U.S. patent number 10,385,339 [Application Number 15/834,383] was granted by the patent office on 2019-08-20 for methods and compositions for enhancing the efficacy and specificity of single and double blunt-ended sirna.
This patent grant is currently assigned to UNIVERSITY OF MASSACHUSETTS. The grantee listed for this patent is UNIVERSITY OF MASSACHUSETTS. Invention is credited to Dianne Schwarz, Phillip D. Zamore.
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United States Patent |
10,385,339 |
Zamore , et al. |
August 20, 2019 |
Methods and compositions for enhancing the efficacy and specificity
of single and double blunt-ended siRNA
Abstract
The present invention provides methods of enhancing the efficacy
and specificity of RNAi using single or double blunt-ended siRNA.
The invention also provides single and double-blunt ended siRNA
compositions, vectors, and transgenes containing the same for
mediating silencing of a target gene. Therapeutic methods are also
featured.
Inventors: |
Zamore; Phillip D. (Northboro,
MA), Schwarz; Dianne (Watertown, MA) |
Applicant: |
Name |
City |
State |
Country |
Type |
UNIVERSITY OF MASSACHUSETTS |
Boston |
MA |
US |
|
|
Assignee: |
UNIVERSITY OF MASSACHUSETTS
(Boston, MA)
|
Family
ID: |
36596445 |
Appl.
No.: |
15/834,383 |
Filed: |
December 7, 2017 |
Prior Publication Data
|
|
|
|
Document
Identifier |
Publication Date |
|
US 20180187194 A1 |
Jul 5, 2018 |
|
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
|
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14271038 |
Jan 30, 2018 |
9879253 |
|
|
|
13270920 |
Oct 11, 2011 |
|
|
|
|
11022055 |
Dec 22, 2004 |
|
|
|
|
60532116 |
Dec 22, 2003 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N
15/111 (20130101); C12N 15/113 (20130101); C12Y
115/01001 (20130101); C12N 2320/50 (20130101); C12N
2310/14 (20130101) |
Current International
Class: |
C12N
15/11 (20060101); C12N 15/113 (20100101) |
References Cited
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CA |
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2432350 |
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CA |
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10160151 |
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Feb 2004 |
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EP |
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05/001043 |
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06/015389 |
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Feb 2006 |
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WO |
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08/136902 |
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Nov 2008 |
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WO |
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|
Primary Examiner: Shin; Dana H
Attorney, Agent or Firm: Lathrop Gage LLP Velema, Esq.;
James H.
Parent Case Text
RELATED APPLICATIONS
This application is a continuation of U.S. patent application Ser.
No. 14/271,038, filed May 6, 2014, which is a continuation of U.S.
patent application Ser. No. 13/270,920, filed Oct. 11, 2011, which
is a continuation of U.S. patent application Ser. No. 11/022,055,
filed Dec. 22, 2004, which claims priority to U.S. Provisional
Patent Application Ser. No. 60/532,116, filed Dec. 22, 2003, the
entire disclosures of which are hereby incorporated herein by
reference.
Claims
What is claimed:
1. A method of silencing a target mRNA comprising providing a
double stranded RNA (dsRNA) comprising a sense strand and an
antisense strand, wherein the base pair strength between the
antisense strand 5' end (AS 5') and the sense strand 3' end (S 3')
is less than the base pair strength between the antisense strand 3'
end (AS 3') and the sense strand 5' end (S 5'), wherein said dsRNA
comprises a frayed AS 5', such that the antisense strand
preferentially guides cleavage of a target mRNA, and wherein said S
3' is lacking an overhang and wherein said AS 3' comprises an
overhang.
2. The method of claim 1, wherein the base pair strength is less
due to at least one mismatched base pair between the AS 5' and the
S 3' than between the AS 3' and the S 5'.
3. The method of claim 1, wherein the mismatched base pair is
selected from the group consisting of G:A, C:A, C:U, G:G, A:A, C:C,
U:U, I:A, I:U, and I:C.
4. The method of claim 1, wherein the base pair strength is less
due to at least one wobble base pair between the AS 5' and the S 3'
than between the AS 3' and the S 5'.
5. The method of claim 4, wherein the wobble base pair is G:U.
6. The method of claim 1, wherein each strand of the dsRNA has a
length of between about 19 nucleotides and about 22
nucleotides.
7. The method of claim 1, wherein said dsRNA is provided as an
shRNA which is processed by a cell to yield the dsRNA.
8. The method of claim 7, wherein a viral construct encodes the
shRNA.
9. The method of claim 1, wherein the AS 3' comprises a two
nucleotide overhang.
10. The method of claim 1, wherein the AS 3' comprises a dTdT
overhang.
Description
RELATED INFORMATION
The contents of any patents, patent applications, and references
cited throughout this specification are hereby incorporated by
reference in their entireties.
BACKGROUND OF THE INVENTION
Small interfering RNAs (siRNAs) are produced by the cleavage of
double-stranded RNA (dsRNA) precursors by Dicer, a member of the
RNase III family of dsRNA-specific endonucleases. Typically, siRNAs
result when transposons, viruses, or endogenous genes express long
dsRNA or when dsRNA is introduced experimentally into plant or
animal cells to trigger gene silencing, a process known as RNA
interference (RNAi).
siRNAs were first identified as the specificity determinants of the
RNA interference (RNAi) pathway, where they act as guides to direct
endonucleolytic cleavage of their target RNAs. Prototypical siRNA
duplexes are 21 nucleotide, double-stranded RNAs that contain 19
base pairs, with two-nucleotide, 3' overhanging ends. Active siRNAs
contain 5' phosphates and 3' hydroxyls.
siRNAs are typically found in the RNA-induced silencing complex
(RISC) that mediates both cleavage and translational control. siRNA
duplexes can assemble into RISC in the absence of target mRNA, both
in vivo and in vitro. Each RISC contains only one of the two
strands of the siRNA duplex. Since siRNA duplexes have no
foreknowledge of which siRNA strand will guide target cleavage,
both strands must assemble with the appropriate proteins to form a
RISC.
It has been observed that both siRNA strands are competent to
direct RNAi (Tuschl et al., Genes Dev 13, 3191-3197 (1999); Hammond
et al., Nature 404, 293-296 (2000); Zamore et al., Cell 101, 25-33
(2000); Elbashir et al., Genes Dev 15, 188-200 (2001); Elbashir et
al., EMBO J 20, 6877-6888 (2001); Nykanen et al., Cell 107, 309-321
(2001). That is, the antisense strand of an siRNA can direct
cleavage of a corresponding sense RNA target, whereas the sense
siRNA strand directs cleavage of an antisense target. In this way,
siRNA duplexes appear to be functionally symmetric.
The ability to control which strand of an siRNA duplex enters into
the RISC complex to direct cleavage of a corresponding RNA target
would provide a significant advance for both research and
therapeutic applications of RNAi technology.
SUMMARY OF THE INVENTION
The invention solves the foregoing problems of siRNA gene targeting
by determining the structural and functional characteristics of
single and blunt-ended siRNAs and in particular, their strand
specificity for a gene target. Accordingly, an entirely new
constellation of single and double blunt-ended siRNA agents, e.g.,
siRNA duplexes, can be designed to efficiently and specifically
modulate a sense and/or antisense gene target.
In addition, the invention provides a method for introducing
alterations in either the 5', 3', or both the 5' and 3' of a single
or double blunt-ended siRNA such that either the sense, the
antisense, or both the sense and antisense strand will enter the
RNAi pathway (e.g., RISC) and target a cognate gene target(s) for
cleavage and destruction. Typically, the alteration takes the form
of a mismatched base pair that allows for a portion of the siRNA
duplex, e.g., the 5' end of the antisense strand, to separate or
fray.
Accordingly, the invention has several advantages which include,
but are not limited to, the following: providing methods for
designing single and double blunt-ended siRNA agents, e.g., siRNA
duplexes, have a characteristic strand specificity; providing
single and double blunt-ended siRNA agents, e.g., siRNA duplexes or
small hairpin RNAs (shRNAs) with at least one blunt end, suitable
for gene modulation in plant or animal cells; and methods for
modulating gene expression in a subject in need thereof using the
single or double blunt-ended siRNA compositions of the invention,
e.g., in the form of a pharmaceutical composition suitable for
administering to a patient.
