WO2007133730A2 - Materials and methods for control of infections - Google Patents

Abstract

The subject invention provides materials and methods for providing immediate and long-term protection against pathogenic microorganisms. One aspect of the subject invention provides a vector, such as a lentiviral vector carrying a nucleic acid sequence encoding an antimicrobial peptide, such as dermcidin (DCD), as an efficient means of delivery of the peptide. Preferably, the vector has a tetracycline-dependent transcriptional regulatory system such that expression of the nucleic acid sequence encoding the antimicrobial peptide is effectively turned 'off' in the absence of tetracycline or its derivatives. Another aspect of the invention provides host cells that have been genetically modified with a vector of the invention to produce an antimicrobial peptide. Another aspect of the invention provides compositions comprising the vectors or host cells of the invention and a pharmaceutically acceptable carrier. Another aspect of the subject invention provides a method for treating or inhibiting the growth of a pathogenic microorganism by administering a vector or host cell of the invention to a subject suffering from, or susceptible to, infection by the pathogenic microorganism. Another aspect of the subject invention provides a method for detecting the presence of an antimicrobial peptide, such as DCD, within a male or female subject's urogenital tract.

Description

DESCRIPTION

MATERIALS AND METHODS FOR CONTROL OF INFECTIONS

CROSS-REFERENCE TO RELATED APPLICATION

The present application claims the benefit of U.S. Provisional Application Serial No. 60/799,569, filed May 11, 2006, which is hereby incorporated by reference herein in its entirety; including any figures, tables, nucleic acid sequences, amino acid sequences, and drawings.

GOVERNMENT SUPPORT

The subject matter of this application has been supported by a research grant from the National Institutes of Health under grant number RO1HD37432. Accordingly, the government may have certain rights in this invention.

BACKGROUND OF THE INVENTION

The surface epithelial cell-layer in multi-cellular organisms represents a major barrier to the environment, providing a first line of defense against invading microorganisms. This protective barrier is operational in many organisms, including plants, insects, and amphibians. Among the critical components of this defense mechanism is the mucosal and systemic immune response mechanisms and local production of antimicrobial peptides.

In recent years, researchers have come to recognize that many organisms use antimicrobial, peptides as part of their host defense systems. These organisms include a full range of species from prokaryotes to humans. The antimicrobial peptides can be subdivided into a number of groups based on their amino acid content, structure and source (Vizioli J. and Salzet M., Trends Pharmacol. Sci, 2002, 23:494-496; Gennaro R. and Zanetti M., Biopofymers, 2000, 55:31-49; Hancock, R.E.W., Lancet, 1997, 349:418- 422; Boman H.G., Annu. Rev. Immunol., 1995, 13:61-92; Brogden K.A., Nat Rev Microbiol., 2005, 3(3):238-50; Maloy and Kari, Biopofymers, 1995, 37:105-122). Two classes of the antimicrobial peptides identified are cathelicidins and defensins, whose production increases following injury and during wound healing, providing an immediate protection against microbial growth (De Smet, K. and Contreras, R. Biotechnol Lett., 2005, 27:1337-1347; Kougias, P. et al J Cell MoI Med., 2005, 9:3-10; Mahida, Y.R. and Cunliffe, R.N. Novartis Found Symp., 2004, 263:71-78; Barak, O. et al Adv Dermatol., 2005, 21:357-374).

In human skin, cathelicidins and defensins are expressed in keratinocytes and in response to inflammatory stimuli and function primarily in the response to injury. Cathelicidins are detected in dermal wound fluid and are expressed by keratinocytes at the site of inflammation in individuals with disorders such as psoriasis, contact dermatitis and systemic lupus erythematosus.

Defensins, which consist of α and β families, are produced by various cell types, including neutrophils, paneth cells, keratinocytes, and the mucosal epithelial cells of the digestive, respiratory, and genital tracts. Defensin levels are elevated in bacterial infections such as sepsis, bacterial meningitis, and obstetric intrauterine infections (De Smet, K. and Contreras, R. Biotechnol Lett., 2005, 27:1337-1347; Kougias, P. et al. JCeIl MoI Med., 2005, 9:3-10; Mahida, Y.R. and Cunliffe, R.N. Novartis Found Symp., 2004, 263:71-78; Barak, O. et al. Adv Dermatol, 2005, 21:357-374; Murakami, M. et al J Invest Dermatol, 2002, 119:1090-1095; Panyutich, A. V. et al J Lab Clin Med, 1993, 122:20-27; Maffei, F.A. et al. Pediatrics, 1999, 103:987-992) and have a broad range of antimicrobial activity against both gram-positive and gram-negative organisms.

In addition to direct antimicrobial properties, defensins can up-regulate host immunity by inducing the degranulation of mast cells, increasing macrophage phagocytosis, stimulating interleukin production, acting as a chemotactic factor for neutrophils to the site of infection, and modulating complement activation (Yang, D. et al Cell MoI Life Sci, 2001, 58:978-989; Foster, TJ. Nat Rev Microbiol, 2005, 3:948-958; Chen, H. et al Peptides, 2006, 27:931-940).

Human β -defensins 1 and 2 show antimicrobial activity predominantly against gram-negative bacteria such as E. coli and yeasts, whereas human β-defensin 3 is also effective against gram-positive bacteria such as Staphylococcus aureus, a major cause of skin infections, particularly in atopic dermatitis. Collectively, local expression of antimicrobial peptides, along with mucosal and systemic immune response, is critical in providing a defense mechanism protecting organisms from the harmful action of microorganisms.

The female urogenital tract is among the organs that are exposed to the outside environment, specifically the harmful action of sexually transmitted microorganisms. As such, the fallopian tube, endometrial, cervical and vaginal surface epithelial cells provide a critical protective barrier against invading bacteria, fungi and viruses and contribute to mucosal immune responses at the epithelial sites. The state of health of the human female urogenital tract is largely a function of the mixture of microbes present. Pelvic inflammatory disease (PID), resulting from the ascent of these pathogenic microorganisms from the lower genital tract to the uterus, fallopian tubes, and ultimately into the peritoneal cavity, is a major cause of reproductive disorders. The organisms commonly involved in the pathogenesis of PID are Neisseria gonorrhea, Chlamydia trachomatis, and the aerobic and anaerobic organisms associated with bacterial vaginosis (Hillier, S.L. et al. Am JObstet Gynecol, 1996, 175:435-441). Symptoms of PID include, but are not limited to, upper genital tract inflammation, fallopian tube damage, peritoneal inflammation and scarring, infertility and tubal pregnancy as well as preterm labor. Acute PID develops in 9% to 47% of women infected with N. gonorrhea and in 8% to 25% of women infected with Chlamydia; however, many women infected with these sexually transmitted diseases do not develop PID.

Evidence indicates that a significant proportion of women who suffer from tubal factor infertility and/or ectopic pregnancy, result from sub-clinical upper genital tract infections (Panyutich, A. V. et al. J Lab Clin Med, 1993, 122:20-27; Maffei, F.A. et al. Pediatrics, 1999, 103:987-992). Serologic evidence of prior infection with C. trachomatis or N. gonorrhea is found in 23% to 91% of women with tubal-factor infertility (Tjiam, K.H. et al Genitourin Med, 1985, 61 :175-178; Sellors, J. W. et al. Fertil Steril, 1988, 49:451-457; "Tubal infertility: serologic relationship to post chlamydial and gonococcal infection" World Health Organization Task Force on the Prevention and Management of Infertility Sex Transm Dis, 1995, 22:71-77). Women with gonorrhea, Chlamydia, or bacterial vaginosis who do not have symptoms of acute PID are more likely to have endometriosis at the time that the lower genital tract infection is diagnosed (Korn, A.P. et al. Am JObstet Gynecol, 1998, 178:987-990). Various host defenses, including mucosal and systemic immunity, cervical barriers and hormonal environment play important roles in the prevention of PID and preserving fertility and successful pregnancy. With respect to antimicrobial peptides, human reproductive tract epithelial cells express several defensins (King, A.E. et al MoI Hum Reprod, 2000, 6:191-196; Quayle, AJ. et al. Am J Pathol, 1998, 152:1247-1258; Svinarich, D.M. et al. Am J Obstet Gynecol, 1997, 176:470-475), recognized as mediators of innate host defense (Schneider, JJ. et al J MoI Med., 2005, 83:587-595). Although the role of defensins in pathogenesis of reproductive tract infections is unclear, in women at risk for PID, vaginal neutrophil α-defensin has been identified as a marker of upper genital tract inflammation. A strong and independent relationship has also been found between defensins and endometritis among women who do not have acute PID.

Dermcidin (DCD) is a recently-identified antimicrobial peptide that is expressed in the pons of the brain and the sweat glands, secreted into the sweat and transported to the epidermal surface. DCD has no homology to any known antimicrobial peptides and displays antimicrobial activity against a variety of pathogenic microorganisms (Schittek, B. et al. Nat Immunol, 2001, 2:1133-1137; Steffen, H. et al. Antimicrobial Agents and Chemotherapy, 2006, 50(8):2608-2620).

The antimicrobial activity of DCD is maintained over a broad range of pH and in high salt concentrations that resemble the conditions in human sweat (Schneider, JJ. et al JMoI Med., 2005, 83:587-595). Approximately 1-10 g/ml of the DCD is detected in sweat, a concentration that proved toxic to most microorganisms tested (Schneider, JJ. et al J. MoI Med., 2005, 83:587-595; Rieg, S. et al. J Immunol, 2005, 174:8003-10; Rieg, S. et al. J Invest Dermatol, 2006, 126:354-65). In contrast, defensins are only active in the presence of low salt concentrations, and like most antimicrobial peptides that are enriched in arginine or lysine residues and cationic, whereas DCD has a net negative charge (Schneider, JJ. et al. J MoI Med, 2005, 83:587-595; Schittek, B. et al. Nat Immunol, 2001, 2:1133-1137; Rieg, S. et al. Br J Dermatol, 2004, 151 :534-539; Rieg, S. et al. J Immunol, 2005, 174:8003-10; Rieg, S. et al J Invest Dermatol, 2006, 126:354- 65; Lai, Y.P. et al. Biochem Biophys Res Commun., 2005, 328:243-250; Landgraf, P. et al FASEB J, 2005, 19:225-227). These finding indicate that DCD present in human sweat represent an antimicrobial protein with the ability to regulate skin flora. BRIEF SUMMARY OF THE INVENTION

The subject invention provides materials and methods for providing immediate and long-term protection against infection by microorganisms. One aspect of the subject invention provides a vector expressing an antimicrobial peptide as an efficient mode of local delivery of the peptide. Preferably, the antimicrobial peptide-encoding polynucleotide that is carried by the vector is under the control of a regulatory system that is effectively "shut off in the resting state, but which has a rapid and repeatable induction in response to an appropriate inducer molecule. Preferably, the inducer is tetracycline or a tetracycline analog, such as doxycycline. In one embodiment of the invention, the vector is a viral vector, such as lentivirus. Preferably, expression of the antimicrobial peptide-encoding polynucleotide carried by the lentiviral vector is inducible by a tetracycline analog such as doxycycline.

Another aspect of the invention provides host cells that have been genetically modified to produce and secrete an antimicrobial peptide. Preferably, the genetic modification is carried out using a vector of the invention, such as a lentivirus, wherein expression of the antimicrobial peptide-encoding polynucleotide that is carried by the lentivirus is inducible by a tetracycline analog such as doxycyline.

Another aspect of the subject invention provides a method for treating or inhibiting a microbial infection by administering an effective amount of an antimicrobial peptide or polynucleotide encoding an antimicrobial peptide to a subject in order to delay onset of one or more symptoms associated with a microbial infection. Polynucleotides encoding the antimicrobial peptides may be carried by a vector or host cell of the invention, which are administered to a subject suffering from, or susceptible to, infection by the microbe. Optionally, the method of the invention includes identifying a patient suffering from an infection of one or more pathogenic microorganisms. The subject can be identified by a clinician (e.g., medical doctor or doctor of osteopathy) or other medical practitioner licensed to make such diagnosis.

In one embodiment of the invention, an effective amount of the antimicrobial peptide or its encoding polynucleotide is administered locally to the mucosa (the moist, inner lining of some organs and body cavities (such as the nose, mouth, lungs, and stomach)), wherein the subject is suffering from, or susceptible to, the microbial infection. In another embodiment of the invention, an effective amount of the antimicrobial peptide or its encoding polynucleotide is administered locally to a male or female subject's urogenital tract, wherein the subject is suffering from, or susceptible to, the microbial infection. In another embodiment of the invention, an effective amount of the antimicrobial peptide or its encoding polynucleotide is administered locally to an acute or chronic wound, wherein the subject is suffering from, or susceptible to, the microbial infection.

In another embodiment of the invention, an effective amount of the antimicrobial peptide or its encoding polynucleotide is administered locally to the subject's respiratory tract, wherein the subject is suffering from, or susceptible to, the microbial infection.

In another embodiment of the invention, an effective amount of the antimicrobial peptide or its encoding polynucleotide is administered to the subject's skin, for local and/or system delivery of an antimicrobial peptide, wherein the subject is suffering from, or susceptible to, the microbial infection. Specifically exemplified herein is the local delivery of the antimicrobial peptide dermcidin (DCD) at the site of microbial infection or a site of inflammation resulting from the infection. The full-length product of the DCD gene is 110 amino acid residues with an N-terminal 19 amino acid signal peptide characteristic of secreted proteins. It is proteolytically processed to form C-terminal peptides of 48 amino acid residues (DCD- IL), 47 amino acid residues (DCD-I), and shorter fragments that can be detected by surface-enhanced laser desorption/ionization technology. Both DCD-I and DCD-IL have the ability to kill pathogenic microorganisms such as Staphylococcus aureus, Escherichia coli, Enterococcus faecalis, and Candida albicans. Any antimicrobial active form of DCD may be used in the present invention. In a preferred embodiment, a polynucleotide encoding an antimicrobial peptide, such as DCD, is locally or systemically administered, and the antimicrobial peptide- encoding polynucleotide is under the control of a regulatory system that is effectively "shut off in the resting state, but which has a rapid and repeatable induction in response to an appropriate inducer molecule. Preferably, the inducer is tetracycline or a tetracycline analog, such as doxycycline. In a preferred embodiment, the vector is a viral vector. In a particularly preferred embodiment, the viral vector is a lentivirus. In those embodiments of the method utilizing an antimicrobial peptide-encoding polynucleotide under the control of a regulatory system that is induced by an inducer molecule (such as tetracycline or a tetracycline derivative), the method may further comprise administering the inducer molecule to the subject systernically or locally at the target anatomical site (e.g., at the site of the vector containing the antimicrobial peptide-encoding polynucleotide). The inducer molecule can be administered before, during, or after administration of the antimicrobial peptide-encoding polynucleotide.

Lentiviral vectors are efficient vehicles for the delivery of genes to both dividing and non-dividing cells in vitro and in vivo. In one embodiment, a lentiviral vector can be constructed in accordance with the subject invention to express DCD and can be used to transfect endometrial and cervical cells as well as keratinocytes in vitro or in vivo.

