KR20080032199A - A transgenic mouse for diagnosis or treatment of age-related disorder or disease comprising a knock-out p62 gene and a method of screening for a compound that counters age-related disorder or disease using the same - Google Patents

Abstract

A transgenic mouse for diagnosis or treatment of age-related disorder or disease comprising a knock-out p62 gene and a method of screening for a compound that counters age-related disorder or disease using the same are provided to be useful for the screening in a research for age-related disorder. A method of screening for a compound that counters age-related disorder or disease using the same includes the steps of: treating a test compound to be screened in a mouse somatic cell; measuring the degree of disorder after processing the compound; and comparing the measured degree of disorder as a control group with the degree of disorder in the somatic cell that is not treated with a test compound. The reduction in the degree of the disorder that is related to aging that is compared with the control group in the second step indicates that the compound counters the disorder relating to aging.

Description

Transformed mouse comprising a knocked out # 62 gene for the diagnosis or treatment of an aging-related disorder or disease and a method for screening a compound corresponding to the aging-related disorder or disease using the same or disease comprising a knock-out p62 gene and a method of screening for a compound that counters age-related disorder or disease using the same}

Reference to related application

The present invention claims priority to U.S. Provisional Patent Application No. 60 / 378,159, filed May 14, 2002, which is hereby incorporated by reference in its entirety.

The present invention relates to a method for detecting the formation of inclusion bodies such as sequestosomes. The present invention also relates to a method for detecting inclusion bodies in neurodegenerative diseases. The present invention further relates to a screening method for a therapeutic agent which disperses the inclusion body.

The present invention relates to transgenic non-human and transgenic non-human animal cells having a transformed gene with a mutation in the p62 gene and having a functionally disrupted p62 locus. The invention further relates to transformed genes and targeting constructs used to produce such transgenic animals and cells; Using the animal to model aging-related disorders; And a method of using said animal to produce a transgenic non-human animal and cell comprising one or more additional transformed genes. The invention further develops obesity, type 2 diabetes mellitus, non-alcoholic fatty liver and various tumors, such as increased male (male) mortality, intracellular inclusions called sequetosomes, and redox regulation, such as It relates to the function of p62 first revealed by phenotypes generated in animals and cells.

The invention further relates to a method for diagnosing the early stages of susceptibility or constitution of a mammalian subject to an age-related disorder or disease.

Methods for cloning and sequencing human p62 genes are described in US Pat. Nos. 6,291,645B1 and 5,962,224, the entire contents of which are incorporated herein by reference. The gene product in humans, the protein is p62 and the gene name is SQSTM localized on human chromosome 5q35. The same gene products in mice and rats are named differently as A170 and ZIP, respectively, and the gene encoding A170 is localized on mouse chromosome 11, sqstm1. For convenience, these human and mouse genes and proteins will be named p62 gene and p62, respectively, in this context.

Aging is associated with most physiological as well as degeneration of cells, tissues and organs, leading to diseases such as cancer, cardiovascular insufficiency, obesity, type 2 diabetes, non-alcoholic fatty liver, and many neurodegenerative diseases. This is related to the declining performance limit. It has been clarified that the redox status of cells has a significant impact on several basic cellular processes, such as signal transduction, gene expression and thus cell proliferation and apoptotic cell death. Accumulation of somatic cell damage is considered a major cause of the aging process. Among many causes of somatic cell damage, reactive oxygen species (ROS), a natural byproduct of oxidative energy metabolism, are often considered to be the decisive factor in aging.

Obesity causes many health problems. Obesity is the presence of excessive amounts of fatty tissue, which has become a serious health problem for man in modern society. Excessive adipose tissue, independently and in association with the development of type 2 diabetes, leads to coronary heart disease (CHD), increased incidence of certain forms of cancer, respiratory complications and osteoarthritis. Obesity can be caused by confusion in one or more of the three components of energy balance: energy absorption, energy consumption and energy distribution. Regulatory cooperation between these three components is achieved through neural networks and neuroendocrine systems, and any defects in these components are sufficient to cause obesity.

Diabetes is defined as a condition in which carbohydrate and lipid metabolism are improperly regulated by insulin. This leads to an increase in fasting and postprandial serum glucose levels, which can lead to complications if left untreated. There are two main categories of disease, namely types 1 and 2. Type 2 diabetes is much more common, which is the result of a combination of insulin secretion and functional deficits. Type 2 diabetes is characterized by a gradual decrease in insulin action, after which β cells cannot compensate for insulin resistance. Because of metabolic changes caused by interactions between genes, aging, and obesity, insulin resistance is the first dysfunction. Insulin resistance in visceral fat induces increased fatty acid production, worsening insulin resistance in liver and muscle. β cells compensate for insulin resistance by secreting higher amounts of insulin. Ultimately, impaired glucose tolerance and diabetes are caused because β cells can no longer compensate.

The majority of individuals suffering from type 2 diabetes are obese with an imbalance between energy absorption and consumption that causes numerous metabolic abnormalities, and central visceral hyperlipidemia. Insulin resistance may be the result of obesity, but it may also be the cause of obesity. Recent consideration of adipocyte biology as an endocrine organ supported this latter theory. This cell type is more than a storage site for lipids; It also secretes a number of important circulating factors, including leptin, TNFα, angiotensin and PAI-1, and is currently known to be a major source for endogenous production of non-esterified fatty acids (NEFAs) through lipolysis. Indeed, the dynamic interactions between adipocytes, specific nuclei of the hypothalamus during abdominal measurements, and insulin synthesis and action organs ensure regulatory coordination on insulin sensitivity as well as energy absorption and consumption.

Non-alcoholic fatty liver disease (NAFLD) encompasses two histological lesions, fatty liver and hepatitis. Insulin resistance is suggested to be a major pathophysiological abnormality in NAFLD patients. Insulin resistance results from the complex interactions between the main targets of insulin, namely muscle, adipose tissue and liver, for the ability of pancreatic islet beta cells. As such, the metabolic and clinical profiles associated with insulin resistance are defined as factors that produce and maintain insulin resistance, and insulin sensitivity lowering effects on various insulin-dependent pathways. The major metabolic defects associated with insulin resistance are increased peripheral lipolysis, increased hepatic glucose output due to increased uveogenesis, and increased lipid oxidation. This is associated with oxidative stress in the liver that can be multiplied by additional pathophysiological abnormalities. Increased fatty acid beta oxidation as well as increased peroxysomal fatty acid oxidation can all increase reactive oxygen species generation and increase subsequent lipid peroxidation reactions. Oxidative stress caused by increased fatty acid oxidation may have additional detrimental effects on amplifying disease processes in the liver.

Increased oxidative stress and the resulting change in cellular redox status are also important factors in the development of numerous neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, and Amyotrophic lateral sclerosis. Known. Intracellular inclusion bodies (ubiquitin positive and / or negative) are ultrastructural features of this neurodegenerative disorder. The fact that proteasome immunoreactivity is enhanced in most ditrophic neuritis in various diseases suggests that the ubiquitin-proteasome pathway is critically involved in the formation of this pathologically insoluble substance. In general, ubiquitated or oxidized proteins do not accumulate in normal cells, but are rapidly degraded in proteasomes. Ubiquitinylated or oxidized protein inclusions in cells must result from a malfunction or overload of the proteasome pathway or from structural changes on the protein substrate that stop their degradation. However, the question of what determines the fate of a protein in relation to the formation of such abnormal inclusion bodies and what the inclusion bodies have had a pathologically close effect in the course of neurodegeneration is still unclear. Thus, clues to these questions will provide a better understanding of the neurodegeneration process, thereby providing a diagnostic and therapeutic tool.

Aging-related disorders such as the formation of intracellular inclusions, obesity, diabetes, cancer, particularly liver cancer, fatty liver, bone diseases of the bone, and premature death of males continue to have significant health problems. There is a need for living animal models that can be used in these diseases and other aging-related disorders studies to screen and evaluate potent therapeutic agents as well as potent diagnostic / prognostic probes useful for treating these disorders. The present invention addresses these and other needs in the art.

Summary of the Invention

Accordingly, it is an object of the present invention to provide a non-human model that inhibits the expression of the p62 gene.

It is an object of the present invention to provide non-human cells and non-human animals containing heterozygous or homozygous null mutations of the p62 locus.

It is an object of the present invention to provide constructs and vectors for producing such cells and animals containing p62 heterozygous or homozygous nonfunctional mutations.

In accordance with the aforementioned objectives, the invention in one aspect is a targeting construct for functional disruption in the p62 gene. Such targeting constructs are substantially homologous to sequences present at or flanking the p62 locus and have sequences that functionally disrupt the expression of p62 protein from this locus when integrated at the corresponding p62 locus. Polynucleotides containing at least one moiety. Such targeting constructs or portions thereof are integrated at the p62 locus by homologous recombination between the endogenous locus and the targeting construct.

In one embodiment, the p62 gene is functionally disrupted by a targeting construct, typically inserting a particular sequence into the coding sequence (ie exon), such that the resulting disrupted p62 gene is capable of substantially expressing the functional p62 protein. none. In one such embodiment, the targeting construct has a top homology region, a non-homologous replacement portion, and a second endogenous p62 gene located below from the first endogenous p62 sequence having a sequence substantially the same as the first endogenous p62 gene sequence. A bottom homology region having a sequence substantially the same as the sequence, wherein the top homology region and the bottom homology region flank the non-homologous replacement portion.

Non-homologous replacement portions of the targeting constructs advantageously include a positive selective expression cassette, such as neo. The targeting construct further advantageously comprises a negative selection cassette distal to the top homology region or the bottom homology region. Such a negative selection cassette can include, for example, the tk gene.

According to another aspect, the invention comprises transferring the aforementioned targeting construct to pluripotent stem cells, and selecting stem cells with precisely targeted homologous recombination between said targeting construct and the endogenous p62 gene sequence. Thus, there is provided a method for generating stem cells having a functionally disrupted endogenous p62 gene.

According to another aspect, the present invention provides a method of metastatic transfer of stem cells into non-human blastocysts (vesicles) with precisely targeted homologous recombination between the aforementioned targeting construct and the endogenous p62 gene sequence; Transplanting the resulting vesicles into a fertility female; And collecting progeny in which the endogenous p62 allele with precisely targeted homologous recombination has been settled, thereby producing a non-human animal having a functionally disrupted endogenous p62 gene.

According to another aspect, the invention provides transgenic non-human animals and stem cells having a genome comprising one or more functionally disrupted p62 genes. Such animal or stem cells are preferably homozygous for the functionally disrupted p62 gene. Such homozygous transgenic animals or stem cells are substantially incapable of directing efficient expression of endogenous p62. For example, in a preferred embodiment, the transgenic mouse is homozygous for the inactivated endogenous (ie native) p62 gene.

Transgenic non-human animals or stem cells homozygous for a functionally disrupted p62 gene include the p62 gene disrupted by an integrated targeting construct, eg, an integrated targeting construct comprising a neo gene. .

According to a preferred embodiment of the invention, the transgenic animal is a mouse comprising a genome having a functionally disrupted murine p62 allele. Preferably such mice are homozygous for the functionally disrupted p62 allele. The mice do not produce functional p62 protein.

According to another aspect of the invention, mammalian cells and cell lines obtained and derived from said transgenic animals can be used to screen for agents that modulate aging-related disorders.

The invention also provides for the treatment of aging-related diseases or disorders in the development of diseases resulting from a defect in the p62 locus. Such treatment includes delivering a therapeutically effective DNA encoding a functional p62 polypeptide to a therapeutically effective site or administering a functional p62 polypeptide directly to a therapeutically effective site. The invention also provides for the treatment and detection of aging related disorders.

Increasing cellular p62 levels in animals or cell lines for the purpose of generating diagnostic tools or therapeutic molecules, or utilizing p62-induced inclusion body formation by oxidative stress or by proteasome inhibition, Another aspect.

Intracellular ubiquitin-positive inclusion bodies can be detected using polypeptides containing the ubiquitin chain binding region of p62 with certain hydrophobic residue (s). When these hydrophobic residues are mutated to small amino acids, these mutants can be used as negative controls in p62 binding with ubiquitin, which is expected to contact samples labeled with inclusions with the labeled p62 and the presence of a label. May be useful for inclusion detection assays that indicate the presence of inclusion bodies. Mutants can serve as negative controls. In this assay, other unlabeled p62 polypeptides can also be used as competitive inhibitors (see Table 1).

The present invention

(a) contacting a particular compound with a sample containing p62 and ubiquitin;

(b) determining the binding level of p62 and ubiquitin under conditions in which p62 and ubiquitin usually bind specifically to each other; And

(c) determining the binding level of p62 and ubiquitin in the presence of the compound and comparing it with the binding level of p62 and ubiquitin mentioned in steps (a) and (b), wherein the level is greater than in (b) ( lower in c), the compound is an inhibitor of p62-ubiquitin binding]

It relates to a method for screening a compound that inhibits p62 / ubiquitin bonds, including.

The present invention also provides

(a) generating a recombinant viral or plasmid vector comprising a DNA sequence encoding a ubiquitin binding fragment of a p62 polypeptide operably linked to a promoter; And

(b) administering such viral or plasmid vector to a patient in need thereof such that the DNA sequence is expressed in the brain such that binding occurs between the ubiquitin binding fragment of p62 and ubiquitin.

It relates to a method for preventing the binding between ubiquitin and p62, preferably containing no toxic side effects.

In addition, the present invention

(a) contacting a sample suspected of containing an inclusion body with a labeled p62 polypeptide; And

(b) assaying for the presence of a label indicating the presence of such inclusions

It relates to a detection method of the inclusion body, including.

The present invention encompasses p62 genes whose functional cells and germ cells have been functionally disrupted, which are introduced into embryonic mouse or mouse ancestors and are aged if homozygous for the disrupted genes. It relates to a transgenic mouse exhibiting a related disorder. In such transgenic mice, the aging-related disorder may be obesity, diabetes, liver cancer, fatty liver, Paget's disease of bone, or premature death of males. In transgenic mice, the collapsed p62 gene is disrupted by the integrated targeting construct. In one aspect, transgenic mice do not produce p62 protein.

The invention also relates to mouse embryonic stem cells having a genome comprising a functionally disrupted p62 gene of heterozygous or homozygous, which embryonic stem cells can be transgenic mice as mentioned above. The mouse embryonic stem cell may have a p62 gene disrupted by an integrated targeting construct.

The invention further relates to a mouse somatic cell having a genome comprising a functionally disrupted p62 gene of heterozygous or homozygous, which is isolated from the transgenic mice mentioned above, wherein the p62 gene is an integrated targeting construct. Can be collapsed by The invention also relates to a mouse somatic cell line comprising the cells mentioned above.

The present invention also relates to the use of the transgenic mouse as a tester animal,

(a) administering a test compound to a transgenic mouse according to claim 4;

(b) measuring the level of disorder after administering such a compound; And

(c) comparing the disorder level measured in (b) with the disorder level in p62 deficient mice, wherein if the aging-related disorder level in (b) is reduced compared to p62 deficient mice, this compound is To fight this age-related disorder]

It includes a method for screening a compound against the age-related disorders or diseases of the mammal, including.

The invention also relates to aging in mammalian subjects, comprising assaying for mutations in the genes of a mammalian subject encoding p62, where detection of such mutations indicates that the subject is at risk for aging-related diseases. And methods for facilitating the diagnosis of the disease. Aging-related diseases can be obesity, diabetes, liver cancer, fatty liver, Paget's disease of bone or premature death of males. The mutation may be, but is not limited to, a functional disruption, or a single nucleotide polymorphic mutation, that results in the expression of functional p62. As long as p62 is mutated and lacks its expression, other types of mutations are possible. In one aspect of the invention, the mammalian subject may be an infant human, and such subjects are expected to be prone to age-related diseases in the future.

These and other objects of the present invention will be better understood from the following detailed description, the drawings appended hereto, and the claims.

As used herein, the term “a.or an” is used to refer to a single, plural and both of an individual.

As used herein, "about" or "substantially" generally provides room (tolerance) from being limited to the exact number. For example, "about" or "substantially" as used in the context of the length of a polypeptide sequence indicates that the polypeptide should not be limited to the number of amino acids mentioned. As long as functional activity such as binding activity is present, several amino acids added to or subtracted from the N-terminus or C-terminus may also be included.

The term "formulation" as used herein refers to any molecule, such as a protein, nucleic acid or pharmaceutical compound, that can affect the molecular and clinical phenomena associated with aging-related disorders. Generally, multiple assay mixtures are run in parallel with different formulation concentrations to obtain differential reactions for various concentrations. Typically, one of these concentrations serves as a negative control, ie below zero concentration or detection level.

As used herein, "aging related" disorders or diseases are cells, tissues, and organs that lead to diseases such as cancer, cardiovascular insufficiency, obesity, type 2 diabetes, non-alcoholic fatty liver, and many neurodegenerative diseases. As well as the degradation of most physiological performance.

As used herein, “amino acid” and “amino acids” refer to all natural L-α-amino acids. This definition also includes norleucine, ornithine and homocysteine.

As used herein, the term “amino acid sequence variant” refers to a molecule with some differences in amino acid sequence compared to a reference (eg original sequence) polypeptide. Such amino acid modifications can be substitutions, insertions, deletions or any combination of such changes on the original amino acid sequence.

