NZ743588A - Exon skipping compositions for treating muscular dystrophy - Google Patents
Exon skipping compositions for treating muscular dystrophy Download PDFInfo
- Publication number
- NZ743588A NZ743588A NZ743588A NZ74358814A NZ743588A NZ 743588 A NZ743588 A NZ 743588A NZ 743588 A NZ743588 A NZ 743588A NZ 74358814 A NZ74358814 A NZ 74358814A NZ 743588 A NZ743588 A NZ 743588A
- Authority
- NZ
- New Zealand
- Prior art keywords
- antisense
- mezn
- seq
- dystrophin
- exon
- Prior art date
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- 229940001584 sodium metabisulfite Drugs 0.000 description 1
- 235000010262 sodium metabisulphite Nutrition 0.000 description 1
- 239000008109 sodium starch glycolate Substances 0.000 description 1
- 229940079832 sodium starch glycolate Drugs 0.000 description 1
- 229920003109 sodium starch glycolate Polymers 0.000 description 1
- 235000010265 sodium sulphite Nutrition 0.000 description 1
- 238000000527 sonication Methods 0.000 description 1
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- 239000004334 sorbic acid Substances 0.000 description 1
- 229940075582 sorbic acid Drugs 0.000 description 1
- 239000003549 soybean oil Substances 0.000 description 1
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- 241000894007 species Species 0.000 description 1
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- 239000003381 stabilizer Substances 0.000 description 1
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- 230000004936 stimulating effect Effects 0.000 description 1
- 229960005322 streptomycin Drugs 0.000 description 1
- 125000005346 substituted cycloalkyl group Chemical group 0.000 description 1
- KDYFGRWQOYBRFD-UHFFFAOYSA-L succinate(2-) Chemical compound [O-]C(=O)CCC([O-])=O KDYFGRWQOYBRFD-UHFFFAOYSA-L 0.000 description 1
- 150000003460 sulfonic acids Chemical class 0.000 description 1
- 239000003765 sweetening agent Substances 0.000 description 1
- 238000010189 synthetic method Methods 0.000 description 1
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- 229940095064 tartrate Drugs 0.000 description 1
- 238000011191 terminal modification Methods 0.000 description 1
- 125000000999 tert-butyl group Chemical group [H]C([H])([H])C(*)(C([H])([H])[H])C([H])([H])[H] 0.000 description 1
- YLQBMQCUIZJEEH-UHFFFAOYSA-N tetrahydrofuran Natural products C=1C=COC=1 YLQBMQCUIZJEEH-UHFFFAOYSA-N 0.000 description 1
- 125000003718 tetrahydrofuranyl group Chemical group 0.000 description 1
- 125000004853 tetrahydropyridinyl group Chemical group N1(CCCC=C1)* 0.000 description 1
- 125000003507 tetrahydrothiofenyl group Chemical group 0.000 description 1
- 238000002560 therapeutic procedure Methods 0.000 description 1
- 230000004797 therapeutic response Effects 0.000 description 1
- 125000000335 thiazolyl group Chemical group 0.000 description 1
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- 229940104230 thymidine Drugs 0.000 description 1
- AOBORMOPSGHCAX-DGHZZKTQSA-N tocofersolan Chemical compound OCCOC(=O)CCC(=O)OC1=C(C)C(C)=C2O[C@](CCC[C@H](C)CCC[C@H](C)CCCC(C)C)(C)CCC2=C1C AOBORMOPSGHCAX-DGHZZKTQSA-N 0.000 description 1
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- JBWKIWSBJXDJDT-UHFFFAOYSA-N triphenylmethyl chloride Chemical compound C=1C=CC=CC=1C(C=1C=CC=CC=1)(Cl)C1=CC=CC=C1 JBWKIWSBJXDJDT-UHFFFAOYSA-N 0.000 description 1
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Landscapes
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
Disclosed is an antisense oligonucleotide of 22 bases comprising the base sequence GAT CTG TCA AAT CGC CTG CAG G (SEQ ID NO: 5), in which thymine bases are optionally uracil bases; wherein the oligonucleotide is chemically linked to one or more moieties or conjugates that enhance the activity, cellular distribution, or cellular uptake of the antisense oligonucleotide (arginine-rich peptide); or a pharmaceutically acceptable salt thereof. Also disclosed is the use of the antisense nucleotide in the treatment of Duchenne’s muscular dystrophy.
Description
EXON SKIPPING COMPOSITIONS FOR NG MUSCULAR DYSTROPHY
RELATED APPLICATIONS
This patent application claims the benefit of U.S. Provisional Patent Application Serial No.
61/784547, filed March 14, 2013. The entire contents of the above-referenced provisional patent
application are incorporate herein by reference. This is a divisional of New Zealand Patent
Application No. 731587, which is a divisional of New Zealand Patent Application No. 631245, the
entire contents of which are orated herein by reference.
FIELD OF THE INVENTION
The present invention relates to novel antisense compounds and compositions le for
facilitating exon skipping in the human dystrophin gene. It also provides methods for ng
exon skipping using the novel antisense compositions adapted for use in the methods of the
invention.
OUND OF THE INVENTION
Antisense technologies are being ped using a range of chemistries to affect gene
expression at a variety of different levels (transcription, splicing, stability, translation). Much of
that research has focused on the use of antisense compounds to correct or compensate for
abnormal or disease-associated genes in a wide range of indications. Antisense molecules are able
to t gene expression with specificity, and because of this, many ch efforts concerning
oligonucleotides as modulators of gene expression have focused on inhibiting the expression of
targeted genes or the function of cis-acting elements. The anti sense oligonucleotides are typically
directed against RNA, either the sense strand (e.g., mRNA), or minus-strand in the case of some
viral RNA s. To achieve a desired effect of specific gene down-regulation, the
oligonucleotides generally either promote the decay of the ed mRNA, block translation of the
mRNA or block the function of cis-acting RNA elements, thereby effectively preventing either de
novo synthesis of the target n or replication of the viral RNA.
However, such techniques are not useful where the object is to up-regulate production of
the native protein or sate for mutations that induce premature termination of translation,
such as se or frame-shifting mutations. In these cases, the defective gene transcript should
not be subjected to targeted degradation or steric inhibition, so the antisense oligonucleotide
try should not promote target mRNA decay or block translation.
In a variety of genetic diseases, the effects of mutations on the eventual expression of a
gene can be modulated through a process of targeted exon skipping during the splicing process.
