Compositions and methods relating to colon specific genes and proteins

The present invention relates to newly identified nucleic acids and polypeptides present in normal and neoplastic colon cells, including fragments, variants and derivatives of the nucleic acids and polypeptides. The present invention also relates to antibodies to the polypeptides of the invention, as well as agonists and antagonists of the polypeptides of the invention. The invention also relates to compositions comprising the nucleic acids, polypeptides, antibodies, variants, derivatives, agonists and antagonists of the invention and methods for the use of these compositions. These uses include identifying, diagnosing, monitoring, staging, imaging and treating colon cancer and non-cancerous disease states in colon tissue, identifying colon tissue, monitoring and identifying and/or designing agonists and antagonists of polypeptides of the invention. The uses also include gene therapy, production of transgenic animals and cells, and production of engineered colon tissue for treatment and research.

EXAMPLES 
 Example 1 
 Gene Expression Analysis CSGs were identified by MRNA subtraction analysis using standard methods. The sequences were extended using GeneBank sequences, Incyte's proprietary database. From the nucleotide sequences, predicted amino acid sequences were prepared. DEX0289 — 1, DEX0289 — 2 correspond to SEQ ID NO.1, 2 etc. DEX0133 was the parent sequence found in the mRNA subtractions. 2 DEX0289_1 DEX0133_1  DEX0289_75 DEX0289_2 DEX0133_2  DEX0289 —76 DEX0289_3 flex DEX0133_2 DEX0289_4 DEX0133_3  DEX0289_77 DEX0289_5 flex DEX0133_3  DEX0289_78 DEX0289_6 DEX0133_4 DEX0289_7 DEX0133_5  DEX0289_79 DEX0289_8 DEX0133_6  DEX0289_80 DEX0289_9 flex DEX0133_6 DEX0289_10 DEX0133_7  DEX0289_81 DEX0289_11 flex DEX0133_7 DEX0289_12 DEX0133_8  DEX0289_82 DEX0289_13 flex DEX0133_8 DEX0289_14 DEX0133_9  DEX0289_83 DEX0289_15 flex DEX0133_9  DEX0289_84 DEX0289_16 DEX0133_10  DEX0289_85 DEX0289_17 flex DEX0133_10 DEX0289_18 DEX0133_11  DEX0289_86 DEX0289_19 flex DEX0133_11 DEX0289_20 DEX0133_12  DEX0289_87 DEX0289_21 flex DEX0133_12 DEX0289_22 DEX0133_13  DEX0289_88 DEX0289_23 DEX0133_14  DEX0289_89 DEX0289_24 flex DEX0133_14 DEX0289_25 DEX0133_15  DEX0289_90 DEX0289_26 flex DEX0133_15  DEX0289_91 DEX0289_27 DEX0133_17  DEX0289_92 DEX0289_28 flex DEX0133_17  DEX0289_93 DEX0289_29 DEX0133_19  DEX0289_94 DEX0289_30 flex DEX0133_19 DEX0289_31 DEX0133_20  DEX0289_95 DEX0289_32 flex DEX0133_20 DEX0289_33 DEX0133_21  DEX0289_96 DEX0289_34 flex DEX0133_21  DEX0289_97 DEX0289_35 DEX0133_22  DEX0289_98 DEX0289_36 flex DEX0133_22 DEX0289_37 DEX0133_24  DEX0289_99 DEX0289_38 flex DEX0133_24 DEX0289_39 DEX0133_25  DEX0289_100 DEX0289_40 flex DEX0133_25 DEX0289_41 DEX0133_26  DEX0289_101 DEX0289_42 flex DEX0133_26 DEX0289_43 DEX0133_27  DEX0289_102 DEX0289_44 flex DEX0133_27 DEX0289_45 DEX0133_28 DEX0289_46 DEX0133_29 DEX0289_103 DEX0289_47 flex DEX0133_29 DEX0289_48 DEX0133_30  DEX0289_104 DEX0289_49 flex DEX0133_30  DEX0289_105 DEX0289_50 DEX0133_33  DEX0289_106 DEX0289_51 DEX0133_34  DEX0289_107 DEX0289_52 flex DEX0133_34  DEX0289_108 DEX0289_53 DEX0133_35  DEX0289_109 DEX0289_54 flex DEX0133_35 DEX0289_55 DEX0133_36  DEX0289_110 DEX0289_56 flex DEX0133_36  DEX0289_111 DEX0289_57 DEX0133_37  DEX0289_112 DEX0289_58 flex DEX0133_37  DEX0289_113 DEX0289_59 DEX0133_38  DEX0289_114 DEX0289_60 flex DEX0133_38 DEX0289_61 DEX0133_39  DEX0289_115 DEX0289_62 flex DEX0133_39 DEX0289_63 DEX0133_41  DEX0289_116 DEX0289_64 flex DEX0133_41 DEX0289_65 DEX0133_42  DEX0289_117 DEX0289_66 flex DEX0133_42  DEX0289_118 DEX0289_67 DEX0133_43  DEX0289_119 DEX0289_68 DEX0133_44  DEX0289_120 DEX0289_69 flex DEX0133_44  DEX0289_121 DEX0289_70 DEX0133_45  DEX0289_122 DEX0289_71 flex DEX0133_45 DEX0289_72 DEX0133_46  DEX0289_123 DEX0289_73 flex DEX0133_46 DEX0289_74 CLN113  DEX0289_124 The expression levels from the lncyte LifeSeq database are listed below: 3 DEX0289_10 SEQ ID NO:10 MAM .0019 LIV .0019 KID .0026 PRO .0028 DEX0289_11 SEQ ID NO:11 MAM .0019 LIV .0019 KID .0026 PRO .0028 DEX0289_13 SEQ ID NO:13 BRN .0002 BRN .0002 CON .0011 CON .0011 DEX0289_14 SEQ ID NO:14 KID .0103 BON .0169 OVR .0195 DEX0289_15 SEQ ID NO:15 KID .0103 BON .0169 OVR .0195 DEX0289_18 SEQ ID NO:18 PRO .0006 DEX0289_19 SEQ ID NO:19 PRO .0006 DEX0289_20 SEQ ID NO:20 NOS .022 INL .0224 DEX0289_22 SEQ ID NO:22 PAN .0012 BLD .0016 BMR .0064 DEX0289 25 SEQ ID NO:25 KID .009 LIV .0151 BON .0169 ESO .0204 DEX0289_26 SEQ ID NO:26 KID .009 LIV .0151 BON .0169 ESO .0204 DEX0289_3 SEQ ID NO:3 OVR .0041 KID .0051 PRO .0056 THR .0068 DEX0289_33 SEQ ID NO:33 LIV .0057 SPL .0063 UNC .008 OVR .0082 DEX0289_34 SEQ ID NO:34 LIV .0057 SPL .0063 UNC .008 OVR .0082 DEX0289 35 SEQ ID NO:35 THR .0091 UTR .0132 TON .0299 DEX0289_36 SEQ ID NO:36 THR .0091 UTR .0132 TON .0299 DEX0289_37 SEQ ID NO:37 THR .0091 BMR .0129 LMN .0139 DEX0289_39 SEQ ID NO:39 INL .0006 GLB .0185 DEX0289_4 SEQ ID NO:4 ADR .0015 DEX0289 40 SEQ ID NO:40 INL .0006 GLB .0185 DEX0289_43 SEQ ID NO:43 LMN .0028 UNC .004 LIV .0057 INT .015 DEX0289_44 SEQ ID NO:44 LMN .0028 UNC .004 LIV .0057 INT .015 DEX0289_46 SEQ ID NO:46 INS .001 INS .001 UTR .0013 BLV .0016 DEX0289_47 SEQ ID NO:47 INS .001 INS .001 UTR .0013 BLV .0016 DEX0289_48 SEQ ID NO:48 INL .0051 LIV .0057 DEX0289_49 SEQ ID NO:49 INL .0051 LIV .0057 DEX0289_5 SEQ ID NO:5 ADR .0015 DEX0289_51 SEQ ID NO:51 UNC .008 DEX0289_52 SEQ ID NO:52 UNC .008 DEX0289_53 SEQ ID NO:53 SAG .079 SAG .079 PIT .3246 PIT .3246 DEX0289_54 SEQ ID NO:54 SAG .079 SAG .079 PIT .3246 PIT .3246 DEX0289_55 SEQ ID NO:55 CRD .0023 TST .0027 INS .0048 CON .0068 DEX0289_59 SEQ ID NO:59 UNC .004 ESO .0051 LIV .0094 SYN .0112 DEX0289_60 SEQ ID NO:60 UNC .004 ESO .0051 LIV .0094 SYN .0112 DEX0289_61 SEQ ID NO:61 LNG .0006 DEXO2S9_62 SEQ ID NO:62 LNG .0006 DEX0289_68 SEQ ID NO:68 INS .0789 DEX0289_70 SEQ ID NO:70 OVR .0031 DEX0289_71 SEQ ID NO:71 OVR .0031 DEX0289_72 SEQ ID NO:72 PRO .0017 OVR .0021 DEX0289_74 SEQ ID NO:74 FTS .0003 CON .0011 LIV .0019 OVR .0021 DEX0289_8 SEQ ID NO:8 BRN .0004 PRO .0006 CON .0011 LIV .0019 DEX0289_9 SEQ ID NO:9 BRN .0004 PRO .0006 CON .0011 LIV .0019 Abbreviation for Tissues: BLO Blood; BRN Brain; CON Connective Tissue; CRD Heart; FTS Fetus; INL Intestine, Large; INS Intestine, Small; KID Kidney; LIV Liver; LNG Lung; MAM Breast; MSL Muscles; NRV Nervous Tissue; OVR Ovary; PRO Prostate; STO Stomach; THR Thyroid Gland; TNS Tonsil/Adenoids; UTR Uterus 
