U.S. patent application number 13/101942 was filed with the patent office on 2011-11-03 for compound and method for treating myotonic dystrophy.
This patent application is currently assigned to AVI BIOPHARMA, INC.. Invention is credited to Ryszard Kole.
Application Number | 20110269665 13/101942 |
Document ID | / |
Family ID | 44858698 |
Filed Date | 2011-11-03 |
United States Patent
Application |
20110269665 |
Kind Code |
A1 |
Kole; Ryszard |
November 3, 2011 |
COMPOUND AND METHOD FOR TREATING MYOTONIC DYSTROPHY
Abstract
Provided are 9-base morpholino antisense compounds targeted to
polyCUG repeats in the 3'UTR region of dystrophia myotonica protein
kinase (DMPK) mRNA, and related methods for treating myotonic
dystrophy DM1.
Inventors: |
Kole; Ryszard; (Chapel Hill,
NC) |
Assignee: |
AVI BIOPHARMA, INC.
Corvallis
OR
|
Family ID: |
44858698 |
Appl. No.: |
13/101942 |
Filed: |
May 5, 2011 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
12493140 |
Jun 26, 2009 |
|
|
|
13101942 |
|
|
|
|
Current U.S.
Class: |
514/1.1 ;
514/44A; 530/300; 536/24.5 |
Current CPC
Class: |
A61P 21/00 20180101;
C12N 15/1137 20130101; C12N 15/113 20130101; C07K 2319/33 20130101;
C12N 15/87 20130101; C12N 2810/40 20130101; C12N 2310/3233
20130101; C12N 2310/113 20130101 |
Class at
Publication: |
514/1.1 ;
536/24.5; 530/300; 514/44.A |
International
Class: |
A61K 31/7088 20060101
A61K031/7088; A61K 38/02 20060101 A61K038/02; A61P 21/00 20060101
A61P021/00; C07H 21/04 20060101 C07H021/04; C07K 17/02 20060101
C07K017/02 |
Claims
1. An antisense compound for treating myotonic dystrophy DM1,
comprising a 9-base morpholino antisense oligonucleotide, where the
9 bases are complementary to polyCUG repeats in the 3'UTR region of
dystrophia myotonica protein kinase (DMPK) mRNA.
2. The antisense compound of claim 1, wherein the oligonucleotide
is a phosphorodiamidate morpholino oligonucleotide (PMO).
3. The antisense compound of claim 2, wherein at least one and up
to about 1 per every 2 intersubunit linkage(s) contains a pendant
cationic group.
4. The antisense compound of claim 3, wherein the cationic group
comprises an optionally substituted piperazino group.
5. The antisense compound of claim 3, wherein the oligonucleotide
is conjugated to a cell-penetrating peptide.
6. A method of treating myotonic dystrophy DM1 in a mammalian
subject, comprising administering to the subject a 9-base
morpholino antisense oligonucleotide, where the 9 bases are
complementary to polyCUG repeats in the 3'UTR region of dystrophia
myotonica protein kinase (DMPK) mRNA, and repeating said
administering at least once every one week to 3 months.
7. The method of claim 6, wherein the oligonucleotide is a
phosphorodiamidate morpholino oligonucleotide (PMO).
8. The method of claim 7, wherein at least one and up to about 1
per every 2 intersubunit linkage(s) contains a pendant cationic
group.
9. The method of claim 8, wherein the cationic group comprises an
optionally substituted piperazino group.
10. The method of claim 6, wherein the oligonucleotide is
conjugated to a cell-penetrating peptide.
11. The method of claim 6, wherein said administering is by
intravenous or subcutaneous injection to the subject, at a dose
between 1-20 mg/kg body weight antisense compound.
12. The method of claim 6, wherein said administering is continued
at regular intervals of every one to three months, and further
includes monitoring the patient during the treatment period for
improvement in skeletal or heart muscle performance.
13. The method of claim 6, wherein said administering is continued
at regular intervals of every one to three months, and further
includes monitoring the patient during the treatment period for
improvement in heart conduction properties.
14. The method of claim 6, wherein said administering is continued
at regular intervals of every one to three months, and further
includes monitoring the patient during the treatment period for
reduction in serum creatine kinase.
Description
CROSS REFERENCE TO RELATED APPLICATION
[0001] This application is a continuation-in-part of U.S.
application Ser. No. 12/493,140, filed Jun. 26, 2009, now pending,
which is incorporated by reference in its entirety.
STATEMENT REGARDING SEQUENCE LISTING
[0002] The sequence listing associated with this application is
provided in text format in lieu of a paper copy, and is hereby
incorporated by reference into the specification. The name of the
text file containing the Sequence Listing is
120178.sub.--492_SEQUENCE_LISTING_txt. The text file is about 13
KB, was created on May 5, 2011, and is being submitted
electronically via EFS-Web.
FIELD OF THE INVENTION
[0003] The present invention relates to antisense oligonucleotides
targeted to polyCUG repeats in the 3'UTR region of dystrophia
myotonica protein kinase (DMPK) mRNA, and methods for treating
myotonic dystrophy DM1.
BACKGROUND OF THE INVENTION
[0004] Myotonic dystrophy type 1 (DM1) and type 2 (DM2) are
associated with long polyCUG and polyCCUG repeats in the 3'-UTR and
intron 1 regions of the transcript dystrophia myotonica protein
kinase (DMPK) and zinc finger protein 9 (ZNF9), respectively
(Wheeler and Thornton 2007). While normal individuals have as many
as 30 CTG repeats, DM1 patients carry a larger number of repeats
ranging from 50 to thousands. The severity of the disease and the
age of onset correlates with the number of repeats. Patients with
adult onsets show milder symptoms and have less than 100 repeats,
juvenile onset DM1 patients carry as many as 500 repeats and
congenital cases usually have around a thousand CTG repeats. The
expanded transcripts containing CUG repeats form a secondary
structure, accumulate in the nucleus in the form of nuclear foci
and sequester RNA-binding proteins (RNA-BP).
[0005] Several RNA-BP have been implicated in the disease,
including muscleblind-like (MBNL) proteins and CUG-binding protein
(CUGBP). MBNL proteins are homologous to Drosophila muscleblind
(Mbl) proteins necessary for photoreceptor and muscle
differentiation. MBNL and CUGBP have been identified as
antagonistic splicing regulators of transcripts affected in DM1
such as cardiac troponin T (cTNT), insulin receptor (IR) and
muscle-specific chloride channel (ClC-1).
[0006] Myotonic dystrophy type 2 (DM2) is associated with repeats
in the first intron of the ZNF9 gene on chromosome 3. CNBP (ZNF9)
is the only gene known to be associated with myotonic dystrophy
type 2. CNBP intron 1 contains a complex repeat motif,
(TG)n(TCTG)n(CCTG)n, and expansion of the CCTG repeat causes DM2.
The number of CCTG repeats in expanded alleles can range from
approximately 75 to more than 11,000, with a mean of approximately
5000 repeats.
[0007] DM1 and DM2 are associated with a variety of serious
pathologies including muscle abnormalities and weakness, and in the
heart, conduction abnormalities.
SUMMARY OF THE INVENTION
[0008] The present invention is based on the unexpected antisense
activity of 9-base phosphorodiamidate morpholino oligomers (PMOs),
relative, for example, to longer PMOs, for reducing or ameliorating
one or more symptoms of myotonic dystrophy type 1 (DM1) or type 2
(DM2). These 9-base antisense oligomers described herein can employ
a variety of PMO-based chemistries, including PMO, PMO+, PPMO, and
PPMO+ chemistries, as described herein.
[0009] Embodiments of the present invention therefore include
antisense compounds for treating myotonic dystrophy type 1 (DM1),
comprising a morpholino antisense oligonucleotide of 9 bases, where
the 9 bases are complementary to polyCUG repeats in the 3'UTR
region of dystrophia myotonica protein kinase (DMPK) mRNA. In
certain embodiments, the oligonucleotide is a phosphorodiamidate
morpholino oligonucleotide (PMO). In some embodiments, at least one
and up to about 1 per every 2 intersubunit linkage(s) of the
oligonucleotide contains a pendant cationic group. In specific
embodiments, the cationic group comprises an optionally substituted
piperazino group (PMO+). In certain embodiments, the
oligonucleotide is conjugated to a cell-penetrating peptide, such
as an arginine-rich peptide (PPMO or PPMO+).
