Patent Grant
7622271
The present invention relates to methods for identifying genes that confer longevity, methods for utilizing the identified genes to screen pharmacological agents useful for extending life spans, and related compositions comprising identified gene sequences.
In general, the life span of an organism is defined by measuring its chronological age. Alternatively, the life span of yeasts can be determined by measuring the number of mitotic divisions completed by a mother cell prior to senescence, defined as the replicative life span (“RLS”). To determine the RLS for a yeast mother cell, each daughter cell needs to be physically removed from a mother cell after each mitotic cycle. Yeast strains that are genetically predisposed to a long life span are identified, for example, by measuring a mean RLS for a statistically reliable number of mother cells for each strain, and by comparing the determined mean RLS value to a mean RLS value of a reference population. Alternatively, life spans can be measured by determining the median RLS value observed for a given strain, or by determining the maximum RLS value observed for a strain.
The labor-intensive nature of the RLS assay and the vast number of individual RLS determinations that need to be performed for a statistically significant set of each variant in a library have precluded attempts to comprehensively approach the identification of aging mutations by implementing a genome-wide characterization. For example, because laboratory yeast strains are highly variable, approximately 40 to 50 cells of each genotype are evaluated in order to determine a statistically reliable mean RLS for a particular strain of interest. When 50 cells of each genetic variant from a hypothetical genomic library containing 5,000 variants are used to determine the mean RLS, then a total number of 250,000 RLS determinations would need to be performed. Determinations of such large numbers of RLSs is impractical, and, therefore, comprehensive analysis of a large collection of strains representing variants of a yeast genome have not been carried out.
Prior to the present invention, RLS determinations have been made utilizing yeast strains of highly disparate backgrounds, including different short-lived strains that have a mean RLS less than the mean RLS of other wildtype yeast strains. Since laboratory yeast strains are highly divergent at the genomic level, many mutations identified from these variant strains may affect RLS in a strain-specific manner. For example, mutations that increase the life span of short-lived strains may result from suppression or reversion events that compensate for strain-specific mutations, and such longevity-promoting mutations may be specific to only that particular strain.
Analysis of yeast variants that are predisposed to longevity can yield previously uncharacterized genes that confer long life spans. An improved method for efficiently evaluating a large collection of genetic variants with differential life spans in order to identify genes that confer longevity is highly desirable. Gene products that regulate the life spans of eukaryotes can be targeted by pharmaceutical agents in order to decrease the rate of aging. Pharmaceutical agents and methods for screening such pharmaceutical agents that can increase the life expectancy of mammals, including humans, are highly desirable. In addition, by slowing the rate of aging, it may be possible to delay the onset of various diseases/conditions associated with aging, including various types of cancers, diabetes, cataracts, heart diseases, and neurodegenerative diseases, such as Parkinson's disease, Huntington disease, and amyloid diseases.
In one aspect, the present invention provides a high throughput method for screening genetic variants that are phenotypically distinguishable. Various embodiments of the present invention are directed to methods for identifying “long-lived” genetic variants among a set of variants. Each variant of a set is evaluated, in turn, to determine whether the variant exhibits a long life span based on a phenotypic measurement. Long-lived variants identified by various methods of the present invention enable the identification of life-span-regulating genes conserved among eukaryotes.
In another aspect, the present invention relates to methods for identifying pharmaceutical compounds that are useful for prolonging the life spans of mammals and for delaying the onset of various mammalian diseases. Various vectors and host cells containing identified genes and gene products of the present invention are useful as an assay for screening compounds that can suppress, inactivate, or modulate the expression of identified genes. In another aspect, the present invention relates to pharmaceutical compositions that can modulate the activities of identified genes/gene products of the present invention.
The present invention therefore provides nucleic acids encoding replicative life span proteins. The invention therefore provides methods of screening for variants. The invention further provides compounds, e.g., small organic molecules, antibodies, peptides, lipids, peptides, cyclic peptides, nucleic acids, antisense molecules, RNAi molecules, and ribozymes, that are capable of modulating replicative life span genes and gene products, e.g., inhibiting replicative life span genes. Therapeutic and diagnostic methods and reagents are also provided.
In one aspect, a method for screening and for sorting a set of genetic variants to determine whether a genetic variant within the set of genetic variants exhibits a phenotype of interest, the method comprising: providing a set of genetic variants; iteratively for each genetic variant in the set of genetic variants, selecting a number N of cells for the genetic variant; quantitatively measuring a phenotype for the N cells of the genetic variant; classifying the genetic variant as positive, negative, or ambiguous based on quantitatively measuring the phenotype; and removing the genetic variant from the set of genetic variants, when the genetic variant is classified as positive or negative; and incrementing a number of iterations until either the number of iterations equals a maximum number of iterations or the set of genetic variants is empty. In some aspects, the N is same for each variant. In other aspects, the N is variable for different variants selected. In another aspect, the classifying the variant further comprises: establishing a positive threshold value; establishing a negative threshold value; comparing the phenotypic measurement to the positive threshold value and to the negative threshold value; evaluating whether the determined phenotypic measurement is greater than the positive threshold value, or less than the negative threshold value; classifying the variant as positive when the determined phenotypic measurement is greater than the positive threshold value; classifying the variant as negative when the determined phenotypic measurement is less than the negative threshold value; and classifying the variant as ambiguous when the determined phenotypic measurement is not greater than the positive threshold value nor less than the negative threshold value. In some aspects, the measuring a phenotype is determining a mean replicative life span for N cells of the variant. In some such aspects, the negative threshold value is established by determining the mean life span value of a statistically reliable set of a wildtype reference, and wherein the positive threshold value is established by determining the mean life span value of a statistically reliable set of variants exhibiting a life span substantially greater than that of the wildtype reference. In some methods, the sample size N is an integer greater than 3 and less than 20, when the variant is a yeast strain containing a mutation that affects the expression of at least one gene, and wherein the maximum number of iterations is between 2 and 5. In some aspects, the positive variant has a mean replicative life span substantially greater than the mean replicative life span of a wildtype reference, and wherein the negative variant has a mean replicative life span less than the mean replicative life span of the wildtype reference. In some such methods, the mean replicative life span of the positive variant is at least about 20% greater than the mean replicative life span of the wildtype reference. In some such aspects, the determining the N further comprises minimizing the misclassification of variants that further includes: minimizing the classification of a positive variant having a mean replicative life span substantially greater than that of a wildtype reference as a negative variant; and minimizing the classification of a negative variant having a mean replicative life span less than that of the wildtype reference as a positive variant. In some aspects, measuring a phenotype further comprises: establishing a first dataset that includes replicative life span values for N cells of a variant, wherein the replicative life span value for each cell of N is included; establishing a second dataset by selecting a subset of the first dataset, wherein the second dataset includes highest replicative life span values observed for the first dataset; determining a mean replicative life span from values included in the second dataset; and utilizing the determined mean replicative life span as the phenotypic measurement for the classification of variants. In some aspects, the classifying the variant further comprises: iteratively for each variant of the set, computing a mean replicative life span for each variant; computing a median replicative life span for the set of variants; computing an average median mean replicative life span for the set; and normalizing the mean replicative life span for each variant. In other such aspects, the normalizing further includes multiplying the computed mean replicative life span for each variant by a coefficient value, wherein the coefficient value is computed by dividing the median replicative life span for the set of variants by the average median mean replicative life span for the set.
In another aspect, the invention provides a method for identifying genes having life-span-regulating activity, the method comprising: identifying a variant having substantially greater life span than the life span of a wildtype reference, according to the method of described herein; and identifying a gene having life-span-regulating activity from the variant.
In another aspect, the invention provides a method for inhibiting the activity of a replicative life span protein, wherein the replicative life span protein is a gene product of a gene set forth in Table 5 or ortholog thereof, or a fragment thereof, the method comprising binding an inhibitor to the replicative life span protein. In some aspects the replicative life span genes identified by the methods of the invention include BRE5, FOB1, IDH2, REI1. ROM2, RPL31A, RPL6B, TOR1, YBR238C, YBR255W, YBR266C, YOR135C, SCH9, or URE2 or any ortholog thereof, or fragment thereof.
In another aspect, the invention provides a vector comprising: a sequence having a life-span-regulating activity, and encoding a polypeptide that has at least about 40% sequence similarity to at least one at least one sequence for a gene indicated in Table 5 or ortholog thereof; and a promoter operably-linked to the sequence. In some aspects, the sequence hybridizes to at least one of the sequences for the genes indicated in Table 5 or ortholog thereof, or complementary sequences of at least one sequence for a gene indicated in Table 5 or ortholog thereof, under moderately stringent hybridization conditions. In some such aspects the sequence is mammalian. In other such aspects, the sequence comprises at least one of sequence for a gene indicated in Table 5 or ortholog thereof. In some aspects, a host cell comprising the vector discussed above.
In another aspect, the invention provides a method for identifying a compound that prolongs a life span of a host, the method comprising: providing a set of target molecules that includes one or more sequences having life-span-regulating activity, and the target molecules having at least about 40% sequence similarity to the sequences for the genes indicated in Table 5 or 6, or having a complementary sequence to molecules that have 40% sequence similarity the sequences for the genes indicated in Table 5 or 6; exposing a library of compounds to the set of target molecules; determining an experimental value correlating with the extent of a biochemical reaction between the compound and the target molecule; comparing the experimental value against a pre-established threshold value; and determining that the compound has a longevity-promoting activity when the experimental value exceeds the pre-established threshold value. In some aspects, the target molecules have at least about 70% sequence similarity to the sequences for the genes indicated in Table 5 or 6, or is a complementary sequence to molecules having 70% sequence similarity to the sequences for the genes indicated in Table 5 or 6.
In another aspect, the invention provides a method for identifying a compound that prolongs a life span of a host, the method comprising: providing a first eukaryotic host deficient in the expression of sir2 and fob1; determining a life span of the first host that has, not been exposed to a test compound; exposing the test compound to a second eukaryotic host of the same genotype as the first host; determining a life span of the second host that has been exposed to the test compound; comparing the life spans of the first and second hosts; and determining that the compound has a longevity-promoting activity when the life span of the second host exceeds the life span of the first host.
In another aspect, the invention provides a compound that increases a life span of a host, the compound comprising an oligonucleotide that interacts with a gene having at least about 40% sequence similarity to at least one of the sequences for the genes indicated in Table 5 or 6, or a gene having at least 70% sequence similarity to at least one the sequences for the genes indicated in Table 5 or 6. In some aspects, the oligonucleotide interacts with a gene product encoded by the gene having at least about 40% sequence similarity to at least one of the sequences for the genes indicated in Table 5 or 6. In some such aspects, the oligonucleotide interacts with a gene product encoded by the gene having at least about 70% sequence similarity to at least one of the sequences for the genes indicated in Table 5 or 6. In other such aspects, the compound is at least one of: a single-stranded DNA oligonucleotide, double-stranded DNA oligonucleotide, a single-stranded RNA oligonucleotide, double-stranded RNA oligonucleotide, and modified variants of these. In some such aspects, the compound includes an anti-sense strand that hybridizes to an endogenous messenger RNA that encodes a protein having life-span-regulating activity, and that inhibits the translation of the messenger RNA.
In another aspect, the invention provides an antibody that increases a life span of a host, the antibody comprising: an antigen-binding domain that reacts with a polypeptide having at least about 40% sequence similarity to at least one least one of the sequences for the genes indicated in Table 5 or 6; and a constant region. In some aspects, the antigen-binding domain reacts with a polypeptide having at least about 70% sequence similarity to at least one least one of the sequences for the genes indicated in Table 5 or 6.
In another aspect, the invention provides a ribozyme that increases a life span of a host, the ribozyme comprising a sub-sequence that is complementary to a target molecule encoded by a gene having at least about 40% sequence similarity to at least one least one of the sequences for the genes indicated in Table 5 or 6. In some aspects, the target molecule is encoded by a gene having at least about 70% sequence similarity to at least one least one of the sequences for the genes indicated in Table 5 or 6.
In another aspect, the invention provides a pharmaceutical composition comprising: a compound as described herein; and a pharmaceutical carrier.
In another aspect, the invention provides a method for extending the life span of a eukaryotic organism, the method comprising administering to a subject, a pharmaceutical composition of as described herein containing an effective dose of a compound determined to have longevity-promoting activity.
In another aspect, the invention provides a non-human transgenic animal that exhibits a life span longer than a non-transgenic reference animal, and that has a genome comprising an inactivated or suppressed endogenous gene having at least about 40% sequence similarity to at least one least one of the sequences for the genes indicated in Table 5 or 6, or a gene having at least about 70% sequence similarity to at least one least one of the sequences for the genes indicated in Table 5 or 6.
In another aspect, the invention provides a method of evaluating the effect of a replicative life span bioactive agent comprising: a) administering the bioactive agent to a mammal; b) removing a cell sample from the mammal; and c) determining the expression profile of the cell sample. In some aspects, the method further comprises comparing the expression profile to an expression profile of a healthy individual. In other aspects, the expression profile includes at least one BRE5, FOB1, IDH2, REI1. ROM2, RPL31A, RPL6B, TOR1, YBR238C, YBR255W, YBR266C, YOR135C, SCH9, or URE2 gene, or ortholog thereof.
In another aspect, the invention provides an array of probes, comprising a support bearing a plurality of nucleic acid probes complementary to a plurality of mRNAs fewer than 1000 in number, wherein the plurality of mRNA probes includes an mRNA expressed by at least one BRE5, FOB1, IDH2, REI1. ROM2, RPL31A, RPL6B, TOR1, YBR238C, YBR255W, YBR266C, YOR135C, SCH9, or URE2 gene, or ortholog thereof. In some aspects, the invention provides a replicative mRNA expressed by akt-1, a homolog of sch9. In some aspects, the probes are cDNA sequences. In some such aspects, the array comprises a plurality of sets of probes, each set of probes complementary to subsequences from a mRNA.
In another aspect, the invention provides a biochip comprising one or more nucleic acid segments encoding the genes as shown in Table 5 or ortholog thereof, or a fragment thereof, wherein the biochip comprises fewer than 1000 nucleic acid probes. In some aspects, probes are cDNA sequences. In some aspects, biochip comprises a plurality of sets of probes, each set of probes complementary to subsequences from a mRNA.
In another aspects, the invention provides a replicative life span nucleic acid having a sequence at least 95% homologous to a sequence of a nucleic acid of Table 5 or ortholog thereof, or its complement. In some aspects, the a vector is provided comprising the nucleic acid molecule described above. In other aspects, the invention provides an isolated host cell comprising the vector described above.
In another aspect, the invention provides a method for producing a replicative life span protein, the method comprising the steps of: a) culturing the host cell as described above under conditions suitable for the expression of the polypeptide; and b) recovering the polypeptide from the host cell culture. In some aspects the host cell is a eukaryotic cell. In other aspects the host cell is a prokaryotic cell.
I. Introduction
The invention provides a number of methods, reagents, and compounds that can be used either for the treatment of a replicative life span disease or disorder or a disease or disorder associated with aging (e.g., various types of cancers, diabetes mellitus, cataracts, heart diseases, and neurodegenerative diseases, such as Alzheimer's disease, Pick's disease, Huntington's disease, Parkinson's disease, adult onset myotonic dystrophy, multiple sclerosis, and adult onset leukodystrophy disease), the development of treatments for life span disorders or related disorders (e.g., various types of cancers, diabetes mellitus, cataracts, heart diseases, and neurodegenerative diseases, such as Alzheimer's disease, Pick's disease, Huntington's disease, Parkinson's disease, adult onset myotonic dystrophy, multiple sclerosis, and adult onset leukodystrophy disease), the practice of the other inventive methods described herein, or for a variety of other purposes.
It is to be understood that this invention is not limited to particular methods, reagents, compounds compositions or biological systems, which can, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting. As used in this specification and the appended claims, the singular forms “a”, “an” and “the” include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to “a cell” includes a combination of two or more cells, and the like.
“About” as used herein when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ±20% or ±10%, more preferably ±5%, even more preferably ±1%, and still more preferably ±0.1% from the specified value, as such variations are appropriate to perform the disclosed methods.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Although any methods and materials similar or equivalent to those described herein can be used in the practice for testing of the present invention, the preferred materials and methods are described herein. In describing and claiming the present invention, the following terminology will be used.
“Replicative life span,” abbreviated as “RLS,” refers to a total number of mitotic divisions completed by a cell of interest prior to senescence or death. The determination of replicative life span is one measure of a yeast life span. An “RLS assay” is typically used to determine “replicative life spans.” Replicative capacity of mammalian cell cultures can be estimated in vitro by enumerating the number of population doublings completed by a population of cells prior to mitotic arrest. Typically, cells of a known population are cultured under conditions that encourage cell division. For example, one method for determining life span of mammalian cultures is to count the number of times that such a population that has been reduced to half-density can mitotically divide to produce the original population density.
“Replicative life span protein” or “RLS” protein or fragment thereof, or nucleic acid encoding “replicative life span” or “RLS” or a fragment thereof refer to nucleic acids and polypeptide polymorphic variants, alleles, mutants, and interspecies homologs that: (1) have an amino acid sequence that has greater than about 40% amino acid sequence identity, 45%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, preferably 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or greater amino acid sequence identity, preferably over a region of at least about 25, 50, 100, 200, 500, 1000, or more amino acids, to an amino acid sequence encoded by a RLS nucleic acid or amino acid sequence of an RLS protein, e.g., a RLS protein as shown in Table 5 or ortholog as shown Table 6; (2) specifically bind to antibodies, e.g., polyclonal antibodies, raised against an immunogen comprising an amino acid sequence of a RLS protein, e.g., a RLS protein as shown in Table 5 or ortholog as shown Table 6, immunogenic fragments thereof, and conservatively modified variants thereof; (3) specifically hybridize under stringent hybridization conditions to an anti-sense strand corresponding to a nucleic acid sequence encoding a RLS protein, e.g., RLS protein (Table 5) or ortholog as shown Table 6, and conservatively modified variants thereof; (4) have a nucleic acid sequence that has greater than about 40% sequence identity, 45%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, preferably 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, or higher nucleotide sequence identity, preferably over a region of at least about 25, 50, 100, 200, 500, 1000, or more nucleotides, to a RLS nucleic acid, e.g., a RLS protein as shown in Table 1 or ortholog as shown Table 2.
A RLS polynucleotide or polypeptide sequence is typically from a mammal including, but not limited to, primate, e.g., human; rodent, e.g., rat, mouse, hamster; cow, pig, horse, sheep, or any mammal. Other RLS polynucleotide or polypeptide sequences are from other organisms, including yeast (e.g., Saccharomyces cerevisiae; also referred to as S. cerevisiae), worms, and insects. The nucleic acids and proteins of the invention include both naturally occurring or recombinant molecules.
The terms “RLS” protein or a fragment thereof, or a nucleic acid encoding “RLS” protein or a fragment thereof refer to nucleic acid and polypeptide polymorphic variants, alleles, mutants, and interspecies homologs that: (1) have an amino acid sequence that has greater than about 40% amino acid sequence identity, 45%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, preferably 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or greater amino acid sequence identity, preferably over a region of over a region of at least about 25, 50, 100, 200, 500, 1000, or more amino acids, to an amino acid sequence encoded by as shown in Table 5 or Table 6; (2) specifically bind to antibodies, e.g., polyclonal antibodies, raised against an immunogen comprising an amino acid sequence encoded by a RLS protein as shown in Table 5 or ortholog as shown Table 6, immunogenic fragments thereof, and conservatively modified variants thereof; (3) specifically hybridize under stringent hybridization conditions to an anti-sense strand corresponding to a nucleic acid sequence encoding a RLS protein, e.g., a RLS protein as shown in Table 5 or ortholog as shown Table 6, or their complements, and conservatively modified variants thereof; (4) have a nucleic acid sequence that has greater than about 40% sequence identity, 45%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, preferably 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, or higher nucleotide sequence identity, preferably over a region of at least about 25, 50, 100, 200, 500, 1000, or more nucleotides, to the genes and their representative sequences as a RLS protein as shown in Table 5 or ortholog as shown Table 6 or their complements.
Exemplary replicative life span genes are listed in Table 5 and interspecies orthologs for these genes are listed in Table 6.
The phrases “identified sequences of the present invention” and “identified genes of the present invention” are used interchangeably in this disclosure. Such “identified sequences” are identified by methods of the present invention, which include genes and gene products; fragments of genes and gene products; sequences of genes and gene products, and any modifications of these genes and gene products that have life-span-regulating function. Such “identified sequences” that regulate eukaryotic life spans, includes yeast genes/gene products identified in “long-lived” variants, and related orthologous genes/gene products having conserved life-span-regulating activity. Such “identified sequences” include yeast gene sequences, corresponding yeast polypeptide sequences, and mammalian orthologous gene sequences; and corresponding mammalian polypeptide sequences, as can be determined by the referenced genes in Table 5 and 6 disclosed herein. Exemplary set of mammalian orthologs is provided in Tables 6 and in Example 3.
“Ortholog” refers to an evolutionarily conserved bio-molecule represented in a species other than the organism in which a reference sequence is identified, and contains a nucleic-acid or amino-acid sequence that is homologous to the reference sequence. To determine the degree of homology between a reference sequence and a sequence in question, two nucleic-acid sequences or two amino-acid sequences are compared. Homology can be defined by percentage identity or by percentage similarity. Percentage identity correlates with the proportion of identical amino-acid residues shared between two sequences compared in an alignment. Percentage similarity correlates with the proportion of amino-acid residues having similar structural properties that is shared between two sequences compared in an alignment. Percentages of similarity and identity can be calculated over a portion of the primary structure and not over the entire gene/protein sequence. For example, amino-acid residues having similar structural properties can be substituted for one another, such as the substitutions of analogous hydrophilic amino-acid residues, and the substitution of analogous hydrophobic amino-acid residues. Percentages of similarity and identity can be calculated over a portion of the primary structure and not over the entire gene/protein sequence. For the present disclosure, an ortholog or an orthologous sequence is defined as a homologous molecule or a sequence having life-span-regulating activity and a sequence identity of at least about 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, and 95%. Alternatively, an ortholog is defined as a homologous molecule or sequence having life-span-regulating activity and a sequence similarity of at least about 40%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, and 95%.
It is further contemplated that “ortholog” is a polypeptide or nucleic acid molecule of an organism that is highly related to a reference protein, or nucleic acid sequence, from another organism. An ortholog is functionally related to the reference gene, protein or nucleic acid sequence. In other words, the ortholog and its reference molecule would be expected to fulfill similar, if not equivalent, functional roles in their respective organisms. It is not required that an ortholog, when aligned with a reference sequence, have a particular degree of amino acid sequence identity to the reference sequence. A protein ortholog might share significant amino acid sequence identity over the entire length of the protein, for example, or, alternatively, might share significant amino acid sequence identity over only a single functionally important domain of the protein. Such functionally important domains may be defined by genetic mutations or by structure-function assays. Orthologs can be identified using methods provided herein. The functional role of an ortholog may be assayed using methods well known to the skilled artisan, and described herein. For example, function might be assayed in vivo or in vitro using a biochemical, immunological, or enzymatic assay; transformation rescue, or for example, in a nematode bioassay for the effect of gene inactivation on nematode phenotype. Alternatively, bioassays may be carried out in tissue culture; function can also be assayed by gene inactivation (e.g., by RNAi, siRNA, or gene knockout), or gene over-expression, as well as by other methods. Exemplary orthologs for the genes of the invention are shown in Table 6.
