CA2080336A1 - Rejuvenation compositions and methods for their use - Google Patents

Abstract

This invention provides a method for restoring the proliferative potential of senescent cells by decreasing the concentration of intranuclear interleukin-I. This invention also provides antisense oligonucleotides that, when added to senescent cell cultures, restore the proliferative potential of these cells. This invention, thus, provides means and methods for rejuvenating cells.

Description

WO 91/1~226 PCr/US9t/0~343 -1- 2~80~3~

TITLE OF THE INVENTION:

REJUVENATION COHPOSITIONS
METHODS FOR THEIR USE

CROSS-REFERENCE TO RELATED APPLICATIONS:

This application is a continuation-in-part application of .U.S. patent application serial no. 07/505,681, filed on April 9, 1990. `- -FI~LD OF THE INVENTION:

This invention relates to nucleic acid molecules, and their equivalents which are capable of restoring the proliferative potential to senescent cells. The invention includes anti-senescence nucleic acid molecules, their equivalents, as well as therapeutic and non-therapeutic compositions which contain these molecules. This invention was made, in part, with GoYernment funds; the Government has certain rights in this invention.

BACKGROUND OF INVENTlON:
~' :
I. Senescence of Cells In Vitro .
Normal human diploid cells have a finite potential for proliferative growth (Hayflick, L. et al., Exp. Cell Res.
25:585 (1961); Hayflick, L., EXD. Cell Res. 37:614 (1965)).
Indeed, under controlled conditions in vitro cultured human cells can maximally proliferate only to about 60 cumulative population doublings. The proliferative potential of such . ,: . . . , ,...... , . :

W o 91/15~26 PCT/US91/0234~

20~033~ -2-cells has been found to be a function of the number of cumulative population doublings which the cell has undergone (Hayflick, L. et al., EXD. Cell Res. 25: 585 (1961);
Hayflick, L. et al., EXD. Cell Res. 37: 614 (1985)). This potential is also inversely proportional to the in vivo age of the cell donor (Martin, G.M. et al., Lab. Invest. 23:86 (1979); Goldstein, S. et al., Proc. Natl. Acad. Sci. (U.S.A.l 64:155 (1969)i Schneider, E.L., Proc. Natl. Acad. Sci.
(U.S.A.) 73:3584 (1976)i LeGuilty, Y. et al., Gereontoloqia 19:303 (1973)).
Cells that have exhausted their potential for proliferative growth are said to have undergone "senescence."
Cellular senescence in vitro is exhibited by morphological r changes and is accompanied by the failure of a cell to respond to exogenous growth factors. Cellular senescence, thus, represents a loss of the proliferative potential of the cell. Although a variety of theories have been proposed to explain the phenomenon of cellular senescence in vitro~
experimental evidence suggests that the age-dependent loss of proliferative potential may be the function of a genetic program (Orgel, ~.E., Proc. Natl~ Acad. Sci. ~U.S.A.) 49:517 (1963); De Mars, R. et al., Human Genet. 16:87 (1972); M.
Buchwald, Mutat. Res. 44:401 (1977); Mart;n, G.M. et al., Amer. J. Pathol. 74:137 (1974); Smith, J.R. et al., Mech. Aqe.
Dev. 13:387 (1980); Kirkwood, T.B.L. et al., Theor. B;ol.
53:481 (1975).
Cell fusion studies with human fibroblasts in vitro have demonstrated that the quiescent phenotype of cellular senescence is dominant over the proliferative phenotype (Pereira-Smith, O.M et al., Somat. Cell Genet. 8:731 (1982);
Norwood, T.H. et al., Proc. Natl. Acad. Sci. (U.S.A.~ 71:223 (1974); Stein, 5.H. et al., EXD. Cell Res. 130:155 (1979)).
Insight into the phenomenon of senescence has been gained from studies in which senescent and young (i.e. non-..
-.: , ', .

WO 91/15226 PCr'/US91/02343 2~a33~

senescent) cells have been fused to form heterodikaryons. In order to induce senescence in the "young" nucleus of the heterodikaryon (as determined by an inhibition in the synthesis of DNA), protein synthesis must occur in the senescent cell prior to fusion (Burmer, G.C. et al., J. Cell.
Biol. 94:187 (1982); Drescher-Lincoln, C.K. et al., EXP. Cell Res. 144:455 (1983); Burner, G.C. et al., EXD. Cell Res.
145:708 (1983); Drescher-Lincoln, C.K. et al., EXD. Cell Res.
~ 208 (1984).
Likewise, microinjection of senescent fibroblast mRNA
into young fibroblasts has been found to inhibit both the ability of the young cells to synthesize DNA (Lumpkin, C.K. et .. . . .
al., Science 232:393 (1986)) and the ability of the cells to enter into the S (stationary) phase of the cell cycle (Lumpkin, C.K. et al., EXP. Cell Res. 160:544 (1985)).
Researchers have identified unique mRNAs that are amplified in senescent fibroblasts in vitro (Roy, A.K. et al , J. Biol. Chem. 258:10123 (1983); Webster, G.C. et al., ~ech. Aqe Dev. 24:335 (1984); Wang, E. et al., J. Cell Biol.
lQl:1695 (1985); Wellinger, R. et al., J. Cell Biol. 34:203 (1986); Flemm~ng, J.E. ~ , Proc. ~!atl. Acad~ Sci. (U.S.A.) 85:4099 (1988); West, M.D. et al., EXP. Cell Res. 184:138 (1989); Giordano, T. et al., EXD. Cell Res. 185:399 (1989)).
For example, expression of the T-kininogen gene is amplified in the liver of old rats (Sierra, F. et al., Molec. Cell Biol. 9:5610 (1989)). It has also been suggested that an altered genetic program exists in senescent human fibroblasts, which involves the repression o~ c-fos expression at the transcriptional level (Seshadri, T. et al., Science 247:205 (1990)).
The human diploid endothelial cell presents an alternative cell type for the study of cellular senescence because such fibroblast cells mimic cellular senescence in Yitro (Maciag, T. et al., 8. Cell. 8iol. 91 420 (1981);

. . . . . ; :
~ . .~ . .
t ~ , ,,: .~., , . `, . , ,'.'',~ ' .. , `. ` ;

W() '~1/15226 PCT/US91/02343 20~033~ 4 Gordon, P.B. et al., In Vitro 19:661 (1983); Johnson, A. et al., Mech Aqe. Dev. 18:1 (1982); Thornton, S.C. et al., Science 222:623 (1983); Van Hinsbergh, V.W.M. et al., Eur. ?.
Cell Biol. 42:101 (1986); Nichols, W.W. et al., J. Cell.
PhYsiol. 132:453 (1987)).
In addition, the human endothelial cell is capable of expressing a variety of functional and reversible phenotypes.
The endothelial cell exhibits several quiescent and non-terminal differentiation phenotypes (Folkman, J. et al., Nature 288:551 (1980); Maciag, T. et al., J. Cell Biol. 94:511 (1982); Madri. J.A. et al., J. Cell Biol. 97:153 (1983);
Montesano, R., J. Cell Biol. 99:1706 (1984), Montesano, R. et al., J. Cell Phvsiol. 34:460 (1988)).
It has been suggested that the pathway of human endothelial cell differentiation .in vitro involves the induction of cellular quiescence mecliated by cytokines that inhibit growth factor-induced endothelial cell proliferation in vitro (Jay, M. et al., Science ~ :882 (1985); Madri, J.A.
~ ~L. In Vitro 23:387 (1987); Ku~ota, Y. et al., J. Cell ~Q~ 107:1589 (1988); Ingher, D.E. et al., J. Cell B~ol.
317 (1989)).
Inhibitors of endothel;al cell proliferation also function as regulators of immediate-early transcriptional events induced during the endothelial cell differentiation in vitro, which involves formation of the capillary-like, tubular endothelial cell phenotype (Maciag, T., In: Imp. Adv. Oncol.
(De Vita, ~.T. et al., eds., J.B. Lippincott. Philadelphia, 42 (1990~; Goldgaber, D. et al., Proc. Natl. Acad. Sci.
(U.S.A.) 86:7606 (1990); Hla, T. et al., Biochem. BioPhvs.
Res. Commun. 167:637 (1990)). The inhibitors of cell proliferation that include:
1. Interleukin-1~ tIL-1~) (Montesano, R. et al., J.
Cell Biol. 99:1706 (1984); Montesano, R. et al., J.
Cell Phvsiol. 122:424 (1985)~;

