WO2000053630A2

WO2000053630A2 - COMPOUNDS THAT DISRUPT MAMMALIAN Rad51 ACTIVITY

Info

Publication number: WO2000053630A2
Application number: PCT/US2000/006125
Authority: WO
Inventors: Paul Hasty; Xiaohai Gong
Original assignee: Lexicon Genetics Inc
Current assignee: Lexicon Pharmaceuticals Inc
Priority date: 1999-03-09
Filing date: 2000-03-09
Publication date: 2000-09-14
Anticipated expiration: 2001-09-09
Also published as: AU3733100A; WO2000053630A3

Abstract

Several peptides that interact with MmRad51 have been identified including, for example Brca2 and M96. Additionally, Rad51 self-associates via the N-terminal region. When a single residue was changed from a conserved lysine to an alanine, the alteration proved toxic to cells. Moreover, a rad51 allele that lacked the RecA homology region was also deleterious to cells. In view of the above, it is clear that inhibiting Rad51 function or the function of any molecule that associates with MmRad51, or any molecule in the Rad51 or Rad52 pathways, hinders cell proliferation and/or viability. Accordingly, the described class of molecules, that are capable of blocking critical DNA repair pathways represent a new class of therapeutics for inhibiting the growth and maintenance of human cancer cells, as well as other cell proliferation disorders.

Description

COMPOUNDS THAT DISRUPT MAMMALIAN RadSl ACTIVITY

This application claims priority under 35 U.S.C. §1 19 (e) to U.S. provisional patent application no. 60/123.438 filed March 9. 1999 and U.S. provisional patent application no. 60/128.214 filed April 7. 1999, which are hereby incorporated by reference in their entirety.

1. FIELD OF THE INVENTION

The present invention relates to molecules that modulate (e.g. disrupt) mammalian Rad51 or Rad52 function or that modulate (e.g. disrupt) the function of other molecules that are involved in the Rad51 or Rad52 pathways. Such molecules are useful as a means to hinder cell proliferation or to promote programmed cell death, and define a novel class of therapeutic agents for use in the treatment of proliferative disorders such as autoimmune disease and cancer.

2. BACKGROUND OF THE INVENTION

DNA repair and recombination are required by organisms to prevent the accumulation of mutations and to maintain the integrity of genetic information. Compromised genetic material may result in cell cycle arrest, programmed cell death, chromosome loss, or cell senescence. Alternatively, compromised genetic information may result in dysregulation of the cell cycle ultimately leading to increased cellular growth and tumor formation.

The repair of double-strand breaks (DSB) in DNA is an essential cellular process. DSBs may occur during general cellular functions such as cell division and proliferation (Friedberg et al.. 1995. DNA Repair and Mutagenesis. American Society for Microbiology, Washington. D.C). One of the cellular molecules that repairs DSBs is Rad51. Increasing concentrations of human Rad51. HsRadSl . localize to the nucleus after exposure to DNA damaging agents which also suggests a repair function (Terasawa et al. 1995), and HsRad51 forms filaments on DNA when HsRadS 1 bind to ssDNA which demonstrates a potential for strand exchange during DSB repair by homologous recombination or gene conversion (Benson et al.. 1994. EMBO 75:5764-71 ). Moreover, mouse cells with a radSl mutation, designated rad5X" . display features that are known to be characteristic of unrepaired DSB in yeast cells such as reduced proliferation, hypersensitivity to γ-radiation, chromosome loss, and programmed cell death. Further evidence of Rad51's essential role in the body is provided by the observation that homozygous Rad51 knockout mice display an embryonic lethal phenotype.

3. SUMMARY OF THE INVENTION

An object of the present invention is to hinder cell proliferation or reduce cell viability by disrupting mammalian RadS l function.

An additional object is to hinder cell proliferation or reduce cell viability by disrupting mammalian Rad52 function. Another object of the present invention is to hinder cell proliferation or reduce cell viability by disrupting proteins that associate with mammalian Rad51.

Another object of the present invention is to hinder cell proliferation or reduce cell viability by disrupting proteins that associate with mammalian Rad52.

Another object of the present invention is to hinder cell proliferation or reduce cell viability by disrupting any proteins involved in the mammalian Rad51 or mammalian

Rad52 pathways.

Another object of the present invention is to hinder cell proliferation or reduce cell viability by disrupting mammalian Rad51 protein interactions.

Another object of the present invention is to hinder cell proliferation or reduce cell viability by disrupting mammalian Rad52 protein interactions.

Another object of the present invention is to hinder cell proliferation or reduce cell viability by disrupting protein-protein interactions that are involved in the mammalian Rad51 or mammalian Rad52 pathways.

Yet another embodiment of the present invention involves methods of identifying compounds that are capable of inhibiting the binding or function of any protein involved in the Rad51 pathway, and. in particular, compounds capable of binding or inhibiting the function of Rad51 protein. Accordingly, an additional embodiment of the present invention involves methods of screening for compounds that disrupt double-stranded break repair by: assaying for decreased cell proliferation: assaying for microsatellite formation in cells; assaying for chromosome loss in cells: assaying for the disruption of strand exchange, etc.. in an in vitro assay; assaying for premature replicative cellular senescence; and/or assaying for increased cell death.

Another object of the invention is to identify compounds capable of interfering with protein-protein interactions involved in DSB repair by screening large numbers of compounds in assays that allow the detection of a decrease in protein-protein interactions.

In a further object of the invention, structural analysis of proteins, peptides. and compounds useful for modulating DSB repair is used to improve the modulation of DSB repair by new or known proteins, peptides. and compounds.

Additional objects of the present invention include compounds that hinder cell proliferation or reduce cell viability by disrupting mammalian Rad51 function.

Additional object of the present invention include compounds that hinder cell proliferation or reduce cell viability by disrupting mammalian Rad52 function.

Other object of the invention are methods to target the compounds of the invention to particular tissues or cells in an animal, including a human. These compounds, in a preferred embodiment, are peptides or polypeptides that hinder cell proliferation or reduce cell viability by disrupting mammalian Rad51 function or by disrupting mammalian Rad52 function. A compound of the invention is targeted to a tissue or cell, in one aspect, by linking the compound to another molecule, i.e.. a targeting agent, which preferably binds to, or localizes in. a tissue or cell of interest. A targeting agent to which the compound of the invention may be linked is. in one aspect, an antibody, a monoclonal antibody, an antibody fragment, a ligand. an agonist, an antagonist, etc.

Additional objects of the present invention are complexes of compounds of the present invention, for example, peptides or polypeptides of the invention, and a targeting agent, for example, an antibody, a monoclonal antibody, an antibody fragment, a ligand, an agonist, an antagonist, etc.

4. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1. mRNA structure of \fmRAD51. The predicted amino acids of the mammalian mRNA for Rαd51 are numbered according to Shinohara et αi. 1993. The shaded box represents the recA homology region. The open boxes represent regions that are not conserved across species. The thick vertical lines represent the ATP binding domains. Figure 2. MmRadS 1 self-association as demonstrated by the yeast two-hybrid system. The self-association is restricted to the most N-terminal region (e.g.. amino acids 1- 131). The shaded box is the RecA core homology region (Shinohara et al, 1993). The thick vertical lines represent the ATP-binding sites. The open boxes represent regions that are not conserved between species. The relative β-galactosidase (β-gal) activities are presented, right panel. Full length wild-type MmRadS 1 is considered to be 100%. E12 served as a negative control and had 1% relative activity.

Figures 3A-3B. Disruption of mammalian RadS l function in cells. A) Conditional expression of mammalian RadS l 1-43 in ES cells increases sensitivity to gamma-radiation. Transgene turned on without Dox and turned off with Dox. Ten clones were observed and the averages are presented. B) Brca2 peptide decreases proliferation of p53-/- fibroblasts. 50 micromolar concentration used. Colony size is based on cell number. The average of two experiments is shown when 100 cells were plated onto a 6 cm plate. The control is either no peptide or the 16 amino acid peptide derived from Antennapedia.

5. DETAILED DESCRIPTION OF THE INVENTION

As discussed above, one embodiment of the present invention is the expression of altered mammalian radSl alleles that disrupt mammalian Rad51 function, mammalian Rad52 function, or the function of any other protein in the mammalian Rad51 or Rad52 pathways. The function of MmRadS 1 is not entireh' known; however, it is likely that it has the same function as ScRad51 which is recombinational repair. The recombinational repair pathway appears to be at least partially conserved between yeast and mammals. Mammalian homologues were found for members of the Rad52 epistasis group (Rad51, Rad52) and to other yeast proteins (Dmcl) implicated in recombinational repair (Malkova et al, 1996. Proc. Natl. Acad. Sci. USA 95:7131 -36: Resnick et al.. 1989, Proc. Natl. Acad. Sci. USA 5(5:2276-80; Tsuzuki et al.. 1996. Proc. Natl. Acad. Sci USA 95:6236-40).

The most compelling evidence that MmRadS 1 and ScRad51 function is conserved comes from analysis of rad5 J mutant cells, the mutation was designated rad51 ^ι (Lim and Hasty. 1996). rad5X" cells exhibited reduced proliferation, hypersensitivity to γ-radiation, chromosome loss, and cell death. These characteristics were similar to veast cells deficient for recombinational repair either due to sequence divergence or due to a mutation in rad51 . or rad52.

For the purposes of the present application the term ionizing radiation shall mean all forms of radiation, including but not limited to α. β. and γ radiation and UN. light, which are capable of directly or indirectly damaging the genetic material of a cell or virus. The term irradiation shall mean the exposure of a sample of interest to ionizing radiation, and the term radiosensitive shall refer to cells or individuals which display unusually adverse consequences after receiving moderate, or medically acceptable (i.e., nonlethal diagnostic or therapeutic doses), exposure to ionizing irradiation. MmRad51 may perform a novel role in DΝA replication, repair, or chromosomal disjunction. MmRADSl expression is restricted during the cell cycle to late G,/S/G₂ and MmRADSl expression was activated by mitogens that induced T and B cell proliferation suggesting a role in replication and repair (Yamamoto et al. 1996. 257: 1-12). MmRad51 may take part in disjunction because it localizes to the kinetochores of diakinesis, and metaphase 1 chromosomes (Ashley et al. , 1995).

The exact function or functions performed by MmRad51 are unimportant with regard to developing anti-proliferative drugs and cancer therapeutics as long as the disruption of the MmRad51 function provides a benefit to the patient. For the purposes of the present invention, it is assumed that the function of RadS l is the repair of DSB; however, it is likely that Rad51 performs additional functions in the cell. However, it is important to note that at least some feature of MmRad51 function is essential for cell proliferation and/or viability, and that molecules capable of disrupting MmRad51 function thus hinder cell proliferation or reduce cell viability. As such, any molecule that disrupts the MmRad51 pathway should prove useful for cancer therapy (for example). Furthermore, disruption of any protein-protein interaction that involves either MmRadS 1 or any other molecule in the MmRadS 1 pathway should also prove useful for cancer therapy.

Although the presently described invention has been specifically exemplified using a species exemplary of the order mammalia, given the relatively high level of interspecies sequence similarity (and functional similarity) observed in the Rad51 proteins, it is clear that the present invention may be broadly applied to other mammalian species, including humans, as well as non-mammalian animals such as birds, and fish. In addition to mice, examples of mammalian species that may be used in the practice of the present invention include, but are not limited to. humans, non-human primates (such as chimpanzees), pigs, rats (or other rodents), rabbits, cattle, goats, sheep, canines, felines, and guinea pigs. Given the critical importance of mammalian RadSl function, any disruption of the mammalian Rad51 or Rad52 complexes, or any member in their pathway will necessarily hinder cell proliferation or viability. When the RadS 1 and Rad52 pathways were disrupted by introducing altered mouse rad51 into mouse cells, nonproductive protein-protein associations resulted. The altered forms of mouse rad51 were generated by disrupting a conserved nucleotide binding motif while preserving the protein association domain. The expression of these transgenes resulted in cellular toxicity. Presumably, the resulting nonproductive protein associations were responsible for the drastically reduced viability of these cells. In view of this result, it is clear that one may reduce cell proliferation by disrupting mammalian Rad51 function, or the function of any protein in the Rad51 repair pathway by hindering protein association by using defective proteins or other means such as small molecules.

5.1. DSB-Re ated Proteins, Polypeptides And Nucleic Acids

Proteins and nucleic acids (sense and antisense) that are involved with DSB repair can be utilized as part of the therapeutic, diagnostic, prognostic and screening methods of the present invention. These proteins and nucleic acids are referred to herein as DSB- related proteins and nucleic acids. DSB-related proteins, polypeptides and peptide fragments, and mutated, truncated or deleted forms of a DSB-related fusion protein product (such as Rad51-Ig fusion proteins, that is. fusions of Rad51 to an IgFc domain) can be utilized.

For example, peptides and polypeptides corresponding to RadS l . truncated or deleted Rad51s. as well as fusion proteins in which the full length RadS l or a Rad51 peptide or truncated Rad51 is fused to a heterologous. unrelated protein are also within the scope of the invention and can be utilized and designed on the basis of such Rad51 nucleotide and amino acid sequences which are shown herein or known to those of skill in the art. The mammalian Rad51 amino acid sequence is shown at SEQ ID NO: l . Similarly, the Rad52 amino acid sequence is shown at SEQ ID NO:2.

Fusion proteins include, but are not limited to. IgFc fusions which stabilize the DSB-related protein or peptide (e.g. RadS l ) and prolong half-life in vivo; or fusions to any amino acid sequence that allows the fusion protein to be anchored to the cell membrane; or fusions to an enzyme, fluorescent protein, or luminescent protein which provide a marker or reporter function, useful e.g, in screening and/or diagnostic methods of the invention. The proteins and peptides which may be used in the methods of the invention include synthetic (e.g.. recombinant or chemically synthesized) proteins and peptides, as well as naturally occurring proteins and peptides. The proteins and peptides may have both naturally occurring and non-naturally occurring amino acid residues (e.g., D-amino acid residues) and/or one or more non-peptide bonds (e.g., imino. ester, hydrazide, semicarbazide, and azo bonds). The proteins or peptides may also contain additional chemical groups (i.e., functional groups) present at the amino and/or carboxy termini, such that, for example, the stability, bioavailability. and/or inhibitory activity of the peptide is enhanced. Exemplary functional groups include hydrophobic groups (e.g. carbobenzoxyl, dansyl, and t-butyloxycarbonyl, groups), an acetyl group, a 9-fluorenylmethoxy-carbonyl group and macromolecular carrier groups (e.g.. lipid-fatty acid conjugates, polyethylene glycol. or carbohydrates) including peptide groups. Additional proteins and peptides which may be used in the methods of the invention include those described in WO 99/59615, which is herein incorporated by reference in its entirety.

While the DSB-related polypeptides and peptides can be chemically synthesized (e.g., see Creighton. 1983, Proteins: Structures and Molecular Principles, W. H. Freeman & Co., N.Y.), large polypeptides derived from DSB-related polypeptides and peptides may advantageously be produced by recombinant DNA technology using techniques well known in the art for expressing nucleic acid containing DSB-related gene sequences and/or coding sequences. DSB-related encoding polynucleotides does not refer only to sequences encoding open reading frames, but also to upstream and downstream sequences within the DSB-related gene (e.g. RadS l). Such methods also can be used to construct expression vectors containing the Rad51 nucleotide sequences and nucleotide sequences encoding other proteins involved in DSB repair. These methods include, for example, in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. See, Sambrook el al.. 1989. Molecular Cloning. A Laboratory Manual. Second Edition. Cold Spring Harbor Press, N.Y.. and Ausabel et al.. 1989. Current Protocols in Molecular Biology, Green Publishing Associates and Wiley Interscience. N.Y.. each of which is incorporated herein by reference in its entirety. Alternatively. RNA capable of encoding

Rad51 nucleotide sequences and nucleotide sequences encoding other proteins involved in DSB repair may be chemically synthesized using, for example, synthesizers. See, for example, the techniques described in "Oligonucleotide Synthesis", 1984, Gait, M. J. ed., IRL Press, Oxford, which is incorporated by reference herein in its entirety. An additional embodiment of the invention relates to nucleic acid sequences that are capable of hybridizing to sequences encoding the proteins that are associated with DSB repair. Such nucleic acid sequences include, but are not limited to, (a) any nucleic acid which hybridizes to a nucleic acid molecule of the invention under moderately stringent conditions, e.g., hybridization to filter-bound DNA in 6x sodium chloride/sodium citrate (SSC) at about 45°C followed by one or more washes in 0.2xSSC/0.1% SDS at about 50-

65°C, or (b) under highly stringent conditions, e.g., hybridization to filter-bound nucleic acid in 6xSSC at about 45°C followed by one or more washes in O.lxSSC/0.2% SDS at about 68°C, or under other hybridization conditions which are apparent to those of skill in the art (see, for example, Ausubel F.M. et al.. eds.. 1989, Current Protocols in Molecular Biology, Vol. I. Green Publishing Associates. Inc.. and John Wiley & sons. Inc.. New York, at pp. 6.3.1-6.3.6 and 2.10.3).