Substitution variants are those in which one or more amino acid residues in the original sequence are removed, and instead different amino acids are inserted at the same position. Such substitution may be a single substitution in which only one amino acid in the molecule is substituted or multiple substitutions in which two or more amino acids are substituted in the same molecule.

Substituents for amino acids in the sequence may be selected from other members of the class to which they belong. For example, nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, phenylalanine, tryptophan and methionine. Polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine and glutamine. Positively charged (basic) amino acids include arginine, lysine and histidine. Negatively charged (acidic) amino acids include aspartic acid and glutamic acid. Also within the scope of the present invention are proteins or fragments or derivatives thereof exhibiting the same or similar biological activity, and during translation by, for example, glycosylation, proteolytic cleavage, linkage to antibody molecules or other cellular ligands, or the like. Derivatives that are later modified in time are included.

Insertional variants are those in which one or more amino acids are inserted immediately adjacent to an amino acid at a specific position within the original amino acid sequence. Immediately adjacent to an amino acid means linked to the α-carboxy or α-amino functional group of such amino acid.

A deletion variant is one or more amino acids in the original amino acid sequence removed. Typically, a deletion variant is one or two amino acids deleted in a specific region of the molecule.

As used herein, the term "capable of hybridization under highly stringent conditions" refers to the annealing of strands of DNA complementary to the DNA of interest under highly stringent conditions. Likewise, "capable of hybridization under low stringent conditions" refers to annealing the strands of DNA complementary to the DNA of interest under low stringent conditions. “Highly stringent conditions” for the annealing process may include, for example, high temperature and / or low salt concentrations that dislike hydrogen-bonded contact between mismatched base pairs. "Low stringent conditions" will include lower temperatures and / or higher salt concentrations than highly stringent conditions. These conditions can anneal two DNA chains when they exist, but there is almost no complete complementarity between these two chains, which encodes the same protein but differs in the sequence of the DNA strands that differ in sequence due to genetic code degeneracy. Same as the case. Moderately stringent conditions that enhance DNA hybridization, such as 6x SSC washing at about 45 ° C., followed by 2x SSC washing at 50 ° C., are known to those skilled in the art or can be found in Current Protocols in Molecular Biology, John Wiley & Sons, NY (1989), 6.31-6.3.6. For example, the salt concentration in the washing step can be selected from a low stringency of about 2 × SSC at 50 ° C. to a high stringency of about 0.2 × SSC at 50 ° C. In addition, the temperature in the washing step can be raised from room temperature, low stringency under about 22 ° C. to highly stringent conditions of about 75 ° C. Other stringency parameters are described in Maniatis, T., et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring N.Y., (1982), at pp. 387-389; Sambrook J. et al., Molecular Cloning: A Laboratory Manual, Second Edition, Volume 2, Cold Spring Harbor Laboratory Press, Cold Spring, N.Y. at pp. 8.46-8.47 (1989)].

As used herein, “carrier” includes pharmaceutically acceptable carriers, excipients or stabilizers which are non-toxic to the cells or mammals exposed to it at the dosages and concentrations employed. Often, the pharmaceutically acceptable carrier is an aqueous pH buffer solution. Examples of pharmaceutically acceptable salts include buffers such as phosphate, citrate and other organic acids; Antioxidants including ascorbic acid; Low molecular weight (less than about 10 residues) polypeptides; Proteins such as serum albumin, gelatin or immunoglobulins; Hydrophilic polymers such as polyvinylpyrrolidone; Amino acids such as glycine, glutamine, asparagine, arginine or lysine; Monosaccharides, disaccharides and other carbohydrates such as glucose, mannose or dextrins; Chelating agents such as EDTA; Sugar alcohols such as mannitol or sorbitol; Salt-forming counterions such as sodium; And / or nonionic surfactants such as, but not limited to, TWEEN ^® , polyethylene glycol (PEG) and PLURONICS ^® .

As used herein, “covalent derivatives” include modifications of the original polypeptide or fragment thereof using an organic proteinaceous or non-proteinaceous derivatizing agent, and post-translational modifications. Covalent modifications are traditionally introduced by reacting an organic derivatizing agent that can react with a selected side chain or terminal residue with a targeting amino acid residue, or by solidifying the post-translational modification mechanism that acts on selected recombinant host cells. Certain post-translational modifications are the result of the action of recombinant host cells on the expressed polypeptide. Glutaminyl and asparaginyl residues are often deamidated after detoxification to the corresponding glutamyl and aspartyl residues. On the other hand, these residues are deamidated under mildly acidic conditions. Any form of these residues may be present in the β-amyloid or VEGF-binding polypeptide of the present invention. Other post-translational modifications include the hydroxylation of proline and lysine; Phosphorylation of hydroxyl groups of seryl, tyrosine or threonyl residues; Methylation of the α-amino groups of lysine, arginine and histidine side chains is included. T. E. Creighton, Proteins: Structure and Molecular Properties, W. H. Freeman & Co., San Francisco, pp. 79-86 (1983).

As used herein, “corresponding to” means that a particular polynucleotide sequence is homologous (ie, identically not strictly evolutionarily related) to all or a portion of a reference polynucleotide sequence, or a particular polynucleotide It means that the sequence is identical to the reference polynucleotide sequence.

The term "cross target sequence" or "endogenous target sequence" as used herein refers to a p62 gene sequence substantially corresponding to or substantially complementary to a transformed gene homology region.

An “effective amount” as used herein is an amount sufficient to produce a beneficial or desired clinical or biochemical result. An effective amount may be administered one or more times. For the purposes of the present invention, an effective amount of an inhibitor compound is an amount sufficient to alleviate, ameliorate, stabilize, reverse or delay or slow the progression of the disease state.

As used herein, “fragment” refers to a portion of a polypeptide that has usable and functional features. For example, in one aspect, polypeptide fragments as used in the context of the present invention may have a binding function to p62 or ubiquitin.

As used herein, the term "functional disruption" or "functionally disrupted" includes one or more mutations or structural changes in a particular locus, such that a functionally disrupted gene is capable of substantially efficient expression of the functional gene product. It means not to be able to dictate. Non-limiting example, the endogenous p62 gene with a neo gene cassette integrated into the exon of the p62 gene is unable to encode a functional p62 protein, so it is a functionally disrupted p62 locus. Deletion or blocking of essential transcriptional regulatory elements, polyadenylation signal (s), splicing site sequences, will also yield functionally-constructed genes. Functional disruption of the endogenous p62 gene may also be caused by other methods, such as antisense polynucleotide gene inhibition. The term “structurally disrupted” refers to a targeting gene in which one or more structural (ie exon) sequences have been modified by homologous gene targeting (eg, insertions, deletions, point mutation (s) and / or translocations). . Typically, structurally disrupted alleles result in functional disruptions. However, the p62 allele can also be functionally disrupted without simultaneously structurally disrupting, ie functionally disrupted by targeted modification of non-exon sequences such as excision of the promoter. Alleles that include targeting modifications that interfere with efficient expression of functional gene products from alleles are referred to as "null alleles."

As used herein, the expression “functional p62 polypeptide” refers to a polypeptide that, when expressed in or administered to an individual p62 − / −, is sufficient to substantially repair or counteract the effect of a p62 nonfunctional mutation in that individual. . In addition, the category of “functional p62” polypeptides includes fusion products comprising native p62 polypeptides or their analogs and one or more attached amino acid sequences that enhance cellular uptake or penetration of p62 polypeptides into various cells. Such fusion products can be prepared by relying on commercial expression vectors that provide for incorporation of the DNA sequence of interest at the bottom from the DNA segment encoding the amino acid sequence with the desired transport properties. The resulting p62 fusion protein can be used as an externally supplied functional p62 protein for treating a variety of individuals.

As used herein, the terms “homologous region” and “homologous clamp” refer to sequences substantially corresponding to or substantially complementary to a predetermined p62 gene sequence, which may include sequences flanking p62. Refers to a segment (ie, a portion) of a targeting construct with Homologous regions are generally at least about 100 nucleotides in length, preferably at least about 250 to 500 nucleotides, typically at least about 1000 nucleotides.

As used herein, "mammal" for therapeutic purposes is any animal classified as a mammal, eg, humans, livestock and farm animals, zoos, sporting or pet animals, such as dogs, cats, cattle, horses. , Sheep, pig, and the like. Preferably, the mammal is a human.

By "knock-out" of a particular gene is meant a modification on the gene sequence that results in a decrease in the function of the target gene, whereby the target gene expression is not detectable or meaningless. Knockout of the endogenous p62 gene means that the function of the p62 gene is substantially degraded so that expression cannot be detected or is present only at insignificant levels. A "knockout" transformant may be a transgenic animal having a heterozygous knockout of the p62 gene or a homozygous knockout of the p62 gene. "Knockout" includes, for example, exposing the animal to a substance that enhances the target genetic modification, introducing an enzyme that enhances recombination at the target gene site, or other method of directing the target genetic modification after birth. Also included are conditional knockouts in which modification of the gene can occur.

As used herein, “natural” as applied to a particular subject refers to the fact that the specific subject can be found in nature. For example, polypeptides or polynucleotide sequences present in organisms (including viruses) that can be isolated from natural sources and have not been intentionally modified by humans in the laboratory are natural. As used herein, laboratory rodent species, which may be selectively bred according to traditional genetics, are considered natural animals.

As used herein, the term “non-homologous sequence” has both a generic and specific meaning. It generally refers to a sequence that is not substantially identical to a specified reference sequence, and if a particular reference sequence is not explicitly identified, it is specifically not substantially identical to at least about 50 consecutive base sequences in the endogenous p62 gene. Refers to a sequence.

"Normal expression" as used herein is defined as the expression level present in a wild-type animal or cell line. Thus, a mouse or cell line as used herein is "defective in normal expression" when such mouse or cell line expresses lower levels of functional p62 (including its total absence) as compared to those present in wild-type animals or cell lines. Is considered to be ". Various techniques known in the art can be used to quantify the expression level of a given protein. Such techniques include immunological techniques such as ELISA, RIA, Western blot or flow cytometry / FACS, or quantitative analysis techniques such as spectroscopy or chromatographic methods (including HPLC, FPLC, affinity or ignition chromatography). Include, but are not limited to.

As used herein, the term “p62 gene” or “p62 locus” extends to all exons that potentially encode p62 polypeptides and flanking sequences that participate in p62 protein expression (eg, promoters, enhancers, etc.) Chromosome region extending through). Thus, the p62 locus includes a region that extends from the first exon through the last exon and also includes adjacent flanking sequences (eg polyadenylation signals) that can participate in p62 gene expression.

As used herein, “p62 polypeptide” or “ubiquitin polypeptide” refers to a polypeptide that specifically binds ubiquitin and p62, respectively. DNA encoding a p62 polypeptide can hybridize with wild type p62 encoding DNA under stringent hybridization conditions. Likewise, DNA encoding ubiquitin polypeptides can hybridize with wild type ubiquitin encoding DNA under stringent hybridization conditions.

As used herein, “pharmaceutically acceptable carriers and / or diluents” include all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. The use of such media and agents for pharmaceutically active substances is well known in the art. Except where conventional media or agents are incompatible with the active ingredient, their use in the therapeutic compositions is contemplated. Supplementary active ingredients may also be incorporated into the compositions.

If administration of a particular composition is tolerated by the recipient animal and it is suitable to administer the composition to the animal, such composition is considered "pharmacologically or physiologically acceptable". The formulation is considered to be administered in a "therapeutically effective amount" when the amount administered is physiologically significant (significant). Such agents are physiologically important if the presence of certain agents results in detectable changes in the physiology of the recipient patient.

The term “phenotype” is the physical, biochemical and physiological structure of a particular animal as determined both genetically and environmentally. In particular, “phenotype generated” refers to a wild type animal or an animal exhibiting a disease state associated with an aging-related disorder.

Agents as used herein are considered "reasonably designed" when they are selected based on a computer model of the ligand binding site of the p62 protein.

As used herein, the term “alternate region” refers to a portion of a targeting construct flanked by homologous regions. In double-cross homologous recombination between the flanking homology regions and their corresponding endogenous p62 gene cross target sequences, the replacement region is integrated into the host cell chromosome between endogenous cross target sequences. The replacement region may be homologous (eg, having a sequence similar to the endogenous p62 gene sequence but with point mutations or missense mutations), non-homologous regions (eg, neo gene expression cassette), or homologous regions May be a combination of homologous regions.

As used herein, “sample” or “biological sample” is referred to in its broadest sense, and includes all obtained from an individual, body fluid, cell line, tissue culture, or other source, depending on the type of assay to be performed. Biological samples are included. As indicated, biological samples include body fluids such as semen, lymph, serum, plasma, urine, synovial fluid, spinal fluid, and the like. Methods of obtaining tissue biopsies and body fluids from mammals are well known in the art. Tissue biopsy of the brain is the preferred source.

In biological samples, certain polypeptides can be detected in vivo by imaging. Labels or markers for in vivo imaging of proteins include those detected by X-radiography, NMR or ESR. Suitable labels for X-radiography include radioisotopes such as barium or cesium, which emit detectable radiation but are not very harmful to the subject. Markers suitable for NMR or ESR include those having a detectable characteristic spin (eg, double hydrogen) that can be incorporated into the polypeptide by labeling using conventional techniques.

As used herein, “sequence identity” refers to the ratio of amino acid or nucleic acid residues in a candidate sequence that is identical to the amino acid residues in the original polypeptide sequence after aligning the sequences and introducing gaps as needed to achieve maximum sequence identity. Further defined as (%), no conservative substitutions are considered as part of the sequence identity. Sequence identity percentage values are described in Altschul et al., (1997), "Gapped BLAST and PSI-BLAST: a new generation of protein database search programs", Nucleic Acids Res., 25: 3389-3402. It is generated by the NCBI BLAST2.0 software as defined. The exceptions are penalties for mismatches in which the parameter is set to an omit value, and mismatches set to -1.

As used herein, the term “specifically binds” refers to an antibody molecule that is immunoreactive with two molecules, eg an antigen, or a non-antibody ligand that is reactive with another polypeptide, eg, a p62 polypeptide. It refers to a non-random, non-covalent reaction between ubiquitin. Moreover, specific binding may occur between chemical compounds or small peptides and p62 or ubiquitin.

A “subject” as used herein is a vertebrate, preferably a mammal, more preferably a human.

As used herein, “substantially complementary” refers to a sequence that is complementary to a sequence that substantially corresponds to a reference sequence. In general, targeting efficiency increases with the length of the targeted transformed gene portion (ie, homologous region) that is substantially complementary to the reference sequence (ie cross target sequence) present in the target DNA. In general, targeting efficiency is optimized when using allogeneic DNA homology clamps, but it is recognized that the presence of various recombinases can reduce the sequence identity required for efficient recombination.

As used herein, the terms "substantially corresponding", "substantially homologous" or "substantially identical" mean that a particular nucleic acid sequence is at least about 70%, 75%, 80%, 85%, By characteristic of a nucleic acid sequence, showing an identity of 90%, 95%, 96%, 97%, 98%, 99% or 100%. % Sequence identity is calculated excluding small deletions or additions which are less than 25% total of reference sequence. The reference sequence may be an acet or larger sequence, eg, a portion of a particular gene or flanking sequence, or a repeating portion of a chromosome. However, the reference sequence is at least 18 nucleotides in length, typically at least about 30 nucleotides, preferably at least about 50 to 100 nucleotides.

As used herein, the term “targeting construct” refers to (1) one or more homologous regions having a sequence substantially identical to or substantially complementary to a sequence present at the host cell p62 locus; And (2) a targeting region that integrates into the host cell p62 locus by homologous recombination between the targeting construct homology region and the p62 locus sequence. Targeting constructs are “hit-and-run” or “in-and-out” type constructs. Valancius and Smithies (1991) Mol. Cell. Biol. 11: 1402; Donehower et al. (1992) Nature 356: 215; (1991) J. NIH Res. 3: 59; Incorporated herein by reference, the targeting region is only temporarily incorporated into the endogenous p62 locus and removed from the host genome by selection. The targeting region may comprise a sequence that is substantially homologous to the endogenous p62 gene sequence and / or may comprise non-homologous sequences, eg, selectable markers (ie neo, tk, gkt). The term “targeting construct” does not necessarily indicate that a polynucleotide includes a gene that integrates into the host genome, nor does it necessarily indicate that the polynucleotide comprises a complete structural gene sequence. The term "targeting construct" as used in the art is synonymous with the term "targeting transformed gene".

As used herein, the term “targeting region” refers to a portion of a target construct that integrates into an endogenous chromosomal location after homologous recombination between a homologous clamp and an endogenous p62 gene sequence. Typically, the targeting regions are flanked on each side by homologous clamps, so that due to the double-cross recombination between each homologous clamp and their corresponding endogenous p62 gene sequence, a portion of the endogenous p62 locus is targeted to Replaced by; Such targeting regions may be referred to as "alternative regions" in such double-crossing gene replacement targeting constructs. However, some targeting constructs can only use a single homology clamp (eg, some “heat and run” type vectors). Bradley et al. (1992) Bio / Technology 10: 534; Incorporated herein by reference.

As used herein, a "transformed gene" is used herein to describe a genetic material that has been artificially inserted into or is intended to be inserted into the genome of mammalian cells, particularly mammalian cells of living animals. The transformed gene is used to transform the cell, which means that permanent or temporary genetic changes, preferably permanent genetic changes, are induced in the cell after incorporation of exogenous DNA. Permanent genetic changes are generally achieved by introducing the DNA into the genome of the cell. Vectors for stable integration include plasmids, retroviruses and other animal viruses, YACs, and the like. Transgenic mammals such as cattle, pigs, goats, horses, and the like, and certain rodents, such as rats, mice and the like, are of interest. Preferably, the transgenic animal is a mouse.

