Recombinase Mediated DNA modifications

Details Recombinase Mediated DNA modifications Recombinase Mediated DNA modifications

1999-04-28

2001-06-12

Ketter, James (Department: 1636)

Chemistry: molecular biology and microbiology

Animal cell, per se ; composition thereof; process of...

Method of regulating cell metabolism or physiology

C475S194000, C536S023500

Reexamination Certificate

active

06245565

ABSTRACT:

INTRODUCTION
1. Technical Field
The field of this invention is the inactivation of target DNA in vivo.
2. Background
The ability to selectively inhibit the growth of a subset of cells in a mixture of cells has many applications in culture and in vivo. Where two sets of cells have distinguishing characteristics, such as tumor cells which require expression of one or more genes, which are not expressed in normal cells or only expressed at a low level, there is substantial interest in being able to selectively inhibit the proliferation of the tumor cells. Where groups of cells are differentiating, and at one level of differentiation, expression of a particular gene is required, the ability to inhibit the expression of that gene can be of interest. Where cells are infected by viruses, parasite or mycoplasmas, the selective ability to inhibit the growth of the virus or mycoplasma can be an important goal.
In the studies of metabolic processes, differentiation, activation, and the like, there are many situations where it is desirable to be able to selectively inhibit the transcription of a particular gene. In this way, one can study the effect of a reduction in the transcription of the gene and expression of the gene on the phenotype of the cell. In the extensive efforts to understand embryonic and fetal development, to define segmental polarity genes and their function, there is also interest in being able to selectively inhibit particular genes during various phases of the development of the fetus.
As in the case of the studies in culture, selective inhibition of particular genes can also be of interest in vivo. In many situations, cellular proliferation can be injurious to the host. The proliferation can be as a result of neoplasia, inflammation, or other process where increased number of cells has an adverse effect upon the health of the host.
There is, therefore, substantial interest in finding techniques and reagents which allow for selective inhibition of particular genes, so as to control intracellular molecular processes.
Relevant Literature
WO93/05178 provides an extensive description of double D-loop formation, with an extended bibliography of references. Sena and Zarling, (1993) Nature Genetics 3:365-372and Révet, Sena and Zarling, J. Mol. Biol.232:779-791 describe double D-loop formation. Golub et al., (1992) Nucleic Acids Res. 20:3121-5; Golub et al., (1993) Proc. Natl. Acad. Sci. USA. 90:7186-90; Ferrin and Camerini-Otero, (1991) Science 254:1494-7.; Koob et al. (1992) Nucleic Acids Res. 20:5831-6; Revet et al. (1993) J. Mol. Biol. 232:779-91; Sena (1993) Nature Genetics 3:365-371and Jayasena and Jonnston (1993) J. Molec Biol. 230:1015-1024.
SUMMARY OF THE INVENTION
Recombinase coated pairs of single-stranded probes having a region of complementarity are introduced into cells. One or both probes may be modified with an agent capable of inhibiting transcription. The pairs of recombinase coated probes are introduced into cells, where the probes are directed to a DNA target sequence, particularly genes, primarily directed to the 5′-transcriptional initiation region or other essential transcriptional initiation sequence, such as an enhancer. The formation of the double D-loop inhibits copying, e.g. transcription, and by providing for a DNA modifying agent bound to one or both of the probes, further inhibition can be achieved.
DESCRIPTION OF THE SPECIFIC EMBODIMENTS
Methods and compositions are provided for inhibiting copying of a DNA sequence, where copying intends transcription and replication. Of particular interest is selectively inhibiting the transcription of at least one gene in a cellular host. The method comprises introducing a pair of probes having a complementary region, where the pair of probes is coated with a recombinase. One or both of the probes may be further modified with an agent which can react with DNA to further inhibit copying. The probes are introduced into the cells by any convenient means, in culture or in vivo. As a result of the introduction of the probes into the cell, with formation of double D-loops, copying of the target DNA is substantially diminished, resulting in a change in the phenotype of the cells or mortality. The subject compositions may be used by themselves or in conjunction with other agents, depending upon the purpose for which the subject compositions are employed.
The probes may be any sequences which have substantial homology with each other and with a target DNA sequence. Usually the homology between the two probes and the target sequence will be at least about 70% between target and probe, more usually at least about 90% and preferably 100% (complementarity). By homology is intended sequences having substantial identity and the percent homology is defined in accordance with FASTP (Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85, 2444-8 (1988).
For the most part, the DNA sequences will be the naturally occurring nucleotides, although some modifications may be made, where a proportion of the nucleotides may be modified to enhance stability. Thus, various oxygens of the phosphate in the backbone may be substituted with sulfur, nitrogen or carbon (methylene), or an unnatural sugar may be employed, e.g. replacing ribose with arabinose. To enhance stability, one or both termini of the sequence may be modified, particularly by functionalization. The modification may serve to inhibit exonuclease, to link to another moiety, or the like. Thus, ethers, amino groups, esters, or other functionality may be provided at one or both termini. Reactive moieties which affect the ability for copying may include single stranded or double stranded DNA scission inducing molecules, intercalating molecules, cross linking molecules, photoactive molecules, and the like. Scission inducing molecules provide for cleavage of one or both of the DNA strands of the target sequence. These may include chelated metal ions, such as iron and copper. Chelating agents may include ethylenediamine tetraacetic acid (EDTA), nitrilo triacetic acid (NTA), 2,9-dimethyl-1,10-phenanthroline, etc. Alternative molecules may include electron accepting molecules, such as quinones, and the like.
Depending upon the particular purpose of the subject composition, various agents which provide for cross linking may be employed. Photoactivatable cross linkers include coumarin, psoralen, and other commercial cross-linking molecules available from Pierce and Pharmacia. Other compounds include oxetanes, or other unstable dioxacyclic compounds.
A large variety of intercalating agents are known, including many dyes, such as thiazole orange, ethidium bromide, phenanthridines, actinomycin D, etc. By intercalating into the DNA, these molecules will further stabilize the complex and reactivity and if chemically modified can interact with the DNA strands to form covalent bonds.
In addition, one can provide for compounds which are activated intracellularly to react with nucleotides. These compounds include quinones having various substituents.
The various active moieties may be tethered to the DNA sequence by a bond or any convenient linking group, which may be a single atom or a chain of about fifty atoms, where the atoms may include carbon, nitrogen, oxygen, sulfur, phosphorous, and the like. Thus, the chain may be an oligonucleotide chain, peptide nucleic acid chain (nucleic acid chain, where phosphate is substituted with glycine), oligosaccharide, polyoxyalkylene, polyamide, e.g. polypeptide, alkylene, arylene, combinations thereof, or the like. The particular linking group is not critical, but one may be selected over another for synthetic convenience, in relation to the particular moiety, to provide solubility, flexibility, hydrophobicity, enhanced binding to DNA, or to remove secondary structure. Usually, there will be from 0 to 3 active moieties per probe, more usually from 0 to 2, particularly 0 to 2 for the combination of probes, more usually 0 to 1 for the combination of probes. The size of the probes, extent of homology, and areas of non-homology may vary widely. Thus, one m

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