U.S. patent application number 14/796112 was filed with the patent office on 2016-01-07 for nucleic acids involved in viral infection.
The applicant listed for this patent is Rosetta Genomics Ltd.. Invention is credited to Ranit Aharonov, Amir Avniel, Issac Bentwich, Yael Karov, Asaf Levy, Yonat Shemer-Avni.
Application Number | 20160002639 14/796112 |
Document ID | / |
Family ID | 40378751 |
Filed Date | 2016-01-07 |
United States Patent
Application |
20160002639 |
Kind Code |
A1 |
Bentwich; Issac ; et
al. |
January 7, 2016 |
NUCLEIC ACIDS INVOLVED IN VIRAL INFECTION
Abstract
The invention provides isolated viral and human nucleic acids
associated with viral infection and various nucleic acid molecules
relating thereto or derived therefrom. The nucleic acids are useful
for prevention, treatment and diagnosis of viral infections.
Inventors: |
Bentwich; Issac; (DN Misgav,
IL) ; Avniel; Amir; (Tel-Aviv, IL) ; Aharonov;
Ranit; (Tel-Aviv, IL) ; Karov; Yael;
(Tel-Aviv, IL) ; Shemer-Avni; Yonat; (Beer Sheva,
IL) ; Levy; Asaf; (Nes-Ziona, IL) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Rosetta Genomics Ltd. |
Rehovot |
|
IL |
|
|
Family ID: |
40378751 |
Appl. No.: |
14/796112 |
Filed: |
July 10, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13986802 |
Jun 6, 2013 |
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14796112 |
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12517760 |
Apr 5, 2010 |
8481506 |
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PCT/IB2007/004718 |
Dec 5, 2007 |
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13986802 |
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60971265 |
Sep 11, 2007 |
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60868666 |
Dec 5, 2006 |
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Current U.S.
Class: |
514/44A ;
435/236; 435/375 |
Current CPC
Class: |
C12N 2310/141 20130101;
C12N 15/1133 20130101; C12N 15/1132 20130101; C12N 15/1131
20130101; C12N 2710/16022 20130101; C12N 2330/10 20130101; C12N
15/113 20130101; A61K 48/00 20130101; C12N 2320/11 20130101; C12N
2330/31 20130101; C12N 2760/18522 20130101; C12N 2760/16122
20130101 |
International
Class: |
C12N 15/113 20060101
C12N015/113 |
Claims
1-6. (canceled)
7. A method of preventing or treating viral infection or a
condition associated with a viral infection in a subject in need
thereof comprising administering to the subject an effective amount
of a composition comprising a nucleic acid sequence selected from
the group consisting of SEQ ID NOS: 1-9,221, 15,366-15,392 and
15,516-15,527; a complementary sequence thereof; and a sequence at
least about 80% identical thereto.
8. The method of claim .sub.7, wherein the viral infection is
selected from the group consisting of HSV1, HSV2, RSV, EBV,
Influenza A, HCV, HPV, HIV, HBV and Vaccinia virus infection.
9. A method of inhibiting expression of a target gene in a cell
comprising introducing a nucleic acid into the cell in an amount
sufficient to inhibit expression of the target gene, wherein the
target gene comprises a binding site substantially identical to a
binding site as set forth in any one of SEQ ID NOS: 9,222-15,365,
15,393-15,515; and wherein the nucleic acid is a nucleic acid
selected from the group consisting of SEQ ID NOS: 1-9,221,
15,366-15,392, and 15,516-15,527; a complementary sequence thereof;
and a sequence at least about 80% identical thereto.
10-18. (canceled)
19. A method of reducing the amount of virus replication in a
target cell, said method comprising: introducing into a target cell
infected with the virus an effective amount of a composition
comprising a nucleic acid sequence selected from the group
consisting of SEQ ID NOS: 1-9,221, 15,366-15,392 and 15,516-15,527;
a complementary sequence thereof; and a sequence at least about 80%
identical thereto.
20. The method of claim 19, wherein said target cell is in
vitro.
21. The method of claim 19, wherein said target cell is in
vivo.
22. The method of claim 21, wherein said method comprises
administering the composition to a subject comprising said target
cell.
23. The method of claim 19, wherein said virus is selected from the
group consisting of HSVi, HSV2, RSV, EBV, Influenza A, HCV, HPV,
HIV, HBV and Vaccinia.
24. The method of claim 19, wherein said method comprises treating
said subject for viral mediated disease condition.
25. The method of claim 24, wherein said disease condition is
selected from the group consisting of Burkitt's lymphoma,
nasopharingal carcinoma, ovarian carcinoma, cervical cancer,
hepatitis, mononucleosis, influenza, genital herpes, encephalitis,
and bronchiolitis.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. Provisional App.
No. 60/868,666, filed Dec. 5, 2006 and U.S. Provisional App. No.
60/971,265, filed Sep. 11, 2007, the contents each of which are
incorporated herein by reference.
FIELD OF THE INVENTION
[0002] Described herein are viral and host microRNA molecules
associated with viral infections, as well as various nucleic acid
molecules relating thereto or derived therefrom.
BACKGROUND OF THE INVENTION
[0003] MicroRNAs (miRNAs) are a family of 18-24 nucleotide long
non-coding small RNAs, that suppress translation of target genes by
binding to their mRNA, thereby regulating the expression of at
least 30% of all human genes. Although miRNAs are present in a wide
range of species including C. elegans, Drosophila and humans, they
have only recently been identified. More importantly, the role of
miRNAs in the development and progression of disease has only
recently become appreciated. There are currently about 500 known
human microRNAs, and their number probably exceeds 800.
[0004] As a result of their small size, miRNAs have been difficult
to identify using standard methodologies. A limited number of
miRNAs have been identified by extracting large quantities of RNA.
miRNAs have been identified that contribute to the presentation of
visibly discernable phenotypes. Expression array data show that
miRNAs are expressed in different developmental stages or in
different tissues. The restriction of miRNAs to certain tissues or
at limited developmental stages indicates that the miRNAs
identified to date are likely only a small fraction of the total
miRNAs.
[0005] Computational approaches have recently been developed to
identify the remainder of miRNAs in the genome. Tools such as
MiRscan and MiRseeker have identified miRNAs that were later
experimentally confirmed. Based on the fundamental importance of
miRNAs in mammalian biology and disease, the art needs to identify
unknown miRNAs.
[0006] Viruses can establish a variety of types of infection. These
infections can be generally classified as lytic or persistent,
though some lytic infections are considered persistent. Generally,
persistent infections fall into two categories: (1) chronic
(productive) infection, i.e., infection wherein infectious virus is
present and can be recovered by traditional biological methods and
(2) latent infection, i.e., infection wherein viral genome is
present in the cell but infectious virus is generally not produced
except during intermittent episodes of reactivation. Persistence
generally involves stages of both productive and latent
infection.
[0007] Lytic infections can also persist under conditions where
only a small fraction of the total cells are infected (smoldering
(cycling) infection). The few infected cells release virus and are
killed, but the progeny virus again only infect a small number of
the total cells.
[0008] Traditional treatments for viral infection include
pharmaceuticals aimed at specific virus derived proteins, or
recombinant (cloned) immune modulators (host derived), such as the
interferons. However, the current methods have several limitations
and drawbacks which include high rates of viral mutations which
render anti-viral pharmaceuticals ineffective. For immune
modulators, limited effectiveness, limiting side effects, a lack of
specificity all limit the general applicability of these agents.
Also the rate of success with current antivirals and
immune-modulators has been disappointing.
[0009] Viral infections are a continuing medical problem because,
like any rapidly-dividing infectious agent, there are continuing
mutations that help some sub-populations of viruses continue to be
resistant to current treatment regimens. Many virally-based
diseases do not have effective anti-viral treatments, because such
treatments address the symptoms of the viral disease and not the
root cause of the disease. There is a need in the art to discover
and develop new anti-viral therapies.
SUMMARY OF THE INVENTION
[0010] Provided herein are compositions and methods for the
identification, suppression and modulation of viral infection in a
target cell. Also provided are pharmaceutical compositions and kits
for use in practicing the methods. The compositions and methods may
be used in a variety of applications, including the treatment of
subjects suffering from a viral mediated disease condition.
[0011] Also provided is an isolated nucleic acid comprising a
sequence selected from the group consisting of SEQ ID NOS: 1-9,221,
15,366-15,392 and 15,516-15,527; the complementary sequence
thereof; and a sequence at least about 80% identical thereto. The
nucleic acid may be from 5-250 nucleotides in length. The nucleic
acid may comprise a modified base.
[0012] Further provided is a probe comprising the nucleic acid. The
probe may comprise at least 8-22 contiguous nucleotides
complementary to SEQ ID NOS: 1-9,221, 15,366-15,392 and
15,516-15,527, or a variant thereof. The probe may also comprise at
least 8-22 contiguous nucleotides complementary to a host microRNA
differentially expressed in viral infection, or a variant
thereof.
[0013] Also provided is a composition comprising the nucleic
acid.
[0014] Further provided is a biochip comprising the nucleic
acid.
[0015] Also provided is a vector comprising the nucleic acid.
[0016] Further provided is a host cell comprising the nucleic
acid.
[0017] Also provided is a pharmaceutical composition comprising the
nucleic acid as an active ingredient, and a composition comprising
the vector.
[0018] Further provided is a method of preventing or treating viral
infection or a condition associated with a viral infection in a
subject in need thereof. The method may comprise administering to
the subject an effective amount of a.composition comprising a
nucleic acid sequence selected from the group consisting of SEQ ID
NOS: 1-9,221, 15,366-15,392 and 15,516-15,527; a complementary
sequence thereof; and a sequence at least about 80% identical
thereto. The viral infection may be caused by a virus selected from
the group consisting of: HSV1, HSV2, RSV, EBV, Influenza A, HCV,
HPV, HIV, HBV and Vaccinia virus. The condition associated with the
viral infection may be selected from the group consisting of:
Burkitt's lymphoma, nasopharingal carcinoma, ovarian carcinoma,
cervical cancer, hepatitis, mononucleosis, genital herpes,
encephalitis, influenza and bronchiolitis.
[0019] Also provided is a method for reducing the amount of virus
replication in a target cell, where the target cell may be present
in vitro or in vivo.
[0020] Further provided is the use of a nucleic acid comprising a
nucleic acid sequence selected from the group consisting of SEQ ID
NOS: 1-9,221, 15,366-15,392 and 15,516-15,527; a complementary
sequence thereof; and a sequence at least about 80% identical
thereto for the manufacture of a medicament for the treatment or
prevention of viral infection.
[0021] Also provided is a method for modulating a nucleic acid. The
method may comprise modulating a first nucleic acid comprising the
nucleotide sequence selected from the group consisting of (a) any
one of SEQ ID NOS: 9,222-15,365 and 15,393-15,515, (b) a
complementary sequence of (a), and (c) sequence at least about 80%
identical to (a) or (b).
[0022] The method may further comprise introducing a second nucleic
acid to the first nucleic acid wherein the second nucleic acid is
selected from the group consisting of (a) SEQ ID NOS: 1-9,221,
15,366-15,392, 15,516-15,527, and (b) sequence at least about 80%
identical to (a), wherein the second nucleic acid modulates
expression of the first nucleic acid. The first nucleic acid may be
a miRNA target-gene. The second nucleic acid may be a miRNA or
siRNA.
[0023] Further provided is a method of inhibiting expression of a
target gene in a cell. A nucleic acid may be introduced into the
cell in an amount sufficient to inhibit expression of the target
gene. The target gene may comprise a binding site substantially
identical to SEQ ID NOS: 9,222-15,365 and 15,393-15,515, or a
variant thereof. The nucleic acid may comprise a portion of SEQ ID
NOS: 1-9,221, 15,366-15,392 and 15,516-15,527 or a variant thereof.
Expression of a target gene may be inhibited in vitro or in
vivo.
[0024] Also provided is a method of detecting viral infection of a
cell comprising determining the expression level of a nucleic acid
sequence selected from the group consisting of SEQ ID NOS: 1-9,221,
15,366-15,392 and 15,516-15,527; a complementary sequence thereto
or a sequence at least about 80% identical thereto. The method of
detecting viral infection may comprise a microRNA array, RT-PCR, or
Northern blot analysis.
[0025] Further provided is a kit comprising the nucleic acid.
[0026] Also provided is a method of reducing the amount of virus
replication in a target cell, which may comprise introducing an
effective amount of a composition into a target cell infected with
a virus. The composition may comprise a nucleic acid sequence
selected from the group consisting of SEQ ID NOS: 1-9,221,
15,366-15,392 and 15,516-15,527; a complementary sequence thereof;
and a sequence at least about 80% identical thereto. The target
cell may be in vitro or in vivo. A subject may comprise said target
cell. The virus may be HSV1, HSV2, RSV, EBV, Influenza A, HCV, HPV,
HIV, HBV, or Vaccinia virus. The method may be used to treat a
viral mediated disease condition in a subject in need thereof. The
disease condition may be Burkitt's lymphoma, nasopharingal
carcinoma, ovarian carcinoma, cervical cancer, hepatitis,
mononucleosis, influenza, genital herpes, encephalitis, or
bronchiolitis.
BRIEF DESCRIPTION OF THE DRAWINGS
[0027] FIG. 1 shows microRNA expression profiling of respiratory
syncytial virus (RSV) infected HEp2 cells as compared to uninfected
control cells using miRdicator.TM. microRNA array. The expressed
host microRNA AMB-10594 (SEQ ID NO: 15,374) is indicated.
[0028] FIGS. 2A-2B show microRNA expression profiling of MDCK cells
infected with Influenza A (FluA) as compared to uninfected control
cells using miRdicator.TM. microRNA array. The expressed microRNA
of FluA-12 (SEQ ID NO: 15,378) is circled. Two independent
experiments are shown.
