U.S. patent application number 14/639312 was filed with the patent office on 2015-11-05 for compounds and methods for improving cellular uptake of oligomeric compounds.
This patent application is currently assigned to ISIS PHARMACEUTICALS, INC.. The applicant listed for this patent is Isis Pharmaceuticals, Inc.. Invention is credited to C. Frank Bennett, Erich Koller.
Application Number | 20150315580 14/639312 |
Document ID | / |
Family ID | 40521685 |
Filed Date | 2015-11-05 |
United States Patent
Application |
20150315580 |
Kind Code |
A1 |
Koller; Erich ; et
al. |
November 5, 2015 |
COMPOUNDS AND METHODS FOR IMPROVING CELLULAR UPTAKE OF OLIGOMERIC
COMPOUNDS
Abstract
The present invention provides method of optimizing the efficacy
and potency of antisense drugs. In certain embodiments, the
invention provides assays useful for determining favorable
oligonucleotide characteristics and excipeints for improved
cellular uptake.
Inventors: |
Koller; Erich; (Basel,
CH) ; Bennett; C. Frank; (Carlsbad, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Isis Pharmaceuticals, Inc. |
Carlsbad |
CA |
US |
|
|
Assignee: |
ISIS PHARMACEUTICALS, INC.
Carlsbad
CA
|
Family ID: |
40521685 |
Appl. No.: |
14/639312 |
Filed: |
March 5, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13658544 |
Oct 23, 2012 |
8999951 |
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14639312 |
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12681593 |
Jul 30, 2010 |
8318496 |
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PCT/US2008/078956 |
Oct 6, 2008 |
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13658544 |
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60977598 |
Oct 4, 2007 |
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Current U.S.
Class: |
514/44A |
Current CPC
Class: |
A61P 1/16 20180101; C12Q
1/025 20130101; C12N 2320/10 20130101; C12N 2320/32 20130101; C12N
2310/315 20130101; C12N 15/113 20130101; C12N 2310/3517 20130101;
C12N 2310/11 20130101; C12N 15/111 20130101; C12N 2310/321
20130101 |
International
Class: |
C12N 15/113 20060101
C12N015/113 |
Claims
1.-20. (canceled)
21. A formulation comprising an antisense oligomeric compound and
an excipient, wherein the excipient increases cellular uptake of
the oligomeric compound.
22. The formulation of claim 21 wherein the excipient
preferentially binds to a non-productive mechanism of cellular
accumulation.
23.-26. (canceled)
27. The method of claim 21, wherein the oligomeric compound is a
modified oligonucleotide.
28. The method of claim 27, wherein the oligomeric compound
consists of 17 linked nucleosides.
29. The method of claim 27, wherein the oligomeric compound
consists of 18 linked nucleosides.
30. The method of claim 27, wherein the oligomeric compound
consists of 19 linked nucleosides.
31. The method of claim 27, wherein the oligomeric compound
consists of 20 linked nucleosides.
32. The method of claim 27, wherein the oligomeric compound
consists of 21 linked nucleosides.
33. The method of claim 27, wherein the oligomeric compound
comprises a nucleoside comprising a 2' modification.
34. The method of claim 33, wherein the 2'-modification is selected
from among: allyl, amino, azido, thio, O-allyl, O--C.sub.1-C.sub.10
alkyl, --OCF.sub.3, O--(CH.sub.2).sub.2-O--CH.sub.3,
2'-O(CH.sub.2).sub.2SCH.sub.3,
O--(CH.sub.2).sub.2--O--N(R.sub.m)(R.sub.n), or
O--CH.sub.2--C(.dbd.O)--N(R.sub.m)(R.sub.n), where each R.sub.m and
R.sub.n is, independently, H or substituted or unsubstituted
C.sub.1-C.sub.10 alkyl.
35. The method of claim 33, wherein the 2'-modification is
2'-O--(CH.sub.2).sub.2--O--CH.sub.3.
36. The method of claim 33, wherein the 2'-modification is
2'-OCH.sub.3.
37. The method of claim 27, wherein the oligomeric compound
comprises a bicyclic nucleic acid.
38. The method of claim 27, wherein the oligomeric compound is a
gapmer.
39. The method of claim 38, wherein the oligomeric compound is a
2-8-3 MOE-DNA gapmer.
40. The method of claim 27, wherein the oligomeric compound is
fully modified.
41. The method of claim 21, wherein the excipient is not a cationic
lipid.
42. The method of claim 41, wherein the excipient is dextran.
43. The method of claim 41, wherein the excipient is dextran
sulfate.
44. The method of claim 27, wherein the oligomeric compound
comprises at least 1 phosphorothioate internucleoside linkage.
Description
FIELD OF THE INVENTION
[0001] The present invention provides a method of optimizing the
efficacy and potency of antisense drugs. This method is achieved by
implementing assays described herein useful for determining
favorable oligonucleotide characteristics for cellular uptake.
BACKGROUND OF THE INVENTION
[0002] There are desirable molecular targets for drug discovery
that are considered "undruggable" by traditional small molecule
technology. Such targets are often members of families of closely
related proteins that are too similar in structure for traditional
drugs to distinguish amongst, the biological function of the
protein is unknown, or it is difficult to develop an assay for drug
screening. Antisense drugs discriminate between targets based on
their genetic sequence, so certain such "undruggable" targets may
be amenable to antisense.
SUMMARY OF THE INVENTION
[0003] In certain embodiments, the present invention provides
methods comprising contacting a cell with a reporter oligomeric
compound and a competitor oligomeric compound and detecting
antisense activity of the reporter oligomeric compound.
[0004] In certain embodiments, the present invention provides
methods comprising preparing two or more test samples wherein each
test sample comprises a cell, a concentration of a reporter
oligomeric compound, and a concentration of a competitor oligomeric
compound, wherein;
[0005] the concentration of the reporter oligomeric compound is the
same in each of the two or more test samples and the concentration
of the competitor oligomeric compound is different in each of the
two or more test samples or the concentration of the reporter
oligomeric compound is different in each two or more test samples
and the concentration of the competitor oligomeric compound is
different in each of the two or more test samples; and
[0006] detecting antisense activity of the reporter oligomeric
compound in each of the two or more test samples.
[0007] In certain such embodiments, the concentration of the
reporter oligomeric compound is the same in each of the two or more
test samples and the concentration of the competitor oligomeric
compound is different in each of the two or more test samples.
[0008] In certain such embodiments, the concentration of the
reporter oligomeric compound is different in each two or more test
samples and the concentration of the competitor oligomeric compound
is different in each of the two or more test sample.
[0009] In certain embodiments, the invention provides methods of
assessing the relative uptake of a first and a second competitor
oligomeric compounds comprising:
[0010] preparing two or more first test samples wherein each first
test sample comprises a cell, a concentration of a reporter
oligomeric compound, and a concentration of a first competitor
oligomeric compound, wherein;
[0011] the concentration of the reporter oligomeric compound is the
same in each of the two or more first test samples and the
concentration of the first competitor oligomeric compound is
different in each of the two or more first test samples or the
concentration of the reporter oligomeric compound is different in
each two or more first test samples and the concentration of the
first competitor oligomeric compound is different in each of the
two or more first test samples;
[0012] preparing two or more second test samples wherein each
second test sample comprises a cell, a concentration of the
reporter oligomeric compound, and a concentration of a second
competitor oligomeric compound, wherein;
[0013] the concentration of the reporter oligomeric compound is the
same in each of the two or more second test samples and the
concentration of the second competitor oligomeric compound is
different in each of the two or more second test samples or the
concentration of the reporter oligomeric compound is different in
each two or more second test samples and the concentration of the
second competitor oligomeric compound is different in each of the
two or more second test samples;
[0014] detecting antisense activity of the reporter oligomeric
compound in each of the two or more first test samples and each of
the two or more second test samples; and
[0015] thereby assessing relative uptake of the first and second
competitor oligomeric compound.