WO ~1/15226 PC~/US91tO2343 2~33~

2. Tumor necrosis factor (Frater-Schroder, M. et al~, Proc. Natl. Acad. _Sci. (U.S.A.l 84:5277 (1987);
Sato, N. et al., J. Natl. Cancer Inst. 76:1113 (1986); Pber, J.P., Amer. J. Pathol. 133:426 (1988);
Shimada, Y. et al., J. Cell PhYsiol. 142:31 (1990));
3. Transforming growth factor-~ (Baird, A. et al., Biochem. BioPhYs. Res. Commun. 138:476 (1986);
Mullew, G. et al., Proc. Natl. Acad. Sci. (U.S`.A.) 84:5600 (1987); Mairi, J.A. et al., J. Cell Biol.
106:1375 (1988));

4. Gamma-interferon (Friesel, R. et al., J. Cell Biol.
104:689 (1987); Tsuruoka, N. et al., 8iochem.
BjODhYS. RES. Commun. 155:429 (1988)) and 5. The tumor promoter, phorbol myristic acid (PMA) (Montesano, R. et al., Cell 42:469 (1985); Doctrow, S.R. et al., J. Cell B~ol. 104:679 (1987);
Montesano, R. et al., J. Cell. Phvsiol. 130:284 (1987); Hoshi, H. et al., FASAB J. 2:2797 (1988)).

Il. Physiological Functions of Endothelial Cells Endothelial cells, which form the inner lining of blood vessels participate in a multiplic;ty of physiological functions, including the formation of a selective barrier for the translocation of blood constituents and macromolecules to underlying tissues and the maintenance of a non-thrombogenic interface between blood and tissue. Endothelial cells are also an important component in the development of new capillaries and blood vessels.
Blood vessel development ("angiogenesis"), occurs during developmental periods, such as during development of the vascular system, and as part of the pathophysiology of a variety of disease states, such as psoriasis, arthritis, . . .: ;,. . ~ ..

6 PCI/US91/0'343 2~033~ -6-chronic inflammatory conditions, diabetic retinopathy, and tumor development.
Angiogenesis involves the organized migration, proliferation, and differentiation of the endothelial cells.
S It is initiated by the endothelial cell in response to angiogenic stimuli. ~hese stimuli can be separated into three distinct events: cell migration, cell proliferation and cell differentiation, whereby the cells organize into a tubular structure.

III. Mitogens and Cytoklnes The above-described events are mediated in vitro. and most likely in vivo, by mitogenic polypeptides. The migration of endothel;al cells is induced by factors, including the heparin binding growth factors and angiotropin. Proliferation is induced by the heparin binding growth factors (hereinafter HBGFs) and differentiation and cellular organization is induced by polypeptides, including interleukin-l ("IL-l"), tumor necrosis factor ("TNF"), gamma-interferon, transforming growth factor alpha and beta ("TGF-a" and "TGF-B", respectively~ and phorbol myristic acetate ("PMA").
As cells age they become refractory to mitogens, such as HBGF-1, that induce proliferation. Cytokines, such as IL-l~
inhibit cell proliferation. IL-I, which is produced by activated macrophages, exhibits a variety of biological activities. These activ;ties reside in two interleukin proteins, IL-l~ and IL-l~, which share only distant homology (March, C.J., et al. Nature 315:641 (1985)).
IL-l~ is a potent modulator of endothelial cell function.
It inhibits endothelial cell growth and alters their phenotype in vitro. In the presence of IL-l, endothelial cells assume an elongated fibroblast-like phenotype, which resembles the phenotype that is present during the early stages of the .. .. . :., . - -; . .. , . ~ :,: . , , . ~.

WC) 91/1522fi P~IUS91/02343 2~336 endothelial differentiation pathway in vitro. IL-l~ induces the expression of activities, such as tissue factor procoagulant activity, increases plasminogen activator inhibitor-l activity and decreases tissue plasminogen activator activity. It induces the production of the vasodilator and inhibition of platelet aggregation, prostacyclin.
IL-l~ shares certain features in common with other mitogens, such as HBGF-I and HB~F-2. The precursor to IL-l~
lacks a signal se4uence for secretion and IL-Ia contains a nuclear translocation sequence, which is responsible for transport across the nuclear membrane. The nucleotide sequence is presented in Figure 4, herein (March, C.J., et al.
Nature 315:641 (1985)).

IV. Summary Thus, because development and differentiation of the human endothelial cell can be manipulated in vitro by the addition of various mitogens, because of its important phys;ological role, and because it e~hibits a finite lifespan that is accompanied by morphological changes and the loss of the ability to respond to mitogens, it represents a good model for the study of senescence.
The prospect of reversing senescence and restoring the proliferative potential of endothelial cells has implications in many fields of endeavor. Many of the diseases of old age are associated with the loss of this potential. Also the tragic disease, progeria, which is characterized by accelerated aging is associated with the loss of proliferative potential af endothelial cells. Restoration of this ability would have far-reaching implications for the treatment of this disease, of other age-related disorders, and, of aging per se.

WO "1/1522fi PCT/US91/02343 2 ~ ~ 9 3 3 ~ `

In addition, the restoration of proliferative potential of cultured cells has uses in medicine and in the pharmaceutical industry. The ability to immortalize nontransformed cells can be used to generate an endless 5supply of certain tissues and also of cellular products.

SUMMARY OF THE INVENTTON

It is one object of this invention to provide a method for rejuvenating senescent cells, comprising decreasing the concentration of IL-1~ in the nucleus of said cells, wherein 10said level is decreased to at most about that observed in the . . . .
nucleus of younger cells that have proliferative potential.
In detail, the invention provides a method for rejuvenating a senescent cell, which comprises providing to the cell an effective amount of an agent capable of impairing 15the expression of a gene which encodes interleukin~
The invention further provides the embodiments of the above-described method wherein (1) the expression is impaired by an agent capable of impairing the translation of mRNA that encodes IL-1~, or wherein (2) the expression is impaired by an 20agent capable of impairing transcription of an mRNA molecule that encodes IL-1~.
The invention further provides the embodiments of the abo~e-described method wherein the agent is an antisense oligonucleotide characterized in:
25(a) having a sequence of nucleotides complementary to an mRNA which encodes interleukin-1~ (IL-1~);
(b) being capable of hybridizing to the mRNA and thereby impairing expression of the mRNA.
The invention further a method for in vitro tissue cell 30culture of a non-immortal cell, comprising culturing the cell in the presence of an effective amount of an agent capable of :

WO 91/1522fi PCT/US91/02343 9 2~033~

impairing the expression of a gene which encodes interleukin-The invention further provides the embodiments of the above-described method wherein (1) the agent is an oligonucleotide capable of impairing the translation of mRNA
that encodes IL-1~ or (2) wherein the agent is capable of specifically blocking transcription of DNA encoding IL-1~ mRNA
in the cell.
The invention further provides the embodiments of the above-described method wherein the agent is an antisense oligonucleotide characterized in:
(a) having a sequence of nucleotides complementary to an mRNA which encodes interleukin-1~ (IL-1~);
(b) being capable of hybr;dizing to the mRNA and thereby inhibiting translation of the mRNA.
The invention further provides a method for rejuvenating a senescent cell, which comprises providing to the cell an effective amount of an agent capable of impairing the translocation of an expression producl; of an IL-1~ gene, as by culturing the cells in the presence of an effective amount of a mutated IL-1~ polypeptide, wherein the mutation is in the nuclear translocation region of the polypeptide, whereby the mutated IL-l~ polypeptide is able to bind to cellular receptors but is unable to translocate to the nucleus and wherein the effective amount is effective to impair nuclear translocation of endogenous IL-1~.
The invention further ~rovides a method for treating an age-related disorder in an afflicted individual which comprises treating the afflicted individual ~ith an effective amount of an agent capable of impairing the expression of a gene which encodes interleukin-1~.
The invention further provides an antisense oligonucleo-tide characterized in:

. ~ , .

