U.S. patent application number 11/679159 was filed with the patent office on 2007-12-27 for backbone modifications to modulate oligonucleotide targeting in vivo.
Invention is credited to Richard S. Geary, Richard H. Griffey, Mausumee Guha, Scott Henry, Thomas A. Leedom, Arthur Levin, Brett P. Monia, Andrew M. Siwkowski, Edward Wancewicz, Lynnetta Watts.
Application Number | 20070299028 11/679159 |
Document ID | / |
Family ID | 34467957 |
Filed Date | 2007-12-27 |
United States Patent
Application |
20070299028 |
Kind Code |
A1 |
Siwkowski; Andrew M. ; et
al. |
December 27, 2007 |
BACKBONE MODIFICATIONS TO MODULATE OLIGONUCLEOTIDE TARGETING IN
VIVO
Abstract
The present invention provides antisense compounds and methods
for modulating the expression of target genes expressed in the
kidney. In particular, this invention provides antisense
oligonucleotide compounds optimized for targeting nucleic acid
molecules expressed in the kidney. Such compounds are shown herein
to efficiently modulate the expression of target genes PTEN, SGLT2
and connective tissue growth factor (CTGF) in the kidney.
Inventors: |
Siwkowski; Andrew M.;
(Carlsbad, CA) ; Wancewicz; Edward; (Poway,
CA) ; Leedom; Thomas A.; (Fallbrook, CA) ;
Watts; Lynnetta; (Carlsbad, CA) ; Guha; Mausumee;
(Carlsbad, CA) ; Monia; Brett P.; (Encinitas,
CA) ; Griffey; Richard H.; (Vista, CA) ;
Geary; Richard S.; (Carlsbad, CA) ; Henry; Scott;
(Cardiff by the Sea, CA) ; Levin; Arthur; (Rancho
Santa Fe, CA) |
Correspondence
Address: |
McDermott Will & Emery
4370 La Jolla Village Drive
Suite 700
San Diego
CA
92122
US
|
Family ID: |
34467957 |
Appl. No.: |
11/679159 |
Filed: |
February 26, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10946498 |
Sep 21, 2004 |
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11679159 |
Feb 26, 2007 |
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60509450 |
Oct 7, 2003 |
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60517334 |
Nov 3, 2003 |
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60776550 |
Feb 24, 2006 |
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Current U.S.
Class: |
514/44A ;
536/23.1 |
Current CPC
Class: |
A61P 3/10 20180101; C12N
2310/3341 20130101; C12N 2310/341 20130101; A61P 3/00 20180101;
C12N 2310/315 20130101; C12N 2310/346 20130101; C07H 21/04
20130101; C12N 2320/32 20130101; C12N 2310/321 20130101; A61K 31/70
20130101; C12N 2310/11 20130101; C12N 2310/3527 20130101; C12N
2310/321 20130101; C12N 2310/3525 20130101; C12N 2500/40 20130101;
C12N 15/1136 20130101; C07H 21/02 20130101; C12N 15/111 20130101;
C12N 15/1138 20130101; C12N 2310/321 20130101 |
Class at
Publication: |
514/044 ;
536/023.1 |
International
Class: |
A61K 31/7088 20060101
A61K031/7088; A61P 3/00 20060101 A61P003/00; A61P 3/10 20060101
A61P003/10; C07H 21/00 20060101 C07H021/00 |
Claims
1. A method of modulating pharmacokinetic and pharmacodynamic
properties of a gapmer antisense compound by modulating the number
of phosphodiester linkages.
2. The method of claim 2, wherein the gapmer comprises two wings
and wherein the antisense compound has at least one phosphodiester
linkage.
3. The method of claim 2, wherein the at least one phosphodiester
linkage is in a wing.
4. The method of claim 3, wherein the wings have one or more
regions of alternating phosphorothioate and phosphodiester linkages
in the wings.
5. The method of claim 4, wherein each region comprise up to 2
nucleobases.
6. The method of claim 4, wherein each region comprises 1
nucleobase.
7. The method of claim 1, wherein the gapmer antisense compound
comprises a first central region consisting of at least 5
contiguous 2'-deoxy nucleosides flanked by a second 5' region and a
third 3' region, each of said second and third regions
independently consisting of at least one 2'-O-methoxyethyl
nucleoside.
8. The method of claim 1, wherein the pharmacodynamic property is
tissue distribution.
9. The method of claim 1, wherein the pharamacodynamic property is
serum protein binding.
10. A method of enhancing antisense inhibition of expression of a
preselected cellular RNA target in a kidney cell or kidney tissue
comprising contacting a kidney cell or tissue with an antisense
compound 8 to 80 nucleobases in length which is substantially
complementary to a preselected cellular RNA target, wherein said
antisense compound comprises a first central region consisting of
at least 5 contiguous 2'-deoxy nucleosides flanked by a second 5'
region and a third 3' region, each of said second and third regions
independently consisting of at least one 2'-O-methoxyethyl
nucleoside, and wherein the internucleoside linkages of the first
region are phosphorothioate linkages and the internucleoside
linkages of the second and third regions comprise at least one
phosphodiester linkage, so that expression of said RNA target is
inhibited.
11. The method of claim 10, wherein the antisense compound is an
antisense oligonucleotide.
12. The method of claim 10, wherein the antisense compound
comprises 10 to 50 nucleobases.
13. The method of claim 10, wherein the antisense compound
comprises 13 to 30 nucleobases.
14. The method of claim 10, wherein the antisense compound
comprises 15 to 25 nucleobases.
15. The method of claim 10, wherein the antisense compound
comprises 18 to 22 nucleobases.
16. The method of claim 10, wherein the antisense compound has at
least 70% complementarity with a nucleic acid molecule encoding
said preselected cellular RNA target.
17. The method of claim 10, wherein the antisense compound has at
least 80% complementarity with a nucleic acid molecule encoding
said preselected cellular RNA target.
18. The method of claim 10, wherein the antisense compound has at
least 90% complementarity with a nucleic acid molecule encoding
said preselected cellular RNA target.
19. The method of claim 10, wherein the antisense compound has at
least 95% complementarity with a nucleic acid molecule encoding
said preselected cellular RNA target.
20. A gapped oligomeric compound comprising: a contiguous sequence
of nucleosides linked by internucleoside linking groups comprising
an internal region of .beta.-D-deoxyribonucleosides linked by
phosphorothioate internucleoside linkages and flanked on each side
by external regions of 2'-sugar modified nucleosides; the
oligomeric compound further comprising phosphorothioate
internucleoside linkages at least between the two nucleosides
located at the 5' terminus, the two nucleosides located at the 3'
terminus and between the junctions located between each external
region and the internal region; and wherein each of the external
regions independently comprises at least one phosphodiester
internucleoside linkage.
21. The gapped oligomeric compound of claim 20 wherein each of the
external regions comprises at least two phosphodiester
internucleoside linkages.
22. The gapped oligomeric compound of claim 20 wherein each
external region has alternating phosphorothioate and phosphodiester
internucleoside linkages.
23. The gapped oligomeric compound of claim 20 wherein each of the
2'-sugar modified nucleosides comprises a 2'-substituent group,
independently, selected from halo, amino, azido, O-allyl, O--C1-10
alkyl, OCF3, O--(CH2)2-O--CH3, O(CH2)2SCH3, O--(CH2)2-O--N(Rm)(Rn)
or O--CH2-C(.dbd.O)--N(Rm)(Rn), wherein each Rm and Rn is,
independently, H, an amino protecting group or substituted or
unsubstituted C1-10 alkyl.
24. The gapped oligomeric compound of claim 23 wherein each
2'-substituent group is, independently, fluoro, O--CH3, OCF3 or
O--(CH2)2-O--CH3.
25. The gapped oligomeric compound of claim 24 wherein each
2'-substituent group is O--(CH2)2-O--CH3.
26. The gapped oligomeric compound of claim 20 wherein each
external region is, independently from 3 to about 6 nucleosides in
length and the internal region is from about 7 to about 14
nucleosides in length.
27. The gapped oligomeric compound of claim 20 wherein each
external region is, independently from 3 to about 5 nucleosides in
length and the internal region is from about 7 to about 12
nucleosides in length.
28. The gapped oligomeric compound of claim 20 having from about 12
to about 30 nucleosides in length.
29. The gapped oligomeric compound of claim 20 having from about 12
to about 24 nucleosides in length.
30. The gapped oligomeric compound of claim 20 having the formula:
5'-Ms(Mj)nMs-(Ns)r-(Mj)mMsM-3' wherein: each M is a is a 2'-sugar
modified nucleoside; each N is a .beta.-D-deoxyribonucleoside; each
s is a phosphorothioate internucleoside linkage; each j is,
independently, a phosphorothioate or phosphodiester internucleoside
linkage; n and m are each, independently, from 1 to 4; r is from
about 6 to about 14; and wherein at least one j in each external
region is a phosphodiester internucleoside linkage.
31. The gapped oligomeric compound of claim 30 wherein n and m are
each 1.
32. The gapped oligomeric compound of claim 31 wherein n and m are
each, independently, from 2 to 4.
33. The gapped oligomeric compound of claim 30 wherein n and m are
each, independently, 3 and each external region independently
comprises two phosphodiester internucleoside linkages.
34. A method of preventing or delaying the onset of a disease or
condition in an animal, wherein said disease or condition is
associated with expression of a preselected cellular RNA target
expressed in the kidney, said method comprising administering to an
animal an effective amount of the antisense compound of claim
20.
35. The method of claim 34, wherein said disease or condition is a
metabolic disease or condition.
36. The method of claim 34, wherein said disease or condition is
diabetes.
37. The method of claim 34, wherein said disease or condition is
type 2 diabetes.
Description
[0001] This application is a continuation-in-part of U.S. patent
application Ser. No. 10/946,498, filed Sep. 21, 2004, which claims
the benefit of priority to U.S. provisional patent application Ser.
No. 60/509,450, filed Oct. 7, 2003 and U.S. provisional patent
application Ser. No. 60/517,334, filed Nov. 3, 2003, this
application also claims the benefit of priority to U.S. provisional
patent application Ser. No. 60/776,550, filed Feb. 24, 2006, each
of which are hereby incorporated by reference in their
entirety.
FIELD OF THE INVENTION
[0002] The present invention provides antisense compounds and
methods for modulating the expression of target genes expressed in
the kidney. In particular, this invention relates to antisense
oligonucleotide compounds optimized for targeting nucleic acid
molecules expressed in the kidney. Such compounds are shown herein
to efficiently modulate the expression of target genes expressed in
the kidney.
BACKGROUND OF THE INVENTION
[0003] One of the challenges of drug development is to direct
compounds to the appropriate site of action in the body. For
topical agents, this difficulty is readily overcome by application
of the drug to the desired site of action. For systemically
delivered agents, delivered by parenteral or non-parenteral routes,
modifications are often made to active compounds identified using
in vitro screening methods to promote desired tissue localization.
These modifications must obviously be made without disrupting
activity of the compound.
[0004] Pharmacokinetic and pharmacodynamic properties of antisense
oligonucleotides (ASOs) are dependent substantially on the
chemistry of the compound (e.g., sugar and backbone modifications,
non-nucleic acid substituent groups) rather than the sequence of
the compound.
[0005] Efficacy and sequence specific behavior of antisense
compounds in biological systems depend upon a variety of factors,
which include their resistance to enzymatic degradation, binding
affinity for the target, susceptibility to RNase H cleavage when
bound to a target mRNA and efficiency of cellular uptake. In order
to achieve the proper balance of these features for efficient
modulation of target gene expression, chemical modifications are
made to the antisense compound. For example, unmodified
phosphodiester antisense oligonucleotides are degraded rapidly in
biological fluids containing hydrolytic enzymes (Shaw et al.,
Nucleic Acids Res. 1991, 19, 747-750; Woolf et al., Nucleic Acids
Res. 1990, 18, 1763-1769) and first generation modified antisense
compounds (i.e. 2'-deoxyphosphorothioate oligonucleotides) also are
subject to activity-limiting degradation (Maier et al., Biomed.
Pept., Proteins Nucleic Acids 1995, 1, 235-241; Agrawal et al.,
Proc. Natl. Acad. Sci. U.S.A. 1991, 88, 7595-7599). Thus,
modifications that render the oligonucleotide more resistant to
nuclease activity are desirable in order to enhance antisense
activity of the compound. Sugar moieties of antisense compounds
also have been modified to increase such properties as
lipophilicity, binding affinity for the target mRNA, chemical
stability and nuclease resistance.
[0006] Distribution to peripheral tissues and ultimate uptake into
the cells of target organs also is critical to the effectiveness of
antisense compounds for treatment of a wide range of diseases.
[0007] The highest concentrations of antisense compounds are
typically found in the liver, kidney, spleen and lymph nodes, but
can be detected in nearly all organs except for the brain (Geary et
al., Curr. Opin. Investig. Drugs, 2001, 2, 562-573; Geary et al.,
J. Pharm. Exp. Therap., 2001, 296, 890-897). Despite the ability of
current antisense compounds to be delivered to the kidney, there is
a need for development of improved antisense compounds that effect
target mRNA reduction in the kidney at lower doses and without
toxicity.
[0008] The kidney is an important target for antisense therapeutics
due to its role in controlling many metabolic processes. A number
of genes expressed in the kidney have been associated with the
development of metabolic disease. Two such examples are the
sodium-dependent glucose cotransporter 2 (SGLT2) and connective
tissue growth factor (CTGF), both of which have been linked to the
development and/or progression of diabetes.
Sodium-Dependent Glucose Cotransporter 2 (SGLT2)
[0009] Diabetes is a disorder characterized by hyperglycemia due to
deficient insulin action. Chronic hyperglycemia is a major risk
factor for diabetes-associated complications, including heart
disease, retinopathy, nephropathy and neuropathy. As the kidneys
play a major role in the regulation of plasma glucose levels, renal
glucose transporters are becoming attractive drug targets (Wright,
Am. J. Physiol. Renal Physiol., 2001, 280, F10-18).
[0010] Synthetic agents that are derived from phlorizin, a specific
inhibitor of sodium/glucose transporters, have been designed and
include T-1095, and its metabolically active form T-1095A
(Tsujihara et al., J. Med. Chem., 1999, 42, 5311-5324). Phlorizin,
T-1095 and T-1095A all inhibited sodium-dependent glucose uptake in
brush border membranes prepared from normal and diabetic rat
kidney, rat small intestine, mouse kidney and dog kidney, as well
as in Xenopus oocytes injected with human SGLT mRNA (Oku et al.,
Diabetes, 1999, 48, 1794-1800; Oku et al., Eur. J. Pharmacol.,
2000, 391, 183-192). These agents have been tested as antidiabetic
compounds in laboratory animals with genetic and
streptozotocin-induced diabetes. In these models, administration of
these compounds inhibited renal SGLT activity, increased urinary
glucose excretion and improved glucose tolerance, hyperglycemia and
hypoinsulemia (Arakawa et al., Br. J. Pharmacol., 2001, 132,
578-586; Oku et al., Diabetes, 1999, 48, 1794-1800; Oku et al.,
Eur. J. Pharmacol., 2000, 391, 183-192). Prolonged treatment of
db/db mice with T-1095 yielded similar results and also almost
completely suppressed the increase of urinary albumin and improved
renal glomeruli pathology, indicating a beneficial influence on
renal dysfunction and a protective effect against nephropathy,
respectively (Arakawa et al., Br. J. Pharmacol., 2001, 132,
578-586). Diabetic nephropathy is the most common cause of
end-stage renal disease that develops in many patients with
diabetes. In Zucker diabetic fatty rats, long-term treatment with
T-1095 lowered both fed and fasting glucose levels to near normal
ranges. Also observed were recovered hepatic glucose production and
glucose utilization rates without a significant improvement in
skeletal muscle glucose utilization rate, indicating that
hyperglycemia contributes to insulin resistance in hepatic and
adipose tissue in this rat model of diabetes. These results further
suggest that glucotoxicity, which results from long-term
hyperglycemia, induces tissue-dependent insulin resistance in
diabetic patients (Nawano et al., Am. J. Physiol. Endocrinol.
Metab., 2000, 278, E535-543).
[0011] Other SGLT2 inhibiting compounds are known in the art, such
as the c-aryl glucosides disclosed and claimed in U.S. Pat. No.
6,414,126, which are inhibitors of sodium dependent glucose
transporters found in the intestine and kidney and are proposed to
treat diabetes, hyperglycemia and related diseases when used alone
or in combination with other antidiabetic agents (Ellsworth et al.,
2002).
[0012] The US pre-grant publication 20030055019 claims and
discloses isolated mutant proteins selected from a group which
includes SGLT2, the corresponding nucleic acid molecules encoding
said mutant proteins, isolated antisense derivatives of the nucleic
acid sequences encoding said mutant proteins, as well as methods of
delivering said antisense nucleic acid derivatives to treat or
prevent hypertension, diabetes, insulin sensitivity, obesity,
dyslipidemia and stroke. This application also discloses the
antisense molecules may be DNA or RNA or a chimeric mixture,
single-stranded or double-stranded or may comprise a ribozyme or
catalytic RNA (Shimkets, 2003).
[0013] The European Patent Applications EP 1 293 569 and EP 1 308
459 claim and disclose a polynucleotide comprising a protein-coding
region of the nucleotide sequence of any one of a group of
sequences which includes a nucleic acid sequence encoding human
SGLT2, an oligonucleotide comprising at least 15 nucleotides
complementary to the nucleotide sequence or to a complementary
strand thereof and an antisense polynucleotide against the claimed
polynucleotide or a part thereof. These applications disclose the
use of said antisense polynucleotides for suppressing the
expression of a polypeptide of the invention and for gene therapy
(Isogai et al., 2003; Isogai et al., 2003).
[0014] Although phlorizin and its derivatives are potent inhibitors
of sodium-glucose cotransporters, these agents do not specifically
inhibit a single species of SGLT, thus all SGLTs in all tissues are
affected. Thus, there remains a need for therapeutic compounds that
target specific SGLT species. Antisense technology is an effective
means for reducing the expression of specific gene products and may
therefore prove to be uniquely useful in a number of therapeutic,
diagnostic and research applications for the modulation of SGLT2
expression. Furthermore, given the role of SGLT2 in the development
of diabetes, antisense compounds with the ability to be delivered
to the kidney and specifically inhibit SGLT2 are highly
desirable.
Connective Tissue Growth Factor (CTGF)
[0015] Connective tissue growth factor (CTGF; also known as
ctgrofact, fibroblast inducible secreted protein, fisp-12, NOV2,
insulin-like growth factor-binding protein-related protein 2,
IGFBP-rP2, IGFBP-8, HBGF-0.8, Hcs24, and ecogenin) is a member of
the CCN (CTGF/CYR61/NOV) family of modular proteins, named for the
first family members identified, connective tissue growth factor,
cysteine-rich (CYR61), and nephroblastoma overexpressed (NOV), but
the family also includes the proteins ELM-1 (expressed in
low-metastatic cells), WISP-3 (Wnt-1-induced secreted protein), and
COP-1 (WISP-2). CCN proteins have been found to be secreted,
extracellular matrix-associated proteins that regulate cellular
processes such as adhesion, migration, mitogenesis,
differentiation, survival, angiogenesis, atherosclerosis,
chondrogenesis, wound healing, tumorigenesis, and vascular and
fibrotic diseases like scleroderma (Lau and Lam, Exp. Cell Res.,
1999, 248, 44-57).
[0016] Connective tissue growth factor is expressed in fibroblasts
during normal differentiation processes that involve extracellular
matrix (ECM) production and remodeling, such as embryogenesis and
uterine decidualization following implantation. Connective tissue
growth factor is also frequently overexpressed in fibrotic skin
disorders such as systemic sclerosis, localized skin sclerosis,
keloids, scar tissue, eosinophilic fasciitis, nodular fasciitis,
and Dupuytren's contracture. Connective tissue growth factor mRNA
or protein levels are elevated in fibrotic lesions of major organs
and tissues including the liver, kidney, lung, cardiovascular
system, pancreas, bowel, eye, and gingiva. In mammary, pancreatic
and fibrohistiocytic tumors characterized by significant connective
tissue involvement, connective tissue growth factor is
overexpressed in the stromal compartment. In many cases, connective
tissue growth factor expression is linked spatially and temporally
to the profibrogenic cytokine transforming growth factor-beta
(TGF-.quadrature.) (Moussad and Brigstock, Mol. Genet. Metab.,
2000, 71, 276-292).
[0017] Expansion of ECM with fibrosis occurs in many tissues as
part of the end-organ complications of diabetes (i.e. diabetic
nephropathy), and advanced glycosylation end products (AGE) are
implicated as one causitive factor in diabetic tissue fibrosis. In
addition to being a potent inducer of ECM synthesis and
angiogenesis, connective tissue growth factor is increased in
tissues from rodent models of diabetes. AGE treatment of primary
cultures of CRL-2097 and CRL-1474 nonfetal human dermal fibroblasts
resulted in an increase in steady state levels of connective tissue
growth factor mRNA as well as protein levels in conditioned medium
and cell-associated connective tissue growth factor, while other
IGFBP-related proteins were not upregulated by AGE. Thus, AGE
upregulates the profibrotic and proangiogenic protein connective
tissue growth factor, which may play a role in diabetic
complications (Twigg et al., Endocrinology, 2001, 142,
1760-1769).
[0018] Connective tissue growth factor has been associated with the
development of diabetes-related conditions, including diabetic
nephropathy. Diabetic nephropathy is a common complication in
patients with either type 1 or type 2 diabetes mellitus and is
recognized to cause severe morbidity and mortality. Structural
hallmarks of advanced diabetic nephropathy are glomerulosclerosis
and tubulointerstitial fibrosis leading to kidney failure. Current
therapies include ACE inhibitors and angiotensin II receptor
blockers, both of which are not justified for blanket use among all
newly diagnosed patients since only 30-40% will develop progressive
renal disease and the long term side effects of these drugs are
unknown.
[0019] In addition to the need for safe and effective treatments
for diabetes is a need for a reliable method to accurately predict,
at early stages of disease, which diabetic patients will develop
nephropathy and progress to kidney failure. Persistent
microalbuminuria is regarded as a predictor of established vascular
damage and an indicator of incipient nephropathy. Studies of renal
biopsies from patients with type 1 diabetic nephropathy demonstrate
an increase in expression of CTGF in renal tissue exhibiting
microalbuminuria and nephropathy, relative to normal control
tissues (Adler et al., Kidney Int., 2001, 60, 2330-2336),
suggesting CTGF is not only a mediator of diabetic nephropathy, but
could be used as a marker for the development of disease (Riser et
al., Kidney Int., 2003, 64, 451-458).
[0020] Disclosed and claimed in U.S. Pat. No. 5,876,730 is a
substantially pure or isolated polypeptide characterized as having
an amino acid sequence corresponding to the carboxy terminal amino
acids of a connective tissue growth factor (CTGF) protein, wherein
the polypeptide has an amino acid sequence beginning at amino acid
residue 247 or 248 from the N-terminus of connective tissue growth
factor, an isolated polynucleotide sequence encoding the connective
tissue growth factor polypeptide, a recombinant expression vector
which contains said polynucleotide, a host cell containing said
expression vector, and a pharmaceutical composition comprising a
therapeutically effective amount of connective tissue growth factor
polypeptide in a pharmaceutically acceptable carrier. Antisense
oligonucleotides are generally disclosed (Brigstock and Harding,
1999).
[0021] Disclosed and claimed in U.S. Pat. Nos. 5,783,187;
5,585,270; 6,232,064; 6,150,101; 6,069,006 and PCT Publication WO
00/35936 are an isolated polynucleotide encoding the connective
tissue growth factor polypeptide, expression vectors, host cells
stably transformed or transfected with said vectors; an isolated
polynucleotide comprising 5' untranslated regulatory nucleotide
sequences isolated from upstream of connective tissue growth
factor, wherein said untranslated regulatory nucleotide sequences
comprises a transcriptional and translational initiation region and
wherein said sequence is a TGF-beta responsive element; an isolated
nucleic acid construct comprising a non-coding regulatory sequence
isolated upstream from a connective tissue growth factor (CTGF)
gene, wherein said non-coding regulatory sequence is operably
associated with a nucleic acid sequence which expresses a protein
of interest or antisense RNA, wherein said nucleic acid sequence is
heterologous to said non-coding sequence; and a fragment of
connective tissue growth factor (CTGF) polypeptide having the
ability to induce ECM synthesis, collagen synthesis and/or
myofibroblast differentiation, comprising an amino acid sequence
encoded by at least exon 2 or exon 3 of said polypeptide. Further
claimed is a method for identifying a composition which affects
TGF-beta-induced connective tissue growth factor expression, and a
method of diagnosing a pathological state in a subject suspected of
having a pathology selected from the group consisting of fibrotic
disease and atherosclerosis, the method comprising obtaining a
sample suspected of containing connective tissue growth factor,
whereby detecting a difference in the level of connective tissue
growth factor in the sample from the subject as compared to the
level of connective tissue growth factor in the normal standard
sample is diagnostic of a pathology characterized by a cell
proliferative disorder associated with connective tissue growth
factor in the subject. Further claimed is a method for ameliorating
a cell proliferative disorder associated with connective tissue
growth factor, comprising administering to a subject having said
disorder, at the site of the disorder, a composition comprising a
therapeutically effective amount of an antibody or fragment thereof
that binds to connective tissue growth factor, wherein said
antibody or fragment thereof does not bind to PDGF. Antisense
oligonucleotides are generally disclosed (Grotendorst, 2000;
Grotendorst and Bradham, 2001; Grotendorst and Bradham, 2000;
Grotendorst and Bradham, 1996; Grotendorst and Bradham, 1998;
Grotendorst and Bradham, 2000).
[0022] Disclosed and claimed in PCT Publication WO 99/66959 is a
device for promoting neuronal regeneration, comprising a gene
activated matrix comprising a biocompatible matrix and at least one
neuronal therapeutic encoding agent having an operably linked
promoter device, wherein the neuronal therapeutic encoding agent
encodes an inhibitor of neuronal cell growth, and wherein the
inhibitor of neuronal cell growth is selected from the group
consisting of NFB42, TGF-beta, connective tissue growth factor
(CTGF), and macrophage migration inhibitory factor (MIF), and
wherein the neuronal therapeutic encoding agent is selected from
the group consisting of a nucleic acid molecule, a vector, an
antisense nucleic acid molecule and a ribozyme (Baird et al.,
1999).
[0023] Disclosed and claimed in PCT Publication WO 00/27868 is a
substantially pure connective tissue growth factor polypeptide or
functional fragments thereof, an isolated polynucleotide sequence
encoding said polypeptide, said polynucleotide sequence wherein T
can also be U, a nucleic acid sequence complementary to said
polynucleotide sequence, and fragments of said sequences that are
at least 15 bases in length and that will hybridize to DNA which
encodes the amino acid sequence of the connective tissue growth
factor protein under moderate to highly stringent conditions.
Further claimed is an expression vector including said
polynucleotide, a host cell stably transformed with said vector, an
antibody that binds to said polypeptide, and a method for producing
said polypeptide. Further claimed is a method for inhibiting the
expression of connective tissue growth factor in a cell comprising
contacting the cell with a polynucleotide which binds to a target
nucleic acid in the cell, wherein the polynucleotide inhibits the
expression of connective tissue growth factor in the cell, wherein
the polynucleotide is an antisense polynucleotide, as well as a kit
for the detection of connective tissue growth factor expression
comprising a carrier means being compartmentalized to receive one
or more containers, comprising at least one container containing at
least one antisense oligonucleotide that binds to connective tissue
growth factor (Schmidt et al., 2000).
[0024] Disclosed and claimed in PCT Publication WO 00/13706 is a
method for treating or preventing fibrosis, the method comprising
administering to a subject in need an effective amount of an agent
that modulates, regulates or inhibits the expression or activity of
connective tissue growth factor or fragments thereof, and wherein
the agent is an antibody, an antisense oligonucleotide, or a small
molecule. The method is directed to treating kidney fibrosis and
associated renal disorders, in particular, complications associated
with diabetes and hypertension (Riser and Denichili, 2000).
[0025] Disclosed and claimed in PCT Publication WO 01/29217 is an
isolated nucleic acid molecule comprising a nucleic acid sequence
encoding a polypeptide comprising an amino acid sequence selected
from a group comprising NOV1, NOV2 (connective tissue growth
factor), and NOV3, a mature form or variant of an amino acid
sequence selected from said group, as well as a nucleic acid
molecule comprising a nucleic acid sequence encoding a polypeptide
comprising an amino acid sequence selected from said group as well
as mature and variant forms or fragments of said polypeptides, and
the complement of said nucleic acid molecule. Antisense
oligonucleotides are generally disclosed (Prayaga et al.,
2001).
[0026] A phosphorothioate antisense oligonucleotide, 16 nucleotides
in length and targeted to the translation initiation start site,
was used to inhibit expression of connective tissue growth factor
and suppress proliferation and migration of bovine aorta vascular
endothelial cells in culture (Shimo et al., J. Biochem. (Tokyo),
1998, 124, 130-140). This antisense oligonucleotide was also used
to show that connective tissue growth factor induces apoptosis in
MCF-7 human breast cancer cells and that TGF-beta-induced apoptosis
is mediated, in part, by connective tissue growth factor (Hishikawa
et al., J. Biol. Chem., 1999, 274, 37461-37466). The same antisense
oligonucleotide was also found to inhibit the TGF-beta-mediated
activation of caspase 3 and thus to inhibit induction of
TGF-beta-mediated apoptosis in human aortic smooth muscle cells
(HASC) (Hishikawa et al., Eur. J. Pharmacol., 1999, 385, 287-290).
This antisense oligonucleotide was also used to block connective
tissue growth factor expression and demonstrate that high blood
pressure upregulates expression of connective tissue growth factor
in mesangial cells, which in turn enhances ECM protein production
and induces apoptosis, contributing to the remodeling of mesangium
and ultimately glomerulosclerosis (Hishikawa et al., J. Biol.
Chem., 2001, 276, 16797-16803).
[0027] Currently, there are no known therapeutic agents that
effectively inhibit the synthesis of connective tissue growth
factor and thus far. Consequently, there remains a long felt need
for additional agents capable of effectively inhibiting connective
tissue growth factor function, which for the treatment of diseases
like diabetes, requires compounds that can be effectively delivered
to the kidney.
[0028] Antisense technology is emerging as an effective means for
reducing the expression of specific gene products and may therefore
prove to be uniquely useful in a number of therapeutic, diagnostic,
and research applications for the modulation of connective tissue
growth factor expression.
[0029] The present invention provides antisense compounds and
methods for optimized kidney targeting as well as methods for
preventing diseases and conditions associated with expression of
selected target genes in the kidney. Further provided are compounds
and methods for modulating blood glucose levels and for inhibiting
the development of diabetic nephropathy. Also provided are
compounds and methods for modulating SGLT2 and CTGF expression in
kidney cells and tissues. Further identified herein are motifs and
modifications to modulate tissue distribution and half-life of
oligonucleotides.
SUMMARY OF THE INVENTION
[0030] The present invention is directed to antisense compounds
that are optimized for modulating expression of target genes in the
kidney. Provided are methods of enhancing antisense inhibition of
expression of preselected cellular RNA targets in kidney cells and
kidney tissue using antisense compounds of the invention. Also
provided are methods of preventing or delaying the onset of a
disease or condition in an animal, wherein said disease or
condition is associated with expression of a preselected cellular
RNA target, particularly SGLT2 or CTGF. Methods of lowering blood
glucose levels in an animal and methods of delaying or preventing
the onset of diabetic nephropathy also are set forth herein. Such
methods comprise administering a therapeutically or
prophylactically effective amount of one or more of the compounds
of the invention to the animal in need of treatment. Provided
herein are methods of enhancing antisense inhibition of expression
of SGLT2 in kidney cells or kidney tissues, comprising contacting
said cells or tissues with one or more of the compounds of the
invention. Also provided are methods of enhancing antisense
inhibition of expression of CTGF in kidney cells or kidney tissues,
comprising contacting said cells or tissues with one or more of the
compounds of the invention.
[0031] The invention includes a gapped oligomeric compound
comprising a contiguous sequence of nucleosides linked by
internucleoside linking groups comprising an internal region of
.beta.-D-deoxyribonucleosides linked by phosphorothioate
internucleoside linkages and flanked on each side by external
regions of 2'-sugar modified nucleosides. The oligomeric compound
further comprises phosphorothioate internucleoside linkages at
least between the two nucleosides located at the 5' terminus, the
two nucleosides located at the 3' terminus and between the
junctions located between each external region and the internal
region; and wherein each of the external regions independently
comprises at least one phosphodiester internucleoside linkage.
Preferably, each of the external regions comprises at least two
phosphodiester internucleoside linkages wherein the phosphodiester
and phosphorothioate linkages can be alternating. The compound can
include nucleosides with modified sugar moieties, wherein the
2'-sugar modified nucleosides comprises a 2'-substituent group,
independently, selected from halo, amino, azido, O-allyl, O--C1-10
alkyl, OCF3, O--(CH2)2-O--CH3, O--(CH2)2SCH3,
O--(CH2)2-O--N(Rm)(Rn) or O--CH2-C(.dbd.O)--N(Rm)(Rn), wherein each
Rm and Rn is, independently, H, an amino protecting group or
substituted or unsubstituted C1-C10 alkyl. In a preferred
embodiment, the 2'-substituent group is, independently, fluoro,
O--CH3, OCF3 or O--(CH2)2-O--CH3. Preferably, each external region
of the compound is, independently from 3 to about 6 nucleosides in
length, preferably about 3 to about 5 nucleosides in length; and
the internal region is from about 7 to about 14 nucleosides in
length, preferably about 7 to 12 nucleosides in length; with the
overall length of the compound being about 12 to 30 nucleosides in
length, preferably about 12 to 24 nucleosides in length.