The splicing process is directed by complex multi-component machinery that brings adjacent
AVN-013BPC
exon-intron junctions in pre-mRNA into close proximity and performs cleavage of phosphodiester
bonds at the ends of the introns with their subsequent reformation between exons that are to be
spliced er. This complex and highly e process is mediated by sequence motifs in the
pre-mRNA that are relatively short, semi—conserved RNA segments to which s nuclear
splicing s that are then involved in the splicing reactions bind. By changing the way the
splicing machinery reads or izes the motifs involved in pre—mRNA processing, it is possible
to create differentially spliced mRNA molecules. It has now been recognized that the majority of
human genes are alternatively spliced during normal gene expression, although the mechanisms
involved have not been identified. Bennett et al. (U.S. Patent No. 6,210,892) describe antisense
modulation of wild-type cellular mRNA processing using antisense oligonucleotide analogs that
do not induce RNAse ated cleavage of the target RNA. This finds utility in being able to
generate alternatively spliced mRNAs that lack specific exons (e.g., as described by (Sazani, Kole,
et a1. 2007) for the generation of soluble TNF superfamily receptors that lack exons encoding
membrane spanning s.
In cases where a normally functional n is prematurely terminated because of
ons therein, a means for ing some functional protein production through antisense
technology has been shown to be possible through ention during the splicing ses, and
that if exons associated with disease—causing mutations can be specifically deleted from some
genes, a shortened protein product can sometimes be produced that has similar biological
ties of the native protein or has sufficient ical activity to ameliorate the disease caused
by mutations associated with the exon (see e. g., Sierakowska, e et al. 1996; Wilton, Lloyd
et al. 1999; van Deutekom, Bremmer-Bout et al. 2001; Lu, Mann et al. 2003; Aartsma-Rus, Janson
et al. 2004). Kole et al. (U.S. Patent Nos. 5,627,274; 5,916,808; 5,976,879; and 5,665,593)
disclose s of combating aberrant splicing using modified antisense oligonucleotide analogs
that do not promote decay of the targeted NA. Bennett et al. (U.S. Patent No. 6,210,892)
describe antisense modulation of wild-type cellular mRNA sing also using antisense
oligonucleotide analogs that do not induce RNAse H—mediated cleavage of the target RNA.
The process of targeted exon skipping is likely to be particularly useful in long genes
where there are many exons and introns, where there is redundancy in the genetic constitution of
the exons or where a protein is able to function without one or more particular exons. Efforts to
redirect gene processing for the treatment of c diseases associated with truncations caused by
mutations in various genes have focused on the use of antisense oligonucleotides that either: (1)
fully or partially overlap with the elements involved in the splicing process; or (2) bind to the pre-
mRNA at a position sufficiently close to the element to disrupt the binding and function of the
splicing factors that would normally mediate a particular splicing reaction which occurs at that
element.
AVN-013BPC
Duchenne muscular dystrophy (DMD) is caused by a defect in the expression of the
protein dystrophin. The gene encoding the protein contains 79 exons spread out over more than 2
million nucleotides of DNA. Any exonic mutation that changes the reading frame of the exon, or
introduces a stop codon, or is characterized by removal of an entire out of frame exon or exons, or
duplications of one or more exons, has the ial to t production of functional dystrophin,
ing in DMD.
A less severe form of muscular dystrophy, Becker muscular dystrophy (BMD) has been
found to arise where a mutation, typically a deletion of one or more exons, results in a correct
reading frame along the entire phin transcript, such that translation of mRNA into protein is
not prematurely terminated. If the joining of the am and downstream exons in the
processing of a mutated dystrophin pre—mRNA ins the correct reading frame of the gene, the
result is an mRNA coding for a protein with a short internal deletion that retains some activity,
resulting in a Becker phenotype.
For many years it has been known that ons of an exon or exons which do not alter the
reading frame of a dystrophin protein would give rise to a BMD phenotype, whereas an exon
deletion that causes a shift will give rise to DMD (Monaco, Bertelson et al. 1988). In
general, dystrophin mutations including point mutations and exon deletions that change the
reading frame and thus interrupt proper protein translation result in DMD. It should also be noted
that some BMD and DMD patients have exon deletions covering multiple exons.
Modulation of mutant dystrophin pre—mRNA splicing with antisense oligoribonucleotides
has been reported both in vitro and in vivo (see e.g., Matsuo, Masumura et al. 1991; Takeshima,
Nishio et al. 1995; Pramono, Takeshima et al. 1996; Dunckley, Eperon et al. 1997; Dunckley,
Manoharan et al. 1998; Errington, Mann et al. 2003).
The first example of specific and reproducible exon skipping in the mdx mouse model was
reported by Wilton et al. (Wilton, Lloyd et al. 1999). By ing an antisense molecule to the
donor splice site, consistent and efficient exon 23 skipping was induced in the dystrophin mRNA
within 6 hours of ent of the cultured cells. Wilton et al. also describe targeting the acceptor
region of the mouse dystrophin pre—mRNA with longer antisense ucleotides. While the first
antisense oligonucleotide directed at the intron 23 donor splice site induced consistent exon
skipping in primary cultured myoblasts, this compound was found to be much less ent in
immortalized cell cultures expressing higher levels of dystrophin. However, with refined targeting
and antisense oligonucleotide design, the efficiency of specific exon removal was increased by
almost an order of ude (Mann, an et al. 2002).
Recent studies have begun to address the challenge of achieving sustained dystrophin
expression accompanied by minimal adverse effects in tissues affected by the e of
dystrophin. Intramuscular injection of an antisense ucleotide targeted to exon 51 (PR0051)
AVN-013BPC
into the tibialis anterior muscle in four patients with DMD resulted in specific skipping of exon 51
without any clinically apparent adverse effects (Mann, Honeyman et al. 2002; van Deutekom,
Janson et al. 2007). Studies looking at systemic delivery of an antisense phosphorodiamidate
morpholino oligomer conjugated to a enetrating peptide (PPMO) targeted to exon 23 in mdx
mice produced high and sustained dystrophin protein production in skeletal and cardiac muscles
without detectable toxicity (Jearawiriyapaisarn, Moulton et al. 2008; Wu, Moulton et al. 2008;
Yin, n et al. 2008).
Recent clinical trials testing the safety and efficacy of splice switching oligonucleotides
(SSOs) for the treatment of DMD are based on SSO technology to induce alternative ng of
NAs by steric blockade of the spliceosome (Cirak er al., 2011; Goemans et al., 2011;
Kinali er al., 2009; van Deutekom er al., 2007).
Despite these successes, there remains a need for improved antisense oligomers targeted to
multiple dystrophin exons and ed muscle delivery compositions and methods for DMD
therapeutic applications.
Y OF THE INVENTION
In one aspect, the invention provide an antisense oligomer of 20—50 nucleotides in length
capable of g a selected target to induce exon skipping in the human dystrophin gene,
wherein the antisense oligomer comprises a sequence of bases that specifically hybridizes to an
exon 44 target region selected from the group ting of H44A(—07+15), H44A(—08+15),
H44A(—06+15), H44A(—08+17), H44A(—07+17), and H44A(—06+17), n the bases of the
oligomer are linked to morpholino ring structures, and wherein the lino ring ures are
joined by phosphorous-containing intersubunit linkages joining a morpholino nitrogen of one ring
structure to a 5’ exocyclic carbon of an adjacent ring structure. In one embodiment, the antisense
oligomer ses a sequence of bases selected from the group consisting of SEQ ID NOs: 1 and
4-8. In another embodiment, the antisense oligomer is about 20 to 30 nucleotides in length or
about 22 to 28 nucleotides in length. In yet another embodiment, the sequence consists of a
sequence ed from SEQ ID NOs: 1 and 4—8. In one embodiment, the invention provides an
antisense oligomer that does not activate RNase H.