[0029] FIG. 3 shows microRNA expression profiling of herpes simplex
virus typel (HSV1) microRNAs in Vero cells infected with HSV1 and
HSV2 as compared to uninfected control cells. The expressed
microRNA HSV1-miR-H1 (SEQ ID NO: 15,375) is indicated.
[0030] FIG. 4 shows microRNA expression profiling of HSV2 microRNAs
in Vero cells infected with HSV2. The expressed microRNAs:
HSV2-Pred13 (SEQ ID NO: 15,376) and HSV2-8 (SEQ ID NO: 15,377) are
indicated.
[0031] FIGS. 5A-5B show up-regulation of host microRNA AMB-10594
(SEQ ID NO: 15,374) (circled) following various viral infections of
various cells: RSV infected HEp2 cells (FIG. 5A), HSV2 infected HF
cells (FIG. 5B), HSV1 infected HF cells (FIG. 5D), and following
interferon treatment of HF cells (FIG. 5C).
[0032] FIG. 6 shows Northern blot analysis of hsa-miR-181a (SEQ ID
NO: 15,366) differentially expressed in HEp2 uninfected (-) and RSV
infected cells.
[0033] FIGS. 7A-7B show real time PCR analysis of HSV2
microRNA-pred-13 (SEQ ID NO: 15,376), HSV2 microRNA-8 (SEQ ID NO:
15,377) and their bulge.
[0034] FIG. 8 shows higher expression of hsa-mir-21 (SEQ ID NO:
15,373) in Vero cells infected with HSV1 in comparison with HSV2
infected cells.
[0035] FIG. 9 shows Northern blot analysis of HSV2 microRNA-pred-13
(SEQ ID NO: 15,376). The lower part of the figure depicts Ethidium
Bromide staining of the gel.
[0036] FIG. 10 shows Northern blot analysis of HSV2 microRNA-8 (SEQ
ID NO: 15,377). The lower part of the figure depicts Ethidium
Bromide staining of the gel.
[0037] FIG. 11 shows the results of quantitative RT-PCR of EBV
viral load in B95-8 cells transfected with EBV anti-mir
oligonucleotides. B95-8 cells persistently infected with EBV were
transfected with various antagonists (2-O-Methyl antimir
oligonucleotides, SEQ ID NOs: 15,517, 15,519, 15,521, 15,523,
15,525, and 15,527) to EBV microRNAs. After 120 hrs, cells were
harvested, DNA was extracted, and EBV DNA copies/ml was determined
by qRTPCR, using commercial standards for EBV viral load.
DETAILED DESCRIPTION
[0038] During viral infection, viruses express specific miRNAs and
alter host miRNA expression. Provided herein are compositions and
methods to prevent or treat viral infection. Identification of
specific miRNA signatures and the targets of these miRNAs induced
by viruses may be used to identify cellulai and viral genes
required for viral infection.
[0039] The inventors have made the surprising discovery that the
expression levels of several viral microRNAs (SEQ ID NOS:
15,375-15,387) and host microRNAs (SEQ ID NOS: 15,366-15,374) were
altered following viral infection. Furthermore, different viruses
produce distinct microRNAs expression patterns in various viral
infected cells.
[0040] Specific viral and host miRNA nucleic acids may be used as
novel therapeutics in the treatment of viral infections. The
nucleic acids also be used in diagnostics for clinical and research
settings including detection of latent infections. The disclosed
viral and host miRNAs may be used to discover new cellular and
viral drugs and drug targets.
[0041] Described herein is the expression of viral and host encoded
microRNAs in viral infections by HSV1, HSV2, RSV, EBV, Influenza A,
HCV, HPV and Vaccinia viruses.
[0042] Provided herein are nucleotide sequences of viral, and human
miRNAs, precursors thereto, targets thereof and related sequences.
Such nucleic acids may be used for diagnostic purposes, therapeutic
purposes, and also for modifying target gene expression.
1. Definitions
[0043] Before the present compositions and methods are disclosed
and described, it is to be understood that the terminology used
herein is for the purpose of describing particular embodiments only
and is not intended to be limiting. It must be noted that, as used
in the specification and the appended claims, the singular forms
"a," "an" and "the" include plural referents unless the context
clearly dictates otherwise. It must further be noted that the terms
"and" and "or" may encompass both conjunctive and disjunctive
meaning unless the context clearly dictates otherwise.
[0044] For the recitation of numeric ranges herein, each
intervening number there between with the same degree of precision
is explicitly contemplated. For example, for the range of 6-9, the
numbers 7 and 8 are contemplated in addition to 6 and 9, and for
the range 6.0-7.0, the numbers 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6,
6.7, 6.8, 6.9, and 7.0 are explicitly contemplated.
[0045] "Animal" as used herein may mean fish, amphibians, reptiles,
birds, and mammals, such as mice, rats, rabbits, goats, cats, dogs,
cows, apes and humans.
[0046] "Attached" or "immobilized" as used herein to refer to a
probe and a solid support may mean that the binding between the
probe and the solid support is sufficient to be stable under
conditions of binding, washing, analysis, and removal. The binding
may be covalent or non-covalent. Covalent bonds may be formed
directly between the probe and the solid support or may be formed
by a cross linker or by inclusion of a specific reactive group on
either the solid support or the probe or both molecules.
Non-covalent binding may be one or more of electrostatic,
hydrophilic, and hydrophobic interactions. Included in non-covalent
binding is the covalent attachment of a molecule, such as
streptavidin, to the support and the non-covalent binding of a
biotinylated probe to the streptavidin. Immobilization may also
involve a combination of covalent and non-covalent
interactions.
[0047] "Biological sample" as used herein may mean a sample of
biological tissue or fluid that comprises nucleic acids. Such
samples include, but are not limited to, tissue isolated from
animals. Biological samples may also include sections of tissues
such as biopsy and autopsy samples, frozen sections taken for
histological purposes, blood, plasma, serum, sputum, stool, tears,
mucus, urine, effusions, amniotic fluid, ascitic fluid, hair, and
skin. Biological samples also include explants and primary and/or
transformed cell cultures derived from patient tissues. A
biological sample may be provided by removing a sample of cells
from an animal, but can also be accomplished by using previously
isolated cells (e.g., isolated by another person, at another time,
and/or for another purpose), or by performing the methods described
herein in vivo. Archival tissues, such as those having treatment or
outcome history, may also be used.
[0048] "Complement" or "complementary" as used herein may mean
Watson-Crick (e.g., A-T/U and C-G) or Hoogsteen base pairing
between nucleotides or nucleotide analogs of nucleic acid
molecules. A complement may be identical in length to a nucleic
acid disclosed herein.
[0049] "Differential expression" may mean qualitative or
quantitative differences in the temporal and/or cellular gene
expression patterns within and among cells and tissue. Thus, a
differentially expressed gene can qualitatively have its expression
altered, including an activation or inactivation, in, e.g., normal
versus disease tissue. Genes may be turned on or turned off in a
particular state, relative to another state thus permitting
comparison of two or more states. A qualitatively regulated gene
will exhibit an expression pattern within a state or cell type that
may be detectable by standard techniques. Some genes will be
expressed in one state or cell type, but not in both.
Alternatively, the difference in expression may be quantitative,
e.g., in that expression is modulated, up-regulated, resulting in
an increased amount of transcript, or down-regulated, resulting in
a decreased amount of transcript. The degree to which expression
differs need only be large enough to quantify via standard
characterization techniques such as expression arrays, quantitative
reverse transcriptase PCR, northern analysis, and RNase
protection.
[0050] "Expression profile" as used herein may mean a genomic
expression profile, e.g., an expression profile of microRNAs.
Profiles may be generated by any convenient means for determining a
level of a nucleic acid sequence e.g. quantitative hybridization of
microRNA, labeled microRNA, amplified microRNA, cRNA, etc.,
quantitative PCR, ELISA for quantitation, and the like, and allow
the analysis of differential gene expression between two samples. A
subject or patient tumor sample, e.g., cells or collections
thereof, e.g., tissues, is assayed. Samples are collected by any
convenient method, as known in the art. Nucleic acid sequences of
interest are nucleic acid sequences that are found to be
predictive, including the nucleic acid sequences provided above,
where the expression profile may include expression data for 5, 10,
20, 25, 50, 100 or more of, including all of the listed nucleic
acid sequences. The term "expression profile" may also mean
measuring the abundance of the nucleic acid sequences in the
measured samples.
[0051] "Gene" used herein may be a genomic gene comprising
transcriptional and/or translational regulatory sequences and/or a
coding region and/or non-translated sequences (e.g., introns, 5'-
and 3'-untranslated sequences). The coding region of a gene may be
a nucleotide sequence coding for an amino acid sequence or a
functional RNA, such as tRNA, rRNA, catalytic RNA, siRNA, miRNA and
antisense RNA. A gene may also be an miRNA or cDNA corresponding to
the coding regions (e.g., exons and miRNA) optionally comprising
5'- or 3'-untranslated sequences linked thereto. A gene may also be
an amplified nucleic acid molecule produced in vitro comprising all
or a part of the coding region and/or 5'- or 3'-untranslated
sequences linked thereto.
[0052] "Host cell" used herein may mean a naturally occurring cell
or a transformed cell that may contain a vector and may support the
replication of the vector. Host cells may be cultured cells,
explants, cells in vivo, and the like. Host cells may be
prokaryotic cells such as E. coli, or eukaryotic cells such as
yeast, insect, amphibian, or mammalian cells, such as CHO,
HeLa.
[0053] "Identical" or "identity" used herein in the context of two
or more nucleic acids or polypeptide sequences, may mean that the
sequences have a specified percentage of residues that are the same
over a specified region. The percentage may be calculated by
comparing optimally aligning the two sequences, comparing the two
sequences over the specified region, determining the number of
positions at which the identical residue occurs in both sequences
to yield the number of matched positions, dividing the number of
matched positions by the total number of positions in the specified
region, and multiplying the result by 100 to yield the percentage
of sequence identity. In cases where the two sequences are of
different lengths or the alignment produces staggered ends and the
specified region of comparison includes only a single sequence, the
residues of single sequence are included in the denominator but not
the numerator of the calculation. When comparing DNA and RNA,
thymine (T) and uracil (U) are considered equivalent. Identity may
be performed manually or by using computer sequence algorithm such
as BLAST or BLAST 2.0.
[0054] "Inhibit" as used herein may mean prevent, suppress,
repress, reduce or eliminate.
[0055] "Label" as used herein may mean a composition detectable by
spectroscopic, photochemical, biochemical, immunochemical,
chemical, or other physical means. For example, useful labels
include P.sup.32, fluorescent dyes, electron-dense reagents,
enzymes (e.g., as commonly used in an ELISA), biotin, digoxigenin,
or haptens and other entities which can be made detectable. A label
may be incorporated into nucleic acids and proteins at any
position.
[0056] "Nucleic acid", "oligonucleotide" or "polynucleotide" used
herein may mean at least two nucleotides covalently linked
together. The depiction of a single strand also defines the
sequence of the complementary strand. Thus, a nucleic acid also
encompasses the complementary strand of a depicted single strand.
Many variants of a nucleic acid may be used for the same purpose as
a given nucleic acid. Thus, a nucleic acid also encompasses
substantially identical nucleic acids and complements thereof. A
single strand provides a probe that may hybridize to the target
sequence under stringent hybridization conditions. Thus, a nucleic
acid also encompasses a probe that hybridizes under stringent
hybridization conditions.
[0057] Nucleic acids may be single stranded or double stranded, or
may contain portions of both double stranded and single stranded
sequence. The nucleic acid may be DNA, both genomic and cDNA, RNA,
or a hybrid, where the nucleic acid may contain combinations of
deoxyribo- and ribo-nucleotides, and combinations of bases
including uracil, adenine, thymine, cytosine, guanine, inosine,
xanthine hypoxanthine, isocytosine and isoguanine. Nucleic acids
may be obtained by chemical synthesis methods or by recombinant
methods.
[0058] A nucleic acid will generally contain phosphodiester bonds,
although nucleic acid analogs may be included that may have at
least one different linkage, e.g., phosphoramidate,
phosphorothioate, phosphorodithioate, or O-methylphosphoroamidite
linkages and peptide nucleic acid backbones and linkages. Other
analog nucleic acids include those with positive backbones;
non-ionic backbones, and non-ribose backbones, including those
described in U.S. Pat. Nos. 5,235,033 and 5,034,506, which are
incorporated by reference. Nucleic acids containing one or more
non-naturally occurring or modified nucleotides are also included
within one definition of nucleic acids. The modified nucleotide
analog may be located for example at the 5'-end and/or the 3'-end
of the nucleic acid molecule. Representative examples of nucleotide
analogs may be selected from sugar- or backbone-modified
ribonucleotides. It should be noted, however, that also
nucleobase-modified ribonucleotides, i.e. ribonucleotides,
containing a non-naturally occurring nucleobase instead of a
naturally occurring nucleobase such as uridines or cytidines
modified at the 5-position, e.g. 5-(2-amino)propyl uridine, 5-bromo
uridine; adenosines and guanosines modified at the 8-position, e.g.
8-bromo guanosine; deaza nucleotides, e.g. 7-deaza-adenosine; O-
and N-alkylated nucleotides, e.g. N6-methyl adenosine are suitable.
The 2'-OH-group may be replaced by a group selected from H, OR, R,
halo, SH, SR, NH.sub.2, NHR, NR.sub.2 or CN, wherein R is
C.sub.1-C.sub.6 alkyl, alkenyl or alkynyl and halo is F, Cl, Br or
I. The nucleic acid may comprise a 2'-ribose replacement such as a
2'-O-methyl and 2'-fluoro group, as described in U.S. Pat. No.
7,138,517, the contents of which are incorporated herein by
reference. Modified nucleotides also include nucleotides conjugated
with cholesterol through a hydroxyprolinol linkage as described in
Krutzfeldt et al., Nature 438:685-689 (2005), Soutschek et al.,
Nature 432:173-178 (2004), and U.S. Patent Publication No.
