U.S. patent application number 13/016896 was filed with the patent office on 2011-05-26 for compositions and methods for inhibiting expression of nav1.8 gene.
This patent application is currently assigned to ALNYLAM PHARMACEUTICALS, INC.. Invention is credited to Maria Frank-Kamenetsky, Anke Geick, Philipp Hadwiger, Ingo Roehl, Dinah Sah, Pamela Tan, Hans-Peter Vornlocher.
Application Number | 20110124711 13/016896 |
Document ID | / |
Family ID | 37745850 |
Filed Date | 2011-05-26 |
United States Patent
Application |
20110124711 |
Kind Code |
A1 |
Sah; Dinah ; et al. |
May 26, 2011 |
COMPOSITIONS AND METHODS FOR INHIBITING EXPRESSION OF Nav1.8
GENE
Abstract
The invention relates to a double-stranded ribonucleic acid
(dsRNA) for inhibiting the expression of the Nav1.8 gene (Nav1.8
gene), comprising an antisense strand having a nucleotide sequence
which is less that 25 nucleotides in length and which is
substantially complementary to at least a part of the Nav1.8 gene.
The invention also relates to a pharmaceutical composition
comprising the dsRNA together with a pharmaceutically acceptable
carrier; methods for treating diseases caused by the expression of
the Nav1.8 gene using the pharmaceutical composition; and methods
for inhibiting the expression of the Nav1.8 gene in a cell.
Inventors: |
Sah; Dinah; (Boston, MA)
; Frank-Kamenetsky; Maria; (Brookline, MA) ;
Geick; Anke; (Bayreuth, DE) ; Hadwiger; Philipp;
(Alenkunstadt, DE) ; Roehl; Ingo; (Memmelsdorf,
DE) ; Tan; Pamela; (Kulmbach, DE) ;
Vornlocher; Hans-Peter; (Bayreuth, DE) |
Assignee: |
ALNYLAM PHARMACEUTICALS,
INC.
Cambridge
MA
|
Family ID: |
37745850 |
Appl. No.: |
13/016896 |
Filed: |
January 28, 2011 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
12487605 |
Jun 18, 2009 |
7902168 |
|
|
13016896 |
|
|
|
|
11593099 |
Nov 3, 2006 |
7582745 |
|
|
12487605 |
|
|
|
|
60733816 |
Nov 4, 2005 |
|
|
|
60741586 |
Dec 2, 2005 |
|
|
|
60763202 |
Jan 26, 2006 |
|
|
|
60795443 |
Apr 27, 2006 |
|
|
|
60849364 |
Oct 4, 2006 |
|
|
|
Current U.S.
Class: |
514/44A ;
435/320.1; 435/325; 435/375; 536/24.5 |
Current CPC
Class: |
A61P 29/00 20180101;
A61P 43/00 20180101; C12N 2310/3515 20130101; C12N 2310/14
20130101; A61P 25/04 20180101; C12N 2310/321 20130101; C12N
2310/315 20130101; A61P 25/00 20180101; C12N 2310/322 20130101;
C12N 15/1138 20130101; C12N 2310/321 20130101; C12N 2310/3521
20130101 |
Class at
Publication: |
514/44.A ;
536/24.5; 435/325; 435/375; 435/320.1 |
International
Class: |
A61K 31/713 20060101
A61K031/713; C07H 21/02 20060101 C07H021/02; A61P 29/00 20060101
A61P029/00; C12N 5/00 20060101 C12N005/00; C12N 5/02 20060101
C12N005/02; C12N 15/63 20060101 C12N015/63; C12N 5/10 20060101
C12N005/10 |
Claims
1. A double-stranded ribonucleic acid (dsRNA) for inhibiting the
expression of a human Nav1.8 gene in a cell, wherein said dsRNA
comprises at least two sequences that are complementary to each
other and wherein a sense strand comprises a first sequence and an
antisense strand comprises a second sequence comprising a region of
complementarity which is substantially complementary to at least a
part of a mRNA encoding Nav1.8, and wherein said region of
complementarity is less than 30 nucleotides in length and wherein
said dsRNA, upon contact with a cell expressing said Nav1.8,
inhibits expression of said Nav1.8 gene by at least 20%.
2. The dsRNA of claim 1, wherein said first sequence is selected
from the group consisting of Tables 1, 4 and 6 and said second
sequence is selected from the group consisting of Tables 1, 4 and
6.
3. The dsRNA of claim 1, wherein said dsRNA comprises at least one
modified nucleotide.
4. The dsRNA of claim 2, wherein said dsRNA comprises at least one
modified nucleotide.
5. The dsRNA of claim 3, wherein said modified nucleotide is chosen
from the group of: a 2'-O-methyl modified nucleotide, a nucleotide
comprising a 5'-phosphorothioate group, and a terminal nucleotide
linked to a cholesteryl derivative or dodecanoic acid bisdecylamide
group.
6. The dsRNA of claim 3, wherein said modified nucleotide is chosen
from the group of: a 2'-deoxy-2'-fluoro modified nucleotide, a
2'-deoxy-modified nucleotide, a locked nucleotide, an abasic
nucleotide, 2'-amino-modified nucleotide, 2'-O-alkyl-modified
nucleotide, morpholino nucleotide, a phosphoramidate, and a
non-natural base comprising nucleotide.
7. A cell comprising the dsRNA of claim 1.
8. A pharmaceutical composition for inhibiting the expression of
the Nav1.8 gene in an organism, comprising a dsRNA and a
pharmaceutically acceptable carrier, wherein the dsRNA comprises at
least two sequences that are complementary to each other and
wherein a sense strand comprises a first sequence and an antisense
strand comprises a second sequence comprising a region of
complementarity which is substantially complementary to at least a
part of a mRNA encoding Nav1.8, and wherein said region of
complementarity is less than 30 nucleotides in length and wherein
said dsRNA, upon contact with a cell expressing said Nav1.8,
inhibits expression of said Nav1.8 gene by at least 20%.
9. The pharmaceutical composition of claim 8, wherein said first
sequence of said dsRNA is selected from the group consisting of
Tables 1, 4 and 6 and said second sequence of said dsRNA is
selected from the group consisting of Tables 1, 4 and 6.
10. The pharmaceutical composition of claim 9, wherein said
composition is formulated for administration selected from the
group consisting of intrathecal infusion or injection, or
intravenous infusion or injection.
11. A method for inhibiting the expression of the Nav1.8 gene in a
cell, the method comprising: (a) introducing into the cell a
double-stranded ribonucleic acid (dsRNA), wherein the dsRNA
comprises at least two sequences that are complementary to each
other and wherein a sense strand comprises a first sequence and an
antisense strand comprises a second sequence comprising a region of
complementarity which is substantially complementary to at least a
part of a mRNA encoding Nav1.8, and wherein said region of
complementarity is less than 30 nucleotides in length and wherein
said dsRNA, upon contact with a cell expressing said Nav1.8,
inhibits expression of said Nav1.8 gene by at least 20%; and (b)
maintaining the cell produced in step (a) for a time sufficient to
obtain degradation of the mRNA transcript of the Nav1.8 gene,
thereby inhibiting expression of the Nav1.8 gene in the cell.
12. The method of claim 11, wherein said first sequence of said
dsRNA is selected from the group consisting of Tables 1, 4 and 6
and said second sequence of said dsRNA is selected from the group
consisting of Tables 1, 4 and 6.
13. A method of treating, preventing or managing pain comprising
administering to a patient in need of such treatment, prevention or
management a therapeutically or prophylactically effective amount
of a dsRNA, wherein the dsRNA comprises at least two sequences that
are complementary to each other and wherein a sense strand
comprises a first sequence and an antisense strand comprises a
second sequence comprising a region of complementarity which is
substantially complementary to at least a part of a mRNA encoding
Nav1.8, and wherein said region of complementarity is less than 30
nucleotides in length and wherein said dsRNA, upon contact with a
cell expressing said Nav1.8, inhibits expression of said Nav1.8
gene by at least 20%.
14. The method of claim 13, wherein said first sequence of said
dsRNA is selected from the group consisting of Tables 1, 4 and 6
and said second sequence of said dsRNA is selected from the group
consisting of Tables 1, 4 and 6.
15. The method of claim 14, wherein said pain is selected from the
group consisting of neuropathic pain and inflammatory pain.
16. A vector for inhibiting the expression of the Nav1.8 gene in a
cell, said vector comprising a regulatory sequence operably linked
to a nucleotide sequence that encodes at least one strand of a
dsRNA, wherein one of the strands of said dsRNA is substantially
complementary to at least a part of a mRNA encoding Nav1.8 and
wherein said dsRNA is less than 30 base pairs in length and wherein
said dsRNA, upon contact with a cell expressing said Nav1.8,
inhibits the expression of said Nav1.8 gene by at least 20%.
17. The vector of claim 16, wherein said first sequence of said
dsRNA is selected from the group consisting of Tables 1, 4 and 6
and said second sequence of said dsRNA is selected from the group
consisting of Tables 1, 4 and 6.
18. A cell comprising the vector of claim 16.
19. A cell comprising the vector of claim 17.
Description
RELATED APPLICATIONS
[0001] This application is a divisional of U.S. patent application
Ser. No. 12/487,605, filed Jun. 18, 2009, which is a divisional of
U.S. patent application Ser. No. 11/593,099, filed Nov. 3, 2006,
which claims the benefit of U.S. Provisional Application No.
60/733,816, filed Nov. 4, 2005; U.S. Provisional Application No.
60/741,586, filed Dec. 2, 2005; U.S. Provisional Application No.
60/763,202, filed Jan. 26, 2006; U.S. Provisional Application No.
60/795,443, filed Apr. 27, 2006; and U.S. Provisional Application
No. 60/849,364, filed Oct. 4, 2006. The contents of these prior
applications are incorporated herein by reference in their
entirety.
SEQUENCE LISTING
[0002] The instant application contains a Sequence Listing which
has been submitted in ASCII format via EFS-Web and is hereby
incorporated by reference in its entirety. Said ASCII copy, created
on Jun. 17, 2009, is named 18217_US_sequencelisting.txt, is 225
kilobytes in size, and consists of 609 sequences.
FIELD OF THE INVENTION
[0003] This invention relates to double-stranded ribonucleic acid
(dsRNA), and its use in mediating RNA interference to inhibit the
expression of the Nav1.8 gene and the use of the dsRNA to treat
pain.
BACKGROUND OF THE INVENTION
[0004] Neuropathic pain can be classified as peripheral and central
neuropathic pain. Peripheral neuropathic pain is caused by injury
or infection of peripheral sensory nerves, whereas central
neuropathic pain is caused by damage to the CNS and/or the spinal
cord. Both peripheral and central neuropathic pain can occur
without obvious initial nerve damage.
[0005] A similar definition is given by the International
Association for the Study of Pain (IASP, Seattle, Wash., USA):
peripheral neuropathic pain is pain initiated or caused by a
primary lesion or dysfunction in the peripheral nervous system.
Central neuropathic pain is pain initiated or caused by a primary
lesion or dysfunction in the central nervous system.
[0006] Peripheral lesions may be lesions of peripheral nerves, e.g.
diabetic neuropathy, drug-inducted neuropathy, e.g. upon
chemotherapy, lesions of nerve roots and posterior ganglia, e.g.
post-herpetic neuralgia or nerve root avulsions, neuropathic cancer
pain due to compression of peripheral nerves, nerve plexuses and
nerve roots, etc. Central lesions may be lesions due to infarction,
compressive tumors or abscesses, e.g. in the brainstem, or may be
spinal cord lesions due to injury or operations (Jain K K, Emerging
Drugs, 2000, 5:241-257; McQuay, 2002, European Journal of Pain 6
(Suppl. A): 11-18).
[0007] The above examples of peripheral and central neuropathic
pain demonstrate that peripheral and central neuropathic pain are
distinguished not only by the anatomical location of the lesion or
dysfunction, but they also demonstrate that peripheral and central
neuropathic pain can be distinguished by its mechanisms (McQuay,
supra). Consequently, there is no clear relation between drug
action mechanism and the effect in distinct pain conditions or for
single drug classes and different pain conditions (Sindrup S H,
Jensen T S, Pain 1999, 83:389-400).
[0008] Common analgesics like opioids and non-steroidal
anti-inflammatory drugs (NSAIDs) improve only insufficiently
chronic abnormal pain syndromes as peripheral and central
neuropathic pain due to insufficient efficacy and/or dose-limiting
side effects. In the search for alternative treatment regimes to
produce satisfactory and sustained pain relief, corticosteroids,
conduction blockade, glycerol, anti-convulsants, anti-arrhythmics,
antidepressants, local anesthetics, topical agents such as
capsaicin, gangliosides and electrostimulation have been tried, but
only a subset of patients with neuropathic pain respond to such
treatments and typically, significant pain remains even in these
responders. The critical issue with current therapies is the
therapeutic window; a particular treatment might have potential for
efficacy but the patients are not `treated to effect` because of
limiting side effects upon dose escalation.
[0009] Central neuropathic pain is a form of neuropathic pain which
is a particularly difficult form to be treated (Yezierski and
Burchiel, 2002). Due to lesions in the spinothalamocortical
pathways, ectopic neuronal discharges can occur in different
neurons of the spinal cord and brain. Hyperexcitability in damaged
areas of the central nervous system plays a major role in the
development of central neuropathic pain. Patients with central
neuropathic pain almost always have stimulus-independent pain. In
addition, in the case of spinal cord injury, for example,
stimulus-dependent pain may be present, usually because skin areas
or viscera below the lesions are allodynic. Thus, partial spinal
lesions may tend to produce pain to a greater extent than do
complete lesions.
[0010] Other accepted forms of central neuropathic pain or diseases
associated with central neuropathic pain exist. Examples include
inflammatory CNS diseases such as multiple sclerosis, myelitis or
syphilis, ischemia, hemorrhages or asteriovenous malformations
(e.g. post-stroke neuropathic pain) located in the thalamus,
spinothalamic pathway or thalamocortical projections, and
syrnigomyelia (Koltzenburg, Pain 2002--An Updated Review: Refresher
Course Syllabus; IASP Press, Seattle, 2002).
[0011] Na.sup.+-channels are central to the generation of action
potentials in all excitable cells such as neurons and myocytes. As
such they play key roles in a variety of disease states such as
pain (See, Waxman, S. G., S. Dib-Hajj, et al. (1999) "Sodium
channels and pain" Proc Natl Acad Sci USA 96(14): 7635-9 and
Waxman, S. G., T. R. Cummins, et al. (2000) "Voltage-gated sodium
channels and the molecular pathogenesis of pain: a review" J
Rehabil Res Dev 37(5): 517-28). Three members of the gene family
(NaV1.8, 1.9, 1.5) are resistant to block by the well-known Na
channel blocker TTX, demonstrating subtype specificity within this
gene family. Mutational analysis has identified glutamate 387 as a
critical residue for TTX binding (See, Noda, M., H. Suzuki, et al.
(1989) "A single point mutation confers tetrodotoxin and saxitoxin
insensitivity on the sodium channel II" FEBS Lett 259(1):
213-6).
[0012] In general, voltage-gated sodium channels (NaVs) are
responsible for initiating the rapid upstroke of action potentials
in excitable tissue in nervous system, which transmit the
electrical signals that compose and encode normal and aberrant pain
sensations. Antagonists of NaV channels can attenuate these pain
signals and are useful for treating a variety of pain conditions,
including but not limited to acute, chronic, inflammatory, and
neuropathic pain. Known NaV antagonists include TTX, lidocaine (See
Mao, J. and L. L. Chen (2000) "Systemic lidocaine for neuropathic
pain relief" Pain 87(1): 7-17.) bupivacaine, carbamazepine,
mexilitene, phenyloin (See Jensen, T. S. (2002) "Anticonvulsants in
neuropathic pain: rationale and clinical evidence" Eur J Pain 6
(Suppl A): 61-8), and lamotrigine (See Rozen, T. D. (2001)
"Antiepileptic drugs in the management of cluster headache and
trigeminal neuralgia" Headache 41 Suppl 1: S25-32 and Jensen, T. S.
(2002). However, side effects include dizziness, somnolence, nausea
and vomiting (See Tremont-Lukats, I. W., C. Megeff, and M. M.
Backonja (2000) "Anticonvulsants for neuropathic pain syndromes:
mechanisms of action and place in therapy" Drugs 60:1029-1052) that
limit the utility of these known NaV antagonists for the treatment
of pain. These side effects are thought to result at least in part
from the block of multiple NaV subtypes. An agent that inhibits the
NaV1.8 channel selectively would provide a much great therapeutic
window than these non-selective, known NaV antagonists.
[0013] The detection and transmission of nociceptive stimuli is
mediated by small sensory neurons. Immunohistochemical, in-situ
hybridization and electrophysiology experiments have all shown that
the sodium channel NaV1.8 is selectively localized to the small
sensory neurons of the dorsal root ganglion and trigeminal ganglion
(see Akopian, A. N., L. Sivilotti, et al. (1996) "A
tetrodotoxin-resistant voltage-gated sodium channel expressed by
sensory neurons" Nature 379(6562): 257-62.). After experimental
nerve injury, immunohistochemical data demonstrated an accumulation
of Nav1.8 at the site of nerve injury, concomitant with an
upregulation in TTX-resistant sodium current, consistent with
NaV1.8 as a mechanism underlying hyperalgesia (see Gold, M. S., D.
Weinreich, et al. (2003) "Redistribution of Nav1.8 in uninjured
axons enables neuropathic pain" J. Neurosci. 23:158-166).
Attenuation of NaV1.8 expression with antisense
oligodeoxynucleotides administered by intrathecal injection
prevented experimental nerve-injury induced redistribution of
Nav1.8 in the sciatic nerve and reversed neuropathic pain (tactile
and thermal hyperalgesia), demonstrating a causal role of Nav1.8 in
nerve-injury induced pain (see also Lai, J., M. S. Gold, et al.
(2002) "Inhibition of neuropathic pain by decreased expression of
the tetrodotoxin-resistant sodium channel, NaV1.8" Pain 95(1-2):
143-52, and Lai, J., J. C. Hunter, et al. (2000) "Blockade of
neuropathic pain by antisense targeting of tetrodotoxin-resistant
sodium channels in sensory neurons" Methods Enzymol 314: 201-13.).
In inflammatory pain models, intrathecal administration of
antisense oligodeoxynucleotides against NaV1.8 resulted in a
significant reduction in PGE.sub.2-induced hyperalgesia (see
Khasar, S. G., M. S. Gold, et al. (1998) "A tetrodotoxin-resistant
sodium current mediates inflammatory pain in the rat" Neurosci Lett
256(1): 17-20) and in CFA (complete Freund's adjuvant)-induced
hyperalgesia (Porreca, F., J. Lai, et al. (1999) "A comparison of
the potential role of the tetrodotoxin-insensitive sodium channels,
PN3/SNS and NaN/SNS2, in rat models of chronic pain" Proc. Nat'l.
Acad. Sci. 96: 7640-7644). In addition, in a rat model of visceral
pain, induced by intravesical infusion of acetic acid, bladder
hyperactivity was reduced by intrathecal injection of antisense
oligodeoxynucleotides against NaV1.8, showing that Nav1.8
contributes to the activation of sensory nerves in visceral pain
(Yoshimura, N., S. Seki, et al. (2001) "The involvement of the
tetrodotoxin-resistant sodium channel Nav1.8 (PN3/SNS) in a rat
model of visceral pain" J. Neurosci. 21: 8690-8696).
[0014] Taken together, these data support a role for NaV1.8 in the
detection and transmission of inflammatory and neuropathic
pain.
[0015] Recently, double-stranded RNA molecules (dsRNA) have been
shown to block gene expression in a highly conserved regulatory
mechanism known as RNA interference (RNAi). WO 99/32619 (Fire et
al.) discloses the use of a dsRNA of at least 25 nucleotides in
length to inhibit the expression of the Nav1.8 gene in C. elegans.
dsRNA has also been shown to degrade target RNA in other organisms,
including plants (see, e.g., WO 99/53050, Waterhouse et al.; and WO
99/61631, Heifetz et al.), Drosophila (see, e.g., Yang, D., et al.,
Curr. Biol. (2000) 10:1191-1200), and mammals (see WO 00/44895,
Limmer; and DE 101 00 586.5, Kreutzer et al.). This natural
mechanism has now become the focus for the development of a new
class of pharmaceutical agents for treating disorders that are
caused by the aberrant or unwanted regulation of a gene.
[0016] Despite significant advances in the field of RNAi and
advances in the treatment of pain, there remains a need for an
agent that can selectively and efficiently silence the Nav1.8 gene
using the cell's own RNAi machinery that has both high biological
activity and in vivo stability, and that can effectively inhibit
expression of a target Nav1.8 gene for use in treating pain.
SUMMARY OF THE INVENTION
[0017] The invention provides double-stranded ribonucleic acid
(dsRNA), as well as compositions and methods for inhibiting the
expression of the Nav1.8 gene in a cell or mammal using such dsRNA.
The invention also provides compositions and methods for treating
pathological conditions and diseases caused by the expression of
the Nav1.8 gene, such as in the propagation of pain signals in
neuropathic and inflammatory pain. The dsRNA of the invention
comprises an RNA strand (the antisense strand) having a region
which is less than 30 nucleotides in length and is substantially
complementary to at least part of an mRNA transcript of the Nav1.8
gene.
[0018] In one embodiment, the invention provides double-stranded
ribonucleic acid (dsRNA) molecules for inhibiting the expression of
the Nav1.8 gene. The dsRNA comprises at least two sequences that
are complementary to each other. The dsRNA comprises a sense strand
comprising a first sequence and an antisense strand comprising a
second sequence. The antisense strand comprises a nucleotide
sequence which is substantially complementary to at least part of
an mRNA encoding Nav1.8, and the region of complementarity is less
than 30 nucleotides in length. The dsRNA, upon contacting with a
cell expressing the Nav1.8, inhibits the expression of the Nav1.8
gene by at least 20%.
[0019] For example, the dsRNA molecules of the invention can be
comprised of a first sequence of the dsRNA that is selected from
the group consisting of the sense sequences of Tables 1, 4 and 6
and the second sequence is selected from the group consisting of
the antisense sequences of Tables 1, 4 and 6. The dsRNA molecules
of the invention can be comprised of naturally occurring
nucleotides or can be comprised of at least one modified
nucleotide, such as a 2'-O-methyl modified nucleotide, a nucleotide
comprising a 5'-phosphorothioate group, and a terminal nucleotide
linked to a cholesteryl derivative or dodecanoic acid bisdecylamide
group. Alternatively, the modified nucleotide may be chosen from
the group of: a 2'-deoxy-2'-fluoro modified nucleotide, a
2'-deoxy-modified nucleotide, a locked nucleotide, an abasic
nucleotide, 2'-amino-modified nucleotide, 2'-alkyl-modified
nucleotide, morpholino nucleotide, a phosphoramidate, and a
non-natural base comprising nucleotide. Preferably, the first
sequence of said dsRNA is selected from the group consisting of the
sense sequences of Tables 1, 4 and 6 and the second sequence is
selected from the group consisting of the antisense sequences of
Tables 1, 4 and 6.
[0020] In another embodiment, the invention provides a cell
comprising one of the dsRNAs of the invention. The cell is
preferably a mammalian cell, such as a human cell.
[0021] In another embodiment, the invention provides a
pharmaceutical composition for inhibiting the expression of the
Nav1.8 gene in an organism, comprising one or more of the dsRNA of
the invention and a pharmaceutically acceptable carrier.
[0022] In another embodiment, the invention provides a method for
inhibiting the expression of the Nav1.8 gene in a cell, comprising
the following steps: [0023] (a) introducing into the cell a
double-stranded ribonucleic acid (dsRNA), wherein the dsRNA
comprises at least two sequences that are complementary to each
other. The dsRNA comprises a sense strand comprising a first
sequence and an antisense strand comprising a second sequence. The
antisense strand comprises a region of complementarity which is
substantially complementary to at least a part of an mRNA encoding
Nav1.8, and wherein the region of complementarity is less than 30
nucleotides in length and wherein the dsRNA, upon contact with a
cell expressing the Nav1.8, inhibits expression of the Nav1.8 gene
by at least 20%; and [0024] (b) maintaining the cell produced in
step (a) for a time sufficient to obtain degradation of the mRNA
transcript of the Nav1.8 gene, thereby inhibiting expression of the
Nav1.8 gene in the cell.
[0025] In another embodiment, the invention provides methods for
treating, preventing or managing pain comprising administering to a
patient in need of such treatment, prevention or management a
therapeutically or prophylactically effective amount of one or more
of the dsRNAs of the invention.
[0026] In another embodiment, the invention provides vectors for
inhibiting the expression of the Nav1.8 gene in a cell, comprising
a regulatory sequence operably linked to a nucleotide sequence that
encodes at least one strand of one of the dsRNA of the
invention.
[0027] In another embodiment, the invention provides a cell
comprising a vector for inhibiting the expression of the Nav1.8
gene in a cell. The vector comprises a regulatory sequence operably
linked to a nucleotide sequence that encodes at least one strand of
one of the dsRNA of the invention.
BRIEF DESCRIPTION OF THE FIGURES
[0028] FIG. 1. In vitro activity of the dsRNAs provided in Table 1
against mRNA expression of transfected human Nav1.8 in Cos-7
cells.
[0029] FIG. 2. Lack of cross reactivity of Nav1.8 dsRNAs to
endogenous Nav1.5 mRNA in SW620 cells in vitro.
[0030] FIG. 3. In vitro activity of the dsRNAs provided in Table 1
against endogenous Nav1.8 mRNA in primary cultures of rat dorsal
root ganglion cells.
[0031] FIG. 4. Dose response of dsRNA AL-DP-6209 against endogenous
Nav1.8 mRNA in primary cultures of dorsal root ganglion cells
[0032] FIG. 5. In vivo efficacy of dsRNA AL-DP-6209 with iFECT
against complete Freund's adjuvant-induced tactile hyperalgesia in
rats.
[0033] FIG. 6. In vivo efficacy of dsRNA AL-DP-4459 and AL-DP-4461
against complete Freund's adjuvant-induced tactile hyperalgesia in
rats.
[0034] FIG. 7. In vivo efficacy of dsRNA AL-DP-6050 against
complete Freund's adjuvant--induced tactile hyperalgesia in
rats.
[0035] FIG. 8. In vivo efficacy of dsRNAs AL-DP-6050 and AL-DP-4459
against complete Freund's adjuvant-induced thermal hyperalgesia in
rats.
[0036] FIG. 9. In vivo efficacy of dsRNA AL-DP-4459 against spinal
nerve ligation-induced tactile and thermal hyperalgesia in
rats.
[0037] FIG. 10. Stability of unconjugated dsRNAs AL-DP-6050,
AL-DP-6209, AL-DP-6217, AL-DP-6218 and AL-DP-6219 in human
cerebrospinal fluid at 37.degree. C.
[0038] FIG. 11. Stability of cholesterol-conjugated dsRNA
AL-DP-4459 in human cerebrospinal fluid at 37.degree. C.
[0039] FIG. 12. Dose response of siRNA AL-DP-6050 and its
cholesterol conjugate AL-DP-4459 against mRNA expression of
endogenous Nav1.8 in primary cultures of rat dorsal root ganglion
cells.
[0040] FIG. 13. In vivo efficacy of dsRNA AL-DP-6050 against spinal
nerve ligation-induced thermal hyperalgesia in rats.
[0041] FIG. 14. In vivo efficacy of ND98-2.7 liposomal formulation
of dsRNA AL-DP-6050 against spinal nerve ligation-induced thermal
hyperalgesia in rats.
[0042] FIG. 15. Structure of ND98 lipid
DETAILED DESCRIPTION OF THE INVENTION
[0043] The invention provides double-stranded ribonucleic acid
(dsRNA), as well as compositions and methods for inhibiting the
expression of the Nav1.8 gene in a cell or mammal using the dsRNA.
The invention also provides compositions and methods for treating
pathological conditions and diseases in a mammal caused by the
expression of the Nav1.8 gene using dsRNA. dsRNA directs the
sequence-specific degradation of mRNA through a process known as
RNA interference (RNAi). The process occurs in a wide variety of
organisms, including mammals and other vertebrates.
[0044] The dsRNA of the invention comprises an RNA strand (the
antisense strand) having a region which is less than 30 nucleotides
in length and which is substantially complementary to at least part
of an mRNA transcript of the Nav1.8 gene. The use of these dsRNAs
enables the targeted degradation of mRNAs of genes that are
implicated in pain response in mammals. Using cell-based and animal
assays, the present inventors have demonstrated that very low
dosages of these dsRNA can specifically and efficiently mediate
RNAi, resulting in significant inhibition of expression of the
Nav1.8 gene. Thus, the methods and compositions of the invention
comprising these dsRNAs are useful for treating pain.
[0045] The following detailed description discloses how to make and
use the dsRNA and compositions containing dsRNA to inhibit the
expression of a target Nav1.8 gene, as well as compositions and
methods for treating diseases and disorders caused by the
expression of Nav1.8, such as neuropathic and inflammatory pain.
The pharmaceutical compositions of the invention comprise a dsRNA
having an antisense strand comprising a region of complementarity
which is less than 30 nucleotides in length and is substantially
complementary to at least part of an RNA transcript of the Nav1.8
gene, together with a pharmaceutically acceptable carrier.
[0046] Accordingly, certain aspects of the invention provide
pharmaceutical compositions comprising the dsRNA of the invention
together with a pharmaceutically acceptable carrier, methods of
using the compositions to inhibit expression of the Nav1.8 gene,
and methods of using the pharmaceutical compositions to treat
diseases caused by expression of the Nav1.8 gene.
I. DEFINITIONS
[0047] For convenience, the meaning of certain terms and phrases
used in the specification, examples, and appended claims, are
provided below. If there is an apparent discrepancy between the
usage of a term in other parts of this specification and its
definition provided in this section, the definition in this section
shall prevail.
[0048] "G," "C," "A" and "U" each generally stand for a nucleotide
that contains guanine, cytosine, adenine, and uracil as a base,
respectively. However, it will be understood that the term
"ribonucleotide" or "nucleotide" can also refer to a modified
nucleotide, as further detailed below, or a surrogate replacement
moiety. The skilled person is well aware that guanine, cytosine,
adenine, and uracil may be replaced by other moieties without
substantially altering the base pairing properties of an
oligonucleotide comprising a nucleotide bearing such replacement
moiety. For example, without limitation, a nucleotide comprising
inosine as its base may base pair with nucleotides containing
adenine, cytosine, or uracil. Hence, nucleotides containing uracil,
guanine, or adenine may be replaced in the nucleotide sequences of
the invention by a nucleotide containing, for example, inosine.
Sequences comprising such replacement moieties are embodiments of
the invention.
[0049] By "Nav1.8" as used herein is meant, any Nav1.8 protein,
peptide, or polypeptide associated with the development or
maintenance of an ion channel. The terms "Nav1.8" also refer to
nucleic acid sequences encoding any Nav1.8 protein, peptide, or
polypeptide. For the Examples, the Nav1.8 mRNA sequences used were
human (NM.sub.--006514), mouse (NM.sub.--009134), rat
(NM.sub.--017247) and dog (NM001003203) mRNA sequences.
[0050] As used herein, "target sequence" refers to a contiguous
portion of the nucleotide sequence of an mRNA molecule formed
during the transcription of the Nav1.8 gene, including mRNA that is
a product of RNA processing of a primary transcription product.
[0051] As used herein, the term "strand comprising a sequence"
refers to an oligonucleotide comprising a chain of nucleotides that
is described by the sequence referred to using the standard
nucleotide nomenclature.
[0052] As used herein, and unless otherwise indicated, the term
"complementary," when used to describe a first nucleotide sequence
in relation to a second nucleotide sequence, refers to the ability
of an oligonucleotide or polynucleotide comprising the first
nucleotide sequence to hybridize and form a duplex structure under
certain conditions with an oligonucleotide or polynucleotide
comprising the second nucleotide sequence, as will be understood by
the skilled person. Such conditions can, for example, be stringent
conditions, where stringent conditions may include: 400 mM NaCl, 40
mM PIPES pH 6.4, 1 mM EDTA, 50.degree. C. or 70.degree. C. for
12-16 hours followed by washing. Other conditions, such as
physiologically relevant conditions as may be encountered inside an
organism, can apply. The skilled person will be able to determine
the set of conditions most appropriate for a test of
complementarity of two sequences in accordance with the ultimate
application of the hybridized nucleotides.
