Modified triple-helix forming oligonucleotides for targeted mutagenesis
Inventors
Glazer, Peter M. • Siedman, Michael M.
Assignees
Concord GmbH • Yale University • US Department of Health and Human Services
Publication Number
US-8658608-B2
Publication Date
2014-02-25
Expiration Date
2026-11-22
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Abstract
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 (Kd≦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 Kd 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.
Core Innovation
The invention discloses high affinity, chemically modified triplex-forming oligonucleotides (TFOs) that bind as third strands to duplex DNA in a sequence-specific manner. These TFOs can comprise any combination of nucleotides and chemically modified nucleotides, where modifications can occur in the heterocyclic base, sugar moiety, or phosphate moiety. The TFOs are designed to have a low dissociation constant (Kd) with a preferred Kd below 2×10⁻⁸, more preferably below 2×10⁻¹⁰, allowing for stable triplex formation with specific DNA segments of target genes.
The binding of the TFO to the target DNA region forms a triple-stranded structure that stimulates mutations within or adjacent to the target site by leveraging cellular DNA synthesis, recombination, and repair mechanisms. These mutations can activate, inactivate, or alter the activity and function of the target gene, enabling targeted mutagenesis in vivo and in vitro. The TFOs can be used alone or in conjunction with donor oligonucleotides to promote targeted recombination with higher efficiency without the need for prior modification of the target. Chemical modifications, such as substitutions of cytosines with pseudocytosine or pseudoisocytosine and sugar or backbone modifications including 2′-O-aminoethoxy or diethylethlenediamine, increase triplex stability and binding affinity at neutral pH, addressing limitations of unmodified TFOs.
The problem addressed by the invention relates to the inefficiency and complexity of existing gene therapy and targeted mutagenesis methods, including low frequency of homologous recombination, requirement for selection procedures, and dependence on viral vectors for delivery. Standard triple helix formation methods have limitations under physiological conditions due to instability of the triplex, especially in sequences with adjacent cytosines. The invention solves these problems by providing chemically modified TFOs with enhanced binding affinity and stability for effective targeted mutagenesis and recombination, offering improved gene therapy alternatives without the need for viral vectors or prior target modification.
Claims Coverage
The patent contains 23 claims, with claim 1 being the independent claim defining the core inventive features. Subsequent claims detail compositions, cell applications, and methods of use.
Single-stranded oligonucleotide binding with enhanced triplex stability
A single-stranded oligonucleotide that binds specifically to a polypyrimidine:polypurine target motif within a double stranded nucleic acid molecule in a beta globin gene forming a triple-stranded nucleic acid. The polypyrimidine strand is from specified nucleotide regions of SEQ ID NO:59 or SEQ ID NO:60. The oligonucleotide comprises one or more chemically modified cytosine nucleotides substituted for cytosines, resulting in increased triplex stability at neutral pH compared to oligonucleotides without such substitutions.
Combination with donor nucleic acids
The composition may further comprise a donor nucleic acid that is single or double stranded, which optionally contains phosphorothioate linkages, and may be tethered or separate from the single stranded oligonucleotide to promote targeted recombination.
Chemically modified sugar moieties in oligonucleotides
The single stranded oligonucleotide comprises chemically modified sugar moieties selected from 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) to increase triplex stability and binding affinity.
Chemically modified phosphate moieties in oligonucleotides
The single stranded oligonucleotide comprises chemically modified phosphate backbones, such as diethyl-ethylenediamide (DEED) or dimethyl-aminopropylamine (DMAP), to reduce electrostatic repulsion and increase binding affinity.
Oligonucleotides comprising peptide nucleic acid monomers
The single stranded oligonucleotide can incorporate peptide nucleic acid monomers, including formations of bis-peptide nucleic acids that form stable triplex clamps with the target DNA strand.
Cell applications
A cell can comprise the recombinagenic or mutagenic composition for targeted genetic modifications.
Targeted recombination or mutation method
A method for targeted recombination or mutation in cells or individuals involving administration of the recombinagenic or mutagenic composition to induce mutation or recombination in double stranded nucleic acid molecules.
Correction of point mutations in the human β-globin gene
The method specifically targets correction of point mutations in the human β-globin gene, particularly those associated with sickle cell anemia or β-thalassemia, restoring the gene to normal sequence.
The claims define chemically modified single-stranded oligonucleotides that bind specifically to beta globin gene targets with enhanced triplex stability, optionally combined with donor nucleic acids, and methods for targeted mutation or recombination correcting disease-related point mutations. The claims also cover peptide nucleic acid-based oligonucleotides and their use in cells for gene therapy.
Stated Advantages
Chemical modifications increase binding affinity and stability of triplex formation under physiological conditions, overcoming limitations of prior TFOs, especially in sequences with adjacent cytosines.
TFOs and bis-PNAs promote efficient targeted mutagenesis and recombination without requiring prior target modification or viral vectors, simplifying gene therapy approaches.
The method enables highly specific and efficient mutation or recombination at desired genomic sites, improving precision in genetic engineering and therapeutic gene correction.
Triplex-forming oligonucleotides can be used in vivo, expanding their utility for therapeutic gene correction and research applications.
Documented Applications
Targeted mutagenesis and recombination in human and animal cells for gene therapy applications.
Treatment of genetic disorders caused by point mutations, including globinopathies such as sickle cell anemia and β-thalassemia.
Targeted gene correction of mutations in the human β-globin gene, specifically at intron two, position one.
Anti-cancer therapy by inactivating oncogenes or activating repressor genes via targeted mutagenesis.
Antiviral therapy by targeting viral genes essential for viral survival or reproduction.
Molecular biology research tool for targeted mutagenesis, genetic engineering, and studying DNA repair mechanisms.
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