+

WO2001079467A2 - Identification d'une nouvelle nadph-oxydase renale - Google Patents

Identification d'une nouvelle nadph-oxydase renale

Info

Publication number
WO2001079467A2
WO2001079467A2 PCT/US2001/010866 US0110866W WO0179467A2 WO 2001079467 A2 WO2001079467 A2 WO 2001079467A2 US 0110866 W US0110866 W US 0110866W WO 0179467 A2 WO0179467 A2 WO 0179467A2
Authority
WO
WIPO (PCT)
Prior art keywords
renox
nucleic acid
nucleotide sequence
cells
seq
Prior art date
Application number
PCT/US2001/010866
Other languages
English (en)
Other versions
WO2001079467A3 (fr
Inventor
Thomas L. Leto
Miklos Geiszt
Original Assignee
The United States Of America, As Represented By Secretary Of The Department Of Health & Human Services
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by The United States Of America, As Represented By Secretary Of The Department Of Health & Human Services filed Critical The United States Of America, As Represented By Secretary Of The Department Of Health & Human Services
Priority to AU2001253123A priority Critical patent/AU2001253123A1/en
Publication of WO2001079467A2 publication Critical patent/WO2001079467A2/fr
Publication of WO2001079467A3 publication Critical patent/WO2001079467A3/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)