[0016] In certain such embodiments, the concentration of the
reporter oligomeric compound is different in each of the two or
more first test samples and in each of the two or more second test
samples and the concentration of the first competitor oligomeric
compound is different in each of the two or more first test samples
and the concentration second competitor oligomeric compound is
different in each of the two or more test samples.
[0017] In certain embodiments, the concentrations of first
competitor oligomeric compound in the first test samples are the
same as the concentrations of the second competitor oligomeric
compound in the second test samples.
[0018] In certain embodiments, the concentration of the reporter
oligomeric compound is the same in each of the two or more first
test samples and in each of the two or more second test samples and
the concentration of the first competitor oligomeric compound is
the same in each of the two or more first test samples and the
concentration second competitor oligomeric compound is the same in
each of the two or more test samples.
[0019] In certain embodiments, the concentration of first
competitor oligomeric compound in the first test samples is the
same as the concentration of the second competitor oligomeric
compound in the second test samples.
[0020] In certain embodiments, the cell is a hepatocyte; a primary
hepatocyte, or MHT cell.
[0021] In certain embodiments, the contacting occurs in the absence
of cationic lipid.
[0022] In certain embodiments, the contacting occurs in the
presence of dextran sulfate.
[0023] In certain embodiments, the present invention provides
methods for assessing cellular uptake of an oligomeric compound
comprising contacting an MHT cell with the oligomeric compound in
the absence of cationic lipid.
[0024] In certain embodiments, the present invention provides kits
comprising cells, and one or more oligomeric compounds. In certain
such embodiments, the cells are MHT cells. In certain embodiments,
the present invention provides formulations comprising an antisense
oligomeric compound and an excipient, wherein the excipient
saturates a mechanism of unproductive accumulation in a cell. In
certain such embodiments, the excipient comprises an oligomeric
compound. In certain embodiments, the oligomeric compound of the
excipient is not an antisense oligomeric compound.
DETAILED DESCRIPTION OF THE INVENTION
[0025] It is to be understood that both the foregoing general
description and the following detailed description are exemplary
and explanatory only and are not restrictive of the invention, as
claimed. Herein, the use of the singular includes the plural unless
specifically stated otherwise. As used herein, the use of "or"
means "and/or" unless stated otherwise. Furthermore, the use of the
term "including" as well as other forms, such as "includes" and
"included", is not limiting. Also, terms such as "element" or
"component" encompass both elements and components comprising one
unit and elements and components that comprise more than one
subunit, unless specifically stated otherwise.
[0026] The section headings used herein are for organizational
purposes only and are not to be construed as limiting the subject
matter described. All documents, or portions of documents, cited in
this application, including, but not limited to, patents, patent
applications, articles, books, and treatises, are hereby expressly
incorporated by reference in their entirety for any purpose.
[0027] A. Definitions
[0028] Unless specific definitions are provided, the nomenclature
utilized in connection with, and the procedures and techniques of,
analytical chemistry, synthetic organic chemistry, and medicinal
and pharmaceutical chemistry described herein are those well known
and commonly used in the art. Standard techniques may be used for
chemical synthesis, and chemical analysis. Certain such techniques
and procedures may be found for example in "Carbohydrate
Modifications in Antisense Research" Edited by Sangvi and Cook,
American Chemical Society , Washington D.C., 1994; "Remington's
Pharmaceutical Sciences," Mack Publishing Co., Easton, Pa., 18th
edition, 1990; and "Antisense Drug Technology, Principles,
Strategies, and Applications" Edited by Stanley T. Crooke, CRC
Press, Boca Raton, Fla.; and Sambrook et al., "Molecular Cloning, A
laboratory Manual," 2.sup.nd Edition, Cold Spring Harbor Laboratory
Press, 1989, which are hereby incorporated by reference for any
purpose. Where permitted, all patents, applications, published
applications and other publications and other data referred to
throughout in the disclosure herein are incorporated by reference
in their entirety.
[0029] Unless otherwise indicated, the following terms have the
following meanings:
[0030] As used herein, the term "nucleoside" means a glycosylamine
comprising a nucleobase and a sugar. Nucleosides include, but are
not limited to, naturally occurring nucleosides, abasic
nucleosides, modified nucleosides, and nucleosides having mimetic
bases and/or sugar groups.
[0031] As used herein, the term "nucleobase" refers to the base
portion of a nucleoside. A nucleobase may comprise any atom or
group of atoms capable of hydrogen bonding to a base of another
nucleic acid.
[0032] As used herein, the term "deoxyribonucleotide" means a
nucleotide having a hydrogen at the 2' position of the sugar
portion of the nucleotide. Deoxyribonucleotides may be modified
with any of a variety of substituents.
[0033] As used herein, the term "ribonucleotide" means a nucleotide
having a hydroxy at the 2' position of the sugar portion of the
nucleotide. Ribonucleotides may be modified with any of a variety
of substituents.
[0034] As used herein, the term "oligomeric compound" refers to a
polymeric structure comprising two or more sub-structures and
capable of hybridizing to a region of a nucleic acid molecule. In
certain embodiments, oligomeric compounds are oligonucleotides. In
certain embodiments, oligomeric compounds are antisense compounds.
In certain embodiments, oligomeric compounds are antisense
oligonucleotides. In certain embodiments, oligomeric compounds are
chimeric oligonucleotides.
[0035] As used herein, the term "reporter oligomeric compound"
refers to an oligomeric compound that that is at least partially
complementary to and capable of hybridizing with a target nucleic
acid molecule, wherein if hybridization occurs, some detectable
change results. In certain embodiments, hybridization of a reporter
oligomeric compound to its target nucleic acid molecule modulates
(increases or decreases) expression of a target nucleic acid,
resulting in more or less target protein. In certain such
embodiments, the presence or amount of target protein is detectable
and/or measurable. In certain embodiments, hybridization of a
reporter oligomeric compound to a target nucleic acid is detectable
by Northern Blot, by fluorescence or by the presence of a splice
variant that would not be present or would be present at a
different amount in the absence of hybridization of the reporter
oligomeric compound with its target nucleic acid molecule. Reporter
oligomeric compounds include, but are not limited to, compounds
that are oligonucleotides, oligonucleosides, oligonucleotide
analogs, oligonucleotide mimetics, and chimeric combinations of
these. In certain embodiments, a reporter oligomeric compound
comprises an oligonucleotide linked to a conjugate. In certain
embodiments, the reporter oligomeric compound comprises a sequence
or motif the cellular uptake of which is to be assessed.
[0036] The term "competitor oligomeric compound" refers to an
oligomeric compound that is not identical to and does not hybridize
with the reporter oligomeric compound. In certain embodiments, the
competitor oligomeric compound comprises a sequence or motif the
cellular uptake of which is to be assessed.
[0037] As used herein, the term "oligonucleotide" refers to an
oligomeric compound comprising a plurality of linked nucleosides.