[0053] This includes base-pairing of the oligonucleotide or
polynucleotide comprising the first nucleotide sequence to the
oligonucleotide or polynucleotide comprising the second nucleotide
sequence over the entire length of the first and second nucleotide
sequence. Such sequences can be referred to as "fully
complementary" with respect to each other herein. However, where a
first sequence is referred to as "substantially complementary" with
respect to a second sequence herein, the two sequences can be fully
complementary, or they may form one or more, but preferably not
more than 4, 3 or 2 mismatched base pairs upon hybridization, while
retaining the ability to hybridize under the conditions most
relevant to their ultimate application. However, where two
oligonucleotides are designed to form, upon hybridization, one or
more single stranded overhangs, such overhangs shall not be
regarded as mismatches with regard to the determination of
complementarity. For example, a dsRNA comprising one
oligonucleotide 21 nucleotides in length and another
oligonucleotide 23 nucleotides in length, wherein the longer
oligonucleotide comprises a sequence of 21 nucleotides that is
fully complementary to the shorter oligonucleotide, may yet be
referred to as "fully complementary" for the purposes of the
invention.
[0054] "Complementary" sequences, as used herein, may also include,
or be formed entirely from, non-Watson-Crick base pairs and/or base
pairs formed from non-natural and modified nucleotides, in as far
as the above requirements with respect to their ability to
hybridize are fulfilled.
[0055] The terms "complementary", "fully complementary" and
"substantially complementary" herein may be used with respect to
the base matching between the sense strand and the antisense strand
of a dsRNA, or between the antisense strand of a dsRNA and a target
sequence, as will be understood from the context of their use.
[0056] As used herein, a polynucleotide which is "substantially
complementary to at least part of a messenger RNA (mRNA) refers to
a polynucleotide which is substantially complementary to a
contiguous portion of the mRNA of interest (e.g., encoding Nav1.8).
For example, a polynucleotide is complementary to at least a part
of a Nav1.8 mRNA if the sequence is substantially complementary to
a non-interrupted portion of an mRNA encoding Nav1.8.
[0057] The term "double-stranded RNA" or "dsRNA", as used herein,
refers to a ribonucleic acid molecule, or complex of ribonucleic
acid molecules, having a duplex structure comprising two
anti-parallel and substantially complementary, as defined above,
nucleic acid strands. The two strands forming the duplex structure
may be different portions of one larger RNA molecule, or they may
be separate RNA molecules. Where the two strands are part of one
larger molecule, and therefore are connected by an uninterrupted
chain of nucleotides between the 3'-end of one strand and the 5'
end of the respective other strand forming the duplex structure,
the connecting RNA chain is referred to as a "hairpin loop". Where
the two strands are connected covalently by means other than an
uninterrupted chain of nucleotides between the 3'-end of one strand
and the 5' end of the respective other strand forming the duplex
structure, the connecting structure is referred to as a "linker".
The RNA strands may have the same or a different number of
nucleotides. The maximum number of base pairs is the number of
nucleotides in the shortest strand of the dsRNA. In addition to the
duplex structure, a dsRNA may comprise one or more nucleotide
overhangs.
[0058] As used herein, a "nucleotide overhang" refers to the
unpaired nucleotide or nucleotides that protrude from the duplex
structure of a dsRNA when a 3'-end of one strand of the dsRNA
extends beyond the 5'-end of the other strand, or vice versa.
"Blunt" or "blunt end" means that there are no unpaired nucleotides
at that end of the dsRNA, i.e., no nucleotide overhang. A "blunt
ended" dsRNA is a dsRNA that is double-stranded over its entire
length, i.e., no nucleotide overhang at either end of the
molecule.
[0059] The term "antisense strand" refers to the strand of a dsRNA
which includes a region that is substantially complementary to a
target sequence. As used herein, the term "region of
complementarity" refers to the region on the antisense strand that
is substantially complementary to a sequence, for example a target
sequence, as defined herein. Where the region of complementarity is
not fully complementary to the target sequence, the mismatches are
most tolerated in the terminal regions and, if present, are
preferably in a terminal region or regions, e.g., within 6, 5, 4,
3, or 2 nucleotides of the 5' and/or 3' terminus.
[0060] The term "sense strand," as used herein, refers to the
strand of a dsRNA that includes a region that is substantially
complementary to a region of the antisense strand.
[0061] "Introducing into a cell", when referring to a dsRNA, means
facilitating uptake or absorption into the cell, as is understood
by those skilled in the art. Absorption or uptake of dsRNA can
occur through unaided diffusive or active cellular processes, or by
auxiliary agents or devices. The meaning of this term is not
limited to cells in vitro; a dsRNA may also be "introduced into a
cell", wherein the cell is part of a living organism. In such
instance, introduction into the cell will include the delivery to
the organism. For example, for in vivo delivery, dsRNA can be
injected into a tissue site or administered systemically. In vitro
introduction into a cell includes methods known in the art such as
electroporation and lipofection.
[0062] The terms "silence" and "inhibit the expression of", in as
far as they refer to the Nav1.8 gene, herein refer to the at least
partial suppression of the expression of the Nav1.8 gene, as
manifested by a reduction of the amount of mRNA transcribed from
the Nav1.8 gene which may be isolated from a first cell or group of
cells in which the Nav1.8 gene is transcribed and which has or have
been treated such that the expression of the Nav1.8 gene is
inhibited, as compared to a second cell or group of cells
substantially identical to the first cell or group of cells but
which has or have not been so treated (control cells). The degree
of inhibition is usually expressed in terms of
( mRNA in control cells ) - ( mRNA in treated cells ) ( mRNA in
control cells ) 100 % ##EQU00001##
[0063] Alternatively, the degree of inhibition may be given in
terms of a reduction of a parameter that is functionally linked to
Nav1.8 gene transcription, e.g. the amount of protein encoded by
the Nav1.8 gene which is secreted by a cell, or the number of cells
displaying a certain phenotype, e.g apoptosis. In principle, Nav1.8
gene silencing may be determined in any cell expressing the target,
either constitutively or by genomic engineering, and by any
appropriate assay. However, when a reference is needed in order to
determine whether a given siRNA inhibits the expression of the
Nav1.8 gene by a certain degree and therefore is encompassed by the
instant invention, the assay provided in the Examples below shall
serve as such reference.
[0064] For example, in certain instances, expression of the Nav1.8
gene is suppressed by at least about 20%, 25%, 35%, or 50% by
administration of the double-stranded oligonucleotide of the
invention. In a preferred embodiment, the Nav1.8 gene is suppressed
by at least about 60%, 70%, or 80% by administration of the
double-stranded oligonucleotide of the invention. In a more
preferred embodiment, the Nav1.8 gene is suppressed by at least
about 85%, 90%, or 95% by administration of the double-stranded
oligonucleotide of the invention. In a most preferred embodiment,
the Nav1.8 gene is suppressed by at least about 98%, 99% or more by
administration of the double-stranded oligonucleotide of the
invention.
[0065] The terms "treat", "treatment", and the like, refer to
relief from or alleviation of the perception of pain, including the
relief from or alleviation of the intensity and/or duration of a
pain (e.g., burning sensation, tingling, electric-shock-like
feelings, etc.) experienced by a subject in response to a given
stimulus (e.g., pressure, tissue injury, cold temperature, etc.).
Relief from or alleviation of the perception of pain can be any
detectable decrease in the intensity or duration of pain. Treatment
can occur in a subject (e.g., a human or companion animal)
suffering from a pain condition or having one or more symptoms of a
pain-related disorder that can be treated according to the present
invention, or in an animal model of pain, such as the SNL rat model
of neuropathic pain or CFA rat model of chronic pain described
herein, or another animal model of pain. In the context of the
present invention insofar as it relates to any of the other
conditions recited herein below (other than pain), the terms
"treat", "treatment", and the like mean to relieve or alleviate at
least one symptom associated with such condition, or to slow or
reverse the progression of such condition.
[0066] As used herein, the phrases "therapeutically effective
amount" and "prophylactically effective amount" refer to an amount
that provides a therapeutic benefit in the treatment, prevention,
or management of pain or an overt symptom of pain. The specific
amount that is therapeutically effective can be readily determined
by ordinary medical practitioner, and may vary depending on factors
known in the art, such as, e.g. the type of pain, the patient's
history and age, the stage of pain, and the administration of other
anti-pain agents.
[0067] As used herein, a "pharmaceutical composition" comprises a
pharmacologically effective amount of a dsRNA and a
pharmaceutically acceptable carrier. As used herein,
"pharmacologically effective amount," "therapeutically effective
amount" or simply "effective amount" refers to that amount of an
RNA effective to produce the intended pharmacological, therapeutic
or preventive result. For example, if a given clinical treatment is
considered effective when there is at least a 25% reduction in a
measurable parameter associated with a disease or disorder, a
therapeutically effective amount of a drug for the treatment of
that disease or disorder is the amount necessary to effect at least
a 25% reduction in that parameter.
[0068] The term "pharmaceutically acceptable carrier" refers to a
carrier for administration of a therapeutic agent. Such carriers
include, but are not limited to, saline, buffered saline, dextrose,
water, glycerol, ethanol, and combinations thereof. The term
specifically excludes cell culture medium. For drugs administered
orally, pharmaceutically acceptable carriers include, but are not
limited to pharmaceutically acceptable excipients such as inert
diluents, disintegrating agents, binding agents, lubricating
agents, sweetening agents, flavoring agents, coloring agents and
preservatives. Suitable inert diluents include sodium and calcium
carbonate, sodium and calcium phosphate, and lactose, while corn
starch and alginic acid are suitable disintegrating agents. Binding
agents may include starch and gelatin, while the lubricating agent,
if present, will generally be magnesium stearate, stearic acid or
talc. If desired, the tablets may be coated with a material such as
glyceryl monostearate or glyceryl distearate, to delay absorption
in the gastrointestinal tract.
[0069] As used herein, a "transformed cell" is a cell into which a
vector has been introduced from which a dsRNA molecule may be
expressed.
II. Double-Stranded Ribonucleic Acid (dsRNA)
[0070] In one embodiment, the invention provides double-stranded
ribonucleic acid (dsRNA) molecules for inhibiting the expression of
the Nav1.8 gene in a cell or mammal, wherein the dsRNA comprises an
antisense strand comprising a region of complementarity which is
complementary to at least a part of an mRNA formed in the
expression of the Nav1.8 gene, and wherein the region of
complementarity is less than 30 nucleotides in length and wherein
said dsRNA, upon contact with a cell expressing said Nav1.8 gene,
inhibits the expression of said Nav1.8 gene by at least 20%. The
dsRNA comprises two RNA strands that are sufficiently complementary
to hybridize to form a duplex structure. One strand of the dsRNA
(the antisense strand) comprises a region of complementarity that
is substantially complementary, and preferably fully complementary,
to a target sequence, derived from the sequence of an mRNA formed
during the expression of the Nav1.8 gene, the other strand (the
sense strand) comprises a region which is complementary to the
antisense strand, such that the two strands hybridize and form a
duplex structure when combined under suitable conditions.
Preferably, the duplex structure is between 15 and 30, more
preferably between 18 and 25, yet more preferably between 19 and
24, and most preferably between 21 and 23 base pairs in length.
Similarly, the region of complementarity to the target sequence is
between 15 and 30, more preferably between 18 and 25, yet more
preferably between 19 and 24, and most preferably between 21 and 23
nucleotides in length. The dsRNA of the invention may further
comprise one or more single-stranded nucleotide overhang(s). The
dsRNA can be synthesized by standard methods known in the art as
further discussed below, e.g., by use of an automated DNA
synthesizer, such as are commercially available from, for example,
Biosearch, Applied Biosystems, Inc. In a preferred embodiment, the
Nav1.8 gene is the human Nav1.8 gene. In specific embodiments, the
antisense strand of the dsRNA comprises the antisense sequences of
Tables 1, 4 and 6 and the second sequence is selected from the
group consisting of the sense sequences of Tables 1, 4 and 6.
[0071] In further embodiments, the dsRNA comprises at least one
nucleotide sequence selected from the groups of sequences provided
in Tables 1, 4 and 6. In other embodiments, the dsRNA comprises at
least two sequences selected from this group, wherein one of the at
least two sequences is complementary to another of the at least two
sequences, and one of the at least two sequences is substantially
complementary to a sequence of an mRNA generated in the expression
of the Nav1.8 gene. Preferably, the dsRNA comprises two
oligonucleotides, wherein one oligonucleotide is described by
Tables 1, 4 and 6 and the second oligonucleotide is described by
Tables 1, 4 and 6.
[0072] The skilled person is well aware that dsRNAs comprising a
duplex structure of between 20 and 23, but specifically 21, base
pairs have been hailed as particularly effective in inducing RNA
interference (Elbashir et al., EMBO 2001, 20:6877-6888). However,
others have found that shorter or longer dsRNAs can be effective as
well. In the embodiments described above, by virtue of the nature
of the oligonucleotide sequences provided in Tables 1, 4 and 6, the
dsRNAs of the invention can comprise at least one strand of a
length of minimally 21 nt. It can be reasonably expected that
shorter dsRNAs comprising one of the sequences of Tables 1, 4 and 6
minus only a few nucleotides on one or both ends may be similarly
effective as compared to the dsRNAs described above. Hence, dsRNAs
comprising a partial sequence of at least 15, 16, 17, 18, 19, 20,
or more contiguous nucleotides from one of the sequences of Tables
1, 4 and 6, and differing in their ability to inhibit the
expression of the Nav1.8 gene in a FACS assay as described herein
below by not more than 5, 10, 15, 20, 25, or 30% inhibition from a
dsRNA comprising the full sequence, are contemplated by the
invention.
[0073] The dsRNA of the invention can contain one or more
mismatches to the target sequence.
[0074] In a preferred embodiment, the dsRNA of the invention
contains no more than 3 mismatches. If the antisense strand of the
dsRNA contains mismatches to a target sequence, it is preferable
that the area of mismatch not be located in the center of the
region of complementarity. If the antisense strand of the dsRNA
contains mismatches to the target sequence, it is preferable that
the mismatch be restricted to 5 nucleotides from either end, for
example 5, 4, 3, 2, or 1 nucleotide from either the 5' or 3' end of
the region of complementarity. For example, for a 23 nucleotide
dsRNA strand which is complementary to a region of the Nav1.8 gene,
the dsRNA preferably does not contain any mismatch within the
central 13 nucleotides. The methods described within the invention
can be used to determine whether a dsRNA containing a mismatch to a
target sequence is effective in inhibiting the expression of the
Nav1.8 gene. Consideration of the efficacy of dsRNAs with
mismatches in inhibiting expression of the Nav1.8 gene is
important, especially if the particular region of complementarity
in the Nav1.8 gene is known to have polymorphic sequence variation
within the population.
[0075] In one embodiment, at least one end of the dsRNA has a
single-stranded nucleotide overhang of 1 to 4, preferably 1 or 2
nucleotides. dsRNAs having at least one nucleotide overhang have
unexpectedly superior inhibitory properties than their blunt-ended
counterparts. Moreover, the present inventors have discovered that
the presence of only one nucleotide overhang strengthens the
interference activity of the dsRNA, without affecting its overall
stability. dsRNA having only one overhang has proven particularly
stable and effective in vivo, as well as in a variety of cells,
cell culture mediums, blood, and serum. Preferably, the
single-stranded overhang is located at the 3'-terminal end of the
antisense strand or, alternatively, at the 3'-terminal end of the
sense strand. The dsRNA may also have a blunt end, preferably
located at the 5'-end of the antisense strand. Such dsRNAs have
improved stability and inhibitory activity, thus allowing
administration at low dosages, i.e., less than 5 mg/kg body weight
of the recipient per day. Preferably, the antisense strand of the
dsRNA has a nucleotide overhang at the 3'-end, and the 5'-end is
blunt. In another embodiment, one or more of the nucleotides in the
overhang is replaced with a nucleoside thiophosphate.
[0076] In yet another embodiment, the dsRNA is chemically modified
to enhance stability. The nucleic acids of the invention may be
synthesized and/or modified by methods well established in the art,
such as those described in "Current protocols in nucleic acid
chemistry", Beaucage, S. L. et al. (Edrs.), John Wiley & Sons,
Inc., New York, N.Y., USA, which is hereby incorporated herein by
reference. Chemical modifications may include, but are not limited
to 2' modifications, introduction of non-natural bases, covalent
attachment to a ligand, and replacement of phosphate linkages with
thiophosphate linkages. In this embodiment, the integrity of the
duplex structure is strengthened by at least one, and preferably
two, chemical linkages. Chemical linking may be achieved by any of
a variety of well-known techniques, for example by introducing
covalent, ionic or hydrogen bonds; hydrophobic interactions, van
der Waals or stacking interactions; by means of metal-ion
coordination, or through use of purine analogues. Preferably, the
chemical groups that can be used to modify the dsRNA include,
without limitation, methylene blue; bifunctional groups, preferably
bis-(2-chloroethyl)amine; N-acetyl-N'-(p-glyoxyl benzoyl)cystamine;
4-thiouracil; and psoralen. In one preferred embodiment, the linker
is a hexa-ethylene glycol linker. In this case, the dsRNA are
produced by solid phase synthesis and the hexa-ethylene glycol
linker is incorporated according to standard methods (e.g.,
Williams, D. J., and K. B. Hall, Biochem. (1996) 35:14665-14670).
In a particular embodiment, the 5'-end of the antisense strand and
the 3'-end of the sense strand are chemically linked via a
hexaethylene glycol linker. In another embodiment, at least one
nucleotide of the dsRNA comprises a phosphorothioate or
phosphorodithioate groups. The chemical bond at the ends of the
dsRNA is preferably formed by triple-helix bonds. Table 1 provides
examples of modified RNAi agents of the invention.
[0077] In certain embodiments, a chemical bond may be formed by
means of one or several bonding groups, wherein such bonding groups
are preferably poly-(oxyphosphinicooxy-1,3-propandiol)- and/or
polyethylene glycol chains. In other embodiments, a chemical bond
may also be formed by means of purine analogs introduced into the
double-stranded structure instead of purines. In further
embodiments, a chemical bond may be formed by azabenzene units
introduced into the double-stranded structure. In still further
embodiments, a chemical bond may be formed by branched nucleotide
analogs instead of nucleotides introduced into the double-stranded
structure. In certain embodiments, a chemical bond may be induced
by ultraviolet light.
[0078] In yet another embodiment, the nucleotides at one or both of
the two single strands may be modified to prevent or inhibit the
activation of cellular enzymes, such as, for example, without
limitation, certain nucleases. Techniques for inhibiting the
activation of cellular enzymes are known in the art including, but
not limited to, 2'-amino modifications, 2'-fluoro modifications,
2'-alkyl modifications, uncharged backbone modifications,
morpholino modifications, 2'-O-methyl modifications, and
phosphoramidate (see, e.g., Wagner, Nat. Med. (1995) 1:1116-8).
Thus, at least one 2'-hydroxyl group of the nucleotides on a dsRNA
is replaced by a chemical group, preferably by a 2'-fluoro or a
2'-O-methyl group. Also, at least one nucleotide may be modified to
form a locked nucleotide. Such locked nucleotide contains a
methylene or ethylene bridge that connects the 2'-oxygen of ribose
with the 4'-carbon of ribose. Oligonucleotides containing the
locked nucleotide are described in Koshkin, A. A., et al.,
Tetrahedron (1998), 54: 3607-3630) and Obika, S. et al.,
Tetrahedron Lett. (1998), 39: 5401-5404). Introduction of a locked
nucleotide into an oligonucleotide improves the affinity for
complementary sequences and increases the melting temperature by
several degrees (Braasch, D. A. and D. R. Corey, Chem. Biol.
(2001), 8:1-7).
[0079] Conjugating a ligand to a dsRNA can enhance its cellular
absorption. In certain instances, a hydrophobic ligand is
conjugated to the dsRNA to facilitate direct permeation of the
cellular membrane. Alternatively, the ligand conjugated to the
dsRNA is a substrate for receptor-mediated endocytosis. These
approaches have been used to facilitate cell permeation of
antisense oligonucleotides. For example, cholesterol has been
conjugated to various antisense oligonucleotides resulting in
compounds that are substantially more active compared to their
non-conjugated analogs. See M. Manoharan Antisense & Nucleic
Acid Drug Development 2002, 12, 103. Other lipophilic compounds
that have been conjugated to oligonucleotides include 1-pyrene
butyric acid, 1,3-bis-O-(hexadecyl)glycerol, and menthol. One
example of a ligand for receptor-mediated endocytosis is folic
acid. Folic acid enters the cell by folate-receptor-mediated
endocytosis. dsRNA compounds bearing folic acid would be
efficiently transported into the cell via the
folate-receptor-mediated endocytosis. Li and coworkers report that
attachment of folic acid to the 3'-terminus of an oligonucleotide
resulted in an 8-fold increase in cellular uptake of the
oligonucleotide. Li, S.; Deshmukh, H. M.; Huang, L. Pharm. Res.
1998, 15, 1540. Other ligands that have been conjugated to
oligonucleotides include polyethylene glycols, carbohydrate
clusters, cross-linking agents, porphyrin conjugates, and delivery
peptides.
[0080] In certain instances, conjugation of a cationic ligand to
oligonucleotides often results in improved resistance to nucleases.
Representative examples of cationic ligands are propylammonium and
dimethylpropylammonium. Interestingly, antisense oligonucleotides
were reported to retain their high binding affinity to mRNA when
the cationic ligand was dispersed throughout the oligonucleotide.
See M. Manoharan Antisense & Nucleic Acid Drug Development
2002, 12, 103 and references therein.
[0081] The ligand-conjugated dsRNA of the invention may be
synthesized by the use of a dsRNA that bears a pendant reactive
functionality, such as that derived from the attachment of a
linking molecule onto the dsRNA. This reactive oligonucleotide may
be reacted directly with commercially-available ligands, ligands
that are synthesized bearing any of a variety of protecting groups,
or ligands that have a linking moiety attached thereto. The methods
of the invention facilitate the synthesis of ligand-conjugated
dsRNA by the use of, in some preferred embodiments, nucleoside
monomers that have been appropriately conjugated with ligands and
that may further be attached to a solid-support material. Such
ligand-nucleoside conjugates, optionally attached to a
solid-support material, are prepared according to some preferred
embodiments of the methods of the invention via reaction of a
selected serum-binding ligand with a linking moiety located on the
5' position of a nucleoside or oligonucleotide. In certain
instances, an dsRNA bearing an aralkyl ligand attached to the
3'-terminus of the dsRNA is prepared by first covalently attaching
a monomer building block to a controlled-pore-glass support via a
long-chain aminoalkyl group. Then, nucleotides are bonded via
standard solid-phase synthesis techniques to the monomer
building-block bound to the solid support. The monomer building
block may be a nucleoside or other organic compound that is
compatible with solid-phase synthesis.
[0082] The dsRNA used in the conjugates of the invention may be
conveniently and routinely made through the well-known technique of
solid-phase synthesis. Equipment for such synthesis is sold by
several vendors including, for example, Applied Biosystems (Foster
City, Calif.). Any other means for such synthesis known in the art
may additionally or alternatively be employed. It is also known to
use similar techniques to prepare other oligonucleotides, such as
the phosphorothioates and alkylated derivatives.
[0083] Teachings regarding the synthesis of particular modified
oligonucleotides may be found in the following U.S. Pat. Nos.
5,138,045 and 5,218,105, drawn to polyamine conjugated
oligonucleotides; U.S. Pat. No. 5,212,295, drawn to monomers for
the preparation of oligonucleotides having chiral phosphorus
linkages; U.S. Pat. Nos. 5,378,825 and 5,541,307, drawn to
oligonucleotides having modified backbones; U.S. Pat. No.
5,386,023, drawn to backbone-modified oligonucleotides and the
preparation thereof through reductive coupling; U.S. Pat. No.
5,457,191, drawn to modified nucleobases based on the 3-deazapurine
ring system and methods of synthesis thereof; U.S. Pat. No.
5,459,255, drawn to modified nucleobases based on N-2 substituted
purines; U.S. Pat. No. 5,521,302, drawn to processes for preparing
oligonucleotides having chiral phosphorus linkages; U.S. Pat. No.
5,539,082, drawn to peptide nucleic acids; U.S. Pat. No. 5,554,746,
drawn to oligonucleotides having .beta.-lactam backbones; U.S. Pat.
No. 5,571,902, drawn to methods and materials for the synthesis of
oligonucleotides; U.S. Pat. No. 5,578,718, drawn to nucleosides
having alkylthio groups, wherein such groups may be used as linkers
to other moieties attached at any of a variety of positions of the
nucleoside; U.S. Pat. Nos. 5,587,361 and 5,599,797, drawn to
oligonucleotides having phosphorothioate linkages of high chiral
purity; U.S. Pat. No. 5,506,351, drawn to processes for the
preparation of 2'-O-alkyl guanosine and related compounds,
including 2,6-diaminopurine compounds; U.S. Pat. No. 5,587,469,
drawn to oligonucleotides having N-2 substituted purines; U.S. Pat.
No. 5,587,470, drawn to oligonucleotides having 3-deazapurines;
U.S. Pat. No. 5,223,168, and U.S. Pat. No. 5,608,046, both drawn to
conjugated 4'-desmethyl nucleoside analogs; U.S. Pat. Nos.
5,602,240, and 5,610,289, drawn to backbone-modified
oligonucleotide analogs; U.S. Pat. Nos. 6,262,241, and 5,459,255,
drawn to, inter alia, methods of synthesizing
2'-fluoro-oligonucleotides.
[0084] In the ligand-conjugated dsRNA and ligand-molecule bearing
sequence-specific linked nucleosides of the invention, the
oligonucleotides and oligonucleosides may be assembled on a
suitable DNA synthesizer utilizing standard nucleotide or
nucleoside precursors, or nucleotide or nucleoside conjugate
precursors that already bear the linking moiety, ligand-nucleotide
or nucleoside-conjugate precursors that already bear the ligand
molecule, or non-nucleoside ligand-bearing building blocks.
[0085] When using nucleotide-conjugate precursors that already bear
a linking moiety, the synthesis of the sequence-specific linked
nucleosides is typically completed, and the ligand molecule is then
reacted with the linking moiety to form the ligand-conjugated
oligonucleotide. Oligonucleotide conjugates bearing a variety of
molecules such as steroids, vitamins, lipids and reporter
molecules, has previously been described (see Manoharan et al., PCT
Application WO 93/07883). In a preferred embodiment, the
oligonucleotides or linked nucleosides of the invention are
synthesized by an automated synthesizer using phosphoramidites
derived from ligand-nucleoside conjugates in addition to the
standard phosphoramidites and non-standard phosphoramidites that
are commercially available and routinely used in oligonucleotide
synthesis.
[0086] The incorporation of a 2'-O-methyl, 2'-O-ethyl, 2'-O-propyl,
2'-O-allyl, 2'-O-aminoalkyl or 2'-deoxy-2'-fluoro group in
nucleosides of an oligonucleotide confers enhanced hybridization
properties to the oligonucleotide. Further, oligonucleotides
containing phosphorothioate backbones have enhanced nuclease
stability. Thus, functionalized, linked nucleosides of the
invention can be augmented to include either or both a
phosphorothioate backbone or a 2'-O-methyl, 2'-O-ethyl,
2'-O-propyl, 2'-O-aminoalkyl, 2'-O-allyl or 2'-deoxy-2'-fluoro
group.
[0087] In some preferred embodiments, functionalized nucleoside
sequences of the invention possessing an amino group at the
5'-terminus are prepared using a DNA synthesizer, and then reacted
with an active ester derivative of a selected ligand. Active ester
derivatives are well known to those skilled in the art.
Representative active esters include N-hydrosuccinimide esters,
tetrafluorophenolic esters, pentafluorophenolic esters and
pentachlorophenolic esters. The reaction of the amino group and the
active ester produces an oligonucleotide in which the selected
ligand is attached to the 5'-position through a linking group. The
amino group at the 5'-terminus can be prepared utilizing a
5'-Amino-Modifier C6 reagent. In a preferred embodiment, ligand
molecules may be conjugated to oligonucleotides at the 5'-position
by the use of a ligand-nucleoside phosphoramidite wherein the
ligand is linked to the 5'-hydroxy group directly or indirectly via
a linker. Such ligand-nucleoside phosphoramidites are typically
used at the end of an automated synthesis procedure to provide a
ligand-conjugated oligonucleotide bearing the ligand at the
5'-terminus.
[0088] In one preferred embodiment of the methods of the invention,
the preparation of ligand conjugated oligonucleotides commences
with the selection of appropriate precursor molecules upon which to
construct the ligand molecule. Typically, the precursor is an
appropriately-protected derivative of the commonly-used
nucleosides. For example, the synthetic precursors for the
synthesis of the ligand-conjugated oligonucleotides of the
invention include, but are not limited to,
2'-aminoalkoxy-5'-ODMT-nucleosides,
2'-6-aminoalkylamino-5'-ODMT-nucleosides,
5'-6-aminoalkoxy-2'-deoxy-nucleosides,
5'-6-aminoalkoxy-2-protected-nucleosides,
3'-6-aminoalkoxy-5'-ODMT-nucleosides, and
3'-aminoalkylamino-5'-ODMT-nucleosides that may be protected in the
nucleobase portion of the molecule. Methods for the synthesis of
such amino-linked protected nucleoside precursors are known to
those of ordinary skill in the art.
[0089] In many cases, protecting groups are used during the
preparation of the compounds of the invention. As used herein, the
term "protected" means that the indicated moiety has a protecting
group appended thereon. In some preferred embodiments of the
invention, compounds contain one or more protecting groups. A wide
variety of protecting groups can be employed in the methods of the
invention. In general, protecting groups render chemical
functionalities inert to specific reaction conditions, and can be
appended to and removed from such functionalities in a molecule
without substantially damaging the remainder of the molecule.
[0090] Representative hydroxylprotecting groups, for example, are
disclosed by Beaucage et al. (Tetrahedron, 1992, 48:2223-2311).
Further hydroxylprotecting groups, as well as other representative
protecting groups, are disclosed in Greene and Wuts, Protective
Groups in Organic Synthesis, Chapter 2, 2d ed., John Wiley &
Sons, New York, 1991, and Oligonucleotides And Analogues A
Practical Approach, Ekstein, F. Ed., IRL Press, N.Y, 1991.
[0091] Examples of hydroxyl protecting groups include, but are not
limited to, t-butyl, t-butoxymethyl, methoxymethyl,
tetrahydropyranyl, 1-ethoxyethyl, 1-(2-chloroethoxy)ethyl,
2-trimethylsilylethyl, p-chlorophenyl, 2,4-dinitrophenyl, benzyl,
2,6-dichlorobenzyl, diphenylmethyl, p,p'-dinitrobenzhydryl,
p-nitrobenzyl, triphenylmethyl, trimethylsilyl, triethylsilyl,
t-butyldimethylsilyl, t-butyldiphenylsilyl, triphenylsilyl,
benzoylformate, acetate, chloroacetate, trichloroacetate,
trifluoroacetate, pivaloate, benzoate, p-phenylbenzoate,
9-fluorenylmethyl carbonate, mesylate and tosylate.
[0092] Amino-protecting groups stable to acid treatment are
selectively removed with base treatment, and are used to make
reactive amino groups selectively available for substitution.
Examples of such groups are the Fmoc (E. Atherton and R. C.
Sheppard in The Peptides, S. Udenfriend, J. Meienhofer, Eds.,
Academic Press, Orlando, 1987, volume 9, p. 1) and various
substituted sulfonylethyl carbamates exemplified by the Nsc group
(Samukov et al., Tetrahedron Lett., 1994, 35:7821; Verhart and
Tesser, Rec. Trav. Chim. Pays-Bas, 1987, 107:621).
[0093] Additional amino-protecting groups include, but are not
limited to, carbamate protecting groups, such as
2-trimethylsilylethoxycarbonyl (Teoc),
1-methyl-1-(4-biphenylyl)ethoxycarbonyl (Bpoc), t-butoxycarbonyl
(BOC), allyloxycarbonyl (Alloc), 9-fluorenylmethyloxycarbonyl
(Fmoc), and benzyloxycarbonyl (Cbz); amide protecting groups, such
as formyl, acetyl, trihaloacetyl, benzoyl, and nitrophenylacetyl;
sulfonamide protecting groups, such as 2-nitrobenzenesulfonyl; and
imine and cyclic imide protecting groups, such as phthalimido and
dithiasuccinoyl. Equivalents of these amino-protecting groups are
also encompassed by the compounds and methods of the invention.
