WO2002074907A2

WO2002074907A2 - Methods of modulating surfactant production

Info

Publication number: WO2002074907A2
Application number: PCT/US2002/005134
Authority: WO
Inventors: Robert James Mason; Steven Neben; Michael R. Eckart; Malinda Longphre
Original assignee: Bayer Pharmaceuticals Corporation
Priority date: 2001-02-02
Filing date: 2002-01-30
Publication date: 2002-09-26
Also published as: AU2002258403A1; WO2002074907A3

Abstract

The present invention provides methods of modulating surfactant production using FAS polypeptides, and agonists or antagonists that are capable of modulating FAS activity. More particularly, the present invention encompasses the use of FAS polypeptides, or agonist or antagonist polypeptides capable of modulating FAS activity, for the treatment of a disease or clinical condition where surfactant production is relevant to the causation or treatment of the disease or clinical condition, such as lung diseases, including adult/infant respiratory distress syndrome, acute lung injury, chronic obstructive pulmonary disease (emphysema and chronic bronchitis), asthma, alveolar proteinosis, small airway disease and maintaining airway patency, and related pulmonary conditions. The present invention also encompasses pharmaceutical compositions containing the FAS polypeptides, or agonist or antagonist polypeptides capable of modulating FAS activity, and the use of such pharmaceutical compositions for the treatment of the above-mentioned diseases or clinical conditions.

Description

TITLE OF THE INVENTION

METHODS OF MODULATING SURFACTANT PRODUCTION

BACKGROUND OF THE INVENTION

This invention relates to methods of regulating surfactant production by modulating Fatty Acid synthase (FAS) activity. More particularly, this invention relates to the use of FAS, or agonist or antagonist compounds capable of modulating FAS activity, to regulate surfactant production as a treatment for lung disease. FAS in vertebrates is a dimerized complex consisting of seven active sites coordinated to catalyze the synthesis of palmitic acid from acetyl Co A and malonyl Co A. Palmitic acid is a major fatty acid building block for surfactant lipids, thus, FAS is an important regulator of surfactant phospholipid synthesis in Type II airway epithelial cells.

Pulmonary surfactant is synthesized in and secreted by the Type π epithelial cells in the alveolar regions ofthe lungs and consists of 80-90%) phospholipids and 10-20%> surfactant proteins. It functions to reduce surface tension in the alveoli thus preventing their collapse. In addition, pulmonary surfactant maintains a key role in reducing the likelihood of bacterial or viral infection in the lungs and is crucial in protecting the lungs from inflammation due to exposure to atmospheric pollutants and antigens. As a result of its key physiological functions, alterations of surfactant levels in the lung are thought to be causal or aggravating factors in many lung pathologies. Alveolar proteinosis is characterized by an alveolar accumulation of proteinaceous materials also rich in phospholipids. (Fishman et al. 1998. Pulmonary Diseases and Disorders 3rd Ed. McGraw-Hill Publishers, New York). Infantile respiratory distress syndrome (IRDS) is characterized by a lack of surfactant present in the lungs of pre-term infants leading to alveolar collapse and hypoxemia. Synthetic surfactants consisting of various combinations of phospholipids and surfactant proteins have been used extensively to prevent and to treat RDS in neonates (Avery, M.E. and Merritt, T.A. 1991. New EnglJ Med 324:910-12). Adult respiratory distress syndrome (ARDS) occurs as a result of trauma, long bone fractures, burns, sepsis and lung injury due to aspiration of gastric contents, inhalation of toxic gases and pneumonia (Fishman et al. 1998. Pulmonary Diseases and Disorders 3^rd Ed. McGraw- Hill Publishers, New York). These phenomena cause a dramatic increase in the permeability ofthe pulmonary micro vasculature leading to leakage of fluids into the lung and dilution and/or inactivation of surfactant. Additionally, inadequate surfactant levels may play a role in chronic obstructive pulmonary disease (emphysema and chronic bronchitis) and asthma resulting in airway collapse and reduced gas exchange.

In light ofthe foregoing, it is clear that the identification and characterization of factors that play a role in modulating surfactant production may facilitate the development of treatments for several lung diseases, including but not limited to infant/adult respiratory distress syndrome, acute lung injury, chronic obstructive pulmonary disease (emphysema and chronic bronchitis), asthma, small airway disease and maintaining airway patency, and related pulmonary conditions.

BRIEF SUMMARY OF THE INVENTION The present invention provides methods for modulating FAS activity in order to regulate surfactant production. One aspect ofthe present invention involves using FAS polypeptides, as well as biologically active and diagnostically or therapeutically useful fragments, analogs and derivatives thereof, to regulate surfactant production. Another aspect ofthe present invention involves using FAS polypeptides to screen test compounds for the ability to modulate FAS activity, and using these agonist and antagonist compounds, as well as biologically active and diagnostically or therapeutically useful fragments, analogs and derivatives thereof, to regulate surfactant production. The present invention further encompasses the use of FAS polypeptides and agonist or antagonist polypeptides for the treatment of a disease or clinical condition where surfactant production is relevant to the causation or treatment ofthe disease or clinical condition. In one embodiment, such diseases or conditions include lung diseases, such as infant/adult respiratory distress syndrome, acute lung injury, chronic obstructive pulmonary disease (emphysema and chronic bronchitis), asthma, alveolar proteinosis, small airway disease and maintaining airway patency, and related pulmonary conditions.

In accordance with one aspect ofthe present invention, there are provided isolated nucleic acid molecules encoding the polypeptides ofthe present invention, including niRNAs, DNAs, cDNAs, genomic DNA, as well as antisense analogs thereof and biologically active and diagnostically or therapeutically useful fragments thereof.

In accordance with another aspect ofthe present invention, there are provided processes for producing such polypeptides by recombinant techniques through the use of recombinant vectors, such as cloning and expression plasmids useful as reagents in the recombinant production ofthe polypeptides ofthe present invention, as well as recombinant prokaryotic and/or eukaryotic host cells comprising a nucleic acid sequence encoding a polypeptide ofthe present invention.

In accordance with a still another aspect ofthe present invention, there is provided a process for utilizing such polypeptides, or polynucleotides encoding such polypeptides, for screening for agonists and antagonists thereto and for therapeutic purposes, for example, in the treatment of a disease or clinical condition where surfactant production is relevant to the causation or treatment ofthe disease or clinical condition, such as various lung diseases. In accordance with yet another aspect ofthe present invention, there are provided diagnostic assays for detecting diseases or clinical conditions, or susceptibility to diseases or clinical conditions, related to mutations in a nucleic acid sequence ofthe present invention and for detecting over-expression or underexpression ofthe polypeptides encoded by such sequences.

In accordance with another aspect ofthe present invention, there are provided pharmaceutical compositions containing the FAS polypeptides and agonist or antagonist polypeptides, and the use of such pharmaceutical compositions for the treatment of a disease or clinical condition where surfactant production is relevant to the causation or treatment of the disease or clinical condition, such as various lung diseases.

