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Title:  Protein specific for cardiac and skeletal muscle
United States Patent: 
July 11, 2006

D'Azzo; Alessandra (Memphis, TN); Bongiovanni; Antonella (Memphis, TN); Nastasi; Tommaso (Memphis, TN)
St. Jude Children's Research Hospital (Memphis, TN)
Appl. No.:  014774
October 29, 2001


Covidien Pharmaceuticals Outsourcing


The present invention relates to a muscle-specific protein, Ozz, and nucleic acids encoding the protein, that regulates development and function of muscle cells. The invention further relates to muscle-specific regulated expression of the protein, and of heterologous genes under control of the same regulatory sequences. In a specific example, a murine Ozz protein of 285 amino acids is preferentially expressed by a 1.0 kb mRNA in heart and skeletal muscle. This protein shares significant homology with neuralized proteins, and associates with a number of muscle proteins, including .beta.-catenin.


Analysis of the 5' regions of the human and mouse PPCA genes led to the identification of their minimal promoter, which bears characteristics of housekeeping gene promoters and drives the transcription of an ubiquitously expressed mRNA. In addition, an alternative upstream promoter, present only in the mouse gene, that controls expression of a larger transcript of 2 kb present only in some tissues was found (Rottier and d'Azzo, DNA Cell Biol., 16:599 610, 1997). Functional analysis of the second PPCA transcript in mouse tissues brought the discovery of a new transcriptional unit, overlapping with exon Ia of the mouse PPCA gene. Thus, the present invention is based, in part, on the isolation and characterization of the corresponding cDNA and encoded protein, which has been named Ozz.

The primary structure of Ozz shows homology to the product of a developmental gene of Drosophila, called neuralized (neu), also found in C. elegans and humans. The Drosophila neu is a member of the neurogenic gene family that determine the cell fate in the developing central nervous system of the fly embryo (Corbin, et al., Cell, 67:311 23, 1991; Martin-Bermundo, et al., Development, 121:219 24, 1995; Hartenstein, et al., Development, 116:203 20, 1992). Absence of neu in the embryo results in an excess of neuroblasts, suggesting that neuralized plays a role in determining cell fate in the neurogenic region of the embryo. The neuralized protein contains four domains: a nuclear localization signal, a homeodomain similarity, a helix-turn-helix motif, a zinc-finger region, as well as a potential DNA binding domain, the RING zinc-finger domain (Price, et al., EMBO J., 12:2411 18, 1993). In addition, neuralized includes a stretch of 100 amino acids which is repeated twice in the Drosophila, human and C. elegans proteins, and is called NHR (Neuralized Homologous Region) (Nakamura, et al., Oncogene, 16:1009 19, 1998). The NHR domain is present in the N-terminal region of the Ozz protein. Ozz mRNA and protein are preferentially expressed in embryonal and adult muscle tissues. Ozz is likely to be involved in muscle differentiation.


Ozz protein, as defined herein, refers to a polypeptide having about 285 amino acids. In a specific embodiment, human Ozz has 285 amino acids. In another specific embodiment, murine Ozz also has 285 amino acids. Ozz can have a calculated molecular weight of about 31.5 kilo-Daltons (kDa); when murine Ozz is expressed in a recombinant cell line, for example, the apparent molecular weight is about 29 30 kDa, as measured by SDS-polyacrylamide gel electrophoresis. Because they are highly homologous, human Ozz can be expected to have very similar properties. Indeed, human and murine Ozz share 90% sequence identity, and 92% sequence similarity. Thus, the term Ozz encompasses polypeptides having about 90% sequence identity or about 92% sequence similarity with SEQ ID NO:2 or 4 (murine or human Ozz). The N-terminal portion of Ozz has significant homology with Neuralized protein. In a specific embodiment, there is about 40% sequence identity between the N-terminal portion of Ozz and a duplicated repeat of Drosophila neuralized protein. In addition, there is a stretch of about 30 amino acids at the C-terminus of Ozz that shows homology to two regions of neuralized protein. In a specific embodiment, the regions of homology of both the N-terminal and C-terminal regions of Ozz with Drosophila neuralized are shown in FIGS. 5 and 6.

Ozz can be further characterized by a tissue-specific expression pattern. Both Ozz mRNA and Ozz protein are only observed in heart and skeletal muscle, using routine assays (Northern analysis for mRNA and Western analysis for protein). This tissue-specific expression pattern has been observed for both mice and humans. It has also been found to be expressed in mice starting at embryonic day 12.5 (E12.5).

Ozz can also be characterized by the proteins to which it binds. In specific embodiments, using a yeast two-hybrid screen, Ozz was found to associate with .beta.-catenin, myosin, c-Nap, and Alix proteins. Further evidence of Ozz association with .beta.-catenin was found by co-immunoprecipitation analysis. In another embodiment, Ozz binds to an Ozz-specific antibody, e.g., as exemplified below.

In a specific embodiment, in order to develop the specific C-terminal and N-terminal Ozz antibodies, antibodies can be raised against the two halves of Ozz protein. The two peptides are produced from two truncated forms of mouse Ozz cDNA (corresponding to the nt 1-483 and nt 478-1036, respectively) fused with the GST coding sequence. The N terminus has neuralized homology.

Ozz fragments, derivatives, and analogs can be characterized by one or more of the characteristics of Ozz protein. For example, an Ozz fragment, also termed herein an Ozz peptide or polypeptide, can have an amino acid sequence corresponding to a homology region of neuralized protein, and in particular one of the fragments having SEQ ID NO:5, 7, 9, or 11 (the homologous fragments of Ozz shown in FIG. 6). In addition, an Ozz peptide can have an amino acid sequence of the SOCS box having the sequence PSLQTLCRLVIQRSMVHRLAIDGLHLPKELKDFCKYE (SEQ ID NO:23), or the amino acid sequence of a BC box having the sequence SLxxxCxxxI (SEQ ID NO:24). In another embodiment, an Ozz fragment comprises a putative phosphorylation site, e.g., a site for casein II (in a specific embodiment, such as site has a sequence as depicted in SEQ ID NO:19), protein kinase C (in a specific embodiment, such a site has a sequence as depicted in SEQ ID NO:20), or tyrosine kinase (in a specific embodiment, such a site has a sequence as depicted in SEQ ID NO:22). In yet another embodiment, an Ozz fragment can contain an alternative modification site, for example a myristoylation site (in a specific embodiment, such a site has a sequence as depicted in SEQ ID NO:21).

