Methods for treating skin cancer with carboxypeptidase vitellogenesis-like (CPVL) inhibitors

Abstract

The present disclosure provides methods of treating a subject with skin cancer or preventing a subject from developing skin cancer, as well as methods of identifying subjects at increased risk of developing skin cancer.

Description

[Technical field]

[0001] Reference to sequence listing This application contains a Sequence Listing that has been submitted electronically as a text file entitled 18923807002SEQ, created on July 2, 2022, having a size of 440 kilobytes. The Sequence Listing is incorporated herein by reference.

[0002] The present disclosure relates generally to treatment of subjects with skin cancer with carboxypeptidase vitellogenesis-like (CPVL) inhibitors, and methods of identifying subjects at increased risk for developing skin cancer. [Background technology]

[0003] Skin cancer refers to any cancer that develops in the skin. These relatively common cancers are often mistaken by patients for non-malignant skin abnormalities, which can lead to delayed detection leading to difficulties in treating the disease and fatal outcomes. The most common type of skin cancer is basal cell carcinoma (BCC), which accounts for about 80% of all skin cancers. Other types of skin cancer are squamous cell carcinoma (SCC), which accounts for approximately 16% of all skin cancers, and melanoma, which accounts for about 4%. BCC and SCC are collectively referred to as non-melanoma skin cancer (NMSC). Melanoma arises from melanocytes in the epidermis and is a carcinoma that often leads to metastasis or death. In 2000, it was reported that 47,000 people were identified with new melanomas, of which 7,700 died (Non-Patent Document 1). UV-induced melanoma is caused by intermittent exposure to UV rays, e.g., intense sunburn, rather than chronic exposure (Non-Patent Document 2). Another rare form of aggressive skin cancer is Merkel cell carcinoma (MCC), which is similar to melanoma.

[0004] Carboxypeptidase vitellogenic-like (CPVL) is a carboxypeptidase that shares strong sequence similarity with serine carboxypeptidases. Carboxypeptidases are a large class of proteases that act to cleave single amino acids from the carboxy terminus of proteins or peptides. However, the exact function of this protein remains to be elucidated. CPVL may be involved in the digestion of phagocytosed particles in lysosomes, in participation in the inflammatory protease cascade, and in trimming peptides for antigen presentation. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Greenlee et al.,Cancer J.Clin.,2000,50,7-33 [Non-Patent Document 2] Gilchrest et al., New Engl. J. Med., 1999, 340, 1341-1348 Summary of the Invention

[0006] The present disclosure provides a method of treating a subject having skin cancer or preventing a subject from developing skin cancer, the method comprising administering a CPVL inhibitor to the subject.

[0007] The disclosure also provides a method of treating a subject having non-melanoma skin cancer or preventing a subject from developing non-melanoma skin cancer, the method comprising administering a CPVL inhibitor to the subject.

[0008] The present disclosure also provides a method of treating a subject having basal cell carcinoma or preventing a subject from developing basal cell carcinoma, the method comprising administering a CPVL inhibitor to the subject.

[0009] The disclosure also provides a method of treating a subject having squamous cell carcinoma or preventing a subject from developing squamous cell carcinoma, the method comprising administering CPVL to the subject.

[0010] The disclosure also provides a method of treating a subject having melanoma or preventing a subject from developing melanoma, the method comprising administering to the subject a CPVL inhibitor.

[0011] The present disclosure also provides a method of treating a subject having Merkel cell carcinoma or preventing a subject from developing Merkel cell carcinoma, the method comprising administering a CPVL inhibitor to the subject.

[0012] The disclosure also provides a method of treating a subject having dermatofibrosarcoma protuberans or preventing a subject from developing dermatofibrosarcoma protuberans, the method comprising administering a CPVL inhibitor to the subject.

[0013] The disclosure also provides a method of treating a subject having sebaceous gland carcinoma or preventing a subject from developing sebaceous gland carcinoma, the method comprising administering a CPVL inhibitor to the subject.

[0014] The disclosure also provides a method of treating a subject with a therapeutic agent that treats or inhibits skin cancer, the subject having skin cancer, the method comprising: obtaining or obtaining a biological sample from the subject; and performing or performing a sequence analysis on the biological sample to determine whether the subject has a genotype that includes a CPVL missense variant nucleic acid molecule that encodes a predicted loss-of-function polypeptide of CPVL, thereby determining whether the subject has a CPVL missense variant nucleic acid molecule that encodes a predicted loss-of-function polypeptide of CPVL; and: i) administering or continuing to administer a therapeutic agent that treats or inhibits skin cancer at a standard dosage to the subject, the CPVL reference, and / or administering to the subject a CPVL reference. ii) administering or continuing to administer to a subject who is heterozygous for a CPVL missense variant nucleic acid molecule a therapeutic agent that treats or inhibits skin cancer at or below a standard dosage, and / or administering a CPVL inhibitor to the subject; or iii) administering or continuing to administer to a subject who is homozygous for a CPVL missense variant nucleic acid molecule a therapeutic agent that treats or inhibits skin cancer at or below a standard dosage; wherein the presence of a genotype having a CPVL missense variant nucleic acid molecule encoding a predicted loss-of-function polypeptide of CPVL indicates a reduced risk of developing skin cancer.

[0015] The disclosure also provides a method of treating a subject with a therapeutic agent that prevents skin cancer, the method comprising: obtaining or obtaining a biological sample from the subject; and performing or performing a sequence analysis on the biological sample to determine whether the subject has a genotype that includes a CPVL missense variant nucleic acid molecule that encodes a predicted loss-of-function polypeptide of CPVL; and determining whether the subject has a CPVL missense variant nucleic acid molecule that encodes a predicted loss-of-function polypeptide of CPVL; and: i) administering or continuing to administer a therapeutic agent that prevents skin cancer at a standard dosage to the subject, the CPVL reference, and / or administering a CPVL inhibitor to the subject. ii) administering or continuing to administer to a subject who is heterozygous for the CPVL missense variant nucleic acid molecule a skin cancer preventive therapeutic agent at or below a standard dosage, and / or administering a CPVL inhibitor to the subject; or iii) administering or continuing to administer to a subject who is homozygous for the CPVL missense variant nucleic acid molecule a skin cancer preventive therapeutic agent at or below a standard dosage; wherein the presence of a genotype having a CPVL missense variant nucleic acid molecule encoding a predicted loss-of-function polypeptide of CPVL indicates a reduced risk of developing skin cancer.

[0016] The disclosure also provides a method of identifying a subject having an increased risk of developing skin cancer, the method comprising determining or having determined the presence or absence of a CPVL missense variant nucleic acid molecule encoding a predicted loss-of-function polypeptide of CPVL in a biological sample obtained from the subject; if the subject is a CPVL reference, then the subject has an increased risk of developing skin cancer; and if the subject is heterozygous or homozygous for a CPVL missense variant nucleic acid molecule encoding a predicted loss-of-function polypeptide of CPVL, then the subject has a decreased risk of developing skin cancer.

[0017] The disclosure also provides a therapeutic agent for treating or inhibiting or preventing skin cancer for use in treating or preventing skin cancer in a subject, where the subject a) is a reference for a CPVL genomic nucleic acid molecule, a CPVL mRNA molecule, or a CPVL cDNA molecule; or b) is heterozygous for i) a CPVL missense variant genomic nucleic acid molecule encoding a predicted loss-of-function polypeptide of CPVL; ii) a CPVL missense variant mRNA molecule encoding a predicted loss-of-function polypeptide of CPVL; or iii) a CPVL missense variant cDNA molecule encoding a predicted loss-of-function polypeptide of CPVL.

[0018] The disclosure also provides a CPVL inhibitor for use in treating or preventing skin cancer in a subject, wherein the subject is a) a reference for a CPVL genomic nucleic acid molecule, a CPVL mRNA molecule, or a CPVL cDNA molecule; or b) heterozygous for i) a CPVL missense variant genomic nucleic acid molecule encoding a predicted loss-of-function polypeptide of CPVL; ii) a CPVL missense variant mRNA molecule encoding a predicted loss-of-function polypeptide of CPVL; or iii) a CPVL missense variant cDNA molecule encoding a predicted loss-of-function polypeptide of CPVL.

[0019] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee. [Brief description of the drawings]

[0020] [Figure 1] CPVL expression (recovered molecules / cells) in melanoma tumors is shown. [Diagram 2] CPVL expression (recovered molecules / cell) in basal cell carcinoma is shown. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0021] Various terms relating to aspects of the present disclosure are used throughout the specification and claims. Such terms are to be given their ordinary meaning in the art unless otherwise indicated. Other specifically defined terms are to be interpreted in a manner consistent with the definitions provided herein.

[0022] Unless expressly stated otherwise, it is in no way intended that any method or aspect set forth herein be construed as requiring that its steps be performed in a particular order. Thus, unless a method claim specifically states in the claims or specification that the steps are to be limited to a particular order, no order is intended to be implied in any way. This also applies to any possible unstated criteria for interpretation, including questions of logic regarding the arrangement of steps or operational flow, the plain meaning derived from grammatical construction or punctuation, or the number or type of aspects described herein.

[0023] As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly indicates otherwise. As used herein, the term "about" means that the numerical value described is approximate and that small variations will not significantly affect the practice of the disclosed embodiments. When numerical values ​​are used, unless otherwise indicated by the context, the term "about" means the numerical value may vary by ±10% and remain within the scope of the disclosed embodiments.

[0024] As used herein, the term "comprising" may, in certain embodiments, optionally be replaced with "consisting of" or "consisting essentially of." As used herein, the term "isolated" in reference to a nucleic acid molecule or polypeptide means that the nucleic acid molecule or polypeptide is in a state other than its native environment, e.g., apart from blood and / or animal tissue. In some embodiments, an isolated nucleic acid molecule or polypeptide is substantially free of other nucleic acid molecules or other polypeptides, particularly other nucleic acid molecules or polypeptides of animal origin. In some embodiments, the nucleic acid molecule or polypeptide may be in a highly purified form, i.e., more than 95% pure or more than 99% pure. When used in this context, the term "isolated" does not exclude the presence of the same nucleic acid molecule or polypeptide in alternative physical forms, such as dimers or alternatively phosphorylated or derivatized forms.

[0025] As used herein, the terms "nucleic acid," "nucleic acid molecule," "nucleic acid sequence," "polynucleotide," or "oligonucleotide" can include a polymeric form of nucleotides of any length, can include DNA and / or RNA, and can be single-stranded, double-stranded, or multistranded. A strand of a nucleic acid also refers to its complement.

[0026] As used herein, the term "subject" includes any animal, including mammals. Mammals include, but are not limited to, farm animals (e.g., horses, cows, pigs, etc.), companion animals (e.g., dogs, cats, etc.), laboratory animals (e.g., mice, rats, rabbits, etc.), and non-human primates. In some embodiments, the subject is a human. In some embodiments, the human is a patient under the care of a physician.

[0027] It has been observed according to the present disclosure that CPVL missense variant nucleic acid molecules encoding predicted loss-of-function polypeptides of CPVL (whether these variations are homozygous or heterozygous in a particular subject) are associated with a reduced risk of developing skin cancer. It is believed that CPVL missense variant nucleic acid molecules encoding predicted loss-of-function polypeptides of CPVL are not associated with melanoma. Moreover, the identification according to the present disclosure of the association between additional variants and gene burden masks indicates that CPVL itself (not linkage disequilibrium with variants in another gene) is responsible for the protective effect in non-melanoma skin cancer and melanoma.

[0028] Thus, subjects who are heterozygous for a CPVL missense variant nucleic acid molecule encoding a CPVL reference or predicted loss-of-function polypeptide of CPVL can be treated with a CPVL inhibitor such that skin cancer is inhibited or prevented, its symptoms are reduced or prevented, and / or the onset of symptoms is inhibited or prevented. It is also contemplated that such subjects with skin cancer can be further treated with a therapeutic agent that treats or inhibits skin cancer.

[0029] For the purpose of this disclosure, any particular subject, for example, a human, can be classified as having one of three CPVL genotypes: i) CPVL reference; ii) heterozygous for the CPVL missense variant nucleic acid molecule encoding the predicted loss-of-function polypeptide of CPVL; or iii) homozygous for the CPVL missense variant nucleic acid molecule encoding the predicted loss-of-function polypeptide of CPVL.If the subject does not have a copy of the CPVL missense variant nucleic acid molecule encoding the predicted loss-of-function polypeptide of CPVL, the subject is CPVL reference.If the subject has a single copy of the CPVL missense variant nucleic acid molecule encoding the predicted loss-of-function polypeptide of CPVL, the subject is heterozygous for the CPVL missense variant nucleic acid molecule encoding the predicted loss-of-function polypeptide of CPVL. The CPVL missense variant nucleic acid molecule encoding the predicted loss-of-function polypeptide of CPVL is any nucleic acid molecule (e.g., genomic nucleic acid molecule, mRNA molecule, or cDNA molecule) encoding a variant CPVL polypeptide with partial loss-of-function, complete loss-of-function, predicted partial loss-of-function, or predicted complete loss-of-function.Subjects with CPVL polypeptides with partial loss-of-function (or predicted partial loss-of-function) are hypomorphic for CPVL.Subjects with two copies (same or different) of the CPVL missense variant nucleic acid molecule encoding the predicted loss-of-function polypeptide of CPVL are homozygous for the CPVL missense variant nucleic acid molecule encoding the predicted loss-of-function polypeptide of CPVL.

[0030] For subjects genotyped or determined to be CPVL reference, such subjects have an increased risk of developing skin cancer, e.g., non-melanoma skin cancer, basal cell carcinoma, squamous cell carcinoma, melanoma, Merkel cell carcinoma, dermatofibrosarcoma protuberans, and / or sebaceous carcinoma. For subjects genotyped or determined to be either CPVL reference or heterozygous for a CPVL missense variant nucleic acid molecule encoding a predicted loss-of-function polypeptide of CPVL, such subject or subjects may be treated with a CPVL inhibitor.