In accordance with still another aspect ofthe present invention, there is provided a process involving expression FAS polypeptide, or polypeptides capable of modulating FAS activity, or polynucleotides encoding such polypeptides, for purposes of gene therapy. As used herein, gene therapy is defined as the process of providing for the expression of nucleic acid sequences of exogenous origin in an individual for the treatment of a disease condition within that individual.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 depicts the cDNA sequence (SEQ ID NO:l) and corresponding deduced amino acid sequence (SEQ ID NO: 2) of human FAS.

FIG. 2 illustrates the amino acid sequence homology between human FAS and FAS from other species (SEQ ID NO:3-5). Conserved amino acids are readily ascertainable. FIG. 3A illustrates early changes in FAS mRNA expression in rat alveolar type II cells in response to treatment with Keratinocyte Growth Factor (KGF, 20 ng/ml) for 1,3, 6, and 24 hours, a stimulus known to dramatically increase surfactant production after 7days. RNA expression was determined by hybridizing 5 ug of total RNA from treated cells to Affmetrix GeneChips. Data shown is the average of 3 separate experiments.

FIG. 3B illustrates FAS mRNA expression in rat alveolar type II cells in response to treatment with KGF (10 ng/ml) for 7 days. RNA expression was determined by hybridizing 5 ug of total RNA from treated cells to Affmetrix GeneChips. Data shown is the average of 3 separate experiments.

DETAILED DESCRIPTION OF THE INVENTION The present invention provides methods for modulating surfactant production using FAS polypeptides, or polynucleotides encoding such polypeptides, as well as biologically active and diagnostically or therapeutically useful fragments, analogs and derivatives thereof. The present invention also provides methods for modulating surfactant production using agonist or antagonist polypeptides that are capable of modulating FAS activity, or polynucleotides encoding such polypeptides, as well as biologically active and diagnostically or therapeutically useful fragments, analogs and derivatives thereof. Polypeptides having FAS activity, and polynucleotide sequences encoding such polypeptides, have been identified in International Patent Publication WO 00/51430, the disclosure of which is incorporated in its entirety by reference herein.

One aspect ofthe present invention therefore relates to isolated nucleic acid molecules (polynucleotides) which encode for the mature FAS polypeptide having the deduced amino acid sequence of FIG. 1 (SEQ ID NO:2). The polynucleotide encoding human FAS is structurally related to the polynucleotide encoding rat FAS, as well as the polynucleotide encoding mouse FAS and the polynucleotide encoding chicken FAS. Human FAS is comprised of a 7602 base pair coding region that encodes a putative protein of 2534 amino acids with calculated molecular weight of 304.1 kDa. Rat FAS is comprised of a 7488 base pair coding region that encodes a putative protein of 2496 amino acids with a calculated molecular weight of 299.5 kDa. Rat FAS has approximately 77% nucleic acid identity, 77% amino acid identity and 84% similarity over the full length nucleic acid and amino acid sequences of human FAS. Mouse FAS is comprised of an 7488 base pair coding region that encodes a putative protein of 2496 amino acids with a calculated molecular weight of 299.5 kDa. Mouse FAS has approximately 76%> nucleic acid identity, 74% amino acid identity and 81%) similarity over the full length nucleic acid and amino acid sequences of human FAS. Chicken FAS is comprised of a 7500 base pair coding region that encodes a putative protein of 2500 amino acids with a calculated molecular weight of 300 kDa. Chicken FAS has approximately 61%> nucleic acid identity, 61 > amino acid identity and 74%> similarity over the full length nucleic acid and amino acid sequences of human FAS. The homology of human FAS to rat FAS, mouse FAS and chicken FAS is depicted by the shaded boxes in FIG. 2.

The polynucleotide ofthe present invention may be in the form of RNA or in the form of DNA, which DNA includes cDNA, genomic DNA, and synthetic DNA. The DNA may be double-stranded or single-stranded. The coding sequence that encodes the mature FAS polypeptide may be identical to the coding sequence shown in FIG. 1 (SEQ ID NO: 1) or may be a different coding sequence as a result ofthe redundancy or degeneracy ofthe genetic code, encoding the same, mature FAS polypeptide as the DNA of FIG. 1, (SEQ ID NO:l). The polynucleotides which encode for the mature FAS polypeptide of FIG. 1 (SEQ

ID NO: 2) may include: only the coding sequence for the mature polypeptide; the coding sequence for the mature polypeptide and additional coding sequence such as a leader or secretory sequence or a proprotein sequence; the coding sequence for the mature polypeptide (and optionally additional coding sequence) and non-coding sequence, such as introns or non- coding sequence 5' and/or 3' ofthe coding sequence for the mature polypeptide.

Thus, the term "polynucleotide encoding a polypeptide" encompasses a polynucleotide which includes only coding sequence for the polypeptide as well as a polynucleotide which includes additional coding and/or non-coding sequence.

The present invention further relates to variants ofthe herein above-described polynucleotides which encode for fragments, analogs and derivatives ofthe polypeptides having the deduced amino acid sequence of FIG. 1 (SEQ ID NO: 2). The variants ofthe polynucleotide may be a naturally occurring allelic variant ofthe polynucleotide or a non- naturally occurring variant ofthe polynucleotide. Thus, the present invention includes polynucleotides encoding the same mature FAS polypeptide as shown in FIG. 1 (SEQ ID NO:2) as well as variants of such polynucleotides which variants encode for a fragment, derivative or analog ofthe polypeptide of FIG. 1 (SEQ ID NO:2). Such nucleotide variants include deletion variants, substitution variants and addition or insertion- variants.

As hereinabove indicated, the polynucleotide may have a coding sequence that is a naturally occurring allelic variant ofthe coding sequence shown in FIG. 1 (SEQ ID NO:l). As known in the art, an allelic variant is an alternate form of a polynucleotide sequence which may have a substitution, deletion or addition of one or more nucleotides, which does not substantially alter the function ofthe encoded polypeptides.

The present invention also includes polynucleotides, wherein the coding sequence for the mature polypeptides may be fused in the same reading frame to a polynucleotide sequence which aids in expression and secretion of a polypeptide from a host cell, for example, a leader sequence which functions as a secretory sequence for controlling transport of a polypeptide from the cell. The polypeptide having a leader sequence is a preprotein and may have the leader sequence cleaved by the host cell to form the mature form ofthe polypeptide. The polynucleotides may also encode for a proprotein which is the mature protein plus additional 5'amino acid residues. A mature protein having a prosequence is a proprotein and is an inactive form ofthe protein. Once the prosequence is cleaved an active mature protein remains.

For example, the polynucleotides ofthe present invention may encode a mature protein, or for a protein having a prosequence or for a protein having both a prosequence and a presequence (leader sequence).

The polynucleotides ofthe present invention may also have the coding sequence fused in frame to a marker sequence which allows for purification ofthe polypeptide ofthe present invention. The marker sequence may be a hexa-histidine tag supplied by a pQE-9 vector to provide for purification ofthe mature polypeptide fused to the marker in the case of a bacterial host, or, for example, the marker sequence may be a hemagglutinin (HA) tag when a mammalian host, e.g. COS-7 cells, is used. The HA tag corresponds to an epitope derived from the influenza hemagglutinin protein (Wilson, I., et al., Cell, 37:767 (1984)).

