COMPOSITIONS AND METHODS FOR THE TREATMENT OF CANCER

SEQUENCE LISTING

This application is being filed electronically via EFS-Web and includes an electronically submitted sequence listing in .txt format. The .txt file contains a sequence listing entitled MOMA_001_01WO_ST25.txt created on Jun. 22, 2021 and having a size of 430 kilobytes. The sequence listing contained in this .txt file is part of the specification and is incorporated herein by reference in its entirety.

FIELD OF THE DISCLOSURE

The disclosure is directed to compositions and methods for the targeting of DNA repair pathway(s) to treat cancer.

BACKGROUND

There is a long-felt and unmet need in the art for an effective and safe cancer therapeutic. The disclosure provides compositions and methods for the selective targeting of components of a first or a second DNA repair pathway in cancer cells in which a first DNA repair pathway is impaired, defective or deregulated.

SUMMARY

The disclosure provides a composition comprising a DNA ligase 1 (LIG1) blocking agent, wherein in a target cell comprising an impaired homologous recombination (HR) repair pathway, the blocking agent reduces or inhibits a function of LIG1.

The disclosure provides a composition comprising a DNA ligase 1 (LIG1) blocking agent, wherein in a target cell comprising a defective homologous recombination (HR) repair pathway, the blocking agent reduces or inhibits a function of LIG1.

The disclosure provides a composition comprising a DNA ligase 1 (LIG1) blocking agent, wherein in a target cell comprising deregulated homologous recombination (HR) repair pathway, the blocking agent reduces or inhibits a function of LIG1.

In some embodiments of the compositions of the disclosure, a LIG1 blocking agent comprises a LIG1 inhibitor.

The disclosure provides a composition comprising a Fanconi Anemia Group M protein (FANCM) blocking agent, wherein in a target cell comprising an impaired homologous recombination (HR) repair pathway, the blocking agent reduces or inhibits a function of FANCM.

The disclosure provides a composition comprising a Fanconi Anemia Group M protein (FANCM) blocking agent, wherein in a target cell comprising a defective homologous recombination (HR) repair pathway, the blocking agent reduces or inhibits a function of FANCM.

The disclosure provides a composition comprising a Fanconi Anemia Group M protein (FANCM) blocking agent, wherein in a target cell comprising a deregulated homologous recombination (HR) repair pathway, the blocking agent reduces or inhibits a function of FANCM.

In some embodiments of the compositions of the disclosure, a FANCM blocking agent comprises a FANCM inhibitor.

Impaired homologous recombination (HR) repair pathways of the disclosure include those pathways wherein one or more components (e.g. signaling proteins/enzymes) of the pathway are impaired. In some embodiments, an HR repair pathway or a component thereof is a variant component that demonstrates an altered function or an altered activity compared to a non-variant or wild type component and which, in the context of the HR repair pathway, impairs a function or an activity of the HR repair pathway. In some embodiments, when a variant component demonstrates an altered function or an altered activity compared to a non-variant or wild type component the altered function or altered activity may be a decrease or loss of the function or the activity. In some embodiments, when a variant component demonstrates an altered function or an altered activity compared to a non-variant or wild type component the altered function or altered activity may be an increase or a gain of the function or of an activity. In some embodiments, an impairment of a pathway (including an HR pathway) or a component thereof comprises a complete loss of function, rendering the pathway or component defective. In some embodiments, an impairment of a pathway (including an HR pathway) or a component thereof comprises a deregulation of the pathway or a component thereof. In some embodiments, an impaired, defective or deregulated pathway or component thereof induces a disease or disorder of the disclosure. In some embodiments, an impaired, defective or deregulated HR pathway or component thereof induces a disease or disorder of the disclosure. In some embodiments, an impaired, defective or deregulated pathway or component thereof induces a malignant transformation of cell and the onset of a cancer. In some embodiments, an impaired, defective or deregulated HR pathway or component thereof induces a malignant transformation of cell and the onset of a cancer. In some embodiments, an HR pathway of a cell is impaired, defective or deregulated. In some embodiments, a component of an HR pathway of a cell is impaired, defective or deregulated. In some embodiments, the component comprises a variant protein of the disclosure.

In some embodiments of the compositions of the disclosure, the target cell does not comprise a variant Breast Cancer (BRCA) BRCA protein or a sequence encoding a variant BRCA protein, and the variant BRCA protein induces a loss or reduction in a function of the HR pathway. In some embodiments, the target cell is a proliferating cell. In some embodiments, the target cell is a tumor cell. In some embodiments, the target cell is a malignant cell. In some embodiments, the target cell is a metastatic cell. In some embodiments, the target cell is produced or derived from a Ewing Sarcoma or sarcoma cell.

In some embodiments of the compositions of the disclosure, the target cell comprises a variant BRCA protein or a sequence encoding a variant BRCA protein and the variant BRCA protein induces a loss or reduction in a function of the HR pathway. In some embodiments, the variant BRCA protein comprises a variant BRCA1 protein or wherein the sequence encoding the variant BRCA protein comprises a sequence encoding a variant BRCA1 protein. In some embodiments, the variant BRCA protein comprises a variant BRCA2 protein or wherein the sequence encoding the variant BRCA protein comprises a sequence encoding a variant BRCA2 protein. In some embodiments, the target cell is a proliferating cell. In some embodiments, the target cell is a tumor cell. In some embodiments, the target cell is a malignant cell. In some embodiments, the target cell is a metastatic cell. In some embodiments of the compositions of the disclosure, the variant protein or the sequence encoding the variant protein comprises one or more of a mutation, a deletion, a promotor methylation, a silencing event and a splicing event. In some embodiments, the mutation comprises one or more of a substitution, an insertion, a deletion, an inversion, and a translocation of a nucleic acid sequence or an amino acid sequence encoding the variant protein. In some embodiments, the mutation introduces a stop codon into a nucleic acid sequence encoding the variant protein, thereby generating one or more of a truncated protein, an inactivated protein and a protein fragment. In some embodiments, the variant protein or the sequence encoding the variant protein comprises a promoter sequence and wherein the promoter controls expression of the variant protein or the sequence encoding the variant protein. In some embodiments, the silencing event comprises a silencing of the promoter sequence, function, or activity. In some embodiments, the promoter sequence controlling expression of the variant protein or the sequence encoding the variant protein comprises a mutation. In some embodiments, the mutation comprises one or more of a substitution, an insertion, a deletion, an inversion, and a translocation of the promoter sequence.

In some embodiments of the compositions of the disclosure, the target cell comprises a nucleic acid or an amino acid encoding a variant DNA repair protein RAD51 homolog 1 (RAD51) or a variant homolog of RAD51 and the variant RAD51 induces a loss or reduction in a function of the HR pathway. In some embodiments of the compositions of the disclosure, the variant protein or the sequence encoding the variant protein comprises one or more of a mutation, a deletion, a promotor methylation, a silencing event and a splicing event. In some embodiments, the mutation comprises one or more of a substitution, an insertion, a deletion, an inversion, and a translocation of a nucleic acid sequence or an amino acid sequence encoding the variant protein. In some embodiments, the mutation introduces a stop codon into a nucleic acid sequence encoding the variant protein, thereby generating one or more of a truncated protein, an inactivated protein and a protein fragment. In some embodiments, the variant protein or the sequence encoding the variant protein comprises a promoter sequence and wherein the promoter controls expression of the variant protein or the sequence encoding the variant protein. In some embodiments, the silencing event comprises a silencing of the promoter sequence, function, or activity. In some embodiments, the promoter sequence controlling expression of the variant protein or the sequence encoding the variant protein comprises a mutation. In some embodiments, the mutation comprises one or more of a substitution, an insertion, a deletion, an inversion, and a translocation of the promoter sequence.

In some embodiments of the compositions of the disclosure, the target cell comprises a nucleic acid or an amino acid encoding a variant DNA repair protein RAD51 homolog 3 (RAD51C) or a variant homolog of RAD51C and the variant RAD51C induces a loss or reduction in a function of the HR pathway. In some embodiments of the compositions of the disclosure, the variant protein or the sequence encoding the variant protein comprises one or more of a mutation, a deletion, a promotor methylation, a silencing event and a splicing event. In some embodiments, the mutation comprises one or more of a substitution, an insertion, a deletion, an inversion, and a translocation of a nucleic acid sequence or an amino acid sequence encoding the variant protein. In some embodiments, the mutation introduces a stop codon into a nucleic acid sequence encoding the variant protein, thereby generating one or more of a truncated protein, an inactivated protein and a protein fragment. In some embodiments, the variant protein or the sequence encoding the variant protein comprises a promoter sequence and wherein the promoter controls expression of the variant protein or the sequence encoding the variant protein. In some embodiments, the silencing event comprises a silencing of the promoter sequence, function, or activity. In some embodiments, the promoter sequence controlling expression of the variant protein or the sequence encoding the variant protein comprises a mutation. In some embodiments, the mutation comprises one or more of a substitution, an insertion, a deletion, an inversion, and a translocation of the promoter sequence.

In some embodiments of the compositions of the disclosure, the target cell comprises a nucleic acid or an amino acid encoding a variant DNA repair protein RAD51 homolog 4 (RAD51D) or a variant homolog of RAD51D and the variant RAD51D induces a loss or reduction in a function of the HR pathway. In some embodiments of the compositions of the disclosure, the variant protein or the sequence encoding the variant protein comprises one or more of a mutation, a deletion, a promotor methylation, a silencing event and a splicing event. In some embodiments, the mutation comprises one or more of a substitution, an insertion, a deletion, an inversion, and a translocation of a nucleic acid sequence or an amino acid sequence encoding the variant protein. In some embodiments, the mutation introduces a stop codon into a nucleic acid sequence encoding the variant protein, thereby generating one or more of a truncated protein, an inactivated protein and a protein fragment. In some embodiments, the variant protein or the sequence encoding the variant protein comprises a promoter sequence and wherein the promoter controls expression of the variant protein or the sequence encoding the variant protein. In some embodiments, the silencing event comprises a silencing of the promoter sequence, function, or activity. In some embodiments, the promoter sequence controlling expression of the variant protein or the sequence encoding the variant protein comprises a mutation. In some embodiments, the mutation comprises one or more of a substitution, an insertion, a deletion, an inversion, and a translocation of the promoter sequence.

In some embodiments of the compositions of the disclosure, the target cell comprises a nucleic acid or an amino acid encoding a variant X-ray repair cross-complementing 2 (XRCC2) or a variant homolog of XRCC2 and the variant XRCC2 induces a loss or reduction in a function of the HR pathway. In some embodiments of the compositions of the disclosure, the variant protein or the sequence encoding the variant protein comprises one or more of a mutation, a deletion, a promotor methylation, a silencing event and a splicing event. In some embodiments, the mutation comprises one or more of a substitution, an insertion, a deletion, an inversion, and a translocation of a nucleic acid sequence or an amino acid sequence encoding the variant protein. In some embodiments, the mutation introduces a stop codon into a nucleic acid sequence encoding the variant protein, thereby generating one or more of a truncated protein, an inactivated protein and a protein fragment. In some embodiments, the variant protein or the sequence encoding the variant protein comprises a promoter sequence and wherein the promoter controls expression of the variant protein or the sequence encoding the variant protein. In some embodiments, the silencing event comprises a silencing of the promoter sequence, function, or activity. In some embodiments, the promoter sequence controlling expression of the variant protein or the sequence encoding the variant protein comprises a mutation. In some embodiments, the mutation comprises one or more of a substitution, an insertion, a deletion, an inversion, and a translocation of the promoter sequence.

In some embodiments of the compositions of the disclosure, the target cell comprises a nucleic acid or an amino acid encoding a variant DNA repair endonuclease XPF or a variant homolog of XPF and the variant XPF induces a loss or reduction in a function of the HR pathway. In some embodiments of the compositions of the disclosure, the variant protein or the sequence encoding the variant protein comprises one or more of a mutation, a deletion, a promotor methylation, a silencing event and a splicing event. In some embodiments, the mutation comprises one or more of a substitution, an insertion, a deletion, an inversion, and a translocation of a nucleic acid sequence or an amino acid sequence encoding the variant protein. In some embodiments, the mutation introduces a stop codon into a nucleic acid sequence encoding the variant protein, thereby generating one or more of a truncated protein, an inactivated protein and a protein fragment. In some embodiments, the variant protein or the sequence encoding the variant protein comprises a promoter sequence and wherein the promoter controls expression of the variant protein or the sequence encoding the variant protein. In some embodiments, the silencing event comprises a silencing of the promoter sequence, function, or activity. In some embodiments, the promoter sequence controlling expression of the variant protein or the sequence encoding the variant protein comprises a mutation. In some embodiments, the mutation comprises one or more of a substitution, an insertion, a deletion, an inversion, and a translocation of the promoter sequence.

In some embodiments of the compositions of the disclosure, the target cell comprises a nucleic acid or an amino acid encoding a variant Meiotic recombination 11 homolog 1 (MRE11A) or a variant homolog of MRE11A and the variant MRE11A induces a loss or reduction in a function of the HR pathway. In some embodiments of the compositions of the disclosure, the variant protein or the sequence encoding the variant protein comprises one or more of a mutation, a deletion, a promotor methylation, a silencing event and a splicing event. In some embodiments, the mutation comprises one or more of a substitution, an insertion, a deletion, an inversion, and a translocation of a nucleic acid sequence or an amino acid sequence encoding the variant protein. In some embodiments, the mutation introduces a stop codon into a nucleic acid sequence encoding the variant protein, thereby generating one or more of a truncated protein, an inactivated protein and a protein fragment. In some embodiments, the variant protein or the sequence encoding the variant protein comprises a promoter sequence and wherein the promoter controls expression of the variant protein or the sequence encoding the variant protein. In some embodiments, the silencing event comprises a silencing of the promoter sequence, function, or activity. In some embodiments, the promoter sequence controlling expression of the variant protein or the sequence encoding the variant protein comprises a mutation. In some embodiments, the mutation comprises one or more of a substitution, an insertion, a deletion, an inversion, and a translocation of the promoter sequence.

In some embodiments of the compositions of the disclosure, the target cell comprises a nucleic acid or an amino acid encoding a variant Ataxia telangiectasia mutated (ATM) or a variant homolog of ATM and the variant ATM induces a loss or reduction in a function of the HR pathway. In some embodiments of the compositions of the disclosure, the variant protein or the sequence encoding the variant protein comprises one or more of a mutation, a deletion, a promotor methylation, a silencing event and a splicing event. In some embodiments, the mutation comprises one or more of a substitution, an insertion, a deletion, an inversion, and a translocation of a nucleic acid sequence or an amino acid sequence encoding the variant protein. In some embodiments, the mutation introduces a stop codon into a nucleic acid sequence encoding the variant protein, thereby generating one or more of a truncated protein, an inactivated protein and a protein fragment. In some embodiments, the variant protein or the sequence encoding the variant protein comprises a promoter sequence and wherein the promoter controls expression of the variant protein or the sequence encoding the variant protein. In some embodiments, the silencing event comprises a silencing of the promoter sequence, function, or activity. In some embodiments, the promoter sequence controlling expression of the variant protein or the sequence encoding the variant protein comprises a mutation. In some embodiments, the mutation comprises one or more of a substitution, an insertion, a deletion, an inversion, and a translocation of the promoter sequence.

In some embodiments of the compositions of the disclosure, the target cell comprises a nucleic acid or an amino acid encoding a variant BRCA1-associated RING domain protein 1 (BARD1) or a variant homolog of BARD1 and the variant BARD1 induces a loss or reduction in a function of the HR pathway. In some embodiments of the compositions of the disclosure, the variant protein or the sequence encoding the variant protein comprises one or more of a mutation, a deletion, a promotor methylation, a silencing event and a splicing event. In some embodiments, the mutation comprises one or more of a substitution, an insertion, a deletion, an inversion, and a translocation of a nucleic acid sequence or an amino acid sequence encoding the variant protein. In some embodiments, the mutation introduces a stop codon into a nucleic acid sequence encoding the variant protein, thereby generating one or more of a truncated protein, an inactivated protein and a protein fragment. In some embodiments, the variant protein or the sequence encoding the variant protein comprises a promoter sequence and wherein the promoter controls expression of the variant protein or the sequence encoding the variant protein. In some embodiments, the silencing event comprises a silencing of the promoter sequence, function, or activity. In some embodiments, the promoter sequence controlling expression of the variant protein or the sequence encoding the variant protein comprises a mutation. In some embodiments, the mutation comprises one or more of a substitution, an insertion, a deletion, an inversion, and a translocation of the promoter sequence.

In some embodiments of the compositions of the disclosure, the target cell comprises a nucleic acid or an amino acid encoding a variant BRCA1-interacting protein C-terminal helicase 1 (BRIP1) or a variant homolog of BRIP1 and the variant BRIP1 induces a loss or reduction in a function of the HR pathway. In some embodiments of the compositions of the disclosure, the variant protein or the sequence encoding the variant protein comprises one or more of a mutation, a deletion, a promotor methylation, a silencing event and a splicing event. In some embodiments, the mutation comprises one or more of a substitution, an insertion, a deletion, an inversion, and a translocation of a nucleic acid sequence or an amino acid sequence encoding the variant protein. In some embodiments, the mutation introduces a stop codon into a nucleic acid sequence encoding the variant protein, thereby generating one or more of a truncated protein, an inactivated protein and a protein fragment. In some embodiments, the variant protein or the sequence encoding the variant protein comprises a promoter sequence and wherein the promoter controls expression of the variant protein or the sequence encoding the variant protein. In some embodiments, the silencing event comprises a silencing of the promoter sequence, function, or activity. In some embodiments, the promoter sequence controlling expression of the variant protein or the sequence encoding the variant protein comprises a mutation. In some embodiments, the mutation comprises one or more of a substitution, an insertion, a deletion, an inversion, and a translocation of the promoter sequence.

In some embodiments of the compositions of the disclosure, the target cell comprises a nucleic acid or an amino acid encoding a variant Cell cycle checkpoint kinase (CHEK1) or a variant homolog of CHEK1 and the variant CHEK1 induces a loss or reduction in a function of the HR pathway. In some embodiments of the compositions of the disclosure, the variant protein or the sequence encoding the variant protein comprises one or more of a mutation, a deletion, a promotor methylation, a silencing event and a splicing event. In some embodiments, the mutation comprises one or more of a substitution, an insertion, a deletion, an inversion, and a translocation of a nucleic acid sequence or an amino acid sequence encoding the variant protein. In some embodiments, the mutation introduces a stop codon into a nucleic acid sequence encoding the variant protein, thereby generating one or more of a truncated protein, an inactivated protein and a protein fragment. In some embodiments, the variant protein or the sequence encoding the variant protein comprises a promoter sequence and wherein the promoter controls expression of the variant protein or the sequence encoding the variant protein. In some embodiments, the silencing event comprises a silencing of the promoter sequence, function, or activity. In some embodiments, the promoter sequence controlling expression of the variant protein or the sequence encoding the variant protein comprises a mutation. In some embodiments, the mutation comprises one or more of a substitution, an insertion, a deletion, an inversion, and a translocation of the promoter sequence.

In some embodiments of the compositions of the disclosure, the target cell comprises a nucleic acid or an amino acid encoding a variant CHEK1 checkpoint homolog (CHEK2) or a variant homolog of CHEK2 and the variant CHEK2 induces a loss or reduction in a function of the HR pathway. In some embodiments of the compositions of the disclosure, the variant protein or the sequence encoding the variant protein comprises one or more of a mutation, a deletion, a promotor methylation, a silencing event and a splicing event. In some embodiments, the mutation comprises one or more of a substitution, an insertion, a deletion, an inversion, and a translocation of a nucleic acid sequence or an amino acid sequence encoding the variant protein. In some embodiments, the mutation introduces a stop codon into a nucleic acid sequence encoding the variant protein, thereby generating one or more of a truncated protein, an inactivated protein and a protein fragment. In some embodiments, the variant protein or the sequence encoding the variant protein comprises a promoter sequence and wherein the promoter controls expression of the variant protein or the sequence encoding the variant protein. In some embodiments, the silencing event comprises a silencing of the promoter sequence, function, or activity. In some embodiments, the promoter sequence controlling expression of the variant protein or the sequence encoding the variant protein comprises a mutation. In some embodiments, the mutation comprises one or more of a substitution, an insertion, a deletion, an inversion, and a translocation of the promoter sequence.

In some embodiments of the compositions of the disclosure, the target cell comprises a nucleic acid or an amino acid encoding a variant Nibrin (NBN) or a variant homolog of NBN and wherein the variant NBN induces a loss or reduction in a function of the HR pathway. In some embodiments of the compositions of the disclosure, the variant protein or the sequence encoding the variant protein comprises one or more of a mutation, a deletion, a promotor methylation, a silencing event and a splicing event. In some embodiments, the mutation comprises one or more of a substitution, an insertion, a deletion, an inversion, and a translocation of a nucleic acid sequence or an amino acid sequence encoding the variant protein. In some embodiments, the mutation introduces a stop codon into a nucleic acid sequence encoding the variant protein, thereby generating one or more of a truncated protein, an inactivated protein and a protein fragment. In some embodiments, the variant protein or the sequence encoding the variant protein comprises a promoter sequence and wherein the promoter controls expression of the variant protein or the sequence encoding the variant protein. In some embodiments, the silencing event comprises a silencing of the promoter sequence, function, or activity. In some embodiments, the promoter sequence controlling expression of the variant protein or the sequence encoding the variant protein comprises a mutation. In some embodiments, the mutation comprises one or more of a substitution, an insertion, a deletion, an inversion, and a translocation of the promoter sequence.

In some embodiments of the compositions of the disclosure, the target cell comprises a nucleic acid or an amino acid encoding a variant Partner and localizer of BRCA2 (PALB2) or a variant homolog of PALB2 and wherein the variant PALB2 induces a loss or reduction in a function of the HR pathway. In some embodiments of the compositions of the disclosure, the variant protein or the sequence encoding the variant protein comprises one or more of a mutation, a deletion, a promotor methylation, a silencing event and a splicing event. In some embodiments, the mutation comprises one or more of a substitution, an insertion, a deletion, an inversion, and a translocation of a nucleic acid sequence or an amino acid sequence encoding the variant protein. In some embodiments, the mutation introduces a stop codon into a nucleic acid sequence encoding the variant protein, thereby generating one or more of a truncated protein, an inactivated protein and a protein fragment. In some embodiments, the variant protein or the sequence encoding the variant protein comprises a promoter sequence and wherein the promoter controls expression of the variant protein or the sequence encoding the variant protein. In some embodiments, the silencing event comprises a silencing of the promoter sequence, function, or activity. In some embodiments, the promoter sequence controlling expression of the variant protein or the sequence encoding the variant protein comprises a mutation. In some embodiments, the mutation comprises one or more of a substitution, an insertion, a deletion, an inversion, and a translocation of the promoter sequence.

In some embodiments of the compositions of the disclosure, the target cell comprises a nucleic acid or an amino acid encoding a variant Structure-specific endonuclease subunit SLX4 (SLX4) or a variant homolog of SLX4 and the variant SLX4 induces a loss or reduction in a function of the HR pathway. In some embodiments of the compositions of the disclosure, the variant protein or the sequence encoding the variant protein comprises one or more of a mutation, a deletion, a promotor methylation, a silencing event and a splicing event. In some embodiments, the mutation comprises one or more of a substitution, an insertion, a deletion, an inversion, and a translocation of a nucleic acid sequence or an amino acid sequence encoding the variant protein. In some embodiments, the mutation introduces a stop codon into a nucleic acid sequence encoding the variant protein, thereby generating one or more of a truncated protein, an inactivated protein and a protein fragment. In some embodiments, the variant protein or the sequence encoding the variant protein comprises a promoter sequence and wherein the promoter controls expression of the variant protein or the sequence encoding the variant protein. In some embodiments, the silencing event comprises a silencing of the promoter sequence, function, or activity. In some embodiments, the promoter sequence controlling expression of the variant protein or the sequence encoding the variant protein comprises a mutation. In some embodiments, the mutation comprises one or more of a substitution, an insertion, a deletion, an inversion, and a translocation of the promoter sequence.

In some embodiments of the compositions of the disclosure, including those embodiments in which the target cell comprises a variant protein or a sequence encoding a variant protein and in which the variant protein transduces an intracellular signal in an HR pathway, the variant protein or the sequence encoding the variant protein comprises one or more of a mutation, a deletion, a promotor methylation, a silencing event and a splicing event. In some embodiments, the mutation comprises one or more of a substitution, an insertion, a deletion, an inversion, and a translocation of a nucleic acid sequence or an amino acid sequence encoding the variant protein. In some embodiments, the mutation introduces a stop codon into a nucleic acid sequence encoding the variant protein, thereby generating one or more of a truncated protein, an inactivated protein and a protein fragment.

In some embodiments of the compositions of the disclosure, including those embodiments in which the target cell comprises a variant protein or a sequence encoding a variant protein and in which the variant protein transduces an intracellular signal in an HR pathway, the variant protein or the sequence encoding the variant protein comprises a promoter sequence and wherein the promoter controls expression of the variant protein or the sequence encoding the variant protein. In some embodiments, the silencing event comprises a silencing of the promoter sequence, function, or activity. In some embodiments, the promoter sequence controlling expression of the variant protein or the sequence encoding the variant protein comprises a mutation. In some embodiments, the mutation comprises one or more of a substitution, an insertion, a deletion, an inversion, and a translocation of the promoter sequence.

In some embodiments of the compositions of the disclosure, the impairment, defect or deregulation of the HR pathway increases a dependence of the target cell upon a function of LIG1. In some embodiments, the function of LIG1 comprises one or more of ATP-binding, magnesium-binding, metal-binding, nucleotide-binding and nucleotide ligation. In some embodiments, LIG1 functions as a ligase. In some embodiments, an activity of LIG1 comprises an increase or a decrease in a function of LIG1. In some embodiments, the variant protein increases a function of LIG1. In some embodiments, the variant protein decreases a function of LIG1.

In some embodiments of the compositions of the disclosure, the impairment, defect or deregulation of the HR pathway increases a dependence of the target cell upon a function of FANCM. In some embodiments, the function of FANCM comprises one or more of ATP-binding, nucleotide-binding, DNA-binding, DNA remodeling, DNA strand separation, DNA-RNA strand separation and catalyzing the break of a chemical bond using water. In some embodiments, FANCM functions as a helicase. In some embodiments, FANCM functions as a hydrolase. In some embodiments, FANCM functions as a translocase. In some embodiments, an activity of FANCM comprises an increase or a decrease in a function of FANCM. In some embodiments, the variant protein increases a function of FANCM. In some embodiments, the variant protein decreases a function of FANCM.

In some embodiments of the compositions of the disclosure, the function of the HR pathway comprises one or more of i) recognizing nucleotide or DNA damage; ii) recruiting a protein to a site of nucleotide or DNA damage; iii) configuring or remodeling a sequence comprising a site of nucleotide or DNA damage; iv) configuring or remodeling a sequence complementary to a site of nucleotide or DNA damage; v) inducing a break in a sequence within a site of nucleotide or DNA damage; vi) inducing a break in a sequence comprising the site of nucleotide or DNA damage; vii) inducing a break in a sequence complementary to a site of nucleotide or DNA damage; viii) removing a sequence within a site of nucleotide or DNA damage; ix) removing a sequence comprising a site of nucleotide or DNA damage; x) synthesizing a new sequence within a site of nucleotide or DNA damage; xi) synthesizing a new sequence comprising a site of nucleotide or DNA damage; xii) resecting a portion of a synthesized sequence within a site of nucleotide or DNA damage; xiii) resecting a portion of a synthesized sequence comprising the site of nucleotide or DNA damage; xiv) stabilizing a site of DNA synthesis or replication within a site of nucleotide or DNA damage; xv) stabilizing a site of DNA synthesis or replication comprising a site of nucleotide or DNA damage; xvi) stabilizing a site of DNA synthesis or replication comprising a target site; xvii) stabilizing a site of DNA synthesis or replication comprising a stalled replication fork; xviii) inducing or facilitating invasion of a synthesized sequence within the site of nucleotide or DNA damage; xix) inducing or facilitating invasion of a synthesized sequence comprising the site of nucleotide or DNA damage; xx) inducing or facilitating insertion of a synthesized sequence within the site of nucleotide or DNA damage by recombination; and xxi) inducing or facilitating insertion of a synthesized sequence comprising the site of nucleotide or DNA damage by recombination.

In some embodiments of the compositions of the disclosure, an activity of the HR pathway comprises an increase or a decrease in a function of a component of the HR pathway. In some embodiments, the variant protein increases a function of a component of the HR pathway. In some embodiments, the variant protein decreases a function of a component of the HR pathway.

In some embodiments of the compositions of the disclosure, the composition further comprises a pharmaceutically acceptable carrier.

In some embodiments of the compositions of the disclosure, the blocking agent comprises an effector moiety that binds to a LIG-1 protein. In some embodiments, the effector moiety comprises one or more of an ion, a small molecule, a single-stranded nucleic acid molecule, a double-stranded nucleic acid molecule, an aptamer, an RNA-guided nuclease, a DNA-guided nuclease, a polypeptide, an antibody, a functional fragment of an antibody, an antibody mimetic, a scaffold, a matrix, or any combination thereof.

In some embodiments of the compositions of the disclosure, the blocking agent comprises an effector moiety that binds to a FANCM protein or a nucleic acid sequence encoding the FANCM protein. In some embodiments, the effector moiety comprises one or more of an ion, a small molecule, a single-stranded nucleic acid molecule, a double-stranded nucleic acid molecule, an aptamer, an RNA-guided nuclease, a DNA-guided nuclease, a polypeptide, an antibody, a functional fragment of an antibody, an antibody mimetic, a scaffold, a matrix, or any combination thereof.

In some embodiments of the compositions of the disclosure, including those in which the blocking agent comprises an effector moiety, the blocking agent further comprises a targeting moiety operably linked to the effector moiety. In some embodiments, the targeting moiety is reversibly linked to the effector moiety. In some embodiments, the targeting moiety specifically binds a component of the target cell. In some embodiments, the target cell is a proliferating cell. In some embodiments, the target cell is a tumor cell. In some embodiments, the target cell is a malignant cell. In some embodiments, the target cell is a metastatic cell. In some embodiments, the target cell is produced or derived from a non-hematological tissue. In some embodiments, the target cell is produced or derived from an epithelial tissue. In some embodiments, the target cell is produced or derived from an organ or a structure comprising an epithelial tissue. In some embodiments, the target cell is produced or derived from a skin area, a skin layer, a lung, a lymph node, a breast, an ovary, a prostate, a mouth, a nose, a nasal passage, an esophagus, an intestine, a small intestine, a large intestine, a stomach, a kidney, a liver, a spleen, a heart, an artery, a vein, a bladder and a colon. In some embodiments, the target cell is produced or derived from a bone or a connective tissue.

In some embodiments of the compositions of the disclosure, including those in which the blocking agent comprises one or more of an effector moiety and a targeting moiety, the blocking agent further comprises a regulation moiety. In some embodiments, the regulation moiety is operably linked to one or more of the effector moiety and the targeting moiety. In some embodiments, the regulation moiety is reversibly linked to one or more of the effector moiety and the targeting moiety. In some embodiments, the regulation moiety selectively binds a component not present in a target cell. In some embodiments, the component not present in a target cell is present in a healthy cell. In some embodiments, the component decreases or inhibits an activity of the effector moiety. In some embodiments, the regulation moiety comprises a microRNA (miRNA) binding site and selectively binds a miRNA.

The disclosure provides a method of inducing cell death in a proliferating cell, comprising contacting the proliferating cell and the composition of the disclosure. In some embodiments, the cell is in vitro or ex vivo. In some embodiments, the cell is in vivo. In some embodiments, the proliferating cell is a target cell of the disclosure.

The disclosure provides a method of inducing cell cycle arrest in a proliferating cell, comprising contacting the proliferating cell and a composition of the disclosure. In some embodiments, the cell is in vitro or ex vivo. In some embodiments, the cell is in vivo. In some embodiments, the proliferating cell is a target cell of the disclosure.

The disclosure provides a method of arresting growth of a proliferating cell, comprising contacting the proliferating cell and a composition of the disclosure. In some embodiments, the cell is in vitro or ex vivo. In some embodiments, the cell is in vivo. In some embodiments, the proliferating cell is a target cell of the disclosure. In some embodiments, arrest growth comprises delaying the onset, reducing a probability or inhibiting the entry of the cell entering the cell cycle following contact with a composition of the disclosure. In some embodiments, prior to contacting the composition, the cell had undergone at least one proliferation, however, subsequent to contacting the composition, the proliferating or target cell does not enter the cell cycle again. In some embodiments, the proliferating or target cell never enters the cell cycle for a second proliferation. In some embodiments, the proliferating or target cell delays entry into the cell cycle for a period of time. In some embodiments, the proliferating or target cell delays entry into the cell cycle for at least 30 minutes, at least 1 hour, at least 12 hours, at least one day (24 hours), at least 2 days, at least 1 week, at least 1 month or at least any period of time in between. In some embodiments, contact with a composition of the disclosure decreases or reduces a probability that the proliferating or target cell will ever enter the cell cycle or that the proliferating or target cell will enter the cell cycle within a period of time. In some embodiments, contact with a composition of the disclosure decreases or reduces a probability that the proliferating or target cell will enter the cell cycle for at least 30 minutes, at least 1 hour, at least 12 hours, at least one day (24 hours), at least 2 days, at least 1 week, at least 1 month or at least any period of time in between. In some embodiments, following contact with a composition of the disclosure, the target cell undergoes necrosis, apoptosis or another form of cell death. In some embodiments, following contact with a composition of the disclosure, the target cell enters senescence.

The disclosure provides a method of inhibiting proliferation of a proliferating cell, comprising contacting the proliferating cell and a composition of the disclosure. In some embodiments, the cell is in vitro or ex vivo. In some embodiments, the cell is in vivo. In some embodiments, the proliferating cell is a target cell of the disclosure. In some embodiments, prior to contacting the composition, the cell had undergone at least one proliferation, however, subsequent to contacting the composition, the proliferating or target cell does not proliferate again. In some embodiments, following contact with a composition of the disclosure, the target cell undergoes necrosis, apoptosis or another form of cell death. In some embodiments, following contact with a composition of the disclosure, the target cell enters senescence.