“Paralogs” are distinct but structurally related proteins made by an organism. Paralogs are believed to arise through gene duplication.
The GenPept accession number for bre5 is CAA96332.1, and GenBank accession number for exemplary nucleotide and amino acid sequences is Z71666.1.
The GenPept accession number for fob1 is CAA88664.1, and GenBank accession number for exemplary nucleotide and amino acid sequences is Z48758.1.
The GenPept accession number for idh2 is CAA99335.1, and GenBank accession number for exemplary nucleotide and amino acid sequences is Z75043.1.
The GenPept accession number for rei1 is CAA85229.1, and GenBank accession number for exemplary nucleotide and amino acid sequences is Z36135.1. (see, e.g., Feldmann et al., EMBO J. 13: 5795-5809, 1994).
The GenPept accession number for rom2 is AAB67564.1, and GenBank accession number for exemplary nucleotide and amino acid sequences is U19103.1. (see, e.g., Johnston et al., Nature 387(6632): 87-90, 1997).
The GenPept accession number for rpl31a is CAA98641.1, and GenBank accession number for exemplary nucleotide and amino acid sequences is Z74123.1.
The GenPept accession number for rpl6b is AAB67529.1, and GenBank accession number for exemplary nucleotide and amino acid sequences is U22382.1 (see, e.g., Johnston et al. 1997, supra).
The GenPept accession number for tor1 is AAB39292.1, and GenBank accession number for exemplary nucleotide and amino acid sequences is L47993.1. (see, e.g., Huang et al., Yeast 12: 869-875, 1996). “TOR” refers to a 280-300 kD peptide belonging to the phosphoinositide (PI) 3-kinase family, which phosphorylate proteins on serine or threonine residues. TOR is a highly conserved protein kinase found in both prokaryotes and eukaryotes. For example, Raught et al., Proc. Natl. Acad. Sci U.S.A. 98: 7037, 2001, describe homologues of TOR protein found in Saccharomyces cerevisiae, Schizosaccharomyces pombe, Drosophila melanogaster and other Metazoans, and mammals. A single mammalian TOR protein has been cloned from several species. (Raught et al., Proc. Natl. Acad. Sci U.S.A. 98: 7037, 2001). In a preferred embodiment, TOR is isolated from rat brain tissue using an ion-exchange column, and fraction purification. However, native TOR is easily isolated from a variety of tissues using a variety of techniques by those of skill in the art. By way of example only, bovine testes is another source for isolating native TOR protein. Additionally, one of skill in the art will readily isolate TOR proteins from other species for use within the spirit of the present invention. As used in the following description, “TOR” refers to any and all proteins in this described family, including but not limited to dTOR, mTOR, TOR1, TOR2, RAFT and others. Many key signaling molecules are conserved from yeast to man. mTOR is a protein kinase involved in nutrient and growth factor signaling in humans. Signaling downstream of the TOR kinase pathways in yeast and humans regulates the nuclear localization of several transcription factors in response to the carbon and nitrogen sources in the nutritional environment.
The GenPept accession number for ybr238c is CAA85201.1, and GenBank accession number for exemplary nucleotide and amino acid sequences is Z36107.1. (see, e.g., Feldmann et al., 1994, supra).
The GenPept accession number for ybr255w is CAA85218.1, and GenBank accession number for exemplary nucleotide and amino acid sequences is Z36124.1. (see, e.g., Feldmann et al., 1994, supra).
The GenPept accession number for ybr266c is CAA85230.1, and GenBank accession number for exemplary nucleotide and amino acid sequences is Z36135.1.
The GenPept accession number for yor135c is CAA99334.1, and GenBank accession number for exemplary nucleotide and amino acid sequences is Z75043.1.
The GenPept accession number for sch9 is NP—012075.1, and GenBank accession number for exemplary nucleotide and amino acid sequences is NC—001140.4. (see, e.g., Goffeau et al., Science 274: 546-547, 1996). A preferred ortholog of this gene is akt-1.
The GenPept accession number for ure2 is NP—014170.1, and GenBank accession number for exemplary nucleotide and amino acid sequences is NC—001146.3. (see, e.g., Philippsen et al., Nature 387(6632): 93-98, 1997).
“Senescence” refers to a mitotically-arrested state in which a cell or an organism may be metabolically active but is incapable of further cell division. In yeast, one cause of senescence is the accumulation of ribosomal DNA (“rDNA”) circles. In mammals, telomere shortening is one mechanism by which cells senesce. Markers for senescence in multicellular organisms include: increase in cell size, shortening in telomere-length, increase in senescence-associated beta-galactosidase (“SA-beta-gal”) expression, and altered patterns of gene expression. Assays that can detect such markers are well-known in the art. For example, senescence can be detected in cultured cells and tissue sections of organisms at pH 6 by histochemical detection of SA-beta-gal activity present only in senescent cells and not in pre-senescent, quiescent, or immortal cells. Various methods for detecting telomeres and for measuring telomere length are known, including Southern analysis of terminal restriction fragments (“TRF”) obtained by digestion of genomic DNA using frequently cutting restriction enzymes. The TRFs containing DNA with uniform telomeric repeats (TTAGGG) and degenerate repeats are separated by gel electrophoresis, blotted, and visualized directly or indirectly by hybridization with labeled oligonucleotides complementary to the telomeric-repeat sequence. “Quiescence,” differs from “senescence” in that cells can retain the ability to re-enter the cell cycle during quiescence.
“Cell culture” refers generally to cells taken from a living organism and grown under controlled condition (“in culture” or “cultured”). A primary cell culture is a culture of cells, tissues, or organs taken directly from an organism(s) before the first subculture. Cells are expanded in culture when they are placed in a growth medium under conditions that facilitate cell growth and/or division, resulting in a larger population of the cells. When cells are expanded in culture, the rate of cell proliferation is sometimes measured by the amount of time needed for the cells to double in number. This is referred to as doubling time.
“Standard growth conditions”, as used herein, refers to culturing of cells (e.g., mammalian cells) at 37° C., in a standard atmosphere comprising 5% CO2. Relative humidity is maintained at about 100%. While the foregoing the conditions are useful for culturing, it is to be understood that such conditions are capable of being varied by the skilled artisan who will appreciate the options available in the art for culturing cells, for example, varying the temperature, CO2, relative humidity, oxygen, growth medium, and the like. For example, “standard growth conditions” for yeast (e.g., S. cerevisiae) include 30° C. and generally under regular atmospheric conditions (less than 0.5% CO2, approximately 20% O2, approximately 80% N2) at a relative humidity at about 100%.
“Gene” refers to a unit of inheritable genetic material found in a chromosome, such as in a human chromosome. Each gene is composed of a linear chain of deoxyribonucleotides which can be referred to by the sequence of nucleotides forming the chain. Thus, “sequence” is used to indicate both the ordered listing of the nucleotides which form the chain, and the chain which has that sequence of nucleotides. The term “sequence” is used in the same way in referring to RNA chains, linear chains made of ribonucleotides. The gene includes regulatory and control sequences, sequences which can be transcribed into an RNA molecule, and can contain sequences with unknown function. Some of the RNA products (products of transcription from DNA) are messenger RNAs (mRNAs) which initially include ribonucleotide sequences (or sequence) which are translated into a polypeptide and ribonucleotide sequences which are not translated. The sequences which are not translated include control sequences, introns and sequences with unknowns function. It can be recognized that small differences in nucleotide sequence for the same gene can exist between different persons, or between normal cells and cancerous cells, without altering the identity of the gene.
“Gene expression pattern” means the set of genes of a specific tissue or cell type that are transcribed or “expressed” to form RNA molecules. Which genes are expressed in a specific cell line or tissue can depend on factors such as tissue or cell type, stage of development or the cell, tissue, or target organism and whether the cells are normal or transformed cells, such as cancerous cells. For example, a gene can be expressed at the embryonic or fetal stage in the development of a specific target organism and then become non-expressed as the target organism matures. Alternatively, a gene can be expressed in liver tissue but not in brain tissue of an adult human.
“Differential expression” refers to both quantitative as well as qualitative differences in the temporal and tissue expression patterns of a gene or a protein. For example, a differentially expressed gene can have its expression activated or completely inactivated in normal versus disease conditions. Such a qualitatively regulated gene can exhibit an expression pattern within a given tissue or cell type that is detectable in either control or disease conditions, but is not detectable in both. Differentially expressed genes can represent “profile genes,” or “target genes” and the like.
Similarly, a differentially expressed protein can have its expression activated or completely inactivated in normal versus disease conditions. Such a qualitatively regulated protein can exhibit an expression pattern within a given tissue or cell type that is detectable in either control or disease conditions, but is not detectable in both. Moreover, differentially expressed genes can represent “profile proteins”, “target proteins” and the like.
Differentially expressed genes can represent “expression profile genes”, which includes “target genes”. “Expression profile gene,” as used herein, refers to a differentially expressed gene whose expression pattern can be used in methods for identifying compounds useful in the modulation of lifespan extension or activity, or the treatment of disorders, or alternatively, the gene can be used as part of a prognostic or diagnostic evaluation of lifespan disorders, e.g., diseases or disorders associated with aging including various types of cancers, diabetes mellitus, cataracts, heart diseases, and neurodegenerative diseases, such as Alzheimer's disease, Pick's disease, Huntington's disease, Parkinson's disease, adult onset myotonic dystrophy, multiple sclerosis, and adult onset leukodystrophy disease. For example, the effect of the compound on the expression profile gene normally displayed in connection with a particular state, for example, can be used to evaluate the efficacy of the compound to modulate that state, or preferably, to induce or maintain that state. Such assays are further described below. Alternatively, the gene can be used as a diagnostic or in the treatment of lifespan disorders as also further described below. In some instances, only a fragment of an expression profile gene is used, as further described below.
“Expression profile,” as used herein, refers to the pattern of gene expression generated from two up to all of the expression profile genes which exist for a given state. As outlined above, an expression profile is in a sense a “fingerprint” or “blueprint” of a particular cellular state; while two or more states have genes that are similarly expressed, the total expression profile of the state will be unique to that state. A “fingerprint pattern”, as used herein, refers to a pattern generated when the expression pattern of a series (which can range from two up to all the fingerprint genes that exist for a given state) of fingerprint genes is determined. A fingerprint pattern also can be referred to as an “expression profile”. A fingerprint pattern or expression profile can be used in the same diagnostic, prognostic, and compound identification methods as the expression of a single fingerprint gene. The gene expression profile obtained for a given state can be useful for a variety of applications, including diagnosis of a particular disease or condition and evaluation of various treatment regimes. In addition, comparisons between the expression profiles of different lifespan disorders can be similarly informative. An expression profile can include genes which do not appreciably change between two states, so long as at least two genes which are differentially expressed are represented. The gene expression profile can also include at least one target gene, as defined below. Alternatively, the profile can include all of the genes which represent one or more states. Specific expression profiles are described below.
Gene expression profiles can be defined in several ways. For example, a gene expression profile can be the relative transcript level of any number of particular set of genes. Alternatively, a gene expression profile can be defined by comparing the level of expression of a variety of genes in one state to the level of expression of the same genes in another state. For example, genes can be either upregulated, downregulated, or remain substantially at the same level in both states.
A “target gene” refers to a nucleic acid, often derived from a biological sample, to which an oligonucleotide probe is designed to specifically hybridize. It is either the presence or absence of the target nucleic acid that is to be detected, or the amount of the target nucleic acid that is to be quantified. The target nucleic acid has a sequence that is complementary to the nucleic acid sequence of the corresponding probe directed to the target. The target nucleic acid can also refer to the specific subsequence of a larger nucleic acid to which the probe is directed or to the overall sequence (e.g., gene or mRNA) whose expression level it is desired to detect. A “target gene”, therefore, refers to a differentially expressed gene in which modulation of the level of gene expression or of gene product activity prevents and/or ameliorates a lifespan disease or disorder. Thus, compounds that modulate the expression of a target gene, the target gene, or the activity of a target gene product can be used in the diagnosis, treatment or prevention lifespan diseases. Particular target genes of the present invention is shown in
A “target protein” refers to an amino acid or protein, often derived from a biological sample, to which a protein-capture agent specifically hybridizes or binds. It is either the presence or absence of the target protein that is to be detected, or the amount of the target protein that is to be quantified. The target protein has a structure that is recognized by the corresponding protein-capture agent directed to the target. The target protein or amino acid can also refer to the specific substructure of a larger protein to which the protein-capture agent is directed or to the overall structure (e.g., gene or mRNA) whose expression level it is desired to detect.
A “differentially expressed gene transcript”, as used herein, refers to a gene, including an chronological life span gene, transcript that is found in different numbers of copies in different cell or tissue types of an organism having a chronological life span disease or disorder or a disease or disorder associated with aging, including various types of cancers, diabetes mellitus, cataracts, heart diseases, and neurodegenerative diseases, such as Alzheimer's disease, Pick's disease, Huntington's disease, Parkinson's disease, adult onset myotonic dystrophy, multiple sclerosis, and adult onset leukodystrophy disease, compared to the numbers of copies or state of the gene transcript found in the cells of the same tissue in a healthy organism, or in the cells of the same tissue in the same organism. Multiple copies of gene transcripts can be found in an organism having a chronological life span disease or disorder or a disease or disorder associated with aging, while fewer copies of the same gene transcript are found in a healthy organism or healthy cells of the same tissue in the same organism, or vice-versa.
A “differentially expressed gene,” can be a target, fingerprint, or pathway gene. For example, a “fingerprint gene”, as used herein, refers to a differentially expressed gene whose expression pattern can be used as a prognostic or diagnostic marker for the evaluation of chronological life span diseases or disorders, or which can be used to identify compounds useful for the treatment of such diseases or disorders or a disease or disorder associated with aging. For example, the effect of a compound on the fingerprint gene expression pattern normally displayed in connection with chronological life span diseases or disorders or diseases or disorders associated with aging, can be used to evaluate the efficacy, such as potency, of the compound as chronological life span treatment, or can be used to monitor patients undergoing clinical evaluation for the treatment of such a disease or disorder.
“variant” may refer to an organism with a particular genotype in singular form, a set of organisms with different genotypes in plural form, and also to alleles of any gene identifiable by methods of the present invention. For example, the term “variants” includes various alleles that may occur at high frequency at a polymorphic locus, and includes organisms containing such allelic variants. The term “variant” includes various “strains” and various “mutants.”
“Strains” refers to genetic variants that arise in a population, spontaneously and non-spontaneously, by acquiring a mutation or change in genomic DNA. Different strains are genotypically different with respect to at least one gene, gene regulatory element, or other non-coding element. The term “strain” can be used to refer to different laboratory-generated strains and to various mutant lines that arise spontaneously in a population.
“Wildtype” refers to a reference genotype, which is arbitrarily defined by a practitioner of the invention. For screening a large collection of genetic variants to identify long-lived variants, the BY4742 strain may be defined as a “wildtype” to screen a deletion variant set as one example. For screening longevity-promoting compounds that interact with components of the CR pathway, a sir2-fob1-double-deletion strain that is unexposed to a test compound may be employed as a “wildtype” reference strain.
“positive” refers to a variant exhibiting a life span greater than a positive threshold, established according to statistical methods of the present invention.
“Pegative” refers to a variant exhibiting a life span less than a negative threshold, established according to statistical methods of the present invention.
“Ambiguous” refers to a variant that cannot be classified as “positive” or “negative.” An example of an “ambiguous” variant is a variant that cannot be classified as “long-lived” or “not-long-lived.”
“Sample size” or “N” refers to a number of genetically identical cells of a variant of interest for which a phenotypic measurement is determined. As an example, for yeast life span determinations, the term “sample size” or “N” refers to genetically identical mother cells of a variant for which μRLS is determined. Suitable values for N depend on a particular organism of interest for which mean life spans are determined.
“Mean replicative life span,” “μRLS,” and “μRLSv” refer to a computed average for RLS values determined for a given N-cell variant set.
“Median mean replicative life span” or “MμRLS” refers to a computed median for μRLS values of a set, where the μRLS is determined for each variant of a set.
“Average median mean replicative life span” or “AvMμRLS” refers to a computed average for MμRLS values determined for each set.
“Substantially greater” refers to a measured life span of an organism, such as a variant, that is at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% greater than that of a reference organism, such as a wildtype. “Substantially less” refers to a measured life span of an organism, such as a variant, that is at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% less than that of a reference organism, such as a wildtype.
“Longevity-promoting” can refer to a substantial increase in a life span of an organism by at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, from an exposure to a compound. “Longevity-inhibiting” can refer to a substantial decrease in a life span of an organism by at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, from an exposure to a compound.
“Patient”, “subject” or “mammal” are used interchangeably and refer to mammals such as human patients and non-human primates, as well as experimental animals such as rabbits, rats, and mice, and other animals. Animals include all vertebrates, e.g., mammals and non-mammals, such as sheep, dogs, cows, chickens, amphibians, and reptiles.
“Treating” or “treatment” includes the administration of the compositions, compounds or agents of the present invention to prevent or delay the onset of the symptoms, complications, or biochemical indicia of a disease, alleviating or ameliorating the symptoms or arresting or inhibiting further development of the disease, condition, or disorder (e.g., diseases/conditions associated with aging, including various types of cancers, diabetes mellitus, cataracts, heart diseases, and neurodegenerative diseases, such as Alzheimer's disease, Pick's disease, Huntington's disease, Parkinson's disease, adult onset myotonic dystrophy, multiple sclerosis, and adult onset leukodystrophy disease). “Treating” further refers to any indicia of success in the treatment or amelioration or prevention of the disease, condition, or disorder, including any objective or subjective parameter such as abatement; remission; diminishing of symptoms or making the disease condition more tolerable to the patient; slowing in the rate of degeneration or decline; or making the final point of degeneration less debilitating. The treatment or amelioration of symptoms can be based on objective or subjective parameters; including the results of an examination by a physician. Accordingly, the term “treating” includes the administration of the compounds or agents of the present invention to prevent or delay, to alleviate, or to arrest or inhibit development of the symptoms or conditions associated with a replicative life span disease or related disease or a disease or disorder associated with aging. The term “therapeutic effect” refers to the reduction, elimination, or prevention of the disease, symptoms of the disease, or side effects of the disease in the subject. “Treating” or “treatment” using the methods of the present invention includes preventing the onset of symptoms in a subject that can be at increased risk of a disease or disorder associated with aging but does not yet experience or exhibit symptoms, inhibiting the symptoms of a disease or disorder (slowing or arresting its development), providing relief from the symptoms or side-effects of a disease (including palliative treatment), and relieving the symptoms of a disease (causing regression). Treatment can be prophylactic (to prevent or delay the onset of the disease, or to prevent the manifestation of clinical or subclinical symptoms thereof) or therapeutic suppression or alleviation of symptoms after the manifestation of the disease or condition.
“Concomitant administration” of a known drug with a compound of the present invention means administration of the drug and the compound at such time that both the known drug and the compound will have a therapeutic effect or diagnostic effect. Such concomitant administration can involve concurrent (i.e., at the same time), prior, or subsequent administration of the drug with respect to the administration of a compound of the present invention. A person of ordinary skill in the art, would have no difficulty determining the appropriate timing, sequence and dosages of administration for particular drugs and compounds of the present invention.
In general, the phrase “well tolerated” refers to the absence of adverse changes in health status that occur as a result of the treatment and would affect treatment decisions.
“Inhibitors,” “activators,” and “modulators” of replicative life span genes and their gene products in cells are used to refer to inhibitory, activating, or modulating molecules, respectively, identified using in vitro and in vivo assays for binding or signaling, e.g., ligands, agonists, antagonists, and their homologs and mimetics. The term “modulator” includes inhibitors and activators. Inhibitors are agents that, e.g., bind to, partially or totally block stimulation, decrease, prevent, delay activation, inactivate, desensitize, or down regulate the activity of replicative life span genes, e.g., antagonists. Activators are agents that, e.g., bind to, stimulate, increase, open, activate, facilitate, enhance activation, sensitize or up regulate the activity of replicative life span genes, e.g., agonists. Modulators include agents that, e.g., alter the interaction of replicative life span gene or gene product with: proteins that bind activators or inhibitors, receptors, including proteins, peptides, lipids, carbohydrates, polysaccharides, or combinations of the above, e.g., lipoproteins, glycoproteins, and the like. Modulators include genetically modified versions of naturally-occurring activated replicative life span disorder ligands, e.g., with altered activity, as well as naturally occurring and synthetic ligands, antagonists, agonists, small chemical molecules and the like. Such assays for inhibitors and activators include, e.g., applying putative modulator compounds to a cell expressing a replicative life span receptor and then determining the functional effects on replicative life span receptor signaling. Samples or assays comprising activated replicative life span receptor that are treated with a potential activator, inhibitor, or modulator are compared to control samples without the inhibitor, activator, or modulator to examine the extent of inhibition. Control samples (untreated with inhibitors) can be assigned a activity value of 100%. Inhibition of activated samples is achieved when the activity value relative to the control is about 80%, optionally 50% or 25-0%. Activation of sample is achieved when the activity value relative to the control is 110%, optionally 150%, optionally 200-500%, or 1000-3000% higher.
An intact “antibody” comprises at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds. Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region is comprised of three domains, CH1, CH2 and CH3. Each light chain is comprised of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region is comprised of one domain, CL. The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR). Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxyl-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The constant regions of the antibodies can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) through cellular receptors such as Fc receptors (e.g., FcγRI, FcγRIIa, FcγRIIb, FcγRIII, and FcRη) and the first component (Clq) of the classical complement system. The term antibody includes antigen-binding portions of an intact antibody that retain capacity to bind the antigen. Examples of antigen binding portions include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CH1 domains; (ii) a F(ab′)2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CH1 domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment (Ward et al., Nature 341: 544-546, 1989), which consists of a VH domain; and (vi) an isolated complementarity determining region (CDR). Furthermore, although the two domains of the Fv fragment, VL and VH, are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv (scFv); See, e.g., Bird et al., Science 242: 423-426, 1988; and Huston et al., Proc. Natl. Acad. Sci. U.S.A. 85: 5879-5883, 1988). Such single chain antibodies are included by reference to the term “antibody” Fragments can be prepared by recombinant techniques or enzymatic or chemical cleavage of intact antibodies.
“Human sequence antibody” includes antibodies having variable and constant regions (if present) derived from human immunoglobulin sequences. The human sequence antibodies of the invention can include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, the term “human sequence antibody”, as used herein, is not intended to include antibodies in which entire CDR sequences sufficient to confer antigen specificity and derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences (i.e., humanized antibodies).