Accordingly, in one aspect, the invention provides methods for
improving the efficiency (or specificity) of an RNAi reaction
comprising modifying (e.g., increasing) the asymmetry of an RNAi
agent (i.e., an RNA duplex having at least one blunt end) such that
the ability of the sense or second strand to mediate RNAi (e.g.,
mediate cleavage of a target RNA) is lessened.
In one embodiment, the asymmetry is increased in favor of the 5'
end of the first strand, e.g., by lessening the bond strength
(e.g., the strength of the interaction) between the 5' end of the
first strand and 3' end of the second strand relative to the bond
strength (e.g., the strength of the interaction) between the 5' end
of the second strand and the 3' end of the first strand.
In another embodiment, the asymmetry is increased in favor of the
5' end of the first strand by increasing bond strength (e.g., the
strength of the interaction) between the 5' end of the second or
sense strand and the 3' end of the first or antisense strand,
relative to the bond strength (e.g., the strength of the
interaction) between the 5' end of the first and the 3' end of the
second strand.
In another embodiment, the bond strength is increased, e.g., the
hydrogen bonding is increased between nucleotides or analogs at the
5' end, e.g., within 5 nucleotides of the second or sense strand
(numbered from the 5' end of the second strand) and complementary
nucleotides of the first or antisense strand. It is understood that
the asymmetry can be zero (i.e., no asymmetry), for example, when
the bonds or base pairs between the 5' and 3' terminal bases are of
the same nature, strength or structure. More routinely, however,
there exists some asymmetry due to the different nature, strength
or structure of at least one nucleotide (often one or more
nucleotides) between terminal nucleotides or nucleotide
analogs.
Accordingly, in one aspect, the instant invention provides a method
of enhancing the ability of a first strand of a single or double
blunt-ended RNAi agent to act as a guide strand in mediating RNAi,
involving lessening the base pair strength between the 5' end of
the first strand and the 3' end of a second strand of the duplex as
compared to the base pair strength between the 3' end of the first
strand and the 5' end of the second strand.
In a related aspect, the invention provides a method of enhancing
the efficacy of a single or double blunt-ended siRNA duplex, the
siRNA duplex comprising a sense and an antisense strand, involving
lessening the base pair strength between the antisense strand 5'
end (AS 5') and the sense strand 3' end (S 3') as compared to the
base pair strength between the antisense strand 3' end (AS 3') and
the sense strand 5' end (S'5), such that efficacy is enhanced.
In another aspect of the invention, a method is provided for
promoting entry of a desired strand of an single or double
blunt-ended siRNA duplex into a RISC complex, comprising enhancing
the asymmetry of the single or double blunt-ended siRNA duplex,
such that entry of the desired strand is promoted. In one
embodiment of this aspect of the invention, the asymmetry is
enhanced by lessening the base pair strength between the 5' end of
the desired strand and the 3' end of a complementary strand of the
duplex as compared to the base pair strength between the 3' end of
the desired strand and the 5' end of the complementary strand.
In another aspect of the invention, a single or double blunt-ended
siRNA duplex is provided comprising a sense strand and an antisense
strand, wherein the base pair strength between the antisense strand
5' end (AS 5') and the sense strand 3' end (S 3') is less than the
base pair strength between the antisense strand 3' end (AS 3') and
the sense strand 5' end (S'5), such that the antisense strand
preferentially guides cleavage of a target mRNA.
In one embodiment of these aspects of the invention, the base-pair
strength is less due to fewer G:C base pairs between the 5' end of
the first or antisense strand and the 3' end of the second or sense
strand than between the 3' end of the first or antisense strand and
the 5' end of the second or sense strand.
In another embodiment, the base pair strength is less due to at
least one mismatched base pair between the 5' end of the first or
antisense strand and the 3' end of the second or sense strand.
Preferably, the mismatched or wobble base pair is selected from the
group consisting of G:A, C:A, C:U, G:G, A:A, C:C, U:U, I:A, I:U,
and I:C.
In yet another embodiment, the base pair strength is less due to at
least one base pair comprising a modified nucleotide. In preferred
embodiments, the modified nucleotide is selected from the group
consisting of 2-amino-G (e.g.,
2,2-diamino-1,2-dihydro-purin-6-one), 2-amino-A, 2,6-diamino-G, and
2,6-diamino-A.
In other embodiments of the above aspects, the single or double
blunt-ended RNAi agent or siRNA duplex is derived from an
engineered precursor, and can be chemically synthesized or
enzymatically synthesized.
In another aspect of the instant invention, compositions are
provided comprising a single or double blunt-ended siRNA duplex of
the invention formulated to facilitate entry of the siRNA duplex
into a cell. Also provided are pharmaceutical composition
comprising a siRNA duplex of the invention.
Further provided are an engineered pre-miRNA comprising the siRNA
duplex of any one of the preceding claims, as well as a vector
encoding the pre-miRNA. In related aspects, the invention provides
a pre-miRNA comprising the pre-miRNA, as well as a vector encoding
the pre-miRNA.
Also featured in the instant invention are small hairpin RNA
(shRNA) capable of forming at least a single blunt end comprising
nucleotide sequence identical to the sense and antisense strand of
the siRNA duplex as described above.
In one embodiment, the nucleotide sequence identical to the sense
strand is upstream of the nucleotide sequence identical to the
antisense strand. In another embodiment, the nucleotide sequence
identical to the antisense strand is upstream of the nucleotide
sequence identical to the sense strand. Further provided are
vectors and transgenes encoding the shRNAs of the invention.
In yet another aspect, the invention provides cells comprising the
vectors featured in the instant invention. Preferably, the cell is
a mammalian cell, e.g., a human cell.
In other aspects of the invention, methods of enhancing silencing
of a target mRNA, comprising contacting a cell having an RNAi
pathway with any of the foregoing single or double blunt-ended RNAi
agents such that silencing is enhanced.
Also provided are methods of enhancing silencing of a target mRNA
in a subject, comprising administering to the subject a
pharmaceutical composition comprising any of the foregoing single
or double blunt-ended RNAi agents such that silencing is
enhanced.
Further provided is a method of decreasing silencing of an
inadvertent target mRNA by a single or double blunt-ended RNAi
agents the RNAi agent comprising a sense strand and an antisense
strand involving the steps of: (a) detecting a significant degree
of complementarity between the sense strand and the inadvertent
target; and (b) enhancing the base pair strength between the 5' end
of the sense strand and the 3' end of the antisense strand relative
to the base pair strength between the 3' end of the sense strand
and the 5' end of the antisense strand; such that silencing of the
inadvertent target mRNA is decreased. In a preferred embodiment,
the silencing of the inadvertent target mRNA is decreased relative
to silencing of a desired target mRNA.
Other features and advantages of the invention will be apparent
from the following detailed description and claims.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows a schematic of the structural and functional
characteristics of classical siRNA (i.e., having 3' dinucleotide
overhangs) with either a 5' or 3' frayed end as compared to the
siRNAs of the invention having at least one blunt end. Selected
single blunt-ended siRNAs with either a 5' or 3' frayed end are
shown as well as their corresponding ability to target cleavage of
a test sense and/or antisense target. Numbers on the left
correspond to the siRNA shown in further detail structurally in
FIG. 4 and as tested for target specificity in FIG. 5.
FIG. 2 shows a schematic of the structural and functional
characteristics of siRNAs of the invention having both 5' and 3'
blunt ends. Selected double blunt-ended siRNAs with either a 5' or
3' frayed end are shown as well as their corresponding ability to
target cleavage of a sense and/or antisense gene target. Numbers on
the left correspond to the siRNA shown in further detail
structurally in FIG. 4 and tested for target specificity in FIG.
5.
FIG. 3-1-FIG. 3-7 shows the structure of all siRNA duplexes tested,
in particular, the single and double blunt-ended siRNA duplexes of
the invention and their correspondence with sense or antisense gene
targets to determine their efficacy and specificity. Each siRNA
duplex tested is identified by a number which corresponds to
functional target specificity results obtained in vitro using
Drosophila extracts (and shown in FIG. 5). Single blunt-ended siRNA
duplexes and double blunted-ended siRNA duplexes and their
alignment with sense targets are numbered, respectively, 1-17 and
18-21. The foregoing and their alignment with antisense targets are
numbered, respectively, 22-43.
