U.S. patent application number 12/566549 was filed with the patent office on 2010-04-01 for methods for slowing familial als disease progression.
Invention is credited to C. Frank Bennett, Don W. Cleveland, Thomas P. Condon, Susan M. Freier, Richard Alan Smith.
Application Number | 20100081705 12/566549 |
Document ID | / |
Family ID | 34520475 |
Filed Date | 2010-04-01 |
United States Patent
Application |
20100081705 |
Kind Code |
A1 |
Bennett; C. Frank ; et
al. |
April 1, 2010 |
METHODS FOR SLOWING FAMILIAL ALS DISEASE PROGRESSION
Abstract
Methods for slowing disease progression in an individual
suffering from familial ALS are provided. Also provided are methods
of increasing the survival time of an individual suffering from
familial ALS. These methods employ antisense oligonucleotides
targeted to SOD1, for use in inhibiting the expression of SOD1 in
the central nervous system of an individual suffering from familial
ALS.
Inventors: |
Bennett; C. Frank;
(Carlsbad, CA) ; Cleveland; Don W.; (Del Mar,
CA) ; Smith; Richard Alan; (La Jolla, CA) ;
Freier; Susan M.; (San Diego, CA) ; Condon; Thomas
P.; (Singapore, SG) |
Correspondence
Address: |
JONES DAY for;Isis Pharmaceuticals, Inc.
222 East 41st Street
New York
NY
10017-6702
US
|
Family ID: |
34520475 |
Appl. No.: |
12/566549 |
Filed: |
September 24, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11526134 |
Sep 21, 2006 |
7622455 |
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12566549 |
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11449446 |
Jun 7, 2006 |
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11526134 |
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10672866 |
Sep 26, 2003 |
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11449446 |
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10633843 |
Aug 4, 2003 |
7132530 |
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10672866 |
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09888360 |
Jun 21, 2001 |
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10633843 |
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60719936 |
Sep 21, 2005 |
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Current U.S.
Class: |
514/44A |
Current CPC
Class: |
C12Y 115/01001 20130101;
C12N 2310/315 20130101; C12N 2310/321 20130101; C12N 2310/341
20130101; C12N 2310/346 20130101; C12N 15/1137 20130101; C12N
2310/11 20130101; C12N 2310/14 20130101; C12N 2310/3341 20130101;
A61K 38/00 20130101; A61P 25/00 20180101; Y02P 20/582 20151101;
C12N 2310/321 20130101; C12N 2310/3525 20130101 |
Class at
Publication: |
514/44.A |
International
Class: |
A61K 31/7088 20060101
A61K031/7088; A61P 25/00 20060101 A61P025/00 |
Claims
1. A method of treating amyotrophic lateral sclerosis (ALS)
comprising administering to the cerebrospinal fluid of a subject in
need thereof a therapeutically or prophylactically effective amount
of a composition comprising: a modified oligonucleotide consisting
of 12 to 30 linked nucleosides that is specifically hybridizable
with SEQ ID NO: 1, and a pharmaceutically acceptable diluents or
carrier, wherein the treatment increases lifespan in the
subject.
2. The method of claim 1, wherein administering is intrathecal
administration.
3. The method of claim 1, wherein administering is intraventricular
administration.
4. The method of claim 1, wherein the modified oligonucleotide
consists of 12 to 30 linked nucleosides having a nucleobase
sequence comprising an 8-nucleobase portion of SEQ ID NO: 3.
5. A method of slowing progression of amyotrophic lateral sclerosis
(ALS) comprising administering to the cerebrospinal fluid of a
subject in need thereof a therapeutically or prophylactically
effective amount of a composition comprising: a modified
oligonucleotide consisting of 12 to 30 linked nucleosides that is
specifically hybridizable with SEQ ID NO: 1, and a pharmaceutically
acceptable diluents or carrier, wherein the treatment slows disease
progression.
6. The method of claim 5, wherein administering is intrathecal
administration.
7. The method of claim 5, wherein administering is intraventricular
administration.
8. The method of claim 5, wherein the modified oligonucleotide
consists of 12 to 30 linked nucleosides having a nucleobase
sequence comprising an 8-nucleobase portion of SEQ ID NO: 3.
Description
RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 11/526,134, filed Sep. 21, 2006, which is a
continuation-in-part of U.S. application Ser. No. 11/449,446, filed
Jun. 7, 2006, which is a continuation of U.S. application Ser. No.
10/672,866, filed Sep. 26, 2003, now abandoned, which is a
continuation-in-part of U.S. application Ser. No. 10/633,843, filed
Aug. 4, 2003, now U.S. Pat. No. 7,132,530, issued Nov. 7, 2006,
which is a continuation of U.S. application Ser. No. 09/888,360,
filed Jun. 21, 2001, now abandoned. Further, U.S. application Ser.
No. 11/526,134 claims priority under 35 U.S.C. .sctn.119(e) to U.S.
Provisional Application Ser. No. 60/719,936, filed Sep. 21, 2005.
The entire contents of these documents are incorporated herein by
reference.
SEQUENCE LISTING
[0002] The present application is being filed along with a Sequence
Listing in electronic format. The Sequence Listing is provided as a
file entitled RTS0242USC4.txt, created on Sep. 24, 2009 which is 4
Kb in size. The information in the electronic format of the
sequence listing is incorporated herein by reference in its
entirety.
FIELD OF THE INVENTION
[0003] The present invention provides compositions and methods for
slowing disease progression in an individual suffering from
familial amyotrophic lateral sclerosis. In particular, this
invention relates to antisense compounds, particularly antisense
oligonucleotides, complementary to SOD1 nucleic acids. Such
antisense oligonucleotides have been shown to inhibit the
expression of SOD1.
BACKGROUND OF THE INVENTION
[0004] The superoxide anion (O.sub.2.sup.-) is a potentially
harmful cellular by-product produced primarily by errors of
oxidative phosphorylation in mitochondria (Cleveland and Liu, Nat.
Med., 2000, 6, 1320-1321). Some of the targets for oxidation by
superoxide in biological systems include the iron-sulfur
dehydratases, aconitase and fumarases. Release of Fe (II) from
these superoxide-inactivated enzymes results in Fenton-type
production of hydroxyl radicals which are capable of attacking
virtually any cellular target, most notably DNA (Fridovich, Annu.
Rev. Biochem., 1995, 64, 97-112).
[0005] The enzymes known as the superoxide dismutases (SODs)
provide defense against oxidative damage of biomolecules by
catalyzing the dismutation of superoxide to hydrogen peroxide
(H.sub.2O.sub.2) (Fridovich, Annu. Rev. Biochem., 1995, 64,
97-112). Two major classes of superoxide dismutases exist. One
consists of a group of enzymes with active sites containing copper
and zinc while the other class has either manganese or iron at the
active site (Fridovich, Annu. Rev. Biochem., 1995, 64, 97-112).
[0006] The soluble superoxide dismutase 1 enzyme (also known as
SOD1 and Cu/Zn superoxide dismutase) contains a zinc- and
copper-type active site (Fridovich, Annu. Rev. Biochem., 1995, 64,
97-112). Lee et al. reported the molecular cloning and high-level
expression of human cytoplasmic superoxide dismutase gene in E.
coli in 1990 (Lee et al., Misaengmul Hakhoechi, 1990, 28, 91-97).
Studies of transgenic mice carrying a mutant human superoxide
dismutase 1 gene, for example, transgenic mice expressing a human
SOD1 gene bearing glycine 93 to alanine (G93A) mutation.
