U.S. patent application number 11/057276 was filed with the patent office on 2005-12-22 for inhibition of gene expression using polynucleotide analogues.
This patent application is currently assigned to Regents of the University of Minnesota, a Minnesota corporation. Invention is credited to Ekker, Stephen C., Kim, Hyon, Nasevicius, Aidas, Sumanas, Saulius.
Application Number | 20050283847 11/057276 |
Document ID | / |
Family ID | 27499242 |
Filed Date | 2005-12-22 |
United States Patent
Application |
20050283847 |
Kind Code |
A1 |
Ekker, Stephen C. ; et
al. |
December 22, 2005 |
Inhibition of gene expression using polynucleotide analogues
Abstract
The invention provides sequence specific polynucleotide
analogues and methods for determining the function of a nucleic
acid of known sequence.
Inventors: |
Ekker, Stephen C.; (St.
Paul, MN) ; Nasevicius, Aidas; (St. Paul, MN)
; Kim, Hyon; (Minneapolis, MN) ; Sumanas,
Saulius; (Minneapolis, MN) |
Correspondence
Address: |
FISH & RICHARDSON P.C.
PO BOX 1022
MINNEAPOLIS
MN
55440-1022
US
|
Assignee: |
Regents of the University of
Minnesota, a Minnesota corporation
|
Family ID: |
27499242 |
Appl. No.: |
11/057276 |
Filed: |
February 11, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11057276 |
Feb 11, 2005 |
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09918242 |
Jul 30, 2001 |
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6867349 |
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60221722 |
Jul 31, 2000 |
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60284974 |
Apr 19, 2001 |
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60252864 |
Nov 22, 2000 |
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Current U.S.
Class: |
800/20 ;
435/6.1 |
Current CPC
Class: |
A01K 2227/40 20130101;
A01K 2217/05 20130101; A01K 2227/50 20130101; A01K 2217/075
20130101; A01K 2267/03 20130101; C12N 15/8509 20130101; A01K
67/0275 20130101 |
Class at
Publication: |
800/020 ;
435/006 |
International
Class: |
A01K 067/027; C12Q
001/68 |
Goverment Interests
[0002] Funding for the work described herein was provided by the
federal government, which may have certain rights in the invention.
Claims
1-31. (canceled)
32. A method for determining a phenotype associated with a selected
nucleic acid in a teleost embryo or egg giving rise to said embryo,
wherein said embryo or egg is of a teleost species that undergoes
meroblastic cleavage, said method comprising: (a) contacting said
teleost embryo or egg giving rise to said embryo with a
morpholino-modified polynucleotide analogue that targets said
selected nucleic acid; and (b) detecting an altered phenotype in
said teleost embryo or egg, or embryo developing from said egg,
wherein said altered phenotype is associated with reduced
expression or altered function of said selected nucleic acid.
33. The method of claim 32, wherein said selected nucleic acid is a
maternal or zygotic nucleic acid.
34. The method of claim 32, wherein said altered phenotype is
observed from fertilization, through organogenesis, to the
completion of embryogenesis.
35. The method of claim 32, said method further comprising
contacting said embryo or egg giving rise to said embryo with a
rescue mRNA, wherein said rescue mRNA encodes a polypeptide whose
expression is reduced by said analogue, and wherein said rescue
mRNA is present in an amount sufficient for expression of said
polypeptide at a level comparable to that of a teleost embryo, or
egg giving rise to said embryo, that is free of said analogue.
36. A method for determining if a phenotype mediated by a
polynucleotide analogue in a teleost organism is sequence-specific,
said method comprising: a) contacting a first teleost embryo or
teleost egg with said polynucleotide analogue; b) assessing the
phenotype of said first teleost embryo or egg, or a teleost embryo
developing from said egg, subsequent to step (a); c) contacting a
second teleost embryo or teleost egg with (i) said polynucleotide
analogue and (ii) a rescue mRNA molecule; d) assessing the
phenotype of said second teleost embryo or egg, or a teleost embryo
developing from said egg, subsequent to step (c); and e) comparing
the results of (b) and (d), wherein a phenotype detected in (b)
that is not detected in (d) indicates that said phenotype detected
in (b) is sequence-specific.
37-67. (canceled)
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority from U.S. Provisional
Application Ser. No. 60/221 722, filed Jul. 31, 2000; U.S.
Provisional Application Ser. No. 60/252,864, filed Nov. 22, 2000;
and U.S. Provisional Application Ser. No. 60/284,974, filed Apr.
19, 2001.
BACKGROUND
[0003] 1. Technical Field
[0004] The invention relates to methods and materials involved in
the determination of the function of a nucleic acid based on its
sequence.
[0005] 2. Background Information
[0006] The ability of the various genome projects to acquire gene
sequence data has far outpaced the ability to ascribe biological
functions to these new genes. This dilemma has led to the concept
of "functional genomics" which can be defined as the attempt to
match biological function with gene sequence on a genome scale.
Since many biological processes are well conserved in evolution,
model organisms for which rapid genetic tools have been developed
can be used as model systems to identify human genes. For example,
genes with specific biological roles are identified first in the
model organism, and then genome databases are used to identify
human homologues.
[0007] A useful model system for the study of vertebrate biology is
the zebrafish Danio rerio. Aspects of the zebrafish developmental
process that render the zebrafish useful as a model system include
rapid development of the organ systems, transparent embryos,
embryos that develop outside the womb, and availability of a large
number of embryos. Furthermore, the sequence of the zebrafish
genome is expected to be completed by the end of 2002. Assignment
of function based on sequence information would be greatly
facilitated by the development of a rapid, targeted, knockdown
technology in this model vertebrate.
[0008] Recently, a number of strategies have been developed for
selectively repressing the expression of specific genes. These
methods are based on inducing degradation of cognate mRNAs by
introduction of double-stranded RNA into cells or addition of
single stranded antisense RNAs that trigger mRNA degradation by
RNase H. Although these methods have been used in D. melanogaster
and C. elegans, they have been unsuccessful in fish due to
toxicity, nonspecific effects, or the inability to achieve uniform
distribution for gene repression.
SUMMARY
[0009] The invention provides methods and materials for determining
the function of a nucleic acid of known sequence. More
specifically, the invention provides methods and materials for
determining the function of, or a phenotype associated with, a
selected nucleic acid of known sequence by specifically reducing
expression from the selected nucleic acid in a teleost. The
invention provides sequence specific polynucleotide analogues that
can be used to specifically reduce expression from selected nucleic
acids as well as methods of using sequence-specific polynucleotide
analogues to reduce expression from selected nucleic acids. The
function of, or phenotype associated with, a selected nucleic acid
can be determined by examining morphological or other phenotypic
alterations associated with the presence of a sequence-specific
polynucleotide analogue in an organism.
[0010] The invention provides a teleost embryo containing a
polynucleotide analogue in an amount effective to reduce expression
from a selected nucleic acid in the embryo. The embryo is of a
teleost species that undergoes meroblastic cleavage. The embryo can
be, for example, a zebrafish embryo, a puffer fish embryo, a medaka
embryo, or a stickleback embryo.
[0011] The polynucleotide analogue can be used to reduce expression
from a selected nucleic acid that is an mRNA. The analogue can be
complementary to a region of the mRNA that includes (1) the 5'
untranslated region of the mRNA, (2) part of or the entire AUG
start codon of the mRNA, (3) the coding region of the mRNA, or (4)
various combinations of the above. The length of the analogue can
be, for example, 9 to 90 bases in length, 15 to 50 bases in length,
or 20 to 30 bases in length. The analogue can be a
morpholino-modified polynucleotide, a 3'-5' phosphoroamidate, a
peptide nucleic acid, or a polynucleotide containing a ribose
moiety that has a 2' O-methyl group. The analogue can have at least
15% non-complementary nucleotides compared to the corresponding
nucleotides in the selected nucleic acid. The analogue can be
complementary to a nucleic acid in the embryo that has a homologue
or orthologue in another species. The analogue can be used to
reduce expression from the selected nucleic acid through larval or
post-hatching stages of development.
[0012] The invention also provides for an embryo containing an
exogeneous rescue mRNA that encodes a polypeptide whose expression
is reduced by the polynucleotide analogue. The rescue mRNA is
present in an amount sufficient for expression of the polypeptide
at a level comparable to that in embryos free of the analogue.
[0013] The invention also provides for an embryo that has at least
one additional polynucleotide analogue that is complementary to a
different region of the same selected nucleic acid. Both analogues
are present in amounts effective to reduce expression from the
selected nucleic acid.
[0014] The invention also provides for an embryo that has at least
one additional polynucleotide analogue that is complementary to at
least one other nucleic acid that is different from the first. All
analogues are present in amounts effective to reduce expression
from each of the different nucleic acids.
[0015] In another embodiment, the invention provides a method for
producing a teleost embryo containing a polynucleotide analogue.
The embryo can be a teleost embryo that undergoes meroblastic
cleavage. The analogue is present in an amount effective to reduce
expression from a selected nucleic acid in the embryo. The method
involves contacting the embryo, or an egg giving rise to the
embryo, with the polynucleotide analogue. For example, the embryo
or egg giving rise to the embryo can be injected with the analogue
or the analogue can be added to the surface of the embryo or egg
giving rise to the embryo. The embryo or egg giving rise to the
embryo can be a zebrafish embryo or egg giving rise to the
zebrafish embryo, a puffer fish embryo or egg giving rise to the
puffer fish embryo, a medaka embryo or egg giving rise to the
medaka embryo, or a stickleback embryo or egg giving rise to the
stickleback embryo.
[0016] In another embodiment, the invention also provides a
composition comprising a morpholino-modified polynucleotide that is
complementary to a selected nucleic acid and a buffer having a pH
similar to the physiological pH within a teleost egg or embryo. The
buffer can be isotonic to the teleost egg or embryo. The buffer can
be Danieau buffer. The teleost egg or embryo can be that of a
species that undergoes meroblastic cleavage. The teleost egg or
embryo can be a zebrafish egg or embryo, a puffer fish egg or
embryo, a medaka egg or embryo, or a stickleback egg or embryo. In
addition, the composition also can contain a rescue mRNA that
encodes a polypeptide whose expression is reduced by the
morpholino-modified analogue. The composition also can contain at
least one additional polynucleotide analogue that is complementary
to different regions of the selected nucleic acid.
[0017] In another embodiment, the invention provides method for
determining a phenotype associated with a selected nucleic acid in
a teleost embryo or egg giving rise to the embryo. The embryo or
egg is that of teleost species that undergoes meroblastic cleavage.
The method involves contacting the teleost embryo or egg giving
rise to the embryo with a morpholino-modified polynucleotide
analogue that targets the selected nucleic acid and then detecting
an altered phenotype in the teleost embryo or egg, or embryo
developing from said egg. The altered phenotype is one that is
associated with reduced expression or altered function of said
selected nucleic acid. The selected nucleic acid can be a maternal
or zygotic nucleic acid and the altered phenotype can be observed
from fertilization, through organogenesis, to the completion of
embryogenesis.
[0018] The invention also provides a method for determining a
phenotype associated with a selected nucleic acid in a teleost
embryo or egg giving rise to the embryo. The embryo or egg is that
of teleost species that undergoes meroblastic cleavage. The method
involves contacting the teleost embryo or egg giving rise to the
embryo with a morpholino-modified polynucleotide and a rescue mRNA
and then detecting an altered phenotype in the teleost embryo or
egg, or embryo developing from said egg. The morpholino-modified
polynucleotide is present in an amount effective to reduce
expression from the nucleic acid. The rescue mRNA encodes a
polypeptide whose expression is reduced by the analogue and is
present in an amount sufficient for expression of the polypeptide
at a level comparable to that of a teleost embryo, or egg giving
rise to said embryo, that is free of the analogue.
[0019] In another embodiment, the invention provides a method for
determining if a phenotype mediated by a polynucleotide analogue in
a teleost organism is sequence-specific. The method involves
contacting a first teleost embryo or teleost egg with the
polynucleotide analogue and assessing the phenotype of the first
teleost embryo or egg, or a teleost embryo developing from the egg,
subsequent to contacting with the polynucleotide analogue. A second
teleost embryo or teleost egg is contacted with (i) the
polynucleotide analogue and (ii) a rescue mRNA molecule and the
phenotype of the second teleost embryo or egg, or a teleost embryo
developing from the egg, is subsequently assessed. The method then
involves comparing the phenotype of the first embryo or egg to the
phenotype of the second embryo or egg. The analogue is
sequence-specific if the phenotype of the first embryo or egg is
not found in the second embryo or egg.
[0020] In another embodiment, the invention provides a method of
determining if first and second polypeptides are genetic
interactors. The method involves contacting a first teleost embryo
or teleost egg with a first polynucleotide analogue that targets a
nucleic acid encoding a first polypeptide, and assessing the
phenotype of the resulting teleost embryo or egg, or a teleost
embryo developing from the egg. The method also involves contacting
a second teleost embryo or egg giving rise to the embryo with a
second polynucleotide analogue that targets a nucleic acid encoding
the second polypeptide, and assessing the phenotype of the
resulting teleost embryo or egg, or a teleost embryo developing
from the egg. The method also involves contacting a third teleost
embryo or egg giving rise to the embryo with the first and second
polynucletide analogues, and assessing the phenotype of the
resulting teleost embryo or egg, or a teleost embryo developing
from the egg. The phenotypes of the resulting first, second, and
third teleost embryos or eggs, or teleost embryos developing from
such eggs, are compared. The two polypeptides are genetic
interactors if the phenotype observed in the third embryo or egg is
different from the sum of the individual phenotypes observed in the
first and second embryos or eggs. The phenotype observed when both
polynucleotide analogues are used can be more or less extensive
than the sum of the individual phenotypes observed when one of the
two analogues is used.
[0021] In another embodiment, the invention provides a kit
comprising a collection of different morpholino-modified
polynucleotides. The different morpholino-modified polynucleotides
are effective to reduce expression from different nucleic acids
that are involved in a common metabolic process.
[0022] In another embodiment, the invention provides a collection
of morphants, each morphant generated by a different
morpholino-modified polynucleotide selected from a collection of
morpholino-modified polynucleotides effective to reduce expression
from different nucleic acids that are involved in a common
metabolic process.
[0023] In another embodiment, the invention provides a teleost
morphant defective in development of a differentiated tissue. The
differentiated tissue can be pancreas, vasculature tissue, blood,
eye, the central neural system, muscle, the backbone, the head, a
limb, or a pigment cell.
[0024] In another embodiment, the invention provides a teleost
morphant that has a phenotype characteristic of a disease
condition. The disease condition can be, for example, porphyria or
cyclopia.
[0025] In another embodiment, the invention provides a method of
identifying a nucleic acid associated with a disease condition. The
method involves generating a teleost morphant having a morphant
phenotype that corresponds to a phenotype characteristic of the
disease condition, and identifying the nucleic acid target of the
morpholino-modified polynucleotide in the teleost morphant. The
nucleic acid target of the morpholino-modified polynucleotide is a
nucleic acid associated with the disease condition.
[0026] In another embodiment, the invention provides a method for
assessing the effect of a drug on a morphant. The method involves
contacting the morphant with the drug and assessing the phenotype
of the morphant subsequent to contact with the drug. The phenotype
of the morphant can be unaltered subsequent to contact with the
drug or replaced by a less severe phenotype subsequent to contact
with the drug. The phenotype of the morphant, subsequent to contact
with said drug, can be correlated with a change in the activity of
a biomarker.