Analogs and derivatives of Ozz of the invention have the same or homologous characteristics of Ozz as set forth above. For example, a truncated form of Ozz can be provided. Such a truncated form includes Ozz with a deletion. In a specific embodiment, the derivative is functionally active, i.e., capable of exhibiting one or more functional activities associated with a full-length, wild-type Ozz of the invention. Such functions include binding .beta.-catenin.

Alternatively, an Ozz chimeric fusion protein can be prepared in which the Ozz portion of the fusion protein has one or more characteristics of Ozz. Such fusion proteins include fusions of Ozz polypeptide with a marker polypeptide, such as FLAG, a histidine tag, or, as exemplified herein, glutathione-S-transferase (GST). Ozz can also be fused with a unique phosphorylation site for labeling. In another embodiment, Ozz can be expressed as a fusion with a bacterial protein, such as .beta.-galactosidase.

Ozz analogs can be made by altering encoding nucleic acid sequences by substitutions, additions or deletions that provide for functionally similar molecules, i.e., molecules that perform one or more Ozz functions. In a specific embodiment, an analog of Ozz is a sequence-conservative variant of Ozz. In another embodiment, an analog of Ozz is a function-conservative variant. In yet another embodiment, an analog of Ozz is an allelic variant or a homologous variant from another species. In a specific embodiment, human and murine variants of Ozz are described.

Ozz derivatives include, but are by no means limited to, phosphorylated Ozz, myristylated Ozz, methylated Ozz, and other Ozz proteins that are chemically modified. Ozz derivatives also include labeled variants, e.g., radio-labeled with iodine (or, as pointed out above, phosphorous); a detectable molecule, such as but by no means limited to biotin, a chelating group complexed with a metal ion, a chromophore or fluorophore, a gold colloid, or a particle such as a latex bead; or attached to a water soluble polymer.

Chemical modification of biologically active component or components of Ozz may provide additional advantages under certain circumstances, such as increasing the stability and circulation time of the component or components and decreasing immunogenicity. See U.S. Pat. No. 4,179,337, Davis et al., issued Dec. 18, 1979. For a review, see Abuchowski et al., in Enzymes as Drugs (J. S. Holcerberg and J. Roberts, eds. pp. 367 383 (1981)). A review article describing protein modification and fusion proteins is Francis, 1992, Focus on Growth Factors 3:4 10, Mediscript: Mountview Court, Friern Barnet Lane, London N20, OLD, UK.

The chemical moieties suitable for derivatization may be selected from among water soluble polymers. The polymer selected should be water soluble so that the component to which it is attached does not precipitate in an aqueous environment, such as a physiological environment. Preferably, for therapeutic use of the end-product preparation, the polymer will be pharmaceutically acceptable. One skilled in the art will be able to select the desired polymer based on such considerations as whether the polymer/component conjugate will be used therapeutically, and if so, the desired dosage, circulation time, resistance to proteolysis, and other considerations. For the present component or components, these may be ascertained using the assays provided herein.

The water soluble polymer may be selected from the group consisting of, for example, polyethylene glycol, copolymers of ethylene glycol/propylene glycol, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene/maleic anhydride copolymer, polyaminoacids (either homopolymers or random copolymers), and dextran or poly(n-vinyl pyrrolidone)polyethylene glycol, propropylene glycol homopolymers, prolypropylene oxide/ethylene oxide co-polymers, polyoxyethylated polyols and polyvinyl alcohol. Polyethylene glycol propionaldenhyde may advantages in manufacturing due to its stability in water.

Cloning and Expression of Ozz

The present invention contemplates analysis and isolation of a gene encoding a functional or mutant Ozz, including a full length, or naturally occurring form of Ozz, and any antigenic fragments thereof from any source, preferably human. It further contemplates expression of functional or mutant Ozz protein for evaluation, diagnosis, or therapy.

In accordance with the present invention there may be employed conventional molecular biology, microbiology, and recombinant DNA techniques within the skill of the art. Such techniques are explained fully in the literature. See, e.g., Sambrook, Fritsch & Maniatis, Molecular Cloning: A Laboratory Manual, Second Edition (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (herein "Sambrook et al., 1989"); DNA Cloning: A Practical Approach, Volumes I and II (D. N. Glover ed. 1985); Oligonucleotide Synthesis (M. J. Gait ed. 1984); Nucleic Acid Hybridization [B. D. Hames & S. J. Higgins eds. (1985)]; Transcription And Translation [B. D. Hames & S. J. Higgins, eds. (1984)]; Animal Cell Culture [R. I. Freshney, ed. (1986)]; Immobilized Cells And Enzymes [IRL Press, (1986)]; B. EPerbal, A Practical Guide To Molecular Cloning (1984); F. M. Ausubel et al. (eds.), Current Protocols in Molecular Biology, John Wiley & Sons, Inc. (1994).

