Compositions and methods for treatment of netherton syndrome

Abstract

To provide novel treatment options targeting molecular correction of SPINK5 deficiencies.SOLUTION: The present disclosure provides: recombinant nucleic acids comprising one or more polynucleotides encoding a Serine Protease Inhibitor Kazal-type (SPINK) polypeptide (e.g., a SPINKS polypeptide); viruses comprising the recombinant nucleic acids; compositions and formulations comprising the recombinant nucleic acids and / or viruses; methods of their use (e.g., for the treatment of Netherton Syndrome); and articles of manufacture or kits thereof.SELECTED DRAWING: Figure 4

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Application No. 62 / 735,582, filed Sep. 24, 2018, which is incorporated by reference in its entirety.

[0002] Submitting a sequence listing as an ASCII text file The contents of the following submission in an ASCII text file are incorporated herein by reference in their entirety: Computer Readable Form (CRF) of the Sequence Listing (Filename: 761342000440SEQLIST.txt, Recorded: September 19, 2019, Size: 60 KB).

[0003] FIELD OF THEINVENTION The present disclosure relates, in part, to recombinant nucleic acids comprising one or more polynucleotides encoding a serine protease inhibitor Kazal type (SPINK) polypeptide (e.g., a human SPINK5 polypeptide), viruses comprising same, pharmaceutical compositions, formulations, and medicaments thereof, and methods of use thereof (e.g., for the treatment of Netherton syndrome). [Background technology]

[0004] background Netherton syndrome (NS), also called Comel-Netherton syndrome, is a debilitating autosomal recessive skin disorder that causes defective keratinization, severe skin barrier defects, and recurrent infections. Patients develop a generalized rash shortly after birth that progresses to severe ichthyosis (Comel, 1949. Dermatologica. 98(3):133-6). Infants with more severe NS symptoms are associated with failure to thrive, hypernatremic dehydration with excessive fluid loss, growth retardation, dwarfism, and recurrent infections (Jones et al., 1986. Br J Dermatol. 114(6):741-3; Hausser et al., 1996. Pediatr Dermatol. 13(3):183-99). Netherton syndrome has an extremely high postnatal mortality rate, with up to 20% of patients not surviving their first birthday (Renner et al., 2009. J Allergy Clin Immunol. 124(3):536-43 (Non-Patent Document 4)).

[0005] Clinically, Netherton syndrome is characterized by congenital ichthyosa-like erythroderma, hair shaft defects, recurrent infections, and skin barrier defects (Judge et al., 1994. Br J Dermatol. 131(5):615-21 (Non-Patent Document 5); Schmuth et al., 2013. Eur J Hum Genet. 21(2):123-33 (Non-Patent Document 6)). Hair shafts are short and thin, brittle and prone to breakage due to intussusceptible trichorrhexis, i.e., "bamboo hair." A tendency to allergies, asthma, and eczema also characterize NS. Finally, individuals with Netherton syndrome often experience chronic skin inflammation, severe dehydration, and poor growth.

[0006] The disease is caused by mutations in the serine protease inhibitor Kazal type 5 (SPINK5) gene, resulting in loss of activity of its encoded serine protease inhibitor protein SPINK5 (also known as lymphoepithelial Kazal type-related inhibitor (LEKTI)) (Bitoun et al., 2002. J Invest Dermatol. 118(2):352-61). In healthy individuals, SPINK5 is one of the serine protease inhibitors expressed in the outermost layer of the skin, where it plays a key role in regulating serine proteases that hydrolyze the extracellular proteins that hold keratinocytes together. In patients suffering from Netherton syndrome, the inhibitory effect of SPINK5 on these serine proteases is abolished due to the underlying genetic mutation in the SPINK5 gene (Komatsu et al., 2008. J Invest Dermatol. 128(5):1148-59 (Non-Patent Document 8)). As a result, hyperactive serine proteases in the skin cause uncontrolled desquamation and lead to skin barrier defects (Descargues et al., 2005. Nat Genet. 37(1):56-65 (Non-Patent Document 9)).

[0007] Currently, there is no known cure for Netherton syndrome, and patients have limited treatment options. Current treatment focuses on managing the symptoms of the disease, including using moisturizing products to minimize scaling / cracking of the skin, and providing anti-infective treatment when appropriate. In addition, intravenous administration of immunoglobulin to reduce skin infections is widespread. Unfortunately, while steroid and retinoid products have shown some success in treating other ichthyosis-related disorders, these products have proven ineffective for NS and may actually make things worse for affected individuals (Braun et al., 1997. Dermatology. 195(1):75). Thus, there is a clear need for novel treatment options that target the molecular correction of the SPINK5 deficiency observed in this susceptible patient population.

[0008] All references cited herein, including patent applications, patent publications, non-patent literature, and NCBI / UniProtKB / Swiss-Prot accession numbers, are hereby incorporated by reference in their entirety as if each individual reference was specifically and individually indicated to be incorporated by reference. [Prior art documents] [Non-patent literature]

[0009] [Non-Patent Document 1] Comel,1949.Dermatologica.98(3):133-6 [Non-Patent Document 2] Jones et al.,1986.Br J Dermatol.114(6):741-3 [Non-Patent Document 3] Hausser et al.,1996.Pediatr Dermatol.13(3):183-99 [Non-Patent Document 4] Renner et al.,2009.J Allergy Clin Immunol.124(3):536-43 [Non-Patent Document 5] Judge et al.,1994.Br J Dermatol.131(5):615-21 [Non-Patent Document 6] Schmuth et al.,2013.Eur J Hum Genet.21(2):123-33 [Non-Patent Document 7] Bitoun et al.,2002.J Invest Dermatol.118(2):352-61 [Non-Patent Document 8] Komatsu et al.,2008.J Invest Dermatol.128(5):1148-59 [Non-Patent Document 9] Descargues et al.,2005.Nat Genet.37(1):56-65 [Non-Patent Document 10] Braun et al.,1997.Dermatology.195(1):75 Summary of the Invention

[0010] Quick Overview To meet these and other needs, provided herein are recombinant nucleic acids (e.g., recombinant herpes simplex virus genomes) encoding serine protease inhibitor Kazal type (SPINK) polypeptides (e.g., SPINK5 polypeptides) for use in viruses (e.g., herpes viruses), pharmaceutical compositions and formulations, medicaments, and / or methods useful for treating SPINK deficiency and / or useful for treating individuals having or at risk for developing a SPINK-associated disorder (e.g., Netherton syndrome, atopic dermatitis, hereditary pancreatitis, tropical calcific pancreatitis, spermatogenesis impair29, etc.). The inventors have shown that the recombinant viruses described herein were capable of transducing human epidermal cells and successfully expressing the encoded exogenous SPINK (RNA and protein), and that the exogenous polypeptide was properly secreted and fully functional (e.g., (See, e.g., Example 2). Furthermore, the inventors have shown that the viruses described herein may be successfully administered in vivo either topically or intradermally without significant cytotoxicity, allowing the encoded human SPINK polypeptide to be expressed and localized to the appropriate regions of the skin (See, e.g., Example 3). Without wishing to be bound by theory, it is believed that increasing, enhancing, and / or supplementing levels of SPINK polypeptide (e.g., human SPINK5) in a subject in need thereof by administering one or more of the recombinant nucleic acids, viruses, compositions, and / or agents described herein will 1) enhance anti-inflammatory and / or antimicrobial protection of mucosal epithelia, 2) reduce transepidermal water loss (TEWL), 3) inhibit desquamation, and 4) reduce or treat skin barrier defects in the subject.Additionally, without wishing to be bound by theory, it is believed that increasing, enhancing, and / or replenishing levels of a SPINK polypeptide (e.g., human SPINK5) in one or more cells of a subject (by administering any of the recombinant nucleic acids, viruses, agents, and / or pharmaceutical compositions described herein) will result in treatment of existing skin abnormalities in individuals suffering from a SPINK deficiency (e.g., Netherton syndrome, atopic dermatitis, etc.).

[0011] Thus, certain aspects of the present disclosure relate to a recombinant herpesvirus genome comprising one or more polynucleotides encoding a serine protease inhibitor Kazal type (SPINK) polypeptide. In some embodiments, the recombinant herpesvirus genome comprises two or more polynucleotides encoding a SPINK polypeptide. In some embodiments, the recombinant herpesvirus genome is replication competent. In some embodiments, the recombinant herpesvirus genome is replication defective. In some embodiments that may be combined with any of the preceding embodiments, the recombinant herpesvirus genome is selected from a recombinant herpes simplex virus genome, a recombinant varicella zoster virus genome, a recombinant human cytomegalovirus genome, a recombinant herpesvirus 6A genome, a recombinant herpesvirus 6B genome, a recombinant herpesvirus 7 genome, a recombinant Kaposi's sarcoma-associated herpesvirus genome, and any combination or derivative thereof. In some embodiments that may be combined with any of the preceding embodiments, the recombinant herpesvirus genome is a recombinant herpes simplex virus genome. In some embodiments, the recombinant herpes simplex virus genome is a recombinant herpes simplex virus type 1 (HSV-1) genome, a recombinant herpes simplex virus type 2 (HSV-2) genome, or any derivative thereof. In some embodiments, the recombinant herpes simplex virus genome is a recombinant HSV-1 genome.

[0012] In some embodiments that may be combined with any of the preceding embodiments, the recombinant herpes simplex virus genome comprises an inactivating mutation. In some embodiments, the inactivating mutation is present in a herpes simplex virus gene. In some embodiments, the inactivating mutation is a deletion of the coding sequence of the herpes simplex virus gene. In some embodiments, the herpes simplex virus gene is selected from infected cell protein (ICP)0 (one or both copies), ICP4 (one or both copies), ICP22, ICP27, ICP47, thymidine kinase (tk), long unique region (UL)41, and UL55. In some embodiments, the recombinant herpes simplex virus genome comprises an inactivating mutation in one or both copies of the ICP4 gene. In some embodiments, the recombinant herpes simplex virus genome comprises an inactivating mutation in the ICP22 gene. In some embodiments, the recombinant herpes simplex virus genome comprises an inactivating mutation in the UL41 gene. In some embodiments, the recombinant herpes simplex virus genome comprises an inactivating mutation in one or both copies of the ICP0 gene. In some embodiments, the recombinant herpes simplex virus genome comprises an inactivating mutation in the ICP27 gene. In some embodiments, the recombinant herpes simplex virus genome comprises an inactivating mutation in the UL55 gene. In some embodiments, the recombinant herpes simplex virus genome comprises an inactivating mutation in the junction region. In some embodiments, the recombinant herpes simplex virus genome comprises a deletion of the junction region.

[0013] In some embodiments that may be combined with any of the preceding embodiments, the recombinant herpes virus genome comprises one or more polynucleotides encoding a SPINK polypeptide in one or more viral loci. In some embodiments that may be combined with any of the preceding embodiments, the recombinant herpes simplex virus genome comprises one or more polynucleotides encoding a SPINK polypeptide in one or both of the ICP4 viral loci. In some embodiments that may be combined with any of the preceding embodiments, the recombinant herpes simplex virus genome comprises one or more polynucleotides encoding a SPINK polypeptide in the ICP22 viral locus. In some embodiments that may be combined with any of the preceding embodiments, the recombinant herpes simplex virus genome comprises one or more polynucleotides encoding a SPINK polypeptide in the UL41 viral locus.

[0014] In some embodiments that may be combined with any of the preceding embodiments, the SPINK polypeptide is a human SPINK polypeptide. In some embodiments that may be combined with any of the preceding embodiments, the SPINK polypeptide is a serine protease inhibitor Kazal type 5 (SPINK5) polypeptide. In some embodiments that may be combined with any of the preceding embodiments, the SPINK polypeptide is a human SPINK5 polypeptide. In some embodiments that may be combined with any of the preceding embodiments, the SPINK polypeptide comprises a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence selected from SEQ ID NOs: 7-25. In some embodiments that may be combined with any of the preceding embodiments, the SPINK polypeptide comprises a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence selected from SEQ ID NOs: 7-9. In some embodiments that may be combined with any of the preceding embodiments, the SPINK polypeptide comprises a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 7.In some embodiments that may be combined with any of the preceding embodiments, the SPINK polypeptide comprises a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 8. In some embodiments that may be combined with any of the preceding embodiments, the SPINK polypeptide comprises a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:9.

[0015] In some embodiments that may be combined with any of the preceding embodiments, the recombinant herpesvirus genome has reduced cytotoxicity when introduced into a target cell compared to the corresponding wild-type herpesvirus genome. In some embodiments, the target cell is a human cell. In some embodiments, the target cell is an epidermal and / or dermal cell. In some embodiments, the target cell is a keratinocyte or a fibroblast.

[0016] Another aspect of the present disclosure relates to a herpesvirus comprising any of the recombinant herpesvirus genomes described herein. In some embodiments, the herpesvirus is replication-competent. In some embodiments, the herpesvirus is replication-deficient. In some embodiments that may be combined with any of the preceding embodiments, the herpesvirus has reduced cytotoxicity compared to the corresponding wild-type herpesvirus. In some embodiments that may be combined with any of the preceding embodiments, the herpesvirus is selected from herpes simplex virus, varicella zoster virus, human cytomegalovirus, herpesvirus 6A, herpesvirus 6B, herpesvirus 7, Kaposi's sarcoma-associated herpesvirus, and any combination or derivative thereof. In some embodiments that may be combined with any of the preceding embodiments, the herpesvirus is a herpes simplex virus. In some embodiments, the herpes simplex virus is HSV-1, HSV-2, or any derivative thereof. In some embodiments, the herpes simplex virus is HSV-1.

[0017] Another aspect of the present disclosure relates to a pharmaceutical composition comprising any of the recombinant herpesvirus genomes described herein and / or any of the herpesviruses described herein and a pharma- ceutically acceptable excipient. In some embodiments, the pharmaceutical composition is suitable for topical, transdermal, subcutaneous, intradermal, oral, sublingual, buccal, rectal, intravaginal, inhalation, intravenous, intraarterial, intramuscular, intracardiac, intraosseous, intraperitoneal, transmucosal, intravitreal, subretinal, intraarticular, periarticular, local, epicutaneous administration, or any combination thereof. In some embodiments that may be combined with any of the preceding embodiments, the pharmaceutical composition is suitable for topical, transdermal, subcutaneous, intradermal, and / or transmucosal administration. In some embodiments that may be combined with any of the preceding embodiments, the pharmaceutical composition is suitable for topical, transdermal, and / or intradermal administration. In some embodiments that may be combined with any of the preceding embodiments, the pharmaceutical composition is suitable for topical and / or intradermal administration. In some embodiments that may be combined with any of the preceding embodiments, the pharmaceutical composition is suitable for topical administration. In some embodiments that may be combined with any of the preceding embodiments, the pharmaceutical composition comprises a methylcellulose gel (e.g., a carboxymethylcellulose gel, a hydroxypropylmethylcellulose gel, etc.). In some embodiments that may be combined with any of the preceding embodiments, the pharmaceutical composition comprises a phosphate buffer. In some embodiments that may be combined with any of the preceding embodiments, the pharmaceutical composition comprises glycerol. In some embodiments that may be combined with any of the preceding embodiments, the pharmaceutical composition comprises a lipid carrier. In some embodiments that may be combined with any of the preceding embodiments, the pharmaceutical composition comprises a nanoparticle carrier.

[0018] Other aspects of the present disclosure relate to the use of any of the recombinant nucleic acids, herpes viruses, and / or pharmaceutical compositions described herein as a medicament.

[0019] Other aspects of the present disclosure relate to the use of any of the recombinant nucleic acids, herpes viruses, and / or pharmaceutical compositions described herein in therapy.

[0020] Other aspects of the present disclosure relate to the use of any of the recombinant nucleic acids, herpes viruses, and / or pharmaceutical compositions described herein in the production or manufacture of a medicament for treating one or more signs or symptoms of a SPINK polypeptide deficiency (e.g., Netherton syndrome, atopic dermatitis, etc.).

[0021] Another aspect of the disclosure relates to a method of enhancing, increasing, enhancing, and / or replenishing SPINK polypeptide levels in one or more cells of a subject, comprising administering to the subject any of the herpesviruses described herein and / or any of the pharmaceutical compositions described herein. In some embodiments, the SPINK polypeptide is a SPINK5 polypeptide (e.g., a human SPINK5 polypeptide). In some embodiments that may be combined with any of the preceding embodiments, the subject is a human. In some embodiments that may be combined with any of the preceding embodiments, the subject's genome comprises a loss-of-function mutation in the SPINK5 gene. In some embodiments that may be combined with any of the preceding embodiments, the herpesvirus and / or pharmaceutical composition is administered to the subject topically, transdermally, subcutaneously, epicutaneously, intradermally, orally, sublingually, buccally, rectally, intravaginally, intravenously, intraarterially, intramuscularly, intraosseously, intracardially, intraperitoneally, transmucosally, intravitreally, subretinally, intra-articularly, peri-articularly, topically, or by inhalation. In some embodiments that may be combined with any of the preceding embodiments, the herpes virus and / or pharmaceutical composition is administered topically, transdermally, subcutaneously, intradermally, or transmucosally to the subject. In some embodiments that may be combined with any of the preceding embodiments, the herpes virus and / or pharmaceutical composition is administered topically to the subject. In some embodiments that may be combined with any of the preceding embodiments, the subject's skin is abraded prior to administration.

[0022] Another aspect of the present disclosure is a method of enhancing, increasing, enhancing, and / or replenishing anti-inflammatory and / or antimicrobial protection of mucosal epithelium in a subject in need thereof, comprising administering to the subject an effective amount of any of the herpesviruses described herein and / or any of the pharmaceutical compositions described herein. In some embodiments, the subject is a human. In some embodiments that may be combined with any of the preceding embodiments, the subject's genome comprises a loss-of-function mutation in the SPINK5 gene. In some embodiments that may be combined with any of the preceding embodiments, the herpesvirus and / or pharmaceutical composition is administered to the subject topically, transdermally, subcutaneously, epicutaneously, intradermally, orally, sublingually, buccally, rectally, intravaginally, intravenously, intraarterially, intramuscularly, intraosseously, intracardially, intraperitoneally, transmucosally, intravitreally, subretinally, intraarticularly, periarticularly, topically, or by inhalation. In some embodiments that may be combined with any of the preceding embodiments, the herpesvirus and / or pharmaceutical composition is administered to the subject topically, transdermally, subcutaneously, intradermally, or transmucosally. In some embodiments that may be combined with any of the preceding embodiments, the herpes virus and / or pharmaceutical composition is administered topically to the subject. In some embodiments that may be combined with any of the preceding embodiments, the skin of the subject is abraded prior to administration.

[0023] Another aspect of the present disclosure relates to a method of inhibiting desquamation in a subject in need thereof, comprising administering to the subject an effective amount of any of the herpesviruses described herein and / or any of the pharmaceutical compositions described herein. In some embodiments, the subject is a human. In some embodiments that may be combined with any of the preceding embodiments, the subject's genome comprises a loss-of-function mutation in the SPINK5 gene. In some embodiments that may be combined with any of the preceding embodiments, the herpesvirus and / or pharmaceutical composition is administered to the subject topically, transdermally, subcutaneously, epicutaneously, intradermally, orally, sublingually, buccally, rectally, intravaginally, intravenously, intraarterially, intramuscularly, intraosseously, intracardially, intraperitoneally, transmucosally, intravitreally, subretinally, intraarticularly, periarticularly, topically, or by inhalation. In some embodiments that may be combined with any of the preceding embodiments, the herpesvirus and / or pharmaceutical composition is administered to the subject topically, transdermally, subcutaneously, intradermally, or transmucosally. In some embodiments that may be combined with any of the preceding embodiments, the herpes virus and / or pharmaceutical composition is administered topically to the subject. In some embodiments that may be combined with any of the preceding embodiments, the skin of the subject is abraded prior to administration.

[0024] Another aspect of the present disclosure is a method of reducing or treating a skin barrier defect in a subject in need thereof, comprising administering to the subject an effective amount of any of the herpesviruses described herein and / or any of the pharmaceutical compositions described herein. In some embodiments, the skin barrier defect is transepithelial water loss (TEWL). In some embodiments that may be combined with any of the preceding embodiments, the subject is a human. In some embodiments that may be combined with any of the preceding embodiments, the subject's genome comprises a loss-of-function mutation in the SPINK5 gene. In some embodiments that may be combined with any of the preceding embodiments, the herpesvirus and / or pharmaceutical composition is administered to the subject topically, transdermally, subcutaneously, epicutaneously, intradermally, orally, sublingually, buccally, rectally, intravaginally, intravenously, intraarterially, intramuscularly, intraosseously, intracardially, intraperitoneally, transmucosally, intravitreally, subretinally, intraarticularly, periarticularly, topically, or by inhalation. In some embodiments that may be combined with any of the preceding embodiments, the herpes virus and / or pharmaceutical composition is administered topically, transdermally, subcutaneously, intradermally, or transmucosally to the subject. In some embodiments that may be combined with any of the preceding embodiments, the herpes virus and / or pharmaceutical composition is administered topically to the subject. In some embodiments that may be combined with any of the preceding embodiments, the subject's skin is abraded prior to administration.

[0025] Another aspect of the present disclosure relates to a method of providing prophylactic, palliative, or therapeutic relief of one or more signs or symptoms of Netherton syndrome (NS) in a subject in need thereof, comprising administering to the subject an effective amount of any of the herpesviruses described herein and / or any of the pharmaceutical compositions described herein. In some embodiments, the one or more signs or symptoms of NS are selected from defective keratinization, defective skin barrier, chronic skin inflammation, generalized pruritus, severe dehydration, poor growth, intussusceptible trichorrhexis and / or trichorrhexis nodularis, fluid leakage from the skin, development of annular lesions in the skin, eczema, increased susceptibility to infections, recurrent skin infections, increased susceptibility to allergies, development of scaly / red skin, development of flexural linear ichthyosis and / or ichthyosiform erythroderma, altered immunoglobulin levels, immature natural killer cells with reduced lytic function, difficulty regulating body temperature, and any combination thereof. In some embodiments that may be combined with any of the preceding embodiments, the one or more signs or symptoms of NS are selected from defective keratinization, defective skin barrier, recurrent skin infections, congenital ichthyosiform erythroderma, flexural linear ichthyosis, intussusceptible trichorrhexis, chronic skin inflammation, and any combination thereof. In some embodiments that may be combined with any of the preceding embodiments, the subject is a human. In some embodiments that may be combined with any of the preceding embodiments, the subject's genome comprises a loss-of-function mutation in the SPINK5 gene. In some embodiments that may be combined with any of the preceding embodiments, the herpesvirus and / or pharmaceutical composition is administered to the subject topically, transdermally, subcutaneously, epicutaneously, intradermally, orally, sublingually, buccally, rectally, intravaginally, intravenously, intraarterially, intramuscularly, intraosseously, intracardially, intraperitoneally, transmucosally, intravitreally, subretinally, intraarticularly, periarticularly, topically, or by inhalation. In some embodiments that may be combined with any of the preceding embodiments, the herpes virus and / or pharmaceutical composition is administered to the subject topically, transdermally, subcutaneously, intradermally, or transmucosally. In some embodiments that may be combined with any of the preceding embodiments, the herpes virus and / or pharmaceutical composition is administered to the subject topically, transdermally, or intradermally.In some embodiments that may be combined with any of the preceding embodiments, the herpes virus and / or pharmaceutical composition is administered topically or intradermally to the subject. In some embodiments that may be combined with any of the preceding embodiments, the herpes virus and / or pharmaceutical composition is administered intradermally to the subject. In some embodiments that may be combined with any of the preceding embodiments, the herpes virus and / or pharmaceutical composition is administered topically to the subject. In some embodiments that may be combined with any of the preceding embodiments, the skin of the subject is abraded prior to administration.

[0026] Another aspect of the present disclosure relates to a method of providing preventative, palliative, or therapeutic relief of one or more signs or symptoms of atopic dermatitis in a subject in need thereof, comprising administering to the subject an effective amount of any of the herpes viruses described herein and / or any of the pharmaceutical compositions described herein. In some embodiments, the one or more signs or symptoms of atopic dermatitis are selected from the group consisting of itchy skin, dry skin, red to brownish-gray spots on the skin, small raised bumps on the skin, thickened skin, cracked skin, scaly skin, swollen skin, weeping sores, skin infections, eyelid dermatitis, cataracts, and any combination thereof. In some embodiments that may be combined with any of the preceding embodiments, the subject is a human. In some embodiments that may be combined with any of the preceding embodiments, the subject's genome comprises a loss-of-function mutation in the SPINK5 gene. In some embodiments that may be combined with any of the preceding embodiments, the herpes virus and / or pharmaceutical composition is administered to the subject topically, transdermally, subcutaneously, epicutaneously, intradermally, orally, sublingually, buccally, rectally, intravaginally, intravenously, intraarterially, intramuscularly, intraosseously, intracardially, intraperitoneally, transmucosally, intravitreally, subretinally, intraarticularly, periarticularly, locally, or by inhalation. In some embodiments that may be combined with any of the preceding embodiments, the herpes virus and / or pharmaceutical composition is administered to the subject topically, transdermally, subcutaneously, intradermally, or transmucosally. In some embodiments that may be combined with any of the preceding embodiments, the herpes virus and / or pharmaceutical composition is administered to the subject topically, transdermally, or intradermally. In some embodiments that may be combined with any of the preceding embodiments, the herpes virus and / or pharmaceutical composition is administered to the subject topically or intradermally. In some embodiments that may be combined with any of the preceding embodiments, the herpes virus and / or pharmaceutical composition is administered to the subject intradermally. In some embodiments that may be combined with any of the preceding embodiments, the herpes virus and / or pharmaceutical composition is administered locally to the subject.In some embodiments that may be combined with any of the preceding embodiments, the subject's skin is abraded prior to administration.

[0027] Other aspects of the disclosure relate to articles of manufacture or kits that include any of the recombinant nucleic acids, viruses, agents, and / or pharmaceutical compositions or formulations described herein and instructions for their administration. [The present invention 1001] A recombinant herpesvirus genome comprising one or more polynucleotides encoding a serine protease inhibitor Kazal type (SPINK) polypeptide. [The present invention 1002] A recombinant herpesvirus genome of the present invention comprising two or more polynucleotides encoding a SPINK polypeptide. [The present invention 1003] A recombinant herpesvirus genome of the present invention 1001 or 1002, which is replicable. [The present invention 1004] A recombinant herpesvirus genome according to claim 1001 or 1002, which is replication-deficient. [The present invention 1005] The recombinant herpes virus genome of any of the present inventions 1001 to 1004, which is selected from the group consisting of a recombinant herpes simplex virus genome, a recombinant varicella-zoster virus genome, a recombinant human cytomegalovirus genome, a recombinant herpes virus 6A genome, a recombinant herpes virus 6B genome, a recombinant herpes virus 7 genome, a recombinant Kaposi's sarcoma-associated herpes virus genome, and any derivatives thereof. [The present invention 1006] The recombinant herpes virus genome of any one of 1001 to 1005 of the present invention, which is a recombinant herpes simplex virus genome. [The present invention 1007] The recombinant herpes virus genome of the present invention 1006, wherein said recombinant herpes simplex virus genome is a recombinant herpes simplex virus type 1 (HSV-1) genome, a recombinant herpes simplex virus type 2 (HSV-2) genome, or any derivative thereof. [The present invention 1008] The recombinant herpesvirus genome of the present invention 1006 or 1007, wherein the recombinant herpes simplex virus genome is a recombinant HSV-1 genome. [The present invention 1009] The recombinant herpes virus genome of any of claims 1006 to 1008, wherein the recombinant herpes simplex virus genome comprises an inactivating mutation. [The present invention 1010] The recombinant herpesvirus genome of the present invention, wherein said inactivating mutation is present in a herpes simplex virus gene. [The present invention 1011] The recombinant herpesvirus genome of the present invention, wherein the inactivating mutation is a deletion in the coding sequence of the herpes simplex virus gene. [The present invention 1012] The recombinant herpesvirus genome of the present invention 1010 or 1011, wherein the herpes simplex virus gene is selected from the group consisting of infected cell protein (ICP) 0, ICP4, ICP22, ICP27, ICP47, thymidine kinase (tk), long unique region (UL) 41, and UL55. [The present invention 1013] 1012. The recombinant herpes simplex virus genome of the present invention, wherein said recombinant herpes simplex virus genome comprises an inactivating mutation in one or both copies of the ICP4 gene. [The present invention 1014] The recombinant herpesvirus genome of the present invention 1012 or 1013, wherein the recombinant herpes simplex virus genome comprises an inactivating mutation in the ICP22 gene. [The present invention 1015] The recombinant herpes virus genome of any of claims 1012 to 1014, wherein the recombinant herpes simplex virus genome contains an inactivating mutation in a UL41 gene. [The present invention 1016] The recombinant herpes virus genome of any of claims 1012 to 1015, wherein the recombinant herpes simplex virus genome contains an inactivating mutation in one or both copies of the ICP0 gene. [The present invention 1017] The recombinant herpes virus genome of any one of claims 1012 to 1016, wherein the recombinant herpes simplex virus genome contains an inactivating mutation in an ICP27 gene. [The present invention 1018] The recombinant herpes simplex virus genome of any of claims 1006 to 1017, wherein the recombinant herpes simplex virus genome comprises the one or more polynucleotides encoding a SPINK polypeptide in one or both of the ICP4 viral loci. [The present invention 1019] The recombinant herpesvirus genome of any of claims 1001 to 1018, wherein the SPINK polypeptide is a human SPINK polypeptide. [The present invention 1020] The recombinant herpesvirus genome of any of claims 1001 to 1018, wherein the SPINK polypeptide is a serine protease inhibitor Kazal type 5 (SPINK5) polypeptide. [The present invention 1021] The recombinant herpesvirus genome of any of claims 1001 to 1020, wherein the SPINK polypeptide is a human SPINK5 polypeptide. [The present invention 1022] The recombinant herpesvirus genome of any of the present inventions 1001 to 1021, wherein the SPINK polypeptide comprises a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 7 to 25. [The present invention 1023] The recombinant herpesvirus genome of any of the present inventions 1001 to 1022, wherein the SPINK polypeptide comprises a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 7 to 9. [The present invention 1024] A recombinant herpesvirus genome according to any one of claims 1001 to 1023, wherein the SPINK polypeptide comprises a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:9. [The present invention 1025] The recombinant herpesvirus genome of any of claims 1001 to 1024, which has reduced cytotoxicity when introduced into a target cell as compared with the corresponding wild-type herpesvirus genome. [The present invention 1026] The recombinant herpesvirus genome of the present invention, wherein the target cell is a human cell. [The present invention 1027] The recombinant herpesvirus genome of claim 1025 or 1026, wherein the target cell is an epidermal cell and / or a dermal cell. [The present invention 1028] The recombinant herpesvirus genome of any one of claims 1025 to 1027, wherein the target cell is a keratinocyte or a fibroblast. [The present invention 1029] A herpesvirus comprising a recombinant herpesvirus genome of any one of the present inventions 1001 to 1028. [The present invention 1030] 1029 Herpesvirus of the present invention, which is replication-competent. [The present invention 1031] 1029 A herpesvirus of the invention which is replication-deficient. [The present invention 1032] The herpesvirus of any of the present inventions 1029 to 1031, which has reduced cytotoxicity as compared with the corresponding wild-type herpesvirus. [The present invention 1033] Any of the herpesviruses of the present invention 1029 to 1032, which is selected from the group consisting of herpes simplex virus, varicella-zoster virus, human cytomegalovirus, herpesvirus 6A, herpesvirus 6B, herpesvirus 7, Kaposi's sarcoma-associated herpesvirus, and any derivatives thereof. [The present invention 1034] The herpesvirus of any one of claims 1029 to 1033, which is a herpes simplex virus. [The present invention 1035] The herpesvirus of the present invention 1033 or 1034, wherein the herpes simplex virus is HSV-1, HSV-2, or any derivative thereof. [The present invention 1036] The herpesvirus of any one of claims 1033 to 1035, wherein the herpes simplex virus is HSV-1. [The present invention 1037] A pharmaceutical composition comprising a recombinant herpesvirus genome of any one of claims 1001 to 1028 or a herpesvirus of any one of claims 1029 to 1036, and a pharma- ceutically acceptable excipient. [The present invention 1038] The pharmaceutical composition of the present invention 1037 is suitable for topical, transdermal, subcutaneous, intradermal, oral, sublingual, buccal, rectal, intravaginal, inhalation, intravenous, intraarterial, intramuscular, intracardiac, intraosseous, intraperitoneal, transmucosal, intravitreal, subretinal, intraarticular, periarticular, local, or epicutaneous administration. [The present invention 1039] A pharmaceutical composition of invention 1037 or invention 1038, which is suitable for topical, transdermal, subcutaneous, intradermal, or transmucosal administration. [The present invention 1040] 1037-1039. The pharmaceutical composition of any of claims 1037-1039, which is suitable for topical, transdermal, or intradermal administration. [The present invention 1041] The pharmaceutical composition of any one of claims 1037 to 1040, which is suitable for topical administration. [The present invention 1042] Any of the pharmaceutical compositions of claims 1037 to 1041, comprising a methylcellulose gel. [The present invention 1043] The pharmaceutical composition according to any one of claims 1037 to 1042, comprising a phosphate buffer. [The present invention 1044] Any of the pharmaceutical compositions according to claims 1037 to 1043, comprising glycerol. [The present invention 1045] The pharmaceutical composition of any one of claims 1037 to 1044, comprising a lipid carrier. [The present invention 1046] Any of the pharmaceutical compositions according to claims 1037 to 1045, comprising a nanoparticle carrier. [The present invention 1047] A herpesvirus according to any one of 1029 to 1036 or a pharmaceutical composition according to any one of 1037 to 1046 for use as a medicament. [The present invention 1048] A herpesvirus according to any of claims 1029 to 1036 or a pharmaceutical composition according to any of claims 1037 to 1046 for use in therapy. [The present invention 1049] Use of any of the herpesviruses according to claims 1029 to 1036 or any of the pharmaceutical compositions according to claims 1037 to 1046 in the manufacture of a medicament for treating one or more signs or symptoms of Netherton syndrome and / or atopic dermatitis. [The present invention 1050] A method for enhancing, increasing, enhancing and / or replenishing SPINK polypeptide levels in one or more cells of a subject, the method comprising administering to the subject an effective amount of any of the herpesviruses of the present inventions 1029 to 1036 or any of the pharmaceutical compositions of the present inventions 1037 to 1046. [The present invention 1051] The method of claim 1050, wherein said SPINK polypeptide is a SPINK5 polypeptide. [The present invention 1052] A method for enhancing, increasing, enhancing and / or replenishing anti-inflammatory and / or antimicrobial protection of mucosal epithelium in a subject in need thereof, comprising administering to the subject an effective amount of any of the herpesviruses of the present inventions 1029 to 1036 or any of the pharmaceutical compositions of the present inventions 1037 to 1046. [The present invention 1053] A method for inhibiting desquamation in a subject in need thereof, comprising administering to the subject an effective amount of any of the herpesviruses of the present inventions 1029 to 1036 or any of the pharmaceutical compositions of the present inventions 1037 to 1046. [The present invention 1054] A method for reducing or treating a skin barrier defect in a subject in need thereof, comprising administering to the subject an effective amount of any of the herpesviruses of the present inventions 1029 to 1036 or any of the pharmaceutical compositions of the present inventions 1037 to 1046. [The present invention 1055] The method of claim 1054, wherein the skin barrier defect is transepidermal water loss. [The present invention 1056] A method for providing preventive, palliative, or therapeutic alleviation of one or more signs or symptoms of Netherton Syndrome (NS) in a subject in need thereof, comprising administering to the subject an effective amount of any of the herpesviruses of the present inventions 1029 to 1036 or any of the pharmaceutical compositions of the present inventions 1037 to 1046. [The present invention 1057] The method of claim 1056, wherein said one or more signs or symptoms of NS are selected from the group consisting of defective keratinization, skin barrier defects, recurrent skin infections, congenital ichthyosiform erythroderma, flexural linear ichthyosis, intussusceptum trichorrhexis, chronic skin inflammation, and any combination thereof. [The present invention 1058] A method for providing preventive, palliative, or therapeutic relief of one or more signs or symptoms of atopic dermatitis in a subject in need thereof, comprising administering to the subject an effective amount of a herpesvirus of any of claims 1029 to 1036 or a pharmaceutical composition of any of claims 1037 to 1046. [The present invention 1059] The method of claim 1058, wherein said one or more signs or symptoms of atopic dermatitis are selected from the group consisting of itchy skin, dry skin, red to brownish-gray spots on the skin, small raised bumps on the skin, thickened skin, cracked skin, scaly skin, swollen skin, weeping sores, skin infections, eyelid dermatitis, cataracts, and any combination thereof. [The present invention 1060] The method of any one of claims 1050 to 1059, wherein the subject is a human. [The present invention 1061] The method according to any one of claims 1050 to 1060, wherein the subject's genome comprises a loss-of-function mutation in the SPINK5 gene. [The present invention 1062] The method of any of claims 1050 to 1061, wherein said herpes virus or said pharmaceutical composition is administered to said subject topically, transdermally, subcutaneously, epicutaneously, intradermally, orally, sublingually, buccally, rectally, intravaginally, intravenously, intraarterially, intramuscularly, intraosseously, intracardially, intraperitoneally, transmucosally, intravitreally, subretinally, intraarticularly, periarticularly, locally, or by inhalation. [The present invention 1063] The method of any of claims 1050 to 1062, wherein said herpesvirus or said pharmaceutical composition is administered to said subject topically, transdermally, subcutaneously, intradermally, or transmucosally. [The present invention 1064] The method of any of claims 1050 to 1063, wherein the herpes virus or the pharmaceutical composition is administered to the subject topically, transdermally, or intradermally. [The present invention 1065] The method of any one of claims 1050 to 1064, wherein the herpes virus or the pharmaceutical composition is administered locally to the subject. [The present invention 1066] The method of any one of claims 1050 to 1065, wherein the skin of the subject is abraded prior to administration. [Brief description of the drawings]