W(~ ~1/1522fi PCI`/US91/02343 20~a33~ -lO-(a) having a sequence of nucleotides complementary to an mRNA which encodes interleukin-l~ (IL-l~);
(b) being capable of hybridizing to the mRNA and thereby inhibiting translation of the mRNA.
Of particular interest to the present invention is an oligonucleotide that comprises the sequence: 5' TTT GGC CAT
CTT GAC TTC 3', or its equivalents.
Unless defined otherwise, all technical and scientific terms used herein have the same mean;ng as is commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials simllar or equivalent to those described herein can be used in the practice of testing of the present invention, the preferred methods and materials are now descri~ed. All publications and patents mentioned hereunder are incorporated by reference thereto.

BRIEF DESCRIP~ION OF T~IE FIGURES

Figure 1 shows the expression of cyclooxygenase (cox) and IL-l~ by young and senescent human endothelial cells in vitro.
Figure lA shows IL-l~ induction of the cox transcript. Figure lB shows PMA induction of the cox transcript. Figure IC
shows expression of IL-l~ mRNA by human endothelial cells.
Figure lD shows induction of IL-l~ by IL-l~ in human endothelial cells. Figure lE shows expression of IL-l~ mRNA
by progeric fibroblasts and age-matched control human fibroblasts.
Figure 2 shows an extension of human endothelial cell lifespan by an antisense oligonucleotide targetPd against human IL~
Figure 3 shows phase contrast photomicrographs of human endothelial cells at different population doublings. Panel (A) shows antisense IL-l~ oligomer-treated cells (PD 88).

,, " . , " ':. ~ ''.
' : `: `'.. `.. '` ': , , :; .:' WO ~1/15226 PCI`/US91/02343 2 ~ 3 ~
-Il^

Panel (B) shows senescPnt cells (PD 50). Panel (C~ shows cells identical to (A) except oligomer was removed from the cultured media for 16 days. Magnification is 200X.
Figure 4 sets forth the nucleotide sequence of the sense strand of DNA that encodes IL-1~ (see, March, C.J., et al.
Nature 315:641 (1985)).

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention derives, in part, from the recognition that the proliferative potential of non-proliferating (i.e. "senescent") cells may be restored by impairing the expression (i.e. transcription and translation) of the IL-1~ gene in such cells.
The present invention thus concerns chemical agents capable of restoring the proliferative potential of mature senescent cells. This restoration of proliferative potential in senescent cells and of a phenotype typical of younger proliferating cells is termed "rejuvenation."
The term "proliferative potential" refers to the potential ability of cells to either grow and divide or to respond to a mitogen that would induce proliferation of younger cells or the same type to proliferate.

A. IL-l~

As used here;n, the term "IL-1~" includes any protein that exhibits the biological act;vit;es and properties of the IL-1~ whose sequence is set forth in Figure 4. It includes alleles thereof and corresponding proteins from any mammalian species. It includes IL-1~ whose transcript is expressed at elevated levels in senescent cells.

;~ .. ..

~ . ~

WO ~ltl5'~26 PCr/US9~/02343 20~3~

~he term "IL-l~" further includes the proteins resulting from the expression of a set of immediate-early genes during the early stage of differentiation in vitro. The ability of IL-l~ to induce expression of at least one of these transcripts, cyclooxygenase (cox) mRNA, is influenced by the number of population doublings that the cells have undergone.
For example, in young cells the cox transcript is inducible by IL-1~.
IL-l~ like the heparin binding growth factors, HBGF-l and HBGF-2, is expressed as a polypeptide lacking a signal sequence (Burgess, W.H. et al., Ann. Rev. Biochem. 58:575 (1989)). _The extracellular secretion of IL-l~ and HBGF-l by anchored dependent cells remains controversial. The description of the nuclear translocation sequence in HBGF-l (U.S. Patent Application Ser. No. 07/505,124 to Imamura et al., which was filed on April 4, 1990, wh;ch is herein incorporated in its entirety by reference thereto) and the detection of the HBGFs (Bouche, G. et al., Proc. Natl. Acad.
Sci. (U.S.A.) 84:6770 (1987); Kardami, E. et al., ~. Cell Biol. 109:1865 (1987)) and IL-l~ `(Grenfell, S. et al., Biochem. ~ :813 (1989); Curtis, B.M. et al., J. Immunol.
1295 (1990)) as intranuclear polypeptides indicates that these proteins may be functional as intracellular regulators of gene expression.

B. Agents Capable of Restoring the ProliferatiYe Potential of Senescent Cells The chemical agents which may be used to rejuvenate cells comprise: (1) an oligonucleotide, (2) a nucleic acid binding protein, or (3) a compound whose structure mimics that of either an oligonucleotide or a nucleic acid binding molecule (i.e. a "peptidomimetic" agent). In particular, the chemical agents of the present inYention have the ability to specifi-.

. . .. . ,... , , . . :

. , ;
. , .. ;. -. . .. . ;. . :, ... ~ .
. . . . . , ,:

WO ~1/15226 PCI/US91/û2343 -13- 2~33~

cally impair (i.e. attenuate or prevent) the translation of the IL-l~ gene.
Oligonucleotides are the preferred chemical agents of the invention. Of particular interest to the present invention are "antisense" oligonucleotides. In general, an "antisense oligonucleotide" is a nucleic acid (either DNA or RNA) whose sequence is complementary to the sequence of a target mRNA
molecule (or its corresponding gene) such that it is capable of binding to, or hybridizing with, the mRNA molecule (or the gene), and thereby impairing (i.e. attenuating or preventing) the translation of the mRNA molecule into a gene product. To act as an antisense oligonucleotide, the nucleic acid molecule must be capable of binding to or hybridizing with that portion of target mRNA molecule (or gene) which mediates the lS translation of the target mRNA. Antisense oligonucleotides are disclosed in European Patent Application Publlcation Nos.
263,740; 335,451; and 329,882, and in PCT Publication No.
WO90/00624, all of which references are incorporated herein by reference~
The present invention is particularly concerned with those antisense oligonucleotides which are capable of binding to or hybridizing with mRNA molecules that encode the IL-l~
gene product.
Thus, in one embodiment of this invention, an antisense oligonuclentide that is designed to specifically block translation of an IL-l~ mRNA transcript can be used to rejuvenate senescent cells. Other means whereby intranuclear or endogenous levels of IL-l~ can be reduced include, but are not limited to, specifically blocking transcription of the IL-1~ gene or impairing nuclear translocation of IL-l~.
The invention further provides a method for rejuvenating a senescent cell, which comprises providing to the cell an effective amount of an agent capable of impairing the translocation of an expression product of an IL-l~ gene, as by - .. . .

' ~'' "`"` '' ` ` ~,' i WC) ~1/1522~ PCrlUS91/02343 ~a~033~ _14_ culturing the cells in the ?resence of an effective amount of a mutated IL-1~ polypeptide, wherein the mutation is in the nuclear translocation region of the polypeptide, whereby the mutated IL-l~ polypeptide is able to bind to cellular receptors but is unable to translocate to the nucleus and wherein the ef~ective amount is effective to impair nuclear translocation of endogenous IL-1~.
One manner in which an anti-IL-1~ antisense oligonucleo-tide may achieve these goals is by having a sequence complementary to that of the translation initiation region Ot the IL-1~ mRNA and of sufficient length to be able to hybridize to the mRNA transcript of the IL-l~ gene. The size of such an oligomer can be any length that is effective for this purpose. Preferably, the antisense oligonucleotide will be about 10-30 nucleotides in length, most preferably, about 15-24 nucleotides in lPngth.
Alternatively, one may use antisense oligonucleotides that are oF a length that is too short to be capable of stably hybridizing to IL-1~ mRNA under physiologic, in vivo conditions. Such an oligonucleotide may be form about 6-10, or more nucleotides in length. To be used in accordance with the present invention, such an oligonucleotide is preferably modified to permit it to bind to locus of the translation region of the IL-1~-encoding mRNA. Examples of such modified molecules include oligonucleotides bound to an antibody (or antibody fragment), or other ligand (such as a divalent crosslinking agent (such as, for example, trimethylpsoralin, 8-methoxypsoralin, etc.)) capable of binding to single-stranded iL 1~ mRNA molecules.
An anti-IL-l~ antisense oligonucleotide bound to one reactive group of a divalent crosslinking agent (such as psoralin (for example, trimethylpsoralin, or 8-methoxy-psoralin) adduct would be capable of crosslinking to IL-1~
mRNA upon activation with 350-420 nm W light. Thus, by , . . ; : ~ : ; :
- . : - . .i . :: .