[0094] Many solid supports are commercially available and one of
ordinary skill in the art can readily select a solid support to be
used in the solid-phase synthesis steps. In certain embodiments, a
universal support is used. A universal support allows for
preparation of oligonucleotides having unusual or modified
nucleotides located at the 3'-terminus of the oligonucleotide.
Universal Support 500 and Universal Support II are universal
supports that are commercially available from Glen Research, 22825
Davis Drive, Sterling, Va. For further details about universal
supports see Scott et al., Innovations and Perspectives in
solid-phase Synthesis, 3rd International Symposium, 1994, Ed. Roger
Epton, Mayflower Worldwide, 115-124]; Azhayev, A. V. Tetrahedron
1999, 55, 787-800; and Azhayev and Antopolsky Tetrahedron 2001, 57,
4977-4986. In addition, it has been reported that the
oligonucleotide can be cleaved from the universal support under
milder reaction conditions when oligonucleotide is bonded to the
solid support via a syn-1,2-acetoxyphosphate group which more
readily undergoes basic hydrolysis. See Guzaev, A. I.; Manoharan,
M. J. Am. Chem. Soc. 2003, 125, 2380.
[0095] The nucleosides are linked by phosphorus-containing or
non-phosphorus-containing covalent internucleoside linkages. For
the purposes of identification, such conjugated nucleosides can be
characterized as ligand-bearing nucleosides or ligand-nucleoside
conjugates. The linked nucleosides having an aralkyl ligand
conjugated to a nucleoside within their sequence will demonstrate
enhanced dsRNA activity when compared to like dsRNA compounds that
are not conjugated.
[0096] The aralkyl-ligand-conjugated oligonucleotides of the
invention also include conjugates of oligonucleotides and linked
nucleosides wherein the ligand is attached directly to the
nucleoside or nucleotide without the intermediacy of a linker
group. The ligand may preferably be attached, via linking groups,
at a carboxyl, amino or oxo group of the ligand. Typical linking
groups may be ester, amide or carbamate groups.
[0097] Specific examples of preferred modified oligonucleotides
envisioned for use in the ligand-conjugated oligonucleotides of the
invention include oligonucleotides containing modified backbones or
non-natural internucleoside linkages. As defined here,
oligonucleotides having modified backbones or internucleoside
linkages include those that retain a phosphorus atom in the
backbone and those that do not have a phosphorus atom in the
backbone. For the purposes of the invention, modified
oligonucleotides that do not have a phosphorus atom in their
intersugar backbone can also be considered to be
oligonucleosides.
[0098] Specific oligonucleotide chemical modifications are
described below. It is not necessary for all positions in a given
compound to be uniformly modified. Conversely, more than one
modifications may be incorporated in a single dsRNA compound or
even in a single nucleotide thereof.
[0099] Preferred modified internucleoside linkages or backbones
include, for example, phosphorothioates, chiral phosphorothioates,
phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters,
methyl and other alkyl phosphonates including 3'-alkylene
phosphonates and chiral phosphonates, phosphinates,
phosphoramidates including 3'-amino phosphoramidate and
aminoalkylphosphoramidates, thionophosphoramidates,
thionoalkylphosphonates, thionoalkylphosphotriesters, and
boranophosphates having normal 3'-5' linkages, 2'-5' linked analogs
of these, and those having inverted polarity wherein the adjacent
pairs of nucleoside units are linked 3'-5' to 5'-3' or 2'-5' to
5'-2'. Various salts, mixed salts and free-acid forms are also
included.
[0100] Representative United States patents relating to the
preparation of the above phosphorus-atom-containing linkages
include, but are not limited to, U.S. Pat. Nos. 3,687,808;
4,469,863; 4,476,301; 5,023,243; 5,177,196; 5,188,897; 5,264,423;
5,276,019; 5,278,302; 5,286,717; 5,321,131; 5,399,676; 5,405,939;
5,453,496; 5,455,233; 5,466,677; 5,476,925; 5,519,126; 5,536,821;
5,541,306; 5,550,111; 5,563,253; 5,571,799; 5,587,361; 5,625,050;
and 5,697,248, each of which is herein incorporated by
reference.
[0101] Preferred modified internucleoside linkages or backbones
that do not include a phosphorus atom therein (i.e.,
oligonucleosides) have backbones that are formed by short chain
alkyl or cycloalkyl intersugar linkages, mixed heteroatom and alkyl
or cycloalkyl intersugar linkages, or one or more short chain
heteroatomic or heterocyclic intersugar linkages. These include
those having morpholino linkages (formed in part from the sugar
portion of a nucleoside); siloxane backbones; sulfide, sulfoxide
and sulfone backbones; formacetyl and thioformacetyl backbones;
methylene formacetyl and thioformacetyl backbones; alkene
containing backbones; sulfamate backbones; methyleneimino and
methylenehydrazino backbones; sulfonate and sulfonamide backbones;
amide backbones; and others having mixed N, O, S and CH.sub.2
component parts.
[0102] Representative United States patents relating to the
preparation of the above oligonucleosides include, but are not
limited to, U.S. Pat. Nos. 5,034,506; 5,166,315; 5,185,444;
5,214,134; 5,216,141; 5,235,033; 5,264,562; 5,264,564; 5,405,938;
5,434,257; 5,466,677; 5,470,967; 5,489,677; 5,541,307; 5,561,225;
5,596,086; 5,602,240; 5,610,289; 5,602,240; 5,608,046; 5,610,289;
5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; and
5,677,439, each of which is herein incorporated by reference.
[0103] In other preferred oligonucleotide mimetics, both the sugar
and the internucleoside linkage, i.e., the backbone, of the
nucleoside units are replaced with novel groups. The nucleobase
units are maintained for hybridization with an appropriate nucleic
acid target compound. One such oligonucleotide, an oligonucleotide
mimetic, that has been shown to have excellent hybridization
properties, is referred to as a peptide nucleic acid (PNA). In PNA
compounds, the sugar-backbone of an oligonucleotide is replaced
with an amide-containing backbone, in particular an
aminoethylglycine backbone. The nucleobases are retained and are
bound directly or indirectly to atoms of the amide portion of the
backbone. Representative United States patents that teach the
preparation of PNA compounds include, but are not limited to, U.S.
Pat. Nos. 5,539,082; 5,714,331; and 5,719,262, each of which is
herein incorporated by reference. Further teaching of PNA compounds
can be found in Nielsen et al., Science, 1991, 254, 1497.
[0104] Some preferred embodiments of the invention employ
oligonucleotides with phosphorothioate linkages and
oligonucleosides with heteroatom backbones, and in particular
--CH.sub.2--NH--O--CH.sub.2--,
--CH.sub.2--N(CH.sub.3)--O--CH.sub.2--[known as a methylene
(methylimino) or MMI backbone],
--CH.sub.2--O--N(CH.sub.3)--CH.sub.2--,
--CH.sub.2--N(CH.sub.3)--N(CH.sub.3)--CH.sub.2--, and
--O--N(CH.sub.3)--CH.sub.2--CH.sub.2-- [wherein the native
phosphodiester backbone is represented as --O--P--O--CH.sub.2--] of
the above referenced U.S. Pat. No. 5,489,677, and the amide
backbones of the above referenced U.S. Pat. No. 5,602,240. Also
preferred are oligonucleotides having morpholino backbone
structures of the above-referenced U.S. Pat. No. 5,034,506.
[0105] The oligonucleotides employed in the ligand-conjugated
oligonucleotides of the invention may additionally or alternatively
comprise nucleobase (often referred to in the art simply as "base")
modifications or substitutions. As used herein, "unmodified" or
"natural" nucleobases include the purine bases adenine (A) and
guanine (G), and the pyrimidine bases thymine (T), cytosine (C),
and uracil (U). Modified nucleobases include other synthetic and
natural nucleobases, such as 5-methylcytosine (5-me-C),
5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine,
6-methyl and other alkyl derivatives of adenine and guanine,
2-propyl and other alkyl derivatives of adenine and guanine,
2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and
cytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine
and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo,
8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted
adenines and guanines, 5-halo particularly 5-bromo,
5-trifluoromethyl and other 5-substituted uracils and cytosines,
7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine,
7-deazaguanine and 7-deazaadenine and 3-deazaguanine and
3-deazaadenine.
[0106] Further nucleobases include those disclosed in U.S. Pat. No.
3,687,808, those disclosed in the Concise Encyclopedia Of Polymer
Science And Engineering, pages 858-859, Kroschwitz, J. I., ed. John
Wiley & Sons, 1990, those disclosed by Englisch et al.,
Angewandte Chemie, International Edition, 1991, 30, 613, and those
disclosed by Sanghvi, Y. S., Chapter 15, Antisense Research and
Applications, pages 289-302, Crooke, S. T. and Lebleu, B., ed., CRC
Press, 1993. Certain of these nucleobases are particularly useful
for increasing the binding affinity of the oligonucleotides of the
invention. These include 5-substituted pyrimidines,
6-azapyrimidines and N-2, N-6 and O-6 substituted purines,
including 2-aminopropyladenine, 5-propynyluracil and
5-propynylcytosine. 5-Methylcytosine substitutions have been shown
to increase nucleic acid duplex stability by 0.6-1.2.degree. C.
(Id., pages 276-278) and are presently preferred base
substitutions, even more particularly when combined with
2'-methoxyethyl sugar modifications.
[0107] Representative United States patents relating to the
preparation of certain of the above-noted modified nucleobases as
well as other modified nucleobases include, but are not limited to,
the above noted U.S. Pat. No. 3,687,808, as well as U.S. Pat. Nos.
4,845,205; 5,130,302; 5,134,066; 5,175,273; 5,367,066; 5,432,272;
5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540;
5,587,469; 5,594,121, 5,596,091; 5,614,617; 5,681,941; and
5,808,027; all of which are hereby incorporated by reference.
[0108] In certain embodiments, the oligonucleotides employed in the
ligand-conjugated oligonucleotides of the invention may
additionally or alternatively comprise one or more substituted
sugar moieties. Preferred oligonucleotides comprise one of the
following at the 2' position: OH; F; O-, S-, or N-alkyl, O-, S-, or
N-alkenyl, or O, S- or N-alkynyl, wherein the alkyl, alkenyl and
alkynyl may be substituted or unsubstituted C.sub.1 to C.sub.10
alkyl or C.sub.2 to C.sub.10 alkenyl and alkynyl. Particularly
preferred are O[(CH.sub.2).sub.nO].sub.mCH.sub.3,
O(CH.sub.2).sub.nOCH.sub.3, O(CH.sub.2).sub.nNH.sub.2,
O(CH.sub.2).sub.nCH.sub.3, O(CH.sub.2).sub.nONH.sub.2, and
O(CH.sub.2).sub.nON[(CH.sub.2).sub.nCH.sub.3)].sub.2, where n and m
are from 1 to about 10. Other preferred oligonucleotides comprise
one of the following at the 2' position: C.sub.1 to C.sub.10 lower
alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or
O-aralkyl, SH, SCH.sub.3, OCN, Cl, Br, CN, CF.sub.3, OCF.sub.3,
SOCH.sub.3, SO.sub.2 CH.sub.3, ONO.sub.2, NO.sub.2, N.sub.3,
NH.sub.2, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino,
polyalkylamino, substituted silyl, an RNA cleaving group, a
reporter group, an intercalator, a group for improving the
pharmacokinetic properties of an oligonucleotide, or a group for
improving the pharmacodynamic properties of an oligonucleotide, and
other substituents having similar properties. a preferred
modification includes 2'-methoxyethoxy
[2'-O--CH.sub.2CH.sub.2OCH.sub.3, also known as
2'-O-(2-methoxyethyl) or 2'-MOE] (Martin et al., Helv. Chim. Acta,
1995, 78, 486), i.e., an alkoxyalkoxy group. A further preferred
modification includes 2'-dimethylaminooxyethoxy, i.e., a
O(CH.sub.2).sub.2ON(CH.sub.3).sub.2 group, also known as 2'-DMAOE,
as described in U.S. Pat. No. 6,127,533, filed on Jan. 30, 1998,
the contents of which are incorporated by reference.
[0109] Other preferred modifications include 2'-methoxy
(2'-O--CH.sub.3), 2'-aminopropoxy
(2'-OCH.sub.2CH.sub.2CH.sub.2NH.sub.2) and 2'-fluoro (2'-F).
Similar modifications may also be made at other positions on the
oligonucleotide, particularly the 3' position of the sugar on the
3' terminal nucleotide or in 2'-5' linked oligonucleotides.
[0110] As used herein, the term "sugar substituent group" or
"2'-substituent group" includes groups attached to the 2'-position
of the ribofuranosyl moiety with or without an oxygen atom. Sugar
substituent groups include, but are not limited to, fluoro,
O-alkyl, O-alkylamino, O-alkylalkoxy, protected O-alkylamino,
O-alkylaminoalkyl, O-alkyl imidazole and polyethers of the formula
(O-alkyl).sub.m, wherein m is 1 to about 10. Preferred among these
polyethers are linear and cyclic polyethylene glycols (PEGs), and
(PEG)-containing groups, such as crown ethers and those which are
disclosed by Ouchi et al. (Drug Design and Discovery 1992, 9:93);
Ravasio et al. (J. Org. Chem. 1991, 56:4329); and Delgardo et. al.
(Critical Reviews in Therapeutic Drug Carrier Systems 1992, 9:249),
each of which is hereby incorporated by reference in its entirety.
Further sugar modifications are disclosed by Cook (Anti-pain Drug
Design, 1991, 6:585-607). Fluoro, O-alkyl, O-alkylamino, O-alkyl
imidazole, O-alkylaminoalkyl, and alkyl amino substitution is
described in U.S. Pat. No. 6,166,197, entitled "Oligomeric
Compounds having Pyrimidine Nucleotide(s) with 2' and 5'
Substitutions," hereby incorporated by reference in its
entirety.
[0111] Additional sugar substituent groups amenable to the
invention include 2'-SR and 2'-NR.sub.2 groups, wherein each R is,
independently, hydrogen, a protecting group or substituted or
unsubstituted alkyl, alkenyl, or alkynyl. 2'-SR Nucleosides are
disclosed in U.S. Pat. No. 5,670,633, hereby incorporated by
reference in its entirety. The incorporation of 2'-SR monomer
synthons is disclosed by Hamm et al. (J. Org. Chem., 1997,
62:3415-3420). 2'-NR nucleosides are disclosed by Goettingen, M.,
J. Org. Chem., 1996, 61, 6273-6281; and Polushin et al.,
Tetrahedron Lett., 1996, 37, 3227-3230. Further representative
2'-substituent groups amenable to the invention include those
having one of formula I or II:
##STR00001##
[0112] wherein,
[0113] E is C.sub.1-C.sub.10 alkyl, N(Q.sub.3)(Q.sub.4) or N.dbd.C
(Q.sub.3)(Q.sub.4); each Q.sub.3 and Q.sub.4 is, independently, H,
C.sub.1-C.sub.10 alkyl, dialkylaminoalkyl, a nitrogen protecting
group, a tethered or untethered conjugate group, a linker to a
solid support; or Q.sub.3 and Q.sub.4, together, form a nitrogen
protecting group or a ring structure optionally including at least
one additional heteroatom selected from N and O;
[0114] q.sub.1 is an integer from 1 to 10;
[0115] q.sub.2 is an integer from 1 to 10;
[0116] q.sub.3 is 0 or 1;
[0117] q.sub.4 is 0, 1 or 2;
[0118] each Z.sub.1, Z.sub.2 and Z.sub.3 is, independently,
C.sub.4-C.sub.7 cycloalkyl, C.sub.5-C.sub.14 aryl or
C.sub.3-C.sub.15 heterocyclyl, wherein the heteroatom in said
heterocyclyl group is selected from oxygen, nitrogen and
sulfur;
[0119] Z.sub.4 is OM.sub.1, SM.sub.1, or N(M.sub.1).sub.2; each
M.sub.1 is, independently, H, C.sub.1-C.sub.8 alkyl,
C.sub.1-C.sub.8 haloalkyl, C(.dbd.NH)N(H)M.sub.2,
C(.dbd.O)N(H)M.sub.2 or OC(.dbd.O)N(H)M.sub.2; M.sub.2 is H or
C.sub.1-C.sub.8 alkyl; and
[0120] Z.sub.5 is C.sub.1-C.sub.10 alkyl, C.sub.1-C.sub.10
haloalkyl, C.sub.2-C.sub.10 alkenyl, C.sub.2-C.sub.10 alkynyl,
C.sub.6-C.sub.14 aryl, N(Q.sub.3)(Q.sub.4), OQ.sub.3, halo,
SQ.sub.3 or CN.
[0121] Representative 2'-O-sugar substituent groups of formula I
are disclosed in U.S. Pat. No. 6,172,209, entitled "Capped
2'-Oxyethoxy Oligonucleotides," hereby incorporated by reference in
its entirety. Representative cyclic 2'-O-sugar substituent groups
of formula II are disclosed in U.S. Pat. No. 6,271,358, entitled
"RNA Targeted 2'-Modified Oligonucleotides that are
Conformationally Preorganized," hereby incorporated by reference in
its entirety.
[0122] Sugars having O-substitutions on the ribosyl ring are also
amenable to the invention. Representative substitutions for ring O
include, but are not limited to, S, CH.sub.2, CHF, and CF.sub.2.
See, e.g., Secrist et al., Abstract 21, Program & Abstracts,
Tenth International Roundtable, Nucleosides, Nucleotides and their
Biological Applications, Park City, Utah, Sep. 16-20, 1992.
[0123] Oligonucleotides may also have sugar mimetics, such as
cyclobutyl moieties, in place of the pentofuranosyl sugar.
Representative United States patents relating to the preparation of
such modified sugars include, but are not limited to, U.S. Pat.
Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878;
5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576,427;
5,591,722; 5,597,909; 5,610,300; 5,627,0531 5,639,873; 5,646,265;
5,658,873; 5,670,633; 5,700,920; and 5,859,221, all of which are
hereby incorporated by reference.
[0124] Additional modifications may also be made at other positions
on the oligonucleotide, particularly the 3' position of the sugar
on the 3' terminal nucleotide. For example, one additional
modification of the ligand-conjugated oligonucleotides of the
invention involves chemically linking to the oligonucleotide one or
more additional non-ligand moieties or conjugates which enhance the
activity, cellular distribution or cellular uptake of the
oligonucleotide. Such moieties include but are not limited to lipid
moieties, such as a cholesterol moiety (Letsinger et al., Proc.
Natl. Acad. Sci. USA, 1989, 86, 6553), cholic acid (Manoharan et
al., Bioorg. Med. Chem. Lett., 1994, 4, 1053), a thioether, e.g.,
hexyl-5-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci., 1992,
660, 306; Manoharan et al., Bioorg. Med. Chem. Let., 1993, 3,
2765), a thiocholesterol (Oberhauser et al., Nucl. Acids Res.,
1992, 20, 533), an aliphatic chain, e.g., dodecandiol or undecyl
residues (Saison-Behmoaras et al., EMBO J., 1991, 10, 111; Kabanov
et al., FEBS Lett., 1990, 259, 327; Svinarchuk et al., Biochimie,
1993, 75, 49), a phospholipid, e.g., di-hexadecyl-rac-glycerol or
triethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate
(Manoharan et al., Tetrahedron Lett., 1995, 36, 3651; Shea et al.,
Nucl. Acids Res., 1990, 18, 3777), a polyamine or a polyethylene
glycol chain (Manoharan et al., Nucleosides & Nucleotides,
1995, 14, 969), or adamantane acetic acid (Manoharan et al.,
Tetrahedron Lett., 1995, 36, 3651), a palmityl moiety (Mishra et
al., Biochim. Biophys. Acta, 1995, 1264, 229), or an octadecylamine
or hexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J.
Pharmacol. Exp. Ther., 1996, 277, 923).
[0125] Representative United States patents relating to the
preparation of such oligonucleotide conjugates include, but are not
limited to, U.S. Pat. Nos. 4,828,979; 4,948,882; 5,218,105;
5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717, 5,580,731;
5,580,731; 5,591,584; 5,109,124; 5,118,802; 5,138,045; 5,414,077;
5,486,603; 5,512,439; 5,578,718; 5,608,046; 4,587,044; 4,605,735;
4,667,025; 4,762,779; 4,789,737; 4,824,941; 4,835,263; 4,876,335;
4,904,582; 4,958,013; 5,082,830; 5,112,963; 5,214,136; 5,082,830;
5,112,963; 5,214,136; 5,245,022; 5,254,469; 5,258,506; 5,262,536;
5,272,250; 5,292,873; 5,317,098; 5,371,241, 5,391,723; 5,416,203,
5,451,463; 5,510,475; 5,512,667; 5,514,785; 5,565,552; 5,567,810;
5,574,142; 5,585,481; 5,587,371; 5,595,726; 5,597,696; 5,599,923;
5,599,928; and 5,688,941, each of which is herein incorporated by
reference.
[0126] The invention also includes compositions employing
oligonucleotides that are substantially chirally pure with regard
to particular positions within the oligonucleotides. Examples of
substantially chirally pure oligonucleotides include, but are not
limited to, those having phosphorothioate linkages that are at
least 75% Sp or Rp (Cook et al., U.S. Pat. No. 5,587,361) and those
having substantially chirally pure (Sp or Rp) alkylphosphonate,
phosphoramidate or phosphotriester linkages (Cook, U.S. Pat. Nos.
5,212,295 and 5,521,302).
[0127] In certain instances, the oligonucleotide may be modified by
a non-ligand group. A number of non-ligand molecules have been
conjugated to oligonucleotides in order to enhance the activity,
cellular distribution or cellular uptake of the oligonucleotide,
and procedures for performing such conjugations are available in
the scientific literature. Such non-ligand moieties have included
lipid moieties, such as cholesterol (Letsinger et al., Proc. Natl.
Acad. Sci. USA, 1989, 86:6553), cholic acid (Manoharan et al.,
Bioorg. Med. Chem. Lett., 1994, 4:1053), a thioether, e.g.,
hexyl-5-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci., 1992,
660:306; Manoharan et al., Bioorg. Med. Chem. Let., 1993, 3:2765),
a thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992,
20:533), an aliphatic chain, e.g., dodecandiol or undecyl residues
(Saison-Behmoaras et al., EMBO J., 1991, 10:111; Kabanov et al.,
FEBS Lett., 1990, 259:327; Svinarchuk et al., Biochimie, 1993,
75:49), a phospholipid, e.g., di-hexadecyl-rac-glycerol or
triethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate
(Manoharan et al., Tetrahedron Lett., 1995, 36:3651; Shea et al.,
Nucl. Acids Res., 1990, 18:3777), a polyamine or a polyethylene
glycol chain (Manoharan et al., Nucleosides & Nucleotides,
1995, 14:969), or adamantane acetic acid (Manoharan et al.,
Tetrahedron Lett., 1995, 36:3651), a palmityl moiety (Mishra et
al., Biochim. Biophys. Acta, 1995, 1264:229), or an octadecylamine
or hexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J.
Pharmacol. Exp. Ther., 1996, 277:923). Representative United States
patents that teach the preparation of such oligonucleotide
conjugates have been listed above. Typical conjugation protocols
involve the synthesis of oligonucleotides bearing an aminolinker at
one or more positions of the sequence. The amino group is then
reacted with the molecule being conjugated using appropriate
coupling or activating reagents. The conjugation reaction may be
performed either with the oligonucleotide still bound to the solid
support or following cleavage of the oligonucleotide in solution
phase. Purification of the oligonucleotide conjugate by HPLC
typically affords the pure conjugate.
[0128] Alternatively, the molecule being conjugated may be
converted into a building block, such as a phosphoramidite, via an
alcohol group present in the molecule or by attachment of a linker
bearing an alcohol group that may be phosphitylated.
[0129] Importantly, each of these approaches may be used for the
synthesis of ligand conjugated oligonucleotides. Aminolinked
oligonucleotides may be coupled directly with ligand via the use of
coupling reagents or following activation of the ligand as an NHS
or pentfluorophenolate ester. Ligand phosphoramidites may be
synthesized via the attachment of an aminohexanol linker to one of
the carboxyl groups followed by phosphitylation of the terminal
alcohol functionality. Other linkers, such as cysteamine, may also
be utilized for conjugation to a chloroacetyl linker present on a
synthesized oligonucleotide.
III. Pharmaceutical Compositions Comprising dsRNA
[0130] In one embodiment, the invention provides pharmaceutical
compositions comprising a dsRNA, as described herein, and a
pharmaceutically acceptable carrier. The pharmaceutical composition
comprising the dsRNA is useful for treating a disease or disorder
associated with the expression or activity of the Nav1.8 gene, such
as neuropathic or inflammatory pain.
[0131] In another embodiment, the invention provides pharmaceutical
compositions comprising at least two dsRNAs, designed to target
different regions of the Nav1.8 gene, and a pharmaceutically
acceptable carrier. In this embodiment, the individual dsRNAs are
prepared as described in the preceding section, which is
incorporated by reference herein. One dsRNA can have a nucleotide
sequence which is substantially complementary to at least one part
of the Nav1.8 gene; additional dsRNAs are prepared, each of which
has a nucleotide sequence that is substantially complementary to
different part of the Nav1.8 gene. The multiple dsRNAs may be
combined in the same pharmaceutical composition, or formulated
separately. If formulated individually, the compositions containing
the separate dsRNAs may comprise the same or different carriers,
and may be administered using the same or different routes of
administration. Moreover, the pharmaceutical compositions
comprising the individual dsRNAs may be administered substantially
simultaneously, sequentially, or at preset intervals throughout the
day or treatment period.
[0132] The pharmaceutical compositions of the invention are
administered in dosages sufficient to inhibit expression of the
Nav1.8 gene. The present inventors have found that, because of
their improved efficiency, compositions comprising the dsRNA of the
invention can be administered at surprisingly low dosages. A
maximum dosage of 5 mg dsRNA per kilogram body weight of recipient
per day is sufficient to inhibit or completely suppress expression
of the Nav1.8 gene, or alleviate chronic pain.
[0133] In general, a suitable dose of dsRNA will be in the range of
0.01 to 5.0 milligrams per kilogram body weight of the recipient
per day, preferably in the range of 0.1 to 200 micrograms per
kilogram body weight per day, more preferably in the range of 0.1
to 100 micrograms per kilogram body weight per day, even more
preferably in the range of 1.0 to 50 micrograms per kilogram body
weight per day, and most preferably in the range of 1.0 to 25
micrograms per kilogram body weight per day. The pharmaceutical
composition may be administered once daily, or the dsRNA may be
administered as two, three, four, five, six or more sub-doses at
appropriate intervals throughout the day or even using continuous
infusion. In that case, the dsRNA contained in each sub-dose must
be correspondingly smaller in order to achieve the total daily
dosage. The dosage unit can also be compounded for delivery over
several days, e.g., using a conventional sustained release
formulation which provides sustained release of the dsRNA over a
several day period. Sustained release formulations are well known
in the art. In this embodiment, the dosage unit contains a
corresponding multiple of the daily dose.
[0134] The skilled artisan will appreciate that certain factors may
influence the dosage and timing 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. Moreover, treatment of a
subject with a therapeutically effective amount of a composition
can include a single treatment or a series of treatments. Estimates
of effective dosages and in vivo half-lives for the individual
dsRNAs encompassed by the invention can be made using conventional
methodologies or on the basis of in vivo testing using an
appropriate animal model, as described elsewhere herein.
[0135] Advances in mouse genetics have generated a number of mouse
models for the study of various human diseases, such as pain. Such
models are used for in vivo testing of dsRNA, as well as for
determining a therapeutically effective dose.
[0136] The pharmaceutical compositions encompassed by the invention
may be administered by any means known in the art including, but
not limited to oral or parenteral routes, including intravenous,
intramuscular, intraperitoneal, epidural, intrathecal,
intracerebroventricular, intraparenchymal (within the peripheral or
central nervous system), subcutaneous, transdermal, intranasal,
airway (aerosol), rectal, vaginal and topical (including buccal and
sublingual) administration. In preferred embodiments, the
pharmaceutical compositions are administered intrathecally by
continuous infusion such as with a pump, or intrathecally by bolus
injection. In other preferred embodiments, the pharmaceutical
compositions are administered intravenously by continuous infusion
such as with a pump, or intravenously by bolus injection.
[0137] For intrathecal, intracerebroventricular, intramuscular,
intraparenchymal (within the peripheral or central nervous system),
subcutaneous and intravenous use, the pharmaceutical compositions
of the invention will generally be provided in sterile aqueous
solutions or suspensions, buffered to an appropriate pH and
isotonicity. Suitable aqueous vehicles include Ringer's solution
and isotonic sodium chloride. In a preferred embodiment, the
carrier consists exclusively of an aqueous buffer. In this context,
"exclusively" means no auxiliary agents or encapsulating substances
are present which might affect or mediate uptake of dsRNA in the
cells that express the Nav1.8 gene. Such substances include, for
example, micellar structures, such as liposomes or capsids, as
described below. Surprisingly, the present inventors have
discovered that compositions containing only naked dsRNA and a
physiologically acceptable solvent are taken up by cells, where the
dsRNA effectively inhibits expression of the Nav1.8 gene. Although
microinjection, lipofection, viruses, viroids, capsids, capsoids,
or other auxiliary agents are required to introduce dsRNA into cell
cultures, surprisingly these methods and agents are not necessary
for uptake of dsRNA in vivo. Aqueous suspensions according to the
invention may include suspending agents such as cellulose
derivatives, sodium alginate, polyvinyl-pyrrolidone and gum
tragacanth, and a wetting agent such as lecithin. Suitable
preservatives for aqueous suspensions include ethyl and n-propyl
p-hydroxybenzoate.
[0138] The pharmaceutical compositions useful according to the
invention also include encapsulated formulations to protect the
dsRNA against rapid elimination from the body, such as a controlled
release formulation, including implants and microencapsulated
delivery systems. Biodegradable, biocompatible polymers can be
used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic
acid, collagen, polyorthoesters, and polylactic acid. Methods for
preparation of such formulations will be apparent to those skilled
in the art. The materials can also be obtained commercially from
Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal
suspensions (including liposomes targeted to infected cells with
monoclonal antibodies to viral antigens) can also be used as
pharmaceutically acceptable carriers. These can be prepared
according to methods known to those skilled in the art, for
example, as described in U.S. Pat. No. 4,522,811; PCT publication
WO 91/06309; and European patent publication EP-A-43075, which are
incorporated by reference herein.
[0139] Using the small interfering RNA vectors previously
described, the invention also provides devices, systems, and
methods for delivery of small interfering RNA to target locations
in the nervous system and or the brain. The envisioned route of
delivery is through the use of implanted, indwelling, intrathecal,
intracerebroventricular or intraparenchymal catheters that provide
a means for injecting small volumes of fluid containing the dsRNA
of the invention directly into local nerves or local brain tissue,
or into bodily fluids surrounding these tissues. The proximal end
of these catheters may be connected to an implanted, intrathecal or
intracerebral access port surgically affixed to the patient's body
or cranium, or to an implanted drug pump located in the patient's
torso.
[0140] Alternatively, implantable delivery devices, such as an
implantable pump may be employed. Examples of the delivery devices
within the scope of the invention include the Model 8506
investigational device (by Medtronic, Inc. of Minneapolis, Minn.),
which can be implanted subcutaneously in the body or on the
cranium, and provides an access port through which therapeutic
agents may be delivered to the nerves or brain. Delivery occurs
through a stereotactically implanted polyurethane catheter. Two
models of catheters that can function with the Model 8506 access
port include the Model 8770 ventricular catheter by Medtronic,
Inc., for delivery to the intracerebral ventricles, which is
disclosed in U.S. Pat. No. 6,093,180, incorporated herein by
reference, and the IPA1 catheter by Medtronic, Inc., for delivery
to the brain tissue itself (i.e., intraparenchymal delivery),
disclosed in U.S. Ser. Nos. 09/540,444 and 09/625,751, which are
incorporated herein by reference. The latter catheter has multiple
outlets on its distal end to deliver the therapeutic agent to
multiple sites along the catheter path. In addition to the
aforementioned device, the delivery of the small interfering RNA
vectors in accordance with the invention can be accomplished with a
wide variety of devices, including but not limited to U.S. Pat.
