Chemistry: molecular biology and microbiology – Process of mutation – cell fusion – or genetic modification
Reexamination Certificate
1999-10-04
2001-10-16
Myers, Carla J. (Department: 1655)
Chemistry: molecular biology and microbiology
Process of mutation, cell fusion, or genetic modification
C435S006120, C435S005000, C435S091100, C435S091200, C435S443000, C435S444000, C435S455000, C435S471000, C536S024500, C514S04400A
Reexamination Certificate
active
06303376
ABSTRACT:
BACKGROUND OF THE INVENTION
This relates to the fields of genetics, and more particularly relates to site-directed mutagenesis of a gene of interest.
Triple-stranded DNA Since the initial observation of triple-stranded DNA many years ago by Felsenfeld et al.,
J. Am. Chem. Soc
. 79:2023 (1957), oligonucleotide-directed triple helix formation has emerged as a valuable tool in molecular biology. Current knowledge suggests that oligonucleotides can bind as third strands of DNA in a sequence specific manner in the major groove in polypurine/polypyrimidine stretches in duplex DNA. In one motif, a polypyrimidine oligonucleotide binds in a direction parallel to the purine strand in the duplex, as described by Moser and Dervan,
Science
238:645 (1987), Praseuth et al.,
Proc. Natl. Acad. Sci. USA
85:1349 (1988), and Mergny et al.,
Biochemistry
30:9791 (1991). In the alternate purine motif, a polypurine strand binds anti-parallel to the purine strand, as described by Beal and Dervan,
Science
251:1360 (1991). The specificity of triplex formation arises from base triplets (AAT and GGC in the purine motif) formed by hydrogen bonding; mismatches destabilize the triple helix, as described by Mergny et al.,
Biochemistry
30:9791 (1991) and Beal and Dervan,
Nuc. Acids Res
. 11:2773 (1992).
Triplex forming oligonucleotides have been found useful for several molecular biology techniques. For example, triplex forming oligonucleotides designed to bind to sites in gene promoters have been used to block DNA binding proteins and to block transcription both in vitro and in vivo. (Maher et al.,
Science
245:725 (1989), Orson et al.,
Nucleic Acids Res
. 19:3435 (1991), Postal et al.,
Proc. Natl. Acad. Sci. USA
88:8227 (1991), Cooney et al.,
Science
241:456 (1988), Young et al.,
Proc. Natl. Acad. Sci. USA
88:10023 (1991), Maher et al.,
Biochemistry
31:70 (1992), Duval-Valentin et al.,
Proc. Natl. Acad. Sci. USA
89:504 (1992), Blume et al.,
Nucleic Acids Res
. 20:1777 (1992), Durland et al.,
Biochemistry
30:9246 (1991), Grigoriev et al.,
J. of Biological Chem
. 267:3389 (1992), and Takasugi et al.,
Proc. Natl. Acad. Sci. USA
88:5602 (1991)). Site specific cleavage of DNA has been achieved by using triplex forming oligonucleotides linked to reactive moieties such as EDTA-Fe(II) or by using triplex forming oligonucleotides in conjunction with DNA modifying enzymes (Perrouault et al.,
Nature
344:358 (1990), Francois et al.,
Proc. Natl. Acad. Sci. USA
86:9702 (1989), Lin et al.,
Biochemistry
28:1054 (1989), Pei et al.,
Proc. Natl. Acad. Sci. USA
87:9858 (1990), Strobel et al.,
Science
254:1639 (1991), and Posvic and Dervan,
J. Am. Chem Soc
. 112:9428 (1992)). Sequence specific DNA purification using triplex affinity capture has also been demonstrated. (Ito et al.,
Proc. Natl. Acad. Sci. USA
89:495 (1992)). Triplex forming oligonucleotides linked to intercalating agents such as acridine, or to cross-linking agents, such as p-azidophenacyl and psoralen, have been utilized, but only to enhance the stability of triplex binding. (Praseuth et al.,
Proc. Natl. Acad. Sci. USA
85:1349 (1988), Grigoriev et al.,
J. of Biological Chem
. 267:3389 (1992), Takasugi et al.,
Proc. Natl. Acad. Sci. USA
88:5602 (1991).