The disclosure provides a method of inducing senescence of a proliferating cell, comprising contacting the proliferating cell and a composition of the disclosure. In some embodiments, the cell is in vitro or ex vivo. In some embodiments, the cell is in vivo. In some embodiments, the proliferating cell is a target cell of the disclosure. In some embodiments, prior to contacting the composition, the cell had undergone at least one proliferation, however, subsequent to contacting the composition, the proliferating or target cell enters senescence. In some embodiments, senescence comprises an irreversible cell cycle arrest. In some embodiments, senescence further comprises one or more of telomere shortening, oxidative stress, genotoxic stress, increase in the expression or activity of one or more inflammatory agents (e.g. mitogens or inflammatory cytokines), an activation of the p53 tumor suppressor, and an activation of the cyclin-dependent kinase inhibitor p16. In some embodiments, oxidative stress comprises an imbalance between a production of free radicals and a neutralization of the free radicals by antioxidants. In some embodiments, genotoxic stress comprises cellular exposure to DNA-damaging agents and, optionally, increased energy expenditures by the cell to repair DNA damage.

The disclosure provides a method of treating cancer, comprising administrating to a subject an effective amount of the composition of the disclosure. In some embodiments, the method further comprises administering a second therapy. In some embodiments, the second therapy comprises radiation and/or a chemotherapy. In some embodiments, the chemotherapy comprises a Poly (ADP-ribose) polymerase (PARP) inhibitor or a platinum-based therapy. In some embodiments, the cancer is resistant to treatment with a PARP inhibitor as a monotherapy. In some embodiments, prior to administration of the composition, the subject has been identified as resistant to treatment with a PARP inhibitor as a monotherapy. In some embodiments, prior to administration of the composition, the subject has been treated with a PARP inhibitor as a monotherapy.

In some embodiments of the methods of the disclosure, the administration is systemic. In some embodiments, the composition is administered by one or more of an oral route, an inhaled route, an intravenous route, an intraperitoneal route, and a subcutaneous route.

In some embodiments of the methods of the disclosure, the administration is local. In some embodiments, the composition is administered by one or more of an intraocular route, an intraspinal route, an intracerebellar route, an intrathecal route, an intramuscular route and an intraosseous route.

In some embodiments of the methods of the disclosure, the composition is administered once per day, twice per day or three times per day. In some embodiments, the composition is administered once per week, twice per week or three times per week. In some embodiments, the composition is administered once per month, twice per month or three times per month.

In some embodiments of the methods of the disclosure, treating comprises a reduction in a severity of a sign or symptom of the cancer. In some embodiments, treating comprises a reduction in a volume of a tumor. In some embodiments, treating comprises a reduction in a number of tumor cells per volume of blood or mass of tissue. In some embodiments, treating comprises a remission. In some embodiments, treating comprises an increased duration of progression free survival.

In some embodiments of the methods of the disclosure, including the methods of treating a cancer, the cancer comprises cancer cells comprising an impaired, defective or deregulated DNA repair pathway. In some embodiments, the cancer cells comprise an impaired, defective or deregulated homologous repair (homologous recombination (HR) repair pathway. In some embodiments, the cancer cells comprise a variant BRCA protein or a sequence encoding a variant BRCA protein, wherein the variant BRCA protein induces a loss or reduction in a function of the HR pathway. In some embodiments, the variant BRCA protein comprises a variant BRCA1 protein or a variant BRCA2 protein, or the sequence encoding the variant BRCA protein comprises a sequence encoding a variant BRCA1 protein or a variant BRCA2 protein. In some embodiments, the cancer cells are ALT+ cells. In some embodiments, the cancer cells are not an ALT+ cells. In some embodiments, the cancer cells are resistant to a PARP inhibitor. In some embodiments, the cancer is an ovarian cancer or a breast cancer, optionally a BRCA1^−/−cancer or a cancer comprising tumor cells comprising a variant BRCA1 protein, or a sequence encoding the variant BRCA protein. In some embodiments, the method comprises administering to a subject having a BRCA1−/− ovarian cancer or BRCA1−/− breast cancer a Fanconi Anemia Group M protein (FANCM) blocking agent. In some embodiments, the method results in DNA damage and/or cell cycle arrest of BRCA1−/− ovarian cancer cells or BRCA1−/− breast cancer cells. In some embodiments, the method decreases survival or growth of BRCA1−/− ovarian cancer cells or BRCA1−/− breast cancer cells.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic diagram depicting a mechanism by which a FANCM blocking agent induces uncontrolled ALT and DNA damage in ALT+ cells.

FIG. 2 is a schematic diagram depicting the ligation mechanism of LIG1 that is common to all LIG1 isoforms.

FIGS. 3A-3B show that CRISPR/Cas9-mediated knockout of FANCM in BRCA1−/− cancer cell lines leads to an increase in DNA damage and G2 arrest. FIG. 3A is an overlay of flow cytometry contour plots of DNA damage marker γH2AX in TOV21G (BRCA1-proficient ovarian cancer), MDAMB436 (BRCA1−/− breast cancer) and UWB1289 (BRCA1−/− ovarian cancer) on day 7 post nucleofection with CRISPR RNP with sgRNA targeting FANCM. Cells, positive for γH2AX are selected with the gate and their percentage is indicated in the table above the plot. FIG. 3B provides cell cycle profiles of TOV21G and UWB1289 on day 7, following CRISPR/Cas9-mediated knockout of FANCM. The percentage of G2 cells in each sample is shown in the table.; NT=non-targeting gRNA control.

FIGS. 4A-4B show that CRISPR/Cas9-mediated knockout of FANCM in BRCA1−/− cancer cell lines leads to a reduction of growth in 2D clonogenic assay. FIG. 4A shows the results of a 2D clonogenic growth assay of UWB1.289 (BRCA1−/− ovarian cancer), COV362 (BRCA1−/− ovarian cancer) MDAMB436 (BRCA1−/− breast cancer), and TOV21G (BRCA1 wild-type ovarian cancer) on between 14-21 days post nucleofection with CRISPR RNP, targeting FANCM. FIG. 4B is a Western blot demonstrating loss of FANCM protein with FANCM sgRNA #3 and #4.

FIGS. 5A-5B show that shRNA mediated knockdown of FANCM in BRCA1−/− cancer cell lines leads reduction of growth in 2D clonogenic assay. FIG. 5A shows the results of a clonogenic growth assay of UWB1.289 (BRCA1−/− ovarian cancer) and MDAMB436 (BRCA1−/− breast cancer), and TOV21G (BRCA1 wild-type ovarian cancer) between 14-21 days after doxycycline induced shRNA expression. FIG. 5B shows qPCR results demonstrating loss of FANCM mRNA transcript in UWB1.289 with FANCM shRNA #1307 and #1858 follow 3 days of doxycycline treatment.

FIGS. 6A-6C show that CRISPR/Cas9-mediated knockout of FANCM in BRCA1−/− cancer cell line UWB1.289 leads to a reduction in colony formation and cell viability and is partially rescued by wild-type BRCA1 add back. FIGS. 6A and 6B show the results of short- and long-term viability assay demonstrates rescue of FANCM dependency when wild-type BRCA1 is present in UWB1.289 cells. FIG. 6C shows nuclear enriched FANCM is undetectable in both UWB1.289 null and BRCA1 add-back lines via western blot. In FIG. 6A, for each treatment, the bars from left to right correspond to UWB1.289 and UWB1.289+BRCA1, respectively.

DETAILED DESCRIPTION

Disclosed are compositions and methods for the selective targeting of components of a first or a second DNA repair pathway in cancer cells in which a first DNA repair pathway is impaired, defective or deregulated. In certain embodiments, selective targeting refers to the use of blocking agents that specifically or selectively target a particular DNA repair pathway or component thereof, e.g., to inhibit the DNA repair pathway. In certain embodiments, the selectively targeted blocking agent does not substantially or significantly target, e.g., inhibit, other DNA repair pathways.

DNA Repair

DNA repair may be accomplished by a number of pathways including, but not limited to, base excision repair (BER), nucleotide excision repair (NER), mismatch repair (MMR), homologous recombination (HR), and non-homologous end joining (NHEJ). These diverse pathways are active throughout the stages of the cell cycle in normal or healthy cells. In healthy cells (a cell in which all DNA repair pathways are fully active), at checkpoints within the cell cycle, DNA damage or replication mistakes are assessed and corrected prior to the next phase or to completion of the cycle. When DNA damage is present and DNA repair pathways are impaired, defective or deregulated, a healthy cell may initiate a programmed cell death or a cell cycle arrest.

Because there is redundancy in the DNA repair system, the impairment of one pathway among several pathways may not lead to apoptosis, but, rather, may lead to increased mutagenesis. Alternatively, or in addition, a loss of function of one component within a pathway may not be sufficient to completely inhibit that DNA repair pathway, leading instead to a functional impairment. In some circumstances, the impairment of a DNA repair pathway may lead to the generation of a malignant cell. Malignant cells of the disclosure may have one or more impaired, defective or deregulated DNA repair pathways. Alternatively, or in addition, malignant cells of the disclosure may have one or more components of the same DNA repair pathway in which a function is lost or reduced or the activity of that function is changed compared to a level of function or activity of that component in a non-malignant cell.

In various embodiments, an impaired, defective, or deregulated DNA repair pathway may be the result of one or more deletions or mutations in a gene encoding a protein in a DNA repair pathway. In certain embodiments, at least a portion of the coding region of a gene encoding a protein in a DNA repair pathway is partially or entirely deleted, resulting in expression of no protein, a truncated protein, or a mutant protein. In certain embodiments, a gene encoding a protein in a DNA repair pathway comprises one or more nucleic acid modification, such as, e.g., an insertion, deletion, or substitution, which may result in the encoded protein comprising one or more amino acid modification, such as a deletion, insertion, or substitution. The encoded protein may result from a frameshift mutation that results in at least a portion of the encoded protein being different than the wild type protein. In certain embodiments, an impaired, defective, or deregulated DNA repair pathway may be the result of a mutation in a regulatory region of a gene encoding a protein in a DNA repair pathway. For example, a promoter region may comprise one or more nucleic acid modification, such as a deletion, insertion or nucleotide substitution that results in reduced or no expression of the protein in the DNA repair pathway. In certain embodiments, the impaired, defective, or deregulated DNA repair pathway results in reduced or no activity of one or more protein in the DNA repair pathay, e.g., less than 90%, less than 80%, less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, or less than 10% activity as compared to the wild type protein. In certain embodiments, the impaired, defective, or deregulated DNA repair pathway results in reduced or no expression of one or more protein in the DNA repair pathay, e.g., less than 90%, less than 80%, less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, les than 20%, or less than 10% protein expression as compared to the amount expressed in a corresponding wild type cell.

In some embodiments, a malignant cell is targeted by a composition of the disclosure (i.e., a target cell) and the malignant or target cell may have an impaired, defective or deregulated homologous recombination (HR) pathway. In some embodiments, a malignant cell is targeted by a composition of the disclosure (i.e., a target cell) and the malignant or target cell may have an impaired, defective or deregulated base excision repair (BER) pathway. In some embodiments, a malignant cell is targeted by a composition of the disclosure (i.e., a target cell) and the malignant or target cell may have an impaired, defective or deregulated nucleotide excision repair (NER) pathway. In some embodiments, a malignant cell is targeted by a composition of the disclosure (i.e., a target cell) and the malignant or target cell may have an impaired, defective or deregulated mismatch repair (MMR) pathway. In some embodiments, a malignant cell is targeted by a composition of the disclosure (i.e., a target cell) and the malignant or target cell may have an impaired, defective or deregulated non-homologous end joining (NHEJ) pathway.

In some embodiments, compositions and methods of the disclosure target a second DNA repair pathway in a cell having an impaired, defective or deregulated first DNA repair pathway. The loss of two DNA repair pathways induces a synergistic effect on selectively and specifically targeting malignant cells to induce cell death or a cell cycle arrest in the malignant cell.

In certain embodiments, compositions and methods of the disclosure may target one of or any two components of the homologous recombination pathway. In some embodiments, compositions and methods of the disclosure may target one of or any two components of the homologous recombination pathway, including, but not limited to, BRCA1, BRCA2, LIG1, FANCM, RAD51, RAD51C, RAD51D, XRCC2, XPF, MRE11A, ATM, BARD1, BRIP1, CHEK1, CHEK2, NBN, PALB2 and SLX4. In some embodiments, compositions and methods of the disclosure may target LIG1 in a cell comprising a variant protein that impairs a function or an activity of the function of the homologous recombination (HR) pathway wherein the variant protein is a variant of one or more of: BRCA1, BRCA2, RAD51, RAD51C, RAD51D, XRCC2, XPF, MRE11A, ATM, BARD1, BRIP1, CHEK1, CHEK2, NBN, PALB2 and SLX4. In some embodiments, compositions and methods of the disclosure may target FANCM in a cell comprising a variant protein that impairs a function or an activity of the function of the homologous recombination (HR) pathway wherein the variant protein is a variant of one or more of: BRCA1, BRCA2, RAD51, RAD51C, RAD51D, XRCC2, XPF, MRE11A, ATM, BARD1, BRIP1, CHEK1, CHEK2, NBN, PALB2 and SLX4. In some embodiments, compositions and methods of the disclosure may target FANCM in a cell comprising a variant protein that impairs a function or an activity of the function of the homologous recombination (HR) repair pathway wherein the variant protein is a variant of BRCA1 or BRCA2. In some embodiments, compositions and methods of the disclosure may target LIG1 and FANCM in a cell comprising a variant protein that impairs a function or an activity of the function of the homologous recombination (HR) pathway wherein the variant protein is a variant of one or more of: BRCA1, BRCA2, RAD51, RAD51C, RAD51D, XRCC2, XPF, MRE11A, ATM, BARD1, BRIP1, CHEK1, CHEK2, NBN, PALB2 and SLX4.

Homologous Recombination

Homologous recombination (HR) is a process interrelated with one or more DNA repair pathways that target sites including, but not limited to, DNA double-stranded breaks (DSBs) and interstrand crosslinks (ICLs). Additionally, the HR pathway stabilizes replication forks during DNA synthesis.

In some embodiments of the disclosure, LIG1 blocking agents of the disclosure block the ability of LIG1 to join unligated DNA fragments, which results in an elevation of single strand breaks that causes replication fork collapse, DNA damage and reduced cell growth in cells that are deficient in homologous recombination. In some embodiments, the LIG1 blocking agent is a selective LIG1 blocking agent.

In some embodiments of the disclosure, FANCM blocking agents of the disclosure block the ability of FANCM to resolve stalled replication forks, which in the context of homologous recombination deficiency results in elevated DNA damage and reduced cell growth. In some embodiments, the FANCM blocking agent is a selective FANCM blocking agent.

Target cells of the disclosure may have an impaired, defective or deregulated HR pathway (which may also be referred to as HR repair pathway).

Target cells of the disclosure may express one or more variant proteins including, but not limited to, one or more BRCA1, BRCA2, RAD51, RAD51C, RAD51D, XRCC2, XPF, MRE11A, ATM, BARD1, BRIP1, CHEK1, CHEK2, NBN, PALB2 and SLX4 variants.

Target cells of the disclosure may contain a sequence encoding one or more variant proteins including, but not limited to, BRCA1, BRCA2, RAD51, RAD51C, RAD51D, XRCC2, XPF, MRE11A, ATM, BARD1, BRIP1, CHEK1, CHEK2, NBN, PALB2 and SLX4 variants. In certain embodiments, the sequence encoding the variant protein comprises a promoter sequence and coding sequence, e.g., wherein the promoter sequence is operative linked to the coding sequence.

Target cells of the disclosure may contain a promoter sequence controlling the expression of one or more wild type or variant proteins including, but not limited to, BRCA1, BRCA2, RAD51, RAD51C, RAD51D, XRCC2, XPF, MRE11A, ATM, BARD1, BRIP1, CHEK1, CHEK2, NBN, PALB2 and SLX4. In some embodiments, the promoter sequence is silenced. Alternatively or in addition, in some embodiments, the promoter sequence comprises a mutation that disrupts a function or an activity of the promoter. Alternatively or in addition, in some embodiments, the promoter sequence comprises a mutation that induce silencing of the promoter.

Compositions and methods of the disclosure may inhibit a component of the same pathway in which a variant protein signals. In some embodiments, the variant protein comprises one or more of BRCA1, BRCA2, RAD51, RAD51C, RAD51D, XRCC2, XPF, MRE11A, ATM, BARD1, BRIP1, CHEK1, CHEK2, NBN, PALB2 and SLX4.

Compositions and methods of the disclosure may inhibit a component of the distinct pathway from which a variant protein signals. In some embodiments, the variant protein comprises one or more of BRCA1, BRCA2, RAD51, RAD51C, RAD51D, XRCC2, XPF, MRE11A, ATM, BARD1, BRIP1, CHEK1, CHEK2, NBN, PALB2 and SLX4.

Target cells of the disclosure may comprise a modification, such as a gene deletion or mutation, that results in no expression or reduced expression of BRCA1. Target cells of the disclosure may comprise a modification, such as an insertion, deletion, or substitution in the gene encoding BRCA1 or the encoded BRCA1 protein.

Target cells of the disclosure may express a BRCA1 protein according to SEQ ID NO: 13-20. Target cells of the disclosure may express a variant BRCA1 having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99% or any percentage in between of identity to SEQ ID NOs: 13-20.

In some embodiments of the compositions and methods of the disclosure, a BRCA1 protein of the disclosure comprises or consists of the amino acid sequence of (UniProt Accession No. P38398-1 and SEQ ID NO: 13):