“Monoclonal antibody” or “monoclonal antibody composition” refer to a preparation of antibody molecules of single molecular composition. A monoclonal antibody composition displays a single binding specificity and affinity for a particular epitope. Accordingly, the term “human monoclonal antibody” refers to antibodies displaying a single binding specificity which have variable and constant regions (if present) derived from human germline immunoglobulin sequences. In one embodiment, the human monoclonal antibodies are produced by a hybridoma which includes a B cell obtained from a transgenic non-human animal, e.g., a transgenic mouse, having a genome comprising a human heavy chain transgene and a light chain transgene fused to an immortalized cell.
“Diclonal antibody” refers to a preparation of at least two antibodies to an antigen. Typically, the different antibodies bind different epitopes.
“Oligoclonal antibody” refers to a preparation of 3 to 100 different antibodies to an antigen. Typically, the antibodies in such a preparation bind to a range of different epitopes.
“Polyclonal antibody” refers to a preparation of more than 1 (two or more) different antibodies to an antigen. Such a preparation includes antibodies binding to a range of different epitopes.
“Recombinant human antibody” includes all human sequence antibodies of the invention that are prepared, expressed, created or isolated by recombinant means, such as antibodies isolated from an animal (e.g., a mouse) that is transgenic for human immunoglobulin genes (described further below); antibodies expressed using a recombinant expression vector transfected into a host cell, antibodies isolated from a recombinant, combinatorial human antibody library, or antibodies prepared, expressed, created or isolated by any other means that involves splicing of human immunoglobulin gene sequences to other DNA sequences. Such recombinant human antibodies have variable and constant regions (if present) derived from human germline immunoglobulin sequences. Such antibodies can, however, be subjected to in vitro mutagenesis (or, when an animal transgenic for human Ig sequences is used, in vivo somatic mutagenesis) and thus the amino acid sequences of the VH and VL regions of the recombinant antibodies are sequences that, while derived from and related to human gerinline VH and VL sequences, may not naturally exist within the human antibody germline repertoire in vivo.
A “heterologous antibody” is defined in relation to the transgenic non-human organism producing such an antibody. This term refers to an antibody having an amino acid sequence or an encoding nucleic acid sequence corresponding to that found in an organism not consisting of the transgenic non-human animal, and generally from a species other than that of the transgenic non-human animal.
A “heterohybrid antibody” refers to an antibody having a light and heavy chains of different organismal origins. For example, an antibody having a human heavy chain associated with a murine light chain is a heterohybrid antibody.
“Substantially pure” or “isolated” means an object species (e.g., an antibody of the invention) has been identified and separated and/or recovered from a component of its natural environment such that the object species is the predominant species present (i.e., on a molar basis it is more abundant than any other individual species in the composition); a “substantially pure” or “isolated” composition also means where the object species comprises at least about 50 percent (on a molar basis) of all macromolecular species present. A substantially pure or isolated composition can also comprise more than about 80 to 90 percent by weight of all macromolecular species present in the composition. An isolated object species (e.g., antibodies of the invention) can also be purified to essential homogeneity (contaminant species cannot be detected in the composition by conventional detection methods) wherein the composition consists essentially of derivatives of a single macromolecular species. For example, an isolated antibody to any one chronological gene product as shown in
“Specific binding” refers to preferential binding of an antibody to a specified antigen relative to other non-specified antigens. The phrase “specifically (or selectively) binds” to an antibody refers to a binding reaction that is determinative of the presence of the protein in a heterogeneous population of proteins and other biologics. Typically, the antibody binds with an association constant (Ka) of at least about 1×106 M−1 or 107 M−1, or about 108 M−1 to 109 M−1, or about 1010 M−1 to 1011 M−1 or higher, and binds to the specified antigen with an affinity that is at least two-fold greater than its affinity for binding to a non-specific antigen (e.g., BSA, casein) other than the specified antigen or a closely-related antigen. The phrases “an antibody recognizing an antigen” and “an antibody specific for an antigen” are used interchangeably herein with the term “an antibody which binds specifically to an antigen”. A predetermined antigen is an antigen that is chosen prior to the selection of an antibody that binds to that antigen.
“Specifically bind(s)” or “bind(s) specifically” when referring to a peptide refers to a peptide molecule which has intermediate or high binding affinity, exclusively or predominately, to a target molecule. The phrases “specifically binds to” refers to a binding reaction which is determinative of the presence of a target protein in the presence of a heterogeneous population of proteins and other biologics. Thus, under designated assay conditions, the specified binding moieties bind preferentially to a particular target protein and do not bind in a significant amount to other components present in a test sample. Specific binding to a target protein under such conditions can require a binding moiety that is selected for its specificity for a particular target antigen. A variety of assay formats can be used to select ligands that are specifically reactive with a particular protein. For example, solid-phase ELISA immunoassays, immunoprecipitation, Biacore and Western blot are used to identify peptides that specifically react with the antigen. Typically a specific or selective reaction will be at least twice background signal or noise and more typically more than 10 times background.
“High affinity” for an antibody refers to an equilibrium association constant (Ka) of at least about 107M−1, at least about 108M−1, at least about 109M−1, at least about 1010M−1, at least about 1011M−1, or at least about 1012M−1 or greater, e.g., up to 1013M−1 or 1014M−1 or greater. However, “high affinity” binding can vary for other antibody isotypes.
“Ka”, as used herein, is intended to refer to the equilibrium association constant of a particular antibody-antigen interaction. This constant has units of 1/M.
“Kd”, as used herein, is intended to refer to the equilibrium dissociation constant of a particular antibody-antigen interaction. This constant has units of M.
The term “ka”, as used herein, is intended to refer to the kinetic association constant of a particular antibody-antigen interaction. This constant has units of 1/Ms.
The term “kd”, as used herein, is intended to refer to the kinetic dissociation constant of a particular antibody-antigen interaction. This constant has units of 1/s.
“Particular antibody-antigen interactions” refers to the experimental conditions under which the equilibrium and kinetic constants are measured.
“Isotype” refers to the antibody class that is encoded by heavy chain constant region genes. Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, and define the antibody's isotype as IgG, IgM, IgA, IgD and IgE, respectively. Additional structural variations characterize distinct subtypes of IgG (e.g., IgG1, IgG2, IgG3 and IgG4) and IgA (e.g., IgA1 and IgA2)
“Isotype switching” refers to the phenomenon by which the class, or isotype, of an antibody changes from one Ig class to one of the other Ig classes/
“Nonswitched isotype” refers to the isotypic class of heavy chain that is produced when no isotype switching has taken place; the CH gene encoding the nonswitched isotype is typically the first CH gene immediately downstream from the functionally rearranged VDJ gene. Isotype switching has been classified as classical or non-classical isotype switching. Classical isotype switching occurs by recombination events which involve at least one switch sequence region in the transgene. Non-classical isotype switching can occur by, for example, homologous recombination between human σμ and human Σμ (δ-associated deletion). Alternative non-classical switching mechanisms, such as intertransgene and/or interchromosomal recombination, among others, can occur and effectuate isotype switching.
“Switch sequence” refers to those DNA sequences responsible for switch recombination. A “switch donor” sequence, typically a μ switch region, are 5′ (i.e., upstream) of the construct region to be deleted during the switch recombination. The “switch acceptor” region are between the construct region to be deleted and the replacement constant region (e.g., γ, ε, and alike). As there is no specific site where recombination always occurs, the final gene sequence is not typically predictable from the construct.
“Glycosylation pattern” is defined as the pattern of carbohydrate units that are covalently attached to a protein, more specifically to an immunoglobulin protein. A glycosylation pattern of a heterologous antibody can be characterized as being substantially similar to glycosylation patterns which occur naturally on antibodies produced by the species of the non-human transgenic animal, when one of ordinary skill in the art would recognize the glycosylation pattern of the heterologous antibody as being more similar to said pattern of glycosylation in the species of the non-human transgenic animal than to the species from which the CH genes of the transgene were derived.
“Naturally-occurring” as applied to an object refers to the fact that an object can be found in nature. For example, a polypeptide or polynucleotide sequence that is present in an organism (including viruses) that can be isolated from a source in nature and which has not been intentionally modified by man in the laboratory is naturally-occurring.
“Immunoglobulin locus” refers to a genetic element or set of linked genetic elements that comprise information that can be used by a B cell or B cell precursor to express an immunoglobulin peptide. This peptide can be a heavy chain peptide, a light chain peptide, or the fusion of a heavy and a light chain peptide. In the case of an unrearranged locus, the genetic elements are assembled by a B cell precursor to form the gene encoding an immunoglobulin peptide. In the case of a rearranged locus, a gene encoding an immunoglobulin peptide is contained within the locus.
“Rearranged” refers to a configuration of a heavy chain or light chain immunoglobulin locus wherein a V segment is positioned immediately adjacent to a D-J or J segment in a conformation encoding essentially a complete VH or VL domain, respectively. A rearranged immunoglobulin gene locus can be identified by comparison to germline DNA; a rearranged locus has at least one recombined heptamer/nonamer homology element.
“Unrearranged” or “germline configuration” in reference to a V segment refers to the configuration wherein the V segment is not recombined so as to be immediately adjacent to a D or J segment.
“Nucleic acid” or “nucleic acid molecule” refer to a deoxyribonucleotide or ribonucleotide polymer in either single- or double-stranded form, and unless otherwise limited, can encompass known analogs of natural nucleotides that can function in a similar manner as naturally occurring nucleotides.
“Isolated nucleic acid” in reference to nucleic acids encoding antibodies or antibody portions (e.g., VH, VL, CDR3) that bind to the antigen, is intended to refer to a nucleic acid in which the nucleotide sequences encoding the antibody or antibody portion are free of other nucleotide sequences encoding antibodies or antibody portions that bind antigens other than which other sequences can naturally flank the nucleic acid in human genomic DNA.
The nucleic acids of the invention be present in whole cells, in a cell lysate, or in a partially purified or substantially pure form. A nucleic acid is “isolated” or “rendered substantially pure” when purified away from other cellular components or other contaminants, e.g., other cellular nucleic acids or proteins, by standard techniques, including alkaline/SDS treatment, CsCl banding, column chromatography, agarose gel electrophoresis and others well known in the art (See, e.g., Sambrook, Tijssen and Ausubel discussed herein and incorporated by reference for all purposes). The nucleic acid sequences of the invention and other nucleic acids used to practice this invention, whether RNA, cDNA, genomic DNA, or hybrids thereof, can be isolated from a variety of sources, genetically engineered, amplified, and/or expressed recombinantly. Any recombinant expression system can be used, including, in addition to bacterial, e.g., yeast, insect or mammalian systems. Alternatively, these nucleic acids can be chemically synthesized in vitro. Techniques for the manipulation of nucleic acids, such as, e.g., subcloning into expression vectors, labeling probes, sequencing, and hybridization are well described in the scientific and patent literature, see, e.g., Sambrook, Tijssen and Ausubel. Nucleic acids can be analyzed and quantified by any of a number of general means well known to those of skill in the art. These include, e.g., analytical biochemical methods such as NMR, spectrophotometry, radiography, electrophoresis, capillary electrophoresis, high performance liquid chromatography (HPLC), thin layer chromatography (TLC), and hyperdiffusion chromatography, various immunological methods, such as fluid or gel precipitin reactions, immunodiffusion (single or double), immunoelectrophoresis, □ adioimmunoassay (RIAs), enzyme-linked immunosorbent assays (ELISAs), immuno-fluorescent assays, Southern analysis, Northern analysis, dot-blot analysis, gel electrophoresis (e.g., SDS-PAGE), RT-PCR, quantitative PCR, other nucleic acid or target or signal amplification methods, radiolabeling, scintillation counting, and affinity chromatography.
The nucleic acid compositions of the present invention, while often in a native sequence (except for modified restriction sites and the like), from either cDNA, genomic or mixtures can be mutated, thereof in accordance with standard techniques to provide gene sequences. For coding sequences, these mutations, can affect amino acid sequence as desired. In particular, DNA sequences substantially homologous to or derived from native V, D, J, constant, switches and other such sequences described herein are contemplated (where “derived” indicates that a sequence is identical or modified from another sequence).
“Recombinant host cell” (or simply “host cell”) refers to a cell into which a recombinant expression vector has been introduced. It should be understood that such terms are intended to refer not only to the particular subject cell but to the progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term “host cell” as used herein.
“Polypeptide,” “peptide” and “protein” are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymer.
“Amino acid” refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Amino acid analogs refers to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., an a carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. Amino acid mimetics refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally occurring amino acid.
Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IWPAC-IUB Biochemical Nomenclature Comrnrission. Nucleotides, likewise, can be referred to by their commonly accepted single-letter codes.
“Conservatively modified variants” applies to both amino acid and nucleic acid sequences. With respect to particular nucleic acid sequences, conservatively modified variants refers to those nucleic acids which encode identical or essentially identical amino acid sequences, or where the nucleic acid does not encode an amino acid sequence, to essentially identical sequences. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For instance, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, at every position where an alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide. Such nucleic acid variations are “silent variations,” which are one species of conservatively modified variations. Every nucleic acid sequence herein which encodes a polypeptide also describes every possible silent variation of the nucleic acid. One of skill will recognize that each codon in a nucleic acid (except AUG, which is ordinarily the only codon for methionine, and TGG, which is ordinarily the only codon for tryptophan) can be modified to yield a functionally identical molecule. Accordingly, each silent variation of a nucleic acid which encodes a polypeptide is implicit in each described sequence with respect to the expression product, but not with respect to actual probe sequences.
As to amino acid sequences, one of skill will recognize that individual substitutions, deletions or additions to a nucleic acid, peptide, polypeptide, or protein sequence which alters, adds or deletes a single amino acid or a small percentage of amino acids in the encoded sequence is a “conservatively modified variant” where the alteration results in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. Such conservatively modified variants are in addition to and do not exclude polymorphic variants, interspecies homologs, and alleles of the invention.
The following eight groups each contain amino acids that are conservative substitutions for one another: 1) Alanine (A), Glycine (G); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W); 7) Serine (S), Threonine (T); and 8) Cysteine (C), Methionine (M) (see, e.g., Creighton, Proteins (1984)).
Macromolecular structures such as polypeptide structures can be described in terms of various levels of organization. For a general discussion of this organization, see, e.g., Alberts et al., Molecular Biology of the Cell (3rd ed., 1994) and Cantor and Schimmel, Biophysical Chemistry Part I: The Conformation of Biological Macromolecules (1980). “Primary structure” refers to the amino acid sequence of a particular peptide. “Secondary structure” refers to locally ordered, three dimensional structures within a polypeptide. These structures are commonly known as domains, e.g., enzymatic domains, extracellular domains, transmembrane domains, pore domains, and cytoplasmic tail domains. Domains are portions of a polypeptide that form a compact unit of the polypeptide and are typically 15 to 350 amino acids long. Exemplary domains include domains with enzymatic activity, e.g., a kinase domain. Typical domains are made up of sections of lesser organization such as stretches of β-sheet and α-helices. “Tertiary structure” refers to the complete three dimensional structure of a polypeptide monomer. “Quaternary structure” refers to the three dimensional structure formed by the noncovalent association of independent tertiary units. Anisotropic terms are also known as energy terms.
A particular nucleic acid sequence also implicitly encompasses “splice variants.” Similarly, a particular protein encoded by a nucleic acid implicitly encompasses any protein encoded by a splice variant of that nucleic acid. “Splice variants,” as the name suggests, are products of alternative splicing of a gene. After transcription, an initial nucleic acid transcript can be spliced such that different (alternate) nucleic acid splice products encode different polypeptides. Mechanisms for the production of splice variants vary, but include alternate splicing of exons. Alternate polypeptides derived from the same nucleic acid by read-through transcription are also encompassed by this definition. Any products of a splicing reaction, including recombinant forms of the splice products, are contemplated here.
A “label” is a composition detectable by spectroscopic, photochemical, biochemical, immunochemical, or chemical means. For example, useful labels include 32P, fluorescent dyes, electron-dense reagents, enzymes (e.g., as commonly used in an ELISA), biotin, digoxigenin, or haptens and proteins for which antisera or monoclonal antibodies are available (e.g., the polypeptides of the invention can be made detectable, e.g., by incorporating a radiolabel into the peptide, and used to detect antibodies specifically reactive with the peptide).
“Rapamycin” is a bacterial macrolide and a potent immunosuppressant with realized or potential clinical applications in the prevention of graft rejection after organ transplantation and the treatment of autoimmune disorders. This drug acts by forming a complex with the immunophillin FKBP12, and then inhibiting activity of TOR. (Abraham et al., Annu. Rev. Immuno. 14: 483, 1996). Rapamycin treatment of cells has been shown to lead to the dephosphorylation and inactivation of TOR substrates such as P70 S6 Kinase and 4E-BP1/PHAS1. (Dumont et al., J. Immunol 144: 251, 1990; Brown et al., Nature 369: 756, 1994; Kunz et al., Cell 73: 585, 1993; Jefferies et al., EMBO J. 15: 3693, 1997; Beretta et al., EMBO J. 15: 658, 1996). Numerous derivatives of rapamycin are known. Certain 40-O-substituted rapamycins are described in, e.g., in U.S. Pat. No. 5,258,389 and WO 94/09010 (O-alkyl rapamycins); WO 92/05179 (carboxylic acid esters), U.S. Pat. No. 5,118,677 (amide esters), U.S. Pat. No. 5,118,678 (carbamates), U.S. Pat. No. 5,100,883 (fluorinated esters), U.S. Pat. No. 5,151,413 (acetals), and U.S. Pat. No. 5,120,842 (silyl ethers). As described herein, rapamycin (or a rapamycin analog, derivative or related compound thereof) is shown to extend life span by inhibiting the TOR pathway.
This invention relies on routine techniques in the field of recombinant genetics. Basic texts disclosing the general methods of use in this invention include Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd ed., 1989; Kriegler, Gene Transfer and Expression: A Laboratory Manual, 1990; and Ausubel et al., eds., Current Protocols in Molecular Biology, 1994; all of which are herein incorporated by reference for all purposes.
In the following, aspects of the present invention are directed to: (1) high throughput methods for classifying genetic variants, and for identifying “long-lived” variants; (2) methods for identifying genes that regulate eukaryotic life spans; (3) various vectors and host cells comprising the identified genes, related orthologs, and related gene products; and (4) pharmaceutical compositions that can modulate, or modify, the function of the identified genes/gene products.
II. High throughput Methods for Identifying Yeast Sequences that Regulate Life Spans
A. Replicative Life Span (RLS) Assay
B. A High throughput Method for Identifying Long-Lived Mutants
1. Screening a Large Set of Genetic Variants by Minimizing Sample Size (N)
The RLS assay described in
Various statistical methods of the present invention can be used to classify genetic variants of any organism, by measuring a phenotype of interest (referred to as “a phenotypic measurement”). As examples, various embodiments of the present invention described below are directed to statistical methods for the classification of “long-lived” (“LL”) variants and “not-long-lived” (“NLL”) variants in an unsorted set. The methods of the present invention can be used to determine a minimal sample size that needs to be evaluated for determining a statistically significant phenotypic measurement.
Various statistical methods of the present invention are useful for making phenotypic analysis of an organism for which a phenotypic measurement requires a statistically significant number of organisms, and assays for measuring a phenotype are inherently labor-intensive and/or costly. For example, methods of the present invention are well-suited for life span determinations that require a statistically significant number of organisms and life-span-determinative assays that are inherently labor-intensive. Examples of suitable hosts include various plants, various fungi, such as yeasts, various invertebrate organisms, such as worrns and flies, and various vertebrates, such as mammals. For the present disclosure, embodiments of the present invention will be described in the context of yeast variants to describe unique features of the invention. Exemplary data include life span determinations for yeast variants, however, the present invention is not intended to be limited only to the analysis of yeast variants.
2. Classification Based on Determination of a Mean Replicative Life Span (μRLS) for N Cells of Each Variant
In one embodiment, the present invention provides a statistical method for screening a large number of genetic variants in order to identify “long-lived” (“LL”) variants by distinguishing variants based on calculated mean RLS (“μRLS”) as a phenotypic measurement. LL variants exhibit μRLS substantially greater than the μRLS of a “wildtype” reference strain.
One embodiment of the present invention provides a high throughput method for classifying genetic variants that exhibit long life spans based on calculated μRLS values. Described below is an exemplary high throughput method for efficiently screening a large collection of variants, including a large genomic library of genetic variants. Identified LL variants enable the identification of genes that affect life-span regulation. In the following, various embodiments relating to the methods of the present invention for classifying genetic variants are described in a computational-like manner to facilitate comprehension of inter-related processes. The automation of the following methods, in part or in whole, is contemplated.
If the phenotypic measurement described, in step 301 of
In step 405, the variant is classified as “positive,” “negative,” or “ambiguous,” depending on the μRLS computed in step 404. If the variant has been classified as ambiguous, as determined in step 406, then the variant is placed into the set of ambiguous variants T in step 407. The for-loop of steps 401-408 continues until the set S is empty, as determined in step 408, indicating that all variants initially present in set S at the beginning of the for-loop have been classified.
3. Classification Based on Determination of a Mean RLS for a Subset of N-Cell Set Exhibiting Maximum RLS for Each Variant
Alternatively, in another embodiment, the present invention provides a statistical method for screening a large number of genetic variants in order to identify “long-lived” variants by distinguishing variants, based on μRLS determined for a subset of N cells of a variant, that exhibit the highest RLS values for the N-cell set. As previously described in
Alternatively, μRLS may be computed by selecting the highest RLS values for a given N-cell set, excluding the lowest RLS values of the N-cell set. For example, RLS values for a subset of N cells that are less than optimal in shape, size, or fitness may be excluded for μRLS determinations. As N value decreases, each cell of a N-cell set is more likely to increase the variance for the set. A classification method that can selectively exclude individual RLS values determined for a N-cell set that vary substantially from a computed μRLS based on N cells, for example, should increase the statistical reliability of a computed μRLS based on a smaller subset of the N-cell set.
4. Classification Based on Determination of a Normalized μRLS for Each Variant of Set S
Alternatively, a classification scheme can be based on the computation of a mean RLS (“μRLSv”) for a variant, a median mean RLS (“MμRLSS”) for a set of variants in Set S, and an average median mean RLS (“AvMμRLS”) for multiple sets of variants, that can be utilized to normalize the mean RLS (“μRLSv”) for each variant.
Statistical methods for establishing thresholds and for reducing a sample size are provided below.
5. Determining Thresholds as a Function of Sample Size (N)
One embodiment of the present invention provides a statistical method for determining a minimal number of cells (Nmin) that need to be evaluated to obtain a statistically reliable mean RLS (μRLS) value for a given variant. In another embodiment, the present invention provides a statistical method for establishing threshold values that may be used to discriminate LL and NLL variants. Positive and negative thresholds described in
In step 902 of
In step 902 of
Threshold values established according to a method described in
6. Determining Thresholds as a Function of Probabilities for Misclassification of Variants
In
Probability distributions may be used to predict probabilities for Type I and Type II errors associated with a hypothetical sample size. For example, when N=3, and the determined μRLS>23, then the probability that a variant would be correctly classified as LL is approximately 95% (column 3). However, when sample size is very small, such as N=3, then it is predicted that approximately 73% of NLL variants are likely to be misclassified as LL. Since numbers of NLL variants are likely to be substantially greater than numbers of LL variants, generally in any given population of naturally-occurring variants and in any randomly-generated variant library, the frequency of Type I error for this sample size is not desirable.