The term "gene" means the segment of DNA involved in producing a polypeptide chain; it includes regions preceding and following the coding region (leader and trailer) as well as intervening sequences (introns) between individual coding segments (exons). Fragments ofthe full length FAS gene may be used as a hybridization probe for a cDNA library to isolate the full length gene and to isolate other genes which have a high sequence similarity to the gene or similar biological activity. Probes of this type preferably have at least 20 bases and may contain, for example, 50 or more bases. The probe may also be used to identify a cDNA clone corresponding to a full length transcript and a genomic clone or clones that contain the complete FAS gene including regulatory and promotor regions, exons, and introns. An example of a screen comprises isolating the coding region of the FAS gene by using the known DNA sequence to synthesize an oligonucleotide probe. Labeled oligonucleotides having a sequence complementary to that ofthe gene ofthe present invention are used to screen a library of human cDNA, genomic DNA or mRNA to determine which members ofthe library the probe hybridizes to.

The present invention further relates to polynucleotides that hybridize to the hereinabove-described sequences if there is at least 60%, preferably at least 70%, more preferably at least 90%>, and still more preferably at least 95%> identity between the sequences. The present invention particularly relates to polynucleotides that hybridize under stringent conditions to the hereinabove-described polynucleotides. As herein used, the term "stringent conditions" means hybridization will occur only if there is at least 90%> and preferably 95%o and more preferably at least 97% identity between the sequences. The polynucleotides which hybridize to the hereinabove described polynucleotides in a preferred embodiment encode polypeptides which either retain substantially the same biological function or activity as the mature polypeptide encoded by the cDNAs of FIG. 1 (SEQ ID NO: 1). Alternatively, the polynucleotide may have at least 20 bases, preferably 30 bases, and more preferably at least 50 bases which hybridize to a polynucleotide ofthe present invention and which has an identity thereto, as hereinabove described, and which may or may not retain activity. For example, such polynucleotides may be employed as probes for the polynucleotide of SEQ ID NO:l, for example, for recovery ofthe polynucleotide or as a diagnostic probe or as a PCR primer.

The present invention is directed to polynucleotides having at least a 50% identity, preferably at least 70%> identity, more preferably at least 90% and still more preferably at least a 95%o identity to a polynucleotide which encodes the polypeptide of SEQ ID NO:2 as well as fragments thereof, which fragments have at least 20 bases and preferably 30 bases and more preferably at least 50 bases and to polypeptides encoded by such polynucleotides. The present invention further relates to a FAS polypeptide having the deduced amino acid sequence of FIG. 1 (SEQ ID NO:2), as well as fragments, analogs and derivatives of such polypeptides.

The terms "fragment," "derivative" and "analog" when referring to the polypeptide of FIG. 1 (SEQ ID NO:2) means polypeptides which retains essentially the same biological function or activity as such polypeptides. Thus, an analog includes a proprotein which can be activated by cleavage ofthe proprotein portion to produce an active mature polypeptide.

The polypeptides ofthe present invention maybe recombinant polypeptides, natural polypeptides or synthetic polypeptides, preferably recombinant polypeptides. The fragment, derivative or analog ofthe polypeptide of FIG. 1 (SEQ ID NO:2) may be (i) one in which one or more ofthe amino acid residues are substituted with a conserved or non-conserved amino acid residue (preferably a conserved amino acid residue) and such substituted amino acid residue may or may not be one encoded by the genetic code, or (ii) one in which one or more ofthe amino acid residues includes a substituent group, or (iii) one in which the mature polypeptide is fused with another compound, such as a compound to increase the half-life of the polypeptide (for example, polyethyleneglycol), or (iv) one in which the additional amino acids are fused to the mature polypeptide, such as a leader or secretory sequence or a sequence which is employed for purification ofthe mature polypeptide or a proprotein sequence. Such fragments, derivatives and analogs are deemed to be within the scope of those skilled in the art from the teachings herein.

The polypeptides and polynucleotides ofthe present invention are preferably provided in an isolated form, and preferably are purified to homogeneity. The term "isolated" means that the material is removed from its original environment (e.g., the natural environment if it is naturally occurring). For example, a naturally-occurring polynucleotide or polypeptide present in a living animal is not isolated, but the same polynucleotide or DNA or polypeptide, separated from some or all ofthe coexisting materials in the natural system, is isolated. Such polynucleotide could be part of a vector and/or such polynucleotide or polypeptide could be part of a composition, and still be isolated in that such vector or composition is not part of its natural environment. The polypeptides ofthe present invention include the polypeptide of SEQ ID NO:2 (in particular the mature polypeptide) as well as polypeptides which have at least 60% similarity (preferably 60% identity) to the polypeptide of SEQ ID NO:2, preferably at least 70% similarity (preferably 70%> identity) to the polypeptide of SEQ ID NO:2 and more preferably at least a 85% similarity (more preferably at least a 85% identity) to the polypeptide of SEQ ID NO:2 and still more preferably at least a 90% similarity (still more preferably a 90% identity) to the polypeptide of SEQ ID NO:2, and even still more preferably at least a 95%> similarity (more preferably at least a 95% identity) to the polypeptide of SEQ ID NO:2, and also include portions of such polypeptides with such portion of the polypeptide generally containing at least 30 amino acids and more preferably at least 50 amino acids. As known in the art "similarity" between two polypeptides is determined by comparing the amino acid sequence and its conserved amino acid substitutes of one polypeptide to the sequence of a second polypeptide. Such conservative substitutions include those described by Dayhoff in The Atlas of Protein Sequence and Structure 5 (1978), and by Argos in EMBO J., 8:779-785 (1989). For example, amino acids belonging to one ofthe following groups represent conservative changes:

- ala, pro, gly, gin, asn, ser, thr;

- cys, ser, tyr, thr; - val, ile, leu, met, ala, phe;

- lys, arg, his;

- phe, tyr, trp, his; and -asp, glu.

Fragments or portions ofthe polypeptides ofthe present invention maybe employed for producing the corresponding full-length polypeptide by peptide synthesis; therefore, the fragments may be employed as intermediates for producing the full-length polypeptides. Fragments or portions ofthe polynucleotides ofthe present invention may be used to synthesize full-length polynucleotides ofthe present invention.