Definitions

  • the present invention relates to nucleic acids and polypeptides related to novel mammalian renal NADPH oxidases and uses of these nucleic acids and polypeptides.
  • ROS Reactive oxygen species
  • ROS produced at low levels by non- immune cells have been implicated in growth factor signaling, mitogenic responses, apoptosis, and oxygen sensing (Irani, K., et al., "Mitogenic signaling mediated by oxidants in Ras-transformed fibroblasts", Science 275, 1649 (1997); Adler, V. et al., “Role of redox potential and reactive oxygen species in stress signaling," Oncogene 18:6104 (1999); and Ebert, B. L., et al., “Regulation of the erythropoietin gene," Blood 94:1864 (1999)).
  • ROS superoxide
  • the heme-binding component of cytochrome b 558 is gp91 phox , a glycosylated flavoprotein associated with p22 phox .
  • Mox1 is highly expressed in the colon and is detected at lower levels in uterus, prostate, and vascular smooth muscle. Over-expression of Mox1 in NIH 3T3 fibroblasts results in increased superoxide production and mitogenic activity. These data suggest the involvement of Mox1 in regulation of the cell cycle; its exact physiological function, however, remains elusive. Gp91 p ⁇ °*, Mox1 , and an alternatively spliced variant of Mox1 encompassing a truncated, four-transmembrane segment-containing portion of this protein all apparently demonstrate electrogenic proton channel activity (Henderson, L. M., et al., "The arachidonate-activable, NADPH oxidase-associated H+ channel.
  • the kidney is susceptible to oxidative damage induced by ischemia- reperfusion, inflammatory, and toxic drug reactions that can lead to renal diseases such as acute ischemic renal failure, acute glomerulonephritis, and chronic or acute tubular disease (Baud, L., et al., "Reactive oxygen species.-production and role in the kidney,” Am. J. Physiol. 251:F765 (1986)). While circulating leukocytes are known to be important mediators of oxidative damage to renal tissues, particularly the glomerular basement membrane, several resident renal cell types are also recognized for their capacity for significant superoxide release.
  • the kidney was noted for high levels of p22 phox expression based on reverse transcription-polymerase chain reactions and immunochemical methods (Radeke, H. H., et al., "Functional expression of NADPH oxidase components by reverse transcription-polymerase chain reactions and immunochemical methods (alpha- and beta-subunits of cytochrome b558 and 45-kDa flavoprotein) by intrinsic human glomerular mesangial cells," J. Biol. Chem.
  • phagocyte-specific oxidase components have been detected in glomerular messangial cells (Greiber, S., et al., "NADPH oxidase activity in cultured human podocytes:effects of adenosine triphosphate, Kidney Int. 53:654 (1998)) or podocytes (Cui, X. L., et al., "Arachidonic acid activates c-jun N-terminal kinase through NADPH oxidase in rabbit proximal tubular epithelial cells," Proc. Natl. Acad. Sci. U S A 94, 3771 (1997)).
  • EPO erythropoietin
  • the current invention meets this need by identifying and characterizing a novel source of superoxide in the kidney referred to as a renal oxidase or Renox.
  • Renox is highly expressed in the proximal tubules of the renal cortex and may fulfill the function of the putative oxygen sensor in the kidney.
  • the present invention includes nucleic acids and their encoded polypeptides related to novel mammalian renal NADPH oxidases, and uses of these nucleic acids and polypeptides.
  • the invention includes expression vectors containing the nucleic acids, recombinant cell lines containing the expression vectors, and methods for producing the polypeptides of the invention using the expression vectors and recombinant cell lines.
  • the invention includes therapeutic compositions related to the novel mammalian renal NADPH oxidases and methods for treating a patient with the therapeutic compositions.
  • the invention includes methods for discovering reagents that effect the function of mammalian Renox.
  • the invention provides a method for treating a mammal in need of Renox therapy.
  • the invention provides a method for sequencing a polynucleotide encoding the novel mammalian renal NADPH oxidases in a biological sample.
  • the invention also provides a method for detecting polymorphisms and/or mutations in a mammalian Renox gene by sequencing the gene using nucleic acids of the current invention.
  • FIG. 1 is a comparison of the deduced amino acid sequences of murine (M.) (SEQ ID NO:1) and human (H.) Renox (SEQ ID NO:2) with that of the murine phagocyte NADPH oxidase homolog gp91 phox (SEQ ID NO:13) (Bjorgvinsdottir, H., et al., "Cloning of murine gp91 phox cDNA and functional expression in a human X-linked chronic granulomatous disease cell line," Blood 87:2005 (1996)).
  • Renox contains all the conserved structural features considered essential for NADPH oxidase activity in gp91 phox , including the six proposed membrane spanning segments (black boxes); FAD binding site (gray box), NADPH binding motifs (open boxes) and proposed heme binding histidines (*) (Leto, T. L., (1999), and references therein; Finegold, A. A., et al., "Intramembrane bis-heme motif for transmembrane electron transport conserved in a yeast iron reductase and the human NADPH oxidase," J. Biol. Chem. 271 :31021 (1996)). Conservative amino acid substitutions in all sequences are indicated in the consensus line as +.
  • FIG. 2 is a Northern blot experiment in which various murine tissue RNAs were probed with an oligonucleotide corresponding to the murine Renox cDNA SEQ ID NO:10. The results reveal high levels of this transcript in the kidney.
  • FIG. 3 shows detection of Renox mRNA in proximal convoluted tubule cells by in situ hybridization. Antisense (A, C, and E) and sense (B) probes demonstrated specific expression of Renox transcripts within the proximal convoluted tubule cells of the renal cortex (CO).
  • Frames A, B, and C represent dark-field images
  • D shows hematoxylin and eosin staining of the field shown in C
  • E represents superimposed polarized epi-illumination and bright-field images in which the signal appear as small lucent dots, ie. small, substantially circular spots which are lighter gray than the surrounding area.
  • High magnification in E shows a strong positive signal in proximal tubule (PT) epithelial cells, while glomeruli (GL; marked by arrowhead in C) and distal tubule (DT) epithelial cells are negative for Renox mRNA expression.
  • PT proximal tubule
  • GL glomeruli
  • DT distal tubule
  • FIG. 4 is a set of bar graphs demonstrating increased production of superoxide when NIH 3T3 cells are transfected with pCDNA3.1 -Renox.
  • A Detection of the Renox message by Northern blot in transfected NIH 3T3 fibroblasts. Lane C1 represents a control cell line transfected with the empty vector, while R10, R15, and R16 correspond to cloned Renox-transfected cell lines.
  • B Detection of superoxide production in Renox-transfected cell lines.
  • the control bar represents cells transfected with empty pCDNA 3.1 vector.
  • the data represent the average response of three control and three Renox- transfected cell lines (shown in A), analyzed in two separate assays.
  • FIG. 5 is a set of bar graphs of proliferation information regarding Renox transfected NIH 3T3 cells versus control cells. On day 1, wells were seeded with 10,000 cells per well of either control (empty vector-transfected) or Renox-transfected cells, allowed to grow for 96 hours, and then counted on day 4.
  • FIG. 6 is a series of micrographs showing the scenescent phenotype that is induced by Renox transfection of NIH 3T3 cells.
  • a and C Control (empty vector-transfected) cells grew faster and exhibited uniform spindle- shaped morphology.
  • B and D Renox transfected cells were heterogeneous, flattened, and enlarged, frequently containing multiple nuclei.
  • FIG. 7 is a model for regulation of erythropoietin synthesis in the kidney showing the proposed role of Renox as an oxygen sensor. In this proposed model, Renox serves as a constitutive oxygen sensor by generating superoxide levels proportional to available oxygen concentrations.
  • HIF-1 At high oxygen tension, downstream reactive oxygen species oxidize HIF-1 and render it susceptible to degradation by schooleasomes. Under hypoxic conditions where reactive oxygen species are generated at lower levels, HIF- 1 ⁇ is stabilized, migrates to the nucleus, and signals the expression of hypoxia inducible genes such as erythropoietin.
  • the current invention consists of isolated nucleic acids encoding mammalian Renox.
  • nucleic acid encoding a Renox polypeptide comprising a nucleotide sequence selected from the group consisting of: a) a first nucleotide sequence selected from the group consisting of SEQ ID NO:3 and SEQ ID NO:4; b) a second nucleotide sequence of a nucleic acid that selectively hybridizes to a nucleic acid comprising a nucleotide sequence complementary to the first nucleotide sequence; c) a third nucleotide sequence that is at least 70% identical to the first nucleotide sequence; and d) a fourth nucleotide sequence of a nucleic acid that encodes the polypeptide encoded by the first, second, or third nucleotide sequence.
  • the isolated nucleic acid encodes the polypeptide of SEQ ID NO:1.
  • the first nucleotide sequence is SEQ ID NO:3.
  • the isolated nucleic acid encodes the polypeptide of SEQ ID NO:2.
  • the first nucleotide sequence is SEQ ID NO:4.
  • nucleic acid and nucleic acid molecule primarily refer to the physical nucleic acid molecule and the phrase “nucleic acid sequence” primarily refers to the sequence of nucleotides on the nucleic acid molecule, the two phrases can be used interchangeably, especially with respect to a nucleic acid molecule, or a nucleic acid sequence encoding Renox.
  • isolated nucleic acid molecule(s) is intended a nucleic acid molecule, DNA, or RNA, which has been removed from its native environment. For example, recombinant DNA molecules contained in a vector are considered isolated for the purposes of the present invention.
  • isolated DNA molecules include recombinant DNA molecules maintained in heterologous host cells or purified (partially or substantially) DNA molecules in solution.
  • Isolated RNA molecules include in vivo or in vitro RNA transcripts of the DNA molecules of the present invention.
  • Isolated nucleic acid molecules according to the present invention further include such molecules produced synthetically.
  • the term "Renox" means a protein comprising a polypeptide backbone related in amino acid sequence to SEQ ID NO:1 and SEQ ID NO:2, wherein the polypeptide backbone comprises the first four hydrophobic segments of SEQ ID NO:1 and SEQ ID NO:2, the minimum size Renox polypeptide sufficient for proton channel activity, based on observations with gp91phox and Mox1.
  • the polypeptide comprises a polypeptide backbone related in amino acid sequence to SEQ ID NO:1 and SEQ ID NO:2, wherein the polypeptide backbone comprises six hydrophobic segments, two of which contain two heme binding histidines, and wherein the polypeptide backbone comprises sequence motifs related to binding sites for flavin and NADPH.
  • Renox proteins of the current invention when expressed recombinantly at high levels in NIH 3T3 cells, significantly increase superoxide production. Additionally, Renox proteins of the current invention are expressed in the kidney at higher levels than in the heart, brain, spleen, lung, liver, skeletal muscle, or testes.
  • Renox is derived from “renal oxidase", since Renox is an oxidase specifically detected in the kidney; its apparent physiological function concerns production of superoxide and related reactive oxygen species within the kidney.
  • Renox nucleic acid means a nucleic acid that encodes a Renox protein.
  • Renox polypeptide means a polypeptide backbone of a Renox protein.
  • nucleic acid refers to a linear array of nucleotides and nucleosides, such as genomic DNA, cDNA, and DNA prepared by partial or total chemical synthesis from nucleotides.
  • encoding means that the nucleic acid may be transcribed and translated into the desired polypeptide.
  • Polypeptide refers to amino acid sequences which comprise both full-length proteins and fragments thereof.
  • An isolated Renox nucleic acid of the present invention can be isolated from its natural source or produced using recombinant DNA technology (e.g., polymerase chain reaction (PCR) amplification or cloning) or chemical synthesis.
  • isolated Renox nucleic acids include a nucleic acid comprising the nucleotide sequence of all or part of SEQ ID NO:3, as well as homologues thereof, and natural allelic variants.
  • the present invention includes nucleic acid molecules modified by nucleotide insertions, deletions, substitutions, and/or inversions of a Renox nucleic acid molecule.
  • a “complementary" nucleic acid sequence refers to the nucleic acid sequence of the nucleic acid strand that is complementary to (i.e., can form a double helix with) the cited nucleic acid molecule.
  • the complementary sequence can easily be determined by those skilled in the art.
  • “Selectively hybridize” in this specification means that two nucleic acids hybridize under stringent conditions.
  • Stringent hybridization conditions are experimental parameters that allow an individual skilled in the art to identify significant similarities between heterologous nucleic acid molecules. These conditions are well known to those skilled in the art. See, for example, Sambrook, et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Labs Press (1989), and Meinkoth, et al., Anal. Biochem. 138, 267 (1984).
  • nucleic acid is comprised of one or more modified nucleotides, such as inosine.
  • Stringent hybridization conditions can be defined mathematically. As explained in detail in the cited references, determination of hybridization conditions involves the manipulation of a set of variables including the ionic strength (M, in moles/liter), the hybridization temperature (°C), the concentration of nucleic acid helix destabilizing agents (such as formamide), the average length of the shortest hybrid duplex (n), and the percent G + C composition of the fragment to which an unknown nucleic acid molecule is being hybridized. For nucleic acid molecules of at least about 150 nucleotides, these variables are inserted into a standard mathematical formula to calculate the melting temperature T m of a given nucleic acid molecule. As defined in the formula below, T m is the temperature at which two complementary nucleic acid molecule strands will disassociate, assuming 100% complementarity between the two strands:
  • T m 81.5°C + 16.6 log M + 0.41 (%G + C) - 500/n - 0.61 (% formamide).
  • hybrid stability is defined by the dissociation temperature T d which is the temperature at which 50% of the duplexes dissociate.
  • T d the stability at which 50% of the duplexes dissociate.
  • T d 4(G + C) + 2(A + T).
  • T d A temperature of 5°C below T d is used to detect hybridization between perfectly matched molecules.
  • Conditions for hybrids between about 50 and about 150 base-pairs can be determined empirically and without undue experimentation using standard laboratory procedures well known to those skilled in the art. (For example, see Sambrook et al., "Molecular Cloning: A Laboratory Manual," eds. N. Ford and C. Nolan, 2d ed., 11.55 1989). In fact, these procedures can be used to determine the T m of any nucleic acid emperically.
  • T m T m
  • these simple procedures for calculating T m allow one skilled in the art to set the hybridization conditions (e.g., altering the salt concentration, the formamide concentration, or the temperature) so that only nucleic acid hybrids with greater than a specified percent base-pair mismatch will hybridize. (Id. at 11.47 and 11.55).
  • T m decreases about 1 °C for each 1% of mismatched base-pairs for hybrids greater than about 150 bp
  • T d decreases about 5°C for each mismatched base-pair for hybrids below about 50 bp.
  • Preferred hybridization solutions such as ExpressHybTM (Clontech
  • hybridizing under either high or low stringency conditions would involve hybridizing a nucleic acid sequence (e.g., the complementary sequence to SEQ ID NO:3 or a portion thereof) with a second target nucleic acid sequence.
  • “High stringency conditions” for the annealing process may involve, for example, high temperature and/or low salt content, which disfavor hydrogen bonding contacts among mismatched base pairs.
  • “Low stringency conditions” would involve lower temperature, and/or higher salt concentration than that of high stringency conditions. Such conditions allow two DNA strands to anneal if substantial, though not complete, complementarity exists between the two strands, as is the case among DNA strands that code for the same protein but differ in sequence due to the degeneracy of the genetic code.
  • Appropriate stringency conditions which promote DNA hybridization are well-known to those skilled in the art.
  • 6X SSC at about 45°C, followed by a wash of 2X SSC at 50°C are known to those skilled in the art. See for example, Current Protocols in Molecular Biology, John Wiley & Sons, NY (1989), 6.31-6.3.6.
  • the salt concentration in the wash step can be selected from a low stringency of about 2X SSC at 50 °C to a high stringency of about 0.2X SSC at 50°C.
  • the temperature in the wash step can be increased from low stringency at room temperature, about 22°C, to high stringency conditions, at about 65°C.
  • Other stringency parameters are described in Sambrook J.
  • the minimum size of an isolated nucleic acid of this invention is a size sufficient to form a stable hybrid (i.e., hybridizing under stringent hybridization conditions) with the complementary sequence of a Renox nucleic acid molecule, and sufficient to produce a Renox polypeptide with the required structural motifs, as defined above.
  • nucleic acid molecule of the present invention can include a portion of a polypeptide-coding sequence, a portion of a gene, an entire polypeptide coding sequence, an entire gene, multiple polypeptide-coding sequences, or multiple genes.
  • the nucleic acids falling within the invention are defined solely in terms of their sequence relatedness to a Renox nucleic acid molecule of the present invention, independent of hybridization stringencies.
  • the nucleic acids of the present invention are greater than about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% identical with a nucleic acid related to Renox nucleic acid molecule of the present invention. It is known in the art that there are commercially available computer programs for determining the degree of similarity between two nucleic acid sequences. These computer programs include various known methods to determine the percentage identity and the number and length of gaps between hybrid nucleic acid molecules.
  • Preferred methods to determine the percent identity among nucleic acid or amino acid sequences include analysis using one or more of the commercially available computer programs designed to compare and analyze nucleic acid or amino acid sequences. These computer programs include, but are not limited to, GCG (available from Genetics Computer Group, Madison, Wl), BLAST sequence alignment algorithms (available on the internet from the National Center for Biological Information currently at "www.ncbi.nlm.nih.gov” and described in Altschul, Stephen F., Thomas L. Madden, Alejandro A. Schaaffer, Jinghui Zhang, Zheng Zhang, Webb Miller, and David J. Lipman (1997), "Gapped BLAST and PSI-BLAST: a new generation of protein database search programs", Nucleic Acids Res.
  • GCG available from Genetics Computer Group, Madison, Wl
  • BLAST sequence alignment algorithms available on the internet from the National Center for Biological Information currently at "www.ncbi.nlm.nih.gov” and described in Altschul, Stephen F
  • a preferred method to determine percent identity among amino acid sequences and also among nucleic acid sequences includes using the Gapped BLAST sequence alignment algorithm using default parameters. Unless otherwise indicated, all nucleotide sequences determined by sequencing a DNA molecule herein were determined using an automated DNA sequencer (e.g., Model 377 from Applied Biosystems, Inc.) and all amino acid sequences of polypeptides encoded by DNA molecules determined herein were predicted by translation of a DNA sequence determined as above. Therefore, as is known in the art for any DNA sequence determined by this automated approach, any nucleotide sequence determined herein may contain some errors.
  • Nucleotide sequences determined by automation are typically at least about 90% identical, more typically at least about 95% to at least about 99.99% identical to the actual nucleotide sequence of the sequenced DNA molecule.
  • the actual sequence can be more precisely determined by other approaches including manual DNA sequencing methods well known in the art.
  • a single insertion or deletion in a determined nucleotide sequence compared to the actual sequence will cause a frame shift in translation of the nucleotide sequence such that the expected amino acid sequence encoded by a determined nucleotide sequence will be completely different from the amino acid sequence actually encoded by the sequenced DNA molecule, beginning at the point of such an insertion or deletion.
  • the Renox nucleic acids of the current invention have numerous utilities including, but not limited to, production of Renox polypeptides, gene therapy of patients with altered Renox function or hyperprol iterative disorders, screening assays for compounds that bind to or effect the activity of Renox, and development of cell lines with altered Renox expression.
  • nucleic acid molecules can be modified using a variety of techniques including, but not limited to, classic mutagenesis and recombinant DNA techniques such as site-directed mutagenesis, chemical treatment, restriction enzyme cleavage, ligation of nucleic acid fragments, PCR amplification, synthesis of oligonucleotides with modified nucleotide sequences, ligation of oligonucleotides with modified nucleotide sequences, and combinations thereof.
  • classic mutagenesis and recombinant DNA techniques such as site-directed mutagenesis, chemical treatment, restriction enzyme cleavage, ligation of nucleic acid fragments, PCR amplification, synthesis of oligonucleotides with modified nucleotide sequences, ligation of oligonucleotides with modified nucleotide sequences, and combinations thereof.
  • Nucleic acid molecule homologues can be selected by selective hybridization with all or a portion of a nucleic acid molecule encoding a mammalian Renox or by screening the function of a protein encoded by the nucleic acid molecule (e.g., ability to stimulate production of superoxide when expressed in NIH 3T3 fibroblasts).
  • the nucleic acids of this aspect of the current invention include allelic variants of mammalian Renox genes.
  • an allelic variant of a mammalian, mouse, or human Renox gene is a gene that occurs at essentially the same locus (or loci) in the genome as the mammalian, mouse, or human Renox polypeptide-coding region described herein, but which, due to natural variations caused by, for example, mutation or recombination, has a similar but not identical sequence. Because natural selection typically selects against alterations that affect function, allelic variants (i.e., alleles corresponding to, or of, cited nucleic acid sequences) usually encode proteins having similar activity to that of the protein encoded by the gene to which they are being compared.
  • Allelic variants of operons or nucleic acid molecules can also comprise alterations in the 5' or 3' untranslated regions of a gene located in the operon (e.g., in regulatory control regions), or can involve alternative splicing of a nascent transcript, thereby bringing alternative exons into juxtaposition. Allelic variants are well known to those skilled in the art and would be expected to occur naturally within a given mammalian Renox gene. Recombinant Expression Vectors.
  • the current invention is a recombinant expression vector comprising a nucleic acid encoding a Renox polypeptide with a nucleic acid sequence selected from the group consisting of: a) a first nucleotide sequence selected from the group consisting of SEQ ID NO:3 and SEQ ID NO:4; b) a second nucleotide sequence of a nucleic acid that selectively hybridizes to a sequence that is complementary to the first nucleotide sequence; c) a third nucleotide sequence that is at least 70% identical to the first nucleotide sequence; and d) a fourth nucleotide sequence of a nucleic acid that encodes the polypeptide encoded by the first, second, or third nucleotide sequence.
  • the nucleic acid encodes the polypeptide of SEQ ID NO:1.
  • the first nucleotide sequence is SEQ ID NO:3.
  • the nucleic acid encodes the polypeptide of SEQ ID NO:2.
  • the first nucleotide sequence is SEQ ID NO:4.
  • This embodiment of the present invention relates to a recombinant vector wherein at least one isolated nucleic acid molecule is inserted into a vector capable of delivering the nucleic acid molecule into a host cell.
  • a vector contains heterologous nucleic acid sequences (i.e., nucleic acid sequences that are not naturally found adjacent to nucleic acid molecules of the present invention) and are preferably derived from a species other than the species from which the nucleic acid molecule(s) are derived.