Although increasing a sample size is one way to decrease Type I error, total number of N cells that need to be analyzed can be reduced to enable more efficient identification of LL variants. In one embodiment, it is desirable to select a sample size and a threshold value that would enable the correct classification of LL variants at frequencies of at least 50%. In another embodiment, it is also desirable to select a sample size and a negative threshold value that would enable the correct classification of NLL variants at frequencies of at least 95%. In the preferred embodiment, sample size (N) of yeast mother cells of any strain is 5. An exemplary method for classifying variants when sample size equals five is described in Example 2.
7. High Throughput Format
An assay performed in a “homogeneous format” means that the assay can be performed in a single container, with no manipulation or purification of any components being required to determine the result of the assay, e.g., a test agent can be added to an assay system and any effects directly measured. Often, such “homogeneous format” assays will comprise at least one component that is “quenched” or otherwise modified in the presence or absence of a test agent.
A “secondary screening step” refers to a screening step whereby a test agent is assessed for a secondary property in order to determine the specificity or mode of action of a compound identified using the methods provided herein. Such secondary screening steps can be performed on all of the test agents, or, e.g., on only those that are found to be positive in a primary screening step, and can be performed subsequently, simultaneously, or prior to a primary screening step.
“High throughput screening” refers to a method of rapidly assessing a large number of test agents for a specific activity. Typically, the plurality of test agents will be assessed in parallel, for example by simultaneously assessing 96 or 384 agents using a 96-well or 384-well plate, 96-well or 384-well dispensers, and detection methods capable of detecting 96 or 384 samples simultaneously. Often, such methods will be automated, e.g., using robotics.
“Robotic high throughput screening” refers to high throughput screening that involves at least one robotic element, thereby eliminating a requirement for human manipulation in at least one step of the screening process. For example, a robotic arm can dispense a plurality of test agents to a multi-well plate.
A “multi-well plate” refers to any container, receptacle, or device that can hold a plurality of samples, e.g., for use in high throughput screening. Typically, such “multi-well plates” will be part of an integrated and preferably automated system that enables the rapid and efficient screening or manipulation of a large number of samples. Such plates can include, e.g., 24, 48, 96, 384, or more wells, and are typically used in conjunction with a 24, 48, 96, 384, or more tip pipettors, samplers, detectors, and the like.
In some assays, it will be desirable to have positive controls to ensure that the components of the assays are working properly.
In the high throughput assays of the invention, it is possible to screen up to several thousand different modulators in a single day. In particular, each well of a microtiter plate can be used to run a separate assay against a selected potential modulator, or if concentration or incubation time effects are to be observed, every 5-10 wells can test a single modulator. Thus, a single standard microtiter plate can assay about 100 (96) modulators. If 1536 well plates are used, then a single plate can easily assay from about 100 to about 1500 different compounds. It is possible to assay many different plates per day; assay screens for up to about 6,000-20,000, and even up to about 100,000-1,000,000 different compounds are possible using the integrated systems of the invention.
8. Solid State and Soluble High throughput Assays
The invention provides soluble assays using a replicative life span gene or gene product, or a cell or tissue expressing a replicative life span gene product, either naturally occurring or recombinant. The invention further provides solid phase based in vitro assays in a high throughput format, where a replicative life span protein or fragment thereof, is attached to a solid phase substrate.
In the high throughput assays of the invention, either soluble or solid state, it is possible to screen up to several thousand different modulators or ligands in a single day. This methodology can be used for the replicative life span proteins in vitro, or for cell-based or membrane-based assays comprising a replicative life span protein. In particular, each well of a microtiter plate can be used to run a separate assay against a selected potential modulator, or, if concentration or incubation time effects are to be observed, every 5-10 wells can test a single modulator. Thus, a single standard microtiter plate can assay about 100 (e.g., 96) modulators. If 1536 well plates are used, then a single plate can easily assay from about 100- about 1500 different compounds. It is possible to assay many plates per day; assay screens for up to about 6,000, 20,000, 50,000, or more than 100,000 different compounds are possible using the integrated systems of the invention.
For a solid state reaction, the protein of interest or a fragment thereof, e.g., an extracellular domain, or a cell or membrane comprising the replicative life span protein of interest or a fragment thereof as part of a fusion protein can be bound to the solid state component, directly or indirectly, via covalent or non covalent linkage, e.g., via a tag. The tag can be any of a variety of components. In general, a molecule which binds the tag (a tag binder) is fixed to a solid support, and the tagged molecule of interest is attached to the solid support by interaction of the tag and the tag binder.
A number of tags and tag binders can be used, based upon known molecular interactions well described in the literature. For example, where a tag has a natural binder, for example, biotin, protein A, or protein G, it can be used in conjunction with appropriate tag binders (avidin, streptavidin, neutravidin, the Fc region of an immunoglobulin, and the like) Antibodies to molecules with natural binders such as biotin are also widely available and appropriate tag binders; see, SIGMA Immunochemicals 1998 catalogue SIGMA, St. Louis Mo.).
Similarly, any haptenic or antigenic compound can be used in combination with an appropriate antibody to form a tag/tag binder pair. Thousands of specific antibodies are commercially available and many additional antibodies are described in the literature. For example, in one common configuration, the tag is a first antibody and the tag binder is a second antibody which recognizes the first antibody. In addition to antibody-antigen interactions, receptor-ligand interactions are also appropriate as tag and tag-binder pairs. For example, agonists and antagonists of cell membrane receptors (e.g., cell receptor-ligand interactions such as transferrin, c-kit, viral receptor ligands, cytokine receptors, chemokine receptors, interleukin receptors, immunoglobulin receptors and antibodies, the cadherein family, the integrin family, the selectin family, and the like; see, e.g., Pigott & Power, The Adhesion Molecule Facts Book I (1993). Similarly, toxins and venoms, viral epitopes, hormones (e.g., opiates, steroids, and the like), intracellular receptors (e.g., which mediate the effects of various small ligands, including steroids, thyroid hormone, retinoids and vitamin D; peptides), drugs, lectins, sugars, nucleic acids (both linear and cyclic polymer configurations), oligosaccharides, proteins, phospholipids and antibodies can all interact with various cell receptors.
Synthetic polymers, such as polyurethanes, polyesters, polycarbonates, polyureas, polyamides, polyethyleneimines, polyarylene sulfides, polysiloxanes, polyimides, and polyacetates can also form an appropriate tag or tag binder. Many other tag/tag binder pairs are also useful in assay systems described herein, as would be apparent to one of skill upon review of this disclosure.
Common linkers such as peptides, polyethers, and the like can also serve as tags, and include polypeptide sequences, such as poly gly sequences of between about 5 and 200 amino acids. Such flexible linkers are known to persons of skill in the art. For example, poly(ethelyne glycol) linkers are available from Shearwater Polymers, Inc. Huntsville, Ala. These linkers optionally have amide linkages, sulfhydryl linkages, or heterofunctional linkages.
Tag binders are fixed to solid substrates using any of a variety of methods currently available. Solid substrates are commonly derivatized or functionalized by exposing all or a portion of the substrate to a chemical reagent which fixes a chemical group to the surface which is reactive with a portion of the tag binder. For example, groups which are suitable for attachment to a longer chain portion would include amines, hydroxyl, thiol, and carboxyl groups. Aminoalkylsilanes and hydroxyalkylsilanes can be used to functionalize a variety of surfaces, such as glass surfaces. The construction of such solid phase biopolymer arrays is well described in the literature. See, e.g., Merrifield, J. Am. Chem. Soc. 85: 2149-2154, 1963 (describing solid phase synthesis of, e.g., peptides); Geysen et al., J. Immun. Meth. 102: 259-274, 1987 (describing synthesis of solid phase components on pins); Frank & Doring, Tetrahedron 44:60316040 (1988) (describing synthesis of various peptide sequences on cellulose disks); Fodor et al., Science, 251: 767-777, 1991; Sheldon et al., Clinical Chemistry 39(4): 718-719, 1993; and Kozal et al., Nature Medicine 2(7):753-759, 1996 (all describing arrays of biopolymers fixed to solid substrates). Non-chemical approaches for fixing tag binders to substrates include other common methods, such as heat, cross-linking by UV radiation, and the like.
C. Mutations and Libraries
For the analysis of yeast variants, examples of suitable libraries include various yeast haploid deletion collections, such as ORF-deletion collections made in various suitable backgrounds. For example, a S. cerevisiae Genome Deletion and Parallel collection (“SCGDP”) contains an almost complete set of genetic variants in which a single-ORF is replaced with a KanMX selectable marker. Several isogenic, S288C-derived, designer deletion variants containing different genetic backgrounds for SCGDP exists, including the BY4741 (MATa), BY4742 (MATα), and BY4743 (MATa/MATα) strains. (Winzeler et al., Science Vol. 285: 901-906, 1999). Four deletion collections (haploid MATa, haploid MATα, heterozygous diploid, and homozygous diploid) representing greater than 6000 unique gene disruptions may be employed for the identification of any subset exhibiting a phenotype of interest, including a long life span.
Variants for analysis by methods of the present invention include naturally occurring variants that arise spontaneously in a laboratory and in nature, and genetic variants that are generated in a laboratory using various mutation-inducing methods known to persons skilled in the art. Variants can be generated in any gene, by various methods, including chemical mutagenesis induced by exposure to a mutagen, such as ethane methyl sulfonate (“EMS”), radiation-induced mutagenesis, and various genetic-engineering techniques, such as PCR-mediated mutations, transposon mutagenesis, site-directed mutagenesis, or gene over-expression techniques. Suitable mutations include point mutations, gene deletions, gene insertions, and any modification of genomic sequences that results in a change in gene expression, such as the over-expression, modification, or inactivation of at least one gene or gene product. Contemplated variants include various species of plants; invertebrates, such as yeasts, insects, and worms; and vertebrates, such as mammals or mammalian cells.
III. Isolation of Life-Span Regulating Yeast Genes and Functionally-Related Orthologs
A. General Techniques
The nucleic acids used to practice this invention, whether RNA, iRNA, antisense nucleic acid, cDNA, genomic DNA, vectors, viruses or hybrids thereof, can be isolated from a variety of sources, genetically engineered, amplified, and/or expressed/generated recombinantly. Recombinant polypeptides generated from these nucleic acids can be individually isolated or cloned and tested for a desired activity. Any recombinant expression system can be used, including bacterial, mammalian, yeast, insect or plant cell expression systems.
Alternatively, these nucleic acids can be synthesized in vitro by well-known chemical synthesis techniques, as described in, e.g., Adams, J. Am. Chem. Soc. 105: 661, 1983; Belousov, Nucleic Acids Res. 25: 3440-3444, 1997; Frenkel, Free Radic. Biol. Med. 19: 373-380, 1995; Blommers, Biochemistry 33: 7886-7896, 1994; Narang, Meth. Enzymol. 68: 90, 1979; Brown Meth. Enzymol. 68: 109, 1979; Beaucage, Tetra. Lett. 22: 1859, 1981; U.S. Pat. No. 4,458,066.
The invention provides oligonucleotides comprising sequences of the invention, e.g., subsequences of the exemplary sequences of the invention. Oligonucleotides can include, e.g., single stranded poly-deoxynucleotides or two complementary polydeoxynucleotide strands which can be chemically synthesized.
Techniques for the manipulation of nucleic acids, such as, e.g., subcloning, labeling probes (e.g., random-primer labeling using Klenow polymerase, nick translation, amplification), sequencing, hybridization and the like are well described in the scientific and patent literature, see, e.g., Sambrook, ed., MOLECULAR CLONING: A LABORATORY MANUAL (2ND ED.), Vols. 1-3, Cold Spring Harbor Laboratory, 1989; CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, Ausubel, ed. John Wiley & Sons, Inc., New York, 1997; LABORATORY TECHNIQUES IN BIOCHEMISTRY AND MOLECULAR BIOLOGY: HYBRIDIZATION WITH NUCLEIC ACID PROBES, Part I. Theory and Nucleic Acid Preparation, Tijssen, ed. Elsevier, N.Y., 1993.
Nucleic acids, vectors, capsids, polypeptides, and the like can be analyzed and quantified by any of a number of general means well known to those of skill in the art. These include, e.g., analytical biochemical methods such as NMR, spectrophotometry, radiography, electrophoresis, capillary electrophoresis, high performance liquid chromatography (HPLC), thin layer chromatography (TLC), and hyperdiffusion chromatography, various immunological methods, e.g., fluid or gel precipitin reactions, immunodiffusion, immuno-electrophoresis, radioimmunoassays (RIAs), enzyme-linked immunosorbent assays (ELISAs), immuno-fluorescent assays, Southern analysis, Northern analysis, dot-blot analysis, gel electrophoresis (e.g., SDS-PAGE), nucleic acid or target or signal amplification methods, radiolabeling, scintillation counting, and affinity chromatography.
Obtaining and manipulating nucleic acids used to practice the methods of the invention can be done by cloning from genomic samples, and, if desired, screening and re-cloning inserts isolated or amplified from, e.g., genomic clones or cDNA clones. Sources of nucleic acid used in the methods of the invention include genomic or cDNA libraries contained in, e.g., mammalian artificial chromosomes (MACs), see, e.g., U.S. Pat. Nos. 5,721,118; 6,025,155; human artificial chromosomes, see, e.g., Rosenfeld, Nat. Genet. 15: 333-335, 1997; yeast artificial chromosomes (YAC); bacterial artificial chromosomes (BAC); P1 artificial chromosomes, see, e.g., Woon, Genomics 50: 306-316, 1998; P1-derived vectors (PACs), see, e.g., Kern, Biotechniques 23: 120-124, 1997; cosmids, recombinant viruses, phages or plasmids.
The invention provides fusion proteins and nucleic acids encoding them. A chronological life span polypeptide of the invention can be fused to a heterologous peptide or polypeptide, such as N-terminal identification peptides which impart desired characteristics, such as increased stability or simplified purification. Peptides and polypeptides of the invention can also be synthesized and expressed as fusion proteins with one or more additional domains linked thereto for, e.g., producing a more immunogenic peptide, to more readily isolate a recombinantly synthesized peptide, to identify and isolate antibodies and antibody-expressing B cells, and the like. Detection and purification facilitating domains include, e.g., metal chelating peptides such as polyhistidine tracts and histidine-tryptophan modules that allow purification on immobilized metals, protein A domains that allow purification on immobilized immunoglobulin, and the domain utilized in the FLAGS extension/affinity purification system (Immunex Corp, Seattle Wash.). The inclusion of a cleavable linker sequences such as Factor Xa or enterokinase (Invitrogen, San Diego, Calif.) between a purification domain and the motif-comprising peptide or polypeptide to facilitate purification. For example, an expression vector can include an epitope-encoding nucleic acid sequence linked to six histidine residues followed by a thioredoxin and an enterokinase cleavage site (see e.g., Williams, Biochemistry 34: 1787-1797, 1995; Dobeli, Protein Expr. Purif 12: 404-414, 1998). The histidine residues facilitate detection and purification while the enterokinase cleavage site provides a means for purifying the epitope from the remainder of the fusion protein. In one aspect, a nucleic acid encoding a polypeptide of the invention is assembled in appropriate phase with a leader sequence capable of directing secretion of the translated polypeptide or fragment thereof. Technology pertaining to vectors encoding fusion proteins and application of fusion proteins are well described in the scientific and patent literature, see e.g., Kroll, DNA Cell. Biol. 12: 441-53, 1993.
B. Verification of Genes Isolated from LL Variants
Genes that confer longevity within identified LL variants can be functionally retested to determine whether the longevity effect observed in LL-classified variants is reproducible. For example, new deletion strains can be re-created by standard homologous recombination methods, and mean RLS for re-created deletion strains can be determined. If a deletion of the gene in question results in life spans substantially higher than that of a “wildtype” reference, then the deleted gene is correctly identified. If the re-created deletion strain is determined to exhibit a life span similar to that of a “wildtype” reference, then it is possible that a genetic change unrelated to a particular known deletion mutation may cause a “long-lived” phenotype observed initially in a variant classified as LL. Various methods known to persons skilled in the art can be utilized to confirm bonafide genes that regulate life spans, and exclude genes that are not related to life-span regulation but are falsely detected.
C. Cloning Genes of Interest from Selected LL Variants
In one aspect, methods of the present invention can be employed for the identification, isolation, and cloning of genes of interest. In one embodiment, methods of the present invention, such as methods described in
Various methods for gene isolation and gene cloning are well-known by persons skilled in the art that enable the identification of a gene of interest having an unknown sequence that may be expressed in one variant and not expressed in a reference variant, or that may not be expressed in one variant and expressed in a reference variant. Exemplary methods for gene isolation and gene cloning include: subtractive or differential hybridization, RT-PCR-mediated differential display (U.S. Pat. No. 5,599,672), and other functionally-equivalent or related methods for gene-cloning known by persons skilled in the art. These methods and many others enable the synthesis of cDNAs corresponding to desirable mRNA transcripts that encode life-span-regulating proteins. Cloned cDNAs of interest can be sequenced so that the determined sequence can be entered into a genomic database in order to identify the gene. In particular, polymerase chain reaction (“PCR”) or other in vitro amplification methods may be used for cloning nucleic acid sequences of interest when a partial or complete sequence is known, as in the characterization of mutants from a known “bar-coded” genomic library.
For in vivo amplification, genes of interest may be subcloned into suitable vectors, such as phage vectors and prokaryotic vectors. Exemplary components of a suitable cloning vector include a replicon recognizable by a prokaryotic host, a gene encoding antibiotic resistance to permit selection of recombinant hosts, and a multiple cloning region containing unique restriction sites within non-essential regions of a plasmid vector so that a gene of interest may be inserted or removed. Examples of cloning plasmids include pBR322-derived plasmids, pSKF, and pET23D.
D. Identification of Life-Span-Regulating Orthologs
1. By Screening Libraries Containing Genomic Sequences
a. Hybridization Conditions
Identified yeast sequences of the present invention can be employed for identifying homologous sequences contained within various invertebrates, such as worms and flies; and vertebrates, including mammals, such as mice, rats, dogs, cats, cows, pigs, horses, and humans. For example, the nucleic acid sequences derived from identified “long-lived” variants can be used as probes for identifying homologous nucleic acid sequences derived from various organisms of diverse species. Identified sequences include genes having at least about 40% sequence similarity to the sequences for the genes indicated in Table 5 or 6, or at least about 70% sequence similarity to these genes. Various methods for assaying sequence homology include hybridization techniques conducted at variable degrees of stringency. To assay for highly-conserved sequences with substantial homology, hybridizations may be conducted under stringent conditions. Under stringent conditions, hybridized pairs of nucleic acids molecules having high thermodynamic stability are selected, in which the hybridized pairs usually consist of highly complementary subsequences. Exemplary stringent hybridization conditions include 50% formamide, 5×SSC and 1% SDS incubated at 42° C., or 5×SSC and 1% SDS incubating at 65° C., which is followed by a wash in 0.2×SSC and 0.1% SDS at 65° C.
For identification of orthologous sequences, including polymorphic alleles and non-polymorphic alleles, that are related to the identified yeast sequences, hybridization conditions that are within a range of low-to-moderately stringent may be more suitable since the percentage of sequence similarity shared between orthologous sequences can be as low as 35%, especially if entire lengths of compared gene sequences are considered. Examples of moderately stringent hybridization conditions include hybridizations performed at 40% formamide, 1 M NaCl, 1% SDS at 37° C., followed by a wash in 1×SSC at 45° C. Generally, decreasing the concentrations of salt, SDS, formamide, and temperature will decrease the stringency of hybridization conditions. Alternative hybridization buffers, wash conditions, and temperature parameters are known by persons skilled in the art of Southern and Northern techniques, and general molecular biology. A hybridization signal intensity of at least twice the intensity of a control probe can be interpreted as a positive identification. Sambrook et al., Molecular Cloning, A Laboratory Manual (2nd ed. 1989) is incorporated by reference in its entirety.
b. Nucleic-Acid Libraries
By utilizing a sub-sequence derived from the identified yeast sequences as probe molecules, various libraries containing desired sequences may be screened. For example, human-derived cellular mRNAs, or human cDNA libraries derived from cellular mRNAs, that presumably contain human orthologous sequences can be screened with a probe containing complementary yeast sequence. Methods for making and screening cDNA libraries are known by persons skilled in the art. Alternatively, a human genomic library presumably containing human orthologous sequences can be screen by utilizing the identified yeast sequence as a probe. Methods for making and screening a genomic library, and methods for sequencing nucleic acid molecules are well-established and known by persons skilled in the art. Although the present examples are based on human libraries, analogous libraries and reagents can be constructed for any organism of interest, including various mammals, such as mice, rats, dogs, cats, cows, pigs, sheep, horses, and other primates. For example, analogous libraries and reagents can be used to identify life-span regulating orthologous sequences represented in various endangered species of fishes, amphibians, reptiles, birds, and mammals. Screening genomic libraries of live-stock animals that are substantially valuable for agricultural use and for recombinant-protein production are also contemplated.
c. Expression Libraries
Alternatively, expression libraries presumably containing orthologs of interest, such as polypeptides having at least about 40% sequence similarity to the sequences for the genes indicated in Table 5 or 6, or at least about 70% sequence similarity to the sequences for the genes indicated in Table 5 or 6, can be screened by utilizing antibodies made against identified yeast homologs, such as the sequences for the genes indicated in Table 6. Orthologs generally contain one or more sub-regions, including regulatory domains and catalytic domains, that are highly-conserved. Antibodies that recognize such highly conserved, functional domains of a conserved homolog are likely to recognize similar domains within a related ortholog. Orthologs identified by such antibodies can be sequenced by various methods known in the art in order to determine the corresponding polypeptide sequences. Expression libraries can be constructed for any organism of interest, including mammalian species, such as mice, rats, dogs, cows, pigs, horses, primates, and human.
d. Degenerate Primers
Alternatively, by utilizing degenerate-oligonucleotide primers based on identified yeast sequences, such as the sequences for the genes indicated in Table 5, various nucleic-acid-amplification techniques, such as polymerase-chain-reaction (PCR), can be used to amplify orthologous sequences from various libraries containing nucleic-acid molecules, including mRNAs, cDNAs, genomic DNA, organelle-specific DNA, and others, that are derived from cells of any organism of interest, including mammalian species, such as mice, rats, dogs, cows, pigs, horses, primates, and human. Various methods for utilizing degenerate-oligonucleotide primers to amplify desired sequences are well-known in the art.