[0028] [Figure 1-1]1A-I show schematic diagrams of wild-type and modified herpes simplex virus genomes. FIG. 1A shows a wild-type herpes simplex virus genome. FIG. 1B shows a modified herpes simplex virus genome including a deletion of the coding sequence of ICP4 (both copies), where an expression cassette including a nucleic acid encoding a human SPINK5 polypeptide has been integrated into each of the ICP4 loci. FIG. 1C shows a modified herpes simplex virus genome including a deletion of the coding sequence of ICP4 (both copies) and ICP22, where an expression cassette including a nucleic acid encoding a human SPINK5 polypeptide has been integrated into each of the ICP4 loci. FIG. 1D shows a modified herpes simplex virus genome including a deletion of the coding sequence of ICP4 (both copies) and ICP22, where an expression cassette including a nucleic acid encoding a human SPINK5 polypeptide has been integrated into the ICP22 locus. Figure 1E shows a modified herpes simplex virus genome including deletions of the coding sequences for ICP4 (both copies) and UL41, where an expression cassette including a nucleic acid encoding a human SPINK5 polypeptide has been integrated into each of the ICP4 loci. Figure 1F shows a modified herpes simplex virus genome including deletions of the coding sequences for ICP4 (both copies) and UL41, where an expression cassette including a nucleic acid encoding a human SPINK5 polypeptide has been integrated into the UL41 locus. Figure 1G shows a modified herpes simplex virus genome including deletions of the coding sequences for ICP4 (both copies), ICP22, and UL41, where an expression cassette including a nucleic acid encoding a human SPINK5 polypeptide has been integrated into each of the ICP4 loci. Figure 1H shows a modified herpes simplex virus genome including deletions of the coding sequences for ICP4 (both copies), ICP22, and UL41, where an expression cassette including a nucleic acid encoding a human SPINK5 polypeptide has been integrated into the UL41 locus. FIG. 1I shows a modified herpes simplex virus genome containing deletions of the coding sequences for ICP4 (both copies), ICP22, and UL41, in which an expression cassette containing a nucleic acid encoding a human SPINK5 polypeptide has been integrated into the ICP22 locus. [Figure 1-2]See description of Figure 1-1. [Figure 1-3] See description of Figure 1-1. [Figure 1-4] See description of Figure 1-1. [Figure 1-5] See description of Figure 1-1. [Diagram 2] Figures 2A-D show human SPINK5 nucleic acid and protein analysis in immortalized normal keratinocytes infected with HSV-S5. Figure 2A shows the DNA levels of codon-optimized human SPINK5 present in immortalized normal keratinocytes 48 hours after infection with HSV-S5 at the indicated MOI, as determined by qPCR analysis. Figure 2B shows the transcript levels of codon-optimized human SPINK5 present in immortalized normal keratinocytes 48 hours after infection with HSV-S5 at the indicated MOI, as determined by qRT-PCR analysis. Figure 2C shows Western blot analysis of human SPINK5 protein expression in immortalized normal keratinocytes 48 hours after infection with HSV-S5 at the indicated MOI. GAPDH was used as a loading control. Figure 2D shows representative immunofluorescence images of human SPINK5 protein expression in immortalized normal keratinocytes 48 hours after infection with HSV-S5 at the indicated MOI. DAPI staining was used to visualize nuclei. For all experiments, non-infected (mock) cells were used as negative controls. [Diagram 3] Figure 1 shows the concentration of secreted human SPINK5 in the supernatant of immortalized normal keratinocytes cultured 48 hours after infection with HSV-S5 at the indicated MOI, as assessed by ELISA. Cell supernatants collected from uninfected (mock) immortalized normal keratinocytes were used as negative controls. [Figure 4]Figure 1 shows the percent inhibition in the proteolytic activity of recombinant human kallikrein 5 (KLK5) following incubation with cell culture supernatants collected from immortalized normal keratinocytes infected with HSV-S5 at the indicated MOI, as assessed by a fluorimetric assay using a synthetic non-natural peptide substrate of human KLK5. Cell supernatants collected from uninfected (mock) immortalized normal keratinocytes were used as negative controls. Data are presented as mean ± SEM. [Figure 5A] Figures 5A-F show nucleic acid and protein analysis and histology of skin biopsies taken from control or HSV-S5-treated BALB / c mice. Figure 5A shows the levels of human SPINK5 DNA present in the skin of immunocompetent animals taken 48 hours after topical application of HSV-S5 or vehicle control, as assessed by qPCR analysis. [Figure 5B] Figures 5A-F show nucleic acid and protein analysis and histology of skin biopsies taken from control or HSV-S5-treated BALB / c mice, and Figure 5B shows transcript levels of human SPINK5 present in the skin of immunocompetent animals taken 48 hours after topical application of HSV-S5 or vehicle control, as assessed by qRT-PCR analysis. [Figure 5C] Figures 5A-F show nucleic acid and protein analysis and histology of skin biopsies taken from control or HSV-S5-treated BALB / c mice, and Figure 5C shows human SPINK5 DNA levels present in the skin of immunocompetent animals taken 48 hours after intradermal injection of HSV-S5 or vehicle control, as assessed by qPCR analysis. [Figure 5D]Figures 5A-F show nucleic acid and protein analysis and histology of skin biopsies taken from control or HSV-S5-treated BALB / c mice. Figure 5D shows transcript levels of human SPINK5 present in the skin of immunocompetent animals taken 48 hours after intradermal injection of HSV-S5 or vehicle control, as assessed by qRT-PCR analysis. For qPCR and qRT-PCR analyses, vehicle control data are presented as the mean ± standard error of two tissue samples (two replicates / tissue sample) and HSV-S5 data are presented as the mean ± standard error of six tissue samples (two replicates / tissue sample). [Figure 5E] Figures 5A-F show nucleic acid and protein analysis, as well as histology, of skin biopsies taken from control or HSV-S5-treated BALB / c mice. Figure 5E shows representative immunofluorescence images of human SPINK5 and mouse filaggrin protein expression in skin biopsies taken from BALB / c mice 48 hours after topical application of HSV-S5 or vehicle control. DAPI staining was used to visualize nuclei. [Figure 5F] Figures 5A-F show nucleic acid and protein analysis and histology of skin biopsies taken from control or HSV-S5-treated BALB / c mice, and Figure 5F shows representative histology of mouse skin biopsies taken from BALB / c mice 48 hours after topical application of HSV-S5 or vehicle control. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0029] Detailed Description In some embodiments, the disclosure relates to recombinant nucleic acids (e.g., recombinant herpes virus genomes) comprising one or more polynucleotides encoding a serine protease inhibitor Kazal type (SPINK) polypeptide (e.g., human SPINK5 polypeptide), and / or the use of those recombinant nucleic acids in viruses (e.g., herpes viruses), compositions, formulations, medicaments, and / or methods to supplement and / or treat a SPINK gene deficiency (e.g., in a subject whose genome naturally harbors a loss-of-function mutation and / or pathogenic variant in the SPINK gene) and / or provide medical intervention to a subject in need thereof (e.g., providing preventative, palliative, and / or therapeutic relief of one or more diseases or disorders resulting from a SPINK gene deficiency (e.g., Netherton syndrome, atopic dermatitis, etc.)). Without wishing to be bound by theory, it is believed that the recombinant nucleic acids, viruses, compositions, formulations, agents, and / or methods described herein will aid in the treatment of existing skin abnormalities in individuals suffering from Netherton syndrome and / or atopic dermatitis, as well as preventing or delaying the re-formation of skin abnormalities in treated subjects.

[0030] The following description sets forth example methods, parameters, etc. However, it should be recognized that such description is not intended to limit the scope of the present disclosure, but is instead provided as a description of example embodiments.

[0031] I. General Techniques The techniques and procedures described or referenced herein are generally well understood by those of skill in the art and may be implemented using conventional methodology, e.g., those described in Sambrook et al., 2001. Molecular Cloning: A Laboratory Manual 3d edition Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, Current Protocols in Molecular Biology (FMA Usubel, et al. eds., (2003)), the series Methods in Enzymology (Academic Press, Inc.): PCR 2: A Practical Approach (MJ MacPherson, BD Hames and GR Taylor eds. (1995)), Harlow and Lane, eds. (1988), Oligonucleotide Synthesis (MJ Gait, ed., 1984), Methods in Molecular Biology, Humana Press, Cell Biology: A Laboratory Notebook (JE Elis, ed., 1998) Academic Press, Animal Cell Culture (RIFreshney), ed., 1987), Introduction to Cell and Tissue Culture (JP Mather and PE Roberts, 1998) Plenum Press, Cell and Tissue Culture: Laboratory Procedures (A. Doyle, JBGriffiths, and DG Newell, eds., 1993-8) J. Wiley and Sons, Gene Transfer Vectors for Mammalian Cells (JMMiller and MP Calos, eds., 1987), PCR: The Polymerase Chain Reaction (Mullis et al., eds., 1994) and is commonly used using widely available methodologies described in Short Protocols in Molecular Biology (Wiley and Sons, 1999).

[0032] II. Definition Before describing the present disclosure in detail, it is to be understood that this disclosure is not limited to particular compositions or biological systems, which can, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[0033] As used herein, the singular forms "a," "an," and "the" include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to "a molecule" optionally includes a combination of two or more such molecules, and so forth.

[0034] As used herein, the term "and / or" may include any and all combinations of one or more of the associated listed items. For example, the term "a and / or b" may refer to "a only," "b only," "a or b," or "a and b," while the term "a, b, and / or c" may refer to "a only," "b only," "c only," "a or b," "a or c," "b or c," "a, b, or c," "a and b," "a and c," "b and c," or "a, b, and c," etc.

[0035] As used herein, the term "about" refers to the normal range of error for the respective value, which is readily known to one of ordinary skill in the art. Reference herein to "about" a value or parameter includes (and describes) embodiments that are directed to the value or parameter itself.

[0036] It is understood that aspects and embodiments of the present disclosure include "comprising," "consisting," and "consisting essentially of" aspects and embodiments.

[0037] As used herein, the terms "polynucleotide", "nucleic acid sequence", "nucleic acid" and variations thereof are intended to be inclusive of polydeoxyribonucleotides (containing 2-deoxy-D-ribose), polyribonucleotides (containing D-ribose), any other type of polynucleotide that is an N-glycoside of a purine or pyrimidine base, and other polymers containing a non-nucleotide backbone, provided that the polymer contains nucleobases in a configuration that allows for base pairing and base stacking, as found in DNA and RNA. Thus, these terms include known types of nucleic acid sequence modifications, such as substitution of one or more analogs of naturally occurring nucleotides, and internucleotide modifications.

[0038] As used herein, a nucleic acid is "operably linked" or "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the coding sequence, and a ribosome binding site is operably linked to a coding sequence if it is positioned to promote translation. In general, "operably linked" or "operably linked" means that the DNA sequences being linked are contiguous.

[0039] As used herein, the term "vector" refers to a separate element used to introduce a heterologous nucleic acid into a cell for either expression or replication thereof. Expression vectors include vectors capable of expressing a nucleic acid operably linked to a regulatory sequence, such as a promoter region, capable of effecting expression of such a nucleic acid. Thus, an expression vector can refer to a DNA or RNA construct, such as a plasmid, phage, recombinant virus, or other vector, that, when introduced into a suitable host cell, results in expression of a nucleic acid. Suitable expression vectors are well known to those skilled in the art and include those that are replicable in eukaryotic cells and those that remain episomal or integrate into the host cell genome.

[0040] As used herein, "open reading frame" or "ORF" refers to a contiguous stretch of either DNA or RNA that encodes a protein or polypeptide. Typically, the nucleic acid includes a translation initiation signal or start codon, such as ATG or AUG, and a stop codon.

[0041] As used herein, "untranslated region" or "UTR" refers to untranslated nucleic acid at the 5' and / or 3' end of an open reading frame. Inclusion of one or more UTRs in a polynucleotide can affect post-transcriptional regulation, mRNA stability, and / or translation of the polynucleotide.

[0042] As used herein, the term "transgene" refers to a polynucleotide that, after being introduced into a cell, can be transcribed into RNA and translated and / or expressed under appropriate conditions, which in some embodiments confers a desired property to the cell into which it is introduced, or otherwise results in a desired therapeutic or diagnostic outcome.

[0043] As used herein, the terms "polypeptide," "protein," and "peptide" are used interchangeably and may refer to a polymer of two or more amino acids.

[0044] As used herein, "subject," "host," or "individual" refers to any animal classified as a mammal, including humans, domestic and farm animals, as well as zoo, sports, or pet animals, such as dogs, horses, cats, cows, and animals used in research, such as mice, rats, hamsters, rabbits, and non-human primates. In some embodiments, the mammal is a human.

[0045] As used herein, the term "pharmaceutical formulation" or "pharmaceutical composition" refers to a preparation that is in a form that allows the biological activity of the active ingredient(s) to be effective and does not contain additional ingredients that are unacceptably toxic to the subject to which the composition or formulation is administered. A "pharmaceutical acceptable" excipient (e.g., vehicle, additive) is one that may be reasonably administered to a target mammal to provide an effective dosage of the active ingredient(s) employed.

[0046] As used herein, an "effective amount" is at least the minimum amount necessary to affect a measurable improvement or prevention of one or more symptoms of a particular disorder. An "effective amount" may vary depending on factors such as the disease state, age, sex, and weight of the patient. An effective amount is also an amount in which any toxic or adverse effects of the treatment are outweighed by the therapeutically beneficial effects. In the case of prophylactic use, beneficial or desired results include results such as eliminating or reducing the risk of, reducing the severity of, or delaying the onset of, the disease, its complications, and intermediate pathological phenotypes presented during the development of the disease. In the case of therapeutic use, beneficial or desired results include clinical results such as reducing one or more symptoms caused by the disease, improving the quality of life of those suffering from the disease, reducing the dose of other drugs used to treat the symptoms of the disease, delaying the progression of the disease, and / or increasing survival time. An effective amount may be administered in one or more administrations. For purposes of this disclosure, an effective amount of a recombinant nucleic acid, virus, and / or pharmaceutical composition is an amount sufficient to achieve prophylactic or therapeutic treatment, either directly or indirectly. As will be understood in a clinical context, an effective amount of a recombinant nucleic acid, virus, and / or pharmaceutical composition may or may not be achieved in conjunction with another drug, compound, or pharmaceutical composition. Thus, an "effective amount" may be considered in the context of administering one or more therapeutic agents, and a single agent may be considered to be given in an effective amount if a desired result can or is achieved in conjunction with one or more other agents.

[0047] As used herein, "treatment" refers to a clinical intervention designed to change the natural course of the treated individual or cell in the course of clinical pathology. The desired effects of treatment include slowing the rate of progression of the disease / disorder / deficiency, improving or alleviating the disease / disorder / deficiency condition, and remission or improved prognosis. For example, an individual is successfully "treated" if one or more symptoms associated with Netherton syndrome and / or atopic dermatitis are reduced or eliminated.

[0048] As used herein, the term "delaying progression" of a disease / disorder / deficiency refers to prolonging, preventing, delaying, retarding, stabilizing, and / or postponing the onset of the disease / disorder / deficiency. This delay can be of varying length or time, depending on the disease / disorder / deficiency and / or the medical history of the individual being treated. As will be apparent to one of skill in the art, a sufficient or significant delay can actually encompass prevention, in that the individual does not develop the disease.

[0049] III. Recombinant Nucleic Acids Certain aspects of the present disclosure relate to recombinant nucleic acids (e.g., isolated recombinant nucleic acids) that include one or more (e.g., one or more, two or more, three or more, four or more, five or more, ten or more, etc.) polynucleotides encoding a serine protease inhibitor Kazal type (SPINK) polypeptide. In some embodiments, the recombinant nucleic acid includes one polynucleotide encoding a SPINK polypeptide. In some embodiments, the recombinant nucleic acid includes two polynucleotides encoding a SPINK polypeptide. In some embodiments, the SPINK polypeptide is a human SPINK polypeptide. In some embodiments, the SPINK polypeptide is a serine protease inhibitor Kazal type 5 (SPINK5) polypeptide. In some embodiments, the SPINK5 polypeptide is a human SPINK5 polypeptide.

[0050] In some embodiments, the recombinant nucleic acid is a vector. In some embodiments, the recombinant nucleic acid is a viral vector. In some embodiments, the recombinant nucleic acid is a herpes virus vector. In some embodiments, the recombinant nucleic acid is a herpes simplex virus amplicon. In some embodiments, the recombinant nucleic acid is a recombinant herpes virus genome. In some embodiments, the recombinant nucleic acid is a recombinant herpes simplex virus genome. In some embodiments, the recombinant nucleic acid is a recombinant herpes simplex virus type 1 (HSV-1) genome.

[0051] Polynucleotides encoding SPINK polypeptides In some embodiments, the disclosure relates to recombinant nucleic acids comprising one or more polynucleotides comprising a coding sequence of a SPINK gene or a portion thereof (e.g., a coding sequence corresponding to one or more Kazal-type domains resulting from proteolytic cleavage of a full-length SPINK polypeptide (e.g., by furin cleavage)). For example, the SPINK1 gene (e.g., the human SPINK1 gene (see, e.g., NCBI Gene ID: 6690)), the SPINK2 gene (e.g., the human SPINK2 gene (see, e.g., NCBI Gene ID: 6691)), the SPINK4 gene (e.g., the human SPINK4 gene (see, e.g., NCBI Gene ID: 27920)), the SPINK5 gene (e.g., the human SPINK5 gene (see, e.g., NCBI Gene ID: 11005)), the SPINK6 gene (e.g., the human SPINK6 gene (see, e.g., NCBI Gene ID: 404203)), the SPINK7 gene (e.g., the human SPINK7 gene (see, e.g., NCBI Gene ID: 11005)), the SPINK8 gene (e.g., the human SPINK8 gene (see, e.g., NCBI Gene ID: 11005)), the SPINK9 gene (e.g., the human SPINK9 gene (see, e.g., NCBI Gene ID: 11005)), the SPINK1 gene (e.g., the human SPINK1 gene (see, e.g., NCBI Gene ID: 11005)), the SPINK2 gene (e.g., the human SPINK2 gene (see, e.g., NCBI Gene ID: 11005)), the SPINK3 gene (e.g. Any suitable SPINK known in the art (including any isoforms thereof) may be encoded by the polynucleotides of the present disclosure, including the SPINK8 gene (e.g., the human SPINK8 gene (see, e.g., NCBI Gene ID: 84651)), the SPINK9 gene (e.g., the human SPINK9 gene (see, e.g., NCBI Gene ID: 643394)), the SPINK13 gene (e.g., the human SPINK13 gene (see, e.g., NCBI Gene ID: 153218)), the SPINK14 gene (e.g., the human SPINK14 gene (see, e.g., NCBI Gene ID: 408187)), and the like.In some embodiments, the polynucleotides of the present disclosure comprise a sequence having at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence of any SPINK gene (and / or its coding sequence) described herein or known in the art. Methods for identifying SPINK gene homologs / orthologs from additional species are known to those of skill in the art, including, for example, the use of nucleic acid sequence alignment programs such as the BLAST® blastn suite. In some embodiments, one or more polynucleotides of the present disclosure comprise a coding sequence of a human SPINK gene.

[0052] In some embodiments, polynucleotides of the disclosure include codon-optimized variants of the SPINK gene described herein or known in the art. In some embodiments, the use of a codon-optimized variant of the SPINK gene increases the stability and / or yield of heterologous expression (RNA and / or protein) of the encoded SPINK polypeptide in a target cell (e.g., a target human cell such as a human keratinocyte or fibroblast) compared to the stability and / or yield of heterologous expression of the corresponding non-codon-optimized wild-type sequence. Any suitable method known in the art for codon-optimizing a sequence for expression in one or more target cells (e.g., one or more human cells) may be used, including, for example, the method described by Fath et al. (PLoS One. 2011 Mar 3;6(3):e17596).

[0053] In some embodiments, the disclosure relates to recombinant nucleic acids comprising one or more polynucleotides comprising a coding sequence of a SPINK5 gene (e.g., a human SPINK5 gene) or a portion thereof (e.g., a coding sequence corresponding to one or more Kazal-type domains resulting from proteolytic cleavage of a full-length SPINK5 polypeptide (e.g., by furin cleavage). Any suitable SPINK5 gene known in the art (including any isoforms thereof) can be encoded by the polynucleotides of the present disclosure, including, for example, the human SPINK5 gene (see, e.g., NCBI Gene ID: 11005, SEQ ID NOs: 1, 3, or 5), the chimpanzee SPINK5 gene (see, e.g., NCBI Gene ID: 462173), the mouse SPINK5 gene (see, e.g., NCBI Gene ID: 72432), the rat SPINK5 gene (see, e.g., NCBI Gene ID: 361319), the dog SPINK5 gene (see, e.g., NCBI Gene ID: 478055), the bovine SPINK5 gene (see, e.g., NCBI Gene ID: 526637), the horse SPINK5 gene (see, e.g., NCBI Gene ID: 100071873), the porcine SPINK5 gene (see, e.g., NCBI Gene ID: 100512160), and the like. In some embodiments, polynucleotides of the present disclosure include sequences having at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence of any SPINK5 gene (and / or its coding sequence) described herein or known in the art. Methods for identifying SPINK5 gene homologs / orthologs from additional species are known to those of skill in the art, including, for example, the use of nucleic acid sequence alignment programs such as the BLAST® blastn suite.

[0054] In some embodiments, polynucleotides of the disclosure comprise a codon-optimized variant of the SPINK5 gene described herein or known in the art (see, e.g., SEQ ID NO: 2, 4, 6, or 26). In some embodiments, the use of a codon-optimized variant of the SPINK5 gene (e.g., a codon-optimized variant of the human SPINK5 gene) increases the stability and / or yield of heterologous expression (RNA and / or protein) of the encoded SPINK5 polypeptide in a target cell (e.g., a target human cell such as a human keratinocyte or fibroblast) compared to the stability and / or yield of heterologous expression of a corresponding non-codon-optimized wild-type sequence. Any suitable method known in the art for codon-optimizing a sequence for expression in one or more target cells (e.g., one or more human cells) may be used, including, for example, the method described by Fath et al. (PLoS One. 2011 Mar 3;6(3):e17596).

[0055] In some embodiments, one or more polynucleotides of the disclosure comprise a coding sequence of the human SPINK5 gene (or a codon-optimized variant thereof) or a portion thereof (e.g., a coding sequence corresponding to one or more Kazal-type domains resulting from proteolytic cleavage of a full-length human SPINK5 polypeptide (e.g., by human Furin cleavage). In some embodiments, a polynucleotide of the disclosure comprises a sequence having at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to a sequence selected from SEQ ID NOs: 1-6 and 26. In some embodiments, a polynucleotide of the disclosure comprises a sequence selected from SEQ ID NOs: 1-6 and 26.

[0056] In some embodiments, the polynucleotides of the disclosure comprise a sequence having at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence of SEQ ID NO:1 or SEQ ID NO:2. In some embodiments, the polynucleotides of the disclosure comprise the sequence of SEQ ID NO:1 or SEQ ID NO:2.

[0057] In some embodiments, polynucleotides of the disclosure include a 5' truncation, 3' truncation, or fragment of the sequence of SEQ ID NO:1 or SEQ ID NO:2. In some embodiments, the 5' truncation, 3' truncation, or fragment of the sequence of SEQ ID NO:1 or SEQ ID NO:2 is a polynucleotide having at least 25, at least 50, at least 75, at least 100, at least 125, at least 150, at least 175, at least 200, at least 250, at least 300, or at least 350, at least 400, at least 450, at least 500, at least 750, at least 1000, at least 1250, at least 1500, at least 1750, at least 2000, at least 2250, at least 2500, at least 2750, at least 3000, at least 3250, but fewer than 3285 contiguous nucleotides of SEQ ID NO:1 or SEQ ID NO:2. In some embodiments, the polynucleotides of the disclosure comprise a sequence having at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence of nucleic acids 1-3282 of SEQ ID NO: 1 or SEQ ID NO: 2. In some embodiments, the polynucleotides of the disclosure comprise the sequence of nucleic acids 1-3282 of SEQ ID NO: 1 or SEQ ID NO: 2.

[0058] In some embodiments, a polynucleotide of the disclosure comprises a sequence having at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence of nucleic acid 82-198 of SEQ ID NO: 1 or SEQ ID NO: 2. In some embodiments, a polynucleotide of the disclosure comprises the sequence of nucleic acid 82-198 of SEQ ID NO: 1 or SEQ ID NO: 2.

[0059] In some embodiments, the polynucleotides of the disclosure comprise a sequence having at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence of nucleic acids 271-459 of SEQ ID NO: 1 or SEQ ID NO: 2. In some embodiments, the polynucleotides of the disclosure comprise the sequence of nucleic acids 271-459 of SEQ ID NO: 1 or SEQ ID NO: 2.

[0060] In some embodiments, the polynucleotides of the disclosure comprise a sequence having at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence of nucleic acids 463-648 of SEQ ID NO: 1 or SEQ ID NO: 2. In some embodiments, the polynucleotides of the disclosure comprise the sequence of nucleic acids 463-648 of SEQ ID NO: 1 or SEQ ID NO: 2.

[0061] In some embodiments, the polynucleotides of the disclosure comprise a sequence having at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence of nucleic acid 655-855 of SEQ ID NO: 1 or SEQ ID NO: 2. In some embodiments, the polynucleotides of the disclosure comprise a sequence of nucleic acid 655-855 of SEQ ID NO: 1 or SEQ ID NO: 2.

[0062] In some embodiments, the polynucleotides of the disclosure comprise a sequence having at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence of nucleic acids 871-1056 of SEQ ID NO: 1 or SEQ ID NO: 2. In some embodiments, the polynucleotides of the disclosure comprise the sequence of nucleic acids 871-1056 of SEQ ID NO: 1 or SEQ ID NO: 2.

[0063] In some embodiments, the polynucleotides of the disclosure comprise a sequence having at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence of nucleic acids 1081-1269 of SEQ ID NO: 1 or SEQ ID NO: 2. In some embodiments, the polynucleotides of the disclosure comprise the sequence of nucleic acids 1081-1269 of SEQ ID NO: 1 or SEQ ID NO: 2.

[0064] In some embodiments, the polynucleotides of the disclosure comprise sequences having at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequences of nucleic acids 1291-1467 of SEQ ID NO: 1 or SEQ ID NO: 2. In some embodiments, the polynucleotides of the disclosure comprise sequences of nucleic acids 1291-1467 of SEQ ID NO: 1 or SEQ ID NO: 2.

[0065] In some embodiments, the polynucleotide of the present disclosure comprises a sequence having at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence of nucleic acid 1468-1653 of SEQ ID NO: 1 or SEQ ID NO: 2. In some embodiments, the polynucleotide of the present disclosure comprises the sequence of nucleic acid 1468-1653 of SEQ ID NO: 1 or SEQ ID NO: 2.

