TITLE
SHORT INTERFERING RNA DUPLEXES TARGETING AN IRES SEQUENCE AND
USES THEREFOR
Related Applications
[0001 ] This application claims the benefit of priority to U.S. Provisional Patent Application No
60/792,968, filed April 19, 2006, which is hereby incorporated by reference herein in its entirety.
BACKGROUND OF THE INVENTION
Field of the Invention
[0002] The present invention relates to the use of inhibitory polynucleotides, particularly short
interfering RNA (siRNA) duplexes, that target an internal ribosome entry site in methods of
inhibiting gene expression, e.g., screening assays.
Related Background Art
[0003] It is well known in the art that the transcription of a gene usually requires a promoter that
is upstream of the gene, that transcription usually results in a monocistronic mRNA (i.e., an
mRNA transcript that comprises only one protein-coding region), and that translation of the
resulting monocistronic mRNA is usually initiated by a translation initiation complex in a cap-
dependent mechanism that involves recognition of a 5' terminal cap-structure on the

monocistronic mRNA (see, e.g., Merrick and Hershey (1996) "The pathway and mechanism of
eukaryotic protein synthesis." In Translational Control, J.W.B. Hershey, M.B. Mathews, and N.
Sonenberg, Eds. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY), pp. 31-69).
The translation initiation complex usually moves along the monocistronic mRNA until it reaches
a first initiation codon (AUG), usually within 50-100 nucleotides of the cap-structure, whereby
translation of the mRNA into protein would usually commence. In other words, translation of
the mRNA into protein generally commences at the first initiation AUG codon. This canonical
model of monocistronic transcription and translation, found in most eukaryotic and some
prokaryotic cells, poses a problem in the utilization of recombinant DNA technology, e.g., for
gene therapy, because it is sometimes advantageous to transfer and express multiple transgenes
within a single host cell.
[0004] Early in the development of recombinant DNA technology, when an investigater was
interested in expressing more than one protein in a single host cell, the genes to be transferred
(transgenes), e.g., those encoding each protein of interest, were placed on different expression
vectors, necessitating that the host cell be successfully modified with each such expression
vector. As expected, modification of a host cell with more than one expression vector often
proved difficult and laborious. Alternatively, a single expression vector comprising each
transgene that encoded a protein(s) of interest was created such that the transcription of each
transgene was controlled by its own individual promoter, i.e., several monocistronic mRNAs
were transcribed from the single expression vector. However, the presence of several promoters
within one expression vector often resulted in reduction or loss of expression over time, likely
due to interference between the promoter sequences. These problems were solved by the
discovery and subsequent utilization of internal ribosome entry sites (IRESes). An IRES is
generally placed downstream of a protein-coding region, the translation of which is initiated by a
first initiation codon. The sequence of an IRES allows protein translation to commence from an
internal (i.e., second) initiation codon (AUG), i.e., an AUG codon downstream of the IRES and
the first initiation AUG codon, and thus allows an mRNA transcript to be polycistronic, i.e.,
capable of comprising more than one protein-coding region.
[0005] To date, IRESes have been identified in the 5' region of noncapped viral mRNAs, such as
members of the Picornaviridae family, e.g., poliomyelitis virus (Pelletier et al. (1988) Mol. Cell.
Biol. 8(3): 1103-12), poliovirus (PV), encephalomyocarditis virus (EMCV) (Jang et al. (1988)

J. Virol. 62(8):2636-43), and foot-and-mouth disease virus (FMDV) (reviewed in Belsham and
Sonenberg (1996) Microbiol. Rev. 60(3):499-511; Robertson et al. (1999) RNA 5(9): 3167-79;
Jackson and Kaminski (1995) RNA l(10):985-1000; Herman (1989) Trends Biochem Sci.
14(6):219-22). IRESes have also been detected in transcripts from other viruses, such as
VL30-type murine retrotransposons (Berlioz et al. (1995)J. Virol. 69(10):6400-07), cardiovirus,
rhinovirus, aphthovirus, hepatitis C virus (HCV), and more recently, in mRNAs encoding the gag
precursor of the Friend (FMLV) and Moloney (MoMLV) murine leukemia viruses (Berlioz and
Darlix (1995)J. Virol. 69(4):2214-22; Vagner et al. (1995)7. Biol. Chem. 270(35):20376-83).
The presence of IRESes in cellular RNAs has also been described. Examples of cellular mRNAs
that comprise IRESes include those encoding immunoglobulin heavy-chain binding protein (BiP)
(Macejak and Sarnow (1991) Nature 353:90-94); certain growth factors such as vascular
endothelial growth factor (VEGF), fibroblast growth factor 2 and insubs like growth factor
(Teerink et al. (1995) Biochim. Biophys. Acta 1264(3):403-08; Vagner et al. (1995) Mol. Cell
Biol. 15(l):35-44); translational initiation factor eIF4G (Gan and Rhoads (1996) J. Biol. Chem.
271(2):623-26), and the yeast transcription factors TFIID and HAP4 (lizuka et al. (1994) Mol.
Cell. Biol. 14(11):7322-30) (see also, Oh et al. (1992) Genes Dev. 6(9): 1643-53; He et al. (1996)
Proc. Natl. Acad. Sci. USA 93(14):7274-78; He et al. (1996) Gene 175(1-2): 121-25:, Tomanin et
al. (1997) Gene 193(2): 129-40; Gambotto et al. (1999) Cancer Gene Ther. 6(l):45-53; Qiao et al.
(1999) Cancer Gene Ther. 6(4):373-79)).
[0006] In the context of recombinant DNA technology, expression vectors comprising IRESes
have been described (see, e.g., International Published Patent Application Nos. WO 98/37189;
WO 99/25860; and WO 93/03143). Generally, these expression vectors would allow the
placement of an IRES between at least two transgenes, and subsequently would allow the
expression of at least two transgenes from a single promoter. In particular, transcription from the
single promoter would result in an mRNA that could be polycistronic, e.g., wherein the at least
two protein-coding regions were separated by at least one IRES, and translation would begin at
both the first initiation AUG codon, and an internal AUG codon(s) downstream of the IRES(es).
[0007] IRESes are powerful tools in the field of recombinant DNA technology because they
allow the translation of several genes from a single mRNA transcript. In other words, use of an
IRES for the expression of multiple different transgenes by a single host cell obviates the need to
modify a host cell with either multiple expression vectors, or with an expression vector

comprising several promoters that may interfere with one another. Additionally, several groups
have reported the stability and functionality of the EMCV-IRES in chicken and mouse embryos,
and in many organs of adult mice (Ghattas et al. (1991) Mol. Cell. Biol. 11(12):5848-59; Kim et
al. (1992) Mol. Cell. Biol. 12(8):3636-43; Creancier et al. (2000)J. Cell. Biol. 150(l):275-81).
[0008] Although IRESes have been incorporated in recombinant DNA technology, the utility of
this technology, e.g., in gene therapy, may be advanced upon investigation into 1) the effects of
expression of such transgenes on the modified host cell or organism, e.g., the effect of the
transgene on the metabolism of the modified host, and 2) the functions of the proteins encoded
by transgenes. A popular method of investigating the effect(s) and function(s) of transgene
expression on a host cell or organism is to inhibit (e.g., reduce, interfere, downregulate, knock
down, etc.) expression of the transgene after it has been successfully introduced into and
expressed by the host cell or organism.
[0009] Several approaches have been developed to inhibit the expression of a gene of interest
(e.g., a transgene, endogenous gene, etc.), including antisense, triple-helix, cosuppression, and
RNAi methods. These methods have involved the utilization of targeting nucleic acid molecules
that are the reverse complement of the targeted gene mRNA transcript (or portions thereof), form
triple-helical structures with the targeted gene, are exact duplicates of the targeted gene, or are
duplex molecules of short interfering RNA (siRNA) comprising a nucleotide sequence of the
targeted gene (or portions thereof), respectively. To date, these approaches have been used to
specifically target a single gene of interest, and as such, require that the sequence of the targeting
molecule (e.g., the antisense molecule, triple-helix forming molecule, the cosuppression
transgene molecule, and the siRNA molecule) correspond to (i.e., specifically hybridize to at
least a portion of one, the other, or both strands of) at least a portion of the targeted gene of
interest. As such, the application of these approaches has heretofore required the investigator to
know the sequence of the targeted gene of interest, and/or the portion of the targeted gene
sequence that has the greatest susceptibility to being targeted, and to create a unique targeting
molecule for each targeted gene. To date, there is neither a mechanism by which to inhibit
expression of a targeted gene without first knowing the sequence of the gene of interest, nor, if
the sequence of the gene of interest is known, is there an efficient assay to determine which
portion of the gene sequence is more susceptible to inhibition.