Among the nucleic acid molecules of the invention are deoxyoligonucleotides ("oligos") which hybridize under highly stringent or moderately stringent conditions to the nucleic acid molecules described above. In general, for probes between 14 and 70 nucleotides in length the melting temperature (TM) is calculated using the formula: Tm

(°C)=81.5+16.6(log[monovalent cations (molar)])+0.41 (% G+C)-(500/N) where N is the length of the probe. If the hybridization is carried out in a solution containing formamide, the melting temperature is calculated using the equation Tm (°C)=81.5+16.6(log[monovalent cations (molar)])+0.41(% G+C)-(0.61% formamide)- (500/N) where N is the length of the probe. In general, hybridization is carried out at about 20-25 degrees below Tm (for DNA-DNA hybrids) or 10- 15 degrees below Tm (for RNA- DNA hybrids).

Exemplary highly stringent conditions may refer, e.g.. to washing in 6xSSC/0.05% sodium pyrophosphate at 37°C (for about 14-base oligos), 48 °C (for about 17-base oligos), 55 °C (for about 20-base oligos) and 60 "C (for about 23-base oligos).

The invention also encompasses (a) DNA vectors which contain any of the foregoing coding sequences and/or their complements (i.e.. antisense); (b) DNA expression vectors which contain any of the foregoing coding sequences operatively associated with a regulatory element which directs the expression of the coding sequences; and (c) genetically engineered host cells which contain such vectors or have been engineered to contain and/or express a nucleic acid sequence of the invention, e.g., any of the foregoing coding sequences operatively associated with a regulatory element which directs the expression of the coding sequences in the host cell. As used herein, regulatory elements include but are not limited to inducible and non-inducible promoters, enhancers, operators and other elements known to those skilled in the art which drive and regulate expression. The invention further includes fragments of any of the DNA sequences disclosed herein.

The nucleic acid molecules may encode or act as antisense molecules, useful, for example, in sequence regulation, and/or as hybridization probes and/or as primers in amplification reactions of nucleic acid sequences. Further, such sequences may be used as part of ribozyme and/or triple helix sequences, also useful for sequence regulation. Still further, such molecules may be used as components of diagnostic methods whereby, for example, the presence of a particular allele involved in a condition, disorder, or disease involving cell death may be detected.

As used herein, the terms "gene" and "recombinant gene" refer to nucleic acid molecules comprising any of up to six open reading frames which may or may not encode a polypeptide of the invention. The term can further include nucleic acid molecules comprising upstream and/or exon/intron sequences and structures.

A variety of host-expression vector systems may be utilized to express the nucleotide sequences of the invention. Where the peptide or polypeptide of the invention is a soluble derivative, the peptide or polypeptide can be recovered from the culture, ie.. from the host cell in cases where the peptide or polypeptide is not secreted, and from the culture media in cases where the peptide or polypeptide is secreted by the cells. However, the expression systems also encompass engineered host cells that express nucleotide sequences involved in DSB repair, or functional equivalents in situ, i.e., anchored in the cell membrane. Purification or enrichment of the expressed protein products from such expression systems can be accomplished using appropriate detergents and lipid micelles and methods well known to those skilled in the art. However, such engineered host cells themselves may be used in situations where it is important not only to retain the structural and functional characteristics of the expressed recombinant protein, but to assess biological activity, e.g., in drug screening assays. Once purified, the proteins of interest may be partially sequenced. and these data may be used to design degenerate oligonucleotide probes for use in cloning the genes encoding the various proteins that are associated with DSB repair. Alternatively, any of a variety of public or private sequence data bases may be searched for nucleic acid or peptide sequences that share homology with genes and proteins associated with Rad51 -mediated DSB repair. Once a similar sequence is identified, peptides may be produced and screened for inhibitory activity. Where a nucleic acid library is involved, one could synthesize a probe corresponding to the nucleic acid sequence of interest, and use the probe to clone a full-length version of the corresponding gene (if necessary). Accordingly, an additional embodiment of the presently claimed invention are nucleic acid sequences that are capable of hybridizing to sequences encoding the proteins that are associated with DSB repair under stringent conditions. For the purposes of the present invention, the term "stringent conditions" generally refers to hybridization conditions that (1) employ low ionic strength and high temperature for washing, for example. 0.015 M NaCl/0.0015 M sodium citrate/0.1% SDS at 50°C; or (2) employ during hybridization a denaturing agent such as formamide. for example, 50% (vol/vol) formamide with 0.1 % bovine serum albumin/0.1 %

Ficoll/0.1% polyvinylpyrrolidone/50 mM sodium phosphate buffer at pH 6.5 with 750 mM NaCI, 75 mM sodium citrate at 42 °C; or (3) employ 50% formamide. 5 x SSC (0.75 M NaCI, 0.075 M Sodium pyrophosphate. 5 x Denhardt's solution, sonicated salmon sperm DNA (50 g/ml). 0.1% SDS. and 10% dextran sulfate at 42°C. with washes at 42°C in 0.2 x SSC and 0.1% SDS. The above examples of hybridization conditions are merely provided for purposes of exemplification and not limitation. A more thorough treatise of the such routine molecular biology techniques may be found in Sambrook et al.. Molecular Cloning, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York. Vols. 1-3: (1989), and periodic updates thereof, herein incorporated by reference.

The expression systems that may be used for purposes of the invention include, but are not limited to. microorganisms such as bacteria (e.g. , E. coli. B. subtilis) transformed with recombinant bacteriophage DNA. plasmid DNA or cosmid DNA expression vectors containing nucleotide sequences involved in DSB-repair; yeast (e.g., Saccharomyces. Pichia) transformed with recombinant yeast expression vectors containing the nucleotide sequences; insect cell systems infected with recombinant virus expression vectors (e.g., baculovirus) containing the sequences: plant cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus. CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing the nucleotide sequences; or mammalian cell systems (e.g.. COS. CHO, BHK. 293, 3T3) harboring recombinant expression constructs containing promoters derived from the genome of mammalian cells (e.g., metallothionein promoter) or from mammalian viruses

(e.g., the adenovirus late promoter; the vaccinia virus 7.5K promoter).

In bacterial systems, a number of expression vectors may be advantageously selected depending upon the use intended for the gene product being expressed. For example, when a large quantity of such a protein is to be produced, for the generation of pharmaceutical compositions of protein or for raising antibodies to protein, for example, vectors which direct the expression of high levels of fusion protein products that are readily purified may be desirable. Such vectors include, but are not limited to. the E. coli expression vector pUR278 (Ruther et al.. 1983. EMBO J. 2: 1791), in which the coding sequence for the protein involved in DSB repair may be ligated individually into the vector in frame with the lacZ coding region so that a fusion protein is produced; pIN vectors

(Inouye & Inouye, 1985. Nucleic Acids Res. 13:3101-3109; Van Heeke & Schuster. 1989, J. Biol. Chem. 264:5503-5509); and the like. pGEX vectors may also be used to express foreign polypeptides as fusion proteins with glutathione S-transferase (GST). In general, such fusion proteins are soluble and can easily be purified from lysed cells by adsorption to glutathione-agarose beads followed by elution in the presence of free glutathione. The

PGEX vectors are designed to include thrombin or factor Xa protease cleavage sites so that the cloned target gene product can be released from the GST moiety.

In an insect system. Autographa californica nuclear polyhidrosis virus (AcNPV) is used as a vector to express foreign genes. The virus grows in Spodoptera frugiperda cells. The coding sequence for the protein involved in DSB repair may be cloned individually into non-essential regions (for example the polyhedrin gene) of the virus and placed under control of an AcNPV promoter (for example the polyhedrin promoter). Successful insertion of a gene coding sequence will result in inactivation of the polyhedrin gene and production of non-occluded recombinant virus, (/^' e.. virus lacking the proteinaceous coat coded for by the polyhedrin gene). These recombinant viruses are then used to infect Spodoptera frugiperda cells in which the inserted gene is expressed. (E.g.. see Smith et al., 1983, J.

Virol. 46: 584: Smith, U.S. Pat. No. 4.215.051 ).

In mammalian host cells, a number of viral-based expression systems may be utilized. In cases where an adenovirus is used as an expression vector, the nucleotide sequence of interest may be ligated to an adenovirus transcription/translation control complex, e.g., the late promoter and tripartite leader sequence. This chimeric gene may then be inserted in the adenovirus genome by in vitro or in vivo recombination. Insertion in a non-essential region of the viral genome (e.g.. region El or E3) will result in a recombinant virus that is viable and capable of expressing the Rad51 gene product in infected hosts. (E.g., See Logan & Shenk. 1984, Proc. Natl. Acad. Sci. USA 81 :3655-3659). Specific initiation signals may also be required for efficient translation of inserted nucleotide sequences. These signals include the ATG initiation codon and adjacent sequences. In cases where entire genes or cDNAs. including their own initiation codons and adjacent sequences, are inserted into the appropriate expression vector, no additional translational control signals may be needed. However, in cases where only a portion of the coding sequence is inserted, exogenous translational control signals, including, perhaps, the ATG initiation codon, must be provided. Furthermore, the initiation codon must be in phase with the reading frame of the desired coding sequence to ensure translation of the entire insert. These exogenous translational control signals and initiation codons can be of a variety of origins, both natural and synthetic. The efficiency of expression may be enhanced by the inclusion of appropriate transcription enhancer elements, transcription terminators, etc. (See Bittner et al., 1987. Methods in Enzymol. 153:516-544). In addition, a host cell strain may be chosen which modulates the expression of the inserted sequences, or modifies and processes the gene product in the specific fashion desired. Such modifications (e.g.. g cosylation) and processing (e.g.. cleavage) of protein products may be important for the function of the protein. Different host cells have characteristic and specific mechanisms for the post-translational processing and modification of proteins and gene products. Appropriate cell lines or host systems can be chosen to ensure the correct modification and processing of the foreign protein expressed. To this end. eukaryotic host cells which possess the cellular machinery for proper processing of the primary transcript, glycosylation. and phosphorylation of the gene product may be used. Such mammalian host cells include but are not limited to CHO, VERO, BHK,

FleLa, COS. MDCK. 293, 3T3. WI38. and in particular, choroid plexus cell lines.

For long-term, high-yield production of recombinant proteins, stable expression is preferred. For example, cell lines which stably express the Rad51 sequences, or other sequences involved in DSB repair may be engineered. Rather than using expression vectors which contain viral origins of replication, host cells can be transformed with DNA controlled by appropriate expression control elements (e.g., promoter, enhancer sequences, transcription terminators, polyadenylation sites, etc.), and a selectable marker. Following the introduction of the foreign DNA. engineered cells may be allowed to grow for 1-2 days in an enriched media, and then are switched to a selective media. The selectable marker in the recombinant plasmid confers resistance to the selection and allows cells to stably integrate the plasmid into their chromosomes and grow to form foci which in turn can be cloned and expanded into cell lines. This method may advantageously be used to engineer cell lines which express DSB-related gene products. Such engineered cell lines may be particularly useful in screening and evaluation of compounds that affect the endogenous activity of RadSl gene products and other gene products involved in DSB-repair.

A number of selection systems may be used, including but not limited to, the herpes simplex virus thymidine kinase (Wigler. et al.. 1977. Cell 1 1 :223). hypoxanthine-guanine phosphoribosyltransferase (Szybalska & Szybalski. 1962. Proc. Natl. Acad. Sci. USA 48:2026). and adenine phosphoribosyltransferase (Lowy. et al, 1980. Cell 22:817) genes can be employed in tk^", hgprt^" or aprt^" cells, respectively. Also, antimetabolite resistance can be used as the basis of selection for the following genes: dhfr. which confers resistance to methotrexate (Wigler. et al.. 1980. Natl. Acad. Sci. USA 77:3567; O'Hare, et al. 1981, Proc. Natl. Acad. Sci. USA 78:1527); gpt. which confers resistance to mycophenolic acid (Mulligan & Berg, 1981. Proc. Natl. Acad. Sci. USA 78:2072): neo. which confers resistance to the aminoglycoside G-418 (Colberre-Garapin, et al., 1981. J. Mol. Biol. 150:1); and hygro. which confers resistance to hygromycin (Santerre. et al., 1984. Gene

30:147).

The gene products of the invention can also be expressed in transgenic animals. Animals of any species, including, but not limited to. mice. rats, rabbits, guinea pigs, pigs, micro-pigs, goats, and non-human primates, e.g., baboons, monkeys, and chimpanzees may be used to generate the transgenic animals.

Any technique known in the art may be used to introduce the transgene into animals or to "knock-out^" or inactivate endogenous genes to produce the founder lines of transgenic animals. Such animals can be utilized as part of the screening methods of the invention, and cells and/or tissues from such animals can be obtained for generation of additional compositions (e.g., cell lines, tissue culture systems) that can also be utilized as part of the screening methods of the invention.

Techniques for generation of such animals are well known to those of skill in the art and include, but are not limited to, pronuclear microinjection (Hoppe. P. C. and Wagner, T. E., 1989. U.S. Pat. No. 4,873.191); retrovirus mediated gene transfer into germ lines (Van der Putten et α/.. 1985, Proc. Natl. Acad. Sci.. USA 82:6148-6152); gene targeting in embryonic stem cells (Thompson et al.. 1989. Cell 56:313-321); electroporafion of embryos (Lo, 1983. Mol Cell. Biol. 3: 1803-1814); and sperm-mediated gene transfer (Lavitrano et al., 1989. Cell 57:717-723): etc. For a review of such techniques, see Gordon, 1989, Transgenic Animals. Intl. Rev. Cytol. 1 15:171-229. which is incorporated by reference herein in its entirety.

With respect to transgenic animals containing a DSB-related transgene (e.g. Rad51), such animals can carry the transgene in all their cells. Alternatively, such animals can carry the transgene or transgenes in some, but not all their cells, i.e.. mosaic animals. The transgene may be integrated as a single transgene or in concatamers, e.g.. head-to-head tandems or head-to-tail tandems. The transgene may also be selectively introduced into and activated in a particular cell type by following, for example, the teaching of Lasko et al. (Lasko. M. et al., 1992. Proc. Natl.Acad. Sci. USA 89: 6232-6236). The regulatory- sequences required for such a cell-type specific activation will depend upon the particular cell type of interest, and will be apparent to those of skill in the art. When it is desired that the transgene be integrated into the chromosomal site of the endogenous gene, gene targeting is preferred. Briefly, when such a technique is to be utilized, vectors containing some nucleotide sequences homologous to the endogenous gene are designed for the purpose of integrating, via homologous recombination with chromosomal sequences, into and disrupting the function of the nucleotide sequence of the endogenous gene, respectively. The transgene may also be selectively introduced into a particular cell type, thus inactivating the endogenous Rad51 gene in only that cell type, by following, for example, the teaching of Gu et al. (Gu, et al, 1994. Science 265: 103-106). The regulatory sequences required for such a cell-type specific inactivation will depend upon the particular cell type of interest, and will be apparent to those of skill in the art.

Once transgenic animals have been generated, the expression of the recombinant gene may be assayed utilizing standard techniques. Initial screening may be accomplished by Southern blot analysis or PCR techniques to analyze animal tissues to assay whether integration of the transgene has taken place. The level of mRNA expression of the transgene in the tissues of the transgenic animals may also be assessed using techniques which include, but are not limited to, Northern blot analysis of tissue samples obtained from the animal, in situ hybridization analysis, and RT-PCR. Samples of DSB-related gene-expressing tissue may also be evaluated immunocytochemically using antibodies specific for the transgene product.