  • the vector can be either RNA or DNA, either prokaryotic or eukaryotic, and typically is a virus or a plasmid.
  • Recombinant vectors can be used in the cloning, sequencing, producing, and/or otherwise manipulating nucleic acids that encode a mammalian Renox of the present invention.
  • One type of recombinant vector referred to herein as a recombinant molecule, comprises a nucleic acid molecule of the present invention operatively linked to an expression vector.
  • the phrase operatively linked refers to insertion of a nucleic acid molecule into an expression vector in a manner such that the molecule is expressed when present within a host cell.
  • an "expression vector” is a DNA or RNA vector that can be introduced into a host cell and can effect expression of a specified nucleic acid molecule.
  • the expression vector is also capable of replicating within the host cell.
  • Expression vectors of the present invention include any vectors that function (i.e., direct gene expression) in recombinant cells of the present invention, including in bacterial, fungal, parasite, insect, other animal, and/or plant cells.
  • Preferred expression vectors of the present invention can direct gene expression in bacterial, yeast, insect, and mammalian cells.
  • Expression vectors of the present invention contain regulatory sequences such as transcription control sequences, translation control sequences, origins of replication, and other regulatory sequences that are compatible with the recombinant cell and that control the expression of nucleic acid molecules of the present invention.
  • recombinant molecules of the present invention include transcription control sequences, which control the initiation, elongation, and termination of transcription.
  • transcription control sequences are those which control transcription initiation, such as promoter, enhancer, operator, and repressor sequences.
  • Suitable transcription control sequences include any transcription control sequence that can function in at least one of the recombinant cells of the present invention. A variety of such transcription control sequences are known to those skilled in the art.
  • Preferred transcription control sequences include those that function in bacterial, yeast, insect, and/or mammalian cells, such as, but not limited to, tac, lac, trp, trc, oxy-pro, omp/lpp, rrnB, bacteriophage lambda (such as lambda pL and lambda pR and fusions that include such promoters), bacteriophage T7, T7lac, bacteriophage T3, bacteriophage SP6, bacteriophage SP01 , metallothionein, alpha-mating factor, Pichia alcohol oxidase, alphavirus subgenomic promoter, antibiotic resistance gene, baculovirus, Heliothis zea insect virus, vaccinia virus, herpesvirus, raccoon poxvirus, other poxvirus, adenovirus, cytomegalovirus (such as immediate early promoter), simian virus 40, retrovirus, actin, retroviral long terminal repeat,
  • transcription control sequences include tissue-specific promoters and enhancers as well as lymphokine-inducible promoters (e.g., promoters inducible by interferons or interleukins).
  • Transcription control sequences of the present invention can also include naturally occurring transcription control sequences naturally associated with a mammalian Renox gene, such as human Renox and/or mouse Renox transcription control sequences.
  • Recombinant molecules of the present invention can also (a) contain secretory signals (i.e., signal segment nucleic acid sequences) to enable an expressed mammalian Renox protein of the present invention to be secreted from the cell that produces the protein and/or (b) contain fusion sequences that lead to the expression of nucleic acid molecules of the present invention as fusion proteins.
  • suitable signal segments include any signal segment capable of directing the secretion of a protein of the present invention.
  • Preferred signal segments include, but are not limited to, tissue plasminogen activator (t-PA), interferon, interleukin, growth hormone, histocompatibility, and viral envelope glycoprotein signal segments.
  • t-PA tissue plasminogen activator
  • Suitable fusion segments encoded by fusion segment nucleic acids are disclosed herein.
  • a nucleic acid molecule of the present invention can be joined to a fusion segment that directs the encoded protein to the proteosome, such as a ubiquitin fusion segment.
  • Eukaryotic recombinant molecules can also include intervening and/or untranslated sequences surrounding and/or within the nucleic acid sequences of nucleic acid molecules of the present invention.
  • the current invention is a host cell transformed with a nucleic acid comprising a nucleic acid sequence selected from the group consisting of: a) a first nucleotide sequence selected from the group consisting of
  • SEQ ID NO:3 and SEQ ID NO:4 b) a second nucleotide sequence of a nucleic acid that selectively hybridizes to a nucleic acid comprising a nucleotide sequence complementary to the first nucleotide sequence; c) a third nucleotide sequence that is at least 70% identical to the first nucleotide sequence; and d) a fourth nucleotide sequence of a nucleic acid that encodes the polypeptide encoded by the first, second, or third nucleotide sequence.
  • the first nucleotide sequence encodes the polypeptide of SEQ ID NO:1. In another embodiment, the first nucleotide sequence is SEQ ID NO:3. In another embodiment, the first nucleotide sequence encodes the polypeptide of SEQ ID NO:2. In another embodiment, the first nucleotide sequence is SEQ ID NO:4.
  • a transformed host cell may be procaryotic or eukaryotic and may be transformed with one or more nucleic acids.
  • a cell can be "transformed," as the term is used in this specification, with a nucleic acid molecule, such as a recombinant expression vector, by any method by which a nucleic acid molecule can be introduced into the cell. Transformation techniques include, but are not limited to, transfection, infection, electroporation, microinjection, lipofection, adsorption, and protoplast fusion. Transformation may be stable or transient.
  • a recombinant cell can remain unicellular or can grow into a tissue, organ, or a multicellular organism.
  • a cell line refers to any immortalized recombinant cell of the present invention that is not a transgenic animal.
  • Transformed nucleic acid molecules of the present invention can remain extrachromosomal or can integrate into one or more sites within a chromosome of the transformed (i.e., recombinant) cell in such a manner that their ability to be expressed is retained.
  • Suitable host cells include any cell that can be transformed with a nucleic acid molecule of the present invention.
  • Host cells can be either untransformed cells or cells that are already transformed with at least one nucleic acid molecule (e.g., nucleic acid molecules of the present invention and/or other proteins useful in the production of multivalent vaccines).
  • Host cells of the present invention either can be endogenously (i.e., naturally) capable of producing Renox proteins or can be capable of producing such protein after being transformed with at least one nucleic acid molecule of the present invention.
  • Host cells of the present invention can be any cell capable of producing at least one polypeptide of the present invention, and include bacterial, fungal (including yeast), parasite (including helminth, protozoa, and ectoparasite), insect, animal, and plant cells.
  • Preferred host cells include bacterial, mycobacterial, yeast, insect, and mammalian cells.
  • More preferred host cells include Salmonella, Escherichia, Bacillus, Listeria, Saccharomyces, Spodoptera, Mycobacteria, Trichoplusia, BHK (baby hamster kidney) cells, MDCK cells (Madin-Darby canine kidney cell line), CRFK cells (Crandell feline kidney cell line), CV-1 cells (African monkey kidney cell line used, for example, to culture raccoon poxvirus), COS (e.g., COS-7) cells, and Vero cells.
  • Particularly preferred host cells are Escherichia coli, including E. coli K-12 derivatives; Salmonella typhr ' ,
  • Salmonella typhimurium including attenuated strains such as UK-1 3987 and SR-11 4072; Spodoptera frugiperda; Trichoplusia ni; BHK cells; MDCK cells; CRFK cells; CV-1 cells; COS cells; Vero cells; non-tumorigenic mouse myoblast G8 cells (e.g., ATCC CRL 1246), K562 erythroleukemia cells, and mouse NIH/3T3 cells.
  • Additional appropriate mammalian cell hosts include other kidney cell lines, other fibroblast cell lines (e.g., human, murine, or chicken embryo fibroblast cell lines), myeloma cell lines, Chinese hamster ovary cells, LMTK31 cells, and/or HeLa cells.
  • the proteins can be expressed as heterologous proteins in myeloma cell lines employing immunoglobulin promoters.
  • a recombinant cell is preferably produced by transforming a host cell with one or more recombinant molecules, each comprising one or more nucleic acid molecules of the present invention operatively linked to an expression vector containing one or more transcription control sequences, . examples of which are disclosed herein.
  • a recombinant cell of the present invention includes any cell transformed with at least one of any nucleic acid molecule of the present invention. Suitable and preferred nucleic acid molecules as well as suitable and preferred recombinant molecules with which to transfer cells are disclosed herein.
  • Recombinant cells of the present invention can also be co-transformed with one or more recombinant molecules including nucleic acid molecules encoding one or more Renox proteins of the present invention and one or more other nucleic acid molecules encoding other protective compounds, as disclosed herein (e.g., to produce multivalent vaccines).
  • Recombinant DNA technologies can be used to improve expression of transformed nucleic acid molecules by manipulating, for example, the number of copies of the nucleic acid molecules within a host cell, the efficiency with which those nucleic acid molecules are transcribed, the efficiency with which the resultant transcripts are translated, and the efficiency of post-translational modifications.
  • Recombinant techniques useful for increasing the expression of nucleic acid molecules of the present invention include, but are not limited to, operatively linking nucleic acid molecules to high-copy number plasmids, integration of the nucleic acid molecules into one or more host cell chromosomes, addition of vector stability sequences to plasmids, substitutions or modifications of transcription control signals (e.g., promoters, operators, enhancers), substitutions or modifications of translational control signals (e.g., ribosome binding sites, Shine-Dalgamo sequences), modification of nucleic acid molecules of the present invention to correspond to the codon usage of the host cell, deletion of sequences that destabilize transcripts, and use of control signals that temporally separate recombinant cell growth from recombinant enzyme production during fermentation.
  • the activity of an expressed recombinant protein of the present invention can be improved by fragmenting, modifying, or derivatizing nucleic acid molecules encoding such a protein.
  • the transformed host cells are immortalized cell lines capable of expressing high levels of Renox mRNA.
  • a detailed procedure for establishing an immortalized cell line capable of expressing high levels of Renox mRNA is given in the Examples section below.
  • a preferred embodiment of the present invention is an NIH 3T3 cell line stably transfected or infected with a Renox expression vector, wherein expression of Renox is controlled by an inducible promoter. This preferred embodiment allows normal proliferation rates of NIH 3T3 cells until expression of Renox is necessary, whereby proliferation rates of NIH 3T3 cells typically decrease.
  • the current invention is an isolated Renox polypeptide or a fragment thereof, comprising an amino acid sequence encoded by a nucleic acid sequence selected from the group consisting of:
  • the isolated polypeptide comprises SEQ ID NO:1.
  • the isolated polypeptide is a fragment of a polypeptide of SEQ ID NO:1.
  • the isolated polypeptide is SEQ ID NO:1.
  • the isolated polypeptide comprises SEQ ID NO:2. In another embodiment, the isolated polypeptide is a fragment of a polypeptide of SEQ ID NO:2. In yet another embodiment, the isolated polypeptide is SEQ ID NO:2.
  • the minimum size of a Renox polypeptide of the present invention is a size sufficient to provide six hydrophobic segments, two of which contain two heme binding histidines, and sequence motifs related to binding sites for flavin and NADPH. These structures are believed to be required for Renox to function as a source of superoxide and protons.
  • the minimum size Renox polypeptide sufficient for the proton channel activity of Renox is expected to encompass the first four hydrophobic segments, based on observations with gp91phox and Mox1. There is no limit, other than a practical limit, on the maximal size of a Renox polypeptide of the present invention because a Renox polypeptide can include sequences in addition to those composing the required motifs listed above.
  • Renox polypeptides of the present invention is a fusion protein that includes domains characteristic of a Renox protein, as described above, attached to one or more fusion segments.
  • Suitable fusion segments for use with the present invention include, but are not limited to, segments that can enhance a protein's stability, act as an immunopotentiator to enhance an immune response against a Renox polypeptide, and/or assist in purification of a Renox polypeptide (e.g., by affinity chromatography).
  • a suitable fusion segment can be a domain of any size that has the desired function (e.g., imparts increased stability or increased immunogenicity on a protein, and/or simplifies purification of a protein).
  • Fusion segments can be joined to the amino and/or carboxyl termini of a Renox polypeptide and can be susceptible to cleavage in order to enable straightforward recovery of the Renox polypeptide.
  • Fusion proteins are preferably produced by culturing a recombinant cell transformed with a fusion nucleic acid molecule that encodes a protein including the fusion segment attached to either the carboxyl and/or amino terminal end of a Renox polypeptide.
  • Preferred fusion segments include a metal binding domain (e.g., a poly-histidine segment), an immunoglobulin binding domain (e.g., Protein A; Protein G; T cell; B cell; Fc receptor or complement protein antibody-binding domains), a sugar binding domain (e.g., a maltose binding domain), and/or a "tag" domain (e.g., at least a portion of ⁇ -galactosidase, a strep tag peptide, a T7 tag peptide, a Flag peptide, or other domains that can be purified using compounds that bind to the domain, such as monoclonal antibodies). More preferred fusion segments include metal binding domains, such as a poly-histidine segment; a maltose binding domain; a strep tag peptide, such as that available from Biometra in Tampa, FL; and an S10 peptide.
  • a metal binding domain e.g., a poly-histidine segment
  • Renox polypeptides can be the result of natural allelic variation or natural mutation.
  • Renox polypeptides of the present invention can also be produced using techniques known in the art including, but not limited to, direct modifications to the polypeptides or modifications to the nucleic acid molecule encoding the polypeptide using, for example, classic or recombinant DNA techniques to effect random or targeted mutagenesis.
  • a Renox polypeptide of the present invention can be used in, for example, assays to detect pharmaceutical compounds that interact with the Renox polypeptide, or inhibit enzymatic activity of the Renox polypeptide.
  • Pharmaceuticals identified in this way are candidate compounds to regulate the following physiological processes: 1) in vivo production of erythropoietin (EPO) or other hypoxia-responsive gene products, 2) inflammatory signaling processes in the kidney related to superoxide production by Renox, 3) antimicrobial activity of ROS metabolites derived from superoxide, which is produced in the kidney by Renox, and 4) functions of Renox related to its effects on proton and sodium transport in kidney tubule cells.
  • EPO erythropoietin
  • Polypeptides of the invention can also be used in therapeutic compositions to treat disorders, especially disorders involving reduced Renox function, as in auto-immune disorders where antibodies against Renox may affect its function.
  • the polypeptides are delivered in a form that facilitates uptake by kidney cells, although delivery to other cells may also provide therapeutic benefit (e.g., tumor cells, vascular cells).
  • the polypeptides of the invention also can be used to generate anti-Renox antibodies, which are useful for detecting cells derived from the kidney or for screening an individual's polypeptides to detect the presence of a polymorphism or mutant version of the polypeptide.
  • the polypeptides of the invention can be used as controls in assays that utilize the anti-Renox antibodies.
  • Renox polypeptides and “Renox nucleic acids” refer to polypeptides and nucleic acid molecules, respectively, that can be isolated from an organism or prepared recombinantly or synthetically.
  • An isolated Renox polypeptide of the present invention includes a polypeptide that is removed from its natural milieu. As such, the term “isolated” does not describe any specific level of purity of the isolated polypeptide.
  • the present invention also includes mimetopes of Renox polypeptides.
  • a "mimetope" of a Renox polypeptide of the present invention refers to a compound that is able to mimic the activity of such a Renox polypeptide, often because the mimetope has a structure that mimics the particular Renox polypeptide.
  • Mimetopes can be, but are not limited to: peptides that have been modified to decrease their susceptibility to degradation such as all-D retro peptides; anti-idiotypic and/or catalytic antibodies, or fragments thereof; non-proteinaceous immunogenic portions of an isolated protein (e.g., carbohydrate structures); and synthetic or natural
  • mimetopes can be designed using computer-generated structures of polypeptides of the present invention. Mimetopes can also be obtained by generating random samples of molecules, such as oligonucleotides, peptides or other organic molecules, and screening such samples by affinity chromatography techniques using the corresponding binding partner.
  • Renox polypeptides of the present invention can be produced in a variety of ways, including methods known in the art involving production and recovery of natural proteins, methods involving production and recovery of recombinant proteins, and chemical synthesis of the polypeptides.
  • One method of producing the Renox polypeptides is a method for producing polypeptides in a host cell, described below.
  • polypeptides of the present invention can be produced from natural sources.
  • Mammalian kidney cells or kidney cell lines can be propagated by standard cell culture methodologies, and Renox polypeptides can be purified therefrom using methods well-known to those skilled in the art.
  • mammalian kidneys can be used as starting material for the isolation of Renox polypeptides.
  • Standard protein purification protocols can be used and fractions containing Renox activity can be identified and used in the development of a purification regime.
  • Protein production strategies from natural sources may utilize an enhancement of Renox production or function to obtain larger quantities and more enriched fractions of Renox proteins.
  • the enhancement of Renox production may be accomplished by targeted gene transfer or by induction of endogenous expression by drugs affecting the promoter activity of the Renox gene which can be identified utilizing the teaching of the current invention.
  • the current invention is a method of producing a Renox polypeptide in a host cell comprising the steps of: a) introducing a Renox nucleic acid which expresses a Renox polypeptide in the host cell into a vector, thereby producing a Renox expression vector; b) introducing the Renox expression vector into the host cell to produce an engineered host cell; c) maintaining the engineering host cell under conditions suitable for the expression of the Renox polypeptide by the engineered host cell; and d) collecting the Renox polypeptide produced by the engineered host cell.
  • the Renox polypeptide comprises SEQ ID NO:1. In another preferred embodiment, the Renox polypeptide comprises SEQ ID NO:2. In another preferred embodiment of this aspect of the invention, the Renox polypeptide is post- translationally modified to form a Renox protein.
  • the host cell may be procaryotic or eukaryotic.
  • a preferred host cell is a host cell that post-translationally modifies a Renox polypeptide to form a Renox protein.
  • Effective culture conditions include, but are not limited to, effective media, bioreactor, temperature, pH, and oxygen conditions that permit protein production.
  • An effective medium refers to any medium in which a cell is cultured to produce a Renox polypeptide of the present invention.
  • Such medium typically comprises an aqueous medium having assimilable carbon, nitrogen, and phosphate sources, and appropriate salts, minerals, metals, and other nutrients, such as vitamins.
  • Cells of the present invention can be cultured in conventional fermentation bioreactors, shake flasks, test tubes, microtiter dishes, and petri plates. Culturing can be carried out at a temperature, pH, and oxygen content optimal for a particular host cell. Such culturing conditions are within the expertise of one of ordinary skill in the art.
  • resultant polypeptides of the present invention can either remain within the recombinant cell; be secreted into the fermentation medium; be secreted into a space between two cellular membranes, such as the periplasmic space in E. coli; or be retained on the outer surface of a cell or viral membrane.
  • polypeptides of the present invention can be purified using a variety of standard protein purification techniques, such as, but not limited to, affinity chromatography, ion exchange chromatography, filtration, electrophoresis, hydrophobic interaction chromatography, gel filtration chromatography, reverse phase chromatography, chromatofocusing, and differential solubilization. Polypeptides of the present invention are preferably retrieved in substantially pure form.
  • substantially pure refers to a purity that allows for the effective use of the protein as a therapeutic composition or diagnostic.
  • a therapeutic composition for animals for example, should exhibit no substantial toxicity and preferably should be capable of stimulating the production of antibodies in a treated animal.
  • Methods for producing the polypeptides of the present invention have utility in that they provide polypeptides for the uses described above, such as, for example, screening for compounds that bind or regulate the function of Renox polypeptides. Additionally, the methods for producing Renox polypeptides provide polypeptides for antibody production and for controls in assays which measure Renox concentration or activity. Method for Detecting a Mammalian Renox Gene, or Portion
  • This aspect of the invention typically involve selectively hybridizing a detection Renox oligonucleotide to a target nucleic acid in a sample to allow detection or sequencing of the sample Renox nucleic acid.
  • the detection or sequencing involves a downstream oligonucleotide that selectively hybridizes to the Renox gene or fragment thereof in an orientation and location that facilitates amplification of a region of the Renox gene or fragment thereof between the detection oligonucleotide and the downstream oligonucleotide using an amplification procedure.
  • Amplification procedures are well-known in the art, and include, but are not limited to, the polymerase chain reaction (PCR) (Saiki, et al., "Enzymatic amplification of beta-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia," Science 239:487 (1985) Sambrook, et al., Molecular Cloning: A Laboratory Manual, CSH Press 1989, pp.14.2-14.33) and the ligase chain reaction (LCR).
  • PCR polymerase chain reaction
  • LCR ligase chain reaction
  • “Selectively hybridizing with a mammalian Renox nucleic acid” means that a detection oligonucleotide hybridizes with a mammalian Renox nucleic acid in a sample under certain hybridizations conditions under which it does not hybridize with other nucleic acids in the sample to an extent which interferes with detection or sequencing of the Renox nucleic acid.
  • the detection oligonucleotide selectively hybridizes with a Renox nucleic acid because it is more identical in sequence to a Renox nucleic acid than any other nucleic acid in the sample.
  • any nucleic acid related to a mammalian Renox nucleic acid which is capable of selectively hybridizing to a target nucleic acid sequence may be regarded as an appropriate detection oligonucleotide.
  • the detection oligonucleotide and downstream oligonucleotide, when present, are typically at least about 15 nt. Where the detection oligonucleotide is used as a probe for a target sequence, it is preferably at least about 20 nt, more preferably at least about 30 nt, and even more preferably, at least about 50 nt in length.
