Note: Descriptions are shown in the official language in which they were submitted.
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Extension of Cellular Lifespan, Methods and Reagents
Background of the Invention
The linear chromosomes of eukaryotic cells offer the biological advantages of
rapid
recombination, assortment, and genetic diversification. However, linear DNA is
inherently more
unstable than circular forms. To address this difficulty, the eukaryotic
chromosome has evolved
to include a DNA-protein structure, the telomere, which caps chromosome ends
and protects
them from degradation and end-to-end fusion (Blackburn (1984) Annu Rev Biochem
53:163-
194; Blackburn (1991) Nature 350:569-573; Zakian (1995) Science 270:1601-
1607).
The DNA component of telomeres consists of tandem repeats of guanine-rich
sequences
tha re essential for telomere function {Blackburn, supra; Zakian, su ra .
These repeats are
replicated by conventional DNA polymerases and by a specialized enzyme,
telomerase (Greider
{1995) "Telomerase Biochemistry and Regulation" In: Telomeres, E.H. Blackburn
and C.W.
Greider, Eds. Cold Spring Harbor Press, Cold Spring Harbor, NY, pp.35-68),
first identified in
the ciliate Tetrahymena (Greider and Blackburn {1985) Cell 43:405-413). The
telomerase
enzyme is essential for complete replication of telomeric DNA because the
cellular DNA-
dependent DNA polymerases ar unable to replicate the ultimate ends of the
telomeres due to
their requirement for a 5' RNA primer and their unidirectional mode of
synthesis. Removal of
the most terminal RNA primer following priming of DNA synthesis leaves a gap
that cannot be
replicated by these polymerases {Olovnikov (1971) Dokl. Akad. Nauk SSSR
201:1496-1499;
Watson (1972) Nat New Biol 239:197-201). Telomerase surmounts this problem by
do novo
addition of single-stranded telomeric DNA to the ends of chromosomes (Greider
and Blackburn
(1985) supra; Greider and Blackburn (1989) Nature 337:331-337; Yu, et al.
(1990) Nature
344:126-132; Greider (1995) su ra .
The telomerase enzymes that have been charcterized to date are RNA-dependent
DNA
polymerases that synthesize the telomeric DNA repeats by using an RNA template
that exists as
a subunit of the telomerase holoenzyme (Greider (1995), su ra). The genes
specifying the RNA
subunits of telomerases have been cloned from a wide variety of species,
including humans
(Feng, et al. (1995) Science 269:1236-1241; Greider (1995), su ra), and have
been shown in
several instances to be essential for telomerase function in vivo (Yu, et al.
supra; Yu and
Blackburn (1991) Cell 67:823-832; Singer and Gottschling (1994) Science
266:404-409; Cohn
and Blackburn (1995) S fence 269:396-400; McEachern and Blackburn (1995)
Nature 376:403-
409). In addition, three proteins have been identified to date that rae
associated with telomerase
activity. P80 and p95 were purified from the ciliate Tetrahymena (Collins, et
al. (1995) Cell
81:677-686), and the gene encoding a mammalian homolog of p80, TP1/TLP1, has
also been
cloned (Harrington, et al. (1997) Science 275:973-977; Nakayama, et al. (1997)
Cell 88:875-
884). The specific mechanism by which these proteins participate in telomerase
function has not
been defined.
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Most recently, two related proteins, Est2p from the yeast Saccharomyces
cerevisiae, and
p 123 from the ciliate Euplotes aediculatus, were identified as the catalytic
subunits of telomerase
in their respective species (Counter, et al. (1997) PNAS USA 94:9202-9207;
Lingner, et al.
(1997) Science 276:561-567). EST2 was first identifed as a gene required for
telomere
maintenance in yeast (Lendvay, et al. {1996) Genetics 144:1399-1412) and is
essential for
telomerase activity {Counter, et al. supra; Lingner, et al. su ra . Both the
yeast and Euplotes
proteins harbor several sequence motifs that are hallmarks of the catlaytic
regions of reverse
transcriptases; substitution of several such residues in Est2p abolishes
telomerase activity
{Counter, et al. supra; Lingner, et al. su ra . The mammalian homolog of these
telomerase
subunits has not yet been reported.
As might be expected from the known enzymatic properties of telomerase,
perturbing the
function of this enzyme in the ciliate Tetrahymena, through the overexpression
of an inactive
form of the telomerase RNA, or in yeast, through the mutation of genes
encoding either the
catalytic protein or template RNA subunit, leads to progressive telomere
shortening as cells pass
through successive cycles of replication (Yu, et al. supra; Singer and
Gottschling supra;
McEachern and Blackburn s., upra; Lendvay, et al. supra; Counter, et al.
s_u~ra; Lingner, et al.
su ra . This loss of telomeric DNA is ultimately lethal if it is not overcome.
The lethality seems
to be triggered when telomeres have been truncated below a critical threshold
level. Hence, in
the absence of compoensating mechanisms, yeast cell lineages that lack
telomerase activity have
a lifespan dictated by the Tenths of their telomeres.
In humans, telomerase activity is readily detectable in germline cells and in
certain stem
cell compartments. However, enzyme activity is not dtectable in most somatic
cell lineages
(Harley, et al. (1994) Cold Sprine Harbor Svmp. Ouant. Biol. 59:307-315; Kim,
et al. (1994)
Science 266:2011-2015; Broccoli, et al. (1995) PNAS USA 92:9082-9086; Counter,
et al. (1995)
Blood 85:2315-2320; Hiyama, et al. (1995) J Immunol 155:3711-3715). Consistent
with this,
telomeres of most types of human somatic cells shorten with increasing
organisrnic age and with
repeated passaging in culture, similar to the situation seen in protozoan and
yeast cells that have
been deprived experimentally of a functional telomerase enzyme {Harley, et al.
( 1990) Nature
345:458-460; Hastie, et al. (1990) Nature 346:866-868). Eventually, the
proliferation of cultured
human cells will halt at a point termed senescence (Hayflick and Moorhead
(1961) Exp Cell Res
25:585-621; Goldstein (1990) Science 249:1129-1133), apparently before the
telomeres of these
cells have become critically short.
Cultured normal human cells can circumvent senscence and thereby continue to
proliferate when transformed by a variety of agents. In such cultures,
telomere shortening
continues until a subsequent point is reached that is termed crisis, where
telomeres have become
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extremely short (Counter, et al. {1992) EMBO J 11:1921-1929; Counter, et al.
(1994a) J Virol
68:3410-3414; Shay, et al. (1993) Oncog_ene 8:1407-1413; Klingehutz, et al.
(1994)). Crisis,
perhaps best described in SV40-transformed cells, is characterized by
karyotypic instability,
particularly the types of instability observed in chromosomes lacking
functional telomeres, and
by significant levels of cell death (Sack (1981) In Vitro 17:1-19). The crisis
phenotype is
reminiscent of that observed in yeast and Tetrahymena cells in which
telomerase function
hasbeen experimentally perturbed.
The simplest interpretation of these data is that the lifespan of telomerase-
negative
human cells, like that of their yeast and ciliate counterparts, is ultimately
limited by the length of
telomeres. Rare human cells that have acquired the ability to grow
indefinitely emerge from
crisis populations with a frequency of 10-6-10-' (Huschtscha and Holliday
(1983) J Cell Sci
63:77-99; Shay and Wright (1989) Exp Cell Res 184:109-118). This implies that
amutational
event is required to confer the immortal phenotype on these cells. The
immortal cells that escape
crisis are characterized by readily detectable levels of telomerase activity
and by stable telomeres
(Counter, et al. (1992) su ra; Counter, et al. (1994a) supra; Shay, et al.
(1995) Mol Cell Biol
15:425-432; Whitaker, et al. (1995) Oncogene 11:971-976; Gollahon and Shay
(1996) Oncogene
12:715-725; Klingelhutz, et al. (1996) Nature 380:79-82). This suggests that
activation of
telomerase can overcome the limitations imposed by telomere length of the
lifespan of cell
lineages.
Activation of telomerase also appears to be a major step in the progression of
human
cancers. Unlike normal human cells, cancer cells can be established as
permanent cell lines and
thus are presumed to have undergone immportalization during the process of
tumorigenesis.
Moreover, telomerase activity is readily detected in the great majority of
human tumor srnaples
analyzed to date (Counter, et al. ( 1994b) PNAS USA 91:2900-2904; Kim, et al.
1994 su ra ;
Shay and Bacchetti { 1997) Eur J Cancer 33:787-791 ).
Taken together, these various observations have been incorporated into a model
that
proposes that the limitation on prolonged cell replication imposed by telomere
shortening serves
as an important antineoplasdc mechanism used by the body to block the
expansion of pre-
cancerous cell clones. According to such a model, tumor cells transcend the
crisis barrier and
emerge as immortalized cell populations by activating previously unexpressed
telomerase,
enabling them to restore and maintain the integrity of their telomeres
(Counter, et al. (1992)
supra; Counter, et al. ( 1994a) supra; Harley, et al. ( I 994) supra).
A major question provoked by this model is the mechanism used to resurrect
telomerase
expression during tumor progression. Expression of the telomerase-associated
protein
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TP 1 /TLP 1 does not reflect the level of telomerase activity (Harnngton, et
al. supra; Nakayama,
et al. su ra . It is also clear that the levels of the human telomerase RNA
component, hTR,
cannot completely explain the regulation of telomerase activity. Although the
levels of hTR and
its mouse counterpart, mTR, increase with tumor progression (Feng, et al.
(1995) Science
269:1236-1241; Blasco, et al. ( 1996) Nat Genet 12:200-204; Broccoli, et al. (
1996) Mol Cell
Biol 16:3765-3772; Soder, et al. (1997) Onco~~ene 14:1013-1021), the amounts
of these
transcripts do not always correlate with enzymatic activity. Indeed, hTR or
mtr transcript levels
can be significantly higher in telomerase-negative cells and tissues than in
telomerase-positive
cancer cells (Avilion, et al. (1996) Cancer Res 56:645-650; Bestilny, et al.
(1996) Cancer Res
56:3796-3802; Blasco, et al. supra). Similarly, even though telomerase levels
increase 100- to
2000-fold during the immortalization of huma n cells, the level of hTR message
increases, at
most, two-fold (Avilion, et al. su ra . Therefore, depression of the hTR and
TPI subunits cannot
easily be invoked to explain the appearance of telomerase activity in the
great majority of human
tumor samples. Thus far, the rate-limiting step in telomerase activation has
remained elusive.
Summary of the Invention
One aspect of the present invention relates to methods and reagents for
extending the life-
span, e.g., the number of mitotic divisions, of a cell. In general, the
subject method relies on the
activation of a telomerase activity, such as by ectopic expression of the
telomerase catalytic
subunit EST2, or a bioactive fragment thereof, or the ectopic expression of
myc, or a bioactive
fragment thereof, or by contacting the cell with an agent (such as a small
organic molecule)
which activates expression of EST2 or myc or relieves an inhibitory signal
(antagonism) of myc.
By "ectopic expression", it is meant that a cell is caused to express, e.g.,
by expression of a
heterologous or endogenous gene or by transcellular uptake of a protein or
inhibition of
degradation of the EST2 or myc protein, a higher than normal level of EST2 or
myc than the cell
normally would for the particular starting phenotype. The subject method is
useful both in vivo,
ex vivo and in situ. Exemplary uses include, merely to illustrate, the
extension of stem cell or
progenitor cell cultures or implants, the extension of skin or other
epithelial cell cultures or
grafts, the expansion of mesenchymal cell cultures or grafts, and the
expansion of chondrocyte or
osteocyte cultures or grafts. Exemplary stem and progenitor cells which can be
extended by the
subject method include neuronal, hematopoietic, epithelial, pancreatic,
hepatic, chondrocytic and
osteocytic stem and progenitor cells. The subject method can be used for wound
healing and
other tissue repair, as well as cosemetic uses. It can be applied for
prolonging the Iifespan of a
culture of normal cells or tissue being used to secrete therapeutic or other
commercially
significant proteins and products.
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The practice of the present invention will employ, unless otherwise indicated,
conventional techniques of cell biology, cell culture, molecular biology,
transgenic biology,
microbiology, recombinant DNA, and immunology, which are within the skill of
the art. Such
techniques are described in the literature. See, for example, Molecular
Cloning: A
Laborator~r Manual, 2nd Ed., ed. by Sambrook, Fritsch and Maniatis (Cold
Spring Harbor
Laboratory Press: 1989); DNA Cloning, Volumes I and II (D. N. Glover ed.,
1985);
Olig_onucleotide Synthesis (M. J. Gait ed., 1984); Mullis et al. U.S. Patent
No: 4,683,195;
Nucleic Acid Hybridization (B. D. Hames & S. J. Higgins eds. 1984);
Transcription And
Translation (B. D. Hames & S. J. Higgins eds. 1984); Culture Of Animal Cells
(R. I. Freshney,
Alan R. Liss, Inc., 1987); Immobilized Cells And Enzymes (IRL Press, 1986); B.
Perbal, A
Practical Guide To Molecular Cloning (1984}; the treatise, Methods In
Enzymology (Academic
Press, Inc., N.Y.); Gene Transfer Vectors For Mammalian Cells (J. H. Miller
and M. P. Calos
eds., 1987, Cold Spring Harbor Laboratory); Methods In Enzvmology, Vols. 154
and 155 (Wu et
al. eds.), Immunochemical Methods In Cell And Molecular Biologv_ (Mayer and
Walker, eds.,
Academic Press, London, 1987); Handbook Of Experimental Immunolo~y, Volumes I-
IV (D. M.
Weir and C. C. Blackwell, eds., 1986); Marupulatin~e Mouse Embryo, (Cold
Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y., 1986).
Brief Description of the Drawings
Figure 1. HEST2 encodes a human homolog of Est2p and p123. Alignment of the
predicted amino acid sequence of HEST2 with the yeast Est2p and Euplotes p I23
homologs.
Amino residues within shaded and closed blocks are indentical between at least
two proteins.
Indentical amino acids within the RT motifs are in closed boxes, an example of
a telomerase-
specific motif in an outlined shaded box, and all identical amino acids in
shaded boxes. RT
motifs are extended in some cases to include other adjacent invariant or
conserved amino acids.
The sequence of the expressed tag AA281296 is underlined.
Figure 2. Alignment of RT motifs 1-6 of telomerase subunits HEST2, p123 and
Est2p
with S Cerevisiae group II intron-encoded RTs a2-Sc and al-Sc. The consensus
sequence of
each RT motif is shon (h=hydrophobic, p=small polar, c=charged). Amino acids
that are
invariant among the telomerases and the RT consensus are in shaded boxes. Open
boxes identify
highly conserved residues unique to either telomerases or to nontelomerase
RTs. Astericks
denote amino acids essential for polymerase catalytic function.
Figure 3. Myc activation of telomerase in HMEC cells. Primary HMEC cells at
passage
12 were infected with empty vector (lanes 1-5), E6 (lanes 6-10), c-myc (lanes
11-15) or cdc25A
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(lanes 16-20) viruses. Two breast cancer cell lines BT549 (lanes 21-25) and
T47D (lanes 26-30)
were included for comparison. The cells were lysed and TRAP assays were
performed using
extract corresponding to 10,000 cells (lanes 2, 6, 7, 11,12, 17, 21, 22, 26
and 27), 1,000 cells
(lanes 3, 8, 13, 18, 23 and 28), 100 cells (lanes 4, 9, 14, 19, 24 and 29) or
10 cells (lanes 5, 10,
15, 20, 25 and 30). Telomerase activity was shown to be sensitive to RNase by
the addition of
RNase A prior to the telomerase assay ("=', without RNase A; "+", with RNase
A). To rule out
the presence of inhibitors in apparently negative lysates, lanes labelled
"Mix" (lanes 1 and 16)
are assays containing lysate from 10,000 of the indicated cells mixed with
Iysate from 10,000
positive {c-myc-expressing) cells .
Figure 4. Myc activaton of telomerase in IMR90 fibroblasts. IMR90 cells at
passage 14
were infected with empty vector (lanes 1-5), c-myc (lanes 6-10) and E6 (lanes
11-15) viruses.
HT1080 cells (lanes 15-20) were included for comparison. TRAP assays contained
10,000 cells
(lanes 2, 6, 7, 12, 16 and 17), 1,000 cells (lanes 3, 8, 13 and 18), 100 cells
(lanes 4, 9, 14 and 19)
or 10 cells (lanes 5, 10, 15 and 20). Telomerase activity was shown to be
sensitive to RNase by
the addition of RNase A prior to extention reaction ("-", without RNase A;
"+", with RNase A).
"Mix" lanes (1 and 11) are assays containing lysate from 10,000 of the
indicated cells mixed
with lysate from 10,000 positive (c-myc-expressing) cells.
Figure 5. E6 increases c-myc protein level in HMEC. A. Levels of myc protein
were
determined by western blotting with a polyclonal myc antibody. Cell lysates
from E6 (lane 1 )
and vector (lane 2) infected IMR90 cells and lysates from c-myc (lane 3), E6
(lane 4) and vector
(lane 5) infected HMEC cells were analyzed. Tumor cell lines, HT1080 (lane 6),
HBL100
(Lane 7), BT549 (lane 8) and T47D (lane 9), were included for comparison. The
expression of
TFIIB was used to normalize loading. B. Total RNA prepared in parallel with
the protein
extracts used in A. was used in northern blots to determine myc mRNA levels.
Equal quantities
of total RNA, as indicated, were probed with a human c-myc cDNA.
Figure 6. Extention of telomere length and cellular lifespan by telomerase
activation. A.
Total RNA was prepared from normal HMEC and from HMEC that had been infected
with a
myc retrovirus. hEST2 transcript was visualized in equal quantities of RNA (10
fig) using a
probe derived from the hEST2 cDNA. B. HMEC and IMR90 cells were infected with
either
empty vector (lanes 1-5 and 11-15) or hEST2 (lanes 6-10 and 16-20) viruses.
TRAP assays were
performed using Iysate equivalent to 10,000 cells (lanes 2, 6, 7, 12, 16
andl7), 1,000 cells (lanes
3, 8, 13 and 18), 100 cells (lanes 4, 9, 14 and 19) or 10 cells (lanes S, 10,
I S and 20).
Telomerase activity was shown to be sensitive to RNase by the addition of
RNase A prior to
assay (' =", without RNase A; "+", with RNase A). To rule out the presence of
inhibitors in
apparently negative Iysates, lanes labelled "Mix" (lanes 1 and 16) are assays
containing lysate
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WO 99135243 PCTIUS99/00682
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from 10,000 of the indicated cells mixed with lysate from 10,000 positive
(HT1080) cells. C.
Genomic DNA from early passage HMEC (passage 12, lane 1 ), late passage HMEC
(passage 22,
lane 2), HMEC/hEST2 (cells infected at passage 12 with hEST2.and subsequently
cultured for
additional passages, lane 3) and HMEC/vector (cells infected at passage 12
with empty vector
5 and subsequently cultured for 10 additional passages, lane 4) were digested
with Rsa I and Hinf
I. Fragments were separated on a 0.8% agarose gel, and telomeric restriction
fragments were
visualized using a 32P-Tabled human telomeric sequence (TTAGGG)3 as a probe.
D. HMEC
cells were transduced at passage 12 with either empty vector, c-Myc or hEST2
retroviruses (as
indicated). These cells were continuously subcultured at a density of 4-Sx105
cells per 100 cmz
10 once per week. After 12 passages following transduction, vector-infected
cells could no longer
be subcultured at this frequency and adopted a classic senescent phenotype. In
contrast, cells
expressing myc and hEST2 continue to proliferate and showed a virtual absence
of sensescent
cells in the population.