[0007] Mutations in the superoxide dismutase 1 gene are associated
with a dominantly-inherited form of amyotrophic lateral sclerosis
(ALS, also known as Lou Gehrig's disease) a disorder characterized
by a selective degeneration of upper and lower motor neurons
(Cleveland and Liu, Nat. Med., 2000, 6, 1320-1321). The deleterious
effects of various mutations on superoxide dismutase 1 are most
likely mediated through a gain of toxic function rather than a loss
of superoxide dismutase 1 activity, as the complete absence of
superoxide dismutase 1 in mice neither diminishes life nor provokes
overt disease (Al-Chalabi and Leigh, Curr. Opin. Neuroi., 2000, 13,
397-405; Alisky and Davidson, Hum. Gene Ther., 2000, 11,
2315-2329).
[0008] Cleveland and Liu proposed two models for mutant superoxide
dismutase 1 toxicity (Cleveland and Liu, Nat. Med., 2000, 6,
1320-1321). The "oxidative hypothesis" ascribes toxicity to binding
of aberrant substrates such as peroxynitrite or hydrogen peroxide
which gain access to the catalytic copper ion through
mutation-dependent loosening of the native superoxide dismutase 1
protein conformation (Cleveland and Liu, Nat. Med., 2000, 6,
1320-1321). A second possible mechanism for mutant superoxide
dismutase 1 toxicity involves the misfolding and aggregation of
mutant superoxide dismutase 1 proteins (Cleveland and Liu, Nat.
Med., 2000, 6, 1320-1321). The idea that aggregates contribute to
ALS has received major support from the observation that murine
models of superoxide dismutase 1 mutant-mediated disease feature
prominent intracellular inclusions in motor neurons and, in some
cases, in the astrocytes surrounding them as well (Bruijn et al.,
Science, 1998, 281, 1851-1854). Furthermore, Brujin et al. also
demonstrate that neither elimination nor elevation of wild-type
superoxide dismutase 1 was found to affect disease induced by
mutant superoxide dismutase 1 in mice (Bruijn et al., Science,
1998, 281, 1851-1854).
[0009] Riluzole, a glutamate regulatory drug, is approved for use
in ALS patients in some countries, but has only a modest effect on
survival. Accordingly, there remains an unmet need for therapeutic
regimens that slow familial ALS disease progression and increase
survival of familial ALS patients.
SUMMARY OF THE INVENTION
[0010] The present invention provides methods of slowing disease
progression in an individual suffering from familial amyotrophic
lateral sclerosis (ALS), comprising administering to the individual
a therapeutically effective amount of a pharmaceutical composition
comprising an antisense oligonucleotide 17 to 25 nucleobases in
length complementary to nucleobases 66 to 102 of SEQ ID NO: 1,
thereby slowing disease progression. The administering may comprise
delivery to the cerebrospinal fluid of the individual, and may
further comprise intrathecal infusion. A slowing of disease
progression is measured by an improvement in one or more indicators
of ALS disease progression selected from ALSFRS-R, FEV.sub.1, FVC,
or muscle strength measurements. The methods further comprise
increasing the survival time of an individual suffering from
familial ALS.
[0011] The methods provided herein comprise the administration of
an antisense oligonucleotide complementary to nucleobases 83 to 102
of SEQ ID NO: 1. The antisense oligonucleotide may be fully
complementary to nucleotides 83 to 102 of SEQ ID NO: 1. Further,
the antisense oligonucleotide may consist essentially of ISIS
333611. Additionally, the antisense oligonucleotide may consist of
ISIS 333611.
[0012] The antisense oligonucleotides employed in the methods
provided herein comprise at least one modified internucleoside
linkage, such as a phosphorothioate internucleoside linkage, a
modified sugar moiety, such as a 2'-O-methoxyethyl sugar moiety or
a bicyclic nucleic acid sugar moiety, or a modified nucleobase,
such as a 5-methylcytosine.
[0013] The present invention provides methods of slowing disease
progression in an individual suffering from familial ALS comprising
administering an antisense oligonucleotide that is a chimeric
oligonucleotide. The chimeric oligonucleotide comprises a
2'-deoxynucleotide gap segment positioned between 5' and 3' wing
segments. The wing segments are comprised of nucleosides containing
a sugar moiety selected from a 2'-O-methoxyethyl sugar moiety or a
bicyclic nucleic acid sugar moiety. The gap segment may be ten
2'-deoxynucleotides in length and each of the wing segments may be
five 2'-O-methoxyethyl nucleotides in length. The chimeric
oligonucleotide may be uniformly comprised of phosphorthioate
internucleoside linkages. Further, each cytosine of the chimeric
oligonucleotide may be a 5'-methylcytosine.
[0014] Further provided are methods comprising selecting an
individual who has received a diagnosis of familial amyotrophic
lateral sclerosis (ALS), administering to the individual a
therapeutically effective amount of a pharmaceutical composition
comprising an antisense oligonucleotide 17 to 25 nucleobases in
length complementary to nucleobases 66 to 102 of SEQ ID NO: 1, and
monitoring ALS disease progression in the individual.
DETAILED DESCRIPTION OF THE INVENTION
[0015] Over 100 mutations of the human SOD1 gene have been
identified, and altogether account for approximately 20% of
familial amyotrophic lateral sclerosis (ALS) cases. Some mutations,
such as the A4V mutation most commonly found in the United States,
are highly lethal and result in survival only nine months from the
onset of disease symptoms. Other mutations of SOD1 manifest in a
slower disease course.
[0016] It has been discovered that antisense inhibition of
superoxide dismutase 1 (SOD1) in an animal model of familial ALS
reduces both SOD1 mRNA and protein, and further results in a
slowing of disease progression and, importantly, increased survival
time. Accordingly, the present invention provides methods for the
slowing of disease progression in an individual suffering from
familial ALS by delivering to the cerebrospinal fluid of the
individual a therapeutically effective amount of a pharmaceutical
composition comprising an antisense oligonucleotide targeted to
SOD1. Such methods further comprise increasing survival time of an
individual suffering from familial ALS. Slowing of disease
progression is indicated by an improvement in one or more
indicators of ALS disease progression, including, without
limitation, the revised ALS functional rating scale, pulmonary
function tests, and muscle strength measurements.
[0017] The present invention employs antisense compounds,
particularly antisense oligonucleotides, for use in modulating the
function of nucleic acid molecules encoding SOD1, ultimately
modulating the amount of SOD1 protein produced. This is
accomplished by providing antisense oligonucleotides which
hybridize to and inhibit the expression of one or more nucleic
acids encoding SOD1. Such antisense oligonucleotides are considered
to be "targeted to SOD1." Antisense oligonucleotides of the present
invention do not necessarily distinguish between wild-type SOD1
mRNA and SOD1 mRNAs bearing mutations. While an object of the
present invention is to reduce SOD1 mRNAs bearing mutations, the
conconmitant reduction of wild-type SOD1 appear to be safe, as
reductions or complete loss of SOD1 does not produce overt disease
or compromise life span in experimental animal models.
[0018] In certain embodiments, an antisense oligonucleotide
targeted to SOD1 is complementary to nucleobases 66 to 102 of a
nucleic acid molecule encoding SOD1 (GENBANK.RTM. accession no.
X02317.1, incorporated herein as SEQ ID NO: 1). In additional
embodiments, an antisense oligonucleotide targeted to SOD1 is
complementary to nucleotides 83 to 102 of SEQ ID NO: 1. In
preferred embodiments, an antisense oligonucleotide targeted to
SOD1 is fully complementary to nucleotides 83 to 102 of SEQ ID NO:
1. In further preferred embodiments, the antisense oligonucleotide
is ISIS 333611.
[0019] As used herein, an "individual suffering from familial ALS"
is an individual who has received from a health professional, such
as a physician, a diagnosis of familial ALS. Relevant diagnostic
tests are well known in the art and are understood to include,
without limitation, genetic testing to determine the presence of a
mutation in the SOD1 gene, neurological examination, and the El
Escorial criteria (see, for example, Brooks et al., Amyothoph.
Lateral Scler. other Motor Neuron Disorders, 2000, 293-299). An
"individual prone to familial ALS" is understood to include an
individual who, based on a physician's assessment, is not yet
suffering from familial ALS but is likely to develop familial
ALS.