[0066] In some embodiments, the polynucleotides of the disclosure comprise a sequence having at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence of nucleic acids 1681-1866 of SEQ ID NO: 1 or SEQ ID NO: 2. In some embodiments, the polynucleotides of the disclosure comprise the sequence of nucleic acids 1681-1866 of SEQ ID NO: 1 or SEQ ID NO: 2.

[0067] In some embodiments, the polynucleotides of the disclosure comprise a sequence having at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence of nucleic acid 1876-2064 of SEQ ID NO: 1 or SEQ ID NO: 2. In some embodiments, the polynucleotides of the disclosure comprise the sequence of nucleic acid 1876-2064 of SEQ ID NO: 1 or SEQ ID NO: 2.

[0068] In some embodiments, the polynucleotides of the disclosure comprise a sequence having at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence of nucleic acids 2101-2271 of SEQ ID NO: 1 or SEQ ID NO: 2. In some embodiments, the polynucleotides of the disclosure comprise the sequence of nucleic acids 2101-2271 of SEQ ID NO: 1 or SEQ ID NO: 2.

[0069] In some embodiments, the polynucleotides of the disclosure include sequences having at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequences of nucleic acids 2302-2490 of SEQ ID NO: 1 or SEQ ID NO: 2. In some embodiments, the polynucleotides of the disclosure include sequences of nucleic acids 2302-2490 of SEQ ID NO: 1 or SEQ ID NO: 2.

[0070] In some embodiments, the polynucleotides of the disclosure comprise a sequence having at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence of nucleic acid 2527-2715 of SEQ ID NO: 1 or SEQ ID NO: 2. In some embodiments, the polynucleotides of the disclosure comprise the sequence of nucleic acid 2527-2715 of SEQ ID NO: 1 or SEQ ID NO: 2.

[0071] In some embodiments, the polynucleotides of the disclosure comprise a sequence having at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence of nucleic acid 2728-3003 of SEQ ID NO: 1 or SEQ ID NO: 2. In some embodiments, the polynucleotides of the disclosure comprise the sequence of nucleic acid 2728-3003 of SEQ ID NO: 1 or SEQ ID NO: 2.

[0072] In some embodiments, the polynucleotides of the disclosure comprise sequences having at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequences of nucleic acids 3049-3234 of SEQ ID NO: 1 or SEQ ID NO: 2. In some embodiments, the polynucleotides of the disclosure comprise sequences of nucleic acids 3049-3234 of SEQ ID NO: 1 or SEQ ID NO: 2.

[0073] In some embodiments, the polynucleotide of the present disclosure comprises a sequence having at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence of SEQ ID NO:3 or SEQ ID NO:4. In some embodiments, the polynucleotide of the present disclosure comprises the sequence of SEQ ID NO:3 or SEQ ID NO:4. In some embodiments, the polynucleotide of the present disclosure comprises the sequence of SEQ ID NO:4.

[0074] In some embodiments, polynucleotides of the disclosure include a 5' truncation, 3' truncation, or fragment of the sequence of SEQ ID NO:3 or SEQ ID NO:4. In some embodiments, the 5' truncation, 3' truncation, or fragment of the sequence of SEQ ID NO:3 or SEQ ID NO:4 is a polynucleotide having at least 25, at least 50, at least 75, at least 100, at least 125, at least 150, at least 175, at least 200, at least 250, at least 300, or at least 350, at least 400, at least 450, at least 500, at least 750, at least 1000, at least 1250, at least 1500, at least 1750, at least 2000, 2250, at least 2500, at least 2750, but fewer than 2751 contiguous nucleotides of SEQ ID NO:3 or SEQ ID NO:4. In some embodiments, the polynucleotides of the disclosure comprise a sequence having at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence of nucleic acids 1-2748 of SEQ ID NO: 3 or SEQ ID NO: 4. In some embodiments, the polynucleotides of the disclosure comprise the sequence of nucleic acids 1-2748 of SEQ ID NO: 3 or SEQ ID NO: 4.

[0075] In some embodiments, the polynucleotide of the present disclosure comprises a sequence having at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence of SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:26. In some embodiments, the polynucleotide of the present disclosure comprises the sequence of SEQ ID NO:6. In some embodiments, the polynucleotide of the present disclosure comprises the sequence of SEQ ID NO:26.

[0076] In some embodiments, polynucleotides of the disclosure include 5' truncations, 3' truncations, or fragments of the sequence of SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:26. In some embodiments, the 5' truncations, 3' truncations, or fragments of the sequence of SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:26 are polynucleotides having at least 25, at least 50, at least 75, at least 100, at least 125, at least 150, at least 175, at least 200, at least 250, at least 300, or at least 350, at least 400, at least 450, at least 500, at least 750, at least 1000, at least 1250, at least 1500, at least 1750, at least 2000, at least 2250, at least 2500, at least 2750, at least 3000, but fewer than 3195 contiguous nucleotides of SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:26. In some embodiments, the polynucleotides of the disclosure comprise a sequence having at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence of nucleic acids 1-3192 of SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:26. In some embodiments, the polynucleotides of the disclosure comprise the sequence of nucleic acids 1-3192 of SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:26.

[0077] Polynucleotides of the present disclosure that encode a SPINK polypeptide (e.g., a human SPINK5 polypeptide) may further encode additional coding and non-coding sequences, including, but not limited to, sequences encoding additional polypeptide tags (e.g., encoded in frame with the SPINK protein to produce a fusion protein), introns (e.g., natural, modified, or heterologous introns), 5'UTRs and / or 3'UTRs (e.g., natural, modified, or heterologous 5'UTRs and / or 3'UTRs), and the like. Examples of suitable polypeptide tags include, but are not limited to, purification tags, such as his-tags, flag tags, maltose binding protein and glutathione-S-transferase tags, detection tags, such as tags that can be photometrically detected (e.g., green fluorescent protein, red fluorescent protein, etc.) and tags with detectable enzymatic activity (e.g., alkaline phosphatase, etc.), any combination of tags including secretion sequences, signal sequences, leader sequences, and / or stabilization sequences, protease cleavage sites (e.g., furin cleavage sites, TEV cleavage sites, thrombin cleavage sites, etc.), etc. In some embodiments, the 5'UTR and / or 3'UTR increase the stability, localization, and / or translation efficiency of the polynucleotide. In some embodiments, the 5'UTR and / or 3'UTR improve protein expression levels and / or duration. In some embodiments, the 5'UTR and / or 3'UTR contain elements (e.g., one or more miRNA binding sites, etc.) that can block or reduce off-target expression (e.g., inhibit expression in a particular cell type (e.g., neuronal cells) at a particular time in the cell cycle or at a particular developmental stage). In some embodiments, the 5'UTR and / or 3'UTR contain elements (e.g., one or more miRNA binding sites, etc.) that can enhance effector protein expression in a particular cell type (e.g., human keratinocytes and / or fibroblasts).

[0078] In some embodiments, the polynucleotides of the present disclosure are operably linked to one or more (e.g., one or more, two or more, three or more, four or more, five or more, ten or more, etc.) regulatory sequences. The term "regulatory sequence" can include enhancers, insulators, promoters, and other expression control elements (e.g., polyadenylation signals). Any suitable enhancer(s) known in the art can be used, including, for example, enhancer sequences from mammalian genes (e.g., globin, elastase, albumin, alpha-fetoprotein, insulin, etc.), enhancer sequences from eukaryotic cell viruses (e.g., SV40 enhancer on the late side of the replication origin (bp 100-270), cytomegalovirus early promoter enhancer, polyoma enhancer on the late side of the replication origin, adenovirus enhancer, etc.), and any combination thereof. Any suitable insulator(s) known in the art may be used, including, for example, the herpes simplex virus (HSV) chromatin boundary (CTRL / CTCF binding / insulator) elements CTRL1 and / or CTRL2 from the human interferon beta gene (IFNB1), chicken hypersensitive site 4 insulator (cHS4), human HNRPA2B1-CBX3 ubiquitous chromatin opening element (UCOE), scaffold / matrix attachment region (S / MAR), and any combination thereof.Any suitable promoter known in the art (e.g., a promoter suitable for transcription in a mammalian host cell) may be used, including, for example, promoters obtained from the genome of a virus (e.g., polyoma virus, fowlpox virus, adenovirus (such as adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, retrovirus, hepatitis B virus, simian virus 40 (SV40), etc.), promoters from heterologous mammalian genes (e.g., actin promoters (e.g., β-actin promoter), ubiquitin promoters (e.g., ubiquitin C (UbC) promoter), phosphoglycerate kinase (PGK) promoter, immunoglobulin promoters, heat shock protein promoters, etc.), promoters from natural and / or homologous mammalian genes (e.g., human SPINK gene promoter), synthetic promoters (e.g., CAGG promoter, etc.), and any combination thereof. Regulatory sequences may include sequences that direct constitutive expression of the nucleic acid, as well as tissue-specific regulatory sequences and / or inducible or repressible sequences.

[0079] In some embodiments, the polynucleotide of the present disclosure is operably linked to one or more heterologous promoters. In some embodiments, the one or more heterologous promoters are one or more of a constitutive promoter, a tissue-specific promoter, a temporal promoter, a spatial promoter, an inducible promoter, and a repressible promoter. In some embodiments, the one or more heterologous promoters are one or more of a human cytomegalovirus (HCMV) immediate early promoter, a human elongation factor-1 (EF1) promoter, a human β-actin promoter, a human UbC promoter, a human PGK promoter, a synthetic CAGG promoter, and any combination thereof. In some embodiments, the polynucleotide of the present disclosure is operably linked to an HCMV promoter.

[0080] In some embodiments, a recombinant nucleic acid of the disclosure does not include a polynucleotide that includes a coding sequence for a collagen alpha-1 (VII) chain polypeptide (COL7) (e.g., a transgene encoding the same). In some embodiments, a recombinant nucleic acid of the disclosure does not include a polynucleotide that includes a coding sequence for a lysyl hydroxylase 3 polypeptide (LH3) (e.g., a transgene encoding the same). In some embodiments, a recombinant nucleic acid of the disclosure does not include a polynucleotide that includes a coding sequence for a keratin type I cytoskeletal 17 polypeptide (KRT17) (e.g., a transgene encoding the same). In some embodiments, a recombinant nucleic acid of the disclosure does not include a polynucleotide that includes a coding sequence for a transglutaminase (TGM) polypeptide (e.g., a human transglutaminase polypeptide, such as a human TGM1 polypeptide) (e.g., a transgene encoding the same). In some embodiments, a recombinant nucleic acid of the disclosure does not include a polynucleotide that includes a coding sequence for a cosmetic protein (e.g., a collagen protein, fibronectin, elastin, lumican, vitronectin / vitronectin receptor, laminin, neuromodulators, fibrillin, additional dermal extracellular matrix proteins, etc.) (e.g., a transgene encoding the same). In some embodiments, a recombinant nucleic acid of the disclosure does not include a polynucleotide that includes a coding sequence for an antibody (e.g., a full-length antibody, an antibody fragment, etc.) (e.g., a transgene encoding the same). In some embodiments, a recombinant nucleic acid of the disclosure does not include a polynucleotide that includes a coding sequence for a collagen alpha-1 (VII) chain polypeptide, a lysyl hydroxylase 3 polypeptide, a keratin type I cytoskeleton 17 polypeptide, and / or any chimeric polypeptides thereof (e.g., a transgene encoding the same). In some embodiments, a recombinant nucleic acid of the present disclosure does not include a polynucleotide comprising a coding sequence for a collagen alpha-1 (VII) chain polypeptide, a lysyl hydroxylase 3 polypeptide, a keratin type I cytoskeletal 17 polypeptide, a transglutaminase (TGM) polypeptide, a cosmetic protein, an antibody, and / or any chimeric polypeptide thereof (e.g., a transgene encoding the same).

[0081] SPINK Polypeptides In some embodiments, the disclosure relates to one or more polynucleotides encoding a SPINK polypeptide or any portion thereof (e.g., the amino acid sequence of one or more Kazal-type domains resulting from proteolytic cleavage of a full-length SPINK polypeptide (e.g., by furin cleavage). For example, a SPINK1 polypeptide (e.g., a human SPINK1 polypeptide (see, e.g., UniProt Accession No.: P00995)), a SPINK2 polypeptide (e.g., a human SPINK2 polypeptide (see, e.g., UniProt Accession No.: P20155)), a SPINK4 polypeptide (e.g., a human SPINK4 polypeptide (see, e.g., UniProt Accession No.: O60575)), a SPINK5 polypeptide (e.g., a human SPINK5 polypeptide (see, e.g., UniProt Accession No.: Q9NQ38)), a SPINK6 polypeptide (e.g., a human SPINK6 polypeptide (see, e.g., UniProt Accession No.: Q6UWN8)), a SPINK7 polypeptide (e.g., a human SPINK7 polypeptide (see, e.g., UniProt Accession No.: Any suitable SPINK polypeptide (or a portion thereof) known in the art can be encoded by the polynucleotides of the present disclosure, including, for example, a SPINK polypeptide (e.g., see UniProt Accession No. P58062), a SPINK8 polypeptide (e.g., human SPINK8 polypeptide (e.g., see UniProt Accession No. P0C7L1), a SPINK9 polypeptide (e.g., human SPINK9 polypeptide (e.g., see UniProt Accession No. Q5DT21), a SPINK13 polypeptide (e.g., human SPINK13 polypeptide (e.g., see UniProt Accession No. Q1W4C9), a SPINK14 polypeptide (e.g., human SPINK14 polypeptide (e.g., see UniProt Accession No. Q6IE38)), and the like.In some embodiments, the SPINK polypeptide of the present disclosure comprises a sequence having at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of any SPINK polypeptide described herein or known in the art. Methods for identifying SPINK polypeptide homologs / orthologs from additional species are known to those of skill in the art, including, for example, the use of amino acid sequence alignment programs such as the BLAST® blastp suite or OrthoDB. In some embodiments, the SPINK polypeptide is a human SPINK polypeptide.

[0082] In some embodiments, the disclosure relates to one or more polynucleotides encoding a SPINK5 polypeptide (e.g., SEQ ID NOs: 7-9) or any portion thereof (e.g., the amino acid sequence of one or more Kazal-type domains resulting from proteolytic cleavage (e.g., by furin cleavage) of a full-length SPINK5 polypeptide (e.g., SEQ ID NOs: 10-25)). Any suitable SPINK5 polypeptide (or a portion thereof) known in the art can be encoded by the polynucleotides of the present disclosure, including, for example, human SPINK5 polypeptide (see, e.g., UniProt Accession No.: 11005), chimpanzee SPINK5 polypeptide (see, e.g., UniProt Accession No.: 462173), mouse SPINK5 polypeptide (see, e.g., UniProt Accession No.: Q5K5D4), rat SPINK5 polypeptide (see, e.g., UniProt Accession No.: D3ZET2), dog SPINK5 polypeptide (see, e.g., UniProt Accession No.: F1PR80), bovine SPINK5 polypeptide (see, e.g., UniProt Accession No.: F1MJH0), horse SPINK5 polypeptide (see, e.g., UniProt Accession No.: B9VJ40), porcine SPINK5 polypeptide (see, e.g., UniProt Accession No.: F1RLZ2), and the like. In some embodiments, a SPINK5 polypeptide of the present disclosure comprises a sequence having at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of any SPINK5 polypeptide described herein or known in the art. Methods for identifying SPINK5 polypeptide homologs / orthologs from additional species are known to those of skill in the art, including, for example, the use of amino acid sequence alignment programs such as the BLAST® blastp suite or OrthoDB.

[0083] In some embodiments, a SPINK5 polypeptide of the disclosure is a human SPINK5 polypeptide or a portion thereof (e.g., a polypeptide comprising an amino acid sequence corresponding to one or more Kazal-type domains resulting from proteolytic cleavage of a full-length human SPINK5 polypeptide (e.g., by human Furin cleavage). In some embodiments, a polynucleotide encoding a human SPINK5 polypeptide is a polynucleotide encoding a polypeptide comprising an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to a sequence selected from SEQ ID NOs: 7-25. In some embodiments, a polynucleotide encoding a human SPINK5 polypeptide is a polynucleotide encoding a polypeptide comprising an amino acid sequence selected from SEQ ID NOs: 7-25. In some embodiments, a polynucleotide encoding a human SPINK5 polypeptide is a polynucleotide encoding a polypeptide comprising an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to a sequence selected from SEQ ID NOs: 7 to 9. In some embodiments, a polynucleotide encoding a human SPINK5 polypeptide is a polynucleotide encoding a polypeptide comprising an amino acid sequence selected from SEQ ID NOs: 7 to 9.

[0084] In some embodiments, a SPINK5 polypeptide of the disclosure comprises, consists essentially of, or consists of a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence selected from SEQ ID NOs: 7-25. In some embodiments, a SPINK5 polypeptide of the disclosure comprises, consists essentially of, or consists of a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence selected from SEQ ID NOs: 7-9. In some embodiments, a SPINK5 polypeptide of the disclosure comprises, consists essentially of, or consists of a sequence selected from SEQ ID NOs:7-9.

[0085] In some embodiments, a polynucleotide encoding a human SPINK5 polypeptide is a polynucleotide that encodes a polypeptide comprising an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequence of SEQ ID NO: 7. In some embodiments, a polynucleotide encoding a human SPINK5 polypeptide is a polynucleotide that encodes a polypeptide comprising the amino acid sequence of SEQ ID NO:7.

[0086] In some embodiments, a polynucleotide encoding a human SPINK5 polypeptide is a polynucleotide encoding an N-terminal truncation, C-terminal truncation, or fragment of the amino acid sequence of SEQ ID NO: 7. The N-terminal truncation, C-terminal truncation, or fragment may comprise at least 10, at least 12, at least 14, at least 16, at least 18, at least 20, at least 30, at least 40, at least 50, at least 75, at least 100, at least 200, at least 300, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, at least 1000, but less than 1094 contiguous amino acids of SEQ ID NO:7.

[0087] In some embodiments, a polynucleotide encoding a human SPINK5 polypeptide is a polynucleotide that encodes a polypeptide comprising an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequence of SEQ ID NO: 8. In some embodiments, a polynucleotide encoding a human SPINK5 polypeptide is a polynucleotide that encodes a polypeptide comprising the amino acid sequence of SEQ ID NO:8.

[0088] In some embodiments, a polynucleotide encoding a human SPINK5 polypeptide is a polynucleotide encoding an N-terminal truncation, C-terminal truncation, or fragment of the amino acid sequence of SEQ ID NO: 8. The N-terminal truncation, C-terminal truncation, or fragment may comprise at least 10, at least 12, at least 14, at least 16, at least 18, at least 20, at least 30, at least 40, at least 50, at least 75, at least 100, at least 200, at least 300, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, but fewer than 916 contiguous amino acids of SEQ ID NO:8.

[0089] In some embodiments, a polynucleotide encoding a human SPINK5 polypeptide is a polynucleotide that encodes a polypeptide comprising an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequence of SEQ ID NO: 9. In some embodiments, a polynucleotide encoding a human SPINK5 polypeptide is a polynucleotide that encodes a polypeptide comprising the amino acid sequence of SEQ ID NO:9.

[0090] In some embodiments, a polynucleotide encoding a human SPINK5 polypeptide is a polynucleotide encoding an N-terminal truncation, C-terminal truncation, or fragment of the amino acid sequence of SEQ ID NO: 9. The N-terminal truncation, C-terminal truncation, or fragment may comprise at least 10, at least 12, at least 14, at least 16, at least 18, at least 20, at least 30, at least 40, at least 50, at least 75, at least 100, at least 200, at least 300, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, at least 1000, but less than 1064 contiguous amino acids of SEQ ID NO:9.

[0091] In some embodiments, a polynucleotide of the disclosure encodes a Kazal-type domain derived from a human SPINK5 polypeptide (eg, a Kazal-type domain resulting from proteolytic cleavage of human SPINK5 (eg, via furin cleavage)).

[0092] In some embodiments, a SPINK5 polypeptide of the disclosure comprises or consists of an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequence of SEQ ID NO: 10. In some embodiments, a SPINK5 polypeptide of the disclosure comprises or consists of the amino acid sequence of SEQ ID NO:10.

[0093] In some embodiments, a SPINK5 polypeptide of the disclosure comprises or consists of an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequence of SEQ ID NO: 11. In some embodiments, a SPINK5 polypeptide of the disclosure comprises or consists of the amino acid sequence of SEQ ID NO:11.

[0094] In some embodiments, a SPINK5 polypeptide of the disclosure comprises or consists of an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequence of SEQ ID NO: 12. In some embodiments, a SPINK5 polypeptide of the disclosure comprises or consists of the amino acid sequence of SEQ ID NO:12.

[0095] In some embodiments, a SPINK5 polypeptide of the disclosure comprises or consists of an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequence of SEQ ID NO: 13. In some embodiments, a SPINK5 polypeptide of the disclosure comprises or consists of the amino acid sequence of SEQ ID NO:13.

[0096] In some embodiments, a SPINK5 polypeptide of the disclosure comprises or consists of an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequence of SEQ ID NO: 14. In some embodiments, a SPINK5 polypeptide of the disclosure comprises or consists of the amino acid sequence of SEQ ID NO:14.

[0097] In some embodiments, a SPINK5 polypeptide of the disclosure comprises or consists of an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequence of SEQ ID NO: 15. In some embodiments, a SPINK5 polypeptide of the disclosure comprises or consists of the amino acid sequence of SEQ ID NO:15.

[0098] In some embodiments, a SPINK5 polypeptide of the disclosure comprises or consists of an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequence of SEQ ID NO: 16. In some embodiments, a SPINK5 polypeptide of the disclosure comprises or consists of the amino acid sequence of SEQ ID NO:16.

[0099] In some embodiments, a SPINK5 polypeptide of the disclosure comprises or consists of an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequence of SEQ ID NO: 17. In some embodiments, a SPINK5 polypeptide of the disclosure comprises or consists of the amino acid sequence of SEQ ID NO:17.

[0100] In some embodiments, a SPINK5 polypeptide of the disclosure comprises or consists of an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequence of SEQ ID NO: 18. In some embodiments, a SPINK5 polypeptide of the disclosure comprises or consists of the amino acid sequence of SEQ ID NO:18.

[0101] In some embodiments, a SPINK5 polypeptide of the disclosure comprises or consists of an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequence of SEQ ID NO: 19. In some embodiments, a SPINK5 polypeptide of the disclosure comprises or consists of the amino acid sequence of SEQ ID NO:19.

[0102] In some embodiments, a SPINK5 polypeptide of the disclosure comprises or consists of an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequence of SEQ ID NO: 20. In some embodiments, a SPINK5 polypeptide of the disclosure comprises or consists of the amino acid sequence of SEQ ID NO:20.

[0103] In some embodiments, a SPINK5 polypeptide of the disclosure comprises or consists of an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequence of SEQ ID NO: 21. In some embodiments, a SPINK5 polypeptide of the disclosure comprises or consists of the amino acid sequence of SEQ ID NO:21.

[0104] In some embodiments, a SPINK5 polypeptide of the disclosure comprises or consists of an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequence of SEQ ID NO: 22. In some embodiments, a SPINK5 polypeptide of the disclosure comprises or consists of the amino acid sequence of SEQ ID NO:22.

[0105] In some embodiments, a SPINK5 polypeptide of the disclosure comprises or consists of an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequence of SEQ ID NO: 23. In some embodiments, a SPINK5 polypeptide of the disclosure comprises or consists of the amino acid sequence of SEQ ID NO:23.

[0106] In some embodiments, a SPINK5 polypeptide of the disclosure comprises or consists of an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequence of SEQ ID NO: 24. In some embodiments, a SPINK5 polypeptide of the disclosure comprises or consists of the amino acid sequence of SEQ ID NO:24.

[0107] In some embodiments, a SPINK5 polypeptide of the disclosure comprises or consists of an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequence of SEQ ID NO: 25. In some embodiments, a SPINK5 polypeptide of the disclosure comprises or consists of the amino acid sequence of SEQ ID NO:25.

[0108] In some embodiments, a polynucleotide of the disclosure encoding a SPINK polypeptide (e.g., a SPINK5 polypeptide) expresses the SPINK polypeptide when the polynucleotide is delivered to one or more target cells of a subject. In some embodiments, expression of a SPINK polypeptide (e.g., a SPINK5 polypeptide) enhances, increases, enhances, and / or replenishes the level, function, and / or activity of the SPINK polypeptide in one or more target cells of a subject (e.g., compared to before expression of an exogenous SPINK polypeptide). In some embodiments, expression of a SPINK polypeptide (e.g., a SPINK5 polypeptide) enhances, increases, enhances, and / or replenishes anti-inflammatory and / or antimicrobial protection of mucosal epithelia in a subject. In some embodiments, expression of a SPINK polypeptide (e.g., a SPINK5 polypeptide) enhances, increases, enhances, and / or replenishes the integrity and / or protective barrier function of the skin of a subject. In some embodiments, expression of a SPINK polypeptide (e.g., a SPINK5 polypeptide) reduces, enhances, and / or inhibits one or more of kallikrein-5 (KLK5), kallikrein-7 (KLK7), kallikrein-14 (KLK14), caspase-14 (CASP14), and / or trypsin in a subject (e.g., reduces, enhances, and / or inhibits the activity of one or more of KLK5, KLK7, KLK14, CASP14, and / or trypsin).

[0109] Recombinant Nucleic Acids In some embodiments, the disclosure relates to a recombinant nucleic acid comprising any one or more of the polynucleotides described herein. In some embodiments, the recombinant nucleic acid is a vector (e.g., an expression vector, a display vector, etc.). In some embodiments, the vector is a DNA vector or an RNA vector. In general, a vector suitable for maintaining, propagating, and / or expressing a polynucleotide to produce one or more polypeptides in a subject may be used. Examples of suitable vectors may include, for example, plasmids, cosmids, episomes, transposons, and viral vectors (e.g., adenovirus vectors, adeno-associated virus vectors, vaccinia virus vectors, Sindbis virus vectors, measles vectors, herpes virus vectors, lentivirus vectors, retrovirus vectors, etc.). In some embodiments, the vector is a herpes virus vector. In some embodiments, the vector is capable of autonomous replication in a host cell. In some embodiments, the vector is not capable of autonomous replication in a host cell. In some embodiments, the vector is capable of integration into host DNA. In some embodiments, the vector is not capable of integration into host DNA (e.g., is episomal). Methods for constructing vectors containing one or more polynucleotides of interest, such as by chemical synthesis or by the artificial manipulation of isolated segments of nucleic acid (eg, by genetic engineering techniques), are well known to those of skill in the art.

[0110] In some embodiments, the recombinant nucleic acid of the present disclosure is a herpes simplex virus (HSV) amplicon. Herpes virus amplicons, including structural features, and methods of making same are generally known to those of skill in the art (see, for example, de Silva S. and Bowers W. "Herpes Virus Amplicon Vectors". Viruses 2009, 1, 594-629). In some embodiments, the herpes simplex virus amplicon is an HSV-1 amplicon. In some embodiments, the herpes simplex virus amplicon is an HSV-1 hybrid amplicon. Examples of HSV-1 hybrid amplicons may include, but are not limited to, HSV / AAV hybrid amplicons, HSV / EBV hybrid amplicons, HSV / EBV / RV hybrid amplicons, and / or HSV / Sleeping Beauty hybrid amplicons. In some embodiments, the amplicon is an HSV / AAV hybrid amplicon. In some embodiments, the amplicon is an HSV / Sleeping Beauty hybrid amplicon.

[0111] In some embodiments, the recombinant nucleic acid of the present disclosure is a recombinant herpesvirus genome. The recombinant herpesvirus genome can be a recombinant genome from any member of the Herpesviridae family of DNA viruses known in the art, including, for example, recombinant herpes simplex virus genome, recombinant varicella zoster virus genome, recombinant human cytomegalovirus genome, recombinant herpesvirus 6A genome, recombinant herpesvirus 6B genome, recombinant herpesvirus 7 genome, recombinant Kaposi's sarcoma-associated herpesvirus genome, and any combination or derivative thereof. In some embodiments, the recombinant herpesvirus genome comprises one or more (e.g., one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, etc.) inactivating mutations. In some embodiments, the one or more inactivating mutations are present in one or more (e.g., one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, etc.) herpesvirus genes. In some embodiments, the recombinant herpesvirus genome is attenuated (e.g., compared to a corresponding wild-type herpesvirus genome). In some embodiments, the recombinant herpesvirus genome is replication competent. In some embodiments, the recombinant herpesvirus genome is replication defective.

[0112] In some embodiments, the recombinant nucleic acid is a recombinant herpes simplex virus (HSV) genome. In some embodiments, the recombinant herpes simplex virus genome is a recombinant herpes simplex virus type 1 (HSV-1) genome, a recombinant herpes simplex virus type 2 (HSV-2) genome, or any derivative thereof. In some embodiments, the recombinant herpes simplex virus genome is a recombinant HSV-1 genome. In some embodiments, the recombinant HSV-1 genome is a recombinant HSV-1 genome, such as strain 17, Ty25, R62, S25, Ku86, S23, R11, Ty148, Ku47, H166syn, 1319-2005, F-13, M-12, 90237, F-17, KOS, 3083-2008, F12g, L2, CD38, H193, M-15, India 2011, 0116209, F-11I, 66-207, 2762, 369-2007, 3355, MacIntyre, McKrae, 7862, 7-h se, HF10, 1394,2005, 270-2007, OD4, SC16, M-19, 4J1037, 5J1060, J1060, KOS79, 132- The recombinant HSV-1 genome may be derived from any HSV-1 strain known in the art, including 1988, 160-1982, H166, 2158-2007, RE, 78326, F18g, F11, 172-2010, H129, F, E4, CJ994, F14g, E03, E22, E10, E06, E11, E25, E23, E35, E15, E07, E12, E14, E08, E19, E13, ATCC 2011, and the like (see, e.g., Bowen et al. J Virol. 2019 Apr 3;93(8)). In some embodiments, the recombinant HSV-1 genome is derived from a KOS strain. In some embodiments, the recombinant HSV-1 genome is not derived from a McKrae strain. In some embodiments, the recombinant herpes simplex virus genome is attenuated. In some embodiments, the recombinant herpes simplex virus genome is replication competent. In some embodiments, the recombinant herpes simplex virus genome is replication defective. In some embodiments, the recombinant herpes simplex virus genome comprises one or more (e.g., one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, etc.) inactivating mutations.In some embodiments, one or more inactivating mutations are present in one or more (e.g., one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, etc.) herpes simplex virus genes. As used herein, "inactivating mutation" may refer to any mutation that results in a gene or regulon product (RNA or protein) with reduced, undetectable, or eliminated amount and / or function (e.g., compared to a corresponding sequence lacking the inactivating mutation). Examples of inactivating mutations may include, but are not limited to, deletions, insertions, point mutations, and rearrangements of the transcriptional control sequences (promoters, enhancers, insulators, etc.) and / or coding sequences of a given gene or regulon. Any suitable method of measuring the amount of a gene or regulon product known in the art may be used, including, for example, qPCR, Northern blot, RNAseq, Western blot, ELISA, etc.

[0113] In some embodiments, the recombinant herpes simplex virus genome comprises an inactivating mutation in at least one, at least two, at least three, at least four, at least five, at least six, at least seven, or all eight of the infected cell protein (or infected cell polypeptide) (ICP)0, ICP4, ICP22, ICP27, ICP47, thymidine kinase (tk), long unique region (UL)41, and / or UL55 herpes simplex virus genes. In some embodiments, the recombinant herpes simplex virus genome does not comprise an inactivating mutation in the ICP34.5 (one or both copies) herpes simplex virus gene and / or the ICP47 herpes simplex virus gene (e.g., to avoid production of immunostimulatory virus). In some embodiments, the recombinant herpes simplex virus genome does not comprise an inactivating mutation in the ICP34.5 (one or both copies) herpes simplex virus gene. In some embodiments, the recombinant herpes simplex virus genome does not comprise an inactivating mutation in the ICP47 herpes simplex virus gene. In some embodiments, the recombinant herpes simplex virus genome does not contain inactivating mutations in the ICP34.5 (one or both copies) herpes simplex virus gene and the ICP47 herpes simplex virus gene. In some embodiments, the recombinant herpes simplex virus genome is not oncolytic.

