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  Pharmaceutical Patents  

 

Title:  Role of p62 in aging-related disease
United States Patent: 
7,435,872
Issued: 
October 14, 2008

Inventors: 
Shin; Jaekyoon (Keumkok-Dong, Kwanson-Ku, Suwon-Si, Kyonggi-Do, KR), Lee; Han-Woong (Seongnam, KR), Oh; Goo Taeg (Daejeon, KR)
Assignee: 
Shin; Jaekyoon (Suwon-si, Gyeonggi-do, KR), Samsung Electronics, Co., Ltd. (Suwon-si, Gyeonggi-do, KR)
Appl. No.: 
10/438,516
Filed: 
May 14, 2003


 

Patheon


Abstract

The application discloses a role of p62 in aging-related disease, such as development of obesity, type 2 diabetes mellitus, non-alcoholic fatty liver, various tumors, increased male mortality, intracellular inclusion named sequestosome, and redox regulation. In particular the application discloses a method of detecting the formation of inclusion bodies in neurodegenerative diseases. The invention further relates to a method of screening for therapeutic agents that disperse the inclusions. Further, transgenic mice containing a mutation in the p62 gene and having a functionally disrupted p62 gene locus are also disclosed.

Description of the Invention

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

p62/Ubiquitin Co-Accumulation

The presence of intracellular inclusion bodies is the hallmark of various neurodegenerative disorders including without limitation, Pick's Disease (Pick's body), Amyotrophic Lateral Sclerosis (ALS) spinal cord, Huntington's Disease, Parkinson's Disease, Alzheimer's Disease neurofibrillar tangle, among others. p62 co-accumulates and co-localizes heavily with ubiquitin to form inclusion bodies in the brain and spinal cord of patients with neurodegenerative diseases. p62 binds directly to ubiquitin with specificity. In vitro experiments showed that p62 bound to ubiquitin with specificity and Applicants have discovered that inclusion bodies resulting from sequestosomes associated with these diseases are p62 positive and ubiquitin positive. Applicants have also discovered that p62 binds to ubiquiin with specificity and that certain regions on p62 bind ubiquitin. Moreover, mutations of certain hydrophobic amino acids residues in p62 enhance binding to ubiquitin.

In one aspect of the invention, the p62 polypeptide and ubiquitin polypeptide retain the capability of specifically binding to ubiquitin and p62, respectively. The length of the p62 polypeptide and ubiquitin polypeptide may vary and may include other sequences attached thereto, as well as variant sequences, which may retain the capability of specifically binding to ubiquitin and p62, respectively.

In one aspect of the invention, the p62 polypeptide may be used as a trap to immobilize ubiquitin areas where there is undesired activity of ubiquitin. The p62 polypeptide may be full length or longer or shorter or variant or derivatized, so long as the polypeptide retains the capability of specifically binding to ubiquitin.

Inhibitor of p62/Ubiquitin Binding

In one embodiment, the invention is directed to screening for a compound such as a polypeptide or chemical compound that inhibits binding of p62 to ubiquitin. It is expected that the inhibitor compound will treat persons suffering from diseases that are at least in part associated with the presence of inclusion bodies. If an inhibitor is used, ubiquinated p62 would not accumulate in the inclusion bodies, and may prevent the formation of inclusion bodies.

In this regard, in one aspect, the invention is directed to any inhibitor molecule that is capable of interacting with p62 to block the binding of p62 to ubiquitin. And alternatively, the molecule may bind to ubiquitin thus disrupting the p62/ubiquitin binding.

One such inhibitor may be modified p62 polypeptide, which retains the ubiquitin binding function but lacks or has decreased level of some or all of the other features associated with p62 function, such as capability of forming aggregates.

It is understood that the inhibitor compound may impair the interaction between the p62 polypeptide and ubiquitin by any number of biochemical or enzymatic inhibition kinetics, such as competitive, non-competitive, or uncompetitive inhibition, so long as the compound impairs the binding of p62 polypeptide to ubiquitin.

Thus, in one particular aspect, the invention is directed to using any polymer or monomer compound that is capable of inhibiting binding between p62 and ubiquitin. In another embodiment of the invention, extracts of natural foods or `functional foods` are contemplated as comprising inhibitors of p62/ubiquitin complex.

Nucleic Acid Encoding Polypeptide that Binds to Ubiquitin or p62

By "isolated" polynucleotide sequence, it is intended to encompass a nucleic acid molecule, DNA or RNA, which has been removed from its native environment. This includes segments of DNA encoding p62 polypeptide or ubiquitin polypeptide of the present invention, and may further comprise heterologous sequences such as vector sequences or other foreign DNA. For example, recombinant DNA molecules contained in a vector are considered isolated for the purposes of the present invention, which may be partially or substantially purified.

In addition, isolated nucleic acid molecules of the invention include DNA molecules, which comprise a sequence substantially different from those described above but which, either due to the degeneracy of the genetic code or other variability, still encode p62 polypeptide or ubiquitin polypeptide and their peptides. Thus, it would be routine for one skilled in the art to generate the variants described above, for instance, to optimize codon expression or general function for a particular host.

In another aspect, the invention provides an isolated nucleic acid molecule comprising a polynucleotide which hybridizes under stringent hybridization conditions to a portion of a polynucleotide in a nucleic acid molecule of the invention described above. Hybridizing polynucleotides are useful as diagnostic probes and primers as discussed above. Portions of a polynucleotide which hybridizes to the p62 polypeptide, which can be used as probes and primers, may be precisely specified by 5' and 3' base positions or by size in nucleotide bases as described above or precisely excluded in the same manner. Similarly, portions of a polynucleotide which hybridize to the ubiquitin polypeptide may be used as probes and primers as well. Preferred hybridizing polynucleotides of the present invention are those that, when labeled and used in a hybridization assay known in the art (e.g. Southern and Northern blot analysis), display the greatest signal strength regardless of other heterologous sequences present in equimolar amounts.

Variant and Mutant Polynucleotides

The present invention further relates to variants of the nucleic acid molecules, which encode portions, analogs or derivatives of p62 polypeptide or ubiquitin polypeptide, which retain the capability of specifically binding to p62 and ubiquitin, respectively, which is also optionally non-functional in at least one other aspect, so long as the variant competitively or non-competitively disrupts p62/ubiquitin binding, without causing toxic side effects. Non-naturally occurring variants may be produced using art-known mutagenesis techniques.

Such nucleic acid variants include those produced by nucleotide substitutions, deletions, or additions. The substitutions, deletions, or additions may involve one or more nucleotides. Alterations in the amino acid sequence may produce conservative or non-conservative amino acid substitutions, deletions or additions. Especially preferred among these are silent substitutions, additions and deletions, which do not alter the properties and activities of the polypeptides of the present invention or portions thereof. Also preferred in this regard are conservative substitutions.

The invention allows for the use of sequences in expression vectors, as well as to transfect host cells and cell lines, be these prokaryotic or eukaryotic cells. The invention also allows for purification of the polypeptides expressed from the expression vector. The expression vector may contain various molecular tags for easy purification. Subsequently obtained expression construct may be transformed into any host cell of choice. Cell lysates from the host cell is isolated by established methods well known in the field. GFP- or GST-containing expression vector may be used to localize p62 polypeptide or ubiquitin polypeptide in the host cell. The expression vector may contain an inducible or constitutive promoter.

Variant and Mutant Polypeptides

To improve or alter the characteristics of p62 polypeptide or ubiquitin polypeptide of the present invention, amino acid engineering may be employed. Recombinant DNA technology known to those skilled in the art can be used to create novel mutant polypeptides including single or multiple amino acid substitutions, deletions, additions, or fusion proteins. Such modified polypeptides can show, e.g., increased/decreased binding activity or increased/decreased stability. In addition, they may be purified in higher yields and show better binding activity than the corresponding natural polypeptide, at least under certain purification and storage conditions.