In another aspect, the invention provide an antisense oligomer that is ally linked to
one or more moieties or conjugates that enhance the activity, cellular bution, or cellular
uptake of the nse oligomer, such as, for example a polyethylene glycol molecule. In other
embodiments, the antisense oligomer is conjugated to an arginine—rich peptide such as a sequence
selected from SEQ ID NOS: 24—39.
AVN-013BPC
In yet another aspect, the invention provides an antisense oligomer of 20-50 nucleotides in
length capable of binding a selected target to induce exon skipping in the human dystrophin gene,
wherein the antisense oligomer ses a sequence of bases that specifically izes to an
exon 44 target region selected from the group ting of H44A(-O7+15), H44A(-08+15),
H44A(-06+15), H44A(-08+17), H44A(-07+17), and H44A(-06+l7), wherein the bases of the
oligomer are linked to lino ring structures, and wherein the morpholino ring structures are
joined by substantially uncharged phosphorus—containing intersubunit linkages joining a
morpholino nitrogen of one ring structure to a 5’ exocyclic carbon of an adjacent ring structure. In
one embodiment, 5%-35% of the linkages of the antisense oligomer are positively charged. In
another embodiment, the intersubunit linkages of the antisense er are uncharged and
interspersed with linkages that are positively charged at physiological pH, wherein the total
number of positively charged linkages is n 2 and no more than half of the total number of
linkages. In some embodiments, the antisense oligomer comprises morpholino ring structures and
phosphorodiamidate intersubunit es. In some embodiments, the antisense oligomer is
modified with a t cationic group.
In another aspect, the invention provides an antisense oligomer selected from the group
consisting of:
AVN-013BPC
E}reak A Il3reak B Brelak C
l i
l/NMeZ
A :/NMe2 A
o A I/NMe2
Yo\/\ /\/o0 $0“ O‘P\ 0”P\ o”P\
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MezN KEOj/C KEOj/A KEOD/G
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A T ’0 A ,,o ,,o
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AFLO
MezN/ \O MezN
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AVN-013BPC
Ofi/O\/\O/\/o\/\OH
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Brag-k B Brea'k c
H44A(—7+17); and
AVN-013BPC
Break A Il3reak B Brelak C
E NMe E NMe2 : /NMe2
:/ 2 A /
OYOV\O/\/O\/\OH I“:
O:P\O O”P\O O’ \O
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T N N N
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KEOJ/C KEOJ/G Kon/0 Kon/A
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A|,,o FLO [LO A Lo
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‘5” K63” Yrs Yr
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M92N O MezN/ \O MezN/ O
°j’ K103”
N N N
éreak A Break B Brea'k
H44A(—6+17),
AVN-013BPC
wherein
NH2 0 0
leaf“! c= fl: e=<Nfr T=m5u= r 3:“
N ‘,(J N o N N‘ NH2 N o
/ l / l
, , ,and ,and
n A = the stereochemistry of the phosphorous center is not defined.
In some embodiments, uracil can be substituted for thymine in the above structures.
In one aspect, the antisense compound is composed of phosphorus-containing intersubunit
linkages joining a morpholino nitrogen of one subunit to a 5' exocyclic carbon of an adjacent
subunit, wherein the linkages are phosphorodiamidate linkages, in accordance with the structure:
EP—X
ill—till
where Y1=O, Z=O, Pj is a purine or pyrimidine airing moiety effective to bind, by
base-specific hydrogen bonding, to a base in a polynucleotide, and X is alkyl, alkoxy, thioalkoxy,
or alkyl amino e.g., wherein X=NR2, where each R is independently hydrogen or methyl. The
above intersubunit linkages, which are uncharged, may be interspersed with linkages that are
positively charged at physiological pH, where the total number of positively charged linkages is
between 1 and up to all of the total number of ubunit es.
In another exemplary embodiment, the nd is sed of intersubunit linkage and
terminal modifications as described in US Application No: 13/ 1 18,298, which is incorporated
herein in its entirety.
According to one aspect, the ion provides antisense molecules capable of binding to
a ed target in human dystrophin pre—mRNA to induce exon skipping. In another aspect, the
invention es two or more antisense oligonucleotides which are used together to induce
single or multiple exon skipping. For example, exon skipping of a single or multiple exons can be
achieved by linking er two or more antisense oligonucleotide molecules.
In another aspect, the invention relates to an isolated antisense oligonucleotide of 20 to 50
nucleotides in length, including at least 10, 12, 15, 17, 20 or more consecutive nucleotides
complementary to an exon 44 target region of the dystrophin gene designated as an annealing site
AVN-013BPC
selected from the group consisting of: H44A(—07+17), H44A(-07+20), H44A(-07+22), H44A(-
8+15), H44A(-7+15), H44A(-6+15), 8+17), H44A(—6+17), H44A(+77+101),
H44A(+64+91), H44A(+62+89), H44A(+62+85), H44A(—13+14), H44A(—14+15), wherein the
antisense oligonucleotide specifically hybridizes to the annealing site inducing exon 44 skipping.
In one embodiment, the antisense oligonucleotide is 25 to 28 nucleotides in length.
In another aspect, the invention relates to an isolated antisense oligonucleotide of 20 to 50
tides in length, including at least 10, 12, 15, 17, 20 or more nucleotides of a nucleotide
sequence selected from the group consisting of: SEQ ID NOS: 1-12, 46 and 47, wherein the
oligonucleotide specifically hybridizes to an exon 44 target region of the Dystrophin gene and
induces exon 44 skipping. In one embodiment, thymine bases in SEQ ID NOs: 1—12, 46, and 47
are optionally uracil.
ary antisense sequences targeted to exon 44 include those identified below.