FIG. 4 shows the efficacy and specificity of the single blunt-ended
siRNA duplexes and their ability to cleave sense and antisense gene
targets using Drosophila extracts that provide a functional
RISC-mediated RNAi pathway. Black (x) data points show % antisense
gene target cleaved (SOD1 sense target; i.e., gene knockdown)
whereas red (o) data points show % sense gene target cleaved.
FIG. 5 shows the efficacy and specificity of the double blunt-ended
siRNA duplexes and their ability to cleave sense and antisense gene
targets using Drosophila extracts that provide a functional
RISC-mediated RNAi pathway. Black (x) data points show % antisense
gene target cleaved (SOD1 sense target; i.e., gene knockdown)
whereas red (o) data points show % sense gene target cleaved.
DETAILED DESCRIPTION OF THE INVENTION
In order to provide a clear understanding of the specification and
claims, the following definitions are conveniently provided
below.
Definitions
As used herein the term "blunt end", for example, "single
blunt-end" or "double blunt-ended siRNA" refers to, e.g., an siRNA
duplex where at least one end of the duplex lacks any overhang,
e.g., a 3' dinucleotide overhang, such that both the 5' and 3'
strand end together, i.e., are flush or as referred to herein, are
blunt. The molecules of the invention have at least one blunt end
and, preferably, two blunt ends, i.e., are double blunt-ended (See
FIGS. 1-3 which show schematically classical siRNA duplexes having
3' dinucleotide overhangs as compared with the single and double
blunt-ended siRNAs of the invention).
The term "small interfering RNA" ("siRNA") (also referred to in the
art as "short interfering RNAs") refers to an RNA (or RNA analog)
comprising between about 10-50 nucleotides (or nucleotide analogs)
which is capable of directing or mediating RNA interference.
Preferably, an siRNA comprises between about 15-30 nucleotides or
nucleotide analogs, more preferably between about 16-25 nucleotides
(or nucleotide analogs), even more preferably between about 18-23
nucleotides (or nucleotide analogs), and even more preferably
between about 19-22 nucleotides (or nucleotide analogs) (e.g., 19,
20, 21 or 22 nucleotides or nucleotide analogs). As mentioned
above, at least one end if not both ends of the siRNA of the
invention, is blunt. Preferred single blunt-ended siRNA molecules
comprise a 21 nucleotide (nt) strand paired with a strand that is
19 nt, 18 nt, or 17 nt. In another embodiment, single blunt-ended
siRNA molecules comprise a 19 nt strand paired with a 18 nt strand
or, preferably, a 17 nt strand, wherein the 19 nt strand is favored
to enter the RISC pathway. It is also understood that a blunt ended
siRNA, if base paired or matched, is more prone to separating or
fraying, then an end that is matched but also has a one or more
nucleotide overhang, e.g., a dinucleotide overhang, because of the
unpaired helical nature of the overhang and the stacking forces
which contribute to maintaining the base pairs immediately
downstream.
The term "RNA interference" ("RNAi") (also referred to in the art
as "gene silencing" and/or "target silencing", e.g., "target mRNA
silencing") refers to a selective intracellular degradation of RNA.
RNAi occurs in cells naturally to remove foreign RNAs (e.g., viral
RNAs). Natural RNAi proceeds via fragments cleaved from free dsRNA
which direct the degradative mechanism to other similar RNA
sequences. Alternatively, RNAi can be initiated by the hand of man,
for example, to silence the expression of target genes.
The term "antisense strand" of an siRNA or RNAi agent refers to a
strand that is substantially complementary to a section of about
10-50 nucleotides, e.g., about 15-30, 16-25, 18-23 or 19-22
nucleotides of the mRNA of the gene targeted for silencing. The
antisense strand or first strand has sequence sufficiently
complementary to the desired target mRNA sequence to direct
target-specific RNA interference (RNAi), e.g., complementarity
sufficient to trigger the destruction of the desired target mRNA by
the RNAi machinery or process.
The term "sense strand" or "second strand" of an siRNA or RNAi
agent refers to a strand that is complementary to the antisense
strand or first strand. Antisense and sense strands can also be
referred to as first or second strands, the first or second strand
having complementarity to the target sequence and the respective
second or first strand having complementarity to said first or
second strand.
The term "guide strand" refers to a strand of an RNAi agent, e.g.,
an antisense strand of an siRNA duplex, that enters into the RISC
complex and directs cleavage of the target mRNA.
The term "target gene" is a gene whose expression is to be
selectively inhibited or "silenced". This silencing is achieved by
cleaving the mRNA of the target gene by an siRNA or miRNA, e.g., an
siRNA or miRNA that is created from an engineered RNA precursor by
a cell's RNAi system. One portion or segment of a duplex stem of
the RNA precursor is an antisense strand that is complementary,
e.g., sufficiently complementary to trigger the destruction of the
desired target mRNA by the RNAi machinery or process, to a section
of about 18 to about 40 or more nucleotides of the mRNA of the
target gene.
The term "asymmetry", as in the asymmetry of a single or double
blunt-ended siRNA duplex, refers to an inequality of bond strength
or base pairing strength between the siRNA termini (e.g., between
terminal nucleotides on a first strand and terminal nucleotides on
an opposing second strand, e.g., a base pair mismatch that allows
for a separation or fraying of the end(s)), such that the 5' end of
one strand of the duplex is more frequently in a transient
unpaired, e.g., single-stranded, state than the 5' end of the
complementary strand. This structural difference determines that
one strand of the duplex is preferentially incorporated into a RISC
complex. The strand whose 5' end is less tightly paired to the
complementary strand will preferentially be incorporated into RISC
and mediate RNAi.
The term "bond strength" or "base pair strength" refers to the
strength of the interaction between pairs of nucleotides (or
nucleotide analogs) on opposing strands of an oligonucleotide
duplex (e.g., an siRNA duplex), due primarily to hydrogen-bonding,
Van der Waals interactions, and the like between such nucleotides
(or nucleotide analogs).
The term "fray" or "fraying" refers to the ability of a portion of
the siRNA duplex of the invention to separate, typically at the
end, preferably at the 5' end of the first or antisense strand, due
to a base pair mismatch. For determining the thermodynamic
stability or local thermodynamic stability of such ends, energy
rules can be based on nearest neighbor analysis and/or amount of
stacking.
DETAILED DESCRIPTION
Overview
The present invention features "small interfering RNA molecules"
("siRNA molecules" or "siRNA") having at least one blunt end,
methods of making such siRNA molecules and methods for using the
single or double blunt-ended siRNA molecules (e.g., research and/or
therapeutic methods). A blunt-ended siRNA molecule of the invention
is a duplex consisting of a sense strand and complementary
antisense strand, the antisense strand having sufficient
complementarity to a target mRNA to mediate RNAi and having at
least one end (5', 3', or both 5' and 3') without an overhang.
Accordingly, the molecules of the invention are distinguished from
typical siRNA molecules which have a 3' dinucleotide overhang at
each end of the molecule.
Preferably, the strands are aligned such that, at one end,
preferably at both ends, there are no bases at the end of the
strands which do not align (i.e., for which no complementary bases
occur in the opposing strand) such that no overhang occurs at one
or both ends of the duplex when the strands are annealed.
Preferably, the single or double blunt-ended siRNA molecule has a
length from about 10-50 or more nucleotides, i.e., each strand
comprises 10-50 nucleotides (or nucleotide analogs). More
preferably, the siRNA molecule has a length from about 15-45 or
15-30 nucleotides. Even more preferably, the siRNA molecule has a
length from about 16-25 nucleotides, 18-23 nucleotides, or 19
nucleotides. The single or double blunt-ended siRNA molecules of
the invention further have a sequence that is "sufficiently
complementary" to a target mRNA sequence to direct target-specific
RNA interference (RNAi), as defined herein, i.e., the single or
double blunt-ended siRNA has a sequence sufficient to trigger the
destruction of the target mRNA by the RNAi machinery or
process.