[0027] In another embodiment, the invention provides a method of
reducing expression from a selected nucleic acid in an animal. The
method involves administering at least two polynucleotide analogues
to the animal. The analogues are complementary to different regions
of the selected nucleic acid. The polynucleotide analogues can act
synergistically to reduce expression from the selected nucleic
acid. Such expression can be reduced by a synergy factor of 3, 5,
or 10. The polynucleotide analogues can be morpholino-modified
polynucleotides.
[0028] In another embodiment, the invention provides a composition
comprising at least two different morpholino-modified
polynucleotides and a pharmaceutically acceptable carrier. The
morpholino-modified polynucleotides can target the same selected
nucleic acid and can be complementary to non-overlapping regions of
the selected nucleic acid. The non-overlapping regions can be
separated by more than 1 000 nucleotides.
[0029] Unless otherwise defined, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention pertains.
Although methods and materials similar or equivalent to those
described herein can be used in the practice or testing of the
present invention, suitable methods and materials are described
below. All publications, patent applications, patents, and other
references mentioned herein are incorporated by reference in their
entirety. In case of conflict, the present specification, including
definitions, will control. In addition, the materials, methods, and
examples are illustrative only and not intended to be limiting.
[0030] Other features and advantages of the invention will be
apparent from the following detailed description, and from the
claims.
DESCRIPTION OF DRAWINGS
[0031] FIG. 1 is a GFP fluorescence inhibition graph demonstrating
sequence-specific and dose-dependent inhibition of GFP
expression.
[0032] FIG. 2 is a graph demonstrating that two chordin-MO
phenotypes, weak and strong, were achieved with increasing doses of
chordin-MO injected.
[0033] FIG. 3 is a bar graph demonstrating that the chordin-MO
phenotype was partially rescued by Xenopus chordin mRNA injection,
thereby illustrating specificity of chordin-MO targeting.
[0034] FIG. 4 is a bar graph demonstrating dose-dependent reduction
in the frequency of the oep phenotype in response to oep mRNA
injections.
[0035] FIG. 5 is a bar graph comparing the frequencies of nt1, oep,
and nt1 and oep phenotypes observed in embryos injected with
nt1-MO, oep-MO, or both MOs.
[0036] FIG. 6 is a bar graph comparing the frequencies of cyclopia,
u-somites, and reduced fins in embryos injected with a control-MO
and twhh-MO, a control-MO and shh-MO, or both twhh- and
shh-MOs.
[0037] FIGS. 7A and 7B are bar graphs demonstrating synergy between
two shh-MOs.
[0038] FIG. 8 is a bar graph demonstrating synergy between two
VEGF-MOs.
[0039] FIG. 9 is a bar graph demonstrating synergy between two
zfz-MOs.
[0040] FIG. 10 is a bar graph demonstrating the synergistic effects
of ztsg1- and chordin-MO on blood island expansion.
DETAILED DESCRIPTION
[0041] The invention provides methods and materials for determining
the function of a nucleic acid of known sequence. More
specifically, the invention provides methods and materials for
determining the function of, or a phenotype associated with, a
selected nucleic acid of known sequence by specifically reducing
expression from the selected nucleic acid in a teleost. The
invention provides sequence specific polynucleotide analogues that
can be used to specifically reduce expression from selected nucleic
acids as well as methods of using sequence-specific polynucleotide
analogues to reduce expression from selected nucleic acids. For
example, the invention provides morpholino-modified polynucleotides
and methods of using morpholino-modified polynucleotides for
reducing expression of selected nucleic acids whose sequences are
known. Reduction in expression is reflected in the level of
specific RNA or polypeptide produced as well as in morphological or
other phenotypic changes. The function of, or phenotype associated
with, a selected nucleic acid can be determined by examining
morphological or other phenotypic alterations associated with the
presence of a sequence-specific polynucleotide analogue in an
organism.
[0042] 1. Polynucleotide and Polynucleotide Analogues
[0043] Polynucleotides are linear polymers consisting of monomeric
subunits called nucleotides. A nucleotide has three components: a
phosphate group, an organic base, and a five-carbon sugar that
links the phosphate group and the organic base. The nucleotide
subunits of a polynucleotide are linked by phophodiester bonds,
i.e. the five-carbon sugar of one nucleotide forms an ester bond
with the phosphate of an adjacent nucleotide. The resulting
sugar-phosphates form the backbone of a polynucleotide, while the
organic bases determine the sequence of the polynucleotide and
allow for interaction with a second polynucleotide. When the
sequence of the bases of one polynucleotide is complementary to the
sequence of the bases of a second polynucleotide, the two
polynucleotides can anneal to form a duplex held together by
hydrogen bonds and hydrophobic interactions. Two polynucleotides
are said to have complementary sequences if the bases in one
polynucleotide are able to pair, through hydrogen bonding, with the
bases in the second polynucleotide according to known Watson-Crick
type base pairing rules, (see DNA in Molecular Cell Biology,
Darnell et al. (1990) Scientific American Books. 2.sup.nd Edition,
pages 68-74). The strength of the interaction between the two
polynucleotides is determined by the degree of complementarity. For
example, the strength of interaction between two polynucleotides is
greatest if the base sequences are 100% complementary. Duplexes
formed between two polynucleotides with 4%, 8%, 16%, 25%, or more
than 25% mismatch bases are successively less strong and will
separate at successively lower temperatures. As used herein, the
term "polynucleotide" refers to a DNA or an RNA polymer having at
least three nucleotides.
[0044] Polynucleotide analogues are chemically modified
polynucleotides. Typically, polynucleotide analogues are formed by
replacing all or portions of the five-carbon sugar-phosphate
backbone of a polynucleotide with alternative functional groups in
such a way that base pairing with a selected nucleic acid is
maintained. As used herein, the term "selected nucleic acid" refers
to a DNA or an RNA having a region that is complementary to the
polynucleotide analogue. The region of the selected nucleic acid
that is complementary to the polynucleotide analogue can be 100%
complementary or less than 100% complementary to the entire
polynucleotide analogue sequence, as long as the polynucleotide
analogue can anneal and form a stable duplex with the selected
nucleic acid under physiological conditions. For example, a
polynucleotide sequence of 25 nucleotides can have as many as three
non-complementary bases distributed throughout the polynucleotide
and still anneal with the selected nucleic acid.
[0045] Some examples of polynucleotide analogues include: analogues
in which the bases are linked by a polyvinyl backbone (Pitha et al.
(1970) Biochem Biophys Acta 204:39 and Pitha et al. (1970)
Biopolymers 9: 965); peptide nucleic acids (PNAs) in which the
bases are linked by amide bonds formed by pseudopeptide
2-aminoethyl-glycine groups; analogues in which the nucleoside
subunits (i.e. base and sugar) are linked by methylphosphonate
groups (Miller et al. (1979) Biochem 18: 5134; Miller et al. (1980)
J Biol Chem 255: 6959); analogues in which the phosphate residues
linking nucleoside subunits are replaced by phosphoroamidate groups
(Froehler et al. (1988) Nucleic Acids Res 156: 4831);
phosphorothioated DNAs, analogues containing sugar moieties that
have 2' O-methyl groups (Cook (1998) CHAPTER 2: Antisense Medicinal
Chemistry in The Medicinal Chemistry of Oligonucleotides. Springer,
New York. pages 51-101); and morpholino-modified analogues,
analogues in which the bases are linked by a
morpholino-phosphorodiamidate backbone (U.S. Pat. Nos. 5,142,047
and 5,185,444). Polynucleotide analogues can be obtained
commercially, produced using commercially available monomeric
subunits, or synthesized using known methods. (See Braasch and
Corey (2001) Chemistry and Biology, pages 1-7.)
[0046] Useful polynucleotide analogues are those that (1) can form
duplexes with selected cellular nucleic acids in a sequence
specific manner, (2) form duplexes that are relatively insensitive
to ionic concentration or relatively resistant to cellular
strand-separating mechanisms, (3) have low nuclease sensitivity,
and (4) have low cellular toxicity and non-specific effects. Useful
polynucleotide analogues typically are specific, can distribute
uniformly throughout most or all cells of an organism, are
functional in many or all cell types, are efficient at reducing
expression from different nucleic acids and have little or no
non-specific effects. Furthermore, the technique for use is
straightforward to perform and reproducible.
[0047] Typically, useful polynucleotide analogues are single
stranded, and can be various lengths such as 8 to more than 112
bases in length. Polynucleotide analogues can be 12 to 72 bases in
length. For example, polynucleotide analogues can be 15 to 45 bases
in length. Ideally, polynucleotide analogues are 18-30 bases in
length.
[0048] A useful polynucleotide analogue can be complementary to a
sense or an antisense nucleic acid. When complementary to a sense
nucleic acid, the polynucleotide analogue is said to be antisense.
When complementary to an antisense nucleic acid, the analogue is
said to be sense. The nucleic acid can be RNA (e.g. a pre-mRNA or
an mRNA) or DNA. For example, a useful polynucleotide analogue can
be antisense to a pre-mRNA or an mRNA moleculeor sense to the DNA
molecule from which an mRNA is transcribed.
[0049] A useful polynucleotide analogue can be complementary to the
non-coding region of a nucleic acid. A non-coding region, for
example, can be a region upstream of a transcriptional start point
or a region downstream of a transcriptional end-point in a DNA
molecule. A non-coding region also can be a region upstream of the
translational start codon or downstream of the stop codon in a
pre-mRNA or an mRNA molecule. A non-coding region also can be the
intronic sequences within a pre-mRNA (see Ekker & Larson (2001)
Genesis 30:89-93). Furthermore, a useful polynucleotide analogue
can be complementary to the coding region of a pre-mRNA molecule or
an mRNA molecule, or the region corresponding to the coding region
on the antisense DNA strand. As used herein, the term "coding
sequence" refers to the region of DNA or RNA that encodes an RNA
molecule or a polypeptide having a cellular function. A useful
polynucleotide analogue also can be complementary to both coding
and non-coding regions of a selected nucleic acid. A polynucleotide
analogue that is complementary to both coding and non-coding
regions of a selected nucleic acid, for example, is one that is
complementary to a region that includes a portion of the 5'
untranslated region leading up to the start codon, the start codon,
and coding sequences immediately following the start codon of a
selected mRNA. A polynucleotide analogue that is complementary to
both coding and non-coding regions of a selected nucleic acid also
includes one that is complementary an intron/exon junction of a
selected pre-mRNA molecule (see Ekker & Larson (2001) Genesis
30:89-93).
[0050] 2. Reduction of Expression
[0051] A polynucleotide analogue can be used to reduce expression
from a selected nucleic acid of known sequence. As used herein,
"reduction" or "reduce" with respect to expression from a nucleic
acid refers to a decrease in expression, or to decrease expression,
in an amount that can be detected by assessing changes in RNA
level, protein level, and phenotype. For example, reduction can
refer to a 5%, 10%, 25%, 50%, 75%, or more than 75% decrease in
expression. A reduction in expression also includes complete
inhibition of expression, whereby greater than 95% reduction of
expression from a nucleic acid is achieved.
[0052] As used herein, the term "expression," with respect to
expression of a gene or expression from a nucleic acid, refers to
production of a functional RNA molecule from a DNA molecule as well
as production of a functional polypeptide from an mRNA molecule.
Expression from a selected nucleic acid can be examined using
standard methods known in the art. For example, RNA levels can be
determined by Northern hybridization and in situ hybridization
using the appropriate nucleic acid hybridization probes, while
polypeptide levels can be determine by antibody staining and
western hybridization. Development of organs, differentiated
tissues, and other cellular structures that are affected by
reduction in expression of selected nucleic acids can be assessed
using various methods. For example, vasculature can be visualized
with FITC-dextran injections; cartilage can be visualized using
Alcian Blue staining; and muscles can be visualized using
fluorescent-phalloidin staining. Alternatively, the expression of
tissue-specific genes can be used to assess development of organs,
differentiated tissues, and particular cellular structures. For
example, expression of a thymus specific marker such as Rag-1 can
be used to assess thymus development; and expression of
pancreas-specific markers such as Fspondin and islet-1 can be used
to assess pancreas development. (For standard methodologies, see H.
W. Detrich III, M. Westerfield, and L. I. Zon, Methods in Cell
Biology Vol. 59: The Zebrafish Biology, Academic Press. San
Diego)
[0053] Expression from a nucleic acid can be reduced by interfering
with (1) any process necessary for RNA transcription, (2) RNA
processing, (3) RNA transport across the nuclear membrane, (4) any
process necessary for RNA translation, or (5) RNA degradation.
[0054] Expression from a nucleic acid such as a DNA molecule can be
reduced by interfering with processes necessary for formation of a
functional RNA molecule or transport of the RNA into the cytoplasm.
Processes necessary for formation of a functional RNA molecule
include, for example, RNA polymerase binding to promoter regions,
binding of transcriptional activator to its recognition sequence,
and transcription. A polynucleotide analogue that anneals to DNA
and interferes with processes necessary for formation of a
functional RNA molecule generally, though not necessarily, has a
sequence that is complementary to the antisense DNA strand from
which mRNA is transcribed. Such polynucleotide analogues are
referred to as "antigene" molecules.
[0055] Expression from a nucleic acid such as an RNA molecule can
be reduced by interfering with any process necessary for formation
of a functional RNA molecule or proper translation of an mRNA
molecule into a functional polypeptide. Expression from an RNA
molecule, for example, can be reduced by interfering with RNA
processing, ribosome binding to the ribosome-binding site of mRNAs,
interfering with initiation of translation, interfering with the
translation process, or interfering with proper termination of
translation (see Ekker and Larson (2001) Genesis 30:89-93 and
Nasevicius & Ekker (2001) Curr Opin in Mol Therapeutics 3:224).
A polynucleotide analogue that anneals to a region of an mRNA
molecule and interferes with translation has a sequence that is
complementary to that region of the mRNA molecule and is referred
to as an antisense molecule. Antisense molecules, for example, can
bind and sterically inhibit scanning of the mRNA by the 40s
ribosomal subunit. Antisense molecules also can reduce expression
by inducing the cellular nuclease system that degrades cognate
mRNAs. In the RNaseH dependent mechanism, the double stranded
mRNA/antisense RNA that is formed is degraded by RNaseH.
[0056] Reduction of expression from a selected nucleic acid can be
achieved using one polynucleotide analogue. Reduction of expression
from a selected nucleic acid also can be achieved using two or more
polynucleotide analogues that are complementary to different
regions of the same selected nucleic acid. When two or more
polynucleotide analogues are used to reduce expression from a
selected nucleic acid, the polynucleotide analogues can be
complementary to non-overlapping regions or to overlapping regions
of the selected nucleic acid. When non-overlapping, polynucleotide
analogues can be complementary to regions of the selected nucleic
acid that are 0, 1, 2, 5, 10, 25, 50, 100, 500, 1000, or more than
1000 nucleotides apart.