[0020] In order for antisense inhibition of SOD1 to have a
clinically desirable effect on familial ALS progression, it is
beneficial to deliver an antisense oligonucleotide targeted to SOD1
to the central nervous system (CNS) of an individual suffering from
familial ALS, and in particular to the regions of the CNS affected
by familial ALS. As the blood-brain barrier is generally
impermeable to antisense oligonucleotides administered
systemically, a preferred method of providing antisense
oligonucleotides targeted to SOD1 to the tissues of the CNS is via
administration of the antisense oligonucleotides directly into the
cerebrospinal fluid (CSF). As is known in the art, means for
delivery to the CSF include intrathecal (IT) and
intracerebroventricular (ICV) administration. As is further known
in the art, IT or ICV administration may be achieved through the
use of surgically implanted pumps that infuse a therapeutic agent
into the cerebrospinal fluid. As used herein, "delivery to the CSF"
and "administration to the CSF" encompass the IT infusion or ICV
infusion of antisense oligonucleotides targeted to SOD1 through the
use of an infusion pump. In some embodiments, IT infusion is a
preferred means for delivery to the CSF. In preferred embodiments,
the antisense oligonucleotide is continuously infused into the CSF
for the entire course of treatment; such administration is referred
to as "continuous infusion" or, in the case of IT infusion,
"continuous IT infusion."
[0021] In the context of the present invention, a preferred means
for delivery of antisense oligonucleotide to the CSF employs an
infusion pump such as Medtronic SyncroMed.RTM. II pump. The
SyncroMed.RTM. II pump is surgically implanted according the
procedures set forth by the manufacturer. The pump contains a
reservoir for retaining a drug solution, which is pumped at a
programmed dose into a catheter that is surgically implanted. For
intrathecal administration of a drug, the catheter is surgically
intrathecally implanted. In the context of the present invention,
the drug is the pharmaceutical composition comprising an antisense
oligonucleotide targeted to SOD1.
[0022] As used herein, a "pharmaceutical composition comprising an
antisense oligonucleotide" refers to a composition comprising an
antisense oligonucleotide targeted to SOD1 in a pharmaceutically
acceptable diluent. By way of example, a suitable pharmaceutically
acceptable diluent is phosphate-buffered saline. In some
embodiments, the pharmaceutical composition comprises an antisense
oligonucleotide complementary to nucleotides 66 to 102 of SOD1 in
phosphate-buffered saline. In preferred embodiments, the
pharmaceutical composition comprises an antisense oligonucleotide
complementary to nucleotides 83 to 102 of SEQ ID NO: 1 in
phosphate-buffered saline. In further preferred embodiments, the
pharmaceutical composition comprises an antisense oligonucleotide
fully complementary to nucleotides 83 to 102 of SEQ ID NO: 1 in
phosphate-buffered saline. In more preferred embodiments, the
pharmaceutical composition comprises ISIS 333611 in
phosphate-buffered saline. ISIS 333611 is the nonadecasodium salt
of the antisense oligonucleotide having the nucleobase sequence
CCGTCGCCCTTCAGCACGCA (SEQ ID NO: 2), where nucleosides 1 to 5 and
16 to 20 have 2'-O-methoxyethyl sugar moieties, nucleosides 6 to 15
are 2'-deoxynucleotides, each internucleoside linkage is a
phosphorothioate linkage, and each cytosine is a
5-methylcytosine.
[0023] As used herein, a "therapeutically effective amount" is an
amount of an antisense oligonucleotide targeted to SOD1 required to
produce a slowing of disease progression and/or an increase in
survival time in an individual suffering from familial ALS.
Accordingly, a therapeutically effect amount is an amount that will
result in an improvement in one or more indicators of ALS
progression, such as, for example, the revised ALSFSR, FEV.sub.1,
FCV, and muscle strength measurements. In some embodiments, a
therapeutically effective amount of an antisense oligonucleotide
targeted to SOD1 ranges from 8 mg to 12 mg of antisense
oligonucleotide. In preferred embodiments, a therapeutically effect
amount of an antisense oligonucleotide targeted to SOD1 is 10 mg.
In one embodiment, a therapeutically effective amount of an
antisense oligonucleotide targeted to SOD1 is administered via
continuous infusion for a minimum of 28 days. In preferred
embodiments, antisense oligonucleotide is delivered via IT
infusion. In further preferred embodiments, the antisense
oligonucleotide administered is ISIS 333611.
[0024] As used herein, "slowing disease progression" means the
prevention of a clinically undesirable change in one or more
disabilities in an individual suffering from familial ALS, and is
assessed by methods routinely practiced in the art, for example,
the revised ALSFSR, pulmonary function tests, and muscle strength
measurements. Such methods are herein referred to as "indicators of
ALS disease progression."
[0025] As used herein, an "improvement in a indicator of ALS
disease progression" refers to slowing of the rate of change in one
or more of the indicators of ALS disease progression described
herein. An improvement in an indicator of ALS disease progression
further includes a lack of a measurable change in one or more of
the indicators of ALS disease progression described herein. An
improvement in an indicator of ALS disease progression additionally
includes a positive change in one of the indicators of ALS disease
progression described herein, such as, for example, an increase in
an ALSFSR-R score. One of skill in the art will appreciate that is
well within the abilities of a physician to identify a slowing of
disease progression in an individual suffering from familial ALS,
using one or more of the disease assessment tests described herein.
Additionally, it is understood that a physician may administer to
the individual diagnostic tests other than those described herein,
such as additional pulmonary function tests or muscle strength
measurement tests, to assess the rate of disease progression in an
individual suffering from familial ALS.
[0026] A slowing of disease progression may further comprise an
"increase in survival time" in an individual suffering from
familial ALS. It is understood that a physician can use one or more
of the disease assessment tests described herein to predict an
approximate survival time of an individual suffering from familial
ALS. A physician may additionally use the known disease course of a
particular familial ALS mutation to predict survival time.
[0027] The "revised ALS functional rating scale" or "ALSFRS-R" is
routinely used by physicians and is a validated rating instrument
for monitoring the progression of disability in ALS patients. The
ALSFRS-R includes 12 questions that ask a physician to rate his or
her impression of an ALS patient's level of functional impairment
in performing one of ten common tasks, for example, climbing
stairs. Each task is rated on a five-point scale, where a score of
zero indicates an inability to perform a task and a score of four
indicates normal ability in performing a task. Individual item
scores are summed to produce a reported score of between zero
(worst) and 48 (best).
[0028] ALS eventually results in progressive muscle weakness.
Pulmonary function tests (PFTs) are employed to reveal the extent
and progression respiratory muscle weakness. Pulmonary function
tests include measurements of the "forced expiratory volume in one
second" (FEV.sub.1), which is the amount of air than an individual
can forcefully exhale during the first second of exhalation
following inhalation, as well as measurements of "forced vital
capacity" (FVC), which is the total amount of air that an
individual can forcefully exhale following inhalation. FEV.sub.1
and FVC are typically reduced in individuals with neuromuscular
diseases, such as ALS. Pulmonary function tests may be administered
using instrument that directly measures the volume of air displaced
during exhalation, or measures airflow during exhalation by a flow
sensing device. An example of such an instrument is a spirometer.
ALS disease progression is also evaluated by assessing the extent
and progression of whole body muscle weakness. Muscle strength
measurements, include, but are not limited to, hand held
dynamometry, maximum voluntary isometric contraction (MVIC) strain
gauge measurements, and manual muscle testing. Muscle strength
tests routinely use an instrument that measures how much force (for
example, pounds of force) an individual can apply to the instrument
using a selected group of muscles, such as the hand muscles. Such
an instrument includes a dynamometer.
Antisense Compounds
[0029] In the context of the present invention, the term
"oligomeric compound(s)" refers to polymeric structures which are
capable of hybridizing to at least a region of an RNA molecule.