1
MDLSALRVEE VQNVINAMQK ILECPICLEL IKEPVSTKCD HIFCKFCMLK LLNQKKGPSQ

61
CPLCKNDITK RSLQESTRES QLVEELLKII CAFQLDTGLE YANSYNFAKK ENNSPEHLKD

121
EVSIIQSMGY RNRAKRLLQS EPENPSLQET SLSVQLSNLG TVRTLRTKQR IQPQKTSVYI

181
ELGSDSSEDT VNKATYCSVG DQELLQITPQ GTRDEISLDS AKKAACEFSE TDVTNTEHHQ

241
PSNNDLNTTE KRAAERHPEK YQGSSVSNLH VEPCGTNTHA SSLQHENSSL LLTKDRMNVE

301
KAEFCNKSKQ PGLARSQHNR WAGSKETCND RRTPSTEKKV DLNADPLCER KEWNKQKLPC

361
SENPRDTEDV PWITLNSSIQ KVNEWFSRSD ELLGSDDSHD GESESNAKVA DVLDVLNEVD

421
EYSGSSEKID LLASDPHEAL ICKSERVHSK SVESNIEDKI FGKTYRKKAS LPNLSHVTEN

481
LIIGAFVTEP QIIQERPLIN KLKRKRRPTS GLHPEDFIKK ADLAVQKTPE MINQGTNQTE

541
QNGQVMNITN SCHENKTKGD SIQNEKNPNP IESLEKESAF KTKAEPISSS ISNMELELNI

601
HNSKAPKKNR LRRKSSTRHI HALELVVSRN LSPPNCTELQ IDSCSSSEEI KKKKYNQMPV

661
RHSRNLQLME GKEPATGAKK SNKPNEQTSK RHDSDTFPEL KLTNAPGSFT KCSNTSELKE

721
FVNPSLPREE KEEKLETVKV SNNAEDPKDL MLSGERVLQT ERSVESSSIS LVPGTDYGTQ

781
ESISLLEVST LGKAKTEPNK CVSQCAAFEN PKGLIHGCSK DNRNDTEGFK YPLGHEVNHS

841
RETSIEMEES ELDAQYLQNT FKVSKRQSFA PFSNPGNAEE ECATFSAHSG SLKKQSPKVT

901
FECEQKEENQ GKNESNIKPV QTVNITAGFP VVGQKDKPVD NAKCSIKGGS RFCLSSQFRG

961
NETGLITPNK HGLLQNPYRI PPLFPIKSFV KTKCKKNLLE ENFEEHSMSP EREMGNENIP

1021
STVSTISRNN IRENVFKEAS SSNINEVGSS TNEVGSSINE IGSSDENIQA ELGRNRGPKL

1081
NAMLRLGVLQ PEVYKQSLPG SNCKHPEIKK QEYEEVVQTV NTDFSPYLIS DNLEQPMGSS

1141
HASQVCSETP DDLLDDGEIK EDTSFAENDI KESSAVESKS VQKGELSRSP SPFTHTHLAQ

1201
GYRRGAKKLE SSEENLSSED EELPCFQHLL FGKVNNIPSQ STRHSTVATE CLSKNTEENL

1261
LSLKNSLNDC SNQVILAKAS QEHHLSEETK CSASLFSSQC SELEDLTANT NTQDPFLIGS

1321
SKQMRHQSES QGVGLSDKEL VSDDEERGTG LEENNQEEQS MDSNLGEAAS GCESETSVSE

1381
DCSGLSSQSD ILTTQQRDTM QHNLIKLQQE MAELEAVLEQ HGSQPSNSYP SIISDSSALE

1441
DLRNPEQSTS EKAVLTSQKS SEYPISQNPE GLSADKFEVS ADSSTSKNKE PGVERSSPSK

1501
CPSLDDRWYM HSCSGSLQNR NYPSQEELIK VVDVEEQQLE ESGPHDLTET SYLPRQDLEG

1561
TPYLESGISL FSDDPESDPS EDRAPESARV GNIPSSTSAL KVPQLKVAES AQSPAAAHTT

1621
DTAGYNAMEE SVSREKPELT ASTERVNKRM SMVVSGLTPE EFMLVYKFAR KHHITLTNLI

1681
TEETTHVVMK TDAEFVCERT LKYFLGIAGG KWVVSYFWVT QSIKERKMLN EHDFEVRGDV

1741
VNGRNHQGPK RARESQDRKI FRGLEICCYG PFTNMPTDQL EWMVQLCGAS VVKELSSFTL

1861
GTGVHPIVVV QPDAWTEDNG FHAIGQMCEA PVVTREWVLD SVALYQCQEL DTYLIPQIPH

1801
SHY.

1
MDLSALRVEE VQNVINAMQK ILECPICLEL IKEPVSTKCD HIFCKFCMLK LLNQKKGPSQ

61
CPL.

1
MDLSALRVEE VQNVINAMQK ILECPICLEL IKEPVSTKCD HIFCKFCMLK LLNQKKGPSQ

61
CPLCKNDITK RSLQESTRFS QLVEELLKII CAFQLDTGLE YANSYNFAKK ENNSPEHLKD

121
EVSIIQSMGY RNRAKRLLQS EPENPSLQET SLSVQLSNLG TVRTLRTKQR IQPQKTSVYI

181
ELGSDSSEDT VNKATYCSVG DQELLQITPQ GTRDEISLDS AKKAACEFSE TDVTNTEHHQ

241
PSNNDLNTTE KRAAERHPEK YQGEAASGCE SETSVSEDCS GLSSQSDILT TQQRDTMQHN

301
LIKLQQEMAE LEAVLEQHGS QPSNSYPSII SDSSALEDLR NPEQSTSEKV LTSQKSSEYP

361
ISQNPEGLSA DKFEVSADSS TSKNKEPGVE RSSPSKCPSL DDRWYMHSCS GSLQNRNYPS

421
QEELIKVVDV EEQQLEESGP HDLTETSYLP RQDLEGTPYL ESGISLFSDD PESDPSEDRA

481
PESARVGNIP SSTSALKVPQ LKVAESAQSP AAAHTTDTAG YNAMEESVSR EKPELTASTE

541
RVNKRMSMVV SGLTPEEFML VYKFARKHHI TLTNLITEET THVVMKTDAE FVCERTLKYF

601
LGIAGGKWVV SYFWVTQSIK ERKMLNEHDF EVRGDVVNGR NHQGPKRARE SQDRKIFRGL

661
EICCYGPFTN MPTDQLEWMV QLCGASVVKE LSSFTLGTGV HPIVVVQPDA WTEDNGFHAI

721
GQMCEAPVVT REWVLDSVAL YQCQELDTYL IPQIPHSHY.

1
MQKILECPIC LELIKEPVST KCDHIFCKFC MLKLLNQKKG PSQCPLCKND ITKRSLQEST

61
RFSQLVEELL KIICAFQLDT GLEYANSYNF AKKENNSPEH LKDEVSIIQS MGYRNRAKRL

121
LQSEPENPSL QETSLSVQLS NLGTVRTLRT KQRIQPQKTS VYIELGSDSS EDTVNKATYC

181
SVGDQELLQI TPQGTRDEIS LDSAKKAACE FSETDVTNTE HHQPSNNDLN TTEKRAAERH

241
PEKYQGSSVS NLHVEPCGTN THASSLQHEN SSLLLTKDRM NVEKAEFCNK SKQPGLARSQ

301
HNRWAGSKET CNDRRTPSTE KKVDLNADPL CERKEWNKQK LPCSENPRDT EDVPWITLNS

361
SIQKVNEWFS RSDELLGSDD SHDGESESNA KVADVLDVLN EVDEYSGSSE KIDLLASDPH

421
EALICKSERV HSKSVESNIE DKIFGKTYRK KASLPNLSHV TENLIIGAFV TEPQIIQERP

481
LTNKLKRKRR PTSGLHPEDF IKKADLAVQK TPEMINQGTN QTEQNGQVMN ITNSGHENKT

541
KGDSIQNEKN PNPIESLEKE SAFKTKAEPI SSSISNMELE LNIHNSKAPK KNRLRRKSST

601
RHIHALELVV SRNLSPPNCT ELQIDSCSSS EEIKKKKYNQ MPVRHSRNLQ LMEGKEPATG

661
AKKSNKPNEQ TSKRHDSDTF PELKLTNAPG SFTKCSNTSE LKEFVNPSLP REEKEEKLET

721
VKVSNNAEDP KDLMLSGERV LQTERSVESS SISLVPGTDY GTQESISLLE VSTLGKAKTE

781
PNKCVSQCAA FENPKGLIHG CSKDNRNDTE GFKYPLGHEV NHSRETSIEM EESELDAQYL

841
QNTFKVSKRQ SFAPFSNPGN AEEECATFSA HSGSLKKQSP KVTFECEQKE ENQGKNESNI

901
KPVQTVNITA GFPVVGQKDK PVDNAKCSIK GGSRFCLSSQ FRGNETGLIT PNKHGLLQNP

961
YRIPPLFPIK SFVKTKCKKN LLEENFEEHS MSPEREMGNE NIPSTVSTIS RNNIRENVFK

1021
EASSSNINEV GSSTNEVGSS INEIGSSDEN IQAELGRNRG PKLNAMLRLG VLQPEVYKQS

1081
LPGSNCKHPE IKKQEYEEVV QTVNTDFSPY LISDNLEQPM GSSHASQVCS ETPDDLLDDG

1141
EIKEDTSFAE NDIKESSAVF SKSVQKGELS RSPSPFTHTH LAQGYRRGAK KLESSEENLS

1201
SEDEELPCFQ HLLFGKVNNI PSQSTRHSTV ATECLSKNTE ENLLSLKNSL NDCSNQVILA

1261
KASQEHHLSE ETKCSASLFS SQCSELEDLT ANTNTQDPFL IGSSKQMRHQ SESQGVGLSD

1321
KELVSDDEER GTGLEENNQE EQSMDSNLGE AASGCESETS VSEDCSGLSS QSDILTTQQR

1381
DTMQHNLIKL QQEMAELEAV LEQHGSQPSN SYPSIISDSS ALEDLRNPEQ STSEKAVLTS

1441
QKSSEYPISQ NPEGLSADKF EVSADSSTSK NKEPGVERSS PSKCPSLDDR WYMHSCSGSL

1501
QNRNYPSQEE LIKVVDVEEQ QLEESGPHDL TETSYLPRQD LEGTPYLESG ISLFSDDPES

1561
DPSEDRAPES ARVGNIPSST SALKVPQLKV AESAQSPAAA HTTDTAGYNA MEESVSREKP

1621
ELTASTERVN KRMSMVVSGL TPEEFMLVYK FARKHHITLT NLITEETTHV VMKTDAEFVC

1681
ERTLKYFLGI AGGKWVVSYF WVTQSIKERK MLNEHDFEVR GDVVNGRNHQ GPKRARESQD

1741
RKIFRGLEIC CYGPFTNMPT DQLEWMVQLC GASVVKELSS FTLGTGVHPI VVVQPDAWTE

1801
DNGFHAIGQM CEAPVVTREW VLDSVALYQC QELDTYLIPQ IPHSHY.

1
MDLSALRVEE VQNVINAMQK ILECPICLEL IKEPVSTKCD HIFCKFCMLK LLNQKKGPSQ

61
CPLCKNDITK RSLQESTRFS QLVEELLKII CAFQLDTGLE YANSYNFAKK ENNSPEHLKD

121
EVSIIQSMGY RNRAKRLLQS EPENPSLQET SLSVQLSNLG TVRTLRTKQR IQPQKTSVYI

181
ELGSDSSEDT VNKATYCSVG DQELLQITPQ GTRDEISLDS AKKGEAASGC ESETSVSEDC

241
SGLSSQSDIL TTQQRDTMQH NLIKLQQEMA ELEAVLEQHG SQPSNSYPSI ISDSSALEDL

301
RNPEQSTSEK AVLTSQKSSE YPISQNPEGL SADKFEVSAD SSTSKNKEPG VERSSPSKCP

361
SLDDRWYMHS CSGSLQNRNY PSQEELIKVV DVEEQQLEES GPHDLTETSY LPRQDLEGTP

421
YLESGISLES DDPESDPSED RAPESARVGN IPSSTSALKV PQLKVAESAQ SPAAAHTTDT

481
AGYNAMEESV SREKPELTAS TERVNKRMSM VVSGLTPEEF MLVYKFARKH HITLTNLITE

541
ETTHVVMKTD AEFVCERTLK YFLGIAGGKW VVSYFWVTQS IKERKMLNEH DFEVRGDVVN

601
GRNHQGPKRA RESQDRKIFR GLEICCYGPF TNMPTDQLEW MVQLCGASVV KELSSFTLGT

721
GVHPIVVVQP DAWTEDNGFH AIGQMCEAPV VTREWVLDSV ALYQCQELDT YLIPQIPHSH

661
Y.

1
MDLSALRVEE VQNVINAMQK ILECPICLEL IKEPVSTKCD HIFCKFCMLK LLNQKKGPSQ

61
CPLCKNDITK RSLQESTRES QLVEELLKII CAFQLDTGLE YANSYNFAKK ENNSPEHLKD

121
EVSIIQSMGY RNRAKRLLQS EPENPSLQET SLSVQLSNLG TVRTLRTKQR IQPQKTSVYI

181
ELGSDSSEDT VNKATYCSVG DQELLQITPQ GTRDEISLDS AKKAACEFSE TDVTNTEHHQ

241
PSNNDLNTTE KRAAERHPEK YQGSSVSNLH VEPCGTNTHA SSLQHENSSL LLTKDRMNVE

301
KAEFCNKSKQ PGLARSQHNR WAGSKETCND RRTPSTEKKV DLNADPLCER KEWNKQKLPC

361
SENPRDTEDV PWITLNSSIQ KVNEWFSRSD ELLGSDDSHD GESESNAKVA DVLDVLNEVD

421
EYSGSSEKID LLASDPHEAL ICKSERVHSK SVESNIEDKI FGKTYRKKAS LPNLSHVTEN

481
LIIGAFVTEP QIIQERPLIN KLKRKRRPTS GLHPEDFIKK ADLAVQKTPE MINQGTNQTE

541
QNGQVMNITN SGHENKTKGD SIQNEKNPNP IESLEKESAF KTKAEPISSS ISNMELELNI

601
HNSKAPKKNR LRRKSSTRHI HALELVVSRN LSPPNCTELQ IDSCSSSEEI KKKKYNQMPV

661
RHSRNLQLME GKEPATGAKK SNKPNEQTSK RHDSDTFPEL KLTNAPGSFT KCSNTSELKE

721
FVNPSLPREE KEEKLETVKV SNNAEDPKDL MLSGERVLQT ERSVESSSIS LVPGTDYGTQ

781
ESISLLEVST LGKAKTEPNK CVSQCAAFEN PKGLIHGCSK DNRNDTEGFK YPLGHEVNHS

841
RETSIEMEES ELDAQYLQNT FKVSKRQSFA PFSNPGNAEE ECATFSAHSG SLKKQSPKVT

901
FECEQKEENQ GKNESNIKPV QTVNITAGFP VVGQKDKPVD NAKCSIKGGS RFCLSSQFRG

961
NETGLITPNK HGLLQNPYRI PPLFPIKSFV KTKCKKNLLE ENFEEHSMSP EREMGNENIP

1021
STVSTISRNN IRENVFKEAS SSNINEVGSS TNEVGSSINE IGSSDENIQA ELGRNRGPKL

1081
NAMLRLGVLQ PEVYKQSLPG SNCKHPEIKK QEYEEVVQTV NTDFSPYLIS DNLEQPMGSS

1141
HASQVCSETP DDLLDDGEIK EDTSFAENDI KESSAVESKS VQKGELSRSP SPFTHTHLAQ

1201
GYRRGAKKLE SSEENLSSED EELPCFQHLL FGKVNNIPSQ STRHSTVATE CLSKNTEENL

1261
LSLKNSINDC SNQVILAKAS QEHHLSEETK CSASLFSSQC SELEDLTANT NTQDPFLIGS

1321
SKQMRHQSES QGVGLSDKEL VSDDEERGTG LEENNQEEQS MDSNLGEAAS GCESETSVSE

1381
DCSGLSSQSD ILTTQQRDTM QHNLIKLQQE MAELEAVLEQ HGSQPSNSYP SIISDSSALE

1441
DLRNPEQSTS EKDSHIHGQR NNSMFSKRPR EHISVLTSQK SSEYPISQNP EGLSADKFEV

1501
SADSSTSKNK EPGVERSSPS KCPSLDDRWY MHSCSGSLQN RNYPSQEELI KVVDVEEQQL

1561
EESGPHDLTE TSYLPRQDLE GTPYLESGIS LFSDDPESDP SEDRAPESAR VGNIPSSTSA

1621
LKVPQLKVAE SAQSPAAAHT TDTAGYNAME ESVSREKPEL TASTERVNKR MSMVVSGLTP

1681
EEFMLVYKFA RKHHITLTNL ITEETTHVVM KTDAEFVCER TLKYFLGIAG GKWVVSYFWV

1741
TQSIKERKML NEHDFEVRGD VVNGRNHQGP KRARESQDRK IFRGLEICCY GPFTNMPTDQ

1801
LEWMVQLCGA SVVKELSSFT LGTGVHPIVV VQPDAWTEDN GFHAIGQMCE APVVTREWVL

1861
DSVALYQCQE LDTYLIPQIP HSHY.

1
MLKLLNQKKG PSQCPLCKND ITKRSLQEST RFSQLVEELL KIICAFQLDT GLEYANSYNF

61
AKKENNSPEH LKDEVSIIQS MGYRNRAKRL LQSEPENPSL QETSLSVQLS NLGTVRTLRT

121
KQRIQPQKTS VYIELGSDSS EDTVNKATYC SVGDQELLQI TPQGTRDEIS LDSAKKAACE

181
FSETDVTNTE HHQPSNNDLN TTEKRAAERH PEKYQGSSVS NLHVEPCGTN THASSLQHEN

241
SSLLLTKDRM NVEKAEFCNK SKQPGLARSQ HNRWAGSKET CNDRRTPSTE KKVDLNADPL

301
CERKEWNKQK LPCSENPRDT EDVPWITLNS SIQKVNEWFS RSDELLGSDD SHDGESESNA

361
KVADVLDVLN EVDEYSGSSE KIDLLASDPH EALICKSERV HSKSVESNIE DKIFGKTYRK

421
KASLPNLSHV TENLIIGAFV TEPQIIQERP LTNKLKRKRR PTSGLHPEDF IKKADLAVQK

481
TPEMINQGTN QTEQNGQVMN ITNSGHENKT KGDSIQNEKN PNPIESLEKE SAFKTKAEPI

541
SSSISNMELE LNIHNSKAPK KNRLRRKSST RHIHALELVV SRNLSPPNCT ELQIDSCSSS

601
EEIKKKKYNQ MPVRHSRNLQ LMEGKEPATG AKKSNKPNEQ TSKRHDSDTF PELKLTNAPG

661
SFTKCSNTSE LKEFVNPSLP REEKEEKLET VKVSNNAEDP KDLMLSGERV LQTERSVESS

721
SISLVPGTDY GTQESISLLE VSTLGKAKTE PNKCVSQCAA FENPKGLIHG CSKDNRNDTE

781
GFKYPLGHEV NHSRETSIEM EESELDAQYL QNTFKVSKRQ SFAPFSNPGN AEEECATFSA

841
HSGSLKKQSP KVTFECEQKE ENQGKNESNI KPVQTVNITA GFPVVGQKDK PVDNAKCSIK

901
GGSRFCLSSQ FRGNETGLIT PNKHGLLQNP YRIPPLFPIK SFVKTKCKKN LLEENFEEHS

961
MSPEREMGNE NIPSTVSTIS RNNIRENVFK EASSSNINEV GSSTNEVGSS INEIGSSDEN

1021
IQAELGRNRG PKLNAMLRLG VLQPEVYKQS LPGSNCKHPE IKKQEYEEVV QTVNTDFSPY

1081
LISDNLEQPM GSSHASQVCS ETPDDLLDDG EIKEDTSFAE NDIKESSAVF SKSVQKGELS

1141
RSPSPFTHTH LAQGYRRGAK KLESSEENLS SEDEELPCFQ HLLFGKVNNI PSQSTRHSTV

1201
ATECLSKNTE ENLLSLKNSL NDCSNQVILA KASQEHHLSE ETKCSASLFS SQCSELEDLT

1261
ANTNTQDPFL IGSSKQMRHQ SESQGVGLSD KELVSDDEER GTGLEENNQE EQSMDSNLGE

1321
AASGCESETS VSEDCSGLSS QSDILTTQQR DTMQHNLIKL QQEMAELEAV LEQHGSQPSN

1381
SYPSIISDSS ALEDLRNPEQ STSEKAVLTS QKSSEYPISQ NPEGLSADKF EVSADSSTSK

1441
NKEPGVERSS PSKCPSLDDR WYMHSCSGSL QNRNYPSQEE LIKVVDVEEQ QLEESGPHDL

1501
TETSYLPRQD LEGTPYLESG ISLFSDDPES DPSEDRAPES ARVGNIPSST SALKVPQLKV

1561
AESAQSPAAA HTTDTAGYNA MEESVSREKP ELTASTERVN KRMSMVVSGL TPEEFMLVYK

1621
FARKHHITLT NLITEETTHV VMKTDAEFVC ERTLKYFLGI AGGKWVVSYF WVTQSIKERK

1681
MLNEHDFEVR GDVVNGRNHQ GPKRARESQD RKIFRGLEIC CYGPFTNMPT DQLEWMVQLC

1801
GASVVKELSS FTLGTGVHPI VVVQPDAWTE DNGFHAIGQM CEAPVVTREW VLDSVALYQC

1741
QELDTYLIPQ IPHSHY.

Target cells of the disclosure may comprise a modification, such as a gene deletion or mutation, that results in no expression or reduced expression of BRCA2. Target cells of the disclosure may comprise a modification, such as an insertion, deletion, or substitution in the gene encoding BRCA2 or the encoded BRCA2 protein. Target cells of the disclosure may express a BRCA2 protein according to SEQ ID NO: 21. Target cells of the disclosure may express a variant BRCA2 having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99% or any percentage in between of identity to SEQ ID NO: 21.

In some embodiments of the compositions and methods of the disclosure, a BRCA2 protein of the disclosure comprises or consists of the amino acid sequence of (UniProt Accession No. P51587-1 and SEQ ID NO: 21):

1
MPIGSKERPT FFEIFKTRCN KADLGPISLN WFEELSSEAP PYNSEPAEES EHKNNNYEPN

61
LFKTPQRKPS YNQLASTPII FKEQGLTLPL YQSPVKELDK FKLDLGRNVP NSRHKSLRTV

121
KTKMDQADDV SCPLLNSCLS ESPVVLQCTH VTPQRDKSVV CGSLFHTPKF VKGRQTPKHI

181
SESLGAEVDP DMSWSSSLAT PPTLSSTVLI VRNEEASETV FPHDTTANVK SYFSNHDESL

241
KKNDRFIASV TDSENTNQRE AASHGFGKTS GNSFKVNSCK DHIGKSMPNV LEDEVYETVV

301
DTSEEDSFSL CFSKCRTKNL QKVRTSKTRK KIFHEANADE CEKSKNQVKE KYSFVSEVEP

361
NDTDPLDSNV ANQKPFESGS DKISKEVVPS LACEWSQLTL SGLNGAQMEK IPLLHISSCD

421
QNISEKDLLD TENKRKKDFL TSENSLPRIS SLPKSEKPLN EETVVNKRDE EQHLESHTDC

481
ILAVKQAISG TSPVASSFQG IKKSIFRIRE SPKETFNASF SGHMTDPNFK KETEASESGL

541
EIHTVCSQKE DSLCPNLIDN GSWPATTTQN SVALKNAGLI STLKKKTNKF IYAIHDETSY

601
KGKKIPKDQK SELINCSAQF EANAFEAPLT FANADSGLLH SSVKRSCSQN DSEEPTLSLT

661
SSFGTILRKC SRNETCSNNT VISQDLDYKE AKCNKEKLQL FITPEADSLS CLQEGQCEND

721
PKSKKVSDIK EEVLAAACHP VQHSKVEYSD TDFQSQKSLL YDHENASTLI LTPTSKDVLS

781
NLVMISRGKE SYKMSDKLKG NNYESDVELT KNIPMEKNQD VCALNENYKN VELLPPEKYM

841
RVASPSRKVQ FNQNTNLRVI QKNQEETTSI SKITVNPDSE ELFSDNENNF VFQVANERNN

901
LALGNTKELH ETDLTCVNEP IFKNSTMVLY GDTGDKQATQ VSIKKDLVYV LAEENKNSVK

961
QHIKMTLGQD LKSDISLNID KIPEKNNDYM NKWAGLLGPI SNHSFGGSFR TASNKEIKLS

1021
EHNIKKSKMF FKDIEEQYPT SLACVEIVNT LALDNQKKLS KPQSINTVSA HLQSSVVVSD

1081
CKNSHITPQM LFSKQDFNSN HNLTPSQKAE ITELSTILEE SGSQFEFTQF RKPSYILQKS

1141
TFEVPENQMT ILKTTSEECR DADLHVIMNA PSIGQVDSSK QFEGTVEIKR KFAGLLKNDC

1201
NKSASGYLTD ENEVGFRGFY SAHGTKLNVS TEALQKAVKL FSDIENISEE TSAEVHPISL

1261
SSSKCHDSVV SMFKIENHND KTVSEKNNKC QLILQNNIEM TTGTFVEEIT ENYKRNTENE

1321
DNKYTAASRN SHNLEFDGSD SSKNDTVCIH KDETDLLFTD QHNICLKLSG QFMKEGNTQI

1381
KEDLSDLTFL EVAKAQEACH GNTSNKEQLT ATKTEQNIKD FETSDTFFQT ASGKNISVAK

1441
ESFNKIVNFF DQKPEELHNF SLNSELHSDI RKNKMDILSY EETDIVKHKI LKESVPVGTG

1501
NQLVTFQGQP ERDEKIKEPT LLGFHTASGK KVKIAKESLD KVKNLFDEKE QGTSEITSFS

1561
HQWAKTLKYR EACKDLELAC ETIEITAAPK CKEMQNSLNN DKNLVSIETV VPPKLLSDNL

1621
CRQTENLKTS KSIFLKVKVH ENVEKETAKS PATCYTNQSP YSVIENSALA FYTSCSRKTS

1681
VSQTSLLEAK KWLREGIFDG QPERINTADY VGNYLYENNS NSTIAENDKN HLSEKQDTYL

1741
SNSSMSNSYS YHSDEVYNDS GYLSKNKLDS GIEPVLKNVE DQKNTSFSKV ISNVKDANAY

1801
PQTVNEDICV EELVTSSSPC KNKNAAIKLS ISNSNNFEVG PPAFRIASGK IVCVSHETIK

1861
KVKDIFTDSF SKVIKENNEN KSKICQTKIM AGCYEALDDS EDILHNSLDN DECSTHSHKV

1921
FADIQSEEIL QHNQNMSGLE KVSKISPCDV SLETSDICKC SIGKLHKSVS SANTCGIFST

1981
ASGKSVQVSD ASLQNARQVF SEIEDSTKQV FSKVLFKSNE HSDQLTREEN TAIRTPEHLI

2041
SQKGFSYNVV NSSAFSGFST ASGKQVSILE SSLHKVKGVL EEFDLIRTEH SLHYSPTSRQ

2101
NVSKILPRVD KRNPEHCVNS EMEKTCSKEF KLSNNLNVEG GSSENNHSIK VSPYLSQFQQ

2161
DKQQLVLGTK VSLVENIHVL GKEQASPKNV KMEIGKTETF SDVPVKTNIE VCSTYSKDSE

2221
NYFETEAVEI AKAFMEDDEL TDSKLPSHAT HSLFTCPENE EMVLSNSRIG KRRGEPLILV

2281
GEPSIKRNLL NEFDRIIENQ EKSLKASKST PDGTIKDRRL FMHHVSLEPI TCVPFRTTKE

2341
RQEIQNPNFT APGQEFLSKS HLYEHLTLEK SSSNLAVSGH PFYQVSATRN EKMRHLITTG

2401
RPTKVFVPPF KTKSHFHRVE QCVRNINLEE NRQKQNIDGH GSDDSKNKIN DNEIHQFNKN

2461
NSNQAAAVTF TKCEEEPLDL ITSLQNARDI QDMRIKKKQR QRVFPQPGSL YLAKTSTLPR

2521
ISLKAAVGGQ VPSACSHKQL YTYGVSKHCI KINSKNAESF QFHTEDYFGK ESLWTGKGIQ

2581
LADGGWLIPS NDGKAGKEEF YRALCDTPGV DPKLISRIWV YNHYRWIIWK LAAMECAFPK

2641
EFANRCLSPE RVLLQLKYRY DTEIDRSRRS AIKKIMERDD TAAKTLVLCV SDIISLSANI

2701
SETSSNKTSS ADTQKVAIIE LTDGWYAVKA QLDPPLLAVL KNGRLTVGQK IILHGAELVG

2761
SPDACTPLEA PESLMLKISA NSTRPARWYT KLGFFPDPRP FPLPLSSLFS DGGNVGCVDV

2821
IIQRAYPIQW MEKTSSGLYI FRNEREEEKE AAKYVEAQQK RLEALFTKIQ EEFEEHEENT

2881
TKPYLPSRAL TRQQVRALQD GAELYEAVKN AADPAYLEGY FSEEQLRALN NHRQMLNDKK

2941
QAQIQLEIRK AMESAEQKEQ GLSRDVTTVW KLRIVSYSKK EKDSVILSIW RPSSDLYSLL

3001
TEGKRYRIYH LATSKSKSKS ERANIQLAAT KKTQYQQLPV SDEILFQIYQ PREPLHFSKF

3061
LDPDFQPSCS EVDLIGFVVS VVKKTGLAPF VYLSDECYNL LAIKFWIDLN EDIIKPHMLI

3121
AASNLQWRPE SKSGLLTLFA GDFSVFSASP KEGHFQETFN KMKNTVENID ILCNEAENKL

3181
MHILHANDPK WSTPTKDCTS GPYTAQIIPG TGNKLLMSSP NCEIYYQSPL SLCMAKRKSV

3241
STPVSAQMTS KSCKGEKEID DQKNCKKRRA LDFLSRLPLP PPVSPICTFV SPAAQKAFQP

3301
PRSCGTKYET PIKKKELNSP QMTPFKKFNE ISLLESNSIA DEELALINTQ ALLSGSTGEK

3361
QFISVSESTR TAPTSSEDYL RLKRRCTTSL IKEQESSQAS TEECEKNKQD TITTKKYI.

In certain embodiments, a target cell comprises a BRCA gene with a mutation disclosed in Fatemeh Karami and Parvin Mehdipour, A Comprehensive Focus on Global Spectrum of BRCA1 and BRCA2 Mutations in Breast Cancer, Hindawi Publishing Corporation, BioMed Research International, Volume 2013, Article ID 928562, or Babita Sharma, MSc, Raman Preet Kaur, PhD, Sonali Raut, MSc, and Anjana Munshi, PhD, BRCA1 mutation spectrum, functions, and therapeutic strategies: The story so far, Current Problems in Cancer, 42, 2018, 189-207.

Target cells of the disclosure may comprise a modification, such as a gene deletion or mutation, that results in no expression or reduced expression of RAD51. Target cells of the disclosure may comprise a modification, such as an insertion, deletion, or substitution in the gene encoding RAD51 or the encoded RAD51 protein. Target cells of the disclosure may express a RAD51 protein according to SEQ ID NOs: 22-25. Target cells of the disclosure may express a variant RAD51 having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99% or any percentage in between of identity to SEQ ID NOs: 22-25.

In some embodiments of the compositions and methods of the disclosure, a RAD51 protein of the disclosure comprises or consists of the amino acid sequence of (UniProt Accession No. Q06609-1 and SEQ ID NO: 22):

1
MAMQMQLEAN ADTSVEEESF GPQPISRLEQ EFHQRRSEII QITTGSKELD AVAYAPKKEL

61
INIKGISEAK ADKILAEAAK LVPMGFTTAT CGINANDVKK LEEAGFHTVE KLLQGGIETG

121
SITEMFGEFR TGKTQICHTL AVTCQLPIDR GGGEGKAMYI DTEGTFRPER LLAVAERYGL

181
SGSDVLDNVA YARAFNTDHQ TQLLYQASAM MVESRYALLI VDSATALYRT DYSGRGELSA

241
RQMHLARFLR MLLRLADEFG VAVVITNQVV AQVDGAAMFA ADPKKPIGGN IIAHASTTRL

301
YLRKGRGETR ICKIYDSPCL PEAEAMFAIN ADGVGDAKD.

1
MAMQMQLEAN ADTSVEEESF GPQPISRLEQ CGINANDVKK LEEAGFHTVE AVAYAPKKEL

61
INIKGISEAK ADKILAVAER YGLSGSDVLD NVAYARAFNT DHQTQLLYQA SAMMVESRYA

121
LLIVDSATAL YRTDYSGRGE LSARQMHLAR FLRMLLRLAD EFGVAVVITN QVVAQVDGAA

181
MFAADPKKPI GGNIIAHAST TRLYLRKGRG ETRICKIYDS PCLPEAEAMF AINADGVGDA

241
KD.

1
MAMQMQLEAN ADTSVEEESF GPQPISRLEQ CGINANDVKK LEEAGFHTVE AVAYAPKKEL

61
INIKGISEAK ADKILAEAAK LVPMGFTTAT EFHQRRSEII QITTGSKELD KLLQGGIETG

121
SITEMFGEFR TGKTQICHTL AVTCQLPIDR GGGEGKAMYI DTEGTFRPER LLAVAERYGL

181
SGSDVLDNVA YARAFNTDHQ TQLLYQASAM MVESRYALLI VDSATALYRT DYSGRGELSA

241
RQMHLARFLR MLLRLADEIV SEERKRGNQN LQNLRLSLSS.

1
MAMQMQLEAN ADTSVEEESF GPQPISRLEQ CGINANDVKK LEEAGFHTVE AVAYAPKKEL

61
INIKGISEAK ADKILTESRS VARLECNSVI LVYCTLRLSG SSDSPASASR VVGTTGGIET

121
GSITEMFGEF RTGKTQICHT LAVTCQLPID RGGGEGKAMY IDTEGTFRPE RLLAVAERYG

181
LSGSDVLDNV AYARAFNTDH QTQLLYQASA MMVESRYALL IVDSATALYR TDYSGRGELS

241
ARQMHLARFL RMLLRLADEF GVAVVITNQV VAQVDGAAMF AADPKKPIGG NIIAHASTTR

301
LYLRKGRGET RICKIYDSPC LPEAEAMFAI NADGVGDAKD.

Target cells of the disclosure may comprise a modification, such as a gene deletion or mutation, that results in no expression or reduced expression of RAD51C. Target cells of the disclosure may comprise a modification, such as an insertion, deletion, or substitution in the gene encoding RAD51C or the encoded RAD51C protein. Target cells of the disclosure may express a RAD51C protein according to SEQ ID NOs: 26 or 27. Target cells of the disclosure may express a variant RAD51C having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99% or any percentage in between of identity to SEQ ID NOs: 26 or 27.

In some embodiments of the compositions and methods of the disclosure, a RAD51C protein of the disclosure comprises or consists of the amino acid sequence of (UniProt Accession No. 043502-1 and SEQ ID NO: 26):

1
MRGKTFRFEM QRDLVSFPLS PAVRVKLVSA GFQTAEELLE VKPSELSKEV GISKAEALET

61
LQIIRRECLT NKPRYAGTSE SHKKCTALEL LEQEHTQGFI ITFCSALDDI LGGGVPLMKT

121
TEICGAPGVG KTQLCMQLAV DVQIPECFGG VAGEAVFIDT EGSFMVDRVV DLATACIQHL

181
QLIAEKHKGE EHRKALEDFT LDNILSHIYY FRCRDYTELL AQVYLLPDFL SEHSKVRLVI

241
VDGIAFPFRH DLDDLSLRTR LLNGLAQQMI SLANNHRLAV ILTNQMTTKI DRNQALLVPA

301
LGESWGHAAT IRLIFHWDRK QRLATLYKSP SQKECTVLFQ IKPQGFRDTV VTSACSLQTE

361
GSLSTRKRSR DPEEEL.

1
MRGKTFRFEM QRDLVSFPLS PAVRVKLVSA GFQTAEELLE VKPSELSKEV GISKAEALET

61
LQIIRRECLT NKPRYAGTSE SHKKCTALEL LEQEHTQGFI ITFCSALDDI LGGGVPLMKT

121
TEICGAPGVG KTQLW.

Target cells of the disclosure may express a RAD51D protein according to SEQ ID NOs: 28-35. Target cells of the disclosure may express a variant RAD51D having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99% or any percentage in between of identity to SEQ ID NOs: 28-35.

Target cells of the disclosure may comprise a modification, such as a gene deletion or mutation, that results in no expression or reduced expression of RAD51D. Target cells of the disclosure may comprise a modification, such as an insertion, deletion, or substitution in the gene encoding RAD51D or the encoded RAD51D protein. In some embodiments of the compositions and methods of the disclosure, a RAD51D protein of the disclosure comprises or consists of the amino acid sequence of (UniProt Accession No. 075771-1 and SEQ ID NO: 28):

1
MGVLRVGLCP GLTEEMIQLL RSHRIKTVVD LVSADLEEVA QKCGLSYKAL VALRRVLLAQ

61
FSAFPVNGAD LYEELKTSTA ILSTGIGSLD KLLDAGLYTG EVTEIVGGPG SGKTQVCLCM

121
AANVAHGLQQ NVLYVDSNGG LTASRLLQLL QAKTQDEEEQ AEALRRIQVV HAFDIFQMLD

181
VLQELRGTVA QQVTGSSGTV KVVVVDSVTA VVSPLLGGQQ REGLALMMQL ARELKTLARD

241
LGMAVVVTNH ITRDRDSGRL KPALGRSWSF VPSTRILLDT IEGAGASGGR RMACLAKSSR

301
QPTGFQEMVD IGTWGTSEQS ATLQGDQT.

In some embodiments of the compositions and methods of the disclosure, a RAD51D protein of the disclosure comprises or consists of the amino acid sequence of (UniProt Accession No. 075771-2 and SEQ ID NO: 29):

1
MGVLRVGLCP GLTEEMIQLL RSHRIKTVVD LVSADLEEVA QKCGLSYKS.

1
MGVLRVGLCP GLTEEMIQLL RSHRIKTVVD LVSADLEEVA QKCGLSYKAE ALRRIQVVHA

61
FDIFQMLDVL QELRGTVAQQ VTGSSGTVKV VVVDSVTAVV SPLLGGQQRE GLALMMQLAR

121
ELKTLARDLG MAVVVTNHIT RDRDSGRLKP ALGRSWSFVP STRILLDTIE GAGASGGRRM

181
ACLAKSSRQP TGFQEMVDIG TWGTSEQSAT LQGDQT.

1
MGVLRVGLCP GLTEEMIQLL RSHRIKTVVD LVSADLEEVA QKCGLSYKAL VALRRVLLAQ

61
FSAFPVNGAD LYEELKTSTA ILSTGIGSLD KLLDAGLYTG EVTEIVGGPG SGKTQAEALR

121
RIQVVHAFDI FQMLDVLQEL RGTVAQQVTG SSGTVKVVVV DSVTAVVSPL LGGQQREGLA

181
LMMQLARELK TLARDLGMAV VVTNHITRDR DSGRLKPALG RSWSFVPSTR ILLDTIEGAG

241
ASGGRRMACL AKSSRQPTGF QEMVDIGTWG TSEQSATLQG DQT.

1
MGVLRVGLCP GLTEEMIQLL RSHRIKTVVD LVSADLEEVA QKCGLSYKAL VALRRVLLAQ

61
FSAFPVNGAD LYEELKTSTA ILSTGIGRQK LSGGSRWCMH LVTEIVGGPG SGKTQVCLCM

121
AANVAHGLQQ NVLYVDSNGG LTASRLLQLL QAKTQDEEEQ AEALRRIQVV HAFDIFQMLD

181
VLQELRGTVA QQVTGSSGTV KVVVVDSVTA VVSPLLGGQQ REGLALMMQL ARELKTLARD

241
LGMAVVVTNH ITRDRDSGRL KPALGRSWSF VPSTRILLDT IEGAGASGGR RMACLAKSSR

301
QPTGFQEMVD IGTWGTSEQS ATLQGDQT.

1
MGVLRVGLCP GLTEEMIQLL RSHRIKTVVD LVSADLEEVA QKCGLSYKAL VALRRVLLAQ

61
FSAFPVNGAD LYEELKTSTA ILSTGIGRHG GRTQVGTWED CSCLRSPQGD RGVGSGML.

1
MGVLRVGLCP GLTEEMIQLL RSHRIKTVVD LVSADLEEVA QKCGLSYKTW RAHSSGNLGG

61
LQLPQVPAGR SWSGVRNALK KAGLGHGGTD GLSLNAFDER GTAVSTSRLD KLLDAGLYTG

121
EVTEIVGGPG SGKTQVCLCM AANVAHGLQQ NVLYVDSNGG LTASRLLQLL QAKTQDEEEQ

181
AEALRRIQVV HAFDIFQMLD VLQELRGTVA QQVTGSSGTV KVVVVDSVTA VVSPLLGGQQ

241
REGLALMMQL ARELKTLARD LGMAVVVTNH ITRDRDSGRL KPALGRSWSF VPSTRILLDT

301
IEGAGASGGR RMACLAKSSR QPTGFQEMVD IGTWGTSEQS ATLQGDQT.

Target cells of the disclosure may comprise a modification, such as a gene deletion or mutation, that results in no expression or reduced expression of XRCC2. Target cells of the disclosure may comprise a modification, such as an insertion, deletion, or substitution in the gene encoding XRCC2 or the encoded XRCC2 protein. Target cells of the disclosure may express a XRCC2 protein according to SEQ ID NO: 36. Target cells of the disclosure may express a variant XRCC2 having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99% or any percentage in between of identity to SEQ ID NO: 36.

In some embodiments of the compositions and methods of the disclosure, a XRCC2 protein of the disclosure comprises or consists of the amino acid sequence of (UniProt Accession No. 043543-1 and SEQ ID NO: 36):

1
MCSAFHRAES GTELLARLEG RSSLKEIEPN LFADEDSPVH GDILEFHGPE GTGKTEMLYH

61
LTARCILPKS EGGLEVEVLF IDTDYHFDML RLVTILEHRL SQSSEEIIKY CLGRFFLVYC

121
SSSTHLLLTL YSLESMFCSH PSLCLLILDS LSAFYWIDRV NGGESVNLQE STLRKCSQCL

181
EKLVNDYRLV LFATTQTIMQ KASSSSEEPS HASRRLCDVD IDYRPYLCKA WQQLVKHRMF

241
FSKQDDSQSS NQFSLVSRCL KSNSLKKHFF IIGESGVEFC.

Target cells of the disclosure may comprise a modification, such as a gene deletion or mutation, that results in no expression or reduced expression of XPF. Target cells of the disclosure may comprise a modification, such as an insertion, deletion, or substitution in the gene encoding XPF or the encoded XPF protein. Target cells of the disclosure may express a XPF protein according to SEQ ID NO: 37 or 38. Target cells of the disclosure may express a variant XPF having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99% or any percentage in between of identity to SEQ ID NO: 37 or 38.

In some embodiments of the compositions and methods of the disclosure, a XPF protein of the disclosure comprises or consists of the amino acid sequence of (UniProt Accession No. Q92889-1 and SEQ ID NO: 37):

1
MESGQPARRI AMAPLLEYER QLVLELLDTD GLVVCARGLG ADRLLYHFLQ LHCHPACLVL

61
VLNTQPAEEE YFINQLKIEG VEHLPRRVTN EITSNSRYEV YTQGGVIFAT SRILVVDFLT

121
DRIPSDLITG ILVYRAHRII ESCQEAFILR LFRQKNKRGF IKAFTDNAVA FDTGFCHVER

181
VMRNLFVRKL YLWPRFHVAV NSFLEQHKPE VVEIHVSMTP TMLAIQTAIL DILNACLKEL

241
KCHNPSLEVE DLSLENAIGK PFDKTIRHYL DPLWHQLGAK TKSLVQDLKI LRTLLQYLSQ

301
YDCVTFLNLL ESLRATEKAF GQNSGWLFLD SSTSMFINAR ARVYHLPDAK MSKKEKISEK

361
MEIKEGEETK KELVLESNPK WEALTEVLKE IEAENKESEA LGGPGQVLIC ASDDRTCSQL

421
RDYITLGAEA FLLRLYRKTF EKDSKAEEVW MKFRKEDSSK RIRKSHKRPK DPQNKERAST

481
KERTLKKKKR KLTLTQMVGK PEELEEEGDV EEGYRREISS SPESCPEEIK HEEFDVNLSS

541
DAAFGILKEP LTIIHPLLGC SDPYALTRVL HEVEPRYVVL YDAELTFVRQ LEIYRASRPG

601
KPLRVYFLIY GGSTEEQRYL TALRKEKEAF EKLIREKASM VVPEEREGRD ETNLDLVRGT

661
ASADVSTDTR KAGGQEQNGT QQSIVVDMRE FRSELPSLIH RRGIDIEPVT LEVGDYILTP

721
EMCVERKSIS DLIGSLNNGR LYSQCISMSR YYKRPVLLIE FDPSKPFSLT SRGALFQEIS

781
SNDISSKLTL LTLHFPRLRI LWCPSPHATA ELFEELKQSK PQPDAATALA ITADSETLPE

841
SEKYNPGPQD FLLKMPGVNA KNCRSLMHHV KNIAELAALS QDELTSILGN AANAKQLYDF

901
IHTSFAEVVS KGKGKK.

Target cells of the disclosure may comprise a modification, such as a gene deletion or mutation, that results in no expression or reduced expression of MRE11A. Target cells of the disclosure may comprise a modification, such as an insertion, deletion, or substitution in the gene encoding MRE11A or the encoded MRE11A protein. Target cells of the disclosure may express a MRE11A protein according to SEQ ID NO: 39-41. Target cells of the disclosure may express a variant MRE11A having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99% or any percentage in between of identity to SEQ ID NO: 39-41.

In some embodiments of the compositions and methods of the disclosure, a MRE11A protein of the disclosure comprises or consists of the amino acid sequence of (UniProt Accession No. P49959-1 and SEQ ID NO: 39):

1
MSTADALDDE NTFKILVATD IHLGFMEKDA VRGNDTFVTL

DEILRLAQEN EVDFILLGGD

61
LFHENKPSRK TLHTCLELLR KYCMGDRPVQ FEILSDQSVN

FGFSKFPWVN YQDGNLNISI

121
PVFSIHGNHD DPTGADALCA LDILSCAGFV NHFGRSMSVE

KIDISPVLLQ KGSTKIALYG

181
LGSIPDERLY RMFVNKKVTM LRPKEDENSW FNLFVIHQNR

SKHGSTNFIP EQFLDDFIDL

241
VIWGHEHECK IAPTKNEQQL FYISQPGSSV VTSLSPGEAV

KKHVGLLRIK GRKMNMHKIP

301
LHTVRQFFME DIVLANHPDI FNPDNPKVTQ AIQSFCLEKI

EEMLENAERE RLGNSHQPEK

361
PLVRLRVDYS GGFEPFSVLR FSQKFVDRVA NPKDIIHFFR

HREQKEKTGE EINFGKLITK

421
PSEGTTLRVE DLVKQYFQTA EKNVQLSLLT ERGMGEAVQE

FVDKEEKDAI EELVKYQLEK

481
TQRFLKERHI DALEDKIDEE VRRFRETRQK NTNEEDDEVR

EAMTRARALR SQSEESASAF

541
SADDLMSIDL AEQMANDSDD SISAATNKGR GRGRGRRGGR

GQNSASRGGS QRGRADTGLE

601
TSTRSRNSKT AVSASRNMSI IDAFKSTRQQ PSRNVTTKNY

SEVIEVDESD VEEDIFPTTS

661
KTDQRWSSTS SSKIMSQSQV SKGVDFESSE DDDDDPFMNT

SSLRRNRR.

1
MSTADALDDE NTFKILVATD IHLGFMEKDA VRGNDTFVTL

DEILRLAQEN EVDFILLGGD

61
LFHENKPSRK TLHTCLELLR KYCMGDRPVQ FEILSDQSVN

FGFSKFPWVN YQDGNLNISI

121
PVFSIHGNHD DPTGADALCA LDILSCAGFV NHFGRSMSVE

KIDISPVLLQ KGSTKIALYG

181
LGSIPDERLY RMFVNKKVTM LRPKEDENSW FNLFVIHQNR

SKHGSTNFIP EQFLDDFIDL

241
VIWGHEHECK IAPTKNEQQL FYISQPGSSV VTSLSPGEAV

KKHVGLLRIK GRKMNMHKIP

301
LHTVRQFFME DIVLANHPDI FNPDNPKVTQ AIQSFCLEKI

EEMLENAERE RLGNSHQPEK

361
PLVRLRVDYS GGFEPFSVLR FSQKFVDRVA NPKDIIHFFR

HREQKEKTGE EINFGKLITK

421
PSEGTTLRVE DLVKQYFQTA EKNVQLSLLT ERGMGEAVQE

FVDKEEKDAI EELVKYQLEK

481
TQRFLKERHI DALEDKIDEE VRRFRETRQK NTNEEDDEVR

EAMTRARALR SQSEESASAF

541
SADDLMSIDL AEQMANDSDD SISAATNKGR GRGRGRRGGR

GQNSASRGGS QRGRAFKSTR

601
QQPSRNVTTK NYSEVIEVDE SDVEEDIFPT TSKTDQRWSS

TSSSKIMSQS QVSKGVDFES

661
SEDDDDDPFM NTSSLRRNRR.

1
MNRNISHQKG DDENTFKILV ATDIHLGFME KDAVRGNDTF

VTLDEILRLA QENEVDFILL

61
GGDLFHENKP SRKTLHTCLE LLRKYCMGDR PVQFEILSDQ

SVNFGFSKFP WVNYQDGNLN

121
ISIPVFSIHG NHDDPTGADA LCALDILSCA GFVNHFGRSM

SVEKIDISPV LLQKGSTKIA

181
LYGLGSIPDE RLYRMFVNKK VTMLRPKEDE NSWFNLFVIH

QNRSKHGSTN FIPEQFLDDF

241
IDLVIWGHEH ECKIAPTKNE QQLFYISQPG SSVVTSLSPG

EAVKKHVGLL RIKGRKMNMH

301
KIPLHTVRQF FMEDIVLANH PDIFNPDNPK VTQAIQSFCL

EKIEEMLENA ERERLGNSHQ

361
PEKPLVRLRV DYSGGFEPFS VLRFSQKFVD RVANPKDIIH

FFRHREQKEK TGEEINFGKL

421
ITKPSEGTTL RVEDLVKQYF QTAEKNVQLS LLTERGMGEA

VQEFVDKEEK DAIEELVKYQ

481
LEKTQRFLKE RHIDALEDKI DEEVRRFRET RQKNTNEEDD

EVREAMTRAR ALRSQSEESA

541
SAFSADDLMS IDLAEQMAND SDDSISAATN KGRGRGRGRR

GGRGQNSASR GGSQRGRADT

601
GLETSTRSRN SKTAVSASRN MSIIDAFKST RQQPSRNVTT

KNYSEVIEVD ESDVEEDIFP

661
TTSKTDQRWS STSSSKIMSQ SQVSKGVDFE SSEDDDDDPF

MNTSSLRRNR R.