[0057] When two or more polynucleotide analogues are used to reduce
expression from a selected nucleic acid, the two or more
polynucleotide analogues can have an additive or synergistic effect
on the phenotype of the organism. A phenotype that results from
introduction of polynucleotide analogues into a selected organism
is herein referred to as a phenotype mediated by the polynucleotide
analogues. The effect of two or more analogues can be described as
additive or synergistic based, for example, on the penetrance
frequency of a phenotype mediated by the analogues. The penetrance
frequency used to determine whether the effect of two or more
analogues is additive or synergistic is the frequency determined
for a 1.times. dose of each polynucleotide analogue used. As used
herein, penetrance frequency is the percent of organisms exhibiting
a particular phenotype when contacted with a particular analogue.
For example, if 100 organisms are contacted with analogue-1 and 80
of these exhibit phenotype-1, then the penetrance frequency is 80%.
A 1.times. dose of an analogue can be any amount needed to achieve
a penetrance frequency of less than 50%. To determine a 1.times.
dose for an analogue, a dose response curve is generated for each
analogue of interest. From the dose response curve, any amount of
an analogue that results in a penetrance frequency less than 50%
and with minimal toxic effects can be used as the 1.times. dose.
Two analogues are considered to have an additive effect if the
penetrance frequency, obtained when both analogues are used, each
at 1.times. dose, is equal to the sum of the penetrance frequencies
of individual analogues at 1.times. doses. For example, two
analogues are considered to have an additive effect if the
penetrance frequencies are (1) 10% for a 1.times. dose of
analogue-1, (2) 20% for a 1.times. dose of analogue-2, and (3) 30%
for a 1.times. dose of analogue-1 and a 1.times. dose of analogue-2
when used together. In contrast, two analogues are considered to
have a synergistic effect if the penetrance frequency, obtained
when both analogues are used, each at 1.times. dose, is greater
than the sum of the penetrance frequencies of individual analogues
at 1.times. doses. For example, two analogues are considered to
have a synergistic effect if the penetrance frequencies are (1) 10%
for a 1.times. dose of analogue-1, (2) 20% for a 1.times. dose of
analogue-2, and (3) 90% for a 1.times. dose of analogue-1 and a
1.times. dose of analogue-2 when used together.
[0058] The effect of two or more analogues also can be described
as, without limitation, additive or synergistic based on the
severity of the phenotype mediated by the analogues compared to
"the sum of the individual phenotypes" mediated by each of the
analogue used. A phenotype that is "the sum of individual
phenotypes" is one that results from an additive effect of each
analogue. Two or more analogues are considered to have a
synergistic effect if the phenotype mediated by the analogues is
more severe than "the sum of the individual phenotypes." For
example, two analogues are considered to have a synergistic effect
if (1) 100% of the organisms contacted with analogue-1 exhibit a
10% reduction in blood vessel formation, (2) 100% of the organisms
contacted with analogue-2 exhibit a 20% reduction in blood vessel
formation, and (3) 100% of organisms contacted with analogue-1 and
analogue-2 exhibit a 90% reduction in blood vessel formation. In
this example, the percent of reduction in blood vessel formation is
the severity of the phenotype mediated by the polynucleotide
analogues. In contrast, two analogues are considered to have an
additive effect on the phenotype of an organism if the phenotype
mediated by the analogues is not more severe than "the sum of the
individual phenotypes." For example, two analogues are considered
to have a synergistic effect if (1) 100% of the organisms contacted
with analogue-1 exhibit a 10% reduction in blood vessel formation,
(2) 100% of the organisms contacted with analogue-2 exhibit a 20%
reduction in blood vessel formation, and (3) 100% of organisms
contacted with analogue-1 and analogue-2 exhibit a 30% reduction in
blood vessel formation.
[0059] Phenotype severity can, in some instances, be measured by
the extent of reduction in expression from the nucleic acid
targeted by the analogues. The amount of reduction in expression
can be quantitated by standard methodologies and then compared to
determine whether two or more analogues have an additive or
synergistic effect on reduction of expression.
[0060] A useful measure of the synergy of two analogues is the
synergy factor. As used herein, the term "synergy factor" is
defined as the penetrance frequency, or the severity of the
phenotype, observed for two or more analogues divided by the
expected additive penetrance frequency or "sum of the individual
phenotypes," respectively. The synergy factor can be determined by
comparing the actual penetrance frequency obtained when both
analogues are used (each at 1.times. dose) with the penetrance
frequency expected of an additive effect. For example, the synergy
factor is 3 (90%/30%) if the penetrance frequencies are (1) 10% for
a 1.times. dose of analogue-1, (2) 20% for a 1.times. dose of
analogue-2, and (3) 90% for a 1.times. dose of analogue-1 and a
1.times. dose of analogue-2 when used together. The synergy factor
also can be determined by comparing the severity of the phenotype
observed with "the sum of the individual phenotypes" when two or
more analogues are used. For example, the synergy factor also is 3
(90%/30%) if (1) 10% reduction in blood vessel formation is
observed in 100% of the organisms contacted with analogue-1, (2)
20% reduction in blood vessel formation is observed in 100% of the
organisms contacted with analogue-2, and (3) 90% reduction in blood
vessel formation is observed in 100% of organisms contacted with
analogue-1 and analogue-2 together. A synergy factor can be
determined for two or more polynucleotide analogues. For example, a
synergy factor can be determined for two, three, or more than three
analogues. A synergy factor can be any value greater than 0. For
example, a synergy factor can be 0.2, 0.4, 0.8, 1.5, 2, 4, 6, 8,
10, 15, 20, or more than 20. A synergistic effect is indicated when
the synergy factor for a particular group of analogues has a value
greater than 1, for example, 1.2, 1.5, 1.8, 2.1, 5, 10, 20, or more
than 20. A synergy factor of 1 represents an additive effect.
Synergy factors between 0 and 1 indicate an interference
effect.
[0061] Polynucleotide analogues also can be used to reduce
expression from two or more different selected nucleic acids in an
organism. For example, multiple polynucleotide analogues, i.e. at
least two, having sequences complementary to multiple selected
nucleic acids can be used to reduce expression from the selected
nucleic acids. When two or more polynucleotide analogues are used
to reduce expression from two or more nucleic acids, reduction in
expression of the various nucleic acids can result in phenotypes of
two classes. The first class is representative of "the sum of the
individual phenotypes" mediated by each of the analogues used,
while the second class consists of those phenotypes that do not
fall into the first class. A phenotype that is "the sum of
individual phenotypes" is one that results from an additive effect
of each analogue. The additive effect of multiple polynucleotide
analogues is as described for multiple polynucleotide analogues
that target one selected nucleic acid. An organism that has been
contacted with two or more analogues also is described as having a
phenotype that is "the sum of individual phenotypes" if the
individual phenotype mediated by each analogue is distinct and
present in that organism. Alternatively, organisms that have been
contacted with two or more analogues can exhibit phenotypes that
are not considered "the sum of individual phenotypes." These
organisms exhibit phenotypes that are more or less extensive than,
or distinctly different from, a phenotype that is "the sum of
individual phenotypes." Typically, phenotypes that are not "the sum
of individual phenotypes" represent synergistic or interference
effects of multiple analogues.
[0062] To be considered additive, synergistic, interference, one
that represents "the sum of other phenotypes," or one that is
distinctly different from a phenotype that is the "sum of
individual phenotypes," a phenotype mediated by a polynucleotide
analogue must be sequence-specific, i.e., the phenotype must result
from sequence-specific reduction of expression from a selected
nucleic acid.
[0063] 3. Determination of Sequence-Specific Reduction of
Expression
[0064] A phenotype mediated by a polynucleotide analogue is said to
be "sequence-specific" if the phenotype is primarily or exclusively
associated with, or results from, reduction of expression from the
selected nucleic acid. To determine if a phenotype mediated by a
polynucleotide analogue is sequence-specific, a second
polynucleotide analogue of unrelated sequence that targets the same
nucleic acid can be used. Alternatively, a control polynucleotide
analogue that does not target the same nucleic acid or a rescue
mRNA that compensates for the reduction in expression from the
selected nucleic acid can be used.
[0065] To show that a phenotype mediated by a first polynucleotide
analogue is sequence-specific, a second polynucleotide analogue
that also targets the same selected nucleic acid is introduced into
a model organism. A phenotype mediated by the second polynucleotide
analogue that is the same as the phenotype mediated by the first
polynucleotide analogue indicates that the phenotype mediated by
either polynucleotide analogue is sequence-specific.
[0066] Alternatively, a control polynucleotide analogue also can be
used to confirm that a phenotype mediated by a first polynucleotide
analogue is sequence-specific. A control polynucleotide analogue is
somewhat similar in sequence to the first polynucleotide analogue.
The control polynucleotide analogue, however, has a number of bases
that are dissimilar to the first polynucleotide analogue such that
the control polynucleotide analogue is not sufficiently
complementary and so will not anneal to the selected nucleic acid
targeted by the first polynucleotide analogue. Therefore, no
phenotype mediated by a control polynucleotide analogue is
observed. A phenotype mediated by the first polynucleotide analogue
that is not observed when the control polynucleotide analogue is
used indicates that the phenotype mediated by the first
polynucleotide analogue is sequence-specific. If a phenotype
mediated by the control polynucleotide analogue is observed, then
the phenotype mediated by the first polynucleotide analogue cannot
be concluded to be sequence-specific.
[0067] In addition, a rescue mRNA encoding the polypeptide whose
expression is reduced by a polynucleotide analogue can be used to
show that a phenotype mediated by the polynucleotide analogue is
sequence-specific. The rescue mRNA is introduced into the organism
exhibiting the phenotype mediated by the polynucleotide analogue.
Restoration of a wild type phenotype in place of the phenotype
mediated by the polynucleotide analogue indicates that the
phenotype mediated by the polynucleotide analogue is
sequence-specific. A phenotype mediated by a morpholino-modified
polynucleotide analogue that has been confirmed to be
sequence-specific by a rescue mRNA or targeting with a second
morpholino of unrelated sequence is referred to as a morphant
phenotype. As used herein, the term "morphant" refers to an
organism exhibiting a sequence-specific phenotype mediated by a
morpholino-modified polynucleotide analogue.
[0068] 4. Applications of Polynucleotide Analogues
[0069] Polynucleotide analogues can be used to determine the
function of a coding sequence of, or a phenotype associated with, a
selected nucleic acid of known sequence. Nucleic acids can be
maternal or zygotic nucleic acids and can be involved in any
biological process, for example embryogenesis and development, gene
expression, regulation of gene expression, formation of particular
differentiated tissues, cell signaling, and metabolic processes
necessary for (1) embryogenesis and development, (2) gene
expression and its regulation, (3) formation of differentiated
tissues, and (4) cell signaling. Nucleic acids of interest also are
those associated with a disease condition. A nucleic acid that is
associated with a disease condition can be one in which reduction
in expression leads to a disease condition or alleviates a disease
condition. A disease condition can result from any change, for
example an increase or decrease, in the level of a biomarker such
as a polypeptide, a nucleic acid, a lipid, any intracellular or
exocellular molecule or compound, and the phophorylated and
unphosphorylated forms of a cellular molecule. A disease condition
can result from, for example, excessive expression of a nucleic
acid or expression of a mutated form of a nucleic acid thereby
forming a product with aberrant activity.
[0070] To determine the function of the coding sequence of, or
phenotype associated with, a selected nucleic acid of known
sequence, a polynucleotide analogue is synthesized having a
sequence complementary to the sequence of the selected nucleic
acid. A polynucleotide analogue designed to have a sequence that is
complementary to the sequence of a selected nucleic acid is said to
"target" the selected nucleic acid. When the polynucleotide
analogue is introduced into an organism, effects of the
polynucleotide analogue on expression from the selected nucleic
acid as well as associated phenotypes are examined.
[0071] Organisms useful for functional studies with polynucleotide
analogues are those in which a polynucleotide analogue is able to
distribute uniformly and inhibit expression from a selected nucleic
acid in cells that express the selected nucleic acid. Organisms can
be a fertilized or unfertilized egg, a cell in culture, an embryo,
or a juvenile or an adult animal. An animal, for example, can be a
fish, a frog, a mouse, a guinea pig, a sheep, a chimpanzee, or a
human. Vertebrate organisms such as teleost eggs and embryos that
undergo meroblastic cleavage can be used for functional studies
with polynucleotide analogues. Cleavage refers to a series of
mitotic divisions that occur in rapid succession as a fertilized
egg is transformed into a multicellular embryo. The multicellular
embryo consists of smaller nucleated cells referred to as
blastomeres. In meroblastic cleavage, only a part of the egg is
subdivided into blastomeres, in contrast to organisms that undergo
holoblastic cleavage in which the entire egg is subdivided into
blastomeres. The blastomeres generated from meroblastic cleavage
are continuous with the remaining uncleaved cytoplasm of the egg
(see Balinsky et al. (1981) Cleavage in An Introduction to
Embryology, 5.sup.th Edt. CBS College Publishing, pages 135-152).
Examples of vertebrate organisms that undergo meroblastic cleavage,
and therefore are useful model organisms for nucleic acid
functional studies using polynucleotide analogues include the eggs
and embryos of a zebrafish, a medaka, a pufferfish, and a
stickleback.
[0072] Polynucleotide analogues can be used to determine the
function of, or phenotype associated with, any nucleic acid of
known sequence but unknown function. The nucleic acid can be one
that is present, or one that is not present, in the model organism.
To determine the function of, or phenotype associated with, any
nucleic acid of known sequence but unknown function, the sequence
of a homologue, orthologue, or paralogue that is present in the
model organism is used. Homologues refer to nucleic acids encoding
polypeptides having similar domains or structures that can be
identified by nucleotide or amino acid sequence comparison.
Homologues also can have similar activities. Orthologues refer to
homologues that are from different species, while paralogues refer
to homologues within one organism that have distinct expression
patterns and therefore distinct biological roles. From the known
sequence of a homologue, orthologue, or paralogue, a polynucleotide
analogue targeting the homologue, orthologue, or paralogue is
generated. The polynucleotide analogue targeting the homologue,
orthologue, or paralogue is introduced into the model organism and
the organism is assessed for a phenotype associated with reduction
of expression of the homologue, paralogue, or orthologue. From the
phenotype observed, the function of the homologue, paralogue, or
orthologue can be determined, and in turn, the function of the
newly discovered nucleic acid or its involvement in a particular
aspect of the biology of the organism can be inferred.
[0073] Polynucleotide analogues also can be used to determine
whether two nucleic acids are, or encode, genetic interactors. As
used herein, the term "genetic interactors" refers to nucleic acids
or polypeptides that function in a common metabolic process. As
used herein, the term "metabolic process" refers to particular sets
of metabolic processes involved in (1) embryogenesis and
development, (2) gene expression and its regulation, (3) formation
of differentiated tissues, (4) cell signaling, and (5) any other
cellular and physiological processes. Genetic interactors, for
example, can be nucleic acids such as DNA or RNA, or the
polypeptides encoded by nucleic acids. Genetic interactors can
interact directly or indirectly.
[0074] To determine if two different nucleic acids are, or encode,
genetic interactors, polynucleotide analogues that target the two
nucleic acids can be used to reduce expression from the nucleic
acids. A sequence-specific phenotype mediated by both
polynucleotide analogues is compared to sequence-specific
phenotypes mediated by each of the polynucleotide analogues. If the
two different nucleic acids are genetic interactors, the phenotype
mediated by both analogues is expected to be different than the
"sum of the individual phenotypes" mediated by each of the
analogues. That is, if the targeted nucleic acids encode two
polypeptides that are genetic interactors, reduction in expression
from both nucleic acids in an organism will result in either a
synergistic effect on the phenotype mediated by the individual
nucleic acids or a phenotype that is distinctly different from the
"sum of the individual phenotypes." In contrast, the targeted
nucleic acids encode two polypeptides that are not genetic
interactors if reductions in expression from both nucleic acids
give rise to a phenotype that is "the sum of the individual
phenotypes."