Nos. 5,735,814, 5,814,014, and 6,042,579, all of which are
incorporated herein by reference. Using the teachings of the
invention and those of skill in the art will recognize that these
and other devices and systems may be suitable for delivery of small
interfering RNA vectors for the treatment of pain in accordance
with the invention.
[0141] In one such embodiment, the method further comprises the
steps of implanting a pump outside the body or brain, the pump
coupled to a proximal end of the catheter, and operating the pump
to deliver the predetermined dosage of the at least one small
interfering RNA or small interfering RNA vector through the
discharge portion of the catheter. A further embodiment comprises
the further step of periodically refreshing a supply of the at
least one small interfering RNA or small interfering RNA vector to
the pump outside said body or brain.
[0142] Thus, the invention includes the delivery of small
interfering RNA vectors using an implantable pump and catheter,
like that taught in U.S. Pat. Nos. 5,735,814 and 6,042,579, and
further using a sensor as part of the infusion system to regulate
the amount of small interfering RNA vectors delivered to the nerves
or brain, like that taught in U.S. Pat. No. 5,814,014. Other
devices and systems can be used in accordance with the method of
the invention, for example, the devices and systems disclosed in
U.S. Ser. Nos. 09/872,698 (filed Jun. 1, 2001) and 09/864,646
(filed May 23, 2001), which are incorporated herein by
reference.
[0143] Toxicity and therapeutic efficacy of such compounds can be
determined by standard pharmaceutical procedures in cell cultures
or experimental animals, e.g., for determining the LD50 (the dose
lethal to 50% of the population) and the ED50 (the dose
therapeutically effective in 50% of the population). The dose ratio
between toxic and therapeutic effects is the therapeutic index and
it can be expressed as the ratio LD50/ED50. Compounds which exhibit
high therapeutic indices are preferred.
[0144] The data obtained from cell culture assays and animal
studies can be used in formulation a range of dosage for use in
humans. The dosage of compositions of the invention lies preferably
within a range of circulating concentrations that include the ED50
with little or no toxicity. The dosage may vary within this range
depending upon the dosage form employed and the route of
administration utilized. For any compound used in the method of the
invention, the therapeutically effective dose can be estimated
initially from cell culture assays. A dose may be formulated in
animal models to achieve a circulating plasma concentration range
of the compound or, when appropriate, of the polypeptide product of
a target sequence (e.g., achieving a decreased concentration of the
polypeptide) that includes the IC50 (i.e., the concentration of the
test compound which achieves a half-maximal inhibition of symptoms)
as determined in cell culture. Such information can be used to more
accurately determine useful doses in humans. Levels in plasma may
be measured, for example, by high performance liquid
chromatography.
[0145] In addition to their administration individually or as a
plurality, as discussed above, the dsRNAs of the invention can be
administered in combination with other known agents effective in
treatment of pain. In any event, the administering physician can
adjust the amount and timing of dsRNA administration on the basis
of results observed using standard measures of efficacy known in
the art or described herein.
[0146] Methods for Treating Diseases Caused by Expression of the
Nav1.8 Gene
[0147] In one embodiment, the invention provides a method for
treating a subject having a pathological condition mediated by the
expression of the Nav1.8 gene, such as neuropathic or inflammatory
pain. In this embodiment, the dsRNA acts as a therapeutic agent for
controlling the expression of the Nav1.8 protein. The method
comprises administering a pharmaceutical composition of the
invention to the patient (e.g., human), such that expression of the
Nav1.8 gene is silenced. Because of their high specificity, the
dsRNAs of the invention specifically target mRNAs of the Nav1.8
gene.
[0148] Pain
[0149] As used herein, the term "pain" is art recognized and
includes a bodily sensation elicited by noxious chemical,
mechanical, or thermal stimuli, in a subject, e.g., a mammal such
as a human. The term "pain" includes chronic pain such as lower
back pain; pain due to arthritis, e.g., osteoarthritis; joint pain,
e.g., knee pain or carpal tunnel syndrome; myofascial pain, and
neuropathic pain. The term "pain" further includes acute pain, such
as pain associated with muscle strains and sprains; tooth pain;
headaches; pain associated with surgery; and pain associated with
various forms of tissue injury, e.g., inflammation, infection, and
ischemia.
[0150] "Neuropathic pain" refers to pain caused by injury or
disease of the central or peripheral nervous system. In contrast to
the immediate (acute) pain caused by tissue injury, neuropathic
pain can develop days or months after a traumatic injury.
Neuropathic pain frequently is long lasting or chronic, and is not
limited in duration to the period of tissue repair. Neuropathic
pain can occur spontaneously, or as a result of stimulation that
normally is not painful. Neuropathic pain is caused by aberrant
somatosensory processing, and is associated with chronic sensory
disturbances, including spontaneous pain, hyperalgesia (i.e.,
sensation of more pain than the stimulus would warrant) and
allodynia (i.e., a condition in which ordinarily painless stimuli
induce the experience of pain). Neuropathic pain includes, but is
not limited to, pain caused by peripheral nerve trauma, viral
infection, diabetes mellitus, chemotherapy, causalgia,
plexus-avulsion, spinal cord injury, neuroma, limb amputation,
vasculitis, nerve damage from surgery, nerve damage from chronic
alcoholism, hypothyroidism, uremia, and vitamin deficiencies, among
other causes. Neuropathic pain is one type of pain associated with
cancer. Cancer pain can also be "nociceptive" or "mixed."
[0151] "Chronic pain" can be defined as pain lasting longer than
three months (Bonica, Semin. Anesth. 1986, 5:82-99), and may be
characterized by unrelenting persistent pain that is not fully
amenable to routine pain control methods. Chronic pain includes,
but is not limited to, inflammatory pain, post-operative pain,
cancer pain, osteoarthritis pain associated with metastatic cancer,
chemotherapy-induced pain, trigeminal neuralgia, acute herpetic and
post-herpetic neuralgia, diabetic neuropathy, pain due to
arthritis, joint pain, myofascial pain, causalgia, brachial plexus
avulsion, occipital neuralgia, reflex sympathetic dystrophy,
fibromyalgia, gout, phantom limb pain, burn pain, pain associated
with spinal cord injury, multiple sclerosis, reflex sympathetic
dystrophy and lower back pain and other forms of neuralgia,
neuropathic, and idiopathic pain syndromes.
[0152] "Nociceptive pain" is due to activation of pain-sensitive
nerve fibers, either somatic or visceral. Nociceptive pain is
generally a response to direct tissue damage. The initial trauma
causes the release of several chemicals including bradykinin,
serotonin, substance P, histamine, and prostaglandin. When somatic
nerves are involved, the pain is typically experienced as an aching
or pressure-like sensation.
[0153] In the phrase "pain and related disorders", the term
"related disorders" refers to disorders that either cause or are
associated with pain, or have been shown to have similar mechanisms
to pain. These disorders include addiction, seizure, stroke,
ischemia, a neurodegenerative disorder, anxiety, depression,
headache, asthma, rheumatic disease, osteoarthritis, retinopathy,
inflammatory eye disorders, pruritis, ulcer, gastric lesions,
uncontrollable urination, an inflammatory or unstable bladder
disorder, inflammatory bowel disease, irritable bowel syndrome
(IBS), irritable bowel disease (IBD), gastroesophageal reflux
disease (GERD), functional dyspepsia, functional chest pain of
presumed oesophageal origin, functional dysphagia, non-cardiac
chest pain, symptomatic gastroesophageal disease, gastritis,
aerophagia, functional constipation, functional diarrhea,
burbulence, chronic functional abdominal pain, recurrent abdominal
pain (RAP), functional abdominal bloating, functional biliary pain,
functional incontinence, functional ano-rectal pain, chronic pelvic
pain, pelvic floor dyssenergia, unspecified functional ano-rectal
disorder, cholecystalgia, interstitial cystitis, dysmenorrhea, and
dyspareunia.
[0154] The invention thus provides the use of an anti-Nav1.8 dsRNA
administered to a human, particularly by intrathecal infusion or
injection, or by intravenous infusion or injection, for the
treatment of pain, including neuropathic and inflammatory pain.
[0155] The pharmaceutical compositions encompassed by the invention
may be administered by any means known in the art including, but
not limited to oral or parenteral routes, including intravenous,
intramuscular, intraperitoneal, epidural, intrathecal,
intracerebroventricular, intraparenchymal (within the peripheral or
central nervous system), subcutaneous, transdermal, intranasal,
airway (aerosol), nasal, rectal, vaginal and topical (including
buccal and sublingual) administration, and epidural administration.
In preferred embodiments, the pharmaceutical compositions are
administered intrathecally by continuous infusion such as with a
pump, or intrathecally by bolus injection. In other preferred
embodiments, the pharmaceutical compositions are administered
intravenously by continuous infusion such as with a pump, or
intravenously by bolus injection.
[0156] Methods for Inhibiting Expression of the Nav1.8 Gene
[0157] In yet another aspect, the invention provides a method for
inhibiting the expression of the Nav1.8 gene in a mammal. The
method comprises administering a composition of the invention to
the mammal such that expression of the target Nav1.8 gene is
silenced. Because of their high specificity, the dsRNAs of the
invention specifically target RNAs (primary or processed) of the
target Nav1.8 gene. Compositions and methods for inhibiting the
expression of these Nav1.8 genes using dsRNAs can be performed as
described elsewhere herein.
[0158] In one embodiment, the method comprises administering a
composition comprising a dsRNA, wherein the dsRNA comprises a
nucleotide sequence which is complementary to at least a part of an
RNA transcript of the Nav1.8 gene of the mammal to be treated. When
the organism to be treated is a mammal such as a human, the
composition may be administered by any means known in the art
including, but not limited to oral or parenteral routes, including
intravenous, intramuscular, intraperitoneal, epidural, intrathecal,
intracerebroventricular, intraparenchymal (within the peripheral or
central nervous system), subcutaneous, transdermal, intranasal,
airway (aerosol), rectal, vaginal and topical (including buccal and
sublingual) administration. In preferred embodiments, the
compositions are administered by intrathecal infusion or injection,
or by intravenous infusion or injection.
[0159] Unless otherwise defined, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. Although
methods and materials similar or equivalent to those described
herein can be used in the practice or testing of the invention,
suitable methods and materials are described below. All
publications, patent applications, patents, and other references
mentioned herein are incorporated by reference in their entirety.
In case of conflict, the present specification, including
definitions, will control. In addition, the materials, methods, and
examples are illustrative only and not intended to be limiting.
EXAMPLES
Example 1
Gene Walking of the Nav1.8 Gene
[0160] siRNAs were identified in a multi step sequence analysis
process in order to design siRNAs targeting the Nav1.8 gene in 4
species of interest.
[0161] ClustalW multiple alignment function of BioEdit Sequence
Alignment Editor (version 7.0.4.1) was used to generate a global
alignment of human (NM.sub.--006514), mouse (NM.sub.--009134), rat
(NM.sub.--017247) and dog (NM001003203) Nav1.8 mRNA sequences.
[0162] Conserved regions were identified by embedded sequence
analysis function of the software. Conserved regions were defined
as sequence stretches with a minimum length of 19 bases for all
aligned sequences containing no internal gaps. Sequence positions
of conserved regions were counted according to the human
sequence.
[0163] The siRNA design web interface at Whitehead Institute for
Biomedical Research was used to identify all potential siRNAs
targeting the conserved regions as well as their respective
off-target hits to sequences in the human, mouse and rat RefSeq
database. siRNAs satisfying the crossreactivity criteria were
selected out of the candidates pool and subjected to the software
embedded off-target analysis. For this, all selected siRNAs were
analyzed in 3 rounds by the NCBI blast algorithm against the NCBI
human, mouse and rat RefSeq database.
[0164] Blast results were downloaded and analyzed by a perl script
in order to extract the identity of the best off-target hit for the
antisense strain as well as the positions of occurring
mismatches.
[0165] All siRNA candidates were ranked according to predicted
properties. For this, different criteria were applied in order to
identify siRNAs with the following properties: [0166] reactivity
criterium: targeting human, mouse, rat and dog sequences [0167]
specificity criteria: highly specific for human, at least specific
for rat and mouse
[0168] The 2 siRNAs that satisfied the applied criteria were
referred to as "multi-species targeting siRNAs".
[0169] In order to identify more siRNAs a second round of siRNA
identification steps were conducted correspondingly with additional
regions that had been previously eliminated due to missing
reactivity with dog sequences.
[0170] The resulting pool of 10 siRNAs matching the above mentioned
criteria were referred to as `human/rat/mouse targeting
siRNAs`.
[0171] A third round of the siRNA design process was conducted
while disregarding cross-reactivity to mouse.
[0172] All candidate siRNAs were again extracted and ranked in 3
steps according to the following criteria:
[0173] Step 1: [0174] reactivity criterium: targeting human, rat
and mouse sequences [0175] specificity criterium: highly specific
for human and rat, moderately specific for mouse [0176] 4 siRNAs
(added to pool of `human/rat/mouse targeting siRNAs`)
[0177] Step 2: [0178] reactivity criterium: targeting human and rat
sequences [0179] sequence-embedded criterium: absense of stretches
with more than 3 Gs in a row [0180] specificity criterium: highly
specific for human [0181] 19 siRNAs
[0182] Step 3: [0183] reactivity criterium: targeting human and rat
sequences [0184] specificity criterium: highly specific for rat,
specific for human and favorable ddG value [0185] 1 siRNA
[0186] The pool resulting form steps 2 and 3 (20 siRNAs) were
referred to as `human/rat targeting siRNAs`.
[0187] The in silico selected 36 siRNAs were synthesized (Table
1).
[0188] Additional sequence selections were performed using the
above generalized methods except that cross reactivity between
species was not used as a selection criterea: the sequence
selection was based solely on the human Nav1.8 sequence. In
addition, ranking was based on off-target scores based on the
closest FASTA hit as was as the number of potential off target
genes. These siRNAs are provided in Table 4.
TABLE-US-00001 TABLE 1 siRNAs specific for Nav1.8 SEQ SEQ ID
antisense strand sequence ID duplex name sense strand sequence
(5'-3') NO: (5'-3') NO: AL-DP-6042
cmcmcmggaumumumumaacmumacmacmcmTT 1 ggugumaguumaaaauccgggTT 2
AL-DP-6043 cmcmggaumumumumaacmumacmacmcmaTT 3
uggugumaguumaaaauccggTT 4 AL-DP-6044
gacmaacmcmcmggaumumumumaacmumTT 5 aguumaaaauccggguugucTT 6
AL-DP-6045 cmcmumumacmaacmcmagcmgcmaggaTT 7 uccugcgcugguugumaaggTT
8 AL-DP-6046 aacmcmcmggaumumumumaacmumacmaTT 9
ugumaguumaaaauccggguuTT 10 AL-DP-6047
umgumgcmaumgacmcmcmgaacmumgaTT 11 ucmaguucgggucmaugcmacmaTT 12
AL-DP-6048 acmaacmcmagcmgcmaggaumgumcmTT 13 gacmauccugcgcugguuguTT
14 AL-DP-6049 acmcmcmggaumumumumaacmumacmTT 15
gugumaguumaaaauccggguTT 16 AL-DP-6050
cmaumcmcmumaumgaacmcmaaumagcmTT 17 gcumauugguucmaumaggaugTT 18
AL-DP-6051 cmumumacmaacmcmagcmgcmaggaumTT 19 auccugcgcugguugumaagTT
20 AL-DP-6052 umumumaacmumacmacmcmagcmumumumgTT 21
cmaaagcuggugumaguumaaaTT 22 AL-DP-6053
gumgumgcmaumgacmcmcmgaacmumgTT 23 cmaguucgggucmaugcmacmacTT 24
AL-DP-6202 cmggaumumumumaacmumacmacmcmagTT 25
cuggugumaguumaaaauccgTT 26 AL-DP-6203 umacmaacmcmagcmgcmaggaumgumTT
27 acmauccugcgcugguugumaTT 28 AL-DP-6204
ggumcmumcmumgumgcmcmcmaumumgcmumTT 29 agcmaaugggcmacmagagaccTT 30
AL-DP-6205 cmcmcmaagumumcmumaumggumgagcmTT 31
gcucmaccmaumagaacuugggTT 32 AL-DP-6206
cmaagumumcmumaumggumgagcmumcmTT 33 gagcucmaccmaumagaacuugTT 34
AL-DP-6207 cmumacmagcmacmacmacmcmggacmaTT 35 uguccggugugugcugumagTT
36 AL-DP-6208 aagumumcmumaumggumgagcmumcmcmTT 37
ggagcucmaccmaumagaacuuTT 38 AL-DP-6209
umumumumgumcmumaaaumgagumumcmaTT 39 ugaacucmauuumagacmaaaaTT 40
AL-DP-6210 cmcmumcmumcmacmumgumumcmcmgcmcmumcmTT 41
gaggcggaacmagugagaggTT 42 AL-DP-6211
aacmcmagumumcmumumumgumggcmcmgTT 43 cggccmacmaaagaacugguuTT 44
AL-DP-6212 cmumcmacmumgumumcmcmgcmcmumcmaumgTT 45
cmaugaggcggaacmagugagTT 46 AL-DP-6213
cmcmaagumumcmumaumggumgagcmumTT 47 agcucmaccmaumagaacuuggTT 48
AL-DP-6214 cmagumumcmumumumgumggcmcmgumcmumTT 49
agacggccmacmaaagaacugTT 50 AL-DP-6215 ggcmumggcmaggumgcmgcmaagaTT
51 ucuugcgcmaccugccmagccTT 52 AL-DP-6216
cmumcmcmumcmumgagggcmagcmacmgTT 53 cgugcugcccucmagaggagTT 54
AL-DP-6217 gumcmumumcmacmumgummcaumumumacmaTT 55
ugumaaaugacmagugaagacTT 56 AL-DP-6218
cmaacmcmagcmgcmaggaumgumcmumTT 57 agacmauccugcgcugguugTT 58
AL-DP-6219 agaagumaumcmumgaumcmumgggaTT 59 ucccmagaucmagaumacuucuTT
60 AL-DP-6220 umcmumacmagcmacmacmacmcmggacmTT 61
guccggugugugcugumagaTT 62 AL-DP-6221
agumumcmumaumggumgagcmumcmcmcmTT 63 gggagcucmaccmaumagaacuTT 64
AL-DP-6222 umcmumaumggumgagcmumcmcmcmagcmTT 65
gcugggagcucmaccmaumagaTT 66 AL-DP-6223
agumumcmumumumgumggcmcmgumcmumumTT 67 aagacggccmacmaaagaacuTT 68
AL-DP-6224 umcmacmumgumumcmcmgcmcmumcmaumgaTT 69
ucmaugaggcggaacmagugaTT 70 AL-DP-6225
ggaumumumumaacmumacmacmcmagcmTT 71 gcuggugumaguumaaaauccTT 72
TABLE-US-00002 TABLE 4 Further siRNAs specific for Nav1.8 SEQ SEQ
Remaining Luc ID Antisense strand sequence ID activity [% of duplex
name Sense strand sequence (5'-3') NO: (5'-3') NO: controls]
AD-11159 ccgcuugcugcgcguauucTT 73 gaauacgcgcagcaagcggTT 74 26 .+-.
2 AD-11160 cuugcuaccguaucguggaTT 75 uccacgauacgguagcaagTT 76 52
.+-. 4 AD-11161 gacuucaucgcuaauccgaTT 77 ucggauuagcgaugaagucTT 78
29 .+-. 4 AD-11162 uggauuuuagcgucauuacTT 79 guaaugacgcuaaaauccaTT
80 70 .+-. 6 AD-11163 cgcuugcugcgcguauucaTT 81
ugaauacgcgcagcaagcgTT 82 31 .+-. 5 AD-11164 caacuuccgucgcuuuacuTT
83 aguaaagcgacggaaguugTT 84 24 .+-. 2 AD-11165
gucgcuuuacuccggagucTT 85 gacuccggaguaaagcgacTT 86 20 .+-. 3
AD-11166 aaugaguucacguaccugaTT 87 ucagguacgugaacucauuTT 88 17 .+-.
3 AD-11167 agacuugcuaccguaucguTT 89 acgauacgguagcaagucuTT 90 38
.+-. 2 AD-11168 aguuuauuuauuacggucaTT 91 ugaccguaauaaauaaacuTT 92
63 .+-. 7 AD-11169 aaaugaguucacguaccugTT 93 cagguacgugaacucauuuTT
94 46 .+-. 4 AD-11170 ugucucggcauucgaugcaTT 95
ugcaucgaaugccgagacaTT 96 72 .+-. 5 AD-11171 ucaaagcccuucgaacccuTT
97 aggguucgaagggcuuugaTT 97 96 .+-. 4 AD-11172
gcgggcucuuucucgauuuTT 99 aaaucgagaaagagcccgcTT 100 34 .+-. 4
AD-11173 ccuuguaccuuugucgauuTT 101 aaucgacaaagguacaaggTT 102 17
.+-. 2 AD-11174 gugguucucuccauugcgaTT 103 ucgcaauggagagaaccacTT 104
24 .+-. 3 AD-11175 caaaaggccuaucggagcuTT 105 agcuccgauaggccuuuugTT
106 46 .+-. 3 AD-11176 aaaggccuaucggagcuauTT 107
auagcuccgauaggccuuuTT 108 48 .+-. 5 AD-11177 ggugcaucaacuauaccgaTT
109 ucgguauaguugaugcaccTT 110 21 .+-. 2 AD-11178
aacuaccguaacaaccgaaTT 111 uucgguuguuacgguaguuTT 112 42 .+-. 8
AD-11179 ucgcuuuacuccggagucaTT 113 ugacuccggaguaaagcgaTT 114 17
.+-. 1 AD-11180 gaaaacgccgggcuagucaTT 115 ugacuagcccggcguuuucTT 116
36 .+-. 8 AD-11181 accgaaaaaauaucuccgcTT 117 gcggagauauuuuuucgguTT
118 105 .+-. 15 AD-11182 uucaucgcuaauccgacugTT 119
cagucggauuagcgaugaaTT 120 87 .+-. 14 AD-11183 uccgucgcuuuacuccggaTT
121 uccggaguaaagcgacggaTT 122 33 .+-. 3 AD-11184
gcuuuacuccggagucacuTT 123 agugacuccggaguaaagcTT 124 14 .+-. 1
AD-11185 ggaaaacgccgggcuagucTT 125 gacuagcccggcguuuuccTT 126 50
.+-. 5 AD-11186 acuaccguaacaaccgaaaTT 127 uuucgguuguuacgguaguTT 128
41 .+-. 5 AD-11187 uggccaucguaccaaacagTT 129 cuguuugguacgauggccaTT
130 85 .+-. 7 AD-11188 acuugcuaccguaucguggTT 131
ccacgauacgguagcaaguTT 132 109 .+-. 15 AD-11189
gauaagucucacagcgaagTT 133 cuucgcugugagacuuaucTT 134 29 .+-. 4
AD-11190 auaagucucacagcgaagaTT 135 ucuucgcugugagacuuauTT 136 34
.+-. 4 AD-11191 aagcccuucgaacccuucgTT 137 cgaaggguucgaagggcuuTT 138
86 .+-. 10 AD-11192 agcccuucgaacccuucgcTT 139 gcgaaggguucgaagggcuTT
140 98 .+-. 16 AD-11193 cccuucgaacccuucgcgcTT 141
gcgcgaaggguucgaagggTT 142 64 .+-. 4 AD-11194 aacccaaucgaaauauacuTT
143 aguauauuucgauuggguuTT 144 40 .+-. 4 AD-11195
cgcuuuacuccggagucacTT 145 gugacuccggaguaaagcgTT 146 17 .+-. 2
AD-11196 gaccauuucccgguuuaguTT 147 acuaaaccgggaaauggucTT 148 77
.+-. 4 AD-11197 cgguuuagugccacucgggTT 149 cccgaguggcacuaaaccgTT 150
58 .+-. 6 AD-11198 cacagcaauagaucuccguTT 151 acggagaucuauugcugugTT
152 34 .+-. 4 AD-11199 acuagggauugacacaaccTT 153
gguugugucaaucccuaguTT 154 86 .+-. 3 AD-11200 cccacaauggaucaccuuuTT
155 aaaggugauccauugugggTT 156 22 .+-. 0 AD-11201
ugucuuuucuaggccucgcTT 157 gcgaggccuagaaaagacaTT 158 91 .+-. 10
AD-11202 agcugucgaugucucggcaTT 159 ugccgagacaucgacagcuTT 160 42
.+-. 2 AD-11203 gucucggcauucgaugcagTT 161 cugcaucgaaugccgagacTT 162
62 .+-. 5 AD-11204 ucaaaaucauugccuucgaTT 163 ucgaaggcaaugauuuugaTT
164 42 .+-. 7 AD-11205 cccgcuggcacaugcacgaTT 165
ucgugcaugugccagcgggTT 166 42 .+-. 4 AD-11206 ucauugucuuccguauccuTT
167 aggauacggaagacaaugaTT 168 79 .+-. 11 AD-11207
uuggccaucguaccaaacaTT 169 uguuugguacgauggccaaTT 170 59 .+-. 8
AD-11208 gcacgguggacugccuagaTT 171 ucuaggcaguccaccgugcTT 172 95
.+-. 12 AD-11209 gcccuucgaacccuucgcgTT 173 cgcgaaggguucgaagggcTT
174 48 .+-. 4 AD-11210 ugcgggcucuuucucgauuTT 175
aaucgagaaagagcccgcaTT 176 35 .+-. 5 AD-11211 gaggugcaucaacuauaccTT
177 gguauaguugaugcaccucTT 178 36 .+-. 4 AD-11212
caucaacuauaccgauggaTT 179 uccaucgguauaguugaugTT 180 58 .+-. 4
AD-11213 aaaucauccuaugaaccaaTT 181 uugguucauaggaugauuuTT 182 26
.+-. 2 AD-11214 aaggccuaucggagcuaugTT 183 cauagcuccgauaggccuuTT 184
63 .+-. 4 AD-11215 uguacucccagacaaaucuTT 185 agauuugucugggaguacaTT
186 40 .+-. 3 AD-11216 aggacaucuagcucaauacTT 187
guauugagcuagauguccuTT 188 29 .+-. 2 AD-11217 ccgguuuagugccacucggTT
189 ccgaguggcacuaaaccggTT 190 22 .+-. 2 AD-11218
caucgcuaauccgacugugTT 191 cacagucggauuagcgaugTT 192 51 .+-. 2
AD-11219 aaacuaccguaacaaccgaTT 193 ucgguuguuacgguaguuuTT 194 29
.+-. 3 AD-11220 aucgcuaauccgacuguguTT 195 acacagucggauuagcgauTT 196
52 .+-. 1 AD-11221 cccgguuuagugccacucgTT 197 cgaguggcacuaaaccgggTT
198 60 .+-. 1 AD-11222 uuuagcgucauuacccuggTT 199
ccaggguaaugacgcuaaaTT 200 105 .+-. 3 AD-11223 aauaagcgaggcacuucugTT
201 cagaagugccucgcuuauuTT 202 66 .+-. 5 AD-11224
cuaccguaacaaccgaaaaTT 203 uuuucgguuguuacgguagTT 204 25 .+-. 3
AD-11225 ucgcuaauccgacugugugTT 205 cacacagucggauuagcgaTT 206 70
.+-. 5 AD-11226 ucccucgaaacuaacaacuTT 207 aguuguuaguuucgagggaTT 208
24 .+-. 3 AD-11227 cuuccgucgcuuuacuccgTT 209 cggaguaaagcgacggaagTT
210 31 .+-. 0 AD-11228 uuucccgguuuagugccacTT 211
guggcacuaaaccgggaaaTT 212 101 .+-. 2 AD-11229 gguuuagugccacucgggcTT
213 gcccgaguggcacuaaaccTT 214 67 .+-. 1 AD-11230
acaacccggauuuuaacuaTT 215 uaguuaaaauccggguuguTT 216 48 .+-. 3
AD-11231 cuagggauugacacaaccuTT 217 agguugugucaaucccuagTT 218 39
.+-. 2 AD-11232 ugucgauugugaauaacaaTT 219 uuguuauucacaaucgacaTT 220
25 .+-. 3 AD-11233 caucgugaccagacaagcuTT 221 agcuugucuggucacgaugTT
222 47 .+-. 4 AD-11234 ccuaucggagcuaugugcuTT 223
agcacauagcuccgauaggTT 224 58 .+-. 7 AD-11235 uuagcgucauuacccuggcTT
225 gccaggguaaugacgcuaaTT 226 92 .+-. 7 AD-11236
agcaauagaucuccgugggTT 227 cccacggagaucuauugcuTT 228 83 .+-. 4
AD-11237 aucgaaauauacugauccaTT 229 uggaucaguauauuucgauTT 230 57
.+-. 3 AD-11238 ggaucccucgaaacuaacaTT 231 uguuaguuucgagggauccTT 232
18 .+-. 1 AD-11239 acaccggacauuuauggugTT 233 caccauaaauguccgguguTT
234 96 .+-. 2 AD-11240 guuuagugccacucgggccTT 235
ggcccgaguggcacuaaacTT 236 95 .+-. 17 AD-11241 augacccgaacugaccuucTT
237 gaaggucaguucgggucauTT 238 80 .+-. 7 AD-11242
auuuuagcgucauuacccuTT 239 aggguaaugacgcuaaaauTT 240 70 .+-. 5
AD-11243 uuuuagcgucauuacccugTT 241 caggguaaugacgcuaaaaTT 242 77
.+-. 8 AD-11244 gcaauagaucuccgugggaTT 243 ucccacggagaucuauugcTT 244
23 .+-. 1 AD-11245 aaauaagcgaggcacuucuTT 245 agaagugccucgcuuauuuTT
246 38 .+-. 3 AD-11246 auaagcgaggcacuucugaTT 247
ucagaagugccucgcuuauTT 248 32 .+-. 2 AD-11247 ugauccuuacaaccagcgcTT
249 gcgcugguuguaaggaucaTT 250 78 .+-. 2 AD-11248
uggccgagauaucucacucTT 251 gagugagauaucucggccaTT 252 66 .+-. 1
AD-11249 caaccgccgcccacuagugTT 253 cacuagugggcggcgguugTT 254 71
.+-. 4 AD-11250 uuagaugaaccuuuccgggTT 255 cccggaaagguucaucuaaTT 256
90 .+-. 3 AD-11251 aaccuuuccgggcccaaagTT 257 cuuugggcccggaaagguuTT
258 99 .+-. 7 AD-11252 auaaccuccguccuugaggTT 259
ccucaaggacggagguuauTT 260 105 .+-. 2 AD-11253 cuugugacggaucccuuugTT
