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  Polycystic xidney disease pkd2 gene and uses thereopUSPTO Application #: 20080057506Title: Polycystic xidney disease pkd2 gene and uses thereop Abstract: The present invention provides a purified and isolated wild type PKD2 gene, as well as mutated forms of this gene. The present invention also provides one or more single-stranded nucleic acid probes which specifically hybridize to the wild type PKD2 gene or the mutated PKD2 gene, and mixtures thereof, which may be formulated in kits, and used in the diagnosis of ADPKD associated with the mutated PKD2 gene. The present invention also provides a method for diagnosing ADPKD caused by a mutated PKD2 gene, as well as a method for treating autosomal dominant polycystic kidney disease caused by a mutated PKD2 gene. (end of abstract) Agent: Amster, Rothstein & Ebenstein LLP - New York, NY, US Inventors: Stefan Somlo, Toshio Mochizuki USPTO Applicaton #: 20080057506 - Class: 435006000 (USPTO) Related Patent Categories: Chemistry: Molecular Biology And Microbiology, Measuring Or Testing Process Involving Enzymes Or Micro-organisms; Composition Or Test Strip Therefore; Processes Of Forming Such Composition Or Test Strip, Involving Nucleic Acid The Patent Description & Claims data below is from USPTO Patent Application 20080057506. Brief Patent Description - Full Patent Description - Patent Application Claims
[0001] This application claims priority of and is a continuation of co-pending U.S. patent application Ser. No. 11/040,384, filed Jan. 21, 2005, which claims priority of and is a divisional of U.S. patent application Ser. No. 09/753,008, filed Jan. 2, 2001, now U.S. Pat. No. 7,083,915, which claims priority of and is a continuation of U.S. patent application Ser. No. 09/385,752, filed Aug. 30, 1999, now U.S. Pat. No. 6,228,591, which claims priority of and is a continuation of U.S. patent application Ser. No. 08/651,999, filed May 23, 1996, now U.S. Pat. No. 6,031,088, the entire contents of which are hereby incorporated by reference. BACKGROUND OF THE INVENTION [0003] This invention is based upon the discovery by the inventors of the PKD2 gene associated with Autosomal Dominant Polycystic Kidney Disease ("ADPKD"), the "PKD2 gene" or "PKD2", and a novel protein encoded by this gene. The discovery of the PKD2 gene and the protein encoded by the gene will have important implications in the diagnosis and treatment of ADPKD caused by defects in the PKD2 gene. [0004] ADPKD is a genetically heterogeneous disorder that affects approximately 500,000 Americans and five million individuals world wide, and accounts for 8 to 10% of all end stage renal disease (ESRD) worldwide (Gabow, P. A. N. Eng. J. Med. 329:332 (1993)). Its principal clinical manifestation is bilateral renal cysts that result in chronic renal failure in about 45% of affected individuals by age 60 (Gabow, P. A., supra). Hypertension and liver cysts are common, and the involvement of other organ systems (Gabow, P. A., et al. Kidney Int. 38:1177 (1990); Chapman, A. B., et al. N. Eng. J. Med. 327:916 (1992); Hossack, K. F., et al. N. Eng. J. Med. 319:907 (1988); Torres, V. E., et al. Am. J. Kidney Dis. 22:513 (1993); Huston, J., et al. J. Am. Soc. Nephrol. 3:1871 (1993); Somlo, S., et al. J. Am. Soc. Nephrol. 4:1371 (1993)) lends support to the view that polycystic kidney disease is a systemic disorder (Gabow, P. A., supra). [0005] To date, most forms of ADPKD have been associated with two genes, PKD1 and PKD2. The full genomic structure and cDNA sequence for the PKD1 gene has been identified (The International Polycystic Kidney Disease Consortium, Cell 81:289 (1995); The American PKD1 Consortium, Hum. Mol. Genet. 4:575 (1995)). Mutations in the PKD1 gene are suspected of causing 80-90% of all cases of ADPKD. The PKD2 gene has been localized on chromosome 4q21-23 and accounts for approximately 15% of affected families (Kimberling, W. J., et al. Genomics 18:467 (1993); Peters, D. J. M. and L. A. Sandkuijl Contrib. Nephrol. 