Ozz Nucleic Acids

A gene encoding Ozz, whether genomic DNA or cDNA, can be isolated from any source, particularly from a human cDNA or genomic library. Methods for obtaining Ozz gene are well known in the art, as described above (see, e.g., Sambrook et al., 1989, supra). The DNA may be obtained by standard procedures known in the art from cloned DNA (e.g., a DNA "library"), and preferably is obtained from a cDNA library prepared from tissues with high level expression of the protein (e.g., a muscle cell library, since these are the cells that evidence highest levels of expression of Ozz), by chemical synthesis, by cDNA cloning, or by the cloning of genomic DNA, or fragments thereof, purified from the desired cell (See, for example, Sambrook et al., 1989, supra; Glover, D. M. (ed.), 1985, DNA Cloning: A Practical Approach, MRL Press, Ltd., Oxford, U.K. Vol. I, II). Clones derived from genomic DNA may contain regulatory and intron DNA regions in addition to coding regions; clones derived from cDNA will not contain intron sequences. Whatever the source, the gene should be molecularly cloned into a suitable vector for propagation of the gene. Identification of the specific DNA fragment containing the desired Ozz gene may be accomplished in a number of ways. For example, a portion of an Ozz gene exemplified infra can be purified and labeled to prepare a labeled probe, and the generated DNA may be screened by nucleic acid hybridization to the labeled probe (Benton and Davis, Science 196:180, 1977; Grunstein and Hogness, Proc. Natl. Acad. Sci. U.S.A. 72:3961, 1975). Those DNA fragments with substantial homology to the probe, such as an allelic variant from another individual, will hybridize. In a specific embodiment, highest stringency hybridization conditions are used to identify a homologous Ozz gene.

Further selection can be carried out on the basis of the properties of the gene, e.g., if the gene encodes a protein product having the isoelectric, electrophoretic, amino acid composition, partial or complete amino acid sequence, antibody binding activity, or ligand binding profile of Ozz protein as disclosed herein. Thus, the presence of the gene may be detected by assays based on the physical, chemical, immunological, or functional properties of its expressed product.

Other DNA sequences which encode substantially the same amino acid sequence as an Ozz gene may be used in the practice of the present invention. These include but are not limited to allelic variants, species variants, sequence conservative variants, and functional variants.

Amino acid substitutions may also be introduced to substitute an amino acid with a particularly preferable property. For example, a Cys may be introduced a potential site for disulfide bridges with another Cys.

The genes encoding Ozz derivatives and analogs of the invention can be produced by various methods known in the art. The manipulations which result in their production can occur at the gene or protein level. For example, the cloned Ozz gene sequence can be modified by any of numerous strategies known in the art (Sambrook et al., 1989, supra). The sequence can be cleaved at appropriate sites with restriction endonuclease(s), followed by further enzymatic modification if desired, isolated, and ligated in vitro. In the production of the gene encoding a derivative or analog of Ozz, care should be taken to ensure that the modified gene remains within the same translational reading frame as the Ozz gene, uninterrupted by translational stop signals, in the gene region where the desired activity is encoded.

Additionally, the Ozz-encoding nucleic acid sequence can be mutated in vitro or in vivo, to create and/or destroy translation, initiation, and/or termination sequences, or to create variations in coding regions and/or form new restriction endonuclease sites or destroy preexisting ones, to facilitate further in vitro modification. Such modifications can be made to introduce restriction sites and facilitate cloning the Ozz gene into an expression vector. Any technique for mutagenesis known in the art can be used, including but not limited to, in vitro site-directed mutagenesis (Hutchinson, C., et al., J. Biol. Chem. 253:6551, 1978; Zoller and Smith, DNA 3:479 488, 1984; Oliphant et al., Gene 44:177, 1986; Hutchinson et al., Proc. Natl. Acad. Sci. U.S.A. 83:710, 1986), use of TAB linkers (Pharmacia), etc. PCR techniques are preferred for site directed mutagenesis (see Higuchi, 1989, "Using PCR to Engineer DNA", in PCR Technology: Principles and Applications for DNA Amplification, H. Erlich, ed., Stockton Press, Chapter 6, pp. 61 70).

The identified and isolated gene can then be inserted into an appropriate cloning vector. A large number of vector-host systems known in the art may be used. Possible vectors include, but are not limited to, plasmids or modified viruses, but the vector system must be compatible with the host cell used. Examples of vectors include, but are not limited to, E. coli, bacteriophages such as lambda derivatives, or plasmids such as pBR322 derivatives or pUC plasmid derivatives, e.g., pGEX vectors, pmal-c, pFLAG, etc. The insertion into a cloning vector can, for example, be accomplished by ligating the DNA fragment into a cloning vector which has complementary cohesive termini. However, if the complementary restriction sites used to fragment the DNA are not present in the cloning vector, the ends of the DNA molecules may be enzymatically modified. Alternatively, any site desired may be produced by ligating nucleotide sequences (linkers) onto the DNA termini; these ligated linkers may comprise specific chemically synthesized oligonucleotides encoding restriction endonuclease recognition sequences.

Recombinant molecules can be introduced into host cells via transformation, transfection, infection, electroporation, etc., so that many copies of the gene sequence are generated. Preferably, the cloned gene is contained on a shuttle vector plasmid, which provides for expansion in a cloning cell, e.g., E. coli, and facile purification for subsequent insertion into an appropriate expression cell line, if such is desired. For example, a shuttle vector, which is a vector that can replicate in more than one type of organism, can be prepared for replication in both E. coli and Saccharomyces cerevisiae by linking sequences from an E. coli plasmid with sequences form the yeast 2m plasmid.

Ozz Regulatory Nucleic Acids

A particular advantage of the present invention is the identification of the heart and muscle-specific promoter of Ozz. This discovery has important implications in the field of gene therapy, since therapeutic vectors, as described in the sub-section entitled "Vectors", infra, can be modified to employ the Ozz promoter for tissue-specific expression of a therapeutic protein. For example, expression of an angiogenic factor, such as basic fibroblast growth factor, VEGF, VEGf2, angiopoietin, etc., can be limited to target ischemic muscle (heart or skeletal muscle).