20050107325, which are incorporated herein by reference. Modified
nucleotides and nucleic acids may also include locked nucleic acids
(LNA), as described in U.S. Pat. No. 20020115080, which is
incorporated herein by reference. Additional modified nucleotides
and nucleic acids are described in U.S. Patent Publication No.
20050182005, which is incorporated herein by reference.
Modifications of the ribose-phosphate backbone may be done for a
variety of reasons, e.g., to increase the stability and half-life
of such molecules in physiological environments or as probes on a
biochip. Mixtures of naturally occurring nucleic acids and analogs
may be made; alternatively, mixtures of different nucleic acid
analogs, and mixtures of naturally occurring nucleic acids and
analogs may be made.
[0059] "Operably linked" used herein may mean that expression of a
gene is under the control of a promoter with which it is spatially
connected. A promoter may be positioned 5' (upstream) or 3'
(downstream) of a gene under its control. The distance between the
promoter and the gene may be approximately the same as the distance
between that promoter and the gene it controls in the gene from
which the promoter is derived. As is known in the art, variation in
this distance may be accommodated without loss of promoter
function.
[0060] "Probe" as used herein may mean an oligonucleotide capable
of binding to a target nucleic acid of complementary sequence
through one or more types of chemical bonds, usually through
complementary base pairing, usually through hydrogen bond
formation. Probes may bind target sequences lacking complete
complementarity with the probe sequence depending upon the
stringency of the hybridization conditions. There may be any number
of base pair mismatches that will interfere with hybridization
between the target sequence and a single stranded nucleic acid
described herein. However, if the number of mismatches is so great
that no hybridization can occur under even the least stringent of
hybridization conditions, the sequence is not a complementary
target sequence. A probe may be single stranded or partially single
and partially double stranded. The strandedness of the probe is
dictated by the structure, composition, and properties of the
target sequence. Probes may be directly labeled or indirectly
labeled such as with biotin to which a streptavidin complex may
later bind.
[0061] "Promoter" as used herein may mean a synthetic or
naturally-derived molecule which is capable of conferring,
activating or enhancing expression of a nucleic acid in a cell. A
promoter may comprise one or more specific regulatory elements to
further enhance expression and/or to alter the spatial expression
and/or temporal expression of same. A promoter may also comprise
distal enhancer or repressor elements, which can be located as much
as several thousand base pairs from the start site of
transcription. A promoter may be derived from sources including
viral, bacterial, fungal, plants, insects, and animals. A promoter
may regulate the expression of a gene component constitutively or
differentially with respect to cell, the tissue or organ in which
expression occurs or, with respect to the developmental stage at
which expression occurs, or in response to external stimuli such as
physiological stresses, pathogens, metal ions, or inducing agents.
Representative examples of promoters include the bacteriophage 17
promoter, bacteriophage T3 promoter, SP6 promoter, lac
operator-promoter, tac promoter, SV40 late promoter, SV40 early
promoter, RSV-LTR promoter, CMV IE promoter, SV40 early promoter or
SV40 late promoter and the CMV IE promoter.
[0062] "Reducing the amount of virus replication" used herein may
mean that the level or quantity of the target viral genome in the
target cell is reduced by at least about 2-fold to 100-fold or
more, as compared to a control, i.e., an identical target cell not
treated according to the subject methods.
[0063] "Selectable marker" used herein may mean any gene which
confers a phenotype on a cell in which it is expressed to
facilitate the identification and/or selection of cells which are
transfected or transformed with a genetic construct. Representative
examples of selectable markers include the ampicillin-resistance
gene (Amp.sup.r), tetracycline-resistance gene (Tc.sup.r),
bacterial kanamycin-resistance gene (Kan.sup.r), zeocin resistance
gene, the AURI-C gene which confers resistance to the antibiotic
aureobasidin A, phosphinothricin-resistance gene, neomycin
phosphotransferase gene (nptII), hygromycin-resistance gene,
beta-glucuronidase (GUS) gene, chloramphenicol acetyltransferase
(CAT) gene, green fluorescent protein-encoding gene and luciferase
gene.
[0064] "Stringent hybridization conditions" used herein may mean
conditions under which a first nucleic acid sequence (e.g., probe)
will hybridize to a second nucleic acid sequence (e.g., target),
such as in a complex mixture of nucleic acids. Stringent conditions
are sequence-dependent and will be different in different
circumstances. Generally, stringent conditions may be selected to
be about 5-10.degree. C. lower than the thermal melting point
(T.sub.m) for the specific sequence at a defined ionic strength pH.
The T.sub.m may be the temperature (under defined ionic strength,
pH, and nucleic concentration) at which 50% of the probes
complementary to the target hybridize to the target sequence at
equilibrium (as the target sequences are present in excess, at
T.sub.m, 50% of the probes are occupied at equilibrium). Stringent
conditions may be those in which the salt concentration is less
than about 1.0 M sodium ion, such as about 0.01-1.0 M sodium ion
concentration (or other salts) at pH 7.0 to 8.3 and the temperature
is at least about 30.degree. C. for short probes (e.g., about 10-50
nucleotides) and at least about 60.degree. C. for long probes
(e.g., greater than about 50 nucleotides). Stringent conditions may
also be achieved with the addition of destabilizing agents such as
formamide. For selective or specific hybridization, a positive
signal may be at least 2 to 10 times background hybridization.
Exemplary stringent hybridization conditions include the following:
50% formamide, 5.times.SSC, and 1% SDS, incubating at 42.degree.
C., or, 5.times.SSC, 1% SDS, incubating at 65.degree. C., with wash
in 0.2.times.SSC, and 0.1% SDS at 65.degree. C.
[0065] "Substantially complementary" used herein may mean that a
first sequence is at least 60%-99% identical to the complement of a
second sequence over a region of 8-50 or more nucleotides, or that
the two sequences hybridize under stringent hybridization
conditions. [0.066] "Substantially identical" used herein may mean
that a first and second sequence are at least 60%-99% identical
over a region of 8-50 or more nucleotides or amino acids, or with
respect to nucleic acids, if the first sequence is substantially
complementary to the complement of the second sequence.
[0066] "Subject" used herein may mean a mammal, such as a
human.
[0067] "Target" as used herein may mean a polynucleotide that may
be bound by one or more probes under stringent hybridization
conditions.
[0068] "Terminator" used herein may mean a sequence at the end of a
transcriptional unit which signals termination of transcription. A
terminator may be a 3'-non-translated DNA sequence containing a
polyadenylation signal, which may facilitate the addition of
polyadenylate sequences to the 3'-end of a primary transcript. A
terminator may be derived from sources including viral, bacterial,
fungal, plants, insects, and animals. Representative examples of
terminators include the SV40 polyadenylation signal, HSV TK
polyadenylation signal, CYC1 terminator, ADH terminator, SPA
terminator, nopaline synthase (NOS) gene terminator of
Agrobacterium tumefaciens, the terminator of the Cauliflower mosaic
virus (CaMV) 35S gene, the zein gene terminator from Zea mays, the
Rubisco small subunit gene (SSU) gene terminator sequences,
subclover stunt virus (SCSV) gene sequence terminators,
rho-independent E. coli terminators, and the lacZ alpha
terminator.
[0069] "Treat" or "treating" used herein when referring to
protection of an animal from a condition may mean preventing,
suppressing, repressing, or eliminating the condition. Preventing
the condition involves administering a composition described herein
to an animal prior to onset of the condition. Suppressing the
condition involves administering the composition to an animal after
induction of the condition but before its clinical appearance.
Repressing the condition involves administering the composition to
an animal after clinical appearance of the condition such that the
condition is reduced or prevented from worsening. Elimination of
the condition involves administering the composition to an animal
after clinical appearance of the condition such that the animal no
longer suffers from the condition.
[0070] "Therapeutically effective amount" used herein or
"therapeutically efficient" as to a drug dosage may refer to dosage
that provides the specific pharmacological response for which the
drug is administered in a significant number of subjects in need of
such treatment. The "therapeutically effective amount" may vary
according, for example, the physical condition of the patient, the
age of the patient and the severity of the disease.
[0071] "Unit dosage form," used herein may refer to a physically
discrete unit suitable as a unitary dosage for a human or animal
subject. Each unit may contain a predetermined quantity of a
composition described herein, calculated in an amount sufficient to
produce a desired effect in association with a pharmaceutically
acceptable diluent, carrier or vehicle. The specifications for a
unit dosage form may depend on the particular composition employed
and the effect to be achieved, and the pharmacodynamics associated
with the composition in the host.
[0072] "Variant" used herein to refer to a nucleic acid may mean
(i) a portion of a referenced nucleotide sequence; (ii) the
complement of a referenced nucleotide sequence or portion thereof;
(iii) a nucleic acid that is substantially identical to a
referenced nucleic acid or the complement thereof; or (iv) a
nucleic acid that hybridizes under stringent conditions to the
referenced nucleic acid, complement thereof, or a sequences
substantially identical thereto.
[0073] "Vector" used herein may mean a nucleic acid sequence
containing an origin of replication. A vector may be a plasmid,
bacteriophage, bacterial artificial chromosome or yeast artificial
chromosome. A vector may be a DNA or RNA vector. A vector may be
either a self-replicating extrachromosomal vector or a vector that
integrates into a host genome.
[0074] "Wild type" used herein to refer to a sequence may mean a
coding, non-coding or interface sequence that may be an allelic
form of a sequence that performs the natural or normal function for
that sequence. Wild type sequences may include multiple allelic
forms of a cognate sequence, for example, multiple alleles of a
wild type sequence may encode silent or conservative changes to the
protein sequence that a coding sequence encodes.
2. MicroRNA
[0075] While not being bound by theory, a gene coding for a miRNA
may be transcribed leading to production of an miRNA precursor
known as the pri-miRNA. The pri-miRNA may be part of a
polycistronic RNA comprising multiple pri-miRNAs. The pri-miRNA may
form a hairpin with a stem and loop. The stem may comprise
mismatched bases.
[0076] The hairpin structure of the pri-miRNA may be recognized by
Drosha, which is an RNase III endonuclease. Drosha may recognize
terminal loops in the pri-miRNA and cleave approximately two
helical turns into the stem to produce a 30 200 nt precursor known
as the pre-miRNA. Drosha may cleave the pri-miRNA with a staggered
cut typical of RNase III endonucleases yielding a pre-miRNA stem
loop with a 5' phosphate and -2 nucleotide 3' overhang.
Approximately one helical turn of the stem (.about.10 nucleotides)
extending beyond the Drosha cleavage site may be essential for
efficient processing. The pre-miRNA may then be actively
transported from the nucleus to the cytoplasm by Ran-GTP and the
export receptor Ex-portin-5.
[0077] The pre-miRNA may be recognized by Dicer, which is also an
RNase III endonuclease. Dicer may recognize the double-stranded
stem of the pre-miRNA. Dicer may also recognize the 5' phosphate
and 3' overhang at the base of the stem loop. Dicer may cleave off
the terminal loop two helical turns away from the base of the stem
loop leaving an additional 5' phosphate and .about.2 nucleotide 3'
overhang. The resulting siRNA-like duplex, which may comprise
mismatches, comprises the mature miRNA and a similar-sized fragment
known as the miRNA*. The miRNA and miRNA* may be derived from
opposing arms of the pri-miRNA and pre-miRNA. MiRNA* sequences may
be found in libraries of cloned miRNAs but typically at lower
frequency than the miRNAs.
[0078] Although initially present as a double-stranded species with
miRNA*, the miRNA may eventually become incorporated as a
single-stranded RNA into a ribonucleoprotein complex known as the
RNA-induced silencing complex (RISC). Various proteins can form the
RISC, which can lead to variability in the specificity for
miRNA/miRNA* duplexes, the binding site of the target gene, the
activity of the miRNA (repress or activate), and which strand of
the miRNA/miRNA* duplex is loaded in to the RISC.
[0079] When the miRNA strand of the miRNA:miRNA* duplex is loaded
into the RISC, the miRNA* may be removed and degraded. The strand
of the miRNA:miRNA* duplex that is loaded into the RISC may be the
strand whose 5' end is less tightly paired. In cases where both
ends of the miRNA:miRNA* have roughly equivalent 5' pairing, both
miRNA and miRNA* may have gene silencing activity.
[0080] The RISC may identify target nucleic acids based on high
levels of complementarity between the miRNA and the mRNA,
especially by nucleotides 2-8 of the miRNA. Only one case has been
reported in animals where the interaction between the miRNA and its
target was along the entire length of the miRNA. This was shown for
mir-196 and Hox B8 and it was further shown that mir-196 mediates
the cleavage of the Hox B8 mRNA (Yekta et al 2004, Science
304-594). Otherwise, such interactions are known only in plants
(Bartel & Bartel 2003, Plant Physiol 132-709).
[0081] A number of studies have looked at the base-pairing
requirement between miRNA and its mRNA target for achieving
efficient inhibition of translation (reviewed by Bartel 2004, Cell
116-281). In mammalian cells, the first 8 nucleotides of the miRNA
may be important (Doench & Sharp 2004 GenesDev 2004-504).
However, other parts of the microRNA may also participate in mRNA
binding. Moreover, sufficient base pairing at the 3' end can
compensate for insufficient pairing at the 5' end (Brennecke et al,
2005 PLoS 3-e85). Computation studies, analyzing miRNA binding on
whole genomes have suggested a specific role for bases 2-7 at the
5' end of the miRNA in target binding but the role of the first
nucleotide, found usually to be "A" was also recognized (Lewis et
at 2005 Cell 120-15). Similarly, nucleotides 1-7 or 2-8 were used
to identify and validate targets by Krek et al (2005, Nat Genet
37-495).
[0082] The target sites in the mRNA may be in the 5' UTR, the 3'
UTR or in the coding region. Interestingly, multiple miRNAs may
regulate the same mRNA target by recognizing the same or multiple
sites. The presence of multiple miRNA binding sites in most
genetically identified targets may indicate that the cooperative
action of multiple RISCs provides the most efficient translational
inhibition.