The present invention also relates to vectors that include polynucleotides ofthe present invention, host cells which are genetically engineered with vectors ofthe invention and the production of polypeptides ofthe invention by recombinant techniques. Host cells may be genetically engineered (transduced or transformed or transfected) with the vectors of this invention which may be, for example, a cloning vector or an expression vector. The vector may be, for example, in the form of a plasmid, a viral particle, a phage, etc. The engineered host cells can be cultured in conventional nutrient media modified as appropriate for activating promoters, selecting transformants or amplifying the FAS genes. The culture conditions, such as temperature, pH and the like, are those previously used with the host cell selected for expression, and will be apparent to the ordinarily skilled artisan. The polynucleotide ofthe present invention may be employed for producing a polypeptide by recombinant techniques. Thus, for example, the polynucleotide sequence may be included in any one of a variety of expression vehicles, in particular vectors or plasmids for expressing a polypeptide. Such vectors include chromosomal, non-chromosomal and synthetic DNA sequences, e.g., derivatives of SV40; bacterial plasmids; phage DNA; yeast plasmids; vectors derived from combinations of plasmids and phage DNA, viral DNA such as vaccinia, adenovirus, fowl pox virus, and pseudorabies. However, any other vector or plasmid may be used as long as they are replicable and viable in the host.

The appropriate DNA sequence may be inserted into the vector by a variety of procedures. In general, the DNA sequence is inserted into an appropriate restriction endonuclease sites by procedures known in the art. Such procedures and others are deemed to be within the scope of those skilled in the art. The DNA sequence in the expression vector is operatively linked to an appropriate expression control sequence(s) (promoter) to direct mRNA synthesis. As representative examples of such promoters, there may be mentioned: LTR or SV40 promoter, the E. coli. lac or trp, the phage lambda P_L promoter and other promoters known to control expression of genes in prokaryotic or eukaryotic cells or their viruses. The expression vector also contains a ribosome binding site for translation initiation and a transcription terminator. The vector may also include appropriate sequences for amplifying expression. In addition, the expression vectors preferably contain a gene to provide a phenotypic trait for selection of transformed host cells such as dihydrofolate reductase or neomycin resistance for eukaryotic cell culture, or such as tetracycline or ampicillin resistance in E. coli. The vector containing the appropriate DNA sequence as herein above described, as well as an appropriate promoter or control sequence, may be employed to transform an appropriate host to permit the host to express the protein. As representative examples of appropriate hosts, there may be mentioned: bacterial cells, such as E. coli, Salmonella typhimurium, Streptomyces; fungal cells, such as yeast; insect cells, such as Drosophila S2 and Spodoptera Sf9; animal cells such as CHO, COS or Bowes melanoma; plant cells, etc. The selection of an appropriate host is deemed to be within the scope of those skilled in the art from the teachings herein. The present invention also includes recombinant constructs comprising one or more of the sequences as broadly described above. The constructs comprise a vector, such as a plasmid or viral vector, into which a sequence ofthe invention has been inserted, in a forward or reverse orientation. In a preferred aspect of this embodiment, the construct further comprises regulatory sequences, including, for example, a promoter, operably linked to the sequence. Large numbers of suitable vectors and promoters are known to those of skill in the art, and are commercially available. The following vectors are provided by way of example. Bacterial: ρQE70, pQE60, pQE-9 (Qiagen), pBS, phagescript, psiX174, pBluescript SK, pBsKS, pNH8a, pNH16a, pNH18a, pNH46a (Stratagene); pTRC99A, pKK223-3, pKK233-3, pDR540, PRIT5 (Pharmacia). Eukaryotic: pWLneo, pSV2cat, pOG44, pXTl, pSG (Stratagene) pSNK3, pBPV, pMSG, PSVL (Pharmacia). However, any other plasmid or vector may be used as long as they are replicable and viable in the host. Promoter regions can be selected from any desired gene using CAT(chloramphenicol transferase) vectors or other vectors with selectable markers. Two appropriate vectors are pKK232-8 and pCM7.

Particular named bacterial promoters include laci, lacZ, T3, T7, gpt, lambda P_R, P and trp. Eukaryotic promoters include CMV immediate early, HSV thymidine kinase, early and late SV40, LTRs from retro virus, and mouse metallothionein-I. Selection ofthe appropriate vector and promoter is well within the level of ordinary skill in the art. The present invention also relates to host cells containing the above-described construct. The host cell can be a higher eukaryotic cell, such as a mammalian cell, or a lower eukaryotic cell, such as a yeast cell, or the host cell can be a prokaryotic cell, such as a bacterial cell. Introduction ofthe construct into the host cell can be effected by calcium phosphate or liposome-based transfection, DEAE-Dextran mediated transfection, or electroporation (Davis, L., Dibner, M., Battey, I., Basic Methods in Molecular Biology, (1986)). The constructs in host cells can be used in a conventional manner to produce the gene product encoded by the recombinant sequence. Alternatively, the polypeptides ofthe invention can be synthetically produced by conventional peptide synthesizers.

Mature proteins can be expressed in mammalian cells, yeast, bacteria, or other cells under the control of appropriate promoters. Cell-free translation systems can also be employed to produce such proteins using RΝAs derived from the DΝA constructs ofthe present invention. Appropriate cloning and expression vectors for use with prokaryotic and eukaryotic hosts are described by Sambrook, et al., Molecular Cloning: A Laboratory Manual, Second Edition, (Cold Spring Harbor, Ν.Y., 1989), the disclosure of which is hereby incorporated by reference.

Transcription of a DNA encoding the polypeptides ofthe present invention by higher eukaryotes is increased by inserting an enhancer sequence into the vector. Enhancers are cis- acting elements of DNA, usually from about 10 to 300 bp, that act on a promoter to increase its transcription. Examples include the SV40 enhancer on the late side ofthe replication origin (bp 100 to 270), a cytomegalo virus early promoter enhancer, a polyoma enhancer on the late side ofthe replication origin, and adenovirus enhancers. Generally, recombinant expression vectors will include origins of replication and selectable markers permitting transformation ofthe host cell, e.g., the ampicillin resistance gene of E. coli and S. cerevisiae TRP1 gene, and a promoter derived from a highly-expressed gene to direct transcription of a downstream structural sequence. Such promoters can be derived from operons encoding glycolytic enzymes such as 3-phosphoglycerate kinase (PGK), α factor, acid phosphatase, or heat shock proteins, among others. The heterologous structural sequence is assembled in appropriate phase with translation, initiation and termination sequences, and preferably, a leader sequence capable of directing secretion of translated protein into the periplasmic space or extracellular medium. Optionally, the heterologous sequence can encode a fusion protein including an N-terminal identification peptide imparting desired characteristics, e.g., stabilization or simplified purification of expressed recombinant product. Useful expression vectors for bacterial use are constructed by inserting a structural

DNA sequence encoding a desired protein together with suitable translation, initiation and termination signals in operable reading phase with a functional promoter. The vector will comprise one or more phenotypic selectable markers and an origin of replication to ensure maintenance ofthe vector and to, if desirable, provide amplification within the host. Suitable prokaryotic hosts for transformation include E.coli, Bacillus subtilis, Salmonella typhimurium and various species within the genera Pseudomonas, Streptomyces, and Staphylococcus, although others may also be employed as a matter of choice. Useful expression vectors for bacterial use can comprise a selectable marker and bacterial origin of replication derived from commercially available plasmids comprising genetic elements ofthe well known cloning vector pBR322 (ATCC 37017). Such commercial vectors include, for example, pKK223-3 (Pharmacia Fine Chemicals, Uppsala, Sweden) and GEM1 (Promega Biotec, Madison, Wis., USA). These pBR322 "backbone" sections are combined with an appropriate promoter and the structural sequence to be expressed.

