Back to results
US 6,936,467 B2Granted
Targeted chromosomal genomic alterations with modified single stranded oligonucleotides
AssigneeUniversity of Delaware
InventorsEric B. Kmiec, Howard B. Gamper, Michael C. Rice
Life of this patent
Estimating the term…
Abstract
Presented are methods and compositions for targeted chromosomal genomic alterations using modified single-stranded oligonucleotides. The oligonucleotides of the invention have at least one modified nuclease-resistant terminal region comprising phosphorothioate linkages, LNA analogs or 2′-O-Me base analogs.
Claims129
A method of targeted alteration of a chromosomal sequence present within a cell in vitro, the method comprising:
- introducing a sequence-altering oligonucleotide into a cell in vitro,
- wherein said sequence-altering oligonucleotide:
- is a single-stranded nonhairpin oligonucleotide 17-121 nucleotides in length;
- has an unmodified DNA domain of at least 8 contiguous deoxyribonucleotides;
- is fully complementary in sequence to a first strand of the cell's chromosomal DNA at a chromosomal target sequence, except for one or two mismatches positioned (i) within said oligonucleotide's unmodified DNA domain and (ii) at least 8 nucleotides from said oligonucleotide's 5′ and 3′ termini;
- and
- has chemical modifications consisting essentially of at least one terminal locked nucleic acid (LNA), or at least one terminal 2′-O-Me base analog, or at least three terminal phosphorothioate linkages, or combinations thereof, and
- wherein said cell is not a human embryonic stem cell,
- whereby said introduced oligonucleotide directs sequence alteration at said chromosomal target sequence by the cellular repair enzyme machinery.
6 more elements
A method of targeted sequence alteration of a nucleic acid present within selectively enriched cells in vitro, cells in culture, or cell-free extracts, comprising:
- contacting the targeted nucleic acid in the presence of cellular repair proteins with a single-stranded nonhairpin oligonucleotide 17-121 nucleotides in length, said oligonucleotide having a domain of at least B contiguous deoxyribonucleotides,
- wherein said oligonucleotide is fully complementary in sequence to the sequence of a first strand of the targeted nucleic acid, but for one or more mismatches as between the sequences of said deoxyribonucleotide domain and its complement on the target nucleic acid first strand, each of said mismatches positioned at least B nucleotides from said oligonucleotide's 5′ and 3′ termini;
- wherein said oligonucleotide has at least one terminal modification selected from the group consisting of:
- at least one terminal locked nucleic acid (LNA), at least one terminal 2′-O-Me base analog, and at least three terminal phosphorothioate linkages;
- wherein said cultured or selectively enriched cells are not human embryonic stem cells, and
- wherein said targeted nucleic acid is selected from the group of human genes consisting of:
- ADA, p53, beta-globin, RB, BRCA1, BRCA2, CFTR, CDKN2A, APC, Factor V, Factor VIII, Factor IX, hemoglobin alpha 1, hemoglobin alpha 2, MLH1, MSH2, MSH6, ApoE, LDL receptor, UGT1, APP, PSEN1, and PSEN2.
4 more elements
A method of targeted sequence alteration of a nucleic acid present within selectively enriched cells in vitro, cells in culture, or cell-free extracts, comprising:
- contacting the targeted nucleic acid in the presence of cellular repair proteins with a single-stranded nonhairpin oligonucleotide 17-121 nucleotides in length, said oligonucleotide having a domain of at least 8 contiguous deoxyribonucleotides,
- wherein said oligonucleotide is fully complementary in sequence to the sequence of a first strand of the targeted nucleic acid, but for one or more mismatches as between the sequences of said deoxyribonucleotide domain and its complement on the target nucleic acid first strand, each of said mismatches positioned at least 8 nucleotides from said oligonucleotide's 5′ and 3′ termini;
- wherein said oligonucleotide has at least one terminal modification selected from the group consisting of:
- at least one terminal locked nucleic acid (LNA), at least one terminal 2′-O-Me base analog, and at least three terminal phosphorothioate linkages;
- wherein said cultured or selectively enriched cells are not human embryonic stem cells, and
- wherein said oligonucleotide includes the sequence of any one of SEQ ID NOs:
- 1-4340.
4 more elements
A method of targeted sequence alteration of a nucleic acid present within selectively enriched cells in vitro, cells in culture, or cell-free extracts, comprising:
- contacting the targeted nucleic acid in the presence of cellular repair proteins with a single-stranded nonhairpin oligonucleotide 17-121 nucleotides in length, said oligonucleotide having a domain of at least 8 contiguous deoxyribonucleotides,
- wherein said oligonucleotide is fully complementary in sequence to the sequence of a first strand of the targeted nucleic acid, but for one or more mismatches as between the sequences of said deoxyribonucleotide domain and its complement on the target nucleic acid first strand, each of said mismatches positioned at least 8 nucleotides from said oligonucleotide's 5′ and 3′ termini;
- wherein said oligonucleotide has at least one terminal modification, said oligonucleotide includes the sequence of any one of SEQ ID NOs:
- 1-4340, and said cultured or selectively enriched cells are not human embryonic stem cells.
1 more element
A method of targeted sequence alteration of a nucleic acid present within selectively enriched hematopoietic stem cells in vitro or hematopoietic stem cells in culture, comprising:
- contacting the targeted nucleic acid in the presence of cellular repair proteins with a single-stranded nonhairpin oligonucleotide 17-121 nucleotides in length, said oligonucleotide having a domain of at least 8 contiguous deoxyribonucleotides,
- wherein said oligonucleotide is fully complementary in sequence to the sequence of a first strand of the targeted nucleic acid, but for one or more mismatches as between the sequences of said deoxyribonucleotide domain and its complement on the target nucleic acid first strand, each of said mismatches positioned at least B nucleotides from said oligonucleotide's 5′ and 3′ termini;
- and
- wherein said oligonucleotide has at least one terminal modification selected from the group consisting of:
- at least one terminal locked nucleic acid (LNA), at least one terminal 2′-O-Me base analog, and at least three terminal phosphorothioate linkages.
2 more elements
Dependencies are inferred from the claim text.