U.S. patent application number 11/286624 was filed with the patent office on 2006-12-21 for rnai modulation of the bcr-abl fusion gene and uses thereof.
Invention is credited to Philipp Hadwiger, Heiko Van Der Kuip, Hans-Peter Vornlocher.
Application Number | 20060287264 11/286624 |
Document ID | / |
Family ID | 36647951 |
Filed Date | 2006-12-21 |
United States Patent
Application |
20060287264 |
Kind Code |
A1 |
Hadwiger; Philipp ; et
al. |
December 21, 2006 |
RNAi modulation of the BCR-ABL fusion gene and uses thereof
Abstract
The invention relates to compositions and methods for modulating
the expression of Bcr-Abl, and more particularly to the
down-regulation of Bcr-Abl mRNA and Bcr-Abl protein levels by
oligonucleotides via RNA interference, e.g., chemically modified
oligonucleotides.
Inventors: |
Hadwiger; Philipp;
(Altenkunstadt, DE) ; Vornlocher; Hans-Peter;
(Bayreuth, DE) ; Van Der Kuip; Heiko; (Ammerbuch,
DE) |
Correspondence
Address: |
FISH & RICHARDSON PC
P.O. BOX 1022
MINNEAPOLIS
MN
55440-1022
US
|
Family ID: |
36647951 |
Appl. No.: |
11/286624 |
Filed: |
November 23, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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60630878 |
Nov 24, 2004 |
|
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60632403 |
Dec 1, 2004 |
|
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Current U.S.
Class: |
514/44A ; 514/81;
536/23.1; 544/243 |
Current CPC
Class: |
A61P 31/00 20180101;
A61P 35/02 20180101; C12N 15/1135 20130101; C12N 2310/315 20130101;
A61P 35/00 20180101; C12N 2310/33 20130101; A61P 43/00 20180101;
C12N 2310/32 20130101; C12N 2310/14 20130101 |
Class at
Publication: |
514/044 ;
514/081; 536/023.1; 544/243 |
International
Class: |
A61K 48/00 20060101
A61K048/00; C07H 21/02 20060101 C07H021/02; C07F 9/6512 20060101
C07F009/6512 |
Claims
1. An iRNA agent comprising an antisense strand which differs at no
more than 1, 2, or 3 nucleotides from a sequence of 15 or more
contiguous nucleotides of one of: SEQ ID No. 2, 6, 12, 14,
16,or20.
2. The iRNA agent of claim 1, wherein the antisense strand has at
least 15 contiguous nucleotides of one of: SEQ ID No. 2, 6, 12, 14,
16, or 20.
3. The iRNA agent of claim 1, further comprising a sense strand
which differs at no more than 1, 2, or 3 nucleotides from a
sequence of 15 or more contiguous nucleotides of one of: SEQ ID No.
1, 5, 11, 13, 15, or 19.
4. The iRNA agent of claim 1, further comprising a sense strand
having a sequence of 15 contiguous nucleotides of one of: SEQ ID
No. 1, 5, 11, 13, 15, or 19.
5. The iRNA agent of claim 1, wherein the agent is one of: agent
no. 3, 4 or 5.
6. The iRNA agent of claim 1, wherein the sense strand has at least
15 contiguous nucleotides of the sense sequences of agent no. 3, 4
or 5, and the antisense strand has at least 15 contiguous
nucleotides of the antisense sequences of agent no. 3, 4 or 5,
respectively.
7. The iRNA agent of claim 1, wherein the iRNA agent significantly
reduces the amount of BCR-ABL fusion protein levels present in
cultured human cells after incubation with the agent compared to
cells which have not been incubated with the agent, wherein the
cells are preferably 32Dp210/e14a2, 32Dp210-T315I, 32Dp210-H396P,
32Dp210/e13a2, 32Dp190/e1a2, M07p210/e14a2, K562, MEG-01, or
SUP-B15, or have been isolated from a leukemic patient.
8. The iRNA agent of claim 1, wherein the antisense RNA strand is
30 or fewer nucleotides in length, and the duplex region of the
iRNA agent is 15-30 nucleotide pairs in length.
9. The iRNA agent of claim 1, comprising a modification that causes
the iRNA agent to have increased stability in a biological
sample.
10. The iRNA agent of claim 9, comprising a phosphorothioate or a
2'-modified nucleotide.
11. The iRNA agent of claim 10, comprising at least one
5'-uridine-adenine-3' (5'-ua-3') dinucleotide wherein the uridine
is a 2'-modified nucleotide; at least one 5'-uridine-guanine-3'
(5'-ug-3') dinucleotide, wherein the 5'-uridine is a 2'-modified
nucleotide; at least one 5'-cytidine-adenine-3' (5'-ca-3')
dinucleotide, wherein the 5'-cytidine is a 2'-modified nucleotide;
or at least one 5'-uridine-uridine-3' (5'-uu-3') dinucleotide,
wherein the 5'-uridine is a 2'-modified nucleotide.
12. The iRNA agent of claim 10, wherein the 2'-modified nucleotide
comprises a modification selected from the group consisting of:
2'-deoxy, 2'-deoxy-2'-fluoro, 2'-O-methyl, 2'-O-methoxyethyl
(2'-O-MOE), 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl
(2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP),
2'-O-dimethylaminoethyloxyethyl (2'-O-DMAEOE), and
2'-O-N-methylacetamido (2'-O-NMA).
13. The iRNA agent of any one of the preceding claims, comprising a
nucleotide overhang having 1 to 4 unpaired nucleotides.
14. The iRNA agent of claim 13, wherein the nucleotide overhang has
2 or 3 unpaired nucleotides.
15. The iRNA agent of claim 13, wherein the nucleotide overhang is
at the 3'-end of the antisense strand of the iRNA agent.
16. The iRNA agent of any one of the preceding claims, comprising a
ligand.
17. The iRNA agent of claim 16, wherein the ligand is conjugated to
the 3'-end of the sense strand of the iRNA agent.
18. A pharmaceutical composition, comprising: an iRNA agent of any
one of the preceding claims, and a pharmaceutically acceptable
carrier.
19. A method of reducing the amount of BCR-ABL RNA in a cell of a
subject, comprising contacting the cell with an iRNA agent of any
one of the preceding claims.
20. A method of making an iRNA agent of claim 1, the method
comprising the synthesis of the iRNA agent, wherein the sense and
antisense strands comprise at least one modification that
stabilizes the iRNA agent against nucleolytic degradation.
21. A method of treating a human having or at risk for developing a
proliferative disorder comprising administering an iRNA agent of
claim 1, and preferably wherein the iRNA agent comprises a sense
strand having at least 15 contiguous nucleotides of the sense
strand sequences of the iRNA agents, agent numbers, 1, 2, 3, 4, 5,
or 6, and an antisense strand having at least 15 contiguous
nucleotides of the antisense sequences of the iRNA agents, agent
numbers, 1, 2, 3, 4, 5, or 6.
22. The method of claim 21, wherein the sense strand of the iRNA
agent comprises at least 15 contiguous nucleotides of the sense
sequence of agent no. 3, 4 or 5, and the antisense strand of the
iRNA agent comprises at least 15 contiguous nucleotides of the
antisense sequence of agent no. 3, 4 or 5, respectively.
23. The method of claim 22, wherein the sense strand of the iRNA
agent comprises at least 15 contiguous nucleotides of the sense
sequence of agent no. 3, or 4, and the antisense strand of the iRNA
agent comprises at least 15 contiguous nucleotides of the antisense
sequence of agent no. 3, or 4, respectively, and the proliferative
disorder is a Chronic Myeloid Leukemia.
24. The method of claim 22, wherein the sense strand of the iRNA
agent comprises at least 15 contiguous nucleotides of the sense
sequence of agent no. 5, and the antisense strand of the iRNA agent
comprises at least 15 contiguous nucleotides of the antisense
sequence of agent no. 5, and the proliferative disorder is a
Ph+acute lymphoblastic leukemia.
25. The method of claim 21, wherein the iRNA agent is administered
in an amount sufficient to reduce the level of BCR-ABL RNA in a
cell or tissue of the human.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority to U.S. application Ser.
No. 60/630,878, filed on Nov. 24, 2004, and to U.S. application
Ser. No. 60/632,403, filed on Dec. 1, 2004. The entire contents of
these provisional applications are hereby incorporated by reference
in the present application.
TECHNICAL FIELD
[0002] The invention relates to compositions and methods for
modulating the expression of Bcr-Abl, and more particularly to the
down-regulation of Bcr-Abl mRNA and Bcr-Abl protein levels by
oligonucleotides via RNA interference, e.g., chemically modified
oligonucleotides.
BACKGROUND
[0003] RNA interference or "RNAI" is a term initially coined by
Fire and co-workers to describe the observation that
double-stranded RNA (dsRNA) can block gene expression when it is
introduced into worms (Fire et al, Nature 391:806-811, 1998). Short
dsRNA directs gene-specific, post-transcriptional silencing in many
organisms, including vertebrates, and has provided a new tool for
studying gene function.
[0004] The discovery of the Philadelphia Chromosome (Ph)
represented the first consistent chromosomal abnormality causing a
specific human cancer (Nowell PC et al., 1960, Science 132:1467).
The Ph Chromosome is generated by a reciprocal translocation
between the long arms of Chromosome 9 and Chromosome 22 (Rowley JD,
1973, Nature, 243:290-293.). It occurs in almost all patients with
chronic myelogenous leukemia (CML), in about 10-20% of the adults
with acute lymphoblastic leukemia (ALL) (Westbrook CA et al., 1992
Blood, 80:2983) and about 2 % of patients with acute myelogenous
leukemia (AML). The t(9;22) translocation fuses the Bcr gene from
Chromosome 22 and the Abl gene from chromosome 9, resulting in the
oncogenic Bcr-Abl fusion-gene (Heisterkamp N et al., 1983, Nature,
306:239). Variable breakpoints within the Bcr gene on chromosome 22
lead to the formation of different Bcr-Abl fusion gene variants
which encode for different proteins p190.sup.Bcr-Abl (Mr 190,000),
p210.sup.Bcr-Abl (Mr 210,000) and p230.sup.Bcr-Abl (Mr 230,000). In
about 95% of the CML-patients the Bcr-Abl fusion transcripts e14a2
(former b3a2) and e13a2 (former b2a2) can be detected (reviewed in
Barnes et al., 2002, Acta Haematologica, 108:180-202). The
translated product is in each case a p210 kD Bcr-Abl protein. In
patients with Ph+ ALL a shorter transcript version called
Bcr-Abl-e1a2, predominates (reviewed in Faderl et al., 2003,
Cancer, 98:1337). Translation of this variant results in the
somewhat lighter p190.sup.Bcr-Abl protein. Both Bcr-Abl proteins
p190.sup.Bcr-Abl and p210.sup.Bcr-Abl are characterised by a
dramatically increased tyrosine-kinase activity, as compared to
that of normal Abl protein, leading to aberrant phosphorylation of
downstream target molecules.
[0005] The kinase activity of Bcr-Abl can be inhibited by a
specific tyrosine kinase inhibitor, Imatinib mesylate (ST1571,
Glivec), which is effective for treatment of Ph+ leukemia (reviewed
in Kurzrock et al., 2003, Ann. Intern. Med., 138:819).
Nevertheless, both ALL and advanced CML patients frequently develop
drug resistance after initial response predominantly caused by
genetic abnormalities such as point mutations in the Bcr-Abl kinase
domain or overexpression of Bcr-Abl (for review: Rothberg, 2003,
Leukemia Res., 27:977). Therefore the development of alternative
strategies to inhibit Bcr-Abl becomes increasingly important.
[0006] The breakpoint of the Bcr-Abl mRNA represents a unique and
leukemia-specific nucleotide sequence. Such fusion transcripts
encoding oncogenic proteins represent ideal targets for a
disease-specific RNAi approach. The possibility to use RNAi for the
specific degradation of the Bcr-Abl-e14a2 transcript variant, as
well as other oncogenic fusion proteins, has been demonstrated
recently (Wilda et al., Oncogene 2002, 21:5716; Scherr et al.,
Blood 2003, 101:1566; Heidenreich et al., Blood. 2003, 101:3157,
Wohlbold et al., Blood 2003, 102:2236; RitterU, et al.,
Oligonucleotides 2003, 13:365; Li et al., Oligonucleotides 2003,
13:401; Chen J, et al., J Clin Invest. 2004, 113:1784). The results
presented are inconclusive, as Wilda et al. did not observe a
sensitizing effect towards imatinib mesylate on Bcr-Abl-expressing
cells by treatment with Bcr-Abl-specific siRNAs, whereas others did
observe such effects. It was therefore unclear so far, whether the
expression of two relevant Bcr-Abl transcripts other than the e14a2
transcript variant (e13a2 and e1a2) can be downregulated by an RNAi
approach.
[0007] The present invention advances the art by providing methods
and medicaments encompassing short dsRNAs leading to the
down-regulation of p210.sup.Bcr-Abl and p190.sup.Bcr-Abl protein
levels in murine 32D cells expressing the respective Bcr-Abl gene
variants, in human leukemic MEG-01, K562 and SUP-B15 cells, and in
cells freshly isolated from human subjects suffering from leukemia.
These methods and medicaments may be used in research into, and in
the treatment of, certain cancers.
SUMMARY
[0008] The present invention is based on an investigation of the
Bcr-Abl fusion gene using iRNA agents and further testing of the
iRNA agents that target the fusion sites of Bcr-Abl breakpoint
variants. Based on these findings, the present invention provides
compositions and methods that are useful in reducing Bcr-Abl mRNA
levels, Bcr-Abl fusion protein levels and undesirable cell
proliferation in a subject, e.g., a mammal, such as a human.
[0009] The present invention specifically provides iRNA agents
consisting of or comprising at least 15 contiguous nucleotides of
one of the agents described in Table 1, agent numbers 1-6. The iRNA
agent preferably comprises less than 30 nucleotides per strand,
e.g., 21-23 nucleotides. The double stranded iRNA agent can either
have blunt ends or more preferably have overhangs of 1-4
nucleotides from one or both 3' ends of the agent.
[0010] Further, the iRNA agent can either contain only naturally
occurring ribonucleotide subunits, or can be synthesized so as to
contain one or more modifications to the sugar or base of one or
more of the ribonucleotide subunits that is included in the agent.
The iRNA agent can be further modified so at to be attached to a
ligand that is selected to improve stability, distribution or
cellular uptake of the agent, e.g. cholesterol. The agents can
further be in isolated form or can be part of a pharmaceutical
composition used for the methods described herein.
[0011] The present invention further provides methods for reducing
the level of Bcr-Abl fusion mRNA in a cell. The present methods
utilize the cellular mechanisms involved in RNA interference to
selectively degrade Bcr-Abl fusion mRNA in a cell and are comprised
of the step of contacting a cell with one of the iRNA agents of the
present invention. Such methods can be performed directly on a cell
or can be performed on a mammalian subject by administering to a
subject one of the iRNA agents of the present invention. Reduction
of Bcr-Abl fusion mRNA in a cell results in a reduction in the
amount of Bcr-Abl fusion protein produced, and in an organism, may
result in a decrease in undesirable cell proliferation, or it may
sensitize proliferating cells towards the activity of another
agent, e.g. a cytostatic or cytotoxic agent, e.g. imatinib mesylate
or gamma radiation.