[0114] In some embodiments, the recombinant herpes simplex virus genome comprises an inactivating mutation in the ICP0 gene (one or both copies). In some embodiments, the recombinant herpes simplex virus genome comprises an inactivating mutation in the ICP0 gene (one or both copies) and further comprises an inactivating mutation in the ICP4 (one or both copies), ICP22, ICP27, ICP47, UL41, and / or UL55 gene. In some embodiments, the recombinant herpes simplex virus genome comprises an inactivating mutation in the ICP0 gene (one or both copies) and comprises an inactivating mutation in the ICP4 gene (one or both copies). In some embodiments, the recombinant herpes simplex virus genome comprises an inactivating mutation in the ICP0 gene (one or both copies) and comprises an inactivating mutation in the ICP22 gene. In some embodiments, the recombinant herpes simplex virus genome comprises an inactivating mutation in the ICP0 gene (one or both copies) and comprises an inactivating mutation in the UL41 gene. In some embodiments, the recombinant herpes simplex virus genome comprises an inactivating mutation in the ICP0 gene (one or both copies), an inactivating mutation in the ICP4 gene (one or both copies), and an inactivating mutation in the ICP22 gene. In some embodiments, the recombinant herpes simplex virus genome comprises an inactivating mutation in the ICP0 gene (one or both copies), an inactivating mutation in the ICP4 gene (one or both copies), and an inactivating mutation in the UL41 gene. In some embodiments, the recombinant herpes simplex virus genome comprises an inactivating mutation in the ICP0 gene (one or both copies), an inactivating mutation in the ICP22 gene, and an inactivating mutation in the UL41 gene. In some embodiments, the recombinant herpes simplex virus genome comprises an inactivating mutation in the ICP0 gene (one or both copies), an inactivating mutation in the ICP4 gene (one or both copies), an inactivating mutation in the ICP22 gene, and an inactivating mutation in the UL41 gene. In some embodiments, the inactivating mutation is a deletion in the coding sequence of the ICP0 (one or both copies), ICP4 (one or both copies), ICP22, and / or UL41 genes.In some embodiments, the recombinant herpes simplex virus genome further comprises an inactivating mutation in the ICP27, ICP47, and / or UL55 gene.

[0115] In some embodiments, the recombinant herpes simplex virus genome comprises an inactivating mutation in the ICP4 gene (one or both copies). In some embodiments, the recombinant herpes simplex virus genome comprises an inactivating mutation in the ICP4 gene (one or both copies) and further comprises an inactivating mutation in the ICP0 (one or both copies), ICP22, ICP27, ICP47, UL41, and / or UL55 genes. In some embodiments, the recombinant herpes simplex virus genome comprises an inactivating mutation in the ICP4 gene (one or both copies) and comprises an inactivating mutation in the ICP22 gene. In some embodiments, the recombinant herpes simplex virus genome comprises an inactivating mutation in the ICP4 gene (one or both copies) and comprises an inactivating mutation in the UL41 gene. In some embodiments, the recombinant herpes simplex virus genome comprises an inactivating mutation in the ICP4 gene (one or both copies), comprises an inactivating mutation in the ICP22 gene, and comprises an inactivating mutation in the UL41 gene. In some embodiments, the inactivating mutation is a deletion of the coding sequence of the ICP4 (one or both copies), ICP22, and / or UL41 genes. In some embodiments, the recombinant herpes simplex virus genome further comprises an inactivating mutation in the ICP0 (one or both copies), ICP27, ICP47, and / or UL55 genes.

[0116] In some embodiments, the recombinant herpes simplex virus genome comprises an inactivating mutation in the ICP22 gene. In some embodiments, the recombinant herpes simplex virus genome comprises an inactivating mutation in the ICP22 gene and further comprises an inactivating mutation in the ICP0 (one or both copies), ICP4 (one or both copies), ICP27, ICP47, UL41, and / or UL55 genes. In some embodiments, the recombinant herpes simplex virus genome comprises an inactivating mutation in the ICP22 gene and further comprises an inactivating mutation in the UL41 gene. In some embodiments, the inactivating mutation is a deletion of the coding sequence of the ICP22 and / or UL41 genes. In some embodiments, the recombinant herpes simplex virus genome further comprises an inactivating mutation in the ICP0 (one or both copies), ICP4 (one or both copies), ICP27, ICP47, and / or UL55 genes.

[0117] In some embodiments, the recombinant herpes simplex virus genome comprises an inactivating mutation in the ICP27 gene. In some embodiments, the recombinant herpes simplex virus genome comprises an inactivating mutation in the ICP27 gene and further comprises an inactivating mutation in an ICP0 (one or both copies), ICP4 (one or both copies), ICP22, ICP47, UL41, and / or UL55 gene. In some embodiments, the inactivating mutation is a deletion of the coding sequence of the ICP27 gene.

[0118] In some embodiments, the recombinant herpes simplex virus genome comprises an inactivating mutation in the ICP47 gene. In some embodiments, the recombinant herpes simplex virus genome comprises an inactivating mutation in the ICP47 gene and further comprises an inactivating mutation in an ICP0 (one or both copies), ICP4 (one or both copies), ICP22, ICP27, UL41, and / or UL55 gene. In some embodiments, the inactivating mutation is a deletion of the coding sequence of the ICP47 gene.

[0119] In some embodiments, the recombinant herpes simplex virus genome comprises an inactivating mutation in the UL41 gene. In some embodiments, the recombinant herpes simplex virus genome comprises an inactivating mutation in the UL41 gene and further comprises an inactivating mutation in an ICP0 (one or both copies), ICP4 (one or both copies), ICP22, ICP27, ICP47, and / or UL55 gene. In some embodiments, the inactivating mutation is a deletion of the coding sequence of the UL41 gene.

[0120] In some embodiments, the recombinant herpes simplex virus genome comprises an inactivating mutation in the UL55 gene. In some embodiments, the recombinant herpes simplex virus genome comprises an inactivating mutation in the UL55 gene and further comprises an inactivating mutation in an ICP0 (one or both copies), ICP4 (one or both copies), ICP22, ICP27, ICP47, and / or UL41 gene. In some embodiments, the inactivating mutation is a deletion of the coding sequence of the UL55 gene.

[0121] In some embodiments, the recombinant herpes simplex virus genome comprises an internal repeat long (IR L ) region and internal repeat short (IR SIn some embodiments, the inactivation (e.g., deletion) of the joint region removes one copy each of the ICP4 and ICP0 genes. In some embodiments, the inactivation (e.g., deletion) of the joint region further inactivates (e.g., deletes) the promoters of the ICP22 and ICP47 genes. If desired, expression of one or both of these genes can be restored by insertion of an immediate early promoter into a recombinant herpes simplex virus genome (e.g., Hill et al. (1995). Nature 375(6530):411-415; Goldsmith et al. (1998). J Exp Med 187(3):341-348). Without wishing to be bound by theory, it is believed that inactivation (e.g., deletion) of the joint region may contribute to the stability of the recombinant herpes simplex virus genome and / or allow the recombinant herpes simplex virus genome to accommodate more and / or larger transgenes.

[0122] In some embodiments, the recombinant herpes simplex virus genome comprises an inactivating mutation in the ICP4 (one or both copies), ICP22, and ICP27 genes. In some embodiments, the recombinant herpes simplex virus genome comprises an inactivating mutation in the ICP4 (one or both copies), ICP27, and UL55 genes. In some embodiments, the recombinant herpes simplex virus genome comprises an inactivating mutation in the ICP4 (one or both copies), ICP22, ICP27, ICP47, and UL55 genes. In some embodiments, the inactivating mutation in the ICP4 (one or both copies), ICP27, and / or UL55 genes is a deletion of the coding sequence of the ICP4 (one or both copies), ICP27, and / or UL55 gene. In some embodiments, the inactivating mutations in the ICP22 and ICP47 genes are deletions of the promoter regions of the ICP22 and ICP47 genes (e.g., the ICP22 and ICP47 coding sequences are intact but not transcriptionally active). In some embodiments, the recombinant herpes simplex virus genome comprises a deletion of the coding sequences of the ICP4 (one or both copies), ICP27, and UL55 genes, and a deletion of the promoter regions of the ICP22 and ICP47 genes. In some embodiments, the recombinant herpes simplex virus genome further comprises an inactivating mutation in the ICP0 gene (one or both copies) and / or the UL41 gene.

[0123] In some embodiments, the recombinant herpes simplex virus genome comprises inactivating mutations in the ICP0 (one or both copies) and ICP4 (one or both copies) genes. In some embodiments, the recombinant herpes simplex virus genome comprises inactivating mutations in the ICP0 (one or both copies), ICP4 (one or both copies), and ICP22 genes. In some embodiments, the recombinant herpes simplex virus genome comprises inactivating mutations in the ICP0 (one or both copies), ICP4 (one or both copies), ICP22, and ICP27 genes. In some embodiments, the recombinant herpes simplex virus genome comprises inactivating mutations in the ICP0 (one or both copies), ICP4 (one or both copies), ICP22, ICP27, and UL55 genes. In some embodiments, the inactivating mutation in the ICP0 (one or both copies), ICP4 (one or both copies), ICP22, ICP27, and / or UL55 gene comprises a deletion of the coding sequence of the ICP0 (one or both copies), ICP4 (one or both copies), ICP22, ICP27, and / or UL55 gene. In some embodiments, the recombinant herpes simplex virus genome further comprises an inactivating mutation in the ICP47 gene and / or the UL41 gene.

[0124] In some embodiments, the recombinant herpes simplex virus genome comprises one or more polynucleotides of the present disclosure within one, two, three, four, five, six, seven, or more viral loci. Examples of suitable viral loci may include, but are not limited to, ICP0 (one or both copies), ICP4 (one or both copies), ICP22, ICP27, ICP47, tk, UL41, and / or UL55 herpes simplex virus loci. In some embodiments, the recombinant herpes simplex virus genome comprises one or more polynucleotides of the present disclosure within one or both viral ICP4 loci (e.g., a recombinant virus comprising a polynucleotide encoding a SPINK polypeptide within one or both ICP4 loci). In some embodiments, the recombinant herpes simplex virus genome comprises one or more polynucleotides of the present disclosure within the viral ICP22 locus (e.g., a recombinant virus comprising a polynucleotide encoding a SPINK polypeptide within the ICP22 locus). In some embodiments, a recombinant herpes simplex virus genome comprises one or more polynucleotides of this disclosure within the viral UL41 locus (e.g., a recombinant virus comprising a polynucleotide encoding a SPINK polypeptide within the UL41 locus). In some embodiments, a recombinant herpes simplex virus genome comprises one or more polynucleotides of this disclosure within the viral ICP47 locus (e.g., a recombinant virus comprising a polynucleotide encoding a SPINK polypeptide within the ICP47 locus). In some embodiments, a recombinant herpes simplex virus genome comprises one or more polynucleotides of this disclosure within one or both of the viral ICP4 loci and one or more polynucleotides of this disclosure within the viral ICP22 locus (e.g., a recombinant virus comprising a polynucleotide encoding a SPINK polypeptide in one or both of the ICP4 loci and a polynucleotide encoding the same or different SPINK polypeptide in the ICP22 locus).In some embodiments, a recombinant herpes simplex virus genome comprises one or more polynucleotides of this disclosure within one or both of the viral ICP4 loci and comprises one or more polynucleotides of this disclosure within the viral UL41 locus (e.g., a recombinant virus comprising a polynucleotide encoding a SPINK polypeptide at one or both of the ICP4 loci and a polynucleotide encoding the same or different SPINK polypeptide at the UL41 locus). In some embodiments, a recombinant herpes simplex virus genome comprises one or more polynucleotides of this disclosure within the viral ICP22 locus and comprises one or more polynucleotides of this disclosure within the viral UL41 locus (e.g., a recombinant virus comprising a polynucleotide encoding a SPINK polypeptide at the ICP22 locus and a polynucleotide encoding the same or different SPINK polypeptide at the UL41 locus). In some embodiments, a recombinant herpes simplex virus genome comprises one or more polynucleotides of the disclosure within one or both of the viral ICP4 loci, comprises one or more polynucleotides of the disclosure within the viral ICP22 locus, and comprises one or more polynucleotides of the disclosure within the viral UL41 locus (e.g., a recombinant virus that comprises a polynucleotide encoding a SPINK polypeptide at one or both of the ICP4 loci, a polynucleotide encoding the same or a different SPINK polypeptide at the ICP22 locus, and a polynucleotide encoding a SPINK polypeptide at the UL41 locus (which may be the same SPINK polypeptide as the SPINK polypeptide encoded at the ICP4 locus and / or the ICP22 locus, or may be a different SPINK polypeptide than the SPINK polypeptide encoded at the ICP4 locus and / or the ICP22 locus).

[0125] In some embodiments, the recombinant herpesvirus genome (e.g., recombinant herpes simplex virus genome) is engineered to reduce or eliminate expression of one or more herpesvirus genes (e.g., one or more toxic herpesvirus genes), such as one or both copies of the HSV ICP0 gene, one or both copies of the HSV ICP4 gene, the HSV ICP22 gene, the HSV UL41 gene, the HSV ICP27 gene, etc. In some embodiments, the recombinant herpesvirus genome (e.g., recombinant herpes simplex virus genome) is engineered to reduce the cytotoxicity of the recombinant genome compared to the corresponding wild-type herpesvirus genome (e.g., wild-type herpes simplex virus genome) (e.g., when introduced into a target cell). In some embodiments, the target cell is a human cell. In some embodiments, the target cell is an epidermal and / or dermal cell (e.g., a human epidermal cell and / or a human dermal cell). In some embodiments, the target cell is a keratinocyte or a fibroblast (e.g., a human keratinocyte or a human fibroblast).In some embodiments, the cytotoxicity (e.g., in human keratinocytes and / or fibroblasts) of the recombinant genome (e.g., a recombinant herpes simplex virus genome) is at least about 5%, at least about 10%, at least about 15%, at least about 20%, 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 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80% compared to the corresponding wild-type herpes virus genome. , is reduced by at least about 85%, at least about 90%, at least about 95%, or at least about 99% (e.g., measuring the relative cytotoxicity of recombinant ΔICP4 (one or both copies) herpes simplex virus genome versus wild-type herpes simplex virus genome in human keratinocytes or fibroblasts (primary cells or cell lines), measuring the relative cytotoxicity of recombinant ΔICP4 (one or both copies) / ΔICP22 herpes simplex virus genome versus wild-type herpes simplex virus genome in human keratinocytes or fibroblasts (primary cells or cell lines), etc.).In some embodiments, the cytotoxicity (e.g., in human keratinocytes and / or fibroblasts) of the recombinant genome (e.g., a recombinant herpes simplex virus genome) is at least about 1.5-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, at least about 25-fold, at least about 50-fold, at least about 75-fold, at least about 100-fold, at least about 250-fold, at least about 500-fold, at least about 750-fold, at least about 1000-fold, or more (e.g., measuring the relative cytotoxicity of recombinant ΔICP4 (one or both copies) herpes simplex virus genome versus wild-type herpes simplex virus genome in human keratinocytes or fibroblasts (primary cells or cell lines), measuring the relative cytotoxicity of recombinant ΔICP4 (one or both copies) / ΔICP22 herpes simplex virus genome versus wild-type herpes simplex virus genome in human keratinocytes or fibroblasts (primary cells or cell lines), etc.). Methods for measuring cytotoxicity are known to those of skill in the art, including, for example, the use of vital dyes (formazan dyes), protease biomarkers, MTT assays (or assays using related tetrazolium salts such as XTT, MTS, water-soluble tetrazolium salts), measuring ATP content, etc.

[0126] In some embodiments, the recombinant herpesvirus genome (e.g., a recombinant herpes simplex virus genome) is engineered to reduce its effect on host cell proliferation following exposure of a target cell to the recombinant genome, as compared to a corresponding wild-type herpesvirus genome (e.g., a wild-type herpes simplex virus genome). In some embodiments, the target cell is a human cell. In some embodiments, the target cell is an epidermal and / or dermal cell (e.g., a human epidermal cell and / or a human dermal cell). In some embodiments, the target cell is a keratinocyte or a fibroblast (e.g., a human keratinocyte or a human fibroblast). In some embodiments, host cell proliferation (e.g., human keratinocytes and / or fibroblasts) following exposure to the recombinant genome is at least about 5%, at least about 10%, at least about 15%, at least about 20%, 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 55%, 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%, or at least about 99% faster (e.g., measuring the relative cell proliferation following exposure to a recombinant ΔICP4 (one or both copies) herpes simplex virus genome relative to the cell proliferation following exposure to a wild-type herpes simplex virus genome in human keratinocytes or fibroblasts (primary cells or cell lines), measuring the relative cell proliferation following exposure to a recombinant ΔICP4 (one or both copies) / ΔICP22 herpes simplex virus genome relative to the cell proliferation following exposure to a wild-type herpes simplex virus genome in human keratinocytes or fibroblasts (primary cells or cell lines), etc.).In some embodiments, host cell proliferation (e.g., human keratinocytes and / or fibroblasts) following exposure to the recombinant genome is at least about 1.5-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, at least about 25-fold, at least about 50-fold, at least about 75-fold, at least about 100-fold, at least about 250-fold, at least about 500-fold, , at least about 750 times, or at least about 1000 times faster (e.g., measuring the relative cell proliferation following exposure to recombinant ΔICP4 (one or both copies) herpes simplex virus genome versus the cell proliferation following exposure to wild-type herpes simplex virus genome in human keratinocytes or fibroblasts (primary cells or cell lines), measuring the relative cell proliferation following exposure to recombinant ΔICP4 (one or both copies) / ΔICP22 herpes simplex virus genome versus the cell proliferation following exposure to wild-type herpes simplex virus genome in human keratinocytes or fibroblasts (primary cells or cell lines), etc.). Methods for measuring cell proliferation are known to those of skill in the art, including, for example, using Ki67 cell proliferation assays, BrdU cell proliferation assays, etc.

[0127] A vector (e.g., a herpes virus vector) can include one or more polynucleotides of the present disclosure in a form suitable for expression of the polynucleotide in a host cell. The vector can include one or more regulatory sequences operably linked to the polynucleotide to be expressed (e.g., as described above).

[0128] In some embodiments, a recombinant nucleic acid of the present disclosure (e.g., a recombinant herpesvirus genome, such as a recombinant herpes simplex virus genome) comprises one or more of the polynucleotides described herein inserted into the recombinant nucleic acid in any orientation. When a recombinant nucleic acid comprises two or more (e.g., two or more, three or more, etc.) polynucleotides described herein, the polynucleotides may be inserted in the same orientation or in opposite orientation to each other. Without wishing to be bound by theory, incorporating two polynucleotides (e.g., two transgenes) into a recombinant nucleic acid (e.g., a vector) in antisense orientation may help avoid read-through and ensure proper expression of each polynucleotide.

[0129] In some embodiments, the disclosure relates to one or more heterologous polynucleotides (eg, bacterial artificial chromosomes (BACs)) that comprise any of the recombinant nucleic acids described herein.

[0130] IV. Viruses Certain aspects of the disclosure relate to viruses comprising any of the polynucleotides and / or recombinant nucleic acids described herein. In some embodiments, the viruses are capable of infecting one or more target cells of a subject (e.g., a human). In some embodiments, the viruses are suitable for delivering the polynucleotides and / or recombinant nucleic acids to one or more target cells of a subject (e.g., a human). In some embodiments, the disclosure relates to one or more viral particles comprising any of the polynucleotides and / or recombinant nucleic acids described herein. In some embodiments, the one or more target cells are one or more human cells. In some embodiments, the one or more target cells are one or more cells having a SPINK deficiency (e.g., one or more cells comprising a loss-of-function mutation in a native SPINK gene, such as the SPINK5 gene, or a pathogenic variant thereof). In some embodiments, the one or more target cells are one or more skin cells (e.g., one or more epidermal, dermal, and / or subcutaneous cells). In some embodiments, the one or more target cells are epidermal and / or dermal cells (e.g., human epidermal and / or dermal cells). In some embodiments, the one or more target cells are selected from keratinocytes, melanocytes, Langerhans cells, Merkel cells, mast cells, fibroblasts, and / or adipocytes. In some embodiments, the one or more target cells are keratinocytes. In some embodiments, the one or more target cells are present in the stratum corneum, stratum granulosum, stratum spinosum, stratum basale, and / or basement membrane. In some embodiments, the one or more target cells are one or more epidermal cells. In some embodiments, the one or more target cells are one or more dermal cells.

[0131] Any suitable virus known in the art may be used, including, for example, adenovirus, adeno-associated virus, retrovirus, lentivirus, Sendai virus, papilloma virus, herpes virus (e.g., herpes simplex virus), vaccinia virus, and / or any hybrid or derivative virus thereof. In some embodiments, the virus is attenuated. In some embodiments, the virus is replication-deficient. In some embodiments, the virus is replication-competent. In some embodiments, the virus is modified to alter its tissue tropism compared to the tissue tropism of the corresponding unmodified wild-type virus. In some embodiments, the virus has reduced cytotoxicity compared to the corresponding wild-type virus. Methods for producing viruses containing recombinant nucleic acids are well known to those of skill in the art.

[0132] In some embodiments, the virus is a member of the Herpesviridae family of DNA viruses, including, for example, herpes simplex virus, varicella zoster virus, human cytomegalovirus, herpes virus 6A, herpes virus 6B, herpes virus 7, and Kaposi's sarcoma-associated herpes virus. In some embodiments, the herpes virus is attenuated. In some embodiments, the herpes virus is replication-deficient. In some embodiments, the herpes virus is replication-competent. In some embodiments, the herpes virus has reduced cytotoxicity compared to the corresponding wild-type herpes virus. In some embodiments, the herpes virus is not oncolytic.

[0133] In some embodiments, the herpes virus is a herpes simplex virus. The herpes simplex virus comprising the recombinant nucleic acid may be produced, for example, by the processes disclosed in WO2015 / 009952 and / or WO2017 / 176336. In some embodiments, the herpes simplex virus is attenuated. In some embodiments, the herpes simplex virus is replication-deficient. In some embodiments, the herpes simplex virus is replication-competent. In some embodiments, the herpes simplex virus is an HSV-1 virus, an HSV-2, or any derivative thereof. In some embodiments, the herpes simplex virus is an HSV-1 virus. In some embodiments, the HSV-1 is attenuated. In some embodiments, the HSV-1 is replication-deficient. In some embodiments, the HSV-1 is replication-competent. In some embodiments, the herpes simplex virus (e.g., HSV-1) has reduced cytotoxicity compared to the corresponding wild-type herpes simplex virus (e.g., wild-type HSV-1). In some embodiments, the herpes simplex virus (eg, HSV-1) is not oncolytic.

[0134] In some embodiments, the herpes simplex virus is modified to alter its tissue tropism compared to the tissue tropism of the unmodified wild-type herpes simplex virus. In some embodiments, the herpes simplex virus comprises a modified envelope. In some embodiments, the modified envelope comprises one or more (e.g., one or more, two or more, three or more, four or more, etc.) mutant herpes simplex virus glycoproteins. Examples of herpes simplex virus glycoproteins may include, but are not limited to, glycoproteins gB, gC, gD, gH, and gL. In some embodiments, the modified envelope alters herpes simplex virus tissue tropism compared to the wild-type herpes simplex virus.

[0135] In some embodiments, the transduction efficiency (in vitro and / or in vivo) of a virus of the present disclosure (e.g., a herpes virus, such as a herpes simplex virus) on one or more target cells (e.g., one or more human keratinocytes and / or fibroblasts) is at least about 25%. For example, the transduction efficiency of the virus on one or more target cells can be 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 55%, 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 99%, at least about 99.5%, or more. In some embodiments, the virus is a herpes simplex virus and the transduction efficiency of the virus on one or more target cells (e.g., one or more human keratinocytes and / or fibroblasts) is about 85% to about 100%. In some embodiments, the virus is a herpes simplex virus, and the viral transduction efficiency for one or more target cells (e.g., one or more human keratinocytes and / or fibroblasts) is 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%, or 100%. Methods for measuring viral transduction efficiency in vitro or in vivo are well known to those skilled in the art, including, for example, qPCR analysis, deep sequencing, Western blotting, quantitative fluorescence analysis (fluorescent in situ hybridization (FISH), fluorescent reporter gene expression, immunofluorescence, FACS, etc.), and the like.

[0136] V. Pharmaceutical Compositions and Formulations Certain aspects of the present disclosure relate to pharmaceutical compositions and formulations comprising any of the recombinant nucleic acids (e.g., recombinant herpesvirus genomes) and / or viruses (e.g., herpes viruses comprising a recombinant genome, such as herpes simplex viruses comprising a recombinant herpes simplex virus genome) described herein, and a pharma- ceutically acceptable excipient or carrier.

[0137] In some embodiments, the pharmaceutical composition or formulation comprises any one or more of the viruses (e.g., herpes viruses) described herein. In some embodiments, the pharmaceutical composition or formulation comprises about 10 4 ~about 10 12 For example, the pharmaceutical composition or formulation may contain about 10 plaque forming units (PFU) / mL of virus. 4 ~about 10 12 , about 10 5 ~about 10 12 , about 10 6 ~about 10 12 , about 10 7 ~about 10 12 , about 10 8 ~about 10 12 , about 10 9 ~about 10 12 , about 10 10 ~about 10 12 , about 10 11 ~about 10 12 , about 10 4 ~about 10 11 , about 10 5 ~about 10 11 , about 10 6 ~about 10 11 , about 10 7 ~about 10 11 , about 10 8 ~about 10 11 , about 10 9 ~about 10 11 , about 10 10 ~about 10 11 , about 10 4 ~about 10 10 , about 10 5 ~about 10 10 , about 10 6 ~about 10 10 , about 10 7 ~about 1010 , about 10 8 ~about 10 10 , about 10 9 ~about 10 10 , about 10 4 ~about 10 9 , about 10 5 ~about 10 9 , about 10 6 ~about 10 9 , about 10 7 ~about 10 9 , about 10 8 ~about 10 9 , about 10 4 ~about 10 8 , about 10 5 ~about 10 8 , about 10 6 ~ approx. 108, approx. 10 7 ~about 10 8 , about 10 4 ~about 10 7 , about 10 5 ~about 10 7 , about 10 6 ~about 10 7 , about 10 4 ~about 10 6 , about 10 5 ~about 10 6 , or about 10 4 ~about 10 5 In some embodiments, the pharmaceutical composition or formulation may contain about 10 PFU / mL of virus. 4 , about 10 5 , about 10 6 , about 10 7 , about 10 8 , about 10 9 , about 10 10 , about 10 11 , or about 10 12 Contains PFU / mL of virus.

[0138] Pharmaceutical compositions and formulations can be prepared by mixing the active ingredient(s) (such as recombinant nucleic acids and / or viruses) having the desired degree of purity with one or more pharma- ceutically acceptable carriers or excipients. Pharmaceutically acceptable carriers or excipients are generally non-toxic to recipients at the dosages and concentrations employed, and may include buffers (such as phosphate, citric acid, acetic acid, and other organic acids), antioxidants (such as ascorbic acid and methionine), preservatives (such as octadecyldimethylbenzyl ammonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butyl or benzyl alcohol, alkyl parabens, catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol), amino acids (such as glycine, glutamine, asparagine, histidine, arginine, or lysine), low molecular weight (less than about 10 residues) polypeptides, and the like. , proteins (such as serum albumin, gelatin, or immunoglobulins), polyols (such as glycerol, e.g., formulations containing 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, etc. glycerol), hydrophilic polymers (such as polyvinylpyrrolidone), monosaccharides, disaccharides, and other carbohydrates (including glucose, mannose, or dextrins), chelating agents (such as EDTA), sugars (such as sucrose, mannitol, trehalose, or sorbitol), salt-forming counterions (such as sodium), metal complexes (such as Zn-protein complexes), and / or non-ionic surfactants (such as polyethylene glycol (PEG)). A thorough discussion of pharma- ceutically acceptable carriers is available in REMINGTON'S PHARMACEUTICAL SCIENCES (Mack Pub. Co., NJ 1991).

[0139] In some embodiments, the pharmaceutical composition or formulation comprises one or more lipid (e.g., cationic lipid) carriers. In some embodiments, the pharmaceutical composition or formulation comprises one or more nanoparticle carriers. Nanoparticles are submicron (less than about 1000 nm) sized drug delivery vehicles that can carry encapsulated drugs (such as synthetic small molecules, proteins, peptides, cells, viruses, and nucleic acid-based biological therapeutics) for immediate or controlled release. A variety of molecules (e.g., proteins, peptides, recombinant nucleic acids, etc.) can be efficiently encapsulated in nanoparticles using processes well known in the art. In some embodiments, a molecule "encapsulated" in a nanoparticle can refer to a molecule (such as a virus) that is contained in the nanoparticle, attached to and / or associated with the surface of the nanoparticle, or any combination thereof. Nanoparticles for use in the compositions or formulations described herein can be any type of biocompatible nanoparticle known in the art, including, for example, nanoparticles comprising poly(lactic acid), poly(glycolic acid), PLGA, PLA, PGA, and any combination thereof (see, e.g., Vauthier et al. Adv Drug Del Rev. (2003) 55:519-48, US2007 / 0148074, US2007 / 0092575, US2006 / 0246139, US5753234, US7081483, and WO2006 / 052285).

[0140] In some embodiments, the pharma- ceutically acceptable carrier or excipient may be adapted for or suitable for any route of administration known in the art, including, for example, intravenous, intramuscular, subcutaneous, dermal, oral, nasal, intratracheal, sublingual, buccal, topical, transdermal, intradermal, intraperitoneal, intraorbital, intravitreal, subretinal, transmucosal, intraarticular, by implant, by inhalation, intrathecal, intraventricular, and / or intranasal administration. In some embodiments, the pharmaceutical composition or formulation is adapted for or suitable for any route of administration known in the art, including, for example, intravenous, intramuscular, subcutaneous, dermal, oral, nasal, intratracheal, sublingual, buccal, topical, transdermal, intradermal, intraperitoneal, intraorbital, intravitreal, subretinal, transmucosal, intraarticular, by implant, by inhalation, intrathecal, intraventricular, and / or intranasal administration. In some embodiments, the pharma- ceutically acceptable carrier or excipient is adapted or suitable for topical, transdermal, subcutaneous, intradermal, and / or transmucosal administration. In some embodiments, the pharmaceutical composition or formulation is adapted or suitable for topical, transdermal, subcutaneous, intradermal, and / or transmucosal administration. In some embodiments, the pharma- ceutical composition or formulation is adapted or suitable for topical, transdermal, and / or intradermal administration. In some embodiments, the pharmaceutical composition or formulation is adapted or suitable for topical, transdermal, and / or intradermal administration. In some embodiments, the pharma- ceutical acceptable carrier or excipient is adapted or suitable for topical administration. In some embodiments, the pharmaceutical composition or formulation is adapted or suitable for topical administration.

[0141] Examples of carriers or excipients adapted or suitable for use in pharmaceutical compositions or formulations of the present disclosure may include, but are not limited to, ointments, oils, pastes, creams, aerosols, suspensions, emulsions, fatty ointments, gels (e.g., methylcellulose gels such as carboxymethylcellulose, hydroxypropylmethylcellulose, etc.), powders, liquids, lotions, solutions, sprays, patches (e.g., transdermal or microneedle patches), adhesive strips, microneedles or microneedle arrays, and inhalants. In some embodiments, the carrier or excipient (e.g., a pharma- ceutically acceptable carrier or excipient) comprises one or more (e.g., one or more, two or more, three or more, four or more, five or more, etc.) of ointments, oils, pastes, creams, aerosols, suspensions, emulsions, fatty ointments, gels, powders, liquids, lotions, solutions, sprays, patches, adhesive strips, and inhalants. In some embodiments, the carrier comprises a patch (e.g., a patch that adheres to the skin), such as a transdermal or microneedle patch. In some embodiments, the carrier comprises a microneedle or a microneedle array. Methods for making and using microneedle arrays suitable for delivery of compositions are generally known in the art (see, for example, Kim Y. et al. "Microneedles for drug and vaccine delivery". Advanced Drug Delivery Reviews 2012, 64(14):1547-68).