[0032] The invention includes a gapped oligomeric compound having
the formula: 5'-Ms(Mj)nMs-(Ns)r-(Mj)mMsM-3' wherein, each M is a is
a 2'-sugar modified nucleoside; each N is a
.beta.-D-deoxyribonucleoside; each s is a phosphorothioate
internucleoside linkage; each j is, independently, a
phosphorothioate or phosphodiester internucleoside linkage; n and m
are each, independently, either 1, 2, 3, or 4; r is from about 6 to
about 14; and wherein at least one j in each external region is a
phosphodiester internucleoside linkage.
[0033] The invention further includes the modulation of
pharmacokinetic and pharmacodynamic properties of an ASO comprising
the use of the motifs of the instant invention. Preferably, the
invention includes a method of altering the pharmacokinetic and
pharmacodynamic properties of a fully phosphorothioate backbone
MOE-gapmer, preferably a 5-10-5 MOE gapmer (e.g., compound 116847)
by insertion of phosphodiester linkages into at least one of the
wings of the MOE-gapmer. Pharmacokinetic and pharmacodynamic
properties include, but are not limited to, tissue targeting, serum
protein binding, and tissue and systemic half-life of the ASO. The
invention includes the method of modulating relative targeting
and/or activity of an ASO in liver, kidney, and/or fat by changing
at least one phosphorothioate backbone linkage to a phosphodiester
linkage in a compound with a fully phosphorothioate backbone. In a
preferred embodiment, the backbone linkage that is changed is in
the wing of a MOE-gapmer. The invention further includes the method
of modulating relative serum protein binding by changing at least
one phosphorothioate backbone linkage to a phosphodiester linkage
in a compound with a fully phosphorothioate backbone. In a
preferred embodiment, the backbone linkage that is changed is in
the wing of a MOE-gapmer.
DETAILED DESCRIPTION OF THE INVENTION
A. Overview of the Invention
[0034] In accordance with the present invention, oligomeric
compounds that efficiently modulate expression of target genes in
the kidney are provided. The oligomeric compounds of the invention
are chimeric oligonucleotides having mixed phosphorothioate and
phosphodiester backbones, referred to herein as "mixed backbone
compounds." Preferably, the compounds of the invention have a
central "gap" region consisting of at least 5 contiguous 2'-deoxy
nucleosides flanked by two "wing" regions consisting of at least
one 2'-O-methoxyethyl nucleoside in each region. The
internucleoside linkages of the preferred compounds are
phosphorothioate linkages in the central "gap" region and
phosphodiester linkages in the two "wing" regions. In another
embodiment, mixed backbone compounds have phosphodiester linkages
in the "wing" regions except for one phosphodiester linkage at one
or both of the extreme 5' and 3' ends of the oligonucleotide.
[0035] It is shown herein that mixed backbone compounds are
efficiently delivered to the kidney and treatment with the mixed
backbone compounds results in efficient modulation of target gene
expression in the kidney without liver or kidney toxicity. It is
further shown herein that treatment with mixed backbone compounds
in mouse models of type 2 diabetes reduces blood glucose levels and
the development of diabetic nephropathy. Data from two molecular
targets are provided as illustrations of the invention.
[0036] The pharmacokinetics and in vivo activity of antisense
oligonucleotideds (ASOs) targeting putative protein tyrosine
phosphatase (PTEN) were characterized in mice and rats. All of the
ASOs had the same nucleotide sequence and differed only by selected
replacement of phosphorothioate with phosphodiester linkages (i.e.,
mixed backbone). Doses of 5 to 30 mg/kg (i.p. or s.c.) were
administered twice per week for 2 and 4 weeks in mice and rats,
respectively. Biodistribution and metabolism of the modified
oligonucleotides were quantifed using capillary gel electrophoresis
(CGE-UV). Quantitative RT-PCR was used to measure PTEN mRNA in
liver, kidney, and fat tissues. Plasma protein binding of the
oligonucleotides was conducted using an ultrafiltration method.
Partial backbone modification in the 2'-MOE portions of the
oligonucleotides did not reduce the metabolic stability of the
oligonucleotide. Distribution of the partially modified ASO shifted
somewhat more to kidney in rat but not mouse, with liver
concentrations being somewhat reduced in both rodent species. This
shift in distribution was shown to correlate with a decrease in
plasma protein binding. Importantly, selected phosphodiester
substitutions resulted in equivalent or better antisense activity
in liver and fat even though total concentrations of drug was
somewhat reduced in these tissues. In contrast, placement of only
one phosphodiester in the -deoxy portion of the oligonucleotide
significantly reduced stability and activity in liver and fat.
Also, complete replacement of phosphorothioate in the 2'-MOE
portion of the ASO significantly decreased the stability of the
compound in tissue and resulted in a concomitant loss of antisense
activity.
[0037] As used herein, the terms "target nucleic acid" and "nucleic
acid molecule encoding the target gene" have been used for
convenience to encompass DNA encoding the target gene, RNA
(including pre-mRNA and mRNA or portions thereof) transcribed from
such DNA, and also cDNA derived from such RNA. The hybridization of
a compound of this invention with its target nucleic acid is
generally referred to as "antisense". Consequently, the preferred
mechanism believed to be included in the practice of some preferred
embodiments of the invention is referred to herein as "antisense
inhibition." Such antisense inhibition is typically based upon
hydrogen bonding-based hybridization of oligonucleotide strands or
segments such that at least one strand or segment is cleaved,
degraded, or otherwise rendered inoperable. In this regard, it is
presently preferred to target specific nucleic acid molecules and
their functions for such antisense inhibition.
[0038] The present invention describes chemically modified
antisense oligonucleotides that are optimized for targeting the
kidney. As used herein, "optimize" means to modify in order to
achieve maximum efficiency. In the context of the present
invention, optimized antisense compounds are those that are
efficiently delivered to the kidney and result in inhibition of
target gene mRNA expression in the kidney while causing minimal
toxicity.
[0039] The functions of DNA to be interfered with can include
replication and transcription. Replication and transcription, for
example, can be from an endogenous cellular template, a vector, a
plasmid construct or otherwise. The functions of RNA to be
interfered with can include functions such as translocation of the
RNA to a site of protein translation, translocation of the RNA to
sites within the cell which are distant from the site of RNA
synthesis, translation of protein from the RNA, splicing of the RNA
to yield one or more RNA species, and catalytic activity or complex
formation involving the RNA which may be engaged in or facilitated
by the RNA. One preferred result of such interference with target
nucleic acid function is modulation of the expression of target
genes in the kidney. In the context of the present invention,
"modulation" and "modulation of expression" mean either an increase
(stimulation) or a decrease (inhibition) in the amount or levels of
a nucleic acid molecule encoding the gene, e.g., DNA or RNA.
Inhibition is often the preferred form of modulation of expression
and mRNA is often a preferred target nucleic acid.
[0040] In the context of this invention, "hybridization" means the
pairing of complementary strands of oligomeric compounds. In the
present invention, the preferred mechanism of pairing involves
hydrogen bonding, which may be Watson-Crick, Hoogsteen or reversed
Hoogsteen hydrogen bonding, between complementary nucleoside or
nucleotide bases (nucleobases) of the strands of oligomeric
compounds. For example, adenine and thymine are complementary
nucleobases which pair through the formation of hydrogen bonds.
Hybridization can occur under varying circumstances.
[0041] An antisense compound is specifically hybridizable when
binding of the compound to the target nucleic acid interferes with
the normal function of the target nucleic acid to cause a loss of
activity, and there is a sufficient degree of complementarity to
avoid non-specific binding of the antisense compound to non-target
nucleic acid sequences under conditions in which specific binding
is desired, i.e., under physiological conditions in the case of in
vivo assays or therapeutic treatment, and under conditions in which
assays are performed in the case of in vitro assays.
[0042] In the present invention the phrase "stringent hybridization
conditions" or "stringent conditions" refers to conditions under
which a compound of the invention will hybridize to its target
sequence, but to a minimal number of other sequences. Stringent
conditions are sequence-dependent and will be different in
different circumstances and in the context of this invention,
"stringent conditions" under which oligomeric compounds hybridize
to a target sequence are determined by the nature and composition
of the oligomeric compounds and the assays in which they are being
investigated.
[0043] "Complementary," as used herein, refers to the capacity for
precise pairing between two nucleobases of an oligomeric compound.
For example, if a nucleobase at a certain position of an
oligonucleotide (an oligomeric compound), is capable of hydrogen
bonding with a nucleobase at a certain position of a target nucleic
acid, said target nucleic acid being a DNA, RNA, or oligonucleotide
molecule, then the position of hydrogen bonding between the
oligonucleotide and the target nucleic acid is considered to be a
complementary position. The oligonucleotide and the DNA, RNA, or
oligonucleotide molecule are complementary to each other when a
sufficient number of complementary positions in each molecule are
occupied by nucleobases which can hydrogen bond with each other.
Thus, "specifically hybridizable" and "complementary" are terms
which are used to indicate a sufficient degree of precise pairing
or complementarity over a sufficient number of nucleobases such
that stable and specific binding occurs between the oligonucleotide
and a target nucleic acid.
[0044] It is understood in the art that the sequence of an
antisense compound need not be 100% complementary to that of its
target nucleic acid to be specifically hybridizable. Moreover, an
oligonucleotide may hybridize over one or more segments such that
intervening or adjacent segments are not involved in the
hybridization event (e.g., a loop structure, mismatch or hairpin
structure). It is preferred that the antisense compounds of the
present invention comprise at least 70%, or at least 75%, or at
least 80%, or at least 85% sequence complementarity to a target
region within the target nucleic acid, more preferably that they
comprise at least 90% sequence complementarity and even more
preferably comprise at least 95% or at least 99% sequence
complementarity to the target region within the target nucleic acid
sequence to which they are targeted. For example, an antisense
compound in which 18 of 20 nucleobases of the antisense compound
are complementary to a target region, and would therefore
specifically hybridize, would represent 90 percent complementarity.
In this example, the remaining noncomplementary nucleobases may be
clustered or interspersed with complementary nucleobases and need
not be contiguous to each other or to complementary nucleobases. As
such, an antisense compound which is 18 nucleobases in length
having 4 (four) noncomplementary nucleobases which are flanked by
two regions of complete complementarity with the target nucleic
acid would have 77.8% overall complementarity with the target
nucleic acid and would thus fall within the scope of the present
invention. Percent complementarity of an antisense compound with a
region of a target nucleic acid can be determined routinely using
BLAST programs (basic local alignment search tools) and PowerBLAST
programs known in the art (Altschul et al., J. Mol. Biol., 1990,
215, 403-410; Zhang and Madden, Genome Res., 1997, 7, 649-656).
[0045] Percent homology, sequence identity or complementarity, can
be determined by, for example, using the default settings of the
Gap program (Wisconsin Sequence Analysis Package, Version 8 for
Unix, Genetics Computer Group, University Research Park, Madison
Wis.), which uses the algorithm of Smith and Waterman (Adv. Appl.
Math., 1981, 2, 482-489). In some preferred embodiments, homology,
sequence identity or complementarity, between the oligomeric
compound and target is between about 50% to about 60%. In some
embodiments, homology, sequence identity or complementarity, is
between about 60% to about 70%. In preferred embodiments, homology,
sequence identity or complementarity, is between about 70% and
about 80%. In more preferred embodiments, homology, sequence
identity or complementarity, is between about 80% and about 90%. In
some preferred embodiments, homology, sequence identity or
complementarity, is about 90%, about 92%, about 94%, about 95%,
about 96%, about 97%, about 98%, about 99% or about 100%.
B. Compounds of the Invention
[0046] The antisense compounds of the present invention include
modified compounds in which a different base is present at one or
more of the nucleotide positions in the compound. For example, if
the first nucleotide is an adenosine, modified compounds may be
produced which contain thymidine, guanosine or cytidine at this
position. This may be done at any of the positions of the antisense
compound. These compounds are then tested using the methods
described herein to determine their ability to inhibit expression
of target gene mRNA.
[0047] In the context of this invention, the term "oligomeric
compound" refers to a polymer or oligomer comprising a plurality of
monomeric units. In the context of this invention, the term
"oligonucleotide" refers to an oligomer or polymer of ribonucleic
acid (RNA) or deoxyribonucleic acid (DNA) or mimetics, chimeras,
analogs and homologs thereof. This term includes oligonucleotides
composed of naturally occurring nucleobases, sugars and covalent
internucleoside (backbone) linkages as well as oligonucleotides
having non-naturally occurring portions which function similarly.
Such modified or substituted oligonucleotides are often preferred
over native forms because of desirable properties such as, for
example, enhanced cellular uptake, enhanced affinity for a target
nucleic acid and increased stability in the presence of
nucleases.
[0048] The antisense compounds in accordance with this invention
preferably comprise from about 8 to about 80 nucleobases (i.e. from
about 8 to about 80 linked nucleosides). One of ordinary skill in
the art will appreciate that the invention embodies compounds of 8,
9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,
26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42,
43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59,
60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76,
77, 78, 79, or 80 nucleobases in length.
[0049] In one preferred embodiment, the antisense compounds of the
invention are 10 to 50 nucleobases in length. One having ordinary
skill in the art will appreciate that this embodies compounds of
10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,
27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,
44, 45, 46, 47, 48, 49 or 50 nucleobases in length.
[0050] In another preferred embodiment, the antisense compounds of
the invention are 13 to 30 nucleobases in length. One having
ordinary skill in the art will appreciate that this embodies
compounds of 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,
26, 27, 28, 29 or 30 nucleobases in length.
[0051] In another preferred embodiment, the antisense compounds of
the invention are 15 to 25 nucleobases in length. One having
ordinary skill in the art will appreciate that this embodies
compounds of 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25
nucleobases in length.
[0052] In another preferred embodiment, the antisense compounds of
the invention are 18 to 22 nucleobases in length. One having
ordinary skill in the art will appreciate that this embodies
compounds of 18, 19, 20, 21 or 22 nucleobases in length.
[0053] Particularly preferred compounds are oligonucleotides from
about 10 to about 50 nucleobases, preferably those comprising from
about 13 to about 30 nucleobases, more preferably those comprising
from about 15 to about 25, and most preferred from about 18 to
about 22 nucleobases.
[0054] Antisense compounds are commonly used as research reagents
and diagnostics. For example, antisense oligonucleotides, which are
able to inhibit gene expression with exquisite specificity, are
often used by those of ordinary skill to elucidate the function of
particular genes. Antisense compounds are also used, for example,
to distinguish between functions of various members of a biological
pathway. Antisense modulation has, therefore, been harnessed for
research use.
[0055] The specificity and sensitivity of antisense is also
harnessed by those of skill in the art for therapeutic uses.
Antisense compounds are employed as therapeutic moieties in the
treatment of disease states in animals, including humans. Antisense
oligonucleotide drugs have been safely and effectively administered
to humans and numerous clinical trials are presently underway. It
is thus established that antisense compounds are useful therapeutic
modalities that can be configured to be useful
in treatment regimes for the treatment of cells, tissues and
animals, especially humans.
[0056] In one embodiment, the antisense compounds of the invention
are targeted to genes expressed in the kidney that have been
associated with a disease or disorder. Such diseases and/or
disorders include, but are not limited to, metabolic diseases, such
as type 1 or type 2 diabetes. Target genes include, but are not
limited to, SGLT2 and connective tissue growth factor.
[0057] The antisense compounds of the invention can be utilized in
pharmaceutical compositions by adding an effective amount of a
compound to a suitable pharmaceutically acceptable diluent or
carrier. Use of the compounds and methods of the invention may also
be useful prophylactically.
C. Modifications
[0058] As is known in the art, a nucleoside is a base-sugar
combination. The base portion of the nucleoside is normally a
heterocyclic base sometimes referred to as a "nucleobase" or simply
a "base". The two most common classes of such heterocyclic bases
are the purines and the pyrimidines. Nucleotides are nucleosides
that further include a phosphate group covalently linked to the
sugar portion of the nucleoside. For those nucleosides that include
a pentofuranosyl sugar, the phosphate group can be linked to either
the 2', 3' or 5' hydroxyl moiety of the sugar. In forming
oligonucleotides, the phosphate groups covalently link adjacent
nucleosides to one another to form a linear polymeric compound. In
turn, the respective ends of this linear polymeric compound can be
further joined to form a circular compound, however, linear
compounds are generally preferred. In addition, linear compounds
may have internal nucleobase complementarity and may therefore fold
in a manner as to produce a fully or partially double-stranded
compound. Within oligonucleotides, the phosphate groups are
commonly referred to as forming the internucleoside backbone of the
oligonucleotide. The normal linkage or backbone of RNA and DNA is a
3' to 5' phosphodiester linkage.
Chimeric Compounds
[0059] The present invention includes antisense compounds which are
chimeric compounds. "Chimeric" antisense compounds or "chimeras,"
in the context of this invention, are antisense compounds,
particularly oligonucleotides, which contain two or more chemically
distinct regions, each made up of at least one monomer unit (i.e.,
a nucleotide). Antisense compounds of the present invention are
chimeric oligonucleotides with a central "gap" region containing
2'-deoxy nucleosides flanked by two "wing" regions containing
2'-O-methoxyethyl nucleosides. The oligomeric compounds have a
mixed backbone of phosphorothioate linkages in the central "gap"
region and phosphodiester linkages in the flanking "wing" regions.
Mixed backbone compounds of the invention may also contain one
phosphorothioate linkage at one or both of the extreme 5' or 3'
ends of the oligonucleotide.
[0060] Oligonucleotides such as these are also known in the art as
"gapmers" or gapped oligonucleotides. In a gapmer that is 20
nucleotides in length, a gap or wing can be 1, 2, 3, 4, 5, 6, 7, 8,
9, 10, 11, 12, 13, 14, 15, 16, 17 or 18 nucleotides in length. In
one embodiment, a 20-nucleotide gapmer is comprised of a gap 8
nucleotides in length, flanked on both the 5' and 3' sides by wings
6 nucleotides in length. In another embodiment, a 20-nucleotide
gapmer is comprised of a gap 10 nucleotides in length, flanked on
both the 5' and 3' sides by wings 5 nucleotides in length. In
another embodiment, a 20-nucleotide gapmer is comprised of a gap 12
nucleotides in length flanked on both the 5' and 3' sides by wings
4 nucleotides in length. In a further embodiment, a 20-nucleotide
gapmer is comprised of a gap 14 nucleotides in length flanked on
both the 5' and 3' sides by wings 3 nucleotides in length. In
another embodiment, a 20-nucleotide gapmer is comprised of a gap 16
nucleotides in length flanked on both the 5' and 3' sides by wings
2 nucleotides in length. In a further embodiment, a 20-nucleotide
gapmer is comprised of a gap 18 nucleotides in length flanked on
both the 5' and 3' ends by wings 1 nucleotide in length.
Alternatively, the wings are of different lengths, for example, a
20-nucleotide gapmer may be comprised of a gap 10 nucleotides in
length, flanked by a 6-nucleotide wing on one side (5' or 3') and a
4-nucleotide wing on the other side (5' or 3'). Antisense
oligonucleotides with greater than or less than 20 nucleotides are
also contemplated. One of skill in the art can select an
appropriate number of nucleotides for the gap and wing regions in
accordance with the present invention.
[0061] Representative United States patents that teach the
preparation of such hybrid structures include, but are not limited
to, U.S. Pat. Nos. 5,013,830; 5,149,797; 5,220,007; 5,256,775;
5,366,878; 5,403,711; 5,491,133; 5,565,350; 5,623,065; 5,652,355;
5,652,356; and 5,700,922, certain of which are commonly owned with
the instant application, and each of which is herein incorporated
by reference in its entirety.
Natural and Modified Nucleobases
[0062] Antisense compounds of the invention may include nucleobase
(often referred to in the art as heterocyclic base or 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 (--C.ident.C--CH.sub.3)
uracil and cytosine and other alkynyl derivatives of pyrimidine
bases, 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, 2-F-adenine,
2-amino-adenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and
7-deazaadenine and 3-deazaguanine and 3-deazaadenine. Further
modified nucleobases include tricyclic pyrimidines such as
phenoxazine cytidine (1H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one),
phenothiazine cytidine
(1H-pyrimido[5,4-b][1,4]benzothiazin-2(3H)-one), G-clamps such as a
substituted phenoxazine cytidine (e.g.
9-(2-aminoethoxy)-H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one),
carbazole cytidine (2H-pyrimido[4,5-b]indol-2-one), pyridoindole
cytidine (H-pyrido[3',2':4,5]pyrrolo[2,3-d]pyrimidin-2-one).
Modified nucleobases may also include those in which the purine or
pyrimidine base is replaced with other heterocycles, for example
7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine and 2-pyridone.
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 compounds 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. and
are presently preferred base substitutions, even more particularly
when combined with 2'-O-methoxyethyl sugar modifications.
[0063] Representative United States patents that teach 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; 5,645,985; 5,830,653;
5,763,588; 6,005,096; and 5,681,941, certain of which are commonly
owned with the instant application, and each of which is herein
incorporated by reference, and U.S. Pat. No. 5,750,692, which is
commonly owned with the instant application and also herein
incorporated by reference.
Conjugates
[0064] Another modification of the antisense compounds of the
invention involves chemically linking to the antisense compound one
or more moieties or conjugates which enhance the activity, cellular
distribution or cellular uptake of the oligonucleotide. These
moieties or conjugates can include conjugate groups covalently
bound to functional groups such as primary or secondary hydroxyl
groups. Conjugate groups of the invention include intercalators,
reporter molecules, polyamines, polyamides, polyethylene glycols,
polyethers, groups that enhance the pharmacodynamic properties of
oligomers, and groups that enhance the pharmacokinetic properties
of oligomers. Typical conjugate groups include cholesterols,
lipids, phospholipids, biotin, phenazine, folate, phenanthridine,
anthraquinone, acridine, fluoresceins, rhodamines, coumarins, and
dyes. Groups that enhance the pharmacodynamic properties, in the
context of this invention, include groups that improve uptake,
enhance resistance to degradation, and/or strengthen
sequence-specific hybridization with the target nucleic acid.
Groups that enhance the pharmacokinetic properties, in the context
of this invention, include groups that improve uptake,
distribution, metabolism or excretion of the compounds of the
present invention. Representative conjugate groups are disclosed in
International Patent Application PCT/US92/09196, filed Oct. 23,
1992, and U.S. Pat. No. 6,287,860, the entire disclosures of which
are incorporated herein by reference. Conjugate moieties include
but are not limited to lipid moieties such as a cholesterol moiety,
cholic acid, a thioether, e.g., hexyl-5-tritylthiol, a
thiocholesterol, an aliphatic chain, e.g., dodecandiol or undecyl
residues, a phospholipid, e.g., di-hexadecyl-rac-glycerol or
triethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate, a
polyamine or a polyethylene glycol chain, or adamantane acetic
acid, a palmityl moiety, or an octadecylamine or
hexylamino-carbonyl-oxycholesterol moiety. Antisense compounds of
the invention may also be conjugated to active drug substances, for
example, aspirin, warfarin, phenylbutazone, ibuprofen, suprofen,
fenbufen, ketoprofen, (S)-(+)-pranoprofen, carprofen,
dansylsarcosine, 2,3,5-triiodo-benzoic acid, flufenamic acid,
folinic acid, a benzothiadiazide, chlorothiazide, a diazepine,
indo-methicin, a barbiturate, a cephalosporin, a sulfa drug, an
antidiabetic, an antibacterial or an antibiotic.
Oligonucleotide-drug conjugates and their preparation are described
in U.S. patent application Ser. No. 09/334,130 (filed Jun. 15,
1999) which is incorporated herein by reference in its
entirety.
[0065] Representative United States patents that teach 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, certain of which are commonly owned with
the instant application, and each of which is herein incorporated
by reference.
[0066] Oligomeric compounds used in the compositions of the present
invention can also be modified to have one or more stabilizing
groups that are generally attached to one or both termini of
oligomeric compounds to enhance properties such as for example
nuclease stability. Included in stabilizing groups are cap
structures. By "cap structure or terminal cap moiety" is meant
chemical modifications, which have been incorporated at either
terminus of oligonucleotides (see for example Wincott et al., WO
97/26270, incorporated by reference herein). These terminal
modifications protect the oligomeric compounds having terminal
nucleic acid molecules from exonuclease degradation, and can help
in delivery and/or localization within a cell. The cap can be
present at the 5'-terminus (5'-cap) or at the 3'-terminus (3'-cap)
or can be present on both termini. In non-limiting examples, the
5'-cap includes inverted abasic residue (moiety), 4',5'-methylene
nucleotide; 1-(beta-D-erythrofuranosyl) nucleotide, 4'-thio
nucleotide, carbocyclic nucleotide; 1,5-anhydrohexitol nucleotide;
L-nucleotides; alpha-nucleotides; modified base nucleotide;
phosphorodithioate linkage; threo-pentofuranosyl nucleotide;
acyclic 3',4'-seco nucleotide; acyclic 3,4-dihydroxybutyl
nucleotide; acyclic 3,5-dihydroxypentyl nucleotide, 3'-3'-inverted
nucleotide moiety; 3'-3'-inverted abasic moiety; 3'-2'-inverted
nucleotide moiety; 3'-2'-inverted abasic moiety; 1,4-butanediol
phosphate; 3'-phosphoramidate; hexylphosphate; aminohexyl
phosphate; 3'-phosphate; 3'-phosphorothioate; phosphorodithioate;
or bridging or non-bridging methylphosphonate moiety (for more
details see Wincott et al., International PCT publication No. WO
97/26270, incorporated by reference herein).
[0067] Particularly preferred 3'-cap structures of the present
invention include, for example 4',5'-methylene nucleotide;
1-(beta-D-erythrofuranosyl) nucleotide; 4'-thio nucleotide,
carbocyclic nucleotide; 5'-amino-alkyl phosphate;
1,3-diamino-2-propyl phosphate, 3-aminopropyl phosphate;
6-aminohexyl phosphate; 1,2-aminododecyl phosphate; hydroxypropyl
phosphate; 1,5-anhydrohexitol nucleotide; L-nucleotide;
alpha-nucleotide; modified base nucleotide; phosphorodithioate;
threo-pentofuranosyl nucleotide; acyclic 3',4'-seco nucleotide;
3,4-dihydroxybutyl nucleotide; 3,5-dihydroxypentyl nucleotide,
5'-5'-inverted nucleotide moiety; 5'-5'-inverted abasic moiety;
5'-phosphoramidate; 5'-phosphorothioate; 1,4-butanediol phosphate;
5'-amino; bridging and/or non-bridging 5'-phosphoramidate,
phosphorothioate and/or phosphorodithioate, bridging or non
bridging methylphosphonate and 5'-mercapto moieties (for more
details see Beaucage and Tyer, 1993, Tetrahedron 49, 1925;
incorporated by reference herein).
[0068] Further 3' and 5'-stabilizing groups that can be used to cap
one or both ends of an oligomeric compound to impart nuclease
stability include those disclosed in WO 03/004602 published on Jan.
16, 2003.
D. Formulations
[0069] The compounds of the invention may also be admixed,
encapsulated, conjugated or otherwise associated with other
molecules, molecule structures or mixtures of compounds, as for
example, liposomes, receptor-targeted molecules, oral, rectal,
topical or other formulations, for assisting in uptake,
distribution and/or absorption. Representative United States
patents that teach the preparation of such uptake, distribution
and/or absorption-assisting formulations include, but are not
limited to, U.S. Pat. Nos. 5,108,921; 5,354,844; 5,416,016;
5,459,127; 5,521,291; 5,543,158; 5,547,932; 5,583,020; 5,591,721;
4,426,330; 4,534,899; 5,013,556; 5,108,921; 5,213,804; 5,227,170;
5,264,221; 5,356,633; 5,395,619; 5,416,016; 5,417,978; 5,462,854;
5,469,854; 5,512,295; 5,527,528; 5,534,259; 5,543,152; 5,556,948;
5,580,575; and 5,595,756, each of which is herein incorporated by
reference.
[0070] The antisense compounds of the invention encompass any
pharmaceutically acceptable salts, esters, or salts of such esters,
or any other compound which, upon administration to an animal,
including a human, is capable of providing (directly or indirectly)
the biologically active metabolite or residue thereof.
[0071] The term "pharmaceutically acceptable salts" refers to
physiologically and pharmaceutically acceptable salts of the
compounds of the invention: i.e., salts that retain the desired
biological activity of the parent compound and do not impart
undesired toxicological effects thereto. For oligonucleotides,
preferred examples of pharmaceutically acceptable salts and their
uses are further described in U.S. Pat. No. 6,287,860, which is
incorporated herein in its entirety.
[0072] The present invention also includes pharmaceutical
compositions and formulations which include the antisense compounds
of the invention. The pharmaceutical compositions of the present
invention may be administered in a number of ways depending upon
whether local or systemic treatment is desired and upon the area to
be treated. Administration may be topical (including ophthalmic and
to mucous membranes including vaginal and rectal delivery),
pulmonary, e.g., by inhalation or insufflation of powders or
aerosols, including by nebulizer; intratracheal, intranasal,
epidermal and transdermal), oral or parenteral. Parenteral
administration includes intravenous, intraarterial, subcutaneous,
intraperitoneal or intramuscular injection or infusion; or
intracranial, e.g., intrathecal or intraventricular,
administration. Oligonucleotides with at least one
2'-O-methoxyethyl modification are believed to be particularly
useful for oral administration. Pharmaceutical compositions and
formulations for topical administration may include transdermal
patches, ointments, lotions, creams, gels, drops, suppositories,
sprays, liquids and powders. Conventional pharmaceutical carriers,
aqueous, powder or oily bases, thickeners and the like may be
necessary or desirable. Coated condoms, gloves and the like may
also be useful.
[0073] The pharmaceutical formulations of the present invention,
which may conveniently be presented in unit dosage form, may be
prepared according to conventional techniques well known in the
pharmaceutical industry. Such techniques include the step of
bringing into association the active ingredients with the
pharmaceutical carrier(s) or excipient(s). In general, the
formulations are prepared by uniformly and intimately bringing into
association the active ingredients with liquid carriers or finely
divided solid carriers or both, and then, if necessary, shaping the
product.
[0074] The compositions of the present invention may be formulated
into any of many possible dosage forms such as, but not limited to,
tablets, capsules, gel capsules, liquid syrups, soft gels,
suppositories, and enemas. The compositions of the present
invention may also be formulated as suspensions in aqueous,
non-aqueous or mixed media. Aqueous suspensions may further contain
substances which increase the viscosity of the suspension
including, for example, sodium carboxymethylcellulose, sorbitol
and/or dextran. The suspension may also contain stabilizers.
[0075] Pharmaceutical compositions of the present invention
include, but are not limited to, solutions, emulsions, foams and
liposome-containing formulations. The pharmaceutical compositions
and formulations of the present invention may comprise one or more
penetration enhancers, carriers, excipients or other active or
inactive ingredients.
[0076] Emulsions are typically heterogenous systems of one liquid
dispersed in another in the form of droplets usually exceeding 0.1
.mu.m in diameter. Emulsions may contain additional components in
addition to the dispersed phases, and the active drug which may be
present as a solution in either the aqueous phase, oily phase or
itself as a separate phase. Microemulsions are included as an
embodiment of the present invention. Emulsions and their uses are
well known in the art and are further described in U.S. Pat. No.
6,287,860, which is incorporated herein in its entirety.
[0077] Formulations of the present invention include liposomal
formulations. As used in the present invention, the term "liposome"
means a vesicle composed of amphiphilic lipids arranged in a
spherical bilayer or bilayers. Liposomes are unilamellar or
multilamellar vesicles which have a membrane formed from a
lipophilic material and an aqueous interior that contains the
composition to be delivered. Cationic liposomes are positively
charged liposomes which are believed to interact with negatively
charged DNA molecules to form a stable complex. Liposomes that are
pH-sensitive or negatively-charged are believed to entrap DNA
rather than complex with it. Both cationic and noncationic
liposomes have been used to deliver DNA to cells.
[0078] Liposomes also include "sterically stabilized" liposomes, a
term which, as used herein, refers to liposomes comprising one or
more specialized lipids that, when incorporated into liposomes,
result in enhanced circulation lifetimes relative to liposomes
lacking such specialized lipids. Examples of sterically stabilized
liposomes are those in which part of the vesicle-forming lipid
portion of the liposome comprises one or more glycolipids or is
derivatized with one or more hydrophilic polymers, such as a
polyethylene glycol (PEG) moiety. Liposomes and their uses are
further described in U.S. Pat. No. 6,287,860, which is incorporated
herein in its entirety.
[0079] The pharmaceutical formulations and compositions of the
present invention may also include surfactants. The use of
surfactants in drug products, formulations and in emulsions is well
known in the art. Surfactants and their uses are further described
in U.S. Pat. No. 6,287,860, which is incorporated herein in its
entirety.
[0080] In one embodiment, the present invention employs various
penetration enhancers to effect the efficient delivery of nucleic
acids, particularly oligonucleotides. In addition to aiding the
diffusion of non-lipophilic drugs across cell membranes,
penetration enhancers also enhance the permeability of lipophilic
drugs. Penetration enhancers may be classified as belonging to one
of five broad categories, i.e., surfactants, fatty acids, bile
salts, chelating agents, and non-chelating non-surfactants.