[0012] The methods and compositions of the invention, e.g., the
methods and iRNA compositions can be used with any dosage and/or
formulation described herein, as well as with any route of
administration described herein.
[0013] The details of one or more embodiments of the invention are
set forth in the accompanying drawings and the description below.
Other features, objects, and advantages of the invention will be
apparent from this description, the drawings, and from the claims.
This application incorporates all cited references, patents, and
patent applications by references in their entirety for all
purposes.
BRIEF DESCRIPTION OF DRAWINGS
[0014] FIGS. 1A-1B:.about.2.5 Mio 32Dp210/e13a2 cells have been
electroporated 1-3 x at intervals of 24 h with the indicated siRNA.
BAF3/BAF15/BAF17/BAF19: bcr-abl-e13a2-specific siRNAs; BAF9:
bcr-abl-e14a2-specific siRNA, served as control; EPC:
electroporation control. FIG. 1A: Western blot analysis of
p210.sup.Bcr-abl (e13a2) in 32Dp210e13a2.about.24 h following siRNA
treatment; the level of GAPDH served as loading control. Lane 1:
EPC, Lane 2: BAF9, Lane 3: BAF3, Lane 4: BAF15, Lane 5: BAF17, Lane
6: BAF19. FIG. 1B: Prolonged treatment with the siRNAs BAF15 as
well as BAF19 led to a reduction of viability in 32Dp210/e13a2
cells..about.40 h following the indicated number of siRNA
treatments, viability of cells was determined by means of MTT.
Values are means+/-SD of triplicates.
[0015] FIGS. 2A-2B:.about.5 Mio 32Dp190/e1a2cells have been
electroporated twice at intervals of 24 h with the indicated siRNA.
BAF22/BAF24: bcr-abl-e1a2-specific siRNAs; BAF9:
bcr-abl-e14a2-specific siRNA, served as control; BAF19:
bcr-abl-e13a2-specific siRNA, served as control; EPC:
electroporation control. FIG. 2A: Western blot analysis of
p190.sup.Bcr-abl (e1a2) in 32Dp190/e1a2 cells about 24 h following
second siRNA treatment; the level of GAPDH served as loading
control. Lane 1: EPC, Lane 2: BAF9, Lane 3: BAF19, Lane 4: BAF22,
Lane 5: BAF24. FIG. 2B: repeated treatment with the siRNA BAF22 led
to a reduction of viability in 32Dp190/e1a2 cells..about.40 h
following the second siRNA treatment, viability of cells was
determined by means of MTT. Values are means +/-SD of
triplicates.
[0016] FIG. 3: Cells from the human B cell precursor leukemia cell
line SUP-B15 (ACC 389; DSMZ, Braunschweig, breakpoint variant e1a2)
were treated as described for FIG. 3 with an el a2-specific siRNA
(BAF22) or with siRNA directed to another breakpoint variant
(BAF19, specific for e13a2) as a control. BAF22 treatment at
intervals of 24 hours for 3 times led to significantly reduced
p190Bcr-Abl protein levels compared to the electroporation-control
(EPC) or to the BAF19 control.
[0017] FIG. 4: CD34 positive cells isolated from 3 newly diagnosed
and untreated Philadelphia chromosome-positive CML patients in
chronic phase and positive for bcr-abl-e14a2 by Ficoll-Hypaque
density gradient centrifugation and affinity column purification
were treated with siRNAs BAF7 (e14a2 specific, Patient 1), BAF8
(mismatch control, Patient 1), BAF12 (el4a2 specific, Patient 2
+3), and BAF16 (e13a2 specific, Patient 2 +3). Cells were diluted
to a density of 2.5 .times.10.sup.6 in 800 .mu.l growth medium,
mixed with 12.8 .mu.l of a 50 .mu.M solution of the respective
siRNA in a 4-mm electroporation cuvette, and electroporated using a
single pulse protocol (250V, 1800 .mu.F). This treatment was
repeated after 24 hours, the cells were washed, incubated for
another 24 hours, and harvested for western blot analysis. BAF7 or
BAF12 treatment resulted in a significant reduction of Bcr-Abl
protein levels compared to cells treated with the mismatch control
(BAF8) or with the siRNA homologous to e13a2 (BAF16). Additionally,
BAF12 treatment compromised Bcr-Abl activity. Phosphorylation of
CRKL, the direct downstream substrate of Bcr-Abl, was significantly
reduced in cells treated with BAF12.
DETAILED DESCRIPTION
[0018] For ease of exposition the term "nucleotide" or
"ribonucleotide" is sometimes used herein in reference to one or
more monomeric subunits of an RNA agent. It will be understood that
the usage of the term "ribonucleotide" or "nucleotide" herein can,
in the case of a modified RNA or nucleotide surrogate, also refer
to a modified nucleotide, or surrogate replacement moiety, as
further described below, at one or more positions.
[0019] An "RNA agent" as used herein, is an unmodified RNA,
modified RNA, or nucleoside surrogates, all of which are described
herein or are well known in the RNA synthetic art. While numerous
modified RNAs and nucleoside surrogates are described, preferred
examples include those which have greater resistance to nuclease
degradation than do unmodified RNAs. Preferred examples include
those that have a 2' sugar modification, a modification in a single
strand overhang, preferably a 3' single strand overhang, or,
particularly if single stranded, a 5'-modification which includes
one or more phosphate groups or one or more analogs of a phosphate
group.
[0020] An "iRNA agent" (abbreviation for "interfering RNA agent")
as used herein, is an RNA agent, which can down-regulate the
expression of a target gene, e.g., a Bcr-Abl fusion gene. While not
wishing to be bound by theory, an iRNA agent may act by one or more
of a number of mechanisms, including post-transcriptional cleavage
of a target mRNA sometimes referred to in the art as RNAi, or
pre-transcriptional or pre-translational mechanisms. An iRNA agent
can be a double stranded iRNA agent.
[0021] A "ds iRNA agent" (abbreviation for "double stranded iRNA
agent"), as used herein, is an iRNA agent which includes more than
one, and preferably two, strands in which interstrand hybridization
can form a region of duplex structure. A "strand" herein refers to
a contigouous sequence of nucleotides (including non-naturally
occurring or modified nucleotides). The two or more strands may be,
or each form a part of, separate molecules, or they may be
covalently interconnected, e.g. by a linker, e.g. a
polyethyleneglycol linker, to form but one molecule. At least one
strand can include a region which is sufficiently complementary to
a target RNA. Such strand is termed the "antisense strand". A
second strand comprised in the dsRNA agent which comprises a region
complementary to the antisense strand is termed the "sense strand".
However, a ds iRNA agent can also be formed from a single RNA
molecule which is, at least partly; self-complementary, forming,
e.g., a hairpin or panhandle structure, including a duplex region.
In such case, the term "strand" refers to one of the regions of the
RNA molecule that is complementary to another region of the same
RNA molecule.
[0022] Although, in mammalian cells, long ds iRNA agents can induce
the interferon response which is frequently deleterious, short ds
iRNA agents do not trigger the interferon response, at least not to
an extent that is deleterious to the cell and/or host (Manche, L.,
et al., Mol. Cell. Biol. 1992, 12:5238; Lee. SB, Esteban, M,
Virology 1994, 199:491; Castelli, JC, et al., J. Exp. Med. 1997,
186:967; Zheng, X., Bevilacqua, PC, RNA 2004, 10:1934; Heidel et
al., "Lack of interferon response in animals to naked siRNAs",
Nature Biotechn. advance online publication, Nov. 21, 2004,
doi:10.1038/nbt1038). The iRNA agents of the present invention
include molecules which are sufficiently short that they do not
trigger a deleterious non-specific interferon response in normal
mammalian cells. Thus, the administration of a composition of an
iRNA agent (e.g., formulated as described herein) to a subject can
be used to silence expression of the Bcr-Abl fusion gene in Bcr-Abl
expressing cells comprised in the subject, while circumventing an
interferon response, especially in other cells not expressing
Bcr-Abl. Molecules that are short enough that they do not trigger a
deleterious interferon response are termed siRNA agents or siRNAs
herein. "siRNA agent" or "siRNA" as used herein, refers to an iRNA
agent, e.g., a ds iRNA agent, that is sufficiently short that it
does not induce a deleterious interferon response in a human cell,
e.g., it has a duplexed region of less than 60 but preferably less
than 50, 40, or 30 nucleotide pairs.
[0023] The isolated iRNA agents described herein, including ds iRNA
agents and siRNA agents, can mediate silencing of an Bcr-Abl fusion
gene, e.g., by RNA degradation. For convenience, such RNA is also
referred to herein as the RNA to be silenced. Such a gene is also
referred to as a target gene. Preferably, the RNA to be silenced is
a gene product of an endogenous Bcr-Abl fusion gene.
[0024] As used herein, the phrase "mediates RNAi" refers to the
ability of an agent to silence, in a sequence specific manner, a
target gene. "Silencing a target gene" means the process whereby a
cell containing and/or secreting a certain product of the target
gene when not in contact with the agent, will contain and/or secret
at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% less of
such gene product when contacted with the agent, as compared to a
similar cell which has not been contacted with the agent. Such
product of the target gene can, for example, be a messenger RNA
(mRNA), a protein, or a regulatory element.
[0025] As used herein, the term "complementary" is used to indicate
a sufficient degree of complementarity such that stable and
specific binding occurs between a compound of the invention and a
target RNA molecule, e.g. an Bcr-Abl fusion mRNA molecule. Specific
binding requires a sufficient degree of complementarity to avoid
non-specific binding of the oligomeric compound to non-target
sequences under conditions in which specific binding is desired,
i.e., under physiological conditions in the case of in vivo assays
or therapeutic treatment, or in the case of in vitro assays, under
conditions in which the assays are performed. The non-target
sequences typically differ by at least 4 nucleotides.
[0026] As used herein, an iRNA agent is "sufficiently
complementary" to a target RNA, e.g., a target mRNA (e.g., a target
Bcr-Abl fusion mRNA) if the iRNA agent reduces the production of a
protein encoded by the target RNA in a cell. The iRNA agent may
also be "exactly complementary" to the target RNA, e.g., the target
RNA and the iRNA agent anneal, preferably to form a hybrid made
exclusively of Watson-Crick basepairs in the region of exact
complementarity. A "sufficiently complementary" iRNA agent can
include an internal region (e.g., of at least 10 nucleotides) that
is exactly complementary to a target Bcr-Abl fusion RNA. Moreover,
in some embodiments, the iRNA agent specifically discriminates a
single-nucleotide difference. In this case, the iRNA agent only
mediates RNAi if exact complementarity is found in the region
(e.g., within 7 nucleotides of) the single-nucleotide difference.
Preferred iRNA agents will be based on or consist or comprise the
sense and antisense sequences provided in Table 1, agent numbers
1-6.
[0027] As used herein, "essentially identical" when used referring
to a first nucleotide sequence in comparison to a second nucleotide
sequence means that the first nucleotide sequence is identical to
the second nucleotide sequence except for up to one, two or three
nucleotide substitutions (e.g. adenosine replaced by uracil).
"Essentially retaining the ability to inhibit Bcr-Abl fusion
expression in cultured human Bcr-Abl expressing cells", as used
herein referring to an iRNA agent not identical to but derived from
one of the iRNA agents of Table 1, agent numbers 1-6, by deletion,
addition or substitution of nucleotides, means that the derived
iRNA agent possesses an inhibitory activity lower by not more than
20% inhibition compared to the iRNA agent of Table 1, agent numbers
1-6, it was derived from. E.g. an iRNA agent derived from an iRNA
agent of Table 1, agent numbers 1-6, which lowers the amount of
Bcr-Abl fusion mRNA present in cultured human Bcr-Abl expressing
cells by 70% may itself lower the amount of Bcr-Abl fusion mRNA
present in cultured human Bcr-Abl expressing cells by at least 50%
in order to be considered as essentially retaining the ability to
inhibit Bcr-Abl fusion expression in cultured human Bcr-Abl
expressing cells. Optionally, an iRNA agent of the invention may
lower the amount of Bcr-Abl fusion mRNA present in cultured human
Bcr-Abl expressing cells by at least 50%.
[0028] As used herein, a "subject" refers to a mammalian organism
undergoing treatment for a disorder mediated by Bcr-Abl fusion
protein expression. The subject can be any mammal, such as a cow,
horse, mouse, rat, dog, pig, goat, or a primate. In the preferred
embodiment, the subject is a human.
[0029] As used herein, disorders associated with Bcr-Abl fusion
expression refers to any biological or pathological state that 1)
is mediated in part by the presence of Bcr-Abl fusion protein and
2) whose outcome can be affected by reducing the level of Bcr-Abl
fusion protein present. Specific disorder associated with Bcr-Abl
fusion expression are noted below.
[0030] 1 Design and Selection of iRNA agents TABLE-US-00001 TABLE 1
Exemplary iRNA agents to target Bcr-Abl fusion mRNA SEQ. SEQ.
Specific for ID Sequence ID Sequence Duplex Bcr-Abl fusion Agent
No. sense strand.sup.a,b No. antisense strand.sup.a,b descriptor
gene variant number 1 agaguucaa|aagcccuucag 2
cgucucaaguu|uucgggaaguc BAF7 Bcr-Abl-e14a2 1 5
gaguucaa|aagcccuucagc 6 gucucaaguu|uucgggaagucg BAF9 Bcr-Abl-e14a2
2 11 auaaggaag|aagcccuucag 12 guuauuccuuc|uucgggaaguc BAF15
Bcr-Abl-e13a2 3 13 gaag|aagcccuucagcggcc 14 uccuuc|uucgggaagucgccgg
BAF17 Bcr-Abl-e13a2 4 15 ucaauaaggaag|aagcccuu 16
guaguuauuccuuc|uucgggaa BAF19 Bcr-Abl-e13a2 5 19
gagacgcag|aagcccuucag 20 accucugcguc|uucgggaaguc BAF22 Bcr-Abl-e1a2
6 .sup.aSee Table 2 for an explanation of nucleotide representation
(e.g., lower case letters, bold and italicized letters).
.sup.bexact Bcr-Abl fusion site is marked by hyphen;
[0031] The present invention is based on the demonstration of
silencing of an Bcr-Abl fusion gene in vitro in cultured cells
after incubation with an iRNA agent, and the resulting reduction in
cell prolifearation.
[0032] An iRNA agent can be rationally designed based on sequence
information and desired characteristics. For example, an iRNA agent
can be designed according to the relative melting temperature of
the candidate duplex. Generally, the duplex should have a lower
melting temperature at the 5' end of the antisense strand than at
the 3' end of the antisense strand.
[0033] Candidate iRNA agents can also be designed by performing,
for example, a gene walk analysis of the genes that will serve as
the target gene. Overlapping, adjacent, or closely spaced candidate
agents corresponding to all or some of the transcribed region can
be generated and tested. Each of the iRNA agents can be tested and
evaluated for the ability to down regulate the target gene
expression (see below, "Evaluation of Candidate iRNA agents").
[0034] Herein, potential iRNA agents targeting the Bcr-Abl fusion
variants Bcr-Abl-e14a2, Bcr-Abl-e13a2, and Bcr-Abl-e1a2 were
designed using the known sequences of the respective fusion sites
(Barnes et al., 2002, Acta Haematologica, 108:180-202; Faderl et
al., 2003, Cancer, 98:1337). Based on the results provided, the
present invention provides iRNA agents that silence these Bcr-Abl
fusion gene breakpoint variants.