After transformation of a suitable host strain and growth ofthe host strain to an appropriate cell density, the selected promoter is derepressed by appropriate means (e.g., temperature shift or chemical induction) and cells are cultured for an additional period. Cells are typically harvested by centrifugation, disrupted by physical or chemical means, and the resulting crude extract retained for further purification. Microbial cells employed in expression of proteins can be disrupted by any convenient method, including freeze-thaw cycling, sonication, mechanical disruption, or use of cell lysing agents.

Various mammalian cell culture systems can also be employed to express recombinant protein. Examples of mammalian expression systems include the COS-7 lines of monkey kidney fibroblasts, described by Gluzman, Cell 23: 175 (1981), and other cell lines capable of expressing a compatible vector, for example, the C127, 3T3, CHO, HeLa and BHK cell lines. Mammalian expression vectors will comprise an origin of replication, a suitable promoter and enhancer, and also any necessary ribosome binding sites, polyadenylation site, splice donor and acceptor sites, transcriptional termination sequences, and 5' flanking nontranscribed sequences. DNA sequences derived from the SV40 viral genome, for example, SV40 origin, early promoter, enhancer, splice, and polyadenylation sites may be used to provide the required non-transcribed genetic elements.

The polypeptide ofthe present invention may be recovered and purified from recombinant cell cultures by methods used heretofore, including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxyapatite chromatography and lectin chromatography. Protein refolding steps can be used, as necessary, in completing configuration ofthe mature protein. Finally, high performance liquid chromatography (HPLC) can be employed for final purification steps.

The polypeptide ofthe present invention may be a naturally purified product, or a product of chemical synthetic-procedures, or produced by recombinant techniques from a prokaryotic or eukaryotic host (for example, by bacterial, yeast, higher plant, insect and mammalian cells in culture). Depending upon the host employed in a recombinant production procedure, the polypeptides ofthe present invention may be glycosylated with mammalian or other eukaryotic carbohydrates or may be non-glycosylated. Polypeptides of the invention may also include an initial methionine amino acid residue.

The polypeptide ofthe present invention, as a result of its ability to modulate surfactant production, may be employed in treatment of various disease or clinical conditions where surfactant production is relevant to the causation or treatment ofthe disease or clinical condition, such as various lung diseases, including adult/infant respiratory distress syndrome, acute lung injury, chronic obstructive pulmonary disease (emphysema and chronic bronchitis), asthma, small airway disease and maintaining airway patency, and related pulmonary conditions.

This invention provides a method of screening compounds to identify those that modulate the action ofthe polypeptide ofthe present invention. This method comprises incubating the FAS polypeptides or a cell transfected with cDNA encoding FAS under conditions sufficient to allow the components to interact, and then measuring the effect ofthe compound or composition on FAS activity. Both agonist and antagonist compounds may be identified by this procedure; however agonist compounds are preferred.

Potential agonist and antagonist compounds include small molecules which stimulate or inhibit FAS polypeptides which in turn stimulates or inhibits surfactant production.

Examples of small molecules include, but are not limited to, small peptides or peptide-like molecules.

Potential antagonist compounds further include antisense constructs prepared using antisense technology. Antisense technology can be used to control gene expression through triple-helix formation or antisense DNA or RNA, both of which methods are based on binding of a polynucleotide to DNA or RNA. For example, the 5' coding portion ofthe polynucleotide sequence, which encodes for the mature polypeptides ofthe present invention, is used to design an antisense RNA oligonucleotide of from about 10 to 40 base pairs in length. A DNA oligonucleotide is designed to be complementary to a region ofthe gene involved in transcription (triple helix—see Lee et al., Nucl. Acids Res. 6:3073 (1979); Cooney et al, Science, 241:456 (1988); and Dervan et al., Science, 251: 1360 (1991)), thereby preventing transcription and the production ofthe polypeptides ofthe present invention. The antisense RNA oligonucleotide hybridizes to the mRNA in vivo and blocks translation ofthe mRNA molecule into the polypeptide (Antisense— Okano, J. Neurochem., 56:560 (1991); Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton, Fla. (1988)). The oligonucleotides described above can also be delivered to cells such that the antisense RNA or DNA may be expressed in vivo to inhibit production ofthe polypeptide. Additional antagonist compounds include ribozymes. Ribozymes are RNA molecules with catalytic activity. See, e.g., Cech, Science 236, 1532-1539; 1987; Cech, Ann. Rev. Biochem. 59, 543-568; 1990, Cech, Curr. Opin. Struct. Biol. 2, 605-609; 1992, Couture & Stinchcomb, Trends Genet. 12, 510-515, 1996. Ribozymes can be used to inhibit gene function by cleaving an RNA sequence, as is known in the art (e.g., Haseloff et al., U.S. Patent 5,641,673). The mechanism of ribozyme action involves sequence-specific hybridization ofthe ribozyme molecule to the complementary target RNA, followed by endonucleolytic cleavage, thereby preventing transcription and the production ofthe polypeptides ofthe present invention. Examples include engineered hammerhead motif ribozyme molecules that can specifically and efficiently catalyze endonucleolytic cleavage of specific nucleotide sequences.

The polypeptides, agonists and antagonists ofthe present invention may be employed in combination with a suitable pharmaceutical carrier to comprise a pharmaceutical composition for parenteral administration. Such compositions comprise a therapeutically effective amount ofthe polypeptide, agonist or antagonist and a pharmaceutically acceptable carrier or excipient. Such a carrier includes but is not limited to saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof. The formulation should suit the mode of administration. The invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more ofthe ingredients ofthe pharmaceutical compositions ofthe invention. Associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration. In addition, the polypeptides, agonists and antagonists ofthe present invention may be employed in conjunction with other therapeutic compounds.

The pharmaceutical compositions may be administered in a convenient manner such as by the oral, topical, sublingual, intratracheal, intravenous, intraperitoneal, intramuscular, subcutaneous, intranasal or intradermal routes. The pharmaceutical compositions can be administered in an amount which is effective for treating and/or prophylaxis ofthe specific indication. In general, they are administered in an amount of at least about 10 μg/kg body weight and in most cases they will be administered in an amount not in excess of about 10 mg/Kg body weight per day. In most cases, the dosage is from about 0.1 μg/kg to about 100 mg/kg body weight daily, taking into account the routes of administration, symptoms, etc. In the specific case of topical administration, dosages are preferably administered from about 0.1 μg to 9 mg per cm².

The FAS polypeptides ofthe invention and agonist and antagonist polypeptides capable of modulating FAS activity may be employed in accordance with the present invention by expression of such polypeptide in vivo, which is often referred to as "gene therapy." Thus, for example, cells may be engineered with a polynucleotide (DNA or RNA) encoding for the polypeptide ex vivo, the engineered cells are then provided to a patient to be treated with the polypeptide. Such methods are well-known in the art. For example, cells may be engineered by procedures known in the art by use of a retro viral particle containing RNA encoding for the FAS polypeptides ofthe present invention, or agonist and antagonist polypeptides capable of modulating FAS activity.