5.1.1. Antibodies to RadSl Proteins Purified preparations of the presently described DNA repair proteins, associated proteins, or fragments thereof, may be used to generate antisera specific for a given agent. Accordingly, additional embodiments of the present invention include polyclonal and monoclonal antibodies that recognize epitopes of the presently described DNA repair complex proteins. The factors used to induce the antibodies of interest need not be biologically active; however, the factors should induce immunological activity in the animal used to generate the antibodies. Given that similar methodologies may be applied to the generation of antibodies to the various factors, for purposes of convenience, only the Rad51 factor antibodies will be discussed further.

Polypeptides for use in the induction of RadS l -specific antibodies may have an amino acid sequence consisting of at least three amino acids, preferably at least about 5 amino acids, and more preferably at least about 10 amino acids that mimic a portion of the amino acid sequence of Rad51. and may contain the entire amino acid sequence of naturally occurring RadS 1 or a Rad51 -derivative.

Anti-RadS 1 antibodies are expected to have a variety of medically useful applications, several of which are described generally below. More detailed and specific descriptions of various uses for anti-RadS 1 antibodies are provided in the sections and subsections which follow. Briefly, anti-Rad51 antibodies may be used for the detection and quantification of Rad51 polypeptide expression in cultured cells, tissue samples, and in vivo. Such immunological detection of RadS 1 may be used, for example, to identify, monitor, and assist in the prognosis of neoplasms that have been treated with factors that inhibit DSB repair. Additionally, monoclonal antibodies recognizing epitopes from different parts of the Rad51 structure may be used to detect and/or distinguish between native Rad51 and various subcomponent and/or mutant forms of the molecule. Additionally, anti-Rad51 monoclonal antibodies may be used to test preparations of agents or factors that mimic segments of RadS 1. or are designed to impair protein association with

Rad51, or to competitively inhibit DNA binding. In addition to the various diagnostic and therapeutic utilities of anti-Rad51 antibodies, a number of industrial and research applications will be obvious to those skilled in the art. including, for example, the use of anti-Rad51 antibodies as affinity reagents for the isolation of Rad51 -associated polypeptides. and as immunological probes for elucidating the biosynthesis, metabolism and biological functions of RadS 1. RadS 1 antibodies may also be used to purify Rad51 or Rad51 -associated factors by affinity chromatography.

Antibodies that specifically recognize and bind to one or more epitopes of Rad51, or epitopes of conserved variants of RadS l . or peptide fragments of RadS l can be utilized as part of the methods of the present invention. Such antibodies include, but are not limited to, polyclonal antibodies, monoclonal antibodies (mAbs). human, humanized or chimeric antibodies, single chain antibodies. Fab fragments. F(ab')₃ fragments, fragments produced by a Fab expression library, anti-idiotypic (anti-Id) antibodies and epitope-binding fragments of any of the above.

Such antibodies may be used, for example, as part of the diagnostic or prognostic methods of the invention by measuring Rad51 levels in the mammal. Such antibodies may also be utilized in conjunction with, for example, compound screening schemes, as described below, for the evaluation of the effect of test compounds on expression and/or activity of the RadSl gene product. Additionally, such antibodies can be used in therapeutic and preventative methods of the invention. For example, such antibodies can correspond to Rad51 receptor agonists or antagonists. Further, such antibodies can be administered to lower RadSl levels. In addition, such antibodies can be utilized to lower Rad51 levels by increasing the rate at which Rad51 is removed from circulation (e.g.. can speed Rad51 breakdown).

For the production of antibodies, various host animals may be immunized by injection with RadSl. a Rad51 peptide. truncated RadS l polypeptides, functional equivalents of Rad51 or mutants of Rad51. Such host animals may include, but are not limited to. rabbits, mice, and rats, to name but a few. Various adjuvants may be used to increase the immunological response, depending on the host species, including but not limited to. Freund's (complete and incomplete), mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin. pluronic polyols. polyanions, peptides, oil emulsions, keyhole limpet hemocyanin. dinitrophenol. and potentially useful human adjuvants such as BCG (bacille Calmette-Guerin) and Corvnebacterium parvum. Polyclonal antibodies are heterogeneous populations of antibody molecules derived from the sera of the immunized animals. Monoclonal antibodies, which are homogeneous populations of antibodies to a particular antigen, may be obtained by any technique which provides for the production of antibody molecules by continuous cell lines in culture. These include, but are not limited to, the hybridoma technique of Kohler and Milstein. (1975. Nature 256:495-497; and U.S. Pat. No. 4,376.1 10). the human B-cell hybridoma technique (Kosbor et al.. 1983, Immunology Today 4:72: Cole et al.. 1983. Proc. Natl. Acad. Sci. USA 80:2026-2030). and the

EBV-hybridoma technique (Cole et al. 1985. Monoclonal Antibodies And Cancer Therapy, Alan R. Liss. Inc.. pp. 77-96). Such antibodies may be of any immunoglobulin class including IgG. IgM. IgE. IgA. IgD and any subclass thereof. The hybridoma producing the mAb of this in\ ention may be cultivated in vitro or in vivo. Production of high titers of mAbs in vivo makes this the presently preferred method of production. Additionally, recombinant antibodies, such as chimeric and humanized monoclonal antibodies, comprising both human and non-human portions, which can be made using standard recombinant DNA techniques, are within the scope of the invention. A chimeric antibody is a molecule in which different portions are derived from different animal species, such as those having a variable region derived from a murine mAb and a human immunoglobulin constant region. (See. e.g.. Cabilly et al.. U.S. Patent No. 4.816,567; and

Boss et al., U.S. Patent No. 4.816397, which are incorporated herein by reference in their entirety.) Humanized antibodies are antibody molecules from non-human species having one or more complementarily determining regions (CDRs) from the non-human species and a framework region from a human immunoglobulin molecule. (See, e.g., Queen. U.S. Patent No. 5,585.089, which is incorporated herein by reference in its entirety.) Such chimeric and humanized monoclonal antibodies can be produced by recombinant DNA techniques known in the art, for example using methods described in PCT Publication No. WO 87/02671 ; European Patent Application 184.187; European Patent Application 171 ,496; European Patent Application 173.494: PCT Publication No. WO 86/01533; U.S. Patent No. 4,816.567; European Patent Application 125.023; Better et al. (1988) Science

240: 1041 -1043: Liu et al. ( 1987) Proc. Xatl. Acad. Sci. USA 84:3439-3443; Liu et al. (1987) J. Immunol. 139:3521 -3526: Sun et al. (1987) Proc. Xatl. Acad. Sci. USA 84:214-218; Nishimura et al. (1987) Cane. Res. 47:999-1005: Wood et al. (1985) Nature 314:446-449; and Shaw et al. (1988) J. Xatl. Cancer lnst. 80: 1553-1559); Morrison (1985) Science 229: 1202-1207; Oi et al. ( 1986) Bio/Techniques 4:214: U.S. Patent 5.225,539;

Jones et al. (1986) Nature 321 :552-525; Verhoeyan et al. (1988) Science 239:1534; and Beidler et α/. (1988) J. Immunol. 141 :4053-4060.

Completely human antibodies are particularly desirable for therapeutic treatment of human patients. Such antibodies can be produced, for example, using transgenic mice which are incapable of expressing endogenous immunoglobulin heavy and light chains genes, but which can express human heavy and light chain genes. The transgenic mice are immunized in the normal fashion with a selected antigen, e.g.. all or a portion of a polypeptide of the invention. Monoclonal antibodies directed against the antigen can be obtained using conventional hybridoma technology. The human immunoglobulin transgenes harbored by the transgenic mice rearrange during B cell differentiation, and subsequently undergo class switching and somatic mutation. Thus, using such a technique, it is possible to produce therapeutically useful IgG. IgA and IgE antibodies. For an overview of this technology for producing human antibodies, see Lonberg and Huszar (1995, Int. Rev. Immunol. 13:65-93). For a detailed discussion of this technology for producing human antibodies and human monoclonal antibodies and protocols for producing such antibodies, see, e.g., U.S. Patent 5.625.126; U.S. Patent 5.633,425; U.S. Patent

5.569,825; U.S. Patent 5.661.016; and U.S. Patent 5.545,806. In addition, companies such as Abgenix, Inc. (Fremont. CA), can be engaged to provide human antibodies directed against a selected antigen using technology similar to that described above.

Completely human antibodies which recognize a selected epitope can be generated using a technique referred to as "guided selection." In this approach a selected non-human monoclonal antibody, e.g., a mouse antibody, is used to guide the selection of a completely human antibody recognizing the same epitope. (Jespers et al. (1994) Bio/technology 12:899-903).

Alternatively, techniques described for the production of single chain antibodies (U.S. Pat. No. 4.946,778; Bird, 1988. Science 242:423-426; Huston et al., 1988, Proc. Natl.

Acad. Sci. USA 85:5879-5883; and Ward et al.. 1989. Nature 334:544-546) can be adapted to produce single chain antibodies against RadSl gene products. Single chain antibodies are formed by linking the heavy and light chain fragments of the Fv region via an amino acid bridge, resulting in a single chain polypeptide. Antibody fragments which recognize specific epitopes may be generated by known techniques. For example, such fragments include, but are not limited to: the F(ab')₂ fragments which can be produced by pepsin digestion of the antibody molecule and the Fab fragments which can be generated by reducing the disulfide bridges of the F(ab')₂ fragments. Alternatively. Fab expression libraries may be constructed (Huse et al.. 1989. Science, 246:1275-1281) to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity. Antibodies to RadS l can. in turn, be utilized to generate anti-idiotype antibodies that "mimic" RadS l . using techniques well known to those skilled in the art. (See. e.g., Greenspan & Bona. 1993. FASEB J 7(5):437-444; and Nissinoff. 1991. J. Immunol. 147(8):2429-2438). Such neutralizing anti-idiotypes or Fab fragments of such anti-idiotypes can be used in therapeutic regimens to neutralize RadSl and treat hyperproliferative disorders.

5.2 Screening Assays

Given that an important aspect of DSB repair is the interaction of different proteins, additional aspects of the invention include the use of screening assays to detect interactions, or the lack of such interactions, of proteins involved in DSB repair.

The following assays are designed to identify compounds that interact with (e.g., bind to) proteins involved in DSB repair. The compounds which may be screened in accordance with the invention include but are not limited to peptides, antibodies and fragments thereof, prostaglandins. lipids and other organic compounds (e.g., terpines, peptidomimetics) that bind to or mimic the activity triggered by a natural ligand (i.e., agonists) or inhibit the activity triggered by a natural ligand (i.e., antagonists) of a protein involved in DSB repair: as well as peptides. antibodies or fragments thereof, and other organic compounds that mimic the natural ligand for a given protein involved in DSB repair.

Such compounds may include, but are not limited to. peptides such as. for example, soluble peptides. including but not limited to members of random peptide libraries (see, e.g., Lam. K.S. et al.. 1991, Nature. 557:82-84; Houghten. R. et al., 1991, Nature, 55 :84-86), and combinatorial chemistry-derived molecular library peptides made of D- and/or L- configuration amino acids, phosphopeptides (including, but not limited to members of random or partially degenerate, directed phosphopepfide libraries; see. e.g., Songyang, Z. et al., 1993. Cell, 72:767-778); antibodies (including, but not limited to. polyclonal, monoclonal, human, humanized, anti-idiotypic. chimeric or single chain antibodies, and FAb, F(ab)₂ and FAb expression library fragments, and epitope-binding fragments thereof): and small organic or inorganic molecules.

Other compounds which can be screened in accordance with the invention include but are not limited to small organic molecules that are able to gain entry into an appropriate cell and affect DSB repair by. for example, modulating protein-protein interactions important for DSB repair (e.g.. by interacting with a protein involved in DSB repair); or such compounds that affect the activity of a gene encoding a protein involved in DSB repair.

Computer modeling and searching technologies permit identification of compounds, or the improvement of already identified compounds, that can modulate DSB repair by, for example, modulating protein-protein interactions involved in DSB repair. Having identified such a compound or composition, the active sites or regions are identified. Such active sites might typically be the binding partner sites, such as. for example, the interaction domains of a protein important for DSB repair with its cognate ligand. The active site can be identified using methods known in the art including, for example, from the amino acid sequences of peptides. from the nucleotide sequences of nucleic acids, or from study of complexes of the relevant compound or composition with its natural ligand. In the latter case, chemical or X- ray crystallographic methods can be used to find the active site by finding where on the factor the complexed ligand is found.

Next, the three dimensional geometric structure of the active site is determined. This can be done by known methods, including X-ray crystallography, which can determine a complete molecular structure. On the other hand, solid or liquid phase NMR can be used to determine certain intra-molecular distances. Any other experimental method of structure determination can be used to obtain partial or complete geometric structures. The geometric structures may be measured with a complexed ligand. natural or artificial, which may increase the accuracy of the active site structure determined.

If an incomplete or insufficiently accurate structure is determined, the methods of computer based numerical modeling can be used to complete the structure or improve its accuracy. Any recognized modeling method may be used, including parameterized models specific to particular biopolymers such as proteins or nucleic acids, molecular dynamics models based on computing molecular motions, statistical mechanics models based on thermal ensembles, or combined models. For most types of models, standard molecular force fields, representing the forces between constituent atoms and groups, are necessary, and can be selected from force fields known in physical chemistry. The incomplete or less accurate experimental structures can serve as constraints on the complete and more accurate structures computed by these modeling methods.

Finally, having determined the structure of the active site, either experimentally, by modeling, or by a combination thereof, candidate modulating compounds can be identified by searching databases containing compounds along with information on their molecular structure. Such a search seeks compounds having structures that match the determined active site structure and that interact with the groups defining the active site. Such a search can be manual, but is preferably computer assisted. The compounds found from such a search generally identify modulating compounds, or genes encoding the same, that are selected for further study or gene targeting.

Alternatively, these methods can be used to identify improved modulating compounds from an already known modulating compound or ligand. The composition of the known compound can be modified and the structural effects of modification can be determined using the experimental and computer modeling methods described above applied to the new composition. The altered structure is then compared to the active site structure of the compound to determine if an improved fit or interaction results. In this manner systematic variations in composition, such as by varying side groups, can be quickly evaluated to obtain modified modulating compounds or ligands of improved specificity or activity. Further experimental and computer modeling methods useful to identify modulating compounds based upon identification of the active sites of regulatory protein interactions, and related transduction factors will be apparent to those of skill in the art.

Representative examples of molecular modeling systems include the CHARMm and QUANTA programs (Polygen Corporation. Waltham. MA). CHARMm performs the energy minimization and molecular dynamics functions. QUANTA performs the construction, graphic modeling and analysis of molecular structure. QUANTA allows interactive construction, modification, visualization, and analysis of the behavior of molecules with each other.

A number of articles review computer modeling of drugs interactive with specific-proteins, such as Rotivinen. et al . 1988. Acta Pharmaceutical Fennica 97: 159-166;

Ripka, New Scientist 54-57 (Jun. 16. 1988); McKinaly and Rossmann. 1989. Annu. Rev.

11 . Pharmacol. Toxiciol. 29: 1 1 1 -122; Perry and Davies. OSAR: Quantitative Structure-Activity Relationships in Drug Design pp. 189-193 (Alan R. Liss. Inc. 1989); Lewis and Dean. 1989 Proc. R. Soc. Lond. 236: 125-140 and 141-162: and. with respect to a model receptor for nucleic acid components. Askew, et al.. 1989. J. Am. Chem. Soc. 1 11 :1082-1090. Other computer programs that screen and graphically depict chemicals are available from companies such as BioDesign. Inc. (Pasadena. Calif). Allelix. Inc. (Mississauga. Ontario, Canada), and Hypercube. Inc. (Cambridge. Ontario). Although these are primarily designed for application to drugs specific to particular proteins, they can be adapted to design of drugs specific to regions of DNA or RNA. once that region is identified. Although described above with reference to design and generation of compounds which could alter binding, one could also screen libraries of known compounds, including natural products or synthetic chemicals, and biologically active materials, including proteins, for compounds which are inhibitors or activators of the proteins and genes that are important for any aspect of DSB repair.

5.2.1 In Vitro Screening Assays In vitro systems can be designed to identify compounds capable of interacting with (e.g., binding to) the regulatory proteins identified using the subject methods. The identified compounds may be useful, for example, in modulating the activity of wild type and/or mutant proteins important for DSB repair. In vitro systems may also be utilized to screen for compounds that disrupt normal interactions important for DSB repair.