1. Preferred RNA Molecules
The single or double blunt-ended siRNAs featured in the invention
provide enhanced specificity and efficacy for mediating
RISC-mediated cleavage of a desired target gene. In a preferred
aspect, the base pair strength between the antisense strand 5' end
(AS 5') and the sense strand 3' end (S 3') of the siRNAs is less
than the bond strength or base pair strength between the antisense
strand 3' end (AS 3') and the sense strand 5' end (S'5), such that
the antisense strand preferentially guides cleavage of a target
mRNA. In one embodiment, the bond strength or base-pair strength is
less due to fewer G:C base pairs between the 5' end of the first or
antisense strand and the 3' end of the second or sense strand than
between the 3' end of the first or antisense strand and the 5' end
of the second or sense strand.
In another embodiment, the bond strength or base pair strength is
less due to at least one mismatched base pair between the 5' end of
the first or antisense strand and the 3' end of the second or sense
strand. Preferably, the mismatched base pair is selected from the
group consisting of G:A, C:A, C:U, G:G, A:A, C:C and U:U. In a
related embodiment, the bond strength or base pair strength is less
due to at least one wobble base pair, e.g., G:U, between the 5' end
of the first or antisense strand and the 3' end of the second or
sense strand.
In yet another embodiment, the bond strength or base pair strength
is less due to at least one base pair comprising a rare nucleotide,
e.g., inosine (I). Preferably, the base pair is selected from the
group consisting of an I:A, I:U, and I:C.
In yet another embodiment, the bond strength or base pair strength
is less due to at least one base pair comprising a modified
nucleotide. In preferred embodiments, the modified nucleotide is
selected from the group consisting of 2-amino-G, 2-amino-A,
2,6-diamino-G, and 2,6-diamino-A.
In general, single or double blunt-ended siRNAs containing
nucleotide sequences sufficiently identical to a portion of the
target gene to effect RISC-mediated cleavage of the target gene are
preferred.
2. Gene Target Sequence Identity
Typically, 100% sequence identity between the single or double
blunt-ended siRNA and the target gene is not required to practice
the present invention. The invention has the advantage of being
able to tolerate preferred sequence variations of the methods and
compositions of the invention in order to enhance efficiency and
specificity of RNAi. For example, single or double blunt-ended
siRNA sequences with insertions, deletions, and single point
mutations relative to the target sequence can also be effective for
inhibition. Alternatively, single or double blunt-ended siRNA
sequences with nucleotide analog substitutions or insertions can be
effective for inhibition.
Sequence identity may be determined by sequence comparison and
alignment algorithms known in the art. To determine the percent
identity of two nucleic acid sequences (or of two amino acid
sequences), the sequences are aligned for optimal comparison
purposes (e.g., gaps can be introduced in the first sequence or
second sequence for optimal alignment). The nucleotides (or amino
acid residues) at corresponding nucleotide (or amino acid)
positions are then compared. When a position in the first sequence
is occupied by the same residue as the corresponding position in
the second sequence, then the molecules are identical at that
position. The percent identity between the two sequences is a
function of the number of identical positions shared by the
sequences (i.e., % homology=# of identical positions/total # of
positions.times.100), optionally penalizing the score for the
number of gaps introduced and/or length of gaps introduced.
The comparison of sequences and determination of percent identity
between two sequences can be accomplished using a mathematical
algorithm. In one embodiment, the alignment generated over a
certain portion of the sequence aligned having sufficient identity
but not over portions having low degree of identity (i.e., a local
alignment). A preferred, non-limiting example of a local alignment
algorithm utilized for the comparison of sequences is the algorithm
of Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA
87:2264-68, modified as in Karlin and Altschul (1993) Proc. Natl.
Acad. Sci. USA 90:5873-77. Such an algorithm is incorporated into
the BLAST programs (version 2.0) of Altschul, et al. (1990) J. Mol.
Biol. 215:403-10.
In another embodiment, the alignment is optimized by introducing
appropriate gaps and percent identity is determined over the length
of the aligned sequences (i.e., a gapped alignment). To obtain
gapped alignments for comparison purposes, Gapped BLAST can be
utilized as described in Altschul et al., (1997) Nucleic Acids Res.
25(17):3389-3402. In another embodiment, the alignment is optimized
by introducing appropriate gaps and percent identity is determined
over the entire length of the sequences aligned (i.e., a global
alignment). A preferred, non-limiting example of a mathematical
algorithm utilized for the global comparison of sequences is the
algorithm of Myers and Miller, CABIOS (1989). Such an algorithm is
incorporated into the ALIGN program (version 2.0) which is part of
the GCG sequence alignment software package. When utilizing the
ALIGN program for comparing amino acid sequences, a PAM120 weight
residue table, a gap length penalty of 12, and a gap penalty of 4
can be used.
Greater than 80% sequence identity, e.g., 80%, 81%, 82%, 83%, 84%,
85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98%, 99% or even 100% sequence identity, between the siRNA
antisense strand and the portion of the target gene is preferred.
Alternatively, the siRNA may be defined functionally as a
nucleotide sequence (or oligonucleotide sequence) that is capable
of hybridizing with a portion of the target gene transcript (e.g.,
400 mM NaCl, 40 mM PIPES pH 6.4, 1 mM EDTA, 50.degree. C. or
70.degree. C. hybridization for 12-16 hours; followed by washing).
Additional preferred hybridization conditions include hybridization
at 70.degree. C. in 1.times.SSC or 50.degree. C. in 1.times.SSC,
50% formamide followed by washing at 70.degree. C. in 0.3.times.SSC
or hybridization at 70.degree. C. in 4.times.SSC or 50.degree. C.
in 4.times.SSC, 50% formamide followed by washing at 67.degree. C.
in 1.times.SSC. The hybridization temperature for hybrids
anticipated to be less than 50 base pairs in length should be
5-10.degree. C. less than the melting temperature (Tm) of the
hybrid, where Tm is determined according to the following
equations. For hybrids less than 18 base pairs in length,
Tm(.degree. C.)=2(# of A+T bases)+4(# of G+C bases). For hybrids
between 18 and 49 base pairs in length, Tm(.degree. C.)=81.5+16.6
(log 10[Na+])+0.41 (% G+C)-(600/N), where N is the number of bases
in the hybrid, and [Na+] is the concentration of sodium ions in the
hybridization buffer ([Na+] for 1.times.SSC=0.165 M). Additional
examples of stringency conditions for polynucleotide hybridization
are provided in Sambrook, J., E. F. Fritsch, and T. Maniatis, 1989,
Molecular Cloning: A Laboratory Manual, Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y., chapters 9 and 11, and
Current Protocols in Molecular Biology, 1995, F. M. Ausubel et al.,
eds., John Wiley & Sons, Inc., sections 2.10 and 6.3-6.4,
incorporated herein by reference. The length of the identical
nucleotide sequences may be at least about 10, 12, 15, 17, 20, 22,
25, 27, 30, 32, 35, 37, 40, 42, 45, 47 or 50 bases.
3. Other Modifications for RNA Stability
The RNA molecules of the present invention can be modified to
improve stability in serum or in growth medium for cell cultures.
In order to enhance the stability, the 3'-residues may be
stabilized against degradation, e.g., they may be selected such
that they consist of purine nucleotides, particularly adenosine or
guanosine nucleotides. Alternatively, substitution of pyrimidine
nucleotides by modified analogues, e.g., substitution of uridine by
2'-deoxythymidine is tolerated and does not affect the efficiency
of RNA interference.
In a preferred aspect, the invention features small interfering
RNAs (siRNAs) that include a sense strand and an antisense strand,
wherein the antisense strand has a sequence sufficiently
complementary to a target mRNA sequence to direct target-specific
RNA interference (RNAi) and wherein the sense strand and/or
antisense strand is modified by the substitution of internal
nucleotides with modified nucleotides, such that in vivo stability
is enhanced as compared to a corresponding unmodified siRNA. As
defined herein, an "internal" nucleotide is one occurring at any
position other than the 5' end or 3' end of nucleic acid molecule,
polynucleotide or oligonucleotide. An internal nucleotide can be
within a single-stranded molecule or within a strand of a duplex or
double-stranded molecule. In one embodiment, the sense strand
and/or antisense strand is modified by the substitution of at least
one internal nucleotide. In another embodiment, the sense strand
and/or antisense strand is modified by the substitution of at least
2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,
21, 22, 23, 24, 25 or more internal nucleotides. In another
embodiment, the sense strand and/or antisense strand is modified by
the substitution of at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%,
45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more of
the internal nucleotides. In yet another embodiment, the sense
strand and/or antisense strand is modified by the substitution of
all of the internal nucleotides.