W o ~1/152~ PCT/US9t/02343 15- 2~33~

regulating the intensity of such light (as by varying the wattage of the UV lamp, by increasing the distance bet~een the cells and the lamp, etc.) one may control the extent of binding between the antisense oligonucleotide and the IL-1 mRNA of a cell. This, in turn, permits one to control the degree of attenuation of IL-1~ gene expression in a recipient cell.
In general, the antisense oligomer is prepared in accordance wlth the nucleotide sequence of the IL-1~ gene (Figure 4) of a portion of the IL-1~ transcript that includes the translation initiation codon and contains a sufficient number of complementary nucleotides to block translation.
As shown in Figure 4, the translation locus of the IL-1 gene contains the following sequence:
5' .... GAA GTC AAG ATG GCC AAA ... 3' where translation begins with the codon Ql~, and where the translated codons are shown in boldface and underlined. This region of the IL-1~ gene is transcribed to form an mRNA
molecule having the sequence:
5' .... GAA GUC AAG AUG 6C~: MA ... 3' Thus, in accordance with the present invention, an anti-IL-1 ant~sense oligonucleotide having the sequence:
3' ... CTT CAG TTC TAC CGG TTT... 5' will be able to bind to the IL-1~ mRNA and impair its translation. This antisense oligonucleotide is the preferred chemical agent of the present invention. As stated above, the antisense oligonucleotide may be of shorter or longer length.
The sequence of the antisense oligonucleotide may contain one or more insertions, substitutions, or deletions of one or more nucleotides provided that the resulting oligonucleotide is capable of binding to or hybridizing with the above-described translation locus of either the IL-1~ mRNA, or the IL-l~ gene itself.

. .
. :
;- - , ~ .; .
- . ' .. ', :,: .;
: :: - : : .
~ . . : , .

W o 91/15226 P~T/US~1/02343 '2 ~ ~ 0 3 3 ~ -16-Any means known in the art to synthesize the antisense oligonucleotides of the present invention may be used (Zamechik et al., Proc. Natl. Acad. Sci. (U.S.A.) 83:4143 (1986~; Goodchild et al., Proc. Natl. Acad. Sci. (U.S.A.) 85:5507 (1988); Wickstrom et al., Proc. Natl. Acad. Sci.
(U.S.A.) 85:1028; Holt, J.T. et al., Mol. Cell. Biol. 8:963 (1988); Gerwirtz, A.M. et al., Science 242:1303 (1988);
Anfossi, G., et al., Proc. Natl. Acad. Sci. ~U.S.A.) 86:3379 (1989); Becker, D., et al., EMBO J. 8:3679 (1989); all of which references are incorporated herein by reference).
Automated nucleic acid synthesizers may be employed for this purpose. In addition, desired nucleotides of any sequence can be obtained from any commercial supplier of such custom molecules.
Most preferably, the antisense oligonucleotides of the present invention may be prepared using solid phase "phosphoramidite synthesis." The synl:hesis is performed with the growing nucleotide chain attached to a solid support derivatized with the nucleotide which will be the 3'-hydroxyl end of the oligonucleotide. The method~involves the cyclical synthesis of DNA using monomer units whose 5'~hydroxyl group is blocked (preferably with a 5'-OMT (dimethoxytrityl) group), and whose amino groups.are blocked with either a benzoyl group (for the amino groups of cytosine and adenosine) or an isobutyryl group (to protect guanosine). Methods for producing such derivatives are well known in the art. ~o form the preferred oligonucleotide of the present invention:
3' ... CTT CAG TTC TAC CGG TTT... 5' the first step of synthesis cycle is treatment of the derivatitized solid support with acid to remove the trityl group of the derivatized 3' terminal C. This frees the 5'- .
hydroxyl group for the addition reaction. The next step, activation, creates a highly reactive nucleoside derivative which reacts with the hydroxyl group. This intermediate is - , .
~;~ ,. ~ . , .
..
,- , . ~ , . .. .
- ~ . - .. :

W O 91t15~26 2 ~ 31f~2343 created by simultaneously adding the phosphoramidite derivative of the next nucleotide (i.e. "T" in the preferred embodiment), and a weak acid (tetrazole) to the reaction chamber. The tetrazole protonates the nitrogen of the phosphoramidite, making it susceptible to nucleophilic attack.
Since this intermediate is highly reactive, the addition reaction is complete in less than 30 seconds at room temperature. The phosphoramidite is blocked at the 5'hydroxy with the DMT group.
The next step ("capping step") terminates any chains which did not undergo addition. This reaction is accomplished with acetic anhydride and dimethylaminopyridine.
The internucleotide linkage is then converted from the phosphite to the more stable phosphate ("oxidation step").
Most preferably, iodine is used as the oxidizing agent, and water as the oxygen donor. This reaction is complete in less than 30 seconds.
After the oxidation step, the DMT group is removed and the cycle is repeated until chain elongation has produced an oligonucleotide of the preferred sequence. At this point, the oligonucleotide is still bound to the support and has protecting groups on the phosphates and the exocycl;c amines of the bases A, G, and C. To produce biologically active DNA, the methyl groups on the phosphates are removed by a 30-minute 2~ treatment with triphenol. The chain is then clea~ed from the support by a one-hour treatement with concentrated ammonium hydroxide. The protecting groups on the exocyclic amines of the bases are cleaved by a 6-12 hour treatment with ammonium hydoxide at 55-C.
The yield of oligonucleotide obtained from synthesis decreases ,with the length of the oligonucleotide in accordance with the formula: XY = Yield, where X is the efficiency of each coupling step (typically 95%), and y is the , : . . . -: .
. .~ - . . .. .:

.. . - : ; , , - , . . ~ ; . :
.. : ,. , ,. ~,. - . .

W O ~1/1522fi PCT/US91/Ot343 2 ~ 8 0 3 3 ~ -18-number of residues. For the preferred oligonucleotide, overall yield is approximately 40% (o.9518 = .~0).

C. Uses of the Rejuvenating Agents of the Present Invention Since the agents of the present invention are capable of restoring the proliferative potential to senescent cells, they may be used for a wide range of therapies and applications.
The agents of the present invention may be used to impair senescence of a desired cell type. Thus, they may be used to immortalize valuable cell types (such as primary tissue culture cells, etc.) which would otherwise have a transient period of proliferative viability. The agents of the present invention may be used for research or to permit the accumulation of large numbers o~ cells, as for organ or tissue grafts or transplants. In one embodiment, therefore, the agents of the present invention may be used in conjunction with methods for organ or tissue clllture to facilitate such methods.
A use is said to be therapeutic if it alters a physiologic condition. A non-theralpeutic use is one which -alters the appearance of a user.
The agents of the present invention may be used topically for a therapeutic or non-therapeutic purpose, such as, ~for example, to counter the effects of aging on skin tone, color, texture, etc. The agents of the present invention may be employed to rejuvenate tissue, particularly skin. Thus, they may be used in skin conditioners, and the like, capable of therapeutic use, or in cosmetic preparations (i.e. make-up, skin creams, skin lotions, and the like) or in cleansers (i.e.
soap, shampoo, hair conditioners, and the like).
Cosmetic preparations may, for example, comprise the antisense oligonucleotide of the present invention, or its equivalent, and a lipophylic carrier or adjunct, preferably . .. . ~ ; ~ , . . ., . -.. ` `: ' -. `. `';,', ` :, , ` "' ' :~' : ' . '" ' , :- ' :, ~ ': : ' ' , .:

WO ~l/lS22fi PCl-/U~;91/0'3~3 -19- 2~8~33~

dissolved in an appropriate solvent. Such a solvent may be, for example, a water-ethanol mixture (containing 10% to 30%
v/v or more ethanol). Cosmetic preparations may contain 000.1% to 1.0% of the antisense oligonucleotide in ointment, cream, or lotion compositions. Suitable cosmetic carriers, adjuncts and solvents are described in Remington's Pharmaceutical Sciences (16th ed., Osol, A., Ed., Mack, Easton PA (1980), which reference is incorporated herein by reference).
Since the agents of the present in~ention stimulate cellular proliferation, they may be used to promote wound healing, recovery from burns, or after surgery, or to restore .. . .
atrophied tissue, etc. For such an embodiment, the agents of the present invention may be formulated with antibiotics, anti-fungal agents, or the like, for topical or systemic administration.
The agents of the present inYention may be used to stimulate the proliferation of spermatocytes, or the maturàtion of oocytes in humans or animals. Thus, the agents of the present invention may be used l.o increase the fertility of a recipient.
Since the agents of the present invention are able to rejuvenate cells, they may be used therapeutically in the treatment of diseases such as: progeria (Badame, A.J.7 Arch.
2S Dermatol. 125:540 (1989); Hamer, L. et al., Orthoped. 11:763 (1988); Martin, G.M., Natl. Canc. Inst. Monoqr. 60:241 (1982); all of which references are incorporated herein by reference); age-related disorders (Martin, G.M., Genome 31:390 (1989); Roe, D.A., Clin. Geriatr. Med. 6:319 (1990);
Mooradian, A.D., J. Amer. Geriat. Soc. 36:831 (1988); Alpertt J.S., Amer. J. Cardiol. 65:23j (1990); all of which references are incorporated herein by reference); Alzheimer's disease (Terry, R.D., Monoqr. Pathol. 32:41 (1990); Costall, B. et al., PharmacoPsychiatry 23:85 (199~ hich references .. . .
. . - , . ~

: ,.:,: ; :.,.
: - . -; .~ ,: .
- .. ;
- ~- -, WO ~1/15226 PCIIUS91/02343 2~)~033~ -20-are incorporated herein by reference); asthenia and cachexia (Verdery, R.B., Geriatrics 45:~6 (1990)i which reference is incorporated herein by reference), etc.
The capacity of the agents of the present invention to mediate cellular proliferation and rejuvenation may be used to identify agents capable of reversing these processes. Thus, for example, one may incubate cells in the presence of both an antisense oligonucleotide and a suspected antagonist compound.
The cells would be monitored in order to determine whether the compound is able to impair the ability of the antisense oligonucleotide to mediate any of the above-described effects. Thus, the present invention includes a "screening assay" capable of identifying antagonists of the antisense oligonucleotides.
Among the antagonist compounds whi`ch could be identified through the use of such a screening assay are compounds which could be used to induce infertility. Similarly, the assay will permit the identification of compounds capable of suppressing tissue regeneration or vascularization. Such compounds may be useful in the treatml3nt of cancer.

D. Methods of Administration The agents of the present invention can be formulated according to known methods to prepare pharmaceutically useful compositions, whereby these materials, or their functional derivatives, are combined in admixture with a pharmaceutically acceptable carrier vehicle. Suitable vehicles and their formulation, inclusive of other human proteins, e.g., human serum albumin, are described, for example, in Remington's Pharmaceutical Sciences (16th ed., Osol, A., Ed., Mack, Easton PA (1980)). In order to form a pharmaceutically acceptable composition suitable for effective administration, such compositions will contain an effective amount of an antisense : : ., ;;
.. - , , : .. . . ...
.. , ., . . ,., . - . . ~; .

, ~ , . , ;::. :

W O 91/15226 PCT/US9t/02343 -21- 2~033~

oligonucleotide, or its equivalent, or their functional derivatives, together with a suitable amount of carrier vehicle.
Additional pharmaceutical methods may be employed to control the duration of action. Controlled release preparations may be achieved through the use of polymers to complex or absorb an antisense oligonucleotide, or its equivalent, or their functional derivatives. The controlled delivery may be exercised by selecting appropriate macromolecules (for example polyesters, polyamino acids~
polyvinyl, pyrrolidone, ethylenevinylacetate, methylcellulose, carboxymethylcellulose, or protamine sulfate) and the concentration of macromolecules as well as the methods of incorporation in order to control release. Another possible method to con.rol the duration of action by controlled release preparations is to incorporate an antisense oligonucleotide, or its equivalent, or their functional derivatives, into particles of a polymeric material such as polyesters, polyamino acids, hydrogels, poly(lactic acid) or ethylene vinylacetate copolymers. Alternatively, instead of incorporating these agents into polymeric particles, it is possible to entrap these materials in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatine-microcapsules and poly(methylmethacylate) microcap-sules, respectively, or in colloidal drug delivery systems, for example, liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules or in macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences (l980).
In one embodiment, the compositions of the present invention may be formulated as a cream, lotion, ointment, or the like, for-topical administration to the skin. Such compositions may optionally contain wetting agents, ... :. , ., ... -;

, . . .

WO 91/15226 PCr/US91/0 '343 20'~33~ -22-emulsifying and suspending agents, or sweetening, flavoring, coloring or perfuming agents.
~he compositions of the present invention can also be formulated for administration orally or parenterally by injection, rapid infusion, nasopharyngeal absorption (intranasopharangeally), or dermoabsorption. The compositions may alternatively be administered intramuscularly, or intravenously. Compositions for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Carriers, adjuncts or occlusive dressings can be used to increase skin permeability and enhance absorption. Liquid dosage forms for oral administration may generally comprise a liposome solution contain;ng the liquid dosage form. Sllitable forms for suspending liposomes include emulsions, suspensions, solutions, syrups, and elixirs con~aining inert diluents commonly used in the art, such as purified water. Besides the inert diluents, such compositions can also include wetting agents, emulsifying and suspending agents, or sweetening, flavoring, coloring or perfuming agents.
A composition is said to be "pharmacologically accept-able" if its administration can be tolerated by a recipient patient. Such an agent is said to be administered in a "therapeutically effective amount" if the amount administered is physiologically significant. An agent is physiologically significant if its presence results in a detectable change in the physiology of a recipient patient.
Generally, the dosage, which can be adjusted by one of ordinary skill in the art, needed to provide an effective amount of the composition will vary depending upon such .
.... . . .

: : . : .
.

WO 91/15226 PCI`/US91/02343 -23- 20~3~

factors as the recipient's age, condition, sex, and extent of disease, if any, and other variables.
Effective amounts of the compositions of the invention can vary from 0.01-1,000 ~g/ml per dose or application, although lesser or greater amounts can be used.
Having now generally described the invention, the same will be more readily understood through reference to the following examples which are provided by way of illustration, and are not intended to be limiting of the present invention, unless specified.

EXAMPLE I
Materials and Cell Culture Recombinant human interleukin-1~ (IL-1~) was obtained from ~offman La Roche, Nutley, NJ. Recombinant human HBGF-1 was obtained from the American Red Cross, Rockville, MD.
Porcine TGF-S was purchased from R & D Systems.
Primary cultures of human umbilical vein endothelial cells (huvec) were obtained from Harvard Medical School, Boston, MA, and were grown on fibronectin coated plates in Medium 199 supplemented with 10% (v/v) fetal bovine serum (FBS), 1x antibiotic and antimycotic mixture (GIBC0, Grand Island, NY). Cells were subcultured at a 1:5 split ratio.

RNA Pre~aration Total RNA was obtained from confluent cultures of HUVEC
that were.starved in Medium 199 with 5% (v/v) FBS for 20 hours and the cells incubated with various compounds (indicated below). Total RNA was isolated by the guanididium isothiocyanate procedure, and was poly A+ purified by affinity chromatography on oligo dT cellulose (Maniatis, T. et al.
In:Molecular Cloninq: A Laboratory Manual, Cold Spring Harbor Press, NY (1982)).

. . .

. . . : , .. : . . . .
.. i , , .

2~336 -24-Northern Blot Analysis Total RNA (10 ~9) or poly A+ purified RNA (5 ~9) was electrophoresed on a 1% agarose gel containing 2.2 M
formaldehyde capillary-blotted onto nylon membrane filters (Zeta-prob (TM) membrane, Biorad, CA) and probed with the desired 32P-labelled probe (Maniatis, T. et al. In:Molecular Cloninq: A Laboratory Manual, Cold Spring Harbor Press, NY
(1982)). The cDNA probe were labeled to high specific activity (>108 cpm/~g) using random primer labeling kit (BRL) and was used to hybridize filters in Church-Gilbert buffer (0.5 M sodium phosphate p 7.2, containing 7% SDS and 1% bovine serum albumin, l mM EDTA and 20% formamide at 65 C for 16-20 . .
hrs). Filters were washed at high-stringency (0.1xSSC, 65C) and exposed to Kodak X-AR film for 24-72 hours at -80C.

