Note: Descriptions are shown in the official language in which they were submitted.
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Title: Methods and means for inducing apoptosis by
interference in RNA processing.
The present invention relates to the field of
apoptosis, as well as to the field of cancer diagnosis and
treatment and diagnosis and treatment of auto-immune
diseases and other diseases. In particular the invention
relates to improved methods and means for inducing apoptosis
in cells to be eliminated. In particular the invention
relates to novel means and methods for inducing apoptosis by
interfering with the RNA processing machinery of a cell. In
particular it relates to inhibiting or modifying the
function of RNA-protein complexes involved in RNA processing
such as snRNP's and hnRNP's. Both complexes are shown herein
to be components of the apoptotic pathway that can be
induced by chicken anemia virus proteins VP2 and/or VP3
(also called apoptin), both the hnRNP-like and snRNPs
compounds are shown to associate to CAV-derived proteins
Apoptin and VP2, which both are known to be involved in the
apoptotic process. Apoptin and VP2 as stated, are proteins
originally found in chicken anemia virus (CAV; Noteborn et
al., 1991; apoptin was originally called VP3. The apoptotic
activity of these proteins was discovered by the group of
the present inventors (Noteborn et al., 1994, 1997).
Apoptosis is an active and programmed physiological
process for eliminating superfluous, altered or malignant
cells (Earnshaw, 1995, Duke et al., 1996). Apoptosis is
characterized by shrinkage of cells, segmentation of the
nucleus, condensation and cleavage of DNA into domain-sized
fragments, in most cells followed by internucleosomal
degradation. The apoptotic cells fragment into membrane-
enclosed apoptotic bodies. Finally, neighbouring cells
and/or macrophages will rapidly phagocytose these dying
cells (Wyllie et al., 1980, White, 1996). Cells grown under
tissue-culture conditions and cells from tissue material can
be analysed for being apoptotic with agents staining DNA, as
e.g. DAPI, which stains normal DNA strongly and regularly,
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whereas apoptotic DNA is stained weakly and/or irregularly
(Noteborn et al., 1994, Telford et al., 1992).
The apoptotic process can be initiated by a variety of
regulatory stimuli (Wyllie, 1995, White 1996, Levine,
1997). Changes in the cell survival rate play an important
role in human pathogenesis, e.g. in cancer development,
which is caused by enhanced proliferation but also by
decreased cell death (Kerr et al., 1994, Paulovich, 1997). A
variety of chemotherapeutic compounds and radiation have
been demonstrated to induce apoptosis in tumor cells, in
many instances via wild-type p53 protein (Thompson, 1995,
Bellamy et al., 1995, Steller, 1995, McDonell et al., 1995).
Many tumors, however, acquire a mutation in p53 during
their development, often correlating with poor response to
cancer therapy. Transforming genes of tumorigenic DNA
viruses inactivate p53 by directly binding to it (Teodoro,
1997). An example of such an agent is the large T antigen of
the tumor DNA virus SV40. For several (leukemic) tumors, a
high expression level of the proto-oncogene Bcl-2 or Bcr-abl
is associated with a strong resistance to various apoptosis-
inducing chemotherapeutic agents (Hockenberry 1994, Sachs
and Lotem, 1997).
For such cancers (representing more than half of the
tumors) alternative anti-tumor therapies are under
development based on induction of apoptosis independent of
p53 (Thompson 1995, Paulovich et al., 1997). One has to
search for the factors involved in induction of apoptosis,
which do not need p53 and/or can not be blocked by Bcl-
2/Bcr-abl-like anti-apoptotic activities. These factors
might be part of a distinct apoptosis pathway or being (far)
downstream to the apoptosis inhibiting compounds.
Apoptin is a small protein derived from chicken anemia
virus (CAV; Noteborn and De Boer, 1995, Noteborn et al.,
1991, Noteborn et al., 1994), which can induce apoptosis in
human malignant and transformed cell lines, but not in
untransformed human cell lines. r_n v; ro, apoptin fails to
induce programmed cell death in normal lymphoid, dermal,
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epidermal, endothelial and smooth-muscle cells. However,
when normal cells are transformed they become susceptible to
apoptosis by apoptin. (Danen-van Ooschot, 1997 and Noteborn,
1996). Long-term expression of apoptin in normal human
fibroblasts revealed that apoptin has no toxic or
transforming activity in these cells.
In normal cells, apoptin was found predominantly in the
cytoplasm, whereas in transformed or malignant cells i.e.
characterized by hyperplasia, metaplasia or dysplasia, it
was located in the nucleus, suggesting that the localization
of apoptin is related to its activity (Danen-van Oorschot et
al. 1997).
Apoptin-induced apoptosis occurs in the absence of
functional p53 (Zhuang et al., 1995a), and cannot be blocked
by Bcl-2, Bcr-abl (Zhuang et al., 1995), the Bcl-2-
associating protein BAG-1 and not by the caspase-inhibitor
cowpox protein CrmA (Danen-Van Oorschot, 1997a, Noteborn,
1996) .
Therefore, apoptin is a potent agent for the
destruction of tumor cells, or other hyperplasia, metaplasia
or dysplasia which have become resistant to
(chemo)therapeutic induction of apoptosis, due to the lack
of functional p53 and (over)-expression of Bcl-2 and other
apoptosis-inhibiting agents (Noteborn et al., 1997).