[0075] Similarly, polynucleotide analogues also can be used to
generate model organisms for the study of diseases. Disease
conditions associated with loss or reduction of function of a
particular nucleic acid in a higher organism such as a human can be
generated in a model organism using a polynucleotide analogue if
the model organism has a homologous or orthologous nucleic acid.
For example, a polynucleotide analogue that targets the homologue
or orthologue in the model organism is generated and introduced
into the model organism. Reduction in expression of the homologue
or orthologue results in a morphant organism that exhibits the
disease condition. Morphants exhibiting a disease condition can be
used to screen for compounds that are useful for treating the
disease condition or alleviating the severity of the disease
condition. To screen for compounds that are useful for treating a
disease condition or alleviating the severity of the disease
condition, morphants exhibiting the disease condition can be
contacted with candidate compounds and then assessed to determine
whether the disease condition is lessened.
[0076] Polynucleotide analogues also can be used to identify
nucleic acids not known to be associated with a disease condition.
To identify nucleic acids not known to be associated with a disease
condition, various polynucleotide analogues are introduced into
wild type organisms and morphants exhibiting any particular disease
condition are chosen for further analysis. A collection of
polynucleotide analogues, for example, can be used to generate a
collection of morphants. Those morphants that exhibit a particular
disease condition can be used to identify drug targets as well as
to develop novel treatments for the disease condition. A potential
drug target, for example, is identified as the nucleic acid whose
reduction in expression led to the disease condition. A morphant
that exhibits a particular disease condition can be used to screen
for novel treatments for the particular disease condition as
described earlier.
[0077] To identify nucleic acids whose excessive activity leads to
a disease condition, polynucleotide analogues are introduced into
organisms exhibiting the disease condition. Those organisms whose
disease state is lessened by the polynucleotide analogue are
further examined to identify nucleic acids whose expressions have
been reduced. These nucleic acids are identified as useful targets
for the development of novel treatments for the particular
disease.
[0078] Polynucleotide analogues also can be used therapeutically as
treatments for disease conditions. For example, a polynucleotide
analogue can be used to treat disease conditions associated with
excessive expression of a particular nucleic acid or expression of
a culprit nucleic acid. Multiple polynucleotide analogues, for
example, that target the same culprit nucleic acid and have
synergistic effects in alleviating the disease phenotype can be
useful for circumventing toxicity associated with treatment using
one analogue, since lower amounts of analogues can be used.
[0079] 5. The Morpholino-Modified Polynucleotide Analogue/Zebrafish
System
[0080] An example of a system that is useful for determining
function or phenotype associated with a selected nucleic acid of
known sequence is the morpholino-modified polynucleotide
analogue/zebrafish system.
[0081] Morpholinos (morpholino-modified polynucleotide analogues)
are not subjected to any known endogenous enzymatic degradation
activity. Morpholinos have been shown to bind to and block
translation of mRNA both in vitro and in tissue culture (Summerton
(1990) Biochim Biophys Acta 1489:141-158; Summerton and Weller
(1997) Antisense Nucleic Acid Drug Dev 7:187-195). This approach
makes morpholino targeting highly predictable for polynucleotide
design and significantly reduces non-specific effects. In contrast,
traditional antisense polynucleotide approaches utilize
RNAse-H-based degradation of mRNA as a mechanism of action. RNAse-H
mediated strategies, however, have been tried with only modest
success (Barabino et al. (1997) Mech Dev 63:133-143). Furthermore,
in fish, single stranded polynucleotides tend to be toxic or there
is an inability to achieve uniform distribution among all cells of
the organism.
[0082] In the following sections, the examples describing the use
of morpholinos in zebrafish show these compounds to be (1) sequence
specific and (2) extremely potent in all cells for at least the
first 50 hours of development in F0 zebrafish embryos as targeted
gene `knockdown` agents. This period in the zebrafish embryonic
development includes the fundamental vertebrate processes of
segmentation and organogenesis. This tool offers the opportunity to
pursue sequence-specific gene targeting studies without the
necessity of laborious, time consuming, and expensive F3 vertebrate
genetic testing. Morpholinos, thus, offer a high throughput F0
vertebrate assay system for vertebrate functional genomics
applications.
[0083] The use of morpholino-based gene targeting represents a new
tool in the genetic repertoire of vertebrate biologists and,
combined with the excellent embryology of the zebrafish, is
extremely powerful in the elaboration of gene function for
similarly conserved developmental processes.
[0084] 6. Methods of Administration
[0085] Polynucleotide analogues can be introduced into a model
organism by methods used to introduce single stranded mRNA into the
model organism. (See Hyatt and Ekker (1999) Methods in Cell biology
59:117-126). Examples of delivery methods include (1)
microinjection and (2) simply exposing the model organism to the
polynucleotide analogue. Polynucleotide analogues can be delivered
in water or a suitable buffer. A suitable buffer is one in which
the polynucleotide analogue can be dissolved and that is non-toxic
to the model organism to which the polynucleotide analogue is to be
delivered. A non-toxic buffer can be one that is isotonic to, or
one that has a pH similar to the physiological pH of, an organism
to which the polynucleotide analogue is to be delivered. A buffer
that is isotonic with an organism is one that has the same
osmolarity as the organism. Danieau solution, for example, is
isotonic with the model organism zebrafish. A pH similar to the
physiological pH of the organism can be 2.5 pH units below or above
the pH of the organism. A polynucleotide solution prepared for
delivery into zebrafish, for example, can have a pH that is 5, 5.4,
5.7, 6, 6.2, 6.6, 7, 7.4, 7.8, 8, 8.6, or any value in between
these.
[0086] Different polynucleotide analogues can be introduced into
the model organism as a mixture. Alternatively, different
polynucleotide analogues can be introduced sequentially by multiple
exposures or injections.
[0087] 7. Compositions and Kits
[0088] The invention provides compositions of individual
polynucleotide analogues in a suitable buffer. The invention also
provides for compositions of at least two polynucleotide analogues
in a pharmaceutically acceptable carrier.
[0089] Compositions can be in the form of tablets, capsules,
powders, solutions, suspensions, or emulsions depending on the
route of administration. Compositions can contain sterile
pharmaceutically acceptable carriers or excipients. Common
pharmaceutically acceptable carriers or excipients can be aqueous
or non-aqueous. Aqueous carriers include, without limitation,
water, alcohol, saline, and buffered solutions. Examples of
non-aqueous carriers include, without limitation, propylene glycol,
polyethylene glycol, vegetable oils, and injectable organic esters.
Preservatives, flavorings, sugars, and other additives such as
antimicrobials, antioxidants, chelating agents, inert gases, and
the like also may be present.
[0090] For administration by injection, a solution can be prepared
using a suitable non-toxic buffer, i.e. one that is similar in pH,
is isotonic, or both similar in pH and isotonic, to a selected
organism.
[0091] For oral administration, tablets or capsules can be prepared
by conventional means with pharmaceutically acceptable excipients
such as binding agents (e.g., pregelatinized maize starch,
polyvinylpyrrolidone or hydroxypropyl methylcellulose); fillers
(e.g., lactose, microcrystalline cellulose or calcium hydrogen
phosphate); lubricants (e.g. magnesium stearate, talc or silica);
disintegrants (e.g., potato starch or sodium starch glycolate); or
wetting agents (e.g., sodium lauryl sulfate). Tablets can be coated
by methods known in the art. Preparations for oral administration
also can be formulated to give controlled release of a
polynucleotide analogue. Biocompatible, biodegradable lactide
polymer, lactide/glycolide copolymer, or
polyoxethylene-polyoxypropylene copolymers are examples of
excipients for controlling the release of a polynucleotide analogue
of the invention in vivo.
[0092] For nasal administration, preparations can be in the form of
a liquid solution, a gel, or a dry product. Inhalation formulations
may be aqueous solutions containing, for example,
polyoxyethylene-9-lauryl ether, glycocholate and deoxycholate, and
may contain excipients such as lactose, if desired. Nebulised
aqueous suspensions or solutions can include carriers or excipients
to adjust pH and/or tonicity. Nasal drops can be administered in
the form of oily solutions.
[0093] For parenteral administration, liquid solutions or
suspensions in aqueous physiological buffer solutions can be
prepared as desired using standard methods. Formulations may
contain common excipients as well as glycocholate for buccal
administration. Suitable parenteral delivery systems include
ethylene-vinyl acetate copolymer particles, osmotic pumps,
implantable infusion systems, and liposomes.
[0094] The invention also provides kits comprising a collection of
different polynucleotide analogues, for example,
morpholino-modified polynucleotides. The collection of
polynucleotide analogues can be analogues directed to nucleic acids
of the entire genome of an organism. The collection of
polynucleotide analogues also can be directed to nucleic acids
involved in a common biological process such as, for example, a
disease condition, regulation of nucleic acid expression, or a
common metabolic, developmental, or signaling pathway.
[0095] The invention also provides a collection of morphants that
can be generated using the collection of different polynucleotide
analogues. Morphants can be models of human diseases such as
prophyria or cyclopia. Morphants also can be defective in a
differentiated tissue, for example, vasculature, blood, an organ,
or a specialized cell type such as fibroblasts, neurons, and
epithelial cells.
[0096] The invention will be further described in the following
examples, which do not limit the scope of the invention described
in the claims.
EXAMPLES
Example 1--Zebrafish care and egg collection
[0097] Standard zebrafish care protocols are described in
Westerfield (1995) The Zebrafish Book: A Guide for the Laboratory
Use of Zebrafish (Brachydanio rerio) 3.sup.rd Edition, University
of Oregon Press.
[0098] Zebrafish were kept in 6.5 gallon (26 liters) and 20 gallon
(76 liters) plastic tanks at 28.degree. C. A 6.5 gallon tank housed
25 fish and a 20 gallon tank housed 70 fish. Tank water was
constantly changed with carbon-filtered and UV-sterilized tap water
(system water) at a rate of 15 to 40 mL/min. Alternatively, tank
water was replaced each day by siphoning up debris from the bottom
of the tank. Tap water, aged a day or more in an open (heated) tank
to release chlorine, was adequate. More consistent conditions,
however, were obtained by adding commercial sea salts to deionized
or distilled water (60 mg of `Instant Ocean` salt per liter of
water, see Westerfield (1995) The Zebrafish Book: A Guide for the
Laboratory Use of Zebrafish (Brachydanio rerio) 3.sup.rd Edition,
University of Oregon Press). A 10-hour dark and 14-hour light day
cycle was maintained in zebrafish facility.
[0099] Fish were fed brine shrimp twice a day. To make shrimp, 100
mL of brine shrimp eggs were added to 18 L of salt water (400 mL of
`Instant Ocean` salt per 18 L of water) and aerated vigorously.
After 2 days at 28.degree. C., the shrimp were filtered through a
fine net, washed with system water, suspended in system water, and
fed to fish. Alternatively, fish could also be fed with `Tetra`
brand dry flake food.
[0100] Zebrafish spawning was induced every morning shortly after
sunrise. To collect the eggs, a `false bottom container` system was
used (Westerfield (1995) The Zebrafish Book: A Guide for the
Laboratory Use of Zebrafish (Brachydanio rerio) 3.sup.rd Edition,
University of Oregon Press). The system consisted of two containers
of approximately 1.5 L, one slightly smaller than the other. The
bottom of the smaller container was replaced with a stainless steel
mesh with holes bigger than the diameter of zebrafish eggs. The
smaller container was then placed into the bigger container, and
the setup was filled with system water. Up to eight zebrafish were
placed inside the smaller container. When the fish spawn, the eggs
fall through the mesh into the bigger container, and in this way,
the eggs cannot be reached by the fish and eaten. About 10-15
minutes were allowed for spawning, after which time the smaller
container with the fish was transferred into another bigger
container. The eggs were collected by filtering using a mesh with
the holes smaller than the diameter of the eggs. Fish were used
once a week for optimal embryo production.
Example 2--Zebrafish and Xenopus strains
[0101] The zebrafish E-line transgenic line contained a single copy
of the pT-EF1.alpha.-GFP-pA transposon at the E-line locus
(Nasevicius and Ekker (2000) Nature Genetics 26:216-220).
Heterozygous E-line embryos were obtained from an outcross of
homozygous E-line adults and can be obtained S. C. Ekker,
University of Minnesota Medical School, Minneapolis, Minn.
Example 3--Polynucleotide Analogues
[0102] Morpholino phosphorodiamidates antisense oligonucleotides
(morpholinos or MOs) were purchased from Gene-Tools, LLC
(Corvallis, Oreg.). MO sequences were designed based on parameters
recommended by the company. MOs were 21 to 25-bases in length, had
no predicted internal hairpins, and consisted of approximately 50%
G/C and 50% A/T residues. Sequences having four consecutive G
nucleotides were avoided. All MOs were designed to bind to 5'
untranslated regions (UTRs), or regions flanking and including
sequence encoding the initiating methionine.
[0103] FITC-labeled 14-mer peptide nucleic acids (PNAs) were
obtained from Applied Biosystems. Rhodamine-labeled 25-mer PNAs
were a gift from Dr. D. Corey, University of Texas Southwestern.
2'-O methyl RNAs were obtained from Integrated DNA Technologies,
Inc. Phosphoramidates were obtained from Annovis, Inc.
[0104] Solutions of polynucleotide analogues were prepared and
injected as described in Example 4.
[0105] Sequences of the polynucleotide analogues used were as
follows. Residues complementary to the predicted start codon are
underlined in all cases.