Advantageously, the subject invention provides a reliable method to control existing and emerging infectious disease. Specifically exemplified herein is the use of antimicrobial peptides to control infections of the male or female urogenital tract. However, in additional embodiments, the materials and methods of the subject invention can also be used to deliver antimicrobial peptides to anatomical targets, such as the respiratory tract or to acute or chronic wounds.

Another aspect of the subject invention provides a method for detecting the presence of one or more antimicrobial peptides, such as DCD, within a male or female subject's urogenital tract. Preferably, the antimicrobial peptides is measured qualitatively, quantitatively, or semi-quantitatively, and compared to a control. Impaired production of the antimicrobial peptide is indicative of microbial colonization and/or infection, or particular susceptibility to microbial infection.

BRIEF DESCRIPTION OF THE DRAWINGS Figures IA and IB show relative dermcidin expression in skin, endometrium, fallopian tubes, endocervix, and scar tissue. As shown in Figure IA, the lowest relative level of dermcidin mRNA expression was detected in the endometrium, followed by endocervix and scar tissues, although variable levels were detected in each of the tissue categories. The relative level of dermcidin mRNA expressed was the highest in skin followed by the fallopian tubes, as shown in Figure IB. For comparative analysis, note the level of expression of dermcidin in the same endometrial tissues shown in Figure IA with skin and fallopian tubes. Figure 2 shows Western blot analysis of dermcidin protein in skin (SK), fallopian tubes (FT), endometrium (EM)₃ subcutaneous incisional scars (SR) and endocervix (CX).

Figures 3A-3R show immunohistochemical analysis of dermcidin in skin (Figures 3A-3D), peritoneal wall (Figure 3E) fetal membranes (Figures 3F and 3G), placenta (Figures 3H and 31), fallopian tube (Figures 3J and 3K), endometrium (Figures 3 L and 3M), and endocervix (Figures 3N and 3Q). Tissue sections incubated with normal IgG instead of the primary antibodies, or deletion of the primary antibody during immunostaining, served as controls (Figure 3D as well as Figures 3P, 3Q, and 3R showing fallopian tube, endometrium and cervix). Figures 4 A and 4B show expression of the green fluorescent protein (GFP) gene in TE671 cells following delivery by lenti virus carrying Tet-On tetracycline inducible constructs.

BRIEF DESCRIPTION OF THE SEQUENCES SEQ ID NO: 1 is the 47-amino acid sequence of the DCD-derived peptide DCD- 1.

SEQ ID NO:2 is the 48-amino acid sequence of the DCD-derived peptide DCD-IL.

SEQ ID NO:3 is the 46-amino acid sequence of the DCD-derived peptide SSL-46.

SEQ ID NO:4 is the 45-amino acid sequence of the DCD-derived peptide SSL-45.

SEQ ID NO:5 is the 29-amino acid sequence of the DCD-derived peptide SSL-29. SEQ ID NO:6 is the 25-amino acid sequence of the DCD-derived peptide SSL-25.

SEQ ID NO:7 is the 45-amino acid sequence of the DCD-derived peptide LEK-45.

SEQ ID NO: 8 is the 44-amino acid sequence of the DCD-derived peptide LEK-44.

SEQ ID NO: 9 is the 43 -amino acid sequence of the DCD-derived peptide LEK-43.

SEQ ID NO: 10 is the 42-amino acid sequence of the DCD-derived peptide LEK-42. SEQ ID NO : 11 is the 41 -amino acid sequence of the DCD-derived peptide LEK-41.

SEQ ID NO:12 is the 26-amino acid sequence of the DCD-derived peptide LEK-26.

SEQ ID NO: 13 is the 24-amino acid sequence of the DCD-derived peptide LEK-24.

SEQ ID NO:14 is the 42-amino acid sequence of the DCD-derived peptide YDP-42.

SEQ ID NO: 15 is the 110-amino acid sequence of the full-length DCD gene product (precursor protein with a 19-amino acid N-terminal signal peptide). DETAILED DESCRIPTION OF THE INVENTION

The subject invention provides materials and methods for delivering effective antimicrobial treatment in a controlled and efficient manner. Advantageously, the invention can be used to provide immediate and long-term protection against infection by microorganisms. Advantageously, the subject invention provides reliable means to control existing and emerging infectious disease. Specifically exemplified herein is the use of antimicrobial peptides to control infections of the female reproductive tract. In additional embodiments, the materials and methods of the subject invention can also be used to deliver therapeutic agents to the respiratory tract and to wound environments. According to the methods of the present invention, antimicrobial peptides or polynucleotides encoding antimicrobial peptides are administered to a subject in order to alleviate (e.g., reduce or eliminate) or delay onset of one or more symptoms associated with a microbial infection. Treatment with antimicrobial peptides or nucleic acid sequences encoding them is intended to include prophylactic intervention to prevent or reduce microbial cell growth and onset of the symptoms associated with microbial cell growth, such as inflammation, pain, etc. The nucleic acid sequences and pharmaceutical compositions of the invention can be co-administered (concurrently or consecutively) to a patient with other therapeutic agents, such as agents useful for treating infections.

The methods of the invention may include further steps in addition to delivery of the antimicrobial peptide. In some embodiments, a subject with the relevant inflammatory disorder and/or cell proliferation disorder is identified or a patient at risk for the disorder is identified. A patient may be someone who has not been diagnosed with the disease or condition (diagnosis, prognosis, and/or staging) or someone diagnosed with the disease or condition (diagnosis, prognosis, monitoring, and/or staging), including someone treated for the disease or condition (prognosis, staging, and/or monitoring). Optionally, diagnosis can include identifying a microbial infection using methods such as culturing a sample obtained from the subject. Alternatively, the person may not have been diagnosed with the disease or condition but suspected of having the disease or condition based either on patient history or family history, or the exhibition or observation of characteristic symptoms .

The subject may be suffering from infection by a single microbe, or suffering from a polymicrobial infection. In the latter, the presence of one microorganism generates a niche for other pathogenic microorganisms to colonize, one microorganism predisposes the subject to colonization by other microorganisms, or two or more nonpathogenic microorganisms together cause disease (Brogden K. A. et ah, Lancet, 2005, 365(9455):253-255). In one embodiment, the subject is suffering from a urogenital disorder, such as an infection of the urinary or reproductive systems.

Optionally, in accordance with the method of the invention, the antimicrobial peptide or antimicrobial peptide-encoding polynucleotide can be administered to a subject to take advantage of a non-antimicrobial activity of the peptide. Thus, the subject may be suffering from, or susceptible to, a genetic or acquired disorder in which delivery of the antimicrobial peptide would be of therapeutic benefit (/. e. , other than microbial infection). For example, defensins have been reported to orchestrate chemotaxis and activation of effector immune cells, including immature dendritic cells (Biragyn A., Curr. Protein Pept. ScI, 2005, 6(l):53-60; Durr M. and A. Pesehel, Infect. Immun., 2002, 70(12):6515- 6517, which are each incorporated herein by reference in its entirety). Thus, the antimicrobial peptide or antimicrobial peptide-encoding polynucleotide can be administered to a subject in order to take advantage of its immunomodulatory and/or immunoenhancing activities.

In general, any known or later discovered antimicrobial peptide can be used for the methods and compositions of the present invention. The antimicrobial peptide can be of any of the various classes of antimicrobial peptide (e.g., anionic peptides, such as DCD; linear cationic alpha-helical peptides; cationic peptides enriched for specific amino acids, anionic or cationic peptide that contains cysteine and forms disulphide bonds; anionic or cationic peptide fragments of larger proteins) (Brogden KA. et ah, Nat Rev Microbiol.;, 2005, 3(3):238-250, which is incorporated herein by reference in its entirety). Naturally occurring antimicrobial peptides generally contain fewer than 100 amino acids. It is generally believed that these peptides' antimicrobial efficacy is in their ability to penetrate and disrupt the microbial membranes, thereby killing the microbe or inhibiting its growth. The antimicrobial activities of the antimicrobial peptides of the present invention can include, without limitation, antibacterial, antiviral, or antifungal activities. In a preferred embodiment, the antimicrobial peptide is a cathelicidin or a defensin {e.g., alpha- or beta-defensin). Examples of antimicrobial peptides and their potential uses in treating various conditions are described in Niyonsaba and H. Ogawa, J. Dermatol. ScL, 2005, 40(3): 157- 168; Selsted M.E. and AJ. OuUette, Nat. Immunol., 2005, 6(6):551-557; Kougias P. et ai, J. Cell MoI. Med., 2005, 9(l):3-10; Mahida Y.R. and R.N. Cuniiffe, Novartis Found Symp., 2004, 263:71-77; Cole A.M., Protein Pept. Lett., 2005, 12(l):41-47; Sahl H.G. et al., J. Leukoc Biol, 2005, 77(4):466-475; Yoshio H. et al, Semin. Perinatol, 2004, 28(4):304-311; Steinstraesser L. et al, Burns, 2004, 30(7):619-627; Marshall R.I., Periodontol 2000, 2004, 35:14-20; Bardan A. et al, Expert Opin. Biol. Then, 2004, 4(4):543-549; King A.E. et al, Reprod. Biol Endocrinol, 2003, 1:116; Cuniiffe R.N. and Y.R. Mahida, J. Leukoc. Biol, 75(l):49-58; Ganz T., Nat. Rev. Immunol, 2003, 3(9):710- 720; Boman H.G., J. Intern. Med., 2003, 254(3): 197-215; Gallo R.L. and V. Nizet, Curr. Allergy Asthma Rep., 2003, 3(5):402-409; Gallo R.L. et al, J. Allergy CHn. Immunol , 2002, 110(6): 823-831; Kunin CM. et al, J. Urol, 2002, 168(2):4134-419; Lupetti A. et al, Expert Opin. Investig. Drugs, 2002, ll(2):309-318; Bos J.D. et al, Clin Dermatol, 2001, 19(5):563-572; Ganz T., J. Infect. Dis., 2001, 183 Suppl. l:S41-42; Kaiser V. and G. Diamond, J. Leukoc Biol, 2000, 68(6):779-784; and Zhang G. et al, Vet. Res., 2000, 31(3). -277-296, which are each incorporated by reference in their entirety.

In one embodiment, the antimicrobial peptide administered to the subject, or the antimicrobial peptide encoded by the polynucleotide administered to the subject, is the full-length dermcidin (DCD) gene product or a DCD-derived peptide, such as one or more peptides listed in Table 1 (SEQ ID NO: 1-14), referred to herein collectively as DCD, DCD peptide, or DCD-derived peptide. In a specific embodiment, DCD, or a polynucleotide encoding DCD, is administered to a mucosal surface on or in a subject, such as an acute or chronic would, or in the subject's urogenital tract. In a preferred embodiment, a polynucleotide encoding DCD is locally administered, and the DCD- encoding polynucleotide is under the control of a regulatory system that is effectively "shut off in the resting state, but which has a rapid and repeatable induction in response to an appropriate inducer molecule. Preferably, the inducer is tetracycline or a tetracycline analog, such as doxycycline. In a preferred embodiment, the vector is a viral vector. In a particularly preferred embodiment, the viral vector is a lenti virus. Table 1 lists several DCD-derived peptides and their amino acid positions within the full-length DCD.

Table 1. DCD-derived pe tides

The sequence of full-length DCD is:

MRFMTLLFLTALAGALVCA YDPEAASAPGSGNPCHEASAAQKENAGEDPGLARQA PKPRKQRSSLLEKGLDGAKKAVGGLGKLGKDAVEDLESVGKGAVHDVKDV LDSVL (SEQ ID NO:15). The sequence of DCD-IL (amino acid positions 63-110) (SEQ ID NO:1) is marked in bold, and the signal peptide (amino acid positions 1-19) is marked in italics (Rieg, S. et ctl, J Invest Dermatol, 2006, Feb;126(2):354-65, which is incorporated herein by reference in its entirety).

DCD-IL and DCD-I exhibit antimicrobial activity against Gram-positive organisms including S. aureus, E. faecalis, and Gram-negative organisms including E. coli (Schittek et al, Nat. Immunol., 2001, 2:1133-1137). Further investigations revealed an extended antimicrobial spectrum including S. epidermidis (Vuong et al, Cell Microbiol., 2004, 6:269-275), Pseudomonas putida, methicillin-resistant £ aureus, as well as rifampicin- and isoniazid-resistant Mycobacterium tuberculosis (Lai et al, Biochem. Biophys. Res. Commun., 2005, 328:243-250). Additionally, shorter DCD fragments like SSL-46 (amino acid positions 63-108) (Rieg et al., J. Immunol, 2005, 174:8003-8010) and SSL-25 exhibit antimicrobial activity. Thus, analogous to the processing of other human antimicrobial peptides like cathelicidin LL-37, proteolytically derived peptides exhibiting antimicrobial activity originate from the C-terminus of the full-length DCD peptide.

I. Production of Antimicrobial Peptides

The antimicrobial peptides utilized in the compositions and methods of the invention can be naturally or non-naturally occurring peptides. For example, the antimicrobial peptides can be recombinantly made, chemically synthesized, or naturally existing and isolated from the natural source. Antimicrobial peptides can be prepared by well-known synthetic procedures. For example, the peptides can be prepared by the well- known Merrifield solid support method. See Merrifield, J. Amer. Chem. Soc, 1963, 85:2149-2154 and Merrifield (1965) Science 150:178-185. This procedure, using many of the same chemical reactions and blocking groups of classical peptide synthesis, provides a growing peptide chain anchored by its carboxyl terminus to a solid support, usually cross-linked polystyrene or styrenedivinylbenzene copolymer. This method conveniently simplifies the number of procedural manipulations since removal of the excess reagents at each step is effected simply by washing of the polymer. Alternatively, antimicrobial peptides can be prepared by use of well-known molecular biology procedures. Polynucleotides, such as DNA sequences, encoding the antimicrobial peptides can be readily synthesized. Such polynucleotides are a further aspect of the present invention. These polynucleotides can be used to genetically engineer eukaryotic or prokaryotic cells, for example, bacteria cells, insect cells, algae cells, plant cells, mammalian cells, yeast cells or fungi cells for synthesis of the peptides of the invention. Preferably, the genetically engineered cells are not susceptible to the antimicrobial peptide(s) to be produced. The recombinant production of a 48-amino acid DCD variant with C-terminal homoserine lactone (DCD-I HsI) by E. coli has been reported in the literature (Cipakova I. et ah, Protein Expression and Purification, 2006, 45:269-274, which is incorporated herein by reference in its entirety).