Of special interest are substitutions of charged amino acids with other charged or neutral amino acids which may produce proteins with highly desirable improved characteristics, such as less aggregation of the produced polypeptides. Aggregation may not only reduce activity but may also be problematic when preparing pharmaceutical formulations, because aggregates can be immunogenic.

Antibodies

In one embodiment, the present invention is directed to detecting inclusion body or sequestosome formation using a variety of detection methods. One way to detect binding of p62 polypeptide to ubiquitin is to label the p62 polypeptide directly and assay for its binding using labeling and separation techniques that are routine to a person of skill in the art. Other methods include using a labeled ligand that specifically binds to either the p62 polypeptide, ubiquitin or p62 polypeptide/ubiquitin complex. Such a ligand may be an antibody.

Purified p62 polypeptide, ubiquitin or p62 polypeptide/ubiquitin complex can be used to produce monoclonal or polyclonal antibody. Fragments of p62 polypeptide also can be used to produce monoclonal or polyclonal antibody. Subsequently obtained monoclonal or polyclonal antibody can be used to determine the binding of p62 polypeptide to ubiquitin and the formation of a p62 polypeptide, ubiquitin or p62 polypeptide/ubiquitin complex in various samples including cells, tissues, and body fluids such as but not limited to serum, plasma, and urine. p62 polypeptide, ubiquitin or p62 polypeptide/ubiquitin complex may be assayed using a variety of molecular biological methods, which include but are not limited to in situ hybridization, immunoprecipitation, immunofluorescence staining, Western blot analysis and so on. One can carry out ELISA by using monoclonal antibody against p62 polypeptide, ubiquitin or p62 polypeptide/ ubiquitin complex, to determine the amount of inclusion body in the brain tissue or other parts of the body, including body fluids of human subjects believed to have an indicated disorder in which p62 and ubiquitin is aggregated and is accumulated and form inclusion bodies.

Antibodies of the invention include, but are not limited to, polyclonal, monoclonal, multispecific, human, humanized or chimeric antibodies, single chain antibodies, Fab fragments, F(ab') fragments, fragments produced by a Fab expression library, anti-idiotypic (anti-Id) antibodies (including, e.g., anti-Id antibodies to antibodies of the invention), and epitope-binding fragments of any of the above. The term "antibody," as used herein, refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding site that immunospecifically binds an antigen. The immunoglobulin molecules of the invention can be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass of immunoglobulin molecule.

The antibodies of the present invention may be monospecific, bispecific, trispecific or of greater multispecificity. Multispecific antibodies may be specific for different epitopes of a polypeptide of the present invention or may be specific for both a polypeptide of the present invention as well as for a heterologous epitope, such as a heterologous polypeptide or solid support material.

Antibodies of the present invention may be described or specified in terms of the epitope(s) or portion(s) of a polypeptide of the present invention, which they recognize or specifically bind. The epitope(s) or polypeptide portion(s) may be specified as described herein, e.g., by N-terminal and C-terminal positions, by size in contiguous amino acid residues.

Antibodies of the present invention may be used, for example, but not limited to, to purify, detect, and target the polypeptides of the present invention, including both in vitro and in vivo diagnostic and therapeutic methods. For example, the antibodies have use in immunoassays for qualitatively and quantitatively measuring levels of p62 polypeptide, ubiquitin or p62 polypeptide/ubiquitin complex of the present invention in biological samples.

As discussed in more detail below, the antibodies of the present invention may be used either alone or in combination with other compositions. The antibodies may further be recombinantly fused to a heterologous polypeptide at the N- or C-terminus or chemically conjugated (including covalent and non-covalent conjugations) to polypeptides or other compositions. For example, antibodies of the present invention may be recombinantly fused or conjugated to molecules useful as labels in detection assays and effector molecules such as heterologous polypeptides, drugs, radionuclides, or toxins.

The antibodies of the present invention may be generated by any suitable method known in the art. Polyclonal antibodies to an antigen of interest can be produced by various procedures well known in the art. For example, a polypeptide of the invention can be administered to various host animals including, but not limited to, rabbits, mice, rats, etc. to induce the production of sera containing polyclonal antibodies specific for the antigen. Various adjuvants may be used to increase the immunological response, depending on the host species, and include but are 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. Such adjuvants are also well known in the art.

Monoclonal antibodies can be prepared using a wide variety of techniques known in the art including the use of hybridoma, recombinant, and phage display technologies, or a combination thereof. For example, monoclonal antibodies can be produced using hybridoma techniques including those known in the art. The term "monoclonal antibody" as used herein is not limited to antibodies produced through hybridoma technology. The term "monoclonal antibody" refers to an antibody that is derived from a single clone, including any eukaryotic, prokaryotic, or phage clone, and not the method by which it is produced.

Methods for producing and screening for specific antibodies using hybridoma technology are routine and well known in the art. In a non-limiting example, mice can be immunized with a p62 polypeptide, ubiquitin or p62 polypeptide/ubiquitin complex or a cell expressing such entity. Once an immune response is detected, e.g., antibodies specific for the antigen are detected in the mouse serum, the mouse spleen is harvested and splenocytes isolated. The splenocytes are then fused by well known techniques to any suitable myeloma cells, for example cells from cell line SP20 available from the ATCC. Hybridomas are selected and cloned by limited dilution. The hybridoma clones are then assayed by methods known in the art for cells that secrete antibodies capable of binding a polypeptide of the invention or its complex with its binding partner. Ascites fluid, which generally contains high levels of antibodies, can be generated by immunizing mice with positive hybridoma clones.

Antibodies may also be attached to solid supports, which are particularly useful for immunoassays or purification of the target antigen. Such solid supports include, but are not limited to, glass, cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride or polypropylene.

Assays for Antibody Binding

The antibodies of the invention may be assayed for immunospecific binding by any method known in the art. The immunoassays which can be used include but are not limited to competitive and non-competitive assay systems using techniques such as Western blots, radioimmunoassays, ELISA (enzyme linked immunosorbent assay), "sandwich" immunoassays, immunoprecipitation assays, precipitin reactions, gel diffusion precipitin reactions, immunodiffusion assays, agglutination assays, complement-fixation assays, immunoradiometric assays, fluorescent immunoassays, protein A immunoassays, to name but a few. Such assays are routine and well known in the art (see, e.g., Ausubel et al, eds, 1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York, which is incorporated by reference herein in its entirety). Exemplary immunoassays are described briefly below but are not intended by way of limitation.

Immunoprecipitation protocols generally comprise lysing a population of cells in a lysis buffer such as RIPA buffer (1% NP-40 or Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 0.15 M NaCl, 0.01 M sodium phosphate at pH 7.2, 1% Trasylol) supplemented with protein phosphatase and/or protease inhibitors (e.g., EDTA, PMSF, aprotinin, sodium vanadate), adding the antibody of interest to the cell lysate, incubating for a period of time (e.g., 1-4 hours) at 4.degree. C., adding protein A and/or protein G sepharose beads to the cell lysate, incubating for about an hour or more at 4.degree. C., washing the beads in lysis buffer and resuspending the beads in SDS/sample buffer. The ability of the antibody of interest to immunoprecipitate a particular antigen can be assessed by, e.g., Western blot analysis. One of skill in the art would be knowledgeable as to the parameters that can be modified to increase the binding of the antibody to an antigen and decrease the background (e.g., pre-clearing the cell lysate with sepharose beads). For further discussion regarding immunoprecipitation protocols see, e.g., Ausubel et al, eds, 1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York at 10.16.1.