 Example 2 
 Relative Quantitation of Gene Expression Real-Time quantitative PCR with fluorescent Taqman probes is a quantitation detection system utilizing the 5′-3′ nuclease activity of Taq DNA polymerase. The method uses an internal fluorescent oligonucleotide probe (Taqman) labeled with a 5′ reporter dye and a downstream, 3′ quencher dye. During PCR, the 5′-3′ nuclease activity of Taq DNA polymerase releases the reporter, whose fluorescence can then be detected by the laser detector of the Model 7700 Sequence Detection System (PE Applied Biosystems, Foster City, Calif., USA). Amplification of an endogenous control is used to standardize the amount of sample RNA added to the reaction and normalize for Reverse Transcriptase (RT) efficiency. Either cyclophilin, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ATPase, or 18S ribosomal RNA (rRNA) is used as this endogenous control. To calculate relative quantitation between all the samples studied, the target RNA levels for one sample were used as the basis for comparative results (calibrator). Quantitation relative to the “calibrator” can be obtained using the standard curve method or the comparative method (User Bulletin &num;2: ABI PRISM 7700 Sequence Detection System). The tissue distribution and the level of the target gene are evaluated for every sample in normal and cancer tissues. Total RNA is extracted from normal tissues, cancer tissues, and from cancers and the corresponding matched adjacent tissues. Subsequently, first strand cDNA is prepared with reverse transcriptase and the polymerase chain reaction is done using primers and Taqman probes specific to each target gene. The results are analyzed using the ABI PRISM 7700 Sequence Detector. The absolute numbers are relative levels of expression of the target gene in a particular tissue compared to the calibrator tissue. One of ordinary skill can design appropriate primers. The relative levels of expression of the CSNA versus normal tissues and other cancer tissues can then be determined. All the values are compared to normal tissue (calibrator). These RNA samples are commercially available pools, originated by pooling samples of a particular tissue from different individuals. The relative levels of expression of the CSNA in pairs of matching samples and 1 cancer and 1 normal/normal adjacent of tissue may also be determined. All the values are compared to normal tissue (calibrator). A matching pair is formed by mRNA from the cancer sample for a particular tissue and mRNA from the normal adjacent sample for that same tissue from the same individual. In the analysis of matching samples, CSNAs show a high degree of tissue specificity for the tissue of interest. These results confirm the tissue specificity results obtained with normal pooled samples. Further, the level of mRNA expression in cancer samples and the isogenic normal adjacent tissue from the same individual are compared. This comparison provides an indication of specificity for the cancer stage (e.g. higher levels of mRNA expression in the cancer sample compared to the normal adjacent). Altogether, the high level of tissue specificity, plus the mRNA overexpression in matching samples tested are indicative of SEQ ID NO: 1 through 74 being diagnostic markers for cancer. Cln113; DEX0289 — 74; DEX0289 — 124; LifeSeq Gold Gene ID&num; 1394652 X73079 g456345 Human encoding Polymeric Ig receptor “Cln113” DNA sequence for Cln113 Sequence available from GenBank database Homo sapiens encoding Polymeric immunoglobulin receptor. Ac&num; X73079 DEX0289 — 74 Experiments on Cln113 are carried out using standard techniques, which are well known and routine to those of skill in the art, except where otherwise described in detail. Routine molecular biology techniques of the following example can be carried out as described in standard laboratory manuals, such as Sambrook et al., MOLECULAR CLONING: A LABORATORY MANUAL, 2nd Ed.; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989). Relative Quantitation of Gene Expression. Real-Time quantitative PCR with fluorescent Taqman probes is a quantitation detection system utilizing the 5′-3′ nuclease activity of Taq DNA polymerase. The method uses an internal fluorescent oligonucleotide probe (Taqman) labeled with a 5′ reporter dye and a downstream, 3′ quencher dye. During PCR, the 5′-3′ nuclease activity of Taq DNA polymerase releases the reporter, whose fluorescence can then be detected by the laser detector of the Model 7700 Sequence Detection System (PE Applied Biosystems, Foster City, Calif., USA). We use amplification of an endogenous control to standardize the amount of sample RNA added to the reaction and normalize for Reverse Transcriptase (RT) efficiency. We either use cyclophilin, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) or 18S ribosomal RNA (rRNA) as this endogenous control. To calculate relative quantitation between all the samples studied, we used the target RNA levels for one sample as the basis for comparative results (calibrator). Quantitation relative to the “calibrator” can be obtained using the standard curve method or the comparative method (User Bulletin &num;2: ABI PRISM 7700 Sequence Detection System). We evaluated the tissue distribution, and the level of the target gene for every example in normal and cancer tissue. Total RNA was extracted from normal tissues, cancer tissues, and from cancers and the corresponding matched adjacent tissues. Subsequently, first strand cDNA was prepared with reverse transcriptase and the polymerase chain reaction was done using primers and Taqman probe specific to each target gene. The results are analyzed using the ABI PRISM 7700 Sequence Detector. The absolute numbers are relative levels of expression of the target gene in a particular tissue compared to the calibrator tissue. Table 1. The absolute numbers are relative levels of expression of Cln113 in 12 normal different tissues. All the values are compared to normal colon (calibrator). These RNA samples are commercially available pools, originated by pooling samples of a particular tissue from different individuals. 