[0142] In some embodiments, the pharmaceutical composition or formulation further comprises one or more additional ingredients. Examples of additional ingredients include binders (such as pregelatinized maize starch, polyvinylpyrrolidone, or hydroxypropyl methylcellulose), fillers (such as lactose and other sugars, microcrystalline cellulose, pectin, gelatin, calcium sulfate, ethylcellulose, polyacrylates, or calcium hydrogen phosphate), lubricants (such as magnesium stearate, talc, silica, colloidal silicon dioxide, stearic acid, metallic stearates, hydrogenated vegetable oils, maize starch, polyethylene glycol, sodium benzoate, etc.), and mixtures thereof. Examples of suitable polynucleotides that may be used include, but are not limited to, disintegrants (e.g., sodium acetate, etc.), disintegrants (e.g., starch, sodium starch glycolate, etc.), wetting agents (e.g., sodium lauryl sulfate, etc.), salt solutions, alcohol, polyethylene glycol, gelatin, lactose, amylase, magnesium stearate, talc, silicic acid, viscous paraffin, methylcellulose, polyvinylpyrrolidone, sweeteners, flavorings, fragrances, colorants, moisturizers, sunscreens, antibacterial agents, agents that can stabilize polynucleotides or prevent their degradation, and the like. In some embodiments, the pharmaceutical composition or formulation comprises a methylcellulose gel, such as a carboxymethylcellulose gel, a hydroxypropylmethylcellulose gel, etc. (e.g., about 0.5%, about 1%, about 1.5%, about 2%, about 2.5%, about 3%, about 3.5%, about 4%, about 4.5%, about 5%, about 5.5%, about 6%, about 6.5%, about 7%, about 7.5%, about 8%, about 8.5%, about 9%, about 9.5%, about 10%, about 10.5%, about 11%, about 11.5%, about 12%, etc.). In some embodiments, the pharmaceutical composition or formulation comprises a phosphate buffer. In some embodiments, the pharmaceutical composition or formulation comprises glycerol (e.g., about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, etc.). In some embodiments, the pharmaceutical composition or formulation comprises a methylcellulose gel (such as a carboxymethylcellulose gel, a hydroxypropylmethylcellulose gel), a phosphate buffer, and / or glycerol.

[0143] Compositions and formulations (e.g., pharmaceutical compositions and formulations) to be used for in vivo administration are generally sterile. Sterilization may be readily accomplished, for example, by filtration through sterile filtration membranes.

[0144] In some embodiments, any of the recombinant nucleic acids, viruses, and / or pharmaceutical compositions or formulations described herein may be used to deliver one or more polynucleotides encoding a SPINK polypeptide to one or more cells of a subject (e.g., one or more SPINK-deficient cells, one or more cells having a SPINK gene mutation, etc.). In some embodiments, any of the recombinant nucleic acids, viruses, and / or pharmaceutical compositions or formulations may be used in therapy. In some embodiments, any of the recombinant nucleic acids, viruses, and / or pharmaceutical compositions or formulations described herein may be used in the treatment of a disease or condition that would benefit from expression of a SPINK polypeptide (e.g., a disease associated with a SPINK deficiency and / or a disease associated with a SPINK gene mutation). In some embodiments, any of the recombinant nucleic acids, viruses, and / or pharmaceutical compositions or formulations described herein may be used to provide preventative, palliative, or therapeutic relief of one or more signs or symptoms of Netherton syndrome (e.g., by delivery of a SPINK5 polypeptide), atopic dermatitis (e.g., by delivery of a SPINK5 polypeptide), hereditary pancreatitis (PCTT) (e.g., by delivery of a SPINK1 polypeptide), tropical calcific pancreatitis (e.g., by delivery of a SPINK1 polypeptide), and / or spermatogenesis failure 29 (SPGF29) (e.g., by delivery of a SPINK2 polypeptide). In some embodiments, any of the recombinant nucleic acids, viruses, and / or pharmaceutical compositions or formulations described herein may be used to treat Netherton syndrome (e.g., by delivery of a human SPINK5 polypeptide). In some embodiments, any of the recombinant nucleic acids, viruses, and / or pharmaceutical compositions or formulations described herein may be used to treat atopic dermatitis (e.g., by delivery of a human SPINK5 polypeptide).

[0145] In some embodiments, any of the recombinant nucleic acids, viruses, and / or pharmaceutical compositions or formulations described herein may be used in the preparation or manufacture of a medicament. In some embodiments, any of the recombinant nucleic acids, viruses, and / or pharmaceutical compositions or formulations described herein may be used in the preparation or manufacture of a medicament useful for delivering one or more polynucleotides encoding a SPINK polypeptide to one or more cells of a subject (e.g., one or more SPINK-deficient cells, one or more cells having a SPINK gene mutation, etc.). In some embodiments, any of the recombinant nucleic acids, viruses, and / or pharmaceutical compositions or formulations described herein may be used in the preparation or manufacture of a medicament useful for treating a disease or condition that would benefit from expression of a SPINK polypeptide (e.g., a disease associated with a SPINK deficiency and / or a disease associated with a SPINK gene mutation). In some embodiments, any of the recombinant nucleic acids, viruses, and / or pharmaceutical compositions or formulations described herein may be used in the preparation or manufacture of a medicament useful for treating Netherton syndrome (e.g., by delivery of a SPINK5 polypeptide), atopic dermatitis (e.g., by delivery of a SPINK5 polypeptide), hereditary pancreatitis (PCTT) (e.g., by delivery of a SPINK1 polypeptide), tropical calcific pancreatitis (e.g., by delivery of a SPINK1 polypeptide), and / or spermatogenesis failure 29 (SPGF29) (e.g., by delivery of a SPINK2 polypeptide). In some embodiments, any of the recombinant nucleic acids, viruses, and / or pharmaceutical compositions or formulations described herein may be used in the preparation or manufacture of a medicament useful for treating Netherton syndrome (e.g., by delivery of a human SPINK5 polypeptide). In some embodiments, any of the recombinant nucleic acids, viruses, and / or pharmaceutical compositions or formulations described herein may be used in the preparation or manufacture of a medicament useful for treating atopic dermatitis (e.g., by delivery of a human SPINK5 polypeptide).

[0146] VI. Method Certain aspects of the present disclosure relate to enhancing, increasing, increasing, enhancing, and / or replenishing levels of a SPINK polypeptide in one or more cells of a subject, comprising administering to the subject an effective amount of any of the recombinant nucleic acids, viruses, agents, and / or pharmaceutical compositions or formulations described herein. In some embodiments, the SPINK polypeptide is a human SPINK polypeptide. In some embodiments, the SPINK polypeptide is a SPINK5 polypeptide. In some embodiments, the SPINK5 polypeptide is a human SPINK5 polypeptide. In some embodiments, the subject is human. In some embodiments, the subject's genome comprises a mutation (e.g., loss-of-function mutation, pathogenic variant) in an endogenous SPINK gene (e.g., an endogenous SPINK5 gene). In some embodiments, the subject is afflicted with Netherton syndrome. In some embodiments, the subject is afflicted with atopic dermatitis.

[0147] In some embodiments, administration of a recombinant nucleic acid, virus, agent, and / or pharmaceutical composition or formulation to a subject increases SPINK (e.g., SPINK5) levels (transcript or protein levels) in one or more contacted or treated cells of the subject by at least about two-fold compared to endogenous SPINK levels in one or more corresponding untreated cells of the subject. For example, administration of a recombinant nucleic acid, virus, agent, and / or pharmaceutical composition or formulation may increase SPINK (e.g., SPINK5) levels (transcript or protein levels) in one or more contacted or treated cells of a subject by at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, at least about 25-fold, at least about 50-fold, at least about 75-fold, at least about 100-fold, at least about 250-fold, at least about 500-fold, at least about 750-fold, at least about 1000-fold, or more, compared to endogenous SPINK levels in one or more corresponding untreated cells of the subject. In some embodiments, the one or more contacted or treated cells are one or more epidermal and / or dermal cells (e.g., keratinocytes). Methods for measuring transcript or protein levels from a sample are well known to those of skill in the art, including, for example, qPCR, Western blot, mass spectrometry, and the like.

[0148] In some embodiments, administering to an individual an effective amount of any of the recombinant nucleic acids, viruses, agents, and / or pharmaceutical compositions or formulations described herein strengthens, increases, enhances, and / or replenishes the skin integrity and / or protective barrier function of the subject. In some embodiments, administering to an individual an effective amount of any of the recombinant nucleic acids, viruses, agents, and / or pharmaceutical compositions or formulations described herein reduces, enhances, and / or inhibits one or more of kallikrein-5 (KLK5), kallikrein-7 (KLK7), kallikrein-14 (KLK14), caspase-14 (CASP14), and / or trypsin in the subject (e.g., reduces, enhances, and / or inhibits one or more activities (e.g., proteolytic activity) of KLK5, KLK7, KLK14, CASP14, and / or trypsin).

[0149] Other aspects of the present disclosure relate to enhancing, increasing, enhancing, and / or replenishing anti-inflammatory and / or antimicrobial protection of mucosal epithelia in a subject in need thereof, comprising administering to the subject an effective amount of any of the recombinant nucleic acids, viruses, agents, and / or pharmaceutical compositions or formulations described herein. In some embodiments, administration of the recombinant nucleic acid, virus, agent, and / or pharmaceutical composition reduces the subject's susceptibility to skin infections.

[0150] Other aspects of the present disclosure relate to suppressing or inhibiting desquamation in a subject in need thereof, comprising administering to the subject an effective amount of any of the recombinant nucleic acids, viruses, agents, and / or pharmaceutical compositions or formulations described herein.

[0151] Another aspect of the present disclosure relates to reducing or treating skin barrier defects in a subject in need thereof, comprising administering to the subject an effective amount of any of the recombinant nucleic acids, viruses, agents, and / or pharmaceutical compositions or formulations described herein. In some embodiments, the skin barrier defect is transepidermal water loss (TEWL), also referred to as transepithelial water loss. In some embodiments, the method of the present disclosure reduces transepidermal water loss in a subject in need thereof. Methods for measuring skin barrier function, including TEWL, are well known to those skilled in the art, including, for example, any of the methods described by Antonov et al. (Curr Probl Dermatol. 2016; 49: 61-70).

[0152] Other aspects of the disclosure relate to providing prophylactic, palliative, or therapeutic alleviation of one or more signs or symptoms of Netherton syndrome in a subject in need thereof, comprising administering to the subject an effective amount of any of the recombinant nucleic acids, viruses, agents, and / or pharmaceutical compositions or formulations described herein. In some embodiments, the subject's genome comprises a mutation (e.g., a loss-of-function mutation, a pathogenic variant) in the endogenous SPINK5 gene. In some embodiments, the recombinant nucleic acid, virus, agent, and / or pharmaceutical composition or formulation comprises one or more polynucleotides encoding a human SPINK5 polypeptide.

[0153] Signs or symptoms of Netherton syndrome may include, but are not limited to, defective keratinization, skin barrier defects, chronic skin inflammation, generalized pruritus (itching), severe dehydration, poor growth, intussusceptible trichorrhexis and / or trichorrhexis nodosa (hair shaft defects, also known as bamboo hairs), fluid leakage from the skin, development of annular lesions on the skin, eczema, increased susceptibility to infections (especially infections of the skin, including recurrent skin infections with Staphylococcus), increased susceptibility to allergies, development of scaly / red skin (similar to atopic dermatitis), development of flexural linear ichthyosis and / or ichthyosa-like erythroderma, changes in immunoglobulin levels (typically high IgE and low to normal IgG immunoglobulins), immature natural killer cells (which have reduced lytic function), and difficulty regulating body temperature.

[0154] Other aspects of the present disclosure relate to providing preventative, palliative, or therapeutic relief of one or more signs or symptoms of atopic dermatitis in a subject in need thereof, comprising administering to the subject an effective amount of any of the recombinant nucleic acids, viruses, agents, and / or pharmaceutical compositions or formulations described herein. In some embodiments, the subject's genome comprises a mutation (e.g., a loss-of-function mutation, a pathogenic variant) in the endogenous SPINK5 gene. In some embodiments, the recombinant nucleic acid, virus, agent, and / or pharmaceutical composition or formulation comprises one or more polynucleotides encoding a human SPINK5 polypeptide.

[0155] Signs and symptoms of atopic dermatitis may include, but are not limited to, dry skin; itching, which may be severe especially at night; red to brownish-gray spots especially on the hands, feet, ankles, wrists, neck, upper chest, eyelids, inside the crooks of the elbows and knees, and in infants, the face and scalp; small raised bumps on the skin, which may be moist; skin infections; eyelid dermatitis; cataracts; elevated IgE levels; thickened, cracked, or scaly skin; swollen skin that is raw and sensitive from scratching.

[0156] The recombinant nucleic acid, virus, agent, and / or pharmaceutical composition or formulation described herein may be administered by any suitable method or route known in the art, including, but not limited to, orally, sublingually, buccally, topically, rectally, by inhalation, transdermally, subcutaneously, intradermally, intravenously, intraarterially, intramuscularly, intracardially, intraosseously, intraperitoneally, transmucosally, intravaginally, intravitreally, intraorbitally, subretinally, intraarticularly, periarticularly, topically, epicutaneously, or any combination thereof. Thus, the present disclosure encompasses methods of delivering any of the recombinant nucleic acid, virus, agent, and / or pharmaceutical composition or formulation described herein to an individual (e.g., an individual having or at risk of developing Netherton syndrome and / or atopic dermatitis).

[0157] In some embodiments, a recombinant nucleic acid, virus, agent, and / or pharmaceutical composition or formulation described herein is administered topically, transdermally, subcutaneously, intradermally, or transmucosally to a subject. In some embodiments, a recombinant nucleic acid, virus, agent, and / or pharmaceutical composition or formulation is administered topically, transdermally, or intradermally to a subject. In some embodiments, a recombinant nucleic acid, virus, agent, and / or pharmaceutical composition or formulation is administered topically or intradermally to a subject. In some embodiments, a recombinant nucleic acid, virus, agent, and / or pharmaceutical composition or formulation is administered topically to a subject.

[0158] In some embodiments, the recombinant nucleic acid, virus, agent, and / or pharmaceutical composition or formulation is administered to the subject once, hi some embodiments, the recombinant nucleic acid, virus, agent, and / or pharmaceutical composition or formulation is administered to the subject at least twice (e.g., at least twice, at least three times, at least four times, at least five times, at least ten times, etc.). In some embodiments, at least one hour (e.g., at least about 1 hour, at least about 6 hours, at least about 12 hours, at least about 18 hours, at least about 1 day, at least about 2 days, at least about 3 days, at least about 4 days, at least about 5 days, at least about 6 days, at least about 7 days, at least about 15 days, at least about 20 days, at least about 30 days, at least about 40 days, at least about 50 days, at least about 60 days, at least about 70 days, at least about 80 days, at least about 90 days, at least about 100 days, at least about 120 days, etc.) passes between administrations (e.g., between a first administration and a second administration, between a second administration and a third administration, etc.). In some embodiments, the recombinant nucleic acid, virus, agent, and / or pharmaceutical composition or formulation is administered to a subject once, twice, three times, four times, five times, or more per day. In some embodiments, the recombinant nucleic acid, virus, agent, and / or pharmaceutical composition or formulation is administered to one or more affected areas (e.g., one or more areas of the skin exhibiting one or more signs or symptoms of Netherton syndrome and / or atopic dermatitis) and / or non-affected areas of a subject.

[0159] In some embodiments, one or more parts of the skin of the target are abraded or made more permeable before treatment.Any suitable method of abrading or increasing the permeability of skin can be used, including, for example, using a skin roller, repeatedly using adhesive strips to remove skin cell layers (tape stripping), scraping with a scalpel or blade, using sandpaper, using chemical permeation enhancers or electrical energy, using sonic or ultrasonic energy, using light (e.g., laser) energy, using micron-sized needles or blades that do not completely penetrate the epidermis but have suitable length to pierce it, etc.

[0160] VII.Host cells Certain aspects of the present disclosure relate to one or more host cells that contain any of the recombinant nucleic acids described herein, such as prokaryotic cells, including eubacteria, such as gram-negative or gram-positive organisms, such as Escherichia (e.g., E. coli), Enterobacter, Erminia, Klebsiella, Proteus, Salmonella (e.g., S. typhimurium), Serratia (e.g., Serratia marcescens), and the like. Enterobacteriaceae such as S. marcescans and Shigella, and Bacilli such as B. subtilis and B. licheniformis, fungal cells (e.g., S. cerevisiae), insect cells (e.g., S2 cells), and mammalian cells (e.g., SV40 transformed monkey kidney strain CV1 (COS-7, ATCC 612626, USA)). CRL 1651), human embryonic kidney line (293 or 293 cells subcloned for growth in suspension culture), baby hamster kidney cells (BHK, ATCC CCL 10), mouse Sertoli cells (TM4), monkey kidney cells (CV1 ATCC CCL 70), African green monkey kidney cells (VERO-76, ATCC CRL-1587), human cervical carcinoma cells (HELA, ATCC CCL 2), canine kidney cells (MDCK, ATCC CCL 34), buffalo rat hepatocytes (BRL 3A, ATCC CRL 1442), human lung cells (W138, ATCC CCL 75), human hepatocytes (Hep G2, HB 8065), mouse mammary tumor (MMT 060562, ATCC CCL51), TRI cells, MRC5 cells, FS4 cells, human hepatoma (Hep Any suitable host cell (prokaryotic or eukaryotic) known in the art may be used, including human or non-human primate cells, including human hamster ovary (CHO) cells (e.g., DHFR"CHO cells), and myeloma cell lines such as NS0 and Sp2 / 0. In some embodiments, the host cell is a human or non-human primate cell.In some embodiments, the host cell is a cell from a cell line.Suitable host cells or cell line examples may include, but are not limited to, 293, HeLa, SH-Sy5y, Hep G2, CACO-2, A549, L929, 3T3, K562, CHO-K1, MDCK, HUVEC, Vero, N20, COS-7, PSN1, VCaP, CHO cells, etc.

[0161] In some embodiments, the recombinant nucleic acid is a herpes simplex virus vector. In some embodiments, the recombinant nucleic acid is a herpes simplex virus amplicon. In some embodiments, the recombinant nucleic acid is an HSV-1 amplicon or an HSV-1 hybrid amplicon. In some embodiments, a host cell containing a helper virus is contacted with an HSV-1 amplicon or an HSV-1 hybrid amplicon described herein, resulting in the production of a virus containing one or more recombinant nucleic acids described herein. In some embodiments, the virus is collected from the supernatant of the contacted host cell. Methods for producing a virus by contacting a host cell containing a helper virus with an HSV-1 amplicon or an HSV-1 hybrid amplicon are known in the art.

[0162] In some embodiments, the host cell is a complementing host cell. In some embodiments, the complementing host cell expresses one or more genes inactivated in any of the viral vectors described herein. In some embodiments, the complementing host cell is contacted with a recombinant herpesvirus genome (e.g., a recombinant herpes simplex virus genome) described herein. In some embodiments, contacting the complementing host cell with the recombinant herpesvirus genome results in the production of a herpesvirus comprising one or more recombinant nucleic acids described herein. In some embodiments, the virus is collected from the supernatant of the contacted host cell. Methods of producing a virus by contacting a complementing host cell with a recombinant herpes simplex virus are generally described in WO2015 / 009952 and / or WO2017 / 176336.

[0163] VIII. Products or Kits Certain aspects of the present disclosure relate to articles of manufacture or kits that include any of the recombinant nucleic acids, viruses, agents, and / or pharmaceutical compositions or formulations described herein. In some embodiments, the articles of manufacture or kits include a package insert that includes instructions for administration of the recombinant nucleic acid, virus, agent, and / or pharmaceutical composition or formulation to treat SPINK deficiency (e.g., in a subject with a SPINK gene mutation) and / or to provide prophylactic, palliative, or therapeutic relief of one or more signs or symptoms of a disease associated with SPINK deficiency (such as Netherton syndrome and / or atopic dermatitis).

[0164] Suitable containers for the recombinant nucleic acid, virus, drug, and / or pharmaceutical compositions or formulations may include, for example, bottles, vials, bags, tubes, and syringes. The containers may be formed from a variety of materials, such as glass, plastic (such as polyvinyl chloride or polyolefin), or metal alloys (such as stainless steel or Hastelloy). In some embodiments, the containers include a label on or associated with the container, which indicates instructions for use. The article or kit may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, package inserts, and the like.

[0165] IX. Enumerated Embodiments Embodiment 1: A recombinant herpesvirus genome comprising one or more polynucleotides encoding a serine protease inhibitor Kazal type (SPINK) polypeptide.

[0166] Embodiment 2: A recombinant herpesvirus genome as described in embodiment 1, which is capable of replication.

[0167] Embodiment 3: A recombinant herpesvirus genome as described in embodiment 1, which is replication-deficient.

[0168] Embodiment 4: A recombinant herpesvirus genome according to any one of embodiments 1 to 3, wherein the recombinant herpes simplex virus genome comprises the one or more polynucleotides encoding the SPINK polypeptide within one or more viral loci.

[0169] Embodiment 5: The recombinant herpesvirus genome according to any one of embodiments 1 to 4, selected from a recombinant herpes simplex virus genome, a recombinant varicella zoster virus genome, a recombinant human cytomegalovirus genome, a recombinant herpesvirus 6A genome, a recombinant herpesvirus 6B genome, a recombinant herpesvirus 7 genome, a recombinant Kaposi's sarcoma-associated herpesvirus genome, and any derivatives thereof.

[0170] Embodiment 6: A recombinant herpesvirus genome according to any one of embodiments 1 to 5, which is a recombinant herpes simplex virus genome.

[0171] Embodiment 7: The recombinant herpesvirus genome of embodiment 5 or embodiment 6, wherein the recombinant herpes simplex virus genome is a recombinant herpes simplex virus type 1 (HSV-1) genome, a recombinant herpes simplex virus type 2 (HSV-2) genome, or any derivative thereof.

[0172] Embodiment 8: A recombinant herpesvirus genome according to any one of embodiments 1 to 7, wherein the SPINK polypeptide is a human SPINK polypeptide.

[0173] Embodiment 9: A recombinant herpesvirus genome according to any one of embodiments 1 to 7, wherein the SPINK polypeptide is a serine protease inhibitor Kazal type 5 (SPINK5) polypeptide.

[0174] Embodiment 10: A recombinant herpesvirus genome according to any one of embodiments 1 to 9, wherein the SPINK polypeptide is a human SPINK5 polypeptide.

[0175] Embodiment 11: A recombinant herpesvirus genome according to embodiment 9 or embodiment 10, wherein the SPINK5 polypeptide comprises a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence selected from SEQ ID NOs: 7-25.

[0176] Embodiment 12: A recombinant herpesvirus genome according to any one of embodiments 9 to 11, wherein the SPINK5 polypeptide comprises a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence selected from SEQ ID NOs: 7 to 9.

[0177] Embodiment 13: A recombinant herpesvirus genome according to any one of embodiments 9 to 12, wherein the SPINK5 polypeptide comprises a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:7.

[0178] Embodiment 14: The recombinant herpesvirus genome of any one of embodiments 5 to 13, wherein the recombinant herpes simplex virus genome comprises an inactivating mutation.

[0179] Embodiment 15: The recombinant herpesvirus genome of embodiment 14, wherein the inactivating mutation is present in a herpes simplex virus gene.

[0180] Embodiment 16: The recombinant herpesvirus genome of embodiment 15, wherein the inactivating mutation is a deletion of the coding sequence of the herpes simplex virus gene.

[0181] Embodiment 17: The recombinant herpesvirus genome of embodiment 15 or embodiment 16, wherein the herpes simplex virus gene is selected from infected cell protein (ICP) 0, ICP4, ICP22, ICP27, ICP47, thymidine kinase (tk), long unique region (UL) 41, and UL55.

[0182] Embodiment 18: The recombinant herpesvirus genome of embodiment 17, wherein the recombinant herpes simplex virus genome comprises an inactivating mutation in one or both copies of the ICP4 gene.

[0183] Embodiment 19: The recombinant herpesvirus genome of embodiment 17 or embodiment 18, wherein the recombinant herpes simplex virus genome comprises an inactivating mutation in the ICP22 gene.

[0184] Embodiment 20: The recombinant herpesvirus genome of any one of embodiments 17 to 19, wherein the recombinant herpes simplex virus genome comprises an inactivating mutation in the UL41 gene.

[0185] Embodiment 21: The recombinant herpesvirus genome of any one of embodiments 17 to 20, wherein the recombinant herpes simplex virus genome comprises an inactivating mutation in one or both copies of the ICP0 gene.

[0186] Embodiment 22: The recombinant herpesvirus genome of any one of embodiments 17 to 21, wherein the recombinant herpes simplex virus genome comprises an inactivating mutation in the ICP27 gene.

[0187] Embodiment 23: A recombinant herpesvirus genome according to any one of embodiments 5 to 22, wherein the recombinant herpes simplex virus genome comprises the one or more polynucleotides encoding the SPINK polypeptide within one or both of the ICP4 viral loci.

[0188] Embodiment 24: A recombinant herpesvirus genome according to any one or embodiments 5 to 23, wherein the recombinant herpes simplex virus genome comprises the one or more polynucleotides encoding the SPINK polypeptide within the ICP22 viral locus.

[0189] Embodiment 25: A recombinant herpesvirus genome according to any one of embodiments 5 to 24, wherein the recombinant herpes simplex virus genome comprises the one or more polynucleotides encoding the SPINK polypeptide within the UL41 viral locus.

[0190] Embodiment 26: A recombinant herpesvirus genome according to any one of embodiments 1 to 25, which has reduced cytotoxicity when introduced into a target cell compared to the corresponding wild-type herpesvirus genome.

[0191] Embodiment 27: The recombinant herpesvirus genome of embodiment 26, wherein the target cell is a human cell.

[0192] Embodiment 28: The recombinant herpesvirus genome of embodiment 26 or embodiment 27, wherein the target cell is an epidermal cell and / or a dermal cell.

[0193] Embodiment 29: A recombinant herpesvirus genome according to any one of embodiments 26 to 28, wherein the target cell is a keratinocyte or a fibroblast.

[0194] Embodiment 30: A herpesvirus comprising a recombinant herpesvirus genome according to any one of embodiments 1 to 29.

[0195] Embodiment 31: A herpesvirus according to embodiment 30, which is replication competent.

[0196] Embodiment 32: The herpesvirus of embodiment 30, which is replication-deficient.

[0197] Embodiment 33: A herpesvirus according to any one of embodiments 30 to 32, having reduced cytotoxicity compared to the corresponding wild-type herpesvirus.

[0198] Embodiment 34: A herpesvirus according to any one of embodiments 30 to 33, selected from herpes simplex virus, varicella zoster virus, human cytomegalovirus, herpesvirus 6A, herpesvirus 6B, herpesvirus 7, Kaposi's sarcoma-associated herpesvirus, and any derivatives thereof.

[0199] Embodiment 35: A herpesvirus according to any one of embodiments 30 to 34, which is a herpes simplex virus.

[0200] Embodiment 36: The herpes virus of embodiment 34 or embodiment 35, wherein the herpes simplex virus is an HSV-1 virus, an HSV-2 virus, or any derivative thereof.

[0201] Embodiment 37: A pharmaceutical composition comprising a recombinant herpesvirus genome according to any one of embodiments 1 to 29 and / or a herpesvirus according to any one of embodiments 30 to 36, and a pharma- ceutically acceptable excipient.

[0202] Embodiment 38: The pharmaceutical composition of embodiment 37, which is suitable for topical, transdermal, subcutaneous, intradermal, oral, sublingual, buccal, rectal, intravaginal, inhalation, intravenous, intraarterial, intramuscular, intracardiac, intraosseous, intraperitoneal, transmucosal, intravitreal, subretinal, intraarticular, periarticular, local, or epicutaneous administration.

[0203] Embodiment 39: A pharmaceutical composition according to embodiment 37 or embodiment 38, which is suitable for topical, transdermal, subcutaneous, intradermal, or transmucosal administration.

[0204] Embodiment 40: A pharmaceutical composition according to any one of embodiments 37 to 39, which is suitable for topical administration.

[0205] Embodiment 41: A pharmaceutical composition according to any one of embodiments 37 to 40, comprising a hydroxypropyl methylcellulose gel.

[0206] Embodiment 42: A pharmaceutical composition according to any one of embodiments 37 to 41, comprising a phosphate buffer.

[0207] Embodiment 43: A pharmaceutical composition according to any one of embodiments 37 to 42, comprising glycerol.

[0208] Embodiment 44: A pharmaceutical composition according to any one of embodiments 37 to 43, comprising a lipid carrier.

[0209] Embodiment 45: A pharmaceutical composition according to any one of embodiments 37 to 44, comprising a nanoparticle carrier.

[0210] Embodiment 46: A method of enhancing, increasing, enhancing and / or replenishing the level of a SPINK polypeptide in one or more cells of a subject, comprising administering to the subject an effective amount of a herpesvirus described in any one of embodiments 30-36 or a pharmaceutical composition described in any one of embodiments 37-45.

[0211] Embodiment 47: The method of embodiment 46, wherein the SPINK polypeptide is a SPINK5 polypeptide.

[0212] Embodiment 48: A method for enhancing, increasing, enhancing and / or replenishing anti-inflammatory and / or antimicrobial protection of mucosal epithelium in a subject in need thereof, comprising administering to the subject an effective amount of a herpesvirus according to any one of embodiments 30-36 or a pharmaceutical composition according to any one of embodiments 37-45.

[0213] Embodiment 49: A method for inhibiting desquamation in a subject in need thereof, comprising administering to the subject an effective amount of a herpesvirus described in any one of embodiments 30-36 or a pharmaceutical composition described in any one of embodiments 37-45.

[0214] Embodiment 50: A method of reducing or treating a skin barrier defect in a subject in need thereof, comprising administering to the subject an effective amount of a herpesvirus described in any one of embodiments 30-36 or a pharmaceutical composition described in any one of embodiments 37-45.

[0215] Embodiment 51: The method of embodiment 50, wherein the skin barrier defect is transepithelial water loss (TEWL).

[0216] Embodiment 52: A method for providing preventive, palliative, or therapeutic alleviation of one or more signs or symptoms of Netherton Syndrome (NS) in a subject in need thereof, comprising administering to the subject an effective amount of a herpesvirus described in any one of embodiments 30-36 or a pharmaceutical composition described in any one of embodiments 37-45.

[0217] Embodiment 53: The method of embodiment 52, wherein the one or more signs or symptoms of NS are selected from the group consisting of defective keratinization, skin barrier defects, recurrent skin infections, congenital ichthyosiform erythroderma, flexural linear ichthyosis, intussusceptum trichorrhexis, chronic skin inflammation, and any combination thereof.

[0218] Embodiment 54: The method of any one of embodiments 46 to 53, wherein the subject is a human.

[0219] Embodiment 55: The method of any one of embodiments 46 to 54, wherein the subject's genome comprises a loss-of-function mutation in the SPINK5 gene.

[0220] Embodiment 56: The method of any one of embodiments 46 to 55, wherein the herpes virus or the pharmaceutical composition is administered to the subject topically, transdermally, subcutaneously, epicutaneously, intradermally, orally, sublingually, buccally, rectally, intravaginally, intravenously, intraarterially, intramuscularly, intraosseously, intracardially, intraperitoneally, transmucosally, intravitreally, subretinally, intraarticularly, periarticularly, locally, or by inhalation.

[0221] Embodiment 57: The method of any one of embodiments 46 to 56, wherein the herpes virus or the pharmaceutical composition is administered to the subject topically, transdermally, subcutaneously, intradermally, or transmucosally.

[0222] Embodiment 58: The method of any one of embodiments 46 to 57, wherein the herpes virus or the pharmaceutical composition is administered locally to the subject.

[0223] Embodiment 59: The method of any one of embodiments 46-58, wherein the subject's skin is abraded prior to administration.

[0224] The specification is considered to be sufficient to enable one skilled in the art to practice the disclosure. Various modifications of the disclosure, in addition to those shown and described herein, will become apparent to those skilled in the art from the foregoing description and fall within the scope of the appended claims. EXAMPLES

[0225] The present disclosure will be more fully understood by referring to the following examples. However, they should not be interpreted as limiting the scope of the present disclosure. It should be understood that the examples and embodiments described herein are for illustrative purposes only, and in light thereof, various modifications or changes will be suggested to those skilled in the art and will fall within the spirit and scope of the present application and the scope of the appended claims.