The antimicrobial peptide (such as DCD) can be delivered as a single polypeptide, a fusion protein (fused to another copy of the antimicrobial peptide, a different antimicrobial peptide, or a non-antimicrobial peptide), or as a multimer (comprising multiple copies of the antimicrobial peptide {e.g., DCD) monomer. Monomer units of antimicrobial peptides are typically joined end to end until the desired minimum size is reached {e.g., 15-20 amino acids, 20-30 amino acids, 40-50 amino acids, 60-70 amino acids, 70-80 amino acids, etc.). Optionally, each antimicrobial peptide monomer is separated from the next by an intervening proteolytic cleavage site. Preferably, the proteolytic cleavage site is one susceptible to proteolysis by an enzyme present at the intended anatomical site (target site) of the subject (such as a wound or infection site). Alternatively, the proteolytic cleavage site can be susceptible to proteolysis by an enzyme that is to administered to the subject systemically or locally at the target site before, during, or after delivery of the multimer. U.S. patent publication no. 20050187151 (Strom R.M. et al.) describes antimicrobial peptides made by polymerizing identical monomer units of four or fewer amino acids. The fusion protein can include a targeting moiety, such as in U.S. patent publication no. 20040137482 (Eckert R. et al.\ which recognizes a target microbial organism. The antimicrobial peptide can be administered as a single polypeptide, fusion protein, or in multimeric form, for example, by delivery of a viral or non-viral vector comprising nucleic acid sequences encoding the single polypeptide, fusion protein, or multimer.

Antimicrobial peptides utilized in the invention can be composed of one or more D-amino acid residues (dextrorotatory isomers), and may contain non-peptide linkages between two or more amino acid residues. Amino-peptidases present in serum are capable of removing one or more residues from the N-terminus of peptides (see, for example, Hooper, N. M. Ectopeptidases, in Biological Barriers to Protein Delivery, pp.23-50, eds., Audus and Raub, Plenum Press, New York, 1993). Some amino- peptidases have a broad substrate specificity, releasing the N-terminal amino acid from unblocked peptides. Based on sites of potential hydrolysis, peptides can be designed to minimize certain degradation pathways. Serum degradation at specific amino acids within a peptide may be avoided by incorporation of D-amino acids or other atypical amino acids, and/or by cyclization to prevent protease recognition.

Amino acid residues are referred to herein by their standard single letter notations: A, alanine; C, cysteine; D, aspartic acid; E, glutamic acid; F, phenylalanine; G, glycine; H, histidine; I, isoleucine; K, lysine; L, leucine; M, methionine; N, asparagine; O, ornithine; P₅ proline; Q, glutamine; R, arginine; S, serine; T, threonine; V, valine; W, tryptophan; X, hydroxyproline; Y, tyrosine. Naturally occurring amino acids are generally L-enantiomers; D-amino acids are so-designated (such as "D-lysine").

II. Polynucleotides Encoding Antimicrobial Peptides and their Expression

Typically, the nucleic acid sequence encoding the antimicrobial peptide will be incorporated into a vector (e.g., a plasmid or virus, such as lentivirus) containing other transcription or translational elements.

Nucleic acids encoding antimicrobial peptides can be introduced into a cell by viral vectors, direct DNA transfection, or non-viral vectors, such as lipofection, particle- mediated gene transfer, calcium phosphate transfection, DEAE-dextran, electroporation, microinjection, cationic lipid-mediated transfection, transduction, scrape loading, ballistic introduction and infection (see, for example, Sambrook et αi, Molecular Cloning: A Laboratory Manual, 2^nd Ed., 1989, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N. Y.). When transfers are made to host cells in vivo, the preferred method of transformation utilizes a viral vector, such as lentivirus. Cells that have incorporated constructs can be identified using hybridization techniques well known in the art or by- using the polymerase chain reaction (PCR) to amplify specific recombinant sequences.

Vectors carrying the polynucleotide encoding the antimicrobial peptide can be administered to cells in vitro or in vivo. For example, vectors can be administered systemically or locally at a target anatomical site. Alternatively, cells can be genetically modified in vitro with vectors carrying the polynucleotide encoding the antimicrobial peptide (e.g., with vectors of the invention) and the cells or their progeny can be administered to a subject systemically or locally at a target anatomical site. Preferably, the subject's own cells are obtained and genetically modified with the polynucleotide encoding the antimicrobial peptide in vitro and subsequently administered (returned) to the subject (ex vivo). The host cells may be returned to the subject in the same or adjacent anatomical region from which they were taken or administered at a remote anatomical region, depending upon where production of the antimicrobial peptide is desired.

A. Vectors

In accordance with the method of the invention, various viral or non-viral vectors may be used to deliver polynucleotides encoding antimicrobial peptides to cells in vitro or in vivo, resulting in expression and production of the antimicrobial peptide. Suitable expression vectors for antimicrobial peptides include any that are known in the art or yet to be identified that will cause expression of antimicrobial peptide-encoding nucleic acid sequences in vertebrate cells such as mammalian cells. Nucleic acids encoding antimicrobial peptides can be introduced into a cell by viral vectors, such as lentivirus, retrovirus, modified herpes virus, herpes virus, adenovirus, adeno-associated virus, DNA conjugates, and the like. Nucleic acids encoding antimicrobial peptides can be introduced into a cell by non- viral vectors such as liposomes, microparticles, or plasmids. A wealth of publications known to those of skill in the art discusses the use of a variety of such vectors for delivery of genes (see, e.g., Ausubel et al, Current Protocols in Molecular Biology, John Wiley & Sons, New York, 1989; Kay, M. A. et al, 2001 Nat. Medic, 7(l):33-40; and Walther W. and Stein U., Drugs, 2000, 60(2):249-71, which are each incorporated herein by reference in their entirety).

Lentiviral vectors are efficient vehicles for the delivery of genes to both dividing and non-dividing cells in vitro and in vivo. In one embodiment, a lentiviral vector can be constructed in accordance with the subject invention to express an antimicrobial peptide such as DCD and can be used to transfect endometrial and cervical cells as well as keratinocytes in vitro or in vivo. Examples of lentiviral viruses that may be utilized are described in U.S. Patent No. 6,207,455 (Chang) and 6,531,123 (Chang), which are each incorporated herein by reference in their entirety.

B. Promoters Preferably, the polynucleotide encoding the antimicrobial is operably linked to a promoter sequence that permits expression of the polynucleotide in a desired tissue within the patient. Vectors typically include one or more promoters. Suitable promoters and other regulatory sequences can be selected as is desirable for a particular application.

The term "promoter" as used herein refers to any sequence that regulates the expression of a coding sequence, such as a gene. Promoters may be constitutive, inducible, repressible, or tissue-specific, for example. A "promoter" is a control sequence that is a region of a polynucleotide sequence at which initiation and rate of transcription are controlled. It may contain genetic elements at which regulatory proteins and molecules may bind such as RNA polymerase and other transcription factors. The phrases "operable linked", "operatively positioned", "operatively linked", "under control", and "under transcriptional control' mean that a promoter is in a correct functional location and/or orientation in relation to a nucleic acid sequence to control transcriptional initiation and/or expression of that sequence. A promoter may or may not be used in conjunction with an "enhancer," which refers to a cis-acting regulatory sequence involved in the transcriptional activation of a nucleic acid sequence. A promoter may be one naturally associated with a gene or sequence, as may be obtained by isolating the 5' non-coding sequences located upstream of the coding segment and/or exon. Such a promoter can be referred to as "endogenous". Similarly, an enhancer may be one naturally associated with a nucleic acid sequence, located either downstream or upstream of that sequence. Alternatively, certain advantages will be gained by positioning the coding nucleic acid segment under the control of a recombinant or heterologous promoter, which refers to a promoter that is not normally associated with a nucleic acid sequence in its natural environment. A recombinant or heterologous enhancer refers also to an enhancer not normally associated with a nucleic acid sequence in its natural environment. Such promoters or enhancers may include promoters or enhancers of other genes, and promoters or enhancers isolated from any other prokaryotic, viral, or eukaryotic cell, and promoters or enhancers not "naturally occurring," i.e., containing different elements of different transcriptional regulatory regions, and/or mutations that alter expression. In addition to producing nucleic acid sequences of promoters and enhancers synthetically, sequences may be produced using recombinant cloning and/or nucleic acid amplification technology, including PCR, in connection with the compositions disclosed herein (see U.S. Patent No. 4,683,202, U.S. Patent No. 5,928,906, each incorporated herein by reference). Furthermore, it is contemplated the control sequences that direct transcription and/or expression of sequences within non-nuclear organelles such as mitochondria, chloroplasts, and the like, can be employed as well.

The promoters can be inducible, tissue-specific, and/or event-specific, as necessary. Tissue-specific promoters or event-specific promoters may be utilized with polynucleotides encoding antimicrobial peptides to further optimize and localize expression at target sites, such as within diseased tissues (e.g., sites of microbial infection or sites of inflammation caused by the infection). For example, the cytomegalovirus (CMV) promoter (Boshart et al, Cell, 1985, 41:521-530) and SV40 promoter (Subramani et al. , MoI Cell. Biol. , 1981, 1 :854-864) have been found to be suitable, but others can be used as well. Suitable promoters that may be employed also include the retroviral LTR; or any other promoter (e.g., cellular promoters such as eukaryotic cellular promoters including, but not limited to, the histone, pol III, and .beta.-actin promoters). Other viral promoters that may be employed include, but are not limited to, adenovirus promoters, TK promoters, and B 19 parvovirus promoters.

Optionally, the antimicrobial peptide-encoding nucleic acid sequences used in the subject invention include a sequence encoding a signal peptide upstream of the antimicrobial peptide-encoding sequence, thereby permitting secretion of the antimicrobial peptide from a host cell. Also, various promoters may be used to limit the expression of the peptide in specific cells or tissues.

A tissue-specific and/or event-specific promoter or transcription element that responds to the target microenvironment and physiology can also be utilized for increased transgene expression at the desired site. There has been an immense amount of research activity directed at strategies for enhancing the transcriptional activity of weak tissue- specific promoters or otherwise increasing transgene expression with viral vectors. It is possible for such strategies to provide enhancement of gene expression equal to one or two orders of magnitude (see, for example, Nettelbeck et al, Gene Ther., 1998, 5(12):1656-1664 and Qin et al, Hum. Gene Ther., 1997, 8(17):2019-2019). Examples of cardiac-specific promoters are the ventricular form of MLC-2v promoter (see, Zhu et al., MoI Cell Biol, 1993, 13:4432-4444, Navankasattusas et al, MoI Cell Biol, 1992, 12:1469-1479, 1992) and myosin light chain-2 promoter (Franz et al, Circ. Res., 1993, 73:629-638). The E-cadherin promoter directs expression specific to epithelial cells (Behrens et al, PNAS, 199I₅ 88:11495-11499), while the estrogen receptor (ER) 3 gene promoter directs expression specifically to the breast epithelium (Hopp et al, J. Mammary Gland Biol. Neoplasia, 1998, 3:73-83). The human C-reactive protein (CRP) gene promoter (Ruther et al, Oncogene 8:87-93, 1993) is a liver-specific promoter. An example of a muscle-specific gene promoter is human enolase (ENO3) (Peshavaria et al, Biochem. J., 1993, 292(Pt 3):701-704). A number of brain-specific promoters are available such as the thy-1 antigen and gamma-enolase promoters (Vibert et al, Eur. J. Biochem. 181:33-39, 1989). The prostate-specific antigen promoter provides prostate tissue specificity (Pang et al, Gene Ther., 1995, 6(11):1417-1426; Lee et al, Anticancer Res., 1996, 16(4A): 1805-1811). The surfactant protein B promoter provides lung specificity (Strayer et al, Am. J. Respir. Cell MoI. Biol, 1998, 18(1):1-11). Any of the aforementioned promoters may be selected for targeted or regulated expression of the antimicrobial peptide-encoding polynucleotide.

In some embodiments, promoters may govern spatial and/or temporal expression. Thus, it is contemplated that promoters useful with the invention are tissue-specific or developmentally-specific (promoting transcription only at certain developmental stages or periods), while in other embodiments, a promoter is inducible, or it is constitutive.

Naturally, it may be important to employ a promoter and/or enhancer that effectively directs the expression of the DNA segment in the cell type, organelle, and organism chosen for expression. Those of skill in the art of molecular biology generally know the use of promoters, enhancers, and cell type combinations for protein expression, for example, see Sambrook et al (1989), incorporated herein by reference. The promoters employed may be constitutive, tissue-specific, inducible, and/or useful under the appropriate conditions to direct high level expression of the introduced DNA segment, such as is advantageous in the large-scale production of recombinant proteins and/or peptides. The promoter may be heterologous or endogenous. In certain embodiments, the promoters employed in the present invention are tissue-specific promoters.

C- Inducible Systems In a preferred embodiment, the polynucleotide encoding the antimicrobial peptide such as DCD is under the control of a regulatory system that is off in the resting state but which has a rapid and repeatable induction in response to an appropriate inducer molecule. In one embodiment, the inducer molecule is doxycycline. In a particularly preferred embodiment, the viral vector is a lentivirus.

A preferred inducible promoter is the tetracycline-controlled transactivator (tTA)- responsive promoter (tet system), a prokaryotic inducible promotor system which has been adapted for use in mammalian cells. The tet system was organized within a retroviral vector so that high levels of constitutively-produced tTA mRNA function not only for production of tTA protein but also the decreased basal expression of the response unit by antisense inhibition. See, Paulus, W. et al. J of Virology, January 1996, 70(l):62~ 67. If the recombinant DNA transferred into the cells produces a protein that can be detected, e.g. , by means of an immunological or enzymatic assay, then the presence of recombinant protein can be confirmed by introducing tetracycline into cells and then performing the assays either on the medium surrounding the cells or on cellular lysates.

In the absence of tetracycline, host cells transformed with the constructs should not express substantial amounts of recombinant DNA. Expression of recombinant DNA sequences incorporated into host cells is induced using either tetracycline per se or a tetracycline analog. The latter is broadly defined as any compound that is related to tetracycline in the sense that it maintains the ability to bind with specificity to the tet repressor. The dissociation constants of such analogs are preferably at least 1 x 10^"6 M and more preferably greater than 1 x 10^"9 M. Examples of analogues that can be used include, but are not limited to, those discussed by Hlavka, et al. ("The Tetracyclines," in Handbook of Experimental Pharmacology 78, Blackwood, et al. - (eds.), New York (1985)) and Mitschef ("The Chemistry of Tetracycline Antibiotics", Medicinal Res. 9, New York (1978)). Similarly, minor modifications in the sequence of the repressor or the operator will not affect the invention provided that such modifications do not substantially reduce either the affinity or specificity of the repressor/operator interaction.

As indicated above, a "tetracycline analog" is any one of a number of compounds that are closely related to tetracycline (Tc) and which bind to the tet repressor, preferably with a K_a of at least about 10⁶ M^"1. More preferably, the tetracycline analog binds with an affinity of about 10⁹ M^"1 or greater, e.g., 10⁹ M^"1. Tetracycline analogs having the ability to bind the tet repressor may be tetracycline derivatives (i.e., derived from tetracycline) or non-derivatives. Examples of such tetracycline analogs include, but are not limited to those disclosed by Hlavka and Boothe, "The Tetracyclines," in Handbook of Experimental Pharmacology 78, R. K. Blackwood et al. (eds.), Springer Verlag, Berlin- New York, 1985; L. A. Mitscher "The Chemistry of the Tetracycline Antibiotics", Medicinal Research 9, Dekker, New York, 1978; Noyee Development Corporation, "Tetracycline Manufacturing Processes," Chemical Process Reviews, Park Ridge, N.J., 2 volumes, 1969; R. C. Evans, "The Technology of the Tetracyclines," Biochemical Reference Series 1, Quadrangle Press, New York, 1968; and H. F. Dowling, "Tetracycline," Antibiotics Monographs, no. 3, Medical Encyclopedia, New York, 1955; the contents of each of which are fully incorporated by reference herein. Non-limiting examples of tetracycline analogs include anhydrotetracycline, doxycycline, chlorotetracycline, epioxy tetracycline, and the like. Certain Tc analogs, such as anhydrotetracycline and epioxy tetracycline, have reduced antibiotic activity compared to Tc. Concentrations of the tetracycline or tetracycline analog useful in the present invention are known in the art or are determined by standard means in the art. In specific embodiments, a doxycycline concentration greater than about 10 ng/mL is utilized.