Target cells of the disclosure may comprise a modification, such as a gene deletion or mutation, that results in no expression or reduced expression of ATM. Target cells of the disclosure may comprise a modification, such as an insertion, deletion, or substitution in the gene encoding ATM or the encoded ATM protein. Target cells of the disclosure may express an ATM protein according to SEQ ID NO: 42. Target cells of the disclosure may express a variant ATM having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99% or any percentage in between of identity to SEQ ID NO: 42.

In some embodiments of the compositions and methods of the disclosure, an ATM protein of the disclosure comprises or consists of the amino acid sequence of (UniProt Accession No. Q13315-1 and SEQ ID NO: 42):

1
MSLVLNDLLI CCRQLEHDRA TERKKEVEKF KRLIRDPETI

KHLDRHSDSK QGKYLNWDAV

61
FRFLQKYIQK ETECLRIAKP NVSASTQASR QKKMQEISSL

VKYFIKCANR RAPRLKCQEL

121
LNYIMDTVKD SSNGAIYGAD CSNILLKDIL SVRKYWCEIS

QQQWLELFSV YFRLYLKPSQ

181
DVHRVLVARI IHAVTKGCCS QTDGLNSKFL DFFSKAIQCA

RQEKSSSGLN HILAALTIFL

241
KTLAVNFRIR VCELGDEILP TLLYIWTQHR LNDSLKEVII

ELFQLQIYIH HPKGAKTQEK

301
GAYESTKWRS ILYNLYDLLV NEISHIGSRG KYSSGFRNIA

VKENLIELMA DICHQVFNED

361
TRSLEISQSY TTTQRESSDY SVPCKRKKIE LGWEVIKDHL

QKSQNDFDLV PWLQIATQLI

421
SKYPASLPNC ELSPLLMILS QLLPQQRHGE RTPYVLRCLT

EVALCQDKRS NLESSQKSDL

481
LKLWNKIWCI TFRGISSEQI QAENFGLLGA IIQGSLVEVD

REFWKLFTGS ACRPSCPAVC

541
CLTLALTTSI VPGTVKMGIE QNMCEVNRSF SLKESIMKWL

LFYQLEGDLE NSTEVPPILH

601
SNFPHLVLEK ILVSLTMKNC KAAMNFFQSV PECEHHQKDK

EELSFSEVEE LFLQTTFDKM

661
DFLTIVRECG IEKHQSSIGF SVHQNLKESL DRCLLGLSEQ

LLNNYSSEIT NSETLVRCSR

721
LLVGVLGCYC YMGVIAEEEA YKSELFQKAK SLMQCAGESI

TLFKNKTNEE FRIGSLRNMM

781
QLCTRCLSNC TKKSPNKIAS GFFLRLLTSK LMNDIADICK

SLASFIKKPF DRGEVESMED

841
DTNGNLMEVE DQSSMNLFND YPDSSVSDAN EPGESQSTIG

AINPLAEEYL SKQDLLFLDM

901
LKFLCLCVTT AQTNTVSFRA ADIRRKLLML IDSSTLEPTK

SLHLHMYLML LKELPGEEYP

961
LPMEDVLELL KPLSNVCSLY RRDQDVCKTI LNHVLHVVKN

LGQSNMDSEN TRDAQGQFLT

1021
VIGAFWHLTK ERKYIFSVRM ALVNCLKTLL EADPYSKWAI

LNVMGKDFPV NEVFTQFLAD

1081
NHHQVRMLAA ESINRLFQDT KGDSSRLLKA LPLKLQQTAF

ENAYLKAQEG MREMSHSAEN

1141
PETLDEIYNR KSVLLTLIAV VLSCSPICEK QALFALCKSV

KENGLEPHLV KKVLEKVSET

1201
FGYRRLEDFM ASHLDYLVLE WLNLQDTEYN LSSFPFILLN

YTNIEDFYRS CYKVLIPHLV

1261
IRSHFDEVKS IANQIQEDWK SLLTDCFPKI LVNILPYFAY

EGTRDSGMAQ QRETATKVYD

1321
MLKSENLLGK QIDHLFISNL PEIVVELLMT LHEPANSSAS

QSTDLCDFSG DLDPAPNPPH

1381
FPSHVIKATF AYISNCHKTK LKSILEILSK SPDSYQKILL

AICEQAAETN NVYKKHRILK

1441
IYHLFVSLLL KDIKSGLGGA WAFVLRDVIY TLIHYINQRP

SCIMDVSLRS FSLCCDLLSQ

1501
VCQTAVTYCK DALENHLHVI VGTLIPLVYE QVEVQKQVLD

LLKYLVIDNK DNENLYITIK

1561
LLDPFPDHVV FKDLRITQQK IKYSRGPFSL LEEINHFLSV

SVYDALPLTR LEGLKDLRRQ

1621
LELHKDQMVD IMRASQDNPQ DGIMVKLVVN LLQLSKMAIN

HTGEKEVLEA VGSCLGEVGP

1681
IDFSTIAIQH SKDASYTKAL KLFEDKELQW TFIMLTYLNN

TLVEDCVKVR SAAVTCLKNI

1741
LATKTGHSFW EIYKMTTDPM LAYLQPFRTS RKKFLEVPRF

DKENPFEGLD DINLWIPLSE

1801
NHDIWIKTLT CAFLDSGGTK CEILQLLKPM CEVKTDFCQT

VLPYLIHDIL LQDTNESWRN

1861
LLSTHVQGFF TSCLRHFSQT SRSTTPANLD SESEHFFRCC

LDKKSQRTML AVVDYMRRQK

1921
RPSSGTIFND AFWLDLNYLE VAKVAQSCAA HFTALLYAEI

YADKKSMDDQ EKRSLAFEEG

1981
SQSTTISSLS EKSKEETGIS LQDLLLEIYR SIGEPDSLYG

CGGGKMLQPI TRLRTYEHEA

2041
MWGKALVTYD LETAIPSSTR QAGIIQALQN LGLCHILSVY

LKGLDYENKD WCPELEELHY

2101
QAAWRNMQWD HCTSVSKEVE GTSYHESLYN ALQSLRDREF

STFYESLKYA RVKEVEEMCK

2161
RSLESVYSLY PTLSRLQAIG ELESIGELFS RSVTHRQLSE

VYIKWQKHSQ LLKDSDFSFQ

2221
EPIMALRTVI LEILMEKEMD NSQRECIKDI LTKHLVELSI

LARTFKNTQL PERAIFQIKQ

2281
YNSVSCGVSE WQLEEAQVFW AKKEQSLALS ILKQMIKKLD

ASCAANNPSL KLTYTECLRV

2341
CGNWLAETCL ENPAVIMQTY LEKAVEVAGN YDGESSDELR

NGKMKAFLSL ARFSDTQYQR

2401
IENYMKSSEF ENKQALLKRA KEEVGLLREH KIQTNRYTVK

VQRELELDEL ALRALKEDRK

2461
RFLCKAVENY INCLLSGEEH DMWVFRLCSL WLENSGVSEV

NGMMKRDGMK IPTYKFLPLM

2521
YQLAARMGTK MMGGLGFHEV LNNLISRISM DHPHHTLFII

LALANANRDE FLTKPEVARR

2581
SRITKNVPKQ SSQLDEDRTE AANRIICTIR SRRPQMVRSV

EALCDAYIIL ANLDATQWKT

2641
QRKGINIPAD QPITKLKNLE DVVVPTMEIK VDHTGEYGNL

VTIQSFKAEF RLAGGVNLPK

2701
IIDCVGSDGK ERRQLVKGRD DLRQDAVMQQ VFQMCNTLLQ

RNTETRKRKL TICTYKVVPL

2761
SQRSGVLEWC TGTVPIGEFL VNNEDGAHKR YRPNDFSAFQ

CQKKMMEVQK KSFEEKYEVF

2821
MDVCQNFQPV FRYFCMEKFL DPAIWFEKRL AYTRSVATSS

IVGYILGLGD RHVQNILINE

2881
QSAELVHIDL GVAFEQGKIL PTPETVPFRL TRDIVDGMGI

TGVEGVFRRC CEKTMEVMRN

2941
SQETLLTIVE VLLYDPLFDW TMNPLKALYL QQRPEDETEL

HPTLNADDQE CKRNLSDIDQ

3001
SFNKVAERVL MRLQEKLKGV EEGTVLSVGG QVNLLIQQAI

DPKNLSRLFP GWKAWV.

Target cells of the disclosure may comprise a modification, such as a gene deletion or mutation, that results in no expression or reduced expression of BARD1. Target cells of the disclosure may comprise a modification, such as an insertion, deletion, or substitution in the gene encoding BARD1 or the encoded BARD1 protein. Target cells of the disclosure may express a BARD1 protein according to SEQ ID NOs 43-46. Target cells of the disclosure may express a variant BARD1 having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99% or any percentage in between of identity to SEQ ID NO: 43-46.

In some embodiments of the compositions and methods of the disclosure, a BARD1 protein of the disclosure comprises or consists of the amino acid sequence of (UniProt Accession No. Q99728-1 and SEQ ID NO: 43):

1
MPDNRQPRNR QPRIRSGNEP RSAPAMEPDG RGAWAHSRAA

LDRLEKLLRC SRCTNILREP

61
VCLGGCEHIF CSNCVSDCIG TGCPVCYTPA WIQDLKINRQ

LDSMIQLCSK LRNLLHDNEL

121
SDLKEDKPRK SLFNDAGNKK NSIKMWFSPR SKKVRYVVSK

ASVQTQPAIK KDASAQQDSY

181
EFVSPSPPAD VSERAKKASA RSGKKQKKKT LAEINQKWNL

EAEKEDGEFD SKEESKQKLV

241
SFCSQPSVIS SPQINGEIDL LASGSLTESE CFGSLTEVSL

PLAEQIESPD TKSRNEVVTP

301
EKVCKNYLTS KKSLPLENNG KRGHHNRLSS PISKRCRTSI

LSTSGDFVKQ TVPSENIPLP

361
ECSSPPSCKR KVGGTSGRKN SNMSDEFISL SPGTPPSTLS

SSSYRRVMSS PSAMKLLPNM

421
AVKRNHRGET LLHIASIKGD IPSVEYLLQN GSDPNVKDHA

GWTPLHEACN HGHLKVVELL

481
LQHKALVNTT GYQNDSPLHD AAKNGHVDIV KLLLSYGASR

NAVNIFGLRP VDYTDDESMK

541
SLLLLPEKNE SSSASHCSVM NTGQRRDGPL VLIGSGLSSE

QQKMLSELAV ILKAKKYTEF

601
DSTVTHVVVP GDAVQSTLKC MLGILNGCWI LKFEWVKACL

RRKVCEQEEK YEIPEGPRRS

661
RLNREQLLPK LFDGCYFYLW GTFKHHPKDN LIKLVTAGGG

QILSRKPKPD SDVTQTINTV

721
AYHARPDSDQ RFCTQYIIYE DLCNYHPERV RQGKVWKAPS

SWFIDCVMSF ELLPLDS.

1
MPDNRQPRNR QPRIRSGNEP RSAPAMEPDG RGAWAHSRAA

LDRLEKLLRC SRCNCVSDCI

61
GTGCPVCYTP AWIQDLKINR QLDSMIQLCS KLRNLLHDNE

LSDLKEDKPR KSLFNDAGNK

121
KNSIKMWFSP RSKKVRYVVS KASVQTQPAI KKDASAQQDS

YEFVSPSPPA DVSERAKKAS

181
ARSGKKQKKK TLAEINQKWN LEAEKEDGEF DSKEESKQKL

VSFCSQPSVI SSPQINGEID

241
LLASGSLTES ECFGSLTEVS LPLAEQIESP DTKSRNEVVT

PEKVCKNYLT SKKSLPLENN

301
GKRGHHNRLS SPISKRCRTS ILSTSGDFVK QTVPSENIPL

PECSSPPSCK RKVGGTSGRK

361
NSNMSDEFIS LSPGTPPSTL SSSSYRRVMS SPSAMKLLPN

MAVKRNHRGE TLLHIASIKG

421
DIPSVEYLLQ NGSDPNVKDH AGWTPLHEAC NHGHLKVVEL

LLQHKALVNT TGYQNDSPLH

481
DAAKNGHVDI VKLLLSYGAS RNAVNIFGLR PVDYTDDESM

KSLLLLPEKN ESSSASHCSV

541
MNTGQRRDGP LVLIGSGLSS EQQKMLSELA VILKAKKYTE

FDSTVTHVVV PGDAVQSTLK

601
CMLGILNGCW ILKFEWVKAC LRRKVCEQEE KYEIPEGPRR

SRLNREQLLP KLFDGCYFYL

661
WGTFKHHPKD NLIKLVTAGG GQILSRKPKP DSDVTQTINT

VAYHARPDSD QRECTQYIIY

721
EDLCNYHPER VRQGKVWKAP SSWFIDCVMS FELLPLDS.

1
MVAVPGPTVA PRSTAWRSCC AARVDLKEDK PRKSLFNDAG

NKKNSIKMWF SPRSKKVRYV

61
VSKASVQTQP AIKKDASAQQ DSYEFVSPSP PADVSERAKK

ASARSGKKQK KKTLAEINQK

121
WNLEAEKEDG EFDSKEESKQ KLVSFCSQPS VISSPQINGE

IDLLASGSLT ESECFGSLTE

181
VSLPLAEQIE SPDTKSRNEV VTPEKVCKNY LTSKKSLPLE

NNGKRGHHNR LSSPISKRCR

241
TSILSTSGDF VKQTVPSENI PLPECSSPPS CKRKVGGTSG

RKNSNMSDEF ISLSPGTPPS

301
TLSSSSYRRV MSSPSAMKLL PNMAVKRNHR GETLLHIASI

KGDIPSVEYL LQNGSDPNVK

361
DHAGWTPLHE ACNHGHLKVV ELLLQHKALV NTTGYQNDSP

LHDAAKNGHV DIVKLLLSYG

421
ASRNAVNIFG LRPVDYTDDE SMKSLLLLPE KNESSSASHC

SVMNTGQRRD GPLVLIGSGL

481
SSEQQKMLSE LAVILKAKKY TEFDSTVTHV VVPGDAVQST

LKCMLGILNG CWILKFEWVK

541
ACLRRKVCEQ EEKYEIPEGP RRSRLNREQL LPKLFDGCYF

YLWGTFKHHP KDNLIKLVTA

601
GGGQILSRKP KPDSDVTQTI NTVAYHARPD SDQRFCTQYI

IYEDLCNYHP ERVRQGKVWK

661
APSSWFIDCV MSFELLPLDS.

1
MPDNRQPRNR QPRIRSGNEP RSAPAMEPDG RGAWAHSRAA

LDRLEKLLRC SRCTNILREP

61
VCLGGCEHIF CSNCVSDCIG TGCPVCYTPA WIQDLKINRQ

LDSMIQLCSK LRNLLHDNEL

121
SGRHTFC.

Target cells of the disclosure may comprise a modification, such as a gene deletion or mutation, that results in no expression or reduced expression of BRIP1. Target cells of the disclosure may comprise a modification, such as an insertion, deletion, or substitution in the gene encoding BRIP1 or the encoded BRIP1 protein. Target cells of the disclosure may express a BRIP1 protein according to SEQ ID NOs 47-48. Target cells of the disclosure may express a variant BRIP1 having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99% or any percentage in between of identity to SEQ ID NO: 47-48.

In some embodiments of the compositions and methods of the disclosure, a BRIP1 protein of the disclosure comprises or consists of the amino acid sequence of (UniProt Accession No. Q9BX63-1 and SEQ ID NO: 47):

1
MSSMWSEYTI GGVKIYFPYK AYPSQLAMMN SILRGLNSKQ

HCLLESPTGS GKSLALLCSA

61
LAWQQSLSGK PADEGVSEKA EVQLSCCCAC HSKDFTNNDM

NQGTSRHFNY PSTPPSERNG

121
TSSTCQDSPE KTTLAAKLSA KKQASIYRDE NDDFQVEKKR

IRPLETTQQI RKRHCFGTEV

181
HNLDAKVDSG KTVKLNSPLE KINSFSPQKP PGHCSRCCCS

TKQGNSQESS NTIKKDHTGK

241
SKIPKIYFGT RTHKQIAQIT RELRRTAYSG VPMTILSSRD

HTCVHPEVVG NFNRNEKCME

301
LLDGKNGKSC YFYHGVHKIS DQHTLQTFQG MCKAWDIEEL

VSLGKKLKAC PYYTARELIQ

361
DADIIFCPYN YLLDAQIRES MDLNLKEQVV ILDEAHNIED

CARESASYSV TEVQLRFARD

421
ELDSMVNNNI RKKDHEPLRA VCCSLINWLE ANAEYLVERD

YESACKIWSG NEMLLTLHKM

481
GITTATFPIL QGHFSAVLQK EEKISPIYGK EEAREVPVIS

ASTQIMLKGL FMVLDYLFRQ

541
NSRFADDYKI AIQQTYSWTN QIDISDKNGL LVLPKNKKRS

RQKTAVHVLN FWCLNPAVAF

601
SDINGKVQTI VLTSGTLSPM KSFSSELGVT FTIQLEANHI

IKNSQVWVGT IGSGPKGRNL

661
CATFQNTETF EFQDEVGALL LSVCQTVSQG ILCFLPSYKL

LEKLKERWLS TGLWHNLELV

721
KTVIVEPQGG EKTNFDELLQ VYYDAIKYKG EKDGALLVAV

CRGKVSEGLD FSDDNARAVI

781
TIGIPFPNVK DLQVELKRQY NDHHSKLRGL LPGRQWYEIQ

AYRALNQALG RCIRHRNDWG

841
ALILVDDRFR NNPSRYISGL SKWVRQQIQH HSTFESALES

LAEFSKKHQK VLNVSIKDRT

901
NIQDNESTLE VTSLKYSTSP YLLEAASHLS PENFVEDEAK

ICVQELQCPK IITKNSPLPS

961
SIISRKEKND PVFLEEAGKA EKIVISRSTS PTFNKQTKRV

SWSSFNSLGQ YFTGKIPKAT

1021
PELGSSENSA SSPPRFKTEK MESKTVLPFT DKCESSNLTV

NTSFGSCPQS ETIISSLKID

1081
ATLTRKNHSE HPLCSEEALD PDIELSLVSE EDKQSTSNRD

FETEAEDESI YFTPELYDPE

1141
DTDEEKNDLA ETDRGNRLAN NSDCILAKDL FEIRTIKEVD

SAREVKAEDC IDTKLNGILH

1201
IEESKIDDID GNVKTTWINE LELGKTHEIE IKNFKPSPSK

NKGMFPGFK.

In some embodiments of the compositions and methods of the disclosure, a BRIM protein of the disclosure comprises or consists of the amino acid sequence of (UniProt Accession No. Q9BX63-2 and SEQ ID NO: 48):

Target cells of the disclosure may comprise a modification, such as a gene deletion or mutation, that results in no expression or reduced expression of CHEK1. Target cells of the disclosure may comprise a modification, such as an insertion, deletion, or substitution in the gene encoding CHEK1 or the encoded CHEK1 protein. Target cells of the disclosure may express a CHEK1 protein according to SEQ ID NOs 49-51. Target cells of the disclosure may express a variant CHEK1 having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99% or any percentage in between of identity to SEQ ID NO: 49-51.

In some embodiments of the compositions and methods of the disclosure, a CHEK1 protein of the disclosure comprises or consists of the amino acid sequence of (UniProt Accession No. 014757-1 and SEQ ID NO: 49):

1
MAVPFVEDWD LVQTLGEGAY GEVQLAVNRV TEEAVAVKIV

DMKRAVDCPE NIKKEICINK

61
MLNHENVVKF YGHRREGNIQ YLFLEYCSGG ELFDRIEPDI

GMPEPDAQRF FHQLMAGVVY

121
LHGIGITHRD IKPENLLLDE RDNLKISDFG LATVFRYNNR

ERLLNKMCGT LPYVAPELLK

181
RREFHAEPVD VWSCGIVLTA MLAGELPWDQ PSDSCQEYSD

WKEKKTYLNP WKKIDSAPLA

241
LLHKILVENP SARITIPDIK KDRWYNKPLK KGAKRPRVTS

GGVSESPSGF SKHIQSNLDF

301
SPVNSASSEE NVKYSSSQPE PRTGLSLWDT SPSYIDKLVQ

GISFSQPTCP DHMLLNSQLL

361
GTPGSSQNPW QRLVKRMTRF FTKLDADKSY QCLKETCEKL

GYQWKKSCMN QVTISTTDRR

421
NNKLIFKVNL LEMDDKILVD FRISKGDGLE FKRHFLKIKG

KLIDIVSSQK IWLPAT.

1
MEKPDIGMPE PDAQRFFHQL MAGVVYLHGI GITHRDIKPE

NLLLDERDNL KISDFGLATV

61
FRYNNRERLL NKMCGTLPYV APELLKRREF HAEPVDVWSC

GIVLTAMLAG ELPWDQPSDS

121
CQEYSDWKEK KTYLNPWKKI DSAPLALLHK ILVENPSARI

TIPDIKKDRW YNKPLKKGAK

181
RPRVTSGGVS ESPSGFSKHI QSNLDFSPVN SASSEENVKY

SSSQPEPRTG LSLWDTSPSY

241
IDKLVQGISF SQPTCPDHML LNSQLLGTPG SSQNPWQRLV

KRMTRFFTKL DADKSYQCLK

301
ETCEKLGYQW KKSCMNQVTI STTDRRNNKL IFKVNLLEMD

DKILVDFRLS KGDGLEFKRH

361
FLKIKGKLID IVSSQKIWLP AT.

Target cells of the disclosure may comprise a modification, such as a gene deletion or mutation, that results in no expression or reduced expression of CHEK2. Target cells of the disclosure may comprise a modification, such as an insertion, deletion, or substitution in the gene encoding CHEK2 or the encoded CHEK2 protein. Target cells of the disclosure may express a CHEK2 protein according to SEQ ID NOs 52-64. Target cells of the disclosure may express a variant CHEK2 having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99% or any percentage in between of identity to SEQ ID NO: 52-64.

In some embodiments of the compositions and methods of the disclosure, a CHEK2 protein of the disclosure comprises or consists of the amino acid sequence of (UniProt Accession No. 096017-1 and SEQ ID NO: 52):

1
MSRESDVEAQ QSHGSSACSQ PHGSVTQSQG SSSQSQGISS

SSTSTMPNSS QSSHSSSGTL

61
SSLETVSTQE LYSIPEDQEP EDQEPEEPTP APWARLWALQ

DGFANLECVN DNYWFGRDKS

121
CEYCFDEPLL KRTDKYRTYS KKHFRIFREV GPKNSYIAYI

EDHSGNGTFV NTELVGKGKR

181
RPLNNNSEIA LSLSRNKVFV FFDLTVDDQS VYPKALRDEY

IMSKTLGSGA CGEVKLAFER

241
KTCKKVAIKI ISKRKFAIGS AREADPALNV ETEIEILKKL

NHPCIIKIKN FFDAEDYYIV

301
LELMEGGELF DKVVGNKRLK EATCKLYFYQ MLLAVQYLHE

NGIIHRDLKP ENVLLSSQEE

361
DCLIKITDFG HSKILGETSL MRTLCGTPTY LAPEVLVSVG

TAGYNRAVDC WSLGVILFIC

421
LSGYPPFSEH RTQVSLKDQI TSGKYNFIPE VWAEVSEKAL

DLVKKLLVVD PKARFTTEEA

481
LRHPWLQDED MKRKFQDLLS EENESTALPQ VLAQPSTSRK

RPREGEAEGA ETTKRPAVCA

541
AVL.

1
MSRESDVEAQ QSHGSSACSQ PHGSVTQSQG SSSQSQGISS SSTSTMPNSS QSSHSSSGTL

61
SSLETVSTQE LYSIPEDQEP EDQEPEEPTP APWARLWALQ DGFANLDEDM KRKFQDLLSE

121
ENESTALPQV LAQPSTSRKR PREGEAEGAE TTKRPAVCAA VL.

1
MSRESDVEAQ QSHGSSACSQ PHGSVTQSQG SSSQSQGISS SSTSTMPNSS QSSHSSSGTL

61
SSLETVSTQE LYSIPEDQEP EDQEPEEPTP APWARLWALQ DGFANLVFVF FDLTVDDQSV

121
YPKALRDEYI MSKTLGSGAC GEVKLAFERK TCKKVAIKII SKRKFAIGSA READPALNVE

181
TEIEILKKLN HPCIIKIKNF FDAEDYYIVL ELMEGGELFD KVVGNKRLKE ATCKLYFYQM

241
LLAVQYLHEN GIIHRDLKPE NVLLSSQEED CLIKITDFGH SKILGETSLM RTLCGTPTYL

301
APEVLVSVGT AGYNRAVDCW SLGVILFICL SGYPPFSEHR TQVSLKDQIT SGKYNFIPEV

361
WAEVSEKALD LVKKLLVVDP KARFTTEEAL RHPWLQDEDM KRKFQDLLSE ENESTALPQV

421
LAQPSTSRKR PREGEAEGAE TTKRPAVCAA VL.

1
MSRESDVEAQ QSHGSSACSQ PHGSVTQSQG SSSQSQGISS SSTSTMPNSS QSSHSSSGTL

61
SSLETVSTQE LYSIPEDQEP EDQEPEEPTP APWARLWALQ DGFANLECVN DNYWFGRDKS

12
CEYCFDEPLL KRTDKYRTYS KKHFRIFREE NLSCPYRIWF NFCLF.

1
MSRESDVEAQ QSHGSSACSQ PHGSVTQSQG SSSQSQGISS SSTSTMPNSS QSSHSSSGTL

61
SSLETVSTQE LYSIPEDQEP EDQEPEEPTP APWARLWALQ DGFANLETES GHVTQSDLEL

121
LLSSDPPASA SQSAGIRGVR HHPRPVCSLK CVNDNYWFGR DKSCEYCFDE PLLKRTDKYR

181
TYSKKHFRIF REVGPKNSYI AYIEDHSGNG TFVNTELVGK GKRRPLNNNS EIALSLSRNK

241
VFVFFDLTVD DQSVYPKALR DEYIMSKTLG SGACGEVKLA FERKTCKKVA IKIISKRKFA

301
IGSAREADPA LNVETEIEIL KKLNHPCIIK IKNFFDAEDY YIVLELMEGG ELFDKVVGNK

361
RLKEATCKLY FYQMLLAVQY LHENGIIHRD LKPENVLLSS QEEDCLIKIT DFGHSKILGE

421
TSLMRTLCGT PTYLAPEVLV SVGTAGYNRA VDCWSLGVIL FICLSGYPPF SEHRTQVSLK

481
DQITSGKYNF IPEVWAEVSE KALDLVKKLL VVDPKARFTT EEALRHPWLQ DEDMKRKFQD

541
LLSEENESTA LPQVLAQPST SRKRPREGEA EGAETTKRPA VCAAVL.

1
MSRESDVEAQ QSHGSSACSQ PHGSVTQSQG SSSQSQGISS SSTSTMPNSS QSSHSSSGTL

61
SSLETVSTQE LYSIPEDQEP EDQEPEEPTP APWARLWALQ DGFANLECVN DNYWFGRDKS

121
CEYCFDEPLL EFRSYSFYLP.

1
MSRESDVEAQ QSHGSSACSQ PHGSVTQSQG SSSQSQGISS SSTSTMPNSS QSSHSSSGTL

61
SSLETVSTQE LYSIPEVLVS VGTAGYNRAV DCWSLGVILF ICLSGYPPFS EHRTQVSLKD

121
QITSGKYNFI PEVWAEVSEK ALDLVKKLLV VDPKARFTTE EALRHPWLQD EDMKRKFQDL

181
LSEENESTAL PQVLAQPSTS RKRPREGEAE GAETTKRPAV CAAVL.

1
MSRESDVEAQ QSHGSSACSQ PHGSVTQSQG SSSQSQGISS SSTSTMPNSS QSSHSSSGTL

61
SSLETVSTQE LYSIPEDQEP EDQEPEEPTP APWARLWALQ DGFANLECVN DNYWFGRDKS

121
CEYCFDEPLL KRTDKYRTYS KKHFRIFREV GPKNSYIAYI EDHSGNGTFV NTELVGKGKR

181
RPLNNNSEIA LSLSRNKVFV FFDLTVDDQS VYPKALRDEY IMSKTLGSGA CGEVKLAFER

241
KTCKKVAIKI ISKRKFAIGS AREADPALNV ETEIEILKKL NHPCIIKIKN FFDAEDYYIV

301
LELMEGGELF DKVVGNKRLK EATCKLYFYQ MLLAVQITDF GHSKILGETS LMRTLCGTPT

361
YLAPEVLVSV GTAGYNRAVD CWSLGVILFI CLSGYPPFSE HRTQVSLKDQ ITSGKYNFIP

421
EVWAEVSEKA LDLVKKLLVV DPKARFTTEE ALRHPWLQDE DMKRKFQDLL SEENESTALP

481
QVLAQPSTSR KRPREGEAEG AETTKRPAVC AAVL.

1
MSKTLGSGAC GEVKLAFERK TCKKVAIKII SKRKFAIGSA READPALNVE TEIEILKKLN

61
HPCIIKIKNF FDAEDYYIVL ELMEGGELFD KVVGNKRLKE ATCKLYFYQM LLAVQYLHEN

121
GIIHRDLKPE NVLLSSQEED CLIKITDFGH SKILGETSLM RTLCGTPTYL APEVLVSVGT

181
AGYNRAVDCW SLGVILFICL SGYPPFSEHR TQVSLKDQIT SGKYNFIPEV WAEVSEKALD

241
LVKKLLVVDP KARFTTEEAL RHPWLQDEDM KRKFQDLLSE ENESTALPQV LAQPSTSRKR

301
PREGEAEGAE TTKRPAVCAA VL.

Target cells of the disclosure may comprise a modification, such as a gene deletion or mutation, that results in no expression or reduced expression of NBN. Target cells of the disclosure may comprise a modification, such as an insertion, deletion, or substitution in the gene encoding NBN or the encoded NBN protein. Target cells of the disclosure may express a NBN protein according to SEQ ID NO 65. Target cells of the disclosure may express a variant NBN having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99% or any percentage in between of identity to SEQ ID NO: 65.

In some embodiments of the compositions and methods of the disclosure, a NBN protein of the disclosure comprises or consists of the amino acid sequence of (UniProt Accession No. 060934-1 and SEQ ID NO: 65):

1
MWKLLPAAGP AGGEPYRLLT GVEYVVGRKN CAILIENDQS ISRNHAVLTA NFSVTNLSQT

61
DEIPVLTLKD NSKYGTFVNE EKMQNGFSRT LKSGDGITFG VFGSKFRIEY EPLVACSSCL

121
DVSGKTALNQ AILQLGGFTV NNWTEECTHL VMVSVKVTIK TICALICGRP IVKPEYFTEF

181
LKAVESKKQP PQIESFYPPL DEPSIGSKNV DLSGRQERKQ IFKGKTFIFL NAKQHKKLSS

241
AVVFGGGEAR LITEENEEEH NFFLAPGTCV VDTGITNSQT LIPDCQKKWI QSIMDMLQRQ

301
GLRPIPEAEI GLAVIFMTTK NYCDPQGHPS TGLKTTTPGP SLSQGVSVDE KLMPSAPVNT

361
TTYVADTESE QADTWDLSER PKEIKVSKME QKFRMLSQDA PTVKESCKTS SNNNSMVSNT

421
LAKMRIPNYQ LSPTKLPSIN KSKDRASQQQ QTNSIRNYFQ PSTKKRERDE ENQEMSSCKS

481
ARIETSCSLL EQTQPATPSL WKNKEQHLSE NEPVDTNSDN NLFTDTDLKS IVKNSASKSH

541
AAEKLRSNKK REMDDVAIED EVLEQLFKDT KPELEIDVKV QKQEEDVNVR KRPRMDIETN

601
DTFSDEAVPE SSKISQENEI GKKRELKEDS LWSAKEISNN DKLQDDSEML PKKLLLTEFR

661
SLVIKNSTSR NPSGINDDYG QLKNFKKFKK VTYPGAGKLP HIIGGSDLIA HHARKNTELE

721
EWLRQEMEVQ NQHAKEESLA DDLFRYNPYL KRRR.

Target cells of the disclosure may comprise a modification, such as a gene deletion or mutation, that results in no expression or reduced expression of PALB2. Target cells of the disclosure may comprise a modification, such as an insertion, deletion, or substitution in the gene encoding PALB2 or the encoded PALB2 protein. Target cells of the disclosure may express a PALB2 protein according to SEQ ID NO 66. Target cells of the disclosure may express a variant PALB2 having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99% or any percentage in between of identity to SEQ ID NO: 66.