[0083] MiRNAs may direct the RISC to downregulate gene expression
by either of two mechanisms: mRNA cleavage or translational
repression. The miRNA may specify cleavage of the mRNA if the mRNA
has a certain degree of complementarity to the miRNA. When a miRNA
guides cleavage, the cut may be between the nucleotides pairing to
residues 10 and 11 of the miRNA. Alternatively, the miRNA may
repress translation if the miRNA does not have the requisite degree
of complementarity to the miRNA. Translational repression may be
more prevalent in animals since animals may have a lower degree of
complementarity between the miRNA and the binding site.
[0084] It should be noted that there may be variability in the 5'
and 3' ends of any pair of miRNA and miRNA*. This variability may
be due to variability in the enzymatic processing of Drosha and
Dicer with respect to the site of cleavage. Variability at the 5'
and 3' ends of miRNA and miRNA* may also be due to mismatches in
the stem structures of the pri-miRNA and pre-miRNA. The mismatches
of the stem strands may lead to a population of different hairpin
structures. Variability in the stem structures may also lead to
variability in the products of cleavage by Drosha and Dicer.
3. Nucleic Acid
[0085] A nucleic acid is provided herein. The nucleic acid may
comprise the sequence of any one of SEQ ID NOS: 1-15,527 or a
variant thereof. The variant may be a complement of the referenced
nucleotide sequence. The variant may also be a nucleotide sequence
that is substantially identical to the referenced nucleotide
sequence or the complement thereof. The variant may also be a
nucleotide sequence which hybridizes under stringent conditions to
the referenced nucleotide sequence, complements thereof, or
nucleotide sequences substantially identical thereto. The nucleic
acid may have a length of at least 10-250 nucleotides. The nucleic
acid may comprise a modified base. The nucleic acid may be
synthesized or expressed in a cell (in vitro or in vivo) using a
synthetic gene described herein. The nucleic acid may be
synthesized as a single strand molecule and hybridized to a
substantially complementary nucleic acid to form a duplex. [0086]
a. Nucleic acid complex
[0087] The nucleic acid may further comprise one or more of the
following: a peptide, a protein, a RNA-DNA hybrid, an antibody, an
antibody fragment, a Fab fragment, and an aptamer. [0088] b.
Pri-miRNA
[0089] The nucleic acid may comprise a sequence of a pri-miRNA or a
variant thereof. The pri-miRNA sequence may comprise from
45-30,000, 50-25,000, 100-20,000, 1,000-1,500 or 80-100
nucleotides. The sequence of the pri-miRNA may comprise a
pre-miRNA, miRNA and miRNA*, as set forth herein, and variants
thereof. The sequence of the pri-miRNA may comprise the sequence of
any one of SEQ ID NOS: 1-15,527 or a variant thereof.
[0090] The pri-miRNA may form a hairpin structure. The hairpin may
comprise first and second nucleic acid sequence that are
substantially complementary. The first and second nucleic acid
sequence may be from 37-50 nucleotides. The first and second
nucleic acid sequence may be separated by a third sequence of from
8-12 nucleotides. The hairpin structure may have a free energy less
than -25 Kcal/mole as calculated by the Vienna algorithm with
default parameters, as described in Hofacker et al., Monatshefte f.
Chemie 125: 167-188 (1994), the contents of which are incorporated
herein. The hairpin may comprise a terminal loop of 4-20, 8-12 or
10 nucleotides. The pri-miRNA may comprise at least 19% adenosine
nucleotides, at least 16% cytosine nucleotides, at least 23%
thymine nucleotides and at least 19% guanine nucleotides. [0091] c.
Pre-miRNA
[0092] The nucleic acid may also comprise a sequence of a pre-miRNA
or a variant thereof. The pre-miRNA sequence may comprise from
45-200, 60-80 or 60-70 nucleotides. The sequence of the pre-miRNA
may comprise a miRNA and a miRNA* as set forth herein. The sequence
of the pre-miRNA may also be that of a pri-miRNA excluding from
0-160 nucleotides from the 5' and 3' ends of the pri-miRNA. The
sequence of the pre-miRNA may comprise the sequence of any one of
SEQ ID NOS: 1-15,527 or a variant thereof. [0093] d. MiRNA
[0094] The nucleic acid may also comprise a sequence of a miRNA
(including miRNA*) or a variant thereof. The miRNA sequence may
comprise from 13-33, 18-24 or 21-23 nucleotides. The miRNA may also
comprise a total of at least 5-40 nucleotides. The sequence of the
miRNA may be the first 13-33 nucleotides of the pre-miRNA. The
sequence of the miRNA may also be the last 13-33 nucleotides of the
pre-miRNA. The sequence of the miRNA may be differentially
expressed during a viral infection, and may comprise the sequence
of any one of SEQ ID NOS: 1-9,221, 15,366-15,392, 15,516-15,527, or
a variant thereof as indicated in Table 1 and Table 8.
TABLE-US-00001 TABLE 8 The host and viral microRNAs which were
differentially expressed upon viral infection. MID HIDs microRNA
name Organism 15,366 15,379 hsa-miR-181a Homo sapiens 15,367 15,380
hsa-miR-193a Homo sapiens 15,368 15,381 hsa-miR-107 Homo sapiens
15,369 15,382 hsa-miR-103 Homo sapiens 15,370 15,383 hsa-miR-296
Homo sapiens 15,371 15,384 hsa-miR-574 Homo sapiens 15,372 15,385
hsa-miR-210 Homo sapiens 15,373 15,386 hsa-miR-21 Homo sapiens
15,374 15,387 RG_AMB_10594 Homo sapiens 15,375 15,388 hsv1-miR-H1
Human herpesvirus 1 15,376 15,389 RG_HSV2_Pred13 Human herpesvirus
2 15,392 15,377 15,389 RG_HSV2_8 Human herpesvirus 2 15,392 15,378
15,390 RG_fluA12 Influenza A virus 15,391 MID: SEQ ID NO of the
mature microRNA sequence HIDs: SEQ ID NO(S) of the hairpin microRNA
precursor microRNA name: The miRBase registry Sanger 9.2 microRNA
name, excluding cases in which the names begins with "RG". In these
cases the names are internal miRNA names of Rosetta Genomics.
Organism: The microRNA organism.
[0095] e. Anti-miRNA
[0096] The nucleic acid may also comprise a sequence of an
anti-miRNA that is capable of blocking the activity of a miRNA or
miRNA*, such as by binding to the pri-miRNA, pre-miRNA, miRNA or
miRNA* (e.g. antisense or RNA silencing), or by binding to the
target binding site. The anti-miRNA may comprise a total of 5-100
or 10-60 nucleotides. The anti-miRNA may also comprise a total of
at least 5-40 nucleotides. The sequence of the anti-miRNA may
comprise (a) at least 5 nucleotides that are substantially
identical or complementary to the 5' of a miRNA and at least 5-12
nucleotides that are substantially complementary to the flanking
regions of the target site from the 5' end of the miRNA, or (b) at
least 5-12 nucleotides that are substantially identical or
complementary to the 3' of a miRNA and at least 5 nucleotide that
are substantially complementary to the flanking region of the
target site from the 3' end of the miRNA. The sequence of the
anti-miRNA may comprise the complement of any one of SEQ ID NOS:
1-15,527 or a variant thereof. [0097] f. Binding Site of Target
[0098] The nucleic acid may also comprise a sequence of a target
microRNA binding site, or a variant thereof. The target site
sequence may comprise a total of 5-100 or 10-60 nucleotides. The
target site sequence may also comprise a total of at least 5-63
nucleotides. The target site sequence may comprise at least 5
nucleotides of the sequence of any one of SEQ ID NOS: 9,222-15,365
and 15,393-15,515 as indicated in Tables 3 and 9.
TABLE-US-00002 TABLE 9 The miRNAs and their predicted binding sites
microRNA Target gene BS SEQ Gene name MID Organism name ID NO
Ontology ID hsa-miR-181a 15,366 Homo sapiens BCL2 15,436 GO:
0006959 hsa-miR-181a 15,366 Homo sapiens BCL2 15,436 GO: 0051607
hsa-miR-181a 15,366 Homo sapiens CARD11 15,416 GO: 0050776
hsa-miR-181a 15,366 Homo sapiens CBLB 15,506 GO: 0006955
hsa-miR-181a 15,366 Homo sapiens CCL8 15,477 GO: 0006955
hsa-miR-181a 15,366 Homo sapiens CCL8 15,477 GO: 0009615
hsa-miR-181a 15,366 Homo sapiens CD4 15,471 GO: 0006955
hsa-miR-181a 15,366 Homo sapiens CD59 15,450 GO: 0006955
hsa-miR-181a 15,366 Homo sapiens CXCL5 15,419 GO: 0006955
hsa-miR-181a 15,366 Homo sapiens ETS1 15,499 GO: 0006955
hsa-miR-181a 15,366 Homo sapiens FAS 15,502 GO: 0006955
hsa-miR-181a 15,366 Homo sapiens GBP6 15,489 GO: 0006955
hsa-miR-181a 15,366 Homo sapiens IFNA17 15,511 GO: 0009615
hsa-miR-181a 15,366 Homo sapiens IL2 15,483 GO: 0006955
hsa-miR-181a 15,366 Homo sapiens LIF 15,400 GO: 0006955
hsa-miR-181a 15,366 Homo sapiens MS4A1 15,494 GO: 0006955
hsa-miR-181a 15,366 Homo sapiens MS4A1 15,497 GO: 0006955
hsa-miR-181a 15,366 Homo sapiens OPRK1 15,425 GO: 0006955
hsa-miR-181a 15,366 Homo sapiens POLA 15,453 GO: 0009615
hsa-miR-181a 15,366 Homo sapiens SAMHD1 15,399 GO: 0006955
hsa-miR-181a 15,366 Homo sapiens SEMA3C 15,393 GO: 0006955
hsa-miR-181a 15,366 Homo sapiens TNF 15,428 GO: 0006959
hsa-miR-181a 15,366 Homo sapiens TNF 15,428 GO: 0009615
hsa-miR-181a 15,366 Homo sapiens TNFAIP1 15,455 GO: 0006955
hsa-miR-181a 15,366 Homo sapiens TNFSF4 15,496 GO: 0006955
hsa-miR-193a 15,367 Homo sapiens CD97 15,468 GO: 0006955
hsa-miR-193a 15,367 Homo sapiens TNFAIP1 15,495 GO: 0006955
hsa-miR-107 15,368 Homo sapiens ARL6IP2 15,446 GO: 0006955
hsa-miR-107 15,368 Homo sapiens BST1 15,395 GO: 0006959 hsa-miR-107
15,368 Homo sapiens CCL13 15,460 GO: 0006955 hsa-miR-107 15,368
Homo sapiens EBI2 15,456 GO: 0006955 hsa-miR-107 15,368 Homo
sapiens FCGR2A 15,487 GO: 0006955 hsa-miR-107 15,368 Homo sapiens
FCGR2B 15,488 GO: 0006955 hsa-miR-107 15,368 Homo sapiens IFIT1L
15,426 GO: 0006955 hsa-miR-107 15,368 Homo sapiens IFNAR1 15,516
GO: 0009615 hsa-miR-107 15,368 Homo sapiens IL15 15,421 GO: 0006955
hsa-miR-107 15,368 Homo sapiens IL16 15,447 GO: 0006955 hsa-miR-107
15,368 Homo sapiens IL1RAP 15,402 GO: 0006955 hsa-miR-107 15,368
Homo sapiens MICB 15,433 GO: 0006955 hsa-miR-107 15,368 Homo
sapiens OAS3 15,444 GO: 0006955 hsa-miR-107 15,368 Homo sapiens
SPON2 15,462 GO: 0006955 hsa-miR-107 15,368 Homo sapiens TNF 15,424
GO: 0006959 hsa-miR-107 15,368 Homo sapiens TNF 15,424 GO: 0009615
hsa-miR-103 15,369 Homo sapiens ARL6IP2 15,446 GO: 0006955
hsa-miR-103 15,369 Homo sapiens BST1 15,395 GO: 0006959 hsa-miR-103
15,369 Homo sapiens CCL13 15,460 GO: 0006955 hsa-miR-103 15,369
Homo sapiens EBI2 15,456 GO: 0006955 hsa-miR-103 15,369 Homo
sapiens FCGR2A 15,487 GO: 0006955 hsa-miR-103 15,369 Homo sapiens
FCGR2B 15,488 GO: 0006955 hsa-miR-103 15,369 Homo sapiens IFIT1L
15,426 GO: 0006955 hsa-miR-103 15,369 Homo sapiens IFNAR1 15,516
GO: 0009615 hsa-miR-103 15,369 Homo sapiens IL15 15,421 GO: 0006955
hsa-miR-103 15,369 Homo sapiens IL16 15,447 GO: 0006955 hsa-miR-103
15,369 Homo sapiens IL1RAP 15,402 GO: 0006955 hsa-miR-103 15,369
Homo sapiens MICB 15,433 GO: 0006955 hsa-miR-103 15,369 Homo
sapiens OAS3 15,444 GO: 0006955 hsa-miR-103 15,369 Homo sapiens
SPON2 15,462 GO: 0006955 hsa-miR-103 15,369 Homo sapiens TNF 15,424
GO: 0006959 hsa-miR-103 15,369 Homo sapiens TNF 15,424 GO: 0009615
hsa-miR-296 15,370 Homo sapiens CD22 15,397 GO: 0006955 hsa-miR-296
15,370 Homo sapiens CD6 15,405 GO: 0006955 hsa-miR-296 15,370 Homo
sapiens CD6 15,440 GO: 0006955 hsa-miR-296 15,370 Homo sapiens CD8A
15,466 GO: 0006955 hsa-miR-296 15,370 Homo sapiens CXCL10 15,501
GO: 0006955 hsa-miR-296 15,370 Homo sapiens GBP4 15,490 GO: 0006955
hsa-miR-296 15,370 Homo sapiens GCK 15,464 GO: 0006955 hsa-miR-296
15,370 Homo sapiens HLA-DOA 15,415 GO: 0006955 hsa-miR-296 15,370
Homo sapiens LAT2 15,396 GO: 0006955 hsa-miR-296 15,370 Homo
sapiens LIF 15,470 GO: 0006955 hsa-miR-296 15,370 Homo sapiens
MAP4K2 15,439 GO: 0006955 hsa-miR-296 15,370 Homo sapiens PVRL1