In specific embodiments of the invention, the antimicrobial peptide is a fusion or chimeric protein. A chimeric protein is a polypeptide that contains all or a discrete part of two or more polypeptides. A discrete part of a polypeptide refers to an amino acid region that contains an identifiable function or activity. A fusion protein is a type of chimeric protein in which a first polypeptide or part of the first polypeptide is linked end-to-end to a second polypeptide or a part of the second polypeptide. In specific embodiments of the invention, there is a chimeric protein that is a regulatable transcriptional modulator. In some cases, the regulatable transcriptional modulator is a fusion protein with a DNA binding domain from one polypeptide and a transcription repression or activation domain from another. In specific embodiments, the regulatable transcription modulator can be negatively regulated or modified, such as by the binding of a drug. In further embodiments, the regulatable transcription modulator can be negatively regulated by tetracycline or a tetracycline analog, such as doxycycline.

The term "transcription modulator" refers to a polypeptide with an activity that directly or indirectly affects transcription, which activity includes, but is not limited to, nucleic acid binding activity, transcriptional activation activity, and/or transcriptional repression activity. Furthermore, the transcription modulator can be "regulatable" in some embodiments of the invention, which means that its activity can be regulated, that is, inhibited, eliminated, reduced, increased, activated, or altered. Regulation may be temporally or spatially limited as well. It is contemplated that an activity of the transcription modulator may be regulated or modified by altering, for example, one or more of the following transcription; translation; mRNA half-life; protein half-life; post- translational modification; localization; nucleic acid or polypeptide binding specificity, rate of dissociation, or affinity; and/or transcriptional activity. Negative regulation or modification refers to a reduction or elimination of activity, while positive regulation or modification refers to an increase or induction of activity. For example, negative modification of a transcriptional repressor may result in alleviation of the repression it is exerting.

Expression of the transcription modulator may be regulated. Its expression may be under the control of a regulatable promoter, such as one that is tissue-specific or inducible. The term "inducible" refers to an activity that can be activated only in response to a specific stimulus, in contrast to a "constitutive" activity. In the context of a "promoter," the term "inducible" means the promoter will promote transcription only under certain conditions, unlike constitutive promoters. A promoter that is inducible is understood to allow for conditional expression. The term "tissue-specific" means that an activity is present only in a specific tissue as opposed being present ubiquitously. In particular embodiments, a promoter used in the present invention is an externally controllable promoter, which may be defined as any promoter (conditional, tissue-specific, regulatable, constitutive, etc.) operably linked to at least one polynucleotide sequence bindable by the binding domain of a conditional repressor fusion protein that comprises a DNA binding domain and a transcription repression domain, and positioned such that the transcription repression domain acts to repress transcription of a coding sequence.

Inducible promoters are characterized by resulting in additional transcription activity when in the presence of, influenced by, or contacted by the inducer than when not in the presence of, under the influence of, or in contact with the promoter. The inducer may be endogenous, or a normally exogenous compound or protein that is administered in such a way as to be active in inducing the inducible promoter. Provision of the inducer, i.e., a compound or protein, may itself be the result of transcription or expression of a polynucleotide, which itself may be under the control or an inducible or repressible promoter. Examples of inducible promoters include but are not limited to: tetracycline or tetracycline analogs, metallothionine, ecdysone, mammalian viruses {e.g., the adenovirus late promoter; and the mouse mammary tumor virus long terminal repeat (MMTV-LTR)) and other steroid-responsive promoters, rapamycin responsive promoters and the like.

The inducible promoters used in the present invention are capable of functioning in a eukaryotic host organism. Preferred embodiments include mammalian inducible promoters, although inducible promoters from other organisms as well as synthetic promoters designed to function in a eukaryotic host may be used. The important functional characteristic of the inducible promoters of the present invention is their ultimate inducibility by exposure to an externally applied agent, such as an environmental inducing agent. Appropriate environmental inducing agents include exposure to heat, various steroidal compounds, divalent cations (including Cu⁺² and Zn⁺²), galactose, tetracycline, IPTG (isopropyl beta-D thiogalactoside), as well as other naturally occurring and synthetic inducing agents and gratuitous inducers. It is important to note that, in certain modes of the invention, the environmental inducing signal can correspond to the removal of any of the above listed agents which are otherwise continuously supplied in the uninduced state. ^' The inducibility of a eukaryotic promoter can be achieved by various mechanisms. Suitable inducible promoters can be dependent upon transcriptional activators that, in turn, are reliant upon an environmental inducing agent. Also, the inducible promoters can be repressed by a transcriptional repressor which itself is rendered inactive by an environmental inducing agent. Thus, the inducible promoter can be either one that is induced by an environmental agent that positively activates a transcriptional activator, or one which is de-repressed by an environmental agent which negatively regulates a transcriptional repressor.

The inducible promoters used in the present invention include those controlled by the action of latent transcriptional activators that are subject to induction by the action of environmental inducing agents. Preferred examples include the copper-inducible promoters of the yeast genes CUPl, CRS5, and SODl that are subject to copper- dependent activation by the yeast ACEl transcriptional activator. Alternatively, the copper inducible promoter of the yeast gene CTTl (encoding cytosolic catalase T), which operates independently of the ACEl transcriptional activator, can be utilized. The copper concentrations required for effective induction of these genes are suitably low so as to be tolerated by most cell systems, including yeast and Drosophila cells. Alternatively, other naturally occurring inducible promoters can be used in the present invention including: steroid inducible gene promoters (see, for example, Oligino et al. Gene Ther., 1998, 5: 491-6); galactose inducible promoters from yeast (see, for example, Johnston, Microbiol Rev, 1987, 51: 458-76; Ruzzi et al, MoI Cell Biol, 1987, 7: 991-7); and various heat shock gene promoters. Many eukaryotic transcriptional activators have been shown to function in a broad range of eukaryotic host cells, and so, for example, many of the inducible promoters identified in yeast can be adapted for use in a mammalian host cell as well. For example, a unique synthetic transcriptional induction system for mammalian cells has been developed based upon a GAL4-estrogen receptor fusion protein that induces mammalian promoters containing GAL4 binding sites (Braselmann et al., Proc Natl Acad Sci, 1993, USA 90: 1657-61). These and other inducible promoters responsive to transcriptional activators that are dependent upon specific inducing agents are suitable for use with the present invention.

The inducible promoters of the present invention also include those that are repressed by repressors that are subject to inactivation by the action of environmental inducing agents. Examples include prokaryotic repressors that can transcriptionally repress eukaryotic promoters that have been engineered to incorporate appropriate repressor-binding operator sequences. Preferred repressors for use in the present invention are sensitive to inactivation by physiologically benign inducing agent. Thus, where the lac repressor protein is used to control the expression of a eukaryotic promoter that has been engineered to contain a lacO operator sequence, treatment of the host cell with IPTG will cause the dissociation of the lac repressor from the engineered promoter and allow transcription to occur. Similarly, where the tet repressor is used to control the expression of a eukaryotic promoter that has been engineered to contain a tetO operator sequence, treatment of the host cell with tetracycline will cause the dissociation of the tet repressor from the engineered promoter and allow transcription to occur.

The promoter may be induced by one or more physiological conditions, such as changes in pH, temperature, radiation, osmotic pressure, saline gradients, cell surface binding and the concentration of one or more extrinsic or intrinsic agents. The extrinsic agent may comprise amino acids and amino acid analogs, saccharides and polysaccharides, nucleic acids, transcriptional activators and repressors, cytokines, toxins, petroleum-based compounds, metal containing compounds, salts, ions, enzyme substrate analogs and combinations thereof. In specific embodiments, the inducible promoter is activated or repressed in response to a change of an environmental condition, such as the change in concentration of a chemical, metal, radiation or nutrient or change in pH.

The inducible promoter may be a phage inducible promoter, nutrient inducible promoter, temperature inducible promoter, radiation inducible promoter, metal inducible promoter, hormone inducible promoter, steroid inducible promoter, and/or hybrids and combinations thereof. Promoters that are inducible by ionizing radiation may be used in certain embodiments, particularly in gene therapy of cancer, where gene expression is induced locally in the cancer cells by exposure to ionizing radiation such as UV or x-rays. Radiation inducible promoters include the non-limiting examples of fos promoter, c-jun promoter or at least one CArG domain of an Egr-1 promoter. Examples of inducible promoters include promoters from genes such as cytochrome P450 genes, heat shock protein genes, metallothionein genes, hormone-inducible genes, such as the estrogen gene promoter, and such.

The inducible promoter may be Zn²⁺ metallothionein promoter, metallothionein- 1 promoter, human metallothionein HA promoter, lac promoter, laco promoter, mouse mammary tumor virus early promoter, mouse mammary tumor virus LTR promoter, triose dehydrogenase promoter, herpes simplex virus thymidine kinase promoter, simian virus 40 early promoter or retroviral myeloproliferative sarcoma virus promoter.

Examples of inducible promoters include mammalian probasin promoter, lactalbumin promoter, GRP78 promoter, or the bacterial tetracycline-inducible promoter. Other examples include heat shock, steroid hormone, heavy metal, phorbol ester, adenovirus ElA element, interferon, and serum inducible promoters.

Inducible promoters for in vivo uses may include those responsive to biologically compatible agents, such as those that are usually encountered in defined animal tissues. An example is the human PAI-I promoter, which is inducible by tumor necrosis factor. Further examples of inducible promoters for use in vivo include cytochrome P450 gene promoters, inducible by various toxins and other agents; heat shock protein genes, inducible by various stresses; hormone-inducible genes, such as the estrogen gene promoter, and such. In some embodiments, the inducer molecule is a molecule that is heterologous to the subject (does not naturally occur in the subject), is not normally found in the target anatomical site, or is normally found at a relatively low level at the target anatomical site.

In those embodiments of the method utilizing an antimicrobial peptide-encoding polynucleotide under the control of a regulatory system that is induced by an inducer molecule (such as tetracycline or a tetracycline derivative), the method may further comprise administering the inducer molecule to the subject systemically or locally at the target anatomical site (e.g., at the site of the vector containing the antimicrobial peptide- encoding polynucleotide). The inducer molecule can be administered before, during, or after administration of the antimicrobial peptide-encoding polynucleotide.

D. Host Cells

The vectors of the present invention can be administered in vitro or in vivo to any cells of the subject's body. As will be understood by one of skill in the art, there are over 200 cell types in the human body. It is believed that the methods of the subject invention can be used to deliver polynucleotides encoding antimicrobial peptides to any of these cell types in vitro or in vivo, for therapeutic or other purposes. For example, any cell arising from the ectoderm, mesoderm, or endoderm germ cell layers can be a target cell for administration of the vectors of the invention. The terms "recombinant host cells", "host cells", "genetically modified cells",

"genetically modified host cells" "cells", "cell lines", "cell cultures", and other such terms denoting microorganisms or higher eukaryotic cells refer to cells that can be, or have been, used as recipients for vectors or expression cassettes carrying a nucleic acid sequence encoding an antimicrobial peptide, immaterial of the method by which the nucleic acid sequence is introduced into the cell or the subsequent disposition of the cell. The terms include the progeny of the original cell that has been transfected. Cells in primary culture can also be used as recipients. Host cells can range in plasticity and proliferation potential.

Host cells can be differentiated cells, progenitor cells, or stem cells, for example. Thus, the cells can range in plasticity from totipotent or pluripotent stem cells (e.g., adult or embryonic), precursor or progenitor cells, to highly specialized cells, such as those of the central nervous system (e.g., neurons and glia). Stem cells can be obtained from a variety of sources, including embryonic tissue, fetal tissue, adult tissue, umbilical cord blood, peripheral blood, bone marrow, and brain, for example.

Host cells can be genetically modified with the vectors of the present invention. The vector may be in the form of a plasmid, a virus, (e.g., AAV, lentivirus, or other virus), a viral particle, a phage, etc. Preferably, the vector has a tetracycline-dependent transcriptional regulatory system such that the expression of the nucleic acid sequence encoding the antimicrobial peptide is effectively "turned off in the absence of an inducer molecule such as tetracycline or its analogs and "turned on" in the presence of the inducer molecule such as tetracycline or its analogs in vitro or in vivo. Thus, the inducer molecule can be administered to a subject any time before, during, or after the host cells or vectors are administered to the subject to trigger expression of the nucleic acid sequence encoding the antimicrobial peptide (and, optionally, triggering expression of other nucleic acid sequences carried by the vectors or host cells).

The genetically modified host cells can be cultured in conventional nutrient media modified as appropriate for activating promoters, selecting transformants/transfectants or amplifying the subunit-encoding polynucleotide. The culture conditions, such as temperature, pH and the like, generally are similar to those previously used with the host cell selected for expression, and will be apparent to those of skill in the art.

In one embodiment, the host cell is a human cell. In another embodiment, the host cell is a non-human mammalian cell. It will be understood by one of skill in the art that the methods of the present invention are also applicable for veterinary purposes. For example, host cells of non-human animals can find application either in human or animal patients (e.g., veterinary uses).

Both prokaryotic and eukaryotic host cells may be used for expression of desired coding sequences when appropriate control sequences (e.g. , promoter sequences) that are compatible with the designated host are used. For example, among prokaryotic hosts, Escherichia coli may be used. Transfer vectors compatible with prokaryotic hosts can be derived from, for example, the plasmid pBR322 that contains operons conferring ampicillin and tetracycline resistance, and the various pUC vectors, that also contain sequences conferring antibiotic resistance markers. These markers may be used to obtain successful transformants by selection. Eukaryotic hosts include yeast and mammalian cells in culture systems. Pichiapastoris, Saccharomyces cerevisiae and S. carlsbergensis are commonly used yeast hosts. Yeast-compatible vectors carry markers that permit selection of successful transformants by conferring protrophy to auxotrophic mutants or resistance to heavy metals on wild-type strains. Yeast compatible vectors may employ the 2-μ origin of replication (Broach et al. Meth. Enzγmol. 101 :307, 1983), the combination of CEN3 and ARSl or other means for assuring replication, such as sequences that will result in incorporation of an appropriate fragment into the host cell genome.

The invention encompasses the host cells transformed by a vector according to the invention. These cells may be obtained by introducing (in vitro or in vivo) into host cells a nucleotide sequence carried by a vector as defined above. Optionally, if the cells are isolated, the cells can be cultured under conditions allowing the replication and/or the expression of the polynucleotide sequences carried by the vector.