The assays used to identify compounds that bind to proteins important for DSB repair involve preparing a reaction mixture of a given protein and the test compound under conditions and for a time sufficient to allow the two components to interact and bind, thus forming a complex which can be removed and/or detected in the reaction mixture. The protein used can vary depending upon the goal of the screening assay. For example, where agonists of the natural ligand are sought, a full length protein, or a fusion protein containing a protein or polypeptide that affords advantages in the assay system (e.g., labeling, isolation of the resulting complex, etc.) can be utilized. The screening assays can be conducted in a variety of ways. For example, one method to conduct such an assay would involve anchoring the protein, polypeptide, peptide or fusion protein or the test substance onto a solid phase and detecting binding between the protein and test compound. In one embodiment of such a method, the protein reactant may be anchored onto a solid surface, and the test compound, which is not anchored, may be labeled, either directly or indirectly. In another embodiment of the method, the test protein is anchored on the solid phase and is complexed with a labeled antibody (and where a monoclonal antibody is used, it is preferably specific for a given region of the protein). Then, a test compound could be assayed for its ability to disrupt the association of the protein/antibody complex.

In practice, microtiter plates, or any modernized iteration thereof, may conveniently be utilized as the solid phase. The anchored component may be immobilized by non- covalent or covalent attachments. Non-covalent attachment may be accomplished by simply coating the solid surface with a solution of the protein and drying. Alternatively, an immobilized antibody, preferably a monoclonal antibody, specific for the protein to be immobilized may be used to anchor the protein to the solid surface. The surfaces may be prepared in advance and stored.

In order to conduct the assay, the nonimmobilized component is added to the coated surface containing the anchored component. After the reaction is complete, unreacted components are removed (e.g., by washing) under conditions such that any complexes formed will remain immobilized on the solid surface. The detection of complexes anchored on the solid surface can be accomplished in a number of ways. Where the previously nonimmobilized component is pre-labeled. the detection of label immobilized on the surface indicates that complexes were formed. Where the previously nonimmobilized component is not pre-labeled. an indirect label can be used to detect complexes anchored on the surface; e.g., using a labeled antibody specific for the previously nonimmobilized component (the antibody, in turn, may be directly labeled or indirectly labeled with a labeled anti-Ig antibody).

Alternatively, a reaction can be conducted in a liquid phase, the reaction products separated from unreacted components, and complexes detected: e.g., using an immobilized antibody specific for the test protein, polypeptide. peptide or fusion protein, or the test compound to anchor any complexes formed in solution, and a labeled antibody specific for the other component of the possible complex to detect anchored complexes. 5.2.2 Screening Assays For Compounds That Interact With Rad51

Protein-protein interactions are critical for recombinational repair in yeast cells, including interactions that involve ScRadS l and ScRad52 (Donovan et al.. 1994; Milne et al, 1993). In addition, the human RadS 1 and Rad52 proteins were shown to associate like their yeast homologues (Shen et α/.. 1996. J. Biol. Chem. 277:148-152).

Any method suitable for detecting protein-protein interactions may be employed for identifying proteins that interact with MmRadS 1. One method which detects protein interactions in vivo, the two-hybrid system, is described in detail for illustration only and not by way of limitation. One version of this system has been described (Chien et al.. 1991,

Proc. Natl. Acad. Sci. USA. 88:9578-9582) and is commercially available from Clontech (Palo Alto. Calif).

Briefly, utilizing such a system, plasmids are constructed that encode two hybrid proteins: one plasmid consists of nucleotides encoding the DNA-binding domain of a transcription activator protein fused to an MmRad51 nucleotide sequence encoding

MmRad51. an MmRad51 polypeptide, peptide or fusion protein, and the other plasmid consists of nucleotides encoding the transcription activator protein's activation domain fused to a cDNA encoding an unknown protein which has been recombined into this plasmid as part of a cDNA library. The DNA-binding domain fusion plasmid and the cDNA library are transformed into a strain of the yeast Saccharomyces cerevisiae that contains a reporter gene (e.g., HBS or lacZ) whose regulatory region contains the transcription activator's binding site. Either hybrid protein alone cannot activate transcription of the reporter gene: the DNA-binding domain hybrid cannot because it does not provide activation function and the activation domain hybrid cannot because it cannot localize to the activator's binding sites. Interaction of the two hybrid proteins reconstitutes the functional activator protein and results in expression of the reporter gene, which is detected by an assay for the reporter gene product.

To isolate proteins that associate with MmRadS 1 , a yeast two-hybrid screen was performed with MmRadS 1 as the "bait" and a T cell library and an embryonic cell library as the "prey". Among other proteins identified using this screen. MmRadS 1 and Brca2 were isolated, and the interactions identified using this screen may prove critical for in vivo

lb function.

Additional biochemical binding assays that can prove useful for identifying compounds that are able to associate with MmRadS 1 (or any other target protein) are well known in the art including, but not limited to equilibrium or membrane flow dialysis, antibody binding assays, gel-shift assays, in vitro binding assays, filter binding assays, enzyme-linked immunoabsorbent assays (ELISA). western blots, co-immunoprecipitation, immunogold co-immunoprecipitation. coimmunolocalization. co-crystallization, fluorescence energy transfer, competition binding assays, chemical crosslinking, and affinity purification. In addition, genetic analysis may be used to identify accessory proteins that interact with MmRadS 1 or are peripherally involved in MmRad51 function.

Where the MmRad51 accessory protein is essential to MmRadS 1 function, mutation in the genes encoding these proteins should typically result in phenotypes similar to those associated with MmRad51 mutations. Similarly, where the MmRad51 accessory proteins function to inhibit or retard MmRadS 1 activity, mutations in the genes encoding these factors shall generally mimic antagonist phenotypes.

When MmRad51 self-association was investigated further, deletion analysis revealed that MmRad51 self-association occurred in the N-terminal region which further demonstrated conservation of function with ScRadS l and RecA since both were shown to self-associate via the N-terminal region of the protein (Donovan et al.. 1994; Horii. 1992; Story et al., 1992, 1993; Tateishi et al.. 1992: Yarranton and Sedgwick. 1982).

5.2.3. Screening Assays For Compounds That Interfere With DSB-Related

Macromolecular Interactions

Macromolecules that interact with a given protein important for DSB repair are referred to, for purposes of this discussion, as "binding partners". Therefore, it is desirable to identify compounds that interfere with or disrupt the interaction with such binding partners which may be useful in modulating DSB repair.

The basic principle of the assay systems used to identify compounds that interfere with the interaction between a protein and its binding partner or partners involves preparing a reaction mixture containing the test protein, polypeptide. peptide or fusion protein as described above, and the binding partner under conditions and for a time sufficient to allow

16 - the two to interact and bind, thus forming a complex. In order to test a compound for inhibitory activity, the reaction mixture is prepared in the presence and absence of the test compound. The test compound may be initially included in the reaction mixture, or may be added at a time subsequent to the addition of the test protein and its binding partner. Control reaction mixtures are incubated without the test compound or with a placebo. The formation of any complexes between the test protein and the binding partner is then detected. The formation of a complex in the control reaction, but not in the reaction mixture containing the test compound, indicates that the compound interferes with the interaction of the test protein and the binding partner. The assay for compounds that interfere with protein binding can be conducted in a heterogeneous or homogeneous format. Heterogeneous assays involve anchoring either the test protein or the binding partner onto a solid phase and detecting complexes anchored on the solid phase at the end of the reaction. In homogeneous assays, the entire reaction is carried out in a liquid phase. The examples below describe similar assays which may be easily modified to screen for compounds which disrupt or enhance the interaction. In either approach, the order of addition of reactants can be varied to obtain different information about the compounds being tested. For example, test compounds that interfere with the interaction by competition can be identified by conducting the reaction in the presence of the test substance; i.e., by adding the test substance to the reaction mixture prior to or simultaneously with the test protein and interactive binding partner. Alternatively, test compounds that disrupt preformed complexes, e.g., compounds with higher binding constants that displace one of the components from the complex, can be tested by adding the test compound to the reaction mixture after complexes have been formed. The various formats are described briefly below. In a heterogeneous assay system, either the test protein, or the interactive binding partner, is anchored onto a solid surface, while the non-anchored species is labeled, either directly or indirectly. In practice, microtiter plates are conveniently utilized. The anchored species may be immobilized by non-covalent or covalent attachments. Non-covalent attachment may be accomplished simply by coating the solid surface with a solution of the test protein or binding partner and drying. Alternatively, an immobilized antibody specific for the species to be anchored may be used to anchor the species to the solid surface. The surfaces may be prepared in advance and stored.

In order to conduct the assay, the partner of the immobilized species is exposed to the coated surface with or without the test compound. After the reaction is complete, unreacted components are removed (e.g.. by washing) and any complexes formed will remain immobilized on the solid surface. The detection of complexes anchored on the solid surface can be accomplished in a number of ways. Where the non-immobilized species is pre-labeled, the detection of label immobilized on the surface indicates that complexes were formed. Where the non-immobilized species is not pre-labeled. an indirect label can be used to detect complexes anchored on the surface: e.g.. using a labeled antibody specific for the initially non-immobilized species (the antibody, in turn, may be directly labeled or indirectly labeled with a labeled anti-Ig antibody). Depending upon the order of addition of reaction components, test compounds which inhibit complex formation or which disrupt preformed complexes can be detected.

Alternatively, the reaction can be conducted in a liquid phase in the presence or absence of the test compound, the reaction products separated from unreacted components, and complexes detected; e.g., using an immobilized antibody specific for one of the binding components to anchor any complexes formed in solution, and a labeled antibody specific for the other partner to detect anchored complexes. Again, depending upon the order of addition of reactants to the liquid phase, test compounds which inhibit complex or which disrupt preformed complexes can be identified.

In an alternate embodiment of the invention, a homogeneous assay can be used. In this approach, a preformed complex of the test protein and the interactive binding partner is prepared in which either protein is labeled, but the signal generated by the label is quenched due to formation of the complex (see. e.g.. U.S. Patent No. 4.109.496 by Rubenstein which utilizes this approach for immunoassays). The addition of a test substance that competes with and displaces one of the species from the preformed complex will result in the generation of a signal above background. In this way. test substances which disrupt the binding interaction can be identified.

For an example of a typical labeling procedure, a test protein or a peptide fragment, e.g., corresponding to the relevant binding domain, can be fused to a glutathione-S- transferase (GST) gene using a fusion vector, such as pGEX-5X-l , in such a manner that its binding activity is maintained in the resulting fusion protein. The interactive binding partner can be labeled with radioactive isotope, for example, by methods routinely practiced in the art. In a heterogeneous assay, e.g.. the GST-fusion protein can be anchored to glutathione-agarose beads. The interactive binding partner can then be added in the presence or absence of the test compound in a manner that allows interaction and binding to occur. At the end of the reaction period, unbound material can be washed away. The interaction between the fusion product and the labeled interactive binding partner can be detected by measuring the amount of radioactivity that remains associated with the glutathione-agarose beads. The successful inhibition of binding by the test compound will result in a decrease in measured radioactivity.

Alternatively, the GST-fusion protein and the labeled interactive binding partner can be mixed together in liquid in the absence of the solid glutathione-agarose beads. The test compound can be added either during or after the species are allowed to interact. This mixture can then be added to the glutathione-agarose beads and unbound material is washed away. Again the extent of binding inhibition can be measured by determining the amount of radioactivity associated with the beads.

In another embodiment of the invention, these same techniques can be employed using peptide fragments that correspond to the binding domains of the test proteins, in place of the full length proteins. Any number of methods routinely practiced in the art can be used to identify and isolate the binding sites. These methods include, but are not limited to, mutagenesis of the gene encoding the protein and screening for disruption of binding in a co-immunoprecipitation assay. Sequence analysis of the gene encoding the protein will reveal the mutations that correspond to the region of the protein involved in interactive binding. The invention encompasses cell-based and animal model-based assays for the identification of compounds exhibiting the ability to alter or correct phenotypes associated with the various genotypes identified and constructed using the present methods. Such cell- based assays can also be used as the standard to assay for purity and potency of the compounds, including recombinantly or synthetically produced proteins or compounds. Given that Rad51 proteins are known to self-associate, the RadS l protein provides a template for the identification and genesis of peptides or factors that disrupt Rad51 function or activity. For the purposes of the present invention a "peptide" is any sequence of at least about three amino acids up to about 100 amino acids. Typically, the peptides of the present invention can encompass enzymatic domains. DNA. RNA. or protein binding domains, or any fragment of a protein or amino acid sequence that directly or indirectly provide the desired function of disrupting cellular RadS 1 or Rad52 activity. Accordingly, an additional embodiment of the present invention are peptides or polypeptides that comprise at least about three, or more preferably about four or five, contiguous amino acids of the mammalian RadS l amino acid sequence (SEQ ID NO. 1), the human Rad51 amino acid sequence (SEQ ID NO. 2), or the mammalian or human BRCA2 amino acid sequences that retain the property of being capable of binding a mammalian Rad51 and/or inhibiting Rad51 function (as detected using a suitable biochemical, genetic, or cellular assay).

Additionally, the blocking of normal RadS 1 function may induce programmed cell death. Thus, one aspect of the present invention are a novel class of therapeutic agents, factors, or compounds that have been engineered, or are otherwise capable of disrupting the essential processes that are mediated by, or associated with, normal Rad51 or Rad52 activity. Accordingly, it is contemplated that this novel class of therapeutics agents may be used to treat diseases including, but not limited to. autoimmune disorders and diseases, inflammation, cancer, graft rejection, and any of a variety of proliferative or hyperproliferative disorders. Typical examples of therapeutic agents based on the above presently described molecules include, but are not limited to. defective (either engineered or naturally occurring) forms of the proteins that associate with the protein complexes, inhibitory fragments of the proteins, wild type and altered genes that code for proteins that disrupt mammalian Rad51 function, small organic molecules, antisense nucleic acid sequences, oligonucleotides that inhibit expression or activity via a triplex mechanism, peptides, aptameric oligonucleotides. and the like.

More particularly, examples of engineered proteins may include, but are not limited to, proteins that comprise inactivating mutations in conserved active sites (e.g., ATP binding motifs. DNA or protein binding domains, catalytic sites, etc.). fusion proteins that comprise at least one inhibitory domain, and the like.

The above agents may be obtained from a wide variety of sources. For example. standard methods of organic synthesis may be used to generate small organic molecules that mimic the desired regions of the target DNA repair proteins. In addition, combinatorial libraries comprising a vast number of compounds (organic, peptide. or nucleic acid, reviewed in Gallop et al. 1994. J. Med. Chem. 57(9): 1233- 1251 ; Gordon et al., 1994. J. Med. Chem. J7r70J:1385-140T ; and U.S. Patent No. 5.424.186 all of which are herein incorporated by reference) may be screened for the ability to bind and inhibit the activity of proteins involved in DSB repair or any other potential mammalian Rad51 function.

In particular, inhibitory peptides should prove very useful. Such compounds may include, but are not limited to, peptides such as. for example, soluble peptides. including but not limited to members of random peptide libraries: (see. e.g., Lam et al, 1991, Nature

55-7:82-84; Houghten et al. 1991 , Nature 55-7:84-86). and combinatorial chemistry-derived molecular library made of D- and/or L- configuration amino acids, phosphopeptides (including, but not limited to members of random or partially degenerate, directed phosphopepfide libraries; see, e.g., Songyang et al, 1993. Cell 72:767-778). In order to increase the specificity of the described peptides. targeting agents can be covalently or noncovalently affixed to the peptides. For the purposes of this disclosure, the term targeting agent shall refer to any and all molecules that can function as ligands or ligand receptors and that can be affixed to the described peptides. Such ligands may include, but are not limited to, polyclonal or monoclonal antibodies such as IgM, IgG, IgA, IgD, and the like, or any portions or subsets thereof, cell factors, cell surface receptors.

