MXPA99001707A - Compositions and methods for use of defensin - Google Patents

Abstract

The invention relates to mammalian beta defensin and methods of use thereof for treatment of microbial infection.

Description

COMPOSITIONS AND METHODS FOR THE USE OF DEFENSINE FIELD OF THE INVENTION The field of the invention is antimicrobial therapy for bacterial infection.

BACKGROUND OF THE INVENTION The respiratory tract of mammals constitutes a barrier between the internal and external environment. This barrier serves as a first line of defense against the invasion of microbial agents. In humans, this defense comprises physical barriers such as mucociliary evacuation, biochemical defenses including complement, lysozyme and antibodies, and cells that are capable of generating an inflammatory immune response, such as alveolar macrophages (Canto et al .: In: Pulmonary Infections and Immunity, H. Chmel et al., Eds., Plenum, New York, 1994, pp. 1-27). A component of this defense line is a family of peptides known as defensins. Mammalian defensins are small antimicrobial peptides (3.5-4.5 kDa) that are characterized by the presence of six cysteines, which form three disulfide bonds, whose ordered arrangement determines whether these proteins are classified as a or ß defensins. A defensins are found in granuiocytes (Ganz et al., 1995, Pharm Therap 66: 191-205) and Paneth cells of the small intestine (Jones et al., 1992, J. Biol. Chem.267: 23216- 23225; Mallow et al., 1996, J. Biol. Chem. 271: 4038-4045) in several species, while ß defensins occur in leukocytes of cattle (Selsted et al., 1993, J. Biol.

Chem. 268: 6641-6648; Tang et al., 1993, J. Biol. Chem. 268: 6649-6653) and domestic poultry (Harwig et al., 1994, FEBS Letters 342: 281-285, Evans et al., 1994. J. Leuc. Biol. 56: 661-665), where they contribute to the host defense system of mucosal surfaces. Lingual and tracheal antimicrobial peptides are ß defensins expressed in bovine epithelial cells of the tongue and trachea (Diamond et al., 1991, Proc. Nati, Acad. Sci. USA 88: 3952-3956; Diamond et al., 1993, Proc. Nati, Acad. Sci. USA 90: 4596-4600, Schonwetter et al., 1995, Science 267: 1645-1648). The first ß defensin isolated from humans, the human ß-defensin 1 (hBD-1), is found in the salivary glands, respiratory tract, prostate and placenta, among other tissues (Bensch et al., 1995, FEBS Letters 368: 331-335, Goldman et al., 1994, Cell 88: 553-560; McCray et al., 1997, Am. J. Respir Cell, Mol. Biol. 16: 343-349; Zhao et al., 1996, FEBS Letters 396: 319-322). The expression of mRNA specifying hBD-1 is detected in the colon, small intestine, kidney, prostate, liver and pancreas. Originally, hBD-1 was isolated from the dialysis hemofiltrate of patients (Bensch et al., Supra). A cDNA sequence specifying only 38 amino acids of mature hBD-1 was identified by Bensch et al. (supra). Figure 1 illustrates this partial cDNA sequence. The upper line represents the sequence of amino acids in simple letter code. The BDNT and UNIP-2 primers used for cloning are represented as arrows. The first seventeen nucleotides originate from the degenerate primer BDNT, so that all possible nucleotides for the third codon position are listed in the sequence (Y = C or T). Cystic fibrosus (CF) is an obstructive disease of multiple systems characterized by chronic infection of the respiratory tract (Decker ef al., 1993, En: Cystic fibrosis, pp. 193-218, PB Davis, Ed., Dekker, New York ). The disease is caused by a defect in the cystic fibrosis transmembrane conductance regulator (CFTR), leading to impaired transport of Na * and CI "and abnormal composition of the airway surface fluid (Collins et al., 1992, Science 256 : 774-779; Joris et al., 1993, Am. Rev. Resplr. Dis. 148: 1633-1637; Quinton, 1990, FASEB J.4: 2709-2717) Many hypotheses have been exposed to explain the propensity of the respiratory tract of CF to be infected (Lehrer et al., 1983, Infect. Immun.42: 10-14; Zasloff, 1987, Proc. Nati. Acad. Sci. USA 84: 5449-5453; Konstan et al., 1993, In: Cystic Fibrosis, pp. 219-276; P.B. DAVIS, Ed. Dekker, New York; Imundo ef al., 1995, Proc. Nati Acad. Sci. USA 92: 3019-3023; Pier et al., 1996, Science 271: 64-67). Despite this, the causal chain of events leading from genetic lesions in CFTR to devastating lung infections suffered by CF patients remains unclear. It has been proposed that dysfunction of antimicrobial peptides of the respiratory tract may be a critical contributor to lung infection (Lehrer et al., 1983, supra; Zasloff et al., 1987, supra). Very recently, antimicrobial activity has been detected in the healthy cultured human respiratory airway epithelial surface fluid (Smith et al., 1996, Cell 85: 229-236). Although hBD-1 is widely expressed, antimicrobial activity was absent in the high salt environment of the cystic fibrosis epithelium. The activity of a molecule similar to the defensin returned when the salt concentration was decreased. The infection associated with cystic fibrosis can be limited to the respiratory system due to its direct association with the external environment. In addition to high salt, which is the result of defective chloride transport, the evaporation of fluid from the airway surface can also concentrate the luminal airway fluid. Both the increase in salt and evaporation of fluid can decrease the effects of hBD-1 in CF patients. There is still a need for antimicrobial therapy directed against bacterial infections in patients who have cystic fibrosis and other infectious diseases. There remains also a need for a full-length cDNA precursor sequence isolated for hBD-1, ie, a defensin molecule which can be used for the prevention and treatment of infection and for other therapeutic purposes. In addition, there remains the need for the development of animal models suitable for the study of the role of defensins in the protection against microbial disease and for the discovery of therapeutic agents to combat said disease.

BRIEF DESCRIPTION OF THE INVENTION The invention relates to a cell comprising an isolated nucleic acid encoding human beta defensin-1.

The invention also relates to a cell comprising an isolated nucleic acid encoding rodent beta defensin-1. In one aspect, the rodent defensin is mouse beta defensin-1. In addition, the nucleic acid may further comprise a promoter / regulatory sequence placed at the 5 'end of human beta defensin-1 or beta defensin-1 of rodent. The invention also includes a vector comprising an isolated nucleic acid encoding human beta defensin-1. In one aspect, the vector can be selected from the group consisting of a plasmid, a virus and a non-viral vector and in another aspect, the vector can be suspended in a pharmaceutical composition. In yet another aspect, the nucleic acid isolated in the vector further comprises a promoter / regulatory sequence placed at the 5 'end of human beta defensin. Also included in the invention is a vector comprising an isolated nucleic acid encoding rodent beta defensin-1. In one aspect of this aspect of the invention, the defensin is mouse beta defensin-1. In another aspect, the vector is selected from the group consisting of a plasmid, a virus and a non-viral vector, and still in another aspect, the isolated nucleic acid further comprises a promoter / regulatory sequence placed at the 5 'end of the beta Rodent defensin-1. Also included in the invention is a nucleic acid encoding human beta defensin-1. Preferably, the isolated nucleic acid is cDNA and more preferably, the isolated nucleic acid is the sequence shown in Figure 2 [SEQ ID NO: 3]. In one aspect, the isolated nucleic acid comprises a promoter / regulatory sequence positioned at the 5 'end of the human beta-defensin-1 coding region. The invention further includes an isolated nucleic acid encoding mouse beta defensin-1. Preferably, the isolated nucleic acid is cDNA. More preferably, the isolated nucleic acid is the sequence shown in Figure 1 2 [SEQ ID NO: 6]. In one aspect, the isolated nucleic acid may further comprise a promoter / regulatory sequence positioned at the 5 'end of mouse beta defensin-1. The invention also includes an isolated nucleic acid encoding a salt insensitive mammalian beta defensin-1. Mammalian beta defensin-1 can be selected from the group consisting of human beta defensin-1 and mouse beta defensin-1, and the isolated nucleic acid may further comprise a promoter / regulatory sequence placed at the 5 'end of the beta mammalian defensin-1. Also included in the invention is an isolated nucleic acid encoding a mammalian beta defensin-1, having enhanced antimicrobial activity when compared to a counterpart of wild-type mammalian defensin-1. In one aspect, mammalian beta defensin-1 is selected from the group consisting of human beta defensin-1 and mouse beta defensin-1, and in another aspect, the isolated nucleic acid further comprises a promoter / regulatory sequence placed on the 5 'end of mammalian beta defensin-1. Also included in the invention is a salt insensitive mammalian beta-defensin-1, which can be selected from the group consisting of human beta defensin-1 and mouse beta defensin-1. In addition, the invention includes mammalian beta defensin-1 with mutation having enhanced antimicrobial activity, when compared to a counterpart of wild-type mammalian defensin-1. In one aspect of this aspect of the invention, mutant mammalian beta defensin-1 is selected from the group consisting of human beta defensin-1 and mouse beta defensin-1. Also provided in the invention is a method for enhancing antimicrobial activity in a tissue sample comprising adding a mammalian beta defensin-1 to the sample. In a modality, the tissue sample is selected from the group consisting of a sample of mammalian lung tissue, a sample of mammalian skin tissue and a sample of mammalian blood tissue. In another embodiment, mammalian beta defensin-1 is selected from the group consisting of human beta defensin-1 and mouse beta defensin-1. In one aspect, mammalian beta defensin-1 is added to the tissue sample in vivo in a mammal. In another aspect, the tissue sample is a sample of lung tissue and the mammalian beta defensin is human beta defensin-1, which is added to the lung tissue sample by means of a nebulizer or bronchoscope.

In yet another aspect, the tissue sample is a sample of lung tissue and the mammalian beta defensin is a human beta defensin-1, which is added to the lung tissue sample in the form of a vector comprising a isolated nucleic acid encoding human beta defensin-1. When the vector is administered to the human, human beta defensin-1 is expressed from there to effect the addition of human beta defensin-1 to the lung tissue sample. In one aspect, the human has a respiratory disease, which is preferably emphysema or cystic fibrosis. However, when the respiratory disease is cystic fibrosis, the vector encoding human beta defensin may be insensitive to the salt or may have added thereto a compound capable of absorbing salt. Still in another aspect, the human has a respiratory disease, which predisposes the human to a pulmonary microbial infection. Preferably, the respiratory disease is emphysema or cystic fibrosis. However, when the respiratory disease is cystic fibrosis, the human beta defensin may be insensitive to salt or may have added to it a compound capable of absorbing salt. Also included in the invention is a method for treating a human having a respiratory disease, which predisposes the human to pulmonary microbial infection, the method comprising administering to the human lungs a pharmaceutical composition comprising human beta defensin-1. Furthermore, the invention relates to a method for treating a human having a respiratory disease, which predisposes the human to pulmonary microbial infection, the method comprising administering to the human lungs a pharmaceutical composition comprising an isolated nucleic acid encoding beta defensin. 1, wherein human beta defensin-1 is expressed from the isolated nucleic acid in cells of the lungs, thereby treating the human. The invention also includes a method for treating a human having a lung infection, the method comprising administering to human lungs, a pharmaceutical composition comprising human beta defensin-1. A method for treating a human having a pulmonary microbial infection is also provided, the method comprising administering to the human lungs a pharmaceutical composition comprising an isolated nucleic acid encoding human beta defensin-1, wherein beta defensin-1 Human is expressed from the isolated nucleic acid in cells of the lungs, thereby treating the human. In addition, a method for treating a human having a microbial infection of the skin is provided, comprising administering to the human skin a composition comprising human beta defensin-1. Additionally, a topical composition for administration to the skin of a mammal is included, comprising mammalian beta defensin-1 suspended in a pharmaceutically acceptable carrier. In one aspect, mammalian beta defensin is selected from the group consisting of human beta defensin-1 and mouse beta defensin-1. A pharmaceutical composition comprising human beta defensin-1 is also provided.

In addition, the invention includes a transgenic mammal comprising an isolated nucleic acid encoding human beta defensin-1. Also included is a method for synthesizing human beta defensin-1 using solid phase 9-fluorenyimethyloxycarbonyl synthesis. The method comprises the regioselective formation of Cys5-Cys34, Cys12-Cys27 and Cys17-Cys35 by protecting Cys5-Cys34 with triphenyl (triphenylmethyl), protecting Cys12-Cys27 with Acm (acetamidomethyl), and protecting Cys 7 and Cys35 with MOL (p- methoxy benzyl).

BRIEF DESCRIPTION OF THE PRIOR ART PCI Figure 1 is the amino acid sequence of the prior art and partial cDNA of hBD-1 [SEQ ID NO: 2 and 1] as described in Bensch et al. (supra) Figure 2 shows the nucleic acid (cDNA) and amino acid sequence of h BD-1. Figure 2A illustrates the cDNA and deduced amino acid sequence of hBD-1 [SEQ ID NO: 3 and 4, respectively]. Double underlined, putative signal sequence; its solid brayado, mature peptide; hyphen, stop codon; and, underlined in bold, polyadenylation signal. Figure 2B illustrates the putative prepro-peptide sequences of hB D-1 and tracheal antimicrobial peptide (TAP) [SEQ ID NO: 5].