Host cells useful for expression of polynucleotides encoding antimicrobial peptides may be primary cells or cells of cell lines. The host cells may be tumor cells or non-tumor cells. Mammalian cell lines available as hosts for expression are known in the art and are available from depositories such as the American Type Culture Collection. These include but are not limited to HeLa cells, human embryonic kidney (HEK) cells, Chinese hamster ovary (CHO) cells, baby hamster kidney (BHK) cells, and others.

Selection of the type of host cell for genetic modification will likely depend upon the intended use of the cell. For example, if a polynucleotide encoding the antimicrobial peptide is to be delivered to a wound in vivo, or if cells are to be genetically modified with the antimicrobial peptide-encoding polynucleotide and administered to a wound, tissue repair cells (e.g., fibroblasts, endothelial cells, inflammatory cells) may be the desirable target cells. In one embodiment, the host cell to which the antimicrobial peptide-encoding polynucleotide is administered is a keratinocyte. Keratinocytes are a major source of antimicrobial peptides in human skin (Harder, J. and Schroder, J.M. J. Biol. Chem., 2002, 277:46779-46784; Glaser, R. et al. Nat. Immunol, 2005, 6:57-64; Wiedow, O. et al. Biochem. Biophys. Res. Commun., 1998, 248:904-909; Frohm, M. et al J. Biol Chem., 1997, 272:15258-15263; Harder, J. et al Nature, 1997, 387:861; Harder, J. et al. J. Biol. Chem., 2001, 276:5707-5713;). Extensive information concerning the introduction and expression of recombinant genes in keratinocytes for the purpose of treating disease is described in Garlick J. A. and Fenjves E.S., Crit. Rev. Oral Biol Med., 7(3):204-221; Arango M. et al, Dermatology Online Journal, 2005, 11(2), which are each incorporated herein by reference in its entirety. Keratinocytes can be genetically modified in vitro with a viral or non-viral vector of the invention to produce an antimicrobial peptide. The genetically modified keratinocyte can then be administered to a subject in need thereof {e.g., ex vivo). Alternatively, antimicrobial peptides or polynucleotides encoding them can be administered to keratinocytes in vivo. Epidermal keratinocytes can secrete polypeptides into the bloodstream, and they can be easily expanded in culture and genetically modified. It is thus possible to use epidermal keratinocytes for the local or systemic delivery of transgene products (Cao T. et al., Human Gene Therapy, 2000, l l(16):2297-2300; Bajaj B. et al, Human Gene Therapy, 2002, 13(15):1821-1831; Cao T. et al, Human Gene Therapy, 2002, 13(9): 1075-1080; and Baek S-C et al, Human Gene Therapy, 2001, 12(12):1551=-1558, which are each incorporated herein by reference in their entirety).

Optionally, the host cells of the invention can be genetically modified with additional nucleic acid sequences encoding desired products, such as growth factors. For example, the observation that non-healing wounds have decreased levels of growth factors has given rise to the approach of treating such wounds with growth factor proteins. Examples of such growth factors include PDGF, TGF-beta, FGG, EGF, keratinocyte growth factor (KGF), vascular endothelial growth factor (VEGF), platelet- derived growth factor (PDGF), and insulin-like growth factor (IGF). Limitations of this approach have been attributed to their short half-lives, degradation by wound proteases, and failure to maintain local protein levels above the therapeutic threshold. In light of these limitations, gene therapy has attracted significant attention as an alternative, cost- effective approach for wound therapy.

III. Antimicrobial Formulations and Therapeutic/Prophylactic Administration Thereof

Mammalian species which benefit from the disclosed methods of treatment include, and are not limited to, apes, chimpanzees, orangutans, humans, monkeys; domesticated animals (e.g., pets) such as dogs, cats, guinea pigs, hamsters, Vietnamese pot-bellied pigs, rabbits, and ferrets; domesticated farm animals such as cows, buffalo, bison, horses, donkey, swine, sheep, and goats; exotic animals typically found in zoos, such as bear, lions, tigers, panthers, elephants, hippopotamus, rhinoceros, giraffes, antelopes, sloth, gazelles, zebras, wildebeests, prairie dogs, koala bears, kangaroo, opossums, raccoons, pandas, hyena, seals, sea lions, elephant seals, otters, porpoises, dolphins, and whales. The terms "patient" and "subject" are used interchangeably herein are intended to include such human and non-human mammalian species. According to the methods of the present invention, human or non-human mammalian antimicrobial peptides (or nucleic acid sequences encoding human or non-human mammalian antimicrobial peptides) can be administered to the subject. The antimicrobial peptide may be naturally occurring within the subject's species or a different mammalian species. The expression vectors used in the subject invention can comprise nucleic acid sequences encoding any human or non-human mammalian antimicrobial peptide. In instances where genetically modified cells carrying the antimicrobial peptide-encoding nucleic acid sequences are administered to a subject, the cells may be autogenic, allogeneic, or xenogeneic, for example.

Pharmaceutical compositions of the subject invention include antimicrobial peptide or polynucleotide encoding the antimicrobial peptide, and a pharmaceutically acceptable carrier. In one embodiment, the pharmaceutical composition comprises a viral or non- viral vector of the invention and a pharmaceutically acceptable carrier. In another embodiment, the pharmaceutical composition comprises host cells of the invention and pharmaceutically acceptable carrier. Pharmaceutical compositions of the invention can be solid, liquid, semi-solid, etc.

The pharmaceutical compositions of the present invention can be formulated according to known methods for preparing pharmaceutically useful compositions. Furthermore, as used herein, the phrase "pharmaceutically acceptable carrier" includes any of the standard pharmaceutically acceptable carriers. The pharmaceutically acceptable carrier can include diluents, adjuvants, and vehicles, as well as implant carriers, and inert, non-toxic solid or liquid fillers, diluents, or encapsulating material that does not react with the active ingredients of the invention. Examples include, but are not limited to, phosphate buffered saline, physiological saline, water, and emulsions, such as oil/water emulsions. The carrier can be a solvent or dispersing medium containing, for example, ethanol, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. Formulations containing pharmaceutically acceptable carriers are described in a number of sources which are well known and readily available to those skilled in the art. For example, Remington 's Pharmaceutical Sciences (Martin E. W., 1995, Easton Pennsylvania, Mack Publishing Company, 19^th ed.), which is incorporated herein by reference in its entirety, describes formulations that can be used in connection with the subject invention. Pharmaceutical compositions of the present invention useful for parenteral injection can include pharmaceutically acceptable sterile aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, as well as sterile powders for reconstitution into sterile injectable solutions or dispersions just prior to use. Examples of suitable aqueous and nonaqueous carriers, diluents, solvents, or vehicles include water, ethanol, polyols (such as glycerol_^ propylene glycol, polyethylene, lycol, and the like), carboxymethylcellulose and suitable mixtures thereof, vegetable oils (such as olive oil), and injectable organic esters such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. Formulations suitable for parenteral administration include, for example, aqueous injectable solutions that may contain antioxidants, buffers, and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and nonaqueous sterile suspensions, which may include suspending agents and thickening agents. The formulations may be presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze dried (lyophilized) condition requiring only the condition of the sterile liquid carrier, for example, water for injections, prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powder, granules, tablets, etc. It should be understood that, in addition to the ingredients particularly mentioned above, the formulations of the subject invention can include other agents conventional in the art having regard to the type of formulation in question.

The pharmaceutical compositions used in the methods of the present invention may also contain adjuvants such as preservatives, wetting agents, emulsifying agents, and dispersing agents. Prevention of the action of microorganisms may be ensured by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents such as sugars, sodium chloride, and the like. Prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents that delay absorption, such as aluminum monostearate and gelatin.

In some cases, in order to prolong the effect of the active agent (e.g. , antimicrobial peptide), it is desirable to slow the absorption from subcutaneous or intramuscular injection. This may be accomplished by the use of a liquid suspension of crystalline or amorphous material with poor water solubility. The rate of absorption of the antimicrobial peptide or antimicrobial peptide-encoding polynucleotide then depends upon its rate of dissolution which, in turn, may depend upon crystal size and crystalline form. Altern atively, delayed absorption of a parenterally administered antimicrobial peptide or antimicrobial peptide-encoding polynucleotide is accomplished by dissolving or suspending the antimicrobial peptide in an oil vehicle.

Injectable depot forms are made by forming microencapsule matrices of the agent (e.g., antimicrobial peptide or antimicrobial peptide-encoding polynucleotide) in biodegradable polymers such as polylactide-polyglycolide. Depending upon the ratio of active agent (e.g., antimicrobial peptide or antimicrobial peptide-encoding polynucleotide) to polymer and the nature of the particular polymer employed, the rate of release can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot injectable formulations are also prepared by entrapping the drug in liposomes or microemulsions which are compatible with body tissues.

The injectable formulations can be sterilized, for example, by filtration through a bacterial-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium just prior to use. Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In such solid dosage forms, the active agents (antimicrobial peptide or antimicrobial peptide-encoding polynucleotide) are mixed with it least one pharmaceutically acceptable excipient or carrier such as sodium nitrate or dicalcium phosphate and/or a) fillers or extenders such as starches, lactose, sucrose, glucose, mannitol, and silicic acid; b) binders such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose, and acacia; c) humectants such as glycerol; d) disintegrating agents such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; e) solution retarding agents such as paraffin; f) absorption accelerators such as quaternary ammonium compounds; g) wetting agents such as, for example, cetyl alcohol and glycerol monostearate; h) absorbents such as kaolin and bentonite clay; and i) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof. In the case of capsules, tablets and pills, the dosage form may also comprise buffering agents.

Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethylene glycols and the like. The solid dosage forms of tablets, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings and other coatings well known in the pharmaceutical formulating art. Optionally, the solid dosage forms contain opacifying agents, and can be of a composition that releases the antimicrobial peptide or antimicrobial peptide-encoding polynucleotide only, or preferentially, in a certain part of the intestinal tract or other target anatomical site, optionally, in a delayed manner. Examples of embedding compositions that can be used include polymeric substances and waxes.

The pharmaceutical composition of the invention can be an article for delivery of the antimicrobial peptide or encoding nucleic acids. Various articles (devices) are available that may be used to deliver the antimicrobial peptides or nucleic acid sequences encoding them to a target anatomical site, such as an acute or chronic wound or tissues of the urogenital tract. In one embodiment, the delivery device is one that delivers the antimicrobial peptide to tissues of the urogenital tract, such as a vaginal tampon, vaginal ring, vaginal cup, vaginal tablet, vaginal sponge, or vaginal bioadhesive tablet, condom, prostheses, or intrauterine device. For example, devices useful for delivery of pharmacological agents to the female urogenital tract include U.S. Patent No. 6,951,654 (Malcolm el al.); U.S. Patent No. 6,416,779 (D'Augustine et al); U.S. Patent No. 6,030,375 (Anderson et al.), which are incorporated herein by reference in their entireties. The antimicrobial peptide can be incorporated into the delivery device as a microemulsion, cream, lotion, self-emulsifying oil, foam, ointment, gel, or solution, for example. Wound dressings, artificial or biological grafts (such as dermal and mucosal grafts), or other articles may be utilized to deliver the antimicrobial peptide or antimicrobial peptide-encoding polynucleotide to an acute or chronic wound (Bhargava S. and Chappie C.R., BJU International, 2004, 93:1191-1193; Laur G. and Schimming R., J. Oral Maxillofac. Surg., 2001, 59:169-177; Laur G. et al, Plast. Reconstr. Surg., 2001, 108(6): 1564-1572; Lauer G. et al, Plast. Reconstr. Surg., 2001, 1078(l):25-33; Shapshay S.M. et al, Ann. Otol. Rhinol. Laryngol, 1995, 104(12):919-923; Linn W.C. et al, Human Reproduction, 2003, 18 (3): 604-607, Moriyama T. et al, Tissue Engineering, 2001, 7(4):415-427; U.S. Patent No. 7,022,890 (Sessions, R. W., filed September 22, 2004), which are each incorporated herein by reference in their entirety). The antimicrobial peptide or encoding nucleic acids can be applied to or placed within such articles. For example, grafts (such as artificial skin) and wound dressings can include keratinocytes or sheets of epithelial cells that have been genetically modified to express nucleic acid sequences encoding antimicrobial peptides.

Antimicrobial articles of the invention exhibit antimicrobial functionality, wherein microbes are killed, and/or microbial growth is reduced or prevented. Antimicrobial activity of the antimicrobial article can be determined by using any number of methods well known in the art, such as those methods for determining antimicrobial activity discussed in Tenover et al (eds.), Manual of Clinical Microbiology, 7^th Edition, Section VIII, 1999, American Society for Microbiology, Washington, D.C., which is incorporated herein by reference in its entirety. The active agents (antimicrobial peptide or antimicrobial peptide-encoding polynucleotide) can also be in micro-encapsulated form, if appropriate, with one or more of the above-mentioned excipients.

Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups and elixirs. In addition to the antimicrobial peptide or antimicrobial peptide-encoding polynucleotide, the liquid dosage forms may contain inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3- butylene glycol, dimethyl formamide, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof. Besides inert diluents, the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents.

Suspensions, in addition to the active compounds, may contain suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar, and tragacanth, and mixtures thereof.

Topical administration includes administration to the skin or mucosa, including surfaces of the lung, eye, tissues of the urogenital tract, peritoneal cavity, wound bed, etc. Compositions for topical administration, including those for inhalation, may be prepared as a dry powder, which may be pressurized or non-pressurized. In non-pressurized powder compositions, the active ingredients in finely divided form may be used in admixture with a larger-sized pharmaceutically acceptable inert carrier comprising particles having a size, for example, of up to 100 μm in diameter. Suitable inert carriers include sugars such as lactose. Desirably, at least 95% by weight of the particles of the active ingredient have an effective particle size in the range of 0.01 to 10 μm.

Many techniques for delivery of drugs and proteins are available in the art to reduce the effects of enzymatic degradation, to facilitate cell uptake, and to reduce any potential toxicity to the patient. Such methods and reagents can be utilized for administration of antimicrobial peptides to cells in vitro or in vivo. For example, peptides known as "cell penetrating peptides" (CPP) or "protein transduction domains" (PTD) have an ability to cross the cell membrane and enter the cell. PTDs can be linked to a cargo moiety such as a drug, peptide, or full-length protein, and can transport the moiety across the cell membrane. One well characterized PTD is the human immunodeficient virus (HIV)-I Tat peptide (see, for example, Frankel et al, U.S. Patent Nos. 5,804,604; 5,747,641; 6,674,980; 5,670,617; and 5,652,122; Fawell, S. et al, Proc. Natl. Acad. Sci. U.S.A., 1994, 91 :664-668). Peptides such as the homeodomaiπ of Drosophila antennapedia (ANTp) and arginine-rich peptides display similar properties (Derossi, D. et al., J. Biol. Chem., 1994, 269:10444-10450; Derossi, D. et al, Trends Cell Biol, 1998, 8:84-87; Rojas, M. et al, Nat. Biotechnol, 1998, 16:370-375; Futaki, S. et al, J. Biol. Chem., 2001, 276:5836-5840). VP22, a tegument protein from Herpes simplex virus type 1 (HSV-I), also has the ability to transport proteins across a cell membrane (Elliot et al, Cell, 1997, 88:223-233; Schwarze S.R. et al, Trends Pharmacol. Set, 2000, 21 :45-48). A common feature of these carriers is that they are highly basic and hydrophilic (Schwarze S.R. et al, Trends Cell Biol, 2000, 10:290-295). Coupling of these carriers to marker proteins such as beta-galactosidase has been shown to confer efficient internalization of the marker protein into cells. More recently, chimeric, in-frame fusion proteins containing these carriers have been used to deliver proteins to a wide spectrum of cell types both in vitro and in vivo. For example, VP22-p53 chimeric protein retained its ability to spread between cells and its pro-apoptotic activity, and had a widespread cytotoxic effect in p53 negative human osteosarcoma cells in vitro (Phelan, A. et al, Nature Biotechnol, 1998, 16:440-443). Intraperitoneal injection of the beta-galactosidase protein fused to the HIV-I Tat peptide resulted in delivery of the biologically active fusion protein to all tissues in mice, including the brain (Schwarze S.R. et al., Science, 1999, 285:1569-1572).