[0010] The present invention solves these problems by providing inhibitory polynucleotides and
methods of using these inhibitory polynucleotides in, methods of e.g., 1) inhibiting (e.g.,
reducing, interfering with, downregulating, knocking down, etc.) the expression of at least one
transgene of interest that does not require the investigator to know or determine the sequence of
the transgene and/or 2) screening libraries of targeting polynucleotides to inhibit expression of a
gene of interest, regardless of whether the gene of interest is a transgene or an endogenous gene.
SUMMARY OF THE INVENTION
[0011] The present invention is related to the discovery that inhibitory polynucleotides that target
an IRES may be used to downregulate (e.g., inhibit) the expression of at least one gene of interest
that is transcribed with its protein-coding region as part of an mRNA transcript comprising a
nucleotide sequence corresponding to the targeted IRES. Accordingly, the present invention
provides an inhibitory polynucleotide directed against an IRES, e.g., an IRES that has the
nucleotide sequence of SEQ ID NO: 1.
[0012] An inhibitory polynucleotide of the invention may be an siRNA molecule, e.g., in one
embodiment of the invention, an inhibitory polynucleotide of the invention comprises a first
strand of an siRNA. In another embodiment of the invention, the first strand of the siRNA has
and/or consists essentially of the RNA equivalent of a nucleotide sequence selected from the
group consisting of the nucleotide sequence of SEQ ID NO:1, a portion of the nucleotide
sequence of SEQ ID NO:1, the complement of the nucleotide sequence of SEQ ID NO: 1, and a
portion of the complement of the nucleotide sequence of SEQ ID NO:1. In another embodiment
of the invention, the first strand of the siRNA is between 5 and 548 nucleotides in length. In
another embodiment of the invention, the first strand of the siRNA has and/or consists essentially
of the RNA equivalent of a nucleotide sequence selected from the group consisting of the
nucleotide sequence of SEQ ID NO:2, the nucleotide sequence of SEQ ID NO:3, the nucleotide
sequence of SEQ ID NO:4, and the nucleotide sequence of subsequences thereof. In another
embodiment of the invention, the first strand of the siRNA is self-complementary and further
comprises a hairpin loop, e.g., an siRNA of the invention may comprise the RNA equivalent of a
nucleotide sequence selected from the group consisting of the nucleotide sequence
complementary to the nucleotide sequence of SEQ ID NO:2, the nucleotide sequence
complementary to the nucleotide sequence of SEQ ID NO:3, and the nucleotide sequence

complementary to the nucleotide sequence of SEQ ID NO:4. In yet another embodiment of the
invention, the inhibitory polynucleotide is an antisense molecule.
[0013] The present invention also provides isolated DNA molecules that encode the inhibitory
polynucleotides of the invention, e.g., as described herein. In one embodiment of the invention,
the DNA molecule is operably linked to at least one expression control sequence. The present
invention also provides a host cell transformed or transfected with such DNA molecules that
encode the inhibitory polynucleotides of the invention. Further, the invention also provides a
microorganism that contains a DNA molecule(s) that encodes an inhibitory polynucleotide of the
invention. In one embodiment, the invention provides a nonhuman transgenic animal in which
the somatic and germ cells contain DNA that encodes an inhibitory polynucleotide of the
invention. In another embodiment, the invention provides a transgenic plant in which the
somatic and germ cells contain DNA that that encodes an inhibitory polynucleotide of the
invention.
[0014] In one embodiment of the invention, an siRNA of the invention (e.g., as described above)
further comprises a second strand of that is complementary to the first strand of the siRNA.
Also, the present invention provides an isolated DNA molecule that encodes a second strand of
an siRNA molecule of the invention. In one embodiment of the invention, the isolated DNA
molecule may be operably linked to at least one expression control sequence. The invention also
provides a host cell transformed with such operably linked DNA molecule(s). In one
embodiment, the invention provides a microorganism that contains DNA that encodes the second
strand of an siRNA of the invention. The invention also provides a nonhuman transgenic animal
in which the somatic and germ cells contain DNA that encodes a second strand of an siRNA
molecule of the invention. In another embodiment, the invention provides a transgenic plant in
which the somatic and germ cells contain DNA that encodes a second strand of an siRNA of the
invention.
[0015] The invention also provides a kit comprising an inhibitory polynucleotide of the invention
and methods of using an inhibitory polynucleotide of the invention.
[0016] The invention also provides a method of downregulating the expression of a transgene by
a host cell, wherein the transgene is transcribed as part of an mRNA transcript comprising a
nucleotide sequence corresponding to an IRES, the method comprising the step of introducing

into the host cell an inhibitory polynucleotide that targets the IRES. In one embodiment of the
invention the IRES has and/or consists essentially of the nucleotide sequence of SEQ ID NO:1
BRIEF DESCRIPTION OF THE DRAWINGS
[0017] FIG. 1 shows the nucleotide sequence of the EMCV-IRES (equivalent to SEQ ID NO:1).
Indicated in bold within the EMCV-IRES sequence are examples of three portions of the
EMCV-IRES sequence (IRES1, IRES2, and IRES3; SEQ ID NOs:2, 3, and 4, respectively) that
may be optimally targeted by siRNA molecules. Also shown in boxes are examples of three
siRNA molecules (siRNAl, siRNA2, and siRNA3; SEQ ID NOs:5, 6, and 7, respectively) that
may be used to target the EMCV-IRES (at IRES1, IRES2, and IRES3, respectively).
[0018] FIG. 2A demonstrates the antibody titer (fig/ml; y-axis) produced by CHO cells
genetically modified to express antibodies from a polycistronic mRNA comprising at least one
EMCV-IRES sequence after transfection with the following (x-axis): control transfection
reagents (control), siRNA1 molecules directed against IRES1 (IRES1), siRNA2 molecules
directed against IRES2 (IRES2), siRNA3 molecules directed against IRES3 (IRES3), or a pool of
siRNA1, siRNA2 and siRNA3 molecules (Pool). Bars represent the average ± SEM antibody
titer of three experiments (n=3), either three days after transfection (day3; ) or six days after
transfection (day6; ). FIG. 2B demonstrates the cell-specific productivity (titer/cell #/day; y-
axis) of recombinant antibody produced by CHO cells genetically modified to express antibodies
from a polycistronic mRNA comprising at least one EMCV-IRES sequence cells after
transfection with the following (x-axis): control transfection reagents (control), siRNA1
molecules directed against IRES1 (IRES1), siRNA2 molecules directed against IRES2 (IRES2),
siRNA3 molecules directed against IRES3 (IRES3), or a pool of siRNA 1, siRNA2 and siRNA3
molecules (Pool). Bars represent the average ± SEM antibody titer of three experiments (n=3),
either three days after transfection (day3: ) or six days after transfection (day6: ).
DETAILED DISCRETION OF THE INVENTION
[0019] The present invention relates to the discovery of internal ribosome entry sites (IRESes)
and the subsequent use of IRESes in recombinant DNA technology to initiate and control the
translation of a protein-coding region within a polycistronic mRNA transcript. The invention is
based on the discovery that targeting a targeted IRES with inhibitory polynucleotides efficiently
prevents translation of at least the protein-coding region upstream of the targeted IRES, and

perhaps all protein-coding regions of the mRNA transcript comprising a nucleotide sequence
corresponding to the targeted IRES. Consequently, provided herein are inhibitory
polynucleotides directed toward an IRES (i.e., a targeted IRES) and methods of using them in
nonspecific approaches to knock down the expression of a gene of interest. As it is the IRES that
is being targeted, this method does not require directing inhibitory polynucleotides precisely
against the gene of interest; i.e., methods provided herein do not require that that the sequence of
the gene of interest be known or determined, and allows the targeting molecules to be used to
inhibit the expression of many transgenes. The method only requires that the protein-coding
region transcribed from the gene of interest be within an mRNA transcript comprising a
nucleotide sequence corresponding to a targeted IRES. In other words, it is likely that the entire
mRNA transcript comprising the IRES will be targeted by an inhibitory polynucleotide of the
invention, e.g.. an siRNA molecule, for inhibition. For example, an siRNA molecule fargeting
the IRES on an mRNA transcript comprising (from 5' to 3') a first transgene, the IRES, and a
second transgene, may be used to knock down expression of both the second and first transgenes.
An mRNA transcript need not be polycistronic to be successfully targeted by an inhibitory
polynucleotide of the invention. For example, an siRNA targeting an IRES sequence on an
mRNA transcript that comprises only one transgene, which is either upstream or downstream of
the IRES, may be used to knock down expression of the one transgene. In a preferred
embodiment, the mRNA transcribed from the gene of interest, i.e., containing the protein-coding
region of the gene of interest, is upstream of an IRES.
[0020] In particular, the present invention is based on the discovery that inhibitory
polynucleotides directed against an IRES may inhibit translation of a protein-coding region that
is part of an mRNA transcript comprising a nucleotide sequence corresponding to the targeted
IRES. It will be apparent to one of skill in the art that use of such inhibitory polynucleotides
directed toward an IRES allows methods of knocking down expression of a gene of interest, the
protein-coding region of which is within an mRNA transcript comprising a nucleotide sequence
corresponding to the targeted IRES, and that such methods do not require modification or
targeting of the transgene itself. A skilled artisan will recognize that the inhibiting
polynucleotides provided herein will not only enable the downregulation of a gene of interest, but
also may be used in methods of screening siRNA libraries, e.g., as positive controls.