The fact that apoptin does not induce apoptosis in
normal transformed human cells, at least not in v; ro,
suggests that a toxic effect of apoptin treatment in v;vo
will be very low. Noteborn et al. (1997) have provided
evidence that adenovirus expressed apoptin does not have an
acute toxic effect in v;vo. In addition, in nude mice it was
shown that apoptin has a strong anti-tumor activity.
It appears, that even pre-malignant, minimally
transformed cells, may be sensitive to the death-inducing
effect of apoptin. In addition, Noteborn and Zhang (1997)
have shown that apoptin-induced apoptosis can be used as
diagnosis of cancer-prone cells and treatment of cancer-
prone cells.
CA 02290031 2001-03-06
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Knowing that apoptin is quite safe in normal cells, but
that as soon as a cell becomes transformed and/or
immortalized (the terms may be used interchangeable herein)
the present inventors designed novel menans and methoids for
induction of apoptosis based on the identification of
compounds involved in the apoptin-induced apoptotic cascade.
These compounds are factors of an apoptosis pathway, which
is specific for transformed cells. Therefore, these proteins
are very important compounds in new treatments and diagnosis
for diseases related with aberrancies in the apoptotic
process, such as cancer, and (auto-)immune diseases.
A group of proteins found to be associated with apoptin is
the family of hnRNP-like proteins.
The invention provides an apoptin-associating hnRNP-
like protein, which is needed for RNA processing. When
apoptin associates with such proteins it interferes with
normal RNA processing, thus leading to apoptosis.
The invention thus further provides a method for
inducing apoptosis through interference with hnRNP-like
proteins (interchangeably referred to as hnRNP or hnRNP-like
proteins) or other parts of hnRNP's.
The invention provides an anti-tumor therapy based on
the interference with hnRNP-like proteins or other parts of
hnRNP's.
As an additional mechanism hnRNP can shuttle apoptin or
apoptin-like compounds to the nucleus where these compounds
can induce apoptosis.
The invention thus provides hnRNP as mediator of apoptin-
induced apoptosis, which is tumor-specific.
The present inventors have also shown a colocalization
of VP2 with snRNP's another compound also involved in RNA
processing.
The invention provides a VP2-associating snRNP-like
protein or component, which is needed for RNA processing.
The invention further provides a method for inducing
apoptosis through interference with snRNP-like proteins or
CA 02290031 2001-03-06
components (interchangeably referred to as snRNP or snRNP-
like proteins).
The invention provides an anti-tumor therapy based on
the interference with snRNP-like proteins.
The invention provides snRNP as mediator of VP2-induced
apoptosis.
The invention further provides a method for inducing
apoptosis through interference with hnRNP-like and snRNP-
like proteins.
The invention provides an anti-tumor therapy based on
the interference with either or both snRNP- and hnRNP-like
proteins.
The invention provides hnRNP and snRNP as mediators of
VP2-induced apoptosis.
More in detail the invention provides a recombinant
and/or isolated nucleic acid molecule encoding a member of
the family of hnRNP proteins involved in RNA processing
comprising at least a functional part of the sequence of
figure 1 or a sequence having at least 60, preferably 70,
preferably 80, more preferably 90a homology with said
sequence. In cells where a particular hnRNP is not used for
RNA processing such hnRNP activity can be used to shuttle
apoptotic agents such as apoptin to the nucleus. It is then
preferred to have such activity in an expression vector.
hnRNP (-like) activity is defined as any molecule directly
or indirectly providing the same kind of activity as an
hnRNP or an hnRNP-like protein.
Such a vector preferably also encodes apoptotic
activity, preferably apoptin-like acctivity which is defined
analogous to hnRNP-like activity.
In this definition functional equivalents and/or fragments
of apoptin are also encompassed.
In the case where hnRNP's are involved in RNA
processing these compounds can be inhibited by apoptin-like
activity, but also by for instance antisense molecules for
hnRNP components. The invention thus also provides a
recombinant and/or isolated nucleic acid molecule encoding
CA 02290031 2001-03-06
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an antisense recombinant molecule which can hybridize with a
recombinant acid molecule according to claim 1. Preferably
again such a molecule is present in an expression vector.
Apoptosis is preferably induced in a gene therapy
setting, so that it is preferred to deliver all vectors to
cells making use of gene delivery vehicle. Gene delivery
vehicle are known in the art and our capable of transporting
nucleic acid molecules of interest to cells. They include
recombinatn viruses (such as adenoviruses and retroviruses)
as well as polymers and liposomes and the like.
It is preferred to also block the snRNP involvement in RNA
processing. This can be done by VP2 (or VP2-like activity
(same defintion as hnRNP-like activity)) or by a further
antisense molecule hybridizing with a nucleic acid molecule
encoding a snRNP component.
Both options are provided by the present invention. The
invention thus provides an expression vector encoding an
antisense molecule for a nucleic acid encoding a component
of an snRNP, preferably together with an hnRNP antisense
molecule.
The invention also provides a method for identifying
apoptotic agents comprising the use of nucleic acid
molecules encoding members of the hnRNP-like family and the
snRNP-like family.
Apoptotic agents identified by such a route are also
considered part of this invention. These agents will
typically be hnRNP antagonists or snRNP antagonists of which
apoptin and VP2 are the first examples.
The most preferred method of inducing apoptosis is using
antagonists to both snRNP and hnRNP, but often singel
anatagonists will suffice.