261 caaagggauccgucacaagTT 262 62 .+-. 5 AD-11254
ccuaccuucgaagccaugcTT 263 gcauggcuucgaagguaggTT 264 84 .+-. 6
AD-11255 aaaucauugccuucgacccTT 265 gggucgaaggcaaugauuuTT 266 92
.+-. 6 AD-11256 cauugccuucgacccauacTT 267 guaugggucgaaggcaaugTT 268
42 .+-. 2 AD-11257 cuucgacccauacuauuauTT 269 auaauaguaugggucgaagTT
270 35 .+-. 2 AD-11258 ucaucgcuaauccgacuguTT 271
acagucggauuagcgaugaTT 272 76 .+-. 2 AD-11259 aauccgacugugugggucuTT
273 agacccacacagucggauuTT 274 87 .+-. 5 AD-11260
agcacgguggacugccuagTT 275 cuaggcaguccaccgugcuTT 276 75 .+-. 7
AD-11261 ggugcgcaagacuugcuacTT 277 guagcaagucuugcgcaccTT 278 34
.+-. 3 AD-11262 aggugcaucaacuauaccgTT 279 cgguauaguugaugcaccuTT 280
59 .+-. 5 AD-11263 aucaacuauaccgauggagTT 281 cuccaucgguauaguugauTT
282 113 .+-. 10 AD-11264 uugucgauugugaauaacaTT 283
uguuauucacaaucgacaaTT 284 36 .+-. 5 AD-11265 aauggguuaccuugcacuuTT
285 aagugcaagguaacccauuTT 286 70 .+-. 6 AD-11266
aggccuaucggagcuauguTT 287 acauagcuccgauaggccuTT 288 72 .+-. 4
AD-11267 ggccuaucggagcuaugugTT 289 cacauagcuccgauaggccTT 290 65
.+-. 7 AD-11268 uaucggagcuaugugcugcTT 291 gcagcacauagcuccgauaTT 292
110 .+-. 10 AD-11269 ccguccuaugagagugucaTT 293
ugacacucucauaggacggTT 294 50 .+-. 3 AD-11270 ccauuggaucccucgaaacTT
295 guuucgagggauccaauggTT 296 18 .+-. 3 AD-11271
cauuggaucccucgaaacuTT 297 aguuucgagggauccaaugTT 298 23 .+-. 3
AD-11272 aucccucgaaacuaacaacTT 299 guuguuaguuucgagggauTT 300 76
.+-. 5 AD-11273 gaaacuaacaacuuccgucTT 301 gacggaaguuguuaguuucTT 302
21 .+-. 3 AD-11274 uuccgucgcuuuacuccggTT 303 ccggaguaaagcgacggaaTT
304 87 .+-. 6 AD-11275 ccgucgcuuuacuccggagTT 305
cuccggaguaaagcgacggTT 306 29 .+-. 2 AD-11276 ggaucuagauccguucuacTT
307 guagaacggaucuagauccTT 308 22 .+-. 3 AD-11277
cacacaccggacauuuaugTT 309 cauaaauguccggugugugTT 310 57 .+-. 2
AD-11278 cacaccggacauuuaugguTT 311 accauaaauguccggugugTT 312 101
.+-. 1 AD-11279 ggaccauuucccgguuuagTT 313 cuaaaccgggaaaugguccTT 314
45 .+-. 4 AD-11280 accauuucccgguuuagugTT 315 cacuaaaccgggaaaugguTT
316 89 .+-. 2
AD-11281 auuucccgguuuagugccaTT 317 uggcacuaaaccgggaaauTT 318 100
.+-. 4 AD-11282 aaccugaucagaagaacggTT 319 ccguucuucugaucagguuTT 320
66 .+-. 5 AD-11283 ucaguuuauuuauuacgguTT 321 accguaauaaauaaacugaTT
322 76 .+-. 4 AD-11284 gugcaugacccgaacugacTT 323
gucaguucgggucaugcacTT 324 29 .+-. 1 AD-11285 augaguucacguaccugagTT
325 cucagguacgugaacucauTT 326 37 .+-. 4 AD-11286
agcgucauuacccuggcauTT 327 augccaggguaaugacgcuTT 328 31 .+-. 1
AD-11287 gcgucauuacccuggcauaTT 329 uaugccaggguaaugacgcTT 330 16
.+-. 1 AD-11288 gucauuacccuggcauaugTT 331 cauaugccaggguaaugacTT 332
21 .+-. 1 AD-11289 acagcaauagaucuccgugTT 333 cacggagaucuauugcuguTT
334 64 .+-. 4 AD-11290 ggacauucagaguucuuagTT 335
cuaagaacucugaauguccTT 336 26 .+-. 1 AD-11291 ucuacauaaauaagcgaggTT
337 ccucgcuuauuuauguagaTT 338 88 .+-. 15 AD-11292
uaaauaagcgaggcacuucTT 339 gaagugccucgcuuauuuaTT 340 68 .+-. 2
AD-11293 ugcccugaugguuauaucuTT 341 agauauaaccaucagggcaTT 342 39
.+-. 3 AD-11294 caacccggauuuuaacuacTT 343 guaguuaaaauccggguugTT 344
26 .+-. 2 AD-11295 gggaaaaucuauaugaucuTT 345 agaucauauagauuuucccTT
346 15 .+-. 1 AD-11296 gcccucgagaugcuccggaTT 347
uccggagcaucucgagggcTT 348 54 .+-. 9 AD-11297 agggauugacacaaccucuTT
349 agagguugugucaaucccuTT 350 22 .+-. 1 AD-11298
gggauugacacaaccucucTT 351 gagagguugugucaaucccTT 352 22 .+-. 2
AD-11299 cacaauggaucaccuuuaaTT 353 uuaaaggugauccauugugTT 354 23
.+-. 0 AD-11300 ccgcucugauccuuacaacTT 355 guuguaaggaucagagcggTT 356
34 .+-. 2 AD-11301 auccuuacaaccagcgcagTT 357 cugcgcugguuguaaggauTT
358 90 .+-. 5 AD-11302 agcgcaggaugucuuuucuTT 359
agaaaagacauccugcgcuTT 360 46 .+-. 3 AD-11303 cuuuucuaggccucgccucTT
361 gaggcgaggccuagaaaagTT 362 94 .+-. 10 AD-11304
cuggaaaacgccgggcuagTT 363 cuagcccggcguuuuccagTT 364 57 .+-. 2
AD-11305 aaacgccgggcuagucaugTT 365 caugacuagcccggcguuuTT 366 95
.+-. 7 AD-11306 aacgccgggcuagucauggTT 367 ccaugacuagcccggcguuTT 368
101 .+-. 5 AD-11307 agaccacgaaagccaucggTT 369 ccgauggcuuucguggucuTT
370 89 .+-. 6 AD-11308 cucccuagaagcccucuucTT 371
gaagagggcuucuagggagTT 372 50 .+-. 3 AD-11309 agaugaacaccaaccgccgTT
373 cggcgguugguguucaucuTT 374 62 .+-. 7 AD-11310
augaacaccaaccgccgccTT 375 ggcggcgguugguguucauTT 376 99 .+-. 6
AD-11311 aaccgccgcccacuagugaTT 377 ucacuagugggcggcgguuTT 378 72
.+-. 2 AD-11312 accgccgcccacuagugagTT 379 cucacuagugggcggcgguTT 380
99 .+-. 13 AD-11313 gcugucgaugucucggcauTT 381 augccgagacaucgacagcTT
382 67 .+-. 8 AD-11314 ucgaugucucggcauucgaTT 383
ucgaaugccgagacaucgaTT 384 36 .+-. 2 AD-11315 ucucggcauucgaugcaggTT
385 ccugcaucgaaugccgagaTT 386 99 .+-. 5 AD-11316
ggcauucgaugcaggacaaTT 387 uuguccugcaucgaaugccTT 388 48 .+-. 5
AD-11317 gcauucgaugcaggacaaaTT 389 uuuguccugcaucgaaugcTT 390 56
.+-. 5 AD-11318 cuuagaugaaccuuuccggTT 391 ccggaaagguucaucuaagTT 392
47 .+-. 3 AD-11319 gaguguugucaguaucauaTT 393 uaugauacugacaacacucTT
394 22 .+-. 2 AD-11320 caguaucauaaccuccgucTT 395
gacggagguuaugauacugTT 396 27 .+-. 2 AD-11321 cucgaggagucugaacagaTT
397 ucuguucagacuccucgagTT 398 32 .+-. 2 AD-11322
guaucugaucugggauugcTT 399 gcaaucccagaucagauacTT 400 65 .+-. 3
AD-11323 agacaauucucuuugggcuTT 401 agcccaaagagaauugucuTT 402 73
.+-. 2 AD-11324 caccuugugcaucguggugTT 403 caccacgaugcacaaggugTT 404
47 .+-. 4 AD-11325 gcaugagcccuaccuucgaTT 405 ucgaagguagggcucaugcTT
406 25 .+-. 1 AD-11326 uaccuucgaagccaugcucTT 407
gagcauggcuucgaagguaTT 408 71 .+-. 2 AD-11327 caaaaucauugccuucgacTT
409 gucgaaggcaaugauuuugTT 410 33 .+-. 3 AD-11328
aucauugccuucgacccauTT 411 augggucgaaggcaaugauTT 412 38 .+-. 2
AD-11329 ucauugccuucgacccauaTT 413 uaugggucgaaggcaaugaTT 414 29
.+-. 1 AD-11330 auugccuucgacccauacuTT 415 aguaugggucgaaggcaauTT 416
77 .+-. 7 AD-11331 ucgacccauacuauuauuuTT 417 aaauaauaguaugggucgaTT
418 55 .+-. 3 AD-11332 caucaucgucacugugaguTT 419
acucacagugacgaugaugTT 420 31 .+-. 3 AD-11333 caucgucacugugagucugTT
421 cagacucacagugacgaugTT 422 33 .+-. 4 AD-11334
ucgucacugugagucugcuTT 423 agcagacucacagugacgaTT 424 44 .+-. 4
AD-11335 ggaaaacuaccguaacaacTT 425 guuguuacgguaguuuuccTT 426 34
.+-. 1 AD-11336 uaccguaacaaccgaaaaaTT 427 uuuuucgguuguuacgguaTT 428
21 .+-. 3 AD-11337 cguaacaaccgaaaaaauaTT 429 uauuuuuucgguuguuacgTT
430 29 .+-. 2 AD-11338 aaaauccauaugccucaucTT 431
gaugaggcauauggauuuuTT 432 87 .+-. 4 AD-11339 cuguucaucgcccugcuauTT
433 auagcagggcgaugaacagTT 434 40 .+-. 2 AD-11340
uguucaucgcccugcuauuTT 435 aauagcagggcgaugaacaTT 436 26 .+-. 1
AD-11341 cccugcuauugaacucuuuTT 437 aaagaguucaauagcagggTT 438 29
.+-. 1 AD-11342 ggccaucguaccaaacaggTT 439 ccuguuugguacgauggccTT 440
41 .+-. 2 AD-11343 ccaucguaccaaacaggcuTT 441 agccuguuugguacgauggTT
442 47 .+-. 2 AD-11344 cagugacuucaucgcuaauTT 443
auuagcgaugaagucacugTT 444 33 .+-. 2 AD-11345 cuucaucgcuaauccgacuTT
445 agucggauuagcgaugaagTT 446 36 .+-. 2 AD-11346
uaauccgacugugugggucTT 447 gacccacacagucggauuaTT 448 97 .+-. 3
AD-11347 gaaucugaucuugaugacuTT 449 agucaucaagaucagauucTT 450 45
.+-. 2 AD-11348 aagaguccaugggauguggTT 451 ccacaucccauggacucuuTT 452
100 .+-. 2 AD-11349 gcaggugcgcaagacuugcTT 453 gcaagucuugcgcaccugcTT
454 39 .+-. 3 AD-11350 gcgcaagacuugcuaccguTT 455
acgguagcaagucuugcgcTT 456 29 .+-. 1 AD-11351 aagacuugcuaccguaucgTT
457 cgauacgguagcaagucuuTT 458 82 .+-. 2 AD-11352
gacuugcuaccguaucgugTT 459 cacgauacgguagcaagucTT 460 56 .+-. 2
AD-11353 cucauugugaauaucucacTT 461 gugagauauucacaaugagTT 462 54
.+-. 3 AD-11354 ugauaagucucacagcgaaTT 463 uucgcugugagacuuaucaTT 464
31 .+-. 1 AD-11355 aucaaagcccuucgaacccTT 465 ggguucgaagggcuuugauTT
466 87 .+-. 8 AD-11356 aaagcccuucgaacccuucTT 467
gaaggguucgaagggcuuuTT 468 83 .+-. 3 AD-11357 ccuucgaacccuucgcgcuTT
469 agcgcgaaggguucgaaggTT 470 50 .+-. 1 AD-11358
cuucgaacccuucgcgcucTT 471 gagcgcgaaggguucgaagTT 472 55 .+-. 4
AD-11359 ugcaucaacuauaccgaugTT 473 caucgguauaguugaugcaTT 474 59
.+-. 7 AD-11360 cccuuguaccuuugucgauTT 475 aucgacaaagguacaagggTT 476
24 .+-. 3 AD-11361 uuguaccuuugucgauuguTT 477 acaaucgacaaagguacaaTT
478 39 .+-. 6 AD-11362 uuugauaauguugcaauggTT 479
ccauugcaacauuaucaaaTT 480 110 .+-. 14 AD-11363
gcaauggguuaccuugcacTT 481 gugcaagguaacccauugcTT 482 93 .+-. 6
AD-11364 uggguuaccuugcacuucuTT 483 agaagugcaagguaacccaTT 484 54
.+-. 11 AD-11365 gcuguugauucccgggaggTT 485 ccucccgggaaucaacagcTT
486 36 .+-. 1 AD-11366 aucgugaccagacaagcuuTT 487
aagcuugucuggucacgauTT 488 36 .+-. 5 AD-11367 cgucuucacaggcgaauguTT
489 acauucgccugugaagacgTT 490 57 .+-. 2 AD-11368
ucacaggcgaaugugucauTT 491 augacacauucgccugugaTT 492 76 .+-. 5
AD-11369 cacaggcgaaugugucaugTT 493 caugacacauucgccugugTT 494 75
.+-. 4 AD-11370 caggcgaaugugucaugaaTT 495 uucaugacacauucgccugTT 496
32 .+-. 5 AD-11371 aggcgaaugugucaugaagTT 497 cuucaugacacauucgccuTT
498 48 .+-. 6 AD-11372 uguucgcuuugaggcaguaTT 499
uacugccucaaagcgaacaTT 500 36 .+-. 4 AD-11373 gcaguacuacuucacaaauTT
501 auuugugaaguaguacugcTT 502 27 .+-. 2 AD-11374
uccauugcgagccugauuuTT 503 aaaucaggcucgcaauggaTT 504 24 .+-. 2
AD-11375 ccauugcgagccugauuuuTT 505 aaaaucaggcucgcaauggTT 506 21
.+-. 3 AD-11376 aucgggcuguugcuauuccTT 507 ggaauagcaacagcccgauTT 508
78 .+-. 11 AD-11377 aaaacccaaucgaaauauaTT 509 uauauuucgauuggguuuuTT
510 33 .+-. 4 AD-11378 caaucgaaauauacugaucTT 511
gaucaguauauuucgauugTT 512 66 .+-. 8 AD-11379 aaucgaaauauacugauccTT
513 ggaucaguauauuucgauuTT 514 128 .+-. 8 AD-11380
ucgaaauauacugauccagTT 515 cuggaucaguauauuucgaTT 516 119 .+-. 7
AD-11381 aaucauccuaugaaccaauTT 517 auugguucauaggaugauuTT 518 39
.+-. 3 AD-11382 uaugaaccaauagcaaccaTT 519 ugguugcuauugguucauaTT 520
61 .+-. 4 AD-11383 cacucuccgauggaagcaaTT 521 uugcuuccaucggagagugTT
522 69 .+-. 5 AD-11384 cuaucggagcuaugugcugTT 523
cagcacauagcuccgauagTT 524 95 .+-. 10 AD-11385 agcaaaugaaaauuguguaTT
525 uacacaauuuucauuugcuTT 526 79 .+-. 6 AD-11386
uacucccagacaaaucugaTT 527 ucagauuugucugggaguaTT 528 36 .+-. 4
AD-11387 agagugucacuagaggccuTT 529 aggccucuagugacacucuTT 530 36
.+-. 1 AD-11388 ggccuuagugauagagucaTT 531 ugacucuaucacuaaggccTT 532
35 .+-. 4 AD-11389 agugauagagucaacaugaTT 533 ucauguugacucuaucacuTT
534 29 .+-. 5 AD-11390 gauagagucaacaugaggaTT 535
uccucauguugacucuaucTT 536 32 .+-. 3 AD-11391 caucuagcucaauacaaaaTT
537 uuuuguauugagcuagaugTT 538 23 .+-. 3 AD-11392
cuagcucaauacaaaaugaTT 539 ucauuuuguauugagcuagTT 540 28 .+-. 5
AD-11393 aguauggagcugauugcccTT 541 gggcaaucagcuccauacuTT 542 108
.+-. 8
Example 2
Optimization of siRNAs by Chemical Modification
[0189] As has been experienced by those working in the antisense
field, ribonucleic acids are often quickly degraded by a range of
nucleases present in virtually all biological environments, e.g.
endonucleases, exonucleases etc. This vulnerability may be
circumvented by chemically modifying these oligonucleotides such
that nucleases may no longer attack. Consequently, siRNAs in Table
1 represent chemically modified oligonucleotides; these chemically
modified siRNAs were tested for inhibitory activity on Nav1.8 gene
expression (Nav1.8 mRNA levels).
[0190] dsRNA Synthesis
[0191] Source of Reagents
[0192] 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.
[0193] siRNA Synthesis
[0194] Single-stranded RNAs were produced by solid phase synthesis
on a scale of 1 .mu.mole using an Expedite 8909 synthesizer
(Applied Biosystems, Applera Deutschland GmbH, Darmstadt, Germany)
and controlled pore glass (CPG, 500A, Proligo Biochemie GmbH,
Hamburg, Germany) 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).
[0195] Deprotection and purification of the crude
oligoribonucleotides by anion exchange HPLC 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, UnterschleiBheim, 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 annealed RNA solution was stored at -20.degree. C. until
use.
[0196] For the synthesis of 3'-cholesterol-conjugated siRNAs
(herein referred to as -Chol or -sChol, depending on whether the
link to the cholesteryl group is effected via a phosphodiester or a
phosphorothioate diester group), an appropriately modified solid
support was used for RNA synthesis. The modified solid support was
prepared as follows:
Diethyl-2-azabutane-1,4-dicarboxylate AA
##STR00002##
[0198] A 4.7 M aqueous solution of sodium hydroxide (50 mL) was
added into a stirred, ice-cooled solution of ethyl glycinate
hydrochloride (32.19 g, 0.23 mole) in water (50 mL). Then, ethyl
acrylate (23.1 g, 0.23 mole) was added and the mixture was stirred
at room temperature until completion of the reaction was
ascertained by TLC. After 19 h the solution was partitioned with
dichloromethane (3.times.100 mL). The organic layer was dried with
anhydrous sodium sulfate, filtered and evaporated. The residue was
distilled to afford AA (28.8 g, 61%).
3-{Ethoxycarbonylmethyl-[6-(9H-fluoren-9-ylmethoxycarbonyl-amino)-hexanoyl-
]-amino}-propionic acid ethyl ester AB
##STR00003##
[0200] Fmoc-6-amino-hexanoic acid (9.12 g, 25.83 mmol) was
dissolved in dichloromethane (50 mL) and cooled with ice.
Diisopropylcarbodiimde (3.25 g, 3.99 mL, 25.83 mmol) was added to
the solution at 0.degree. C. It was then followed by the addition
of Diethyl-azabutane-1,4-dicarboxylate (5 g, 24.6 mmol) and
dimethylamino pyridine (0.305 g, 2.5 mmol). The solution was
brought to room temperature and stirred further for 6 h. Completion
of the reaction was ascertained by TLC. The reaction mixture was
concentrated under vacuum and ethyl acetate was added to
precipitate diisopropyl urea. The suspension was filtered. The
filtrate was washed with 5% aqueous hydrochloric acid, 5% sodium
bicarbonate and water. The combined organic layer was dried over
sodium sulfate and concentrated to give the crude product which was
purified by column chromatography (50% EtOAC/Hexanes) to yield
11.87 g (88%) of AB.
3-[(6-Amino-hexanoyl)-ethoxycarbonylmethyl-amino]-propionic acid
ethyl ester AC
##STR00004##
[0202]
3-{Ethoxycarbonylmethyl-[6-(9H-fluoren-9-ylmethoxycarbonylamino)-he-
xanoyl]-amino}-propionic acid ethyl ester AB (11.5 g, 21.3 mmol)
was dissolved in 20% piperidine in dimethylformamide at 0.degree.
C. The solution was continued stifling for 1 h. The reaction
mixture was concentrated under vacuum, water was added to the
residue, and the product was extracted with ethyl acetate. The
crude product was purified by conversion into its hydrochloride
salt.
3-({6-[17-(1,5-Dimethyl-hexyl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,1-
5,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yloxycarbonylamino]-h-
exanoyl}ethoxycarbonylmethyl-amino)-propionic acid ethyl ester
AD
##STR00005##
[0204] The hydrochloride salt of
3-[(6-Amino-hexanoyl)-ethoxycarbonylmethyl-amino]-propionic acid
ethyl ester AC (4.7 g, 14.8 mmol) was taken up in dichloromethane.
The suspension was cooled to 0.degree. C. on ice. To the suspension
diisopropylethylamine (3.87 g, 5.2 mL, 30 mmol) was added. To the
resulting solution cholesteryl chloroformate (6.675 g, 14.8 mmol)
was added. The reaction mixture was stirred overnight. The reaction
mixture was diluted with dichloromethane and washed with 10%
hydrochloric acid. The product was purified by flash chromatography
(10.3 g, 92%).
1-{6-[17-(1,5-Dimethyl-hexyl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15-
,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yloxycarbonylamino]-he-
xanoyl}-4-oxo-pyrrolidine-3-carboxylic acid ethyl ester AE
##STR00006##
[0206] Potassium t-butoxide (1.1 g, 9.8 mmol) was slurried in 30 mL
of dry toluene. The mixture was cooled to 0.degree. C. on ice and 5
g (6.6 mmol) of diester AD was added slowly with stirring within 20
mins. The temperature was kept below 5.degree. C. during the
addition. The stifling was continued for 30 mins at 0.degree. C.
and 1 mL of glacial acetic acid was added, immediately followed by
4 g of NaH.sub.2PO.sub.4.H.sub.2O in 40 mL of water The resultant
mixture was extracted twice with 100 mL of dichloromethane each and
the combined organic extracts were washed twice with 10 mL of
phosphate buffer each, dried, and evaporated to dryness. The
residue was dissolved in 60 mL of toluene, cooled to 0.degree. C.
and extracted with three 50 mL portions of cold pH 9.5 carbonate
buffer. The aqueous extracts were adjusted to pH 3 with phosphoric
acid, and extracted with five 40 mL portions of chloroform which
were combined, dried and evaporated to dryness. The residue was
purified by column chromatography using 25% ethylacetate/hexane to
afford 1.9 g of b-ketoester (39%).
[6-(3-Hydroxy-4-hydroxymethyl-pyrrolidin-1-yl)-6-oxo-hexyl]-carbamic
acid
17-(1,5-dimethyl-hexyl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,1-
7-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl ester AF
##STR00007##
[0208] Methanol (2 mL) was added dropwise over a period of 1 h to a
refluxing mixture of b-ketoester AE (1.5 g, 2.2 mmol) and sodium
borohydride (0.226 g, 6 mmol) in tetrahydrofuran (10 mL). Stirring
was continued at reflux temperature for 1 h. After cooling to room
temperature, 1 N HCl (12.5 mL) was added, the mixture was extracted
with ethylacetate (3.times.40 mL). The combined ethylacetate layer
was dried over anhydrous sodium sulfate and concentrated under
vacuum to yield the product which was purified by column
chromatography (10% MeOH/CHCl.sub.3) (89%).
(6-{3-[Bis-(4-methoxy-phenyl)-phenyl-methoxymethyl]-4-hydroxy-pyrrolidin-1-
-yl}-6-oxo-hexyl)-carbamic acid
17-(1,5-dimethyl-hexyl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,1-
7-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl ester AG
##STR00008##
[0210] Diol AF (1.25 gm 1.994 mmol) was dried by evaporating with
pyridine (2.times.5 mL) in vacuo. Anhydrous pyridine (10 mL) and
4,4'-dimethoxytritylchloride (0.724 g, 2.13 mmol) were added with
stirring. The reaction was carried out at room temperature
overnight. The reaction was quenched by the addition of methanol.
The reaction mixture was concentrated under vacuum and to the
residue dichloromethane (50 mL) was added. The organic layer was
washed with 1M aqueous sodium bicarbonate. The organic layer was
dried over anhydrous sodium sulfate, filtered and concentrated. The
residual pyridine was removed by evaporating with toluene. The
crude product was purified by column chromatography (2%
MeOH/Chloroform, Rf=0.5 in 5% MeOH/CHCl.sub.3) (1.75 g, 95%).
Succinic acid
mono-(4-[bis-(4-methoxy-phenyl)-phenyl-methoxymethyl]-1-{6-[17-(1,5-dimet-
hyl-hexyl)-10,13-dimethyl
2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H
cyclopenta[a]phenanthren-3-yloxycarbonylamino]-hexanoyl}-pyrrolidin-3-yl)-
ester AH
##STR00009##
[0212] Compound AG (1.0 g, 1.05 mmol) was mixed with succinic
anhydride (0.150 g, 1.5 mmol) and DMAP (0.073 g, 0.6 mmol) and
dried in a vacuum at 40.degree. C. overnight. The mixture was
dissolved in anhydrous dichloroethane (3 mL), triethylamine (0.318
g, 0.440 mL, 3.15 mmol) was added and the solution was stirred at
room temperature under argon atmosphere for 16 h. It was then
diluted with dichloromethane (40 mL) and washed with ice cold
aqueous citric acid (5 wt %, 30 mL) and water (2.times.20 mL). The
organic phase was dried over anhydrous sodium sulfate and
concentrated to dryness. The residue was used as such for the next
step.
[0213] Cholesterol Derivatised CPG AI
##STR00010##
[0214] Succinate AH (0.254 g, 0.242 mmol) was dissolved in a
mixture of dichloromethane/acetonitrile (3:2, 3 mL). To that
solution DMAP (0.0296 g, 0.242 mmol) in acetonitrile (1.25 mL),
2,2'-Dithio-bis(5-nitropyridine) (0.075 g, 0.242 mmol) in
acetonitrile/dichloroethane (3:1, 1.25 mL) were added successively.
To the resulting solution triphenylphosphine (0.064 g, 0.242 mmol)
in acetonitrile (0.6 ml) was added. The reaction mixture turned
bright orange in color. The solution was agitated briefly using a
wrist-action shaker (5 mins). Long chain alkyl amine-CPG (LCAA-CPG)
(1.5 g, 61 mM) was added. The suspension was agitated for 2 h. The
CPG was filtered through a sintered funnel and washed with
acetonitrile, dichloromethane and ether successively. Unreacted
amino groups were masked using acetic anhydride/pyridine. The
achieved loading of the CPG was measured by taking UV measurement
(37 mM/g).
[0215] The synthesis of siRNAs bearing a 5'-12-dodecanoic acid
bisdecylamide group (herein referred to as "5'-C32-") or a
5'-cholesteryl derivative group (herein referred to as "5'-Chol-")
was performed as described in WO 2004/065601, except that, for the
cholesteryl derivative, the oxidation step was performed using the
Beaucage reagent in order to introduce a phosphorothioate linkage
at the 5'-end of the nucleic acid oligomer.
[0216] Nucleic acid sequences are represented below using standard
nomenclature, and specifically the abbreviations of Table 2.
TABLE-US-00003 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 N, n any 2'-deoxy-nucleotide/nucleotide (G, A,
C, or T, g, a, c or u) Am 2'-O-methyladenosine-5'-phosphate Cm
2'-O-methylcytidine-5'-phosphate Gm
2'-O-methylguanosine-5'-phosphate Tm
2'-O-methyl-thymidine-5'-phosphate Um
2'-O-methyluridine-5'-phosphate Af
2'-fluoro-2'-deoxy-adenosine-5'-phosphate Cf
2'-fluoro-2'-deoxy-cytidine-5'-phosphate Gf
2'-fluoro-2'-deoxy-guanosine-5'-phosphate Tf
2'-fluoro-2'-deoxy-thymidine-5'-phosphate Uf
2'-fluoro-2'-deoxy-uridine-5'-phosphate A, C, G, T, U, underlined:
nucleoside-5'-phosphorothioate a, c, g, t, u am, cm, gm,
underlined: 2-O-methyl-nucleoside-5'-phosphorothioate tm, um
.sup.acapital letters represent 2'-deoxyribonucleotides (DNA),
lower case letters represent ribonucleotides (RNA)
[0217] Single-Dose Screen of Nav1.8 siRNAs Against mRNA Expression
of Transfected Human Nav1.8 in Cos-7 Cells.
[0218] All Nav1.8 siRNAs in Table 1 and Table 4 were tested
initially at a single dose of 100 nM (Table 1) or 50 nM (Table 4)
for activity in reducing mRNA expression of transfected human
Nav1.8 in Cos-7 cells. One day before transfection, Cos-7 cells
(DSMZ, Braunschweig, Germany) were seeded at 1.5.times.10.sup.4
cells/well on 96-well plates (Greiner Bio-One GmbH, Frickenhausen,
Germany) in 100 .mu.l of growth medium (Dulbecco's MEM, 10% fetal
calf serum, 2 mM L-glutamine, 1.2 .mu.g/ml sodium bicarbonate, 100
u penicillin/100 .mu.g/ml streptomycin, all from Biochrom AG,
Berlin, Germany). Four hours prior to siRNA transfection, 20 ng of
plasmid/well (Table 1) or 50 ng/well (Table 4) were transfected
with Lipofectamine-2000 (Invitrogen) as described below for the
siRNAs, with the plasmid diluted in Opti-MEM to a final volume of
12.5 .mu.l/well, prepared as a mastermix for the whole plate.
[0219] siRNA transfections were performed in triplicate. For each
well, 0.5 .mu.l Lipofectamine2000 (Invitrogen GmbH, Karlsruhe,
Germany) was mixed with 12 .mu.l Opti-MEM (Invitrogen) and
incubated for 15 min at room temperature. For an siRNA
concentration of 100 nM in a transfection volume of 100 .mu.l, 2
.mu.l of a 5 .mu.M siRNA were mixed with 10.5 .mu.l Opti-MEM per
well, combined with the Lipofectamine2000-Opti-MEM mixture and
again incubated for 15 minutes at room temperature. During that
incubation time, growth medium was removed from cells and replaced
by 75 .mu.l/well of fresh medium. In six wells, growth medium was
replaced by 100 .mu.l of fresh medium and cells were lysed
immediately by adding lysis mixture, as described below, in order
to analyse the background value in the bDNA-assay caused by the
Nav1.8-cDNA in the plasmid. siRNA-Lipofectamine2000-complexes were
applied completely (25 .mu.l each per well) to the cells and cells
were incubated for 24 h at 37.degree. C. and 5% CO.sub.2 in a
humidified incubator (Heraeus GmbH, Hanau, Germany).
[0220] Cells were harvested by applying 50 .mu.l of lysis mixture
(from the QuantiGene bDNA-kit from Genospectra, Fremont, USA) to
each well and were lysed at 53.degree. C. for 30 min. Afterwards,
50 .mu.l of the lysates were incubated with probesets specific to
human Nav1.8 and human GAPDH (sequence of probesets see below) and
processed according to the manufacturer's protocol for QuantiGene.
Chemoluminescence was measured in a Victor2-Light (Perkin Elmer,
Wiesbaden, Germany) as RLUs (relative light units) and values
obtained with the human Nav1.8 probeset were normalized to the
respective human GAPDH (GAPDH sequence of Cercopithecus aethiops so
far unknown) values for each well. Values obtained with cells lysed
4 h after plasmid transfection were subtracted from the values
obtained with cells lysed 24 h after siRNA transfection. Values
acquired with siRNAs directed against Nav1.8 were further
normalized relative to the value obtained with an unrelated siRNA
(directed against hepatitis C virus) which was set to 100%.
[0221] FIG. 1 provides the results from a representative experiment
where siRNAs from Table 1 were tested at a single dose of 100 nM
and Table 4 provides the results for additional siRNAs at a dose of
50 nM. Several siRNAs (in Tables 1 and 4) were effective at the
dose tested in reducing Nav1.8 mRNA levels by at least 50% in COS-7
cells transfected with Nav1.8.
[0222] Dose-Response Curves for Selected siRNAs Against mRNA
Expression of Transfected Human Nav1.8 in Cos-7 Cells
[0223] Several effective siRNAs against Nav1.8 from the single dose
screen (results in FIG. 1) were further characterized by dose
response curves. For dose response curves, transfections were
performed as for the single dose screen above, but with the
following concentrations of siRNA (nM): 100, 33, 11, 3.7, 1.2, 0.4,
1, 0.14, 0.05, 0.015, 0.005 and mock (no siRNA). siRNAs were
diluted with Opti-MEM to a final volume of 12.5 .mu.l according to
the above protocol.
[0224] Three independent dose response experiments were carried out
to generate dose response curves (DRCs). The dose response curves
were repeated, and a summary of the results are provided in Table
3.