Reverse TranscriDtase-PolYmerase Chain Reaction AnalYsis (RT-PCR~
Because of the low levels of the transcripts of interest in senescent human endothelial cells required, it was necessary to use RT-PCR methods for detection.
Total RNA from HUVEC was purified as described abo~e, was converted to cDNA by treatment with 21)0 units of MMLV reverse transcriptase (Bethesda Research Labs, MD) using antisense pr;mers (0.5, ~9) in 50 mM Tris-HCl, pH 8.0, 1 mM
dithiothreitol, 15 mM NaCl, 3 mM MgCl2 1 unit RNAsin (Promega), 0.8 mM dNTPs and incubated for 1 hour at 37C.
The reaction was terminated by heating at 95-C for 5 to 10 minutes and diluted to 1 ml with distilled water.
Enzymatic amplification was done on a 10 ~l aliquot of the cDNA mix. PCR was performed in 50 mM Tris-HCl, pH, 8.0, 1.5 mM MgCl2, 10 mM KCl, 0.2 mM dNTPs, 0.5 ~9 of each sense and antisense primer, and 1 unit of Taq DNA polymerase (Cetus, CA) (Saiki, et al., Science 239:487-491 (1988), Mullis, K. et al., Cold SPrinq Harbor symD~ quant. Biol. 51:263-273 (1986);

,.-.,. . ~.

:: , . . .

W ~ /15226 PCTIUS91/02343 -25- 2~3~

Erlich H. et al., EP 50,424; EP 84,796, EP 258,017, EP
237,362; Mullis, K., EP 201,184i Mullis K. et al.! US
4,683,20Z; Erlich, H., US 4,582,788; and Saiki, R. çt al., US
4,683,194), which references are incorporated herein by reference). The reaction mixture was heated at 94 C for 1 minute, annealed at 55 C for 2 minutes, and extended at 72' C
for 3 minutes for the indicated number of repetitive cycles.

The expression of the transcript for cox was measured by reverse transcriptase polymerase chain reaction (RT-PCR).
The results of this experiment are shown in Figure 1. Figure 1 shows the expression of cyclooxygenase (cox) and IL-1~ by young and senescent human endothelial cells in vitro. Figure lA shows IL-1~ induction of the cox transcript. For this experiment, young or senescent human endothelial cells (HUVEC;
105 cells) (number of population doublings ("PD") 20 and 53, respectively) were incubated with IL-l~ (1 ng/ml) for 4 hrs.
Total RNA was extracted and reverse-transcribed to form cDNA
(1 ~9) using the methods of Maniatis (Maniatis, T. et al.
In:Molecular Cloninq: A Laboratory Manual, Cold Spring Harbor Press, NY (19B2)).
The cDNA fragments were diluted 1:20 in water and 10 ~l were amplified by polymerase chain reaction (PCR) for 40 cycles (Saiki, et_al., Science 239:487-491 (1988), Mullis, K.
et al., Cold SPrinq Harbor Symp. Quant. Biol. 51:263-273 (1986); Erlich H. et al., EP 50,424; EP 84,796, EP 258,017, EP
237,362; Mullis, K., EP 201,184; Mullis K. et al., US
4,683,202; Erlich, H., US 4,582,788; and Saiki, R. et al., US
4,683,194), which references are incorporated herein by reference). The products of the amplification were fractionated on a 1.2% agarose gel and stained with ethidium -. ~ .., ..; .
:,` ,''.. '; ,.'.; ~ :

W~ 91/152~6 PCI/US91/02343 2~ 336 -26-bromide. Amplification of the same RNA and GAPDH primers confirmed that equal amounts of RNA were reverse transcribed.
The sequence of the sense and antisense primers for cox are: 5'-GC~ GGG AGT CTT TCT CCA ACG TGA G-3' and 5'-GGC AAT
GCG GTT GCG GTA TTG GAA CT-3', respectively. The sense and antisense primers for GAPDH are: 5' -CCA CCC ATG GCA AAT TCC
ATG GCA-3' and 5' -TCT AGA CGG CAG GTC AGG TCC ACC-3', respectively. In Figure lA, lanes 1-2 show the results with young cells; lanes 3-4, show the results with senescent cells.
Figure lB shows PMA induction of the cox transcript.
Young (PD 20, lane 1-2) and senescent tPD 53, lane 3-4) HUVEC
were treated with PMA (20 ng/ml) for 1 hr. RNA was extracted, reverse-transcribed and amplified by PCR as described above.
The~ data demonstrate that, while cox mRNA is readily induc;ble by IL- 1~ in young endothelial cells (F;gures lA), the levels of the cox transcript appear to be amplified in senescent human endothelial cells not exposed to exogenous IL-1~. In addition, the levels of cox mRNA in the senescent human endothelial cell population did not change in response to exogenous IL-1~ (Figure lA). Similar cox expression data were also obtained with human endothelial cells exposed to PMA (Figure 18).

IL-1~ has been shown to be a potent inhibitor of endothelial cell proliferation in Yitro (Montesano, R., J.
Cell Biol. 99:1706 (1984); Montesano, R. et al., J. Cell Phvsiol. 34:460 (1988)). It has also been shown that IL-1 can induce the expression of IL-1~ transcript in human endothelial cells (Pierce, J.H. et al. Blood 71:684 (1988);
Mantovani, A. et al., Immunol Todav 10:370 (1989)).
Because cox mRNA expression appeared to be amplified in senescent human endothelial cells, and because endothelial cells were able to express the transcript of IL-1~, young and .. ; : . .. , ~ ,, W O~ 5~2fi PCT/US91/02343 -27- 2~

senescent human endothelial cells were examined for the presence of the IL-l~ transcript.
Figure lD shows induction of IL-l~ by IL-l~ in human endothelial cells. For this experiment, young cells (P0 20), senescent cells (PD 53) and antisense IL-l~ oligomer-treated (PD 97) cells were stimulated with IL-l~ (10 ng/ml) for 6 hrs.
Total RNA was extracted, reverse-transcribed and amplified by PCR for 30 cycles as above. Lanes 1-2 show results for young cells. Lanes 3-4 show results for senescent cells. Lanes 5-6 show results for antisense IL-l~ oligomer-treated cells.
Figure lD shows that young endothelial cells were able to express the IL-1~ mRNA in response to IL-l~ but that senescent endothelial cells contained elevated levels of the IL-l~
transcript and were not responsive to IL-l~.
lS The levels of IL-l~ mRNA in human endothelial cells exposed to the antisense lL-1~ oligomer were examined an found to be low (Figure lD). Further, it was found that the oligomer-exposed cells were responsive to exogenous IL-l~.
Upon addition of exogenous IL-l~, a significant increase in the IL-1~ transcript was observed (Figure lD). While this response was also observed in young human endothelial cells the IL-l~ mRNA levels in the senescent population remained elevated and non-responsiYe to the addition of exogenous IL-1~ (Figure lD). Further, in contrast with the levels of the IL-l~ polypeptide in the senescent population, it was not possible to detect the expression of the IL-lh polypeptide in the population of human endothelial cells treated with the antisense IL-l~ oligomer using immunoprecipitation methods.
Thus, the inability of the population of human endothelial cells with extended population doublings to sustain the - elev~ted levels ~f IL-1~ mRNA may be a result of the low levels of the IL-1~ polypeptide in these cells.
Also the IL-l~ translation product was detected in senescent but not young human endothelial cells, which .
.
, . . .

.

WO 91/15226 P~T/US91/02343 ~a33~ -28-suggests that the elevated levels of cox mRNA in senescent human endothelial cells and the failure of senescent human endothelial cells to proliferate in vitro could be a result of elevated levels of the IL-l~ mRNA.