4 Tissue NORMAL Colon-Ascending 1.00 Endometrium 0.00 Kidney 0.07 Liver 0.00 Ovary 0.00 Pancreas 0.00 Prostate 0.00 Small Intestine 0.28 Spleen 0.00 Stomach 0.30 Testis 0.00 Uterus 0.00 The relative levels of expression in Table 1 show that overall gene expression levels of Cln113 in the RNA samples from different normal tissues are low. All the normal tissues analyzed had lower levels of expression compared to normal colon which was used as a calibrator with a relative expression level of 1. These results demonstrated that Cln113 mRNA expression is highly specific for colon. The absolute numbers in Table 1 were obtained analyzing pools of samples of a particular tissue from different individuals. They can not be compared to the absolute numbers originated from RNA obtained from tissue samples of a single individual in Table 2. 5 TABLE 2 The absolute numbers are relative levels of expression of Cln113 in 48 pairs of matching samples. All the values are compared to normal colon (calibrator). A matching pair is formed by mRNA from the cancer sample for a particular tissue and mRNA from the normal adjacent sample for that same tissue from the same individual. MATCHING NORMAL Sample ID Cancer Type Tissue CANCER ADJACENT Stomach StoMT54 Stomach-1 0.12 0.00 Small SmInt21XA Small 0.05 0.00 Intestine Intestine -1 Colon- ClnAS45 Colon-1 0.00 0.23 Ascending (A) Colon-Cecum ClnCM67 Colon-2 0.29 0.00 (B) Colon-Cecum ClnB56 Colon-3 0.8 0.00 (C) Colon- ClnAS67 Colon-4 0.7 0.3 Ascending (B) Colon- ClnAS12 Colon-5 0.19 0.55 Ascending (B) Colon- ClnAS43 Colon-6 4.71 0.00 Ascending (C) Colon- ClnAS46 Colon-7 0.00 3.06 Ascending (C) Colon- ClnAS89 Colon-8 1.1 0.00 Ascending (D) Colon- ClnAS19 Colon-9 0.00 0.99 Ascending (D) Colon- ClnTX01 Colon-10 0.08 0.73 Transverse (B) Colon- ClnTX89 Colon-11 1.3 0.00 Transverse (B) Colon- ClnTX67 Colon-12 0.00 0.4 Transverse (C) Colon- ClnDC63 Colon-13 1.02 0.00 Descending (C) Colon-Sigmoid ClnSG27 Colon-14 0.35 0.41 (C) Colon-Sigmoid ClnSG20 Colon-15 1.59 1.33 (B) Colon-Sigmoid ClnSG45 Colon-16 0.00 0.9 (D) Colon- ClnB34 Colon-17 0.1 0.00 Rectosigmoid (A) Colon-Rectum ClnCXGA Colon-18 1.97 0.37 (A) Colon-Rectum ClnRC67 Colon-19 0.00 0.00 (B) Colon - ClnC9XR Colon-20 0.00 0.00 Rectosigmoid (C) Colon-Rectum ClnRC01 Colon-21 1.0 0.2 (C) Colon-Rectum ClnRC89 Colon-22 0.00 0.4 (D) Bladder Bld32XK Bladder-1 0.00 0.00 Bladder Bld46XK Bladder-2 0.00 0.00 Cervix CvxKS83 Cervix-1 0.00 0.00 Cervix CvxKS52 Cervix-2 0.00 0.00 Endometrium endo10479 Endometrium-1 0.00 0.00 Endometrium endo12XA Endometrium-2 0.00 0.0 Kidney Kid11XD Kidney-1 0.00 0.00 Kidney Kid10XD Kidney-2 0.00 0.00 Kidney Kid107XD Kidney-3 0.00 0.01 Kidney Kid109XD Kidney-4 0.19 0.11 Kidney Kid106XD Kidney-5 0.05 0.02 Liver Liv42X Liver-1 0.00 0.00 Liver Liv15XA Liver-2 0.00 0.00 Liver Liv94XA Liver-3 0.00 0.00 Lung LngAC11 Lung-1 0.00 0.00 Lung Lng90X Lung-2 0.00 0.00 Lung Lng60XL Lung-3 0.00 0.00 Lung LNG47XQ Lung-4 0.00 0.00 Mammary Gland Mam12X Mammary 0.00 0.00 gland-1 Mammary Gland Mam14DN Mammary 0.00 0.00 gland-2 Prostate Pro12B Prostate-1 0.00 0.00 Testis Tst39X Testis-1 0.00 0.00 Uterus Utr85XU Uterus-1 0.08 0.00 Uterus Utr135XO Uterus-2 0.00 0.00 0 &equals; Negative In the analysis of matching samples, the higher levels of expression were in colon showing a high degree of tissue specificity for colon tissue. These results confirm the tissue specificity results obtained with normal pooled samples (Table 1). Furthermore, we compared the level of mRNA expression in cancer samples and the isogenic normal adjacent tissue from the same individual. This comparison provides an indication of specificity for the cancer stage (e.g. higher levels of mRNA expression in the cancer sample compared to the normal adjacent). Table 2 shows overexpression of Cln113 in 10 of the colon cancer tissues compared with their respective normal adjacent (colon samples &num;2, 3, 4, 6, 8, 11, 13, 17, 18, and 21). There is overexpression in the cancer tissue for 45% of the colon matching samples tested (total of 22 colon matching samples). Altogether, the high level of tissue specificity, plus the mRNA overexpression in 45% of the colon adenocarcinoma matching samples tested are believed to make Cln113 a good diagnostic marker for colon cancer. forward: hitting DEX0289 — 74 SEQ ID NO. 74 (bp1441-1462) reverse: hitting DEX0289 — 74 SEQ ID NO. 74 (bp1603-1585) 
 Example 3 
 Protein Expression The CSNA is amplified by polymerase chain reaction (PCR) and the amplified DNA fragment encoding the CSNA is subcloned in pET-21d for expression in E. coli . In addition to the CSNA coding sequence, codons for two amino acids, Met-Ala, flanking the NH 2 -terminus of the coding sequence of CSNA, and six histidines, flanking the COOH-terminus of the coding sequence of CSNA, are incorporated to serve as initiating Met/restriction site and purification tag, respectively. An over-expressed protein band of the appropriate molecular weight may be observed on a Coomassie blue stained polyacrylamide gel. This protein band is confirmed by Western blot analysis using monoclonal antibody against 6× Histidine tag. Large-scale purification of CSP was achieved using cell paste generated from 6-liter bacterial cultures, and purified using immobilized metal affinity chromatography (IMAC). Soluble fractions that had been separated from total cell lysate were incubated with a nickle chelating resin. The column was packed and washed with five column volumes of wash buffer. CSP was eluted stepwise with various concentration imidazole buffers. 
 Example 4 
 Protein Fusions Briefly, the human Fc portion of the IgG molecule can be PCR amplified, using primers that span the 5′and 3′ ends of the sequence described below. These primers also should have convenient restriction enzyme sites that will facilitate cloning into an expression vector, preferably a mammalian expression vector. For example, if pC4 (Accession No. 209646) is used, the human Fc portion can be ligated into the BamHI cloning site. Note that the 3′ BamHI site should be destroyed. Next, the vector containing the human Fc portion is re-restricted with BamHI, linearizing the vector, and a polynucleotide of the present invention, isolated by the PCR protocol described in Example 2, is ligated into this BamHI site. Note that the polynucleotide is cloned without a stop codon, otherwise a fusion protein will not be produced. If the naturally occurring signal sequence is used to produce the secreted protein, pC4 does not need a second signal peptide. Alternatively, if the naturally occurring signal sequence is not used, the vector can be modified to include a heterologous signal sequence. See, e. g., WO 96/34891. 