[0031] In any of the embodiments described herein, the subject in whom skin cancer is prevented by administering a CPVL inhibitor may be any at risk of developing skin cancer, including, but not limited to, subjects with familial or genetic risk, elderly subjects, subjects with European ancestry, and subjects with lighter skin pigmentation. Also, in some embodiments, any subject may be at risk of developing skin cancer. In some embodiments, administering a CPVL inhibitor may be performed to prevent the development of additional skin cancer(s) in a subject who already has one or more skin cancers.

[0032] In any of the embodiments described herein, the CPVL missense variant nucleic acid molecule encoding a predicted loss-of-function polypeptide of CPVL can be any nucleic acid molecule (e.g., a genomic nucleic acid molecule, an mRNA molecule, or a cDNA molecule, etc.) encoding a CPVL variant polypeptide with partial loss-of-function, complete loss-of-function, predicted partial loss-of-function, or predicted complete loss-of-function. In some embodiments, the CPVL missense variant nucleic acid molecule encoding a predicted loss-of-function polypeptide of CPVL is associated with a reduced in vitro response to a CPVL ligand compared to a reference CPVL. In some embodiments, the CPVL missense variant nucleic acid molecule encoding a predicted loss-of-function polypeptide of CPVL is a CPVL variant that causes or is predicted to cause premature cleavage of the CPVL polypeptide compared to a human reference genome sequence. In some embodiments, the CPVL missense variant nucleic acid molecule encoding a predicted loss-of-function polypeptide of CPVL is a variant predicted to be damaging by an in vitro prediction algorithm, such as Polyphen, SIFT, or a similar algorithm. In some embodiments, the CPVL missense variant nucleic acid molecule encoding a predicted loss-of-function polypeptide of CPVL is a variant that causes or is predicted to cause a nonsynonymous amino acid substitution in CPVL and has an allele frequency of less than 1 / 100 alleles in the population from which the subject is selected. In some embodiments, the CPVL missense variant nucleic acid molecule encoding a predicted loss-of-function polypeptide of CPVL is any rare missense variant (allele frequency <0.1%; or 1 in 1,000 alleles), or any splice site, stop gain, start loss, stop loss, frameshift, or in-frame indel, or other frameshift CPVL variant.

[0033] In any of the embodiments described herein, the predicted loss-of-function polypeptide of CPVL can be any CPVL polypeptide having a partial loss of function, a complete loss of function, a predicted partial loss of function, or a predicted complete loss of function.

[0034] In any of the embodiments described herein, a CPVL missense variant nucleic acid molecule encoding a predicted loss-of-function polypeptide of CPVL can include a variation at a location on chromosome 7 using the nucleotide sequence of the CPVL reference genome nucleic acid molecule (SEQ ID NO:1; ENSG00000106066.15 chr7:28,995,637 to 29,195,276 in the GRCh38 / hg38 human genome assembly) as the reference sequence.

[0035] There are a number of genetic variants in CPVL that cause subsequent changes in the CPVL polypeptide sequence, including but not limited to rs117744081 (Tyr168His), rs147771477 (Arg464Gln), and rs138216401 (Ser61Asn).

[0036] Any one or more (i.e., any combination) of CPVL missense variant nucleic acid molecules encoding predicted loss-of-function polypeptides of CPVL may be used in any of the methods described herein to determine whether a subject has an increased risk of developing skin cancer. Particular variant combinations may form a mask used for statistical analysis of a particular correlation between CPVL and increased risk of developing skin cancer.

[0037] In any of the embodiments described herein, the skin cancer is non-melanoma skin cancer, basal cell carcinoma, squamous cell carcinoma (including cutaneous squamous cell carcinoma), melanoma, Merkel cell carcinoma, dermatofibrosarcoma protuberans, and / or sebaceous gland carcinoma. In some embodiments, the skin cancer is non-melanoma skin cancer. In some embodiments, the skin cancer is basal cell carcinoma. In some embodiments, the skin cancer is squamous cell carcinoma. In some embodiments, the skin cancer is melanoma. In some embodiments, the skin cancer is Merkel cell carcinoma. In some embodiments, the skin cancer is dermatofibrosarcoma protuberans. In some embodiments, the skin cancer is sebaceous gland carcinoma.

[0038] Symptoms of basal cell carcinoma include, but are not limited to, raised, smooth, pearly bumps on the sun-exposed skin of an individual's head, neck, or shoulders. Small blood vessels can often be seen within the tumor. Clustering of the tumor and bleeding can occur. Individuals sometimes mistake basal cell carcinoma for a sore that will not heal. Basal cell carcinoma is the least deadly form of skin cancer and can often be completely eliminated with proper treatment.

[0039] Symptoms of squamous cell carcinoma include, but are not limited to, red, scaly, thickened spots on an individual's sun-exposed skin. Some forms of squamous cell carcinoma appear as hard nodules and as dome-shaped. Breaking and bleeding of the nodules may occur. If left untreated, squamous cell carcinoma can develop into large lumps. Squamous cell carcinoma is the second most common form of skin cancer.

[0040] Symptoms of melanoma include, but are not limited to, shadows or brown to black lesions. There are also some melanomas that appear pink, red or flesh-colored, and these are called amelanotic melanomas. Amelanotic melanomas are a more aggressive form of melanoma. Some of the warning signs of malignant melanoma may include changes in size, shape, color, and prominence of moles, development of new moles during the transition from puberty to adulthood, itching, ulcers, or bleeding. Melanoma is the most deadly form of skin cancer.

[0041] Symptoms of Merkel cell carcinoma include, but are not limited to, fast-growing, non-tender flesh-colored to red / purple bumps that are usually not painful or itchy. These bumps appear on areas of the head, neck, and arms that are highly exposed to the sun. Individuals often mistake Merkel cell carcinoma for a cyst or other types of cancer.

[0042] Symptoms of dermatofibrosarcoma protuberans include, but are not limited to, small, slightly raised red or purple spots on the skin 1 to 5 centimeters wide that may become raised nodules and, in some cases, may cause redness, opening or bleeding.

[0043] Symptoms of sebaceous carcinoma include, but are not limited to, a slow-growing, sometimes yellow, painless lump on the eyelid where the eyelid and eyelashes meet. The bump may bleed or ooze and may also have a thickened or yellow or reddish scab.

[0044] The present disclosure provides a method of treating a subject having skin cancer, the method comprising administering a CPVL inhibitor to the subject. The disclosure also provides a method of treating a subject having non-melanoma skin cancer, the method comprising administering a CPVL inhibitor to the subject.

[0045] The disclosure also provides a method of treating a subject having basal cell carcinoma, the method comprising administering a CPVL inhibitor to the subject. The disclosure also provides a method of treating a subject having squamous cell carcinoma, the method comprising administering a CPVL inhibitor to the subject.

[0046] The disclosure also provides a method of treating a subject having melanoma, the method comprising administering a CPVL inhibitor to the subject. The disclosure also provides a method of treating a subject having Merkel cell carcinoma, the method comprising administering a CPVL inhibitor to the subject.

[0047] The disclosure also provides a method of treating a subject having dermatofibrosarcoma protuberans, the method comprising administering a CPVL inhibitor to the subject. The disclosure also provides a method of treating a subject having sebaceous carcinoma, the method comprising administering a CPVL inhibitor to the subject.

[0048] The disclosure also provides a method of preventing a subject from developing skin cancer, the method comprising administering a CPVL inhibitor to the subject. The disclosure also provides a method of preventing a subject from developing non-melanoma skin cancer, the method comprising administering a CPVL inhibitor to the subject.

[0049] The disclosure also provides a method of preventing a subject from developing basal cell carcinoma, the method comprising administering a CPVL inhibitor to the subject. The disclosure also provides a method of preventing a subject from developing squamous cell carcinoma, the method comprising administering CPVL to the subject.

[0050] The disclosure also provides a method of preventing a subject from developing melanoma, the method comprising administering a CPVL inhibitor to the subject. The present disclosure also provides a method of preventing a subject from developing Merkel cell carcinoma, the method comprising administering a CPVL inhibitor to the subject.

[0051] The disclosure also provides a method of preventing a subject from developing dermatofibrosarcoma protuberans, the method comprising administering a CPVL inhibitor to the subject. The disclosure also provides a method of preventing a subject from developing sebaceous carcinoma, the method comprising administering a CPVL inhibitor to the subject.

[0052] In some embodiments, the CPVL inhibitor comprises an inhibitory nucleic acid molecule. Examples of inhibitory nucleic acid molecules include, but are not limited to, antisense nucleic acid molecules, small interfering RNA (siRNA), and short hairpin RNA (shRNA). Such inhibitory nucleic acid molecules can be designed to target any region of the CPVL nucleic acid molecule. In some embodiments, the antisense RNA, siRNA, or shRNA hybridizes to a sequence in the CPVL genomic nucleic acid molecule or mRNA molecule and reduces the expression of the CPVL polypeptide in cells in the subject. In some embodiments, the CPVL inhibitor comprises an antisense molecule that hybridizes to a sequence in the CPVL genomic nucleic acid molecule or mRNA molecule and reduces the expression of the CPVL polypeptide in cells in the subject. In some embodiments, the CPVL inhibitor comprises an siRNA that hybridizes to a sequence in the CPVL genomic nucleic acid molecule or mRNA molecule and reduces the expression of the CPVL polypeptide in cells in the subject. In some embodiments, the CPVL inhibitor comprises an shRNA that hybridizes to a sequence in the CPVL genomic nucleic acid molecule or mRNA molecule and reduces the expression of the CPVL polypeptide in cells in the subject.

[0053] The inhibitory nucleic acid molecule may comprise RNA, DNA, or both RNA and DNA. The inhibitory nucleic acid molecule may also be linked or fused to a heterologous nucleic acid sequence, such as in a vector, or to a heterologous label. For example, the inhibitory nucleic acid molecule may be present as an exogenous donor sequence in or containing a vector that contains the inhibitory nucleic acid molecule and a heterologous nucleic acid sequence. The inhibitory nucleic acid molecule may also be linked or fused to a heterologous label. The label may be directly detectable (e.g., a fluorophore, etc.) or indirectly detectable (e.g., a hapten, an enzyme, or a fluorophore quencher, etc.). Such labels may be detectable by spectroscopic, photochemical, biochemical, immunochemical, or chemical means. Such labels include, for example, radioactive labels, pigments, dyes, chromogens, spin labels, and fluorescent labels. The label may also be, for example, a chemiluminescent substance; a metal-containing substance; or an enzyme, in which case an enzyme-dependent secondary generation of a signal occurs. The term "label" may also refer to a "tag" or hapten that can selectively bind to a conjugated molecule such that the conjugated molecule is subsequently added with a substrate and used to generate a detectable signal. For example, biotin can be used as a tag with an avidin or streptavidin conjugate of horseradish peroxidase (HRP) to bind to the tag and examined using a colorimetric substrate (e.g., tetramethylbenzidine (TMB) or the like) or a fluorogenic substrate to detect the presence of HRP. Exemplary labels that can be used as tags to facilitate purification include, but are not limited to, myc, HA, FLAG or 3XFLAG, 6XHis or polyhistidine, glutathione-S-transferase (GST), maltose binding protein, epitope tags, or the Fc portion of an immunoglobulin. Numerous labels include, for example, particles, fluorophores, haptens, enzymes and their calorimetric, fluorescent and chemiluminescent substrates and other labels.

[0054] Inhibitory nucleic acid molecules can include, for example, nucleotides or non-natural or modified nucleotides, such as nucleotide analogs or nucleotide substitutes. Such nucleotides include nucleotides that contain modified bases, sugars, or phosphate groups, or incorporate non-natural sites in their structure. Examples of non-natural nucleotides include, but are not limited to, dideoxynucleotides, biotinylated, aminated, deaminated, alkylated, benzylated, and fluorophore-labeled nucleotides.

[0055] The inhibitory nucleic acid molecule may also contain one or more nucleotide analogs or substitutions. A nucleotide analog is a nucleotide that contains a modification to either the base, sugar, or phosphate moiety. Modifications to the base moiety include, but are not limited to, natural and synthetic modifications of A, C, G, and T / U, as well as different purine or pyrimidine bases, such as pseudouridine, uracil-5-yl, hypoxanthine-9-yl (I), and 2-aminoadenine-9-yl. Modified bases include 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyluracil and cytosine, 6-azouracil, cytosine and thymine, 5-uracil (sulfonyluracil), and 5-uracil (sulfonyluracil). These include, but are not limited to, 8-substituted adenines and guanines, 5-halo (e.g., 5-bromo), 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine, 7-methyladenine, 8-azaguanine, 8-azaadenine, 7-deazaguanine, 7-deazaadenine, 3-deazaguanine, and 3-deazaadenine.

[0056] Nucleotide analogs can also include modifications at the sugar moiety. Modifications to the sugar moiety include, but are not limited to, natural and synthetic modifications of the ribose and deoxyribose. Sugar modifications include, but are not limited to, the following modifications at the 2' position: OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S-, or N-alkynyl; or O-alkyl-O-alkyl, where alkyl, alkenyl, and alkynyl are substituted or unsubstituted C 1-10 Alkyl or C 2-10 Alkenyl, and C 2-10 Exemplary 2' sugar modifications also include -O[(CH2) n O] m CH3, -O(CH2) n OCH3, -O(CH2) n NH2, -O(CH2) n CH3, -O(CH2) n -ONH2 and -O(CH2) n ON [(CH2) n CH3)]2, where n and m are independently 1 to about 10. Other modifications at the 2' position include, but are not limited to, C 1-10 These include, but are not limited to, alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH3, OCN, Cl, Br, CN, CF3, OCF3, SOCH3, SO2CH3, ONO2, NO2, N3, NH2, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, RNA cleaving groups, reporter groups, intercalators, groups for improving the pharmacokinetic properties of oligonucleotides, or groups for improving the pharmacodynamic properties of oligonucleotides, and other substituents with similar properties. Similar modifications can also be made at other positions on the sugar, particularly the 3' position of the sugar on the 3' terminal nucleotide or in 2'-5' linked oligonucleotides, and the 5' position of the 5' terminal nucleotide. Modified sugars can also include those containing modifications at the bridging ring oxygen, such as CH2 and S. Nucleotide sugar analogs can also have sugar mimetics, such as a cyclobutyl moiety in place of the pentofuranosyl sugar.