An antisense oligonucleotide for IL-l~ was designed and synthesized in order to evaluate whether senescence was a result of elevated levels of the IL-l~ mRNA. Antisense oligomers have found increased utility as selective repressors of translation in vitro (Zamechik et al., Proc. Natl Acad.
Sci. (U.S.A.) 83:4143 (1986); Goodchild et al., Proc.- Natl.
Acad. Sci. (U.S.A.I 85:5507 (1988); Wickstrom et al., Proc.
Natl. Acad. Sci. (U.S.A.l 85:1028; Holt, J.T. et al., Mol.
Cell. Biol. 8:963 (1988); Gerwirtz, A.M. et al., Science ~ 1303 (1988); Anfossi, G., et al., Proc. Natl. Acad. Sci.
(U.S.A.~ 86:3379 (1989); Becker, D., et al., EMB0 ~. 8:3679 ` (1989), all of which references are incorporated herein by reference).
An antisense AUG IL-I~ oligonucleotide was prepared which had the sequence: 5'-TTT GGC CAT CTT GAC TTC-3 and spanned 3 codons on each side of the ATG start codon of the IL-l~
transcript (March, C.J., et al., NaturQ 315:340 (1985); Figure 4).
Daily addition of this antisense IL-l~ oligomer to populations of human endothelial cells near senescence resulted in a significant extension of human endothelial cell proliferation in vitro. The results are depicted in Figures 2 and 3.
F;gure 2 shows an extension of the human endothelial cell lifespan by an antisense oligonucleotide targeted against human IL-l~. For this experiment, cultured human endothelial ` cells were treated daily with the above-described antisense cligonucleotide (50 ug/ml). Population doublings were `

.

.. . . : , :~ . :. . , :: . .:
.
'.:,...

WO ~1/15226 PCI/US91/02343 29 2~33~

calculated after each passage. ~iable cell counts were obtained for the control population (closed circles), for the antisense IL-1~ oligomer population (open triangles) and for the population generated after removal of the antisense IL-1 S oligomer (closed triangles). No effect was observed when cells were exposed to sense or antisense oligonucleotides complementary to regions of the human IL-1~ that did not interfere with translation.
Figure 3 shows phase contrast photomicrographs of human endothelial cells at different population doublings. Cells were fixed in methanol and stained with Giemsa (Maciag, T., J.
Cell. Biol. 91:420 (1981)). Panel (A) shows antisense IL-I~
oligomer-treated cells (PD 88). Panel (B) shows senescent cells (PD 50). Panel (C) shows cells identical to (A) except oligomer was removed from the cultured media for 16 days.
Magnification is 200X.
As shown, the senescent phenotype occurs in B and C but not in A. Rejuvenated human endothelial cells, which look like typical young human endothelial cells, are shown in the first panel; senescent cells, are shown in the second panel, and cells, which had been rejuvenal:ed, and then allowed to revert are shown in the third panel.
Cell density was measured as a function of the number of generations (population doublings), using untreated and antisense oligonucleotide-treated human endothelial cells.
As shown in Figure 2, the untreated cells rapidly senesced after about 40 to 60 generations, whereas the treated cells maintained a high density for 90 and more generations.
It can also be seen that if the antisense oligonucleotide was removed from the medium, the cells reverted to the senescent phenotype.
The control population declined in proliferative capacity near 50 population doublings (Figure 2) resulting in an increase in the size of the human endothelial cell (Figure ,~

, ~ ~ . ' .
1 . ' ' ' '~' ` ''', ,, ~''" ' ', ' w o gl/15226 PCT~US91/02343 20~3~ -30-3B), which is characteristic of the human endothelial cell senescent phenotype. In contrast, the population of human endothelial cells treated with a daily supplement of the antisense IL-l~ oligomer continued to proliferate beyond the normal boundary of population doublings and it was possible to double the in vitro lifespan of the human endothelial cell (Figure 2). The in vitro phenotype of the human endothelial cell appeared to resemble the phenotype of a population of endothelial cells with a reduced population doubling number (Figure 3A) and only a small increase in cell size was observed over the duration of the experiment.
In addition, the extended proliferative capacity of the human endothelial cell population exposed to the antisense IL-l~ oligomer and the maintenance of a normal phenotype were dependent upon the presence of the antisense IL-l~ oligomer.
Removal of the antisense IL-l~ oligomer from the population of human endothelial cells in vitro with an extended population of doubling level resulted in a reduction in the proliferative capacity of the monolayer (Figure 2) and a significant increase in the size of the human endothelial cell population (Figure 3C).
The extension of the number of population doublings was reversed by removal of the antisense IL-1~ oligomer. Thus, the human endothelial cell senescent phenotype may represent a non-terminal differentiation phenotype. Cellular senescence is a dynamic process with a reversible component regulated by the potential intracellular activity of a well recognized cytokine.

The expression of IL-1~ mRNA by human endothelial cells that were grown in the presence of the antisense oligonu-cleotide was compared with that in senescent and young endothelial cells and in a spontaneously transformed human ::. . : :. . ::

. .
~: . , .

WO ~1/152~6 PCT/US91/02343 2~033~

endothelial cell line, which does not require the additlon of growth factor supplements for proliferation.
This experiment is illustrated in Figure lC. Figure lC
shows expression of IL-1~ mRNA by human endothelial cells.
For this experiment, RNA was prepared ~rom confluent cultures of human endothelial cells. Total RNA was reverse transcribed and amplified for 30 cycles by PCR. The sequence of the sense and antisense primers for IL-1~ are 5'-GTT CCA
GAC ATG TTT GAA GAC CTG-3' and 5'-TGG ATG GGC AAC TGA TGT GAA
ATA-3'. Lane 1 shows results with transformed human endothelial cells. Lane 2 shows results with young human endotheliaI cells (PD 30). Lane 3 shows results with senescent human endothelial cells (PD 53). Lane 4 shows results with antisense IL-1~ oligomer-treated HUVEC (PD 95).
IL-1~ mRNA was not observed in the young and oligomer-treated cells, but was observed in the senescent cells. No IL-1~ transcript was detected (Figure lC, lane I) in the spontaneously transformed cells.
.....
EXAMPlE 6 Fibroblasts derived from human progerics, an autosomal recessive disease of premature a~ling (Debusk, F.L., J.
Pediatrics B0:697 (1972); Brown, ~.T., et al., Molecular Bioloqv of Aqinq, Woodhead, A.D., et al., eds., Plenum Press, NY, 375 (1984)) were examined.
The expression of IL-l~ mRNA by progeric fibroblasts was compared with that of age-matched control human fibroblasts.
This experiment is shown in Figure lE. Total RNA was reverse-transcribed and amplified for 30 cycles by PCR. Lane 1 contained cDNA produced control human fibroblasts and lane 2 contained cDNA from progeric fibroblasts. As shown in Figure lE, progeric fibroblasts, unlike age-matched control human fibroblasts, contain exaggerated levels of the IL-1 transcript.

', ~ , . , ' ' ' ' ' .~ . . "' '~ ;~
, ~ ~ , .. . ` . ~

- ,. . .

WO 91~15226 PCT/~S9t/02343 208~33~ -32-In summary, in order to elucidate the biological bases for senescence, human umbilical vein endothelial cells ~ere serially propagated in vitro (Maciag, T. et al., J. Cell.
Biol. 91:420 (1981); Gordon, P.B. et al., In Yitro 19:661 (1983); Johnson, A. et al., Mech Aqe. Dev. 18:1 (1982);
Thornton, S.C. et al., Science 222:623 (1983); Van Hinsbergh, V.W.M. et al., Eur. J. Cell Biol. 42:101 (1986); Nichols, W.W.
et al., J. Cell. Phvsiol. 1~:453 (1987); all of which references are incorporated herein by reference). Cell cultures representing different population doublings were exposed to IL-l~ and the induction of specific transcripts was measured by reverse transcriptase polymerase chain reaction.
~he data demonstrated that while cox mRNA is readily inducible by IL-1¢ in young endothelial cells (Figure lA), the levels of the cox transcript appears to be ampllfied in senescent endothelial cells not expos~d to exogenous IL~
In addition, the levels of cox mRNA in the senescent human endothelial cell population did not change in response to 2Q exogenous IL-l~ (Figure 2A). Similar cox expression data were also obtain~d with human endothelial cells exposed to PMA
(Figure lB).
Although human endothelial cells express low levels of the transcripts for heparin-binding growth factor (HBGF-l and HBGF-2), there were no significan~ differences in levels of these mRNAs in young and senescent populations of human endothelial cells. In addition, the levels of a potent inhibitor of IL-l~ did not vary among the different populations of human endothelial cells (the nucleotide sequence of the DNA encoding this inhibitor of IL-l¢ was recently reported; Hannum, C.H. et al., Nature 3~3:336 (1990); and Eisenberg, S.P. et al., Nature 343:341 (1990), :
:

` ~. : . ; ,, .

