Methods and compositions to facilitate D-loop formation by...

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Reexamination Certificate

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C435S091400

Reexamination Certificate

active

06312925

ABSTRACT:

STATEMENT OF RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED RESEARCH
Not applicable.
TECHNICAL FIELD
The invention is in the field of genetic modification. More particularly, it is in the field of modified oligonucleotides for use in gene targeting, gene modification and genetic therapy.
BACKGROUND
Targeted modification of a chromosomal gene in a living cell is central to the development of gene therapy. To be maximally effective, such targeted modification results in the change of one or more nucleotides in the sequence of a chromosomal gene. Specific examples include conversion of a mutant allele into its wild-type counterpart and inactivation of a deleterious gene by creating a nucleotide sequence specifying premature transcriptional or translational termination, or altered RNA processing.
A serious challenge to the development of effective compositions and methods for targeted modification has been the difficulty in designing modifying agents which are capable of stable interaction with a target sequence, but retain the specificity necessary for targeted modification. For example, certain intercalating agents have a high affinity for DNA, but react non-specifically with numerous different DNA sequences. On the other hand, reagents that are highly specific for a particular nucleotide sequence, such as complementary oligonucleotides, often do not have sufficient affinity for a target sequence to allow efficient targeted modification to proceed on a reasonable time scale.
Several approaches to sequence-specific modification of a target double-stranded nucleotide sequence have been attempted. The use of triplex-forming oligonucleotides with attached modifying groups has been described in WO 94/17092 and WO 96/40711. These reagents are capable of recognizing a target sequence comprising base-paired, double-stranded DNA, and forming a triple-stranded structure that is mediated by a type of base-pairing different than Watson-Crick type base-pairing. Fresco, U.S. Pat. No. 5,422,251. Attachment of a suitable chemical modifying agent to such an oligonucleotide makes it possible to generate a lesion at or near a target sequence in a gene of interest. Subsequent cellular processes related to DNA replication, recombination and/or repair can result in either restoration of the original sequence by repair of the lesion, or mutagenesis, for example by misrepair, resulting in a base change at the site of the lesion. However, formation of triplexes that are sufficiently stable to achieve modification of a target sequence require sequences containing at least about 12 consecutive purine residues on one strand. Consequently, targeting strategies utilizing modified triplex-forming oligonucleotides are restricted to genes having the requisite homopurine runs.
An alternative approach to targeted modification involves the use of modified oligonucleotides having traditional Watson-Crick complementarity to a target sequence, in concert with a recombinase enzyme. The recombinase enzyme facilitates strand invasion at the target sequence by the complementary oligonucleotide, with the formation of a D-loop-type structure. See WO 93/03736 and WO 96/40711. Efficient formation of this structure and hence, efficient modification, requires at least approximately 26 nucleotides of homology between the oligonucleotide and its target sequence, as described in WO 96/40711. In addition, the method depends on either deliberate or fortuitous interaction between the oligonucleotide and a recombinase enzyme, which may be difficult to control.
Thus, a facile method for non-enzymatic targeting of specific sequences in double-stranded DNA that is more broadly applicable than conventional triplex targeting, along with compositions for use in such a method, would greatly enhance the field of gene therapy. Methods and compositions designed to facilitate the interaction of a complementary oligonucleotide with a target sequence have heretofore relied on attaching the oligonucleotide to an agent having non-specific affinity for DNA, such as an intercalating agent, staphylococcal nuclease or short synthetic positively-charged peptides. U.S. Pat. No. 4,835,263; Mouscadet et al. (1994)
Biochemistry
33:4187-4196; Corey et al. (1995)
Bioconjug. Chem.
6:93-100; and Iyer et al. (1995)
J. Biol. Chem.
270:14712-14717. However, these agents possess only a weak general affinity for DNA and thus are not able to localize the oligonucleotide to the vicinity of its target sequence.
Displacement loop (D-loop) formation offers, in principle, no limits on targeting sequence but faces significant thermodynamic and topological issues. Peptide nucleic acids can form D-loop like structures by strand invasion, but only at homopurine runs. The versatility of Watson-Crick sequence targeting might be realized if: (a) D-loop formation could be facilitated and (b) the unstable D-loop could be stabilized.
DISCLOSURE OF THE INVENTION
The present invention provides new methods and compositions for the targeting and/or modification of a specific sequence in a double-stranded DNA molecule, thereby increasing the number and variety of such sequences that are amenable to targeting and/or modification by a complementary oligonucleotide. Recognition of the target sequence occurs with high affinity and with a high degree of selectivity as a result of two types of sequence specificity, which are provided by an oligonucleotide or oligonucleotide composition comprising two functional domains.
The first functional domain comprises an entity capable of recognizing a double-stranded DNA sequence. This can be a protein, peptide, antibiotic, minor groove binding agent or a nucleotide sequence capable of triplex formation. The first domain may also optionally carry one or more modifying groups. The second functional domain, which is covalently joined to the first, is capable of recognizing a single-stranded DNA sequence. This second domain will most often be substantially complementary, in the Watson-Crick sense, to a target sequence in a double-stranded nucleic acid. The second functional domain can optionally carry one or more modifying groups, capable of causing a mutation, a pre-mutagenic lesion, or some other type of heritable change in the target sequence. Either of the two domains can also include moieties which facilitate their sequence-specific interaction with a double-stranded DNA molecule.
By providing a non-enzymatic targeting method that does not rely exclusively upon triplex homology, the practice of the present invention significantly broadens the repertoire of sequences in double-stranded DNA which can be targeted and/or modified. In the practice of the invention, D-loop formation is facilitated by the first domain, which tethers the D-loop-forming domain to the vicinity of the target sequence. Furthermore, potentially unstable D-loops, once formed, can be stabilized through the action of reactive groups attached to the D-loop-forming domain.
In one embodiment, the present invention provides oligonucleotides and oligonucleotide compositions comprising a triplex-forming domain and a D-loop-forming domain, wherein the former is capable of facilitating strand invasion by the latter. Optionally, a reactive group in the D-loop-forming domain can permanently stabilize that structure, once formed.
In another embodiment, the present invention provides a method for modifying a target nucleotide sequence in a double-stranded DNA molecule, by contacting the double-stranded DNA molecule with an oligonucleotide composition containing a first functional domain and a second functional domain wherein the first functional domain recognizes a region of double-stranded DNA adjacent to or in the vicinity of the target sequence, the second functional domain is substantially complementary, in the Watson-Crick sense, to the target nucleotide sequence and wherein the second functional domain carries an attached modifying agent capable of modifying the target nucleotide sequence.
In another embodiment, the present invention provides a method for modifying a target nu

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