[0021] As such, the invention provides inhibitory polynucleotides that target an IRES.
Additionally, the invention provides methods of modifying a host cell or organism to express
inhibitory polynucleotides of the invention, and also provides such modified host cells or
organisms. The invention also provides methods of using the inhibitory polynucleotides to alter
the expression of genes of interest and as positive controls in screening assays, e.g., siRNA
screening assays.
[0022] In accordance with the invention, a gene of interest may encode a therapeutic protein. A
therapeutic protein, as used herein, is a protein or peptide that has a biological effect on a region
in the body on which it acts or on a region of the body on which it remotely acts via
intermediates. A therapeutic protein can be, for example, a secreted protein, such as, an
antibody, an antigen-binding fragment of an antibody, a soluble receptor, a receptor fusion, a
cytokine, a growth factor, an enzyme, or a clotting factor, as described in more detail herein. The
above list of proteins is merely exemplary in nature, and is not intended to be a limiting
recitation. One of ordinary skill in the art will understand that any protein may be used in
accordance with the present invention and will be able to select the particular protein to be
produced based as needed.
[0023] As used in the specification, the terms polypeptide, protein and peptide are synonymous
and are used interchangeably. Accordingly, as used herein, the size of a protein, peptide or
polypeptide generally comprises more than 2 amino acids. For example, a protein, peptide or
polypeptide can comprise from about 2 to about 20 amino acids, from about 20 to about 40
amino acids, from about 40 to about 100 amino acids, from about 100 amino acids to about 200
amino acids, from about 200 amino acids to about 300 amino acids, and so on.
[0024] As used herein, an amino acid refers to any naturally occurring amino acid, any amino
acid derivative or any amino acid mimic known in the art. In certain embodiments, the residues
of the protein or peptide are sequential, without any non-amino acid interrupting the sequence of
amino acid residues. In other embodiments, the sequence may comprise one or more non-amino
acid moieties. In particular embodiments, the sequence of residues of the protein or peptide may
be interrupted by one or more non-amino acid moieties.
[0025] As used herein, an antibody refers to any antibody-like molecule that has an antigen
binding region, and includes antibody fragments such as Fab', Fab, F(ab')2, single domain

antibodies (DABs), Fv, scFv (single chain Fv), and the like. Techniques for preparing and using
various antibody-based constructs and fragments are well known in the art. Means for preparing
and characterizing antibodies are also well known in the art (see, e.g., Harlow and Lane,
Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988; incorporated herein by
reference in its entirety). For example, an antibody can include at least one, and preferably two
full-length heavy chains, and at least one, and preferably two light chains. The term "antibody'
as used herein includes an antibody fragment or a variant molecule such as an antigen-binding
fragment (e.g., an Fab, F(ab')2, Fv, a single chain Fv fragment, a heavy chain fragment (e.g., a
camelid VHH) and a binding domain-immunoglobulin fusion (e.g., SMIPTM).
[0026] The antibody can be a monoclonal or single-specificity antibody. The antibody can also
be a human, humanized, chimeric, CDR-grafted, or in vitro-generated antibody. Tn yet other
embodiments, the antibody has a heavy chain constant region chosen from, e.g., IgG i, igG2,
IgG3, or IgG4. In another embodiment, the antibody has a light chain chosen from, e.g., kappa
or lambda. In one embodiment, the constant region is altered, e.g., mutated, to modify the
properties of the antibody (e.g., to increase or decrease one or more of: Fc receptor binding,
antibody glycosylation, the number of cysteine residues, effector cell function, or complement
function). Typically, the antibody specifically binds to a predetermined antigen, e.g., an antigen
associated with a disorder, e.g., a neurodegenerative, metabolic, inflammatory, autoimmune,
and/or malignant disorder.
[0027] Small Modular ImmunoPharmaceuticals (SMIP™) provide an example of a variant
molecule comprising a binding domain polypeptide. SMIPs and their uses and applications are
disclosed in, e.g., U.S. Published Patent Application. Nos. 2003/0118592, 2003/0133939,
2004/0058445, 2005/0136049, 2005/0175614,2005/0180970, 2005/0186216, 2005/0202012,
2005/0202023,2005/0202028, 2005/0202534, and 2005/0238646, and related patent family
members thereof, all of which are hereby incorporated by reference herein in their entireties.
[0028] Single domain antibodies can include antibodies whose complementary determining
regions are part of a single domain polypeptide. Examples include, but are not limited to, heavy
chain antibodies, antibodies naturally devoid of light chains, single domain antibodies derived
from conventional four-chain antibodies, engineered antibodies and single domain scaffolds
other than those derived from antibodies. Single domain antibodies may be any of the art, or any

future single domain antibodies. Single domain antibodies may be derived from any species
including, but not limited to mouse, human, camel, llama, goat, rabbit, and bovine. According to
one aspect of the invention, a single domain antibody as used herein is a naturally occurring
single domain antibody known as heavy chain antibody devoid of light chains. Such single
domain antibodies are disclosed in International Published Application No. WO 9404678, for
example. For reasons of clarity, this variable domain derived from a heavy chain antibody
naturally devoid of light chain is known herein as a VHH or nanobody to distinguish it from the
conventional VH of four-chain immunoglobulins. Such a VHH molecule can be derived from
antibodies raised in Camelidae species, for example in camel, llama, dromedary, alpaca and
guanaco. Other species besides Camelidae may produce heavy chain antibodies naturally devoid
of light chain; such VHHs are within the scope of the invention.
[0029] Examples of binding fragments encompassed within the term ''antigen-binding fragment"
of an antibody include (i) a Fab fragment, a monovalent fragment consisting of the VI., VH, CL
and CH1 domains; (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments
linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CH1
domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an
antibody, (v) a dAb fragment, which consists of a VH domain; (vi) a camelid or camelized
variable domain, e.g., a VHH domain; (vii) a single chain Fv (scFv); (viii) a bispecific antibody;
and (ix) one or more fragments of an immunoglobulin molecule fused to an Fc region.
Furthermore, although the two domains of the Fv fragment, VL and VH, are coded for by
separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables
them to be made as a single protein chain in which the VL and VH regions pair to form
monovalent molecules (known as single chain Fv (scFv); see, e.g., Bird et al. (1988) Science
242:423-26; Huston et al. (1988) Proc. Natl. Acad. Sci. U.S.A. 85:5879-83). Such single chain
antibodies are also intended to be encompassed within the term "antigen-binding fragment" of an
antibody. These antibody fragments are obtained using conventional techniques known to those
skilled in the art, and the fragments are evaluated for function in the same manner as are intact
antibodies.
[0030] Other than "bispecific" or "bifunctional" antibodies, an antibody is understood to have
each of its binding sites identical. A "bispecific" or "bifunctional" antibody is an artificial hybrid
antibody having two different heavy/light chain pairs and two different binding sites. Bispecific