Generally, an oligomeric compound is "antisense" to a target
nucleic acid when it comprises the reverse complement of the
corresponding region of the target nucleic acid. Such oligomeric
compounds are known as "antisense compounds", which include,
without limitation, oligonucleotides (i.e. antisense
oligonucleotides), oligonucleosides, oligonucleotide analogs,
oligonucleotide mimetics and combinations of these. In general, an
antisense compound comprises a backbone of linked monomeric
subunits (sugar moieties) where each linked monomeric subunit is
directly or indirectly attached to a heterocyclic base moiety.
Modifications to antisense compounds encompass substitutions or
changes to internucleoside linkages, sugar moieties, or
heterocyclic base moieties, such as those described below. As used
herein, the term "modification" includes substitution and/or any
change from a starting or natural nucleoside or nucleotide.
Modified antisense compounds are often preferred over native forms
because of desirable properties such as, for example, enhanced
cellular uptake, enhanced affinity for nucleic acid target,
increased stability in the presence of nucleases, or increased
inhibitory activity. Antisense compounds are often defined in the
art to comprise the further limitation of, once hybridized to a
target, being able to induce or trigger a reduction in target gene
expression or target gene levels. In one embodiment, the antisense
compounds, e.g. antisense oligonucleotides, trigger a reduction in
the levels of a nucleic acid encoding SOD1.
[0030] "Targeting" an antisense oligonucleotide to a nucleic acid
encoding SOD1 includes the determination of at least one target
segment within a nucleic acid encoding SOD1 for hybridization to
occur such that the desired effect, e.g., inhibition of SOD1 mRNA
expression, will result. As used herein, the terms "SOD1 target
nucleic acid" and "nucleic acid encoding SOD1" encompass RNA
(including pre-mRNA and mRNA) transcribed from DNA encoding SOD1,
and also cDNA derived from such RNA. The inhibition of gene
expression that results from the hybridization of an antisense
oligonucleotide with a target nucleic acid is generally referred to
as "antisense inhibition". The functions of RNA to be interfered
with include, for example, translocation of the RNA to the site of
protein translation, translation of protein from the RNA, splicing
of the RNA to yield one or more mRNA species, and catalytic
activity which may be engaged in or facilitated by the RNA. The
overall effect of such interference with target nucleic acid
function is modulation of the SOD1 protein. In the context of the
present invention, "modulation" means either an increase
(stimulation) or a decrease (inhibition) in the expression of a
gene. In the context of the present invention, inhibition is the
preferred form of modulation of gene expression and SOD1 mRNA (e.g.
SEQ ID NO: 1) is a preferred target.
[0031] As used herein, a "target segment" means a sequence of an
SOD1 target nucleic acid to which one or more antisense
oligonucleotides are complementary. Multiple antisense
oligonucleotides complementary to a given target segment may or may
not have overlapping sequences. Within the context of the present
invention, the term "target site" is defined as a sequence of an
SOD1 nucleic acid to which one antisense oligonucleotide is
complementary. For example, the nucleobase sequence of ISIS 333611
is complementary to nucleobases 83 to 102 of SEQ ID NO: 1, thus
these nucleobases represent a target site of an SOD1 nucleic acid.
Several antisense oligonucleotides of the invention have target
sites within nucleobases 66 to 102 of SEQ ID NO: 1, thus these
nucleobases represent a target segment of an SOD1 nucleic acid. In
some embodiments, a target segment and target site are represented
by the same nucleobase sequence.
[0032] In the practice of the methods of the present invention,
particularly preferred SOD1 target segments include, without
limitation, nucleobases 66 to 102 of SEQ ID NO: 1; nucleobases 73
to 102 of SEQ ID NO: 1; and nucleobases 79 to 102 of SEQ ID NO: 1.
Particularly preferred target sites include nucleobases 81 to 100
of SEQ ID NO: 1, to which ISIS 146145 is complementary; and
nucleobases 83 to 102 of SEQ ID NO: 1, to which ISIS 333611 is
complementary.
[0033] The antisense oligonucleotides in accordance with this
invention comprise from 15 to 30 nucleosides in length, i.e., from
15 to 30 linked nucleosides. One of skill in the art will
appreciate that this embodies antisense oligonucleotides of 15, 16,
17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30
nucleosides in length.
[0034] In one embodiment, the antisense oligonucleotides of the
invention are 17 to 25 nucleosides in length, as exemplified
herein.
[0035] In preferred embodiments, the antisense oligonucleotides of
the invention are 19, 20, 21, 22 or 23 nucleosides in length.
[0036] "Base complementarity" as used herein, refers to the
capacity for the precise base pairing of nucleobases of an
antisense oligonucleotide with corresponding nucleobases in a
target nucleic acid (i.e., hybridization). In the context of the
present invention, the mechanism of pairing involves hydrogen
bonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteen
hydrogen bonding, between corresponding nucleobases. Both natural
and modified nucleobases are capable of participating in hydrogen
bonding. Hybridization can occur under varying circumstances.
[0037] As used herein, an antisense oligonucleotide is "fully
complementary" to a target nucleic acid when each nucleobase of the
antisense oligonucleotide is capable of undergoing precise base
pairing with an equal number of nucleobases in the target nucleic
acid. It is understood in the art that the sequence of the
antisense oligonucleotide need not be fully complementary to that
of its target nucleic acid to be active in inhibiting the activity
of the target nucleic acid. In some embodiments there are
"non-complementary" positions, also known as "mismatches", between
the antisense oligonucleotide and the target nucleic acid, and such
non-complementary positions may be tolerated between an antisense
oligonucleotide and the target nucleic acid provided that the
antisense oligonucleotide remains specifically hybridizable to the
target nucleic acid. For example, ISIS 333611, having two
non-complementary nucleobases with respect to monkey SOD1, is
capable of reducing monkey SOD1 mRNA levels in cultured cells. A
"non-complementary nucleobase" means a nucleobase of an antisense
oligonucleotide that is unable to undergo precise base pairing with
a nucleobase at a corresponding position in a target nucleic acid.
As used herein, the terms "non-complementary" and "mismatch" are
interchangable. In the context of the present invention, antisense
oligonucleotides having no more than three non-complementary
nucleobases with respect to a nucleic acid encoding SOD1 are
considered "complementary" to a nucleic acid encoding SOD1. In
preferred embodiments, the antisense oligonucleotide contains no
more than two non-complementary nucleobases with respect to a
nucleic acid encoding SOD1. In further preferred embodiments, the
antisense oligonucleotide contains no more than one
non-complementary nucleobases with respect to a nucleic acid
encoding SOD1.
[0038] The location of a non-complementary nucleobase may be at the
5' end or 3' end of the antisense oligonucleotide. Alternatively,
the non-complementary nucleobase may be at an internal position in
the antisense oligonucleotide. When two or more non-complementary
nucleobases are present, they may be contiguous (i.e. linked) or
non-contiguous.
[0039] In other embodiments of the invention, the antisense
oligonucleotides comprise at least 90% sequence complementarity to
an SOD1 target nucleic acid. In further embodiments of the
invention, the antisense oligonucleotides comprise at least 95%
sequence complementarity to an SOD1 target nucleic acid. Percent
complementarity of an antisense oligonucleotide with a region of a
target nucleic acid can be determined routinely by those having
ordinary skill in the art, and may be accomplished 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).
[0040] Antisense oligonucleotides may have a defined percent
identity to a SEQ ID NO, or an antisense oligonucleotide having a
specific ISIS number. This identity may be over the entire length
of the antisense oligonucleotide, or over less than the entire
length of the antisense oligonucleotide. Calculating percent
identity is well within the ability of those skilled in the art. It
is understood by those skilled in the art that an antisense
oligonucleotide need not have an identical sequence to those
described herein to function similarly to the antisense
oligonucleotides described herein. For example, antisense
oligonucleotides having at least 90%, or at least 95%, identity to
antisense oligonucleotides taught herein are contemplated in the
present invention.
