Note: Descriptions are shown in the official language in which they were submitted.
CA 02242382 2004-09-17
COMPOSITIONS AND METHODS FOR MEDIAT1NG
CELL CYCLE PROGRESSION
Government Suooort
The U.S. government may have certain rights in the
invention pursuant to Grant No. CA 61352 received from the
U.S. National Institutes of Health.
is Cross-Referepcg. To Re~~~gd An~ali~a ion
This application is a continuation-in-part of
U.S. Patent Serial No. 5,958,769,
Backqround of the Invention
Mammalian cells can shift from a proliferating
state to a quiescent state only during a brief window of the
cell cycle. Temin, J. Cell. PrEys. 78:161 (1971}. Thus,
depending on their position in the cell cycle, cells deprived
of mitogens such as those present in serum will undergo
immediate cell cycle arrest, or they will complete mitosis and
arrest in the next cell cycle. The transition from mitogen-
dependence to mitogen-independence occurs in the mid- to late-
G1 phase of the cell cycle. Pardee, Proc, Natl. Arad. Sci.
71:1286 (1974), showed that many different anti-mitogenic
signals cause the cell cycle to arrest at a kinetically common
point, and further showed that the cell cycle becomes
unresponsive to all of these signals at approximately the same
time in mid- to late-G1. This point was named the restriction
point, or R point.
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2
Time-lapse cinematography of mitotically
proliferating single cells has also been used to precisely map
the timing of the cell cycle transition to mitogen-
independence. This confirmed that mitogen depletion or other '
growth inhibitory signals cause post-mitotic, early-G1 cells
to immediately exit the cell cycle, and that cell cycle
commitment (autonomy from mitogenic signals), occurs in mid-G1
(Larsson et al., J. Cell. Phys. 139:47? (1989), and Zetterberg
et al., Proc. Natl. Acad. Sci. USA 82:5365 (1985)). Together
these observations show that the mitogen-dependent controls on
cell proliferation are linked to cell cycle progression.
Transit through G1 and entry into S phase requires
the action of cyclin-dependent kinases (Cdks) (Sherr, Cell
79:551 (1994)). Growth inhibitory signals have been shown to
prevent activation of these Cdks during G1 (Serrano et al.,
Nature 366:704 (1993); Hannon and Beach, Nature 371: 257
(1994); E1-Deiry et al., Cell 75:89 (1993); Xiong et al.,
Nature 366:701 (1993); Polyak et al., Cell 78:59 (1994);
Toyashima and Hunter, ibid., p. 67; Lee et al., Genes & Dev.
9:639 (1995); Matsuoka et al., ibid., p. 650; Koff et al.,
Science 260:536 (1993)). The catalytic activity of Cdks is
known to be regulated by two general mechanisms, protein
phosphorylation and association with regulatory subunits
(could et al., EMBO J. 10:3297 (3.991); Solomon et al., ibid.,
12:3133 (1993); Solomon et al., Mol. Biol. Cell 3:13 (1992);
Jeffrey et al., Nature 376:313 (1995); Morgan, Nature 374:131
(1995)). Among the regulatory subunits, the association of
Cdks with inhibitory CKI subunits (Cyclin-dependent Kinase
Inhibitors) has been most closely correlated with the effect
of mitogen depletion on cell proliferation and Cdk activity.
The CKI directly implicated in mitogen-dependent
Cdk regulation is p27Kip1 (Polyak et al., Cell 78:59 (1994);
Toyashima and Hunter, ibid., p. 677). The p27 protein
accumulates to high levels in quiescent cells, and is rapidly
destroyed after quiescent cells axe re-stimulated with
specific mitogens (Nourse et al., ure 372:570 (1994); Kato
et al., Cell 79:487 (1994)). Moreover, constitutive
_ ~
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3
expression of p27 in cultured cells causes the cell cycle to
arrest in G1 (Polyak supra, Toyashima and Hunter, supra).
Gene therapy is proposed for treating and
preventing a wide variety of acquired and hereditary diseasea,
such as infectious diseasEas, cancer, etc. and relies on the
. efficient delivery of therapeutic genes to target cells. Most
of the somatic cells that have been targeted for gene therapy,
e.g., hematopoietic cells,, skin fibroblasts and keratinocytes,
hepatocytes, endothelial cells, muscle cells and lymphocytes,
are normally non-dividing.. Retroviral vectors, which are the
most widely used vectors for gene therapy, unfortunately
require cell division for effective transduction (Miller et
al., ~tol. Cell. Biol. 10:4239-4242 (1990)). This is also true
with other gene therapy vectors such as the adeno-associated
vectors (Russell et al., Proc. Natl. Acad. Sci. USA 91: 8915-
8919 (1994); Alexander et al., J. Virol. 68: 8282-8287 (1994);
Srivastrava, Mood Cells Z0: 531-538 (1994)). The majority of
stem cells, a preferred target for many gene therapy
treatments, are normally not proliferating. Thus, the
efficiency of transduction is often relatively low, and the
gene product may not be expressed in therapeutically or
prophylactically effective amounts. This has led
investigators to develop techniques such as pretreatment with
5-fluorouracil, infection in the presence of cytokines, and
extending the vector infection period to increase the
likelihood that stem cells are dividing during infection, but
these have met with limited success.
In one aspect, what is needed in the art is a
method for improving the efficiency of gene transfer that is
useful for a wide variety of gene therapy applications. For
example, what is needed is a means to improve transduction
efficiency into a wide variety of vertebrate cells with
vectors that can transduce only dividing cells by controlling
key molecular events in the cell cycle commitment through the
Restriction point and thus cell cycle progression.
Gene targeting, mediated by homologous
recombination between a targeting polynucleotide construct and
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4
a homologous chromosomal sequence, has been used to disrupt
several genes, including the HPRT gene, f32-microglobulin gene,
int-2 proto-oncogene, and the fos proto-oncogene (Thomas and
Cappechi (1987) Cell 51: 503; Zijl.stra et al. (1989} ature '
X42: 435; Mansour et al. (1988) Nature 336: 348; and Johnson
et al. (1989) Science 245: 1234: Adair et al. (1989) Proc. '
~1atl. Acad. Sci (U S A ) 86:4574; Capecchi, M. (1989)- TIG
,x:70; Capecchi, M. (1989) Science 244:1288}. Mansour et al.
(1988) op.cit. have described homologous targeting constructs
that include a HSV tk gene that permits negative selection
against nonhomologous integration events in conjunction with
positive selection for integrated transgenes.
Transgenic nonhuman mammalian cells and
transgenic nonhuman animals which harbor one or more
inactivated cyclin inhibitor genes required for induction or
inhibition of cell proliferation, such as the cyclin regulator
proteins p27, p16, p14, p18, p21, and the like are desirable
as experimental model systems and as hosts for expression of
transgenes encoding heterologous (e. g., human) cyclin-related
proteins. Such cells and animals also have cell proliferation
advantages which are desired in industry and agriculture, such
as increased cell proliferation, increased animal size, and
increased growth rate. Lonberg (W092/03918) describes
construction of vectors for targeting endogenous
immunoglobulin loci and inactivation of endogenous
immunoglobulin genes with such targeting vectors. Rahemtulla
et al. (1991) ature 353: 180, describes disruption of an
endogenous murine CD4 gene by homologous gene targeting in
embryonic stem cells. Jasin et al. {I990) Genes Devel. 4:
157, report targeting the human CD4 gene in a T lymphoma cell
line by epitope addition. Koh et al. (1992) Science 256:
1210, report disruption of an endogenous murine CD8 gene by
homologous gene targeting in ES cells. Molina et al. (1992)
op.cit., describes disruption of the murine Zck gene, which
encodes a tyrosine kinase implicated in signal transduction by
CD4 and CDB. Grusby et al. (1991) Science 253: 1417,
describes disruption of the MHC Class II Ab beta gene by gene
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targeting in mice; the resultant targeted mice are reported to
be depleted of CD4+ lymphocytes. Nakayama et al. (1993)
Science 261: 1584 report making chimeric knockout mice wherein
some somatic cells of the chimera lack functional bc1-2 genes,
5 and germline transmission of the knockout allele.
~ organisms having a functionally inactivated
endogenous cyclin inhibitor gene (and optionally also
harboring a transgene which expresses a heterologous (i.e.,
derived from a different species) or mutant variant cyclin
inhibitor gene product} would be useful as models for studying
disease pathogenesis and fundamental cell biology, as well as
providing useful models for screening far novel therapeutic
agents to treat diseases related to abnormal cell
proliferation.
Based on the foregoing, it is clear that a
need exists for nonhuman cells and organisms harboring one or
more functionally inactivated endogenous cyclin inhibitor
genes, and optionally also harboring a transgene encoding a
heterologous cyclin inhibitor polypeptide or mutant variant
cyclin inhibitor polypeptide which is expressed in at least a
subset of host cells. Thus, it is an object of the invention
herein to provide targeting transgenes for inactivating, by
homologous recombination, endogenous cyclin inhibitor genes,.
particularly the p27 gene. It is also an object of the
invention to provide methods to produce transgenic nonhuman
cells and transgenic nonhuman animals harboring correctly
targeted homologously recombined transgenes of the invention.
The methods may also be used to inactivate p27 genes and/or
other cyclin inhibitor genes in cells explanted from a patient
(e.g., for ex vivo gene therapy), such as to impart to the
resultant targeted cells an altered cell proliferation
phenotype.
Methods for controlling the expression of
certain plant genes can be used to modify a plant's phenotyX>e
as desired, such as controlling the rate or time at which
fruit ripening occurs or potentially even the growth rate of: a
plant. One way to control. expression of endogenous plant
~l, lii.i I i
CA 02242382 2004-09-17
6
genes is the inhibition-of specific gene expression by
antisense suppression (U. S. Patents 5,457,281, 5,453,566,
5,365,015, 5,254,$00, 5,107,065, and 5,073,676), and an
alternative method to inhibit expression of specific genes is
sense suppression (U.S. Patents 5,283,184, 5,231,020, and
5,034,323).
Summary of the Inve
The present invention provides compositions
which comprise inhibitors of p27 that specifically increase
the proportion of dividing cells to non-dividing cells in a
population of cells. The inhibitors can substantially
decrease or eliminate expression of p27 protein, thereby
penaitting activation of eyclfin Cdk complexes, for example,
cyclin E-Cdk2 and/or cyclin A-Cdk2 complexes. Particularly
useful are oligonucleotide inhibitors of p27, such as triplex
fonaing oligonucleotides, antisense oligonucleotides, and
ribozymes.
In another embodiment the invention also
provides isolated vertebrate cell populations which have been
treated with a p27 inhibitor and have an increased proportion
of dividing cells to non-dividing cells relative to the same
proportion in a population of untreated cells. The dividing
cells, e.g., hematopoietic progenitor cells, are particularly
useful as targets of gene therapy, including the use of viral
vectors that preferentially transduce dividing cells. Thus,
the invention provides a method for increasing the efficiency
of gene therapy techniques by increasing the number of cells
Which can be transduced and thereby increasing the
availability of a desired gene product.
In other embodiments the invention provides
methods for increasing the proportion of dividing cells in a
vertebrate cell population. A population of cells is exposed
to a p27 inhibitor in an amount sufficient to increase the
proportion of dividing cells to non-dividing cells relative to
the same proportion in a population of untreated cells. Such
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a cell population can be a substantially non-dividing or
terminally differentiated primary cell population, including,
e.g., fibroblasts, osteoblasts, myeloblasts, neurons or
epithelial cells. Isolated hematopoietic progenitor cells are
particularly useful in the present methods. The cells can be
exposed to the inhibitor either in vitro or in vivo. When
performed .zn vitro, the method can further comprise the step
of administering the exposed cells to a host, particularly
when the exposed cells have been transduced to express a
desired gene. Thus, the method provides for increasing the
efficiency of transducing a vertebrate cell population with a
viral vector encoding a gene product of interest. The target
cells, e.g., mammalian hematopoietic progenitor cells, are
exposed to a p27 inhibitor in an amount sufficient to increase
the percentage of dividing cells, and contacting the treated
cells to a viral vector encoding the gene product of intereat.
In a broad aspect of the invention is provided
a method for producing hypertrophic organisms (i.e., organi:gyms
of enhanced size, including organisms exhibiting
hypercellularity and/or hyperplasticity) comprising
functionally inactivating expression of at least one cyclin
inhibitor gene (which inc:Ludes CDK inhibitor genes) in the
organism. In a related aspect the invention provides a method
for increasing the growth rate of an organism such that a
desired size is attained snore quickly than as compared to
nonvariant organisms. In one embodiment, the non-human
organism is an animal, such as a nonhuman mammal (e. g., mouse,
rat, sheep, pig, cows, rabbit, and the like), fish (e. g.,
trout, salmon, catfish and the like), birds (e. g., poultry)
etc., or a plant. In an embodiment, the cyclin inhibitor gene
is a mammalian p27 gene. Generally, the method employs
germline transgenes or germline structurally disrupted cyclin
inhibitor gene alleles generated by homologous recombination
with a targeting construct:.
In one aspect of the invention, targeting
constructs are provided which contain at least one portion
having a sequence that is substantially homologous to a
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_.
sequence present in or .flanking a cyciin inhibitor gene locus
(which includes CDK inhibitor gene loci) and which, when
integrated at the corresponding cyclin inhibitor gene locus,
functionally inactivate expression of cyclin inhibitor protein
encoded by the gene locus. Such targeting constructs, or
portions thereof, integrate at the cyclin inhibitor gene locus
by homologous recombination between the endogenous gene locus
and the targeting construct, and cells harboring correctly
integrated targeting constructs are selected for and
identified by screening according to the methods described
herein. Tn one embodiment, the targeting constructs delete
all or a portion of an endogenous cyclin inhibitor gene by a
"hit-and-run" strategy, wherein the resultant functionally
inactivated cyclin inhibitor locus comprises a deletion and
does not comprise an integrated selectable marker. In an
alternative embodiment, an endogenous cyclin inhibitor gene is
functionally inactivated by a targeting construct which
inserts a sequence, typically into a coding sequence (i.e.,
exon), wherein the resultant inactivated cyclin inhibitor gene
2o is substantially incapable of expressing a functional cyclin
inhibitor protein. The invention also provides targeting
constructs which functionally inactivate an endogenous cyclin
inhibitor gene by targeted site-specific point mutation(s),
such as to create a missense or nonsense codon in a coding
sequence or ablate a splice signal or transcriptional element
sequence. In a preferred embodiment of the invention, an
endogenous cyclin inhibitor locus, such as a cyclin inhibitor
locus, e.g., that encoding p27, is functionally inactivated.
The invention also provides targeting
constructs that contain at least one portion having a sequence
that is substantially homologous to a sequence present in or
flanking a cyclin inhibitor gene locus, and which serves as a
template for gene conversion of the corresponding endogenous
cyclin inhibitor gene locus. Such targeted gene conversion
results in the converted (i.e., mutated by gene conversion)
endogenous cyclin inhibitor locus being functionally
inactivated and incapable of directing the efficient
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expression of functional cyclin inhibitor protein. The
invention also provides cells and nonhuman animals and planter
harboring inactivated cycain inhibitor genes that result from
correctly targeted gene conversion. Nucleotide sequences that
result from correctly targeted gene conversion generally area
~ not naturally-occurring sequences in the genome(s) of mamma7_s,
so a sequence resulting f~_om targeted gene conversion is
generally distinguishable from naturally-occurring mutant
cyclin inhibitor alleles in the host cell or host animal
s0 species. A preferable cyc:lin inhibitor gene for functional
disruption by gene conver:aion is a p27 gene.
The.invent.ion also provides targeting
constructs which replace, by homologous recombination, at
least a portion of an endogenous cyclin inhibitor gene with a
corresponding portion of a heterologous cyclin inhibitor gene.
Such replacements may be partial, yielding a hybrid cyclin
inhibitor gene composed partially of~endogenous coding and/or
regulatory sequences and partially of heterologous cyclin
inhibitor gene sequences, or total, wherein the endogenous
cyclin inhibitor gene is replaced by a heterologous cyclin
inhibitor gene. In some embodiments, the heterologous cycli.n
inhibitor gene sequences comprise deletions of nonessential
sequences, such as intronic sequences, and are referred to as
cyclin inhibitor minigenes. For example, the invention
provides a human or murine cyclin inhibitor minigene which can
be transcribed and translated in a nonhuman host to produce a
functional human cyclin inhibitor protein which is
developmentally expressed in the same way as an endogenous
host cyclin inhibitor gene in a naturally occurring, non-
transgenic animal. Such a human cyclin inhibitor minigene may
comprise part of a targeting construct or may be separately
introduced as a transgene.
The invention also provides nonhuman animals
and cells which harbor at least one integrated targeting
' 35 construct that functionally inactivates an endogenous cyclin
inhibitor gene locus, typically by deleting or mutating a
genetic element (e. g., exon sequence, splicing signal,
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promoter, enhancer) that is required for efficient functional
expression of a complete gene product. In one embodiment,
disruption of an endogenous cyclin inhibitor gene locus may be
accomplished by replacement of apportion of the endogenous '
5 cyclin inhibitor gene with a portion of a heterologous cyclin
inhibitor gene (e.g., a human p27 gene sequence) by homologous '
recombination or gene conversion. In an alternative
embodiment, a targeting construct is employed to functionally
disrupt an endogenous cyclin inhibitor gene by homologous
10 recombination, and a transgene encoding and expressing a
heteroiogous molecule is separately introduced into the host
genome at a nonhomologous site.
The invention also provides transgenic
nonhuman animals and plants harboring at least one endogenous
cyclin inhibitor gene that is inactivated by a targeted
genetic modification produced by contacting the endogenous
cyclin inhibitor gene with a targeting construct of the
invention. Such contacting of a targeting construct with an
endogenous cyclin inhibitor sequence generally involves
electroporation, lipofection, microinjection, calcium
phosphate precipitation, biolistics, or other polynucleotide
transfer method known in the art.
The invention also provides cells that express
an endogenous cyclin inhibitor gene, but which have portions
of the expressed endogenous cyclin inhibitor gene deleted or
mutated. For example but not limitation, an endogenous cyclin
inhibitor gene can be modified by deleting specific,
predetermined exons from germline DNA with one or more
targeting constructs, with preferable deletions being those
having boundaries approximately the same as boundaries for
structural and/or functional domains of the cyclin inhibitor
protein. In an alternative embodiment, predetermined exons or
structural domains of an endogenous cyclin inhibitor gene may
be replaced, by homologous targeting, with corresponding
portions of a heterologous cyclin inizibitor gene to generate a
hybrid cyclin inhibitor gene.
_.
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The invention also provides organisms, such as
transgenic nonhuman animals, that have at least one
inactivated endogenous cyclin inhibitor gene, and preferably
are homozygous for inactivated cyclin inhibitor alleles, and
which are substantially ~:ncapable of directing the efficient
expression of endogenous cyclin inhibitor. For example, in a
preferred embodiment, a t:ransgenic nonhuman mammal is
homozygous for inactivated endogenous cyclin inhibitor alleles
and is substantially incapable of producing cyclin inhibitor
encoded by an endogenous (i.e., naturally-occurring) cyclin
inhibitor gene.