As used herein, “wild type” refers to an animal or cell line that has not been genetically altered.

p62 Of Ubiquitin  Co-accumulation

The presence of intracellular inclusion bodies is particularly characteristic of various neurodegenerative disorders including but not limited to peak disease (peak body), amyotrophic lateral sclerosis (ALS) spinal cord, Huntington's disease, Parkinson's disease, Alzheimer's disease neurofibrillary tangles. p62 co-accumulates and localizes in large quantities with ubiquitin to form inclusion bodies in the brain and spinal cord of patients with neurodegenerative diseases. p62 specifically binds specifically to ubiquitin. In vitro experiments indicate that p62 specifically binds to ubiquitin, and we have found that inclusion bodies derived from the sequestosomes associated with the disease are p62 positive and ubiquitin positive. Applicants have also found that p62 specifically binds ubiquitin and certain regions on p62 bind ubiquitin. Moreover, mutations in certain hydrophobic amino acid residues in p62 enhance binding to ubiquitin.

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

In one aspect of the invention, a p62 polypeptide can be used as a trap for immobilizing ubiquitin in the region of undesired activity of ubiquitin. The p62 polypeptide may be full length or longer, shorter or mutated or derivatized so long as it retains the ability to specifically bind ubiquitin.

p62 Of Ubiquitin  Binding inhibitor

In one aspect, the invention relates to screening for compounds such as polypeptides or chemical compounds that inhibit the binding of p62 to ubiquitin. Such inhibitor compounds are expected to treat a person suffering from a disease that is at least partially associated with the presence of inclusion bodies. In the case of using an inhibitor, ubiquitination p62 will not accumulate in the inclusion body, thus preventing formation of the inclusion body.

In this regard, in one aspect, the present invention relates to all inhibitor molecules capable of interacting with p62 to block the binding of p62 to ubiquitin. On the other hand, such molecules can disrupt p62 / ubiquitin bonds by binding to ubiquitin.

One such inhibitor is a modified p62 polypeptide, which retains ubiquitin binding function but lacks some or all of the other features associated with p62 function (eg, the ability to form aggregates) or are at a reduced level.

Inhibitor compounds are those compounds that interact with the p62 polypeptide and ubiquitin by any biochemical or enzymatic inhibition kinetics such as competitive, non-competitive or non-competitive inhibition, so long as they damage the binding of the p62 polypeptide to ubiquitin. It should be recognized that it can be damaged.

Thus, in one particular aspect, the present invention relates to the use of any polymer or monomeric compound capable of inhibiting the binding between p62 and ubiquitin. In another aspect of the invention, it is contemplated that extracts of natural foods or 'functional foods' comprise inhibitors of the p62 / ubiquitin complex.

Ubiquitin  or p62 Nucleic Acids Encoding Polypeptides That Bind with

"Isolated" polynucleotide sequence encompasses nucleic acid molecules, DNA or RNA, removed from their natural environment. This includes segments of DNA encoding p62 polypeptides or ubiquitin polypeptides of the 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 can be partially or substantially purified.

In addition, isolated nucleic acid molecules of the present invention include DNA molecules encoding sequences that are substantially different from those previously described, but are still p62 polypeptides or ubiquitin polypeptides and peptides thereof due to genetic coding deprivation or other variability. Thus, for example, it will be common for one skilled in the art to generate the aforementioned variants to optimize codon expression or generic function for a particular host.

In another aspect, the present invention provides isolated nucleic acid molecules comprising polynucleotides that hybridize with a portion of the polynucleotides in the above-mentioned nucleic acid molecules of the present invention under stringent hybridization conditions. Hybridized polynucleotides are useful as diagnostic probes and primers as discussed above. Certain portions of the polynucleotide that hybridize with the p62 polypeptide, which can be used as a probe or primer, can be precisely specified by the 5 'and 3' base positions or by the nucleotide base size as mentioned above, or in the same manner. May be excluded. Similarly, certain portions of polynucleotides that hybridize with ubiquitin polypeptides can also be used as probes and primers. Preferred hybridization polynucleotides of the present invention, when labeled and used in hybridization assays known in the art, are those that exhibit the greatest signal intensity regardless of other heterologous sequences present in equimolar amounts.

Variant  And mutant polynucleotides

The invention further provides nucleic acids encoding p62 polypeptides or portions, homologues or derivatives of p62 polypeptides or ubiquitin polypeptides, which possess the ability to specifically bind p62 and ubiquitin, each of which is also optionally non-functional in at least one other aspect. It is directed to variants of the molecule, provided that such variants disrupt competitive or non-competitive p62 / ubiquitin bonds without causing toxic side effects. Non-natural variants can be generated using mutagenesis techniques known in the art.

Such nucleic acid variants include those produced by nucleotide substitutions, deletions or additions. Such substitutions, deletions or additions may involve one or more nucleotides. Changes in amino acid sequence can lead to conservative or non-conservative amino acid substitutions, deletions or additions. Particularly preferred are silent substitutions, additions and deletions, which do not alter the properties and activities of the polypeptides or portions thereof of the invention. Conservative substitutions are also preferred in this regard.

The present invention not only allows the use of sequences in expression vectors but also transfects host cells and cell lines that are prokaryotic or eukaryotic cells. The invention also allows for the purification of polypeptides expressed from expression vectors. This expression vector may contain various molecular tags to facilitate purification. Expression constructs obtained in succession can be transformed into any host cell of choice. Cell lysates from such host cells are isolated by established methods well known in the art. GFP- or GST-containing expression vectors may be used to localize p62 polypeptides or ubiquitin polypeptides to host cells. Expression vectors can be inducible or constitutive promoters.

Variant  And mutant polypeptides

Amino acid engineering can be used to improve or change the characteristics of the p62 polypeptides or ubiquitin polypeptides of the invention. Recombinant DNA techniques known to those skilled in the art can be used to create novel mutant polypeptides, including single or multiple amino acid substitutions, deletions, adducts or fusion proteins. Such modified polypeptides can, for example, exhibit increased / decreased binding activity or increased / decreased stability. In addition, they may be purified in higher yields and may exhibit superior binding activity than the corresponding natural polypeptides, at least under certain purification and storage conditions.

Of particular interest is the substitution of charged amino acids with neutral or other charged amino acids that can produce proteins that exhibit highly desirable improved features, eg, reduced aggregation of the resulting polypeptide. Aggregation can not only reduce activity but can also cause problems when preparing pharmaceutical formulations, since the aggregate can be immunogenic.

Antibodies

In one aspect, the present invention relates to detecting inclusion body or sequetosome formation using various detection methods. One method of detecting binding of p62 polypeptides to ubiquitin is to label the p62 polypeptides directly and assay for their binding using labeling and separation techniques conventional to those skilled in the art. Other methods include the use of labeled ligands that specifically bind to a p62 polypeptide, ubiquitin or p62 polypeptide / ubiquitin complex. Such ligand may be an antibody.

Purified p62 polypeptides, ubiquitin or p62 polypeptide / ubiquitin complexes can be used to generate monoclonal or polyclonal antibodies. Fragments of p62 polypeptides can also be used to generate monoclonal or polyclonal antibodies. Binding of p62 polypeptides to ubiquitin using monoclonal or polyclonal antibodies obtained in succession and various, including, but not limited to, cells, tissues and body fluids (eg, but not limited to serum, plasma and urine) Formation of p62 polypeptide, ubiquitin or p62 polypeptide / ubiquitin complex in a sample can be determined. P62 polypeptides, ubiquitin or p62 polypeptide / ubiquitin complexes can be assayed using various molecular biological methods, including but not limited to in situ hybridization, immunoprecipitation, immunofluorescence staining, western blot analysis, and the like. p62 polypeptide, ubiquitin or p62 polypeptide / ubiquitin to determine the amount of inclusion bodies in other parts of the body or brain tissue, including the body fluids of human subjects, believed to have an indicated disorder in which p62 and ubiquitin aggregate and accumulate to form inclusion bodies. ELISA can be performed by using monoclonal antibodies to the complex.

Antibodies of the invention include polyclonal, monoclonal, multispecific, human, human adaptation or chimeric antibodies, single chain antibodies, Fab fragments, F (ab ') fragments, fragments generated by Fab expression libraries, anti- Genotype (anti-Id) antibodies (including, for example, anti-Id antibodies to antibodies of the invention), and epitope-binding fragments of those mentioned above are included, but are not limited to these. As used herein, the term “antibody” refers to an immunoglobulin molecule and a molecule containing an immunologically active portion of an immunoglobulin molecule, ie, an antigen binding site that immunospecifically binds an antigen. The immunoglobulin molecules of the invention may be of any type (eg, IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclasses of immunoglobulin molecules. have.

Antibodies of the invention may be monospecific, bispecific, triple specific or higher order multispecific. Multispecific antibodies may be specific for different epitopes of polypeptides of the invention, or may be specific for both polypeptides of the invention as well as heterologous epitopes, eg, heterologous polypeptides or solid support materials.

Antibodies of the invention may be described or specified in terms of epitope (s) or portion (s) of the polypeptides of the invention to which they recognize or specifically bind. Such epitope (s) or polypeptide portion (s) can be specified as described herein, eg, by the N-terminal and C-terminal positions, the size of consecutive amino acid residues, and the like.

Antibodies of the invention can be used to purify, detect, and target polypeptides of the invention, including but not limited to, for example, in vitro and in vivo diagnostic and therapeutic methods. For example, the antibodies are used in immunoassays to qualitatively and quantitatively determine the level of a p62 polypeptide, ubiquitin or p62 polypeptide / ubiquitin complex of the invention in a biological sample.

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

Antibodies of the invention can be prepared by any suitable method known in the art. Polyclonal antibodies directed against the antigen of interest can be produced by a variety of procedures well known in the art. For example, the polypeptide of the present invention can be administered to a variety of host animals, including but not limited to rabbits, mice, rats, and the like, to induce the production of serum containing polyclonal antibodies specific for the antigen. have. Various adjuvants can be used to increase the immunological response depending on the host species, including Freund (complete and incomplete), mineral gels (e.g. aluminum hydroxide), surfactants (e.g. lysocithin, fluo) Nick polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, dinitrophenol) and potentially useful human adjuvants such as BCG (bacille Calmette-Guerin and corynebacterium parvum) Include, but are not limited to. Such adjuvants are also well known in the art.

Monoclonal antibodies can be prepared using a wide range of techniques known in the art, including the use of hybridoma, recombinant and phage display techniques, or combinations thereof. For example, monoclonal antibodies can be produced using hybridoma techniques, including those known in the art. As used herein, the term “monoclonal antibody” is not limited to antibodies produced through hybridoma technology. The term "monoclonal antibody" refers to an antibody derived from a single clone, including all eukaryotic, prokaryotic or phage clones, not to antibodies produced by the method of production thereof.

Methods of generating and screening specific antibodies using hybridoma technology are conventional and well known in the art. In a non-limiting example, mice can be immunized with p62 polypeptide, ubiquitin or p62 polypeptide / ubiquitin complex or cells expressing it. Once an immune response is detected, for example, an antibody specific for an antigen is detected in mouse serum, the mouse spleen is harvested and the spleen cells are isolated. Spleen cells are then fused to suitable myeloma cells, such as cells of cell line SP20, available from ATCC using well known techniques. Hybridomas are selected and cloned at limited dilution. The hybridoma clones are then assayed for cells secreting antibodies capable of binding to the polypeptide of the present invention or a complex with a binding partner thereof by methods known in the art. By immunizing mice with positive hybridoma clones, ascites fluids generally containing high levels of antibodies can be generated.

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

Assay for Antibody Binding

Antibodies of the invention can be assayed for immunospecific binding by any method known in the art. Immunoassays that may be used include western blots, radioimmunoassays, ELISA (enzyme-linked immunosorbent assays), "sandwich" immunoassays, immunoprecipitation assays, precipitin reactions, gel diffusion sedimentation antibody responses, immunodiffusion reactions, aggregation Competitive and non-competitive assay systems using techniques such as response assays, complement-fixation assays, immunoradiometric assays, fluorescence immunoassays, Protein A immunoassays, and the like. Such assays are conventional and well known in the art. See Ausubel et al, eds, 1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York; The entirety of which is incorporated herein by reference]. Examples of immunoassays are briefly described below, but are not limited thereto.

Immunoprecipitation protocols generally include RIPA buffer (1% NP-40 or Triton X-100, 1% sodium deoxy) supplemented with protein phosphatase and / or protein inhibitors (e.g. EDTA, PMSF, aprotinin, sodium vanadate). Lysing the cell population in lysis buffer such as cholate, 0.1% SDS, 0.15M NaCl, 0.01M sodium phosphate, pH 7.2, 1% trasylol); The antibody of interest is added to the cell lysate; Incubate at 4 ° C. for a period of time (eg, 1 to 4 hours); Protein A and / or Protein G Sepharose beads are added to the cell lysate; Incubate at 4 ° C. for at least about 1 hour; Washing the beads in lysis buffer; Resuspending these beads in SDS / sample buffer. The ability of the antibody of interest to immunoprecipitate a particular antigen can be assessed, for example, by western blot analysis. Those skilled in the art will be knowledgeable about parameters that can be modified to increase binding of the antibody to antigen and to reduce background (eg, pre-clean cell lysates with Sepharose beads). For further discussion of immunoprecipitation protocols, reference may be made to 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 produces protein samples; Such protein samples are electrophoresed on polyacrylamide gels (eg 8% -20% SDS-PAGE depending on the molecular weight of the antigen); Protein samples are transferred from polyacrylamide gels to membranes such as nitrocellulose, PVDF or nylon; Blocking the membrane in a blocking solution (eg 3% BSA or PBS with nonfat milk); The membrane is washed in a wash buffer (eg PBS-Tween 20); Blocking the membrane with a primary antibody (antibody of interest) diluted in blocking buffer; Washing the membrane in wash buffer; The membrane is a secondary antibody (an antibody that recognizes a primary antibody) conjugated to an enzyme substrate (eg horseradish peroxidase or alkaline phosphatase) or radioactive molecule (eg ³² P or ¹²⁵ I) diluted in blocking buffer; For example, anti-human antibodies); Washing the membrane in wash buffer; Detecting the presence of an antigen. Those skilled in the art will be knowledgeable about parameters that can be modified to increase the detected signal and reduce background noise. For further discussion of Western blot protocols, reference may be made to Ausubel et al, eds, 1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York at 10.8.1].

ELISAs produce antigens that may include a sample comprising p62 polypeptide, ubiquitin or p62 polypeptide / ubiquitin; Coating wells of a 96 well microtiter plate with said antigen; Adding an antibody of interest conjugated to a detectable compound, such as an enzyme substrate (eg horseradish peroxidase or alkaline phosphatase) to the wells, incubating for a period of time, and then detecting the presence of the antigen. do. In ELISAs, the antibody of interest should not be conjugated to a detectable compound; Instead, a second antibody conjugated to a detectable compound (which recognizes the antibody of interest) can be added to the well. In addition, instead of coating the wells with the antigen, the antibodies may be coated with the wells. In such cases, a second antibody conjugated to a detectable compound can be added at the same time or after addition of the antigen of interest to the coated well. Those skilled in the art will be knowledgeable about parameters that can be modified to increase the signal detected as well as to increase other variables of ELISAs known in the art. For further discussion of ELISAs, reference may be made to Ausubel et al, eds, 1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York at 11.2.1].

Inclusion Diagnostic Test

The invention also provides a diagnostic method for detecting the presence of inclusion bodies in a biological sample. This can be done directly by assaying (eg, by assaying polypeptide levels using antibodies derived in response to a p62 polypeptide or fragment thereof) or indirectly by assaying (eg, having specificity for a p62 polypeptide or fragment thereof). By assaying for antibodies).

If a diagnosis of a disease state has already been made, the present invention is useful for monitoring the progression or regression of the disease state by measuring the amount of ubiquitin / p62 complex present in the patient, thereby lowering the levels of patients exhibiting increased inclusion body production. As a result, patients will experience worse clinical outcomes than patients who produce inclusion bodies.

The presence of labeled molecules can be detected in the patient using methods known in the art for in vivo scanning. These methods depend on the type of label used. The skilled person will be able to determine the appropriate method for detecting a particular label. Methods and apparatus that can be used in the diagnostic methods of the invention include computerized tomography (CT), whole body scans such as positron emission tomography (PET), magnetic resonance imaging (MRI), and ultrasonography as well as electron paramagnetic resonance ( EPR) and nuclear magnetic resonance (NMR), but are not limited thereto.

In certain embodiments, molecules such as polypeptides that specifically bind ubiquitin are labeled with a radioisotope and detected in a patient using a radioactive surgical instrument (Thurston et al. US Pat. No. 5,441,050). In another embodiment, the molecule is labeled with a fluorescent compound and detected in the patient using a fluorescent reactive scanning instrument. In another embodiment, the molecule is labeled with a positron emitting metal and detected in the patient using positron emission tomography. In another embodiment, the molecule is labeled with a paramagnetic label and detected in the patient using magnetic resonance imaging (MRI).