Figure 3 is a series of graphs showing the antibiotic activity of extracts derived from transformed human embryonic kidney cells transfected with hBD-1 cDNA. The filled circles represent the results of duplicate antibiotic assays performed on five independent transfected cell extracts; the unfilled circles indicate the values obtained in extracts of simulated transfected cells. Figure 4 is a photograph of a gel showing the mRNA distribution of hBD-1 in epithelial cells (three traces on the left) and non-epithelial cells (three traces on the right). Figure 5 is a photograph of a gel showing the distribution of h-BD-1 mRNA in human tissue. Figure 6 is a series of images showing in situ hybridization of a hBD-1 probe for normal lung tissue and CF. The antisense oligonucleotides (central panels) and sense oligonucleotides (right panels) for the gene encoding hBD-1 were hybridized to sections of human lung tissues and exposed to photoemulsion for six days.

Proximal non-cartilage airways (AC and Gl) and distal airways (DF and JL) obtained from normal lungs (upper rows [AF] and CF (central rows [GL]) are shown.) Representative cross-sections obtained from xenografts are also shown. CF (MO) and CF (PR) (lower rows) Bright-field photomicrographs (left panels) and dark field (central panels) of the same region are presented No mRNA was seen encoding hBD-1 in serial sections hybridized to The antisense probe after treatment of the section with RNase, confirming the specificity of the test Arrowheads indicate epithelium, stars denote submucosal glands Increase: proximal airways, 8x, distal airways and xenografts, Figure 7 is a graph showing salt sensitivity of antimicrobial activity of h BD-1 against Pseudomonas aeruginosa Synthetic hBD-1 was incubated with 5 x 1 04 colonic forming units P. aeruginosa in 1 00 μl of 1 mM phosphate buffer (pH 7.4) and the indicated concentrations of NaCl. The reactions were then incubated at 37 ° C for 20 minutes. Serial dilutions were platinized and colony counts were performed the next day. Serious bacterial death was observed in low NaCl solution with concentrations of hBD-1 varying from 60 to 500 μg / ml. Fig. 8 is a panel of graphs showing the fact that the correction of the CF defect in xenografts normalizes the salt concentration of the airway surface fluid (ASF) and restores its bactericidal activity. ASF was collected from non-CF (left panel), CD (central panel) and bronchial CF xenografts treated with a recombinant adenovirus expressing CFTR (right panel) In this fluid, sodium concentrations were measured (inverted triangle without rel lenar) and chloride (filled inverted triangle) and antibacterial liquid broth tests were carried out against 1 03 cfu of P. aeruginosa (central and lower rows, respectively) Each point shown represents the results obtained from an individual graft (n = 1 2) Measurements of transepithelial potential difference were obtained from xenografts both before and after gene transfer The presented data (upper row) illustrate the change in potential transepithelial difference in epithelium treated with amiloride in response to both a substitution of chloride and cAM P. agonist treatment. As controls, non-CF (n = 6) and CF (n = 6) xenografts were treated with an ad recombinant enovirus expressing β-galactosidase. The ion measurements and antibacterial properties of ASF from the grafts treated with the β-galactosidase vector were similar to untreated controls. Figure 9 is a series of images showing a xenograft analysis of CF for the expression of recombinant mRNA encoding CFTR. The transfer of genes for CF bronchial xenografts treated with a recombinant adenovirus expressing CFTR was analyzed by in situ hybridization. Screened antisense sections (panels B and E) and sense (panels C and F) of tissue from xenograft to photoemulsion were exposed for one week. Bright field images (panels A and D) and dark field (panels B and E) of the same section are shown. The efficiency of gene transfer was determined by dividing the number of positive signals by the total number of cells present in multiple sections of an individual graft. Increase: (panels A-C) 4x; (panels D-F) 1 5x. Fig. 10 is a series of graphs showing the under-regulation of hBD-1 RNA by antisense oligonucleotide. Airway surface fluid was collected from non-CF xenografts before and after the instillation of phosphorothioate oligonucleotides. Antibacterial activity was uniformly presented in the ASF of all grafts before the administration of any oligonucleotide. Antibacterial broth assays were performed against P aeruginosa (1 03 cfu) by incubating the organisms with 30 μl of ASF for 2 h at 37 ° C, plating serial dilutions of the mixture, and completing colonies the following day. obtained during the examination of an individual graft Figure 11 is a series of images showing the RT-PCR analysis of RNA encoding subunits of hBD-1 and β1-integrin of xenografts treated with oligonucleotides with phosphorothioate. local administration of the oligonucleotide, explanted and RNA was isolated, RT-PCR was performed using specific primers of hBD-1 or β-integrin subunits, and an aliquot of the reaction mixture was stained on a nitrocellulose membrane and hybridized with Specific probes Figure 12 is the cDNA and corresponding amino acid sequence of murine ß defensin (mBD-1) Figure 12A is the sequence of cDNA and deduced amino acids of mBD-1 [SEQ ID NOS: 6 and 7, respectively]. The first underline indicates the putative mature peptide; the hyphen represents the termination codon; the second underlining indicates the polyadenylation signal. Figure 12B is a comparison of the putative prepropeptide sequences of mBD-1 [SEQ ID NO: 7], hBD-1 [SEQ ID NO: 4], tracheal antimicrobial peptide (TAP) [SEQ ID NO: 5] and peptide lingual antimicrobial (LAP) [SEQ ID NO: 8], all derived from cDNA sequences as well as the peptides sequences of bovine neutrophil β-defensin 1 and 11 (BNBD-1, BNBD-11) [SEQ ID NOS: 9 and 10, respectively] and Gallinacina 1 (Gal-1). { SEQ ID NO: 11]. The lower line presents the consensus sequence of β-defensins.

Figure 13 is a diagram illustrating the structure of the mouse ß-defensin 1 gene. A restriction map of the m-BD-1 gene is shown together with a schematic drawing of the gene, the cDNA sequence and the predicted structure of the prepropeptide. The gene is represented schematically by having the following individual components: the untranslated region 5 '(5'UTR), open box; signal sequence, shaded box; interrupted prosecution, black box; mature peptide, shaded box; and 3 'UTR, open box. Figure 14 is a series of graphs showing the results of antibacterial assays in mBD-1. In Figures 14A, 14B and 14C, the results of coincubation assays of antibacterial fluid liquid are shown. E. coli (Figure 14A), S. aureus (Figure 14B) and P. aeruginosa (Figure 14C) were added at a concentration of 1 03 to 1 04 cfu to 50 μl of lysates obtained from cells transfected with vector containing mBD cDNA -1 full length (empty circles) and cells transfected with the vector alone (filled circles). Each circle shown represents the results of an individual transfection experiment. The sensitivity to salt of the antimicrobial activity of m BD-1 is shown in Figure 14D. Extracts from transfected cells of m BD-1 were incubated with 5 x 1 04 colony forming units of E. coli D31 in 1 00 mM phosphate buffer (pH 7.4), and the indicated concentrations of NaCl. After incubation at 37 ° C for 1 hour, serial dilutions of bacteria were platinized and the colonies counted the next day.

Fig. 15 is a series of gel images showing an expression analysis of m BD-1 in RNase protection assays and RT-PCR. Figure 1A shows the measurement of mBD-1 expression in an RNase protection assay. The total RNA obtained from kidney (k), skeletal muscle (m), and lung / trachea (I) was examined. Hybridization to a labeled riboprobe specific for β-actin (a) or mBD-1 (d) transcripts was performed in separate tubes. Figure 1 5B shows the detection of mBD-1 expression in various mouse tissues using nested RT-PCR. PolyA + RNA was isolated from mouse tissues, reverse transcribed and the cDNAs were amplified using two pairs of mBD-1 specific primers. A single 250 bp band was used as a positive control and was generated during the amplification. The mRNA encoding β-actin was amplified using gene-specific primers. 1 = trachea, 2 = lung, 3 = tongue, 4 = esophagus, 5 = small intestine, 6 = large intestine, 7 = gallbladder, 8 = pancreas, 9 = skeletal muscle, 1 0 = heart, 1 1 = horn Fallopian, 1 2 = ovary, 1 3 = vagina, and 14 = brain. Figure 1 6 is a series of images comprising the A-P panels, which illustrate the detection of transcripts encoding mBD-1 in the respiratory tract of mice. Antisense probes (two columns on the left) and sense (two columns on the right) were used to examine the tissue distribution of the mBD-1 transcripts. Representative sections are shown in a dark and bright field obtained from the nose (panels A-D), trachea (E-H panels), large bronchioles (panels L-L), terminal bronchioles, and lung parenchyma (M-P panels). The bar in panels A-D represents 0.7 mm, and the bar in panels E-P represents 270 μm. Figure 17 is a series of images comprising panels A-P, showing the detection of transcripts encoding mBD-1 in extrapulmonary organs. Antisense probes (two columns on the left) and sense (two columns on the right) were used to examine the tissue distribution of mBD-1 transcripts. Representative sections are shown in dark and bright field obtained from kidney (panels A-D), tongue (panels E-H), liver (panels L-L), heart muscle (panels M-P). The bar in panels A-H represents 0.7 mm; the panel bar l-L represents 270 μm; and the bar in the panels M-P represents 1 30 μm.

DETAILED DESCRIPTION OF THE INVENTION In accordance with the present invention, a full-length cDNA has been isolated and characterized by encoding a human defensin protein. The data provided herein establishes that the gene of the present invention, encoding said human defensin gene, is expressed in the human respiratory tract. The results of the experiments presented herein further establish that the human defensin is an antimicrobial peptide, which is highly sensitive to salt. These data strongly support a CF lung pathology model, in which elevated NaCl in the airway surface fluid compromises the activity of the defensin, leading to infection.

