Note: Descriptions are shown in the official language in which they were submitted.
WO 91/1~226 PCr/US9t/0~343
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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
. ,: . . . , ,...... , . :
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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-
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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);
. . . . . ; :
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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
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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,
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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
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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
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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
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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:
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(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).
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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.
;~ .. ..
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~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-
.
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WO ~1/15226 PCI/US91/û2343
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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
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~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
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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.
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W o 91/15226 P~T/US~1/02343
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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
.. . .
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: ,.:,: ; :.,.
: - . -; .~ ,: .
- .. ;
- ~- -,
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
: : ., ;;
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: .. . . ...
.. , ., . . ,., . - . . ~; .
, ~ , . , ;::. :
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 ,
, .
WO 91/15226 PCI/US91/02343
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.
EXAMPLE 2
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).
EXAMPLE 3
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.
EXAMPLE 4
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
`
.
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.
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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
,~
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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.
EXAMPLE 5
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
::. . : :. . ::
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WO ~1/152~6 PCT/US91/02343
2~033~
-31-
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.
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WO 91~15226 PCT/~S9t/02343
208~33~ -32-
EXAMPLE 7
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),
:
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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
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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.
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