Penetration enhancers and their uses are further described in U.S.
Pat. No. 6,287,860, which is incorporated herein in its
entirety.
[0081] One of skill in the art will recognize that formulations are
routinely designed according to their intended use, i.e. route of
administration.
[0082] Preferred formulations for topical administration include
those in which the oligonucleotides of the invention are in
admixture with a topical delivery agent such as lipids, liposomes,
fatty acids, fatty acid esters, steroids, chelating agents and
surfactants. Preferred lipids and liposomes include neutral (e.g.
dioleoylphosphatidyl DOPE ethanolamine, dimyristoylphosphatidyl
choline DMPC, distearolyphosphatidyl choline) negative (e.g.
dimyristoylphosphatidyl glycerol DMPG) and cationic (e.g.
dioleoyltetramethylaminopropyl DOTAP and dioleoylphosphatidyl
ethanolamine DOTMA).
[0083] For topical or other administration, oligonucleotides of the
invention may be encapsulated within liposomes or may form
complexes thereto, in particular to cationic liposomes.
Alternatively, oligonucleotides may be complexed to lipids, in
particular to cationic lipids. Preferred fatty acids and esters,
pharmaceutically acceptable salts thereof, and their uses are
further described in U.S. Pat. No. 6,287,860, which is incorporated
herein in its entirety. Topical formulations are described in
detail in U.S. patent application Ser. No. 09/315,298 filed on May
20, 1999, which is incorporated herein by reference in its
entirety.
[0084] Compositions and formulations for oral administration
include powders or granules, microparticulates, nanoparticulates,
suspensions or solutions in water or non-aqueous media, capsules,
gel capsules, sachets, tablets or minitablets. Thickeners,
flavoring agents, diluents, emulsifiers, dispersing aids or binders
may be desirable. Preferred oral formulations are those in which
oligonucleotides of the invention are administered in conjunction
with one or more penetration enhancers surfactants and chelators.
Preferred surfactants include fatty acids and/or esters or salts
thereof, bile acids and/or salts thereof. Preferred bile
acids/salts and fatty acids and their uses are further described in
U.S. Pat. No. 6,287,860, which is incorporated herein in its
entirety. Also preferred are combinations of penetration enhancers,
for example, fatty acids/salts in combination with bile
acids/salts. A particularly preferred combination is the sodium
salt of lauric acid, capric acid and UDCA. Further penetration
enhancers include polyoxyethylene-9-lauryl ether,
polyoxyethylene-20-cetyl ether. Oligonucleotides of the invention
may be delivered orally, in granular form including sprayed dried
particles, or complexed to form micro or nanoparticles.
Oligonucleotide complexing agents and their uses are further
described in U.S. Pat. No. 6,287,860, which is incorporated herein
in its entirety. Oral formulations for oligonucleotides and their
preparation are described in detail in U.S. application Ser. Nos.
09/108,673 (filed Jul. 1, 1998), 09/315,298 (filed May 20, 1999)
and 10/071,822, (filed Feb. 8, 2002; published as US2003-0027780),
each of which is incorporated herein by reference in their
entirety.
[0085] Compositions and formulations for parenteral, intrathecal or
intraventricular administration may include sterile aqueous
solutions which may also contain buffers, diluents and other
suitable additives such as, but not limited to, penetration
enhancers, carrier compounds and other pharmaceutically acceptable
carriers or excipients.
[0086] Oligonucleotides may be formulated for delivery in vivo in
an acceptable dosage form, e.g. as parenteral or non-parenteral
formulations. Parenteral formulations include intravenous (IV),
subcutaneous (SC), intraperitoneal (IP), intravitreal and
intramuscular (IM) formulations, as well as formulations for
delivery via pulmonary inhalation, intranasal administration,
topical administration, etc. Non-parenteral formulations include
formulations for delivery via the alimentary canal, e.g. oral
administration, rectal administration, intrajejunal instillation,
etc. Rectal administration includes administration as an enema or a
suppository. Oral administration includes administration as a
capsule, a gel capsule, a pill, an elixir, etc.
[0087] In some embodiments, an oligonucleotide may be administered
to a subject via an oral route of administration. The subject may
be an animal or a human (man). An animal subject may be a mammal,
such as a mouse, a rat, a dog, a guinea pig, a monkey, a non-human
primate, a cat or a pig. Non-human primates include monkeys and
chimpanzees. A suitable animal subject may be an experimental
animal, such as a mouse, rat, mouse, a rat, a dog, a monkey, a
non-human primate, a cat or a pig.
[0088] In some embodiments, the subject may be a human. In certain
embodiments, the subject may be a human patient in need of
therapeutic treatment as discussed in more detail herein. In
certain embodiments, the subject may be in need of modulation of
expression of one or more genes as discussed in more detail herein.
In some particular embodiments, the subject may be in need of
inhibition of expression of one or more genes as discussed in more
detail herein. In particular embodiments, the subject may be in
need of modulation, i.e. inhibition or enhancement, of HIF1-beta in
order to obtain therapeutic indications discussed in more detail
herein.
[0089] In some embodiments, non-parenteral (e.g. oral)
oligonucleotide formulations according to the present invention
result in enhanced bioavailability of the oligonucleotide. In this
context, the term "bioavailability" refers to a measurement of that
portion of an administered drug which reaches the circulatory
system (e.g. blood, especially blood plasma) when a particular mode
of administration is used to deliver the drug. Enhanced
bioavailability refers to a particular mode of administration's
ability to deliver oligonucleotide to the peripheral blood plasma
of a subject relative to another mode of administration. For
example, when a non-parenteral mode of administration (e.g. an oral
mode) is used to introduce the drug into a subject, the
bioavailability for that mode of administration may be compared to
a different mode of administration, e.g. an IV mode of
administration. In some embodiments, the area under a compound's
blood plasma concentration curve (AUC.sub.0) after non-parenteral
(e.g. oral, rectal, intrajejunal) administration may be divided by
the area under the drug's plasma concentration curve after
intravenous (i.v.) administration (AUC.sub.iv) to provide a
dimensionless quotient (relative bioavailability, RB) that
represents fraction of compound absorbed via the non-parenteral
route as compared to the IV route. A composition's bioavailability
is said to be enhanced in comparison to another composition's
bioavailability when the first composition's relative
bioavailability (RB.sub.1) is greater than the second composition's
relative bioavailability (RB.sub.2).
[0090] In general, bioavailability correlates with therapeutic
efficacy when a compound's therapeutic efficacy is related to the
blood concentration achieved, even if the drug's ultimate site of
action is intracellular (van Berge-Henegouwen et al.,
Gastroenterol., 1977, 73, 300). Bioavailability studies have been
used to determine the degree of intestinal absorption of a drug by
measuring the change in peripheral blood levels of the drug after
an oral dose (DiSanto, Chapter 76 In: Remington's Pharmaceutical
Sciences, 18th Ed., Gennaro, ed., Mack Publishing Co., Easton, Pa.,
1990, pages 1451-1458).
[0091] In general, an oral composition's bioavailability is said to
be "enhanced" when its relative bioavailability is greater than the
bioavailability of a composition substantially consisting of pure
oligonucleotide, i.e. oligonucleotide in the absence of a
penetration enhancer.
[0092] Organ bioavailability refers to the concentration of
compound in an organ. Organ bioavailability may be measured in test
subjects by a number of means, such as by whole-body radiography.
Organ bioavailability may be modified, e.g. enhanced, by one or
more modifications to the oligonucleotide, by use of one or more
carrier compounds or excipients, etc. as discussed in more detail
herein. In general, an increase in bioavailability will result in
an increase in organ bioavailability.
[0093] Oral oligonucleotide compositions according to the present
invention may comprise one or more "mucosal penetration enhancers,"
also known as "absorption enhancers" or simply as "penetration
enhancers." Accordingly, some embodiments of the invention comprise
at least one oligonucleotide in combination with at least one
penetration enhancer. In general, a penetration enhancer is a
substance that facilitates the transport of a drug across mucous
membrane(s) associated with the desired mode of administration,
e.g. intestinal epithelial membranes. Accordingly it is desirable
to select one or more penetration enhancers that facilitate the
uptake of an oligonucleotide, without interfering with the activity
of the oligonucleotide, and in a such a manner the oligonucleotide
can be introduced into the body of an animal without unacceptable
side-effects such as toxicity, irritation or allergic response.
[0094] Embodiments of the present invention provide compositions
comprising one or more pharmaceutically acceptable penetration
enhancers, and methods of using such compositions, which result in
the improved bioavailability of oligonucleotides administered via
non-parenteral modes of administration. Heretofore, certain
penetration enhancers have been used to improve the bioavailability
of certain drugs. See Muranishi, Crit. Rev. Ther. Drug Carrier
Systems, 1990, 7, 1 and Lee et al., Crit. Rev. Ther. Drug Carrier
Systems, 1991, 8, 91. It has been found that the uptake and
delivery of oligonucleotides, relatively complex molecules which
are known to be difficult to administer to animals and man, can be
greatly improved even when administered by non-parenteral means
through the use of a number of different classes of penetration
enhancers.
[0095] In some embodiments, compositions for non-parenteral
administration include one or more modifications from
naturally-occurring oligonucleotides (i.e. full-phosphodiester
deoxyribosyl or full-phosphodiester ribosyl oligonucleotides). Such
modifications may increase binding affinity, nuclease stability,
cell or tissue permeability, tissue distribution, or other
biological or pharmacokinetic property. Modifications may be made
to the base, the linker, or the sugar, in general, as discussed in
more detail herein with regards to oligonucleotide chemistry. In
some embodiments of the invention, compositions for administration
to a subject, and in particular oral compositions for
administration to an animal or human subject, will comprise
modified oligonucleotides having one or more modifications for
enhancing affinity, stability, tissue distribution, or other
biological property.
[0096] Suitable modified linkers include phosphorothioate linkers.
In some embodiments according to the invention, the oligonucleotide
has at least one phosphorothioate linker. Phosphorothioate linkers
provide nuclease stability as well as plasma protein binding
characteristics to the oligonucleotide. Nuclease stability is
useful for increasing the in vivo lifetime of oligonucleotides,
while plasma protein binding decreases the rate of first pass
clearance of oligonucleotide via renal excretion. In some
embodiments according to the present invention, the oligonucleotide
has at least two phosphorothioate linkers. In some embodiments,
wherein the oligonucleotide has exactly n nucleosides, the
oligonucleotide has from one to n-1 phosphorothioate linkages. In
some embodiments, wherein the oligonucleotide has exactly n
nucleosides, the oligonucleotide has n-1 phosphorothioate linkages.
In other embodiments wherein the oligonucleotide has exactly n
nucleosides, and n is even, the oligonucleotide has from 1 to n/2
phosphorothioate linkages, or, when n is odd, from 1 to (n-1)/2
phosphorothioate linkages. In some embodiments, the oligonucleotide
has alternating phosphodiester (PO) and phosphorothioate (PS)
linkages. In other embodiments, the oligonucleotide has at least
one stretch of two or more consecutive PO linkages and at least one
stretch of two or more PS linkages. In other embodiments, the
oligonucleotide has at least two stretches of PO linkages
interrupted by at least on PS linkage.
[0097] In some embodiments, at least one of the nucleosides is
modified on the ribosyl sugar unit by a modification that imparts
nuclease stability, binding affinity or some other beneficial
biological property to the sugar. In some cases, the sugar
modification includes a 2'-modification, e.g. the 2'-OH of the
ribosyl sugar is replaced or substituted. Suitable replacements for
2'-OH include 2'-F and 2'-arabino-F. Suitable substitutions for OH
include 2'-O-alkyl, e.g. 2-O-methyl, and 2'-O-substituted alkyl,
e.g. 2'-O-methoxyethyl, 2'-O-aminopropyl, etc. In some embodiments,
the oligonucleotide contains at least one 2'-modification. In some
embodiments, the oligonucleotide contains at least 2
2'-modifications. In some embodiments, the oligonucleotide has at
least one 2'-modification at each of the termini (i.e. the 3'- and
5'-terminal nucleosides each have the same or different
2'-modifications). In some embodiments, the oligonucleotide has at
least two sequential 2'-modifications at each end of the
oligonucleotide. In some embodiments, oligonucleotides further
comprise at least one deoxynucleoside. In particular embodiments,
oligonucleotides comprise a stretch of deoxynucleosides such that
the stretch is capable of activating RNase (e.g. RNase H) cleavage
of an RNA to which the oligonucleotide is capable of hybridizing.
In some embodiments, a stretch of deoxynucleosides capable of
activating RNase-mediated cleavage of RNA comprises about 6 to
about 16, e.g. about 8 to about 16 consecutive deoxynucleosides. In
further embodiments, oligonucleotides are capable of eliciting
cleaveage by dsRNAse enzymes.
[0098] Oral compositions for administration of non-parenteral
oligonucleotide compositions of the present invention may be
formulated in various dosage forms such as, but not limited to,
tablets, capsules, liquid syrups, soft gels, suppositories, and
enemas. The term "alimentary delivery" encompasses e.g. oral,
rectal, endoscopic and sublingual/buccal administration. A common
requirement for these modes of administration is absorption over
some portion or all of the alimentary tract and a need for
efficient mucosal penetration of the nucleic acid(s) so
administered.
[0099] Delivery of a drug via the oral mucosa, as in the case of
buccal and sublingual administration, has several desirable
features, including, in many instances, a more rapid rise in plasma
concentration of the drug than via oral delivery (Harvey, Chapter
35 In: Remington's Pharmaceutical Sciences, 18th Ed., Gennaro, ed.,
Mack Publishing Co., Easton, Pa., 1990, page 711).
[0100] Endoscopy may be used for drug delivery directly to an
interior portion of the alimentary tract. For example, endoscopic
retrograde cystopancreatography (ERCP) takes advantage of extended
gastroscopy and permits selective access to the biliary tract and
the pancreatic duct (Hirahata et al., Gan To Kagaku Ryoho, 1992,
19(10 Suppl.), 1591). Pharmaceutical compositions, including
liposomal formulations, can be delivered directly into portions of
the alimentary canal, such as, e.g., the duodenum (Somogyi et al.,
Pharm. Res., 1995, 12, 149) or the gastric submucosa (Akamo et al.,
Japanese J. Cancer Res., 1994, 85, 652) via endoscopic means.
Gastric lavage devices (Inoue et al., Artif. Organs, 1997, 21, 28)
and percutaneous endoscopic feeding devices (Pennington et al.,
Ailment Pharmacol. Ther., 1995, 9, 471) can also be used for direct
alimentary delivery of pharmaceutical compositions.
[0101] In some embodiments, oligonucleotide formulations may be
administered through the anus into the rectum or lower intestine.
Rectal suppositories, retention enemas or rectal catheters can be
used for this purpose and may be preferred when patient compliance
might otherwise be difficult to achieve (e.g., in pediatric and
geriatric applications, or when the patient is vomiting or
unconscious). Rectal administration can result in more prompt and
higher blood levels than the oral route. (Harvey, Chapter 35 In:
Remington's Pharmaceutical Sciences, 18th Ed., Gennaro, ed., Mack
Publishing Co., Easton, Pa., 1990, page 711). Because about 50% of
the drug that is absorbed from the rectum will bypass the liver,
administration by this route significantly reduces the potential
for first-pass metabolism (Benet et al., Chapter 1 In: Goodman
& Gilman's The Pharmacological Basis of Therapeutics, 9th Ed.,
Hardman et al., eds., McGraw-Hill, New York, N.Y., 1996).
[0102] One advantageous method of non-parenteral administration of
oligonucleotide compositions is oral delivery. Some embodiments
employ various penetration enhancers in order to effect transport
of oligonucleotides and other nucleic acids across mucosal and
epithelial membranes. Penetration enhancers may be classified as
belonging to one of five broad categories--surfactants, fatty
acids, bile salts, chelating agents, and non-chelating
non-surfactants (Lee et al., Critical Reviews in Therapeutic Drug
Carrier Systems, 1991, p. 92). Accordingly, some embodiments
comprise oral oligonucleotide compositions comprising at least one
member of the group consisting of surfactants, fatty acids, bile
salts, chelating agents, and non-chelating surfactants. Further
embodiments comprise oral oligonucleotide comprising at least one
fatty acid, e.g. capric or lauric acid, or combinations or salts
thereof. Other embodiments comprise methods of enhancing the oral
bioavailability of an oligonucleotide, the method comprising
co-administering the oligonucleotide and at least one penetration
enhancer.
[0103] Other excipients that may be added to oral oligonucleotide
compositions include surfactants (or "surface-active agents"),
which are chemical entities which, when dissolved in an aqueous
solution, reduce the surface tension of the solution or the
interfacial tension between the aqueous solution and another
liquid, with the result that absorption of oligonucleotides through
the alimentary mucosa and other epithelial membranes is enhanced.
In addition to bile salts and fatty acids, surfactants include, for
example, sodium lauryl sulfate, polyoxyethylene-9-lauryl ether and
polyoxyethylene-20-cetyl ether (Lee et al., Critical Reviews in
Therapeutic Drug Carrier Systems, 1991, page 92); and
perfluorohemical emulsions, such as FC-43 (Takahashi et al., J.
Pharm. Phamacol., 1988, 40, 252).
[0104] Fatty acids and their derivatives which act as penetration
enhancers and may be used in compositions of the present invention
include, for example, oleic acid, lauric acid, capric acid
(n-decanoic acid), myristic acid, palmitic acid, stearic acid,
linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein
(1-monooleoyl-rac-glycerol), dilaurin, caprylic acid, arachidonic
acid, glyceryl 1-monocaprate, 1-dodecylazacycloheptan-2-one,
acylcarnitines, acylcholines and mono- and di-glycerides thereof
and/or physiologically acceptable salts thereof (i.e., oleate,
laurate, caprate, myristate, palmitate, stearate, linoleate, etc.)
(Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems,
1991, page 92; Muranishi, Critical Reviews in Therapeutic Drug
Carrier Systems, 1990, 7, 1; El-Hariri et al., J. Pharm.
Pharmacol., 1992, 44, 651).
[0105] In some embodiments, oligonucleotide compositions for oral
delivery comprise at least two discrete phases, which phases may
comprise particles, capsules, gel-capsules, microspheres, etc. Each
phase may contain one or more oligonucleotides, penetration
enhancers, surfactants, bioadhesives, effervescent agents, or other
adjuvant, excipient or diluent. In some embodiments, one phase
comprises at least one oligonucleotide and at least one penetration
enhancer. In some embodiments, a first phase comprises at least one
oligonucleotide and at least one penetration enhancer, while a
second phase comprises at least one penetration enhancer. In some
embodiments, a first phase comprises at least one oligonucleotide
and at least one penetration enhancer, while a second phase
comprises at least one penetration enhancer and substantially no
oligonucleotide. In some embodiments, at least one phase is
compounded with at least one degradation retardant, such as a
coating or a matrix, which delays release of the contents of that
phase. In some embodiments, a first phase comprises at least one
oligonucleotide, at least one penetration enhancer, while a second
phase comprises at least one penetration enhancer and a
release-retardant. In particular embodiments, an oral
oligonucleotide comprises a first phase comprising particles
containing an oligonucleotide and a penetration enhancer, and a
second phase comprising particles coated with a release-retarding
agent and containing penetration enhancer.
[0106] A variety of bile salts also function as penetration
enhancers to facilitate the uptake and bioavailability of drugs.
The physiological roles of bile include the facilitation of
dispersion and absorption of lipids and fat-soluble vitamins
(Brunton, Chapter 38 In: Goodman & Gilman's The Pharmacological
Basis of Therapeutics, 9th Ed., Hardman et al., eds., McGraw-Hill,
New York, N.Y., 1996, pages 934-935). Various natural bile salts,
and their synthetic derivatives, act as penetration enhancers.
Thus, the term "bile salt" includes any of the naturally occurring
components of bile as well as any of their synthetic derivatives.
The bile salts of the invention include, for example, cholic acid
(or its pharmaceutically acceptable sodium salt, sodium cholate),
dehydrocholic acid (sodium dehydrocholate), deoxycholic acid
(sodium deoxycholate), glucholic acid (sodium glucholate),
glycholic acid (sodium glycocholate), glycodeoxycholic acid (sodium
glycodeoxycholate), taurocholic acid (sodium taurocholate),
taurodeoxycholic acid (sodium taurodeoxycholate), chenodeoxycholic
acid (CDCA, sodium chenodeoxycholate), ursodeoxycholic acid (UDCA),
sodium tauro-24,25-dihydro-fusidate (STDHF), sodium
glycodihydrofusidate and polyoxyethylene-9-lauryl ether (POE) (Lee
et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991,
page 92; Swinyard, Chapter 39 In: Remington's Pharmaceutical
Sciences, 18th Ed., Gennaro, ed., Mack Publishing Co., Easton, Pa.,
1990, pages 782-783; Muranishi, Critical Reviews in Therapeutic
Drug Carrier Systems, 1990, 7, 1; Yamamoto et al., J. Pharm. Exp.
Ther., 1992, 263, 25; Yamashita et al., J. Pharm. Sci., 1990, 79,
579).
[0107] In some embodiments, penetration enhancers useful in some
embodiments of present invention are mixtures of penetration
enhancing compounds. One such penetration enhancer is a mixture of
UDCA (and/or CDCA) with capric and/or lauric acids or salts thereof
e.g. sodium. Such mixtures are useful for enhancing the delivery of
biologically active substances across mucosal membranes, in
particular intestinal mucosa. Other penetration enhancer mixtures
comprise about 5-95% of bile acid or salt(s) UDCA and/or CDCA with
5-95% capric and/or lauric acid. Particular penetration enhancers
are mixtures of the sodium salts of UDCA, capric acid and lauric
acid in a ratio of about 1:2:2 respectively. Anther such
penetration enhancer is a mixture of capric and lauric acid (or
salts thereof) in a 0.01:1 to 1:0.01 ratio (mole basis). In
particular embodiments capric acid and lauric acid are present in
molar ratios of e.g. about 0.1:1 to about 1:0.1, in particular
about 0.5:1 to about 1:0.5.
[0108] Other excipients include chelating agents, i.e. compounds
that remove metallic ions from solution by forming complexes
therewith, with the result that absorption of oligonucleotides
through the alimentary and other mucosa is enhanced. With regards
to their use as penetration enhancers in the present invention,
chelating agents have the added advantage of also serving as DNase
inhibitors, as most characterized DNA nucleases require a divalent
metal ion for catalysis and are thus inhibited by chelating agents
(Jarrett, J. Chromatogr., 1993, 618, 315). Chelating agents of the
invention include, but are not limited to, disodium
ethylenediaminetetraacetate (EDTA), citric acid, salicylates (e.g.,
sodium salicylate, 5-methoxysalicylate and homovanilate), N-acyl
derivatives of collagen, laureth-9 and N-amino acyl derivatives of
beta-diketones (enamines) (Lee et al., Critical Reviews in
Therapeutic Drug Carrier Systems, 1991, page 92; Muranishi,
Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1;
Buur et al., J. Control Rel., 1990, 14, 43).
[0109] As used herein, non-chelating non-surfactant penetration
enhancers may be defined as compounds that demonstrate
insignificant activity as chelating agents or as surfactants but
that nonetheless enhance absorption of oligonucleotides through the
alimentary and other mucosal membranes (Muranishi, Critical Reviews
in Therapeutic Drug Carrier Systems, 1990, 7, 1). This class of
penetration enhancers includes, but is not limited to, unsaturated
cyclic ureas, 1-alkyl- and 1-alkenylazacyclo-alkanone derivatives
(Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems,
1991, page 92); and non-steroidal anti-inflammatory agents such as
diclofenac sodium, indomethacin and phenylbutazone (Yamashita et
al., J. Pharm. Pharmacol., 1987, 39, 621).
[0110] Agents that enhance uptake of oligonucleotides at the
cellular level may also be added to the pharmaceutical and other
compositions of the present invention. For example, cationic
lipids, such as lipofectin (Junichi et al, U.S. Pat. No.
5,705,188), cationic glycerol derivatives, and polycationic
molecules, such as polylysine (Lollo et al., PCT Application WO
97/30731), can be used.
[0111] Some oral oligonucleotide compositions also incorporate
carrier compounds in the formulation. As used herein, "carrier
compound" or "carrier" can refer to a nucleic acid, or analog
thereof, which may be inert (i.e., does not possess biological
activity per se) or may be necessary for transport, recognition or
pathway activation or mediation, or is recognized as a nucleic acid
by in vivo processes that reduce the bioavailability of a nucleic
acid having biological activity by, for example, degrading the
biologically active nucleic acid or promoting its removal from
circulation. The coadministration of a nucleic acid and a carrier
compound, typically with an excess of the latter substance, can
result in a substantial reduction of the amount of nucleic acid
recovered in the liver, kidney or other extracirculatory
reservoirs, presumably due to competition between the carrier
compound and the nucleic acid for a common receptor. For example,
the recovery of a partially phosphorothioate oligonucleotide in
hepatic tissue can be reduced when it is coadministered with
polyinosinic acid, dextran sulfate, polycytidic acid or
4-acetamido-4'isothiocyano-stilbene-2,2'-disulfonic acid (Miyao et
al., Antisense Res. Dev., 1995, 5, 115; Takakura et al., Antisense
& Nucl. Acid Drug Dev., 1996, 6, 177).
[0112] A "pharmaceutical carrier" or "excipient" may be a
pharmaceutically acceptable solvent, suspending agent or any other
pharmacologically inert vehicle for delivering one or more nucleic
acids to an animal. The excipient may be liquid or solid and is
selected, with the planned manner of administration in mind, so as
to provide for the desired bulk, consistency, etc., when combined
with a nucleic acid and the other components of a given
pharmaceutical composition. Typical pharmaceutical carriers
include, but are not limited to, binding agents (e.g.,
pregelatinised maize starch, polyvinylpyrrolidone or hydroxypropyl
methylcellulose, etc.); fillers (e.g., lactose and other sugars,
microcrystalline cellulose, pectin, gelatin, calcium sulfate, ethyl
cellulose, polyacrylates or calcium hydrogen phosphate, etc.);
lubricants (e.g., magnesium stearate, talc, silica, colloidal
silicon dioxide, stearic acid, metallic stearates, hydrogenated
vegetable oils, corn starch, polyethylene glycols, sodium benzoate,
sodium acetate, etc.); disintegrants (e.g., starch, sodium starch
glycolate, EXPLOTAB); and wetting agents (e.g., sodium lauryl
sulphate, etc.).
[0113] Oral oligonucleotide compositions may additionally contain
other adjunct components conventionally found in pharmaceutical
compositions, at their art-established usage levels. Thus, for
example, the compositions may contain additional, compatible,
pharmaceutically-active materials such as, for example,
antipruritics, astringents, local anesthetics or anti-inflammatory
agents, or may contain additional materials useful in physically
formulating various dosage forms of the composition of present
invention, such as dyes, flavoring agents, preservatives,
antioxidants, opacifiers, thickening agents and stabilizers.
However, such materials, when added, should not unduly interfere
with the biological activities of the components of the
compositions of the present invention.
[0114] Certain embodiments of the invention provide pharmaceutical
compositions containing one or more oligomeric compounds and one or
more other chemotherapeutic agents which function by a
non-antisense mechanism. Examples of such chemotherapeutic agents
include but are not limited to cancer chemotherapeutic drugs such
as daunorubicin, daunomycin, dactinomycin, doxorubicin, epirubicin,
idarubicin, esorubicin, bleomycin, mafosfamide, ifosfamide,
cytosine arabinoside, bis-chloroethylnitrosurea, busulfan,
mitomycin C, actinomycin D, mithramycin, prednisone,
hydroxyprogesterone, testosterone, tamoxifen, dacarbazine,
procarbazine, hexamethylmelamine, pentamethylmelamine,
mitoxantrone, amsacrine, chlorambucil, methylcyclohexylnitrosurea,
nitrogen mustards, melphalan, cyclophosphamide, 6-mercaptopurine,
6-thioguanine, cytarabine, 5-azacytidine, hydroxyurea,
deoxycoformycin, 4-hydroxyperoxycyclophosphoramide, 5-fluorouracil
(5-FU), 5-fluorodeoxyuridine (5-FUdR), methotrexate (MTX),
colchicine, taxol, vincristine, vinblastine, etoposide (VP-16),
trimetrexate, irinotecan, topotecan, gemcitabine, teniposide,
cisplatin and diethylstilbestrol (DES). When used with the
compounds of the invention, such chemotherapeutic agents may be
used individually (e.g., 5-FU and oligonucleotide), sequentially
(e.g., 5-FU and oligonucleotide for a period of time followed by
MTX and oligonucleotide), or in combination with one or more other
such chemotherapeutic agents (e.g., 5-FU, MTX and oligonucleotide,
or 5-FU, radiotherapy and oligonucleotide). Anti-inflammatory
drugs, including but not limited to nonsteroidal anti-inflammatory
drugs and corticosteroids, and antiviral drugs, including but not
limited to ribivirin, vidarabine, acyclovir and ganciclovir, may
also be combined in compositions of the invention. Combinations of
antisense compounds and other non-antisense drugs are also within
the scope of this invention. Two or more combined compounds may be
used together or sequentially.
[0115] In another related embodiment, compositions of the invention
may contain one or more antisense compounds, particularly
oligonucleotides, targeted to a first nucleic acid and one or more
additional antisense compounds targeted to a second nucleic acid
target. Alternatively, compositions of the invention may contain
two or more antisense compounds targeted to different regions of
the same nucleic acid target. Numerous examples of antisense
compounds are known in the art. Two or more combined compounds may
be used together or sequentially.
E. Dosing
[0116] The formulation of therapeutic compositions and their
subsequent administration (dosing) is believed to be within the
skill of those in the art. Dosing is dependent on severity and
responsiveness of the disease state to be treated, with the course
of treatment lasting from several days to several months, or until
a cure is effected or a diminution of the disease state is
achieved. Optimal dosing schedules can be calculated from
measurements of drug accumulation in the body of the patient.
Persons of ordinary skill can easily determine optimum dosages,
dosing methodologies and repetition rates. Optimum dosages may vary
depending on the relative potency of individual oligonucleotides,
and can generally be estimated based on EC.sub.50s found to be
effective in in vitro and in vivo animal models. In general, dosage
is from 0.0001 .mu.g to 100 g per kg of body weight, and may be
given once or more daily, weekly, monthly or yearly, or even once
every 2 to 20 years. Persons of ordinary skill in the art can
easily estimate repetition rates for dosing based on measured
residence times and concentrations of the drug in bodily fluids or
tissues. Following successful treatment, it may be desirable to
have the patient undergo maintenance therapy to prevent the
recurrence of the disease state, wherein the oligonucleotide is
administered in maintenance doses, ranging from 0.0001 .mu.g to 100
g per kg of body weight, once or more daily, to once every 20
years.
[0117] While the present invention has been described with
specificity in accordance with certain of its preferred
embodiments, the following examples serve only to illustrate the
invention and are not intended to limit the same. Each of the
references, GenBank accession numbers, and the like recited in the
present application is incorporated herein by reference in its
entirety.