[0010] Also included are methods of treating myotonic dystrophy DM1
in a mammalian subject, comprising administering to the subject, a
morpholino antisense compound of 9 bases, where the 9 bases are
complementary to polyCUG repeats in the 3'UTR region of dystrophia
myotonica protein kinase (DMPK) mRNA, and optionally repeating said
administering at least once every one week to 3 months. In some
embodiments, at least one and up to about 1 per every 2
intersubunit linkage(s) of the oligonucleotide contains a pendant
cationic group. In specific embodiments, the cationic group
comprises an optionally substituted piperazino group. In certain
embodiments, the oligonucleotide is conjugated to an arginine-rich
peptide.
[0011] The compounds may be administered by intravenous or
subcutaneous injection to the subject, at a dose between 1-5 or
1-20 mg/kg body weight antisense compound, and the administering
step may be continued at regular intervals of every two weeks to
three months. The subject may be monitored during the treatment for
improvement in muscle performance, heart conduction properties,
and/or for a reduction in serum creatine kinase.
[0012] Certain embodiments include antisense compounds for treating
myotonic dystrophy type 2 (DM2), comprising a morpholino antisense
oligonucleotide of 9 bases, where the 9 bases are complementary to
polyCCUG repeats in the first intron region of zinc finger protein
9 (ZNF9) pre-mRNA. Also included are methods of treating myotonic
dystrophy DM1 in a mammalian subject, comprising administering to
the subject, a morpholino antisense oligonucleotide of 9 bases,
where the 9 bases are complementary to polyCCUG repeats in the
first intron region of zinc finger protein 9 (ZNF9) pre-mRNA, and
optionally repeating said administering at least once every one
week to 3 months.
BRIEF DESCRIPTION OF THE FIGURES
[0013] FIGS. 1A-C show exemplary structures of a
phosphorodiamidate-linked morpholino oligomer (PMO), a
peptide-conjugated PMO (PPMO), and a peptide-conjugated PMO having
cationic intersubunit linkages (PPMO+), respectively. Though
multiple cationic linkage types are illustrated in FIG. 1C, a PMO+
or PPMO+oligomer will typically include just one type of cationic
linkage.
DETAILED DESCRIPTION
Definitions
[0014] The terms below, as used herein, have the following
meanings, unless indicated otherwise:
[0015] The terms "cell penetrating peptide" or "CPP" are used
interchangeably and refer to cationic cell penetrating peptides,
also called transport peptides, carrier peptides, or peptide
transduction domains. Examples of cell-penetrating peptides include
arginine-rich peptides. The peptides, as shown herein, typically
have the capability of inducing cell penetration within 100% of
cells of a given cell culture population and allow macromolecular
translocation within multiple tissues in vivo upon systemic
administration.
[0016] The terms "antisense oligomer" or "antisense
oligonucleotide" or "oligonucleotide" are used interchangeably and
refer to a sequence of cyclic subunits, each bearing a base-pairing
moiety, linked by intersubunit linkages that allow the base-pairing
moieties to hybridize to a target sequence in a nucleic acid
(typically an RNA) by Watson-Crick base pairing, to form a nucleic
acid:oligomer heteroduplex within the target sequence. The cyclic
subunits are based on ribose or another pentose sugar or, in a
preferred embodiment, a morpholino group (see description of
morpholino oligomers below). The oligomer may have exact or near
sequence complementarity to the target sequence; variations in
sequence near the termini of an oligomer are generally preferable
to variations in the interior.
[0017] In one aspect of the invention, for the treatment of DM1,
the antisense oligonucleotide is complementary to at least 8,
optionally 9-12 or more contiguous bases in polyCUG repeats within
the 3' UTR regions of the transcript for dystrophia myotonica
protein kinase (DMPK) in muscle cells, and is designed to bind by
hybridization to these repeats, blocking binding of
splice-associated proteins, such as one or more muscleblind family
proteins, e.g., MBNL1, or CUGBP to the transcript. The
oligonucleotide may be said to be "directed to" or "targeted
against" 3'UTR polyCUG repeats with which it hybridizes. The target
sequence may include a polyCUG region of at least 8 contiguous
bases, preferably at least 9-25, and up to 40 bases or more.
[0018] In another aspect of the invention, for the treatment of
DM2, the antisense oligonucleotide is complementary to at least 8,
optionally 9-12 or more contiguous bases in polyCUG repeats within
intron 1 of the pre-mRNA transcript for zinc finger protein 9
(ZNF9) in muscle cells, and is designed to bind by hybridization to
these repeats, blocking binding of splice-associated proteins, such
as one or more muscleblind family proteins, e.g., MBNL1, or CUGBP
to the pre-mRNA transcript or the excised intron 1 resulting from
ZNF9 pre-mRNA processing. The oligonucleotide may be said to be
"directed to" or "targeted against" polyCCUG repeats with which it
hybridizes. The target sequence may include a polyCCUG region of at
least 8 contiguous bases, preferably at least 9-25, and up to 40
bases or more.
[0019] Specific embodiments include 9 base antisense oligomers such
as PMO, PMO+, PPMO, or PPMO+ antisense oligonucleotides/compounds
that are fully complementary to polyCUG repeats within the 3' UTR
regions of the RNA transcript for DMPK, and antisense
oligonucleotides/compounds that are fully complementary to polyCCUG
repeats within intron 1 of the pre-mRNA transcript for ZNF9.
Examples include the antisense oligomers of SEQ ID NOS:1, 5, 9, and
15-18 targeted to polyCUG repeats, and SEQ ID NOS: 19, 21, 23,
26-29 targeted to polyCCUG repeats.
[0020] The terms "morpholino oligomer" or "PMO" (phosphoramidate-
or phosphorodiamidate morpholino oligomer) refer to an
oligonucleotide composed of morpholino subunit structures, where
(i) the structures are linked together by phosphorus-containing
linkages, one to three atoms long, preferably two atoms long, and
preferably uncharged or cationic, joining the morpholino nitrogen
of one subunit to a 5' exocyclic carbon of an adjacent subunit, and
(ii) each morpholino ring bears a purine or pyrimidine base-pairing
moiety effective to bind, by base specific hydrogen bonding, to a
base in a polynucleotide. See, for example, the structure in FIG.
1A, which shows a preferred phosphorodiamidate linkage type.
Variations can be made to this linkage as long as they do not
interfere with binding or activity. For example, the oxygen
attached to phosphorus may be substituted with sulfur
(thiophosphorodiamidate). The 5' oxygen may be substituted with
amino or lower alkyl substituted amino. The pendant nitrogen
attached to phosphorus may be unsubstituted, monosubstituted, or
disubstituted with (optionally substituted) lower alkyl. See also
the discussion of cationic linkages below. The purine or pyrimidine
base pairing moiety is typically adenine, cytosine, guanine,
uracil, thymine or inosine. The synthesis, structures, and binding
characteristics of morpholino oligomers are detailed in U.S. Pat.
Nos. 5,698,685, 5,217,866, 5,142,047, 5,034,506, 5,166,315,
5,521,063, and 5,506,337, and PCT Pubn. No. WO 2008036127 (cationic
linkages), all of which are incorporated herein by reference.
[0021] An "amino acid subunit" or "amino acid residue" can refer to
an .alpha.-amino acid residue (--CO--CHR--NH--) or a .beta.- or
other amino acid residue (e.g. --CO--(CH.sub.2).sub.nCHR--NH--),
where R is a side chain (which may include hydrogen) and n is 1 to
6, preferably 1 to 4.
[0022] The term "naturally occurring amino acid" refers to an amino
acid present in proteins found in nature. The term "non-natural
amino acids" refers to those amino acids not present in proteins
found in nature, examples include beta-alanine (.beta.-Ala),
6-aminohexanoic acid (Ahx) and 6-aminopentanoic acid.
[0023] A "marker compound" refers to a detectable compound attached
to a transport peptide for evaluation of transport of the resulting
conjugate into a cell. The compound may be visually or
spectrophotometrically detected, e.g. a fluorescent compound or
fluorescently labeled compound, which may include a fluorescently
labeled oligomer. Preferably, the marker compound is a labeled or
unlabeled antisense oligomer. In this case, detection of transport
involves detection of a product resulting from modulation of
splicing and/or transcription of a nucleic acid by an antisense
oligomeric compound. Exemplary methods, such as a splice correction
assay or exon skipping assay, are described in Materials and
Methods below.
[0024] An "effective amount" or "therapeutically effective amount"
refers to an amount of therapeutic compound, such as an antisense
oligomer, administered to a mammalian subject, either as a single
dose or as part of a series of doses, which is effective to produce
a desired therapeutic effect.