The invention also provides vectors, method:,
and compositions useful for suppressing the expression of one
or more species of cyclin. inhibitor gene products, without
1.5 disrupting an endogenous cyclin inhibitor locus. Such methods
are useful for suppressing expression of one or more
endogenous cyclin inhibitor gene products; and in a variation
cari be conditionally controlled by use of an operably-linked
transcriptional regulatory sequence which can conditionally
2o express (e. g., in the presence of an inducer, in a tissue-
specific mariner, in a developmental stage-specific manner, or
the like) the suppression antisense transcript, permitting the
regulated expression (or suppression) of one or more cyclin
inhibitor gene products. Unlike genetic disruption of an
25 endogenous cyclin inhibitor locus, suppression of cyclin
inhibitor gene product ex~~ression does not require the time--
consuming breeding that is needed to establish transgenic
animals homozygous for a disrupted endogenous locus. An
additional advantage of suppression as compared to endogenous
30 cyclin inhibitor gene dis~__°uption is that, in certain
embodiments, suppression is reversible within an individual
animal. For example, cyclin inhibitor suppression may be
accomplished with: (1) transgenes encoding and expressing
antisense RNA that specifically hybridizes to an endogenous
35 cyclin inhibitor gene sequence, (2) antisense oligonucleotides
that specifically hybridize to an endogenous cyclin inhibitor
gene sequence, and (3) intracellular proteins that bind
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12
specifically to an endogenous cyclin inhibitor polypeptide and
inhibit its function.
In an aspect, the invention provides a method
for producing organisms having reduced size and/or cell
number, comprising effecting hyperphysiological expression of
at least one cyclin inhibitor gene. In a related embodiment, -
the invention provides expression transgenes which comprise a
transcriptional regulatory sequence operably linked to a
cyclin inhibitor gene encoding sequence, which can affect
expression of the cyclin inhibitor gene product and retard or
inhibit sell proliferation. Such transgenes, when expressed
in a nonhuman animal, can yield animals having reduced
morphologic characteristics (e. g., smaller body size, reduced
cellularity of organs, atypical body plan dimensions), and
other related cell proliferation phenotypes.
The invention also provides transgenes which
encode a cyclin inhibitor gene product in a nonhuman host
species. Such transgenes typically comprise a cyclin
inhibitor gene expression cassette, wherein a linked promoter
and, preferably, an enhancer drive expression of structural
sequences encoding the cyclin inhibitor protein. For example,
the invention provides transgenes which comprise a
constitutive murine enhancer and promoter linked to structural
sequences that encode a cyclin inhibitor protein. Transgenic
mice harboring such transgenes express cyclin inhibitor in
developmental patterns and at levels which are comparable with
expression patterns and levels of the mouse gene from which
the promoter and enhancer were derived in normal nontransgenic
mice. In one aspect, the polynucleotide sequence encoding the
heterologous cyclin inhibitor molecule is operably linked to
cis-acting transcriptional regulatory regions (e. g., promoter,
enhancer) so that a cyclin inhibitor protein is expressed in a
subset of cells. Transgenes encoding cyclin inhibitor
proteins may be targeted adjacent to endogenous
transcriptions! regulatory sequences, so that the operable
linkage of a regulatory sequence occurs upon integration of
the transgene into.a targeted endogenous chromosomal location.
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In embodiments where it is desired to
overexpress a cyclin inhilbitor gene, at least one cyclin
inhibitor protein may be encoded and expressed from a
transgene(s) in transgenic nonhuman organisms. Such
transgenes may be integrated in a nonhomologous location in a
chromosome of the nonhuman animal, or may be integrated by
homologous recombination or gene conversion into a nonhuman
gene locus.
Brief Description of the Drawings
Fig. 1 shows that p27 is required for cell cycle
withdrawal, where Fig. 1A is a p27 immunoblot analysis of
extracts from control proliferating Balb/c-3T3 cells (Hi),
subconfluent serum starved Balb/c-3T3 cells (Low) and
subconfluent Balb/c-3T3 cells serum starved for 24 h following
lipofection with either p2.7 mismatch (MS) or antisense (AS)
oiigonucleotides. Fig. 1E3 is a p27 immunoblot analysis of
cyclin A, cyclin E or Cdk2 immunoprecipitates from
proliferating Balb/c-3T3 cells (HI), subconfluent serum
starved Balb/c-3T3 cells (Lo) or Balb/c-3T3 cells serum
starved for 24 h following lipofection with either mismatch
(MS) or p27 antisense oiigonucleotides (AS).
Fig. 2 shows that enforced p27 expression reverses
the p27 antisense effect i.n serum starved cells, where Fig. 2A
is a p27 immunoblot analysis of proliferating Balb/c-3T3 cells
24 h after iipofection in the presence (+) or absence (-) of
p27 antisense oligonucleot:ides with plasmid encoding either
wild type p27 or tagged (p27*) p27 wobble mutant. Fig. 2B
3o shows results obtained when proliferating Balb/c-3T3
fibroblasts (Hi) were lipofected with p27 mismatch (MSM) or
antisense (AS) oligonucleotides for 6 h in high serum.
'Fig. 3A shows the mean and 95% confidence
interval of organ weights from 20 control and p27-~- mice at
6-7 weeks of age, and plotted is percent increase in weight in
knockout mice compared with control mice. Fig. 3B shows mean
and 95% confidence interval of weights of 30 control, p27-~-,,
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14
and p27-~- female mice as a function of age, where the inset
shows weights of a separate group of 20 male plus female mice
weighed at birth and at 10 days. Data from 21 days is the
mean of the results from the first group. Fig. 3C depicts the
same as Fig. 3B, but data were obtained from male mice.
DETAILED DESCRIPTION OF THE NVENTION
I. Definitions
Unless defined otherwise, all technical and
scientific terms used herein have the same meaning as commonly
understood by one of ordinary skill in the art to which this
invention belongs. Although any methods and materials similar
or equivalent to those described herein can be used in the
practice or testing of the present invention, the preferred
methods and materials are described. For purposes of the
present invention, the following terms are defined below.
"Cyclin inhibitor protein" as used herein,
refers to a protein which binds to and inactivates a cyciin-
dependent kinase (CDK) or a related protein in the cyclin
pathway in a cell. The p27 protein is an example of a cyclin
inhibitor protein. A cyclin inhibitor gene as used herein is
a polynucleotide sequence which encodes a cyclin inhibitor
protein.
As used herein, the term "cyclin inhibitor
gene" or "cyclin inhibitor gene locus" refers to a region of a
chromosome spanning all of the exons which potentially encode
a cyclin inhibitor polypeptide and extending through flanking
sequences (e. g., including promoters, enhancers, etc.) that
participate in cyclin inhibitor protein expression.
Essentially any gene encoding a cyclin inhibitor protein may
be targeted. A particularly preferred gene is the p27 gene,
which can be targeted, and, if desired, replaced with a
cognate heterologous gene or minigene.
The term "structurally disrupted" as used
herein means that a gene locus comprises at least one mutation
or structural alteration such that the disrupted gene is
incapable of directing the efficient expression of a
_ _ _ _
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functional gene product. The term "functionally inactivated"
means a gene locus that is either not expressed or is
incapable of expressing a gene product. Functional
inactivation may result from structural disruption and/or
5 interruption of expression at either the level of
transcription or translation. Functional inactivation of an
endogenous cyclin inhibitor gene, such as a p27 gene, may also
be produced by other methods, e.g., antisense polynucleotide
gene suppression.
10 The term ~°corresponds to" is used herein to
mean that a polynucleotide sequence that shares identity to
all or a portion of a reference polynucleotide sequence. The
term "complementary to" is used herein to mean that the
sequence is complementary to all or a portion of a reference
15 polynucleotide sequence.
The terms °'substantially corresponds to",
"substantially homologous", or "substantial identity" as used
herein denotes a characteristic of a nucleic acid sequence,
wherein a nucleic acid sequence has at least about 70 percent
sequence identity as compared to a.reference sequence,
typically at least about E35 percent sequence identity, and
preferably at least about 95 percent sequence identity as
compared to a reference sequence. The percentage of sequence
identity is calculated excluding small deletions or additions
which total less than 25 percent of the reference sequence.
The reference sequence may be a subset of a larger sequence,
such as a portion of a gene or flanking sequence, or a
repetitive portion of a chromosome. However, the reference
sequence is at least 18 nucleotides long, typically at least:
about 30 nucleotides long, and preferably at least about 50 to
100 nucleotides long. "Substantially complementary" as used
herein refers to a sequence that is complementary to a
sequence that substantially corresponds to a reference
sequence. In general, targeting efficiency increases with t:he
length of the targeting transgene portion (i.e., homology
region) that is substantially complementary to a reference
sequence present in the target DNA (i.e., crossover target
CA 02242382 2004-09-17
16
sequence). In general,-targeting efficiency is optimized with
the use of isogenic DNA homology clamps, although it is
recognized that the presence of various recombinases may
reduce the degree of sequence identity required for efficient
recombination.
The term "nonhomologous sequence", as used
herein, has both a general and a specific meaning; it refers
generally to a sequence that is not substantially identical to
a specified reference sequence, and, where no particular
reference sequence is explicitly identified, it refers
specifically to a sequence that is not substantially identical
to a sequence of at least about 50 contiguous bases at a
targeted endogenous cyclin inhibitor gene, such as a p27 gene.
Specific hybridization is defined herein as
the formation of hybrids between a targeting transgene
sequence (e.g., a polynucleotide of the invention which may
include substitutions, deletion, and/or additions) and a
specific target DNA sequence (e. g., a p27 gene sequence),
wherein a labeled targeting transgene sequence preferentially
hybridizes to the target such that, for example, a single band
corresponding to a restriction fragment of a genomic cyclin
inhibitor gene can be identified on a Southern blot of DNA
prepared from cells using said labeled targeting transgene
sequence as a probe. It is evident that optimal hybridization
conditions will vary depending upon the sequence composition
and lengths) of the targeting transgene(s) and endogenous
target(s), and the experimental method selected by the
practitioner. Various guidelines may be used to select
appropriate hybridization conditions (fee, Maniatis et al.,
Molecular Cloning: A Laboratory Manual (1989), 2nd Ed., Cold
Spring Harbor, N.Y. and Berger and Kimmel, Methods in
V me C1 in chni
(1987), Academic Press, Inc., San Diego, CA.
The term ''naturally-occurring" as used herein
as applied to an object refers to the fact that an object can
be found in nature. For example, a polypeptide or
CA 02242382 2004-09-17
I7
polynucleotide sequence that is present in an organism
{including viruses) that can be isolated from a source fn
nature and which has not been intentionally modified by man in
the laboratory is naturally-occurring. As used herein,
laboratory strains of rodents which may have been selectively
bred according to classical genetics are considered naturally-
occurring animals.
The term "homologue" as used herein refers to
a gene sequence that is evolutionarily and functionally
related between species.
As used herein, the term "targeting construct"
refers to a polynucleotide which comprises: (1) at least one
homology region having a sequence that is substantially
identical to or substantially complementary to a sequence
present in a host cell endogenous cyclin inhibitor gene locus,
and (2) a targeting region which becomes integrated into an
host cell endogenous cyclin inhibitor gene locus by homologous
recombination between a targeting construct homology region
and said endogenous cyclin inhibitor gene locus sequence. If
the targeting construct is a "hit-and-run" or "in-and-out"
type construct (valancius and Smithies (1991) Mol. Cell. Byol.
,~: i402; Donehower et al. (1992) ~ 356: 215; (1991)
NIH R~ ~: 59) .
the targeting region is only transiently incorporated into the
endogenous cyclin inhibitor gene locus and is eliminated from
the host genome by selection. A targeting region may comprise
a sequence that is substantially homologous to an endogenous
cyclin inhibitor gene sequence and/or may comprise a
nonhomologous sequence, such as a selectable marker (e. g.,
aeo, tk, gpt). The term "targeting construct" does not
necessarily indicate that the polynucleotide comprises a gene
which becomes integrated into the host genome, nor does it.
necessarily indicate that the polynucleotide comprises a
complete structural gene sequence. As used in the art, the
term "targeting construct" is synonymous with the term
"targeting transgene" as used herein.
CA 02242382 1998-07-20
WO 97/26327 PCT/US97/00831
- 1$
The terms "homology region°° and "homology
clamp" as used herein refer to a segment (i.e., a portion) of
a targeting construct having a sequence that substantially
corresponds to, or is substantially complementary to, a
predetermined endogenous cyclin inhibitor gene sequence, which
can include sequences flanking said cyclin inhibitor gene. A
homology region is generally at least about 100 nucleotides
fang, preferably at least about 250 to 500 nucleotides long,
typically at least about 1000 nucleotides long or longer.
Although there is no demonstrated theoretical minimum length
for a homology clamp to mediate homologous recombination, it
is believed that homologous recombination efficiency generally
increases with the length of the homology clamp. Similarly,
the recombination efficiency increases with the degree of
sequence homology between a targeting construct homology
region and the endogenous target sequence, with optimal
recombination efficiency occurring when a homology clamp is
isogenic with the endogenous target sequence. The terms
"homology clamp" and "homology region" are interchangeable as
used herein, and the alternative terminology is offered for
clarity, in view of the inconsistent usage of similar terms in
the art. A homology clamp does nat necessarily connote
formation of a base-paired hybrid structure with an endogenous
sequence. Endogenous cyclin inhibitor gene sequences that
substantially correspond to, or are substantially
complementary to, a transgene homology region are referred to
herein as "crossover target sequences" or "endogenous target
sequences."
As used herein, the term "correctly targeted
construct" refers to a portion of the targeting construct
which is integrated~within or adjacent to an endogenous
crossover target sequence, such as a portion of an endogenous
p27 gene locus. Tt is possible to generate cells having both
a correctly targeted transgene(s) and an incorrectly targeted
transgene(s). Cells and animals having a correctly targeted
transgene(s) and/or an incorrectly targeted transgene(s) may
CA 02242382 2004-09-17
19
be identified and resolved by PCR and/or southern blot
analysis of genomic DNA.
As used herein, the term "targeting region"
refers to a portion of a targeting construct which becomes
integrated into an endogenous chromosomal location following
homologous recombination between a homology clamp and an
endogenous cyclin inhibitor gene, such as a p27 gene sequence.
Typically, a targeting region is flanked on each side by a
homology clamp, such that a double-crossover recombination
between each of the homology clamps and their corresponding
endogenous cyclin inhibitor gene sequences results in
replacement of the portion of the endogenous cyclin inhibitor
gene locus by the targeting region; in such double-crossover
gene replacement targeting constructs the targeting region can
be referred to as a "replacement region". However, some
targeting constructs may employ only a single homology clamp
(e.g., some "hit-and-run"-type vectors, see, Bradley et al.
Bio~ITechnoloav 10: 534 (1992)).
The term "agent" is used herein to denote a
chemical compound, a mixture of chemical compounds, a
biological macromolecule, or an extract made from biological
materials such as bacteria, plants, fungi, or animal
(particularly mammalian) cells or tissues.
The term "cyclin inhibitor knockout phenotype"
refers to a phenotypic characteristic present in cyclin
inhibitor gene -/- animals (e.g., mice homozygous for
functionally inactivated cyclin inhibitor alleles) and absent
in Wild--type animals of the same species, strain, sex, and age
when raised under the same conditions. Examples include those
described herein, for example: hyperpiasia, overall
hypertrophy, hypercellular and other phenotypic
characteristics noted herein.
As used herein, "plant" refers to either a
whole plant, a plant part, a plant cell, or a group of plant
cells. The class of plants which can be used in the method of
the invention is generally as broad as the class of higher
CA 02242382 1998-07-20
WO 97/26327 PCTIUS97/00831
plants amenable to transformation techniques, including both
monocotyledonous and dicotyledonous plants. It includes
plants of a variety of ploidy levels, including polyploid,
diploid and haploid.
5 An "isolated" polynucleotide or polypeptide is
a polynucleotide or polypeptide which is substantially
separated from other contaminants that naturally accompany it,
e.g., protein, lipids, and other polynucleotide sequences.
The term embraces polynucleotide sequences which have been
10 removed or purified from their naturally-occurring environment
or clone library, and include recombinant or cloned DNA
isolates and chemically synthesized analogues or analogues
biologically synthesized by heterologous systems.
15 II. general Methods and Overview
The present invention provides compositions
and methods for increasing the proportion of proliferating
cells in a cell population by exposing the cell population to
an inhibitor of p27 activity. The mediator can be directed to
20 a nucleic acid molecule which encodes the p27 protein, i.e.,
the p27 gene or RNA transcripts thereof, or to the p27 protein
itself, or subunits thereof. The inhibitor is provided to the
cell population under conditions and in an amount sufficient
to permitting progression of the cell cycle in the treated
cells, thereby increasing the percentage of dividing cells in
the cell population relative to an untreated cell population.
Modulating cell cycle regulation may be used
to effect organism size and growth rate. Methods for
modulating cell cycle include modulating the expression or
activity of cyclin inhibitors, the expression or activity of
cyclin activators, the expression of cyclin proteins and
modulation of cyclin degradation, e.g., by regulating the
ubiquitin pathway, e.g., human CDC34. Thus, in one aspect
modulation of p27 affects the growth rate and size of an
organism. In another aspect modulation of cyclin E is
employed to affect organism size or growth rate. It may be
advantageous to combine the modulation of various cell cycle
CA 02242382 2004-09-17
21
regulators as described herein to amplify the effect on the
rate of cell cycle progression and thus organism size or
growth rate. For example, inhibition of p27 can be coupled
with inhibition of other cyclin inhibitors, such as p21, p57,
16, p15, p18, and p19 to achieve increased growth rate and
increased size.
p27 is a cellular protein having a molecular
weight of about 27 kD that inhibits progression of the cell
cycle through the Restriction point in early to mid-G1 phase.
p27 acts by binding to and inhibiting the activation of cyclin
E-Cdk2 and cyclin A-Cdk2 complexes. Characterization of the
p27 protein and cloning and sequencing of the gene encoding
the p27 protein are described in more detail in co-pending PCT
application WO 96/02140.
Inhibitors of p2? are useful in the present
invention to permit the activation of cyclin E-Cdk2 and cyclin
A-Cdk2 complexes and the ensuing progression of the cell cycle
through cell division. Hy maintaining p27 at sufficiently low
levels repetitive cell cycling can be achieved. As the
proportion of dividing cells in a given cell population
increases, among other things the efficiency of transduction
increases for viral vectors encoding desired gene products.
Thus, the inhibitors are useful to overcome obstacles that
have plagued gene therapy efforts. The inhibitors are
particularly useful for increasing the population of dividing
cells among hematopoietic stem cells, which represent a
preferred target cell population for many gene therapy
protocols.
Generally, the nomenclature used hereafter and
the laboratory procedures in cell culture, molecular genetics,
and nucleic acid chemistry and hybridization described below
are those well known and commonly employed in the art.
Standard techniques are used for recombinant nucleic acid
methods, polynucleotide synthesis, cell culture, and transgene
incorporation (e. g., electroporation, microinjection,
lipofectian~. Generally enzymatic reactions, oligonucleotide
i~ ,,~ ~ .