EXAMPLES
Example 1
Synthesis of Nucleoside Phosphoramidites
[0118] The following compounds, including amidites and their
intermediates were prepared as described in U.S. Pat. No. 6,426,220
and published PCT WO 02/36743; 5'-O-Dimethoxytrityl-thymidine
intermediate for 5-methyl dC amidite,
5'-O-Dimethoxytrityl-2'-deoxy-5-methylcytidine intermediate for
5-methyl-dC amidite,
5'-O-Dimethoxytrityl-2'-deoxy-N4-benzoyl-5-methylcytidine
penultimate intermediate for 5-methyl dC amidite,
[5'-O-(4,4'-Dimethoxytriphenylmethyl)-2'-deoxy-N.sup.4-benzoyl-5-methylcy-
tidin-3'-O-yl]-2-cyanoethyl-N,N-diisopropylphosphoramidite
(5-methyl dC amidite), 2'-Fluorodeoxyadenosine,
2'-Fluorodeoxyguanosine, 2'-Fluorouridine, 2'-Fluorodeoxycytidine,
2'-O-(2-Methoxyethyl) modified amidites,
2'-O-(2-methoxyethyl)-5-methyluridine intermediate,
5'-O-DMT-2'-O-(2-methoxyethyl)-5-methyluridine penultimate
intermediate,
[5'-O-(4,4'-Dimethoxytriphenylmethyl)-2'-O-(2-methoxyethyl)-5-methyluridi-
n-3'-O-yl]-2-cyanoethyl-N,N-diisopropylphosphoramidite (MOE T
amidite),
5'-O-Dimethoxytrityl-2'-O-(2-methoxyethyl)-5-methylcytidine
intermediate,
5'-O-dimethoxytrityl-2'-O-(2-methoxyethyl)-N.sup.4-benzoyl-5-methylcytidi-
ne penultimate intermediate,
[5'-O-(4,4'-Dimethoxytriphenylmethyl)-2'-O-(2-methoxyethyl)-N.sup.4-benzo-
yl-5-methylcytidin-3'-O-yl]-2-cyanoethyl-N,N-diisopropylphosphoramidite
(MOE 5-Me-C amidite),
[5'-O-(4,4'-Dimethoxytriphenylmethyl)-2'-O-(2-methoxyethyl)-N.sup.6-benzo-
yladenosin-3'-O-yl]-2-cyanoethyl-N,N-diisopropylphosphoramidite
(MOE A amdite),
[5'-O-(4,4'-Dimethoxytriphenylmethyl)-2'-O-(2-methoxyethyl)-N.su-
p.4-isobutyrylguanosin-3'-O-yl]-2-cyanoethyl-N,N-diisopropylphosphoramidit-
e (MOE G amidite), 2'-O-(Aminooxyethyl) nucleoside amidites and
2'-O-(dimethylaminooxyethyl) nucleoside amidites,
2'-(Dimethylaminooxyethoxy) nucleoside amidites,
5'-O-tert-Butyldiphenylsilyl-O.sup.2-2'-anhydro-5-methyluridine,
5'-O-tert-Butyldiphenylsilyl-2'-O-(2-hydroxyethyl)-5-methyluridine,
2'-O-([2-phthalimidoxy)ethyl]-5'-t-butyldiphenylsilyl-5-methyluridine,
5'-O-tert-butyldiphenylsilyl-2'-O-[(2-formadoximinooxy)ethyl]-5-methyluri-
dine, 5'-O-tert-Butyldiphenylsilyl-2'-O-[N,N
dimethylaminooxyethyl]-5-methyluridine,
2'-O-(dimethylaminooxyethyl)-5-methyluridine,
5'-O-DMT-2'-O-(dimethylaminooxyethyl)-5-methyluridine,
5'-O-DMT-2'-O-(2-N,N-dimethylaminooxyethyl)-5-methyluridine-3'-[(2-cyanoe-
thyl)-N,N-diisopropylphosphoramidite], 2'-(Aminooxyethoxy)
nucleoside amidites,
N2-isobutyryl-6-O-diphenylcarbamoyl-2'-O-(2-ethylacetyl)-5'-O-(-
4,4'-dimethoxytrityl)guanosine-3'-[(2-cyanoethyl)-N,N-diisopropylphosphora-
midite], 2'-dimethylaminoethoxyethoxy (2'-DMAEOE) nucleoside
amidites, 2'-O-[2(2-N,N-dimethylaminoethoxy)ethyl]-5-methyl
uridine,
5'-O-dimethoxytrityl-2'-O-[2(2-N,N-dimethylaminoethoxy)-ethyl)]-5-methyl
uridine and
5'-O-Dimethoxytrityl-2'-O-[2(2-N,N-dimethylaminoethoxy)-ethyl)]-5-methyl
uridine-3'-O-(cyanoethyl-N,N-diisopropyl)phosphoramidite.
Example 2
Oligonucleotide and Oligonucleoside Synthesis
[0119] The antisense compounds used in accordance with this
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 well known to use similar techniques to prepare
oligonucleotides such as the phosphorothioates and alkylated
derivatives.
Oligonucleotides
[0120] Unsubstituted and substituted phosphodiester (P.dbd.O)
oligonucleotides are synthesized on an automated DNA synthesizer
(Applied Biosystems model 394) using standard phosphoramidite
chemistry with oxidation by iodine.
[0121] Phosphorothioates (P.dbd.S) are synthesized similar to
phosphodiester oligonucleotides with the following exceptions:
thiation was effected by utilizing a 10% w/v solution of
3,H-1,2-benzodithiole-3-one 1,1-dioxide in acetonitrile for the
oxidation of the phosphite linkages. The thiation reaction step
time was increased to 180 sec and preceded by the normal capping
step. After cleavage from the CPG column and deblocking in
concentrated ammonium hydroxide at 55.degree. C. (12-16 hr), the
oligonucleotides were recovered by precipitating with >3 volumes
of ethanol from a 1 M NH.sub.4OAc solution. Phosphinate
oligonucleotides are prepared as described in U.S. Pat. No.
5,508,270, herein incorporated by reference.
[0122] Alkyl phosphonate oligonucleotides are prepared as described
in U.S. Pat. No. 4,469,863, herein incorporated by reference.
[0123] 3'-Deoxy-3'-methylene phosphonate oligonucleotides are
prepared as described in U.S. Pat. No. 5,610,289 or 5,625,050,
herein incorporated by reference.
[0124] Phosphoramidite oligonucleotides are prepared as described
in U.S. Pat. No. 5,256,775 or U.S. Pat. No. 5,366,878, herein
incorporated by reference.
[0125] Alkylphosphonothioate oligonucleotides are prepared as
described in published PCT applications PCT/US94/00902 and
PCT/US93/06976 (published as WO 94/17093 and WO 94/02499,
respectively), herein incorporated by reference.
[0126] 3'-Deoxy-3'-amino phosphoramidate oligonucleotides are
prepared as described in U.S. Pat. No. 5,476,925, herein
incorporated by reference.
[0127] Phosphotriester oligonucleotides are prepared as described
in U.S. Pat. No. 5,023,243, herein incorporated by reference.
[0128] Borano phosphate oligonucleotides are prepared as described
in U.S. Pat. Nos. 5,130,302 and 5,177,198, both herein incorporated
by reference.
Oligonucleosides
[0129] Methylenemethylimino linked oligonucleosides, also
identified as MMI linked oligonucleosides, methylenedimethylhydrazo
linked oligonucleosides, also identified as MDH linked
oligonucleosides, and methylenecarbonylamino linked
oligonucleosides, also identified as amide-3 linked
oligonucleosides, and methyleneaminocarbonyl linked
oligonucleosides, also identified as amide-4 linked
oligonucleosides, as well as mixed backbone compounds having, for
instance, alternating MMI and P.dbd.O or P.dbd.S linkages are
prepared as described in U.S. Pat. Nos. 5,378,825, 5,386,023,
5,489,677, 5,602,240 and 5,610,289, all of which are herein
incorporated by reference.
[0130] Formacetal and thioformacetal linked oligonucleosides are
prepared as described in U.S. Pat. Nos. 5,264,562 and 5,264,564,
herein incorporated by reference.
[0131] Ethylene oxide linked oligonucleosides are prepared as
described in U.S. Pat. No. 5,223,618, herein incorporated by
reference.
Example 3
RNA Synthesis
[0132] In general, RNA synthesis chemistry is based on the
selective incorporation of various protecting groups at strategic
intermediary reactions. Although one of ordinary skill in the art
will understand the use of protecting groups in organic synthesis,
a useful class of protecting groups includes silyl ethers. In
particular bulky silyl ethers are used to protect the 5'-hydroxyl
in combination with an acid-labile orthoester protecting group on
the 2'-hydroxyl. This set of protecting groups is then used with
standard solid-phase synthesis technology. It is important to
lastly remove the acid labile orthoester protecting group after all
other synthetic steps. Moreover, the early use of the silyl
protecting groups during synthesis ensures facile removal when
desired, without undesired deprotection of 2' hydroxyl.
[0133] Following this procedure for the sequential protection of
the 5'-hydroxyl in combination with protection of the 2'-hydroxyl
by protecting groups that are differentially removed and are
differentially chemically labile, RNA oligonucleotides were
synthesized.
[0134] RNA oligonucleotides are synthesized in a stepwise fashion.
Each nucleotide is added sequentially (3'- to 5'-direction) to a
solid support-bound oligonucleotide. The first nucleoside at the
3'-end of the chain is covalently attached to a solid support. The
nucleotide precursor, a ribonucleoside phosphoramidite, and
activator are added, coupling the second base onto the 5'-end of
the first nucleoside. The support is washed and any unreacted
5'-hydroxyl groups are capped with acetic anhydride to yield
5'-acetyl moieties. The linkage is then oxidized to the more stable
and ultimately desired P(V) linkage. At the end of the nucleotide
addition cycle, the 5'-silyl group is cleaved with fluoride. The
cycle is repeated for each subsequent nucleotide.
[0135] Following synthesis, the methyl protecting groups on the
phosphates are cleaved in 30 minutes utilizing 1 M
disodium-2-carbamoyl-2-cyanoethylene-1,1-dithiolate trihydrate
(S.sub.2Na.sub.2) in DMF. The deprotection solution is washed from
the solid support-bound oligonucleotide using water. The support is
then treated with 40% methylamine in water for 10 minutes at
55.degree. C. This releases the RNA oligonucleotides into solution,
deprotects the exocyclic amines, and modifies the 2'-groups. The
oligonucleotides can be analyzed by anion exchange HPLC at this
stage.
[0136] The 2'-orthoester groups are the last protecting groups to
be removed. The ethylene glycol monoacetate orthoester protecting
group developed by Dharmacon Research, Inc. (Lafayette, Colo.), is
one example of a useful orthoester protecting group which, has the
following important properties. It is stable to the conditions of
nucleoside phosphoramidite synthesis and oligonucleotide synthesis.
However, after oligonucleotide synthesis the oligonucleotide is
treated with methylamine which not only cleaves the oligonucleotide
from the solid support but also removes the acetyl groups from the
orthoesters. The resulting 2-ethyl-hydroxyl substituents on the
orthoester are less electron withdrawing than the acetylated
precursor. As a result, the modified orthoester becomes more labile
to acid-catalyzed hydrolysis. Specifically, the rate of cleavage is
approximately 10 times faster after the acetyl groups are removed.
Therefore, this orthoester possesses sufficient stability in order
to be compatible with oligonucleotide synthesis and yet, when
subsequently modified, permits deprotection to be carried out under
relatively mild aqueous conditions compatible with the final RNA
oligonucleotide product.
[0137] Additionally, methods of RNA synthesis are well known in the
art (Scaringe, S. A. Ph.D. Thesis, University of Colorado, 1996;
Scaringe, S. A., et al., J. Am. Chem. Soc., 1998, 120, 11820-11821;
Matteucci, M. D. and Caruthers, M. H. J. Am. Chem. Soc., 1981, 103,
3185-3191; Beaucage, S. L. and Caruthers, M. H. Tetrahedron Lett.,
1981, 22, 1859-1862; Dahl, B. J., et al., Acta Chem. Scand,. 1990,
44, 639-641; Reddy, M. P., et al., Tetrahedrom Lett., 1994, 25,
4311-4314; Wincott, F. et al., Nucleic Acids Res., 1995, 23,
2677-2684; Griffin, B. E., et al., Tetrahedron, 1967, 23,
2301-2313; Griffin, B. E., et al., Tetrahedron, 1967, 23,
2315-2331).
[0138] RNA antisense compounds (RNA oligonucleotides) of the
present invention can be synthesized by the methods herein or
purchased from Dharmacon Research, Inc (Lafayette, Colo.). Once
synthesized, complementary RNA antisense compounds can then be
annealed by methods known in the art to form double stranded
(duplexed) antisense compounds. For example, duplexes can be formed
by combining 30 .mu.l of each of the complementary strands of RNA
oligonucleotides (50 .mu.M RNA oligonucleotide solution) and 15
.mu.l of 5.times. annealing buffer (100 mM potassium acetate, 30 mM
HEPES-KOH pH 7.4, 2 mM magnesium acetate) followed by heating for 1
minute at 90.degree. C., then 1 hour at 37.degree. C. The resulting
duplexed antisense compounds can be used in kits, assays, screens,
or other methods to investigate the role of a target nucleic acid,
or for diagnostic or therapeutic purposes.
Example 4
Synthesis of Chimeric Compounds
[0139] Chimeric oligonucleotides, oligonucleosides or mixed
oligonucleotides/oligonucleosides of the invention can be of
several different types. These include a first type wherein the
"gap" segment of linked nucleosides is positioned between 5' and 3'
"wing" segments of linked nucleosides and a second "open end" type
wherein the "gap" segment is located at either the 3' or the 5'
terminus of the oligomeric compound. Oligonucleotides of the first
type are also known in the art as "gapmers" or gapped
oligonucleotides. Oligonucleotides of the second type are also
known in the art as "hemimers" or "wingmers".
[2'-O-Me]-[2'-deoxy]-[2'-O-Me] Chimeric Phosphorothioate
Oligonucleotides
[0140] Chimeric oligonucleotides having 2'-O-alkyl phosphorothioate
and 2'-deoxy phosphorothioate oligonucleotide segments are
synthesized using an Applied Biosystems automated DNA synthesizer
Model 394, as above. Oligonucleotides are synthesized using the
automated synthesizer and
2'-deoxy-5'-dimethoxytrityl-3'-O-phosphoramidite for the DNA
portion and 5'-dimethoxytrityl-2'-O-methyl-3'-O-phosphoramidite for
5' and 3' wings. The standard synthesis cycle is modified by
incorporating coupling steps with increased reaction times for the
5'-dimethoxytrityl-2'-O-methyl-3'-O-phosphoramidite. The fully
protected oligonucleotide is cleaved from the support and
deprotected in concentrated ammonia (NH.sub.4OH) for 12-16 hr at
55.degree. C. The deprotected oligo is then recovered by an
appropriate method (precipitation, column chromatography, volume
reduced in vacuo and analyzed spectrophotometrically for yield and
for purity by capillary electrophoresis and by mass
spectrometry.
[2'-O-(2-Methoxyethyl)]-[2'-deoxy]-[2'-O-(Methoxyethyl)] Chimeric
Phosphorothioate Oligonucleotides
[0141] [2'-O-(2-methoxyethyl)]-[2'-deoxy]-[-2'-O-(methoxyethyl)]
chimeric phosphorothioate oligonucleotides were prepared as per the
procedure above for the 2'-O-methyl chimeric oligonucleotide, with
the substitution of 2'-O-(methoxyethyl) amidites for the
2'-O-methyl amidites.
[2'-O-(2-Methoxyethyl)Phosphodiester]-[2'-deoxy
Phosphorothioate]-[2'-O-(2-Methoxyethyl)Phosphodiester] Chimeric
Oligonucleotides
[0142] [2'-O-(2-methoxyethyl phosphodiester]-[2'-deoxy
phosphorothioate]-[2'-O-(methoxyethyl)phosphodiester] chimeric
oligonucleotides are prepared as per the above procedure for the
2'-O-methyl chimeric oligonucleotide with the substitution of
2'-O-(methoxyethyl) amidites for the 2'-O-methyl amidites,
oxidation with iodine to generate the phosphodiester
internucleotide linkages within the wing portions of the chimeric
structures and sulfurization utilizing 3,H-1,2 benzodithiole-3-one
1,1 dioxide (Beaucage Reagent) to generate the phosphorothioate
internucleotide linkages for the center gap.
[0143] Other chimeric oligonucleotides, chimeric oligonucleosides
and mixed chimeric oligonucleotides/oligonucleosides are
synthesized according to U.S. Pat. No. 5,623,065, herein
incorporated by reference.
Example 5
Oligonucleotide Isolation
[0144] After cleavage from the controlled pore glass solid support
and deblocking in concentrated ammonium hydroxide at 55.degree. C.
for 12-16 hours, the oligonucleotides or oligonucleosides are
recovered by precipitation out of 1 M NH.sub.4OAc with >3
volumes of ethanol. Synthesized oligonucleotides were analyzed by
electrospray mass spectroscopy (molecular weight determination) and
by capillary gel electrophoresis and judged to be at least 70% full
length material. The relative amounts of phosphorothioate and
phosphodiester linkages obtained in the synthesis was determined by
the ratio of correct molecular weight relative to the -16 amu
product (+/-32 +/-48). For some studies oligonucleotides were
purified by HPLC, as described by Chiang et al., J. Biol. Chem.
1991, 266, 18162-18171. Results obtained with HPLC-purified
material were similar to those obtained with non-HPLC purified
material.
Example 6
Oligonucleotide Synthesis
96 Well Plate Format
[0145] Oligonucleotides were synthesized via solid phase P(III)
phosphoramidite chemistry on an automated synthesizer capable of
assembling 96 sequences simultaneously in a 96-well format.
Phosphodiester internucleotide linkages were afforded by oxidation
with aqueous iodine. Phosphorothioate internucleotide linkages were
generated by sulfurization utilizing 3,H-1,2 benzodithiole-3-one
1,1 dioxide (Beaucage Reagent) in anhydrous acetonitrile. Standard
base-protected beta-cyanoethyl-diiso-propyl phosphoramidites were
purchased from commercial vendors (e.g. PE-Applied Biosystems,
Foster City, Calif., or Pharmacia, Piscataway, N.J.). Non-standard
nucleosides are synthesized as per standard or patented methods.
They are utilized as base protected beta-cyanoethyldiisopropyl
phosphoramidites.
[0146] Oligonucleotides were cleaved from support and deprotected
with concentrated NH.sub.4OH at elevated temperature (55-60.degree.
C.) for 12-16 hours and the released product then dried in vacuo.
The dried product was then re-suspended in sterile water to afford
a master plate from which all analytical and test plate samples are
then diluted utilizing robotic pipettors.
Example 7
Oligonucleotide Analysis
96-Well Plate Format
[0147] The concentration of oligonucleotide in each well was
assessed by dilution of samples and UV absorption spectroscopy. The
full-length integrity of the individual products was evaluated by
capillary electrophoresis (CE) in either the 96-well format
(Beckman P/ACE.TM. MDQ) or, for individually prepared samples, on a
commercial CE apparatus (e.g., Beckman P/ACE.TM. 5000, ABI 270).
Base and backbone composition was confirmed by mass analysis of the
compounds utilizing electrospray-mass spectroscopy. All assay test
plates were diluted from the master plate using single and
multi-channel robotic pipettors. Plates were judged to be
acceptable if at least 85% of the compounds on the plate were at
least 85% full length.
Example 8
Cell Culture and Oligonucleotide Treatment
[0148] The effect of antisense compounds on target nucleic acid
expression can be tested in any of a variety of cell types provided
that the target nucleic acid is present at measurable levels. This
can be routinely determined using, for example, PCR or Northern
blot analysis. The following cell types are provided for
illustrative purposes, but other cell types can be routinely used,
provided that the target is expressed in the cell type chosen. This
can be readily determined by methods routine in the art, for
example Northern blot analysis, ribonuclease protection assays, or
RT-PCR.
T-24 Cells:
[0149] The human transitional cell bladder carcinoma cell line T-24
was obtained from the American Type Culture Collection (ATCC)
(Manassas, Va.). T-24 cells were routinely cultured in complete
McCoy's 5A basal media (Invitrogen Corporation, Carlsbad, Calif.)
supplemented with 10% fetal calf serum (Invitrogen Corporation,
Carlsbad, Calif.), penicillin 100 units per mL, and streptomycin
100 micrograms per mL (Invitrogen Corporation, Carlsbad, Calif.).
Cells were routinely passaged by trypsinization and dilution when
they reached 90% confluence. Cells were seeded into 96-well plates
(Falcon-Primaria #353872) at a density of 7000 cells/well for use
in RT-PCR analysis.
A549 Cells:
[0150] The human lung carcinoma cell line A549 was obtained from
the American Type Culture Collection (ATCC) (Manassas, Va.). A549
cells were routinely cultured in DMEM basal media (Invitrogen
Corporation, Carlsbad, Calif.) supplemented with 10% fetal calf
serum (Invitrogen Corporation, Carlsbad, Calif.), penicillin 100
units per mL, and streptomycin 100 micrograms per mL (Invitrogen
Corporation, Carlsbad, Calif.). Cells were routinely passaged by
trypsinization and dilution when they reached 90% confluence.
NHDF Cells:
[0151] Human neonatal dermal fibroblast (NHDF) were obtained from
the Clonetics Corporation (Walkersville, Md.). NHDFs were routinely
maintained in Fibroblast Growth Medium (Clonetics Corporation,
Walkersville, Md.) supplemented as recommended by the supplier.
Cells were maintained for up to 10 passages as recommended by the
supplier.
HEK Cells:
[0152] Human embryonic keratinocytes (HEK) were obtained from the
Clonetics Corporation (Walkersville, Md.). HEKs were routinely
maintained in Keratinocyte Growth Medium (Clonetics Corporation,
Walkersville, Md.) formulated as recommended by the supplier. Cells
were routinely maintained for up to 10 passages as recommended by
the supplier.
HK-2 Cells:
[0153] HK-2 (human kidney 2) is a proximal tubular cell (PTC) line
derived from normal kidney cells immortalized by transduction with
human papilloma virus 16 (HPV-16) E6/E7 genes (CRL-2190, American
Type Culture Collection, Manassus, Va.). HK-2 cells were routinely
cultured in Keratinocyte-Serum Free Medium (17005-042, Invitrogen
Corporation, Carlsbad, Calif.) which includes 5 ng/ml recombinant
epidermal growth factor and 0.05 mg/ml bovine pituitary extract.
Cells were routinely passaged by trypsinization and split at a
ratio of 1:4 when they reached 70-80% confluence. One day prior to
transfection, cells were seeded into 96-well plates
(Falcon-Primaria #353872, BD Biosciences, Bedford, Mass.) at a
density of 10,000 cells/well.
b.END Cells:
[0154] The mouse brain endothelial cell line b.END was obtained
from Dr. Werner Risau at the Max Plank Institute (Bad Nauheim,
Germany). b.END cells were routinely cultured in DMEM, high glucose
(Gibco/Life Technologies, Gaithersburg, Md.) supplemented with 10%
fetal calf serum (Gibco/Life Technologies, Gaithersburg, Md.).
Cells were routinely passaged by trypsinization and dilution when
they reached 90% confluence. Cells were seeded into 96-well plates
(Falcon-Primaria #3872) at a density of 3000 cells/well for use in
RT-PCR analysis.
Treatment with Antisense Compounds:
[0155] When cells reached 65-75% confluency, they were treated with
oligonucleotide. For cells grown in 96-well plates, wells were
washed once with 100 .mu.L OPTI-MEM.TM.-1 reduced-serum medium
(Invitrogen Corporation, Carlsbad, Calif.) and then treated with
130 .mu.L of OPTI-MEM.TM.-1 containing 3.75 .mu.g/mL LIPOFECTIN.TM.
(Invitrogen Corporation, Carlsbad, Calif.) and the desired
concentration of oligonucleotide. Cells are treated and data are
obtained in triplicate. After 4-7 hours of treatment at 37.degree.
C., the medium was replaced with fresh medium. Cells were harvested
16-24 hours after oligonucleotide treatment.
[0156] For Northern blotting or other analysis, cells may be seeded
onto 100 mm or other standard tissue culture plates and treated
similarly, using appropriate volumes of medium and
oligonucleotide.
[0157] The concentration of oligonucleotide used varies from cell
line to cell line. To determine the optimal oligonucleotide
concentration for a particular cell line, the cells are treated
with a positive control oligonucleotide at a range of
concentrations. For human cells the positive control
oligonucleotide is selected from either ISIS 13920
(TCCGTCATCGCTCCTCAGGG; SEQ ID NO: 1) which is targeted to human
H-ras, or ISIS 18078, (GTGCGCGCGAGCCCGAAATC; SEQ ID NO: 2) which is
targeted to human Jun-N-terminal kinase-2 (JNK2). Both controls are
2'-O-methoxyethyl gapmers (2'-O-methoxyethyls shown in bold) with a
phosphorothioate backbone. For mouse or rat cells the positive
control oligonucleotide is ISIS 15770 (ATGCATTCTGCCCCCAAGGA; SEQ ID
NO: 3), a 2'-O-methoxyethyl gapmer (2'-O-methoxyethyls shown in
bold) with a phosphorothioate backbone which is targeted to both
mouse and rat c-raf. The concentration of positive control
oligonucleotide that results in 80% inhibition of c-H-ras (for ISIS
13920), JNK2 (for ISIS 18078) or c-raf (for ISIS 15770) mRNA is
then utilized as the screening concentration for new
oligonucleotides in subsequent experiments for that cell line. If
80% inhibition is not achieved, the lowest concentration of
positive control oligonucleotide that results in 60% inhibition of
c-H-ras, JNK2 or c-raf mRNA is then utilized as the oligonucleotide
screening concentration in subsequent experiments for that cell
line. If 60% inhibition is not achieved, that particular cell line
is deemed as unsuitable for oligonucleotide transfection
experiments. The concentrations of antisense oligonucleotides used
herein are from 50 nM to 300 nM.
Example 9
Analysis of Oligonucleotide Inhibition of Target Gene
Expression
[0158] Antisense modulation of target gene expression can be
assayed in a variety of ways known in the art. For example, target
gene mRNA levels can be quantitated by, e.g., Northern blot
analysis, competitive polymerase chain reaction (PCR), or real-time
PCR (RT-PCR). Real-time quantitative PCR is presently preferred.
RNA analysis can be performed on total cellular RNA or poly(A)+
mRNA. The preferred method of RNA analysis of the present invention
is the use of total cellular RNA as described in other examples
herein. Methods of RNA isolation are well known in the art.
Northern blot analysis is also routine in the art. Real-time
quantitative (PCR) can be conveniently accomplished using the
commercially available ABI PRISM.TM. 7600, 7700, or 7900 Sequence
Detection System, available from PE-Applied Biosystems, Foster
City, Calif. and used according to manufacturer's instructions.
[0159] Protein levels of target genes can be quantitated in a
variety of ways well known in the art, such as immunoprecipitation,
Western blot analysis (immunoblotting), enzyme-linked immunosorbent
assay (ELISA) or fluorescence-activated cell sorting (FACS).
Antibodies directed to a target gene can be identified and obtained
from a variety of sources, such as the MSRS catalog of antibodies
(Aerie Corporation, Birmingham, Mich.), or can be prepared via
conventional monoclonal or polyclonal antibody generation methods
well known in the art.
Example 10
RNA Isolation
Poly(A)+ mRNA Isolation
[0160] Poly(A)+ mRNA was isolated according to Miura et al., (Clin.
Chem., 1996, 42, 1758-1764). Other methods for poly(A)+ mRNA
isolation are routine in the art. Briefly, for cells grown on
96-well plates, growth medium was removed from the cells and each
well was washed with 200 .mu.L cold PBS. 60 .mu.L lysis buffer (10
mM Tris-HCl, pH 7.6, 1 mM EDTA, 0.5 M NaCl, 0.5% NP-40, 20 mM
vanadyl-ribonucleoside complex) was added to each well, the plate
was gently agitated and then incubated at room temperature for five
minutes. 55 .mu.L of lysate was transferred to Oligo d(T) coated
96-well plates (AGCT Inc., Irvine Calif.). Plates were incubated
for 60 minutes at room temperature, washed 3 times with 200 .mu.L
of wash buffer (10 mM Tris-HCl pH 7.6, 1 mM EDTA, 0.3 M NaCl).
After the final wash, the plate was blotted on paper towels to
remove excess wash buffer and then air-dried for 5 minutes. 60
.mu.L of elution buffer (5 mM Tris-HCl pH 7.6), preheated to
70.degree. C., was added to each well, the plate was incubated on a
90.degree. C. hot plate for 5 minutes, and the eluate was then
transferred to a fresh 96-well plate.
[0161] Cells grown on 100 mm or other standard plates may be
treated similarly, using appropriate volumes of all solutions.
Total RNA Isolation
[0162] Total RNA was isolated using an RNEASY 96.TM. kit and
buffers purchased from Qiagen Inc. (Valencia, Calif.) following the
manufacturer's recommended procedures. Briefly, for cells grown on
96-well plates, growth medium was removed from the cells and each
well was washed with 200 .mu.L cold PBS. 150 .mu.L Buffer RLT was
added to each well and the plate vigorously agitated for 20
seconds. 150 .mu.L of 70% ethanol was then added to each well and
the contents mixed by pipetting three times up and down. The
samples were then transferred to the RNEASY 96.TM. well plate
attached to a QIAVAC.TM. manifold fitted with a waste collection
tray and attached to a vacuum source. Vacuum was applied for 1
minute. 500 .mu.L of Buffer RW1 was added to each well of the
RNEASY 96.TM. plate and incubated for 15 minutes and the vacuum was
again applied for 1 minute. An additional 500 .mu.L of Buffer RW1
was added to each well of the RNEASY 96.TM. plate and the vacuum
was applied for 2 minutes. 1 mL of Buffer RPE was then added to
each well of the RNEASY 96.TM. plate and the vacuum applied for a
period of 90 seconds. The Buffer RPE wash was then repeated and the
vacuum was applied for an additional 3 minutes. The plate was then
removed from the QIAVAC.TM. manifold and blotted dry on paper
towels. The plate was then re-attached to the QIAVAC.TM. manifold
fitted with a collection tube rack containing 1.2 mL collection
tubes. RNA was then eluted by pipetting 140 .mu.L of RNAse free
water into each well, incubating 1 minute, and then applying the
vacuum for 3 minutes.
[0163] The repetitive pipetting and elution steps may be automated
using a QIAGEN Bio-Robot 9604 (Qiagen, Inc., Valencia Calif.).
Essentially, after lysing of the cells on the culture plate, the
plate is transferred to the robot deck where the pipetting, DNase
treatment and elution steps are carried out.
Example 11
Real-time Quantitative PCR Analysis of Target Gene mRNA Levels
[0164] Quantitation of target gene mRNA levels was accomplished by
real-time quantitative PCR using the ABI PRISM.TM. 7600, 7700, or
7900 Sequence Detection System (PE-Applied Biosystems, Foster City,
Calif.) according to manufacturer's instructions. This is a
closed-tube, non-gel-based, fluorescence detection system which
allows high-throughput quantitation of polymerase chain reaction
(PCR) products in real-time. As opposed to standard PCR in which
amplification products are quantitated after the PCR is completed,
products in real-time quantitative PCR are quantitated as they
accumulate. This is accomplished by including in the PCR reaction
an oligonucleotide probe that anneals specifically between the
forward and reverse PCR primers, and contains two fluorescent dyes.
A reporter dye (e.g., FAM or JOE, obtained from either PE-Applied
Biosystems, Foster City, Calif., Operon Technologies Inc., Alameda,
Calif. or Integrated DNA Technologies Inc., Coralville, Iowa) is
attached to the 5' end of the probe and a quencher dye (e.g.,
TAMRA, obtained from either PE-Applied Biosystems, Foster City,
Calif., Operon Technologies Inc., Alameda, Calif. or Integrated DNA
Technologies Inc., Coralville, Iowa) is attached to the 3' end of
the probe. When the probe and dyes are intact, reporter dye
emission is quenched by the proximity of the 3' quencher dye.
During amplification, annealing of the probe to the target sequence
creates a substrate that can be cleaved by the 5'-exonuclease
activity of Taq polymerase. During the extension phase of the PCR
amplification cycle, cleavage of the probe by Taq polymerase
releases the reporter dye from the remainder of the probe (and
hence from the quencher moiety) and a sequence-specific fluorescent
signal is generated. With each cycle, additional reporter dye
molecules are cleaved from their respective probes, and the
fluorescence intensity is monitored at regular intervals by laser
optics built into the ABI PRISM.TM. Sequence Detection System. In
each assay, a series of parallel reactions containing serial
dilutions of mRNA from untreated control samples generates a
standard curve that is used to quantitate the percent inhibition
after antisense oligonucleotide treatment of test samples.
[0165] Prior to quantitative PCR analysis, primer-probe sets
specific to the target gene being measured are evaluated for their
ability to be "multiplexed" with a GAPDH amplification reaction. In
multiplexing, both the target gene and the internal standard gene
GAPDH are amplified concurrently in a single sample. In this
analysis, mRNA isolated from untreated cells is serially diluted.
Each dilution is amplified in the presence of primer-probe sets
specific for GAPDH only, target gene only ("single-plexing"), or
both (multiplexing). Following PCR amplification, standard curves
of GAPDH and target mRNA signal as a function of dilution are
generated from both the single-plexed and multiplexed samples. If
both the slope and correlation coefficient of the GAPDH and target
signals generated from the multiplexed samples fall within 10% of
their corresponding values generated from the single-plexed
samples, the primer-probe set specific for that target is deemed
multiplexable. Other methods of PCR are also known in the art.
[0166] PCR reagents were obtained from Invitrogen Corporation,
(Carlsbad, Calif.). RT-PCR reactions were carried out by adding 20
.mu.L PCR cocktail (2.5.times.PCR buffer minus MgCl.sub.2, 6.6 mM
MgCl.sub.2, 375 .mu.M each of dATP, dCTP, dCTP and dGTP, 375 nM
each of forward primer and reverse primer, 125 nM of probe, 4 Units
RNAse inhibitor, 1.25 Units PLATINUM.RTM. Taq, 5 Units MuLV reverse
transcriptase, and 2.5.times.ROX dye) to 96-well plates containing
30 .mu.L total RNA solution (20-200 ng). The RT reaction was
carried out by incubation for 30 minutes at 48.degree. C. Following
a 10 minute incubation at 95.degree. C. to activate the
PLATINUM.RTM. Taq, 40 cycles of a two-step PCR protocol were
carried out: 95.degree. C. for 15 seconds (denaturation) followed
by 60.degree. C. for 1.5 minutes (annealing/extension).
[0167] Gene target quantities obtained by real time RT-PCR are
normalized using either the expression level of GAPDH, a gene whose
expression is constant, or by quantifying total RNA using
RiboGreen.TM. (Molecular Probes, Inc. Eugene, Oreg.). GAPDH
expression is quantified by real time RT-PCR, by being run
simultaneously with the target, multiplexing, or separately. Total
RNA is quantified using RiboGreen.TM. RNA quantification reagent
(Molecular Probes, Inc. Eugene, Oreg.). Methods of RNA
quantification by RiboGreen.TM. are taught in Jones, L. J., et al,
(Analytical Biochemistry, 1998, 265, 368-374).
[0168] In this assay, 170 .mu.L of RiboGreen.TM. working reagent
(RiboGreen.TM. reagent diluted 1:350 in 10 mM Tris-HCl, 1 mM EDTA,
pH 7.5) is pipetted into a 96-well plate containing 30 .mu.L
purified, cellular RNA. The plate is read in a CytoFluor 4000 (PE
Applied Biosystems) with excitation at 485 nm and emission at 530
nm.