07+17): 5'-CAGATCTGTCAAATCGCCTGCAGG-3' (SEQ ID NO: 1)
H44A(-07+20): 5'-CAACAGATCTGTCAAATCGCCTGCAGG-3' (SEQ ID NO: 2)
H44A(—07+22): 5'—CTCAACAGATCTGTCAAATCGCCTGCAGG—3' (SEQ ID NO: 3)
H44A(-8+15): 5'—GATCTGTCAAATCGCCTGCAGGT-3' (SEQ ID NO: 4)
H44A(-7+15): 5'-GATCTGTCAAATCGCCTGCAGG-3' (SEQ ID NO: 5)
H44A(-6+15): 5'-GATCTGTCAAATCGCCTGCAG—3' (SEQ ID NO: 6)
H44A(—8+17): 5'—CAGATCTGTCAAATCGCCTGCAGGT-3' (SEQ ID NO: 7)
H44A(-6+17): 5'-CAGATCTGTCAAATCGCCTGCAG-3' (SEQ ID NO: 8)
H44A(+77+101): 5'-GTGTCTTTCTGAGAAACTGTTCAGC-3' (SEQ ID NO: 9)
H44A(+64+91): 5'-GAGAAACTGTTCAGCTTCTGTTAGCCAC—3' (SEQ ID NO: 10)
H44A(+62+89): 5'—GAAACTGTTCAGCTTCTGTTAGCCACTG-3' (SEQ ID NO: 11)
H44A(+62+85): 5'-CTGTTCAGCTTCTGTTAGCCACTG-3' (SEQ ID NO: 12)
H44A(-13+14): 5'-ATCTGTCAAATCGCCTGCAGGTAAAAG—3' (SEQ ID NO: 46)
H44A(—14+15): CTGTCAAATCGCCTGCAGGTAAAAGC-3' (SEQ ID NO: 47)
In one embodiment, the nse oligomer specifically izes to annealing site
H44A(-07+17), such as SEQ ID NO:1. In another embodiment, the antisense er
specifically hybridizes to annealing site H44A(-07+20), such as SEQ ID NO: 2. In another
embodiment, the nse oligomer specifically hybridizes to annealing site H44A(-07+22), such
as SEQ ID NO: 3. In another embodiment, the antisense er specifically izes to
annealing site H44A(-8+15), such as SEQ ID NO: 4. In another embodiment, the antisense
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oligomer specifically hybridizes to annealing site H44A(-7+15), such as SEQ ID NO: 5. In another
embodiment, the antisense oligomer specifically hybridizes to annealing site H44A(-6+15), such
as SEQ ID NO: 6. In another embodiment, the nse oligomer specifically hybridizes to
ing site 8+17), such as SEQ ID NO: 7. In another embodiment, the antisense
oligomer specifically hybridizes to annealing site H44A(-6+17), such as SEQ ID NO: 8. In another
embodiment, the antisense oligomer specifically hybridizes to annealing site H44A(+77+101),
such as SEQ ID NO: 9. In another embodiment, the nse oligomer specifically hybridizes to
annealing site H44A(+64+91), such as SEQ ID NO: 10. In another embodiment, the antisense
oligomer specifically hybridizes to annealing site H44A(+62+89), such as SEQ ID NO: 11. In
another embodiment, the antisense oligomer specifically hybridizes to annealing site
62+85), such as SEQ ID NO: 12. In another embodiment, the antisense oligomer
specifically izes to annealing site H44A(-13+14), such as SEQ ID NO: 46. In another
embodiment, the antisense oligomer specifically hybridizes to annealing site H44A(—14+15), such
as SEQ ID NO: 47.
In some embodiments, the antisense ucleotides of the invention n one or more
modifications to minimize or prevent cleavage by RNase H. In some embodiments, the antisense
oligonucleotides of the invention do not activate RNase H. In some embodiments, the antisense
oligonucleotides comprise a non—natural backbone. In some embodiments, the sugar moieties of
the oligonucleotide backbone are replaced with tural moieties, such as morpholinos. In
some embodiments, the antisense oligonucleotides have the inter—nucleotide linkages of the
oligonucleotide backbone replaced with non—natural inter—nucleotide linkages, such as modified
phosphates. Exemplary modified phosphates include methyl phosphonates, methyl
phosphorothioates, phosphoromorpholidates, hropiperazidates, and phosphoroamidates. In
some embodiments, the antisense oligonucleotide is a 2’—O—methyl—oligoribonucleotide or a
peptide nucleic acid.
In some embodiments, the nse ucleotides contain base modifications or
substitutions. For example, certain nucleo—bases may be selected to increase the binding affinity of
the antisense oligonucleotides described herein. These include 5-substituted pyrimidines, 6-
azapyrimidines and N-2, N-6 and 0—6 substituted s, including Z-aminopropyladenine, 5-
propynyluracil, 5-propynylcytosine and 2, inopurine. 5-methylcytosine substitutions have
been shown to increase nucleic acid duplex stability by 20C, and may be incorporated into
the antisense oligonucleotides bed herein. In one embodiment, at least one pyrimidine base
of the ucleotide comprises a 5-substituted pyrimidine base, wherein the pyrimidine base is
selected from the group ting of cytosine, thymine and uracil. In one embodiment, the 5-
tuted pyrimidine base is 5—methylcytosine. In another embodiment, at least one purine base
3BPC
of the oligonucleotide comprises an N-2, N—6 substituted purine base. In one embodiment, the N-
2, N-6 tuted purine base is 2, 6-diaminopurine.
In one embodiment, the antisense oligonucleotide includes one or more 5—methylcytosine
substitutions alone or in combination with another modification, such as 2'—O-methoxyethyl sugar
modifications. In yet another embodiment, the antisense oligonucleotide includes one or more 2,
6-diaminopurine substitutions alone or in combination with another cation.
In some embodiments, the antisense oligonucleotide is chemically linked to one or more
moieties, such as a polyethylene glycol moiety, or ates, such as a arginine-rich cell
penetrating peptide (e.g., SEQ ID NOs: 24—39), that enhance the activity, cellular distribution, or
cellular uptake of the nse oligonucleotide. In one exemplary embodiment, the arginine—rich
polypeptide is covalently coupled at its N—terminal or C—terminal residue to the 3' or 5’ end of the
antisense compound. Also in an exemplary embodiment, the antisense compound is composed of
morpholino subunits and phosphorus-containing intersubunit linkages joining a morpholino
en of one t to a 5' exocyclic carbon of an adjacent subunit.
In another aspect, the invention provides sion vectors that incorporate the antisense
oligonucleotides described above, e.g., the antisense ucleotides of SEQ ID NOs: 1-12, 46
and 47. In some ments, the expression vector is a modified retrovirus or non—retroviral
vector, such as a adeno—associated viral vector.
In another aspect, the invention provides pharmaceutical compositions that include the
antisense oligonucleotides described above, and a saline solution that es a phosphate buffer.
In another aspect, the invention provides antisense molecules ed and or adapted to
aid in the lactic or therapeutic treatment of a genetic disorder comprising at least an
antisense molecule in a form suitable for delivery to a patient.
In another aspect, the invention provides a method for treating a patient ing from a
genetic disease wherein there is a mutation in a gene encoding a particular protein and the affect of
the mutation can be abrogated by exon skipping, comprising the steps of: (a) selecting an antisense
molecule in accordance with the methods described herein; and (b) administering the molecule to a
patient in need of such treatment. The invention also ses the use of purified and isolated
antisense oligonucleotides of the invention, for the manufacture of a medicament for treatment of a
genetic disease.
In another aspect, the invention es a method of treating a condition characterized by
Duchenne muscular dystrophy, which includes administering to a patient an effective amount of an
riately designed antisense ucleotide of the invention, relevant to the particular genetic
lesion in that patient. Further, the invention provides a method for prophylactically treating a
patient to prevent or minimize Duchenne muscular dystrophy, by administering to the patient an
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ive amount of an antisense oligonucleotide or a pharmaceutical composition comprising one
or more of these biological molecules.