[0035] Table 1 provides active iRNA agents targeting Bcr-Abl
fusion, specifically agent numbers 1-6,. As shown in the Examples
below, the iRNA agents of Table 1, agent numbers 1-6, possess the
advantageous and surprising ability to reduce the amount of Bcr-Abl
fusion mRNA present in cultured human Bcr-Abl expressing cells
after incubation with these agents by more than 50 % (and with some
agents, more than 80%) compared to cells which have not been
incubated with the agent, and/or to reduce the amount of Bcr-Abl
fusion protein secreted into cell culture supernatant by cultured
human Bcr-Abl expressing cells by more than 50 %.
[0036] Based on these results, the invention specifically provides
an iRNA agent that includes a sense strand having at least 15
contiguous nucleotides of the sense strand sequences of the agents
provided in Table 1, agent numbers 1-6, and an antisense strand
having at least 15 contiguous nucleotides of the antisense
sequences of the agents provided in Table 1, agent numbers 1-6.
[0037] The iRNA agents shown in Table 1 are composed of a sense
strand of 21 nucleotides in length, and an antisense strand of 23
nucleotides in length and the present invention provides agents
that comprise 15 contiguous nucleotides from these agents. However,
while these lengths may potentially be optimal, the iRNA agents are
not meant to be limited to these lengths. The skilled person is
well aware that shorter or longer iRNA agents may be similarly
effective, since, within certain length ranges, the efficacy is
rather a function of the nucleotide sequence than strand length.
For example, Yang, D., et al., PNAS 2002, 99:9942-9947,
demonstrated similar efficacies for iRNA agents of lengths between
21 and 30 base pairs. Others have shown effective silencing of
genes by iRNA agents down to a length of approx. 15 base pairs
(Byrom, W.M., et al., Inducing RNAi with siRNA Cocktails Generated
by RNase III; Tech Notes 10(1), Ambion, Inc., Austin, Tex.,
USA).
[0038] Therefore, it is possible and contemplated by the instant
invention to select from the sequences provided in Table 1, agent
numbers 1-6, a partial sequence of between 15 to 22 nucleotides for
the generation of an iRNA agent derived from one of the sequences
provided in Table 1, agent numbers 1-6,. Alternatively, one may add
one or several nucleotides to one of the sequences provided in
Table 1, agent numbers 1-6, or an agent comprising 15 contiguous
nucleotides from one of these agents, preferably, but not
necessarily, in such a fashion that the added nucleotides are
complementary to the respective sequence of the target gene, e.g.
Bcr-Abl fusion. For example, the first 15 nucleotides from one of
the agents can be combined with the 8 nucleotides found 5' to these
sequence in the Bcr-Abl fusion mRNA to obtain an agent with 23
nucleotides in the sense and antisense strands. All such derived
iRNA agents are included in the iRNA agents of the present
invention, provided they essentially retain the ability to inhibit
Bcr-Abl fusion expression in cultured human Bcr-Abl expressing
cells.
[0039] The antisense strand of an iRNA agent should be equal to or
at least, 14, 15, 16 17, 18, 19, 25, 29, 40, or 50 nucleotides in
length. It should be equal to or less than 60, 50, 40, or 30,
nucleotides in length. Preferred ranges are 15-30, 17 to 25, 19 to
23, and 19 to 21 nucleotides in length.
[0040] The sense strand of an iRNA agent should be equal to or at
least 14, 15, 16 17, 18, 19, 25, 29, 40, or 50 nucleotides in
length. It should be equal to or less than 60, 50, 40, or 30
nucleotides in length. Preferred ranges are 15-30, 17 to 25, 19 to
23, and 19 to 21 nucleotides in length.
[0041] The double stranded portion of an iRNA agent should be equal
to or at least, 15, 16 17, 18, 19, 20, 21, 22, 23, 24, 25, 29, 40,
or 50 nucleotide pairs in length. It should be equal to or less
than 60, 50, 40, or 30 nucleotides pairs in length. Preferred
ranges are 15-30, 17. to 25, 19 to 23, and 19 to 21 nucleotides
pairs in length.
[0042] Generally, the iRNA agents of the instant invention include
a region of sufficient complementarity to the respective Bcr-Abl
fusion gene, and are of sufficient length in terms of nucleotides,
that the iRNA agent, or a fragment thereof, can mediate down
regulation of the Bcr-Abl fusion gene. The antisense strands of the
iRNA agents of Table 1, agent numbers 1-6, are fully complementary
to the mRNA sequences of the respective Bcr-Abl fusion gene, and
their sense strands are fully complementary to the antisense
strands except for the two 3'-terminal nucleotides on the antisense
strand. However, it is not necessary that there be perfect
complementarity between the iRNA agent and the target, but the
correspondence must be sufficient to enable the iRNA agent, or a
cleavage product thereof, to direct sequence specific silencing,
e.g., by RNAi cleavage of an Bcr-Abl fusion mRNA.
[0043] Therefore, the iRNA agents of the instant invention include
agents comprising a sense strand and antisense strand each
comprising a sequence of at least 16, 17 or 18 nucleotides which is
essentially identical, as defined below, to one of the sequences of
Table 1, agent numbers 1-6, except that not more than 1, 2 or 3
nucleotides per strand, respectively, have been substituted by
other nucleotides (e.g. adenosine replaced by uracil), while
essentially retaining the ability to inhibit Bcr-Abl expression in
cultured human Bcr-Abl expressing cells. These agents will
therefore possess at least 15 nucleotides identical to one of the
sequences of Table 1, agent numbers 1-6, but 1, 2 or 3 base
mismatches with respect to either the target Bcr-Abl fusion mRNA
sequence or between the sense and antisense strand are introduced.
Mismatches to the target Bcr-Abl fusion mRNA sequence, particularly
in the antisense strand, are most tolerated in the terminal regions
and if present are preferably in a terminal region or regions,
e.g., within 6, 5, 4, or 3 nucleotides of a 5' and/or 3' terminus,
most preferably within 6, 5, 4, or 3 nucleotides of the 5'-terninus
of the sense strand or the 3'-terminus of the antisense strand. The
sense strand need only be sufficiently complementary with the
antisense strand to maintain the overall double stranded character
of the molecule.
[0044] It is preferred that the sense and antisense strands be
chosen such that the iRNA agent includes a single strand or
unpaired region at one or both ends of the molecule. Thus, an iRNA
agent contains sense and antisense strands, preferably paired to
contain an overhang, e.g., one or two 5' or 3' overhangs but
preferably a 3' overhang of 2-3 nucleotides. Most embodiments will
have a 3' overhang. Preferred siRNA agents will have
single-stranded overhangs, preferably 3' overhangs, of 1 to 4, or
preferably 2 or 3 nucleotides, in length, at one or both ends of
the iRNA agent. The overhangs can be the result of one strand being
longer than the other, or the result of two strands of the same
length being staggered. 5'-ends are preferably phosphorylated.
[0045] Preferred lengths for the duplexed region is between 15 and
30, most preferably 18, 19, 20, 21, 22, and 23 nucleotides in
length, e.g., in the siRNA agent range discussed above. siRNA
agents can resemble in length and structure the natural Dicer
processed products from long dsRNAs. Embodiments in which the two
strands of the siRNA agent are linked, e.g., covalently linked, are
also included. Hairpin, or other single strand structures which
provide the required double stranded region, and preferably a 3'
overhang are also within the invention.
[0046] 2 Evaluation of Candidate iRNA Agents
[0047] A candidate iRNA agent can be evaluated for its ability to
downregulate target gene expression. For example, a candidate iRNA
agent can be provided, and contacted with a cell, that expresses
the target gene, e.g., the Bcr-Abl fusion gene, either endogenously
or because it has been transfected with a construct from which a
Bcr-Abl fusion protein can be expressed. The level of target gene
expression prior to and following contact with the candidate iRNA
agent can be compared, e.g. on an mRNA or protein level. If it is
determined that the amount of RNA or protein expressed from the
target gene is lower following contact with the iRNA agent, then it
can be concluded that the iRNA agent downregulates target gene
expression. The level of target Bcr-Abl fusion RNA or Bcr-Abl
fusion protein in the cell can be determined by any method desired.
For example, the level of target RNA can be determined by Northern
blot analysis, reverse transcription coupled with polymerase chain
reaction (RT-PCR), or RNAse protection assay. The level of protein
can be determined, for example, by Western blot analysis or
immuno-fluorescence.
[0048] Stability Testing Modification and Retesting of iRNA
Agents
[0049] A candidate iRNA agent can be evaluated with respect to
stability, e.g., its susceptibility to cleavage by an endonuclease
or exonuclease, such as when the iRNA agent is introduced into the
body of a subject. Methods can be employed to identify sites that
are susceptible to modification, particularly cleavage, e.g.,
cleavage by a component found in the body of a subject.
[0050] When sites susceptible to cleavage are identified, a further
iRNA agent can be designed and/or synthesized wherein the potential
cleavage site is made resistant to cleavage, e.g. by introduction
of a 2'-modification on the site of cleavage, e.g. a 2'-O-methyl
group. This further iRNA agent can be retested for stability, and
this process may be iterated until an iRNA agent is found
exhibiting the desired stability.
[0051] In Vivo Testing
[0052] An iRNA agent identified as being capable of inhibiting
Bcr-Abl fusion gene expression can be tested for functionality in
vivo in an animal model (e.g., in a mammal, such as in mouse or
rat). For example, the iRNA agent can be administered to an animal,
and the iRNA agent evaluated with respect to its biodistribution,
stability, and its ability to inhibit a Bcr-Abl fusion gene
expression or reduce undesirable cell proliferation.
[0053] The iRNA agent can be administered directly to the target
tissue, such as by injection, or the iRNA agent can be administered
to the animal model in the same manner that it would be
administered to a human.
[0054] The iRNA agent can also be evaluated for its intracellular
distribution. The evaluation can include determining whether the
iRNA agent was taken up into the cell. The evaluation can also
include determining the stability (e.g., the half-life) of the iRNA
agent. Evaluation of an iRNA agent in vivo can be facilitated by
use of an iRNA agent conjugated to a traceable marker (e.g., a
fluorescent marker such as fluorescein; a radioactive label, such
as .sup.35S, .sup.32P, .sup.33P, or .sup.3H; gold particles; or
antigen particles for immunohistochemistry).
[0055] The iRNA agent can be evaluated with respect to its ability
to down regulate Bcr-Abl fusion gene expression. Levels of Bcr-Abl
fusion gene expression in vivo can be measured, for example, by in
situ hybridization, or by the isolation of RNA from tissue prior to
and following exposure to the iRNA agent. Where the animal needs to
be sacrificed in order to harvest the tissue, an untreated control
animal will serve for comparison. Target Bcr-Abl fusion mRNA can be
detected by any desired method, including but not limited to
RT-PCR, Northern blot, branched-DNA assay, or RNAase protection
assay. Alternatively, or additionally, Bcr-Abl fusion gene
expression can be monitored by performing Western blot analysis on
tissue extracts treated with the iRNA agent.
[0056] Animal models may be used to establish the concentration
necessary to achieve a certain desired effect (e.g., EC50). Such
animal models may include transgenic animals that express a human
gene, e.g., a gene that produces a target human Bcr-Abl fusion RNA.
In another embodiment, the composition for testing includes an iRNA
agent that is complementary, at least in an internal region, to a
sequence that is conserved between the target Bcr-Abl fusion RNA in
the animal model and the target Bcr-Abl fusion RNA in a human.
[0057] 3 iRNA Chemistry
[0058] Described herein are isolated iRNA agents, e.g., ds RNA
agents that mediate RNAi to inhibit expression of an Bcr-Abl fusion
gene.
[0059] RNA agents discussed herein include otherwise unmodified RNA
as well as RNA which have been modified, e.g., to improve efficacy,
and polymers of nucleoside surrogates. Unmodified RNA refers to a
molecule in which the components of the nucleic acid, namely
sugars, bases, and phosphate moieties, are the same or essentially
the same as that which occur in nature, preferably as occur
naturally in the human body. The art has referred to rare or
unusual, but naturally occurring, RNAs as modified RNAs, see, e.g.,
Limbach et al., (1994) Nucleic Acids Res. 22: 2183-2196. Such rare
or unusual RNAs, often termed modified RNAs (apparently because the
are typically the result of a post-transcriptional modification)
are within the term unmodified RNA, as used herein. Modified RNA as
used herein refers to a molecule in which one or more of the
components of the nucleic acid, namely sugars, bases, and phosphate
moieties, are different from that which occur in nature, preferably
different from that which occurs in the human body. While they are
referred to as modified "RNAs," they will of course, because of the
modification, include molecules which are not RNAs. Nucleoside
surrogates are molecules in which the ribophosphate backbone is
replaced with a non-ribophosphate construct that allows the bases
to the presented in the correct spatial relationship such that
hybridization is substantially similar to what is seen with a
ribophosphate backbone, e.g., non-charged mimics of the
ribophosphate backbone. Examples of all of the above are discussed
herein.
[0060] Modifications described herein can be incorporated into any
double-stranded RNA and RNA-like molecule described herein, e.g.,
an iRNA agent. It may be desirable to modify one or both of the
antisense and sense strands of an iRNA agent. As nucleic acids are
polymers of subunits or monomers, many of the modifications
described below occur at a position which is repeated within a
nucleic acid, e.g., a modification of a base, or a phosphate
moiety, or the non-linking 0 of a phosphate moiety. In some cases
the modification will occur at all of the subject positions in the
nucleic acid but in many, and in fact in most, cases it will not.
By way of example, a modification may only occur at a 3' or 5'
terminal position, may only occur in a terminal region, e.g. at a
position on a terminal nucleotide or in the last 2, 3, 4, 5, or 10
nucleotides of a strand. A modification may occur in a double
strand region, a single strand region, or in both. E.g., a
phosphorothioate modification at a non-linking O position may only
occur at one or both termini, may only occur in a terminal regions,
e.g., at a position on a terminal nucleotide or in the last 2, 3,
4, 5, or 10 nucleotides of a strand, or may occur in double strand
and single strand regions, particularly at termini. Similarly, a
modification may occur on the sense strand, antisense strand, or
both. In some cases, the sense and antisense strand will have the
same modifications or the same class of modifications, but in other
cases the sense and antisense strand will have different
modifications, e.g., in some cases it may be desirable to modify
only one strand, e.g. the sense strand.
[0061] Two prime objectives for the introduction of modifications
into iRNA agents is their stabilization towards degradation in
biological environments and the improvement of pharmacological
properties, e.g. pharmacodynamic properties, which are further
discussed below. Other suitable modifications to a sugar, base, or
backbone of an iRNA agent are described in co-owned PCT Application
No. PCT/US2004/01193, filed Jan. 16, 2004. An iRNA agent can
include a non-naturally occurring base, such as the bases described
in co-owned PCT Application No. PCT/US2004/011822, filed Apr. 16,
2004. An iRNA agent can include a non-naturally occurring sugar,
such as a non-carbohydrate cyclic carrier molecule. Exemplary
features of non-naturally occurring sugars for use in iRNA agents
are described in co-owned PCT Application No. PCT/US2004/11829
filed Apr. 16, 2003.
[0062] An iRNA agent can include an internucleotide linkage (e.g.,
the chiral phosphorothioate linkage) useful for increasing nuclease
resistance. In addition, or in the alternative, an iRNA agent can
include a ribose mimic for increased nuclease resistance. Exemplary
internucleotide linkages and ribose mimics for increased nuclease
resistance are described in co-owned PCT Application No.