antibodies can be produced by a variety of methods including fusion of hybridomas or linking of
Fab' fragments (see, e.g., Songsivilai and Lachmann (1990) Clin. Exp. Immunol. 79:315-21;
Kostelny etal. (1992)J. Immunol. 148:1547-53).
Target Sequences
[0031] The invention may be applied to most, if not all, well-known IRESes (particularly those
routinely used in recombinant DNA methods), without undue experimentation. Thus, it is part of
the invention that a target sequence related to the invention is an IRES sequence derived from
any viral or cellular gene. The sequences of most IRESes are available from public databases,
e.g., www.ncbi.nlm.nih.gov, www.rangueiI.inserm.fr/IRESdatabase, etc. As a nonlimiting
example, the present invention relates to the use of an IRES isolated from the
encephalomyocarditis virus (EMCV) genome. As such, in one embodiment, the present
invention relates to isolated and purified polynucleotides of the EMCV-1RES.
[0032] The nucleotide sequence of a cDNA encoding EMCV-IRES is set forth in SEQ ID NO: 1
Polynucleotides related to the present invention also include polynucleotides that hybridize under
stringent conditions to SEQ ID NO.l, or complements thereof, and/or encode mRNAs that retain
substantial biological activity of EMCV-IRES. Polynucleotides related to the present invention
also include continuous portions of the sequence set forth in SEQ ID NO:1 comprising at least
about 15 to 30 nucleotides, e.g., 19-27 nucleotides. In one embodiment, polynucleotides related
to the present invention also include continuous portions of the sequence set forth in SEQ ID
NO:1 comprising about 19 or 21 consecutive nucleotides.
[0033] The isolated polynucleotides related to the present invention (e.g., SEQ ID NO: 1,
complements thereof, and continuous portions thereof) may be used as hybridization probes and
primers to identify and isolate nucleic acids having sequences identical to, or similar to, those
encoding the disclosed polynucleotides. Hybridization methods for identifying and isolating
nucleic acids include polymerase chain reaction (PCR), Southern hybridization, and Northern
hybridization, and are well known to those skilled in the art.
[0034] Hybridization reactions may be performed under conditions of different stringencies. The
stringency of a hybridization reaction includes the difficulty with which any two nucleic acid
molecules will hybridize to one another. Preferably, each hybridizing polynucleotide hybridizes
to its corresponding polynucleotide under reduced stringency conditions, more preferably

stringent conditions, and most preferably highly stringent conditions. Examples of stringency
conditions are shown in Table 1 below: highly stringent conditions are those that are at least as
stringent as, for example, conditions A-F; stringent conditions are at least as stringent as, for
example, conditions G-L; and reduced stringency conditions are at least as stringent as, for
example, conditions M-R.
TABLE 1



21 The hybrid length is that anticipated for the hybridized region(s) of the hybridizing polynucleotides. When hybridizing a
polynucleotide to a target polynucleotide of unknown sequence, the hybrid length is assumed to be that of the hybridizing
polynucleotide. When polynucleotides of known sequence are hybridized, the hybrid length can be determined by aligning the
sequences of the polynucleotides and identifying the region or regions of optimal sequence complementarity.
2SSPF, (1xSSPH is 0.15M NaCl 10mM NaH2PO1, and 1.25mM EDTA, pH 7.4) can be substituted for SSC (1 x SSC is 0.15M
NaCl and 15mM sodium citrate) in the hybridizat on and wash buffers; washes are performed for 13 minutes after hybridization is
complete.
TB* - TR*: The hybridization temperature for hybrids anticipated to be less than 50 base pairs in length should be 5-10°C less
than the melting temperature (Tm) of the hybrid, where Tm is determined according to the following equations. For hybrids less
than 18 base pairs in length, Tm,(°C) = 2(# of A + T bases) + 4(# of G + C bases). For hybrids between 18 and 49 base pairs in
length, Tm(°C) = 81.5 + 16.6(log10Na+) + 0.41(%G + C) - (600/N), where N is the number of bases in the hybrid, and Na+ is the
concentration of sodium ions in the hybridization buffer (Na+ for lxSSC = 0.165M).
Additional examples of stringency conditions for polynucleotide hybridization are provided in Sambrook et al. (1989) Molecular
Cloning: A Laboratory Manual, Chs. 9 & 11, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, and Ausubel et al.,
eds. (1995) Current Protocols in Molecular Biology, Sects. 2.10 & 6.3-6.4, John Wiley & Sons, Inc., herein incorporated by
reference.
[0035] The isolated polynucleotides related to the present invention may also be used as
hybridization probes and primers to identify and isolate DNAs having sequences homologous to
the disclosed polynucleotides. These homologs are polynucleotides isolated from different
species than those of the disclosed polynucleotides, or within the same species, but with
significant sequence similarity to the disclosed polynucleotides. Preferably, polynucleotide
homologs have at least 60% sequence identity; more preferably at least 75% identity; and most
preferably at least 90% identity, with the disclosed polynucleotides. Preferably, homologs of the
disclosed polynucleotides are those isolated from a virus, e.g., a virus of the Picornaviridae
family.
[0036] The isolated polynucleotides related to the present invention may also be used as
hybridization probes and primers to identify cells and tissues that express the inhibitory
polynucleotides of the present invention, as described below, and the conditions under which
they are expressed.

[0037] Generally, a polynucleotide according to the present invention is provided as an isolate, in
isolated and/or purified form, or free or substantially free of material with which it is naturally
associated, such as free or substantially free of a nucleic acid(s) flanking the sequence in a
genome (e.g., a picornavirus genome), except possibly one or more regulatory sequence(s) for
expression. A polynucleotide of the invention may be wholly or partially synthetic and may
include genomic DNA, cDNA or RNA. Where a polynucleotide according to the invention
includes RNA, reference to the sequence shown should be construed as reference to the RNA
equivalent, e.g., with U substituted for T.
Inhibitory Polynucleotides
[0038] It is an object of the invention to provide inhibitory polynucleotides directed against a
targeted IRES that may be used in methods of downregulating the expression of a gene of interest
(e.g., endogenous gene, transgene, etc.), the transcription of which results in its protein-coding
region being within an mRNA transcript comprising a nucleotide sequence corresponding to the
targeted IRES, and wherein the methods do not require modification or targeting of the gene of
interest itself. It is another object of the invention to provide methods of screening inhibitory
polynucleotide libraries using the inhibitory polynucleotides of the invention as positive controls.
To this end, the inventors have demonstrated that inhibited (i.e., reduced, interfered with,
downregulated, knocked down, etc.) expression of a transgene of interest may be achieved in a
cell or organism through the use of inhibitory polynucleotides, e.g., siRNA molecules, that target
(e.g., bind and/or cleave) IRES mRNA (e.g., EMCV-IRES mRNA), thus preventing translation
of any protein-coding region found on the same mRNA transcript as the IRES mRNA.
[0039] Altered expression of the IRES sequences related to the invention in a cell or organism
may be achieved through the use of various inhibitory polynucleotides, such as antisense
polynucleotides, ribozymes that bind and/or cleave the mRNA transcribed from the genes of the
invention, triplex-forming oligonucleotides that target regulatory regions of the genes, and short
interfering RNA that causes sequence-specific degradation of target mRNA (e.g., Galderisi et al.
(1999) J. Cell. Physiol. 181:251-57; Sioud (2001) Curr. Mol. Med. 1:575-88; Knauert and Glazer
(2001) Hum. Mol. Genet. 10:2243-51; Bass (2001) Nature 411:428-29).
[0040] The inhibitory antisense or ribozyme polynucleotides of the invention can be
complementary to an entire coding strand of an IRES sequence related to the invention, or to
only a portion thereof. Alternatively, inhibitory polynucleotides can be complementary to a

noncoding region of the coding strand of an IRES sequence related to the invention. The
inhibitory polynucleotides of the invention can be constructed using chemical synthesis and/or
enzymatic ligation reactions using procedures well known in the art. The nucleoside linkages of
chemically synthesized polynucleotides can be modified to enhance their ability to resist
nuclease-mediated degradation, as well as to increase their sequence specificity. Such linkage
modifications include, but are not limited to, phosphorothioate, methylphosphonate,
phosphoroamidate, boranophosphate. morpholino, and peptide nucleic acid (PNA) linkages
(Galderisi et al., supra; Heasman (2002) Dev. Biol. 243:209-14; Mickelfield (2001) Curr. Med..
Chem. 8:1157-79). Alternatively, antisense molecules can be produced biologically using an
expression vector into which a polynucleotide of the present invention has been subcloned in an
antisense (i.e., reverse) orientation.
[0041] In yet another embodiment, the antisense polynucleotide molecule of the invention is an
α-anomeric polynucleotide molecule. An a-anomeric polynucleotide molecule forms specific
double-stranded hybrids with complementary RNA in which, contrary to the usual (3-units, the
strands run parallel to each other. The antisense polynucleotide molecule can also comprise a
2'-o-methylribonucleotide or a chimeric RNA-DNA analogue, according to techniques that are
known in the art.
[0042] The inhibitory triplex-forming oligonucleotides (TFOs) encompassed by the present
invention bind in the major groove of duplex DNA with high specificity and affinity (Knauert
and Glazer, supra). Expression of the genes of the present invention can be inhibited by
targeting TFOs complementary to the regulatory regions of the genes (i.e., the promoter and/or
enhancer sequences) to form triple helical structures that prevent transcription of the genes.
[0043] In one embodiment of the invention, the inhibitory polynucleotides of the present
invention are short interfering RNA (siRNA) molecules (see, e.g., Galderisi et al. (1999) J. Cell
Physiol. 181:251-57; Sioud (2001) Curr. Mol. Med. 1:575-88). These siRNA molecules are short
(preferably 19-25 nucleotides, more preferably 19 or 21 nucleotides) double-stranded RNA
molecules that cause sequence-specific degradation of the targeted mRNA. This degradation is
known as RNA interference (RNAi) (e.g., Bass (2001) Nature 411:428-29). Originally identified
in lower organisms, RNAi has been effectively applied to mammalian cells and has recently been
shown to prevent fulminant hepatitis in mice treated with siRNA molecules targeted to Fas