CA 02242382 2004-09-17
22
synthesis, and purification steps are performed according to
the manufacturer's specifications. The techniques and
procedures are generally performed according to conventional
methods in the art and various general references which are
provided throughout this document. The procedures therein are
believed to be well known in the art and are provided for the
convenience of the reader.
Chimeric targeted mice are derived according
to Hogan, et al., ManiDUla~~na the Mouse Embryo~ A Laboratory
Manual, Cold Spring Harbor Laboratory (1988) and
a is
BS~,roach, E.J. Robertson, ed., IRL Press, Washington, D.C.,
(1987).
Embryonic stem cells are manipulated according
to published procedures (Teratocarci~~gas and Embryonic tend
Cel s: A Practical Agproach, E.J. Robertson, ed., IRL Press,
Washington, D.C. (1987); Zjilstra et al., Nature ,x:435-438
(1989); and Schwartzberg et al., ~S,,S.~pnce ~:?99-803 (1989)).
Oligonucleotides can be synthesized on an
Applied Bio Systems oligonucleotide synthesizer according to
specifications provided by the manufacturer.
In general, the invention encompasses methods
and polynucleotide constructs which are employed for
generating nonhuman transgenic organisms having at least one
endogenous cyclin inhibitor gene,, such as p27, functionally
inactivated and, in some embodiments, also harboring at least
one heterologous cyclin inhibitor gene capable of expression.
In addition to being useful in the various applications
described above, such organisms are also useful in screening
for other mediators of cell cycle progression.
III. Gene Taraetina
Gene targeting, which is a method of using,
homologous recombination to modify a mammalian genome, can be
used to introduce changes into cultured cells. Hy targeting a
CA 02242382 2004-09-17
23
gene of interest i» embryonic stem (ES) cells, these changes
can be introduced into the germlines of laboratory animals to
study the effects of the modifications on whole organisms,
among other uses. The gene targeting procedure is
accoiaplished by introducing into tissue culture cells a DNA
targeting construct that has a segment homologous to a target
locus and which also comprises an intended sequence
modification (e.g., insertion, deletion, point mutation). The
treated cells are then screened for accurate targeting to
identify and isolate those which have been properly targeted.
A common scheme to disrupt gene function by gene targeting in
ES cells is to construct a targeting construct which is
designed to undergo a homologous recombination with its
chromosomal counterpart in the ES cell genome. The targeting
constructs are typically arranged so that they insert
additional sequences, such as a positive selection marker,
into coding elements of the target gene, thereby functionally
inactivating it. Targeting constructs usually are insertion-
type or replacement-type constructs (Hasty et al. (1991) Mol.
~g,~~ Biol. ~,: 4509.
IV. Targeting of ~d_$aenous Cvclin,. Inhib,~tor Genes
The invention encompasses methods to produce
nonhuman organisms that have endogenous cyclin inhibitor genes
(i.e., at least one cyclin inhibitor locus) inactivated by
gene targeting with a homologous recombination targeting
construct. Typically, such nonhuman organisms have at least
one functionally inactivated cyclin inhibitor gene.
Typically, a cyclin inhibitor gene sequence is used as a basis
for producing PCR primers that fiank~a region that will be
used as a homology clamp in a targeting construct. The PCR
primers are then used to amplify, by high fidelity FC.R
amplification (Mattila et al. (1991) Nucleic Ac~~~,Aes. .~9:
4967; Eckert, K.A. and Kunkel, T.A. (1991) pcR ~gthods and
Applications ~: 17; U.S. Patent 4,683,202),
a genomic sequence from a
genomic clone library or from a preparation of genamic DNA,
CA 02242382 2004-09-17
24
preferably from the strain of nonhuman animal that is to be
targeted with the targeting construct. The amplified DNA is
then used as a homology clamp and/or targeting region. Thus,
homology clamps for targeting essentially any cyclin inhibitor
gene may be readily produced on the basis of nucleotide
sequence information available in the art and/or by routine
cloning. General principles regarding the construction of
targeting constructs and selection methods are reviewed in
Bradley et al. (1992) Ej.o/Tecbnoloav "1_0: 534.
Targeting constructs can be transferred into
pluripotent stem cells, such as murine embryonal stem cells,
wherein the targeting constructs homologousiy recombine with a
portion of an endogenous cyclin inhibitor gave locus and
create mutations) (i.e., insertions, deletions,
rearrangements, sequence replacements, and/or point mutations)
which prevent the functional expression of the endogenous
cyclin inhibitor gene.
A preferred method of the invention is to
delete, by targeted homologous recombination, essential
structural elements of an endogenous cyclin inhibitor gene.
For example, a targeting construct can homologously recombine
with an endogenous p27 gene and delete a portion spanning
substantially all of one or more of the axons to create an
axon-depleted allele, typically by inserting a replacement
region lacking the corresponding exon(s). Transgenic animals
homozygous for the axon-depleted allele (e.g., by breeding of
heterozygotes to each other) produce cells which are
essentially incapable of expressing a functional endogenous
p27 molecule. Similarly, homologous gene targeting can be
used, if desired, to functionally inactivate a cyclin
inhibitor gene by deleting only a portion of an axon of an
endogenous cyclin inhibitor gene.
Targeting constructs can also be used to
delete essential regulatory elements of a cyclin inhibitor
gene, such as promoters, enhancers, splice sites,
polyadenylation sites, and other regulatory sequences,
.n.nl, . . L irvNd . . n
CA 02242382 2004-09-17
including sequences that occur upstream or downstream of the
cyclin inhibitor structural gene but which participate in
cyclin inhibitor gene expression. Deletion of regulatory
elements is typically accomplished by inserting, by homologous
5 double-crossover recombination, a replacement region lacking
the corresponding regulatory element{s).
.An alternative preferred method of the
invention is to interrupt essential structural and/or
regulatory elements of an endogenous cyclin inhibitor gene by
l0 targeted insertion of a polynucleotide sequence, and thereby
functionally inactivate the endogenous cyclin inhibitor gene.
For example, a targeting construct can homologously recombine
with an endogenous p27 gene and insert a nonhomologous
sequence, such as a neo expression cassette, into a structural
15 element (e. g., an axon) and/or regulatory element (e. g.,
enhancer, promoter, splice site, polyadenylation site) to
yield a targeted p27 allele having an insertional
interruption. The inserted sequence can range in size from
about 1 nucleotide (e. g., to produce a frameshift in an axon
20 sequence) to several kilobases or more, as limited by
efficiency of homologous gene targeting with targeting
constructs having a long nonhomologous replacement region.
Targeting constructs of the invention can also
be employed to replace a portion of an endogenous cyclin
25 inhibitor gene with an exogenous sequence (i.e., a portion of
a targeting transgene); for example, the first axon of a
cyclin inhibitor gene may be replaced with a substantially
identical portion that contains a nonsense or missense
mutation.
V. Targ~etina Constructs
Several gene targeting techniques have been
described, including but not limited to: co-electroporation,
"hit-and-run", single-crossover integration, and double-
crossover recombination {Bradley et al. (I992) B_io_,/Teg~noloov
~,Q: 534. The invention can
be practiced using essentially any applicable homologous gene
CA 02242382 1998-07-20
WO 97/26327 PCT/US97100831
26
targeting strategy known in the art. The configuration of a
targeting construct depends upon the specific targeting
technique chosen. For example, a targeting construct for
single-crossover integration or "hit-and-run" targeting need
only have a single homology clamp linked to the targeting
region, whereas a double-crossover replacement-type targeting
construct requires two homology clamps, one flanking each side
of the replacement region.
For example and not limitation, an embodiment
is a targeting construct comprising, in order: (1) a first
homology clamp having a sequence substantially identical to a
sequence within about 3 kilobases upstream (i.e., in the
direction opposite to the translational reading frame of the
cyclin inhibitor gene axons) of an axon of an endogenous
cyclin inhibitor gene, (2) a replacement region comprising a
positive selection cassette having a pgk promoter driving
transcription of a neo gene, (3) a second homology clamp
having a sequence substantially identical to a sequence within
about 3 kilobases downstream of said axon of said endogenous
cyciin inhibitor gene, and (4) a negative selection cassette,
comprising a HSV tk promoter driving transcription of an HSV
tk gene. Such a targeting construct is suitable for double-
crossover replacement recombination which deletes a portion of
the endogenous cyclin inhibitor locus spanning said axon and
replaces it with the replacement region having the positive
selection cassette. If the deleted axon is essential for
expression of a functional cyclin inhibitor gene product, the
resultant axon-depleted allele is functionally inactivated and
is termed a null allele.
Targeting constructs of the invention comprise
at least one homology clamp linked in polynucleotide linkage
(i.e., by phosphodiester bonds) to a targeting region. A
'homology clamp has a sequence which substantially corresponds
to, or is substantially complementary to, a predetermined
endogenous cyclin inhibitor gene sequence of a nonhuman host
organism, and may comprise sequences flanking the
predetermined cyclin inhibitor gene.
i~ ~,~,~ ,l
CA 02242382 2004-09-17
27
Although no lower or upper size boundaries for
recombinogenic homology clamps for gene targeting have been
conclusively determined iri the art, the best mode far homology
clamps is believed to be in the range between about 50
basepairs and several tens of kilobases. Consequently,
targeting constructs are generally at least about 50 to 100
nucleotides long, preferably at least about 250 to 500
nucleotides long, more preferably at least about 1000 to 2000
nucleotides long, or longer. Construct homology regions
(homology clamps) are generally at least about 50 to 100 bases
tong, preferably at least about 100 to 500 bases long, and
more preferably at least about 750 to 2400 bases long. It is
believed that homology regions of about 7 to 8 kilobases in
length are preferred, with one preferred embodiment having a
first homology region of about 7 kilobases flanking one side
of a replacement region and a second homology region of about
1 kilabase flanking the other side of said replacement region.
The length of homology (i.e., substantial identity) for a
homology region may be selected at the discretion of the
practitioner on the basis of the sequence composition and
complexity of the predetermined endogenous cyclin inhibitor
gene target sequences) and guidance provided in the art
(Hasty et al. (1991) ~Iol. Cell. Biol. 11: 5586; Shulman et al.
(1990) ~!ol. ,C~~1. Biol. 10: 4466) .
The homology region which substantially
corresponds to, or is substantially complementary to, a
predetermined sequence (e.g., an exon sequence, an enhancer, a
promoter, an intronic sequence, or a flanking sequence within
about 3-20 kb of a cyclin inhibitor gene) serves as a template
for homologous pairing and recombination with substantially
identical endogenous cyclin inhibitor gene sequence(s). In
targeting constructs, such homology regions typically flank
the replacement region, which is a region of the targeting
construct that is to undergo replacement with the targeted
endogenous cyclin inhibitor gene sequence (Berinstein et al.
0~7.,~~~,liE,~ o~ . 12: 3fi0 (1992) ) .
Thus, a segment of the targeting construct
CA 02242382 1998-07-20
WO 97/26327 PCTlUS97/00831
28
flanked by homology regions can replace a segment of an
endogenous cyclin inhibitor gene sequence by double-crossover
homologous recombination. Homology regions and targeting
regions are linked together in conventional linear
polynucieotide linkage (5'to 3' phosphodiester backbone).
Targeting constructs are generally double-stranded DNA
molecules, most usually linear.
Without wishing to be bound by any particular
theory of homologous recombination or gene conversion, it is
believed that in such a double-crossover replacement
recombination, a first homologous recombination (e. g., strand
exchange, strand pairing, strand scission, strand ligation)
between a first targeting construct homology region and a
first endogenous cyclin inhibitor gene sequence is accompanied
I5 by a second homologous recombination between a second
targeting construct homology region and a second endogenous
cyclin inhibitor gene sequence, thereby resulting in the
portion of the targeting construct that was located between
the two homology regions replacing the portion of the
endogenous cyclin inhibitor gene that was located between the
first and second endogenous cyclin inhibitor gene sequences.
For this reason, homology regions are generally used in the
same orientation (i.e., the upstream direction is the same for
each homology region of a transgene to avoid rearrangements).
Double-crossover replacement recombination thus can be used to
delete a portion of an endogenous cyclin inhibitor gene and
concomitantly transfer a nonhomologous portion (e.g., a neo
gene expression cassette) into the corresponding chromosomal
location. Double-crossover recombination can also be used to
add a nonhomologous portion into an endogenous cyclin
inhibitor gene without deleting endogenous chromosomal
portions. However, double-crossover recombination can also be
employed simply to delete a portion of an endogenous gene
sequence without transferring a nonhomologous portion into the
endogenous cyclin inhibitor gene (see Jasin et al. (1988)
genes Deyel. 2:1353). Upstream and/or downstream from the
nonhomologous portion may be a gene which provides for
_
CA 02242382 2004-09-17
29
identification of whether a double-crossover homologous
recombination has occurred; such a gene is typically the HSV
tk gene which may be used for negative selection.
The positive selection expression casa~ette
encodes a selectable marker which affords a means for
selecting cells which have integrated targeting transgene
sequences spanning the positive selection expression cassette.
The negative selection expression cassette encodes a
selectable marker which affords a means for selecting cells
which do not have an integrated copy of the negative selection
expression cassette. Thus, by a combination positive-negative
selection protocol, it is possible to select cells that have
undergone homologous replacement recombination and
incorporated the portion of the transgene between the homology
regions (i.e., the replacement region) into a chromosomal
location by selecting for the presence of the positive marker
and for the absence of the negative marker. Selectable
markers typically are also be used for hit-and-run targeting
constructs and selection schemes (Valancius arid Smithies,
on,,cit.).
An expression cassette typically comprises a
promoter which is operational in the targeted host cell (e. g.,
ES cell) linked to a structural sequence that encodes a
protein or polypeptide that confers a selectable phenotype on
the targeted host cell, and a polyadenylativn signal. A
promoter included in an expression cassette may be
constitutive, cell type-specific, stage-specific, and/or
modnlatable (e.g., by hormones such as giucocorticoids; 1~ITV
promoter), but is expressed prior to and/or during selection.
34 An expression cassette can optionally include one or more
enhancers, typically linked upstream of the promoter and
within about 3-10 kilobases. However, when homologous
recombination at the targeted endogenous sites) places the
nvnhomologous sequence downstream of a functional endogenous
promoter, it may be possible for the targeting construct
replacement region to comprise only a structural sequence
encoding the selectable marker, and rely upon the endogenous
CA 02242382 2004-09-17
promoter to drive transcription (Doetschman et al. (1988)
Proc. N~,tl. Ac~pd~.~,,Sci. (U. S ;7~,. ) ~: 8583 ) .
similarly, an endogenous enhancer located near
the targeted endogenous site may be relied on to enhance
5 transcription of transgene sequences in enhancerless transgene
constructs. Preferred expression cassettes of the invention
encode and express a selectable drug resistance marker and/or
a HSV thymidine kinase enzyme. Suitable drug resistance genes
include, for example: gpt (xanthine-guanine
10 phospheribosyltransferase), which can be selected for with
mycophenolic acid; neo (neomycin phosphotransferase), which
can be selected for with 6418 or hygromycin; and DFHR
(dihydrofolate reductase), which can be selected for with
methotrexate (Mulligan and Berg (1981) Proc. N~tl. Acad~c_i._
15 (U.S.g.1 7~: 2072; Southern and Berg (1982) J. Mol. ioel.
~,: 327 ) .
selection for correctly targeted recombinants
will generally employ at least positive selection, wherein a
nonhomologous expression cassette encodes and expresses a
20 functional protein (e.g., nea or gpt) that confers a
selectable phenotype to targeted cells harboring the
endogenously integrated expression cassette, so that, by
addition of a selection agent (e. g., 6418 or mycophenolic
acid) such targeted cells have a growth or survival advantage
25 over cells which do not have an integrated expression
cassette.
It is preferable that selection for correctly
targeted homologous recombinants also employ negative
selection, so that cells bearing only nonhomologous
30 integration of the transgene are selected against. Typically,
such negative selection employs an expression cassette
encoding the herpes simplex virus thymidine kinase gene (HSV
tk) positioned in the transgene so that it should integrate
only by nonhomologous recombination. Such positioning
generally is accomplished by linking the HSV tk expression
cassette (or other negative selection cassette) distal to the
recombinogenic homology regions so that double-crossover
CA 02242382 2004-09-17
3I
replacement recombination of the homology regions transfers
the positive selection expression cassette to a chromosomal
location but does not transfer the HSV tk gene (or other
negative selection cassette) to a chromosomal location. A
8 nucleoside analog, gancyclovir, which is preferentially toxic
to cells expressing HSV tk, can be used as the negative
selection agent, as it selects for cells which do not have an
integrated HSV tk expression cassette. FIAU may also be used
as a selective agent to select for cells lacking HSV tk.
1o In order to reduce the background of cells
having incorrectly integrated targeting construct sequences, a
combination positive-negative selection scheme is typically
used (Mansour et al. (1988) pp,,cit.}.
Positive-negative selection involves the use of
15 two active selection cassettes: (1) a positive one (e.g., the
neo gene), that can be stably expressed following either
random integration or homologous targeting, and (Z) a negative
one (a. g., the HSV tk gene), that can only be stably expressed
following random integration, and cannot be expressed after
20 correctly targeted double-crossover homologous recombination.
By combining both positive and negative selection steps, host
. cells having the correctly targeted homologous recombination
between the transgene and the endogenous cyclin inhibitor gene
can be obtained.
25 Generally, targeting constructs of the
invention preferably include: (1) a positive selection
expression cassette flanked by two'homology regions that are
substantially identical to host cell endogenous cyclin
inhibitor gene sequences, and (2) a distal negative selection
30 expression cassette. However, targeting constructs which
include only a positive selection expression cassette can also
be used. Typically, a targeting construct will contain a
positive selection expression cassette which includes a neo
gene linked downstream (i.e., towards the carboxy-terminus of
35 the encoded polypeptide in translational reading frame
orientation) of a promoter such as the HSV tk promoter or the
pgk promoter. More typically, the targeting transgene will
CA 02242382 1998-07-20
WO 97/26327 PCT/US97/00831
32
also contain a negative-selection expression cassette which
includes an HSV tk gene linked downstream of a HSV tk
promoter.
It is preferred that targeting constructs of -
the invention have homology regions that are highly homologous
to the predetermined target endogenous DNA sequence(s), -
preferably isogenic (i.e., identical sequence). Isogenic or
nearly isogenic sequences may be obtained by genomic cloning
or high-fidelity PCR amplification of genomic DNA from the
strain of nonhuman mammals which are the source of the ES
cells used in the gene targeting procedure. Therefore, both
homology region length and the degree of sequence homology can
only be determined with reference to a particular
predetermined sequence, but homology regions generally must be
at least about 50 nucleotides long and must also substantially
correspond or be substantially complementary to a
predetermined endogenous target sequence. Preferably, a
homology region is at least about 100 nucleotides long and is
identical to or complementary to a predetermined target
sequence in or flanking a cyclin inhibitor gene. If it is
desired that correctly targeted homologous recombinants are
generated at high efficiency, it is preferable that at least
one homology region is isogenic (i.e., has exact sequence
identity with the crossover target sequences) of the
endogenous cyclin inhibitor gene), and is more preferred that
isogenic homology regions flank the exogenous targeting
construct sequence that is to replace the targeted endogenous
cyclin inhibitor sequence.
