Modified triple-helix forming oligonucleotides for targeted mutagenesis
View Patent ↗High affinity, chemically modified triplex-forming oligonucleotides (TFOs) and methods for use thereof are disclosed. TFOs are defined as triplex-forming oligonucleotides which bind as third strands to duplex DNA in a sequence specific manner. Triplex-forming oligonucleotides may be comprised of any possible combination of nucleotides and modified nucleotides. Modified nucleotides may contain chemical modifications of the heterocyclic base, sugar moiety or phosphate moiety. A high affinity oligonucleotide (K d ≦2×10 −8 ) which forms a triple strand with a specific DNA segment of a target gene DNA is generated. It is preferable that the K d for the high affinity oligonucleotide is below 2×10 −10 . The nucleotide binds or hybridizes to a target sequence within a target gene or target region of a chromosome, forming a triplex region. The binding of the oligonucleotide to the target region stimulates mutations within or adjacent to the target region using cellular DNA synthesis, recombination, and repair mechanisms. The mutation generated activates, inactivates, or alters the activity and function of the target gene.
1. A recombinagenic or mutagenic composition comprising
a single-stranded oligonucleotide that binds to a polypyrimidine:polypurine target motif in a double stranded nucleic acid molecule in a beta globin gene to form a triple-stranded nucleic acid molecule,
wherein the polypyrimidine strand of the polypyrimidine:polypurine target motif comprises from nucleotide 837 to nucleotide 846 of SEQ ID NO:59 or nucleotide 774 to nucleotide 787 of SEQ ID NO:60,
wherein the single-stranded oligonucleotide comprises a sequence substantially complementary to the polypurine strand of the polypyrimidine:polypurine target motif, and
wherein the single stranded oligonucleotide comprises one or more chemically modified cytosine nucleotides substituted for one or more cytosine nucleotides and has increased triplex stability at neutral pH relative to the single-stranded oligonucleotide in the absence of the substituted cytosine nucleotides.
2. The recombinagenic or mutagenic composition of claim 1 further comprising a donor nucleic acid.
3. The recombinagenic or mutagenic composition of claim 2 wherein the donor nucleic acid is single stranded or double stranded.
4. The composition of claim 3 wherein the donor nucleic acid comprises one or more phosphorothioate linkages.
5. The composition of claim 2 wherein the donor nucleic acid is tethered to the single stranded oligonucleotide.
6. The composition of claim 2 wherein the donor nucleic acid is separate from the single stranded oligonucleotide.
7. The composition of claim 1 wherein the single stranded oligonucleotide is between 10 and 60 nucleotides residues in length.
8. The composition of claim 1 wherein the single stranded oligonucleotide comprises a chemically modified sugar moiety selected from the group consisting of 2′-O-aminoethoxy, 2′-O-amonioethyl, 2′-O-methoxy, 2′-O-methyl, 2-guanidoethyl, 2′-O,4′-C-methylene, 2′-O-(methoxyethyl) and 2′-O—(N-(methyl)acetamido).
9. The composition of claim 1 wherein the single stranded oligonucleotide comprises a chemically modified phosphate moiety selected from the group consisting of diethyl-ethylenediamide and dimethyl-aminopropylamine.
10. The composition of claim 1 wherein the single stranded oligonucleotide comprises a peptide nucleic acid monomer.
11. The composition of claim 10 wherein the single stranded oligonucleotide comprises peptide nucleic acid monomers positioned in the oligonucleotide to form a bis-peptide nucleic acid.
12. A cell comprising the recombinagenic or mutagenic composition of claim 1 .
13. A method for targeted recombination or mutation of a nucleic acid molecule comprising administering to cells or an individual an effective amount of the recombinagenic or mutagenic composition of claim 1 to induce mutation or recombination in a double stranded nucleic acid molecule in the cells or individual.
14. The method of claim 13 wherein the targeted recombination or mutation corrects a point mutation in the human β-globin gene and restores the DNA sequence of the human β-globin gene to normal.
15. The method of claim 14 wherein the point mutation in the human β-globin gene is associated with sickle cell anemia or β-thalassemia.
16. The recombinagenic or mutagenic composition of claim 1 wherein the double-stranded nucleic acid molecule is a defective β-hemoglobin gene.
17. The recombinogenic or mutagenic composition of claim 1 wherein the one or more chemically modified cytosines are selected from the group consisting of pseudocytosine, pseudoisocytosine, and 5-methylcytosine.
18. The recombinagenic or mutagenic composition of claim 11 wherein the bis-peptide nucleic acid comprises SEQ ID NO:49.
19. The recombinagenic or mutagenic composition of claim 2 wherein the donor oligonucleotide is selected from the group consisting of SEQ ID NO:50 and SEQ ID NO:51.
20. The recombinagenic or mutagenic composition of claim 16 wherein the defect in the β-hemoglobin gene is caused by a point mutation in intron two of the human β-hemoglobin gene.
21. The recombinagenic or mutagenic composition of claim 20 wherein the point mutation is in position one of intron two of the human β-hemoglobin gene.
22. The method of claim 15 wherein the defect in the β-hemoglobin gene is caused by a point mutation in intron two of the human β-hemoglobin gene.
23. The method of claim 22 wherein the point mutation is in position one of intron two of the human β-hemoglobin gene.