Ozz appears to share the PPCA proximal promoter (see FIG. 3), which contains three E-boxes. These E-boxes, which can function in either orientation, are target sites for muscle-specific transcription factors belonging to the Myo-D family. (It is, therefore, surprising to find them in the PPCA promoter, since PPCA is expressed ubiquitously). Other elements of the Ozz promoter can be identified by scanning the human genomic region upstream of the Ozz start site, e.g., by creating deletion mutants and checking for expression, or with the TRANSFAC algorithm. Sequences up to about 6 kb or more upstream from the Ozz start site can contain tissue-specific regulatory elements. In particular, in intron X of the human PPGB gene (the human homolog of murine PPCA), at 5.5 kb and 4.5 kb upstream of the Ozz start site, recognition sites for a cardiac-specific transcription factor nkxc-2.5 characterized in mouse (Chen, et al., J. Biol. Chem., 270:15628 33, 1995) have been identified. These may function to regulate tissue-specific expression of Ozz.

The term "Ozz promoter" encompasses artificial promoters. Such promoters can be prepared by deleting non-essentially intervening sequences from the upstream region of the Ozz promoter, or by joining upstream regulatory elements from the Ozz promoter with a heterologous minimal promoter, such as the CMV immediate early promoter.

Expression of Ozz Polypeptides

The nucleotide sequence coding for Ozz, or antigenic fragment, derivative or analog thereof, or a functionally active derivative, including a chimeric protein, thereof, can be inserted into an appropriate expression vector, i.e., a vector which contains the necessary elements for the transcription and translation of the inserted protein-coding sequence. Thus, a nucleic acid encoding Ozz of the invention can be operationally associated with a promoter in an expression vector of the invention. Both cDNA and genomic sequences can be cloned and expressed under control of such regulatory sequences. Such vectors can be used to express functional or functionally inactivated Ozz polypeptides.

The necessary transcriptional and translational signals can be provided on a recombinant expression vector, or they may be supplied by the native gene encoding Ozz and/or its flanking regions.

Potential host-vector systems include but are not limited to mammalian cell systems transfected with expression plasmids or infected with virus (e.g., vaccinia virus, adenovirus, adeno-associated virus, herpes virus, etc.); insect cell systems infected with virus (e.g., baculovirus); microorganisms such as yeast containing yeast vectors; or bacteria transformed with bacteriophage, DNA, plasmid DNA, or cosmid DNA. The expression elements of vectors vary in their strengths and specificities. Depending on the host-vector system utilized, any one of a number of suitable transcription and translation elements may be used.

Expression of Ozz protein may be controlled by any promoter/enhancer element known in the art, but these regulatory elements must be functional in the host selected for expression. Promoters which may be used to control Ozz gene expression include, but are not limited to, cytomegalovirus (CMV) promoter, the SV40 early promoter region (Benoist and Chambon, 1981, Nature 290:304 310), the promoter contained in the 3' long terminal repeat of Rous sarcoma virus (Yamamoto, et al., Cell 22:787 797, 1980), the herpes thymidine kinase promoter (Wagner et al., Proc. Natl. Acad. Sci. U.S.A. 78:1441 1445, 1981), the regulatory sequences of the metallothionein gene (Brinster et al., Nature 296:39 42, 1982); prokaryotic expression vectors such as the b-lactamase promoter (Villa-Komaroff, et al., Proc. Natl. Acad. Sci. U.S.A. 75:3727 3731, 1978), or the tac promoter (DeBoer, et al., Proc. Natl. Acad. Sci. U.S.A. 80:21 25, 1983); see also "Useful proteins from recombinant bacteria" in Scientific American, 242:74 94, 1980; promoter elements from yeast or other fungi such as the Gal 4 promoter, the ADC (alcohol dehydrogenase) promoter, PGK (phosphoglycerol kinase) promoter, alkaline phosphatase promoter; and transcriptional control regions that exhibit hematopoietic tissue specificity, in particular: beta-globin gene control region which is active in myeloid cells (Mogram et al., Nature 315:338 340, 1985; Kollias et al., Cell 46:89 94, 1986), hematopoietic stem cell differentiation factor promoters, erythropoietin receptor promoter (Maouche et al., Blood, 15:2557, 1991), etc.

Soluble forms of the protein can be obtained by collecting culture fluid, or solubilizing inclusion bodies, e.g., by treatment with detergent, and if desired sonication or other mechanical processes, as described above. The solubilized or soluble protein can be isolated using various techniques, such as polyacrylamide gel electrophoresis (PAGE), isoelectric focusing, 2-dimensional gel electrophoresis, chromatography (e.g., ion exchange, affinity, immunoaffinity, and sizing column chromatography), centrifugation, differential solubility, immunoprecipitation, or by any other standard technique for the purification of proteins.


A wide variety of host/expression vector combinations may be employed in expressing the DNA sequences of this invention. Useful expression vectors, for example, may consist of segments of chromosomal, non-chromosomal and synthetic DNA sequences. Suitable vectors include derivatives of SV40 and known bacterial plasmids, e.g., E. coli plasmids col El, pCR1, pBR322, pMal-C2, pET, pGEX (Smith et al., Gene 67:31 40, 1988), pMB9 and their derivatives, plasmids such as RP4; phage DNAS, e.g., the numerous derivatives of phage 1, e.g., NM989, and other phage DNA, e.g., M13 and filamentous single stranded phage DNA; yeast plasmids such as the 2m plasmid or derivatives thereof, vectors useful in eukaryotic cells, such as vectors useful in insect or mammalian cells; vectors derived from combinations of plasmids and phage DNAs, such as plasmids that have been modified to employ phage DNA or other expression control sequences; and the like.

Preferred vectors are viral vectors, such as lentiviruses, retroviruses, herpes viruses, adenoviruses, adeno-associated viruses, vaccinia virus, baculovirus, and other recombinant viruses with desirable cellular tropism. Thus, a gene encoding a functional or mutant Ozz protein or polypeptide domain fragment thereof can be introduced in vivo, ex vivo, or in vitro using a viral vector or through direct introduction of DNA. Expression in targeted tissues can be effected by targeting the transgenic vector to specific cells, such as with a viral vector or a receptor ligand, or by using a tissue-specific promoter, or both. Targeted gene delivery is described in International Patent Publication WO 95/28494, published October 1995.