[0226] Example 1: Modified herpes simplex virus vector encoding the human SPINK5 protein To generate modified recombinant herpes simplex virus vectors capable of expressing SPINK5 polypeptides in target mammalian cells (such as human keratinocytes or fibroblasts), the herpes simplex virus genome (FIG. 1A) is first modified to inactivate one or more herpes simplex virus genes. Such modifications may reduce the toxicity of the genome in mammalian cells. Variants of these modified / attenuated recombinant virus constructs are then generated such that they carry one or more polynucleotides encoding the desired SPINK5 polypeptides.These variants include: 1) a recombinant ΔICP4-modified HSV-1 genome comprising an expression cassette comprising a coding sequence (e.g., SEQ ID NO:2) for a human SPINK5 polypeptide (e.g., SEQ ID NO:7) under the control of a heterologous promoter integrated into each ICP4 locus (FIG. 1B); 2) a recombinant ΔICP4 / ΔICP22-modified HSV-1 genome comprising an expression cassette comprising a coding sequence (e.g., SEQ ID NO:2) for a human SPINK5 polypeptide (e.g., SEQ ID NO:4) under the control of a heterologous promoter integrated into each ICP4 locus (FIG. 1C); ) a recombinant ΔICP4 / ΔICP22 modified HSV-1 genome comprising an expression cassette comprising a coding sequence (e.g., SEQ ID NO:2) of a human SPINK5 polypeptide (e.g., SEQ ID NO:4) under the control of a heterologous promoter integrated into the ICP22 locus (FIG. 1D); 4) a recombinant ΔICP4 / ΔUL41 modified HSV-1 genome comprising an expression cassette comprising a coding sequence (e.g., SEQ ID NO:2) of a human SPINK5 polypeptide (e.g., SEQ ID NO:4) under the control of a heterologous promoter integrated into each ICP4 locus (FIG. 1E); 5) a UL41 gene 1) a recombinant ΔICP4 / ΔUL41 modified HSV-1 genome comprising an expression cassette comprising a coding sequence (e.g., SEQ ID NO:2) of a human SPINK5 polypeptide (e.g., SEQ ID NO:4) under the control of a heterologous promoter integrated into each ICP4 locus (Figure 1F); 2) a recombinant ΔICP4 / ΔICP22 / ΔUL41 modified HSV-1 genome comprising an expression cassette comprising a coding sequence (e.g., SEQ ID NO:2) of a human SPINK5 polypeptide (e.g., SEQ ID NO:4) under the control of a heterologous promoter integrated into each ICP4 locus (Figure 1G); 3) a recombinant ΔICP4 / ΔICP22 / ΔUL41 modified HSV-1 genome comprising an expression cassette comprising a coding sequence (e.g., SEQ ID NO:2) of a human SPINK5 polypeptide (e.g., SEQ ID NO:4) under the control of a heterologous promoter integrated into each ICP4 locus (Figure 1G); 8) a recombinant ΔICP4 / ΔICP22 / ΔUL41 modified HSV-1 genome comprising an expression cassette comprising a coding sequence (e.g., SEQ ID NO:2) for a human SPINK5 polypeptide (e.g., SEQ ID NO:4) under the control of a heterologous promoter integrated into the ICP22 locus (FIG. 1I).

[0227] These modified herpes simplex virus genome vectors are transfected into engineered Vero cells that have been modified to express one or more herpes virus genes. These engineered Vero cells then secrete the replication-defective herpes simplex virus with the modified genome packaged inside into the supernatant of the cell culture. The supernatant is then collected, concentrated, and sterile-filtered through a 5 μm filter.

[0228] Example 2: Construction and in vitro analysis of HSV candidates encoding human SPINK5 The following examples describe the construction of a recombinant herpes simplex virus type 1 (HSV-1) modified to express human SPINK5, and further provide experiments showing that this recombinant virus was capable of expressing functional human SPINK5 in vitro in infected human cells.

[0229] Materials and Methods Virus construction The "HSV-S5" vector was generated as follows: recombinant HSV-1 was engineered to incorporate a codon-optimized human SPINK5 expression cassette containing a heterologous promoter and polyA sequence into each of the ICP4 loci. Multiple plaques of the engineered viruses containing the putative human SPINK5 cassette were picked and screened by infection of Vero cells to test for SPINK5 expression (data not shown). The highest SPINK5 producing virus, designated "HSV-S5", was isolated and purified.

[0230] cell culture All cells were incubated in 5% CO 2 Immortalized normal keratinocytes were cultured in DMEM supplemented with 10% FBS and 1% HEPES buffer. For qPCR / qRT-PCR analysis, Western blotting, and effector secretion and functionality assays, cells were grown in 6-well plates. For immunofluorescence, cells were grown in 8-chamber slides.

[0231] Viral infection Viral aliquots were removed from the -80°C freezer and thawed under a tissue culture laminar flow hood. The multiplicity of infection was calculated from the viral titer and the number of target cells, and an appropriate volume of the viral stock was diluted in cell culture medium to a final volume of 100 μL, and the prepared virus was incubated with the target cells for 1 h at 37°C. After 1 h of infection, 2 mL of the appropriate cell culture medium was added to the wells of a 6-well plate, and 400 μL of medium was added for an 8-chamber slide.

[0232] qPCR / qRT-PCR analysis Immortalized keratinocytes were cultured at 7 × 10 in a 6-well plate. 5 Cells / well were plated and reached 90-100% confluence the next day. 48 h after mock infection or infection with HSV-S5, cells were resuspended in 350 μL of RLT buffer containing DTT and homogenized by passing through a QiaShredder column according to the manufacturer's protocol. DNA and RNA were isolated using AllPrep DNA / RNA Mini Kit (Qiagen) according to the manufacturer's protocol. For qPCR / qRT-PCR analysis, 50 ng of DNA or RNA was used per reaction in a total reaction volume of 25 μL. DNA quantification was determined by qPCR analysis using Taqman® Fast Advanced Master Mix (Applied Biosystems) and RNA quantification was determined by qRT-PCR analysis using Quantabio 1-Step RT-qPCR ToughMix. All samples were run in duplicate.

[0233] Western blotting Immortalized keratinocytes were cultured at 7 × 10 in a 6-well plate. 5Cells / well were plated and reached 90–100% confluence the next day. 48 h after mock infection or infection with HSV-S5, the medium was aspirated, cells were harvested by gentle scraping, and cell pellets were generated by centrifugation. Cells were lysed in RIPA buffer and incubated with intermittent agitation. Benzonase (10 U) was then added to each lysate for 10 min at room temperature. Lysates were then mixed with 4x LDS sample buffer (Invitrogen) containing 5% 2-mercaptoethanol. Before loading, samples were first boiled at 95°C for 10 min, allowed to cool to room temperature, and then briefly centrifuged. Lysates were then run on a 4–20% Tris-glycine gel and then transferred to PVDF. Primary antibodies (rabbit anti-human SPINK5, R&D Systems catalog no. AF8515; rabbit anti-human GAPDH, Abcam catalog no. ab9485) were incubated with the blocked blots. The blots were then incubated with secondary antibody (goat anti-rabbit IgG-AP conjugate, Sigma) for 1 h at room temperature, the blots were washed three times with TBS for 5 min each, and the washed blots were then incubated with AP chromogenic substrate (Invitrogen) to allow development of the bands.

[0234] immunocytochemistry Immortalized keratinocytes were cultured at 1.3 x 10 in an 8-chamber slide. 5 Cells / well were plated and reached 90-100% confluence the next day. 48 hours after mock infection or infection with HSV-S5, cells were fixed with 2% paraformaldehyde and permeabilized with 0.2% Triton™ X-100 for 5 min. Cells were incubated with Powerblock (BioGenex) for 30 min and then incubated overnight with rabbit anti-human SPINK5 primary antibody (Novus Biologicals catalog no. NBP1-90509). Slides were then incubated with Alexa Fluor® 488-conjugated secondary antibody and mounted with ProLong Gold antifade reagent containing DAPI.

[0235] SPINK5 ELISA Human SPINK5 protein expression and secretion was assessed using a commercial ELISA kit according to the manufacturer's protocol (LSBio). Briefly, standards or samples (medium from cell culture) were added to the wells, unbound standards or samples were washed away, and then biotin-conjugated detection antibodies were added. Avidin-horseradish peroxidase conjugate was incubated in the wells to bind to biotin, and then TMB substrate was added, resulting in quantitative colorimetric development. Color development was terminated by adding sulfuric acid stop solution, and the optical density of each well was measured at a wavelength of 450 nm using a Synergy H1 Hybrid Multi-Mode Reader.

[0236] SPINK5 activity assay Inhibition of recombinant human kallikrein 5 (R&D Systems) protease activity by HSV-S5-infected immortalized keratinocyte cell supernatants was assessed using a modified method provided by R&D Systems. Briefly, rhKLK5 (2 ng / μL) was preincubated with culture supernatants from HSV-S5-infected (or mock-infected) cells for 5 min at room temperature. After preincubation, the fluorogenic peptide Boc-VPR-AMC was added to a final concentration of 20 μM. Immediately after addition of the substrate, cleavage of the substrate (relative fluorescence units, RFU) was measured every 70 s for 15 min at excitation 380 nm / emission 460 nm using a Synergy H1 Hybrid Multi-Mode Reader.

[0237] result Immortalized normal human keratinocytes (HaCaT) were infected with HSV-S5 at various multiplicities of infection (MOI) ranging from 0.3 to 3.0. SPINK5 expression was assessed 48 h postinfection by quantitative polymerase chain reaction (qPCR), quantitative reverse transcription PCR (qRT-PCR), Western blot analysis, and enzyme-linked immunosorbent assay (ELISA).

[0238] SPINK5 was detected in normal keratinocytes infected at an MOI as low as 0.3 and appeared to show a dose-dependent increase in both DNA (Fig. 2A) and transcript levels (Fig. 2B) up to an MOI of 3.0. A concomitant dose-dependent increase in SPINK5 protein expression, as assessed by Western blot, was observed in the cytoplasm of infected normal keratinocytes (Fig. 2C).

[0239] SPINK5 expression in HSV-S5-infected keratinocytes was also assessed by immunocytochemistry (ICC). Consistent with the nucleic acid and Western blot analyses, a dose-dependent increase in SPINK5 protein expression was observed by ICC analysis (Figure 2D). While 7.36% of mock-infected cells were SPINK5 positive, 27.3%, 68.48%, and 95.20% were SPINK5 positive when HSV-S5-transduced cells were infected at MOIs of 0.3, 1.0, and 3.0, respectively. Notably, even at high doses of HSV-S5, there was no significant effect of infection on either cell morphology or cell survival.

[0240] Since endogenous SPINK5 is a naturally secreted protein, it was important to show that HSV-S5-expressed exogenous human SPINK5 was also effectively secreted from infected cells and therefore could support the use of HSV-S5 in SPINK5-deficient patients. Therefore, SPINK5 protein levels were quantified in cell culture medium harvested from infected HaCaT. SPINK5 was successfully secreted from infected normal keratinocytes in a dose-dependent manner (Figure 3).

[0241] Finally, the functionality of HSV-S5-expressed human SPINK5 secreted from infected keratinocytes was confirmed using an enzyme inhibition assay measuring the ability of SPINK5 to inhibit the human serine protease kallikrein 5 (KLK5), the natural target of SPINK5. Briefly, recombinant human KLK5 was preincubated with cell culture supernatants collected from HSV-S5-infected HaCaT (supernatants from mock-infected cells were used as controls). After preincubation, a synthetic non-natural peptide substrate of human KLK5 (Boc-VPR-AMC) was added to the samples, and KLK5-mediated cleavage of the substrate was assessed in each sample. Using this experimental approach, the proteolytic activity of KLK5 (and its inhibition by SPINK5) could be directly quantified by determining the fluorescence resulting from the release of the previously quenched fluorescent AMC moiety of Boc-VPR-AMC following hydrolysis of the Arg-AMC amide bond by KLK5.

[0242] As expected, no inhibition of KLK5 activity was observed when the protease was preincubated with cell culture supernatants from mock-infected keratinocytes. In contrast, infection with HSV-S5 led to a dose-dependent increase in functional SPINK5 expression, as observed by increased KLK5 inhibition when the protease was pretreated with cell culture supernatants collected from keratinocytes infected with increasing MOIs of HSV-S5 (Figure 4).

[0243] Taken together, the data presented in this example show that engineered herpes simplex viruses can efficiently transduce normal human keratinocytes and produce high levels of exogenous SPINK5 after transduction. Importantly, while HSV-S5 was able to rescue / replenish SPINK5 expression in human cells, the vector did not induce any significant toxicity in any of these studies. In addition, the data presented herein show that HSV-S5 can induce the secretion of functional SPINK5 at therapeutically relevant levels.

[0244] Example 3: In vivo characterization of HSV-S5 The following examples describe in vivo experiments establishing multiple delivery routes for HSV-S5 in healthy immunocompetent animals. These studies were performed in BALB / c mice, as there are no practical disease animal models of SPINK5 deficiency because homozygous deletion of SPINK5 is neonatal lethal in animals.

[0245] Materials and Methods All procedures performed in this example complied with applicable animal welfare laws and were approved by the local Institutional Animal Care and Use Committee (IACUC).

[0246] Topical administration of HSV-S5 Before further manipulation, the backs of the mice were shaved and hair follicles were removed with a depilatory product. Tape stripping (using Tegaderm™) was performed as previously described (Ekanayake-Mudiyanselage et al. J Invest Dermatol (1998), 111(3):517-23). ​​After tape stripping, two sites on the back of each mouse were topically treated with the appropriate test article. To contain the topical formulation at the treatment sites, a sterile plastic well covered with a clear adhesive dressing was attached to the skin of the animal using surgical adhesive. HSV-S5 (or vehicle control) formulated in a methylcellulose gel carrier was then applied to the treatment sites by injection through the clear adhesive dressing.

[0247] HSV-S5 intradermal injection Before further manipulation, the backs of the mice were shaved, after which HSV-S5 (or vehicle control) was injected intradermally at two sites on the back of each mouse.

[0248] Tissue collection After the infection period and subsequent recovery period, animals were cultured in a CO 2Mice were euthanized by inhalation followed by cervical dislocation and treated sites were removed using 8 mm punch biopsies. Half of each biopsy was snap frozen in liquid nitrogen for qPCR / qRT-PCR analysis and the other half was processed for immunofluorescence analysis and H&E staining.

[0249] qPCR / qRT-PCR analysis Snap-frozen half biopsies were stored at -80°C until analysis. For processing and analysis, samples were resuspended in 350 μL of RLT buffer prepared fresh with DTT according to the manufacturer's protocol (Qiagen), sonicated three times at 25% amplitude, and incubated intermittently on ice for 1 min. DNA and RNA extraction was performed using the Qiagen AllPrep DNA / RNA extraction kit according to the manufacturer's protocol (including an optional DNase treatment step for RNA samples). Both DNA and RNA samples were resuspended in RNase-free deionized distilled water and quantified spectrophotometrically on a SynergyTM H1 microplate reader (BioTek).

[0250] Absolute quantification of SPINK5 DNA and RNA copies was performed by TaqMan real-time PCR analysis using custom transgene-specific primer / probe pairs. Taqman® Fast Advanced Master Mix (Applied Biosystems) was used for DNA quantification (qPCR) and Quantabio 1-Step RT-qPCR ToughMix was used for RNA quantification (qRT-PCR). All samples were run in duplicate and copy numbers were determined using a standard curve obtained from a dilution series of a plasmid standard containing a known copy number of the SPINK5 transgene.

[0251] Immunofluorescence staining 5 μm sections were taken from OCT frozen tissues, mounted on slides, and air-dried for up to 1 h. Slides were then immersed in 100% methanol (MeOH) for 10 min at -20°C and air-dried. Methanol-fixed sections were washed three times (5 min each) at room temperature in PBS, followed by 3% H 2 O2 The sections were rehydrated by incubating in PBS for 10 min at room temperature and washing three times with PBS. The samples were then incubated with blocking solution (Power Block) for 10 min at room temperature in a humidified chamber. Excess blocking solution was removed and the sections were stained with a drop of primary Ab solution (final dilution 1:200) prepared in antibody diluent buffer (30-50 μL primary antibody (Ab) solution / section). The sections were incubated with primary antibody for 16 h at 4 °C or 1 h at room temperature, washed three times at room temperature for 5 min in TBST (TBS + 0.025% Triton X-100), and secondary Ab was applied at a dilution of 1:200 in antibody diluent buffer for 30 min at room temperature in a humidified chamber. Slides were washed again three times with TBST and stained sections were mounted with mounting medium (ProLong™ Gold antifade mounting medium with DAPI, ThermoFisher, Cat. No. P36931) and covered with a coverslip. Sections were imaged after dehydration (approximately 24 hours) using an ECHO fluorescence microscope. Primary and secondary antibodies used in this study are presented in Table 1.

[0252] Table 1. Antibodies used for immunofluorescence TIFF2025004042000002.tif19128

[0253] Hematoxylin and eosin (H&E) staining 5 μm sections were taken from cryopreserved tissues, mounted on slides, and air-dried for up to 1 h. Dried slides were rehydrated by immersion in double distilled water for 2 min at room temperature. Sections were then incubated in Hematoxylin Gill 2x (VWR) for 2 min at room temperature, followed by immersion 2-3 times in acid alcohol, 3-4 times in blue ammonia water, and incubation in Eosin (Eosin Y solution 1%, VWR) for 2 min. Between each step, samples were rinsed 3-4 times with tap water. Stained and rinsed sections were gradually dehydrated in EtOH by first rinsing twice in 95% EtOH (2 min each) and then twice in 100% EtOH (2 min each). Sections were then cleared by rinsing three times in Histo-Clear (2 min each), mounted in mounting medium (Permount™ mounting medium), and covered with a coverslip. Sections were imaged approximately 24 hours after dehydration using a bright field microscope.

[0254] result A single-dose pharmacology study was performed in immunocompetent BALB / c mice to determine the feasibility of administering human SPINK5 via topical and / or intradermal administration of HSV-S5. A total of four BALB / c mice were used in this study. Prior to administration of the test article, the backs of the mice were shaved and hair follicles were removed using a chemical depilatory product. At the topical treatment sites, the exposed skin was then tape stripped nine times to disrupt / remove the stratum corneum and 1 × 10 SPINK5 formulated in a gel carrier was applied. 8 Plaque-forming units (PFU) of HSV-S5 (or vehicle control) were administered topically to two areas of tape-stripped skin on each mouse. For intradermal administration, 1 × 10 8 PFU of HSV-S5 (or vehicle control) was injected into two parallel sites on the back of each animal. Table 2 below provides an overview of the experimental design.

[0255] Table 2. Study plan and test substance administration TIFF2025004042000003.tif16141

[0256] Forty-eight hours after dosing, 8 mm full-thickness biopsies were taken from each treatment site and divided in half. Half of each section was snap frozen in liquid nitrogen and then processed for qPCR and qRT-PCR analysis to quantify SPINK5 DNA copy number and transcript levels, respectively. The other half of each biopsy was embedded in OCT for immunofluorescence (IF).

[0257] qPCR analysis of topically treated tape-stripped skin demonstrated that the engineered HSV genome encoding the human SPINK5 transgene efficiently transduced the disrupted skin of immunocompetent animals (Figure 5A). Not only did the genome efficiently enter the target tissue, but recombinant human SPINK5 was robustly expressed after infection, as assessed by qRT-PCR analysis (Figure 5B). Similar results were seen at both the DNA (Figure 5C) and RNA levels (Figure 5D) after intradermal injection of HSV-S5 into fully intact skin. Neither SPINK5 DNA nor SPINK5 RNA was detected in vehicle-treated tissue, demonstrating the specificity of the assay for the HSV-S5 test article.

[0258] SPINK5 expression in frozen sections taken from topically treated animals was determined by immunofluorescence analysis using an anti-human SPINK5 antibody. To confirm that SPINK5 expressed from HSV-S5 was correctly localized to the cornified layer of the epidermis, samples were counterstained for mouse filaggrin, another structural protein that was localized to this skin region (Figure 5E). This data demonstrated that topical application of HSV-S5 resulted in successful transduction of mouse skin and induced robust expression of the encoded human transgene in the correct layers of the epidermis.

[0259] Histological evaluation of topically treated skin showed no inflammatory infiltrates at the treatment sites, and HSV-S5-treated skin appeared morphologically normal (comparable to vehicle-treated skin) ( Fig. 5<em>F ), demonstrating the safety of this therapy.

[0260] Taken together, the in vivo data presented herein clearly show that (1) HSV-S5 successfully expresses human SPINK5 in vivo when administered topically and intradermally to immunocompetent mice, and (2) recombinant SPINK5 is expressed and localized in the appropriate regions of the epidermis. In addition, no inflammatory infiltrates or gross structural changes to the skin were observed at the HSV-S5 treatment sites, confirming that HSV-S5 is well tolerated. Without wishing to be bound by theory, it is believed that these data, combined with the results of in vitro studies, strongly support the safe and effective use of topical or intradermal HSV-S5 for the transient repeated delivery of human SPINK5. In addition, without wishing to be bound by theory, it is believed that HSV-S5 may be a clinically beneficial non-invasive gene therapy candidate for the treatment of Netherton syndrome.