TABLE-US-00004 TABLE 3 IC50 values for selected siRNAs targeting
Nav1.8 IC50-values [nM]: 1st DRC screen 2nd DRC screen AL-DP-6218
0.0068 0.021 AL-DP-6217 0.036 0.0021 AL-DP-6050 0.013 0.16
AL-DP-6209 0.060 0.012 AL-DP-6042 nd 0.016 AL-DP-6049 0.24 0.0010
AL-DP-6219 0.13 0.0069 AL-DP-6223 73140 22 AL-DP-6225 0.70 8.07
[0225] Specificity Testing of the Nav1.8 siRNAs Against Nav1.5
mRNA
[0226] Nav1.5 is an NaV subtype that is closely related to Nav1.8,
but has an important role in normal cardiac function. Therefore, it
is important to confirm that siRNAs selective for Nav1.8 do not
inhibit Nav1.5 expression. Several siRNAs were tested for
specificity towards Nav1.8 by transfecting SW620 cells, which
express endogenous human Nav1.5, with these siRNAs, and assessing
Nav1.5 mRNA levels. A control unrelated siRNA (AL-DP-5002) was also
transfected into SW620 cells. siRNAs targeting Nav1.8 were tested
at the following doses: 1200 nM, 400 nM, 133.3 nM, 44.4 nM, 14.8
nM, 4.9 nM, 1.6 nM, and mock (without siRNA). One siRNA targeting
Nav1.8 (AL-DP-6217) was tested at 1 nM, 10 nM, 100 nM and 1 uM. The
expression of Nav1.5 mRNA, which encodes the protein with the
highest homology to Nav1.8 in the Nav-family, was then
quantified.
[0227] One day before transfection, SW620 cells (LCG Promochem,
Wesel, Germany) were seeded at 1.5.times.10.sup.4 cells/well on
96-well plates (Greiner Bio-One GmbH, Frickenhausen, Germany) in
100 .mu.l of growth medium (Leibowitz L-15 Medium, 10% fetal calf
serum, 2 mM L-glutamine, 100 u penicillin/100 .mu.g/ml
streptomycin, all from Biochrom AG, Berlin, Germany).
[0228] siRNA transfections were performed in triplicate. For each
well, 0.6 .mu.l Oligofectamine (Invitrogen GmbH, Karlsruhe,
Germany) was mixed with 2.4 .mu.l Opti-MEM (Invitrogen) and
incubated for 10 min at room temperature. For an siRNA
concentration of 1200 nM in 100 .mu.l transfection volume, 5 .mu.l
of 24 .mu.M siRNA was mixed with 12 .mu.l Opti-MEM per well,
combined with the Oligofectamine-Opti-MEM mixture and again
incubated for 20 minutes at room temperature. During that
incubation time, growth medium was removed from cells and replaced
by 80 .mu.l/well of fresh growth medium without serum.
siRNA-Oligofectamine-complexes were applied completely (20 .mu.l
each per well) to the cells and cells were incubated for 24 h at
37.degree. C. without CO.sub.2 in a humidified incubator (Heraeus
GmbH, Hanau, Germany).
[0229] Cells were harvested by applying 50 .mu.l of lysis mixture
(content of the QuantiGene bDNA-kit from Genospectra, Fremont, USA)
to each well and were lysed at 53.degree. C. for 30 min.
Afterwards, 50 .mu.l of the lysates were incubated with probesets
specific to human Nav1.5 and human GAPDH (sequence of probesets see
below) and processed according to the manufacturer's protocol for
QuantiGene. Chemoluminescence was measured in a Victor2-Light
(Perkin Elmer, Wiesbaden, Germany) as RLUs (relative light units)
and values obtained with the human Nav1.5 probeset were normalized
to the respective human GAPDH values for each well. An unrelated
control siRNA (directed against hepatitis C virus) was used as a
negative control.
[0230] FIG. 2 provides the results. At the concentrations tested,
the selected siRNAs targeting Nav1.8, did not exhibit significant
dose-dependent inhibition of Nav1.5 mRNA as compared with the
unrelated control siRNA (AL-DP-5002), confirming the specificity of
these Nav1.8 siRNAs for Nav1.8 over Nav1.5.
[0231] In addition, at concentrations up to 1 uM, AL-DP-6217
exhibited no significant inhibition of Nav1.5 mRNA (data not
shown), confirming the specificity of this Nav1.8 siRNA for Nav1.8
over Nav1.5.
TABLE-US-00005 TABLE 5 bDNA Probesets for the quantitation of human
Nav1.8, Nav1.5 and GAPDH SEQ ID FPL Name Function Sequence NO:
Human Nav1.8 probeset: hNa182001 CE
TTTGGAGGTTAAAGGTGATCCATTTTTTCTCTTGGAAAGAAAGT 543 hNa182002 CE
GGCGTTTTCCAGAGGCGAGTTTTTCTCTTGGAAAGAAAGT 544 hNa182003 CE
CCAGCAGCAGAGAGCCCCTTTTTCTCTTGGAAAGAAAGT 545 hNa182004 CE
CTGGGTTGAGGAAGAGGGCTTTTTTCTCTTGGAAAGAAAGT 546 hNa182005 CE
AACACTCATTGCCCTTTGGGTTTTTCTCTTGGAAAGAAAGT 547 hNa182006 CE
AAGGACGGAGGTTATGATACTGACTTTTTCTCTTGGAAAGAAAGT 548 hNa182007 CE
TGTTCAGACTCCTCGAGTTCCTCTTTTTCTCTTGGAAAGAAAGT 549 hNa182008 LE
CTATGCCTTCTCTCACTGGCATTTTTTTAGGCATAGGACCCGTGTCT 550 hNa182009 LE
CTGGTTGTAAGGATCAGAGCGGTTTTTAGGCATAGGACCCGTGTCT 551 hNa182010 LE
AACACACTGCCATGACTAGCCCTTTTTAGGCATAGGACCCGTGTCT 552 hNa182011 LE
GATGGCTTTCGTGGTCTCCATTTTTAGGCATAGGACCCGTGTCT 553 hNa182012 LE
CTGGCCAGCACCCCCACTTTTTAGGCATAGGACCCGTGTCT 554 hNa182013 LE
CATGCCTGGAGTCAGGGTTGTTTTTAGGCATAGGACCCGTGTCT 555 hNa182014 LE
AGACATCGACAGCTCCAGGGTTTTTAGGCATAGGACCCGTGTCT 556 hNa182015 LE
GTCCTGCATCGAATGCCGTTTTTAGGCATAGGACCCGTGTCT 557 hNa182016 LE
CAAGCAGGGTGGGCACTTCTTTTTAGGCATAGGACCCGTGTCT 558 hNa182017 BL
TGTGGGAGTGGAGAGAGGTTG 559 hNa182018 BL CCCTCTGACACTCTTGGCTTTATT 560
hNa182019 BL GGTGATTTGTTGTCTTCTGTGGAG 561 hNa182020 BL
GCCTAGAAAAGACATCCTGCG 562 hNa182021 BL GCCAGGGGACCGGAAATGG 563
hNa182022 BL CCCTCAGGGAGTGAGATATCTCG 564 hNa182023 BL
GGAAAGACTCCATCATCTGTGACT 565 hNa182024 BL TCTAGGGAGGGGGCCTTG 566
hNa182025 BL GCGGTTGGTGTTCATCTTCTC 567 hNa182026 BL
GCAAGCTCACTAGTGGGCG 568 hNa182027 BL TCTGCTGACAAGAAAGTCTTCTTTT 569
hNa182028 BL CCCGGAAAGGTTCATCTAAGTAT 570 Human GAPDH probeset:
hGAP001 CE GAATTTGCCATGGGTGGAATTTTTTCTCTTGGAAAGAAAGT 571 hGAP002 CE
GGAGGGATCTCGCTCCTGGATTTTTCTCTTGGAAAGAAAGT 572 hGAP003 CE
CCCCAGCCTTCTCCATGGTTTTTTCTCTTGGAAAGAAAGT 573 hGAP004 CE
GCTCCCCCCTGCAAATGAGTTTTTCTCTTGGAAAGAAAGT 574 hGAP005 LE
AGCCTTGACGGTGCCATGTTTTTAGGCATAGGACCCGTGTCT 575 hGAP006 LE
GATGACAAGCTTCCCGTTCTCTTTTTAGGCATAGGACCCGTGTCT 576 hGAP007 LE
AGATGGTGATGGGATTTCCATTTTTTTAGGCATAGGACCCGTGTCT 577 hGAP008 LE
GCATCGCCCCACTTGATTTTTTTTTAGGCATAGGACCCGTGTCT 578 hGAP009 LE
CACGACGTACTCAGCGCCATTTTTAGGCATAGGACCCGTGTCT 579 hGAP010 LE
GGCAGAGATGATGACCCTTTTGTTTTTAGGCATAGGACCCGTGTCT 580 hGAP011 BL
GGTGAAGACGCCAGTGGACTC 581 Human Nav1.5 probeset: hNa15001 CE
CGTTTTCTCCTCTTGCTTCTTCTCTTTTTCTCTTGGAAAGAAAGT 582 hNa15002 CE
GGGAGCCTGTCCTCCCCATTTTTCTCTTGGAAAGAAAGT 583 hNa15003 CE
TCGCCTGCGAAAGGTGAATTTTTCTCTTGGAAAGAAAGT 584 hNa15004 CE
CTCTCGCTCTCCCCCGCTTTTTCTCTTGGAAAGAAAGT 585 hNa15005 CE
CCGGGACTGGGCTGTCCTTTTTCTCTTGGAAAGAAAGT 586 hNa15006 CE
TTTTGCCATGGAGGGCGTTTTTTCTCTTGGAAAGAAAGT 587 hNa15007 LE
GGCTGAGATGATTCATTGCTCTGTTTTTAGGCATAGGACCCGTGTCT 588 hNa15008 LE
GCTGAGGCCACGGGTGATTTTTAGGCATAGGACCCGTGTCT 589 hNa15009 LE
AATGCTCCCGCGGCTGGTTTTTAGGCATAGGACCCGTGTCT 590 hNa15010 LE
CTGGGCACTGGTCCGGCTTTTTAGGCATAGGACCCGTGTCT 591 hNa15011 LE
GGCCAGGAGCCGAGGTTTTTTTAGGCATAGGACCCGTGTCT 592 hNa15012 LE
CCCAGTAATGAGACCACCCCATTTTTTAGGCATAGGACCCGTGTCT 593 hNa15013 LE
CCTCTGGGTCGCCTGCCTTTTTAGGCATAGGACCCGTGTCT 594 hNa15014 BL
CACTCCTCAGTTCCTGAAGACATC 595 hNa15015 BL GGACCATCTTCTGAGTCAGACTTG
596 hNa15016 BL AACGTGGCTTCATAGAAGTCCT 597 hNa15017 BL
AATCTGCTTCAGAACCCAGGTC 598 hNa15018 BL TGTGCTGTTTTCATCATCTGCAA 599
hNa15019 BL CCAGCAGTGATGTGTGGTGG 600 hNa15020 BL GCAGGGGCCAGGGCA
601 hNa15021 BL TGCAGTCCACAGTGCTGTTCT 602 hNa15022 BL
GGCTTCCTGGGGATGTGG 603
[0232] Single-Dose Screen of Nav1.8 siRNAs Against mRNA Expression
of Endogenous Nav1.8 in Primary Cultures of Rat Dorsal Root
Ganglion Cells.
[0233] To confirm and extend the results obtained on mRNA
expression of transfected human Nav1.8 in Cos-7 cells, Nav1.8
siRNAs from Table 1 were tested at a single dose of 200 nM for
activity in reducing mRNA expression of endogenous Nav1.8 in
primary cultures of rat dorsal root ganglion (DRG) cells.
[0234] DRG cells were isolated from Sprague-Dawley rats at
postnatal day 3 to 6. DRGs were dissected and cells dissociated
into single cells by incubation with 0.28 Wunsch units/ml Liberase
Blendzyme (Roche) in S-MEM (Gibco) at 37.degree. C. for 35 min. The
cell suspension was pre-plated on tissue-culture plates to remove
non-neuronal cells. Neurons were then plated onto tissue-culture
Biocoat.TM. PDL Poly-D-Lysine/Laminin 96-well plates (BD
Biosciences, Bedford Mass., USA) in F12-HAM's Medium containing
glutamine (Invitrogen Gibco, Carlsbad Calif., USA) with 5% fetal
bovine serum (FBS, heat inactivated) and 5% horse serum (heat
inactivated) (both Invitrogen Gibco, Carlsbad Calif., USA)
supplemented with 50 ng/ml mouse nerve growth factor 2.5S (NGF;
Promega Corp., Madison Wis., USA) and kept at 37.degree. C., 5%
CO.sub.2 in a humidified incubator until transfection.
[0235] Nav1.8 siRNAs were screened in DRG cultures at 200 nM in
duplicate using TransMessenger.TM. Transfection reagent (Qiagen
GmbH, Hilden, Germany, cat. no. 301525) which is based on a lipid
formulation, a specific RNA-condensing reagent (Enhancer R.TM.) and
an RNA-condensing buffer (Buffer EC-R.TM.), keeping siRNA:Enhancer
R.TM. ratio (.mu.g:.mu.l) constant at 1:2, and
siRNA:TransMessenger.TM. ratio (.mu.g:.mu.l) constant at 1:12.
[0236] DRG neurons were transfected 24 h post-plating. For each
well, 0.52 .mu.l Enhancer R.TM. were first mixed with 13.68 .mu.l
Buffer EC-R.TM.. 0.8 .mu.l of a 25 .mu.M solution of AL-DP-5987
(0.26 .mu.g) in annealing buffer (20 mM sodium phosphate, pH 6.8;
100 mM sodium chloride), or 0.8 .mu.l of annealing buffer
(siRNA-free control) were added and the mixture incubated for 5 min
at RT. 3.12 .mu.l TransMessenger.TM. Transfection Reagent were
diluted with 6.88 .mu.l Buffer EC-R.TM., added to the mixture, and
the mixture incubated for another 10 min at room temperature to
allow transfection-complex formation. 75 .mu.l serum free F12-HAM's
Medium containing glutamine (Invitrogen Gibco, Carlsbad Calif.,
USA) supplemented with 50 ng/ml NGF 2.5S (Promega Corp., Madison
Wis., USA) and 1:50 B27 supplement (Invitrogen Gibco, Carlsbad
Calif., USA) were added to the transfection complexes and complete
mixing achieved by gently pipetting up and down. The growth medium
was removed from the DRG cells, and 90 .mu.l of the above
transfection complex mixture were added onto the cells. After 7 to
8 h of incubation at 37.degree. C., 5% CO.sub.2 in a humidified
incubator supernatant was removed from the cells, fresh F12-HAM's
medium containing glutamine supplemented with 5% FBS, 5% horse
serum (both Invitrogen Gibco, Carlsbad Calif., USA), 50 ng/ml mouse
NGF 2.5S (Promega Corp., Madison Wis., USA) and 1:100
Penicillin/Streptomycin (Invitrogen Gibco, Carlsbad Calif., USA)
was added, the cells were incubated for another 16 h at 37.degree.
C., 5% CO.sub.2 in a humidified incubator, and Nav1.8 mRNA was
quantified.
[0237] Nav1.8 mRNA levels were measured using the QuantiGene.TM.
bDNA kit (Genospectra, Fremont, USA) according to manufacturer's
protocol. Briefly, the supernatant was removed from the DRG cells,
and the cells were lysed by addition of 150 .mu.l of Lysis Working
Reagent (1 volume of Lysis Mixture plus 2 volumes of medium) and
incubation at 52.degree. C. for 30 min. 40 .mu.l of the lysates
were incubated at 52.degree. C. for 40 min with the probe sets
specific to rat Nav1.8 and mouse synuclein (SNCL).
Chemoluminescence was read on a Victor.sup.2-Light.TM. (PerkinElmer
Life And Analytical Sciences, Inc., Boston Mass., USA) as Relative
Light Units (RLU). RLU for Nav1.8 were normalized to SNCL RLU for
each well. Normalized Nav1.8/SNCL ratios were then compared to the
siRNA-free control, which was set as 100%.
[0238] FIG. 3 provides the results. At 200 nM, at least 13
(indicated by `*`) of the Nav1.8 siRNAs tested showed at least 50%
Nav1.8 mRNA knock down compared to the siRNA-free control
(TransMessenger.TM. only; .TM. only), while an unrelated control
siRNA against RhoA had no effect.
[0239] Dose Response of dsRNA AL-DP-6209 Against mRNA Expression of
Endogenous Nav1.8 in Primary Cultures of Rat Dorsal Root Ganglion
Cells
[0240] One effective siRNA (AL-DP-6209) against Nav1.8 from the
single dose screen in primary cultures of rat dorsal root ganglion
cells was further characterized for dose dependence. For the dose
response curve, experiments were performed as for the single dose
screen in DRG cultures above, but with the following concentrations
of siRNA: 175, 88, 44, 22, 11 and 5.5 nM. For all siRNA
concentrations, siRNA:Enhancer R ratio (ug:ul) was kept constant at
1:2 and siRNA:TransMessenger ratio (ug/ul) was kept constant at
1:12 (FIG. 4).
[0241] FIG. 4 provides the result for the selected siRNA AL-DP-6209
from a dose response experiment. At 5.5 and 11 nM, AL-DP-6209 did
not inhibit Nav1.8 mRNA expression relative to SNCL, whereas at 88
and 175 nM, AL-DP-6209 inhibited Nav1.8 mRNA expression relative to
SNCL by >40%. Maximal inhibition of Nav1.8 mRNA expression
relative to SNCL occurred in this experiment at 175 nM.
[0242] Intrathecal Bolus Administration of siRNAs Against Nav1.8
with iFECT Prevents Inflammatory Pain
[0243] The effect of siRNAs against Nav1.8, formulated with iFECT,
on complete Freund's adjuvant-induced tactile hypersensitivity was
evaluated in rats (FIG. 5). Adult male Sprague-Dawley rats received
an injection of CFA (150 uL) into the hindpaw on day 0. siRNAs
against Nav1.8 were then administered by intrathecal bolus to the
lumbar region of the spinal cord on days 1, 2 and 3; specifically,
for each bolus injection, 2 ug of siRNA was complexed with iFECT
transfection reagent (Neuromics, Minneapolis Minn., USA) at a ratio
of 1:4 (w:v) in a total volume of 10 uL. Five groups of rats (with
5 rats per group) were treated with either siRNA (AL-DP-6049,
AL-DP-6209, AL-DP-6217 or AL-DP-6218; Table 1), or PBS, in the
presence of iFECT. Tactile hypersensitivity was expressed as
tactile withdrawal thresholds which were measured by probing the
hindpaw with 8 calibrated von Frey filaments (Stoelting, Wood Dale
Ill., USA) (0.41 g to 15 g). Each filament was applied to the
plantar surface of the paw. Withdrawal threshold was determined by
sequentially increasing and decreasing the stimulus strength and
calculated with a Dixon non-parametric test (see Dixon, W. J.
(1980) "Efficient analysis of experimental observations" Annu Rev
Pharmacol Toxicol 20:441-462; Chaplan, S.R., F. W. Bach, et
al.(1994) "Quantitative assessment of tactile allodynia in the rat
paw" J Neurosci Methods 53:55-63). Tactile thresholds were measured
before CFA injection to assess baseline thresholds, and then on day
4 after CFA and treatment with test articles. In rats treated with
PBS, tactile hypersensitivity was pronounced on day 4, as evidenced
by reduced paw withdrawal threshold, as expected. In rats treated
with AL-DP-6209, tactile thresholds were nearly normalized on day
4, demonstrating that the Nav1.8 siRNA, AL-DP-6209, is efficacious
in vivo against inflammation-induced hyperalgesia. Treatment with
the Nav1.8 siRNA, AL-DP-6217, resulted in the average tactile
threshold trending towards baseline, with one of five rats
demonstrating a normal tactile response. AL-DP-6049 and AL-DP-6218
did not significantly alter tactile thresholds compared to PBS
treatment, in this experimental paradigm.
[0244] These results demonstrate that siRNAs targeting Nav1.8,
formulated with transfection reagent and administered
intrathecally, alleviate CFA-induced tactile hyperalgesia, and
therefore represent a novel approach to providing effective
treatment of clinical inflammatory pain.
[0245] Intrathecal Bolus Administration of siRNAs Against Nav1.8
without Transfection Reagent Alleviates Inflammatory Pain
[0246] The effect of siRNAs against Nav1.8, formulated in phosphate
buffered saline (PBS), on complete Freund's adjuvant (CFA)-induced
tactile hypersensitivity was evaluated in rats (FIG. 6). Of 3
Nav1.8 siRNAs tested, 2 siRNAs were efficacious against CFA-induced
tactile hypersensitivity: AL-DP-4461, an unconjugated siRNA with
different chemical modifications but the same sequence as
AL-DP-6050, and AL-DP-4459, a cholesterol-conjugated siRNA with the
same chemical modifications and sequence as AL-DP-6050. With the
dosing paradigms evaluated in this experiment, AL-DP-6980
(cholesterol-conjugated siRNA with the same chemical modifications
and sequence as AL-DP-6209) was not efficacious against CFA-induced
tactile hypersensitivity. The sequences of AL-DP-4461, AL-DP-4459
and AL-DP-6980 are shown in Table 6.
TABLE-US-00006 TABLE 6 Further modified siRNAs specific for Nav1.8
SEQ SEQ Duplex sense strand ID anti-sense strand ID name sequence
(5'-3') NO: sequence (5'-3') NO: AL-DP- cauccuaugaaccaauagceTeT 604
gcuauugguucauaggaugeceT 605 4461 AL-DP- cmaumcmcmumaumgaacmcmaa 606
gcumauugguucmaumaggaugTT 607 4459 umagcmTT-sChol AL-DP-
umumumumgumcmumaaaumgag 608 ugaacucmauuumagacmaaaaTT 609 6980
umumcmaTT-sChol Note: Prefix e represents 2'-O-(2-methoxyethyl)
modified nucleotides
[0247] Adult male Sprague-Dawley rats received an injection of CFA
into the hindpaw on day 0. Four groups of rats (with 5 rats per
group) were treated starting on day 1 after CFA injection with
either siRNA against Nav1.8 (AL-DP-4461, AL-DP-4459, or
AL-DP-6980), or PBS. siRNAs against Nav1.8 (AL-DP-4461, AL-DP-4459
or AL-DP-6980) or PBS were administered by intrathecal bolus
injections (10 uL per injection) to the lumbar level of the spinal
cord twice per day (BID) on days 1, 2 and 3. Tactile
hypersensitivity was measured as above, both before CFA injection
to assess baseline thresholds, and then on day 4 after CFA and
treatment with test articles. In rats treated with PBS, tactile
hypersensitivity was pronounced on day 4, as evidenced by reduced
paw withdrawal threshold, as expected. In rats treated with
AL-DP-4461 by bolus BID (0.5 mg/bolus), tactile thresholds were
moderately normalized on day 4, demonstrating that the Nav1.8
siRNA, AL-DP-4461, is moderately efficacious with this dosing
paradigm in vivo against CFA-induced tactile hyperalgesia.
Treatment with the Nav1.8 siRNA, AL-DP-4459, by bolus BID (0.15
mg/bolus), resulted in nearly complete normalization of tactile
threshold, with all 5 rats demonstrating substantial recovery of
tactile thresholds to more than 10.2 g by day 4. In contract, the
Nav1.8 siRNA AL-DP-6980, by bolus BID (0.15 mg/bolus), did not
affect tactile hypersensitivity, showing that not all
cholesterol-conjugated siRNAs against a target are equally
efficacious or potent, even if efficacious when transfected into
cultured cells.
[0248] These results suggest that cholesterol conjugation enhances
in vivo efficacy of siRNAs for neurological disorders, including
chronic pain. Furthermore, these results demonstrate that siRNAs
targeting Nav1.8, either with or without cholesterol-conjugation,
formulated in saline and administered intrathecally by bolus
injection, alleviate CFA-induced tactile hyperalgesia, and
therefore represent a novel approach to providing effective
treatment of clinical inflammatory pain.
[0249] Intrathecal pump or bolus administration of siRNAs against
Nav1.8 without transfection reagent alleviates inflammatory
pain
[0250] The effect of siRNAs against Nav1.8, formulated in phosphate
buffered saline (PBS), on complete Freund's adjuvant (CFA)-induced
tactile hypersensitivity was evaluated in rats after intrathecal
pump infusion (FIG. 7, left) or intrathecal BID bolus injection
(FIG. 7, right). Of 3 Nav1.8 siRNAs tested by continuous
intrathecal pump infusion at 0.4 mg/day, 1 siRNA (AL-DP-6050, Table
1) was modestly efficacious against CFA-induced tactile
hypersensitivity. AL-DP-6050, when tested by intrathecal BID bolus
injection at 0.5 mg/bolus, was efficacious against CFA-induced
tactile hypersensitivity.
[0251] For evaluating effects of siRNAs against NaV1.8 with
continuous intrathecal pump infusion, adult male Sprague-Dawley
rats received an injection of CFA into the hindpaw on day 0. Four
groups of rats (with 5 rats per group) were treated starting on day
1 after CFA injection with either siRNA against Nav1.8 (AL-DP-6050,
AL-DP-6218, or AL-DP-6219), or PBS. In all rats, test articles were
intrathecally administered by continuous osmotic mini-pump infusion
with an infusion rate of 0.5 uL/hour, beginning on day 1. siRNAs
were infused at 0.4 mg/day. Tactile hypersensitivity was measured
as above, both before CFA injection to assess baseline thresholds,
and then on day 4 after CFA and treatment with test articles (FIG.
7, left). In rats treated with PBS, tactile hypersensitivity was
pronounced on day 4, as evidenced by reduced paw withdrawal
threshold, as expected. In rats treated with AL-DP-6050 by
continuous intrathecal pump infusion (0.4 mg/day), tactile
thresholds were modestly normalized on day 4, demonstrating that
the Nav1.8 siRNA, AL-DP-6050, is modestly efficacious with this
dosing paradigm in vivo against inflammatory pain.
[0252] For evaluating effects of siRNAs against NaV1.8 with BID
intrathecal bolus injection, adult male Sprague-Dawley rats
received an injection of CFA into the hindpaw on day 0. Two groups
of rats (with 4 to 5 rats per group) were treated starting on day 1
after CFA injection with either siRNA against Nav1.8 (AL-DP-6050,
0.5 mg/bolus) or PBS. Tactile hypersensitivity was assessed as
described above. Tactile thresholds were measured before CFA
injection to assess baseline thresholds, and then on day 4 after
CFA injection and treatment with test articles (FIG. 7, right). As
expected, in rats treated with PBS, tactile hypersensitivity was
pronounced on day 4, as evidenced by reduced paw withdrawal
threshold. In rats treated with AL-DP-6050 by BID intrathecal bolus
injection (0.5 mg/bolus), tactile thresholds were substantially
normalized on day 4, demonstrating that the Nav1.8 siRNA,
AL-DP-6050, is modestly efficacious with this dosing paradigm in
vivo against inflammatory pain.
[0253] These results further demonstrate that siRNAs targeting
Nav1.8 without cholesterol-conjugation, formulated in saline and
administered intrathecally by either bolus injection or continuous
pump infusion, alleviate CFA-induced tactile hyperalgesia, and
therefore represent a novel approach to providing effective
treatment of clinical inflammatory pain.
[0254] Intrathecal Bolus Administration of siRNAs Against Nav1.8
without Transfection Reagent Alleviates Inflammatory Pain
[0255] The effect of siRNAs against Nav1.8, formulated in phosphate
buffered saline (PBS), on complete Freund's adjuvant (CFA)-induced
thermal hypersensitivity was evaluated in rats (FIG. 8). Both
unconjugated dsRNA AL-DP-6050 (Table 1) and cholesterol-conjugated
dsRNA AL-DP-4459 (Table 6) were efficacious against CFA-induced
thermal hypersensitivity with the BID bolus intrathecal dosing
paradigms evaluated.
[0256] Adult male Sprague-Dawley rats received an injection of CFA
into the hindpaw on day 0. Three groups of rats (with 4 to 5 rats
per group) were treated starting on day 1 after CFA injection with
either siRNA against Nav1.8 (AL-DP-6050 or AL-DP-4459), or PBS. In
all rats, test articles were administered intrathecally by BID
bolus injection (10 uL per bolus) beginning on day 1. AL-DP-4459
was dosed at 0.15 mg/bolus whereas AL-DP-6050 was dosed at 0.5
mg/bolus. Thermal hypersensitivity was measured by assessing paw
withdrawal latency to a noxious thermal stimulus as described by
Hargreaves and colleagues (Hargreaves, K., R. Dubner, et al. (1988)
"A new and sensitive method for measuring thermal nociception in
cutaneous hyperalgesia" Pain 32:77-88). Latency to withdrawal of a
hindpaw in response to noxious radiant heat was determined. A
maximal cut-off of 40 sec prevented tissue damage.
[0257] Thermal responses were measured before CFA injection to
assess baseline thresholds, and then on day 4 after CFA injection
and treatment with test articles. As expected, in rats treated with
PBS, thermal hypersensitivity was pronounced on day 4, as evidenced
by reduced paw withdrawal latency. In rats treated with AL-DP-6050
(0.5 mg/bolus) or AL-DP-4459 (0.15 mg/bolus) by intrathecal BID
bolus injection, thermal latencies were normalized on day 4,
demonstrating that the unconjugated Nav1.8 siRNA, AL-DP-6050, and
the cholesterol-conjugated Nav1.8 siRNA AL-DP-4459 are efficacious
with this dosing paradigm in vivo against inflammatory pain.
[0258] These results demonstrate that siRNAs targeting Nav1.8,
either with or without cholesterol-conjugation, formulated in
saline and administered intrathecally by bolus injection, alleviate
CFA-induced thermal hyperalgesia, in addition to tactile
hyperalgesia (above), and therefore represent a novel approach to
providing effective treatment of multiple types of hyperalgesia in
clinical inflammatory pain.
[0259] Intrathecal Bolus Administration of Cholesterol-Conjugated
siRNA Against Nav1.8 without Transfection Reagent Alleviates
Neuropathic Pain
[0260] The effect of AL-DP-4459 (Table 6) against Nav1.8,
formulated in phosphate buffered saline (PBS), on spinal nerve
ligation (SNL)-induced tactile and thermal hypersensitivity was
evaluated in rats (FIG. 9). With the bolus intrathecal dosing
paradigm evaluated (0.15 mg/bolus, BID), AL-DP-4459 was efficacious
against SNL-induced tactile and thermal hypersensitivity.
[0261] Adult male Sprague-Dawley rats received unilateral ligation
of the L5 and L6 spinal nerves on day 0 (SNL surgery). Three groups
of rats (with 6 to 8 rats per group) were treated starting on day 3
after SNL surgery by intrathecal administration of either the siRNA
AL-DP-4459 against Nav1.8, or PBS. In 2 of these groups, AL-DP-4459
or PBS was administered by intrathecal bolus injections (5 uL per
injection) to the lumbar level of the spinal cord twice per day
(BID) on post-SNL days 3 through 7. In one group of rats,
AL-DP-4459 was intrathecally administered by continuous osmotic
mini-pump infusion at 0.18 mg/day, with an infusion rate of 0.5
uL/hour, on post-SNL days 3 through 7. Tactile and thermal
hypersensitivities were assessed as described above.
[0262] Tactile (FIG. 9, left) and thermal (FIG. 9, right) responses
were measured before SNL surgery to assess baseline responses (BL),
and on post-SNL day 3 before treatment with test articles to verify
that tactile and thermal hyperalgesia had developed fully. In rats
treated with PBS, tactile and thermal hypersensitivities were
pronounced, as expected, on post-SNL days 3, 5 and 7 as evidenced
by reduced paw withdrawal thresholds and reduced thermal latencies,
respectively. The Nav1.8 siRNA AL-DP-4459, when intrathecally
administered by continuous pump infusion at 0.18 mg/day, did not
affect tactile or hypersensitivity significantly over the
time-frame shown. In contrast, in rats treated with AL-DP-4459 by
bolus BID (0.15 mg/bolus), tactile thresholds and thermal latencies
were substantially normalized on post-SNL day 7 (day 5 of
treatment), demonstrating that the Nav1.8 siRNA, AL-DP-4459, is
efficacious in vivo against SNL-induced tactile and thermal
hyperalgesia.
[0263] These results demonstrate that intrathecally administered
cholesterol-conjugated siRNAs targeting Nav1.8, formulated in
saline, are efficacious against experimental nerve injury-induced
chronic pain in rats, and therefore, represent a novel approach to
providing effective treatment of clinical neuropathic pain.
[0264] Unconjugated and Cholesterol-Conjugated siRNAs Targeting
Nav1.8 are Stable in Human Cerebrospinal Fluid at 37.degree. C.