MHC or HLA markers, viral envelope proteins, peptides or small organic ligands or derivatives thereof, agonists, antagonists, and the like. Generally, the targeting agent is capable of preferentially binding to epitopes or receptors present on the targeted cells or tissues. For example, where hepatocytes are the preferred target cell or tissue, molecules such as fetuin may prove useful. Hepatocytes contain a galactose receptor. After treatment with neuraminidase, the protein fetuin is converted to asialofetuin which displays a number of galactose residues on its surface. Consequently, the asialofetuin-associated complexes are targeted to hepatocytes by virtue of the exposed galactose residues on the protein. Fetuin can also be readily attached to the described peptides using established chemical linkers. Alternatively, asialofetuin can be directly attached to the peptides. For example, iodate can be used to convert at least a portion of the hydroxyl groups on galactose to aldehydes, the aldehydes react with primary amino groups to form Schiff bases, which may be subsequently be reduced with lithium aluminum hydride. Alternatively, the aldehydes may be reacted with hydrazide to attach heterobifunctional cross-linking reagents (which have been linked to the described peptides and/or other targeting ligands/receptors). A brief list that is exemplary of such targeting proteins/receptors includes: CDl(a-c), CD4. CD8- 1 l(a-c). CD15. CDwl7. CD18, CD21-25. CD27. CD30-45(R(O. A, and B)), CD46-48, CDw49(b.d,f), CDw50. CD51. CD53-54. CDw60. CD61-64, CDw65. CD66-69, CDw70, CD71, CD73-74, CDw75. CD76-77. LAMP-1 and LAMP-2. and the T-cell receptor, integrin receptors, endoglin for proliferative endothelium. or antibodies against the same.

The above strategy is simply illustrative of the many possible ways asialofetuin may be derivatized with practically any targeting ligand as well as the described peptides, and should not be construed as limiting the invention in any way. In another aspect, the compounds of the present invention may be targeted to particular tissues, organs or cells in an animal, including a human. Such targeting can be accomplished in any way known in the art. for example, by linking the compound of the present invention to a molecule that preferably binds to cells of a particular type or that binds to cells or molecules which are preferably located in a particular tissue or organ, i.e., a targeting agent. Such preferred binding may result from an ability of the targeting agent to recognize a molecular component of the targeted tissue, organ, cell type or extracellular space {e.g., extracellular matrix). Examples of targeting agents are antibodies of any kind, for example, polyclonal antibodies, monoclonal antibodies, antibody fragments, etc. Such antibodies may be specific to cell surface receptors, extracellular matrix components, cell membrane components (e.g., glycolipids). or any antigen that is found excusively or preferably in a particular tissue, organ, cell type or extracellular space. Other examples of targeting agents are ligands of cellular receptors, ligands of extracellular matrix components, lectins. or any kind of molecule, ligand. agonist, antagonist, etc. that has a binding partner which is found exclusively or preferably in a particular tissue, organ, cell type or extracellular space.

In a preferred embodiment, a peptide or a polypeptide of the present invention is linked to a targeting agent. For example, a peptide of the present invention may be linked to an antibody which recognizes a tumor specific antigen. Thus, the complex of the peptide and the antibody would bind the antigen and the peptide would therefore be targeted to the tumor, i.e., the peptide would preferably localize close to or in the tumor cells which express the antigen. As a result of linking a peptide or a polypeptide of the invention to a targeting agent, the peptide or polypeptide can be used at a lower concentration when compared to using the peptide or polypeptide without a targeting molecule, or the peptide would be more potent when used at an equal concentration but while linked to a targeting agent. The compounds of the present invention may be linked to a targeting agent in any way known in the art. For example, such linkage may be accomplished through complex formation, through hydrogen bonds, through a covalent bond, or through any other kind of link or bond. The link between the compound of the present invention and the targeting agent may be a direct link or it may be accomplished through a linker molecule. Any molecule known to the skilled artisan may be used as a linker molecule, for example, a peptide. General background on linking a compound of the present invention to a targeting agent is provided in U.S. Patent Nos. 5,877,289: 5.844,094; 5,837,242: 5,834,589, and in Rogers et al., 1997, Gene Therapy -7: 1387-1392. all of which, and all references cited therein, are incorporated herein by reference in their entirety. Given that they will serve as templates for the rational design of agents for disrupting DSB repair activity in the cell, it would be advantageous to purify each of the individual proteins that are directly or indirectly involved in DSB repair of any other potential mammalian Rad51 function. The various proteins involved in the DSB repair pathways may be purified using any of a number of variations of well established biochemical, and molecular biology techniques. Such techniques are well known to those of ordinary skill in the biochemical arts and have been extensively described in references such as Berger and Kimmel. Guide to Molecular Cloning Techniques. Methods in Enzymology. Volume 152. Academic Press. San Diego, CA (1987; Molecular Cloning: A Laboratory Manual, 2d ed.. Sambrook. J.. Fritsch. E.F.. and Maniatis. T. (1989); Current Protocols in Molecular Biology. John Wiley & Sons, all Vols., 1989. and periodic updates thereof); New Protein Techniques: Methods in Molecular Biology. Walker. J.M., ed.. Humana Press. Clifton. N.J.. 1988: and Protein Purification: Principles and Practice. 3rd. Ed., Scopes. R.K.. Springer-Verlag. New York. N.Y.. 1987. In general, techniques including, but not limited to. ammonium sulfate precipitation: centrifugation. ion exchange, gel filtration, and reverse-phase chromatography (and the HPLC or FPLC forms thereof) may be used to purify the various proteins of the DSB repair complex.

5.3. Uses of The Nucleic Acid Sequences. Polypeptides And Antibodies

The presently described DSB repair antagonists are particularly useful for the treatment of cancer. Cancers that may be treated by the methods of the invention include, but are not limited to: Cardiac: sarcoma (angiosarcoma. fibrosarcoma. rhabdomyosarcoma, liposarcoma). myxoma, rhabdomyoma. fibroma, lipoma and teratoma: Lung: bronchogenic carcinoma (squamous cell, undifferentiated small cell, undifferentiated large cell, adenocarcinoma). alveolar (bronchiolar) carcinoma, bronchial adenoma, sarcoma, lymphoma, chondromatous hamartoma, mesothelioma; Gastrointestinal: esophagus (squamous cell carcinoma, adenocarcinoma. leiomyosarcoma. lymphoma). stomach

(carcinoma, lymphoma, leiomyosarcoma), pancreas (ductal adenocarcinoma, insulinoma, glucagonoma, gastrinoma, carcinoid tumors, vipoma). small bowel (adenocarcinoma, lymphoma, carcinoid tumors, Karposi's sarcoma, leiomyoma. hemangioma. lipoma, neurofibroma. fibroma), large bowel (adenocarcinoma. tubular adenoma, villous adenoma, hamartoma. leiomyoma); Genitourinary tract: kidney (adenocarcinoma. Wilm's tumor

[nephroblastoma], lymphoma. leukemia), bladder and urethra (squamous cell carcinoma, transitional cell carcinoma, adenocarcinoma). prostate (adenocarcinoma. sarcoma), testis (seminoma. teratoma, embryonal carcinoma, teratocarcinoma. choriocarcinoma. sarcoma, interstitial cell carcinoma, fibroma, fibroadenoma. adenomatoid tumors, lipoma); Liver: hepatoma (hepatocellular carcinoma), cholangiocarcinoma, hepatoblastom. angiosarcoma, hepatocellular adenoma, hemangioma; Bone: osteogenic sarcoma (osteosarcoma), fibrosarcoma. malignant fibrous histiocytoma. chondrosarcoma. Ewing's sarcoma, malignant lymphoma (reticulum cell sarcoma), multiple myeloma, malignant giant cell tumor, chordoma. osteochronfroma (osteocartilaginous exostoses). benign chondroma, chondroblastoma. chondromyxofibroma. osteoid osteoma and giant cell tumors; Nervous system: skull (osteoma. hemangioma. granuloma. xanthoma. osteitis deformans), meninges (meningioma. meningiosarcoma. gliomatosis). brain (astrocytoma. medulloblastoma. glioma. ependymoma. germinoma [pinealoma]. glioblastoma multiforme. oligodendroglioma. schwannoma. retinoblastoma. congenital tumors), spinal cord (neurofibroma. meningioma, glioma. sarcoma): Gynecological: uterus (endometrial carcinoma), cervix (cervical carcinoma, pre-tumor cervical dysplasia), ovaries (ovarian carcinoma [serous cystadenocarcinoma. mucinous cystadenocarcinoma, endometrioid tumors, celioblastoma, clear cell carcinoma, unclassified carcinoma], granulosa-thecal cell tumors, Sertoli-Leydig cell tumors, dysgerminoma. malignant teratoma), vulva (squamous cell carcinoma, intraepithelial carcinoma, adenocarcinoma. fibrosarcoma. melanoma), vagina (clear cell carcinoma, squamous cell carcinoma, botryoid sarcoma [embryonal rhabdomyosarcoma], fallopian tubes (carcinoma); Hematologic: blood (myeloid leukemia [acute and chronic], acute lymphoblastic leukemia, chronic lymphocytic leukemia, myeloproliferative diseases, multiple myeloma, myelodysplastic syndrome). Hodgkin's disease, non-Hodgkin's lymphoma [malignant lymphoma]; Skin: malignant melanoma. basal cell carcinoma, squamous cell carcinoma. Karposi's sarcoma, moles, dysplastic nevi, lipoma, angioma. dermatofibroma. keloids. psoriasis; and Adrenal glands: neuroblastoma.

In addition to cancer, the presently disclosed compounds are effective against any of a wide variety of hyperproliferative disorders including, but not limited to: autoimmune disease, arthritis, inflammatory bowel disease, and/or proliferation induced after medical procedures, including, but not limited to. surgery, angioplasty. and the like.

The anti-cancer application of agents that functionally disrupt mammalian Rad51, Rad52 or any member in the DSB repair pathway, is premised upon the demonstration that DSB repair remains equally critical in cancer cells. Cancer cells lack many of the normal cell cycle regulatory mechanisms that are critical to controlling proliferation, and inducing programmed cell death, and it is possible that, in some cancer cells, the absence of these mechanisms renders Rad51 and/or Rad52 function nonessential. The protein p53 is central to regulation of the cell cycle, and stimulation of cell death in response to DNA damage including DNA damaged by ionizing radiation (reviewed by Ko and Prives, 1996. Genes & Develop. 7(9:1054-72). p53 is the most commonly mutated gene in cancer cells (Donehower et al, 1992, Nature 356:215-21 ; Vogelstein. 1990. Nature 5^:681 -682) and mutations in p53 are known to increase cell proliferation and promote chromosomal instability (Harvey et al., 1993. Oncogene 5:2457-67).

The early lethal phenotype in rad5X" mutant embryos and cells may be stimulated by a cell cycle response to unrepaired DNA damage. DNA damage was shown to inhibit progression through the cell cycle, demonstrating a relationship between DNA lesions and cell cycle proteins (Carr and Hoekstra. 1995. Trends in Cell Biology 5:32-40). In mitotically dividing budding yeast cells, a single DSB in a dispensable plasmid was sufficient to induce cell death, partly under the control of Rad9 (Bennett et al., 1993, Proc. Natl. Acad. Sci. USA 90:5613-17: Schiestl et al.. 1989, Mol. Cell. Biol. 9:1882-9654. Weinert and Hartwell. 1988. Science 277:317-22). In mammalian cells, the tumor suppressor gene, p53, responded to DNA damage induced by γ-radiation by delaying the cell cycle, or inducing programmed cell death (Kastan et al.. 1991. Cancer Research 57:6304-11; Kuerbitz et al.. 1992. Proc. Natl. Acad. Sci. USA 59:7491-95). These responses may be the critical tumor suppressor function of p53 (Baker et al., 1990, Science 279:912-15; Lowe et al.. 1994, Science 256:807-10. Symonds et al., 1994, Cell 75:703-11). Induction of p53 after exposure to ionizing radiation and restriction endonuclease suggest that the formation of DSB may initiate a p53 response (Lu and Lane, 1993, Cell 75:765-78). p53 was at least partly responsible for regulating the rad51 ^! phenotype because development was extended from the early egg cylinder stage to the head fold stage in ap53- mutant background. However, the double-mutant embryos died from either accumulation of DNA damage resulting in metabolic incompetence and mitotic failure, or p53- independent regulation. Murine embryonic fibroblasts. generated from double-mutant embryos, failed to proliferate and were completely senescent in tissue culture; thus, demonstrating that MmRadS 1 function was critical in cells that exhibit chromosomal instability and accelerated proliferation. It is therefore likely that disruption of MmRad51 or any other protein in its pathway or disruption of any protein-protein interaction important in the DSB repair pathway results in reduced proliferation or decreased cell viability. This feature remains true even in cells with reduced capacity to regulate the cell cycle.

5.3.1. Modulatorv, Antisense, Ribozvme and Triple Helix Approaches In another embodiment, the levels of proteins involved in DSB repair (e.g. Rad51) can be reduced by using well-known antisense. gene "knock-out." ribozvme and/or triple helix methods Such molecules ma\ be designed to modulate reduce or inhibit either unimpaired, or if appropriate, mutant sequence Techniques lor the production and use of such molecules are well known to those of skill in the art

Antisense RNA and DNA molecules act to directly block the translation of mRNA by hybridizing to targeted mRNA and prev enting protein translation Antisense approaches involve the design of oligonucleotides hich are complementary to an mRNA sequence The antisense oligonucleotides ill bind to the complementan mRNA sequence transcripts and prevent translation Absolute complementaπt\ . although preferred, is not required.

A sequence "complementa " to a portion of an RNA. as referred to herein, means a sequence having sufficient complementarity to be able to hybridize with the RNA, forming a stable duplex, in the case of double-stranded antisense nucleic acids, a single strand of the duplex DNA may thus be tested, or triplex formation may be assayed The ability to hybridize will depend on both the degree of complementarity and the length of the antisense nucleic acid Generally, the longer the hybridizing nucleic acid, the more base mismatches with an RNA it may contain and still form a stable duplex (or triplex, as the case may be)

One skilled in the art can ascertain a tolerable degree of mismatch by use of standard procedures to determine the melting point of the hybridized complex

In one embodiment, oligonucleotides complementary to non-coding regions of the sequence of interest could be used in an antisense approach to inhibit translation of endogenous mRNA Antisense nucleic acids should be at least six nucleotides in length, and are preferably oligonucleotides ranging from 6 to about 50 nucleotides in length In specific aspects, the oligonucleotide is at least 10 nucleotides. at least 17 nucleotides, at least 25 nucleotides or at least 50 nucleotides

Regardless of the choice of target sequence, it is preferred that in vitro studies are first performed to quantitate the ability of the antisense oligonucleotide to inhibit mRNA expression It is preferred that these studies utilize controls that distinguish between antisense gene inhibition and nonspecific biological effects of oligonucleotides Additionally, it is envisioned that results obtained using the antisense oligonucleotide are compared with those obtained using a control oligonucleotide It is preferred that the control oligonucleotide is of approximately the same length as the test oligonucleotide and that the nucleic acid of the oligonucleotide differs from the antisense sequence no more than is necessary to prevent specific hybridization to the target sequence The oligonucleotides can be DNA or RNA or chimeric mixtures or derivatives or modified versions thereof, single-stranded or double-stranded. The oligonucleotide can be modified at the base moiety, sugar moiety, or phosphate backbone, for example, to improve stability of the molecule, hybridization, etc. The oligonucleotide may include other appended groups such as peptides (e.g.. for targeting host cell receptors in vivo), or agents facilitating transport across the cell membrane (see. e.g., Letsinger. et al.. 1989, Proc. Natl. Acad. Sci. U.S.A. 86:6553-6556; Lemaitre. el al.. 1987, Proc. Natl. Acad. Sci. U.S.A. 84:648-652; PCT Publication No. WO88/09810. published December 15. 1988) or the blood-brain barrier (see. e.g.. PCT Publication No. WO89/10134, published April 25, 1988), hybridization-triggered cleavage agents (see. e.g.. Krol et al.. 1988. BioTechniques

6:958-976) or intercalating agents (see. e.g. Zon, 1988. Pharm. Res. 5:539-549). To this end, the oligonucleotide may be conjugated to another molecule, e.g., a peptide, hybridization triggered cross-linking agent, transport agent, hybridization-triggered cleavage agent, etc. The antisense oligonucleotide may comprise at least one modified base moiety which is selected from the group including but not limited to 5-fluorouracil. 5-bromouracil, 5-chlorouracil. 5-iodouracil. hypoxanthine. xanthine. 4-acetylcytosine, 5-(carboxyhydroxylmethyl) uracil. 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil. dihydrouracil. beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine. 1-methylinosine. 2,2-dimethylguanine,

2-methyladenine, 2-methylguanine. 3-methylcytosine, 5-methylcytosine. N6-adenine, 7-methylguanine. 5-methylaminomethyluracil. 5-methoxyaminomethyl-2-thiouracil, beta- D-mannosylqueosine. 5^"-methoxycarboxymethyluracil, 5-methoxyuracil. 2-methylthio-N6- isopentenyladenine, uracil-5-oxyacetic acid (v). wybutoxosine. pseudouracil. queosine, 2-thiocytosine. 5-methyl-2-thiouracil. 2-thiouracil. 4-thiouracil. 5-methyluracil, uracil-

5-oxyacetic acid methylester. uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil, 3-(3-amino- 3-N-2-carboxypropyl) uracil. (acp3)w. and 2.6-diaminopurine.