[0057] Nucleotide analogs can also be modified at the phosphate site. Modified phosphate sites include, but are not limited to, those in which the bond between two nucleotides can be modified to contain phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkyl phosphotriesters, methyl phosphonates and other alkyl phosphonates (including 3'-alkylene phosphonates and chiral phosphonates), phosphinates, phosphoramidates (including 3'-amino phosphoramidates and aminoalkyl phosphoramidates), thionophosphoramidates, thionoalkyl phosphonates, thionoalkyl phosphotriesters, and boranophosphates. These phosphate or modified phosphate bonds between two nucleotides can be through 3'-5' or 2'-5' bonds, and the bonds can contain reverse polarity, such as 3'-5' to 5'-3' or 2'-5' to 5'-2'. Various salts, mixed salts, and free acid forms are also included. Nucleotide substitutes also include peptide nucleic acids (PNA).

[0058] In some embodiments, the antisense nucleic acid molecule is a gapmer, whereby the first 1-7 nucleotides at the 5' and 3' termini, respectively, have a 2'-methoxyethyl (2'-MOE) modification. In some embodiments, the first 5 nucleotides at the 5' and 3' termini, respectively, have a 2'-MOE modification. In some embodiments, the first 1-7 nucleotides at the 5' and 3' termini are RNA nucleotides. In some embodiments, the first 5 nucleotides at the 5' and 3' termini are RNA nucleotides. In some embodiments, each of the internucleotide backbone linkages is a phosphorothioate linkage.

[0059] In some embodiments, the siRNA molecule has a terminal modification. In some embodiments, the 5' end of the antisense strand is phosphorylated. In some embodiments, A 5'-phosphate analog that cannot be hydrolyzed is used, such as a 5'-(E)-vinyl-phosphonate.

[0060] In some embodiments, the siRNA molecule has a backbone modification. In some embodiments, modified phosphodiester groups linking consecutive ribose nucleosides have been shown to improve the stability and in vivo bioavailability of siRNA. The non-ester group (-OH, =O) of the phosphodiester bond can be replaced with sulfur, boron, or acetate to provide phosphorothioate, boranophosphate, and phosphonoacetate linkages. Replacing the phosphodiester group with a phosphotriester can also facilitate cellular uptake of siRNA and retention on serum components by eliminating their negative charge. In some embodiments, the siRNA molecule has a sugar modification. In some embodiments, the sugar is deprotonated (a reaction catalyzed by exo- and endonucleases), which allows the 2'-hydroxyl to act as a nucleophile and attack the adjacent phosphorus in the phosphodiester bond. Such alternatives include 2'-O-methyl, 2'-O-methoxyethyl, and 2'-fluoro modifications.

[0061] In some embodiments, the siRNA molecule has base modifications, in some embodiments, the bases can be replaced with modified bases, such as pseudouridine, 5'-methylcytidine, N6-methyladenosine, inosine, and N7-methylguanosine.

[0062] In some embodiments, siRNA molecules are conjugated to lipids.Lipids can be conjugated to the 5' or 3' end of siRNA to improve their in vivo bioavailability by allowing them to associate with serum lipoproteins.Representative lipids include, but are not limited to, cholesterol and vitamin E, and fatty acids such as palmitate and tocopherol.

[0063] In some embodiments, an exemplary siRNA has the following formula: Sense: mN*mN* / i2FN / mN / i2FN / mN / i2FN / mN / i2FN / mN / i2FN / mN / i2FN / mN / i2FN / mN / i2FN / *mN* / 32FN / Antisense: / 52FN / * / i2FN / *mN / i2FN / mN / i2FN / mN / i2FN / mN / i2FN / mN / i2FN / mN / i2FN / mN / i2FN / mN / i2FN / mN*N*N where "N" is a base; "2F" is a 2'-F modification; "m" is a 2'-O-methyl modification; "I" is an internal base; and "*" is a phosphorothioate backbone linkage.

[0064] The present disclosure also provides vectors comprising any one or more of the inhibitory nucleic acid molecules. In some embodiments, the vector comprises any one or more of the inhibitory nucleic acid molecules and a heterologous nucleic acid. The vector can be a viral or non-viral vector capable of transporting the nucleic acid molecule. In some embodiments, the vector is a plasmid or cosmid (e.g., a circular double-stranded DNA into which additional DNA segments can be ligated, etc.). In some embodiments, the vector is a viral vector into which additional DNA segments can be ligated into the viral genome. Expression vectors include, but are not limited to, plasmids, cosmids, retroviruses, adenoviruses, adeno-associated viruses (AAV), plant viruses such as cauliflower mosaic virus and tobacco mosaic virus, yeast artificial chromosomes (YACs), Epstein-Barr (EBV) derived episomes, and other expression vectors known in the art.

[0065] The present disclosure also provides compositions comprising any one or more of the inhibitory nucleic acid molecules. In some embodiments, the compositions are pharmaceutical compositions. In some embodiments, the compositions comprise a carrier and / or excipient. Examples of carriers include, but are not limited to, poly(lactic acid) (PLA) microspheres, poly(D,L-lactic-co-glycolic acid) (PLGA) microspheres, liposomes, micelles, reverse micelles, lipid cochleates, and lipid microtubules. The carrier may comprise a buffered salt solution, such as PBS, HBSS, and the like.

[0066] In some embodiments, the CPVL inhibitor comprises a nuclease agent that induces one or more nicks or double-strand breaks in the recognition sequence(s) or a DNA binding protein that binds to the recognition sequence in the CPVL genomic nucleic acid molecule. The recognition sequence can be located in the coding region of the CPVL gene or in a regulatory region that affects the expression of the gene. The recognition sequence of the DNA binding protein or nuclease agent can be located in an intron, exon, promoter, enhancer, regulatory region, or any non-protein coding region. The recognition sequence can include or be close to the start codon of the CPVL gene. For example, the recognition sequence can be located about 10, about 20, about 30, about 40, about 50, about 100, about 200, about 300, about 400, about 500, or about 1,000 nucleotides from the start codon. As another example, two or more nuclease agents can be used, each of which targets a nuclease recognition sequence that includes or is close to the start codon. As another example, two nuclease agents can be used, one targeting a nuclease recognition sequence that includes or is close to the start codon and one targeting a nuclease recognition sequence that includes or is close to the stop codon, and cleavage by the nuclease agents can result in the deletion of the coding region between the two nuclease recognition sequences. Any nuclease agent that induces a nick or double-stranded break within the desired recognition sequence can be used in the methods and compositions disclosed herein. Any DNA binding protein that binds to a desired recognition sequence can be used in the methods and compositions disclosed herein.

[0067] Suitable nuclease agents and DNA binding proteins for use herein include, but are not limited to, zinc finger proteins or zinc finger nuclease (ZFN) pairs, transcription activator-like effector (TALE) proteins or transcription activator-like effector nucleases (TALEN), or clustered regularly interspersed short palindromic repeats (CRISPR) / CRISPR-associated (Cas) systems. The length of the recognition sequence can vary, including, for example, recognition sequences that are about 30-36 bp for zinc finger proteins or ZFN pairs, about 15-18 bp for each ZFN, about 36 bp for TALE proteins or TALEN, and about 20 bp for CRISPR / Cas guide RNAs.

[0068] In some embodiments, the CRISPR / Cas system can be used to modify the CPVL genomic nucleic acid molecule in a cell. The methods and compositions disclosed herein can use the CRISPR-Cas system by utilizing a CRISPR complex (including a guide RNA (gRNA) complexed to a Cas protein) for site-specific cleavage of the CPVL nucleic acid molecule.

[0069] Cas proteins usually contain at least one RNA recognition or binding domain that can interact with gRNA. Cas proteins can also contain nuclease domains (such as DNase or RNase domains), DNA binding domains, helicase domains, protein-protein interaction domains, dimerization domains, and other domains. Suitable Cas proteins include, for example, wild-type Cas9 proteins and wild-type Cpf1 proteins (such as FnCpf1). Cas proteins can have sufficient cleavage activity to generate double-stranded breaks in CPVL genomic nucleic acid molecules, or can be nickases that generate single-stranded breaks in CPVL genomic nucleic acid molecules. Additional examples of Cas proteins include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas5e (CasD), Cas6, Cas6e, Cas6f, Cas7, Cas8a1, Cas8a2, Cas8b, Cas8c, Cas9 (Csn1 or Csx12), Cas10, Cas10d, CasF, CasG, CasH, Csy1, Csy2, Csy3, Cse1 (CasA), Cse2 (CasB), Cse3 (CasE), Cse4 (CasF), Cse5 (CasF), Cse6 (CasF), Cse7 (CasF), Cse8 (CasF), Cse9 (CasF), Cse10 (CasF), Cse11 (CasF), Cse12 (CasF), Cse13 (CasF), Cse14 (CasF), Cse15 (CasF), Cse16 (CasF), Cse17 (CasF), Cse18 (CasF), Cse19 (CasF), Cse20 (CasF), Cse21 (CasF), Cse22 (CasF), Cse23 (CasF), Cse24 (CasF), Cse25 (CasF), Cse26 (CasF), Cse27 (CasF), Cse28 (CasF), Cse29 ...9 (CasF), Cse21 (CasF), Cse22 (CasF), Cse23 (CasF), Cse24 (CasF), Cse25 (CasF), Cse26 (CasF), Cse27 (Ca Cas proteins include, but are not limited to, CasC 4, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, and Cu1966, as well as homologs or modified versions thereof. Cas proteins can also be operably linked to heterologous polypeptides as fusion proteins. For example, Cas proteins can be fused to a cleavage domain, an epigenetic modification domain, a transcription activation domain, or a transcription repressor domain. Cas proteins can be provided in any form. For example, the Cas protein may be provided in the form of a protein, such as a Cas protein complexed with a gRNA. Alternatively, the Cas protein may be provided in the form of a nucleic acid molecule, such as RNA or DNA, that encodes the Cas protein.

[0070] In some embodiments, targeted genetic modification of a CPVL genomic nucleic acid molecule can be generated by contacting a cell with a Cas protein and one or more gRNAs that hybridize to one or more gRNA recognition sequences in a target genomic locus in a CPVL genomic nucleic acid molecule. For example, the gRNA recognition sequence can be located within the region of SEQ ID NO: 1. The gRNA recognition sequence can include or be close to the start codon of the CPVL genomic nucleic acid molecule or the stop codon of the CPVL genomic nucleic acid molecule. For example, the gRNA recognition sequence can be located about 10, about 20, about 30, about 40, about 50, about 100, about 200, about 300, about 400, about 500, or about 1,000 nucleotides from the start codon or the stop codon.

[0071] The gRNA recognition sequence in the target genomic locus in the CPVL genomic nucleic acid molecule is located near a protospacer adjacent motif (PAM) sequence, which is a 2-6 base pair DNA sequence immediately following the DNA sequence targeted by the Cas9 nuclease. A canonical PAM is the sequence 5'-NGG-3', where "N" is any nucleobase followed by two guanine ("G") nucleobases. The gRNA can transport Cas9 anywhere in the genome for gene editing, but editing cannot occur at any site other than the site where Cas9 recognizes the PAM. 5'-NGA-3' can also be a non-canonical PAM that is highly efficient for human cells. Typically, the PAM is about 2-6 nucleotides downstream of the DNA sequence targeted by the gRNA. The PAM can be adjacent to the gRNA recognition sequence. In some embodiments, the gRNA recognition sequence can be adjacent to the PAM at the 3' end. In some embodiments, the gRNA recognition sequence can be adjacent to the PAM at the 5' end. For example, the cleavage site of the Cas protein can be about 1 to about 10, about 2 to about 5 base pairs, or 3 base pairs upstream or downstream of the PAM sequence. In some embodiments (such as when Cas9 from S. pyogenes or a closely related Cas9 is used), the PAM sequence of the non-complementary strand can be 5'-NGG-3', where N is any DNA nucleotide and is immediately 3' to the gRNA recognition sequence of the non-complementary strand of the target DNA. Thus, the PAM sequence of the complementary strand will be 5'-CCN-3', where N is any DNA nucleotide and is immediately 5' to the gRNA recognition sequence of the complementary strand of the target DNA.

[0072] gRNA is an RNA molecule that binds to Cas protein and targets Cas protein to a specific location in CPVL genomic nucleic acid molecule.Exemplary gRNA is an effective gRNA for inducing Cas enzyme to bind to or cleave CPVL genomic nucleic acid molecule, and the gRNA comprises a DNA targeting segment that hybridizes to a gRNA recognition sequence in CPVL genomic nucleic acid molecule.Exemplary gRNA comprises a DNA targeting segment that hybridizes to a gRNA recognition sequence present in CPVL genomic nucleic acid molecule that includes or is close to a start codon or a stop codon. For example, a gRNA can be selected to hybridize to a gRNA recognition sequence located about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 100, about 200, about 300, about 400, about 500, or about 1,000 nucleotides from the start codon, or about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 100, about 200, about 300, about 400, about 500, or about 1,000 nucleotides from the stop codon. Suitable gRNAs can include about 17 to about 25 nucleotides, about 17 to about 23 nucleotides, about 18 to about 22 nucleotides, or about 19 to about 21 nucleotides. In some embodiments, the gRNA can include 20 nucleotides.