Similarly, cells may be engineered in vivo for expression ofthe polypeptide in vivo, for example, by procedures known in the art. As known in the art, a producer cell for producing a retroviral particle containing RNA encoding the polypeptide ofthe present invention may be administered to a patient for engineering cells in vivo and expression ofthe polypeptide in vivo. These and other methods for administering a polypeptide ofthe present invention by such methods should be apparent to those skilled in the art from the teachings of the present invention. For example, the expression vehicle for engineering cells may be other than a simple retroviral particle, for example, a lentivirus, an adenovirus or an adeno- associated virus, which may be used to engineer cells in vivo after combination with a suitable delivery vehicle.

Retroviruses from which the retroviral plasmid vectors hereinabove mentioned may be derived include, but are not limited to, B-type viruses represented by the mouse mammary tumor virus as well as mammalian C-type retroviruses such as Moloney murine leukemia virus, spleen necrosis virus, gibbon ape leukemia virus, Harvey sarcoma virus and myeloproliferative sarcoma virus. In addition, plasmid vectors may be based on avian sarcoma/leukemia viruses such as Rous sarcoma virus and avian leukosis virus. Retroviral constructs may also be generated from lentiviruses exemplified by the human immunodeficiency virus and spumaretroviruses such as the human foamy virus.

The vector includes one or more promoters. Suitable promoters which may be employed include, but are not limited to, the retroviral LTR; the SV40 promoter; and the human cytomegalovirus (CMV) promoter described in Miller, et al., Biotechniques, 7(9): 980-990 (1989), the T7 promoter, or RSV promoter, or any other promoter (e.g., cellular promoters such as eukaryotic cellular promoters including, but not limited to, the histone, pol IH, and β-actin promoters). Other viral promoters which may be employed include, but are not limited to, adenovirus promoters, thymidine kinase (TK) promoters, and B19 parvo virus promoters. The selection of a suitable promoter will be apparent to those skilled in the art from the teachings contained herein.

The nucleic acid sequences encoding the polypeptides ofthe present invention are under the control of a suitable promoter. Suitable promoters which may be employed include, but are not limited to, adenoviral promoters, such as the adenoviral major late promoter; or hetorologous promoters, such as the cytomegalovirus (CMV) promoter; the respiratory syncytial virus (RS V) promoter; the albumin promoter; the ApoAI promoter; human globin promoters; viral thymidine kinase promoters, such as the Herpes Simplex thymidine kinase promoter; retroviral LTRs (including the modified retroviral LTRs hereinabove described); the β-actin promoter; and human growth hormone promoters. Potentially useful inducible promoters include the lac operator-repressor system, the human and mouse metallothionein promoter, the heat-shock-inducible promoter, the tetR-based promoter and the mifepristone- activated promoter. The promoter also may be the native promoter which controls the gene encoding the polypeptide.

The retroviral plasmid vector is employed to transduce packaging cell lines to form producer cell lines. Examples of packaging cells which maybe transfected include, but are not limited to, the PE501, PA317, ψ-2, ψ-AM, PA12, T19-14X, VT-19-17-H2, ψCRE, ψCRIP, GP+E-86,GP+envAml2, and DAN cell lines as described in Miller, Human Gene Therapy, 1: 5-14 (1990), which is incorporated herein by reference in its entirety. The vector may transduce the packaging cells through any means known in the art. Such means include, but are not limited to, electroporation, the use of liposomes, and CaPO₄ precipitation. In one alternative, the retroviral plasmid vector may be encapsulated into a liposome, or coupled to a lipid, and then administered to a host. The producer cell line generates infectious retroviral vector particles which include the nucleic acid sequence(s) encoding the polypeptides. Such retroviral vector particles then may be employed, to transduce eukaryotic cells, either in vitro or in vivo. The transduced eukaryotic cells will express the nucleic acid sequence(s) encoding the polypeptide. Eukaryotic cells which may be transduced include, but are not limited to, embryonic stem cells, embryonic carcinoma cells, as well as hematopoietic stem cells, hepatocytes, fibroblasts, myoblasts, keratinocytes, endothelial cells, and bronchial epithelial cells.

This invention is also related to the use ofthe genes ofthe present invention as part of a diagnostic assay for detecting diseases or susceptibility to diseases related to the presence of mutations in the nucleic acid sequences encoding the polypeptide ofthe present invention. Individuals carrying mutations in a gene ofthe present invention may be detected at the DNA level by a variety of techniques. Nucleic acids for diagnosis may be obtained from a patient's cells, such as from blood, urine, saliva, tissue biopsy and autopsy material. The genomic DNA may be used directly for detection or may be amplified enzymatically by using PCR (Saiki et al., Nature, 324: 163-166 (1986)) prior to analysis. RNA or cDNA may also be used for the same purpose. As an example, PCR primers complementary to the nucleic acid encoding a polypeptide ofthe present invention can be used to identify and analyze mutations. For example, deletions and insertions can be detected by a change in size ofthe amplified product in comparison to the normal genotype. Point mutations can be identified by hybridizing amplified DNA to radiolabeled RNA or alternatively, radiolabeled antisense DNA sequences. Perfectly matched sequences can be distinguished from mismatched duplexes by RNase A digestion or by differences in melting temperatures.

Genetic testing based on DNA sequence differences may be achieved by detection of alteration in electrophoretic mobility of DNA fragments in gels with or without denaturing agents. Small sequence deletions and insertions can be visualized by high resolution gel electrophoresis. DNA fragments of different sequences may be distinguished on denaturing formamide gradient gels in which the mobilities of different DNA fragments are retarded in the gel at different positions according to their specific melting or partial melting temperatures (see, e.g., Myers et al., Science, 230:1242 (1985)). Sequence changes at specific locations may also be revealed by nuclease protection assays, such as RNase and Si protection or the chemical cleavage method (e.g., Cotton et al., PNAS, USA, 85:4397-4401 (1985)). Thus, the detection of a specific DNA sequence may be achieved by methods such as hybridization, RNase protection, chemical cleavage, direct DNA sequencing or the use of restriction enzymes, (e.g., Restriction Fragment Length Polymorphisms (RFLP)) and Southern blotting of genomic DNA. In addition to more conventional gel-electrophoresis and DNA sequencing, mutations can also be detected by in situ analysis.