The antisense oligonucleotide may also comprise at least one modified sugar moiety selected from the group including but not limited to arabinose. 2-fluoroarabinose, xylulose. and hexose.

In yet another embodiment, the antisense oligonucleotide comprises at least one modified phosphate backbone selected from the group consisting of a phosphorothioate, a phosphorodithioate. a phosphoramidothioate. a phosphoramidate, a phosphordiamidate, a methylphosphonate. an alkyl phosphotriester. and a formacetal or analog thereof. In yet another embodiment, the antisense oligonucleotide is an α-anomeric oligonucleotide. An α-anomeric oligonucleotide forms specific double-stranded hybrids with complementary RNA in which, contrary to the usual β-units. the strands run parallel to each other (Gamier, et al . 1987, Nucl. Acids Res. 15 :6625-6641 ). The oligonucleotide is a 2'-0-methylribonucleotide (Inoue. et al.. 1987. Nucl. Acids Res. 15:6131 -6148). or a chimeric RNA-DNA analogue (Inoue. et al. , 1987. FEBS Lett. 215:327-330).

Oligonucleotides of the invention may be synthesized by standard methods known in the art. e.g., by use of an automated DNA synthesizer (such as are commercially available from Biosearch. Applied Biosystems. etc.). As examples, phosphorothioate oligonucleotides may be synthesized by the method of Stein, et al. (1988, Nucl. Acids Res.

16:3209), mefhylphosphonate oligonucleotides can be prepared by use of controlled pore glass polymer supports (Sarin. et al., 1988, Proc. Natl. Acad. Sci. U.S.A. 85:7448-7451), ^' etc.

While antisense nucleotides complementan' to a coding region sequence could be used, those complementary to the transcribed, untranslated region are most preferred.

Antisense molecules should be delivered to cells that express the sequence in vivo. A number of methods have been developed for delivering antisense DNA or RNA to cells; e.g., antisense molecules can be injected directly into the tissue site, or modified antisense molecules, designed to target the desired cells (e.g.. antisense linked to peptides or antibodies which specifically bind receptors or antigens expressed on the target cell surface) can be administered systemically.

A preferred approach to achieve intracellular concentrations of the antisense sufficient to suppress translation of endogenous mRNAs utilizes a recombinant DNA construct in which the antisense oligonucleotide is placed under the control of a strong pol III or pol II promoter. The use of such a construct to transfect target cells in the patient will result in the transcription of sufficient amounts of single stranded RNAs which will form complementary base pairs with the endogenous sequence transcripts and thereby prevent translation of the mRNA sequence. For example, a vector can be introduced e.g., such that it is taken up by a cell and directs the transcription of an antisense RNA. Such a vector can remain episomal or become chromosomally integrated, as long as it can be transcribed to produce the desired antisense RNA. Such vectors can be constructed by recombinant DNA technology methods standard in the art. Vectors can be plasmid. viral, or others known in the art, used for replication and expression in mammalian cells. Expression of the sequence encoding the antisense RNA can be by any promoter known in the art to act in mammalian. preferably human cells. Such promoters can be inducible or constitutive. Such promoters include but are not limited to: the SV40 early promoter region (Bernoist and Chambon, 1981. Nature 290:304-310). the promoter contained in the 3 ^"-long terminal repeat of Rous sarcoma virus ( Yamamoto. et al.. 1980. Cell 22:787-797), the herpes thymidine kinase promoter (Wagner, et al.. 1981. Proc. Natl. Acad. Sci. U.S.A. 78: 1441-1445). the regulatory sequences of the metallothionein gene (Brinster. et al.. 1982. Nature 296:39-42), etc. Any type of plasmid. cosmid, YAC or viral vector can be used to prepare the recombinant DNA construct that can be introduced directly into the tissue site. Alternatively, viral vectors can be used that selectively infect the desired tissue, in which case administration may be accomplished by another route (e.g.. systemically). Ribozyme molecules designed to catalytically cleave target gene mRNA transcripts can also be used to prevent translation of target gene mRNA and. therefore, expression of target gene product. (See, e.g., PCT International Publication WO90/1 1364. published October 4. 1990: Sarver. et al.. 1990. Science 247. 1222-1225).

Ribozymes are enzymatic RNA molecules capable of catalyzing the specific cleavage of RNA. (For a review, see Rossi. 1994. Current Biology 4:469-471). The mechanism of ribozyme action involves sequence specific hybridization of the ribozyme molecule to complementary target RNA. followed by an endonucleolytic cleavage event. The composition of ribozyme molecules must include one or more sequences complementary to the target gene mRNA. and must include the well known catalytic sequence responsible for mRNA cleavage. For this sequence, see. e.g.. U.S. Patent No.

5,093.246, which is incorporated herein by reference in its entirety.

While ribozymes that cleave mRNA at site specific recognition sequences can be used to destroy target gene mRNAs. the use of hammerhead ribozymes is preferred. Hammerhead ribozymes cleave mRNAs at locations dictated by flanking regions which form complementary base pairs with the target mRNA. The sole requirement is that the target mRNA have the following sequence of two bases: 5'-UG-3'. The construction and production of hammerhead ribozymes is well known in the art and is described more fully in Myers. 1995. Molecular Biology and Biotechnology: A Comprehensive Desk Reference. VCH Publishers. New York, (see especially Figure 4. page 833) and in Haseloff and Gerlach. 1988. Nature, 334:585-591. which is incorporated herein by reference in its entirety.

Preferably the ribozyme is engineered so that the cleavage recognition site is located near the 5' end of the target gene mRNA. i.e.. to increase efficiency and minimize the intracellular accumulation of non-functional mRNA transcripts. The ribozymes of the present invention also include RNA endoribonucleases (hereinafter "Cech-type ribozymes" ) such as the one which occurs naturally in Tetrahvmena thermophila (known as the I VS. or L-19 IVS RNA) and which has been extensively described by Thomas Cech and collaborators (Zaug. et al.. 1984. Science. 224:574-578; Zaug and Cech. 1986, Science. 231 :470-475: Zaug. et al , 1986. Nature, 324:429-433; published International patent application No. WO 88/04300 by University Patents Inc.; Been and Cech. 1986, Cell. 47:207-216). The Cech-type ribozymes have an eight base pair active site that hybridizes to a target RNA sequence whereafter cleavage of the target RNA takes place. The invention encompasses those Cech-type ribozymes that target eight base- pair active site sequences that are present in the target gene.

As in the antisense approach, the ribozymes can be composed of modified oligonucleotides (e.g.. for improved stability, targeting, etc.) and should be delivered to cells that express the target gene in vivo. A preferred method of delivery involves using a DNA construct "encoding" the ribozyme under the control of a strong constitutive pol III or pol II promoter, so that transfected cells will produce sufficient quantities of the ribozyme to destroy endogenous target gene messages and inhibit translation. Because ribozymes, unlike antisense molecules, are catalytic, a lower intracellular concentration is required for efficiency.

Endogenous target gene expression can also be reduced by inactivating or "knocking out" the target gene or its promoter using targeted homologous recombination (e.g., see

Smithies, et al. 1985, Nature 317:230-234; Thomas and Capecchi, 1987, Cell 51 :503-512; Thompson, et al, 1989, Cell 5:313-321 : each of which is incorporated by reference herein in its entirety). For example, a mutant, non-functional target gene (or a completely unrelated DNA sequence) flanked by DNA homologous to the endogenous target gene (either the coding regions or regulatory regions of the target gene) can be used, with or without a selectable marker and/or a negative selectable marker, to transfect cells which express the target gene in vivo. Insertion of the DNA construct, via targeted homologous recombination, results in inactivation of the target gene. Such approaches are particularly suited in the agricultural field where modifications to ES (embryonic stem) cells can be used to generate animal offspring with an inactive target gene (e.g., see Thomas and

Capecchi. 1987 and Thompson. 1989. supra). However this approach can be adapted for use in humans provided the recombinant DNA constructs are directly administered or targeted to the required site in vivo using appropriate viral vectors.

Alternatively, endogenous target gene expression can be reduced by targeting deoxyribonucleotide sequences complementary to the regulatory region of the target gene (i.e.. the target gene promoter and/or enhancers ) to form triple helical structures which prevent transcription of the target gene in target cells in the body. (See generally. Helene, 1991. Anticancer Drug Des.. 6(6):569-584; Helene. et al.. 1992. Ann. N. Y. Acad. Sci.. 660:27-36: and Maher. 1992. Bioassays 14(12):807-815). Nucleic acid molecules to be used in triple helix formation for the inhibition of transcription should be single stranded and composed of deoxynucleotides. The base composition of these oligonucleotides must be designed to promote triple helix formation via Hoogsteen base pairing rules, which generally require sizable stretches of either purines or pyrimidines to be present on one strand of a duplex. Nucleic acids may be pyrimidine- based, which will result in TAT and CGC^{^} triplets across the three associated strands of the resulting triple helix. The pyrimidine-rich molecules provide base complementarity to a purine-rich region of a single strand of the duplex in a parallel orientation to that strand. In addition, nucleic acid molecules may be chosen which are purine-rich. for example, contain a stretch of G residues. These molecules will form a triple helix with a DNA duplex that is rich in GC pairs, in which the majority of the purine residues are located on a single strand of the targeted duplex, resulting in GGC triplets across the three strands in the triplex.

Alternatively, the potential sequences that can be targeted for triple helix formation may be increased by creating a so-called "switchback" nucleic acid molecule. Switchback molecules are synthesized in an alternating 5'-3', 3'-5' manner, such that they base pair with first one strand of a duplex and then the other, eliminating the necessity for a sizable stretch of either purines or pyrimidines to be present on one strand of a duplex.

In instances wherein the antisense. ribozyme. and/or triple helix molecules described herein are utilized to inhibit mutant gene expression, it is possible that the technique may so efficiently reduce or inhibit the transcription (triple helix) and/or translation (antisense, ribozyme) of mRNA produced by normal target gene alleles which the possibility may arise wherein the concentration of normal target gene product present may be lower than is necessary for a normal phenotype. In such cases, to ensure that substantially normal levels of target gene activity are maintained, therefore, nucleic acid molecules which encode and express target gene polypeptides exhibiting normal target gene activity may. be introduced into cells via gene therapy methods such as those described, below, which do not contain sequences susceptible to whatever antisense. ribozv me. or triple helix treatments are being utilized. Alternatively, in instances whereby the target gene encodes an extracellular protein, it may be preferable to co-administer normal target gene protein in order to maintain the requisite level of target gene activity.

Anti-sense RNA and DNA. ribozyme. and triple helix molecules of the invention may be prepared by any method known in the art for the synthesis of DNA and RNA molecules, as discussed above. These include techniques for chemically synthesizing oligodeoxyribonucleotides and oligoribonucleotides well known in the art such as for example solid-phase phosphoramidite chemical synthesis. Alternatively. RNA molecules may be generated by in vitro and in vivo transcription of DNA sequences encoding the antisense RNA molecule. Such DNA sequences may be incorporated into a wide variety of vectors that incorporate suitable RNA polymerase promoters such as the T7 or SP6 polymerase promoters. Alternatively, antisense cDNA constructs that synthesize antisense RNA constitutively or inducibly, depending on the promoter used, can be introduced stably into cell lines.

5.3.2 Gene Replacement Therapy

An alternative means for employing the presently disclosed anti-proliferation agents includes the use of vectors to directly insert genes encoding the agents into target cells (e.g., gene therapy). For example, when the tumor cells express the genes encoding the desired sequences. DSB repair will be disrupted and the tumor cell will die. Alternatively, one could attack tumor cells using a strategy conceptually similar to that disclosed in U.S.

Patent No. 5,529,774 herein incorporated by reference. In brief, cells that produce transducing virus encoding sequence that disrupts DSB repair may be implanted at or near the tumor mass. As the producer cells continue to elaborate virus, the growing tumor cells are infected and effectively killed as they express the agent that blocks DSB repair. The above methodology has proven useful in the treatment of glioblastomas and other tumors of the brain by using retroviral vectors to selectively target actively replicating tumor cells. A similar methodology can be used to deliver antisense sequences that target (and thus inhibit) the expression of Rad51 or any of the proteins involved in the Rad51 or Rad52 pathways. The mammalian Rad51 - or Rad52-mediated repair pathways, and the associated proteins, are essential for cell proliferation or viabilitv . These DNA repair pathways most likely function by repairing DSB via homologous recombination between sister chromatids during S/G₂ (recombinational repair): however, during G,. the repair of DSB may also occur via nonhomologous recombination (nonhomologous end joining). The nucleic acid sequences which disrupt DSB repair mechanisms can be utilized for transferring recombinant nucleic acid sequences to cells and expressing said sequences in recipient cells. Such techniques can be used, for example, in marking cells or for the treatment of a condition, disorder, or disease involving hyperproliferation. Such treatment can be in the form of gene replacement therapy. Specifically, one or more copies of a normal sequence or a portion of the sequence which directs the production of a sequence product exhibiting normal sequence function, may be inserted into the appropriate cells within a patient, using vectors which include, but are not limited to adenovirus, adeno- associated virus and retrovirus vectors, in addition to other particles which introduce DNA into cells, such as liposomes.

In another embodiment, techniques for delivery involve direct administration, e.g., by stereotactic delivery of such sequences to the site of the cells in which the sequences are to be expressed.

Methods for introducing genes for expression in mammalian cells are well known in the field. Generally, for such gene therapy methods, the nucleic acid is directly administered in vivo into a target cell or a transgenic mouse that expresses SP-10 promoter operably linked to a reporter gene. This can be accomplished by any methods known in the art, e.g., by constructing it as part of an appropriate nucleic acid expression vector and administering it so that it becomes intracellular. e.g.. by infection using a defective or attenuated retroviral or other viral vector (see U.S. Patent No. 4.980,286), by direct injection of naked DNA, by use of microparticle bombardment (e.g., a gene gun: Biolistic, Dupont), by coating with lipids or cell-surface receptors or transfecting agents, by encapsulation in liposomes. microparticles. or microcapsules. by administering it in linkage to a peptide which is known to enter the nucleus, or by administering it in linkage to a ligand subject to receptor-mediated endocytosis (see e.g.. Wu and Wu. 1987. J. Biol. Chem. 262:4429-4432). which can be used to target cell types specifically expressing the receptors. In another embodiment, a nucleic acid-ligand complex can be formed in which the ligand comprises a fusogenic viral peptide to disrupt endosomes. allowing the nucleic acid to avoid lysosomal degradation. In v et another embodiment, the nucleic acid can be targeted in vivo for cell specific uptake and expression, by targeting a specific receptor (see, e.g.. PCT Publications

WO 92/06180 dated April 16. 1992: WO 92/22635 dated December 23. 1992; WO92/20316 dated November 26. 1992: WO93/14188 dated July 22. 1993: WO 93/20221 dated October 14, 1993).

Additional methods which may be utilized to increase the overall level of expression of sequences which disrupt DSB repair activity include using targeted homologous recombination methods, discussed, above, to modify the expression characteristics of an endogenous sequence in a cell or microorganism by inserting a heterologous DNA regulatory element such that the inserted regulatory element is operatively linked with the endogenous sequence in question. Targeted homologous recombination can thus be used to activate transcription of an endogenous sequence which is "transcriptionally silent", i.e., is not normally expressed or is normallv expressed at very low levels, or to enhance the expression of an endogenous sequence which is normally expressed.