In another aspect, the invention also es kits for ng a genetic disease, which kits
comprise at least an antisense oligonucleotide of the present invention, ed in a suitable
container and instructions for its use.
These and other s and features will be more fully understood when the following
ed description of the invention is read in conjunction with the figures.
BRIEF DESCRIPTION OF THE FIGURES
shows an exemplary morpholino er structure with a phosphorodiamidate
linkage.
shows a conjugate of an arginine—rich peptide and an antisense oligomer, in
accordance with an embodiment of the invention.
shows a ate as in , wherein the backbone linkages contain one or
more positively charged groups.
FIGS. 1D-G show the repeating subunit segment of exemplary morpholino
oligonucleotides, designated D through G.
FIGS. 2A-2B depicts a reaction scheme for the preparation of a linker for solid phase
synthesis and a solid support for oligomer synthesis.
FIGS. 3 and 4 depict graphs corresponding to experiments showing relative activities of
exemplary antisense oligomers for inducing exon 44 skipping in cultured human rhabdomyosarcoma
cells. RNA isolated from rhabdomyosarcoma cells treated with the indicated oligomers was
subjected to exon cific nested RT-PCR amplification, followed by gel electrophoresis and
band intensity quantification. Data are plotted as % exon skipping as assessed by PCR, i.e., the band
intensity of the exon—skipped product relative to the full—length PCR product. In Figure 3, H44A(-
06+14), H44A(-06+20) and H44A(-09+17) (SEQ ID NOS: 13, 19 and 20, respectively) are published
ers. In Figure 4, H44A(+85+104), H44A(+59+85) and H44A(+65+90) (SEQ ID NOs: 14, 21
and 23, respectively) are published oligomers.
FIGS. 5 and 6 depict graphs corresponding to experiments showing relative activities of
exemplary antisense oligomers for inducing exon 44 skipping in primary human myoblasts.
DETAILED DESCRIPTION OF THE INVENTION
Embodiments of the t ion relate generally to ed antisense compounds,
and s of use f, which are specifically designed to induce exon skipping in the human
dystrophin gene. Dystrophin plays a Vital role in muscle function, and various muscle-related
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diseases are characterized by mutated forms of this gene. Hence, in certain embodiments, the
improved antisense nds described herein induce exon skipping in mutated forms of the
human dystrophin gene, such as the mutated phin genes found in Duchenne muscular
dystrophy (DMD) and Becker muscular dystrophy (BMD).
Due to aberrant mRNA splicing events caused by mutations, these mutated human
dystrophin genes either express defective phin protein or express no measurable dystrophin
at all, a condition that leads to various forms of ar dystrophy. To remedy this ion, the
antisense compounds of the present invention hybridize to selected regions of a pre-processed
RNA of a mutated human dystrophin gene, induce exon skipping and differential splicing in that
otherwise ntly spliced dystrophin mRNA, and thereby allow muscle cells to produce an
mRNA transcript that encodes a functional dystrophin protein. In certain embodiments, the
resulting phin protein is not arily the "wild-type" form of dystrophin, but is rather a
truncated, yet functional or semi-functional, form of dystrophin.
By increasing the levels of functional dystrophin n in muscle cells, these and related
ments may be useful in the prophylaxis and treatment of muscular dystrophy, especially
those forms of muscular phy, such as DMD and BMD, that are characterized by the
expression of defective dystrophin proteins due to aberrant mRNA splicing. The specific
oligomers described herein further provide ed, dystrophin-exon-specific targeting over
other oligomers in use, and thereby offer icant and practical advantages over alternate
methods of treating relevant forms of muscular dystrophy.
In one aspect, the invention provide an nse oligomer of 20-50 nucleotides in length
capable of binding a selected target to induce exon skipping in the human dystrophin gene,
wherein the antisense oligomer comprises a sequence of bases that specifically hybridizes to an
exon 44 target region selected from the group consisting of H44A(—07+15), H44A(—08+15),
06+15), H44A(—08+17), H44A(—07+17), and 06+17), wherein the bases of the
oligomer are linked to lino ring structures, and wherein the morpholino ring structures are
joined by phosphorous-containing intersubunit linkages joining a morpholino nitrogen of one ring
structure to a 5’ exocyclic carbon of an adjacent ring structure. In one embodiment, the antisense
oligomer comprises a sequence of bases selected from the group ting of SEQ ID NOS: 1 and
4-8. In another embodiment, the antisense oligomer is about 20 to 30 nucleotides in length or
about 22 to 28 nucleotides in length. In yet another embodiment, the sequence consists of a
sequence selected from SEQ ID NOs: 1 and 4—8. In one embodiment, the invention provides an
antisense oligomer that does not activate RNase H.
In another aspect, the invention provide an antisense oligomer that is chemically linked to
one or more moieties or conjugates that enhance the activity, cellular bution, or cellular
uptake of the antisense oligomer, such as, for example a polyethylene glycol molecule. In other
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embodiments, the antisense oligomer is conjugated to an arginine-rich peptide such as a sequence
selected from SEQ ID NOS: 24-39.
In yet another , the invention provides an antisense oligomer of 20—50 tides in
length capable of binding a selected target to induce exon skipping in the human dystrophin gene,
wherein the antisense oligomer comprises a sequence of bases that specifically hybridizes to an
exon 44 target region selected from the group consisting of H44A(—07+15), H44A(-08+15),
H44A(—06+15), H44A(—08+17), H44A(—07+17), and H44A(—06+17), wherein the bases of the
oligomer are linked to morpholino ring structures, and wherein the morpholino ring structures are
joined by substantially uncharged phosphorus—containing ubunit linkages joining a
morpholino nitrogen of one ring structure to a 5’ exocyclic carbon of an adjacent ring structure. In
one embodiment, 5%—35% of the linkages of the antisense oligomer are positively charged. In
another embodiment, the intersubunit linkages of the antisense oligomer are uncharged and
interspersed with linkages that are positively charged at physiological pH, n the total
number of vely charged es is between 2 and no more than half of the total number of
linkages. In some embodiments, the antisense oligomer ses morpholino ring ures and
phosphorodiamidate ubunit linkages. In some ments, the antisense oligomer is
modified with a pendant cationic group.
In another aspect, the invention provides the antisense oligomers described in Examples 2-
7. In some embodiments, uracil can be tuted for thymine in the antisense ers
described in Examples 2-7.
In one aspect, the invention relates to isolated antisense oligonucleotides of 20 to 50
nucleotides in length, including at least 10, 12, 15, 17, 20 or more, nucleotides complementary to
an exon 44 target region of the dystrophin gene designated as an annealing site selected from the
group consisting of: H44A(-07+17), H44A(—07+20), H44A(—07+22), H44A(—8+15), H44A(—7+15),
H44A(-6+15), H44A(—8+17), H44A(—6+17), 77+101), H44A(+64+91), H44A(+62+89),
H44A(+62+85), H44A(-13+14), H44A(-14+15). Antisense oligonucleotides specifically ize
to the annealing site, inducing exon 44 skipping.