  • fragments 100, 150, 200, 250, 300, 350, 400, 450, and 500 nt in length are also useful as detection oligonucleotides according to the present invention, as are fragments corresponding to most, if not all, of the nucleotide sequence of SEQ ID NO:14 and/or SEQ ID NO:4.
  • detection oligonucleotides and downstream oligonucleotides will include at least 8 or 9, more preferably 10- 50, and most preferably 18-24 consecutive nucleotides from the mammalian Renox gene and related to SEQ ID NO: 14, or SEQ ID NO:4.
  • oligonucleotides which include 20 or more contiguous bases from a Renox nucleic acid. Since the nucleotide sequence of several mammalian Renox nucleic acids is disclosed in SEQ ID NO: 14 and SEQ ID NO:4, generating such DNA fragments would be routine to the skilled artisan. For example, restriction endonuclease cleavage or shearing by sonication could easily be used to generate fragments of various sizes. Alternatively, such fragments could be generated synthetically.
  • a detection oligonucleotide of the current aspect of the invention may be labeled with a detectable moiety.
  • Suitable labels include fluorochromes, (e.g., fluorescein isothiocyanate (FITC)), rhodamine, Texas Red, phycoerythrin, allophycocyanin, 6-carboxyfluorescein (6-FAM), 2 ⁇ 7 , -dimethoxy-4 , ,5'-dichloro-6-carboxyfluorescein (JOE), 6-carboxy-X-rhodamine (ROX), ⁇ -carboxy ⁇ ' ⁇ ' ' ⁇ -hexachlorofluorescein (HEX), 5-carboxyfluorescein (5-FAM) or N,N,N ⁇ N'-tetramethyl-6- carboxyrhodamine (TAMRA), radioactive labels (e.g., 32 P, 35 S, 3 H), and the like.
  • fluorochromes e.
  • the label may be a component of a binding pair detection system, wherein the label is biotin, haptens, and the like, having a high affinity binding partner (e.g., avidin, specific antibodies, and the like) where the binding partner is conjugated to a detectable label.
  • a mammalian Renox nucleic acid any sample can be analyzed, providing it contains, or is suspected of containing, a mammalian Renox nucleic acid. If the sample is impure, it may be treated before amplification with an amount of a reagent effective to open cells, or animal cell membranes of the sample, and to expose and/or separate the strand(s) of the nucleic acid(s). This lysing and nucleic acid denaturing step to expose and separate the strands will allow hybridization to occur much more readily.
  • the target nucleic acid may be mRNA, cDNA, or genomic DNA.
  • genomic DNA utilized for this aspect of the invention may be extracted from a body sample, such as blood, tissue material, and the like by a variety of techniques such as that described by Maniatis, et. al. in Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, N.Y., p 280-281 , (1982)).
  • the target nucleic acid may be a fraction of a larger molecule or can be present initially as a discrete molecule, so that the specific sequence constitutes the entire nucleic acid. It is not necessary that the sequence to be analyzed be present in a pure form; it may be a minor fraction of a complex mixture, such as contained in a preparation of whole human DNA.
  • the method comprises: a) contacting the sample with a detection oligonucleotide capable of selectively hybridizing with a target mammalian Renox nucleic acid or portion thereof, wherein said detection oligonucleotide comprises contiguous nucleotides of a nucleic acid selected from the group consisting of: i) a first nucleotide sequence selected from the group consisting of SEQ ID NO:3 and SEQ ID NO:4; ii) a second nucleotide sequence that is fully complementary to the first nucleotide sequence; and iii) a third nucleotide sequence of a nucleic acid that selectively hybridizes to the first or second nucleotide sequence; and iv) a fourth nucleotide sequence that is at least 70% identical to the first, second, or third nucleotide sequence; and b) detecting the detection nucleotide, thereby detecting the target mammalian Renox nucleic acid or portion thereof, wherein
  • This method for detecting a mammalian Renox nucleic acid, or portion thereof has many utilities.
  • the method can be used to detect Renox messenger RNA expression in patients with abnormal kidney function to determine whether the abnormal kidney function correlates with abnormal Renox expression.
  • the method can also be used to identify an unidentified adult mammalian cell as a kidney cell.
  • the method can be used to localize precise genomic location of mammalian Renox genes within the genome of a mammalian species to assist in determining whether a particular mammalian Renox gene is involved with certain inherited disorders such as polycythemia.
  • tissue of a mammal is analyzed with Northern blotting using a detection oligonucleotide selected from SEQ ID NO:10 or SEQ ID NO:11.
  • the presence of a product after an amplification reaction indicates hybridization of the detection oligonucleotide and downstream oligonucleotide to the target nucleic acid.
  • Visualization of such products can be effected by methods known in the art (e.g., autoradiography, fluorometry, colorimetry, and the like).
  • in situ hybridization may be used to detect binding of a detection oligonucleotide to a target nucleic acid.
  • the detection oligonucleotide is an RNA transcript, such as illustrated in the Examples.
  • the invention is a general method for detecting polymorphisms or mutations in a mammalian Renox nucleic acid or portions thereof, preferably a mammalian Renox gene.
  • this aspect may involve methods analyzing protein products of mammalian Renox nucleic acids, preferably, the general method comprises a method determining the nucleotide sequence of a mammalian Renox nucleic acid or portion thereof in a sample.
  • the steps of this method include: a) contacting the sample with a detection oligonucleotide capable of selectively hybridizing with a target mammalian Renox nucleic acid or portion thereof, wherein said detection oligonucleotide comprises contiguous nucleotides of a nucleic acid selected from the group consisting of: i) a first nucleotide sequence selected from the group consisting of SEQ ID NO:14 and SEQ ID NO:4; ii) a second nucleotide sequence that is fully complementary to the first nucleotide sequence; and iii) a third nucleotide sequence of a nucleic acid that selectively hybridizes to the first nucleotide or second sequence; and iv) a fourth nucleotide sequence that is at least 70% identical to the first, second, or third nucleotide sequence; b) incubating the sample with the detection oligonucleotide under selective hybridization conditions, thereby allowing selective hybridization of
  • the nucleotide sequence of the detection oligonucleotide sequence is SEQ IN NO: 14. In another embodiment the nucleotide sequence of the detection oligonucleotide is SEQ ID NO:4.
  • the sequences for this aspect of the invention include single nucleotide polymorphisms or other sequence variants that comprise polymorphisms and mutant versions of mammalian Renox genes.
  • sequence determination assay means any method that utilizes selective hybridization of an oligonucleotide to a target nucleic acid to provide information regarding the nucleotide sequence of the target nucleic acid. Many sequence determination assays are known in the art.
  • sequence determination assays include, hybridization using sequence-specific oligonucleotides, restriction enzyme digests and mapping, PCR reactions modified for sequence determination, RNase protection, ligase-mediated detection, and various other methods. Where additional amounts of target nucleic acids are required for a sequence determination, an amplification or cloning method can be employed as part of, or before, the sequence determination assay.
  • the DNA may be isolated and used directly for detection of a specific sequence, or may be amplified by an amplification method, such as the polymerase chain reaction (PCR), or a cloning method prior to analysis. Similarly, RNA or cDNA may also be used, with or without amplification.
  • PCR polymerase chain reaction
  • RNA or cDNA may also be used, with or without amplification.
  • This sequencing embodiment of this aspect of the invention permits analysis of a patient sample for the presence of polymorphisms or mutations associated with a disease state or genetic predisposition to a disease state.
  • polymorphisms is meant naturally-occurring variants of a gene that involve minor alterations in nucleotide sequence that are not believed to be directly involved in pathology.
  • mutants relatively substantial changes in a nucleotide sequence of a gene, typically a base change or a gain or loss of base pair(s) in a nucleic acid sequence, which results in a nucleic acid which codes for a non-functioning protein or a protein with substantially reduced or altered function. Analysis may be performed to determine whether a sequence polymorphism or mutation in a Renox coding region or control region is associated with disease. Disease associated mutations may also include deletion or truncation of the gene, nucleotide sequence changes that alter expression level, and the like.
  • Diseases that are most likely to be associated with Renox mutations are diseases that involve altered function of the kidney. Such diseases include anemia associated with decreased production of erythropoietin, such as in chronic renal failure or other acute or chronic inflammatory disease states. Other diseases involving the kidney that may be correlated with polymorphisms and mutations in a mammalian Renox gene include polycythemia, diseases of enhanced susceptibility to microbial infections of the urogenital tract, or other conditions where normal proton, sodium, and potassium transport properties of kidney tubule cells are affected. Diagnosis of inherited cases of these diseases can be accomplished based on the methods of the current aspect of the invention.
  • One embodiment of this aspect of the invention utilizes a detection oligonucleotide that hybridizes near the 5' or 3' end of SEQ ID NO:14 or SEQ ID NO:4. Hybridization of an oligonucleotide to an end of SEQ ID NO:14 or SEQ ID NO:4 permits sequence analysis of regions of a mammalian Renox gene outside the coding sequence.
  • Such non-coding sequences are well known in the art generally to be involved with regulation of expression of a given gene. Important non-coding sequences that are involved in the regulation of expression are promoter and enhancer sequences.
  • changes in the nucleotide sequence of mammalian Renox promoter or enhancer sequences can be determined.
  • genomic DNA is the source for target nucleic acid in the sample where non-coding sequences, and more typically, untranscribed sequences are targeted for analysis.
  • Changes that may affect expression levels of a mammalian Renox gene can be identified using this aspect of the invention.
  • Expression levels of different allelic versions of promoter or enhancer sequences of a mammalian Renox gene can be compared by various methods known in the art.
  • Methods for determining promoter or enhancer strength include quantitation of the expressed natural protein and quantitation of a reporter gene after insertion of the variant promoter or enhancer element into a vector with the reporter gene and transfection of the vector into a host cell.
  • reporter genes included, but are not limited to, ⁇ -galactosidase, luciferase, chloramphenicol acetyltransferase, and the like that provide for convenient quantitation.
  • Target oligonucleotides and downstream oligonucleotides for this aspect of the current invention preferably are subsequences of a mammalian Renox gene in which mutations and polymorphisms occur. Particularly contemplated as useful will be sequences from a mammalian Renox gene in which disease-causing mutations are known to be present, or sequences which flank these disease-causing mutations. Therefore, the detection oligonucleotides of this aspect of the present invention can be directed to any portion of a mammalian Renox gene corresponding to normal and/or mutant sequences, including introns and 5' and 3' untranslated regions, which may be shown to be associated with the development of disease.
  • the detection oligonucleotide may include specific mutated nucleotides.
  • the sequence determination assay comprises detecting selective Renox-subtype hybridization, wherein the detection oligonucleotide is capable of selectively hybridizing to a wild-type or first polymorphic Renox nucleic acid but not a mutant or second polymorphic Renox nucleic acid.
  • a blotting procedure can be used to detect binding, such as the blotting procedures discussed above.
  • the sequence determination assay utilizes the detection oligonucleotide as a primer for a sequencing reaction.
  • Sequencing reactions such as, but not limited to, the Sanger dideoxy-mediated chain-termination method, are well-known in the art. See Sambrook et al., Molecular Cloning: A Laboratory Manual, (Cold Spring Harbor Laboratory Press): 13.42-13.104 (1989).
  • sequence determination assay comprises an amplification assay
  • sequence determination is solely performed using the amplification assay, or the sequence determination is performed using another methodology that requires an amplified target nucleic acid.
  • Well- known methods have been developed to use PCR to sequence a target nucleic acid (e.g., CyclistTM DNA Sequencing Kits, Stratagene, La Jolla, CA).
  • methods have been developed for using the ligase chain reaction for sequence analysis and the identification of polymorphisms and mutations. See, e.g., Edelstein R. E.
  • the detection oligonucleotide and downstream oligonucleotide are derived from the regions flanking a mutation, and correspond to positions anywhere from 1 to 1000 bp, but preferably 1 to 200 bp, removed from the site of the mutation.
  • PCR primers which are 5' to the mutation site (on the coding strand) should correspond in sequence to the coding strand of the Renox gene whereas PCR primers which are 3' to the mutation site (on the coding strand) should correspond to the non-coding or antisense strand.
  • SSCP denaturing gradient gel electrophoresis
  • DGGE denaturing gradient gel electrophoresis
  • heteroduplex analysis in gel matrices.
  • the sample can digested with that endonuclease, and the products size fractionated to determine whether the fragment was digested. Fractionation can be performed by gel or capillary electrophoresis, particularly acrylamide or agarose gels.
  • the current invention is a diagnostic kit for detecting nucleic acids of a mammalian Renox nucleic acid or portion thereof, wherein the kit comprises: a) a detection oligonucleotide comprising a contiguous nucleotide sequence selected from the group consisting of: i) a first nucleotide sequence selected from the group consisting of SEQ ID NO:3 and SEQ ID NO:4; ii) a second nucleotide sequence that is fully complementary to the first nucleotide sequence; and iii) a third nucleotide sequence of a nucleic acid that selectively hybridizes to the first or second nucleotide sequence; and iv) a fourth nucleotide sequence that is at least 70% identical to the first, second, or third nucleotide sequence; and b) a reagent for detecting binding of the detection oligonucleotide to target mamm
  • the reagent for detecting binding of the nucleic acid is a reagent for performing a hybridization or amplification method (e.g., Southern hybridization, dot blot hybridization, PCR, SDA, and the like) such as a second nucleic acid that can complement the nucleic acid of the invention as a primer in an amplification reaction (i.e., the downstream nucleic acid described above), or an enzyme for amplifying a target nucleic acid upon binding of a primer.
  • the kit may further include other components and reagents for performing a hybridization or amplification method as disclosed above. This kit aspect of the invention is especially useful for performing the method of detecting polymorphisms and mutations in a mammalian Renox gene described above.
  • kit for this aspect of the invention are typically packaged together in a common container, including written instructions for performing selected specific embodiments of the methods disclosed herein.
  • Components for detection methods may optionally be included in the kit, for example, a second probe, and/or reagents and means for performing label detection (e.g., radiolabel, enzyme substrates, antibodies, and the like).
  • the current invention is a therapeutic composition wherein the active ingredient is related to mammalian Renox.
  • the therapeutic comprises a mammalian Renox protein, polypeptide, or nucleic acid, most preferably a nucleic acid.
  • the therapeutic composition includes a pharmaceutically acceptable carrier.
  • the invention provides for treatment or prevention of various diseases and disorders by administration of a therapeutic compound or compounds.
  • therapeutics include but are not limited to: Renox proteins and analogs and derivatives (including fragments) thereof; antibodies thereto (as described herein); Renox nucleic acids; mammalian Renox antisense nucleic acids, and mammalian Renox agonists and antagonists.
  • Renox proteins and analogs and derivatives including fragments thereof; antibodies thereto (as described herein); Renox nucleic acids; mammalian Renox antisense nucleic acids, and mammalian Renox agonists and antagonists.
  • a human Renox protein, derivative, or analog, or human Renox nucleic acid, or an antibody to a human Renox protein is therapeutically or prophylactically administered to a human patient.
  • the invention provides methods of treatment (and prophylaxis) by administering to a subject an effective amount of a therapeutic of the invention.
  • the therapeutic is substantially purified.
  • the subject is preferably an animal, including but not limited to, cows, pigs, horses, chickens, cats, dogs, and the like.
  • the subject is a human.
  • Various delivery systems are known and can be used to administer a therapeutic of the invention. Such systems include, for example, encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing the therapeutic (see, e.g., Wu and Wu, "Receptor- mediated in vitro gene transformation by a soluble DNA carrier system," J. Biol. Chem.
  • Methods of introduction include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes.
  • the compounds may be administered by any convenient route, including, for example, infusion or bolus injection, absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, and the like) and may be administered together with other biologically active agents. Administration can be systemic or local.
  • intraventricular injection may be facilitated by an intraventricular catheter, for example, attached to a reservoir, such as an Ommaya reservoir.
  • Pulmonary administration can also be employed (e.g., by an inhaler or nebulizer using a formulation containing an aerosolizing agent).
  • compositions of the invention may be desirable to administer the pharmaceutical compositions of the invention locally to the area in need of treatment; this may be achieved by, for example, and not by way of limitation, local infusion during surgery, topical application (e.g., wound dressing after surgery), injection, catheter, suppository, or implant (e.g., implants formed from porous, non-porous, or gelatinous materials, including membranes, such as sialastic membranes, or fibers), and the like.
  • administration can be by direct injection at the site (or former site) of a malignant tumor or neoplastic or preneoplastic tissue.
  • administration can be by direct application to the kidney.
  • the therapeutic can be delivered in a vesicle, in particular a liposome (see Langer, ⁇ ew methods of drug delivery, " Science 249:1527 (1990); Treat et al., in Liposomes in the Therapy of Infectious Disease and Cancer, Lopez-Berestein and Fidler (eds.), Liss, N.Y., pp. 353-365 (1989)).
  • a liposome see Langer, ⁇ ew methods of drug delivery, " Science 249:1527 (1990); Treat et al., in Liposomes in the Therapy of Infectious Disease and Cancer, Lopez-Berestein and Fidler (eds.), Liss, N.Y., pp. 353-365 (1989)).
  • the therapeutic can be delivered in a controlled release system.
  • a pump may be used (see Langer, (1990); Sefton, "Implantable pumps,” Crit. Rev. Biomed. Eng. 14:201 (1987); Buchwald et al., “Long-term, continuous intravenous heparin administration by an implantable infusion pump in ambulatory patients with recurrent venous thrombosis," Surgery 88:507 (1980); and Saudek et al., "A preliminary trial of the programmable implantable medication system for insulin delivery," N. Engl. J. Med. 321 :574 (1989)).
  • polymeric materials can be used (see Ranger et al., Macromol. Sci. Rev.
  • This aspect of the present invention typically includes a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable means approved by a regulatory agency of the federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans.
  • carrier refers to a diluent, adjuvant, excipient, or vehicle with which the therapeutic is administered.
  • Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. Water is a preferred carrier when the pharmaceutical composition is administered intravenously.
  • Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions.
  • suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol, and the like.
  • the therapeutic if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. These therapeutics can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations, and the like.
  • the therapeutic can be formulated as a suppository, with traditional binders and carriers such as triglycerides.
  • Oral formulation can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, and the like. Examples of suitable pharmaceutical carriers are described in "Remington's
  • Such therapeutics will contain a therapeutically effective amount of the active ingredient, preferably in purified form, together with a suitable amount of carrier so as to provide proper administration to the patient.
  • the formulation should suit the mode of administration.
  • the composition is formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous administration to human beings.
  • compositions for intravenous administration are solutions in sterile isotonic aqueous buffer.
  • the composition may also include a solubilizing agent and a local anesthetic such as lignocaine to ease pain at the site of the injection.
  • the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent.
  • composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline.
  • an ampule of sterile water or saline can be provided so that the ingredients may be mixed prior to administration.
  • the amount of the therapeutic of the invention which will be effective in the treatment of a particular disorder or condition will depend on the nature of the disorder or condition, as well as the stage of the disorder or condition. Effective amounts can be determined by standard clinical techniques. In addition, in vitro assays, such as those described below, may optionally be employed to help identify optimal dosage ranges. The precise dose to be employed in the formulation will also depend on the route of administration, and should be decided according to the judgment of the practitioner and each patient's circumstances. However, suitable dosage ranges for intravenous administration are generally about 20 to about 500 micrograms of active compound per kilogram body weight. Suitable dosage ranges for intranasal administration are generally about 0.01 pg/kg body weight to about 1 mg/kg body weight.
  • Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems.
  • Suppositories generally contain active ingredient in the range of about 0.5% to about 10% by weight; oral formulations preferably contain about 10% to about 95% active ingredient.
  • the invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the therapeutics of the invention.
  • the therapeutics of the current invention promote Renox function and are used to treat diseases and disorders involving cell overproliferation.
  • therapeutics include, but are not limited to, nucleic acids encoding mammalian Renox under the control of a strong inducible promoter, particularly that are active in inhibiting cell proliferation (e.g., as demonstrated in in vitro assays (as described below) or in animal models).
  • Other therapeutics e.g., Renox proteins
  • therapeutics that promote Renox function are administered therapeutically (including prophylactically): (1) in diseases or disorders involving a decreased (relative to normal or desired) level of Renox protein or function, for example, in patients where Renox protein is under- expressed, genetically defective, biologically hypoactive, or depleted by autoimmune antibodies; or (2) in diseases or disorders wherein in vitro (or in vivo) assays indicate the utility of Renox agonist administration.
  • Decreased level in Renox protein or function can be readily detected by obtaining a patient tissue sample (e.g., from biopsy tissue) and assaying it in vitro for RNA or protein levels, structure, and/or activity of the expressed Renox or protein.
  • Renox protein e.g., Western blot, immunoprecipitation followed by sodium dodecyl sulfate polyacrylamide gel electrophoresis, immunocytochemistry, and the like
  • hybridization assays to detect Renox expression by detecting and/or visualizing Renox mRNA (e.g., Northern assays, dot blots, in situ hybridization, and the like).
  • ectopic expression of Renox may be a means for limiting cell growth in disorders involving hyperproliferation, such as cancer, either by inducing cancer cell senescence or inhibiting angiogenesis.