In certain embodiment, one or more nucleosides of an
oligonucleotide is modified. In certain embodiments, an
oligonucleotide comprises ribonucleic acid (RNA) or
deoxyribonucleic acid (DNA). In certain embodiments,
oligonucleotides are composed of naturally-and/or
non-naturally-occurring nucleobases, sugars and covalent
internucleoside linkages, and may further include non-nucleic acid
conjugates.
[0038] As used herein "oligonucleoside" refers to an
oligonucleotide in which the internucleoside linkages do not
contain a phosphorus atom.
[0039] As used herein "internucleoside linkage" refers to a
covalent linkage between adjacent nucleosides.
[0040] As used herein, the term "antisense compound" refers to an
oligomeric compound that is at least partially complementary to a
target nucleic acid molecule to which it hybridizes. In certain
embodiments, an antisense compound modulates (increases or
decreases) expression of a target nucleic acid. Antisense compounds
include, but are not limited to, compounds that are
oligonucleotides, oligonucleosides, oligonucleotide analogs,
oligonucleotide mimetics, and chimeric combinations of these.
Consequently, while all antisense compounds are oligomeric
compounds, not all oligomeric compounds are antisense
compounds.
[0041] As used herein, the term "antisense activity" refers to any
detectable and/or measurable activity attributable to the
hybridization of an antisense compound to its target nucleic acid.
Such detection and or measuring may be direct or indirect. For
example, in certain embodiments, antisense activity is assessed by
detecting and or measuring the amount of target protein. In certain
embodiments, antisense activity is assessed by detecting and/or
measuring the amount of target nucleic acids and/or cleaved target
nucleic acids and/or alternatively spliced target nucleic
acids.
[0042] As used herein the term "detecting antisense activity" or
"measuring antisense activity" means that a test for detecting or
measuring antisense activity is performed on a particular sample
and compared to that of a control sample. Such detection and/or
measuring may include values of zero. Thus, if a test for detection
of antisense activity results in a finding of no antisense activity
(antisense activity of zero), the step of "detecting antisense
activity" has nevertheless been performed.
[0043] As used herein the term "control sample" refers to a sample
that has not been contacted with a reporter oligomeric
compound.
[0044] As used herein, the term "various concentrations" or
"different concentrations" can include concentrations of zero.
Thus, the statement that an assay component was tested at various
concentrations may include samples where that component is
absent.
[0045] As used herein, the term "uptake" or "taken up" refers to
the ability of an oligomeric compound to enter a cell in a way that
allows antisense activity.
[0046] As used herein, the term "accumulate" refers to the ability
of an oligomeric compound to enter a cell, whether or not it is
available for antisense activity. For example, if an oligomeric
compound enters a cell, but is localized such that it is shielded
from its target nucleic acid and no antisense activity is detected,
the oligomeric compound has "accumulated" in the cell, but has not
been "taken up."
[0047] As used herein, the term "antisense oligonucleotide" refers
to an antisense compound that is an oligonucleotide.
[0048] As used herein, the term "test sample" refers to a sample
suitable for testing and includes, but is not limited to samples in
wells of multiwell plates, petri dishes, test tubes, or assay
kits.
[0049] As used herein, the term "motif" refers to a pattern of
unmodified and modified nucleosides and/or linkages in an
oligomeric compound. In certain embodiments, a motif can be
described using a shorthand nomenclature comprising a series of
numbers where each number represents the number of nucleosides of
an oligomeric compound comprising a particular modification, where
the first number represents the number of nucleosides of a type
starting at the 5' end of the oligomeric compound. For example: a
2-8-3 MOE-DNA gapmer is an oligonucleotide wherein the two 5'
terminal nucleosides are MOE-substituted nucleosides, the next
eight nucleosides are unsubstituted DNA, and the three 3' terminal
nucleosides are MOE-substituted nucleosides. Linkage modifications
can likewise be identified, for example the above 2-8-3 MOE gapmer
could also have a 3-2-3-2-2 alternating
phosphorothioate/phosphodiester, mixed backbone, wherein the first
three linkages starting at the 5' end (i.e., the linkages between
the first and second nucleoside, the second and third nucleoside,
and the third and fourth nucleoside) are each phosphorothioate, the
next two are phosphodiester, the next three are phosphorothioate,
the next two are phosphodiester, and the final two are
phosphorothioate.
[0050] As used herein, the term "target protein" refers to a
protein, the modulation of which is desired or which is suitable
for testing.
[0051] As used herein, the term "target gene" refers to a gene
encoding a target protein.
[0052] As used herein, the terms "target nucleic acid" refers to
any nucleic acid molecule the expression or activity of which is
capable of being modulated by an antisense compound or a reporter
oligomeric compound, including endogenous and exogenously
introduced nucleic acids. Target nucleic acids include, but are not
limited to, RNA (including, but not limited to pre-mRNA and mRNA or
portions thereof) transcribed from DNA encoding a target, and also
cDNA derived from such RNA, and miRNA. For example, the target
nucleic acid can be a cellular gene (or mRNA transcribed from the
gene) whose expression is associated with a particular disorder or
disease state, or a nucleic acid molecule from an infectious
agent.
[0053] As used herein, the term "nucleobase complementarity" refers
to a nucleobase that is capable of base pairing with another
nucleobase. For example, in DNA, adenine (A) is complementary to
thymine (T). For example, in RNA, adenine (A) is complementary to
uracil (U). In certain embodiments, complementary nucleobase refers
to a nucleobase of an antisense compound that is capable of base
pairing with a nucleobase of its target nucleic acid. For example,
if a nucleobase at a certain position of an antisense compound is
capable of hydrogen bonding with a nucleobase at a certain position
of a target nucleic acid, then the position of hydrogen bonding
between the oligonucleotide and the target nucleic acid is
considered to be complementary at that nucleobase pair.
[0054] As used herein, the term "non-complementary nucleobase"
refers to a pair of nucleobases that do not form hydrogen bonds
with one another or otherwise support hybridization.
[0055] As used herein, the term "complementary" refers to the
capacity of an oligomeric compound to hybridize to another
oligomeric compound or nucleic acid through nucleobase
complementarity. In certain embodiments, an antisense compound and
its target are complementary to each other when a sufficient number
of corresponding positions in each molecule are occupied by
nucleobases that can bond with each other to allow stable
association between the antisense compound and the target. One
skilled in the art recognizes that the inclusion of mismatches is
possible without eliminating the ability of the oligomeric
compounds to remain in association. Therefore, described herein are
antisense compounds that may comprise up to about 20% nucleotides
that are mismatched (i.e., are not nucleobase complementary to the
corresponding nucleotides of the target). Preferably the antisense
compounds contain no more than about 15%, more preferably not more
than about 10%, most preferably not more than 5% or no mismatches.
The remaining nucleotides are nucleobase complementary or otherwise
do not disrupt hybridization (e.g., universal bases). One of
ordinary skill in the art would recognize the compounds provided
herein are at least 80%, at least 85%, at least 90%, at least 95%,
at least 96%, at least 97%, at least 98%, at least 99% or 100%
complementary to a target nucleic acid.