15,465 GO: 0006955 hsa-miR-296 15,370 Homo sapiens SQSTM1 15,475
GO: 0006955 hsa-miR-296 15,370 Homo sapiens TNFSF15 15,504 GO:
0006955 hsa-miR-296 15,370 Homo sapiens VIPR1 15,458 GO: 0006955
hsa-miR-574 15,371 Homo sapiens IL28RA 15,486 GO: 0050691
hsa-miR-210 15,372 Homo sapiens CD59 15,406 GO: 0006955 hsa-miR-21
15,373 Homo sapiens CCL1 15,513 GO: 0006955 hsa-miR-21 15,373 Homo
sapiens CCL20 15,512 GO: 0006955 hsa-miR-21 15,373 Homo sapiens
CTSC 15,418 GO: 0006955 hsa-miR-21 15,373 Homo sapiens FASLG 15,401
GO: 0006955 hsa-miR-21 15,373 Homo sapiens IL12A 15,515 GO: 0006955
hsa-miR-21 15,373 Homo sapiens LILRB4 15,430 GO: 0006955 hsa-miR-21
15,373 Homo sapiens PAG1 15,482 GO: 0006955 hsa-miR-21 15,373 Homo
sapiens ST6GAL1 15,484 GO: 0006959 AMB_10594 15,374 Homo sapiens
C5AR1 15,445 GO: 0006955 AMB_10594 15,374 Homo sapiens CD74 15,461
GO: 0006955 AMB_10594 15,374 Homo sapiens CD79B 15,437 GO: 0006955
AMB_10594 15,374 Homo sapiens CX3CL1 15,413 GO: 0006955 AMB_10594
15,374 Homo sapiens DBNL 15,451 GO: 0006955 AMB_10594 15,374 Homo
sapiens HLA-DOA 15,435 GO: 0006955 AMB_10594 15,374 Homo sapiens
IFITM1 15,442 GO: 0006955 AMB_10594 15,374 Homo sapiens IL1R1
15,474 GO: 0006955 AMB_10594 15,374 Homo sapiens IL6ST 15,427 GO:
0006955 AMB_10594 15,374 Homo sapiens MBP 15,443 GO: 0006955
AMB_10594 15,374 Homo sapiens MS4A2 15,478 GO: 0006959 AMB_10594
15,374 Homo sapiens NCR3 15,459 GO: 0006955 AMB_10594 15,374 Homo
sapiens TNFSF8 15,414 GO: 0006955 AMB_10594 15,374 Homo sapiens
TOLLIP 15,411 GO: 0006955 hsv1-miR-H1 15,375 Human herpesvirus 1
ADA 15,448 GO: 0006955 hsv1-miR-H1 15,375 Human herpesvirus 1
ANXA11 15,409 GO: 0006955 hsv1-miR-H1 15,375 Human herpesvirus 1
CXCL9 15,473 GO: 0006955 hsv1-miR-H1 15,375 Human herpesvirus 1
HLA-DOB 15,469 GO: 0006955 hsv1-miR-H1 15,375 Human herpesvirus 1
IFIT5 15,423 GO: 0006955 hsv1-miR-H1 15,375 Human herpesvirus 1
OASL 15,412 GO: 0006955 hsv1-miR-H1 15,375 Human herpesvirus 1
POU2AF1 15,441 GO: 0006959 hsv1-miR-H1 15,375 Human herpesvirus 1
PTGER4 15,420 GO: 0006955 hsv1-miR-H1 15,375 Human herpesvirus 1
SERPINB4 15,505 GO: 0006955 hsv1-miR-H1 15,375 Human herpesvirus 1
SLA2 15,454 GO: 0050776 hsv1-miR-H1 15,375 Human herpesvirus 1
ST6GAL1 15,422 GO: 0006959 hsv1-miR-H1 15,375 Human herpesvirus 1
TCF1 15,498 GO: 0006955 HSV2_Pred13 15,376 Human herpesvirus 2 CD24
15,480 GO: 0006959 HSV2_Pred13 15,376 Human herpesvirus 2 IL16
15,493 GO: 0006955 HSV2_Pred13 15,376 Human herpesvirus 2 IL1A
15,431 GO: 0006955 HSV2_Pred13 15,376 Human herpesvirus 2 IL1RL1
15,434 GO: 0006955 HSV2_Pred13 15,376 Human herpesvirus 2 MBP
15,507 GO: 0006955 HSV2_Pred13 15,376 Human herpesvirus 2 OLR1
15,394 GO: 0006955 HSV2_Pred13 15,376 Human herpesvirus 2 TNFAIP1
15,403 GO: 0006955 HSV2_Pred13 15,376 Human herpesvirus 2 TRIM5
15,404 GO: 0009615 HSV2_Pred13 15,376 Human herpesvirus 2 VIPR1
15,410 GO: 0006955 HSV2_8 15,377 Human herpesvirus 2 ABCE1 15,472
GO: 0009615 HSV2_8 15,377 Human herpesvirus 2 CAST 15,514 GO:
0006955 HSV2_8 15,377 Human herpesvirus 2 CCL20 15,429 GO: 0006955
HSV2_8 15,377 Human herpesvirus 2 CCR2 15,476 GO: 0006955 HSV2_8
15,377 Human herpesvirus 2 CXCL3 15,417 GO: 0006955 HSV2_8 15,377
Human herpesvirus 2 DUOX2 15,408 GO: 0009615 fluA12 15,378
Influenza A virus C19orf2 15,479 GO: 0009615 fluA12 15,378
Influenza A virus CAST 15,467 GO: 0006955 fluA12 15,378 Influenza A
virus CBLB 15,509 GO: 0006955 fluA12 15,378 Influenza A virus CCL8
15,438 GO: 0006955 fluA12 15,378 Influenza A virus CCL8 15,438 GO:
0009615 fluA12 15,378 Influenza A virus CCR9 15,432 GO: 0006955
fluA12 15,378 Influenza A virus CLEC6A 15,463 GO: 0006955 fluA12
15,378 Influenza A virus CTSS 15,481 GO: 0006955 fluA12 15,378
Influenza A virus CXCL12 15,485 GO: 0006955 fluA12 15,378 Influenza
A virus CXCL12 15,485 GO: 0009615 fluA12 15,378 Influenza A virus
CXCL5 15,510 GO: 0006955 fluA12 15,378 Influenza A virus CXCL6
15,500 GO: 0006955 fluA12 15,378 Influenza A virus IFNGR2 15,451
GO: 0009615 fluA12 15,378 Influenza A virus IL1RAP 15,508 GO:
0006955 fluA12 15,378 Influenza A virus MADCAM1 15,407 GO: 0006955
fluA12 15,378 Influenza A virus MS4A1 15,492 GO: 0006955 fluA12
15,378 Influenza A virus OAS3 15,457 GO: 0006955 fluA12 15,378
Influenza A virus SMAD3 15,503 GO: 0050776 fluA12 15,378 Influenza
A virus TNFRSF11A 15,491 GO: 0006955 fluA12 15,378 Influenza A
virus ZF 15,398 GO: 0009615 microRNA name: The miRBase registry
(Release 9.2) microRNA name, excluding cases in which the names
begins with "RG". In these cases the names are internal miRNA names
of Rosetta Genomics. MID: SEQ ID NO of the mature microRNA Target
gene name: Target gene name according to RefSeq database BS SEQ ID
NO: The SEQ ID NO of the binding site of the microRNA to the 3' UTR
of the target gene. Gene Ontology ID: Gene Ontology (GO) ID. All
are related to viral infection and to the immune response, as
described in Table 10.
TABLE-US-00003 TABLE 10 The description of Gene Ontology IDs GOid
Description GO: 0009615 response to virus GO: 0051607 defense
response to virus GO: 0050691 regulation of antiviral response by
host GO: 0006959 humoral immune response GO: 0006955 immune
response GO: 0050776 regulation of immune response Description: The
Gene Ontology description of GO ID
4. Synthetic Gene
[0099] A synthetic gene is also provided comprising a nucleic acid
described herein operably linked to a transcriptional and/or
translational regulatory sequence. The synthetic gene may be
capable of modifying the expression of a target gene with a binding
site for a nucleic acid described herein. Expression of the target
gene may be modified in a cell, tissue or organ. The synthetic gene
may be synthesized or derived from naturally-occurring genes by
standard recombinant techniques. The synthetic gene may also
comprise terminators at the 3'-end of the transcriptional unit of
the synthetic gene sequence. The synthetic gene may also comprise a
selectable marker.
5. Vector
[0100] A vector is also provided comprising a nucleic acid
described herein, such as a pri-miRNA, pre-miRNA, miRNA,
anti-miRNA, target gene binding site, or synthetic gene. The vector
may be an expression vector. An expression vector may comprise
additional elements. For example, the expression vector may have
two replication systems allowing it to be maintained in two
organisms, e.g., in one host cell for expression and in a second
host cell (e.g., bacteria) for cloning and amplification. For
integrating expression vectors, the expression vector may contain
at least one sequence homologous to the host cell genome, and
preferably two homologous sequences which flank the expression
construct. The integrating vector may be directed to a specific
locus in the host cell by selecting the appropriate homologous
sequence for inclusion in the vector. The vector may also comprise
a selectable marker gene to allow the selection of transformed host
cells.
6. Host Cell
[0101] A host cell is also provided comprising a vector, synthetic
gene or nucleic acid described herein. The cell may be a bacterial,
fungal, plant, insect or animal cell. For example, the host cell
line may be DG44 and DUXB 11 (Chinese Hamster Ovary lines, DHFR
minus), HELA (human cervical carcinoma), CVI (monkey kidney line),
COS (a derivative of CVI with SV40 T antigen), 81610 (Chinese
hamster fibroblast) BALBC/3T3 (mouse fibroblast), HAK (hamster
kidney line), SP2/O (mouse myeloma), P3.times.63-Ag3.653 (mouse
myeloma), BFA-1c1BPT (bovine endothelial cells), RAJI (human
lymphocyte) and 293 (human kidney). Host cell lines may be
available from commercial services, the American Tissue Culture
Collection or from published literature.
7. Probes
[0102] A probe is also provided comprising a nucleic acid described
herein. Probes may be used for screening and diagnostic methods, as
outlined herein. The probe may be attached or immobilized to a
solid substrate, such as a biochip.
[0103] The probe may have a length of from 8 to 500, 10 to 100 or
20 to 60 nucleotides. The probe may also have a length of at least
8-300 nucleotides. The probe may further comprise a linker sequence
of from 10-60 nucleotides.
8. Biochip
[0104] A biochip is also provided. The biochip may comprise a solid
substrate comprising an attached probe or plurality of probes
described herein. The probes may be capable of hybridizing to a
target sequence under stringent hybridization conditions. The
probes may be attached at spatially defined addresses on the
substrate. More than one probe per target sequence may be used,
with either overlapping probes or probes to different sections of a
particular target sequence. The probes may be capable of
hybridizing to target sequences associated with a single disorder.
The probes may either be synthesized first, with subsequent
attachment to the biochip, or may be directly synthesized on the
biochip.
[0105] The solid substrate may be a material that may be modified
to contain discrete individual sites appropriate for the attachment
or association of the probes and is amenable to at least one
detection method. Representative examples of substrates include
glass and modified or functionalized glass, plastics (including
acrylics, polystyrene and copolymers of styrene and other
materials, polypropylene, polyethylene, polybutylene,
polyurethanes, TeflonJ, etc.), polysaccharides, nylon or
nitrocellulose, resins, silica or silica-based materials including
silicon and modified silicon, carbon, metals, inorganic glasses and
plastics. The substrates may allow optical detection without
appreciably fluorescing.
[0106] The substrate may be planar, although other configurations
of substrates may be used as well. For example, probes may be
placed on the inside surface of a tube, for flow-through sample
analysis to minimize sample volume. Similarly, the substrate may be
flexible, such as flexible foam, including closed cell foams made
of particular plastics.
[0107] The biochip and the probe may be derivatized with chemical
functional groups for subsequent attachment of the two. For
example, the biochip may be derivatized with a chemical functional
group including, but not limited to, amino groups, carboxyl groups,
oxo groups or thiol groups. Using these functional groups, the
probes may be attached using functional groups on the probes either
directly or indirectly using a linker. The probes may be attached
to the solid support by either the 5' terminus, 3' terminus, or via
an internal nucleotide.
[0108] The probe may also be attached to the solid support
non-covalently. For example, biotinylated oligonucleotides can be
made, which may bind to surfaces covalently coated with
streptavidin, resulting in attachment. Alternatively, probes may be
synthesized on the surface using techniques such as
photopolymerization and photolithography.
9. miRNA Expression Analysis
[0109] A method of identifying miRNAs that are associated with
disease or a pathological condition, such as viral infection is
also provided, comprising contacting a biological sample with a
probe or biochip provided herein and detecting the amount of
hybridization. PCR may be used to amplify nucleic acids in the
sample, which may provide higher sensitivity. A bioinformatic
method may be used to identify a specific miRNA target or target
pattern that is common among different viruses, and to identify a
target of human miRNA in a viral genome. The method may be used in
a system to identify a mRNA target of a host or viral miRNA. The
target may be useful to evaluate the role of a miRNA in a
virus-host interaction by up or down regulation, or for the
development of a therapeutic use of a miRNA.