mRNA (Song et al. (2003) Nat. Med. 9:347-51). In addition, intrathecally delivered siRNA has
recently been reported to block pain responses in two models (agonist-induced pain model and
neuropathic pain model) in the rat (Dorn et al. (2004) Nucleic Acids Res. 32(5):e49).
[0044] The duplex structure of siRNA molecules of the invention may comprise one or more
strands of polymerized RNA, i.e., the duplex structure may be formed by a single self-
complementary RNA strand comprising a hairpin loop or two complementary strands. siRNA
sequences with insertions, deletions, and single point mutations relative to the targeted sequence
have also been found to be effective in inhibiting the expression of the targeted sequence (Fire et
al., U.S. Patent No. 6,506,559). Accordingly, it is preferred that siRNA molecules of the
invention comprise a nucleotide sequence with substantial sequence identity to at least a portion
of the mRNA corresponding to the targeted IRES. For example, the duplex region of an siRNA
molecule of the invention may have greater than 90% sequence identity, and preferably 100%
sequence identity, to at least of portion of the mRNA corresponding to the targeted IRES.
Alternatively, substantial sequence identity may be defined as the ability of at least one strand of
the duplex region of the siRNA molecule to hybridize to at least a portion of the targeted IRES
under at least, e.g., stringent conditions as defined as conditions G-L in Table 1, above. In a
preferred embodiment, the siRNA molecule hybridizes to at least of a portion of the targeted
IRES under highly stringent conditions, e.g., those that are at least as stringent as, for example,
conditions A-F in Table 1, above. Since 100% sequence identity between at least one strand of
the duplex region of an siRNA molecule of the invention and at least a portion of a targeted
sequence is not required, siRNAs directed toward, e.g., an IRES sequence having and/or
consisting essentially of SEQ ID NO:1, may also inhibit the expression of any protein-coding
region located on an mRNA transcript that comprises an IRES sequence that differs from SEQ
ID NO:1 due to mutations, polymorphisms, the redundancy of the genetic code, evolutionary
divergence, etc. (see, e.g., Fire et al., supra). The length of the substantially identical nucleotide
sequences may be at least 10, 15, 19, 21, 23, 25, 27, 50, 100, 200, 300, 400, or 500 nucleotides, is
preferably 19-27 nucleotides, and is most preferably 19 or 21 nucleotides (see Fire et al., supra),
[0045] The inhibitory polynucleotides of the invention may be designed based on criteria well
known in the art (e.g., Elbashir et al. (2001) EMBO J. 20:6877-88) and/or by using well-known
algorithms (e.g., publicly available algorithms). For example, the targeting portion of an
inhibitory polynucleotide of the invention (e.g., the duplex region of an siRNA molecule)

preferably should begin with AA (most preferred), TA, GA, or CA; an siRNA molecule of the
invention preferably should comprise a sequence whereby the GC ratio is 45-55%; an siRNA
molecule of the invention preferably should not contain three of the same nucleotides in a row.
and an siRNA molecule of the invention preferably should not contain seven mixed G/Cs in a
row. Based on these criteria, or on other known criteria (e.g., Reynolds et al. (2004) Nat.
Biotechnol. 22:326-30), siRNA molecules of the present invention that target an IRES, e.g., the
EMCV-IRES having and/or consisting essentially of the nucleotide sequence of SEQ ID NO:1,
may be designed by one of ordinary skill in the art. For example, in one embodiment, an siRNA
molecule of the invention may have and/or consist essentially of a nucleotide sequence selected
from the group consisting of the nucleotide sequence of SEQ ID NO:2, the nucleotide sequence
of SEQ ID NO:3, and the nucleotide sequence of SEQ ID NO:4. In this embodiment, an siRNA
molecule of iiie invention further comprises the complement of the nucleotide sequence of SEQ
ID NO:2, the complement of the nucleotide sequence of SEQ ID NO:3, and the complement of
the nucleotide sequence of SEQ ID NO:4, respectively.
[0046] For example, the siRNA molecules of the present invention may be generated by
annealing two complementary single-stranded RNA molecules together (Fire et al., supra) or
through the use of a single hairpin RNA molecule that folds back on itself to produce the
requisite double-stranded portion (Yu et al. (2002) Proc. Natl. Acad. Sci. USA 99:6047-52). The
siRNA molecules may be chemically synthesized (Elbashir et al. (2001) Nature 411:494-98) or
produced by in vitro transcription using single-stranded DNA templates (Yu et al., supra).
Alternatively, the siRNA molecules can be produced biologically, either transiently (Yu et al.,
supra; Sui et al. (2002) Proc. Natl. Acad. Sci. USA 99:5515-20) or stably (Paddison et al. (2002)
Proc. Natl. Acad. Sci. USA 99:1443-48), using an expression vector(s), e.g., as described below,
comprising polynucleotides related to the present invention in sense and/or antisense orientation
relative to their promoter. Recombinant RNA polymerase may be used for transcription in vivo
or in vitro, or endogenous RNA polymerase of a modified cell may mediate transcription in vivo.
Recently, reduction of levels of target mRNA in primary human cells, in an efficient and
sequence-specific manner, was demonstrated using adenoviral vectors that express hairpin
RNAs, which are further processed into siRNA molecules (Arts et al. (2003) Genome Res.
13:2325-32).

[0047] The inhibitory polynucleotides of the invention may be constructed using chemical
synthesis and enzymatic ligation reactions including procedures well known in the art. The
nucleoside linkages of chemically synthesized polynucleotides may be modified to enhance their
ability to resist nuclease-mediated degradation, avoid a general panic response in some
organisms that is generated by duplex RNA, and/or to increase their sequence specificity. Such
linkage modifications include, but are not limited to, phosphorothioate, methylphosphonate,
phosphoroamidate, boranophosphate, morpholino, and peptide nucleic acid (PNA) linkages
(Galderisi et al, supra; Heasman, supra; Micklefield, supra).
[0048] As described above, the isolated polynucleotides, or continuous portions thereof, related
to the present invention may be operably linked in sense or antisense orientation to an expression
control sequence and/or ligafed into an expression vector for recombinant expression of the
inhibitory polynucleotides (e.g., siRNA molecules) of the invention. General methods of
recombinant expression of inhibitory polynucleotides are well known in the art.
[0049] An expression vector, as used herein, is intended to refer to a nucleic acid molecule
capable of transporting another nucleic acid to which it has been linked. One type of vector is a
plasmid, which refers to a circular double stranded DNA loop into which additional DNA
segments may be ligated. Another type of vector is a viral vector, wherein additional DNA
segments may be ligated into the viral genome. Certain vectors are capable of autonomous
replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial
origin of replication and episomal mammalian vectors). Other vectors (e.g., nonepisomal
mammalian vectors) can be integrated into the genome of a host cell upon introduction into the
host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are
capable of directing the expression of the inhibitory polynucleotides to which they are operably
linked. Such vectors are referred to herein as recombinant expression vectors (or simply,
expression vectors). In general, expression vectors of utility in recombinant DNA techniques are
often in the form of plasmids. In the present specification, plasmid and vector may be used
interchangeably as the plasmid is the most commonly used form of vector. However, the
invention is intended to include other forms of expression vectors, such as viral vectors (e.g.,
replication defective retroviruses, adenoviruses and adeno-associated viruses) that serve
equivalent functions.