The present invention also relates to a diagnostic assay for detecting altered levels of FAS proteins in various tissues since an over-expression or under-expression ofthe proteins compared to normal control tissue samples may detect the presence of abnormal surfactant production, which in turn may indicate the presence of certain lung diseases. Assays used to detect levels of protein in a sample derived from a host are well-known to those of skill in the art and include radioimmunoassays, competitive-binding assays, Western Blot analysis, ELISA assays and "sandwich" type assays. An ELISA assay (Coligan, et al., Current Protocols in Immunology, 1(2), Chapter 6, (1991)) initially comprises preparing an antibody specific to an antigen to the polypeptides ofthe present invention, preferably a monoclonal antibody. In addition a reporter antibody is prepared against the monoclonal antibody. To the reporter antibody is attached a detectable reagent such as radioactivity, fluorescence or, in this example, a horseradish peroxidase enzyme. A sample is removed from a host and incubated on a solid support, e.g. a polystyrene dish, that binds the proteins in the sample. Any free protein binding sites on the dish are then covered by incubating with a non-specific protein like bovine serum albumen. Next, the monoclonal antibody is incubated in the dish during which time the monoclonal antibodies attach to any polypeptides ofthe present invention attached to the polystyrene dish. All unbound monoclonal antibody is washed out with buffer. The reporter antibody linked to horseradish peroxidase is now placed in the dish resulting in binding ofthe reporter antibody to any monoclonal antibody bound to the protein of interest. Unattached reporter antibody is then washed out. Peroxidase substrates are then added to the dish and the amount of color developed in a given time period is a measurement ofthe amount of a polypeptide ofthe present invention present in a given volume of patient sample when compared against a standard curve.

A competition assay may be employed wherein antibodies specific to a polypeptide of the present invention are attached to a solid support and labeled FAS and a sample derived from the host are passed over the solid support and the amount of label detected, for example by liquid scintillation chromatography, can be correlated to a quantity of a polypeptide ofthe present invention in the sample.

A "sandwich" assay is similar to an ELISA assay. In a "sandwich" assay a polypeptide ofthe present invention is passed over a solid support and binds to antibody attached to a solid support. A second antibody is then bound to the polypeptide of interest. A third antibody which is labeled and specific to the second antibody is then passed over the solid support and binds to the second antibody and an amount can then be quantified.

The sequences ofthe present invention are also valuable for chromosome identification. The sequence is specifically targeted to and can hybridize with a particular location on an individual human chromosome. Moreover, there is a current need for identifying particular sites on the chromosome. Few chromosome marking reagents based on actual sequence data (repeat polymorphisms) are presently available for marking chromosomal location. The mapping of DNAs to chromosomes according to the present invention is an important first step in correlating those sequences with genes associated with disease. Briefly, sequences can be mapped to chromosomes by preparing PCR primers (preferably 15-25 bp) from the cDNA. Computer analysis ofthe 3' untranslated region is used to rapidly select primers that do not span more than one exon in the genomic DNA, thus complicating the amplification process. These primers are then used for PCR screening of somatic cell hybrids containing individual human chromosomes. Only those hybrids containing the human gene corresponding to the primer will yield an amplified fragment.

PCR mapping of somatic cell hybrids is a rapid procedure for assigning a particular DNA to a particular chromosome. Using the present invention with the same oligonucleotide primers, sub-localization can be achieved with panels of fragments from specific chromosomes or pools of large genomic clones in an analogous manner. Other mapping strategies that can similarly be used to map to its chromosome include in situ hybridization, prescreening with labeled flow-sorted chromosomes and preselection by hybridization to construct chromosome specific-cDNA libraries.

Fluorescence in situ hybridization (FISH) of a cDNA clone to a metaphase chromosomal spread can be used to provide a precise chromosomal location in one step. This technique can be used with cDNA as short as 50 or 60 bases. For a review of this technique, see Verma et al., Human Chromosomes: a Manual of Basic Techniques, Pergamon Press, New York (1988).

Once a sequence has been mapped to a precise chromosomal location, the physical position ofthe sequence on the chromosome can be correlated with genetic map data. Such data are found, for example, in V. McKusick, Mendelian Inheritance in Man (available on line through ohns Hopkins University Welch Medical Library). The relationship between genes and diseases that have been mapped to the same chromosomal region are then identified through linkage analysis (coinheritance of physically adjacent genes). Next, it is necessary to determine the differences in the cDNA or genomic sequence between affected and unaffected individuals. If a mutation is observed in some or all ofthe affected individuals but not in any normal individuals, then the mutation is likely to be the causative agent ofthe disease.

With current resolution of physical mapping and genetic mapping techniques, a cDNA precisely localized to a chromosomal region associated with the disease could be one of between 50 and 500 potential causative genes. (This assumes 1 megabase mapping resolution and one gene per 20kb). The polypeptides, their fragments or other derivatives, or analogs thereof, or cells expressing them can be used as an immunogen to produce antibodies thereto. These antibodies can be, for example, polyclonal or monoclonal antibodies. The present invention also includes chimeric, single chain, and humanized antibodies, as well as Fab fragments, or the product of an Fab expression library. Various procedures known in the art may be used for the production of such antibodies and fragments.

Antibodies generated against the polypeptides corresponding to a sequence ofthe present invention can be obtained by direct injection ofthe polypeptides into an animal or by administering the polypeptides to an animal, preferably a nonhuman. The antibody so obtained will then bind the polypeptides itself. In this manner, even a sequence encoding only a fragment ofthe polypeptides can be used to generate antibodies binding the whole native polypeptides. Such antibodies can then be used to isolate the polypeptide from tissue expressing that polypeptide.

For preparation of monoclonal antibodies, any technique which provides antibodies produced by continuous cell line cultures can be used. See generally Monoclonal Antibodies, a Laboratory Manual, Harlow and Lane, eds. 1988. Examples include the hybridoma technique (Kohler and Milstein, Nature, 256:495-497 (1975)), the trioma technique, the human B-cell hybridoma technique (Kozbor et al., Immunology Today, 4:72 (1983)), and the EBV-hybridoma technique to produce human monoclonal antibodies (Cole, et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96 (1985)).

Techniques described for the production of single chain antibodies (U.S. Pat. No. 4,946,778) can be adapted to produce single chain antibodies to immunogenic polypeptide products of this invention. Also, transgenic mice may be used to express humanized antibodies to immunogenic polypeptide products of this invention. Humanized antibodies may also be produced by methods described in U.S. Pat. Nos. 5,530,101; 5,585,089; 5,693,761; and 5,693,762, incoφorated herein by reference.

The present invention will be further described with reference to the following examples; however, it is to be understood that the present invention is not limited to such examples. All parts or amounts, unless otherwise specified, are by weight.

Example 1. Differential gene expression by rat alveolar type II cells using Affymetrix GeneChip technology Cell culture and KGF Treatment Methods:

Primary rat type II cells were cultured on a substratum composed on extracellular matrix from Engelbreth-Holm-Swarm (EHS) tumors and type I collagen. Cells underwent either a short (l-24h) treatment with KGF (20ng/ml) or a longer (7 days) treatment with KGF (10 ng/ml), KGF + 5% rat serum, KGF + 5% rat serum + lOnM dexamethasone (Dex), or 5% rat serum.

RN4 Preparation Methods:

Total RΝA was isolated from the cells using TRIzol Reagent (cat. No. 15596) from Gibco BRL/Life Technologies (Rockville, MD) according to the manufacturers instructions which are based on the method of Chomczynski and Sacchi (Anal. Biochem. 162:156, 1987). Poly A+ RNA was then isolated from the total RNA using FastTrack 2.0 Kit (cat. No. K1593- 02) from Invitrogen (Carlsbad, CA) according to the manufacturers protocol which utilizes an oligo dT cellulose extraction purification procedure.