 Example 5 
 Production of an Antibody from a Polypeptide In general, such procedures involve immunizing an animal (preferably a mouse) with polypeptide or, more preferably, with a secreted polypeptide-expressing cell. Such cells may be cultured in any suitable tissue culture medium; however, it is preferable to culture cells in Earle's modified Eagle's medium supplemented with 10% fetal bovine serum (inactivated at about 56° C.), and supplemented with about 10 g/l of nonessential amino acids, about 1,000 U/ml of penicillin, and about 100 &mgr;g/ml of streptomycin. The splenocytes of such mice are extracted and fused with a suitable myeloma cell line. Any suitable myeloma cell line may be employed in accordance with the present invention; however, it is preferable to employ the parent myeloma cell line (SP20), available from the ATCC. After fusion, the resulting hybridoma cells are selectively maintained in HAT medium, and then cloned by limiting dilution as described by Wands et al., Gastroenterology 80: 225-232 (1981). The hybridoma cells obtained through such a selection are then assayed to identify clones which secrete antibodies capable of binding the polypeptide. Alternatively, additional antibodies capable of binding to the polypeptide can be produced in a two-step procedure using anti-idiotypic antibodies. Such a method makes use of the fact that antibodies are themselves antigens, and therefore, it is possible to obtain an antibody which binds to a second antibody. In accordance with this method, protein specific antibodies are used to inununize an animal, preferably a mouse. The splenocytes of such an animal are then used to produce hybridoma cells, and the hybridoma cells are screened to identify clones which produce an antibody whose ability to bind to the protein-specific antibody can be blocked by the polypeptide. Such antibodies comprise anti-idiotypic antibodies to the protein specific antibody and can be used to immunize an animal to induce formation of further protein-specific antibodies. Using the Jameson-Wolf methods the following epitopes were predicted. (Jameson and Wolf, CABIOS, 4(1), 181-186, 1988, the contents of which are incorporated by reference). Based on the underlying sequences found by mRNA subtractions the following extended nucleotide sequences and predicted amino acid sequences were determined. The chromosomal locations were determined for several of the sequences. Specifically: 6 DEX0289_3  chromosome 6 DEX0289_6  chromosome 7 DEX0289_10  chromosome 13 DEX0289_16  chromosome 9 DEX0289_17  chromosome 9 DBX0289_20  chromosome 3 DEX0289_22  chromosome 4 DEX0289_23  chromosome 6 DEX0289_26  chromosome 3 DEX0289_29  chromosome 9 DEX0289_30  chromosome 9 DEX0289_31  chromosome 7 DEX0289_32  chromosome 7 DEX0289_34  chromosome 13 DEX0289_43  chromosome 1 DEX0289_44  chromosome 1 DEX0289_45  chromosome 3 DEX0289_49  chromosome 9 DEX0289_51  chromosome 7 DEX0289_52  chromosome 7 DEX0289_55  chromosome 3 DEX0289_56  chromosome 3 DEX0289_57  chromosome 9 DEX0289_58  chromosome 9 DEX0289_63  chromosome 7 DEX0289_64  chromosome 7 DEX0289_66  chromosome 4 DEX0289_67  chromosome 3 DEX0289_68  chromosome 16 DEX0289_69  chromosome 16 DEX0289_72  chromosome 4 DEX0289_74  chromosome 1 The predicted antigenicity for the amino acid sequences is as follows: 7 Antigenicity Index (Jameson-Wolf) positions AI avg length DEX0289_78 +TC,19 135-144 1.06 10 DEX0289_84 5-45 1.12 41 98-108 1.05 11 DEX0289_85 29-44 1.14 16 DEX0289_91 66-82 1.00 17 DEX0289_98 22-35 1.04 14 DEX0289_101 5-14 1.04 10 DEX0289_108 618-627 1.10 10 576-611 1.10 36 330-341 1.07 12 488-498 1.06 11 DEX0289_113 47-56 1.15 10 DEX0289_115 16-30 1.03 15 DEX0289_120 12-24 1.10 13 DEX0289_121 54-67 1.27 14 DEX0289_124 372-382 1.30 11 99-110 1.26 12 42-63 1.15 22 506-516 1.15 11 270-279 1.12 10 385-394 1.12 10 484-504 1.03 21 179-193 1.00 15 The predicted helicity for the amino acid sequences is listed below: 8 DEX0289_80 PredHel&equals;1 Topology&equals;i9-31o DEX0289_88 PredHel&equals;1 Topology&equals;o15-32i DEX0289_108 PredHel&equals;9 Topology&equals;i35-57o63-85i92-109o170- 192i199-216o226-248i261-283o298- 320i359-378o DEX0289_112 PredHehl&equals;1 Topology&equals;i2-19o DEX0289_124 PredHel&equals;1 Topology&equals;o639-661i Examples of post-translational modifications (PTMs) of the BSPs of this invention are listed below. In addition, antibodies that specifically bind such post-translational modifications may be useful as a diagnostic or as therapeutic. Using the ProSite database (Bairoch et al., Nucleic Acids Res. 25(1):217-221 (1997), the contents of which are incorporated by reference), the following PTMs were predicted for the LSPs of the invention (http ://npsa-pbil.ibcp.fr/cgi-bin/npsa_automat.p1&quest;page&equals;npsa_prosite.html most recently accessed Oct. 23, 2001). For full definitions of the PTMs see http://www.expasy.org/cgi-bin/prosite-list.pl most recently accessed Oct. 23, 2001. 9 DEX0289_100 Myristyl 13-18;36-41; Pkc_Phospho_Site 26-28; DEX0289_101 Ck2_Phospho_Site 46-49; Myristyl 42-47; Pkc_Phospho_Site 20-22;46-48;58-60; DEX0289_102 Amidation 29-32; DEX0289_103 Ck2_Phospho_Site 25-28; Myristyl 33-38; DEX0289_104 Asn_Glycosylation 35-38; Myristyl 39-44; Pkc_Phospho_Site 42-44; DEX0289_105 Asn_Glycosylation 18-21; Ck2_Phospho_Site 76-79; Myristyl 19-24;39-44; Pkc_Phospho_Site 4-6; DEX0289_106 Ck2_Phospho_Site 27-30; DEX0289_107 Myristyl 5-10;11-16; DEX0289_108 Asn_Glycosylation 111-114;533-536;598-601; Camp_Phospho_Site 263-266;620-623; Ck2_Phospho_Site 18-21;138-141;155-158; 215-218;247-250;524-527;631-634;684-687; Myristyl 245-250;380-385;404-409;409-414;446-51; 464-469;534-539;632-637; Pkc_Phospho_Site 4-6;32-34;113-115; 122-124;219-221;249-251;332-334;519-521;606- 608;611-613;619-621; DEX0289_109 Asn_Glycosylation 20-23; Pkc_Phospho_Site 2-4;11-13;22-24;34-36; DEX0289_111 Asn_Glycosylation 105-108; Camp_Phospho_Site 48-51; Ck2_Phospho_Site 13-16; Glycosaminoglycan 93-96; Myristyl 108-113; Pkc_Phospho_Site 87-89; DEX0289_113 Ck2_Phospho_Site 97-100; Myristyl 20-25; Pkc_Phospho_Site 48-50;67-69;97-99; DEX0289_114 Ck2_Phospho_Site 6-9; Pkc_Phospho_Site 6-8; DEX0289_115 Ck2_Phospho_Site 29-32; DEX0289_116 Camp_Phospho_Site 11-14;15-18; Ck2_Phospho_Site 