[0073] Examples of suitable gRNA recognition sequences located within the human CPVL reference gene are shown in Table 1 as SEQ ID NOs: 57-76.

[0074] [Table 1]

[0075] The Cas protein and gRNA form a complex, and the Cas protein cuts the target CPVL genomic nucleic acid molecule. The Cas protein can cut the nucleic acid molecule at a site within or outside the nucleic acid sequence present in the target CPVL genomic nucleic acid molecule to which the DNA targeting segment of the gRNA binds. For example, the formation of a CRISPR complex (including the gRNA hybridized to the gRNA recognition sequence and complexed with the Cas protein) can result in the cleavage of one or both strands within or near (e.g., within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50 or more base pairs from the nucleic acid sequence) the nucleic acid sequence present in the CPVL genomic nucleic acid molecule to which the DNA targeting segment of the gRNA binds.

[0076] Such a method may result in a CPVL genomic nucleic acid molecule in which, for example, the region of SEQ ID NO:1 is destroyed, the start codon is destroyed, the stop codon is destroyed, or the coding sequence is destroyed or deleted. Optionally, the cell may be further contacted with one or more additional gRNAs that hybridize with additional gRNA recognition sequences in the target genomic locus in the CPVL genomic nucleic acid molecule. By contacting the cell with one or more additional gRNAs (such as a second gRNA that hybridizes with a second gRNA recognition sequence), the cleavage by Cas protein may generate two or more double-strand breaks or two or more single-strand breaks.

[0077] In some embodiments, the methods of treatment and / or prevention further comprise detecting the presence or absence of a CPVL missense variant nucleic acid molecule encoding a predicted loss-of-function polypeptide of CPVL in a biological sample from the subject. As used throughout this disclosure, a "CPVL missense variant nucleic acid molecule encoding a predicted loss-of-function polypeptide of CPVL" is any CPVL nucleic acid molecule (e.g., a genomic nucleic acid molecule, an mRNA molecule, or a cDNA molecule, etc.) that encodes a CPVL polypeptide having partial loss-of-function, complete loss-of-function, predicted partial loss-of-function, or predicted complete loss-of-function.

[0078] The present disclosure also provides a method of treating a subject with a therapeutic agent that treats or inhibits skin cancer, the subject having skin cancer. The present disclosure also provides a method of preventing a subject from developing skin cancer by administering a therapeutic agent that prevents skin cancer. In some embodiments, the method includes obtaining or obtaining a biological sample from the subject, and performing or performing a sequence analysis on the biological sample to determine whether the subject has a genotype that includes a CPVL missense variant nucleic acid molecule that encodes a predicted loss-of-function polypeptide of CPVL, thereby determining whether the subject has a CPVL missense variant nucleic acid molecule that encodes a predicted loss-of-function polypeptide of CPVL. In some embodiments, the method further includes administering or continuing administration of a therapeutic agent that treats, prevents, or inhibits skin cancer at a standard dosage to the CPVL reference subject, and / or administering a CPVL inhibitor to the subject. In some embodiments, the method further comprises administering or continuing to administer a therapeutic agent for treating, preventing, or inhibiting skin cancer to a subject who is heterozygous for a CPVL missense variant nucleic acid molecule at a standard dosage or less, and / or administering a CPVL inhibitor to the subject. In some embodiments, the method further comprises administering or continuing to administer a therapeutic agent for treating, preventing, or inhibiting skin cancer to a subject who is homozygous for a CPVL missense variant nucleic acid molecule at a standard dosage or less. The presence of a genotype with a CPVL missense variant nucleic acid molecule encoding a predicted loss-of-function polypeptide of CPVL indicates that the subject has a reduced risk of developing skin cancer. In some embodiments, the subject is a CPVL reference. In some embodiments, the subject is heterozygous for a CPVL missense variant nucleic acid molecule encoding a predicted loss-of-function polypeptide of CPVL.

[0079] For subjects who are genotyped or determined to be heterozygous for a CPVL reference or CPVL missense variant nucleic acid molecule encoding a predicted loss-of-function polypeptide of CPVL, such subjects may be administered a CPVL inhibitor as described herein.

[0080] Detecting the presence or absence of a CPVL missense variant nucleic acid molecule encoding a predicted loss-of-function polypeptide of CPVL in a biological sample from a subject and / or determining whether a subject has a CPVL missense variant nucleic acid molecule encoding a predicted loss-of-function polypeptide of CPVL may be performed by any of the methods described herein. In some embodiments, these methods may be performed in vitro. In some embodiments, these methods may be performed in situ. In some embodiments, these methods may be performed in vivo. In any of these embodiments, the nucleic acid molecule may be present in a cell obtained from the subject.

[0081] In some embodiments, if the subject is a CPVL reference, the subject is administered a standard dosage of a therapeutic agent that treats, prevents, or inhibits skin cancer. In some embodiments, if the subject is heterozygous for a CPVL missense variant nucleic acid molecule that encodes a predicted loss-of-function polypeptide of CPVL, the subject is administered a standard dosage or a lower dosage of a therapeutic agent that treats, prevents, or inhibits skin cancer.

[0082] In some embodiments, the treatment and / or prevention methods further include detecting the presence or absence of a predicted loss-of-function polypeptide of CPVL in a biological sample from the subject. In some embodiments, if the subject does not have a predicted loss-of-function polypeptide of CPVL, the subject is also administered a standard dosage of a therapeutic agent that treats, prevents, or inhibits skin cancer. In some embodiments, if the subject has a predicted loss-of-function polypeptide of CPVL, the subject is also administered a standard dosage or a lower dosage of a therapeutic agent that treats, prevents, or inhibits skin cancer.

[0083] The present disclosure also provides a method of treating a subject with a therapeutic agent that treats or inhibits skin cancer, the subject having skin cancer. In some embodiments, the method includes determining whether the subject has a predicted loss-of-function polypeptide of CPVL by obtaining or obtaining a biological sample from the subject and performing or performing an assay on the biological sample to determine whether the subject has a predicted loss-of-function polypeptide of CPVL. If the subject does not have a predicted loss-of-function polypeptide of CPVL, the therapeutic agent that treats or inhibits skin cancer is administered or continues to be administered to the subject at a standard dosage, and / or a CPVL inhibitor is administered to the subject. If the subject has a predicted loss-of-function polypeptide of CPVL, the therapeutic agent that treats or inhibits skin cancer is administered or continues to be administered to the subject at a standard dosage or less, and / or a CPVL inhibitor is administered to the subject. The presence of a predicted loss-of-function polypeptide of CPVL indicates that the subject has a reduced risk of developing skin cancer. In some embodiments, the subject has a predicted loss-of-function polypeptide of CPVL. In some embodiments, the subject does not have a predicted loss-of-function polypeptide of CPVL.

[0084] The present disclosure also provides a method of preventing a subject from developing skin cancer by administering a therapeutic agent that prevents skin cancer. In some embodiments, the method includes determining whether the subject has a predicted loss-of-function polypeptide of CPVL by obtaining or obtaining a biological sample from the subject and performing or performing an assay on the biological sample to determine whether the subject has a predicted loss-of-function polypeptide of CPVL. If the subject does not have a predicted loss-of-function polypeptide of CPVL, the therapeutic agent that prevents skin cancer is administered or continues to be administered to the subject at a standard dosage, and / or a CPVL inhibitor is administered to the subject. If the subject has a predicted loss-of-function polypeptide of CPVL, the therapeutic agent that prevents skin cancer is administered or continues to be administered to the subject at a standard dosage or less, and / or a CPVL inhibitor is administered to the subject. The presence of a predicted loss-of-function polypeptide of CPVL indicates that the subject has a reduced risk of developing skin cancer. In some embodiments, the subject has a predicted loss-of-function polypeptide of CPVL. In some embodiments, the subject does not have a predicted loss-of-function polypeptide of CPVL.

[0085] Detecting the presence or absence of a predicted loss-of-function polypeptide of CPVL in a biological sample from a subject and / or determining whether a subject has a predicted loss-of-function polypeptide of CPVL may be performed by any of the methods described herein. In some embodiments, these methods may be performed in vitro. In some embodiments, these methods may be performed in situ. In some embodiments, these methods may be performed in vivo. In any of these embodiments, the polypeptide may be present in a cell obtained from the subject.

[0086] In some embodiments, the CPVL inhibitor is a small molecule. In some embodiments, the CPVL inhibitor is a hydroxymethyl(N-methyliminodiacetic acid) boronate (hydroxymethyl(MIDA) boronate), azidomethyl(N-methyliminodiacetic acid) boronate (azidomethyl(MIDA) boronate), or an α-functionalized alkyl(MIDA) boronate compound (see Table 1 in Adachi et al., Chem. Commun., 2015, 51, 3608-3611). Additional inhibitors include, but are not limited to, the following compounds:

[0087] [ka]

[0088] In some embodiments, the CPVL inhibitor is an immuno-oncology agent or an immune checkpoint inhibitor. In some embodiments, the immune checkpoint inhibitor is an anti-PD-1 agent, an anti-PD-L1 agent, or an anti-CTLA-4 agent. In some embodiments, the immune checkpoint inhibitor is an anti-PD-1 agent, such as KEYTRUDA® (pembrolizumab), OPDIVO® (nivolumab), and LIBTAYO® (cemiplimab). In some embodiments, the immune checkpoint inhibitor is KEYTRUDA® (pembrolizumab). In some embodiments, the immune checkpoint inhibitor is pembrolizumab. In some embodiments, the immune checkpoint inhibitor is OPDIVO® (nivolumab). In some embodiments, the immune checkpoint inhibitor is nivolumab. In some embodiments, the immune checkpoint inhibitor is LIBTAYO® (cemiplimab). In some embodiments, the immune checkpoint inhibitor is cemiplimab. In some embodiments, the immune checkpoint inhibitor is an anti-PD-L1 agent, such as, for example, TECENTRIQ® (atezolizumab), BAVENCIO® (avelumab), and IMFINZI® (durvalumab). In some embodiments, the immune checkpoint inhibitor is TECENTRIQ® (atezolizumab). In some embodiments, the immune checkpoint inhibitor is atezolizumab. In some embodiments, the immune checkpoint inhibitor is BAVENCIO® (avelumab). In some embodiments, the immune checkpoint inhibitor is avelumab. In some embodiments, the immune checkpoint inhibitor is IMFINZI® (durvalumab). In some embodiments, the immune checkpoint inhibitor is durvalumab. In some embodiments, the immune checkpoint inhibitor is an anti-CTLA-4 agent, such as, for example, YERVOY® (ipilimumab) and tremelimumab.In some embodiments, the immune checkpoint inhibitor is YERVOY® (ipilimumab) or tremelimumab. In some embodiments, the immune checkpoint inhibitor is YERVOY® (ipilimumab). In some embodiments, the immune checkpoint inhibitor is ipilimumab. In some embodiments, the immune checkpoint inhibitor is tremelimumab. In some embodiments, the CPVL inhibitor is a combination of any of the CPVL inhibitors described herein and any of the immune checkpoint inhibitors described herein. In some embodiments, the CPVL inhibitor is a combination of any of the CPVL inhibitors described herein and an anti-PD-1 agent and an anti-CTLA-4 agent.

[0089] Examples of therapeutic agents that treat or inhibit basal cell carcinoma include, but are not limited to, imiquimod, fluorouracil, cemiplimab-rwlc, sonidegib, and vismodegib, or any combination thereof. In some embodiments, the therapeutic agent is imiquimod. In some embodiments, the therapeutic agent is fluorouracil. In some embodiments, the therapeutic agent is cemiplimab-rwlc. In some embodiments, the therapeutic agent is sonidegib. In some embodiments, the therapeutic agent is vismodegib.

[0090] Examples of therapeutic agents that treat or inhibit squamous cell carcinoma include, but are not limited to, cemiplimab-rwlc and pembrolizumab, or a combination thereof. In some embodiments, the therapeutic agent is cemiplimab-rwlc. In some embodiments, the therapeutic agent is pembrolizumab.

[0091] Examples of therapeutic agents that treat or inhibit melanoma include, but are not limited to, aldesleukin, cobimetinib, dabrafenib, dacarbazine, recombinant interferon alpha-2b, ipilimumab, nivolumab, nivolumab, peginterferon alpha-2b, pembrolizumab, talimogene laherparepvec, trametinib dimethylsulfoxide, and vemurafenib, or any combination thereof. In some embodiments, the therapeutic agent is aldesleukin. In some embodiments, the therapeutic agent is cobimetinib. In some embodiments, the therapeutic agent is dabrafenib. In some embodiments, the therapeutic agent is dacarbazine. In some embodiments, the therapeutic agent is recombinant interferon alpha-2b. In some embodiments, the therapeutic agent is ipilimumab. In some embodiments, the therapeutic agent is nivolumab. In some embodiments, the therapeutic agent is nivolumab. In some embodiments, the therapeutic agent is peginterferon alpha-2b. In some embodiments, the therapeutic agent is pembrolizumab. In some embodiments, the therapeutic agent is talimogene laherparepvec. In some embodiments, the therapeutic agent is trametinib dimethylsulfoxide. In some embodiments, the therapeutic agent is vemurafenib.

[0092] Examples of therapeutic agents for treating or inhibiting Merkel cell carcinoma include, but are not limited to, avelumab, pembrolizumab, etoposide (VP16), and carboplatin combination regimens, or any combination thereof. In some embodiments, the therapeutic agent is avelumab. In some embodiments, the therapeutic agent is pembrolizumab. In some embodiments, the therapeutic agent is etoposide (VP16). In some embodiments, the therapeutic agent is a carboplatin combination regimen.