PCT/US2004/07070 filed on Mar. 8, 2004.
[0063] An iRNA agent can include ligand-conjugated monomer subunits
and monomers for oligonucleotide synthesis. Exemplary monomers are
described in co-owned U.S. application Ser. No. 10/916,185, filed
on Aug. 10, 2004.
[0064] An iRNA agent can have a ZXY structure, such as is described
in co-owned PCT Application No. PCT/US2004/07070 filed on Mar. 8,
2004.
[0065] An iRNA agent can be complexed with an amphipathic moiety.
Exemplary amphipathic moieties for use with iRNA agents are
described in co-owned PCT Application No. PCT/US2004/07070 filed on
Mar. 8, 2004.
[0066] In another embodiment, the iRNA agent can be complexed to a
delivery agent that features a modular complex. The complex can
include a carrier agent linked to one or more of (preferably two or
more, more preferably all three of): (a) a condensing agent (e.g.,
an agent capable of attracting, e.g., binding, a nucleic acid,
e.g., through ionic or electrostatic interactions); (b) a fusogenic
agent (e.g., an agent capable of fusing and/or being transported
through a cell membrane); and (c) a targeting group, e.g., a cell
or tissue targeting agent, e.g., a lectin, glycoprotein, lipid or
protein, e.g., an antibody, that binds to a specified cell type.
iRNA agents complexed to a delivery agent are described in co-owned
PCT Application No. PCT/US2004/07070 filed on Mar. 8, 2004.
[0067] An iRNA agent can have non-canonical pairings, such as
between the sense and antisense sequences of the iRNA duplex.
Exemplary features of non-canonical iRNA agents are described in
co-owned PCT Application No. PCT/US2004/07070 filed on Mar. 8,
2004.
[0068] Enhanced Nuclease Resistance
[0069] An iRNA agent, e.g., an iRNA agent that targets Bcr-Abl
fusion, can have enhanced resistance to nucleases.
[0070] One way to increase resistance is to identify cleavage sites
and modify such sites to inhibit cleavage, as described in co-owned
and co-pending applications U.S. Ser. No. 60/574,744 and
PCT/US2005/018931. For example, the dinucleotides 5'-ua-3',
5'-ca-3', 5'-ug-3', 5'-uu-3', or 5'-cc-3' can serve as cleavage
sites. In certain embodiments, all the pyrimidines of an iRNA agent
carry a 2'-modification in either the sense strand, the antisense
strand, or both strands, and the iRNA agent therefore has enhanced
resistance to endonucleases. Enhanced nuclease resistance can also
be achieved by modifying the 5' nucleotide, resulting, for example,
in at least one 5'-uridine-adenine-3' (5'-ua-3') dinucleotide
wherein the uridine is a 2'-modified nucleotide; at least one
5'-cytidine-adenine-3' (5'-ca-3') dinucleotide, wherein the
5'-cytidine is a 2'-modified nucleotide; at least one
5'-uridine-guanine-3' (5'-ug-3') dinucleotide, wherein the
5'-uridine is a 2'-modified nucleotide; at least one
5'-uridine-uridine-3' (5'-uu-3') dinucleotide, wherein the
5'-uridine is a 2'-modified nucleotide; or at least one
5'-cytidine-cytidine-3' (5'-cc-3') dinucleotide, wherein the
5'-cytidine is a 2'-modified nucleotide, as described in co-owned
International Application No. PCT/US2005/018931, filed on May 27,
2005. The iRNA agent can include at least 2, at least 3, at least 4
or at least 5 of such dinucleotides. In a particularly preferred
embodiment, the 5' nucleotide in all occurrences of the sequence
motifs 5'-ua-3' and 5'-ca-3' in either the sense strand, the
antisense strand, or both strands is a modified nucleotide.
Preferably, the 5' nucleotide in all occurrences of the sequence
motifs 5'-ua-3', 5'-ca-3' and 5'-ug-3' in either the sense strand,
the antisense strand, or both strands is a modified nucleotide.
More preferably, all pyrimidine nucleotides in the sense strand are
modified nucleotides, and the 5' nucleotide in all occurrences of
the sequence motifs 5'-ua-3' and 5'-ca-3' in the antisense strand
are modified nucleotides, or where the antisense strand does
comprise neither of a 5'-ua-3' and a 5'-ca-3' motif, in all
occurrences of the sequence motif 5'-ug-3'.
[0071] For increased nuclease resistance and/or binding affinity to
the target, an iRNA agent, e.g., the sense and/or antisense strands
of the iRNA agent, can include, for example, 2'-modified ribose
units and/or phosphorothioate linkages. E.g., the 2' hydroxyl group
(OH) can be modified or replaced with a number of different "oxy"
or "deoxy" substituents.
[0072] Examples of "oxy"-2' hydroxyl group modifications include
alkoxy or aryloxy (OR, e.g., R =H, alkyl, cycloalkyl, aryl,
aralkyl, heteroaryl or sugar); polyethyleneglycols (PEG),
O(CH.sub.2CH.sub.2O).sub.nCH.sub.2CH.sub.2OR; "locked" nucleic
acids (LNA) in which the 2' hydroxyl is connected, e.g., by a
methylene bridge, to the 4' carbon of the same ribose sugar;
O-AMINE and aminoalkoxy, O(CH.sub.2).sub.nAMINE, (e.g.,
AMINE=NH.sub.2; alkylamino, dialkylamino, heterocyclyl amino,
arylamino, diaryl amino, heteroaryl amino, or diheteroaryl amino,
ethylene diamine, polyamino). It is noteworthy that
oligonucleotides containing only the methoxyethyl group (MOE),
(OCH.sub.2CH.sub.2OCH.sub.3, a PEG derivative), exhibit nuclease
stabilities comparable to those modified with the robust
phosphorothioate modification.
[0073] "Deoxy" modifications include hydrogen (i.e. deoxyribose
sugars, which are of particular relevance to the overhang portions
of partially ds RNA); halo (e.g., fluoro); amino (e.g. NH.sub.2;
alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino,
heteroaryl amino, diheteroaryl amino, or amino acid);
NH(CH.sub.2CH.sub.2NH).sub.nCH.sub.2CH.sub.2-AMINE (AMINE=NH.sub.2;
alkylamino, dialkylamino, heterocyclyl amino, arylamino, diaryl
amino, heteroaryl amino,or diheteroaryl amino), --NHC(O)R (R=alkyl,
cycloalkyl, aryl, aralkyl, heteroaryl or sugar), cyano; mercapto;
alkyl-thio-alkyl; thioalkoxy; and alkyl, cycloalkyl, aryl, alkenyl
and alkynyl, which may be optionally substituted with e.g., an
amino functionality.
[0074] Preferred substitutents are 2'-methoxyethyl, 2'-OCH.sub.3,
2'-O-allyl, 2'-C- allyl, and 2'-fluoro.
[0075] To maximize nuclease resistance, the 2' modifications can be
used in combination with one or more phosphate linker modifications
(e.g., phosphorothioate). The so-called "chimeric" oligonucleotides
are those that contain two or more different modifications.
[0076] The inclusion of furanose sugars in the oligonucleotide
backbone can also decrease endonucleolytic cleavage. An iRNA agent
can be further modified by including a 3' cationic group, or by
inverting the nucleoside at the 3'-terminus with a 3'-3' linkage.
In another alternative, the 3'-terminus can be blocked with an
aminoalkyl group, e.g., a 3.degree. C5-aminoalkyl dT. Other 3'
conjugates can inhibit 3'-5' exonucleolytic cleavage. While not
being bound by theory, a 3' conjugate, such as naproxen or
ibuprofen, may inhibit exonucleolytic cleavage by sterically
blocking the exonuclease from binding to the 3'-end of
oligonucleotide. Even small alkyl chains, aryl groups, or
heterocyclic conjugates or modified sugars (D-ribose, deoxyribose,
glucose etc.) can block 3'-5'-exonucleases.
[0077] Similarly, 5' conjugates can inhibit 5'-3' exonucleolytic
cleavage. While not being bound by theory, a 5' conjugate, such as
naproxen or ibuprofen, may inhibit exonucleolytic cleavage by
sterically blocking the exonuclease from binding to the 5'-end of
oligonucleotide. Even small alkyl chains, aryl groups, or
heterocyclic conjugates or modified sugars (D-ribose, deoxyribose,
glucose etc.) can block 3'-5'-exonucleases.
[0078] An iRNA agent can have increased resistance to nucleases
when a duplexed iRNA agent includes a single-stranded nucleotide
overhang on at least one end. In preferred embodiments, the
nucleotide overhang includes 1 to 4, preferably 2 to 3, unpaired
nucleotides. In a preferred embodiment, the unpaired nucleotide of
the single-stranded overhang that is directly adjacent to the
terminal nucleotide pair contains a purine base, and the terminal
nucleotide pair is a G-C pair, or at least two of the last four
complementary nucleotide pairs are G-C pairs. In further
embodiments, the nucleotide overhang may have 1 or 2 unpaired
nucleotides, and in an exemplary embodiment the nucleotide overhang
is 5'-GC-3'. In preferred embodiments, the nucleotide overhang is
on the 3'-end of the antisense strand. In one embodiment, the iRNA
agent includes the motif 5'-CGC-3' on the 3'-end of the antisense
strand, such that a 2-nt overhang 5'-GC-3' is formed.
[0079] Thus, an iRNA agent can include monomers which have been
modified so as to inhibit degradation, e.g., by nucleases, e.g.,
endonucleases or exonucleases, found in the body of a subject.
These monomers are referred to herein as NRMs, or Nuclease
Resistance promoting Monomers, the corresponding modifications as
NRM modifications. In many cases these modifications will modulate
other properties of the iRNA agent as well, e.g., the ability to
interact with a protein, e.g., a transport protein, e.g., serum
albumin, or a member of the RISC, or the ability of the first and
second sequences to form a duplex with one another or to form a
duplex with another sequence, e.g., a target molecule.
[0080] Modifications that can be useful for producing iRNA agents
that meet the preferred nuclease resistance criteria delineated
above can include one or more of the following chemical and/or
stereochemical modifications of the sugar, base, and/or phosphate
backbone:
[0081] (i) chiral (S.sub.p) thioates. Thus, preferred NRMs include
nucleotide dimers enriched or pure for a particular chiral form of
a modified phosphate group containing a heteroatom at the
nonbridging position, e.g., Sp or Rp, at the position X, where this
is the position normally occupied by the oxygen. The atom at X can
also be S, Se, Nr.sub.2, or Br.sub.3. When X is S, enriched or
chirally pure Sp linkage is preferred. Enriched means at least 70,
80, 90, 95, or 99% of the preferred form.;
[0082] (ii) attachment of one or more cationic groups to the sugar,
base, and/or the phosphorus atom of a phosphate or modified
phosphate backbone moiety. Thus, preferred NRMs include monomers at
the terminal position derivatized at a cationic group. As the
5'-end of an antisense sequence should have a terminal --OH or
phosphate group this NRM is preferably not used at the 5'-end of an
antisense sequence. The group should be attached at a position on
the base which minimizes interference with H-bond formation and
hybridization, e.g., away from the face which interacts with the
complementary base on the other strand, e.g, at the 5' position of
a pyrimidine or a 7-position of a purine.;
[0083] (iii) nonphosphate linkages at the termini. Thus, preferred
NRMs include non-phosphate linkages, e.g., a linkage of 4 atoms
which confers greater resistance to cleavage than does a phosphate
bond. Examples include 3' CH.sub.2-NCH.sub.3-O-CH.sub.2-5' and 3'
CH.sub.2-NH-(O=)-CH.sub.2-5'.;
[0084] (iv) 3'-bridging thiophosphates and 5'-bridging
thiophosphates. Thus, preferred NRM's can include these
structures;
[0085] (v) L-RNA, 2'-5' linkages, inverted linkages, a-nucleosides.
Thus, other preferred NRM's include: L nucleosides and dimeric
nucleotides derived from L-nucleosides; 2'-5' phosphate,
non-phosphate and modified phosphate linkages (e.g.,
thiophosphates, phosphoramidates and boronophosphates); dimers
having inverted linkages, e.g., 3'-3' or 5'-5' linkages; monomers
having an alpha linkage at the 1' site on the sugar, e.g., the
structures described herein having an alpha linkage;
[0086] (vi) conjugate groups. Thus, preferred NRM's can include,
e.g., a targeting moiety or a conjugated ligand described herein
conjugated with the monomer, e.g., through the sugar, base, or
backbone. These are discussed in more detail below;
[0087] (vi) abasic linkages. Thus, preferred NRM's can include an
abasic monomer, e.g., an abasic monomer (e.g., a nucleobaseless
monomer); an aromatic or heterocyclic or polyheterocyclic aromatic
monomer; and
[0088] (vii) 5'-phosphonates and 5'-phosphate prodrugs. Thus,
preferred NRM's include monomers, preferably at the terminal
position, e.g., the 5' position, in which one or more atoms of the
phosphate group is derivatized with a protecting group, which
protecting group or groups, are removed as a result of the action
of a component in the subject's body, e.g, a carboxyesterase or an
enzyme present in the subject's body. E.g., a phosphate prodrug in
which a carboxy esterase cleaves the protected molecule resulting
in the production of a thioate anion which attacks a carbon
adjacent to the O of a phosphate and resulting in the production of
an unprotected phosphate.
[0089] One or more different NRM modifications can be introduced
into an iRNA agent or into a sequence of an iRNA agent. An NRM
modification can be used more than once in a sequence or in an iRNA
agent.
[0090] NRM modifications include some which can be placed only at
the terminus and others which can go at any position. Some NRM
modifications can inhibit hybridization so it is preferable to use
them only in terminal regions, and preferable to not use them at
the cleavage site or in the cleavage region of a sequence which
targets a subject sequence or gene, particularly on the antisense
strand. They can be used anywhere in a sense strand, provided that
sufficient hybridization between the two strands of the ds iRNA
agent is maintained. In some embodiments it is desirable to put the
NRM at the cleavage site or in the cleavage region of a sense
strand, as it can minimize off-target silencing.
[0091] In most cases, NRM modifications will be distributed
differently depending on whether they are comprised on a sense or
antisense strand. If on an antisense strand, modifications which
interfere with or inhibit endonuclease cleavage should not be
inserted in the region which is subject to RISC mediated cleavage,
e.g., the cleavage site or the cleavage region (As described in
Elbashir et al., 2001, Genes and Dev. 15: 188, hereby incorporated
by reference). Cleavage of the target occurs about in the middle of
a 20 or 21 nt antisense strand, or about 10 or 11 nucleotides
upstream of the first nucleotide on the target mRNA which is
complementary to the antisense strand. As used herein cleavage site
refers to the nucleotides on either side of the cleavage site, on
the target or on the iRNA agent strand which hybridizes to it.
Cleavage region means the nucleotides within 1, 2, or 3 nucleotides
of the cleavagee site, in either direction.
[0092] Such modifications can be introduced into the terminal
regions, e.g., at the terminal position or with 2, 3, 4, or 5
positions of the terminus, of a sense or antisense strand.
[0093] Tethered Ligands
[0094] The properties of an iRNA agent, including its
pharmacological properties, can be influenced and tailored, for
example, by the introduction of ligands, e.g. tethered ligands.