Other antisense oligonucleotides of the invention are 20 to 50 nucleotides in length and
include at least 10, 12, 15, 17, 20, 22, 25 or more tides of SEQ ID NOs: 1-12, 46, and 47. In
some embodiments, thymine bases in SEQ ID NOs: 1—12, 46, and 47 are optionally uracil.
Exemplary antisense oligomers of the invention are set forth below:
H44A(-07+17): 5'-CAGATCTGTCAAATCGCCTGCAGG-3' (SEQ ID NO: 1)
H44A(-07+20): 5'-CAACAGATCTGTCAAATCGCCTGCAGG—3' (SEQ ID NO: 2)
H44A(—07+22): AACAGATCTGTCAAATCGCCTGCAGG-3' (SEQ ID NO: 3)
H44A(-8+15): 5'-GATCTGTCAAATCGCCTGCAGGT-3' (SEQ ID NO: 4)
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H44A(-7+15): 5'-GATCTGTCAAATCGCCTGCAGG-3' (SEQ ID NO: 5)
H44A(-6+15): 5'-GATCTGTCAAATCGCCTGCAG—3' (SEQ ID NO: 6)
H44A(—8+17): 5'—CAGATCTGTCAAATCGCCTGCAGGT—3' (SEQ ID NO: 7)
H44A(-6+17): 5'-CAGATCTGTCAAATCGCCTGCAG-3' (SEQ ID NO: 8)
H44A(+77+101): 5'-GTGTCTTTCTGAGAAACTGTTCAGC-3' (SEQ ID NO: 9)
H44A(+64+91): 5'-GAGAAACTGTTCAGCTTCTGTTAGCCAC—3' (SEQ ID NO: 10)
H44A(+62+89): 5'—GAAACTGTTCAGCTTCTGTTAGCCACTG-3' (SEQ ID NO: 11)
H44A(+62+85): 5'-CTGTTCAGCTTCTGTTAGCCACTG-3' (SEQ ID NO: 12)
H44A(-13+14): 5'-ATCTGTCAAATCGCCTGCAGGTAAAAG-3' (SEQ ID NO: 46)
H44A(-14+15): 5'-GATCTGTCAAATCGCCTGCAGGTAAAAGC—3' (SEQ ID NO: 47)
In one embodiment, the antisense oligomer specifically hybridizes to annealing site
07+17), such as SEQ ID NO:1. In another embodiment, the nse oligomer
specifically hybridizes to ing site H44A(—07+20), such as SEQ ID NO: 2. In r
embodiment, the antisense oligomer specifically hybridizes to annealing site H44A(-07+22), such
as SEQ ID NO: 3. In another ment, the antisense oligomer specifically hybridizes to
ing site H44A(—8+15), such as SEQ ID NO: 4. In another embodiment, the antisense
er specifically hybridizes to annealing site H44A(—7+15), such as SEQ ID NO: 5. In another
embodiment, the antisense oligomer specifically hybridizes to annealing site H44A(-6+15), such
as SEQ ID NO: 6. In another embodiment, the antisense oligomer specifically hybridizes to
annealing site H44A(—8+17), such as SEQ ID NO: 7. In another ment, the antisense
oligomer specifically hybridizes to annealing site H44A(-6+l7), such as SEQ ID NO: 8. In
r embodiment, the antisense oligomer specifically hybridizes to annealing site
77+101), such as SEQ ID NO: 9. In another embodiment, the antisense oligomer
specifically hybridizes to annealing site H44A(+64-l-91), such as SEQ ID NO: 10. In another
embodiment, the antisense oligomer specifically hybridizes to annealing site H44A(+62+89), such
as SEQ ID NO: 11. In another embodiment, the antisense oligomer specifically hybridizes to
annealing site H44A(+62+85), such as SEQ ID NO: 12. In another embodiment, the antisense
oligomer specifically hybridizes to annealing site H44A(-13+l4), such as SEQ ID NO: 46. In
another embodiment, the antisense oligomer specifically hybridizes to annealing site H44A(-
14+15), such as SEQ ID NO: 47.
Unless defined otherwise, all technical and scientific terms used herein have the same
meaning as commonly understood by those of ordinary skill in the art to which the ion
belongs. Although any methods and materials similar or equivalent to those described herein can
be used in the ce or g of the present invention, preferred methods and materials are
described. For the purposes of the t invention, the ing terms are defined below.
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1. Definitions
By "about" is meant a quantity, level, value, number, frequency, percentage, dimension,
size, amount, weight or length that varies by as much as 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2 or
1% to a reference quantity, level, value, number, frequency, percentage, dimension, size, amount,
weight or length.
The terms "complementary" and "complementarity" refer to polynucleotides (i.e., a
sequence of nucleotides) d by base—pairing rules. For example, the sequence "T-G-A (5’-
3’)," is complementary to the sequence "T—C—A )." Complementarity may be "partial," in
which only some of the nucleic acids‘ bases are matched according to base g rules. Or, there
may be "complete" or "total" complementarity between the nucleic acids. The degree of
complementarity between nucleic acid strands has significant effects on the efficiency and th
of hybridization between nucleic acid strands. While perfect complementarity is often d,
some embodiments can include one or more but preferably 6, 5, 4, 3, 2, or 1 mismatches with
respect to the target RNA. ions at any location within the oligomer are included. In certain
embodiments, variations in sequence near the termini of an oligomer are generally able to
variations in the interior, and if t are typically within about 6, 5, 4, 3, 2, or 1 nucleotides of
the 5' and/or 3' terminus.
The terms “cell penetrating peptide” and “CPP” are used interchangeably and refer to
cationic cell penetrating peptides, also called transport peptides, carrier peptides, or peptide
transduction domains. The peptides, as shown herein, 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. A red CPP
embodiment is an arginine-rich peptide as described r below.
The terms “antisense oligomer” and “antisense compound” and ense
oligonucleotide” are used interchangeably and refer to a ce of cyclic subunits, each bearing
a base-pairing moiety, linked by intersubunit linkages that allow the airing 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 e sugar or, in a preferred embodiment, a morpholino group (see
ption 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.
Such an antisense oligomer can be designed to block or inhibit translation of mRNA or to
inhibit natural pre—mRNA splice processing, and may be said to be ted to” or ted
against” a target sequence with which it hybridizes. The target sequence is typically a region
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including an AUG start codon of an mRNA, a Translation Suppressing Oligomer, or splice site of
a pre-processed mRNA, a Splice Suppressing Oligomer (SSO). The target sequence for a splice
site may include an mRNA sequence having its 5' end 1 to about 25 base pairs downstream of a
normal splice acceptor junction in a preprocessed mRNA. A preferred target sequence is any
region of a preprocessed mRNA that includes a splice site or is contained entirely within an exon
coding ce or spans a splice acceptor or donor site. An er is more generally said to be
“targeted against” a biologically nt target, such as a protein, virus, or bacteria, when it is
targeted against the nucleic acid of the target in the manner described above.