97:128 (1992)). Prior to the present invention, however, the PKD2 gene had not been identified. SUMMARY OF THE INVENTION [0006] The present invention provides a purified and isolated wild type PKD2 gene, as well as mutated forms of this gene. The present invention also provides one or more single-stranded nucleic acid probes which specifically hybridize to the wild type PKD2 gene or the mutated PKD2 gene, and mixtures thereof, which may be formulated in kits, and used in the diagnosis of ADPKD associated with the mutated PKD2 gene. [0007] The present invention also provides a vector comprising nucleic acid encoding an active PKD2 protein, a cell stably transformed with this vector, as well as a method for producing recombinant, active PKD2 protein. A purified, active PKD2 protein is also provided by the present invention. In addition, the present invention provides an antibody immunoreactive with a wild type PKD2 protein, as well as an antibody immunoreactive with a mutant PKD2 protein, which may be formulated in kits, and used in the diagnosis of ADPKD associated with the mutated PKD2 gene. [0008] The present invention further provides a method for diagnosing ADPKD caused by a mutated PKD2 gene in an adult subject suspected of having the disease comprising detecting the presence of a mutated PKD2 gene in nucleic acid of the subject. The present invention still further provides a method for treating ADPKD caused by a mutated PKD2 gene in a subject in need of such treatment comprising the delivery and expression of a functional PKD2 gene into a sufficient number of cells of the subject to treat the disease. A stem cell which expresses the PKD2 gene introduced therein through viral transduction, homologous recombination or transfection is also provided by the invention. [0009] In addition, the present invention provides a recombinant viral vector for treating a defect in the PKD2 gene in a target cell comprising (a) the nucleic acid of or corresponding to at least a portion of the genome of a virus, which portion is capable of directing the infection of the target cell, and (b) a PKD2 gene operably linked to the viral nucleic acid and capable of being expressed as a functional gene product in the target cell. [0010] Finally, the present invention provides a vector and an embryonic stem cell each of which comprises a mutated PKD2 gene, a non-human, transgenic animal whose germ and somatic cells contain a mutated PKD2 gene sequence introduced into said animal, or an ancestor thereof, at an embryonic stage, as well as a method for producing the non-human, transgenic animal. [0011] Additional objects of the invention will be apparent from the description which follows. BRIEF DESCRIPTION OF THE FIGURES [0012] FIG. 1A represents the subset of STSs from the high density map of the PKD2 region showing polymorphic loci flanking the interval. JSTG3 and AICA1 are two of nine microsatellite markers in this region developed previously. SPP1 (osteopontin, STS4-1078). and D4S1171 were used to screen the P1 library as described in Materials and Methods. Other sources of STSs include published linkage maps and genome center databases. cen, centromere; tel telomere. Distances are in Morgans along chromosome 4. [0013] FIG. 1B shows representative mega-YACs (Cohen, D., et al. Nature 366:698 (1993)), and their STS content, forming a contig around the PKD2 region. [0014] FIG. 1C shows the minimum tiling path of the cosmid and P1 contig in the PKD2 region. Clone names beginning with "c" and "p" refer to cosmid and P1 clones, respectively; addresses are from the. original arrayed libraries. The clones containing JSTG3 and AICA1 are shown; a single gap of <40 kb is indicated by the arrow. [0015] FIG. 1D shows the detail of the portion of the contig containing the PKD2 candidate gene, cTM-4. [0016] FIG. 1E shows overlapping map of nine cDNA clones for cTM-4 and a composite schematic at the bottom. Clones K1-1 and K1-5 are from the adult kidney