Gene Therapy
Gene therapy can be defined by the methods used to introduce heterologous DNA into a host cell or by the methods used to alter the expression of endogenous genes within a cell. As such, gene therapy methods can be used to alter the phenotype and/or genotype of a cell.
Methods which alter the genotype of a cell typically rely on the introduction into the cell of an entire replacement copy of a defective gene, a heterologous gene, or a small nucleic acid molecule such as an oligonucleotide, to treat human, animal and plant genetic disorders. The introduced gene or nucleic acid molecule, via genetic recombination, replaces the endogenous gene. This approach requires complex delivery systems to introduce the replacement gene into the cell, such as genetically engineered viruses, or viral vectors.
Alternatively, gene therapy methods can be used to alter the expression of an endogenous gene. One example of this type of method is the field of antisense therapy. In antisense therapy, a nucleic acid molecule is introduced into a cell, the nucleic acid molecule being of a specific nucleic acid sequence so as to hybridize or bind to the mRNA encoding a specific protein. The binding of the antisense molecule to an mRNA species decreases the efficiency and rate of translation of the mRNA.
Gene therapy is being used on an experimental basis to treat well known genetic disorders of humans such as retinoblastoma, cystic fibrosis, and sickle cell anemia. However, in vivo efficiency is low due to the limited number of recombination events actually resulting in replacement of the defective gene.
A method for targeted mutagenesis of a target DNA molecule would be useful as another means of gene therapy which can be carried out in vivo. Such a method would also be a useful research tool for genetic engineering or for studying genetic mechanisms such as DNA repair.
Therefore, it is an object of the present invention to provide a method for in vivo and in vitro targeted mutagenesis of a target DNA molecule.
It is a further object of the present invention to provide a method for mutagenesis of a target DNA molecule that is highly specific and efficient.
It is a further object of the present invention to provide a method for treating genetic disorders by gene therapy without the need for a viral vector.
It is a further object of the present invention to provide a method for treating cancer.
It is a further object of the present invention to provide oligonucleotides for use in therapy and research.
SUMMARY OF THE INVENTION
High affinity, triplex-forming oligonucleotides and methods for use thereof are described herein. 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. The oligonucleotide binds/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.
If the target gene contains a mutation that is the cause of a genetic disorder, then the oligonucleotide is useful for mutagenic repair that restores the DNA sequence of the target gene to normal. If the target gene is a viral gene needed for viral survival or reproduction or an oncogene causing unregulated proliferation, such as in a cancer cell, then the mutagenic oligonucleotide is useful for causing a mutation that inactivates the gene to incapacitate or prevent reproduction of the virus or to terminate or reduce the uncontrolled proliferation of the cancer cell. The mutagenic oligonucleotide is also a useful anti-cancer agent for activating a repressor gene that has lost its ability to repress proliferation.
The triplex-forming oligonucleotide is also particularly useful as a molecular biology research tool to cause targeted mutagenesis. Targeted mutagenesis is useful for targeting a normal gene and for the study of mechanisms such as DNA repair. Targeted mutagenesis of a specific gene in an animal oocyte, such as a mouse oocyte, provides a useful and powerful tool for genetic engineering for research and therapy and for generation of new strains of “transmutated” animals and plants for research and agriculture.
REFERENCES:
patent: 5176996 (1993-01-01), Hogan et al.
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patent: 0 375 408 A1 (1990-06-01), None
patent: WO 95/01364 A1 (1995-01-01), None
patent: WO 95/01365 A1 (1995-01-01), None
Agrawal, et al., “Pharmacokinetics, biodistribution, and stability of oligoeoxynucleotide phosphorothioates in mice,”Proc Natl Acad Sci U S A.88(17):7595-9 (1991).
*
Holland & Knight LLP
Myers Carla J.
Yale University
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