[0025] "Treatment" of an individual (e.g. a mammal, such as a
human) or a cell is any type of intervention used in an attempt to
alter the natural course of the individual or cell. Treatment
includes, but is not limited to, administration of a pharmaceutical
composition, and may be performed either prophylactically or
subsequent to the initiation of a pathologic event or contact with
an etiologic agent.
[0026] The terms "antisense compound" or "compound" or "conjugate
compound" include stand-alone antisense oligonucleotides (e.g.,
PMO, PMO+), and compounds formed by conjugating a cell-penetrating
peptide (e.g., arginine-rich peptide) to an antisense
oligonucleotide (e.g., PPMO, PPMO+). Examples of arginine-rich
peptides include SEQ ID NOS:30-44, including the
(RXRR(X/B)R).sub.2XB (SEQ ID NO:55) cell-penetrating peptides,
which can be conjugated, for example, to an oligonucleotide
targeted against a region of polyCUG or polyCCUG repeats.
[0027] "Systemic administration" of a compound refers to
administration, such as intravenous (iv) subcutaneous (subQ),
intramuscular (IM), and intraperitoneal (IP) that delivers the
compound directly into the bloodstream.
[0028] A systemically administered antisense oligonucleotide can be
targeted, for example, to heart muscle tissue by conjugation to the
CPP(RXRRBR).sub.2 (SEQ ID NO:42) with an XB linkage, or other
cell-penetrating peptide. In certain instances, the compound, when
administered systemically to a DM1 subject in accordance with the
method herein, produces a measurable improvement in heart muscle
performance and/or improvement in conduction properties of the
heart, as measured by known methods.
Structural Features of Transport Peptides
[0029] Exemplary cell-penetrating peptides that can employed in the
invention include a class of a transport peptide having 8 to 30
amino acid residues in length and consisting of subsequences
selected from the group consisting of RXR, RX, RB, and RBR; where R
is arginine (which may include D-arginine, represented in the
sequences herein by r), B is .beta.-alanine, and each X is
independently --C(O)--(CHR.sup.1).sub.n--NH--, where n is 4-6 and
each R.sup.1 is independently H or methyl, such that at most two
R.sup.1's are methyl. Preferably, each R.sup.1 is hydrogen. These
peptides have the generic formula (RXRRR(B/X)R).sub.2XB (SEQ ID
NO:55), where R is arginine; B is .beta.-alanine; and each X is
--C(O)--(CH.sub.2).sub.n--NH--, where n is 4-6, preferably 6, and
include both (RXRRBR).sub.2 (SEQ ID NO:42) with an XB linkage, and
(RXRRXR).sub.2 (SEQ ID NO:40) with an XB linkage, and where R is
arginine; B is .beta.-alanine; and each X is
--C(O)--(CH.sub.2).sub.n--NH--, where n is 4-6. As discussed below,
these peptides have been discovered to selectively target an
oligonucleotide, including a PMO, to muscle tissue, including heart
muscle tissue.
[0030] Table 1 below includes certain transport peptides in this
class that have been evaluated, in conjugation with suitable
antisense oligonucleotides, for their ability to selectively target
various tissues, including heart and skeletal muscle. See, e.g.,
U.S. application Ser. No. 12/493,140, incorporated by reference in
its entirety. The peptides have been evaluated for cellular uptake,
as determined by flow cytometry; for antisense activity, as
determined by a splice correction assay (Kang, Cho et al. 1998);
and for cellular toxicity, as determined by MTT cell viability,
propidium iodide membrane integrity and hemolysis assays, and
microscopic imaging, and their uptake and functional activity in
muscle tissue relative to a variety of non-muscle tissue were
compared. The (RXRRXR).sub.2 peptide (SEQ ID NO:40) with an XB
linkage was among the most active in antisense activity, as
determined by the splice correction assay, both in the presence and
absence of added serum. Both (RXRR(B/X)R).sub.2XB (SEQ ID NO:55)
peptides were effective in selectively targeting oligonucleotides
to heart and skeletal tissue, while showing relatively low-level
targeting to a variety of other tissues, including mammary gland
tissue, ovary/prostate (particularly (RXRRXRR).sub.2 (SEQ ID NO:40)
with an XB linkage), and brain. Embodiments of the present
invention may employ any one or more of these cell-penetrating or
arginine-rich peptides.
TABLE-US-00001 TABLE 1 Cell-Penetrating Peptides Name SEQ ID
(Designation) Sequence NO..sup.a rTAT RRRQRRKKR 30 Tat RKKRRQRRR 31
R.sub.9F.sub.2 RRRRRRRRRFF 32 R.sub.5F.sub.2R.sub.4 RRRRRFFRRRR 33
R.sub.4 RRRR 34 R.sub.5 RRRRR 35 R.sub.6 RRRRRR 36 R.sub.7 RRRRRRR
37 R.sub.8 RRRRRRRR 38 R.sub.9 RRRRRRRRR 39 (RAhxR).sub.4;
RAhxRRAhxRRAhxRRAhxR 40 (P007) (RAhxR).sub.5;
RAhxRRAhxRRAhxRRAhxRRAhxR 41 (CP04057) (RAhxRRBR).sub.2;
RAhxRRBRRAhxRRBR 42 (CP06062) (RAR).sub.4F.sub.2 RARRARRARRARFFC 43
(RGR).sub.4F.sub.2 RGRRGRRGRRGRFFC 44 .sup.aSequences assigned to
SEQ ID NOs do not include the linkage portion (e.g., C, G, Ahx, B,
AhxB where Ahx and B refer to 6-aminohexanoic acid and
beta-alanine, respectively).
Therapeutic Applications
[0031] The phosphorodiamidate morpholino oligomers (e.g., PMO,
PMO+) and other antisense oligomers described herein are useful for
treating myotonic dystrophy type 1 (DM1), and the conjugate
compounds (e.g., PPMO, PPMO+) of the present invention are further
useful for targeting and delivering these antisense oligomers
across both the cell and nuclear membranes to the nucleus of muscle
cells in skeletal and heart muscle tissue, by exposing the cell to
an antisense oligomer or conjugate comprising the oligomer
covalently linked to a carrier peptide, as described herein.
[0032] Treatment of Myotonic Dystrophy. As the name of the disorder
implies, the characteristic clinical manifestation in DM is
myotonia (muscle hyperexcitability) and muscle degeneration.
Affected individuals will also develop insulin resistance,
cataracts, heart conduction defects, testicular atrophy,
hypogammaglobulinemia and sleep disorders. Symptoms of DM can
manifest in the adult or in childhood. The childhood onset form of
the disease is often associated with mental retardation. In
addition, there is a form of the disease referred to as congenital
myotonic dystrophy. This latter form of the disease is frequently
fatal and is seen almost exclusively in children born of mothers
who themselves are mildly affected by the disease. In congenital DM
the facial manifestations are distinctive due to bilateral facial
palsy and marked jaw weakness. Many infants with congenital DM die
due to respiratory insufficiency before a proper diagnosis of the
disease is made.
[0033] DM1 initially involves the distal muscles of the extremities
and only as the disease progresses do proximal muscles become
affected. In addition, muscles of the head and neck are affected
early in the course of the disease. Weakness in eyelid closure,
limited extraocular movement and ptosis results from involvement of
the extraocular muscles. Many individuals with DM1 exhibit a
characteristic "haggard" appearance that is the result of atrophy
of the masseters (large muscles that raise and lower the jaw),
sternocleidomastoids (large, thick muscles that pass obliquely
across each side of the neck and contribute to arm movement) and
the temporalis muscle (muscle involved in chewing).
[0034] Treatment of DM1, in accordance with general embodiments of
the invention, may comprise, for example: (i) administering to the
subject with DM1, an antisense compound comprising an antisense
oligonucleotide having 8-30 bases, with at least 8 contiguous bases
being complementary to the polyCUG repeats in the 3'UTR region of
dystrophia myotonica protein kinase (DMPK) mRNA in DM1 and
optionally conjugated to the oligonucleotide, a cell-penetrating
peptide, and (ii) optionally repeating the compound administration
at least once every one week to once every three months or longer.
Examples of cell-penetrating peptides include the peptides of SEQ
ID NOS:30-44. In specific embodiments, the cell-penetrating peptide
may have the sequence (RXRRR(B/X)R).sub.2XB (SEQ ID NO:55), where R
is arginine; B is .beta.-alanine; and each X is
--C(O)--(CH.sub.2).sub.n--NH--, where n is 4-6.