Viral vectors commonly used for in vivo or ex vivo targeting and therapy procedures are DNA-based vectors and retroviral vectors. Methods for constructing and using viral vectors are known in the art (see, e.g., Miller and Rosman, BioTechniques, 7:980 990, 1992). Preferably, the viral vectors are replication defective, that is, they are unable to replicate autonomously in the target cell. In general, the genome of the replication defective viral vectors which are used within the scope of the present invention lack at least one region which is necessary for the replication of the virus in the infected cell. These regions can either be eliminated (in whole or in part), be rendered non-functional by any technique known to a person skilled in the art. These techniques include the total removal, substitution (by other sequences, in particular by the inserted nucleic acid), partial deletion or addition of one or more bases to an essential (for replication) region. Such techniques may be performed in vitro (on the isolated DNA) or in situ, using the techniques of genetic manipulation or by treatment with mutagenic agents. Preferably, the replication defective virus retains the sequences of its genome which are necessary for encapsidating the viral particles.

DNA viral vectors include an attenuated or defective DNA virus, such as but not limited to herpes simplex virus (HSV), papillomavirus, Epstein Barr virus (EBV), adenovirus, adeno-associated virus (AAV), and the like. Defective viruses, which entirely or almost entirely lack viral genes, are preferred. Defective virus is not infective after introduction into a cell. Use of defective viral vectors allows for administration to cells in a specific, localized area, without concern that the vector can infect other cells. Thus, a specific tissue can be specifically targeted. Examples of particular vectors include, but are not limited to, a defective herpes virus 1 (HSV 1) vector (Kaplitt et al., Molec. Cell. Neurosci. 2:320 330, 1991), defective herpes virus vector lacking a glyco-protein L gene (Patent Publication RD 371005 A), or other defective herpes virus vectors (International Patent Publication No. WO 94/21807, published Sep. 29, 1994; International Patent Publication No. WO 92/05263, published Apr. 2, 1994); an attenuated adenovirus vector, such as the vector described by Stratford-Perricaudet et al. (J. Clin. Invest. 90:626 630, 1992; see also La Salle et al., Science 259:988 990, 1993); and a defective adeno-associated virus vector (Samulski et al., J. Virol. 61:3096 3101, 1987; Samulski et al., J. Virol. 63:3822 3828, 1989; Lebkowski et al., Mol. Cell. Biol. 8:3988 3996, 1988).

Various companies produce viral vectors commercially, including but by no means limited to Avigen, Inc. (Alameda, Calif.; AAV vectors), Cell Genesys (Foster City, Calif.; retroviral, adenoviral, AAV vectors, and lentiviral vectors), Clontech (retroviral and baculoviral vectors), Genovo, Inc. (Sharon Hill, Pa.; adenoviral and AAV vectors), Genvec (adenoviral vectors), Intro Gene (Lei den, Netherlands; adenoviral vectors), Molecular Medicine (retroviral, adenoviral, AAV, and herpes viral vectors), Norgen (adenoviral vectors), Oxford BioMedica (Oxford, United Kingdom; lentiviral vectors), and Transgene (Strasbourg, France; adenoviral, vaccinia, retroviral, and lentiviral vectors).

Preferably, for in vivo administration, an appropriate immunosuppressive treatment is employed in conjunction with the viral vector, e.g., adenovirus vector, to avoid immuno-deactivation of the viral vector and transfected cells. For example, immunosuppressive cytokines, such as interleukin-12 (IL-12), interferon-g (IFN-g), or anti-CD4 antibody, can be administered to block humoral or cellular immune responses to the viral vectors (see, e.g., Wilson, Nature Medicine, 1995). In that regard, it is advantageous to employ a viral vector that is engineered to express a minimal number of antigens.

Ozz Binding Partners

The present invention further permits identification of physiological binding partners of Ozz. For example, as shown below, the invention exemplifies reagents to investigate the interaction between Ozz and .beta.-catenin. Similar experiments can be done with myosin and the less known c-Nap and Alix, in order to understand their possible role in the Ozz pathway.

One method for evaluating and identifying Ozz binding partners is the yeast two-hybrid screen. Preferably, the yeast two-hybrid screen would be performed using a muscle cell library with yeast that are transformed with recombinant Ozz, e.g., as shown in the Example, infra. Alternatively, Ozz can be used as a capture or affinity purification reagent. Again, the preferred source material for such preparations is muscle cells. In another alternative, labeled Ozz can be used as a probe for binding, e.g., immunoprecipitation or Western analysis.

Generally, binding interactions between Ozz and any of its binding partners will be strongest under conditions approximating those found in the cytoplasm, i.e., physiological conditions of ionic strength, pH and temperature. Perturbation of these conditions will tend to disrupt the stability of a binding interaction.

Antibodies to Ozz

Antibodies to Ozz are useful, inter alia, for diagnostics and intracellular regulation of Ozz activity, as set forth below. According to the invention, Ozz polypeptides produced recombinantly or by chemical synthesis, and fragments or other derivatives or analogs thereof, including fusion proteins, may be used as an immunogen to generate antibodies that recognize the Ozz polypeptide. Such antibodies include but are not limited to polyclonal, monoclonal, chimeric, single chain, Fab fragments, and an Fab expression library. Such an antibody is specific for human Ozz; it may recognize a mutant form of Ozz, or wild-type Ozz.

Various procedures known in the art may be used for the production of polyclonal antibodies to Ozz polypeptide or derivative or analog thereof. For the production of antibody, various host animals can be immunized by injection with the Ozz polypeptide, or a derivative (e.g., fragment or fusion protein) thereof, including but not limited to rabbits, mice, rats, sheep, goats, etc. In one embodiment, the Ozz polypeptide or fragment thereof can be conjugated to an immunogenic carrier, e.g., bovine serum albumin (BSA) or keyhole limpet hemocyanin (KLH). Various adjuvants may be used to increase the immunological response, depending on the host species, including but not limited to Freund's (complete and incomplete), mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanins, dinitrophenol, and potentially useful human adjuvants such as BCG (bacille Calmette-Guerin) and Corynebacterium parvum.