[0261] Sequence information SEQUENCE LISTING <110> Krystal Biotech, Inc. <120> COMPOSITIONS AND METHODS FOR THE TREATMENT OF NETHERTON SYNDROME <150> US 62 / 735,582 <151> 2018-09-24 <160> 26 <170> FastSEQ for Windows Version 4.0 <210> 1 <211> 3285 <212> DNA <213> Homo sapiens <400> 1 atgaagatag ccacagtgtc agtgcttctg cccttggctc tttgcctcat acaagatgct 60 120 aaactgttct gtccccagga taagaaattt tttcaaagtc ttgatggaat aatgttcatc 180 aataatgtg ccacgtgcaa aatgatactg gaaaagaag caaaatcaca gaagggcc 240 aggcatttag caagagctcc caaggctact gccccaacag agctgaattg tgatgatttt 300 aaaaaaggag aaagagatgg ggattttatc tgtcctgatt attatgaagc tgtttgtggc 360 420 tcccaaattg gtgtaaaaag tgaagggaa tgtaagagca gtaatccaga gcaggatgta 480 tgcagtgctt ttcggccctt tgttagagat ggaagaacttg gatgcacaag ggaaaatgat 540 cctgttcttg gtcctgatgg gaagacgcat ggcaataagt gtgcaatgtg tgctgagctg 600 tttttaaaag aagctgaaaa tgccaagcga gagggtgaaa ctagaattcg acgaaatgct 660 gaaaggatt tttgcaagga atatgaaaaa gaagagagaa atggaaggct ttttgtaca 720 cgggagagtg atccagtccg tggccctgac ggcaggatgc atggcaacaa atgtgccctg 780 tgtgctgaaa ttttcaagca gcgtttttca gaggaaaaca gtaaaacaga tcaaaatttg 840 ggaaaagctg aagaaaaaac taaagttaaa agaaattg tgaaactctg cagtcaatat 900 caaaatcagg caaagaatgg aatacttttc tgtaccagag aaaatgaccc tattcgtggt 960 ccagatggga aaatgcatgg caacttgtgt tccatgtgtc aagcctactt ccaagcagaa 1020 aatgaaa agaaaaaggc tgaagcacga gctagaaaca aaagagaatc tggaaaagca 1080 acctcatatg cagagctttg cagtgaatat cgaaagcttg tgaggaacgg aaaacttgct 1140 tgcaccagag agaacgatcc tatccagggc ccagatggga aagtgcatgg caacacctgc 1200 tccatgtgtg aggtcttctt ccaagcagaa gaagagaaa agaaaaagaa ggaaggtaa 1260 tcaagaaaca aaagacaatc taagagtaca gcttcctttg aggagttgtg tagtgaatac 1320 cgcaaatcca ggaaaaacgg acggcttttt tgcaccagag agaatgaccc catccagggc 1380 ccagatggaa aaatgcatgg caacacctgc tccatgtgtg aggccttctt tcaacaagaa 1440 gaagagcaa gagcaaaggc taaaagagaa gctgcaaagg aaatctgcag tgaattcgg 1500 gaccaagtga ggaatggaac acttatatgc accagggagc ataatcctgt ccgtggccca 1560 gatggcaaaa tgcatggaaa caagtgtgcc atgtgtgcca gtgtgttcaa acttgaagaa 1620 gaagagaaga aaaatgataa agaagaaaaa gggaaagtcg aggctgaaaa agttaagaga 1680 gaagcagttc aggagctgtg cagtgaatat cgtcattatg tgaggaatgg acgactcccc 1740 tgtaccagag agaatgatcc tattgagggt ctagatggga aaatccacgg caacacctgc 1800 tccatgtgtg aagccttctt ccagcaagaa gcaaaagaaa aagaaagagc tgaacccaga 1860 gcaaaagtca aaagagaagc tgaaaaggag acatgcgatg aatttcggag acttttgcaa 1920 aatggaaaac ttttctgcac aagagaaaat gatcctgtgc gtggcccaga tggcaagacc 1980 catggcaaca agtgtgccat gtgtaaggca gtcttccaga aagaaaatga ggaaagaaag 2040 aggaaagaag aggaagatca gagaaatgct gcaggacatg gttccagtgg tggtggagga 2100 ggaaacactc aggacgaatg tgctgagtat cgggaacaaa tgaaaaatgg aagactcagc 2160 tgtactcggg agagtgatcc tgtacgtgat gctgatggca aatcgtacaa caatcagtgt 2220 accatgtgta aagcaaattt ggaaagagaa gcagagagaa aaaatgagta ttctcgctcc 2280 agatcaatg ggactggatc agatcagggg aagatacat gtgatgagtt tagagccaa 2340 atgaaaaatg gaaaactcat ctgcactcga gaagtgacc ctgtccgggg tccagatggc 2400 aagacacatg gcaatagtg tactatgt aaggaaaac tggaaggga agcagctgaa 2460 aaaaaaaaga agaggaaaaaaaaaaaaaaaaaaaaaaaaaaaaaagggga 2520 aggagcaatg aaagagga tctgtgtcgt gatttcgaa gcatgcagag aaatggaaag 2580 cttatctgca ccagagaaaaaccctgtt cgaggcccat atggcagat gcacatcaat 2640 aaatgtgcta tgtgtcagag catctttgat cgagaagcta atgaaagaa aaagaagat 2700 gagagaaat caagtagca gccctcaat aatgcaagg accagtgcag acaggttcag 2760 aatgaagcgg aggatgcaa atttagacaa cctggggcgtt ccttggccctc tgttgccagg 2820 actcatctgc actcatctgc 2880 cctagagaga atgacccagt gcacggtgct gatgaaagt tctatacaa caagtgctac 2940 atgtgcagag ctgtctttct aacagaagct ttggaaaggg caaagcttca agaaaagcca 3000 tcccatgtta gagctctca agaggagac agcccagact ctttcagttc tctggattct 3060 gagatgtgca aagactaccg agtattgccc agataggtt atctttgtcc aaaggattta 3120 aagcctgtct gtggtgacga tggccaacc tacacaatc cttgcatgct ctgtcatgaa 3180 aacctgatac gccaacaaa tacacacatc cgcagtacag ggaagtgtga ggagagcagc 3240 accccaggaa ccaccgcagc cagcatgccc ccgtctgacg atga 3285 <210> 2 <211> 3285 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Construct <400> 2 atgaagatcg ccaccgtgtc tgtgctgctg cctctggctc tgtgcctgat tcaggatgcc 60 gccagcaaga acgaggacca agagatgtgc cacgagttcc aggcctttat gaagaacggc 120 aagctgttct gccctcaaga caagaagttc ttccagagcc tggacggcat catgttcatc 180 aacaagtgcg ccacatgca gatgatcctg gaaaagagg ccaagagcca gaagcgggcc 240 agacaccttg ctagagcccc taaagccacc gctcctaccg agctgaactg cgacgacttc 300 aagaaaggcg agcgcgacgg cgacttcatc tgccccgatt attacgaggc cgtgtgcggc 360 accgatggca agacctacga taacagatgc gccctgtgcg ccgagaatgc caagacagga 420 tctcagatcg gcgtgaagtc tgagggcgag tgcaagagca gcaaccccga gcaggatgtg 480 tgcagcgcct tcagaccctt cgtcagagat ggcagactgg gctgcaccag agaaaacgat 540 cctgtgctgg gccctgacgg caagacacac ggaaacaagt gtgctatgtg tgccgagctg 600 ttcctgaaag aggccgaaaa cgccaagaga gagggcgaga cacggatcag aagaaacgcc 660 gagaaggact tctgcaaaga atacgagaag caagtgcgga acggacgcct gttctgtacc 720 cgggaaagcg atccagtcag aggccccgat ggaagaatgc acggcaacaa atgcgcactg 780 tgtgctgaga tcttcaagca gcggttcagc gaggaaaaca gcaagaccga ccagaacctg 840 ggcaaagccg aagaaaagac caaagtgaag cgcgagatcg tgaagctgtg cagccagtac 900 cagaaccagg ccaagaatgg catcctgttc tgcacacgcg agaacgaccc catcagagga 960 cccgacggaa agatgcatgg aaacctgtgc agcatgtgcc aggcctactt ccaggccgag 1020 aacgaagaga agagaaggc cgaagccagg gccagaaaca agagagaaag cggcaaggcc 1080 accagctacg ccgaactgtg tagcgagtac agaaagctcg tgcggaatgg caagctggcc 1140 tgcactaggg aaaatgaccc tattcaggga ccagacggca aggtgcacgg gaatacctgt 1200 tccatgtgcg aggtgttctt tcaggccgag gaaggaaa aaaagaagaa agaggggcaag 1260 tcccggaaca agcggcagag caagagcacc gccagcttcg aggaactgtg ctccgagtac 1320 cggaagtcca gaagaatgg ccggcttttc tgcacgagag agaatgatcc catccagggg 1380 cctgatggca aaatgcatgg caatacttgc tctatgtgcg aggcattttt ccagcaagag 1440 gaacgggcca gagccaaggc caaaagagag gccgccaaag agatctgcag cgagtttagg 1500 gaccaagtcc gcaacggcac cctgatctgc acagggaac aaaccctgt cagagggcca 1560 gacgggaaaa tgcacgggaa caagtgtgcc atgtgcgcct ccgtgttcaa gctggaagaa 1620 gaaaaaga aaaacgacaa agagagaag ggaaaagtcg aagccgagaa agtgaagg 1680 gaagccgtgc aagaactctg ttctgagtac cggcactacg tccggaacgg cagactgcct 1740 tgtactcggg aaaacgaccc aatcgaagga ctggacggga agatccatgg caacacctgt 1800 agtatgtgcg aggcattctt tcaacaagaa gccaaagaaa aagagcgggc cgagcctcgg 1860 gccaaagtca aaagagaagc tgagaaagag acatgcgacg agttccggcg gctgctgcag 1920 aatggaaaac tcttctgtac acgtgaaaat gatcccgtgc ggggaccaga tggaaagacc 1980 catgggaaca aatgcgctat gtgcaaggcc gtgttccaaa aagagaatga ggaacgcaag 2040 cggaaagagg aagaagatca gcggaacgcc gctggccacg gatctagtgg cggaggtggt 2100 ggaaataccc aggacgagtg tgccgaatac cgggaacaga tgaagaatgg aaggctgagc 2160 tgcacacggg aatctgaccc cgttagagat gccgacggca agtcctacaa caaccagtgc 2220 accatgtgta aagccaagct cgagcgcgag gccgagcgga agaatgagta cagcagaagc 2280 cggtctaacg gcaccggctc tgagtctggc aaggatacct gtgacgagtt cagatcccag 2340 atgaagaacg ggaaactcat ctgcacaaga gaatcagacc cagtccgggg acctgacggg 2400 aaaactcatg gcaacaagtg caaatgtgc aaagaagaac tggaacgcga gggctccgag 2460 aagaaaaaga aagaggacga ggatcggagc aacaccggcg agagatctaa taccggcgag 2520 cggagcaatg acaaagagga cctctgccgg gaattccggt ccatgcagag aaacggaaag 2580 ctgatatgta cgcgcgagaa caatcccgtt cggggccctt atgggaagat gcacattaac 2640 aaatgtgcaa tgtgccagag catcttcgac agagaggcca acgaacgcaa aaagaaggat 2700 gaagagaagt ccagcagcaa gcccagcaac aacgccaagg accagtgcag acaggtgcag 2760 aacgaggctg aggacgccaa gtttagacag cccggtagaa gcctggcctc tgtggccaga 2820 atgagcaccg atgagtgcag cgaattccgg aactatatcc ggaacga gctgatctgt 2880 ccgcgagaga atgaccctgt gcatggcgcc gatggaaagt tctacaccaa caagtgttat 2940 atgtgcagag ccgtctttct gacagaggcc ctggaaagag ccaagctgca agaaagcct 3000 tctcacgtgc gggcctctca agaagaggac agccctgata gcttcagcag cctggacagc 3060 gagatgtgca aggactacag agtgctgccc cggatcggct atctgtgccc caaggatctg 3120 aagcctgtgt gtggcgacga cggccagaca tacaacaatc cctgcatgct gtgtcacgag 3180 aacctgatcc ggcagaccaa cacacacatc cggtccaccg gcaagtgcga agagtctagc 3240 3285 <210> 3 <211> 2751 <212> DNA <213> Homo sapiens <400> 3 atgaagatag ccacagtgtc agtgcttctg cccttggctc tttgcctcat acaagatgct 60 120 aaactgttct gtccccagga taagaaattt tttcaaagtc ttgatggaat aatgttcatc 180 aataatgtg ccacgtgcaa aatgatactg gaaaagaag caaaatcaca gaagggcc 240 aggcatttag caagagctcc caaggctact gccccaacag agctgaattg tgatgatttt 300 aaaaaaggag aaagagatgg ggattttatc tgtcctgatt attatgaagc tgtttgtggc 360 420 tcccaaattg gtgtaaaaag tgaagggaa tgtaagagca gtaatccaga gcaggatgta 480 tgcagtgctt ttcggccctt tgttagagat ggaagaacttg gatgcacaag ggaaaatgat 540 cctgttcttg gtcctgatgg gaagacgcat ggcaataagt gtgcaatgtg tgctgagctg 600 tttttaaaag aagctgaaaa tgccaagcga gagggtgaaa ctagaattcg acgaaatgct 660 gaaaggatt tttgcaagga atatgaaaaa gaagagagaa atggaaggct ttttgtaca 720 cgggagagtg atccagtccg tggccctgac ggcaggatgc atggcaacaa atgtgccctg 780 tgtgctgaaa ttttcaagca gcgtttttca gaggaaaaca gtaaaacaga tcaaaatttg 840 ggaaaagctg aagaaaaaac taaagttaaa agaaattg tgaaactctg cagtcaatat 900 caaaatcagg caaagaatgg aatacttttc tgtaccagag aaaatgaccc tattcgtggt 960 ccagatggga aaatgcatgg caacttgtgt tccatgtgtc aagcctactt ccaagcagaa 1020 aatgaagaaa agaaaaaggc tgaagcacga gctagaaaca aaagagaatc tggaaaagca 1080 acctcatatg cagagctttg cagtgaatat cgaaagcttg tgaggaacgg aaaacttgct 1140 tgcaccagag agaacgatcc tatccagggc ccagatggga aagtgcatgg caacacctgc 1200 tccatgtgtg aggtcttctt ccaagcagaa gaagagaaa agaaaaagaa ggaaggtaa 1260 tcaagaaaca aaagacaatc taagagtaca gcttcctttg aggagttgtg tagtgaatac 1320 cgcaaatcca ggaaaaacgg acggcttttt tgcaccagag agaatgaccc catccagggc 1380 ccagatggaa aaatgcatgg caacacctgc tccatgtgtg aggccttctt tcaacaagaa 1440 gaagagcaa gagcaaaggc taaaagagaa gctgcaaagg aaatctgcag tgaattcgg 1500 1560 1620 gaagaagaa aaaatgataa agaagaaaaa gggaaaagtcg aggctgaaaa agttaagaaga 1680 gaagcagttc aggagctgtg cagtgaatat cgtcattatg tgaggaatgg acgactcccc 1740 tgtaccagag agaatgatcc tattgagggt ctagatggga aaatccacgg caacacctgc 1800 tccatgtgtg aagccttctt ccagcaagaa gcaaaagaaa aagaaagagc tgaacccaga 1860 gcaaagtca aaagagaagc tgaaaggag acatgcgatg aatttcggag acttttgcaa 1920 aatggaaaac ttttctgcac aagagaaat gatcctgtgc gtggcccaga tggcagacc 1980 catggcaaca agtgtgccat gtgtaggca gtcttccaga aagaaatga ggaaagaaag 2040 aggaagaag aggaagatca gagaaatgct gcaggacatg gttccagtgg tggtggagga 2100 ggaaacactc aggacgaatg tgctgagtat cgggaacaaa tgaaaaatgg agactcagc 2160 tgtactcggg agagtgatcc tgtacgtgat gctgatggca aatcgtacaa caatcagtgt 2220 accatgtgta aagcaaattt ggaaagagaa gcagagagaa aaaatgagta ttctcgctcc 2280 agatcaatg ggactggatc agatcagggg aagatacat gtgatgagtt tagagccaa 2340 atgaaaaatg gaaaactcat ctgcactcga gaagtgacc ctgtccgggg tccagatggc 2400 aagacacatg gcaatagtg tactatgt aaggaaaac tggaaggga agcagctgaa 2460 aaaaaaaaga agaggaaaaaaaaaaaaaaaaaaaaaaaaaaaaaagggga 2520 aggagcaatg aaagagga tctgtgtcgt gatttcgaa gcatgcagag aaatggaaag 2580 cttatctgca ccagagaaaaaccctgtt cgaggcccat atggcagat gcacatcaat 2640 aaatgtgcta tgtgtcagag catctttgat cgagaagcta atgaaagaa aaagaaagat 2700 gagagaaat caagtagca gccctcaat atgcaagg tttatta a 2751 <210> 4 <211> 2751 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Construct <400> 4 atgaagatcg ccaccgtgtc tgtgctgctg cctctggctc tgtgcctgat tcaggatgcc 60 gccagcaaga acgaggacca agagatgtgc cacgagttcc aggcctttat gaagaacggc 120 aagctgttct gccctcaaga caagaagttc ttccagagcc tggacggcat catgttcatc 180 aacaagtgcg ccacatgca gatgatcctg gaaaaagagg ccaagagcca gaagcgggcc 240 agacaccttg ctagagcccc taaagccacc gctcctaccg agctgaacctg cgacgactc 300 aagaaaggcg agcggacgg cgactcatc tgccccgatt attacgaggc cgtgtgcggc 360 accgatggca agacctacga taacagatgc gccctgtgcg ccgagaatgc caagacagga 420 tctcagatcg gcgtgaagtc tgaggcgag tgcagagca gcaccccga gcaggatgtg 480 tgcagcgcct tcagaccct cgtcagagat ggcagactgg gctgcaccag agaaaacgat 540 cctgtgctgg gccctgacgg caacacac ggaacaagt gtgctatgtg tgccgagctg 600 ttcctgaaag aggccgaaaa cgccaagaga gagggcgaga cacggatcag aagaaacgcc 660 gagaaggact tctgcaaga atacgagaag caagtgcgga acggacgcct gttctgtacc 720 cgggaaagcg atccagtcag aggccccgat ggaagaatgc acggcacaa atgcgcactg 780 tgtgctgaga tcttcaagca gcggttcagc gaggaaaaca gcaagaccga ccagaacctg 840 ggcaagccg aagaaagac caagtgaag cgcgagatcg tgaagctgtg cagccagtac 900 cagaaccagg ccaagaatgg catcctgttc tgcacacgcg agaacgaccc catcagagga 960 cccgacggaa agatgcatgg aaacctgtgc agcatgtgcc agcctactt ccaggccgag 1020 aacgaagaga agagaaggc cgaagccagg gccagaaaagagagaaag cggcaaggcc 1080 accagctacg ccgaacctgtg tagcgagtac agaagctcg tgcggaatgg caagctggcc 1140 tgcactaggg aaaatgaccc tattcaggga ccagacggca aggtgcacgg gaatacctgt 1200 tccatgtgcg aggtgttctt tcaggccgag gaaggaaa aaaagaagaa agaggggcaag 1260 tcccggaaca agcggcagag caagagcacc gccagcttcg aggaactgtg ctccgagtac 1320 cggaagtcca gaagaatgg ccggcttttc tgcacgagag agaatgatcc catccagggg 1380 cctgatggca aaatgcatgg caatacttgc tctatgtgcg aggcattttt ccagcaagag 1440 gaacgggcca gagccaaggc caaaagagag gccgccaaag agatctgcag cgagtttagg 1500 gaccaagtcc gcaacggcac cctgatctgc acagggaac aaaccctgt cagagggcca 1560 gacgggaaaa tgcacgggaa caagtgtgcc atgtgcgcct ccgtgttcaa gctggaagaa 1620 gaaaaaga aaaacgacaa agagagaag ggaaaagtcg aagccgagaa agtgaagg 1680 gaagccgtgc aagaactctg ttctgagtac cggcactacg tccggaacgg cagactgcct 1740 tgtactcggg aaaacgaccc aatcgaagga ctggacggga agatccatgg caacacctgt 1800 agtatgtgcg aggcattctt tcaacaagaa gccaaagaaa aagagcgggc cgagcctcgg 1860 gccaaagtca aaagaagagc tgaagaagag acatgcgacg agttccggcg gctgctgcag 1920 aatggaaaac tcttctgtac acgtgaaaat gatcccgtgc ggggaccaga tggaaagacc 1980 catgggaaca aatgcgctat gtgcaaggcc gtgttccaaa aagagaatga ggaacgcaag 2040 cggaagg aagagatca gcggaacgcc gctggccacg gatctagtgg cggaggtggt 2100 ggaataccc agcacgagtg tgccgaatac cgggaacaga tgaagaatgg aaggtgagc 2160 tgcacacggg aatctgaccc cgttagagat gccgacggca agtcctacaa caaccagtgc 2220 2280 cggtctaacg gcaccggctc tgagtctggc areatacct gtgacgagtt cagatcccag 2340 atgaagaacg ggaactcat ctgcacaaga gaatcagacc cagtccgggg acctgacggg 2400 aaaactcatg gcaacaagtg caaatgtgc aaagaagaac tggaacgcga gggctccgag 2460 aagaaaaaga aagaggacga ggatcggagc aacaccggcg agagatctaa taccggcgag 2520 cggagcaatg acaaagagga cctctgccgg gaattccggt ccatgcagag aaacggaaag 2580 ctgatatgta cgcgcgagaa caatcccgtt cggggccctt atgggaagat gcacattaac 2640 aaatgtgcaa tgtgccagag catcttcgac agagaggcca acgaacgcaa aaagaaggat 2700 2751 <210> 5 <211> 3195 <212> DNA <213> Homo sapiens <400> 5 atgaagatag ccacagtgtc agtgcttctg cccttggctc tttgcctcat acaagatgct 60 120 aaactgttct gtccccagga taagaaattt tttcaaagtc ttgatggaat aatgttcatc 180 aataatgtg ccacgtgcaa aatgatactg gaaaagaag caaaatcaca gaagggcc 240 aggcatttag caagagctcc caaggctact gccccaacag agctgaattg tgatgatttt 300 aaaaaaggag aaagagatgg ggattttatc tgtcctgatt attatgaagc tgtttgtggc 360 420 tcccaaattg gtgtaaaaag tgaagggaa tgtaagagca gtaatccaga gcaggatgta 480 tgcagtgctt ttcggccctt tgttagagat ggaagaacttg gatgcacaag ggaaaatgat 540 cctgttcttg gtcctgatgg gaagacgcat ggcaataagt gtgcaatgtg tgctgagctg 600 tttttaaaag aagctgaaaa tgccaagcga gagggtgaaa ctagaattcg acgaaatgct 660 gaaaggatt tttgcaagga atatgaaaaa gaagagagaa atggaaggct ttttgtaca 720 cgggagagtg atccagtccg tggccctgac ggcaggatgc atggcaacaa atgtgccctg 780 tgtgctgaaa ttttcaagca gcgtttttca gaggaaaaca gtaaaacaga tcaaaatttg 840 ggaaaagctg aagaaaaaac taaagttaaa agaaattg tgaaactctg cagtcaatat 900 caaaatcagg caaagaatgg aatacttttc tgtaccagag aaaatgaccc tattcgtggt 960 ccagatggga aaatgcatgg caacttgtgt tccatgtgtc aagcctactt ccaagcagaa 1020 aatgaaa agaaaaaggc tgaagcacga gctagaaaca aaagagaatc tggaaaagca 1080 acctcatatg cagagctttg cagtgaatat cgaaagcttg tgaggaacgg aaaacttgct 1140 tgcaccagag agaacgatcc tatccagggc ccagatggga aagtgcatgg caacacctgc 1200 tccatgtgtg aggtcttctt ccaagcagaa gaagagaaa agaaaaagaa ggaaggtaa 1260 tcaagaaaca aaagacaatc taagagtaca gcttcctttg aggagttgtg tagtgaatac 1320 cgcaaatcca ggaaaaacgg acggcttttt tgcaccagag agaatgaccc catccagggc 1380 ccagatggaa aaatgcatgg caacacctgc tccatgtgtg aggccttctt tcaacaagaa 1440 gaagagcaa gagcaaaggc taaaagagaa gctgcaaagg aaatctgcag tgaattcgg 1500 1560 1620 gaagaagaa aaaatgataa agaagaaaaa gggaaaagtcg aggctgaaaa agttaagaaga 1680 gaagcagttc aggagctgtg cagtgaatat cgtcattatg tgaggaatgg acgactcccc 1740 tgtaccagag agaatgatcc tattgagggt ctagatggga aaatccacgg caacacctgc 1800 tccatgtgtg aagccttctt ccagcaagaa gcaaaagaaa aagaaagagc tgaacccaga 1860 gcaaagtca aaagagaagc tgaaaggag acatgcgatg aatttcggag acttttgcaa 1920 aatggaaaac ttttctgcac aagagaaat gatcctgtgc gtggcccaga tggcagacc 1980 catggcaaca agtgtgccat gtgtaggca gtcttccaga aagaaatga ggaaagaaag 2040 aggaagaag aggaagatca gagaaatgct gcaggacatg gttccagtgg tggtggagga 2100 ggaaacactc aggacgaatg tgctgagtat cgggaacaaa tgaaaaatgg agactcagc 2160 tgtactcggg agagtgatcc tgtacgtgat gctgatggca aatcgtacaa caatcagtgt 2220 accatgtgta aagcaaattt ggaaagagaa gcagagagaa aaaatgagta ttctcgctcc 2280 agatcaatg ggactggatc agatcagggg aagatacat gtgatgagtt tagagccaa 2340 atgaaaaatg gaaaactcat ctgcactcga gaagtgacc ctgtccgggg tccagatggc 2400 aagacacatg gcaatagtg tactatgt aaggaaaac tggaaggga agcagctgaa 2460 aaaaaaaaga agaggaaaaaaaaaaaaaaaaaaaaaaaaaaaaaagggga 2520 aggagcaatg aaagagga tctgtgtcgt gatttcgaa gcatgcagag aaatggaaag 2580 cttatctgca ccagagaaaaaccctgtt cgaggcccat atggcagat gcacatcaat 2640 aaatgtgcta tgtgtcagag catctttgat cgagaagcta atgaaagaa aaagaaagat 2700 gagagaaat caagtagcaa gccctcaat atgcaagg atgagtgcag tgaatttcga 2760 aactatataa ggaacaatga actcatctgc cctagagaga atgacccagt gcacggtgct 2820 gatgaaagt tctatacaa caagtgctac atgtgcagag ctgtctttct aacagaagct 2880 ttggaaaggg caaagcttca agaaaagcca tcccatgtta gagctctca agaggaagac 2940 agcccagact ctttcagttc tctggattct gagatgtgca agactaccg agtattgccc 3000 aggataggtt atctttgtcc aaaggattta aagctgtct gtggtgacga tggccaacc 3060 tacaacaatc cttgcatgct ctgtcatgaa aacctgatac gccaacaaa tacacacatc 3120 cgcagtacag ggaagtgtga ggagagcagc acchaggaa ccaccgcagc cagcatgccc 3180 ccgtctgacg atga 3195 <210> 6 <211> 3195 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Construct <400> 6 atgaagatcg ccaccgtgtc tgtgctgctg cctctggctc tgtgcctgat tcaggatgcc 60 gccagcaaga acgaggacca agagatgtgc cacgagttcc aggcctttat gaagaacggc 120 aagctgttct gccctcaaga caagaagttc ttccagagcc tggacggcat catgttcatc 180 aacaagtgcg ccacatgca gatgatcctg gaaaaagagg ccaagagcca gaagcgggcc 240 agacaccttg ctagagcccc taaagccacc gctcctaccg agctgaacctg cgacgactc 300 aagaaaggcg agcggacgg cgactcatc tgccccgatt attacgaggc cgtgtgcggc 360 accgatggca agacctacga taacagatgc gccctgtgcg ccgagaatgc caagacagga 420 tctcagatcg gcgtgaagtc tgaggcgag tgcagagca gcaccccga gcaggatgtg 480 tgcagcgcct tcagaccct cgtcagagat ggcagactgg gctgcaccag agaaaacgat 540 cctgtgctgg gccctgacgg caacacac ggaacaagt gtgctatgtg tgccgagctg 600 ttcctgaaag aggccgaaaa cgccaagaga gagggcgaga cacggatcag aagaaacgcc 660 gagaagact tctgcaaaga atacgagaag gaaggcgga acggacgcct gttctgtacc 720 cgggaaagcg atccagtcag aggccccgat ggagaatgc acggcaacaa atgcgcactg 780 tgtgctgaga tcttcaagca gcggttcagc gaggaaaaca gcaagaccga ccagaacctg 840 ggcaaagccg aagaaaagac caaagtgaag cgcgagatcg tgaagctgtg cagccagtac 900 cagaaccagg ccaagaatgg catcctgttc tgcacacgcg agaacgaccc catcagagga 960 cccgacggaa agatgcatgg aaacctgtgc agcatgtgcc aggcctactt ccaggccgag 1020 aacgaagaga agagaaggc cgaagccagg gccagaaaca agagagaaag cggcaaggcc 1080 accagctacg ccgaactgtg tagcgagtac agaaagctcg tgcggaatgg caagctggcc 1140 tgcactaggg aaaatgaccc tattcaggga ccagacggca aggtgcacgg gaatacctgt 1200 tccatgtgcg aggtgttctt tcaggccgag gaaggaaa aaaagaagaa agaggggcaag 1260 tcccggaaca agcggcagag caagagcacc gccagcttcg aggaactgtg ctccgagtac 1320 cggaagtcca gaagaatgg ccggcttttc tgcacgagag agaatgatcc catccagggg 1380 cctgatggca aaatgcatgg caatacttgc tctatgtgcg aggcattttt ccagcaagag 1440 gaacgggcca gagccaaggc caaaagagag gccgccaaag agatctgcag cgagtttagg 1500 gaccaagtcc gcaacggcac cctgatctgc acagggaac aaaccctgt cagagggcca 1560 gacgggaaaa tgcacgggaa caagtgtgcc atgtgcgcct ccgtgttcaa gctggaagaa 1620 gaaaaaga aaaacgacaa agagagaag ggaaaagtcg aagccgagaa agtgaagg 1680 gaagccgtgc aagaactctg ttctgagtac cggcactacg tccggaacgg cagactgcct 1740 tgtactcggg aaaacgaccc aatcgaagga ctggacggga agatccatgg caacacctgt 1800 agtatgtgcg aggcattctt tcaacaagaa gccaaagaaa aagagcgggc cgagcctcgg 1860 gccaaagtca aaagaagagc tgaagaagag acatgcgacg agttccggcg gctgctgcag 1920 aatggaaaac tcttctgtac acgtgaaaat gatcccgtgc ggggaccaga tggaaagacc 1980 catgggaaca aatgcgctat gtgcaaggcc gtgttccaaa aagagaatga ggaacgcaag 2040 cggaagg aagagatca gcggaacgcc gctggccacg gatctagtgg cggaggtggt 2100 ggaataccc agcacgagtg tgccgaatac cgggaacaga tgaagaatgg aaggtgagc 2160 tgcacacggg aatctgaccc cgttagagat gccgacggca agtcctacaa caaccagtgc 2220 2280 cggtctaacg gcaccggctc tgagtctggc areatacct gtgacgagtt cagatcccag 2340 atgaagaacg ggaactcat ctgcacaaga gaatcagacc cagtccgggg acctgacggg 2400 aaaactcatg gcaacaagtg caaatgtgc aaagaagaac tggaacgcga gggctccgag 2460 aagaaaaaga aagaggacga ggatcggagc aacaccggcg agagatctaa taccggcgag 2520 cggagcaatg acaaagagga cctctgccgg gaattccggt ccatgcagag aaacggaaag 2580 ctgatatgta cgcgcgagaa caatcccgtt cggggccctt atgggaagat gcacattaac 2640 aaatgtgcaa tgtgccagag catcttcgac agagaggcca acgaacgcaa aaagaaggat 2700 gaagagaagt ccagcagcaa gcccagcaac aacgccaagg acgaatgcag cgaattccgg 2760 aactatatcc ggaacga gctgatctgt ccgcgagaga atgaccctgt gcatggcgcc 2820 gatggaaagt tctacaccaa caagtgttat atgtgcagag ccgtctttct gacagaggcc 2880 ctggaaagag ccaagctgca agagaagcct tctcacgtgc gggcctctca agaagaggac 2940 agccctgata gcttcagcag cctggacagc gagatgtgca aggactacag agtgctgccc 3000 cggatcggct atctgtgccc caaggatctg aagcctgtgt gtggcgacga cggccagaca 3060 tacaacaatc cctgcatgct gtgtcacgag aacctgatcc ggcagaccaa cacacacatc 3120 cggtccaccg gcaagtgcga agagtctagc acacctggaa caaccgccgc ctctatgcct 3180 cctagcgacg aatga 3195 <210> 7 <211> 1094 <212> PRT <213> Homo sapiens <400> 7 Met Lys Ile Ala Thr Val Ser Val Leu Leu Pro Leu Ala Leu Cys Leu 1 5 10 15 Ile Gln Asp Ala Ala Ser Lys Asn Glu Asp Gln Glu Met Cys His Glu 20 25 30 Phe Gln Ala Phe Met Lys Asn Gly Lys Leu Phe Cys Pro Gln Asp Lys 35 40 45 Lys Phe Phe Gln Ser Leu Asp Gly Ile Met Phe Ile Asn Lys Cys Ala 50 55 60 Thr Cys Lys Met Ile Leu Glu Lys Glu Ala Lys Ser Gln Lys Arg Ala 65 70 75 80 Arg His Leu Ala Arg Ala Pro Lys Ala Thr Ala Pro Thr Glu Leu Asn 85 90 95 Cys Asp Asp Phe Lys Lys Gly Glu Arg Asp Gly Asp Phe Ile Cys Pro 100 105 110 Asp Tyr Tyr Glu Ala Val Cys Gly Thr Asp Gly Lys Thr Tyr Asp Asn 115 120 125 Arg Cys Ala Leu Cys Ala Glu Asn Ala Lys Thr Gly Ser Gln Ile Gly 130 135 140 Val Lys Ser Glu Gly Glu Cys Lys Ser Ser Asn Pro Glu Gln Asp Val 145 150 155 160 Cys Ser Ala Phe Arg Pro Phe Val Arg Asp Gly Arg Leu Gly Cys Thr 165 170 175 Arg Glu Asn Asp Pro Val Leu Gly Pro Asp Gly Lys Thr His Gly Asn 180 185 190 Lys Cys Ala Met Cys Ala Glu Leu Phe Leu Lys Glu Ala Glu Asn Ala 195 200 205 Lys Arg Glu Gly Glu Thr Arg Ile Arg Arg Asn Ala Glu Lys Asp Phe 210 215 220 Cys Lys Glu Tyr Glu Lys Gln Val Arg Asn Gly Arg Leu Phe Cys Thr 225 230 235 240 Arg Glu Ser Asp Pro Val Arg Gly Pro Asp Gly Arg Met His Gly Asn 245 250 255 Lys Cys Ala Leu Cys Ala Glu Ile Phe Lys Gln Arg Phe Ser Glu Glu 260 265 270 Asn Ser Lys Thr Asp Gln Asn Leu Gly Lys Ala Glu Glu Lys Thr Lys 275 280 285 Val Lys Arg Glu Ile Val Lys Leu Cys Ser Gln Tyr Gln Asn Gln Ala 290 295 300 Lys Asn Gly Ile Leu Phe Cys Thr Arg Glu Asn Asp Pro Ile Arg Gly 305 310 315 320 Pro Asp Gly Lys Met His Gly Asn Leu Cys Ser Met Cys Gln Ala Tyr 325 330 335 Phe Gln Ala Glu Asn Glu Glu Lys Lys Lys Ala Glu Ala Arg Ala Arg 340 345 350 Asn Lys Arg Glu Ser Gly Lys Ala Thr Ser Tyr Ala Glu Leu Cys Ser 355 360 365 Glu Tyr Arg Lys Leu Val Arg Asn Gly Lys Leu Ala Cys Thr Arg Glu 370 375 380 Asn Asp Pro Ile Gln Gly Pro Asp Gly Lys Val His Gly Asn Thr Cys 385 390 395 400 Ser Met Cys Glu Val Phe Phe Gln Ala Glu Glu Glu Glu Lys Lys Lys 405 410 415 Lys Glu Gly Lys Ser Arg Asn Lys Arg Gln Ser Lys Ser Thr Ala Ser 420 425 430 Phe Glu Glu Leu Cys Ser Glu Tyr Arg Lys Ser Arg Lys Asn Gly Arg 435 440 445 Leu Phe Cys Thr Arg Glu Asn Asp Pro Ile Gln Gly Pro Asp Gly Lys 450 455 460 Met His Gly Asn Thr Cys Ser Met Cys Glu Ala Phe Phe Gln Gln Glu 465 470 475 480 Glu Arg Ala Arg Ala Lys Ala Lys Arg Glu Ala Ala Lys Glu Ile Cys 485 490 495 Ser Glu Phe Arg Asp Gln Val Arg Asn Gly Thr Leu Ile Cys Thr Arg 500 505 510 Glu His Asn Pro Val Arg Gly Pro Asp Gly Lys Met His Gly Asn Lys 515 520 525 Cys Ala Met Cys Ala Ser Val Phe Lys Leu Glu Glu Glu Glu Lys Lys 530 535 540 Asn Asp Lys Glu Glu Lys Gly Lys Val Glu Ala Glu Lys Val Lys Arg 545 550 555 560 Glu Ala Val Gln Glu Leu Cys Ser Glu Tyr Arg His Tyr Val Arg Asn 565 570 575 Gly Arg Leu Pro Cys Thr Arg Glu Asn Asp Pro Ile Glu Gly Leu Asp 580 585 590 Gly Lys Ile His Gly Asn Thr Cys Ser Met Cys Glu Ala Phe Phe Gln 595 600 605 Gln Glu Ala Lys Glu Lys Glu Arg Ala Glu Pro Arg Ala Lys Val Lys 610 615 620 Arg Glu Ala Glu Lys Glu Thr Cys Asp Glu Phe Arg Arg Leu Leu Gln 625 630 635 640 Asn Gly Lys Leu Phe Cys Thr Arg Glu Asn Asp Pro Val Arg Gly Pro 645 650 655 Asp Gly Lys Thr His Gly Asn Lys Cys Ala Met Cys Lys Ala Val Phe 660 665 670 Gln Lys Glu Asn Glu Glu Arg Lys Arg Lys Glu Glu Glu Asp Gln Arg 675 680 685 Asn Ala Ala Gly His Gly Ser Ser Gly Gly Gly Gly Gly Asn Thr Gln 690 695 700 Asp Glu Cys Ala Glu Tyr Arg Glu Gln Met Lys Asn Gly Arg Leu Ser 705 710 715 720 Cys Thr Arg Glu Ser Asp Pro Val Arg Asp Ala Asp Gly Lys Ser Tyr 725 730 735 Asn Asn Gln Cys Thr Met Cys Lys Ala Lys Leu Glu Arg Glu Ala Glu 740 745 750 Arg Lys Asn Glu Tyr Ser Arg Ser Arg Ser Asn Gly Thr Gly Ser Glu 755 760 765 Ser Gly Lys Asp Thr Cys Asp Glu Phe Arg Ser Gln Met Lys Asn Gly 770 775 780 Lys Leu Ile Cys Thr Arg Glu Ser Asp Pro Val Arg Gly Pro Asp Gly 785 790 795 800 Lys Thr His Gly Asn Lys Cys Thr Met Cys Lys Glu Lys Leu Glu Arg 805 810 815 Glu Ala Ala Glu Lys Lys Lys Lys Glu Asp Glu Asp Arg Ser Asn Thr 820 825 830 Gly Glu Arg Ser Asn Thr Gly Glu Arg Ser Asn Asp Lys Glu Asp Leu 835 840 845 Cys Arg Glu Phe Arg Ser Met Gln Arg Asn Gly Lys Leu Ile Cys Thr 850 855 860 Arg Glu Asn Asn Pro Val Arg Gly Pro Tyr Gly Lys Met His Ile Asn 865 870 875 880 Lys Cys Ala Met Cys Gln Ser Ile Phe Asp Arg Glu Ala Asn Glu Arg 885 890 895 Lys Lys Lys Asp Glu Glu Lys Ser Ser Ser Lys Pro Ser Asn Asn Ala 900 905 910 Lys Asp Gln Cys Arg Gln Val Gln Asn Glu Ala Glu Asp Ala Lys Phe 915 920 925 Arg Gln Pro Gly Arg Ser Leu Ala Ser Val Ala Arg Met Ser Thr Asp 930 935 940 Glu Cys Ser Glu Phe Arg Asn Tyr Ile Arg Asn Asn Glu Leu Ile Cys 945 950 955 960 Pro Arg Glu Asn Asp Pro Val His Gly Ala Asp Gly Lys Phe Tyr Thr 965 970 975 Asn Lys Cys Tyr Met Cys Arg Ala Val Phe Leu Thr Glu Ala Leu Glu 980 985 990 Arg Ala Lys Leu Gln Glu Lys Pro Ser His Val Arg Ala Ser Gln Glu 995 1000 1005 Glu Asp Ser Pro Asp Ser Phe Ser Ser Leu Asp Ser Glu Met Cys Lys 1010 1015 1020 Asp Tyr Arg Val Leu Pro Arg Ile Gly Tyr Leu Cys Pro Lys Asp Leu 1025 1030 1035 1040 Lys Pro Val Cys Gly Asp Asp Gly Gln Thr Tyr Asn Asn Pro Cys Met 1045 1050 1055 Leu Cys His Glu Asn Leu Ile Arg Gln Thr Asn Thr His Ile Arg Ser 1060 1065 1070 Thr Gly Lys Cys Glu Glu Ser Ser Thr Pro Gly Thr Thr Ala Ala Ser 1075 1080 1085 Met Pro Pro Ser Asp Glu 1090 <210> 8 <211> 916 <212> PRT <213> Homo sapiens <400> 8 Met Lys Ile Ala Thr Val Ser Val Leu Leu Pro Leu Ala Leu Cys Leu 1 5 10 15 Ile Gln Asp Ala Ala Ser Lys Asn Glu Asp Gln Glu Met Cys His Glu 20 25 30 Phe Gln Ala Phe Met Lys Asn Gly Lys Leu Phe Cys Pro Gln Asp Lys 35 40 45 Lys Phe Phe Gln Ser Leu Asp Gly Ile Met Phe Ile Asn Lys Cys Ala 50 55 60 Thr Cys Lys Met Ile Leu Glu Lys Glu Ala Lys Ser Gln Lys Arg Ala 65 70 75 80 Arg His Leu Ala Arg Ala Pro Lys Ala Thr Ala Pro Thr Glu Leu Asn 85 90 95 Cys Asp Asp Phe Lys Lys Gly Glu Arg Asp Gly Asp Phe Ile Cys Pro 100 105 110 Asp Tyr Tyr Glu Ala Val Cys Gly Thr Asp Gly Lys Thr Tyr Asp Asn 115 120 125 Arg Cys Ala Leu Cys Ala Glu Asn Ala Lys Thr Gly Ser Gln Ile Gly 130 135 140 Val Lys Ser Glu Gly Glu Cys Lys Ser Ser Asn Pro Glu Gln Asp Val 145 150 155 160 Cys Ser Ala Phe Arg Pro Phe Val Arg Asp Gly Arg Leu Gly Cys Thr 165 170 175 Arg Glu Asn Asp Pro Val Leu Gly Pro Asp Gly Lys Thr His Gly Asn 180 185 190 Lys Cys Ala Met Cys Ala Glu Leu Phe Leu Lys Glu Ala Glu Asn Ala 195 200 205 Lys Arg Glu Gly Glu Thr Arg Ile Arg Arg Asn Ala Glu Lys Asp Phe 210 215 220 Cys Lys Glu Tyr Glu Lys Gln Val Arg Asn Gly Arg Leu Phe Cys Thr 225 230 235 240 Arg Glu Ser Asp Pro Val Arg Gly Pro Asp Gly Arg Met His Gly Asn 245 250 255 Lys Cys Ala Leu Cys Ala Glu Ile Phe Lys Gln Arg Phe Ser Glu Glu 260 265 270 Asn Ser Lys Thr Asp Gln Asn Leu Gly Lys Ala Glu Glu Lys Thr Lys 275 280 285 Val Lys Arg Glu Ile Val Lys Leu Cys Ser Gln Tyr Gln Asn Gln Ala 290 295 300 Lys Asn Gly Ile Leu Phe Cys Thr Arg Glu Asn Asp Pro Ile Arg Gly 305 310 315 320 Pro Asp Gly Lys Met His Gly Asn Leu Cys Ser Met Cys Gln Ala Tyr 325 330 335 Phe Gln Ala Glu Asn Glu Glu Lys Lys Lys Ala Glu Ala Arg Ala Arg 340 345 350 Asn Lys Arg Glu Ser Gly Lys Ala Thr Ser Tyr Ala Glu Leu Cys Ser 355 360 365 Glu Tyr Arg Lys Leu Val Arg Asn Gly Lys Leu Ala Cys Thr Arg Glu 370 375 380 Asn Asp Pro Ile Gln Gly Pro Asp Gly Lys Val His Gly Asn Thr Cys 385 390 395 400 Ser Met Cys Glu Val Phe Phe Gln Ala Glu Glu Glu Glu Lys Lys Lys 405 410 415 Lys Glu Gly Lys Ser Arg Asn Lys Arg Gln Ser Lys Ser Thr Ala Ser 420 425 430 Phe Glu Glu Leu Cys Ser Glu Tyr Arg Lys Ser Arg Lys Asn Gly Arg 435 440 445 Leu Phe Cys Thr Arg Glu Asn Asp Pro Ile Gln Gly Pro Asp Gly Lys 450 455 460 Met His Gly Asn Thr Cys Ser Met Cys Glu Ala Phe Phe Gln Gln Glu 465 470 475 480 Glu Arg Ala Arg Ala Lys Ala Lys Arg Glu Ala Ala Lys Glu Ile Cys 485 490 495 Ser Glu Phe Arg Asp Gln Val Arg Asn Gly Thr Leu Ile Cys Thr Arg 500 505 510 Glu His Asn Pro Val Arg Gly Pro Asp Gly Lys Met His Gly Asn Lys 515 520 525 Cys Ala Met Cys Ala Ser Val Phe Lys Leu Glu Glu Glu Glu Lys Lys 530 535 540 Asn Asp Lys Glu Glu Lys Gly Lys Val Glu Ala Glu Lys Val Lys Arg 545 550 555 560 Glu Ala Val Gln Glu Leu Cys Ser Glu Tyr Arg His Tyr Val Arg Asn 565 570 575 Gly Arg Leu Pro Cys Thr Arg Glu Asn Asp Pro Ile Glu Gly Leu Asp 580 585 590 Gly Lys Ile His Gly Asn Thr Cys Ser Met Cys Glu Ala Phe Phe Gln 595 600 605 Gln Glu Ala Lys Glu Lys Glu Arg Ala Glu Pro Arg Ala Lys Val Lys 610 615 620 Arg Glu Ala Glu Lys Glu Thr Cys Asp Glu Phe Arg Arg Leu Leu Gln 625 630 635 640 Asn Gly Lys Leu Phe Cys Thr Arg Glu Asn Asp Pro Val Arg Gly Pro 645 650 655 Asp Gly Lys Thr His Gly Asn Lys Cys Ala Met Cys Lys Ala Val Phe 660 665 670 Gln Lys Glu Asn Glu Glu Arg Lys Arg Lys Glu Glu Glu Asp Gln Arg 675 680 685 Asn Ala Ala Gly His Gly Ser Ser Gly Gly Gly Gly Gly Asn Thr Gln 690 695 700 Asp Glu Cys Ala Glu Tyr Arg Glu Gln Met Lys Asn Gly Arg Leu Ser 705 710 715 720 Cys Thr Arg Glu Ser Asp Pro Val Arg Asp Ala Asp Gly Lys Ser Tyr 725 730 735 Asn Asn Gln Cys Thr Met Cys Lys Ala Lys Leu Glu Arg Glu Ala Glu 740 745 750 Arg Lys Asn Glu Tyr Ser Arg Ser Arg Ser Asn Gly Thr Gly Ser Glu 755 760 765 Ser Gly Lys Asp Thr Cys Asp Glu Phe Arg Ser Gln Met Lys Asn Gly 770 775 780 Lys Leu Ile Cys Thr Arg Glu Ser Asp Pro Val Arg Gly Pro Asp Gly 785 790 795 800 Lys Thr His Gly Asn Lys Cys Thr Met Cys Lys Glu Lys Leu Glu Arg 805 810 815 Glu Ala Ala Glu Lys Lys Lys Lys Glu Asp Glu Asp Arg Ser Asn Thr 820 825 830 Gly Glu Arg Ser Asn Thr Gly Glu Arg Ser Asn Asp Lys Glu Asp Leu 835 840 845 Cys Arg Glu Phe Arg Ser Met Gln Arg Asn Gly Lys Leu Ile Cys Thr 850 855 860 Arg Glu Asn Asn Pro Val Arg Gly Pro Tyr Gly Lys Met His Ile Asn 865 870 875 880 Lys Cys Ala Met Cys Gln Ser Ile Phe Asp Arg Glu Ala Asn Glu Arg 885 890 895 Lys Lys Lys Asp Glu Glu Lys Ser Ser Ser Lys Pro Ser Asn Asn Ala 900 905 910 Lys Val Ile Tyr 915 <210> 9 <211> 1064 <212> PRT <213> Homo sapiens <400> 9 Met Lys Ile Ala Thr Val Ser Val Leu Leu Pro Leu Ala Leu Cys Leu 1 5 10 15 Ile Gln Asp Ala Ala Ser Lys Asn Glu Asp Gln Glu Met Cys His Glu 20 25 30 Phe Gln Ala Phe Met Lys Asn Gly Lys Leu Phe Cys Pro Gln Asp Lys 35 40 45 Lys Phe Phe Gln Ser Leu Asp Gly Ile Met Phe Ile Asn Lys Cys Ala 50 55 60 Thr Cys Lys Met Ile Leu Glu Lys Glu Ala Lys Ser Gln Lys Arg Ala 65 70 75 80 Arg His Leu Ala Arg Ala Pro Lys Ala Thr Ala Pro Thr Glu Leu Asn 85 90 95 Cys Asp Asp Phe Lys Lys Gly Glu Arg Asp Gly Asp Phe Ile Cys Pro 100 105 110 Asp Tyr Tyr Glu Ala Val Cys Gly Thr Asp Gly Lys Thr Tyr Asp Asn 115 120 125 Arg Cys Ala Leu Cys Ala Glu Asn Ala Lys Thr Gly Ser Gln Ile Gly 130 135 140 Val Lys Ser Glu Gly Glu Cys Lys Ser Ser Asn Pro Glu Gln Asp Val 145 150 155 160 Cys Ser Ala Phe Arg Pro Phe Val Arg Asp Gly Arg Leu Gly Cys Thr 165 170 175 Arg Glu Asn Asp Pro Val Leu Gly Pro Asp Gly Lys Thr His Gly Asn 180 185 190 Lys Cys Ala Met Cys Ala Glu Leu Phe Leu Lys Glu Ala Glu Asn Ala 195 200 205 Lys Arg Glu Gly Glu Thr Arg Ile Arg Arg Asn Ala Glu Lys Asp Phe 210 215 220 Cys Lys Glu Tyr Glu Lys Gln Val Arg Asn Gly Arg Leu Phe Cys Thr 225 230 235 240 Arg Glu Ser Asp Pro Val Arg Gly Pro Asp Gly Arg Met His Gly Asn 245 250 255 Lys Cys Ala Leu Cys Ala Glu Ile Phe Lys Gln Arg Phe Ser Glu Glu 260 265 270 Asn Ser Lys Thr Asp Gln Asn Leu Gly Lys Ala Glu Glu Lys Thr Lys 275 280 285 Val Lys Arg Glu Ile Val Lys Leu Cys Ser Gln Tyr Gln Asn Gln Ala 290 295 300 Lys Asn Gly Ile Leu Phe Cys Thr Arg Glu Asn Asp Pro Ile Arg Gly 305 310 315 320 Pro Asp Gly Lys Met His Gly Asn Leu Cys Ser Met Cys Gln Ala Tyr 325 330 335 Phe Gln Ala Glu Asn Glu Glu Lys Lys Lys Ala Glu Ala Arg Ala Arg 340 345 350 Asn Lys Arg Glu Ser Gly Lys Ala Thr Ser Tyr Ala Glu Leu Cys Ser 355 360 365 Glu Tyr Arg Lys Leu Val Arg Asn Gly Lys Leu Ala Cys Thr Arg Glu 370 375 380 Asn Asp Pro Ile Gln Gly Pro Asp Gly Lys Val His Gly Asn Thr Cys 385 390 395 400 Ser Met Cys Glu Val Phe Phe Gln Ala Glu Glu Glu Glu Lys Lys Lys 405 410 415 Lys Glu Gly Lys Ser Arg Asn Lys Arg Gln Ser Lys Ser Thr Ala Ser 420 425 430 Phe Glu Glu Leu Cys Ser Glu Tyr Arg Lys Ser Arg Lys Asn Gly Arg 435 440 445 Leu Phe Cys Thr Arg Glu Asn Asp Pro Ile Gln Gly Pro Asp Gly Lys 450 455 460 Met His Gly Asn Thr Cys Ser Met Cys Glu Ala Phe Phe Gln Gln Glu 465 470 475 480 Glu Arg Ala Arg Ala Lys Ala Lys Arg Glu Ala Ala Lys Glu Ile Cys 485 490 495 Ser Glu Phe Arg Asp Gln Val Arg Asn Gly Thr Leu Ile Cys Thr Arg 500 505 510 Glu His Asn Pro Val Arg Gly Pro Asp Gly Lys Met His Gly Asn Lys 515 520 525 Cys Ala Met Cys Ala Ser Val Phe Lys Leu Glu Glu Glu Glu Lys Lys 530 535 540 Asn Asp Lys Glu Glu Lys Gly Lys Val Glu Ala Glu Lys Val Lys Arg 545 550 555 560 Glu Ala Val Gln Glu Leu Cys Ser Glu Tyr Arg His Tyr Val Arg Asn 565 570 575 Gly Arg Leu Pro Cys Thr Arg Glu Asn Asp Pro Ile Glu Gly Leu Asp 580 585 590 Gly Lys Ile His Gly Asn Thr Cys Ser Met Cys Glu Ala Phe Phe Gln 595 600 605 Gln Glu Ala Lys Glu Lys Glu Arg Ala Glu Pro Arg Ala Lys Val Lys 610 615 620 Arg Glu Ala Glu Lys Glu Thr Cys Asp Glu Phe Arg Arg Leu Leu Gln 625 630 635 640 Asn Gly Lys Leu Phe Cys Thr Arg Glu Asn Asp Pro Val Arg Gly Pro 645 650 655 Asp Gly Lys Thr His Gly Asn Lys Cys Ala Met Cys Lys Ala Val Phe 660 665 670 Gln Lys Glu Asn Glu Glu Arg Lys Arg Lys Glu Glu Glu Asp Gln Arg 675 680 685 Asn Ala Ala Gly His Gly Ser Ser Gly Gly Gly Gly Gly Asn Thr Gln 690 695 700 Asp Glu Cys Ala Glu Tyr Arg Glu Gln Met Lys Asn Gly Arg Leu Ser 705 710 715 720 Cys Thr Arg Glu Ser Asp Pro Val Arg Asp Ala Asp Gly Lys Ser Tyr 725 730 735 Asn Asn Gln Cys Thr Met Cys Lys Ala Lys Leu Glu Arg Glu Ala Glu 740 745 750 Arg Lys Asn Glu Tyr Ser Arg Ser Arg Ser Asn Gly Thr Gly Ser Glu 755 760 765 Ser Gly Lys Asp Thr Cys Asp Glu Phe Arg Ser Gln Met Lys Asn Gly 770 775 780 Lys Leu Ile Cys Thr Arg Glu Ser Asp Pro Val Arg Gly Pro Asp Gly 785 790 795 800 Lys Thr His Gly Asn Lys Cys Thr Met Cys Lys Glu Lys Leu Glu Arg 805 810 815 Glu Ala Ala Glu Lys Lys Lys Lys Glu Asp Glu Asp Arg Ser Asn Thr 820 825 830 Gly Glu Arg Ser Asn Thr Gly Glu Arg Ser Asn Asp Lys Glu Asp Leu 835 840 845 Cys Arg Glu Phe Arg Ser Met Gln Arg Asn Gly Lys Leu Ile Cys Thr 850 855 860 Arg Glu Asn Asn Pro Val Arg Gly Pro Tyr Gly Lys Met His Ile Asn 865 870 875 880 Lys Cys Ala Met Cys Gln Ser Ile Phe Asp Arg Glu Ala Asn Glu Arg 885 890 895 Lys Lys Lys Asp Glu Glu Lys Ser Ser Ser Lys Pro Ser Asn Asn Ala 900 905 910 Lys Asp Glu Cys Ser Glu Phe Arg Asn Tyr Ile Arg Asn Asn Glu Leu 915 920 925 Ile Cys Pro Arg Glu Asn Asp Pro Val His Gly Ala Asp Gly Lys Phe 930 935 940 Tyr Thr Asn Lys Cys Tyr Met Cys Arg Ala Val Phe Leu Thr Glu Ala 945 950 955 960 Leu Glu Arg Ala Lys Leu Gln Glu Lys Pro Ser His Val Arg Ala Ser 965 970 975 Gln Glu Glu Asp Ser Pro Asp Ser Phe Ser Ser Leu Asp Ser Glu Met 980 985 990 Cys Lys Asp Tyr Arg Val Leu Pro Arg Ile Gly Tyr Leu Cys Pro Lys 995 1000 1005 Asp Leu Lys Pro Val Cys Gly Asp Asp Gly Gln Thr Tyr Asn Asn Pro 1010 1015 1020 Cys Met Leu Cys His Glu Asn Leu Ile Arg Gln Thr Asn Thr His Ile 1025 1030 1035 1040 Arg Ser Thr Gly Lys Cys Glu Glu Ser Ser Thr Pro Gly Thr Thr Ala 1045 1050 1055 Ala Ser Met Pro Pro Ser Asp Glu 1060 <210> 10 <211> 39 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 10 Glu Met Cys His Glu Phe Gln Ala Phe Met Lys Asn Gly Lys Leu Phe 5 10 15 Cys Pro Gln Asp Lys Lys Phe Phe Gln Ser Leu Asp Gly Ile Met Phe 20 25 30 Ile Asn Lys Cys Ala Thr Cys 35 <210> 11 <211> 63 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 11 Ala Pro Thr Glu Leu Asn Cys Asp Asp Phe Lys Lys Gly Glu Arg Asp 5 10 15 Gly Asp Phe Ile Cys Pro Asp Tyr Tyr Glu Ala Val Cys Gly Thr Asp 20 25 30 Gly Lys Thr Tyr Asp Asn Arg Cys Ala Leu Cys Ala Glu Asn Ala Lys 35 40 45 Thr Gly Ser Gln Ile Gly Val Lys Ser Glu Gly Glu Cys Lys Ser 50 55 60 <210> 12 <211> 62 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 12 Asn Pro Glu Gln Asp Val Cys Ser Ala Phe Arg Pro Phe Val Arg Asp 5 10 15 Gly Arg Leu Gly Cys Thr Arg Glu Asn Asp Pro Val Leu Gly Pro Asp 20 25 30 Gly Lys Thr His Gly Asn Lys Cys Ala Met Cys Ala Glu Leu Phe Leu 35 40 45 Lys Glu Ala Glu Asn Ala Lys Arg Glu Gly Glu Thr Arg Ile 50 55 60 <210> 13 <211> 67 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 13 Asn Ala Glu Lys Asp Phe Cys Lys Glu Tyr Glu Lys Gln Val Arg Asn 5 10 15 Gly Arg Leu Phe Cys Thr Arg Glu Ser Asp Pro Val Arg Gly Pro Asp 20 25 30 Gly Arg Met His Gly Asn Lys Cys Ala Leu Cys Ala Glu Ile Phe Lys 35 40 45 Gln Arg Phe Ser Glu Glu Asn Ser Lys Thr Asp Gln Asn Leu Gly Lys 50 55 60 Ala Glu Glu 65 <210> 14 <211> 62 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 14 Arg Glu Ile Val Lys Leu Cys Ser Gln Tyr Gln Asn Gln Ala Lys Asn 5 10 15 Gly Ile Leu Phe Cys Thr Arg Glu Asn Asp Pro Ile Arg Gly Pro Asp 20 25 30 Gly Lys Met His Gly Asn Leu Cys Ser Met Cys Gln Ala Tyr Phe Gln 35 40 45 Ala Glu Asn Glu Glu Lys Lys Lys Ala Glu Ala Arg Ala Arg 50 55 60 <210> 15 <211> 63 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 15 Thr Ser Tyr Ala Glu Leu Cys Ser Glu Tyr Arg Lys Leu Val Arg Asn 5 10 15 Gly Lys Leu Ala Cys Thr Arg Glu Asn Asp Pro Ile Gln Gly Pro Asp 20 25 30 Gly Lys Val His Gly Asn Thr Cys Ser Met Cys Glu Val Phe Phe Gln 35 40 45 Ala Glu Glu Glu Glu Lys Lys Lys Lys Glu Gly Lys Ser Arg Asn 50 55 60 <210> 16 <211> 59 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 16 Ala Ser Phe Glu Glu Leu Cys Ser Glu Tyr Arg Lys Ser Arg Lys Asn 5 10 15 Gly Arg Leu Phe Cys Thr Arg Glu Asn Asp Pro Ile Gln Gly Pro Asp 20 25 30 Gly Lys Met His Gly Asn Thr Cys Ser Met Cys Glu Ala Phe Phe Gln 35 40 45 Gln Glu Glu Arg Ala Arg Ala Lys Ala Lys Arg 50 55 <210> 17 <211> 62 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 17 Glu Ala Ala Lys Glu Ile Cys Ser Glu Phe Arg Asp Gln Val Arg Asn 5 10 15 Gly Thr Leu Ile Cys Thr Arg Glu His Asn Pro Val Arg Gly Pro Asp 20 25 30 Gly Lys Met His Gly Asn Lys Cys Ala Met Cys Ala Ser Val Phe Lys 35 40 45 Leu Glu Glu Glu Glu Lys Lys Asn Asp Lys Glu Glu Lys Gly 50 55 60 <210> 18 <211> 62 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 18 Glu Ala Val Gln Glu Leu Cys Ser Glu Tyr Arg His Tyr Val Arg Asn 5 10 15 Gly Arg Leu Pro Cys Thr Arg Glu Asn Asp Pro Ile Glu Gly Leu Asp 20 25 30 Gly Lys Ile His Gly Asn Thr Cys Ser Met Cys Glu Ala Phe Phe Gln 35 40 45 Gln Glu Ala Lys Glu Lys Glu Arg Ala Glu Pro Arg Ala Lys 50 55 60 <210> 19 <211> 63 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 19 Glu Ala Glu Lys Glu Thr Cys Asp Glu Phe Arg Arg Leu Leu Gln Asn 5 10 15 Gly Lys Leu Phe Cys Thr Arg Glu Asn Asp Pro Val Arg Gly Pro Asp 20 25 30 Gly Lys Thr His Gly Asn Lys Cys Ala Met Cys Lys Ala Val Phe Gln 35 40 45 Lys Glu Asn Glu Glu Arg Lys Arg Lys Glu Glu Glu Asp Gln Arg 50 55 60 <210> 20 <211> 57 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 20 Gly Asn Thr Gln Asp Glu Cys Ala Glu Tyr Arg Glu Gln Met Lys Asn 5 10 15 Gly Arg Leu Ser Cys Thr Arg Glu Ser Asp Pro Val Arg Asp Ala Asp 20 25 30 Gly Lys Ser Tyr Asn Asn Gln Cys Thr Met Cys Lys Ala Lys Leu Glu 35 40 45 Arg Glu Ala Glu Arg Lys Asn Glu Tyr 50 55 <210> 21 <211> 63 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 21 Glu Ser Gly Lys Asp Thr Cys Asp Glu Phe Arg Ser Gln Met Lys Asn 5 10 15 Gly Lys Leu Ile Cys Thr Arg Glu Ser Asp Pro Val Arg Gly Pro Asp 20 25 30 Gly Lys Thr His Gly Asn Lys Cys Thr Met Cys Lys Glu Lys Leu Glu 35 40 45 Arg Glu Ala Ala Glu Lys Lys Lys Lys Glu Asp Glu Asp Arg Ser 50 55 60 <210> 22 <211> 63 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 22 Asn Asp Lys Glu Asp Leu Cys Arg Glu Phe Arg Ser Met Gln Arg Asn 5 10 15 Gly Lys Leu Ile Cys Thr Arg Glu Asn Asn Pro Val Arg Gly Pro Tyr 20 25 30 Gly Lys Met His Ile Asn Lys Cys Ala Met Cys Gln Ser Ile Phe Asp 35 40 45 Arg Glu Ala Asn Glu Arg Lys Lys Lys Asp Glu Glu Lys Ser Ser 50 55 60 <210> 23 <211> 92 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 23 Asn Asn Ala Lys Asp Gln Cys Arg Gln Val Gln Asn Glu Ala Glu Asp 5 10 15 Ala Lys Phe Arg Gln Pro Gly Arg Ser Leu Ala Ser Val Ala Arg Met 20 25 30 Ser Thr Asp Glu Cys Ser Glu Phe Arg Asn Tyr Ile Arg Asn Asn Glu 35 40 45 Leu Ile Cys Pro Arg Glu Asn Asp Pro Val His Gly Ala Asp Gly Lys 50 55 60 Phe Tyr Thr Asn Lys Cys Tyr Met Cys Arg Ala Val Phe Leu Thr Glu 65 70 75 80 Ala Leu Glu Arg Ala Lys Leu Gln Glu Lys Pro Ser 85 90 <210> 24 <211> 62 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 24 Asn Asn Ala Lys Asp Glu Cys Ser Glu Phe Arg Asn Tyr Ile Arg Asn 5 10 15 Asn Glu Leu Ile Cys Pro Arg Glu Asn Asp Pro Val His Gly Ala Asp 20 25 30 Gly Lys Phe Tyr Thr Asn Lys Cys Tyr Met Cys Arg Ala Val Phe Leu 35 40 45 Thr Glu Ala Leu Glu Arg Ala Lys Leu Gln Glu Lys Pro Ser 50 55 60 <210> 25 <211> 62 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 25 Ser Leu Asp Ser Glu Met Cys Lys Asp Tyr Arg Val Leu Pro Arg Ile 5 10 15 Gly Tyr Leu Cys Pro Lys Asp Leu Lys Pro Val Cys Gly Asp Asp Gly 20 25 30 Gln Thr Tyr Asn Asn Pro Cys Met Leu Cys His Glu Asn Leu Ile Arg 35 40 45 Gln Thr Asn Thr His Ile Arg Ser Thr Gly Lys Cys Glu Glu 50 55 60 <210> 26 <211> 3195 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Construct <400> 26 atgaagatcg ccaccgtgtc tgtgctgctg cctctggctc tgtgcctgat tcaggatgcc 60 gccagcaaga acgaggacca agagatgtgc cacgagttcc aggcctttat gaagaacggc 120 aagctgttct gccctcaaga caagaagttc ttccagagcc tggacggcat catgttcatc 180 aacaagtgcg ccacatgca gatgatcctg gaaaagagg ccaagagcca gaagcgggcc 240 agacaccttg ctagagcccc taaagccacc gctcctaccg agctgaacctg cgacgactc 300 aagaaaggcg agcggacgg cgactcatc tgccccgatt attacgaggc cgtgtgcggc 360 accgatggca agacctacga taacagatgc gccctgtgcg ccgagaatgc caagacagga 420 tctcagatcg gcgtgaagtc tgaggcgag tgcagagca gcaccccga gcaggatgtg 480 tgcagcgcct tcagaccctt cgtcagagat ggcagactgg gctgcaccag agaaaacgat 540 cctgtgctgg gccctgacgg caacacac ggaacaagt gtgctatgtg tgccgagctg 600 ttcctgaaag aggccgaaaa cgccaagaga gagggcgaga cacggatcag aagaaacgcc 660 gagaagact tctgcaaaga atacgagaag gaaggcgga acggacgcct gttctgtacc 720 cgggaaagcg atccagtcag aggccccgat ggagaatgc acggcaacaa atgcgcactg 780 tgtgctgaga tcttcaagca gcggttcagc gaggaaaaca gcaagaccga ccagaacctg 840 ggcaaagccg aagaaaagac caaagtgaag cgcgagatcg tgaagctgtg cagccagtac 900 cagaaccagg ccaagaatgg catcctgttc tgcacacgcg agaacgaccc catcagagga 960 cccgacggaa agatgcatgg aaacctgtgc agcatgtgcc aggcctactt ccaggccgag 1020 aacgaagaga agagaaggc cgaagccagg gccagaaaca agagagaaag cggcaaggcc 1080 accagctacg ccgaactgtg tagcgagtac agaaagctcg tgcggaatgg caagctggcc 1140 tgcactaggg aaaatgaccc tattcaggga ccagacggca aggtgcacgg gaatacctgt 1200 tccatgtgcg aggtgttctt tcaggccgag gaaggaaa aaaagaagaa agaggggcaag 1260 tcccggaaca agcggcagag caagagcacc gccagcttcg aggaactgtg ctccgagtac 1320 cggaagtcca gaagaatgg ccggcttttc tgcacgagag agaatgatcc catccagggg 1380 cctgatggca aaatgcatgg caatacttgc tctatgtgcg aggcattttt ccagcaagag 1440 gaacgggcca gagccaaggc caaaagagag gccgccaaag agatctgcag cgagtttagg 1500 gaccaagtcc gcaacggcac cctgatctgc acagggaac aaaccctgt cagagggcca 1560 gacgggaaaa tgcacgggaa caagtgtgcc atgtgcgcct ccgtgttcaa gctggaagaa 1620 gaaaaaga aaaacgacaa agagagaag ggaaaagtcg aagccgagaa agtgaagg 1680 gaagccgtgc aagaactctg ttctgagtac cggcactacg tccggaacgg cagactgcct 1740 tgtactcggg aaaacgaccc aatcgaagga ctggacggga agatccatgg caacacctgt 1800 agtatgtgcg aggcattctt tcaacaagaa gccaaagaaa aagagcgggc cgagcctcgg 1860 gccaaagtca aaagaagagc tgaagaagag acatgcgacg agttccggcg gctgctgcag 1920 aatggaaaac tcttctgtac acgtgaaaat gatcccgtgc ggggaccaga tggaaagacc 1980 catgggaaca aatgcgctat gtgcaaggcc gtgttccaaa aagagaatga ggaacgcaag 2040 cggaagg aagagatca gcggaacgcc gctggccacg gatctagtgg cggaggtggt 2100 ggaataccc agcacgagtg tgccgaatac cgggaacaga tgaagaatgg aaggtgagc 2160 tgcacacggg aatctgaccc cgttagagat gccgacggca agtcctacaa caaccagtgc 2220 2280 cggtctaacg gcaccggctc tgagtctggc areatacct gtgacgagtt cagatcccag 2340 atgaagaacg ggaactcat ctgcacaaga gaatcagacc cagtccgggg acctgacggg 2400 aaaactcatg gcaacaagtg caaatgtgc aaagaagaac tggaacgcga gggctccgag 2460 aagaaaaaga aagaggacga ggatcggagc aacaccggcg agagatctaa taccggcgag 2520 cggagcaatg acaaagaga cctctgccgg gaatttcgga gcatgcagag aacggaaag 2580 ctcatatgta cgcgcgagaa caatcccgtt cggggccctt atgggaagat gcacattaac 2640 aaatgtgcaa tgtgccagag catcttcgac agagaggcca acgaacgcaa aaagaaggat 2700 gaagagaagt ccagcagcaa gcccagcaac aacgccaagg acgaatgctc tgagttccgc 2760 aattacatcc ggagaacga gctgatctgt ccgcgagaga acgatccagt tcatggcgcc 2820 gatgggaagt tctacaccaa caagtgttat atgtgcagag ccgtctttct gacagaggcc 2880 ctggaaagag ccaagctgca agaaagcct tctcacgtgc gggcctctca agaagaggac 2940 agccctgata gcttcagcag cctggacagc gagatgtgca aggactacag agtgctgccc 3000 cggatcggct atctgtgccc caaggatctg aagcctgtgt gtggcgacga cggccagaca 3060 tacaacaatc cctgcatgct gtgtcacgag aacctgatcc ggcagaccaa cacacacatc 3120 cggtccaccg gcaagtgcga agagtctagc acacctggaa caaccgccgc ctctatgcct 3180 cctagcgacg aatga 3195