Western blot analysis generally comprises preparing protein samples, electrophoresis of the protein samples in a polyacrylamide gel (e.g., 8%-20% SDS-PAGE depending on the molecular weight of the antigen), transferring the protein sample from the polyacrylamide gel to a membrane such as nitrocellulose, PVDF or nylon, blocking the membrane in blocking solution (e.g., PBS with 3% BSA or non-fat milk), washing the membrane in washing buffer (e.g., PBS-Tween 20), blocking the membrane with primary antibody (the antibody of interest) diluted in blocking buffer, washing the membrane in washing buffer, blocking the membrane with a secondary antibody (which recognizes the primary antibody, e.g., an anti-human antibody) conjugated to an enzymatic substrate (e.g., horse radish peroxidase or alkaline phosphatase) or radioactive molecule (e.g., .sup.32P or .sup.125I) diluted in blocking buffer, washing the membrane in wash buffer, and detecting the presence of the antigen. One of skill in the art would be knowledgeable as to the parameters that can be modified to increase the signal detected and to reduce the background noise. For further discussion regarding western blot protocols see, e.g., Ausubel et al, eds, 1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York at 10.8.1.

ELISAs comprise preparing antigen, which may include a sample comprising p62 polypeptide, ubiquitin or p62 polypeptide/ubiquitin, coating the well of a 96 well microtiter plate with the antigen, adding the antibody of interest conjugated to a detectable compound such as an enzymatic substrate (e.g., horseradish peroxidase or alkaline phosphatase) to the well and incubating for a period of time, and detecting the presence of the antigen. In ELISAs the antibody of interest does not have to be conjugated to a detectable compound; instead, a second antibody (which recognizes the antibody of interest) conjugated to a detectable compound may be added to the well. Further, instead of coating the well with the antigen, the antibody may be coated to the well. In this case, a second antibody conjugated to a detectable compound may be added simultaneously or following the addition of the antigen of interest to the coated well. One of skill in the art would be knowledgeable as to the parameters that can be modified to increase the signal detected as well as other variations of ELISAs known in the art. For further discussion regarding ELISAs see, e.g., Ausubel et al, eds, 1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York at 11.2.1.

Inclusion Body Diagnostic Assay

The invention also provides diagnostic methods for detecting the presence of inclusion body in a biological sample. This may be assayed either directly (e.g., by assaying polypeptide levels using antibodies elicited in response to p62 polypeptide or fragments thereof) or indirectly (e.g., by assaying for antibodies having specificity for p62 polypeptide or fragments thereof).

Where diagnosis of a diseased state has already been made, the present invention is useful for monitoring progression or regression of the disease state by measuring the amount of ubiquitin/p62 complex present in a patient or whereby patients exhibiting enhanced inclusion body production will experience a worse clinical outcome relative to patients producing inclusion body at a lower level.

Presence of the labeled molecule can be detected in the patient using methods known in the art for in vivo scanning. These methods depend upon the type of label used. Skilled artisans will be able to determine the appropriate method for detecting a particular label. Methods and devices that may be used in the diagnostic methods of the invention include, but are not limited to, computed tomography (CT), whole body scan such as position emission tomography (PET), magnetic resonance imaging (MRI), and sonography, as well as electron paramagnetic resonance (EPR) and nuclear magnetic resonance (NMR).

In a specific embodiment, a molecule such as a polypeptide that specifically binds to ubiquitin is labeled with a radioisotope and is detected in the patient using a radiation responsive surgical instrument (Thurston et al., U.S. Pat. No. 5,441,050). In another embodiment, the molecule is labeled with a fluorescent compound and is detected in the patient using a fluorescence responsive scanning instrument. In another embodiment, the molecule is labeled with a positron emitting metal and is detected in the patient using positron emission-tomography. In yet another embodiment, the molecule is labeled with a paramagnetic label and is detected in a patient using magnetic resonance imaging (MRI).

In one aspect, the iii vivo diagnosis may be made by using a label that is detectable by the above described methods or some other mechanism, which is non-toxic to the subject, injecting the labeled polypeptide into a subject such that the polypeptide travels and binds to its binding target, such as an aggregated region, whereby the presence of the target mass, such as ubiquitin or inclusion body aggregate is detected.

Labels

Suitable enzyme labels include, for example, those from the oxidase group, which catalyze the production of hydrogen peroxide by reacting with substrate. Glucose oxidase is particularly preferred as it has good stability and its substrate (glucose) is readily available. Activity of an oxidase label may be assayed by measuring the concentration of hydrogen peroxide formed by the enzyme-labeled antibody/substrate reaction. Besides enzymes, other suitable labels include radioisotopes, such as iodine (.sup.125I, .sup.121I), carbon (.sup.14C), sulphur (.sup.35S), tritium (.sup.3H), indium (.sup.112In), and technetium (.sup.99mTc), and fluorescent labels, such as fluorescein and rhodamine, and biotin.

Further suitable labels for the p62 polypeptide-, ubiquitin- or p62 polypeptide/ubiquitin complex-specific antibodies of the present invention are provided below. Examples of suitable enzyme labels include malate dehydrogenase, .delta.-5-steroid isomerase, yeast-alcohol dehydrogenase, .alpha.-glycerol phosphate dehydrogenase, triose phosphate isomerase, peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, .beta.-galactosidase, ribonuclease, urease, catalase, glucose-6-phosphate dehydrogenase, glucoamylase, and acetylcholine esterase.

Examples of suitable radioisotopic labels include 3H, .sup.111In, .sup.125I, .sup.131I, .sup.32P, .sup.35S, .sup.14C, .sup.51Cr, .sup.57To, .sup.58Co, .sup.59Fe, .sup.75Se, .sup.152Eu, .sup.90Y, .sup.67Cu, .sup.217Ci, .sup.211At, .sup.212Pb, .sup.47Sc, .sup.109Pd, etc. .sup.111In is preferred isotope where in vivo imaging is used since its avoids the problem of dehalogenation of the .sup.125I or .sup.131I-labeled polypeptide by the liver. In addition, this radionucleotide has a more favorable gamma emission energy for imaging. For example, .sup.111In coupled to monoclonal antibodies with 1-(P-isothiocyanatobenzyl)-DPTA has shown little uptake in non-tumors tissues, particularly the liver, and therefore enhances specificity of tumor localization.

Examples of suitable non-radioactive isotopic labels include .sup.157Gd, .sup.55Mn, .sup.162Dy, 52.sup.Tr, and .sup.56Fe.

Examples of suitable fluorescent labels include an .sup.152Eu label, a fluorescein label, an isothiocyanate label, a rhodamine label, a phycoerythrin label, a phycocyanin label, an allophycocyanin label, an o-phthaldehyde label, and a fluorescamine label.

Examples of suitable toxin labels include, Pseudomonas toxin, diphtheria toxin, ricin, and cholera toxin.

Examples of chemiluminescent labels include a luminal label, an isoluminal label, an aromatic acridinium ester label, an imidazole label, an acridinium salt label, an oxalate ester label, a luciferin label, a luciferase label, and an aequorin label.

Examples of nuclear magnetic resonance contrasting agents include heavy metal nuclei such as Gd, Mn, and iron. Deuterium may also be used. Other contrasting agents also exist for EPR, PET or other imaging mechanisms, which are known to persons of skill in the art.

Typical techniques for binding the above-described labels to polypeptides are provided by Kennedy et al. (1976) Clin. Chim. Acta 70:1-31, and Schurs et al. (1977) Clin. Chim. Acta 81:1-40. Coupling techniques include the glutaraldehyde method, the periodate method, the dimaleimide method, the m-maleimidobenzyl-N-hydroxy-succinimide ester method, all of which methods are incorporated by reference herein.

The polypeptides and antibodies of the present invention, including fragments thereof, may be used to detect p62 polypeptide, ubiquitin or p62 polypeptide/ubiquitin complex using biochip and biosensor technology. Biochip and biosensors of the present invention may comprise the polypeptides of the present invention to detect antibodies, which specifically recognize p62 polypeptide/ubiquitin complex. Bio chip and biosensors of the present invention may also comprise antibodies which specifically recognize the polypeptides of the present invention to detect p62 polypeptide/ubiquitin complex. In addition, the invention also contemplates an array, such as a microarray or macroarray chip, which comprises p62 nucleic acid, preferably oligonucleotides, and preferably antisense oligonucleotides that may be used to detect or hybridize to wild-type or mutant p62 transcript that may be present in an assayable sample such as a biological sample. Such array structures are well-known.