[0035] Treatment of DM1, in accordance with specific embodiments of
the invention, may comprise: (i) administering to the subject with
DM1 a 9-base morpholino antisense oligonucleotide, where the 9
bases are complementary to polyCUG repeats in the 3'UTR region of
dystrophia myotonica protein kinase (DMPK) mRNA, and (ii)
optionally repeating said administering at least once every one
week to 3 months. The morpholino antisense oligomer may be a
phosphorodiamidate morpholino oligonucleotide (PMO), and/or it may
be a PMO where at least one and up to about 1 per every 2
intersubunit linkage(s) contains a pendant cationic group, such as
an optionally piperazino group (PMO+). The PMO or PMO+ compound may
be optionally conjugated to a cell-penetrating peptide, such as an
arginine-rich peptide (e.g., PPMO, PPMO+).
[0036] The compound is preferably administered by intravenous or
subcutaneous injection to the subject, at a dose between 1-5 or
1-20 mg/kg body weight antisense compound, at a dosing schedule of
once a month to once every 2-3 months. For subQ administration, the
dose required may be roughly twice that for IV administration.
During the course of treatment, the patient is monitored for
improvement or stabilization of muscle performance, improvement in
heart conduction properties and/or reduction in serum reduction in
serum creatine kinase. Because myotonic dystrophy is a chronic
disease, the treatment method will be applied over the subject's
lifetime, with dose adjustments being made during the treatment
period to achieve a desired level of muscle function and to
accommodate patient growth.
[0037] The treatment methods offer a number of important advantages
over earlier proposed antisense methods of treating DM1. First,
targeting, uptake and antisense activity of the antisense compounds
described herein into skeletal muscle, heart muscle, or both, is
efficient. This allows effective treatment with relatively modest
compound doses, e.g., in the range 1-5 mg/kg subject weight.
Second, little or no compound toxicity has been observed, as
evidenced, for example, by no microscopically observable increases
in muscle damage, inflammatory cellular infiltrates, or necrotic
fibers in muscles injected with PPMOs and/or PMOs. Finally, in
certain instances, the effect of a single dose may be effective for
up to three months or more, allowing the patient to be effectively
treated by dosing at intervals of no less than one month, and up to
3 months or more between successive treatments.
Combination with Homing Peptides
[0038] The antisense oligonucleotides and conjugate compounds of
the invention may be used in conjunction with homing peptides
selective for the target tissue, to further enhance muscle-specific
delivery. An example of this approach can be found in the
application of muscle-binding peptides (Samoylova and Smith, 1999;
Vodyanoy et al., U.S. Appn. Pubn. No. 20030640466) coupled to
antisense oligomers designed to be therapeutic treatments for
Duchenne muscular dystrophy (DMD) (Gebski, Mann et al. 2003; Alter,
Lou et al. 2006) (PCT Pubn. No. WO2006000057). The heptapeptide
sequence ASSLNIA (SEQ ID NO:45) has enhanced in vivo skeletal and
cardiac muscle binding properties, as described by Samoylova and
Smith. As a further example, a pancreas-homing peptide, CRVASVLPC
(SEQ ID NO:56), mimics the natural prolactin receptor ligand
(Kolonin, Sun et al. 2006).
[0039] An exemplary dual peptide molecule has a cell penetrating
peptide to one terminus, e.g. at the 5' end of the antisense
oligomer, as described herein, and a homing peptide coupled to the
other terminus, i.e. the 3' terminus. The homing peptide localizes
the peptide-conjugated PMO to the target tissue, where the
cell-penetrating peptide moiety effects transport into the cells of
the tissue.
[0040] Alternatively, a preferred exemplary dual peptide molecule
would have both a homing peptide (HP) and cell-penetrating peptide
(CPP) conjugated to one end, e.g. the 5' terminus of the antisense
oligomer, in either a HP-CPP-PMO configuration or, more preferably,
a CPP-HP-PMO configuration.
TABLE-US-00002 TABLE 2 Examples of Muscle-specific Homing Peptides
(HP) Peptide Sequence SEQ ID Target Tissue (NH.sub.2 to COOH) NO.
Skeletal Muscle-SMP1 ASSLNIA 45 SMP2 SLGSFP 46 SMP3 SGASAV 47 SMP4
GRSGAR 48 SMP5 TARGEHKEEELI 49 Cardiac Muscle-CMP1
WLSEAGPVVTVRALRGTGSW 50 CMP2 VTVRALRGTSW 51 CMP3 VVTVRALRGTGSW 52
CMP4 CRPPR 53 CMP5 SKTFNTHPQSTP 54 CRVASVLPC 56
Peptide-Antisense Oligomer Conjugate Compositions
Conjugates for Specific Muscle Treatments
[0041] Therapeutic conjugates comprising selected transport peptide
sequences are also provided by the invention. These include
conjugates comprising a carrier peptide (RXRR(B/X)R).sub.2XB (SEQ
ID NO:55), as described herein, conjugated to an oligonucleotide,
e.g., PMO, designed for therapeutic action within muscle tissue.
Also included are conjugates comprising an oligonucleotide
conjugated to any one of SEQ ID NOS:30-44.
[0042] The conjugates may further comprise a targeting moiety
effective to bind to tissue specific receptors of a target tissue
type, linked to the therapeutic compound or, preferably, to another
terminus of the carrier peptide. In particularly preferred
embodiments, a homing peptide such as described above is conjugated
to therapeutic compound or to the cell-penetrating peptide.
[0043] For use in treating myotonic dystrophy DM1, the conjugate
compound may comprise an antisense oligonucleotide, having 8-30
bases, preferably 9 bases, with at least 8 or 9 or more contiguous
bases being complementary to the polyCUG repeats in the 3'UTR
region of dystrophia myotonica protein kinase (DMPK) mRNA, and
conjugated to the oligonucleotide, a cell-penetrating peptide of
any one of SEQ ID NOS:30-44, including, for example, a peptide
having the sequence (RXRR(B/X)R).sub.2XB (SEQ ID NO:55), where R is
arginine; B is .beta.-alanine; and each X is
--C(O)--(CH.sub.2).sub.n--NH--, where n is 4-6. Such compounds are
effective to selectively block the sequestration of
muscleblind-like 1 protein (MBNL1) and/or CUGBP in heart and
quadricep muscle in a myotonic dystrophy animal model.
Morpholino Oligomers Having Cationic and Other Intersubunit
Linkages
[0044] In preferred embodiments, as noted above, the antisense
oligomer is a phosphorodiamidate morpholino oligonucleotide (PMO).
Certain PMOs may include between about 10-50% or 20-50% positively
charged or cationic backbone linkages, as described below and
further in WO120081036127, which is incorporated by reference.
[0045] Certain cationic PMOs (e.g., PMO+) include morpholino
oligomers in which at least one intersubunit linkage between two
consecutive morpholino ring structures contains a pendant cationic
group. The pendant group bears a distal nitrogen atom that can bear
a positive charge at neutral or near-neutral (e.g., physiological)
pH. Examples are shown in FIGS. 1B-C.
[0046] The intersubunit linkages in these oligomers are preferably
phosphorus-containing linkages, having the structure:
##STR00001##
where W is S or O, and is preferably O,
X=NR.sup.1R.sup.2 or OR.sup.6,
Y=O or NR.sup.7,
[0047] and each said linkage in the oligomer is selected from:
[0048] (a) uncharged linkage (a), where each of R.sup.1, R.sup.2,
R.sup.6 and R.sup.7 is independently selected from hydrogen and
lower alkyl;
[0049] (b1) cationic linkage (b1), where X=NR.sup.1R.sup.2 and Y=O,
and NR.sup.1R.sup.2 represents an optionally substituted piperazino
group, such that
R.sup.1R.sup.2=--CHRCHRN(R.sup.3)(R.sup.4)CHRCHR--, where
[0050] each R is independently H or CH.sub.3,
[0051] R.sup.4 is H, CH.sub.3, or an electron pair, and
[0052] R.sup.3 is selected from H, lower alkyl, e.g. CH.sub.3,
C(.dbd.NH)NH.sub.2, Z-L-NHC(.dbd.NH)NH.sub.2, and
{C(O)CHR'NH}.sub.mH, where: Z is C(O) or a direct bond, L is an
optional linker up to 18 atoms in length, preferably up to 12
atoms, and more preferably up to 8 atoms in length, having bonds
selected from alkyl, alkoxy, and alkylamino, R' is a side chain of
a naturally occurring amino acid or a one- or two-carbon homolog
thereof, and m is 1 to 6, preferably 1 to 4;
[0053] (b2) cationic linkage (b2), where X=NR.sup.1R.sup.2 and Y=O,
R.sup.1=H or CH.sub.3, and R.sup.2=LNR.sup.3R.sup.4R.sup.5, where
L, R.sup.3, and R.sup.4 are as defined above, and R.sup.5 is H,
lower alkyl, or lower (alkoxy)alkyl; and
[0054] (b3) cationic linkage (b3), where Y=NR.sup.7 and X=OR.sup.6,
and R.sup.7=LNR.sup.3R.sup.4R.sup.5, where L, R.sup.3, R.sup.4 and
R.sup.5 are as defined above, and R.sup.6 is H or lower alkyl;
[0055] and at least one said linkage is selected from cationic
linkages (b1), (b2), and (b3).