WO ~1/152~6 PCI/US91/02343 2~336 both of which references are incorporated herein by reference).
Although similar data were not obtained for transformins growth factor (TGF-~), which is another potent inhibitor of human endothelial cell proliferation in vitro (Baird, A. et al., Biochem. BioPhYs. Res. Commun. 138:476 (1986); Mullew, G.
et al., Proc. Natl. Acad. Sci. (U.S.A.) 84:5600 (1987); Mairi, J.A. et al., J. Cell Biol. 106:1375 (1988)), it is unlikely that TGF-~ participates as an inhibitor of cell proliferation during senescence since TGF-~ is expressed as an inactive extracellular precursor requiring proteolytic activation by plasmin (Laiho, M. et al., J. Biol. Chem. 262:17467 (1987);
Noses, H.L. et al., J. Cell Phvsiol. 5:1 (1987); Moscaelli, D.
et al., Biochem. BioDh~s. Acta 948:67 (1988); Saksela, 0. et ~, J. Cell Biol. 110:767 (1990)).
~ecause fetal bovine serum ~contains relatively high levels of plasmin inhibitors and the expression of plasminogen activator inhibitor-1 is induced by IL-1~ in endothelial cells, if TGF-~ expression is amplified during senescence, proteolytic activation of the precursor is an unlikely event.
Further, cell culture media conditioned by senescent human endothelial cells in vitro do n~t repress the prolifer-ative capacity of young endothelial cells in vitro (Libby, P.
et al., ~. Clin. Invest. 78:1432 (1986); Miossec, P. et al., J. Immunol. 136:2486 (1986); Stern, D.M. et al., J. EXD. Med.
162:1223 (1985); Malone, D.G. et al., Blood 71:684 (1988)).
The most significant observation, however, is that senescent cells exhibited enhanced expression of IL-l~. It was found that, if the levels of the IL-1~ transcript were reduced, the proliferative potential of the senescent cells was restored and the cells exhibited a morphology and phenotype characteristic of younger cells.
Thus, in accordance with this invention there is provided a method to rejuvenate senescent cells by reducing the .. , ..
. . ~ . . .-. .

,' ~

WO ~1tlS22fi PCl`IUS91/02343 ', i ~ 3$ -3~-intranuclear levels of IL-l~. Any method known to those in the art whereby this reduction may be effected may be used.
While the invention has been described in connection with specific embodiments thereof, it will be understood that it is S capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains and as may be applied to the essential features hereinbefore set forth and as follows in the scope of the appended claims.

,: . ~ `. .. :
.: . ., . ~ , . , ; -. .... , .. . , ,, - i,.

. .
: ...

Claims (34)

What is claimed is:

1. A method for rejuvenating a senescent cell, which comprises providing to said cell an effective amount of an agent capable of impairing the expression of a gene which encodes interleukin-1.alpha..

2. The method of claim 1, wherein said expression is impaired by an agent capable of impairing the translation of mRNA that encodes IL-1.alpha..

3. The method of either claim 1 or claim 2 wherein said agent is an antisense oligonucleotide characterized in:
(a) having a sequence of nucleotides complementary to an mRNA which encodes interleukin-1.alpha. (IL-l.alpha.);
(b) being capable of hybridizing to said mRNA and thereby impairing translation of said mRNA.

4. The method of claim 3, wherein said oligonucleotide comprises the sequence: 5' TTT GGC CAT CTT GAC TTC 3', or its equivalents.

5. The method of claim 3 wherein said oligonucleotide consists essentially of nucleotides that have the sequence:
5' TTT GGC CAT CTT GAC TTC 3', or its equivalents.

6. The method of claim 1, wherein said expression is impaired by an agent capable of impairing transcription of an mRNA molecule that encodes IL-1.alpha..

7. The method of claim 1, wherein said cell is an endothelial cell or a fibroblast.

8. A method for in vitro tissue cell culture of a non-immortal cell, comprising culturing said cell in the presence of an effective amount of an agent capable of impairing the expression of a gene which encodes interleukin-1.alpha..

9. The method of claim 8 wherein said agent is an antisense oligonucleotide characterized in:
(a) having a sequence of nucleotides complementary to an mRNA which encodes interleukin-1.alpha. (IL-1.alpha.);
(b) being capable of hybridizing to said mRNA and thereby impairing translation of said mRNA.

10. The method of claim 8, wherein said oligonucleotide comprises the sequence: 5' TTT GGC CAT CTT GAC TTC 3', or its equivalents.

11. The method of claim 8 wherein said oligonucleotide consists essentially of nucleotides that have the sequence:
5' TTT GGC CAT CTT GAC TTC 3', or its equivalents.

12. The method of claim 8, wherein said expression is impaired by an agent capable of impairing transcription of an mRNA molecule that encodes IL-1.alpha..

13. The method of claim 8, wherein said cell is an endothelial cell or a fibroblast.

14. A method for rejuvenating a senescent cell, which comprises providing to said cell an effective amount of an agent capable of impairing the translocation of an expression product of an IL-1.alpha. gene.

15. The method of claim 14, comprising culturing said cells in the presence of an effective amount of a mutated IL-1.alpha. polypeptide, wherein said mutation is in the nuclear translocation region of said polypeptide, whereby said mutated IL-1.alpha. polypeptide is able to bind to cellular receptors but is unable to translocate to the nucleus and wherein said effective amount is effective to impair nuclear translocation of endogenous IL-1.alpha..

16. The method of claim 14, wherein said impairment is effected by impairing nuclear translocation of IL-1.alpha. in said cells.

17. A method for treating an age-related disorder in an afflicted individual which comprises administering an effective amount of an agent capable of impairing the expression of a gene which encodes interleukin-1.alpha. to said afflicted individual.

18. The method of claim 17, wherein said agent is administered topically.

19. The method of claim 18, wherein said agent is a skin conditioner.

20. The method of claim 17, wherein said agent is administered systemically.

21. The method of claim 20, wherein said systemic administration is parenteral.

22. The method of claim 21, wherein said parenteral administration is by intramuscular or intravenous injection, rapid infusion, nasopharyngeal absorption, or dermoabsorption.

23. The method of claim 20, wherein said systemic administration is enteral.

24. The method of claim 23, wherein said enteral administration is oral.

25. The method of claim 18 wherein said agent is formulated as a cosmetic, or as a cleanser.

26. The method of claim 17 wherein said agent is an antisense oligonucleotide characterized in:
(a) having a sequence of nucleotides complementary to an mRNA which encodes interleukin-1.alpha. (IL-1.alpha.);
(b) being capable of hybridizing to said mRNA and thereby impairing translation of said mRNA.

27. The method of claim 26, wherein said oligonucleotide comprises the sequence: 5' TTT GGC CAT CTT GAC TTC 3', or its equivalents.

28. The method of claim 26, wherein said oligonucleotide consists essentially of nucleotides that have the sequence:
5' TTT GGC CAT CTT GAC TTC 3', or its equivalents.

29. An antisense oligonucleotide characterized in:
(a) having a sequence of nucleotides complementary to an mRNA which encodes interleukin-1.alpha. (IL-1.alpha.);
(b) being capable of hybridizing to said mRNA and thereby inhibiting translation of said mRNA.

30. The oligonucleotide of claim 29 that rejuvenates a senescent cell, a skin cell, or tissue, which comprises the sequence: 5' TTT GGC CAT CTT GAC TTC 3', or its equivalents.

31. The oligonucleotide of claim 29 that rejuvenates a senescent cell, a skin cell, or tissue, which consists essentially of nucleotides that have the sequence:
5' TTT GGC CAT CTT GAC TTC 3', or its equivalents.

32. A cosmetic composition comprising the oligo-nucleotide of claim 29 in an amount sufficient to rejuvenate a senescent cell, a skin cell, or tissue; and a cosmetically suitable carrier or adjunct.

33. A cosmetic composition comprising the oligo-nucleotide of claim 30 in an amount sufficient to rejuvenate a senescent cell, a skin cell, or tissue; and a cosmetically suitable carrier or adjunct.

34. A cosmetic composition comprising the oligo-nucleotide of claim 31 in an amount sufficient to rejuvenate a senescent cell, a skin cell, or tissue; and a cosmetically suitable carrier or adjunct.