Figure 6. Illustrates a MarxII vector including the coding sequence for hEST2.
The
long terminal repeats (LTRs) include, though not shown, recombinase sites such
that, upon
treatment of a cell in which the MarxII-hEST2 vector is integrated, the
proviral vector including
the hEST2 coding sequence is excised.
Detailed Description of the Invention
Normal mammalian diploid cells placed in culture have a finite proliferative
life-span and
enter a nondividing state termed senescence, which is characterized by altered
gene expression
(Hayflick et al. (1961) Exp. Cell Res. 25:585; Wright et al. (1989) Mol. Cell.
Biol. 9:3088;
Goldstein, ( 1990) Science 249:112; Campisi, ( 1996) Cell 84:497; Campisi (
1997) Eur. J. Cancer
33:703; Faragher et al. (1997) Drug Discoverer Today 2:64). Replicative
senescence is dependent
upon cumulative cell divisions and not chronologic or metabolic time,
indicating that
proliferation is limited by a "mitotic clock" (Dell'Orco et al. (1973) Exp.
Cell Res. 77:356;
Hadey et al. ( 1978) J. Cell. Physiol. 97:509). The reduction in proliferative
capacity of cells from
old donors and patients with premature aging syndromes (Martin et al. ( 1970)
Lab. Invest 23:86;
Schneider et al. (1976) PNAS 73:3584; Schneider et al. (1972) Proc. Soc. Exp.
Biol. Med.
141:1092; Elmore et al. (1976) Cell Phvsiol. 87:229), and the accumulation in
vivo of senescent
cells with altered patterns of gene expression (Stanulis-Praeger et al. (1987)
Mech. Ageing Dev.
38:1; and Dimri et al. (1995) PNAS 92:9363), implicate cellular senescence in
aging and age-
related pathologies ((Hayflick et al. (1961) Exp. Cell Res. 25:585; Wright et
al. (1989) Mol.
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Cell. Biol. 9:3088; Goldstein, (1990) Science 249:112; Campisi, (1996) Cell
84:497; Campisi
(1997) Eur. J. Cancer 33:703; Faragher et al. (1997) DritQ Discovery Today
2:64).
Telomere loss is thought to control entry into senescence. Human telomeres
consist of
repeats of the sequence TTAGGG/CCCTAA at chromosome ends; these repeats are
synthesized
S by the ribonucleoprotein enzyme telomerase. Telomerase is active in germline
cells and, in
humans, telomeres in these cells are maintained at about 1 S kilobase pairs
(kbp). In contrast,
telomerase is not expressed in most human somatic tissues, and telomere length
is significantly
shorter. The telomere hypothesis of cellular aging proposes that cells become
senescent when
progressive telomere shortening during each division produces a threshold
telomere length.
The human telomerase reverse transcriptase subunit (hTRT) has been cloned. See
Nakamura et al., (1997) Science 277:955; Meyerson et al., (1997) Cell 90:78;
and Kilian et al.,
(1997) Hum. Mol. Genet. 6:2011. It has recently been demonstrated that
telomerase activity can
be reconstituted by transient expression of hTRT in normal human diploid
cells, which express
the template RNA component of telomerase (hTR) but do not express hTRT. See,
for example,
Wang et al. (1998) Genes Dev 12:1769; and Weinrich et al., (1997) Nature
Genet. 17:498. This
provided the opportunity to manipulate telomere length and test the hypothesis
that telomere
shortening causes cellular senescence.
The reported results indicate that telomere loss in the absence of telomerase
is the
intrinsic timing mechanism that controls the number of cell divisions prior to
senescence. The
long-term effects of exogenous telomerase expression on telomere maintenance
and the life-span
of these cells remain to be determined in studies of longer duration.
Telomere homeostasis is likely to result from a balance of lengthening and
shortening
activities. Very low levels of telomerase activity are apparently insufficient
to prevent telomere
shortening. This is consistent with the observation that stem cells have low
but detectable
telomerase activity, yet continue to exhibit shortening of their telomeres
throughout life. Thus, a
threshold level of telomerase activity is likely required for life-span
extension.
Cellular senescence is believed to contribute to multiple conditions in the
elderly that
could in principle be remedied by cell life-span extension in situ. Examples
include atrophy of
the skin through loss of extracellular matrix homeostasis in dermal
fibroblasts; age-related
macular degeneration caused by accumulation of lipofuscin and downregulation
of a neuronal
survival factor in RPE cells; and atherosclerosis caused by loss of
proliferadve capacity and
overexpression of hypertensive and thrombotic factors in endothelial cells.
Extended Iife-span cells also have potential applications ex vivo. Cloned
normal diploid
cells could replace established tumor cell lines in studies of biochemical and
physiological
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aspects of growth and differentiation; long-lived normal human cells could be
used for the
production of normal or engineered biotechnology products; and expanded
populations of
normal or genetically engineered rejuvenated cells could be used for
autologous or allogeneic
cell and gene therapy. Thus the ability to extend cellular life-span, while
maintaining the diploid
S status, growth characteristics, and gene expression pattern typical of young
normal cells, has
important implications for biological research, the pharmaceutical industry,
and medicine.
(i) Overview
One aspect of the present invention relates to methods and reagents for
extending the life-
span, e.g., the number of mitotic divisions, of a cell. In preferred
embodiments, the cells are
isolated in culture for at least a portion of the treatment.
In general, the invention provides a method for increasing the proliferative
capacity of
metazoan cells, preferably mammalian cells, and more preferably normal
mammalian cells, by
contacting the cell with an agent that activates telomerase activity in cell.
In certain
embodiments, the subject method relies on the ectopic expression of the
telomerase catalytic
subunit EST2, or a bioactive fragment thereof. By "ectopic expression", it is
meant that a cell is
caused to express, e.g., by expression of a heterologous or endogenous gene or
by transcellular
uptake of a protein, a higher than normal level of EST2 than the cell normally
would for the
particular starting phenotype.
In other embodiments, the subject method can be carned out by the ectopic
expression of
an activator of telomerase activity (collectively herein "telomerase
activator") such as a myc
gene product of a papillomavirus E6 protein. In preferred embodiments wherein
the ectopic
expression of the telomerase or telomerase activator involves a recombinant
gene, expression of
the gene in the host cell is inducible (or otherwise conditionally regulated)
and/or the genetic
construct including the gene can be readily removed from thehost cell.
In still other embodiments, the subject method can be carried out by
contacting the cell
with an agent that inhibits degradation (ubiquitin-dependent or independent)
of the EST2 protein
or telomerase activator in order to increase the cellular half life of the
protein. For example, the
method can utilize an agent which inhibits ubiquitination of to increase the
cellular half life of
the protein. For example, the method can utilize an agent which inhibits
ubiquitination of myc
and thereby increases the cellular concentration of myc. In preferred
embodiments, such agents
are small, organic molecules, e.g., having molecular weights of less than 5000
amu (more
preferably less than 1000 amu), and which are membrane permeant.
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In still other embodiments, cellular prolifeartive capacity can be incrased by
contacting
the cell with an agent, e.g. a small molecule, which relieves or otherwise
inhibits a signal which
antagonizes myc-induced activation of telomerase activity. For instance,
agents can be used
which disrupt protein-protein interactions involved in inhibition of myc
activity by, e.g., mad
max heterodimers.
The subject method is useful both in vivo, ex vivo and in situ. Exemplary uses
include,
merely to illustrate, the extension of stem cell or progenitor cell cultures
or implants, the
extension of skin or other epithelial cell cultures or grafts, the expansion
of mesenchymal cell
cultures or grafts, and the expansion of chondrocyte or osteocyte cultures or
grafts. Exemplary
stem and progenitor cells which can be extended by the subject method include
neuronal,
hematopoietic, pancreatic, and hepatic stem and progenitor cells.
An important feature of certain preferred embodiments of the subject method is
the
reversibility of activation of telomerase activity, rather than constitutive
activation. For
example, where a vector is used to ectopically express an EST2 protein or
telomerase activator,
the vector can be configured so as to be excisable from the cell. Thus, for ex
vivo therapies, cells
can be treated ex vivo with a vector encoding EST2 of a telomerase activator,
and prior to
implantation, the vector can be excised to inhibit further recombinant
expression of the construct
in vivo. In preferred embodiments, the vector can be excised so as to have
little to no
heterologous nucleic acid sequences in the host cell.
Another aspect of the present invention relates to in vitro preparations of
cells which
have been treated by the subject method. Such cell compositions can be used,
e.g., to generate a
medicament for transplantation to an animal.
(ii) Definitions
For convenience, certain terms used herein as defined below.
As used herein, the term "fusion protein" is art recognized and refer to a
chimeric protein
which is at least initially expressed as single chain protein comprised of
amino acid sequences
derived from two or more different proteins, e.g., the fusion protein is a
gene product of a fusion
gene.
The art term "fusion gene" refers to a nucleic acid in which two or more genes
are fused
resulting in a single open reading frame for coding two or more proteins that
as a result of this
fusion are joined by one or more peptide bonds.
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As used herein, the term "nucleic acid" refers to polynucleotides such as
deoxyribonucleic acid (DNA), and, where appropriate, ribonucleic acid (RNA).
The term should
also be understood to include, as equivalents, analogs of either RNA or DNA
made from
nucleotide analogs, and, as applicable to the embodiment being described,
single-stranded (such
S as sense or antisense) and double-stranded polynucleotides.
As used herein, the term "gene" or "recombinant gene" refers to a nucleic acid
comprising an open reading frame encoding a polypeptide, including both exonic
and
(optionally) intronic sequences. A gene, according to the present invention,
can be in the form
of a DNA construct which is transcribed or an RNA construct which is directly
translatable. An
exemplary recombinant gene encoding a subject EST2 protein is represented by
SEQ. ID NO: 1.
As used herein, the term "transfection" means the introduction of a
heterologous nucleic
acid, e.g., an expression vector, into a recipient cell by nucleic acid-
mediated gene transfer.
"Transformation", as used herein with respect to transfected nucleic acid,
refers to a process in
which a cell's genotype is changed as a result of the cellular uptake of
exogenous DNA or RNA,
and, for example, the transformed cell expresses a recombinant form of an EST2
or myc
polypeptide.
"Expression vector" refers to a replicable nucelic acid construct used to
express a gene
which encodes the desired protein and which includes a transcriptional unit
comprising an
assembly of ( 1 ) genetic elements) having a regulatory role in gene
expression, for example,
promoters, operators, or enhancers, operatively linked to (2) a sequence
encoding a desired
protein (e.g. an EST2 or myc protein), and (3) as necessary, appropriate
transcription and
translation initiation and termination sequences. The choice of promoter and
other regulatory
elements generally varies according to the intended host cell. In general,
expression vectors of
utility in recombinant techniques are often in the form of "plasmids" which
refer to circular
double stranded DNA loops which, in their vector form are not bound to the
chromosome. In the
present specification, "plasmid" and "vector" are used interchangeably as the
plasmid is the most
commonly used form of vector. However, the invention is intended to include
such other forms
of expression vectors which serve equivalent functions and which become known
in the art
subsequently hereto.
In the expression vectors, regulatory elements controlling transcription or
translation can
be generally derived from mammalian, microbial, viral or insect genes The
ability to replicate in
a host, usually conferred by an origin of replication, and a selection gene to
facilitate recognition
of transformants may additionally be incorporated. Vectors derived from
viruses, such as
retroviruses, adenoviruses, and the like, may be employed.
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"Transcriptional regulatory sequence" is a generic term used throughout the
specification
to refer to nucleic acid sequences, such as initiation signals, enhancers, and
promoters and the
like which induce or control transcription of protein coding sequences with
which they are
operably linked. In preferred embodiments, transcription of the EST2 or other
telomerase
activator gene is under the control of a promoter sequence (or other
transcriptional regulatory
sequence) which controls the expression of the recombinant gene in a cell-type
in which
expression is intended. It will also be understood that the recombinant gene
can be under the
control of transcriptional regulatory sequences which are the same or which
are different from
those sequences which control transcription of one of the natwally-occurring
forms of a protein.
As used herein, the term "tissue-specific promoter" means a DNA sequence that
serves as
a promoter, i.e., regulates expression of a selected DNA sequence operably
linked to the
promoter, and which effects expression of the selected DNA sequence in
specific cells of a
tissue, such as cells of a urogenital origin, e.g. renal cells, or cells of a
neural origin, e.g.
neuronal cells. The term also covers so-called "leaky" promoters, which
regulate expression of
a selected DNA primarily in one tissue, but cause expression in other tissues
as well.
"Operably linked" when describing the relationship between two DNA regions
simply
means that they are functionally related to each other. For example, a
promoter or other
transcriptional regulatory sequence is operably linked to a coding sequence if
it controls the
transcription of the coding sequence.
The terms "EST2 proteins" and "EST2 polypeptides" refer to catalytic subunits
of
telomerase, preferably of a mammalian telomerase, and even more preferably of
a human
telomerase. Exemplary EST2 proteins are encoded by the nucleic acid of SEQ ID
NO:1, or by a
nucleic acid which hybridizes thereto. Thus, the EST2 proteins useful in the
subject method can
be at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, or even at least 95% identical
to the human
EST2 of SEQ ID N0:2, or a fragment thereof which reconsitutes a telomerase
elongation
enzyme in a host cell (such as a human cell). A variety of different
techniques are available in
the art for assessing the activity of a particular EST2 polypeptide, e.g.,
which may vary in
sequence and/or length relative to SEQ ID NO: 1.
The term "telomerase-activating therapeutic agent" refers to any agent which
can be used
to activation of telomerase activity in a cell, e.g., a mammalian cell. Far
example, it includes
expression vectors encoding EST2, myc, E6 or the like, formulations of such
polypeptides, small
molecule activators of expression of an endogenous telomerase activator gene,
inhibitors of
degradation of a telomerase activator, to name but a few.
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The term "EST2 therapeutic agent" refers to any telomerase-activating
therapeutic agent
which can be used to cause ectopic expression of an EST2 polypeptide in a
cell. For example, it
includes EST2 expression vectors, formulations of EST2 polypeptides, and small
molecule
activators of expression of an endogenous EST2 gene, to name but a few.
The term "derepresses myc" refers to the ability of an agent to overcome an
antagonism
of myc, e.g., it may prevent mad/max inactivation of myc and thereby activates
myc.
The term "progenitor cell" refers to an undifferentiated cell which is capable
of
proliferation and giving rise to more progenitor cells having the ability to
generate a large
number of mother cells that can in turn give rise to differentiated, or
differentiable daughter cells.
As used herein, the term "progenitor cell" is also intended to encompass a
cell which is
sometimes referred to in the art as a "stem cell". In a preferred embodiment,
the term "progenitor
cell" refers to a generalized mother cell whose descendants (progeny)
specialize, often in
different directions, by differentiation, e.g., by acquiring completely
individual characters, as
occurs in progressive diversification of embryonic cells and tissues.
As used herein the term "substantially pure", with respect to progenitor
cells, refers to a
population of progenitor cells that is at least about 75%, preferably at least
about 85%, more
preferably at least about 90%, and most preferably at least about 95% pure,
with respect to
progenitor cells making up a total cell population. Recast, the term
"substantially pure" refers to
a population of progenitor cell of the present invention that contain fewer
than about 20%, more
preferably fewer than about 10%, most preferably fewer than about 5%, of
lineage committed
cells in the original unamplified and isolated population prior to subsequent
culturing and
amplification.
The term "cosmetic preparation" refers to a form of a pharmaceutical
preparation which
is formulated for topical administration.
As used herein, the term "cellular composition" refers to a preparation of
cells, which
preparation may include, in addition to the cells, non-cellular components
such as cell culture
media, e.g. proteins, amino acids, nucleic acids, nucleotides, co-enzyme, anti-
oxidants, metals
and the like. Furthermore, the cellular composition can have components which
do not affect the
growth or viability of the cellular component, but which are used to provide
the cells in a
particular format, e.g., as polymeric matrix for encapsulation or a
pharmaceutical preparation.
As used herein the term "animal" refers to mammals, preferably mammals such as
humans. Likewise, a "patient" or "subject" to be treated by the method of the
invention can
mean either a human or non-human animal.
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(iii) Illustrative Embodiments
(A7 Exem~larv Telomease Activators
In one embodiment, the subject involves the administration of an expression
vector
S encoding an EST2 polypeptide or other telomerase activator polypeptide.
The isolation of a gene the represents the human homolog, EST2, of the yeast
and ciliate
genes encoding the telomerase catalytic subunits has recently been reported.
See Meyerson, et
al. (1997) Cell 90:785; and Nakamwa et al. (1997) Science 277:955.
The predicted 127 kDa protein shares extensive sequence similarity with the
entire
sequences of the Euplotes and yeast telomerase subunits (Figwe 1) and extends
beyond the
amino and carboxyl termini of these proteins. A BLAST search reveals that the
probabilities of
these smilarieites occurring by chance are 1.3 x 10''g and 3 x 10-'3,
respectively. By way of
comparison, the probability of similarity between the yeast and Euplotes
telomerases in a protein
BLAST search is 6.9 x 10'x. We have named the hiuman gene hEST2 (human EST2
homology
to reflect its clear relationship with the yeast gene, the first of these
genes to be described. EST2
was named because of the phenotype of Ever Shortening Telomerase catalytic
subunit (Counter
et al. (1997} supra; Lingner et al. (1997)).
Like the yeast and ciliate telomerase proteins, hEST2 is a member of the
reverse
transcriptase (RT) family of enzymes (Figwes l and 2). Seven conserved
sequence motifs,
which define the polymerase domains of these enzymes, are shared among the
otherwise highly
divergent RT family (Poch et al. (1989) EMBO J 8:3867-3874; Xiong and Eickbush
(1990)
EMBO J 9:3353-3362). P123 and Est2p share six of these motifs with, most
prominently, the
a2-Sc enzyme, an RT that is encoded within the second intron of the yeast COX1
gene (Kennell
et al. (1993) Cell 133-146). These six motifs, includiung the invariant
aspartic acid residues
known to be required for telomerase enzymatic function (Counter et al. (1997)
supra; Lingner et
al. supra), are found at the appropriate positions of the predicted sequence
of hEST2 (Figwes 1
and 2). Thus, the proposed human telomerase catalytic subunit, like its yeast
and ciliate
counterparts, belongs to the RT superfamily of enzymes.
Exemplary human EST coding sequence and protein for use in the subject method
is
provided at GenBank accession AF018167, AF043739 and AF015950. Exemplary EST
constructs are also decribed in PCT application W098/I4593 and Ulaner et al.
(1998) Cancer
Res 58:4168-72, Counter et L. (1998) Onco~ene 161217-22, and Vaziri et al.
(1998) Curr Biol 8:
279-82. In a preferred embodiment, the EST construct includes an EST coding
sequence which
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hybridizes under stringent conditions to SEQ ID No: 1, or a coding sequence
set forth in
GenBank accession AF018167, AF043739 or AF015950. The EST coding sequence can
encode
an EST protein, or fragment thereof which retains a telomerase activity, which
is is at least, for
example, 60, 70 , 80, 85, 90 , 95 or 98 percent identical with a sequence of
SEQ ID No. 2 or
GenBank accession AF018167, AF043739 and AF015950, or identical with one of
the
enumerated sequences.
in other illustrative embodiments, telomerase activation can be caused by
ectopic
expression of a myc protein, e.g., c-myc. An exemplary human myc coding
sequence is provided
at the SWISS-PROT locus MYC_HUMAN, accession P01106. In a preferred
embodiment, the
myc construct includes an myc coding sequence which hybridizes under stringent
conditions to a
coding sequence set forth in SWISS-PROT locus MYC HUMAN, accession P01106. The
myc
coding sequence can encode a myc protein, or fragment thereof which retains
the ability to
activate a telomerase activity, which is is at least, for example, 60, 70 ,
80, 85, 90 , 95 or 98
percent identical with the protein sequence set forth in SWISS-PROT locus MYC
HUMAN,
accession P01106, or identical thereto.