  • malignancy or dysproliferative changes such as metaplasias and dysplasias
  • hyperproliferative disorders are treated or prevented in the brain, breast, colon, prostate, lung, kidney, skin, or other tissues or organs in which undesirable proliferative changes occur.
  • carcinoma, melanoma, or leukemia can be treated or prevented using the components of this invention.
  • the therapeutics of the invention that agonize and promote Renox activity can also be administered to treat premalignant conditions and to prevent progression to a neoplastic or malignant state.
  • Such prophylactic or therapeutic use is indicated in conditions known or suspected of preceding progression to neoplasia or cancer, in particular, where non-neoplastic cell growth consisting of hyperplasia, metaplasia, or most particularly, dysplasia has occurred.
  • Hyperplasia is a form of controlled cell proliferation involving an increase in cell number in a tissue or organ, without significant alteration in structure or function. As but one example, endometrial hyperplasia often precedes endometrial cancer. Metaplasia is a form of controlled cell growth in which one type of adult or fully differentiated cell substitutes for another type of adult cell. Metaplasia can occur in epithelial or connective tissue cells. Atypical metaplasia involves a somewhat disorderly metaplastic epithelium.
  • Dysplasia is frequently a forerunner of cancer, and is found mainly in the epithelia; it is the most disorderly form of non-neoplastic cell growth, involving a loss in individual cell uniformity and in the architectural orientation of cells.
  • Dysplastic cells often have abnormally large, deeply stained nuclei, and exhibit pleomorphism.
  • Dysplasia characteristically occurs where there exists chronic irritation or inflammation, and is often found in the cervix, respiratory passages, oral cavity, and gall bladder.
  • the presence of one or more characteristics of a transformed phenotype, or of a malignant phenotype, displayed in vivo or displayed in vitro by a cell sample from a patient can indicate the desirability of prophylactic/therapeutic administration of a therapeutic that promotes Renox function.
  • characteristics of a transformed phenotype include morphology changes, looser substratum attachment, loss of contact inhibition, loss of anchorage dependence, protease release, increased sugar transport, decreased serum requirement, and expression of fetal antigens.
  • a patient which exhibits one or more of the following predisposing factors for malignancy is treated by administration of an effective amount of a therapeutic of this embodiment of the present invention: a chromosomal translocation associated with a malignancy (e.g., the Philadelphia chromosome for chronic myelogenous leukemia, t(14;18) for follicular lymphoma, and the like), familial polyposis or Gardner's syndrome (possible forerunners of colon cancer), benign monoclonal gammopathy (a possible forerunner of multiple myeloma), and a first degree kinship with persons having a cancer or precancerous disease showing a Mendelian (genetic) inheritance pattern (e.g., familial polyposis of the colon, Gardner's syndrome, hereditary exostosis, polyendocrine adenomatosis, medullary thyroid carcinoma with amyloid production and pheochromocytoma, Peutz-Jeghers syndrome
  • the therapeutics of the invention that agonize and promote Renox activity can also be administered to treat high blood pressure, particularly related to Na+ retention, since Renox activation is associated with both intracellular H+ generation and transport of protons from the cytoplasm.
  • This proposed function is based on the high expression of Renox described in this invention within renal tubule cells (which have a key role in proton excretion and Na+ uptake ( Na+/H+ exchange)).
  • This proposed application is further supported by the demonstrated function of Renox-related oxidases (Mox1 and gp91phox) as H+ channels (Henderson et al., (1995), Henderson et al., (1998); Banfi et al., (1999), Banfi et al., (2000)).
  • a prophylactic/therapeutic administration of a therapeutic that promotes Renox function would be desirable in treatment of hypertension.
  • prophylactic/therapeutic administration of a therapeutic that promotes Renox function would be desirable in treatment of microbial infections of the urinary tract.
  • This proposed application is based on the demonstration of superoxide release by Renox and the site of high Renox expression with renal cells described in this invention, as well as the well known role of ROS as antimicrobial agents clearly demonstrated by the activity of the phagocyte oxidase (Leto (1999)).
  • the therapeutic comprises an effective amount of a recombinant vector containing a Renox nucleic acid insert, and a pharmaceutically acceptable carrier.
  • the Renox nucleic acid inserted into the vector comprises a nucleotide sequence selected from the group consisting of: a) a first nucleotide sequence selected from the group consisting of SEQ ID NO:3 and SEQ ID NO:4; b) a second nucleotide sequence of a nucleic acid that selectively hybridizes to the first nucleotide sequence; c) a third nucleotide sequence that is at least 70% identical to the first nucleotide sequence; d) a fourth nucleotide sequence of a nucleic acid that selectively hybridizes to a nucleic acid comprising a nucleotide sequence complementary to the first nucleotide sequence; and e) a fifth nucleotide sequence of a nucleic acid that encodes the polypeptide encoded by the first, third, or fourth nucleotide sequence.
  • the Renox nucleic acid comprises the nucleotide sequence of SEQ ID NO:3.
  • the nucleotide sequence is SEQ ID NO:4.
  • the nucleic acid can be administered in vivo to promote expression of its encoded protein, by constructing it as part of an appropriate Renox nucleic acid expression vector (as described in the section above that describes the Renox nucleic acid expression vector) and administering it so that it becomes intracellular.
  • nucleic acid so that it becomes intracellular.
  • viral vectors such as a retroviral vector (see Morgan, et al., "In vivo introduction and expression of foreign genetic material in epithelial cells," U.S. Pat. No. 4,980,286 (1990)).
  • direct injection e.g., a gene gun; Biolistic, Dupont
  • lipid coating can be used.
  • the methods utilize cell-surface receptors, transfecting agents, administration in linkage to a homeobox-like peptide which is known to enter the nucleus (see, e.g., Joliot et al., "Antennapedia homeobox peptide regulates neural morphogenesis,” Proc. Natl. Acad. Sci. U.S.A. 88:1864 (1991)), or administration in linkage to a ligand subject to receptor-mediated endocytosis (see, e.g., Wu and Wu, "Receptor-mediated in vitro gene transformation by a soluble DNA carrier system," J. Biol. Chem. 262:4429 (1987)).
  • a homeobox-like peptide which is known to enter the nucleus
  • administration in linkage to a ligand subject to receptor-mediated endocytosis see, e.g., Wu and Wu, "Receptor-mediated in vitro gene transformation by a soluble DNA carrier system," J. Bio
  • the methods utilize a nucleic acid-ligand complex in which the ligand comprises a fusogenic viral peptide to disrupt endosomes, allowing the nucleic acid to avoid lysosomal degradation.
  • the method targets the nucleic acid in vivo for cell specific uptake and expression.
  • the method may target a specific receptor (see, e.g.,Wu et al., “Targeting Viruses And Cells For Selective Internalization by Cells," WO 92/06180 (1992); Wilson et al., “Targeted Delivery of Genes Encoding Secretory Proteins," WO 92/22635 (1992); Findeis et al., “Targeted Delivery of Genes Encoding Immunogenic Proteins," WO 92/20316 (1992); Clarke et al., “Targeted Virus,” WO 93/14188 (1993); and Young, “Gene Therapy Using Targeted Viral Vecors,” WO 93/20221 (1993)).
  • a specific receptor see, e.g.,Wu et al., "Targeting Viruses And Cells For Selective Internalization by Cells," WO 92/06180 (1992); Wilson et al., “Targeted Delivery of Genes Encoding Secretory Protein
  • colloidal dispersion systems include macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes.
  • the preferred colloidal system of this invention is a liposome.
  • Liposomes are artificial membrane vesicles which are useful as delivery vehicles in vitro and in vivo. It has been shown that large unilamellar vesicles (LUV), which range in size from 0.2-4.0 urn can encapsulate a substantial percentage of an aqueous buffer containing large macromolecules.
  • LUV large unilamellar vesicles
  • RNA, DNA, and intact virions can be encapsulated within the aqueous interior and be delivered to cells in a biologically active form (Fraley, et al., "Liposome-mediated delivery of deoxyribonucleic acid to cells: enhanced efficiency of delivery related to lipid composition and incubation conditions," Biochemistry, 20:6978, (1981)).
  • liposomes In addition to mammalian cells, liposomes have been used for delivery of polynucleotides in plant, yeast, and bacterial cells.
  • a liposome In order for a liposome to be an efficient gene transfer vehicle, the following characteristics are desired: (1) encapsulation of the gene or genes of interest at high efficiency without compromising their biological activity; (2) preferential and substantial binding to a target cell in comparison to non-target cells; (3) delivery of the aqueous contents of the vesicle to the target cell cytoplasm at high efficiency; and (4) accurate and effective expression of genetic information (Mannino, et al., "Liposome mediated gene transfer," Biotechniques, 6:682, 1988).
  • Still other methods for introducing the nucleic acid intracellularly involves incorporating the nucleic acid within host cell DNA by homologous recombination (Koller et al., "Inactivating the beta 2-microglobulin locus in mouse embryonic stem cells by homologous recombination," Proc. Natl. Acad. Sci. U.S.A. 86:8932 (1989); Zijlstra et al., "Germ-line transmission of a disrupted beta 2-microglobulin gene produced by homologous recombination in embryonic stem cells," Nature 342:435-438 (1989)).
  • transcription control sequences can be used with the Renox nucleic acid therapeutics of the present invention. These control sequences are described in the "Production of Renox Polypeptide" and "Renox Expression Vector” sections of this specification. As discussed in those sections, particularly preferred transcription control sequences include cytomegalovirus immediate early (preferably in conjunction with Intron-A), Rous sarcoma virus long terminal repeat, and tissue-specific transcription control sequences, as well as transcription control sequences endogenous to viral vectors if viral vectors are used. The incorporation of a "strong" polyadenylation signal is also preferred.
  • Nucleic acids for use as therapeutics can be tested in suitable animal model systems prior to testing in humans, including, but not limited to, rats, mice, chicken, cows, monkeys, rabbits, and the like.
  • suitable animal model systems including, but not limited to, rats, mice, chicken, cows, monkeys, rabbits, and the like.
  • any animal model system known in the art may be used.
  • this aspect of the invention comprises a therapeutic composition and a method for providing Renox therapy to a mammal in need of Renox therapy, by administering an effective amount of the therapeutic composition, comprising a Renox polypeptide encoded by a nucleic acid comprising a sequence selected from the group consisting of: a) a first nucleotide sequence selected from the group consisting of SEQ ID NO:3 and SEQ ID NO:4; b) a second nucleotide sequence of a nucleic acid that selectively hybridizes to a nucleic acid comprising a nucleotide sequence complementary to the first nucleotide sequence; c) a third nucleotide sequence that is at least 70% identical to the first nucleotide sequence; and d) a fourth nucleotide sequence of a nucleic acid that encodes the polypeptide encoded by the first, second, or third nucleotide sequence.
  • the mammal is afflicted with autoimmune antibodies against
  • the method comprises treating a mammal with an effective amount of a therapeutic composition comprising a recombinant vector and a pharmaceutically acceptable carrier.
  • the recombinant vector contains a Renox nucleic acid comprising a nucleotide sequence selected from the group consisting of: a) a first nucleotide sequence selected from the group consisting of
  • SEQ ID NO:3 and SEQ ID NO:4 b) a second nucleotide sequence of a nucleic acid that selectively hybridizes to the first nucleotide sequence; c) a third nucleotide sequence that is at least 70% identical to the first nucleotide sequence; d) a fourth nucleotide sequence that hybridizes to a nucleic acid that is complementary to the second nucleotide sequence; and e) a fifth nucleotide sequence of a nucleic acid that encodes the polypeptide encoded by the first, third, or fourth nucleotide sequence.
  • the nucleotide sequence encodes a polypeptide comprising an amino acid sequence of SEQ ID NO:1. In another embodiment, the nucleotide sequence encodes a polypeptide comprising an amino acid sequence of SEQ ID NO:2.
  • Renox expression vectors and therapeutic compositions related to Renox are described in previous sections of this specification.
  • the mammal may be afflicted by a hyper-proliferative disorder such as cancer or conditions treatable by altering the activity of the kidney oxygen sensor such as conditions involving low erythropoietin activity.
  • a hyper-proliferative disorder such as cancer or conditions treatable by altering the activity of the kidney oxygen sensor such as conditions involving low erythropoietin activity.
  • These conditions include renal failure, chronic inflammatory diseases such as rheumatoid arthritis and inflammatory bowel disease, HIV, HIV with AZT therapy, cancer, and cancer chemotherapy, where suppression of Renox activity by Renox antisense may be desirable.
  • Renox gene therapy could also be used as a method for affecting kidney H+/Na+/K+ transport properties, as a means of altering a patient's blood pressure.
  • Renox gene therapy aspects of the invention would involve either suppressing or enhancing Renox levels in order to alter inflammatory responses or host defense (antimicrobial) capabilities within the kidney or the urinary tract.
  • the mammal is afflicted with a disorder involving autoimmune antibodies against Renox.
  • the mammal is a human afflicted with polycythemia.
  • the recombinant vector contains a Renox nucleic acid comprising a nucleotide sequence selected from the group consisting of: a) a first nucleotide sequence selected from the group consisting of SEQ ID NO:3 and SEQ ID NO:4; b) a second nucleotide sequence that is at least 70% identical to the first nucleotide sequence; c) a third nucleotide sequence that hybridizes to a nucleic acid that is complementary to the second nucleotide sequence; and d) a fourth nucleotide sequence of a nucleic acid that encodes the polypeptide encoded by the first, second or third nucleotide sequence.
  • the nucleic acid comprises a Renox nucleic acid or antisense nucleic acid that is part of an expression vector that expresses a Renox protein (as described above), or segment, or chimeric protein thereof in a suitable host.
  • a nucleic acid has a promoter operably linked to the Renox coding region, said promoter being inducible or constitutive, and, optionally, tissue-specific.
  • a nucleic acid molecule is used in which the Renox coding sequences and any other desired sequences are flanked by regions that promote homologous recombination at a desired site in the genome, thus providing for intrachromosomal expression of the Renox nucleic acid (Koller and Smithies, 'Inactivating the beta 2-microglobulin locus in mouse embryonic stem cells by homologous recombination, " Proc. Natl. Acad. Sci. USA 86:8932 (1989); and Zijlstra et al., "Germ-line transmission of a disrupted beta 2-microglobulin gene produced by homologous recombination in embryonic stem cells," Nature 342:435 (1989)).
  • Delivery of the nucleic acid into a patient may be either direct, in which case the patient is directly exposed to the nucleic acid or nucleic acid-carrying vector, or indirect, in which case, cells are first transformed with the nucleic acid in vitro, and then transplanted into the patient. These two approaches are known, respectively, as in vivo or ex vivo gene therapy.
  • the nucleic acid is directly administered in vivo, where it is expressed to produce the encoded product. This can be accomplished by any of numerous methods known in the art, as described in the "Therapeutic Compositions" section of this specification in the context of intracellular introduction of a nucleic acid.
  • a viral vector that contains a mammalian Renox nucleic acid is used.
  • a retroviral vector can be used (see Miller et al., "Use of retroviral vectors for gene transfer and expression," Meth. Enzymol. 217:581 (1993)). These retroviral vectors have been modified to delete retroviral sequences that are not necessary for packaging of the viral genome and integration into host cell DNA.
  • the mammalian Renox nucleic acid to be used in gene therapy is cloned into the vector, which facilitates delivery of the gene into a patient.
  • retroviral vectors More detail about retroviral vectors can be found in Boesen et al., "Circumvention of chemotherapy-induced myelosuppression by transfer of the mdrl gene," Biotherapy 6:291 (1994), which describes the use of a retroviral vector to deliver the mdrl gene to hematopoietic stem cells in order to make the stem cells more resistant to chemotherapy.
  • Other references illustrating the use of retroviral vectors in gene therapy are: Clowes et al., "Long-term biological response of injured rat carotid artery seeded with smooth muscle cells expressing retrovirally introduced human genes," J. Clin. Invest.
  • Adenoviruses can be used in gene therapy and are especially attractive vehicles for delivering genes to respiratory epithelia since adenoviruses naturally infect respiratory epithelia where they cause a mild disease.
  • adenovirus-based delivery systems include liver, the central nervous system, endothelial cells, and muscle.
  • Adenoviruses have the advantage of being capable of infecting non-dividing cells.
  • Kozarsky and Wilson present a review of adenovirus-based gene therapy ("Gene Therapy: adenovirus vectors," Current Opinion in Genetics and Development 3:499 (1993).
  • Bout et al. demonstrated the use of adenovirus vectors to transfer genes to the respiratory epithelia of rhesus monkeys (Human Gene Therapy 5:3 (1994)).
  • Adeno-associated virus has also been proposed for use in gene therapy (Walsh et al., "Gene therapy for human hemoglobinopathies,” Proc. Soc. Exp. Biol. Med. 204:289 (1993)).
  • Another approach to gene therapy involves transferring a gene to cells in tissue culture prior to administration in vivo of the resulting recombinant cell.
  • introduction can be carried out by any method known in the art, including but not limited to transfection, electroporation, microinjection, infection with a viral or bacteriophage vector containing the nucleic acid sequences, cell fusion, chromosome-mediated gene transfer, microcell-mediated gene transfer, spheroplast fusion, and the like.
  • Numerous techniques are known in the art for the introduction of foreign genes into cells (see, e.g., Loeffler et al., "Gene transfer into primary and established mammalian cell lines with lipopolyamine-coated DNA'", Meth. Enzymol.
  • the technique should provide for the stable transfer of the nucleic acid to the cell, so that the nucleic acid is expressible by the cell and preferably heritable and expressible by its cell progeny.
  • the method of transfer includes the transfer of a selectable marker to the cells. The cells are then placed in a selective environment to isolate those cells that have taken up and are expressing the transferred gene. Those cells are then delivered to a patient.
  • the resulting recombinant cells can be delivered to a patient by various methods known in the art.
  • epithelial cells are injected (e.g., subcutaneously).
  • recombinant skin cells may be applied as a skin graft onto the patient.
  • Recombinant blood cells e.g., hematopoietic stem or progenitor cells
  • the amount of cells envisioned for use depends on the desired effect, patient state, and the like, and can be determined by one skilled in the art.
  • Cells into which a nucleic acid can be introduced for purposes of gene therapy encompass any desired, available cell type, and include, but are not limited to, epithelial cells, endothelial cells, keratinocytes, fibroblasts, muscle cells, hepatocytes; blood cells such as T lymphocytes, B lymphocytes, monocytes, macrophages, neutrophils, eosinophils, megakaryocytes, and granulocytes.
  • a Renox nucleic acid is introduced into cells of the kidney.
  • the cell used for gene therapy is autologous to the patient.
  • a Renox nucleic acid is introduced into the cells such that it is expressible by the cells or their progeny, and the recombinant cells are then administered in vivo for therapeutic effect.
  • stem or progenitor cells are used. Any stem and/or progenitor cells which can be isolated and maintained in vitro can potentially be used in accordance with this embodiment of the present invention.
  • Such stem cells include, but are not limited to, hematopoietic stem cells (HSC), stem cells of epithelial tissues such as the skin and the lining of the gut, embryonic heart muscle cells, liver stem cells (e.g., Naughton et al., “Liver Reserve Cells,” WO 94/08598, (1994)), and neural stem cells (e.g., Stemple et al., "Isolation of a stem cell for neurons and glia from the mammalian neural crest,” Cell 71 :973 (1992)).
  • HSC hematopoietic stem cells
  • stem cells of epithelial tissues such as the skin and the lining of the gut
  • embryonic heart muscle cells e.g., embryonic heart muscle cells
  • liver stem cells e.g., Naughton et al., "Liver Reserve Cells," WO 94/08598, (1994)
  • neural stem cells e.g., Stemple et al., "Isolation
  • the nucleic acid to be introduced for purposes of gene therapy comprises an inducible promoter operably linked to the coding region, such that expression of the nucleic acid is controllable by controlling the presence or absence of the appropriate inducer of transcription.
  • Antisense nucleic acids of the invention can be expressed from an expression vector, as described herein (see, e.g., Ushio-Fukai M. et al., "p22phox is a critical component of the superoxide-generating NADH/NADPH oxidase system and regulates angiotensin ll-induced hypertrophy in vascular smooth muscle cells," J. Biol. Chem., 20:23317 (1996)).
  • antisense nucleic acids of the current invention may be double-stranded or single-stranded oligonucleotides, RNA or DNA, or a modification or derivative thereof, which can be directly administered to an orgnanism or a cell, as is known in the art (see, e.g., Dorseuil O. et al., "Inhibition of superoxide production in B lymphoocytes by rac antisense oligonucleotides," J. Biol. Chem. 267:20540 (1992); Dorseuil O., et al., “Inhibition of Rac function using antisense oligonucleotides," Methods Enzymol.
  • Anti-Renox antisense oligonucleotides are typically at least six nucleotides in length and are antisense to a gene or cDNA encoding Renox or a portion thereof.
  • a Renox “antisense” nucleic acid as used herein refers to a nucleic acid capable of hybridizing to a portion of a Renox RNA (preferably mRNA) by virtue of some sequence complementarity.
  • the antisense nucleic acid may be complementary to a coding and/or noncoding region of a Renox mRNA.
  • Such antisense nucleic acids have utility as therapeutics that inhibit Renox function, and can be used in the treatment or prevention of disorders as described herein.
  • Antisense oligonucleotides are well-suited for anti-renox therapy because they are polar molecules that will be readily directed to the kidney.
  • the method includes administering to the mammal, an effective amount of an antisense therapeutic composition comprising an antisense nucleic acid and a pharmaceutically acceptable carrier.
  • the antisense nucleic acid comprises a nucleotide sequence of at least six nucleotides, where the nucleotide sequence is selected from the group consisting of: a) a first nucleotide sequence of an antisense nucleic acid that selectively hybridizes to a first nucleotide sequence selected from the group consisting of SEQ ID NO:3 and SEQ ID NO:4; and b) a second nucleotide sequence that is at least 70% identical to the first nucleotide sequence.
  • the gene therapy method described above utilizes an antisense nucleic acid comprises at least fifty nucleotides. In another embodiment, the gene therapy method described above utilizes an antisense nucleic acid comprises at least ten antisense nucleotides of SEQ ID NO:3 or SEQ ID NO:4.
  • the anti-Renox antisense nucleic acids are administered therapeutically (including prophylactically): (1) in diseases or disorders involving an increased (relative to normal or desired) level of Renox protein or function, for example, in patients where Renox protein is overactive or overexpressed; or (2) in diseases or disorders wherein in vitro (or in vivo) assays indicate the utility of Renox antagonist administration.
  • the increased levels in Renox protein or function can be readily detected by, for example, quantifying protein and/or RNA, by obtaining a patient tissue sample (e.g., from biopsy tissue) and assaying it in vitro for RNA or protein levels, structure and/or activity of the expressed Renox RNA or protein.