In one aspect, the use of a label that can be detected by the aforementioned method or some other mechanism, which is non-toxic to the subject; In vivo diagnostics can be performed by injecting such a polypeptide into a subject so that the labeled polypeptide circulates and binds to its binding target, thereby detecting the presence of a target mass, such as ubiquitin or inclusion aggregates.

Cover

Suitable enzyme labels include, for example, those derived from oxidase groups that catalyze the production of hydrogen peroxide by reacting with a substrate. Glucose oxidase is particularly preferred because of its excellent stability and the availability of its substrate (glocos). The activity of the oxidase label can be assayed by measuring the concentration of hydrogen peroxide formed by the enzyme-labeled antibody / substrate reaction. In addition to enzymes, other suitable labels include radioactive isotopes such as iodine ( ¹²⁵ I, ¹²¹ I), carbon ( ¹⁴ C), sulfur ( ³⁵ S), tritium ( ³ H), indium ( ¹¹² In) and technetium ( ^{99 m} Tc), and fluorescent labels such as fluorescein and rhodamine, and biotin.

Further labels suitable for the p62 polypeptide-, ubiquitin- or p62 polypeptide / ubiquitin complex-specific antibodies of the invention are provided below. Examples of suitable enzyme labels include maleate dehydrogenase, δ-5-steroid isomerase, yeast-alcohol dehydrogenase, α-glycerol phosphate dehydrogenase, trios phosphate isomerase, peroxidase, Alkaline phosphatase, asparaginase, glucose oxidase, β-galactosidase, ribonucleases, urease, catalase, glucose-6-phosphate dehydrogenase, glucoamylase and acetylcholine esterase.

Examples of suitable radioisotope labels include ³ H, ¹¹¹ In, ¹²⁵ I, ¹³¹ I, ³² P, ³⁵ S, ¹⁴ C, ⁵¹ Cr, ⁵⁷ To, ⁵⁸ Co, ⁵⁹ Fe, ⁷⁵ Se, ¹⁵² Eu, ⁹⁰ Y, ⁶⁷ Cu, ²¹⁷ Ci, ²¹¹ At, ²¹² Pb, ⁴⁷ Sc, ¹⁰⁹ Pd and the like. ¹¹¹ In, where in vivo imaging is used, is the preferred isotope because it avoids the problem of dehalogenation of ¹²⁵ I or ¹³¹ I-labeled polypeptides by the liver. In addition, such radionucleotides have a more preferred gamma emission energy for imaging. For example, ¹¹¹ In coupled with a monoclonal antibody with 1- (P-isothiocyanatobenzyl) -DPTA is hardly absorbed by non-tumor tissues, especially the liver, thus enhancing the specificity of tumor localization. It turned out.

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

Examples of suitable fluorescent labels include ¹⁵² Eu labels, fluorescein labels, isothiocyanate labels, rhodamine labels, phycoerythrin labels, phycocyanin labels, allophycocyanin labels, o-phthalaldehyde labels and fluorescamine Labels are included.

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

Examples of chemiluminescent labels include luminal labels, isoluminal labels, aromatic acridinium ester labels, imidazole labels, acridinium salt labels, oxalate ester labels, luciferin labels, luciferase labels and aqueous labels.

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

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

Polypeptides and antibodies of the invention, including fragments thereof, can be used to detect p62 polypeptides, ubiquitin or p62 polypeptide / ubiquitin complexes using biochip and biosensor techniques. Biochips and biosensors of the invention can include polypeptides of the invention to detect antibodies that specifically recognize the p62 polypeptide / ubiquitin complex. Biochips and biosensors of the invention may also comprise an antibody that specifically recognizes a polypeptide of the invention to detect a p62 polypeptide / ubiquitin complex. In addition, the invention provides antisense oligonucleotides that can be used to hybridize or detect wild type or mutant p62 transcripts that may be present in p62 nucleic acids, preferably oligonucleotides, and preferably assayable samples such as biological samples. Consider an array including, for example, a microarray or a macroarray chip. Such array structures are well known.

Inclusion Detection Kit

The invention also includes a kit for analyzing a sample to determine the presence of a p62 polypeptide / ubiquitin complex in a biological sample. In a general embodiment, the kit is preferably one or more containers of ligands that specifically bind to a p62 polypeptide, ubiquitin or p62 polypeptide / ubiquitin complex, which may be a p62 polypeptide, or a purified antibody against a p62 polypeptide / ubiquitin complex. Include in. In certain embodiments, the kits of the present invention contain substantially isolated polypeptides comprising epitopes that are specifically immunoreactive with the antibodies contained in such kits. Preferably, the kit of the present invention further comprises a control antibody that does not react with the polypeptide of interest. The kit further includes a label and instructions for its use.

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

Transformability  Animal and Cell Lines

The present invention provides a variety of animals produced using germline foreign DNA or using specific genes of modified expression levels. These animals typically have foreign or mutated genes incorporated into their genomes. In this class of transgenic animals, so-called homozygous nonfunctional or "knockout" mutants, gene manipulation has been used to inhibit expression of endogenous genes.

Also provided herein are mammalian cell lines that are heterozygous or homozygous that are defective in 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 cell lines which are defective in the normal synthesis of p62. Such cell lines can be derived from pluripotent cell lines.

The transgenic animal can be homozygous or heterozygous for genetic modification. The subject animal is useful for testing candidate agents for prophylactically or after onset of an individual diagnosed with an age-related disorder.

Transgenic animals generally have at least one copy of a transformed gene that is homologous or non-homologously integrated into an endogenous chromosomal location to encode a foreign or mutant protein. Such transgenic animals are typically produced by introducing a transformed gene or targeting construct into fertilized egg or embryonic stem (ES) cells and then injecting it into an embryo. Introduction of the transformed gene into fertilized eggs or ES cells is typically performed by microinjection, retroviral infection, electroporation, lipofection or bioolistic. The fertilized egg or embryo is then transferred to the appropriate fertility female during pregnancy. According to this method knockout mutants can be obtained, wherein the non-native DNA introduced includes a nucleic acid construct that will be used to inhibit expression of a particular gene. Such knockout constructs are typically introduced into ES cells.

Transgenic animals comprise exogenous nucleic acid sequences present as extrachromosomal elements or stably integrated into all or part of their cells, in particular germ cells. Unless stated otherwise, transgenic animals will be assumed to contain stable changes to germline sequences. During the initial construction of such animals, "chimeras" or "chimeric animals" are produced, in which only cells of the acet have a modified genome. Chimeras are primarily used for breeding purposes to produce the desired transgenic animals. Animals with heterozygous modifications are produced by breeding chimeras. Male and female heterozygotes are typically bred to produce homozygous animals.

Hogan, et al., Manipulating the Mouse Embryo: A Laboratory Manual, Cold Spring Harbor Laboratory (1988) and Teratocarcinomas and Embryonic Stem Cells: A Practical Approach, EJ Robertson, ed., IRL Press, Washington, DC, (1987); Incorporated herein by reference, the chimeric targeting mouse can be induced.

Embryonic stem cells are described in the literature, 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.

In accordance with the practice of the present invention, the endogenous p62 allele of a particular cell line or non-human animal is functionally disrupted to inhibit or eliminate the expression of endogenously encoded p62.

Embryonic stem cells (ES cells) are preferred for preparing transgenic non-human animals (including homologously targeted non-human animals). See Robertson, E. J. (1987) in Teratocarcinomas and Embryonic Stein Cells: A Practical Approach. E. J. Robertson, ed. (Oxford: IRL Press), p. 71-112, murine ES cells, such as AB-1 strains grown on mitotically inactive SNL76 / 7 cell feed layers (McMahon and Bradley (1990) Cell 62: 1073), as described essentially Can be used for homologous gene targeting. Other suitable ES states include E14 week [Hooper et al. (1987) Nature 326: 292-295, D3 week [Doetschman et al. (1985) J. Embryol. Exp. Morphi. 87: 27-45, and the CCE note (Robertson et al. (1986) Nature 323: 445-448, but is not limited to such. The practice of the present invention is specifically illustrated below using ES cells of the murine species 129 / J (Jackson Laboratories). Success regarding the generation of mouse strains from ES cells carrying specific targeting mutations is dependent on the pluripotency of the ES cells (ie, once injected into the host vesicles, participate in embryogenesis and the resulting germ cells of the resulting animals). The ability of the cells to contribute to Embryos containing ES cells injected as above are generated in the uterus of nonpregnant non-human females and born as chimeric mice. The resulting transgenic mice are chimeric to cells with inactivated endogenous p62 locus, and backcrossed, and then the tail biopsy DNA of the offspring to identify transgenic mice heterozygous for the inactivated p62. Screen for the presence of precisely targeted transformed gene (s) by PCR or Southern blot analysis. By performing appropriate breeding, one can produce transgenic non-human animals homozygous for the functionally disrupted p62 allele. Such transgenic animals are unable to produce substantially endogenous p62 gene products.

Functionally disrupted p62 homozygous nonfunctional mutant transgenic animals will typically include rats or mice, but include non-murine species, such as dogs, cattle, sheep, goats, pigs, and non-human primates. You can also use

The p62-/-mammals of the invention can be utilized as models for studying age-related disorders or diseases. In addition, p62-/-animals can be used for the screening of potentially therapeutic synthetic p62 peptides. Such peptides can be screened for their ability to induce anti-aging related disorder phenotype upon topical administration to p62 − / − mice. Candidate peptides may be administered locally by injection into p62 − / − mice.

This practice of the invention is illustrated herein using the neo gene as a transformed gene. It will be appreciated that it is possible to produce non-human animals in which any transformed gene of interest is established, provided that such transformed gene can be contained in a construct further comprising a wild type p62 gene. .

gene Targeting Construct

Genetic changes can be introduced into cultured cells using gene targeting, a method using homologous recombination to modify the mammalian genome. By targeting the gene of interest to embryonic stem (ES) cells, these changes can be introduced into the germline of a laboratory animal, in particular studying the effect of the modification on the whole organism. The gene targeting process is performed by introducing DNA targeting constructs into tissue culture cells that have segments homologous to the target locus and that also include intended sequence modifications (eg, insertions, deletions, point mutations). Cells thus treated are then screened for correct targeting, identifying and isolating the appropriately targeted. A common scheme of disrupting gene function by gene targeting in ES cells is to construct targeting constructs designed to undergo homologous recombination with their chromosomal counterparts in the ES cell genome. Such targeting constructs are typically arranged such that they functionally disrupt it by inserting additional sequences, eg, a positive selection marker, into the coding element of the target gene. Targeting constructs are typically insertion-type or replacement-type constructs. Hasty et al. (1991) Mol. Cell. Biol. 11: 4509.

The present invention encompasses the generation of stem cells and non-human animals having endogenous p62 genes inactivated by gene targeting with homologous recombinant targeting constructs. The p62 gene sequence can be used as a reference for generating PCR primers flanking the region that will be used as a homology clamp in the targeting construct. The PCR primers are then used to amplify genomic sequences from preparations of genomic clone libraries or genomic DNA, preferably from non-human animal species to be targeted with targeting constructs. The DNA thus amplified is then used as a homology clamp and / or targeting region. Generic principles regarding the construction and screening methods of targeting constructs are described in Bradley et al. (1992) Bio / Technology 10: 534; Incorporated herein by reference.

Isolation of p62 genomic DNA useful for this purpose is described herein. Suitable probes can 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 described in US Pat. Nos. 6,291,645B1 and 5,962,224, the entire contents of which are incorporated herein by reference.

Targeting constructs can be transferred into pluripotent stem cells, such as ES cells, where the targeting construct is homologously recombined with a portion of the endogenous p62 locus and prevents the functional expression of the endogenous p62 gene (water) (Ie, insertion (water), deletion (water), translocation (water), sequence replacement (water) and / or point mutation (water)).

One method is to delete essential structural elements of the endogenous p62 gene by targeted homologous recombination. For example, the targeting construct can be homologously recombined with the endogenous p62 gene and the extensified portion of one or more exons removed to create an exon-depleted allele, typically corresponding By inserting a replacement region lacking exon (s). Transgenic animals that are homozygous for exon-depleted alleles (eg, by breeding heterozygotes for each other) are essentially incapable of expressing functional endogenous p62 polypeptides. Similarly, homologous gene targeting can optionally be used to functionally disrupt the p62 gene by deleting only a specific portion of the exon.

Targeting constructs may also be used to generate essential regulatory elements of the endogenous p62 gene, such as promoters, enhancers, splice sites, polyadenylation sites, and other regulatory sequences [top or bottom of the p62 structural gene. But may include cis-acting sequences that participate in endogenous p62 gene expression. Deletion of regulatory elements is typically accomplished by inserting a replacement region lacking the corresponding regulatory element (s) by homologous double-cross recombination.

A preferred method is to block the essential structural and / or regulatory elements of the endogenous p62 gene by targeting insertion of the polynucleotide sequence, thereby functionally disrupting the endogenous p62 gene. For example, the targeting construct may be homologously recombined with an endogenous p62 gene and a non-homologous sequence, eg, a neo expression cassette, may be incorporated into structural elements (eg exons) and / or regulatory elements (eg enhancers). , Promoters, splice sites, polyadenylation sites) can be used to yield targeted p62 alleles exhibiting interstitial blocking. Such inserted sequences can range from about 1 nucleotide in size (eg, to generate frameshifts in exon sequences), as limited by the efficiency of homologous gene targeting using targeting constructs with long non-homologous replacement regions. It may range from several kilobases or more.

One preferred target site is indicated in FIG. 1 and identified as shown in FIG. 2.

Targeting constructs may also be used to replace a portion of the endogenous p62 gene with an exogenous sequence (ie, a portion of the targeted transformed gene); For example, the first exon of the p62 gene can be replaced with a substantially identical portion containing a nonsense or missense mutation.

Targeting constructs can be transferred into totipotent ES cell lines by electroporation or microinjection. The targeting construct recombines homologously with either the endogenous sequence in the p62 locus or the endogenous sequence flanking the p62 locus and functionally disrupts one or more alleles of the p62 gene. Typically, due to homologous recombination of the endogenous p62 locus sequence and the targeting construct, non-homologous sequences encoding and expressing a selectable marker (eg neo) will typically be integrated in the form of a positive selection cassette. By propagating the cells in a medium that preferentially propagates cells expressing a selectable marker, ES cells having one or more of the p62 nonfunctional alleles are selected. ES cells thus screened are examined by PCR analysis and / or Southern blot analysis to confirm the presence of precisely targeted p62 alleles. Breeding of non-human animals heterozygous for a nonfunctional function allele can be performed to produce non-human animals homozygous for the nonfunctional allele, so-called "knockout" animals. See Donehower et. al. (1992) Nature 256: 215; Science 256: 1392; Incorporated herein by reference. On the other hand, ES cells homozygous for a functioning allele with integrated selectable markers can be produced in culture by selecting in a medium containing high levels of selection agent (eg G418 or hygromycin). have. Heterozygous and / or homozygous for correctly targeted functioning alleles can be confirmed using PCR analysis and / or Southern blot analysis of DNA isolated from aliquots and / or tail biopsies of selected ES cell clones.

Gene targeting techniques that have been reported include, but are not limited to, co-electroporation, "heat and run", single-cross integration and double-cross recombination. Bradley et al. (1992) Bio / Technology 10: 534. Preparation of homozygous p62 nonfunctional mutants can be carried out using any of the essentially applicable homologous gene targeting strategies known in the art. The steric configuration of the targeting construct depends on the particular targeting technique chosen. For example, targeting constructs for single-crossing integration or “heat and run” targeting require only a single homology clamp coupled to the targeting region, whereas dual-crossing alternative-type targeting constructs each side of the alternate region. It requires two homology clamps to flank.

For example (but not limited to), the targeting construct may comprise: (1) within about 3 kilobases of the exon of the endogenous p62 gene (ie, in the direction facing the readout frame of the exon); ) A first homology clamp having a sequence substantially the same as the sequence; (2) a replacement region comprising a positive selection cassette with a pgk promoter that drives transcription of the neo gene; (3) a second homology clamp having a sequence substantially the same as the sequence within the bottom of about 3 kilobases of the exon of the endogenous p62 gene; And (4) a negative selection cassette with a pgk promoter that drives the transcription of the HSV tk gene. Such targeting constructs are suitable for double-crossover replacement recombination, which deletes a portion of the endogenous p62 locus extended to the exon and replaces it with an alternative region with a positive selection cassette. Exons deleted as described above are essential for the expression of the functional p62 gene product. Thus, the resulting exon-depleted alleles are functionally disrupted and are therefore termed nonfunctional alleles.

The targeting construct includes one or more homology clamps linked by nucleotide chains (ie, by phosphodiester bonds) to the targeting region. Homologous clamps may have a sequence substantially corresponding to or substantially complementary to the endogenous p62 gene sequence of a non-human host animal and may comprise a sequence flanking the p62 gene.