[0265] To determine the stability in human cerebrospinal fluid
(CSF) of unconjugated and cholesterol-conjugated siRNAs targeting
NaV1.8, siRNA duplexes were incubated in human CSF for 48 hours at
37.degree. C., and the single strands were measured by quantitative
ion exchange chromatography. In this example, unconjugated siRNAs
AL-DP-6050, AL-DP-6209, AL-DP-6217, AL-DP-6218 and AL-DP-6219
(Table 1), and cholesterol-conjugated siRNA AL-DP-4459 (Table 6)
were evaluated. 30 .mu.l of human CSF was mixed with 30 .mu.l of 50
.mu.M siRNA (150 pmole/well) in a 96-well plate, sealed to avoid
evaporation, and incubated for 1 to 48 hours at 37.degree. C.
Incubation in 30 .mu.l PBS for 48 hours at 37.degree. C. served as
a control for nonspecific degradation. Reactions were stopped by
the addition of 4 .mu.l proteinase K (20 mg/ml) and 25 .mu.l of
proteinase K buffer, and incubation of this mixture for 20 min at
42.degree. C. Samples were spin filtered through a 0.2 .mu.m
96-well filter plate at 3000 rpm for 20 min. Incubation wells were
washed with 50 .mu.l Millipore water twice and the combined washing
solutions were spin filtered also. A 5 .mu.l aliquot of 50 .mu.M
40-mer RNA was added to each sample to act as an internal standard
(IS) for normalization of volume changes in the filtration volume.
Samples were analyzed by ion exchange HPLC under denaturing
conditions.
[0266] 1. HPLC System for analysis of cholesterol-conjugated siRNA
AL-DP-4459
TABLE-US-00007 Column: Dionex DNAPac PA200 (4 .times. 250 mm
analytical column) Temp.: 80.degree. C. (denaturing conditions)
Flow: 1 ml/min Injection: 50 ul Detection: 260 (reference
wavelength 600 nm) HPLC Eluent A: 25 mM TRIS-HCl; 1 mM EDTA; 50%
ACN; pH = 8 HPLC Eluent B: 600 mM NaBr in A
[0267] Gradient Table:
TABLE-US-00008 Time % A % B 0.00 min 80 20 1.00 min 80 20 15.0 min
30 70 15.5 min 0 100 17.5 min 0 100 18.0 min 80 20 21.0 min 80
20
[0268] 2. HPLC System for unconjugated siRNAs AL-DP-6050, 6209,
6217, 6218, 6219
TABLE-US-00009 Column: Dionex DNAPac PA200 (4 .times. 250 mm
analytical column) Temp.: 30.degree. C. (denaturing conditions by
pH = 11) Flow: 1 ml/min Injection: 50 ul Detection: 260 nm
(reference wavelength 600 nm) HPLC Eluent A: 20 mM Na.sub.3PO.sub.4
in 10% ACN; pH = 11 HPLC Eluent B: 1M NaBr in A
[0269] Gradient Table:
TABLE-US-00010 Time % A % B 0.00 min 75 25 1.00 min 75 25 21.0 min
40 60 21.5 min 0 100 23.0 min 0 100 24.5 min 75 25 28.0 min 75
25
[0270] Under the denaturing IEX-HPLC conditions, the duplexes
eluted as two separated single strands. The internal standard
eluted at higher retention times than the single strands of the
duplex, and did not interfere with the analysis.
[0271] For every injection, the chromatograms were integrated
automatically by the Dionex Chromeleon 6.60 HPLC software, but were
adjusted manually as necessary. All peak areas were corrected by
the following equation to the internal standard (IS) peak:
CF.sub.(IS)=100/PeakArea.sub.(IS); Corrected
PeakArea.sub.(s/as)=CF*PeakArea.sub.(a/as)
[0272] The %-values for the remaining intact FLP at all time points
are calculated by the following equation:
%-FLP.sub.(s/as)=(Corrected PeakArea.sub.(s/as)/Corrected
PeakArea.sub.(s/as); t=0min)*100%
[0273] All values were normalized to the incubation at t=0 min.
[0274] The results for the unconjugated siRNAs are shown in FIG.
10. After 48 hours incubation at 37.degree. C. in PBS (PBS-48), all
five siRNAs were completely stable, with no loss detectable. After
48 hours incubation at 37.degree. C. in human CSF, three
(AL-DP-6050, AL-DP-6209 and AL-DP-6217) of the five siRNAs
exhibited less than 20% loss, whereas two (AL-DP-6218 and
AL-DP-6219) of the siRNAs exhibited greater than 50% loss. The
results for the cholesterol-conjugated siRNA AL-DP-4459 are shown
in FIG. 11. After 48 hours incubation at 37.degree. C. in PBS
(PBS-48), AL-DP-4459 was completely stable, with no loss
detectable. When assessed for stability in human CSF, AL-DP-4459
was found to be stable at 37.degree. C. for 48 hours, with less
than 20% variation detected over this time period.
[0275] Unconjugated and Cholesterol-Conjugated siRNAs Targeting
Nav1.8 Reduce Nav1.8 mRNA in a Dose-Dependent Manner in Primary Rat
Sensory Neuronal Cultures
[0276] Another effective siRNA (AL-DP-6050, Table 1) against Nav1.8
from the single dose screen in primary cultures of rat dorsal root
ganglion cells and its cholesterol conjugate AL-DP-4459 (Table 6)
were further characterized for dose dependence. For the dose
response curves, transfections were performed using Lipofectamine
2000 (see below) with the following concentrations of siRNA: 200,
80, 32, 12.8, 5.12, 2.05, 0.82 and 0.33 nM (FIG. 12).
[0277] DRG neurons were transfected 24 h post-plating. For each
well, 0.4 .mu.l Lipofectamine.TM. 2000 (Invitrogen Corporation,
Carlsbad, Calif.) was used and transfections were performed
according to the manufacturer's protocol. Specifically, the siRNA:
Lipofectamine.TM. 2000 complexes were prepared as follows. The
appropriate amount of siRNA was diluted in Opti-MEM I Reduced Serum
Medium and mixed gently. The Lipofectamine.TM. 2000 was vortexed
before use; then for each well of a 96 well plate, 0.4 ul
Lipofectamine.TM. 2000 was diluted in 25 .mu.l of Opti-MEM I
Reduced Serum Medium, mixed gently and incubated for 5 minutes at
room temperature. After the 5 minute incubation, 1 ul of the
diluted siRNA was combined with the diluted Lipofectamine.TM. 2000
(total volume, 26.4 .mu.l). The complex was mixed gently and
incubated for 20 minutes at room temperature to allow the siRNA:
Lipofectamine.TM. 2000 complexes to form. Then 100 .mu.l of
serum-free F12-HAM's Medium containing glutamine (Invitrogen Gibco,
Carlsbad Calif., USA) supplemented with 50 ng/ml NGF 2.5S (Promega
Corp., Madison Wis., USA) and 1:50 B27 supplement (Invitrogen
Gibco, Carlsbad Calif., USA) was added to the transfection
complexes, and mixing was achieved by gently pipetting up and down.
The growth medium was removed from the DRG cells and 100 .mu.l of
the above transfection complex mixture was added to each well of a
96-well plate. After 20 h of incubation at 37.degree. C., 5%
CO.sub.2 in a humidified incubator, supernatant was removed from
the cells, fresh F12-HAM's medium containing glutamine supplemented
with 5% FBS, 5% horse serum (both Invitrogen Gibco, Carlsbad
Calif., USA), 50 ng/ml mouse NGF 2.5S (Promega Corp., Madison Wis.,
USA) and 1:100 Penicillin/Streptomycin (Invitrogen Gibco, Carlsbad
Calif., USA) was added. The cells were incubated for another 20-24
h at 37.degree. C., 5% CO.sub.2 in a humidified incubator, and
Nav1.8 mRNA was quantified by the bDNA assay, as described
previously.
[0278] FIG. 12 provides the result for the selected siRNA
AL-DP-6050 and its cholesterol conjugate AL-DP-4459 from a dose
response experiment. AL-DP-6050 and its cholesterol conjugate
AL-DP-4459 inhibited Nav1.8 mRNA expression relative to
alpha-synuclein (SNCA) in a dose-dependent manner, with maximal
inhibitions of .about.40% at >30 nM siRNA, in this
experiment.
[0279] Intrathecal Bolus Administration of Unconjugated siRNA
Against Nav1.8 without Transfection Reagent Alleviates Neuropathic
Pain
[0280] The effect of AL-DP-6050 (Table 1) against Nav1.8,
formulated in phosphate buffered saline (PBS), on spinal nerve
ligation (SNL)-induced thermal hypersensitivity was evaluated in
rats (FIG. 13). With the bolus intrathecal dosing paradigm
evaluated (0.15 mg/bolus, BID), AL-DP-6050 was efficacious against
SNL-induced thermal hypersensitivity.
[0281] Adult male Sprague-Dawley rats received unilateral ligation
of the L5 and L6 spinal nerves on day 0 (SNL surgery). Two groups
of rats (with 8 rats per group) were treated starting on day 3
after SNL surgery by intrathecal bolus injection with either the
siRNA AL-DP-6050 against Nav1.8, or PBS. Intrathecal bolus
injections (0.15 mg in 5 uL per injection) were administered to the
lumbar level of the spinal cord twice per day (BID) on post-SNL
days 3 through 7. Thermal hypersensitivity was assessed as
described above.
[0282] Thermal responses were measured before SNL surgery to assess
baseline responses (BL), and on post-SNL day 3 before treatment
with test articles to verify that thermal hyperalgesia had
developed fully. In rats treated with PBS, thermal hypersensitivity
was pronounced, as expected, on post-SNL days 3, 5 and 7 as
evidenced by reduced thermal latencies. In contrast, in rats
treated with AL-DP-6050 by bolus BID (0.15 mg/bolus), thermal
latencies were substantially normalized on post-SNL days 5 (day 3
of treatment) and 7 (day 5 of treatment), demonstrating that the
Nav1.8 siRNA, AL-DP-6050, is efficacious in vivo against
SNL-induced thermal hyperalgesia.
[0283] These results demonstrate that siRNAs targeting Nav1.8,
formulated in saline and administered by intrathecal bolus
injection, are efficacious against experimental nerve
injury-induced chronic pain in rats, and therefore, represent a
novel approach to providing effective treatment of clinical
neuropathic pain.
[0284] Intrathecal Bolus Administration of ND98-2.7 Liposomal
Formulation of siRNA Against Nav1.8 Alleviates Neuropathic Pain
[0285] The effect of AL-DP-6050 (Table 1) against Nav1.8,
formulated in ND98-2.7 (described below), and administered by
intrathecal bolus injection, on spinal nerve ligation (SNL)-induced
thermal hypersensitivity was evaluated in rats (FIG. 14). With the
bolus intrathecal dosing paradigm evaluated (5 micrograms/bolus,
daily), AL-DP-6050 was efficacious against SNL-induced thermal
hypersensitivity.
[0286] Adult male Sprague-Dawley rats received unilateral ligation
of the L5 and L6 spinal nerves on day 0 (SNL surgery). Two groups
of rats (with 6 rats per group) were treated starting on day 3
after SNL surgery by intrathecal bolus injection with either the
siRNA AL-DP-6050 against Nav1.8, formulated in ND98-2.7, or PBS.
Intrathecal bolus injections (5 micrograms in 5 uL per injection)
were administered to the lumbar level of the spinal cord daily on
post-SNL days 3 through 5. Thermal hypersensitivity was assessed as
described above.
[0287] Thermal responses were measured before SNL surgery to assess
baseline responses (BL), and on post-SNL day 3 before treatment
with test articles to verify that thermal hyperalgesia had
developed fully. In rats treated with PBS, thermal hypersensitivity
was pronounced, as expected, on post-SNL days 3, 5, 7 and 10 as
evidenced by reduced thermal latencies. In contrast, in rats
treated with AL-DP-6050 formulated in ND98-2.7 liposomes, by
intrathecal bolus daily injection (5 micrograms/bolus), thermal
latencies were substantially normalized on post-SNL day 5 (day 3 of
treatment), demonstrating that the Nav1.8 siRNA, AL-DP-6050,
formulated in ND98-2.7 liposomes and administered by intrathecal
injection, is efficacious in vivo against SNL-induced thermal
hyperalgesia. Moreover, the dose level of siRNA required for
efficacy with the ND98-2.7 liposomal formulation (5 micrograms per
day) was much lower than that observed for efficacy with the PBS
formulation (300 micrograms per day).
[0288] These results demonstrate that siRNAs targeting Nav1.8,
administered by intrathecal bolus injection and formulated in
ND98-2.7 liposomes, are efficacious against experimental nerve
injury-induced chronic pain in rats, and therefore, represent a
novel approach to providing effective treatment of clinical
neuropathic pain.
[0289] Intravenous Bolus and Continuous Pump Administration of
ND98-2.7 Liposomal Formulation of siRNA Against Nav1.8 Alleviates
Neuropathic Pain
[0290] The effect of AL-DP-6050 (Table 1) against Nav1.8,
formulated in ND98-2.7, and administered by intravenous bolus
injection or intravenous continuous pump infusion, on spinal nerve
ligation (SNL)-induced thermal hypersensitivity was evaluated in
rats (FIG. 14). With the bolus intravenous dosing paradigm
evaluated (daily 0.5 mg/bolus, equivalent to approximately 2
mg/kg/bolus), and with the continuous intravenous pump paradigm
evaluated (0.24 mg/day, equivalent to approximately 1 mg/kg/day),
AL-DP-6050, formulated in ND98-2.7 liposomes, was efficacious
against SNL-induced thermal hypersensitivity.
[0291] Adult male Sprague-Dawley rats received unilateral ligation
of the L5 and L6 spinal nerves on day 0 (SNL surgery). Two groups
of rats (with 6 rats per group) were treated starting on day 3
after SNL surgery by intravenous bolus injection or intravenous
continuous pump infusion with the siRNA AL-DP-6050 against Nav1.8,
formulated in ND98-2.7. Intravenous bolus injections (0.5 mg in 1
mL per bolus) were administered daily on post-SNL days 3 through 5.
Intravenous continuous pump infusion (10 uL/hour, equivalent to
0.24 mg/day) was administered on post-SNL days 3 through 10 via a
cannula in the jugular vein. Thermal hypersensitivity was assessed
as described above.
[0292] Thermal responses were measured before SNL surgery to assess
baseline responses (BL), and on post-SNL day 3 before treatment
with test articles to verify that thermal hyperalgesia had
developed fully. Thermal hypersensitivity was pronounced, as
expected, on post-SNL day 3, before treatment, as evidenced by
reduced paw withdrawal latencies. In contrast, in rats treated with
AL-DP-6050 formulated in ND98-2.7 liposomes, by intravenous bolus
daily injection (0.5 mg/bolus), thermal latencies were
substantially normalized on post-SNL day 5 (day 3 of treatment),
and post-SNL day 7 (2 days after the last intravenous bolus
treatment) demonstrating that the Nav1.8 siRNA, AL-DP-6050,
formulated in ND98-2.7 liposomes and administered by intravenous
bolus injection, is efficacious in vivo against SNL-induced thermal
hyperalgesia. Furthermore, in rats treated with AL-DP-6050
formulated in ND98-2.7 liposomes, by intravenous continuous pump
infusion (0.24 mg/day), thermal latencies were substantially
normalized on post-SNL days 5 (day 3 of treatment), 7 (day 5 of
treatment) and 10 (day 8 of treatment).
[0293] These results demonstrate that siRNAs targeting Nav1.8,
formulated in ND98-2.7 liposomes, and administered by intravenous
bolus injection or intravenous continuous pump infusion, are
efficacious against experimental nerve injury-induced chronic pain
in rats, and therefore, represent a novel approach to providing
effective treatment of clinical neuropathic pain. Moreover, these
results demonstrate that ND98-2.7 liposomal formulation of siRNA
can provide effective delivery of siRNA to sensory neurons of the
dorsal root ganglion, with systemic administration.
[0294] Formulation Procedure
[0295] The lipidoid ND98.4HCl (MW 1487), Cholesterol
(Sigma-Aldrich), and PEG-Ceramide C16 (Avanti Polar Lipids) were
used to prepare lipid-siRNA nanoparticles.
[0296] Stock solutions of each in ethanol were prepared: ND98 (FIG.
15, ND98 Isomer I), 133 mg/mL; Cholesterol, 25 mg/mL, PEG-Ceramide
C16, 100 mg/mL. ND98, Cholesterol, and PEG-Ceramide C16 stock
solutions were then combined in a 42:48:10 molar ratio. Combined
lipid solution was mixed rapidly with aqueous siRNA (in sodium
acetate pH 5) such that the final ethanol concentration was 35-45%
and the final sodium acetate concentration was 100-300 mM.
Lipid-siRNA nanoparticles formed spontaneously upon mixing.
Depending on the desired particle size distribution, the resultant
nanoparticle mixture was in some cases extruded through a
polycarbonate membrane (100 nm cut-off) using a thermobarrel
extruder (Lipex Extruder, Northern Lipids, Inc). In other cases,
the extrusion step was omitted. Ethanol removal and simultaneous
buffer exchange was accomplished by either dialysis or tangential
flow filtration. Buffer was exchanged to phosphate buffered saline
(PBS) pH 7.2.
[0297] Characterization of Formulations
[0298] Formulations prepared by either the standard or
extrusion-free method are characterized in a similar manner.
Formulations are first characterized by visual inspection. They
should be whitish translucent solutions free from aggregates or
sediment. Particle size and particle size distribution of
lipid-nanoparticles are measured by dynamic light scattering using
a Malvern Zetasizer Nano ZS (Malvern, USA). Particles should be
20-300 nm, and ideally, 40-100 nm in size. The particle size
distribution should be unimodal. The total siRNA concentration in
the formulation, as well as the entrapped fraction, is estimated
using a dye exclusion assay. A sample of the formulated siRNA is
incubated with the RNA-binding dye Ribogreen (Molecular Probes) in
the presence or absence of a formulation disrupting surfactant,
0.5% Triton-X100. The total siRNA in the formulation is determined
by the signal from the sample containing the surfactant, relative
to a standard curve. The entrapped fraction is determined by
subtracting the "free" siRNA content (as measured by the signal in
the absence of surfactant) from the total siRNA content. Percent
entrapped siRNA is typically >85%.
[0299] dsRNA Expression Vectors
[0300] In another aspect of the invention, Nav1.8 specific dsRNA
molecules that modulate Nav1.8 gene expression activity are
expressed from transcription units inserted into DNA or RNA vectors
(see, e.g., Couture, A, et al., TIG. (1996), 12:5-10; Skillern, A.,
et al., International PCT Publication No. WO 00/22113, Conrad,
International PCT Publication No. WO 00/22114, and Conrad, U.S.
Pat. No. 6,054,299). These transgenes can be introduced as a linear
construct, a circular plasmid, or a viral vector, which can be
incorporated and inherited as a transgene integrated into the host
genome. The transgene can also be constructed to permit it to be
inherited as an extrachromosomal plasmid (Gassmann, et al., Proc.
Natl. Acad. Sci. USA (1995) 92:1292).
[0301] The individual strands of a dsRNA can be transcribed by
promoters on two separate expression vectors and co-transfected
into a target cell. Alternatively each individual strand of the
dsRNA can be transcribed by promoters both of which are located on
the same expression plasmid. In a preferred embodiment, a dsRNA is
expressed as an inverted repeat joined by a linker polynucleotide
sequence such that the dsRNA has a stem and loop structure.
[0302] The recombinant dsRNA expression vectors are preferably DNA
plasmids or viral vectors. dsRNA expressing viral vectors can be
constructed based on, but not limited to, adeno-associated virus
(for a review, see Muzyczka, et al., Curr. Topics Micro. Immunol.
(1992) 158:97-129)); adenovirus (see, for example, Berkner, et al.,
BioTechniques (1998) 6:616), Rosenfeld et al. (1991, Science
252:431-434), and Rosenfeld et al. (1992), Cell 68:143-155)); or
alphavirus as well as others known in the art. Retroviruses have
been used to introduce a variety of genes into many different cell
types, including epithelial cells, in vitro and/or in vivo (see,
e.g., Eglitis, et al., Science (1985) 230:1395-1398; Danos and
Mulligan, Proc. Natl. Acad. Sci. USA (1998) 85:6460-6464; Wilson et
al., 1988, Proc. Natl. Acad. Sci. USA 85:3014-3018; Armentano et
al., 1990, Proc. Natl. Acad. Sci. USA 87:61416145; Huber et al.,
1991, Proc. Natl. Acad. Sci. USA 88:8039-8043; Ferry et al., 1991,
Proc. Natl. Acad. Sci. USA 88:8377-8381; Chowdhury et al., 1991,
Science 254:1802-1805; van Beusechem. et al., 1992, Proc. Natl.
Acad. Sci. USA 89:7640-19; Kay et al., 1992, Human Gene Therapy
3:641-647; Dai et al., 1992, Proc. Natl. Acad. Sci. USA
89:10892-10895; Hwu et al., 1993, J. Immunol. 150:4104-4115; U.S.
Pat. No. 4,868,116; U.S. Pat. No. 4,980,286; PCT Application WO
89/07136; PCT Application WO 89/02468; PCT Application WO 89/05345;
and PCT Application WO 92/07573). Recombinant retroviral vectors
capable of transducing and expressing genes inserted into the
genome of a cell can be produced by transfecting the recombinant
retroviral genome into suitable packaging cell lines such as PA317
and Psi-CRIP (Comette et al., 1991, Human Gene Therapy 2:5-10; Cone
et al., 1984, Proc. Natl. Acad. Sci. USA 81:6349). Recombinant
adenoviral vectors can be used to infect a wide variety of cells
and tissues in susceptible hosts (e.g., rat, hamster, dog, and
chimpanzee) (Hsu et al., 1992, J. Infectious Disease, 166:769), and
also have the advantage of not requiring mitotically active cells
for infection.
[0303] The promoter driving dsRNA expression in either a DNA
plasmid or viral vector of the invention may be a eukaryotic RNA
polymerase I (e.g. ribosomal RNA promoter), RNA polymerase II (e.g.
CMV early promoter or actin promoter or U1 snRNA promoter) or
preferably RNA polymerase III promoter (e.g. U6 snRNA or 7SK RNA
promoter) or a prokaryotic promoter, for example the T7 promoter,
provided the expression plasmid also encodes T7 RNA polymerase
required for transcription from a T7 promoter. The promoter can
also direct transgene expression to the pancreas (see, e.g. the
insulin regulatory sequence for pancreas (Bucchini et al., 1986,
Proc. Natl. Acad. Sci. USA 83:2511-2515)).
[0304] In addition, expression of the transgene can be precisely
regulated, for example, by using an inducible regulatory sequence
and expression systems such as a regulatory sequence that is
sensitive to certain physiological regulators, e.g., circulating
glucose levels, or hormones (Docherty et al., 1994, FASEB J.
8:20-24). Such inducible expression systems, suitable for the
control of transgene expression in cells or in mammals include
regulation by ecdysone, by estrogen, progesterone, tetracycline,
chemical inducers of dimerization, and
isopropyl-beta-D1-thiogalactopyranoside (EPTG). A person skilled in
the art would be able to choose the appropriate regulatory/promoter
sequence based on the intended use of the dsRNA transgene.
[0305] Preferably, recombinant vectors capable of expressing dsRNA
molecules are delivered as described below, and persist in target
cells. Alternatively, viral vectors can be used that provide for
transient expression of dsRNA molecules. Such vectors can be
repeatedly administered as necessary. Once expressed, the dsRNAs
bind to target RNA and modulate its function or expression.
Delivery of dsRNA expressing vectors can be systemic, such as by
intravenous or intramuscular administration, by administration to
target cells ex-planted from the patient followed by reintroduction
into the patient, or by any other means that allows for
introduction into a desired target cell.
[0306] dsRNA expression DNA plasmids are typically transfected into
target cells as a complex with cationic lipid carriers (e.g.
Oligofectamine) or non-cationic lipid-based carriers (e.g.
Transit-TKO.TM.). Multiple lipid transfections for dsRNA-mediated
knockdowns targeting different regions of a single Nav1.8 gene or
multiple Nav1.8 genes over a period of a week or more are also
contemplated by the invention. Successful introduction of the
vectors of the invention into host cells can be monitored using
various known methods. For example, transient transfection. can be
signaled with a reporter, such as a fluorescent marker, such as
Green Fluorescent Protein (GFP). Stable transfection of ex vivo
cells can be ensured using markers that provide the transfected
cell with resistance to specific environmental factors (e.g.,
antibiotics and drugs), such as hygromycin B resistance.
[0307] The Nav1.8 specific dsRNA molecules can also be inserted
into vectors and used as gene therapy vectors for human patients.
Gene therapy vectors can be delivered to a subject by, for example,
intravenous injection, local administration (see U.S. Pat. No.
5,328,470) or by stereotactic injection (see e.g., Chen et al.
(1994) Proc. Natl. Acad. Sci. USA 91:3054-3057). 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.
Alternatively, where the complete gene delivery vector can be
produced intact from recombinant cells, e.g., retroviral vectors,
the pharmaceutical preparation can include one or more cells which
produce the gene delivery system.