[0056] As used herein, "hybridization" means the pairing of
complementary oligomeric compounds (e.g., an antisense compound and
its target nucleic acid). While not limited to a particular
mechanism, the most common mechanism of pairing involves hydrogen
bonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteen
hydrogen bonding, between complementary nucleoside or nucleotide
bases (nucleobases). For example, the natural base adenine is
nucleobase complementary to the natural nucleobases thymidine and
uracil which pair through the formation of hydrogen bonds. The
natural base guanine is nucleobase complementary to the natural
bases cytosine and 5-methyl cytosine. Hybridization can occur under
varying circumstances.
[0057] As used herein, the term "specifically hybridizes" refers to
the ability of an oligomeric compound to hybridize to one nucleic
acid site with greater affinity than it hybridizes to another
nucleic acid site. In certain embodiments, an antisense
oligonucleotide specifically hybridizes to more than one target
site.
[0058] As used herein, the term "expression" refers to all the
functions and steps by which a gene's coded information is
converted into structures present and operating in a cell. Such
structures include, but are not limited to the products of
transcription and translation.
[0059] As used herein, the term "2'-modified" or "2'-substituted"
means a sugar comprising substituent at the 2' position other than
H or OH. 2'-modified monomers, include, but are not limited to,
BNA's and monomers (e.g., nucleosides and nucleotides) with
2'-substituents, such as allyl, amino, azido, thio, O-allyl
O--C.sub.1-C.sub.10 alkyl --OCF.sub.3,
O--(CH.sub.2).sub.2--O--CH.sub.3, 2'-O(CH.sub.2).sub.2SCH.sub.3,
O--(CH.sub.2).sub.2--O--N(R.sub.m)(R.sub.n), or
O--CH.sub.2--C(.dbd.O)--N(R.sub.m)(R.sub.n), where each R.sub.m and
R.sub.n is, independently, H or substituted or unsubstituted
C.sub.1-C.sub.10 alkyl. In certain embodiments, oligomeric
compounds comprise a 2' modified monomer that does not have the
formula 2'-O(CH.sub.2).sub.nH, wherein n is one to six. In certain
embodiments, oligomeric compounds comprise a 2' modified monomer
that does not have the formula 2'-OCH.sub.3 In certain embodiments,
oligomeric compounds comprise a 2' modified monomer that does not
have the formula or, in the alternative,
2'-O(CH.sub.2).sub.2OCH.sub.3.
[0060] As used herein, the term "bicyclic nucleic acid" or "BNA" or
"bicyclic nucleoside" or "bicyclic nucleotide" refers to a
nucleoside or nucleotide wherein the furanose portion of the
nucleoside includes a bridge connecting two carbon atoms on the
furanose ring, thereby forming a bicyclic ring system.
[0061] As used herein, unless otherwise indicated, the term
"methyleneoxy BNA" alone refers to .beta.-D-methyleneoxy BNA. As
used herein, the term "MOE" refers to a 2'-O-methoxyethyl
substituent.
[0062] As used herein, the term "gapmer" refers to a chimeric
oligomeric compound comprising a central region (a "gap") and a
region on either side of the central region (the "wings"), wherein
the gap comprises at least one modification that is different from
that of each wing. Such modifications include nucleobase, monomeric
linkage, and sugar modifications as well as the absence of
modification (unmodified). Thus, in certain embodiments, the
nucleotide linkages in each of the wings are different than the
nucleotide linkages in the gap. In certain embodiments, each wing
comprises nucleotides with high affinity modifications and the gap
comprises nucleotides that do not comprise that modification. In
certain embodiments the nucleotides in the gap and the nucleotides
in the wings all comprise high affinity modifications, but the high
affinity modifications in the gap are different than the high
affinity modifications in the wings. In certain embodiments, the
modifications in the wings are the same as one another. In certain
embodiments, the modifications in the wings are different from each
other. In certain embodiments, nucleotides in the gap are
unmodified and nucleotides in the wings are modified. In certain
embodiments, the modification(s) in each wing are the same. In
certain embodiments, the modification(s) in one wing are different
from the modification(s) in the other wing. In certain embodiments,
oligomeric antisense compounds are gapmers having
2'-deoxynucleotides in the gap and nucleotides with high-affinity
modifications in the wing.
[0063] As used herein, the term "cap structure" or "terminal cap
moiety" refers to chemical modifications, which have been
incorporated at either terminus of an antisense compound.
[0064] B. Cellular Uptake of Oligomeric Compounds
[0065] Oligomeric compounds designed to hybridize to specific mRNA
sequences have been used to inhibit the function of a number of
cellular and viral proteins. Thus, if the DNA sequence of a protein
of interest is known, it is possible to design complementary
oligomeric compounds that bind the target RNA and inhibit
expression of the protein. Unmodified oligo-deoxyribonucleotides
have been used to inhibit the expression of several viral and
cellular encoded proteins in cell culture. However, these compounds
are unstable in biological fluids and display poor cellular uptake
characteristics (Bennett et al., Molecular Pharmacology, 41, 1023
(1992)). Cellular uptake of unmodified oligo-deoxynucleotides is
poorly understood, but appears to involve binding to acceptor sites
on a tissue surface responsible for facilitating uptake (Crooke S.
T., Antisense Drug Technology, Second Ed. (2008), Chapter 6, CRC
Press, Florida).
[0066] To address uptake limitations, numerous modifications of the
oligonucleotide phosphodiester backbone have been performed to
decrease the sensitivity of the oligonucleotides to nucleases
and/or to increase cellular uptake. Examples of such chemistry
modifications include, for example, phosphorothioates and
2'-methoxyethyl (2'MOE) (Bennett et al., Molecular Pharmacology,
41, 1023 (1992).
[0067] It is known that uptake varies according to oligonucleotide
type (Crooke, S. T., Current Opinion in Biotechnology, 3, 656
(1992)). For example, the data suggest that phosphorothioate
oligonucleotides are taken up by receptor-mediated endocytosis.
Localization studies using fluorescently labeled oligonucleotides
support this notion (Crooke, S. T., Current Opinion in
Biotechnology, 3, 656 (1992)).
[0068] Uptake affects the pharmacological activity of oligomeric
compounds. Pharmacological activity depends on a number of factors
that influence the effective concentration of these agents at
specific nuclear or cytoplasmic receptors. A key factor is the
ability of antisense compounds to traverse the plasma membrane of
specific cells involved in disease processes (Crooke, R. M.,
Anti-cancer Drug Design, 6, 609 (1992)).
[0069] In certain embodiments, the present invention provides
methods for determining favorable characteristics for cellular
uptake. Variables such as chemical modifications and sequence may
affect cellular uptake. It is advantageous to ascertain the
particular characteristics favored by a cell in order to optimize
cellular uptake. By optimizing cellular uptake, the efficacy and
potency of antisense drugs can be increased.
[0070] C. Certain Cells
[0071] In certain embodiments, the present invention provides cells
useful for assessing uptake of oligomeric compounds. Many mammalian
cell lines accumulate oligomeric compounds when such oligomeric
compounds are added to culture media. However, in those cultured
cell lines, antisense oligomeric compounds typically do not
demonstrate antisense activity, perhaps resulting from localization
within the cells that prevents the antisense oligomeric compounds
from contacting their target nucleic acids. To observe and test
antisense activity in cell lines, investigators typically must add
cationic lipids. See e.g., Bennett et al., Molecular Pharmacology,
41:1023-1033(1992). In vivo, though, administration of antisense
oligomeric compounds in only saline has produced sequence specific
antisense activity. Thus, it appears that there is a difference
between uptake of oligomeric compounds in cell lines and in cells
in vivo. Accordingly, it is difficult to assess, for example, the
effect of different sequences, chemical modifications, or motifs on
uptake of oligomeric compounds, since assays using cell lines are
not expected to yield meaningful results.