In yet other illustrative embodiments, telomerase activation is accomplished
by
expression of a papillomavirus E6 protein, preferably an E6 protein from a
human
papillomavirus (HPV), and more preferably an E6 protein from a high risk HPV
(e.g., HPV-16
or -18). It may desirable to use an E6 protein which has been mutated so as to
be incapable of
effecting p53 degradation. In a preferred embodiment, the E6 construct
includes an Eb coding
sequence which hybridizes under stringent conditions to a coding sequence set
forth in EMBL:
locus A06324, accession A06324. The E6 coding sequence can encode an E6
protein, or
fragment thereof which retains the ability to activate a telomerase activity,
which is is at least,
for example, 60, 70 , 80, 85, 90 , 95 or 98 percent identical with the protein
sequence set forth in
EMBL: locus A06324, accession A06324, or identical thereto
In accordance with the subject method, expression constructs of the subject
polypeptides
may be administered in any biologically effective carrier, e.g. any
formulation or composition
capable of effectively transfecting cells in vitro or in vivo with a
recombinant gene. Approaches
include insertion of the subject EST2 or telomerase activator gene in viral
vectors including
recombinant retroviruses, adenovirus, adeno-associated virus, and herpes
simplex virus-1, or
recombinant bacterial or eukaryotic plasmids. Viral vectors can be used to
transfect cells
directly; plasmid DNA can be delivered with the help of, for example, cationic
liposomes
(lipofectin) or derivatized (e.g. antibody conjugated), polylysine conjugates,
gramacidin S,
artificial viral envelopes or other such intracellular Garners, as well as
direct injection of the gene
construct or CaP04 precipitation carned out in vivo. It will be appreciated
that because
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transduction of appropriate target cells represents the critical first step in
gene therapy, choice of
the particular gene delivery system will depend on such factors as the
phenotype of the intended
target and the route of administration, e.g. locally or systemically.
A preferred approach for introduction of nucleic acid encoding a telomerase
activator
into a cell is by use of a viral vector containing nucleic acid, e.g. a cDNA,
encoding the gene
product. Infection of cells with a viral vector has the advantage that a large
proportion of the
targeted cells can receive the nucleic acid. Additionally, molecules encoded
within the viral
vector, e.g., by a cDNA contained in the viral vector, are expressed
efficiently in cells which
have taken up viral vector nucleic acid.
Retrovirus vectors and adeno-associated virus vectors are generally understood
to be the
recombinant gene delivery system of choice for the transfer of exogenous genes
in vivo,
particularly into humans. These vectors provide efficient delivery of genes
into cells, and the
transferred nucleic acids are stably integrated into the chromosomal DNA of
the host. A major
prerequisite for the use of retroviruses is to ensure the safety of their use,
particularly with regard
to the possibility of the spread of wild-type virus in the cell population.
The development of
specialized cell lines (termed "packaging cells") which produce only
replication-defective
retroviruses has increased the utility of retroviruses for gene therapy, and
defective retroviruses
are well characterized for use in gene transfer for gene therapy purposes (for
a review see Miller,
A.D. (1990) Blood 76:271). Thus, recombinant retrovirus can be constructed in
which part of
the retroviral coding sequence (gag, pol, env) has been replaced by nucleic
acid encoding, e.g.,
an EST2 or myc polypeptide, rendering the retrovirus replication defective.
The replication
defective retrovirus is then packaged into virions which can be used to infect
a target cell
through the use of a helper virus by standard techniques. Protocols for
producing recombinant
retroviruses and for infecting cells in vitro or in vivo with such viruses can
be found in Current
Protocols in Molecular Biolosy, Ausubel, F.M. et al. (eds.) Greene Publishing
Associates,
( 1989), Sections 9.10-9.14 and other standard laboratory manuals. Examples of
suitable
retroviruses include pL3, pZIP, pWE and pEM which are well known to those
skilled in the art.
Examples of suitable packaging virus lines for preparing both ecotropic and
amphotropic
retroviral systems include yrCrip, yrCre, yr2 and yrAm. Retroviruses have been
used to introduce
a variety of genes into many different cell types, including neural cells,
epithelial cells,
endothelial cells, lymphocytes, myoblasts, hepatocytes, bone marrow cells, in
vitro and/or in
vivo (see for example Eglitis, et al. (1985) Science 230:1395-1398; Danos and
Mulligan (1988)
Proc. Natl. Acad. Sci. USA 85:6460-6464; Wilson et al. (1988) Proc. Natl.
Acad. Sci. USA
85:3014-3018; Armentano et al. (1990) Proc. Natl. Acad. Sci. USA 87:6141-6145;
Huber et al.
(1991) PrcZc. Natl. Acad. Sci. USA 88:8039-8043; Ferry et al. (1991) Proc.
Natl. Acad. Sci. USA
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88:8377-8381; Chowdhury et al. (1991) Science 254:1802-1805; van Beusechem et
al. (1992)
Proc. Natl. Acad. Sci. USA 89:7640-7644; Kay et al. (1992) Human Gene Theranv
3:641-647;
Dai et al. (1992) Proc. Natl. Acad. Sci. USA 89:10892-10895; Hwu et al. (1993)
J. Immunol.
150:4104-4115; U.S. Patent No. 4,868,116; U.S. Patent No. 4,980,286; PCT
Application WO
89/07136; PCT Application WO 89/02468; PCT Application WO 89105345; and PCT
Application WO 92/07573).
In choosing retroviral vectors as a gene delivery system for the subject
telomerase
activator proteins, it is important to note that a prerequisite for the
successful infection of target
cells by most retroviruses, and therefore of stable introduction of the
recombinant gene, is that
the target cells must be dividing. In general, this requirement will not be a
hindrance to use of
retroviral vectors to deliver the subject gene constructs.
Furthermore, it has been shown that it is possible to limit the infection
spectrum of
retroviruses and consequently of retroviral-based vectors, by modifying the
viral packaging
proteins on the surface of the viral particle (see, for example PCT
publications W093/25234,
W094/06920, and W094/11524). For instance, strategies for the modification of
the infection
spectrum of retroviral vectors include: coupling antibodies specific for cell
surface antigens to
the viral env protein (Roux et al. (1989) PNAS 86:9079-9083; Julan et al.
(1992) J. Gen Virol
73:3251-3255; and Goud et al. (1983) Viroloev 163:251-254); or coupling cell
surface ligands
to the viral env proteins (Veda et al. (1991) J Biol Chem 266:14143-14146).
Coupling can be in
the form of the chemical cross-linking with a protein or other variety (e.g.
lactose to convert the
env protein to an asialoglycoprotein), as well as by generating fusion
proteins (e.g. single-chain
antibody/env fusion proteins). This technique, while useful to limit or
otherwise direct the
infection to certain tissue types, and can also be used to convert an
ecotropic vector in to an
amphotropic vector.
Moreover, use of retroviral gene delivery can be further enhanced by the use
of tissue- or
cell-specific transcriptional regulatory sequences which control expression of
the recombinant
gene of the retroviral vector.
Another viral gene delivery system useful in the present invention utilitizes
adenovirus-
derived vectors. The genome of an adenovirus can be manipulated such that it
encodes a gene
product of interest, but is inactivate in terms of its ability to replicate in
a non;nal lytic viral life
cycle (see, for example, Berkner et al. (1988) BioTechnic~,ues 6:616;
Rosenfeld et al. (1991)
Science 252:431-434; and Rosenfeld et al. (1992) Cell 68:143-155). Suitable
adenoviral vectors
derived from the adenovirus strain Ad type 5 d1324 or other strains of
adenovirus (e.g., Ad2,
Ad3, Ad7 etc.) are well known to those skilled in the art. Recombinant
adenoviruses can be
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advantageous in certain circumstances in that they are not capable of
infecting nondividing cells
and can be used to infect a wide variety of cell types, including endothelial
cells (Lemarchand et
al. (1992) Proc. Natl. Acad. Sci. USA 89:6482-6486), and smooth muscle cells
(Quantin et al.
(1992) Proc. Natl. Acad. Sci. USA 89:2581-2584). Furthermore, the virus
particle is relatively
stable and amenable to purification and concentration, and as above, can be
modified so as to
affect the spectrum of infectivity. Additionally, introduced adenoviral DNA
(and foreign DNA
contained therein) is not integrated into the genome of a host cell but
remains episomal, thereby
avoiding potential problems that can occur as a result of insertional
mutagenesis in situations
where introduced DNA becomes integrated into the host genome (e.g., retroviral
DNA).
Moreover, the carrying capacity of the adenoviral genome for foreign DNA is
large (up to 8
kilobases) relative to other gene delivery vectors (Berkner et al., supra; Haj-
Ahmand and
Graham (1986) J. Virol. 57:267). Most replication-defective adenoviral vectors
currently in use
and therefore favored by the present invention are deleted for ail or parts of
the viral E1 and E3
genes but retain as much as 80% of the adenoviral genetic material (see, e.g.,
Jones et al. (1979)
Cell 16:683; Berkner et al., supra; and Graham et al. in Methods in Molecular
Biolosv, E.J.
Murray, Ed. (Humana, Clifton, NJ, 1991) vol. 7. pp. 109-127). Expression of
the inserted gene
can be under control of, for example, the E 1 A promoter, the maj or late
promoter (MLP) and
associated leader sequences, the E3 promoter, or exogenously added promoter
sequences.
Yet another viral vector system useful for delivery of the subject telomerase
activator
constructs is the adeno-associated virus (AAV). Adeno-associated virus is a
naturally occurring
defective virus that requires another virus, such as an adenovirus or a herpes
virus, as a helper
virus for efficient replication and a productive life cycle. (For a review see
Muzyczka et al. Curr.
Topics in Micro. and Immunol. (1992) 158:97-129). It is also one of the few
viruses that may
integrate its DNA into non-dividing cells, and exhibits a high frequency of
stable integration (see
for example Flotte et al. (1992) Am. J. Respir. Cell. Mol. Biol. 7:349-356;
Samulski et al. (1989)
J. Virol. 63:3822-3828; and McLaughlin et al. (1989) J. Virol. 62:1963-1973).
Vectors
containing as little as 300 base pairs of AAV can be packaged and can
integrate. Space for
exogenous DNA is limited to about 4.5 kb. An AAV vector such as that described
in Tratschin
et al. (1985) Mol. Cell. Biol. 5:3251-3260 can be used to introduce DNA into
cells. A variety of
nucleic acids have been introduced into different cell types using AAV vectors
(see for example
Hermonat et al. (1984) Proc. Natl. Acad. Sci. USA 81:6466-6470; Tratschin et
al. (1985) Mol.
Cell. Biol. 4:2072-2081; Wondisford et al. (1988) Mol. Endocrinol. 2:32-39;
Tratschin et al.
(1984) J. Virol. 51:611-619; and Flotte et al. (1993) J. Biol. Chem. 268:3781-
3790).
Other viral vector systems that may have application in gene therapy have been
derived
from herpes virus, vaccinia virus, and several RNA viruses. In particular,
herpes virus vectors
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may provide a unique strategy for persistent expression of the subject
telomerase activator
proteins in cells of the central nervous system, such as neuronal stem cells,
and ocular tissue
(Pepose et al. (1994) Invest Ophthalmol Vis Sci 35:2662-2666)
In addition to viral transfer methods, such as those illustrated above, non-
viral methods
can also be employed to cause expression of a the subject proteins in the
tissue of an animal.
Most nonviral methods of gene transfer rely on normal mechanisms used by
mammalian cells for
the uptake and intracellular transport of macromolecules. In preferred
embodiments, non-viral
gene delivery systems of the present invention rely on endocytic pathways for
the uptake of the
gene by the targeted cell. Exemplary gene delivery systems of this type
include liposomal
derived systems, poly-lysine conjugates; and artificial viral envelopes.
In a representative embodiment, a gene encoding one of the subject proteins
can be
entrapped in liposomes bearing positive charges on their surface (e.g.,
lipofectins) and
(optionally) which are tagged with antibodies against cell surface antigens of
the target tissue
(Mizuno et al. ( 1992) No Shinkei Geka 20:547-551; PCT publication W091
/06309; Japanese
patent application 1047381; and European patent publication EP-A-43075). For
example,
Iipofection of neuroglioma cells can be carried out using liposomes tagged
with monoclonal
antibodies against glioma-associated antigen (Mizuno et al. ( 1992) Neurol.
Med. Chir. 32:873-
876).
In yet another illustrative embodiment, the gene delivery system comprises an
antibody
or cell surface ligand which is cross-linked with a gene binding agent such as
poly-lysine (see,
for example, PCT publications W093/04701, W092/22635, W092/20316, W092/19749,
and
W092/06180). For example, the subject gene construct can be used to transfect
hepatocytic
cells in vivo using a soluble polynucleotide carrier comprising an
asialoglycoprotein conjugated
to a polycation, e.g. poly-lysine (see U.S. Patent 5,166,320). It will also be
appreciated that
effective delivery of the subject nucleic acid constructs via receptor-
mediated endocytosis can be
improved using agents which enhance escape of the gene from the endosomal
structures. For
instance, whole adenovirus or fusogenic peptides of the influenza HA gene
product can be used
as part of the delivery system to induce efficient disruption of DNA-
containing endosomes
(Mulligan et al. (1993) Science 260-926; Wagner et al. (1992) PNAS 89:7934;
and Christiano et
al. (1993) PNAS 90:2122).
While the repair of telomers, e.g., by the activation of telomerase activty,
can be enough
for extending the replicative capacity of a cell, it can be a transforming
event (e.g., to cause crisis
and emergence of cancer cells), particularly where activation persists.
Therefore, in one aspect,
the present invention provides a method for increasing the proliferative
capacity of cells,
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preferably normal cells, which method comprises delivering into the cell a
gene construct which
can selectively and reversibly activate telomerase activity in the cell.
In one embodiment, the coding sequence for the telomerase activator is
provided as part
of a vector which can be partially or completely excised from the host cell is
an inducible
S manner. For instance, the vector can include:
(i) one or more transposition elements for integration of the vector into
chromosomal
DNA of a eukaryotic host cell;
(ii) a coding sequence of a telomerase activator; and
(iii) excision elements for removing, upon contact of the cell with an
excision agent
(which activates the excision element) all or at least the portion of an
integrated
form of the vector from chromosomal DNA in a manner which is results in loss-
of
function of the heterologous telomerase activator.
For example, the excision elements can be provided in the vector so as flank
at least the coding
sequence of a telomerase activator, though they may flank only a portion of
the coding sequence
such that the sequence resulting after excision does not encode a functional
activator, or they
may flank a sufficient portion of a transcriptional regulatory sequence for
the telomerase
activator such that resulting construct does not express the telomerase
activator.
In preferred embodiments, the exicision elements are disposed in the vector
such that,
upon excision of the integrated form of the vector, no or substantially no
portion (e.g., less than
50 nucleotides) of the vector DNA is left in the chromosomal DNA of the host
cell.
In preferred embodiments, the transposition elements are viral transposition
elements,
e.g, retroviral or lentiviral transposition elements, such as may be provided
where the vector is a
replication-deficient virus.
In preferred embodiments, the excision elements comprise enzyme-assisted site-
specific
integration sequences. For instance, the excision elements may include
recombinase target sites,
e.g., recombinase target sites for Cre recombinase, Flp recombinase, Pin
recombinase, lamda
integrase, Gin recombinase or R recombinase. The excision elements may also be
restriction
enzyme sites.
In preferred embodiments, the vector is a retroviral vector which recombinase
sites which
are located in the LTRs such that excision of a proviral sequence occurs,
e.g., the viral vector is
completely, or nearly completely excised from the chromosomal DNA of the host
cell.
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The vector can include such other elements as: transcriptional regulatory
sequences for
directing transcription of the coding sequence for the telemorase activator
cnucleic; a packaging
signal for packaging the vector in an infectious viral particle;
Exemplary vectors of this type, e.g., readily excisable, are descibed in the
appended
examples as well as PCT publication WO 98/12339. On advantage that certain of
these vectors
have, e.g., those which can be substantially excised, can be realized for
embodiments wherein
the method is part of an ex vivo therapy. In such embodiments, the cells can
be treated ex vivo
with the constructs. Prior to implantation in a host, the cells are treated
with an agent, such as a
recombinase, which results in exicision of the vector from the genomic DNA of
the hast cell.
Thus, the cells which are implanted are no longer genetically engineered. In
such embodiments,
it may be desirable to include one or more detectable genes (markers) on the
vector in order to be
able to identify cells which still retained the vector, e.g., by FACS sorting,
affinity purification
or other techniques.
The reversibility of telomerase activation can also be generated by use of an
expression
1 S system which is inducible because of the presence of an inducible
transcriptional regulatory
sequence controlling the expression of the coding sequence of the EST or
telomerase activator.
Inciucihic ~ru,»«tcrs are ~romotcrs that initiate increased levels of
transcription from DNA
under their control in response to some change in culture conditions, e.g.,
the presence or
absence of a nutrient or a change in temperature. Where the cells are to be
transplanted into a
patient, the inducible promoter is preferably one which is regulated by a
small molecule or other
factor which is not endogenous to the host animal.
Exemplary regulatable promoters include the tct:cwc,-clim responsive
pri,mi~tcrs, ,vch as
clcscriUccl in. f'or ex~.m~le. Gossen et al. (1992) PNAS 89:5547-5551; and
Pescini et al., (1994)
Biochem. Bi~hvs. Res. Comm. 202:1664-1667.
In another another embodiment, the subject method utilizes the multimerization
technology first pioneered by Schreiber and Crabtree. This technique permits
the regulation of
expression of an endogenous or heterologous gene, in this case a coding
sequence for EST or a
telomerase activator, by use of chimeric transcription factors which are
dependent on small
molecules "dimerizers" to assemble transcriptionally active complexes. See,
for example, PCT
publications WO 9612796; WO 9505389; WO 9502684; WO 9418317; WO 9606097; and
WO
9606110. Moreover, a number of techniques have been deveolped more recently
which permit
the recruitment of endogenous DNA binding and activation domains to the
transcriptional
regulatory sequences by use of artificial dimerization molecules. See, for
example, PCT
publication WO 9613613.
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-22-
In other embodiments, the reversibility of telomerase activation can be
accomplished by
use of conditionally active (or conditionally inactivable) forms of EST or of
the telomerase
activators. For instance, temperature-sensitive mutants of telomerase or myc
can be employed in
the subject method. In embodiments wherein the cells are to be transplanted
into an animal, the
S is mutant can be inactive at body temperature (the non-permissive
temperature) and active at a
lower or higher cell culture temperature.
To illustrate, one strategy for producing temperature-sensitive EST or myc
mutants, that
does not require a search for a is mutation in a gene of interest, is based on
a portable, heat-
inducible N-degron. The N-degron is an intracellular degradation signal whose
essential
determinant is a "destabilizing" N-terminal residue of a protein. A set of N-
degrons containing
different destabilizing residues is manifested as the N-end rule, which
relates the in vivo half life
of a protein to the identity of its N-terminal residue. In eukaryotes, the N-
degron consists of at
least two determinants: a destabilizing N-terminal residue and a specific
internal Lys residue (or
residues) of a substrate. The Lys residue is the site of attachment of a
multiubiquitin chain.