Sequence CWU 1
1
609121DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 1cccggauuuu aacuacacct t
21221DNAArtificial SequenceDescription of Combined DNA/RNA Molecule
Synthetic oligonucleotide 2gguguaguua aaauccgggt t
21321DNAArtificial SequenceDescription of Combined DNA/RNA Molecule
Synthetic oligonucleotide 3ccggauuuua acuacaccat t
21421DNAArtificial SequenceDescription of Combined DNA/RNA Molecule
Synthetic oligonucleotide 4ugguguaguu aaaauccggt t
21521DNAArtificial SequenceDescription of Combined DNA/RNA Molecule
Synthetic oligonucleotide 5gacaacccgg auuuuaacut t
21621DNAArtificial SequenceDescription of Combined DNA/RNA Molecule
Synthetic oligonucleotide 6aguuaaaauc cggguuguct t
21721DNAArtificial SequenceDescription of Combined DNA/RNA Molecule
Synthetic oligonucleotide 7ccuuacaacc agcgcaggat t
21821DNAArtificial SequenceDescription of Combined DNA/RNA Molecule
Synthetic oligonucleotide 8uccugcgcug guuguaaggt t
21921DNAArtificial SequenceDescription of Combined DNA/RNA Molecule
Synthetic oligonucleotide 9aacccggauu uuaacuacat t
211021DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 10uguaguuaaa auccggguut t
211121DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 11ugugcaugac ccgaacugat t
211221DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 12ucaguucggg ucaugcacat t
211321DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 13acaaccagcg caggauguct t
211421DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 14gacauccugc gcugguugut t
211521DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 15acccggauuu uaacuacact t
211621DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 16guguaguuaa aauccgggut t
211721DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 17cauccuauga accaauagct t
211821DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 18gcuauugguu cauaggaugt t
211921DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 19cuuacaacca gcgcaggaut t
212021DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 20auccugcgcu gguuguaagt t
212121DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 21uuuaacuaca ccagcuuugt t
212221DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 22caaagcuggu guaguuaaat t
212321DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 23gugugcauga cccgaacugt t
212421DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 24caguucgggu caugcacact t
212521DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 25cggauuuuaa cuacaccagt t
212621DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 26cugguguagu uaaaauccgt t
212721DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 27uacaaccagc gcaggaugut t
212821DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 28acauccugcg cugguuguat t
212921DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 29ggucucugug cccauugcut t
213021DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 30agcaaugggc acagagacct t
213121DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 31cccaaguucu auggugagct t
213221DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 32gcucaccaua gaacuugggt t
213321DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 33caaguucuau ggugagcuct t
213421DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 34gagcucacca uagaacuugt t
213521DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 35cuacagcaca caccggacat t
213621DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 36uguccggugu gugcuguagt t
213721DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 37aaguucuaug gugagcucct t
213821DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 38ggagcucacc auagaacuut t
213921DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 39uuuugucuaa augaguucat t
214021DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 40ugaacucauu uagacaaaat t
214121DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 41ccucucacug uuccgccuct t
214221DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 42gaggcggaac agugagaggt t
214321DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 43aaccaguucu uuguggccgt t
214421DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 44cggccacaaa gaacugguut t
214521DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 45cucacuguuc cgccucaugt t
214621DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 46caugaggcgg aacagugagt t
214721DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 47ccaaguucua uggugagcut t
214821DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 48agcucaccau agaacuuggt t
214921DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 49caguucuuug uggccgucut t
215021DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 50agacggccac aaagaacugt t
215121DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 51ggcuggcagg ugcgcaagat t
215221DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 52ucuugcgcac cugccagcct t
215321DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 53cuccucugag ggcagcacgt t
215421DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 54cgugcugccc ucagaggagt t
215521DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 55gucuucacug ucauuuacat t
215621DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 56uguaaaugac agugaagact t
215721DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 57caaccagcgc aggaugucut t
215821DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 58agacauccug cgcugguugt t
215921DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 59agaaguaucu gaucugggat t
216021DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 60ucccagauca gauacuucut t
216121DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 61ucuacagcac acaccggact t
216221DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 62guccggugug ugcuguagat t
216321DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 63aguucuaugg ugagcuccct t
216421DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 64gggagcucac cauagaacut t
216521DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 65ucuaugguga gcucccagct t
216621DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 66gcugggagcu caccauagat t
216721DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 67aguucuuugu ggccgucuut t
216821DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 68aagacggcca caaagaacut t
216921DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 69ucacuguucc gccucaugat t
217021DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 70ucaugaggcg gaacagugat t
217121DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 71ggauuuuaac uacaccagct t
217221DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 72gcugguguag uuaaaaucct t
217321DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 73ccgcuugcug cgcguauuct t
217421DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 74gaauacgcgc agcaagcggt t
217521DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 75cuugcuaccg uaucguggat t
217621DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 76uccacgauac gguagcaagt t
217721DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 77gacuucaucg cuaauccgat t
217821DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 78ucggauuagc gaugaaguct t
217921DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 79uggauuuuag cgucauuact t
218021DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 80guaaugacgc uaaaauccat t
218121DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 81cgcuugcugc gcguauucat t
218221DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 82ugaauacgcg cagcaagcgt t
218321DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 83caacuuccgu cgcuuuacut t
218421DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 84aguaaagcga cggaaguugt t
218521DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 85gucgcuuuac uccggaguct t
218621DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 86gacuccggag uaaagcgact t
218721DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 87aaugaguuca cguaccugat t
218821DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 88ucagguacgu gaacucauut t
218921DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 89agacuugcua ccguaucgut t
219021DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 90acgauacggu agcaagucut t
219121DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 91aguuuauuua uuacggucat t
219221DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 92ugaccguaau aaauaaacut t
219321DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 93aaaugaguuc acguaccugt t
219421DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 94cagguacgug aacucauuut t
219521DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 95ugucucggca uucgaugcat t
219621DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 96ugcaucgaau gccgagacat t
219721DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 97ucaaagcccu ucgaacccut t
219821DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 98aggguucgaa gggcuuugat t
219921DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 99gcgggcucuu ucucgauuut t
2110021DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 100aaaucgagaa agagcccgct t
2110121DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic
oligonucleotide 101ccuuguaccu uugucgauut t 2110221DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 102aaucgacaaa gguacaaggt t 2110321DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 103gugguucucu ccauugcgat t 2110421DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 104ucgcaaugga gagaaccact t 2110521DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 105caaaaggccu aucggagcut t 2110621DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 106agcuccgaua ggccuuuugt t 2110721DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 107aaaggccuau cggagcuaut t 2110821DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 108auagcuccga uaggccuuut t 2110921DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 109ggugcaucaa cuauaccgat t 2111021DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 110ucgguauagu ugaugcacct t 2111121DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 111aacuaccgua acaaccgaat t 2111221DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 112uucgguuguu acgguaguut t 2111321DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 113ucgcuuuacu ccggagucat t 2111421DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 114ugacuccgga guaaagcgat t 2111521DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 115gaaaacgccg ggcuagucat t 2111621DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 116ugacuagccc ggcguuuuct t 2111721DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 117accgaaaaaa uaucuccgct t 2111821DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 118gcggagauau uuuuucggut t 2111921DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 119uucaucgcua auccgacugt t 2112021DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 120cagucggauu agcgaugaat t 2112121DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 121uccgucgcuu uacuccggat t 2112221DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 122uccggaguaa agcgacggat t 2112321DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 123gcuuuacucc ggagucacut t 2112421DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 124agugacuccg gaguaaagct t 2112521DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 125ggaaaacgcc gggcuaguct t 2112621DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 126gacuagcccg gcguuuucct t 2112721DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 127acuaccguaa caaccgaaat t 2112821DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 128uuucgguugu uacgguagut t 2112921DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 129uggccaucgu accaaacagt t 2113021DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 130cuguuuggua cgauggccat t 2113121DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 131acuugcuacc guaucguggt t 2113221DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 132ccacgauacg guagcaagut t 2113321DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 133gauaagucuc acagcgaagt t 2113421DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 134cuucgcugug agacuuauct t 2113521DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 135auaagucuca cagcgaagat t 2113621DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 136ucuucgcugu gagacuuaut t 2113721DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 137aagcccuucg aacccuucgt t 2113821DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 138cgaaggguuc gaagggcuut t 2113921DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 139agcccuucga acccuucgct t 2114021DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 140gcgaaggguu cgaagggcut t 2114121DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 141cccuucgaac ccuucgcgct t 2114221DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 142gcgcgaaggg uucgaagggt t 2114321DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 143aacccaaucg aaauauacut t 2114421DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 144aguauauuuc gauuggguut t 2114521DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 145cgcuuuacuc cggagucact t 2114621DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 146gugacuccgg aguaaagcgt t 2114721DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 147gaccauuucc cgguuuagut t 2114821DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 148acuaaaccgg gaaaugguct t 2114921DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 149cgguuuagug ccacucgggt t 2115021DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 150cccgaguggc acuaaaccgt t 2115121DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 151cacagcaaua gaucuccgut t 2115221DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 152acggagaucu auugcugugt t 2115321DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 153acuagggauu gacacaacct t 2115421DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 154gguuguguca aucccuagut t 2115521DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 155cccacaaugg aucaccuuut t 2115621DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 156aaaggugauc cauugugggt t 2115721DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 157ugucuuuucu aggccucgct t 2115821DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 158gcgaggccua gaaaagacat t 2115921DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 159agcugucgau gucucggcat t 2116021DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 160ugccgagaca ucgacagcut t 2116121DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 161gucucggcau ucgaugcagt t 2116221DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 162cugcaucgaa ugccgagact t 2116321DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 163ucaaaaucau ugccuucgat t 2116421DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 164ucgaaggcaa ugauuuugat t 2116521DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 165cccgcuggca caugcacgat t 2116621DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 166ucgugcaugu gccagcgggt t 2116721DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 167ucauugucuu ccguauccut t 2116821DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 168aggauacgga agacaaugat t 2116921DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 169uuggccaucg uaccaaacat t 2117021DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 170uguuugguac gauggccaat t 2117121DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 171gcacggugga cugccuagat t 2117221DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 172ucuaggcagu ccaccgugct t 2117321DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 173gcccuucgaa cccuucgcgt t 2117421DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 174cgcgaagggu ucgaagggct t 2117521DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 175ugcgggcucu uucucgauut t 2117621DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 176aaucgagaaa gagcccgcat t 2117721DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 177gaggugcauc aacuauacct t 2117821DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 178gguauaguug augcaccuct t 2117921DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 179caucaacuau accgauggat t 2118021DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 180uccaucggua uaguugaugt t 2118121DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 181aaaucauccu augaaccaat t 2118221DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 182uugguucaua ggaugauuut t 2118321DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 183aaggccuauc ggagcuaugt t 2118421DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 184cauagcuccg auaggccuut t 2118521DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 185uguacuccca gacaaaucut t 2118621DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 186agauuugucu gggaguacat t 2118721DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 187aggacaucua gcucaauact t 2118821DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 188guauugagcu agauguccut t 2118921DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 189ccgguuuagu gccacucggt t 2119021DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 190ccgaguggca cuaaaccggt t 2119121DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 191caucgcuaau ccgacugugt t 2119221DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 192cacagucgga uuagcgaugt t 2119321DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 193aaacuaccgu aacaaccgat t 2119421DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 194ucgguuguua cgguaguuut t 2119521DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 195aucgcuaauc cgacugugut t 2119621DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 196acacagucgg auuagcgaut t 2119721DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 197cccgguuuag ugccacucgt t 2119821DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 198cgaguggcac uaaaccgggt t 2119921DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 199uuuagcguca uuacccuggt t 2120021DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 200ccaggguaau gacgcuaaat t 2120121DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 201aauaagcgag gcacuucugt t
2120221DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 202cagaagugcc ucgcuuauut t
2120321DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 203cuaccguaac aaccgaaaat t
2120421DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 204uuuucgguug uuacgguagt t
2120521DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 205ucgcuaaucc gacugugugt t
2120621DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 206cacacagucg gauuagcgat t
2120721DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 207ucccucgaaa cuaacaacut t
2120821DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 208aguuguuagu uucgagggat t
2120921DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 209cuuccgucgc uuuacuccgt t
2121021DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 210cggaguaaag cgacggaagt t
2121121DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 211uuucccgguu uagugccact t
2121221DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 212guggcacuaa accgggaaat t
2121321DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 213gguuuagugc cacucgggct t
2121421DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 214gcccgagugg cacuaaacct t
2121521DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 215acaacccgga uuuuaacuat t
2121621DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 216uaguuaaaau ccggguugut t
2121721DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 217cuagggauug acacaaccut t
2121821DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 218agguuguguc aaucccuagt t
2121921DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 219ugucgauugu gaauaacaat t
2122021DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 220uuguuauuca caaucgacat t
2122121DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 221caucgugacc agacaagcut t
2122221DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 222agcuugucug gucacgaugt t
2122321DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 223ccuaucggag cuaugugcut t
2122421DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 224agcacauagc uccgauaggt t
2122521DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 225uuagcgucau uacccuggct t
2122621DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 226gccaggguaa ugacgcuaat t
2122721DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 227agcaauagau cuccgugggt t
2122821DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 228cccacggaga ucuauugcut t
2122921DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 229aucgaaauau acugauccat t
2123021DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 230uggaucagua uauuucgaut t
2123121DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 231ggaucccucg aaacuaacat t
2123221DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 232uguuaguuuc gagggaucct t
2123321DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 233acaccggaca uuuauggugt t
2123421DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 234caccauaaau guccggugut t
2123521DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 235guuuagugcc acucgggcct t
2123621DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 236ggcccgagug gcacuaaact t
2123721DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 237augacccgaa cugaccuuct t
2123821DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 238gaaggucagu ucgggucaut t
2123921DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 239auuuuagcgu cauuacccut t
2124021DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 240aggguaauga cgcuaaaaut t
2124121DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 241uuuuagcguc auuacccugt t
2124221DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 242caggguaaug acgcuaaaat t
2124321DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 243gcaauagauc uccgugggat t
2124421DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 244ucccacggag aucuauugct t
2124521DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 245aaauaagcga ggcacuucut t
2124621DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 246agaagugccu cgcuuauuut t
2124721DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 247auaagcgagg cacuucugat t
2124821DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 248ucagaagugc cucgcuuaut t
2124921DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 249ugauccuuac aaccagcgct t
2125021DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 250gcgcugguug uaaggaucat t
2125121DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 251uggccgagau aucucacuct t
2125221DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 252gagugagaua ucucggccat t
2125321DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 253caaccgccgc ccacuagugt t
2125421DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 254cacuaguggg cggcgguugt t
2125521DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 255uuagaugaac cuuuccgggt t
2125621DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 256cccggaaagg uucaucuaat t
2125721DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 257aaccuuuccg ggcccaaagt t
2125821DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 258cuuugggccc ggaaagguut t
2125921DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 259auaaccuccg uccuugaggt t
2126021DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 260ccucaaggac ggagguuaut t
2126121DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 261cuugugacgg aucccuuugt t
2126221DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 262caaagggauc cgucacaagt t
2126321DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 263ccuaccuucg aagccaugct t
2126421DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 264gcauggcuuc gaagguaggt t
2126521DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 265aaaucauugc cuucgaccct t
2126621DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 266gggucgaagg caaugauuut t
2126721DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 267cauugccuuc gacccauact t
2126821DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 268guaugggucg aaggcaaugt t
2126921DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 269cuucgaccca uacuauuaut t
2127021DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 270auaauaguau gggucgaagt t
2127121DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 271ucaucgcuaa uccgacugut t
2127221DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 272acagucggau uagcgaugat t
2127321DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 273aauccgacug ugugggucut t
2127421DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 274agacccacac agucggauut t
2127521DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 275agcacggugg acugccuagt t
2127621DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 276cuaggcaguc caccgugcut t
2127721DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 277ggugcgcaag acuugcuact t
2127821DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 278guagcaaguc uugcgcacct t
2127921DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 279aggugcauca acuauaccgt t
2128021DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 280cgguauaguu gaugcaccut t
2128121DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 281aucaacuaua ccgauggagt t
2128221DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 282cuccaucggu auaguugaut t
2128321DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 283uugucgauug ugaauaacat t
2128421DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 284uguuauucac aaucgacaat t
2128521DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 285aauggguuac cuugcacuut t
2128621DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 286aagugcaagg uaacccauut t
2128721DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 287aggccuaucg gagcuaugut t
2128821DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 288acauagcucc gauaggccut t
2128921DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 289ggccuaucgg agcuaugugt t
2129021DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 290cacauagcuc cgauaggcct t
2129121DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 291uaucggagcu augugcugct t
2129221DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 292gcagcacaua gcuccgauat t
2129321DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 293ccguccuaug agagugucat t
2129421DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 294ugacacucuc auaggacggt t
2129521DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 295ccauuggauc ccucgaaact t
2129621DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 296guuucgaggg auccaauggt t
2129721DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 297cauuggaucc cucgaaacut t
2129821DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 298aguuucgagg gauccaaugt t
2129921DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 299aucccucgaa acuaacaact t
2130021DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 300guuguuaguu ucgagggaut t
2130121DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 301gaaacuaaca acuuccguct t
2130221DNAArtificial SequenceDescription of Combined
DNA/RNA Molecule Synthetic oligonucleotide 302gacggaaguu guuaguuuct
t 2130321DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 303uuccgucgcu uuacuccggt t
2130421DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 304ccggaguaaa gcgacggaat t
2130521DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 305ccgucgcuuu acuccggagt t
2130621DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 306cuccggagua aagcgacggt t
2130721DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 307ggaucuagau ccguucuact t
2130821DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 308guagaacgga ucuagaucct t
2130921DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 309cacacaccgg acauuuaugt t
2131021DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 310cauaaauguc cggugugugt t
2131121DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 311cacaccggac auuuauggut t
2131221DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 312accauaaaug uccggugugt t
2131321DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 313ggaccauuuc ccgguuuagt t
2131421DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 314cuaaaccggg aaauggucct t
2131521DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 315accauuuccc gguuuagugt t
2131621DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 316cacuaaaccg ggaaauggut t
2131721DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 317auuucccggu uuagugccat t
2131821DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 318uggcacuaaa ccgggaaaut t
2131921DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 319aaccugauca gaagaacggt t
2132021DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 320ccguucuucu gaucagguut t
2132121DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 321ucaguuuauu uauuacggut t
2132221DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 322accguaauaa auaaacugat t
2132321DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 323gugcaugacc cgaacugact t
2132421DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 324gucaguucgg gucaugcact t
2132521DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 325augaguucac guaccugagt t
2132621DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 326cucagguacg ugaacucaut t
2132721DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 327agcgucauua cccuggcaut t
2132821DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 328augccagggu aaugacgcut t
2132921DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 329gcgucauuac ccuggcauat t
2133021DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 330uaugccaggg uaaugacgct t
2133121DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 331gucauuaccc uggcauaugt t
2133221DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 332cauaugccag gguaaugact t
2133321DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 333acagcaauag aucuccgugt t
2133421DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 334cacggagauc uauugcugut t
2133521DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 335ggacauucag aguucuuagt t
2133621DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 336cuaagaacuc ugaaugucct t
2133721DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 337ucuacauaaa uaagcgaggt t
2133821DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 338ccucgcuuau uuauguagat t
2133921DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 339uaaauaagcg aggcacuuct t
2134021DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 340gaagugccuc gcuuauuuat t
2134121DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 341ugcccugaug guuauaucut t
2134221DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 342agauauaacc aucagggcat t
2134321DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 343caacccggau uuuaacuact t
2134421DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 344guaguuaaaa uccggguugt t
2134521DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 345gggaaaaucu auaugaucut t
2134621DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 346agaucauaua gauuuuccct t
2134721DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 347gcccucgaga ugcuccggat t
2134821DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 348uccggagcau cucgagggct t
2134921DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 349agggauugac acaaccucut t
2135021DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 350agagguugug ucaaucccut t
2135121DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 351gggauugaca caaccucuct t
2135221DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 352gagagguugu gucaauccct t
2135321DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 353cacaauggau caccuuuaat t
2135421DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 354uuaaagguga uccauugugt t
2135521DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 355ccgcucugau ccuuacaact t
2135621DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 356guuguaagga ucagagcggt t
2135721DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 357auccuuacaa ccagcgcagt t
2135821DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 358cugcgcuggu uguaaggaut t
2135921DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 359agcgcaggau gucuuuucut t
2136021DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 360agaaaagaca uccugcgcut t
2136121DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 361cuuuucuagg ccucgccuct t
2136221DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 362gaggcgaggc cuagaaaagt t
2136321DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 363cuggaaaacg ccgggcuagt t
2136421DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 364cuagcccggc guuuuccagt t
2136521DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 365aaacgccggg cuagucaugt t
2136621DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 366caugacuagc ccggcguuut t
2136721DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 367aacgccgggc uagucauggt t
2136821DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 368ccaugacuag cccggcguut t
2136921DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 369agaccacgaa agccaucggt t
2137021DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 370ccgauggcuu ucguggucut t
2137121DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 371cucccuagaa gcccucuuct t
2137221DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 372gaagagggcu ucuagggagt t
2137321DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 373agaugaacac caaccgccgt t
2137421DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 374cggcgguugg uguucaucut t
2137521DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 375augaacacca accgccgcct t
2137621DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 376ggcggcgguu gguguucaut t
2137721DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 377aaccgccgcc cacuagugat t
2137821DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 378ucacuagugg gcggcgguut t
2137921DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 379accgccgccc acuagugagt t
2138021DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 380cucacuagug ggcggcggut t
2138121DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 381gcugucgaug ucucggcaut t
2138221DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 382augccgagac aucgacagct t
2138321DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 383ucgaugucuc ggcauucgat t
2138421DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 384ucgaaugccg agacaucgat t
2138521DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 385ucucggcauu cgaugcaggt t
2138621DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 386ccugcaucga augccgagat t
2138721DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 387ggcauucgau gcaggacaat t
2138821DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 388uuguccugca ucgaaugcct t
2138921DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 389gcauucgaug caggacaaat t
2139021DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 390uuuguccugc aucgaaugct t
2139121DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 391cuuagaugaa ccuuuccggt t
2139221DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 392ccggaaaggu ucaucuaagt t
2139321DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 393gaguguuguc aguaucauat t
2139421DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 394uaugauacug acaacacuct t
2139521DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 395caguaucaua accuccguct t
2139621DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 396gacggagguu augauacugt t
2139721DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 397cucgaggagu cugaacagat t
2139821DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 398ucuguucaga cuccucgagt t
2139921DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 399guaucugauc ugggauugct t
2140021DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 400gcaaucccag aucagauact t
2140121DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 401agacaauucu cuuugggcut t
2140221DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 402agcccaaaga gaauugucut
t 2140321DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 403caccuugugc aucguggugt t
2140421DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 404caccacgaug cacaaggugt t
2140521DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 405gcaugagccc uaccuucgat t
2140621DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 406ucgaagguag ggcucaugct t
2140721DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 407uaccuucgaa gccaugcuct t
2140821DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 408gagcauggcu ucgaagguat t
2140921DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 409caaaaucauu gccuucgact t
2141021DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 410gucgaaggca augauuuugt t
2141121DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 411aucauugccu ucgacccaut t
2141221DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 412augggucgaa ggcaaugaut t
2141321DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 413ucauugccuu cgacccauat t
2141421DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 414uaugggucga aggcaaugat t
2141521DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 415auugccuucg acccauacut t
2141621DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 416aguauggguc gaaggcaaut t
2141721DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 417ucgacccaua cuauuauuut t
2141821DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 418aaauaauagu augggucgat t
2141921DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 419caucaucguc acugugagut t
2142021DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 420acucacagug acgaugaugt t
2142121DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 421caucgucacu gugagucugt t
2142221DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 422cagacucaca gugacgaugt t
2142321DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 423ucgucacugu gagucugcut t
2142421DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 424agcagacuca cagugacgat t
2142521DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 425ggaaaacuac cguaacaact t
2142621DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 426guuguuacgg uaguuuucct t
2142721DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 427uaccguaaca accgaaaaat t
2142821DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 428uuuuucgguu guuacgguat t
2142921DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 429cguaacaacc gaaaaaauat t
2143021DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 430uauuuuuucg guuguuacgt t
2143121DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 431aaaauccaua ugccucauct t
2143221DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 432gaugaggcau auggauuuut t
2143321DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 433cuguucaucg cccugcuaut t
2143421DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 434auagcagggc gaugaacagt t
2143521DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 435uguucaucgc ccugcuauut t
2143621DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 436aauagcaggg cgaugaacat t
2143721DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 437cccugcuauu gaacucuuut t
2143821DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 438aaagaguuca auagcagggt t
2143921DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 439ggccaucgua ccaaacaggt t
2144021DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 440ccuguuuggu acgauggcct t
2144121DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 441ccaucguacc aaacaggcut t
2144221DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 442agccuguuug guacgauggt t
2144321DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 443cagugacuuc aucgcuaaut t
2144421DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 444auuagcgaug aagucacugt t
2144521DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 445cuucaucgcu aauccgacut t
2144621DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 446agucggauua gcgaugaagt t
2144721DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 447uaauccgacu guguggguct t
2144821DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 448gacccacaca gucggauuat t
2144921DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 449gaaucugauc uugaugacut t
2145021DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 450agucaucaag aucagauuct t
2145121DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 451aagaguccau gggauguggt t
2145221DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 452ccacauccca uggacucuut t
2145321DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 453gcaggugcgc aagacuugct t
2145421DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 454gcaagucuug cgcaccugct t
2145521DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 455gcgcaagacu ugcuaccgut t
2145621DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 456acgguagcaa gucuugcgct t
2145721DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 457aagacuugcu accguaucgt t
2145821DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 458cgauacggua gcaagucuut t
2145921DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 459gacuugcuac cguaucgugt t
2146021DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 460cacgauacgg uagcaaguct t
2146121DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 461cucauuguga auaucucact t
2146221DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 462gugagauauu cacaaugagt t
2146321DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 463ugauaagucu cacagcgaat t
2146421DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 464uucgcuguga gacuuaucat t
2146521DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 465aucaaagccc uucgaaccct t
2146621DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 466ggguucgaag ggcuuugaut t
2146721DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 467aaagcccuuc gaacccuuct t
2146821DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 468gaaggguucg aagggcuuut t
2146921DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 469ccuucgaacc cuucgcgcut t
2147021DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 470agcgcgaagg guucgaaggt t
2147121DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 471cuucgaaccc uucgcgcuct t
2147221DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 472gagcgcgaag gguucgaagt t
2147321DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 473ugcaucaacu auaccgaugt t
2147421DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 474caucgguaua guugaugcat t
2147521DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 475cccuuguacc uuugucgaut t
2147621DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 476aucgacaaag guacaagggt t
2147721DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 477uuguaccuuu gucgauugut t
2147821DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 478acaaucgaca aagguacaat t
2147921DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 479uuugauaaug uugcaauggt t
2148021DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 480ccauugcaac auuaucaaat t
2148121DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 481gcaauggguu accuugcact t
2148221DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 482gugcaaggua acccauugct t
2148321DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 483uggguuaccu ugcacuucut t
2148421DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 484agaagugcaa gguaacccat t
2148521DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 485gcuguugauu cccgggaggt t
2148621DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 486ccucccggga aucaacagct t
2148721DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 487aucgugacca gacaagcuut t
2148821DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 488aagcuugucu ggucacgaut t
2148921DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 489cgucuucaca ggcgaaugut t
2149021DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 490acauucgccu gugaagacgt t
2149121DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 491ucacaggcga augugucaut t
2149221DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 492augacacauu cgccugugat t
2149321DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 493cacaggcgaa ugugucaugt t
2149421DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 494caugacacau ucgccugugt t
2149521DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 495caggcgaaug ugucaugaat t
2149621DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 496uucaugacac auucgccugt t
2149721DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 497aggcgaaugu gucaugaagt t
2149821DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 498cuucaugaca cauucgccut t
2149921DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 499uguucgcuuu gaggcaguat t
2150021DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 500uacugccuca aagcgaacat t
2150121DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 501gcaguacuac uucacaaaut t
2150221DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 502auuugugaag uaguacugct t
2150321DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 503uccauugcga gccugauuut t 2150421DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 504aaaucaggcu cgcaauggat t 2150521DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 505ccauugcgag ccugauuuut t 2150621DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 506aaaaucaggc ucgcaauggt t 2150721DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 507aucgggcugu ugcuauucct t 2150821DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 508ggaauagcaa cagcccgaut t 2150921DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 509aaaacccaau cgaaauauat t 2151021DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 510uauauuucga uuggguuuut t 2151121DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 511caaucgaaau auacugauct t 2151221DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 512gaucaguaua uuucgauugt t 2151321DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 513aaucgaaaua uacugaucct t 2151421DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 514ggaucaguau auuucgauut t 2151521DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 515ucgaaauaua cugauccagt t 2151621DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 516cuggaucagu auauuucgat t 2151721DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 517aaucauccua ugaaccaaut t 2151821DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 518auugguucau aggaugauut t 2151921DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 519uaugaaccaa uagcaaccat t 2152021DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 520ugguugcuau ugguucauat t 2152121DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 521cacucuccga uggaagcaat t 2152221DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 522uugcuuccau cggagagugt t 2152321DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 523cuaucggagc uaugugcugt t 2152421DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 524cagcacauag cuccgauagt t 2152521DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 525agcaaaugaa aauuguguat t 2152621DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 526uacacaauuu ucauuugcut t 2152721DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 527uacucccaga caaaucugat t 2152821DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 528ucagauuugu cugggaguat t 2152921DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 529agagugucac uagaggccut t 2153021DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 530aggccucuag ugacacucut t 2153121DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 531ggccuuagug auagagucat t 2153221DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 532ugacucuauc acuaaggcct t 2153321DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 533agugauagag ucaacaugat t 2153421DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 534ucauguugac ucuaucacut t 2153521DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 535gauagaguca acaugaggat t 2153621DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 536uccucauguu gacucuauct t 2153721DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 537caucuagcuc aauacaaaat t 2153821DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 538uuuuguauug agcuagaugt t 2153921DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 539cuagcucaau acaaaaugat t 2154021DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 540ucauuuugua uugagcuagt t 2154121DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 541aguauggagc ugauugccct t 2154221DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 542gggcaaucag cuccauacut t 2154344DNAArtificial
SequenceDescription of Artificial Sequence Synthetic probe
543tttggaggtt aaaggtgatc cattttttct cttggaaaga aagt
4454440DNAArtificial SequenceDescription of Artificial Sequence
Synthetic probe 544ggcgttttcc agaggcgagt ttttctcttg gaaagaaagt
4054539DNAArtificial SequenceDescription of Artificial Sequence
Synthetic probe 545ccagcagcag agagcccctt tttctcttgg aaagaaagt
3954641DNAArtificial SequenceDescription of Artificial Sequence
Synthetic probe 546ctgggttgag gaagagggct tttttctctt ggaaagaaag t
4154741DNAArtificial SequenceDescription of Artificial Sequence
Synthetic probe 547aacactcatt gccctttggg tttttctctt ggaaagaaag t
4154845DNAArtificial SequenceDescription of Artificial Sequence
Synthetic probe 548aaggacggag gttatgatac tgactttttc tcttggaaag
aaagt 4554944DNAArtificial SequenceDescription of Artificial
Sequence Synthetic probe 549tgttcagact cctcgagttc ctctttttct
cttggaaaga aagt 4455047DNAArtificial SequenceDescription of
Artificial Sequence Synthetic probe 550ctatgccttc tctcactggc
atttttttag gcataggacc cgtgtct 4755146DNAArtificial
SequenceDescription of Artificial Sequence Synthetic probe
551ctggttgtaa ggatcagagc ggtttttagg cataggaccc gtgtct
4655246DNAArtificial SequenceDescription of Artificial Sequence
Synthetic probe 552aacacactgc catgactagc cctttttagg cataggaccc
gtgtct 4655344DNAArtificial SequenceDescription of Artificial
Sequence Synthetic probe 553gatggctttc gtggtctcca tttttaggca
taggacccgt gtct 4455441DNAArtificial SequenceDescription of
Artificial Sequence Synthetic probe 554ctggccagca cccccacttt
ttaggcatag gacccgtgtc t 4155544DNAArtificial SequenceDescription of
Artificial Sequence Synthetic probe 555catgcctgga gtcagggttg
tttttaggca taggacccgt gtct 4455644DNAArtificial SequenceDescription
of Artificial Sequence Synthetic probe 556agacatcgac agctccaggg
tttttaggca taggacccgt gtct 4455742DNAArtificial SequenceDescription
of Artificial Sequence Synthetic probe 557gtcctgcatc gaatgccgtt
tttaggcata ggacccgtgt ct 4255843DNAArtificial SequenceDescription
of Artificial Sequence Synthetic probe 558caagcagggt gggcacttct
ttttaggcat aggacccgtg tct 4355921DNAArtificial SequenceDescription
of Artificial Sequence Synthetic probe 559tgtgggagtg gagagaggtt g
2156024DNAArtificial SequenceDescription of Artificial Sequence
Synthetic probe 560ccctctgaca ctcttggctt tatt 2456124DNAArtificial
SequenceDescription of Artificial Sequence Synthetic probe
561ggtgatttgt tgtcttctgt ggag 2456221DNAArtificial
SequenceDescription of Artificial Sequence Synthetic probe
562gcctagaaaa gacatcctgc g 2156319DNAArtificial SequenceDescription
of Artificial Sequence Synthetic probe 563gccaggggac cggaaatgg
1956423DNAArtificial SequenceDescription of Artificial Sequence
Synthetic probe 564ccctcaggga gtgagatatc tcg 2356524DNAArtificial
SequenceDescription of Artificial Sequence Synthetic probe
565ggaaagactc catcatctgt gact 2456618DNAArtificial
SequenceDescription of Artificial Sequence Synthetic probe
566tctagggagg gggccttg 1856721DNAArtificial SequenceDescription of
Artificial Sequence Synthetic probe 567gcggttggtg ttcatcttct c
2156819DNAArtificial SequenceDescription of Artificial Sequence
Synthetic probe 568gcaagctcac tagtgggcg 1956925DNAArtificial
SequenceDescription of Artificial Sequence Synthetic probe
569tctgctgaca agaaagtctt ctttt 2557023DNAArtificial
SequenceDescription of Artificial Sequence Synthetic probe
570cccggaaagg ttcatctaag tat 2357141DNAArtificial
SequenceDescription of Artificial Sequence Synthetic probe
571gaatttgcca tgggtggaat tttttctctt ggaaagaaag t
4157241DNAArtificial SequenceDescription of Artificial Sequence
Synthetic probe 572ggagggatct cgctcctgga tttttctctt ggaaagaaag t
4157340DNAArtificial SequenceDescription of Artificial Sequence
Synthetic probe 573ccccagcctt ctccatggtt ttttctcttg gaaagaaagt
4057440DNAArtificial SequenceDescription of Artificial Sequence
Synthetic probe 574gctcccccct gcaaatgagt ttttctcttg gaaagaaagt
4057542DNAArtificial SequenceDescription of Artificial Sequence
Synthetic probe 575agccttgacg gtgccatgtt tttaggcata ggacccgtgt ct
4257645DNAArtificial SequenceDescription of Artificial Sequence
Synthetic probe 576gatgacaagc ttcccgttct ctttttaggc ataggacccg
tgtct 4557746DNAArtificial SequenceDescription of Artificial
Sequence Synthetic probe 577agatggtgat gggatttcca tttttttagg
cataggaccc gtgtct 4657844DNAArtificial SequenceDescription of
Artificial Sequence Synthetic probe 578gcatcgcccc acttgatttt
tttttaggca taggacccgt gtct 4457943DNAArtificial SequenceDescription
of Artificial Sequence Synthetic probe 579cacgacgtac tcagcgccat
ttttaggcat aggacccgtg tct 4358046DNAArtificial SequenceDescription
of Artificial Sequence Synthetic probe 580ggcagagatg atgacccttt
tgtttttagg cataggaccc gtgtct 4658121DNAArtificial
SequenceDescription of Artificial Sequence Synthetic probe
581ggtgaagacg ccagtggact c 2158245DNAArtificial SequenceDescription
of Artificial Sequence Synthetic probe 582cgttttctcc tcttgcttct
tctctttttc tcttggaaag aaagt 4558339DNAArtificial
SequenceDescription of Artificial Sequence Synthetic probe
583gggagcctgt cctccccatt tttctcttgg aaagaaagt 3958439DNAArtificial
SequenceDescription of Artificial Sequence Synthetic probe
584tcgcctgcga aaggtgaatt tttctcttgg aaagaaagt 3958538DNAArtificial
SequenceDescription of Artificial Sequence Synthetic probe
585ctctcgctct cccccgcttt ttctcttgga aagaaagt 3858638DNAArtificial
SequenceDescription of Artificial Sequence Synthetic probe
586ccgggactgg gctgtccttt ttctcttgga aagaaagt 3858739DNAArtificial
SequenceDescription of Artificial Sequence Synthetic probe
587ttttgccatg gagggcgttt tttctcttgg aaagaaagt 3958847DNAArtificial
SequenceDescription of Artificial Sequence Synthetic probe
588ggctgagatg attcattgct ctgtttttag gcataggacc cgtgtct
4758941DNAArtificial SequenceDescription of Artificial Sequence
Synthetic probe 589gctgaggcca cgggtgattt ttaggcatag gacccgtgtc t
4159041DNAArtificial SequenceDescription of Artificial Sequence
Synthetic probe 590aatgctcccg cggctggttt ttaggcatag gacccgtgtc t
4159141DNAArtificial SequenceDescription of Artificial Sequence
Synthetic probe 591ctgggcactg gtccggcttt ttaggcatag gacccgtgtc t
4159241DNAArtificial SequenceDescription of Artificial Sequence
Synthetic probe 592ggccaggagc cgaggttttt ttaggcatag gacccgtgtc t
4159346DNAArtificial SequenceDescription of Artificial Sequence
Synthetic probe 593cccagtaatg agaccacccc attttttagg cataggaccc
gtgtct 4659441DNAArtificial SequenceDescription of Artificial
Sequence Synthetic probe 594cctctgggtc gcctgccttt ttaggcatag
gacccgtgtc t 4159524DNAArtificial SequenceDescription of Artificial
Sequence Synthetic probe 595cactcctcag ttcctgaaga catc
2459624DNAArtificial SequenceDescription of Artificial Sequence
Synthetic probe 596ggaccatctt ctgagtcaga cttg 2459722DNAArtificial
SequenceDescription of Artificial Sequence Synthetic probe
597aacgtggctt catagaagtc ct 2259822DNAArtificial
SequenceDescription of Artificial Sequence Synthetic probe
598aatctgcttc agaacccagg tc 2259923DNAArtificial
SequenceDescription of Artificial Sequence Synthetic probe
599tgtgctgttt tcatcatctg caa 2360020DNAArtificial
SequenceDescription of Artificial Sequence Synthetic probe
600ccagcagtga tgtgtggtgg 2060115DNAArtificial SequenceDescription
of Artificial Sequence Synthetic probe 601gcaggggcca gggca
1560221DNAArtificial SequenceDescription of Artificial Sequence
Synthetic probe 602tgcagtccac agtgctgttc t 2160318DNAArtificial
SequenceDescription of Artificial Sequence Synthetic probe
603ggcttcctgg ggatgtgg 1860421DNAArtificial SequenceDescription of
Combined DNA/RNA Molecule Synthetic oligonucleotide 604cauccuauga
accaauagct t 2160521DNAArtificial SequenceDescription of Combined
DNA/RNA Molecule Synthetic oligonucleotide 605gcuauugguu cauaggaugc
t 2160621DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 606cauccuauga accaauagct t
2160721DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 607gcuauugguu cauaggaugt t
2160821DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 608uuuugucuaa augaguucat t
2160921DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 609ugaacucauu
uagacaaaat t 21
* * * * *