Inclusion Body Detection Kit

The invention also includes a kit for analyzing samples for the presence of p62 polypeptide/ubiquitin complex in a biological sample. In a general embodiment, the kit comprises ligand which binds specifically to p62 polypeptide, ubiquitin or p62 polypeptide/ubiquitin complex, which may be preferably a purified antibody to p62 polypeptide, or p62 polypeptide/ubiquitin complex, in one or more containers. In a specific embodiment, the kit of the present invention contains a substantially isolated polypeptide comprising an epitope which is specifically immunoreactive with an antibody included in the kit. Preferably, the kit of the present invention further comprises a control antibody which does not react with the polypeptide of interest. The kit further comprises instructions and labels on its use.

In another specific embodiment, the kit of the present invention contains a means for detecting the binding of an antibody to a polypeptide of interest (e.g., the antibody may be conjugated to a detectable substrate such as a fluorescent compound, an enzymatic substrate, a radioactive compound or a luminescent compound, or a second antibody which recognizes the first antibody may be conjugated to a detectable substrate), and instructions and labels on its use. The kit may also contain labeled p62 polypeptide with instructions for its use as an inclusion body detector.

Transgenic Animals and Cell Lines

The present invention provides for various animals that have been produced with germ line foreign DNA, or with altered levels of expression of certain genes. These animals typically have a foreign or mutated gene incorporated into their genome. In one such class of transgenic animal, the so-called homozygous null or "knockout" mutants, expression of an endogenous gene has been suppressed through genetic manipulation.

Also provided in the present invention is a mammalian cell line, which is heterozygous or homozygous for a deficiency in the normal synthesis of p62. In one embodiment of the invention, the cell line does not synthesize detectable levels of p62. In another embodiment, the cell line does not synthesize functional p62. The invention further provides for a cell line that is deficient in the normal synthesis of p62. The cell line may be derived from a pluripotent cell line.

The transgenic animals may be either homozygous or heterozygous for the genetic alteration. The subject animals are useful for testing candidate agents for treatment of individuals diagnosed with aging related disorder, either prophylactically or after disease onset.

Transgenic animals generally harbor at least one copy of a transgene either homologously or nonhomologously integrated into an endogenous chromosomal location so as to encode a foreign or mutant protein. Such transgenic animals are usually produced by introducing the transgene or targeting construct into a fertilized egg, or into an embryonic stem (ES) cell which is then injected into an embryo. Introduction of the transgene into the fertilized egg or ES cell is typically performed by microinjection, retroviral infection, electroporation, lipofection, or biolistics. The fertilized egg or embryo is then transferred to an appropriate pseudopregnant female for the duration of gestation. Knockout mutants may be obtained according to this method where the non-native DNA which is introduced comprises a nucleic acid construct that will be used to suppress expression of a particular gene. Such knockout constructs are typically introduced into ES cells.

Transgenic animals comprise an exogenous nucleic acid sequence present as an extrachromosomal element or stably integrated in all or a portion of its cells, especially in germ cells. Unless otherwise indicated, it will be assumed that a transgenic animal comprises stable changes to the genline sequence. During the initial construction of the animal, "chimeras" or "chimeric animals" are generated, in which only a subset of cells have the altered genome. Chimeras are primarily used for breeding purposes in order to generate the desired transgenic animal. Animals having a heterozygous alteration are generated by breeding of chimeras. Male and female heterozygotes are typically bred to generate homozygous animals.

Chimeric targeted mice may be derived according to Hogan, et al., Manipulating the Mouse Embryo: A Laboratory Manual, Cold Spring Harbor Laboratory (1988) and Teratocarcinomas and Embryonic Stem Cells: A Practical Approach, E. J. Robertson, ed., IRL Press, Washington, D.C., (1987) which are incorporated herein by reference.

Embryonic stem cells may be manipulated according to published procedures (Teratocarcinomas and Embryonic Stem Cells: A Practical Approach, E. J. Robertson, ed., IRL Press, Washington, D.C. (1987); Zjilstra et al., Nature 342:435-438 (1989); and Schwartzberg et al., Science 246:799-803 (1989), each of which is incorporated herein by reference).

According to the practice of the invention, the endogenous p62 alleles of a cell line or nonhuman animal are functionally disrupted so that expression of endogenously encoded p62 gene is suppressed or eliminated.

For making transgenic non-human animals (which include homologously targeted non-human animals), embryonal stem cells (ES cells) are preferred. Murine ES cells, such as AB-1 line grown on mitotically inactive SNL76/7 cell feeder layers (McMahon and Bradley (1990) Cell 62: 1073) essentially as described (Robertson, E. J. (1987) in Teratocarcinomas and Embryonic Stein Cells: A Practical Approach. E. J. Robertson, ed. (Oxford: IRL Press), p. 71-112) may be used for homologous gene targeting. Other suitable ES lines include but are not limited to, the E14 line (Hooper et al. (1987) Nature 326: 292-295), the D3 line (Doetschman et al. (1985) J. Embryol. Exp. Morphi. 87: 27-45), and the CCE line (Robertson et al. (1986) Nature 323: 445-448). The practice of the present invention is specifically exemplified hereinafter using ES cells of mouse strain 129/J (Jackson Laboratories). The success of generating a mouse line from ES cells bearing a specific targeted mutation depends on the pluripotence of the ES cells (i.e., their ability, once injected into a host blastocyst, to participate in embryogenesis and contribute to the germ cells of the resulting animal). The blastocysts containing the injected ES cells are allowed to develop in the uteri of pseudopregnant nonhuman females and are born as chimeric mice. The resultant transgenic mice are chimeric for cells having an inactivated endogenous p62 locus and are backcrossed and screened for the presence of the correctly targeted transgene(s) by PCR or Southern blot analysis on tail biopsy DNA of offspring so as to identify transgenic mice-heterozygous for the inactivated p62. By performing the appropriate crosses, it is possible to produce a transgenic nonhuman animal homozygous for functionally disrupted p62 alleles. Such transgenic animals are substantially incapable of making an endogenous p62 gene product.

The functionally disrupted p62 homozygous null mutant transgenic animals will typically comprise rats or mice, but nonmurine species such as dogs, cattle, sheep, goats, pigs and nonhuman primates, for example, may be utilized.

The p62-/- mammals of the invention may be utilized as a model for studying aging related disorders or diseases. In addition, the p62-/- animals may be used in the screening of potential therapeutic synthetic p62 peptides. Such peptides could be screened for the ability to induce the anti-aging related disorder phenotype upon local administration to the p62-/- mice. The candidate peptide would be administered locally by injection into the p62-/- mice.

The practice of the present invention is exemplified herein using the neo gene as the transgene. It may be appreciated that it is possible to generate nonhuman animals which harbor any desired transgene, provided the transgene may be contained in a construct further including a wild type p62 gene.

Gene Targeting Construct

Gene targeting, which is a method of using homologous recombination to modify a mammalian genome, can be used to introduce genetic changes into cultured cells. By targeting a gene of interest in embryonic stern (ES) cells, these changes can be introduced into the germlines of laboratory animals to study the effects of the modifications on whole organisms, among other uses. The gene targeting procedure is accomplished by introducing into tissue culture cells a DNA targeting construct that has a segment homologous to a target locus and which also comprises an intended sequence modification (e.g., insertion, deletion, point mutation). The treated cells are then screened for accurate targeting to identify and isolate those which have been properly targeted. A common scheme to disrupt gene function by gene targeting in ES cells is to construct a targeting construct which is designed to undergo a homologous recombination with its cliromosomal counterpart in the ES cell genome. The targeting constructs are typically arranged so that they insert an additional sequence, such as a positive selection marker, into coding elements of the target gene, thereby functionally disrupting it. Targeting constructs usually are insertion-type or replacement-type constructs (Hasty et al. (1991) Mol. Cell. Biol. 11: 4509).