Ubiquidn is a protein whose covalent conjugation to other proteins plays a
role in a number of
cellular processes, primarily through routes that involve protein degradation.
For a description
of exemplary heat-inducible N-degron modules which can be adapted for
generating conditional
mutants of EST, myc or other telomerase activators, see US Patents 5,705,387
and 5,538,862,
and Dohmen et al. ( 1994) Science 263:1273-6.
In yet other embodiments, the multimerization technology referred to above can
be used
to generate small molecule inducible forms of EST or a telomerase activator.
To illustrate, a
first gene construct can be provided which encodes a fusion protein including
a DNA binding
domain (and optionally oligomerization domains) of myc and a ligand binding
domain which
binds to a small organic molecule, e.g., a domain which will bind to a
dimerizing agent. A
second gene construct is also provided, which construct encodes a fusion
protein including an
activation domain, e.g., a VP16 activation domain, and a ligand binding domain
which will also
bind the dimerizing agent when it is already bound to the first fusion
protein. Expression of
these two fusion proteins in a host cell, in the absence of the dimerizing
agent, will not activate
telomerase. Upon addition of the dimerizing agent, the fusion proteins
associate, and activate
transcription of genes which include myc responsive elements, which causes
activation of
telomerase activity.
In yet another embodiment, ectopic expression of EST2 or other telomerase
activator can
be by way of a "gene activation" construct which, by homologous recombination
with a genomic
DNA, alters the transcriptional regulatory sequences of an endogenous
telomearse activator
gene. For instance, the gene activation construct can replace the endogenous
promoter of an
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EST2 gene with a heterologous promoter, e.g., one which causes consitutive
expression of the
EST2 gene or which causes inducible expression of the gene under conditions
different from the
normal expression pattern of the gene. A vareity of different formats for the
gene activation
constructs are available. See, for example, the Transkaryotic Therapies, Inc
PCT publications
W093/09222, W095/31560, W096/29411, W095/31560 and W094/12650.
In preferred embodiments, the nucleotide sequence used as the gene activation
construct
can be comprised of ( 1 ) DNA from some portion of the endogenous gene (exon
sequence, intron
sequence, promoter sequences, etc.) which direct recombination and (2)
heterologous
transcriptional regulatory sequences) which is to be operably linked to the
coding sequence for
the genomic gene upon recombination of the gene activation construct. The
construct may
further include a reporter gene to detect the presence of the knockout
construct in the cell.
The gene activation construct is inserted into a cell, and integrates with the
genomic
DNA of the cell in such a position so as to provide the heterologous
regulatory sequences in
operative association with, e.g., the native EST2 gene. Such insertion occurs
by homologous
recombination, i.e., recombination regions of the activation construct that
are homologous to the
endogenous EST2 gene sequence hybridize to the genomic DNA and recombine with
the
genomic sequences so that the construct is incorporated into the corresponding
position of the
genomic DNA.
The terms "recombination region" or "targeting sequence" refer to a segment
(i.e., a
portion) of a gene activation construct having a sequence that is
substantially identical to or
substantially complementary to a genomic gene sequence, e.g., including 5'
flanking sequences
of the genomic gene, and can facilitate homologous recombination between the
genomic
sequence and the targeting transgene construct.
As used herein, the term "replacement region" refers to a portion of a
activation construct
which becomes integrated into an endogenous chromosomal location following
homologous
recombination between a recombination region and a genomic sequence.
The heterologous regulatory sequences, e.g., which are provided in the
replacement
region, can include one or more of a variety elements, including: promoters
(such as constitutive
or inducible promoters), enhancers, negative regualtory elements, locus
control regions,
transcription factor binding sites, or combinations thereof.
Promoters/enhancers which may be
used to control the expression of the targeted gene in vivo include, but are
not limited to, the
cytomegalovirus (CMV) promoter/enhancer (I~arasuyama et al., 1989, J. Exp.
Med., 169:13), the
human ~-actin promoter (Gunning et al. (1987) PNAS 84:4831-4835), the
glucocorticoid-
inducible promoter present in the mouse mammary tumor virus long terminal
repeat (MMTV
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LTR) (Klessig et al. (1984) Mol. Cell Biol. 4:1354-1362), the long terminal
repeat sequences of
Moloney marine leukemia virus (MuLV LTR) (Weiss et al. (1985) RNA Tumor
Viruses, Cold
Spring Harbor Laboratory, Cold Spring Harbor, New York), the SV40 early or
late region
promoter (Bernoist et al. (1981) Nature 290:304-310; Templeton et aI. (1984)
Mol. Cell Biol.,
4:817; and Sprague et al. (1983) J. Virol., 45:773), the promoter contained in
the 3' long terminal
repeat of Rous sarcoma virus (RSV) (Yamamoto et al., 1980, Cell, 22:787-797),
the herpes
simplex virus (HSV) thymidine kinase promoter/enhancer (Wagner et al. (1981)
PNAS 82:3567-
71), and the herpes simplex virus LAT promoter (Wolfe et al. (1992) Nature
Genetics, 1:379-
384).
In still other embodiments, the replacement region merely deletes a negative
transcriptional control element of the native gene, e.g., to activate
expression.
In yet another embodiment, membrane permeable drugs (e.g., preferably small
organic
molecules) can be identified which activate the expression of an endogenous
EST2 gene. In
light of the availability of the genomic EST2 gene, it will be possible to
produce reporter
constructs in which a reporter gene is operably linked to the transcriptional
regulatory seqence of
the EST2 gene. When transfected into cells which possess the appropriate
intracellular
machinery for activation of the reporter construct through the EST2 regulatory
sequence, the
resulting cells can be used in a cell-based approach for identifying such
compounds.
In embodiments wherein the cells are treated in culture, RNA encoding EST2,
myc or
another telomerase activator can be introduced directly into the cell, e.g.,
from RNA generated
by in vitro transciption. In preferred embodiments, the RNA is preferably a
modified
polynucleotide which is resistant to endogenous nucleases, e.g. exonucleases
and/or
endonucleases. Exemplary nucleic acid modifications which can be used to
generate such RNA
polynucleotides include phosphoramidate, phosphothioate and methylphosphonate
analogs of
nucleic acids (see also U.S. Patents 5,176,996; 5,264,564; and 5,256,775), or
peptide nucleic
acids (PNAs).
In still another embodiment of the subject method, the telomerase activator
polypeptide
can be contacted with a cell under conditions wherein the protein is taken up
by the cell, e.g.,
internalized, without the need for recombinant expression in the cell. For
instance, in the
application of the subject method to skin, mucosa and the like, a variety of
techniques have been
developed for the transcytotic delivery of ectopically added proteins.
In an exemplary embodiment, the EST2 or myc protein is provided for
transmucosal or
transdermal delivery. For such administration, penetrants appropriate to the
barrier to be
permeated are used in the formulation with the polypeptide. Such penetrants
are generally
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known in the art, and include, for example, for transmucosal administration
bile salts and fusidic
acid derivatives. In addition, detergents may be used to facilitate
permeation. Transmucosal
administration may be through nasal sprays or using suppositories. For topical
administration,
the proteins of the invention are formulated into ointments, salves, gels, or
creams as generally
known in the art. For example, Chien et al. (1989) J. Pharm. Sci. 78:376-383
describes direct
current iontophoretic transdermal delivery of peptide and protein drugs.
Srinivasan et al.,
(1989) J. of Pharm. Sci. 78:370-375 describes the transdermal iontophoretic
drug delivery
Mechanistic analysis and application to polypeptide delivery. See also USSN
4,940,456.
USSN 5,459,127 describes the use of cationic lipids for intracellular delivery
of
biologically active molecules.
USSN 5,190,762 describes methods of administering proteins to living skin
cell.
In another embodiment, the polypeptide is provided as a chimeric polypeptide
which
includes a heterologous peptide sequence ("internalizing peptide") which
drives the translocation
of an extracellular form of a thereapeutic polypeptide sequence across a cell
membrane in order
to facilitate intracellular localization of the thereapeutic polypeptide. In
this regard, the
therapeutic polypeptide sequence is one which is active intracellularly, such
as a tumor
suppressor polypeptide, transcription factor or the like. The internalizing
peptide, by itself, is
capable of crossing a cellular membrane by, e.g., transcytosis, at a
relatively high rate. The
internalizing peptide is conjugated, e.g., as a fusion protein, to the
telomerase activator
polypeptide. The resulting chimeric polypeptide is transported into cells at a
higher rate relative
to the activator polypeptide alone to thereby provide an means for enhancing
its introduction into
cells to which it is applied, e.g., to enhance topical applications of the
EST2 polypeptide.
In one embodiment, the internalizing peptide is derived from the drosopholia
antepennepedia protein, or homologs thereof. The 60 amino acid long long
homeodomain of
the homeo-protein antepennepedia has been demonstrated to translocate through
biological
membranes and can facilitate the translocation of heterologous polypeptides to
which it is
couples. See for example Derossi et al. (1994) J Biol Chem 269:10444-10450;
and Perez et al.
(1992) J Cell Sci 102:717-722. Recently, it has been demonstrated that
fragments as small as 16
amino acids long of this protein are sufficient to drive internalization. See
Derossi et al. (1996) J
Biol Chem 271:18188-18193. The present invention contemplates a chimeric
protein
comprising at least one EST2 or myc polypeptide sequence and at least a
portion of the
antepennepedia protein (or homolog thereof) sufficient to increase the
transmembrane transport
of the chimeric protein, relative to the EST2 or myc polypeptide, by a
statistically significant
amount.
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Another example of an internalizing peptide is the HIV transactivator (TAT)
protein.
This protein appears to be divided into four domains (Kuppuswamy et al. (1989)
Nucl. Acids
Res. 17:3551-3561). Purified TAT protein is taken up by cells in tissue
culture (Frankel and
Pabo, (1989) Cell 55:1189-1193), and peptides, such as the fragment
corresponding to residues
37 -62 of TAT, are rapidly taken up by cell in vitro (Green and Loewenstein,
(1989) Cell
55:1179-1188). The highly basic region mediates internalization and targeting
of the
internalizing moiety to the nucleus (Ruben et al., (1989) J. Virol. 63:1-8).
Peptides or analogs
that include a sequence present in the highly basic region, such as
CFITKALGISYGRKKRRQRRRPPQGS, are conjugated to EST2 or myc polypeptides to aid
in
internalization and targeting those proteins to the intracellular milleau.
Another exemplary transcellular polypeptide can be generated to include a
sufficient
portion of mastoparan (T. Higashijima et al., (1990) J. Biol. Chem. 265:14176)
to increase the
transmembrane transport of the chimeric protein.
While not wishing to be bound by any particular theory, it is noted that
hydrophilic
polypeptides may be also be physiologically transported across the membrane
barriers by
coupling or conjugating the polypeptide to a transportable peptide which is
capable of crossing
the membrane by receptor-mediated transcytosis. Suitable internalizing
peptides of this type can
be generated using all or a portion of, e.g., a histone, insulin, transferrin,
basic albumin,
prolactin and insulin-like growth factor I (IGF-I), insulin-like growth factor
II (IGF-II) or other
growth factors. For instance, it has been found that an insulin fragment,
showing affinity for the
insulin receptor on capillary cells, and being less effective than insulin in
blood sugar reduction,
is capable of transmembrane transport by receptor-mediated transcytosis and
can therefor serve
as an internalizing peptide for the subject transcellular polypeptides.
Preferred growth factor-
derived internalizing peptides include EGF (epidermal growth factor)-derived
peptides, such as
CMHIESLDSYTC and CMYIEALDKYAC; TGF- beta (transforming growth factor beta )-
derived peptides; peptides derived from PDGF (platelet-derived growth factor)
or PDGF-2;
peptides derived from IGF-I (insulin-like growth factor) or IGF-II; and FGF
(fibroblast growth
factor)-derived peptides.
Another class of translocating/internalizing peptides exhibits pH-dependent
membrane
binding. For an internalizing peptide that assumes a helical conformation at
an acidic pH, the
internalizing peptide acquires the property of amphiphilicity, e.g., it has
both hydrophobic and
hydrophilic interfaces. More specifically, within a pH range of approximately
5.0-5.5, an
internalizing peptide forms an alpha-helical, amphiphilic structure that
facilitates insertion of the
moiety into a target membrane. An alpha-helix-inducing acidic pH environment
may be found,
for example, in the low pH environment present within cellular endosomes. Such
internalizing
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peptides can be used to facilitate transport of telomerase activator
polypeptides, taken up by an
endocytic mechanism, from endosomal compartments to the cytoplasm.
A preferred pH-dependent membrane-binding internalizing peptide includes a
high
percentage of helix-forming residues, such as glutamate, methionine, alanine
and leucine. In
addition, a preferred internalizing peptide sequence includes ionizable
residues having pKa's
within the range of pH 5-7, so that a sufficient uncharged membrane-binding
domain will be
present within the peptide at pH 5 to allow insertion into the target cell
membrane.
A particularly preferred pH-dependent membrane-binding internalizing peptide
in this
regard is aal-aa2-aa3-EAALA(EALA~-EALEALAA-amide, which represents a
modification of
the peptide sequence of Subbarao et al. (Biochemistry 26:2964, 1987). Within
this peptide
sequence, the first amino acid residue (aal ) is preferably a unique residue,
such as cysteine or
lysine, that facilitates chemical conjugation of the internalizing peptide to
a targeting protein
conjugate. Amino acid residues 2-3 may be selected to modulate the affinity of
the internalizing
peptide for different membranes. For instance, if both residues 2 and 3 are
lys or arg, the
internalizing peptide will have the capacity to bind to membranes or patches
of lipids having a
negative surface charge. If residues 2-3 are neutral amino acids, the
internalizing peptide will
insert into neutral membranes.
Yet other preferred internalizing peptides include peptides of apo-lipoprotein
A-1 and B;
peptide toxins, such as melittin, bombolittin, delta hemolysin and the
pardaxins; antibiotic
peptides, such as alamethicin; peptide hormones, such as calcitonin,
corticotrophin releasing
factor, beta endorphin, glucagon, parathyroid hormone, pancreatic polypeptide;
and peptides
corresponding to signal sequences of numerous secreted proteins. in addition,
exemplary
internalizing peptides may be modified through attachment of substituents that
enhance the
alpha-helical character of the internalizing peptide at acidic pH.
Yet another class of internalizing peptides suitable for use within the
present invention
include hydrophobic domains that are "hidden" at physiological pH, but are
exposed in the low
pH environment of the target cell endosome. Upon pH-induced unfolding and
exposure of the
hydrophobic domain, the moiety binds to lipid bilayers and effects
translocation of the
covalently linked polypeptide into the cell cytoplasm. Such internalizing
peptides may be
modeled after sequences identified in, e.g., Pseudomonas exotoxin A, clathrin,
or Diphtheria
toxin.
Pore-forming proteins or peptides may also serve as internalizing peptides
herein. Pore-
forming proteins or peptides may be obtained or derived from, for example, C9
complement
protein, cytolytic T-cell molecules or NK-cell molecules. These moieties are
capable of forming
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ring-like structures in membranes, thereby allowing transport of attached
polypeptide through
the membrane and into the cell interior.
Mere membrane intercalation of an internalizing peptide may be sufficient for
translocation of the polypeptide, e.g. EST2 or myc, across cell membranes.
However,
translocation may be improved by attaching to the internalizing peptide a
substrate for
intracellular enzymes (i.e., an "accessory peptide"). It is preferred that an
accessory peptide be
attached to a portions) of the internalizing peptide that protrudes through
the cell membrane to
the cytoplasmic face. The accessory peptide may be advantageously attached to
one terminus of
a translocating/internalizing moiety or anchoring peptide. An accessory moiety
of the present
invention may contain one or more amino acid residues. In one embodiment, an
accessory
moiety may provide a substrate for cellular phosphorylation (for instance, the
accessory peptide
may contain a tyrosine residue).
An exemplary accessory moiety in this regard would be a peptide substrate for
N-
myristoyl transferase, such as GNAAAARR (Eubanks et al., in: Peptides.
Chemistr~,and
Biolouy, Garland Marshall (ed.), ESCOM, Leiden, 1988, pp. 566-69) In this
construct, an
internalizing, peptide would be attached to the C-terminus of the accessory
peptide, since the N
terminal glycine is critical for the accessory moiety's activity. This hybrid
peptide, upon
attachment to an EST2 or myc polypeptide at its C-terminus, is N-rnyristylated
and further
anchored to the target cell membrane, e.g., it serves to increase the local
concentration of the
polypeptide at the cell membrane.
To further illustrate use of an accessory peptide, a phosphorylatable
accessory peptide is
first covalently attached to the C-terminus of an internalizing peptide and
then incorporated into
a fusion protein with an EST2 or myc polypeptide. The peptide component of the
fusion protein
intercalates into the target cell plasma membrane and, as a result, the
accessory peptide is
translocated across the membrane and protrudes into the cytoplasm of the
target cell. On the
cytoplasmic side of the plasma membrane, the accessory peptide is
phosphorylated by cellular
kinases at neutral pH. Once phosphorylated, the accessory peptide acts to
irreversibly anchor the
fusion protein into the membrane. Localization to the cell surface membrane
can enhance the
translocation of the polypeptide into the cell cytoplasm.
Suitable accessory peptides include peptides that are kinase substrates,
peptides that
possess a single positive charge, and peptides that contain sequences which
are glycosylated by
membrane-bound glycotransferases. Accessory peptides that are glycosylated by
membrane-
bound glycotransferases may include the sequence x-NLT-x, where "x" may be
another peptide,
an amino acid, coupling agent or hydrophobic molecule, for example. When this
hydrophobic
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tripeptide is incubated with microsomal vesicles, it crosses vesicular
membranes, is glycosylated
on the luminal side, and is entrapped within the vesicles due to its
hydrophilicity (C. Hirschberg
et al., (1987) Ann. Rev. Biochem. 56:63-87). Accessory peptides that contain
the sequence x-
NLT-x thus will enhance target cell retention of corresponding polypeptide.
In another embodiment of this aspect of the invention, an accessory peptide
can be used
to enhance interaction of the telomerase activator polypeptide with the target
cell. Exemplary
accessory peptides in this regard include peptides derived from cell adhesion
proteins containing
the sequence "RGD", or peptides derived from laminin containing the sequence
CDPGYIGSRC.
Extracellular matrix glycoproteins, such as fibronectin and laminin, bind to
cell surfaces through
receptor-mediated processes. A tripeptide sequence, RGD, has been identified
as necessary for
binding to cell surface receptors. This sequence is present in fibronectin,
vitronectin, C3bi of
complement, von-Willebrand factor, EGF receptor, transforming growth factor
beta , collagen
type I, lambda receptor of E. coli, fibrinogen and Sindbis coat protein (E.
Ruoslahti, Ann. Rev.
Biochem. 57:375-413, 1988). Cell surface receptors that recognize RGD
sequences have been
grouped into a superfamily of related proteins designated "integrins". Binding
of "RGD
peptides" to cell surface integrins will promote cell-surface retention, and
ultimately
translocation, of the polypeptide.
As described above, the internalizing and accessory peptides can each,
independently, be
added to an EST2 or myc polypeptide by either chemical cross-linking or in the
form of a fusion
protein. In the instance of fusion proteins, unstructured polypeptide linkers
can be included
between each of the peptide moieties.