14-17;19-22; Pkc_Phospho_Site 14-16; DEX0289_117 Ck2_Phospho_Site 30-33; Pkc_Phospho_Site 35-37; DEX0289_118 Pkc_Phospho_Site 15-17; DEX0289_120 Pkc_Phospho_Site 27-29; DEX0289_121 Ck2_Phospho_Site 55-58; Myristyl 77-82; DEX0289_122 Camp_Phospho_Site 19-22; DEX0289_123 Asn_Glycosylation 11-14; DEX0289_124 Amidation 286-289; Asn_Glycosylation 83-86;90-93; 135-138;186-189;421-424;469-472;499-502; Camp — Phospho_Site 161-164;375-378;732—735; Ck2 — Phospho_Site 101-104;274-277;414-417;432-435; 443-446;451-454;489-492;508-511;535-538;629-632; 682-685;684-687;721-724;734-737;735-738; Myristyl 33-38;59-64;62-67;111-116;216-221;361-366; 364-369;389-394;468-473;545-550;620-625;635-640; 648-653;659-664;693-698; Pkc_Phospho — Site 17-19;51-53;75-77;105-107;188-190;228-230; 380-382;432-434;477-479;636-638;673-675;676-678; 734-736; Tyr_Phospho_Site 211 —218; 239-246;316-323;536-542;736-743; DEX0289_75 Asn_Glycosylation 4-7; Pkc_Phospho_Site 18-20; DEX0289_76 Pkc_Phospho_Site 24-26; DEX0289_77 Camp_Phospho_Site 5-8; DEX0289_78 Ck2_Phospho_Site 138-141; Myristyl 26-31; DEX0289_79 Ck2_Phospho_Site 30-33; Pkc_Phospho_Site 5-7;53-55; DEX0289_81 Pkc_Phospho_Site 14-16; DEX0289_82 Asn_Glycosylation 11-14; Ck2_Phospho_Site 13-16; DEX0289_83 Myristyl 13-18; Pkc_Phospho_Site 18-20 DEX0289_84 Asn_Glycosylation 13-16;73-76;166-169; Ck2_Phospho_Site 18-21; Pkc_Phospho_Site 132-134; DEX0289_85 Camp_Phospho_Site 23-26; Myristyl 33-38;39-44; Pkc_Phospho_Site 21-23;34-36; DEX0289_86 Asn_Glycosylation 16-19; Ck2_Phospho_Site 18-21; Myristyl 5-10; DEX0289_87 Asn_Glycosylation 23-26; Pkc_Phospho_Site 20-22; 25-27; DEX0289_88 Asn_Glycosylation 10-13; Pkc_Phospho_Site 7-9; DEX0289_91 Ck2_Phospho_Site 57-60; Myristyl 69-74; Pkc_Phospho_Site 5-7;73-75; DEX0289_92 Myristyl 2-7; DEX0289_93 Amidation 65-68; Camp_Phospho_Site 37-40;72-75; Ck2_Phospho_Site 26-29;46-49;75-78; Pkc_Phospho_Site 25-27;65-67;75-77; DEX0289_94 Asn_Glycosylation 38-41; Ck2_Phospho_Site 5-8; Pkc_Phospho_Site 21-23; DEX0289_95 Ck2_Phospho_Site 15-18; DEX0289_96 Pkc_Phospho_Site 31-33; DEX0289_97 Myristyl 20-25; Pkc_Phospho_Site 13-15;40-42; DEX0289_98 Myristyl 16-21; Pkc_Phospho_Site 33-35 
 Example 6 
 Method of Determining Alterations in a Gene Corresponding to a Polynucleotide RNA is isolated from individual patients or from a family of individuals that have a phenotype of interest. cDNA is then generated from these RNA samples using protocols known in the art. See, Sambrook (2001), supra. The cDNA is then used as a template for PCR, employing primers surrounding regions of interest in SEQ ID NO: 1 through 74. Suggested PCR conditions consist of 35 cycles at 95° C. for 30 seconds; 60-120 seconds at 52-58° C.; and 60-120 seconds at 70° C., using buffer solutions described in Sidransky et al., Science 252(5006): 706-9 (1991). See also Sidransky et al., Science 278(5340): 1054-9 (1997). PCR products are then sequenced using primers labeled at their 5′ end with T4 polynucleotide kinase, employing SequiTherm Polymerase. (Epicentre Technologies). The intron-exon borders of selected exons is also determined and genomic PCR products analyzed to confirm the results. PCR products harboring suspected mutations are then cloned and sequenced to validate the results of the direct sequencing. PCR products is cloned into T-tailed vectors as described in Holton et al., Nucleic Acids Res., 19: 1156 (1991) and sequenced with T7 polymerase (United States Biochemical). Affected individuals are identified by mutations not present in unaffected individuals. Genomic rearrangements may also be determined. Genomic clones are nick-translated with digoxigenin deoxyuridine 5′ triphosphate (Boehringer Manheim), and FISH is performed as described in Johnson et al., Methods Cell Biol. 35: 73-99 (1991). Hybridization with the labeled probe is carried out using a vast excess of human cot-1 DNA for specific hybridization to the corresponding genomic locus. Chromosomes are counterstained with 4,6-diamino-2-phenylidole and propidium iodide, producing a combination of C-and R-bands. Aligned images for precise mapping are obtained using a triple-band filter set (Chroma Technology, Brattleboro, Vt.) in combination with a cooled charge-coupled device camera (Photometrics, Tucson, Ariz.) and variable excitation wavelength filters. Id. hnage collection, analysis and chromosomal fractional length measurements are performed using the ISee Graphical Program System. (Inovision Corporation, Durham, N.C.) Chromosome alterations of the genomic region hybridized by the probe are identified as insertions, deletions, and translocations. These alterations are used as a diagnostic marker for an associated disease. 
 Example 7 
 Method of Detecting Abnormal Levels of a Polypeptide in a Biological Sample Antibody-sandwich ELISAs are used to detect polypeptides in a sample, preferably a biological sample. Wells of a microtiter plate are coated with specific antibodies, at a final concentration of 0.2 to 10 &mgr;g/ml. The antibodies are either monoclonal or polyclonal and are produced by the method described above. The wells are blocked so that non-specific binding of the polypeptide to the well is reduced. The coated wells are then incubated for >2 hours at RT with a sample containing the polypeptide. Preferably, serial dilutions of the sample should be used to validate results. The plates are then washed three times with deionized or distilled water to remove unbound polypeptide. Next, 50 &mgr;l of specific antibody-alkaline phosphatase conjugate, at a concentration of 25-400 ng, is added and incubated for 2 hours at room temperature. The plates are again washed three times with deionized or distilled water to remove unbound conjugate. 75 &mgr;l of 4-methylumbelliferyl phosphate (MUP) or p-nitrophenyl phosphate (NPP) substrate solution are added to each well and incubated 1 hour at room temperature. The reaction is measured by a microtiter plate reader. A standard curve is prepared, using serial dilutions of a control sample, and polypeptide concentrations are plotted on the X-axis (log scale) and fluorescence or absorbance on the Y-axis (linear scale). The concentration of the polypeptide in the sample is calculated using the standard curve. 