The invention will be explained in more detail in the
following experimental part. This only serves for the
purpose of illustration and should not be interpreted as a
limitation of the scope of the invention.
CA 02290031 2001-03-06
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EXPERIMENTAL PART
The inventors have used the yeast-2 hybrid system
(Durfee et al., 1993) to identify apoptin-associating
cellular compounds, that are essential in the induction of
apoptosis. The used system is an in-vivo strategy to
identify human proteins capable of physically associating
with apoptin. It has been used to screen cDNA libraries for
clones encoding proteins capable of binding to a protein of
interest (Fields and Song, 1989, Yang et al., 1992).
Construction of pGBT9-VP3
For the construction of the bait plasmid, which enables
the identification of apoptin-associating proteins by means
of a yeast-two-hybrid system, plasmid pET-16b-VP3 (Noteborn,
unpublished results) was treated with N~.I and B~mHI. The
0.4 kb NdeI-B~mHI DNA fragment was isolated from low-
melting-point agarose.
Plasmid pGBT9 (Clontech Laboratories, Inc, Palo Alto,
USA) was treated with the restriction enzymes EcoRI and
~mHI. The about 5.4 kb DNA fragment was isolated and
ligated with an EcoRI-NdeI linker and 0.4-kb NdeI-BamHI DNA
fragment containing the apoptin-encoding sequences starting
from its own ATG-initiation codon. The final construct
containing a fusion gene of the GAL4-binding domain sequence
and apoptin under the regulation of the yeast promoter ADH
was called pGBT-VP3 and was proven to be correct by
restriction-enzyme analysis and DNA-sequencing according to
the Sanger method (1977).
All cloning steps were essentially carried out as
described by Maniatis et al. (1992). The plasmid pGBT-VP3
was prurified by centrifugation in a CsCl gradient and
column chromatography in Sephacryl 5500 (Pharmacia).
GAL4-activation domain-tagged cDNA library.
The expression vector pACT, containing the cDNAs from
Epstein-Bar-virus-transformed human B cells fused to
sequences for the GAL4 transcriptional activation domain,
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was used for detecting apoptin-associating proteins. The
pACT c-DNA library is derived from the lambda-ACT cDNA
library, as described by Durfee et al. 1993.
Bacterial and Yeast strains
The E.coli strain JM109 was the transformation
recipient for the plasmid pGBT9 and pGBT-VP3. The bacterial
strain Electromax/DH10B was used for the transformation
needed for the recovery the apoptin-associating pACT-cDNAs,
and was obtained from GIBCO-BRL, USA.
The yeast strain Y190 was used for screening the cDNA
library, and all transformations which are part of the used
yeast-two-hybrid system.
Media
For drug selections Luria Broth (LB) plates for E.coli
were supplemented with ampicillin (50 microgram per ml).
Yeast YPD and SC media were prepared as described by Rose et
al. (1990).
Transformation of competent yeast strain Y190 with plasmids
pGBT-VP3 and pACT-cDNA and screening for beta-galactosidase
activity.
The yeast strain Y190 was made competent and
transformed according to the methods described by Klebe et
al. (1983). The yeast cells were first transformed with
pGBT-VP3 and subsequently transformed with pACT-cDNA, and
these transformed yeast cells were grown on histidine-minus
plates, also lacking leucine and tryptophan.
Hybond-N filters were layed on yeast colonies , which
were histidine-positive and allowed to wet completely. The
filters were lifted and submerged in liquid nitrogen to
permeabilize the yeast cells. The filters were thawed and
layed with the colony side up on Whattman 3MM paper in a
petridish with Z-buffer (Per liter: 16.1 gr Na2HP04.7H20, 5.5
gr NaH2P04.H20, 0.75 gr KCl and 0,246 gr MgS04.7HZ0, pH 7.0)
containing 0.27% beta-mercapto-ethanol and 1 mg/ml X-gal.
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The filters were incubated for at least 15 minutes or during
night.
Recovery of plasmids from yeast
Total DNA from yeast cells, which were histidine- and
beta-galactosidase-positive was prepared by using the
glusulase-alkaline lysis method as described by Hoffman and
Winston (1987) and used to transform Electromax/DH10B
bacteria via electroporation using a Bio-Rad GenePulser
according the manufacturer's specifications.
Transformants were plated on LB media containing
ampicillin.
Isolation of apoptin-associating pACT clones
By means of colony-filter assay the colonies were lysed
and hybridized to a radioactive-labeled 17-mer oligomer,
which is specific for PACT (see also section Sequence
analysis).
Plasmid DNA was isolated from the pACT-positive clones,
and by means of XhQI digestion analysed for the presence of
a cDNA insert.
Sequence analysis
The subclones containing the sequences encoding
apoptin-associating proteins were sequenced using dideoxy
NTPs according to the Sanger method which was performed by
Eurogentec, Nederland BV (Maastricht, The Netherlands). The
used sequencing primer was a pACT-specific 17-mer comprising
of the DNA-sequence 5'-TACCACTACAATGGATG-3'.
The sequences of the apoptin-associating proteins were
compared with known gene sequences from the EMBL/Genbank.
Results and discussion
Apoptin induces specifically apoptosis in transformed
cells, such as cell lines derived from human tumors. To
identify the essential compounds in this cell-
CA 02290031 2001-03-06
transformation-specific and/or tumor-specific apoptosis
pathway, a yeast genetic screen was carried out.