[0095] A wide variety of entities, e.g., ligands, can be tethered
to an iRNA agent, e.g., to the carrier of a ligand-conjugated
monomer subunit. Examples are described below in the context of a
ligand-conjugated monomer subunit but that is only preferred,
entities can be coupled at other points to an iRNA agent.
[0096] Preferred moieties are ligands, which are coupled,
preferably covalently, either directly or indirectly via an
intervening tether, to the carrier. In preferred embodiments, the
ligand is attached to the carrier via an intervening tether. The
ligand or tethered ligand may be present on the ligand-conjugated
monomer when the ligand-conjugated monomer is incorporated into the
growing strand. In some embodiments, the ligand may be incorporated
into a "precursor" ligand-conjugated monomer subunit after a
"precursor" ligand-conjugated monomer subunit has been incorporated
into the growing strand. For example, a monomer having, e.g., an
amino-terminated tether, e.g., TAP-(CH.sub.2).sub.nNH.sub.2 may be
incorporated into a growing sense or antisense strand. In a
subsequent operation, i.e., after incorporation of the precursor
monomer subunit into the strand, a ligand having an electrophilic
group, e.g., a pentafluorophenyl ester or aldehyde group, can
subsequently be attached to the precursor ligand-conjugated monomer
by coupling the electrophilic group of the ligand with the terminal
nucleophilic group of the precursor ligand-conjugated monomer
subunit tether.
[0097] In preferred embodiments, a ligand alters the distribution,
targeting or lifetime of an iRNA agent into which it is
incorporated. In preferred embodiments a ligand provides an
enhanced affinity for a selected target, e.g, molecule, cell or
cell type, compartment, e.g., a cellular or organ compartment,
tissue, organ or region of the body, as, e.g., compared to a
species absent such a ligand.
[0098] Preferred ligands can improve transport, hybridization, and
specificity properties and may also improve nuclease resistance of
the resultant natural or modified oligoribonucleotide, or a
polymeric molecule comprising any combination of monomers described
herein and/or natural or modified ribonucleotides.
[0099] Ligands in general can include therapeutic modifiers, e.g.,
for enhancing uptake; diagnostic compounds or reporter groups e.g.,
for monitoring distribution; cross-linking agents;
nuclease-resistance conferring moieties; and natural or unusual
nucleobases. General examples include lipophilic moleculeses,
lipids, steroids (e.g.,uvaol, hecigenin, diosgenin), terpenes
(e.g., triterpenes, e.g., sarsasapogenin, Friedelin, epifriedelanol
derivatized lithocholic acid), vitamins, carbohydrates (e.g., a
dextran, pullulan, chitin, chitosan, inulin, cyclodextrin or
hyaluronic acid), proteins, protein binding agents, integrin
targeting molecules, polycationics, peptides, polyamines, and
peptide mimics.
[0100] The ligand may be a naturally occurring substance or a
recombinant or synthetic molecule, such as a synthetic polymer,
e.g., a synthetic polyamino acid. Examples of polyamino acids
include polyamino acid is a polylysine (PLL), poly L-aspartic acid,
poly L-glutamic acid, styrene-maleic acid anhydride copolymer,
poly(L-lactide-co-glycolied) copolymer, divinyl ether-maleic
anhydride copolymer, N-(2-hydroxypropyl)methacrylamide copolymer
(HMPA), polyethylene glycol (PEG), polyvinyl alcohol (PVA),
polyurethane, poly(2-ethylacrylic acid), N-isopropylacrylamide
polymers, or polyphosphazine. Example of polyamines include:
polyethylenimine, polylysine (PLL), spermine, spermidine,
polyamine, pseudopeptide-polyamine, peptidomimetic polyamine,
dendrimer polyamine, arginine, amidine, protamine, cationic
moieties, e.g., cationic lipid, cationic porphyrin, quaternary salt
of a polyamine, or an alpha helical peptide.
[0101] Ligands can also include targeting groups, e.g., a cell or
tissue targeting agent, e.g., an antibody that binds to a specified
cell type such as a liver cell or a cell of the jejunum, a
thyrotropin, melanotropin, lectin, glycoprotein, surfactant protein
A, Mucin carbohydrate, glycosylated polyaminoacids, transferrin,
bisphosphonate, polyglutamate, polyaspartate, or an RGD peptide or
RGD peptide mimetic.
[0102] Other examples of ligands include dyes, intercalating agents
(e.g. acridines), cross-linkers (e.g. psoralene, mitomycin C),
porphyrins (TPPC4, texaphyrin, Sapphyrin), polycyclic aromatic
hydrocarbons (e.g., phenazine, dihydrophenazine), artificial
endonucleases (e.g. EDTA), lipophilic molecules, e.g, cholesterol,
cholic acid, adamantane acetic acid, 1-pyrene butyric acid,
dihydrotestosterone, glycerol (e.g., esters and ethers thereof,
e.g., C.sub.10, C.sub.11, C.sub.12, C.sub.13,C.sub.14, C.sub.15,
C.sub.16, C.sub.17, C.sub.18, C.sub.19, or C.sub.20 alkyl; e.g.,
1,3-bis-O(hexadecyl)glycerol, 1,3-bis-O(octaadecyl)glycerol),
geranyloxyhexyl group, hexadecylglycerol, borneol, menthol,
1,3-propanediol, heptadecyl group, palmitic acid, myristic acid,
O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid,
dimethoxytrityl, or phenoxazine) and protein or peptide conjugates
(e.g., an antibody, a lipoprotein, e.g., low density lipoprotein,
an albumin, e.g., human serum albumin(HSA)), alkylating agents,
phosphate, amino, mercapto, PEG (e.g., PEG-40K), MPEG,
[MPEG].sub.2, polyamino, alkyl, substituted alkyl, radiolabeled
markers, enzymes, haptens (e.g. biotin), transport/absorption
facilitators (e.g., aspirin, folic acid), synthetic ribonucleases
(e.g., imidazole, bisimidazole, histamine, imidazole clusters,
acridine-imidazole conjugates, Eu3+ complexes of
tetraazamacrocycles), dinitrophenyl, HRP, or AP.
[0103] The ligand can be a substance, e.g, a drug, which can
increase the uptake of the iRNA agent into the cell, for example,
by disrupting the cell's cytoskeleton, e.g., by disrupting the
cell's microtubules, microfilaments, and/or intermediate filaments.
The drug can be, for example, taxon, vincristine, vinblastine,
cytochalasin, nocodazole, japlakinolide, latrunculin A, phalloidin,
swinholide A, indanocine, or myoservin.
[0104] The ligand can increase the uptake of the iRNA agent into
the cell by activating an inflammatory response, for example.
Exemplary ligands that would have such an effect include tumor
necrosis factor alpha (TNFalpha), interleukin-1 beta, or gamma
interferon.
[0105] In one aspect, the ligand is a lipid or lipid-based
molecule. Such a lipid or lipid-based molecule preferably binds a
serum protein, e.g., human serum albumin (HSA). An HSA binding
ligand allows for distribution of the conjugate to a target tissue,
e.g., liver tissue, including parenchymal cells of the liver. Other
molecules that can bind HSA can also be used as ligands. For
example, neproxin or aspirin can be used. A lipid or lipid-based
ligand can (a) increase resistance to degradation of the conjugate,
(b) increase targeting or transport into a target cell or cell
membrane, and/or (c) can be used to adjust binding to a serum
protein, e.g., HSA.
[0106] A lipid based ligand can be used to modulate, e.g., control
the binding of the conjugate to a target tissue. For example, a
lipid or lipid-based ligand that binds to HSA more strongly will be
less likely to be targeted to the kidney and therefore less likely
to be cleared from the body. A lipid or lipid-based ligand that
binds to HSA less strongly can be used to target the conjugate to
the kidney.
[0107] In a preferred embodiment, the lipid based ligand binds HSA.
Preferably, it binds HSA with a sufficient affinity such that the
conjugate will be preferably distributed to a non-kidney tissue.
However, it is preferred that the affinity not be so strong that
the HSA-ligand binding cannot be reversed.
[0108] In another aspect, the ligand is a moiety, e.g., a vitamin
or nutrient, which is taken up by a target cell, e.g., a
proliferating cell. These are particularly useful for treating
disorders characterized by unwanted cell proliferation, e.g., of
the malignant or non-malignant type, e.g., cancer cells. Exemplary
vitamins include vitamin A, E, and K. Other exemplary vitamins are
the B vitamins, e.g., folic acid, B12, riboflavin, biotin,
pyridoxal or other vitamins or nutrients taken up by cancer
cells.
[0109] In another aspect, the ligand is a cell-permeation agent,
preferably a helical cell-permeation agent. Preferably, the agent
is amphipathic. An exemplary agent is a peptide such as tat or
antennopedia. If the agent is a peptide, it can be modified,
including a peptidylmimetic, invertomers, non-peptide or
pseudo-peptide linkages, and use of D-amino acids. The helical
agent is preferably an alpha-helical agent, which preferably has a
lipophilic and a lipophobic phase.
[0110] A targeting agent that incorporates a sugar, e.g., galactose
and/or analogues thereof, is particularly useful. These agents
target, in particular, the parenchymal cells of the liver. For
example, a targeting moiety can include more than one or preferably
two or three galactose moieties, spaced about 15 angstroms from
each other. The targeting moiety can alternatively be lactose
(e.g., three lactose moieties), which is glucose coupled to a
galactose. The targeting moiety can also be N-Acetyl-Galactosamine,
N-Ac-Glucosamine, multivalent lactose, multivalent galactose,
multivalent mannose, or multivalent fucose. A mannose or
mannose-6-phosphate targeting moiety can be used for macrophage
targeting.
[0111] 5'-Phosphate Modifications
[0112] In preferred embodiments, iRNA agents are 5' phosphorylated
or include a phosphoryl analog at the 5' prime terminus.
5'-phosphate modifications of the antisense strand include those
which are compatible with RISC mediated gene silencing. Suitable
modifications include: 5'-monophosphate ((HO)2(O)P-O-5');
5'-diphosphate ((HO)2(O)P-O-P(HO)(O)-O-5'); 5'-triphosphate
((HO)2(O)P-O-(HO)(O)P-O-P(HO)(O)-O-5'); 5'-guanosine cap
(7-methylated or non-methylated)
(7m-G-O-5'-(HO)(O)P-O-(HO)(O)P-O-P(HO)(O)-O-5'); 5'-adenosine cap
(Appp), and any modified or unmodified nucleotide cap structure
(N-O-5'-(HO)(O)P-O-(HO)(O)P-O-P(HO)(O)-O-5'); 5'-monothiophosphate
(phosphorothioate; (HO)2(S)P-O-5'); 5'-monodithiophosphate
(phosphorodithioate; (HO)(HS)(S)P-O-5'), 5'-phosphorothiolate
((HO)2(O)P-S-5'); any additional combination of oxygen/sulfur
replaced monophosphate, diphosphate and triphosphates (e.g.
5'-alpha-thiotriphosphate, 5'-gamma-thiotriphosphate, etc.),
5'-phosphoramidates ((HO)2(O)P-NH-5', (HO)(NH2)(O)P-O-5'),
5'-alkylphosphonates (R=alkyl=methyl, ethyl, isopropyl, propyl,
etc., e.g. RP(OH)(O)-O-5'-, (OH)2(O)P-5'-CH2-),
5'-alkyletherphosphonates (R=alkylether=methoxymethyl (MeOCH2-),
ethoxymethyl, etc., e.g. RP(OH)(O)-O-5'-).
[0113] The sense strand can be modified in order to inactivate the
sense strand and prevent formation of an active RISC, therby
potentially reducing off-target effects. This can be accomplished
by a modification which prevents 5'-phosphorylation of the sense
strand, e.g., by modification with a 5'-O-methyl ribonucleotide
(see Nykanen et al., (2001) ATP requirements and small interfering
RNA structure in the RNA interference pathway. Cell 107, 309-321.)
Other modifications which prevent phosphorylation can also be used,
e.g., simply substituting the 5'-OH by H rather than O-Me.
Alternatively, a large bulky group may be added to the 5'-phosphate
turning it into a phosphodiester linkage.
[0114] Transport of iRNA Aagents into Cells
[0115] Not wishing to be bound by any theory, the chemical
similarity between cholesterol-conjugated iRNA agents and certain
constituents of lipoproteins (e.g. cholesterol, cholesteryl esters,
phospholipids) may lead to the association of iRNA agents with
lipoproteins (e.g. LDL, HDL) in blood and/or the interaction of the
iRNA agent with cellular components having an affinity for
cholesterol, e.g. components of the cholesterol transport pathway.
Lipoproteins as well as their constituents are taken up and
processed by cells by various active and passive transport
mechanisms, for example, without limitation, endocytosis of
LDL-receptor bound LDL, endocytosis of oxidized or otherwise
modified LDLs through interaction with Scavenger receptor A,
Scavenger receptor B1-mediated uptake of HDL cholesterol in the
liver, pinocytosis, or transport of cholesterol across membranes by
ABC (ATP-binding cassette) transporter proteins, e.g. ABC-A1,
ABC-G1 or ABC-G4. Hence, cholesterol-conjugated iRNA agents could
enjoy facilitated uptake by cells possessing such transport
mechanisms, e.g. cells of the liver. As such, the present invention
provides evidence and general methods for targeting iRNA agents to
cells expressing certain cell surface components, e.g. receptors,
by conjugating a natural ligand for such component (e.g.
cholesterol) to the iRNA agent, or by conjugating a chemical moiety
(e.g. cholesterol) to the iRNA agent which associates with or binds
to a natural ligand for the component (e.g. LDL, HDL).
[0116] 4 Other Embodiments
[0117] An RNA, e.g., an iRNA agent, can be produced in a cell in
vivo, e.g., from exogenous DNA templates that are delivered into
the cell. For example, the DNA templates can be inserted into
vectors and used as gene therapy vectors. Gene therapy vectors can
be delivered to a subject by, for example, intravenous injection,
local administration (U.S. Pat. No. 5,328,470), or by stereotactic
injection (see, e.g., Chen et al., Proc. Natl. Acad. Sci. USA
91:3054-3057, 1994). The pharmaceutical preparation of the gene
therapy vector can include the gene therapy vector in an acceptable
diluent, or can comprise a slow release matrix in which the gene
delivery vehicle is imbedded. The DNA templates, for example, can
include two transcription units, one that produces a transcript
that includes the top strand of an iRNA agent and one that produces
a transcript that includes the bottom strand of an iRNA agent. When
the templates are transcribed, the iRNA agent is produced, and
processed into siRNA agent fragments that mediate gene
silencing.
[0118] 5 Formulation
[0119] The iRNA agents described herein can be formulated for
administration to a subject.
[0120] For ease of exposition, the formulations, compositions, and
methods in this section are discussed largely with regard to
unmodified iRNA agents. It should be understood, however, that
these formulations, compositions, and methods can be practiced with
other iRNA agents, e.g., modified iRNA agents, and such practice is
within the invention.
[0121] A formulated iRNA agent composition can assume a variety of
states. In some examples, the composition is at least partially
crystalline, uniformly crystalline, and/or anhydrous (e.g., less
than 80, 50, 30, 20, or 10% water). In another example, the iRNA
agent is in an aqueous phase, e.g., in a solution that includes
water.