Example 12
Northern Blot Analysis of Target Gene mRNA Levels
[0169] For in vivo studies, total RNA was prepared from procured
tissues of sacrificed mice by homogenization in GITC buffer
(Invitrogen, Carlsbad, Calif.) containing 2-mercaptoethanol
(Sigma-Aldrich, St. Louis, Mo.) following manufacturer's
recommended protocols followed by ultracentrifugation through a
CsCl cushion. For cell culture studies, eighteen hours after
antisense treatment, cell monolayers were washed twice with cold
PBS and lysed in 1 mL RNAZOL.TM. (TEL-TEST "B" Inc., Friendswood,
Tex.). Total RNA was prepared following manufacturer's recommended
protocols.
[0170] Twenty micrograms of total RNA was fractionated by
electrophoresis through 1.2% agarose gels containing 1.1%
formaldehyde using a MOPS buffer system (AMRESCO, Inc. Solon,
Ohio). RNA was transferred from the gel to HYBOND.TM.-N+ nylon
membranes (Amersham Pharmacia Biotech, Piscataway, N.J.) by
overnight capillary transfer. RNA transfer was confirmed by UV
visualization. Membranes were fixed by UV cross-linking using a
STRATALINKER.TM. UV Crosslinker 2400 (Stratagene, Inc, La Jolla,
Calif.) and then probed using RapidHYB.TM. hybridization solution
(Amersham Pharmacia Biotech, Piscataway, N.J.) using manufacturer's
recommendations for stringent conditions.
[0171] Hybridized membranes were visualized and quantitated using a
PHOSPHORIMAGER.TM. and IMAGEQUANT.TM. Software V3.3 (Molecular
Dynamics, Sunnyvale, Calif.). Data was normalized to GAPDH levels
in untreated controls.
Example 13
Design of Chemically Modified Antisense Compounds for Optimized
Kidney Targeting
[0172] A series of chemically modified antisense compounds were
designed using the sequence of ISIS 145733 (SEQ ID NO: 4), ISIS
145742 (SEQ ID NO: 5) or ISIS 145746 (SEQ ID NO: 6). Modifications
were made to the internucleoside linkages such that the
oligonucleotides consisted of either full phosphorothioate
backbones or mixed phosphorothioate and phosphodiester backbones
(mixed backbone compounds). Modified antisense compounds also
contained sugar moiety substitutions at the 2' position, comprising
a 2'-methoxyethyl (2'-MOE) or a 2'-0-dimethylaminoethoxyethyl
(2'-DMAEOE). Further modifications included nucleobase
substitutions, wherein the unmodified cytosine nucleobase was used
in place of the modified 5-methylcytosine at one position in the
antisense compound. The compounds are shown in Table 1.
[0173] ISIS 145733 (SEQ ID NO: 4), ISIS 145742 (SEQ ID NO: 5) and
ISIS 145746 (SEQ ID NO: 6) are chimeric oligonucleotides having
2'-MOE wings and a deoxy gap with phosphorothioate linkages
throughout the oligonucleotide. ISIS 257016 (SEQ ID NO: 4), ISIS
341699 (SEQ ID NO: 5) and ISIS 351642 (SEQ ID NO: 6) are chimeric
oligonucleotides having 2'-MOE wings and a deoxy gap, with
phosphodiester linkages in the wings and phosphorothioate linkages
in the gap. ISIS 351641 (SEQ ID NO: 4), ISIS 360886 (SEQ ID NO: 4)
and ISIS 360887 (SEQ ID NO: 4) are chimeric oligonucleotides having
2'-MOE wings and a deoxy gap, with phosphorothioate linkages in the
gap and phosphodiester linkages in the wings, except for one
phosphorothioate linkage in the wing(s) at either the extreme 5'
end (ISIS 360886), the extreme 3' end (ISIS 360887) or both of the
extreme 5' and 3' ends (ISIS 351641).
[0174] ISIS 323294 (SEQ ID NO: 4) consists of 2'-MOE nucleotides at
positions 1, 2, 3, 4, 17 and 19, 2'-nucleotides at positions 5, 16,
18 and 20 and 2'-deoxynucleotides at positions 6 through 15 with
phosphorothioate linkages throughout the oligonucleotide. ISIS
323295 (SEQ ID NO: 4) consists of 2'-MOE nucleotides at positions
1, 2, 3, 4, 17 and 19, 2'-DMAEOE nucleotides at positions 5, 16, 18
and 20 and 2'-deoxynucleotides at positions 6 through 15, wherein
the first and last 4 internucleoside linkages are phosphodiester
and the central internucleotide linkages are phosphorothioate.
[0175] The nucleotides in the 3' most positions in ISIS 251017 and
257018 are cytosine residues (indicated by an asterisk in Table 4).
All other cytosine residues of the oligonucleotides listed above
are 5-methylcytosines. The compounds are shown in Table 1.
Phosphodiester (P.dbd.O) internucleotide linkages are indicated by
an "o" between nucleotide positions. Phosphorothioate (P.dbd.S)
internucleoside linkages are indicated by an "s" between nucleotide
positions. 2'-MOE nucleotides are underscored and 2'-DMAEOE
nucleotides are emboldened. All compounds in Table 1 target the
coding region of murine SGLT2 (provided herein as SEQ ID NO: 7).
TABLE-US-00001 TABLE 1 Chemical modifications of antisense
compounds targeting SGLT2 SEQ ID ISIS # Sequence NO 145733
GsAsAsGsTsAsGsCsCsAsCsCsAsAsCsTsGsTs 4 GsC 257016
GoAoAoGoTsAsGsCsCsAsCsCsAsAsCsToGoTo 4 GoC 257017
GsAsAsGsTsAsGsCsCsAsCsCsAsAsCsTsGsTs 4 GsC* 257018
GoAoAoGoTsAsGsCsCsAsCsCsAsAsCsToGoTo 4 GoC* 145742
GsAsGsAsAsCsAsTsAsTsCsCsAsCsCsGsAsGs 5 AsT 341699
GoAoGoAoAsCsAsTsAsTsCsCsAsCsCsGoAoGo 5 AoT 145746
CsTsGsCsAsCsAsGsTsGsTsCsTsGsTsGsTsAs 6 CsA 351642
CoToGoCoAsCsAsGsTsGsTsCsTsGsTsGoToAo 6 CoA 351641
GsAoAoGoTsAsGsCsCsAsCsCsAsAsCsToGoTo 4 GsC 360886
GsAoAoGoTsAsGsCsCsAsCsCsAsAsCsToGoTo 4 GoC 360887
GoAoAoGoTsAsGsCsCsAsCsCsAsAsCsToGoTo 4 GsC 323294
GsAsAsGsTsAsGsCsCsAsCsCsAsAsCsTsGsTs 4 GsC 323295
GoAoAoGoTsAsGsCsCsAsCsCsAsAsCsToGoTo 4 GoC
Example 14
Antisense Inhibition of SGLT2 in Murine Kidney
Comparison of Various Oligonucleotide Chemistries
[0176] In accordance with the present invention, modified SGLT2
antisense compounds were investigated for their activity in vivo.
ISIS 29837 (TCGATCTCCTTTTATGCCCG, SEQ ID NO: 8) served as a control
compound and is a chimeric oligonucleotide ("gapmer") 20
nucleotides in length, composed of a central "gap" region
consisting of ten 2'-deoxynucleotides, which is flanked on both
sides (5' and 3' directions) by five-nucleotide "wings". The wings
are composed of 2'-methoxyethyl (2'-MOE) nucleotides. The
internucleoside (backbone) linkages are phosphorothioate (P.dbd.S)
throughout the oligonucleotide. All cytidine residues are
5-methylcytidines.
[0177] Male 6-week old Balb/c mice (Charles River Laboratories,
Wilmington, Mass.) were given intraperitoneal injections of ISIS
145733, ISIS 257016, ISIS 323294, ISIS 323295 or ISIS 29837 at a
dose of 25 mg/kg twice per week for two weeks. Saline-injected
animals served as controls. Each treatment group contained four
animals. The mice were sacrificed 2 days following administration
of the fourth and final dose of oligonucleotide or saline.
[0178] Mice were evaluated for SGLT2 levels in kidney. Target
levels were determined by quantitative real-time PCR as described
by other examples herein. PCR results were normalized to the
ubiquitously expressed mouse cyclophilin A gene.
[0179] Probes and primers to mouse SGLT2 were designed to hybridize
to a mouse SGLT2 sequence, using published sequence information
(incorporated herein as SEQ ID NO: 7). For mouse SGLT2 the PCR
primers were: TABLE-US-00002 forward primer:
CTCGTCTCATACCCGAGTTCTTCT (SEQ ID NO: 9) reverse primer:
AATGATGGCGAAATAGAGGTAGTGTAC (SEQ ID NO: 10)
and the PCR probe was: FAM-TGCGACCCTCAGCGTGCCC-TAMRA (SEQ ID NO:
11) where FAM is the fluorescent dye and TAMRA is the quencher dye.
For mouse cyclophilin A the PCR primers were: forward primer:
TCGCCGCTTGCTGCA (SEQ ID NO: 12) reverse primer: ATCGGCCGTGATGTCGA
(SEQ ID NO: 13) and the PCR probe was: 5'
JOE-CCATGGTCAACCCCACCGTGTTC-3' (SEQ ID NO: 14) where JOE is the
fluorescent reporter dye and TAMRA is the quencher dye.
[0180] The data are expressed as percent change ("-" indicates a
decrease) relative to saline treated animals and are shown in Table
2. TABLE-US-00003 TABLE 2 Antisense inhibition of SGLT2 mRNA
expression in vivo by modified SGLT2 antisense compounds Percent
change in SGLT2 expression relative to saline ISIS ISIS ISIS ISIS
ISIS 145733 257016 323294 323295 29837 -44 -82 -40 -31 -23
[0181] These data illustrate that antisense compounds of different
chemistries inhibit the expression of SGLT2 mRNA in mouse kidney.
Greatest inhibition of SGLT2 is observed in kidneys from mice
treated with ISIS 257016, which is a mixed backbone antisense
compound.
[0182] Mice were further evaluated for total body weight, liver
weight and spleen weight. Significant changes in spleen, liver or
body weight can indicate that a particular compound causes toxic
effects. The data are expressed as percent change in body or organ
weight ("+" indicates an increase, "-" indicates a decrease). The
results are presented in Table 3. TABLE-US-00004 TABLE 3 Effects of
antisense compounds on total body weight, liver weight and spleen
weight in mice Percent change in weight 145733 257016 323294 323295
29837 Total Body 0 0 -1 -3 0 Liver +1 +1 +9 +4 +12 Spleen +4 +1 +19
+8 +1
[0183] All changes in body weight and organ weight were within the
margin of error of the experiment. No significant changes in body
weight were observed during the treatment or at study termination.
No significant changes in liver or spleen weights were
observed.
[0184] Toxic effects of compounds administered in vivo can also be
assessed by measuring the levels of enzymes and proteins associated
with disease or injury of the liver or kidney. Elevations in the
levels of the serum transaminases aspartate aminotransferase (AST)
and alanine aminotransferase (ALT) are often indicators of liver
disease or injury. Serum total bilirubin is an indicator of liver
and biliary function, and albumin and blood urea nitrogen (BUN) are
indicators of renal function. Glucose and triglyceride levels are
sometimes altered due to toxicity of a treatment. Serum glucose
also depends in part upon the activity of SGLT2.
[0185] In accordance with the present invention, the levels of ALT,
AST, total bilirubin, albumin, BUN, glucose and triglyceride were
measured in mice treated with the compounds of the invention. Serum
was analyzed by LabCorp Testing Facility (San Diego, Calif.). The
results are expressed as units measured and are shown in Table 4.
TABLE-US-00005 TABLE 4 Effects of antisense compounds targeting
SGLT2 on liver and kidney function in mice Treatment Normal and
units measured Serum indicator Range Saline 145733 257016 323294
323295 29837 BUN 15-40 27 29 33 29 30 30 mg/dL Albumin 2.5-4.0 3 3
3 3 3 3 g/dL Bilirubin mg/dL 0.1-1.0 0.1 0.1 0.1 0.1 0.1 0.1 AST
30-300 124 83 129 174 89 114 IU/L ALT 30-200 33 26 47 61 32 31 IU/L
Triglycerides 25-100* 179 154 157 160 209 198 mg/dL Glucose 80-150*
242 270 222 284 271 235 mg/dL *Triglyceride and glucose levels are
routinely higher in the Balb/c strain of mice than in other strains
of mice.
[0186] The levels of routine clinical indicators of liver and
kidney injury and disease are within normal ranges and are not
significantly changed relative to saline-treated animals,
demonstrating that the compounds of the invention do not
significantly affect renal or hepatic function. Triglyceride and
glucose levels, while outside the normal range for most mice as is
common in the Balb/c strain, are not significantly elevated
relative to saline-treated animals.
[0187] Mice injected with ISIS 145733, ISIS 257016, ISIS 323294 and
ISIS 323295 were also evaluated histologically following routine
procedures. Liver, spleen, kidney, intestine, pancreas, lung, skin,
heart and muscle samples were procured, fixed in 10%
neutral-buffered formalin and processed for staining with
hematoxylin and eosin, to visualize nuclei and cytoplasm, or with
the anti-oligonucleotide IgG1 antibody 2E1-B5 (Berkeley Antibody
Company, Berkeley, Calif.) to assess oligonucleotide staining
patterns. Hematoxylin and eosin staining in most tissues exhibited
no significant difference between saline- and
oligonucleotide-treated animals. Heart sections from animals
treated with ISIS 323294 and ISIS 323295 showed a high amount of
inflammation relative to hearts from saline-treated mice. 2E1-B5
antibody was recognized using an isospecific anti-IgG2 horse-radish
peroxidase-conjugated secondary antibody (Zymed, San Francisco,
Calif.) and immunostaining was developed with 3,3'-diaminobenzidene
(DAKO, Carpenteria, Calif.). 2E1-B5 staining was performed in
duplicate and showed that none of the chemistries significantly
stained the liver, while staining was observed in the kidney
proximal tubules.
[0188] The results illustrated in this example demonstrate that
antisense compounds of different chemistries are delivered to the
kidney, reduce SGLT2 expression in vivo, and that treatment with
these compounds does not result in liver or kidney toxicity. The
mixed backbone compound, ISIS 257016, is particularly efficient at
reducing target mRNA levels in the kidney.
Example 15
Antisense Inhibition of SGLT2 mRNA Expression In Vivo
Dose Response Study Comparing Mixed Backbone and Full
Phosphorothioate Backbone Compounds
[0189] ISIS 145733 and ISIS 257016 were selected for a dose
response study in mice to further evaluate the effectiveness of
mixed backbone antisense compounds for kidney targeting. Male
8-week old Balb/c mice (Charles River Laboratories, Wilmington,
Mass.) were given intraperitoneal injections of ISIS 145733 or ISIS
257016 at doses of 6.25, 12.5, 25 or 50 mg/kg twice per week for
two weeks. Saline-injected animals served as controls. A total of 4
animals were injected per group. The mice were sacrificed 2 days
following administration of the fourth and final dose of
oligonucleotide or saline.
[0190] Mice were evaluated for SGLT2 levels in kidney. Target
levels were determined by quantitative real-time PCR as described
in other examples herein. PCR results were normalized to
cyclophilin as described in Example 14. The data are expressed as
percent change ("+" indicates an increase, "-" indicates a
decrease) relative to saline treated animals and are illustrated in
Table 5. TABLE-US-00006 TABLE 5 Antisense inhibition of SGLT2 mRNA
expression in vivo by antisense compounds with varying chemistries
Percent change in SGLT2 expression relative to saline Dose of
oligonucleotide ISIS ISIS mg/kg 145733 257016 6.25 -3 -58 12.5 -7
-68 25 -37 -68 50 -34 -77
[0191] These results illustrate that the compounds of the
invention, both full phosphorothioate and mixed backbone
oligonucleotides, inhibit the expression of SGLT2 in vivo in a
dose-dependent manner. However, treatment with the mixed backbone
oligonucleotide, ISIS 257016, resulted in the greatest reduction of
target mRNA levels.
[0192] The levels of SGLT2 expression were also evaluated by
Northern blot analysis of both pooled and individual RNA samples,
to validate the target reduction observed by real-time PCR. Total
RNA was prepared from procured tissues of sacrificed mice by
homogenization in GITC buffer (Invitrogen, Carlsbad, Calif.)
containing 2-mercaptoethanol (Sigma-Aldrich, St. Louis, Mo.)
following manufacturer's recommended protocols followed by
ultracentrifugation through a CsCl cushion. Twenty micrograms of
total RNA was fractionated by electrophoresis through 1.2% agarose
gels containing 1.1% formaldehyde using a MOPS buffer system
(AMRESCO, Inc. Solon, Ohio). RNA was transferred from the gel to
HYBOND.TM.-N+ nylon membranes (Amersham Pharmacia Biotech,
Piscataway, N.J.) by overnight capillary transfer. RNA transfer was
confirmed by UV visualization. Membranes were fixed by UV
cross-linking using a STRATALINKER.TM. UV Crosslinker 2400
(Stratagene, Inc, La Jolla, Calif.) and then probed using
RapidHYB.TM. hybridization solution (Amersham Pharmacia Biotech,
Piscataway, N.J.) using manufacturer's recommendations for
stringent conditions.
[0193] To detect mouse SGLT2, a mouse SGLT2 specific template was
prepared by PCR using the forward primer
5'-ATGGAGCAACACGTAGAGGCAGGCT-3' (SEQ ID NO: 15) and the reverse
primer 5'-GAGTGCCGCCAGCCCTCCTGTCACA-3' (SEQ ID NO: 16) and gel
purified. The probe was prepared by asymmetric PCR with the
purified template and the reverse primer incorporating .sup.32P CTP
to label the probe. Following hybridization blots were exposed
overnight to phosphorimager screens (Molecular Dynamics, Amersham)
and quantitated. To normalize for variations in loading and
transfer efficiency membranes were stripped and probed for mouse
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) RNA (Clontech,
Palo Alto, Calif.).
[0194] For pooled sample analysis, equal amounts of RNA isolated
from the kidneys of mice in the same treatment was combined for a
total of 20 .mu.g, and the pooled sample was subjected to Northern
blot analysis. The results of the pooled sample analysis are shown
in Table 6 and are normalized to saline controls ("+" indicates an
increase, "-" indicates a decrease). TABLE-US-00007 TABLE 6
Northern Analysis of SGLT2 message in pooled kidney RNA samples
Percent change in SGLT2 expression Dose of relative to saline
oligonucleotide ISIS ISIS mg/kg 145733 257016 6.25 +21 -57 12.5 +7
-50 25 -35 -75 50 -35 -82
[0195] These results demonstrate that, as determined by Northern
blot analysis of pooled samples, ISIS 257016 inhibits SGLT2
expression inhibits SGLT2 expression at all doses of antisense
compound in a dose-dependent manner, whereas ISIS 145733 inhibits
SLGT2 expression at the two highest doses of antisense
compound.
[0196] Target levels in kidney RNA samples from individual mice
were also measured by Northern blot analysis. Equal amounts of RNA
were individually subjected to Northern blot analysis to determine
the level of SGLT2. Target level measurements for each treatment
group were then averaged. The results are shown in Table 7 and are
normalized to saline controls ("-" indicates a decrease).
TABLE-US-00008 TABLE 7 Northern Analysis of SGLT2 message in
individually measured RNA samples Percent change in SGLT2
expression Dose of relative to saline oligonucleotide ISIS mg/kg
ISIS 145733 257016 6.25 -34 -66 12.5 -38 -68 25 -39 -74 50 -59
-82
[0197] These results again show that treatment with mixed backbone
compound ISIS 257016 results in greater inhibition of SGLT2 mRNA
expression in the kidney relative to treatment with a full
phosphorothioate backbone compound.
[0198] Treated mice were further evaluated at the end of the
treatment period for total body, liver and spleen weight. The data
are expressed as percent change in body or organ weight ("+"
indicates an increase, "-" indicates a decrease). The results are
presented in Table 8. TABLE-US-00009 TABLE 8 Effects of modified
antisense compounds on total body weight, liver weight and spleen
weight in mice Percent change in weight ISIS ISIS Dose of 145733
257016 oligonucleotide Total Total mg/kg Body Liver Spleen Body
Liver Spleen 6.25 -4 -10 -12 -1 -3 +1 12.5 -6 -2 -7 -3 -13 -9 25 1
-1 +10 1 -8 +8 50 -1 +6 +10 -3 -9 +12
[0199] These data demonstrate that no significant changes in total
body, liver or spleen weights are observed following treatment with
ISIS 145733 or ISIS 257016 at 4 different doses. No changes in
total body weight were observed during the treatment period, or at
study termination.
[0200] In addition to the indicators of toxicity listed in Example
14, creatinine levels are also used to evaluate renal function. In
accordance with the present invention, the levels of ALT, AST,
total bilirubin, creatinine, BUN, glucose and triglyceride were
measured in mice treated with the compounds of the invention. Serum
was analyzed by LabCorp Testing Facility (San Diego, Calif.). The
results are expressed as units measured and are shown in Table 9.
TABLE-US-00010 TABLE 9 Effects of modified antisense compounds
targeting SGLT2 on liver and kidney function in mice Units measured
per treatment and dose Normal 145733 145733 257016 257016 Serum
indicator Range Saline 25 mg/kg 50 mg/kg 25 mg/kg 50 mg/kg BUN
15-40 24 24 25 26 26 mg/dL Creatinine 0.0-1.0 0.1 0.1 0.1 0.1 0.1
mg/L Bilirubin mg/dL 0.1-1.0 0.1 0.1 0.1 0.1 0.1 AST 30-300 77 65
96 133 141 IU/L ALT 30-200 24 18 22 34 35 IU/L Triglycerides
25-100* 165 169 230 130 111 mg/dL Glucose 80-150* 236 280 256 244
248 mg/dL *Triglyceride and glucose levels are routinely higher in
the Balb/c strain of mice than in other strains of mice.
[0201] The ALT levels in animals treated with either 25 mg/kg or 50
mg/kg of ISIS 145733 are slightly below the normal range, as is the
ALT level for saline treated mice. Otherwise, the levels of routine
clinical indicators of liver and kidney injury and disease are
within normal ranges and are not significantly changed relative to
saline-treated animals, demonstrating that the compounds of the
invention do not significantly affect renal or hepatic function.
Triglyceride and glucose levels, while outside the normal range as
is common in the Balb/c strain, are not significantly elevated
relative to saline-treated animals.
[0202] Mice injected with ISIS 145733 and ISIS 257016 at doses from
6.25 to 50 mg/kg also were evaluated histologically following
routine procedures. Liver and kidney samples were procured, fixed
in 10% neutral-buffered formalin and processed for staining with
hematoxylin and eosin or with the anti-oligonucleotide IgG1
antibody 2E1-B5 (Berkeley Antibody Company, Berkeley, Calif.) as
described in other examples herein. Hematoxylin and eosin staining
exhibited no significant difference between saline- and
oligonucleotide-treated animals. 2E1 staining showed no detectable
oligonucleotide in the liver, while staining was observed in the
kidney proximal tubules. Staining intensity lessened concomitantly
with a decrease in oligonucleotide dose.
[0203] The results illustrated in this example demonstrate that
antisense compounds of different chemistries are delivered to the
kidney, reduce SGLT2 expression in vivo in a dose-dependent manner,
and that treatment with these compounds does not result in liver or
kidney toxicity. The results further demonstrate that mixed
backbone compound ISIS 257016 is particularly effective at reducing
target mRNA levels in the kidney.
Example 16
Antisense Inhibition of SGLT2 mRNA Expression In Vivo
A Second Dose Response Study Comparing Mixed Backbone and Full
Phosphorothioate Backbones
[0204] ISIS 145733 and ISIS 257016 were selected for a dose
response study in mice using two identical and two lower doses with
respect to the doses used in Example 15.
[0205] Male 8-week old Balb/c mice (Charles River Laboratories,
Wilmington, Mass.) were given intraperitoneal injections of ISIS
145733 or ISIS 257016 at doses of 1, 5, 25 or 50 mg/kg twice per
week for two weeks. Saline-injected animals served as controls.
Each treatment group contained 4 mice. The mice were sacrificed 2
days following administration of the fourth and final dose of
oligonucleotide or saline.
[0206] Mice were evaluated for SGLT2 levels in kidney and liver.
Target levels were determined by quantitative real-time PCR as
described by other examples herein. PCR results were normalized to
cyclophilin. The data are expressed as percent change relative to
saline treated animals ("+" indicates an increase, "-" indicates a
decrease) and are illustrated in Table 10. TABLE-US-00011 TABLE 10
Antisense inhibition of SGLT2 mRNA expression in vivo by antisense
compounds with varying chemistries Percent change in SGLT2
expression relative to saline Dose of Kidney Liver oligonucleotide
ISIS ISIS ISIS ISIS mg/kg 145733 257016 145733 257016 1 +2 -46 -19
+13 5 -15 -64 -39 +1 25 -34 -74 -21 -5 50 -40 -76 -59 -12
[0207] These results illustrate that the compounds of the
invention, both full phosphorothioate and mixed backbone
oligonucleotides, can inhibit the expression of kidney SGLT2 in a
dose-dependent manner. Greater inhibition is observed in kidneys
from mice treated with ISIS 257016, a mixed backbone antisense
compound. SGLT2 is not highly expressed in liver, therefore target
levels are low before treatment and therefore more difficult to
accurately measure. ISIS 145733 and ISIS 257016 lowered liver SGLT2
expression, with 145733 having a greater effect in liver than the
mixed backbone ISIS 257016.
[0208] Treated mice were further evaluated for liver and spleen
weight. The data are expressed as percent change in body or organ
weight ("+" indicates an increase, "-" indicates a decrease). The
results are presented in Table 11. TABLE-US-00012 TABLE 11 Effects
of antisense compounds on total body weight, liver weight and
spleen weight in mice Percent change in weight ISIS ISIS Dose of
145733 257016 oligonucleotide Total Total mg/kg Body Liver Spleen
Body Liver Spleen 1 0 -6 +10 -2 -8 +13 5 +3 +1 +10 -3 -9 +5 25 -1
+2 -4 +2 +2 +12 50 -1 +13 +35 -2 -6 +15
[0209] No significant change was observed in total body weight at
timepoints throughout or at the termination of the study. All
changes in liver and spleen weight were within the margin of error
for the data and are therefore not significant.
[0210] In addition to the other serum markers described herein,
cholesterol levels can be used as a measure of toxicity. In
accordance with the present invention, the levels of ALT, AST,
total bilirubin, albumin, creatinine, BUN, triglyceride,
cholesterol and glucose were measured in mice treated with the
compounds of the invention. Plasma samples were analyzed using the
Olympus AU400e automated chemistry analyzer (Olympus America,
Irving, Tex.). The results, expressed as units measured, are shown
for ISIS 145733 in Table 12 and for ISIS 257016 in Table 13.
TABLE-US-00013 TABLE 12 Effect of the full phosphorothioate
antisense compound ISIS 145733 on indicators of liver and kidney
function Units measured per Normal dose of ISIS 145733 Serum
indicator Range Saline 1 mg/kg 5 mg/kg 25 mg/kg 50 mg/kg BUN 15-40
27 31 31 30 25 mg/dL Creatinine 0.0-1.0 0.2 0.2 0.2 0.2 0.2 mg/L
Bilirubin mg/dL 0.1-1.0 0.3 0.2 0.1 0.3 0.1 AST 30-300 92 91 45 133
56 IU/L ALT 30-200 35 27 26 37 31 IU/L Albumin 2.5-4.0 3 3 3 3 3
g/dL Triglycerides 25-100* 136 188 183 153 224 mg/dL Cholesterol
70-125 122 116 117 120 132 mg/dL Glucose 80-150* 208 202 173 170
161 mg/dL
[0211] TABLE-US-00014 TABLE 13 Effect of the mixed backbone
antisense compound ISIS 257016 on indicators of liver and kidney
function Units measured per Normal dose of ISIS 257016 Serum
indicator Range Saline 1 mg/kg 5 mg/kg 25 mg/kg 50 mg/kg BUN 15-40
27 23 29 25 28 mg/dL Creatinine 0.0-1.0 0.2 0.2 0.2 0.2 0.2 mg/L
Bilirubin mg/dL 0.1-1.0 0.3 0.2 0.2 0.2 0.2 AST 30-300 92 74 73 99
138 IU/L ALT 30-200 35 34 34 46 48 IU/L Albumin 2.5-4.0 3 3 3 3 3
g/dL Triglycerides 25-100* 136 271 233 225 136 mg/dL Cholesterol
70-125 122 116 124 144 137 mg/dL Glucose 80-150* 208 180 178 154
182 mg/dL *Triglyceride and glucose levels are routinely higher in
the Balb/c strain of mice than in other strains of mice.
[0212] Cholesterol levels of animals treated with either 25 or 50
mg/kg of ISIS 257016 were slightly above the normal range; however;
they are not significantly greater than saline control animals
given the margin of error for the experiment. The levels of routine
clinical indicators of liver and kidney injury and disease are
within normal ranges and are not significantly changed relative to
saline-treated animals, demonstrating that the compounds of the
invention do not significantly affect renal or hepatic function.
Triglyceride and glucose levels, while outside the normal range as
is common in the Balb/c strain, are not significantly elevated
relative to saline-treated animals.
[0213] Mice injected ISIS 145733 and ISIS 257016 at 1-50 mg/kg also
were evaluated histologically following routine procedures. Liver
and kidney samples were procured, fixed in 10% neutral-buffered
formalin and processed for staining with hematoxylin and eosin or
with the anti-oligonucleotide IgG1 antibody 2E1-B5 (Berkeley
Antibody Company, Berkeley, Calif.). Hematoxylin and eosin staining
in most tissues exhibited no significant difference between saline-
and 145733-treated animals, with the exception of slight
inflammatory cell infiltration in the liver tissue. Livers from
mice treated with ISIS 257016 showed evidence of nuclear 20
degradation and mitosis at 50 mg/kg and slight mitosis at 25 mg/kg.
Kidneys from mice treated with ISIS 257016 exhibited no significant
differences compared to saline-treated kidneys. Staining with the
2E1 antibody showed weak staining in liver and kidneys from animals
treated with ISIS 145733, whereas staining was strong in liver and
kidney from animals treated with ISIS 257016. Kidney 2E1 staining
appears in a punctate pattern.
[0214] The results illustrated in this example demonstrate that
antisense compounds of different chemistries are delivered to the
kidney, reduce SGLT2 expression in vivo in a dose-dependent manner,
and that treatment with these compounds does not result in liver or
kidney toxicity. The results further demonstrate that mixed
backbone compound ISIS 257016 is particularly effective, even at
low doses, at reducing target mRNA levels in the kidney.
Example 17
Dose Response Study Comparing Mixed Backbone and Full
Phosphorothioate Backbones
A Second SGLT2 Antisense Sequence
[0215] A second mixed backbone SGLT2 oligonucleotide, ISIS 341699
(SEQ ID NO: 5), and control phosphorothioate SGLT2 oligonucleotide,
ISIS 145742 (SEQ ID NO: 5), were selected for a dose response study
in mice. For comparison, ISIS 257016 (mixed backbone) also was
included in this study.
[0216] Male 8-week old Balb/c mice (Charles River Laboratories,
Wilmington, Mass.) were given intraperitoneal injections of ISIS
341699, ISIS 145742 or ISIS 257016 twice per week for two weeks
with the doses shown in Table 14. Saline-injected animals served as
controls. Each treatment group contained 4 mice. The mice were
sacrificed 2 days following administration of the fourth and final
dose of oligonucleotide or saline.
[0217] Mice were evaluated for SGLT2 levels in kidney. Target
levels were determined by quantitative real-time PCR as described
by other examples herein. PCR results were normalized to
cyclophilin. The data are expressed as percent change relative to
saline treated animals ("+" indicates an increase, "-" indicates a
decrease) and are illustrated in Table 14. TABLE-US-00015 TABLE 14
Antisense inhibition of SGLT2 mRNA expression in vivo by mixed
backbone and full phosphorothioate oligonucleotides (expressed as
percent change in SGLT2 mRNA expression relative to saline) Dose of
oligonucleotide ISIS ISIS ISIS mg/kg 145742 341699 257016 0.2 -- --
-18.9 1 -- -1.8 -50.5 5 -0.6 -10.9 -56.7 25 -24.9 -23.9 -- 50 -32.6
-- --
[0218] These results illustrate that the compounds of the
invention, both full phosphorothioate and mixed backbone
oligonucleotides, can inhibit the expression of kidney SGLT2 in a
dose-dependent manner. However, lower doses of the mixed backbone
compound are required to inhibit SGLT2 expression in kidneys from
treated mice.