We have used a human cDNA library, which is based on
the plasmid vector pACT containing the complete cDNA copies
made from Epstein-Barr virus-transformed human B cells
(Durfee et al., 1993).
Construction of a bait plasmid expressing a fusion
gene product of GAL4-DNA-binding domain and apoptin
To examine the existence apoptin-associating proteins
by the human transformed/tumorigenic cDNA library, a so-
called bait plasmid had to be constructed.
To that end, the complete apoptin-encoding region,
flanked by about 40 basepairs downstream from the apoptin
gene, was cloned in the multiple cloning site of plasmid
pGBT9.
The final construct, called pGBT-VP3, was analysed by
restriction-enzyme analysis and sequencing of the fusion
area between apoptin and the GAL4-DNA-binding domain.
A gene(fragment) encoding an apoptin-associating protein is
determined by transactivation of a GAL4-responsive promoter
in yeast
The apoptin gene is fused to the GAL4-DNA-binding
domain of plasmid pGBT-VP3, whereas all cDNAs derived from
the transformed human B cells are fused to the GAL4-
activation domain of plasmid pACT. If one of the cDNAs will
bind to apoptin, the GAL4-DNA-binding domain be in the
vicinity of the GAL4-activation domain resulting in the
activation of the GAL4-responsive promoter, which regulates
the reporter genes HIS3 and LacZ.
The yeast clones containing plasmid expressing apoptin
and a plasmid expressing an apoptin-associating
protein(fragment) can grow on a histidine-minus medium and
will stain blue in a beta-galactosidase assay. Subsequently,
the plasmid with the cDNA insert encoding the apoptin-
associating protein can be isolated and characterized.
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Before we could do so, however, we have determined that
transformation of yeast cells with pGBT-VP3 plasmid only or
in combination with an empty pACT vector, did not result in
the activation of the GAL4-responsive promoter.
Identification of apoptin-associating proteins encoded by
cDNAs derived from a human transformed B cell line
We have found yeast colonies, which upon transformation
with pGBT-VP3 and pACT-CDNA were able to grow on a
histidine-minus medium (also lacking leucine and tryptophan)
and stained blue in a beta-galactosidase assay. These
results indicate that these yeast colonies contain besides
the bait plasmid pGBT-VP3 a PACT plasmid encoding for a
potential apoptin-associating protein.
Plasmid DNA was isolated from these positive yeast
colonies, which were transformed in bacteria. By means of an
filter-hybridization assay using a pACT-specific labeled
DNA-probe, the clones containing pACT plasmid could be
determined. Subsequently, pACT DNA was isolated and
digested with restriction enzyme XhQI, which is indicative
for the presence of a cDNA insert. Finally, the pACT
plasmids with a cDNA insert were sequenced.
Description of apoptin-associating proteins
The yeast genetic screen for apoptin-associating
proteins resulted in the detection of a human homolog of the
hnRNP-H. The determined DNA sequence is shown in Fig. lThe
amino acid sequence of the cloned hnRNP-H homolog is shown
in Fig.2. Most likely, the cloned cDNA insert represents a
new member of the family of (human) hnRNPs.
Characteristics of hnRNP-H
The detected cDNA shows homology to part of hnRNP-H,
which is the abbreviation of heterogenous nuclear
ribonucleoprotein H). hnRNPs bind to primary RNA transcripts
(hnRNA or pre-mRNA), and are among the most abundant
proteins in the nucleus. More than 20 hnRNPs have been
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discovered sofar, differing in size, localization, domains
and nucleic acid binding specificity. Some hnRNPs were found
to be confined to the nucleus, whereas others shuttle
between the nucleus and cytoplasm (Dreyfuss et al., 1993).
Antibody staining shows a general nucleoplasmic
localization, with little staining in nucleoli and electron-
microscopy analysis localized hnRNPs, mainly to
perichromatin fibrils. There is no evidence for free (not
RNA-bound) hnRNPs in the nucleus. Many hnRNPs show
preferential binding to certain RNA sequences, like
stretches of identical bases or intron-splice sites. Almost
all, including hnRNP-H have a common domain with which they
can bind to RNA (Holzmann et al., 1997, Dreyfuss et al.,
1993 ) .
The relationship between hnRNP-like proteins and apoptin.
The hnRNP-H protein interacts with the proteins CBP80
and CBP20, the components of the nuclear cap-binding
complex. The hnRNP-H protein is closely related to hnRNP,
which also binds to mentioned CBPs (Gamberi et al., 1997).
Apoptin association will result in the inhibition of hnRNP
activity. In this respect, it is interesting to mention that
the CAV thereby developed a strategy, which makes the
translation of the capsid protein cap-independent. Synthesis
of VP2 and apoptin preceeds the production of the virus
capsid protein. Most likely, apoptin will cause inhibition
of capping activity by interference with hnRNP-H (Noteborn
et al . , 1991) .
Upon various signals the expression of hnRNP-H can be
up-regulated in transformed fibroblasts but not in normal
cells (Honore et al., 1995). This seems to correlate with
the apoptin activity in (human) transformed cells, whereas
it does not in various normal human cells.
Interestingly, immunofluorescence-microscopy revealed
that hnRNP are concentrated in discrete regions of the
nucleoplasm, in contrast to the general nucleoplasmic
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distribution of previously characterized hnRNP (Matunis et
al., 1994).
Co-localization studies in transformed human
fibroblasts and keratinocytes with apoptin-specific and
hnRNP-H monoclonal antibodies proved that apoptin is
situated in a similar nuclear structure.
