Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2000-08-08
2003-05-27
Horlick, Kenneth R. (Department: 1656)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C536S024300
Reexamination Certificate
active
06569624
ABSTRACT:
BACKGROUND OF THE INVENTION
A common feature of most multicellular organisms is the progressive and irreversible physiological decline that characterizes senescence. Although genetic and environmental factors can influence the aging process, the molecular basis of senescence remains unknown. Postulated mechanisms include cumulative damage to DNA leading to genomic instability, epigenetic alterations that lead to altered gene expression patterns, telomere shortening in replicative cells, oxidative damage to critical macromolecules and nonenzymatic glycation of long-lived proteins (S. M. Jazwinski,
Science
273:54, 1996; G. M. Martin, et al.,
Nature Gen
. 13:25, 1996; F. B. Johnson, et al.,
Cell
96:291, 1996; K. B. Beckman and B. N. Ames,
Physiol. Revs
. 78:547, 1998). Factors which contribute to the difficulty of elucidating mechanisms and testing interventions include the complexity of organismal senescence and the lack of molecular markers of biological age (biomarkers). Aging is complex in that underlying mechanisms in tissues with limited regenerative capacities (e.g., skeletal and cardiac muscle, brain), which are composed mainly of postmitotic (non-dividing) cells, may differ markedly from those operative in proliferative tissues. Accordingly, approaches which provide a global assessment of senescence in specific tissues would greatly increase understanding of the aging process and the possibility of pharmaceutical, genetic or nutritional intervention.
Genetic manipulation of the aging process in multicellular organisms has been achieved in Drosophila, through the over-expression of catalase and Cu/Zn superoxide dismutase (W. C. Orr and R. S. Sohal,
Science
263:1128, 1994; T. L. Parkes, et al.,
Nat. Genet
. 19:171, 1998), in the nematode
C. elegans
, through alterations in the insulin receptor signaling pathway (S. Ogg, et al.,
Nature
389:994, 1997; S. Paradis and G. Ruvkun,
Genes Dev
. 12:2488-2498, 1998; H. A. Tissenbaum and G. Ruvkun,
Genetics
148:703,1998), and through the selection of stress-resistant mutants in either organism (T. E. Johnson,
Science
249:908, 1990; S. Murakami and T. E. Johnson,
Genetics
143:1207, 1996; Y. J. Lin, et al.,
Science
282:943, 1998). In mammals, there has been limited success in the identification of genes that control aging rates. Mutations in the Werner Syndrome locus (WRN) accelerate the onset of a subset of aging-related pathology in humans, but the role of the WRN gene product in the modulation of normal aging is unknown (C. E. Yu, et al.,
Science
272:258, 1996; D. B. Lombard and L. Guanrente,
Trends Genet
. 12:283, 1996).
In contrast to the current lack of genetic interventions to retard the aging process in mammals, caloric restriction (CR) appears to slow the intrinsic rate of aging (R. Weindruch and R. L. Walford,
The Retardation of Aging and Disease by Dietary Restriction
(C C. Thomas, Springfield, Ill., 1988; L. Fishbein, Ed.,
Biological Effects of Dietary Restriction
(Springer-Verlag, N.Y., 1991; B. P. Yu, Ed.,
Modulation of Aging Processes by Dietary Restriction
(CRC Press, Boca Raton, Fla. 1994). Most studies have involved laboratory rodents which, when subjected to a long-term, 25-50% reduction in calorie intake without essential nutrient deficiency, display delayed onset of age-associated pathological and physiological changes and extension of maximum lifespan.
BRIEF SUMMARY OF THE INVENTION
The present invention will allow the evaluation of aging interventions on a molecular and tissue-specific basis through the identification of aging biomarkers. In particular, the use of gene expression profiles allows the measurement of aging rates of target organs, tissues and cells, and to what extent aging is delayed by specific interventions, as determined by quantitative analysis of mRNA abundance. Because aging-related gene expression profiles can be classified in subgroups according to function, the invention also allows for the determination of how function-specific aspects of aging are affected. This particular feature will allow for determination of combination therapies that prevent or reverse most aging related changes in particular organs, tissues, and cells.
In one embodiment, the present invention is a method of measuring the biological age of a multicellular organism comprising the steps of (a) obtaining a sample of nucleic acid isolated from the organism's organ, tissue or cell, wherein the nucleic acid is RNA or a cDNA copy of RNA and (b) determining the expression pattern of a panel of sequences within the nucleic acid that have been predetermined to either increase or decrease in response to biological aging of the organ, tissue or cell. Preferably, the expression patterns of at least ten sequences are determined in step (b) and the organism is a mammal, most preferably a rodent.
In one preferred embodiment of the method described above, the nucleic acid is isolated from a mammalian tissue selected from the group consisting of brain tissue, heart tissue, muscle tissue, skin, liver tissue, blood, skeletal muscle, lymphocytes and mucosa.
In another embodiment the present invention is a method of obtaining biomarkers of aging comprising the steps of: (a) comparing a gene expression profile of a young multicellular organism subject's organ, tissue or cells; a gene expression profile from a chronologically aged (and therefore biologically aged) subject's organ, tissue or cell; and a gene expression profile from a chronologically aged but biologically younger subject's organ, tissue or cell, and (b) identifying gene expression alterations that are observed when comparing the young subjects and the chronologically aged subjects and are not observed or reduced in magnitude when comparing the young subjects and chronologically aged and biologically younger subjects. Preferably, one uses high density oligonucleotide arrays comprising at least 5-10% of the subject's gene expression product to compare the subject's gene expression profile, and caloric restriction to obtain a chronologically aged but biologically younger subject.
In a preferred embodiment of the method described above, the gene expression profile indicates a two-fold or greater increase or decrease in the expression of certain genes in biologically aged subjects. In a more preferred embodiment of the present invention, the gene expression profile indicates a three-fold or greater or, most preferably three-fold or greater, increase or decrease in the expression of certain genes in aged subjects.
In another embodiment, the present invention is a method of measuring biological age of muscle tissue comprising the step of quantifying the mRNA abundance of a panel of biomarkers selected from the group consisting of markers described in the Tables 1, 2, 15 and 16. A method of measuring biological age of brain tissue comprising the step of quantifying the mRNA abundance of a panel of biomarkers selected from the group consisting of markers described in Tables 5, 6, 9, 10, 11, 12, 13 and 14.
In another embodiment, the present invention is a method for screening a compound for the ability to inhibit or retard the aging process in a multicellular organism tissue, organ or cell, preferably mammalian tissue, organ or cell, comprising the steps of: (a) dividing test organisms into first and second samples; (b) administering a test compound to the organisms of the first sample; (c) analyzing tissues, organisms and cells of the first and second samples for the level of expression of a panel of sequences that have been predetermined to either increase or decrease in response to biological aging of the tissue, (d) comparing the analysis of the first and second samples and identifying test compounds that modify the expression of the sequences of step (c) in the first sample such that the expression pattern is indicative of tissue that has an inhibited or retarded biological age.
It is an object of the present invention to evaluate or screen compounds for the ability to inhibit or retard the aging process.
It is also an o
Lee Cheol-Koo
Prolla Tomas A.
Weindruch Richard H.
Horlick Kenneth R.
Strzelecka Teresa
Wisconsin Alumni Research Foundation
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