For preparation of monoclonal antibodies directed toward the Ozz polypeptide, or fragment, analog, or derivative thereof, any technique that provides for the production of antibody molecules by continuous cell lines in culture may be used. These include but are not limited to the hybridoma technique originally developed by Kohler and Milstein (Nature 256:495 497, 1975), as well as the trioma technique, the human B-cell hybridoma technique (Kozbor et al., Immunology Today 4:72, 1983; Cote et al., Proc. Natl. Acad. Sci. U.S.A. 80:2026 2030, 1983), and the EBV-hybridoma technique to produce human monoclonal antibodies (Cole et al., in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77 96, 1985). In an additional embodiment of the invention, monoclonal antibodies can be produced in germ-free animals (International Patent Publication No. WO 89/12690, published Dec. 28, 1989). In fact, according to the invention, techniques developed for the production of "chimeric antibodies" (Morrison et al., J. Bacteriol. 159:870, 1984); Neuberger et al., Nature 312:604 608, 1984; Takeda et al., Nature 314:452 454, 1985) by splicing the genes from a mouse antibody molecule specific for an Ozz polypeptide together with genes from a human antibody molecule of appropriate biological activity can be used; such antibodies are within the scope of this invention. Such human or humanized chimeric antibodies are preferred for use in therapy of human diseases or disorders (described infra), since the human or humanized antibodies are much less likely than xenogenic antibodies to induce an immune response, in particular an allergic response, themselves.

Antibody fragments which contain the idiotype of the antibody molecule can be generated by known techniques. For example, such fragments include but are not limited to: the F(ab').sub.2 fragment which can be produced by pepsin digestion of the antibody molecule; the Fab' fragments which can be generated by reducing the disulfide bridges of the F(ab').sub.2 fragment, and the Fab fragments which can be generated by treating the antibody molecule with papain and a reducing agent.

According to the invention, techniques described for the production of single chain antibodies (U.S. Pat. Nos. 5,476,786 and 5,132,405 to Huston; U.S. Pat, No. 4,946,778) can be adapted to produce Ozz polypeptide-specific single chain antibodies. An additional embodiment of the invention utilizes the techniques described for the construction of Fab expression libraries (Huse et al., Science 246:1275 1281, 1989) to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity for an Ozz polypeptide, or its derivatives, or analogs.

In the production and use of antibodies, screening for or testing with the desired antibody can be accomplished by techniques known in the art, e.g., radioimmunoassay, ELISA (enzyme-linked immunosorbant assay), "sandwich" immunoassays, immunoradiometric assays, gel diffusion precipitin reactions, immunodiffusion assays, in situ immunoassays (using colloidal gold, enzyme or radioisotope labels, for example), western blots, precipitation reactions, agglutination assays (e.g., gel agglutination assays, hemagglutination assays), complement fixation assays, immunofluorescence assays, protein A assays, and immunoelectrophoresis assays, etc. In one embodiment, antibody binding is detected by detecting a label on the primary antibody. In another embodiment, the primary antibody is detected by detecting binding of a secondary antibody or reagent to the primary antibody. In a further embodiment, the secondary antibody is labeled. Many means are known in the art for detecting binding in an immunoassay and are within the scope of the present invention. For example, to select antibodies which recognize a specific epitope of an Ozz polypeptide, one may assay generated hybridomas for a product which binds to an Ozz polypeptide fragment containing such epitope. For selection of an antibody specific to an Ozz polypeptide from a particular species of animal, one can select on the basis of positive binding with Ozz polypeptide expressed by or isolated from cells of that species of animal.

The foregoing antibodies can be used in methods known in the art relating to the localization and activity of the Ozz polypeptide, e.g., for Western blotting, imaging Ozz polypeptide in situ, measuring levels thereof in appropriate physiological samples, etc. using any of the detection techniques mentioned above or known in the art. Such antibodies can also be used in assays for ligand binding, e.g., as described in U.S. Pat. No. 5,679,582. Antibody binding generally occurs most readily under physiological conditions, e.g., pH of between about 7 and 8, and physiological ionic strength. The presence of a carrier protein in the buffer solutions stabilizes the assays. While there is some tolerance of perturbation of optimal conditions, e.g., increasing or decreasing ionic strength, temperature, or pH, or adding detergents or chaotropic salts, such perturbations will decrease binding stability.

In a specific embodiment, antibodies that agonize or antagonize the activity of Ozz polypeptide can be generated. In particular, intracellular single chain Fv antibodies can be used to regulate (inhibit) Ozz. Such antibodies can be tested using the assays described infra for identifying ligands.

Screening and Chemistry

According to the present invention, nucleotide sequences derived from the gene encoding Ozz, and peptide sequences derived from Ozz, are useful targets to identify drugs that are effective in treating myogenesis disorders. Drug targets include without limitation (i) isolated nucleic acids derived from the gene encoding Ozz and (ii) isolated peptides and polypeptides derived from Ozz polypeptides.

In particular, identification and isolation of Ozz provides for development of screening assays, particularly for high throughput screening of molecules that up- or down-regulate the activity of Ozz, e.g., by permitting expression of Ozz in quantities greater than can be isolated from natural sources, or in indicator cells that are specially engineered to indicate the activity of Ozz expressed after transfection or transformation of the cells. Accordingly, the present invention contemplates methods for identifying specific ligands of Ozz using various screening assays known in the art.

Any screening technique known in the art can be used to screen for Ozz agonists or antagonists. The present invention contemplates screens for small molecule ligands or ligand analogs and mimics, as well as screens for natural ligands that bind to and agonize or antagonize Ozz in vivo. Such agonists or antagonists may, for example, interfere in the phosphorylation or dephosphorylation of Ozz, with resulting effects on Ozz function. For example, natural products libraries can be screened using assays of the invention for molecules that agonize or antagonize Ozz activity.