The terms “morpholino oligomer” or “PMO” (phosphoramidate- or phosphorodiamidate
morpholino oligomer) refer to an oligonucleotide analog 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 ged or cationic, joining the morpholino
nitrogen of one t to a 5’ exocyclic carbon of an nt 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 Figure 1A, which shows a
preferred phosphorodiamidate linkage type. The term "morpholino ring structure" may be used
inetrchangably with the term "morpholino subunit." Variations can be made to this linkage as long
as they do not ere with binding or activity. For example, the oxygen attached to phosphorus
may be substituted with sulfur (thiophosphorodiamidate). The 5’ oxygen may be tuted 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 ic linkages below. The purine or dine base pairing moiety is typically
adenine, ne, guanine, uracil, thymine or inosine. The synthesis, structures, and binding
characteristics of morpholino oligomers are detailed in US. Patent Nos. 5,698,685, 5,217,866,
5,142,047, 5,034,506, 5,166,315, 5,521,063, 5,506,337, 8,076,476, 8,299,206 and 7,943,762
(cationic linkages), all of which are incorporated herein by reference. Modified intersubunit linkages
and terminal groups are detailed in PCT ation US2011/038459 and publication
WO/201 1/150408 which are incorporated herein by reference in their entirety.
An “amino acid subunit” or “amino acid residue” can refer to an (x-amino acid e
(-CO-CHR-NH-) or a [5- or other amino acid residue CO—(CH2)nCHR-NH-), where R is a side
chain (which may include hydrogen) and n is 1 t0 6, preferably 1 to 4.
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 t in ns
found in nature, examples include beta—alanine (B—Ala), 6—aminohexanoic acid (Ahx) and
6—aminopentanoic acid.
The term “naturally occurring nucleic acid” refers to a nucleic acid found in nature.
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Typically, naturally occurring nucleic acids are polymers of nucleotides (each containing a purine or
pyrimidine nucleobase and a e sugar) joined together by phosphodiester linkages. Exemplary
naturally occurring nucleic acid les include RNA and DNA. The term “non—naturally
occurring nucleic acid” refers to a nucleic acid that is not present in nature. For example, non-
naturally occurring c acids can include one or more tural base, sugar, and/or intersubunit
e, e.g., a sugar, base, and/or linkage that has been modified or substituted with respect to that
found in a lly ing nucleic acid molecule. Exemplary modifications are described .
In some ments, non-naturally occurring nucleic acids include more than one type of
modification, e. g. , sugar and base modifications, sugar and linkage modifications, base and linkage
modifications, or base, sugar, and linkage modifications. In a preferred embodiment, the antisense
oligonucleotides of the present invention are non—naturally occurring nucleic acid molecules. For
example, in some embodiments, the nse oligonucleotides contain a tural (e. g. modified
or substituted) base. In some ments, the antisense oligonucleotides contain a non—natural
(e.g., modified or substituted) sugar. In some embodiments, the antisense ucleotides contain a
non-natural (e. g. modified or substituted) intersubunit linkage. In some embodiments, the antisense
oligonucleotides contain more than one type of modification or substutution, e. g. a non-natural base
and/or a non—natural sugar, and/or a non—natural intersubuint linkage. In other embodiments,
antisense oligonucleotides have the chemical composition of a naturally occuring nucleic acid
molecule, 122., the antisense oligonucleotides do not include a modified or substituted base, sugar, or
intersubunit linkage. Regardless of al composition, antisense oligonucleotides of the
invention are synthesized in vitro and do not include antisense compositions of biological origin.
An "exon" refers to a defined section of nucleic acid that encodes for a protein, or a
nucleic acid sequence that is represented in the mature form of an RNA molecule after either
ns of a pre-processed (or precursor) RNA have been removed by splicing. The mature RNA
molecule can be a messenger RNA (mRNA) or a functional form of a non-coding RNA, such as
rRNA or tRNA. The human dystrophin gene has about 79 exons.
An "intron" refers to a c acid region (within a gene) that is not translated into a
protein. An intron is a non—coding section that is transcribed into a precursor mRNA (pre-mRNA),
and subsequently removed by splicing during formation of the mature RNA.
An “effective amount” or “therapeutically effective amount” refers to an amount of
therapeutic compound, such as an nse 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.
For an antisense oligomer, this effect is lly brought about by inhibiting translation or natural
splice-processing of a selected target sequence.
"Exon ng" refers generally to the process by which an entire exon, or a portion
thereof, is removed from a given pre-processed RNA, and is thereby excluded from being present
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in the mature RNA, such as the mature mRNA that is translated into a protein. Hence, the portion
of the protein that is otherwise encoded by the skipped exon is not present in the expressed form of
the protein, typically creating an altered, though still functional, form of the n. In certain
embodiments, the exon being skipped is an aberrant exon from the human dystrophin gene, which
may contain a mutation or other tion in its sequence that otherwise causes aberrant splicing.
In certain embodiments, the exon being d is any one or more of exons 1—79 of the phin
gene, though exon 44 of the human phin gene is preferred.
"Dystrophin" is a rod-shaped cytoplasmic protein, and a vital part of the n complex
that connects the cytoskeleton of a muscle fiber to the surrounding ellular matrix h the
cell membrane. Dystrophin contains multiple functional domains. For instance, dystrophin
contains an actin binding domain at about amino acids 14—240 and a central rod domain at about
amino acids 253-3040. This large central domain is formed by 24 spectrin-like triple-helical
elements of about 109 amino acids, which have homology to alpha—actinin and spectrin. The
repeats are lly upted by four proline—rich non—repeat segments, also referred to as hinge
regions. Repeats 15 and 16 are separated by an 18 amino acid stretch that appears to provide a
major site for proteolytic cleavage of dystrophin. The sequence identity between most repeats
ranges from 10—25%. One repeat contains three alpha—helices: 1, 2 and 3. Alpha—helices 1 and 3
are each formed by 7 helix turns, probably interacting as a coiled-coil through a hobic
interface. Alpha-helix 2 has a more complex structure and is formed by segments of four and three
helix turns, separated by a Glycine or Proline residue. Each repeat is d by two exons,
typically upted by an intron between amino acids 47 and 48 in the first part of alpha-helix 2.
The other intron is found at different positions in the repeat, usually scattered over helix-3.
Dystrophin also contains a cysteine-rich domain at about amino acids 3080-3360), including a
cysteine-rich segment (i.e., 15 Cysteines in 280 amino acids) g homology to the C—terminal
domain of the slime mold (Dictyostelium discoideum) alpha-actinin. The carboxy-terminal
domain is at about amino acids 3361-3685.