Further, the overall level of expression of sequences which disrupt DSB repair activity may be increased by the introduction of appropriate sequence-expressing cells, preferably autologous cells, into a patient at positions and in numbers which are sufficient to ameliorate the symptoms of a condition, disorder, or disease involving hyperproliferation.

Such cells may be either recombinant or non-recombinant.

Among the cells that can be administered to increase the overall level of sequence expression in a patient are normal cells which express the sequence. Alternatively, cells, preferably autologous cells, can be engineered to express the sequences, and may then be introduced into a patient in positions appropriate for the amelioration of the symptoms of a condition, disorder, or disease involving hyperproliferation.

When the cells to be administered are non-autologous cells, they can be administered using well-known techniques that prevent a host immune response against the introduced cells from developing. For example, the cells may be introduced in an encapsulated form that, while allowing for an exchange of components with the immediate extracellular environment, does not allow the introduced cells to be recognized by the host immune system.

5.4 Pharmaceutical Formulations And Methods of Administration

The compounds of this invention can be formulated and administered to inhibit a variety of disease states by any means that produces contact of the active ingredient with the agent's site of action in the body of a mammal. They can be administered by any conventional means available for use in conjunction with pharmaceuticals, either as individual therapeutic active ingredients or in a combination of therapeutic active ingredients. They can be administered alone, but are generally administered with a pharmaceutical carrier selected on the basis of the chosen route of administration and standard pharmaceutical practice. The dosage administered will be a therapeutically effective amount of the compound sufficient to result in amelioration of symptoms of the disease and will, of course, vary depending upon known factors such as the pharmacodynamic characteristics of the particular active ingredient and its mode and route of administration; age, sex. health and weight of the recipient; nature and extent of symptoms: kind of concurrent treatment, frequency of treatment and the effect desired.

Preferably, agents that disrupt DSB repair shall be substantially specific for blocking the desired repair pathways. For the purposes of the present invention, the term substantially specific shall mean that a given agent is capable of being dosaged to provide the desired effect while not causing undue cellular toxicity.

One of ordinary skill will appreciate that, from a medical practitioner's or patient's perspective, virtually any alleviation or prevention of an undesirable symptom (e.g.. symptoms related to disease, sensitivity to environmental factors, normal aging, and the like) would be desirable. Thus, for the purposes of this Application, the terms "treatment",

"therapeutic use", or "medicinal use" used herein shall refer to any and all uses of compositions comprising the claimed agents which remedy a disease state or symptoms, or otherwise prevent, hinder, retard, or reverse the progression of disease or other undesirable symptoms in any way whatsoever. When used in the therapeutic treatment of disease, an appropriate dosage of presently described agents, or derivatives thereof, may be determined by any of several well established methodologies. For instance, animal studies are commonly used to determine the maximal tolerable dose, or MTD. of bioactive agent per kilogram weight. In general, at least one of the animal species tested is mammalian. Those skilled in the art regularly extrapolate doses for efficacy and avoiding toxicity to other species, including human.

Before human studies of efficacy are undertaken. Phase I clinical studies in normal subjects help establish safe doses.

Additionally, the bioactive agents may be complexed with a variety of well established compounds or structures that, for instance, enhance the stability of the bioactive agent, or otherwise enhance its pharmacological properties (e.g., increase in vivo half-life, reduce toxicity. etc.). S.6.1. Dose Determinations

Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g. , for determining the LD₅₀ (the dose lethal to 50% of the population) and the ED,,, (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD,₀ /ED,₀. Compounds which exhibit large therapeutic indices are preferred. While compounds that exhibit toxic side effects may be used, care should be taken to design a delivery system that targets such compounds to the site of affected tissue in order to minimize potential damage to uninfected cells and, thereby, reduce side effects.

The data obtained from the cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED₅₀ with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For any compound used in the method of the invention, the therapeutically effective dose can be estimated initially from cell culture assays. A dose may be formulated in animal models to achieve a circulating plasma concentration range that includes the IC₅₀ (i.e.. the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma may be measured, for example, by high performance liquid chromatography.

Specific dosages may also be utilized for antibodies. Typically, the preferred dosage is 0.1 mg/kg to 100 mg/kg of body weight (generally 10 mg/kg to 20 mg/kg), and if the antibody is to act in the brain, a dosage of 50 mg/kg to 100 mg/kg is usually appropriate. If the antibody is partially human or fully human, it generally will have a longer half-life within the human body than other antibodies. Accordingly, lower dosages of partially human and fully human antibodies is often possible. Additional modifications may be used to further stabilize antibodies. For example, lipidation can be used to stabilize antibodies and to enhance uptake and tissue penetration (e.g.. into the brain). A method for lipidation of antibodies is described by Cruikshank et al. (( 1997) J. Acquired Immune Deficiency

Syndromes and Human Retrovirology 14: 193). A therapeutically effective amount of protein or polypeptide (i.e.. an effective dosage) ranges from about 0.001 to 30 mg_'kg body weight, preferably about 0.01 to 25 mg/kg body weight, more preferably about 0.1 to 20 mg/kg body weight, and even more preferably about 1 to 10 mg/kg, 2 to 9 mg/kg, 3 to 8 mg/kg. 4 to 7 mg/kg. or 5 to 6 mg/kg body weight.

Moreover, treatment of a subject with a therapeutically effective amount of a protein, polypeptide or antibody can include a single treatment or. preferably, can include a series of treatments. In a preferred example, a subject is treated with antibody, protein, or polypeptide in the range of between about 0.1 to 20 mg/kg body weight, one time per week for between about 1 to 10 weeks, preferably between 2 to 8 weeks, more preferably between about 3 to 7 weeks, and even more preferably for about 4. 5 or 6 weeks.

The present invention further encompasses agents which modulate expression or activity. An agent may, for example, be a small molecule. For example, such small molecules include, but are not limited to. peptides. peptidomimetics, amino acids, amino acid analogs, polynucleotides. polynucleotide analogs, nucleotides, nucleotide analogs, organic or inorganic compounds (i.e.. including heteroorganic and organometallic compounds) having a molecular weight less than about 10.000 grams per mole, organic or inorganic compounds having a molecular weight less than about 5,000 grams per mole, organic or inorganic compounds having a molecular weight less than about 1.000 grams per mole, organic or inorganic compounds having a molecular weight less than about 500 grams per mole, and salts, esters, and other pharmaceutically acceptable forms of such compounds.

It is understood that appropriate doses of small molecule agents depends upon a number of factors known to those or ordinary skill in the art, e.g., a physician. The dose(s) of the small molecule will vary, for example, depending upon the identity, size, and condition of the subject or sample being treated, further depending upon the route by which the composition is to be administered, if applicable, and the effect which the practitioner desires the small molecule to have upon the nucleic acid or polypeptide of the invention. Exemplary doses include milligram or microgram amounts of the small molecule per kilogram of subject or sample weight (e.g.. about 1 microgram per kilogram to about 500 milligrams per kilogram, about 100 micrograms per kilogram to about 5 milligrams per kilogram, or about 1 microgram per kilogram to about 50 micrograms per kilogram. 5.6.2 Formulations and Use

Pharmaceutical compositions for use in accordance with the present invention may be formulated in conventional manner using one or more physiologically acceptable carriers or excipients. Thus, the compounds and their physiologically acceptable salts and solvates may be formulated for administration by inhalation or insufflation (either through the mouth or the nose) or oral, buccal, parenteral or rectal administration.

For oral administration, the pharmaceutical compositions may take the form of. for example, tablets or capsules prepared by conventional means with pharmaceutically acceptable excipients such as binding agents (e.g., pregelatinised maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystalline cellulose or calcium hydrogen phosphate); lubricants (e.g.. magnesium stearate. talc or silica); disintegrants (e.g.. potato starch or sodium starch glycolate); or wetting agents (e.g., sodium lauryl sulphate). The tablets may be coated by methods well known in the art. Liquid preparations for oral administration may take the form of, for example, solutions, syrups or suspensions, or they may be presented as a dry product for constitution with water or other suitable vehicle before use. Such liquid preparations may be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible fats); emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles (e.g.. almond oil, oily esters, ethyl alcohol or fractionated vegetable oils): and preservatives (e.g.. methyl or propyl-p-hydroxybenzoates or sorbic acid). The preparations may also contain buffer salts, flavoring, coloring and sweetening agents as appropriate.

Preparations for oral administration may be suitably formulated to give controlled release of the active compound.

For buccal administration the compositions may take the form of tablets or lozenges formulated in conventional manner.

For administration by inhalation, the compounds for use according to the present invention are conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebulizer, with the use of a suitable propellant. e.g.. dichlorodifluoromethane, trichlorofluoromethane. dichlorotetrafluoroethane. carbon dioxide or other suitable gas. In the case of a pressurized aerosol the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges of e.g. gelatin for use in an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch. The compounds may be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with an added preservative. The compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents. Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, e.g.. sterile pyrogen-free water, before use. In general, water, a suitable oil, saline, aqueous dextrose (glucose), and related sugar solutions and glycols such as propylene glycol or polyethylene glycols are suitable carriers for parenteral solutions. Solutions for parenteral administration contain preferably a water soluble salt of the active ingredient, suitable stabilizing agents and. if necessary, buffer substances.

Antioxidizing agents such as sodium bisulfate. sodium sulfite or ascorbic acid, either alone or combined, are suitable stabilizing agents. Also used are citric acid and its salts and sodium ethylenediaminetetraacetic acid (EDTA). In addition, parenteral solutions can contain preservatives such as benzalkonium chloride, methyl- or propyl-paraben and chlorobutanol. Suitable pharmaceutical carriers are described in Remington's

Pharmaceutical Sciences, a standard reference text in this field.

The compounds may also be formulated in rectal compositions such as suppositories or retention enemas, e.g., containing conventional suppository bases such as cocoa butter or other glycerides. In addition to the formulations described previously, the compounds may also be formulated as a depot preparation. Such long acting formulations may be administered by implantation (for example subcutaneously or intramuscularly) or by intramuscular injection. Thus, for example, the compounds may be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt. Additionally, standard pharmaceutical methods can be employed to control the duration of action. These are well known in the art and include control release preparations and can include appropriate macromolecules. for example polymers, polyesters, polvamino acids, polyvinyl. pyrolidone. ethylenevinylacetate. methyl cellulose, carboxymethyl cellulose or protamine sulfate. The concentration of macromolecules as well as the methods of incorporation can be adjusted in order to control release. Additionally, the agent can be incorporated into particles of polymeric materials such as polyesters, polyamino acids, hydrogels, poly (lactic acid) or ethylenevinylacetate copolymers. In addition to being incorporated, these agents can also be used to trap the compound in microcapsules. Another aspect of the present invention includes formulations that provide for the sustained release of DSB repair antagonists. Examples of such sustained release formulations include composites of biocompatible polymers, such as poly(lactic acid). poly(lactic-co-glycolic acid), methylcellulose, hyaluronic acid, collagen, and the like. The structure, selection and use of degradable polymers in drug delivery vehicles have been reviewed in several publications, including, A. Domb et al. Polymers for Advanced

Technologies 5:279-292 (1992). Additional guidance in selecting and using polymers in pharmaceutical formulations can be found in the text by M. Chasin and R. Langer (eds.), "Biodegradable Polymers as Drug Delivery Systems, " Vol. 45 of "Drugs and the Pharmaceutical Sciences." M. Dekker. New York. 1990. Liposomes may also be used to provide for the sustained release of DSB repair antagonists. Details concerning how to use and make liposomal formulations of drugs of interest can be found in. among other places, U.S. Pat. No 4.944,948; U.S. Pat. No. 5.008.050: U.S. Pat. No. 4.921.706; U.S. Pat. No. 4,927,637; U.S. Pat. No. 4.452,747: U.S. Pat. No. 4.016.100; U.S. Pat. No. 4,31 1.712; U.S. Pat. No. 4,370.349; U.S. Pat. No. 4.372,949: U.S. Pat. No. 4.529,561 ; U.S. Pat. No. 5,009.956; U.S. Pat. No. 4.725.442; U.S. Pat. No. 4.737.323; U.S. Pat. No. 4,920.016.

Sustained release formulations are of particular interest when it is desirable to provide a high local concentration of DSB repair antagonist, e.g.. near a tumor, site of inflammation, etc.

Where diagnostic, therapeutic or medicinal use of the presently described agents, or derivatives thereof, is contemplated, the bioactive agents may be introduced in vivo by any of a number of established methods. For instance, the agent may be administered by inhalation: by subcutaneous (sub-q): intravenous (IN.), intraperitoneal (I. P.), or intramuscular (I.M.) injection: or as a topically applied agent (transdermal patch, ointments, creams, salves, eye drops, and the like).

The compositions may. if desired, be presented in a pack or dispenser device which may contain one or more unit dosage forms containing the active ingredient. The pack may for example comprise metal or plastic foil, such as a blister pack. The pack or dispenser device may be accompanied by instructions for administration.

Useful pharmaceutical dosage forms, for administration of the compounds of this invention can be illustrated as follows: Capsules: Capsules are prepared by filling standard two-piece hard gelatin capsulates each with the desired amount of powdered active ingredient. 175 milligrams of lactose, 24 milligrams of talc and 6 milligrams magnesium stearate.

Soft Gelatin Capsules: A mixture of active ingredient in soybean oil is prepared and injected by means of a positive displacement pump into gelatin to form soft gelatin capsules containing the desired amount of the active ingredient. The capsules are then washed and dried.

Tablets: Tablets are prepared by conventional procedures so that the dosage unit is the desired amount of active ingredient. 0.2 milligrams of colloidal silicon dioxide, 5 milligrams of magnesium stearate. 275 milligrams of macrocrystalline cellulose, 1 1 milligrams of cornstarch and 98.8 milligrams of lactose. Appropriate coatings may be applied to increase palatability or to delay absorption.

Injectable: A parenteral composition suitable for administration by injection is prepared by stirring 1.5% by weight of active ingredients in 10% by volume propylene glycol and water. The solution is made isotonic with sodium chloride and sterilized. Suspension: An aqueous suspension is prepared for oral administration so that each

5 millimeters contain 100 milligrams of finely divided active ingredient, 200 milligrams of sodium carboxymethyl cellulose. 5 milligrams of sodium benzoate. 1.0 grams of sorbitol solution U.S. P. and 0.025 millimeters of vanillin.

Gene Therapy Administration: Where appropriate, the gene therapy vectors can be formulated into preparations in solid, semisolid. liquid or gaseous forms such as tablets, capsules, powders, granules, ointments, solutions, suppositories, injections, inhalants, and aerosols, in the usual ways for their respective route of administration. Means known in the art can be utilized to prevent release and absorption of the composition until it reaches the target organ or to ensure timed-release of the composition. A pharmaceutically acceptable form should be employed which does not ineffectuate the compositions of the present invention. In pharmaceutical dosage forms, the compositions can be used alone or in appropriate association, as well as in combination, with other pharmaceutically active compounds.

Accordingly, the pharmaceutical composition of the present invention may be delivered via various routes and to various sites in an animal body to achieve a particular effect (see, e.g., Rosenfeld et al. (1991). supra: Rosenfeld et al.. Clin. Res., 3 9(2), 31 1 A

(1991 a); Jaffe et al, supra; Berkner. supra). One skilled in the art will recognize that although more than one route can be used for administration, a particular route can provide a more immediate and more effective reaction than another route. Local or systemic delivery can be accomplished by administration comprising application or instillation of the formulation into body cavities, inhalation or insufflation of an aerosol, or by parenteral introduction, comprising intramuscular, intravenous, peritoneal, subcutaneous, intradermal, as well as topical administration.

The composition of the present invention can be provided in unit dosage form wherein each dosage unit, e.g., a teaspoonful. tablet, solution, or suppository, contains a predetermined amount of the composition, alone or in appropriate combination with other active agents. The term "unit dosage form^" as used herein refers to physically discrete units suitable as unitary dosages for human and animal subjects, each unit containing a predetermined quantity of the compositions of the present invention, alone or in combination with other active agents, calculated in an amount sufficient to produce the desired effect, in association with a pharmaceutically acceptable diluent, carrier, or vehicle, where appropriate. The specifications for the unit dosage forms of the present invention depend on the particular effect to be achieved and the particular pharmacodynamics associated with the pharmaceutical composition in the particular host.