In some embodiments of the compositions and methods of the disclosure, a PALB2 protein of the disclosure comprises or consists of the amino acid sequence of (UniProt Accession No. Q86YC2-1 and SEQ ID NO: 66):

1
MDEPPGKPLS CEEKEKLKEK LAFLKREYSK TLARLQRAQR AEKIKHSIKK TVEEQDCLSQ

61
QDLSPQLKHS EPKNKICVYD KLHIKTHLDE ETGEKTSITL DVGPESFNPG DGPGGLPIQR

121
TDDTQEHFPH RVSDPSGEQK QKLPSRRKKQ QKRTFISQER DCVFGTDSLR LSGKRLKEQE

181
EISSKNPARS PVTEIRTHLL SLKSELPDSP EPVTEINEDS VLIPPTAQPE KGVDTFLRRP

241
NFTRATTVPL QTLSDSGSSQ HLEHIPPKGS SELTTHDLKN IRFTSPVSLE AQGKKMTVST

301
DNLLVNKAIS KSGQLPTSSN LEANISCSLN ELTYNNLPAN ENQNLKEQNQ TEKSLKSPSD

361
TLDGRNENLQ ESEILSQPKS LSLEATSPLS AEKHSCTVPE GLLFPAEYYV RTTRSMSNCQ

421
RKVAVEAVIQ SHLDVKKKGF KNKNKDASKN LNLSNEETDQ SEIRMSGTCT GQPSSRTSQK

481
LLSLTKVSSP AGPTEDNDLS RKAVAQAPGR RYTGKRKSAC TPASDHCEPL LPTSSLSIVN

541
RSKEEVTSHK YQHEKLFIQV KGKKSRHQKE DSLSWSNSAY LSLDDDAFTA PFHRDGMLSL

601
KQLLSFLSIT DFQLPDEDFG PLKLEKVKSC SEKPVEPFES KMFGERHLKE GSCIFPEELS

661
PKRMDTEMED LEEDLIVLPG KSHPKRPNSQ SQHTKTGLSS SILLYTPLNT VAPDDNDRPT

721
TDMCSPAFPI LGTTPAFGPQ GSYEKASTEV AGRTCCTPQL AHLKDSVCLA SDTKQFDSSG

781
SPAKPHTTLQ VSGRQGQPTC DCDSVPPGTP PPIESFTFKE NQLCRNTCQE LHKHSVEQTE

841
TAELPASDSI NPGNLQLVSE LKNPSGSCSV DVSAMFWERA GCKEPCIITA CEDVVSLWKA

901
LDAWQWEKLY TWHFAEVPVL QIVPVPDVYN LVCVALGNLE IREIRALFCS SDDESEKQVL

961
LKSGNIKAVL GLTKRRLVSS SGTLSDQQVE VMTFAEDGGG KENQFLMPPE ETILTFAEVQ

1021
GMQEALLGTT IMNNIVIWNL KTGQLLKKMH IDDSYQASVC HKAYSEMGLL FIVLSHPCAK

1081
ESESLRSPVF QLIVINPKTT LSVGVMLYCL PPGQAGRFLE GDVKDHCAAA ILTSGTIAIW

1141
DLLLGQCTAL LPPVSDQHWS FVKWSGTDSH LLAGQKDGNI FVYHYS.

Target cells of the disclosure may comprise a modification, such as a gene deletion or mutation, that results in no expression or reduced expression of SLX4. Target cells of the disclosure may comprise a modification, such as an insertion, deletion, or substitution in the gene encoding SLX4 or the encoded SLX4 protein. Target cells of the disclosure may express a SLX4 protein according to SEQ ID NO 67 or 68. Target cells of the disclosure may express a variant SLX4 having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99% or any percentage in between of identity to SEQ ID NO: 67 or 68.

In some embodiments of the compositions and methods of the disclosure, a SLX4 protein of the disclosure comprises or consists of the amino acid sequence of (UniProt Accession No. Q8IY92-1 and SEQ ID NO: 67):

1
MKLSVNEAQL GFYLGSLSHL SACPGIDPRS SEDQPESLKT GQMMDESDED FKELCASFFQ

61
RVKKHGIKEV SGERKTQKAA SNGTQIRSKL KRTKQTATKT KTLQGPAEKK PPSGSQAPRT

121
KKQRVTKWQA SEPAHSVNGE GGVLASAPDP PVLRETAQNT QTGNQQEPSP NLSREKTREN

181
VPNSDSQPPP SCLTTAVPSP SKPRTAQLVL QRMQQFKRAD PERLRHASEE CSLEAAREEN

241
VPKDPQEEMM AGNVYGLGPP APESDAAVAL TLQQEFARVG ASAHDDSLEE KGLFFCQICQ

301
KNLSAMNVTR REQHVNRCLD EAEKTLRPSV PQIPECPICG KPFLTLKSRT SHLKQCAVKM

361
EVGPQLLLQA VRLQTAQPEG SSSPPMFSFS DHSRGLKRRG PTSKKEPRKR RKVDEAPSED

421
LLVAMALSRS EMEPGAAVPA LRLESAFSER IRPEAENKSR KKKPPVSPPL LLVQDSETTG

481
RQIEDRVALL LSEEVELSST PPLPASRILK EGWERAGQCP PPPERKQSFL WEGSALTGAW

541
AMEDFYTARL VPPLVPQRPA QGLMQEPVPP LVPPEHSELS ERRSPALHGT PTAGCGSRGP

601
SPSASQREHQ ALQDLVDLAR EGLSASPWPG SGGLAGSEGT AGLDVVPGGL PLTGFVVPSQ

661
DKHPDRGGRT LLSLGLLVAD FGAMVNNPHL SDVQFQTDSG EVLYAHKFVL YARCPLLIQY

721
VNNEGFSAVE DGVLTQRVLL GDVSTEAART FLHYLYTADT GLPPGLSSEL SSLAHRFGVS

781
ELVHLCEQVP IATDSEGKPW EEKEAENCES RAENFQELLR SMWADEEEEA ETLLKSKDHE

841
EDQENVNEAE MEEIYEFAAT QRKLLQEERA AGAGEDADWL EGGSPVSGQL LAGVQVQKQW

901
DKVEEMEPLE PGRDEAATTW EKMGQCALPP PQGQHSGARG AEAPEQEAPE EALGHSSCSS

961
PSRDCQAERK EGSLPHSDDA GDYEQLFSST QGEISEPSQI TSEPEEQSGA VRERGLEVSH

1021
RLAPWQASPP HPCRFLLGPP QGGSPRGSHH TSGSSLSTPR SRGGTSQVGS PTLLSPAVPS

1081
KQKRDRSILT LSKEPGHQKG KERRSVLECR NKGVLMFPEK SPSIDLTQSN PDHSSSRSQK

1141
SSSKLNEEDE VILLLDSDEE LELEQTKMKS ISSDPLEEKK ALEISPRSCE LFSIIDVDAD

1201
QEPSQSPPRS EAVLQQEDEG ALPENRGSLG RRGAPWLFCD RESSPSEAST TDTSWLVPAT

1261
PLASRSRDCS SQTQISSLRS GLAVQAVTQH TPRASVGNRE GNEVAQKFSV IRPQTPPPQT

1321
PSSCLTPVSP GTSDGRRQGH RSPSRPHPGG HPHSSPLAPH PISGDRAHFS RRFLKHSPPG

1381
PSFLNQTPAG EVVEVGDSDD EQEVASHQAN RSPPLDSDPP IPIDDCCWHM EPLSPIPIDH

1441
WNLERTGPLS TSSPSRRMNE AADSRDCRSP GLLDTTPIRG SCTTQRKLQE KSSGAGSLGN

1501
SRPSFLNSAL WDVWDGEEQR PPETPPPAQM PSAGGAQKPE GLETPKGANR KKNLPPKVPI

1561
TPMPQYSIME TPVLKKELDR FGVRPLPKRQ MVLKLKEIFQ YTHQTLDSDS EDESQSSQPL

1621
LQAPHCQTLA SQTYKPSRAG VHAQQEATTG PGAHRPKGPA KTKGPRHQRK HHESITPPSR

1681
SPTKEAPPGL NDDAQIPASQ ESVATSVDGS DSSLSSQSSS SCEFGAAFES AGEEEGEGEV

1741
SASQAAVQAA DTDEALRCYI RSKPALYQKV LLYQPFELRE LQAELRQNGL RVSSRRLLDF

1801
LDTHCITFTT AATRREKLQG RRRQPRGKKK VERN.

1
MFSFRCLDEA EKTLRPSVPQ IPECPICGKP FLTLKSRTSH

LKQCAVKMEV GPQLLLQAVR

61
LQTAQPEGSS SPPMFSFSDH SRGLKRRGPT SKKEPRKRRK

VDEAPSEDLL VAMALSRSEM

121
EPGAAVPALR LESAFSERIR PEAENKSRKK KPPVSPPLLL

VQDSETTGRQ IEDRVALLLS

181
EEVELSSTPP LPASRILKEG WERAGQCPPP PERKQSFLWE

GSALTGAWAM EDFYTARLVP

241
PLVPQRPAQG LMQEPVPPLV PPEHSELSER RSPALHGTPT

AGCGSRGPSP SASQREHQAL

301
QDLVDLAREG LSASPWPGSG GLAGSEGTAG LDVVPGGLPL

TGFVVPSQDK HPDRGGRTLL

361
SLGLLVADFG AMVNNPHLSD VQFQTDSGEV LYAHKFVLYA

RCPLLIQYVN NEGFSAVEDG

421
VLTQRVLLGD VSTEAARTFL HYLYTADTGL PPGLSSELSS

LAHRFGVSEL VHLCEQVPIA

481
TDSEGKPWEE KEAENCESRA ENFQELLRSM WADEEEEAET

LLKSKDHEED QENVNEAEME

541
EIYEFAATQR KLLQEERAAG AGEDADWLEG GSPVSGQLLA

GVQVQKQWDK VEEMEPLEPG

601
RDEAATTWEK MGQCALPPPQ GQHSGARGAE APEQEAPEEA

LGHSSCSSPS RDCQAERKEG

661
SLPHSDDAGD YEQLFSSTQG EISEPSQITS EPEEQSGAVR

ERGLEVSHRL APWQASPPHP

721
CRFLLGPPQG GSPRGSHHTS GSSLSTPRSR GGTSQVGSPT

LLSPAVPSKQ KRDRSILTLS

781
KEPGHQKGKE RRSVLECRNK GVLMFPEKSP SIDLTQSNPD

HSSSRSQKSS SKLNEEDEVI

841
LLLDSDEELE LEQTKMKSIS SDPLEEKKAL EISPRSCELF

SIIDVDADQE PSQSPPRSEA

901
VLQQEDEGAL PENRGSLGRR GAPWLFCDRE SSPSEASTTD

TSWLVPATPL ASRSRDCSSQ

961
TQISSLRSGL AVQAVTQHTP RASVGNREGN EVAQKFSVIR

PQTPPPQTPS SCLTPVSPGT

1021
SDGRRQGHRS PSRPHPGGHP HSSPLAPHPI SGDRAHFSRR

FLKHSPPGPS FLNQTPAGEV

1081
VEVGDSDDEQ EVASHQANRS PPLDSDPPIP IDDCCWHMEP

LSPIPIDHWN LERTGPLSTS

1141
SPSRRMNEAA DSRDCRSPGL LDTTPIRGSC TTQRKLQEKS

SGAGSLGNSR PSFLNSALWD

1201
VWDGEEQRPP ETPPPAQMPS AGGAQKPEGL ETPKGANRKK

NLPPKVPITP MPQYSIMETP

1261
VLKKELDRFG VRPLPKRQMV LKLKEIFQYT HQTLDSDSED

ESQSSQPLLQ APHCQTLASQ

1321
TYKPSRAGVH AQQEATTGPG AHRPKGPAKT KGPRHQRKHH

ESITPPSRSP TKEAPPGLND

1381
DAQIPASQES VATSVDGSDS SLSSQSSSSC EFGAAFESAG

EEEGEGEVSA SQAAVQAADT

1441
DEALRCYIRS KPALYQKVLL YQPFELRELQ AELRQNGLRV

SSRRLLDFLD THCITFTTAA

1501
TRREKLQGRR RQPRGKKKVE RN.

DNA Ligase 1 (LIG1)

LIG1 is an ATP-dependent DNA ligase. Although there are multiple isoforms (shown below), the ligation mechanism is common to all isoforms (see FIG. 2). LIG1 is involved in DNA replication and repair by joining Okazaki fragments and closing single strand nicks during base excision repair. As used throughout the disclosure, the term Okazaki fragments are meant to describe small fragments of the lagging strand of replication. In some embodiments, Okazaki fragments comprise or consist of between 100 and 200 base pairs, inclusive of the endpoints.

LIG1 is the predominant replicative DNA ligase, but other DNA ligases and/or other LIG1 isoforms may have redundant functions (e.g. LIG3). LIG3 is the only mitochondrial DNA ligase and is therefore essential in mitrochondria. LIG4 is involved in NHEJ and V(D)J recombination.

LIG1 is recruited to sites of replication via interaction with PCNA and RCF.

During the cell cycle, LIG1 has a role in DNA replication. LIG1 is the major ligase responsible for closing nicks in lagging strand. In each human S-phase, 30-50 million Okazaki fragments may be generated and LIG1 is responsible for joining them.

In the process of base excision repair (BER), LIG1 removes single damaged bases. This mechanism of repair is active throughout the cell cycle. During the S-phase, LIG1 is responsible for long-patch BER. Throughout the cell cycle, LIG3 is responsible for short-path BER.

With respect to other repair pathways, LIG1 and LIG3 have overlapping roles in microhomology mediated endjoining (MMEJ) and alternative endjoining (Alt-NHEJ).

In target cells lacking a functional BRCA1 protein or otherwise having an impaired, defective or deregulated HR pathway (even in the absence of variant BRCA1), loss of LIG1 leads to elevation of unligated Okazaki fragments and single strand breaks. HR impairment, deficiency or deregulation prevents detection and repair of unligated fragments. Replication forks collapse during the next cell cycle producing single-ended double strand breaks. A LIG1 blocking agent of the disclosure may increase a number of unligated Okazaki fragments and single strand breaks, which in those cells containing a variant protein that prevents detection and repair of the unligated fragments, induces collapse of one or more replication forks during a phase of the cell cycle producing single-ended double strand breaks. Target cells may have a synergistic interaction or an increased synergy with LIG1 that express a variant protein. In some embodiments, a cell having an impaired, defective or deregulated HR pathway comprises one or more of a variant BRCA1, a variant BRCA2, a variant RAD51, a variant RAD51C, a variant RAD51D, a variant XRCC2, a variant XPF, a variant MRE11A, a variant ATM, a variant BARD1, a variant BRIP1, a variant CHEK1, a variant CHEK2, a variant NBN, a variant PALB2 and a variant SLX4. In some embodiments, a target cell having an impaired, defective or deregulated HR pathway is a Ewing sarcoma cell. In some embodiments, the impaired, defective or deregulated HR pathway is isolated or derived from a Ewing's sarcoma cell.

The human DNA ligase 1 (LIG1) protein is encoded by the LIG1 gene. LIG1 (also known as polydeoxyribonucleotide synthase [ATP] 1) has 3 isoforms. LIG1 may have an additional 7 isoforms from computational analyses (see Uniprot Accession No. P18858).

Blocking agents, compositions or formulations of the disclosure may comprise one or more blocking agents of LIG. Blocking agents may inhibit one or more activity or function of LIG1. In particular embodiments, a LIG1 blocking agent inhibits a LIG1 activity or function such as DNA ligase activity. In particular embodiments, the activity is inhibited by at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or about 100%.

Blocking agents, compositions or formulations of the disclosure may comprise one or more isoforms of LIG1. Blocking agents, compositions or formulations of the disclosure may comprise a LIG1 variant encoded by an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99% or any percentage in between of identity to a LIG1 protein of the disclosure. Blocking agents, compositions or formulations of the disclosure may comprise a LIG1 variant encoded by an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99% or any percentage in between of identity to one or more of SEQ ID NO 1, 3 and 5.

Blocking agents, compositions or formulations of the disclosure may comprise a LIG1 variant encoded by a nucleic sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99% or any percentage in between of identity to a nucleic acid sequence encoding a LIG1 protein of the disclosure. Blocking agents, compositions or formulations of the disclosure may comprise a LIG1 variant encoded by a nucleic sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99% or any percentage in between of identity to SEQ ID NO: 2, 4, and 6.

The amino acid sequence of LIG1, isoform 1, is provided below (see also UniProt Accession No. P18858-1 and GenBank Accession No. NP_000225; SEQ ID NO: 1):

1
MQRSIMSFFH PKKEGKAKKP EKEASNSSRE TEPPPKAALK

EWNGVVSESD SPVKRPGRKA

61
ARVLGSEGEE EDEALSPAKG QKPALDCSQV SPPRPATSPE

NNASLSDTSP MDSSPSGIPK

121
RRTARKQLPK RTIQEVLEEQ SEDEDREAKR KKEEEEEETP

KESLTEAEVA TEKEGEDGDQ

181
PTTPPKPLKT SKAETPTESV SEPEVATKQE LQEEEEQTKP

PRRAPKTLSS FFTPRKPAVK

241
KEVKEEEPGA PGKEGAAEGP LDPSGYNPAK NNYHPVEDAC

WKPGQKVPYL AVARTFEKIE

301
EVSARLRMVE TLSNLLRSVV ALSPPDLLPV LYLSLNHLGP

PQQGLELGVG DGVLLKAVAQ

361
ATGRQLESVR AEAAEKGDVG LVAENSRSTQ RLMLPPPPLT

ASGVFSKFRD IARLTGSAST

421
AKKIDIIKGL FVACRHSEAR FIARSLSGRL RLGLAEQSVL

AALSQAVSLT PPGQEFPPAM

481
VDAGKGKTAE ARKTWLEEQG MILKQTFCEV PDLDRIIPVL

LEHGLERLPE HCKLSPGIPL

541
KPMLAHPTRG ISEVLKRFEE AAFTCEYKYD GQRAQIHALE

GGEVKIFSRN QEDNTGKYPD

601
IISRIPKIKL PSVTSFILDT EAVAWDREKK QIQPFQVLTT

RKRKEVDASE IQVQVCLYAF

661
DLIYLNGESL VREPLSRRRQ LLRENFVETE GEFVFATSLD

TKDIEQIAEF LEQSVKDSCE

721
GLMVKTLDVD ATYEIAKRSH NWLKLKKDYL DGVGDTLDLV

VIGAYLGRGK RAGRYGGFLL

781
ASYDEDSEEL QAICKLGTGF SDEELEEHHQ SLKALVLPSP

RPYVRIDGAV IPDHWLDPSA

841
VWEVKCADLS LSPIYPAARG LVDSDKGISL RFPRFIRVRE

DKQPEQATTS AQVACLYRKQ

901
SQIQNQQGED SGSDPEDTY.

The nucleic acid sequence of LIG1, isoform 1, is provided below (see also UniProt Accession No. P18858-1 and GenBank Accession No. NM_000234; SEQ ID NO: 2):

1
ctcgcggggg cgcttccacc gattcctcct ctttccctgc

cagtcactcc tcagaccctc

61
agccacaccc gctcatccag ggcgagggaa agcgcgggca

ttttcccagt gtgctctgcg

121
ggagggctcg ccccacttca ccccttttcc cgccctcctc

ccattcggga gactacgact

181
cccagtgtcc tccgcgcgac ggcggcggtg cggacggtgc

ccaggtcccg cccctaggct

241
ctgccccgcc cccgcccgca gacgtctgcg cgcgaatgcc

gtggcgcgaa cttgggactg

301
cagaggcgcg cctggcggat ctgagtgtgt tgcccgggca

gcggcgcgcg ggaccaacgc

361
aaggagcagc tgacagacga agaaaagtgc tggacaggaa

gggagaattc tgacgccaac

421
atgcagcgaa gtatcatgtc atttttccac cccaagaaag

agggtaaagc aaagaagcct

481
gagaaggagg catccaatag cagcagagag acggagcccc

ctccaaaggc ggcactgaag

541
gagtggaatg gagtggtgtc cgagagtgac tctccggtga

agaggccagg gaggaaggcg

601
gcccgggtcc tgggcagcga aggggaagag gaggatgaag

cccttagccc tgctaaaggc

661
cagaagcctg ccctggactg ctcacaggtc tccccgcccc

gtcctgccac atctcctgag

721
aacaatgctt ccctctctga cacctctccc atggacagtt

ccccatcagg gattccgaag

781
cgtcgcacag ctcggaagca gctcccgaaa cggaccattc

aggaagtcct ggaagagcag

841
agtgaggacg aggacagaga agccaagagg aagaaggagg

aggaagaaga ggagaccccg

901
aaagaaagcc tcacagaggc tgaagtggcc acagagaagg

aaggagaaga cggggaccag

961
cccaccacgc ctcccaagcc cctaaagacc tccaaagcag

agaccccgac ggaaagcgtt

1021
tcagagcctg aggtggccac gaagcaggaa ctgcaggagg

aggaagagca gaccaagcct

1081
ccccgcagag ctcccaagac gctcagcagc ttcttcaccc

cccggaagcc agcagtcaaa

1141
aaagaagtga aggaagagga gccaggggct ccaggaaagg

agggagctgc tgagggaccc

1201
ctggatccat ctggttacaa tcctgccaag aacaactatc

atcccgtgga agatgcctgc

1261
tggaaaccgg gccagaaggt tccttacctg gctgtggccc

ggacgtttga gaagatcgag

1321
gaggtgtctg ctcggctccg gatggtggag acgctgagca

acttgctgcg ctccgtggtg

1381
gccctgtcgc ctccagacct cctccctgtc ctctacctca

gcctcaacca ccttgggcca

1441
ccccagcagg gcctggagct tggcgtgggt gatggtgtcc

ttctcaaggc agtggcccag

1501
gccacaggtc ggcagctgga gtccgtccgg gctgaggcag

ccgagaaagg cgacgtgggg

1561
ctggtggccg agaacagccg cagcacccag aggctcatgc

tgccaccacc tccgctcact

1621
gcctccgggg tcttcagcaa gttccgcgac atcgccaggc

tcactggcag tgcttccaca

1681
gccaagaaga tagacatcat caaaggcctc tttgtggcct

gccgccactc agaagcccgg

1741
ttcatcgcta ggtccctgag cggacggctg cgccttgggc

tggcagagca gtcggtgctg

1801
gctgccctct cccaggcagt gagcctcacg cccccgggcc

aagaattccc accagccatg

1861
gtggatgctg ggaagggcaa gacagcagag gccagaaaga

cgtggctgga ggagcaaggc

1921
atgatcctga agcagacgtt ctgcgaggtt cccgacctgg

accgaattat ccccgtgctg

1981
ctggagcacg gcctggaacg tctcccggag cactgcaagc

tgagcccagg gattcccctg

2041
aaaccaatgt tggcccatcc cacccggggc atcagcgagg

tcctgaaacg ctttgaggag

2101
gcagctttca cctgcgaata caaatatgac gggcagaggg

cacagatcca cgccctggaa

2161
ggcggggagg tgaagatctt cagcaggaat caggaagaca

acactgggaa gtacccggac

2221
atcatcagcc gcatccccaa gattaaactc ccatcggtca

catccttcat cctggacacc

2281
gaagccgtgg cttgggaccg ggaaaagaag cagatccagc

cattccaagt gctcaccacc

2341
cgcaaacgca aggaggtgga tgcgtctgag atccaggtgc

aggtgtgttt gtacgccttc

2401
gacctcatct acctcaatgg agagtccctg gtacgtgagc

ccctttcccg gcgccggcag

2461
ctgctccggg agaactttgt ggagacagag ggcgagtttg

tcttcgccac ctccctggac

2521
accaaggaca tcgagcagat cgccgagttc ctggagcagt

cagtgaaaga ctcctgcgag

2581
gggctgatgg tgaagaccct ggatgttgat gccacctacg

agatcgccaa gagatcgcac

2641
aactggctca agctgaagaa ggactacctt gatggcgtgg

gtgacaccct ggacctggtg

2701
gtgatcggcg cctacctggg ccgggggaag cgggccggcc

ggtacggggg cttcctgctg

2761
gcctcctacg acgaggacag tgaggagctg caggccatat

gcaagcttgg aactggcttc

2821
agtgatgagg agctggagga gcatcaccag agcctcaagg

cgctggtgct gcccagccca

2881
cgcccttacg tgcggataga tggcgctgtg attcccgacc

actggctgga ccccagcgct

2941
gtgtgggagg tgaagtgcgc tgacctctcc ctctctccca

tctaccctgc tgcgcggggc

3001
ctggtggata gtgacaaggg catctccctt cgcttccctc

ggtttattcg agtccgtgaa

3061
gacaagcagc cggagcaggc caccaccagt gctcaggtgg

cctgtttgta ccggaagcaa

3121
agtcagattc agaaccaaca aggcgaggac tcaggctctg

accctgaaga tacctactaa

3181
gccctcgccc tcctagggcc tgggtacagg gcatgagttg

gacggacccc agggttatta

3241
ttgcctttgc tttttagcaa atctgctgtg gcaggctgtg

gattttgaga gtcaggggag

3301
gggtgtgtgt gtgagggggt ggcttactcc ggagtctggg

attcatcccg tcatttcttt

3361
caataaataa ttattggata gctaaaaaaa aaaaaaaaaa

aaaaaaaaaa.

The amino acid sequence of LIG1, isoform 2, is provided below (see also UniProt Accession No. P18858-2 and GenBank Accession No. AAI10623; SEQ ID NO: 3):

The nucleic acid sequence of LIG1, isoform 2, is provided below (see also UniProt Accession No. P18858-2 and GenBank Accession No. BC110622; SEQ ID NO: 4):

1
gaccaacgca aggagcagct gacagacgaa gaaaagtgct

ggacaggaag ggagaattct

61
gatgccaaca tgcagcgaag tatcatgtca tttttccacc

ccaagaaaga gggtaaagca

121
aagaagcctg agaaggaggc atccaatagc agcagagaga

cggagccccc tccaaaggcg

181
gcactgaagg agtggaatgg agtggtgtcc gagagtgact

ctccggtgaa gaggccaggg

241
aggaaggcgg cccgggtcct gggcagcgaa ggggaagagg

aggatgaagc ccttagccct

301
gctaaaggcc agaagcctgc cctggactgc tcacaggtct

ccccgccccg tcctgccaca

361
tctcctgaga acaatgcttc cctctctgac acctctccca

tggacagttc cccatcaggg

421
attccgaagc gtcgcacagc tcggaagcag ctcccgaaac

ggaccattca ggaagtcctg

481
gaagagcaga gtgaggacga ggacagagaa gccaagagga

agaaggagga ggaagaagag

541
gagaccccga aagaaagcct cacagaggct gaagtggcaa

cagagaagga aggagaagac

601
ggggaccagc ccaccacgcc tcccaagccc ctaaagacct

ccaaagcaga gaccccgacg

661
gaaagcgttt cagagcctga ggtggccacg aagcaggaac

tgcaggagga ggaagagcag

721
accaagcctc cccgcagagc tcccaagacg ctcagcagct

tcttcacccc ccggaagcca

781
gcagtcaaaa aagaagtgaa ggaagaggag ccaggggctc

caggaaagga gggagctgct

841
gagggacccc tcgatccatc tggttacaat cctgccaaga

acaactatca tcccgtggaa

901
gatgcctgct ggaaaccggg ccagaaggtt ccttacctgg

ctgtggcccg gacgtttgag

961
aagatcgagg aggtgtctgc tcggctccgg atggtggaga

cgctgagcaa cttgctgcgc

1021
tccgtggtgg ccctgtcgcc tccagacctc ctccctgtcc

tctacctcag cctcaaccac

1081
cttgggccac cccagcaggg cctggagctt ggcgtgggtg

atggtgtcct tctcaaggca

1141
gtggcccagg ccacaggtcg gcagctggag tccgtccggg

ctgaggcagc cgagaaaggc

1201
gacgtggggc tggtggccga gaacagccgc agcacccaga

ggctcatgct gccaccacct

1261
ccgctcactg cctccggggt cttcagcaag ttccgcgaca

tcgccaggct cactggcagt

1321
gcttccacag ccaagaagat agacatcatc aaaggcctct

ttgtggcctg ccgccactca

1381
gaagcccggt tcatcgctag gtccctgagc ggacggctgc

gccttgggct ggcagagcag

1441
tcggtgctgg ctgccctctc ccaggcagtg agcctcacgc

ccccgggcca agaattccca

1501
ccagccatgg tggatgctgg gaagggcaag acagcagagg

ccagaaagac gtggctggag

1561
gagcaaggca tgatcctgaa gcagacgttc tgcgaggttc

ccgacctgga ccgaattatc

1621
cccgtgctgc tggagcacgg cctggaacgt ctcccggagc

actgcaagct gagcccaggg

1681
attcccctga aaccaatgtt ggcccatccc acccggggca

tcagcgaggt cctgaaacgc

1741
tttgaggagg cagctttcac ctgcgaatac aaatatgacg

ggcagagggc acagatccac

1801
gccctggaag gcggggaggt gaagatcttc agcaggaatc

aggaagacaa cactgggaag

1861
tacccggaca tcatcagccg catccccaag attaaactcc

catcggtcac atccttcatc

1921
ctggacaccg aagccgtggc ttgggaccgg gaaaagaagc

agatccagcc attccaagtg

1981
ctcaccaccc gcaaacgcaa ggaggtggat gcgtctgaga

tccaggtgca ggtgtgtttg

2041
tacgccttcg acctcatcta cctcaatgga gagtccctgg

tacgtgagcc cctttcccgg

2101
cgccggcagc tgctccggga gaactttgtg gagacagagg

gcgagtttgt cttcgccacc

2161
tccctggaca ccaaggacat cgagcagatc gccgagttcc

tggagcagtc agtgaaagac

2221
tcctgcgagg ggctgatggt gaagaccctg gatgttgatg

ccacctacga gatcgccaag

2281
agatcgcaca actggctcaa gctgaagaag gactaccttg

atggcgtggg tgacaccctg

2341
gacctggtgg tgatcggcgc ctacctgggc cgggggaagc

gggccggccg gtacgggggc

2401
ttcctgctgg cctcctacga cgaggacagt gaggagctgc

aggccatatg caaggtcctg

2461
gggaactggg gctgatggca gaagcaggag ggaaggagat

tagactcccg ggggtatttc

2521
ctattgcaca acccagaccg aggggtgctc acatccccct

gtgaaggatc cgttctcccc

2581
agtttttatt tactttttct ggttctatga gcaactcttg

cttcatatag aaaaacaggc

2641
tcggatagaa agatgtcagg aatgtgtgtc agtgagagat

tgagattagc tacaaatagt

2701
ggtggctcac ataagaaaag aatttttttg ttatgaaatg

agaagtgtgg agggggcagt

2761
ctgatgccat tagggattca ggttcctcct ctccccacat

ttttttttgt ccccacattt

2821
tttttttctg ccatgtgcct ctggcctcat catcacaatg

tggctgcagg agttccagcc

2881
ctcatggcca tgcctgctcc acgcaccagg gaagagcaaa

gggggttgga ccctgagtca

2941
gcctcagcca gtgaacacct tctgctcaag ttctgttgct

catcactttg ctccatggct

3001
cacccctagc tgcaaggaat tctgggaaat gtaaagtgat

ttactttttt tcaagttaaa

3061
gtttttctta aagctgtgca cattttggac tagattttct

tacgtatttc ctatattccc

3121
ctctccaaca aatttgggtc ttttgccaca gaaacagaac

aaaatatcac ctagaatcgt

3181
accacccaga gacacgcagt agtgacattt gagggaatgt

tttatttaca tttttgcaaa

3241
gttgtgatcc ttctctatga gtaattttct tttctgcctt

tttaccctgg aagtactttg

3301
ttttcagtgt tgtatatgcc cttaagcaat atggcaacag

gacctgaatg cccgcacaag

3361
agttttcaag tgctgtgccc catagcacat agtgctgtgt

cctgaaccag ctccccgtgg

3421
ctgtgtgggg aagttgttgc cggctttgtt ctgatggctt

ggaactggct tcagtgacga

3481
ggagctggag gagcatcacc agagcctcaa ggcgctggtg

ctgcccagcc cacgccctta

3541
cgtgcggata gatggcgctg tgattcccga ccactggctg

gaccccagcg ctgtgtggga

3601
ggtgaagtgc gctgacctct ccctctctcc catctaccct

gctgcgcggg gcctggtgga

3661
tagtgacaag ggcatctccc ttcgcttccc tcggtttatt

cgagtccgtg aagacaagca

3721
gccggagcag gccaccacca gtgctcaggt ggcctgtttg

taccggaagc aaagtcagat

3781
tcagaaccaa caaggcgagg actcaggctc tgaccctgaa

gatacctact aagccctcgc

3841
cctcctaggg cctgggtaca gggcatgagt tggacggacc

ccagggttat tattgccttt

3901
gctttttagc aaatctgctg tggcaggctg tggattttga

gagtcagggg aggggtg.

The amino acid sequence of LIG1, isoform 3, is provided below (see also UniProt Accession No. P18858-3 and GenBank Accession No. NP_001275992; SEQ ID NO: 5):

1
MQRSIMAALK EWNGVVSESD SPVKRPGRKA ARVLGSEGEE

EDEALSPAKG QKPALDCSQV

61
SPPRPATSPE NNASLSDTSP MDSSPSGIPK RRTARKQLPK

RTIQEVLEEQ SEDEDREAKR

121
KKEEEEETPK ESLTEAEVAT EKEGEDGDQP TTPPKPLKTS

KAETPTESVS EPEVATKQEL

181
QEEEEQTKPP RRAPKTLSSF FTPRKPAVKK EVKEEEPGAP

GKEGAAEGPL DPSGYNPAKN

241
NYHPVEDACW KPGQKVPYLA VARTFEKIEE VSARLRMVET

LSNLLRSVVA LSPPDLLPVL

301
YLSLNHLGPP QQGLELGVGD GVLLKAVAQA TGRQLESVRA

EAAEKGDVGL VAENSRSTQR

361
LMLPPPPLTA SGVFSKFRDI ARLTGSASTA KKIDIIKGLF

VACRHSEARF IARSLSGRLR

421
LGLAEQSVLA ALSQAVSLTP PGQEFPPAMV DAGKGKTAEA

RKTWLEEQGM ILKQTFCEVP

481
DLDRIIPVLL EHGLERLPEH CKLSPGIPLK PMLAHPTRGI

SEVLKRFEEA AFTCEYKYDG

541
QRAQIHALEG GEVKIFSRNQ EDNTGKYPDI ISRIPKIKLP

SVTSFILDTE AVAWDREKKQ

601
IQPFQVLTTR KRKEVDASEI QVQVCLYAFD LIYLNGESLV

REPLSRRRQL LRENFVETEG

661
EFVFATSLDT KDIEQIAEFL EQSVKDSCEG LMVKTLDVDA

TYEIAKRSHN WLKLKKDYLD

721
GVGDTLDLVV IGAYLGRGKR AGRYGGFLLA SYDEDSEELQ

AICKLGTGFS DEELEEHHQS

781
LKALVLPSPR PYVRIDGAVI PDHWLDPSAV WEVKCADLSL

SPIYPAARGL VDSDKGISLR

841
FPRFIRVRED KQPEQATTSA QVACLYRKQS QIQNQQGEDS

GSDPEDTY.