[0072] In certain embodiments, the present invention provides
primary cells that demonstrate antisense activity when antisense
oligomeric compounds are added without cationic lipids. In certain
embodiments, such cells are hepatocytes, macrophages, or
keratinocytes. In certain embodiments, such cells are primary liver
cells. In certain embodiments, cells are primary hepatocytes. In
certain embodiments, such cells are mouse or human primary
hepatocytes. In certain embodiments, primary hepatocytes are useful
for assessing uptake of oligomeric compounds for up to 24 hours. In
certain embodiments, primary hepatocytes are useful for assessing
uptake of oligomeric compounds for up to 48 hours. In certain
embodiments, primary hepatocytes lose their ability to demonstrate
antisense activity after about 24 to 30 hours. Certain such uses of
primary hepatocytes may be found, for example, in U.S. application
Ser. No. 11/221,001, which is hereby incorporated by reference in
its entirety for any and all purposes.
[0073] In certain embodiments, the present invention provides a
cell line that is useful for assessing uptake of oligomeric
compounds. In certain embodiments, the invention provides a
hepatocellular carcinoma cell line derived from a primary tumor
that retains the ability to uptake oligomeric compounds. In certain
such embodiments, the cell line is derived from an SV40 large T
antigen tumor. In certain embodiments, the cell line is derived
from a mouse. In certain embodiments, the cell line is MHT. In
certain embodiments, such MHT cells retain the ability to uptake
oligomeric compounds for 20 or more passages. In certain
embodiments, such cells demonstrate antisense activity when
contacted with an antisense oligomeric compound. In certain
embodiments, such cells are contacted with a reporter oligomeric
compound and antisense activity is detected and/or measured.
[0074] D. Certain Assays
[0075] In certain embodiments, the invention provides assays for
assessing uptake of oligomeric compounds. In certain embodiments,
such assays involve (1) plating cells, for example MHT cells, on a
multiwell plate (2) adding varying concentrations of a reporter
oligomeric compound or no oligomeric compound to each plate, and
(3) detecting antisense activity. In certain embodiments, such an
assay can be performed in parallel with several reporter oligomeric
compounds. For example, the several reporter oligomeric compounds
may have the same sequence, but differ in type, number or position
of chemical modifications or in length or presence or type of
conjugate group. Or the such several reporter oligomeric compounds
may have the same target nucleic acid, but different sequences.
Thus, in certain embodiments, the invention provides ways of
comparing oligomeric compounds.
[0076] In certain embodiments, the invention provides a competition
assay useful for assessing uptake of oligomeric compounds. In
certain such embodiments, (1) cells, for example MHT cells, are
plated on a multiwell plate; (2) a competitor oligomeric compound
is added to each plate at the same concentration per well; (3) a
reporter oligomeric compound is added to each of several wells,
typically at several different concentrations; and (4) antisense
activity is detected and/or measured. In certain embodiments, such
assays are useful for assessing the relative uptake of the reporter
oligomeric compound and the competitor oligomeric compound. In
certain embodiments, the concentration of the competitor oligomeric
compound is varied and the concentration of the reporter oligomeric
compound is the same for each well. In certain embodiments, two or
more competitor oligomeric compounds are separately tested against
the same reporter oligomeric compound to assess the relative uptake
of the two or more competitor oligomeric compounds. In certain
embodiments, two or more reporter oligomeric compounds are used and
the antisense activity of each of them is measured. In such
embodiments, the assay may or may not include a competitor
oligomeric compound. One of ordinary skill in the art will readily
appreciate that these components can be manipulated in a variety of
ways.
[0077] E. Certain Oligomeric Compounds
[0078] As described above, in certain embodiments, the invention
provides assays for determining relative uptake of oligomeric
compounds. Such assays are useful for generating a structure
activity relationship to better design oligomeric compounds for use
in vivo, including therapeutic uses. Accordingly, the oligomeric
compounds suitable for assays of the present invention may comprise
any known or newly designed chemical modifications including,
without limitation, nucleoside modifications, including sugar
modifications and base modifications, and modified internucleoside
linkages, including oligomeric compounds with mixed backbones. Also
suitable for testing in assays of the present invention are the
effects of various caps, conjugates or terminal groups. Also
suitable for testing in assays of the present invention are motifs
varying the type, number and/or position of chemical modifications.
Examples of motifs include, but are not limited to, gapmers,
hemimers, blockmers, alternating and positional motifs all with any
number and/or type of modifications in the various regions. Certain
modifications and motifs suitable for oligomeric compounds for use
in the assays of the present invention may be found, for example,
in pending U.S. application Ser. No. 11/745,429; PCT/US2007/068404;
and U.S. application Ser. No. 11/221,001, which are incorporated by
reference in their entirety. Also suitable for testing in assays of
the present invention are oligomeric compounds of differing
lengths. Assays of the present invention may also be used to assess
whether similarly modified oligomeric compounds with different
sequences are preferentially taken up. Such modifications and
combinations of modifications may be incorporated into one or more
reporter oligomeric compound and/or one or more competitor
oligomeric compound.