Generally, any predetermined endogenous cyclin
inhibitor locus can be altered by homologous recombination
(which includes gene conversion) with a targeting transgene
that has at least one homology region which substantially
corresponds to or is substantially complementary to a
predetermined endogenous cyclin inhibitor gene locus sequence
in a mammalian cell having said predetermined endogenous
cyclin inhibitor gene sequence. Typically, a targeting
transgene comprises a portion having a sequence that is not
CA 02242382 1998-07-20
WO 97/26327 PCT/US97/flfl831
33
present in the preselected endogenous targeted cyclin
inhibitor sequences) (i.e., a nonhomologous portion) which
may be as small as a single mismatched nucleotide or may span
- up to about several kilobases or~more of nonhomologous
sequence. Substitutions, additions, and deletions may be as
small as 1 nucleotide or :may range up to~about 2 to to
kilobases or more. Targeting transgenes can be used to
inactivate one or more cyclin inhibitor genes in a cell, such
as in a marine ES cell, a:nd transgenic nonhuman organism
l0 harboring such inactivated genes may be produced.
Once the :specific cyclin inhibitor genes) to
be modified are selected, their sequences will be scanned for
possible disruption sites. Plasmids are engineered to contain
an appropriately sized construct replacement sequence with a
deletion or insertion in the cyclin inhibitor gene of interest
and at least one flanking homology region which substantial7_y
- corresponds or is substantially complementary to an endogenous
target DNA sequence. Typically two flanking homology regions
are used, one on each sides of the~replacement region sequence.
For example, but not to limit the invention, one homology
region may be substantial7~.y identical to a sequence upstream
(i.e., the direction towards the transcription start sites)
of the marine p27 first exon and a second homology region many
be substantially identical. to a sequence downstream of the
marine p27 second exon.
A method of the invention is to transfer a
targeting transgene into a pluripotent stem cell line which
can be used to generate transgenic nonhuman animals following
injection into a host blastocyst. In one embodiment of the
invention is a p27 gene targeting construct containing both
positive (e.g., neo) and, optionally, negative (e.g., HSV tk)
selection expression cassettes. The p27 targeting transgene
" is transferred into mouse ES cells (e. g., by electroporation)
under conditions suitable for the continued viability of the
' 35 electroporated ES cells. The electroporated ES cells are .
cultured under selective conditions for positive selection
(e.g., a selective concentration of G418), and optionally are
I . .alnl. . . L ~~~.~. ~i l . n
CA 02242382 2004-09-17
34
cultured under selective conditions for negative selection
(e. g., a selective concentration of gancyclovir or FIAU),
either simultaneously or sequentially. Selected cells are
then verified as having the correctly targeted transgene
recombination by PCR analysis according to standard PCR or
Southern blotting methods known in the art (U. S. Patent
4,683,202; Erlich et al., c a 252: 1643 (1991)).
Correctly targeted ES
cells are then transferred into suitable blastocyst hosts for
generation of chimeric transgenic animals according to methods
known in the art (Capecchi, M. (1989) OD.~it.).
Briefly, the invention involves regulation of
cell cycle, for example the inactivation of a cyclin inhibitor
gene, usually a p27 gene. Within one example a DNA construct
that contains an altered, copy of a mouse cyclin inhibitor
gene (e.g., a p27 gene) is introduced into the nuclei of
embryonic stem cells. In a portion of the cells, the
introduced DNA recombines with the endogenous copy of the
mouse gene, replacing it with the altered copy. Cells
containing the newly engineered genetic lesion are injected
into a host mouse embryo, which is refmplanted into a
recipient female. Some of these embryos develop into chimeric
mice that possess germ cells derived from the mutant cell
line. Therefore, by breeding the chimeric mice it is possible
to obtain a new line of mice containing the introduced genetic
lesion (reviewed by Capecchi, M. (1989) _op.c t.).
In one example, to disrupt the marine p27rip1
gene, a targeting construct based on the design employed by
Jaenisch and co-workers (Zjilstra, et al. (1989) op.cit.) for
the successful disruption of the mouse ~2-microglobulin gene
can be ussd. The neomycin resistance gene (neo), from the
plasmid pMCiNEO is inserted into the coding region of the
target bcI-2 gene. The pMCiNEO insert uses a hybrid viral
promoter/enhancer sequence to drive neo expression. This
promoter is active in embryonic stem cells. Therefore, neo
can be used as a selectable marker for integration of the
..e.". i~ ",~ a
CA 02242382 2004-09-17
knack-out construct. 'hhe ASV thymidine kinase (tk) gene is
added to the end of the construct as a negative selection
marker against random insertion events (Zjilstra, et al.,
cD~ci~.)
5 Vectors containing a targeting construct are
typically grown in E. coli and then isolated using standard
molecular biology methods, or may be synthesized as
oligonucleotides. Direct targeted inactivation which does not
require prokaryotic. or eukaryotic vectors may also be done.
10 Targeting transgenes can be transferred to host cells by any
suitable technique, including microinjection, electroporation,
lipofection, biolistics, calcium phosphate precipitation, and
viral-based vectors, amo»g others. Other methods used to
transform mammalian cells include the use of Polybrene,
15 protoplast fusion, and others (see, gp~~erallv, Sambrook et al.
Molecular Cloning: A Laboratory Manual, 2d ed., 1989, Cold
Spring Harbor Laboratory press, Coid Spring harbor, N.Y.).
It is preferable to use a transfection
20 technique with linearized transgenes containing only modified
target gene sequences) and without vector sequences. The
modified gene site is such that a homologous recombinant
between the exogenous targeting construct and the endogenous
DNA target sequence can be identified by using carefully
25 chosen primers and PGR or by Southern blot analysis, followed
by analysis to detect if PCR products or Southern blot bands
specific to the desired targeted event are present (Erlich et
al., (1991) oD.cit.).
Several studies have already used PCR to
30 successfully identify the desired transfected cell lines
(Zimmer and Gruss (1989) a ~: 150; Mouel lic et al.
(1990) proc. Natl. Acad. Sci. fU.S.A.~ 87: 4712; Shesely et
al. (1991) P~oc. Natl. Aced, Sci. USA ~: 4294).
This approach is very
35 effective when the number of cells receiving exogenous
targeting transgene(s) is high (i.e., with electroporation or
with liposomes) and the treated cell populations are allowed
CA 02242382 2004-09-17
3s
to expand (Capacchi, M: (1989) o .~).
For making transgenic non-human organisms
(which include hamologously targeted non-human animals),
embryonal stem cells (ES cells} are preferred. Murine ES
cells, such as AH-1 line grown on mitotically inactive SNL7s/7
cell feeder layers (McMahon and Bradley, Cell øx,:1073-1085
(1990)) essentially as described (Robertson, E.J. (1987) in
Te~atocarcinomas aid ~brvon~,.c Stem Cells: A Practical
~,. E.J. Robertson, ed. (Oxford: IRL Press}, p. 71-112)
may be used for homologous gene targeting. Other suitable ES
lines include, but are not limited to, the E14 line (Hooper et
al. (1987) Nature ,~: 292-295), the D3 line (Doetschman et
al. (1985) ,~;'Embrvol. E~q~. Mornh. 87: 27-45), and the CCE
line (Robertson et al. (1985) j~ to ur_e_ ~: 445-448). The
success of generating a mouse line from ES cells bearing a
specific targeted mutation depends on the pluripotence of the
ES cells (i.e., their ability, once injected into a host
blastocyst, to participate in embryogenesis and contribute to
the germ cells of the resulting animal), The blastocysts
containing the injected ES cells are allowed to develop in the
uteri of pseudopregnant nonhuman females and are born as
chimerie mice. The resultant transgenic mice are.chimeric for
cells having inactivated endogenous cyclin inhibitor loci and
are backcrossed and screened for the presence of the correctly
targeted transgene(s) by PCR yr Southern blot analysis on tail
biopsy DNA of offspring so as to identify transgenic mice
heterozygous for the inactivated cyclin inhibitor locus/loci.
By performing the appropriate crosses, it is possible to
produce a transgenic nonhuman animal homozygous for multiple
functionally inactivated cyclin inhibitor loci, and optionally
also for a transgene encoding a heterologous cyclin inhibitor
protein. Such transgenic animals are substantially incapable
of making an endogenous cyclin inhibitor gene product. For
these reasons, such transgenic animals are satisfactory hosts
for introduction of transgenes encoding heterologous cyclin
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37
inhibitor proteins, such as, for example, a transgene encoding
human p27 integrated into a mouse genome.
Inactivation of an endogenous mouse cyclin
inhibitor locus is achieved by targeted disruption of the
appropriate gene by homologous recombination in mouse
embryonic stem cells. For inactivation, any targeting
construct that produces a genetic alteration in the target
cyclin inhibitor gene locus resulting in the prevention of
effective expression of a functional gene product of that
locus may be employed. If only regulatory elements are
targeted, some low-level expression of the targeted gene may
occur (i.e., the targeted allele is '°leaky'~), however the
level of expression may be sufficiently low that the leaky
targeted allele is functionally inactivated.
VI. Knockout Animals
In one embodiment of the invention, an
endogenous cyclin inhibitor gene in a nonhuman host is
functionally inactivated by homologous recombination with a
targeting construct that does not comprise a heterologous
cyclin inhibitor gene segment. In this embodiment, a portion
of the targeting construct integrates into an essential
structural or regulatory element of the endogenous cyclin
inhibitor gene locus, thereby functionally inactivating it to
generate a null allele. Typically, null alleles are produced
by integrating a nonhomolagous sequence encoding a selectable
marker (e.g.; a neo gene expression cassette) into an
essential structural and/or regulatory sequence of a cyclin
inhibitor gene by homologous recombination of the targeting
construct homology clamps with endogenous cyclin inhibitor
gene sequences, although other strategies (see, infra) may be
employed.
Most usually, a targeting construct is
transferred by electroporation or microinjection into a
totipotent embryonal stem (ES) cell line, such as the murine
AB-1 or CCE lines. The targeting construct homologously
recombines with endogenous sequences in or flanking a cyclin
CA 02242382 2004-09-17
38
inhibitor gene locus and functionally inactivates at least one
allele of the cyclin inhibitor gene. Typically, homologous
recombination of the targeting construct with endogenous
cyclin inhibitor locus sequences-results in integration of a
nonhomologous sequence encoding and expressing a selectable
marker, such as neo, usually in the form of a positive
selection cassette (). The functionally inactivated
allele is termed a cyclin inhibitor null~allele. ES cells
having at least one cyclin inhibitor null allele are selected
far by propagating the cells in a medium that permits the
preferential propagation of cells expressing the selectable
marker. Selected ES cells are examined by PCR analysis and/or
Southern blot analysis to verify the presence of a correctly
targeted cyclin inhibitor allele. Breeding of nonhuman
animals which are heterozygous for a null allele may be
performed to produce nonhuman animals homozygous for said null
allele, so-called "knockouts animals (Donehower et al. (1992)
Nature 256: 215; science ,~: 1392).
Alternatively, ES cells homozygous for a null
allele having an integrated selectable marker can be produced
in culture by selection in a medium containing high levels of
the selection agent (e. g., 6418 or hygromycin).
Heterozygosity and/or homozygosity for a correctly targeted
null allele can be verified with PCR analysis and/or Southern
blot analysis of DNA isolated from an aliquot of a selected ES
cell clone and/or from tail biopsies.
If desired, a transgene encoding a
heteralogous cyclin inhibitor protein can be transferred into
a nonhuman host having a cyclin inhibitor null allele,
pr8ferably into a nonhuman E5 cell that is homozygous for the
null allele. It is generally advantageous that the transgene
comprises a promoter and enhancer which drive expression of
structural sequences enevding a functional heterologous cyclin
inhibitor gene product. Thus, for example and not limitation,
a knockout mouse homozygous for null alleles at the p27xipi
locus is preferably a host for a transgene which encodes and
expresses a functional human p27 protein.
CA 02242382 1998-07-20
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39
Nonhuma~ animals comprising germline copies of
a functionally inactivated cyclin inhibitor gene, such as a
structurally disrupted p27 gene, are produced. Preferably the
knockout animals are homozygous for the functionally
inactivated cyclin inhibitor gene.
The knockout organisms can be used with
methods for identifying agents that are cyclin inhibitor gene
product mimetics (i.e., have CDK inhibition activity; can
replace gene function in a cyclin inhibitor gene knockout
background) or cyclin inhibitor agonists (i.e., enhance
function of endogenous p2'7 in a hemizygote) or cyclin
inhibitor antagonists (i.e., inhibit residual p27 function :in
a hemizygote). Transgenic nonhuman animals lacking functional
cyclin inhibitor alleles define whole-animal cyclin inhibitor
knockout phenotypes. Agents that can reverse a whole-anima:L
cyclin inhibitor knockout phenotype (i.e., induce a reversion
to phenotypic characteristics of normal, non-knockout animals)
when administered to a cyclin inhibitor knockout animal are
. identified as cyclin inhibitor mimetics. Agents that can
reverse a whole-animal eyt:1in inhibitor knockout phenotype
(i.e., induce a reversion to phenotypic characteristics of
normal, non-knockout animals) when administered to an animal
which comprises reduced cyclin inhibitor function (e. g.,
partial cyclin inhibitor knockout or reduced cyclin inhibitor
expression animals; such as by hemizygosity or partial
antisense suppression) are identified as cyclin inhibitor
agonists. Agents that can induce a whole-animal cyclin
inhibitor knockout phenotype (i.e., induce phenotypic
characteristics of cyclin inhibitor-knockout animals) when
administered to normal, non-knockout animals are identified as
cyclin inhibitor antagonists. These types of agents can be
used to control cell proliferation for morphologic growth
' regulation to control animal size and body characteristics, to
treat or prevent diseases of abnormal cell proliferation
(e.g., neoplasia, hyperplasia, inflammation, and the like)., as
commercial laboratory reagents which can be sold to the
biotechnology industry and research institutions (akin to
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patented restriction endonucleases, PCR reagents, and the
like), among other uses related to the control of cell
proliferation.
Nonhuman animals comprising knockout alleles
5 of cyclin inhibitor genes, such as p27, can be used
commercially for toxicological evaluation of test agents on
the knockout animals which represent animals compromised in
cell proliferation control pathways. The cyclin inhibitor
gene knockouts of the present invention result in animals that
10 have enhanced cell proliferation and can be predisposed to
developing cell proliferation control diseases as compared to
normal (non-knockout) animals. Such gene knockout animals
have many uses, including but not limited to identifying
compounds that effect or affect cell proliferation control; in
15 one variation, the agents are thereby identified as
toxicological hazards. The knockout animals can also be used
to develop agents that modulate cell proliferation; such
agents can serve as therapeutic agents to treat cell
proliferation-related diseases, such as neoplasia or
20 hyperplasia (e.g., BPIi). The knockout animals of the
invention can also serve as disease models for investigating
cell proliferation-related pathological conditions (e. g., ALS,
Alzheimer's disease, AIDS, and the like).
25 VII. Supt~ressina Expression of Endogenous Cvclin
~nhib~.tor Loci
Suppression is an alternative method for
preventing the expression of an endogenous cyclin inhibitor
locus. Suppression of endogenous cyclin inhibitor genes may
30 be accomplished with antisense RNA produced from one or more
integrated transgenes, by antisense oligonucleotides, and/or
by expression of intracellular polypeptides which inactivate
the cyclin inhibitor gene product.
35 VIII. Antisense Polvnucleotides
Antisense RNA transgenes can be employed to
partially or totally knock-out expression of specific genes
(Pepin et al. (1991) Nature 355: 725; Helene., C. and Toulme,
.al.vl. . . In ~t.n d l ~ n
CA 02242382 2004-09-17
41
J. (1990) ~iochimica H~,S,iDhys. Ar,~a 1049: 99; Stout, J. and
Caskey, T. (1990) Som . Cell l~to~. Gengt. ,~6: 369; Munir et
al. (1990) Somat. ,dell Mol.. enet. ,l,ø: 383) .
"Antisense polynucleotides" are
polynucleotides that: (1) are complementary to all or part of
a reference sequence, such as a sequence of an endogenous
cyclin inhibitor gene region, and (2) which specifically
hybridize to a complementary target sequence, such as a
chromosomal gene locus or a mRNA. Such complementary
antisense polynucleotides may include nucleotide
substitutions, additions, deletions, or transpositions, so
long as specific hybridizatian to the relevant target sequence
is retained as a functional property of the polynucleotide.
Complementary antisense polynucleotides include soluble
antisense RNA or DNA oligonucleotides which can hybridize
specifically to individual mRNA species and prevent
transcription and/or RNA processing of the mRNA species and/or
translation of the encoded polypeptide (Ching et al., grx.
Natl. Acad. Sci, UiS.A. x:10006-10010 (1989); 8roder et al.,
j~,. Int. Med. x:604-618 (1~990j; Loresu et al., FEHS etters
x:53-56 (1990); Holcenberg et al., W091/11535; U.S. Patent
5,256,643 ("New human CRIPTO gene"); W091/09865; W091/04753;
W090/13641; and EP 386563).
An antisense sequence is s
polynucleotide sequence that is complementary to at least one
cyclin inhibitor gene sequence of at least about 1i contiguous
nucleotides in length, typically at least 2o to 30 nucleotides
in length, and preferably more than about 30 nucleotides in
length. However, in some embodiments, antisense sequences aay
have substitutions, additions, or deletions as compared to the
complementary cyciin inhibitor gene sequence, so long as
specific hybridization is retained as a property of the
antisense polynucleotide. Generally, an antisense sequence is
complementary to an endogenous cyclin inhibitor gene sequence
that encodes, or has the potential to encode after DNA
rearrangement, an cyclin inhibitor gene product. In some
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42
cases, sense sequences corresponding to an cyclin inhibitor
gene sequence may function to suppress expression,
particularly by interfering with transcription.
The antisense polynucleotides therefore
inhibit production of the encoded polypeptide(s). In this
regard, antisense polynucleotides that inhibit transcription
and/or translation of one or more endogenous cyclin inhibitor
loci can alter the capacity and/or specificity of a non-human
organism to produce cyclin inhibitor gene products encoded by
ZO endogenous cyclin inhibitor loci, and thereby exhibit an
altered cell proliferation phenotype.
Antisense polynucleotides may be produced from
a heterolagous expression cassette in a transfectant cell or
transgenic cell, such as a transgenic pluripotent
hematopoietic stem cell used to reconstitute all or part of
the stem cell population of an individual, or as a germline
copy integrated (or otherwise episomally replicated) in the
genome of transgenic nonhuman animal. Alternatively, the
antisense polynucleotides may comprise soluble
oligonucleotides that are administered to the external milieu,
either in culture medium ~ vitro or in the circulatory system
or interstitial fluid ~ vivo. Soluble antisense
polynucleotides present in the external milieu have been shown
to gain access to the cytoplasm and inhibit translation of
specific mRNA species. In some embodiments the antisense
polynucleotides comprise methylphosphonate moieties,
alternatively phosphorothiolates or O-methylribonucleotides
may be used, and chimeric oligonucleotides may also be used
{Dagle et al. (1990) Nucleic Acids Res. 18: 4751). Far some
applications, antisense oligohucleotides may comprise
polyamide nucleic acids (Nielsen et al. (1991) Science 254:
1497). For general methods relating to antisense
polynucleotides, see Antisense RNA and DNA, (1988), D.A.