1 FITC-labeled MO, FITC-labeled 3'-5' phosphoroamidate, and
FITC-labeled 2'-O methyl RNA: (SEQ ID NO: 1) 5'-ATC CAC AGC AGC CCC
TCC ATC ATC C-3' FITC-labeled PNA-1: (SEQ ID NO: 2) 5'-AGC AGC CCC
TCC AT-3' FITC-labeled PNA-2: (SEQ ID NO: 3)
5'-TCTCTCTC-O-nJTJTJTJT- -3' Negative control-MO sequence
[unlabeled or FITC-labeled at the 3' end]: (SEQ ID NO: 4)
5'-CCTCTTACCTCAGTTACAATTTATA-3' chordin-MO [FITC-labeled at the 3'
end] and chordin-PNA [rhodamine-labeled]; (SEQ ID NO: 5) 5'
ATCCACAGCAGCCCCTCCATCATCC-3' GFP-MO: (SEQ ID NO: 6)
5'-TCTTCTCCTTTACTCATTTTCTACC-3' GFPD4-MO: (SEQ ID NO: 7)
5'-TCTaCTCgTTTACTCATTaTCTtCC-3- ' oep-MO: (SEQ ID NO: 8)
5'-GCCAATAAACTCCAAAACAACTCGA-3' ntl-MO: (SEQ ID NO: 9)
5'-GACTTGAGGCAGGCATATTTCCGAT-3' shh-MO #1: (SEQ ID NO: 10)
5'-CAGCACTCTCGTCAAAAGCCGCATT-3' shh-MO #2: (SEQ ID NO: 11)
5'-TGTCTAGCAGGGTTTCTCGTTGTCG-3' twhh-MO: (SEQ ID NO: 12)
5'-TTCCATGACGTTTGAATTATCTCTT-3' nacre-MO and nacre-PNA
[rhodamine-labeled]: (SEQ ID NO: 13)
5'-CATGTTCAACTATGTGTTAGCTTCA-3' sparse-MO: (SEQ ID NO: 14)
5'-TATAAGTCCATCTATCTCATGTGTG-3' urod-MO: (SEQ ID NO: 15)
5'-GAATGAAACTGTCCTTATCCATCA-3' VEGF-A-1: (SEQ ID NO: 16)
5'-GTATCAAATAAACAACCAAGTTCAT-3' VEGF-A-1D4 [four base mismatch]:
(SEQ ID NO: 17) 5'-GTAaCAAtTAAACAACCAtGTTgAT-3' VDGF-A-3: (SEQ ID
NO: 18) 5'-TAAGAAAGCGAAGCTGCTGGGTATG-3' ztsg1-MO: (SEQ ID NO: 19)
5'-CTGATGATGATGATGAAGACCCCAT-3' zfz2MO-ATG: (SEQ ID NO: 20)
5'-CACACACACTTCCACTCGCCTGCAT-3- ' zfzMO-UTR: (SEQ ID NO: 21)
5'-CCTGCATTGTCTCGAAAAGTTCCGC-3' Fli-1-UTR MO: (SEQ ID NO: 22)
5'-CAATTTTCAGTGGAGCCCGACAATA-3' Fli-1-ATG MO: (SEQ ID NO: 23)
5'-TAATAGTTCCGTCCATTTTCCGCAA- -3' Fli-1.3 MO: (SEQ ID NO: 24)
5'-TCCGTCCATTTTCCGCAATTTTCAG-3'
Example 4--Injections of Polynucleotide Analogues
[0106] The analogues MOs, 2'O-methyl RNA, and 3'-5'
phosphoroamidate were solubilized in water at a concentration of 8
mM (approximately 65 mg/mil). The resulting stock solution was
diluted to working concentrations of 0.09 to 3.0 mg/ml in water or
1.times. Danieau solution (58 mM NaCl, 0.7 mM KCl, 0.4 mM
MgSO.sub.4, 0.6 mM Ca (NO.sub.3).sub.2, 5 mM HEPES pH 7.6).
Analogue solutions were injected into the yolk as described in
Ekker et al. (1995) Curr Biol 5:944-955. Analogue injections were
preformed using a method similar to that used for mRNA injections.
Briefly, zebrafish eggs were collected and transferred onto agarose
plates as described in Westerfield (1995) The Zebrafish Book: A
Guide for the Laboratory Use of Zebrafish (Brachydanio rerio)
3.sup.rd Edition, University of Oregon Press. While agarose plates
for mRNA injections were kept cold to slow embryo development, the
plates for analogue injections were prewarmed to approximately
20.degree. C., since analogue injection into cold embryos were
found to increase non-specific effects and mortality of the
injected embryos.
[0107] Needles used for analogue injections were the same as for
mRNA injections (Hyatt and Ekker (1999) Methods in Cell Biology
59:117-126). The needles were back-filled with a pipette and
calibrated by injecting the loaded morpholino solution into a glass
capillary tube. The picoinjector volume control was then setup for
1.5 to 15 nL. The injection volume depended on the required dose,
usually 1.5 to 18 ng of analogue was injected. Analogue solutions
were injected through the chorion into the yolk of zebrafish
embryos. The injected embryos were transferred to petri dishes
containing system water and allowed to develop at 28.degree. C.
[0108] Injections of buffered Danieau-MO solutions resulted in
lower mortality rates in injected embryos compared to injections of
water-MO solutions. No difference in the penetrance of the observed
phenotypes, however, was seen betweeen embryos injected with
buffered Danieau or water solution. The injection volume was 1.5 nL
to 15 nL for all analogues depending on the required injected dose.
For wild type or heterozygous E-line embryos of 1 to 16 cell
stages, analogues were injected into the yolk.
[0109] Embryos that received two different MOs were injected twice,
one injection for each MO. In experiments involving injection of
two different MOs, embryos injected with only a single MO were
analyzed and compared with embryos injected with two different
MOs.
[0110] Effective doses were determined separately for each
analogue. For example, at the effective dose of 4.5 ng and lower
for the GFP-MO, reduction of GFP protein was detected, and
.gtoreq.90% of the GFP-MO-injected embryos developed normally as
assayed using standard morphological criteria. Higher doses of the
GFP-MO resulted in a larger average reduction of GFP protein, but
also caused some detectable detrimental effects on development.
These higher doses were not pursued further for this MO. In all
cases shown, the dose used for analysis resulted in embryos of two
classes, those displaying a specific phenotype or those that were
normal when assessed using morphological criteria. A small fraction
of embryos (typically .ltoreq.5%) developed abnormally due to
mechanical damage following microinjection. Penetrance number for a
specific MO and concentration was assessed by examining morphology
of injected embryos. A minimum sample size of 25 was used.
[0111] PNAs (chordin and nacre 25-mer PNAs) were diluted in water
to a concentration of 0.25 mg/ml (resulting in a solution with
final pH=5). The resulting solutions were incubated at 55.degree.
C. for 5 minutes and placed on ice. The PNA solutions were injected
into the yolk of zebrafish embryo as described for morpholino
phosphorodiamidate (MO), 2'O-methyl RNA, and other injections. In
contrast to work with MOs, PNAs display a mosaic distribution if
injected later than the 4-cell stage. Furthermore, precipitation of
a significant fraction of the injected PNA at the site of injection
was observed in the injection of PNA at doses of 1.5 ng per embryo
or higher.
Example 5--mRNA Injections
[0112] Synthetic mRNA was injected, as described in Ekker et al.
(1995) Curr Biol 5:944-955, into the yolk of zebrafish embryos
previously injected with the indicated MO. Siblings from the same
pool of MO-injected embryos served as internal controls for these
experiments.
[0113] For Xenopus, mRNA microinjections were performed at the
4-cell stage using 0.3.times.MMR, 3.5% ficoll. Dorsal-ventral
polarity of early cleavage stage embryos was determined using
pigmentation differences (Cho et al. (1991) Cell 67:111-120).
[0114] Synthetic mRNAs were designed such that any overlap between
the synthetic mRNA and the MO would be insufficient for MO
targeting. The oep mRNA used did not contain any overlap with the
oep-MO. The twhh mRNA used contained only a six base overlap with
twhh-MO, a degree of overlap previously shown to be insufficient
for MO targeting in vitro and in tissue culture studies (Summerton
(1999) Biochim Biophys Acta 1489:141-158 and Summerton et al.
(1997) Antisense Nucleic Acid Drug Dev 7:187-195). Chordin mRNA
from Xenopus was used in order to avoid any sequence homology with
the zebrafish chordin-MO, and because previous studies showed
Xenopus and zebrafish chordin genes encoded equivalent specific
activities in Xenopus embryos (Miller-Bertoglio et al. (1998) Dev
Biol 214:72-86).
Example 6--FITC-Dextran Injections, Tissue Sectioning, and
Visualization
[0115] Microangiography was performed as described in Weinstein et
al. (1995) Nat Med 1:1143-7. Fluorescein isothiocyanate-dextran
(FITC-dextran) having a molecular weight of 2,000,000 Daltons
(SIGMA, catalog #FD-2000S) was used for these studies. The dextran
was solubilized in 1.times. Danieau solution at 2 mg/ml
concentration. Approximately 10 .mu.l of the prepared solution was
injected into sinus venosa/cardinal vein of anesthetized 48-hour
embryos.
[0116] Embryos injected with FITC-dextran were fixed overnight,
embedded into paraffin using standard procedures, and sectioned.
Histological haematoxylin-eosin staining of the sections was
subsequently carried out using standard protocols.
[0117] Both FITC-dextran injected embryos and unprocessed tissue
sections (i.e. unstained sections) were visualized using a ZEISS
Axioskop 2 microscope with a standard FITC filter set.
Example 7--Digital Photography
[0118] Bright field and in situ photography were performed on a
ZEISS Axioplan 2 microscope using Nikon CoolPix 990 (bright field)
or Kodak DCS 420 (in situ) digital cameras. For fluorescent
photography, a ZEISS AxioCam or a Nikon CoolPix 990 digital camera
was used.
Example 8--Fluorescence Analysis
[0119] Embryos injected with FITC-labeled antisense polynucleotide
analogues were analyzed using FITC filters on a Zeiss Axioplan2
fluorescence microscope. Images were obtained using a Kodak DCS420
Digital Camera.
[0120] Fluorescence pictures of groups of embryos were taken using
a MICROIMAGE I30B Low Light Integrating Camera and captured using a
DC30+ analogue to digital video capture board (Pinnacle Systems) at
maximum resolution (640.times.480) settings. Signal intensities
were set to sub-saturation levels for maximal information capture.
Sub-saturation level of signal intensity was determined using
uninjected E-line embryos. Fidelity of capture was confirmed by
simultaneously imaging both analogue and digital video shots. The
resulting images were imported into Adobe Photoshop 5.0 for
quantitative analysis. The background for each image, established
using the "Selective Color" algorithm, was set to the same setting
in all images. The "Mean" value of the "Histogram" algorithm was
used to measure the green channel signal; the other channels were
removed to minimize non-specific background. All scores were
normalized to the values obtained from uninjected E-line (100%) and
wild type (0%) data points.
Example 9--Western, Northern, and in Situ Hybridization
Analyses
[0121] For detection of GFP or NTL protein, standard western
analysis was performed using GFP antibody (Clontech) or NTL
antibody (gift of S. Schulte-Merker). Proteins isolated from pools
of injected embryos were used. The amount of protein analyzed per
sample was equivalent to the amount of protein obtained from five
embryos.
[0122] For antibody staining, rabbit anti-phospho Mad antibody, a
gift from P. ten Dijke, was used at {fraction (1/2000)} dilution.
Staining was visualized using an alkaline phosphatase-coupled
secondary antibody (Promega laboratories).
[0123] For detection of GFP mRNA, Northern blot hybridization was
performed according to standard procedures. A 700 base pair
fragment from a Pst I digest that corresponded to the GFP coding
region was used as probe. Each sample analyzed consisted of 5 .mu.g
total RNA isolated from a pool of 30 embryos. Two independent
analyses were performed.
[0124] For whole-mount in situ hybridization, methods described by
Mason et al. (1994) Genes and Development 8:1489-1501 and Jowett
(1999) Methods in Cell Biol 59:63-85 were used. Hybridization was
performed at 65.degree. C. T7 polymerase was used for riboprobe
synthesis. Riboprobes for fli-1 and flk-1 were synthesized using
plasmids zffli-1 and zfflk-1 (Thompson et al. (1998) Dev Biol
197:248-69) digested with EcoR I and Sma I, respectively.
Example 10--Uniform Distribution of Injected Modified
Polynucleotide Analogues in Early Zebrafish Embryos
[0125] The delivery efficiency of modified polynucleotide analogues
was determined using FITC-labeled analogues. FITC-labeled modified
polynucleotide analogues were injected into embryos at the 1-2-cell
stage. Modified polynucleotide analogues that were injected
included MOs, peptide nucleic acids (PNA), 2'-O methyl RNA, and
3'-5' phosphoroamidate. Distribution of FITC-labeled polynucleotide
analogues was examined by fluorescence microscopy as described in
Examples 7 and 8. Injected embryos were compared with uninjected
control embyros. Results demonstrate that polynucleotide analogues
were completely translocated to blastomeres as early as the 8-cell
stage (less than 1 hour after injection). Polynucleotide analogues
remained uniformly distributed among blastomeres as evident from
samples at the mid-blastula stage.
[0126] In a second distribution study, 90 pg of FITC-labeled
control-MO or FITC-labeled chordin-MO were injected into wild type
zebrafish embryos from 1 to 16 cell stages of development. Uniform
distribution of FITC-labeled-MOs was achieved at sphere and 28 hour
stages. Injection of a chordin-MO dose of 90 pg resulted in embryos
that developed normally.
[0127] In a third distribution study, FITC-labeled PNA-2 (Gene
Therapy Systems) was microinjected into yolks of 1-16 cell
zebrafish embryos. Injection volumes were 6 to 15 nl; approximately
50-200 ng was injected. Buffer from Gene Therapy Systems or
0.5.times. Danieau buffer was used. Toxicity was not observed with
either buffer. From 85-90% of the injected embryos survived at 48
hours of development (usual survival rate for non-toxic
injections). Older embryos were not analyzed. The fluorescence
signal was detected in all tissues at comparative levels. The
distribution was uniform as determined by fluorescence microscopy.
Isolated points of high signal concentration, however, were
observed (<10 per embryo, at approximately 1 cell diameter).
Example 11--Specific Inhibition of GFP Transgene Expression in all
Cells of a Zebrafish Embryo by GFP-MO
[0128] A non-essential ubiquitous GFP transgene was used to test
the applicability of antisense MOs as a general gene knockdown
strategy in zebrafish. A GFP-targeted MO (GFP-MO) was injected into
zebrafish E-line embryos. Fluorescence and western blot assays were
used to examine GFP transgene expression. GFP fluorescence of (1)
uninjected E-line embryos, (2) E-line embryos injected with 4.5 ng
of control-MO, (3) E-line embryos injected with 4.5 ng of GFP-MO,
and (4) wild type embryos was compared by FITC illumination. GFP
fluorescence in uninjected E-line embryos was ubiquitous.
Similarly, GFP fluorescence in E-line embryos injected with 4.5 ng
of a control-MO or a four base mismatch GFP-MO (GFPD4-MO) was near
wild type. In contrast, GFP transgene expression was inhibited in
all cells of the zebrafish E-line embryo injected with 4.5 ng of
GFP-MO. The specific loss of GFP signal in embryos injected with
GFP-MO was noted in nine separate experiments; at least 30 embryos
were assayed in each experiment. The lack of visible GFP
fluorescence indicated that a nearly complete loss of GFP protein
expression was achieved. Furthermore, GFP-MO inhibition of GFP
expression was dose-dependent. FIG. 1 is a GFP fluorescence
inhibition graph demonstrating sequence-specific and dose-dependent
inhibition of GFP expression. GFP activity in embryos injected with
the control-MO and in embryos injected with the GFP-MO was
compared. Fifteen embryos were assayed for each data point shown.
Fluorescence activity data were confirmed by western blotting.
These data demonstrate that specific inhibition of gene expression
in all cells of the 28-hour zebrafish embryo was achieved using
MOs.
Example 12--Inhibition of Chordin Expression by Chordin-MO
[0129] A chordin antisense MO (chordin-MO) was injected into
zebrafish embryos and a highly specific series of phenotypes
dependent upon dose was observed in 28-hour embryos and 3-day old
embryos. Embryos injected with various amounts of chordin-MO were
compared with wild type embryos. The numbers of embryos injected
with particular amounts of chordin-MO were as follows: 169 embryos
were injected with 0.09 ng MO; 97 embryos were injected with 0.9 ng
MO; 399 embryos were injected with 1.5 ng MO; 423 embryos were
injected with 4.5 ng MO; and 224 embryos were injected with 9 ng
MO. FIG. 2 is a graph demonstrating that two chordin-MO phenotypes,
weak and strong, were achieved with increasing doses of chordin-MO
injected. At low doses, a weak chordin-MO phenotype, the equivalent
of a reduced chordin loss of function phenotype, was noted. Embryos
displaying the weak chordin-MO phenotype had partially expanded
blood islands, u-shaped somites, and abnormal tail fins with
multiple folds. At higher doses, chordin-MO injected embryos
exhibited a phenocopy of chordin null mutant embryos (Fisher et al.