[0219] Treated mice were further evaluated for liver and spleen
weight. The data are expressed as percent change in body or organ
weight ("+" indicates an increase, "-" indicates a decrease). The
results are presented in Table 15 and Table 16. TABLE-US-00016
TABLE 15 Effects of antisense compounds on total body weight of
mice (expressed as percent change in body weight) Dose of
oligonucleotide ISIS ISIS ISIS mg/kg 145742 341699 257016 0.2 -- --
+7.9 1 -- +5.7 +5.8 5 +5.0 +5.8 +3.2 25 +2.0 +2.5 -- 50 +7.2 --
--
[0220] TABLE-US-00017 TABLE 16 Effects of antisense compounds on
liver weight and spleen weight of mice (expressed as percent change
in organ weight) Dose of Liver Spleen oligonucleotide ISIS ISIS
ISIS ISIS ISIS ISIS mg/kg 145742 341699 257016 145742 341699 257016
0.2 -- -- -6.0 -- -- -4.7 1 -- +2.3 +14.9 -- -4.2 +1.4 5 +7.1 +2.2
+7.0 +10.6 -2.8 -7.6 25 +7.2 +5.8 -- +0.8 -0.2 -- 50 +12.1 -- --
+9.4 -- --
[0221] No significant change was observed in total body weight,
liver weight or spleen weight at timepoints throughout or at the
termination of the study.
[0222] Levels of BUN, creatinine, AST, ALT, albumin, triglycerides,
cholesterol and glucose were measured in mice treated with the
compounds of the invention. Plasma samples were analyzed using the
Olympus AU400e automated chemistry analyzer (Olympus America,
Irving, Tex.). The results, expressed as units measured, are shown
for ISIS 145742 in Table 17, ISIS 341699 in Table 18 and ISIS
257016 in Table 19. TABLE-US-00018 TABLE 17 Effect of the full
phosphorothioate antisense compound ISIS 145742 on indicators of
liver and kidney function Units measured per Normal dose of ISIS
145742 Serum indicator Range Saline 5 mg/kg 25 mg/kg 50 mg/kg BUN
15-40 20 21.3 25.5 20.8 mg/dL Creatinine 0.0-1.0 0.1 0.2 0.2 0.2
mg/L AST 30-300 113 75.3 83.5 145.3 IU/L ALT 30-200 35.5 29.8 40.3
47.5 IU/L Albumin 2.5-4.0 3.0 3.0 2.9 2.9 g/dL Triglycerides
25-100* 223.8 176.5 192 176.8 mg/dL Cholesterol 70-125 129 119.5
119.5 113.5 mg/dL Glucose 80-150* 176.5 196.5 192 194.8 mg/dL
[0223] TABLE-US-00019 TABLE 18 Effect of mixed backbone antisense
compound ISIS 341699 on indicators of liver and kidney function
Units measured per Normal dose of ISIS 341699 Serum indicator Range
Saline 1 mg/kg 5 mg/kg 25 mg/kg BUN 15-40 20 20 21.8 22 mg/dL
Creatinine 0.0-1.0 0.1 0.2 0.2 0.2 mg/L AST 30-300 113 78.2 119
64.8 IU/L ALT 30-200 35.5 36.2 37.3 33.0 IU/L Albumin 2.5-4.0 3.0
3.3 3.1 3.2 g/dL Triglycerides 25-100* 223.8 206.4 186.8 183.5
mg/dL Cholesterol 70-125 129 135 124 120.8 mg/dL Glucose 80-150*
176.5 203.2 171.5 197 mg/dL
[0224] TABLE-US-00020 TABLE 19 Effect of mixed backbone antisense
compound ISIS 257016 on indicators of liver and kidney function
Units measured per Serum dose of ISIS 257016 indicator Normal Range
Saline 0.2 mg/kg 1 mg/kg 5 mg/kg BUN 15-40 20 21.8 26.3 20.5 mg/dL
Creatinine 0.0-1.0 0.1 0.2 0.2 0.2 mg/L AST 30-300 113 123.8 85.3
69.5 IU/L ALT 30-200 35.5 36.8 44 43 IU/L Albumin 2.5-4.0 3.0 3.1
3.4 3.1 g/dL Triglycerides 25-100* 223.8 138.8 268.3 212.8 mg/dL
Cholesterol 70-125 129 128 152 135.3 mg/dL Glucose 80-150* 176.5
208.8 212.3 164.5 mg/dL *Triglyceride and glucose levels are
routinely higher in the Balb/c strain of mice than in other strains
of mice.
[0225] In some oligonucleotide-treated animals cholesterol levels
were above the normal range; however, this elevation is not
significant since saline-treated animals also exhibited cholesterol
above the normal range. The levels of the remaining routine
clinical indicators of liver and kidney injury and disease are
within normal ranges and are not significantly changed relative to
saline-treated animals, demonstrating that the compounds of the
invention do not significantly affect renal or hepatic function.
Triglyceride and glucose levels, while outside the normal range as
is common in the Balb/c strain, are not significantly elevated
relative to saline-treated animals.
[0226] Mice injected with ISIS 145742, ISIS 341699 and ISIS 257016
at 0.2-50 mg/kg were also evaluated histologically following
routine procedures. Liver and kidney samples were procured, fixed
in 10% neutral-buffered formalin and processed for staining with
hematoxylin and eosin or with the anti-oligonucleotide IgG1
antibody 2E1-B5, as described in other examples herein.
[0227] Hematoxylin and eosin staining in both liver and kidney
tissues exhibited no significant difference between saline- and
antisense oligonucleotide-treated animals. Staining with the 2E1
antibody showed high background in sinusoidal tissues of liver from
the saline-injected animals, therefore making it difficult to
interpret positive staining in the oligonucleotide-treated livers.
Kidney samples from saline-injected animals and animals treated
with 0.2 mg/kg ISIS 257016 showed no positive oligonucleotide
staining; however, the remainder of the oligonucleotide-treated
animals demonstrated high levels of staining in the proximal
tubules, which increased with dose.
[0228] The results illustrated in this example demonstrate that
antisense compounds of different chemistries are delivered to the
kidney, reduce SGLT2 expression in vivo in a dose-dependent manner,
and that treatment with these compounds does not result in liver or
kidney toxicity. The results further demonstrate that mixed
backbone compounds ISIS 341699 and ISIS 257016 are particularly
effective at reducing target mRNA levels in the kidney.
Example 18
Dose Response Study Comparing Mixed Backbone and Full
Phosphorothioate Backbones
A Third SGLT2 Antisense Sequence
[0229] A third mixed backbone SGLT2 oligonucleotide, ISIS 351642
(SEQ ID NO: 6), and control phosphorothioate SGLT2 oligonucleotide,
ISIS 145746 (SEQ ID NO: 6), were selected for a dose response study
in mice.
[0230] Male 7-week old Balb/c mice (Charles River Laboratories,
Wilmington, Mass.) were given intraperitoneal injections of ISIS
145746 or ISIS 351642 twice per week for two weeks with the doses
shown in Table 20. Saline-injected animals served as controls. Each
treatment group contained 4 mice. The mice were sacrificed 2 days
following administration of the fourth and final dose of
oligonucleotide or saline.
[0231] Mice were evaluated of SGLT2 levels in kidney. Target levels
were determined by quantitative real-time PCR as described by other
examples herein. PCR results were normalized to cyclophilin. The
data are expressed as percent change relative to saline treated
animals ("+" indicates an increase, "-" indicates a decrease) and
are illustrated in Table 20. TABLE-US-00021 TABLE 20 Antisense
inhibition of SGLT2 mRNA expression in vivo by mixed backbone and
full phosphorothioate oligonucleotides (expressed as percent change
in SGLT2 mRNA expression relative to saline) Dose of
oligonucleotide ISIS ISIS mg/kg 145746 351642 1 -- -26.7 5 -5.8
-35.1 25 -10.5 -44.3 50 -35.6 -31.8
[0232] These illustrate that the compounds of the invention, both
full phosphorothioate and mixed backbone oligonucleotides, can
inhibit the expression of kidney SGLT2 in a dose-dependent manner.
At doses of 5 and 25 mg/kg, greater inhibition is observed in
kidneys from mice treated with ISIS 351463, suggesting the mixed
backbone antisense compound is a more efficient inhibitor of target
mRNA expression in the kidney.
[0233] Treated mice were further evaluated for body weight, liver
weight and spleen weight. The data are expressed as present change
in body or organ weight ("+" indicates an increase, "-" indicates a
decrease). The results are presented in Table 21. TABLE-US-00022
TABLE 21 Effects of antisense compounds on total body weight, liver
weight and spleen weight of mice Percent change in weight ISIS ISIS
Dose of 145746 351642 oligonucleotide Total Total mg/kg Body Liver
Spleen Body Liver Spleen 1 -- -- -- +6.9 -8.2 +0.8 5 +3.6 -5.7 +6.5
+4.6 -0.6 -7.9 25 +5.4 -2.0 +3.7 +4.7 -10.6 +1.1 50 +12.1 -8.4
+10.0 +7.4 -3.0 +1.3
[0234] No significant change was observed in total body weight,
liver weight or spleen weight at timepoints throughout or at the
termination of the study.
[0235] Levels of BUN, creatinine, AST, ALT, albumin, triglycerides,
cholesterol and glucose were measured in mice treated with the
compounds of the invention. Plasma samples were analyzed using the
Olympus AU400e automated chemistry analyzer (Olympus America,
Irving, Tex.). The results, expressed as units measured, are shown
for ISIS 145746 in Table 22 and ISIS 351642 in Table 23.
TABLE-US-00023 TABLE 22 Effect of the full phosphorothioate
antisense compound ISIS 145746 on indicators of liver and kidney
function Units measured per dose of ISIS 145746 Serum indicator
Normal Range Saline 1 mg/kg 5 mg/kg 25 mg/kg 50 mg/kg Creatinine
0.0-1.0 0.1 -- 0.2 0.2 0.1 mg/L AST 30-300 129 -- 60 84 155 IU/L
ALT 30-200 30 -- 28 26 77 IU/L Albumin 2.5-4.0 2.8 -- 2.9 2.8 2.9
g/dL Triglycerides 25-100* 298 -- 268 259 236 mg/dL Cholesterol
70-125 116 -- 118 108 106 mg/dL Glucose 80-150* 163 -- 162 181 179
mg/dL
[0236] TABLE-US-00024 TABLE 23 Effect of mixed backbone antisense
compound ISIS 351642 on indicators of liver and kidney function
Units measured per dose of ISIS 351642 Serum indicator Normal Range
Saline 1 mg/kg 5 mg/kg 25 mg/kg 50 mg/kg Creatinine 0.0-1.0 0.1 0.1
0.1 0.2 0.2 mg/L AST 30-300 129 132 75 131 160 IU/L ALT 30-200 30
31 28 29 31 IU/L Albumin 2.5-4.0 2.8 2.9 3.0 2.7 2.8 g/dL
Triglycerides 25-100* 298 238 287 240 233 mg/dL Cholesterol 70-125
116 117 122 106 113 mg/dL Glucose 80-150* 163 195 175 164 171 mg/dL
*Triglyceride and glucose levels are routinely higher in the Balb/c
strain of mice than in other strains of mice.
[0237] The levels of routine clinical indicators of liver and
kidney injury and disease are within normal ranges and are not
significantly changed relative to saline-treated animals,
demonstrating that the compounds of the invention do not
significantly affect renal or hepatic function. Triglyceride and
glucose levels, while outside the normal range as is common in the
Balb/c strain, are not significantly elevated relative to
saline-treated animals.
[0238] The results illustrated in this example demonstrate that
antisense compounds of different chemistries are delivered to the
kidney, reduce SGLT2 expression in vivo in a dose-dependent manner,
and that treatment with these compounds does not result in liver or
kidney toxicity. The results further suggest that mixed backbone
compound ISIS 351642 is more effective than full phosphorothioate
oligonucleotides at reducing target mRNA levels in the kidney,
particularly at low doses.
Example 19
Comparison of a Standard Mixed Backbone Compound and a Mixed
Backbone Compound with Phosphorothioate Linkages at the Extreme 5'
and 3' Ends
A Single Dose Study
[0239] In accordance with the present invention, ISIS 257016 and
ISIS 351641 were analyzed for their ability to inhibit SGLT2
expression in vivo. ISIS 257016 is a standard mixed backbone
compound having 2'-MOE wings and a deoxy gap, with phosphodiester
linkages in the wings and phosphorothioate linkages in the gap.
ISIS 351641 differs from the standard mixed backbone compounds by
having one phosphorothioate linkage at each of the extreme 5' and
3' ends of the wings.
[0240] Male 8-week old Balb/c mice (Charles River Laboratories,
Wilmington, Mass.) were given a single intraperitoneal injection of
ISIS 257016 or ISIS 351641 at a dose of 1, 5, 25, or 50 mg/kg.
Saline-injected animals served as controls. Each treatment group
contained 4 mice. The mice were sacrificed 2 days following
administration of the single dose of oligonucleotide or saline.
[0241] Mice were evaluated for SGLT2 levels in kidney. Target
levels were determined by quantitative real-time PCR as described
by other examples herein. PCR results were normalized to
cyclophilin. The data are expressed as percent change relative to
saline treated animals ("+" indicates an increase, "-" indicates a
decrease) and are illustrated in Table 24. TABLE-US-00025 TABLE 24
Antisense inhibition of SGLT2 mRNA expression in vivo by mixed
backbone oligonucleotides (expressed as percent change in SGLT2
mRNA expression relative to saline) Dose of oligonucleotide ISIS
ISIS mg/kg 257016 351641 1 -21.5 -14.0 5 -26.4 -19.3 25 -24.2 -12.5
50 -36.3 -22.0
[0242] These results illustrate that mixed backbone compounds of
the invention, with either complete phosphodiester linkages in the
wings, or with the extreme 5' and 3' ends substituted with
phosphorothioate linkages, inhibit the expression of kidney SGLT2
in a dose-dependent manner. However, greater inhibition is observed
in kidneys from mice treated with ISIS 257016, which contains all
phosphodiester linkages in the wings.
[0243] Treated mice were further evaluated for body weight and
liver and spleen weight. The data are expressed as percent change
in body or organ weight ("+" indicates an increase, "-" indicates a
decrease). The results are presented in Table 25. TABLE-US-00026
TABLE 25 Effects of antisense compounds on total body weight, liver
weight and spleen weight of mice Percent change in weight ISIS ISIS
Dose of 257016 351641 oligonucleotide Total Total mg/kg Body Liver
Spleen Body Liver Spleen 1 -0.9 +1.2 -1.6 +2.8 +3.0 -0.1 5 -5.1
+5.4 +20.1 +4.0 +2.1 +9.7 25 -1.1 +3.5 +3.8 -0.7 +9.3 +5.9 50 -2.5
-2.3 +7.8 +0.9 -0.7 +10.2
[0244] No significant change was observed in total body weight,
liver weight or spleen weight at time points throughout or at the
termination of the study.
[0245] Levels of creatinine, AST, ALT, albumin, triglycerides,
cholesterol and glucose were measured in mice treated with the
compounds of the invention. Plasma samples were analyzed using the
Olympus AU400e automated chemistry analyzer (Olympus America,
Irving, Tex.). The results, expressed as units measured, are shown
for ISIS 257016 in Table 26 and for ISIS 351641 in Table 27.
TABLE-US-00027 TABLE 26 Effect of mixed backbone antisense compound
ISIS 257016 on indicators of liver and kidney function Units
measured per dose of ISIS 257016 Serum indicator Normal Range
Saline 1 mg/kg 5 mg/kg 25 mg/kg 50 mg/kg Creatinine 0.0-1.0 0.0 0.0
0.0 0.0 0.2 mg/L AST 30-300 141 62 77 89 88 IU/L ALT 30-200 30 29
28 27 33 IU/L Albumin 2.5-4.0 2.9 2.8 2.8 3.0 2.9 g/dL
Triglycerides 25-100* 213 253 255 347 245 mg/dL Cholesterol 70-125
118 111 116 125 120 mg/dL Glucose 80-150* 155 186 172 174 169
mg/dL
[0246] TABLE-US-00028 TABLE 27 Effect of mixed backbone antisense
compound ISIS 351641 on indicators of liver and kidney function
Units measured per dose of ISIS 351641 Serum indicator Normal Range
Saline 1 mg/kg 5 mg/kg 25 mg/kg 50 mg/kg Creatinine 0.0-1.0 0.0 0.2
0.1 0.1 0.2 mg/L AST 30-300 141 75 117 68 98 IU/L ALT 30-200 30 25
33 30 27 IU/L Albumin 2.5-4.0 2.9 2.9 2.9 2.9 2.9 g/dL
Triglycerides 25-100* 213 271 280 296 271 mg/dL Cholesterol 70-125
118 120 126 112 117 mg/dL Glucose 80-150* 155 162 171 189 175 mg/dL
*Triglyceride and glucose levels are routinely higher in the Balb/c
strain of mice than in other strains of mice.
[0247] The levels of routine clinical indicators of liver and
kidney injury and disease are within normal ranges and are not
significantly changed relative to saline-treated animals,
demonstrating that the compounds of the invention do not
significantly affect renal or hepatic function. Triglyceride and
glucose levels, while outside the normal range as is common in the
Balb/c strain, are not significantly elevated relative to
saline-treated animals.
[0248] The results illustrated in this example demonstrate that
mixed backbone compounds of varying chemistries are delivered to
the kidney, reduce SGLT2 expression in vivo, and that treatment
with these compounds does not result in liver or kidney toxicity.
The results further indicate that mixed backbone compounds with
wings composed completely of phosphodiester linkages are more
efficient inhibitors of target mRNA.
Example 20
Effects of Modified Antisense Compounds on SGLT2 mRNA Expression In
Vivo
Two and Three Dose Protocols
[0249] In accordance with the present invention, mixed backbone
compound ISIS 257016 was analyzed for its ability to inhibit SGLT2
expression in vivo when administered in either two or three doses.
ISIS 353003 (CCTTCCCTGAAGGTTCCTCC; SEQ ID NO: 17), a mixed backbone
oligonucleotide which targets human PTP1B, was used as a
control.
[0250] Male 8-week old Balb/c mice (Charles River Laboratories,
Wilmington, Mass.) were given two or three intraperitoneal
injections of ISIS 257016 or ISIS 353003 at three day intervals.
ISIS 257016 was administered at doses of 1, 5 or 25 mg/kg and ISIS
353003 was administered at a dose of 25 mg/kg. Saline-injected
animals served as controls. Each treatment group contained 4 mice.
The mice were sacrificed 2 days following administration of the
final dose of oligonucleotide or saline.
[0251] Mice were evaluated for SGLT2 levels in kidney. Target
levels were determined by quantitative real-time PCR as described
in other examples herein. PCR results were normalized to
cyclophilin. The data are expressed as percent change relative to
saline treated animals ("+" indicates an increase, "-" indicates a
decrease) and are illustrated in Table 28. TABLE-US-00029 TABLE 28
Antisense inhibition of SGLT2 mRNA expression in vivo by two doses
or three doses of mixed backbone oligonucleotides (expressed as
percent change in SGLT2 mRNA expression relative to saline control)
Oligonucleotide Three (dose in mg/kg) Two Doses Doses ISIS 257016
(1 mg/kg) -43.2 -39.1 ISIS 257016 (5 mg/kg) -39.7 -42.9 ISIS 257016
(25 mg/kg) -53.8 -65.5 ISIS 353003 (25 mg/kg) -8.0 -6.9
[0252] These results illustrate that the mixed backbone compounds
of the invention efficiently inhibit the expression of kidney SGLT2
in a dose-dependent manner. Furthermore, inhibition increases with
the number of doses administered.
[0253] Treated mice were further evaluated for body weight, kidney
weight, liver weight and spleen weight. The data are expressed as
percent change in body or organ weight ("+" indicates an increase,
"-" indicates a decrease). The results are presented in Table 29
and Table 30. TABLE-US-00030 TABLE 29 Effects of antisense
compounds on total body weight of mice (expressed as percent change
in body weight) Oligonucleotide Two Three (dose in mg/kg) Doses
Doses ISIS 257016 (1 mg/kg) -1.1 0 ISIS 257016 (5 mg/kg) +1.3 +0.8
ISIS 257016 (25 mg/kg) +0.1 +1.3 ISIS 353003 (25 mg/kg) -0.8
+0.8
[0254] TABLE-US-00031 TABLE 30 Effects of antisense compounds on
total kidney weight, liver weight and spleen weight of mice Oligo-
Percent change in weight nucleotide Two Doses Three Doses (dose in
mg/kg) Kidney Liver Spleen Kidney Liver Spleen ISIS 257016 -0.5
-2.2 -4.3 -5.6 -3.8 -5.9 (1 mg/kg) ISIS 257016 -5.4 +2.5 +7.4 -6.6
-7.1 -9.0 (5 mg/kg) ISIS 257016 -7.9 -1.1 +4.2 -8.6 -8.8 -1.2 (25
mg/kg) ISIS 353003 -5.5 +1.2 -2.7 -0.2 -4.0 +6.5 (25 mg/kg)
[0255] No significant change was observed in total body weight,
kidney weight, liver weight or spleen weight at timepoints
throughout or at the termination of the study.
[0256] Levels of BUN, creatinine, bilirubin, AST, ALT, albumin,
triglycerides, cholesterol and glucose were measured in mice
treated with the compounds of the invention. Plasma samples were
analyzed using the Olympus AU400e automated chemistry analyzer
(Olympus America, Irving, Tex.). The results, expressed as units
measured, are shown for the two dose protocol in Table 31 and for
the three dose protocol in Table 32. TABLE-US-00032 TABLE 31 Effect
of mixed backbone antisense compound ISIS 257016 administered
according to the two dose protocol on indicators of liver and
kidney function Units measured per dose of ISIS 257016 Serum
indicator Normal Range Saline 1 mg/kg 5 mg/kg 25 mg/kg ISIS 353003
BUN 15-40 32 34 29 25 28 mg/dL Creatinine 0.0-1.0 0.1 0.1 0.2 0.1
0.1 mg/L Bilirubin mg/dL 0.1-1.0 0.1 0.1 0.1 0.1 0.1 AST 30-300 54
119 156 116 154 IU/L ALT 30-200 27 36 45 30 36 IU/L Albumin 2.5-4.0
2.7 3.2 3.1 3.0 2.8 g/dL Triglycerides 25-100* 221 263 234 264 278
mg/dL Cholesterol 70-125 113 118 117 125 125 mg/dL Glucose 80-150*
170 157 177 163 152 mg/dL
[0257] TABLE-US-00033 TABLE 32 Effect of mixed backbone antisense
compound ISIS 257016 administered according to the three dose
protocol on indicators of liver and kidney function Units measured
per dose of ISIS 257016 Serum indicator Normal Range Saline 1 mg/kg
5 mg/kg 25 mg/kg ISIS 353003 BUN 15-40 30 32 30 27 27 mg/dL
Creatinine 0.0-1.0 0.1 0.1 0.2 0.1 0.1 mg/L Bilirubin mg/dL 0.1-1.0
0.1 0.1 0.1 0.1 0.1 AST 30-300 126 83 81 59 57 IU/L ALT 30-200 35
30 57 27 24 IU/L Albumin 2.5-4.0 3.0 2.8 2.8 2.7 2.8 g/dL
Triglycerides 25-100* 223 236 202 153 188 mg/dL Cholesterol 70-125
112 113 114 116 106 mg/dL Glucose 80-150* 152 169 161 181 192 mg/dL
*Triglyceride and glucose levels are routinely higher in the Balb/c
strain of mice than in other strains of mice.
[0258] The levels of routine clinical indicators of liver and
kidney injury and disease are within normal ranges and are not
significantly changed relative to saline-treated animals,
demonstrating that the compounds of the invention do not
significantly affect renal or hepatic function. Triglyceride and
glucose levels, while outside the normal range as is common in the
Balb/c strain, are not significantly elevated relative to
saline-treated animals.
[0259] Mice injected with ISIS 257016 and control animals were also
evaluated histologically following routine procedures. Liver and
kidney samples were procured, fixed in 10% neutral-buffered
formalin and processed for staining with hematoxylin and eosin.
Hematoxylin and eosin staining exhibited no significant difference
between saline- and oligonucleotide-treated animals. All tissue
samples exhibited normal kidney and liver morphology.
[0260] The results illustrated in this example demonstrate that
mixed backbone compounds are delivered to the kidney, reduce SGLT2
expression in vivo, and that treatment with these compounds does
not result in liver or kidney toxicity. The results further
indicate that inhibition of target mRNA expression in the kidney
increases with the number of doses administered.
Example 21
Effects of Mixed Backbone Antisense Compounds on SGLT2 mRNA
Expression In Vivo
Two to Five Day Consecutive Daily Dosing Protocols
[0261] In accordance with the present invention, mixed backbone
compound ISIS 257016 (SEQ ID NO: 4) was analyzed for its ability to
inhibit SGLT2 expression in vivo when administered in two to five
doses (consecutive daily doses). ISIS 353003 (SEQ ID NO: 17), a
mixed backbone oligonucleotide which targets human PTP1B, was used
as a control.
[0262] Male 9-week old Balb/c mice (Charles River Laboratories,
Wilmington, Mass.) were given two, three, four or five
intraperitoneal injections of ISIS 257016 or ISIS 353003 once a day
for the treatment period. ISIS 257016 was administered at doses of
2.5 or 25 mg/kg and ISIS 353003 was administered at a dose of 25
mg/kg. Saline-injected animals served as controls. Each treatment
group contained 4 mice. The mice were sacrificed 2 days following
administration of the final dose of oligonucleotide or saline.
[0263] Mice were evaluated for SGLT2 levels in kidney. Target
levels were determined by quantitative real-time PCR as described
in other examples herein. PCR results were normalized to
cyclophilin. The data are expressed as percent change relative to
saline treated animals ("+" indicates an increase, "-" indicates a
decrease) and are illustrated in Table 33. TABLE-US-00034 TABLE 33
Antisense inhibition of SGLT2 mRNA expression in vivo by mixed
backbone oligonucleotide (expressed as percent change in SGLT2 mRNA
expression relative to saline control) Oligonucleotide Three Five
(dose in mg/kg) Two Doses Doses Four Doses Doses ISIS 257016 (2.5
mg/kg) -14.2 -35.4 -25.3 -42.0 ISIS 257016 (25 mg/kg) -12.5 -32.9
-39.1 -68.9 ISIS 353003 (25 mg/kg) -4.5 -9.6 +0.5 -11.3
[0264] These results illustrate that the mixed backbone compounds
of the invention efficiently inhibit the expression of kidney SGLT2
and inhibition increases with the number of doses administered.
[0265] Treated mice were further evaluated for body weight, kidney
weight, liver weight and spleen weight. The data are expressed as
percent change in body or organ weight ("+" indicates an increase,
"-" indicates a decrease). The results are presented in Tables
34-37. TABLE-US-00035 TABLE 34 Effects of antisense compounds on
total body weight of mice (expressed as percent change in body
weight) Oligonucleotide Two Three Four Five (dose in mg/kg) Doses
Doses Doses Doses ISIS 257016 (2.5 mg/kg) +2.7 +2.7 +3.2 +1.5 ISIS
257016 (25 mg/kg) +2.0 +2.0 +3.1 -0.7 ISIS 353003 (25 mg/kg) +0.6
+0.8 +2.5 +1.3
[0266] TABLE-US-00036 TABLE 35 Effects of antisense compounds on
total kidney weight (expressed as percent change in kidney weight)
Oligonucleotide Two Three Four Five (dose in mg/kg) Doses Doses
Doses Doses ISIS 257016 (2.5 mg/kg) +8.2 -1.4 +8.9 +1.5 ISIS 257016
(25 mg/kg) +11.5 +3.6 +2.7 -7.7 ISIS 353003 (25 mg/kg) +5.3 -3.6
+4.9 +7.1
[0267] TABLE-US-00037 TABLE 36 Effects of antisense compounds on
total liver weight (expressed as percent change in liver weight)
Oligonucleotide Two Three Four Five (dose in mg/kg) Doses Doses
Doses Doses ISIS 257016 (2.5 mg/kg) +9.2 +7.5 +4.8 +4.8 ISIS 257016
(25 mg/kg) +11.8 +5.2 +0.6 -8.0 ISIS 353003 (25 mg/kg) +7.4 -3.4
+12.9 +9.5
[0268] TABLE-US-00038 TABLE 37 Effects of antisense compounds on
total spleen weight (expressed as percent change in spleen weight)
Oligonucleotide Two Three Four Five (dose in mg/kg) Doses Doses
Doses Doses ISIS 257016 (2.5 mg/kg) +22.2 +10.1 +15.3 +10.7 ISIS
257016 (25 mg/kg) +13.3 +5.1 +6.7 +4.5 ISIS 353003 (25 mg/kg) +7.3
+1.4 +19.8 +8.6
[0269] No significant change was observed in total body weight,
kidney weight, liver weight or spleen weight at timepoints
throughout or at the termination of the study.
[0270] Levels of creatinine, AST, ALT, albumin, triglycerides,
cholesterol and glucose were measured in mice treated with the
compounds of the invention. Plasma samples were analyzed using the
Olympus AU400e automated chemistry analyzer (Olympus America,
Irving, Tex.). The results, expressed as units measured, are shown
in Tables 38-41. TABLE-US-00039 TABLE 38 Effect of mixed backbone
antisense compound ISIS 257016 administered as two consecutive
daily doses on indicators of liver and kidney function Units
measured per dose of oligonucleotide ISIS ISIS ISIS Serum Normal
257016 257016 353003 indicator Range Saline 2.5 mg/kg 25 mg/kg 25
mg/kg Creatinine 0.0-1.0 0.2 0.1 0.1 0.2 mg/L AST 30-300 160 132 75
131 IU/L ALT 30-200 31 31 28 29 IU/L Albumin 2.5-4.0 2.8 2.9 3.0
2.7 g/dL Triglycerides 25-100* 233 238 287 240 mg/dL Cholesterol
70-125 113 117 122 106 mg/dL Glucose 80-150* 171 195 175 164
mg/dL
[0271] TABLE-US-00040 TABLE 39 Effect of mixed backbone antisense
compound ISIS 257016 administered as three consecutive daily doses
on indicators of liver and kidney function Units measured per dose
of oligonucleotide ISIS ISIS ISIS Serum Normal 257016 257016 353003
indicator Range Saline 2.5 mg/kg 25 mg/kg 25 mg/kg Creatinine
0.0-1.0 0.1 0.2 0.2 0.1 mg/L AST 30-300 199 60 84 155 IU/L ALT
30-200 29 28 26 77 IU/L Albumin 2.5-4.0 2.8 2.9 2.8 2.9 g/dL
Triglycerides 25-100* 289 268 259 236 mg/dL Cholesterol 70-125 111
118 108 106 mg/dL Glucose 80-150* 204 162 181 179 mg/dL
[0272] TABLE-US-00041 TABLE 40 Effect of mixed backbone antisense
compound ISIS 257016 administered as four consecutive daily doses
on indicators of liver and kidney function Units measured per dose
of oligonucleotide ISIS ISIS ISIS Serum Normal 257016 257016 353003
indicator Range Saline 2.5 mg/kg 25 mg/kg 25 mg/kg Creatinine
0.0-1.0 0.1 0.1 0.1 0.2 mg/L AST 30-300 199 92 120 144 IU/L ALT
30-200 29 30 30 36 IU/L Albumin 2.5-4.0 2.8 3.0 2.8 3.0 g/dL
Triglycerides 25-100* 289 252 269 294 mg/dL Cholesterol 70-125 111
126 115 120 mg/dL Glucose 80-150* 204 173 198 192 mg/dL
[0273] TABLE-US-00042 TABLE 41 Effect of mixed backbone antisense
compound ISIS 257016 administered as five consecutive daily doses
on indicators of liver and kidney function Units measured per dose
of oligonucleotide ISIS ISIS ISIS Serum Normal 257016 257016 353003
indicator Range Saline 2.5 mg/kg 25 mg/kg 25 mg/kg Creatinine
0.0-1.0 0.1 0.1 0.1 0.1 mg/L AST 30-300 129 121 125 97 IU/L ALT
30-200 30 30 33 29 IU/L Albumin 2.5-4.0 2.8 2.9 2.8 2.9 g/dL
Triglycerides 25-100* 298 298 285 277 mg/dL Cholesterol 70-125 116
126 122 126 mg/dL Glucose 80-150* 163 177 204 185 mg/dL
*Triglyceride and glucose levels are routinely higher in the Balb/c
strain of mice than in other strains of mice.
[0274] The levels of routine clinical indicators of liver and
kidney injury and disease are within normal ranges and are not
significantly changed relative to saline-treated animals,
demonstrating that the compounds of the invention do not
significantly affect renal or hepatic function. Triglyceride and
glucose levels, while outside the normal range as is common in the
Balb/c strain, are not significantly elevated relative to
saline-treated animals.
[0275] The results illustrated in this example demonstrate that
mixed backbone compounds are delivered to the kidney, reduce SGLT2
expression in vivo, and that treatment with these compounds does
not result in liver or kidney toxicity. The results further
indicate that inhibition of target mRNA expression in the kidney
increases with the number of doses administered.
Example 22
Comparison of a Standard Mixed Backbone Compound and Mixed Backbone
Compounds with Phosphorothioate Linkages at Either or Both of the
Extreme 5' and 3' Ends
A Four Dose Protocol
[0276] In accordance with the present invention, ISIS 257016 (SEQ
ID NO: 4), ISIS 351641 (SEQ ID NO: 4), ISIS 360886 (SEQ ID NO: 4)
and ISIS 360887 (SEQ ID NO: 4) were analyzed for their ability to
inhibit SGLT2 expression in vivo. ISIS 257016 is a standard mixed
backbone compound having 2'-MOE wings and a deoxy gap, with
phosphodiester linkages in the wings and phosphorothioate linkages
in the gap. ISIS 351641 differs from the standard mixed backbone
compounds by having one phosphorothioate linkage at each of the
extreme 5' and 3' ends of the wings. ISIS 360886 and ISIS 360887
are mixed backbone compounds with one phosphorothioate linkage at
the extreme 5' end or extreme 3' end, respectively.