[0110] The level of the nucleic acid in the sample may also be
compared to a control sample (e.g., a normal cell) to determine
whether the nucleic acid is differentially expressed (e.g.,
overexpressed or underexpressed). The ability to identify miRNAs
that are differentially expressed in pathological cells compared to
a control can provide high-resolution, high-sensitivity datasets
which may be used in the areas of diagnostics, prognostics,
therapeutics, drug development, pharmacogenetics, biosensor
development, and other related areas. An expression profile
generated by the current methods may be a "fingerprint" of the
state of the sample with respect to a number of miRNAs. While two
states may have any particular miRNA similarly expressed, the
evaluation of a number of miRNAs simultaneously allows the
generation of a gene expression profile that is characteristic of
the state of the cell. That is, normal tissue may be distinguished
from diseased tissue. By comparing expression profiles of tissue in
known different disease states, information regarding which miRNAs
are associated in each of these states may be obtained. Then,
diagnosis may be performed or confirmed to determine whether a
tissue sample has the expression profile of normal or disease
tissue. This may provide for molecular diagnosis of related
conditions.
10. Determining Expression Levels
[0111] The expression level of a viral infection- or
disease-associated nucleic acid may be informative in a number of
ways. For example, differential expression of a viral infection- or
disease-associated nucleic acid compared to a control may be
diagnostic of a patient suffering from the viral infection or
disease. Expression levels of a viral infection- or
disease-associated nucleic acid may also be used to monitor the
treatment and viral infection or disease state of a patient.
Furthermore, expression levels of a viral infection- or
disease-associated miRNA may allow the screening of drug candidates
for altering a particular expression profile or suppressing an
expression profile associated with viral infection or disease.
[0112] A target nucleic acid may be detected and levels of the
target nucleic acid measured by contacting a sample comprising the
target nucleic acid with a biochip comprising an attached probe
sufficiently complementary to the target nucleic acid and detecting
hybridization to the probe above control levels.
[0113] The target nucleic acid may also be detected by immobilizing
the nucleic acid to be examined on a solid support such as nylon
membranes and hybridizing a labeled probe with the sample.
Similarly, the target nucleic may also be detected by immobilizing
the labeled probe to a solid support and hybridizing a sample
comprising a labeled target nucleic acid. Following washing to
remove the non-specific hybridization, the label may be
detected.
[0114] The target nucleic acid may also be detected in situ by
contacting permeabilized cells or tissue samples with a labeled
probe to allow hybridization with the target nucleic acid.
Following washing to remove the non-specifically bound probe, the
label may be detected.
[0115] These assays can be direct hybridization assays or can
comprise sandwich assays, which include the use of multiple probes,
as generally outlined in U.S. Pat. Nos. 5,681,702; 5,597,909;
5,545,730; 5,594,117; 5,591,584; 5,571,670; 5,580,731; 5,571,670;
5,591,584; 5,624,802; 5,635,352; 5,594,118; 5,359,100; 5,124,246;
and 5,681,697, each of which is hereby incorporated by
reference.
[0116] A variety of hybridization conditions may be used, including
high, moderate and low stringency conditions as outlined above. The
assays may be performed under stringency conditions which allow
hybridization of the probe only to the target. Stringency can be
controlled by altering a step parameter that is a thermodynamic
variable, including, but not limited to, temperature, formamide
concentration, salt concentration, chaotropic salt concentration
pH, or organic solvent concentration.
[0117] Hybridization reactions may be accomplished in a variety of
ways. Components of the reaction may be added simultaneously, or
sequentially, in different orders. In addition, the reaction may
include a variety of other reagents. These include salts, buffers,
neutral proteins, e.g., albumin, detergents, etc. which may be used
to facilitate optimal hybridization and detection, and/or reduce
non-specific or background interactions. Reagents that otherwise
improve the efficiency of the assay, such as protease inhibitors,
nuclease inhibitors and anti-microbial agents may also be used as
appropriate, depending on the sample preparation methods and purity
of the target.
11. Diagnostic
[0118] A method of diagnosis is also provided. The method comprises
detecting a differential expression level of a disease-associated
nucleic acid in a biological sample. The sample may be derived from
a patient. Diagnosis of a disease state in a patient may allow for
prognosis and selection of therapeutic strategy. Further, the
developmental stage of cells may be classified by determining
temporarily expressed disease-associated nucleic acids.
[0119] In situ hybridization of labeled probes to tissue arrays may
be performed. When comparing the fingerprints between an individual
and a standard, the skilled artisan can make a diagnosis, a
prognosis, or a prediction based on the findings. It is further
understood that the genes which indicate the diagnosis may differ
from those which indicate the prognosis and molecular profiling of
the condition of the cells may lead to distinctions between
responsive or refractory conditions or may be predictive of
outcomes.
12. Drug Screening
[0120] A method of screening therapeutics is also provided. The
method comprises contacting a pathological cell capable of
expressing a disease related nucleic acid with a candidate
therapeutic and evaluating the effect of a drug candidate on the
expression profile of the disease associated nucleic acid. Having
identified the differentially expressed nucleic acid, a variety of
assays may be executed. Test compounds may be screened for the
ability to modulate gene expression of the disease associated
nucleic acid. Modulation includes both an increase and a decrease
in gene expression.
[0121] The test compound or drug candidate may be any molecule,
e.g., protein, oligopeptide, small organic molecule,
polysaccharide, polynucleotide, etc., to be tested for the capacity
to directly or indirectly alter the disease phenotype or the
expression of the disease associated nucleic acid. Drug candidates
encompass numerous chemical classes, such as small organic
molecules having a molecular weight of more than 100 and less than
about 500, 1,000, 1,500, 2,000 or 2,500 daltons. Candidate
compounds may comprise functional groups necessary for structural
interaction with proteins, particularly hydrogen bonding, and
typically include at least an amine, carbonyl, hydroxyl or carboxyl
group, preferably at least two of the functional chemical groups.
The candidate agents may comprise cyclical carbon or heterocyclic
structures and/or aromatic or polyaromatic structures substituted
with one or more of the above functional groups. Candidate agents
are also found among biomolecules including peptides, saccharides,
fatty acids, steroids, purines, pyrimidines, derivatives,
structural analogs or combinations thereof.
[0122] Combinatorial libraries of potential modulators may be
screened for the ability to bind to the disease associated nucleic
acid or to modulate the activity thereof. The combinatorial library
may be a collection of diverse chemical compounds generated by
either chemical synthesis or biological synthesis by combining a
number of chemical building blocks such as reagents. Preparation
and screening of combinatorial chemical libraries is well known to
those of skill in the art. Such combinatorial chemical libraries
include, but are not limited to, peptide libraries encoded
peptides, benzodiazepines, diversomers such as hydantoins,
benzodiazepines and dipeptide, vinylogous polypeptides, analogous
organic syntheses of small compound libraries, oligocarbamates,
and/or peptidyl phosphonates, nucleic acid libraries, peptide
nucleic acid libraries, antibody libraries, carbohydrate libraries,
and small organic molecule libraries.
13. Gene Silencing
[0123] Also provided is a method of reducing the expression of a
target gene in a cell, tissue or organ. Expression of the target
gene may be reduced by expressing a nucleic acid described herein
that comprises a sequence substantially complementary to one or
more binding sites of the target mRNA. The nucleic acid may be a
miRNA or a variant thereof. The nucleic acid may also be pri-miRNA,
pre-miRNA, or a variant thereof, which may be processed to yield a
miRNA. The expressed miRNA may hybridize to a substantially
complementary binding site on the target mRNA, which may lead to
activation of RISC-mediated gene silencing. An example for a study
employing over-expression of miRNA is provided in Yekta et al 2004,
Science 304-594, which is incorporated herein by reference. The
nucleic acids described herein may also be used to inhibit
expression of target genes using antisense methods well known in
the art, as well as RNAi methods described in U.S. Pat. Nos.
6,506,559 and 6,573,099, which are incorporated by reference.
[0124] The target gene may be a viral gene, the level of which may
be reduced by expressing a viral or human miRNA. The target gene
may also be a human gene that is expressed upon viral infection,
the level of which may be reduced by expressing a viral or human
miRNA. The target of gene silencing may be a protein that causes
the silencing of a second protein. By repressing expression of the
target gene, expression of the second protein may be increased.
Examples for efficient suppression of miRNA expression are the
studies by Esau et al 2004 JBC 275-52361; and Cheng et al 2005
Nucleic Acids Res. 33-1290, which is incorporated herein by
reference.
14. Gene Enhancement
[0125] Also provided is a method of increasing the expression of a
target gene in a cell, tissue or organ. Expression of the target
gene may be increased by expressing a nucleic acid described herein
that comprises a sequence substantially complementary to a
pri-miRNA, pre-miRNA, miRNA or a variant thereof. The nucleic acid
may be an anti-miRNA. The anti-miRNA may hybridize with a
pri-miRNA, pre-miRNA or miRNA, thereby reducing its gene repression
activity. Expression of the target gene may also be increased by
expressing a nucleic acid described herein that is substantially
complementary to a portion of the binding site in the target gene,
such that binding of the nucleic acid to the binding site may
prevent miRNA binding.
[0126] The target gene may be a viral gene, expression of which may
reduce infectivity of the virus. The target gene may also be a
human gene, expression of which may reduce infectivity of the virus
or increase resistance or immunity to the viral infection.
15. Reducing Viral Replication
[0127] A method of reducing the amount of viral replication is
provided, which may occur via gene silencing or gene enhancement
using the nucleic aid as described herein. The nucleic acid may
also be used to reduce the expression of a target gene in a cell
such as a viral gene in a virus-infected cell. Expression of the
viral or target gene may be reduced by expressing the nucleic acid,
which may comprise a sequence substantially complementary to one or
more binding sites of the target gene. The nucleic acid may be a
miRNA or a variant thereof. The nucleic acid may also be a
pri-miRNA, pre-miRNA, or a variant thereof, which may be processed
to yield a miRNA. The expressed miRNA may hybridize to a
substantially complementary binding site on the target mRNA, which
may lead to interruption of the function of the gene. In the case
of a viral target, replication of the virus may be inhibited, and
the viral infection may be reduced or eliminated.
[0128] The target gene may be a viral gene, which may be reduced by
expressing a viral or human miRNA. The target gene may also be a
human gene that is expressed upon viral infection, which may be
reduced by expressing a viral or human miRNA. The target of gene
silencing may be a protein that causes the silencing of a second
protein. By repressing expression of the target gene, expression of
the second protein may be decreased.
16. Therapeutic
[0129] Also provided is a method of modulating a disease or
disorder, which may be associated with a viral infection. In
general, the nucleic acid described herein may be used as a
modulator of the expression of a gene that is at least partially
complementary to the nucleic acid. Further, a miRNA molecule may
act as a target for a therapeutic screening procedure, e.g.
inhibition or activation of a miRNA molecule might modulate a
cellular differentiation process, e.g. apoptosis.
[0130] Furthermore, an existing miRNA molecule may be used as a
starting material for the manufacture of a sequence-modified miRNA
molecule, in order to modify the target-specificity thereof, e.g.
an oncogene, a multidrug-resistance gene or another therapeutic
target gene. Further, a miRNA molecule may be modified, so that it
may be processed and then generated as double-stranded siRNA that
may again be directed against a therapeutically relevant target.
Furthermore, a miRNA molecule may be used for tissue reprogramming
procedures, e.g. a differentiated cell line might be transformed by
expression of a miRNA molecule into a different cell type or a stem
cell.
17. Compositions
[0131] Also provided herein is a pharmaceutical composition, which
may comprise a nucleic acid described herein and optionally a
pharmaceutically acceptable carrier. The nucleic acid may be an
active ingredient of the composition. The composition may be used
for diagnostic or therapeutic applications. The administration of
the pharmaceutical composition may be carried out by known methods,
wherein a nucleic acid is introduced into a desired target cell in
vitro or in vivo.
[0132] The composition may be formulated in combination with
appropriate, pharmaceutically acceptable carriers or diluents, and
can be formulated into preparations in solid, semi-solid, liquid or
gaseous forms, such as tablets, capsules, powders, granules,
ointments, solutions, suppositories, injections, inhalants and
aerosols. As such, administration of the agents can be achieved in
various ways, including oral, buccal, rectal, parenteral,
intraperitoneal, intradermal, transdermal, intracheal, etc.
18. Nucleic Acid Delivery
[0133] The nucleic acid may be introduced to a cell, tissue or
organ in a single- or double-stranded form or capable of being
expressed by a synthetic gene using methods well known to those
skilled in the art, including as described in U.S. Pat. No.
6,506,559 which is incorporated by reference.
[0134] Methods for the delivery of nucleic acid molecules are
described in Akhtar et al., (Trends Cell Bio. 2, 139, 1992). WO
94/02595 describes general methods for delivery of RNA molecules.
These protocols can be utilized for the delivery of virtually any
nucleic acid molecule. Nucleic acid molecules can be administered
to cells by a variety of methods known to those familiar to the
art, including, but not restricted to, encapsulation in liposomes,
by iontophoresis, or by incorporation into other vehicles, such as
hydrogels, cyclodextrins, biodegradable nanocapsules, and
bioadhesive microspheres. Alternatively, the nucleic acid/vehicle
combination is locally delivered by direct injection or by use of
an infusion pump. Other routes of delivery include, but are not
limited to oral (tablet or pill form) and/or intrathecal delivery
(Gold, 1997, Neuroscience, 76, 1153-1158). Other approaches include
the use of various transport and carrier systems, for example,
through the use of conjugates and biodegradable polymers. More
detailed descriptions of nucleic acid delivery and administration
are provided for example in WO93/23569, WO99/05094, and
WO99/04819.