(1997) Development 124:1301-1311; Hammerschmidt et al. (1996)
Development 123:95-102), i.e. the strong chordin-MO phenotype. This
strong chordin-MO phenotype was observed at high frequency in high
dose injections (.gtoreq.75% at 4.5 ng MO injection, n=423), and
consisted of abnormal u-shaped somites, extremely expanded blood
islands, abnormal tail fins, and reduced heads. About 80% of the
epiboly embryos subjected to in situ hybridization for otx-2 showed
reduction of otx-2 consistent with head reduction. About 65% of
epiboly embryos subjected to in situ hybridization for gata-2
showed that the gata-2 expression domain was greatly enlarged and
shifted anteriorly, indicating ventralization of the mesoderm.
(Gata-2 was expanded in 100% of the injected embryos; n=30).
Comparison of injected embryos with uninjected embryos demonstrated
posterior fusion of gata-2 expression stripes. These results
demonstrate that the equivalent of an allelic series for the loss
of the chordin gene was achieved by the use of different doses of
the chordin-MO.
[0130] Whole-mount in situ hybridization for chordin mRNA was
performed. Comparison of injected and uninjected embryos
demonstrated that chordin mRNA levels were similar. Therefore,
inhibition of chordin function by chordin-MO was not mediated by
the conventional RNAseH mediated antisense-targeted degradation of
chordin mRNA. To verify the specificity of gene targeting by
chordin-MO, embryos were injected with synthetic Xenopus chordin
mRNA to determine if effects of chordin-MO can be reversed. When
423 embryos were injected with 4.5 ng of chordin-MO, 76% showed the
strong chordin phenotype. In contrast, when 30 embryos were
injected with 4.5 ng of chordin-MO and 300 pg of Xenopus chordin
mRNA, 10% exhibited the strong chordin phenotype. FIG. 3 is a graph
demonstrating that the chordin-MO phenotype was partially rescued
by Xenopus chordin mRNA injection, thereby illustrating specificity
of chordin-MO targeting.
Example 13--Inhibition of Chordin Expression by Chordin-PNA
[0131] Twenty-five-base chordin-PNA (chd-PNA) and nacre-PNA were
injected into zebrafish embryos as described in Example 4. Injected
embryos were compared to wild type embryos. The resulting
phenotypes were observed in 28-hour old embryos.
[0132] The numbers of embryos injected with particular amounts of
chordin PNA were as follows: 41 embryos were injected with 0.25 ng
chd-PNA; 130 embryos were injected with 0.5 ng chd-PNA; 98 embryos
were injected with 1 ng chd-PNA; 77 embryos were injected with 1.5
ng chd-PNA. The numbers of embryos injected with particular amounts
of nacre-PNA were as follows: 38 embryos were injected with 0.25 ng
nacre-PNA, 71 embryos were injected with 0.5 ng nacre-PNA; 78
embryos were injected with 1 ng nacre-PNA; 75 embryos were injected
with 1.5 ng nacre-PNA.
[0133] A weak ventralization, equivalent to the weak phenotype
observed in chd-MO injection, was observed in 13% of the embryos
injected with 0.25 ng of chd-PNA. Non-specific effects were noted
in 6% of the injected embryos, while mortality rate was 22%. In the
embryos injected with 0.5 ng chd-PNA, 31% mortality rate was
observed. About 38% of the injected embryos exhibited a weak
ventralization phenotype, while 2% exhibited a strong
ventralization phenotype similar to a null chordin mutant
phenotype. Non-specific effects were observed in 43% of the
injected embryos. Injection with 1 ng and 1.5 ng of chd-PNA
resulted in 74% and 76% mortality rates, respectively. The
injection phenotypes were not analyzed.
[0134] Epiboly stage embryos were subjected to in situ
hybridization to confirm morphological results. These analyses
indicated that 28% of the chd-PNA injected embryos had expanded
gata-2 expression, a hallmark of mesoderm ventralization. In situ
hybridization for otx-2 showed that 50% of zebrafish embryos
injected with 0.5 ng chd-PNA and 70% of zebrafish embryos injected
with 1 ng and 1.5 ng chd-PNA had reduced otx-2 expression.
Reduction of otx-2 expression is indicative of embryo
ventralization by the chd-PNA.
[0135] Nacre-PNA injections resulted in low to moderate mortality
rates. Injections of 0.25 ng, 0.5 ng, 1 ng, and 1.5 ng of nacre-PNA
resulted in mortality rates of 12%, 24%, 36%, and 35%,
respectively. Non-specific abnormality rates ranged from 3% for
injections of 0.25 ng and 0.5 ng to 12% for injection of 1.5 ng of
nacre-PNA. No ventralization phenotype was observed in embryos
injected with nacre-PNA. Furthermore, analysis of 2 day-old
zebrafish embryos showed that injection with nacre-PNA had no
effect on the nacre gene activity. In situ hybridization analysis
of injected embryos showed that nacre-PNA injection did not alter
gata-2 or otx-2 expression. These results indicate that the
ventralization phenotype is specific to chordin-PNA.
Example 14--Inhibition of Maternal Gene Expression by oep-MO
[0136] The one-eyed pinhead gene (oep) (Zhang et al. (1998) Cell
92:241-251) was selected to test for maternal gene activity
inhibition by MO. Embryonic oep function is due to both maternal
and zygotic genetic contributions that are distinguishable based on
specific criteria (Gritsman et al. (1999) Cell 97:121-132). Embryos
deficient in oep function are defective in signaling through the
nodal pathway. Embryos were injected with 9 ng of oep-MO, and
phenotypes consistent with loss of zygotic oep function were seen.
Cyclopia and ventral curvature typical of zygotic oep mutants were
observed in 30.+-.5% of embryos (n=291). In 13.+-.5% of embryos
(n=291), severe cyclopia, somite absence in the trunk, misshapen
tail somites, and reduced notochord typical of a maternal-zygotic
oep mutant were observed. Tailbud stage embryos were subjected to
in situ hybridization for pax-2 and axial. Prechordal mesoderm
reduction was seen in 45% of injected embryos when compared to wild
type embryos; 24 embryos were analyzed.
[0137] The oep-MO phenotype was rescued by injection with synthetic
zebrafish oep mRNA. Forty-three percent of embryos (n=291)
displayed oep phenotypes when injected with 9 ng oep-MO, while none
of the 33 embryos analyzed displayed oep phenotypes when injected
with 9 ng oep-MO and 50 pg oep mRNA. FIG. 4 is a graph
demonstrating dose-dependent reduction in the frequency of the oep
phenotype in response to oep mRNA injections. This result
demonstrates that the observed oep phenotype was due to the
specific inhibition of oep gene function.
[0138] Injections with higher concentrations of oep-MO resulted in
a phenocopy of the loss of both zygotic and maternal oep functions.
MOs are thus capable of targeting maternal gene function, albeit at
reduced levels compared to zygotic gene targeting.
Example 15--Use of Morpholinos to Identify Genetic Interactors
[0139] An MO targeted to the no tail (nt1) gene (Schulte-Merker et
al. (1994) Development 120:1009-1015) was used to identify genetic
interactors of nt1. Embryos (n=118) were injected with 9 ng of
nt1-MO. Ninety-eight percent of the injected embryos were
indistinguishable from those caused by a null mutation (Halpern et
al. (1997) Dev Biol 187:154-170) when assessed using molecular and
phenotypic criteria. Normal head, abnormal somites, and extremely
reduced tail were prominent. When nt1-MO was injected into nt1
mutant embryos (n=72), no additional defect was noted in the nt1
mutant embryos due to the injection of nt1-MO. NTL protein was
specifically and quantitatively reduced in wild type embryos
injected with nt1-MO.
[0140] Activation of the somitic mesodermal marker myod requires
input from both oep and nt1 pathways (Schier et al. (1997)
Development 124:327-342). When embryos were co-injected with 9 ng
of nt1-MO and 9 ng of oep-MO, highly reduced head, reduced tail,
and extremely reduced somites and notochord were observed. When
10-12 somite embryos were subjected to in situ hybridization for
myod, embryos reduced in either nt1 or oep function displayed an
altered but robust expression of myod, while embryos reduced of
both functions expressed myod in only a few cells. For example, 92%
of nt1-MO injected embryo (n=25) exhibited adaxial mesoderm
reduction and posterior somite fusion, and 41% of oep-MO injected
embryos (n=22) exhibited posterior fusion of the adaxial mesoderm.
In nt1-MO and oep-MO co-injected embryos (n=24), 52% exhibited no
adaxial mesoderm and extremely reduced somitic mesoderm.
[0141] Whole-mount in situ hybridization of shh in 10-12 somite
stage embryos demonstrated that 80% nt1-MO injected embryos (n=25)
exhibited weak expansion of shh expression, while 50% of eop-MO
injected embryos (n=22) exhibited strong expansion of the shh
expression. Fifty-two percent of embryos co-injected with nt1-and
oep-MO (n=31) exhibited severe reduction of shh expression. FIG. 5
is a graph comparing the frequencies of nt1, oep, and nt1 and oep
phenotypes observed in embryos injected with nt1-MO, oep-MO, or
both MOs. MOs are thus effective tools for the testing of genetic
interactions in vivo.
Example 16--Inhibition of Gene Expression Throughout Somitogenesis
and Organogenesis
[0142] Later-acting genes were used to determine the perdurance of
morpholino effects. One hundred and twelve embryos were injected
with 9 ng of nacre-MO. Ninety-eight percent of injected embryos
exhibited a characteristic and nearly complete loss of body
pigmentation through the first 50 hours of development, a phenotype
indistinguishable from that observed in the nacre mutant (Lister et
al. (1999) Development 126:3757-3767). At later time points,
pigmentation returned at a variable rate. One hundred and
fifty-nine embryos were injected with 9 ng of sparse-MO. Injection
of sparse-MO duplicated a known zebrafish pigment mutation sparse
(Parichy et al. (1999) Development 126:3425-3436). When 65-hour and
10-day old zebrafish embryos were examined, 95% exhibited reduced
numbers of pigmented cells (melanocytes). Dorsal melanocytes were
significantly reduced in these embryos. Therefore, MO-based gene
targeting was completely penetrant throughout the first two days,
and potentially the first 10 days, of development. Furthermore,
MO-based gene targeting was completely penetrant throughout the
critical vertebrate processes of somatogenesis and organogenesis in
the zebrafish embryo.
Example 17--Development of Morpholino-Based Model Systems for Human
Diseases
[0143] Hepatoerythropoietic porphyria (HEP) is caused by a defect
in haem biosynthesis through loss of the uroporphyrinogen
decarboxylase (urod) enzyme (Kappas et al. (1995) The Metabolic
Basis of Inherited Diseases pages 2103-2159). The manifestation of
this syndrome includes fluorescent and photosensitive red blood
cells. One hundred and eithteen embryos were injected with urod-MO,
and then examined using a rhodamine filter set. Control-MO injected
embryos were compared with urod-MO injected embryos. All of the
injected embryos displayed both fluorescent and photosensitive red
blood cells as had been noted in a hypomorphic urod mutation (Wang
et al. (1998) Nature Genet 20:239-243). Intense auto-fluorescence
in the injected embryos indicated accumulation of photosensitive
porphyrins in the circulating blood cells. Photosensitivity of
blood cells in urod-MO injected embryos was evident when embryos
were exposed to light. Exposure to light resulted in depletion of
all red blood cells in urod-MO injected embryos. The complete
phenotypic penetrance of embryos injected with urod-MO demonstrates
that a MO-based animal model of human disease was generated
[0144] Holoprosencephaly (HPE) occurs at high frequency in human
embryos (1:250) and in live births (1:16,000) (Wallis et al. (1999)
Mol Genet Metab 68:126-138). In extreme cases, the phenotype is
cyclopia. The gene sonic hedgehog (shh) is thought to play a
critical role in the development of this disease in humans (Belloni
et al. (1996) Nature Genet 14:353-356; Roessler et al. (1996)
Nature Genet 14:357-360). Zebrafish shh mutations, however, result
in no anterior midline signaling defects (Schauerte et al. (1998)
Development 125:2983-2993). A second shh orthologue expressed in
the anterior midline, tiggy-winkle hedgehog (twhh) (Ekker et al.
(1995) Curr Biol 5:944-955), could explain this lack of phenotype
in a highly conserved vertebrate developmental process. Both shh
and twhh genes were targeted using MOs to test for redundancy and
to develop zebrafish as a genetic model for HPE.
[0145] Embryos were (1) injected with 18 ng twhh-MO and 9 ng of
control-MO; (2) injected with 18 ng shh-MO and 9 ng of control-MO;
(3) co-injected with shh-MO and twhh-MO, 13.5 ng each; or (4)
injected with shh-MO and twhh-MO (13.5 ng each), and 100 pg of twhh
mRNA. Embryos at 3-day old or 10 somite stage were analyzed. FIG. 6
is a graph comparing the frequencies of cyclopia, u-somites, and
reduced fins in embryos injected with a control-MO and twhh-MO, a
control-MO and shh-MO, or both twhh- and shh-MOs.
[0146] Embyros injected with shh-MO exhibited phenotypes
characteristic of a shh mutation (Schauerte et al. (1998)
Development 125:2983-2993). These embryos displayed `u`-shaped
somites, lacked the horizontal myoseptum, and had reduced pectoral
fins. Embryos injected with twhh-MO exhibited phenotypes
indistinguishable from controls. These embyros had normal heads,
`v` shaped somites, and normal myoseptum when compared to wildtype
embryos. Injection of both twhh-MO and shh-MO, however, resulted in
embryos with synergistic defects in somitic patterning in the
trunk, a new phenotype in the forebrain, partial cyclopia, absence
of myoseptum, and loss of transcription of the hedgehog target gene
patched (ptc) (Concordet et al. (1996) Development
122:2835-2846).
[0147] When 9 ng twhh-MO was injected into shh deficient embryos
(n=52), 100% penetrance of partial cyclopia was achieved. In
contrast, twhh-MO failed to cause any cyclopia in sibling embryos
(0%, n=120) (S. Bingham and A. Chandrasekhar, in press). This
suggests that zebrafish embryos contain two functionally redundant
orthologues of the mammalian shh gene with similar roles in
anterior midline patterning.
[0148] Embryos at the 10 somite stage were examined by whole-mount
in situ hybridization for myod. Results demonstrated that shh-MO
injected embryos exhibited a weak reduction of adaxial and somatic
mesoderm when compared to wildtype embryos. Embryos co-injected
with shh-MO and twhh-MO exhibited an extreme reduction of adaxial
mesoderm. This reduction was reversed and adaxial mesoderm was
lightly expanded by injection of zebrafish twhh mRNA.
[0149] These results demonstrate that zebrafish is an effective
model for HPE. In addition, these experiments demonstrate the
utility of morpholinos for both single and multiple gene knockdowns
for the understanding of vertebrate embryonic development and
disease.