The invention encompasses production of stem cells and nonhuman animals that have the endogenous p62 gene inactivated by gene targeting with a homologous recombination targeting construct. The p62 gene sequence may be used as a basis for producing PCR primers that flank a region that will be used as a homology clamp in a targeting construct. The PCR primers are then used to amplify a genomic sequence from a genomic clone library or from a preparation of genomic DNA, preferably from the strain of nonhuman animal that is to be targeted with the targeting construct. The amplified DNA is then used as a homology clamp and/or targeting region. General principles regarding the construction of targeting constructs and selection methods are reviewed in Bradley et al. (1992) Bio/Technology 10: 534, incorporated herein by reference.

The isolation of p62 genomic DNA useful for this purpose is described herein. Appropriate probes may be designed based on known p62 cDNA nucleotide sequences. For example, the complete nucleotide sequence of human p62 cDNA and its deduced amino acid sequence are disclosed in U.S. Pat. Nos. 6,291,645B1 and 5,962,224, which are incorporated by reference herein in their entirety.

Targeting constructs can be transferred into pluripotent stem cells, such as ES cells, wherein the targeting constructs homologously recombine with a portion of the endogenous p62 gene locus and create mutation(s) (i.e., insertions, deletions, rearrangements, sequence replacements, and/or point mutations) which prevent the functional expression of the endogenous p62 gene.

One method is to delete, by targeted homologous recombination, essential structural elements of the endogenous p62 gene. For example, a targeting construct can homologously recombine with an endogenous p62 gene and delete a portion spanning substantially all of one or more exons to create an exon-depleted allele, typically by inserting a replacement region lacking the corresponding exon(s). Transgenic animals homozygous for the exon-depleted allele (e.g., by breeding of heterozygotes to each other) are essentially incapable of expressing a functional endogenous p62 polypeptide. Similarly, homologous gene targeting can be used, if desired, to functionally disrupt the p62 gene by deleting only a portion of an exon.

Targeting constructs can also be used to delete essential regulatory elements of the endogenous p62 gene, such as promoters, enhancers, splice sites, polyadenylation sites, and other regulatory sequences, including cis-acting sequences that may occur upstream or downstream of the p62 structural gene but which participate in endogenous p62 gene expression. Deletion of regulatory elements is typically accomplished by inserting, by homologous double-crossover recombination, a replacement region lacking the corresponding regulatory element(s).

A preferred method is to interrupt essential structural and/or regulatory elements of the endogenous p62 gene by targeted insertion of a polynucleotide sequence, and thereby functionally disrupt the endogenous p62 gene. For example, a targeting construct can homologously recombine with the endogenous p62 gene and insert a nonhomologous sequence, such as a neo expression cassette into a structural element (e.g., an exon) and/or regulatory element (e.g., enhancer, promoter, splice site, polyadenylation site) to yield a targeted p62 allele having an insertional interruption. The inserted sequence can range in size from about 1 nucleotide (e.g., to produce a frameshift in an exon sequence) to several kilobases or more, as limited by efficiency of homologous gene targeting with targeting constructs having a long nonhomologous replacement region.

One preferred target site is as indicated in FIG. 1 (see Original Patent), and confirmed as shown in FIG. 2 (see Original Patent).

Targeting constructs can also be employed to replace a portion of the endogenous p62 gene with an exogenous sequence (i.e., a portion of a targeting transgene); for example, a first exon of a p62 gene may be replaced with a substantially identical portion that contains a nonsense or missense mutation.

A targeting construct may be transferred by electroporation of microinjection into a totipotent ES cell line. The targeting construct homologously recombines with endogenous sequences in or flanking of the p62 gene locus and functionally disrupts at least one allele of the p62 gene. Typically, homologous recombination of the targeting construct with endogenous p62 locus sequences will result in integration of a nonhomologous sequence encoding and expressing a selectable marker, such as neo, usually in the form of a positive selection cassette. ES cells having at least one such p62 null allele are selected for by propagating the cells in a medium that permits the preferential propagation of cells expressing the selectable marker. Selected ES cells are examined by PCR analysis and/or Southern blot analysis to verify the presence of a correctly targeted p62 allele. Breeding of nonhuman animals which are heterozygous for a null allele may be performed to produce nonhuman animals homozygous for said null allele, so-called "knockout" animals (Donehower et al. (1992) Nature 256: 215; Science 256: 1392, incorporated herein by reference). Alternatively, ES cells homozygous for a null allele having an integrated selectable marker can be produced in culture by selection in a medium containing high levels of the selection agent (e.g., G418 or hygromycin). Heterozygosity and/or homozygosity for a correctly targeted null allele can be verified with PCR analysis and/or Southern blot analysis of DNA isolated from an aliquot of a selected ES cell clone and/or from tail biopsies.

Gene targeting techniques which have been described, include but are not limited to: co-electroporation, "hit-and-run", single-crossover integration, and double-crossover recombination (Bradley et al. (1992) Bio/Technology 10: 534). The preparation of the homozygous p62 null mutants can be practiced using essentially any applicable homologous gene targeting strategy known in the art. The configuration of a targeting construct depends upon the specific targeting technique chosen. For example, a targeting construct for single-crossover integration or "hit-and-run" targeting need only have a single homology clamp linked to the targeting region, whereas a double-crossover replacement-type targeting construct requires two homology clamps, one flanking each side of the replacement region.

For example and not by way of limitation, a targeting construct may comprise: (1) a first homology clamp having a sequence substantially identical to a sequence within about 3 kilobases upstream (i.e., in the direction opposite to the translational reading frame of the exons) of an exon of an endogenous p62 gene, (2) a replacement region comprising a positive selection cassette having a pgk promoter driving transcription of a neo gene, (3) a second homology clamp having a sequence substantially identical to a sequence within about 3 kilobases downstream of said exon of said endogenous p62 gene, and (4) a negative selection cassette, comprising a pgk promoter driving transcription of an HSV tk gene. Such a targeting construct is suitable for double-crossover replacement recombination which deletes a portion of the endogenous p62 locus spanning the exon and replaces it with the replacement region having the positive selection cassette. The deleted exon is one which is essential for expression of a functional p62 gene product. Thus, the resultant exon-depleted allele is functionally disrupted and is termed a null allele.

Targeting constructs comprise at least one homology clamp linked in polynucleotide linkage (i.e., by phosphodiester bonds) to a targeting region. A homology clamp has a sequence which substantially corresponds to, or is substantially complementary to, an endogenous p62 gene sequence of a nonhuman host animal, and may comprise sequences flanking the p62 gene.