[0093] Examples of therapeutic agents that treat or inhibit dermatofibrosarcoma protuberans include, but are not limited to, imatinib. In some embodiments, the dose of a therapeutic agent for treating, preventing, or inhibiting skin cancer may be reduced by about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, or about 90% (i.e., less than the standard dose) for a subject who is heterozygous for a CPVL missense variant nucleic acid molecule encoding a predicted loss-of-function polypeptide of CPVL compared to a subject who is a CPVL reference (which may receive a standard dose). In some embodiments, the dose of a therapeutic agent for treating, preventing, or inhibiting skin cancer may be reduced by about 10%, about 20%, about 30%, about 40%, or about 50%. Also, a subject who is heterozygous for a CPVL missense variant nucleic acid molecule encoding a predicted loss-of-function polypeptide of CPVL may be administered less frequently compared to a subject who is a CPVL reference.

[0094] In some embodiments, the dose of a therapeutic agent for treating, preventing, or inhibiting skin cancer may be reduced by about 10%, about 20%, about 30%, about 40%, about 50% in a subject who is homozygous for a CPVL missense variant nucleic acid molecule encoding a predicted loss-of-function polypeptide of CPVL compared to a subject who is heterozygous for a CPVL missense variant nucleic acid molecule encoding a predicted loss-of-function polypeptide of CPVL. In some embodiments, the dose of a therapeutic agent for treating, preventing, or inhibiting skin cancer may be reduced by about 10%, about 20%, about 30%, about 40%, or about 50%. Also, the dose of a therapeutic agent for treating, preventing, or inhibiting skin cancer in a subject who is homozygous for a CPVL missense variant nucleic acid molecule encoding a predicted loss-of-function polypeptide of CPVL may be administered less frequently compared to a subject who is heterozygous for a CPVL missense variant nucleic acid molecule encoding a predicted loss-of-function polypeptide of CPVL.

[0095] Administration of the therapeutic agent and / or CPVL inhibitor for treating, preventing or inhibiting skin cancer can be repeated, for example, after 1 day, 2 days, 3 days, 5 days, 1 week, 2 weeks, 3 weeks, 1 month, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 2 months or 3 months. Repeated administration can be the same dose or different dose. Administration can be repeated 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more times. For example, according to a given dosing regimen, a subject can receive therapy for a long period of time, for example, 6 months, 1 year or more.

[0096] Administration of the therapeutic agent and / or CPVL inhibitor for treating, preventing, or inhibiting skin cancer may be by any suitable route, including, but not limited to, parenteral, intravenous, oral, subcutaneous, intraarterial, intracranial, intrathecal, intraperitoneal, topical, intranasal, or intramuscular. Pharmaceutical compositions for administration are desirably sterile, substantially isotonic, and manufactured under GMP conditions. Pharmaceutical compositions may be provided in unit dosage form (i.e., dosage for a single administration). Pharmaceutical compositions may be formulated using one or more physiologically and pharma- ceutical acceptable carriers, diluents, excipients, or adjuvants. The formulation depends on the route of administration selected. The term "pharmaceutical acceptable" means that the carrier, diluent, excipient, or adjuvant is compatible with the other ingredients of the formulation and is not substantially deleterious to the recipient thereof.

[0097] The terms "treat", "treating", and "treatment" as well as "prevent", "preventing", and "prevention", as used herein, refer to causing a desired biological response, e.g., therapeutic and prophylactic effects, respectively. In some embodiments, the therapeutic effect includes one or more of: reduction / reduction of skin cancer following administration of an agent or a composition comprising an agent, reduction / reduction of the severity of skin cancer (e.g., reduction or inhibition of the onset of skin cancer), reduction / reduction of symptoms and skin cancer-related effects, delaying the onset of symptoms and skin cancer-related effects, reducing the severity of symptoms of skin cancer-related effects, reducing the number of symptoms and skin cancer-related effects, reducing the latency of symptoms and skin cancer-related effects, amelioration of symptoms and skin cancer-related effects, reducing secondary symptoms, reducing secondary infections, preventing relapse of skin cancer, reducing the number or frequency of relapse attacks, increasing the latency between symptomatic attacks, increasing the time to sustained progression, accelerating recovery, or increasing the effectiveness or reducing resistance to alternative therapeutic agents, and / or increasing the survival time of the affected host animal. A prophylactic effect can include complete or partial avoidance and / or inhibition or delay (e.g., complete or partial avoidance / inhibition or delay, etc.) of the onset / progression of skin cancer following administration of a therapeutic protocol, and increased survival time of the affected host animal. Treatment of skin cancer includes treatment of a subject already diagnosed as having any form of skin cancer at any clinical stage or manifestation, or delaying the onset or progression or worsening or deterioration of symptoms or signs of skin cancer, and / or preventing and / or reducing the severity of skin cancer.

[0098] The present disclosure also provides a method for identifying a subject with an increased risk of developing skin cancer. In some embodiments, the method includes determining or having determined the presence or absence of a CPVL missense variant nucleic acid molecule (e.g., a genomic nucleic acid molecule, an mRNA molecule, and / or a cDNA molecule) encoding a predicted loss-of-function polypeptide of CPVL in a biological sample obtained from the subject. If the subject lacks a CPVL missense variant nucleic acid molecule encoding a predicted loss-of-function polypeptide of CPVL (i.e., the subject is genotypically classified as a CPVL reference), then the subject has an increased risk of developing skin cancer. If the subject has a CPVL missense variant nucleic acid molecule encoding a predicted loss-of-function polypeptide of CPVL (i.e., the subject is heterozygous or homozygous for a CPVL missense variant nucleic acid molecule encoding a predicted loss-of-function polypeptide of CPVL), then the subject has a reduced risk of developing skin cancer.

[0099] Having a single copy of a CPVL missense variant nucleic acid molecule encoding a predicted loss-of-function polypeptide of CPVL protects a subject from developing skin cancer more than not having a copy of a CPVL missense variant nucleic acid molecule encoding a predicted loss-of-function polypeptide of CPVL. Without intending to be limited to any particular theory or mechanism of action, it is believed that a single copy of a CPVL missense variant nucleic acid molecule encoding a predicted loss-of-function polypeptide of CPVL (i.e., heterozygous for a CPVL missense variant nucleic acid molecule encoding a predicted loss-of-function polypeptide of CPVL) protects a subject from developing skin cancer, and it is believed that having two copies of a CPVL missense variant nucleic acid molecule encoding a predicted loss-of-function polypeptide of CPVL (i.e., homozygous for a CPVL missense variant nucleic acid molecule encoding a predicted loss-of-function polypeptide of CPVL) may be more protective against a subject developing skin cancer than a subject having a single copy. Thus, in some embodiments, a single copy of a CPVL missense variant nucleic acid molecule encoding a predicted loss-of-function polypeptide of CPVL may not be completely protective, but may instead partially or incompletely protect a subject from developing skin cancer. Without wishing to be bound by any particular theory, there may be additional factors or molecules involved in the development of skin cancer that are still present in a subject having a single copy of a CPVL missense variant nucleic acid molecule encoding a predicted loss-of-function polypeptide of CPVL, thus resulting in less than complete protection from developing skin cancer.

[0100] Determining whether a subject has a CPVL missense variant nucleic acid molecule encoding a predicted loss-of-function polypeptide of CPVL in a biological sample from the subject and / or determining whether a subject has a CPVL missense variant nucleic acid molecule encoding a predicted loss-of-function polypeptide of CPVL may be performed by any of the methods described herein. In some embodiments, these methods may be performed in vitro. In some embodiments, these methods may be performed in situ. In some embodiments, these methods may be performed in vivo. In any of these embodiments, the nucleic acid molecule may be present in a cell obtained from the subject.

[0101] In some embodiments, if a subject is identified as having an increased risk of developing skin cancer, the subject is administered a therapeutic agent for treating, preventing, or inhibiting skin cancer described herein and / or a CPVL inhibitor. For example, if a subject is a CPVL reference and therefore has an increased risk of developing skin cancer, the subject is administered a CPVL inhibitor. In some embodiments, such a subject is also administered a therapeutic agent for treating, preventing, or inhibiting skin cancer. In some embodiments, if a subject is heterozygous for a CPVL missense variant nucleic acid molecule encoding a predicted loss-of-function polypeptide of CPVL, the subject is administered a therapeutic agent for treating, preventing, or inhibiting skin cancer at a dosage equal to or less than the standard dosage, and / or is administered a CPVL inhibitor. In some embodiments, such a subject is also administered a therapeutic agent for treating, preventing, or inhibiting skin cancer. In some embodiments, if the subject is homozygous for the CPVL missense variant nucleic acid molecule encoding the predicted loss-of-function polypeptide of CPVL, the subject is administered the therapeutic agent for treating, preventing or inhibiting skin cancer at a dosage equal to or less than the standard dosage.In some embodiments, the subject is a CPVL reference.In some embodiments, the subject is heterozygous for the CPVL missense variant nucleic acid molecule encoding the predicted loss-of-function polypeptide of CPVL.In some embodiments, the subject is homozygous for the CPVL missense variant nucleic acid molecule encoding the predicted loss-of-function polypeptide of CPVL.

[0102] In some embodiments, any of the methods described herein may further comprise determining the collective burden of subjects with CPVL missense variant nucleic acid molecules encoding predicted loss-of-function polypeptides of CPVL and / or predicted loss-of-function variant polypeptides of CPVL associated with a reduced risk of developing skin cancer. The collective burden is the sum of all variants in the CPVL gene, which may be performed in an association analysis with skin cancer. In some embodiments, the subject is homozygous for one or more CPVL missense variant nucleic acid molecules encoding predicted loss-of-function polypeptides of CPVL associated with a reduced risk of developing skin cancer. In some embodiments, the subject is heterozygous for one or more CPVL missense variant nucleic acid molecules encoding predicted loss-of-function polypeptides of CPVL associated with a reduced risk of developing skin cancer. The results of the association analysis suggest that the CPVL missense variant nucleic acid molecules encoding predicted loss-of-function polypeptides of CPVL are associated with a reduced risk of developing skin cancer. If the subject has a lower collective burden, the subject is at higher risk of developing skin cancer, and the subject is administered or continues to be administered a standard dose of a therapeutic agent for treating, preventing, or inhibiting skin cancer.If the subject has a higher collective burden, the subject is at lower risk of developing skin cancer, and the subject is administered or continues to be administered a standard dose or a lower dose of a therapeutic agent for treating, preventing, or inhibiting skin cancer.The higher the collective burden, the lower the risk of developing skin cancer.CPVL variants that can be used in collective burden analysis include any one or more of the following, or any combination, in Table 2:

[0103] [Table 2-1]

[0104] [Table 2-2]

[0105]

Table 2-3

[0106]

Table 2-4

[0107]

Table 2-5

[0108]

Table 2-6

[0109] In some embodiments, the collective burden of subjects having any one or more CPVL missense variant nucleic acid molecules encoding predicted loss-of-function polypeptides of CPVL represents a weighted sum of any of a plurality of CPVL missense variant nucleic acid molecules encoding predicted loss-of-function polypeptides of CPVL. In some embodiments, the collective burden is calculated using at least about 2, at least about 3, at least about 4, at least about 5, at least about 10, at least about 20, at least about 30, at least about 40, at least about 50, at least about 60, at least about 70, at least about 80, at least about 100, at least about 120, at least about 150, at least about 200, at least about 250, at least about 300, at least about 400, at least about 500, at least about 1,000, at least about 10,000, at least about 100,000, or at least about or more than 1,000,000 genetic variants present in or around (up to 10 Mb) the CPVL gene, where the genetic burden is the number of alleles multiplied by the estimated association with skin cancer or a related outcome for each allele (e.g., a weighted polygenic burden score). This may include any genetic variants close to the CPVL gene (up to 10Mb around the gene) that show non-zero association with skin cancer-related traits in genetic association analysis, regardless of their genomic annotation.In some embodiments, if a subject has a collective load that exceeds a desired threshold score, the subject has a reduced risk of developing skin cancer.In some embodiments, if a subject has a collective load that is below a desired threshold score, the subject has an increased risk of developing skin cancer.

[0110] In some embodiments, the collective burden may be divided into quintiles, e.g., top quintile, middle quintile, and bottom quintile, with the top quintile of collective burden corresponding to the lowest risk group and the bottom quintile of collective burden corresponding to the highest risk group. In some embodiments, the subjects with the larger collective burden comprise the highest weighted collective burden, including but not limited to the top 10%, top 20%, top 30%, top 40%, or top 50% collective burden from the subject population. In some embodiments, the genetic variants comprise genetic variants with association with skin cancer in the top 10%, top 20%, top 30%, top 40%, or top 50% p-value range for association. In some embodiments, each of the identified genetic variants is about 10 -2 , about 10 -3 , about 10 -4 , about 10 -5 , about 10 -6 , about 10 -7 , about 10 -8 , about 10 -9 , about 10 -10 , about 10 -11 , about 10 -12 , about 10 -13 , about 10 -14 , about or 10 -15 In some embodiments, the identified genetic variants include those with an association with skin cancer having a p-value of 5×10 -8In some embodiments, the identified genetic variants include genetic variants having an association with skin cancer in high-risk subjects with an odds ratio (OR) of about 1.5 or more, about 1.75 or more, about 2.0 or more, or about 2.25 or more for the top 20% of the distribution; or about 1.5 or more, about 1.75 or more, about 2.0 or more, about 2.25 or more, about 2.5 or more, or about 2.75 or more compared to the remainder of the reference population. In some embodiments, the odds ratio (OR) may range from about 1.0 to about 1.5, about 1.5 to about 2.0, about 2.0 to about 2.5, about 2.5 to about 3.0, about 3.0 to about 3.5, about 3.5 to about 4.0, about 4.0 to about 4.5, about 4.5 to about 5.0, about 5.0 to about 5.5, about 5.5 to about 6.0, about 6.0 to about 6.5, about 6.5 to about 7.0, or more than 7.0. In some embodiments, high-risk subjects include subjects with a collective burden in the bottom decile, quintile, or tertile in a reference population. The collective burden threshold is determined based on the nature of the intended practical application and the risk difference that would be considered meaningful for that practical application.