2. By Searching Databases and by Sequence Alignments
Alternatively, genomic databases for model organisms of various species can be employed for conducting multi-genome-wide sequence alignments in order to identify homologous sequences of interest. For each identified yeast sequence, such as the sequences for the genes indicated in Table 5, related orthologous sequences can be determined by searching composite genomic databases. The breath of a database search is limited by the scope of representative model organisms for which sequence data is available.
Homology can be determined by various methods, including alignments of open-reading-frames (“ORFs”) contained in private and/or public databases. Any suitable mathematical algorithm may be used to determine percent identities and percent similarities between any two sequences being compared. For example, nucleic acid and protein sequences of the present invention can be used as a “query sequence” to perform a search against sequences deposited within various public databases to identify other family members or evolutionarily-related sequences. Genomic sequences for various organisms are currently available, including fungi, such as the budding yeast, or Saccharomyces cerevisiae; invertebrates, such as Caenorhabditis elegans and Drosophila melangaster; and mammals, such as the mouse, rat, and human. Exemplary databases for identifying orthologs of interest include Genebank, Swiss Protein, EMBL, and National Center for Biotechnology Information (“NCBI”), and many others known in the art. These databases enable a user to set various parameters for a hypothetical search according to the user's preference, or to utilize default settings. Example 3 provides a table of identified sequences, including various exemplary mammalian orthologs.
To determine and identify sequence identities, structural homologies, motifs and the like in silico, the sequence of the invention can be stored, recorded, and manipulated on any medium which can be read and accessed by a computer. Accordingly, the invention provides computers, computer systems, computer readable mediums, computer programs products and the like recorded or stored thereon the nucleic acid and polypeptide sequences of the invention. As used herein, the words “recorded” and “stored” refer to a process for storing information on a computer medium. A skilled artisan can readily adopt any known methods for recording information on a computer readable medium to generate manufactures comprising one or more of the nucleic acid and/or polypeptide sequences of the invention.
Another aspect of the invention is a computer readable medium having recorded thereon at least one nucleic acid and/or polypeptide sequence of the invention. Computer readable media include magnetically readable media, optically readable media, electronically readable media and magnetic/optical media. For example, the computer readable media can be a hard disk, a floppy disk, a magnetic tape, CD-ROM, Digital Versatile Disk (DVD), Random Access Memory (RAM), or Read Only Memory (ROM) as well as other types of other media known to those skilled in the art.
As used herein, the terms “computer,” “computer program” and “processor” are used in their broadest general contexts and incorporate all such devices.
E. Functional Confirmation of Orthologs
Various genes and gene products isolated from LL variants, and orthologous genes/gene products are useful for identifying pharmaceutical agents that can modulate the activities of these genes/gene products. Prior to utilizing orthologous genes/gene products for screening compounds, the life-span-regulating properties of orthologs can be confirmed by various methods known in the art, which are provided below.
1. Genetic Complementation of Long-Lived Variants by Over-Expression of Orthologs
2. Suppression of Orthologs by RNA Interference
Anti-sense RNAi molecules that contain a sequence complementary to a target mRNA, such as “C-RNAi” 2206, includes various forms of RNAi molecules, such as short-interfering RNAs (“siRNAs”) and short-hairpin RNAs (“shRNAs”) that can be introduced into cellular hosts in various ways. For example, if the host is a worm or a fly, then target-specific RNAi can be introduced as double-stranded RNAs via integrated transposons or by replicating viruses. Double-stranded RNAs can be processed in vivo into siRNAs that can activate RNA-induced silencing complexes (“RISCs”) and degrade homologous mRNA molecules resulting in post-transcriptional gene silencing. For gene inactivation in mammalian cells, introduction of long double-stranded RNA molecules results in nonspecific toxicity due to the activation of the gamma-interferon pathway. Thus, for the identified genes/gene products of the present invention having life-span-regulating activity, various siRNAs of 21-23 base-pairs, may be chemically synthesized and introduced into mammalian host cells by various methods, including transfection, without activating the gamma-interferon pathway.
Alternatively, expression vectors that produce siRNA-like molecules in vivo can be constructed so that promoters, such as RNA-polymerase-II and RNA-polymerase-III, are operably-linked to sequences encoding shRNA molecules. Such shRNAs are processed intracellularly into siRNA-like molecules by “Dicer,” a RNase III family member. These siRNAs are incorporated into RNA-inducing silencing complexes (“RISCs”), in which siRNA duplexes are unwound so that single-stranded siRNA molecules can guide RISC to complementary sub-sequences within target MRNA, resulting in endonucleolytic cleavage of target mRNA. Designing effective siRNA and shRNA are known in the art, and are discussed further in section IV.
Alternative methods for achieving gene inactivation are also known, which can be applied in this assay. For example, genetic suppressor elements (“GSE”) are short and biologically-active cDNA fragments that can interfere with the function of an endogenous gene. GSEs can inactivate a target gene by encoding anti-sense RNA molecules that interfere with the function of the complementary mRNA, and by encoding peptide fragments that act as dominant-negative inhibitors of the full-length target protein. In one embodiment, the present invention is directed to compounds that increase a life span of a host, the compounds comprising an oligonucleotide that interacts with a gene having at least about 40% sequence similarity to at least one of the sequences for the genes indicated in Table 5 or 6, or a gene having at least 70% sequence similarity to at least one of the sequences for the genes indicated in Table 5 or 6. Such DNA oligonucleotide compounds can be single-stranded or double-stranded. In another embodiment, the oligonucleotide compound interacts with a coding strand of a gene. In another embodiment, the oligonucleotide compound interacts with a non-coding strand of a gene.
Alternatively, ribozymes, which are catalytic RNA molecules that can cleave RNA targets containing complementary subsequences, can be used for gene inactivation. A discussion of ribozymes that can inactivate life-span-regulatory genes/gene products are discussed further in section IV.
In addition, non-human transgenic animals in which endogenous genes having life-span-regulating activity are inactivated within somatic and/or germ-line cells can be generated by methods known in the art. In one embodiment, the present invention is directed to these “knock-out” animals that cannot produce gene products encoded by genes having life-span regulating activity, and having at least about 40% sequence similarity to the sequences for the genes indicated in Table 5 or 6, or at least about 70% sequence “similarity” to the sequences for the genes indicated in Table 5 or 6. Exemplary non-human transgenic animals include various flies, worms, and mammals, such as rodents and rabbits.
F. Epistasis Analysis of LL Variants to Identify Mammalian Components Acting in Calorie Restriction (CR) Pathway
Second, the ERC-regulation pathway 2304 controls the rate of extra-chromosomal rDNA (ERCs) formation, involving life-span-regulatory proteins, such as Fob1 and Sir2 that act antagonistically. Fob1 is a replication-fork-barrier protein, and Sir2 is a NAD-dependent-histone deacetylase. ERCs formed by recombination within an rDNA repeat continue to replicate through out the life span of a given mother cell, and partition with the mother cell instead of emerging daughter cells. After successive rounds of mitosis, nuclear accumulation of ERCs in mother cells reaches toxic levels that restrict further cell divisions.
The present inventors are the first to identify a Sir2-independent calorie restriction (CR) pathway in yeast, which may be conserved in a broad range of eukaryotes. Data supporting the characterization of this novel pathway, is provided in Example 4. Example 5 provides an exemplary method utilizing epigenetic analysis to identify mammalian genes/gene products that act either in: (1) the Sir2-independent CR pathway, and (2) other putatively uncharacterized life-span-regulatory pathways 2306. In one embodiment, variants deficient in FOB1 and SIR2, such as fob1Δ-sir2Δ-double-deletion strains, can be used in cell-based assays to identify longevity-promoting compounds that can interact with components of the calorie restriction (CR) pathway in mammals. Drug-screening methods utilizing fob-sir2-deficient strains are further described in section IV.
IV. Methods for Identifying Compounds that Promote Life-Span Extension
Genes sequences identified by present methods can be subcloned into various prokaryotic and eukaryotic vectors for various uses. Various embodiments are directed to prokaryotic expression vectors comprising identified sequences of the present invention, including related orthologs of yeast sequences that have life-span-regulating activities. Various embodiments are directed to eukaryotic expression vectors comprising identified sequences of the present invention, including related orthologs of yeast sequences that have life-span-regulating activities. These expression vectors comprising life-span-regulating sequences (referred to as “present expression vectors”) can be used, for example: (1) to assay identified orthologs to confirm life-span-regulating activities, as described in section III, and (2) to screen compounds in order to identify those that can promote life-span extension, or promote longevity.
Various methods for screening such longevity-promoting compounds include: (1) in vitro screening methods, and (2) in vivo screening methods, provided below. For implementing these methods, identified sequences of the present invention, including related orthologs of yeast sequences, need to be expressed within various cellular hosts. Suitable expression vectors and host cells are provided before discussion of screening methods to facilitate discussion.
A. Expression Vectors
Construction of prokaryotic expression systems that are operable in various prokaryotic cells, and eukaryotic expression systems that are operable in yeast, insects, worms, and various mammalian cells are well-known by persons skilled in the art. Generally, suitable expression vectors include a transcriptional regulatory element operably-linked to a transcriptional unit comprising a gene of interest, such as the gene sequences identified by methods of the present invention that regulate eukaryotic-aging processes, including genes having at least about 40% sequence similarity to the sequences for the genes indicated in Table 5 or 6, or at least about 70% sequence similarity to the sequences for the genes indicated in Table 5 or 6. Suitable gene sequences include double-stranded DNA sequence, such as fragments of genomic DNA, cDNAs, or cDNA fragments, that encode any portion of life-span-regulating polypeptides. A gene of interest can be a chimeric molecule, comprising two or more distinct polynucleotide sequences that together encodes a fusion protein. For example, epitope tags can be optionally added to recombinant proteins of interest in order to provide a molecular label, to facilitate isolation, or other uses. Examples of recombinant techniques can be found in various sources, such as standard laboratory molecular biology treatises, such as Sambrook et al., Molecular Cloning: A Laboratory Manual, 3d ed., 2001, Cold Spring Harbor, which is incorporated in its entirety. Conventional techniques for DNA engineering, including restriction-enzyme digestion, nucleic-acid ligation, and PCR are known by persons skilled in the art.
A transcriptional regulatory element of a transcriptional unit may include one or more promoters and enhancers. Generally, a promoter is positioned upstream of a gene of interest. In contrast, an enhancer can be placed upstream, downstream, or both upstream and down stream of a gene of interest. With respect to a gene of interest, promoter and enhancer elements may be derived from a homologous source, for example, derived from a native gene locus. For example, an endogenous promoter of gene A may be operably-linked to gene A within a vector. Alternatively, promoter and enhancer elements may be derived from a heterologous source, such as from the same gene locus of a different species, from a different gene locus of the same species, or from a different gene locus of a different species.
Various constitutive and inducible promoters that are suitable for either prokaryotic or eukaryotic expression vectors are generally known by persons skilled in the art. Constitutive promoters are transcriptionally activated at steady-state. Inducible promoters are transcriptionally activated upon proper stimulus. Examples of constitutive eukaryotic promoters include various promoters derived from infectious viruses, such as retroviruses, herpes virus, lentivirus, adenovirus and adenovirus variants, and mumps and poliovirus viruses. Promoter and enhancer selections are generally independent of a particular gene of interest, and are dependent on the presence or absence of host-specific factors, such as tissue-specific regulatory factors that are present within a host cell. Promoter and enhancer selections can vary depending on the type of eukaryotic host cell in which the present expression vectors need to be operable. Thus, operable promoters of the present invention include a broad range of constitutive, inducible, tissue-specific, non-tissue-specific, and developmentally-specific promoters and enhancers that can be derived from various viruses, prokaryotes, and eukaryotes.
Optionally, the present expression vectors may include one or more additional sequences that can enhance gene expression. For example, the present expression vectors may include a 3′ untranslated sequence (“UTS”), such as a polyadenylation site that can be derived from the 3′ end of most eukaryotic genes. Various polyadenylation sites may be positioned downstream of the gene of interest, including the 3′ UTS derived from any gene locus, including homologous polyadenylation site and heterologous polyadenylation sites. In addition, a 5′ UTS element may be optionally positioned upstream of a gene of interest to enhance long-term gene expression.
The present expression constructs may also include genes that encode a selectable marker that confers a selectable phenotype upon introduction into various mammalian cells. For example, a selectable marker which confers a selectable phenotype, such as drug resistance, nutritional auxotrophy, resistance to a cytotoxic agent, or expression of a surface protein, may be used. Examples of suitable selectable markers include genes that confer resistance to neomycin, dihydrofolate, puromycin, hygromycin, and histidine D. A selectable marker can be introduced into host cells by incorporating the selectable marker gene within present expression vectors comprising identified sequences having life-span-regulating activities, or by incorporating the marker gene into a distinct vector than can be co-transfected with the present expression vectors.
B. Host Cells
The present expression vectors may be introduced into various host cells that include various cell-lines, primary cells, and secondary cells, known by persons skilled in the art. Methods for gene-delivery include receptor-mediated gene delivery, microinjection, protoplast fusion, and conventional methods of transfection, such as liposome fusion, electroporation, calcium-phosphate precipitation, and other methods known in the art. For protein production using insect cell-lines such as the Hi-fives and Sf-9 cells, viral infection is a preferred method for gene delivery. Vectors designed to target insect tissues for making recombinant proteins are contemplated. (Wurm, Nature Biotechnology 21: 34-35, 2003). To produce recombinant proteins using various mammalian cell lines, transfection and receptor-mediated gene delivery methods, such as virus infection, may be preferred.
C. In Vitro Biochemical Screening Assays
In one embodiment, the present invention is directed to high throughput screening methods to screen longevity-promoting compounds by utilizing identified genes/gene products having life-span-regulating activities as target molecules.
In one embodiment, target molecules of interest are coding and/or non-coding strands of DNA molecules, such as genes, or gene fragments, having at least about 40% sequence similarity to the sequences for the genes indicated in Table 5 or 6, or at least about 70% sequence similarity to the sequences for the genes indicated in Table 5 or 6. In another embodiment, target molecules of interest are proteins, such as polypeptides encoded by genes having at least about 40% sequence similarity to the sequences for the genes indicated in Table 5 or 6, or at least about 70% sequence similarity to the sequences for the genes indicated in Table 5 or 6. In another embodiment, target molecules of interest are RNA molecules, such as RNA sequences encoded by genes having at least about 40% sequence similarity to the sequences for the genes indicated in Table 5 or 6, or at least about 70% sequence similarity to the sequences for the genes indicated in Table 5 or 6. In another embodiment, target molecules of interest are sequences that are complementary to messenger RNAs encoded by genes having at least about 40% sequence similarity to the sequences for the genes indicated in Table 5 or 6, or at least about 70% sequence similarity to the sequences for the genes indicated in Table 5 or 6.
In various embodiments discussed above, various assays, including homogenous assays and separation-based assays, may be employed for detecting products derived from various biochemical reactions. Examples of homogeneous assays, in which the detection of the product does not require a separation step, includes: fluorescence polarization assays, time-resolved-fluorescence-energy-transfer (“HTRF/LANCE™”) assays, conventional fluorescence-resonance-energy-transfer (“FRET”) assays, SPA/flashplate assays, alpha screen assays, and coupled-enzyme assays. Examples of separation-based assays, in which the reaction product is detected after separation from starting reagents, includes: filter-binding assays, precipitation/filtration assays, and enzyme-linked-immunosorbent assays (“ELISA”). These methods and others are known in the art, and reviewed in Walters et al., Nature Reviews 2: 259-266, 2003, which is incorporated by reference.
Other embodiments are directed to virtual-screening methods to identify longevity-promoting compounds by utilizing identified genes/gene products having life-span-regulating activities as target molecules. Virtual libraries are structural databases containing candidate compounds. In virtual screening, at the first-dimensional level, a generic filter is first applied to eliminate chemical structures having properties that are statistically predicted to be unlikely drug candidates. At the second and third dimensional levels, additional levels of filters are applied, which takes into consideration ligand information and conformation, respectively. At the highest dimensional level, various highest-ranking molecules are selected for biostructure-based “docking and scoring,” determined with respect to a binding site of a target protein. These and other related methods are known by persons skilled in the art.
D. Screening Longevity-Promoting Compounds in Sir2-Fob1-Deficient Cells
In another embodiment, genetic variants deficient in sir2 and fob1 may be utilized as hosts for screening longevity-promoting compounds that can modulate regulatory proteins in the Calorie Restriction (CR) pathway.
Various embodiments are directed to high throughput screening methods for evaluating candidate compounds having longevity-promoting properties. In the above method, life spans for “G(1)”-deficient host cells can be determined by applying the various statistical methods used to screen genetic variants described in
When a test compound is determined to be effective in life-span extension, gene products encoded by “G(2)” 2906 that act in the yeast CR pathway are presumably targeted. Such compounds with specificity for yeast components of the CR pathway have high probability of reacting with conserved domains of orthologous eukaryotic sequences. The identification of lead candidate compounds with known structures and known life-span-prolonging activities should facilitate rational-drug design, which is preferred over methods involving randomly-selected compounds. Methods for designing pharmaceutical compounds based on pre-selected lead structures are known by persons skilled in the art.
E. Functional Assays
When the function of a gene product encoded by a gene having at least about 40% sequence similarity the sequences for the genes indicated in Table 5 or 6, or at least about 70% sequence similarity to the sequences for the genes indicated in Table 5 or 6 is known, a function-based assay can be designed. For example, if a gene of interest encodes a transcriptional activator, a reporter construct containing binding-sites specific for the transcriptional activator can be positioned upstream of a reporter gene. The effect of a compound on reporter gene expression can be used to assay for altered function of the transcriptional activator. If a compound is able to interact with the transcriptional activator, the level of reporter-protein production should decrease. In contrast, if a compound cannot interact with the transcriptional activator, the level of reporter-protein production should not decrease, and should be comparable to control levels observed in the absence of the compound.
However, if a function is not known, then compounds that can physically interact with gene products encoded by genes having at least about 40% sequence similarity to the sequences for the genes indicated in Table 5 or 6, or at least about 70% sequence similarity to the sequences for the genes indicated in Table 5 or 6 can be identified by employing, for example, an assay described in
F. Arrays or “Biochips”
The invention provides methods for identifying/screening for modulators (e.g., inhibitors, activators) of a chronological life span activity, e.g., chronological life span-signaling activity, using arrays. Potential modulators, including small molecules, nucleic acids, polypeptides (including antibodies) can be immobilized to arrays. Nucleic acids or polypeptides of the invention can be immobilized to or applied to an array. Arrays can be used to screen for or monitor libraries of compositions (e.g., small molecules, antibodies, nucleic acids, and the like) for their ability to bind to or modulate the activity of a nucleic acid or a polypeptide of the invention, e.g., a chronological life span activity. For example, in one aspect of the invention, a monitored parameter is transcript expression of a gene comprising a nucleic acid of the invention. One or more, or, all the transcripts of a cell can be measured by hybridization of a sample comprising transcripts of the cell, or, nucleic acids representative of or complementary to transcripts of a cell, by hybridization to immobilized nucleic acids on an array, or “biochip.” By using an “array” of nucleic acids on a microchip, some or all of the transcripts of a cell can be simultaneously quantified. Alternatively, arrays comprising genomic nucleic acid can also be used to determine the genotype of a newly engineered strain made by the methods of the invention. Polypeptide arrays can be used to simultaneously quantify a plurality of proteins. Small molecule arrays can be used to simultaneously analyze a plurality of chronological life span modulating or binding activities.
The present invention can be practiced with any known “array,” also referred to as a “microarray” or “nucleic acid array” or “polypeptide array” or “antibody array” or “biochip,” or variation thereof. Arrays are generically a plurality of “spots” or “target elements,” each target element comprising a defined amount of one or more biological molecules, e.g., oligonucleotides, immobilized onto a defined area of a substrate surface for specific binding to a sample molecule, e.g., mRNA transcripts. In practicing the methods of the invention, any known array and/or method of making and using arrays can be incorporated in whole or in part, or variations thereof, as described, for example, in U.S. Pat. Nos. 6,277,628; 6,277,489; 6,261,776; 6,258,606; 6,054,270; 6,048,695; 6,045,996; 6,022,963; 6,013,440; 5,965,452; 5,959,098; 5,856,174; 5,830,645; 5,770,456; 5,632,957; 5,556,752; 5,143,854; 5,807,522; 5,800,992; 5,744,305; 5,700,637; 5,556,752; 5,434,049; see also, e.g., WO 99/51773; WO 99/09217; WO 97/46313; WO 96/17958; see also, e.g., Johnston, Curr. Biol. 8: R171-R174, 1998; Schummer, Biotechniques 23: 1087-1092, 1997; Kern, Biotechniques 23: 120-124, 1997; Solinas-Toldo, Genes, Chromosomes & Cancer 20: 399-407, 1997; Bowtell, Nature Genetics Supp. 21: 25-32, 1999. See also published U.S. patent application Nos. 20010018642; 20010019827; 20010016322; 20010014449; 20010014448; 20010012537; 20010008765.
The terms “array” or “microarray” or “biochip” or “chip” as used herein is a plurality of target elements, each target element comprising a defined amount of one or more polypeptides (including antibodies) or nucleic acids immobilized onto a defined area of a substrate surface.
G. Pharmaceutical Compounds that Promote Longevity
Various genes/gene products isolated from “long-lived” (LL) variants, and related orthologous genes/gene products can be targeted for modulation by pharmaceutical agents. Pharmaceutical agents and methods for screening such pharmaceutical agents that can increase the life expectancy of an organism are highly desirable for decreasing the rate of metabolic aging in humans. In addition, by slowing the rate of aging, it may be possible to delay the onset of a variety of diseases associated with the aging process, including various types of cancers, diabetes, cataracts, heart diseases, and neurodegenerative diseases, such as Parkinson's disease, Huntington disease, amyloid diseases, and others. An effective compound can increase the life span of a host by at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%.
1. Sequence-Specific Compounds
a. RNAi Compounds
In one embodiment, various siRNAs that are complementary to identified sequences of the present invention, including mammalian orthologs, can be employed by persons skilled in the art in order to prevent or to decrease the expression of life-span-regulating genes/gene products. Identified sequences include genes having at least about 40% sequence similarity to having at least about 40% sequence similarity the sequences for the genes indicated in Table 5 or 6, or at least about 70% sequence similarity to the sequences for the genes indicated in Table 5 or 6. Silencing or inactivation of life-span-regulating genes/gene products within selective tissues of a host receiving such RNAi compounds can increase the longevity of the host. As discussed briefly in section III, because introduction of double-stranded RNA (“dsRNA”) that are longer than 30 nucleotides into mammalian cells induces a sequence-nonspecific interferon response, alternative methods for delivery of interfering RNA molecules (“RNAi”) may be suitable. For example, most common form of RNAi molecules are short-interfering RNAs (“siRNAs”) of 21-23 base-pairs that are chemically or enzymatically synthesized, which can be introduced into mammalian host cells by various methods, including transfections. However, unlike fungi, plants, and worms that can replicate siRNAs in vivo, transfection of siRNA produces only transient gene-silencing effect in mammalian cells. As an alternative, DNA vectors encoding precursor-like forms of siRNAs may be used for stable production of siRNAs in vivo in various hosts, including mammalian cells.