Accordingly, the present invention also provides a method of transferring a therapeutic gene to a host, which comprises administering the vector of the present invention, preferably as part of a composition, using any of the aforementioned routes of

DJ administration or alternative routes known to those skilled in the art and appropriate for a particular application. The "effective amount^" of the composition is such as to produce the desired effect in a host which can be monitored using several end-points known to those skilled in the art. Effective gene transfer of a vector to a host cell in accordance with the present invention to a host cell can be monitored in terms of a therapeutic effect (e.g. alleviation of some symptom associated with the particular disease being treated) or, further, by evidence of the transferred gene or expression of the gene within the host (e.g., using the polymerase chain reaction in conjunction with sequencing. Northern or Southern hybridizations, or transcription assays to detect the nucleic acid in host cells, or using immunoblot analysis, antibody-mediated detection. mRNA or protein half-life studies, or particularized assays to detect protein or polypeptide encoded by the transferred nucleic acid, or impacted in level or function due to such transfer).

These methods described herein are by no means all-inclusive, and further methods to suit the specific application will be apparent to the ordinary skilled artisan. Moreover, the effective amount of the compositions can be further approximated through analogy to compounds known to exert the desired effect.

Furthermore, the actual dose and schedule can vary depending on whether the compositions are administered in combination with other pharmaceutical compositions, or depending on interindividual differences in pharmacokinetics. drug disposition, and metabolism. Similarly, amounts can van' in in vitro applications depending on the particular cell line utilized (e.g., based on the number of adenoviral receptors present on the cell surface, or the ability of the particular vector employed for gene transfer to replicate in that cell line). Furthermore, the amount of vector to be added per cell will likely vary with the length and stability of the therapeutic gene inserted in the vector, as well as also the nature of the sequence, and is particularly a parameter which needs to be determined empirically, and can be altered due to factors not inherent to the methods of the present invention (for instance, the cost associated with synthesis). One skilled in the art can easily make any necessary adjustments in accordance with the exigencies of the particular situation.

The present invention is further illustrated by the following examples, which are not intended to be limiting in any way whatsoever. 6. EXAMPLES

Initial studies had identified several proteins that interact with Rad51 (see generally U.S. Application Ser. No. 08/964.614. filed November 5, 1997. herein incorporated by- reference in its entirety) as well as the region of RadS 1 that participates in such interactions.

TABLE 1. Clones isolated from the veast two-hybrid screen

Clone Homology Library

1 100% to MmRadS 1 T cell

2 100% to M96 embryo

45% to Histone HI. 48% to XPG embryo

4 100% to Brca2 T cell

5 novel T cell

6 novel embryo

7 novel embryo

8 100%RFP embryo

Clones isolated from a yeast two-hybrid screen with MmRad51 as the "bait" and an embryonic or T cell cDNA library as the "prey".

The inserts obtained from the "prey^" were sequenced and compared to sequences in the GCG database. The measured extent of protein homology is listed. All clones strongly associated with MmRad51 in the N-terminal region (amino acids 1-43). Colonies grew within three days in 3-AT. and cells generally stained blue after about 5 minutes of X-gal exposure.

Initial studies had also indicated that the N-terminal region (amino acids 1-131) of Rad51 was involved in self-association as well as the association of Rad51 with other accessory proteins.

The other seven proteins listed in Table 1 were tested to determine if they interacted with the N-terminal region of MmRadS 1. All seven strongly interacted with the N-terminus of Rad51 were thus deemed responsible for the obsen ed RadS l protein-protein interactions.

- 53 Given the high level of homology shared between the human and murine Rad51 proteins (in the important N-terminal self-association region, the proteins only differ at amino acid positions 10 and 46 where the human sequence respectively contains an asparagine in lieu of the serine, and a phenylalanine in place of the tyrosine encoded by the mouse protein - both relatively conservative replacements), the presently described results should reflect the results expected from similar studies using the human Rad51 protein.

Further studies indicated that the conditional expression of the N-terminus of Rad51 in ES cells markedly increased the cells^" sensitivity to γ-radiation. Since ES cells are immortal and transformed, these data demonstrate that disrupting the Rad51 pathway provides a novel therapeutic approach for treating cancer. Therefore, expression of dominant negative transgenes that code for any protein that disrupts mammalian Rad51 function can serve as a therapeutic for cancer and should not be limited to the N-terminus of Rad51. An additional example of such a peptide is found in the BRCA2-derived amino acid sequence ROIKIWFONRRMKWKKFLSRLPLPSPVSPICTFVSPAAOKAFOPPRS (SEQ ID NO. 3). This peptide contains the region of Brca2 that interacts with Rad51, amino acids 3196-3226 (not underlined). This peptide also contains 16 amino acids derived from the Drosophila Antennapedia protein (underlined) that presumably facilitates translocation through biological membranes. This peptide was added to media after immortalized and transformed p53^~'^~ fibroblasts were plated at low concentration (100 cells/ 6 cm plate). Colonies were counted based on size as determined by the number of cells.

The peptide caused a great reduction of colonies composed of 265 or greater cells (Fig. 3b). Thus, the peptide had a profoundly negative effect on cellular proliferation. The 16 amino acids derived from Antennapedia had no effect on the number of colonies at any size; therefore, the inhibitory affect was due to the Brca2 sequences. Since p53^{' '} cells are highly proliferative and commonly found in cancer, these data demonstrate that disrupting the RadS 1 pathway will serve as a therapeutic for cancer. Therefore, any peptide that interacts with mammalian Rad51 may inhibit proliferation of cells in tissue culture and could be used to inhibit the growth of cancer. Additional studies have further refined the region of the BRCA2 sequence that is responsible for inhibiting Rad51 activity. In particular, the sequence PVSPICTFVSPA (SEQ ID NO. 4). PICTF

(SEQ ID NO. 5). ICTFV (SEQ ID NO. 6). CTFVS (SEQ ID NO. 7). PICSF (SEQ ID NO. 8) were able to trigger apoptosis in targeted Hela (human tumor cells) cells and murine ES cells. Additionally, the sequences ICT (SEQ ID NO. 9). TFVSP (SEQ ID NO. 10), CTF (SEQ ID NO. 1 1 ). ICTF (SEQ ID NO. 12). CTFV (SEQ ID NO. 13), and PICAF (SEQ ID NO. 14) demonstrated reduced, but significant, ability to trigger apoptosis in target cells. The peptide sequences VSPIC (SEQ ID NO. 15), SPICT (SEQ ID NO. 16). and PIMTF

(SEQ ID NO. 17) did not trigger apoptosis and also serve as negative controls for these assays. All of the disclosed peptides were conjugated to the disclosed antennapedia peptide, although other peptide sequences could be used to facilitate the test sequences ability to traverse the cellular and nuclear membranes (e.g., fibroblast growth factor receptor peptide, the protein transduction domain of human immunodeficiency virus TAT protein (Schwarze et al, 1999, Science 285: 1569-72). etc.). As is readily evident, conservative amino acid changes may be made in the above peptides without abolishing activity. Accordingly, the present invention additionally contemplates peptides incorporating conservative amino acid changes (i.e. interchanging T and S; I, L. A, and V: F and Y; hydrophobic amino acids; polar amino acids; and hydrophilic amino acids) and that retain the ability to induce apoptosis in target cells by binding and inhibiting Rad51.

Amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity and/or the amphipathic nature of the residues involved. For example, nonpolar (hydrophobic) amino acids include alanine. leucine, isoleucine, valine, proline, phenylalanine. tryptophan and methionine; polar neutral amino acids include glycine, serine, threonine. cysteine. tyrosine, asparagine and glutamine; positively charged (basic) amino acids include arginine, lysine and histidine; and negatively charged (acidic) amino acids include aspartic acid and glutamic acid. Additionally, non- natural amino acids, including, but not limited to. D-amino acids may be used.

6.1. Application of Molecules that Disrupt Mammalian Rad51 and/or Rad52 Function for Cancer Therapeutics

The rad51 " mutation reduces proliferation and promotes cellular senescence, even in ap53 mutant background. In addition. rad51 dominant negative alleles also display this phenotype by presumably forming nonproductive protein associations with RadS l and other proteins like Rad52, M96 and Brca2. Therefore, it is likely that the disruption of mammalian Rad51. mammalian Rad52 (or any protein in the DSB repair pathway mediated by these proteins) will reduce cell proliferation or induce cell death, and thus be suitable as_. a cancer therapeutic. In addition, the disruption of any protein-protein association important for mammalian Rad51 function or mammalian Rad52 function will also reduce cell proliferation or induce cell death, and thus be suitable as a cancer therapeutic. Additionally, dominant negative alleles oϊradSl may be used to express cancer therapeutics that reduce cell proliferation or induce cell death. An expression vector that codes for a dominant negative rad51 allele. or a dominant negative amino acid sequence from a Rad51 accessory protein (e.g. , BRCA2) may be introduced into cancer cells, or an mRNA that codes for a dominant negative alleles may be introduced into cancer cells, or a dominant negative Rad51 protein may be introduced into cancer cells. Several examples of such dominant negative radSl alleles are presently disclosed. Of the rα-757 alleles, the protein encoded by rad51K-A131 appears to have the strongest self-association, and. toxicity to proliferating cells. In fact, any rø-757 allele that rendered the RecA homology region nonfunctional, but preserved the N-terminal protein association region should reduce cell proliferation or induce cell death and could thus be used as a cancer therapeutic.

In addition to subtle alterations in the RecA core homology region of mammalian Rad51, C-terminal truncations in mammalian ra-757 may also be used to reduce cell proliferation and/or induce cell death. rad51TRl-131 demonstrated a toxic effect on cells even though it had a relatively weak interaction with MmRad51 which suggested that the phenotype might be caused by nonfunctional self-associations, or nonfunctional associations with other proteins such as Rad52. M96 and Brca2. In fact, any C-terminal truncation that preserves the protein interacting region of Rad51 may be used as a dominant negative allele for cancer therapy. Additionally, fusion of the N terminal domain of mammalian Rad51 to the 16 or 60 amino acids of the 3rd helix of the antennapedia protein may promote entry into the nucleus (Derossi et al.. 1994, J. Bio. Chem. 269: 10444- 10450).

Given Rad51's importance in the cell, antisense or ribozyme polynucleotides and oligonucleotides can be used to mediate suppression of Rad51 expression and thus can also be used to provide therapeutic benefit for the treatment of proliferative disorders and cancer. The normal function of mammalian RadSl can also be disrupted by agents that interfere with DNA binding, high energy metabolism (ADP and ATP binding and exchange at the Walker A and/or Walker B motifs). Given Rad51's demonstrated interaction with other proteins (besides itself), the disruption of these interactions could also be used to reduce cell proliferation or induce cell death. Other proteins that interact with Rad51 include but are not limited to mammalian Rad52. Brca2, M96, and RFP.

The identification of additional proteins that interact with RadSl will further elucidate the pathway and present opportunities to better discern how this pathway may be exploited to hinder cell proliferation. Since mammalian Rad52 associates with mammalian Rad51 and other proteins (Park et al. 1996; Shen et al., 1996), dominant alleles of mammalian Rad52 may also hinder cell proliferation or induce cell death. Such alleles could also be used for cancer therapeutics. In fact, dominant alleles of any protein that associates with mammalian Rad51. Rad52 or any other protein in these pathways, may be expected to hinder cell proliferation or induce cell death. Thus, all of the above molecules collectively define a new class of therapeutic agents for the treatment of proliferative disorders, viral infection (especially HIV infection), and cancer.

Genes encoding therapeutic dominant negative peptides and proteins can be isolated or synthesized using conventional techniques. For example, the degenerate nature of the genetic code is well known, and, accordingly, each amino acid present in a given peptide sequence is generically representative of the well known nucleic acid '"triplet" codon, or in many cases codons. that can encode the amino acid. As such, as contemplated herein, the presently described amino acid sequences, when taken together with the genetic code (see, for example. Table 4-1 at page 109 of "Molecular Cell Biology", 1986, J. Darnell et al. eds.,

Scientific American Books, New York. NY, herein incorporated by reference) are generically representative of all the various permutations and combinations of nucleic acid sequences that can encode such amino acid sequences. Genes encoding the disclosed peptides can also be administered by gene delivery technology using viral (e.g., retrovirus, lentivirus, adenovirus , adeno-associated virus, herpes virus, etc.), chemical (lipofection, liposomes, etc.), or other (naked DNA or RNA. electroporation, etc.) means of polynucleotide delivery. Preferably, the expression of such peptides, proteins, and polynucleotides will be largely restricted to the targeted tumor cells by using promoters or control elements that are preferentially, or specifically, expressed in the target tumor cell (e g-, adenoviral or retroviral vectors that preferentially express cloned genes in. for example. p53 minus cells). Alternatively, retroviral. for example, packaging cell lines that produce retrovirus that can express the dominant negative peptides, proteins, and polynucleotides can be implanted near the tumor site where the retrovirus will preferentially integrate into the rapidly growing tumor cells. Optionally, such retroviral producer cell lines are irradiated prior to implantation. Where human cells are targeted, the packaging cell lines will preferably produced virus with amphotropic or pantropic envelopes that allow the packaged virus to infect human cells.

EQUIVALENTS

The foregoing specification is considered to be sufficient to enable one skilled in the art to broadly practice the invention. Indeed, various modifications of the above-described methods for carrying out the invention, which are obvious to those skilled in the field of microbiology, biochemistry, organic chemistry, medicine or related fields, are intended to be within the scope of the following claims. All patents, patents applications, and publications cited herein are incorporated by reference.

Claims

WHAT IS CLAIMED IS:

1. An isolated peptide comprising from about 3 to about 41 amino acids, said peptide being capable of binding mammalian RadSl .

2. The peptide according to Claim 1 further comprising said peptide being from about 3 to about 30 amino acids in length.

3. The peptide according to Claim 2 further comprising said peptide being from about 3 to about 21 amino acids in length.

4. The peptide according to Claim 1. said peptide having a membrane permeability domain fused to an effector domain that inhibits Rad51 function.

5. The peptide according to Claim 4 wherein said effector domain comprises at least about five contiguous amino acids drawn from SEQ ID NO 1.

6. A method for hindering human cell proliferation comprising the administration of a molecule that disrupts double stranded DNA break repair.

7. The method according to Claim 6 wherein said molecule is of biological origin.

8. The method according to Claim 7 wherein said molecule was chemically synthesized.

9. A method for treating a hyperproliferative disorder comprising: administering to a mammal in need of said treatment a therapeutically effective amount of a compound that disrupts DNA break repair.

10. The method of Claim 9. wherein said mammal is a human.

1 1. The method of Claim 9. wherein said compound is a peptide comprising from about 3 to about 41 amino acids and is capable of binding mammalian Rad51.

12. The method of Claim 1 1. wherein said compound is a peptide further comprising from about 3 to about 30 amino acids and is capable of binding mammalian

Rad51.

13. The method of Claim 12. wherein said compound is a peptide further comprising from about 3 to about 21 amino acids and is capable of binding mammalian Rad51.

14. The method of Claim 13. wherein said compound is a peptide comprising at least about five contiguous amino acids drawn from SEQ ID NOT .

15. The method of Claim 9. wherein said compound is an antisense. ribozyme or triple helix sequence of a Rad51 -encoding polynucleotide.

16. A method for identifying a compound to be tested for an ability to dusrupt DNA break repair in a mammal, comprising: (a) contacting a test compound with a Rad51 polypeptide; and

(b) determining whether the test compound binds the Rad51 polypeptide, so that if the test compound binds the RadS 1 polypeptide. then a compound to be tested for an ability to disrupt DNA break repair is identified.

17. The method of Claim 16. wherein said mammal is a human.

18. A method for identifying a compound that inhibits cell proliferation in a mammal, comprising:

(a) contacting a test compound with a Rad51 polypeptide; and (b) identifying a test compound that binds the Rad51 polypeptide. and (c) administering the test compound in (b) to a non-human mammal affected with a hyperproliferative disorder, and determining whether the test compound disrupts double stranded DNA break repair in the mammal relative to that of a corresponding control, untreated, non-human mammal affected with the hyperproliferative disorder, so that if the test compound disrupts double stranded DNA break repair, then a compound that inhibits cell proliferation in a mammal has been identified.

19. The method of Claim 18. wherein said mammal is a human.