[0111] In some embodiments, if a subject is identified as having an increased risk of developing skin cancer, the subject is further administered a therapeutic agent for treating, preventing, or inhibiting skin cancer described herein and / or a CPVL inhibitor. For example, if a subject is a CPVL reference and therefore has an increased risk of developing skin cancer, the subject is administered a CPVL inhibitor. In some embodiments, such a subject is also administered a therapeutic agent for treating, preventing, or inhibiting skin cancer. In some embodiments, if a subject is heterozygous for a CPVL missense variant nucleic acid molecule encoding a predicted loss-of-function polypeptide of CPVL, the subject is administered a therapeutic agent for treating, preventing, or inhibiting skin cancer at a standard dosage or less, and / or is administered a CPVL inhibitor. In some embodiments, the subject is a CPVL reference. In some embodiments, the subject is heterozygous for a CPVL missense variant nucleic acid molecule encoding a predicted loss-of-function polypeptide of CPVL. Furthermore, if a subject has a lower collective burden of CPVL missense variant nucleic acid molecules encoding predicted loss-of-function polypeptides of CPVL and therefore has an increased risk of developing skin cancer, the subject is administered a therapeutic agent that treats, prevents, or inhibits skin cancer. In some embodiments, if a subject has a lower collective burden of CPVL missense variant nucleic acid molecules encoding predicted loss-of-function polypeptides of CPVL, the subject is administered a therapeutic agent that treats, prevents, or inhibits skin cancer at a dosage that is the same as or greater than the standard dosage administered to a subject with a higher collective burden of CPVL missense variant nucleic acid molecules encoding predicted loss-of-function polypeptides of CPVL.

[0112] The present disclosure also provides a method for detecting the presence or absence of a CPVL missense variant nucleic acid molecule (i.e., a genomic nucleic acid molecule, an mRNA molecule, or a cDNA molecule generated from an mRNA molecule) encoding a predicted loss-of-function polypeptide of CPVL in a biological sample from a subject. It is understood that gene sequences in a population and the mRNA molecules encoded by such genes may vary due to polymorphisms, such as single nucleotide polymorphisms. The sequences provided herein for CPVL variant genomic nucleic acid molecules, CPVL variant mRNA molecules, and CPVL variant cDNA molecules are merely exemplary sequences. Other sequences for CPVL variant genomic nucleic acid molecules, variant mRNA molecules, and variant cDNA molecules are also possible.

[0113] The biological sample may be derived from any cell, tissue, or biological fluid from a subject. The biological sample may include any clinically relevant tissue, such as a bone marrow sample, a tumor biopsy, a fine needle aspirate, or a sample of bodily fluids, such as blood, gingival crevicular fluid, plasma, serum, lymph, ascites, cyst fluid, or urine. In some cases, the sample includes a buccal swab. The biological sample used in the methods disclosed herein may vary based on the assay format, the nature of the detection method, and the tissue, cell, or extract used as the sample. The biological sample may be processed differently depending on the assay being used. For example, when detecting any CPVL missense variant nucleic acid molecule that encodes a predicted loss-of-function polypeptide of CPVL, a pre-processing designed to isolate or enrich the biological sample for genomic DNA may be used. A variety of techniques may be used for this purpose. When detecting the level of any CPVL variant mRNA molecule, different techniques may be used to enrich the biological sample with mRNA molecules. A variety of methods may be used to detect the presence or level of mRNA molecules or the presence of a particular variant genomic DNA locus.

[0114] In some embodiments, detecting a CPVL missense variant nucleic acid molecule encoding a predicted loss-of-function polypeptide of CPVL in a subject comprises performing a sequence analysis on a biological sample obtained from the subject to determine whether a CPVL genomic nucleic acid molecule in the biological sample, and / or a CPVL mRNA molecule in the biological sample, and / or a CPVL cDNA molecule generated from the mRNA molecule in the biological sample contains one or more variations that cause or are predicted to cause a loss of function (partial or complete).

[0115] In some embodiments, a method for detecting the presence or absence of a CPVL missense variant nucleic acid molecule (e.g., a genomic nucleic acid molecule, an mRNA molecule, and / or a cDNA molecule generated from an mRNA molecule, etc.) encoding a predicted loss-of-function polypeptide of CPVL in a subject comprises performing an assay on a biological sample obtained from the subject, the assay determining whether a nucleic acid molecule in the biological sample comprises a particular nucleotide sequence.

[0116] In some embodiments, the biological sample comprises a cell or cell lysate. Such a method may further comprise, for example, obtaining a biological sample from a subject that comprises a CPVL genomic nucleic acid molecule or an mRNA molecule, and, in the case of mRNA, optionally reverse transcribing the mRNA into cDNA. Such an assay may comprise, for example, determining the identity of these positions of a particular CPVL nucleic acid molecule. In some embodiments, the method is an in vitro method.

[0117] In some embodiments, the determining, detecting, or sequence analysis comprises sequencing at least a portion of the nucleotide sequence of a CPVL genomic nucleic acid molecule, a CPVL mRNA molecule, or a CPVL cDNA in a biological sample, wherein the sequenced portion contains one or more variations that cause or are predicted to cause a loss of function (partial or complete).

[0118] In some embodiments, the assay comprises sequencing the entire nucleic acid molecule. In some embodiments, only the CPVL genomic nucleic acid molecule is analyzed. In some embodiments, only the CPVL mRNA is analyzed. In some embodiments, only the CPVL cDNA obtained from the CPVL mRNA is analyzed.

[0119] Variation-specific polymerase chain reaction techniques can be used to detect mutations, such as SNPs, in nucleic acid sequences. Variation-specific primers can be used because DNA polymerase will not extend if there is a mismatch with the template.

[0120] In some embodiments, the nucleic acid molecule in the sample is mRNA, and the mRNA is reverse transcribed into cDNA prior to the amplification step. In some embodiments, the nucleic acid molecule is present in a cell obtained from the subject.

[0121] In some embodiments, the assay involves contacting a biological sample under stringent conditions with a primer or probe, such as a mutation-specific primer or mutation-specific probe, that specifically hybridizes to a CPVL variant genomic sequence, variant mRNA sequence, or variant cDNA sequence and does not hybridize to the corresponding CPVL reference sequence, and determining whether hybridization occurs.

[0122] In some embodiments, the determining, detecting, or sequence analyzing step comprises: a) amplifying at least a portion of a nucleic acid molecule encoding a CPVL polypeptide; b) labeling the amplified nucleic acid molecule with a detectable label; c) contacting the labeled nucleic acid molecule with a support comprising a mutation-specific probe; and d) detecting the detectable label.

[0123] In some embodiments, the assay includes RNA sequencing (RNA-Seq). In some embodiments, the assay also includes reverse transcribing mRNA into cDNA, for example, by reverse transcriptase polymerase chain reaction (RT-PCR).

[0124] In some embodiments, the method utilizes probes and primers of sufficient nucleotide length to bind to the target nucleotide sequence and specifically detect and / or identify polynucleotides including CPVL variant genomic nucleic acid molecules, variant mRNA molecules, or variant cDNA molecules. Hybridization or reaction conditions can be determined by the operator to achieve this result. The nucleotide length can be any length sufficient for use in the selected detection method, including any assay described or exemplified herein. Such probes and primers can specifically hybridize to the target nucleotide sequence under high stringency hybridization conditions. Probes and primers can have complete nucleotide sequence identity of consecutive nucleotides within the target nucleotide sequence, but probes that differ from the target nucleotide sequence and retain the ability to specifically detect and / or identify the target nucleotide sequence can be designed by conventional methods. The probes and primers can have about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% sequence identity or complementarity to the nucleotide sequence of the target nucleic acid molecule.

[0125] Illustrative examples of nucleic acid sequencing techniques include, but are not limited to, chain terminator (Sanger) sequencing and dye terminator sequencing. Other methods involve nucleic acid hybridization methods other than sequencing (fluorescence in situ hybridization (FISH)), including using labeled primers or probes directed to purified DNA, amplified DNA, and fixed cell preparations. In some methods, the target nucleic acid molecule can be amplified prior to or simultaneously with detection. Illustrative examples of nucleic acid amplification techniques include, but are not limited to, polymerase chain reaction (PCR), ligase chain reaction (LCR), strand displacement amplification (SDA), and nucleic acid sequence-based amplification (NASBA). Other methods include, but are not limited to, ligase chain reaction, strand displacement amplification, and thermophilic SDA (tSDA).

[0126] Hybridization techniques may use stringent conditions so that the probe or primer hybridizes specifically to its target. In some embodiments, a polynucleotide primer or probe under stringent conditions will hybridize to its target sequence detectably higher than other non-target sequences, for example, at least 2-fold, at least 3-fold, at least 4-fold, or more than background (including more than 10-fold above background). In some embodiments, a polynucleotide primer or probe under stringent conditions will hybridize to its target sequence detectably higher than other nucleotide sequences at least 2-fold. In some embodiments, a polynucleotide primer or probe under stringent conditions will hybridize to its target sequence detectably higher than other nucleotide sequences at least 3-fold. In some embodiments, a polynucleotide primer or probe under stringent conditions will hybridize to its target sequence detectably higher than other nucleotide sequences at least 4-fold. In some embodiments, a polynucleotide primer or probe under stringent conditions will hybridize to its target sequence to a detectably greater extent than other nucleotide sequences, more than 10-fold above background. Stringent conditions are sequence-dependent and will be different in different circumstances.

[0127] Suitable stringency conditions that promote DNA hybridization, such as 6× sodium chloride / sodium citrate (SSC) at about 45° C., followed by a 2× SSC wash at 50° C., are known or can be found in Current Protocols in Molecular Biology, John Wiley & Sons, NY (1989), 6.3.1-6.3.6. Typically, stringent conditions for hybridization and detection include conditions in which the salt concentration is less than about 1.5 M NaCl at pH 7.0-8.3. + ion, typically about 0.01 to 1.0 M Na +The ionic concentration (or other salts) and the temperature will be at least about 30°C for short probes (e.g., 10-50 nucleotides, etc.) and at least about 60°C for longer probes (e.g., more than 50 nucleotides, etc.). Stringent conditions can also be achieved by the addition of destabilizing agents, such as formamide. Optionally, the wash buffer can contain about 0.1% to about 1% SDS. The duration of hybridization is generally less than about 24 hours, usually about 4 to about 12 hours. The duration of the wash period will be at least long enough to reach equilibrium.

[0128] In some embodiments, such isolated nucleic acid molecules comprise at least about 5, at least about 8, at least about 10, at least about 11, at least about 12, at least about 13, at least about 14, at least about 15, at least about 16, at least about 17, at least about 18, at least about 19, at least about 20, at least about 21, at least about 22, at least about 23, at least about 24, at least about 25, at least about 30, at least about 35, at least about 40, at least about 45, at least about 50, at least about 51, at least about 52, at least about 53, at least about 54, at least about 55, at least about 56, at least about 57, at least about 58, at least about 59, at least about 60, at least about 61, at least about 62, at least about 63, at least about 64, at least about 65, at least about 66, at least about 67, at least about 68, at least about 69, at least about 70, at least about 71, at least about 72, at least about 73, at least about 74, at least about 75, at least about 76, at least about 77, at least about 78, at least about 79, at least about 80, at least about 81, at least about 82, at least about 83, at least about 84, at least about 85, at least about 86, at least about 87, at least about 88, at least about 89, at least about 90, at least about 91, at least about 92, at least about 93, at least about 94, at least about 95, at least about 96, at least about 97, at least about 98, at least about 99 at least about 5, at least about 60, at least about 65, at least about 70, at least about 75, at least about 80, at least about 85, at least about 90, at least about 95, at least about 100, at least about 200, at least about 300, at least about 400, at least about 500, at least about 600, at least about 700, at least about 800, at least about 900, at least about 1000, at least about 2000, at least about 3000, at least about 4000, or at least about 5000 nucleotides. In some embodiments, such isolated nucleic acid molecules comprise or consist of at least about 5, at least about 8, at least about 10, at least about 11, at least about 12, at least about 13, at least about 14, at least about 15, at least about 16, at least about 17, at least about 18, at least about 19, at least about 20, at least about 21, at least about 22, at least about 23, at least about 24, or at least about 25 nucleotides. In some embodiments, the isolated nucleic acid molecule comprises or consists of at least about 18 nucleotides. In some embodiments, the isolated nucleic acid molecule comprises or consists of at least about 15 nucleotides. In some embodiments, the isolated nucleic acid molecule comprises or consists of about 10 to about 35, about 10 to about 30, about 10 to about 25, about 12 to about 30, about 12 to about 28, about 12 to about 24, about 15 to about 30, about 15 to about 25, about 18 to about 30, about 18 to about 25, about 18 to about 24, or about 18 to about 22 nucleotides. In some embodiments, the isolated nucleic acid molecule comprises or consists of about 18 to about 30 nucleotides.In some embodiments, the isolated nucleic acid molecule comprises or consists of at least about 15 nucleotides to at least about 35 nucleotides.

[0129] In some embodiments, such isolated nucleic acid molecules hybridize under stringent conditions to CPVL missense variant nucleic acid molecules (e.g., genomic nucleic acid molecules, mRNA molecules, and / or cDNA molecules). Such nucleic acid molecules can be used, for example, as probes, primers, variation-specific probes, or variation-specific primers described or exemplified herein, including, but not limited to, primers, probes, antisense RNA, shRNA, and siRNA, each of which is described in more detail elsewhere herein and can be used in any of the methods described herein.