Melton, Ed., Cold Spring Harbor Laboratory, Cold Spring
Harbor, NY).
Whether as soluble antisense oligonucleotides
or as antisense RNA transcribed from an antisense transgene,
_~
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43
the antisense polynucleotides of this invention are selected
so as to hybridize preferentially to endogenous cyclin
inhibitor gene sequences at physiological conditions in viv~~.
Polynucleotides of this invention may serve as
anti.sense vectors or sense suppression constructs for
introduction into a plant genome or as integrated into a plant
genome at a position other than a naturally-occurring cyclin
inhibitor locus or in place of a naturally-occurring cyclin
inhibitor locus (e. g., by replacement homologous
recombination).
IX. ~vclin Inhi.bitar Transg!enes
Whereas expression of an endogenous cyclin
inhibitor gene and/or the encoded protein can be inhibited by
antisense suppression and/or related methods, the invention
also provides polynucleot:~des which encode a cyclin inhibitor
gene product or variant thereof and which, when introduced
into a suitable animal or plant genome, are expressed as a
functional cyclin inhibitor protein in the host animal or
plant.
For expression or overexpression of a cyclin
inhibitor gene product, a polynucleotide encoding a cyclin
inhibitor polypeptide having detectable CDK inhibition
activity (e.g., p27) is introduced into a suitable animal or
plant genome in a form suitable for expression as desired.
Typically, the cyclin inhibitor encoding polynucleotide is
operably linked to a transcriptionai regulatory sequence
(e. g., promoter, optional enhancer, polyadenylation sequence:,
etc.) capable of driving transcription. of the cyclin inhibitor
encoding sequence such that a translatable mRNA is ultimately
produced (i.e., RNA splicing of the primary transcript can be
required in some embodiments). In a variation, a cyclin
' inhibitor encoding polynucleotide can be targeted, by
homologous recombination gene targeting, into a position
adjacent to an operable endogenous promoter in a genome, such
that the resultant endogenous chromosomal locus comprises a
cyclin inhibitor encoding polynucleotide in operable linkage
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44
to an endogenous promoter, and optionally an endogenous
polyadenylation sequence and transcription termination
sequence. In an embodiment, the cyclin inhibitor encoding
polynucleotide can encode a full-length cyclin inhibitor
protein, although truncated variants or other deletion,
addition, or substitution variants caw be used. In an
embodiment, the cyclin inhibitor encoding polynucleotide
encodes a fusion protein comprising a full-length cyclin
inhibitor protein or active portion thereof in polypeptide
linkage to a fusion partner sequence, such as the sequence of
a naturally-occurring gene other than the cyclin inhibitor
gene.
A cyclin inhibitor encoding polynucleotide
typically in operable linkage to a transcriptional regulatory
sequence (e.g., promoter) and capable of expression is
introduced into a genome of a suitable animal (e. g., nonhuman
mammal, fish, reptile, bird) or plant variety (e. g., pepper,
tomato, tomatillo, etc.). Individuals exhibiting a desired
phenotype characterized by expression of the cyclin inhibitor
protein encoded by the introduced polynucleotide are selected
on the basis of a desired phenotype which is determined, such
as by enzyme assay, visual inspection, pathological condition,
and the like.
Thus, the invention provides a means of
expressing a cyclin inhibitor gene (e. g., p27) under control
of a heterologous promoter for any desired purpose. It can be
advantageous to use cyclin inhibitor gene expression
constructs to produce expression of a hyperphysiological level
of a cyclin inhibitor gene product in a cell, cell type,
tissue, organ, or organism. For example, such animals and
plants exhibit enhanced levels of cyclin inhibitor activity
can possess advantageous properties, such as decreased size
and cellularity, and the like.
X. Constructs and Introduction
In considering the expected temporal stage of
expression of the introduced gene, relevant factors include
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the type of promoter, the temporal pattern of the promoter,
and the operation of the promoter in view of its position
within the genome. A promoter which is expressed concurrently
~ with. or prior to the normal activation of the homologous
5 endogenous sequence is-preferred. A constitutive promoter is
often preferred, such as the CMV promoter. This promoter is
constitutive because its operation is relatively independent.
of the developmental stage of the cell in which it is
contained. A regulated promoter is also suitable. This
20 control may be either temporal with respect to the
developmental stage of the cell, or based upon differential
expression by different parts or organs of the organism.
Another way to regulate.the time of expression
of the introduced sequence is by linking the introduced
15 sequence to an inducible promoter that can be activated by
causing the organism (or part thereof) to be exposed to an
inducing agent (e.g., a steroid hormone in the case of a
steroid-responsive promoter/enhancer), chemical, W or other'
light source, or another activating treatment. It may also be
2o desirable to suppress a gene in one part of an organism only
using promoters that direct transcription in one part or organ
of an organism only (i.e., a fruiting body of a plant).
As referred to above, the operation of a
promoter may vary depending on its location in the genome.
25 Thus, a regulated promoter may operate differently from how it
does in its normal location, e.g., it may become fully or
partially constitutive.
It is preferred to have the DNA sequence
linked to and situated at a distance from the promoter
30 corresponding to the distance at which the promoter is
normally most effective so as to ensure sufficient
transcriptional activity. This distance should be within
about 2000 nucleotides, preferably within about 500
nucleotides and more preferably within about 300 nucleotides
35 of the translation initiation codon.
At the 3~ end of the coding sequence, operab7.y
linked segments may also be included. Thus, it would be
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46
optimum to have a 3' untranslated region containing the
polyadenylation site and any relevant transcription
termination sites. A 3' sequence of less than about 1000
nucleotides is sufficient, about~500 preferred and about 300,
or the length of the 3' untranslated tail of the endogenous
sequence is more preferred.
If the introduced cyclin inhibitor gene is an
intact gene or cDNA a fraction of independent transgenotes,
depending on the gene, may carry the introduced gene in
1.0 locations that result in abnormal expression, i.e., expression
at abnormal times in development. If the introduced gene is a
chimeric gene (meaning that one or more elements, such as a
promoter, from another gene has been substituted for a
component of the intact gene or added to the intact gene,
including coding sequences fused to upstream and downstream
sequences necessary or beneficial for expression) and is
driven by a constitutive (fully or partially) promoter, then
abnormal levels and times of expression will be achieved in a
large fraction of transgenotes. If the introduced gene is a
chimeric gene and is driven by a developmentally regulated
promoter, depending on the promoter, some fraction of
transgenotes will show abnormal levels and times of expression
of the introduced gene. The strength of the promoter or other
cis element can be the same, lower, or higher than the coding
sequence's usual promoter. The timing in development can be
earlier or the same.
Polynucleotides encoding full-length cyclin
inhibitor gene products or fragments or analogs thereof, may
include sequences that facilitate transcription (expression
sequences) and translation of the coding sequences, such that
the encoded polypeptide product is produced. Construction of
such polynucleotides is well known in the art and is described
further in Maniatis et al., Molecular Cloning: A Laboratory
2nd Ed. (1989), Cold Spring Harbor, N.Y. For example,
but not for limitation, such polynucleotides can include a
promoter, a transcription termination site (polyadenylation
site in eukaryotic expression hosts), a ribosome binding site,
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47
and, optionally, an enhancer for use in eukaryotic expression
hosts, and, optionally, sequences necessary for replication. of
a vector. A typical eukaryotic expression cassette will
lriclude a polynucleotide sequence encoding a cyclin inhibitor
polypeptide linked downstream (i.e., in translational reading
frame orientation; polynucleotide linkage) of a promoter such
as the HSV tk promoter or the pgk (phosphoglycerate kinase)
promoter, optionally linked to an enhancer and a downstream
polyadenylation site (e. g., an SV40 large T Ag poly A addition
site).
Additionally, a cyclin inhibitor gene or cDDtA
may be used to construct transgenes for expressing cyclin
inhibitor polypeptides at high levels and/or under the
transcriptional control of transcription control sequences
which do not naturally occur adjacent to the cyclin inhibit«r
gene. For example but not limitation, a constitutive promoi~er
(e. g., a HSV-tk or pgk promoter) or a cell-lineage specific
transcriptional regulatory sequence (e. g., a CD4 or CD8 gene
promoter/enhancer) may be operably linked to a cyclin
inhibitor-encoding polynuc:leotide sequence to form a transgene
(typically in combination with a selectable marker such as a
neo gene expression cassette). Such transgenes can be
introduced into cells (e. g., ES cells, hematopoietic stem
cells) and transgenic cells and transgenic nonhuman animals
may be obtained according to conventional methods.
The likelihood of obtaining a desirable
transgenote will depend upon the number of transgenotes
screened and the efficiency of actual transformation and
expression of the foreign nucleic acid sequence. Typically,
at least about 25 to 50 transgenotes will be screened, but 100
to 500 or more may need to be screened before the described
effect is seen.
Suppression and Ex»ression Transaenes in Plants
In general, a transcribable cyciin'inhibitor
polynucleotide sequence or its reverse complement contain an
operably linked promoter capable of functioning in the cell
CA 02242382 1998-07-20
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48
into which the polynucleotide is to be transferred. The
transcribable cyclin inhibitor polynucleotide sequence is at
least 25 nucleotides long, more usually at least 50-100
nucleotides long, frequently at least 100-250 nucleotides
long, often at least 500 nucleotides long or-longer, up to the
length of the complete endogenous gene (spanning promoter
through transcription termination sequence/polyadenylation
site). The transcribable cyclin inhibitor sequence is
positioned relative to the promoter such that a RNA transcript
to of the transcribable sequence is the same or reverse
complement polarity as the mRNA transcript of the endogenous
gene (i.e., sense or antisense orientation). The suppression
polynucleotide may be part of a larger polynucleotide, such as
a transgene having a selectable marker to identify cells
having integrated the transgene, or a homologous recombination
construct having selectable markers) and homology regions for
targeting the suppression polynucleotide to a predetermined
location in the genome of cells. Suppression polynucleotides
may be in the form of a heterologous expression cassette in a
transfectant cell or transgenic cell. Often, the suppression
polynucleotide is obtained as a vector produced with DNA
isolated from a cloned copy (or portion thereof) of the target
endogenous gene to be suppressed. The suppression
polynucleotide sequence is usually isolated as part of a
genomic gene clone, although in some embodiments a cDNA clone
(or portion thereof) of the target gene to be suppressed can
be employed (for general cDNA methods see, Goodspeed et al.
(1989) Gene 76: 1; Dunn et al. (1989) J. Biol. Chem. X64:
13057).
Vectors containing a suppression
polynucleotide are typically grown in ,F~. co i and then
isolated using standard molecular biology methods, or may be
synthesized as oligonucleotides. Direct polynucleotide
synthesis and ligation (if necessary) which does not require
prokaryotic or eukaryotic vectors may also be done.
Polynucleotides (and transgenes comprising such) can be
transferred to host cells by any suitable technique, including
CA 02242382 2004-09-17
49
microinjection, electroporation, lipofection, biolistics,
calcium phosphate precipitation, and viral-based vectors,
among others (e. g., U.S. Patents 5,442,05x, 5,354,854,
5,2?8,05?, 5,262,316, 5,13?,817, and 4,962,028).
A preferred method of introducing the
nucleic acid segments into plant cells is to infect a plaltt
cell, an explant, a meristem orla seed with Acupbacterium
ty~mefacie~s, transformed with the segment. Under appropriate
conditions known in the art, the transformed plant cells are
grown to form shoots, roots, and develop further into plants.
The nucleic acid segments can be introduced into appropriate
plant cells, for example, by means of the Tf plasmid of
gcZrob~,aerium tnmefac~~ns. The Ti plasmid is transmitted to
plant cells upon infection by Aarobac~erium tumefaciens, and
is stably integrated into the plant genome (Horsch et al.,
(1984) "Inheritance of Functional Foreign Genes in Plants,~~
$ciencg, 233:496--498; Fraley et al. , (1983) Pr~,r. Nazi-Acad.
Sci. USA 80:4803) . One Aqrobac~~r,l,~ method is ,~ pianta
Ac~o ~~cterium-mediated gene transfer by infiltration, e.g., of
2D adult Arabidopsis thaliana plants; Bechtold et al. (1993) C;R.
Acad. Sci. Life Sciences ~: 1194 et seq.).
All plant cells which can be transformed by
Agr?bactirrium and whole plants regenerated from the
transformed cells can also be transformed according to the
invention so as to produce transformed whole plants which
contain the transferred foreign nucleic acid sequence.
Plant cells can be transformed with
Aq~o_~aacterium in various ways, including: co-cultivation of
~ctrobacterium with cultured isolated
protoplasts,
transformation of cells or tissues with AcTrobact~rium, or
transformation of seeds, apices or meristems with
~ro~ra~~cter ium .
A preferred system is the binary system in
which two plasmids are needed: a T-DNA containing plasmid and
a vir plasmid. Any one of a number of T-DNA aantaining
CA 02242382 1998-07-20
WO 97/26327 PCT/US97/00831
plasmids can be used, the only requirement is that one be able
to select independently for each of the two plasmids.
After transformation of the plant cell or
plant, those plant cells or plants transformed by the Ti
5 plasmid so that the desired DNA segment is integrated can be
selected by an appropriate phenotypic marker. These
phenotypic markers include, but are not limited to, antibiotic
resistance, herbicide resistance or visual observation. Other
phenotypic markers are known in the art and may be used in
10 this invention.
If naked nucleic acid introduction methods are
chosen, then the vector need be no more than the minimal
nucleic acid sequences necessary to confer the desired traits,
without the need for additional other sequences. Thus, the
15 possible vectors include the Ti plasmid vectors, shuttle
vectors designed merely to maximally yield high numbers of
copies, episomal vectors containing minimal sequences
necessary for ultimate replication once transformation has
occurred, transposon vectors, homologous recombination
2o vectors, mini-chromosome vectors, and viral vectors, including
the possibility of RNA forms of the gene sequences. The
selection of vectors and methods to construct them are
commonly known to persons of ordinary skill in the art and are
described in general technical references (Methods in
25 Enzymology, supra) ,
However, any additional attached vector
sequences which will confer resistance to degradation of the
nucleic acid fragment to be introduced, which assists in the
process of genomic integration or provides a means to easily
30 select for those cells or plants which are actually, in fact,
transformed are advantageous and greatly decrease the
difficulty of selecting useable transgenotes.
All transformable plants from which whole
regenerated plants can be generated can be used in the present
35 invention. Monocots may be transformed with Aarobacterium by
electroporation (Fromm et al. [1986] Nature 319:791-793;
Rhodes et al. Science [1988] 240: 204-207); by direct gene
CA 02242382 1998-07-20
WO 97!26327 PCT/US97/00831
51
transfer (Baker et al.-[1985] Plant Genetics 201-211); by
using pollen-mediated vectors (EP 0 270 356); and by injection
of DNA into floral tillers (de la Pena et al. [1987], Nature
325:274-276).
- Cvclin Inhibitors
p27 inhibitors that permit the activation of cyclin
E-Cdk2 and/or cyclin A-Cdk2 complexes can be identified in ,a
variety of screening assay formats. Inhibitors of p27-
to mediated activation of cyclin E-Cdk2 and/or cyclin A-Cdk2 in
the presence of p27 can be screened, for example, using an
assay in which test substances are exposed to suitable amounts
of p27 protein, cyclin E and or cyclin A, and Gdk2 under
conditions that permit the formation of active cyclin E- or
cyclin A-Cdk2 complexes in the absence of p27. The active
cyclin E- and/or cyclin AwCdk2 complexes formed are then
quantitated and compared 'to the active complexes formed in the
absence of the test substance.
Substances which can serve as p27 inhibitors
include, but are riot limited to, compounds capable of
inhibiting the p27-mediated inhibition of cyclin E-Cdk2
complex activation, compounds that specifically inhibit the
interaction between p27 and cyclin E-Cdk2 complexes and/or
between p27 and cyclin A-Cdk2 complexes, but not the site-
specific phosphorylation of the Cdk2 moiety of the cyclin-C<ik2
complex in the absence of p27, compounds that degrade or
inactivate the p27 protein, and compounds that interfere wii:h
the expression of p27 protein. Such agents may include
chemical compound inhibitors of p27, protein or peptide p27
antagonists, and molecules that inhibit the expression of p27
such as triplex forming oligonucleatides, antisense
oligonucleotides, ribozymes, etc.
For use as p27 inhibitors in the present invention
to mediate cell cycle progression, the triplex forming
' 35 oligonucleotides are p27 sequence-specific DNA binding drugs
that interfere with p27 transcription. Triplex-forming
oligonucleotides are generally described in Maher, Bioessav:~
l I ~ilinli l Ii. iM,"I" i~ l I
CA 02242382 2004-09-17
52
14: 807-815 (1992); Gee et al., Gene 149: 109-114 (1994);
Noonberg et al., Gene 149: 123-126 (1994); Song et al., nn.
~lfY Acad. Sci. 761: 97-108 (1995); Westin et al., Nuc. Acids.
$es. 23: 2184-2191 (1995); and Wand and Gla$er, J. Biol. Chem.
207: 22595-22901 (1995). These oligonucleotides form triple
helical complexes, under physiological conditions, on double-
stranded DNA selectively inhibiting p2? transcription by
physically blocking RNA polymerise or transcription factor
access to the p27 DNA template. See also, e.g., WO 95/25818;
WO 95/20404; WO 94/15616; WO 94/04550; and WO 93/09788.
The triplex
forming oligonucleotides targeted to the p27 gene may contain
either a nucleotide or non-nucleotide tail to enhance the
inhibition of transcription factor binding.
Antisense oligonucleotides that interfere with the
expression of p27 and permit progression of the cell cycle, as
exemplified in the Examples described hereinbelow, are
particularly useful in the present invention, p2'7 antisense
inhibitors are identified using methods, e.g., as described in
detail in the Examples. The use of antisense oligonucleotides
and their applications are described generally in, for
example, Mol and Van der Krul, ads., Arrtisense Nucleic Acids
and Proteins Fundament$1s and Applications, New York, NY,
1992.
Suitable antisense oligonucleotides are at least 11
nucleotide in length and up to and including the upstream
untranslated and associated coding sequences of p27. As will
be evident to one skilled in the art, the optimal length of
antisense oligonucleotides is dependent on the strength of the
interaction between the antisense oligonucleotides and their
comple:aentary sequence on the mRNA, the temperature and ionic
environment translation takes place, the base sequence of the
antisense oligonucleotide, and the presence of secondary and
tertiary structure in the mRNA and/or in the antisense
oligonucleotide. Suitable target sequences for antisense
oligonucleotides include intron-axon junctions (to prevent
proper splicing), regions in which DNAjRNA hybrids will
CA 02242382 1998-07-20
WO 97/26327 PCT/LTS97/00831
53
prevent transport of mRNA from the nucleus to the cytoplasm,
initiation factor binding sites, ribosome binding sites, and
sites that interfere with, ribosome progression. A
particularly preferred target region for antisense
oligonucleotide is the 5~~untranslated region of the p27 gene.