In one embodiment, the present invention is directed to compounds that increase a life span of a host, the compounds comprising an oligonucleotide that interacts with a gene product encoded by the gene having at least about 40% sequence similarity to at least one the sequences for the genes indicated in Table 5 of 6, or a gene having at least 70% sequence similarity to at least one the sequences for the genes indicated in Table 5 or 6. Such RNA oligonucleotide compounds can be single-stranded or double-stranded. Suitable lengths of RNA oligonucleotides include molecules containing 15-20 nucleotides, 20-30 nucleotides, 30-50 nucleotides, 50-75 nucleotides, 75-100 nucleotides, 100-150 nucleotides, 150-200 nucleotides, and 200-300 nucleotides. In another embodiment, the present invention is directed to compounds that increase a life span of a host, the compounds comprising an oligonucleotide that interacts with sequences that are complementary to gene products encoded by a gene having at least about 40% sequence similarity to at least one the sequences for the genes indicated in Table 5 or 6, or a gene having at least 70% sequence similarity to at least one the sequences for the genes indicated in Table 5 or 6. In another embodiment, the present invention is directed to compounds that increase a life span of a host, the compounds include an anti-sense strand that hybridizes to an endogenous messenger RNA that encodes a protein having life-span-regulating activity, and that inhibits the translation of the messenger RNA.
Selection of efficient siRNAs is an empirical process, but certain rules governing optimal selection of siRNAs are known. The sequence selected for a siRNA appears to be critical. For example, siRNAs containing sequence motifs, such as AAN19TT, NAN19NN, NARN17YNN, and NANN17YNN, are effective, in which N is any nucleotide, R is a purine, and Y is a pyrimidine. In addition, regions of complementary DNA should have non-repetitive sequences, and should avoid intronic sequences. Suitable siRNAs contain approximately 30-70% GC content, contain even representation of all nucleotides on the anti-sense strand, and do not contain stretches of single nucleotide, especially stretches of Gs.
Although any region of mRNA can be theoretically targeted, certain sequences that are known binding sites for mRNA-binding proteins should be avoided, including untranslated regions, such as the “5′UTR” and “3′UTR,” start-codons, and exon-exon boundaries. For some mRNA targets, siRNA-directed silencing may be more effective if the siRNA sequence is selected at least 50-100 nucleotides downstream of a start codon, and preferably directed towards the 3′ end of a target mRNA. In addition, the conformation of a mRNA recognition site within an mRNA target is preferably RNAse-H-sensitive, and preferably not within a highly-structured RNA region. These guidelines are generally applicable since the choice of a siRNA depends on the target mRNA sequence, and persons skilled in the art would need to synthesize several siRNAs to validate the efficiency of each. The specificity of a siRNA for a single gene can be ascertained by performing a multiple-genome-sequence alignment, such as a BLAST search of the selected sequence against sequence databases, including “Unigene” libraries associated with National Center for Biotechnology Information (NCBI). Potential off-target silencing by siRNA may be minimized by choosing a siRNA sequence with maximum sequence divergence from a list of genes with partial-sequence identity to the intended mRNA target. General principles for siRNA selection are taught by the following two review articles, which are incorporated by reference. (Dorsett and Tuschl, Nature Reviews 3: 318-329, 2004; Dykxhoorn et al., Nature Reviews 4: 457-467, 2003).
Various expression vectors can be constructed to enable stable production of siRNA-like molecules in vivo. For example, RNA-pol II promoters may be operably-linked to a hairpin precursor of a siRNA sequence of interest. RNA-pol II promoters represent a broad range of promoters that enable substantial control over parameters governing RNA expression, such as inducible, constitutive, tissue-specific, or developmentally-regulated RNA expression. Alternatively, RNA-pol III promoters may be used to produce short RNA species that do not activate the interferon pathway. Suitable RNA-pol III promoters include class III promoters that lack essential transcriptional elements downstream of a transcription initiation site, such as U6 and H1 promoters, which may be operably-linked to a siRNA-encoding sequence.
In the above, long-hairpin RNAs, imperfect shRNAs, miRNAs, and siRNAs, can be designed as follows. For example, a sub-sequence of a messenger RNA encoded by a gene, including genes having at least about 40% sequence similarity to the sequences for the genes indicated in Table 5 or 6, or at least about 70% sequence similarity to the sequences for the genes indicated in Table 5 or 6, may be selected for targeting. For example, an anti-sense strand of shRNA can be designed by selecting a sub-sequence portion of a complementary RNA sequence to a messenger RNA sequence of interest, which is encoded by any gene having at least about 40% sequence similarity to the sequences for the genes indicated in Table 5 or 6, or at least about 70% sequence similarity to the sequences for the genes indicated in Table 5 or 6.
For designing shRNA, the composition and size of the loop and length of the stem of a hairpin duplex should be considered. Suitable stem lengths for efficient silencing include a broad range, including stem lengths of 19-29 nucleotides. Suitable loop lengths for efficient silencing include a broad range, including loop lengths of 4-23 nucleotides. In certain context, hairpin structures with duplexed regions that are longer than 21 nucleotides may promote effective siRNA-directed silencing, regardless of loop sequence and length.
Various gene-delivery vectors that are practiced by persons skilled in the art can be used to introduce the present expression vectors. Examples of viral vectors that may be used to infect host cells include: improved adenoviral vectors that can target pulmonary tissues (Reynold et al., Nature Biotechnology 19: 838-842, 2001); gene-deleted adenovirus-transposon vectors that can stably maintain virus-encoded gene of interests in vivo through integration into host chromosomes (Yant et al., Nature Biotechnology 20: 999-1005, 2002); recombinant adenoviruses that can target rat brain neurons (Bilang-Bleuel et al., Proc. Natl. Acad. Sci. U.S.A. 94: 8818-8823, 1997); the Moloney-murine-leukemia-virus (“Mo-MuLV”) based retroviral vectors that can target CD4+ and CD+8 Tcells and monocyte-macrophages (Auten et al., Human Gene Therapy 10: 1389-99, 2003); and poliovirus-replicon-based vectors that can target tissues of the central nervous tissue (Bledsoe et al., Nature Biotechnology 18: 964-969, 2000). Examples of other suitable viral vectors include: herpes virus, mumps virus, Sindbis virus, vaccinia virus, such as the canary pox virus, and lentivirus. The usage of viral vectors is well known by persons skilled in the art, and for gene therapy uses, viral infection is preferred generally. Robbins and Ghizzani, Mol. Med. Today 1: 410-417, 1995) is incorporated by reference.
b. Chemically Modified Anti-Sense Oligodeoxyribonucleic Acids (“ODNs”)
In one embodiment, various ODN molecules that are complementary to endogenous mRNA transcripts identified as having life-span-regulating activity, and having at least about 40% sequence similarity to the sequences for the genes indicated in Table 5 or 6, or at least about 70% sequence similarity to the sequences for the genes indicated in Table 5 or 6, can be employed by persons skilled in the art in order to prevent or to decrease the expression of life-span-regulating genes/gene products. Silencing or inactivation of life-span-regulating genes/gene products within selective tissues of a host receiving such ODN compounds can increase the longevity of the host. Various ODN compounds can be screened in assays comparable to the assays described in
c. Ribozymes
In one embodiment, various ribozymes containing sequences that are complementary to mRNAs encoded by genes having at least about 40% sequence similarity to the sequences for the genes indicated in Table 5 or 6, or at least about 70% sequence similarity to the sequences for the genes indicated in Table 5 or 6, can be employed by persons skilled in the art in order to prevent or to decrease the expression of life-span-regulating genes/gene products. Silencing or inactivation of life-span-regulating genes/gene products within selective tissues of a host receiving such ribozyme compounds can increase the longevity of the host. Ribozymes, including the “hammer-head” ribozyme, are RNA molecules that bind target mRNA by assuming a unique secondary structure when hybridized to target mRNA, which enables catalytic hydrolysis of a phosphodiester bond within in the backbone of target mRNA. Efficient cleavage by a ribozyme requires the presence of divalent ions, such as magnesium, and is also dependent on target RNA structure, and relative proximity between ribozyme and target molecule. RNA-localization signals or RNA chaperones may be used so that low concentrations of ribozymes are sufficiently effective in silencing a gene of interest. Ribozymes can be chemically synthesized in vitro, and can be transcribed from expression vectors in vivo. Methods for ribozyme construction and utilization are known by persons skilled in the art. Doudna and Cech, Nature, 418: 222-228, 2002; Kuwabara et al, J. Biochem. 132: 149-155, 2002; Michienzi and Rossi, Methods Enzymol. 341: 581-596, 2001; and Good et al., Gene Ther., 4: 45-54, 1997 are incorporated by reference.
d. Full and Partial Length Antisense RNA Transcripts
Antisense RNA transcripts have a base sequence complementary to part or all of any other RNA transcript in the same cell. Such transcripts have been shown to modulate gene expression through a variety of mechanisms including the modulation of RNA splicing, the modulation of RNA transport and the modulation of the translation of mRNA (Denhardt, Ann N Y Acad. Sci. 660: 70, 1992; Nellen, Trends Biochem. Sci. 18: 419, 1993; Baker and Monia, Biochem. Biophys. Acta, 1489: 3, 1999; Xu et al., Gene Therapy 7: 438, 2000; French and Gerdes, Curr. Opin. Microbiol. 3: 159, 2000; Terryn and Rouze, Trends Plant Sci. 5: 1360, 2000).
2. Other Compounds
In one embodiment, non-sequence-specific compounds that can interact with identified genes/gene products having at least about 40% sequence similarity to the sequences for the genes indicated in Table 5 or 6, or at least about 70% sequence similarity to the sequences for the genes indicated in Table 5 or 6, can be employed by persons skilled in the art in order to prevent or to decrease the expression of life-span-regulating genes/gene products. In addition, compounds that can interact with RNA and DNA molecules that are complementary to sequences having at least about 40% sequence similarity to the sequences for the genes indicated in Table 5 or 6, or at least about 70% sequence similarity to the sequences for the genes indicated in Table 5 or 6 may be used. Silencing or inactivation of life-span-regulating genes/gene products within selective tissues of a host receiving such compounds can increase the longevity of the host. Longevity-promoting compounds can be either naturally-occurring or synthetically-produced. Large combinatorial libraries of chemical/biological compounds can be generated by various chemical and biological synthesis methods known in the art. Such combinatorial chemical libraries include: small organic molecule libraries (benzodiazepines, Baum, C&EN 33, 1993); (Chen et al., J. Amer. Chem. Soc., 116: 2661, 1994,) such as isoprenoids (U.S. Pat. No. 5,569,588), thiazolidinones and metathiazanones (U.S. Pat. No. 5,549,974), pyrrolidines (U.S. Pat. Nos. 5,525,735 and 5,519,134), morpholino compounds (U.S. Pat. No. 5,506,337), and benzodiazepines (U.S. Pat. No. 5,288,514), oligocarbamates (Cho et al., Science 261: 1303, 1993), and peptidyl phosphonates (Campbell et al., J. Org. Chem. 59: 658, 1994). Exemplary combinatorial libraries include: various peptide libraries (U.S. Pat. No. 5,010,175; Furka, Int. J. Pept. Prot. Res. 37: 487-493, 1991); Houghton et al., Nature 354: 84-88, 1991); peptoid libraries (PCT Publication No. WO 91/19735); encoded peptides (PCT Publication No. WO 93/20242); random bio-oligomers (PCT Publication No. WO 92/00091); benzodiazepines (U.S. Pat. No. 5,288,514); diversomers such as hydantoins, benzodiazepines and dipeptides (Hobbs et al., Proc. Nat. Acad. Sci. USA 90: 6909-6913, 1993); vinylogous polypeptides (Hagihara et al., J. Amer. Chem. Soc. 114: 6568, 1992); nonpeptidal peptidomimetics with glucose scaffolding (Hirschmann et al., J. Amer. Chem. Soc. 114: 9217-9218, 1992); various nucleic-acid libraries; various peptide-nucleic acid libraries (U.S. Pat. No. 5,539,083); various carbohydrate libraries (Liang et al., Science 274: 1520-1522, 1996); (U.S. Pat. No. 5,593,853); and various antibody libraries (Vaughn et al., Nature Biotechnology 14: 309-314, 1996).
3. Therapeutic Antibodies
In another embodiment, various antibodies that bind specifically to proteins of the present invention having at least about 40% sequence similarity to the sequences for the genes indicated in Table 5 or 6, or at least about 70% sequence similarity to the sequences for the genes indicated in Table 5 or 6, can be employed by persons skilled in the art in order to prevent or to decrease the expression of life-span-regulating genes/gene products. Suitable antibodies have antigen-binding domains that can react with polypeptides having at least about 40% sequence similarity to the sequences for the genes indicated in Table 5 or 6, or at least about 70% sequence similarity to the sequences for the genes indicated in Table 5 or 6. In addition, constant regions of such antibodies include various isotypic variants, such as kappa and lambda isotypes. Antibodies of the present invention include IgG, IgM, IgA, IgD, and IgE molecules containing different Fc regions that can interact with different effector cells of an immune system. Inactivation of life-span-regulating gene products within selective tissues of a host receiving such antibodies can increase the longevity of the host. Methods for producing polyclonal and monoclonal antibodies that can react specifically with life-span-regulating proteins are well-known in the art. Any fragment of such proteins may be used as an immunogen to produce antibodies with specificity for life-span-regulating proteins identified by the present invention. For example, suitable immunogens can be derived from an isolated endogenous protein or an antigenic fragment thereof, an isolated recombinant protein or an antigenic fragment thereof, and a synthetic peptide representing a portion of a life-span-regulating protein conjugated to a carrier protein. Methods for the preparation of various forms of immunogen are known by persons skilled in the art. In general, immunogens formulated in a standard adjuvant, such as Freund's adjuvant, are injected into warm-blooded animals capable of producing antibodies, such as mice, rabbits, and goats. Methods for polyclonal antibody production are known to persons skilled in the art, in which blood containing high titers of polyclonal antibodies are collected, including antisera enriched for immunogen-specific antibodies. Methods for monoclonal antibody production are known in the art, in which spleen cells that are removed from an animal immunized with an immunogen of interest are fused with an immortalized cell, such as a myeloma cell (Kohler & Milstein, Eur. J. Immunol. 6: 511-519, 1976) or may be transformed with viruses, such as retroviruses containing oncogenes, such as the Epstein Barr Virus. Individual hybridoma cells are screened to identify monoclonal antibodies having desired specificity and affinity. Alternatively, chimeric forms of antibodies containing mouse and human sequences that have specificity to life-span-regulating proteins of the present invention having at least about 40% sequence similarity to the sequences for the genes indicated in Table 5 or 6, or at least about 70% sequence similarity to the sequences for the genes indicated in Table 5 or 6, may be produced (Harlow and Lane Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory (1988) is incorporated by reference). Alternatively, humanized antibodies, containing selective amino-acid residues derived from non-human sources, that have specificity to life-span-regulating proteins of the present invention may be produced. (Verhoeyen et al., Science 239: 1534-1536, 1988). Alternatively, large libraries of human Fabs generated by various combinatorial methods using recombinant molecular biology and phage-display methods can be screened to identify antibodies that have high specificity and affinity for life-span-regulating proteins of the present invention. (de Haard et al., J. Biol. Chem. 274: 18218-18230, 1999).
4. Replicative Life Span Transgenic and “Knockout” Non-Human Animals
The invention provides transgenic non-human animals comprising a nucleic acid, a polypeptide, an expression cassette or vector or a transfected or transformed cell of the invention. The transgenic non-human animals can be, e.g., goats, rabbits, sheep, pigs, cows, rats and mice, comprising the nucleic acids of the invention. A “transgenic animal” is an animal having cells that contain DNA which has been artificially inserted into a cell, which DNA becomes part of the genome of the animal which develops from that cell. Preferred transgenic animals are primates, mice, rats, cows, pigs, horses, goats, sheep, dogs and cats. The transgenic DNA can encode mammalian kinases. Native expression in an animal can be reduced by providing an amount of antisense RNA or DNA effective to reduce expression of the receptor.
These animals can be used, e.g., as in vivo models to study which is modulators of a replicative life span-signaling activity, or, as models to screen for agents that change the replicative life span-signaling activity in vivo.
In one aspect, the inserted transgenic sequence is a sequence of the invention designed such that it does not express a functional replicative life span polypeptide. The defect can be designed to be on the transcriptional, translational and/or the protein level.
The coding sequences for the polypeptides, the replicative life span polypeptides, or mutant polypeptide to be expressed in the transgenic non-human animals can be designed to be constitutive, or, under the control of tissue-specific, developmental-specific or inducible transcriptional regulatory factors. Transgenic non-human animals can be designed and generated using any method known in the art; see, e.g., U.S. Pat. Nos. 6,211,428; 6,187,992; 6,156,952; 6,118,044; 6,111,166; 6,107,541; 5,959,171; 5,922,854; 5,892,070; 5,880,327; 5,891,698; 5,639,940; 5,573,933; 5,387,742; 5,087,571, describing making and using transformed cells and eggs and transgenic mice, rats, rabbits, sheep, pigs and cows. See also, e.g., Pollock, J. Immunol. Methods 231: 147-157, 1999, describing the production of recombinant proteins in the milk of transgenic dairy animals; Baguisi, Nat. Biotechnol. 17: 456-461, 1999, demonstrating the production of transgenic goats. U.S. Pat. No. 6,211,428, describes making and using transgenic non-human mammals which express in their brains a nucleic acid construct comprising a DNA sequence. U.S. Pat. No. 5,387,742, describes injecting cloned recombinant or synthetic DNA sequences into fertilized mouse eggs, implanting the injected eggs in pseudo-pregnant females, and growing to term transgenic mice whose cells express proteins related to the pathology of Alzheimer's disease. U.S. Pat. No. 6,187,992, describes making and using a transgenic mouse whose genome comprises a disruption of the gene encoding amyloid precursor protein (APP). One exemplary method to produce genetically altered non-human animals is to genetically modify embryonic stem cells. The modified cells are injected into the blastocoel of a blastocyst. This is then grown in the uterus of a pseudopregnant female. In order to readily detect chimeric progeny, the blastocysts can be obtained from a different parental line than the embryonic stem cells. For example, the blastocysts and embryonic stem cells can be derived from parental lines with different hair color or other readily observable phenotype. The resulting chimeric animals can be bred in order to obtain non-chimeric animals which have received the modified genes through germ-line transmission. Techniques for the introduction of embryonic stem cells into blastocysts and the resulting generation of transgenic animals are well known.
Because cells contain more than one copy of a gene, the cell lines obtained from a first round of targeting are likely to be heterozygous for the targeted allele. Homozygosity, in which both alleles are modified, can be achieved in a number of ways. In one approach, a number of cells in which one copy has been modified are grown. They are then subjected to another round of targeting using a different selectable marker. Alternatively, homozygotes can be obtained by breeding animals heterozygous for the modified allele, according to traditional Mendelian genetics. In some situations, it may be desirable to have two different modified alleles. This can be achieved by successive rounds of gene targeting or by breeding heterozygotes, each of which carries one of the desired modified alleles. See, e.g., U.S. Pat. No. 5,789,215.
A variety of methods are available for the production of transgenic animals associated with this invention. DNA can be injected into the pronucleus of a fertilized egg before fusion of the male and female pronuclei, or injected into the nucleus of an embryonic cell (e.g., the nucleus of a two-cell embryo) following the initiation of cell division. (Brinster et al., Proc. Nat. Acad. Sci. USA 82: 4438-4442, 1985). Embryos can be infected with viruses, especially retroviruses, modified to carry inorganic-ion receptor nucleotide sequences of the invention.
Pluripotent stem cells derived from the inner cell mass of the embryo and stabilized in culture can be manipulated in culture to incorporate nucleotide sequences of the invention. A transgenic animal can be produced from such cells through implantation into a blastocyst that is implanted into a foster mother and allowed to come to term. Animals suitable for transgenic experiments can be obtained from standard commercial sources such as Charles River (Wilmington, Mass.), Taconic (Germantown, N.Y.), Harlan Sprague Dawley (Indianapolis, Ind.), and the like
The procedures for manipulation of the rodent embryo and for microinjection of DNA into the pronucleus of the zygote are well known to those of ordinary skill in the art (Hogan et al., supra). Microinjection procedures for fish, amphibian eggs and birds are detailed in Houdebine and Chourrout, Experientia 47: 897-905, 1991. Other procedures for introduction of DNA into tissues of animals are described in U.S. Pat. No. 4,945,050 (Sanford et al., Jul. 30, 1990).
By way of example only, to prepare a transgenic mouse, female mice are induced to superovulate. Females are placed with males, and the mated females are sacrificed by CO.sub.2 asphyxiation or cervical dislocation and embryos are recovered from excised oviducts. Surrounding cumulus cells are removed. Pronuclear embryos are then washed and stored until the time of injection. Randomly cycling adult female mice are paired with vasectomized males. Recipient females are mated at the same time as donor females. Embryos then are transferred surgically. The procedure for generating transgenic rats is similar to that of mice. (Hammer et al., Cell 63: 1099-1112, 1990).
Methods for the culturing of embryonic stem (ES) cells and the subsequent production of transgenic animals by the introduction of DNA into ES cells using methods such as electroporation, calcium phosphate/DNA precipitation and direct injection also are well known to those of ordinary skill in the art (Teratocarcinomas and Embryonic Stem Cells, A Practical Approach, E. J. Robertson, ed., IRL Press, 1987).
In cases involving random gene integration, a clone containing the sequence(s) of the invention is co-transfected with a gene encoding resistance. Alternatively, the gene encoding neomycin resistance is physically linked to the sequence(s) of the invention. Transfection and isolation of desired clones are carried out by any one of several methods well known to those of ordinary skill in the art (E. J. Robertson, supra).
DNA molecules introduced into ES cells can also be integrated into the chromosome through the process of homologous recombination. (Capecchi, Science 244: 1288-1292, 1989). Methods for positive selection of the recombination event (i.e., neo resistance) and dual positive-negative selection (i.e., neo resistance and gancyclovir resistance) and the subsequent identification of the desired clones by PCR have been described by Capecchi, supra and Joyner et al., Nature 338: 153-156, 1989, the teachings of which are incorporated herein in their entirety including any drawings. The final phase of the procedure is to inject targeted ES cells into blastocysts and to transfer the blastocysts into pseudopregnant females. The resulting chimeric animals are bred and the offspring are analyzed by Southern blotting to identify individuals that carry the transgene. Procedures for the production of non-rodent mammals and other animals have been discussed by others. (Houdebine and Chourrout, supra; Pursel et al., Science 244: 1281-1288, 1989; and Simms et al., Bio/Technology 6: 179-183, 1988).