The nucleic acid sequence of LIG1, isoform 3, is provided below (see also UniProt Accession No. P18858-3 and GenBank Accession No. NM_001289063; SEQ ID NO: 6):

1
ctcgcggggg cgcttccacc gattcctcct ctttccctgc

cagtcactcc tcagaccctc

61
agccacaccc gctcatccag ggcgagggaa agcgcgggca

ttttcccagt gtgctctgcg

121
ggagggctcg ccccacttca ccccttttcc cgccctcctc

ccattcggga gactacgact

181
cccagtgtcc tccgcgcgac ggcggcggtg cggacggtgc

ccaggtcccg cccctaggct

241
ctgccccgcc cccgcccgca gacgtctgcg cgcgaatgcc

gtggcgcgaa cttgggactg

301
cagaggcgcg cctggcggat ctgagtgtgt tgcccgggca

gcggcgcgcg ggaccaacgc

361
aaggagcagc tgacagacga agaaaagtgc tggacaggaa

gggagaattc tgacgccaac

421
atgcagcgaa gtatcatggc ggcactgaag gagtggaatg

gagtggtgtc cgagagtgac

481
tctccggtga agaggccagg gaggaaggcg gcccgggtcc

tgggcagcga aggggaagag

541
gaggatgaag cccttagccc tgctaaaggc cagaagcctg

ccctggactg ctcacaggtc

601
tccccgcccc gtcctgccac atctcctgag aacaatgctt

ccctctctga cacctctccc

661
atggacagtt ccccatcagg gattccgaag cgtcgcacag

ctcggaagca gctcccgaaa

721
cggaccattc aggaagtcct ggaagagcag agtgaggacg

aggacagaga agccaagagg

781
aagaaggagg aggaagagga gaccccgaaa gaaagcctca

cagaggctga agtggccaca

841
gagaaggaag gagaagacgg ggaccagccc accacgcctc

ccaagcccct aaagacctcc

901
aaagcagaga ccccgacgga aagcgtttca gagcctgagg

tggccacgaa gcaggaactg

961
caggaggagg aagagcagac caagcctccc cgcagagctc

ccaagacgct cagcagcttc

1021
ttcacccccc ggaagccagc agtcaaaaaa gaagtgaagg

aagaggagcc aggggctcca

1081
ggaaaggagg gagctgctga gggacccctg gatccatctg

gttacaatcc tcccaagaac

1141
aactatcatc ccgtggaaga tgcctgctgg aaaccgggcc

agaaggttcc ttacctggct

1201
gtggcccgga cgtttgagaa gatcgaggag gtgtctgctc

ggctccggat ggtggagacg

1261
ctgagcaact tgctgcgctc cgtggtggcc ctgtcgcctc

cagacctcct ccctgtcctc

1321
tacctcagcc tcaaccacct tgggccaccc cagcagggcc

tggagcttgg cgtgggtgat

1381
ggtgtccttc tcaaggcagt ggcccaggcc acaggtcggc

agctggagtc cgtccgggct

1441
gaggcagccg agaaaggcga cgtggggctg gtggccgaga

acagccgcag cacccagagg

1501
ctcatgctgc caccacctcc gctcactgcc tccggggtct

tcagcaagtt ccgcgacatc

1561
gccaggctca ctggcagtgc ttccacagcc aagaagatag

acatcatcaa aggcctcttt

1621
gtggcctgcc gccactcaga agcccggttc atcgctaggt

ccctgagcgg acggctgcgc

1681
cttgggctgg cagagcagtc ggtgctggct gccctctccc

aggcagtgag cctcacgccc

1741
ccgggccaag aattcccacc agccatggtg gatgctggga

agggcaagac agcagaggcc

1801
agaaagacgt ggctggagga gcaaggcatg atcctgaagc

agacgttctg cgaggttccc

1861
gacctggacc gaattatccc cgtgctgctg gagcacggcc

tggaacgtct cccggagcac

1921
tgcaagctga gcccagggat tcccctgaaa ccaatgttgg

cccatcccac ccggggcatc

1981
agcgaggtcc tgaaacgctt tgaggaggca gctttcacct

gcgaatacaa atatgacggg

2041
cagagggcac agatccacgc cctggaaggc ggggaggtga

agatcttcag caggaatcag

2101
gaagacaaca ctgggaagta cccggacatc atcagccgca

tccccaagat taaactccca

2161
tcggtcacat ccttcatcct ggacaccgaa gccgtggctt

gggaccggga aaagaagcag

2221
atccagccat tccaagtgct caccacccgc aaacgcaagg

aggtggatgc gtctgagatc

2281
caggtgcagg tgtgtttgta cgccttcgac ctcatctacc

tcaatggaga gtccctggta

2341
cgtgagcccc tttcccggcg ccggcagctg ctccgggaga

actttgtgga gacagagggc

2401
gagtttgtct tcgccacctc cctggacacc aaggacatcg

agcagatcgc cgagttcctg

2461
gagcagtcag tgaaagactc ctgcgagggg ctgatggtga

agaccctgga tcttgatgcc

2521
acctacgaga tcgccaagag atcgcacaac tggctcaagc

tgaagaagga ctaccttgat

2581
ggcgtgggtg acaccctgga cctggtggtg atcggcgcct

acctgggccg ggggaagcgg

2641
gccggccggt acgggggctt cctgctggcc tcctacgacg

aggacagtga ggagctgcag

2701
gccatatgca agcttggaac tggcttcagt gatgaggagc

tggaggagca tcaccagagc

2761
ctcaaggcgc tggtgctgcc cagcccacgc ccttacgtgc

ggatagatgg cgctgtgatt

2821
cccgaccact ggctggaccc cagcgctgtg tgggaggtga

agtgcgctga cctctccctc

2881
tctcccatct accctgctgc gcggggcctg gtggatagtg

acaagggcat ctcccttcgc

2941
ttccctcggt ttattcgagt ccgtgaagac aagcagccgg

agcaggccac caccagtgct

3001
caggtggcct gtttgtaccg gaagcaaagt cagattcaga

accaacaagg cgaggactca

3061
ggctctgacc ctgaagatac ctactaagcc ctcgccctcc

tagggcctgg gtacagggca

3121
tgagttggac ggaccccagg gttattattg cctttgcttt

ttagcaaatc tgctgtggca

3181
ggctgtggat tttgagagtc aggggagggg tgtgtgtgtg

agggggtggc ttactccgga

3241
gtctgggatt catcccgtca tttctttcaa taaataatta

ttggatagct aaaaaaaaaa

3301
aaaaaaaaaa aaaaaaa.

Fanconi Anemia Group M Protein (FANCM)

FANCM is a DEAD/DEAH box (DExD/H box) helicase. FANCM mediates DNA repair through the stabilization of DNA replication fork structures. FANCM can also resolve DNA-RNA hybrids known as R-loops.

FANCM is a component of the Fanconia Anemia (FA) complex. The Fanconia Anemia (FA) complex prevents, inhibits, minimizes or decreases replication-induced interstrand DNA crosslinks.

Although part of the FA complex, FANCM loss-of-function (LoF) mutations (also referred to herein as variants) do not cause Fanconi anemia in humans; however, an increased risk or predisposition for the development of cancers such as breast cancer have been observed. Additionally, FANCM promotes telomere maintanence in cells utilizing the alternative lengthening of telomere (ALT) pathway. Cells utilizing the ALT pathway are referred to as ALT cells or ALT+ cells, whereas cells not utilizing the ALT pathway are referred to as not being ALT cells or as being ALT− cells.

The human Fanconi Anemia Group M protein (FANCM) protein is encoded by the FANCM gene (also known as KIAA1596). FANCM (also known as Fanconi anemia group M protein (FACM, ATP-dependent RNA helicase FANCM, Fanconi anemia-associated polypeptide of 250 kDa (FAAP250) and Protein Hef ortholog) has 3 isoforms. FANCM may have an additional 4 isoforms from computational analyses (see Uniprot Accession No. Q8IYD8).

Blocking agents, compositions or formulations of the disclosure may comprise one or more blocking agents of FANCM. Blocking agents may inhibit one or more activity or function of FANCM. In particular embodiments, a FANCM blocking agent inhibits a FANCM activity or function selected from ATP-binding, nucleotide-binding, DNA-binding, DNA remodeling, DNA strand separation, DNA-RNA strand separation, or catalyzing the break of a chemical bond using water. In certain embodiments, a FANCM blocking agent inhibits FANCM helicase activity, hydrolase activity, translocase activity, or ATPase activity. In particular embodiments, the activity is inhibited by at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or about 100%.

Blocking agents, compositions or formulations of the disclosure may inhibit one or more isoforms of FANCM in a target cell, including any of those having an amino acid sequence set forth in any of SEQ ID NOs: 7, 9, or 11. Blocking agents, compositions or formulations of the disclosure may comprise one or more isoforms of FANCM. Blocking agents, compositions or formulations of the disclosure may comprise a FANCM variant encoded by an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99% or any percentage in between of identity to a FANCM protein of the disclosure. Blocking agents, compositions or formulations of the disclosure may comprise a FANCM variant encoded by an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99% or any percentage in between of identity to one or more of SEQ ID NOs: 7, 9 and 11.

Blocking agents, compositions or formulations of the disclosure may comprise a FANCM variant encoded by a nucleic sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99% or any percentage in between of identity to a nucleic acid sequence encoding a FANCM protein of the disclosure. Blocking agents, compositions or formulations of the disclosure may comprise a FANCM variant encoded by a nucleic sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99% or any percentage in between of identity to SEQ ID NO: 8, 10 and 12.

The amino acid sequence of FANCM, isoform 1, is provided below (see also UniProt Accession No. Q8IYD8-1 and GenBank Accession No. NP_065988; SEQ ID NO: 7):

1
MSGRQRTLFQ TWGSSISRSS GTPGCSSGTE RPQSPGSSKA

PLPAAAEAQL ESDDDVLLVA

61
AYEAERQLCL ENGGFCTSAG ALWIYPTNCP VRDYQLHISR

AALFCNTLVC LPTGLGKTFI

121
AAVVMYNFYR WFPSGKVVFM APTKPLVTQQ IEACYQVMGI

PQSHMAEMTG STQASTRKEI

181
WCSKRVLFLT PQVMVNDLSR GACPAAEIKC LVIDEAHKAL

GNYAYCQVVR ELVKYTNHFR

241
ILALSATPGS DIKAVQQVIT NLLIGQIELR SEDSPDILTY

SHERKVEKLI VPLGEELAAI

301
QKTYIQILES FARSLIQRNV LMRRDIPNLT KYQIILARDQ

FRKNPSPNIV GIQQGIIEGE

361
FAICISLYHG YELLQQMGMR SLYFFLCGIM DGTKGMTRSK

NELGRNEDFM KLYNHLECMF

421
ARTRSTSANG ISAIQQGDKN KKFVYSHPKL KKLEEVVIEH

FKSWNAENTT EKKRDETRVM

481
IFSSFRDSVQ EIAEMLSQHQ PIIRVMTFVG HASGKSTKGF

TQKEQLEVVK QFRDGGYNTL

541
VSTCVGEEGL DIGEVDLIIC FDSQKSPIRL VQRMGRTGRK

RQGRIVIILS EGREERIYNQ

601
SQSNKRSIYK AISSNRQVLH FYQRSPRMVP DGINPKLHKM

FITHGVYEPE KPSRNLQRKS

661
SIFSYRDGMR QSSLKKDWFL SEEEFKLWNR LYRLRDSDEI

KEITLPQVQF SSLQNEENKP

721
AQESTTGIHQ LSLSEWRLWQ DHPLPTHQVD HSDRCRHFIG

LMQMIEGMRH EEGECSYELE

781
VESYLQMEDV TSTFIAPRNE SNNLASDTFI THKKSSFIKN

INQGSSSSVI ESDEECAEIV

841
KQTHIKPTKI VSLKKKVSKE IKKDQLKKEN NHGIIDSVDN

DRNSTVENIF QEDLPNDKRT

901
SDTDEIAATC TINENVIKEP CVLLTECQFT NKSTSSLAGN

VLDSGYNSFN DEKSVSSNLF

961
LPFEEELYIV RTDDQFYNCH SLTKEVLANV ERFLSYSPPP

LSGLSDLEYE IAKGTALENL

1021
LFLPCAEHLR SDKCTCLLSH SAVNSQQNLE LNSLKCINYP

SEKSCLYDIP NDNISDEPSL

1081
CDCDVHKHNQ NENLVPNNRV QIHRSPAQNL VGENNHDVDN

SDLPVLSTDQ DESLLLFEDV

1141
NTEFDDVSLS PLNSKSESLP VSDKTAISET PLVSQFLISD

ELLLDNNSEL QDQITRDANS

1201
FKSRDQRGVQ EEKVKNHEDI FDCSRDLFSV TFDLGFCSPD

SDDEILEHTS DSNRPLDDLY

1261
GRYLEIKEIS DANYVSNQAL IPRDHSKNFT SGTVIIPSNE

DMQNPNYVHL PLSAAKNEEL

1321
LSPGYSQFSL PVQKKVMSTP LSKSNTLNSF SKIRKEILKT

PDSSKEKVNL QRFKEALNST

1381
FDYSEFSLEK SKSSGPMYLH KSCHSVEDGQ LLTSNESEDD

EIFRRKVKRA KGNVLNSPED

1441
QKNSEVDSPL HAVKKRRFPI NRSELSSSDE SENFPKPCSQ

LEDFKVCNGN ARRGIKVPKR

1501
QSHLKHVARK FLDDEAELSE EDAEYVSSDE NDESENEQDS

SLLDFLNDET QLSQAINDSE

1561
MRAIYMKSLR SPMMNNKYKM IHKTHKNINI FSQIPEQDET

YLEDSFCVDE EESCKGQSSE

1621
EEVCVDFNLI TDDCFANSKK YKTRRAVMLK EMMEQNCAHS

KKKLSRIILP DDSSEEENNV

1681
NDKRESNIAV NPSTVKKNKQ QDHCLNSVPS GSSAQSKVRS

TPRVNPLAKQ SKQTSLNLKD

1741
TISEVSDFKP QNHNEVQSTT PPFTTVDSQK DCRKFPVPQK

DGSALEDSST SGASCSKSRP

1801
HLAGTHTSLR LPQEGKGTCI LVGGHEITSG LEVISSLRAI

HGLQVEVCPL NGCDYIVSNR

1861
MVVERRSQSE MLNSVNKNKF IEQIQHLQSM FERICVIVEK

DREKTGDTSR MFRRTKSYDS

1921
LLTTLIGAGI RILFSSCQEE TADLLKELSL VEQRKNVGIH

VPTVVNSNKS EALQFYLSIP

1981
NISYITALNM CHQFSSVKRM ANSSLQEISM YAQVTHQKAE

EIYRYIHYVF DIQMLPNDLN

2041
QDRLKSDI.

The nucleic acid sequence of FANCM, isoform 1, is provided below (see also UniProt Accession No. Q8IYD8-1 and GenBank Accession No. NM_020937; SEQ ID NO: 8):

1
tgtgcgaagg aaaccgatgg ggatcggaac cgtagcggtt gagctgctgc tgctacggat

61
atctgacaga agccttcggt ggttgtcggc ctaatgagcg gacggcaaag aacgcttttt

121
cagacgtggg gctcaagtat ctcccgatca tctgggactc cgggttgcag ctccggaact

181
gagcgacctc agagccctgg cagctccaag gcgcctttgc cagcagcagc ggaggctcag

241
ctggagtcgg acgatgatgt gttgcttgtc gcggcgtacg aggctgagcg gcagttgtgt

301
ctagagaatg gcgggttctg cacctccgcg ggcgccctgt ggatttaccc taccaattgc

361
ccagtgcggg actaccagct gcacatttcc cgggctgctc tgttttgcaa tacgctggtg

421
tgtctgccta ccggactggg aaagaccttt attgccgccg tggtcatgta caatttctac

481
cgctggttcc cttcaggaaa ggtggtcttc atggccccaa cgaaaccctt ggtgacacag

541
cagatcgagg cttgctacca ggtgatgggt atcccgcaat cccacatggc cgaaatgaca

601
gggtctacac aagcttccac caggaaggaa atatggtgca gtaagagagt gctttttctt

661
acacctcagg tcatggtaaa tgacctttct agaggagctt gtcccgctgc tgaaataaag

721
tctttagtta ttgatgaagc tcataaagct ctcggaaact atgcttattg ccaggttgta

781
agagaactag tcaaatatac aaatcacttt agaatcttgg ctctaagtgc cacaccaggt

841
agtgatataa aggctgtgca acaagttatt actaacctgc taattgggca gatagagctt

901
cgttctgaag attctccaga tattttgaca tattctcatg aaagaaaagt tgaaaagctt

961
attgttccgc ttggtgaaga acttgcagcc atccaaaaga cctatatcca gattttggaa

1021
tcatttgctc gttctttgat tcagaggaat gttttgatga gaagggatat cccaaatcta

1081
acaaaatatc agataattct ggcaagagat cagtttagga aaaacccatc tccgaatatt

1141
gtgggaatac aacaaggcat aatcgaggga gagtttgcta tttgtattag tttatatcat

1201
ggttatgaat tattgcagca aatgggaatg agatcattat atttcttcct ttgtggaatt

1261
atggatggaa ctaaagggat gacacggtca aaaaatgaac ttggccgaaa tgaagacttc

1321
atgaaactct ataatcatct agagtgtatg tttgcacgta cacgtagtac ttcagcaaat

1381
ggtatttctg ctatccaaca aggagataaa aataaaaaat ttgtttatag tcatccaaag

1441
ttaaagaaat tagaagaagt tgtaattgaa cacttcaagt catggaatgc tgaaaacact

1501
actgaaaaga aacgtgatga gacccgagtt atgatcttct cttcatttcg agatagtgtt

1561
caagaaattg cagaaatgct ttcacagcat cagccaatta ttagagtaat gacttttgtc

1621
ggccatgcct cagggaaaag cacgaagggt tttacccaga aggagcaact ggaggtagtg

1681
aaacagtttc gtgacggtgg ttacaacacg ctggtttcta cctgtgtggg tgaagaaggt

1741
ttggatatag gagaagttga tcttataata tgttttgatt cccagaagag cccaattcgt

1801
cttgtacaac gaatgggtag aactggccgt aaacgtcaag gcaggatagt tattatcctt

1861
tctgaaggac gagaggaacg tatttataat cagagtcagt ccaacaaaag aagtatatat

1921
aaagctattt caagtaacag gcaggtcctt catttttacc aaagaagtcc acgaatggtt

1981
cctgatggaa tcaacccaaa attacacaaa atgttcatca cacatggtgt ctatgaacca

2041
gagaagcctt ctcggaactt gcagcgaaag tcatctatct tttcctatag ggatggaatg

2101
aggcaaagta gcctaaagaa agattggttc ttatcagaag aagaatttaa attatggaac

2161
agactttata gattaaggga cagtgatgaa attaaagaga taacattgcc tcaagttcag

2221
ttttcttctt tacaaaatga ggaaaacaaa ccagctcaag aatcaaccac tggaattcat

2281
caactctctc tctctgaatg gagactgtgg caagatcatc ctttgcctac acatcaagtt

2341
gatcactcag atcgatgccg ccattttata ggccttatgc aaatgataga gggaatgaga

2401
cacgaagagg gagaatgcag ctatgaattg gaagttgaat cttatttaca aatggaagat

2461
gttacctcaa catttattgc tcccaggaat gaatctaata atcttgccag tgacaccttt

2521
atcactcaca agaaatcgtc atttataaag aacataaatc aaggcagttc atcctcagtg

2581
atagaatctg atgaagaatg tgctgaaatt gttaaacaaa ctcatatcaa acctactaaa

2641
attgtttctt taaagaaaaa agtgtctaaa gaaataaaaa aagatcagct taaaaaagaa

2701
aataatcacg gtattataga ttctgtagat aatgacagaa attccactgt tgaaaatatt

2761
tttcaagaag acctaccaaa tgataaaagg acatcagata cagatgaaat tgctgccaca

2821
tgtactatta atgaaaatgt tattaaagaa ccgtgtgtgt tattaacaga gtgtcagttt

2881
acaaataaat ccactagttc acttgctgga aatgttttag attctggtta taacagtttc

2941
aatgatgaaa aatctgtttc atctaactta tttcttccat tcgaagaaga gctttatatt

3001
gttagaacag atgaccaatt ttataattgt cactcattga caaaagaggt actagctaat

3061
gtagagagat ttttatctta ttctcctccg cctctcagtg gactctcaga cttggaatat

3121
gaaattgcta agggtactgc acttgagaat ttgcttttct taccctgtgc agagcattta

3181
cgaagtgata aatgcacctg tttgctgtca cattcagctg tgaattctca acagaattta

3241
gaattgaatt cacttaaatg tataaattat ccatctgaaa aaagttgcct ttatgatata

3301
cctaatgata atatttctga tgagccaagt ctctgtgact gtgatgtaca taaacataat

3361
caaaatgaaa atttagtacc taacaatcgt gttcaaatac acagaagccc tgcacagaat

3421
ttagttggag agaacaatca tgatgttgat aacagtgacc tcccagtatt gtccactgat

3481
caagatgaaa gtttgctgtt atttgaagat gttaatacag agttcgacga tgtgagtctt

3541
tcacccttga acagtaaaag cgaatcttta cctgtgtcag acaaaactgc tattagtgaa

3601
acgcctctgg tctctcagtt cttaatttct gatgaacttt tgttggacaa taattctgaa

3661
ctccaagatc aaatcacccg tgatgctaat agttttaaat ctcgtgatca gagaggtgta

3721
caggaagaaa aagtgaagaa tcatgaggat atttttgatt gctctaggga tttattttct

3781
gttacctttg atttaggatt ctgtagtcca gattctgatg atgaaatatt ggaacataca

3841
tcagatagca atagacctct agatgatcta tatggaaggt atttggaaat taaggagata

3901
agtgatgcaa attatgtttc gaatcaagca ctaataccaa gagatcatag taaaaatttt

3961
actagtggaa ctgttattat cccatcaaat gaagatatgc agaatccaaa ttatgtacat

4021
ttgccactga gtgcagcaaa aaatgaagaa ttgttatctc ctggttattc tcagttttct

4081
ttaccagtgc aaaaaaaagt tatgagtaca ccactctcta aatcaaacac attgaactca

4141
ttttctaaga taagaaagga aatacttaag acaccagatt ctagtaagga aaaagtaaac

4201
ctacaaagat tcaaagaagc attgaattca acttttgatt attcagaatt ttctctagaa

4261
aagtctaaaa gcagtggtcc aatgtatctg cataaatcct gtcattctgt tgaagatgga

4321
caattattaa caagtaacga aagtgaagat gacgagattt tccgaagaaa agttaaaaga

4381
gcaaaaggaa atgttttaaa ctctcctgag gatcagaaaa atagtgaagt tgattctcca

4441
cttcatgctg tcaaaaagcg cagatttcct ataaacagat cagaattatc atctagtgat

4501
gagagtgaga attttcccaa accatgttca caattagaag acttcaaggt ttgtaacggg

4561
aatgccagaa gaggcatcaa agtcccaaag agacagagtc acttaaagca tgtagctagg

4621
aagtttttag atgatgaagc agaactttct gaagaagaty cagaatatgt ttcatcagat

4681
gaaaatgatg agtcagaaaa tgaacaagat tcctcattac ttgacttttt aaatgatgaa

4741
actcaacttt cacaggctat aaatgattct gaaatgagag ctatttacat gaaatctttg

4801
cgtagtccaa tgatgaacaa taagtacaaa atgattcata agacacataa aaacataaac

4861
attttctcgc agattcctga acaagatgaa acctatttag aggatagttt ttgtgttgat

4921
gaagaggagt cttgcaaagg ccaatcaagt gaagaagaag tttgtgttga ttttaactta

4981
ataactgatg attgctttgc aaatagtaaa aagtataaaa ctcgacgtgc agtaatgcta

5041
aaagaaatga tggaacaaaa ttgtgcacat tcaaaaaaga aattatccag aattatttta

5101
ccagatgatt caagtgagga ggagaacaat gtaaatgata aaagagaatc taatattgcg

5161
gttaacccaa gcactgttaa gaagaacaaa caacaggacc attgtttaaa ttcagtgcct

5221
tctggatctt ctgcgcagtc caaggtgcgt tctactccaa gagttaatcc attagcaaag

5281
cagagcaaac agacatcgct gaatttaaag gatacaattt ccgaagtctc agacttcaaa

5341
cctcagaatc ataatgaagt ccagtctacc acaccaccct tcactactgt tgattcacag

5401
aaagactgta gaaaatttcc agttccacag aaggatggta gtgctttgga ggattctagc

5461
acttcagggg catcctgttc caagtcaaga ccacatttag ctgggacaca tacttctctt

5521
agacttccgc aggaaggaaa aggaacctgt attcttgtag gtggtcatga aatcacttct

5581
ggattagaag taatttcttc cctaagagca attcatgggt tgcaagtaga agtttgtcct

5641
cttaatggct gtgattacat cgtgagtaat cgcatggtgg tggaaaggag gtctcaatct

5701
gagatgttaa atagtgtcaa taagaacaag ttcattgagc agatccagca cctgcagagt

5761
atgtttgaaa gaatatgtgt gattgtggaa aaggacagag aaaaaacagg agacacatca

5821
aggatgttta ggagaacaaa gagctatgac agcctgctga ctaccttaat tggcgctgga

5881
atccgaattc ttttcagttc ctgccaagaa gaaaccgcag atttgctaaa ggaactgtct

5941
ttagtggaac aaagaaagaa tgttggtatt catgttccaa cagtggtgaa tagtaataaa

6001
agtgaggcac tccagtttta tttaagtatt cccaatataa gttatataac tgcattaaat

6061
atgtgtcacc agttttcatc tgtgaaaagg atggctaaca gctcacttca agaaatctcc

6121
atgtatgcac aagtaactca tcagaaggct gaggagatct atagatatat tcactatgta

6181
tttgacatac aaatgttacc aaatgatctt aaccaagata gactgaaatc tgatatataa

6241
tcaagctgct caagatgggg ttttcaaaga cctctcacaa tattaaatgc acttcaataa

6301
tcattgctgt tttatgttta tttgtaaata agagaatatt ttatttaaat attttatatt

6361
gtatacattt ttatttatag attatagaaa ttattaaaaa agaaaaatct gatgttcagt

6421
gatcattttg actagattat aaaactaatt tttcttatta aataaaacaa ggtttattaa

6481
aagtgttact aaggatagtt taagaaagta aaagctaagc tagagatata ctttggaatg

6541
tttcccaaaa ttaaagttgt actgttgtga taaatagtaa agttgacatg tctatgacta

6601
cagccaactt gtcgattttc cctatgtgta gatagtatac ttttaagtgt actgattcta

6661
aatacatgta cttggtaagg tgtgggtgat gggtgggttg tgagataaat gacccagtaa

6721
ctaggaaagt agaaaactta actgaatgtt tatctgacca aaggtgtgtc ccagttaagt

6781
actgtcaaat ctattaatat gaactctgat atggtttggc tgtgtcccca accaaaatct

6841
catcttgact tgtaatctga attataatcc caatatattg gggagggacc tcctggaacg

6901
tgattagctc atgggggcgg ttcccccatg ctgttctagt gatagttctc agaggatctg

6961
atggttttat aagcttttcc tctgttcact ctgcagttct cttgcctact gccatgtgga

7021
aaaggaaacg tttgcttccc ctccaccatg attgtaagtt cccgaggcct ccccagccat

7081
gcaggactgt gagtcaatta aacatctttt ccttataaat taaaaaaaaa aaaaaaaa

The amino acid sequence of FANCM, isoform 2, is provided below (see also UniProt Accession No. Q8IYD8-2 and GenBank Accession No. NP_001295063; SEQ ID NO: 9):

The nucleic acid sequence of FANCM, isoform 2, is provided below (see also UniProt Accession No. Q8IYD8-2 and GenBank Accession No. NM_001308134; SEQ ID NO: 10):

The amino acid sequence of FANCM, isoform 3, is provided below (see also UniProt Accession No. Q8IYD8-3 and GenBank Accession No. NP_001295062; SEQ ID NO: 11):

1
MSGRQRTLFQ TWGSSISRSS GTPGCSSGTE RPQSPGSSKA

PLPAAAEAQL ESDDDVLLVA

61
AYEAERQLCL ENGGFCTSAG ALWIYPTNCP VRDYQLHISR

AALFCNTLVC LPTGLGKTFI

121
AAVVMYNFYR WFPSGKVVFM APTKPLVTQQ IEACYQVMGI

PQSHMAEMTG STQASTRKEI

181
WCSKRVLFLT PQVMVNDLSR GACPAAEIKC LVIDEAHKAL

GNYAYCQAVQ QVITNLLIGQ

241
IELRSEDSPD ILTYSHERKV EKLIVPLGEE LAAIQKTYIQ

ILESFARSLI QRNVLMRRDI

301
PNLTKYQIIL ARDQFRKNPS PNIVGIQQGI IEGEFAICIS

LYHGYELLQQ MGMRSLYFFL

361
CGIMDGTKGM TRSKNELGRN EDFMKLYNHL ECMFARTRST

SANGISAIQQ GDKNKKFVYS

421
HPKLKKLEEV VIEHFKSWNA ENTTEKKRDE TRVMIFSSFR

DSVQEIAEML SQHQPIIRVM

481
TFVGHASGKS TKGFTQKEQL EVVKQFRDGG YNTLVSTCVG

EEGLDIGEVD LIICFDSQKS

541
PIRLVQRMGR TGRKRQGRIV IILSEGREER IYNQSQSNKR

SIYKAISSNR QVLHFYQRSP

601
RMVPDGINPK LHKMFITHGV YEPEKPSRNL QRKSSIFSYR

DGMRQSSLKK DWFLSEEEFK

661
LWNRLYRLRD SDEIKEITLP QVQFSSLQNE ENKPAQESTT

GIHQLSLSEW RLWQDHPLPT

721
HQVDHSDRCR HFIGLMQMIE GMRHEEGECS YELEVESYLQ

MEDVTSTFIA PRNESNNLAS

781
DTFITHKKSS FIKNINQGSS SSVIESDEEC AEIVKQTHIK

PTKIVSLKKK VSKEIKKDQL

841
KKENNHGIID SVDNDRNSTV ENIFQEDLPN DKRTSDTDEI

AATCTINENV IKEPCVLLTE

901
CQFTNKSTSS LAGNVLDSGY NSFNDEKSVS SNLFLPFEEE

LYIVRTDDQF YNCHSLTKEV

961
LANVERFLSY SPPPLSGLSD LEYEIAKGTA LENLLFLPCA

EHLRSDKCTC LLSHSAVNSQ

1021
QNLELNSLKC INYPSEKSCL YDIPNDNISD EPSLCDCDVH

KHNQNENLVP NNRVQIHRSP

1081
AQNLVGENNH DVDNSDLPVL STDQDESLLL FEDVNTEFDD

VSLSPLNSKS ESLPVSDKTA

1141
ISETPLVSQF LISDELLLDN NSELQDQITR DANSFKSRDQ

RGVQEEKVKN HEDIFDCSRD

1201
LFSVTFDLGF CSPDSDDEIL EHTSDSNRPL DDLYGRYLEI

KEISDANYVS NQALIPRDHS

1261
KNFTSGTVII PSNEDMQNPN YVHLPLSAAK NEELLSPGYS

QFSLPVQKKV MSTPLSKSNT

1321
LNSFSKIRKE ILKTPDSSKE KVNLQRFKEA LNSTFDYSEF

SLEKSKSSGP MYLHKSCHSV

1381
EDGQLLTSNE SEDDEIFRRK VKRAKGNVLN SPEDQKNSEV

DSPLHAVKKR RFPINRSELS

1441
SSDESENFPK PCSQLEDFKV CNGNARRGIK VPKRQSHLKH

VARKFLDDEA ELSEEDAEYV

1501
SSDENDESEN EQDSSLLDFL NDETQLSQAI NDSEMRAIYM

KSLRSPMMNN KYKMIHKTHK

1561
NINIFSQIPE QDETYLEDSF CVDEEESCKG QSSEEEVCVD

FNLITDDCFA NSKKYKTRRA

1621
VMLKEMMEQN CAHSKKKLSR IILPDDSSEE ENNVNDKRES

NIAVNPSTVK KNKQQDHCLN

1681
SVPSGSSAQS KVRSTPRVNP LAKQSKQTSL NLKDTISEVS

DFKPQNHNEV QSTTPPFTTV

1741
DSQKDCRKFP VPQKDGSALE DSSTSGASCS KSRPHLAGTH

TSLRLPQEGK GTCILVGGHE

1801
ITSGLEVISS LRAIHGLQVE VCPLNGCDYI VSNRMVVERR

SQSEMLNSVN KNKFIEQIQH

1861
LQSMFERICV IVEKDREKTG DTSRMFRRTK SYDSLLTTLI

GAGIRILFSS CQEETADLLK

1921
ELSLVEQRKN VGIHVPTVVN SNKSEALQFY LSIPNISYIT

ALNMCHQFSS VKRMANSSLQ

1981
EISMYAQVTH QKAEEIYRYI HYVFDIQMLP NDLNQDRLKS

DI.