Thusfar, hnRNP-H proteins were not linked with the
apoptotic pathway. We provide evidence (apoptin as an
example) that interference with the function of hnRNP-H
results in the induction of apoptosis.
Co-localization of VP2 and snRNPs.
Noteborn et al. (1997) have provided evidence that the
other CAV-derived VP2 protein induces relatively weakly
apoptosis in comparison to apoptin. Interestingly, however,
VP2 has an enhancing effect on apoptin-induced apoptosis.
VP2 is like appoptin present in distinct structures in
the nucleus. The structures of apoptin do not co-loxcalize
with these of VP2. We have examined to which structures VP2
belong. Co-localization studies with an apoptin-specific and
a snRNP-specific monoclonal antibody clearly revealed co-
localization of snRNPs and VP2. In a parallel control
experiment, it was proven that apoptin did not co-localize
with snRNPs.
We have shown that the observed VP2 and apoptin
activities, and their interactive behaviour, can be
explained by the fact that both VP2 and apoptin interfere in
the RNA processing pathway.
It is the first time that such a dualism of
interference within the apoptotic pathway is linked to the
apoptotic process.
Conclusions
In conclusion, we have provided evidence that
interference of specific factors with RNA processing, to be
precise hnRNP-like proteins and/or snRNP-like proteins, will
result in induction of apoptosis.
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Therapies based on induction of apoptosis are possible
if they succeed in the interference with the function of
hnRNP-like and/or snRNP-like proteins. Examples of such
interfering RNA-processing proteins are the CAV-derived
proteins VP2 and apoptin.
Other apoptin-associating proteins
The genetic yeast screen with pGBT-VP3 as bait plasmid and
pACT plasmid containing cDNAs from transformed human B cells
also delivered the protein filamin. The protein filamin is
localized within lamellipodia and filopodia. Filamin is one
of the cross-linking proteins of actin. It may play an
additional role of linking the cytoskeleton to cell-
substratum adhesion sites (Matsudaira, 1994). Two
independent filamin-like clones were found. The found
associating amino acid sequence of the two filamin clones
are shown in Figure 3.
Description of the figures
Figure 1 shows the DNA sequence of the analysed region of
the apoptin-associating clone hnRNP.
Figure 2 shows the amino acid sequence of the detected
hnRNP-like protein, derived from clones No-1 and No-2. In
addition, the three C-terminal amino acids (HEG) of the
multiple cloning site of pACT are given to illustrate that
the hnRNP-like amino acid sequence is in frame with the
GAL4-activation domain. This feature proves that the hnRNP-
like region is indeed synthesized in yeast cells.
Figure 3 shows the amino acids of the sequenced region of
the apoptin-associating filamin clones. In addition, the
three C-terminal amino acids (underlined) of the GAL4
activation domain are given to illustrate that the filamin-
like amino acid sequence is in frame with the GAL4-
CA 02290031 2001-03-06
activation domain. This feature proves that the filamin-like
region is indeed synthesized.
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17
21. Noteborn, M.H.M., and De Boer, G.F. (1996). Patent
USA/no. 030, 335.
22. Noteborn, M.H.M., De Boer, G.F., Van Roozelaar, D.,
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Zantema, A., Hoeben, R., Koch, G., Van Ormondt, H., and Van
der Eb, A.J. (1991). Characterization of cloned chicken
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infectious replication cycle. Journal of Virology 65, 3131-
3139.
23. Noteborn, M.H.M., Hoeben, R.C., and Pietersen, A.
(1997). A gene delivery vehicle expressing the apoptosis-
inducing proteins VP2 and/or apoptin. European Patent
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24. Noteborn, M.H.M., Todd, D., Verschueren, C.A.J., De
Gauw, H.W.F.M., Curran, W.L., Veldkamp, S., Douglas, A.J.,
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for determining the predisposition of cells to become
transformed and prophylactic treatment of cancer using
apoptin-like activity. European Patent Application no. 97439
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CA 02290031 2001-03-06
18
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1739-1746.
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CA 02290031 2001-03-06
19
SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT:
(A) Leadd B.V.