5. Replicative Life Span Functional Knockouts
The invention provides non-human animals that do not express their endogenous replicative life span polypeptides, or, express their endogenous replicative life span polypeptides at lower than wild type levels (thus, while not completely “knocked out” their replicative life span activity is functionally “knocked out”). The invention also provides “knockout animals” and methods for making and using them. For example, in one aspect, the transgenic or modified animals of the invention comprise a “knockout animal,” e.g., a “knockout mouse,” engineered not to express an endogenous gene, e.g., an endogenous replicative life span gene, which is replaced with a gene expressing a polypeptide of the invention, or, a fusion protein comprising a polypeptide of the invention. Thus, in one aspect, the inserted transgenic sequence is a sequence of the invention designed such that it does not express a functional replicative life span polypeptide. The defect can be designed to be on the transcriptional, translational and/or the protein level. Because the endogenous replicative life span gene has been “knocked out,” only the inserted polypeptide of the invention is expressed.
A “knock-out animal” is a specific type of transgenic animal having cells that contain DNA containing an alteration in the nucleic acid sequence that reduces the biological activity of the polypeptide normally encoded therefrom by at least 80% compared to the unaltered gene. The alteration can be an insertion, deletion, frameshift mutation, missense mutation, introduction of stop codons, mutation of critical amino acid residue, removal of an intron junction, and the like. Preferably, the alteration is an insertion or deletion, or is a frameshift mutation that creates a stop codon. Typically, the disruption of specific endogenous genes can be accomplished by deleting some portion of the gene or replacing it with other sequences to generate a null allele. Cross-breeding mammals having the null allele generates a homozygous mammals lacking an active copy of the gene.
A number of such mammals have been developed, and are extremely helpful in medical development. For example, U.S. Pat. No. 5,616,491 describes knock-out mice having suppression of CD28 and CD45. Procedures for preparation and manipulation of cells and embryos are similar to those described above with respect to transgenic animals, and are well known to those of ordinary skill in the art.
A knock out construct refers to a uniquely configured fragment of nucleic acid which is introduced into a stem cell line and allowed to recombine with the genome at the chromosomal locus of the gene of interest to be mutated. Thus, a given knock out construct is specific for a given gene to be targeted for disruption. Nonetheless, many common elements exist among these constructs and these elements are well known in the art. A typical knock out construct contains nucleic acid fragments of about 0.5 kb to about 10.0 kb from both the 5′ and the 3′ ends of the genomic locus which encodes the gene to be mutated. These two fragments are typically separated by an intervening fragment of nucleic acid which encodes a positive selectable marker, such as the neomycin resistance gene. The resulting nucleic acid fragment, consisting of a nucleic acid from the extreme 5′ end of the genomic locus linked to a nucleic acid encoding a positive selectable marker which is in turn linked to a nucleic acid from the extreme 3′ end of the genomic locus of interest, omits most of the coding sequence for the gene of interest to be knocked out. When the resulting construct recombines homologously with the chromosome at this locus, it results in the loss of the omitted coding sequence, otherwise known as the structural gene, from the genomic locus. A stem cell in which such a rare homologous recombination event has taken place can be selected for by virtue of the stable integration into the genome of the nucleic acid of the gene encoding the positive selectable marker and subsequent selection for cells expressing this marker gene in the presence of an appropriate drug.
Variations on this basic technique also exist and are well known in the art. For example, a “knock-in” construct refers to the same basic arrangement of a nucleic acid encoding a 5′ genomic locus fragment linked to nucleic acid encoding a positive selectable marker which in turn is linked to a nucleic acid encoding a 3′ genomic locus fragment, but which differs in that none of the coding sequence is omitted and thus the 5′ and the 3′ genomic fragments used were initially contiguous before being disrupted by the introduction of the nucleic acid encoding the positive selectable marker gene. This “knock-in” type of construct is thus very useful for the construction of mutant transgenic animals when only a limited region of the genomic locus of the gene to be mutated, such as a single exon, is available for cloning and genetic manipulation. Alternatively, the “knock-in” construct can be used to specifically eliminate a single functional domain of the targeted gene, resulting in a transgenic animal which expresses a polypeptide of the targeted gene which is defective in one function, while retaining the function of other domains of the encoded polypeptide. This type of “knock-in” mutant frequently has the characteristic of a so-called “dominant negative” mutant because, especially in the case of proteins which homomultimerize, it can specifically block the action of the polypeptide product of the wild-type gene from which it was derived.
Each knockout construct to be inserted into the cell must first be in the linear form. Therefore, if the knockout construct has been inserted into a vector, linearization is accomplished by digesting the DNA with a suitable restriction endonuclease selected to cut only within the vector sequence and not within the knockout construct sequence. For insertion, the knockout construct is added to the ES cells under appropriate conditions for the insertion method chosen, as is known to the skilled artisan. Where more than one construct is to be introduced into the ES cell, each knockout construct can be introduced simultaneously or one at a time.
After suitable ES cells containing the knockout construct in the proper location have been identified by the selection techniques outlined above, the cells can be inserted into an embryo. Insertion can be accomplished in a variety of ways known to the skilled artisan, however a preferred method is by microinjection. For microinjection, about 10-30 cells are collected into a micropipette and injected into embryos that are at the proper stage of development to permit integration of the foreign ES cell containing the knockout construct into the developing embryo. For instance, the transformed ES cells can be microinjected into blastocytes. The suitable stage of development for the embryo used for insertion of ES cells is very species dependent, however for mice it is about 3.5 days. The embryos are obtained by perfusing the uterus of pregnant females. Suitable methods for accomplishing this are known to the skilled artisan. After the ES cell has been introduced into the embryo, the embryo can be implanted into the uterus of a pseudopregnant foster mother for gestation as described above.
Yet other methods of making knock-out or disruption transgenic animals are also generally known. See, for example, Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986). Recombinase dependent knockouts can also be generated, e.g. by homologous recombination to insert target sequences, such that tissue specific and/or temporal control of inactivation of a target gene can be controlled by recombinase sequences (described infra).
Animals containing more than one knockout construct and/or more than one transgene expression construct are prepared in any of several ways. The preferred manner of preparation is to generate a series of mammals, each containing one of the desired transgenic phenotypes. Such animals are bred together through a series of crosses, backcrosses and selections, to ultimately generate a single animal containing all desired knockout constructs and/or expression constructs, where the animal is otherwise congenic (genetically identical) to the wild type except for the presence of the knockout construct(s) and/or transgene(s).
The functional replicative life span “knockout” non-human animals of the invention are of several types. Some non-human animals of the invention that are functional replicative life span “knockouts” express sufficient levels of a replicative life span inhibitory nucleic acid, e.g., antisense sequences or ribozymes of the invention, to decrease the levels or knockout the expression of functional polypeptide. Some non-human animals of the invention that are functional replicative life span “knockouts” express sufficient levels of a replicative life span dominant negative polypeptide such that the effective amount of free endogenous active replicative life span is decreased. Some non-human animals of the invention that are functional replicative life span “knockouts” express sufficient levels of an antibody of the invention, e.g., a replicative life span antibody, such that the effective amount of free endogenous active replicative life span protein is decreased. Some non-human animals of the invention that are functional replicative life span “knockouts” are “conventional” knockouts in that their endogenous replicative life span gene has been disrupted or mutated.
Functional replicative life span “knockout” non-human animals of the invention also include the inbred mouse strain of the invention and the cells and cell lines derived from these mice.
The invention provides methods for treating a subject with a replicative life span related disease or disorder. The method comprises providing an inhibitor of a replicative life span activity, e.g., a nucleic acid (e.g., antisense, ribozyme) or a polypeptide (e.g., antibody or dominant negative) of the invention. The inhibitor is administered in sufficient amounts to the subject to inhibit the expression of replicative life span polypeptides.
6. Replicative Life Span Inbred Mouse Strains
The invention provides an inbred mouse and an inbred mouse strain that can be generated as described herein and bred by standard techniques, see, e.g., U.S. Pat. Nos. 6,040,495; 5,552,287.
In order to screen for mutations with recessive effects a number of strategies can be used, all involving a further two generations. For example, male G1 mice can be bred to wild-type female mice. The resulting progeny (G2 mice) can be interbred or bred back to the G1 father. The G3 mice that result from these crosses will be homozygotes for mutations in a small number of genes (3-6) in the genome, but the identity of these genes is unknown. With enough G3 mice, a good sampling of the genome should be present.
7. Pharmaceutical Compositions and Therapeutic Dosage and Administration
Pharmaceutical formulations for effective delivery of pharmaceutical compounds of the present invention will vary depending on the pharmaceutical compound of interest and mode of administration. Suitable pharmaceutical carriers are known by persons skilled in the art (Remington's Pharmaceutical Sciences (1989), which is incorporated in its entirety). Pharmaceutical compounds described above can be administered by various methods, including by injection, oral administration, inhalation, transdermal application, or rectal administration. For oral administration, suitable formulations containing a pharmaceutical compound and pharmaceutically-compatible carriers can be delivered in various forms, such as tablets or capsules, liquid solutions, suspensions, emulsions, and the like. For inhalation, suitable formulations containing a pharmaceutical compound and pharmaceutically-compatible carriers can be delivered as aerosol formulations that can be placed into pressurized propellants, such as dichlorodifluoromethane, propane, nitrogen, and the like. For parenteral administration, suitable formulations containing a pharmaceutical compound and pharmaceutically-compatible carriers can be delivered by intra-articular, intra-venous, intramuscular, intra-dermal, intra-peritoneal, and sub-cutaneous routes.
8. Toxicity
Pharmaceutical compositions suitable for use in the present invention include compositions containing active ingredients in an effective amount to achieve its intended purpose. More specifically, a therapeutically-effective amount means an amount effective to prevent pre-mature aging or to delay aging-process in subjects exposed to pharmaceutical compositions of the present invention. Determination of the effective amounts is well within the capability of persons skilled in the art. For example, a therapeutically-effective dose can be estimated initially from cell culture assays described above. A dose can be formulated in animal models to achieve a circulating concentration range that includes IC50 value, defined as a dose in which 50% of cells of a culture show an effect due to the test compound. Such information can be used to more accurately determine useful doses in human subjects.
Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures utilizing cell cultures or experimental animals in order to determine a LD50 value, the dose determined to be lethal to 50% of the exposed population, and to determine a ED50 value, the dose determined to be therapeutically effective in 50% of the exposed population. A dose ratio between toxic effect and therapeutic effect is referred to as the “therapeutic index,” or it can be expressed as the ratio of the LD50 value over the ED50 value. Compounds which exhibit high therapeutic indices are preferred. The data obtained from cell culture assays and animal studies can be used in formulating a range of effective dosage for human usage. Optimal dosage range includes a ED50 dose with minimal toxicity, although the dosage may vary within this range depending on a given pharmaceutical formulation and route of administration. Dosage administered to a subject should be adjusted according to the age of the subject, the weight of the subject, the manner of administration, and other circumstances unique to each subject.
9. Kits
The invention provides kits comprising the compositions, e.g., the differentially expressed protein, agonist or antagonist of the present invention or their homologs and are useful tools for examining expression and regulation of, for example, the genes as disclosed herein. Reagents that specifically hybridize to nucleic acids encoding differentially expressed proteins of the invention (including probes and primers of the differentially expressed proteins), and reagents that specifically bind to the differentially expressed proteins, e.g., antibodies, are used to examine expression and regulation.
Also within the scope of the invention are kits comprising the compositions (e.g., monoclonal antibodies, human sequence antibodies, human antibodies, multispecific and bispecific molecules, nucleic acid compositions, e.g., antisense oligonucleotides, double stranded RNA oligonucleotides (RNAi), or DNA oligonucleotides (vectors) containing nucleotide sequences encoding for the transcription of shRNA molecules) of the invention and instructions for use. The kit can further contain a least one additional reagent, or one or more additional human antibodies of the invention (e.g., a human antibody having a complementary activity which binds to an epitope in the antigen distinct from the first human antibody).
Nucleic acid assays for the presence of differentially expressed proteins in a sample include numerous techniques are known to those skilled in the art, such as Southern analysis, northern analysis, dot blots, RNase protection, S1 analysis, amplification techniques such as PCR and LCR, high density oligonucleotide array analysis, and in situ hybridization. In in situ hybridization, for example, the target nucleic acid is liberated from its cellular surroundings in such as to be available for hybridization within the cell while preserving the cellular morphology for subsequent interpretation and analysis. The following articles provide an overview of the art of in situ hybridization: Singer et al., Biotechniques 4: 230-250, 1986; Haase et al., Methods in Virology 7: 189-226, 1984; and Nucleic Acid Hybridization: A Practical Approach (Hames et al., eds. 1987). In addition, a differentially expressed protein can be detected with the various immunoassay techniques described above. The test sample is typically compared to both a positive control (e.g., a sample expressing recombinant differentially expressed protein) and a negative control.
The present invention also provides for kits for screening drug candidates for treatment of replicative life span diseases or disorders or diseases or disorders associated with aging such as various types of cancers, diabetes mellitus, cataracts, heart diseases, and neurodegenerative diseases, such as Alzheimer's disease, Pick's disease, Huntington's disease, Parkinson's disease, adult onset myotonic dystrophy, multiple sclerosis, and adult onset leukodystrophy disease. Such kits can be prepared from readily available materials and reagents. For example, such kits can comprise any one or more of the following materials: the differentially expressed proteins, agonists, or antagonists of the present invention, pharmaceutical compositions that can modulate, or modify, the function of the identified genes and gene products, reaction tubes, and instructions for testing the activities of differentially expressed genes. A wide variety of kits and components can be prepared according to the present invention, depending upon the intended user of the kit and the particular needs of the user. For example, the kit can be tailored for in vitro or in vivo assays for measuring the activity of drug candidates for treatment of replicative life span diseases or disorders or related to diseases or conditions associated with aging as described herein.
The invention further provides kits comprising probe arrays as described above. The invention further provides oligonucleotide arrays comprising one or more of the inventive probes described above. In particular, the invention provides an oligonucleotide array comprising oligonucleotide probes that are able to detect polymorphic variants of the genes defined and disclosed herein. In a preferred embodiment the genes are defined in
Optional additional components of the kit include, for example, other restriction enzymes, reverse-transcriptase or polymerase, the substrate nucleoside triphosphates, means used to label (for example, an avidin-enzyme conjugate and enzyme substrate and chromogen if the label is biotin), and the appropriate buffers for reverse transcription, PCR, or hybridization reactions. Usually, the kits of the present invention also contain instructions for carrying out the methods.
The invention further provides compositions, kits and integrated systems for practicing the assays described herein. For example, an assay composition having a source of cells. Additional assay components as described above are also provided. For instance, a solid support or substrate in which the assays can be carried out can also be included. Such solid supports include membranes (e.g., nitrocellulose or nylon), a microtiter dish (e.g., PVC, polypropylene, or polystyrene), a test tube (glass or plastic), a dipstick (e.g., glass, PVC, polypropylene, polystyrene, latex, and the like), a microcentrifuge tube, or a glass, silica, plastic, metallic or polymer bead or other substrate such as paper. Most commonly, the assay will use 96, 384 or 1536 well microtiter plates.
The kits can include any of the compositions noted above, and optionally further include additional components such as instructions to practice a high throughput method of screening for replicative life span modulators, one or more containers or compartments (e.g., to hold the cells, test agents, controls, dyes, and the like), a control activity modulator, a robotic armature for mixing kit components, and the like.
The invention further provides compositions, kits and integrated systems for practicing the assays described herein. For example, an assay composition having a source of cells. Additional assay components as described above are also provided. For instance, a solid support or substrate in which the assays can be carried out can also be included. Such solid supports include membranes (e.g., nitrocellulose or nylon), a microtiter dish (e.g., PVC, polypropylene, or polystyrene), a test tube (glass or plastic), a dipstick (e.g., glass, PVC, polypropylene, polystyrene, latex, and the like), a microcentrifuge tube, or a glass, silica, plastic, metallic or polymer bead or other substrate such as paper. Most commonly, the assay will use 96, 384 or 1536 well microtiter plates.
The kits can include any of the compositions noted above, and optionally further include additional components such as instructions to practice a high throughput method of screening for chronological life span modulators, one or more containers or compartments (e.g., to hold the cells, test agents, controls, dyes, and the like), a control activity modulator, a robotic armature for mixing kit components, and the like.
The invention also provides integrated systems for high throughput screening of potential modulators of chronological life span extension. Such systems typically include a robotic armature which transfers fluid from a source to a destination, a controller which controls the robotic armature, a label detector, a data storage unit which records label detection, and an assay component such as a microtiter dish.
A number of well-known robotic systems have also been developed for solution phase chemistries. These systems include automated workstations like the automated synthesis apparatus developed by Takeda Chemical Industries, LTD. (Osaka, Japan) and many robotic systems utilizing robotic arms (Zymate II, Zymark Corporation, Hopkinton, Mass.; Orca, Hewlett-Packard, Palo Alto, Calif.) which mimic the manual synthetic operations performed by a chemist. Any of the above devices are suitable for use with the present invention. The nature and implementation of modifications to these devices (if any) so that they can operate as discussed herein will be apparent to persons skilled in the relevant art. Any of the assays for compounds that modulate activity, as described herein, are amenable to high throughput screening. High throughput screening systems are commercially available (see, e.g., Zymark Corp. (Hopkinton, Mass.); Air Technical Industries (Mentor, Ohio); Beckman Instruments, Inc. (Fullerton, Calif.); Precision Systems, Inc., (Natick, Mass.), and the like). Such systems typically automate entire procedures including all sample and reagent pipetting, liquid dispensing, timed incubations, and final readings of the microplate in detector(s) appropriate for the assay. These configurable systems provide high throughput and rapid start up as well as a high degree of flexibility and customization. The manufacturers of such systems provide detailed protocols for the various high throughput systems.
Optical images viewed (and, optionally, recorded) by a camera or other recording device (e.g., a photodiode and data storage device) are optionally further processed in any of the embodiments described herein, e.g., by digitizing the image and storing and analyzing the image on a computer. A variety of commercially available peripheral equipment and software is available for digitizing, storing and analyzing a digitized video or digitized optical image, e.g., using PC (Intel x86 or Pentium chip-compatible DOS®, OS2® WINDOWS®, WINDOWS NT® or WINDOWS95® based machines), MACINTOSH®, or UNIX based (e.g., SUN® work station) computers.
One conventional system carries light from the specimen field to a cooled charge-coupled device (CCD) camera, in common use in the art. A CCD camera includes an array of picture elements (pixels). The light from the specimen is imaged on the CCD. Particular pixels corresponding to regions of the specimen (e.g., individual hybridization sites on an array of biological polymers) are sampled to obtain light intensity readings for each position. Multiple pixels are processed in parallel to increase speed. The apparatus and methods of the invention are easily used for viewing any sample, e.g., by fluorescent or dark field microscopic techniques.
Establishing WT-μRLS Distributions and LL-μRLS Distributions
A comprehensive yeast-ORF-deletion collection (“SCGDP”) provided an exemplary yeast variant library for conducting a genome-wide screen for long-lived variants. As an exemplary set, the BY4742-derived MATα deletion collection containing approximately 4800 unique single-ORF deletion variants are subjected to RLS analysis to generate a dataset of mean RLS values for each variant. RLS assays are performed to determine mean replicative life span for 42 different deletion variants reported by others to affect either replicative life span (RLS) or chronological life span (CLS) when respective genes are deleted in shorter-lived background variants. In terms of replicative aging, the BY4742 background is the longest-lived, “wildtype” variant tested by the present inventors, and therefore, yeast variants having a BY4742 background, or the equivalent, may be preferred for identifying genes that are involved in the regulation of life span. To generate the data set shown in Table 1-5 described below, over 130,000 daughter cells were removed or approximately 5,000 life spans for various mother cells were determined. The sample size (N) for each variant assayed is provided in the last column.
Table 1 provides mean replicative life spans (μRLS) determined for various exemplary background variants commonly available. All variants are identified by randomly assigned numbers to eliminate any potential bias during the RLS assay. All life spans are performed in sets of 10 cells per variant, with the BY4742 variant, defined as “wildtype” (WT) control, included in each experiment. In total, at least 4 independent 10-cell sets are analyzed for each variant. For this particular set of backgrounds evaluated, the BY4742 and BY4741 backgrounds demonstrate longest life spans with μRLSs of 27.4 and 26.4, respectively. ΔRLS refers to the strain RLS relative to pair-matched BY4742 cells assayed in the same experiment. N is the number of cells assayed.
Table 2 provides mean replicative life span (μRLS) determined for various exemplary single-gene-deletion stains assayed that are reported to have long life spans. The BY4742 variant defined as a “wildtype” (WT) control is included in each experiment. For this particular set, the fob1-deletion variant exhibited the longest life span having a μRLS of 37.5.
Table 3 provides mean replicative life span (μRLS) determined for a selective set of exemplary single-gene-deletion haploids constructed in BY4742 background. The BY4742 variant defined as a “wildtype” (WT) control is included in each experiment. For this particular set, the cpr7::URA3a deletion variant exhibited the longest life span having a μRLS of 33.9. Unexpectedly, several deletions do not exhibit RLS phenotypes similar to those reported in the literature from experiments performed in short-lived variants. In order to address the possibility that the deletion set variants may contain other genetic anomalies such as aneuploidy, new deletion alleles for several genes are generated in the “wildtype” BY4742 variant, including SGS1, CPR7, RAS2, and RPD3. In every case, except SGS1, the newly created deletion variants behave identically to the variants from the deletion set when analyzed by RLS assay. Surprisingly, many of the gene deletions reported to extend RLS have minimal or no effect in a long-lived variant background.
Table 4 provides mean replicative life span (μRLS) determined for a selective set of exemplary multipe-gene deletion variants constructed in the BY4742 background. The BY4742 variant defined as a “wildtype” (WT) control is included in each experiment. For this particular set, the fob1-gpa2-double-deletion variant exhibited the longest life span having a μRLS of 54.5.
Identification Long-Lived Yeast Variants by Screening a Single-Gene Deletion Library in BY4742 Background from a Small Sample Size (N=5)
As proof of principle, approximately 5,000 mother cells are assayed to establish statistically reliable replicative life span (RLS) data sets for wildtype (“WT-RLS data set”) and for long-lived variants (“LL-RLS data set”). Exemplary LL-RLS data set includes RLS values determined for deletion variants fob1Δ, gpa2Δ, gpr1Δ, and Δhxk2 cells that live, on average, 30% longer than a “wildtype” reference (Table 2, Example 1). A WT-RLS data set can be complied from 250 discrete RLS determinations for “wildtype” BY4742 cells.