[0056] Preferably, the oligomer includes at least two consecutive
linkages of type (a) (i.e. uncharged linkages). In further
embodiments, at least 5% of the linkages in the oligomer are
cationic linkages (i.e. type (b1), (b2), or (b3)); for example, 10%
to 80%, 10% to 50%, or 10% to 35% of the linkages may be cationic
linkages.
[0057] In one embodiment, at least one linkage is of type (b1),
where, preferably, each R is H, R.sup.4 is H, CH.sub.3, or an
electron pair, and R.sup.3 is selected from H, lower alkyl, e.g.
CH.sub.3, C(.dbd.NH)NH.sub.2, and C(O)-L-NHC(.dbd.NH)NH.sub.2 The
latter two embodiments of R.sup.3 provide a guanidino moiety,
either attached directly to the piperazine ring, or pendant to a
linker group L, respectively. For ease of synthesis, the variable Z
in R.sup.3 is preferably C(O) (carbonyl), as shown.
[0058] The linker group L, as noted above, contains bonds in its
backbone selected from alkyl (e.g. --CH.sub.2--CH.sub.2--), alkoxy
(--C--O--), and alkylamino (e.g. --CH.sub.2--NH--), with the
proviso that the terminal atoms in L (e.g., those adjacent to
carbonyl or nitrogen) are carbon atoms. Although branched linkages
(e.g. --CH.sub.2--CHCH.sub.3--) are possible, the linker is
preferably unbranched. In one embodiment, the linker is a
hydrocarbon linker. Such a linker may have the structure
--(CH.sub.2).sub.n--, where n is 1-12, preferably 2-8, and more
preferably 2-6.
[0059] The use of embodiments of linkage types (b1), (b2) and (b3)
above to link morpholino subunits may be illustrated graphically as
follows:
##STR00002##
[0060] Preferably, all cationic linkages in the oligomer are of the
same type; i.e. all of type (b1), all of type (b2), or all of type
(b3). The base-pairing moieties Pi may be the same or different,
and are generally designed to provide a sequence which binds to a
target nucleic acid.
[0061] In further embodiments, the cationic linkages are selected
from linkages (b1') and (b1'') as shown below, where (b1 ) is
referred to herein as a "Pip" linkage and (b1'') is referred to
herein as a "GuX" linkage:
##STR00003##
[0062] In the structures above, W is S or O, and is preferably O;
each of R.sup.1 and R.sup.2 is independently selected from hydrogen
and lower alkyl, and is preferably methyl; and A represents
hydrogen or a non-interfering substituent on one or more carbon
atoms in (b1') and (b1''). Preferably, the ring carbons in the
piperazine ring are unsubstituted; however, they may include
non-interfering substituents, such as methyl or fluorine.
Preferably, at most one or two carbon atoms is so substituted.
[0063] In further embodiments, at least 10% of the linkages are of
type (b1') or (b1''); for example, 20% to 80%, 20% to 50%, or 20%
to 30% of the linkages may be of type (b1') or (b1'').
[0064] In other embodiments, the oligomer contains no linkages of
the type (b1') above. Alternatively, the oligomer contains no
linkages of type (b1) where each R is H, R.sup.3 is H or CH.sub.3,
and R.sup.4 is H, CH.sub.3, or an electron pair.
[0065] Oligomers having any number of cationic linkages can be
used, including fully cationic-linked oligomers. Preferably,
however, the oligomers are partially charged, having, for example,
5, 10, 20, 30, 40, 50, 60, 70, 80 or 90 percent cationic linkages.
In selected embodiments, about 10 to 80, 20 to 80, 20 to 60, 20 to
50, 20 to 40, or about 20 to 35 percent of the linkages are
cationic.
[0066] In one embodiment, the cationic linkages are interspersed
along the backbone. The partially charged oligomers preferably
contain at least two consecutive uncharged linkages; that is, the
oligomer preferably does not have a strictly alternating pattern
along its entire length.
[0067] Also considered are oligomers having blocks of cationic
linkages and blocks of uncharged linkages; for example, a central
block of uncharged linkages may be flanked by blocks of cationic
linkages, or vice versa. In one embodiment, the oligomer has
approximately equal-length 5', 3' and center regions, and the
percentage of cationic linkages in the center region is greater
than about 50%, preferably greater than about 70%.
[0068] Oligomers for use in antisense applications generally range
in length from about 10 to about 40 subunits, more preferably about
15 to 25 subunits. For example, a cationic oligomer having 19-20
subunits, a useful length for an antisense oligomer, may ideally
have two to seven, e.g. four to six, or three to five, cationic
linkages, and the remainder uncharged linkages. An oligomer having
14-15 subunits may ideally have two to five, e.g. 3 or 4, cationic
linkages and the remainder uncharged linkages. Specific examples
include a 9 subunit oligomer with about 1, 2, or 3 cationic
linkages, and the remainder uncharged linkages.
[0069] Each morpholino ring structure supports a base pairing
moiety, to form a sequence of base pairing moieties which is
typically designed to hybridize to a selected antisense target in a
cell or in a subject being treated. The base pairing moiety may be
a purine or pyrimidine found in native DNA or RNA (A, G, C, T, or
U) or an analog, such as hypoxanthine (the base component of the
nucleoside inosine) or 5-methyl cytosine.
[0070] As noted above, the substantially uncharged oligonucleotide
may be modified to include one or more charged linkages, e.g. up to
about 1 per every 2-5 uncharged linkages, typically 3-5 per every
10 uncharged linkages. Optimal improvement in antisense activity is
seen where up to about half of the backbone linkages are cationic.
Some, but not maximum enhancement is typically seen with a small
number e.g., 10-20% cationic linkages; where the number of cationic
linkages exceeds 50-60%, the sequence specificity of the antisense
binding to its target may be compromised or lost.
[0071] The enhancement seen with added cationic backbone charges
may, in some case, be further enhanced by distributing the bulk of
the charges close of the "center-region" backbone linkages of the
antisense oligonucleotide, e.g., in a 20-mer oligonucleotide with 8
cationic backbone linkages, having 70%400% of these charged
linkages localized in the 10 centermost linkages.
Other Oligomer Types
[0072] Delivery of alternative antisense chemistries can also
benefit from the disclosed carrier peptide. Specific examples of
other antisense compounds useful in this invention include those in
which at least one, or all, of the internucleotide bridging
phosphate residues are modified phosphates, such as methyl
phosphonates, phosphorothioates, or phosphoramidates. Also included
are molecules wherein at least one, or all, of the nucleotides
contains a 2' lower alkyl moiety (e.g., C1-C4, linear or branched,
saturated or unsaturated alkyl, such as methyl, ethyl, ethenyl,
propyl, 1-propenyl, 2-propenyl, or isopropyl).
[0073] In other oligonucleotide mimetics, both the sugar and the
internucleoside linkage, i.e., the backbone, of the nucleotide
units are modified. The base units are maintained for hybridization
with an appropriate nucleic acid target compound. One such
oligomeric compound, an oligonucleotide mimetic that has been shown
to have excellent hybridization properties, is referred to as a
peptide nucleic acid (PNA). In PNA compounds, the sugar-phosphate
backbone of an oligonucleotide is replaced with an amide containing
backbone, in particular an aminoethylglycine backbone.
[0074] Modified oligonucleotides may be classified as "chimeric,"
e.g., containing at least one region wherein the oligonucleotide is
modified so as to confer increased resistance to nuclease
degradation or increased cellular uptake, and an additional region
for increased binding affinity for the target nucleic acid.
EXAMPLES
[0075] The following examples are intended to illustrate but not to
limit the invention.