[0135] The nucleic acids can be introduced into tissues or host
cells by any number of routes, including viral infection,
microinjection, or fusion of vesicles. Jet injection may also be
used for intra-muscular administration, as described by Furth et
al. (Anal Biochem 115 205:365-368, 1992). The nucleic acids can be
coated onto gold microparticles, and delivered intradermally by a
particle bombardment device, or "gene gun" as described in the
literature (see, for example, Tang et al. Nature 356:152-154,
1992), where gold microprojectiles are coated with the DNA, then
bombarded into skin cells.
19. Kits
[0136] Also provided is a kit comprising a nucleic acid described
herein together with any or all of the following: assay reagents,
buffers, probes and/or primers, and sterile saline or another
pharmaceutically acceptable emulsion and suspension base. In
addition, the kit may include instructional materials containing
directions (e.g., protocols) for the practice of the methods
described herein.
20. Virus
[0137] The methods and nucleic acids described herein may be
associated with any one of a number of different visuses, including
HSV1, HSV2, RSV, EBV, Influenza A, HCV, HPV, HIV, HBV, and
Vaccinia. Influenza virus infection is a major public health
problem, causing millions of cases of severe illness and as many as
500,000 deaths each year worldwide (WHO report, 2004, A56/23).
Influenza virus has A, B and C types, among which the type A can be
further classified into many sub-types according to the variations
in NA and HA genes. Thus far, there have been 15 HA subtypes and 9
NA subtypes and the different combinations between HA and NA
subtypes can form many types of influenza A virus subtypes.
[0138] Although inactivated vaccines are 60-80% effective against
the matched influenza strains, vaccination coverage is a problem
worldwide. Moreover, this strategy provides no protection against
unexpected strains. Currently, antiviral drugs are the best defense
against these outbreaks, but they provide only partial protection
(Nicholson, etc., Lancet, 355:1845-1850, 2000), usually companied
with some side effects, especially to the central nervous system
(Wenzel, JAMA, 283:1057-1059, 2000).
[0139] Epstein Barr Virus (EBV), a large DNA virus of the Herpes
family that infects normal human B cells, is the etiologic agent of
infectious mononucleosis and is strongly associated with Burkitt's
lymphoma and nasopharingal carcinoma.
[0140] Herpes simplex virus type-1 and 2 (HSV1 and HSV2) enter and
reactivate from latency in sensory neurons, although the events
governing these processes are little understood. During latency,
only the latency-associated transcripts are produced.
21. Disease
[0141] The methods and nucleic acids described herein may be
associated with any one of a number of different diseases,
including Burkitt's lymphoma, nasopharingal carcinoma, ovarian
carcinoma, cervical cancer, hepatitis, mononucleosis, influenza,
genital herpes, encephalitis, and bronchiolitis.
EXAMPLES
Example 1
Prediction Of MiRNAs
[0142] We surveyed a number of viral genomes for potential miRNA
coding genes using three computational approaches similar to those
described in U.S. patent application Ser. Nos. 60/522,459,
10/709,577 and 10/709,572, the contents of which are incorporated
herein by reference, for predicting miRNAs. The predicted hairpins
and potential miRNAs were scored by thermodynamic stability, as
well as structural and contextual features. The algorithm was
calibrated by using miRNAs in the Sanger Database which had been
validated.
[0143] 1. Viral Genome Screen
[0144] Table 1 and Table 8 list the SEQ ID NO for each predicted
hairpin ("HID") of a particular viral genome ("V"; See also Table
7). Table 1 also lists the genomic location for each hairpin
("Hairpin_Loc"). The format for the genomic location is a
concatenation of <strand><start position>. For viruses
that have more than one chromosome or segment, such as Influenza A,
the segment number is identified in column "C" (viruses with only
one chromosome have a value of 1 in this column). The genetic
location is based on the NCBI--Entrez Nucleotides database. The
Entrez Nucleotides database is a collection of sequences from
several sources, including GenBank, RefSeq, and PDB. Table 7 shows
the accession number and the build (version) for each of the viral
genomes used in this screen. Two viruses in Table 7 have multiple
accession numbers because each segment (i.e., chromosome analog) of
the virus' genomes had a different accession number. One of the
viruses has no accession number.
[0145] Table 1 also lists the SEQ ID NO ("MID") for each predicted
miRNA and miRNA*. Table 1 also lists the prediction score grade for
each hairpin ("P") on a scale of 0-1 (1 means that the hairpin is
the most reliable), as described in Hofacker et al., Monatshefte f.
Chemie 125: 167-188, 1994. Table 1 also lists the p-value ("Pval")
calculated out of background hairpins for the values of each P
scores. All the p-values were significant, i.e., less than 0.05. If
the Pval is indicated as 0.0, then the Pval is less than 0.0001.
The p-values were calculated by comparing the palgrade of the
tested hairpin to the palgrade of other sequences without
pre-selection of hairpins.
[0146] Table 1 also lists whether the miRNAs were validated by
expression analysis ("E") (Y=Yes, N=No), as detailed in Table 2. It
should be noted that failure to sequence or detect expression of a
miRNA does not necessarily mean that a miRNA does not exist. Such
undetected miRNAs may be expressed in tissues other than those
tested. In addition, such undetected miRNAs may be expressed in the
test tissues, but at a difference stage or under different
conditions compared to the experimental cells.
[0147] Table 1 also lists a genetic location cluster ("LC") for
those hairpins that are within 1,000 nucleotides of each other of a
particular virus. Each miRNA that has the same LC shares the same
genetic cluster. Hairpins that overlap were not clustered.
Example 2
Prediction of Target Genes
[0148] The predicted miRNAs from the computational screen of
Example 1 were used to predict human and viral target genes and
their binding sites using computational approaches for predicting
miRNAs, similar to approaches described in U.S. patent application
Ser. Nos. 60/522,459, 10/709,577 and 10/709,572, the contents of
which are incorporated herein by reference.
1. Human Genome Screen
[0149] a. Human Target Genes
[0150] Table 3 and Table 9 list a predicted human target gene for
each miRNA ("MID") from a particular virus ("V") and its hairpin
("HID") from the viral genome screen. The virus codes listed in "V"
are, as for Table 1, defined in Table 7. The names of the target
genes ("Target_Gene") in Table 3 were taken from NCBI Reference
Sequence release 9 (http://www.ncbi.nlm.nih.gov; Pruitt et al.,
Nucleic Acids Res, 33(1):D501-D504, 2005; Pruitt et al., Trends
Genet., 16(1):44-47, 2000; and Tatusova et al., Bioinformatics,
15(7-8):536-43, 1999). Target genes were identified by having a
mammalian conserved perfect complementary match of a 7 nucleotide
miRNA seed (positions 2-8) and an A on the UTR (total=8
nucleotides). For a discussion on identifying target genes, see
Lewis et al., Cell, 120: 15-20, (2005). For a discussion of the
seed being sufficient for binding of a miRNA to a UTR, see Lim Lau
et al., (Nature 2005) and Brenneck et al, (PLoS Biol 2005).
[0151] The binding site screen only considered the first 4000
nucleotides per UTR and considered the longest transcript when
there were several transcripts per gene. The filtering reduced the
total number of transcripts from 23626 to 14239. Table 3 lists the
SEQ ID NO for the predicted binding sites ("Binding_site") for each
target gene. The sequence of the binding site includes the 20
nucleotides 5' and 3' of the binding site as they are located on
the spliced mRNA. In cases that the binding site is comprised from
2 exons, 20 nucleotides are included from both 5' and 3' ends of
both exons. [0152] b. Viral Target Genes
[0153] Human Herpes virus 1 and 2 are related to any of several
inflammatory diseases caused by a herpesvirus and marked in one
case by groups of watery blisters on the skin or mucous membranes
(as of the mouth and lips) above the waist and in the other by such
blisters on the genitals. Human herpesvirus 4 (Epstein-Barr virus)
is capable of causing infectious mononucleosis and is associated
with Burkitt's lymphoma and nasopharyngeal carcinoma. HIV strains
are related to Acquired Immune Deficiency Syndrome (AIDS).
Hepatitis B and C viruses are capable of causing inflammation of
the liver. Human papillomavirus is capable of causing cervical
cancer, human respiratory syncytial virus (RSV) is capable of
causing respiratory disease, and Influenza A virus is capable of
causing Influenza. Vaccinia virus has not been shown to be capable
of causing disease in humans, and is usually used for the
preparation of vaccines.
Example 3
Validation of miRNAs
[0154] To confirm the hairpins and miRNAs predicted in Example 1,
we detected expression in various tissues using the high-throughput
microarrays similar to those described in U.S. patent application
Ser. Nos. 60/522,459, 10/709,577 and 10/709,572, the contents of
which are incorporated herein by reference. For each predicted
precursor miRNA, mature miRNAs derived from both stems of the
hairpin were tested.
1. Expression Analysis
[0155] Table 2 shows the hairpins ("HID") of the third prediction
set that were validated by detecting expression of related miRNAs
("MID") from a particular virus ("V"), as well as a code for the
tissue ("Tissue") in which expression was detected. In cases where
there was more than one score from the same miRNA in the same
tissue, only the miRNA with the higher score is presented.
[0156] The tissue and disease codes are listed in Table 4 and Table
5, respectively. Table 6 shows the relationship between each gene
and at least one disease, enabling a miRNA described herein to be
connected to a disease. Table 6 condenses data derived from OMIM
and lists for each gene the numeric code(s) of the disease(s)
associated with the gene.
[0157] All the tissues disclosed give an indication of a viral
disease. The fact that significant expression of the virus was
measured implies that in this tissue it may be involve in a viral
disease(s). For example, if a miRNA from HIV is expressed in a T
cell line it may have an effect on AIDS. Of course cell lines
represent only subset of the features of a tissue as its function
in an organ however we can deduce from the expression as it is
measured in the cell line.
[0158] Table 2 also shows the chip expression score grade, which
ranges from 500 to 65000 ("S"). A threshold of 500 was used to
eliminate non-significant signals and the score was normalized by
miRNA microarray probe signals from different experiments.
Variations in the intensities of fluorescence material between
experiments may be due to variability in RNA preparation or
labeling efficiency. Normalization was performed based on the
assumption that the total amount of miRNAs in each sample was
relatively constant. First, the background signal was subtracted
from the raw signal of each probe, where the background signal was
defined as 400. Next, each miRNA probe signal was divided by the
average signal of all miRNAs, multiplied by 10000 and added back
the background signal of 400. Thus, by definition, the sum of all
miRNA probe signals in each experiment was 10400.
[0159] Table 2 also shows a statistical analysis of the normalized
signal ("Spval") calculated on the normalized score. For each
miRNA, a relevant control group was used out of the full predicted
miRNA list. Each miRNA had an internal control of probes with
mismatches. The relevant control group contained probes with
similar C and G percentage (abs diff <5%) in order to have a
similar Tm. The probe signal P value is the ratio over the relevant
control group probes with the same or higher signals. The results
were p-value .ltoreq.0.05 and the score was above 500. In those
cases for which the SPVa1 is listed as 0.0, the value is less than
0.0001.
2. Sequencing
[0160] To further validate the hairpins ("HID") of the second
prediction, a number of miRNAs were validated by sequencing methods
similar to those described in U.S. patent application Ser. Nos.
60/522,459, 10/709,577 and 10/709,572, the contents of which are
incorporated herein by reference.
3. Northern Blot Analysis
[0161] A group of miRNAs were validated by Northern blot analysis,
as shown in FIGS. 6, 9 and 10.
Example 4
Preparation of RNA libraries from Virus-Infected Cells and from
Interferon A Treated Cells
[0162] RNA libraries from virus-infected cells were prepared. For
each library from infected cells, a control library from uninfected
cells was also prepared. Total RNA was extracted from the paired
cell lines (infected and uninfected) and labeled directly with
either Cy5 or Cy3 (mirVanaTM miRNA Labeling Kit, Ambion). Each set
(labeled with either Cy3 or Cy5) was tested on the same micro-array
slide of microRNAs (MIRDICATOR.TM., Rosetta Genomics).
[0163] RNA libraries were prepared from the following cell lines
and viruses: [0164] a. Primary human fibroblasts (HF, from amnion
synthesis), human neuroblastoma cell line (UKF NB4), and Vero
cells, infected with HSV1 or HSV2, including libraries of early
stage and late stage of infection for both viruses. [0165] b. HEp2
cells infected with Respiratory Syncytial Virus (RSV). [0166] c.
MDCK cells (dog cells) infected with Influenza A (FluA). [0167] In
each case a library was also prepared from an uninfected control of
the same cell line.
[0168] RNA libraries were also prepared from interferon a treated
cells and from untreated cells to evaluate the effect of interferon
a on microRNA production.
[0169] MIRDICATOR.TM. microRNA array were used to detect novel host
and viral encoded miRNAs in the libraries (phase-1).
[0170] Glass slides were printed with probes of all the known
hsa-mirs in anti-sense (AS) orientation; as well as various
positive and negative controls (e.g. U6). In addition, AS probes to
the predictions of microRNAs of viral genes were included. The
predicted viral-microRNAs that were probed and analyzed were: HSV1,
HSV2, RSV, and FluA microRNAs. Therefore, for single hybridization,
two sets of positive signals on the microRNA array were obtained:
expression of host (human) microRNAs and expression of viral
microRNAs, in infected versus uninfected cells.
Example 5
Use of MIRDICATOR.TM. microRNA Array to Detect RSV Induced
miRNAs
[0171] Total RNA was extracted from HEp2 cells infected with RSV or
from control non-infected HEp2 cells and labeled with cy3 and cy5.
The probes were hybridized together to the same microRNA array
slide. The results depict signals for all microRNAs for both probes
plotted against each other.
[0172] FIG. 1 shows the results of RSV infected and uninfected HEp2
cells hybridized on microRNA array. The human microRNA
RG_AMB.sub.--10594 (SEQ ID NO: 15,374) was significantly
upregulated in the RSV infected HEp2 cells as compared to
uninfected control cells. The results suggest that host microRNAs
may play a role in RSV replication.