It is believed that the defensin molecule expressed in the human airways is inactive in cystic fibrosis, leading to predisposition to infections with pseudomonas and staphylococci. It is well known that the main problem in cystic fibrosis lung disease is the chronic colonization of the respiratory tract with staphylococci and pseudomonas, organisms that are not normally associated with respiratory infections otherwise in healthy individuals. This suggests that the major defect in cystic fibrosis, which is thought to be an abnormal chloride channel, leads to a problem in the defense of the host. Thus, the invention includes methods and compositions, based on the discovery of the gene that encodes human defensin, which provide effective therapies for the treatment of CF. The human defensin gene (h BD-1) can be obtained by identifying the h-BD-1 nucleic acid in a cDNA library using poly A + RNA obtained from cells suspected of expressing defensin. The hBD-1 gene is amplified by 5 'and 3' PCR or RACE using nucleic acid probes comprising a signal sequence and a portion of the predicted hBD-1 gene of a partial amino acid sequence of the protein. Details of the isolation of the hBD-1 and m BD-1 genes are provided herein in the experimental examples section. However, other equivalent isolation methods known to those skilled in the art can also be used. Any other human defensin gene can also be cloned using molecular techniques to isolate the defensin DNA from a genomic or poly A + RNA library obtained from defensin-expressing cells. In addition, probes derived from the human defensin of the present invention can be generated, which comprise conserved nucleotide sequences of the defensin genes. These probes can be used to identify additional defensin genes in genomic DNA libraries obtained from other human cells and tissues or from cells and tissues of other mammals, using PCR or other recombinant DNA methodology. Such techniques are well known in the art and are described, for example, in Sambrook et al. , (1989, Molecular Cloning, A Laboratory Manual, Cold Spring Harbor, New York). To determine whether the DNA thus selected actually encodes a defensin, the nucleotide sequence of the DNA is obtained. The putative amino acid sequence encoded by the DNA is deduced and this sequence is then compared to the amino acid sequence of a known defensin. Additionally, the putative defensin gene is cloned into an expression vector, the protein is expressed and examined for activated salt-sensitive antimicrobial, as described herein. Thus, although the gene encoding human defensin has been initially discovered according to the present invention in human bronchial epithelium, a gene encoding a defensin obtained from another human tissue is also included in the invention. Additionally, in light of the discovery of a novel mouse ß defensin gene as described herein in the present invention, the invention should be construed to include defensin genes from mammals other than humans, said defensin functions in a manner substantially similar to the human defensin described herein. Preferably, the nucleotide sequence comprising the gene encoding defensin is approximately 50% homologous, more preferably about 70% homologous, even more preferably about 80% homologous and most preferably about 90% homologous to the gene encoding defensin obtained from epithelium human bronchial, that is, hBD-1. "Homolog" as used herein, refers to the similarity of subunit sequence between two polymer molecules, for example, between two nucleic acid molecules, eg, two DNA molecules or two RNA molecules, or between two polypeptide molecules. When a subunit position in both molecules is occupied by the same monomeric subunit, for example, if a position in each of the two DNA molecules is occupied by adenine, then they are homologous in that position. The homology between two sequences is a direct function of the number of equal or homologated positions, for example, if half (for example, five positions in a polymer of ten subunits in length) of the positions in two sequences of compounds are homologous, then the two sequences are 50% homologous, if 90% of the positions, for example 9 out of 10, are equal or homologous, the two sequences share 90% homology. By way of example, the DNA sequences 3 'ATTGCC 5' and 3 'TATGCG 5' share 50% homology. The beta defensin molecules and DNAs encoding them, which are homologous to the beta defensins presented herein, should be interpreted as including a mutant, derivative and variant of beta defensins. An "isolated nucleic acid", as used herein, refers to a fragment, segment or nucleic acid sequence, which has been separated from the sequences that flank it in a state that occurs naturally, for example, a fragment of DNA which has been removed from the sequences that are normally adjacent to the fragment, for example, the sequences adjacent to the fragment in a genome which occurs naturally. The term also applies to nucleic acids which have been substantially purified from other components, which naturally accompany the nucleic acid, for example, RNA or DNA or proteins, which naturally accompany it in the cell. Therefore, the term includes, for example, a recombinant DNA which is incorporated into a vector; in a virus or plasmid replicating autonomously; or in the genomic DNA of a prokaryote or eukaryote; or which exists as a separate molecule (e.g., as a cDNA or a cDNA or genomic fragment produced by PCR or restriction enzyme digestion) independent of other sequences. It also includes a recombinant DNA, which is part of an hybrid gene encoding an additional polypeptide sequence. In other related aspects, the invention includes vectors which contain such isolated nucleic acid, and which are, preferably, capable of directing the expression of the encoded protein by the nucleic acid in a vector-containing cell; and cells containing such vectors, either eukaryotic cells or prokaryotic cells, preferably eukaryotic cells. By the term "vector" as used herein, is meant an autonomously replicating plasmid or a virus. The term should also be interpreted to include non-plasmid and non-viral compounds which facilitate the transfer of nucleic acid into cells, such as, for example, polylysine compounds and the like. Examples of viral vectors include, but are not limited to, adenoviral vectors, adeno-associated virus vectors, retroviral vectors, and the like. The invention also includes an isolated nucleic acid having a sequence which is in the antisense orientation (i.e., is complementary) to a portion or all of the nucleic acid encoding defensin. By "complementary to a portion or the entire defensin gene" is meant a nucleic acid sequence which does not encode defensin protein. Instead, the sequence that is being expressed in the cells is identical to the non-coding strand of the defensin gene and thus does not encode defensin. The terms "complementary" and "antisense" as used herein are not completely synonymous. "Antisense" refers particularly to the nucleic acid sequence of the non-coding strand of a double-stranded DNA molecule encoding a protein, or to a sequence which is substantially homologous to the non-coding strand. "Complementary" as used herein refers to the broad concept of complementarity between two nucleic acids, for example DNA molecules. When a nucleotide position is occupied in both molecules by nucleotides normally capable of making base pairs with one another, then the nucleic acids are considered to be complementary to one another in this position. Thus, two nucleic acids are complementary to each other when a substantial number (at least 50%) of corresponding positions in each of the molecules are occupied by nucleotides, which normally make a pair of bases with one another (for example, pairs of nucleic acids). nucleotides A: T and G: C). As defined herein, an antisense sequence is complementary to the sequence of a double-stranded DNA molecule encoding a protein. It is not necessary that the antisense sequence be solely complementary to the coding portion of the coding strand of the DNA molecule. The antisense sequence may be complementary to regulatory sequences specified in the coding strand of a DNA molecule encoding a protein, said regulatory sequences controlling the expression of the coding sequences. The invention should be construed in this manner to include: nucleic acid encoding defensin and fragments of nucleic acid encoding defensin; and, nucleic acid and nucleic acid fragments that are in antisense orientation to the nucleic acid encoding defensin. The fragments of nucleic acid encoding defensin encode portions of defensin, which have the biological activity, ie, which have the antimicrobial activity of defensin as defined herein., or which encode a polypeptide comprising a portion of defensin, said polypeptide is useful, as discussed in detail herein, for the treatment of microbial infections in mammals in need of such treatment, such as, but not limited to, humans who have CF. The invention also includes an isolated protein encoded by the human defensin gene, which can be isolated by the skilled artisan once armed with the present invention. Preferably, the amino acid sequence of a defensin protein thus discovered is about 70% homologous, more preferably about 80% homologous, even more preferably about 90% homologous, most preferably about 95% homologous and most preferably at least about 995 homologous to the amino acid sequence of hBD-1. The substantially pure defensin obtained as described herein can be purified by the following known methods for protein purification, wherein an immunological, enzymatic or other assay is used to monitor the purification at each step in the process. As used herein, the term "substantially pure" describes a compound, for example, a protein or polypeptide, which has been separated from components from components, which naturally accompany it. Typically, a compound is substantially pure when at least 10%, more preferably at least 20%, more preferably at least 50%, more preferably at least 60%, more preferably at least 75%, more preferably at least 90%, and most preferably at least 99% of the total material (by volume, by weight or dry weight, or by percent mole or molar fraction) in a sample is the compound of interest. The purity can be measured by any suitable method, for example, in the case of polypeptides, by column chromatography, gel electrophoresis or HPLC analysis. A compound, for example, a protein, is also substantially purified when it is essentially free of naturally associated components or when it is separated from the natural contaminants which accompany it in its natural state. The present invention also provides analogs of proteins or peptides encoded by a defensin gene. Analogs may differ from proteins or peptides that occur naturally by conservative amino acid sequence differences or by modifications which do not affect the sequence, or by both. For example, conservative amino acid changes can be made, which although they alter the primary sequence of the protein or peptide, normally do not alter its function. Conservative amino acid substitutions usually include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid; asparagine, glutamine; serine, threonine; lysine, arginine; phenylalanine, tyrosine. Modifications (which normally do not alter the primary sequence) include chemical derivatization in vivo or in vitro of polypeptides, for example, acetylation or carboxylation. Also included are glycosylation modifications, for example, those made by modifying the glycosylation patterns of a polypeptide during its synthesis and processing or in additional processing steps; for example, by exposing the polypeptide to enzymes which affect glycosylation, for example, glycosylating or deglycosylating enzymes of mammals. Also encompassed are sequences which have phosphorylated amino acid residues, for example, phosphotyrosine, phosphoserine, or phosphothreonine. Also included are polypeptides which have been modified using ordinary techniques of molecular biology, in order to improve their resistance to proteolytic degradation or to optimize the properties of sol ubility. Analogs of such polypeptides include those that contain residues other than naturally occurring L-amino acids, for example, D-amino acid or synthetic amino acids that do not occur naturally. The peptides of the invention are not limited to products of any of the specific exemplary processes listed herein. In addition to the substantially full-length polypeptides, the present invention provides biologically active fragments of the polypeptides. A defensin polypeptide is "biologically active" if it possesses antimicrobial activity as defined herein. As noted above, the antimicrobial activity of wild-type BD-1 is sensitive to high salt concentrations. However, the invention also contemplates defensin polypeptides that are either full length or less than full length, which comprise one or more mutations, which make the antimicrobial activity of the salt insensitive defensin. Thus, defensin polypeptides with mutation are biologically active if they possess antimicrobial activity. As used herein, the term "fragment," as applied to a polypeptide, will ordinarily be at least about fifteen contiguous amino acids, typically at least about twenty-five contiguous amino acids, more usually at least about forty contiguous amino acids, usually at least about forty-five contiguous amino acids and preferably at least about fifty contiguous amino acids in length. To identify the defensin region, which confers sensitivity to the salt in the polypeptide, the systematic mutation of the cloned gene can be conducted using ordinary molecular biology techniques. For example, mutant defensin molecules can be generated by linker screening mutation or other deletion and / or insertion mutation, or a series of stop codons can be introduced in a systematic fashion along the length of the gene. Such technology is well known to the skilled artisan and the procedures for practicing it are described in any molecular biology manual, such as, for example, Sambrook et al. (supra).

To assess the function of a defensin gene mutated in that way, the gene can be cloned into an expression vector, and the protein is then expressed in appropriate cells. The expression of the gene can be confirmed by carrying out the hybridization designed to detect RNA, such as "Northern" hybridization or even in situ hybridization. The expression of a defensin polypeptide can be assessed by gel electrophoresis, or by "Western blot" or any other well-known protein or immunological assay. The antimicrobial activity of the defensin thus expressed is assessed by obtaining fluid from the cells and determining their effect on the growth of the microorganisms, such as staphylococci or pseudomonas, as described herein. The salt sensitivity of the defensin is assessed by including salt in selected samples in the microbial growth assay. Defensive polypeptides having antimicrobial activity that is insensitive to salt are considered to be mutation in a defensin region, which is involved in salt sensitivity. Such mutant DNAs are sequenced and the salt sensitive region of defensin DNA can be defined accordingly.