[0041] Shortened or truncated versions of antisense
oligonucleotides taught herein have one, two or more nucleosides
deleted, and are contemplated in the present invention. When an
antisense oligonucleotide has two or more deleted nucleosides, the
deleted nucleosides may be adjacent to each other, for example, in
an antisense oligonucleotide having two nucleosides truncated from
the 5' end (5' truncation), or alternatively from the 3' end (3'
truncation), of the antisense oligonucleotide. Alternatively, the
deleted nucleosides may be dispersed through out the antisense, for
example, in an antisense oligonucleotide having one nucleoside
deleted from the 5' end and one nucleoside deleted from the 3'
end.
[0042] Also falling within the scope of the invention are
lengthened versions of antisense oligonucleotides taught herein,
i.e. antisense oligonucleotides having one or more additional
nucleosides relative to an antisense oligonucleotide disclosed
herein. When two are more additional nucleosides are present, the
added nucleosides may be adjacent to each other, for example, in an
antisense oligonucleotide having two nucleosides added to the 5'
end (5' addition), or alternatively to the 3' end (3' addition), of
the antisense oligonucleotide. Alternatively, the added nucleosides
may be dispersed throughout the antisense oligonucleotide, for
example, in an antisense oligonucleotide having one nucleoside
added to the 5' end and one nucleoside added to the 3' end.
[0043] Antisense oligonucleotides of the invention may be also be
described as complementary to a portion of a target site. A
"portion" is defined as at least 18 contiguous nucleobases of a
target site. In other embodiments, a portion is 19 or 20 contiguous
nucleobases of a target site. By way of example, antisense
oligonucleotides may be complementary, or alternatively fully
complementary, to a 19 nucleobase portion of SEQ ID NO: 1.
[0044] The antisense compounds of the invention are synthesized in
vitro and do not include antisense compositions of biological
origin, or genetic vector constructs designed to direct the in vivo
synthesis of antisense molecules.
Antisense Compound Modifications
[0045] Any of the antisense compounds taught herein, including
antisense oligonucleotides taught herein, may contain modifications
which confer desirable properties to the antisense compound
including, but are not limited to, increased affinity of an
antisense oligonucleotide for its target RNA and increased
resistance to nucleases.
[0046] As is known in the art, a nucleoside is a base-sugar
combination. The base (also known as nucleobase) portion of the
nucleoside is normally a heterocyclic base moiety. 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 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. The respective ends of this linear polymeric structure
can be joined to form a circular structure by hybridization or by
formation of a covalent bond. Within the oligonucleotide structure,
the phosphate groups are commonly referred to as forming the
internucleoside linkages of the oligonucleotide. The normal
internucleoside linkage of RNA and DNA is a 3' to 5' phosphodiester
linkage.
[0047] In the context of this invention, the term "oligonucleotide"
refers generally to an oligomer or polymer of ribonucleic acid
(RNA) or deoxyribonucleic acid (DNA), and may be used to refer to
unmodified oligonucleotides or oligonucleotide analogs. The term
"unmodified oligonucleotide" refers generally to oligonucleotides
composed of naturally occurring nucleobases, sugars, and covalent
internucleoside linkages. The term "oligonucleotide analog" refers
to oligonucleotides that have one or more non-naturally occurring
nucleobases, sugars, and/or internucleoside linkages. Such
non-naturally occurring oligonucleotides are often selected over
naturally occurring forms because of desirable properties such as,
for example, enhanced cellular uptake, enhanced affinity for target
nucleic acids, increased stability in the presence of nucleases, or
increased inhibitory activity.
[0048] Chemically modified nucleosides may also be employed to
increase the binding affinity of a shortened or truncated antisense
compound for its target nucleic acid. Consequently, comparable
results can often be obtained with shorter antisense compounds that
have such chemically modified nucleosides.
Modified Internucleoside Linkages
[0049] Specific examples of antisense compounds useful in this
invention include oligonucleotides containing one or more modified,
i.e. non-naturally occurring, internucleoside linkages. Such
non-naturally internucleoside linkages are often selected over
naturally occurring forms because of desirable properties such as,
for example, enhanced cellular uptake, enhanced affinity for other
oligonucleotides or nucleic acid targets and increased stability in
the presence of nucleases.
[0050] As defined in this specification, oligonucleotides having
modified internucleoside linkages include internucleoside linkages
that retain a phosphorus atom as well as internucleoside linkages
that do not have a phosphorus atom. Representative phosphorus
containing internucleoside linkages include, but are not limited
to, phosphodiesters, phosphotriesters, methylphosphonates,
phosphoramidate, and phosphorothioates. For the purposes of this
specification, and as sometimes referenced in the art, modified
oligonucleotides that do not have a phosphorus atom in their
internucleoside backbone can also be considered to be
"oligonucleosides". Methods of preparation of
phosphorous-containing and non-phosphorous-containing linkages are
well known to those skilled in the art.
Modified Sugar Moieties
[0051] Antisense compounds of the invention may also contain one or
more nucleosides having modified sugar moieties. The base moieties
(natural, modified or a combination thereof) are maintained for
hybridization with an appropriate nucleic acid target. Sugar
modifications may impart nuclease stability, binding affinity or
some other beneficial biological property to the antisense
compounds. The furanosyl sugar ring of a nucleoside can be modified
in a number of ways including, but not limited to, addition of a
substituent group, particularly at the 2' position, bridging of two
non-geminal ring atoms to form a bicyclic nucleic acid (BNA) and
substitution of an atom or group such as --S--, --N(R)-- or
--C(R.sub.1)(R.sub.2) for the ring oxygen at the 4'-position. A
representative list of preferred modified sugars includes but is
not limited to: substituted sugars, especially 2'-substituted
sugars having a 2'-F, 2'-OCH.sub.2 (2'-OMe) or a
2'-O(CH.sub.2).sub.2--OCH.sub.3 (2'-O-methoxyethyl or 2'-MOE)
substituent group; and bicyclic modified sugars (BNAs), having a
4'-(CH.sub.2)--O-2' bridge, where n=1 or n=2. Sugars can also be
replaced with sugar mimetic groups. Methods for the preparations of
modified sugars are well known to those skilled in the art.
Modified Nucleobases
[0052] Antisense compounds of the invention may also contain one or
more nucleobase (often referred to in the art simply as "base")
modifications or substitutions which are structurally
distinguishable from, yet functionally interchangeable with,
naturally occurring or synthetic unmodified nucleobases. Such
nucleobase modifications may impart nuclease stability, binding
affinity or some other beneficial biological property to the
antisense compounds. 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 also referred to herein as heterocyclic base
moieties 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.
[0053] Heterocyclic base moieties 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. Nucleobases that are particularly useful for increasing
the binding affinity of the antisense compounds of the invention
includ 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and
O-6 substituted purines, including 2 aminopropyladenine,
5-propynyluracil and 5-propynylcytosine.
[0054] Certain nucleobase substitutions, including
5-methylcytosinse substitutions, are particularly useful for
increasing the binding affinity of the oligonucleotides of the
invention. For example, 5-methylcytosine substitutions have been
shown to increase nucleic acid duplex stability by 0.6-1.2.degree.
C. (Sanghvi, Y. S., Crooke, S. T. and Lebleu, B., eds., Antisense
Research and Applications, CRC Press, Boca Raton, 1993, pp.
276-278) and are presently preferred base substitutions, even more
particularly when combined with 2'-O-methoxyethyl sugar
modifications.
Oligonucleotide Mimetics
[0055] Antisense compounds can also include an "oligonucleotide
mimetic," which refers to oligonucleotides in which only the
furanose ring or both the furanose ring and the internucleoside
linkage are replaced with novel groups.