 Example 8 
 Formulating a Polypeptide The secreted polypeptide composition will be formulated and dosed in a fashion consistent with good medical practice, taking into account the clinical condition of the individual patient (especially the side effects of treatment with the secreted polypeptide alone), the site of delivery, the method of administration, the scheduling of administration, and other factors known to practitioners. The “effective amount” for purposes herein is thus determined by such considerations. As a general proposition, the total pharmaceutically effective amount of secreted polypeptide administered parenterally per dose will be in the range of about 1, &mgr;g/kg/day to 10 mg/kg/day of patient body weight, although, as noted above, this will be subject to therapeutic discretion. More preferably, this dose is at least 0.01 mg/kg/day, and most preferably for humans between about 0.01 and 1 mg/kg/day for the hormone. If given continuously, the secreted polypeptide is typically administered at a dose rate of about 1 &mgr;g/kg/hour to about 50 mg/kg/hour, either by 1-4 injections per day or by continuous subcutaneous infusions, for example, using a mini-pump. An intravenous bag solution may also be employed. The length of treatment needed to observe changes and the interval following treatment for responses to occur appears to vary depending on the desired effect. Pharmaceutical compositions containing the secreted protein of the invention are administered orally, rectally, parenterally, intracistemally, intravaginally, intraperitoneally, topically (as by powders, ointments, gels, drops or transdermal patch), bucally, or as an oral or nasal spray. “Pharmaceutically acceptable carrier” refers to a non-toxic solid, semisolid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type. The term “parenteral” as used herein refers to modes of administration which include intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous and intraarticular injection and infusion. The secreted polypeptide is also suitably administered by sustained-release systems. Suitable examples of sustained-release compositions include semipermeable polymer matrices in the form of shaped articles, e. g., films, or microcapsules. Sustained-release matrices include polylactides (U.S. Pat. No. 3,773,919, EP 58,481), copolymers of L-glutamic acid and gamma-ethyl-L-glutamate (Sidman, U. et al., Biopolymers 22: 547-556 (1983)), poly (2-hydroxyethyl methacrylate) (R. Langer et al., J. Biomed. Mater. Res. 15: 167-277 (1981), and R. Langer, Chem. Tech. 12: 98-105 (1982)), ethylene vinyl acetate (R. Langer et al.) or poly-D-(−)-3-hydroxybutyric acid (EP 133,988). Sustained-release compositions also include liposomally entrapped polypeptides. Liposomes containing the secreted polypeptide are prepared by methods known per se: DE Epstein et al., Proc. Natl. Acad. Sci. USA 82: 3688-3692 (1985); Hwang et al., Proc. Natl. Acad. Sci. USA 77: 4030-4034 (1980); EP 52,322; EP 36,676; EP 88,046; EP 143,949; EP 142,641; Japanese Pat. Appl. 83-118008; U.S. Pat. Nos. 4,485,045 and 4,544,545; and EP 102,324. Ordinarily, the liposomes are of the small (about 200-800 Angstroms) unilamellar type in which the lipid content is greater than about 30 mol. percent cholesterol, the selected proportion being adjusted for the optimal secreted polypeptide therapy. For parenteral administration, in one embodiment, the secreted polypeptide is formulated generally by mixing it at the desired degree of purity, in a unit dosage injectable form (solution, suspension, or emulsion), with a pharmaceutically acceptable carrier, I. e., one that is non-toxic to recipients at the dosages and concentrations employed and is compatible with other ingredients of the formulation. For example, the formulation preferably does not include oxidizing agents and other compounds that are known to be deleterious to polypeptides. Generally, the formulations are prepared by contacting the polypeptide uniformly and intimately with liquid carriers or finely divided solid carriers or both. Then, if necessary, the product is shaped into the desired formulation. Preferably the carrier is a parenteral carrier, more preferably a solution that is isotonic with the blood of the recipient. Examples of such carrier vehicles include water, saline, Ringer's solution, and dextrose solution. Non-aqueous vehicles such as fixed oils and ethyl oleate are also useful herein, as well as liposomes. The carrier suitably contains minor amounts of additives such as substances that enhance isotonicity and chemical stability. Such materials are non-toxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, succinate, acetic acid, and other organic acids or their salts; antioxidants such as ascorbic acid; low molecular weight (less than about ten residues) polypeptides, e. g., polyarginine or tripeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids, such as glycine, glutamic acid, aspartic acid, or arginine; monosaccharides, disaccharides, and other carbohydrates including cellulose or its derivatives, glucose, manose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; counterions such as sodium; and/or nonionic surfactants such as polysorbates, poloxamers, or PEG. The secreted polypeptide is typically formulated in such vehicles at a concentration of about 0.1 mg/ml to 100 mg/ml, preferably 1-10 mg/ml, at a pH of about 3 to 8. It will be understood that the use of certain of the foregoing excipients, carriers, or stabilizers will result in the formation of polypeptide salts. Any polypeptide to be used for therapeutic administration can be sterile. Sterility is readily accomplished by filtration through sterile filtration membranes (e. g., 0.2 micron membranes). Therapeutic polypeptide compositions generally are placed into a container having a sterile access port, for example, an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle. Polypeptides ordinarily will be stored in unit or multi-dose containers, for example, sealed ampules or vials, as an aqueous solution or as a lyophilized formulation for reconstitution. As an example of a lyophilized formulation, 10-ml vials are filled with 5 ml of sterile-filtered 1% (w/v) aqueous polypeptide solution, and the resulting mixture is lyophilized. The infusion solution is prepared by reconstituting the lyophilized polypeptide using bacteriostatic Water-for-Injection. The invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions of the 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 of the present invention may be employed in conjunction with other therapeutic compounds. 
 Example 9 
 Method of Treating Decreased Levels of the Polypeptide It will be appreciated that conditions caused by a decrease in the standard or normal expression level of a secreted protein in an individual can be treated by administering the polypeptide of the present invention, preferably in the secreted form. Thus, the invention also provides a method of treatment of an individual in need of an increased level of the polypeptide comprising administering to such an individual a pharmaceutical composition comprising an amount of the polypeptide to increase the activity level of the polypeptide in such an individual. For example, a patient with decreased levels of a polypeptide receives a daily dose 0.1-100 &mgr;g/kg of the polypeptide for six consecutive days. Preferably, the polypeptide is in the secreted form. The exact details of the dosing scheme, based on administration and formulation, are provided above. 
 Example 10 
 Method of Treating Increased Levels of the Polypeptide Antisense technology is used to inhibit production of a polypeptide of the present invention. This technology is one example of a method of decreasing levels of a polypeptide, preferably a secreted form, due to a variety of etiologies, such as cancer. For example, a patient diagnosed with abnormally increased levels of a polypeptide is administered intravenously antisense polynucleotides at 0.5, 1.0, 1.5, 2.0 and 3.0 mg/kg day for 21 days. This treatment is repeated after a 7-day rest period if the treatment was well tolerated. The formulation of the antisense polynucleotide is provided above. 
 Example 11 
 Method of Treatment Using Gene Therapy One method of gene therapy transplants fibroblasts, which are capable of expressing a polypeptide, onto a patient. Generally, fibroblasts are obtained from a subject by skin biopsy. The resulting tissue is placed in tissue-culture medium and separated into small pieces. Small chunks of the tissue are placed on a wet surface of a tissue culture flask, approximately ten pieces are placed in each flask. The flask is turned upside down, closed tight and left at room temperature over night. After 24 hours at room temperature, the flask is inverted and the chunks of tissue remain fixed to the bottom of the flask and fresh media (e. g., Ham's F12 media, with 10% FBS, penicillin and streptomycin) is added. The flasks are then incubated at 37° C. for approximately one week. At this time, fresh media is added and subsequently changed every several days. After an additional two weeks in culture, a monolayer of fibroblasts emerge. The monolayer is trypsinized and scaled into larger flasks. pMV-7 (Kirschmeier, P. T. et al., DNA, 7: 219-25 (1988)), flanked by the long terminal repeats of the Moloney murine sarcoma virus, is digested with EcoRI and HindIII and subsequently treated with calf intestinal phosphatase. The linear vector is fractionated on agarose gel and purified, using glass beads. The cDNA encoding a polypeptide of the present invention can be amplified using PCR primers which correspond to the 5′and 3′end sequences respectively as set forth in Example 1. Preferably, the 5′primer contains an EcoRI site and the 3′primer includes a HindIII site. Equal quantities of the Moloney murine sarcoma virus linear backbone and the amplified EcoRI and HindIII fragment are added together, in the presence of T4 DNA ligase. The resulting mixture is maintained under conditions appropriate for ligation of the two fragments. The ligation mixture is then used to transform bacteria HB 101, which are then plated onto agar containing kanamycin for the purpose of confirming that the vector has the gene of interest properly inserted. The amphotropic pA317 or GP&plus;am12 packaging cells are grown in tissue culture to confluent density in Dulbecco's Modified Eagles Medium (DMEM) with 10% calf serum (CS), penicillin and streptomycin. The MSV vector containing the gene is then added to the media and the packaging cells transduced with the vector. The packaging cells now produce infectious viral particles containing the gene (the packaging cells are now referred to as producer cells). Fresh media is added to the transduced producer cells, and subsequently, the media is harvested from a 10 cm plate of confluent producer cells. The spent media, containing the infectious viral particles, is filtered through a millipore filter to remove detached producer cells and this media is then used to infect fibroblast cells. Media is removed from a sub-confluent plate of fibroblasts and quickly replaced with the media from the producer cells. This media is removed and replaced with fresh media. If the titer of virus is high, then virtually all fibroblasts will be infected and no selection is required. If the titer is very low, then it is necessary to use a retroviral vector that has a selectable marker, such as neo or his. Once the fibroblasts have been efficiently infected, the fibroblasts are analyzed to determine whether protein is produced. The engineered fibroblasts are then transplanted onto the host, either alone or after having been grown to confluence on cytodex 3 microcarrier beads. 