Although no lower or upper size boundaries for recombinogenic homology clamps for gene targeting have been conclusively determined in the art, the best mode for homology clamps is believed to be in the range between about 50 bp and several tens of kilobases. Consequently, targeting constructs are generally at least about 50 to 100 nucleotides long, preferably at least about 250 to 500 nucleotides long, more preferably at least about 1000 to 2000 nucleotides long, or longer. Construct homology regions (homology clamps) are generally at least about 50 to 100 bases long, preferably at least about 100 to 500 bases long, and more preferably at least about 750 to 2000 bases long. It is believed that homology regions of about 7 to 8 kilobases in length are preferred with one preferred embodiment having a first homology region of about 7 kilobases flanking one side of a replacement region and a second homology region of abut 1 kilobase flanking the other side of said replacement region. The length of homology (i.e., substantial identity) for a homology region may be selected at the discretion of the practitioner on the basis of the sequence composition and complexity of the endogenous p62 gene target sequence(s) and guidance provided in the art. Targeting constructs have at least one homology region having a sequence that substantially corresponds to, or is substantially complementary to, an endogenous p62 gene sequence (e.g., an exon sequence, an enhancer, a promoter, an intronic sequence, or a flanking sequence within about 3-20 kb of the p62 gene). Such a targeting construct homology region serves as a template for homologous pairing and recombination with substantially identical endogenous p62 gene sequence(s). In targeting constructs, such homology regions typically flank the replacement region, which is a region of the targeting construct that is to undergo replacement with the targeted endogenous p62 gene sequence. Thus, a segment of the targeting construct flanked by homology regions can replace a segment of an endogenous p62 gene sequence by double-crossover homologous recombination. Homology regions and targeting regions are linked together in conventional linear polynucleotide linkage (5' to 3' phosphodiester backbone). Targeting constructs are generally double-stranded DNA molecules, most usually linear.

Homology regions are generally used in the same orientation (i.e., the upstream direction is the same for each homology region of a transgene to avoid rearrangements). Double-crossover replacement recombination thus can be used to delete a portion of the endogenous p62 and concomitantly transfer a nonhomologous portion (i.e., a neo gene expression cassette) into the corresponding chromosomal location. Double-crossover recombination can also be used to add a nonhomologous portion into the endogenous p62 gene without deleting endogenous chromosomal portions. However, double-crossover recombination can also be employed simply to delete a portion of an endogenous gene sequence without transferring a nonhomologous portion into the endogenous p62 gene. Upstream and/or downstream from the nonhomologous portion may be a gene which provides for identification of whether a double-crossover homologous recombination has occurred; such a gene is typically the HSV tk gene which may be used for negative selection.

Typically, targeting constructs used for functionally disrupting endogenous p62 gene will comprise at least two homology regions separated by a nonhomologous sequence which contains an expression cassette encoding a selectable marker, such as neo (Smith and Berg (1984) Cold Spring Harbor Symp. Quant. Biol. 49: 171; Sedivy and Sharp (1989) Proc. Natl. Acad. Sci. (U.S.A.) 86: 227; Thomas and Capechi (1987), Cell 51: 503). However, some targeting transgenes may have the homology region(s) flanking only one side of a nonhomologous sequence. Targeting transgenes of the invention may also be of the type referred to in the art as "hit-and-run" or "in-and-out" transgenes (Valancius and Smithies (1991) Mol. Cell. Biol. 11: 1402; Donehower et al. (1992) Nature 356: 215; (1991) J.NIH Res. 3: 59; which are incorporated herein by reference).

The positive selection expression cassette encodes a selectable marker which affords a means for selecting cells which have integrated targeting transgene sequences spanning the positive selection expression cassette. The negative selection expression cassette encodes a selectable marker which affords a means for selecting cells which do not have an integrated copy of the negative selection expression cassette. Thus, by a combination positive-negative selection protocol, it is possible to select cells that have undergone homologous replacement recombination and incorporated the portion of the transgene between the homology regions (i.e., the replacement region) into a chromosomal location by selecting for the presence of the positive marker and for the absence of the negative marker (Valancius and Smithies, supra).

An expression cassette typically comprises a promoter which is operational in the targeted host cell (e.g., ES cell) linked to a structural sequence that encodes a protein or polypeptide that confers a selectable phenotype on the targeted host cell, and a polyadenylation signal. A promoter included in an expression cassette may be constitutive, cell type-specific, stage-specific, and/or modulatable (e., by hormones such as glucocorticoids; MMTV promoter), but is expressed prior to and/or during selection. An expression cassette can optionally include one or more enhancers, typically linked upstream of the promoter and within about 3-10 kilobases. However, when homologous recombination at the targeted endogenous site(s) places the nonhomologous sequence downstream of a functional endogenous promoter, it may be possible for the targeting construct replacement region to comprise only a structural sequence encoding the selectable marker, and rely upon the endogenous promoter to drive transcription (Doetschman et al. (1988) Proc. Natl. Acad. Sci. (U.S.A.) 85: 8583; incorporated herein by reference). Similarly, an endogenous enhancer located near the targeted endogenous site may be relied on to enhance transcription of transgene sequences in enhancerless transgene constructs.

Preferred expression cassettes for inclusion in the targeting constructs encode and express a selectable drug resistance marker and/or a HSV thymidine kinase (tk) enzyme. Suitable drug resistance genes include, for example: gpt (xanthine-guanine phosphoribosyltransferase), which can be selected for with mycophenolic acid; neo (neomycin phosphotransferase), which can be selected for with G418 or hygromycin; and DFHR (dihydrofolate reductase), which can be selected for with methotrexate (Nulligan and Berg (1981) Proc. Natl. Acad. Sci. (U.S.A.) 78: 2072; Southern and Berg (1982) J. Mol. Appl. Genet. 1: 327; which are incorporated herein by reference).

Selection for correctly targeted recombinants will generally employ at least positive selection, wherein a nonhornologous expression cassette encodes and expresses a functional protein (e.g., neo or gpt) that confers a selectable phenotype to targeted cells harboring the endogenously integrated expression cassette, so that, by addition of a selection agent (e.g., G418 or mycophenolic acid) such targeted cells have a growth or survival advantage over cells which do not have an integrated expression cassette.

It is preferable that selection for correctly targeted homologous recombinants also employ negative selection, so that cells bearing only nonhomologous integration of the transgene are selected against. Typically, such negative selection employs an expression cassette encoding the herpes simplex virus thymidine kinase gene (HSV tk) positioned in the transgene so that it should integrate only by nonhomologous recombination. Such positioning generally is accomplished by linking the HSV tk expression cassette (or other negative selection cassette) distal to the recombinogenic homology regions so that double-crossover replacement recombination of the homology regions transfers the positive selection expression cassette to a chromosomal location but does not transfer the HSV tk gene (or other negative selection cassette) to a chromosomal location. A nucleoside analog, ganciclovir, which is preferentially toxic to cells expressing HSV tk, can be used as the negative selection agent, as it selects for cells which do not have an integrated HSV tk expression cassette. FIAU may also be used as a selective agent to select for cells lacking HSV tk.

In order to reduce the background of cells having incorrectly integrated targeting construct sequences, a combination positive-negative selection scheme is typically used (Mansour et al., Nature 336: 348-352 (1988) incorporated herein by reference). Positive-negative selection involves the use of two active selection cassettes: (1) a positive one (e.g., the neo gene), that can be stably expressed following either random integration or homologous targeting, and (2) a negative one (e.g., the HSV tk gene), that can only be stably expressed following random integration, and cannot be expressed after correctly targeted double-crossover homologous recombination. By combining both positive and negative selection steps, host cells having the correctly targeted homologous recombination between the transgene and the endogenous p62 gene can be obtained.

Generally targeting constructs preferably include: (1) a positive selection expression cassette flanked by two homology regions that are substantially identical to host cell endogenous p62 gene sequences, and (2) a distal negative selection expression cassette. However, targeting constructs which include only a positive selection expression cassette can also be used. Typically, a targeting construct will contain a positive selection expression cassette which includes a neo gene linked downstream (i.e., towards the carboxy-terminus of the encoded polypeptide in translational reading frame orientation) of a promoter such as the HSV tk promoter or the pgk promoter. More typically, the targeting transgene will also contain a negative selection expression cassette which includes an HSV tk gene linked downstream of a pgk promoter.