To generate a stable, salt insensitive defensin polypeptide, one or more mutations can be introduced into the defensin DNA region by governing the salt sensitivity. Such mutant defensin genes are tested for their ability to motivate the defensin polypeptide having antimicrobial activity insensitive to salt. By the term "salt-sensitive defensin" as used herein, it is meant a defensin molecule which has a lower antimicrobial activity in the presence of a salt concentration, which is greater than the physiological concentration salt, when compared to the antimicrobial activity of the same defensin in the presence of a physiological salt concentration. By the term "salt insensitive defensin" as used herein, is meant a defensin molecule which possesses antimicrobial activity in the presence of a salt concentration at which the antimicrobial activity of the corresponding natural type defensin is either diminished or extirpated. To determine if a defensin molecule with mutation is insensitive to salt, samples comprising natural type defensin or mutation by antimicrobial activity are evaluated in the presence of increasing salt concentrations. A defensin molecule which possesses antimicrobial activity in the presence of a salt concentration that decreases or removes the antimicrobial activity of the wild type defensin is considered to be insensitive to salt. A defensin gene can also be generated, which causes high levels of defensin in a tissue, thereby intensifying the antimicrobial activity in the tissue. To express the defensin, defensin coding sequences are cloned under the control of a promoter capable of handling high levels of defensin expression in cells. Many such promoter / regulatory sequences are available in the art including, but not limited to, for example, the immediate early promoter / enhancer sequence of human cytomegalovirus, the SV40 early promoter, the Rous sarcoma virus promoter sequence and other sequences retroviral promoters / intensifiers. By the term "promoter / regulatory sequence" is meant a DNA sequence which is required for the expression of a gene fused to the promoter / regulatory sequence. In some cases, the promoter / regulatory sequence can function in a specific tissue manner, since, the promoter / regulatory sequence alone is capable of driving the expression of genes in a cell of a particular tissue type. In some cases, this sequence may be the core promoter sequence and in other cases, this sequence may also include an enhancer sequence and other regulatory elements, which are required for the expression of the gene in a specific tissue manner. Preferably, when the defensin gene also comprises a promoter / regulatory sequence, the promoter / regulatory sequence is placed at the 5 'end of the defensin gene, so as to drive expression of the defensin gene in a cell. By the term "placed at the 5 'end" as used herein, it is meant that the promoter / regulatory sequence is covalently linked to the 5' end of the gene, whose expression regulates, in a position sufficiently close to the starting site 5 'transcription of the gene, in order to drive the expression of the gene. In another aspect, the defensin coding region undergoes a mutation, so that the polypeptide encoded thereby has enhanced antimicrobial activity, when compared to the corresponding wild-type protein expressed by the same animal. Such a wild-type protein is referred to herein as a wild-type counterpart protein. A defensin molecule having enhanced antimicrobial activity may also be insensitive to salt simply by incorporating a mutation in the defensin DNA, which makes the protein product insensitive to salt along with a mutation, which intensifies the antimicrobial activity of the molecule. To generate a defensin polypeptide having enhanced antimicrobial activity, the procedures described above are followed for the introduction of defensin DNA mounts, except that the gene with the resulting mutation is tested for antimicrobial activity in any quantitative antimicrobial assay. Quantitative microbial assays are well known in the art. In this form, the region of defensin DNA that governs the antimicrobial activity can be located and the mutations can be introduced therein, which provides a defensin molecule having enhanced antimicrobial activity. As is evident from the experimental data provided herein, the mature human defensin polypeptide exemplified herein is not very large, having thirty-six amino acids, the precursor polypeptide having sixty-eight am inoctates. Thus, it is not necessary that the defensin polypeptide be produced solely by cloning and expressing ion. Given their size and the state of the art of synthesis of polypeptides, natural or mutation-type defensin molecules can be generated by direct synthesis using a peptide synthesizer. Similarly, the DNA ding the defensin, which is either wild-type or comprises one or more mutations, but which also has antimicrobial activity, can be synthesized in a nucleic acid synthesizer. Thus, the invention should be interpreted as including synthetic forms of defensin and nucleic acid ding same. The invention should also be construed as including defensin analogs as described above, said analogs may be more soluble or more stable than natural-type defensin, and which also contain the above-described mutations, rendering the defensin insensitive to salt and / or more antimicrobially active. By the term "antimicrobial activity" as used herein, it is meant the ability to inhibit the growth of or actually kill a population of bacteria. Thus, "antimicrobial activity" should be interpreted to mean both bacteriostatic as well as bactericidal activity. The antimicrobial activity should also be interpreted as including a compound, which is capable of inhibiting the pathogenesis, that is, the ability to cause disease, of a bacterium. Additionally, an antimicrobiological assay for defensin is not necessarily limited by the type of bacteria used in the assay. Normally, the type of bacteria to be used includes those bacteria which colonize the respiratory tract in CF patients. For example, the most common infections in CF patient patients are those caused by Staphylococcus and Pseudomonas species. However, since the infections in CF patients are not limited to these bacteria, and even more, since the defensin molecules of the present invention should not be interpreted as being limited solely to the treatment of infections in CF patients, The antimicrobial assay can include any species of bacteria important in the infection of mammals. Although many antimicrobial assays are known in the art, one example of such assays is as follows. Serial dilutions of fluid obtained from cells transfected with an expression vector comprising a defensin with mutation are evaluated by antimicrobial activity against a constant number of microbial cells. Controls may include cells transfected with wild-type defensin and cells which are transfected with vector sequs in the abs of a functional defensin gene. The level of antimicrobial activity in cellular fluid obtained from each set of cells is measured. A gene of defensin with mutation having enhanced antimicrobial activity is identified by its ability to inhibit the growth of test organisms at a concentration which is lower than that of the corresponding wild type defensin. Thus, the invention should be construed as including defensin genes and polypeptides which comprise mutations that affect the salt sensitivity and antimicrobial activity of the defensin. Depending on the location of the salt sensitive region compared to the defensin D NA region that governs the antimicrobial activity, the salt insensitive defensin polypeptides, which also have enhanced antimicrobial activity, can be generated using the procedures described in FIG. present in combination with well-known molecular biology procedures. Such defensin polypeptides and their corresponding DNAs are also considered to be included in the invention. The defensin genes and their corresponding peptides are useful for enhancing antimicrobial activity in a tissue sample. Essentially, a defensin protein or a gene encoding defensin is added to the tissue sample, preferably a sample of lung tissue, wherein the defensin protein exerts its antimicrobial activity. The defensin is added to the tissue sample either in vitro or in vivo, where, when the compound is added to the sample in vivo, it is suspended in a suitable pharmaceutical carrier. When added in vivo, the defensin, or DNA encoding it, can be added to the tissue sample in the form of a nebulizer or bronchoscope. When the defensin is added in the form of DNA encoding defensin, the DNA can be incorporated into any suitable vector, as described herein, for delivery of the DNA to the tissue sample. By the term "tissue sample" as used herein, it is meant to include both cells which are either in or surrounding a tissue, and any and all extracellular fluid that is produced by or which surrounds the cells. cells in or around the tissue. The tissue sample can include any mammalian tissue, including, but not limited to, tissue from the lung, skin tissue, blood tissue (ie, bloodstream and fluid cells contained therein), and the . . In one aspect, the method of the invention includes the addition of hBD-1, or DNA encoding hBD-1, to a sample of lung tissue in a human in vivo, wherein the human has a respiratory disease, which predisposes to human to pulmonary microbial infection. Such respiratory diseases include, but are not limited to, cystic fibrosis and emphysema, wherein the human lungs are compromised so that they are more susceptible to infection by microorganism than the lungs of individuals who do not have the respiratory disease. The invention should also be construed as including any other respiratory disease, such as bronchitis and pneumonia, and any other microbial infection of the lung, whether the infection is acute, chronic, transient or that is the result or is not the result of a predisposition condition. The defensin genes and their corresponding polypeptides are useful for the treatment of a variety of microbial infections in mammals, preferably humans, and more preferably, these molecules are useful for the treatment of infections in humans having CF and in animals that represent animal models of CF. However, the defensins of the present invention are also useful for the treatment of infections in mammals, preferably humans, who are immunosuppressed and therefore, are susceptible to acute or chronic infection by bacteria, which normally do not infect mammals. healthy For example, patients who are H IV positive or who are immunosuppressed as a result of cancer treatment, transplant procedures, etc. , they can also benefit from the present invention. Similarly, patients who have another underlying CF non-CF disease, such as emphysema, for example, and therefore are susceptible to bacterial infection, may also benefit from the present invention. In one aspect of the present invention, the treatment of the infection may involve the administration of a salt-sensitive or salt-insensitive defensin to the infected mammal. Treatment regimens, which are considered to include a simple dose or dosage which is administered per hour, per day, per week or month, or per year. The dosages may vary from 1 μg to 1000 mg / kg body weight of defensin, and will be in a form suitable for delivery of the compound to the mammal. The route of administration may also vary depending on the disorder to be treated. The defensin is prepared for administration by being suspended or dissolved in a pharmaceutically acceptable carrier, such as isotonic saline solution, isotonic salt solution or other formulations, which will be apparent to those skilled in such administration. The compositions of the invention can be administered to a mammal in one of the traditional modes (eg, orally, parenterally, transdermally or transmucosally), in a sustained release formulation using a biocompatible biodegradable polymer, or by delivery to the patient. site using micelles, gels and liposomes, or rectally (eg, by suppositories or enemas). Preferably, for the treatment of patients who have lung infection, the route of administration is intranasal delivery by aerosol or blood route. The appropriate pharmaceutically acceptable carrier will be apparent to those skilled in the art and will largely depend on the route of administration. Although the wild-type defensin can be administered to non-CF patients in any of the pharmaceutical compositions described above, when the wild-type defensin is administered to a patient having CF, the pharmaceutical composition should comprise a compound capable of absorbing salt in the patient. environment of the lung for a sufficient time for the defensin to act without being deactivated by the high concentration of salt present. Such compounds will be apparent to those in the art who are now aware of the present invention. The problems associated with the defensive salt sensitivity of the defensin can be overcome by using a defensin mutation form having enhanced antimicrobial activity. Such a molecule, being more potent than its natural-type counterpart, can act to inhibit bacterial growth before substantial inactivation by salt. However, preferably, when defensin is used to treat a CF patient, a salt-insensitive form is used, thus overriding the deactivation of the antimicrobial activity of the defensin by the salt. DNA encoding natural or mutated defensin can also be used to treat infections in mammals. Examples of methods for delivering DNA to mammals are known in the art and are described, for example, in the following references (WO 94 28938 and U.S. Patent No. 5,240,846), each of which is incorporated herein by reference. present by reference. DNA can be admired as a naked molecule, encapsulated in a protein or lipid formulation, a synthetic formulation or in a number of viral vectors, such as, for example, adenoviruses, adeno-associated viruses, retroviral vectors and the like. When DNA encoding natural type defensin is administered 0 with mutation to the mammal as described herein, the defensin is expressed therein providing therapeutic benefit to the mammal in a manner similar to the administration of the defensin polypeptide. The defensin can also be administered to the skin of a mammal for treatment of an infection therein. For administration to the skin, the defensin can be formulated in any well-known skin formulation described, for example, in Remington's Pharmaceutical Science, or in U.S. Patent No. 4,954,487. Topical administration is useful for the treatment of skin infection causing common acne, or for treatment of other microbial infections of the skin, such as, for example, Staphylococcus aureus infection. The mammalian homologues of the hBD-1 gene exemplified below can also be identified and isolated once armed with the present invention. In this way, these homologs are included in the present invention. Homologs of the BD-1 gene can be obtained from any suitable mammal including higher mammals, such as primates, cows, pigs and lower mammals including rabbits and rodents, such as rats and mice. Preferably, the invention includes the mouse homolog of hBD-1. A mammalian homolog of hBD-1 is isolated using hBD-1 probes in combination with PCR or other hybridization technology well known in the art. Mutations in the mammalian homolog can be introduced by the following methods described herein, said mutations imparting salt insensitivity or enhanced antimicrobial activity to any defensin molecule isolated in that manner. Transgenic animals can be generated, which encode a defensin gene which is incapable of antimicrobial activity. Such animals would be unable to fight infections with staphylococci and pseudomonas, and will therefore have a CF phenotype. The animal model is also useful for classifying antimicrobials directed against staphylococci and pseudomonas. Preferably, the transgenic animal is a mouse. A transgenic mammal that encodes a defensin gene with mutation, which is incapable of antimicrobial activity, can be generated as follows. First, the desired mutation is introduced e? the defensin gene. Next, the muteinized defensin is cloned under the control of an appropriate promoter / regulatory sequence. The promoter / regulatory sequence used is one that is capable of driving gene expression preferably in airway cells. Examples of such promoter / regulatory sequences include, but are not limited to, those derived from mammalian genes, such as β-actin and viruses, such as the retrovirus LTR and the immediate early human cytomegalovirus gene. The introduction of the chimeric gene into the fertilized egg of the mammal is achieved by any number of standard techniques in transgenic technology (Hogan et al., 1986, Manipulating the Mouse Embryo: A Laboratory Manual, Cold Spring Harbor, NY). Most commonly, the chimeric gene is introduced into the embryo as a microinjection. Once the gene is introduced into the egg, the egg is incubated for a short period of time and then transferred to a pseudopregnant mammal of the same species from which the egg was obtained (Hogan et al., 1986, Manipulating the Mouse Embryo: A Laboratory Manual, Cold Spring Harbor, NY). Normally, approximately 125 eggs are injected per experiment, approximately two thirds of which will survive the procedure. Approximately twenty viable eggs are transferred to pseudopregnant animals, of which four to ten will develop into live offspring. Normally, approximately 10-30% of the offspring carry the transgene. Transgenic mammals, preferably mice, which encode defensin with mutation can then be assessed for a CF phenotype by examining their ability to handle infection by bacteria, particularly, species of Staphylococcus and Pseudomonas. The animals are infected with the target bacteria and the degree of infection in all tissues is assessed by pathological, histochemical, immunological and other well-known techniques in the art. The invention is further described in detail by reference to the following experimental examples. These examples are provided for illustrative purposes only, and are not intended to be limiting unless otherwise specified. Thus, the invention should not be construed in any way as being limited to the following examples, but instead should be construed as encompassing any and all variations that become apparent as a result of the teaching provided herein. . The defensin of the present invention is expressed at high levels in human respiratory epithelial cells in both CF and non-CF patients. Using transfection assays, the data described in the experimental examples herein establish that the expression of human defensin cDNA generates a peptide capable of killing staphylococci and pseudomonas, said peptide being sensitive to salt.

Procedure for cloning cDNA from -hBD-1 cDNA from h BD-1 was amplified from poly i (A) + RNA from primary human bronchial epithelium by 5 'and 3' RACE (Frohman et al., 1988). Proc. Nati, Acad. Sci. USA 85: 8998-9002). The primer used for RACE 5 'was 5'-TTTGGTAAAGATCGGGCA-3' [SEQ ID NO: 1 2], corresponding to CPI FTK [SEQ ID NO: 1 3] of the nucleotide sequence of h BD-1 derived from peptide ( Bensch ef al., 1 995, FEBS Letters 368: 331-335). The primer used for RACE 3 'was 5'-ATGAGAACTTCCTACCTT-3' [SEQ ID NO: 14], corresponding to the residues of signal sequence M RTSYL [SEQ ID NO: 1 5]. A two-phase procedure was used. To use the RACE protocol, a short stretch of an exon sequence is required. In the present invention, the full-length cDNA was cloned from cultured human bronchoepithelial primary cell mRNA. From this region, initiators oriented in the 3 'and 5' directions were chosen, which will produce overlapping cDNA when fully extended. During the first phase of reverse transcription, the 3 'ends were amplified as follows. A mixture of RTC buffer, RNasin, dT17 adapter initiator, and AMV reverse transcriptase was created and stored on ice. RNA was heated in H20 for 3 minutes at 65 ° C and then quenched in ice. RNA was added to the previous mixture. The mixture was then incubated first at 42 ° C for 1 hour and then 52 ° C for 30 minutes, and was expressed and stored at 4 ° C as a "cDNA deposit". During the amplification phase, a PCR cocktail of PCR buffer, DMSO, dNTPs, H20, adapter initiator, gene-specific primer and the cDNA deposit of the first phase was formed. This mixture was denatured at 95 ° C for 5 minutes and cooled to 72 ° C. Taq polymerase was added and the solution was covered with mineral oil that had been heated at 72 ° C for 40 minutes. The PCR conditions were as follows: 95 ° C for 5 minutes, followed by 35 cycles of 95 ° C for 40 seconds, 58 ° C for 2 minutes, and 72 ° C for 3 minutes, followed by 72 ° C for 1 minute. 5 minutes.

In the second phase, using the protocol described in the first phase, a gene-specific primer 1 was replaced by the adapter dT? 7. The excess of initiator was removed by using a Centricon 1 00 rotation filter. The reverse transcription mixture was diluted with OJ x TE and centrifuged at 1000 x g for 20 minutes. This process was repeated and the retained liquid was collected. Speed Vac centrifugation was used to concentrate the solution to 10 μl. 5X Tailing buffer, 1 mM dATP and Terminal Transferase were added. The solution was diluted for 5 minutes at 37 ° C and 5 minutes at 65 ° C. The solution was then diluted to 500 μl with TE. The amplification phase of procedure one was then followed by adding adapter initiator dT17 in addition to the adapter primer and the gene-specific primer 2. The amplification products were subcloned into the Bluescript vector (Marchuk et al., 1 991, Nucí. Res. 1 9: 1154) and sequenced. The multiple clones were analyzed to verify the nucleotide sequence. H-BD-1 cDNA was additionally cloned from colon (Caco-2) and tongue (SCC-25) epithelial cells.