In general, the internalization peptide will be sufficient to also direct
export of the
polypeptide. However, where an accessory peptide is provided, such as an RGD
sequence, it
may be necessary to include a secretion signal sequence to direct export of
the fusion protein
from its host cell. In preferred embodiments, the secretion signal sequence is
located at the
extreme N-terminus, and is (optionally) flanked by a proteolytic site between
the secretion signal
and the rest of the fusion protein.
In an exemplary embodiment, an EST2 or myc polypeptide is engineered to
include an
integrin-binding RGD peptide/SV40 nuclear localization signal (see, for
example Hart SL et al.,
1994; J. Biol. Chem.,269:12468-12474), such as encoded by the nucleotide
sequence provided in
the Ndel-EcoRl fragment:
catatgggtggctgccgtggcgatatgttcggttgcggtgctcctccaaaaaagaagagaaag-
gtagctggattc, which encodes the RGD/SV40 nucleotide sequence: MGGCRGDMFGCGAPP-
KKKRICVAGF. In another embodiment, the protein can be engineered with the HIV-
1 tat(1-72)
polypeptide, e.g., as provided by the Ndel-EcoRl
fragment:catatggagccagtagatcctagactagagccc-
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tggaagcatccaggaagtcagcctaaaactgcttgtaccaattgctattgtaaaaagtgttgctttcattgccaagttt
gtttcataacaaaagcc
cttggcatctcctatggcaggaagaagcggagacagcgacgaagacctcctcaaggcagtcagactcatcaagtttctc
taagtaagcaag
gattc, which encodes the HIV-1 tat(1-72) peptide sequence:
MEPVDPRLEPWKHPGSQPKT-
ACTNCYCKKCCFHCQVCFITKALGISYGRKKRRQRRRPPQGSQTHQVSLSKQ. In still
another embodiment, the fusion protein includes the HSV-1 VP22 polypeptide
(Elliott G.,
O'Hare P (1997) Cell. 88:223-233) provided by the Ndel-EcoRl fragment:
cat atg acc tct cgc cgc tcc gtg aag tcg ggt ccg cgg gag gtt ccg cgc gat gag
tac gag gat ctg tac tac
acc ccg tct tca ggt atg gcg agt ccc gat agt ccg cct gac acc tcc cgc cgt ggc
gcc cta cag aca cgc tcg
cgc cag agg ggc gag gtc cgt ttc gtc cag tac gac gag tcg gat tat gcc ctc tac
ggg ggc tcg tca tcc gaa
gac gac gaa cac ccg gag gtc ccc cgg acg cgg cgt ccc gtt tcc ggg gcg gtt ttg
tcc ggc ccg ggg cct
gcg cgg gcg cct ccg cca ccc get ggg tcc gga ggg gcc gga cgc aca ccc acc acc
gcc ccc cgg gcc ccc
cga acc cag cgg gtg gcg act aag gcc ccc gcg gcc ccg gcg gcg gag acc acc cgc
ggc agg aaa tcg gcc
cag cca gaa tcc gcc gca ctc cca gac gcc ccc gcg tcg acg gcg cca acc cga tcc
aag aca ccc gcg cag
ggg ctg gcc aga aag ctg cac ttt agc acc gcc ccc cca aac ccc gac gcg cca tgg
acc ccc cgg gtg gcc
ggc ttt aac aag cgc gtc ttc tgc gcc gcg gtc ggg cgc ctg gcg gcc atg cat gcc
cgg atg gcg gcg gtc cag
ctc tgg gac atg tcg cgt ccg cgc aca gac gaa gac ctc aac gaa ctc ctt ggc atc
acc acc atc cgc gtg acg
gtc tgc gag ggc aaa aac ctg ctt cag cgc gcc aac gag ttg gtg aat cca gac gtg
gtg cag gac gtc gac gcg
gcc acg gcg act cga ggg cgt tct gcg gcg tcg cgc ccc acc gag cga cct cga gcc
cca gcc cgc tcc get tct
cgc ccc aga cgg ccc gtc gag gaa ttc
which encodes the HSV-1 VP22 peptide having the sequence:
MTSRRSVKSGPREVPRDEYEDLYYTPSSGMASPDSPPDTSRRGALQTRSRQRGEVRFVQ
YDESDYALYGGSS SEDDEHPEV PRTRRPV SGAVL S GPGPARAPPPPAGSGGAGRTPTTA
PRAPRTGRVATKAPAAPAAETTRGRKSAQPESAALPDAPASTAPTRSKTPAQGLARKLH
FSTAPPNPDAPWTPRVAGFNKRVFCAAVGRLAAMHARMAAVQLWDMSRPRTDEDLN
ELLGITTIRVTVCEGKNLLQRANELVNPDVVQDVDAATATRGRSAASRPTERPRAPARS
ASRPRRPVE
In still another embodiment, the fusion protein includes the C-terminal domain
of the
VP22 protein from, e.g., the nucleotide sequence (Ndel-EcoRl fragment):
cat atg gac gtc gac gcg gcc acg gcg act cga ggg cgt tct gcg gcg tcg cgc ccc
acc gag cga cct cga
gcc cca gcc cgc tcc get tct cgc ccc aga cgg ccc gtc gag gaa ttc
which encodes the VP22 (C-terminal domain) peptide sequence:
MDVDAATATRGRSAASRPTERPRAPARSASRPRRPVE
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In other embodiments, the subject method employs small, organic molecules,
e.g., having
a molecular weight of less than s00o amu, more preferably less than 1000 amu,
and even more
preferably less than 500 amu. Moreover, such compounds are preferably membrane
permeant,
e.g., able to diffuse acmss the cell membrane into the host cell when added
directly to culture
cells or cells in whole blood.
In this regard, the art provides examples of assays for identifying agents
which are
capable of activating telomerase activity, e.g., see US Patents 5,837,453,
5,830,644, 5,804,380
and 5,686,245.
In yet another embodiment, to the extent it is relevant, the intracellular
level of '1'R'h car a
mlomcrase activati~r (prc~tcinj can be upregulated by inhibiting its natural
turnover rate. For
example, inhibitors of ubiquitin-dependent or independent degradation of the
~rotc:i n can be used
to cause ectopic expression of pnotcin in the sense that the concentration of
the protein in the cell
can be artificially elevated. Assays for detecting inhibitors of
ubiquitination, e:g., which can be
readily adapted for detecting inhibitors of ubiquitination of myc or other
telomerase activators,
are described in the literature, as for example US Patents 5,744,343,
5,847,094, 5,847,076,
5,834,487, 5,817,494, 5,780,454 and 5,766,927. Likewise, to the extent that
other post-
translational modifications, such as phosphorylation, influence protein
stability, the present
invention contemplates the use of inhibitors of such modifications, including,
as appropriate,
kinase or phosphatase inhibitors.
In still other embodiments, cellular prolifeartive capacity can be incrased by
contacting
the cell with an agent, e.g. a small molecule, which relieves or otherwise
inhibits a signal which
antagonizes myc-induced activation of telomerase activity. For instance,
agents can be used
which disrupt protein-protein interactions involved in inhibition of myc
activity by, e.g., mad
max heterodimers.
(B7 Cori oa int Applications
Another aspect of the invention provides a conjoint therapy wherein one or
more other
therapeutic agents are administered with the telomerase-activating therapeutic
agent. Such
conjoint treatment may be achieved by way of the simultaneous, sequential or
separate dosing of
the individual components of the treatment. For example, the telomerase-
activating therapeutic
agent can be administered conjointly with a growth factors and other mitogenic
agents.
Mitogenic agent, as used herein; refers to any compound or composition,
including peptides,
proteins, and glycoproteins, which is capable of stimulating proliferation of
a target cell
population. For example, the telomerase-activating therapeutic agent can be
conjointly
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administered with a T-cell mitogenic agent such as lectins, e.g., concanavalin
A or
phytohemagglutinin. Other exemplary mitogenic agents include insulin-like
growth factor
(IGF), platelet-derived growth factor (PDGF), fibroblast growth factor (FGF),
and certain of the
transforming growth factors (TGFs).
In one embodiment, the subject telomerase-activating therapeutic agent agent
is co-
administered with an agent that relieves "capping" inhibition of EST2 rescue.
We have noticed
that EST2 wiill neither extend telomere length nor lifespan in late-passage
HMEC cells, and
certain other cell lines such as fibroblasts. While not wishing to be bound by
any particular
theory, this inability to extend telomeres in such cells may be the result of
reaction kinetics -e.g.,
telomere binding proteins such as TRF (TTAGGG repeat binding factor) become
abundant
relevant to the telomeric sequences. The increased loading of telomeres with
such proteins
inhibits elongation induced by ectopic EST2. Such relative overabundance of
proteins to
telomers may be the result of, for example, reduction in the number of
telomeric sequences
relative to a constant concentration of associated proteins, increased
expression (or stability) of
the associated proteins, or a combination thereof. To alleviate such kinetic
inhibition of EST2
activity, the cells can be treated with an oligonucleotide which competes
(e.g., as a decoy) with
the telomeres for binding of the telomere binding proteins. See, for example,
Wright et al. (1996)
EMBO J 15: 1734. In other embodiments, a dominant negative mutant of a
telomere binding
protein can be introduced into the cell in order to inhibit the formation of
inhibitory protein
complexes with the telomeric sequences. See, for example, Bianchi et al.
(1997) EMBO J
16:1785-94; Broccoli et al. (1997) Hum Mol Genet 6: 69-76; Smith et al. (1997)
Trends Genet
13:21-26; Zhong et al, (1992) Mol. Cell. Biol. 12:4834-4843; Chong et al.
(1995) Science
270:1663-166). In still other embodiments, the agent can be an inhibitor of
expression of a
telomere binding proteins, such as antisense or a small molecule inhibitor of
transcription of the
gene. In yet other embodiments, such agents, particularly small molecules, can
be identified by
their ability to directly inhibit the formation of telomeric complexes
including telomere binding
proteins.
(Cl Exemplar~r Uses of the Subject Method
The present method can be used to increase the proliferative capacity of cells
in vivo, in
vitro and as part of an ex vivo protocol. While the method of the invention is
applicable to any
normal cell type, the method is preferably practiced using normal cells that
express a low level
of telomerase activity. For purposes of the present invention, the term
"normal" refers to cells
other than tumor cells, cancer calls, or transformed cells. An exemplary cell
is an embryonic
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stem cells, such as disclosed in Thomson et al. ( 1998) Science 282:1 I45 and
Shamblott et al.
(1998) PNAS 95:13726. Especially preferred cells for use in the present method
include
embryonic, fetal, neonatal, and adult stem cells of any organ, and adult
pluripotent hematopoietic
stem cells.
In one embodiment, the cells are stem and/or progenitor cells. These include
hematopoietic stem cells, e.g., which are derived from bone marrow, mobilized
peripheral blood
cells, or cord blood. In other embodiments, the cells are progenitor cells for
pancreatic or
hepatic tissue, or other tissue deriving from the primitive gut. In still
other embodiments, the
stem is a neuronal stem cell, such as neural crest which can be used to form
neurons or smooth
muscle cells.
In other embodiments, the cells are not stem or progenitor cells, e.g., they
are committed
cells, such as pancreatic ~i cells, smooth muscle cells (or other myocytic
cells), fibroblasts,
lymphocytic cells, e.g., B or T cells, osteocytes or chondrocytes, to name but
a few.
While the subject method can be used either in vivo or in vitro, the invention
has
1 S particular application to the cultivation of cells ex vivo, and provides
especially important
benefits to therapeutic methods in which cells are cultured ex vivo and then
reintroduced to a
host. For example, the subject method can be used to extend the proliferative
capacity of cells
which are harvested, or otherwise isolated in culture, which are to be
transplanted to a patient.
Such protocols can find use in bone marrow transplants wherein bone marrow, or
isolated
hematopoietic progenitor cells are treated according to the present invention,
with the activation
of telomerase and inactivation of Rb being reverted to the wild-type phenotype
before, or shortly
after, transplantation.
The subject method can also be used to extend T cell life in HIV and Down's
patients.
It also has application in protocols for the formation of artificial tissues
such as prosthetic
devices, e.g., deriving from stem or committed cells. Exemplary tissues
include pancreatic,
hepatic, neural, myocytic, cartilaginous and osseous tissue.
To illustrate, the subject method can be used to enhance the lifespan of a
hematopoietic
cells and hematopoietic stem/progenitor cells. The term "hematopoietic cells"
herein refers to
fully differentiated myeloid cells such as erythrocytes or red blood cells,
megakaryocytes,
monocytes, granulocytes, and eosinophils, as well as fully differentiated
lymphoid cells such as
B lymphocytes and T lymphocytes. Thus, a hematopoietic stem/progenitor cell
includes the
various hematopoietic precursor cells from which these differentiated cells
develop, such as
BFU-E (burst-forming units-erythroid), CFU-E {colony forming unit-erythroid),
CFU-Meg
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(colony forming twit-megakaryocyte), CFU-GM (colony forming unit-granulocyte-
monocyte),
CFU-Eo (colony forming unit-eosinophil), and CFU-GEMM (colony forming unit-
granulocyte-
erythrocyte-megakaryocyte-monocyte).
In another embodiment, the subject method can be use to extend the lifespan of
a
pancreatic cells and pancreatic stem/progenitor cells. The term "pancreatic
progenitor cell"
refers to a cell which can differentiate into a cell of pancreatic lineage,
e.g. a cell which can
produce a hormone or enzyme normally produced by a pancreatic cell. For
instance, a
pancreatic progenitor cell may be caused to differentiate, at least partially,
into a, (3, 8, or ~ islet
cell, or a cell of exocrine fate. The pancreatic progenitor cells of the
invention can also be
cultured prior to administration to a subject under conditions which promote
cell proliferation
and differentiation. These conditions include culturing the cells to allow
proliferation and
confluence in vitro at which time the cells can be made to form pseudo islet-
like aggregates or
clusters and secrete insulin, glucagon, and somatostatin.
The endocrine portion of the pancreas is composed of the islets of Langerhans.
The
islets of Langerhans appear as rounded clusters of cells embedded within the
exocrine pancreas.
Four different types of cells- a, (3, S, and ~-have been identified in the
islets. The a cells
constitute about 20% of the cells found in pancreatic islets and produce the
hormone glucagon.
Glucagon acts on several tissues to make energy available in the intervals
between feeding. In
the liver, glucagon causes breakdown of glycogen and promotes gluconeogenesis
from amino
acid precursors. The 8 cells produce somatostatin which acts in the pancreas
to inhibit glucagon
release and to decrease pancreatic exocrine secretion. The hormone pancreatic
polypeptide is
produced in the ~ cells. This hormone inhibits pancreatic exocrine secretion
of bicarbonate and
enzymes, causes relaxation of the gallbladder, and decreases bile secretion.
The most abundant
cell in the islets, constituting 60-80% of the cells, is the ~3 cell, which
produces insulin. Insulin
is known to cause the storage of excess nutrients arising during and shortly
after feeding. The
major target organs for insulin are the liver, muscle, and fat-organs
specialized for storage of
energy.
in an exemplary embodiment, the subject telomerase-activating therapeutic
agents can be
used to extend the lifespan of implanted pancreatic tissue, e.g., implanted ~i-
islet cells. Recently,
tissue-engineering approaches to treatment have focused on transplanting
pancreatic islets,
usually encapsulated in a membrane to avoid immune rejection. Many methods for
encapsulating
cells are known in the art. For example, a source of ~i islet cells producing
insulin is
encapsulated in implantable hollow fibers. Such fibers can be pre-spun and
subsequently loaded
with the ~i islet cells (Aebischer et al. U.S. Patent No. 4,892,538; Aebischer
et al. U.S. Patent No.
5,106,627; Hoffman et al. (1990) Expt. Neurobiol. 110:39-44; Jaeger et al.
(1990) Pmg. Brain
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Res. 82:41-46; and Aebischer et al. (1991) J. Biomech. Eng. 113:178-183), or
can be co-
extruded with a polymer which acts to form a polymeric coat about the ~i islet
cells (Lim U.S.
Patent No. 4,391,909; Sefton U.S. Patent No. 4,353,888; Sugamori et al. (1989)
Trans. Am.
Artif. Intern. Orsans 35:791-799; Sefton et al. (1987) Biotehnol. Bioena.
29:1135-1143; and
Aebischer et al. (1991) Biomaterials 12:50-55).
In any of the above-embodiments, the pancreatic cells can be treated by the
subject
method ex vivo, andlor treated by the subject method by subsequent delivery of
an therapeutic to
an animal in which the device is implanted. Such cells can be used for
treatment of diabetes
because they have the ability to differentiate into cells of pancreatic
lineage, e.g., ~i islet cells.
The pancreatic cells of the invention can be cultured in vitro under
conditions which can further
induce these cells to differentiate into mature pancreatic cells, or they can
undergo differentiation
in vivo once introduced into a subject.
Moreover, in addition to providing a source of implantable cells, either in
the form of the
progenitor cell population of the differentiated progeny thereof, the subject
method can be used
to extend the life of normal pancreatic cells used to produce cultures for the
production and
purification of secreted factors. For instance, cultured cells can be provided
as a source of
insulin. Likewise, exocrine cultures can be provided as a source for
pancreatin.
In still another embodiment, the subject method can be used to extend the life
span of
hepatic cells and hepatic stem cells. The term "hepatic progenitor cell" as
used herein refers to a
cell which can differentiate in a cell of hepatic lineage, such a Liver
parenchymal cell, e.g., a
hepatocyte. Hepatocytes are some of the most versatile cells in the body.
Hepatocytes have
both endocrine and exocrine functions, and synthesize and accumulate certain
substance,
detoxify others, and secrete others to perform enzymatic, transport, or
hormonal activities. The
main activities of liver cells include bile secretion, regulation of
carbohydrate, lipid, and protein
metabolism, storage of substances important in metabolism, degradation and
secretion of
hormones, and transformation and excretion of drugs and toxins. The subject
method can be
used to facilitate the long term culture of hepatic cells and hepatic
progenitor cells either in vitro
or subsequent to implantation.
In still another embodiment, the subject method can be used to enhance the
life of
"feeder" cell layers for cell co-cultures.
In another embodiment, the subject method can be used to enhance large-scale
cloning,
e.g., of non-human animals, by enhancing the presence of actively dividing
fetal fibroblasts for
nuclear transfer.
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Prior research in nuclear transplantation has shown that the cell cycle stage
of the donor
cell affects the extent of development of the embryo after nuclear transfer.
When the donor cell
is fused to the recipient oocyte, which is arrested in the second metaphase in
meiosis, the nuclear
envelope breaks down and the chromosomes condense until the oocyte is
activated. This
condensation phase has been shown to cause chromosomal defects in donor cells
that are
undergoing DNA synthesis. Donor cells in the G~ phase of the cell cycle
(before DNA
synthesis), however, condense normally and support a high rate of early
development.
Our rationale in selecting an optimal donor cell for nuclear transplantation
was that the
cell should not have ceased dividing (which is the case in Gp) but be actively
dividing, as an
indication of a relatively undifferentiated state and for compatibility with
the rapid cell divisions
that occur during early embryo development. The cells should also be in G~,
either by artificially
arresting the cell cycle or by choosing a cell type that has an inherently
long G1 phase.
The subject methods are also applicable to general cell culture techniques.
For example,
the method can be used to increase the replicadve capacity of hybrids between
immortal and
I S mortal human cells, such as hybrids between human B-lymphocytes and
myeloma cells, e.g., to
increase the replicative capacity of antibody producing human hybridomas.
More generally, the subject method can be used to increase the replicative
capacity of
cells in culture which have been engineered to produce recombinant proteins.