Antisense polynucieotides targeted to the p27 gene
'are prepared by inserting a DNA molecule containing the target
DNA sequence into a suitable expression vector such that the
DNA molecule is inserted downstream of a promoter in a reverse
orientation as compared to the gene itself. The expression
vector may then be transduced, transformed or transfected into
a suitable cell resulting in the expression of antisense
polynucleotides. Alternatively, antisense oligonucleotides
may be synthesized using standard manual or automated
synthesis techniques. Synthesized oligonucleotides may be
introduced into suitable .cells by a variety of means including
electroporation (e.g., as described in Yang et al., Nuc
Acids. Res. 23:2803-2810 (1995)), calcium phosphate
precipitation, microinjection, poly-L-ornithine/DMSO (Doug et
al., Nucl. Acids. Res. 21:771-772 (1993)). The selection of a
suitable antisense oligonucleotide administration method wiJLl
be evident to one skilled in the art. With respect to
synthesized oligonucleotides, the stability of antisense
oligonucleotides-mRNA hybrids may be increased by the addition
of stabilizing agents to i:he oligonucleotide. Stabilizing
agents include intercalating agents that are covalently
attached to either or both ends of the oligonucleotide.
Oligonucleotides may be made resistant to nucleases by, for
example, modifications to the phosphodiester backbone by thE:
introduction of phosphotr9_esters, phosphonates,
phosphorothioates, phosphoroselenoates, phosphoramidates or
phosphorodithioates. Oligonucleotides may also be made
nuclease resistant by the synthesis of the oligonucleotides
with alpha-anomers of the deoxyribonucieotides, as generally
described in Mol and Van der Krul, supra.
For oligonucleotide-based inhibitors, the choice of
a suitable sequence will be guided by, for example, the type
CA 02242382 1998-07-20
WO 97/26327 PCT/US97/00831
54
of inhibitor (i.e., triplex forming oligonucleotide or
antisense oligonucleotide) and the species to be treated. It
may be preferable to choose sequences that are conserved
between species to permit use in readily available animal
models. As shown in more detail below, antisense
oligonucleotides to sequences within p27 that are conserved
between mouse and human were chosen for use in the mouse
model. Such sequences may then be used in human cells without
reformulation.
The present invention also provides compositions
and methods for inhibiting p27 and thereby permitting cell
cycle progression using ribozymes. The ribozymes can be
administered in a variety of ways, including, by gene therapy
targeted to a desired cell. A ribozyme of the invention
targets the RNA transcripts of the p27 gene. Each ribozyme
molecule contains a catalytically active segment capable of
cleaving the p27 RNA, and further comprises flanking sequences
having a nucleotide sequence complementary to portions of the
targeted RNA. The flanking sequences serve to anneal the
ribozyme to the RNA in a site-specific manner. Absolute
complementarity of the flanking sequences to the target p27
sequence is not necessary, however, as only an amount of
complementarity sufficient to farm a duplex with the target
RNA and to allow the catalytically active segment of the
ribozyme to cleave at the target sites is necessary. Thus,
only sufficient complementarity to permit the ribozyme to be
hybridizable with the target RNA is required.
As used herein, the term °'ribozyme°° means an RNA
molecule having an enzymatic activity that is able to cleave
or splice other separate RNA molecules in a nucleotide base
sequence specific manner. By reference to catalytic or
enzyimatic RNA molecule is meant an RNA molecule which has
complementarity in a substrate binding region to a specific
p27 RNA target, and also has enzymatic activity that is active
to cleave and/or splice RNA in that target, thereby altering
the target molecule. In preferred embodiments of the present
invention the enzymatic RNA molecule is formed in a hammerhead
CA 02242382 2004-09-17
motif, but the ribozyme may also be formed in the motif of a
bairpin, hepatitis delta virus, group I intron or RNAse P RNA
(in association with an RNA guide sequence). Examples of
hammerhead motifs are described by Rossi et al., AIDS Res.
5 gym-Retrovir. 8: 183 (1992), hairpin motifs are described by
Hampel et al., ~~~ychem. 28:4929 (1989) and Hampel et al.,
N~~cl- Ag~.ds Res. 18: 299 (1990), the hepatitis delta virus
motif is exemplified in Perrotta and Heen, ~ochPm. 31: 16
{1992), an RNAseP motif is described in Guerrier-Takada et
l0 al., Cell 35:849 {1983), and examples of the group I intron
motif are described in Cech et al., U.S. Patent 4,987,071.
These specific motifs are not limiting in the
present invention and those of skill in the art will recognize
15 that an enzymatic RNA molecule of the invention has a specific
substrate binding site which is complementary to one or more
of the target p27 RNA regions and that it has nucleotide
sequences within or surrounding that substrate binding site
which impart an RNA cleaving activity to the molecule.
20 The flanking sequences upstream and downstream of
the ribozyme catalytic site may comprise segments of any
length that effectively imparts the desired degree of
targeting specificity for the ribozyme. Preferably a flanking
sequence comprises from about 9 to about 24 nucleotides, more
25 preferably from about 6 to about 15 nucleotides, and typically
about 9 to 12, and results in base pairing to the substrate
sequence immediately upstream and downstream of the p27 RNA
sequences which comprise the cleavage site.
The p2? inhibitors may be used alone or in
30 combination may be formulated for a variety of modes of
administration. Administration of the inhibitors may include
systemic, topical or local administration. Techniques and
formulations are generally described in Reminaton,~s
Pharmar,~uti~;,al Science8, Mack Publishing Co., Easton, PA,
35 latest~edition. The inhibitor is generally combined with a
pharmaceutically acceptable carrier such as a diluent or
excipient. Suitable carriers may include fillers, extenders,
CA 02242382 1998-07-20
WO 97/26327 PCTlITS97/00831
56
binders, wetting agents; disintegrants, surface-active agents
or lubricants. The choice of such ingredients will depend on
the mode of administration and dosage forms. Typical dosage
forms include'tablets, powders, liquid preparation including
suspensions, emulsions, and solutions, granules, capsules and
suppositories. Liquid preparation for injection are also
typical and include liposome preparations.
A sequence comprising or encoding an
oligonucleotide p27 inhibitor, e.g., triplex forming
oligonucleotides, antisense oligonucleotide, ribozyme, etc.,
or a combination of such inhibitors targeted to different
portions of the p27 DNA or corresponding RNA can be delivered
in a wide variety of ways to targeted cells to facilitate
progression of the cell cycle. The oligonucleotides can be
administered as synthetic oligonucleotides or expressed from
an expression vector. The oligonucleotide can be administered
vivo, i.e., contacted with target cells that have been
removed from an individual or other cell source, treated and
returned, or the oligonucleotide molecule can be administered
,~ vivo. When administered ~ vivo typically the target cells
are exposed to mitogens, e.g., serum mitogens (SCF, IL-3, EPO,
TPO, etc.) or the like depending on particular cell
population.
Delivery to the targeted cell population can be via
an appropriate delivery vehicle, e.g., a liposome, a
controlled release vehicle, by use of iontophoresis,
electroporation or ion paired molecules, or covalently
attached adducts, and other pharmacologically acceptable
methods of delivery. Preferably a carrier provides a means to
accumulate the oligonucleotide within or at a desired cell
population. The delivery vehicle~can be designed to serve as
a slow release reservoir or to deliver its contents directly
to the target cell. Examples of oligonucleotide delivery
vehicles include liposomes, hydrogels, cyclodextrins,
biodegradable nanocapsules, and microspheres. Liposomes can
readily be targeted to the various tissues or cell
populations. In another embodiment the anti-p27
_. _...... __.... ___ _.. k __..
CA 02242382 1998-07-20
WO 97!26327 PCT/US97/00831
57
oligonucleotide is administered via an expression vector that
is suitable for delivery and expression of an oligonucleotide
comprising said oligonucl~eotide in a mammalian host cell.
For 'fin vivo use, routes of oligonucleotide
administration include~intramuscular, aerosol, intravenous,
parenteral, intraperitoneal, etc. The specific delivery route
for a selected oligonucleotide will depend on a variety of
factors, such as the form of the oligonucleotide, the intended
target, the condition being treated, etc. For example, while
to unmodified oligonucleotide is taken up by cells, modifications
can be made to enhance cellular uptake, e.g., by reducing the
oligonucleotide's charge to produce a molecule which is able
to diffuse across the cell membrane. The structural
requirements necessary to maintain oligonucleotide activity
are generally recognized in the art. Modifications to enhance
cellular delivery can also be designed to reduce
susceptibility to nuclease degradation.
The dosage of oligonucleotide inhibitor will also
depend on a variety of factors, such as the form of the
oligonucleotide, the route of administration, the stage of the
cell cycle, the percentage of non-dividing cells in a selected
population, whether terminal differentiation has been reached,
etc., and thus can vary widely. Generally the dosage will
result in complete inhibition of p27 activity or levels
sufficiently low within th.e targeted cells sufficient to
permit activation of the cyclin E- and/or cyclin A-Cdk2
complexes and progression of the cell cycle. Establishment of
effective levels of p27 inhibitor within a targeted cell
population depends upon, e.g., the rate of uptake (or
expression by a particular vector), and rate at which the
inhibitor is degraded. The duration of treatment may extend
for a time sufficient to permit, e.g., transduction of a
' relatively high percentage of dividing cells compared to an
untreated control cell population, but usually will be at
least for about 2-4 days, sometimes 6-10 days, although longer
durations may be necessary for quiescent or terminally
differentiated cell populations. The number and timing of
CA 02242382 1998-07-20
WO 97/26327 PCT!(JS97/00831
58
doses can vary consider-ably, depending on the factors
discussed above and the efficacy of a particular inhibitor or
mixture thereof, the delivery vehicle and route of
administration, etc. . ,
For nucleotide inhibitors of p27 such as p27
antisense oligonucleotides or p27-specific triplex forming
oligonucleatides, it may be preferable in include an effective
concentration of a lipid formulation with the oligonucleotide
of the present invention. Suitable lipid formulations and
concentrations are those that enhance the uptake of the
oligonucleotides by cells. Such lipids include cationic
lipids used for lipofection such as N- [1-(2,3-
dioleyloxy)propyl-N,N,N-trimethylammonium chloride (DOTMP.) and
dioleoyl phophatidylethanolamine (DOPE). One skilled in the
art may determine the particular lipid formulation or
concentration that will be effective~for enhancing the uptake
of the oligonucleotide.
Within the methods described in detail herein, the
p27 inhibitors may be used in combination with other compounds
that inhibit cells from entering cell cycle arrest or which
inhibit differentiation that may accompany the proliferation
of certain cells. Retinoic acid receptor antagonists, for
example, may be used in combination with the disclosed methods
and compositions to increase the number of proliferating cells
in a cell population. The retinoic acid receptor a antagonist
Ro 41-5253 (Apfel et al., Proc. Natl. Acad Sci USA 89: 7129-
7133, 1992) has been shown to counteract the retinoic acid-
induced differentiation of the promyelocytic cell line HL-60.
Alternatively, antagonists of mitotic inhibitors such as p14
(Guan et al., Genes Dev. 8: 2939-2952
(1994)), p15 (Hannon and
Beach, ure 371: 257-261 (1994)), p16 (Okamoto et al.,
Cancer Research 55: 1448-151 (1995) and Serrano et al., Nature
366: 704-707 (1993)), p18 (Guan et al.; .ibid.), p19 (Chap et
al., idol. Cell. Biol. 15: 2682-2688 (1995) and Zhang et al.,
Cell 82: 915-925 (1995)) and p21 (Harper et al., Cell 805-816
(1993) may be used in combination with the p27 inhibitors of
the present invention to increase the proportion of
, "nn ~,~,.i.a
CA 02242382 2004-09-17
59
proliferating cells in-a cell population. Antagonists of
these mitotic inhibitors include, but are not limited to,
agents that interfere with the transcription or translation of
the inhibitors, destruction of the protein, and direct
inhibitors of the protein. As such, inhibitors of mitotic
inhibitors may include chemical compound inhibitors of the
mitotic inhibitors, protein or peptide mitotic inhibitor
antagonists, triplex fonaing oligonucleotides and antisense
molecules that inhibit the expression of the mitotic
l0 inhibitors, ribozymes, etc.
The methods of the present invention are
particularly useful for gene therapy. Target cells for gene
therapy are exposed to p27 inhibitors under suitable
conditions and for a time sufficient to increase the
proportion of dividing cells in the target cell population.
The dividing cells are then exposed to a suitable viral vector
comprising a gene of interest. Within one embodiment, the
cells are exposed to the p27 inhibitor and the viral vector
concurrently. Suitable viral vectors include retroviral
vectors (see Miller, ~'urr. Top. Microbi.n~ Immunol 158: 1-24
(1992); Salmons and Gunzburg, Human 5',~ene Theracv 4: 129-141
(1993); Miller et al., Methods i,~, ~naym0loav 217: 581--599,
(1994)) and adeno-associated vectors (reviewed in Carter,
Curr. Ot~inion Biotech. 3: 533-539 (1992); Muzcyzka, Curr. Ton.
Microb,,pl. Immunol. 158: 97-i29 (1992)). Other viral vectors
that may be used within the methods include adenoviral
vectors, herpes viral vectors and Sindbis viral vectors, as
generally described in, e.g., Jolly, cancer Gene Therauv 1:51-
64 (1994); Latchman, Molec, 5fotechnol. 2:179-195 (1994j; and
Johanning et al., Nucl. Acids Res. 23:1495-1501 (1998)).
The choice of vector will
rely in part on the cell type targeted, the disease state
that is being treated and the size of the gene to be
transferred.
Cells which are exposed to a p27 inhibitor in an
amount and for a time sufficient to inhibit exit from the cell
cycle can be treated by a variety of substances that target
....n.n ..,..a n, . ,
CA 02242382 2004-09-17
dividing cells. In one embodiment, for example, a cell
population in which the proportion of dividing cells has been
increased by a p27 inhibitor are more efficiently transduced
or transfected with a nucleotide~sequence encoding a gene
5 product of interest. Thus, the methods described herein
increase the efficiency of gene therapy techniques. For
example, target cells treated with a~p27 inhibitor are
transduced with at least one gene encoding an expression
product of interest, typically an RNA or protein molecule.
10 The encoded RNA or protein is one which confers a benefit to
the cell population or host being treated, either directly or
indirectly. The gene may encode a secreted or non-secreted
protein, or an active portion thereof. The selection of a
suitable gene for the condition being treated will depend on
15 the condition being treated or prevented and other factors
apparent to those skilled in the art. By "gene" is meant DNA
that encodes a desired product, such as, for example, a
cytokine, a clotting factor, a hormone, an enzyme, a transport
protein, a regulatory protein, a structural protein, a
20 receptor, an antigen, ribozyme, antisense molecule, etc.
Representative examples of genes far introducing into humans
are those encoding human erythropoietin (described in U.S.
Patent No. 4,703,008), human G-CSF, human GM-CSF (Anderson et
al., Prod,, Natl. Aca~. Sci. USA 82:6250 {1985)), plasminogen
25 activator, urokinase, insulin (e.g., human insulin as
described in U.S. Patent No. 4,652,525 or proinsulin described
in U.S. Patent No. 4,431,740), interleukins (e. g.,
interleukin-1, interleukin-2 [described in U.S. Patent No.
4,738,927j, interleukin-3 [described in EP Publ. 275,598 and
30 282,185], interleukin-4, interleukin-7 [U.S. Patent No.
4,965,195j, etc.}, interferons, Factor VIII, Factor IX, von
Willebrand Factor, ADA, human growth hormone (described in
U.S. Patent No. 4,342,832), etc., analogs and fusions thereof
(e. g., fusions of GM-CSF and IL-3 [U. S. Patent No. 5,108,910j.
I . ~ ~iil~~p l Ir. n.rl ~a I ~ r
CA 02242382 2004-09-17
6i
It is possible and may be desirable in some
instances to employ a mixture of cells treated with a p27
inhibitor, which include a first group transduced with a gene
of interest arid a second group transduced with a second,
different gene of interest. Alternatively, the treated cells
may be transduced with more than one gene of interest.
The genes are transduced or transfected into the
target cell population which has been treated with a p27
inhibitor using well established protocols. Typically the
gene transfer vector will be a retroviral vector, but other
vectors may al:o be employed, e.g., adenovirus vectors (e. g.,
Rosenfeld et al., Cell 68: 143-155 (1992) and Curiel et al.,
grgc. ,jsatl,"_,~cad. Sci. t~SA 88: 8850-8854 (1991), adenovirus
associated vectors (e. g., Muzyczka, burr. T~. Microbiol.
Immunol. 158: 97-129 (1992), and as reviewed by Miller, Nature
357: 455-460 (1992)).
The construction of retroviral vectors
has been described, e.g., Miller and Rosman, giotechniQUes 7:
980-990 (1989); Adam et al., J. Virol. 65: 4985-4990 (2991);
Miller, C~,~r. fop. Microbio~ Immunol. 158: 1-24 (1992); and
UK Patent publication GH 2,259,175A.
A preferred retroviral
vector is made using PA317 amphotropic retrovirus packaging
cells, as described in Miller, U.S. Patent No. 4,861,719.
When the sell population treated with p27 inhibitor
is transduced or transfected e~ viva with a gene of interest,
cells containing the desired genes) are often cultured,
typically in the presence of a selection agent, e.g., 6418,
neomycin or the like depending on~the selectable marker used
in the vector, and then may be returned to the host or
expanded until a sufficient number of cells are available for
return to the host.
The compositions and methods of the present
invention are used to treat a wide variety of cell types.
Among those most often targeted for gene therapy are
hematopoietic precursor (stem) cells. Other cells include
CA 02242382 1998-07-20
WO 97/26327 PCT/US97/00831
62
those of which a proportion of the targeted cells are
nondividing or slow dividing. These include, for example,
fibroblasts, keratinocytes, endothelial cells, skeletal arid
smooth muscle cells, osteoblasts; neurons, quiescent
lymphocytes, terminally differentiated cells, slow or non-
cycling primary cells, etc. The methods and compositions can '
be employed with cells of a wide variety of vertebrates,
including mammals, and especially those of veterinary
importance, e.g, canine, feline, equine, bovine, ovine,
caprine, rodent, lagomorph, swine, etc., in addition to human
cell populations.
The present invention is particularly preferred for
increasing the proportion of dividing cells in a population of
hematopoietic precursor cells, especially those of human and
other mammals, either e~ vivo or ~ vivo. In an ex vivo
method, hematopoietic precursor cells are separated from a
blood product, such as bone marrow, peripheral blood, or
umbilical cord blood of'a donor, fetal peripheral blood and
other sources. Such separation may be performed, for example,
by immunoselection on the basis of their expression of an
antigen, such as the CD34 antigen which is present on
substantially all human hematopoietic precursor cells, but is
substantially absent from more mature hematopoietic cells.