(B) STREET: Wassenaarseweg 72
(C) CITY: Leiden
(D) PROVINCE:
(E) COUNTRY: THE NETHERLANDS
(F) POSTAL CODE (ZIP): 2333 AL
(ii) TITLE OF INVENTION: Methods and means for inducing apoptosis
by interference in RNA processing
(iii) NUMBER OF SEQUENCES: 7
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: Borden Ladner Gervais LLP
(B) STREET: 60 Queen Street
(C) CITY: Ottawa
(D) PROVINCE: Province
(E) COUNTRY: Canada
(F) POSTAL CODE: K1P 5Y7
(v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Ver. 2.1
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER; 2,290,031
(B) FILING DATE: 1999-12-06
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: DAVID CONN
(B) REGISTRATION NUMBER: 3960
(C) REFERENCE/DOCKET NUMBER: PAT 45497-1
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: (613) 237-5160
(B) TELEFAX: (613) 787-3558
(2) INFORMATION FOR SEQ ID N0:1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 650 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
CA 02290031 2001-03-06
(ii) MOLECULE TYPE: DNA
(iii) ORGANISM: Homo Sapiens
(ix) FEATURE:
(D) OTHER INFORMATION: /note="Sequence of hnRNP-clone,
whereby "n" stands for unknown
nucleic acid"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:1:
CTGAAAATGA CATTGCTAAT TTCTTCTCAC CACTAAATCC AATACGAGTT CATATTGATA 60
TTGGAGCTGA TGGCAGAGCC ACAGGAGAAN CNCCNTGTAG AGTTTGTGAC ACATGAAGAT 120
GCAGTAGCTG CCATGTCTAA AGATAAAAAT AACATGCAAC ATCGATATAT TGAACTCTTC 180
TTGAATTCTA CTCCTGGAGG CGGCTCTGGC ATGGGAGGTT CTGGAATGGG AGGCTACGGA 240
AGAGATGGAA TGGATAATCA GGGAGGCTAT GGATCAGTTG GAAGAATGGG AATGGGGAAC 300
AATTACAGTG GAGGATATGG TACTCCTGAT GGTTTGGGTG GTTATGGCCG TGGTGGTGGA 360
GGCAGTGGAG GTTACTATGG GCANNGCGGC ATGAGTGGAG GTGGATGGCG TGGGATGTAC 420
TGAAAGCAAA AACACCAACA TACAAGTCTT GACAACAGCA TCTGGTCTAC TAGACTTTCT 480
TACAGATTTA ATTTCTTTTG TATTTTAAGA ACTTTATAAT GACTGAAGGA ATGTGTTTTC 540
AANATATTAT TTGNGAAAGC AACAGATTGT GATGGGAAAA TGTTTTCNGT TAGTTTATTT 600
GTTGCATACC TTGACTTAAA AATAAATTTT ATATTCAAAC CNNNAAATTG 650
(2) INFORMATION FOR SEQ ID N0:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 214 amino acids
(B) TYPE: amino acids
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: PRT
(iii) ORGANISM: Homo Sapiens
(ix) FEATURE:
(D) OTHER INFORMATION: /note="Amino acid sequence of
hnRNP-clone, whereby "x" stands for
unknown amino acid"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:2:
CA 02290031 2001-03-06
21
His Glu Gly Pro Glu Asn Asp Ile Ala Asn Phe Phe Ser Pro Leu Asn
1 5 10 15
Pro Ile Arg Val His Ile Asp Ile Gly Ala Asp Gly Arg Ala Thr Gly
20 25 30
Glu Xaa Pro Val Glu Phe Val Thr His Glu Asp Ala Val Ala Ala Met
35 40 45
Ser Lys Asp Lys Asn Asn Met Gln His Arg Tyr Ile Glu Leu Phe Leu
50 55 60
Asn Ser Thr Pro Glu Ala Ala Leu Ala Trp Glu Val Leu Glu Trp Glu
65 70 75 80
Ala Thr Glu Glu Met Glu Trp Ile Ile Arg Glu Ala Met Ile Ser Trp
85 90 95
Lys Asn Gly Asn Gly Glu Gln Leu Gln Trp Arg Ile Trp Tyr Ser Trp
100 105 110
Phe Gly Trp Leu Trp Arg Gly Gly Gly Gly Ser Gly Gly Tyr Tyr Gly
115 120 125
Xaa Xaa Gly Met Ser Gly Gly Gly Trp Arg Gly Met Tyr Ser Lys Asn
130 135 140
Thr Asn Ile Gln Val Leu Thr Thr Ala Ser Gly Leu Leu Asp Phe Leu
145 150 155 160
Thr Asp Leu Ile Ser Phe Tyr Phe Lys Asn Phe Ile Met Thr Glu Gly
165 170 175
Met Cys Phe Gln Xaa Ile Ile Xaa Glu Ser Asn Arg Leu Trp Lys Met
180 185 190
Phe Ser Val Ser Leu Phe Val Ala Tyr Leu Asp Leu Lys Ile Asn Phe
195 200 205
Ile Phe Lys Pro Xaa Asn
210
(2) INFORMATION FOR SEQ ID N0:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 199 amino acids
(B) TYPE: amino acids
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: PRT
(iii) ORGANISM: Homo Sapiens
CA 02290031 2001-03-06
22
(ix) FEATURE:
(D) OTHER INFORMATION: /note="Amino acid sequence of
hnRNP-clone"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:3:
Asp Gly Tyr Gly Phe Gly Ser Asp Arg Phe Gly Arg Asp Leu Asn Tyr
1 5 10 15
Cys Phe Ser Gly Met Ser Asp His Arg Tyr Gly Asp Gly Gly Ser Thr
20 25 30
Phe Gln Ser Thr Thr Gly His Cys Val His Met Arg Gly Leu Pro Tyr
35 40 45
Arg Ala Thr Glu Asn Asp Ile Tyr Asn Phe Phe Ser Pro Leu Asn Pro
50 55 60
Val Arg Val His Ile Glu Ile Gly Pro Asp Gly Arg Val Thr Gly Glu
65 70 75 80
Ala Asp Val Glu Phe Ala Thr His Glu Asp Ala Val Ala Ala Met Ser
85 90 95
Lys Asp Lys Ala Asn Met Gln His Arg Tyr Val Glu Leu Phe Leu Asn
100 105 110
Ser Thr Ala Gly Ala Ser Gly Gly Ala Tyr Glu His Arg Tyr Val Glu
115 120 125
Leu Phe Leu Asn Ser Thr Ala Gly Ala Ser Gly Gly Ala Tyr Gly Ser
130 135 140
Gln Met Met Gly Gly Met Gly Leu Ser Asn Gln Ser Ser Tyr Gly Gly
145 150 155 160
Pro Ala Ser Gln Gln Leu Ser Gly Gly Tyr Gly Gly Gly Tyr Gly Gly
165 170 175
Gln Ser Ser Met Ser Gly Tyr Asp Gln Val Leu Gln Glu Asn Ser Ser
180 185 190
Asp Phe Gln Ser Asn Ile Ala
195
(2) INFORMATION FOR SEQ ID N0:4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 647 amino acids
(B) TYPE: amino acids
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: PRT
CA 02290031 2001-03-06
23
(iii) ORGANISM: Homo sapiens
(ix) FEATURE:
(D) OTHER INFORMATION: /note=~~Amino acid sequence of the
apoptin-associating filamin clones"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:4:
Arg Leu Arg Asn Gly His Val Gly Ile Ser Phe Val Pro Lys Glu Thr
1 5 10 15
Gly Glu His Leu Val His Val Lys Lys Asn Gly Gln His Val Ala Ser
20 25 30
Ser Pro Ile Pro Val Val Ile Ser Gln Ser Glu Ile Gly Asp Ala Ser
35 40 45
Arg Val Arg Val Ser Gly Gln Gly Leu His Glu Gly His Thr Phe Glu
50 55 60
Pro Ala Glu Phe Ile Ile Asp Thr Arg Asp Ala Gly Tyr Gly Gly Leu
65 70 75 80
Ser Leu Ser Ile Glu Gly Pro Ser Lys Val Asp Ile Asn Thr Glu Asp
85 90 95
Leu Glu Asp Gly Thr Cys Arg Val Thr Tyr Cys Pro Thr Glu Pro Gly
100 105 110
Asn Tyr Ile Ile Asn Ile Lys Phe Ala Asp Gln His Val Pro Gly Ser
115 120 125
Pro Phe Ser Val Lys Val Thr Gly Glu Gly Arg Val Lys Glu Ser Ile
130 135 140
Thr Arg Arg Arg Arg Ala Pro Ser Val Ala Asn Val Gly Ser His Cys
145 150 155 160
Asp Leu Ser Leu Lys Ile Pro Glu Ile Ser Ile Gln Asp Met Thr Ala
165 170 175
Gln Val Thr Ser Pro Ser Gly Lys Thr His Glu Ala Glu Ile Val Glu
180 185 190
Gly Glu Asn His Thr Tyr Cys Ile Arg Phe Val Pro Ala Glu Met Gly
195 200 205
Thr His Thr Val Ser Val Lys Tyr Lys Gly Gln His Val Pro Gly Ser
210 215 220
Pro Phe Gln Phe Thr Val Gly Pro Leu Gly Glu Gly Gly Ala His Lys
225 230 235 240
Val Arg Ala Gly Gly Pro Gly Leu Glu Arg Ala Glu Ala Gly Val Pro
245 250 255
CA 02290031 2001-03-06
24
Ala Glu Phe Ser Ile Trp Thr Arg Glu Ala Gly Ala Gly Gly Leu Ala
260 265 270
Ile Ala Val Glu Gly Pro Ser Lys Ala Glu Ile Ser Phe Glu Asp Arg
275 280 285
Lys Asp Gly Ser Cys Gly Val Ala Tyr Val Val Gln Glu Pro Gly Asp
290 295 300
Tyr Glu Val Ser Val Lys Phe Asn Glu Glu His Ile Pro Asp Ser Pro
305 310 315 320
Phe Val Val Pro Val Ala Ser Pro Ser Gly Asp Ala Arg Arg Leu Thr
325 330 335
Val Ser Ser Leu Gln Glu Ser Gly Leu Lys Val Asn Gln Pro Ala Ser
340 345 350
Phe Ala Val Ser Leu Asn Gly Ala Lys Gly Ala Ile Asp Ala Lys Val
355 360 365
His Ser Pro Ser Gly Ala Leu Glu Glu Cys Tyr Val Thr Glu Ile Asp
370 375 380
Gln Asp Lys Tyr Ala Val Arg Phe Ile Pro Arg Glu Asn Gly Val Tyr
385 390 395 400
Leu Ile Asp Val Lys Phe Asn Gly Thr His Ile Pro Gly Ser Pro Phe
405 410 415
Lys Ile Arg Val Gly Glu Pro Gly His Gly Gly Asp Pro Gly Leu Val
420 425 430
Ser Ala Tyr Gly Ala Gly Leu Glu Gly Gly Val Thr Gly Asn Pro Ala
435 440 445
Glu Phe Val Val Asn Thr Ser Asn Ala Gly Ala Gly Ala Leu Ser Val
450 455 460
Thr Ile Asp Gly Pro Ser Lys Val Lys Met Asp Cys Gln Glu Cys Pro
465 470 475 480
Glu Gly Tyr Arg Val Thr Tyr Thr Pro Met Ala Pro Gly Ser Tyr Leu
485 490 495
Ile Ser Ile Lys Tyr Gly Gly Pro Tyr His Ile Gly Gly Ser Pro Phe
500 505 510
Lys Ala Lys Val Thr Gly Pro Arg Leu Val Ser Asn His Ser Leu His