[0079] In certain embodiments, the present invention provides
oligomeric compounds of any of a variety of ranges of lengths. In
certain embodiments, the invention provides oligomeric compounds
comprising oligonucleotides consisting of X to Y linked
nucleosides, where X and Y are each independently selected from 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, and 50; provided that X.ltoreq.Y. For
example, in certain embodiments, the invention provides oligomeric
compounds, including, but not limited to reporter oligomeric
compounds and competitor oligomeric compounds, which comprise
oligonucleotides consisting of 8 to 9, 8 to 10, 8 to 11, 8 to 12, 8
to 13, 8 to 14, 8 to 15, 8 to 16, 8 to 17, 8 to 18, 8 to 19, 8 to
20, 8 to 21, 8 to 22, 8 to 23, 8 to 24, 8 to 25, 8 to 26, 8 to 27,
8 to 28, 8 to 29, 8 to 30, 9 to 10, 9 to 11, 9 to 12, 9 to 13, 9 to
14, 9 to 15, 9 to 16, 9 to 17, 9 to 18, 9 to 19, 9 to 20, 9 to 21,
9 to 22, 9 to 23, 9 to 24, 9 to 25, 9 to 26, 9 to 27, 9 to 28, 9 to
29, 9 to 30, 10 to 11, 10 to 12, 10 to 13, 10 to 14, 10 to 15, 10
to 16, 10 to 17, 10 to 18, 10 to 19, 10 to 20, 10 to 21, 10 to 22,
10 to 23, 10 to 24, 10 to 25, 10 to 26, 10 to 27, 10 to 28, 10 to
29, 10 to 30, 11 to 12, 11 to 13, 11 to 14, 11 to 15, 11 to 16, 11
to 17, 11 to 18, 11 to 19, 11 to 20, 11 to 21, 11 to 22, 11 to 23,
11 to 24, 11 to 25, 11 to 26, 11 to 27, 11 to 28, 11 to 29, 11 to
30, 12 to 13, 12 to 14, 12 to 15, 12 to 16, 12 to 17, 12 to 18, 12
to 19, 12 to 20, 12 to 21, 12 to 22, 12 to 23, 12 to 24, 12 to 25,
12 to 26, 12 to 27, 12 to 28, 12 to 29, 12 to 30, 13 to 14, 13 to
15, 13 to 16, 13 to 17, 13 to 18, 13 to 19, 13 to 20, 13 to 21, 13
to 22, 13 to 23, 13 to 24, 13 to 25, 13 to 26, 13 to 27, 13 to 28,
13 to 29, 13 to 30, 14 to 15, 14 to 16, 14 to 17, 14 to 18, 14 to
19, 14 to 20, 14 to 21, 14 to 22, 14 to 23, 14 to 24, 14 to 25, 14
to 26, 14 to 27, 14 to 28, 14 to 29, 14 to 30, 15 to 16, 15 to 17,
15 to 18, 15 to 19, 15 to 20, 15 to 21, 15 to 22, 15 to 23, 15 to
24, 15 to 25, 15 to 26, 15 to 27, 15 to 28, 15 to 29, 15 to 30, 16
to 17, 16 to 18, 16 to 19, 16 to 20, 16 to 21, 16 to 22, 16 to 23,
16 to 24, 16 to 25, 16 to 26, 16 to 27, 16 to 28, 16 to 29, 16 to
30, 17 to 18, 17 to 19, 17 to 20, 17 to 21, 17 to 22, 17 to 23, 17
to 24, 17 to 25, 17 to 26, 17 to 27, 17 to 28, 17 to 29, 17 to 30,
18 to 19, 18 to 20, 18 to 21, 18 to 22, 18 to 23, 18 to 24, 18 to
25, 18 to 26, 18 to 27, 18 to 28, 18 to 29, 18 to 30, 19 to 20, 19
to 21, 19 to 22, 19 to 23, 19 to 24, 19 to 25, 19 to 26, 19 to 29,
19 to 28, 19 to 29, 19 to 30, 20 to 21, 20 to 22, 20 to 23, 20 to
24, 20 to 25, 20 to 26, 20 to 27, 20 to 28, 20 to 29, 20 to 30, 21
to 22, 21 to 23, 21 to 24, 21 to 25, 21 to 26, 21 to 27, 21 to 28,
21 to 29, 21 to 30, 22 to 23, 22 to 24, 22 to 25, 22 to 26, 22 to
27, 22 to 28, 22 to 29, 22 to 30, 23 to 24, 23 to 25, 23 to 26, 23
to 27, 23 to 28, 23 to 29, 23 to 30, 24 to 25, 24 to 26, 24 to 27,
24 to 28, 24 to 29, 24 to 30, 25 to 26, 25 to 27, 25 to 28, 25 to
29, 25 to 30, 26 to 27, 26 to 28, 26 to 29, 26 to 30, 27 to 28, 27
to 29, 27 to 30, 28 to 29, 28 to 30, or 29 to 30 linked
nucleosides.
[0080] In certain embodiments, in assays using more than one
oligomeric compound, the modifications, motifs, lengths, and
sequences of each oligomeric compound is independent of those
features of the other oligomeric compounds in the assay. In certain
assays using more than one oligomeric compound, certain of those
features or sets of those features are matched. Thus, for example,
in certain embodiments oligomeric compounds used in an assay all
have the same length and types and number of modification, but have
different motifs (i.e., the modifications are differently
positioned on the oligomeric compounds).
[0081] F. Certain Excipients for Administration of Oligomeric
Compounds
[0082] In certain embodiments, the invention further provides
excipients predicted to be useful for administration of oligomeric
compounds in vivo. When certain uptake assays described above are
performed in the presence of low concentrations of dextran sulfate,
cellular accumulation of oligomeric compound is inhibited, but
antisense activity is unchanged. This result suggests that there
are two mechanisms by which oligomeric compounds accumulate in
cells: a mechanism that results in unproductive accumulation and
which is sensitive to low concentrations of dextran sulfate; and a
productive mechanism that results in antisense activity and which
is not sensitive to low concentrations of dextran sulfate.
Moreover, the bulk of the oligomeric compound appears to enter the
unproductive mechanism. When assays are performed in the presence
of chloroquine and separately in the presence of brefeldin A,
antisense activity is inhibited. These data suggest that the
productive uptake mechanism is "endocytotic-like," since those
compounds interfere with such processes.
[0083] Accordingly, in certain embodiments, the present invention
provides excipients and formulations designed to take advantage of
productive uptake and/or avoid unproductive accumulation. In
certain embodiments, the invention provides formulations of
antisense oligomeric compounds for administration comprising
additional components that saturate the unproductive mechanism. In
certain such embodiments, an additional component is one or more
additional oligomeric compounds. In certain embodiments, such
additional oligomeric compounds comprises different characteristics
(modifications, motifs, length) such that the antisense oligomeric
compound preferentially exploits the productive pathway, while the
additional oligomeric compounds preferentially enter the
unproductive pathway. In certain such embodiments, the additional
oligomeric compounds are non-sense compounds (i.e., is not
complementary to any known cellular sequence). Such excipients may
be administered separately or together with an antisense oligomeric
compound. If administered separately they may administered through
the same route of administration or through different routes of
administration. They may be administered at the same time or at
different times. In certain embodiments, the excipient is first
administered and the antisense oligomeric compound is later
administered. Such administration includes, but is not limited to
administration to an animal, including, but not limited to a
human.
[0084] In certain embodiments, the invention provides assays useful
for identifying such excipients. In certain instances, an excipient
is an oligomeric compound. In such instances, a competition assay
described above may be performed where one or more candidate
excipient oligomeric compounds may be tested for its ability to
increase uptake of another oligomeric compound. In such
embodiments, the candidate excipient oligomeric compound may be
either the competitor oligomeric compound or the reporter
oligomeric compound. In certain instances an excipient is not an
oligomeric compound. In such instances, one may perform an assay
similar to the competition assay described above, but where the
competitor oligomeric compound is replaced with a non-oligomeric
candidate excipient.
[0085] G. Kits, Research Reagents and Diagnostics
[0086] The cells and assays provided herein can be utilized as
research reagents and kits. For use in kits, either alone or in
combination with other compounds or reagents, cells and oligomeric
compounds of the present invention can be used as tools useful for
studying uptake and intracellular trafficking of oligomeric
compounds.
[0087] Nonlimiting Disclosure and Incorporation by Reference
[0088] While certain compounds, compositions and methods described
herein have been described with specificity in accordance with
certain embodiments, the following examples serve only to
illustrate the compounds described herein and are not intended to
limit the same. Each of the references recited in the present
application is incorporated herein by reference in its
entirety.
EXAMPLES
Example 1
Development and Characterization of MHT Cells
[0089] Transgenic mice engineered to express the SV40 large T
antigen (SV40 t/T mice) under the control of the liver-specific
C-reactive protein promoter are a source of transformed cells
(Ruther et al., Oncogene, 8, 1993, 87-93). The expression of SV40
large T antigen can be transiently induced by injection of
bacterial lipopolysaccacharide. The cells can be isolated from the
livers of the transgenic mice. The cells are herein referred to as
Mouse Hepatocyte SV40 T-Antigen expressing cells, or MHT cells.