The separated hematopoietic precursor cells may be stored
frozen and thawed at a later date for inoculation into a
suitable vessel containing a culture medium comprising a
nutritive medium. Alternatively, the separated cells may be
inoculated directly into culture without first freezing. In
both cases the resultant cell suspension is cultured with a
p27 inhibitor as described herein under conditions and for a
time sufficient to increase the proportion of dividing
hematopoietic precursor cells relative to the proportion of
such cells present initially in the blood product. The cells
may then be treated with vector capable of expressing the gene
product of interest. The cells may then be infused or
implanted into a host or stored frozen for infusion at a later
date.
CA 02242382 1998-07-20
WO 97/26327 PCTlUS97/00831
63 _
In addition,-the methods of the present invention
may be used 'fin vitro to create novel stem cell lines.
According to this aspect of the invention the p27 inhibitor is
administered to a cell pr~pulation, thereby preventing cells
from exiting the cell cycle and increasing the percentage of
cells in the cell cycle, and may also reduce the need to
include exogenous serum mitogens. The methods may also be
used in combination with, for example, methods for creating
stem cell lines by exposing the cell population to a p27
antagonist under suitable conditions and for a time sufficient
to increase the population of dividing cells, and exposing 'the
dividing cells to a suitable expression vector comprising an
gene encoding a desired gene product such that the resulting
cells express the gene product and are self-renewing.
The following examples are offered by way of
illustration, not by way~of limitation.
EXAMPLES
EXAMPLE I
Subconfluent, exponentially asynchronous
proliferating Balb/c-3T3 fibroblasts (Rb wild type; p53 status
unknown) in media containing 10% fetal calf serum were rins~ad
once with serum-free medium and transferred to low serum
medium containing mitogens (0.1% serum). Flow cytometry
analysis (Firpo et al., l~c~i. Cell. Biol. 14:4889 (1994))
demonstrated that within 24 hours, approximately the length of
one cell cycle, 95% of the cells arrested in G1, indicating
that these cells require a mitogenic signal to proceed through
each division cycle. G1 arrest correlated with a 6 to 8 fold
induction of the p27Kipx protein as determined by immunobiot
analysis (Nourse et al., Nature 372:570 (1994); Kato et al.,,
Cell 79:487 (1994)) of proliferating and serum-starved cell:.
' Similar increases in p27 expression occur in primary human
diplaid fibroblasts deprived of serum mitogens, and in primary
human T lymphocytes following withdrawal of IL-2, indicating
that this is a common pattern of p27 expression in normal,
non-transformed cells (NoL~rse, ibid., Kato, ibid.),
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64
It was then shown that in Balb/c-3T3 cells p27
levels start to increase within 4 hours of serum withdrawal,
reach 60% of maximal levels within 12 hours, and peak by 24
hours. Proliferating Balb/c-3T3 fibroblasts were rinsed in
serum-free medium and re-fed with low serum medium containing
0.1% serum. p27 western blots (ECL, Amersham) were performed
on cells harvested at 4, 8, 12, 16 and 24 hours after re-
feeding. p27 levels started to increase at 4 hours and were
60% of maximal at 12 hours). Thus, the induction of p27
protein parallels the accumulation of the initially
asynchronous cell population in G1, and indicates a critical
role in the early events associated with exit from the cell
cycle.
Histone H1 kinase assays were performed on cyclin
A, cyciin E and Cdk2 (Firpo et al., 141. Cell. Biol. 14: 4889
(1994)) immunoprecipitated from extracts made from
proliferating and serum-starved Baib/c-3T3 cells. The results
showed that cell cycle arrest of Baib/c-3T3 cells was
correlated with downregulation of the cyclin E-Cdk2 and cyclin
A-Cdk2 protein kinases, and this appeared to be related to
induction of p27. Both cyclin E-Cdk2 and cyclin A-Cdk2 were
associated with increased amounts of p27 following mitogen
withdrawal. Immunodepletion experiments were also performed
to determine the amount of cyclin E bound to p27. Cell
extracts from asynchronously proliferating Balb/c-3T3 cells
and Balb/c-3T3 cells that had been serum-starved for 24 hours
were depleted for p27 by incubating 100 ug of each extract
with p27 antiserum and protein A agarose for 1 hour at 4°C,
centrifuging the immunoprecipitates for 5 seconds at 13,000
r.p.m and immunodepleting the remaining unbound supernatant
twice more with p27 antiserum and protein A agarose. The
immunodepleted extracts (a p27) were analyzed by cyclin E
(Ohtsubo and Roberts, Science 259: 1908 (1993); Matsushime et
al., Cell 65: 701 (1991); Koff et al., Bcience 257:1689
(1992)) and p27 immunoblots and compared to undepleted
extracts and extracts depleted with p27 preimmune sera. The
results showed that only a small portion of cyclin E in
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proliferating cells was bound to p27, while ali the cyclin E
in arrested cells was bound to p27. Similar results were
obtained for cyclin A: Experiments were performed as for
cycliri E, except that cyclin A and p27 immunoblots were
5 performed on extracts depleted for p27. A11 of the cyclin A
was bound to p27 in extracts from serum-starved cells while
only a small fraction (5%) of cyclin A was associated with p27
in proliferating cells).
In sum, Balb/c:-3T3 fibroblasts arrest in the first
10 G1 following mitogen withdrawal, and this correlates with
increased expression of ~a27, increased association of. p27 with
cyclins E and A, and inactivation of the cyclin E- and cyclin
A-Cdk2 kinases.
The relationship between p27 expression and cell
15 proliferation was studied by testing the relative abilities of
specific serum mitogens t:o both downregulate p27 and induce:
cell proliferation. Flow cytometry analysis was performed on
both the asynchronously proliferating Balb/c-3T3 cells (Hi
serum) and subconfluent Balb/c-3T3 cells that had been serum-
2o starved for 24 hours (Low serum) in the presence of either
individual growth factors (PDGF, IGF-1 or EGF) or all three
growth factors (PIE) (see Table). p27 immunoblots were
performed on cell extracts (10 ug) from cells treated with
growth factors. Only PDGF was able to.prevent G1 arrest, a.nd
25 only PDGF prevented the induction of p27. Balb/c-3T3
fibroblasts grown at high density have more complex mitogen
requirements than when grown subconfluently; no single mitogen
is able to cause proliferation of cells at high density.
Instead, PDGF initially stimulates the density arrested,
30 quiescent cells to become "competent" to respond to
"progression" factors, IGF-1 and EGF (Pledger et al., oc.
l~latl. Acad. Sci USA 74:4481 (1977); Leof et al., Exp. Cell
' Res. 147:202 (1983)). Therefore, under these conditions
passage through the restriction point does not occur until
- 35 cells have been exposed to all three mitogens.
It was also observed that in density-arrested ce7.ls
PDGF alone was insufficie~rnt to alter p27 abundance; rather X027
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66
levels declined once cells became committed to proliferate in
response to the complete mitogenic signal provided by the
combined action of PDGF, EGF and TGF-1. Density-arrested
Balb/c-3T3 fibroblasts were rinsed in serum-free medium and
were re-fed with low serum medium containing 0.1 % serum and
ng/ml of either PDGF, IGF, EGF, IGF and EGF, or all three
growth factors. Cells were harvested 24 hours later and were
analyzed by flow cytometry for DNA content and also by p27
immunoblot. The results indicated that a combination of all
10 three growth factors was required to stimulate 70% of the
cells to enter the cell cycle and to decrease p27 levels by
ten-fold.
Thus, under two different growth arrest conditions
the ability of specific mitogens to stimulate passage through
the restriction point correlated with their ability to
regulate p27. These results showed that p27 is not
necessarily a downstream effector for any particular mitogen.
Rather, decreased expression of p27 reflects the integrated
action of the collection of mitogens required for cell
2o proliferation.
EXAMPLE II
The observed correlation between p27 regulation and
mitogenic signaling was extended by using anti-sense
oligonucleotides to block expression of the p27 protein. This
showed that regulation of p27 was necessary for cell cycle
control by serum mitogens.
Phosphorothioate oligonucleotides were modified by
the addition of a propyl group to the pyrimidine bases, which
is thought to enhance base stacking and facilitate the sense
antisense interaction (Raviprakash et al., J. Virol. 69:69
(1995)). The oligonucleotides were synthesized by the H-
phosphonate method on an automated synthesizer (model 8750,
Milligen Bioresearch, Bedford, MA) using standard chemistry on
controlled pore glass (CPG) support. The nucleoside analogs
were prepared as previously described (B. Froehler, Protocols
for Oligonucleotides and Analogs: Synthesis and Properties.
i . ~~i~,n , i~ "n~i.,~d~~ i
CA 02242382 2004-09-17
57
Humans, Totowa, NJ (1993); Froehler et al., Te,rahedron ~gtt.
33:5307 (1992); and Froehler et al., Tetrahedron Lett. 34:
1003 (1993)). The antisense oligonucleotides were designed to
target sequences that are identical between the mouse and the
human p27 sequences, which are described in WO 96/02140
and deposited with Genbank under accession nos. U09968 and
U10906, respectively.
The antisense oligonucleotide sequences used in
these experiments oligonucleotide 3163 ([SEQ ID NO:i] 5' UGG
CUC UCC UGC GCC 3~j (targets base pair 306-320 of marine Kipl,
the sequence of which is described in WO 96/02140
and is also deposited with
Genbank under Accession Number U09968j and its mismatch
control oligonucleotide 3436 ([SEQ ID N0:2~ 5' UCC CW UGG CGC
GCC 3'j, and oligonucleotide 3162 ([SEQ ID N0:3] 5~ GCG UCU
GCU CCA CAG 3~) (targets base pair 548-562 of marine Kipl, the
sequence of which is described ~in WO 96/02140
and deposited with Genbank
under Accession Number U09968) and its mismatch control
oligonucleotide 3437 ([SEQ ID N0:4) 5' GCA UCC CCU GUG CAG
3'j. The mismatch control oligonucleotides were designed to
have the same base composition as the antisense
oligonucleatides but with scrambled nucleotide sequences.
Oligonucleotides were efficiently delivered to
cells by association with a lipophilic reagent, dioleoyl
phosphotidylethanolamine (DOPE). For the lipofection
procedure 30 nM of each oligonucleotide was mixed with 2.5
ug/ml of DOPE (2:1j {Gilead Sciences, Inc., Foster City, CAj
in serum-free medium and incubated for l0 minutes at 37°C.
Proliferating Halb/c-3T3 fibroblasts were rinsed once in
serum-free medium and-re-fed with the oligonucleotide/DOPE
solution in low serum medium containing 0.1% serum. The cells
were then incubated for 24 hours in humidified incubators at
37°C with 5% C02.
The percentage of cells that took up the
oligonucleotides was determined by lipofecting proliferating
Balb/c-3T3 cells with an FITC-tagged random oligonucleotide
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68
(Gilead Sciences, Inc.)- for 6 hours with subsequent re-feeding
with low serum medium containing 0.1% serum for 24 hours. The
percentage of cells that were positive for uptake of the FITC-
tagged oligonucleotides was determined by UV fluorescent
microscopy. The use of the FITC-labeled oligonucleotide
control showed that 90-95% of the cells took up and
concentrated the oligonucleotides in the cell nucleus.
Cell extracts from the serum-starved (24 hours in
low serum medium containing 0.1% serum) Balb/c-3T3 fibroblasts
1o transfected with the p27 antisense or mismatch control
oligonucleotides were analyzed by immunoblotting with anti-p27
antiserum. The immunoblots showed that expression of p27
protein was substantially decreased in the antisense treated
cells (Fig. 1A) while the mismatch oligonucleotide had no
effect on accumulation of p27 following serum withdrawal.
While the results were shown for one antisense and one control
oligonucleotide, identical results were obtained with the
other antisense and control oligonucleotides.
p27 antisense treatment did not decrease expression
of the related CKI, p21. Proliferating Balb/c-3T3 fibroblasts
were lipofected with antisense and mismatch oiigonucleotides
as described above. Cells were re-fed with low serum medium
containing 0.1% serum and were analyzed 24 hours later by flow
cytometry and p21 immunoblots. As observed in Firpo et al.,
~Ql. Cell. Biol. 14:4889 {1994), p21 levels were elevated in
proliferating cells as compared to serum-starved cells. Cells
lipofected with either p2? mismatch or antisense
oligonucleotides expressed slightly higher levels of p21 as
compared to serum-starved control cells.
A decrease in the association of p27 with cyclin A
and cyclin E corresponded to the decrease in overall levels of
p27 in the antisense-treated cells (Fig. 1B). This was
associated with restoration of cyclin E and cyclin A-
associated kinase activities in serum-starved cells.
Proliferating Balb/c-3T3 fibroblasts were lipofected with
either p27 mismatch or antisense oligonucleotides for 6 hours
and were then re-fed with low serum medium containing 0.1%
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69
serum. 24 hours later Jthe.cells were harvested, and Histone
H1 kinase assays were performed on cyclin E and cyclin A
immunoprecipitates. Serum-starved cells lipofected with p27
antisense oligonucleotides contained elevated levels of cyclin
E and cyclin A associated. Histone H1 kinase activity as
compared to serum-starved cells.
In a proliferating population of Balb/c-3T3
fibroblasts 27% of the cells are in S phase, and this falls to
about 9% of cells within 24 hours following serum withdrawal
(Table). Flow cytometry of subconfluent Balb/c-3T3 cells
serum-starved for 24 hours after lipofection with either p2'7
mismatch or antisense oligonucleotides as described above
showed that cells exposed to the mismatch oligonucleotide
behaved identically to control cells. However, cells exposed
3.5 to p27 antisense oligonucleotides did not undergo Gl arrest
after serum withdrawal; 23% of the cells remained in S phase
(Table). p27 antisense oligonucleotides also prevented the
osteosarcoma cell line SA~JS-2 (Rb mutated; p53 mutated) from
exiting the cell cycle in response to serum withdrawal
(Table). This demonstrated that the requirement for p27 is
manifest in more than one cell type, and that p27 is required
for mitogen responsiveness independently of the Rb status of
the cell.
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Table: Data for experiments
using flow cytometry.
Flow
cytometry analysis was erformed as in Firpo et al.,
p described
Mol. Cell. Biol. 14:4889 (1994). The data are presented as
the percentage of cells in each phase of the ell cycle.
c
5
Call Type/Condition G1 S G2/M
10 Balb/a-3T3
Hi Serum 63.7 27.4 8,g
Low serum 86.9 9.3* 3.9
15
MSM/Lo 81.7 11.6 ~ 6.7
AS/Lo 62.2 23.4 14.4
20 MSM/Hi 59.2 26.8 14.1
AS/Hi 42.3 35.1 22.6
25 PDGF 69.4 21.4 g,2
IGF 83.2 7.7 9.1
EGF 90.5 3.4 6.1
30
PDGF/IGF/EGF 64.2 23.8 11,9
SAOS-2
35
Hi Serum 54.3 25.8 lg,g
Low Serum 70.6 i3.6 15.8
40 MSM/Lo . 60.5 16.8 22.7
AS/LO 44.2 27.9 27.9
* Flow cytometry analysis overestimated the percentage of
cells in S phase. BrdU staining demonstrated that under low
serum conditions 25~ of the cells were in S phase.
Incorporation of bromodeoxyuridine (BrdU, Amersham)
and tritiated thymidine into nuclear DNA were used as
independent measures of the effect of p27 antisense on cell
cycle progression. Twenty-four hours after serum starvation
_ _ _F _
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71
Balb/c-3T3 cells that had been transfected with either the p27
antisense or mismatch oligonucleotides were pulse-labeled with
BrdU for three hours to measure the fraction of cells
continuing to transit S phase. The percentage of nuclei
stained by uptake by BrdU was determined by immunostaining
with anti-BrdU monoclonal antibodies as described by (Ohtsubo
and Roberts, ibid.; Matsushime et al.,ibid.; and Koff et al.,
ibid.).
The
percent of cells staining positive for HrdU incorporation
(percent labeled nuclei) was determined as a percentage of the
total number of cells present on a 1 mm coverslip. The
transfected cells were labeled with tritiated thymidine
essentially as described above with the serum-starved cells
being subjected to a three-hour pulse labeling with luCi/mo of
tritiated thymidine. The percent of tritiated thymidine
incorporation was determined as the percentage of tritiated
thymidine incorporated (c.p.m.) into serum-starved and
lipofected cells as compared to asynchronously proliferating
cells pulse-labeled for three hours with tritiated thymidine.
This confirmed that cells exposed to p27 antisense
aligonucleotides continued to synthesize DNA for at least 24
hours following serum withdrawal. Of the serum starved cells
treated with p27 antisense oligonucleotides, 35% incorporated
BrdU into nuclear DNA, while only 2-3% of the cells treated
with mismatch control oligonucleotides did so. Analogous
results were obtained by using tritiated thymidine
incorporation to measure DNA synthesis rates.
In sum, these results show that cells treated with p27
antisense oligonucleotides failed to induce p27 protein in
response to mitogen depletion, and were unable to exit the
cell cycle. Although the duration of the effect for this
antisense preparation was limited, cells treated with p27
antisense expressed low levels of p2~ protein and continued to
proliferate for at least 48 hours without serum mitogens.
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72
- EXAMPLE III
The specificity of antisense oligonucleotides was
demonstrated by showing that the effect of the antisense
treatment could be overcome by restoring expression of the
targeted protein.
The degeneracy of the genetic code was used to
construct a p27 expression plasmid which could not be
inhibited by the antisense oligonucleotides, but nevertheless
encoded wild-type p27 protein (the p27 °'wobble" plasmid):
[SEQ ID N0:5] (102) L A Q E S
(106)
[SEQ ID N0:6] p27 Wild type.CTG GCG CAG GAG AGC
[SEQ TD N0:7] p27 Wobble Mutant --T --A --A --A TCA
To construct the p27 "wobble°' expression plasmid, a
"megaprimer" was generated by PCR amplification using a primer
to plasmid sequences (T7 primer) and a primer ([SEQ ID N0:8]
5'TAA AGG CAC CGC CTG GCG ACT ACC GCT GAC GTC CTG TGA TTC TTG
TGC AAG CAC CTT GCA GGC GCT C-3') which contains mutations at
the wobble positions for the amino acid sequence LAQESQD [SEQ
ID N0:9] (amino acids 102-108) of murine p27. The
"megaprimer" was subsequently used with a primer to plasmid
sequences (T3 primer) at the 3' end to PCR amplify a full
length clone which was subcloned into the expression vector
pCS2+. These mutations created a p27 sequence with 7
unmatched bases to the p27 antisense oligonucleotide and
created a unique Aat II site.
A "tagged" version of the p27 wobble plasmid was also
constructed, which encoded an electrophoretic variant of p27
resulting from a single amino acid change outside of the
domain targeted by the antisense oligonucl2otide. In addition
to the base changes listed above for amino acids 102-108, the
p27 "tagged'° wobble mutant also contained mutations at Serine
(111) and Arginine (112). These amino acids were converted to
Threonine and Serine, respectively resulting in a p27 wobble
mutant that migrates slightly slower than endogenous murine
p27 and exogenous wild type p27. The tagged p27 could be
separated and thereby distinguished from endogenous p27,
__
k.
l 1 iilnl~ l ~ Ii iurlJ.~ I
CA 02242382 2004-09-17
73
enabling a simultaneous- test of the effects of p2'7 antisense
oligonucleotides on expression from the wild type and wobble
p27 genes in the same cell.
p27 immunoblot assay were carried out on extracts from
proliferating Balb/c-3T3 cells twenty-four hours after
lipofection in the presence or absence of p27 antisense
oiigonucleotides with plasmid encoding either wild type p27 or
tagged p27 wobble mutant. It Was observed that the p27
antisense oligonucleotides effectively inhibited expression
l0 from both an exogenous wild-type p27 gene, and from the
endogenous p27 gene, but were unable to inhibit p27 protein
expression from the p27 wobble plasmid (Fig. 2A).