Claims

(a) a herpes simplex virus comprising a recombinant herpes simplex virus genome, the recombinant herpes simplex virus genome comprising one or more polynucleotides encoding a serine protease inhibitor Kazal (SPINK) polypeptide; and (b) a pharma- ceutically acceptable excipient 23. A pharmaceutical composition comprising:

2. The pharmaceutical composition described in claim 1, wherein the recombinant herpes simplex virus genome is a recombinant HSV-1 genome.

3. The pharmaceutical composition described in claim 1 or 2, wherein the recombinant herpes simplex virus genome contains an inactivating mutation in a herpes simplex virus gene selected from the group consisting of ICP0, ICP4, ICP22, ICP27, ICP47, tk, UL41, and UL55.

4. A pharmaceutical composition described in any one of claims 1 to 3, wherein the recombinant herpes simplex virus genome contains an inactivating mutation in one or both copies of the ICP4 herpes simplex virus gene.

5. The pharmaceutical composition of any one of claims 1 to 4, wherein the SPINK polypeptide is selected from the group consisting of SPINK1, SPINK2, SPINK4, SPINK5, SPINK6, SPINK7, SPINK8, SPINK9, SPINK13, and SPINK14.

6. The pharmaceutical composition described in any one of claims 1 to 5, wherein the SPINK polypeptide is a SPINK5 polypeptide.

7. The pharmaceutical composition described in any one of claims 1 to 6, wherein the SPINK polypeptide is a human SPINK polypeptide.

8. The pharmaceutical composition of claim 1, wherein the SPINK polypeptide comprises a sequence having at least 95% or 100% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 7-25.

9. The pharmaceutical composition of claim 1, wherein one or more polynucleotides encoding a SPINK polypeptide are operably linked to a promoter suitable for transcription in a mammalian cell.

10. The pharmaceutical composition of any one of claims 1 to 9, wherein the herpes simplex virus is replication-deficient.

11. The pharmaceutical composition of any one of claims 1 to 10, wherein the pharma- ceutically acceptable excipient is suitable for topical, transdermal, subcutaneous, epicutaneous, intradermal, oral, sublingual, buccal, rectal, intravaginal, intravenous, intraarterial, intramuscular, intraosseous, intracardiac, intraperitoneal, transmucosal, intravitreal, subretinal, intra-articular, peri-articular, local, or inhalation administration.

12. Use of a pharmaceutical composition according to any one of claims 1 to 11 in the manufacture of a medicament for enhancing, increasing, enhancing and / or replenishing levels of a serine protease inhibitor Kazal (SPINK) polypeptide in one or more cells of a subject.

13. The pharmaceutical composition of any one of claims 1 to 11, for enhancing, increasing, enhancing and / or replenishing levels of a serine protease inhibitor Kazal (SPINK) polypeptide in one or more cells of a subject.

14. The pharmaceutical composition of claim 13, wherein the one or more cells are selected from the group consisting of keratinocytes, melanocytes, Langerhans cells, Merkel cells, mast cells, fibroblasts, and adipocytes.

15. The pharmaceutical composition of claim 13 or 14, wherein the subject's genome comprises a loss-of-function mutation in the SPINK5 gene.

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