Typically, targeting polynucleotides of the invention have at least one homology region that is at least about 50 nucleotides long, and it is preferable that homology regions are at least about 75 to 100 nucleotides long, and more preferably at least about 200-2000 nucleotides long, although the degree of sequence homology between the homology region and the targeted sequence and the base composition of the targeted sequence will determine the optimal and minimal homology region lengths (e., G--C rich sequences are typically more thermodynamically stable and will generally require shorter homology region length). Therefore, both homology region length and the degree of sequence homology can only be determined with reference to a particular predetermined sequence, but homology regions generally must be at least about 50 nucleotides long and must also substantially correspond or be substantially complementary to a predetermined endogenous target sequence. Preferably, a homology region is at least about 100 nucleotides long and is identical to or complementary to a predetermined target sequence in or flanking the p62 gene. If it is desired that correctly targeted homologous recombinants are generated at high efficiency, it is preferable that at least one homology region is isogeneic (i.e., has exact sequence identity with the crossover target sequence(s) of the endogenous p62 gene), and is more preferred that isogeneic homology regions flank the exogenous targeting construct sequence that is to replace the targeted endogenous p62 sequence.

The p62 sequence may be scanned for possible disruption sites. Plasmids are engineered to contain an appropriately sized construct replacement sequence with a deletion or insertion in the p62 gene and at least one flanking homology region which substantially corresponds or is substantially complementary to an endogenous target DNA sequence. Typically, two flanking homology regions are used, one on each side of the replacement region sequence.

The p62 gene is inactivated by homologous recombination in a pluripotent cell line that is capable of differentiating into germ cell tissue. A DNA construct, as discussed above, that contains an altered copy of a mouse p62 gene is introduced into the nuclei of ES cells. In a portion of the cells, the introduced DNA recombines with the endogenous copy of the mouse p62 gene, replacing it with the altered copy. Cells containing the newly engineered genetic lesion are injected into a host mouse embryo, which is reimplanted into a recipient female. Some of these embryos develop into chimeric mice that possess germ cells derived from the mutant cell line. Therefore, by breeding the chimeric mice it is possible to obtain a new line of mice containing the introduced genetic lesion.

Vectors containing a targeting construct are typically grown in E. coli and then isolated using standard molecular biology methods, or may be synthesized as oligonucleotides. Direct targeted inactivation which does not require prokaryotic or eukaryotic vectors may also be performed. Targeting constructs can be transferred to host cells by any suitable technique, including microinjection, electroporation, lipofection, biolistics, calcium phosphate precipitation, and viral-based vectors, among others. Other methods used to transform mammalian cells include the use of Polybrene, protoplast fusion, and others (e.g., generally, Sambrook et al. Molecular Cloning: A Laboratory Manual, 2d ed., 1989, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., which is incorporated herein by reference).

Screening Methods for p62 Interacting Factors

A number of methods for screening candidate factors that interact with the p62 gene or gene product are well-known in the art, and will allow one of ordinary skill to determine if a compound is useful in the present invention.

The agent can be selected and screened at random, or can be rationally selected or rationally designed using protein modeling techniques.

For random screening, agents such as, but not limited to, peptides, carbohydrates, steroids or vitamin derivatives are selected at random and are assayed, using direct or indirect methods that are routine in the art, for their ability to bind to p62 gene or gene product that is present in mice or cell lines described in the present invention. Alternatively, agents can be assayed for modulation of the aging related disorder.

Routine assays may be used to screen compounds for their effect on p62 gene expression and p62 protein function. For example, a transient expression/gel retardation system may be used to study the effects of agents such as synthetic steroids or natural herbal extracts.

In another screening assay, transgenic animals, e.g., mice, and cell lines, that are altered in their expression of one or more of p62 genes may be made, and may be used to identify factors, which modulate the expression and function of p62 gene product. In such an assay, the agent which is to be tested may be incubated with one or more of the transgenic cell lines or mice or tissues derived therefrom. The level of binding of the agent is then determined, or the effect the agent has on biological effect or gene expression is monitored, by techniques that are routine to those of ordinary skill. As used herein, the term "incubate" is defined as contacting the compound or agent under investigation with the appropriate cell or tissue, or administering the agent or compound to the appropriate animal, e.g., transgenic mouse, via any one of the well-known routes of administration including enteral, intravenous, subcutaneous, and intramuscular.

Other assays may include immunological techniques such as immunofluorescent or immunoelectron microscopy, using antibodies specific for p62 fusion proteins. p62 interacting may also be identified by their abilities to modulate the in vitro aging related phenotypes. The agents screened can be, but are not limited to peptides, carbohydrates, steroids, herbal extracts, and vitamin derivatives.

Molecules which can modulate (induce, enhance, reduce, or abolish) p62-positive inclusions can be screened in the cells or cell lines expressing p62 by treating cells with proteasomal inhibitors or oxidative stress including ROS generators or redox modifiers.

Detection of changes in the p62 locus of human genome including deletion, rearrangement, mutation or single nucleotide polymorphism may be used to predict susceptibility to or diagnose the p62-defect related diseases such as obesity, type-2 diabetes, various cancers and neurodegenerative diseases, and early death of male.

Drug Screening Assays for Treating Aging-Related Disease

The animals of the invention can be used as tester animals for materials of interest, e.g. antioxidants such as Vitamin E, thought to confer protection against the development of aging related disorders. An animal is treated with the material of interest, and a reduced incidence or delayed onset of aging related disease, as compared to untreated animals, is detected as an indication of protection. The indices used preferably are those which can be detected in a live animal. The effectiveness can be confirmed by effects on pathological changes when the animal dies or is sacrificed. The animals further can be used as tester animals for materials of interest thought to improve or cure aging related disorder or disease. For instance, an animal with a disease related to the red-ox state of the body may be treated with the material of interest, and a delayed death, or improvement in body weight, fat deposit or glucose uptake/utilization, as compared to untreated animals with aging related disease, is detected as an indication of amelioration or cure.

Through use of the subject transgenic animals or cells derived therefrom, one can identify ligands or substrates that modulate phenomena associated with aging related disorder. Of particular interest are screening assays for agents that have a low toxicity for human cells.

A wide variety of assays may be used for this purpose, including behavioral studies, determination of the localization of drugs after administration, immunoassays to detect amyloid deposition, and the like. Depending on the particular assay, whole animals may be used, or cells derived therefrom. Cells may be freshly isolated from an animal, or may be immortalized in culture. Cells of particular interest are derived from neural tissue.

Candidate agents encompass numerous chemical classes, though typically they are organic molecules, preferably small organic compounds having a molecular weight of more than 50 and less than about 2,500 daltons. Candidate agents comprise functional groups necessary for structural interaction with proteins, particularly hydrogen bonding, and typically include at least an amine, carbonyl, hydroxyl or carboxyl group, preferably at least two of the functional chemical groups. The candidate agents often comprise cyclical carbon or heterocyclic structures and/or aromatic or polyaromatic structures substituted with one or more of the above functional groups. Candidate agents are also found among biomolecules including, but not limited to: herbal extracts, peptides, saccharides, fatty acids, steroids, purines, pyrimidines, derivatives, structural analogs or combinations thereof.

Candidate agents are 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, etc. to produce structural analogs.

For example, detection may utilize staining of cells or histological sections, performed in accordance with conventional methods. The antibodies of interest are added to the cell sample, and incubated for a period of time sufficient to allow binding to the epitope, usually at least about 10 minutes. The antibody may be labeled with radioisotopes, enzymes, fluorescers, chemiluminescers, or other labels for direct detection. Alternatively, a second stage antibody or reagent is used to amplify the signal. Such reagents are well known in the art. For example, the primary antibody may be conjugated to biotin, with horseradish peroxidase-conjugated avidin added as a second stage reagent. Final detection uses a substrate that undergoes a color change in the presence of the peroxidase. The absence or presence of antibody binding may be determined by various methods, including flow cytometry of dissociated cells, microscopy, radiography, scintillation counting, etc.

A number of assays are known in the art for determining the effect of a drug on animal behavior and other phenomena associated with aging related disorders. Some examples are provided, although it will be understood by one of skill in the art that many other assays may also be used. The subject animals may be used by themselves, or in combination with control animals. The screen using the transgenic animals of the invention can employ any phenomena associated with aging related diseases that can be readily assessed in an animal model. Preferably, the screen will include control values.