Example 1
PMO, PMO+, PPMO and PPMO+ Consisting of (CAG)n Repeats Reverse
Molecular and Physiological Manifestations of DM1 in a Mouse
Model
[0076] To determine whether antisense compositions described herein
(e.g., SEQ ID NOs: 1-18) can influence in vivo expanded CUG
(CUGexp) repeat interactions with MBNL1 splicing factor, their
effects can be examined in a transgenic mouse model of DM1. The
antisense oligonucleotides and conjugates shown in Table A below
can be manufactured according to routine techniques and then tested
in this transgenic mouse model of DM1.
TABLE-US-00003 TABLE A PMO, PMO+, PPMO,and PPMO+ agents targeted to
polyCUG repeats in the 3'UTR region of dystrophia myotonica protein
kinase (DMPK). Sample Name Sequence 5'End 3'End CAG 9 mer CAG CAG
CAG (SEQ ID NO: 1) EG3 H CAG 9 mer-B CAG CAG CAG (SEQ ID NO: 1) EG3
CP06062 CAG 9 mer-R9F2 CAG CAG CAG (SEQ ID NO: 1) EG3 R9F2 CAG 9
mer-rTat CAG CAG CAG (SEQ ID NO: 1) EG3 rTat CAG 12 mer CAG CAG CAG
CAG (SEQ ID NO: 2) EG3 H CAG 12 mer-B CAG CAG CAG CAG (SEQ ID NO:
2) EG3 CP06062 CAG 15 mer CAG CAG CAG CAG CAG (SEQ ID NO: 3) EG3 H
CAG 15 mer-B CAG CAG CAG CAG CAG (SEQ ID NO: 3) EG3 CP06062 CAG 18
mer CAG CAG CAG CAG CAG CAG (SEQ ID NO: 4) EG3 H CAG 18 mer-B CAG
CAG CAG CAG CAG CAG (SEQ ID NO: 4) EG3 CP06062 AGC 9 mer AGC AGC
AGC (SEQ ID NO: 5) EG3 H AGC 9 mer-B AGC AGC AGC (SEQ ID NO: 5) EG3
CP06062 AGC 12 mer AGC AGC AGC AGC (SEQ ID NO: 6) EG3 H AGC 12
mer-B AGC AGC AGC AGC (SEQ ID NO: 6) EG3 CP06062 AGC 15 mer AGC AGC
AGC AGC AGC (SEQ ID NO: 7) EG3 H AGC 15 mer-B AGC AGC AGC AGC AGC
(SEQ ID NO: 7) EG3 CP06062 AGC 18 mer AGC AGC AGC AGC AGC AGC (SEQ
ID NO: 8) EG3 H AGC 18 mer-B AGC AGC AGC AGC AGC AGC (SEQ ID NO: 8)
EG3 CP06062 GCA 9 mer GCA GCA GCA (SEQ ID NO: 9) EG3 H GCA 9 mer-B
GCA GCA GCA (SEQ ID NO: 9) EG3 CP06062 GCA 12 mer GCA GCA GCA GCA
(SEQ ID NO: 10) EG3 H GCA 12 mer-B GCA GCA GCA GCA (SEQ ID NO: 10)
EG3 CP06062 GCA 15 mer GCA GCA GCA GCA GCA (SEQ ID NO: 11) EG3 H
GCA 15 mer-B GCA GCA GCA GCA GCA (SEQ ID NO: 11) EG3 CP06062 GCA 18
mer GCA GCA GCA GCA GCA GCA (SEQ ID NO: 12) EG3 H GCA 18 mer-B GCA
GCA GCA GCA GCA GCA (SEQ ID NO: 12) EG3 CP06062 AGC 25 mer AGC AGC
AGC AGC AGC AGC AGC AGC A EG3 H (SEQ ID NO: 13) AGC 25 mer-B AGC
AGC AGC AGC AGC AGC AGC AGC A EG3 CP06062 (SEQ ID NO: 13) CAG 25
mer CAG CAG CAG CAG CAG CAG CAG CAG C EG3 H (SEQ ID NO: 14) CAG 25
mer-B CAG CAG CAG CAG CAG CAG CAG CAG C EG3 CP06062 (SEQ ID NO: 14)
CAG 25 mer-R9F2 CAG CAG CAG CAG CAG CAG CAG CAG C EG3 R9F2 (SEQ ID
NO: 14) CAG 25 mer-rTat CAG CAG CAG CAG CAG CAG CAG CAG C EG3 rTat
(SEQ ID NO: 14) CAG 9 mer+ C+AG C+AG C+AG (SEQ ID NO: 15) EG3 H CAG
9 mer+B C+AG C+AG C+AG (SEQ ID NO: 15) EG3 CP06062 CAG 9 mer+ C+AG
CAG CAG (SEQ ID NO: 16) EG3 H CAG 9 mer+ CAG CAG C+AG (SEQ ID NO:
17) EG3 H CAG 9 mer+ CAG C+AG CAG (SEQ ID NO: 18) EG3 H * The
linkage(s) between the oligonucleotide and the cell-penetrating
peptide can included a variety of linkages, but preferred linkages
are C, AhxB, G, and B.
[0077] HSA.sup.LR transgenic mice express human skeletal actin
transcripts that have (CUG)250 inserted in the 3' UTR (Mankodi,
Logigian et al. 2000). These mice accumulate CUGexp RNA and MBNL1
protein in nuclear foci in skeletal muscle, a process that depends
on CUGexp-MBNL1 interaction (Dansithong, Paul et al. 2005). The
effect of antisense compositions of the present invention can be
examined in their ability to block foci development in muscle
cells. PMO and PPMO an be delivered intravenously or
intraperitoneally at doses ranging from 30 to 600 micrograms.
Muscle tissue can be examined 1-3 weeks later by fluorescence in
situ hybridization using probes that hybridize to the CUG repeat or
to sequences flanking the repeat. Activity of any given compound
can be measured by the magnitude of reduction of nuclear foci and
redistribution of MBNL1 from a punctate pattern to diffuse
localization in the nucleus.
[0078] Compositions of the present invention can also reverse the
biochemical consequences of MBNL1 sequestration. Accumulation of
CUGexp RNA-MBNL1 complexes in the foci results also in aberrant
mis-splicing of several genes, namely, ClC-1, Serca-1, m-Titin,
Tnnt3 and Zasp genes (Mulders, van den Broek et al. 2009).
HSA.sup.LR transgenic mice show alternative splicing changes
similar to those observed in human DM1 (Wheeler, Sobczak et al.
2009). DM1-affected, aberrantly spliced exons can be examined in
mice treated with compositions of the invention to determine
whether alternative splicing is corrected at three weeks following
injection of compositions of the present invention. Effects of PMO
or PPMO treatment on aberrant splicing can be expected to persist
for at least fourteen weeks.
[0079] It is also expected that compositions of the present
invention can rescue the physiological effects of DM1 (myotonia)
and can be examined by measuring the expression and function of
chloride channel 1 (ClC-1) which is inactivated by mis-splicing in
DM1 and the HSA.sup.LR mouse model. Myotonia can be measured
through determination of delayed muscle relaxation and repetitive
action potentials and are expected to improve in HSA.sup.LR mice
treated with compositions of the present invention.
Example 2
PMO, PPMOplus, PPMO and PMO-X Consisting of (CCAG)n Repeats Reverse
Molecular and Physiological Manifestations of DM2
[0080] To determine whether antisense compositions described herein
(e.g., SEQ ID NOs: 19-29) can influence in vivo expanded CCUG
(CCUGexp) repeat interactions with MBNL1 splicing factor, their
effects can be examined in an analogous transgenic mouse model to
that described above for DM1. The antisense oligonucleotides and
conjugates shown in Table B below can be manufactured according to
routine techniques and then tested in this transgenic mouse model
of DM2. The expected experimental outcomes are similar to those
described for DM1 in Example 1.