Although no lower or upper boundary for recombination homology clamps for gene targeting has been conclusively determined in the art, the best mode for homology clamps is believed to be in the range of about 50 bp to several tens of kilobases. As a result, the targeting construct is generally about 50 to 100 or more nucleotides in length, preferably about 250 to 500 or more nucleotides, more preferably about 1000 to 2000 or more nucleotides. Construct homology clamps (homologous clamps) are generally about 50 to 100 or more bases in length, preferably about 100 to 500 or more bases, more preferably about 750 to 2000 or more bases. Homologous regions of about 7 to 8 kilobases in length are considered preferred, and one preferred embodiment flanks the first homologous region of about 7 kilobases and the other side of the replacement region, which flanks one side of the replacement region. Has a second homology region of about 1 kilobase. Homology (ie, substantial identity) lengths for homologous regions can be selected at the discretion of the practitioner based on sequence composition and complexity of the endogenous p62 gene target sequence (s) and guidelines provided in the art. The targeting construct is one having a sequence substantially corresponding to or substantially complementary to an endogenous p62 gene sequence (eg, an exon sequence, enhancer, promoter, intronic sequence, or flanking sequence within about 3-20 kb of p62 gene). It has the above homology region. Such targeting construct homology regions serve as templates for homologous pairing and recombination with substantially identical endogenous p62 gene sequence (s). In targeting constructs such homologous regions typically flank the replacement region, which is the region of the targeting construct where replacement with the targeted endogenous p62 gene sequence should be performed. Thus, segments of the targeting construct flanked by homologous regions can replace segments of the endogenous p62 gene sequence by double-cross homologous recombination. The homologous region and the targeting region are linked together in a conventional linear polynucleotide chain (5 'to 3' phosphodiester backbone). Targeting constructs are generally double stranded DNA molecules, most of which are typically linear.

Homologous regions are generally used in the same orientation (ie, the top direction is the same for each homology region of the transformed gene to avoid translocations). Thus, double-cross alternative recombination can be used to delete a portion of endogenous p62, while at the same time transferring a non-homologous portion (ie neo gene expression cassette) into the corresponding chromosomal location. Double-cross recombination can also be used to add non-homologous portions to the endogenous p62 gene without deleting endogenous chromosomal positions. However, double-cross recombination may be used to simply delete a portion of the endogenous gene sequence without transferring the non-homologous portion into the endogenous p62 gene. The top and / or bottom from the non-homologous portion can be a gene confirming that double-cross homologous recombination has occurred; Such genes are typically HSV tk genes that can be used for negative selection.

Typically, the targeting construct used to functionally disrupt the endogenous p62 gene will comprise two or more homologous regions sequestered by a non-homologous sequence containing an expression cassette encoding a selectable marker (eg neo). See 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 transformed genes may have homologous region (s) flanking only one side of the non-homologous sequence. Targeted transformed genes of the invention may be of the type referred to in the art as “heat and run” or “in and out” transformed genes. Valancius and Smithies (1991) Mol. Cell. Biol. 11: 1402; Donehower et al. (1992) Nature 356: 215; (1991) J. NIH Res. 3: 59; Incorporated herein by reference.

A positive select expression cassette encodes a selectable marker that provides a means for selecting cells having an integrated targeting transformed gene sequence that is expanded to such a positive select expression cassette. The negative selection expression cassette encodes a selectable marker that provides a means for selecting cells that do not have an integrated copy of this negative selection expression cassette. Thus, by a combination positive-negative selection protocol, by selecting for the presence of positive markers and for the presence of negative markers, the transformed gene portion (ie, replacement region) between homologous regions is incorporated into the chromosomal location and homologous. It is possible to select cells that have undergone alternative recombination (Valancius and Smithies, supra).

Expression cassettes typically include a promoter that is operative in a targeting host cell (eg, ES cell) linked to a structural sequence encoding a protein or polypeptide that confers a selective phenotype on the targeted host cell, and a polyadenylation signal. Promoters included in the expression cassette may be constitutive, cell type-specific, step-specific, and / or modulatory (eg, by hormones such as glucocorticoids; MMTV promoters), but prior to selection and And / or expressed during selection. The expression cassette may optionally include one or more enhancers, typically linked to the top of the promoter and within about 3 to 10 kilobases. However, if homologous recombination at the targeting endogenous site (s) occurs below the non-homologous sequence of the functional endogenous promoter, the targeting construct replacement region may comprise only the structural sequence encoding the selectable marker and drive transcription. May be dependent on endogenous promoters. See 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 rely on enhancing transcription of the transformed gene sequence in the transformed construct without the enhancer.

Preferred expression cassettes for inclusion in the targeting construct encode and express select drug resistance markers and / or HSV thymidine kinase (tk) enzymes. Suitable drug resistance genes include, for example, gpt (xanthine-guanine phosphoribosyltransferase), which can be screened for having mycophenolic acid; Neo (neomycin phosphotransferase), which can be selected for having G418 or hygromycin; And DFHR (dihydrofolate reductase), which can be screened for having methotrexate. Nulligan and Berg (1981) Proc. Natl. Acad. Sci. (U.S.A.) 78: 2072; Southern and Berg (1982) J. Mol. Appl. Genet. 1: 327; Incorporated herein by reference.

Selection for correctly targeted recombinants will generally use at least positive selection, where a non-homologous expression cassette provides a functional protein (eg, neo) that confers a selective phenotype to the targeting cells in which the inherently integrated expression cassette has been anchored. Or, because it encodes and expresses gpt), the addition of a selection agent (eg G418 or mycophenolic acid) such that these targeted cells have a growth or survival advantage over cells that do not have an integrated expression cassette.

It is desirable for precisely targeted homologous recombinants to also use negative selection so that cells bearing only non-homologous integration of the transformed gene are selected against it. Typically, such negative selection should be done only by non-homologous recombination, using an expression cassette encoding the herpes simplex virus thymidine kinase gene (HSV tk) located within the transformed gene. This positioning is generally accomplished by linking the HSV tk expression cassette (or other negative selection cassette) distal to the recombination homology region, such that a double-crossing alternative recombination of the homology region is a positive selection expression cassette. Transfers to the chromosomal location, but the HSV tk gene (or other negative selection cassette) does not transfer to the chromosomal location. Ganciclovir, a nucleoside homologue that is preferentially toxic to cells expressing HSV tk, can be used as a negative selection agent because it selects cells that do not have an integrated HSV tk expression cassette. . FIAU can also be used as a selective agent for selecting cells lacking HSV tk.

To reduce the background of cells with incorrectly integrated targeting construct sequences, combinatorial positive-negative selection schemes are 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) positive selection cassettes (eg neo genes) that can be stably expressed after random integration or homologous targeting; And (2) negative selection cassettes (eg, HSV tk genes) that can be stably expressed only after random integration and cannot be expressed after precisely targeted double-cross homologous recombination. By combining the positive and negative selection steps, a host cell with precisely targeted homologous recombination between the transformed gene and the endogenous p62 gene can be obtained.

In general, the targeting construct preferably comprises (1) a positive selective expression cassette flanked by two homology regions substantially identical to the host cell endogenous p62 gene sequence; And (2) a distal negative selective expression cassette. However, targeting constructs comprising only positive selective expression cassettes may also be used. Typically, the targeting construct will contain a positive selection expression cassette comprising a neo gene linked to the bottom of the promoter, such as the HSV tk promoter or the pgk promoter (ie, towards the carboxy-terminus of the encoded polypeptide in translation read frame orientation). . More typically, the targeted transformed gene will also contain a negative selection expression cassette comprising the HSV tk gene linked to the bottom of the pgk promoter.

Typically, the targeting polynucleotides of the present invention have one or more homology regions of at least about 50 nucleotides in length, with homology regions of at least about 75 to 100 nucleotides in length, and about 200 to 2000 lengths. Homologous regions of the above nucleotides are more preferred, but the sequence homology between the homologous region and the targeted sequence and the base composition of the targeted sequence will determine the optimal and minimum homologous region length (ie, GC rich sequences are typically thermodynamic Will be more stable and will generally require shorter length homology regions). Thus, while both homologous region length and sequence homology can only be determined based on a particular predetermined sequence, the length of the homologous region should generally be at least about 50 nucleotides and is substantially dependent on the predetermined endogenous target sequence. It must be equivalent or substantially complementary thereto. Preferably, the homologous region is at least about 100 nucleotides in length and is identical or complementary to a predetermined target sequence in or on the p62 gene. Where it is desired to produce highly targeted homologous recombinants with high efficiency, it is preferred that the one or more homologous regions are homologous (ie, exhibit precise sequence identity with the cross target sequence (s) of the endogenous p62 gene) and It is even more desirable to flank the exogenous targeting construct sequence where the homologous homologous region should replace the targeted endogenous p62 sequence.

The p62 sequence can be scanned for possible sites of disruption. The plasmid is engineered to contain a construct replacement sequence of the appropriate size indicating deletion or insertion in the p62 gene, and one or more flanking homology regions substantially corresponding to or substantially complementary to the endogenous target DNA sequence. Typically, two flanking homology regions are used, which reside on each side of the replacement region sequence.

The p62 gene is inactivated by homologous recombination in pluripotent cell lines that can differentiate into germ cell tissue. A DNA construct as discussed above containing a modified copy of the mouse p62 gene is introduced into the nucleus of an ES cell. In some parts of these cells, the introduced DNA is recombined with the endogenous copy of the mouse p62 gene, replacing it with a modified copy. Cells containing the newly engineered genetic lesions are injected into host mouse embryos and transplanted into recipient females. Some of these embryos evolve into chimeric mice that contain germ cells derived from transformant cell lines. Thus, by breeding such chimeric mice, it is possible to obtain new mouse strains containing the genetic lesions introduced above.

Vectors containing the targeting construct are typically E. coli. Growth in E. coli can then be isolated using standard molecular biology methods or synthesized as oligonucleotides. Direct targeting inactivation may also be performed that does not require prokaryotic or eukaryotic vectors. Targeting constructs can be transferred to host cells by any suitable technique, including microinjection, electroporation, lipofection, biostick, calcium phosphate precipitation, and virus-utilizing vectors. Other methods used to transform mammalian cells include polybrene, protoplast fusion, and the like. Sambrook et al. Molecular Cloning: A Laboratory Manual, 2d ed., 1989, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y .; Incorporated herein by reference.

p62  Screening Methods for Interactivity Factors

Numerous methods for screening candidate factors for interacting with the p62 gene or gene product are well known in the art and one skilled in the art will be able to determine if a particular compound is useful in the present invention.

The agents can be selected and screened randomly, or can be reasonably selected or reasonably designed using protein modeling techniques.

For random screening, randomly select agents, including but not limited to peptides, carbohydrates, steroids or vitamin derivatives, and determine their ability to bind to the p62 gene or gene product present in a mouse or cell line described herein. To this end, the formulation is assayed using direct or indirect methods conventional in the art. Alternatively, the agent can be assayed to see how to adjust for aging-related disorders.

Conventional assays can be used to screen compounds to determine their effect on p62 gene expression and p62 protein function. For example, transient expression / gel delay systems can be used to study formulation effects such as synthetic steroids or natural herbal extracts.

In another screening assay, transgenic animals (eg mice) and cell lines in which the expression level of one or more p62 genes are modified can be made and used to identify factors that modulate the expression and function of the p62 gene product. . In such assays, the agent to be tested can be incubated with one or more transgenic cell lines or mice or tissues derived therefrom. The level of binding of the agent is then determined by techniques conventional to those skilled in the art or the effect of the agent on biological effects or gene expression is monitored. The term “incubation” as used herein refers to contacting the compound or agent under study with appropriate cells or tissues, or to any of the well-known modes of administration including enteral, intravenous, subcutaneous and intramuscular administration. The formulation or compound is defined as being administered to a suitable animal, eg, a transgenic mouse, via either.

Other assays include immunological techniques using antibodies specific for the p62 fusion protein, such as immunofluorescence or immunoelectron microscopy. p62 interactivity may be identified by their ability to modulate in vitro aging related phenotype. Such screened agents may be, but are not limited to, peptides, carbohydrates, steroids, herbal extracts and vitamin derivatives.

Molecules capable of modulating (inducing, enhancing, decreasing or abrogating p62-positive inclusions) express p62 by treating cells with proteasome inhibitors or oxidative stress (including ROS generators or redox modifiers). Can be screened in cells or cell lines.

Detection of changes in the p62 locus of the human genome, including deletions, translocations, mutations or single nucleotide polymorphisms, can be used to detect p62-defect related diseases such as obesity, type 2 diabetes, various cancers and neurodegenerative diseases, And susceptibility to premature death of males can be predicted or diagnosed.

Drug Screening Assays to Treat Aging-Related Diseases

Animals of the invention can be used as test (tester) animals for substances of interest, such as antioxidants (such as vitamin E), which are believed to provide protection against the development of aging-related disorders. Treatment of animals with substances of interest and a reduction in the incidence of or delay in the onset of aging-related disorders compared to untreated animals are detected as indicative of protection. Indicators preferably used are those which can be detected in living animals. Efficacy can be confirmed as an effect on pathological changes in the event of death or sacrifice of an animal. Animals of the invention can further be used as test animals for substances of interest that are believed to ameliorate or treat aging-related disorders or diseases. For example, animals with diseases related to the redox state of the body can be treated with substances of interest, delayed death or weight, fat accumulation, or glucose absorption / Improvements in utilization are detected to indicate improvement or treatment of the disease.

Through the use of such transgenic animals or cells derived therefrom, one can identify ligands or substrates that mediate phenomena associated with aging-related disorders. Of particular interest are assays for screening agents with low toxicity to human cells.

To this end, various assays can be used, including behavioral studies, local determination of drugs after administration, amyloid deposition, and the like. Depending on the specific assay, complete animals or cells derived therefrom can be used. The cells can be freshly isolated from the animal or immortalized in culture. Of particular interest are cells derived from neural tissue.

Candidate formulations encompass many chemical classes, but they are typically organic molecules, preferably small organic molecules having a molecular weight of at least 50 Daltons to less than about 2,500 Daltons. Candidate agents include functional groups necessary for structural interaction with the protein, in particular hydrogen bonds, and typically comprise at least two of at least amine, carbonyl, hydroxyl or carboxyl groups, preferably functional chemical groups. . Candidate agents often include cyclic carbon or heterocyclic structures and / or aromatic or polyaromatic structures, which are substituted by one or more of these 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 homologs, or combinations thereof.

Candidate preparations are obtained from a wide variety of sources, including libraries of synthetic or natural compounds. For example, numerous methods for the randomized and directed synthesis of a wide range of organic compounds and biomolecules can be used, including expressing random oligonucleotides and oligopeptides. On the other hand, libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts can be used or readily produced. In addition, naturally or synthetically produced libraries and compounds are readily modified through conventional chemical, physical and biochemical means, which can be used to generate combinatorial libraries. Known pharmacological agents can be subjected to directed or random chemical modifications such as acylation, alkylation, esterification, amidation and the like to produce structural homologs.

For example, detection may utilize staining of cells or histological sections, performed according to conventional methods. The antibody of interest is added to the cell sample and incubated for a sufficient time, typically about 10 minutes or more, to bind the epitope. The antibody can be labeled with radioisotopes, enzymes, phosphors, chemilumines, or other labels for direct detection. Alternatively, a second step antibody or reagent is used to amplify the signal. Such reagents are well known in the art. For example, primary antibodies can be conjugated to biotin, with horseradish peroxidase-conjugated avidin added to the second stage reagent. Final detection utilizes a substrate that undergoes color change in the presence of peroxidase. The absence or presence of antibody binding can be determined by a variety of methods including cell flow counting, microscopy, radiographs, scintillation counts, and the like of dissociated cells.

Numerous assays are known in the art to determine the effect of a particular drug on animal behavior and other phenomena associated with aging-related disorders. Although some examples are provided, those skilled in the art will recognize that many other assays may be used. The subject animal can be used as is or in combination with a control animal.

Screens using transgenic animals of the present invention can utilize any phenomenon associated with age-related diseases that can be readily assessed in animal models. Preferably, this screen will contain a control value.

Therapeutic Composition

In one aspect, the present invention relates to the treatment of various diseases characterized as aging-related disorders as defined herein. In this way, a therapeutic compound of the invention may be used to treat a patient suffering from disorders such as premature death of obesity, obesity, diabetes, cancer, especially liver cancer, fatty liver, and Paget's disease of the pock that harbor p62 homozygous mutations. It can be administered to.

Formulations of therapeutic compositions are generally known in the art and may be conveniently referenced by Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing Co., Easton, Pa., USA. For example, about 0.05 μg to about 20 mg may be administered per kg of body weight per day. Dosage regimens may be adjusted to provide the optimum therapeutic response. For example, it may be administered daily in several portions or the dose may be gradually reduced as indicated by the urgency of the therapeutic situation. The active compound may be used in conventional manner, eg, by oral, intravenous (if water-soluble), intramuscular, subcutaneous, nasal, intradermal or suppository route or by implantation (eg intraperitoneal route or by cells, eg For example, by using sustained-release molecules by using monocytes or dendritic cells sensitized in vitro and proton immunotransferred to the recipient). Depending on the route of administration, it may be desirable to coat the peptide with a substance to protect it from the action of enzymes, acids and other natural conditions that may inactivate these components.

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

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

Pharmaceutical formulations suitable for injectable use include sterile aqueous solvents (if water soluble) or dispersants and sterile powders for immediate preparation with sterile injectable solutions or dispersants. In all cases, the form must be sterile and must be fluid to the extent that injection is easy. 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, for example, a dispersion medium or solvent containing water, ethanol, polyols (such as glycerol, propylene glycol and liquid polyethylene glycols, etc.), suitable mixtures thereof, and vegetable oils. For example, proper fluidity can be maintained by using a coating agent such as lecithin, and in the case of a dispersant, by using a surfactant while maintaining the required particle size. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, chlorobutanol, phenol, sorbic acid, theomesal 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 achieved by use in compositions which delay absorption, for example, compositions of aluminum monostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the active compound in the required amount in an appropriate solvent with the various other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersants are prepared by incorporating the various sterilizable active ingredients into a sterile vehicle that contains the basic dispersion medium and the other ingredients required from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, preferred methods of preparation are freeze-drying techniques and vacuum drying techniques which yield in addition to the active ingredient the additional desired component from its sterile-filtered solution as described above.