[0050] A person of ordinary skill in the art will know how to create an expression vector from
which an inhibitory polynucleotide of the invention may be transcribed. First, a skilled artisan
will know that a regulatory region (e.g., promoter, enhancer, silencer, splice donor, acceptor, etc.)
may be used to transcribe an RNA strand or RNA strands of an inhibitory polynucleotide of the
invention from an expression construct. Second, a skilled artisan will recognize that, e.g., in
creating a duplex siRNA molecule of the invention, the sense and antisense strands of the
targeted portion of the targeted IRES may be transcribed as two separate RNA strands that will
anneal together, or as a single RNA strand that will form a hairpin loop and anneal with itself.
For example, a skilled artisan will know how to create an expression construct whereby the
targeted portion of a targeted IRES is inserted between two promoters (e.g., two bacteriophage
T7 promoters, or two different promoters) such that transcription occurs bidirectionally and will
result in complementary RNA strands that may subsequently anneal to form an inhabitory siRNA
of the invention. Alternatively, a targeted portion of a targeted IRES may exist as a first and
second coding region together on a single expression vector, wherein the first coding region of
the targeted portion of a targeted IRES is in sense orientation relative to its controlling promoter,
and wherein the second coding region of the targeted portion of a targeted IRES is in antisense
orientation relative to its controlling promoter. A skilled artisan will recognize that if
transcription of the sense and antisense coding regions of the targeted portion of the targeted
IRES occurs from two separate promoters, the result will be two separate RNA strands that may
subsequently anneal to form an inhibitory siRNA of the invention. On the other hand, if
transcription of the sense and antisense targeted portion of the targeted IRES is controlled by a
single promoter, the resulting transcript will be a single hairpin RNA strand that is self-
complementary, i.e., forms a duplex by folding back on itself to create an siRNA molecule of the
invention. In the latter configuration, a skilled artisan will also recognize that a spacer, e.g., of
nucleotides, between the sense and antisense coding regions of the targeted portion of the
targeted IRES will improve the ability of the single strand RNA to form a hairpin loop, wherein
the hairpin loop comprises the spacer. In a preferred embodiment, the spacer comprises a length
of nucleotides of at least about 5, 9, 11, or 15 nucleotides. Finally, a skilled artisan will
recognize that the sense and antisense coding regions of the targeted portion of the targeted IRES
may each be on a separate expression vector and under the control of its own promoter.
[0051] The recombinant expression vectors of the invention may carry additional sequences,
such as sequences that regulate replication of the vector in host cells (e.g., origins of replication)

and selectable marker genes. The selectable marker gene facilitates selection of host cells into
which the vector has been introduced. For example, typically the selectable marker gene confers
resistance to drugs, such as G418, hygromycin or methotrexate, on a host cell into which the
vector has been introduced. Preferred selectable marker genes include the dihydrofolate
reductase (DHFR) gene (for use in dhfr- host cells with methotrexate selection/amplification) and
the neo gene (for G418 selection).
[0052] Suitable vectors may be chosen or constructed, containing appropriate regulatory
sequences, including promoter sequences, terminator sequences, polyadenylation sequences,
enhancer sequences, marker genes and other sequences, e.g., sequences that regulate replication
of the vector in the host cells (e.g., origins of replication) as appropriate. Vectors may be
plasmids or viral, e.g., phage, or phagemid, as appropriate. For further details see, for example,
Molecular Cloning: a Laboratory Manual: 2nd ed., Sambrook et al., Cold Spring Harbor
Laboratory Press, 1989. Many known techniques and protocols for manipulation of nucleic acid,
for example, in preparation of nucleic acid constructs, mutagenesis, sequencing, introduction of
DNA into cells and gene expression, and analysis of proteins, are described in detail in Current
Protocols in Molecular Biology, 2nd ed., Ausubel et al. eds., John Wiley & Sons, 1992.
[0053] In one embodiment, a recombinant vector of the invention comprises the EMCV-IRES
having and/or consisting essentially of the nucleotide sequence of SEQ ID NO:1 and its
complement, or continuous portions thereof, for the transcription of the inhibitory
polynucleotides of the invention as described above. For example, an expression vector of the
invention may comprise one or two copies of double-stranded DNA, wherein the first DNA
strand comprises a nucleotide sequence selected from the group consisting of the nucleotide
sequence of nucleotides 27-46 of SEQ ID NO:1, the nucleotide sequence of nucleotides 347-366
of SEQ ID NO:1, the nucleotide sequence of nucleotides 472-491 of SEQ ID NO: 1, and
subsequences or portions thereof, and wherein the second DNA strand comprises a nucleotide
sequence complementary to the nucleotide sequence of the first DNA strand. A skilled artisan
will recognize that such a construct may produce an inhibitory polynucleotide of the invention
having or consisting essentially of a nucleotide sequence selected from the group consisting of
the nucleotide sequence of SEQ ID NO:2, the nucleotide sequence of SEQ ID NO:3, the
nucleotide sequence of SEQ ID NO:4, and subsequences thereof, respectively. Thus, nucleotides
27-46 of SEQ ID NO:1 (i.e., SEQ ID NO:2), nucleotides 347-366 of SEQ ID NO.1 (i.e., SEQ ID

N0:3), and nucleotides 472-491 of SEQ ID NO:1 (i.e., SEQ ID NO:4) represent exemplary
siRNA target sites.
Host Cells / Organisms
[0054] A further aspect of the present invention provides a method of modifying a host cell or
organism with an inhibitory polynucleotide of the invention. Additionally, the present invention
provides a host cell or organism comprising an inhibitory polynucleotide as disclosed herein.
[0055] A number of cell lines may act as suitable host cells for introduction or recombinant
expression of the inhibitory polynucleotides of the present invention. The inhibitory
polynucleotides of the present invention (or expression vector(s) from which the inhibitory
polynucleotide of the invention is transcribed) may be introduced into, e.g., a cell line derived
from plant or animal tissue. One of skill in the art will recognize that an inhibitory
polynucleotide of the invention (or expression vector(s) from which an inhibitory polynucleotide
of the invention is transcribed) is preferably introduced into a host cell that comprises an IRES
polynucleotide related to the invention, e.g., SEQ ID NO:1, and more preferably into a host cell
that has been modified to comprise an IRES polynucleotide related to the present invention. A
skilled artisan will recognize that, as part of the invention, mammalian host cells should be
modified to comprise a viral IRES polynucleotide related to the invention to prevent the
inadvertent inhibition of endogenous genes when an inhibitory polynucleotide of the invention
targeting the IRES polynucleotide is introduced to the modified host cell. In the case where the
host cell is not derived from a mammalian cell, IRES sequences derived from mammalian genes
may be preferable. Although these are preferred embodiments, a skilled artisan will also
recognize that an inhibitory polynucleotide of the invention (or expression vector(s) from which
an inhibitory polynucleotide of the invention is transcribed) may be introduced into host cells not
comprising an IRES polynucleotide related to the invention, e.g., for control purposes.
[0056] Mammalian host cell lines include, for example, COS cells, CHO cells, 293 cells, A431
cells, 3T3 cells, CV-1 cells, HeLa cells, L cells, BHK21 cells, HL-60 cells, U937 cells, HaK
cells, Jurkat cells, as well as cell strains derived from in vitro culture of primary tissue and
primary explants. Plant host cell lines include, but are not limited to, corn, tobacco, Arabidopsis,
rapeseed, and Lemna plant cells.

[0057] Alternatively, it may be possible to recombinantly express the inhibitory polynucleotides
of the present invention in lower eukaryotes such as yeast or in prokaryotes. Potentially suitable
yeast strains include Saccharomyces cerevisiae, Schizosaccharomyces pornbe, Kluyveromyces
strains, and Candida strains. Potentially suitable bacterial strains include Escherichia coli,
Bacillus subtilis, and Salmonella typhimurium.
[0058] The inhibitory polynucleotides of the present invention may also be recombinantly
expressed by operably linking the isolated polynucleotides of the present invention to suitable
control sequences in one or more insect expression vectors, such as baculovirus vectors, and
employing an insect cell expression system. Materials and methods for baculovirus/Sf9
expression systems are commercially available in kit form (e.g., the MAXBAC® kit, Invitrogen,
Carlsbad, CA).
[0059] Any available technique for the introduction of the inhibitory polynucleotides of the
invention (or expression vector(s) from which the inhibitory polynucleotides are transcribed) into
host cells or organisms will be well known by one of ordinary skill in the art and may be used
[0060] For example, if synthesized chemically or by in vitro enzymatic synthesis, the inhibitory
polynucleotides of the invention may be purified prior to introduction into a host cell or
organism. For example, RNA may be purified from a mixture by extraction with a solvent or
resin, precipitation, electrophoresis, chromatography, or a combination thereof. Alternatively,
the RNA may be used with no or a minimum of purification to avoid losses due to sample
processing. The RNA may be dried for storage or dissolved in an aqueous solution. The solution
may contain buffers or salts to promote annealing, and/or stabilization of the duplex strands.
After purification, the inhibitory polynucleotides of the invention (or expression vector(s) from
which the inhibitory polynucleotides are transcribed), may be directly introduced into the cell,
introduced extracellularly into a cavity or interstitial space or into the circulation of an organism,
introduced orally, or introduced by bathing a cell or organism in a solution comprising the
inhibitory polynucleotides of the invention. Physical methods of introducing nucleic acids
include injection of a solution comprising the inhibitory polynucleotides of the invention,
bombardment by particles covered by the inhibitory polynucleotides, soaking or bathing the cell
or organism in the solution, or electorporation.