In a preferred embodiment of the present invention the RNA molecule
may contain at least one modified nucleotide analogue. The
nucleotide analogues may be located at positions where the
target-specific activity, e.g., the RNAi mediating activity is not
substantially effected, e.g., in a region at the 5'-end and/or the
3'-end of the RNA molecule. Particularly, the ends may be
stabilized by incorporating modified nucleotide analogues.
Preferred nucleotide analogues include sugar- and/or
backbone-modified ribonucleotides (i.e., include modifications to
the phosphate-sugar backbone). For example, the phosphodiester
linkages of natural RNA may be modified to include at least one of
a nitrogen or sulfur heteroatom. In preferred backbone-modified
ribonucleotides the phosphoester group connecting to adjacent
ribonucleotides is replaced by a modified group, e.g., of
phosphothioate group. In preferred sugar-modified ribonucleotides,
the 2' OH-group is replaced by a group selected from H, OR, R,
halo, SH, SR, NH.sub.2, NHR, NR.sub.2 or ON, wherein R is
C.sub.1-C.sub.6 alkyl, alkenyl or alkynyl and halo is F, Cl, Br or
I.
Also preferred are nucleobase-modified ribonucleotides, i.e.,
ribonucleotides, containing at least one non-naturally occurring
nucleobase instead of a naturally occurring nucleobase. Bases may
be modified to block the activity of adenosine deaminase Exemplary
modified nucleobases include, but are not limited to, uridine
and/or cytidine modified at the 5-position, e.g., 5-(2-amino)propyl
uridine, 5-bromo uridine; adenosine and/or guanosines modified at
the 8 position, e.g., 8-bromo guanosine; deaza nucleotides, e.g.,
7-deaza-adenosine; O- and N-alkylated nucleotides, e.g., N6-methyl
adenosine are suitable. It should be noted that the above
modifications may be combined.
4. RNA Synthesis
RNA may be produced enzymatically or by partial/total organic
synthesis, any modified ribonucleotide can be introduced by in
vitro enzymatic or organic synthesis. In one embodiment, an RNAi
agent is prepared chemically. Methods of synthesizing RNA molecules
are known in the art, in particular, the chemical synthesis methods
as de scribed in Verma and Eckstein (1998) Annul Rev. Biochem.
67:99-134.
In another embodiment, a ss-siRNA is prepared enzymatically. For
example, a ds-siRNA can be prepared by enzymatic processing of a
long ds RNA having sufficient complementarity to the desired target
mRNA. Processing of long ds RNA can be accomplished in vitro, for
example, using appropriate cellular lysates and ds-siRNAs can be
subsequently purified by gel electrophoresis or gel filtration.
ds-siRNA can then be denatured according to art-recognized
methodologies.
In an exemplary embodiment, RNA can be purified from a mixture by
extraction with a solvent or resin, precipitation, electrophoresis,
chromatography, or a combination thereof. Alternatively, the RNA
may be used with no or a minimum of purification to avoid losses
due to sample processing. Alternatively, the siRNA can also be
prepared by enzymatic transcription from synthetic DNA templates or
from DNA plasmids isolated from recombinant bacteria. Typically,
phage RNA polymerases are used such as T7, T3 or SP6 RNA polymerase
(Milligan and Uhlenbeck (1989) Methods Enzymol. 180:51-62). The RNA
may be dried for storage or dissolved in an aqueous solution. The
solution may contain buffers or salts to inhibit annealing, and/or
promote stabilization of the single strands.
In one embodiment, the single or double blunt-ended siRNAs are
synthesized either in vivo, in situ, or in vitro. Endogenous RNA
polymerase of the cell may mediate transcription in vivo or in
situ, or cloned RNA polymerase can be used for transcription in
vivo or in vitro. For transcription from a transgene in vivo or an
expression construct, a regulatory region (e.g., promoter,
enhancer, silencer, splice donor and acceptor, polyadenylation) may
be used to transcribe the ss-siRNA. Inhibition may be targeted by
specific transcription in an organ, tissue, or cell type;
stimulation of an environmental condition (e.g., infection, stress,
temperature, chemical inducers); and/or engineering transcription
at a developmental stage or age. A transgenic organism that
expresses ss-siRNA from a recombinant construct may be produced by
introducing the construct into a zygote, an embryonic stem cell, or
another multipotent cell derived from the appropriate organism.
5. Selecting a Gene Target
In one embodiment, the target mRNA of the invention encodes the
amino acid sequence of a cellular protein, e.g., a protein involved
in cell growth or suppression, e.g., a nuclear, cytoplasmic,
transmembrane, membrane-associated protein, or cellular ligand. In
another embodiment, the target mRNA of the invention specifies the
amino acid sequence of an extracellular protein (e.g., an
extracellular matrix protein or secreted protein). Typical classes
of proteins are listed for illustrative purposes.
Developmental proteins suitable for targeting according to the
invention include e.g., adhesion molecules, cyclin kinase
inhibitors, Wnt family members, Pax family members, Winged helix
family members, Hox family members, cytokines/lymphokines and their
receptors, growth/differentiation factors and their receptors,
neurotransmitters and their receptors).
Oncogene-encoded proteins suitable for targeting according to the
invention include, e.g., ABLI, BCLI, BCL2, BCL6, CBFA2, CBL, CSFIR,
ERBA, ERBB, EBRB2, ETSI, ETSI, ETV6, FGR, FOS, FYN, HCR, HRAS, JUN,
KRAS, LCK, LYN, MDM2, MLL, MYB, MYC, MYCLI, MYCN, NRAS, PIM I, PML,
RET, SRC, TALI, TCL3, and YES).
Tumor suppressor proteins suitable for targeting according to the
invention include e.g., APC, BRCA1, BRCA2, MADH4, MCC, NF I, NF2,
RB I, TP53, and WTI).
Enzymatic proteins suitable for targeting according to the
invention include, e.g., ACC synthases and oxidases, ACP
desaturases and hydroxylases, ADP-glucose pyrophorylases, ATPases,
alcohol dehydrogenases, amylases, amyloglucosidases, catalases,
cellulases, chalcone synthases, chitinases, cyclooxygenases,
decarboxylases, dextriinases, DNA and RNA polymerases,
galactosidases, glucanases, glucose oxidases, granule-bound starch
synthases, GTPases, helicases, hernicellulases, integrases,
inulinases, invertases, isomerases, kinases, lactases, lipases,
lipoxygenases, lysozymes, nopaline synthases, octopine synthases,
pectinesterases, peroxidases, phosphatases, phospholipases,
phosphorylases, phytases, plant growth regulator synthases,
polygalacturonases, proteinases and peptidases, pullanases,
recombinases, reverse transcriptases, RUBISCOs, topoisomerases,
xylanases, and telomerases.
In a preferred aspect of the invention, the target mRNA molecule of
the invention specifies the amino acid sequence of a protein
associated with a pathological condition. For example, the protein
may be a pathogen-associated protein (e.g., a viral protein
involved in immunosuppression of the host, replication of the
pathogen, transmission of the pathogen, or maintenance of the
infection), or a host protein which facilitates entry of the
pathogen into the host, drug metabolism by the pathogen or host,
replication or integration of the pathogen's genome, establishment
or spread of infection in the host, or assembly of the next
generation of pathogen. Alternatively, the protein may be a
tumor-associated protein or an autoimmune disease-associated
protein.
By modulating the expression of the foregoing proteins, valuable
information regarding the function of such proteins and therapeutic
benefits which may be obtained from such modulation can be
obtained.
6. Assay for Testing Engineered RNA Precursors
Drosophila embryo lysates can be used to determine if the
engineered siRNAs of the invention, e.g., single or double
blunt-ended siRNA duplexes (but also, e.g., expressed shRNAs) have
their intended function (see also Examples 1-3). This lysate assay
is described in Tuschl et al., 1999, supra, Zamore et al., 2000,
supra, and Hutvdgner et al., Science 293, 834-838 (2001). These
lysates recapitulate RNAi in vitro, thus permitting investigation
into, e.g., which strand enters the complex, is assembled into
RISC, and is used as a guide strand for target destruction.