If the peptide is properly protected as mentioned above, the active compound can be administered orally, for example with an inert diluent or an assimilable edible carrier, or it can be enclosed in a hard or soft shell gelatin capsule, or It may be compressed into tablets or it may be incorporated with dietary foods. For oral therapeutic administration, the active compound may be incorporated with excipients and used in the form of ingestible tablets, ball 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 percentages of the compositions and formulations may, of course, vary and may conveniently be from about 5 to about 80 weight percent based on unit weight. 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 invention are prepared so that oral dosage unit forms contain from about 0.1 μg to 2000 mg of the active compound.

Tablets, pills, capsules, and the like may contain the following ingredients: binders such as tragacand gum, acacia gum, corn starch or gelatin; Excipients such as dicalcium phosphate; Disintegrants such as corn starch, potato starch, alginic acid and the like; Lubricants such as magnesium stearate; And sweetening agents such as sucrose, lactose or saccharin, or flavoring agents such as peppermint, oil of wintergreen or cherry flavor. When the dosage unit form is a capsule, it may contain a liquid carrier in addition to the above types of substances. Various other materials may be present as coatings or may be present to modify the physical form of the dosage unit. For example, tablets, pills or capsules may be coated with shellac, sugar or both. Syrups or elixirs may contain the active compound, sucrose as a sweetening agent, methyl and propylparabens as preservatives, dyes and flavoring agents such as cherry or orange flavors. Of course, the materials used to prepare any dosage unit form must be pharmaceutically pure and substantially nontoxic in the amounts employed. In addition, the active compounds may be incorporated into sustained release formulations and formulations.

It is especially advantageous to formulate parenteral compositions in dosage unit form in order to facilitate administration and homogeneous dosage. Dosage unit form as used herein refers to physically discrete units suitable as a single administration to a mammalian subject to be treated; Each unit contains a predetermined amount of active substance estimated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. Details of dosage unit forms of the invention include (a) the unique characteristics of the active substance and the particular therapeutic effect to be achieved; And (b) directly and in accordance with the limitations inherent in the field of combining active substances to treat diseases in living subjects with diseases impairing physical health.

The main active ingredient is combined for convenient and effective administration in an effective amount with a pharmaceutically acceptable carrier suitable for the dosage unit form. Unit dosage forms may contain, for example, an active ingredient in an amount of 0.5 μg to about 2000 mg. Expressed in proportions, the active compound is generally present at about 0.5 μg per ml of carrier. In the case of compositions containing supplementary active ingredients, the dosage will be determined with reference to the usual dosages and modes of administration of these ingredients.

Delivery system

Various delivery systems are known and can be used to administer the compounds of the invention, including, for example, liposomes, microparticles, microcapsules, encapsulation in the synthesized cells capable of expressing the compounds, receptor- Mediated endocytosis, construction of nucleic acids as part of retroviral or other vectors, and the like. Methods of introduction include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, nasal, epidural and oral routes. The compound or composition may be administered by all conventional routes, for example by infusion or bolus injection, absorption through epithelial or mucosal linings (eg, oral mucosa, rectal and intestinal mucosa, etc.), and other biologically active agents. It can be administered together with. Administration can be systemic or local. It may also be desirable to introduce the pharmaceutical compounds or compositions of the present invention into the central nervous system by any suitable route, including indoors and intravesicles; Indoor injection can be facilitated by, for example, an indoor catheter attached to a reservoir (eg, Ommaya reservoir). For example, pulmonary administration may be performed using an inhaler or nebulizer and using a formulation with an aerosolized formulation.

In certain embodiments, it may be desirable to administer the pharmaceutical compounds or compositions of the invention locally to the area in need of treatment; This can be achieved, for example, but not limited to, by topical injection during surgery, topical application associated with postoperative wound dressing, injection, via catheter, by suppository, or by using an implant. The sieve is a porous, non-porous or gelatinous material, including membranes (eg sialic membranes) and fibers. Preferably, when administering a protein comprising an antibody or peptide of the present invention, care should be taken to use substances which are not absorbed by such proteins. In another embodiment, the compounds or compositions of the invention can be delivered in endoplasmic reticulum, especially liposomes. In another embodiment, a compound or composition of the present invention can be delivered to a controlled release system. In one aspect, a pump may be used. In another embodiment, polymeric materials can be used. In another embodiment, only a portion of the systemic dose may be needed because the controlled release system can be located proximate to the therapeutic target, ie the brain.

Diagnosis of Aging-Related Diseases

The present invention also includes comparing wild-type genes encoding p62 with p62 genes in mammalian subjects, to premature death of aging-related disorders such as obesity, type II diabetes, liver carcinoma, males in such mammalian subjects. It relates to a method for facilitating the diagnosis of sensitivity. Detection of point mutations, single nucleotide polymorphisms, deletions, frameshift mutations, translocations, truncation or disruption of genes, causing loss of function or loss of gene function, indicates that subject mammals are at risk of developing aging-related disorders or diseases. . Molecular biology methods for identifying and comparing sequences are well known in the art.

The invention also relates to a method for identifying an aging related disorder or disease caused or associated with somatic mutations in the gene encoding p62. In one non-limiting example, the method

i. providing a sequence of p62 gene product;

ii. Identifying the sequence of the mutant p62 protein;

iii. Preparing a probe for a gene encoding the mutant protein or fragment thereof; And

iv. Probing a biological sample from a subject with the aforementioned disorder or disease and a biological sample from a patient without the disease, wherein a mutant protein is present in the biological sample from the patient with the disease, and The absence of a mutant protein in a biological sample from a non-mammalian subject indicates that the disease or susceptibility to such disease is caused or associated with a somatic mutation in the gene.)

It includes.

The present invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention other than those described herein will be apparent to those skilled in the art from the foregoing and the accompanying drawings. Such modifications fall within the scope of the appended claims. The following examples are provided to illustrate the invention and are not so limited.

Example  One - p62  Preparation of Knockout Mice

A. p62  Isolation of Genomic Clones

Human p62 genes are isolated as set forth in US Pat. No. 6,291,645, which is incorporated herein by reference in its entirety, which describes cloning and sequencing of the p62 gene. Genomic DNA clones corresponding to the p62 locus in mice are cloned from the RPCI 22 mouse BAC library prepared from mouse species 129 / svEvTACCfBr (Invitrogen).

B. p62  knockout Targeting  Manufacture of vector

To generate the nonfunctional allele of p62, a gene targeting vector is prepared as shown in FIG. The results of p62 genotyping of litters from Southern blot and immunoblot analysis of extracts from wild-type or non-functional p62 mouse organs, as well as through PCR analysis of the litters derived from heterozygous heterologous crosses, indicated that the targeted genes were disrupted. Indicates. The targeting vector is constructed by inserting 2.3 kb EcoRI-BamHI amplified from the 5 'end of the p62 genomic clone into the EcoRI-BamHI site of pPNT. A 4.0 kb SspI fragment derived from the 3 'end of the p62 genomic clone is inserted into the vector at the XhoI site blunt ended with the Klenow enzyme. Potential recombinants are identified by recombinant fragment assays for gene targeting based on PCR. Genomic DNA is prepared from ES cells, chimeric tissues and their progeny. The digested DNA is electrophoresed on an agarose gel, a Southern blot of the gel is prepared, and hybridized with ³² P-labeled probes specific for the 5 'or 3' flanking region of the p62 gene.

C. ES  Insertion of target vector in cells

The resulting plasmid is linearized with SspI and electroporated into E14TG2a ES cells. By selection in the presence of G418 and gancyclovir, heterozygous ES cell clones are obtained. Genomic DNAs are isolated from ES cells, and the mouse-tail is characterized by Southern blot analysis. Probes are prepared from the 3 'region of the p62 genomic sequence. This probe hybridizes with an 11 kb fragment (endogenous p62 gene) and with an 8 kb fragment (collapsed allele). Electroporation is performed on HPRT-deficient embryonic stem cell line E14TG2a. The cell line was maintained on mouse embryonic fibroblasts examined as described previously. Electroporation in the presence of a linearized targeting construct at a concentration of 2-5 nM used a 1-second discharge from a 250-uF capacitor charged to 300V. Linearization of both plasmid constructs is achieved using the restriction enzyme NotI. Selection is accomplished using G418 and gancyclovir.

D. Homozygous p62 -/- No function  Generation of mouse

Cysts are secreted from 6-7 week old C57BL6 / J. Two to three days after passage, freshly trypsinized embryonic stem (ES) cells are resuspended in M2 medium and introduced into vesicles by microinjection (10-15 cells per bladder bag) at room temperature. The injected bladders are returned to the uterus of a fertility female mated with a male previously presecrated male 2.5 days ago without further incubation.

Pups derived from blastocysts injected with p62-4 cells are judged to exhibit coat color chimeric phenomena by examination. The species 129 / Ola mice that induce the E14TG2a cell line and thus the p62-4 cell line carry mutations at three loci, namely A, c and p, which are known to affect coat color. 129 / Ola mice are homozygous for the dominant allele Aw at the agouti locus, homozygous for the recessive allele at the c locus, and homozygous for the recessive allele p at the p locus Is; These are creamy white with pink eyes. Species C57BL6 / J mice are a source of recipient vesicles; These are dark black colors with black eyes. Chimeras between these two species are identified by the presence of brighter fur patches on a black background. The patch color varies depending on whether the epidermal layer is ES cell-derived or recipient vesicle-derived. Aguti patches show indirect inhibitory effects of the dominant Aw gene in ES cell-induced mesodermal cells on black pigment production by melanocytes. Slightly yellow patches show a direct effect of the Cch and p genes on the production and packaging of the pigment itself. Aguti and / or yellow patches can be found in certain chimeric animals. Animals that are strongly chimeric to coat color may be creamy with black eyes or a nearly homogeneous yellowish brown. The proportion of chimeras thus produced is 28% of the offspring living. Pups are scored for coat color chimeric phenomena and mated when maturated to test for transfer of coat color markers from p62-4 to offspring.

E. Homozygous p62 -/-Mouse phenotype

(i) obesity

3 shows photographs of representative wild type and p62-functional mice. Mice without p62-function are obese. 4 depicts the amount of fat deposition in wild type mice and p62-functional mice.

FIG. 5 depicts population studies in terms of body weight of wild-type (black square) and p62-functional (white square) male mice of various ages. This once again indicates that p62-null mice are generally more obese than wild type mice. In another aspect, FIG. 6 depicts a cohort study measuring the growth of wild type and p62-functional male mice from weaning (4 weeks) until 30 years of age. Curves are generated that indicate that p62-functional mice grow to greater body weight from birth to 30 weeks of age.

Female mice were also studied for weight and growth. FIG. 7 depicts population studies in terms of body weight of wild type (black squares) and p62-functional (white squares) female mice of various ages. This indicates that female mice without p62-function are generally more obese than wild type female mice. FIG. 8 depicts a cohort study measuring growth of wild type and p62-functional female mice from weaning (4 weeks) until 30 years of age. Figure 9 depicts weight gain in wild type and p62-functional female mice at 20 and 40 weeks of age. Brain-to-weight ratios are the same in wild-type and p62-functional mice.

Total cholesterol levels in blood were also measured. FIG. 11 shows total cholesterol levels in blood after 12 hours fasting in wild type and p62-null female mice at 20 and 40 weeks of age. Total cholesterol levels in blood are 134 ± 23 mg / ml and 187 ± 33 mg / ml in 20-week-old wild-type and p62-null mice, respectively. Total cholesterol levels in blood in the 40-week-old wild type and p62-null mice were 135 ± 31 mg / ml and 175 ± 11 mg / ml, respectively.

Blood triglyceride levels were measured. 12 depicts blood triglyceride levels after 12 h fasting in wild type and p62-null female mice at 20 and 40 weeks of age. Serum triglyceride levels are 67 ± 13 mg / ml and 81 ± 10 mg / ml in 20-week-old wild-type and p62-functional mice, respectively. Serum triglyceride levels in 40-week-old wild-type and p62-null mice were 76 ± 17 mg / ml and 96 ± 16 mg / ml, respectively.

Finally, serum leptin levels were measured. FIG. 13 depicts serum leptin levels after 12 h fasting in wild type and p62-null female mice. Serum leptin levels are 5.00 ± 0.84 mg / ml in wild-type mice and 8.37 ± 0.84 mg / ml in p62-functional mice. Thus, mice without p62-function show signs of obesity.

All p62-/-litters have a similar appearance. At birth, p62 − / − mice appear to be indistinguishable from their compatriots. Differences in the size and appearance of the p62-/-mice were observed during the first few weeks of life.

( ii ) diabetes

Blood glucose levels were measured. FIG. 10 shows blood glucose levels after 12 h fasting in wild type and p62-null female mice at 20 and 40 weeks of age. Blood glucose levels are 160 ± 19 mg / ml and 240 ± 30 mg / ml, respectively, in 20-week-old wild-type and p62-functional mice. Blood glucose levels in the 40-week-old wild-type and p62-functional mice were 147 ± 49 mg / ml and 241 ± 33 mg / ml, respectively.

* Mice were measured for signs of diabetes. FIG. 14 shows serum insulin levels after 12 h fasting in wild type and p62-null female mice. Serum insulin levels are 84.60 ± 0.70 mg / ml in wild-type mice and 144.30 ± 47.30 mg / ml in p62-functional mice, significantly higher in p62-functional females. In addition, FIG. 15 depicts glucose levels during the glucose tolerance test in 28-week-old wild type and p62-functional female mice, suggesting that blood glucose levels are higher after 2 hours in knockout female mice. This also indicates that glucose is not readily processed in p62-functional mice. FIG. 16 shows elevated glucose levels during insulin resistance testing in 20-week-old p62-functional male mice compared to wild type mice.

Examination of pancreatic islet size and morphology in 20-week-old wild type and p62-functional female mice indicates that p62-functional female mice have larger sized pancreatic islets. 17A-17B show pictures of pancreatic islets. In addition, FIG. 18 depicts urine glucose testing in wild type and p62-functional mice. Glucose test patches are indicated by arrows and turn green in p62-unfunctional mice. Mice with no p62-function displayed higher amounts of glucose excreted in the urine. All of these phenotypes are typical for the development of human or mouse type 2 diabetes.

( iii A) fatty liver

Mice without p62-function show a fatty liver phenotype. FIG. 19 depicts hepatomegaly and steatosis in mice without p62-function. In addition, FIGS. 20A-20B depict lipoatosis in p62-functional mice. Tissue sections were stained with H & E.

( iv Liver cancer

In addition to the above, the test results for mice show that mice without p62-function suffer from liver cancer. 21A-21D depict hepatocellular carcinoma in 67-week-old p62-functional male mice. 22A-22B depict hepatocellular carcinoma in 36-week-old p62-null male mice. 23A-23B depict hepatocellular carcinoma in 59-week-old p62-functional male mice.

(v) Male  Premature death

An interesting phenotype associated with p62-functional mice is that they indicate male premature death. FIG. 24 depicts Kaplan-Meier survival curves for male mice in wild type group (black) and p62 knockout group (red). Male p62-null mice have a much shorter lifespan than wild type mice. FIG. 25 depicts Kaplan-Meier survival curves for female mice in wild type group (black) and p62 knockout group (red). These curves show that the life spans of female mice without p62-function are approximately the same.

( vi ) Intracellular  Formation of inclusion bodies

Multiubiquitin binds to p62. 26A-26C show multiubiquitin chain binding to p62 and Mcb1. (A) SDS-PAGE for purified recombinant Arabidopsis Mcb1 (Mcb1), GST fused p62 ubiquitin binding region (GST.P62C259), and GST, and transfer to nitrocellulose. The membranes are stained with Ponceau S (left panel) or incubated with ¹²⁵ I-labeled multiubiquitin chains, washed and self-recorded (right panel). (B) GST or GST.p62C259 incubated with ¹²⁵ I-labeled multiubiquitin chains in solution, the bound chains precipitated using glutathione coupled agarose beads, and then the amount of bound chains Measure by counting. (C) eluting ¹²⁵ I-labeled multiubiquitin chains precipitated in (B) from the beads by heating in SDS-containing buffer; This eluate is separated by SDS PAGE and the profile of the multiubiquitin chain is analyzed by autoradiography. A mixture of ¹²⁵ I-labeled multiubiquitin chains is included for comparison (free chain).

p62 interacts with the ubiquitin-protein conjugate that is endogenous to HeLa cells. FIG. 27 depicts the interaction of p62 with the ubiquitin-protein conjugate that is endogenous to HeLa cells. HeLa cells expressing p62.T7 are incubated in the presence or absence of lactacystin and then lysed. p62.T7 was immunoprecipitated from the lysate using anti-T7 monoclonal antibody and immunoprecipitated from co-precipitated p62 and ubiquitin-protein conjugates followed by polyclonal anti-p62 and ubiquitin-conjugate antiserum Detection by immunoblot.