[0090] Transgenic mice were anesthetized, and perfusion into the
portal vein of the liver was performed to introduce collagenase
into the liver tissue. The livers were isolated from the livers of
SV40 t/T mice, the tissue was gently homogenized and a hepatocyte
cell fraction was isolated. The cells were then placed in 12-well
plates, with and without a collagen coating. The culture medium was
either DMEM containing 10% fetal bovine serum (FBS) or William's
Medium E containing 10% FBS, 10 mM HEPES, and glutamine The culture
medium was changed every 3 days, and any growing cells were
transferred to 6-well culture plates for continued culture and
expansion. Distinct populations of cells were present after
approximately one month of culture. SV40 mRNA expression was
monitored using real-time PCR with primers specific for SV40 mRNA;
cells expressing the SV40 mRNA were identified as MHT cells.
[0091] Two groups of MHT cells, one group cultured in DMEM and the
other group cultured in William's Medium E, were selected for
single-cell cloning. Cells were diluted and placed into wells of a
96-well collagen-coated culture plate such that no more than one
cell would be in a single well. Cells were allowed to expand.
Several clones were selected and found to express SV40 mRNA, as
measured by real-time PCR.
Example 2
Lipid-Mediated Transfection of Oligomeric Compounds into MHT
Cells
[0092] A representative MHT cell clone was evaluated for its
ability to internalize oligonucleotide in the presence of a
transfection reagent. Oligomeric compounds having a nucleobase
sequence complementary to SV40 mRNA were introduced into MHT cells
using a lipofection:oligomeric compound ratio of 3 ug/mL:100 nM.
Oligomeric compound concentrations were 25, 50, 100, or 200 nM. An
untreated sample served as a control. After 48 hours, RNA was
isolated from the cells and SV40 mRNA was measured by real-time
PCR. The oligonucleotides were able to significantly reduce SV40
mRNA in a concentration dependent manner. SV40 mRNA was reduced by
at least 60% at concentrations of 50 nM.
[0093] An oligomeric compound having a nucleobase sequence to an
endogenous target was also tested, and was found to reduce target
mRNA in a concentration-dependent manner at doses of 50, 100, and
200 nM.
Example 3
Oligomeric Compound Uptake in the Absence of Transfection
Reagent
[0094] A representative MHT cell clone was evaluated for its
ability to internalize oligomeric compounds in the absence of a
transfection reagent. The endogenous target tested in
lipofectin-mediated transfection (Example 2) was again tested
without a transfection reagent. Cells were plated in
collagen-coated 96-well plates at a density of 5000 cells per well.
After one day in culture, cells were washed with phosphate-buffered
saline (PBS), then overlaid with William's E Medium containing 1%
FBS, 0.1% bovine serum albumin (BSA), and the desired concentration
of oligomeric compound (either 1 uM or 4 uM). Untreated cells
served as a control. After 48 hours, RNA was isolated from the
cells and target mRNA levels were measured by real-time PCR. A
concentration-dependent reduction in target mRNA was observed,
demonstrating that the oligomeric compounds were internalized by
MHT cells, and that the oligomeric compound was able to hybridize
to and reduce the level of the target mRNA.
[0095] Additional oligomeric compounds having nucleobase sequence
complementary to endogenous targets were tested, and were found to
reduce target mRNA in a concentration-dependent manner.
[0096] Oligomeric compound uptake was also found to occur in a
time-dependent manner. An SR-B1 oligo conjugated to a fluorescent
moiety was added to the culture medium of MHT cells, without a
transfection reagent. Cells were harvested at 2, 4, 6.5, and 24
hours, trypsinized, washed, and prepared for flow cytometry.
Fluoresence was measured using a flow cytometer (FACSCALIBUR) to
assess the amount of oligomeric compound uptake at each timepoint.
The amount of oligomeric compound in cells increased in a
time-dependent manner.
[0097] It was further observed that oligomeric compounds are
capable of entering cells within minutes of addition to the culture
medium. An SR-B1 oligo was added to MHT cell culture medium, at
varying concentrations, without a transfection reagent. Cells were
harvested after 15 minutes, 30 minutes, 1 hour, and 2 hours, and
SR-B1 mRNA was measured using real-time PCR. Reduction in SR-B1
occurred as early as 15 minutes following addition of the SR-B1
oligo to the culture medium.
[0098] Accordingly, the present invention provides cells useful for
assessing uptake of oligomeric compounds. In certain embodiments,
the uptake is assessed in the presence of a transfection reagent.
In certain embodiments, the update is assessed in the absence of a
transfection reagent.
Example 3
Competition Assay to Assess the Relative Uptake of Oligomeric
Compounds
[0099] In certain embodiments, the invention provides a competition
assay useful for assessing the uptake of oligomeric compounds. In
certain embodiments, the competition assay is useful for assessing
the relative uptake of oligomeric compounds. The competition assay
employs a competitor oligomeric compound and a reporter oligomeric
compound. In certain embodiments, the concentration of the
competitor oligomeric compound remains constant while the
concentration of the reporter oligomeric compound is varied.
[0100] An oligomeric compound complementary to SR-B1 mRNA (SR-B1
oligo) was used as a reporter oligomeric compound. An oligomeric
compound complementary to PTEN (PTEN oligo) was used as a
competitor oligomeric compound. Cultured MHT cells were cultured in
serum-containing medium.
[0101] One MHT culture received increasing concentrations of SR-B1
oligo: 10, 100, 1000, and 10000 nM. One day after addition of the
oligonucleotides, RNA was isolated from the cells. Real-time PCR
demonstrated that SR-B1 mRNA was reduced in a concentration
dependent manner, to 78%, 56%, 35%, and 62% of untreated control
mRNA levels, respectively.
[0102] Additional cultures received both SR-B1 and PTEN oligos. The
cultures received the combinations of SR-B1 and PTEN oligos shown
in Table 1. After 24 hours, RNA was isolated from the cells.
Real-time PCR was used to measure SR-B1 mRNA levels.
TABLE-US-00001 TABLE 1 MHT cell treatments SR-B1 mRNA SR-B1 oligo
PTEN oligo % untreated control 100 nM 0 nM 62 100 nM 80 nM 59 100
nM 400 nM 72 100 nM 2000 nM 82 100 nM 10000 nM 95 200 nM 0 nM 38
200 nM 80 nM 52 200 nM 400 nM 53 200 nM 2000 nM 75 200 nM 10000 nM
88 400 nM 0 nM 33 400 nM 80 nM 42 400 nM 400 nM 42 400 nM 2000 nM
52 400 nM 10000 nM 78 800 nM 0 nM 34 800 nM 80 nM 41 800 nM 400 nM
41 800 nM 2000 nM 50 800 nM 10000 nM 81 1600 nM 0 nM 31 1600 nM 80
nM 35 1600 nM 400 nM 31 1600 nM 2000 nM 39 1600 nM 10000 nM 67
[0103] As shown in Table 1, an inverse correlation between PTEN
oligo concentration and SR-B1 mRNA levels was observed. While the
SR-B1 oligo concentration is held constant and the PTEN oligo
concentration increases, SR-B1 mRNA levels increase, indicating
that less SR-B1 oligo is available to hybridize to and effect the
reduction of SR-B1 mRNA. This demonstrates that the SR-B1 oligo and
PTEN oligo are competing for uptake into the cells, in other words,
PTEN oligo is entering the cells in place of the SR-B1 oligo. As
the PTEN oligo concentration increases, the amount of PTEN oligo
internalized by the cells increases, effectively preventing the
cells from internalizing SR-B1 oligo. It was observed that the free
uptake is saturable. The competition assay was performed to compare
the uptake of oligomeric compound conjugated to a fluorescent
moiety to that of unconjugated oligomeric compounds. MHT cells were
incubated with 400 nM of SR-B1 oligo and 1, 4, or 16 uM of
fluorescein-conjugated SR-B1 oligo for 24 hours. Cells were then
trypsinized, and the amount of fluorescein-conjugated SR-B1 oligo
was quantitated by flow cytometry (FACSCALIBER instrument). As the
concentration of SR-B1 oligo was increased, the amount of
fluorescence in each sample decreased, indicating that the
unconjugated SR-B1 oligo competed with the fluorescein-conjugated
SR-B1 oligo for cellular uptake.