Example 18--Sonic Hedgehog MO Synergy
[0150] Embryos were injected, as described in Example 4, with two
non-overlapping shh-MOs: shh-MO #1 and shh-MO #2. Sequences of
shh-MO #1 and shh-MO # 2 are provided in Example 3. Embryos were
analyzed at 3 days of development for phenotypic changes resulting
from loss of sonic hedgehog function (Schauerte et al. (1998)
Development 125: 2983-2993). Embryos that displayed effects in
somites as strong as the weakest allele tq252 were scored as
demonstrating a positive phenotype. FIG. 7 compares the frequencies
of embryos that displayed a phenotype associated with sonic
hedgehog loss-of-function when injected with one or two shh-MOs.
Results shown were obtained from three independent experiments.
Twenty or more embryos were scored for each data point. As shown in
FIG. 7A, the number of embryos having a sonic hedgehog
loss-of-function phenotype resulting from morpholino injection was
greater when embryos were injected with two shh-MOs than when
embryos were injected with a single shh-MO. A comparison of the
numbers of embryos having the sonic hedgehog loss-of-function
phenotype resulting from injection of one or two shh-MOs
demonstrates that the increase was more than additive, i.e.,
injection with two shh-MOs had a synergistic effect on the number
of embryos exhibiting sonic hedgehog loss-of-function phenotype.
Furthermore, injection of a half dose of two shh-MOs is much more
potent at knocking down shh gene expression than a full dose of
either MO alone (see FIG. 7B).
Example 19--Efficiency of Gene Inactivation by Morpholinos
[0151] Morpholinos were generated against known genes to determine
an estimate of success rate. Genes targeted included shh, chordin,
no tail, one-eyed-pinhead (oep), sparse, nacre, urod,
bozozok/dharma, an EF1a-GFP transgene, pax 2.1, bmp1, bmp2b, bmp7,
alk8, smad5, wnt5, and wnt11 (see Ekker (2000) Yeast 17:302-306).
With the exception of pax 2.1, all genes targeted resulted in clear
specific gene inactivation with the first MO tried, i.e., 16/17 or
>94% of known genes were inactivated successfully. This rate of
successful gene inactivation can be reduced by MO mistargeting or
other non-specific effects. For example, an MO can inhibit a second
gene resulting in embryos with a combined phenotype. An extreme
example is represented by the bozozok/dharma MO, in which a second
effect (CNS degeneration) is superimposed on the bozozok loss of
function phenotype (Nasevicius et al. (2000) Nature Genetics
26:216-220). If these secondary, non-specific effects result in
loss of embryonic structures or premature death of the embryo, then
the function of the gene under study will not be scorable. The
reduction in the rate of successful MO gene inactivation (16/17) by
the rate of gene mistargeting (2/17) is a lower estimate of MO
screening efficiency, and yields an initial MO screening rate of
82% (14/17). A significant fraction of these `missed` genes can be
recovered by the use of a second MO of unrelated sequence. Assuming
a similar success rate of approximately 80% for the remaining 18%
of `missed` genes, an additional 14% of genes can be targeted by
the second MO, for a combined expected success rate of >95% for
genes screened using two targeted MOs.
[0152] Should the leader sequence in a specific locus be especially
prone to polymorphism, the selected gene might not be inactivated
in all embryos due to the high specificity of MO targeting in vivo.
The one-eyed-pinhead locus is a potential example of this
phenomenon (see Example 14); in one wild-type strain, only
.about.50% of embryos responded to this MO, in another, none.
Other, less direct strain differences also could reduce the
effectiveness of MOs. For example, variations in genetic
backgrounds could alter the penetrance of a given MO effect due to
genetic factors in a second, modulator locus. The characterization
and inclusion of common `wild-type` and other non-isogenic
laboratory strains in the sequencing project is suggested to make
maximum use of MO technology in zebrafish.
[0153] As with many genetic screens, MOs also are limited by
functional redundancy, an issue especially relevant to vertebrates.
Moreover, the zebrafish genome contains an additional set of
incomplete duplicates for an estimated 30% of genes found in
mammals (Postlethwait et al. (1999) Methods Cell Biol 60:149-163;
and Oates et al. (1999) Dev Dyn 215:352-370). This partial genome
duplication occasionally results in two orthologues in zebrafish
for one in humans. One example is the sonic hedgehog locus (Example
17). Comparative expression profiles of likely orthologues,
however, demonstrate only a duplication of a subset of expression
patterns for these genes. Indeed, no two orthologues have identical
expression patterns in zebrafish (Gates et al. (1999) Genome Res
9:334-347). The use of MOs, however, is highly amenable to rapid
tests of redundancy through the simultaneous targeting of genes of
related sequence (see Example 17). Multi-gene targeting strategies
are thus practical using current morphino technology, with an
estimated minimum success rate of (0.82.times.0.82)=67%. For genes
amenable to this strategy, morpholinos will be extremely effective
at identifying and testing molecules with redundant functions in
vivo.
Example 20--Morphological Effects of VEGF-A-1 Morpholino Injection
at 36 Hours
[0154] Signaling by members of the Vascular Endothelial Growth
Factor (VEGF) gene family is implicated in the formation of
vasculature during embryogenesis, during wound healing, and for the
growth of tumor-induced vasculature (See Carmeliet et al. (1996)
Nature 380:435-9; Carmeliet et al. (1997) Am J Physiol
273:H2091-104; and Ferrara (1999) J Mol Med 77:527-43). Pioneering
work in mice with VEGF-A demonstrates the extreme dose
responsiveness of the mouse embryo to VEGF-A signaling during
development. Loss of a single copy of the VEGF-A gene induces
haploinsufficient lethality by day 9.5 pc (Ferrara et al. (1996)
Nature 380:439-42); Carmeliet et al. (1996) Nature 380:435-9). This
biological hurdle to the genetic investigation of VEGF-A
requirements during later development has resulted in a series of
experiments using conditional knockout strategies (Gerber et al.
(1999) Development 126:1149-59; Haigh et al. (2000) Development
127:1445-53) or dominant negative proteins (Gerber et al. (1999)
Development 126:1149-59). A more recent approach to address this
problem used intravenous injection of antisense oligonucleotides in
pregnant mice to reveal loss of function requirements of VEGF-A
function during murine embryogenesis (Driver et al. (1999) Nat
Biotechnol 17:1184-7).
[0155] Zebrafish embryos develop externally and have only limited
requirements for a functioning circulatory system during early
development. For example, embryos with no circulating red blood
cells due to porphyria live through the first three days of
development (Ransom et al. (1996) Development 123:311-9), a period
that includes all of segmentation and organogenesis in the fish
embryo. Multiple mutations in cardiovascular development were
isolated in the initial large-scale chemical mutagenesis screens
(Stainier et al. (1996) Development 123:285-92; Chen et al. (1996)
Development 123:293-302; Weinstein et al. (1995) Nat Med 1:1143-7).
Together, the zebrafish has the potential to rapidly assess the
biological role of angiogenic factors required for this essential
vertebrate process.
[0156] Zebrafish VEGF-A is expressed during embryogenesis in the
anterior nervous system, in mesoderm flanking the prospective heart
fields, and in somitic mesoderm that flanks the developing endoderm
(Liang et al. (1998) Biochim Biophys 1397:14-20). MOs were
generated against VEGF-A to analyze the requirement of this gene
during embryonic development. MOs had been shown to be effective at
gene inactivation during the first two days of zebrafish
development (see Example 16).
[0157] Embryos were injected with 9 ng of VEGF-A-1-MO. The
resulting VEGF-A morphant embryos developed with no overt abnormal
phenotype during the first day of development. At two days of
embryogenesis, the VEGF-A morphant phenotype consisted of an
enlarged pericardium, no circulating red blood cells, a slight
reduction in neural tube and overall body size, and little or no
functioning vasculature. In a subset of embryos, red blood cell
accumulation was observed in the ventral tail. Table 1 summarizes
the frequencies of embryos exhibiting loss of vasculature,
pericardial edema, blood accumulation in the anterior aorta, and
blood accumulation in the tail when injected with the indicated
amounts of MO.
[0158] Four experiments were performed at each MO dose. Average
frequencies of all experiments performed at each MO dose are shown.
The standard error is the mean of the differences between the
average frequency and the frequencies of individual
experiments.
2TABLE 1 Microangiography analysis of embryos injected with
VEGF-A-1-MO at 48 hours. Injected VEGF-A-1 morpholino dose, ng
Observed phenotypes (frequency, %) 3 6 9 12 18 Heart and yolk
vasculature only 3 .+-. 2 3 .+-. 1 21 .+-. 4 38 .+-. 13 39 .+-. 1
No axial or intersegmental vasculature 7 .+-. 4 2 .+-. 1 25 .+-. 3
27 .+-. 13 29 .+-. 9 No/reduced intersegmental vasculature 67 .+-.
5 78 .+-. 13 51 .+-. 2 36 .+-. 20 30 .+-. 6 Normal vasculature 23
.+-. 10 17 .+-. 16 3 .+-. 1 0 .+-. 0 2 .+-. 3 Pericardial edema 15
.+-. 2 35 .+-. 10 49 .+-. 2 55 .+-. 0 57 .+-. 2 Blood accumulation
in anterior hypochord 0 .+-. 0 1 .+-. 1 5 .+-. 3 3 .+-. 0 8 .+-. 2
Blood accumulation in tail 1 .+-. 1 2 .+-. 2 16 .+-. 8 19 .+-. 0 19
.+-. 7 Total embryo number 110 120 105 106 101
Example 21--Microangiography Visualization of Vasculature Defects
in VEGF-A-1 Morphants
[0159] Two separate fluorescent assays were used to assess vascular
function. In the first assay, fluorescently-labeled RBCs were
generated through inactivation of the uroporphyrinogen
decarboxylase gene (urod; Wang et al. (1998) Nature Genet
20:239-243) using 9 ng of urod-MO. In 36-hour embryos injected with
9 ng of urod MO, fluorescing RBCs highlighted the axial
vasculature, head vasculature, yolk sac, and heart. In embryos
injected with 9 ng of VEGF-A-1-MO, RBCs were localized only to
anterior aorta.
[0160] In the second assay, the vasculature was directly analyzed
by injection with FITC-dextran. Injection of FITC-dextran into the
sinus venosa/cardinal vein of an anesthetized 48-hour old embryo
results in labeling of the entire vasculature of the zebrafish
embryo, including the yolk sac, heart, head, axial, and
intersegmental blood vessels (Weinstein et al. (1995) Nat Med
1:1143-7).
[0161] Injection with VEGF-A-1-MO showed that these structures were
differentially sensitive to VEGF-A signaling. Further, three
phenotypic classes were observed when various amounts of
VEGF-A-1-MO were injected. In the most severe phenotypic class,
i.e., at high dose injections of VEGF-A-1-MO, the only vasculature
detectable was in the heart and yolk. Head, axial, and
intersegmental blood vessels were not visible. The vasculature
either failed to form at all or contained no functioning
connections to the heart in these embryos. To distinguish between
these possibilities histological analyses were performed on the
most severely affected embryos. Neither dorsal aorta nor axial vein
could be seen in the injected embryos.
[0162] A frequent but less severe phenotypic class of embryos also
was observed. These embryos were said to exhibit a moderate
VEGF-A-1-MO injection effect. In these embryos, the injected
FITC-dextran highlighted yolk sac, heart, and head blood vessels
only. No axial or intersegmental vasculature was observed.
[0163] The least severe phenotypic classification included embryos
exhibiting reduced intersegmental vasculature, but normal heart,
yolk, head, and axial blood vessels. Embryos exhibiting no or few
intersegmental blood vessels, but normal yolk sac, heart, head
vasculature, and axial blood vessels were said to exhibit a weak
VEGF-A-1-MO effect.
[0164] The penetrance of these phenotypic classes was dependent on
dose (see Table 1). This is consistent with the strong
dose-dependence of VEGF-A function in mouse embryos (Ferrara et al.
(1996) Nature 380:439-42; Carmeliet et al. (1996) Nature
380:435-9). Heterozygous mouse VEGF-A mutants showed a reduced
dorsal aorta detected by histological analysis. Fewer
intersegmental blood vessels were detected by a tissue-specific
lacZ expression. Lack of dorsal aorta was indicated by histological
analysis in homozygous mouse VEGF-A mutants (Carmeliet et al.
(1996) Nature 380:435-9), suggesting that the most severe zebrafish
VEGF-A morphant class represents a nearly complete loss of function
phenotype. Although mouse VEGF-A mutants also displayed heart with
under-developed myoblast (Ferrara et al. (1996) Nature 380:439-42;
Haigh et al. (2000) Development 127:1445-53), the heart in
zebrafish VEGF-A morphants had essentially a normal appearance with
a slightly enlarged atrium and ventricle, possibly due to higher
cardiac pressure.
Example 22--Confirmation of VEGF-A MO Specificity
[0165] Two VEGF-A-MOs, one having a sequence that did not overlap
with VEGF-A-1-MO and one containing a four base mismatch, were used
to confirm specificity of targeting (see Table 2). Four
VEGF-A-D4-MO injection experiments and two VEGF-A-3-MO injection
experiments were performed at each MO dose. Table 2 summarizes the
average frequencies observed in all experiments performed at each
MO dose. The standard error is the mean of the differences between
the average frequency and the frequencies of individual
experiments.
[0166] Injection of embryos with VEGF-A-D4, the mismatched-MO,
demonstrated the sequence-specific nature of the noted effects of
the VEGF-A-1-MO (see Table 2). To independently test for
specificity of targeting to the endogenous VEGF-A gene, a second
VEGF-A-MO of completely independent sequence (VEGF-A-3) was used.
This very potent MO caused the same phenotypic effects on
development as the VEGF-A-1-MO, including a dose-dependent
reduction of vascular function, pericardial edema, and blood
accumulation in the tail (see Table 2). The differential efficacy
might be due to different secondary structures of the MOs or the
targeted mRNA regions. Alternatively, the effect might be caused by
the higher predicted melting temperature of VEGF-A-3 (48% G/C)
compared to VEGF-A-1 (28% G/C). Therefore, phenotypes associated
with injection of VEGF-A-1-MO resulted from MO-based specific
inhibition of translation of VEGF-A transcripts.
[0167] A number of genes whose mutation results in cardiovascular
defects have been observed in chemical mutagenesis screens with
zebrafish (Stainier et al. (1996) Development 123:285-92; Chen et
al. (1996) Development 123:293-302). Although none of the
phenotypes associated with these mutations strongly resemble the
VEGF-A morphant phenotype, the mutant embryos had overlapping
phenotypes. Mutations in multiple loci result in embryos with
cardiac edema, and a similar accumulation of blood in the ventral
tail fin was noted due to disorganized endothelia in the scotch
tape (sco) mutation (Chen et al. (1996) Development 123:293-302).
Several mutations giving rise to altered circulation were noted
(Stainier et al. (1996) Development 123:285-92); Chen et al. (1996)
Development 123:293-302). These mutations included gridlock, a gene
that encodes a bHLH protein required only for arterial development
(Weinstein et al. (1995) Nat Med 1:1143-7; Stainier et al. (1996)
Development 123:285-92); Zhong et al. (2000) Science 287:1820-4).