Briefly, the test siRNA is incubated with Drosophila embryo lysate
for various times, then assayed for the production of the mature
siRNA by primer extension or Northern hybridization. As in the in
vivo setting, mature RNA accumulates in the cell-free reaction.
Thus, an RNA corresponding to the proposed precursor can be shown
to be converted into a siRNA duplex in the Drosophila embryo
lysate.
Furthermore, an engineered RNA precursor can be functionally tested
in the Drosophila embryo lysates. In this case, the engineered RNA
precursor is incubated in the lysate in the presence of a 5'
radiolabeled target mRNA in a standard in vitro RNAi reaction for
various lengths of time. The target mRNA can be 5' radiolabeled
using guanylyl transferase (as described in Tuschl et al., 1999,
supra and references therein) or other suitable methods. The
products of the in vitro reaction are then isolated and analyzed on
a denaturing acrylamide or agarose gel to determine if the target
mRNA has been cleaved in response to the presence of the engineered
RNA precursor in the reaction. The extent and position of such
cleavage of the mRNA target will indicate if the engineering of the
precursor created a pre-siRNA capable of mediating
sequence-specific RNAi.
7. Methods of Introducing RNAs, Vectors, and Host Cells
Physical methods of introducing nucleic acids include injection of
a solution containing the RNA, bombardment by particles covered by
the RNA, soaking the cell or organism in a solution of the RNA, or
electroporation of cell membranes in the presence of the RNA. A
viral construct packaged into a viral particle would accomplish
both efficient introduction of an expression construct into the
cell and transcription of RNA encoded by the expression construct.
Other methods known in the art for introducing nucleic acids to
cells may be used, such as lipid-mediated carrier transport,
chemical-mediated transport, such as calcium phosphate, and the
like. Thus the RNA may be introduced along with components that
perform one or more of the following activities: enhance RNA uptake
by the cell, inhibit annealing of single strands, stabilize the
single strands, or other-wise increase inhibition of the target
gene.
RNA may be directly introduced into the cell (i.e.,
intracellularly); or introduced extracellularly into a cavity,
interstitial space, into the circulation of an organism, introduced
orally, or may be introduced by bathing a cell or organism in a
solution containing the RNA. Vascular or extravascular circulation,
the blood or lymph system, and the cerebrospinal fluid are sites
where the RNA may be introduced.
The cell with the target gene may be derived from or contained in
any organism. The organism may a plant, animal, protozoan,
bacterium, virus, or fungus. The plant may be a monocot, dicot or
gymnosperm; the animal may be a vertebrate or invertebrate.
Preferred microbes are those used in agriculture or by industry,
and those that are pathogenic for plants or animals
Alternatively, vectors, e.g., transgenes encoding a siRNA of the
invention, i.e., having at least one blunt end, can be engineered
into a host cell or transgenic animal using art recognized
techniques.
8. Methods of Treatment:
The present invention provides for both prophylactic and
therapeutic methods of treating a subject at risk of (or
susceptible to) a disorder or having a disorder associated with
aberrant or unwanted target gene expression or activity. It is
understood that "treatment" or "treating" as used herein, is
defined as the application or administration of a therapeutic agent
(e.g., a RNAi agent or vector or transgene encoding same) to a
patient, or application or administration of a therapeutic agent to
an isolated tissue or cell line from a patient, who has a disease
or disorder, a symptom of disease or disorder or a predisposition
toward a disease or disorder, with the purpose to cure, heal,
alleviate, relieve, alter, remedy, ameliorate, improve or affect
the disease or disorder, the symptoms of the disease or disorder,
or the predisposition toward disease.
9. Prophylactic Methods
In another aspect, the invention provides a method for preventing
in a subject, a disease or condition associated with an aberrant or
unwanted target gene expression or activity, by administering to
the subject a therapeutic agent (e.g., a RNAi agent or vector or
transgene encoding same). Subjects at risk for a disease which is
caused or contributed to by aberrant or unwanted target gene
expression or activity can be identified by, for example, any or a
combination of diagnostic or prognostic assays as described herein.
Administration of a prophylactic agent can occur prior to the
manifestation of symptoms characteristic of the target gene
aberrancy, such that a disease or disorder is prevented or,
alternatively, delayed in its progression. Depending on the type of
target gene aberrancy, for example, a target gene, target gene
agonist or target gene antagonist agent can be used for treating
the subject. The appropriate agent can be determined based on
screening assays described herein.
10. Therapeutic Methods
In yet another aspect, the invention pertains to methods of
modulating target gene expression, protein expression or activity
for therapeutic purposes. Accordingly, in an exemplary embodiment,
the modulatory method of the invention involves contacting a cell
capable of expressing target gene with a therapeutic agent (e.g., a
RNAi agent or vector or transgene encoding same) that is specific
for the target gene or protein (e.g., is specific for the mRNA
encoded by said gene or specifying the amino acid sequence of said
protein) such that expression or one or more of the activities of
target protein is modulated. These modulatory methods can be
performed in vitro (e.g., by culturing the cell with the agent) or,
alternatively, in vivo (e.g., by administering the agent to a
subject). As such, the present invention provides methods of
treating an individual afflicted with a disease or disorder
characterized by aberrant or unwanted expression or activity of a
target gene polypeptide or nucleic acid molecule. Inhibition of
target gene activity is desirable in situations in which target
gene is abnormally unregulated and/or in which decreased target
gene activity is likely to have a beneficial effect.
11. Pharmacogenomics
The therapeutic agents (e.g., a RNAi agent or vector or transgene
encoding same) of the invention can be administered to individuals
to treat (prophylactically or therapeutically) disorders associated
with aberrant or unwanted target gene activity. In conjunction with
such treatment, pharmacogenomics (i.e., the study of the
relationship between an individual's genotype and that individual's
response to a foreign compound or drug) may be considered.
Differences in metabolism of therapeutics can lead to severe
toxicity or therapeutic failure by altering the relation between
dose and blood concentration of the pharmacologically active drug.
Thus, a physician or clinician may consider applying knowledge
obtained in relevant pharmacogenomics studies in determining
whether to administer a therapeutic agent as well as tailoring the
dosage and/or therapeutic regimen of treatment with a therapeutic
agent.
Pharmacogenomics deals with clinically significant hereditary
variations in the response to drugs due to altered drug disposition
and abnormal action in affected persons. See, for example,
Eichelbaum, M. et al. (1996) Clin. Exp. Pharmacol. Physiol.
23(10-11): 983-985 and Linder, M. W. et al. (1997) Clin. Chem.
43(2):254-266
12. Pharmaceutical Compositions
The invention pertains to uses of the above-described agents for
therapeutic treatments as described infra. Accordingly, the
modulators of the present invention can be incorporated into
pharmaceutical compositions suitable for administration. Such
compositions typically comprise the nucleic acid molecule, protein,
antibody, or modulatory compound and a pharmaceutically acceptable
carrier. As used herein the language "pharmaceutically acceptable
carrier" is intended to include any and all solvents, dispersion
media, coatings, antibacterial and antifungal agents, isotonic and
absorption delaying agents, and the like, compatible with
pharmaceutical administration.
The use of such media and agents for pharmaceutically active
substances is well known in the art. Except insofar as any
conventional media or agent is incompatible with the active
compound, use thereof in the compositions is contemplated.
Supplementary active compounds can also be incorporated into the
compositions.
13. Knockout and/or Knockdown Cells or Organisms
A further preferred use for the RNAi agents of the present
invention (or vectors or transgenes encoding same) is a functional
analysis to be carried out in eukaryotic cells, or eukaryotic
non-human organisms, preferably mammalian cells or organisms and
most preferably human cells, e.g. cell lines such as HeLa or 293 or
rodents, e.g. rats and mice. By administering a suitable RNAi agent
which is sufficiently complementary to a target mRNA sequence to
direct target-specific RNA interference, a specific knockout or
knockdown phenotype can be obtained in a target cell, e.g. in cell
culture or in a target organism.
Thus, a further subject matter of the invention is a eukaryotic
cell or a eukaryotic non-human organism exhibiting a target
gene-specific knockout or knockdown phenotype comprising a fully or
at least partially deficient expression of at least one endogeneous
target gene wherein said cell or organism is transfected with at
least one vector comprising DNA encoding an RNAi agent capable of
inhibiting the expression of the target gene. It should be noted
that the present invention allows a target-specific knockout or
knockdown of several different endogeneous genes due to the
specificity of the RNAi agent.