[0056] As used herein the term "mimetic" refers to groups that are
substituted for a sugar, a nucleobase, and/or internucleoside
linkage. Generally, a mimetic is used in place of the sugar or
sugar-internucleoside linkage combination, and the nucleobase is
maintained for hybridization to a selected target. Representative
examples of a sugar mimetic include, but are not limited to,
cyclohexenyl or morpholino. Representative examples of a mimetic
for a sugar-internucleoside linkage combination include, but are
not limited to, peptide nucleic acids (PNA) and morpholino groups
linked by uncharged achiral linkages. In some instances a mimetic
is used in place of the nucleobase. Representative nucleobase
mimetics are well known in the art and include, but are not limited
to, tricyclic phenoxazine analogs and universal bases (Berger et
al., Nuc. Acid Res. 2000, 28:2911-14, incorporated herein by
reference). Methods of synthesis of sugar, nucleoside and
nucleobase mimetics are well known to those skilled in the art.
Conjugated Antisense Compounds
[0057] One substitution that can be appended to the antisense
compounds of the invention involves the linkage of one or more
moieties or conjugates which enhance the activity, cellular
distribution or cellular uptake of the resulting antisense
compounds. In one embodiment such modified antisense compounds are
prepared by covalently attaching conjugate groups to functional
groups such as hydroxyl or amino 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 the antisense compounds.
Typical conjugate groups include cholesterol moieties and lipid
moieties. Additional conjugate groups include carbohydrates,
phospholipids, biotin, phenazine, folate, phenanthridine,
anthraquinone, acridine, fluoresceins, rhodamines, coumarins, and
dyes.
[0058] Antisense 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
antisense 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 an antisense compound (see for example Wincott et al.,
WO 97/26270). These terminal modifications protect the antisense
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. For
double-stranded antisense compounds, the cap may be present at
either or both termini of either strand. Cap structures are well
known in the art and include, for example, inverted deoxy a basic
caps. Further 3' and 5'-stabilizing groups that can be used to cap
one or both ends of an antisense compound to impart nuclease
stability include those disclosed in WO 03/004602 published on Jan.
16, 2003.
Antisense Compound Motifs
[0059] Antisense compounds of this invention may have the
chemically modified subunits arranged in patterns enhance the
inhibitory activity of the antisense compounds. These patterns are
described herein as "motifs."
[0060] As used in the present invention the term "gapped motif" or
"gapmer" is meant to include an antisense compound having an
internal region (also referred to as a "gap" or "gap segment")
positioned between two external regions (also referred to as "wing"
or "wing segment"). The regions are differentiated by the types of
sugar moieties comprising each distinct region. The types of sugar
moieties that are used to differentiate the regions of a gapmer may
in some embodiments include .beta.-D-ribonucleosides,
.beta.-D-deoxyribonucleosides, 2'-modified nucleosides (such
2'-modified nucleosides may include 2'-MOE, and 2'-O--CH.sub.3,
among others), and bicyclic sugar modified nucleosides (such
bicyclic sugar modified nucleosides may include LNA.TM. or ENA.TM.,
among others). In general, each distinct region comprises uniform
sugar moieties.
[0061] Gapped motifs or gapmers are further defined as being either
"symmetric" or "asymmetric". A gapmer wherein the nucleosides of
the first wing have the same sugar modifications as the nucleosides
of the second wing is termed a symmetric gapped antisense compound.
Symmetric gapmers can have, for example, an internal region
comprising a first sugar moiety, and external regions each
comprising a second sugar moiety, wherein at least one sugar moiety
is a modified sugar moiety.
[0062] "Chimeric antisense compounds" or "chimeras," in the context
of this invention, are antisense compounds that at least 2
chemically distinct regions, each made up of at least one monomer
unit, i.e., a nucleotide or nucleoside in the case of a nucleic
acid based antisense compound. Accordingly, antisense compounds
having a gapmer motif considered chimeric antisense compounds.
[0063] Chimeric antisense compounds typically contain at least one
region modified so as to confer increased resistance to nuclease
degradation, increased cellular uptake, increased binding affinity
for the target nucleic acid, and/or increased inhibitory activity.
By way of example, an antisense compound may be designed to
comprise a region that serves as a substrate for the cellular
endonuclease RNase H, which cleaves the RNA strand of an RNA:DNA
duplex. In the case of gapmer, the internal region generally serves
as the substrate for endonuclease cleavage.
Compositions and Methods for Formulating Pharmaceutical
Compositions
[0064] The antisense compounds of the invention may also be admixed
with pharmaceutically acceptable substances, active and/or inert,
that are well known to those skilled in the art.
[0065] The present invention also includes pharmaceutical
compositions and formulations that include the antisense compounds
and compositions of the invention. Compositions and methods for the
formulation of pharmaceutical compositions are dependent upon a
number of criteria, including, but not limited to, route of
administration, extent of disease, or dose to be administered. Such
considerations are well understood by those skilled in the art.
[0066] The antisense compounds and compositions of the invention
can be utilized in pharmaceutical compositions by adding an
effective amount of the compound or composition to a suitable
pharmaceutically acceptable diluent or carrier. In the context of
the present invention, a pharmaceutically acceptable diluent
includes phosphate-buffered saline (PBS). PBS is a diluent suitable
for use in compositions to be delivered to the CNS.
[0067] The antisense compounds and compositions 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.
Accordingly, for example, the disclosure is also drawn to prodrugs
and pharmaceutically acceptable salts of the antisense compounds of
the invention, pharmaceutically acceptable salts of such prodrugs,
and other bioequivalents.
[0068] The term "prodrug" indicates a therapeutic agent that is
prepared in an inactive form that is converted to an active form
(i.e., drug) within the body or cells thereof by the action of
endogenous enzymes or other chemicals and/or conditions. This can
include the incorporation of additional nucleosides at one or both
ends of an antisense compound which are cleaved by endogenous
nucleases within the body, to form the active antisense
compound.
[0069] The term "pharmaceutically acceptable salts" refers to
physiologically and pharmaceutically acceptable salts of the
antisense compounds and compositions of the invention: i.e., salts
that retain the desired biological activity of the parent compound
and do not impart undesired toxicological effects thereto. Suitable
examples include, but are not limited to, sodium and potassium
salts.
Cell Culture and Antisense Oligonucleotide Treatment
[0070] The effects of antisense oligonucleotides on the level,
activity or expression of SOD1 target nucleic acids can be tested
in vitro in a variety of cell types. Cell types used for such
analyses are available from commercial vendors (e.g. American Type
Culture Collection, Manassus, Va.; Zen-Bio, Inc., Research Triangle
Park, N.C.; Clonetics Corporation, Walkersville, Md.) and cells are
cultured according to the vendor's instructions using commercially
available reagents (e.g. Invitrogen Life Technologies, Carlsbad,
Calif.). Illustrative cell types include, but are not limited to,
A549 cells, fibroblasts, and neuronal cells.
In Vitro Testing of Antisense Oligonucleotides
[0071] In general, when cells reach approximately 60-80%
confluency, they are treated with antisense oligonucleotides of the
invention.
[0072] One reagent commonly used to introduce antisense
oligonucleotides into cultured cells includes the cationic lipid
transfection reagent LIPOFECTIN.RTM. (Invitrogen, Carlsbad,
Calif.). Antisense oligonucleotides are mixed with LIPOFECTIN.RTM.
in OPTI-MEM.RTM. 1 (Invitrogen, Carlsbad, Calif.) to achieve the
desired final concentration of antisense oligonucleotide and a
LIPOFECTIN.RTM. concentration that typically ranges 2 to 12 ug/mL
per 100 nM antisense oligonucleotide.