Isolated RNA was used to generate first and second strand cDNA (complimentary DNA) using the Superscript Choice kit (cat. No. 18090-019) from Gibco BRL/Life Technologies according to the manufacturers instructions. The double stranded cDNA was then purified using a Phase Lock Gel kit (cat. No. pl-188233) from Eppendorf-5 Prime, Inc.(Boulder, CO).

Purified double-stranded cDNA was then used to generate biotin-labeled cRNA (complimentary RNA) by in vitro transcription using a BioArray High Yield RNA Transcript Labeling kit (cat. No. 900182) from Enzo Biochem (New York, NY) according to the instructions ofthe manufacturer. The resulting in vitro transcription products were then purified using a RNAeasy Mini Kit (Cat. No. 74104) from Qiagen (Valencia, CA) according to the manufacturers protocol and fragmented by heating at 94°C for 35 minutes.

Hybridization Method:

Array hybridization was performed using a GeneChip Eukaryotic Hybrization Control Kit (cat no. 900299) from Affymetrix (Santa Clara, CA). Briefly, the hybridization cocktail containing fragmented cRNA, control oligonucleotides, herring sperm DNA, acetylated bovine serum albumin, and MES hybridization buffer was loaded onto the GeneChip and incubated at 45 °C for 16 hours followed by washing at high stringency. The quality ofthe cRNA was first evaluated by control hybridization to Affymetrix Test Chips containing probes built to match the 5', middle, and 3' sequences of housekeeping genes. Fluorescently labeled cRNA samples from cell preparations were then hybridized to Affymetrix rat U34 oligonucleotide arrays containing approximately 7000 full length genes and 17000 ESTs. All chips were scanned using Affymetrix software. Analysis was performed using the GeneSpring 3.2.8 software from Silicon Genetics (Redwood City, CA). Each chip was normalized to itself.

Short term KGF (20 ng/ml) treatment of cells increased FAS expression by over 2 fold, specifically at lhour of treatment (FIG. 3A). After seven days of treatment, KGF (10 ng/ml) treatment of cells also increased FAS expression, an effect that may have been enhanced by the addition of dexamethasone, but not serum (FIG. 3B).

Claims

CLAIMSWhat is claimed is:

1. A method for modulating surfactant production comprising administering to a mammal a therapeutically effective dose of an isolated FAS polypeptide, a fragment, variant, derivative or analog thereof.

2. The method of claim 1, wherein the FAS polypeptide is human FAS (SEQ ID NO:2).

3. The method of claim 1, wherein the FAS polypeptide has an amino acid sequence which is at least 60% identical to SEQ ID NO:2.

4. The method of claim 1, wherein the FAS polypeptide has an amino acid sequence which is at least 70% identical to SEQ ID NO:2.

5. The method of claim 1, wherein the FAS polypeptide has an amino acid sequence which is at least 90% identical to SEQ ID NO:2.

6. The method of claim 1, wherein the mammal is human.

7. The method of claim 1, wherein the administering is performed in vivo.

8 . The method of claim 1, further comprising administering a pharmaceutical carrier.

9. The method of claim 7, further comprising preparing a recombinant cell transfected with a FAS nucleic acid.

10. The method of claim 9, wherein the FAS nucleic acid is operably linked to a promoter in an expression vector.

11. The method of claim 10, wherein the expression vector is an adenoviral vector, a lentiviral vector or a retroviral vector.

12. The method of claim 1, further comprising preparing a recombinant cell transfected with a FAS nucleic acid and administering the cell to the mammal.

13. The method of claim 1, wherein the FAS polypeptide is recombinantly expressed by culturing a cell containing an FAS nucleic acid under conditions which result in expression ofthe polypeptide, and recovering the FAS polypeptide from the cell culture.

14. The method of claim 13, wherein the FAS polypeptide is expressed in E. coli.

15. The method of claim 13, wherein the FAS polypeptide is expressed mammalian cells.

16. The method of claim 13, wherein the FAS nucleic acid is operably linked to a promoter in an expression vector.

17. The method of claim 16, wherein the expression vector is an adenoviral vector, a lentiviral vector or a retroviral vector.

18. The method of claim 9, wherein the FAS nucleic acid has the sequence of human FAS (SEQ ID NO:l).

19. A method of preventing, treating or ameliorating a medical condition in a subject comprising administering to the subject a therapeutically effective amount of a FAS polypeptide, fragment, variant, derivative or analog thereof.

20. The method of claim 19, wherein the FAS polypeptide is human FAS (SEQ ID NO:2).

21. The method of claim 19 wherein the FAS polypeptide has an amino acid sequence which is at least 60%> identical to SEQ ID NO:2.

22. The method of claim 19, wherein the FAS polypeptide has an amino acid sequence which is at least 70% identical to SEQ ID NO:2.

23. The method of claim 19, wherein the FAS polypeptide has an amino acid sequence which is at least 90% identical to SEQ ID NO:2.

24. The method of claim 19, wherein the mammal is human.

25. The method of claim 19, wherein the administering is performed in vivo.

26 . The method of claim 19, further comprising administering a pharmaceutical carrier.

27. The method of claim 25, further comprising preparing a recombinant cell transfected with a FAS nucleic acid.

28. The method of claim 27, wherein the FAS nucleic acid is operably linked to a promoter in an expression vector.

29. The method of claim 28, wherein the expression vector is an adenoviral vector, a lentiviral vector or a retroviral vector.

30. The method of claim 27, wherein the recombinant cell is administered to the mammal.

31. The method of claim 19, wherein the FAS polypeptide is recombinantly expressed by culturing a cell containing an FAS nucleic acid under conditions which result in expression ofthe polypeptide, and recovering the FAS polypeptide from the cell culture.

32. The method of claim 31, wherein the FAS polypeptide is expressed in E. coli.

33. The method of claim 31, wherein the FAS polypeptide is expressed mammalian cells.

34. The method of claim 31, wherein the FAS nucleic acid is operably linked to a promoter in an expression vector.

35. The method of claim 34, wherein the expression vector is an adenoviral vector, a lentiviral vector or a retroviral vector.

36. The method of claim 27, wherein the FAS nucleic acid has the sequence of human FAS (SEQ ID NO:l).

37. A method for identifying an agonist or antagonist that modulates FAS activity, the method comprising obtaining a FAS polypeptide and contacting the FAS polypeptide with different concentrations of a test agonist or antagonist and a control sample; and measuring the FAS activity with and without different concentrations of the test agonist or antagonist.

38. A method for screening drug candidate compounds that modulate surfactant production, the method comprising obtaining a FAS polypeptide and contacting the FAS polypeptide with different concentrations of a drug candidate and a control sample; and measuring the FAS activity with and without different concentrations ofthe drug candidate.

39. An agonist or antagonist to the FAS polypeptide of claim 1.