Gene-specific knockout or knockdown phenotypes of cells or
non-human organisms, particularly of human cells or non-human
mammals may be used in analytic to procedures, e.g. in the
functional and/or phenotypical analysis of complex physiological
processes such as analysis of gene expression profiles and/or
proteomes. Preferably the analysis is carried out by high
throughput methods using oligonucleotide based chips.
14. Transgenic Organisms
Engineered RNA precursors of the invention can be expressed in
transgenic animals. These animals represent a model system for the
study of disorders that are caused by, or exacerbated by,
overexpression or underexpression (as compared to wildtype or
normal) of nucleic acids (and their encoded polypeptides) targeted
for destruction by the RNAi agents, e.g., siRNAs and shRNAs, and
for the development of therapeutic agents that modulate the
expression or activity of nucleic acids or polypeptides targeted
for destruction.
Transgenic animals can be farm animals (pigs, goats, sheep, cows,
horses, rabbits, and the like), rodents (such as rats, guinea pigs,
and mice), non-human primates (for example, baboons, monkeys, and
chimpanzees), and domestic animals (for example, dogs and cats).
Invertebrates such as Caenorhabditis elegans or Drosophila can be
used as well as non-mammalian vertebrates such as fish (e.g.,
zebrafish) or birds (e.g., chickens).
Engineered RNA precursors with stems of 18 to 30 nucleotides in
length are preferred for use in mammals, such as mice. A transgenic
founder animal can be identified based upon the presence of a
transgene that encodes the new RNA precursors in its genome, and/or
expression of the transgene in tissues or cells of the animals, for
example, using PCR or Northern analysis. Expression is confirmed by
a decrease in the expression (RNA or protein) of the target
sequence.
Methods for generating transgenic animals include introducing the
transgene into the germ line of the animal. One method is by
microinjection of a gene construct into the pronucleus of an early
stage embryo (e.g., before the four-cell stage; Wagner et al.,
1981, Proc. Natl. Acad. Sci. USA 78:5016; Brinster et al., 1985,
Proc. Natl. Acad. Sci. USA 82:4438). Alternatively, the transgene
can be introduced into the pronucleus by retroviral infection. A
detailed procedure for producing such transgenic mice has been
described (see e.g., Hogan et al., Manipulating the Mouse Embryo.
Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1986);
U.S. Pat. No. 5,175,383 (1992)). This procedure has also been
adapted for other animal species (e.g., Hammer et al., 1985, Nature
315:680; Murray et al., 1989, Reprod. Fert. Devl. 1:147; Pursel et
al., 1987, Vet. Immunol. Histopath. 17:303; Rexroad et al., 1990,
J. Reprod. Fert. 41 (suppl): 1 19; Rexroad et al., 1989, Molec.
Reprod. Devl. 1:164; Simons et al., 1988, BioTechnology 6:179; Vize
et al., 1988, J. Cell. Sci. 90:295; and Wagner, 1989, J. Cell.
Biochem. 13B (suppl): 164). Clones of the non-human transgenic
animals described herein can be produced according to the methods
described in Wilmut et al. ((1997) Nature, 385:810-813) and PCT
publication Nos. WO 97/07668 and WO 97/07669.
15. Screening Assays
The methods of the invention are also suitable for use in methods
to identify and/or characterize potential pharmacological agents,
e.g. identifying new pharmacological agents from a collection of
test substances and/or characterizing mechanisms of action and/or
side effects of known pharmacological agents.
Thus, the present invention also relates to a system for
identifying and/or characterizing pharmacological agents acting on
at least one target protein comprising: (a) a eukaryotic cell or a
eukaryotic non-human organism capable of expressing at least one
endogeneous target gene coding for said so target protein, (b) at
least one RNAi agent molecule capable of inhibiting the expression
of said at least one endogeneous target gene, and (c) a test
substance or a collection of test substances wherein
pharmacological properties of said test substance or said
collection are to be identified and/or characterized. Further, the
system as described above preferably comprises: (d) at least one
exogenous target nucleic acid coding for the target protein or a
variant or mutated form of the target protein wherein said
exogenous target nucleic acid differs from the endogeneous target
gene on the nucleic acid level such that the expression of the
exogenous target nucleic acid is substantially less inhibited by
the RNAi agent than the expression of the endogeneous target
gene.
The test compounds of the present invention can be obtained using
any of the numerous approaches in combinatorial library methods
known in the art, including: biological libraries; spatially
addressable parallel solid phase or solution phase libraries;
synthetic library methods requiring deconvolution; the `one-bead
one-compound` library method; and synthetic library methods using
affinity chromatography selection. The biological library approach
is limited to peptide libraries, while the other four approaches
are applicable to peptide, non-peptide oligomer or small molecule
libraries of compounds (Lam, K. S. (1997) Anticancer Drug Des.
12:145).
Examples of methods for the synthesis of molecular libraries can be
found in the art, for example in: DeWitt et al. (1993) Proc. Natl.
Acad. Sci. U.S.A. 90:6909; Erb et al. (1994) Proc. Natl. Acad. Sci.
USA 91:11422; Zuckermann et al. (1994). J. Med. Chem. 37:2678; Cho
et al. (1993) Science 261:1303; Carrell et al. (1994) Angew. Chem.
Int. Ed. Engl. 33:2059; Carell et al. (1994) Angew. Chem. Int. Ed.
Engl. 33:2061; and in Gallop et al. (1994) J. Med. Chem.
37:1233.
Libraries of compounds may be presented in solution (e.g., Houghten
(1992) Biotechniques 13:412-421), or on beads (Lam (1991) Nature
354:82-84), chips (Fodor (1993) Nature 364:555-556), bacteria
(Ladner U.S. Pat. No. 5,223,409), spores (Ladner USP '409),
plasmids (Cull et al. (1992) Proc Natl Acad Sci USA 89:1865-1869)
or on phage (Scott and Smith (1990) Science 249:386-390); (Devlin
(1990) Science 249:404-406); (Cwirla et al. (1990) Proc. Natl.
Acad. Sci. 87:6378-6382); (Felici (1991) J. Mol. Biol.
222:301-310); (Ladner supra.)).
In a preferred embodiment, the library is a natural product
library, e.g., a library produced by a bacterial, fungal, or yeast
culture. In another preferred embodiment, the library is a
synthetic compound library.
This invention is further illustrated by the following examples
which should not be construed as limiting.
EXEMPLIFICATION
Throughout the examples, the following materials and methods were
used unless otherwise stated.
Materials and Methods
In general, the practice of the present invention employs, unless
otherwise indicated, conventional techniques of nucleic acid
chemistry, recombinant DNA technology, molecular biology,
biochemistry, and cell and cell extract preparation. See, e.g., DNA
Cloning, Vols. 1 and 2, (D. N. Glover, Ed. 1985); Oligonucleotide
Synthesis (M. J. Gait, Ed. 1984); Oxford Handbook of Nucleic Acid
Structure, Neidle, Ed., Oxford Univ Press (1999); RNA Interference:
The Nuts & Bolts of siRNA Technology, by D. Engelke, DNA Press,
(2003); Gene Silencing by RNA Interference: Technology and
Application, by M. Sohail, CRC Press (2004); Sambrook, Fritsch and
Maniatis, Molecular Cloning: Cold Spring Harbor Laboratory Press
(1989); and Current Protocols in Molecular Biology, eds. Ausubel et
al., John Wiley & Sons (1992). See also PCT/US03/24768; U.S.
Ser. No. 60/475,331; U.S. Ser. No. 60/507,928; and U.S. Ser. No.
60/475,386, of which all are incorporated in their entireties by
reference herein.
siRNA Preparation
Synthetic RNAs (Dharmacon) were deprotected according to the
manufacturer's protocol. siRNA strands were annealed (Elbashir et
al., Genes Dev 15, 188-200 (2001) and used at 50 nM final
concentration unless otherwise noted. siRNA single strands were
phosphorylated with polynucleotide kinase (New England Biolabs) and
1 mM ATP according to the manufacturer's directions and used at 500
nM final concentration.