library; clones yj63h09 and yc93g07 were identified by GenBank searching and are from the normalized infant brain library (Soares, M. B., et al. Proc. Natl. Acad. Sci. USA 91: 9228 (1994)); all other clones are from the fetal brain library. Shaded areas represent chimeric portions of clones. [0017] FIG. 2 represents expression of the PKD2 candidate gene. Insert from cTM-4B3-3 (FIG. 1E) was used as a hybridization probe on mRNA blots containing human tissues (Clonetech, Palo Alto, Calif.). Hybridization was performed without pre-competition and a final wash stringency of 0.5.times.SSC, 0.1% SDS at 65.degree. C. Tissues in numbered lanes are: (1) heart, (2) brain, (3) placenta, (4) lung, (5) liver, (6) skeletal muscle, (7) kidney, (8) pancreas, (9) spleen, (10) thymus, (11) prostate, (12) testis, (13) ovary, (14) small intestine, (15) colon, (16) leukocytes, (17) fetal brain, (18) fetal lung, (19) fetal liver, (20) fetal kidney. At bottom, .beta.-actin hybridization to the same blots is used to compare relative mRNA loading within each blot. [0018] FIG. 3 depicts the mutations in PKD2 from an analysis of genomic PCR products in three PKD2 families. Left panel shows the results of direct sequencing of genomic PCR products from affected individuals. The arrows denote double peaks, confirmed by sequencing in both directions, indicative of heterozygosity at that nucleotide. Each of the mutant alleles results in a premature stop codon. The right panel demonstrates segregation of the mutated allele with the disease phenotype. In families 97 and 1605, the affected alleles are not digested by Bsr I and Taq I, respectively, since the restriction sites are lost by mutation. Family 1601 shows segregation of the SSCA variant, indicated by the arrow, with the disease phenotype. For each family, only portions of more extensive pedigrees are shown. Filled symbols, affected individuals. Open symbols, unaffected individuals. M, 100 bp ladder. The human PKD2 mutation region from Family 97-SEQ ID NO:13, the human PKD2 mutation region from Family 1605-SEQ ID NO:14, and the human PKD2 mutation region from Family 1601-SEQ ID NO:15. [0019] FIG. 4A depicts the deduced amino acid sequence of PKD2 (cTM-4) (GenBank accession: gb1U50928) in alignment with PKD1 (gb.vertline.U24497), the C. elegans homolog of PKD1 (ZK945.9; swiss.vertline.Q09624) and VACC.alpha.1E-1 (pir.vertline.B54972) using BESTFIT (Program Manual for the Wisconsin Package, Version 8, September 1994): identity to cTM-4, |; similarity to cTM-4, :. Numbers in parentheses refer to amino acids in respective sequences. Putative transmembrane domains, tm1 to tm6. Predicted N-glycosylation sites, *. Potential phosphorylation sites with strong consensus sequences: protein kinase C, +; cGMP dependent kinase, open square (Ser 826 is also consistent with a protein kinase A site); casein kinase, open circle. The sites of the nonsense mutations (FIG. 3) are indicated by arrows labeled with the respective family numbers. The EF-hand domain is indicated by the dashed line. [0020] FIG. 4B shows alignment of the EF-hand domain with the EF-hand test sequence. The residues E, G, I, and E, the latter being a Ca.sup.2+ coordination vertex, are the expected residues at the indicated positions in the EF-hand. Positions indicated as "n" are expected to have hydrophobic amino acids (L, I, V, F, M); those denoted with * should be oxygen-containing amino acids (D, N, E, Q, S, T) comprising the remainder of coordination vertices for Ca.sup.2+ binding; the -Y vertex can be any amino acid. The Leu (L) in PKD2 in place of the Ile (I) is likely a permissible substitution; PKD2 has Gln (Q) in place of the consensus Gly (G) as is the case with EF-hand domains in the .alpha.1 Na.sup.+ channels. [0021] FIG. 5A-5G represents the nucleotide sequence (SEQ ID NO:6) of the PKD2 gene and the deduced amino acid sequence (SEQ ID NO:7) for PKD2. DETAILED DESCRIPTION OF THE INVENTION Continue reading... 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