[0277] Male 7-week old Balb/c mice (Charles River Laboratories,
Wilmington, Mass.) were given intraperitoneal injections of ISIS
257016, ISIS 351641, ISIS 360886 or ISIS 360887 twice a week for
two weeks at doses of 1.56, 6.25 or 25 mg/kg. Saline-injected
animals served as controls. Each treatment group contained 4 mice.
The mice were sacrificed 2 days following administration of the
final dose of oligonucleotide or saline.
[0278] Mice were evaluated for SGLT2 levels in kidney. Target
levels were determined by quantitative real-time PCR as described
in other examples herein. PCR results were normalized to
cyclophilin. The data are expressed as percent change relative to
saline treated animals ("+" indicates an increase, "-" indicates a
decrease) and are illustrated in Table 42. TABLE-US-00043 TABLE 42
Antisense inhibition of SGLT2 mRNA expression in vivo by mixed
backbone oligonucleotides (expressed as percent change in SGLT2
mRNA expression relative to saline) Dose of oligonucleotide ISIS
ISIS ISIS ISIS mg/kg 257016 351641 360886 360887 1.56 -39.1 -4.2
-12.7 -9.7 6.25 -52.8 -4.87 -19.7 -7.3 25 -57.8 -11.0 -29.0
-4.9
[0279] These results illustrate that mixed backbone compounds of
the invention, with either complete phosphodiester linkages in the
wings, or with the extreme 5' and 3' ends substituted with
phosphorothioate linkages, can inhibit the expression of kidney
SGLT2 in a dose-dependent manner. With the exception of ISIS
360887, inhibition of target mRNA was dose-dependent. Although all
mixed backbone compounds inhibited SGLT2 expression, greater
inhibition is observed in kidneys from mice treated with ISIS
257016, which is a mixed backbone compound that contains all
phosphodiester linkages in the wings.
[0280] Treated mice were further evaluated for body weight, kidney
weight, liver weight and spleen weight. The data are expressed as
percent change in body or organ weight ("+" indicates an increase,
"-" indicates a decrease). The results are presented in Table 43.
TABLE-US-00044 TABLE 43 Effects of antisense compounds on total
body weight, kidney weight, liver weight and spleen weight of mice
(expressed as percent change in weight) Dose Body Kidney Liver
Spleen Oligonucleotide mg/kg weight weight weight weight ISIS
257016 1.56 +11.6 -3.5 -4.2 -2.4 ISIS 257016 6.25 +7.9 -3.0 +3.8
-1.3 ISIS 257016 25 +11.7 -4.1 +1.4 +8.9 ISIS 351641 1.56 +7.9 -0.9
-5.4 +9.4 ISIS 351641 6.25 +11.1 +1.3 -2.2 +13.4 ISIS 351641 25
+7.4 -2.1 -0.5 -1.4 ISIS 360886 1.56 +7.6 -1.0 -13.7 -5.0 ISIS
360886 6.25 +8.9 -3.7 -16.6 +1.2 ISIS 360886 25 +11.1 -5.5 -11.6
+0.8 ISIS 360887 1.56 +8.5 +1.0 -10.4 -0.4 ISIS 360887 6.25 +7.5
-1.8 -8.4 +1.1 ISIS 360887 25 +9.8 +2.2 -9.0 +11.8
[0281] No significant change was observed in total body weight,
liver weight or spleen weight at timepoints throughout or at the
termination of the study.
[0282] Levels of BUN, creatinine, bilirubin, AST, ALT, albumin,
triglycerides, cholesterol and glucose were measured in mice
treated with the compounds of the invention. Plasma samples were
analyzed using the Olympus AU400e automated chemistry analyzer
(Olympus America, Irving, Tex.). The results, expressed as units
measured, are shown in Tables 44-47. TABLE-US-00045 TABLE 44 Effect
of mixed backbone antisense compound ISIS 257016 on indicators of
liver and kidney function Units measured per dose of ISIS 257016
Serum Normal 1.56 indicator Range Saline mg/kg 6.25 mg/kg 25 mg/kg
BUN 15-40 23 21 26 22 mg/dL Creatinine 0.0-1.0 0.2 0.2 0.2 0.2 mg/L
Bilirubin 0.1-1.0 0.2 0.2 0.2 0.1 mg/dL AST 30-300 75 61 83 71 IU/L
ALT 30-200 30 30 33 39 IU/L Albumin 2.5-4.0 2.8 2.9 2.9 2.7 g/dL
Triglycerides 25-100* 208 210 243 150 mg/dL Cholesterol 70-125 116
125 130 135 mg/dL Glucose 80-150* 207 184 184 215 mg/dL
[0283] TABLE-US-00046 TABLE 45 Effect of mixed backbone antisense
compound ISIS 351641 on indicators of liver and kidney function
Units measured per dose of ISIS 351641 Normal 1.56 6.25 Serum
indicator Range Saline mg/kg mg/kg 25 mg/kg BUN 15-40 23 23 25 22
mg/dL Creatinine 0.0-1.0 0.2 0.2 0.2 0.2 mg/L Bilirubin mg/dL
0.1-1.0 0.2 0.1 0.2 0.1 AST 30-300 75 61 67 54 IU/L ALT 30-200 30
32 31 30 IU/L Albumin 2.5-4.0 2.8 2.7 2.7 2.8 g/dL Triglycerides
25-100* 208 169 176 185 mg/dL Cholesterol 70-125 116 110 115 107
mg/dL Glucose 80-150* 207 205 199 208 mg/dL
[0284] TABLE-US-00047 TABLE 46 Effect of mixed backbone antisense
compound ISIS 360886 on indicators of liver and kidney function
Units measured per dose of ISIS 360886 Normal 1.56 6.25 Serum
indicator Range Saline mg/kg mg/kg 25 mg/kg BUN 15-40 23 21 23 24
mg/dL Creatinine 0.0-1.0 0.2 0.1 0.2 0.2 mg/L Bilirubin mg/dL
0.1-1.0 0.2 0.2 0.2 0.1 AST 30-300 75 56 77 73 IU/L ALT 30-200 30
26 27 28 IU/L Albumin 2.5-4.0 2.8 2.7 2.7 2.7 g/dL Triglycerides
25-100* 208 164 181 169 mg/dL Cholesterol 70-125 116 105 108 108
mg/dL Glucose 80-150* 207 189 202 200 mg/dL
[0285] TABLE-US-00048 TABLE 47 Effect of mixed backbone antisense
compound ISIS 360887 on indicators of liver and kidney function
Units measured per dose of ISIS 360887 Normal 1.56 6.25 Serum
indicator Range Saline mg/kg mg/kg 25 mg/kg BUN 15-40 23 23 22 23
mg/dL Creatinine 0.0-1.0 0.2 0.2 0.2 0.2 mg/L Bilirubin mg/dL
0.1-1.0 0.2 0.2 0.1 0.2 AST 30-300 75 142 83 108 IU/L ALT 30-200 30
40 39 34 IU/L Albumin 2.5-4.0 2.8 2.7 2.7 2.7 g/dL Triglycerides
25-100* 208 136 157 200 mg/dL Cholesterol 70-125 116 109 107 110
mg/dL Glucose 80-150* 207 199 201 187 mg/dL *Triglyceride and
glucose levels are routinely higher in the Balb/c strain of mice
than in other strains of mice.
[0286] Cholesterol levels of mice treated with either 6.25 or 25
mg/kg were slightly elevated; however, these levels are not
significantly greater than the cholesterol levels observed in
saline-treated control animals. Otherwise, the levels of routine
clinical indicators of liver and kidney injury and disease are
within normal ranges and are not significantly changed relative to
saline-treated animals, demonstrating that the compounds of the
invention do not significantly affect renal or hepatic function.
Triglyceride and glucose levels, while outside the normal range as
is common in the Balb/c strain, are not significantly elevated
relative to saline-treated animals.
[0287] Saline- and oligonucleotide-injected animals also were
evaluated histologically following routine procedures. Liver and
kidney samples were procured, fixed in 10% neutral-buffered
formalin and processed for staining with hematoxylin and eosin.
Hematoxylin and eosin staining exhibited no significant difference
between control and oligonucleotide-treated animals.
[0288] The results illustrated in this example demonstrate that
mixed backbone compounds are delivered to the kidney, reduce SGLT2
expression in vivo, and that treatment with these compounds does
not result in liver or kidney toxicity. The results further
indicate that mixed backbone compounds with complete phosphodiester
linkages in the wings are more effective modulators of target mRNA
expression in the kidney than mixed backbone compounds with a
phosphorothioate linkage at one or both of the extreme 5' and 3'
ends.
Example 23
Comparison of a Standard Mixed Backbone Compound and Mixed Backbone
Compounds with Phosphorothioate Linkages at Either or Both of the
Extreme 5' and 3' Ends
An Eight Dose Protocol
[0289] A second study of SGLT2 antisense oligonucleotides ISIS
257016, ISIS 351641, ISIS 360886 and ISIS 360887 was undertaken in
which mice received eight doses over a four week period. As
described previously, ISIS 257016 is a standard mixed backbone
compound having 2'-MOE wings and a deoxy gap, with phosphodiester
linkages in the wings and phosphorothioate linkages in the gap.
ISIS 351641 differs from the standard mixed backbone compounds by
having one phosphorothioate linkage at each of the extreme 5' and
3' ends of the wings. ISIS 360886 and ISIS 360887 are mixed
backbone compounds with one phosphorothioate linkage at the extreme
5' end and extreme 3' end, respectively.
[0290] Male 8-week old Balb/c mice (Charles River Laboratories,
Wilmington, Mass.) were given intraperitoneal injections of ISIS
257016, ISIS 351641, ISIS 360886 or ISIS 360887 twice a week for
four weeks at doses of 1, 5 or 25 mg/kg. Saline-injected animals
served as controls. Each treatment group contained 4 mice. The mice
were sacrificed 2 days following administration of the final dose
of oligonucleotide or saline.
[0291] Mice were evaluated for SGLT2 levels in kidney. Target
levels were determined by quantitative real-time PCR as described
by other examples herein. PCR results were normalized to
cyclophilin. The data are expressed as percent change relative to
saline treated animals ("+" indicates an increase, "-" indicates a
decrease) and are illustrated in Table 48. TABLE-US-00049 TABLE 48
Antisense inhibition of SGLT2 mRNA expression in vivo by mixed
backbone oligonucleotides (expressed as percent change in SGLT2
mRNA expression relative to saline) Dose of oligonucleotide ISIS
ISIS ISIS ISIS mg/kg 257016 351641 360886 360887 1 -53 -14 -24 -23
5 -64 -23 -30 -26 25 -68 -37 -50 -40
[0292] These results illustrate that mixed backbone compounds of
the invention, with either complete phosphodiester linkages in the
wings, or with the extreme 5' and 3' ends substituted with
phosphorothioate linkages, can inhibit the expression of kidney
SGLT2 in a dose-dependent manner. However, greater inhibition is
observed in kidneys from mice treated with ISIS 257016, which
contains all phosphodiester linkages in the wings.
[0293] Treated mice were further evaluated for body weight and
liver and spleen weight. The data are expressed as percent change
in body or organ weight ("+" indicates an increase, "-" indicates a
decrease). The results are presented in Table 49. TABLE-US-00050
TABLE 49 Effects of antisense compounds on total body weight, liver
weight and spleen weight of mice (expressed as percent change in
weight) Dose Body Liver Spleen Oligonucleotide mg/kg weight weight
weight ISIS 257016 1 +11.8 -6.9 -10.1 ISIS 257016 5 +8.4 -4.3 +4.4
ISIS 257016 25 +5.4 -2.1 +12.5 ISIS 351641 1 +12.3 -2.8 -2.9 ISIS
351641 5 +9.2 -8.7 -5.5 ISIS 351641 25 +9.4 -0.8 +3.3 ISIS 360886 1
+9.2 -5.2 -4.5 ISIS 360886 5 +10.3 -2.7 +15.1 ISIS 360886 25 +9.4
-2.1 -11.4 ISIS 360887 1 +10.0 -7.0 -1.5 ISIS 360887 5 +12.6 -3.2
+4.0 ISIS 360887 25 +11.8 -7.6 +14.7
[0294] No significant change was observed in total body weight,
liver weight or spleen weight at timepoints throughout or at the
termination of the study.
[0295] Levels of BUN, creatinine, bilirubin, AST, ALT, albumin,
triglycerides, cholesterol and glucose were measured in mice
treated with the compounds of the invention. Plasma samples were
analyzed using the Olympus AU400e automated chemistry analyzer
(Olympus America, Irving, Tex.). The results, expressed as units
measured, are shown in Tables 50-53. TABLE-US-00051 TABLE 50 Effect
of mixed backbone antisense compound ISIS 257016 on indicators of
liver and kidney function Units measured per Normal dose of ISIS
257016 Serum indicator Range Saline 1 mg/kg 5 mg/kg 25 mg/kg BUN
15-40 27 31 29 23 mg/dL Creatinine 0.0-1.0 0.2 0.2 0.2 0.2 mg/L
Bilirubin mg/dL 0.1-1.0 0.2 0.2 0.2 0.2 AST 30-300 60 58 82 119
IU/L ALT 30-200 22 27 35 66 IU/L Albumin 2.5-4.0 2.7 2.8 2.7 2.6
g/dL Triglycerides 25-100* 178 263 187 99 mg/dL Cholesterol 70-125
123 142 138 162 mg/dL Glucose 80-150* 193 201 201 185 mg/dL
[0296] TABLE-US-00052 TABLE 51 Effect of mixed backbone antisense
compound ISIS 351641 on indicators of liver and kidney function
Units measured per Normal dose of ISIS 351641 Serum indicator Range
Saline 1 mg/kg 5 mg/kg 25 mg/kg BUN 15-40 27 27 26 28 mg/dL
Creatinine 0.0-1.0 0.2 0.2 0.2 0.2 mg/L Bilirubin mg/dL 0.1-1.0 0.2
0.1 0 0.1 AST 30-300 60 48 49 50 IU/L ALT 30-200 22 23 23 20 IU/L
Albumin 2.5-4.0 2.7 2.8 2.8 2.7 g/dL Triglycerides 25-100* 178 165
197 222 mg/dL Cholesterol 70-125 123 118 120 118 mg/dL Glucose
80-150* 193 192 200 197 mg/dL
[0297] TABLE-US-00053 TABLE 52 Effect of mixed backbone antisense
compound ISIS 360886 on indicators of liver and kidney function
Units measured per Normal dose of ISIS 360886 Serum indicator Range
Saline 1 mg/kg 5 mg/kg 25 mg/kg BUN 15-40 27 27 26 27 mg/dL
Creatinine 0.0-1.0 0.2 0.2 0.2 0.2 mg/L Bilirubin mg/dL 0.1-1.0 0.2
0 0.1 0.1 AST 30-300 60 52 71 90 IU/L ALT 30-200 22 23 23 29 IU/L
Albumin 2.5-4.0 2.7 2.8 2.8 2.8 g/dL Triglycerides 25-100* 178 230
250 227 mg/dL Cholesterol 70-125 123 122 129 133 mg/dL Glucose
80-150* 193 187 182 185 mg/dL
[0298] TABLE-US-00054 TABLE 53 Effect of mixed backbone antisense
compound ISIS 360887 on indicators of liver and kidney function
Units measured per Normal dose of ISIS 360887 Serum indicator Range
Saline 1 mg/kg 5 mg/kg 25 mg/kg BUN 15-40 27 25 24 23 mg/dL
Creatinine 0.0-1.0 0.2 0.2 0.2 0.1 mg/L Bilirubin mg/dL 0.1-1.0 0.2
0.2 0.2 0.2 AST 30-300 60 60 44 92 IU/L ALT 30-200 22 24 22 31 IU/L
Albumin 2.5-4.0 2.7 2.7 2.5 2.7 g/dL Triglycerides 25-100* 178 240
262 171 mg/dL Cholesterol 70-125 123 121 129 134 mg/dL Glucose
80-150* 193 189 186 181 mg/dL *Triglyceride and glucose levels are
routinely higher in the Balb/c strain of mice than in other strains
of mice.
[0299] The levels of routine clinical indicators of liver and
kidney injury and disease are within normal ranges and are not
significantly changed relative to saline-treated animals,
demonstrating that the compounds of the invention do not
significantly affect renal or hepatic function. Triglyceride and
glucose levels, while outside the normal range as is common in the
Balb/c strain, are not significantly elevated relative to
saline-treated animals.
[0300] The results illustrated in this example demonstrate that
mixed backbone compounds are delivered to the kidney, reduce SGLT2
expression in vivo, and that treatment with these compounds does
not result in liver or kidney toxicity. Furthermore, the eight dose
protocol resulted in greater inhibition of target mRNA levels in
the kidney than observed for the four dose protocol shown in
Example 22.
Example 24
Antisense Inhibition of SGLT2 in a Murine Model of Type 2
Diabetes
Comparison of Full Phosphorothioate and Mixed Backbone
Oligonucleotides
[0301] The Animal Models of Diabetic Complications Consortium
(AMDCC) has developed protocols for the induction of diabetes in a
number of animal models. The genetic C57BLKS/J
Lep.sup.db/Lep.sup.db model has been approved by the AMDCC as an
appropriate model system for studies of diabetic nephropathy
associated with type 2 diabetes.
[0302] Leptin is a hormone produced by fat that regulates appetite.
Deficiencies in this hormone in both humans and non-human animals
lead to obesity. Lep.sup.db/Lep.sup.db mice have a mutation in the
leptin receptor gene which results in obesity and hyperglycemia. As
such, these mice are a useful model for the investigation of
obesity and diabetes and treatments designed to treat these
conditions. In accordance with the present invention, oligomeric
compounds of the present invention were tested in the
Lep.sup.db/Lep.sup.db model of type 2 diabetes.
[0303] Male Lep.sup.db/Lep.sup.db mice were given intraperitoneal
injections of either ISIS 257016, which has a mixed backbone, or
ISIS 145733, which has a phosphorothioate backbone, twice a week
for four weeks at doses of 12.5, 25 or 37.5 mg/kg. Saline-injected
animals served as controls. Each treatment group contained 6 mice.
The mice were sacrificed 2 days following administration of the
final dose of oligonucleotide or saline.
[0304] Mice were evaluated for SGLT2 levels in kidney. Target
levels were determined by quantitative real-time PCR as described
by other examples herein. PCR results were normalized to
cyclophilin. The data are expressed as percent change relative to
saline treated animals ("+" indicates an increase, "-" indicates a
decrease) and are illustrated in Table 54. TABLE-US-00055 TABLE 54
Antisense inhibition of SGLT2 mRNA expression in db/db mice
(expressed as percent change in SGLT2 mRNA expression relative to
saline) Dose of oligonucleotide ISIS ISIS mg/kg 145733 257016 12.5
-48 -72 25 -71 -72 37.5 -64 -72
[0305] These results illustrate that both mixed backbone compound
ISIS 257016 and full phosphorothioate compound ISIS 145733
effectively inhibit the expression of kidney SGLT2. However,
greater inhibition is observed in kidneys from mice treated with
ISIS 257016, particularly at the lowest dose of 12.5 mg/kg.
[0306] Treated mice were further evaluated for body weight and
liver and spleen weight. The data are expressed as weight in grams.
The results are presented in Table 55. TABLE-US-00056 TABLE 55
Effects of antisense compounds on total body weight, liver weight
and spleen weight of db/db mice (in grams) Dose Body Kidney Liver
Spleen Oligonucleotide mg/kg weight weight weight weight Saline --
35 0.32 1.5 0.09 ISIS 145733 12.5 34 0.32 1.9 0.12 ISIS 145733 25
37 0.37 2.1 0.15 ISIS 145733 37.5 38 0.35 2.3 0.14 ISIS 257016 12.5
34 0.31 1.6 0.09 ISIS 257016 25 36 0.31 1.7 0.08 ISIS 257016 37.5
34 0.35 1.8 0.11
[0307] No significant change was observed in total body weight,
liver weight or spleen weight at timepoints throughout or at the
termination of the study.
[0308] Levels of AST, ALT, triglycerides, cholesterol and glucose
were measured in mice treated with the compounds of the invention.
Plasma samples were analyzed using the Olympus AU400e automated
chemistry analyzer (Olympus America, Irving, Tex.). The results,
expressed as units measured, are shown in Table 56 and Table 57.
TABLE-US-00057 TABLE 56 Effect of full phosphorothioate backbone
compound ISIS 145733 on indicators of toxicity Units measured per
Serum Normal dose of ISIS 145733 indicator Range Saline 12.5 mg/kg
25 mg/kg 37.5 mg/kg AST 30-300 61 72 80 93 IU/L ALT 30-200 63 87
101 120 IU/L Triglycerides 25-100* 245 216 243 204 mg/dL
Cholesterol 70-125* 182 196 211 224 mg/dL Glucose 80-150* 611 452
391 351 mg/dL
[0309] TABLE-US-00058 TABLE 57 Effect of mixed backbone antisense
compound ISIS 257016 on indicators of toxicity Units measured per
Serum Normal dose of ISIS 257016 indicator Range Saline 12.5 mg/kg
25 mg/kg 37.5 mg/kg AST 30-300 61 120 144 175 IU/L ALT 30-200 63
123 142 154 IU/L Triglycerides 25-100* 245 167 188 183 mg/dL
Cholesterol 70-125* 182 248 264 265 mg/dL Glucose 80-150* 611 281
320 326 mg/dL *Triglyceride, cholesterol and glucose levels are
routinely higher in the Lep.sup.db/Lep.sup.db strain of mice than
in other strains of mice.
[0310] The levels of routine clinical indicators of liver injury
and disease are within normal ranges and are not significantly
changed relative to saline-treated animals, demonstrating that the
compounds of the invention do not significantly affect hepatic
function. Given the genetic defect of the Lep.sup.db/Lep.sup.db
mice and the diabetic phenotype exhibited by these mice, it is
expected that triglyceride, cholesterol and glucose levels will
exceed the normal range. Importantly, treatment with either of the
SGLT2 antisense compounds resulted in a significant decrease in
blood glucose levels, with ISIS 25016, the mixed backbone compound,
achieving greater levels of target mRNA inhibition. Treatment with
ISIS 257016 also resulted in a significant decrease in serum
triglyceride levels.
[0311] The results illustrated in this example demonstrate that
mixed backbone compounds are effectively delivered to the kidney,
reduce SGLT2 expression in vivo, and that treatment with these
compounds does not result in liver or other toxicity. Furthermore,
these results indicate that mixed backbone compounds targeted to
SGLT2 efficiently decrease blood glucose levels and serum
triglyceride levels in a mouse model of type 2 diabetes.
Example 25
Antisense Inhibition of SGLT2 in a Murine Model of Type 2
Diabetes
Low Dose Comparison of Full Phosphorothioate and Mixed Backbone
Oligonucleotides
[0312] Since treatment with ISIS 257016 resulted in significant
reduction in SGLT2 expression levels even at the lowest dose of
12.5 mg/kg, a second dose-response study was conducted using a
lower dose range of 1.56, 3.12 and 6.25 mg/kg. Male
Lep.sup.db/Lep.sup.db mice were given intraperitoneal injections of
either mixed backbone compound ISIS 257016 or full phosphorothioate
compound ISIS 145733 twice a week for four weeks at doses of 1.56,
3.12 or 6.25 mg/kg. Saline-injected animals served as controls.
Each treatment group contained 4 mice. The mice were sacrificed 2
days following administration of the final dose of oligonucleotide
or saline.
[0313] Mice were evaluated for SGLT2 levels in kidney. Target
levels were determined by quantitative real-time PCR as described
by other examples herein. PCR results were normalized to
cyclophilin. The data are expressed as percent change relative to
saline treated animals ("+" indicates an increase, "-" indicates a
decrease) and are illustrated in Table 58. TABLE-US-00059 TABLE 58
Antisense inhibition of SGLT2 mRNA expression in db/db mice
(expressed as percent change in SGLT2 mRNA expression relative to
saline) Dose of oligonucleotide ISIS ISIS mg/kg 145733 257016 1.56
-13 -75 3.12 -14 -83 6.25 -12 -80
[0314] These results illustrate that mixed backbone compound ISIS
257016 is a more effective inhibitor of SGLT2 mRNA expression in
the kidney, particularly at low doses of oligonucleotide.
[0315] Levels of glucose were measured in mice treated with the
compounds of the invention. Plasma samples were analyzed using the
Olympus AU400e automated chemistry analyzer (Olympus America,
Irving, Tex.). The data are expressed as percent change relative to
saline treated animals ("+" indicates an increase, "-" indicates a
decrease) and are illustrated in Table 59. TABLE-US-00060 TABLE 59
Blood glucose levels in db/db mice treated with SGLT2 antisense
compounds (expressed as percent change in blood glucose relative to
saline) Dose of oligonucleotide ISIS ISIS mg/kg 145733 257016 1.56
-5 -41 3.12 -7 -37 6.25 -14 -40
[0316] The results demonstrate that treatment with mixed backbone
compound ISIS 257016 results in a significant decrease in blood
glucose levels and that mixed backbone compounds are more effective
at lowering blood glucose levels than full phosphorothioate
antisense compounds.
[0317] Antisense inhibition of SGLT2 by ISIS 257016 was further
evaluated using a dose range of 0.39, 0.78 and 1.56 mg/kg. As
described above, male Lep.sup.db/Lep.sup.db mice were given
intraperitoneal injections of mixed backbone compound ISIS 257016
twice a week for four weeks. Saline-injected animals served as
controls. Each treatment group contained 4 mice. The mice were
sacrificed 2 days following administration of the final dose of
oligonucleotide or saline.
[0318] Mice were evaluated for SGLT2 levels in kidney. Target
levels were determined by quantitative real-time PCR as described
by other examples herein. PCR results were normalized to
cyclophilin. Blood glucose levels also were determined. Plasma
samples were analyzed using the Olympus AU400e automated chemistry
analyzer (Olympus America, Irving, Tex.). The data are expressed as
percent change relative to saline treated animals ("+" indicates an
increase, "-" indicates a decrease) and are illustrated in Table
60. TABLE-US-00061 TABLE 60 Antisense inhibition of SGLT2 mRNA
expression and blood glucose levels in db/db mice (expressed as
percent change in SGLT2 mRNA expression or blood glucose levels
relative to saline) Dose of oligonucleotide SGLT2 Blood mg/kg mRNA
glucose 0.39 -66 -16 0.78 -68 -21 1.56 -82 -21
[0319] These results further demonstrate the effectiveness of mixed
backbone compounds at inhibiting SGLT2 expression in the kidney and
lowering blood glucose levels when administered at very low doses
of oligonucleotide.
[0320] Mice treated with the compounds of the invention also were
evaluated for liver and kidney toxicity, organ and body weights and
tissue histology. These studies demonstrated no significant level
of toxicity or change in body or organ weight, indicating that
mixed backbone compounds are effective in vivo without toxicity to
the animal.
[0321] The results illustrated in this example demonstrate that
mixed backbone compounds are effectively delivered to the kidney,
reduce SGLT2 expression in vivo, and that treatment with these
compounds lowers blood glucose levels in diabetic animals.
Example 26
Antisense Inhibition of SGLT2 in a Murine Model of Obesity and
Diabetes Using Mixed Backbone Compounds
[0322] Leptin is a hormone produced by fat that regulates appetite.
Deficiencies in this hormone in both humans and non-human animals
leads to obesity. C57Bl/6J-Lep ob/ob mice have a mutation in the
leptin gene which results in obesity and hyperglycemia. As such,
these mice are a useful model for the investigation of obesity and
diabetes and treatments designed to treat these conditions. In
accordance with the present invention, the oligomeric compounds of
the invention were tested in the ob/ob model of obesity and
diabetes.
[0323] Male C57Bl/6J-Lep ob/ob mice (Jackson Laboratory, Bar
Harbor, Me.) were subcutaneously injected with ISIS 257016 at a
dose of 25 mg/kg two times per week for 4 weeks. Saline-injected
animals served as controls. Each treatment group contained 4 mice.
The mice were sacrificed 2 days following administration of the
final dose of oligonucleotide or saline.
[0324] Mice were evaluated for SGLT2 levels in kidney. Target
levels were determined by quantitative real-time PCR as described
by other examples herein. PCR results were normalized to
cyclophilin. Blood glucose levels also were determined. Plasma
samples were analyzed using the Olympus AU400e automated chemistry
analyzer (Olympus America, Irving, Tex.). The data are expressed as
percent change relative to saline treated animals ("+" indicates an
increase, "-" indicates a decrease) and are illustrated in Table
61. TABLE-US-00062 TABLE 61 Antisense inhibition of SGLT2 mRNA
expression and blood glucose levels in db/db mice (expressed as
percent change in SGLT2 mRNA expression or blood glucose levels
relative to saline) Dose of oligonucleotide SGLT2 Blood mg/kg mRNA
glucose 25 -83 -39
[0325] The results demonstrate that treatment with a mixed backbone
SGLT2 antisense compound results in a significant decrease in SGLT2
mRNA expression in the kidney of diabetic mice. Importantly, blood
glucose levels also are significantly decreased in treated
animals.
Example 27
Comparison of Mixed Backbone Compounds 16 to 20 Nucleobases in
Length
[0326] In accordance with the present invention, mixed backbone
compounds with less than 20 nucleobases were evaluated for their
ability to inhibit SGLT2 expression in the kidney. Four compounds
were synthesized based on the sequence of ISIS 257016 (SEQ ID NO:
4). ISIS 366847, ISIS 366848, ISIS 366849 and ISIS 366850 are
comprised of the 5'-most 19, 18, 17 and 16 nucleobases,
respectively, of ISIS 257016. ISIS 257016 has 2'-MOE wings of five
nucleobases each and a deoxy gap of 10 nucleobases. ISIS 366847,
ISIS 366848, ISIS 366849 and ISIS 366850 have a 10 nucleobases gap,
a five nucleobase 2'-MOE wing at the 5' end, but contain a
shortened 3' wing of 1 to 4 nucleobases.
[0327] Male 6-week old Balb/c mice (Charles River Laboratories,
Wilmington, Mass.) were given intraperitoneal injections of ISIS
257016, ISIS 366847, ISIS 366848, ISIS 366849 or ISIS 366850 twice
a week for two weeks at doses of 0.14, 0.7 or 3.5 micromoles per
kilogram (.mu.M/kg). Saline-injected animals served as controls.
Each treatment group contained 4 mice. The mice were sacrificed 2
days following administration of the final dose of oligonucleotide
or saline.
[0328] Mice were evaluated for SGLT2 levels in kidney. Target
levels were determined by quantitative real-time PCR as described
in other examples herein. PCR results were normalized to
cyclophilin. The data are expressed as percent change relative to
saline treated animals ("+" indicates an increase, "-" indicates a
decrease) and are illustrated in Table 62. TABLE-US-00063 TABLE 62
Antisense inhibition of SGLT2 mRNA expression in vivo by mixed
backbone oligonucleotides (expressed as percent change relative to
saline control) Dose of oligonucleotide ISIS ISIS ISIS ISIS ISIS
.mu.M/kg 257016 366847 366848 366849 366850 0.14 -53 -55 -58 -57
-49 0.7 -56 -63 -59 -61 -57 3.5 -70 -64 -72 -69 -69
[0329] These results illustrate that mixed backbone compounds of
the invention, containing 16 to 20 nucleobases, are effective
inhibitors of SGLT2 expression in the kidney.
[0330] Treated mice were further evaluated for body weight, kidney
weight, liver weight and spleen weight. The data are expressed as
percent change in body or organ weight ("+" indicates an increase,
"-" indicates a decrease). The results are presented in Table 63.
TABLE-US-00064 TABLE 63 Effects of antisense compounds on total
body weight, kidney weight, liver weight and spleen weight of mice
(expressed as percent change in weight) Dose Body Kidney Liver
Spleen Oligonucleotide .quadrature.M/kg weight weight weight weight
ISIS 257016 0.14 +9.0 -4.5 -6.1 -8.3 ISIS 257016 0.7 +11.1 -5.3
+4.1 -3.7 ISIS 257016 3.5 +10.2 -3.6 +3.7 +11.9 ISIS 366847 0.14
+15.0 -0.5 +0.2 -6.9 ISIS 366847 0.7 +12.7 +1.2 +6.8 -4.9 ISIS
366847 3.5 +10.3 +3.6 +3.8 +2.9 ISIS 366848 0.17 +8.5 -7.1 -7.9
-2.4 ISIS 366848 0.7 +7.7 +6.4 +5.9 +3.8 ISIS 366848 3.5 +10.8 +3.0
+4.6 +9.3 ISIS 366849 0.14 +6.9 -3.3 -2.6 -7.2 ISIS 366849 0.7 +7.4
+0.1 -4.3 -2.2 ISIS 366849 3.5 +8.4 -2.9 -5.2 -3.9 ISIS 366850 0.14
+11.1 -3.8 -4.6 +2.0 ISIS 366850 0.7 +4.8 -0.8 -1.7 +0.9 ISIS
366850 3.5 11.2 -6.0 +4.5 +9.8
[0331] No significant change was observed in total body weight,
liver weight or spleen weight at timepoints throughout or at the
termination of the study.