[0122] The aqueous phase or the crystalline compositions can, e.g.,
be incorporated into a delivery vehicle, e.g., a liposome
(particularly for the aqueous phase) or a particle (e.g., a
microparticle as can be appropriate for a crystalline composition).
Generally, the iRNA agent composition is formulated in a manner
that is compatible with the intended method of administration.
[0123] An iRNA agent preparation can be formulated in combination
with another agent, e.g., another therapeutic agent or an agent
that stabilizes an iRNA agent, e.g., a protein that complexes with
the iRNA agent to form an iRNP. Still other agents include
chelators, e.g., EDTA (e.g., to remove divalent cations such as
Mg.sup.2+), salts, RNAse inhibitors (e.g., a broad specificity
RNAse inhibitor such as RNAsin) and so forth.
[0124] In one embodiment, the iRNA agent preparation includes two
or more iRNA agent(s), e.g., two or more iRNA agents that can
mediate RNAi with respect to the same gene, or different alleles of
the gene, or with respect to different genes. Such preparations can
include at least three, five, ten, twenty, fifty, or a hundred or
more different iRNA agent species. Such iRNA agents can mediate
RNAi with respect to a similar number of different genes.
[0125] Where the two or more iRNA agents in such preparation target
the same gene, they can have target sequences that are
non-overlapping and non-adjacent, or the target sequences may be
overlapping or adjacent.
[0126] 6 Disorders Associated with Bcr-Abl Fusion Expression
[0127] An iRNA agent that targets Bcr-Abl fusion, e.g., an iRNA
agent described herein, can be used to treat a subject, e.g., a
human having or at risk for developing a disease or disorder
associated with Bcr-Abl fusion gene expression, e.g., undesirable
cell proliferation or cancer, or, more specifically, leukemia.
[0128] For example, an iRNA agent that targets Bcr-Abl fusion mRNA
can be used to treat disorders associated with undesirable cell
proliferation, such as leukemia, e.g., acute myelogenous leukemia
(AML), chronic myelogenous leukemia (CML), or acute lymphoblastic
leukemia (ALL). The subject can be one who is currently being
treated with a cytostatic or cytotoxic agent, one who has been
treated with a cytostatic or cytotoxic agent in the past, or one
who is unsuited for treatment with a cytostatic or cytotoxic
agent.
[0129] 7 Treatment Methods and Routes of Delivery
[0130] A composition that includes an iRNA agent, e.g., an iRNA
agent that targets Bcr-Abl fusion, can be delivered to a subject by
a variety of routes. Exemplary routes include intrathecal,
parenchymal, intravenous, nasal, oral, and ocular delivery. The
preferred means of administering the iRNA agents of the present
invention is through parenteral administration.
[0131] An iRNA agent can be incorporated into pharmaceutical
compositions suitable for administration. For example, compositions
can include one or more species of an iRNA agent 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.
[0132] The pharmaceutical compositions of the present invention may
be administered in a number of ways depending upon whether local or
systemic treatment is desired and upon the area to be treated.
Administration may be topical (including ophthalmic, intranasal,
transdermal), oral or parenteral. Parenteral administration
includes intravenous drip, subcutaneous, intraperitoneal or
intramuscular injection, or intrathecal or intraventricular
administration.
[0133] The route of delivery can be dependent on the disorder of
the patient. In general, the delivery of the iRNA agents of the
present invention is done to achieve systemic delivery into the
subject. The preferred means of achieving this is through parental
administration.
[0134] Formulations for parenteral administration may include
sterile aqueous solutions which may also contain buffers, diluents
and other suitable additives. For intravenous use, the total
concentration of solutes should be controlled to render the
preparation isotonic.
[0135] Administration can be provided by the subject or by another
person, e.g., a caregiver. A caregiver can be any entity involved
with providing care to the human: for example, a hospital, hospice,
doctor's office, outpatient clinic; a healthcare worker such as a
doctor, nurse, or other practitioner; or a spouse or guardian, such
as a parent. The medication can be provided in measured doses or in
a dispenser which delivers a metered dose.
[0136] The term "therapeutically effective amount" is the amount
present in the composition that is needed to provide the desired
level of drug in the subject to be treated to give the anticipated
physiological response.
[0137] The term "physiologically effective amount" is that amount
delivered to a subject to give the desired palliative or curative
effect.
[0138] The term "pharmaceutically acceptable carrier" means that
the carrier can be taken into the lungs with no significant adverse
toxicological effects on the lungs.
[0139] The term "co-administration" refers to administering to a
subject two or more agents, and in particular two or more iRNA
agents. The agents can be contained in a single pharmaceutical
composition and be administered at the same time, or the agents can
be contained in separate formulation and administered serially to a
subject. So long as the two agents can be detected in the subject
at the same time, the two agents are said to be
co-administered.
[0140] The types of pharmaceutical excipients that are useful as
carrier include stabilizers such as human serum albumin (HSA),
bulking agents such as carbohydrates, amino acids and polypeptides;
pH adjusters or buffers; salts such as sodium chloride; and the
like. These carriers may be in a crystalline or amorphous form or
may be a mixture of the two.
[0141] Bulking agents that are particularly valuable include
compatible carbohydrates, polypeptides, amino acids or combinations
thereof. Suitable carbohydrates include monosaccharides such as
galactose, D-mannose, sorbose, and the like; disaccharides, such as
lactose, trehalose, and the like; cyclodextrins, such as
2-hydroxypropyl-.beta.-cyclodextrin; and polysaccharides, such as
raffinose, maltodextrins, dextrans, and the like; alditols, such as
mannitol, xylitol, and the like. A preferred group of carbohydrates
includes lactose, threhalose, raffinose maltodextrins, and
mannitol. Suitable polypeptides include aspartame. Amino acids
include alanine and glycine, with glycine being preferred.
[0142] Suitable pH adjusters or buffers include organic salts
prepared from organic acids and bases, such as sodium citrate,
sodium ascorbate, and the like; sodium citrate is preferred.
[0143] Dosage. An iRNA agent can be administered at a unit dose
less than about 75 mg per kg of bodyweight, or less than about 70,
60, 50, 40, 30, 20, 10, 5, 2, 1, 0.5, 0.1, 0.05, 0.01, 0.005,
0.001, or 0.0005 mg per kg of bodyweight, and less than 200 nmol of
iRNA agent (e.g., about 4.4.times.1016 copies) per kg of
bodyweight, or less than 1500, 750, 300, 150, 75, 15, 7.5, 1.5,
0.75, 0.15, 0.075, 0.015, 0.0075, 0.0015, 0.00075, 0.00015 nmol of
iRNA agent per kg of bodyweight. The unit dose, for example, can be
administered by injection (e.g., intravenous or intramuscular,
intrathecally, or directly into an organ), an inhaled dose, or a
topical application.
[0144] Delivery of an iRNA agent directly to an organ (e.g.,
directly to the liver) can be at a dosage on the order of about
0.00001 mg to about 3 mg per organ, or preferably about
0.0001-0.001 mg per organ, about 0.03- 3.0 mg per organ, about
0.1-3.0 mg per eye or about 0.3-3.0 mg per organ.
[0145] The dosage can be an amount effective to treat or prevent a
disease or disorder.
[0146] In one embodiment, the unit dose is administered less
frequently than once a day, e.g., less than every 2, 4, 8 or 30
days. In another embodiment, the unit dose is not administered with
a frequency (e.g., not a regular frequency). For example, the unit
dose may be administered a single time. Because iRNA agent mediated
silencing can persist for several days after administering the iRNA
agent composition, in many instances, it is possible to administer
the composition with a frequency of less than once per day, or, for
some instances, only once for the entire therapeutic regimen.
[0147] In one embodiment, a subject is administered an initial
dose, and one or more maintenance doses of an iRNA agent, e.g., a
double-stranded iRNA agent, or siRNA agent, (e.g., a precursor,
e.g., a larger iRNA agent which can be processed into an siRNA
agent, or a DNA which encodes an iRNA agent, e.g., a
double-stranded iRNA agent, or siRNA agent, or precursor thereof).
The maintenance dose or doses are generally lower than the initial
dose, e.g., one-half less of the initial dose. A maintenance
regimen can include treating the subject with a dose or doses
ranging from 0.01 to 75 mg/kg of body weight per day, e.g., 70, 60,
50, 40, 30, 20, 10, 5, 2, 1, 0.5, 0.1, 0.05, 0.01, 0.005, 0.001, or
0.0005 mg per kg of body weight per day. The maintenance doses are
preferably administered no more than once every 5, 10, or 30 days.
Further, the treatment regimen may last for a period of time which
will vary depending upon the nature of the particular disease, its
severity and the overall condition of the patient. In preferred
embodiments the dosage may be delivered no more than once per day,
e.g., no more than once per 24, 36, 48, or more hours, e.g., no
more than once every 5 or 8 days. Following treatment, the patient
can be monitored for changes in his condition and for alleviation
of the symptoms of the disease state. The dosage of the compound
may either be increased in the event the patient does not respond
significantly to current dosage levels, or the dose may be
decreased if an alleviation of the symptoms of the disease state is
observed, if the disease state has been ablated, or if undesired
side-effects are observed.
[0148] The effective dose can be administered in a single dose or
in two or more doses, as desired or considered appropriate under
the specific circumstances. If desired to facilitate repeated or
frequent infusions, implantation of a delivery device, e.g., a
pump, semi-permanent stent (e.g., intravenous, intraperitoneal,
intracisternal or intracapsular), or reservoir may be
advisable.
[0149] Following successful treatment, it may be desirable to have
the patient undergo maintenance therapy to prevent the recurrence
of the disease state, wherein the compound of the invention is
administered in maintenance doses, ranging from 0.001 g to 100 g
per kg of body weight (see U.S. Pat. No. 6,107,094).
[0150] The concentration of the iRNA agent composition is an amount
sufficient to be effective in treating or preventing a disorder or
to regulate a physiological condition in humans. The concentration
or amount of iRNA agent administered will depend on the parameters
determined for the agent and the method of administration, e.g.
nasal, buccal, or pulmonary. For example, nasal formulations tend
to require much lower concentrations of some ingredients in order
to avoid irritation or burning of the nasal passages. It is
sometimes desirable to dilute an oral formulation up to 10-100
times in order to provide a suitable nasal formulation.
[0151] Certain factors may influence the dosage required to
effectively treat a subject, including but not limited to the
severity of the disease or disorder, previous treatments, the
general health and/or age of the subject, and other diseases
present. It will also be appreciated that the effective dosage of
an iRNA agent such as an siRNA used for treatment may increase or
decrease over the course of a particular treatment. Changes in
dosage may result and become apparent from the results of
diagnostic assays. For example, the subject can be monitored after
administering an iRNA agent composition. Based on information from
the monitoring, an additional amount of the iRNA agent composition
can be administered.
[0152] Dosing is dependent on severity and responsiveness of the
disease condition to be treated, with the course of treatment
lasting from several days to several months, or until a cure is
effected or a diminution of disease state is achieved. Optimal
dosing schedules can be calculated from measurements of drug
accumulation in the body of the patient. Persons of ordinary skill
can easily determine optimum dosages, dosing methodologies and
repetition rates. Optimum dosages may vary depending on the
relative potency of individual compounds, and can generally be
estimated based on EC50s found to be effective in in vitro and in
vivo animal models as described above.
[0153] The invention is further illustrated by the following
examples, which should not be construed as further limiting.
EXAMPLES
[0154] Source of Reagents
[0155] Where the source of a reagent is not specifically given
herein, such reagent may be obtained from any supplier of reagents
for molecular biology at a quality/purity standard for application
in molecular biology.
Example 1: siRNA Synthesis
[0156] siRNA synthesis
[0157] Single-stranded RNAs were produced by solid phase synthesis
on a scale of 1 Smole using an Expedite 8909 synthesizer (Applied
Biosystems, Applera Deutschland GmbH, Darmstadt, Germany) and
controlled pore glass (CPG, 500.ANG., Glen Research, Sterling Va.)
as solid support. RNA and RNA containing 2'-O-methyl nucleotides
were generated by solid phase synthesis employing the corresponding
phosphoramidites and 2'-O-methyl phosphoramidites, respectively
(Proligo Biochemie GmbH, Hamburg, Germany). These building blocks
were incorporated at selected sites within the sequence of the
oligoribonucleotide chain using standard nucleoside phosphoramidite
chemistry such as described in Current protocols in nucleic acid
chemistry, Beaucage, S.L. et al. (Edrs.), John Wiley & Sons,
Inc., New York, N.Y., USA. Phosphorothioate linkages were
introduced by replacement of the iodine oxidizer solution with a
solution of the Beaucage reagent (Chruachem Ltd, Glasgow, UK) in
acetonitrile (1%). Further ancillary reagents were obtained from
Mallinckrodt Baker (Griesheim, Germany).
[0158] Deprotection and purification by anion exchange HPLC of the
crude oligoribonucleotides were carried out according to
established procedures. Yields and concentrations were determined
by UV absorption of a solution of the respective RNA at a
wavelength of 260 nm using a spectral photometer (DU 640B, Beckman
Coulter GmbH, Unterschlei13heim, Germany). Double stranded RNA was
generated by mixing an equimolar solution of complementary strands
in annealing buffer (20 mM sodium phosphate, pH 6.8; 100 mM sodium
chloride), heated in a water bath at 85-90.degree. C. for 3 minutes
and cooled to room temperature over a period of 3-4 hours. The
purified RNA solution was stored at -20 .degree. C until use.
Example 2: Inhibition of Bcr-Abl Expression in Cells Expressing
Bcr-Abl Breakpoint Variants
[0159] Nucleic acid sequences are represented below using standard
nomenclature, and specifically the abbreviations of Table 2.