Knowledge of the primary sequence of Ozz, and the similarity of that sequence with proteins of known function, can provide an initial clue as the inhibitors or antagonists of the protein. Identification and screening of antagonists is further facilitated by determining structural features of the protein, e.g., using X-ray crystallography, neutron diffraction, nuclear magnetic resonance spectrometry, and other techniques for structure determination. These techniques provide for the rational design or identification of agonists and antagonists.

Another approach uses recombinant bacteriophage to produce large libraries. Using the "phage method" (Scott and Smith, Science 249:386 390, 1990; Cwirla, et al., Proc. Natl. Acad. Sci., 87:6378 6382, 1990; Devlin et al., Science, 49:404 406, 1990), very large libraries can be constructed (10.sup.6 10.sup.8 chemical entities). A second approach uses primarily chemical methods, of which the Geysen method (Geysen et al., Molecular Immunology 23:709 715, 1986; Geysen et al. J. Immunologic Method 102:259 274, 1987; and the method of Fodor et al. (Science 251:767 773, 1991) are examples. Furka et al. (14th International Congress of Biochemistry, Volume #5, Abstract FR:013, 1988; Furka, Int. J. Peptide Protein Res. 37:487 493, 1991), Houghton (U.S. Pat. No. 4,631,211, issued December 1986) and Rutter et al. (U.S. Pat. No. 5,010,175, issued Apr. 23, 1991) describe methods to produce a mixture of peptides that can be tested as agonists or antagonists.

In another aspect, synthetic libraries (Needels et al., Proc. Natl . Acad. Sci. USA 90:10700 4, 1993; Ohlmeyer et al., Proc. Natl. Acad. Sci. USA 90:10922 10926, 1993; Lam et al., International Patent Publication No. WO 92/00252; Kocis et al., International Patent Publication No. WO 9428028) and the like can be used to screen for Ozz ligands according to the present invention.

Test compounds are screened from large libraries of synthetic or natural compounds. Numerous means are currently used for random and directed synthesis of saccharide, peptide, and nucleic acid based compounds. Synthetic compound libraries are commercially available from Maybridge Chemical Co. (Trevillet, Cornwall, UK), Comgenex (Princeton, N.J.), Brandon Associates (Merrimack, N.H.), and Microsource (New Milford, Conn.). A rare chemical library is available from Aldrich (Milwaukee, Wis.). Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are available from e.g. Pan Laboratories (Bothell, Wash.) or MycoSearch (N.C.), or are readily producible. Additionally, natural and synthetically produced libraries and compounds are readily modified through conventional chemical, physical, and biochemical means (Blondelle et al., Tib Tech, 14:60, 1996).

In Vivo Screening Methods

Intact cells or whole animals expressing a gene encoding Ozz can be used in screening methods to identify candidate drugs.

In one series of embodiments, a permanent cell line is established. Alternatively, cells (including without limitation mammalian, insect, yeast, or bacterial cells) are transiently programmed to express an Ozz gene by introduction of appropriate DNA or mRNA. Identification of candidate compounds can be achieved using any suitable assay, including without limitation (i) assays that measure selective binding of test compounds to Ozz (ii) assays that measure the ability of a test compound to modify (i.e., inhibit or enhance) a measurable activity or function of Ozz and (iii) assays that measure the ability of a compound to modify (i.e., inhibit or enhance) the transcriptional activity of sequences derived from the promoter (i.e., regulatory) regions the Ozz gene.

Ozz knockout mammals can be prepared for evaluating the molecular pathology of this defect in greater detail than is possible with human subjects. Such animals also provide excellent models for screening drug candidates. A "knockout mammal" is an mammal (e.g., mouse) that contains within its genome a specific gene that has been inactivated by the method of gene targeting (see, e.g., U.S. Pat. Nos. 5,777,195 and 5,616,491). A knockout mammal includes both a heterozygote knockout (i.e., one defective allele and one wild-type allele) and a homozygous mutant (i.e., two defective alleles; however, in this case a heterologous construct for expression of an Ozz, such as a human Ozz, would be inserted to permit the knockout mammal to live). Preparation of a knockout mammal requires first introducing a nucleic acid construct that will be used to suppress expression of a particular gene into an undifferentiated cell type termed an embryonic stem cell. This cell is then injected into a mammalian embryo. A mammalian embryo with an integrated cell is then implanted into a foster mother for the duration of gestation. Zhou, et al. (Genes and Development, 9:2623 34, 1995) describes PPCA knock-out mice.

The term "knockout" refers to partial or complete suppression of the expression of at least a portion of a protein encoded by an endogenous DNA sequence in a cell. The term "knockout construct" refers to a nucleic acid sequence that is designed to decrease or suppress expression of a protein encoded by endogenous DNA sequences in a cell. The nucleic acid sequence used as the knockout construct is typically comprised of (1) DNA from some portion of the gene (exon sequence, intron sequence, and/or promoter sequence) to be suppressed and (2) a marker sequence used to detect the presence of the knockout construct in the cell. The knockout construct is inserted into a cell, and integrates with the genomic DNA of the cell in such a position so as to prevent or interrupt transcription of the native DNA sequence. Such insertion usually occurs by homologous recombination (i.e., regions of the knockout construct that are homologous to endogenous DNA sequences hybridize to each other when the knockout construct is inserted into the cell and recombine so that the knockout construct is incorporated into the corresponding position of the endogenous DNA). The knockout construct nucleic acid sequence may comprise 1) a full or partial sequence of one or more exons and/or introns of the gene to be suppressed, 2) a full or partial promoter sequence of the gene to be suppressed, or 3) combinations thereof. Typically, the knockout construct is inserted into an embryonic stem cell (ES cell) and is integrated into the ES cell genomic DNA, usually by the process of homologous recombination. This ES cell is then injected into, and integrates with, the developing embryo.