A p27 wobble plasmid was then used to determine
whether expression of p27 protein in the antisense treated
cells renewed their responsiveness to mitogen depletion.
These experiments were designed to study the physiological
effects of p27 expression, and therefore used a wobble plasmid
encoding fully wild type p27, rather than the electrophoretic
variant described above. Balb/c-3T3 cells were lipofected
with mismatch or p27 antisense oligonucleotides, and then
microinjected with a bath plasmid encoding B-galactosidase (to
mark the injected cells) and with the p27 wobble plasmid.
Microinjection, immunofluorescence staining, and fluorescence
microscopy were carried out as described in Fisher et al.,
Nuc. Acid Res. 21: 3857 (1993); Fianvey et al., Science 258:
1481 (1992); Wagner et al., Science 260:1510 (1993); Moulds et
al., ~iochem. 34:5044 (1995),
Cells were rinsed once in serum-free
medium and were then serum-starved in Low serum medium
containing 0.1% serum for 24 hours. As described above, the
cells were pulse-labeled with BrdU for three hours followed by
immunostaining for both SrdU and ~-galactosidase. For
costaining of ~-galactosidase and BrdU, the cells were fixed,
and then first incubated with a polyclonal anti-S
galactosidase antibody (5'3~ Inc. Boulder, CO) for 60 minutes,
followed by incubation with a fluorescein-conjugated goat
anti-rabbit IgG (Jackson Immunoresearch Laboratories, West
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74
Grove, PA) for 30 minutes. The cells were then incubated with
a fluorescein-conjugated rabbit anti-goat IgG antibody for 30
minutes. At the end of this procedure, the slides were fixed
again with 3.7% formaldehyde for '10 minutes followed by -
incubation in acetone for 1 minute. The cells were rehydrated
with TBS followed by a 10 minute treatment with 4 N FiCl and a
final wash with TBS. To visualized the BrdU staining, the
cells were incubated for d hour with a monoclonal anti-BrdU
antibody (Boehringer Mannheim, Germany), followed by a 30
minute incubation with a rhodamine-conjugated donkey anti-
mouse antibody (Jackson Immunoresearch Laboratories, West
Grove, PA)). The percentage of cells in S phase measured by
pulse labeling with BrdU which was carried out as described
above. The percent of a-galactosidase positive cells that
incorporated BrdU was determined and expressed as the percent
of cells in S phase as compared to the total number of cells
staining positive for /3-galactosidase expression. Lipofection
of cells with p27 antisense oligonucleotides markedly
decreased the percentage of cells that withdrew from the cell
cycle ,following mitogen depletion, and this was reversed by
microinjection with the p27 wobble plasmid (Fig. 2B).
These results showed that~the inability of p27
antisense treated cells to~exit the cell cycle after mitogen
depletion is specifically caused by the loss of p27
expression.
EXAMPLE IV
The basal level of p27 expressed in proliferating
cells may contribute to an inhibitory threshold imposed on Cdk
activation during G1 (Sherr and Roberts, Genes & Dev. 9:1149
(1995). In mitotically proliferating cells Cdk activation
would thus occur when the number of cyclin-Cdk complexes
exceeds the CKI threshold. Therefore, the time of Cdk
activation during G1 would depend both upon the rate of cyclin
synthesis and the level of CKI expression. (Over-expression
of Gl cyclins causes early activation of cyclin-Cdk complexes,
and a shorter G1. Ohtsubo and Roberts, Science 259:1908
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(1993); Quelle et al.,-Genes & Dev. 7: 1559 (1993); Resnitxky
and Reed, Mol Cell. Biol= 15:3463 (1995)).
This Example describes experiments which indicate that
. a p27 threshold influences the timing of Cdk activation, anal
5 therefore the duration of G1. At one extreme, high levels of
- p27 have been shown to prevent Cdk activation and arrest th.e
cell cycle in G1 (Polyak et al., Cell 78: 59 (1994), Toyashima
and Hunter, ibid., p. 67).
To determine whether decreased p27 expression allowed
l0 premature Cdk activation and a shortened Gl, exponentially
proliferating Balb/c-3T3 cells were lipofected with p27
antisense or mismatch control oligonucleotides and allowed to
continue to proliferate in high serum for an additional 24
hours.
15 The p27 antisense treatment was observed to decrease
p27 protein expression in. proliferating cells well below the
normal basal level, while no effect was seen on p27 expression
in the mismatch control. Analysis of these cell populations
by flow cytometry revealed that p27 antisense oligonucleotides
20 markedly decreased the percentage of cells in G1, indicating
that the length of G1 has been shortened relative to other
phases of the sell cycle. This supports the conclusion that
the level of p27 expressed in proliferating cells contributes
to the length of G1.
EXAMPLE V
A targeted deletion of the p27 gene was created in
transgenic mice and viable homozygous p27 "knock-out" animals
wherein the p27 locus is functionally inactivated by a
structural disruption of the gene were produced.
The knock-out mice, in which the p27 gene coding
sequence was replaced with the neomycin resistance gene, were
generated to determine the effect of such a deletion in
homozygous and heterozygous mice.' The~genomic p27 sequences
were derived from the 129/Sv strain of mice so that the
homologous recombination could take place in a congenic
background in 129/Sv mouse embryonic stem cells. A p27
CA 02242382 2004-09-17
76
genomic clone was isolated from a genomic library prepared
from 129/Sv mice (Soriano et al., Cell 64: 693-707 {1991))
using a 3ZP-
radiolabeled p27 cDNA probe. Plssmid pPNT (Tybulewicz et al.,
~ 65: 1153-1163 {1991))
containing the neomycin resistance
gene {neo, a positive selection marker) and the Xerpes sfmplex
virus thymidine kinase gene (hsv-tk; a negative selection
marker) under the control of the PGK promoter provided the
vector backbone for the targeting construct. A 7 kb Xho I
fragment containing the genomic 5' untranslated sequence of
p27 was inserted at the Xho I site of the pPNT vectar such
that the 5' end of the p27 fragment was inserted upstream of
the pGK promoter-neo expression cassette. A 1.8 kb Bgl II -
Eco RI fragment containing. the 3' untransiated p27 genomic
sequence was inserted between Bgl II and Eco Ri sites,
downstream of the PGK promoter-neo expression cassette such
that the 5' and 3' of the genamic fragments were in the same
orientation. This resulted in a total of 8.8 kb of homology
from the flanking regions of p27 with the entire p27 coding
region being replaced by the PGK promoter-neo expression
cassette from the pPNT vector. In this construct hsv-tk is
also driven by the PGK promoter but lies 3' to the p27
flanking DNA and provides a means of selection against random
integration events by causing cell death in the presence of
1(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)-5-iodouracil
(FIAU, a nucleoside analog).
The targeting construct was linearized and transfected
by electroporation into mouse embryonic stem (ES) cells. A
129/Sv derived ES cell line, AK-7, described by Zhuang et al.
{Cell 79: 875-884 (1994))
was used for electroporation.
These ES cells were routinely cultured on mitomycin C-treated
(Sigma) 6NL 76/7 STO cells (feeder cells) as described by
McMahon and Bradley (dell 62: 1073-1085 (1990)) in culture
CA 02242382 2004-09-17
77
medium containing high glucose DMEM supplemented with 158
fetal bovine serum (Hycione} and 0.1 mM ~-mercaptoethanol.
To prepare the targeting construct for transfection,
25 ~cg of the targeting construct was linearized by digestion
with Hind III, phenol-chloroform extracted, and ethanol
precipitated. The linearized vector was then electroporated
into lOT ES cells. The electroporated sells were seeded onto
two gelatinized plates with a subconfluent layer of mitomycin-
C inactivated SNL 76/? STO feeder cells. Twenty-four hours
post-electroporation, one plate received medium containing 0.2
a~M G418 and the remaining plate received 0.2 mM G418 and 0.2
mH FIAU. The presence of FIAU provided approximately a 10-
fold reduction in the number of colonies formed in comparison
to control plates with 6418 alone. The culture medium for
each plate was changed every day for the first few days, and
then changed as needed after selection had occurred. Colonies
of ES cells with true homologous recombination (HR} events, in
which p27 gene was replaced with the nee gene, were identified
by the ability to amplify a 2 kb PCR fragment unique to the
p27-knock-out construct. After 10 days of selection, a
portion of each colony was picked microscopically with a drawn
micropipette, and was directly analyzed by PCR as described by
Joynsr et al. (Nature 338: 153-156 (1989)}.
Briefly,
PCR amplification was performed as described (Kogan et al.,
dew. England J. Med. 317: 985-990 (1987})
using 4 cycles of 93°C
for 30 seconds , 36 cycles of 93°C for 30 seconds, 55°C for 30
seconds, and 65°C for 2 minutes. 'To detect the mutant p27
allele, primers neo-1 (CCT TCT ATG GCC TCC TTG ACG} and mgK2
{TTC TTA CCG AAA GGG ACA CTA ATC) [SEQ ID Nos:lO and 11,
respectively] were used in the PCR reaction. Positive
colonies, identified by PCR, were subcloned into 4-well
plates, expanded into 60 mm plates and frozen into 2-3
ampules. Southern blot analysis using probes external to both
the 5~ and 3~ end of the targeting construct confirmed that a
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78
true homologous recombination event had occurred in each of 12
clones surveyed.
To generate chimeric mice, 6 positive clones were
trypsinized into single cells, and blastocysts obtained from -
C57BL/6J mice were each injected with approximately 15 cells
from an individual clone. The injected blastocysts were then
implanted into pseudopregnant F1 mice (C57BL/6J x 129/Sv).
Chimeric pups with predominantly agouti coats (indicating a
major contribution of the ES cells to the somatic tissues)
were~selected for further breeding. Nine complete male
chimeras were subsequently identified representing three
separate ES cell clones. The male chimeras were bred to
C57BL/6J females. The chimeric males were also bred to 129/sv
females to place the knock-out mutation in a congenic
background.
The transmission of the mutant p27 transgene in 50% of
the F1 agouti progeny was again shown with PCR. Briefly,
genomic DNA prepared from tail biopsies was subjected to PCR
as described above using primers mgK-3 (TGG AAC CCT GTG CCA
TCT CTA T) and neo-1 [SEQ ID Nos:l2.and 10] to identify the
mutant (p27 knock-out gene) and primers mgk-3 and mck-5 (GAG
CAG ACG CCC AAG AAG C) [SEQ ID Nos:l2 and 13j to identify the
wild-type gene. Homozygous p27 deletions were obtained in the
F2 generation as confirmed by the absence of a the ability to
PCR a 0.5 kb fragment unique to the mutant transgene and the
absence of a 0.9 kb wildtype fragment. The complete absence
of p27 protein from these mice was confirmed on Western blots
of whole tissue extracts using rabbit polyclonal anti-p27
antisera.
In a comparison of mice of each genotype (the
homozygous knock-out, -/-; the heterozygous knock-out, -/+;
and wildtype, +/+) on the hybrid genetic background (129/Sv x
C57BL/6J), a size difference between the homozygous p27 knock- -
out mice relative to wildtype mice was demonstrated. The
hybrid mice (129/Sv x C57BL/6J) from the F2 generation
displayed a considerable size variation because the wildtype
129/Sv mice are considerably larger than their C57BL/6J
CA 02242382 2004-09-17
79
counterparts. However,- the homozygous knock-out mice
displayed, on average, about 30% greater weight than sex
matched wildtype litter mate controls (See, Figs. 3B and C).
This difference was present at 3 weeks of age and persisted to
adulthood (p<0.05). This size difference has been confirmed
in the inbred (129/Sv) background.
To further examine the size difference between the
knock-out mice and the wildtype~miee, internal organs from
randomly selected knock-out mice and wildtype litter mate
controls were dissected. The weights of internal organs of
the knock-out mice were proportional to body size with the
notable exception of the thymus and spleen, which on the
average were approximately twice as large in the knock-out
animals (Fig. 3A). Counts of nucleated cells from the spleen
and thymus from the knock-out mice confirmed the
hypercellularity of these tissues and were proportional to the
weights of the organs. p27 has been shown to be expressed
both in the cortex and the more mature medullary areas of the
mouse thymus. The increased mass of the thymus and spleen,
however, was small in comparison to the overall body weight of
the animal and therefore did not account for the weight
difference of the animals as a whole. Thus, the p27 deletion
appeared to lead to an overall increase in the animals size,
Without a disproportionate increase in fat or organomegaly.
Splenic cFC1-Meg (megakaryocyte colony forming unit),
CFU--GM (granulocyte/macrophage colony forming unit), HFU-E
(erythroid burst forming unit) were determined on spleens
harvested from two wildtype and two homozygous knock-out mice
(that were less than a factor of two different in size in
weight and total cell number) by colony-forming units assay
essentially as described (Kaushansky et al. Nature 369: 568-
571 (1994); Broudy et al., Blood 85: 1719-1726 (1995);
Kaushansky et al., J.. Clin. Invest. 96: 1683-1687 (1995)).
A comparison of
3s the total number of CFU-Meg, CFU-GM, 8FU-E from the spleens of
the knock-out and wildtype mice demonstrated up to a 10-fold
increase in.the number of each of the cell types in the
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spleens from the knockout mice relative to the number of each
celh type from the spleens of the wildtype mice (Table).
5
Table
Hematopoetic Colony Formations
CFU-GM CFU-E CFU-MK BFU-E
Femur
10 Wiidtype 25.5 1.2 88.0 -1 14.7 4.42 0.73 2.60 0.14
P27 Null 34.2 1.4 120.0 22.7 4.20 0.55 4.61 0
94
bP = 0.02 0.20 0 50 .
0 10
Spleen
Wildtype 2.90 0.61 135 16,3 2.58 0.24 1.03 0.34
15 P27 Null 9.34 0.54 400 144 7.37 0.86 3.11 0.36
P = 0.001 0.05 0.05 0.02
s Total numbers of colony forming units per organ x10'3
20 b Statistics by Mann-Whitney test.
Western blots of normal murine ES cell extracts
reveals p27 expression even at this early stage of mouse
25 development. western blots detected p27 expression in normal
mouse tissues, including a diffuse pattern of expression in
thymic tissue. No detectable p27 expression was seen in
Western blots of tissues from knock-out mice.
30 EXAMPLE VI
Production of Immortalized Cell Lines
An immortalized fibroblast cell line was derived using
standard 3T3 methods. See, e.g., Aaronson and Todaro, J. Cell
Physiol. 72:141-148 (1968) and Todaro and Green, J. Cell Biol.
35 17:299-313 (1963). p27+/- heterozygous mice were crossed and
pregnant females were sacrificed and the e10 to e14 embryos
were harvested. The head and internal organs of each embryo
were removed. The presence of the transgene was determined
using the PCR method described in Example V, above, on DNA
40 prepared from the embryo heads. The remaining tissue from
each embryo was minced and plated in Dulbecco's Modified '
Eagles Medium (DMEM) supplemented with 10~ fetal bovine serum.
The cells were cultured and split to a density of 3 X 105
cells every three days into 60 mm culture dishes. The cells
i ~i,n i~e.i.d i
CA 02242382 2004-09-17
were passaged as described through crisis, at which time the
immmortalized cells were grown, and designated as p27 +/+ 3T3,
p27 +/- 3T3 and p27 -/- 3T3.
Although the foregoing invention has been described in
some detail by way of illustration and example for purposes of
clarity of understanding, it will be obvious that certain
changes and modifications may be practiced within the scope of
the appended claims.
CA 02242382 1998-10-13
82
SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT: Roberts, James M.
Coats, Steven R.
Fero, Matthew L.
(ii) TITLE OF INVENTION: COMPOSITIONS AND METHODS FOR
MEDIATING CELL CYCLE PROGRESSION
(iii) NUMBER OF SEQUENCES: 13
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: Smart & Biggar
(B) STREET: Box 11560, Vancouver Centre, 650 W. Georgia
Street, Suite 2200
(C) CITY: Vancouver
(D) STATE: British Columbia
(E) COUNTRY: CA
(F) ZIP: V6B 4N8
(v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release #1.0, Version #1.25
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER: 2,242,382
(B) FILING DATE: 17-JAN-1997
(vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: 08/588,595
(B) FILING DATE: 18-JAN-1996
(C) APPLICATION NUMBER: 08/656,562
(D) FILING DATE: 31-MAY-1996
(viii) PATENT AGENT INFORMATION:
(A) NAME: Smart & Biggar
(B) REFERENCE NUMBER: 80323-37
(2) INFORMATION FOR SEQ ID NO:1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleotide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:
UGGCUCUCCU GCGCC 15
(2) INFORMATION FOR SEQ ID N0:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
CA 02242382 1998-07-20
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83
(ii) MOLECULE TYPE: other nucleotide
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:2:
UCCCUUUGGC GCGCC 15
(2) INFORMATION FOR SEQ ID N0:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleotide
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:3:
GCGUCUGCUC CACAG 15
(2) INFORMATION FOR SEQ ID N0:4:
(f) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 base pairs
($) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleotide
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:4:
GCAUCCCCUG UGCAG 15
(2) INFORMATION FOR SEQ ID N0:5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 5 amino acids
(B) TYPE.: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:5:
Leu Aia Gln Glu Ser
1 5
(2) INFORMATION FOR SEQ ID NO:fi:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other reucleotide
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84
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:6:
CTGGCGCAGG AGAGC I5
(2) INFORMATION FOR SEQ ID NO:7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleotide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:?:
CTTGCACAAG AATCA 15
(2) INFORMATION FOR SEQ ID N0:8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 66 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D).TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleotide
(xi) SEQUENCE DESCRIPTIONz SEQ ID N0:8:
TAAAGGCACC GCCTGGCGAC TACCGCTGAC GTCCTGTGAT TCTTGTGCAA GCACCTTGCA 60
GGCGCT 66
(2) INFORMATION FOR SEQ ID N0:9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 7 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:9:
Leu Ala Gln Glu Ser Gln Asp
I 5
(2) INFORMATION FOR SEQ ID NO:1Q:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single _
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleotide
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(xi) SEQUENCE DESCRIPTI-0N: SEQ ID NO:10:
CCTTCTATGG CCTCCTTGAC G 21
(2) INFORMATION FOR SEQ ID NO:11:
- (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 base.pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPODOGY: linear
(ii) MOLECULE TYPE: other nucleotide
{xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:
TTCTTACCGA AAGGGACACT AATC 24
(2) INFORMATION FOR SEQ ID N0:12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 22 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleotide
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:12:
TGGAACCCTG TGCCATCTCT AT 22
(2) INFORMATION FOR SEQ ID N0:.13:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 19 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleotide
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:13:
GAGCAGACGC CCAAGAAGC