Indeed, the
subject method can permit the use of "normal" cells as the recombinant cell,
so that problems
which may occur with the use of immortal cells (such as differences in post-
translation
modifications) can be avoided, particularly for producing secreted proteins.
In another aspect, the present invention provides pharmaceutical preparations
and
methods for controlling the proliferation of epithelially-derived tissue
utilizing, as an active
ingredient, a telomerase-activating therapeutic agent. The invention also
relates to methods of
controlling proliferation of epithelial-derived tissue by use of the
pharmaceutical preparations of
the invention. To illustrate, a telomerase-activating therapeutic agent of the
present invention
may be used as part of regimens in the treatment of disorders of; or surgical
or cosmetic repair
of, such epithelial tissues as skin and skin organs; corneal, lens and other
ocular tissue; mucosal
membranes; and periodontal epithelium. The methods and compositions disclosed
herein
provide for the treatment or prevention of a variety of damaged epithelial and
mucosal tissues.
For instance, the subject method can be used to control wound healing
processes, as for example
may be desirable in connection with any surgery involving epithelial tissue,
such as from
dermatological or periodontal surgeries. Exemplary surgical repair for which
use of a
telomerase-activating therapeutic agent is a candidate treatment include
severe burn and skin
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regeneration, skin grafts, pressure sores, dermal ulcers, fissures, post
surgery scar reduction, and
ulcerative colitis.
In another aspect of the present invention, telomerase-activating therapeutic
agents can be
used to effect the growth of hair, as for example in the treatment of alopecia
whereby hair
growth is potentiated or otherwise extended.
Still another aspect of the present invention provides a method of extending
the lifetime of
epithelial tissue in tissue culture.
The terms "epithelia", "epithelial" and "epithelium" refer to the cellular
covering of
internal and external body surfaces (cutaneous, mucous and serous), including
the glands and
other structures derived therefrom, e.g., corneal, esophegeal, epidermal, and
hair follicle
epithelial cells. Other exemplary epithlelial tissue includes: olfactory
epithelium, which is the
pseudostratified epithelium lining the olfactory region of the nasal cavity,
and containing the
receptors for the sense of smell; glandular epithelium, which refers to
epithelium composed of
secreting cells; squamous epithelium, which refers to epithelium composed of
flattened plate-like
cells. The term epithelium can also refer to transitional epithelium, which
that characteristically
found lining hollow organs that are subject to great mechanical change due to
contraction and
distention, e.g. tissue which represents a transition between stratified
squamous and columnar
epithelium.
The term "epithelialization" refers to healing by the growth of epithelial
tissue over a
denuded surface.
The term "skin" refers to the outer protective covering of the body,
consisting of the
corium and the epidermis, and is understood to include sweat and sebaceous
glands, as well as
hair follicle structures. Throughout the present application, the adjective
"cutaneous" may be
used, and should be understood to refer generally to attributes of the skin,
as appropriate to the
context in which they are used.
The term "epidermis" refers to the outermost and nonvascular layer of the
skin, derived
from the embryonic ectoderm, varying in thickness from 0.07-1.4 mm. On the
palmar and
plantar surfaces it comprises, from within outward, five layers: basal layer
composed of
columnar cells arranged perpendicularly; prickle-cell or spinous layer
composed of flattened
polyhedral cells with short processes or spines; granular layer composed of
flattened granular
cells; clear layer composed of several layers of clear, transparent cells in
which the nuclei are
indistinct or absent; and horny layer composed of flattened, connified non-
nucleated cells. In the
epidermis of the general body surface, the clear layer is usually absent.
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The "corium" or "dermis" refers to the layer of the skin deep to the
epidermis, consisting
of a dense bed of vascular connective tissue, and containing the nerves and
terminal organs of
sensation. The hair roots, and sebaceous and sweat glands are structures of
the epidermis which
are deeply embedded in the dermis.
The term "hair" refers to a threadlike structure, especially the specialized
epidermal
structure composed of keratin and developing from a papilla sunk in the
corium, produced only
by mammals and characteristic of that group of animals. Also, the aggregate of
such hairs. A
"hair follicle" refers to one of the tubular-invaginations of the epidermis
enclosing the hairs, and
from which the hairs grow; and "hair follicle epithelial cells" refers to
epithelial cells which
surround the dermal papilla in the hair follicle, e.g., stem cells, outer root
sheath cells, matrix
cells, and inner root sheath cells. Such cells may be normal non-malignant
cells, or
transformed/immortalized cells.
"Excisional wounds" include tears, abrasions, cuts, punctures or lacerations
in the
epithelial layer of the skin and may extend into the dermal layer and even
into subcutaneous fat
I S and beyond. Excisional wounds can result from surgical procedures or from
accidental
penetration of the skin.
"Bum wounds" refer to cases where large surface areas of skin have been
removed or lost
from an individual due to heat andlor chemical agents.
"Dermal skin ulcers" refer to lesions on the skin caused by superficial loss
of tissue,
usually with inflammation. Dermal skin ulcers which can be treated by the
method of the present
invention include decubitus ulcers, diabetic ulcers, venous stasis ulcers and
arterial ulcers.
Decubitus wounds refer to chronic ulcers that result from pressure applied to
areas of the skin for
extended periods of time. Wounds of this type are often called bedsores or
pressure sores.
Venous stasis ulcers result from the stagnation of blood or other fluids from
defective veins.
Arterial ulcers refer to necrotic skin in the area around arteries having poor
blood flow.
"Dental tissue" refers to tissue in the mouth which is similar to epithelial
tissue, for
example gum tissue. The method of the present invention is useful for treating
periodontal
disease.
"Internal epithelial tissue" refers to tissue inside the body which has
characteristics
similar to the epidernlal layer in the skin. Examples include the lining of
the intestine. The
method of the present invention is useful for promoting the healing of certain
internal wounds,
for example wounds resulting from surgery.
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A "wound to eye tissue" refers to severe dry eye syndrome, corneal ulcers and
abrasions
and ophthalmic surgical wounds.
The subject method has wide applicability to the treatment or prophylaxis of
disorders
afflicting epithelial tissue, as well as in cosmetic uses. In general, the
method can be
characterized as including a step of contacting a cell, in vitro or in vivo,
with an amount of an
telomerase-activating therapeutic agent agent sufficient to alter the life
span of the treated
epithelial tissue. For in vivo use, the mode of administration and dosage
regimens will vary
depending on the epithelial tissues) which is to be treated. For example,
topical formulations
will be preferred where the treated tissue is epidermal tissue, such as dermal
or mucosal tissues.
A method which "promotes the healing of a wound" results in the wound healing
more
quickly as a result of the treatment than a similar wound heals in the absence
of the treatment.
"Promotion of wound healing" can also mean that the method causes the extends
the
proliferative and growth phase of, inter alia, keratinocytes, or that the
wound heals with less
scarring, less wound contraction, less collagen deposition and more
superficial surface area. In
certain instances, "promotion of wound healing" can also mean that certain
methods of wound
healing have improved success rates, {e.g. the take rates of skin grafts,)
when used together with
the method of the present invention.
Complications are a constant risk with wounds that have not fully healed and
remain
open. Although most wounds heal quickly without treatment, some types of
wounds resist
healing. Wounds which cover large surface areas also remain open for extended
periods of time.
In one embodiment of the present invention, the subject method can be used to
enhance and/or
otherwise accelerate the healing of wounds involving epithelial tissues, such
as resulting from
surgery, burns, inflammation or irritation. The telomerase-activating
therapeutic agent agents of
the present invention can also be applied prophylactically, such as in the
form of a cosmetic
preparation, to enhance tissue regeneration processes, e.g., of the skin, hair
and/or fingernails.
Full and partial thickness burns are an example of a wound type which often
covers large
surface areas and therefore requires prolonged periods of time to heal. As a
result, life-
threatening complications such as infection and loss of bodily fluids often
arise. In addition,
healing in burns is often disorderly, resulting in scarring and disfigurement.
In some cases
wound contraction due to excessive collagen deposition results in reduced
mobility of muscles in
the vicinity of the wound. The compositions and method of the present
invention can be used to
enhance the healing of burns and to promote healing processes that result in
more desirable
cosmetic outcomes and less wound contraction and scarring.
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Severe burns which cover large areas are often treated by skin autografts
taken from
undamaged areas of the patient's body. The subject method can also be used in
conjunction with
skin grafts to impove the grafts performance and life span in culture, as well
as improve the
"take" rates of the graft by accelerating growth of both the grafted skin and
the patient's skin that
is proximal to the graft.
Dermal ulcers are yet another example of wounds that are amenable to treatment
by the
subject method, e.g., to cause healing of the ulcer and/or to prevent the
ulcer from becoming a
chronic wound. For example, one in seven individuals with diabetes develop
dermal ulcers on
their extremities, which are susceptible to infection. Individuals with
infected diabetic ulcers
often require hospitalization, intensive services, expensive antibiotics, and,
in some cases,
amputation. Dermal ulcers, such as those resulting from venous disease (venous
stasis ulcers),
excessive pressure (decubitus ulcers) and arterial ulcers also resist healing.
The prior art
treatments are generally limited to keeping the wound protected, free of
infection and, in some
cases, to restore blood flow by vascular surgery. According to the present
method, the afflicted
area of skin can be treated by a therapy which includes a telomerase-
activating therapeutic agent
agent which promotes epithelization of the wound, e.g., accelerates the rate
of the healing of the
skin ulcers.
In another exemplary embodiment, the subject method is provided for treating
or
preventing gastrointestinal diseases. Briefly, a wide variety of diseases are
associated with
disruption of the gastrointestinal epithelium or villi, including chemotherapy-
and radiation
therapy-induced enteritis (i.e. gut toxicity) and mucositis, peptic ulcer
disease, gastroenteritis
and colitis, villus atrophic disorders, and the like. For example,
chemotherapeutic agents and
radiation therapy used in bone marrow transplantation and cancer therapy
affect rapidly
proliferating cells in both the hematopoietic tissues and small intestine,
leading to severe and
often dose-limiting toxicities. Damage to the small intestine mucosal barrier
results in serious
complications of bleeding and sepsis. The subject method can be used to
promote proliferation
of gastrointenstinal epithelium and thereby increase the tolerated doses for
radiation and
chemotherapy agents. Effective treatment of gastrointestinal diseases may be
determined by
several criteria, including an enteritis score, other tests well known in the
art.
With age, the epidermis thins and the skin appendages atrophy. Hair becomes
sparse and
sebaceous secretions decrease, with consequent susceptibility to dryness,
chapping, and
fissuring. The dermis diminishes with loss of elastic and collagen fibers.
Moreover, keratinocyte
proliferation (which is indicative of skin thickness and skin proliferative
capacity) decreases
with age. An increase, or proiinged rate of keratinocyte proliferation is
believed to conteract
skin aging, i.e., wrinkles, thickness, elasticity and repair. According to the
present invention, a
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telomerase-activating therapeutic agent can be used either therapeutically or
cosmetically to
counteract, at least for a time, the effects of aging on skin.
The subject method can also be used in treatment of a wound to eye tissue.
Generally,
damage to corneal tissue, whether by disease, surgery or injury, may affect
epithelial and/or
S endothelial cells, depending on the nature of the wound. Corneal epithelial
cells are the non-
keratinized epithelial cells lining the external surface of the cornea and
provide a protective
barner against the external environment. Corneal wound healing has been of
concern to both
clinicians and researchers. Opthomologists are frequently confronted with
corneal dystrophies
and problematic injuries that result in persistent and recurrent epithelial
erosion, often leading to
permanent endothelial loss. The use of telomerase-activating therapeutic
agents can be used in
these instances to promote epithelialization of the affected corneal tissue.
To further illustrate,
specific disorders typically associated with epithelial cell damage in the
eye, and for which the
subject method can provide beneficial treatment, include persistent corneal
epithelial defects,
recurrent erosions, neurotrophic corneal ulcers, keratoconjunctivitis sicca,
microbial corneal
1 S ulcers, viral cornea ulcers, and the like. Moreover, superficial wounds
such as scrapes, surface
erosion, inflammation, etc. can cause lose of epithelial cells. According to
the present invention,
the corneal epithelium is contacted with an amount of a telomerase-activating
therapeutic agent
effective to enhance proliferation of the corneal epithelial cells to
appropriately heal the wound.
The maintenance of tissues and organs ex vivo is also highly desirable. Tissue
replacement therapy is well established in the treatment of human disease. For
example, more
than 40,000 corneal transplants were performed in the United States in 1996.
Human epidermal
cells can be grown in vitro and used to populate burn sites and chronic skin
ulcers and other
dermal wounds. The subject method can be used to enhance the life span of
epithelial tissue in
vitro, as well as to enhance the grafting of the cultured epithelial tissue to
an animal host
2S The present method can be used for improving the "take rate" of a skin
graft. Grafts of
epidermal tissue can, if the take rate of the graft is to long, blister and
shear, decreasing the
likelihood that the autograft will "take", i.e. adhere to the wound and form a
basement membrane
with the underlying granulation tissue. Take rates can be increased by the
subject method by
enhancing the proliferation of the keratinocytes. The method of increasing
take rates comprises
contacting the skin autograft with an effective wound healing amount of a
telomerase-activating
therapeutic agent described in the method of promoting wound healing and in
the method of
promoting the growth and proliferation of keratinocytes, as described above.
Skin equivalents have many uses not only as a replacement for human or animal
skin for
skin grafting, but also as test skin for determining the effects of
pharmaceutical substances and
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cosmetics on skin. A major difficulty in pharmacological, chemical and
cosmetic testing is the
difficulties in determining the efficacy and safety of the products on skin.
One advantage of the
skin equivalents of the invention is their use as an indicator of the effects
produced by such
substances through in vitro testing on test skin.
Thus, in one embodiment of the subject method can be used as part of a
protocol for skin
grafting of, e.g., denuded areas, granulating wounds and burns. The use of
telomerase-activating
therapeutic agents can enhance such grafting techniques as split thickness
autografts and
epidermal autografts (cultured autogenic keratinocytes) and epidermal
allografts (cultured
allogenic keratinocytes). In the instance of the allograft, the use of the
subject method to
enhance the formation of skin equivalents in culture helps to provide/maintain
a ready supply of
such grafts (e.g., in tissue banks) so that the patients might be covered in a
single procedure with
a material which allows permanent healing to occur.
In this regard, the present invention also concerns composite living skin
equivalents
comprising an epidermal layer of cultured keratinocyte cells which have been
expanded in the
presence of a telomerase-activating therapeutic agent. The subject method can
be used as part of
a process for the preparation of composite living skin equivalents. In an
illustrative embodiment,
such a method comprises obtaining a skin sample, treating the skin sample
enzymically to
separate the epidermis from the dermis, treating the epidermis enzymically to
release the
keratinocyte cells, culturing, in the presence of a telomerase-activating
therapeutic agent, the
epidermal keratinocytes until confluence, in parallel, or separately, treating
the dermis
enzymatically to release the fibroblast cells, culturing the fibroblasts cells
until sub-confluence,
inoculating a porous, cross-linked collagen sponge membrane with the cultured
fibroblast cells,
incubating the inoculated collagen sponge on its surface to allow the growth
of the fibroblast
cells throughout the collagen sponge, and then inoculating it with cultured
keratinocyte cells, and
fiirther incubating the composite skin equivalent complex in the presence of a
telomerase-
activating therapeutic agent to enhance the life span of the cells.
In other embodiments, skin sheets containing both epithelial and mesenchymal
layers can
be isolated in culture and expanded with culture media supplemented with a
telomerase-
activating therapeutic agent.
Any skin sample amenable to cell culture techniques can be used in accordance
with the
present invention. The skin samples may be autogenic or allogenic.
In another aspect of the invention, the subject method can be used in
conjunction with
various periodontal procedures in which control of epithelial cell
proliferation in and around
periodontal tissue is desired. In one embodiment, proliferative forms of the
hedgehog and ptc
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therapeutics can be used to enhance reepithelialization around natural and
prosthetic teeth, e.g.,
to promote formation of gum tissue.
In yet another aspect, the subject method can be used to help control guided
tissue
regeneration, such as when used in conjunction with bioresorptable materials.
For example,
incorporation of periodontal implants, such as prosthetic teeth, can be
facilitated by the instant
method. Reattachment of a tooth involves both formation of connective tissue
fibers and re-
epithelization of the tooth pocket. The subject method treatment can be used
to enhance tissue
reattachment by controlling the mitotic capacity of basal epithelial cells in
the wound healing
process.
Exemplification
The invention now being generally described, it will be more readily
understood by
reference to the following examples which are included merely for purposes of
illustration of
certain aspects and embodiments of the present invention, and are not intended
to limit the
invention.
Telomere maintenance has been proposed as an essential prerequisite to human
tumor
development. The telomerase enzyme is itself a specific marker for tumor
cells, but the genetic
alterations that activate the enzyme during neoplastic tranformation have
remained a mystery.
Amplification of the myc oncogene is prevalent in a broad spectrum of human
tumors. Here, we
show that myc induces telomerase both in normal human mammary epithelial cells
(HMEC) and
in normal human diploid fibroblasts. Myc increases expression of hEST2
(hEST/TP2), the
catalytic subunit of telomerase. Since hEST2 limits enzyme activity in normal
cells, myc may
control telomerase solely by regulating hEST2 levels. Activation of telomerase
through hEST2
is sufficient to increase average telomere length and extend lifespan in
normal human mammary
epithelial cells. Since myc can also extend the lifespan of these cells,
activation of telomerase
may be one mechanism by which myc contributes to tumor formation.
Telomerase activity is largely absent from somatic cells in vivo and from
normal human
cells in culturel. As these cells proliferate, telomeric repeats are
progressively lost due to the
incomplete replication of chromosome ends during each division cycle 2-5.
Telomere
shortening has been proposed as the mitotic clock that marks the progress of a
cell toward the
end of its replicative life-span. According to this model, erosion of
chromosome ends triggers
cellular senescence 6. Bypass of senescence through negation of tumor
suppressor pathways
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(e.g. p53 and Rb/pl6) allows continued proliferation and further loss of
telomeric sequences 5, 7.
Indefinite proliferation in the absence of telomere maintenance would result
in chromosomal
destabilization due to complete loss of telomeres8. Since this is probably
incompatible with
survival, cells with an indeterminate life span must adopt strategies for
telomere conservation 1,
9~ 10.
Stabilization of telomeric repeats has been proposed as a prerequisite for
tumorigenesis
11. Circumstantial support for this notion comes from the observation that
telomerase is
activated in a high percentage of late-stage human tumors 1 ~ 11, 12. ~e
possibility that
telomere maintenance might be an essential component of the tumorigenic
phenotype led us to
survey known oncogenes for the ability to activate the telomerase enzyme.
Normal human mammary epithelial cells lack telomerase, whereas immortal HMEC-
derivatives and breast tumor cell lines are almost universally telomerase-
positive 13-15.
Introduction into HMEC of HPV-16 E6 protein stimulates telomerase activity,
suggesting that,
in these cells, a single genetic event can potentiate the enzyme 16, 17 (Fig.
3). HMEC were
therefore used for the oncogene survey. Ectopic expression of mdm-2 failed to
induce
telomerase, consistent with the observation that activation of telomerase by
E6 is separable from
the ability of E6 to promote the degradation of p5316 (data not shown).
Several other cellular
and viral oncogenes, including E7, activated ras {V 12) and all cdc25
isoforms, also failed to
induce telomerase (Fig 3, data not shown). However, introduction of a c-myc
expression cassette
resulted in the appearance of telomerase activity in HMEC ( Fig. 3). The
enzyme was detectable
within one passage after transduction of HMEC with a retrovirus that directs
myc expression.