TABLE-US-00004 TABLE B PMO, PMO+, PPMO, and PPMO+ agents targeted
to polyCCUG repeats in the first intron of zinc finger protein 9
(ZNF9) pre-mRNA. Sample Name Sequence 5'End 3'End CAGG 9 mer CAG
GCA GGC (SEQ ID NO: 19) EG3 H CAGG 9 mer-B CAG GCA GGC (SEQ ID NO:
19) EG3 CP06062 CAGG 9 mer-R9F2 CAG GCA GGC (SEQ ID NO: 19) EG3
R9F2 CCAG 9 mer-rTat CAG GCA GGC (SEQ ID NO: 19) EG3 rTat CCAG 12
mer CAG GCA GGC AGG (SEQ ID NO: 20) EG3 H CCAG 12 mer-B CAG GCA GGC
AGG (SEQ ID NO: 20) EG3 CP06062 AGCC 9 mer AGG CAG GCA (SEQ ID NO:
21) EG3 H AGCC 9 mer-B AGG CAG GCA (SEQ ID NO: 21) EG3 CP06062 AGCC
12 mer AGG CAG GCA GGC (SEQ ID NO: 22) EG3 H AGCC 12 mer-B AGG CAG
GCA GGC (SEQ ID NO: 22) EG3 CP06062 GCCA 9 mer GGC AGG CAG (SEQ ID
NO: 23) EG3 H GCCA 9 mer-B GGC AGG CAG (SEQ ID NO: 23) EG3 CP06062
GCCA 12 mer GGC AGG CAG GCA (SEQ ID NO: 24) EG3 H GCCA 12 mer-B GGC
AGG CAG GCA (SEQ ID NO: 24) EG3 CP06062 CAGG 24 mer CAG GCA GGC AGG
CAG GCA GGC AGG EG3 H (SEQ ID NO: 25) CAGG 24 mer-B CAG GCA GGC AGG
CAG GCA GGC AGG EG3 CP06062 (SEQ ID NO: 25) CAGG 24 mer-R9F2 CAG
GCA GGC AGG CAG GCA GGC AGG EG3 R9F2 (SEQ ID NO: 25) CAGG 24
mer-rTat CAG GCA GGC AGG CAG GCA GGC AGG EG3 rTat (SEQ ID NO: 25)
CAGG 9 mer+ C+AG GC+A GGC (SEQ ID NO: 26) EG3 H CAGG 9 mer+B C+AG
GC+A GGC (SEQ ID NO: 27) EG3 CP06062 CAGG 9 mer+ C+AG GCA GGC (SEQ
ID NO: 28) EG3 H CAGG 9 mer+ CAG GC+A GGC (SEQ ID NO: 29) EG3 H *
The linkage(s) between the oligonucleotide and the cell-penetrating
peptide can included a variety of linkages, but preferred linkages
are C, AhxB, G, and B.
[0081] Although the invention has been described with respect to
certain embodiments and examples, it will be appreciated that
various changes, modifications, and additions may be made without
departing from the claimed invention.
REFERENCES
[0082] Abes, S., H. M. Moulton et al. (2006). "Vectorization of
morpholino oligomers by the (R-Ahx-R).sub.4 peptide allows
efficient splicing correction in the absence of endosomolytic
agents." J Control Release 116(3): 304-13. [0083] Arap, W. et al.
(2004). "Human and mouse targeting peptides identified by phage
display." U.S. Appn. Pubn. No. 20040170955. [0084] Behlke, M. A.
(2006). "Progress towards in vivo use of siRNAs." Mol Ther 13(4):
644-70. [0085] Alter, J., F. Lou et al. (2006). "Systemic delivery
of morpholino oligonucleotide restores dystrophin expression
bodywide and improves dystrophic pathology." Nat Med 12(2): 175-7.
[0086] Chen, C. P., L. R. Zhang et al. (2003). "A concise method
for the preparation of peptide and arginine-rich peptide-conjugated
antisense oligonucleotide." Bioconjug Chem 14(3): 532-8. [0087]
Gebski, B. L., C. J. Mann et al. (2003). "Morpholino antisense
oligonucleotide induced dystrophin exon 23 skipping in mdx mouse
muscle." Hum Mol Genet. 12(15): 1801-11. [0088] Jearawiriyapaisarn,
Moulton et al. (2008). "Sustained Dystrophin Expression Induced by
Peptide-conjugated Morpholino Oligomers in the Muscles of mdx
Mice." Mol Therapy, Jun. 10, 2008 (advance online publication).
[0089] Kang, S. H., M. J. Cho et al. (1998). "Up-regulation of
luciferase gene expression with antisense oligonucleotides:
implications and applications in functional assay development."
Biochemistry 37(18): 6235-9. [0090] Kolonin, M. G., J. Sun et al.
(2006). "Synchronous selection of homing peptides for multiple
tissues by in vivo phage display." FASEB J20(7): 979-81. [0091]
Meade, B. R. and S. F. Dowdy (2007). "Exogenous siRNA delivery
using peptide transduction domains/cell penetrating peptides." Adv
Drug Deliv Rev 59(2-3): 134-40. [0092] Richard, J. P., K. Melikov
et al. (2003). "Cell-penetrating peptides. A reevaluation of the
mechanism of cellular uptake." J Biol Chem 278(1): 585-90. [0093]
Rothbard, J. B., E. Kreider et al. (2002). "Arginine-rich molecular
transporters for drug delivery: role of backbone spacing in
cellular uptake." J Med Chem 45(17): 3612-8. [0094] Samoylova, T.
I. and B. F. Smith (1999). "Elucidation of muscle-binding peptides
by phage display screening." Muscle Nerve 22(4): 460-6. [0095]
Sazani, P., F. Gemignani et al. (2002). "Systemically delivered
antisense oligomers upregulate gene expression in mouse tissues."
Nat Biotechnol 20(12): 1228-33. [0096] Sontheimer, E. J. (2005).
"Assembly and function of RNA silencing complexes." Nat Rev Mol
Cell Biol 6(2): 127-38. [0097] Vodyanoy, V. et al. (2003). "Ligand
sensor devices and uses thereof." U.S. Appn. Pubn. No. 20030640466.
[0098] Wu, R. P., D. S. Youngblood et al. (2007). "Cell-penetrating
peptides as transporters for morpholino oligomers: effects of amino
acid composition on intracellular delivery and cytotoxicity."
Nucleic Acids Res. 35(15):5182-91. (Epub 2007 Aug. 1.) [0099]
Youngblood, D. S., S. A. Hatlevig et al. (2007). "Stability of
cell-penetrating peptide-morpholino oligomer conjugates in human
serum and in cells." Bioconjug Chem 18(1): 50-60. [0100]
Dansithong, W., S. Paul, et al. (2005). "MBNL1 is the primary
determinant of focus formation and aberrant insulin receptor
splicing in DM1." J Biol Chem 280(7): 5773-80. [0101] Mankodi, A.,
E. Logigian, et al. (2000). "Myotonic dystrophy in transgenic mice
expressing an expanded CUG repeat." Science 289 (5485): 1769-73.
[0102] Wheeler, T. M., K. Sobczak, et al. (2009). "Reversal of RNA
dominance by displacement of protein sequestered on triplet repeat
RNA." Science 325 (5938): 336-9. [0103] Mulders, S. A., W. J. van
den Broek, et al. (2009). "Triplet-repeat oligonucleotide-mediated
reversal of RNA toxicity in myotonic dystrophy." Proc Natl Acad Sci
USA 106(33): 13915-20. [0104] Wheeler, T. M. and C. A. Thornton
(2007). "Myotonic dystrophy: RNA-mediated muscle disease." Curr
Opin Neurol 20(5): 572-6.