[0073] Another reagent used to introduce antisense oligonucleotides
into cultured cells includes LIPOFECTAMINE.RTM. (Invitrogen,
Carlsbad, Calif.). Antisense oligonucleotide is mixed with
LIPOFECTAMINE.RTM. in OPTI-MEM.RTM. 1 reduced serum medium
(Invitrogen, Carlsbad, Calif.) to achieve the desired concentration
of antisense oligonucleotide and a LIPOFECTAMINE.RTM. concentration
that typically ranges 2 to 12 ug/mL per 100 nM antisense
compound.
[0074] Cells are treated with antisense oligonucleotides by routine
methods well known to those skilled in the art. Cells are typically
harvested 16-24 hours after antisense oligonucleotide treatment, at
which time RNA or protein levels of target nucleic acids are
measured by methods known in the art and described herein. In
general, when treatments are performed in multiple replicates, the
data are presented as the average of the replicate treatments.
[0075] The concentration of antisense oligonucleotide used varies
from cell line to cell line.
[0076] Methods to determine the optimal antisense oligonucleotide
concentration for a particular cell line are well known in the art.
Antisense oligonucleotides are typically used at concentrations
ranging from 1 nM to 300 nM.
In Vivo Testing of Antisense Oligonucleotides
[0077] Antisense oligonucleotides are tested in animals to assess
their ability to inhibit expression of a target nucleic acid and
produce phenotypic changes. Testing may be performed in normal
animals, or in experimental disease models. For administration to
animals, antisense oligonucleotides are formulated in a
pharmaceutically acceptable diluent, such as phosphate-buffered
saline. Administration includes parenteral routes of
administration, such as intraperitoneal, intravenous, and
subcutaneous, and further includes intrathecal and
intracerebroventricular routes of administration. Calculation of
antisense oligonucleotide dosage and dosing frequency is within the
abilities of those skilled in the art, and depends upon factors
such as route of administration and animal body weight. Following a
period of treatment with antisense oligonucleotides, RNA is
isolated from various tissues and changes in target nucleic acid
expression are measured. Changes in proteins encoded by target
nucleic acids may also be measured. The types of phenotypic changes
selected for monitoring are dependent upon the cellular pathway and
disease with which the target nucleic acid is associated.
RNA Isolation
[0078] RNA analysis can be performed on total cellular RNA or
poly(A)+ mRNA. Methods of RNA isolation are well known in the art.
RNA is prepared using methods well known in the art, for example,
using the TRIZOL.RTM. Reagent (Invitrogen, Carlsbad, Calif.)
according to the manufacturer's recommended protocols.
Analysis of Inhibition of Target Levels or Expression
[0079] Inhibition of levels or expression of an SOD1 nucleic acid
can be assayed in a variety of ways known in the art. For example,
target nucleic acid levels can be quantitated by, e.g., Northern
blot analysis, competitive polymerase chain reaction (PCR), or
quantitaive real-time PCR. RNA analysis can be performed on total
cellular RNA or poly(A)+ mRNA. Methods of RNA isolation are well
known in the art. Northern blot analysis is also routine in the
art. Quantitative real-time PCR can be conveniently accomplished
using the commercially available ABI PRISM.RTM. 7600, 7700, or 7900
Sequence Detection System, available from PE-Applied Biosystems,
Foster City, Calif. and used according to manufacturer's
instructions.
Quantitative Real-Time PCR Analysis of Target RNA Levels
[0080] Quantitation of target RNA levels may be accomplished by
quantitative real-time PCR using the ABI PRISM.RTM. 7600, 7700, or
7900 Sequence Detection System (PE-Applied Biosystems, Foster City,
Calif.) according to manufacturer's instructions. Methods of
quantitative real-time PCR are well known in the art.
[0081] Prior to real-time PCR, the isolated RNA is subjected to a
reverse transcriptase (RT) reaction, which produces complementary
DNA (cDNA) that is then used as the substrate for the real-time PCR
amplification. The RT and real-time PCR reactions are performed
sequentially in the same sample well. RT and real-time PCR reagents
are obtained from Invitrogen (Carlsbad, Calif.). RT, real-time-PCR
reactions are carried out by methods well known to those skilled in
the art.
[0082] Gene (or RNA) target quantities obtained by real time PCR
are normalized using either the expression level of a gene whose
expression is constant, such as GAPDH, or by quantifying total RNA
using RIBOGREEN.RTM. (Molecular Probes, Inc. Eugene, Oreg.). GAPDH
expression is quantified by real time PCR, by being run
simultaneously with the target, multiplexing, or separately. Total
RNA is quantified using RIBOGREEN.RTM. RNA quantification reagent
(Molecular Probes, Inc. Eugene, Oreg.). Methods of RNA
quantification by RIBOGREEN.RTM. are taught in Jones, L. J., et al,
(Analytical Biochemistry, 1998, 265, 368-374). A CYTOFLUOR.RTM.
4000 instrument (PE Applied Biosystems) is used to measure
RIBOGREEN.RTM. fluorescence.
[0083] Probes and primers are designed to hybridize to an SOD1
target nucleic acid. Methods for designing real-time PCR probes and
primers are well known in the art, and may include the use of
software such as PRIMER EXPRESS.RTM. Software (Applied Biosystems,
Foster City, Calif.). Primers and probes useful for detection of
human and rat SOD1 mRNA are described in U.S. application Ser. No.
10/672,866, published as US 2005/0019915, which is herein
incorporated by reference in its entirety.
Analysis of Protein Levels
[0084] Protein levels of SOD1 can be evaluated or quantitated in a
variety of ways well known in the art, such as immunoprecipitation,
Western blot analysis (immunoblotting), enzyme-linked immunosorbent
assay (ELISA), quantitative protein assays, protein activity assays
(for example, caspase activity assays), immunohistochemistry,
immunocytochemistry or fluorescence-activated cell sorting (FACS).
Antibodies directed to a target 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. Antibodies useful for the detection of human
and rat SOD1 are well known in the art.
Example 1
Inhibition of SOD1 mRNA in Rat Brain Following
Intracerebroventricular Administration
[0085] In order to inhibit the gene expression in the central
nervous system, antisense oligonucleotides must be delivered
directly to the cerebrospinal fluid by, for example,
intracerebroventricular (ICV) administration. To evaluate antisense
inhibition of SOD1 in the brains of normal rats, SOD1 mRNA levels
were measured in rat brain following ICV administration. SOD1 mRNA
levels were measured in both rat spinal cord and rat brain
following ICV administration of ISIS 146192, an antisense
oligonucleotide targeted to SOD1. Administration was performed
daily at either 33 .mu.g/day or 50 .mu.g/day for 14 days. ICV
administration of ISIS 146192 significantly reduced SOD1 mRNA
levels in the spinal cord and right temporal parietal section of
the brain. Thus, antisense oligonucleotides that are delivered to
the cerebrospinal fluid via ICV administration are able to inhibit
the expression of SOD1 in central nervous system tissues that are
affected in ALS. Accordingly, an embodiment of the present
invention is the delivery of antisense oligonucleotides to the
cerebrospinal fluid by way of ICV administration.
Example 2
Antisense Inhibition of SOD1 in Human Fibroblasts
[0086] The A4V mutation of SOD1 accounts for 50% of SOD1-mediated
familial ALS in the United States. Antisense oligonucleotides
targeting SOD1 were tested for their ability to inhibit SOD1
expression in fibroblasts isolated from an individual harboring the
A4V mutation. ISIS 333611, ISIS 333624 (complementary to
nucleotides 440 to 459 of SEQ ID NO: 1), and ISIS 333636
(complementary to nucleobases 452 to 471 of SEQ ID NO: 1) inhibited
SOD1 expression in a dose dependent manner when tested at doses of
3, 10, 30, 100, or 300 nM. SOD2 mRNA levels were not affected.