[0061] For eukaryotic cells, suitable techniques for the introduction of an expression vector(s)
that encode for an inhibitory polynucleotide of the invention may include calcium phosphate
transfection, DEAE-Dextran, electroporation, liposome-mediated transfection, and transduction
using retrovirus or other viruses, e.g., vaccinia or, for insect cells, baculovirus. In a preferred
embodiment, a viral construct packaged into a viral particle would accomplish both efficient
introduction of an expression construct(s) of the invention into the cell and transcription of the
inhibitory polynucleotides of the invention that is encoded by the expression construct(s). For
bacterial cells, suitable techniques may include calcium chloride transformation, electroporation
and transfection using bacteriophage. A skilled artisan will recognize that for plant cells, well-
known techniques similar to those used for eukaryotic cells (e.g., Agrobacterium-mediated
transformation and "gene gun" methods using gold particles to physically introduce plastnid
DNA into plant tissue) may be used. Additionally, the inhibitory polynucleotides of the
invention may be introduced along with components that perform one or more of the following
activities: enhance uptake by the cell, promote annealing of duplex strands, stabilize the
hybridization of annealed strands, or otherwise increase targeting of the targeted IRES. Finally,
the introduction may be followed by causing or allowing expression from the nucleic acid, e.g.,
by culturing host cells under conditions for expression of the gene.
[0062] Expression of an inhibitory polynucleotide of the present invention in an organism may
also be achieved through the creation of nonhuman transgenic plants or animals into whose
genomes IRES polynucleotides related to the present invention, or continuous portions thereof,
have been introduced. Such transgenic plants or animals include those that have multiple copies
of an inhibitory polynucleotide of the present invention. A tissue-specific regulatory sequence(s)
may be operably linked to an IRES polynucleotide, or continuous portion thereof, to direct
expression of an inhibitory polynucleotide of the present invention to particular cells or a
particular developmental stage. Methods for generating transgenic plants (e.g., via physical
introduction of the inhibitory nucleotide) or for generating transgenic animals (e.g., via embryo
manipulation and microinjection, including, but not limited to, animals such as mice, goats,
nematodes, etc.) have become conventional and are well known in the art (e.g., Ma et al. (1995)
Science 268:716-19; Smith and Glick (2000) Biotechnol. Adv. 18:85-89; Peeters et al. (2001)
Vaccine 19:2756-61; Bockamp et al. (2002) Physiol. Genomics 11:115-32).

Methods of the Invention
[0063] Instead of the time-consuming and laborious isolation of mutants by traditional genetic
screening, the function of uncharacterized genes may be determined by employing inhibitory
polynucleotides of the invention to inhibit the expression of a gene of interest (e.g., endogenous
gene, transgene, etc.). Such inhibition of expression may be used, e.g., to reduce the amount
and/or to alter the timing of the activity of the gene of interest. The invention may be used in
determining potential targets for pharmaceutics, understanding normal and pathological events
associated with development, determining signaling pathways responsible for postnatal
development / aging, and the like. As a nonlimiting example, a simple assay would be to modify
a host cell to express a transgene of interest (of known or unknown function) such that the
protein-coding region is transcribed within an mRNA transcript comprising a sequence
corresponding to a targeted IRES, and then to use the inhibitory polynucleotides of the invention
that target the targeted IRES to inhibit (reduce, downregulate, knock down, suppress, etc.) the
expression of the transgene of interest. In another nonlimiting embodiment, the inhibitory
polynucleotides of the invention are used as positive controls in screening assays, e.g., siRNA
screening assays.
[0064] Inhibition of expression refers to an observable decrease in the level of gene products
(e.g., mRNA and/or protein), and may be detected by examination of the outward properties of
the host cell or organism, or by biochemical techniques such as hybridization reactions (e.g.,
Northern blot analysis, RNase protection assays, microarray analysis, etc.), reverse transcription
and polymerase chain reactions, binding reactions (e.g., Western blots, ELISA, FACS, etc.),
reporter assays, drug resistance assays, etc. Depending on the method of detection, greater than
5%, 10%, 33%, 50%, 90%, 95% or 99% inhibition of the expression of a gene of interest by a
host cell or organism treated with an inhibitory polynucleotide of the invention compared to a
nontreated host cell or organism may be expected. Additionally, treatment of a population of
host cells according to a method provided herein may result in a fraction of the cells (e.g., at least
2%, 5%, 10%, 20%, 50%, 75%, 90%, 95%, or 99% of treated cells) exhibiting inhibited
expression of a gene of interest. Increasing the dose of inhibitory polynucleotides may increase
the amount of inhibition detected. A skilled artisan will recognize that since the inhibitory
polynucleotides are directed against a targeted IRES, and not a gene of interest, quantitation of
expression of the gene of interest in treated cell(s) or organism(s) may show dissimilar levels of
inhibition at the mRNA level compared to the protein level. As an example, although the

efficiency of inhibition may be determined by detecting the mRNA level of the gene of interest,
e.g., by Northern blot analysis, a preferred method of determining the level of inhibition is by
detecting the level of protein.
[0065] The inhibitory polynucleotides of the invention may be introduced into a host cell or
organism, as described above, in sufficient amounts to allow introduction of at least one copy of
an inhibitory polynucleotide into the cell. Higher doses (e.g., at least 5, 10, 100, 500, or 1000
copies per cell) of an inhibitory polynucleotide may yield more effective inhibition.
Downregulation of a Transgene of Interest
[0066] The present invention provides a method of inhibiting the expression of a transgene of
interest, the protein-coding region of which is transcribed by a host cell or organism within an
mRNA transcnpt comprising a nucleotide sequence corresponding to a targeted IRES. The
method comprises introducing an inhibitory polynucleotide of the invention that targets a
targeted IRES into the host cell or organism comprising the transgene of interest, wherein the
transgene is transcribed into an mRNA transcript comprising a sequence corresponding to the
targeted IRES. A skilled artisan will recognize that although the inhibitory molecules of the
invention specifically target the IRES, introduction of the inhibitory polynucleotides of the
invention will also result in downregulation of any protein-coding region located on the same
mRNA transcript as the IRES.
[0067] Thus, the inhibitory polynucleotides of the invention are particularly useful because they
may be used to inhibit the expression of more than the targeted IRES, i.e., they may be used to
knock down expressions of transgenes with nucleotide sequences that differ, i.e., do not
correspond to the sequence of the inhibitory polynucleotides (see Example 1). Any transgene
may be inhibited using the inhibitory polynucleotides of the invention as long as transcription of
the transgene results in its protein-coding region being within an mRNA transcript comprising a
nucleotide sequence corresponding to an IRES sequence related to the invention.
Screening Assays
[0068] As described above, the inhibitory polynucleotides of the present invention that target a
targeted IRES may be used to inhibit the expression of a gene, e.g., a transgene that is transcribed
as part of an mRNA transcript comprising a sequence corresponding to the targeted IRES. In at
least one other embodiment, the inhibitory polynucleotides of the invention are used as positive

controls in methods of screening libraries of inhibitory polynucleotides directed toward a
particular gene (including endogenous genes, transgenes, etc.).
[0069] The inhibitory polynucleotides of the invention are useful as positive controls in methods
of screening a library of inhibitory polynucleotides, e.g., siRNA molecules, for inhibitory
polynucleotides that optimally inhibit the expression of a gene of interest. For high throughput
assays, a positive control on each assay plate may be used to validate the results from each plate
[0070] For example, a transgene of interest could be placed into an expression vector such that
its protein-coding region is transcribed as part of an mRNA transcript comprising a nucleotide
sequence corresponding to a targeted IRES. The expression vector may then be used to modify a
host cell. Modified host cells may then be subjected to a library of inhibitory polynucleotides
directed against the transgene of interest, wherein the library comprises as a positive control
least one inhibitory polynucleotide of the invention that targets the targeted IRES.
[0071] In another embodiment of the invention, the inhibitory polynucleotides of the invention
are used as positive controls to screen, or optimize the screening of, a library of the inhibitory
polynucleotides directed against an endogenous gene of interest. For example, a host cell may be
modified with an expression vector comprising a reporter nucleic acid, wherein the protein-
coding region of the reporter nucleic acid is part of an mRNA transcript comprising a nucleotide
sequence corresponding to a targeted IRES. In this embodiment, an inhibitory polynucleotide(s)
of the invention is useful as a positive control(s) by inhibiting the expression of the reporter
nucleic acid. Detection of such inhibition of reporter nucleic acid activity via well-known
reporter assays serves as validation of the screening protocol.
[0072] Use of the inhibitory polynucleotides of the invention in methods of downregulating the
expression of a reporter nucleic acid may be useful for screening a library of inhibitory
polynucleotides directed against an endogenous gene of interest, and/or for screening a library of
transgenes for sequences that may induce novel phenotypes. The function of the inhibitory
polynucleotides in the latter method originates from the ability of the inhibitory polynucleotides
of the invention to downregulate the expression of any gene that is transcribed into an mRNA
transcript comprising a sequence that corresponds to an IRES. For example, a library may
comprise a plurality of expression vectors, wherein each expression vector comprises a unique
transgene sequence, an IRES, and a reporter nucleic acid, such that each will be transcribed into