TABLE-US-00005 [0104] Sequence Listing Table Sample Name Sequence
SEQ ID NO: CAG 9 mer CAG CAG CAG 1 CAG 12 mer CAG CAG CAG CAG 2 CAG
15 mer CAG CAG CAG CAG CAG 3 CAG 18 mer CAG CAG CAG CAG CAG CAG 4
AGC 9 mer AGC AGC AGC 5 AGC 12 mer AGC AGC AGC AGC 6 AGC 15 mer AGC
AGC AGC AGC AGC 7 AGC 18 mer AGC AGC AGC AGC AGC AGC 8 GCA 9 mer
GCA GCA GCA 9 GCA 12 mer GCA GCA GCA GCA 10 GCA 15 mer GCA GCA GCA
GCA GCA 11 GCA 18 mer GCA GCA GCA GCA GCA GCA 12 AGC 25 mer AGC AGC
AGC AGC AGC AGC AGC AGC A 13 CAG 25 mer CAG CAG CAG CAG CAG CAG CAG
CAG C 14 CAG 9 mer+ C+AG C+AG C+AG 15 CAG 9 mer+ C+AG CAG CAG 16
CAG 9 mer+ CAG CAG C+AG 17 CAG 9 mer+ CAG C+AG CAG 18 CAGG 9 mer
CAG GCA GGC 19 CAGG 12 mer CAG GCA GGC AGG 20 AGGC 9 mer AGG CAG
GCA 21 AGGC 12 mer AGG CAG GCA GGC 22 GGCA 9 mer GGC AGG CAG 23
GGCA 12 mer GGC AGG CAG GCA 24 CAGG 24 mer CAG GCA GGC AGG CAG GCA
GGC AGG 25 CAGG 9 mer+ C+AG GC+A GGC 26 CAGG 9 mer+B C+AG GC+A GGC
27 CAGG 9 mer+ C+AG GCA GGC 28 CAGG 9 mer+ CAG GC+A GGC 29 rTAT
RRRQRRKKR 30 Tat RKKRRQRRR 31 R.sub.9F.sub.2 RRRRRRRRRFF 32
R.sub.5F.sub.2R.sub.4 RRRRRFFRRRR 33 R.sub.4 RRRR 34 R.sub.5 RRRRR
35 R.sub.6 RRRRRR 36 R.sub.7 RRRRRRR 37 R.sub.8 RRRRRRRR 38 R.sub.9
RRRRRRRRR 39 (RAhxR).sub.4; (P007) RAhxRRAhxRRAhxRRAhxR 40
(RAhxR).sub.5; RAhxRRAhxRRAhxRRAhxRRAhxR 41 (CP04057)
(RAhxRRBR).sub.2; RAhxRRBRRAhxRRBR 42 (CP06062) (RAR).sub.4F.sub.2
RARRARRARRARFFC 43 (RGR).sub.4F.sub.2 RGRRGRRGRRGRFFC 44 SMP1
ASSLNIA 45 SMP2 SLGSFP 46 SMP3 SGASAV 47 SMP4 GRSGAR 48 SMP5
TARGEHKEEELI 49 CMP1 WLSEAGPVVTVRALRGTGSW 50 CMP2 VTVRALRGTSW 51
CMP3 VVTVRALRGTGSW 52 CMP4 CRPPR 53 CMP5 SKTFNTHPQSTP 54
(RXRR(X/B)R).sub.2XB 55 CRVASVLPC 56 *In SEQ ID NOS: 15-18 and
26-29, "+" refers to a cationic linkage, such as 1-piperazinyl.
Sequence CWU 1
1
5619DNAArtificial SequenceAntisense oligomer 1cagcagcag
9212DNAArtificial SequenceAntisense oligomer 2cagcagcagc ag
12315DNAArtificial SequenceAntisense oligomer 3cagcagcagc agcag
15418DNAArtificial SequenceAntisense oligomer 4cagcagcagc agcagcag
1859DNAArtificial SequenceAntisense oligomer 5agcagcagc
9612DNAArtificial SequenceAntisense oligomer 6agcagcagca gc
12715DNAArtificial SequenceAntisense oligomer 7agcagcagca gcagc
15818DNAArtificial SequenceAntisense oligomer 8agcagcagca gcagcagc
1899DNAArtificial SequenceAntisense oligomer 9gcagcagca
91012DNAArtificial SequenceAntisense oligomer 10gcagcagcag ca
121115DNAArtificial SequenceAntisense oligomer 11gcagcagcag cagca
151218DNAArtificial SequenceAntisense oligomer 12gcagcagcag
cagcagca 181325DNAArtificial SequenceAntisense oligomer
13agcagcagca gcagcagcag cagca 251425DNAArtificial SequenceAntisense
oligomer 14cagcagcagc agcagcagca gcagc 25159DNAArtificial
SequenceAntisense oligomer 15cagcagcag 9169DNAArtificial
SequenceAntisense oligomer 16cagcagcag 9179DNAArtificial
SequenceAntisense oligomer 17cagcagcag 9189DNAArtificial
SequenceAntisense oligomer 18cagcagcag 9199DNAArtificial
SequenceAntisense oligomer 19caggcaggc 92012DNAArtificial
SequenceAntisense oligomer 20caggcaggca gg 12219DNAArtificial
SequenceAntisense oligomer 21aggcaggca 92212DNAArtificial
SequenceAntisense oligomer 22aggcaggcag gc 12239DNAArtificial
SequenceAntisense oligomer 23ggcaggcag 92412DNAArtificial
SequenceAntisense oligomer 24ggcaggcagg ca 122524DNAArtificial
SequenceAntisense oligomer 25caggcaggca ggcaggcagg cagg
24269DNAArtificial SequenceAntisense oligomer 26caggcaggc
9279DNAArtificial SequenceAntisense oligomer 27caggcaggc
9289DNAArtificial SequenceAntisense oligomer 28caggcaggc
9299DNAArtificial SequenceAntisense oligomer 29caggcaggc
9309PRTArtificial SequenceCell penetrating peptide 30Arg Arg Arg
Gln Arg Arg Lys Lys Arg1 5319PRTArtificial SequenceCell penetrating
peptide 31Arg Lys Lys Arg Arg Gln Arg Arg Arg1 53211PRTArtificial
SequenceCell penetrating peptide 32Arg Arg Arg Arg Arg Arg Arg Arg
Arg Phe Phe1 5 103311PRTArtificial SequenceCell penetrating peptide
33Arg Arg Arg Arg Arg Phe Phe Arg Arg Arg Arg1 5 10344PRTArtificial
SequenceCell penetrating peptide 34Arg Arg Arg Arg1355PRTArtificial
SequenceCell penetrating peptide 35Arg Arg Arg Arg Arg1
5366PRTArtificial SequenceCell penetrating peptide 36Arg Arg Arg
Arg Arg Arg1 5377PRTArtificial SequenceCell penetrating peptide
37Arg Arg Arg Arg Arg Arg Arg1 5388PRTArtificial SequenceCell
penetrating peptide 38Arg Arg Arg Arg Arg Arg Arg Arg1
5399PRTArtificial SequenceCell penetrating peptide 39Arg Arg Arg
Arg Arg Arg Arg Arg Arg1 54012PRTArtificial SequenceCell
penetrating peptide 40Arg Xaa Arg Arg Xaa Arg Arg Xaa Arg Arg Xaa
Arg1 5 104115PRTArtificial SequenceCell penetrating peptide 41Arg
Xaa Arg Arg Xaa Arg Arg Xaa Arg Arg Xaa Arg Arg Xaa Arg1 5 10
154212PRTArtificial SequenceCell penetrating peptide 42Arg Xaa Arg
Arg Xaa Arg Arg Xaa Arg Arg Xaa Arg1 5 104315PRTArtificial
SequenceCell penetrating peptide 43Arg Ala Arg Arg Ala Arg Arg Ala
Arg Arg Ala Arg Phe Phe Cys1 5 10 154415PRTArtificial SequenceCell
penetrating peptide 44Arg Gly Arg Arg Gly Arg Arg Gly Arg Arg Gly
Arg Phe Phe Cys1 5 10 15457PRTArtificial SequenceMuscle-specific
Homing Peptide 45Ala Ser Ser Leu Asn Ile Ala1 5466PRTArtificial
SequenceMuscle-specific Homing Peptide 46Ser Leu Gly Ser Phe Pro1
5476PRTArtificial SequenceMuscle-specific Homing Peptide 47Ser Gly
Ala Ser Ala Val1 5486PRTArtificial SequenceMuscle-specific Homing
Peptide 48Gly Arg Ser Gly Ala Arg1 54912PRTArtificial
SequenceMuscle-specific Homing Peptide 49Thr Ala Arg Gly Glu His
Lys Glu Glu Glu Leu Ile1 5 105020PRTArtificial
SequenceMuscle-specific Homing Peptide 50Trp Leu Ser Glu Ala Gly
Pro Val Val Thr Val Arg Ala Leu Arg Gly1 5 10 15Thr Gly Ser Trp
205111PRTArtificial SequenceMuscle-specific Homing Peptide 51Val
Thr Val Arg Ala Leu Arg Gly Thr Ser Trp1 5 105213PRTArtificial
SequenceMuscle-specific Homing Peptide 52Val Val Thr Val Arg Ala
Leu Arg Gly Thr Gly Ser Trp1 5 10535PRTArtificial
SequenceMuscle-specific Homing Peptide 53Cys Arg Pro Pro Arg1
55412PRTArtificial SequenceMuscle-specific Homing Peptide 54Ser Lys
Thr Phe Asn Thr His Pro Gln Ser Thr Pro1 5 105514PRTArtificial
SequenceCell penetrating peptide 55Arg Xaa Arg Arg Xaa Arg Arg Xaa
Arg Arg Xaa Arg Xaa Xaa1 5 10569PRTArtificial
SequenceMuscle-specific Homing Peptide 56Cys Arg Val Ala Ser Val
Leu Pro Cys1 5
* * * * *