 Example 12 
 Method of Treatment Using Gene Therapy-In Vivo Another aspect of the present invention is using in vivo gene therapy methods to treat disorders, diseases and conditions. The gene therapy method relates to the introduction of naked nucleic acid (DNA, RNA, and antisense DNA or RNA) sequences into an animal to increase or decrease the expression of the polypeptide. The polynucleotide of the present invention may be operatively linked to a promoter or any other genetic elements necessary for the expression of the polypeptide by the target tissue. Such gene therapy and delivery techniques and methods are known in the art, see, for example, WO 90/11092, WO 98/11779; U.S. Pat. No. 5,693,622; 5,705,151; 5,580,859; Tabata H. et al. (1997) Cardiovasc. Res. 35 (3): 470-479, Chao J et al. (1997) Pharmacol. Res. 35 (6): 517-522, Wolff J. A. (1997) Neuromuscul. Disord. 7 (5): 314-318, Schwartz B. et al. (1996) Gene Ther. 3 (5): 405-411, Tsurumi Y. et al. (1996) Circulation 94 (12): 3281-3290 (incorporated herein by reference). The polynucleotide constructs may be delivered by any method that delivers injectable materials to the cells of an animal, such as, injection into the interstitial space of tissues (heart, muscle, skin, lung, liver, intestine and the like). The polynucleotide constructs can be delivered in a pharmaceutically acceptable liquid or aqueous carrier. The term “naked” polynucleotide, DNA or RNA, refers to sequences that are free from any delivery vehicle that acts to assist, promote, or facilitate entry into the cell, including viral sequences, viral particles, liposome formulations, lipofectin or precipitating agents and the like. However, the polynucleotides of the present invention may also be delivered in liposome formulations (such as those taught in Felgner P. L. et al. (1995) Ann. NY Acad. Sci. 772: 126-139 and Abdallah B. et al. (1995) Biol. Cell 85 (1): 1-7) which can be prepared by methods well known to those skilled in the art. The polynucleotide vector constructs used in the gene therapy method are preferably constructs that will not integrate into the host genome nor will they contain sequences that allow for replication. Any strong promoter known to those skilled in the art can be used for driving the expression of DNA. Unlike other gene therapies techniques, one major advantage of introducing naked nucleic acid sequences into target cells is the transitory nature of the polynucleotide synthesis in the cells. Studies have shown that non-replicating DNA sequences can be introduced into cells to provide production of the desired polypeptide for periods of up to six months. The polynucleotide construct can be delivered to the interstitial space of tissues within the an animal, including of muscle, skin, brain, lung, liver, spleen, bone marrow, thymus, heart, lymph, blood, bone, cartilage, pancreas, kidney, gall bladder, stomach, intestine, testis, ovary, uterus, rectum, nervous system, eye, gland, and connective tissue. Interstitial space of the tissues comprises the intercellular fluid, mucopolysaccharide matrix among the reticular fibers of organ tissues, elastic fibers in the walls of vessels or chambers, collagen fibers of fibrous tissues, or that same matrix within connective tissue ensheathing muscle cells or in the lacunae of bone. It is similarly the space occupied by the plasma of the circulation and the lymph fluid of the lymphatic channels. Delivery to the interstitial space of muscle tissue is preferred for the reasons discussed below. They may be conveniently delivered by injection into the tissues comprising these cells. They are preferably delivered to and expressed in persistent, non-dividing cells which are differentiated, although delivery and expression may be achieved in non-differentiated or less completely differentiated cells, such as, for example, stem cells of blood or skin fibroblasts. In vivo muscle cells are particularly competent in their ability to take up and express polynucleotides. For the naked polynucleotide injection, an effective dosage amount of DNA or RNA will be in the range of from about 0.05 &mgr;g/kg body weight to about 50 mg/kg body weight. Preferably the dosage will be from about 0.005 mg/kg to about 20 mg/kg and more preferably from about 0.05 mg/kg to about 5 mg/kg. Of course, as the artisan of ordinary skill will appreciate, this dosage will vary according to the tissue site of injection. The appropriate and effective dosage of nucleic acid sequence can readily be determined by those of ordinary skill in the art and may depend on the condition being treated and the route of administration. The preferred route of administration is by the parenteral route of injection into the interstitial space of tissues. However, other parenteral routes may also be used, such as, inhalation of an aerosol formulation particularly for delivery to lungs or bronchial tissues, throat or mucous membranes of the nose. In addition, naked polynucleotide constructs can be delivered to arteries during angioplasty by the catheter used in the procedure. The dose response effects of injected polynucleotide in muscle in vivo is determined as follows. Suitable template DNA for production of mRNA coding for polypeptide of the present invention is prepared in accordance with a standard recombinant DNA methodology. The template DNA, which may be either circular or linear, is either used as naked DNA or complexed with liposomes. The quadriceps muscles of mice are then injected with various amounts of the template DNA. Five to six week old female and male Balb/C mice are anesthetized by intraperitoneal injection with 0.3 ml of 2.5% Avertin. A 1.5 cm incision is made on the anterior thigh, and the quadriceps muscle is directly visualized. The template DNA is injected in 0.1 ml of carrier in a 1 cc syringe through a 27 gauge needle over one minute, approximately 0.5 cm from the distal insertion site of the muscle into the knee and about 0.2 cm deep. A suture is placed over the injection site for future localization, and the skin is closed with stainless steel clips. After an appropriate incubation time (e. g., 7 days) muscle extracts are prepared by excising the entire quadriceps. Every fifth 15 um cross-section of the individual quadriceps muscles is histochemically stained for protein expression. A time course for protein expression may be done in a similar fashion except that quadriceps from different mice are harvested at different times. Persistence of DNA in muscle following injection may be determined by Southern blot analysis after preparing total cellular DNA and HIRT supernatants from injected and control mice. The results of the above experimentation in mice can be use to extrapolate proper dosages and other treatment parameters in humans and other animals using naked DNA. 