Procedures for conducting assays for antibiotic activity and salt dependence Human embryonic kidney cells were transfected (293) (ATCC CRL 1 573) that grew in the absence of antibiotics, with the mammalian reporter vector pCMV (Clontech) containing the full-length hBD-1 cDNA. Simulated control and transfected transfected cells were harvested at 24 to 48 hours post-transfection, pelleted by centrifugation, resuspended in 10 mM phosphate buffer (pH 7.4) and used by brief sonication. The cell debris was removed by centrifugation and the resulting supernatant was tested for antibiotic activity. Approximately 1 03 or 1 04 bacteria were exposed to cell extracts for 2 hours in 1 mM phosphate buffer (pH 7.4) and cell viability was measured by platinating several dilutions and counting the colonies the next day. Essentially, for assays involving Pseudomonas and Escherichia coli, approximately 5 x 1 04 bacterial cells were exposed to approximately 50 μg to 500 μg of synthetic hBD-1 in 1 mM phosphate buffer (pH 7.4) in a reaction volume of 1 00 μl. The samples were incubated at 37 ° C for 20 minutes and then diluted to 1 ml of total volume with the corresponding test buffer. A volume of 1000 μl of this solution and two serial 10-fold dilutions thereof were platinized and incubated overnight at 37 ° C to facilitate colony formation. A culture of broth used to assess the antimicrobial activity of hBD-1 directed against Escherichia coli was also used. In this case, 1 x 1 04 bacterial cells were incubated in 200 μl of 0.25X LB broth at 37 ° C in the presence or absence of 500 μg of synthetic hBD-1. After 7 hours, the optical density at 630 nm of the cultures was recorded as a measure of bacterial growth. After the support subtraction due to growth medium, the average optical density of the untreated cultures was 0J 27, while those containing hBD-1 was 0.004, indicating the almost complete suppression of bacterial growth. The salt dependence of antibiotic activity was measured essentially as described for a-defensins (Lehrer et al., 1983, supra).; Selsted I went to. , 1884, supra). Briefly, approximately 5 x 1 04 bacterial cells were exposed to 50 μg of synthetic BD-1 in 10 mM phosphate buffer (pH 7.4) containing varying concentrations of NaCl in a reaction volume of 1 00 μl. The reactions were incubated at 37 ° C for 20 minutes and then diluted to 1 ml of total volume with the corresponding test buffer. A volume of 100 μl of this solution and two serial 10-fold dilutions thereof were platinized and incubated overnight at 37 ° C to facilitate colony formation.

Methods for performing Northern blot analysis Cellular mRNA was prepared by extraction of guanidinium isothiocyanate (Chirgwin et al., 1979, Biochemistry 1 8: 5294-5299), followed by oligo (dT) selection. The tissue mRNA was purchased from Clontech (Palo Alto, CA). Cellular mRNA (3 μg), or tissue mRNA (5 μg) was fractionated by formaldehyde gel electrophoresis, transferred to nylon, and hybridized to 32 P-labeled cDNA probe as follows: An agarose gel was prepared and RNA samples were mixed with a buffer that runs on formaldehyde gel, formaldehyde and formam ida. The samples were then incubated at 65 ° C for 15 minutes and cooled on ice and charged with DEPC-treated formaldehyde gel buffer. Samples were loaded on the gel, electrophoresis was performed and spots were prepared using standard procedures (Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, NY). The RNA spots were prehybridized for 8 to 20 hours at 42 ° C.

Hybridization was performed at 42 ° C in 50% formamide, 5X SSC, 5X Denhardt, 50 mM sodium phosphate, and 259 μg / ml salmon sperm DNA. The washing conditions were 50 ° C in 0.1X SSC and 0.1% SDS. The probe corresponded to a fragment extending over nucleotides 80 to 379 of the cDNA. The filters were then exposed to X-ray film to visualize the RNA bands. The filters were extracted and re-probed for β-actin to confirm the presence of intact RNA. Cell lines in which RNA expression was assessed were obtained from the American Type Culture Collection and include Caco-2 (ATTC # HTB 37), SCC-25 (ATCC # CRL 1628), HS27 (ATTC # CRL 1634), A-204 (ATCC # HTB 82), and SK-N-MC (ATCC # HM 10).

In situ hybridization procedures To perform cytochemical analyzes, lung tissue and / or xenografts were explanted, embedded in OCT (Tissue-TCK, miles, Inc.), and cryosected. The tissue sections (6 μm) were mounted on slides and fixed in 4% paraformaldehyde in PBS (pH 7.4) for 4 hours. Following the fixation, the sections were dehydrated through graduated concentrations of ethanol. The sections were then dried overnight under vacuum and stored at -20 ° C. The next day, the sections were treated with 10 μg / ml proteinase K for 30 minutes at 30 ° C, rinsed twice in 0.2X SSC for 30 seconds each time, fixed in 4% formaldehyde in PBS, rinsed twice in 0.1 M triethanolamine (pH 8.0) for 4 minutes each time, incubated in 0.25% acetic anhydride for 10 minutes at room temperature, and then dehydrated through graduated concentrations of ethanol. Sections were again dried under vacuum before prehybridization for 4 hours at 54 ° C in 10 mM Tris (pH 8.9), 50% formamide, 2.5X Denhardt, 0.6 M NaCI, 1 mM EDTA (pH 8.0), 0.1 % SDS, 500 μg / ml tRNA, and 1 μm dithiothreitol (DTT). The RNase control sections were treated with 200 μg / ml of RNase A for 1 h at 37 ° C before the prehybridization step. The sections were then hybridized with 5 x 1 06 cpm / ml of sense or antisense probes of 35S for 16 hours at 54 ° C. The probes were synthesized using the Promega in vitro transcription system and 35S UTP and 35S CTP as markers. The cRNA probes for h BD-1 were generated from the polymerase promoters of full-length cDNA T7 and T3 RNA encoding hBD-1 and subcloned into a Bluescript vector (Stratagene). The probes for CFTR were synthesized from the polymerase promoters of T7 RNA and SP6 of a PCR template derived from the cDNA for CFTR. Following hybridization, the slides were washed in 4X SSC for 20 minutes (four changes) at room temperature, RNase (20 μg / ml) for 30 minutes at 37 ° C, 2X SSC / 1 mM DTT at room temperature for 10 minutes ( two changes), and finally, three washes of 1 5 minutes in 0.5X SSC / 1 mM DTT at 54 ° C. The slides were dehydrated through ethanol and air dried before immersion in photoemulsion (Kodak). The slides were developed and analyzed by bright and dark field microscopy using a Nikon Microphot-FXA microscope. The efficiency of adenovirus-mediated CFTR gene transfer to xenografts was determined by dividing the number of positive signals by the total number of cells present in 20 sections derived from multiple blocks of each treated graft. The expression of hBD-1 was evaluated in xenografts from uninfected samples.

Peptide Synthesis Procedures hBD-1 was synthesized by solid phase methodology. The regioselective formation of the three disulfide bridges, Cys5-Cys34 (I), Cys- | 2-Cys27 (I I) and Cys17-Cys35 (11) was performed following the procedures described in Kellenberger et al. (1 995, Peptide Res. 8: 321 -327) as modified herein. The disulfide bridges were formed by protection of the disulphide bridges as described below and the peptide as generated was characterized by electrophoretic capillary zone analysis, high performance liquid chromatography, and mass spectrum. The details of the procedure for the generation of hBD-1 are as follows. H BD-1 was prepared by the solid phase method of peptide synthesis using the Fmoc (9-fluorenylmethyloxycarbonyl) strategy. The three disulfide bridges, Cys5-Cys34 (I), Cys12-Cys27 (I I) and Cys17-Cys35 (11) are formed regioselectively. During the synthesis, the sulfide side chains of Cys5 and Cys34 were protected with trifly (triphenylmethyl), those of Cys12 and Cys27 were protected with Acm (acetamidomethyl) and those of Cys1 7 and Cys35 were protected with MOL (p-methoxybenzyl). After the synthesis was finished, the peptide was removed from the resin by treatment with trifluoroacetic acid, the partially protected peptide was purified by preparative H PLC and the first disulfide bridge was formed between Cys5 and Cys34 by oxidation of air. The peptide was purified and the second disulfide bridge formed between Cys 2 and Cys27 when treating with iodine. The peptide was purified once more and the third disulfide bridge was formed by treating the peptide with trifluoromethane sulfonic acid and trifluoroacetic acid followed by air oxidation. The hBD-1 thus obtained was further purified by H PLC using standard means and characterized by mass spectrometry, capillary zone electrophoresis and H PLC.

Recombinant adenovirus The structure, production and measurement of virus titers E 1 -suprimides expressing β-galactosidase (CMV promoter in a sub360 skeleton) and CFTR (CMV-enhanced actin promoter in a skeleton dl7001) have been described previously (Engelhardt ef al., 1 993, Human Gene Therapy 4: 759-769). The viruses were titrated as previously described (Engelhardt et al., Id.).

Generation of bronchial xenografts Human bronchial tissues were obtained from explanted lungs or donors at the time of lung transplantation, and bronchial xenografts were prepared as previously described (U.S. Patent No. 5,625, 1 28) Engelhardt et al. , 1992, J. Clin. I nvest. 90: 2598-2607; Goldman et al. , 1995, Nature Genet. 9: 126-131). Surface epithelial cells were removed by incubating the bronchus in protease 14 (Sigma Chemical coJ, platinized in primary culture, and maintained for 5 to 7 days before being released with trypsin. Tracheas from donor rats were collected from rats. (200 g) Fisher 344 asphyxiated with C02, from which the epithelium was stripped by three rounds of freeze-thawing, these tracheas were ligated to tubing at both ends after seeding of 2 x 10 6 bronchial epithelial cells in 30 μl of medium of hormonally defined growth (Clonetics) The grafts were then implanted subcutaneously in the flanks of male BALB / c nu / nu mice and were maintained for three weeks in vivo to allow the maturation of a fully differentiated bronchial epithelium. in the graft lumen (5 x 1 010 pfu / ml, 1 00 μl) and air-cast 6 hours later The samples from three lungs of CF (homozygous? F508) and six non-CF pu se les were used to generate xenografts for these studies.

Measurements of potential transepithelial difference The xenografts were analyzed for changes in the potential difference (PD) in response to perfusion with different solutions before and after the gene transfer as described (Goldman et al., supra). In preparation for these measurements, animals were anesthetized with ketamine / xylazine administered intraperitoneally (1 00 μl, 10% v / v) in phosphate buffered saline (PBS) (pH 7.4). Agar bridges were prepared by filling 21-gauge butterfly needles and intubation with 1 M KCl in 4% agar. The reference bridge was implanted subcutaneously on the mouse flank, while the scanning electrode bridge was in contact with a pool of buffered solution being delivered at a constant rate of 2 ml / minute by a syringe pump. Each bridge was connected by a calomel cell half to a voltmeter. The measurements were recorded every 10 seconds by a computer interfaced with the voltmeter. Before use, pairs of agar bridges were maintained in a common pool of buffered solution and only electrodes that differed by less than 0.2 mV were used for analysis. Each record measured voltage as a function of time in response to the sequential perfusion of the following: (i) solution of call devices buffered with phosphate H EPES (HPBR) containing H EPES 1 0 mM (pH 7.4), NaCl 140 m M, 5 mM KCl, 1.2 mM MgSO4, 1.0 mM Ca gluconate, 2.4 mM K2HP04, and 0.4 mM K2HP04; (ii) HPBR with 100 μM amiloride; (iii) HPB R chloride free (gluconate replaces chloride) with 1 00 μM amiloride; (iv) chloride-free HPBR with 1 00 μM of amiloride, 200 μM of 8-cpt cAMP, and 1 00 μM of phospholine; and (v) return to H PBR.

ASF analysis xenografts and not of CF CF For AST suficinetes volumes for study, allowed airway secretions in the xenografts accumulate for at least a week and then harvested by expelling air. After a brief centrifugation (1 04 x g) to remove the mucus, the supernatant was recovered for antibacterial tests and measurements of ions. Sodium and chloride concentrations in ASF were measured using ion-specific glass microelectrodes (Londonderry, N H) calibrated to the prepared standards. Antibacterial studies were performed as previously described (Harwig et al., 1994, Meth. Enzymol 236: 160-1 72, Smith et al., 1996, Cell 85: 229-236). Simple bacterial colonies, P. aeruginosa (ATCC # 39324) or a clinical isolate, were inoculated in LB broth and cultured overnight at 37 ° C. An aliquot of this culture was transferred to fresh LB and incubated for an additional 2-3 hours at 37 ° C to obtthe semi-logarithmic phase cells. The organisms were washed with 1 mM sodium phosphate buffer (pH 7.4), and the concentration of colony forming units (cfu) per ml was quantified by measuring its absorbance at 620 nm ASF (30 ml) was mixed. with 1 03 cfu bacterial and the mixture was incubated for 2 hours at 37 ° C. Serial dilutions were then platinized and colony counts were completed the next day. Respiratory surface fluid analyzes were conducted from less 1 2 CF and non-CF individual xenografts before and after gene transfer.Also identical studies were performed on six non-CF xenografts treated with a recombinant adenovirus expressing β-galactosidase.

Delivery of oligonucleotides to xenografts and molecular analysis The phosphorothioate oligonucleotides consisted of 21-mer analogs for the 5 'end of the hBD-1 gene. The inequalities are underlined in the sequences given below. ucleótidos oligon the phosphorothioate 5'- CAGAAGGTAGGAAGTTCTCAT-3 'antisense [S EQ ID IMO.1 6], 5'-TACAGAGGTGCTCACTGGGTA-3' sense [SEQ ID NO: 17] were instilled, 5'-CAGAAGGTAGGAAGTTGTCTT-3'desigualado 1 [SEQ ID nO: 18] and 5'-TCTAAGGTAGGGAGTTCTTTG-3 'desigualado 2 [SEQ ID nO: 1 9] xenografts no CF at a concentration of 20 uM of oligonucleotide in 70 mM phosphate buffer (pH 7.4) . Any remng solution was expelled with air on the next day. The ability of ASF to kill bacteria was measured before and three days after the delivery of oligonucleotide. Antibacterial broth assays were performed as previously described. The grafts were harvested four days after the administration of oligonucleotide and the total RNA was extracted using RNAzoi B (Tel-Test, I nc.). RT-PCR was performed on 1 μg of template RNA using the Titan RT-PCR system (Boehringer Mannheim) and specific primer sets for ß l -integrin or hBD-1 subunit. Aliquots of the PCR reactions were resolved on a 1% agarose gel, std with nitrocellulose, and hybridized to probes labeled with a specific 32 P random primer.