[0332] Levels of BUN, creatinine, bilirubin, AST, ALT, albumin,
triglycerides, cholesterol and glucose were measured in mice
treated with the compounds of the invention. Plasma samples were
analyzed using the Olympus AU400e automated chemistry analyzer
(Olympus America, Irving, Tex.). The results, expressed as units
measured, are shown in Tables 64-68. TABLE-US-00065 TABLE 64 Effect
of mixed backbone antisense compound ISIS 257016 on indicators of
liver and kidney function Units measured per Serum Normal dose of
ISIS 257016 indicator Range Saline 0.14 .mu.M/kg 0.7 .mu.M/kg 3.5
.mu.M/kg BUN 15-40 31 32 32 31 mg/dL Creatinine 0.0-1.0 0.2 0.2 0.2
0.2 mg/L Bilirubin 0.1-1.0 0.2 0.1 0.1 0.1 mg/dL AST 30-300 82 68
85 117 IU/L ALT 30-200 22 24 26 32 IU/L Albumin 2.5-4.0 3.0 3.2 3.1
3.1 g/dL Triglycerides 25-100* 225 266 308 225 mg/dL Cholesterol
70-125 123 128 128 147 mg/dL Glucose 80-150* 181 195 187 183
mg/dL
[0333] TABLE-US-00066 TABLE 65 Effect of mixed backbone antisense
compound ISIS 366847 on indicators of liver and kidney function
Units measured per Serum Normal dose of ISIS 366847 indicator Range
Saline 0.14 .mu.M/kg 0.7 .mu.M/kg 3.5 .mu.M/kg BUN 15-40 31 29 32
29 mg/dL Creatinine 0.0-1.0 0.2 0.2 0.2 0.2 mg/L Bilirubin 0.1-1.0
0.2 0.1 0 0.1 mg/dL AST 30-300 82 53 69 131 IU/L ALT 30-200 22 23
28 50 IU/L Albumin 2.5-4.0 3.0 3.1 3.2 3.0 g/dL Triglycerides
25-100* 225 289 308 184 mg/dL Cholesterol 70-125 123 122 132 145
mg/dL Glucose 80-150* 181 173 193 181 mg/dL
[0334] TABLE-US-00067 TABLE 66 Effect of mixed backbone antisense
compound ISIS 366848 on indicators of liver and kidney function
Units measured per Serum Normal dose of ISIS 366848 indicator Range
Saline 0.14 .mu.M/kg 0.7 .mu.M/kg 3.5 .mu.M/kg BUN 15-40 31 31 29
32 mg/dL Creatinine 0.0-1.0 0.2 0.2 0.2 0.2 mg/L Bilirubin 0.1-1.0
0.2 0.1 0.1 0.1 mg/dL AST 30-300 82 82 105 123 IU/L ALT 30-200 22
23 34 46 IU/L Albumin 2.5-4.0 3.0 3.1 3.1 3.0 g/dL Triglycerides
25-100* 225 320 374 246 mg/dL Cholesterol 70-125 123 132 142 147
mg/dL Glucose 80-150* 181 200 187 190 mg/dL
[0335] TABLE-US-00068 TABLE 67 Effect of mixed backbone antisense
compound ISIS 366849 on indicators of liver and kidney function
Units measured per Serum Normal dose of ISIS 366849 indicator Range
Saline 0.14 .mu.M/kg 0.7 .mu.M/kg 3.5 .mu.M/kg BUN 15-40 31 25 30
33 mg/dL Creatinine 0.0-1.0 0.2 0.2 0.2 0.2 mg/L Bilirubin 0.1-1.0
0.2 0.1 0.1 0.1 mg/dL AST 30-300 82 98 90 92 IU/L ALT 30-200 22 26
24 33 IU/L Albumin 2.5-4.0 3.0 3.0 3.0 3.0 g/dL Triglycerides
25-100* 225 354 308 240 mg/dL Cholesterol 70-125 123 133 129 150
mg/dL Glucose 80-150* 181 170 173 192 mg/dL
[0336] TABLE-US-00069 TABLE 68 Effect of mixed backbone antisense
compound ISIS 366850 on indicators of liver and kidney function
Units measured per Serum Normal dose of ISIS 366850 indicator Range
Saline 0.14 .mu.M/kg 0.7 .mu.M/kg 3.5 .mu.M/kg BUN 15-40 31 26 25
23 mg/dL Creatinine 0.0-1.0 0.2 0.2 0.2 0.2 mg/L Bilirubin 0.1-1.0
0.2 0.1 0.1 0 mg/dL AST 30-300 82 83 69 108 IU/L ALT 30-200 22 21
27 38 IU/L Albumin 2.5-4.0 3.0 3.0 3.0 3.0 g/dL Triglycerides
25-100* 225 320 380 271 mg/dL Cholesterol 70-125 123 127 131 164
mg/dL Glucose 80-150* 181 192 187 179 mg/dL *Triglyceride and
glucose levels are routinely higher in the Balb/c strain of mice
than in other strains of mice.
[0337] Some oligonucleotide treated animals exhibited elevated
levels of cholesterol; however, saline control animals also
demonstrated cholesterol levels at the high end of the normal
range. Thus, the slightly elevated cholesterol levels do not appear
to be significant. Otherwise, the levels of routine clinical
indicators of liver and kidney injury and disease are within normal
ranges and are not significantly changed relative to saline-treated
animals, demonstrating that the compounds of the invention do not
significantly affect renal or hepatic function. Triglyceride and
glucose levels, while outside the normal range as is common in the
Balb/c strain, are not significantly elevated relative to
saline-treated animals.
[0338] The results illustrated in this example demonstrate that
mixed backbone compounds of 16 to 20 nucleobases are delivered to
the kidney, reduce SGLT2 expression in vivo, and that treatment
with these compounds does not result in liver or kidney
toxicity.
Example 28
Antisense Inhibition of SGLT2 in Sprague Dawley Rats
[0339] In accordance with the present invention, 7-week old Sprague
Dawley rats (purchased from Charles River Labs, Wilmington, Mass.)
were treated with SGLT2 mixed backbone compound ISIS 257016 (SEQ ID
NO: 4) or SGLT2 full phosphorothioate compound ISIS 145733 (SEQ ID
NO: 4). Rats were injected i.p. twice a week for three weeks with
10 mg/kg of oligonucleotide. Saline-injected animals served as
controls. The rats were sacrificed 2 days following administration
of the final dose of oligonucleotide or saline.
[0340] Rats were evaluated for SGLT2 levels in kidney. Target
levels were determined by quantitative real-time PCR as described
in other examples herein. PCR results were normalized to
cyclophilin. The data are expressed as percent change relative to
saline treated animals ("+" indicates an increase, "-" indicates a
decrease) and are illustrated in Table 69. TABLE-US-00070 TABLE 69
Antisense inhibition of SGLT2 mRNA expression in Sprague Dawley
rats (expressed as percent change in SGLT2 mRNA expression relative
to saline) % Change Treatment in mRNA Saline 0 ISIS 257016 -83.9
ISIS 145733 -38.5
[0341] These results illustrate that both full phosphorothioate and
mixed backbone compounds inhibit SGLT2 expression in the kidney of
rats. However, the mixed backbone compound is a more effective
inhibitor of SGLT2.
[0342] Treated rats were further evaluated for body weight, kidney
weight, liver weight and spleen weight. For body weight, the data
are expressed as percent change in body weight ("+" indicates an
increase, "-" indicates a decrease). For organ weights, the results
are expressed as percent of saline control normalized to body
weight. The results are presented in Table 70 and Table 71.
TABLE-US-00071 TABLE 70 Effects of antisense compounds on total
body weight of rats (expressed as percent change in weight) Body
Treatment weight Saline +60.7 ISIS 257016 -58.4 ISIS 145733
+57.1
[0343] TABLE-US-00072 TABLE 71 Effects of antisense compounds on
total kidney weight, liver weight and spleen weight of rats
(expressed as percent of saline control normalized to body weight)
Kidney Liver Spleen Treatment weight weight weight ISIS 257016 99.3
93.4 105.8 ISIS 145733 107.2 105.2 123.4
[0344] No significant change was observed in total body weight,
kidney weight, liver weight or spleen weight at timepoints
throughout or at the termination of the study.
[0345] Levels of BUN, creatinine, bilirubin, AST, ALT, albumin,
triglycerides, cholesterol and glucose were measured in rats
treated with the compounds of the invention. Plasma samples were
analyzed using the Olympus AU400e automated chemistry analyzer
(Olympus America, Irving, Tex.). The results, expressed as units
measured, are shown in Table 72. TABLE-US-00073 TABLE 72 Effect of
mixed backbone antisense compound ISIS 257016 and full
phosphorothioate compound ISIS 145733 on indicators of liver and
kidney function (expressed as units measured) ISIS ISIS Serum
Indicator Saline 257016 145733 BUN 19 19 17 mg/dL Creatinine 0.3
0.4 0.2 mg/L Bilirubin mg/dL 0.1 0.1 0.1 AST 157 105 105 IU/L ALT
65 44 36 IU/L Albumin 3.7 3.8 3.6 g/dL Triglycerides 42 47 53 mg/dL
Cholesterol 68 66 54 mg/dL Glucose 189 173 180 mg/dL
[0346] The levels of routine clinical indicators of liver and
kidney injury and disease are not significantly changed relative to
saline-treated animals, demonstrating that the compounds of the
invention do not significantly affect renal or hepatic function in
rats.
[0347] The results illustrated in this example demonstrate that
both full phosphorothioate and mixed backbone compounds are
delivered to the kidney, reduce SGLT2 expression in vivo, and that
treatment with these compounds does not result in liver or kidney
toxicity. The results further indicate that mixed backbone
compounds are more effective inhibitors of SGLT2 expression in
vivo.
Example 29
Antisense Inhibition of Connective Tissue Growth Factor in a Murine
Model of Type 2 Diabetes
Comparison of Full Phosphorothioate and Mixed Backbone
Oligonucleotides
[0348] Three month old C57BLKS/J Lep.sup.db/Lep.sup.db and
age-matched control C57BLKS/J mice were treated twice a week for
six weeks with control oligonucleotide ISIS 141923
(CCTTCCCTGAAGGTTCCTCC; SEQ ID NO: 18), or CTGF antisense
oligonucleotides ISIS 124212 (CCACAAGCTGTCCAGTCTAA; SEQ ID NO: 19)
or ISIS 334157 (CCACAAGCTGTCCAGTCTAA; SEQ ID NO: 19). ISIS 124212,
ISIS 334517 and ISIS 141923 are chimeric oligonucleotides
("gapmers") 20 nucleotides in length, composed of a central "gap"
region consisting of ten 2'-deoxynucleotides, which is flanked on
both sides (5' and 3' directions) by five-nucleotide "wings". The
wings are composed of 2'-methoxyethyl (2'-MOE) nucleotides. ISIS
124212 and ISIS 141923 have phosphorothioate (P.dbd.S)
internucleoside (backbone) linkages throughout the oligonucleotide.
ISIS 334517 has a mixed backbone with phosphorothioate linkages in
the central gap region and phosphodiester linkages in the wings.
All cytidine residues in each oligonucleotide are
5-methylcytidines.
[0349] Oligonucleotides were delivered subcutaneously at a dose of
10 mg/kg or 25 mg/kg. Saline-injected animals served as controls.
Blood and urine chemistries were analyzed prior to treatment, at
three weeks, and post-treatment.
[0350] After the treatment period, mice are sacrificed and CTGF
mRNA levels were evaluated in the kidney. Target levels were
determined by quantitative real-time PCR as described in other
examples herein. For mouse CTGF, the PCR primers were:
TABLE-US-00074 Forward primer: GCTCAGGGTAAGGTCCGATTC (SEQ ID NO:
26) Reverse primer: GCCCCCCACCCCAAA (SEQ ID NO: 27)
[0351] The PCR probe was: FAM-TCATAATCAAAGAAGCAGCAAGCACTTCC-TAMRA
(SEQ ID NO: 28), where FAM is the fluorescent dye and TAMRA is the
quencher dye. The results are illustrated in Table 73.
TABLE-US-00075 TABLE 73 Antisense inhibition of CTGF mRNA in
Lep.sup.db/Lep.sup.db kidney (shown as percent of saline-injected
control mice) Percent expression of CTGF mRNA after treatment with
oligonucleotide at the concentrations shown: Oligonucleotide Saline
10 mg/kg 25 mg/kg 141923 100 -- 82 124212 100 110 71 334157 100 83
53
[0352] Treatment with ISIS 334157 resulted in a significant
decrease in CTGF expression at a dose of 25 mg/kg. ISIS 124212 also
inhibited expression CTGF at the same dose. These results
demonstrate that mixed backbone compounds inhibit expression of
target mRNA in the kidney.
[0353] To assess distribution of CTGF antisense oligonucleotides in
Lep.sup.db/Lep.sup.db kidney, 2E1 staining was performed as
described in other examples herein. The results demonstrated that
ISIS 124212 and ISIS 334157 exhibit a similar pattern of
distribution in the inner and outer cortex.
[0354] To evaluate whether antisense inhibition of CTGF using mixed
backbone compounds alters the development of diabetic nephropathy,
levels of collagen 1A and collagen IV (.quadrature.2) were
determined. Collagen synthesis is a prerequisite for the
development of kidney fibrosis characteristic of diabetic
nephorpathy. Collagen 1A and collagen IV (.quadrature.2) target
mRNA levels were determined by quantitative real-time PCR as
described by other examples herein.
[0355] Probes and primers to mouse collagen 1A and collagen IV
(.quadrature.2) were designed to hybridize to mouse collagen 1A and
collagen IV (.quadrature.2) sequences using published sequence
information. For mouse collagen 1A, the PCR primers were:
TABLE-US-00076 Forward primer: TGGATTCCCGTTCGAGTACG (SEQ ID NO: 20)
Reverse primer: TCAGCTGGATAGCGACATCG (SEQ ID NO: 21)
The PCR probe was: FAM-AAGCGAGGGCTCCGACCCGA-TAMRA (SEQ ID NO: 22)
FAM is the fluorescent dye and TAMRA is the quencher dye.
[0356] For mouse collagen IV, the PCR primers were: TABLE-US-00077
Forward primer: AGACCAACAAGCAAGTGAGTGC (SEQ ID NO: 23) Reverse
primer: CTAGCATGTGAGCCACATTCATCC (SEQ ID NO: 24)
[0357] The PCR probe was: FAM-CTGCTGAGGGCACGCTGAGCT-TAMRA (SEQ ID
NO: 25) FAM is the fluorescent dye and TAMRA is the quencher dye.
TABLE-US-00078 TABLE 74 Inhibition of collagen 1A mRNA expression
in Lep.sup.db/Lep.sup.db mice treated with CTGF antisense
oligonucleotide (shown as percent of saline-injected control mice)
Percent expression of collagen 1A mRNA after treatment with CTGF
antisense oligonucleotide at the concentrations shown:
Oligonucleotide Saline 10 mg/kg 25 mg/kg 141923 100 -- 145 124212
100 60 65 334157 100 78 75
[0358] TABLE-US-00079 TABLE 75 Inhibition of collagen IV mRNA
expression in Lep.sup.db/Lep.sup.db mice treated with CTGF
antisense oligonucleotide (shown as percent of saline-injected
control mice) Percent expression of collagen IV mRNA after
treatment with CTGF antisense oligonucleotide at the concentrations
shown: Oligonucleotide Saline 10 mg/kg 25 mg/kg 141923 100 -- 72
124212 100 50 40 334157 100 37 24
[0359] Treatment with either ISIS 124212 or ISIS 334157 led to a
significant reduction in mRNA expression of both collagen 1A and
collagen IV in Lep.sup.db/Lep.sup.db mice, relative to both
saline-injected control mice and mice treated with control
oligonucleotide. These results indicate that treatment with CTGF
antisense oligonucleotides, including mixed backbone compounds,
inhibits development of nephropathy associated with type 2
diabetes.
[0360] Toxic effects of compounds administered in vivo can be
assessed by measuring the levels of enzymes and proteins associated
with disease or injury of the liver or kidney. Elevations in the
levels of the serum transaminases aspartate aminotransferase (AST)
and alanine aminotransferase (ALT) are often indicators of liver
disease or injury. To assess the physiological effects resulting
from inhibition of target mRNA, the Lep.sup.db/Lep.sup.db mice were
further evaluated at the end of the treatment period for AST and
ALT. Serum was analyzed by LabCorp Testing Facility (San Diego,
Calif.). The levels of AST and ALT were within normal ranges and
were not significantly changed relative to saline-treated animals,
demonstrating that the mixed backbone and full phosphorothioate
antisense compounds of the invention do not significantly affect
hepatic function.
[0361] These results illustrate that both full phosphorothioate
backbone and mixed backbone compounds are effectively delivered to
the kidney, reduce CTGF expression in vivo without toxicity and
that these compounds inhibit the development of diabetic
nephropathy in diabetic animals.
Example 30
Design of Chemically Modified Antisense Compounds for Optimized
Kidney Targeting
[0362] A series of chemically modified antisense compounds were
designed using the sequence of ISIS 116847 (SEQ ID NO: 29), ISIS
331424 (SEQ ID NO: 30), ISIS 342351 (SEQ ID NO: 31), ISIS 331425
(SEQ ID NO: 32), or ISIS 335252 (SEQ ID NO: 33). Modifications were
made to the internucleoside linkages such that the oligonucleotides
consisted of either full phosphorothioate backbones or mixed
phosphorothioate and phosphodiester backbones (mixed backbone
compounds). Modified antisense compounds also contained sugar
moiety substitutions at the 2' position, comprising a
2'-methoxyethyl (2'-MOE). Some of the oligonucleotides contain
further modifications including nucleobase substitutions, wherein
the unmodified cytosine nucleobase was used in place of the
modified 5-methylcytosine at one position in the antisense
compound. The compounds are shown in Table 1.
[0363] ISIS 116847 (SEQ ID NO: 29) is chimeric oligonucleotides
having 2'-MOE wings and a deoxy gap with phosphorothioate linkages
throughout the oligonucleotide. ISIS 33525 (SEQ ID NO: 33) is a
chimeric oligonucleotide having 2'-MOE wings and a deoxy gap, with
phosphodiester linkages in the wings and phosphorothioate linkages
in the gap. TABLE-US-00080 TABLE 76 Chemical modifications of
antisense compounds targeting PTEN SEQ ID ISIS # Sequence NO 116847
CsTsGsCsTs agcctctgga sTsTsTsGsA 29 331424 CsTsGsCsTs agcctoctgga
sTsTsTsGsA 30 342351 CsTsGoCsTs agcctctgga sTsToTsGsA 31 331425
CsToGsCoTs agcctctgga sToTsToGsA 32 335252 CoToGoCoTs agcctctgga
sToToToGoA 33
Example 31
Comparison of a Standard Phosphorothioate Compound and a Mixed
Backbone Compound with Phosphodiester Linkages Inserted in the Gap
and Wings
A Single Dose Study
[0364] In accordance with the present invention, ISIS 116847, ISIS
331424, ISIS 331425, and ISIS 335252 were analyzed for their
ability to inhibit PTEN expression in vivo.
[0365] Male Balb/c mice (Charles River Laboratories, Wilmington,
Mass.) were given single intrapertoneal injections of ISIS 116847,
ISIS 331424, ISIS 331425, or ISIS 335252 at a dose of 25 mg/kg
twice per week for two weeks. Saline-injected animals served as
controls. The mice were sacrificed 2 days following administration
of the fourth and final dose of oligonucleotide or saline.
[0366] Mice were evaluated for PTEN mRNA levels in liver, kidney
and fat tissues. Target levels were determined by quantitative
real-time PCR as described by other examples herein. PCR results
were normalized to the ubiquitously expressed mouse cyclophilin A
gene.
[0367] The data are expressed as percent change ("-" indicates a
decrease) relative to saline treated animals and are shown in Table
2. TABLE-US-00081 TABLE 77 Antisense inhibition of PTEN mRNA
expression in vivo by modified PTEN antisense compounds in liver
Percent change in PTEN expression relative to saline, p < 0.05
ISIS ISIS ISIS ISIS 116847 331424 331425 335252 -80 -38 -65 -43
[0368] These data illustrate that antisense compounds of different
chemistries inhibit the expression of PTEN mRNA in mouse liver.
Greatest inhibition of PTEN is observed in livers from mice treated
with ISIS 116847 and ISIS 331425, which are mixed backbone
antisense compounds. TABLE-US-00082 TABLE 78 Antisense inhibition
of PTEN mRNA expression in vivo by modified PTEN antisense
compounds in fat tissues Percent change in PTEN expression relative
to saline, p < 0.05 ISIS ISIS ISIS ISIS 116847 331424 331425
335252 -39 -10 -45 -30
[0369] These data illustrate that antisense compounds of different
chemistries inhibit the expression of PTEN mRNA in mouse fat
tissues. Greatest inhibition of PTEN is observed in livers from
mice treated with ISIS 331425 and ISIS 116847, which are mixed
backbone antisense compounds.
[0370] Mice were further evaluated for total body weight, liver
weight and spleen weight. Significant changes in spleen, liver or
body weight. The data are expressed as percent change in body or
organ weight ("+" indicates an increase, "-" indicates a decrease).
The results are presented in Table 79. TABLE-US-00083 TABLE 79
Effects of antisense compounds on liver weight and spleen weight in
mice Percent change in weight ISIS ISIS ISIS ISIS 116847 331424
331425 335252 25 5 25 5 25 5 5 mg/kg mg/kg mg/kg mg/kg mg/kg mg/kg
25 mg/kg mg/kg Liver +28 +9 +10 +7 +5 -10 +5 -9 Spleen +12 -2 +8 +9
+6 -12 -2 0
[0371] All changes in organ weight were within the margin of error
of the experiment. No significant change was observed in liver
weight or spleen weight at timepoints throughout or at the
termination of the study. TABLE-US-00084 TABLE 80 Biodistribution
of compounds in mice Accumulation of different ASOs in tissues ug
ASO/g tissue ISIS ISIS ISIS ISIS 116847 331424 331425 335252 Liver
102 38 65 3 Kidney 90 80 100 5 Fat 19 18 18 0
Example 32
Antisense Inhibition of PTEN in rats
[0372] In accordance with the present invention, male Sprague
Dawley rats (purchased from Charles River Labs, Wilmington, Mass.)
were treated with PTEN mixed backbone compound ISIS 116847 (SEQ ID
NO: 29). Rats were given intraperitoneal injections twice a week
for four weeks with 30 mg/kg of oligonucleotide. Saline-injected
animals served as controls. The rats were sacrificed 2 days
following administration of the final dose of oligonucleotide or
saline.
[0373] Rats were evaluated for PTEN levels in liver and adipose
tissue. Target levels were determined by quantitative real-time PCR
as described in other examples herein. PCR results were normalized
to cyclophilin. The data are expressed as percent change relative
to saline treated animals ("+" indicates an increase, "-" indicates
a decrease) and are illustrated in Table 80. TABLE-US-00085 TABLE
81 Antisense inhibition of PTEN mRNA expression in Sprague Dawley
rats (expressed as percent change in PTEN mRNA expression relative
to saline) % Change in % Change in mRNA mRNA in adipose Treatment
in liver tissue Saline 0 0 ISIS 116847 -46 -33 ISIS 342351 -88 -43
ISIS 331425 -65 -47
[0374] These results illustrate that both full phosphorothioate and
mixed backbone compounds inhibit PTEN expression in both the liver
and adipose tissue of rats. However, the mixed backbone compounds
are more effective inhibitor of PTEN.
[0375] Rats were further evaluated for metabolites in kidney. Rats
were given intraperitoneal injections twice a week for four weeks
with 30 mg/kg of oligonucleotide. The rats were sacrificed one day
following administration of the final dose of oligonucleotide. The
results are presented in Table 81. TABLE-US-00086 TABLE 82 Effects
of antisense compounds on metabolites in kidney Parent
Oligonucleotide Total Oligonucleotide Concentrations (.mu.g/g)
Concentrations (.mu.g/g) % FL ISIS 116847 1100 1400 68 ISIS 342351
2100 2900 67 ISIS 331425 1200 1800 66
[0376] The percent FL (parent oligonucleotide concentrations/total
oligonucleotide concentrations) for all of the oligonucleotides are
in similar extent.
[0377] A comparison experiment was conducted to study the total
oligonucleotide concentrations of several modifications to an
antisense oligonucleotide modulating PTEN in rats. Rats were given
intraperitoneal injections twice a week for four weeks with 30
mg/kg of oligonucleotide. The rats were sacrificed one day
following administration of the final dose of oligonucleotide. The
results are presented in Table 82. TABLE-US-00087 TABLE 83 Effects
of antisense compounds on total oligonucleotide concentration of
rats Total Oligonucleotide Concentrations (.mu.g/g) Kidney Liver
ISIS 116847 1450 900 ISIS 342351 1750 600 ISIS 331425 2950 500
[0378] Rats were also evaluated for the percentage of plasma
protein binding. A concentration of 2 .mu.M antisense
oligonucleotides was used in rat plasma. The results are presented
in Table 83. TABLE-US-00088 TABLE 84 Effects of antisense compounds
on plasma protein binding in rats Rat plasma protein binding (%
bound) ISIS 116847 97.1 .+-. 0.2 ISIS 342351 96.6 .+-. 0.1 ISIS
331425 95.5 .+-. 0.3 ISIS 335352 88.8 .+-. 2.0
[0379] All of the cited references including patents and patent
applications, as well as Genbank numbers, are herein incorporated
by reference in their entirety.
Sequence CWU 1
1
33 1 20 DNA Artificial Sequence Synthetic oligonucleotide 1
tccgtcatcg ctcctcaggg 20 2 20 DNA Artificial Sequence Synthetic
oligonucleotide 2 gtgcgcgcga gcccgaaatc 20 3 20 DNA Artificial
Sequence Synthetic oligonucleotide 3 atgcattctg cccccaagga 20 4 20
DNA Artificial Sequence Synthetic oligonucleotide 4 gaagtagcca
ccaactgtgc 20 5 20 DNA Artificial Sequence Synthetic
oligonucleotide 5 gagaacatat ccaccgagat 20 6 20 DNA Artificial
Sequence Synthetic oligonucleotide 6 ctgcacagtg tctgtgtaca 20 7
2454 DNA Mus musculus misc_feature 510, 657, 702, 741, 1231, 1370,
1426, 1432, 1677 n = A,T,C or G 7 agagaatgga gcaacacgta gaggcaggct
ctgaacttgg ggagcagaag gtcctgattg 60 ataatcctgc tgacattctg
gttatcgctg cctatttcct gctggtcatt ggtgttggct 120 tgtggtctat
gttcagaacc aatagaggca cagttggtgg ctacttcctg gcaggacgga 180
acatggtgtg gtggccggtt ggagcctctc tgttcgccag caacatcggc agcggtcatt
240 ttgtgggcct ggcagggact ggtgcagcaa gtggcttggc ggtggctgga
tttgagtgga 300 atgcgctctt cgtggtgctg ctcctcggat ggctttttgt
gccagtgtat ctgaccgctg 360 gcgtgatcac aatgcctcag tacctccgca
agcgctttgg tgggcaccgt attcgcctct 420 acctgtccgt gctctcgctt
tttttgtaca ttttcaccaa gatctcggtg gatatgttct 480 ctggggcagt
attcattcaa caggccctgn gctggaacat ttacgcttcg gtcatcgctc 540
tcttgggcat caccatgatt tatactgtga caggagggct ggcggcactg atgtacacag
600 acactgtgca gaccttcgtc attcttgccg gggcctccat cctcactggt
tatgctntcc 660 atgaagtggg cgggtacttc ggtctcttcg acacatacct
gngagcaatg acttcactga 720 cgggtgtcca ggatccatct ngtgggcaca
tctccagcac ctgctaccag ccgaggcctg 780 actcctatca cctgctgcgt
gaccctgtga caggagacct gccatggcct gcgctgctcc 840 tggggcttac
cattgtctcg ggctggtatt ggtgcagcga tcaggtaata gtgcagcggt 900
gcctggctgg aaagaatctg actcacatca aagctgggtg catcttgtgt ggctacctga
960 agctgatgcc catgttcctc atggtcatgc caggcatgat cagccgcatt
ctctacccag 1020 atgaggtggc atgtgtggta cctgaggtgt gtaagcgggt
gtgtggcact gaggtgggct 1080 gctctaacat cgcctaccca cagctcgtgg
tgaagctcat gcccaatggt ctgcgcggac 1140 tcatgctggc agtcatgctg
gctgccctca tgtcttctct ggcatccatc tttaacagca 1200 gtagcacgct
cttcaccatg gatatctaca ncgcgcctgc ggcccgtgca ggtgataagg 1260
agctgctgct agttggaagg ctctgggtgg tattcatcgt ggcggtgtcc gtggctcggc
1320 tgccagtggt gcaggcagct cagggtgggc agctcttcga ttacatcagn
tctgtctcca 1380 gctatctggc acctcaagtg tctgcggtct ttgtgcttgc
actctntgtg cnccgtgtta 1440 atgagaaggg agccttctgg ggactagttg
ggggcctgct gatgggccta gctcgtctca 1500 tacccgagtt cttctttggc
tcgggcagct gtgtgcgacc ctcagcgtgc ccggcactct 1560 tctgtcgggt
acactacctc tatttcgcca tcattctctt catctgctct ggcatcctca 1620
cactgggaat ctccctgtgc actggcccat cccctcagaa gcatctccat cgctggnttt
1680 tcagtctccg gcacagcaag gaggagcggg aggacctgga tgctgatgag
ttagaaggtc 1740 cagcccctgc tcctgtgcag aacgggggcc aggaatgtgc
aatggagatg gaagaggtcc 1800 agtccccggc tccaggcctg ctccgccggt
gcctgctttg gttctgtggg atgagcaaga 1860 gtgggtcagg gagtcctccg
cccactaccg aggaggtggc ggcaaccacc aggcggctgg 1920 aggacatcag
tgaggatccc cgctgggcac gagtagtcaa cctcaatgcc ctactcatga 1980
tgaccgtggc tgtgttcctc tggggcttct atgcataaag tcgagggtgt tggatgccat
2040 gagctacaac caggccatgt tggaccctca caaagagtaa gggtgagcag
cttggagtgg 2100 atcccagaaa aggaacaggg caagaataca gcaggaagga
accggttccc ttcctcttta 2160 cccggggtcc agtccatttg attggttgtc
acttcccaca agatgatggc caattggtca 2220 tagaggtttg cctatacaaa
aataaaactg ccctcctaac atcctgttgt ggctgaaaca 2280 tcgttgctct
cggcttcatc ctggtctctg ggctcctgtt ctgggtcctg ggcttggagc 2340
acggttgctc ataagacctt cttttctgga gacaagggcc atgtggccct ccactcatcc
2400 acctctagat ggtgtttctc cgtcttccag ccagcagcct gcagtccttt caag
2454 8 20 DNA Artificial Sequence Synthetic oligonucleotide 8
tcgatctcct tttatgcccg 20 9 24 DNA Artificial Sequence Primer 9
ctcgtctcat acccgagttc ttct 24 10 27 DNA Artificial Sequence Primer
10 aatgatggcg aaatagaggt agtgtac 27 11 19 DNA Artificial Sequence
Probe 11 tgcgaccctc agcgtgccc 19 12 15 DNA Artificial Sequence
Primer 12 tcgccgcttg ctgca 15 13 17 DNA Artificial Sequence Primer
13 atcggccgtg atgtcga 17 14 23 DNA Artificial Sequence Probe 14
ccatggtcaa ccccaccgtg ttc 23 15 25 DNA Artificial Sequence
Synthetic oligonucleotide 15 atggagcaac acgtagaggc aggct 25 16 25
DNA Artificial Sequence Synthetic oligonucleotide 16 gagtgccgcc
agccctcctg tcaca 25 17 20 DNA Artificial Sequence Synthetic
oligonucleotide 17 ccttccctga aggttcctcc 20 18 20 DNA Artificial
Sequence Synthetic oligonucleotide 18 ccttccctga aggttcctcc 20 19
20 DNA Artificial Sequence Synthetic oligonucleotide 19 ccacaagctg
tccagtctaa 20 20 20 DNA Artificial Sequence Primer 20 tggattcccg
ttcgagtacg 20 21 20 DNA Artificial Sequence Primer 21 tcagctggat
agcgacatcg 20 22 20 DNA Artificial Sequence Probe 22 aagcgagggc
tccgacccga 20 23 22 DNA Artificial Sequence Primer 23 agaccaacaa
gcaagtgagt gc 22 24 24 DNA Artificial Sequence Primer 24 ctagcatgtg
agccacattc atcc 24 25 21 DNA Artificial Sequence Probe 25
ctgctgaggg cacgctgagc t 21 26 21 DNA Artificial Sequence Primer 26
gctcagggta aggtccgatt c 21 27 15 DNA Artificial Sequence Primer 27
gccccccacc ccaaa 15 28 29 DNA Artificial Sequence Probe 28
tcataatcaa agaagcagca agcacttcc 29 29 20 DNA Artificial Sequence
Synthetic oligonucleotide 29 ctgctagcct ctggatttga 20 30 20 DNA
Artificial Sequence Synthetic oligonucleotide 30 ctgctagcct
ctggatttga 20 31 20 DNA Artificial Sequence Synthetic
oligonucleotide 31 ctgctagcct ctggatttga 20 32 20 DNA Artificial
Sequence Synthetic oligonucleotide 32 ctgctagcct ctggatttga 20 33
20 DNA Artificial Sequence Synthetic oligonucleotide 33 ctgctagcct
ctggatttga 20
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