TABLE-US-00002 TABLE 2 Abbreviations of nucleotide monomers used in
nucleic acid sequence representation. It will be understood that
these monomers, when present in an oligonucleotide, are mutually
linked by 5'-3'-phosphodiester bonds. Abbreviation.sup.a
Nucleotide(s) A, a 2'-deoxy-adenosine-5'-phosphate,
adenosine-5'-phosphate C, c 2'-deoxy-cytidine-5'-phosphate,
cytidine-5'-phosphate G, g 2'-deoxy-guanosine-5'-phosphate,
guanosine-5'-phosphate T, t 2'-deoxy-thymidine-5'-phosphate,
thymidine-5'-phosphate U, u 2'-deoxy-uridine-5'-phosphate,
uridine-5'-phosphate Y, y pyrimidine (C or T, c or u) R, r purine
(A or G, a or g) N, n any (G, A, C, or T, g, a, c or u) bold
italic: 2'-deoxy-adenosine, 2'-deoxy-cytidine, 2'-deoxy-guanosine,
2'- deoxy-thymidine, 2'-deoxy-uridine, adenosine, cytidine,
guanosine, thymidine, uridine (5'-hydroxyl) .sup.acapital letters
represent 2'-deoxyribonucleotides (DNA), lower case letters
represent ribonucleotides (RNA)
[0160] TABLE-US-00003 TABLE 3 Nucleic acid sequences of siRNA
duplexes targeting Bcr-Abl SEQ. SEQ. Specific for ID Sequence ID
Sequence Duplex Bcr-Abl fusion No. sense strand.sup.a,b No.
antisense strand.sup.a,b descriptor gene variant 1
agaguucaa|aagcccuucag 2 ugaagggcuu|uugaacucugcu BAF7 Bcr-Abl-e14a2
3 aguguucau|aagccguucag 4 ugaacggcuu|augaacacugcu BAF8
Bcr-Abl-e14a2, mismatch to BAF7.sup.c 5 gaguucaa|aagcccuucagc 6
cugaagggcuu|uugaacucugc BAF9 Bcr-Abl-e14a2 7 agaguugaa|aagcccuucag
8 ugaagggcuu|uucaacucugcu BAF1 Bcr-Abl-e14a2, mismatch to
BAF7.sup.d 9 aaggaag|aagcccuucagcg 10 gcugaagggcuu|cuuccuuauu BAF3
Bcr-Abl-e13a2 11 auaaggaag|aagcccuucag 12 ugaagggcuu|cuuccuuauuga
BAF15 Bcr-Abl-e13a2 13 gaag|aagcccuucagcggcc 14
gccgcugaagggcuu|cuuccuu BAF17 Bcr-Abl-e13a2 15
ucaauaaggaag|aagcccuu 16 agggcuu|cuuccuuauugaugg BAF19
Bcr-Abl-e13a2 17 ucuauaagcaag|aaccccuu 18 agggguu|cuugcuuauagaugg
BAF28 Bcr-Abl-e13a2, mismatch to BAF19.sup.e 19
gagacgcag|aagcccuucag 20 ugaagggcuu|cugcgucuccau BAF22 Bcr-Abl-e1a2
21 acgcag|aagcccuucagcgg 22 cgcugaagggcuu|cugcgucuc BAF24
Bcr-Abl-e1a2 .sup.aSee Table 2 for an explanation of nucleotide
representation (e.g., lower case letters, bold and italicized
letters). .sup.bexact Bcr-Abl fusion site is marked by vertical
line .sup.cPos. 4 a .gtoreq. u, Pos. 10 a .gtoreq. u, Pos. 16 c
.gtoreq. g in sense strand .sup.dPos. 16 c .gtoreq. g in sense
strand .sup.ePos. 4 a .gtoreq. u, Pos. 10 c .gtoreq. g, Pos. 16 g
.gtoreq. c in sense strand
[0161] Table 3 lists the nucleic acid sequences of siRNAs specific
for Bcr-Abl fusion variants Bcr-Abl-e14a2, Bcr-Abl-e13a2, and
Bcr-Abl-e1a2 which were synthesized.
[0162] The exact fusion site of the Bcr and Abl sequences on the
Bcr-Abl mRNA represents a leukemia specific nucleotide sequence.
Such fusion transcripts encoding disease specific proteins are
ideal targets for a tumor-specific RNAi approach. The aim of the
present study was to develop an optimized in vitro Bcr-Abl RNAi
protocol. Therefore, several chemically synthesized asymmetric
siRNAs, as well as stable expressed shRNAs targeting the fusion
site of the clinically relevant Bcr-Abl transcript variants (e14a2,
e13a2 or e1a2) were evaluated. RNAi efficiency was determined
mainly by Western blot analysis of the Bcr-Abl protein level and by
assessing the impact on the leukemic growth of the treated
cells.
[0163] The results of this work show that repeated transfection
with chemically synthesized 21 nt (sense strand) -23 nt (antisense
strand) siRNAs at intervals of 24 h was much more effective for
both down-regulation of the Bcr-Abl protein and induction of cell
death: A single treatment of 32Dp210/e14a2 with the Bcr-Abl-e14a2
specific siRNA BAF7 resulted in a notable reduction of Bcr-Abl
protein levels followed by a decrease in the viability of
32Dp210/e14a2 cells to approximately 59% relative to
electroporation control cells (EPC, 100%). By contrast, four
consecutive treatments of cells with BAF7 reduced the amount of
Bcr-Abl protein to the detection limit and led to a virtual loss of
viability. Similar conclusions were reached by determination of
total cell numbers 48 h following last treatment: whereas the
number of cells transfected with mismatched control siRNA (BAF8)
increased from 2.5 Mio cells to more than 25 Mio cells within 48 h,
a single transfection with BAF7 was sufficient to reduce
proliferation by approximately 40% and repeating the treatment four
times with the Bcr-Abl specific BAF7 siRNA, resulted in a greater
than 90% inhibition of the increase in cell number.
[0164] The observation that prolonged exposure notably increased
the efficiency of siRNAs may be at least partially explained by the
long half life of Bcr-Abl. For example, Spiller and colleagues
(Spiller DG, et al., Antisense Nucleic Acid Drug Dev. 1998, 8:281)
determined the half life of p210Bcr-Abl (e13a2) in human KYO-1
cells as>48 h. The target protein half life was shown to be
important for the effectiveness of conventional antisense- (as)
-oligodesoxynucleotides (ODN): p210Bcr-Abl (e13a2) protein levels
were unaffected by treatment with an as-ODN targeted to
Bcr-Abl-e13a2 mRNA, even though mRNA levels were substantially
reduced at early time points. Secondly, the target gene
down-regulating effect of siRNAs achieved in mammalian cells is
generally transient (for review: Mittal V, Nat Rev Genet.2004,
5:355). Accordingly, Bcr-Abl protein levels recovered within 48 h
after the last siRNA treatment in the cell lines used in this work.
But the results of the present work show that this limitation can
be overcome, either by repeated treatment with chemically
synthesized siRNA at intervals of 24 h or by stable shRNA
expression. With this optimized protocol the decrease in Bcr-Abl
protein levels achieved was up to 86%, accompanied by a loss of
viability of up to 96%.
[0165] To assess whether RNAi dependent inactivation of Bcr-Abl
leads to sensitization of Bcr-Abl expressing cells to clinical
therapeutics, the sensitivity to imatinib and .gamma.-irradiation
was determined following repeated treatment of 32Dp210/e14a2 cells
with anti-Bcr-Abl siRNA. It could be shown that interference with
Bcr-Abl expression is capable to enhance the sensitivity of the
cells for both .gamma.-irradiation and imatinib mesylate.
[0166] The .gamma.-irradiation dose causing a 50% cell kill was 2.5
Gy in 32Dp210/e14a2 cells treated with Bcr-Abl homologous siRNA,
whereas cells treated with mismatch control tolerated approximately
2.5 times higher doses (6 Gy). The quantity of Bcr-Abl protein also
determined the sensitivity of these cells to imatinib mesylate.
After reduction of the Bcr-Abl protein level with siRNA a 3.4fold
drop of the IC50 of imatinib mesylate was observed in 32Dp210/e14a2
cells when compared to controls. This phenomenon was also observed
in human M07p210/e14a2 cells: 0.05 .mu.M imatinib mesylate caused a
significant induction of apoptosis in BAF7-treated cells whereas
the same concentration had no considerable effect on
electroporation control cells.
[0167] These results are in disagreement with some of the
previously published data, where no additive effect on the
induction of apoptosis was observed in K562 cells upon co-treatment
with imatinib mesylate and an siRNA agent specific for Bcr-Abl
(Wilda M, et al., Oncogene. 2002, 21:5716). The K562 cells employed
by Wilda et al., supra, exhibited a very high level of resistance
to imatinib mesylate as evidenced by the fact that only 8%
underwent apoptosis after a 48 h treatment with imatinib mesylate
alone. Such a highly resistant cell system may be suboptimal for
evaluating possible additive effects with other potential
inhibitors. By contrast, results corroborating our findings were
published recently by Chen and colleagues (Chen J, et al., J Clin
Invest. 2004, 113:1784), illustrating that the down-regulation of
the fusion protein TEL-PDGF.beta.R by RNA interference sensitizes
TEL-PDGF.beta.R expressing cells for imatinib mesylate and
rapamycin, hence antagonizing drug-resistance. As breakpoint
specific siRNAs have to overlap the breakpoint on either side, only
a limited number of approximately 10 different potential siRNA
sequences may be chosen for RNAi directed silencing of fusion
sequences.
[0168] Additionally, siRNA treatment restored imatinib sensitivity
in cells expressing the imatinib resistance conferring Bcr-Abl
variant H396P: Two of the imatinib resistance causing Bcr-Abl
variants found in leukemia patients who relapsed after initial
response to imatinib are Bcr-Abl-T3151 and Bcr-Abl-H396P. These
proteins display a single amino acid change in their kinase domain
compared to Bcr-Abl (wt) rendering them less accessible to
imatinib. Accordingly, expression of Bcr-Abl-T315I in 32D cells
conferred complete resistance to imatinib mesylate and expression
of Bcr-Abl-H396P rendered the respective cells.about.4.7fold less
sensitive to imatinib mesylate when compared to 32Dp210-wt. siRNA
treatment led to a significant downregulation of Bcr-Abl in all 3
cell lines. This down-regulation of Bcr-Abl protein levels using
siRNA agents resulted in a 3.4fold sensitization of 32Dp210wt and a
4fold sensitization of 32Dp210-H396P to imatinib mesylate. By
contrast, imatinib mesylate sensitivity of 32Dp210-T3151 cells
highly resistant to imatinib mesylate was not significantly
affected by siRNA treatment, presumably for the same reason as
given above for the lack of a significant effect as observed by
Wilda et al., Further, Corbin et al. showed that the T3151-mutated
Abl kinase domain exhibited no significant inhibition at imatinib
concentrations 200-fold higher than the IC50 value of the WT
kinase; it also showed a 2-fold increase in its ATP affinity
relative to the wild type protein (Corbin AS, Buchdunger E, Furet
P, Druker BJ. Analysis of the structural basis of specificity of
the Abl kinase by ST1571. J Biol Chem 2001;277:32214-9.). supra:
The effect of imatinib treatment in 32Dp210T315I is itself too
small to observe an increased imatinib sensitivity mediated by
siRNA treatment.
[0169] Any off-target siRNA effects leading to concomitant
down-regulation of the physiological c-Abl and Bcr gene expression
were excluded in the present study. The great specificity of the
RNAi effect was confirmed by the fact that even a single point
mutation in the siRNA sequence led to significant loss of siRNA
efficacy. Also, siRNAs targeting the breakpoint variants e13a2 and
e14a2 affected only their respective target RNAs but not other
breakpoint variants sharing the same a2 portion. This strongly
supports the view that fusion genes resulting from translocations
can be specifically targeted by RNAi. Still, predicting the
effectiveness of siRNA molecules appears to be difficult. The
target regions of the different siRNAs used in this work exhibited
considerable overlap. Yet, siRNA efficiency varied extremely. For
example, the target sequence of the Bcr-Abl-e13a2 specific siRNA
BAF3 was shifted only 2 nt downstream into the Abl part of the
fusion site compared to BAF15. Nevertheless, in contrast to BAF15,
BAF3 was completely ineffective. The same was true for the
Bcr-Abl-e1a2 specific siRNAs BAF22 and BAF24. The sequence of BAF24
was quite similar to that of BAF22, shifted only by 3 nt further
into the Abl region of the fusion transcript. BAF22 actively
silenced Bcr-Abl-e1a2 gene expression whereas BAF24 did not.
Overall, these results show that the efficiency of siRNAs targeted
at the breakpoint sites in oncogenic fusion proteins may not be
predicted. TABLE-US-00004 TABLE 4 Cell lines used in determination
of siRNA activity (DSMZ: Deutsche Sammlung fur M und Zelllinien)
Cell line Description Bcr-Abl expression 32D murine bone marrow;
DSMZ-Nr.: ACC 411 -- 32Dp210-wt bzw. generated by transfection of
32D-cells with retroviral + (e14a2) 32Dp210/e14a2 vector
Migp210-wta (Pear WS, et al.,. Blood. 1998, 92: 3780) 32Dp210-T315I
generated by transfection of 32D-cells with retroviral + (e14a2)
vector Migp210-T315Ia (v. Bubnoff N, et al., Lancet. 2002, 359:
487) 32Dp210-H396P generated by transfection of 32D-cells with
retroviral + (e14a2) vector Migp210-H396Pa (v. Bubnoff et al.,
Lancet. 2002, 359: 487) 32Dp210/e13a2 generated by transfection of
32D-cells with retroviral + (e13a2) vector
pSR.beta.MSVtkneo-p210/e13a2b (Muller AJ, et al., Mol Cell Biol.
1991; 11: 1785) 32Dp190/e1a2 generated by transfection of 32D-cells
with retroviral + (e1a2) vector pSR.beta.MSVtkneo-p190/e1a2d
(Muller AJ, et al., Mol Cell Biol. 1991; 11: 1785) M07p210/e14a2
generated by transfection of M07-cells with retroviral + (e14a2)
vector pGD210 (Daley GQ, et al., Science 1990; 247: 824) K562 human
CML-blast cells, DSMZ-Nr.: ACC 10 Ph+ (e14a2) MEG-01 human
CML-blast cells (megakaryocytic), DSMZ- Ph+ (e13a2) Nr.: ACC 364
SUP-B15 human B cell precursor leukemia cell line; DSMZ- Ph+ (e1a2)
Nr.: ACC 389
[0170] Cells were cultivated in RPMI/10% FCS complemented with
glutamine and Penicillin/Streptomycin. Primary CD34 positive cells
were grown in RPMI medium supplemented with glutamine,
Penicillin/Streptomycin, 20% FCS, recombinant human IL-3 (10
ng/ml), human G-CSF (20 ng/ml), and human FLT3 (100 ng/ml).
[0171] Protein assay: Western blot analysis was performed.about.24h
following the last siRNA-treatment or.about.96 hours following
transfection with the pSUPER siRNA--expression vector as described
(Goetz AW, et al., Cancer Res. 2001, 61:7635)
[0172] Survival Assay: Following the last siRNA treatment cells
were seeded in 96well plates and cultivated for another.about.48
hours. Cell survival was then measured by MTF assay as described
(Van der Kuip H, et al., Blood. 2001, 98:1532)
[0173] siRNA-transfection: siRNAs were transfected into the murine
cell lines 32Dp210Bcr-Abl-e14a2, -e13a2, 32Dp 190Bcr-Abl -e1a2 and
the human leukemic cell line MEG-01 using electroporation. Cell
density was adjusted to 2,5-5.times.10.sup.6/ml in RPMI/10% FCS.
800 .mu.l of this cell suspension were mixed with siRNA in a 4 mm
electroporation cuvette. Cells were electroporated by means of an
EasyJect electroporator (peqlab, Erlangen, Germany) using a
single-pulse protocol (250 V, 1800 .mu.F, 8.). This treatment was
repeated in intervals of 24 hours as indicated.
[0174] All cell lines used exhibit strict dependency on the
activity of Bcr-Abl. In 32Dp210Bcr-Abl-e14a2, -e13a2 and
32Dp190Bcr-Abl-e1a2 inhibition of Bcr-Abl can be compensated by
addition of exogenous growth factor to the medium. Therefor,
recombinant murine IL-3, (1 ng/ml) was added to the growth medium
during siRNA treatment of these cell lines. Following the last
siRNA treatment cells were washed and factor deprived before
starting the different examination procedures (Western
blot/MTT).
[0175] For the transfection of siRNA molecules into the human K562
cells we used LipofectaminTM 2000 (Invitrogen, Karlsruhe). 1,5 Mio.
cells were plated in 1,5 ml RPMI/V10%FCS w/o antibiotics into a 6
well plate. For each well 500 .mu.l transfection solution was
prepared, containing 8,4 .mu..mu.g siRNA and 21 .mu.l Lipofectamin
in OPTI-MEM I reduced serum medium (Gibco, Karlsruhe). The cells
were incubated 5 hours with the transfection solution, then the
medium was changed to RPMI/10%FCS complemented with
Penicillin/Streptomycin. When treated several times, cells were
counted.about.24 h following treatment, diluted to 1 Mio/ml and
seeded again in a 6 well plate (1,5 ml cell suspension per well).