Cloning and characterization of BD-1 cDNA hBD-1 is a member of the ß-defensins family. The protein was originally isolated from hemofiltration of dialysis patients (Bensch et al., 1995, supra). In accordance with the present invention, full-length hD-BD-1 cDNA was cloned from mRNA obtd from cultured human bronchoepithelial primary cells. The sequence of the cloned cDNA is shown in Figure 2A [SEQ ID NO: 3]. The cDNA encodes a predicted 68 amino acid precursor that is similar to TAP [SEQ ID NO: 5] (Figure 2B). The antibiotic activity of h BD-1 was demonstrated by expressing the full-length cDNA in transformed human embryonic kidney cells and then testing the antimicrobial activity of cell extracts thus transformed. Extracts of transfected cells exhibited antibiotic activity agt Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus, while those of transfected cells simulated did not (Figure 3).

Expression in tissue and cells of hBD-1 To determine in which cell types and human tissues hBD-1 is expressed, the following experiments were performed. The expression of hBD-1 was evaluated first in epithelial cells, since it was originally discovered in bronchial epithelial cells (Figure 4). As is evident from the figure, in addition to primary airway cells, hBD-1 mRNA is expressed in colon epithelial cells (Caco-2) and tongue (SCC-25). HBD-1 mRNA is absent in several types of non-epithelial cells, including human foreskin fibroblasts (Hs68), rhabdomyosarcoma (A-204), and neuroblastoma (SK-N-MC). Sequence analysis of cDNA derived from epithelial cell lines confirmed the presence of hBD-1. Northern blot analysis (Figure 5) reveals the expression of hBD-1 at sites with exposed epithelial surfaces and ducts, including colon, small intestine, kidney, prostate, liver and pancreas. All of these are sites associated with the expression of CFTR and CF pathology (Strong et al., 1 993, In: Cystic Fibrosis - Current Topics Dodge et al., Eds., Pp. 1-26, Wiley, Chichester). Such an extended epithelial distribution suggests that hBD-1 may play a general role in mucosal immunity. Additionally, hBD-1 is present in the spinal cord (Figure 5) and in the U937 monocytic cell line. Thus hBD-1 is produced by cells of both epithelial and hematopoietic origin. In order to localize the expression of hBD-1 mRNA in the respiratory tract, in situ hybridization was performed in human respiratory tract tissue. Figure 6 represents a series of. Non-CF lung and CF lung tissue images were sounded antisense and sense of hBD-1. High-level BD-1-specific RNA was present along the non-CD lung surface conduction airways from the proximal bronchi (Figure 6, panels A-C) to distal bronchioles (Figure 6, panels D-F). Expression along the epithelium of glands was also detected in their ucosal bm (Figure 6, panels A and B) and alveolar cells (Figure 6, panels D and E). This differs from the expression of TAP in bovine lung, which was detected mainly in proximal airways (Diamond, et al, 1991, Proc Nati, Acad Sci USA 93:51 56-5160). A similar distribution of expression of hBD-1 was demonstrated in the CF lung (proximal airways, Figure 6, panels G-1, distal airways, Figure 6, panels J-L). Hybridization of serial sections with the sense probe failed to demonstrate specific signals, confirming the specificity of the assay (Figure 6, panels to the right). To test the hypothesis that hBD-1 is a salt-sensitive antibiotic, a synthetic form of the mature peptide was made, which contained the three disulfide bridges (ie, Cys5-Cys3, Cys12-Cys27, Cys17-Cys35). In the presence of low NaCl, synthetic hBD-1 exhibited potent bactericidal activity for an extensive array of organisms, including P. aeruginosa (Figure 7) and E. Coli. The impact of NaCl concentration on hBD-1 was evaluated by incubating the synthetic peptide with 5 x 104 cfu of P. aeruginosa in the presence of varying NaCl concentrations (Figure 7). The antimicrobial activity of hB D-1 exhibited a dramatic salt dependence characterized by an acute loss of activity as the salt concentration increased from 50 μM to 125 μM. Bacterial death was observed in low salt with hBD-1 concentrations varying between 60-500 μg / ml. This titration of hBD-1 activity occurred in the range of NaCl that distinguished AS F from CF and not from CF in natural human proximal lung and the bronchial xenograft model. To further assess the molecular basis of the defect in bacterial death in CF ASF, an authentic model for CF lung biology based on the growth of human bronchial xenografts in nu / nu mice was used (Engelhardt et al., 1992, J. Clin. I nvest. 90: 2598-2607; Engelhardt et al. , 1 993, Nature Genet. 4: 27-34; Goldman ef al. , 1995, Nature Genet. 9: 1 26-1 31; Zhang ef al. , 1 996, Am. J. Physiol. 270: C 1 326-1 335). The primary cultures of epithelial cells derived from the proximal airways of CD and non-CF patients were seeded in stripped rat trachea and implanted subcutaneously in nu / nu mice with the proximal and distal ends open to the surface via ligated tubing. Within two weeks, pseudostratified epithelium of human origin was established. Quantitative morphometric analyzes of electron micrographs of transmission indicated that the epithelium generated in the xenograft was indistinguishable in organization, cell type distribution, and ultrastructure when compared to the epithelium of the natural bronchus from which the epithelial cells were derived (Engelhardt ef al., 1992, J. Clin.Insvest 90: 2598-2607, Engelhardt et al., 1993, Nature Genet 4: 27-34, Zhang et al., supra). Submucosal glands containing ducts with serous tubules and mucus are partially formed (Engelhardt et al., 1 995, Development 1 21: 2031 -2048). Previous studies evaluated the conductive properties of xenograft epithelial ions through the measurement of surface voltage in response to ion transport modulators. In these studies, it was established that the functional properties of these epithelia were identical to that observed in nasal and intrapulmonary human airways (Goldman et al., Supra). The most discriminant measure between non-CF and CF was the change in voltage measured in response to the decrease in luminal Cl and activation with cAMP. A number of xenografts was established for the characterization of the antimicrobial defect in ASF, including 1 2 grafts obtained from 6 non-CF patients and 12 grafts obtained from 3 CF patients. The results of the studies in these xenografts were presented in Figure 8. Non-CF grafts were distinguished from CF grafts based on the measurement of? Vt in response to low chloride and cAMP (no CF: -14.3 ± 1 .1 mV; CF: -0.3 ± 0.3 mV). ASF obtained from xenografts was obtained for antimicrobial assays and ion measurements by expelling the luminal contents with air, followed by a brief centrifugation to remove mucus. Direct ion measurements of AST not CF established that Na and Cl concentrations were 83 + 3 mM. The antimicrobial activity was measured by incubating ASF (30 μl) with 1 03 of P. aeruginosa for 2 hours at 37 ° C before a quantitative assessment of bacterial viability using standard colony counts. ASF obtained from all non-CF xenografts killed the bacteria completely. This antibacterial activity was inactivated in high salt and reducing agents, and fractionated through gel filtration with an apparent molecular weight of less than 1 0. Similar studies performed on CF xenografts revealed a significant increase in Na and Cl content in ASF at 178 ± 9 mM and 172 + 9 mM, respectively. ASF obtained from all CF xenografts failed to kill P. aeruginosa. Diluting AS F of CF in hypotonic solution reconstituted the bactericidal activity.

The relationship between the ionic composition and the bactericidal properties of ASF and the epithelial expression of CFTR was further studied in CF xenografts treated with adenoviral vectors containing a cDNA encoding CFTR. E1-suppressed adenoviral vectors were instilled expressing CFTR or β-galactosidase in the lumen of CF xenografts. In situ hybridization revealed the transduction and expression of high level recombinant CFTR in 9% -1 5% of surface epithelial cells (Figure 9). The transfer of CFTR genes corrected the conductive properties of CF epithelia (? Vt = -1 2.7 ± 0.3 in response to low chloride and cAMP) and normalized the content of Na (89 ± 5 mM) and Cl (87 ± 4) m M) of AS F (see Figure 8). ASF of the CFTR-corrected CF xenografts was able to completely kill P. aeruginosa in 1 0 of 12 grafts, with partial death observed in the remaining 2 grafts (Figure 8). CF xenografts treated with similar doses of LacZ virus were indistinguishable from untreated grafts.

I n antisense hybridization of hBD-1 in brnquial non-CF xenografts removes antimicrobial activity in AS F To understand the pathogenesis of CF lung it is important to identify the molecule in AS F that confers microbial death in non-CF lung, which is inactive in the CF lung. The distribution of expression tissue of hBD-1 and its sensitivity to salt for bacterial death suggests that it is responsible for host defense mediated by ASF. In situ hybridization demonstrated high-level, diffuse hBD-1 expression in epithelia of both non-CF xenografts (Figure 6, M-O panels) and CF (Figure 6, P-R panels). The role of hBD-1 in microbial killing was further studied by genetically excising its expression using a 21-mer phosphorothioate oligonucleotide antisense DNA specific for the 5 'region of mRNA encoding hBD-1. The controls used in these experiments included a nonsense oligonucleotide containing a stirred sequence and two designated oligonucleotides, which were identical to the antisense oligo at nucleotides 1 9-21 (inequality 1) and 14-21 (inequality 2). The oligonucleotides were instilled in the lumen of non-CF xenografts and three days later, ASF was collected for microbial death assays (Figure 10) before the removal of the xenografts in order that RNA could be isolated and an analysis of RT-PCR of the expression of hBD-1 (Fig. 1 1). RNA was detected for h BD-1 by RT-PCR at equivalent levels in all non-CF xenografts that were nonsense, unequal 1 or unequal 2 untreated or pre-incubated oligonucleotides. Preincubation of non-CF xenografts with these control nueclotides had no effect on the antimicrobial activity of ASF; Eight of nine ASF samples completely killed bacteria while one sample produced only 20 colonies. Preincubation of non-CF xenografts with the antisense oligonucleotide of hBD-1 substantially decreased the level of RNA specific for h BD-1 in six of eight xenografts, each of which failed to fully bind the bacteria; the complete antimicrobial activity was retained in two xenografts treated with antisense which were the same grafts in which hBD-1 specific RNA levels did not decrease. The data presented here establish that hBD-1 is expressed in the respiratory tract. These data represent the first demonstration of antibiotic peptide expression in human respiratory epithelium. The causal link between CF dysfunction and repeated airway infection in CF patients has never been adequately explained. The data presented herein provides evidence, which strongly supports a model in which the epithelial defensin activity of human respiratory tract is impaired by the high salt environment characteristic of CF airways., leading to defense of the compromised host and chronic infection. The progressive inhibition of synthetic defensin activity observed in vitro occurs in a range of salt concentrations (50 mM to 100 μM) that is consistent with previously published physiological values of approximately 80 mM Na + and CI "in surface fluid of pathways Respiratory normal against approximately 1 20 mM in airways of CF (Joris ef al., 1993, Am. Rev. Respir Dis 148: 1633-1637) Chronic destructive inflammation that is a hallmark of lung pathology of CF, may represent a compensation response to the lack of a normal antibiotic shield Such a model, in which the inhibition of salt of defensin activity leads to CF lung pathology, has important therapeutic implications as described in FIG. The acute sensitivity to salt observed for the antimicrobial activity of hBD-1 suggests that even relatively modest alterations in ambient ionic strength can intensify od decrease its effectiveness. Therapies, which are designed to alter the salt concentration in airways of CF, or which, as described herein, are designed to alter the activity of defensins such as hBD-1, are predicted to greatly alleviate the CF symptomatology Given the data presented here, it is evident that it would be of great importance to have a suitable animal model, such as a mouse or rat, which is available to examine the role of the ß defensins in host lung defense. The cryotidines, which are expressed in the Paneth cells of the small intestine, are only defensins identified in the mouse (Eisenhauer et al., 1992, Infecí Immu. 60: 3556-3565; Ouellette et al., 1992, FEBS Letters 304: 146-148; Ouellette et al., 1994, Infect. Immu. 62: 5040-5047; Huttner et al., 1994, Genomics 19: 448-453) and notably, mouse neutrophil deficiency defensins (Eisenhauer et al., 1992, Infect.immu.60: 3446-3447). In the experiments presented below, the isolation and characterization of a ß defensin, designated ß mouse defensin 1 (mBD-1), is described. The experimental procedures used in this study are presented below.