  • the anti-Renox antisense nucleic acids provided by the instant invention can be used to increase expression of erythropoietin.
  • This embodiment of the invention further provides pharmaceutical compositions comprising an effective amount of the Renox antisense nucleic acids of the invention in a pharmaceutically acceptable carrier, as described above.
  • anti-Renox antisense nucleic acids are at least six nucleotides, in some embodiments at least 10 nucleotides, in some embodiments at least 15 nucleotides, in some embodiments at least 100 nucleotides, and in certain embodiments at least 200 nucleotides in length.
  • antisense oligonucleotides of this aspect of the current invention are from 6 to about 50 oligonucleotides in length.
  • the oligonucleotide can be DNA or RNA or chimeric mixtures or derivatives or modified versions thereof, single-stranded or double-stranded.
  • the oligonucleotide can be modified at the base moiety, sugar moiety, or phosphate backbone.
  • the oligonucleotide may include other appending groups such as peptides, or agents facilitating transport across the cell membrane (see, e.g., Letsinger et al., "Cholesteryl- conjugated oligonucleotides: synthesis, properties, and activity as inhibitors of replication of human immunodeficiency virus in cell culture," Proc. Natl. Acad. Sci. U.S.A. 86:6553 (1989); Lemaitre et al., Proc. Natl. Acad. Sci.
  • an anti- Renox antisense oligonucleotide is single-stranded DNA.
  • the anti-Renox antisense oligonucleotide may comprise at least one modified base moiety which is selected from the group including, but not limited to, 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl)uracil, 5-carboxymethyl aminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylgu
  • the oligonucleotide comprises at least one modified sugar moiety selected from the group including, but not limited to, arabinose, 2-fluoroarabinose, xylulose, and hexose.
  • the oligonucleotide comprises at least one modified phosphate backbone selected from the group consisting of a phosphorothioate, a phosphorodithioate, a phosphoramidothioate, a phosphoramidate, a phosphordiamidate, a methylphosphonate, an alkyl phosphotriester, a formacetal or analogs thereof.
  • the oligonucleotide is an oc-anomeric oligonucleotide.
  • An ⁇ -anomeric oligonucleotide forms specific double-stranded hybrids with complementary RNA in which, contrary to the usual ⁇ -units, the strands run parallel to each other (Gautier et al., "Alpha- DNA. IV: alpha-anomeric and beta-anomeric tetrathymidylates covalently linked to intercalating oxazolopyridocarbazole. Synthesis, physicochemical properties and poly (rA) binding," Nucl. Acids Res. 15:6625 (1987)).
  • the oligonucleotide may be conjugated to another molecule, including, for example, a peptide, hybridization triggered cross-linking agent, transport agent, hybridization-triggered cleavage agent, and the like.
  • Oligonucleotides of the invention may be synthesized by standard methods known in the art (e.g., automated DNA synthesizer such as are commercially available from Biosearch, Applied Biosystems, and others). Phosphorothioate oligonucleotides may be synthesized by the method of Stein et al. ('Physicochemical properties of phosphorothioate oligodeoxynucleotides," Nucl. Acids Res. 16:3209 (1988)).
  • Methylphosphonate oligonucleotides can be prepared by use of controlled pore glass polymer supports (Sarin et al., "Inhibition of acquired immunodeficiency syndrome virus by oligodeoxynucleoside methylphosphonates," Proc. Natl. Acad. Sci. U.S.A. 85:7448 (1988)).
  • the Renox antisense oligonucleotide comprises catalytic RNA, or a ribozyme (see, e.g., Cech et al., "RNA Ribozyme Restriction Endoribonucleases and Methods," WO 90/11364
  • the oligonucleotide is a 2'-O-methylribonucleotide (Inoue et al., “Synthesis and hybridization studies on two complementary nona(2'-O-methyl)ribonucleotides," Nucl. Acids Res. 15:6131 (1987), or a chimeric RNA-DNA analogue (Inoue et al., "Sequence- dependent hydrolysis of RNA using modified oligonucleotide splints and RNase H," FEBS Lett.
  • the antisense nucleic acids of the invention comprise a sequence complementary to at least a portion of an RNA transcript of a mammalian Renox gene, preferably a human Renox gene. Absolute complementarity, although preferred, is not required.
  • a sequence "complementary to at least a portion of an RNA,” as referred to herein, means a sequence having sufficient complementarity to be able to hybridize under highly stringent conditions with the RNA, forming a stable duplex; in the case of double-stranded Renox antisense nucleic acids, a single strand of the duplex DNA may thus be tested, or triplex formation may be assayed.
  • the ability to hybridize will depend on both the degree of complementarity and the length of the antisense nucleic acid. Generally, the longer the hybridizing nucleic acid, the more base mismatches with a Renox RNA it may contain and still form a stable duplex (or triplex, as the case may be). One skilled in the art can ascertain a tolerable degree of mismatch using standard procedures to determine the melting point of the hybridized complex, as discussed herein. Antisense nucleic acids of the current invention
  • the Renox antisense nucleic acid of the invention is produced intracellularly by transcription from an exogenous sequence.
  • a vector can be introduced in vivo such that it is taken up by a cell, wherein the vector or a portion thereof is transcribed, producing an antisense nucleic acid (RNA) of the invention.
  • RNA antisense nucleic acid
  • Such a vector would contain a sequence encoding the Renox antisense nucleic acid.
  • Such a vector can remain episomal or become chromosomally integrated, as long as it can be transcribed to produce the desired antisense RNA.
  • Such vectors can be constructed by recombinant DNA technology methods standard in the art.
  • Vectors can be plasmid, viral, or others known in the art, used for replication and expression in mammalian cells. Expression of the sequence encoding the Renox antisense RNA can be by any promoter known in the art to act in mammalian, preferably human, cells. Such promoters, which can be inducible or constitutive, are discussed above. Methods for Identifying Reagents That Inhibit Renox. Another aspect of the current invention is a method for identifying a reagent involved in regulating Renox.
  • the method comprises: a) providing a host cell transfected with a Renox nucleic acid; b) contacting the host cell with a sample containing the reagent; and c) detecting a change in a cellular output characteristic of expression of exogenous Renox within a cell, thereby determining whether the reagent is involved in regulating Renox.
  • the transformed host cell is a fibroblast.
  • the fibroblast is an NIH 3T3 cell.
  • the transformed fibroblast is an immortalized cell that stably expresses recombinant Renox.
  • outputs characteristic of expression of exogenous Renox can be determined by comparing results related to a detectable output obtained for host cells successfully transfected with a Renox nucleic acid and similar, but untransfected, host cells, especially with regard to the ability to express the transfected Renox gene. This approach is demonstrated in the Examples to determine several outputs characteristic of expression of exogenous Renox.
  • outputs characteristic of Renox likely exist. Any output that can be identified can be readily screened by one of ordinary skill to determine whether it is a cellular output characteristic of expression of exogenous Renox. Outputs characteristic of expression of exogenous Renox appear to be cell-type specific. Therefore, experiments can be performed in a particular cell-type using the approach above to determine outputs characteristic of expression of exogenous Renox in that cell type. Outputs related to the predicted oxidase activity of Renox are especially preferred. Based on the results disclosed in the Examples section, outputs characteristic of Renox expression in fibroblasts include an altered cellular morphology, particularly a senescent morphology, superoxide production, and a decreased rate of proliferation.
  • Cellular morphology can be readily determined using microscopic methods well-known in the art. As demonstrated in the Examples, NIH 3T3 cells that express high levels of Renox exhibit a senescent morphology. This morphology includes a more flattened and larger size, appearance of long processes extending out from the cell, and often, multiple nuclei. Proliferation rates can be determined by determining cell number changes over time using methods well-known in the art.
  • Outputs related to the predicted oxidase activity of Renox such as, for example, generation of reactive oxygen species (including superoxide) in fibroblasts, are especially preferred.
  • reagent as used herein describes any molecule (e.g., protein, nucleic acid, polypeptide, or pharmaceutical) with the capability of effecting or mimicking the physiological function of Renox. Generally a plurality of assay mixtures are run in parallel with different reagent concentrations to obtain a differential response to the various concentrations. Typically, one of these concentrations serves as a negative control (i.e., zero concentration or below the level of detection).
  • the reagent inhibits the output.
  • the reagent increases, accelerates, or enhances the output, or the reagent induces the output in the absence of expression of transfected Renox.
  • Candidate reagents encompass numerous chemical classes, though typically they are organic molecules, preferably small organic molecules having a molecular weight of more than 50 and less than about 2,500 daltons.
  • Candidate reagents comprise functional groups necessary for structural interaction with proteins, particularly hydrogen bonding, and typically include at least one of the group consisting of an amine, carbonyl, hydroxyl, or carboxyl group, and preferably at least two of such functional chemical groups.
  • the candidate reagents often comprise cyclical carbon or heterocyclic structures and/or aromatic or polyaromatic structures substituted with one or more of the above functional groups.
  • Candidate reagents are also found among biomolecules including peptides, proteins, saccharides, fatty acids, steroids, purines, pyrimidines, derivatives, structural analogs, or combinations thereof.
  • Candidate reagents can be obtained from a wide variety of sources including libraries of synthetic or natural compounds. For example, numerous means are available for random and directed synthesis of a wide variety of organic compounds and biomolecules, including expression of randomized oligonucleotides and oligopeptides. Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant, and animal extracts are available or readily produced. Additionally, natural or synthetically produced libraries and compounds are readily modified through conventional chemical, physical, and biochemical means, and may be used to produce combinatorial libraries. Known pharmacological agents may be subjected to directed or random chemical modifications, such as acylation, alkylation, esterification, amidification, and the like to produce structural analogs. In addition, compounds can be obtained from commercial sources.
  • the methods of this invention are suitable and useful for screening compounds for possible Renox inhibitor activity. This screening is particularly appropriate for commercially available analogs of identified inhibitors of oxidases, such as diphenylene iodonium or phenylarsine oxide (Hancock, J.,
  • Compounds with identified structures from commercial sources can be efficiently screened for activity against a particular target by first restricting the compounds to be screened to those with preferred structural characteristics. As an example, compounds with structural characteristics causing high gross toxicity can be excluded. Similarly, once a number of inhibitors of a specific target have been found, a sub-library may be generated consisting of compounds which have structural features in common with the identified inhibitors.
  • the ISIS computer program (MDL Information Systems, Inc.) is suitable to perform a 2D-substructure search of the Available Chemicals Directory database (MDL Information Systems, Inc.). This database contains structural and ordering information on approximately 175,000 commercially available chemical compounds. Other publicly accessible chemical databases may similarly be used.
  • Gross acute toxicity of an identified inhibitor of a specific gene target, such as Renox may be assessed in a mouse model.
  • the inhibitor is administered at a range of doses, including high doses, (typically 0 - 100 mg/kg, but preferably to at least 100 times the expected therapeutic dose) subcutaneously or orally, as appropriate, to healthy mice.
  • the mice are observed for 3-10 days.
  • a combination of such an inhibitor with any additional therapeutic components is tested for possible acute toxicity.
  • Another class of candidate reagents are antisense nucleic acids that selectively hybridize with mammalian Renox nucleic acids, as disclosed in earlier sections of this specification.
  • a cellular output that is useful for identifying effective antisense nucleic acids and vector constructs is expression of Renox protein. This expression can be measured using methods well-known in the art including immunological methods for determining protein concentrations such as ELISA assays. Examples of immunological assays are described in the Antibodies section of this specification. The current invention is not limited to any particular method of determining Renox expression levels.
  • drug screening using the mammalian Renox nucleic acids, expression vectors, and polypeptides of the current invention can be performed using a genetically altered animal.
  • drug screening protocols can be used to identify reagents that provide a replacement for Renox function in cells which have a mutated Renox gene that encodes a Renox that does not function properly or is not expressed properly.
  • Renox nucleic acid of the invention can be used to transfect host cells for this aspect of the invention.
  • the Renox nucleic acid is inserted into a vector, typically an expression vector.
  • Expression vectors containing Renox nucleic acids are described in an earlier section of this specification.
  • the Renox nucleic acid encodes a polypeptide of SEQ ID NO:1.
  • the Renox nucleic acid encodes a polypeptide of SEQ ID NO:2.
  • reagents may be included in the screening assay. These include reagents like salts, neutral proteins (e.g., albumin), detergents, and the like that are often used to facilitate optimal protein-protein binding and/or reduce non-specific or background interactions. Reagents that improve the efficiency of the assay, such as protease inhibitors, nuclease inhibitors, anti-microbial agents, and the like may be used. The mixture of components are added in any order that provides for the requisite binding. Incubations are performed at any suitable temperature, typically between 4 and 40°C. Incubation periods are selected for optimum activity, but may also be optimized to facilitate rapid high-throughput screening. Typically between 0.1 and 1 hours will be sufficient.
  • Reagents having the desired pharmacological activity may be administered in a physiologically acceptable carrier to a host for treatment. Such reagents are administered with many of the same considerations as described in the Therapeutic Compositions section above. Methods for Identifying Reagents That Bind to Renox
  • the invention includes methods for detecting a reagent that binds to Renox polypeptides.
  • the method comprises: a) contacting a Renox polypeptide or a fragment thereof with a reagent, wherein said polypeptide is encoded by a nucleic acid comprising a nucleotide sequence selected from the group consisting of: i) a first nucleotide sequence selected from the group consisting of SEQ ID NO:3 and SEQ ID NO: 4; ii) a second nucleotide sequence of a nucleic acid that selectively hybridizes to a nucleic acid that is complementary to the first nucleotide sequence; iii) a third nucleotide sequence that is at least 70% identical to the first nucleotide sequence; and iv) a fourth nucleotide sequence of a nucleic acid that encodes the polypeptide encoded by the first, second, or third nucleotide sequence.
  • Renox nucleic acid of the invention can be used to express a polypeptide to be used in this aspect of the invention.
  • the Renox nucleic acid is inserted into a vector, typically an expression vector, and transfected into a host cell, to produce a polypeptide to be used in this aspect of the invention.
  • Expression vectors containing Renox nucleic acids are described in an earlier section of this specification, as are methods for producing such Renox polypeptides.
  • the Renox nucleic acid encodes a polypeptide of SEQ ID NO:1.
  • the Renox nucleic acid encodes a polypeptide of SEQ ID NO:2.
  • one or both of the potential binding pair members may be joined to a label, where the label can directly or indirectly provide a detectable signal.
  • Various labels include radioisotopes, fluorescers, chemiluminescers, enzymes, specific binding molecules, particles (e.g., magnetic particles), and the like.
  • Specific binding molecules may also include pairs, such as biotin and streptavidin, digoxin and antidigoxin, and the like.
  • the complementary member would normally be labeled with a molecule that provides for detection, using known procedures and/or assays.
  • binding assays in screening for compounds active on a specific polypeptide, it is generally preferred that the binding be at a substrate binding site, at a binding site for an allosteric modulator, or at another site which alters the relevant biological activity of the molecule.
  • simple detection of binding is often useful as a preliminary indicator of activity; the initial indication can then be confirmed by other methods.
  • Binding assays can be provided in a variety of different formats. These formats can include, for example, direct determination of the amount of bound molecule (either while bound or after release); indirect detection of binding, such as by determination of a change in a relevant activity; and competitive binding.
  • the assay may use an immobilized support or may be solution- based. Further, often binding assays can be performed using only a portion of a polypeptide which includes the relevant binding site. Such fragments can be constructed, for example, by expressing a gene fragment which includes the sequence coding for a particular polypeptide fragment and isolating the polypeptide fragment. Other methods known to those skilled in the art can also be used. Thus, Renox nucleic acids identified herein provide polypeptides which can be utilized in such binding assays. Those skilled in the art can readily determine the suitable polypeptides, appropriate binding conditions, and appropriate detection methods.
  • the test reagent is a protein or polypeptide.
  • phage display random peptides (up to 20 amino acids long) are expressed with coat proteins of filamentous phage such that they are expressed on the surface of the phage thus generating a library of phage that express random sequences.
  • a library of these random sequences is then selected by incubating the library with the Renox protein or fragments thereof bound to a support matrix. Phage that bind to the protein are then eluted either by cleavage of Renox from the support matrix or by elution using an excess concentration of soluble Renox protein or fragments.
  • the eluted phage are then repropagated and the selection repeated many times to enrich for higher affinity interactions.
  • the random peptides can either by completely random or constrained at certain positions through the introduction of specific residues. After several rounds of selection, the final positive phage are sequenced to determine the sequence of the peptide.
  • Renox or fragments thereof are expressed in yeast as a fusion to a DNA binding domain.
  • This fusion protein is capable of binding to target promoter elements in genes that have been engineered into the yeast. These promoters drive expression of specific reporter genes (typically the auxotrophic marker HIS3 and the enzyme ⁇ - galactosidase).
  • a library of cDNAs can then be constructed from a tissue or cell line and fused to a transcriptional activation domain.
  • HIS3 and ⁇ -galactosidase depend on association of the Renox fusion protein (which contains the DNA binding domain) and the target protein (which carries the activation domain). Yeast survival on specific growth media lacking histidine requires this interaction. This approach allows for the identification of specific proteins that interact with Renox. The approach has
  • Renox or its fragments, are fused with the DNA binding domain and are screened with a library of random peptides or peptides which are constrained at specific positions linked to a transcriptional activation domain. Interaction is detected by growth of the interacting peptides on media lacking histidine and by detection of ⁇ - galactosidase activity using standard techniques.
  • the identification of proteins or small peptides that interact with Renox can provide the basis for the design of small peptide antagonists or agonists of Renox function. Further, the structure of these peptides determined by standard techniques such as protein NMR or X-ray crystallography can provide the structural basis for the design of small molecule drugs.
  • Antibodies Against Renox The knowledge of the amino acid and nucleotide sequence of mammalian Renox allows for the production of antibodies which selectively bind the Renox protein or fragments thereof.
  • the Renox nucleic acids and methods for detecting a Renox nucleic acid which are discussed above, facilitate the identification of mutations in the Renox sequence.
  • Antibodies can be made to selectively bind and/or distinguish mutant from normal protein.
  • fusion proteins containing defined portions or all of the Renox protein can be synthesized in bacteria by expression of corresponding DNA sequences in a suitable cloning vehicle. Fusion proteins are commonly used as a source of antigen for producing antibodies. Alternatively protein may be isolated and purified from Renox expressing cultures, as discussed above, and used as a source of antigen. It is understood that the entire protein or fragments thereof can be used as a source of antigen to produce antibodies.
  • Renox protein is purified and mixed with Freund's adjuvant (to help stimulate the antigenic response by the animal) and injected into rabbits or other appropriate laboratory animals.
  • fragments of the Renox protein can be used as the immunogen.
  • Carrier proteins may be used which are bound to the Renox protein or fragments thereof to assist in antibody production.
  • the rabbits or other laboratory animals are bled and sera isolated. This sera can then be tested by methods well-known in the art to determine whether it is anti-Renox sera (i.e., contains antibodies against Renox).
  • the sera can be used directly or purified prior to use by various methods including affinity chromatography employing Protein A-Sepharose, Antigen Sepharose, or Anti-mouse-lg- Sepharose, to provide polyclonal antibodies.
  • synthetic peptides can be made corresponding to the antigenic portions of the protein, and used to inoculate the animals.
  • a 10 to 15 amino acid residue peptide corresponding to the carboxyl or amino terminal sequence of a protein antigen can be prepared and chemically cross-linked to a carrier molecule such as keyhole limpet hemocyanin or BSA.
  • selection of the peptide is based on the use of algorithms that predict potential antigenic sites. These predictive methods are, in turn, based on predictions of hydrophilicity (Kyte, J. and Doolittle, "A simple method for displaying the hydropathic character of a protein," J. Mol. Biol.
  • the selection process is also limited by constraints imposed by the chemistry of the coupling procedures used to attach peptide to carrier protein.
  • Carboxyl-terminal peptides are frequently chosen because these are often more mobile than the rest of the molecule and the peptide can be coupled to a carrier in a straightforward manner using glutaraldehyde. (The amino- terminal peptide is used less often since it may be modified post- translationally by acetylation or by the removal of a leader sequence.)
  • a comparison of the protein amino acid sequence between species can yield important information. Those regions with sequence differences between species are likely to be immunogenic. Synthetic peptides can also be synthesized as immunogens as long as they mimic the native antigen as closely as possible.
  • Monoclonal Renox antibodies may also be produced.
  • a Renox protein or fragment thereof isolated from cells recombinantly expressing the protein or fragment thereof is injected in Freund's adjuvant into mice.
  • the spleens of the immunized mice are removed and resuspended in phosphate buffered saline (PBS).
  • PBS phosphate buffered saline
  • the spleen cells serve as a source of lymphocytes, some of which produce antibody of a selected specificity.
