence modifies a human CFTR mutant protein to suppress its disease-causing effects.
13. The method of claim 12 , wherein the human CFTR mutant protein causes cystic fibrosis.
14. The method of claim 13 , wherein cystic fibrosis caused by a delta 508 mutation.
15. The method of claim 1 , wherein the chemically modified oligonucleotide further comprises a phosphorothiate modification at the 5′ terminus.
16. The method of claim 15 , wherein the chemically modified oligonucleotide comprises a phosphorothiate modification at the first three nucleotides of the 5′ terminus.
17. The method of claim 1 , wherein the chemically modified oligonucleotide comprises a methylphosphonate end-block.
18. The method of claim 1 , wherein the 2′-O-methyl modifications are 5′ of the editing site and the 2′-fluoro modifications are 3′ of the editing site.