[0130] In some embodiments, the isolated nucleic acid molecule hybridizes to at least about 15 contiguous nucleotides of a nucleic acid molecule that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to a CPVL missense variant genomic nucleic acid molecule, a CPVL missense variant mRNA molecule, and / or a CPVL missense variant cDNA molecule. In some embodiments, the isolated nucleic acid molecule comprises or consists of about 15 to about 100 nucleotides, or about 15 to about 35 nucleotides. In some embodiments, the isolated nucleic acid molecule comprises or consists of about 15 to about 100 nucleotides. In some embodiments, the isolated nucleic acid molecule comprises or consists of about 15 to about 35 nucleotides.

[0131] In some embodiments, the variation-specific probe and the variation-specific primer comprise DNA. In some embodiments, the variation-specific probe and the variation-specific primer comprise RNA.

[0132] In some embodiments, the probes and primers described herein (including mutation-specific probes and mutation-specific primers) have nucleotide sequences that specifically hybridize to any of the nucleic acid molecules disclosed herein, or their complements. In some embodiments, the probes and primers specifically hybridize to any of the nucleic acid molecules disclosed herein under stringent conditions.

[0133] In some embodiments, primers including variation-specific primers can be used in second generation sequencing or high throughput sequencing. In some examples, primers including variation-specific primers can be modified. In particular, primers can include various modifications used in different steps of, for example, massively parallel signature sequencing (MPSS), polony sequencing, and 454 pyrosequencing. Modified primers can be used in several steps of the process, including biotinylated primers in the cloning step, and fluorescently labeled primers used in the bead loading and detection steps. Polony sequencing is usually performed using paired-end tag libraries, where each molecule of DNA template is about 135 bp in length. Biotinylated primers are used in the bead loading step and emulsion PCR. Fluorescently labeled degenerate nonamer oligonucleotides are used in the detection step. Adapters can contain 5'-biotin tags for immobilization of DNA libraries to streptavidin-coated beads.

[0134] The probes and primers described herein can be used to detect nucleotide variations in any of the CPVL variant missense variant genomic nucleic acid molecules, CPVL missense variant mRNA molecules, and / or CPVL missense variant cDNA molecules disclosed herein. The primers described herein can be used to amplify a CPVL missense variant genomic nucleic acid molecule, a CPVL missense variant mRNA molecule, or a CPVL missense variant cDNA molecule, or a fragment thereof.

[0135] In the context of this disclosure, "specifically hybridizes" means that a probe or primer (e.g., a variation-specific probe or variation-specific primer, etc.) does not hybridize to a nucleic acid sequence encoding a CPVL reference genomic nucleic acid molecule, a CPVL reference mRNA molecule, and / or a CPVL reference cDNA molecule.

[0136] In some embodiments, the probe (e.g., the mutation-specific probe, etc.) comprises a label. In some embodiments, the label is a fluorescent label, a radioactive label, or biotin. The present disclosure also provides a support comprising a substrate to which any one or more of the probes disclosed herein are attached. A solid support is a solid-state substrate or support to which molecules such as any of the probes disclosed herein can be associated. A form of solid support is an array. Another form of solid support is an array detector. An array detector is a solid support to which multiple different probes are coupled in an array, grid, or other organized pattern. A form for a solid-state substrate is a microtiter dish, such as a standard 96-well type. In some embodiments, a multi-well glass slide can be used, usually containing one array per well.

[0137] The nucleotide sequence of the CPVL reference genome nucleic acid molecule is set forth in SEQ ID NO:1 (ENSG00000106066.15, encompassing chr7:28,995,637 to 29,195,276 in the GRCh38 / hg38 human genome assembly).

[0138] The nucleotide sequence of a CPVL reference mRNA molecule is set forth in SEQ ID NO:2. The nucleotide sequence of another CPVL reference mRNA molecule is set forth in SEQ ID NO:3. The nucleotide sequence of another CPVL reference mRNA molecule is set forth in SEQ ID NO:4. The nucleotide sequence of another CPVL reference mRNA molecule is set forth in SEQ ID NO:5. The nucleotide sequence of another CPVL reference mRNA molecule is set forth in SEQ ID NO:6. The nucleotide sequence of another CPVL reference mRNA molecule is set forth in SEQ ID NO:7. The nucleotide sequence of another CPVL reference mRNA molecule is set forth in SEQ ID NO:8. The nucleotide sequence of another CPVL reference mRNA molecule is set forth in SEQ ID NO:9. The nucleotide sequence of another CPVL reference mRNA molecule is set forth in SEQ ID NO:10. The nucleotide sequence of another CPVL reference mRNA molecule is set forth in SEQ ID NO:11. The nucleotide sequence of another CPVL reference mRNA molecule is set forth in SEQ ID NO:12. The nucleotide sequence of another CPVL reference mRNA molecule is set forth in SEQ ID NO:13. The nucleotide sequence of another CPVL reference mRNA molecule is set forth in SEQ ID NO:14. The nucleotide sequence of another CPVL reference mRNA molecule is set forth in SEQ ID NO: 15. The nucleotide sequence of another CPVL reference mRNA molecule is set forth in SEQ ID NO: 16. The nucleotide sequence of another CPVL reference mRNA molecule is set forth in SEQ ID NO: 17. The nucleotide sequence of another CPVL reference mRNA molecule is set forth in SEQ ID NO: 18. The nucleotide sequence of another CPVL reference mRNA molecule is set forth in SEQ ID NO: 19. The nucleotide sequence of another CPVL reference mRNA molecule is set forth in SEQ ID NO: 20. The nucleotide sequence of another CPVL reference mRNA molecule is set forth in SEQ ID NO: 21. The nucleotide sequence of another CPVL reference mRNA molecule is set forth in SEQ ID NO: 22. The nucleotide sequence of another CPVL reference mRNA molecule is set forth in SEQ ID NO: 23. The nucleotide sequence of another CPVL reference mRNA molecule is set forth in SEQ ID NO: 24. The nucleotide sequence of another CPVL reference mRNA molecule is set forth in SEQ ID NO: 25. The nucleotide sequence of another CPVL reference mRNA molecule is set forth in SEQ ID NO: 26.

[0139] The nucleotide sequence of a CPVL reference cDNA molecule is set forth in SEQ ID NO:27. The nucleotide sequence of another CPVL reference cDNA molecule is set forth in SEQ ID NO:28. The nucleotide sequence of another CPVL reference cDNA molecule is set forth in SEQ ID NO:29. The nucleotide sequence of another CPVL reference cDNA molecule is set forth in SEQ ID NO:30. The nucleotide sequence of another CPVL reference cDNA molecule is set forth in SEQ ID NO:31. The nucleotide sequence of another CPVL reference cDNA molecule is set forth in SEQ ID NO:32. The nucleotide sequence of another CPVL reference cDNA molecule is set forth in SEQ ID NO:33. The nucleotide sequence of another CPVL reference cDNA molecule is set forth in SEQ ID NO:34. The nucleotide sequence of another CPVL reference cDNA molecule is set forth in SEQ ID NO:35. The nucleotide sequence of another CPVL reference cDNA molecule is set forth in SEQ ID NO:36. The nucleotide sequence of another CPVL reference cDNA molecule is set forth in SEQ ID NO:37. The nucleotide sequence of another CPVL reference cDNA molecule is set forth in SEQ ID NO:38. The nucleotide sequence of another CPVL reference cDNA molecule is set forth in SEQ ID NO:39. The nucleotide sequence of another CPVL reference cDNA molecule is set forth in SEQ ID NO: 40. The nucleotide sequence of another CPVL reference cDNA molecule is set forth in SEQ ID NO: 41. The nucleotide sequence of another CPVL reference cDNA molecule is set forth in SEQ ID NO: 42. The nucleotide sequence of another CPVL reference cDNA molecule is set forth in SEQ ID NO: 43. The nucleotide sequence of another CPVL reference cDNA molecule is set forth in SEQ ID NO: 44. The nucleotide sequence of another CPVL reference cDNA molecule is set forth in SEQ ID NO: 45. The nucleotide sequence of another CPVL reference cDNA molecule is set forth in SEQ ID NO: 46. The nucleotide sequence of another CPVL reference cDNA molecule is set forth in SEQ ID NO: 47. The nucleotide sequence of another CPVL reference cDNA molecule is set forth in SEQ ID NO: 48. The nucleotide sequence of another CPVL reference cDNA molecule is set forth in SEQ ID NO: 49. The nucleotide sequence of another CPVL reference cDNA molecule is set forth in SEQ ID NO: 50. The nucleotide sequence of another CPVL reference cDNA molecule is set forth in SEQ ID NO: 51.

[0140] The amino acid sequence of the CPVL reference polypeptide is set forth in SEQ ID NO:52 and is 476 amino acids in length. The amino acid sequence of another CPVL reference polypeptide is set forth in SEQ ID NO:53 and is 406 amino acids in length. The amino acid sequence of another CPVL reference polypeptide is set forth in SEQ ID NO:54 and is 490 amino acids in length. The amino acid sequence of another CPVL reference polypeptide is set forth in SEQ ID NO:55 and is 298 amino acids in length. The amino acid sequence of another CPVL reference polypeptide is set forth in SEQ ID NO:56 and is 233 amino acids in length.

[0141] The genomic nucleic acid molecule, mRNA molecule, and cDNA molecule can be from any organism. For example, the genomic nucleic acid molecule, mRNA molecule, and cDNA molecule can be orthologs from humans or another organism, such as non-human mammals, rodents, mice, or rats. It is understood that gene sequences within a population can vary due to polymorphisms, such as single nucleotide polymorphisms. The examples provided herein are merely exemplary sequences. Other sequences are possible.

[0142] Also provided herein is a functional polynucleotide that can interact with the disclosed nucleic acid molecule.The examples of functional polynucleotide include, but are not limited to, antisense molecules, aptamers, ribozymes, triplex-forming molecules, and external guide sequences.Functional polynucleotides can act as effectors, inhibitors, regulators, and stimulators of the specific activity possessed by target molecules, or functional polynucleotides can possess new activities independent of any other molecules.

[0143] The isolated nucleic acid molecules disclosed herein may include RNA, DNA, or both RNA and DNA. The isolated nucleic acid molecules may also be linked or fused to heterologous nucleic acid sequences, such as in a vector, or to a heterologous label. For example, the isolated nucleic acid molecules disclosed herein may be present as exogenous donor sequences in or containing a vector that includes the isolated nucleic acid molecule and a heterologous nucleic acid sequence. The isolated nucleic acid molecules may also be linked or fused to a heterologous label. The label may be directly detectable (e.g., a fluorophore, etc.) or indirectly detectable (e.g., a hapten, enzyme, or fluorophore quencher, etc.). Such labels may be detectable by spectroscopic, photochemical, biochemical, immunochemical, or chemical means. Such labels include, for example, radioactive labels, pigments, dyes, chromogens, spin labels, and fluorescent labels. The label may also be, for example, a chemiluminescent material; a metal-containing material; or an enzyme, in which case an enzyme-dependent secondary generation of a signal occurs. The term "label" may also refer to a "tag" or hapten that can selectively bind to a conjugated molecule such that the conjugated molecule is subsequently added with a substrate and used to generate a detectable signal. For example, biotin can be used as a tag with an avidin or streptavidin conjugate of horseradish peroxidase (HRP) to bind to the tag and examined using a colorimetric substrate (e.g., tetramethylbenzidine (TMB), etc.) or a fluorogenic substrate to detect the presence of HRP. Exemplary labels that can be used as tags to facilitate purification include, but are not limited to, myc, HA, FLAG or 3XFLAG, 6Xhis or polyhistidine, glutathione-S-transferase (GST), maltose binding protein, epitope tags, or the Fc portion of an immunoglobulin. Numerous labels include, for example, particles, fluorophores, haptens, enzymes and their calorimetric, fluorescent and chemiluminescent substrates and other labels.

[0144] Percent identity (or complementarity) between particular stretches of nucleotide sequences in nucleic acid molecules or amino acid sequences in polypeptides can be routinely determined using the BLAST program (basic local alignment search tool) and PowerBLAST program (Altschul et al., J. Mol. Biol., 1990, 215, 403-410; Zhang and Madden, Genome Res., 1997, 7, 649-656), or by using the Gap program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, Madison Wis.) using default settings that employ the algorithm of Smith and Waterman (Adv. Appl. Math., 1981, 2, 482-489). When referring to percent sequence identity herein, higher percentages of sequence identity are preferred over lower ones.

[0145] As used herein, the phrase "corresponding to" or grammatical variations thereof, when used in the context of numbering a particular nucleotide or sequence of nucleotides or positions, refers to the numbering of the designated reference sequence when that particular nucleotide or nucleotide sequence is compared to a reference sequence (e.g., SEQ ID NO:1, etc.). That is, the residue (e.g., nucleotide or amino acid, etc.) number or residue (e.g., nucleotide or amino acid, etc.) position of a particular polymer is designated with reference to the reference sequence, not by the actual position number of that residue within that particular nucleotide or nucleotide sequence. For example, a particular nucleotide sequence may be aligned to a reference sequence by introducing gaps to optimize residue matching between the two sequences. In these cases, although gaps are present, the numbering of the residues in a particular nucleotide or nucleotide sequence is done with respect to the reference sequence to which it is aligned.

[0146] The nucleotide and amino acid sequences listed in the accompanying sequence listing are shown using standard letter abbreviations for nucleotide bases and three letter codes for amino acids. The nucleotide sequences follow the standard convention of starting at the 5'-terminus of the sequence and proceeding forward to the 3'-terminus (i.e., from left to right on each line). Only one strand of each nucleotide sequence is shown, but it is understood that the complementary strand is encompassed by any reference to the presented strand. The amino acid sequences follow the standard convention of starting at the amino-terminus of the sequence and proceeding forward to the carboxy-terminus (i.e., from left to right on each sequence).