The nucleic acid sequence of FANCM, isoform 3, is provided below (see also UniProt Accession No. Q8IYD8-3 and GenBank Accession No. NM_001308133; SEQ ID NO: 12):

1
ccagagtttt gtgcgaagga aaccgatggg gatcggaacc gtagcggttg agctgctgct

61
gctacggata tctgacagaa gccttcggtg gttgtcggcc taatgagcgg acggcaaaga

121
acgctttttc agacgtgggg ctcaagtatc tcccgatcat ctgggactcc gggttgcagc

181
tccggaactg agcgacctca gagccctggc agctccaagg cgcctttgcc agcagcagcg

241
gaggctcagc tggagtcgga cgatgatgtg ttgcttgtcg cggcgtacga ggctgagcgg

301
cagttgtgtc tagagaatgg cgggttctgc acctccgcgg gcgccctgtg gatttaccct

361
accaattgcc cagtgcggga ctaccagctg cacatttccc gggctgctct gttttgcaat

421
acgctggtgt gtctgcctac cggactggga aagaccttta ttgccgccgt ggtcatgtac

481
aatttctacc gctggttccc ttcaggaaag gtggtcttca tggccccaac gaaacccttg

541
gtgacacagc agatcgaggc ttgctaccag gtgatgggta tcccgcaatc ccacatggcc

601
gaaatgacag ggtctacaca agcttccacc aggaaggaaa tatggtgcag taagagagtg

661
ctttttctta cacctcaggt catggtaaat gacctttcta gaggagcttg tcccgctgct

721
gaaataaagt gtttagttat tgatgaagct cataaagctc tcggaaacta tgcttattgc

781
caggctgtgc aacaagttat tactaacctg ctaattgggc agatagagct tcgttctgaa

841
gattctccag atattttgac atattctcat gaaagaaaag ttgaaaagct tattgttccg

901
cttggtgaag aacttgcagc catccaaaag acctatatcc agattttgga atcatttgct

961
cgttctttga ttcagaggaa tgttttgatg agaagggata tcccaaatct aacaaaatat

1021
cagataattc tggcaagaga tcagtttagg aaaaacccat ctccgaatat tgtgggaata

1081
caacaaggca taatcgaggg agagtttgct atttgtatta gtttatatca tggttatgaa

1141
ttattgcagc aaatgggaat gagatcatta tatttcttcc tttgtggaat tatggatgga

1201
actaaaggga tgacacggtc aaaaaatgaa cttggccgaa atgaagactt catgaaactc

1261
tataatcatc tagagtgtat gtttgcacgt acacgtagta cttcagcaaa tggtatttct

1321
gctatccaac aaggagataa aaataaaaaa tttgtttata gtcatccaaa gttaaagaaa

1381
ttagaagaag ttgtaattga acacttcaag tcatggaatg ctgaaaacac tactgaaaag

1441
aaacgtgatg agacccgagt tatgatcttc tcttcatttc gagatagtgt tcaagaaatt

1501
gcagaaatgc tttcacagca tcagccaatt attagagtaa tgacttttgt cggccatgcc

1561
tcagggaaaa gcacgaaggg ttttacccag aaggagcaac tggaggtagt gaaacagttt

1621
cgtgacggtg gttacaacac gctggtttct acctgtgtgg gtgaagaagg tttggatata

1681
ggagaagttg atcttataat atgttttgat tcccagaaga gcccaattcg tcttgtacaa

1741
cgaatgggta gaactggccg taaacgtcaa ggcaggatag ttattatcct ttctgaagga

1801
cgagaggaac gtatttataa tcagagtcag tccaacaaaa gaagtatata taaagctatt

1861
tcaagtaaca ggcaggtcct tcatttttac caaagaagtc cacgaatggt tcctgatgga

1921
atcaacccaa aattacacaa aatgttcatc acacatggtg tctatgaacc agagaagcct

1981
tctcggaact tgcagcgaaa gtcatctatc ttttcctata gggatggaat gaggcaaagt

2041
agcctaaaga aagattggtt cttatcagaa gaagaattta aattatggaa cagactttat

2101
agattaaggg acagtgatga aattaaagag ataacattgc ctcaagttca gttttcttct

2161
ttacaaaatg aggaaaacaa accagctcaa gaatcaacca ctggaattca tcaactctct

2221
ctctctgaat ggagactgtg gcaagatcat cctttgccta cacatcaagt tgatcactca

2281
gatcgatgcc gccattttat aggccttatg caaatgatag agggaatgag acacgaagag

2341
ggagaatgca gctatgaatt ggaagttgaa tcttatttac aaatggaaga tgttacctca

2401
acatttattg ctcccaggaa tgaatctaat aatcttgcca gtgacacctt tatcactcac

2461
aagaaatcgt catttataaa gaacataaat caaggcagtt catcctcagt gatagaatct

2521
gatgaagaat gtgctgaaat tgttaaacaa actcatatca aacctactaa aattgtttct

2581
ttaaagaaaa aagtgtctaa agaaataaaa aaagatcagc ttaaaaaaga aaataatcac

2641
ggtattatag attctgtaga taatgacaga aattccactg ttgaaaatat ttttcaagaa

2701
gacctaccaa atgataaaag gacatcagat acagatgaaa ttgctgccac atgtactatt

2761
aatgaaaatg ttattaaaga accgtgtgtg ttattaacag agtgtcagtt tacaaataaa

2821
tccactagtt cacttgctgg aaatgtttta gattctggtt ataacagttt caatgatgaa

2881
aaatctgttt catctaactt atttcttcca ttcgaagaag agctttatat tgttagaaca

2941
gatgaccaat tttataattg tcactcattg acaaaagagg tactagctaa tgtagagaga

3001
tttttatctt attctcctcc gcctctcagt ggactctcag acttggaata tgaaattgct

3061
aagggtactg cacttgagaa tttgcttttc ttaccctgtg cagagcattt acgaagtgat

3121
aaatgcacct gtttgctgtc acattcagct gtgaattctc aacagaattt agaattgaat

3181
tcacttaaat gtataaatta tccatctgaa aaaagttgcc tttatgatat acctaatgat

3241
aatatttctg atgagccaag tctctgtgac tgtgatgtac ataaacataa tcaaaatgaa

3301
aatttagtac ctaacaatcg tgttcaaata cacagaagcc ctgcacagaa tttagttgga

3361
gagaacaatc atgatgttga taacagtgac ctcccagtat tgtccactga tcaagatgaa

3421
agtttgctgt tatttgaaga tgttaataca gagttcgacg atgtgagtct ttcacccttg

3481
aacagtaaaa gcgaatcttt acctgtgtca gacaaaactg ctattagtga aacgcctctg

3541
gtctctcagt tcttaatttc tgatgaactt ttgttggaca ataattctga actccaagat

3601
caaatcaccc gtgatgctaa tagttttaaa tctcgtgatc agagaggtgt acaggaagaa

3661
aaagtgaaga atcatgagga tatttttgat tgctctaggg atttattttc tgttaccttt

3721
gatttaggat tctgtagtcc agattctgat gatgaaatat tggaacatac atcagatagc

3781
aatagacctc tagatgatct atatggaagg tatttggaaa ttaaggagat aagtgatgca

3841
aattatgttt cgaatcaagc actaatacca agagatcata gtaaaaattt tactagtgga

3901
actgttatta tcccatcaaa tgaagatatg cagaatccaa attatgtaca tttgccactg

3961
agtgcagcaa aaaatgaaga attgttatct cctggttatt ctcagttttc tttaccagtg

4021
caaaaaaaag ttatgagtac accactctct aaatcaaaca cattgaactc attttctaag

4081
ataagaaagg aaatacttaa gacaccagat tctagtaagg aaaaagtaaa cctacaaaga

4141
ttcaaagaag cattgaattc aacttttgat tattcagaat tttctctaga aaagtctaaa

4201
agcagtggtc caatgtatct gcataaatcc tgtcattctg ttgaagatgg acaattatta

4261
acaagtaacg aaagtgaaga tgacgagatt ttccgaagaa aagttaaaag agcaaaagga

4321
aatgttttaa actctcctga ggatcagaaa aatagtgaag ttgattctcc acttcatgct

4381
gtcaaaaagc gcagatttcc tataaacaga tcagaattat catctagtga tgagagtgag

4441
aattttccca aaccatgttc acaattagaa gacttcaagg tttgtaacgg gaatgccaga

4501
agaggcatca aagtcccaaa gagacagagt cacttaaagc atgtagctag gaagttttta

4561
gatgatgaag cagaactttc tgaagaagat gcagaatatg tttcatcaga tgaaaatgat

4621
gagtcagaaa atgaacaaga ttcctcatta cttgactttt taaatgatga aactcaactt

4681
tcacaggcta taaatgattc tgaaatgaga gctatttaca tgaaatcttt gcgtagtcca

4741
atgatgaaca ataagtacaa aatgattcat aagacacata aaaacataaa cattttctcg

4801
cagattcctg aacaagatga aacctattta gaggatagtt tttgtgttga tgaagaggag

4861
tcttgcaaag gccaatcaag tgaagaagaa gtttgtgttg attttaactt aataactgat

4921
gattgctttg caaatagtaa aaagtataaa actcgacgtg cagtaatgct aaaagaaatg

4981
atggaacaaa attgtgcaca ttcaaaaaag aaattatcca gaattatttt accagatgat

5041
tcaagtgagg aggagaacaa tgtaaatgat aaaagagaat ctaatattgc ggttaaccca

5101
agcactgtta agaagaacaa acaacaggac cattgtttaa attcagtgcc ttctggatct

5161
tctgcgcagt ccaaggtgcg ttctactcca agagttaatc cattagcaaa gcagagcaaa

5221
cagacatcgc tgaatttaaa ggatacaatt tccgaagtct cagacttcaa acctcagaat

5281
cataatgaag tccagtctac cacaccaccc ttcactactg ttgattcaca gaaagactgt

5341
agaaaatttc cagttccaca gaaggatggt agtgctttgg aggattctag cacttcaggg

5401
gcatcctgtt ccaagtcaag accacattta gctgggacac atacttctct tagacttccg

5461
caggaaggaa aaggaacctg tattcttgta ggtggtcatg aaatcacttc tcgattagaa

5521
gtaatttctt ccctaagagc aattcatggg ttgcaagtag aagtttgtcc tcttaatggc

5581
tgtgattaca tcgtgagtaa tcgcatggtg gtggaaagga ggtctcaatc tgagatgtta

5641
aatagtgtca ataagaacaa gttcattgag cagatccagc acctgcagag tatgtttgaa

5701
agaatatgtg tgattgtgga aaaggacaga gaaaaaacag gagacacatc aaggatgttt

5761
aggagaacaa agagctatga cagcctgctg actaccttaa ttggcgctgg aatccgaatt

5821
cttttcagtt cctgccaaga agaaaccgca gatttgctaa aggaactgtc tttagtggaa

5881
caaagaaaga atgttggtat tcatgttcca acagtggtga atagtaataa aagtgaggca

5941
ctccagtttt atttaagtat tcccaatata agttatataa ctgcattaaa tatgtgtcac

6001
cagttttcat ctgtgaaaag gatggctaac agctcacttc aagaaatctc catgtatgca

6061
caagtaactc atcagaaggc tgaggagatc tatagatata ttcactatgt atttgacata

6121
caaatgttac caaatgatct taaccaagat agactgaaat ctgatatata atcaagctgc

6181
tcaagatggg gttttcaaag acctctcaca atattaaatg cacttcaata atcattgctg

6241
ttttatgttt atttgtaaat aagagaatat tttatttaaa tattttatat tgtatacatt

6301
tttatttata gattatagaa attattaaaa aagaaaaatc tgatgttcag tgatcatttt

6361
gactagatta taaaactaat ttttcttatt aaataaaaca aggtttatta aaagtgttac

6421
taaggatagt ttaagaaagt aaaagctaag ctagagatat actttggaat gtttcccaaa

6481
attaaagttg tactgttgtg ataaatagta aagttgacat gtctatgact acagccaact

6541
tgtcgatttt ccctatgtgt agatagtata cttttaagtg tactgattct aaatacatgt

6601
acttggtaag gtgtgggtga tgggtgggtt gtgagataaa tgacccagta actaggaaag

6661
tagaaaactt aactgaatgt ttatctgacc aaaggtgtgt cccagttaag tactgtcaaa

6721
tctattaata tgaactctga tatggtttgg ctgtgtcccc aaccaaaatc tcatcttgac

6781
ttgtaatctg aattataatc ccaatatatt ggggagggac ctcctggaac gtgattagct

6841
catgggggcg gttcccccat gctgttctag tgatagttct cagaggatct gatggtttta

6901
taagcttttc ctctgttcac tctgcagttc tcttgcctac tgccatgtgg aaaaggaaac

6961
gtttgcttcc cctccaccat gattgtaagt tcccgaggcc tccccagcca tgcaggactg

7021
tgagtcaatt aaacatcttt tccttataaa tta.

Alt+ Background

Alternative lengthening of telomeres (ALT) is a telomerase independent mechanism of maintaining telomeres that utilizes break-induced replication stimulated by DSB's thus inducing sustained replicative stress (see FIG. 1). ALT is independent of telomerase and dependent upon homologous recombination. In some embodiments, ALT is mediated directly or indirectly through a function or an activity of one or more of RAD51, RAD51C, RAD51D, XRCC2, XPF, MRE11A, ATM, BARD1, BRIP1, CHEK1, CHEK2, NBN, PALB2 and SLX4. In some embodiments, ALT is mediated directly or indirectly through a function or an activity of one or more of SLX4, RAD51 and MRE11A.

Measurable ALT hallmarks include, but are not limited to, extrachromosomal telomeric DNA (C-circles) that can be measured via rolling circle PCR; absence of telomerase activity; ALT associated telomeric containing promyelocytic leukemia protein (PML) nuclear bodies that can be measured using immunohistochemistry (IHC) assays; Heterogenous telomere sequences that can be measured using whole genome sequencing (WGS) assay; and ATRX/DAXX mutations.

ALT occurs in about 5-10% of cancer. The prevalence is high in mesenchymal tumors and particularly high in sarcomas.

ALT is a marker of poor prognosis in most tumor types (especially soft tissue sarcomas).

The role of ALT in non-cancerous or healthy cells is unknown.

Target cells of the disclosure may demonstrate ALT in addition to or as an alternative to an impaired, defective or deregulated DNA repair pathway. In some embodiments, sarcoma target cells of the disclosure may demonstrate ALT in addition to or as an alternative to an impaired, defective or deregulated DNA repair pathway. In some embodiments, a sarcoma target cell of the disclosure may demonstrate ALT in addition to or as an alternative to an impaired, defective or deregulated DNA repair pathway. In other embodiments, target cells of the disclosure are not ALT+ cells. In some embodiments, breast cancer or ovarian cancer target cells, e.g., comprising a BRCA1 or BRCA2 mutation, are not ALT+ cells, or are not ALT+ cells prior to treatment with the blocking agent.

Composition and methods of the disclosure may increase or enhance a function or an activity of ALT in a target cell. In some embodiments, a FANCM blocking agent of the disclosure may increase or enhance a function or an activity of ALT in a target cell. In some embodiments, the FANCM blocking agent induces increased or enhanced a function of ALT in a target cell. In some embodiments, the increased or enhanced function of ALT induces DNA damage. In some embodiments, the FANCM blocking agent induces uncontrolled ALT and DNA damage in ALT+ cells. In cells having uncontrolled ALT, the threshold at which the accumulation of DNA damage sufficient to cause cell cycle arrest and/or induce programmed cell death may lower than in a cell not having uncontrolled ALT.

FANCM is a component of multiple repair pathways and is required for genome protection against Interstrand crosslinks (ICLs). ICLs obstruct both replication and transcription. FANCM binding at ICLs recruits a core FA complex and ATR-dependent checkpoint activation. The activation of the cell cycle checkpoint stalls the complex at the stalled replication forks referred to as the replisome. ICLs can lead to double strand breaks in a DNA sequence if they are not resolved. In this context, the HR repair pathway involves DSB end resection, strand invasion and Holliday junction resolution. When a cell having an impaired, defective or deregulated HR pathway contacts a FANCM blocking agent, the synergistic combination results in DNA damage sufficient to cause cell cycle arrest and/or induce programmed cell death.

Target cells of the disclosure having an impaired, defective or deregulated HR pathway may increase their dependence upon FANCM and FANCM-mediated repair. This increased dependence upon FANCM may occur in the absence of one or more ICL(s). This increased dependence upon FANCM neither requires nor depends upon the occurrence of one or more ICL(s).

Methods

Compositions and methods of the disclosure may be used to reduce or inhibit survival or growth of target cells, e.g., cells comprising an impaired, defective or deregulated DNA repair pathway, including any of those described herein. In particular embodiments, the target cells comprise an impaired HR repair pathway. In certain embodiments, the cells are BRCA negative (BRCA^−/−) cells. In some embodiments, the BRCA is BRCA1 and/or BRCA2. Thus, in certain embodiments, target cells are BRCA1^−/− or BRCA2^−/−. In certain embodiments, the cells are tumor cells.

Compositions and methods of the disclosure may be used to induce or increase DNA damage and/or cell cycle arrest of target cells, e.g., cells comprising an impaired, defective or deregulated DNA repair pathway, including any of those described herein. In particular embodiments, the target cells comprise an impaired HR repair pathway. In certain embodiments, the cells are BRCA negative (BRCA^−/−) cells. In some embodiments, the BRCA is BRCA1 and/or BRCA2. Thus, in certain embodiments, target cells are BRCA1^−/− or BRCA2^−/−. In certain embodiments, the cells are tumor cells.

Composition and methods of the disclosure may be used to treat a disease or disorder. In some embodiments, the disease or disorder is a proliferative disorder or a disorder characterized by an aberrant or uncontrolled proliferation of one or more cells or types of cells. In some embodiments, the disease or disorder is a cancer. In some embodiments, the disease or disorder is a sarcoma. In some embodiments, the disease or disorder is a cancer of a bone or connective tissue. In some embodiments, the disease or disorder is a cancer of an epithelial tissue or of an organ or tissue comprising an epithelial cell. In some embodiments, the disease or disorder is an ovarian cancer.

Exemplary proliferative disorders of the disclosure include, but are not limited to, benign proliferative disorders (e.g. benign tumor that causes harm by blockage), oncogenic proliferative disorders (e.g. cancer, metastatic cancer), immunoproliferative disorders (e.g. inflammation and autoimmune disorders), Lymphoproliferative disorders, and myeloproliferative disorders.

Exemplary cancers of the disclosure include, but are not limited to, Acute Lymphocytic Leukemia (ALL) in Adults, Acute Myeloid Leukemia (AML) in Adults, Adrenal Cancer, Anal Cancer, Basal and Squamous Cell Skin Cancer, Bile Duct Cancer, Bladder Cancer, Bone Cancer, Brain and Spinal Cord Tumors in Adults, Brain and Spinal Cord Tumors in Children, Breast Cancer, Breast Cancer in Men, Cancer in Adolescents, Cancer in Children, Cancer in Young Adults, Cancer of Unknown Primary, Castleman Disease, Cervical Cancer, Chronic Lymphocytic Leukemia (CLL), Chronic Myeloid Leukemia (CIVIL), Chronic Myelomonocytic Leukemia (CMML), Colorectal Cancer, Endometrial Cancer, Esophagus Cancer, Ewing Family of Tumors, Ewing Sarcoma, Eye Cancer (Ocular Melanoma), Gallbladder Cancer, Gastrointestinal Neuroendocrine (Carcinoid) Tumors, Gastrointestinal Stromal Tumor (GIST), Gestational Trophoblastic Disease, Hodgkin Lymphoma, Kaposi Sarcoma, Kidney Cancer, Laryngeal and Hypopharyngeal Cancer, Leukemia, Leukemia in Children, Liver Cancer, Lung Cancer, Lung Carcinoid Tumor, Lymphoma, Lymphoma of the Skin, Malignant Mesothelioma, Melanoma Skin Cancer, Merkel Cell Skin Cancer, Multiple Myeloma, Myelodysplastic Syndromes, Nasal Cavity and Paranasal Sinuses Cancer, Nasopharyngeal Cancer, Neuroblastoma, Non-Hodgkin Lymphoma, Oral Cavity and Oropharyngeal Cancer, Osteosarcoma, Ovarian Cancer, Pancreatic Cancer, Pancreatic Neuroendocrine Tumor (NET), Penile Cancer, Pituitary Tumors, Prostate Cancer, Retinoblastoma, Rhabdomyosarcoma, Salivary Gland Cancer, Skin Cancer, Small Intestine Cancer, Soft Tissue Sarcoma, Stomach Cancer, Testicular Cancer, Thymus Cancer, Thyroid Cancer, Uterine Sarcoma, Vaginal Cancer, Vulvar Cancer, Waldenstrom Macroglobulinemia and Wilms Tumor.

In certain embodiments, the method comprises treating a cancer with a FANCM blocking agent. In particular embodiments, the cancer comprises an impaired, defective or deregulated DNA repair pathway. In certain embodiments, the DNA repair pathway is the homologous recombination (HR) repair pathway.

In some embodiments of the methods of the disclosure, the cancer is a BRCA negative (BRCA^−/−) ovarian cancer, e.g., a BRCA1^−/− and/or BRCA2^−/− ovarian cancer. In some embodiments, the BRCA is BRCA1 and/or BRCA2. Thus, in certain embodiments, target cells are BRCA1^−/− and/or BRCA2^−/−. In particular embodiments, the BRCA1 and/or BRCA2 gene comprises a deletion or modification. In some embodiments, the BRCA^−/− ovarian cancer is resistant to treatment with a PARP inhibitor. In some embodiments, the BRCA^−/− ovarian cancer resistance to a PARP inhibitor is acquired resistance following treatment with a PARP inhibitor. In certain embodiments, the disclosure provides a method for treating a BRCA^−/− ovarian cancer in a subject, comprising providing to the subject a FANCM blocking agent. In particular embodiments, the blocking agent is an ion, a small molecule, a single-stranded nucleic acid molecule, a double-stranded nucleic acid molecule, an aptamer, an RNA-guided nuclease, a DNA-guided nuclease, a polypeptide, an antibody, a functional fragment of an antibody, an antibody mimetic, a scaffold, a matrix, or any combination thereof.

In some embodiments of the methods of the disclosure, the cancer is a BRCA negative (BRCA^−/−) breast cancer, e.g., a BRCA1^−/− and/or BRCA2^−/− breast cancer. In some embodiments, the BRCA is BRCA1 and/or BRCA2. In particular embodiments, the BRCA1 and/or BRCA2 gene comprises a deletion or modification. In some embodiments, the BRCA^−/− breast cancer is resistant to treatment with a PARP inhibitor. In some embodiments, the BRCA^−/− breast cancer resistance to a PARP inhibitor is acquired resistance following treatment with a PARP inhibitor. In certain embodiments, the disclosure provides a method for treating a BRCA^−/− breast cancer in a subject, comprising providing to the subject a FANCM blocking agent. In particular embodiments, the blocking agent is an ion, a small molecule, a single-stranded nucleic acid molecule, a double-stranded nucleic acid molecule, an aptamer, an RNA-guided nuclease, a DNA-guided nuclease, a polypeptide, an antibody, a functional fragment of an antibody, an antibody mimetic, a scaffold, a matrix, or any combination thereof.

In some embodiments of the methods of the disclosure, the target cell is in vivo or ex vivo. In some embodiments, compositions of the disclosure are contacted to a target cell in vivo or administered to a subject comprising the target cell to treat a disease or disorder of the disclosure. In some embodiments, compositions of the disclosure are contacted to a target cell ex vivo. The target cell may be cultured or maintained as a culture for use as a companion diagnostic. The target cell may be contacted with a composition of the disclosure ex vivo to confirm the subject's response to a therapeutic administration of the composition prior to the therapeutic administration. The target cell may be used to develop a library of cells for screening compositions of the disclosure as a companion diagnostic method.

In some embodiments of the methods of the disclosure, the method comprises administrating to a subject an effective amount of the composition of the disclosure. In some embodiments, the method further comprises administering a second therapy or a second therapeutic composition to the subject. In some embodiments, the second therapy or the second therapeutic composition is administered simultaneously with a composition of the disclosure. In some embodiments, the second therapy or the second therapeutic composition is administered sequentially with a composition of the disclosure. In some embodiments, a composition of the disclosure is administered to subject and, subsequently, a second therapy or a second therapeutic composition is administered to the subject. In some embodiments, a second therapy or a second therapeutic composition is administered to the subject, and subsequently, a composition of the disclosure is administered to the subject. In some embodiments, a therapeutically effective amount of a second therapy or a second therapeutic composition is administered to the subject

In some embodiments of the methods of the disclosure, a second therapy or a second therapeutic composition comprises a radiation treatment and/or a chemotherapy. In some embodiments, the chemotherapy comprises a Poly (ADP-ribose) polymerase (PARP) inhibitor or a platinum-based therapy. In some embodiments, the disease or disorder is cancer and the cancer is resistant to treatment with a PARP inhibitor as a monotherapy. In some embodiments, prior to administration of the composition, the subject has been identified as resistant to treatment with a PARP inhibitor as a monotherapy. In some embodiments, prior to administration of the composition, the subject has been treated with a PARP inhibitor as a monotherapy.

In some embodiments of the methods of the disclosure, the administration of a composition of the disclosure or of a second therapy or of a second therapeutic composition is systemic. In some embodiments, a composition of the disclosure is administered by one or more of an oral route, an inhaled route, an intravenous route, an intraperitoneal route, and a subcutaneous route. In some embodiments, a second therapy or a second therapeutic composition of the disclosure is administered by one or more of an oral route, an inhaled route, an intravenous route, an intraperitoneal route, and a subcutaneous route.

In some embodiments of the methods of the disclosure, the administration of a composition of the disclosure or of a second therapy or of a second therapeutic composition is local. In some embodiments, a composition of the disclosure is administered by one or more of an intraocular route, an intraspinal route, an intracerebellar route, an intrathecal route, an intramuscular route and an intraosseous route. In some embodiments, a second therapy or a second therapeutic composition of the disclosure is administered by one or more of an intraocular route, an intraspinal route, an intracerebellar route, an intrathecal route, an intramuscular route and an intraosseous route.

In some embodiments of the methods of the disclosure, a composition of the disclosure or of a second therapy or of a second therapeutic composition is administered once per day, twice per day or three times per day. In some embodiments of the methods of the disclosure, a composition of the disclosure or of a second therapy or of a second therapeutic composition is administered once per week, twice per week or three times per week. In some embodiments of the methods of the disclosure, a composition of the disclosure or of a second therapy or of a second therapeutic composition is administered once per month, twice per month or three times per month.

In some embodiments of the methods of the disclosure, including those wherein the disease or disorder is cancer, treating comprises a reduction in a severity of a sign or symptom of the cancer. In some embodiments, a therapeutically effective amount of a composition of the disclosure or of a second therapy or of a second therapeutic composition reduces the severity of a sign or symptom of the cancer.

In some embodiments of the methods of the disclosure, including those wherein the disease or disorder is cancer, treating comprises a reduction in a volume of a tumor. In some embodiments, a therapeutically effective amount of a composition of the disclosure or of a second therapy or of a second therapeutic composition reduces a number of tumor cells per volume of blood or mass of tissue.

In some embodiments of the methods of the disclosure, including those wherein the disease or disorder is cancer, treating comprises a remission. In some embodiments, a therapeutically effective amount of a composition of the disclosure or of a second therapy or of a second therapeutic composition induces a remission.

In some embodiments of the methods of the disclosure, including those wherein the disease or disorder is cancer, treating comprises an increased duration of progression free survival. In some embodiments, a therapeutically effective amount of a composition of the disclosure or of a second therapy or of a second therapeutic composition increases the duration of progression free survival.

Blocking Agents

Blocking agents may selectively inhibit or disrupt one or more DNA repair pathways or components thereof. In certain embodiments, a blocking agent inhibits or disrupts LIG1 or FANCM.

In certain embodiments, the blocking agent specifically, selectively or preferentially binds to or inhibits a targeted DNA repair pathway or component thereof. In certain embodiments, the blocking agent binds to the target with a dissociation constant (KD) of about 1 μM or less, about 100 nM or less, about 40 nM or less, about 20 nM or less, or about 10 nM or less.

Various types of blocking agents are known in the art and may be used according to aspects of the disclosure. In particular embodiments, a blocking agent is an ion, a small molecule, a single-stranded nucleic acid molecule, a double-stranded nucleic acid molecule, an aptamer, an RNA-guided nuclease, a DNA-guided nuclease, a polypeptide, an antibody, a functional fragment of an antibody, an antibody mimetic, a scaffold, a matrix, or any combination thereof.

In certain embodiments, the blocking agent is a small molecule, e.g., a small organic molecule that inhibits a target DNA repair pathway or component thereof, e.g., LIG1 or FANCM.

In certain embodiments, the blocking agent is a polypeptide. In particular embodiments, a blocking agent comprises a variant of a targeted DNA repair pathway protein, such as, e.g. a LIG1 or FANCM variant or fragment, or fragment variant, including but not limited to any disclosed herein. In particular embodiments, the variant comprises a mutation in one or more amino acids required for a function or activity of the targeted DNA repair pathway protein. In certain embodimemts, the variant is a fragment of the targeted DNA repair pathway protein or variant thereof, e.g., a dominant negative inhibitor.

In certain embodiments, the blocking agent is an antibody or functional fragment thereof, which binds to a targeted DNA repair pathway protein, e.g., LIG1 or FANCM, to inhibit one or more functions or activities of the targeted protein. A variety of antibodies and functional fragments thereof are known in the art and may be used according to the disclosure, including but not limited to monoclonal antibodies. The term “monoclonal antibody” encompasses not only intact monoclonal antibodies and full-length monoclonal antibodies, but also fragments thereof (such as Fab, Fab′, F(ab′)2, Fv), single chain Fv (scFv), diabodies, single domain (sdAb) or VHH antibody fragment (also known as a Nanobody®), UniBody®, variants thereof, fusion proteins comprising an antigen-binding fragment of a monoclonal antibody, humanized monoclonal antibodies, chimeric monoclonal antibodies, and any other modified configuration of the immunoglobulin molecule that comprises an antigen-binding fragment (epitope recognition site) of the required specificity and the ability to bind to an epitope. It is not intended to be limited as regards the source of the antibody or the manner in which it is made (e.g., by hybridoma, phage selection, recombinant expression, transgenic animals, etc.). The term includes whole immunoglobulins as well as the fragments etc. described above under the definition of “antibody”.

In certain embodiments, a blocking agent is a component of a gene editing system. As used herein, a gene-editing system is a system comprising one or more proteins or polynucleotides capable of editing an endogenous target gene or locus in a sequence specific manner. In some embodiments, the gene-editing system is a protein-based gene regulating system comprising a protein comprising one or more zinc-finger binding domains and an enzymatic domain. In some embodiments, the protein-based gene regulating system comprises a protein comprising a Transcription activator-like effector nuclease (TALEN) domain and an enzymatic domain. Such embodiments are referred to herein as “TALENs”.

Zinc finger-based systems comprise a fusion protein comprising two protein domains: a zinc finger DNA binding domain and an enzymatic domain. A “zinc finger DNA binding domain”, “zinc finger protein”, or “ZFP” is a protein, or a domain within a larger protein, that binds DNA in a sequence-specific manner through one or more zinc fingers, which are regions of amino acid sequence within the binding domain whose structure is stabilized through coordination of a zinc ion. The zinc finger domain, by binding to a target DNA sequence, directs the activity of the enzymatic domain to the vicinity of the sequence and, hence, induces modification of the endogenous target gene in the vicinity of the target sequence. A zinc finger domain can be engineered to bind to virtually any desired sequence. Accordingly, after identifying a target genetic locus containing a target DNA sequence at which cleavage or recombination is desired (e.g., a target locus in a target gene referenced in Table 1), one or more zinc finger binding domains can be engineered to bind to one or more target DNA sequences in the target genetic locus. Expression of a fusion protein comprising a zinc finger binding domain and an enzymatic domain in a cell, effects modification in the target genetic locus.

TALEN-based systems comprise a protein comprising a TAL effector DNA binding domain and an enzymatic domain. They are made by fusing a TAL effector DNA-binding domain to a DNA cleavage domain (a nuclease which cuts DNA strands). The FokI restriction enzyme is an exemplary enzymatic domain suitable for use in TALEN-based gene regulating systems. Methods and compositions for assembling TAL-effector repeats are known in the art. See e.g., Cermak et al, Nucleic Acids Research, 39:12, 2011, e82. Plasmids for constructions of the TAL-effector repeats are commercially available from Addgene.

In some embodiments, the gene-editing system is a combination gene-regulating system comprising a site-directed modifying polypeptide and a nucleic acid guide molecule. Herein, a “site-directed modifying polypeptide” refers to a polypeptide that binds to a nucleic acid guide molecule, is targeted to a target nucleic acid sequence, such as, for example, a DNA sequence, by the nucleic acid guide molecule to which it is bound, and modifies the target DNA sequence (e.g., cleavage, mutation, or methylation of target DNA). A site-directed modifying polypeptide comprises two portions, a portion that binds the nucleic acid guide and an activity portion. In some embodiments, a site-directed modifying polypeptide comprises an activity portion that exhibits site-directed enzymatic activity (e.g., DNA methylation, DNA cleavage, histone acetylation, histone methylation, etc.), wherein the site of enzymatic activity is determined by the guide nucleic acid.

The nucleic acid guide comprises two portions: a first portion that is complementary to, and capable of binding with, an endogenous target DNA sequence (referred to herein as a “DNA-binding segment”), and a second portion that is capable of interacting with the site-directed modifying polypeptide (referred to herein as a “protein-binding segment”). In some embodiments, the DNA-binding segment and protein-binding segment of a nucleic acid guide are comprised within a single polynucleotide molecule. In some embodiments, the DNA-binding segment and protein-binding segment of a nucleic acid guide are each comprised within separate polynucleotide molecules, such that the nucleic acid guide comprises two polynucleotide molecules that associate with each other to form the functional guide.

The nucleic acid guide mediates the target specificity of the combined protein/nucleic gene regulating systems by specifically hybridizing with a target DNA sequence comprised within the DNA sequence of a target gene. Reference herein to a target gene encompasses the full-length DNA sequence for that particular gene and a full-length DNA sequence for a particular target gene will comprise a plurality of target genetic loci, which refer to portions of a particular target gene sequence (e.g., an exon or an intron). Within each target genetic loci are shorter stretches of DNA sequences referred to herein as “target DNA sequences” or “target sequences” that can be modified by the gene-regulating systems described herein. Further, each target genetic loci comprises a “target modification site,” which refers to the precise location of the modification induced by the gene-regulating system (e.g., the location of an insertion, a deletion, or mutation, the location of a DNA break, or the location of an epigenetic modification). The gene-regulating systems described herein may comprise a single nucleic acid guide, or may comprise a plurality of nucleic acid guides (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more nucleic acid guides).