 Example 13 
 Transgenic Animals The polypeptides of the invention can also be expressed in transgenic animals. Animals of any species, including, but not limited to, mice, rats, rabbits, hamsters, guinea pigs, pigs, micro-pigs, goats, sheep, cows and non-human primates, e. g., baboons, monkeys, and chimpanzees may be used to generate transgenic animals. In a specific embodiment, techniques described herein or otherwise known in the art, are used to express polypeptides of the invention in humans, as part of a gene therapy protocol. Any technique known in the art may be used to introduce the transgene (i. e., polynucleotides of the invention) into animals to produce the founder lines of transgenic animals. Such techniques include, but are not limited to, pronuclear microinjection (Paterson et al., Appl. Microbiol. Biotechnol. 40: 691-698 (1994); Carver et al., Biotechnology (NY) 11: 1263-1270 (1993); Wright et al., Biotechnology (NY) 9: 830-834 (1991); and Hoppe et al., U.S. Pat. No. 4,873,191 (1989)); retrovirus mediated gene transfer into germ lines (Van der Putten et al., Proc. Natl. Acad. Sci., USA 82: 6148-6152 (1985)), blastocysts or embryos; gene targeting in embryonic stem cells (Thompson et al., Cell 56: 313-321 (1989)); electroporation of cells or embryos (Lo, 1983, Mol Cell. Biol. 3: 1803-1814 (1983)); introduction of the polynucleotides of the invention using a gene gun (see, e. g., Ulmer et al., Science 259: 1745 (1993); introducing nucleic acid constructs into embryonic pleuripotent stem cells and transferring the stem cells back into the blastocyst; and sperm mediated gene transfer (Lavitrano et al., Cell 57: 717-723 (1989); etc. For a review of such techniques, see Gordon, “Transgenic Animals,” Intl. Rev. Cytol. 115: 171-229 (1989), which is incorporated by reference herein in its entirety. Any technique known in the art may be used to produce transgenic clones containing polynucleotides of the invention, for example, nuclear transfer into enucleated oocytes of nuclei from cultured embryonic, fetal, or adult cells induced to quiescence (Campell et al., Nature 380: 64-66 (1996); Wilmut et al., Nature 385: 810813 (1997)). The present invention provides for transgenic animals that carry the transgene in all their cells, as well as animals which carry the transgene in some, but not all their cells, I.e., mosaic animals or chimeric. The transgene may be integrated as a single transgene or as multiple copies such as in concatamers, e.g., head-to-head tandems or head-to-tail tandems. The transgene may also be selectively introduced into and activated in a particular cell type by following, for example, the teaching of Lasko et al. (Lasko et al., Proc. Natl. Acad. Sci. USA 89: 6232-6236 (1992)). The regulatory sequences required for such a cell-type specific activation will depend upon the particular cell type of interest, and will be apparent to those of skill in the art. When it is desired that the polynucleotide transgene be integrated into the chromosomal site of the endogenous gene, gene targeting is preferred. Briefly, when such a technique is to be utilized, vectors containing some nucleotide sequences homologous to the endogenous gene are designed for the purpose of integrating, via homologous recombination with chromosomal sequences, into and disrupting the function of the nucleotide sequence of the endogenous gene. The transgene may also be selectively introduced into a particular cell type, thus inactivating the endogenous gene in only that cell type, by following, for example, the teaching of Gu et al. (Gu et al., Science 265: 103-106 (1994)). The regulatory sequences required for such a cell-type specific inactivation will depend upon the particular cell type of interest, and will be apparent to those of skill in the art. Once transgenic animals have been generated, the expression of the recombinant gene may be assayed utilizing standard techniques. Initial screening may be accomplished by Southern blot analysis or PCR techniques to analyze animal tissues to verify that integration of the transgene has taken place. The level of MRNA expression of the transgene in the tissues of the transgenic animals may also be assessed using techniques which include, but are not limited to, Northern blot analysis of tissue samples obtained from the animal, in situ hybridization analysis, and reverse transcriptase-PCR (rt-PCR). Samples of transgenic gene-expressing tissue may also be evaluated immunocytochemically or immunohistochemically using antibodies specific for the transgene product. Once the founder animals are produced, they may be bred, inbred, outbred, or crossbred to produce colonies of the particular animal. Examples of such breeding strategies include, but are not limited to: outbreeding of founder animals with more than one integration site in order to establish separate lines; inbreeding of separate lines in order to produce compound transgenics that express the transgene at higher levels because of the effects of additive expression of each transgene; crossing of heterozygous transgenic animals to produce animals homozygous for a given integration site in order to both augment expression and eliminate the need for screening of animals by DNA analysis; crossing of separate homozygous lines to produce compound heterozygous or homozygous lines; and breeding to place the transgene on a distinct background that is appropriate for an experimental model of interest. Transgenic animals of the invention have uses which include, but are not limited to, animal model systems useful in elaborating the biological function of polypeptides of the present invention, studying conditions and/or disorders associated with aberrant expression, and in screening for compounds effective in ameliorating such conditions and/or disorders. 
 Example 14 
 Knock-Out Animals Endogenous gene expression can also be reduced by inactivating or “knocking out” the gene and/or its promoter using targeted homologous recombination. (E.g., see Smithies et al., Nature 317: 230-234 (1985); Thomas & Capecchi, Cell 51: 503512 (1987); Thompson et al., Cell 5: 313-321(1989); each of which is incorporated by reference herein in its entirety). For example, a mutant, non-functional polynucleotide of the invention (or a completely unrelated DNA sequence) flanked by DNA homologous to the endogenous polynucleotide sequence (either the coding regions or regulatory regions of the gene) can be used, with or without a selectable marker and/or a negative selectable marker, to transfect cells that express polypeptides of the invention in vivo. In another embodiment, techniques known in the art are used to generate knockouts in cells that contain, but do not express the gene of interest. Insertion of the DNA construct, via targeted homologous recombination, results in inactivation of the targeted gene. Such approaches are particularly suited in research and agricultural fields where modifications to embryonic stem cells can be used to generate animal offspring with an inactive targeted gene (e. g., see Thomas & Capecchi 1987 and Thompson 1989, supra). However this approach can be routinely adapted for use in humans provided the recombinant DNA constructs are directly administered or targeted to the required site in vivo using appropriate viral vectors that will be apparent to those of skill in the art. In further embodiments of the invention, cells that are genetically engineered to express the polypeptides of the invention, or alternatively, that are genetically engineered not to express the polypeptides of the invention (e. g., knockouts) are administered to a patient in vivo. Such cells may be obtained from the patient (I. e., animal, including human) or an MHC compatible donor and can include, but are not limited to fibroblasts, bone marrow cells, blood cells (e. g., lymphocytes), adipocytes, muscle cells, endothelial cells etc. The cells are genetically engineered in vitro using recombinant DNA techniques to introduce the coding sequence of polypeptides of the invention into the cells, or alternatively, to disrupt the coding sequence and/or endogenous regulatory sequence associated with the polypeptides of the invention, e. g., by transduction (using viral vectors, and preferably vectors that integrate the transgene into the cell genome) or transfection procedures, including, but not limited to, the use of plasmids, cosmids, YACs, naked DNA, electroporation, liposomes, etc. The coding sequence of the polypeptides of the invention can be placed under the control of a strong constitutive or inducible promoter or promoter/enhancer to achieve expression, and preferably secretion, of the polypeptides of the invention. The engineered cells which express and preferably secrete the polypeptides of the invention can be introduced into the patient systemically, e. g., in the circulation, or intraperitoneally. Alternatively, the cells can be incorporated into a matrix and implanted in the body, e. g., genetically engineered fibroblasts can be implanted as part of a skin graft; genetically engineered endothelial cells can be implanted as part of a lymphatic or vascular graft. (See, for example, Anderson et al. U.S. Pat. No. 5,399,349; and Mulligan & Wilson, U.S. Pat. No. 5,460,959 each of which is incorporated by reference herein in its entirety). When the cells to be administered are non-autologous or non-MHC compatible cells, they can be administered using well known techniques which prevent the development of a host immune response against the introduced cells. For example, the cells may be introduced in an encapsulated form which, while allowing for an exchange of components with the immediate extracellular environment, does not allow the introduced cells to be recognized by the host immune system. Transgenic and “knock-out” animals of the invention have uses which include, but are not limited to, animal model systems useful in elaborating the biological function of polypeptides of the present invention, studying conditions and/or disorders associated with aberrant expression, and in screening for compounds effective in ameliorating such conditions and/or disorders. All patents, patent publications, and other published references mentioned herein are hereby incorporated by reference in their entireties as if each had been individually and specifically incorporated by reference herein. While preferred illustrative embodiments of the present invention are described, one skilled in the art will appreciate that the present invention can be practiced by other than the described embodiments, which are presented for purposes of illustration only and not by way of limitation. The present invention is limited only by the claims that follow.