[0147] The present disclosure also provides a skin cancer treating, preventing, or inhibiting therapeutic agent for use in treating and / or preventing skin cancer in a subject, wherein the subject is a) a reference to a carboxypeptidase vitellogenic-like (CPVL) genomic nucleic acid molecule, a CPVL mRNA molecule, or a CPVL cDNA molecule; or b) a reference to i) a CPVL missense variant genomic nucleic acid molecule encoding a predicted loss-of-function polypeptide of CPVL; ii) a CPVL missense variant mRNA molecule encoding a predicted loss-of-function polypeptide of CPVL; or iii) a CPVL missense variant cDNA molecule encoding a predicted loss-of-function polypeptide of CPVL. Any of the skin cancer treating, preventing, or inhibiting therapeutic agents described herein may be used in these methods.

[0148] The present disclosure also provides the use of a therapeutic agent for treating, preventing, or inhibiting skin cancer for use in the preparation of a medicament for treating and / or preventing skin cancer in a subject, wherein the subject is a) a reference to a carboxypeptidase vitellogenic-like (CPVL) genomic nucleic acid molecule, a CPVL mRNA molecule, or a CPVL cDNA molecule; or b) i) a CPVL missense variant genomic nucleic acid molecule encoding a predicted loss-of-function polypeptide of CPVL; ii) a CPVL missense variant mRNA molecule encoding a predicted loss-of-function polypeptide of CPVL; or iii) a CPVL missense variant cDNA molecule encoding a predicted loss-of-function polypeptide of CPVL. Any of the therapeutic agents for treating, preventing, or inhibiting skin cancer described herein may be used in these methods.

[0149] The present disclosure also provides a CPVL inhibitor for use in treating and / or preventing skin cancer in a subject, the subject being a) a reference to a carboxypeptidase vitellogenic-like (CPVL) genomic nucleic acid molecule, a CPVL mRNA molecule, or a CPVL cDNA molecule; or b) heterozygous for i) a CPVL missense variant genomic nucleic acid molecule encoding a predicted loss-of-function polypeptide of CPVL; ii) a CPVL missense variant mRNA molecule encoding a predicted loss-of-function polypeptide of CPVL; or iii) a CPVL missense variant cDNA molecule encoding a predicted loss-of-function polypeptide of CPVL. Any of the CPVL inhibitors described herein may be used in these methods.

[0150] The present disclosure also provides the use of a CPVL inhibitor in the preparation of a medicament for treating and / or preventing skin cancer in a subject, wherein the subject is a) a reference to a carboxypeptidase vitellogenic-like (CPVL) genomic nucleic acid molecule, a CPVL mRNA molecule, or a CPVL cDNA molecule; or b) a reference to i) a CPVL missense variant genomic nucleic acid molecule encoding a predicted loss-of-function polypeptide of CPVL; ii) a CPVL missense variant mRNA molecule encoding a predicted loss-of-function polypeptide of CPVL; or iii) a CPVL missense variant cDNA molecule encoding a predicted loss-of-function polypeptide of CPVL. Any of the CPVL inhibitors described herein may be used in these methods.

[0151] All patent documents, websites, other publications, accession numbers, etc. cited above or below are incorporated by reference in their entirety for all purposes to the same extent as if each individual item was specifically and individually indicated to be so incorporated by reference. Where different versions of sequences are associated with accession numbers at different times, the version associated with the accession number at the effective filing date of this application is meant. Effective filing date means the earlier of the actual filing date or the filing date of the priority application that references the accession number, if applicable. Similarly, where different versions of publications, websites, etc. are published at different times, the latest published version at the effective filing date of this application is meant unless otherwise indicated. Any feature, step, element, embodiment, or aspect of the present disclosure may be used in combination with any other feature, step, element, embodiment, or aspect unless otherwise specifically indicated. The present disclosure has been described in some detail by illustration and example for purposes of clarity and understanding, but it will be apparent that certain changes and modifications may be practiced within the scope of the appended claims.

[0152] The following examples are provided to more fully describe the embodiments. They are intended to illustrate, not limit, the claimed embodiments. The following examples provide those skilled in the art with a disclosure and description of how the compounds, compositions, articles, devices and / or methods described herein are made and evaluated, are intended to be purely exemplary, and are not intended to limit the scope of any claims. Attempts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperatures, etc.), but some error and deviation can be accounted for. Unless otherwise indicated, parts are parts by weight, temperature is °C or is ambient temperature, and pressure is at or near atmospheric pressure. EXAMPLES

[0153] Example 1: Meta-analysis of GWAS shows protective gene burden associations for CPVL. Regeneron conducted the largest exome-wide cancer association analysis to date, which led to the identification of CPVL. A meta-analysis of genome-wide association studies (GWAS) conducted in the UKB and GHS cohorts demonstrated that CPVL pLOF mutations were associated with a reduced risk of non-melanoma skin cancer (Table 3; phenotype = NMSC; chromosome 7, position 28995771 in CPVL). Also, most of the loci associated with vitiligo in prior GWAS are protective for non-melanoma skin cancer (data not shown).

[0154] [Table 3]

[0155] Mask definition: M3.5 variant rs117744081 encodes a missense p.Tyr168 in the CPVL peptidase domain; M3.1 association is induced by a p.Arg464Gln missense in the same functional domain; p.Ser61Asn is outside the peptidase domain and contributes to the M3.1 mask.

[0156] Moreover, GWAS performed in the UKB and GHS cohorts showed that CPVL pLOF mutations are associated with a reduced risk of melanoma (Table 4 ; phenotype = melanoma; chromosome 7, position 28995771 in CPVL).

[0157] [Table 4]

[0158] Example 2: CPVL expression in macrophages Publicly available single-cell RNA expression studies were analyzed to quantitate cell type specificity of CPVL expression (see Table 5, Jerby-Arnon et al., Cell, 2018, 175, 984-987; Table 6, Yost et al., Nat. Med., 2019, 25, 1251-1259; and Table 7, Hughes et al., Immunity, 2020, 53, 878-894).

[0159] [Table 5]

[0160] [Table 6]

[0161] [Table 7]

[0162] In the tumor microenvironment of melanoma tumors, CPVL was found to be expressed almost exclusively by macrophages. Subpopulations of cancer-associated fibroblasts (CAFs) and endothelial cells also appeared to express the gene (see Figure 1). Also, in the basal cell carcinoma (BCC) dataset, CPVL was found to be highly expressed in macrophages and lightly expressed in melanocytes (see Figure 2; lower panel is from Yost et al., Nat. Med., 2019, 25, 1251-1259 for background).

[0163] In addition to those described herein, various modifications of the described subject matter will be apparent to those skilled in the art from the above description. Such modifications are also intended to fall within the scope of the appended claims. Each reference cited in this application (including but not limited to journal articles, U.S. and non-U.S. patents, patent application publications, international patent application publications, gene bank accession numbers, etc.) is incorporated herein by reference in its entirety for all purposes.

Claims

1. An in vitro method for determining the susceptibility of a subject to developing skin cancer, said method comprising: determining or having determined the presence or absence of a CPVL missense variant nucleic acid molecule encoding a predicted loss-of-function polypeptide of carboxypeptidase vitellogenesis-like (CPVL) in a biological sample obtained from said subject; wherein if said subject is a CPVL reference, said subject is represented as having an increased risk of developing skin cancer; and wherein if said subject is heterozygous or homozygous for said CPVL missense variant nucleic acid molecule encoding a predicted loss-of-function polypeptide of said CPVL, said subject is represented as having a decreased risk of developing skin cancer, said method.

2. The method of claim 1, wherein said CPVL missense variant nucleic acid molecule is a splice site variant, a stop gain variant, a start loss variant, a stop loss variant, a frameshift variant, or an in-frame indel variant, or a variant encoding a predicted loss-of-function polypeptide of truncated CPVL.

3. The method of claim 1, wherein said CPVL missense variant nucleic acid molecule encodes a predicted loss-of-function polypeptide of truncated CPVL.

4. The method according to any one of claims 1 to 3, wherein said skin cancer is non-melanoma skin cancer, basal cell carcinoma, squamous cell carcinoma, melanoma, Merkel cell carcinoma, dermatofibrosarcoma protuberans, or adenocarcinoma.

5. Use of a therapeutic agent for treating, preventing, or inhibiting skin cancer in the preparation of a medicament for treating and / or preventing skin cancer in a subject, said subject being i) a CPVL missense variant genomic nucleic acid molecule encoding a predicted loss-of-function polypeptide of CPVL; or ii) a CPVL missense variant mRNA molecule encoding a predicted loss-of-function polypeptide of CPVL; for use, which is heterozygous for.

6. Use according to claim 5, wherein the skin cancer is non-melanoma skin cancer, basal cell carcinoma, squamous cell carcinoma, melanoma, Merkel cell carcinoma, dermatofibrosarcoma protuberans, or adenocarcinoma.

7. Use according to claim 5, wherein the skin cancer is basal cell carcinoma and the therapeutic agent is selected from imiquimod, fluorouracil, semiprimab-rwlc, sonidegib, and visimodegib, or any combination thereof.

8. Use according to claim 5, wherein the skin cancer is squamous cell carcinoma and the therapeutic agent is selected from semiprimab-rwlc and pembrolizumab, or any combination thereof.

9. Use according to claim 5, wherein the skin cancer is melanoma and the therapeutic agent is selected from aldesleukin, cobimetinib, dabrafenib, dacarbazine, recombinant interferon alpha-2b, ipilimumab, nivolumab, nivolumab, peginterferon alpha-2b, pembrolizumab, talimogene laherparepvec, trametinib dimethyl sulfoxide, and vemurafenib, or any combination thereof.

10. Use according to claim 5, wherein the skin cancer is Merkel cell carcinoma and the therapeutic agent is selected from avelumab, pembrolizumab, and etoposide (VP16), and carboplatin, or any combination thereof.

11. Use according to claim 5, wherein the skin cancer is dermatofibrosarcoma protuberans and the therapeutic agent is imatinib.

12. Use of a carboxypeptidase vitellogenesis-like (CPVL) inhibitor in the preparation of a medicament for the treatment and / or prevention of skin cancer in a subject, wherein the subject is a) A reference to a CPVL genomic nucleic acid molecule or a CPVL mRNA molecule; or b) i) A CPVL missense variant genomic nucleic acid molecule encoding a predicted loss-of-function polypeptide of CPVL; or ii) A CPVL missense variant mRNA molecule encoding a predicted loss-of-function polypeptide of CPVL; which is heterozygous for use. **Claim 13** The use according to claim 12, wherein the skin cancer is non-melanoma skin cancer, basal cell carcinoma, squamous cell carcinoma, melanoma, Merkel cell carcinoma, dermatofibrosarcoma protuberans, or adenocarcinoma. **Claim 14** The use according to claim 12 or 13, wherein the CPVL inhibitor comprises an inhibitory nucleic acid molecule that hybridizes to a CPVL nucleic acid molecule. **Claim 15** The use according to claim 14, wherein the inhibitory nucleic acid molecule comprises an antisense nucleic acid molecule, small interfering RNA (siRNA), or short hairpin RNA (shRNA). **Claim 16** The use according to claim 12 or 13, wherein the CPVL inhibitor comprises a Cas protein and a guide RNA (gRNA) that hybridizes to a gRNA recognition sequence within the CPVL genomic nucleic acid molecule. **Claim 17** The use according to claim 16, wherein the Cas protein is Cas9 or Cpf1. **Claim 18** The use according to claim 12 or 13, wherein the CPVL inhibitor comprises hydroxymethyl (N-methyliminodiacetic acid) boronate (hydroxymethyl (MIDA) boronate), azidomethyl (N-methyliminodiacetic acid) boronate (azidomethyl (MIDA) boronate), or an α-functionalized alkyl (MIDA) boronate compound. **Claim 19** The CPVL inhibitor is **[Chemical Formula 1]** for use according to claim 12 or 13. Use according to claim 12 or 13, wherein the CPVL inhibitor comprises a cancer immunotherapeutic agent or an immune checkpoint inhibitor. Use according to claim 20, wherein the immune checkpoint inhibitor comprises an anti-PD-1 agent, an anti-PD-L1 agent, or an anti-CTLA-4 agent. Use according to claim 21, wherein the anti-PD-1 agent comprises pembrolizumab, nivolumab, or semaprimab. Use according to claim 21, wherein the anti-PD-L1 agent comprises atezolizumab, avelumab, or durvalumab. Use according to claim 21, wherein the anti-CTLA-4 agent comprises ipilimumab or tremelimumab. A pharmaceutical composition for treating skin cancer in a patient who is heterozygous for a CPVL missense variant genomic nucleic acid molecule encoding a predicted loss-of-function polypeptide of carboxypeptidase vitellogenesis-like (CPVL), or a CPVL missense variant mRNA molecule encoding a predicted loss-of-function polypeptide of CPVL, the pharmaceutical composition comprising, as an active ingredient, a therapeutic agent for treating or preventing skin cancer. A pharmaceutical composition for treating skin cancer in a patient, wherein the patient is a) a reference for a carboxypeptidase vitellogenesis-like (CPVL) genomic nucleic acid molecule or a CPVL mRNA molecule; or b) heterozygous for a CPVL missense variant genomic nucleic acid molecule encoding a predicted loss-of-function polypeptide of CPVL, or a CPVL missense variant mRNA molecule encoding a predicted loss-of-function polypeptide of CPVL, and the pharmaceutical composition comprises a CPVL inhibitor as an active ingredient. The pharmaceutical composition according to claim 25 or 26, wherein the skin cancer is non-melanoma skin cancer, basal cell carcinoma, squamous cell carcinoma, melanoma, Merkel cell carcinoma, dermatofibrosarcoma protuberans, or adenocarcinoma.

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