The role of these genes in VEGF signaling awaits molecular genetic
characterization of the remaining loci.
3TABLE 2 Microangiography analysis of embryos injected with
VEGF-A-D4 and VEGF-A-3-MO at 48 hours Injected dose, ng VEGF-A-D4
morpholino 3 6 9 12 18 Heart and yolk vasculature only 1 .+-. 1 0
.+-. 0 2 .+-. 3 3 .+-. 0 3 .+-. 0 No axial or intersegmental
vasculature 0 .+-. 0 0 .+-. 0 0 .+-. 0 0 .+-. 0 0 .+-. 0 No/reduced
intersegmental vasculature 7 .+-. 0 6 .+-. 3 5 .+-. 2 22 .+-. 1 14
.+-. 3 Normal vasculature 92 .+-. 1 94 .+-. 3 93 .+-. 3 75 .+-. 2
84 .+-. 3 Total embryo number 163 116 119 76 73 VEGF-A-3 morpholino
0.5 1.5 3 Heart and yolk vasculature only 0 .+-. 0 13 .+-. 13 27
.+-. 11 No axial or intersegmental vasculature 0 .+-. 0 24 .+-. 24
40 .+-. 13 No/reduced intersegmental vasculature 18.8 37 .+-. 11 24
.+-. 14 Normal vasculature 81.2 26 .+-. 26 10 .+-. 10 Total embryo
number 32 71 69
Example 23--Comparative Expression of fli-1 and flk-1 in 26
Hour-Wild Type and VEGF-A-1 Morpholino Injected Zebrafish
Embryos
[0168] Expressions of two endodermal vascular markers, fli-1 and
flk-1, in VEGF-A morphant embryos were analyzed. The transcription
factor fli-1 is a very early marker of vascular cell fate
specification (Thompson et al. (1998) Dev Biol 197:248-69; Brown et
al. (2000) Mech Dev 92:237-52). In wild-type 26-hour embryos, fli-1
was expressed in the forming dorsal aorta and axial vein and in the
intersegmental vasculature in overlying somites. In embryos that
failed to complete vascular development, only a subset of the
vascular expression patterns of fli-1 was altered. No detectable
intersegmental expression of fli-1 was observed. This coincided
with the exquisite intersegmental vascular endoderm sensitivity to
VEGF signaling (Table 1). The cells either were not properly
specified or failed to migrate during formation of the
intersegmental vessels. The distinct responsiveness of the
expression of the endothelial marker fli-1 in intersegmental
vessels to VEGF-A signaling demonstrates a dual role for VEGF
during vascular development. First, VEGF-A is required for proper
axial vessel formation but not for initial axial vessel patterning.
Second, VEGF-A is required for intersegmental vessel cell
specification or migration, and presumably, for subsequent vascular
formation.
[0169] Similar results were obtained upon analysis of expression of
the tyrosine kinase VEGF receptor flk-1. Distribution of flk-1
transcripts was very similar to that of fli-1 in the trunk and tail
of wild-type embryos. In VEGF-A morphant embryos generated by
injection with 9 ng VEGF-A-1, intersegmental but not axial
expression was absent. A significant reduction in flk-1 gene
expression was noted in mouse embryos with no VEGF-A activity
(Carmeliet et al. (1996) Nature 380:435-9). A less extreme lack of
flk-1 expressing cells in the intersegmental vasculature also was
observed in mouse with the partial and conditional VEGF-A knockout
(Haigh et al. (2000) Development 127:1445-53).
[0170] Zebrafish embryos injected with 18 ng of VEGF-A-1-MO
displayed the same specific loss of expression only in the
intersegmental regions for both fli-1 and flk-1. The lack of a
requirement for VEGF signaling for flk-1 expression is consistent
with previous observations of paracrine modes of VEGF signaling
(reviewed in Ferrara, 1999). The expression of the VEGF receptor
flk-1 is, however, VEGF-dependent during intersegmental
vascularization. This latter observation suggests a possible
autoregulatory loop, functioning during vasculogenesis of the
intersegmental vessels. The strong conservation of VEGF function
from fish to mammals implicates this as a fundamental vertebrate
biological pathway.
[0171] These experiments demonstrate a fundamental distinction
between VEGF-A requirements for axial and intersegmental vascular
structure specification. More specifically, VEGF-A is not required
for the initial establishment of axial vasculature patterning,
whereas all development of intersegmental vasculature is dependent
on VEGF-A signaling. The absence of axial and intersegmental
vasculature in VEGF-A morphant embryos indicates that VEGF-A has a
role beyond establishing flk-1 and fli-1 expression in blood vessel
formation.
Example 24--VEGF-MO Synergy
[0172] Embryos were injected, as described in Example 4, with two
VEGF-MOs: VEGF-A-1-MO and VEGF-A-3-MO. Sequences for the
VEGF-A-1-MO and VEGF-A-3-MO are provided in Example 3. Embryos at 2
days of development were analyzed using microangiography, as
described in Example 21. Embryos that displayed defects in
intersegmental vasculature only were scored as displaying a weak
VEGF-MO phenotype, whereas embryos that displayed defects in both
intersegmental and axial vasculature were scored as a strong
VEGF-MO phenotype. Results, depicted in FIG. 8, demonstrate that
the number of embryos having a weak or strong VEGF-MO phenotype
resulting from morpholino injection was greater when embryos were
injected with two VEGF-MOs than when embryos were injected with a
single VEGF-MO. A comparison of the numbers of embryos having the
weak or strong VEGF-MO phenotype resulting from injection of one or
two VEGF-MOs demonstrates that the increase was more than additive,
i.e., injection with two MOs had a synergistic effect on the
numbers of embryos exhibiting weak or strong VEGF-MO
phenotypes.
Example 25--Zebrafish Frizzled-2 Targeting Analyses
[0173] Embryos were injected, as described in Example 4, with two
zfz2-MOs: zfz2-MO-ATG and zfz2-MO-UTR. Sequences for zfz2-MO-ATG
and zfz2-MO-UTR are provided in Example 3. FIG. 9 is a bar graph
showing the percentages of embryos affected by injection with
either one of the two zfz2-MOs or both zfz2-MOs. The
zfz2-associated developmental defects are undulating notochords and
wider than normal posterior-concentrated somites during
embryosgenesis. Embryos injected with either a single zfz2-MO or
two zfz2-MOs were examined for these morphologically-visible
defects. Both zfz2-MOs were capable of eliciting similar undulated
notochord and somite defects when injected individually into an
embryo. When both of the zfz2-MOs were injected into the same
embryo, defects in notochord and somite developments were
synergistic.
[0174] In addition, development of the zebrafish pancreas was
examined using pancreas-specific markers such as Fspondin and
islet-1. Fspondin and islet-1 are expressed by a subset of cells in
the zebrafish pancreas, and the absence of expression of Fspondin
or islet-1 indicates that pancreas development is defective. When
Fspondin and islet-1 expressions were examined, reduced expression
or a complete lack of expression was found in morpholino-injected
embryos.
Example 26--Vertebrate Tsg Functions to Augment Chordin Inhibitory
Activity
[0175] To examine the function of tsg in vertebrates, the function
of the ztsg1 gene in zebrafish was analyzed using a ztsg1-MO. The
ztsg1 gene is the appropriate counterpart to the early embryonic
Drosophila tsg since ztsg1 is expressed ubiquitously in early
zebrafish embryos, while ztsg2 is only expressed at later
stages.
[0176] MOs were used to reduce the function of the endogenous
ztsg1. To visualize the vasculature, 9 ng of UroD-MO was injected
into embyros. In situ hybridizations were performed for the
following markers at the indicated developmental stages: MyoD (8
somite stage), Krox20 (8 somite), GATA2 (22 somite) and BMP-4 (3
somite stage). Injection of 12 ng of a ztsg1 morpholino (ztsg-MO)
resulted in 50% of the injected embryos giving rise to zebrafish
with phenotypes characteristic of expanded BMP signaling
(Hammerschmidt et al. (1996) Develop 123:95-102; Miller-Bertoglio
et al. (1999) Dev Biol 214:72-86). Embryos developed expansion of
the ventral fin region that corresponded to ectopic blood islands,
a tissue derived from ventral mesoderm. Injected embryos also
showed an expansion of GATA2 at the 22 somite stage of development
(48%, n=21), loss of paraxial mesoderm (visualized with the marker
MyoD) at the 8 somite stage (38%, n=33), and a mild reduction of
anterior ectodermal tissues (detected by staining for Krox-20) at
the 8 somite stage (49%, n=39). Caudal expression of BMP4 also was
expanded in these embryos at the 3 somite stage (48%, n=25), while
the anterior ectodermal marker otx-2 was reduced. Treated embryos
also exhibited an expansion in apoptotic cells ventral to the yolk
extension similar to dino and mercedes mutants (Hammerschmidt et
al. (1996) Develop 123:95-102; Fisher et al. (1997) Develop
124:1301-11). Overall, this phenotype is very similar to that of
ogon/mercedes mutants (Hammerschmidt et al. (1996) Develop
123:95-102; Miller-Bertoglio et al. (1999) Dev Biol 214:72-86) and
moderate chordin loss-of-function mutants, and represents a modest
ventralized phenotype (Nasevicius et al. (2000) Nat Gen 26:216-220;
Hammerschmidt et al. (1996) Develop 123:95-102).
[0177] Injection of ztsg1 mRNA resulted in phenotypes diagnostic of
reduced BMP signaling (Mullins et al. (1996) Develop 123:81-93;
Kishimoto et al. (1997) Develop 124:4457-66; Dick et al. (2000)
Develop 127:343-54). Injection of 75 pg of ztsg1 mRNA resulted in
56% of the surviving animals (164 injected, 108 alive) exhibiting
reduced axial length with loss of ventral fin. This is a phenocopy
of the C3-C4 class of dorsalized mutant embryos observed with the
snailhouse (BMP-7 homologue) and piggytail mutations (Mullins et
al. (1996) Develop 123:81-93; Dick et al. (2000) Develop
127:343-54). Molecular analysis revealed a similar expansion of
MyoD (50%, n=8) and Krox20 (70%, n=10) at the 8 somite stage.
Furthermore, the dorsalizing effect of ztsg1 mRNA partially
reversed the ventralizing effect of the ztsg1-MO (9 ng tsg1-MO
caused 47.+-.2% ventralized embryos (n=376); 9 ng ztsg1-MO plus 30
pg ztsg1 mRNA resulted in 19.+-.8% ventralized embryos (n=270))
indicating that loss of ztsg1 was responsible for the phenotype.
Therefore, loss of ztsg1 led to embryos with a ventralized
phenotype, while ectopic expression of ztsg1 led to a dorsalized
embryonic phenotype.
Example 27--Enhancement of the ztsg1 Loss-of-Function Phenotype by
Sub-Inhibitory Loss of the Chordin Gene
[0178] Drosophila data suggest that one role of Tsg is to
potentiate the inhibition of BMP signaling in conjunction with
Sog/Chordin. To determine if the same is true for vertebrates,
sub-inhibitory levels of chordin-MO and ztsg1-MO were injected into
wild type embryos and the effect on ectopic blood island
development was scored. Embryos were injected with either a low
dose of ztsg1-MO, chordin-MO, or both ztsg1-MO and chordin-MO, and
the resulting embryos were examined for blood island expansion.
Results, depicted in FIG. 10, demonstrate that ztsg1-MO and
chordin-MO synergistically enhanced blood island expansion.
Co-injection of sub-inhibitory levels of MOs directed against both
Tsg and Chordin synergistically enhances the penetrance of the
ventralized phenotype. Therefore, both gene products cooperatively
inhibited BMP signaling. The finding that Tsg and Chordin cooperate
to inhibit BMP-2 signalling was confirmed by results of biochemical
experiments performed in Drosophila as well as in Xenopus (See Ross
et al. (2001) Nature 410:479-83.)
Other Embodiments
[0179] It is to be understood that while the invention has been
described in conjunction with the detailed description thereof, the
foregoing description is intended to illustrate and not limit the
scope of the invention, which is defined by the scope of the
appended claims. Other aspects, advantages, and modifications are
within the scope of the following claims.
Sequence CWU 1
1
25 1 25 DNA Artificial Sequence Synthetically generated
oligonucleotide 1 atccacagca gcccctccat catcc 25 2 14 DNA
Artificial Sequence Synthetically generated oligonucleotide 2
agcagcccct ccat 14 3 17 DNA Artificial Sequence Synthetically
generated oligonucleotide 3 tctctctcnn tntntnt 17 4 25 DNA
Artificial Sequence Synthetically generated oligonucleotide 4
cctcttacct cagttacaat ttata 25 5 25 DNA Artificial Sequence
Synthetically generated oligonucleotide 5 atccacagca gcccctccat
catcc 25 6 25 DNA Artificial Sequence Synthetically generated
oligonucleotide 6 tcttctcctt tactcatttt ctacc 25 7 25 DNA
Artificial Sequence Synthetically generated oligonucleotide 7
tctactcgtt tactcattat cttcc 25 8 25 DNA Artificial Sequence
Synthetically generated oligonucleotide 8 gccaataaac tccaaaacaa
ctcga 25 9 25 DNA Artificial Sequence Synthetically generated
oligonucleotide 9 gacttgaggc aggcatattt ccgat 25 10 25 DNA
Artificial Sequence Synthetically generated oligonucleotide 10
cagcactctc gtcaaaagcc gcatt 25 11 25 DNA Artificial Sequence
Synthetically generated oligonucleotide 11 tgtctagcag ggtttctcgt
tgtcg 25 12 25 DNA Artificial Sequence Synthetically generated
oligonucleotide 12 ttccatgacg tttgaattat ctctt 25 13 25 DNA
Artificial Sequence Synthetically generated oligonucleotide 13
catgttcaac tatgtgttag cttca 25 14 25 DNA Artificial Sequence
Synthetically generated oligonucleotide 14 tataagtcca tctatctcat
gtgtg 25 15 24 DNA Artificial Sequence Synthetically generated
oligonucleotide 15 gaatgaaact gtccttatcc atca 24 16 25 DNA
Artificial Sequence Synthetically generated oligonucleotide 16
gtatcaaata aacaaccaag ttcat 25 17 25 DNA Artificial Sequence
Synthetically generated oligonucleotide 17 gtaacaatta aacaaccatg
ttgat 25 18 25 DNA Artificial Sequence Synthetically generated
oligonucleotide 18 taagaaagcg aagctgctgg gtatg 25 19 25 DNA
Artificial Sequence Synthetically generated oligonucleotide 19
ctgatgatga tgatgaagac cccat 25 20 25 DNA Artificial Sequence
Synthetically generated oligonucleotide 20 cacacacact tccactcgcc
tgcat 25 21 25 DNA Artificial Sequence Synthetically generated
oligonucleotide 21 cctgcattgt ctcgaaaagt tccgc 25 22 25 DNA
Artificial Sequence Synthetically generated oligonucleotide 22
caattttcag tggagcccga caata 25 23 25 DNA Artificial Sequence
Synthetically generated oligonucleotide 23 taatagttcc gtccattttc
cgcaa 25 24 25 DNA Artificial Sequence Synthetically generated
oligonucleotide 24 tccgtccatt ttccgcaatt ttcag 25 25 25 RNA
Artificial Sequence Synthetically generated oligonucleotide 25
auccacagca gccccuccau caucc 25
* * * * *