  • These are then fused with a permanently growing myeloma partner cell, and the products of the fusion are plated into a number of tissue culture wells in the presence of a selective agent such as HAT.
  • the wells are then screened by ELISA to identify those containing cells making binding antibody. These are then plated and after a period of growth, these wells are again screened to identify antibody-producing cells. Cloning procedures are carried out until over 90% of the wells contain single clones which are positive for production of the desired antibody. From this procedure, a stable line of clones which produce the antibody is established.
  • the monoclonal antibody can then be purified by affinity chromatography using Protein A Sepharose, ion-exchange chromatography, as well as variations and combinations of these techniques.
  • Truncated versions of monoclonal antibodies may also be produced by recombinant techniques in which plasmids are generated which express the desired monoclonal antibody fragment(s) in a suitable host.
  • antibodies specific for mutagenised epitopes can also be generated. These antibodies are especially useful to screen for the expression of mutated Renox.
  • Antibodies are also useful in various immunoassays for detecting and quantitating relative amounts of wild type and mutant protein. Enzyme-linked immunosorbant assays (ELISA) may be used to detect both wild type and
  • Antibodies to Renox may also be used for coupling to compounds such as radionuclides or fluorescent compounds, or to liposomes for diagnostic imaging and therapy, in order to target compounds to the kideny.
  • compounds such as radionuclides or fluorescent compounds, or to liposomes for diagnostic imaging and therapy, in order to target compounds to the kideny.
  • the protein can generally be isolated from natural sources, without the necessity for a recombinant coding sequence.
  • assays include those based on antibody binding, enzymatic activity, and competitive binding of substrate analogs or other compounds. Consequently, this invention provides purified, enriched, or isolated mammalian Renox proteins, which may be produced from recombinant coding sequences or by purification from cells naturally expressing this protein.
  • Renox nucleic acid sequences can be used to create transgenic non-human animals to serve as animal models for studying Renox function and the effectiveness of Renox therapy.
  • These transgenic animals can be developed to overexpress Renox (transgenic expression) or can be developed with mutations such as multiple stop codons ("knockouts" or "null alleles") or other mutations which alter Renox activity.
  • transgenic mice having little or no Renox activity due to mutations in one or both alleles of the gene can be developed.
  • the non-human animals of the invention comprise any animal having altered Renox activity as a result of the transgenic alteration of the Renox gene.
  • Such non-human animals include vertebrates such as rodents, non- human primates, sheep, dog, cow, amphibians, reptiles, and the like.
  • Preferred non-human animals are mammals, more preferably, rodents including rats and mice, and most preferably mice.
  • the transgenic animals of the invention are animals into which has been introduced by nonnatural means (i.e., by human manipulation), one or more genes or gene fragments that do not occur naturally in the animal (e.g., foreign renox nucleic acids, genetically engineered endogenous Renox nucleic acids, and the like).
  • transgenes may be from the same or a different species as the animal but not naturally found in the animal in the configuration and/or at the chromosomal locus conferred by the transgene.
  • Transgenes may comprise foreign DNA sequences (i.e., sequences not normally found in the genome of the host animal).
  • transgenes may comprise endogenous DNA sequences that are abnormal in that they have been rearranged or mutated in vitro in order to alter the normal in vivo pattern of expression of the gene, or to alter or eliminate the biological activity of an endogenous gene product encoded by the gene (Watson, J. D., et al., in Recombinant DNA, 2d Ed., W. H. Freeman & Co., New York, 255 (1992); Gordon, J. W., "Transgenic animals,” Intl. Rev. Cytol.
  • the nucleic acids of the invention are used to prepare transgenic constructs to be introduced into non-human animals in order to generate the transgenic animals of the invention.
  • the transgenic non-human animals of the invention are produced by introducing Renox transgenic constructs into the germline of the non- human animal.
  • Embryonic target cells at various developmental stages are used to introduce the transgenes of the invention. Different methods are used depending on the stage of development of the embryonic target cell(s).
  • Microinjection of zygotes i.e., a fertilized ovum that has not undergone pronuclei fusion or subsequent cell division
  • zygotes i.e., a fertilized ovum that has not undergone pronuclei fusion or subsequent cell division
  • the male pronucleus of murine zygote reaches a size of approximately 20 micrometers in diameter, which allows for the reproducible injection of 1-2 picoliters of a solution containing transgenic DNA sequences.
  • the use of a zygote for introduction of transgenes has the advantage that, in most cases, the injected transgenic DNA sequences will be incorporated into the host animal's genome before the first cell division (Brinster, R.L.
  • transgenic allele normally demonstrates Mendelian inheritance.
  • half of the offspring resulting from the cross of a transgenic animal with a non-transgenic animal will inherit the transgenic allele.
  • Viral integration can also be used to introduce the transgenes comprising Renox nucleic acids of the invention into an animal.
  • Developing embryos are cultured in vitro to the blastocyte developmental stage.
  • the blastomeres may be infected with appropriate retroviruses (Jaenisch, R., "Germ line integration and Mendelian transmission of the exogenous Molononey leukemia virus," Proc. Natl. Acad. Sci. (USA) 73:1260 (1976)).
  • Infection of the blastomeres is enhanced by enzymatic removal of the zona pellucida (Hogan et al., in Manipulating the Mouse Embryo, Cold Spring
  • Transgenes are introduced via viral vectors which are typically replication-defective but which remain competent for integration of viral-associated DNA sequences, including transgenic DNA sequences linked to such viral sequences, into the host animal's genome (Jahner, D. et al., "Insertion of the bacterial gpt gene into the germ line of mice by retroviral infection," Proc. Natl. Acad. Sci. (USA) 82:6927 (1985); van der Putten, H. et al., "Efficient insertion of genes into the mouse germ line via retroviral vectors," Proc. Natl. Acad. Sci. (USA) 82:6148 (1985)).
  • infection may be performed at a later stage, such as at a blastocoele stage (Jahner, D., et al., "De nono methylation and expression of retroviral genomes during mouse embryogenesis," Nature 298:623 (1982)).
  • a blastocoele stage Jahner, D., et al., "De nono methylation and expression of retroviral genomes during mouse embryogenesis," Nature 298:623 (1982)
  • most transgenic founder animals produced by viral integration will be mosaics for the transgenic allele; that is, the transgene is incorporated into only a subset of all the cells that form the transgenic founder animal.
  • multiple viral integration events may occur in a single founder animal, generating multiple transgenic alleles which will segregate in future generations of offspring.
  • transgenes into germline cells by this method are possible but probably occurs at a low frequency (Jahner, D., et al., (1982)).
  • offspring may be produced in which the transgenic allele is present in all of the animal's cells (i.e., in both somatic and germline cells).
  • Embryonic stem (ES) cells can also serve as target cells for introduction of the transgenes of the invention into animals.
  • ES cells are obtained from pre-implantation embryos that are cultured in vitro (Evans, M. J., et al., "Establishment in culture of pluripotential cells from mouse embryos," Nature 292:154 (1981); Bradley, M. O., et al., “Formation of germline chimaeras from embryo-derived teratocarcinoma cell lines," Nature 309:255 (1984); Gossler, A. et al., “Transgenesis by means of blastocyst- derived embryonic stem cell lines," Proc. Natl. Acad. Sci. (USA) 83:9065 (1986); Robertson E. et al., "Germ-line transmission of genes introduced into cultured pluripotential cells by retroviral vector," Nature 323:445 (1986);
  • ES cells which are commercially available (from, e.g., Genome Systems, Inc., St. Louis, Mo.), can be transformed with one or more transgenes by established methods (Lovell-Badge, R. H., in Teratocarcinomas and Embryonic Stem Cells: A Practical Approach, Robertson, E. J., ed., IRL Press, Oxford (1987), pages 153-182).
  • Transformed ES cells can be combined with an animal blastocyst, whereafter the ES cells colonize the embryo and contribute to the germline of the resulting animal, which is a chimera (composed of cells derived from two or more animals) (Jaenisch, R., "Transgenic animals," Science 240:1468 (1988); Bradley, A., in Teratocarcinomas and Embryonic Stem Cells: A Practical Approach,
  • transgenic allele is present in all of the animal's cells (i.e., in both somatic and germline cells).
  • the transgenes of the invention may, therefore, be stably integrated into germ line cells and transmitted to offspring of the transgenic animal as Mendelian loci.
  • Other transgenic techniques generally result in mosaic transgenic animals, in which some, but not all, cells carry the transgenes. In mosaic transgenic animals in which germ line cells do not carry the transgenes, transmission of the transgenes to offspring does not occur. Nevertheless, mosaic transgenic animals are capable of demonstrating phenotypes associated with the transgenes.
  • Transgenes may be introduced into animals in order to provide animal models for human diseases such as those associated with altered Renox function.
  • Transgenic animals that are predisposed to a disease may be used to identify compositions that induce or prevent the disease and to evaluate the pathogenic potential of compositions known or suspected to induce or prevent the disease (Berns, A. J. M., U.S. Pat. No. 5,174,986 (Dec. 29, 1992)).
  • Offspring that have inherited the transgenes of the invention are distinguished from littermates that have not inherited transgenes by analysis of genetic material from the offspring for the presence of biomolecules that comprise unique sequences corresponding to sequences of, or encoded by, the transgenes of the invention.
  • a simple and reliable means of identifying transgenic offspring comprises obtaining a tissue sample from an extremity of an animal (e.g., the tail) and analyzing the sample for the presence of nucleic acid sequences corresponding to the DNA sequence of a unique portion or portions of the transgenes of the invention.
  • nucleic acid sequences may be determined by known techniques, including, for example, hybridization ("Southern") assays with DNA sequences corresponding to unique portions of the transgene, assays of the products of PCR reactions using DNA sequences in a sample as substrates and oligonucleotides derived from the transgene's DNA sequence, and the like.
  • a positive selectable marker for example the hygromycin phosphotransferase cassette (van Deursen, J. and Wieringa, "Targeting of the creatine kinase M gene in embryonic stem cells using isogenic and nonisogenic vectors," Nucleic Acids Res. 20:3815 (1992)), is inserted into a 5' portion of a Renox gene.
  • This position for the positive selectable marker is chosen to obtain a genuine null mutant allele (i.e., to avoid translation of a truncated polypeptide).
  • the hygromycin gene is flanked 5' and 3' by several kb of homologous murine genomic sequences.
  • a negative selectable marker for example the Herpes Simplex Virus (HSV) thymidine kinase (TK) gene, is placed in a 3' position flanking the region of homologous sequences in order to enable selection against nonhomologous integrants.
  • HSV Herpes Simplex Virus
  • TK thymidine kinase
  • Both the positive and negative selectable markers are inserted in the antisense orientation with respect to the transcriptional orientation of the Renox, and are expressed due to the TK promoter and Py F441 Polyoma enhancer.
  • Linearized targeting construct is introduced into ES cells by electroporation or other suitable means and selection with hygromycin and FIAU (1-[2-deoxy, 2-fluoro-beta-D- arabinofuranosyl]) is carried out for 7 to 10 days. Resistant colonies are expanded in 24-well plates; half of the cells in each well are cryo-preserved and the other half expanded for genotype analysis. Positive clones are stored in liquid nitrogen and thawed at least 3 days prior to blastocyst injection.
  • blastocysts can be isolated, for example, at day 3.5 postcoitum by flushing the uterine horns of naturally mated pregnant females with DMEM +10% FBS. Approximately 10 to 15 ES cells from each homologous recombinant clone with a normal karyotype are microinjected into recipient blastocysts, and about 10 to 20 embryos are transferred into the uterine horns of F1 pseudopregnant females (Bradley, A., in Teratocarcinomas and Embryonic Stem Cells: A Practical Approach, Robertson, E. J., ed., IRL Press, Oxford (1987), pages 113-151).
  • EXAMPLE 1 Isolation and Characterization of Nucleic Acid Seguences Encoding Mouse and Human Renal NADPH Oxidase.
  • Mouse and human Renal NADPH oxidase (Renox)-encoding nucleic acids were isolated and characterized.
  • BLAST nucleotide searches Altschul, Stephen F., Thomas L. Madden, Alejandro A. Schaaffer, Jinghui Zhang, Zheng Zhang, Webb Miller, and David J. Lipman (1997), "Gapped BLAST and PSI-BLAST: a new generation of protein database search programs", Nucleic Acids Res.
  • mice and human expressed sequence tags were identified that were derived from kidney libraries in the GenBank dbEST database that showed significant nucleic acid homology to gp91 phox .
  • the mouse cDNA EST clone (GenBank Accession Number: Al 226641) (SEQ ID NO:9) was obtained, sequenced, and then the complete cDNA sequence (SEQ ID NO:14), including the complete protein coding region (SEQ ID NO:3), was obtained by rapid amplification of the 5'- and 3'-ends (5'- and 3'-RACE) using mouse kidney poly-A RNA as a template for cDNA synthesis.
  • the human cDNA was also amplified based on sequences derived from two human EST clones (GenBank Accession Numbers: AW237557 (SEQ ID NO:10) and AI241222 (SEQ ID NO:11)), as described above, to obtain the complete protein-coding region (SEQ ID NO:4). These RACE reactions of human nucleic acids used oligonucleotide primers with the sequence of SEQ ID NO:7 and SEQ ID NO:8. A subsequent BLAST search of the unfinished High Throughput Genomic Sequences (HTGS) database using the human cDNA as a query sequence revealed matches with gene sequences located on chromosomes 11 and 15.
  • HGS High Throughput Genomic Sequences
  • the murine open-reading frame encodes a 578 amino acid long protein (SEQ ID NO:1) showing 40% sequence identity and 57% similarity to mouse gp91 ph0X (Fig. 1), while the corresponding human homologue is also a 578 amino acid long protein (SEQ ID NO:2) with 90% identity to its mouse counterpart.
  • the deduced sequences contain conserved features considered critical for NADPH oxidase function (Leto, T. L. (1999)), namely six hydrophobic segments within the amino-terminal segment, proposed as membrane-embedded domains involved in transmembrane electron transport, as well as sequence motifs corresponding to proposed binding sites for heme, flavin, and NADPH.
  • the third and fifth hydrophobic segments each contain two conserved histidines, which are thought to serve as coordination sites for two heme moieties within the corresponding sequences of gp91 hox and ferric reductase (Finegold, A. A., et al., "Intramembrane bis-heme motif for transmembrane electron transport conserved in a yeast iron reductase and the human NADPH oxidase," J Biol. Chem. 271 :31021 (1996)).
  • Other sites exhibiting high homology occur within the C-terminal portion, corresponding to gp91 phox sequences that are thought to represent binding sites for flavin and NADPH ( Figure 1).
  • sequence pattern analysis revealed a nucleotide-binding sequence motif in the C- terminal region (534-AKCNRGKT-543) which is often referred as the "P-loop” present in various ATP- or GTP-binding proteins (Saraste, M., et al., "The P- loop--a common motif in ATP- and GTP-binding proteins," Trends. Biochem. Sci. 15:430 (1990)).
  • EXAMPLE 2 Localization of Renox mRNA. Experiments were performed to determine the tissue-expression profile of mRNA encoding Renox. Mouse multiple-tissue Northern blot membranes (Clontech, Palo Alto, CA) were probed at 50° C for 1 hour with a radiolabeled oligonucleotide probe with the nucleotide sequence listed as SEQ ID NO:12 corresponding to a portion of murine Renox cDNA, in ExpressHybTM (Clontech, Pal Alto, CA) hybridization solution. Following hybridization, the blots were washed in 2X SSC, 0.05% SDS for 30 minutes followed by 0.1 X SSC, 0.1% SDS for 40 minutes.
  • Membranes were probed at 55° C for 1 hour with a 537 bp, randomly radiolabeled (Amersham Pharmacia Biotech, Alameda, CA) Renox cDNA fragment with the nucleotide sequence of SEQ ID NO: 13, corresponding to a portion of murine Renox cDNA, in ExpressHybTM (Clontech, Pal Alto, CA) hybridization solution. Following hybridization, the blots were washed in 2X SSC, 0.05% SDS for 30 minutes followed by 0.1X SSC, 0.1% SDS for 40 minutes.
  • RNA transcripts (sense or antisense) were synthesized by SP6 or T7 RNA polymerases. Preparation and probing of fixed and paraffin-embedded kidney thin section specimens were performed as described in Fox, CH. et al., ("In situ hybridization for detection of HIV RNA" in Current Protocols in Immunology, Coligan, Kruibeek,, Margulies, Shevack, Strober, eds (Wiley and Green, New York) 2:12.8.1-21 (1993))
  • Renox mRNA was detected only in the kidney, and not in other tissues (heart, brain, spleen, lung, liver, skeletal muscle, and testis) (Fig. 2).
  • Renox mRNA was also highly expressed in a mouse inner medullary collecting duct cell line (MIMCD3). Since a phagocyte-type oxidase has been postulated to function in kidney as an oxygen sensor for EPO-synthesis, we were interested in the intrarenal distribution of the Renox message.
  • In situ hybridization experiments with fixed mouse kidney sections revealed that Renox has the highest expression within the renal cortex (Fig. 3), specifically in proximal convoluted tubule cells, while lower expression was detected in tubules within the medulla.
  • EXAMPLE 3 Expression of Renox in Transfected Cells To explore the enzymatic function of Renox, NIH 3T3 fibroblasts were transfected with Renox cDNA constructed in the pcDNA3.1 vector (Invitrogen, Carlsbad, CA). For expression studies, the complete coding sequence of murine Renox was subcloned into pcDNA3.1. NIH 3T3 fibroblasts were maintained in DMEM containing 10% FCS, penicillin (100units/ ml), and streptomycin (100ug/ ml).
  • NIH 3T3 fibroblasts were transfected with pcDNA3.1- Renox or the empty pcDNA3.1 vector (Invitrogen) using GENEPORTER transfection reagent (Gene Therapy Systems, San Diego, CA). After 48 hours, cells were selected with G418 (2 mg/ml) and individual, resistant colonies were isolated after 7 days.
  • NIH 3T3 clones expressing high levels of Renox mRNA were identified by RT-PCR and further analyzed by Northern blotting. Cell lines showing the highest expression of Renox mRNA were selected and assayed in further experiments (Fig. 4A). Superoxide production was measured by chemiluminescence using an enhanced luminol-containing reagent that has high sensitivity and specificity for superoxide. Renox-transfected cells showed significant superoxide production when compared with several control (empty vector) transfected lines (Fig. 4B), a response that was not increased by elevating intracellular calcium concentrations and activating protein kinase C with phorbol esters.
  • Renox- transfected fibroblasts showed drastic changes in cellular morphology and a significantly decreased rate of proliferation (Fig. 5).
  • Renox-transfected fibroblasts became flattened and larger in size, developed long processes, and often contained multiple nuclei.
  • ROS ROS
  • EXAMPLE 4 Theoretical Considerations Regarding the Function of Renox. Not wishing to be limited by theory, the inventors set out the following theoretical considerations related to the present disclosure. In this application we describe the characterization of a novel renal gp91 phox homologue called Renox. Using Northern blot and in situ hybridization techniques we have shown that Renox is highly expressed in the kidney, particularly in the proximal convoluted tubule cells of the renal cortex.
  • Renox as a source of superoxide in proximal convoluted tubules could have important physiological and pathological implications, as ROS play significant roles in tubular hypertrophic responses to angiotensin II (Hannken, T., et al., (1998)) and the nephotoxicity of drugs such as cylosporin and aminoglycosides (Baud et al., (1986)).
  • ROS nken, T., et al., (1998)
  • drugs such as cylosporin and aminoglycosides
  • the invention Based on the well known role of ROS as antimicrobial agents, which is well illustrated in the case of the phagocyte oxidase of immune cells, the invention provides a basis for considering Renox as an important host defense and pro-inflammatory component of renal tubule cells, because Renox was demonstrated to be a source of superoxide and is expressed at high levels in renal tubules. Therefore the invention provides a basis for either augmenting or inhibiting the host defense and proinflammatory capabilities of kidney tubule cells. Another basis for considering Rendox an important host defense and pro-inflammatory component of renal tubule cells is the extensive sequence homology between Renox and gp91phox, the electron transporting subunit of the phagocytic oxidase.
  • the gp91phox subunit of the phagocytic oxidase has a well-defined role in antimicrobial host defense and regulation of inflammatory signals.
  • the absence of this oxidase results in a severe immunedeficiency called chronic granulomatous disease, which is characterized by enhanced susceptibility to microbial infections and dysregulated inflammatory responses.
  • the overproduction of superoxide has been linked to the pathogenesis of several autoimmune diseases and cancer. Therefore, the ability of Renox to produce superoxide in the kidney raises the possibility that this enzyme also has a role in host defense processes in the kidney and could be an important factor in maintaining sterility in the upper urinary tract.
  • Renox activity may be associated with tissue injury and inflammatory reactions affecting tubular and glomerular cell functions (Baud, L., et al., "Reactive oxygen species: production and role in the kidney,” Am. J. Physiol. 251 :F765 (1986)).
  • phagocyte oxidase is induced by inflammatory cytokines such as interferon (Levy, R., et al. "Induction of the respiratory burst in HL-60 cells. Correlation of function and protein expression" J Immunol. 145:2595 (1990)), consistent with the presence of interferon responsive elements in several phox genes, future work should address whether the Renox gene is also directly responsive to inflammatory cytokines. Such responsiveness may account for the diminished EPO synthesis observed in a variety of inflammatory diseases (Ebert et al. (1999)).
  • Renox function would be facilitated by creation of transgenic animal models as described herein, which will provide a better understanding of oxygen sensing and its role in EPO synthesis and may also help explore the role of Renox in experimental models of kidney diseases.
  • Pharmacological inhibitors targeted to Renox may have an important role in stimulation of intrinsic EPO synthesis in certain diseases where anemia is caused by insufficient EPO production.
  • oxidases related to Renox gp91phox, Mox1 , and its spliced variant
  • Renox likely serves a critical role in promoting Na+ reabsorption by the kidney based on several considerations.
  • Both murine and human Renox polypeptide sequences described in the invention contain all of the structural features considered essential for the proton channel activity of gp91phox, namely conserved histidine residues in the third transmembrane domain (His 104, 118, and 123 corresponding to His 104, 118, and 123 of gp 91phox; Henderson, LM, "Role of histidines identified by mutagenesis in the NADPH oxidase-associated H+ channel.” J. Biol. Chem.273: 33216 (1998)).
  • Renox produces protons (H + ) into the intracellular space, thus lowering the pH (increasing H + concentrations) in the cytosol, which would facilitate the activity of the Na + -H + exchanger, thus promoting sodium reabsorption and proton excretion at the sites of Renox expression.
  • Renox is highly expressed in proximal convoluted tubule cells, and is also present in collecting ducts, sites involved in H + -excretion in the kidney. Therefore, both the unique structural features of Renox and the specific expression patterns of this gene described by this invention indicate Renox is a good candidate for therapeutic intervention (Renox therapy) aimed at altering both proton secretion and sodium reabsorption by the kidney.