Procedures for cDNA cloning of mBD-1 The cDNA sequence of human β-defensin 1 (hBD-1) was used to perform a BLAST search at the National Center of Biotechnology Information (NCBI) site. www.ncbi.nlm.nih.gov). A 444 bp EST mouse cDNA sequence presented by Marra ef al. (access number AA065510), which exhibited 51% identity with hBD-1 at the amino acid level including the 6 specific cysteines of ß defensin. The reverse transcriptase polymerase chain reaction was used (RT-PCR) to clone the full-length cDNA sequence. Total RNA from mouse kidney C57BL / 6 was isolated using Trizol (Gibco BRL) and polyA + RNA was extracted from there using oligodT columns (Qiagen). PolyA + RNA (approximately 100 ng) was reverse transcribed using Not l- (dT) 1 8 as an initiator (First-Strand cDNA Synthesis Kit, Pharmacia Biotech) and 10% of the reaction was used for a PCR reaction. The specific primers for mBD-l were designed as follows: forward primer (m-def 1) -5'-CGAAGCTTCACATCCTCTCTGCACTCTGG-3 '[S EQ ID NO: 20] (nucleotides 4-24 of the GenBank sequence plus 9 nucleotide at the 5 'end containing a restriction site for Hind III); reverse primer (m-def 2) -5'-CGACTAGTCCAGGCAGATGTTCTGG-3 '[SEQ ID NO: 21] (nucleotides 433-444 of the GenBank sequence plus 8 nucleotides at the 5' end containing a restriction site for Spe I) . The PCR products were analyzed on a 1.5% agarose gel and a 440 bp band was isolated and subcloned in Bluescript I I SK "- (Stratagene).

Cloning of the genomic sequence of m BD-1 A genomic mouse library constructed in FIX II vector (Stratagene) was classified according to standard procedures (Sambrook et al., Supra) using a probe generated by PCR, where cDNA of mBD -1 served as template for gene-specific primers (forward: 5'-TTTCACATCCTCTCTGCACT-3 '[SEQ ID NO: 22] or 5'-TGCACTCTGGACCCTGGCT-3' [SEQ ID NO: 23]; inverse: 5'-ACCTGGCTCCATCTGGGAGA -3 '[SEQ ID NO: 24] or 5'- CCATCTGGGAGAAAAGAAAACA-3' [S EQ ID NO: 25]). Positive clones were purified and genomic DNA was isolated and subcloned in Bluescript I I KS-. The genomic clones were analyzed by partial sequence and digestion with restriction endonucleases.

Antimicrobial Activity Test of m BD-1 To test the antibacterial activity of m BD-1, lysates obtained from cells transfected with mBD-1 cDNA placed in a transfection vector were analyzed. A clinical isolate of Staphylococcus aureus, Escherichia coli D31 (Steiner et al., 1981, Nature 292: 246-248), and a clinical isolate of Pseudomonas aeruginosa as test organisms were used both in diffusion assays and in broth assays. iquid To obtain the cell lysates, human adrenal adenocarcinoma cells SW 1 3 (ATCC CCL 1 05) grown in DM EM were transfected with 2.5% fetal calf serum in the absence of antibiotics, with pcDNA 3. 1 - (Invitrogen) containing the full-length mBD-1 cDNA using calcium phosphate (Profection Mammalian Transfection Systems, Promega). As a control, cells transfected with the vector which did not contain the insert were used. Simulated transfected and transfected control cells were harvested 48 hours after transfection, pelleted by centrifugation, resuspended in 500 μl of distilled water and used by brief sonication. Tests of antibacterial liquid broth were performed as previously described for a-defensins (Steiner et al., 1981, Nature 292: 246-248). Bacteria (5 x 1 04 cfu) grown in LB broth in the semi-logarithmic phase were exposed to 50 μl of the cell lysates for 2 hours at 37 ° C. Cell viability was measured by platinating several dilutions and counting the colonies on the following day. The antibacterial diffusion assays were performed as described by Lehrer et al. (1 991, J. I mmun. Meth. 1 37: 167-173) with some modification. Essentially, bacteria were grown to semi-logarithmic phase in a solution of yeast extract (5 g / l) and tryptone (1 0 g / l) buffered with 10 mM PI PES at pH 7.5 and 5 x 1 O6 cfu were diluted in 10 ml of solution containing additionally 1% agarose. After pouring over 1 50 mm plates and cooling, the cell lysates (5 μl) were pipetted into cavities formed with a 4 mm cork corer and allowed to incubate at 30 ° C overnight. To analyze the salt dependence of mBD-1 activity against E. coli D31, NaCl was added to aliquots of the agarose solution to obtain final concentrations of 50 mM, 1 00 mM, 200 mM and 500 m NaCl M. The exact concentration of NaCl in the solutions was measured using sensitive Na or Cl (Microelectrodes) electrodes. The pH dependence of the m BD-1 activity was studied by adjusting the pH of the agarose solution to 9.0 or 5.5 by adding NaOH or HCl. As a positive control for antibacterial activity, 5 μl of a solution of magainin 1 (1 mg / ml) was applied (Magainin Pharmaceuticals, I nc.). Additionally, liquid broth assays were performed by adding NaCl (final concentrations: 7 mM, 50 mM, 1 00 mM, 200 mM and 500 mM) to the cell lysates.

Analysis of the expression of mBD-1 Northern blot analysis Norhern blots of polyA + RNA (2 μg) obtained from adult mouse tissues (testis, kidney, muscle, liver, lung, spleen, brain, heart) and fetal mice at 7, 1 1, 1 5, 1 7 days of gestation, were purchased from Clontech. 32P-dCTP randomized labeling cDNA (Rediprima DNA labeling system, Amersham) of m BD-1 cDNA was used as a probe. Hybridization was performed overnight at 42 ° C in 50% formamide, 5X SSC, Denhardt 5X solution, 50 mM sodium phosphate, and 250 μg / ml salmon sperm DNA. The washing conditions were as follows: 42 ° C in 2X SSC / 0. 1% SDS and finally 65 ° C in 0.1 X SSC / 0.1% SDS. After autoradiography, the filters were removed and probed again with a cDNA probe specific for human β-actin.

Ribonuclease protection assay Ribonuclease protection assays were also used to measure the level of transcripts of mBD-1 in the lung. The total RNA obtained from the lung / trachea, skeletal muscle and kidney was isolated as described above. 32P-dCTP labeled antisense riboprobes were prepared by in vitro transcription using T7 RNA polymerase promoters in linearized Bluescript KS II - containing cDNA from m BD-1 (304 bp) or β-actin (265 bp - SP6 / T7 Transcription Kit, Boehringer Mannheim). Hybridizations were performed in separate tubes using 5 x 1 05 cpm either of hBD-1 or β-actin probe and 20 μg of total RNA. The RNA-RNA hybrids were digested with RNase A and RNase T1 to produce protected fragments of 261 bp and 237 bp for mBD-1 and β-actin, respectively (Lysate Ribonuclease Protection Kit, Amersham). The results were visualized by electrophoresing the reaction products in a urea-polyacrylamide gel followed by autoradiography.

Transversal PCR i nverse Nested RT-PCR was used to detect low levels of mBD-1 mRNA in various tissues. PolyA + RNA was isolated from various mouse tissues (trachea, lung, tongue, esophagus, small intestine, large intestine, gallbladder, pancreas, skeletal muscle, heart, fallopian tube, ovary, vagina, brain) and were reverse transcribed as described above. The following primers were used in the first round of PCR: forward primer (m-def 5) -5'- TGGACCCTGGCTGCCACCACTATG-3 '[SEQ I D No.26] and reverse primer (m-def 6) 5'-GCTCATTCTTCAAACTACTGTCAG-3 '[SEQ ID NO: 27]. The products of this PCR were diluted 1: 50 in distilled water and an aliquot (2 μl) was used as a template for a nested PCR using the same reaction conditions as for the first round and including the following primers: forward primer ( m- def 7) -5'-ATGAAAACTCATTACTTTCTCÓTGGTGATG-3 '[SEQ ID NO: 28] and reverse primer (m def 8) -5'-CAATCCATCGCTCGCCTTTTATGCTC-3' [SEQ ID NO: 29]. The predicted size of the PCR product was 252 bp. The reverse transcriptase was omitted in the negative control, whereas, an RT-PCR with specific primers for mouse β-actin was used as a positive control. The PCR products were analyzed on a 1.5% agarose gel.

In situ hybridization Several tissues were collected from adult mice (nose, trachea, lung, kidney, liver, tongue and heart) of animals that were euthanized with C02. Tissues were embedded in OCT (Tissue-TCK, Miles I nc.), And cryosected. The 6 μm thick woven sections were mounted on slides and fixed in 4% paraformaldehyde in PBS (pH 7.4) for 4 hours at 4 ° C. Following dehydration through graduated ethanol concentrations, the sections were dried overnight under vacuum and stored at -20 ° C. On the following day, sections were treated with 10 μg / ml proteinase K for 30 minutes at 30 ° C, rinsed twice in 0.2X SSC for 30 seconds each time and fixed in 4% paraformaldehyde in PBS. The sections were then rinsed twice in 0. 1 M triethanolamine (pH 8.0) for 4 minutes each time, they were incubated with 0.25% acetic anhydride in 0. 1 triethanolamine for 10 minutes at room temperature, and then dehydrated through ethanol. Following drying under vacuum, prehybridization was performed for 4 hours at 54 ° C in 10 mM Tris (pH 8-0), 50% formamide, 2.5X Denhardt's solution, 0.6 M NaCl, 1 mM EDTA, 0.1 SDS %, 500 μg / ml of RNA, and 10 mM dithiothreitol. The RNase control sections were treated with 200 mg / ml of RNase A for 1 hour at 37 ° C before the prehybridization step. The sections were then hybridized with 5 x 106 cpm / ml of sense or antisense probes of 35S for 16 hours at 54 ° C in the prehybridization solution. The probes were synthesized by in vitro transcription of full length mBD-1 cDNA driven by the T7 or T3 RNA polymerase promoters cloned in Bluescript KS II - (Promega In Vitro Transcription System). Following prehybridization, the slides were washed in 4X SSC for 20 minutes at room temperature, treated with RNase (20 μg / ml) for 30 minutes at 37 ° C, and washed 2 × SSC / 1 mM DDT at room temperature for 10 minutes. minutes, followed by three washes in 0.5X SSC / 1 mM DDT at 54 ° C. The slides were dehydrated through ethanol and air dried before immersion in photoemulsion (Kodak). The slides were developed and analyzed by bright and dark field microscopy using a Nikon Microphot-FXA microscope. The results of these experiments are described below.

Cloning of the cDNA and genomic sequence of mBD-1 The EST homologous to the cDNA sequence of hBD-1 was obtained by performing a BLAST search at the National Center of Biotechnology Information (NCBI) web site. .ncbi.nlm.nih.gov) (Figure 12A) [SEQ ID NO: 6]. This cDNA clone exhibited 62% nucleotide identity with the cDNA of hBD-1; the encoded peptide was 51% identical. The murine clone was named mouse β-defensin 1 (mBD-1). The predicted peptide of mBD-1 contained the conserved amino acids specific for β-defensin, including the typical six-cysteine arrangement (Figure 1 2B). A cDNA of m BD-1 was isolated from kidney RNA using RT-PCR. A simple product of the predicted size (440 bp) was obtained and cloned in Bluescript I I SK. The insertion was sequenced and found to be identical to the database clone (Figure 12A) [SEQ ID NO: 6]. The cDNA sequence of m BD-1 consists of an open reading frame of 204 bp which encodes a peptide of 68 amino acids in length with a putative preprosequency (Figures 1 2A and 1 2B). A mouse genomic library was screened using PCR amplified sequences and several positive clones were isolated and further analyzed by hybridization with radiolabeled probes corresponding to the 5 'or 3' regions of the BD-1 m cDNA. Two clones contained the total sequence found in the cDNA. A restriction map of the m-BD-1 gene is shown in Figure 1 3. Comparison of the cDNA of mBD-1 and the corresponding genomic clones indicated that the m-BD-1 gene contains two exons separated by an intron of 1 5 kb. A TATA box is located in the 5 'flanking region and a polyadenylation signal is located at position 309 of the cDNA. The exon-intron splice site sequences conform to the consensus rule (Mount, 1 982, Nucí Acids Res. 1 0: 459-472). Sequence comparison analysis using a BLAST search on non-additional β-defensin-related sequences identified NCI B in mouse or human DNA, although a cDNA clone (access number X89820) obtained from rat was found to be 94% identical to the cDNA of m BD-1.

Analysis of antimicrobial activity To test whether the cloned cDNA sequence encodes an antimicrobial peptide, lysates obtained from cells transfected with the mB D-1 cDNA were used in both diffusion and liquid broth assays and were found to be active against all the bacteria used in both types of tests (Figures 14A, 14B and 14C). Lysates obtained from cells transfected with an empty vector failed to demonstrate annihilating activity, supporting the specificity of the assay. These results indicate that mBD-1 possessed microbial activity directed against both ram-positive and gram-negative bacteria. Additionally, this activity was substantially decreased in the presence of high concentrations of NaCl (Figure 14D) and in acidic pH.