The phrases "disruption of the gene" and "gene disruption" refer to insertion of a nucleic acid sequence into one region of the native DNA sequence (usually one or more exons) and/or the promoter region of a gene so as to decrease or prevent expression of that gene in the cell as compared to the wild-type or naturally occurring sequence of the gene. By way of example, a nucleic acid construct can be prepared containing a DNA sequence encoding an antibiotic resistance gene which is inserted into the DNA sequence that is complementary to the DNA sequence (promoter and/or coding region) to be disrupted. When this nucleic acid construct is then transfected into a cell, the construct will integrate into the genomic DNA. Thus, many progeny of the cell will no longer express the gene at least in some cells, or will express it at a decreased level, as the DNA is now disrupted by the antibiotic resistance gene.

Generally, the DNA will be at least about 1 kilobase (kb) in length and preferably 3 4 kb in length, thereby providing sufficient complementary sequence for recombination when the knockout construct is introduced into the genomic DNA of the ES cell (discussed below).

Included within the scope of this invention is a mammal in which two or more genes have been knocked out. Such mammals can be generated by repeating the procedures set forth herein for generating each knockout construct, or by breeding to mammals, each with a single gene knocked out, to each other, and screening for those with the double knockout genotype.

Regulated knockout animals can be prepared using various systems, such as the tet-repressor system (see U.S. Pat. No. 5,654,168) or the Cre-Lox system (see U.S. Pat. Nos. 4,959,317 and 5,801,030).

In another series of embodiments, transgenic animals are created in which (i) a human Ozz is stably inserted into the genome of the transgenic animal; and/or (ii) the endogenous Ozz genes are inactivated and replaced with human Ozz genes. See, e.g., Coffman, Semin. Nephrol. 17:404, 1997; Esther et al., Lab. Invest. 74:953, 1996; Murakami et al., Blood Press. Suppl. 2:36, 1996. Such animals can be treated with candidate compounds and monitored for muscle weakness, heart defects, or other indicia of muscle dysfunction.

High-Troughput Screen

Agents according to the invention may be identified by screening in high-throughput assays, including without limitation cell-based or cell-free assays. It will be appreciated by those skilled in the art that different types of assays can be used to detect different types of agents. Several methods of automated assays have been developed in recent years so as to permit screening of tens of thousands of compounds in a short period of time. Such high-throughput screening methods are particularly preferred. The use of high-throughput screening assays to test for agents is greatly facilitated by the availability of large amounts of purified polypeptides, as provided by the invention.

Methods of Diagnosis

According to the present invention, genetic variants of Ozz can be detected to diagnose a muscle degenerative disease. The various methods for detecting such variants are described herein. Where such variants impact Ozz function, either as a result of a mutated amino acid sequence or because the mutation results in expression of a truncated protein, or no expression at all, they are expected to result in disregulation of muscle development or function, including, possibly, muscle degeneration or alternatively hyperproliferation (e.g., a muscle sarcoma). In another embodiment, the presence of Ozz in blood or a blood fraction (serum, plasma) indicates muscle tissue damage, e.g., ischemia associated with either unstable angina, myocardial infarction, or both (see U.S. Pat. Nos. 5,747,274 and 5,744,358).

According to the present invention, altered Ozz protein levels and localization can be detected to diagnose diseases associated with altered Ozz protein expression and localization. The methods for detecting such altered protein levels and protein are described herein. When altered protein levels or protein localization are detected, they are expected to be associated with disease states that occur with altered Ozz expression. In one specific embodiment, the altered Ozz protein expression and localization is associated with galactosialidosis. In another embodiment, the altered protein levels or localization are evaluated with muscle cells that are from the atrium of the heart.

A "sample" as used herein refers to a biological sample, such as, for example, tissue (or cells) or fluid isolated from an individual or from in vitro cell culture constituents, as well as samples obtained from the environment or laboratory procedures. Non-limiting examples of cell sources available in clinical practice include without limitation muscle biopsies.

Nucleic Acid Assays

The DNA may be obtained from any cell source. DNA is extracted from the cell source or body fluid using any of the numerous methods that are standard in the art. It will be understood that the particular method used to extract DNA will depend on the nature of the source. Generally, the minimum amount of DNA to be extracted for use in the present invention is about 25 pg (corresponding to about 5 cell equivalents of a genome size of 4.times.10.sup.9 base pairs). Sequencing methods are described in detail, supra.

In another alternate embodiment, RNA is isolated from biopsy tissue using standard methods well known to those of ordinary skill in the art such as guanidium thiocyanate-phenol-chloroform extraction (Chomocyznski et al., Anal. Biochem., 162:156, 1987). The isolated RNA is then subjected to coupled reverse transcription and amplification by polymerase chain reaction (RT-PCR), using specific oligonucleotide primers that are specific for a selected site. Conditions for primer annealing are chosen to ensure specific reverse transcription and amplification; thus, the appearance of an amplification product is diagnostic of the presence of a particular genetic variation. In another embodiment, RNA is reverse-transcribed and amplified, after which the amplified sequences are identified by, e.g., direct sequencing. In still another embodiment, cDNA obtained from the RNA can be cloned and sequenced to identify a mutation.

Protein Assays

In an alternate embodiment, biopsy tissue is obtained from a subject. Antibodies that are capable of specifically binding to Ozz are then contacted with samples of the tissue to determine the presence or absence of a Ozz polypeptide specified by the antibody. The antibodies may be polyclonal or monoclonal, preferably monoclonal. Measurement of specific antibody binding to cells may be accomplished by any known method, e.g., quantitative flow cytometry, enzyme-linked or fluorescence-linked immunoassay, Western analysis, etc.

Immunoassay technology, e.g., as described in U.S. Pat. Nos. 5,747,274 and 5,744,358, and particularly solid phase "chromatographic" format immunoassays, are preferred for detecting proteins in blood or blood fractions.

Claim 1 of 11 Claims

1. An isolated nucleic acid encoding an Ozz protein wherein the Ozz protein is expressed only in cardiac and skeletal muscle, comprises about 285 amino acids, and shares about 90% sequence identity or about 92% sequence similarity with SEQ ID NO:2 or SEQ ID NO:4.

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