Transfection was then repeated exactly as the day before.
[0176] We used three murine hematopoetic 32D cell lines expressing
either p210 Bcr-Abl (e14a2), p210Bcr-Abl (e13a2) or p190Bcr-Abl
(e1a2) to study the effect3iveness of siRNAs to silence each of
these human Bcr-Abl fusion breakpoint variants. In addition,
experiments were performed in human Ph+ leukemia cells (K562,
MEG-01). All siRNAs used were directed to breakpoints of the Bcr
and
[0177] Abl sequence of the respective Bcr-Abl mRNA. RNAi-efficiency
was assayed via analysis of Bcr-Abl protein levels and by
monitoring the biological effect using MTT viability assay.
[0178] In order to determine optimal conditions for RNA
interference, we repeated the siRNA treatment one to four times at
intervals of 24 hours. One single treatment of 32Dp210Bcr-Abl
(e14a2) with the Bcr-Abl-e14a2 specific siRNA BAF7 resulted in a
significant reduction of Bcr-Abl protein levels. However, Bcr-Abl
protein levels quickly recovered to pre-treatment levels. Despite
its short duration, the effect was still sufficient to reduce the
viability of cells to approximately 59% relative to control cells
(EPC, 100%). Repeated treatment was much more effective both for
down-regulation of the Bcr-Abl protein and inducing cell death:
Fourfold BAF-7 treatment of cells resulted in almost complete
disappearance of Bcr-Abl protein and led to virtually complete loss
of viability. The residual viability level of the cells treated
four times with BAF7 measured in MTT assay was not more
than.about.2,5% of the level of the control cells. Identical
results were obtained when cell counts 48 hours following last
treatment were used for assessment of the biological effect. Both
mock-treated and cells treated with a mismatch siRNA still
increased their cell number from 2.5 Mio cells to more than 25 Mio
cells within 48 hours. Cells transfected once with siRNA homologous
to Bcr-Abl-e14a2 (BAF7) reduced proliferation by app. 40%.
Repeating treatment two or four times with the Bcr-Abl specific
BAF7 siRNA more effectively reduced Bcr-Abl dependent cell growth
reaching a growth inhibition of more than 90%. In addition we could
show in these 32Dp210Bcr-Abl (e14a2) cells, that a single point
mutation in the siRNA sequence is capable to impair the silencing
effect. We used a siRNA molecule with a single mismatch compared to
BAF7 (BAF1). Treatment with BAF1 resulted in a less efficient
down-regulation of Bcr-Abl protein levels compared to treatment
with the breakpoint specific siRNA (BAF7) in 32Dp210Bcr-Abl (e14a2)
cells (FIG. 2A). The lower efficiency of BAF1 became even more
obvious when the biological effect of BAF1 was assessed. Even cells
treated four times with BAF1 showed only a moderate reduction in
viability to 52% respective to EPC. This magnitude was comparable
to the effect of a single treatment with the optimal siRNA.
[0179] We confirmed the observation that prolonged siRNA treatment
is more effective for RNA interference by studying the biological
effect of BAF7 in human K562 cells. Since the K562 cells proved to
be more sensitive to our electroporation protocol, cells were
transfected using lipofectamin 2000. 48 hours following one single
treatment with siRNA cells showed only a minimal growth reduction.
In contrast, 48 hours following the third lipofection with siRNAs
at intervals of 24 hours viability was significantly reduced to 45%
in cells treated with siRNA homologous to Bcr-Abl-e14a2 mRNA
(BAF7).
[0180] All three major Bcr-Abl oncogene variants -e14a2,-e13a2 and
-e1a2 can be targeted by RNA interference. To identify effective
siRNAs for inhibition of the second major Bcr-Abl fusion gene
relevant in CML, Bcr-Abl-e13a2, we treated 32Dp210Bcr-Abl (e13a2)
cells with four siRNAs (BAF3, BAF15, BAF17, BAF19) targeting
different sequences of the Bcr-Able13a2 mRNA breakpoint. As a
control we used a siRNA directed to the Bcr-Abl-e14a2 fusion
sequence (BAF9). BAF3, BAF15, BAF17 and BAF19 exhibited
significantly different efficiencies in down-regulating Bcr-Abl.
Repeated electroporation of the siRNAs BAF15 and BAF19 led to
effective down-regulation of Bcr-Abl protein levels. Despite the
considerable overlap in their target sequence, BAF17 and BAF3 had
no effect Bcr-Abl protein levels. Equivalent results were obtained
when studying the biological effect of these siRNAs via the
MTT-viability assay. Three treatments of 32Dp210Bcr-Abl (e13a2)
cells with the effective siRNAs BAF15 and BAF19 resulted in an
almost complete loss of viability. 48 h following 3rd treatment
viability was only.about.10% (BAF15) and -14% (BAF19) of controls
(EPC). Hence, siRNAs that were ineffective in down-regulating
Bcr-Abl protein levels, BAF3 and BAF17, did also not interfere with
the viability of the cells. Using the same protocol, siRNA BAF19
effectively silenced Bcr-Abl-e13a2 expression in the cell line
MEG-01, a human megakaryocytic CML cell line expressing the
Bcr-Abl-e13a2 RNA. When treated three times with the Bcr-Able 13a2-
specific siRNA BAF19 the cells showed a significant loss of Bcr-Abl
protein levels compared to cells that were electroporated with the
respective mismatch control (BAF28). Down-regulation of Bcr-Abl
protein levels by RNAi also caused substantial loss of viability in
MEG-01 cells. In MEG-01 cells treated three times with the
effective BAF19 siRNA viability was reduced to a level of.about.28%
compared to the level in control cells (EPC, 100%).
[0181] The third major Bcr-Abl variant, p190Bcr-Abl (e1a2) can be
detected in 20-50% of the Ph+ patients with adult-ALL and in
approximately 90% of the patients with Ph+ pediatric ALL. To asses
whether this Bcr-Abl fusion site may also serve as a target for
RNAi, we treated 32Dp190Bcr-Abl (e1a2) cells twice with two
different sequence specific siRNAs (BAF22, BAF24). As controls we
used active siRNAs directed to Bcr-Abl-e14a2 (BAF9) and
Bcr-Abl-e13a2 (BAF19). Repeated treatment at intervals of 24 h led
to significant down-regulation of Bcr-Abl protein levels only in
cells treated with the siRNA BAF22. Transfection of the BAF24 siRNA
had no effect at all. In 32Dp190Bcr-Abl (e1a2) effective reduction
of Bcr-Abl Protein levels after 2nd treatment with BAF22 siRNA
resulted in a reduction of viability reaching al level of 15%
compared to EPC (100%).
[0182] siRNA BAF22 also effectively silenced Bcr-Abl-e1a2
expression in the cell line SUP-B 15, human B cell precursor
leukemia cell line expressing the Bcr-Abl-e1a2 RNA. The human B
cell precursor leukemia cell line SUP-B15 was originally
established from the bone marrow of a 9-year-old boy with acute
lymphoblastic leukemia (B cell precursor ALL) and carries the ALL
variant (m-bcr) of the Bcr-Abl fusion gene (e1a2). This cell line
was obtained from the DSMZ cell culture collection (ACC 389; DSMZ,
Braunschweig).
[0183] SUP-B15 cells were treated by electroporation as described
above with an eIa2-specific siRNA (BAF22) or with siRNA directed to
another breakpoint variant (BAF19) as a control. BAF22 treatment at
intervals of 24 hours for 3 times led to significantly reduced
p190Bcr-Abl protein levels compared to the electroporation-control
(EPC) or to the BAF19 control (See FIG. 3).
[0184] The tendency that prolonged siRNA treatment is more
effective in terms of RNAi was also observed in the 32Dp210Bcr-Abl
(e13a2) and 32Dp190Bcr-Abl (e1a2) cell lines. Repeated
electroporation of siRNAs at an interval of 24 hours) was necessary
to achieve a distinct down-regulation of Bcr-Abl protein levels and
led to further loss of viability than single treatment.
[0185] We have therefore proven that the expression of all major
Bcr-Abl breakpoint variants may be influenced by siRNA treatment of
cells in vitro. Testing a panel of siRNA molecules we were able to
identify effective siRNAs for the e14a2, the e13a2 and the ela2
breakpoint variants. These data extend previous experiments
published by others and by our group on the down-regulation of
Bcr-Abl transcripts bearing the e14a2 fusion.
Example 3: Bcr-Abl Down-Regulation in Cells Isolated from Human
Leukemia Patients
[0186] Down-regulation of BCR-ABL in primary patient cells positive
for breakpoint variant e14a2
[0187] CD34 positive cells were isolated from 3 newly diagnosed and
untreated Philadelphia chromosome-positive CML patients in chronic
phase. Informed consent was obtained by the patients prior to
collection of cells. Mononuclear cells were harvested by
Ficoll-Hypaque density gradient centrifugation (Seromed, Berlin,
Germany). CD34 positive cells were isolated using a stem cell
isolation kit and a MACS column (Miltenyi Biotech, Bergisch
Gladbach, Germany) according to manufacturer's instructions. Cell
purity was checked by FACS analysis using a FITC-conjugated
anti-CD34 antibody (BD Biosciences, Immunocytometry Systems, San
Jose, CA, USA). The fraction of CD34 positive cells ranged from 96
to 99%.
[0188] CD34 positive cells were grown to a density of 500.000
cells/ml in RPMI medium supplemented with 20% FCS, recombinant
human IL-3 (10 ng/ml, Stratmann Biotech AG, Hamburg, Germany),
human G-CSF (20 ng/ml, Amgen, Munich, Germany), and human
recombinant FLT3 ligand (100 ng/ml, Research Diagnostics Inc.,
Concord Mass., USA). A small aliquot of the cells was used for
determining the Bcr-Abl breakpoint variant prior to siRNA
treatment. Total RNA was isolated using the RNeasy-Mini Kit
(Qiagen, Hilden, Germany) according to manufacturer's instructions
and 1 .mu.g RNA was used to generate cDNA using SuperScript reverse
transcriptase (Gibco-BRL, Carlsbad Calif., USA) according to the
manufacturer's protocol. One microliter of cDNA was then used for
PCR with the breakpoint specific Bcr-AbI primers 5'-Bcr-Abl
(5'-CTGACATCCGTGGAGCTG-3') and 3'-Bcr-Abl
(5'-CATTGTGATTATAGCCTAAGA-3') generating a 390bp fragment (e14a2)
or a 290bp fragment (e13a2).
[0189] On day 2 of cell culture, cells were treated with siRNA.
Cells were diluted to a density of 2.5.times.10.sup.6 in 800 .mu.l
growth medium and mixed with 12.8 .mu.l of a 50 .mu.M solution of
the respective siRNA in annealing buffer (20 mM NaPO4, 100 mM NaCl;
pH 6.9) in a 4-mm electroporation cuvette. The cells were then
electroporated using an EasyJect-electroporator, single pulse
protocol (250V, 1800 .mu.F). This treatment was repeated after 24
hours. After the second treatment, the cells were washed and factor
deprived. Following a further cultivation period of 24 hours, the
cells were harvested for western blot analysis.
[0190] BAF7 or BAF12 (both e14a2-specific) treatment resulted in a
significant reduction of Bcr-Abl protein levels compared to cells
treated with mismatch control (BAF8) or with siRNA homologous to
e13a2 (BAF16). Additionally, BAF12 treatment compromised Bcr-Abl
activity. Phosphorylation of CRKL, the direct downstream substrate
of Bcr-Abl, was significantly reduced in cells treated with BAF12
(See FIG. 4).
Sequence CWU 1
1
30 1 21 RNA Artificial Sequence Synthetically generated iRNA 1
cagaguucaa aagcccuuca g 21 2 23 RNA Artificial Sequence
Synthetically generated iRNA 2 ucgucucaag uuuucgggaa guc 23 3 21
RNA Artificial Sequence Synthetically generated iRNA 3 caguguucau
aagccguuca g 21 4 23 RNA Artificial Sequence Synthetically
generated iRNA 4 cugaacggcu uaugaacacu gcu 23 5 21 RNA Artificial
Sequence Synthetically generated iRNA 5 agaguucaaa agcccuucag c 21
6 23 RNA Artificial Sequence Synthetically generated iRNA 6
cgucucaagu uuucgggaag ucg 23 7 21 RNA Artificial Sequence
Synthetically generated iRNA 7 cagaguugaa aagcccuuca g 21 8 23 RNA
Artificial Sequence Synthetically generated iRNA 8 cugaagggcu
uuucaacucu gcu 23 9 21 RNA Artificial Sequence Synthetically
generated iRNA 9 uaaggaagaa gcccuucagc g 21 10 23 RNA Artificial
Sequence Synthetically generated iRNA 10 cgcugaaggg cuucuuccuu auu
23 11 21 RNA Artificial Sequence Synthetically generated iRNA 11
aauaaggaag aagcccuuca g 21 12 23 RNA Artificial Sequence
Synthetically generated iRNA 12 aguuauuccu ucuucgggaa guc 23 13 21
RNA Artificial Sequence Synthetically generated iRNA 13 ggaagaagcc
cuucagcggc c 21 14 23 RNA Artificial Sequence Synthetically
generated iRNA 14 uuccuucuuc gggaagucgc cgg 23 15 21 RNA Artificial
Sequence Synthetically generated iRNA 15 aucaauaagg aagaagcccu u 21
16 23 RNA Artificial Sequence Synthetically generated iRNA 16
gguaguuauu ccuucuucgg gaa 23 17 21 RNA Artificial Sequence
Synthetically generated iRNA 17 aucuauaagc aagaaccccu u 21 18 23
RNA Artificial Sequence Synthetically generated iRNA 18 aagggguucu
ugcuuauaga ugg 23 19 21 RNA Artificial Sequence Synthetically
generated iRNA 19 ggagacgcag aagcccuuca g 21 20 23 RNA Artificial
Sequence Synthetically generated iRNA 20 uaccucugcg ucuucgggaa guc
23 21 21 RNA Artificial Sequence Synthetically generated iRNA 21
gacgcagaag cccuucagcg g 21 22 23 RNA Artificial Sequence
Synthetically generated iRNA 22 ccgcugaagg gcuucugcgu cuc 23 23 23
RNA Artificial Sequence Synthetically generated iRNA 23 cugaagggcu
uuugaacucu gcu 23 24 23 RNA Artificial Sequence Synthetically
generated iRNA 24 gcugaagggc uuuugaacuc ugc 23 25 23 RNA Artificial
Sequence Synthetically generated iRNA 25 cugaagggcu ucuuccuuau uga
23 26 23 RNA Artificial Sequence Synthetically generated iRNA 26
ggccgcugaa gggcuucuuc cuu 23 27 23 RNA Artificial Sequence
Synthetically generated iRNA 27 aagggcuucu uccuuauuga ugg 23 28 23
RNA Artificial Sequence Synthetically generated iRNA 28 cugaagggcu
ucugcgucuc cau 23 29 18 DNA Artificial Sequence Primer 29
ctgacatccg tggagctg 18 30 21 DNA Artificial Sequence Primer 30
cattgtgatt atagcctaag a 21
* * * * *