Expression of m BD-1 in mouse tissues Analysis of total RNA cell RNA revealed the expression of m B D-1 as a transcript of approximately 0.5 kb in the kidney and liver of adult mice. This transcript was not detected by hybridization in other tissues, such as lung, testis, muscle, spleen, brain, heart or fetal tissues. RNA obtained from various tissues was also evaluated for the presence of m-BD-1 transcripts in the most sensitive ribonuclease protection assay. Using this assay, mBD-1 mRNA was detected in lung / tracheal homogenates albeit at levels much lower than those detected in the kidney. This assay failed to detect mBD-1 RNA in many other tissues, such as muscle (Figure 1A). A specific probe for β-actin was used as an internal control. Nested RT-PCR was performed using mRNA obtained from various mouse tissues to evaluate the low level of BD-1 m expression. Using this technique, the expression of mBD-1 in lung was detected, trachea, tongue, esophagus, fallopian tube, ovary and vagina (Figure 1 5B). Β-actin was again used as a positive control. No signal was detected when the reverse transcriptase of the reaction was omitted. The tissue distribution of mBD-1 was analyzed at a cellular level by in situ hybridization. The transcripts of mBD-1 were detected in the epithelium of the nose (Figure 1 6A and 1 6B), the large cartilaginous airways (Figure 1 6E and 1 6F) and the major bryocytes (Figure 1 61 and 1 6J) when hybridized tissue sections to the antisense probe specific for mBD-1. Diffusion-specific hybridization signal for mBD-1 appeared virtually throughout all the surface epithelial cells of the conductive airways. This signal was substantially reduced or absent in the small bronchioles or lung parenchyma (Figure 16M and 16N). Positive results were also seen in the distal tubules and collecting ducts of the kidney (Figure 17A and 17 B), the epithelium covering the tongue (Fig. 1 7E and 1 7F), and as a diffuse signal in the liver (Fig. Fig. 1 71 and 1 7J). No signal was detected above that of support levels in the heart muscle (Figure 17M and 17N). Hybridization using sense mBD-1 riboprobes (Figures 16 and 17) or digestion of the tissue with RNase prior to hybridization to the antisense probe resulted in no detectable signal, confirming the sensitivity of the assay. The cDNA sequence of mBD-1 was discovered in a BLAST search using the nucleotide sequence of hBD-1. Although the similarity of mBD-1 to hBD-1 is only 62%, and 51% at the nucleotide and amino acid levels, respectively, the amino acid sequence of the putative mBD-1 prepropeptide revealed the structural contrast marks of the ß- defensins. The amino terminal prepro-portion of the peptide contains several hydrophobic residues, which are characteristic of the β-defensins and are found in other members of the β-defensin family expressed on mucosal surfaces, such as, TAP (Diamond et al. , 1991, Proc. Nati, Acad. Sci. USA 88: 3952-3956; Diamond et al., 1993, Proc. Nati Acad. Sci. 90: 4596-4600), LAP (Schonwetter et al., 1995, Science 267: 1645-1648) or hBD-1 (data presented herein and Bensch et al., 1995, FEBS Letters 368-331-335 ). The putative mature peptide contains six separated cysteine residues in a typical array and other conserved amino acids that may have important roles for the conformation and function of β-defensins, such as G10, P18, G25 and T26 (Zimmermann et al., 1995 , Biochemistry 34: 13663-13671). Several characteristic charged residues are present in the putative mature peptide.

The analysis of the mBD-1 gene revealed the presence of two exons surrounding an intron of 15 kb. The structure of the mBD-1 gene is therefore identical to those of other ß-defensins, such as LAP (Diamond et al., 1993, Proc. Nati, Acad. Sci. USA 90: 4596-4600) or hBD-1 (Liu et al., 1996, J. Invest. Med.44: 294A) each of which are contained in 2 exons. The first exon contains the pre- and one part of the pro-peptide, while the mature peptide and the remaining part of the pro-peptide are encoded by the second exon. These data indicate an evolutionary conservation of the members of the ß-defensins group. An analysis of the genomic clones and cDNA of mBD-1 revealed the presence of typical regulatory elements, such as a TATA box and a polyadenylation signal. However, no binding sites could be found for transcription factors that are involved in the inflammatory response in the regulatory sequence of mBD-1. This region of mBD-1 differs, therefore, from that of the 5 'region of the TAP gene, which contains a NF-KB site upstream of the transcription start site (Schonwetter et al., 1995, Science 267: 1645-1648). While the expression of other mucosal ß-defensins, such as, LAP and TAP (Diamond et al., 1993, Proc. Nati. Acad. Sci. USA 90: 4596-4600; Schonwetter et al., 1995, Science 267 : 1645-1648; Diamond et al., 1996, Proc. Nati, Acad. Sci. USA 93: 5156-5160; Russell et al., 1996, Infect. Immu. 64: 1565-1568), over regulated by mediators Inflammatory and bacterial components, incubation of cultured mouse cells (mouse primary tracheal epithelial cells, mouse primary hepatocytes, and mouse lung adenoma cells) with LPS or TNF exhibited no increase in mBD-1 expression. In this way, it appears that mBD-1 can not be regulated by inflammatory responses, but it can be part of a constitutively expressed host defense system. The cloned cDNA sequence specifying mBD-1 encodes an antimicrobial peptide having antimicrobial activity directed against gram-negative and gram-positive bacteria. In addition, this activity was lost at high salt concentrations and acid pH. The data presented here indicate that mBD-1 is expressed in a pattern similar to the expression pattern of hBD-1. In the case of m BD-1, the organ in which the most abundant expression was observed was the kidney. However, the transcription of mB D-1 was also observed along the surface epithelium of the conductive airways. There appeared to be a g radient expression of mBD-1 along the conductive airways, where the highest expression in proximal structures was noted. This expression pattern differs from that of hBD-1, which is expressed more uniformly throughout the conductive airways (data presented here and McCray et al., 1997, Am. J. Respir. Cell. Mol. Biol. 1 6: 343-349). The expression pattern of m BD-1 in airways is of specific importance in light of the data presented here, which establish a salt-dependent defect of BD-1 in the pathogenesis of CF. This model specifically suggests that an elevated concentration of NaCl in CF airway surface fluid inactivates the antimicrobial activity of defensin and / or other antimicrobial molecules possibly resulting in bacterial colonization and infection. Similar expression patterns of mBD-1 and expression of hBD-1 in lung coupled with identical NaCl-dependent biological activities demonstrate that the mouse is a useful animal model for investigating the role of antimicrobial peptides in defense of host pathways. respiratory The descriptions of each of the patents, patent applications and publications cited herein are incorporated herein by reference in their entirety. Although this invention has been described with reference to the specific embodiments, it is evident that other embodiments and variations of this invention may be devised by others skilled in the art without departing from the true spirit and scope of the invention. The appended claims are intended to be construed as including all these modalities and equivalent variations.

Claims (56)

1. RECIPE N DI CACI ON ES 1 . A cell comprising an isolated nucleic acid encoding human beta defensin-1.
2. 2. A cell comprising an isolated nucleic acid encoding rodent beta defensin-1.
3. 3. The cell of claim 2, wherein said defensin is mouse beta defensin-1.
4. 4. The cell of claim 1, wherein said nucleic acid further comprises a promoter / regulatory sequence positioned at the 5 'end of said human defensin beta. The cell of claim 2, wherein said nucleic acid further comprises a promoter / regulatory sequence positioned at the 5 'end of said rodent beta defensin-1. 6. A vector comprising an isolated nucleic acid encoding human beta defensin-1. 7. The vector of claim 6 being selected from the group consisting of a plasmid, a virus and a non-viral vector. 8. The vector of claim 6 being suspended in a pharmaceutical composition. 9. The vector of claim 7, wherein said asylated nucleic acid further comprises a promoter / regulatory sequence positioned at the 5 'end of said human beta defensin. 1 0. A vector comprising an isolated nucleic acid encoding rodent beta defensin-1. eleven . The vector of claim 10, wherein said defensin is mouse beta defensin-1. 12. The vector of claim 10 being selected from the group consisting of a plasmid, a virus and a non-viral vector. The vector of claim 10, wherein said isolated nucleic acid further comprises a promoter / regulatory sequence positioned at the 5 'end of said rodent beta defensin-1. 14. An isolated nucleic acid encoding human beta defensin-1. The isolated nucleic acid of claim 14, wherein said nucleic acid is cDNA. 16. The isolated nucleic acid of claim 15, the sequence shown in Figure 2 being [SEQ ID NO: 3]. 17. The isolated nucleic acid of claim 14, further comprising a promoter / regulatory sequence positioned at the 5 'end of the coding region of said human beta defensin-1. 1 8. An isolated nucleic acid encoding mouse beta defensin-1. 9. The isolated nucleic acid of claim 18, wherein said nucleic acid is cDNA. 20. The isolated nucleic acid of claim 1, wherein the sequence shown in Figure 1 2 [SEQ ID NO: 6]. twenty-one . The isolated nucleic acid of claim 1 8, further comprising a promoter / regulatory sequence placed at the 5 'end of said mouse defensin beta-1. 22. An isolated nucleic acid encoding a salt insensitive mammalian beta defensin-1. 23. The isolated nucleic acid of claim 23, wherein said mammalian defensin beta-1 is selected from the group consisting of human beta defensin-1 and mouse beta defensin-1. 24. The isolated nucleic acid of claim 22, further comprising a regulatory promoter sequence placed at the 5 'end of said mammalian defensin beta. 25. An isolated nucleic acid encoding a mammalian beta defensin-1, which has enhanced antimicrobial activity when compared to a wild-type mammalian defensin-1 counterpart. 26. The isolated nucleic acid of claim 25, wherein said mammalian beta defensin-1 is selected from the group consisting of human beta defensin-1 and mouse beta defensin-1. 27. The isolated nucleic acid of claim 25, further comprising a promoter / regulatory sequence positioned at the 5 'end of said mammalian defensin beta. 28. A salt insensitive mammalian beta-defensin-1. 29. The mammalian beta defensin-1 of claim 28, being selected from the group consisting of human beta defensin-1 and mouse beta defensin-1. 30. Mutated mammalian defensin-1 beta, which has enhanced antimicrobial activity when compared to a natural-type mammalian defensin-1 counterpart. 31 Beta defensin-1 of mammal with mutation of the claim 30. being selected from the group consisting of human beta defensin-1 and mouse beta defensin-1. 32. A method for enhancing antimicrobial activity in a tissue sample, comprising adding a mammalian beta defensin-1 to said sample. 33. The method of claim 32, wherein said tissue sample is selected from the group consisting of a sample of mammalian lung tissue, a sample of mammalian skin tissue and a sample of mammalian blood tissue. 34. The method of claim 32, wherein said mammalian beta defensin-1 is selected from the group consisting of human beta defensin-1 and mouse beta defensin-1. 35. The method of claim 32, wherein said mammalian beta defensin-1 is added to said tissue sample in vivo in a mammal. 36. The method of claim 35, wherein said mammalian beta defensin-1 is human beta defensin-1 and said mammal is a human. 37. The method of claim 35, wherein said tissue sample is a sample of lung tissue, said mammalian beta defensin is human beta defensin-1, which is added to said lung tissue sample by means of a nebulizer. or a bronchoscope. 38. The method of claim 35, wherein said tissue sample is a sample of lung tissue, said mammalian beta defensin is human beta defensin-1, which is added to said sample of lung tissue in the form of a vector comprising an isolated nucleic acid encoding said human beta defensin-1, wherein when said vector is administered to said human, said human beta defensin-1 is expressed therein to effect the addition of said human beta defensin-1 to said sample of lung tissue. 39. The method of claim 36, wherein said human has a respiratory disease which predisposes said human to pulmonary microbial infection. 40. The method of claim 39, wherein said respiratory disease is emphysema. 41 The method of claim 29, wherein said respiratory disease is cystic fibrosis and said human beta defensin is insensitive to salt. 42. The method of claim 39, wherein said disease is cystic fibrosis and said human beta defensin-1 has added to it a compound capable of absorbing salt. 43. The method of claim 38, wherein said human has a respiratory disease, which predisposes said human to pulmonary microbial infection. 44. The method of claim 43, wherein said respiratory disease is emphysema. 45. The method of claim 43, wherein said respiratory disease is cystic fibrosis and said human beta defensin is insensitive to salt. 46. The method of claim 43, wherein said respiratory disease is cystic fibrosis and said human beta defensin-1 has added to it a compound capable of absorbing salt. 47. A method for treating a human having a respiratory disease, which predisposes said human to pulmonary microbial infection, said method comprising administering to the lungs of said human a pharmaceutical composition comprising human beta defensin-1. 48. A method for treating a human having a respiratory disease, which predisposes said human to pulmonary microbial infection, said method comprising administering to the lungs of said human a pharmaceutical composition comprising an isolated nucleic acid encoding beta defensin. 1 human, wherein said human beta defensin-1 is expressed from said isolated nucleic acid in cells of said lungs, thereby treating said human. 49. A method for treating a human having a lung infection, said method comprising administering to the lungs of said human a pharmaceutical composition comprising human beta defensin-1. 50. A method for treating a human having a pulmonary microbial infection, said method comprising administering to the lungs of said human a pharmaceutical composition comprising an isolated nucleic acid encoding human beta defensin-1, wherein said beta defensin-1 h umana is expressed from said isolated nucleic acid in cells of said lungs, thereby treating said human. 51 A transgenic mammal comprising an isolated nucleic acid encoding human beta defensin-1. 52. A method for treating a human having a microbial infection of the skin, comprising administering to the skin of said human a composition comprising human beta defensin-1. 53. A topical composition for administration to the skin of a mammal, comprising mammalian beta defensin-1 suspended in a pharmaceutically acceptable carrier. 54. The topical composition of claim 53, wherein said mammalian beta defensin is selected from the group consisting of human beta defensin-1 and mouse beta defensin-1. 55. A pharmaceutical composition comprising human beta defensin-1. 56. A method to synthesize human beta defensin-1 using solid phase 9-phenylrenylmethyloxycarbonyl synthesis, comprising the regioselective formation of Cys5-Cys34, Cys1 2-Cys27 and Cys17-Cys35 by protecting Cys5-Cys34 with trifly (triphenylmethyl), protect Cys12-Cys27 with Acm (acetamidomethyl), and protect Cys1 and Cys35 with MOL (p-methoxy benzyl).