In some embodiments, the gene editing systems described herein are CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas (CRISPR Associated) nuclease systems. In such embodiments, the site-directed modifying polypeptide is a CRISPR-associated endonuclease (a “Cas” endonuclease) and the nucleic acid guide molecule is a guide RNA (gRNA).

A Cas polypeptide refers to a polypeptide that can interact with a gRNA molecule and, in concert with the gRNA molecule, homes or localizes to a target DNA sequence and includes naturally occurring Cas proteins and engineered, altered, or otherwise modified Cas proteins that differ by one or more amino acid residues from a naturally-occurring Cas sequence.

In some embodiments, the Cas protein is a Cas9 protein. Cas9 is a multi-domain enzyme that uses an HNH nuclease domain to cleave the target strand of DNA and a RuvC-like domain to cleave the non-target strand. In some embodiments, mutants of Cas9 can be generated by selective domain inactivation enabling the conversion of WT Cas9 into an enzymatically inactive mutant (e.g., dCas9), which is unable to cleave DNA, or a nickase mutant, which is able to produce single-stranded DNA breaks by cleaving one or the other of the target or non-target strand.

A guide RNA (gRNA) comprises two segments, a DNA-binding segment and a protein-binding segment. In some embodiments, the protein-binding segment of a gRNA is comprised in one RNA molecule and the DNA-binding segment is comprised in another separate RNA molecule. Such embodiments are referred to herein as “double-molecule gRNAs” or “two-molecule gRNA” or “dual gRNAs.” In some embodiments, the gRNA is a single RNA molecule and is referred to herein as a “single-guide RNA” or an “sgRNA.” The term “guide RNA” or “gRNA” is inclusive, referring both to two-molecule guide RNAs and sgRNAs.

The protein-binding segment of a gRNA comprises, in part, two complementary stretches of nucleotides that hybridize to one another to form a double stranded RNA duplex (dsRNA duplex), which facilitates binding to the Cas protein.

The DNA-binding segment (or “DNA-binding sequence”) of a gRNA comprises a nucleotide sequence that is complementary to and capable of binding to a specific sequence target DNA sequence. The protein-binding segment of the gRNA interacts with a Cas polypeptide and the interaction of the gRNA molecule and site-directed modifying polypeptide results in Cas binding to the endogenous DNA and produces one or more modifications within or around the target DNA sequence. The precise location of the target modification site is determined by both (i) base-pairing complementarity between the gRNA and the target DNA sequence; and (ii) the location of a short motif, referred to as the protospacer adjacent motif (PAM), in the target DNA sequence. The PAM sequence is required for Cas binding to the target DNA sequence. A variety of PAM sequences are known in the art and are suitable for use with a particular Cas endonuclease (e.g., a Cas9 endonuclease) are known in the art (See e.g., Nat Methods. 2013 November; 10(11): 1116-1121 and Sci Rep. 2014; 4: 5405). In some embodiments, the PAM sequence is located within 50 base pairs of the target modification site. In some embodiments, the PAM sequence is located within 10 base pairs of the target modification site. The DNA sequences that can be targeted by this method are limited only by the relative distance of the PAM sequence to the target modification site and the presence of a unique 20 base pair sequence to mediate sequence-specific, gRNA-mediated Cas binding. In some embodiments, the target modification site is located at the 5′ terminus of the target locus. In some embodiments, the target modification site is located at the 3′ end of the target locus. In some embodiments, the target modification site is located within an intron or an exon of the target locus.

In some embodiments, the present disclosure provides a polynucleotide encoding a gRNA. In some embodiments, a gRNA-encoding nucleic acid is comprised in an expression vector, e.g., a recombinant expression vector. In some embodiments, the present disclosure provides a polynucleotide encoding a site-directed modifying polypeptide. In some embodiments, the polynucleotide encoding a site-directed modifying polypeptide is comprised in an expression vector, e.g., a recombinant expression vector.

In some embodiments, the site-directed modifying polypeptide is a Cas protein. Cas molecules of a variety of species can be used in the methods and compositions described herein.

In some embodiments, the Cas protein is a Cas9 protein or a Cas9 ortholog.

Guide RNAs (gRNAs) direct a site-directed modifying polypeptide to a specific target DNA sequence. A gRNA comprises a DNA-targeting segment and protein-binding segment. The DNA-targeting segment of a gRNA comprises a nucleotide sequence that is complementary to a sequence in the target DNA sequence. As such, the DNA-targeting segment of a gRNA interacts with a target DNA in a sequence-specific manner via hybridization (i.e., base pairing), and the nucleotide sequence of the DNA-targeting segment determines the location within the target DNA that the gRNA will bind. The DNA-targeting segment of a gRNA can be modified (e.g., by genetic engineering) to hybridize to any desired sequence within a target DNA sequence.

The protein-binding segment of a guide RNA interacts with a site-directed modifying polypeptide (e.g. a Cas9 protein) to form a complex. The guide RNA guides the bound polypeptide to a specific nucleotide sequence within target DNA via the above-described DNA-targeting segment. The protein-binding segment of a guide RNA comprises two stretches of nucleotides that are complementary to one another and which form a double stranded RNA duplex.

In some embodiments, a gRNA comprises two separate RNA molecules. In such embodiments, each of the two RNA molecules comprises a stretch of nucleotides that are complementary to one another such that the complementary nucleotides of the two RNA molecules hybridize to form the double-stranded RNA duplex of the protein-binding segment. In some embodiments, a gRNA comprises a single RNA molecule (sgRNA). The specificity of a gRNA for a target loci is mediated by the sequence of the DNA-binding segment, which comprises about 20 nucleotides that are complementary to a target DNA sequence within the target locus. In some embodiments, the corresponding target DNA sequence is approximately 20 nucleotides in length. In some embodiments, the DNA-binding segments of the gRNA sequences of the present invention are at least 90% complementary to a target DNA sequence within a target locus. In some embodiments, the DNA-binding segments of the gRNA sequences of the present invention are at least 95%, 96%, 97%, 98%, or 99% complementary to a target DNA sequence within a target locus. In some embodiments, the DNA-binding segments of the gRNA sequences of the present invention are 100% complementary to a target DNA sequence within a target locus.

In some embodiments, the DNA-binding segments of the gRNA sequences bind to a target DNA sequence that is at least 90% identical to a target DNA sequence of a target disclosed herein. In some embodiments, the DNA-binding segments of the gRNA sequences bind to a target DNA sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to a target DNA sequence disclosed herein for LIG1 or FANCM. In some embodiments, the DNA-binding segments of the gRNA sequences bind to a target DNA sequence that is 100% identical to a target DNA sequence within a target locus of a LIG1 or FANCM gene.

Enumerated Embodiments

Certain non-limiting embodiments of aspects of the disclosure are enumerated below.

1. A composition comprising a Fanconi Anemia Group M protein (FANCM) blocking agent, wherein in a target cell comprising an impaired, defective or deregulated DNA repair pathway, the blocking agent reduces or inhibits a function of FANCM.

2. The composition of 1, wherein the target cell is a proliferating cell.

3. The composition of 1 or 2, wherein the target cell is a tumor cell.

4. The composition of any one of 1-3, wherein the target cell is a malignant cell.

5. The composition of any one of 1-4, wherein the target cell is a metastatic cell.

6. The composition of any one of 1-5, wherein the impaired, defective or deregulated DNA repair pathway is an impaired, defective or deregulated homologous recombination (HR) repair pathway, optionally wherein the target cell comprises a variant protein of the HR pathway.

7. The composition of 6, wherein the target cell comprises a BRCA gene deletion, a variant BRCA protein, or a sequence encoding a variant BRCA protein, wherein the variant BRCA protein induces a loss or reduction in a function of the HR pathway.

8. The composition of 7, wherein the BRCA gene deletion is a BRCA1 gene deletion, and the variant BRCA protein comprises a variant BRCA1 protein, or wherein the sequence encoding the variant BRCA protein comprises a sequence encoding a variant BRCA1 protein.

9. The composition of 7, wherein the BRCA gene deletion is a BRCA2 gene deletion, and the variant BRCA protein comprises a variant BRCA2 protein, or wherein the sequence encoding the variant BRCA protein comprises a sequence encoding a variant BRCA2 protein.

10. The composition of 6, wherein the target cell comprises a nucleic acid or an amino acid encoding a variant DNA repair protein RAD51 homolog 1 (RAD51) or a variant homolog of RAD51 and wherein the variant RAD51 induces a loss or reduction in a function of the HR pathway.

11. The composition of 6, wherein the target cell comprises a nucleic acid or an amino acid encoding a variant DNA repair protein RAD51 homolog 3 (RAD51C) or a variant homolog of RAD51C and wherein the variant RAD51C induces a loss or reduction in a function of the HR pathway.

12. The composition of 6, wherein the target cell comprises a nucleic acid or an amino acid encoding a variant DNA repair protein RAD51 homolog 4 (RAD51D) or a variant homolog of RAD51D and wherein the variant RAD51D induces a loss or reduction in a function of the HR pathway.

13. The composition of 6, wherein the target cell comprises a nucleic acid or an amino acid encoding a variant X-ray repair cross-complementing 2 (XRCC2) or a variant homolog of XRCC2 and wherein the variant XRCC2 induces a loss or reduction in a function of the HR pathway.

14. The composition of 6, wherein the target cell comprises a nucleic acid or an amino acid encoding a variant DNA repair endonuclease XPF or a variant homolog of XPF and wherein the variant XPF induces a loss or reduction in a function of the HR pathway.

15. The composition of 6, wherein the target cell comprises a nucleic acid or an amino acid encoding a variant Meiotic recombination 11 homolog 1 (MRE11A) or a variant homolog of MRE11A and wherein the variant MRE11A induces a loss or reduction in a function of the HR pathway.

16. The composition of 6, wherein the target cell comprises a nucleic acid or an amino acid encoding a variant Ataxia telangiectasia mutated (ATM) or a variant homolog of ATM and wherein the variant ATM induces a loss or reduction in a function of the HR pathway.

17. The composition of 6, wherein the target cell comprises a nucleic acid or an amino acid encoding a variant BRCA1-associated RING domain protein 1 (BARD1) or a variant homolog of BARD1 and wherein the variant BARD1 induces a loss or reduction in a function of the HR pathway.

18. The composition of 6, wherein the target cell comprises a nucleic acid or an amino acid encoding a variant BRCA1-interacting protein C-terminal helicase 1 (BRIP1) or a variant homolog of BRIP1 and wherein the variant BRIP1 induces a loss or reduction in a function of the HR pathway.

19. The composition of 6, wherein the target cell comprises a nucleic acid or an amino acid encoding a variant Cell cycle checkpoint kinase (CHEK1) or a variant homolog of CHEK1 and wherein the variant CHEK1 induces a loss or reduction in a function of the HR pathway.

20. The composition of 6, wherein the target cell comprises a nucleic acid or an amino acid encoding a variant CHK1 checkpoint homolog (CHEK2) or a variant homolog of CHEK2 and wherein the variant CHEK2 induces a loss or reduction in a function of the HR pathway.

21. The composition of 6, wherein the target cell comprises a nucleic acid or an amino acid encoding a variant Nibrin (NBN) or a variant homolog of NBN and wherein the variant NBN induces a loss or reduction in a function of the HR pathway.

22. The composition of 6, wherein the target cell comprises a nucleic acid or an amino acid encoding a variant Partner and localizer of BRCA2 (PALB2) or a variant homolog of PALB2 and wherein the variant PALB2 induces a loss or reduction in a function of the HR pathway.

23. The composition of 6, wherein the target cell comprises a nucleic acid or an amino acid encoding a variant Structure-specific endonuclease subunit SLX4 (SLX4) or a variant homolog of SLX4 and wherein the variant SLX4 induces a loss or reduction in a function of the HR pathway.

24. The composition of any one of 7-23, wherein the variant protein or the sequence encoding the variant protein comprises one or more of a mutation, a deletion, a promotor methylation, a silencing event and a splicing event.

25. The composition of 24, wherein the mutation comprises one or more of a substitution, an insertion, a deletion, an inversion, and a translocation of a nucleic acid sequence or an amino acid sequence encoding the variant BRCA protein, optionally wherein the mutation is BRCA1 or BRCA2 gene deletion, or wherein the variant BRCA protein is a variant BRCA1 protein or a variant BRCA2 protein.

26. The composition of 24 or 25, wherein the mutation introduces a stop codon into a nucleic acid sequence encoding the variant protein, thereby generating one or more of a truncated protein, an inactivated protein and a protein fragment.

27. The composition of any one of 3-28, wherein the variant protein or the sequence encoding the variant protein comprises a promoter sequence, and wherein the promoter controls expression of the variant protein or the sequence encoding the variant protein.

28. The composition of 27, wherein the silencing event comprises a silencing of the promoter sequence, function, or activity.

29. The composition of 27 or 28, wherein the promoter sequence controlling expression of the variant protein or the sequence encoding the variant protein comprises a mutation.

30. The composition of 29, wherein the mutation comprises one or more of a substitution, an insertion, a deletion, an inversion, and a translocation of the promoter sequence.

31. The composition of any one of 1-30, wherein the function of FANCM comprises one or more of ATP-binding, nucleotide-binding, DNA-binding, DNA remodeling, DNA strand separation, DNA-RNA strand separation and catalyzing the break of a chemical bond using water.

32. The composition of any one of 1-30, wherein the function of FANCM comprises a helicase activity.

33. The composition of any one of 1-30, wherein the function of FANCM comprises a hydrolase activity.

34. The composition of any one of 1-30, wherein the function of FANCM comprises a translocase activity.

35. The composition of any one of 1-34, wherein the function of FANCM comprises an ATPase activity.

36. The composition of any one of 6-35, wherein the variant protein increases a function of FANCM.

37. The composition of any one of 6-35, wherein the variant protein decreases a function of FANCM.

38. The composition of any one of 1-30, wherein the impairment, defect or deregulation of the HR pathway increases a dependence of the target cell upon a function of FANCM.

39. The composition of any one of 6-38, wherein the function of the HR pathway comprises one or more of

i) recognizing nucleotide or DNA damage;

ii) recruiting a protein to a site of nucleotide or DNA damage;

iii) configuring or remodeling a sequence comprising a site of nucleotide or DNA damage;

iv) configuring or remodeling a sequence complementary to a site of nucleotide or DNA damage;

v) inducing a break in a sequence within a site of nucleotide or DNA damage;

vi) inducing a break in a sequence comprising the site of nucleotide or DNA damage;

vii) inducing a break in a sequence complementary to a site of nucleotide or DNA damage;

viii) removing a sequence within a site of nucleotide or DNA damage;

ix) removing a sequence comprising a site of nucleotide or DNA damage;

x) synthesizing a new sequence within a site of nucleotide or DNA damage;

xi) synthesizing a new sequence comprising a site of nucleotide or DNA damage;

xii) resecting a portion of a synthesized sequence within a site of nucleotide or DNA damage;

xiii) resecting a portion of a synthesized sequence comprising the site of nucleotide or DNA damage;

xiv) stabilizing a site of DNA synthesis or replication within a site of nucleotide or DNA damage;

xv) stabilizing a site of DNA synthesis or replication comprising a site of nucleotide or DNA damage;

xvi) stabilizing a site of DNA synthesis or replication comprising a target site;

xvii) stabilizing a site of DNA synthesis or replication comprising a stalled replication fork;

xviii) inducing or facilitating invasion of a synthesized sequence within the site of nucleotide or DNA damage;

xix) inducing or facilitating invasion of a synthesized sequence comprising the site of nucleotide or DNA damage;

xx) inducing or facilitating insertion of a synthesized sequence within the site of nucleotide or DNA damage by recombination; and

xxi) inducing or facilitating insertion of a synthesized sequence comprising the site of nucleotide or DNA damage by recombination.

40. The composition of any one of 6-39, wherein an activity of the HR pathway comprises an increase or a decrease in a function of a component of the HR pathway.

41. The composition of any one of 6-40, wherein the variant protein increases a function of a component of the HR pathway.

42. The composition of any one of 6-40, wherein the variant protein decreases a function of a component of the HR pathway.

43. The composition of any one of 1-42, further comprising a pharmaceutically-acceptable carrier.

44. The composition of any one of 1-43, wherein the blocking agent comprises an effector moiety that binds to a FANCM protein or a nucleic acid sequence encoding the FANCM protein.

45. The composition of 44, wherein the effector moiety comprises one or more of an ion, a small molecule, a single-stranded nucleic acid molecule, a double-stranded nucleic acid molecule, an aptamer, an RNA-guided nuclease, a DNA-guided nuclease, a polypeptide, an antibody, a functional fragment of an antibody, an antibody mimetic, a scaffold, a matrix, or any combination thereof.

46. The composition of any one of 1-45, wherein the blocking agent further comprises a targeting moiety operably-linked to the effector moiety.

47. The composition of 46, wherein the targeting moiety is reversibly-linked to the effector moiety.

48. The composition of 46 or 47, wherein the targeting moiety specifically binds a component of the target cell.

49. The composition of any one of 1-48, wherein the target cell is a proliferating cell.

50. The composition of any one of 1-49, wherein the target cell is a tumor cell.

51. The composition of any one of 1-50, wherein the target cell is a malignant cell.

52. The composition of any one of 1-51, wherein the target cell is a metastatic cell.

53. The composition of any one of 1-52, wherein the target cell is produced or derived from a non-hematological tissue.

54. The composition of any one of 1-53, wherein the target cell is produced or derived from an epithelial tissue.

55. The composition of any one of 1-54, wherein the target cell is produced or derived from an organ or a structure comprising an epithelial tissue.

56. The composition of any one of 1-55, wherein the target cell is produced or derived from a skin area, a skin layer, a lung, a lymph node, a breast, an ovary, a prostate, a mouth, a nose, a nasal passage, an esophagus, an intestine, a small intestine, a large intestine, a stomach, a kidney, a liver, a spleen, a heart, an artery, a vein, a bladder and a colon.

57. The composition of any one of 1-52, wherein the target cell is produced or derived from a bone or a connective tissue.

58. The composition of any one of 1-57, wherein the blocking agent further comprises a regulation moiety.

59. The composition of 58, wherein the regulation moiety is operably-linked to one or more of the effector moiety and the targeting moiety.

60. The composition of 58 or 59, wherein the regulation moiety is reversibly-linked to one or more of the effector moiety and the targeting moiety.

61. The composition of any one of 58-60, wherein the regulation moiety selectively binds a component not present in a target cell.

62. The composition of 61, wherein the component not present in a target cell is present in a healthy cell.

63. The composition of 61 or 62, wherein the component decreases or inhibits an activity of the effector moiety.

64. The composition of any one of 58-63, wherein the regulation moiety comprises a microRNA (miRNA) binding site and selectively binds a miRNA.

65. The composition of any one of 1-64, wherein the target cell is an ALT+ cell.

66. The composition of any one of 1-64, wherein the target cell is not an ALT+ cell.

67. A method of inducing cell death in a proliferating cell, comprising contacting the proliferating cell with the composition of any one of 1-66.

68. The method of 67, wherein the cell is in vitro or ex vivo.

69. The method of 67, wherein the cell is in vivo.

70. A method of inducing cell cycle arrest in a proliferating cell, comprising contacting the proliferating cell with the composition of any one of 1-66.

71. The method of 70, wherein the proliferating cell is in vitro or ex vivo.

72. The method of 71, wherein the proliferating cell is in vivo.

73. The method of any one of 67-72, wherein the proliferating cell comprises the impaired, defective or deregulated DNA repair pathway.

74. The method of 73, wherein the proliferating cell comprises the impaired, defective or deregulated homologous recombination (HR) repair pathway.

75. The method of 74, wherein the proliferating cell comprises a variant BRCA protein or a sequence encoding a variant BRCA protein, and wherein the variant BRCA protein induces a loss or reduction in a function of the HR pathway.

76. The method of 75, wherein the variant BRCA protein comprises a variant BRCA1 protein or a variant BRCA2 protein, or wherein the sequence encoding the variant BRCA protein comprises a sequence encoding a variant BRCA1 protein or a variant BRCA2 protein.

77. The method of any one of 70-76, wherein the proliferating cell is an ALT+ cell.

78. The method of any one of 70-76, wherein the proliferating cell is not an ALT+ cell.

79. The method of any one of 70-78, wherein the proliferating cell is resistant to a PARP inhibitor.

80. The method of any one of 70-79, wherein the proliferating cell is an ovarian tumor cell or a breast tumor cell, optionally a BRCA1−/− tumor cell or a tumor cell comprising the variant BRCA1 protein, or a sequence encoding the variant BRCA protein.

81. A method of treating cancer, comprising administrating to a subject an effective amount of the composition of any one of 1-66.

82. The method of 81, wherein the method further comprises administering a second therapy.

83. The method of 82, wherein the second therapy comprises radiation and/or a chemotherapy.

84. The method of 83, wherein the chemotherapy comprises a Poly (ADP-ribose) polymerase (PARP) inhibitor or a platinum-based therapy.

85. The method of any one of 81-84, wherein the cancer is resistant to treatment with a PARP inhibitor as a monotherapy.

86. The method of any one of 81-85, wherein, prior to administration of the composition, the subject has been identified as resistant to treatment with a PARP inhibitor as a monotherapy.

87. The method of any one of 81-86, wherein, prior to administration of the composition, the subject has been treated with a PARP inhibitor as a monotherapy.

88. The method of any one of 81-87, wherein the administration is systemic.

89. The method of 88, wherein the composition is administered by one or more of an oral route, an inhaled route, an intravenous route, an intraperitoneal route, and a subcutaneous route.

90. The method of any one of 81-87, wherein the administration is local.

91. The method of 90, wherein the composition is administered by one or more of an intraocular route, an intraspinal route, an intracerebellar route, an intrathecal route, an intramuscular route and an intraosseous route.

92. The method of any one of 81-91, wherein the composition is administered once per day, twice per day or three times per day.

93. The method of any one of 81-92, wherein the composition is administered once per week, twice per week or three times per week.

94. The method of any one of 81-93, wherein the composition is administered once per month, twice per month or three times per month.

95. The method of any one of 81-94, wherein treating comprises a reduction in a severity of a sign or symptom of the cancer.

96. The method of any one of 81-95, wherein treating comprises a reduction in a volume of a tumor.

97. The method of any one of 81-96, wherein treating comprises a reduction in a number of tumor cells per volume of blood or mass of tissue.

98. The method of any one of 81-97, wherein treating comprises a remission.

99. The method of any one of 81-98, wherein treating comprises an increased duration of progression free survival.

100. The method of any one of 81-99, wherein the cancer comprises cancer cells comprising an impaired, defective or deregulated DNA repair pathway.

101. The method of 100, wherein the cancer cells comprise an impaired, defective or deregulated homologous recombination (HR) repair pathway.

102. The method of 101, wherein the cancer cells comprise a variant BRCA protein or a sequence encoding a variant BRCA protein, and wherein the variant BRCA protein induces a loss or reduction in a function of the HR pathway.

103. The method of 102, wherein the variant BRCA protein comprises a variant BRCA1 protein or a variant BRCA2 protein, or wherein the sequence encoding the variant BRCA protein comprises a sequence encoding a variant BRCA1 protein or a variant BRCA2 protein.

104. The method of any one of 81-103, wherein the cancer cells are ALT+ cells.

105. The method of any one of 81-103, wherein the cancer cells are not an ALT+ cells.

106. The method of any one of 81-105, wherein the cancer cells are resistant to a PARP inhibitor.

107. The method of any one of 81-106, wherein the cancer is an ovarian cancer or a breast cancer, optionally a BRCA1−/− cancer or a cancer comprising tumor cells comprising a variant BRCA1 protein, or a sequence encoding the variant BRCA protein.

108. The method of 81, wherein the method comprises administering to a subject having a BRCA1−/− ovarian cancer or BRCA1−/− breast cancer a Fanconi Anemia Group M protein (FANCM) blocking agent.

109. The method of 108, wherein the method results in DNA damage and/or cell cycle arrest of BRCA1−/− ovarian cancer cells or BRCA1−/− breast cancer cells.

110. The method of 108 or 109, wherein the method decreases survival or growth of BRCA1−/− ovarian cancer cells or BRCA1−/− breast cancer cells.

While the present invention has been described in conjunction with the specific embodiments set forth above, many alternatives, modifications and other variations thereof will be apparent to those of ordinary skill in the art. All such alternatives, modifications and variations are intended to fall within the spirit and scope of the present invention. Accordingly, the invention is not limited except as by the appended claims.

Furthermore, it is intended that any method described in the disclosure may be rewritten into Swiss-type format for the use of any agent described in the disclosure, for the manufacture of a medicament, in treating any of the disorders described in the disclosure. Likewise, it is intended for any method described in the disclosure to be rewritten as a compound for use claim, or as a use of a compound claim.

All U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications and non-patent publications referred to in this specification are incorporated herein by reference, in their entirety.

EXAMPLES

The disclosure is further illustrated by the following examples, which are not to be construed as limiting this disclosure in scope or spirit to the specific procedures described in the disclosure. It is to be understood that the examples are provided to illustrate certain embodiments and that no limitation to the scope of the disclosure is intended thereby. It is to be further understood that resort may be had to various other embodiments, modifications, and equivalents thereof which may suggest themselves to those skilled in the art without departing from the spirit of the present disclosure.

Example 1
Crispr/CAS9-Mediated Knockout of FANCM in BRCA1−/− Cancer Cell Lines Leads to an Increase in DNA Damage and G2 Arrest and a Reduction of Growth in 2D Clonogenic Assay

To determine the molecular consequences of FANCM loss on a BRCA1-null background, we performed CRISPR/Cas9 knockout experiments in two BRCA1−/− cancer cell lines: MDAMD436 (breast) and UWB1289 (ovarian) as well as a BRCA1-proficient ovarian cancer control: TOV21G. We designed three different sgRNAs to target the FANCM locus: gRNA3, gRNA4, gRNA Avana1, and harvested cells on Day 7 post CRISPR RNP nucleofection to measure DNA damage by flow cytometry for DNA double strand break marker pH2AXS139 (γH2AX). We observed a 13-17% increase in γH2AX-positive cells in both BRCA1−/− deficient cell lines MDAMB436 and UWB1289 in the conditions where FANCM was knocked out relative to the baseline levels with the NT control gRNA (FIG. 3A). In contrast, no significant changes in γH2AX-positive cells were observed when FANCM was knocked out in the BRCA1-proficient cell line TOV21G (FIG. 3A). We noted that the increase in DNA damage upon FANCM loss led to accumulation of cells in G2 phase of the cell cycle (FIG. 3B). As shown in FIG. 3B, while the percentage of TOV21G cells in G2 remained unchanged across experimental conditions (around 30%), there was a marked increase in the G2 population of UWB1289 cells in the FANCM knockout conditions (46-48%) relative to the NT control (around 30%). This increase of 16-18% in the G2 population tracks well with the 13-17% increase in γH2AX-positive cells, registered for the FANCM knockout conditions in the BRCA1−/− cell lines MDAMB436 and UWB1289 on FIG. 3A. Thus, loss of FANCM in BRCA1−/− breast and ovarian cancer cell lines leads to an increase in DNA damage and G2 arrest, while BRCA1-proficient cells remain unaffected.

We also examined cell growth via a 2D colony formation assay on between 14-21 days post nucleofection with the CRISPR RNP targeting FANCM. As shown in FIG. 4A, CRISPR/Cas9-mediated knockout of FANCM in BRCA1−/− cancer cell lines, UWB1.289, COV362, and MDAMB436, led to a reduction of growth, which was not observed in the BRCA1 wild-type TOV21G cells. FANCM protein levels were determined by Western blot, and FIG. 4B shows loss of FANCM protein expression when cells were treated with the FANCM sgRNA #3 and #4.

We further examined cell growth via the 2D colony formation assay on between days 14-21 following doxycycline induced shRNA expression of shRNA targeting FANCM for knockdown. As shown in FIG. 5A, shRNA knockdown of FANCM in BRCA1−/− cancer cell lines, UWB1.289 and MDAMB436, led to a reduction of growth. qPCR was performed to measure FANCM mRNA expression, and FIG. 5B shows loss of FANCM mRNA transcripts in UWB1.289 treated with shRNA #1307 and #1858 following three days of doxycline treatment confirming the on-target activity of the shRNAs and specific FANCM mRNA knockdown.

Short term and long term viability assays were conducted in BRCA1−/− cancer cell line UWB1.289 subjected to CRISPR/Cas9-mediated knockout of FANCM. FIGS. 6A and 6B demonstrate rescue of FANCM dependency when wild-type BRCA1 is present in UWB1.289 cells. FANCM protein levels were also determined via western blot, and FIG. 6C shows that nuclear enriched FANCM is undetectable in both UW1.289 null and BRCA1 add-back lines. Together, these data demonstrate a dependency on FANCM activity in BRCA1−/− cancer cell lines of different lineages and indicate that FANCM activity in this context is not redundant with other helicases/translocases but required for resolution of stalled replication forks or DNA damage in the absence of a functional BRCA1 protein.

Materials and Methods
Flow Cytometry:

For pH2AXS139 (γH2AX) staining, cells (up to 500K) were harvested 7 days after nucleofection with CRISPR/Cas9 RNP targeting the FANCM locus and fixed in 4% PFA for 10 min at RT. Following a wash with PBS, cells were permeabilized with 2% FBS in PBST (PBS+0.1% Triton) for 30 min at RT. Staining was performed with a γH2AX-A647 antibody (CST #9720S) at 1:50 in 2% FBS in PBST for 1 h at RT in the dark. Cells were then washed 2× with PBST and resuspended in PBS for analysis on the Attune NxT Flow Cytometer (Thermo Fisher Scientific). Data was plotted and analyzed with FlowJo_v10.7.1.

Cell cycle analysis was performed on day 7 after CRISPR/Cas9-mediated knockout of FANCM. Cells (up to 500K) were collected and fixed in 70% ice-cold EtOH. After a wash with PBS, cells were resuspended in FxCycle PI/RNAse Staining Solution (Thermo Fisher #F10797) and incubated for 15 min at RT in the dark. At least 50K events per sample were acquired on the Attune NxT Flow Cytometer and analyzed with FlowJo_v10.7.1.

2D Colony Formation Assay:

Cells were washed with PBS and trypsinized with TrypLE until loss of adherence. Cells were collected in a 15 mL falcon tube and spun down into a pellet at 1,000 RPM×5 minutes. The supernatant was aspirated and the cells were resuspended by 5 mL of their appropriate media and counted using Luna cell counter. Cells were diluted to 1-2000 cells/well and plated in triplicate in 6-well plates. The length of the assays varied depending on the lines rate of growth, typically 17-21 days, and on the final day the plates were aspirated, washed and stained with PBS containing 0.5% crystal violet and 3.2% paraformaldehyde. Stained colonies were imaged on the Li-Cor Odyssey CLx (700 nm wavelength).

CRISPR RNP and shRNA:

sgRNA Sequences:

FANCM sgRNA #3:

(SEQ ID NO: 69)

CATGACCACGGCGGCAATAA

FANCM sgRNA #4:

(SEQ ID NO: 70)

AAAGACCTTTATTGCCGCCG

FANCM Avana #1:

(SEQ ID NO: 71)

CCTTTCCTGAAGGGAACCAG

HPRT sgRNA:

(SEQ ID NO: 72)

ATTATGCTGAGGATTTGGAA

PSMA4 sgRNA:

(SEQ ID NO: 73)

AGTCTCGAAGATATGACTCC

PCNA sgRNA:

(SEQ ID NO: 74)

CGAAGATAACGCGGATACCT

Non-targeting sgRNA:

(SEQ ID NO: 75)

GTACGTCGGTATAACTCCTC

sgRNA were provided by Synthego (CRISPRevolution sgRNA EZ Kit—modified) as 10 nmol and resuspended to a 120 uM working solution using nuclease free H20.

Truecut Cas9 V2 (Thermo Fisher Cat #A36498) was used for all CRISPR RNP reactions. 1×10{circumflex over ( )}6 cells per RNP reaction were used with 2 ul of Truecut Cas9 VS and 1 uL of sgRNA (120 uM stock). Cas9 and sgRNA's were pre-incubated for 15 minutes before being added to 20 uL solution of cells and then subjected to electroporation using the Lonza Nucelofector 4D system. Electroporated cells were subsequently replated into downstream assays.

shRNA Sequences:

All shRNAs were cloned into pLKO-tet-on vector, packaged into lentivirus and titered for each cell line for an MOI <0.7.

shNT:

(SEQ ID NO: 76)

CAACAAGATGAAGAGCACCAA

shRNA #1307:

(SEQ ID NO: 77)

GACTTCATGAAACTCTATAAT

shRNA #1858:

(SEQ ID NO: 78)

AGGACGAGAGGAACGTATTTA

Western Blots:

Cells were plated for all conditions at a necessary density for analysis (1×10{circumflex over ( )}7 cells per/reaction for nuclear isolation). Following treatment, cells were washed with PBS and trypsinized with TrypLE until loss of adherence. Cells were collected in a 15 mL falcon tube and spun down into a pellet at 1,000 RPM×5 minutes. The supernatant is aspirated and pellets were then placed into downstream protocol (NE-PER cell fractionation kit protocol as provided by vendor). Samples were run on 3-8% Bis-tris gels (Thermo fisher Cat #EA0375PK2) for 45 minutes @ 200 v. Samples were transferred to PVDF membranes using iBlot2 system (IB21001) and subsequently blocked with li-cor blocking buffer (Intercept® (TBS) Blocking Buffer cat #927-60001) for 1 hr at room temp. Membranes were stained overnight at 4 degrees with appropriate antibodies.

Materials:
Cell Lines:

UWB1.289 (ovarian; BRCA1 −/− 2475delC)

UWB1.289+BRCA1

MDA-MB-436 (breast; BRCA1 −/− 5277+1G>A)

COV362 (ovarian; BRCA1 mutant)

TOV21G (ovarian; BRCA1 wild-type)

Antibodies:

FANCM: Anti-FANCM Antibody, clone CV5.1 (MABC545) 1:1000 dilution

Lamin B: Recombinant Anti-Lamin B1 antibody [EPR8985(B)] (ab133741) 1:2000 dilution

COMPOSITIONS AND METHODS FOR THE TREATMENT OF CANCER

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