Therapeutic Composition

In one embodiment, the present invention relates to treatment for various diseases that are characterized as aging related disease as defined in the present application. In this way, the inventive therapeutic compound may be administered to human patients who are either suffering from disorders, such as obesity, diabetes, cancer, in particular, liver cancer, fatty liver, and Paget Disease of Bone, and early mortality for males harboring p62 homozygous mutation.

The formulation of therapeutic compounds is generally known in the art and reference can conveniently be made to Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing Co., Easton, Pa., USA. For example, from about 0.05 .mu.g to about 20 mg per kilogram of body weight per day may be administered. Dosage regime may be adjusted to provide the optimum therapeutic response. For example, several divided doses may be administered daily or the dose may be proportionally reduced as indicated by the exigencies of the therapeutic situation. The active compound may be administered in a convenient manner such as by the oral, intravenous (where water soluble), intramuscular, subcutaneous, intra nasal, intradermal or suppository routes or implanting (eg using slow release molecules by the intraperitoneal route or by using cells e.g. monocytes or dendrite cells sensitised in vitro and adoptively transferred to the recipient). Depending on the route of administration, the peptide may be required to be coated in a material to protect it from the action of enzymes, acids and other natural conditions which may inactivate said ingredients.

For example, the low lipophilicity of the peptides will allow them to be destroyed in the gastrointestinal tract by enzymes capable of cleaving peptide bonds and in the stomach by acid hydrolysis. In order to administer peptides by other than parenteral administration, they will be coated by, or administered with, a material to prevent its inactivation. For example, peptides may be administered in an adjuvant, co-administered with enzyme inhibitors or in liposomes. Adjuvants contemplated herein include resorcinols, non-ionic surfactants such as polyoxyethylene oleyl ether and n-hexadecyl polyethylene ether. Enzyme inhibitors include pancreatic trypsin inhibitor, diisopropylfluorophosphate (DEP) and trasylol. Liposomes include water-in-oil-in-water CGF emulsions as well as conventional liposomes.

The active compounds may also be administered parenterally or intraperitoneally. Dispersions can also be prepared in glycerol liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.

The pharmaceutical forms suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. In all cases the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of superfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, chlorobutanol, phenol, sorbic acid, theomersal and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the composition of agents delaying absorption, for example, aluminium monostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with various other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterile active ingredient into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and the freeze-drying technique which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

When the peptides are suitably protected as described above, the active compound may be orally administered, for example, with an inert diluent or with an assimilable edible carrier, or it may be enclosed in hard or soft shell gelatin capsule, or it may be compressed into tablets, or it may be incorporated directly with the food of the diet. For oral therapeutic administration, the active compound may be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. Such compositions and preparations should contain at least 1% by weight of active compound. The percentage of the compositions and preparations may, of course, be varied and may conveniently be between about 5 to about 80% of the weight of the unit. The amount of active compound in such therapeutically useful compositions is such that a suitable dosage will be obtained. Preferred compositions or preparations according to the present invention are prepared so that an oral dosage unit form contains between about 0.1 .mu.g and 2000 mg of active compound.

The tablets, pills, capsules and the like may also contain the following: A binder such as gum tragacanth, acacia, corn starch or gelatin; excipients such as dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid and the like; a lubricant such as magnesium stearate; and a sweetening agent such as sucrose, lactose or saccharin may be added or a flavoring agent such as peppermint, oil of wintergreen, or cherry flavoring. When the dosage unit form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier. Various other materials may be present as coatings or to otherwise modify the physical form of the dosage unit. For instance, tablets, pills, or capsules may be coated with shellac, sugar or both. A syrup or elixir may contain the active compound, sucrose as a sweetening agent, methyl and propylparabens as preservatives, a dye and flavoring such as cherry or orange flavor. Of course, any material used in preparing any dosage unit form should be pharmaceutically pure and substantially non-toxic in the amounts employed. In addition, the active compound may be incorporated into sustained-release preparations and formulations.

It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the mammalian subjects to be treated; each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the invention are dictated by and directly dependent on (a) the unique characteristics of the active material and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding such an active material for the treatment of disease in living subjects having a diseased condition in which bodily health is impaired.

The principal active ingredient is compounded for convenient and effective administration in effective amounts with a suitable pharmaceutically acceptable carrier in dosage unit form. A unit dosage form can, for example, contain the principal active compound in amounts ranging from 0.5 .mu.g to about 2000 mg. Expressed in proportions, the active compound is generally present in from about 0.5 .mu.g/ml of carrier. In the case of compositions containing supplementary active ingredients, the dosages are determined by reference to the usual dose and marmer of administration of the said ingredients.

Delivery Systems

Various delivery systems arc known and can be used to administer a compound of the invention, e.g., encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing the compound, receptor-mediated endocytosis, construction of a nucleic acid as part of a retroviral or other vector, etc. Methods of introduction include but are not limited to intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The compounds or compositions may be administered by any convenient route, for example by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.) and may be administered together with other biologically active agents. Administration can be systemic or local. In addition, it may be desirable to introduce the pharmaceutical compounds or compositions of the invention into the central nervous system by any suitable route, including intraventricular and intrathecal injection; 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 use of an inhaler or nebulizer, and formulation with an aerosolizing agent.

In a specific embodiment, it may be desirable to administer the pharmaceutical compounds or 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., in conjunction with a wound dressing after surgery, by injection, by means of a catheter, by means of a suppository, or by means of an implant, said implant being of a porous, non-porous, or gelatinous material, including membranes, such as sialastic membranes, or fibers. Preferably, when administering a protein, including an antibody or a peptide of the invention, care must be taken to use materials to which the protein does not absorb. In another embodiment, the compound or composition can be delivered in a vesicle, in particular a liposome. In yet another embodiment, the compound or composition can be delivered in a controlled release system. In one embodiment, a pump may be used. In another embodiment, polymeric materials can be used. In yet another embodiment, a controlled release system can be placed in proximity of the therapeutic target, i.e., the brain, thus requiring only a fraction of the systemic dose.

Diagnosis of Aging Related Disease

The invention is also directed to a method to assist in the diagnosis of subsceptibility to aging-related disease such as obesity, Type II diabetes, hepatocarcinoma, early male mortality, in a mammalian subject, comprising comparing the wild-type gene encoding p62 with the subject's p62 gene. The detection of a point mutation, single nucleotide polymorphism, deletion, frameshift mutation, rearrangement, truncation or disruption of the gene such that its function is lost or decreased, indicates that the subject mammal is at risk for the aging related disorder or disease. Molecular biological methods for identifying and comparing sequences are well-known in the alt.

The invention is also directed to a method for identifying aging-related disease or disorder caused by or associated with a somatic mutation in a gene encoding p62. In one non-limiting example, the method may comprise:

i. providing the sequence of a p62 gene product;

ii. identifying the sequence of a mutant p62 protein;

iii. preparing a probe to the gene encoding the mutant protein or a fragment thereof; and

iv. probing a biological sample from a subject having the above-cited disorder or disease and a biological sample from a patient not having the disease, wherein the presence of the mutant protein in a biological sample from a patient having the disease and the absence of the mutant protein in a biological sample from a mammalian subject not having the disease indicates that the disease or susceptibility to the disease is caused by or associated with the somatic mutation in the gene.
 

Claim 1 of 2 Claims

1. A homozygous p62 mutant transgenic mouse, which does not produce p62 protein and exhibits a phenotype which is selected from the group consisting of obesity, diabetes, fatty liver, and early mortality for male, whose somatic and germ cells comprise a functionally disrupted endogenous p62 gene, wherein said disrupted gene is generated in the mouse or an ancestor of the mouse at an embryonic stage by introduction of a targeting vector.

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