A large-scaled, life-span analysis is performed as follows. Approximately three days before initiating a life-span-determination assay, a set of 95 strains contained in a yeast alpha-haploid-deletion collection (Open Biosystems), is transferred to a 96-well plate containing YPDagar using a FX robot, so that each strain occupies one well of a 96-well plate. Concurrently, “wildtype” reference cells (BY4742) are plated within an empty well of the 96-well test plate. For 1-2 days, plated cells are allowed to grow on rich medium, such as YPD, at 30° C. Cells are patched onto life-span-measuring plates containing YPD so that approximately 12 strains are plated per plate the night, or approximately 12 hours, before initiating life-span analysis. A life-span experiment is initiated by gridding a region of the plate distal to growing cells, to which 5 cells of each strain of 12 strains are transferred by micromanipulation. Cells are incubated for two hours at 30° C. Then, virgin daughter cells of each deletion variant cell are selected, and original mother cells are removed. Such virgin daughter cells are monitored for de novo production of daughters at 2-3 hour intervals. Produced daughters are individually removed, and tabulated. Mother cells are placed back at 30° C. to allow continued growth. During evening hours, cells are moved to 4° C. so that the cell division rate is reduced to one cell division per evening. The following day, cells are placed at 30° C. to resume growth. This process is continued for a period of time, approximately two weeks, to allow cells of each variant strain to exhaust its proliferative potential. The total number of daughter cells produced by each mother variant cell is tabulated, and determined RLS values are entered into a database.
A computational algorithm can be designed to randomly extract a large number of hypothetical RLS datasets. For example, 100,000 hypothetical subsets can be computationally selected from a WT-RLS data set and LL-RLS data set, as a function of hypothetical sample sizes (N). As exemplary sample sizes, four N values are selected where N=3, 5, 10, and 20. For each wildtype or LL hypothetical subsets selected at a particular N value, the mean RLS (μRLS) is calculated. The calculated μRLS values for thousands of subsets determined for 8 groups (“wildtype” at N=3, 5, 10, and 20; “long-lived” at N=3, 5, 10, and 20) are plotted to determine the frequency of mean RLS observed for WT and LL cells for each hypothetical sample size. A cumulative probability distribution curve that defines the probabilities for Type I and Type II error that correlate with a particular μRLS for a particular sample size N is generated. See Table 1 in
In this example, in order to completely evaluate 4800 deletion variants present in the BY4742-derived-MATα-deletion collection, an RLS assay described in
As a preferred embodiment, methods of the present invention are employed for identifying yeast variants exhibiting a long life span by selecting a sample size N=5 cells of each variant, as in step 403 of
RESULT: As an example, μRLS values for each deletion variant from a MATα-haploid-deletion set, contained in one 96-well plate (˜2% of the entire set), is determined. Based on the cumulative probability distribution for N=5 (Table 1,
For this initial dataset, μRLS for 5 additional cells for all 96 variants present in the test plate are evaluated, instead of analyzing only 30 variants classified as “ambiguous” after the first round, in order to determine the efficiency of the sorting algorithm, and to verify that a substantial fraction of LL variants are not being wrongly classified as NLL in the first round. Of the 4 variants classified as LL in the first set, 3 variants are independently scored as LL and the fourth variant (ΔYBR255W scored as “ambiguous” in the second set. In contrast, only 1 variant out of 62 variants that initially scored as NLL retested as LL in the second round. Of the 30 deletion variants categorized as “ambiguous” in the first round, 4 are classified as LL and 14 are classified as NLL in the second round, leaving 12 variants that require further RLS analysis after two rounds. For each 96-well-plate-deletion collection assayed, one empty well contained a “wildtype” reference control. The “wildtype” control classified as “ambiguous” in the first round is correctly classified as NLL in the second round.
Exemplary Orthologous Sequences Identified by Database Search
Approximately 14 unique open reading frames (“ORFs”) of yeast sequences are identified from long-lived variants classified by methods of the present invention. For each yeast sequence identified as conferring “long-lived” or life-span-regulating, the amino-acid sequence of the corresponding protein is used as a “query sequence” to perform a search against sequences deposited within various public databases to identify evolutionarily-related sequences. Coding sequences for each yeast ORF are obtained from a genomic database for Saccharomyces cerevisiae.
A BLAST search of the NCBI database resulted in the identification of various mammalian orthologs that are related to fourteen of these ORFs listed in Table 5. Tables 6 lists exemplary sets of conserved orthologs that correspond to the yeast homolog. For each identified yeast ORF, the respective percent identities, percent similarities, and E values are shown. Default parameters were used to perform the search.
For the present invention, an ortholog is defined as a homologous molecule or sequence having life-span-regulating activity and a sequence identity of at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, and 95%. Alternatively, an ortholog is defined as a homologous molecule or sequence having life-span-regulating activity and a sequence similarity of at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, and 95%:
S. cerevisiae Gcn2p activation
Drosophila), promotes the nuclear localization of
C. elegans CDK-7, which is a putative cyclin-
C. elegans T04B2.2, which functions in lipid storage
C. elegans Y65B4A.9 which is involved in
Identification of Sir2-Independent Calorie Restriction Pathway
A similar effect of additive longevity is observed when FOB1 deletion is combined with a GPA2 deletion mutant which acts in the CR pathway.
Finally, the combined effects of SIR2 over-expression and CR in the BY4742 background is tested. Over-expression of SIR2 results in a significant life span extension, and calorie restriction of SIR2-overexpressing cells results in a further increase in life span, supporting that CR and SIR2 can promote longevity by influencing different pathways.
Finally, the combined effects of SIR2 over-expression and CR in the BY4742 background is tested. Over-expression of SIR2 results in a significant life span extension, and calorie restriction of SIR2-overexpressing cells results in a further increase in life span, supporting that CR and SIR2 can promote longevity by influencing different pathways.
From these experiments, it appears that at least two pathways regulate aging in yeast: one pathway is regulated by ERC accumulation and the other pathway is undefined at a molecular level, but responsive to CR. In a long-lived “wildtype” background, such as the BY4742 strain, both processes influence longevity. The existence of an ERC-independent aging pathway in yeast modulated by CR suggests the existence of similar mechanism for aging in higher eukaryotes. CR is the only intervention shown to extend life span in a wide range of eukaryotes, including mammals. Nevertheless, in C. elegans, increased expression of the sir2 ortholog, sir-2.1, has been found to extend life span in a manner dependent on the Daf-16 transcription factor. Similarly, the mammalian sir2 ortholog, SirT1, has recently been reported to regulate the activity of murine Foxo3A. These experiments confirm a role for sir2 proteins in eukaryotic aging, linking Sirtuin activity to insulin/IGF-1 signaling pathways. Evidence has accumulated in both C. elegans and mice that CR can further extend the life span of long-lived insulin/IGF-1 pathway mutants, suggesting that CR and insulin/IGF-1 act in two distinct pathways to regulate aging. Both pathways may be conserved throughout eukaryotic aging mechanisms.
Epistasis Analysis of LL Variants
Each variant classified as LL by methods of the present invention can be further classified into one or more life-span-regulating pathways. New combinations of deletion strains can be generated so that genes that confer longer life spans in identified LL variants can be sorted into distinct life-span-regulating pathways. Various combination of well-characterized genetic models described above can be used as suitable genetic backgrounds, including fob1Δ strain, sir2Δ strain, fob1Δ-sir2Δ-double-deletion strain, hxk2Δ strain, gpa2Δ strain, gpr1Δ strain, fob1Δ-hxk2Δ-double-deletion strain, fob1Δ-gpa2Δ-double-deletion strain, and fob1Δ-gpr1Δ-double-deletion strain. Methods for generating new genetic variants, such as homologous recombination, yeast mating, and sporulation, are well-known by persons in the art.
For example, for each LL-deletion variant identified by the present methods, such LL variants can be combined with FOB1 deletion strains to produce novel double-deletion strains, referred to as “LLΔ-fob1Δ” variants, and also combined with fob1Δ-sir2Δ-double-deletion strains to produce novel triple-deletion strains, referred to as “LLΔ-fob1Δ-sir2Δ” variants. The RLSs for each “LLΔ-fob1Δ” double variant and “LLΔ-fob1Δ-sir2Δ” triple variant generated can be determined to generate mortality curves for each LL variant. Any “LLΔ-fob1Δ” double variants that exhibit life spans comparable to a “wildtype” strain, and that do not further extend the life span of a fob1Δ, most likely lack a gene that acts in the ERC-dependent pathway. Any LLΔ-fob1Δ-sir2Δ-triple variants that exhibit life spans comparable to a “wildtype” strain, most likely lack a gene that acts in the ERC pathway. For those “LLΔ-fob1Δ” double variants and “LLΔ-fob1Δ-sir2Δ” triple variants that exhibit life spans which substantially exceeds the “wildtype” RLS, then such variants are likely to represent genes in an alternative pathway, such as a sir2-independent CR pathway. A substantial life-span extension includes an increase in RLS of at least 80%, 85%, 90%, 95%, and 100% relative to a “wildtype” reference.
Identification of Sir2 Regulators Using LL Variants
Because Sir2 orthologs in C. elegans are known to regulate aging pathways and to regulate transcription factors that are purported to participate in aging mechanisms in mammalian systems, regulators of Sir2 are very likely to be conserved broadly among diverse eukaryotic systems. Yeast genes and orthologs identifiable by methods of the present invention (“present methods”) can be assayed to determine whether encoded gene products are able to regulate Sir2 histone deacetylase activity. For example, whether an identified gene can alter Sir2 activity can be determined by measuring Sir2-dependent silencing at the telomeres and rDNA within LL variants. Yeast strains containing reporter genes positioned at either loci, telomeres or rDNA, can be crossed for example, with LL-deletion variants so that LL variants containing reporter genes at these loci can be generated. Such LL-deletion variants can be monitored for Sir2-dependent transcriptional silencing, and levels of marker gene expression in LL-deletion variants can be compared to levels of a “wild type” reference to determine the effect of a deleted gene in question on Sir2-dependent transcriptional silencing.
In addition, for each LL-deletion variant identified by present methods, such LL-deletion variants can be combined with FOB1 deletion strains to produce novel double-deletion strains, referred to as “LLΔ-fob1Δ” variants, and also combined with fob1Δ-sir2Δ-double-deletion strains to produce novel triple-deletion strains, referred to as “LLΔ-fob1Δ-sir2Δ” variants. The degree of Sir2-dependent transcriptional silencing for each “LLΔ-fob1Δ” double variant and “LLΔ-fob1Δ-sir2Δ” triple variant can be determined. A “LLΔ-fob1Δ-sir2Δ” variant that does not exhibit an increased life span compared to an increased life span observed for a LL variant with only a single-gene deletion in question (“LLΔ”) indicates that the gene is likely to act within a Sir2-dependent pathway. Genes (LLΔ) identified in LL-deletion variants are candidates for Sir2 regulators, which are predicted to be conserved in higher eukaryotes. Various methods for generating new genetic variants are known by persons skilled in the art. Exemplary reporter genes include ADE2 or URA3 that can be positioned near telomeres, and ADE2, URA3 or MET15 that can be positioned within rDNA sites.
Translation Inhibition and Aging
Many of the genes, which when deleted result in extended replicative life span, encode proteins that regulate or are directly involved in ribosome biogenesis. RPL31A, RPL6B and RPS21A encode components of the ribosome and REI1 is implicated in formation of the 60S subunit of the ribosome. Ribosomal genes are often duplicated in the yeast genome. Thus deletion of RPL31A reduces but does not eliminate formation of functional ribosomes since RPL31B encodes a gene product with redundant function. Thus, in these deletions, we are likely reducing the number of ribosomes, but not eliminating ribosomal function altogether (an event that would be lethal). Polysome profiling, which measures the quantity of ribosomes in yeast cells and their degree of association with mRNA, supports this assertion. Δrpl31b or Δrei1 cells contain less ribosomes associated with mRNA and a striking reduction in the free cytoplasmic 60S (but not the free 40S) subunit of the ribosome. Since Rpl31b is a known component of the 60S subunit this result is not surprising.
Also identified in our genome-wide RLS screen were signaling components that link nutrient levels to ribosome biogenesis, including TOR1 and URE2. Deletion of SCH9 and components of the protein kinase A (PKA) pathway also result in extended RLS. These genes encode products in signaling pathways that also link nutrient levels to ribosome biogenesis. However the downstream effects of PKA and Sch9 signaling are numerous and no one has connected life span extension by reduced PKA or Sch9 activity to ribosome biogenesis. We speculate these two events are linked and moreover that reduction in ribosome biogenesis may be the primary mechanism by which CR (which results in less PKA, Sch9 and TOR activity) extends yeast RLS. Indeed we find that ribosome levels are reduced in Δsch9 cells as measured by polysome profiling, consistent with predictions based on gene expression profiling. Tor1 is a protein kinase intimately linked to yeast cell growth. Yeast in rich media are exposed to high levels of nitrogen and carbon (e.g. glucose), which results in TOR activation, ultimately leading to enhanced ribosome biogenesis and increased growth rates.
A number of drugs antagonize TOR activity including rapamycin and several derivatives thereof. In addition, the drug methionine sulfoximine (MSX), an inhibitor of glutamine synthase, results in decreased glutamine levels. Intracellular glutamine levels reflect nitrogen availability, and intracellular levels of this metabolite are monitored by TOR. Increased glutamine levels therefore result in increased TOR activity. Thus exposure of cell to MSX results in decreased TOR activity. We find that exposure of yeast cells to MSX extends replicative life span in yeast, confirming our observation the Δtor1 strains have increased RLS and suggesting that MSX and derivatives thereof may be effective anti-aging compounds in eukaryotic organisms.
There are approximately 150 yeast genes that are known to be involved in ribosome biogenesis and function. We will perform the following experiments to address their possible roles in yeast replicative aging: (1) perform RLS (replicative life span) analysis on all deletion strains, (2) examine growth rate of all deletion strains in rich and calorie restricted conditions as a measure of their relative contribution to ribosome function, and (3) perform polysome analysis on all deletion strains that exhibit extended replicative life span.
How does decreased ribosomal biogenesis lead to enhanced replicative life span? We consider two general explanations. First, lengthened RLS may result from a general decrease in the synthesis and activity of ribosomes. This model derives from the observation that the yeast cell overproduces ribosomes in times of plenty. Indeed a significant proportion of ribosomes in the yeast cell are not associated with mRNA as indicted by polysome profiling of young wild-type cells. Ribosome production accounts for up to half of the cells energy usage under conditions of rapid growth. One possibility is that there is a tradeoff between reproduction (growth rate) and aging in yeast, as has been speculated for mammals. In other words, the yeast cell overproduces ribosomes to maximize growth rate and thus production of new cells. Therefore, the yield of daughter cells on a population level (clonal reproduction) would be maximized at the expense of aging in single “mother” cells. The ability of a single mother cell to divide more than 15 times may be superfluous under these conditions since rapid division ensures that the vast majority of cells in the population are young. Alternatively, extra ribosomes may be produced and stored in times of plenty, so that they can be used in later times of starvation when energy may not be sufficient to generate new ribosomes. If it is true that enhanced RLS derives from the benefits of reduced ribsome biogenesis, then drugs that precipitate this effect, may prolong life span. Possible drugs include cycloheximide and edeine, which inhibit either translation or translation initiation (see section on drugs).
A second explanation is that the benefit of reduced ribosome biogenesis is specific to decreased or altered translation of one or more messages that encode proteins delimiting yeast replicative aging. If this is the case, it will be important to determine which messages are altered. Even in this case, drugs which generally inhibit translation are likely provide an aging benefit, since mutations with this effect (e.g. Δsch9) increase life span.
Small Molecules that Delay Yeast Aging
Based on our genome-wide screen of yeast replicative aging, we can predict two classes of drugs that are likely to increase life span. The first class consists of inhibitors of upstream components of the TOR, SCH9, or PKA pathways. These include rapamycin and methionine sulfoximine (MSX, an inhibitor of glutamine synthase). The second class consists of inhibitors of the relevant downstream targets of these pathways, speculatively including cycloheximide, edeine, and amino acid alcohols (e.g methioninol and threoninol). These compounds inhibit translation through various known mechanisms. We have determined that MSX administration significantly increases life span at 50 uM, 100 uM, 1 mM, and 2.5 mM. Maximal life span extension in these preliminary experiments is obtained at 1 mM, results in a 25-30% increase in mean replicative life span. We will test the effect of both translation inhibitors on yeast replicative life span as well. Derivatives of these drugs may also delay aging as might any compound found to slow translation. In general, once pathways are determined that regulate yeast replicative aging, we will test any compounds known to regulate that pathway and derivatives thereof.
Any compound found to prolong yeast RLS will be tested in other eukaryotic organisms and in yeast chronological life span analysis. We will administer the compound to C. elegans and determine whether life span is extended in this organism (see section on C. elegans). Also we will perform studies using mice as a model system to determine whether prolonged exposure to a compound in question results in prolonged life span or health span in mammals.
Aging studies in C. elegans. If a yeast strain lacking a gene exhibits extended mean and/or maximum replicative life span, one can infer that the protein encoded by that gene restricts life span in the single-celled eukaryote. This raises the question of whether decreased function of an orthologous protein will result in prolonged life span in another eukaryotic organism. Thus we will test orthologs of yeast aging genes identified in our genome-wide RLS screen in the nematode C. elegans. Over half of the yeast aging genes identified have orthologs in C. elegans. In some cases, more than one C. elegans protein bears significant homology to a yeast aging gene, and in these cases we will examine the effects of each potential ortholog.
One (or both) or two approaches will be taken to examine C. elegans genes. We will use RNAi, with the double-stranded RNA delivered to the worms through expression in their bacterial food source E. coli. This approach is used routinely and interfering RNAs specific to most C. elegans genes have already been created. As a second approach, we will generate or obtain worms with inactivating mutations in potential aging genes. Life span studies will be performed in a variety of manners. For the RNAi approach, we will shift worms in the L4 larval stage to E. coli expressing the double-stranded RNA. This will lead to downregulation of gene expression of the potential aging gene after development and thus avoid most developmental defects and possible dauer formation, which complicates aging studies. Alternatively, we will administer the RNAi to adult worms and monitor life span of their progeny. In this case, worms will be exposed to the RNAi both during development and as adults. Finally, we will administer candidate compounds identified in yeast studies to determine their potential effects on aging in worms.
Aging Studies in a Mitotically Active Simple Eukaryote
Since C. elegans exists as a completely post-mitotic adult (with the exception of the germ line), we recognize that C. elegans may not be an optimal choice for testing candidate aging genes identified from a replicative life span screen in yeast. To address this issue, we may examine orthologs of candidate aging genes in a different, mitotically active simple eukaryote. Possible organisms under consideration are the flatworm Planaria and the fruit fly Drosophila melanogaster.
Aging Studies in Mice
An important question will be to determine whether aging genes identified in yeast also affect life span or health span in mammals. Therefore we will initiate aging studies in mice, where gene knockouts of orthologs of identified yeast/worm aging genes can be created. We will choose a subset of the genes identified in our yeast studies, emphasizing those that also regulate aging in worms or another multicellular model. Given the evolutionary divergence of worms and yeast, we feel strongly that gene sets regulating aging in both organisms, are highly likely to regulate aging in mammals as well.
Experiments will be performed by generating conditional knock-outs of orthologs of yeast and worm aging genes. By flanking the gene in question with lox sites, we can control when the gene is excised by temporal or tissue-specific administration of Cre, an enzyme that excises DNA between two lox sites. Therefore, we can allow mice to undergo fetal development and then generate the gene deletion post-natally by ubiquitous delivery of Cre. This will allow us to avoid developmental defects associated with loss of a candidate aging gene that would impair aging studies. Post-natal administration can be performed in a variety of documented methods including but not limited to the use of a tet-regulated promoter driving expression of Cre present in the germ line of the mouse. Should post-natal administration result in lethality or other phenotypes which preclude aging analysis, we will perform life span analysis in mice heterozygous for the gene in question.
In addition to monitoring mouse life span, we will examing a variety of aging biomarkers including but not limited to changes in cognitive ability, fat mass and body weight, body temperature, strength, as well as alopecia and blood serum levels of a variety of compounds including insulin, IGF, glucose, leptin, DHEA, growth hormone, and molecules diagnostic of immune response. Additionally we will perform gene expression array analysis looking at genome-wide changes in gene expression during the mouse aging process (see specific section). Changes in expression of many genes known to occur during aging and by monitoring these genes, we can measure rates of aging. By using such a biomarkers, we can (1) determine whether a gene deletion is likely to affect life span prior to the completion of aging studies and (2) monitor changes in health span that occur with age.
Finally, we will examine the effects of prolonged administration of drugs on mouse aging and aforementioned biomarkers of aging. Drugs that are effective in yeast and worms will be chosen for studies in mice.
Gene Expression Array Analysis
We will use genome-wide gene expression array analysis in all three organisms (yeast, worms and mice), as well as potentially on human cells in culture, to (1) determine aging rates and (2) identify downstream targets of aging genes that might underlie delayed aging phenotypes. This analysis will be performed on yeast examining gene expression changes in strains lacking genes which exhibit long RLS. We will also perform array analysis on yeast exposed to environmental conditions (e.g., calorie restriction defined as media containing 0.05% glucose) or in the presence of compounds such as MSX which result in extended yeast RLS.
In worms, we will monitor changes in genome-wide gene expression in worms lacking aging genes. This analysis may be performed both in young worms and throughout the aging process. In addition, we will examine environmental conditions that extend life span and compounds as described above. Similar experiments will be performed in young and aging mice. Again, this will allow us to monitor the rate of aging by looking at changes in gene expression known to occur during murine aging and to identify critical targets of aging genes.
Polymorphisms and Human Aging
Loss-of-function mutations resulting in prolonged life span or health span in yeast, worms and mice might be phenocopied by polymorphisms that have arisen in the human population. Thus, we will determine whether aging genes identified in yeast, worms or mice are enriched for particular polymorphisms in unusually old individuals relative to the normal population. An enhancement in the relative proportion of a particular allele of a potential aging gene would provide evidence for the gene regulating aging in humans.
Although the present invention has been described in terms of particular embodiments, it is not intended that the invention be limited to these embodiments. Modifications within the spirit of the invention will be apparent to those skilled in the art. The foregoing description, for purposes of explanation, used specific nomenclature to provide a thorough understanding of the invention. However, it will be apparent to one skilled in the art that the specific details are not required in order to practice the invention. The foregoing descriptions of specific embodiments of the present invention are presented for purpose of illustration and description. They are not intended to be exhaustive or to limit the invention to the precise forms disclosed. Obviously many modifications and variations are possible in view of the above teachings. The embodiments are shown and described in order to best explain the principles of the invention and practical applications, and thereby enable others skilled in the art to best utilize the invention and various embodiments with various modifications as are suited to particular uses contemplated. It is intended that the scope of the invention be defined by the following claims and their equivalents. Each recited range includes all combinations and sub-combinations of ranges, as well as specific numerals contained therein.
All publications and patent applications cited in this specification are herein incorporated by reference in their entirety for all purposes as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference for all purposes.
This application claims the benefit of U.S. Provisional Application No. 60/591,461, filed on Jul. 26, 2004, the disclosure of which is incorporated herein in its entirety.
This invention was made by government support by Grant No. P30 AG0133280 from the National Institutes of Health. The Government has certain rights in this invention.
| Number | Date | Country | |
|---|---|---|---|
| 20060068414 A1 | Mar 2006 | US |
| Number | Date | Country | |
|---|---|---|---|
| 60591461 | Jul 2004 | US |