Liposomes of various compositions can also be used for site-specific delivery of proteins and drugs (Witschi, C. et al, Pharm. Res., 1999, 16:382-390; Yeh, M.K. et al, Pharm. Res., 1996, 1693-1698). The interaction between the liposomes and the protein cargo usually relies on hydrophobic interactions or charge attractions, particularly in the case of cationic lipid delivery systems (Zelphati, O. et al, J. Biol Chem., 2001, 276:35103-35110). Tat peptide-bearing liposomes have also been constructed and used to deliver cargo directly into the cytoplasm, bypassing the endocytotic pathway (Torchilin V.P. et al. , Biochim. Biophys. Acta-Biomembranes, 2001 , 1511 :397-411 ; Torchilin V.P. et al, Proc. Natl. Acad. ScL USA, 2001, 98:8786-8791). When encapsulated in sugar- grafted liposomes, pentamidine isethionate and a derivative have been found to be more potent in comparison to normal liposome-encapsulated drug or to the free drug (Banerjee, G. et al, J. Antimicrob. Chemother., 1996, 38(l):145-150). A thermo-sensitive liposomal taxol formulation (heat-mediated targeted drug delivery) has been administered in vivo to tumor-bearing mice in combination with local hyperthermia, and a significant reduction in tumor volume and an increase in survival time was observed compared to the equivalent dose of free taxol with or without hyperthermia (Sharma, D. et al, Melanoma Res., 1998, 8(3):240-244). Topical application of liposome preparations for delivery of insulin, IFN-alpha, IFN-gamma, and prostaglandin El have met with some success (Cevc G. et al, Biochim. Biophys, Acta, 1998, 1368:201-215; Foldvari M. et al, J. Liposome Res., 1997, 7:115-126; Short S.M. et al, Pharm. Res., 1996, 13:1020-1027; Foldvari M. et al, Urology, 1998, 52(5) :838-843; U.S. Patent No. 5,853,755).

Antibodies represent another targeting device that may make liposome uptake tissue-specific or cell-specific (Mastrobattista, E. et al, Biochim. Biophys. Acta, 1999, 1419(2):353-363; Mastrobattista, E. et al, Adv. Drug Deliv. Rev., 1999, 40(l-2):103- 127). The liposome approach offers several advantages, including the ability to slowly release encapsulated drugs and proteins, the capability of evading the immune system and proteolytic enzymes, and the ability to target tumors and cause preferentially accumulation in tumor tissues and their metastases by extravasation through their leaky neo vasculature. Other carriers have also been used to deliver anti-cancer drugs to neoplastic cells, such as polyvinylpyrrolidone nanopaiticles and maleylated bovine serum albumin (Sharma. D. et al, Oncol. Res., 1996, 8(7-8):281-286; Mukhopadhyay, A. et al, FEBS Lett., 1995, 376(l-2):95-98). Thus, using targeting and encapsulation technologies, which are very versatile and amenable to rational design and modification, delivery of antimicrobial peptides to desired cells can be facilitated. Furthermore, because many liposome compositions are also viable delivery vehicles for genetic material, many of the advantages of liposomes are equally applicable to polynucleotides encoding antimicrobial peptides.

As indicated above, the pharmaceutical composition of the present invention can include a liposome component. According to the present invention, a liposome comprises a lipid composition that is capable of fusing with the plasma membrane of a cell, thereby allowing the liposome to deliver a nucleic acid molecule and/or a protein composition into a cell. Some preferred liposomes include those liposomes commonly used in gene delivery methods known to those of skill in the art. Some preferred liposome delivery vehicles comprise multilamellar vesicle (MLV) lipids and extruded lipids, although the invention is not limited to such liposomes. Methods for preparation of MLVs are well known in the art. "Extruded lipids" are also contemplated. Extruded lipids are lipids that are prepared similarly to MLV lipids, but which are subsequently extruded through filters of decreasing size, as described in Templeton et al, Nature Biotech., 1997, 15:647-652, which is incorporated herein by reference in its entirety. Small unilamellar vesicle (SUV) lipids can also be used in the compositions and methods of the present invention. Other preferred liposome delivery vehicles comprise liposomes having a polycationic lipid composition {i.e., cationic liposomes). For example, cationic liposome compositions include, but are not limited to, any cationic liposome complexed with cholesterol, and without limitation, include DOTMA and cholesterol, DOTAP and cholesterol, DOTIM and cholesterol, and DDAB and cholesterol. Liposomes utilized in the present invention can be any size, including from about 10 to 1000 nanometers (ran), or any size in between.

A liposome delivery vehicle can be modified to target a particular site in a mammal, thereby targeting and making use of an antimicrobial peptide-encoding nucleic acid molecule of the present invention at that site. Suitable modifications include manipulating the chemical formula of the lipid portion of the delivery vehicle. Manipulating the chemical formula of the lipid portion of the delivery vehicle can elicit the extracellular or intracellular targeting of the delivery vehicle. For example, a chemical can be added to the lipid formula of a liposome that alters the charge of the lipid bilayer of the liposome so that the liposome fuses with particular cells having particular charge characteristics. In one embodiment, other targeting mechanisms, such as targeting by addition of exogenous targeting molecules to a liposome (i.e., antibodies) may not be a necessary component of the liposome of the present invention, since effective immune activation at immunologically active organs can already be provided by the composition when the route of delivery is intravenous or intraperitoneal, without the aid of additional targeting mechanisms. However, in some embodiments, a liposome can be directed to a particular target cell or tissue by using a targeting agent, such as an antibody, soluble receptor or ligand, incorporated with the liposome, to target a particular cell or tissue to which the targeting molecule can bind. Targeting liposomes are described, for example, in Ho et al, Biochemistry, 1986, 25: 5500-6; Ho et al, J Biol Chem, 1987a, 262: 13979- 84; Ho et al, J Biol Chem, 1987b, 262: 13973-8; and U.S. Patent No. 4,957,735 to Huang et al, each of which is incorporated herein by reference in its entirety). In one embodiment, if avoidance of the efficient uptake of injected liposomes by reticuloendothelial system cells due to opsonization of liposomes by plasma proteins or other factors is desired, hydrophilic lipids, such as gangliosides (Allen et al., FEBS Lett, 1987, 223 : 42-6) or polyethylene glycol (PEG)-derived lipids (Klibanov et al. , FEBS Lett, 1990, 268: 235-7), can be incorporated into the bilayer of a conventional liposome to form the so-called sterically-stabilized or "stealth" liposomes (Woodle et al., Biochim Biophys Acta, 1992, 1113: 171-99). Variations of such liposomes are described, for example, in U.S. Patent No. 5,705,187 to Unger et al, U.S. Patent No. 5,820,873 to Choi et al, U.S. Patent No. 5,817,856 to Tirosh et al; U.S. Patent No. 5,686,101 to Tagawa et al; U.S. Patent No. 5,043,164 to Huang et al, and U.S. Patent No. 5,013,556 to Woodle et al, all of which are incorporated herein by reference in their entireties).

The methods of this invention, generally speaking, may be practiced using any mode of administration that is medically acceptable, meaning any mode that produces effective levels of response without causing clinically unacceptable adverse effects. Preferred modes of administration include parenteral, injection, infusion, deposition, implantation, anal or vaginal supposition, oral ingestion, inhalation, and topical administration. Injections can be intravenous, intradermal, subcutaneous, intramuscular, or interperitoneal. For example, the pharmaceutical composition comprising the antimicrobial peptide or antimicrobial peptide-encoding polynucleotide can be injected or topically applied directly into or on to a target site in the urogenital tract, wound area, or other anatomical target site. In some embodiments, the doses can be given at multiple locations. Implantation includes inserting implantable drug delivery systems, e.g., microspheres, hydrogels, polymeric reservoirs, cholesterol matrixes, polymeric systems, e.g., matrix erosion and/or diffusion systems and non-polymeric systems, e.g., compressed, fused, or partially fused pellets. Inhalation includes administering the pharmaceutical composition with an aerosol in an inhaler, either alone or attached to a carrier that can be absorbed. For systemic administration, it may be preferred that the pharmaceutical composition is encapsulated in liposomes. The term "parenteral" includes subcutaneous injections, intravenous, intramuscular, intraperitoneal, intrastemal injection or infusion techniques. In certain preferred embodiments of the invention, the administration can be designed so as to result in sequential exposure of the pharmaceutical composition over some period of time, e.g., hours, days, weeks, months or years. This can be accomplished by repeated administrations of the pharmaceutical composition, by one of the methods described above, or alternatively, by a sustained- release delivery system in which the pharmaceutical composition is delivered to the subject for a prolonged period without repeated administrations. By sustained-release delivery system, it is meant that total release of the pharmaceutical composition does not occur immediately upon administration, but rather is delayed for some period of time. Release can occur in bursts or it can occur gradually and continuously. Administration of such a system can be, e.g., by long-lasting oral dosage forms, bolus injections, transdermal patches, and subcutaneous implants.

The antimicrobial peptides or polynucleotides encoding them can be incorporated in a physiologically acceptable carrier or salt, suitable for topical application to the affected area, or for direct injection into the affected areas such as the vagina or peritoneal cavity, or for diffusion from an implanted device. Topical applications include lavage of body cavities or lumens, e.g., pre- or post-surgical peritoneal lavage or pulmonary lavage. Topical applications include use of gels, creams, lotions, supposities, and use of devices and dressings such as dissolving patches and bandages impregnated prior to use with the antimicrobial peptide. Additional routes of delivery include oral, and injection or infusion that is intramuscular, intravenous, subcutaneous, intraperitoneal, intraspinal, and epidural.

The compositions can contain from about 0.1 nM to about 10 mM of antimicrobial peptide, usually containing from about 0.01 μM to about 1 mM of antimicrobial peptide, and more usually containing from about 0.1 μM to about 100 μM of antimicrobial peptide. However, as indicated above, compositions can contain polynucleotides encoding the antimicrobial peptide (e.g., within a viral or non- viral vector). The nature of the carrier depends on the intended area of application. For application to the skin, a cream lotion, or ointment base is usually preferred, with suitable bases including lanolin, SILVADENE, particularly for the treatment of burns; AQUAPHOR (Duke Laboratories, South Norwalk, Conn.), and the like. The antimicrobial peptides can be incorporated into or onto natural and synthetic bandages and other wound dressings to provide for continuous exposure of a wound to the antimicrobial peptide. Aerosol applicators and inhaler devices can be used for delivery to sinuses and deeper portions of the respiratory system. Antimicrobial peptides can also incorporated in or coated on implantable devices, such as heart pacemakers, intralumenal stents, and the like where the antimicrobial activity would be of benefit. Coating can be achieved by non-specific adsorption or covalent attachment. Optionally, an anti-pruritic agent such as an opioid is added to an antimicrobial composition to relieve pain at an infected site. Additional antimicrobial agents can be combined with the antimicrobial peptides or encoding polynucleotides, including but not limited to one or more of beta-lactam antibiotics such as penicillin, macrolides such as erythromycin, aminoglycosides such as lincomycin, tetracyclines such as doxycycline, semi-synthetic antibiotics such as Ceclor, and bacterially-derived peptide antibiotics such as gramicidin and tyrocidin.

IV. Detecting Antimicrobial Peptides

DCD has been found to be highly expressed in human skin, melanocytic nevus tissue, and cutaneous melanoma tissue; however, expression was not detected in spleen, thymus, prostate, testis, ovary, small intestine, colon, peripheral blood lymphocytes, heart, brain, placenta, lung, liver, skeletal muscle, kidney, and pancreas (Schittek B. et al, Nat. Immunol, 2001, 2:1133-1137, which is incorporated herein by reference in its entirety). Deficiency of DCD-derived antimicrobial peptides in sweat of patients with atopic dermatitis correlates with an impaired innate defense of human skin in vivo (Rieg S. et al, J. Immunol., 2005, 174:8003-8010, which is incorporated herein by reference in its entirety). The present inventors are the first to identify DCD expression in human reproductive tract tissues. The expression of DCD was identified through proteomic screening and subsequently verified in fallopian tubes, endometrium, and endocervix using real time polymerase chain reaction (PCR), Western blotting and immunohistochemistry. DCD was specifically localized in association with the surface epithelial cells in these tissues, with skin keratinocytes and sweat gland used as controls. As compared to skin, these tissues express lower levels of DCD. Furthermore, consistent with these findings, DCD has recently been identified in human gestational (placental) tissue (Motoyama J-P. L. et al. , Biochem. Biophys. Res. Commun. , 2007).

Based on the foregoing discoveries, the invention provides a method for detecting antimicrobial peptide impairment (e.g., under-expression) within tissues of a male or female subject's urogenital tract in which the antimicrobial peptide is not normally expressed. Thus, in one embodiment, the urogenital tract tissue tested is not prostate, testis, or ovary. The method is useful for detecting microbial infection or other urogenital disorders, or susceptibility thereto, within in the male or female urogenital tract. The detection method of the includes: (a) providing a biological sample derived from the subject, e.g., endometrial tissue, secretions from the urogenital tract, or peritoneal fluid; (b) analyzing the expression of an antimicrobial peptide, such as a DCD-derived peptide, in the sample; and (c) correlating the expression of the antimicrobial peptide with the presence or absence of the infection or other disorder in the subject. In one embodiment, the antimicrobial peptide expression to be determined is one or more DCD-derived peptides, such as those in Table 1 (SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO-.10, SEQ ID NO:11, SEQ ID NO: 12, SEQ ID NO.13, SEQ ID NO.14, or SEQ ID NO: 15). In another embodiment, the method involves the identification of DCD-derived peptide expression in human reproductive tract tissues.

Suitable subjects for use in the detection method of the invention can be any male or female human or non-human animal having a urogenital tract or peritoneum/peritoneal cavity. For example, the subject can be a female mammal such as a dog, cat, horse, cow, pig, sheep, goat, chicken, primate, rat, or mouse. A preferred subject for the methods of the invention is a human female. Particularly preferred are subjects suspected of having or at risk for developing a urogenital infection or other disorder of the urogenital tract (e.g., infection or other disorder of the upper or lower reproductive tract), based on clinical findings or other diagnostic test results.