Following drug selection of infected cells, the myc-expressing population
contained levels of
telomerase activity that approximated those seen in a random sample of breast
carcinoma cell
lines (Fig. 3; e. g. T47D).
Introduction of E6 into normal human diploid fibroblasts fails to activate
telomerase 16,
17 (Fig. 4). Similar results were observed following transfer of either
activated ras or a
dominant-negative p53 allele (data not shown). However, telomerase was induced
by
transduction of either IMR-90 (Fig. 4) or WI-38 cells (not shown) with a
retrovirus that directs
myc expression. As with HMEC, activity was apparent immediately after
infection, and
following selection of the myc-expressing population, telomerase reached
levels comparable to
those seen in a telomerase-positive fibrosarcoma cell line, HT1080 (Fig. 4).
A recent report suggests that E6 can activate the myc promoter 18. This
prompted us to
ask whether E6 might regulate telomerase through an effect on myc expression.
In HMEC,
expression of E6 resulted in induction of myc to levels approaching those
achieved upon
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transduction of HMEC with a retrovirus that directs myc expression (Fig. SA).
Surprisingly, E6-
induced alterations in myc protein did not reflect changes in the abundance of
myc mRNA (Fig.
SB), suggesting that control of myc expression by E6 must occur at the post-
transcriptional level.
In contrast, myc levels remained unaltered following expression of E6 in IMR-
90 cells wherein
E6 is incapable of activating telomerase (Fig. SA). This result is consistent
with a model in
which E6 regulates telomerase in HMEC by altering the abundance of myc.
The presence of the mRNA encoding hEST2, the catalytic subunit of telomerase,
strictly
correlates with telomerase activity. The mRNA for hEST2 is undetectable in
normal tissue and
in normal cell lines, whereas hEST2 is present in immortal and tumor-derived
cell lines 19-21.
Moreover, hEST2 expression and telomerase are concomitantly suppressed when
cells are
induced to differentiate 20. As expected, hEST2 mRNA was absent from normal
HMEC.
However, hEST2 could be detected in HMEC cells following transduction with a
myc retrovirus
(Fig. 6A). To determine whether increased expression of hEST2 was sufficient
to account for
activation of telomerase by myc, we infected HMEC and IMR-90 with a retrovirus
that directs
expression of hEST2. Delivery of hEST2 resulted in a clear induction of
telomerase in both cell
types (Fig. 6B). Considered together, our results indicate that myc regulates
telomerase by
controlling the expression of a limiting telomerase subunit. Myc is a
transcription factor that can
enhance the expression of responsive genes. Thus, myc could increase hEST2
expression by
directly stimulating the hEST2 promoter. Alternatively, changes in hEST2
expression could
arise as a secondary consequence of the ability of myc to regulate other
genes.
Telomere length is regulated at two distinct levels. First, preservation of
telomeric
repeats requires either the telomerase enzyme or the activation of an
alternative pathway for
telomere maintenance 1 ~ 9, 10, 14, 22. Second, telomere length can be
controlled by telomere
binding proteins 23. To determine whether activation of telomerase in HMEC
cells is sufficient
to stabilize telomere length, we followed telomeric restriction fragment (TRF)
size as HMEC
were passaged either in the presence or absence of telomerase activity. In
normal HMEC,
telomere length diminished slightly as cells underwent multiple rounds of
division (Fig. 6C).
Activation of telomerase by expression of hEST2 not only prevented telomere
shrinkage but also
increased average TRF length over that observed in early-passage cells (Fig.
6C).
Telomere length has been proposed as the counting mechanism that determines
the
replicative lifespan of a cell. Early-passage, normal HMEC which recieved
either hEST2 or myc
expression cassettes display extended lifespan as compared to vector-
transduced cells (Fig. 6D).
This supports the notion that telomere length is one of the criteria used by a
cell to calculate its
proliferative capacity.
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Here we show that ectopic expression of myc can induce telomerase both in
normal
epithelial cells and in normal fibroblasts and can extend the replicative
lifespan of HMEC. The
myc oncogene is activated by gene amplification and possibly by mutation in a
wide variety of
different tumor types 24, 25_ Since myc can elevate telomerase to a level
approximating that
S observed in tumor cell lines, increased myc activity could account for the
presence of telomerase
in many late-stage tumors. In this regard, a study of 100 neuroblastomas
revealed that ~20%
(16/100) had exceptionally high telomerase activity. Of these, 11 showed
amplification of the
N-myc locus 26. Thus, in this case, telomerase levels correlated well with myc
activation.
Although the myc oncogene may induce telomerase in significant proportion of
tumors, the
enzyme may also be regulated by other pathways 27.
Promotion of cell proliferation and oncogenic transformation by myc probably
requires
induction of a number of different target genes 28. As telomere maintenance
may contribute to
the long-term proliferative potential of.tumor cells, telomerase activation
may be an essential
component of the ability of myc to facilitate tumor formation.
Methods
Retroviral plasmids. The following viral plasmids were used for transfection:
pBabe-puro 29,
MarXII-hygro, mouse c-myc/MarXII-hygro (gifts from Dr. P. Sun, CSHL), E6/pBabe-
puro,
cdc25A/MarXII-hygro. The full length hEST2 cDNA (a gift from Dr. R. Weinberg)
was cloned
into pBabe-puro vector at the EcoRI and SaII sites.
Celt culture and retroviral-mediated gene transfer. Human mammary epithelial
cells
(HMEC 184 spiral K) were obtained from Dr. M. Stampher. Normal human diploid
fibroblasts
(IMR90 and WI38) and human breast cancer cell lines (BT549, T47D and HBL100)
were
obtained from ATCC. HT1080 cells were a gift from G. Stark (Cleveland Clinic
Foundation).
The amphotropic packaging line, linX-A, was produced in our laboratory (L. Y.
X, D. B. and G.
H., unpublished). HMEC were cultured in complete MEGM (Clonetics). Fibroblasts
and LinX-
A cells were maitained in DMEM (GIBCO) plus 10% fetal bovine serum (FBS;
Sigma). BT549,
HBL100 and T47D were maintained as directed by the supplier. LinX-A cells were
transfected
by calcium-phosphate precipitation with a mixture containing 15 pg of
retroviral plasmid and 15
~g of sonicated salmon sperm DNA. Transfected cells were incubated at
37°C for 24 hr and then
shifted to 30°C for virus production. After 48 hr, the virus was
collected, and the virus-
containing medium was filtered to remove packaging cells (0.45 ~,m filter;
Millipore). Target
cells were infected with virus supernatants supplemented with 4 p,g/ml
polybrene (Sigma) by
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centrifuging for 1 hr at 1000 g and then incubating at 30°C overnight.
The infected cells were
selected 48 hours after infection using appropriate drugs (hygromycin, 6418 or
puromycin).
TRAP assays. The TRAP assay was performed essentially as described 1 with some
modification. Briefly, extracts were prepared in lysis buffer (10 mM Tris [pH
7.5], 1 mM
MgCl2, 1 mM EGTA, 10% Glycerol), and cleared by centrifugation for 30 min at
SO,OOOxg.
Lysate corresponding to from 10 to 104 cells was used in the assay. Telomeric
repeats were
synthesized onto an oligonucleotide, TS (5' AATCCGTCGAGCAGAGTT3'), in an
extension
reaction that proceeded at 30°C for 1 hr. Extension products were
amplified by polymerase
chain reaction (PCR) in the presence of'zP-dATP using TS in combination with a
downstream
anchor primer (S' GCGCGGCTAACCCTAACCCTAACC 3'). Five microliters of each
reaction
was analyzed on a 6% acrylamide / 8 M urea gel.
Northern blotting. Total RNA was isolated from subconfluent cultures using
Trizol reagent
(GIBCO BRL). Ten micrograms of total RNA was resolved by electrophoresis and
transferred
to Hybond-N+ membranes according to the manufacturer's instructions. hEST2 was
visualized
following hybridization with a labelled Stu I fragment of hEST2 as described
20,
Western blotting, Western blotting was performed essentially as described 30.
Cells were
washed with cold PBS and lysed in Laemmli loading buffer. Lysates were heated
at 95°C for 10
min. Samples were separated on 8% SDS-PAGE gels and transferred to
nitrocellulose
membranes (Schleicher 8c Schuell). The blots were incubated either with a c-
myc rabbit
polyclonal antibody (N-262; Santa Crutz) or with a TFIIB rabbit polyclonal
antibody (a gift from
Dr. B. Tansey). Immune complexes were visualize by secondary incubation with
I25I-protein A
(ICN).
TRF analysis. Telomeric restriction fragment length was measured precisely as
described
previously22,
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All of the above-cited references and publications are hereby incorporated by
reference.
EcLuivalents
Those skilled in the art will recognize, or be able to ascertain using no more
than routine
experimentation, numerous equivalents to the specific polypeptides, nucleic
acids, methods,
assays and reagents described herein. Such equivalents are considered to be
within the scope of
this invention.
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SEQUENCE LISTING
S 1) GENERAL INFORMATION:
(i) APPLICANT:
(A) NAME: COLD SPRING HARBOR LABORATORY
IO (B) STREET: ONE BUNGTOWN ROAD
(C) CITY: COLD SPRING HARBOR
(D) STATE: NEW YORK
(E) COUNTRY: US
(F) POSTAL CODE: 11724
1S
(ii) TITLE OF INVENTION: EXTENSION OF CELLULAR
LIFESPAN,
METHODS AND REAGENTS
{iii) NUMBER OF SEQUENCES: 2
20
(iv) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC compatible
2S (C) OPERATING SYSTEM: PC-DOS/MS-DOS
{D) SOFTWARE:
{2) INFORMATION FOR SEQ ID NO:1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 4027 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: both
3S (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
4O (ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 57..3452
4S (xi)SEQUENCE ID
DESCRIPTION: N0:1:
SEQ
CAGGCAGCGT GGTCCTGCTG AAGCCCTGGC CCCGGCCACC
CGCACGTGGG CCCGCG
56
ATG CCGCGCGCT CCCCGC TGCCGA GCCGTG CGCTCC CTGCTG CGCACC 104
SO Met ProArgAla ProArg CysArg AlaVal ArgSer LeuLeu ArgSer
1 5 10 15
CAC TACCGCGAG GTGCTG CCGCTG GCCACC TTCGTG CGGCGC CTGGGG 152
His TyrArgGlu ValLeu ProLeu AlaThr PheVal ArgArg LeuGly
SS 20 25 30
CCC CAC GGC TGG CGG CTG GTG CAC CGC GGG CAC CCG GCG GCT TTC CGC 200
Pro Gln Gly Trp Arg Leu Val Gln Arg Gly Asp Pro Ala Ala Phe Arg
40 45
GCG CTG GTG GCC CAC TGC CTG GTG TGC GTG CCC TGG CAC GCA CGG CCG 248
Ala Leu Val Ala Gln Cys Leu Val Cys Val Pro Trp Asp Ala Arg Pro
50 55 60
E)S CCC CCC GCC GCC CCC TCC TTC CGC CAC GTG TCC TGC CTG AAG GAG CTG 296
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Pro Pro Ala Ala Pro Ser Phe Arg Gln Val Ser Cys Leu Lys Glu Leu
65 70 75 BO
GTG GCC CGA GTG CTG CAG AGG CTG TGC GAG CGC GGC GCG AAG AAC GTG 344
$ Val Ala Arg Val Leu Gln Arg Leu Cys Glu Arg Gly Ala Lys Asn Val
85 90 95
CTG GCC TTC GGC TTC GCG CTG CTG GAC GGG GCC CGC GGG GGC CCC CCC 392
Leu Ala Phe Gly Phe Ala Leu Leu Asp Gly Ala Arg Gly Gly Pro Pro
100 105 110
1$
GAG GCC TTC ACC ACC AGC GTG CGC AGC TAC CTG CCC AAC ACG GTG ACC 490
Glu Ala Phe Thr Thr Ser Val Arg Ser Tyr Leu Pro Asn Thr Val Thr
115 120 125
GAC GCA CTG CGG GGG AGC GGG GCG TGG GGG CTG CTG TTG CGC CGC GTG 488
Asp Ala Leu Arg Gly Ser Gly Ala Trp Gly Leu Leu Leu Arg Arg Val
130 135 190
ZO GGC GAC GAC GTG CTG GTT CAC CTG CTG GCA CGC TGC GCG CTC TTT GTG 536
Gly Asp Asp Val Leu Val His Leu Leu Ala Arg Cys Ala Leu Phe Val
145 150 155 160
CTG GTG GCT CCC AGC TGC GCC TAC CAG GTG TGC GGG CCG CCG CTG TAC 589
2$ Leu Val Ala Pro Ser Cys Ala Tyr Gln Val Cys Gly Pro Pro Leu Tyr
165 170 175
CAG CTC GGC GCT GCC ACT CAG GCC CGG CCC CCG CCA CAC GCT AGT GGA 632
Gln Leu Gly Ala Ala Thr Gln Ala Arg Pro Pro Pro His Ala Ser Gly
30 leo les 190
3$
CCC CGA AGG CGT CTG GGA TGC GAA CGG GCC TGG AAC CAT AGC GTC AGG 680
Pro Arg Arg Arg Leu Gly Cys Glu Arg Ala Trp Asn His Ser Val Arg
195 200 205
GAG GCC GGG GTC CCC CTG GGC CTG CCA GCC CCG GGT GCG AGG AGG CGC 728
Glu Ala Gly Val Pro Leu Gly Leu Pro Ala Pro Gly Ala Arg Arg Arg
210 215 220
4O GGG GGC AGT GCC AGC CGA AGT CTG CCG TTG CCC AAG AGG CCC AGG CGT 776
Gly Gly Ser Ala Ser Arg Ser Leu Pro Leu Pro Lys Arg Pro Arg Arg
225 230 235 240
GGC GCT GCC CCT GAG CCG GAG CGG ACG CCC GTT GGG CAG GGG TCC TGG 824
4$ Gly Ala Ala Pro Glu Pro Glu Arg Thr Pro Val Gly Gln Gly Ser Trp
245 250 255
GCC CAC CCG GGC AGG ACG CGT GGA CCG AGT GAC CGT GGT TTC TGT GTG 872
Ala His Pro Gly Arg Thr Arg Gly Pro Ser Asp Arg Gly Phe Cys Val
$0 260 265 270
$$
GTG TCA CCT GCC AGA CCC GCC GAA GAA GCC ACC TCT TTG GAG GGT GCG 920
Val Ser Pro Ala Arg Pro Ala Glu Glu Ala Thr Ser Leu Glu Gly Ala
275 280 285
CTC TCT GGC ACG CGC CAC TCC CAC CCA TCC GTG GGC CGC CAG CAC CAC 968
Leu Ser Gly Thr Arg His Ser His Pro Ser Val Gly Arg Gln His His
290 295 300
60 GCG GGC CCC CCA TCC ACA TCG CGG CCA CCA CGT CCC TGG GAC ACG CCT 1016
Ala Gly Pro Pro Ser Thr Ser Arg Pro Pro Arg Pro Trp Asp Thr Pro
305 310 315 320
TGT CCC CCG GTG TAC GCC GAG ACC AAG CAC TTC CTC TAC TCC TCA GGC 1064
fi$ Cys Pro Pro Val Tyr Ala Glu Thr Lys His Phe Leu Tyr Ser Ser Gly
325 330 335