Example 4
Localization of Oligomeric Compounds in Cells
[0104] The localization of oligomeric compounds in MHT cells was
compared following introduction of oligomeric compounds into the
cell culture medium in the presence or absence of a transfection
reagent.
[0105] Cells were treated with fluorescein-conjugated oligo in the
presence or absence of lipofectin. The following day, the cells
were washed and then fixed with 4% formaldehyde for 15 minutes.
Fluorescence microscopy revealed that, when the oligomeric compound
was introduced in the presence of lipofectin, more oligomeric
compound was localized to the nucleus, relative to oligomeric
compound that was introduced in the absence of lipofectin.
[0106] The localization of oligomeric compound was also compared to
the localization of LAMP1, a lysosomal marker. MHT cells were
incubated with 1 uM of Cy3-conjugated SR-B1 oligo for 24 hours.
Cells were then washed with PBS, fixed with 4% formaldehyde for 15
minutes, permeabilized with 0.1% TritonX-100, and then incubated
with FITC-conjugated LAMP1 antibody (1:100 dilution) for one hour.
Cells were then stained with DAPI and prepared for microscopic
analysis. Fluorescence microscopy revealed that the highest
concentration of oligomeric compound is localized to cellular
structures that are also marked by LAMP1 staining This assay
indicates that the highest concentration of oligomeric compound is
co-localized with lysosomes.
Example 5
Comparison of In Vitro and In Vivo Potency
[0107] Cultured cells are often used to screen oligomeric compounds
for those compounds that are likely to reduce levels of the
intended target. The effects of oligomeric compounds on target mRNA
levels were compared to the effects of on target mRNA in vivo, to
assess the correlation between in vitro and in vivo potency.
[0108] MHT cells were treated as described herein with increasing
concentrations of SR-B1 oligo or PTEN oligo, in the presence or
absence of lipofection. An additional oligomeric compound, targeted
to a non-coding RNA, was tested. Each oligomeric compound reduced
respective target mRNA levels in a concentration dependent
manner.
[0109] The same oligomeric compounds were tested in vivo in normal
mice. Mice were injected with a single dose of 1.6, 5, 16, or 50
mg/kg of oligomeric compound. Three days following administration
of the oligomeric compound, the mice were sacrificed and liver
tissue was isolated. RNA was isolated from the liver tissue, and
target mRNA levels were quantitated using real-time PCR. The SR-B1
oligo reduced liver SR-B1 mRNA in a concentration-dependent manner.
The oligomeric compound targeted to the non-coding RNA likewise
reduced RNA levels in a concentration-dependent manner. The PTEN
oligo reduced liver PTEN mRNA levels.
[0110] The ED.sub.50 (dose at which 50% reduction is observed in
vivo) and IC.sub.50 (concentration at which 50% reduction is
observed in vitro) for each oligomeric compound was calculated. For
each oligomeric compound tested, the rank-order potency in vitro,
in the presence or absence of a transfection reagent, correlated
well with the in vivo potency. These data indicate that MHT cells
may be used to identify oligomeric compounds that will exhibit
potency in vivo with respect to mRNA reduction.
Example 6
Oligomeric Compound Uptake in the Presence of Excipients
[0111] In certain embodiments, the invention further provides
excipients that are useful for administration of oligomeric
compounds in vivo. The effects of the excipients on the uptake of
oligomeric compounds can be assessed in vitro in MHT cells, and the
results can be used to identify excipients that may affect
oligomeric compound uptake in vivo.
[0112] To assess the effects of dextran sulfate on oligomeric
compound activity, MHT cells were treated with increasing
concentrations of SR-B1 oligo in the presence and absence of
dextran sulfate. One sample included no excipient, a second sample
was treated with SR-B1 oligo and 1 uM dextran sulfate, and a third
sample was treated with SR-B1 oligo and 10 uM dextran sulfate.
After 24 hours, SR-B1 mRNA levels were quantitated by real-time
PCR. While the lower concentration of dextran sulfate did not
affect antisense activity of the oligomeric compound, the higher
concentration did interfere with the antisense activity of the
SR-B1 oligo.
[0113] To determine whether the uptake of oligomeric compound was
inhibited by dextran sulfate, MHT cells were incubated with 400 nM
of fluorescein-conjugated SR-B1 oligo in the presence of 0.5 uM, 1
uM, 5 uM or 10 uM of dextran sulfate, for a period of 24 hours.
Cells were stained with DAPI, fixed and examined by fluorescence
microscopy as described herein. As the concentration of dextran
sulfate increased, less oligomeric compound was present in the
cells. Thus it was observed that dextran sulfate competes for
oligomeric compound uptake into cells.
[0114] The inhibition of antisense activity in the presence of
dextran sulfate supports the hypothesis that a large portion of
oligomeric compound is present in cells in a non-functional
compartment. For example, the lower concentration of dextran
sulfate inhibited oligomeric compound uptake without interfering
with antisense activity, indicating that a portion of the
oligomeric compound present in the cell is unproductive.
[0115] Additional excipient, chloroquine, was tested for its
effects on antisense activity. Chloroquine accumulates
preferentially in the lysosomes of cells. MHT cells were incubated
with increasing concentrations of SR-B1 oligo in the presence or
absence of chloroquine. One sample included no excipient, a second
sample was treated with SR-B1 oligo and 20 uM chloroquine, and a
third sample was treated with SR-B1 oligo and 40 uM chloroquine.
After 24 hours, SR-B1 mRNA levels were quantitated by real-time
PCR. At each concentration of chloroquine, SR-B1 mRNA levels were
not significantly reduced, even at the highest concentration of
SR-B1 oligo, indicating that chloroquine does interfere with
antisense activity.
[0116] An additional excipient, brefeldin A, was tested for its
effects on antisense activity. Brefeldin A interferes with
anterograde protein transport from the endoplasmic reticulum to the
Golgi apparatus by inhibiting transport in the Golgi apparatus,
which leads to proteins accumulating inside the endoplasmic
reticulum. MHT cells were incubated with increasing concentrations
of SR-B1 oligo in the presence or absence of brefeldin A. One
sample included no excipient, a second sample was treated with
SR-B1 oligo and 2.5 uM brefeldin A, and a third sample was treated
with SR-B1 oligo and 5 uM brefeldin A. After 24 hours, SR-B1 mRNA
levels were quantitated by real-time PCR. SR-B1 mRNA levels were
not significantly reduced following treatment with SR-B1 oligo in
combination with brefeldin A, particularly at the higher dose of
brefeldin A, indicating that brefeldin A does interfere with
antisense activity.
[0117] The inhibition of antisense activity in the presence of
brefeldin A or chloroquine suggests that cellular uptake of
oligomeric compound is facilitated by an `endocytotic-like`
pathway.
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