The ubiquitin binding domain of p62 is described by neural network methods. 28A-28B depict the amino acid sequence (SEQ ID NO: 1) of the ubiquitin binding domain of (A) p62. The neural network method was used to predict the secondary structure of the ubiquitin binding domain of p62. The predicted α-helix region is underlined. (B) The predicted α-helix region is depicted on the helical wheel. Hydrophobic residue is in the box.

Several of these mutant p62 proteins were targeted for binding to the multiubiquitin chain. Table 1 shows multiubiquitin chain binding to mutant p62 protein (p62 protein was tested as GST fusion bound to G-beads). Purified recombinant GST fused p62 ubiquitin binding region (GST.P62C259), GST and various p62 mutant proteins were incubated with ¹²⁵ I-labeled multiubiquitin chains in solution and the bound chains were glutathione coupled agar. Precipitate using rose beads and then measure the amount of bound chain by γ-counting. The results indicate that hydrophobic residues in the ubiquitin chain binding region are important for binding to the ubiquitin chain.

Mutant Bound multiubiquitin chain cpm (%) None ^a 90 (0%) p62 5214 (100%) L ₃₉₄ / L ₃₉₅ → A / A 877 (17%) M ₄₀₁ / L ₄₀₂ → A / A 69 (0%) W ₄₁₂ / L ₄₁₃ → A / A 108 (0%) L ₄₁₆ / L ₄₁₇ → A / A 63 (0%) a: sample contains only G-beads

p62 is not associated with proteasomes. 29A-29C show that p62 is not associated with proteasomes. (A) 26S proteasome enhanced fractions were separated by differential centrifugation of total lysates from HeLa cells. 5 μg of each fraction was used for the peptidase assay and TBP1. (B) 26S proteasomes were purified from P100-2h fractions on DEAE Affi-gel blue column. 20 μl each was used for peptidase assay and Western blot. (C) The 26S proteasome was further purified using a 15% -40% continuous glycerol gradient. Fractions (500 μl) were collected from the top of this gradient and 20 μl each was used for peptidase assay and immunoblot. Other 26S proteasome subunits (ATPase p42, S4.p45 and 20S core complex subunit p31) were also found in the same fraction as TBP1 by immunoblot analysis (data not shown).

p62 localizes simultaneously to the multiubiquitin chain after treatment with proteasome inhibitors. FIG. 30 depicts co-localization of p62 with multi-ubiquitin chains after treatment with LLnL in HEK 293 cells. HEK 293 cells were treated with indicated increasing concentrations of LLnL for 16 hours prior to harvesting. Cells are lysed and immunostained with anti-p62 and anti-ubiquitin antibodies. FIG. 31 shows that various proteasome inhibitors can induce the formation of sequestosomes. COS7 cells are treated with several proteasome inhibitors (20 mM ALLN, 5 mM lactacystin, 10 mM MG132 and 50 nM epoxmycin) for 12 hours and immunostained with anti-p62 antibody. In addition, FIGS. 32A-32B show that p62 forms a cytosolic inclusion construct named sequestosome by the proteasome inhibitor LLnL. (A) p62 accumulates in sequetosomes by treatment with LLnL. COS7 cells are incubated for 12 hours in the presence of 20 mM LLnL and treated with antibodies to p62 for immunofluorescence microscopy. (B) The level of p62 is increased in the detergent-insoluble fraction by exposure to LLnL. COS7 cells were incubated in the presence of carrier (DMSO) or 20 mM LLnL for 12 hours before harvesting. Cells are lysed and separated into Triton X-100 soluble and insoluble fractions. By reprobing the blot with an anti-b-actin antibody, an equivalent load sample was confirmed.

In addition, FIGS. 33A-33B illustrate time-dependent p62-sequestosome formation. (A) COS7 cells are incubated in the presence of 20 mM LLnL for the indicated times and immunostained with an antibody against p62. (B) shows rise of p62 in detergent-insoluble fraction over time. After treatment with 20 mM LLnL for the indicated times, COS7 cells are harvested, lysed and separated into detergent-soluble and -insoluble fractions. By reprobing the blot with an anti-b-actin antibody, an equivalent load sample was confirmed.

Ubiquitin-p62 inclusion bodies are formed in mouse embryonic fibroblasts. FIG. 34 depicts ubiquitin positive p62 inclusion body formation in mouse embryonic fibroblasts (MEFs). MEF cells are incubated for 12 hours in the presence of 20 mM LLnL and immunostained with anti-p62 and ubiquitin antibodies. In addition, FIG. 35 depicts p62 gene dose-dependent UB-inclusion formation. MEFs from p62 wild type, heterozygous or nonfunctional embryos are incubated for 12 hours with or without LLnL (20 mM) and immunostained with anti-ubiquitin antibody. p62 wild-type MEFs well formed ubiquitin positive inclusion bodies, whereas p62-functional MEFs could not form inclusion bodies at all, and p62-heterozygotes could form inclusion bodies less efficiently. FIG. 36 depicts p62 overexpression in p62 − / − MEF rescue ubiquitin inclusion bodies formed by proteasome inhibitors. MEFs from embryos without p62 function are transfected with p62 expression vectors. After 24 hours, these cells are incubated for 12 hours in the presence of LLnL (20 mM) and immunostained with anti-p62 and anti-ubiquitin antibodies.

In addition, p62-positive intracellular inclusion bodies are also formed by oxidative stress. For example, FIGS. 37A-37B depict the formation of p62- or ubiquitin aggregates in MEFs in the presence of glutathione (GSH) -depleting diethylmaleate (DEM). MEFs are incubated for 12 hours in the presence or absence of DEM. p62 wt (A) and p62 nonfunctional (B) MEFs are immunolabeled with antibodies to p62 and ubiquitin. DEMs can induce ubiquitin-positive inclusion bodies only in p62-wild-type MEFs, but not in p62-functional MEFs. Thus, p62 has the function of collecting misfolded and / or ubiquitinated intracellular proteins and making intracellular inclusion bodies.

38A-38B depict activation of the p62 gene in response to proteasome inhibitor LLnL. (A) Cell lysates are analyzed by treating COS7 cells with 20 mM LLnL for the indicated times and immunoblotting with anti-p62 antibodies. Equivalent load was confirmed by anti-beta-actin immunoblotting. (B) COS7 cells are treated with 20 mM LLnL in the absence (left upper panel) or presence (actual and upper right panel) of actinomycin D. Total RNA is extracted from untreated and treated cells and analyzed for equivalent amounts (10 mg) of RNA. Equivalent loads of RNA were identified by staining 28S and 18S ribosomal RNA with ethidium bromide (bottom panel). Since the rate of extinction for p62 mRNA with or without actinomycin in the presence of LLnL is the same, increasing p62 levels by proteasome inhibition is due to activation of the p62 gene.

P62 activation is required to form sequestosomes (FIGS. 39A-39B). (A) COS7 cells are treated with 20 mM LLnL for 12 hours in the presence or absence of cycloheximide and immunostained with an antibody against p62. (B) COS7 cells are exposed to 20 mM LLnL for the time indicated in the presence (top panel) or absence (cyclopanel) of cycloheximide. Cells so treated are harvested, lysed and separated into 0.5% Triton X-100 soluble and insoluble fractions. These lysates are immunoblotted using anti-p62 antibodies. When cells were treated with proteasome inhibitors in the presence of a detoxification inhibitor, cycloheximide, no cellular inclusion bodies were formed. Under these conditions, p62 cellular levels did not increase, and in particular, little p62 in the detergent insoluble fraction was detectable. Therefore, efficient p62 protein synthesis is required for this inclusion body formation following activation of the p62 gene, and the protein in the inclusion body is expected to be insoluble in washing such as Triton X-100.

( vi ) ALS in p62  Role of Benign Inclusions

p62 inclusion bodies are found in ALS model mice. 40A-40B depict ALS model mice showing p62 inclusion bodies in their lumbar spinal cord. Transgenic mice expressing (A) wild type human SOD1 or (B) ALS-related mutant human SOD1 (G93A) genes were sacrificed at 16 weeks of age, and their lumbar spinal cord regions were stained with hematoxylin and anti-p62 antibodies. Let's do it. p62 was mainly detected in motor neurons in the ventral horn region of mutant transgenic mouse spinal cord slices. In contrast, little or no staining with the antibody was observed in the reprinted regions of wild-type transgenic mouse sections. Figure 41 depicts p62 inclusion bodies in the lumbar spinal cord of ALS mice. Transgenic mice expressing wild-type human SOD1 or ALS-related mutant human SOD1 (G93A) genes are sacrificed at age 16 and their lumbar spinal cord regions are stained with hematoxylin and anti-p62 antibodies. p62 immunostaining was used to detect the nucleus inclusions of p62. Additionally, FIGS. 42A-42B show that the p62 inclusion bodies in ALS are ubiquitin positive. Transgenic mice expressing wild-type human SOD1 or ALS-related mutant human SOD1 (G93A) genes were sacrificed at 16 weeks of age, and their lumbar spinal cord regions were stained with (A) anti-p62 antibody and (B) anti- Co-staining with p62 antibody and anti-ubiquitin antibody.

p62 localizes and co-precipitates simultaneously with mutant SOD1 (G93A) (FIGS. 43A-43B). (A) COS7 cells are transfected with flag tagged SOD1 or SOD1G93A mutant expressing plasmids. After 24 hours, these cells are immunostained with anti-flag antibody and anti-p62 antibody with or without treatment with 20 μΜ LLnL for 12 hours. (B) The flag tagged SOD1 or SOD1G93A mutant expressing plasmids are co-transfected with T7 tagged p62 expressing plasmids. These cells are incubated for 12 hours with or without the proteasome inhibitor LLnL (20 mM) and immunoprecipitated with anti-T7 antibody. Precipitated proteins are analyzed by immunoblotting using anti-flag antibodies. The results show that p62 binds directly to the mutant but not to wild type SOD-1, and that p62 and mutant SOD-1G93A are simultaneously localized in the same inclusion body. Because p62 drives intracellular inclusion body formation, inclusion bodies found in various neurodegenerative diseases, including ALS, are expected to be formed by p62.

( viii ) Redox  biochemistry

In addition to the above, FIGS. 44A-44B depict the effect of glutathion depletion on cell viability in p62 + / + and p62 − / − mouse embryonic fibroblasts (MEFs). Cell viability is assessed using the methylthiazolium (MTT) assay, which measures the mitochondrial dehydrogenase activity of living cells by spectrophotometry. MEFs (3 × 10 ³ cells / well) in 96-well plates were treated with different concentrations of DEM (A) or BSO (B), washed with PBS and then 4 hours with 0.5 mg / ml MTT in PBS. Incubate for a while. To solubilize the formazan product, 100 ml DMSO is added. The absorbance at 570 nm is determined. The results show that when glutathione is depleted, p62-functional MEFs die more easily than wild-type MEFs. The death of these p62-functional MEFs is an apoptotic process (FIG. 48). Thus, p62 is believed to have a function of regulating cellular redox status and / or cellular reactive oxygen species (ROS) generation and scavenging system.

The cell death mode was measured. 48A-48B depict annexin V-binding assays in p62 + / + and p62 − / − mouse embryonic fibroblasts (MEFs) 6 hours after exposure to DEM. Annexin V Cy3 was used to assess phosphatidylserine membrane translocation. To identify necrotic and apoptotic cells that also exhibit the translocation of phosphatidylserine, 6-carboxyfluorescein diacetate (6-CFDA) was used in combination with Ann V-Cy3. Non-fluorescent 6-CFDA enters the cell and is converted to the fluorescent compound 6-carboxyfluorescein (6-CF). This conversion is a function of esterases that exist only in living cells. Thus, no fluorescence can be produced in necrotic cells. According to fluorescence microscopy, 6-CF is observed as green fluorescence and Ann V-Cy3 is observed as red fluorescence. p62 wt (A) and p62 null (B) MEFs were incubated on poly-L-lysine-coated slides, treated with diethylmaleate (DEM, 50 mM) for 6 hours and in the presence of calcium Ann Vcy3 / Stain with 6-CFDA solution and incubate for 10 min in the dark. After excess fluorescent compound is washed, the slide is capped and immediately examined with a fluorescence microscope. Annexin V positive cells were immediately found in DEM treated p62-functional MEFs. Thus, the degradation of cell viability of p62-functional MEFs is due to apoptotic cell death.

Experiments are performed to detect reactive oxygen species in p62 + / + and p62 − / − mouse embryonic fibroblasts (MEFs) as measured by DCF fluorescence (FIG. 45). Cultures are treated with or without diethylmaleate (DEM, 50 mM) or butionine sulfoximine (BSO, 1 mM). Cells were harvested after 12 hours, incubated at 37 ° C. for 30 minutes in the presence of 10 mM oxidation-sensitive probe DCFH-DA, followed by flow cytometry. Comparable flow cytometry distributions of DCF fluorescence intensity are shown. The histogram represents the number of cells as a function of fluorescence intensity expressed as the number of channels. The results show that much larger amounts of ROS accumulated in p62-functional MEFs, even without any treatment.

Total glutathione in MEF was measured. FIG. 46 depicts total glutathione concentrations in p62 + / + and p62 − / − mouse embryonic fibroblasts (MEFs). Cultures are treated with or without diethylmaleate (DEM, 50 mM) for 12 hours. The concentration of GSH in MEF (4 × 10 ⁵ cells in 5% metaphosphate) was determined by the colorimetric assay kit according to the manufacturer's instructions. Known amounts of GSH were used to generate standard curves. The results show that only about 60% of glutathione (GSH) is available in p62-functional MEFs compared to wild-type cells, and that DEM treatment further reduces cellular GSH levels in p62-functional MEFs.

In addition, FIG. 47 depicts oxidized proteins in p62 + / + and p62 − / − mouse embryonic fibroblasts (MEFs). Cultures are treated with or without diethylmaleate (DEM) for 12 hours. Oxyblot was achieved using 5 mg of cytosolic protein extracts from p62-wt and p62-null. The extract is treated with 2,4-dinitrophenylhydrazine and then submitted to SDS-PAGE on a 10% (w / v) polyacrylamide gel and transferred onto a nitrocellulose filter. The blot is treated with anti-DNP antibody as described by the manufacturer. As a result of decreased GSH levels and increased ROS in these cells, higher amounts of oxidized protein accumulated in p62-functional MEFs.

In addition to the above, FIGS. 49A-49B depict proteasome peptidase activity in p62 + / + and p62 − / − mouse embryonic fibroblasts (MEF). Cultures are treated with or without diethylmaleate (DEM, 50 mM) for 12 hours. Cells are harvested and homogenized in extraction buffer. The membrane and cellular debris are examined by centrifugation at 10,000 g and the soluble extract is recovered. The peptidase activity of the proteasome was assayed using the fluorogenic peptide LLVY-AMC (SEQ ID NO: 2) to determine chymotrypsin-like activity. Oxidative stress induced by treatment of DEM (ie, glutathione depletion) resulted in a more sensitive degradation of proteasome activity in p62-unfunctional cells. Taken together, p62 is an important cellular component in the defense against oxidative stress generated endogenously or externally. One of the defense mechanisms would be to collect p62 and sequester the misfolded protein (oxidized or ubiquitinated) into the intracellular inclusion body. On the other hand, the accumulation of the misfolded protein relative to cellular capacity will induce cell death, which is a pathological feature of neurodegeneration. In addition, the intrinsic imbalance of the redox system and the accumulation of ROS due to p62 dysfunction may be the cause of various phenotypes observed in mice without p62 function.

All references cited herein are hereby incorporated by reference in their entirety.

Those skilled in the art will recognize, or be able to ascertain many equivalent aspects of the specific embodiments of the invention specifically described herein using only routine experimentation. Such equivalent aspects are encompassed within the scope of the claims of the present invention.

Transgenic animals comprising a nonfunctional p62 gene according to the present invention, such transgenic animals will develop aging-related diseases, and are useful for screening of such diseases and researches and therapeutic agents.

The present invention has been presented by way of example only and will be better understood from the following detailed description and the accompanying drawings, which do not limit the invention.

1 depicts targeted disruption, targeting constructs, and homologous recombination of the p62 gene structure of the mouse p62 gene.

2A-2C show p62 genotyping analysis. (A) PCR analysis of litter induced heterozygous heterologous crosses: + / +, wild type; +/-, heterozygotes; -/-, Homozygous. (B) Southern blot analysis. (C) Immunoblot analysis of extracts from wild type or nonfunctional p62 mouse organs.

3 shows photographs of representative wild type and p62-functional mice.

4 depicts fat deposition in wild-type and p62-functional mice.

FIG. 5 depicts the weight of wild-type (black square) and p62-functional (white square) male mice at population study—various ages.

FIG. 6 shows growth curves of wild-type and p62-functional male mice from weaning (week 4) until cohort study—30 weeks old.

FIG. 7 depicts the weight of wild-type (black squares) and p62-functional (white squares) female mice at population study—various ages.

FIG. 8 depicts growth curves of wild type and p62-functional female mice from weaning (week 4) until cohort study—30th week of age.

FIG. 9 shows weight gain of wild type and p62-functional female mice at 20 and 40 weeks of age. Brain-to-weight ratios are the same in wild-type and p62-functional mice.

FIG. 10 shows blood glucose levels after 12 h fasting in wild type and p62-null female mice at 20 and 40 weeks of age. Blood glucose levels are 160 ± 19 mg / ml and 240 ± 30 mg / ml, respectively, in 20-week-old wild-type and p62-functional mice. Blood glucose levels in the 40-week-old wild-type and p62-functional mice were 147 ± 49 mg / ml and 241 ± 33 mg / ml, respectively.