The step of providing a biological sample derived from the subject can be performed by conventional medical techniques. For example, an endometrial tissue sample can be taken from the subject by biopsy. Samples of secretions of the urogenital tract can be obtained, for example, by using a swab or wipe. As another example, a sample of peritoneal fluid can be taken from a subject by conventional techniques.

The step of analyzing the expression of a antimicrobial peptide in the sample can be performed in a variety of different ways. Numerous suitable techniques are known for analyzing protein expression. For example, antimicrobial peptide expression can be determined directly by assessing protein expression of cells or fluid of a biological sample (e.g., endometrial tissue, secretion, or peritoneal fluid). Protein expression can be detected using immunological techniques, e.g., using antibodies that specifically bind the protein (e.g., antimicrobial peptide) in assays such as immunofluorescence or immunohistochemical staining and analysis, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), immunoblotting (e.g., Western blotting), and like techniques. For example, a polyclonal antiserum to DCD-I (amino acid residues 63-109 of full-length DCD) has been reported (Rieg S. et al, British J. Dermatology, 2004, 151 :534-539; and Schittek B. et al, Nat. Immunol, 2001, 2:1133-1137, each of which is incorporated herein by reference in its entirety). Expression of an antimicrobial peptide can also be determined by directly or indirectly measuring the amount of mRNA encoding the antimicrobial peptide in a cellular sample using known techniques such as Northern blotting and PCR-based methods such as competitive quantitative reverse transcriptase PCR (Q-RT-PCR). Suitable methods for analyzing expression of antimicrobial peptides are described below; nonetheless, other suitable methods might also be employed.

The step of correlating the expression of the antimicrobial peptide with the presence or absence of the infection or other urogenital disorder in the subject involves comparing the level of antimicrobial peptide expression in the test biological sample with levels of the antimicrobial peptide expressed in control samples, e.g., those derived from subjects known to have or not to have the particular disorder (e.g., a healthy subject). Thus, after quantifying antimicrobial peptide expression in a biological sample from a test subject, the test result is compared to levels of antimicrobial peptide expression determined from (a) a panel of tissues derived from subjects (preferably matched to the test subject by age, species, strain or ethnicity, and/or other medically relevant criteria) known to have a particular disorder and (b) a panel of tissues derived from subjects (preferably also matched as above) known not to have a particular disorder. If the test result is closer to the levels (e.g., mean or arithmetic average) from the panel of tissues derived from subjects known to have a particular disorder, then the test result correlates with the test subject having the particular disorder. On the other hand, if the test result is closer to the levels (e.g., mean or arithmetic average) from the panel of tissues derived from subjects known not to have a particular infection or disorder, then the test result correlates with the test subject not having the particular infection or disorder. By way of example, detection and/or analysis of antimicrobial peptide expression in a sample (e.g., a sample from a subject's urogenital or reproductive tract) can be carried out using surface-enhanced laser desorption/ionization (SELDI) technology or other methods, such as those described in Flad, T. et αl, J. Immunol Methods, 2002, 270:53-62; Rieg, S. et αl, J. Investigative Dermatology, 2006, 126:354-365; Rieg S. et al, J. Immunol, 2005, 174:8003-8010; Murakami, M. et al J Invest. Dermatology, 2002, 119(5): 1090-1095; Rieg, S. et al Brit. J Dermatology, 2004, 151(3):534-539; Kimata, H. J. Psychosom. Res., 2007, 62(l):57-59; Steffen, H. et al. Antimicrob. Agents Chemother., 2006, 50(8):2608-2620; Baechle, D. et al. J. Biol. Chem., 2006, 281(9):5406-5415, Epub date: Dec. 14, 2005; and/or Lee, M.J.P. et al. Biochem. Biophys. Res. Commun., 2007, March 28, Epub ahead of print, each of which are incorporated herein by reference in their entirety. DCD was localized in association with fallopian tubes, endometrial and endocervial epithelial cells as well with skin keratinocytes and sweat gland used as control. As compared to skin, these tissues express lower levels of DCD mRNA. Local production of DCD can be used as described herein to provide immediate protection against a wide range of microorganisms in urogenital and reproductive tract tissues. Advantageously, the materials and methods of the subject invention can characterize the expression and regulation of DCD in these tissues and through local delivery provide long-term protection against microorganisms.

V. Definitions As used herein, the term "viral vector" and equivalent terms refer to viruses that are utilized for transferring selected DNA or RNA sequences into a host cell. The vectors may be utilized for the purpose of transferring nucleic acids encodin g antimicrobial peptides into cells either in vitro or in vivo. Viruses that have been commonly used for the transfer of genetic material in vivo include the retroviruses, adenoviruses, parvoviruses and herpes viruses.

As used herein, the term "expression vector" and comparable terms refer to a vector that is capable of inducing the expression of nucleic acids that have been cloned into it after transformation into a host cell. The cloned genetic material is usually placed under the control of (i.e., operably linked to) certain regulatory sequences such a promoters or enhancers. Promoter sequences may be constitutive, inducible or repressible. Any prokaryotic or eukaryotic cell that is the recipient of a vector is the host for that vector. The term encompasses prokaryotic or eukaryotic cells that have been engineered to incorporate a gene in their genome. Cells that can serve as hosts are well known in the art as are techniques for cellular transformation (see e.g., Sambrook, et al., Molecular Cloning: A Laboratory Manual, 2nd ed.₃ Cold Spring Harbor (1989)).

As used herein, the terms "substantially pure" or "purified" mean that the desired product (e.g., antimicrobial peptide) is essentially free from contaminating cellular components. Containments may include, but are not limited to, proteins, carbohydrates and lipids. One method for determining the purity of a protein or nucleic acid is by electrophoresis in a matrix such as polyacrylamide or agarose. Purity is evidence by the appearance of a single band after staining. As indicated above, a nucleic acid sequence that initiates the transcription of a gene is a promoter. Promoters are typically found 5' to the gene and located proximal to the start codon. If a promotor is of the inducible type, then the rate of transcription increases in response to an inducing agent. Expression is the process by which a polypeptide is produced from a nucleic acid sequence. The process involves the transcription of the gene or coding sequence into mRNA and the translation of this mRNA into a polypeptide. Depending on the context in which it is used, the term "expression" may refer to the production of RNA, protein, or both.

As used herein, the term "recombinant" refers to nucleic acid that is formed by combining nucleic acid sequences and sequence elements. A recombinant host is any host receiving a recombinant nucleic acid and the term "recombinant protein" refers to protein produced by such a host.

As used herein, the terms "gene" and "coding sequence" refer to the nucleic acid sequence that undergoes transcription as the result of promoter activity. A gene may code for a particular polypeptide or for an RNA sequence that is of interest in itself, e.g. because it acts as an antisense oligonucleotide or interfering RNA.

The term "antimicrobial" is used herein to refer to the ability of a compound, i.e. an indolicidin, to decrease the population of microscopic flora and/or fauna on a surface, e.g., on a contact lens surface. Antimicrobial activity includes bacteriostatic or antibacterial activity, antifungal activity, anti-algal activity, and the like. An antimicrobial need not eliminate all microbes, but simply decreases the viable population on the treated surface. Similarly, the term "antimicrobial activity" refers to the ability of a compound to inhibit or irreversibly prevent the growth of a microorganism. Such inhibition or prevention can be through a microbicidal action or microbistatic inhibition. The term "antimicrobial selectivity" can also refer to the relative amount of antimicrobial activity of an analog as compared to its cytolytic activity against normal cells in a subject.

The term "microbistatic inhibition", as used herein, refers to the ability of the microbistatic or antimicrobial compound to inhibit or to retard the growth of the target organism without causing death. Microbicidal or microbistatic inhibition can be applied to either an environment either presently exhibiting microbial growth (i.e., therapeutic treatment) or an environment at risk of supporting such growth (i.e., prevention, reducing onset, or prophylaxis). "Broad spectrum antimicrobial activity" refers to the ability of a compound (e.g., a peptide) to inhibit or prevent the survival or growth of various prokaryotic and eukaryotic microorganisms including, for example, protozoans such as Giardia lamblia, fungi such as Cryptococcus, various genera of bacteria such as Escherichia, Salmonella and Staphylococcus, and enveloped viruses. Antimicrobial activity can occur through a microbicidal or a microbistatic inhibition.

The term, "microbicidal inhibition", as used herein, refers to the ability of a compound (e.g., a peptide) to reduce or inhibit the survival of a microorganism by killing or irreversibly damaging it, whereas the term "microbistatic inhibition" refers to the ability of a compound to inhibit the growth of a target microorganism without killing it. A compound having microbicidal or microbistatic inhibition can be applied to an environment that presently allows for the survival or growth of a microorganism (i.e., therapeutic treatment) or to an environment at risk of supporting such survival or growth (/. e. , prevention, delay of onset, or prophylaxis) .

As used herein, the term "urogenital tract" includes organs and tissues associated with reproduction and in the formation and voidance of urine in a male or female subject. These organs and tissues include but are not limited to urinary sphincter, vaginal tissues, fallopian tube, endometrial tissue, cervical tissue, external genitalia, uterus, urinary bladder, urethra, kidney, ureter, and prostate.

The term "urogenital disorder", as used herein, encompasses any infection of the urinary and/or reproductive systems, caused or mediated by one or more microbes. Such disorders can include one or more of the following conditions: vaginitis; vaginal burning, itching, discharge; ulcerative lesions; dysuria; painful urination; prostatitis; urethritis; epidiymitis; urethral stricture; and any other urogenital condition commonly associated with microbial infection. In immunocompromised subjects, urogenital disorders caused or mediated by microbial infection may be more severe and life threatening than the common disorders listed above. Therefore, the term "urogenital disorder" also includes any condition commonly associated with microbial infection in immunocompromised subjects including, but not limited to, foul smelling discharge, bleeding or purulent urogenital lesions, severe pruritus, painful dysuria, and microhematuria.

The term "treating" or "treatment" as used herein encompasses both prophylactic and therapeutic treatment. Unless expressly indicated otherwise, reference herein to delivery or administration of "the antimicrobial peptide" includes delivery or administration of a polynucleotide encoding the antimicrobial peptide (e.g., in a viral or non- viral vector or a host cell carrying the antimicrobial peptide-encoding polynucleotide).

As used herein, the phrase "polynucleotide encoding the polypeptide" (e.g., polynucleotide encoding an antimicrobial peptide) includes the nucleic acid sequence encoding the polypeptide and additional nucleic acid sequences that are coding or non- coding sequences.

The term "protein", "polypeptide", and "peptide" are used herein interchangeably to refer to an amino acid sequence of any length. In one embodiment, the amino acid sequence is between two and thirty amino acids in length. In another embodiment, the amino acid sequence is thirty-one to fifty amino acids in length.

Methods involving conventional molecular biology techniques are described herein. Such techniques are generally known in the art and are described in detail in methodology treatises such as Molecular Cloning: A Laboratory Manual, 2nd ed., vol. 1- 3, ed. Sambrook et ah, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N. Y., 1989; and Current Protocols in Molecular Biology, ed. Ausubel et ah, Greene Publishing and Wiley-Interscience, New York, 1992 (with periodic updates). Various techniques using polymerase chain reaction (PCR) are described, e.g., in Innis et ah, PCR Protocols: A Guide to Methods and Applications, Academic Press: San Diego, 1990. Methods for chemical synthesis of nucleic acids are discussed, for example, in Beaucage and Caπuthers, Tetra. Letts., 1981, 22:1859-1862, and Matteucci et ah, J. Am. Chem. Soc, 1981, 103:3185. Chemical synthesis of nucleic acids can be performed, for example, on commercial automated oligonucleotide synthesizers. Immunological methods (e.g., preparation of antigen-specific antibodies, immunoprecipitation, and immunoblotting) are described, e.g., in Current Protocols in Immunology, ed. Coligan et ah, John Wiley & Sons, New York, 1991; and Methods of Immunological Analysis, ed. Masseyeff et ah, John Wiley & Sons, New York, 1992. Conventional methods of gene transfer and gene therapy can also be adapted for use in the present invention. See, e.g., Gene Therapy: Principles and Applications, ed. T. Blackenstein, Springer Verlag, 1999; Gene Therapy Protocols (Methods in Molecular Medicine), ed. P. D. Robbins, Humana Press, 1997; and Retro-vectors for Human Gene Therapy, ed. C. P. Hodgson, Springer Verlag, 1996. Following are examples that illustrate materials, methods, and procedures for practicing the invention. The examples are illustrative and should not be construed as limiting.

Example 1 — Relative Expression Levels of Dermcidin (PCD^") in Various Tissues Total RNA was isolated from skin, endometrium, fallopian tubes, subcutaneous scar formed at the site of previous surgical incision, and endocervix using Trizol Reagent (invitrogen, Carlsbad, CA). The levels of dermcidin mRNA was determined using Realtime PCR performed on ABI-Prism 7700 Sequence Detection System (Applied Biosystems). Briefly, complimentary DNA was generated from 2 μg of total RNA using Taqman reverse transcription reagent high capacity cDNA Archive kit. Newly synthesized cDNA was used for PCR reaction. PCR was performed in 96-well optical reaction plates with cDNA equivalent to lOOng RNA in a volume of 25 μl system, containing Ix Taqman Universal Master Mix, optimized concentrations of FAM-labeled probe and dermcidin specific forward and reverse primers selected from Assay on Demand (Applied Biosystems). Controls included RNA subjected to RT-PCR without reverse transcriptase and PCR with water replacing cDNA. All the controls gave a Ct value of 40, indicating no detectable PCR product under these cycle conditions. The cycle number at which fluorescence emission crossed the automatically determined threshold level (Ct) was determined using Applied Biosystems software. The results were analyzed using comparative method and the values were normalized to the 18S rRNA expression by subtracting mean Ct of 18S rRNA from mean target Ct for each sample, to obtain the mean ΔCt. The mean ΔCt values were then converted into fold change based on a doubling of PCR product in each PCR cycle, according to the manufacturer's guidelines. As shown in Figure IA, the lowest relative level of dermcidin mRNA expression was detected in the endometrium followed by endocervix and Scar tissues although variable levels were detected in each tissue categories. The relative level of dermcidin mRNA expressed was the highest in skin followed by the fallopian tubes, as shown in Figure IB. For comparative analysis, note the level of expression of dermcidin in the same endometrial tissues shown in Figure IA with skin and fallopian tubes.