Example 6
Use of MIRDICATOR.TM. to Detect FluA Encoded microRNAs
[0173] MDCK cells were infected with FluA and control cells were
left uninfected. Three days after infection RNA was extracted,
labeled with Cy3 and Cy5 and hybridized to the glass-microRNA
array.
[0174] The results shown in FIGS. 2A-2B as a plot of MDCK cells
infected with FluA vs. uninfected cells. Two independent
experiments are shown. Out of 12 FluA probes (predicted by the
bioinformatics-algorithm and spotted on the microRNA array), one,
FluA-12 (SEQ ID NO: 15,378), gave a positive signal as shown
circled in FIGS. 2A-2B. This viral-microRNA is validated by real
time PCR for microRNAs (MIR-PCR, see details of the procedure
below).
Example 7
Use of MIRDICATOR.TM. to Detect HSV-1& HSV-2 Encoded
microRNAs
[0175] Neuroblastoma, Vero cells (cell line from green monkey's
kidney) and human fibroblasts (HF) were infected with either HSV-1
or HSV-2. RNAs were extracted and labeled with cy3 and cy5.
Eighteen samples from Vero cells and human fibroblasts (HF) were
hybridized together to the same type of microRNA array slide. Each
set of samples were either uninfected or infected with HSV-1 or
with HSV-2. The results depict signals for all microRNAs from both
probes, plotted against each other.
[0176] As shown in FIG. 3, the expression of hsv1-miR-H1 (SEQ ID
NO: 15,375) was found in the late stage of HSV-1 infected Vero
cells.
[0177] Two novel microRNAs of HSV-2: hsv2-8 (SEQ ID NO: 15,377) and
hsv2-predl3 (SEQ ID NO: 15,376) were found to be expressed in
HSV2-infected Vero cells as shown in FIG. 4, and to a lesser extent
in human neuroblastoma cell (UKF-NB4). These microRNAs are
originated from the 3' arm and the 5' arm of the same pre-microRNA
and are found twice in the HSV-2 genome. The HSV-2 microRNAs were
found to be expressed at the late stage of infection. These are the
first validated two microRNAs of HSV-2, a main cause of genital
herpes. The data was confirmed further by MIR-PCR (FIG. 7) and by
Northern blot analyses (FIGS. 9-10).
Example 8
Use of microRNA Array miRdicator.TM. to Detect Host microRNA
Differential Expression During Viral Infections
[0178] Up and down regulation of host microRNA (Homo sapiens,
hsa-mirs) during viral infections could be observed in several
infected cell lines. MDCK cells were infected with FluA. An
uninfected control was included. Three days after infection RNA was
extracted, labeled with Cy3 and Cy5 and hybridized to the
glass-microRNA array.
[0179] Table 11 depicts differential expression of host microRNAs
that are common to Homo sapiens and MDCK in FluA infected cells.
The results are average of two experiments. These results are
further validated by miRNA-RT-PCR and Northern blots.
[0180] Host hsa-miR-181a expression (SEQ ID NO: 15,366), is reduced
not only in FluA infected MDCK cells, but also in RSV infected HEp2
cells (see Northern blot analysis in FIG. 6). The results of the
microRNA array for hsa-miR-296 (SEQ ID NO: 15,370), hsa-miR-210
(SEQ ID NO: 15,372), hsa-miR-193a (SEQ ID NO: 15,367),
hsa-miR-181a, hsa-miR-107 (SEQ ID NO: 15,368) and hsa-miR-103 (SEQ
ID NO: 15,369) by Northern blot and miR-PCR analysis are under
validation.
TABLE-US-00004 TABLE 11 Differential expression of host miRs in
MDCK cells infected with FluA Signal from Signal from infected
uninfected Fold probe source cells cells change comments
hsa_miR_296 2674.5 435.5 (+)6 up-regulated in infected cells
hsa_miR_210 461 1746 (-)3.8 down-regulated in infected cells
hsa_miR_193a 2116.5 11879 (-)5.6 down-regulated in infected cells
hsa_miR_181a 1880.5 10124 (-)5.4 down-regulated in infected cells
hsa_miR_107 1160 6898 (-)5.9 down-regulated in infected cells
hsa_miR_103 661 4243 (-)6.4 down-regulated in infected cells
Example 9
Higher Expression of Human hsa-miR-21 in Vero Cells Infected with
HSV1 in Comparison to Vero Cells Infected with HSV2
[0181] Following infection of Vero cells with HSV-1 and HSV-2, RNA
was extracted, labeled with Cy3 and Cy5 and hybridized to the
glass-microRNA array. As demonstrated in
[0182] FIG. 8, hsa-miR-21 (SEQ ID NO: 15,373) is expressed at
higher levels in Vero cells infected with HSV-1 in comparison to
Vero cells infected with HSV2.
Example 10
Expression of Host miRNA AMB-10594 in Viral Infected or Interferon
Treated Cells
[0183] The following tests were carried out: interferon a treated
human fibroblasts (HF), HSV-1 and HSV-2 infected HF cells, and RSV
infected Hep2 cells. All tests included an untreated or uninfected
control. Three days after interferon a treatment or viral infection
RNA was extracted, labeled with Cy3 and Cy5 and hybridized to the
glass-microRNA array. As shown in FIG. 5, increased expression of
host-miR AMB-10594 (SEQ ID NO: 15,374) (shown circled) was found
in: interferon treated HF cells (FIG. 5C), HSV1 infected HF cells
(FIG. 5D), HSV2 infected HF cells (FIG. 5B), and RSV infected HEp2
cells (FIG. 5A). In each of the graphs the treated/infected cells
are plotted against the untreated/uninfected cells.
Example 11
[0184] Validation of the Results of the Microarray screening by
Northern Blot Analysis and by Cloning of Mirnas from Libraries of
Virus Infected and Cytokines Treated Cells
[0185] The results obtained from the microarray analysis were
validated in two procedures:
[0186] I. Northern blot analysis. Northern blot analysis comparing
RSV infected HEp2 cells with uninfected HEp2 cells was performed.
5Oug of RNA/slot were subjected to acrylamide-gel electrophoresis.
Northern blot analysis was performed using a
P.sup.32-labeled-AS-probe to human hsa-miR-181a. The result shown
in FIG. 6 indicates the down-regulation of the host miR in the RSV
infected cells, thus confirming the microarray results.
[0187] II. Real time PCR, aimed at amplification of miRs (miR-PCR),
adopted from Shi and Chiang, (Biotechniques, 2005. 39(4): p.
519-25). As little as 100 pg total RNA is polyadenylated and
reverse-transcribed with a poly(T) adapter into cDNAs for real-time
PCR using the miR-specific forward primer and the sequence
complementary to the poly(T) adapter as the reverse primer. This
real-time PCR method is simple and sensitive for quantifying the
expression of miRs and also reveals miR tissue-specific expression
patterns that cannot be resolved by Northern blot analysis.
[0188] Analysis by miR-PCR demonstrated specific expression of the
two novel HSV2 miRs (HSV2-miR-8 (SEQ ID NO: 15,377) and
HSV2-mir-pred13 (SEQ ID NO: 15,376) in HSV2 infected neuroblastoma
(UKF-NB4) and Vero cells. Total RNA was extracted from infected
cells, polyadenylated and reverse-transcribed with a poly(T)
adapter into cDNAs. The results are shown in FIG. 7: miR-specific
forward primer HSV2-miR-8 (FIG. 7A and 7B), HSV2-miR-pred13 (FIG.
7A) or bulge-primer (FIG. 7B). The reverse primer used was the
sequence complementary to the poly(T) adapter. The products were
subjected to agarose gel electrophoresis to determine their
relative size. Nonspecific bands formed the same pattern in
uninfected and HSV1 infected cells.
[0189] The results of the miR-PCR revealed a bulge probe (as
expected), a higher band of about 100 nucleotides corresponding to
the pre-microRNA precursor of these miRs (FIG. 7).
[0190] For Northern blot analysis, total RNA was extracted from
either Vero cells or human neuroblastoma cells (UKF-NB4):
uninfected, and HSV-1 and HSV-2 infected. The samples were enriched
for small RNAs (Ambion kit). The small RNAs samples were run on
Urea gel, 10 .mu.g/lane, blotted to Nitrocellulose and hybridized
with .sup.32P labeled probe to HSV2-mir-pred-13 or with .sup.32P
labeled probe to HSV2-mir-8 (SEQ ID NO: 15,377). As shown in FIG.
9, after exposure of 30 days the precursor (.about.100 nts) of
HSV2-mir-pred-13 (15,376) was visible. As shown in FIG. 10, the
precursor of HSV2-mir-8 (.about.100 nts) was visible and in
UKF-NB4, a band at the region of .about.22 nts was also detected.
The hybridization was specific to HSV2; no signal was detected
under the same conditions with either uninfected cells or cell
infected with HSV1. Experiments with specific inhibitors to these
HSV2 miRs will help to determine their role in HSV-2 infection.
Example 12
Epstain Barr-Virus (EBV) microRNAs are Differentially Expressed and
Can Block the Viral Replication
[0191] The EBV virus encodes several distinct miRNAs in latently
infected cells that are located in two main clusters. The BART
miRNAs (SEQ ID NOs: 15,516, 15,520, 15,522, 15,524 and 15,526) are
expressed at high levels in stages I and II of latency, whereas the
BHRF1 miRNAs (e.g. SEQ ID NO: 15,518) are expressed at high levels
in stage III latency, while being essentially undetectable in other
stages of virus life cycle. Induction of lytic EBV replication in
B95-8 cells, by Cisplatin and 12-O-tetradecanonoyl
phorbol-13-acetate (TPA) with n-Butyric acid or by TET ON/OFF
regulated expression of immediate early (1E) gene ZEBRA (BZLF1),
resulted in high expression of one of the BHRF-1-microRNA-cluster.
This cluster is localized on the 5' and 3'-UTRs of BHRF1 mRNA.
ZEBRA, is IE key gene in EBV switch to lytic infection. EBV
replication resulted in LMP-1 (latency associated protein 1)
reduction and increased viral load as determined by FACS and
RT-PCR.
[0192] B95-8 cells persistently infected with EBV were transfected
with various antagonists (2-O-Methyl antimir oligonucleotides, SEQ
ID NOs: 15,517, 15,519, 15,521, 15,523, 15,525, and 15,527) to EBV
microRNAs. After 120 hrs, cells were harvested, DNA was extracted,
and EBV DNA copies/ml was determined by qRTPCR, using commercial
standards for EBV viral load (FIG. 11).
[0193] BHRF-1, a viral homologue to Bcl2 oncogene, is also a member
of the IE proteins of EBV. Antisense blockage of BHRF1-miRNA
expression resulted in increased number of viral particles, up to
ten fold, in comparison to GFP control nonspecific antisense
treatment (FIG. 11).
[0194] Anti-microRNA to BART-2 had a moderate effect on viral
replication (FIG. 11). Taken together, the result supports the
notion that microRNAs play a central role in the control of viral
life cycle and that their suppression leads to lytic activation and
escape from latency. These findings also indicate the potential use
of microRNAs as bases for antiviral therapies.
[0195] Over expression of BHRF1-miRNA is achieved by transfection
of oligonucleotide mimicking the BHRF-1 microRNA into B-95-8 cells,
using serial dilutions. The oligonucleotides are comprised of two
small synthetic RNA molecules with 5'-phophorylation
(5'-/phos/rUrArUrCrUrUrUrUrGrCrGrGrCrArGrArArArUrUrGrA-3' (SEQ ID
NO: 4,581); and
5'-/phos/rArArArUrUrCrUrGrUrUrGrCrArGrCrArGrArUrArGrC) (SEQ ID NO:
15,518), hybridized to each other. Immediately after transfection
and at 24 hrs intervals the transfected cells are harvested and EBV
viral load is tested by RT-PCR.
[0196] Differential expression of EBV-mir-BHRF 1-2* (SEQ ID NO:
15,518) is tested in B-95-8, before and after induction with
doxycycline treatment which initiates viral early cycles. RNA is
extracted from the treated and untreated cells for Northern blot
analysis.
Example 13
Testing the Effect of HSV2 microRNAs on HSV2 Replication
[0197] Neuroblastoma and Vero cells that are stably transfected
with a plasmid containing the preMicrRNA of HSV2 (the precursor of
HSV2-mir 8 and HSV2-mir-pred13, SEQ ID NOs: 15,389 and 15,392),
cloned under IE-CMV-promoter in pEGFP-N1, Clontech), are infected
with either HSV1 or HSV2 to test the specific effect of over
expression of these two HSV2 microRNAs on HSV2 replication. Cells
transfected with pEGFP-N1 empty vector and cells transfected with a
vector containing the premicroRNA in AS-orientation are used as
negative controls. Northern blot analysis is carried out to confirm
that the construct produces the desired HSV-2-mirs and viral load
is tested by RT-PCR to evaluate the effect of over expression of
HSV-microRNAs on viral replication.
TABLE-US-LTS-CD-00001 LENGTHY TABLES The patent application
contains a lengthy table section. A copy of the table is available
in electronic form from the USPTO web site
(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20160002639A1).
An electronic copy of the table will also be available from the
USPTO upon request and payment of the fee set forth in 37 CFR
1.19(b)(3).
Sequence CWU 0 SQTB SEQUENCE LISTING The patent application
contains a lengthy "Sequence Listing" section. A copy of the
"Sequence Listing" is available in electronic form from the USPTO
web site
(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20160002639A1).
An electronic copy of the "Sequence Listing" will also be available
from the USPTO upon request and payment of the fee set forth in 37
CFR 1.19(b)(3).
0 SQTB SEQUENCE LISTING The patent application contains a lengthy
"Sequence Listing" section. A copy of the "Sequence Listing" is
available in electronic form from the USPTO web site
(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20160002639A1).
An electronic copy of the "Sequence Listing" will also be available
from the USPTO upon request and payment of the fee set forth in 37
CFR 1.19(b)(3).
* * * * *
References