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Wood Science & Technology (AREA)
  • Microbiology (AREA)
  • Biotechnology (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Enzymes And Modification Thereof (AREA)
  • Investigating Or Analysing Biological Materials (AREA)

Abstract

Cette invention se rapporte à des acides nucléiques et à leurs polypeptides codés correspondant à de nouvelles NADPH-oxydases rénales mammaliennes, et à des utilisations de ces acides nucléiques et polypeptides. Cette invention concerne des compositions thérapeutiques correspondant à ces nouvelles NADPH-oxydases rénales mammaliennes, des vecteurs d'expression contenant ces acides nucléiques et des lignées cellulaires transformées contenant ces vecteurs d'expression, ainsi que des procédés de traitement de patients à l'aide de compositions thérapeutiques correspondant à ces nouvelles NADPH-oxydases rénales mammaliennes et des procédés pour produire les polypeptides faisant l'objet de cette invention. Cette invention concerne en outre des dosages permettant de découvrir des composés qui ont la fonction de protéines codées par des acides nucléiques de ces nouvelles NADPH-oxydases rénales mammaliennes, un procédé pour traiter ou prévenir le cancer, l'hypertension, les maladies rénales inflammatoires et les infections des voies urinaires, et un procédé pour traiter des patients souffrant de polycythémie, ainsi qu'un procédé pour déterminer la séquence d'un gène de NADPH-oxydases rénales mammaliennes dans un échantillon.
PCT/US2001/010866 2000-04-12 2001-04-03 Identification d'une nouvelle nadph-oxydase renale WO2001079467A2 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU2001253123A AU2001253123A1 (en) 2000-04-12 2001-04-03 Identification of a renal nadph oxidase

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US19634100P 2000-04-12 2000-04-12
US60/196,341 2000-04-12
US21262900P 2000-06-19 2000-06-19
US60/212,629 2000-06-19

Publications (2)

Publication Number Publication Date
WO2001079467A2 true WO2001079467A2 (fr) 2001-10-25
WO2001079467A3 WO2001079467A3 (fr) 2003-01-23

Family

ID=26891837

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2001/010866 WO2001079467A2 (fr) 2000-04-12 2001-04-03 Identification d'une nouvelle nadph-oxydase renale

Country Status (2)

Country Link
AU (1) AU2001253123A1 (fr)
WO (1) WO2001079467A2 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002081703A3 (fr) * 2000-11-16 2003-12-24 Univ Emory Regulateurs d'oxygenase mitogeniques

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1242443A4 (fr) * 1999-12-23 2005-06-22 Nuvelo Inc Nouveaux acides nucleiques et polypeptides

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002081703A3 (fr) * 2000-11-16 2003-12-24 Univ Emory Regulateurs d'oxygenase mitogeniques
US6846672B2 (en) 2000-11-16 2005-01-25 Emory University Mitogenic oxygenase regulators
US7202053B2 (en) 2000-11-16 2007-04-10 Emory University Mitogenic oxygenase regulators
US7202052B2 (en) 2000-11-16 2007-04-10 Emory University Mitogenic oxygenase regulators
AU2001297756B2 (en) * 2000-11-16 2007-11-08 Emory University Mitogenic oxygenase regulators
US7524936B2 (en) 2000-11-16 2009-04-28 Emory University Mitogenic oxygenase regulators

Also Published As

Publication number Publication date
AU2001253123A1 (en) 2001-10-30
WO2001079467A3 (fr) 2003-01-23

Similar Documents

Publication Publication Date Title
JP2002525115A (ja) 発作、高血圧、糖尿病および肥満を予報および治療する、遺伝子およびタンパク質
JP2001517439A (ja) ５３ｂｐ２複合体
JP2001527421A (ja) Ｓｍａｄ６及びその使用
WO2001019999A1 (fr) Gene codant une nouvelle threonyl-arnt synthase, ses utilisations et procedes de preparation
JP2003502046A (ja) 新規ヒト環状ヌクレオチドホスホジエステラーゼ
WO1999007893A1 (fr) ISOLEMENT D'UN NOUVEAU GENE p23 DU FACTEUR DE SENESCENCE
JP2002525081A (ja) 低酸素により調節される遺伝子転写に特徴的な配列
EP1124572B1 (fr) Genes et proteines destines a la prevention et therapie de maladies renales et de troubles associes
AU761651B2 (en) CDK2 protein and CDK2 protein complexes
US20040038248A1 (en) Novel polypeptide-human heterogeneous nuclear ribonucleoprotein 32.01 and the polynucleotide encoding said polypeptide
EP1119619A2 (fr) Proteines d'interaction avec nlk1
US7619065B2 (en) Cystine-knot fold protein
US6987004B1 (en) Genes integrating signal transduction pathways
WO2001079467A2 (fr) Identification d'une nouvelle nadph-oxydase renale
WO1999041376A9 (fr) Complexes proteiques de retinoblastome et proteines interagissant avec le retinoblastome
US20090312245A1 (en) SRA binding protein
US20030148331A1 (en) Novel human hepatoma associated protein and the polynucleotide encoding said polypeptide
US6825034B2 (en) Human RRN3 and compositions and methods relating thereto
US6908765B1 (en) Polypeptide—human SR splicing factor 52 and a polynucleotide encoding the same
WO1998009979A1 (fr) Compositions relatives a la voie metabolique lipidique et utilisations therapeutiques et diagnostiques de telles compositions
US6919430B1 (en) Polypeptide—human galectin 15 and a polynucleotide encoding the same
AU2319700A (en) Meg-4 protein
WO2012113022A1 (fr) Utilisations thérapeutiques de slirp
US20040005658A1 (en) Novel polypeptide-human an1-like protein 16 and the polynucleotide encoding the same
US6994996B1 (en) Polypeptide, human vacuolar H+ -ATPase C subunit 42 and polynucleotide encoding it

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A2

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BY BZ CA CH CN CO CR CU CZ DE DK DM DZ EE ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NO NZ PL PT RO RU SD SE SG SI SK SL TJ TM TR TT TZ UA UG US UZ VN YU ZA ZW

AL Designated countries for regional patents

Kind code of ref document: A2

Designated state(s): GH GM KE LS MW MZ SD SL SZ TZ UG ZW AM AZ BY KG KZ MD RU TJ TM AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE TR BF BJ CF CG CI CM GA GN GW ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
122 Ep: pct application non-entry in european phase
NENP Non-entry into the national phase

Ref country code: JP

点击 这是indexloc提供的php浏览器服务，不要输入任何密码和下载