Figure 2 shows Western blot analysis of dermcidin protein in skin (SK), fallopian tubes (FT), endometrium (EM), subcutaneous incisional scars (SR) and endocervix (CX). Small portion of the tissues were homogenized in buffer containing 20 mM Tris (pH 8.0), 137 mM NaCl, 10% glycerol, 1% Nonidet P-40, 0.1% SDS, 0.5% deoxycholate, 2 mM EDTA, 5 mM sodium vanadate, 5 mM benzamidine, and 1 mM phenylmethylsulfonyl fluoride, 5mM NaF and protease inhibitor cocktail. The homogenates were centrifuged at 10,000 xg for 15 min at 4°C. The supernatants were collected, their total protein content was determined using a conventional method (Pierce, Rockford, IL) and aliquots were stored at -80⁰C until assayed. For Western blot analysis, an equal amount of protein from each specimen was resolved using 10% SDS-polyacrylamide gel electrophoresis and transferred into polyvinyldiene difluoride (PVDF) membrane by electroblotting in a buffer containing Tris-HCl (25 mM), glycine (192 mM), SDS (0.1%, w/v) and methanol (15%, v/v). After transfer the blots were incubated in 5% powdered milk in 10 mM Tris- HCl, pH 7.5, 0.15M NaCl, 0.1% Tween 20 overnight at 4°C and then incubated an affinity purified goat polyclonal antibody raised against a peptide mapping at the N- terminus of dermcidin of human origin (Santa Cruz Biotechnology, Santa Cruz, CA) at 1:500 dilution for 2 hours. The membranes were washed with 0.01 M Tris-HCl, 0.15 M NaCl pH 7.5 and twice in the same buffer containing 0.05% Triton X-100, and three times in the same buffer Triton-free and exposed to donkey anti-goat IgG-HRP at 1 :5000 dilution (Santa Cruz) for 60 minutes. Immunostained proteins were visualized using enhanced chemiluminescence reagents (Amersham-Pharmacia Biotech, Piscataway, NJ). Rainbow molecular weight Standard (BioRad, Hercules, CA) was used as marker. To normalize for protein, loading an additional blot was prepared and incubated with monoclonal antibody to β actin (Sigma Chemical, St. Louis, MO).

Figures 3 A-3R show immunohistochemical analysis of dermcidin in skin (Figures 3A-3D), peritoneal wall (Figure 3E) fetal membranes (Figures 3F and 3G), placenta (Figures 3H and 31), fallopian tube (Figures 3J and 3K), endometrium (Figures 3L and 3M), and endocervix (Figures 3N and 3Q). Small portion of the tissues were fixed in Bouins solution, paraffin embedded and tissue sections 3 to 5 μm thick were prepared. Following standard procedures, the sections were pretreated with Triton X-100 (0.001%) and Hyaluronidase (0.03 μg/ml, Sigma Chemical, St. Louis MO) for 15 minutes each. The section were washed with PBS and following inhibition of endogenous proxidae activity with H2O2 and endogenous IgG with normal serum each for 20 to 30 minutes, the sections were incubated with affinity purified goat polyclonal antibody raised against a peptide mapping at the N-terminus of dermcidin of human origin (Santa Cruz Biotechnology, Santa Cruz, CA) at 1 :50 dilution prepared in phosphate buffered saline, pH 7.4, containing 0.1% bovine serum albumin for 2 hours. The sections were than exposed to biotinylated second antibodies and avidin horseradish peroxidase (Vector Laboratories, Burlingame, CA) and chromogenic reaction was developed using 3, 3'diaminobenzidine and counter sections were counter staining with hemotoxalin as indicated. Tissue sections incubated with normal IgG instead of the primary antibodies, or deletion of the primary antibody during immunostaining served as controls (Figure 3D as well as Figures 3P, 3Q, and 3R showing fallopian tube, endometrium and cervix). Magnifications. Dermcidin immunoreactive protein is present in the following areas. Arrows in Figures 3A-3D point to keratinocytes at the epidermis. Arrowheads in Figure 3 C point out dermcidin in sweat glands (SG), and in sebaceous gland (SbG) and hair follicle (HF). Arrows in Figure 3E point out the peritoneal mesothelial cells. In Figures 3E and 3F₅ arrows point out the amniotic epithelial cells in the amnion (AM), as well as dermcidin staining seen in chorionic (CH) villa and decidua (DC) and placental syncitio- trophoblast (arrows in Figures 3H and 31). Dermcidin staining is also present in association with fallopian tubes epithelial cells (arrows in Figures 3J and 3K) both in secretory cells (SEC) and ciliated cells (CEC). In the endometrium, dermcidin is localized with surface epithelial cells (arrow in Figure 3L), glandular epithelial cells (EGEC) and lower intensity in endometrial stromal cells (ESC). In endocervix, immunoreactive dermcidin is present with the epithelial cells (arrows in Figures 3N and 3O), although some regions did not become immunostained.

Example 2 — Lentiviral Vectors with Doxycycline-responsive Transcriptional Regulation of Dermcidin (PCD^') Many vectors for gene therapy use a constitutive promoter to drive the expression of the transgene. However, it can be advantageous for the gene therapy vector to include a regulatory system that is effectively "shut off' in the resting state, exhibiting tight regulation of gene expression, but allowing for rapid and repeatable induction in response to a clinically approved inducer molecule, such as doxycycline, a tetracycline analog (Clackson, T. Gene Ther., 2000, 7:120-125; Toniatti, C. et al Gene Ther., 2004, 11:649- 657; Gossen, M. and Bujard, H. Proc. Natl Acad. Sci. USA, 1992, 89:5547-5551; Agha- Mohammadi, S. and Lotze, M.T. J. Clin. Invest., 2000, 105:1177-1183, each of which is incorporated herein by reference in its entirety).

The tetracycline-dependent transcriptional regulatory system is one of the best- studied systems with proven efficacy in vitro and in vivo. This system is based on the E.coli TnIO tetracycline resistance operator, consisting of the tetracycline repressor protein (TetR) and a specific DNA-binding site, the tetracycline operator sequence (TetO). In the absence of tetracycline, TetR dimerizes and binds to the TetO. Tetracycline or doxycycline can bind and induce a conformational change in the TetR leading to its disassociation from the TetO. A TetR mutant has been identified with a reverse phenotype where binding to the TetO is triggered by doxycycline (Agha- Mohammadi, S. and Lotze, M.T. J. Clin. Invest., 2000, 105:1177-1183). A tetracycline responsive promoter (TRE) for mammalian expression has been constructed by fusing a minimal cytomegalovirus (CMV) promoter to seven TetO repeats, which was combined with either tTA to make the Tet-Off or rtTA to make the Tet- On transcriptional regulatory system (Agha-Mohammadi, S. and Lotze, M.T. J. Clin. Invest., 2000, 105:1177-1183). The Tet-On system depends on the pharmacological administration of doxycycline to induce gene expression such as DCD, which is ideal when used with a lentiviral vector that provides long-term gene expression. These properties make the Tet- On system a better choice for transcriptional regulation in most gene therapy applications. Recent improvements in the rtTA protein including reduced background activity and increased doxycycline sensitivity (Corbel, S. Y. and Rossi, F.M. Curr Opin Biotechnol, 2002, 13:448-452), make this approach ideal for induction of antimicrobial peptides such as DCD in the mucosa, such as urogenital tract tissues, and in the wound environment to provide a treatment and defense against microorganisms.

The 47-amino acid sequence of DCD-I is SSLLEKGLDGAKKAVGGLGKLGKDAVEDLESVGKGAVHDVKDVLDSV (SEQ ID NO:2; Schittek B. el al., Nat. Immunol, 2001, 2(12):1133-1137; Mukami M. et al., J. Immunol, 2004, 172(5):3070-3077. which are each incorporated herein by reference in their entirety). In one embodiment;, the polynucleotide encodes an antimicrobial peptide comprising DCD-I (SEQ ID NO:2).

It has been found that doxycycline, in addition to its well established antimicrobial property, acts through an anti-inflammatory mechanism to inhibit the production of proinflammatory mediators and proteases by the uterine cells. Using lentiviral vectors with doxycycline-responsive transcriptional regulation of DCD is a powerful defense system against microorganisms. Since DCD gene therapy is under doxycycline regulation, DCD is produced whenever the subject is treated with doxycycline and is off following doxycycline withdrawal. Many Tet-On regulated transgene expression systems use two separate constructs, one containing the TRE-regulated transgene and the second containing rtTA expressed by a constitutive promoter. This binary system relies on the co-transduction of cells and requires selection and screening to obtain a homogenously transduced population, which is not possible in vivo. Therefore, in a preferred embodiment the elements required for Tet-On regulation are combined into a single cassette with either a constitutive promoter or an autoregulatory loop for the expression of rtTA. These cassettes can be cloned into a third generation lentiviral vector system for the production of self-inactivating vectors (Das, A.T. et al J. Biol. Chem., 2004, 279:18776-18782; Unsinger, J. Biochem. Biophys. Res. Commun, 2004, 319:879-887). A limited number of studies have evaluated the autoregulatory expression of rtTA in a plasmid and AAV vector (Gould, D.J. et al Gene Ther._y 2000, 7:2061-2070; Chtarto, A. et al. Gene Ther., 2003, 10:84-94) as compared with tTA. Autoregulatory expression of rtTA allows for extremely low levels of both the transactivator and the transgene product in the absence of doxycycline, with sufficient rtTA molecules for initiating the expression upon addition of doxycycline. Restricted expression of rtTA with an autoregulatory system can expand applicability in vitro and in vivo.

Accordingly, in one embodiment, the subject invention provides methods for achieving antimicrobial peptide expression in various tissues of the body, and a means of providing protection against microorganisms in these tissues. Figures 4A and 4B show expression of the green fluorescent protein (GFP) gene in TE671 cells, following delivery of the gene by lentivirus carrying Tet-On tetracycline inducible constructs. An inducible lentiviral expression system is demonstrated with three different Tet-On tetracycline inducible constructs: rtTA-TREdsGFP, rtTATS-TREdsGFP and rtTA-cHSTREdsGFP. These Tet-On constructs contain the fusion protein of TA as activator and TS as suppressor. TE671 cells were infected with the three different lenti viral constructs and the dsGFP expression was induced with doxycycline (Doxy). Within 48 hours after the addition of Doxy, the expression of dsGFP was up-regulated in cells infected with rtTA- TREdsGFP or rtTA-cHSTREdsGFP (Figure 4A), but not rtTATS-TREdsGFP. The insulator-containing lentiviral vector (rtTA-cHSTREdsGFP) expressed the highest level of dsGFP. Kinetics of dsGFP induction is demonstrated in Figure 4B with additions and withdraws of Doxy repetitively at different time points. The graph in Figure 4B shows that up- and down-regulations of the dsGFP are tightly controlled by the lentiviral constructs, and again the insulator-containing construct rtTA-cHSTREdsGFP demonstrated the highest level of dsGFP expression.

All patents, patent applications, provisional applications, and publications referred to or cited herein are incorporated by reference in their entirety, including all figures and tables, to the extent they are not inconsistent with the explicit teachings of this specification.

It should be understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application.

Claims

CLAIMSWe claim:

1. A vector comprising a polynucleotide encoding an antimicrobial peptide operably linked to an inducible promoter.

2. The vector of claim 2, wherein the inducible promoter is induced by an inducer molecule.

3. The vector of claim 2, wherein the inducer molecule comprises tetracycline or a tetracycline analog.

4. The vector of claim 2, wherein the inducer molecule comprises doxycycline.

5. The vector of any of claims 1-4, wherein the inducible promoter comprises the tetracycline-controlled transactivator (tTA)-responsive promoter.

6. The vector of claim 1, wherein the inducible promoter is induced by a change in one or more physiological conditions.

7. The vector of any of claims 1-6, wherein the vector is a viral vector.

8. The vector of any of claims 1-7, wherein the vector is a lentiviral vector.

9. The vector of any of claims 1-8, wherein the antimicrobial peptide is dermcidin.

10. The vector of any of claims 1-9, wherein the antimicrobial peptide is at least one dermcidin-derived peptide selected from the group consisting of DCD-I L_> DCD-I₅ SSL-46, SSL-45, SSL-29, SSL-25, LEK-45, LEK-44, LEK-43, LEK-42, LEK-41, LEK-26, LEK-24, and YDP-42.

11. The vector of any of claims 1-10, wherein the polynucleotide encodes a fusion polypeptide comprising the antimicrobial peptide and a heterologous peptide.

12. The vector of any of claims 1-10, wherein the polynucleotide encodes a multimer comprising a plurality of antimicrobial peptides, wherein the antimicrobial peptides are the same or different.

13. An isolated host cell comprising the vector of any of claims 1-12.

14. A composition comprising the vector of any of claims 1-12 or the isolated host cell of claim 13.

15. A method for treating or delaying the onset of a microbial infection, comprising administering a vector of any of claims 1-12 or host cells of claim 13 to a subject in need thereof.

16. The method of claim 15, wherein the subject is suffering from the infection.

17. The method of claim 15, wherein the subject is at risk of developing the infection.

18. The method of any of claims 15-17, wherein the subject is human.

19. The method of claim 15 or 18, wherein the subject is suffering from the infection, and the vector or host cell is administered locally at the site of infection or a site of inflammation caused by the infection.

20. The method of any of claims 15-19, wherein the vector or host cell is administered to the subject's mucosa.

21. The method of any of claims 15-19, wherein the vector or host cell is administered to the subject's urogenital tract.

22. The method of any of claims 15-19, wherein the subject is suffering from an acute or chronic wound, and the vector or host cell is administered to the wound.

23. The method of any of claims 15-19, wherein the vector or host cell is administered to the respiratory tract.

24. The method of any of claims 15-19, wherein the vector or host cell is administered to the subject's skin.

25. The method of any of claims 15 to 19, wherein the subject is a female subject suffering from pelvic inflammatory disease caused by the microbial infection, and wherein the vector or host cell is administered at the site of infection.

26. The method of any of claims 15-25, wherein the inducible promoter is induced by an inducer molecule, and said method further comprises administering the inducer molecule to the subject before, during, or after administration of the vector or host cell.

27. The method of any of claims 15-26, wherein the vector is administered to the subject, wherein the vector is a lentiviral vector, and wherein the inducible promoter comprises the tetracycline-controlled transactivator (tTA)-responsive promoter.

28. The method of claim 27, wherein the antimicrobial peptide is dermcidin.

29. A method for detecting the presence of one or more antimicrobial peptides within a subject's urogenital or reproductive tract, comprising obtaining a biological sample from the subject's urogenital or reproductive tract; and analyzing the expression of the one or more antimicrobial peptides within the sample.

30. The method of claim 29, wherein the sample comprises endometrial tissue, secretions from the urogenital tract, or peritoneal fluid.

31. The method of claim 29 or 30, wherein said analyzing comprises determining the amount of the one or more antimicrobial peptides in the sample or the amount of mRNA encoding the one or more antimicrobial peptide in the sample.

32. The method of any of claims 29 to 31, further comprising comparing the antimicrobial peptide expression in the sample with antimicrobial peptide expression within a reference sample.

33. The method of any of claims 29 to 32, further comprising correlating the expression of one or more antimicrobial peptides with the presence or absence of a microbial infection within the subject.

34. The method of any of claims 29 to 32, wherein the one or more antimicrobial peptides comprise dermcidin.

35. An article for delivery of one or more antimicrobial peptides, comprising an antimicrobial peptide, a vector of any of claims 1-12 or host cells of claim 13.

36. The article of claim 35, wherein the device is adapted for delivery of the one or more antimicrobial peptides to the reproductive or urogenital tract.

37. The article of claim 35 or 36, wherein the article is an implant.

38. The article of claim 34, comprising a wound dressing.