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GAC AAG GAG CAG CTG CGG CCC TCC TTC CTA CTC AGC TCT CTG AGG CCC 1112
Asp Lys Glu Gln Leu Arg Pro Ser Phe Leu Leu Ser Ser Leu Arg Pro
390 395 350
AGC CTG ACT GGC GCT CGG AGG CTC GTG GAG ACC ATC TTT CTG GGT TCC 1160
Ser Leu Thr Gly Ala Arg Arg Leu Val Glu Thr Ile Phe Leu Gly Ser
355 360 365
lO AGG CCCTGG ATGCCA GGGACTCCC CGCAGGTTG CCCCGC CTGCCCCAG 1208
Arg ProTrp MetPro GlyThrPro ArgArgLeu ProArg LeuProGln
370 375 380
CGC TACTGG CAAATG CGGCCCCTG TTTCTGGAG CTGCTT GGGAACCAC 1256
1$ Arg TyrTrp GlnMet ArgProLeu PheLeuGlu LeuLeu GlyAsnHis
385 390 395 400
GCG CAGTGC CCCTAC GGGGTGCTC CTCAAGACG CACTGC CCGCTGCGA 1304
Ala GlnCys ProTyr GlyValLeu LeuLysThr HisCys ProLeuArg
2O 405 410 415
GCT GCGGTC ACCCCA GCAGCCGGT GTCTGTGCC CGGGAG AAGCCCCAG 1352
Ala AlaVal ThrPro AlaAlaGly ValCysAla ArgGlu LysProGln
420 425 930
2$
GGC TCTGTG GCGGCC CCCGAGGAG GAGGACACA GACCCC CGTCGCCTG 1400
Gly SerVal AlaAla ProGluGlu GluAspThr AspPro ArgArgLeu
435 440 445
3O GTG CAGCTG CTCCGC CAGCACAGC AGCCCCTGG CAGGTG TACGGCTTC 1448
Val GlnLeu LeuArg GlnHisSer SerProTrp GlnVal TyrGlyPhe
450 455 960
GTG CGGGCC TGCCTG CGCCGGCTG GTGCCCCCA GGCCTC TGGGGCTCC 1496
3$ Val ArgAla CysLeu ArgArgLeu ValProPro GlyLeu TrpGlySer
465 470 475 480
AGG CACAAC GAACGC CGCTTCCTC AGGAACACC AAGAAG TTCATCTCC 1594
Arg HisAsn GluArg ArgPheLeu ArgAsnThr LysLys PheIleSer
4O 985 490 495
CTG GGGAAG CATGCC AAGCTCTCG CTGCAGGAG CTGACG TGGAAGATG 1592
Leu GlyLys HisAla LysLeuSer LeuGlnGlu LeuThr TrpLysMet
500 505 510
AGC GTGCGG GGCTGCGCT TGGCTG CGCAGGAGC CCA GTTGGCTGT 1640
GGG
Ser ValArg GlyCysAla TrpLeu ArgArgSer ProGly ValGlyCys
515 520 525
SO GTT CCGGCC GCAGAGCAC CGTCTG CGTGAGGAG ATCCTG GCCAAGTTC 1688
Val ProAla AlaGluHis ArgLeu ArgGluGlu IleLeu AlaLysPhe
530 535 540
CTG CACTGG CTGATGAGT GTGTAC GTCGTCGAG CTGCTC AGGTCTTTC 1736
$$ Leu HisTrp LeuMetSer ValTyr ValValGlu LeuLeu ArgSerPhe
545 550 555 560
TTT TATGTC ACGGAGACC ACGTTT CAAAAGAAC AGGCTC TTTTTCTAC 1784
Phe TyrVal ThrGluThr ThrPhe GlnLysAsn ArgLeu PhePheTyr
6O 565 570 575
CGG AAGAGT GTCTGGAGC AAGTTG CAAAGCATT GGAATC AGACAGCAC 1832
Arg LysSer ValTrpSer LysLeu GlnSerIle GlyIle ArgGlnHis
580 585 590
6$
TTG AAGAGG GTGCAGCTG CGGGAG CTGTCGGAA GCAGAG GTCAGGCAG 1880
CA 02315265 2000-06-14
WO 99I35Z43 PCT/US99/0068Z
4/9
Leu Lys Arg Val Gln Leu Arg Glu Leu Ser Glu Ala Glu Val Arg Gln
s9s 600 605
CAT CGG GAA GCC AGG CCC GCC CTG CTG ACG TCC AGA CTC CGC TTC ATC 1928
His Arg Glu Ala Arg Pro Ala Leu Leu Thr Ser Arg Leu Arg Phe Ile
610 615 620
CCC AAG CCT GAC GGG CTG CGG CCG ATT GTG AAC ATG GAC TAC GTC GTG 1976
Pro Lys Pro Asp Gly Leu Arg Pro Ile Val Asn Met Asp Tyr Val Val
625 630 635 640
GGA GCC AGA ACG TTC CGC AGA GAA AAG AGG GCC GAG CGT CTC ACC TCG 2024
Gly Ala Arg Thr Phe Arg Arg Glu Lys Arg Ala Glu Arg Leu Thr Ser
645 650 655
AGG GTG AAG GCA CTG TTC AGC GTG CTC AAC TAC GAG CGG GCG CGG CGC 2072
Arg Val Lys Ala Leu Phe Ser Val Leu Asn Tyr Glu Arg Ala Arg Arg
660 665 670
ZO CCC GGC CTC CTG GGC GCC TCT GTG CTG GGC CTG GAC GAT ATC CAC AGG 2120
Pro Gly Leu Leu Gly Ala Ser Val Leu Gly Leu Asp Asp Ile His Arg
675 680 685
GCC TGG CGC ACC TTC GTG CTG CGT GTG CGG GCC CAG GAC CCG CCG CCT 2168
Ala Trp Arg Thr Phe Val Leu Arg Val Arg Ala Gln Asp Pro Pro Pro
690 695 700
GAG CTG TAC TTT GTC AAG GTG GAT GTG ACG GGC GCG TAC GAC ACC ATC 2216
Glu Leu Tyr Phe Val Lys Val Asp Val Thr Gly Ala Tyr Asp Thr Ile
705 710 715 720
CCC CAG GAC AGG CTC ACG GAG GTC ATC GCC AGC ATC ATC AAA CCC CAG 2264
Pro Gln Asp Arg Leu Thr Glu Val Ile Ala Ser Ile Ile Lys Pro Gln
725 730 735
AAC ACG TAC TGC GTG CGT CGG TAT GCC GTG GTC CAG AAG GCC GCC CAT 2312
Asn Thr Tyr Cys Val Arg Arg Tyr Ala Val Val Gln Lys Ala Ala His
740 745 750
4O GGG CAC GTC CGC AAG GCC TTC AAG AGC CAC GTC TCT ACC TTG ACA GAC 2360
Gly His Val Arg Lys Ala Phe Lys Ser His Val Ser Thr Leu Thr Asp
755 760 765
CTC CAG CCG TAC ATG CGA CAG TTC GTG GCT CAC CTG CAG GAG ACC AGC 2408
Leu Gln Pro Tyr Met Arg Gln Phe Val Ala His Leu Gln Glu Thr Ser
770 775 780
CCG CTG AGG GAT GCC GTC GTC ATC GAG CAG AGC TCC TCC CTG AAT GAG 2456
Pro Leu Arg Asp Ala Val Val Ile Glu Gln Ser Ser Ser Leu Asn Glu
S0 785 790 795 800
GCC AGC AGT GGC CTC TTC GAC GTC TTC CTA CGC TTC ATG TGC CAC CAC 2504
Ala Ser Ser Gly Leu Phe Asp Val Phe Leu Arg Phe Met Cys His His
805 810 815
GCC GTG CGC ATC AGG GGC AAG TCC TAC GTC CAG TGC CAG GGG ATC CCG 2552
Ala Val Arg Ile Arg Gly Lys Ser Tyr Val Gln Cys Gln Gly Ile Pro
820 825 830
6O CAG GGC TCC ATC CTC TCC ACG CTG CTC TGC AGC CTG TGC TAC GGC GAC 2600
Gln Gly Ser Ile Leu Ser Thr Leu Leu Cys Ser Leu Cys Tyr Gly Asp
835 890 B45
ATG GAG AAC AAG CTG TTT GCG GGG ATT CGG CGG GAC GGG CTG CTC CTG 2648
6$ Met Glu Asn Lys Leu Phe Ala Gly Ile Arg Arg Asp Gly Leu Leu Leu
850 855 860
CA 02315265 2000-06-14
WO 99/35243 PCT/US99/00682
5/9
CGT TTG GTG GAT GAT TTC TTG TTG GTG ACA CCT CAC CTC ACC CAC GCG 2696
Arg Leu Val Asp Asp Phe Leu Leu Val Thr Pro His Leu Thr His Ala
865 870 875 880
AAA ACC TTC CTC AGG ACC CTG GTC CGA GGT GTC CCT GAG TAT GGC TGC 2749
Lys Thr Phe Leu Arg Thr Leu Val Arg Gly Val Pro Glu Tyr Gly Cys
885 B90 895
IO GTG GTG AAC TTG CGG AAG ACA GTG GTG AAC TTC CCT GTA GAA GAC GAG 2792
Val Val Asn Leu Arg Lys Thr Val Val Asn Phe Pro Val Glu Asp Glu
900 905 910
GCC CTG GGT GGC ACG GCT TTT GTT CAG ATG CCG GCC CAC GGC CTA TTC 2840
IS Ala Leu Gly Gly Thr Ala Phe Val Gln Met Pro Ala His Gly Leu Phe
915 920 925
CCC TGG TGC GGC CTG CTG CTG GAT ACC CGG ACC CTG GAG GTG CAG AGC 2888
Pro Trp Cys Gly Leu Leu Leu Asp Thr Arg Thr Leu Glu Val Gln Ser
20 930 935 940
GAC TAC TCC AGC TAT GCC CGG ACC TCC ATC AGA GCC AGT CTC ACC TTC 2936
Asp Tyr Ser Ser Tyr Ala Arg Thr Ser Ile Arg Ala Ser Leu Thr Phe
945 950 955 960
AAC CGC GGC TTC AAG GCT GGG AGG AAC ATG CGT CGC AAA CTC TTT GGG 2989
Asn Arg Gly Phe Lys Ala Gly Arg Asn Met Arg Arg Lys Leu Phe Gly
965 970 975
3O GTC TTG CGG CTG AAG TGT CAC AGC CTG TTT CTG GAT TTG CAG GTG AAC 3032
Val Leu Arg Leu Lys Cys His Ser Leu Phe Leu Asp Leu Gln Val Asn
980 985 990
AGC CTC CAG ACG GTG TGC ACC AAC ATC TAC AAG ATC CTC CTG CTG CAG 3080
3$ Ser Leu Gln Thr Val Cys Thr Asn Ile Tyr Lys Ile Leu Leu Leu Gln
995 1000 1005
GCG TAC AGG TTT CAC GCA TGT GTG CTG CAG CTC CCA TTT CAT CAG CAA 3128
Ala Tyr Arg Phe His Ala Cys Val Leu Gln Leu Pro Phe His Gln Gln
40 logo 1015 1020
GTT TGG AAG AAC CCC ACA TTT TTC CTG CGC GTC ATC TCT GAC ACG GCC 3176
Val Trp Lys Asn Pro Thr Phe Phe Leu Arg Val Ile Ser Asp Thr Ala
1025 1030 1035 1040
TCC CTC TGC TAC TCC ATC CTG AAA GCC AAG AAC GCA GGG ATG TCG CTG 3224
Ser Leu Cys Tyr Ser Ile Leu Lys Ala Lys Asn Ala Gly Met Ser Leu
1095 1050 1055
SO GGG GCC AAG GGC GCC GCC GGC CCT CTG CCC TCC GAG GCC GTG CAG TGG 3272
Gly Ala Lys Gly Ala Ala Gly Pro Leu Pro Ser Glu Ala Val Gln Trp
1060 1065 1070
CTG TGC CAC CAA GCA TTC CTG CTC AAG CTG ACT CGA CAC CGT GTC ACC 3320
SS Leu Cys His Gln Ala Phe Leu Leu Lys Leu Thr Arg His Arg Val Thr
1075 1080 1085
TAC GTG CCA CTC CTG GGG TCA CTC AGG ACA GCC CAG ACG CAG CTG AGT 3368
Tyr Val Pro Leu Leu Gly Ser Leu Arg Thr Ala Gln Thr Gln Leu Ser
60 1090 1095 1100
CGG AAG CTC CCG GGG ACG ACG CTG ACT GCC CTG GAG GCC GCA GCC AAC 3416
Arg Lys Leu Pro Gly Thr Thr Leu Thr Ala Leu Glu Ala Ala Ala Asn
1105 1110 1115 1120
CCG GCA CTG CCC TCA GAC TTC AAG ACC ATC CTG GAC TGATGGCCAC 3462
CA 02315265 2000-06-14
WO 99/35243 PCT/~59910068Z
6/9
Pro Ala Pro Ser Asp
Leu Asp Phe
Lys Thr
Ile Leu
1125 1130
CCGCCCACAG CCAGGCCGAGAGCAGACACCAGCAGCCCTGTCACGCCGGGCTCTACGTCC3522
S
CAGGGAGGGA GGGGCGGCCCACACCCAGGCCCGCACCGCTGGGAGTCTGAGGCCTGAGTG3582
AGTGTTTGGC CGAGGCCTGCATGTCCGGCTGAAGGCTGAGTGTCCGGCTGAGGCCTGAGC3642
IO GAGTGTCCAG CCAAGGGCTGAGTGTCCAGCACACCTGCCGTCTTCACTTCCCCACAGGCT3702
GGCGCTCGGC TCCACCCCAGGGCCAGCTTTTCCTCACCAGGAGCCCGGCTTCCACTCCCC3762
ACATAGGAAT AGTCCATCCCCAGATTCGCCATTGTTCACCCCTCGCCCTGCCCTCCTTTG3822
IS
CCTTCCACCC CCACCATCCAGGTGGAGACCCTGAGAAGGACCCTGGGAGCTCTGGGAATT3882
TGGAGTGACC AAAGGTGTGCCCTGTACACAGGCGAGGACCCTGCACCTGGATGGGGGTCC3992
ZO CTGTGGGTCA AATTGGGGGGAGGTGCTGTGGGAGTAAAATACTGAATATATGAGTTTTTC4002
AGTTTTGAAA 1~,F~~A,F~AAAAAAAAAA 4027
ZS (2) INFORMATION FOR SEQ ID N0:2:
(i) CHARACTERISTICS:
SEQUENCE
(A) LENGTH: 1132 acids
amino
(B) TYPE: mino d
a aci
30 (D) TOPOLOG Y: r
linea
(ii) ULETYPE: n
MOLEC protei
(xi) DESCRIPTION: SEQID N0:2:
SEQUENCE
35
Met ProArgAla ProArg CysArg AlaValArg SerLeuLeu ArgSer
1 5 10 15
His TyrArgGlu ValLeu ProLeu AlaThrPhe ValArgArg LeuGly
20 25 30
Pro GlnGlyTrp ArgLeu ValGln ArgGlyAsp ProAlaAla PheArg
35 40 45
45 Ala LeuValAla GlnCys LeuVal CysValPro TrpAspAla ArgPro
50 55 60
Pro ProAlaAla ProSer PheArg GlnValSer CysLeuLys GluLeu
65 70 75 80
50
Val AlaArgVal LeuGln ArgLeu CysGluArg GlyAlaLys AsnVal
85 90 95
Leu AlaPheGly PheAla LeuLeu AspGlyAla ArgGlyGly ProPro
55 loo l05 llo
Glu AlaPheThr ThrSer ValArg SerTyrLeu ProAsnThr ValThr
115 120 125
60 Asp AlaLeuArg GlySer GlyAla TrpGlyLeu LeuLeuArg ArgVal
130 135 140
Gly AspAspVal LeuVal HisLeu LeuAlaArg CysAlaLeu PheVal
145 150 155 160
65
Leu ValAlaPro SerCys AlaTyr GlnValCys GlyProPro LeuTyr
CA 02315265 2000-06-14
WO 99/35243 PCTIUS99/0068Z
7/9
165 170 175
Gln Leu Gly Ala Ala Thr Gln Ala Arg Pro Pro Pro His Ala Ser Gly
180 185 190
Pro Arg Arg Arg Leu Gly Cys Glu Arg Ala Trp Asn His Ser Val Arg
195 200 205
Glu Ala Gly Val Pro Leu Gly Leu Pro Ala Pro Gly Ala Arg Arg Arg
210 215 220
Gly Gly Ser Ala Ser Arg Ser Leu Pro Leu Pro Lys Arg Pro Arg Arg
225 230 235 240
1$ Gly Ala Ala Pro Glu Pro Glu Arg Thr Pro Val Gly Gln Gly Ser Trp
245 250 255
Ala His Pro Gly Arg Thr Arg Gly Pro Ser Asp Arg Gly Phe Cys Val
260 265 270
Val Ser Pro Ala Arg Pro Ala Glu Glu Ala Thr Ser Leu Glu Gly Ala
275 280 285
Leu Ser Gly Thr Arg His Ser His Pro Ser Val Gly Arg Gln His His
2$ 290 295 300
Ala Gly Pro Pro Ser Thr Ser Arg Pro Pro Arg Pro Trp Asp Thr Pro
305 310 315 320
3fl Cys Pro Pro Val Tyr Ala Glu Thr Lys His Phe Leu Tyr Ser Ser Gly
325 330 335
Asp Lys Glu Gln Leu Arg Pro Ser Phe Leu Leu Ser Ser Leu Arg Pro
390 395 350
Ser Leu Thr Gly Ala Arg Arg Leu Val Glu Thr Ile Phe Leu Gly Ser
355 360 365
Arg Pro Trp Met Pro Gly Thr Pro Arg Arg Leu Pro Arg Leu Pro Gln
370 375 380
Arg Tyr Trp Gln Met Arg Pro Leu Phe Leu Glu Leu Leu Gly Asn His
385 390 395 400
4$ Ala Gln Cys Pro Tyr Gly Val Leu Leu Lys Thr His Cys Pro Leu Arg
405 910 415
Ala Ala Val Thr Pro Ala Ala Gly Val Cys Ala Arg Glu Lys Pro Gln
420 425 430
$0
Gly SerVal AlaAlaPro GluGlu GluAspThr AspPro ArgArgLeu
435 940 445
Val GlnLeu LeuArgGln HisSer SerProTrp GlnVal TyrGlyPhe
$$ 450 455 460
Val ArgAla CysLeuArg ArgLeu ValProPro GlyLeu TrpGlySer
465 470 475 480
60 Arg HisAsn GluArgArg PheLeu ArgAsnThr LysLys PheIleSer
485 990 495
Leu GlyLys HisAlaLys LeuSer LeuGlnGlu LeuThr TrpLysMet
500 505 510
6$
Ser ValArg GlyCysAla TrpLeu ArgArgSer ProGly ValGlyCys
CA 02315265 2000-06-14
WO 99135243
8/9
515 520 525
Val ProAla AlaGlu HisArgLeu ArgGlu GluIleLeu AlaLys Phe
530 535 540
Leu HisTrp LeuMet SerValTyr ValVal GluLeuLeu ArgSer Phe
545 550 555 560
Phe TyrVal ThrGlu ThrThrPhe GlnLys AsnArgLeu PhePhe Tyr
565 570 575
Arg LysSer ValTrp SerLysLeu GlnSer IleGlyIle ArgGln His
580 585 590
1$ Leu LysArg ValGln LeuArgGlu LeuSer GluAlaGlu ValArg Gln
595 600 605
His ArgGlu AlaArg ProAlaLeu LeuThr SerArgLeu ArgPhe Ile
610 615 620
Pro LysPro AspGly LeuArgPro IleVal AsnMetAsp TyrVal Val
625 630 635 640
Gly AlaArg ThrPhe ArgArgGlu LysArg AlaGluArg LeuThr Ser
645 650 655
Arg ValLys AlaLeu PheSerVal LeuAsn TyrGluArg AlaArg Arg
660 665 670
3~ Pro Gly LeuLeu GlyAlaSer ValLeu GlyLeuAsp AspIle HisArg
675 680 685
Ala Trp ArgThr PheValLeu ArgVal ArgAlaGln AspPro ProPro
690 695 700
Glu Leu TyrPhe ValLysVal AspVal ThrGlyAla TyrAsp ThrIle
705 710 715 720
Pro Gln AspArg LeuThrGlu ValIle AlaSerIle IleLys ProGln
725 730 735
Asn Thr TyrCys ValArgArg TyrAla ValValGln LysAla AlaHis
740 745 750
Gly His ValArg LysAlaPhe LysSer HisValSer ThrLeu ThrAsp
755 760 765
Leu Gln ProTyr MetArgGln PheVal AlaHisLeu GlnGlu ThrSer
770 775 780
Pro Leu ArgAsp AlaValVal IleGlu GlnSerSer SerLeu AsnGlu
785 790 795 800
Ala Ser SerGly LeuPheAsp ValPhe LeuArgPhe MetCys HisHis
$5 805 810 815
Ala Val ArgIle ArgGlyLys SerTyr ValGlnCys GlnGly IlePro
B20 825 830
fi0Gln Gly SerIle LeuSerThr LeuLeu CysSerLeu CysTyr GlyAsp
835 890 845
Met Glu AsnLys LeuPheAla GlyIle ArgArgAsp GlyLeu LeuLeu
850 855 860
65
Arg Leu ValAsp AspPheLeu LeuVal ThrProHis LeuThr HisAla
CA 02315265 2000-06-14
WO 99/35243 PCTNS99I00682
9/9
865 870 875 880
Lys Thr Phe Leu Arg Thr Leu Val Arg Gly Val Pro Glu Tyr Gly Cys
885 890 895
Val Val Asn Leu Arg Lys Thr Val Val Asn Phe Pro Val Glu Asp Glu
900 905 910
Ala Leu Gly Gly Thr Ala Phe Val Gln Met Pro Ala His Gly Leu Phe
915 920 925
Pro TrpCys GlyLeu LeuLeuAsp ThrArgThr Leu GluValGln Ser
930 935 940
1$ Asp TyrSer SerTyr AlaArgThr SerIleArg Ala SerLeuThr Phe
945 950 955 960
Asn ArgGly PheLys AlaGlyArg AsnMetArg Arg LysLeuPhe Gly
965 970 975
Val LeuArg LeuLys CysHisSer LeuPheLeu Asp LeuGlnVal Asn
980 985 990
Ser LeuGln ThrVal CysThrAsn IleTyrLys Ile LeuLeuLeu Gln
995 1000 1005
Ala TyrArg PheHis AlaCysVal LeuGlnLeu Pro PheHisGln Gln
1010 1015 1020
Val TrpLys AsnPro ThrPhePhe LeuArgVal Ile SerAspThr Ala
1025 1030 1035 1040
Ser LeuCys TyrSer IleLeuLys AlaLysAsn Ala GlyMetSer Leu
1045 1050 1055
Gly AlaLys GlyAla AlaGlyPro LeuProSer Glu AlaValGln Trp
1060 1065 1070
Leu CysHis GlnAla PheLeuLeu LysLeuThr Arg HisArgVal Thr
1075 1080 1085
Tyr ValPro LeuLeu GlySerLeu ArgThrAla Gln ThrGlnLeu Ser
1090 1095 1100
Arg LysLeu ProGly ThrThrLeu ThrAlaLeu Glu AlaAlaAla Asn
1105 1110 1115 1120
Pro AlaLeu ProSer AspPheLys ThrIleLeu Asp
1125 1130