Example 3
Slowed Disease Progression in a Rat Model of Familial ALS
[0087] Several lines of transgenic mice and rats have been
generated and extensively studied as experimental models of
familial ALS. For example, transgenic mice have been engineered to
express the human G85R SOD1 variant. Transgenic rats expressing the
human SOD1 G93A variant develop symptoms similar to ALS and do not
survive beyond three to five months after birth. As such, these
transgenic rats are useful for the testing of antisense
oligonucleotides targeted to SOD1. The presence of the human G93A
SOD1 variant causes human SOD1 mRNA to accumulate to levels
approximately 5 to 10 times that of endogenous wild-type rat
SOD1.
[0088] Antisense oligonucleotide was infused into the right lateral
ventricle of 65 day old rats expressing the human G93A SOD1 variant
at a dose of 100 ug/day for 28 days, using Alzet minipumps.
Following the treatment period, RNA was isolated from different
regions of the brain, and SOD1 mRNA levels were measured by
real-time PCR. Despite the high level of human SOD1 mRNA, ISIS
146145, ISIS 333611, ISIS 333624, and ISIS 333636 were effective at
reducing human SOD1 mRNA in different regions of the brain. For
example, ISIS 333611 effectively reduced human SOD1 mRNA levels in
the right cortex, cervical spinal cord, thoracic spinal cord, and
lumbar spinal cord by approximately 69%, 45%, 50%, and 42%,
respectively. Human SOD1 protein levels were reduced following
treatment with ISIS 333611 by approximately 40% and 35%,
respectively, in the right cortex and cervical spinal cord.
Reduction of SOD1 protein was greater at one month than at two
weeks, reflecting the known long half-life of SOD1 protein.
[0089] An additional study was performed to test the effects of
antisense inhibition of SOD1 on ALS disease onset in rats
expressing the human G93A SOD1 variant. Animals were treated by ICV
infusion of 100 ug/day of ISIS 333611 (n=12) for a period of 28
days. Saline-treated (n=11) animals and control
oligonucleotide-treated (n=8) animals served as controls. The
timing of disease onset, at approximately 95 days of age, was
similar in each group. While antisense inhibition of SOD1 did not
slow early disease onset, infusion of ISIS 333611 slowed disease
progression, extending survival from 122.+-.8 days to 132.+-.7
days. Infusion of the control oligonucleotide had no effect on
disease progression.
[0090] An embodiment of the present invention is a method of the
slowing of disease progression in an individual suffering from
familial ALS by delivering to the cerebrospinal fluid an antisense
oligonucleotide targeted to SOD1. In other embodiments, the method
further comprises extending the survival of an individual suffering
from familial ALS. In preferred embodiments, the antisense
oligonucleotide is ISIS 333611.
Example 4
Distribution of Antisense Oligonucleotides in Primate Tissues
[0091] To assess the distribution of antisense oligonucleotides
following delivery to the cerebrospinal fluid, ISIS 13920 (an
antisense oligonucleotide having a gapped motif) was infused
intracerebroventricularly or intrathecally into non-human primates
at a dose of 1 mg/day for 14 days. A monoclonal antibody that
recognizes oligonucleotides allowed for the immunohistochemical
detection of antisense oligonucleotide. Following ICV infusion,
ISIS 13920 distributed broadly throughout the central nervous
system, with the highest concentrations found in the cortex and the
lowest concentrations found in the hypothalamus. Following IT
infusion, antisense oligonucleotides was broadly distributed
throughout the central nervous system, with the highest
concentrations of oligonucleotides found in the tissue adjacent to
the site of infusion, the lumbar cord.
[0092] Accordingly, an embodiment of the present invention is the
delivery of antisense oligonucleotide targeted to SOD1 to the
central nervous system, as well as the cerebrospinal fluid, through
intracerebroventricular or intrathecal infusion.
Example 5
Administration of ISIS 333611 to Individuals Suffering from
Familial ALS
[0093] The present invention provides methods of slowing disease
progression in an individual suffering from familial ALS. Such
methods comprise the administration to the cerebrospinal fluid of
the individual a pharmaceutical composition comprising ISIS 333611.
Delivery of the pharmaceutical composition to the cerebrospinal
fluid allows for contact of the antisense oligonucleotide with the
cells of central nervous system tissues, including tissues affected
by ALS.
[0094] Individuals suffering from familial ALS receive a diagnosis
of familial ALS from a physician. The physician's assessment
includes the El Escorial criteria, genetic testing to verify the
presence of a mutation in the SOD1 gene, and a neurological
examination.
[0095] A Medtronic SyncroMed.RTM. II pump is used to deliver a
pharmaceutical composition comprising ISIS 333611 to the
cerebrospinal fluid of an individual suffering from familial ALS.
The pump is surgically implanted per the procedures outlined by the
manufacturer. Drug is retained in the reservoir of the pump, and is
pumped at a programmed dose into a catheter that is surgically
intrathecally implanted.
[0096] The reservoir is loaded with a pharmaceutical composition
comprising ISIS 333611 in phosphate-buffered saline. The
pharmaceutical composition is administered at an amount that yields
an infusion of 8 mg to 12 mg of ISIS 333611 into the cerebrospinal
fluid. In preferred embodiments, the amount of ISIS 333611 infused
is 10 mg. Administration is for a period of at least 28 days.
[0097] Disease progression is measured by methods routine in the
art and described herein, for example, using the ALSFSR-R, and
measurements of FEV.sub.1, FVC, and muscle strength. These methods
are used by a physician to assess disease state at initiation of
treatment, and this assessment serves as a baseline for disease
state. Subsequent assessments are performed at regular intervals
during the pharmaceutical composition delivery period; these
intervals are determined by the physician. Administration of a
pharmaceutical composition comprising ISIS 333611 to the CSF of
individuals suffering from familial ALS slows the progression of
ALS. In one embodiment, the ALSFSR-R score is not reduced relative
to the baseline ALSFSR-R score. In another embodiment, FEV.sub.1 is
not reduced relative to baseline values. In an additional
embodiment, FVC is not reduced relative to baseline values. In a
further embodiment, muscle strength, such as hand grip strength, is
not reduced relative to baseline values.
Sequence CWU 1
1
31874DNAH. sapiens 1ctgcagcgtc tggggtttcc gttgcagtcc tcggaaccag
gacctcggcg tggcctagcg 60agttatggcg acgaaggccg tgtgcgtgct gaagggcgac
ggcccagtgc agggcatcat 120caatttcgag cagaaggaaa gtaatggacc
agtgaaggtg tggggaagca ttaaaggact 180gactgaaggc ctgcatggat
tccatgttca tgagtttgga gataatacag caggctgtac 240cagtgcaggt
cctcacttta atcctctatc cagaaaacac ggtgggccaa aggatgaaga
300gaggcatgtt ggagacttgg gcaatgtgac tgctgacaaa gatggtgtgg
ccgatgtgtc 360tattgaagat tctgtgatct cactctcagg agaccattgc
atcattggcc gcacactggt 420ggtccatgaa aaagcagatg acttgggcaa
aggtggaaat gaagaaagta caaagacagg 480aaacgctgga agtcgtttgg
cttgtggtgt aattgggatc gcccaataaa cattcccttg 540gatgtagtct
gaggcccctt aactcatctg ttatcctgct agctgtagaa atgtatcctg
600ataaacatta aacactgtaa tcttaaaagt gtaattgtgt gactttttca
gagttgcttt 660aaagtacctg tagtgagaaa ctgatttatg atcacttgga
agatttgtat agttttataa 720aactcagtta aaatgtctgt ttcaatgacc
tgtattttgc cagacttaaa tcacagatgg 780gtattaaact tgtcagaatt
tctttgtcat tcaagcctgt gaataaaaac cctgtatggc 840acttattatg
aggctattaa aagaatccaa attc 874220DNAArtificial SequenceSynthetic
Antisense Oligonucleotide 2ccgtcgccct tcagcacgca 20320DNAArtificial
SequenceSynthetic Antisense Oligonucleotide 3gtcgcccttc agcacgcaca
20
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