515 520 525
Glu Thr Ser Ser Val Phe Val Asp Ser Leu Thr Lys Ala Thr Cys Ala
530 535 540
CA 02290031 2001-03-06
Pro Gln His Gly Ala Pro Gly Pro Gly Pro Ala Asp Ala Ser Lys Val
545 550 555
560
Val Ala Lys Gly Leu Gly Leu Ser Lys Ala Tyr Val Gly Gln Lys Ser
565 570 575
Ser Phe Thr Val Asp Cys Ser Lys Ala Gly Asn Asn Met Leu Leu Val
580 585 590
Gly Val His Gly Pro Arg Thr Pro Cys Glu Glu Ile Leu Val Lys His
595 600 605
Val Gly Ser Arg Leu Tyr Ser Val Ser Tyr Leu Leu Lys Asp Lys Gly
610 615 620
Glu Tyr Thr Leu Val Val Lys Trp Gly His Glu His Ile Pro Gly Ser
625 630 635 640
Pro Tyr Arg Val Val Val Pro
645
(2) INFORMATION FOR SEQ ID N0:5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 213 amino acids
(B) TYPE: amino acids
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: PRT
(iii) ORGANISM: Homo Sapiens
(ix) FEATURE:
(D) OTHER INFORMATION: /note="Amino acid sequence of the
apoptin-associating filamin clones"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:5:
His Glu Gly Arg Gly Val Thr Gly Asn Pro Ala Glu Phe Val Val Asn
1 5 10 15
Thr Ser Asn Ala Gly Ala Gly Ala Leu Ser Val Thr Ile Asp Gly Pro
20 25 30
Ser Lys Val Lys Met Asp Cys Gln Glu Cys Pro Glu Gly Tyr Arg Val
40 45
Thr Tyr Thr Pro Met Ala Pro Gly Ser Tyr Leu Ile Ser Ile Lys Tyr
50 55 60
Gly Gly Pro Tyr His Ile Gly Gly Ser Pro Phe Lys Ala Lys Val Thr
65 70 75 80
CA 02290031 2001-03-06
26
GlyProArg LeuValSer AsnHisSer LeuHisGluThr SerSerVal
85 90 95
PheValAsp SerLeuThr LysAlaThr CysAlaProHis HisGlyAla
100 105 110
ProGlyPro GlyProAla AspAlaSer LysValValAla LysGlyLeu
115 120 125
GlyLeuSer LysAlaTyr ValCysHis LysSerSerPhe ThrValAsp
130 135 140
CysSerLys AlaCysIle IleMetLeu LeuValGlyVal HisGlyPro
145 150 155 160
TrpThrPro CysAspGlu IleLeuVal LysAlaArgGly GlnProAla
165 170 175
LeuGlnArg ValLeuThr CysPheLys AspLysGlyGlu ValHisThr
180 185 190
GlyGlyGln AsnGlyGly AspTyrGln IleProCysLys ProLeuPro
195 200 205
LeuCysGly CysPro
210
(2) INFORMATION FOR SEQ ID N0:6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 213 amino acids
(B) TYPE: amino acids
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: PRT
(iii) ORGANISM: Homo Sapiens
(ix) FEATURE:
(D) OTHER INFORMATION: /note="Amino acid sequence of the
apoptin-associating filamin clones,
whereby "x" stands for unknown amino
acid"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:6:
His Glu Gly Arg Pro Thr Glu Pro Gly Asn Tyr Ile Ile Asn Ile Lys
1 5 10 15
Phe Ala Asp Gln His Val Pro Gly Ser Pro Phe Ser Val Lys Val Thr
20 25 30
CA 02290031 2001-03-06
27
Gly Glu Gly Arg Val Lys Glu Ser Ile Thr Arg Arg Arg Arg Ala Pro
35 40 45
Ser Val Ala Asn Va1 Gly Ser His Cys Asp Leu Ser Leu Lys Ile Pro
50 55 60
Glu Ile Ser Ile Gln Asp Met Thr Ala Gln Val Thr Ser Pro Ser Gly
65 70 75 80
Lys Thr His Glu Ala Glu Ile Val Glu Gly Glu Asn His Thr Tyr Cys
85 90 95
Ile Arg Phe Val Pro Ala Glu Met Gly Thr His Thr Val Ser Val Lys
100 105 110
Tyr Lys Gly Gln His Val Pro Gly Ser Pro Phe Gln Phe Thr Val Gly
115 120 125
Pro Leu Gly Glu Gly Gly Ala His Xaa Val Arg Ala Gly Gly Pro Gly
130 135 140
Leu Xaa Lys Ser Ser Trp Ser Ala Ser Arg Ile Gln Tyr Leu Gly Pro
145 150 155 160
Gly Lys Leu Val Leu Glu Ala Trp Pro Leu Leu Ser Xaa Ala Pro Ala
165 170 175
Xaa Leu Xaa Ser Leu Leu Arg Thr Ala Arg Thr Ala Pro Val Val Leu
180 185 190
Leu Met Leu Val Xaa Glu Pro Ser Asp Xaa Asn Pro Xaa Gln Val Ser
195 200 205
Thr Lys Glu His Xaa
210
(2) INFORMATION FOR SEQ ID N0:7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 17 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(iii) ORGANISM: Artificial Sequence
(ix) FEATURE:
(D) OTHER INFORMATION: Description of Artificial Sequence:
primer
CA 02290031 2001-03-06
28
(ix) FEATURE:
(D) OTHER INFORMATION: /note="PACT-specific 17-mer"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:7:
TACCACTACA ATGGATG 17