FIG. 11 shows total cholesterol levels in blood after 12 hours fasting in wild type and p62-null female mice at 20 and 40 weeks of age. Total cholesterol levels in blood are 134 ± 23 mg / ml and 187 ± 33 mg / ml in 20-week-old wild-type and p62-null mice, respectively. Total cholesterol levels in blood in the 40-week-old wild type and p62-null mice were 135 ± 31 mg / ml and 175 ± 11 mg / ml, respectively.

12 depicts blood triglyceride levels after 12 h fasting in wild type and p62-null female mice at 20 and 40 weeks of age. Serum triglyceride levels are 67 ± 13 mg / ml and 81 ± 10 mg / ml in 20-week-old wild-type and p62-functional mice, respectively. Serum triglyceride levels in 40-week-old wild-type and p62-null mice were 76 ± 17 mg / ml and 96 ± 16 mg / ml, respectively.

FIG. 13 depicts serum leptin levels after 12 h fasting in wild type and p62-null female mice. Serum leptin levels are 5.00 ± 0.84 mg / ml in wild-type mice and 8.37 ± 0.84 mg / ml in p62-functional mice.

FIG. 14 shows serum insulin levels after 12 h fasting in wild type and p62-null female mice. Serum insulin levels are 84.60 ± 0.70 mg / ml in wild-type mice and 144.30 ± 47.30 mg / ml in p62-functional mice.

FIG. 15 depicts glucose levels during the glucose tolerance test in 28-week-old wild type and p62-functional female mice.

FIG. 16 depicts glucose levels during the insulin resistance test in 20-week-old wild type and p62-unfunctional male mice.

17A-17B depict pancreatic islet size and morphology in (A) 20-week-old wild type female mice and (B) p62-functional female mice.

FIG. 18 depicts urine glucose test in wild type and p62-functional mice. Glucose test patches are indicated by arrows and turn green in p62-unfunctional mice.

FIG. 19 depicts hepatomegaly and steatosis in p62-null mice.

20A-20B depict lipoatosis in mice without p62-function. Liver samples were obtained from (A) wild type and (B) mice without p62-function. Tissue sections were stained with H & E.

21A-21D depict hepatocellular carcinoma in 67-week-old p62-functional male mice.

22A-22B depict hepatocellular carcinoma in 36-week-old p62-null male mice.

23A-23B depict hepatocellular carcinoma in 59-week-old p62-functional male mice.

FIG. 24 depicts Kaplan-Meier survival curves for male mice in wild type group (black) and p62 knockout group (red).

FIG. 25 depicts Kaplan-Meier survival curves for female mice in wild type group (black) and p62 knockout group (red).

26A-26C show multiubiquitin chain binding to p62 and Mcb1. (A) SDS-PAGE for purified recombinant Arabidopsis Mcb1 (Mcb1), GST fused p62 ubiquitin binding region (GST.P62C259), and GST, and transfer to nitrocellulose. The membranes are stained with Ponceau S (left panel) or incubated with ¹²⁵ I-labeled multiubiquitin chains, washed and self-recorded (right panel). (B) GST or GST.p62C259 incubated with ¹²⁵ I-labeled multiubiquitin chains in solution, the bound chains precipitated using glutathione coupled agarose beads, and then the amount of bound chains Measure by counting. (C) eluting ¹²⁵ I-labeled multiubiquitin chains precipitated in (B) from the beads by heating in SDS-containing buffer; This eluate is separated by SDS PAGE and the profile of the multiubiquitin chain is analyzed by autoradiography. A mixture of ¹²⁵ I-labeled multiubiquitin chains is included for comparison (free chain).

FIG. 27 depicts the interaction of p62 with the ubiquitin-protein conjugate that is endogenous to HeLa cells. HeLa cells expressing p62.T7 are incubated in the presence or absence of lactacystin and then lysed. p62.T7 was immunoprecipitated from the lysate using anti-T7 monoclonal antibody and immunoprecipitated from co-precipitated p62 and ubiquitin-protein conjugates followed by polyclonal anti-p62 and ubiquitin-conjugate antiserum Detection by immunoblot.

28A-28B depict amino acid sequences of the ubiquitin binding domain of (A) p62. The neural network method was used to predict the secondary structure of the ubiquitin binding domain of p62. The predicted α-helix region is underlined. (B) The predicted α-helix region is depicted on the helical wheel. Hydrophobic residue is in the box.

29A-29C show that p62 is not associated with proteasomes. (A) 26S proteasome enhanced fractions were separated by differential centrifugation of total lysates from HeLa cells. 5 μg of each fraction was used for the peptidase assay and TBP1. (B) 26S proteasomes were purified from P100-2h fractions on DEAE Affi-gel blue column. 20 μl each was used for peptidase assay and Western blot. (C) The 26S proteasome was further purified using a 15% -40% continuous glycerol gradient. Fractions (500 μl) were collected from the top of this gradient and 20 μl each was used for peptidase assay and immunoblot. Other 26S proteasome subunits (ATPase p42, S4.p45 and 20S core complex subunit p31) were also found in the same fraction as TBP1 by immunoblot analysis (data not shown).

FIG. 30 depicts co-localization of p62 with multi-ubiquitin chains after treatment with LLnL in HEK 293 cells. HEK 293 cells were treated with indicated increasing concentrations of LLnL for 16 hours prior to harvesting. Cells are lysed and immunostained with anti-p62 and anti-ubiquitin antibodies.

FIG. 31 shows that various proteasome inhibitors can induce the formation of sequestosomes. COS7 cells are treated with several proteasome inhibitors (20 mM ALLN, 5 mM lactacystin, 10 mM MG132 and 50 nM epoxmycin) for 12 hours and immunostained with anti-p62 antibody.

32A-32B show that p62 forms a cytosolic inclusion construct named sequestosome by the proteasome inhibitor LLnL. (A) p62 accumulates in sequetosomes by treatment with LLnL. COS7 cells are incubated for 12 hours in the presence of 20 mM LLnL and treated with antibodies to p62 for immunofluorescence microscopy. (B) The level of p62 is increased in the detergent-insoluble fraction by exposure to LLnL. COS7 cells were incubated in the presence of carrier (DMSO) or 20 mM LLnL for 12 hours before harvesting. Cells are lysed and separated into Triton X-100 soluble and insoluble fractions. By reprobing the blot with an anti-b-actin antibody, an equivalent load sample was confirmed.

33A-33B depict time-dependent p62-Sequestosome formation. (A) COS7 cells are incubated in the presence of 20 mM LLnL for the indicated times and immunostained with an antibody against p62. (B) shows rise of p62 in detergent-insoluble fraction over time. After treatment with 20 mM LLnL for the indicated times, COS7 cells are harvested, lysed and separated into detergent-soluble and -insoluble fractions. By reprobing the blot with an anti-b-actin antibody, an equivalent load sample was confirmed.

FIG. 34 depicts ubiquitin positive p62 inclusion body formation in mouse embryonic fibroblasts (MEFs). MEF cells are incubated for 12 hours in the presence of 20 mM LLnL and immunostained with anti-p62 and ubiquitin antibodies.

35 depicts p62 gene dose-dependent UB-inclusion formation. MEFs from p62 wild type, heterozygous or nonfunctional embryos are incubated for 12 hours with or without LLnL (20 mM) and immunostained with anti-ubiquitin antibody.

FIG. 36 depicts p62 overexpression in p62 − / − MEF rescue ubiquitin inclusion bodies formed by proteasome inhibitors. MEFs from embryos without p62 function are transfected with p62 expression vectors. After 24 hours, these cells are incubated for 12 hours in the presence of LLnL (20 mM) and immunostained with anti-p62 and anti-ubiquitin antibodies.

37A-37B depict p62 aggregates formed in MEFs in the presence of a GSH-depletor. MEFs are incubated for 12 hours in the presence or absence of DEM. p62 wt (A) and p62 null (B) MEFs are immunolabeled with antibodies against p62 and ubiquitin. Bar = 10 μm.

38A-38B depict activation of the p62 gene in response to proteasome inhibitor LLnL. (A) Analyze cell lysates by treating COS7 cells with 20 mM LLnL for the indicated times and immunoblotting using anti-p62 antibodies. Equivalent load was confirmed by anti-beta-actin immunoblotting. (B) COS7 cells are treated with 20 mM LLnL in the absence (left upper panel) or presence (actual and upper right panel) of actinomycin D. Total RNA is extracted from untreated and treated cells and analyzed for equivalent amounts (10 mg) of RNA. Equivalent loads of RNA were identified by staining 28S and 18S ribosomal RNA with ethidium bromide (bottom panel).

39A-39B show that p62 activation is required to form sequestosomes. (A) COS7 cells are treated with 20 mM LLnL for 12 hours in the presence or absence of cycloheximide and immunostained with an antibody against p62. (B) COS7 cells are exposed to 20 mM LLnL for the time indicated in the presence (top panel) or absence (cyclopanel) of cycloheximide. Cells so treated are harvested, lysed and separated into 0.5% Triton X-100 soluble and insoluble fractions. These lysates are immunoblotted using anti-p62 antibodies.

40A-40B depict ALS model mice showing p62 inclusion bodies in their lumbar spinal cord. Transgenic mice expressing (A) wild type human SOD1 or (B) ALS-related mutant human SOD1 (G93A) genes were sacrificed at 16 weeks of age, and their lumbar spinal cord regions were stained with hematoxylin and anti-p62 antibodies. Let's do it. p62 was mainly detected in motor neurons in the ventral horn region of mutant transgenic mouse spinal cord slices. In contrast, little or no staining with the antibody was observed in the reprinted regions of wild-type transgenic mouse sections.

Figure 41 depicts p62 inclusion bodies in the lumbar spinal cord of ALS mice. Transgenic mice expressing wild-type human SOD1 or ALS-related mutant human SOD1 (G93A) genes are sacrificed at age 16 and their lumbar spinal cord regions are stained with hematoxylin and anti-p62 antibodies. p62 immunostaining was used to detect the nucleus inclusions of p62.

42A-42B show that p62 inclusion bodies in ALS are ubiquitin positive. Transgenic mice expressing wild-type human SOD1 or ALS-related mutant human SOD1 (G93A) genes were sacrificed at 16 weeks of age, and their lumbar spinal cord regions were stained with (A) anti-p62 antibody and (B) anti- Co-staining with p62 antibody and anti-ubiquitin antibody.

43A-43B show that p62 localizes and co-precipitates simultaneously with mutant SOD1 (G93A). (A) COS7 cells are transfected with flag tagged SOD1 or SOD1G93A mutant expressing plasmids. After 24 hours, these cells are immunostained with anti-flag antibody and anti-p62 antibody with or without treatment with 20 μΜ LLnL for 12 hours. (B) The flag tagged SOD1 or SOD1G93A mutant expressing plasmids are co-transfected with T7 tagged p62 expressing plasmids. These cells are incubated for 12 hours with or without the proteasome inhibitor LLnL (20 mM) and immunoprecipitated with anti-T7 antibody. Precipitated proteins are analyzed by immunoblotting using anti-flag antibodies.

44A-44B depict the effect of glutathion depletion on cell viability in p62 + / + and p62 − / − mouse embryonic fibroblasts (MEFs). Cell viability is assessed using the methylthiazolium (MTT) assay, which measures the mitochondrial dehydrogenase activity of living cells by spectrophotometry. MEFs (3 × 10 ³ cells / well) in 96-well plates were treated with different concentrations of DEM (A) or BSO (B), washed with PBS and then 4 hours with 0.5 mg / ml MTT in PBS. Incubate for a while. To solubilize the formazan product, 100 ml DMSO is added. The absorbance at 570 nm is determined.

45 depicts ROS detection in p62 + / + and p62 − / − mouse embryonic fibroblasts (MEFs) as measured by DCF fluorescence. Cultures are treated with or without diethylmaleate (DEM, 50 mM) or butionine sulfoximine (BSO, 1 mM). Cells were harvested after 12 hours, incubated at 37 ° C. for 30 minutes in the presence of 10 mM oxidation-sensitive probe DCFH-DA, followed by flow cytometry. Comparable flow cytometry distributions of DCF fluorescence intensity are shown. The histogram represents the number of cells as a function of fluorescence intensity expressed as the number of channels.

FIG. 46 depicts total glutathione concentrations in p62 + / + and p62 − / − mouse embryonic fibroblasts (MEFs). Cultures are treated with or without diethylmaleate (DEM, 50 mM) for 12 hours. The concentration of GSH in MEF (4 × 10 ⁵ cells in 5% metaphosphate) was determined by the colorimetric assay kit according to the manufacturer's instructions. Known amounts of GSH were used to generate standard curves.

FIG. 47 depicts oxidized proteins in p62 + / + and p62 − / − mouse embryonic fibroblasts (MEFs). Cultures are treated with or without diethylmaleate (DEM) for 12 hours. Oxyblot was achieved using 5 mg of cytosolic protein extracts from p62-wt and p62-null. The extract is treated with 2,4-dinitrophenylhydrazine and then submitted to SDS-PAGE on a 10% (w / v) polyacrylamide gel and transferred onto a nitrocellulose filter. The blot is treated with anti-DNP antibody as described by the manufacturer.

48A-48B depict annexin V-binding assays in p62 + / + and p62 − / − mouse embryonic fibroblasts (MEFs) 6 hours after exposure to DEM. Annexin V Cy3 was used to assess phosphatidylserine membrane translocation. To identify necrotic and apoptotic cells that also exhibit the translocation of phosphatidylserine, 6-carboxyfluorescein diacetate (6-CFDA) was used in combination with Ann V-Cy3. Non-fluorescent 6-CFDA enters the cell and is converted to the fluorescent compound 6-carboxyfluorescein (6-CF). This conversion is a function of esterases that exist only in living cells. Thus, no fluorescence can be produced in necrotic cells. According to fluorescence microscopy, 6-CF is observed as green fluorescence and Ann V-Cy3 is observed as red fluorescence. p62 wt (A) and p62 null (B) MEFs were incubated on poly-L-lysine-coated slides, treated with diethylmaleate (DEM, 50 mM) for 6 hours and in the presence of calcium Ann Vcy3 / Stain with 6-CFDA solution and incubate for 10 min in the dark. After excess fluorescent compound is washed, the slide is capped and immediately examined with a fluorescence microscope.

49A-49B depict proteasome peptidase activity in p62 + / + and p62 − / − mouse embryonic fibroblasts (MEFs). Cultures are treated with or without diethylmaleate (DEM, 50 mM). Cells are harvested and homogenized in extraction buffer. The membrane and cellular debris are examined by centrifugation at 10,000 g and the soluble extract is recovered. The peptidase activity of the proteasome was assayed using the fluorogenic peptidase LLVY-AMC to determine chymotrypsin-like activity.

<110> SHIN, Jaekyoon          LEE, Hanwoong          OH, Gootaek <120> A transgenic mouse for diagnosis or treatment of age-related          disorder or disease comprising a konck-out p62 gene and a method          of screening for a compound that counters age-related disorder or          disease using the same <150> US60 / 378159 <151> 2002-05-14 <160> 2 <170> KopatentIn 1.71 <210> 1 <211> 77 <212> PRT <213> Homo sapiens <400> 1 Pro Ser Ser Leu Asp Pro Ser Gln Glu Gly Pro Thr Gly Leu Lys Glu   1 5 10 15 Ala Ala Leu Tyr Pro His Leu Pro Pro Glu Ala Asp Pro Arg Leu Ile              20 25 30 Glu Ser Leu Ser Gln Met Leu Ser Met Gly Phe Ser Asp Glu Gly Gly          35 40 45 Trp Leu Thr Arg Leu Leu Gln Thr Lys Asn Tyr Asp Ile Gly Ala Ala      50 55 60 Leu Asp Thr Ile Gln Tyr Ser Lys His Pro Pro Pro Leu  65 70 75 <210> 2 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> Fluorogenic peptides <400> 2 Leu Leu Val Tyr   One  

Claims (7)

Somatic cells and germ cells include the knocked-out p62 gene, wherein the knocked-out gene is introduced into a mouse ancestor into a mouse or embryo, and is homozygous for the knocked out gene, Transgenic mice showing diseases selected from obesity, diabetes, fatty liver, Paget's disease of bone (PDB) and premature death of males. The transgenic mouse of claim 1, wherein the knocked out p62 gene is knocked out by the targeting vector of FIG. 1. A mouse embryonic stem cell having a genome comprising a heterozygous or homozygous knockout p62 gene, which can be a transgenic mouse of claim 1. The mouse embryonic stem cell of claim 2, wherein the knockout p62 gene is knocked out by the targeting vector of FIG. 1. A mouse somatic cell isolated from the transgenic mouse of claim 1 and having a genome comprising a heterozygous or homozygous knockout p62 gene. The mouse somatic cell of claim 5, wherein the knockout p62 gene is the p62 targeting vector of FIG. 1. (a) treating a mouse somatic cell (treatment somatic cell) according to claim 5 with the test compound to be screened; (b) measuring the level of disorder after treating the compound; And (c) comparing the disorder level measured in (b) with the disorder level in untreated (untreated somatic cells) without the test compound as a control, A method of screening for a compound corresponding to an aging-related disorder or disease in a mammal, wherein the reduction in aging-related disorder level in (b) compared to the control indicates that the compound is against aging-related disorder.

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