the same one polycistronic mRNA. The library may then be used to modify a plurality of a host
cell, wherein each host cell of the plurality is modified with a different expression vector. The
phenotype of each modified host cell may then be observed. The transgene producing a
phenotype of interest may then be further analyzed (e.g., its expression may be inhibited) using
the inhibitory polynucleotides of the invention, wherein downregulation of the reporter nucleic
acid will serve as a positive indication that an observed reverse in phenotype is correlated with
downregulation of the transgene.
[0073] In the above-described assays, many of the well-known reporter nucleic acids and related
assays may be used. In one embodiment, the reporter nucleic acid is green fluorescent protein.
In a second embodiment, the reporter is β-galactosidase. In other embodiments, the reporter
nucleic acid is alkaline phosphatase, β-lactamase, luciferase, or chloramphenicol
acetyltransferase.
[0074] The present invention may be used alone, or as a component of a kit having at least one of
the reagents necessary to cany out the introduction of the inhibitory polynucleotides of the
invention to test samples, i.e., host cells or organisms. Such a kit may also include instructions to
allow a user of the kit to practice the invention.
[0075] The entire contents of all references, patents, and patent applications cited throughout this
application are hereby incorporated by reference herein.
EXAMPLE
[0076] The following Example provides illustrative embodiments of the invention and does not
in any way limit the invention. One of ordinary skill in the art will recognize that numerous
other embodiments are encompassed within the scope of the invention.
EXAMPLE 1
Knocking Down Gene Expression Using siRNA Directed Against
the EMCV-IRES
Example 1.1: Materials and Methods
[0077] The publicly available Dharmacon siRNA design algorithm (see
www.dharmacon.com/sidesign/; see also Reynolds et al., supra) was used to design siRNA
molecules (hereinafter "siRNAs") directed against the EMCV-IRES. Three portions of the IRES

sequence were identified by the Dharmacon algorithm as the optimally targeted sequences
(IRES1, IRES2, IRES3), and were chosen to be targeted by siRNA molecules (FIG. 1). In
particular, siRNA1, siRNA2, and siRNA3 were synthesized by Dharmacon (Lafayette, CO) to
target IRES1, IRES2, and IRES3, respectively (FIG. 1).
[0078] The three synthesized siRNA molecules were used to transfect a CHO cell line that was
stably modified to express a recombinant antibody. The CHO cell line was modified with an
expression vector that encoded the heavy chain of the antibody and an expression vector that
encoded the light chain of the antibody. The expression vectors transcribed either the heavy or
light chain into a polycistronic mRNA, which comprised the heavy or light chain protein-coding
region upstream of a sequence that corresponds to the EMCV-IRES, and a different selectable
marker downstream of the sequence that corresponds to the EMCV-IRES. It was expected that
siRNA-mediated knockdown of EMCV-lRES-containing transcripts would result in knockdown
of expression of the recombinant antibody (i.e., either or both the heavy and light chain genes
upstream of the IRES). Such knockdown of expression is easily assessed by monitoring the
expression of the recombinant monoclonal antibody in the conditioned media, e.g., via methods
of western blotting, ELISA, or automated bead-based capture/detection methods (e.g., IGEN-
based assays (Roche Diagnostics, Alameda, CA)). The modified CHO cells were transfected
with each siRNA individually, or with a pool of all three siRNAs, in 72-hour and 144-hour
secretion assays (n=3 each). Antibody titer and cell numbers were assessed using an IGEN-
based assay at 72 hours (3 days) and 144 hours (6 days) to estimate cell-specific productivity
(liter/cell number/day), which normalizes titer data for differences in seed density and cell
growth during the experiment.
Example 1.2: Results
[0079] FIGs. 2A and 2B demonstrate that all three siRNAs can mediate knockdown of
monoclonal antibody transgene expression in the conditioned medium. The siRNA molecules
directed against IRES2 and IRES3 appear to be more effective than an siRNA molecule directed
against IRES1, and the pool of siRNA molecules was very effective in knocking down
expression. The knockdown was observed at both 72 hours and 144 hours post-transfection.
This effect was observed in several CHO cell lines expressing different monoclonal antibodies
(data not shown). The present invention demonstrates that IRES siRNAs may be used with a cell
line that utilizes an IRES in the expression vector. The ability to monitor knockdown of product

gene expression using a titer-based assay also eliminates the need to develop other validation
assays, such as real time PCR.

WHAT IS CLAIMED IS:
1. An inhibitory polynucleotide directed against an IRES.
2. The inhibitory polynucleotide of claim 1, wherein the IRES has the nucleotide sequence
of SEQ ID NO:l.
3. The inhibitory polynucleotide of claim 2, wherein the inhibitory polynucleotide
comprises a first strand of an siRNA.
4. The inhibitory polynucleotide of claim 3, wherein the first strand of the siRN A has the
RNA equivalent of a nucleotide sequence selected from the group consisting of the nucleotide
sequence of SEQ ID NO: 1, a portion of the nucleotide sequence of SEQ ID NO: 1, the
complement of the nucleotide sequence of SEQ ID NO:1, and a portion of the complement of the
nucleotide sequence of SEQ ID NO: 1.
5. The inhibitory polynucleotide of claim 4, wherein the first strand of the siRNA is between
5 and 548 nucleotides in length.
6. The inhibitory polynucleotide of claim 5, wherein the first strand of the siRNA has the
RNA equivalent of a nucleotide sequence selected from the group consisting of the nucleotide
sequence of SEQ ID NO:2, the nucleotide sequence of SEQ ID NO:3, the nucleotide sequence of
SEQ ID NO:4, and the nucleotide sequence of subsequences thereof.
7. The inhibitory polynucleotide of claim 6, wherein the first strand of the siRNA is self-
complementary and further comprises a hairpin loop.
8. The inhibitory polynucleotide of claim 7, wherein the first strand of the siRNA further
comprises the RNA equivalent of a nucleotide sequence selected from the group consisting of the
nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO:2, the nucleotide
sequence complementary to the nucleotide sequence of SEQ ID NO:3, and the nucleotide
sequence complementary to the nucleotide sequence of SEQ ID NO:4.

9. The inhibitory polynucleotide of claim 2, wherein the inhibitory polynucleotide is an
antisense molecule.
10. The inhibitory polynucleotide of claim 6, wherein the inhibitory polynucleotide further
comprises a second strand of the siRNA that is complementary to the first strand of the siRNA
11. An isolated DNA molecule that encodes the inhibitory polynucleotide as in one of
claims 1-9.
12. The isolated DNA molecule of claim 11, wherein the DNA molecule is operably linked to
at least one expression control sequence.
13. A host cell transformed or transfected with the isolated DNA molecule of claim 12.
14. A microorganism that contains DNA that encodes the inhibitory polynucleotide as in one
of claims 1-9.
15. A nonhuman transgenic animal in which the somatic and germ cells contain DNA that
encodes the inhibitory polynucleotide as in one of claims 1-9.
16. A transgenic plant in which the somatic and germ cells contain DNA that encodes the
inhibitory polynucleotide as in one of claims 1-9.
17. An isolated DNA molecule that encodes the second strand of the siRNA of claim 10.
18. The isolated DNA molecule of claim 17, wherein the DNA molecule is operably linked to
at least one expression control sequence.
19. A host cell transformed or transfected with the isolated DNA molecule of claim 18.
20. A microorganism that contains DNA that encodes the second strand of the siRNA of
claim 10.

21. A nonhuman transgenic animal in which the somatic and germ cells contain DNA that
encodes the second strand of the siRNA of claim 10.
22. A transgenic plant in which the somatic and germ cells contain DNA that encodes the
second strand of the siRNA of claim 10.
23. A kit comprising the inhibitory polynucleotide of claim 1.
24. A method of downregulating the expression of a transgene by a host cell, wherein the
transgene is transcribed as part of an mRNA transcript comprising a nucleotide sequence
corresponding to an IRES, the method comprising the step of introducing into the host cell an
inhibitory polynucleotide that targets the IRFS.
25. The method of claim 24, wherein the IRES consists essentially of the nucleotide sequence
of SEQ ID NO:1.

The invention provides novel inhibitory polynucleotides directed against IRES sequences. The invention also provides genetically engineered expression vectors, host cells, transgenic animals, and transgenic plants comprising the novel inhibitory
polynucleotides of the invention. The invention additionally provides methods of using the inhibitory polynucleotides the inven
tion.