Sense and Anti-Sense Target Preparation
Target RNAs were transcribed with recombinant, histidine-tagged, T7
RNA Polymerase from PCR products as described (Nykanen et al.,
2001, supra; Hutvagner and Zamore, Science 297, 2056-2060 (2002),
except for sense sod1 mRNA, which was transcribed from a plasmid
template (Crow et al., J Neurochem 69, 1936-1944 (1997)) linearized
with Bam HI. PCR templates for htt sense and antisense and sod1
antisense target RNAs were generated by amplifying 0.1 ng/ml (final
concentration) plasmid template encoding htt or sod1 cDNA using the
following primer pairs: htt sense target, 5'-GCG TAA TAC GAC TCA
CTA TAG GAA CAG TAT GTC TCA GAC ATC-3' (SEQ ID NO:1) and 5'-UUCG
AAG UAU UCC GCG UAC GU-3' (SEQ ID NO:4); htt antisense target,
5'-GCG TAA TAC GAC TCA CTA TAG GAC AAG CCT AAT TAG TGATGC-3' (SEQ
ID NO:5) and 5'-GAA CAG TAT GTC TCA GAC ATC-3'; sod1 antisense
target, 5'-GCG TAA TAC GAC TCA CTA TAG GGC TTT GTT AGC AGC CGG
AT-3' and 5'-GGG AGA CCA CAA CGG TTT CCC-3' (SEQ ID NO:6).
RISC Extract Preparation
Drosophila embryo lysate preparation, in vitro RNAi reactions, and
cap-labeling of target RNAs using guanylyl transferase were carried
out as previously described (Tuschl et al., 1999, supra; Zamore et
al., 2000, supra). Target RNAs were used at .about.5 nM
concentration to ensure that reactions occurred under
single-turnover conditions. Target cleavage under these conditions
was proportionate to siRNA concentrations. Cleavage products of
RNAi reactions were analyzed by electrophoresis on 5% or 8%
denaturing acrylamide gels. 5' end labeling and determination of
siRNA unwinding status were according to Nykanen et al. (Nykanen et
al., 2001, supra) except that unlabeled competitor RNA was used at
100-fold molar excess. Gels were dried, then exposed to image
plates (Fuji), which were scanned with a Fuji FLA-5000
phosphorimager. Images were analyzed using Image Reader FLA-5000
version 1.0 (Fuji) and Image Gauge version 3.45 or 4.1 (Fuji). Data
analysis was performed using Excel (Microsoft) and IgorPro 5.0
(Wavemetrics).
Example 1
Functionally Asymmetric siRNA Duplexes Having a Single Blunt
End
The following example describes methods for constructing single
blunt-ended siRNA duplexes capable of selectively entering a
RISC-mediated RNAi pathway and selectively cleaving a test target
for destruction.
Briefly, to assess quantitatively if the two strands of an siRNA
duplex having a single 5' or 3' blunt end are equally competent to
direct RNAi, the individual rates of sense and antisense target
cleavage for a single blunt-ended siRNA duplex directed against the
SOD1 target gene were examined (FIG. 4). The relevant portions of
the sense and antisense target RNA sequences are shown in FIG. 4
and in schematic form in FIG. 1 (see lower panel). The single
blunt-ended siRNA duplex effectively silences SOD1 expression in
Drosophila extracts when having a weakened 5' end (i.e., "frayed
end") (compare 4 with 1 in FIGS. 1 and 4).
Accordingly, these results indicate that 1) a single blunt end
siRNA is functional and 2) that weakening the 5' antisense base
pair interaction with the 3' sense strand dramatically increases
entry of the antisense strand into the complex and subsequent gene
knockdown activity.
Example 2
Functionally Asymmetric siRNA Duplexes Having a Double Blunt
Ends
The following example describes methods for constructing double
blunt-ended siRNA duplexes capable of selectively entering a
RISC-mediated RNAi pathway and selectively cleaving a test target
for destruction.
Briefly, to assess quantitatively if the two strands of an double
blunt-ended siRNA duplex are equally competent to direct RNAi, the
individual rates of sense and antisense target cleavage for a
single blunt-ended siRNA duplex directed against the SOD1 target
gene were examined (FIG. 5). The relevant portions of the sense and
antisense target RNA sequences are shown in FIG. 3 and in schematic
form in FIG. 2 (see lower panel). The double blunt-ended siRNA
duplexes effectively silence SOD1 expression in Drosophila extracts
and this activity is increased in the when having a weakened 5' end
(i.e., "frayed end") (compare 4 with 1 in FIGS. 1 and 4).
Accordingly, these results indicate that 1) double blunt-end siRNA
molecules are functional and 2) that weakening the 5' antisense
base pair interaction with the 3' sense strand or the 5' sense base
pair interaction with the 3' antisense strand modulates the entry
of the antisense and sense strand into the complex and subsequent
gene knockdown activity (see FIGS. 2 and 5).
Example 3
Single and Double Blunt-Ended siRNA Strand Contribution in RISC
Assembly
The following example describes methods for determining
RISC-mediated selectivity regarding the single and double
blunt-ended siRNAs of the invention.
To identify the source of asymmetry in the function of such an
single or double blunt-ended siRNA duplex, the unwinding of the two
siRNA strands when the duplex is incubated in a standard in vitro
RNAi reaction is measured. This assay has been observed to
determine accurately the fraction of siRNA that is unwound in an
ATP-dependent step in the RNAi pathway and that no functional RISC
is assembled in the absence of ATP (Nykanen et al., 2001). Other
observations have noted that siRNA unwinding correlates with
capacity of an siRNA to function in target cleavage (Nykanen et
al., 2001, supra; Martinez et al., Cell 110, 563-574 (2002)),
demonstrating that siRNA duplex unwinding is required to assemble a
RISC competent to base pair with its target RNA.
Accordingly, the accumulation of single stranded siRNA against a
test gene such as luciferase after 1 hour incubation in an in vitro
RNAi reaction in the absence of target RNA is measured. After one
hour of incubation with Drosophila embryo lysate in a standard RNAi
reaction, the antisense strand of the luciferase siRNA is converted
to single-strand. In control experiments, single-stranded RNA is
assayed without incubation in lysate. Since the production of
single-stranded antisense siRNA must be accompanied by an equal
amount of single-stranded sense siRNA, the missing sense-strand is
calculated to have been destroyed after unwinding.
To establish that the observed asymmetry in the accumulation of the
two single-strands is not an artifact of our unwinding assay, an
independent method for measuring the fraction of siRNA present as
single-strands in protein-RNA complexes is performed. In this
assay, double-stranded siRNA is incubated with Drosophila embryo
lysate in a standard RNAi reaction for 1 h, then a 31 nt
2'-O-methyl RNA oligonucleotide containing a 21 nt sequence
complementary to the radiolabeled siRNA strand is added.
2'-O-methyl oligonucleotides are not cleaved by the RNAi machinery,
but can bind stably to complementary siRNA within the RISC. To
allow recovery of RISC, the 2'-O-methyl oligonucleotide is tethered
to a magnetic bead via a biotin-streptavidin linkage. After washing
away unbound RNA and protein, the amount of radioactive siRNA bound
to the bead is measured. The assay is performed with separate siRNA
duplexes in which either the sense or the antisense strand is
5'-.sup.32P-radiolabeled. Capture of .sup.32P-siRNA is observed
when the 2'-O-methyl oligonucleotide contained a 21-nt region
complementary to the radiolabeled siRNA strand, but not when an
unrelated oligonucleotide is used.
Thus, the above assay captures all RISC activity directed by the
siRNA strand complementary to the tethered oligonucleotide,
demonstrating that it measures siRNA present in the lysate as
single-strand complexed with RISC proteins.
Accordingly, this assay can determine the contribution each strand
from a single or double blunt-ended siRNA of the invention makes to
RISC assembly.
EQUIVALENTS
Those skilled in the art will recognize, or be able to ascertain
using no more than routine experimentation, many equivalents to the
specific embodiments of the invention described herein. Such
equivalents are intended to be encompassed by the following
claims.
SEQUENCE LISTINGS
1
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21342DNAArtificial Sequencesource/note="Description of Artificial
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21541DNAArtificial Sequencesource/note="Description of Artificial
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211121DNAArtificial Sequencesource/note="Description of Artificial
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ccaagucuct t 211221DNAArtificial Sequencesource/note="Description
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caaugugact t 211821DNAArtificial Sequencesource/note="Description
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* * * * *
References