The amino-terminus of dystrophin binds to F—actin and the carboxy—terminus binds to the
dystrophin—associated protein complex (DAPC) at the sarcolemma. The DAPC includes the
dystroglycans, sarcoglycans, integrins and caveolin, and ons in any of these components
cause autosomally inherited muscular dystrophies. The DAPC is destabilized when dystrophin is
absent, which results in diminished levels of the member proteins, and in turn leads to progressive
fibre damage and ne leakage. In various forms of muscular dystrophy, such as Duchenne's
muscular dystrophy (DMD) and Becker's muscular dystrophy (BMD), muscle cells produce an
altered and functionally defective form of dystrophin, or no phin at all, mainly due to
mutations in the gene sequence that lead to incorrect splicing. The predominant expression of the
defective dystrophin protein, or the complete lack of dystrophin or a dystrophin-like protein, leads
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to rapid progression of muscle degeneration, as noted above. In this regard, a "defective"
dystrophin protein may be characterized by the forms of dystrophin that are produced in n
subjects with DMD or BMD, as known in the art, or by the absence of detectable dystrophin.
As used herein, the terms "function" and "functional" and the like refer to a biological,
enzymatic, or therapeutic function.
A "functional" dystrophin protein refers generally to a dystrophin protein having sufficient
ical activity to reduce the progressive degradation of muscle tissue that is otherwise
characteristic of muscular dystrophy, lly as compared to the altered or "defective" form of
dystrophin protein that is present in certain ts with DMD or BMD. In certain embodiments,
a functional dystrophin protein may have about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%,
90%, or 100% (including all integers in between) of the in vitro or in vivo biological activity of
wild-type dystrophin, as measured according to routine techniques in the art. As one example,
phin-related activity in muscle cultures in vitro can be measured according to myotube size,
ril organization (or disorganization), contractile activity, and spontaneous clustering of
acetylcholine receptors (see, e. g., Brown et al., Journal of Cell Science. 112:209-216, 1999).
Animal models are also le resources for studying the pathogenesis of disease, and provide a
means to test dystrophin—related activity. Two of the most widely used animal models for DMD
research are the mdx mouse and the golden retriever muscular phy (GRMD) dog, both of
which are dystrophin negative (see, e.g., Collins & Morgan, Int J Exp Pathol 84: 2, 2003).
These and other animal models can be used to measure the onal activity of various
dystrophin proteins. Included are truncated forms of dystrophin, such as those forms that are
ed by certain of the exon-skipping antisense compounds of the present invention.
By ted" is meant material that is ntially or essentially free from components
that normally accompany it in its native state. For example, an "isolated polynucleotide," as used
herein, may refer to a polynucleotide that has been purified or removed from the sequences that
flank it in a naturally-occurring state, e.g., a DNA fragment that has been removed from the
ces that are normally adjacent to the fragment.
As used herein, “sufficient length” refers to an antisense oligonucleotide that is
complementary to at least 8, more typically 8—30, contiguous nucleobases in a target dystrophin
pre-mRNA. In some embodiments, an antisense of sufficient length includes at least 8, 9, 10, 11,
12, 13, 14, 15, 17, 20 or more contiguous bases in the target dystrophin pre—mRNA. In
other embodiments an antisense of sufficient length includes at least 16, 17, 18, 19, 20, 21, 22, 23,
24, or 25 contiguous nucleobases in the target dystrophin pre-mRNA. An antisense
oligonucleotide of sufficient length has at least a minimal number of nucleotides to be capable of
specifically hybridizing to exon 44. Preferably an oligonucleotide of sufficient length is from
about 10 to about 50 tides in length, including oligonucleotides of 10, 11, 12, 13, 14, 15, 16,
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17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 and 40 or
more nucleotides. In one ment, an ucleotide of ient length is from 10 to about
nucleotides in length. In another embodiment, an oligonucleotide of sufficient length is from
to about 25 nucleotides in . In yet another embodiment, an oligonucleotide of sufficient
length is from 20 to 30, or 20 to 50, nucleotides in length. In yet another embodiment, an
oligonucleotide of sufficient length is from 22 to 25, 22 to 28, 24 to 28, 24 to 29, 25 to 28, 20 to
, or 25 to 30 nucleotides in length.
By "enhance" or cing," or "increase" or "increasing," or "stimulate" or
"stimulating," refers generally to the ability of one or antisense compounds or compositions to
produce or cause a greater physiological response (i.e., downstream effects) in a cell or a subject,
as compared to the response caused by either no antisense nd or a l compound. A
measurable physiological response may e increased expression of a functional form of a
dystrophin protein, or increased dystrophin—related biological activity in muscle tissue, among
other responses apparent from the understanding in the art and the description herein. Increased
muscle function can also be measured, including increases or improvements in muscle function by
about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%,10%,11%,12%,13%,14%,15%,16%,17%,18%,
19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%,
or 100%. The percentage of muscle fibres that express a onal dystrophin can also be
measured, including increased dystrophin expression in about 1%, 2%, %, 15%, 16%, 17%, 18%,
19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%,
or 100% of muscle fibres. For instance, it has been shown that around 40% of muscle function
improvement can occur if 25-30% of fibers express dystrophin (see, e. g., DelloRusso et al, Proc
Natl Acad Sci USA 99: 12979-12984, 2002). An "increased" or "enhanced" amount is typically a
"statistically significant" amount, and may include an se that is 1.1, 1.2, 2, 3, 4, 5, 6, 7, 8, 9,
10, 15, 20, 30, 40, 50 or more times (e.g., 500, 1000 times) (including all integers and decimal
points in between and above 1), e.g., 1.5, 1.6, 1.7, 1.8, etc.) the amount produced by no antisense
compound (the absence of an agent) or a control compound.
The term "reduce" or "inhibit" may relate generally to the ability of one or more antisense
compounds of the invention to "decrease" a relevant logical or cellular se, such as a
symptom of a disease or condition described herein, as measured according to routine techniques
in the diagnostic art. Relevant physiological or cellular responses (in vivo or in vitro) will be
apparent to s skilled in the art, and may include reductions in the ms or pathology of
muscular dystrophy, or reductions in the expression of ive forms of dystrophin, such as the
altered forms of dystrophin that are expressed in individuals with DMD or BMD. A ase" in
a response may be statistically significant as compared to the response produced by no antisense
compound or a control composition, and may include a 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%,
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%,11%,12%,13%,14%,15%,16%,17%,18%,19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%,
55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% decrease, including all integers in
between.
Also included are vector delivery systems that are capable of expressing the oligomeric,
dystrophin-targeting sequences of the present invention, such as vectors that express a
polynucleotide sequence comprising any one or more of SEQ ID NOS: 1—12, 46, and 47, as
described herein. By "vector" or "nucleic acid construct" is meant a polynucleotide molecule,
preferably a DNA le derived, for example, from a plasmid, bacteriophage, yeast or Virus,
into which a polynucleotide can be inserted or cloned. A vector preferably contains one or more
unique restriction sites and can be capable of autonomous ation in a defined host cell
including a target cell or tissue or a progenitor cell or tissue thereof, or be integrable with the
genome of the d host such that the cloned sequence is reproducible. Accordingly, the vector
can be an mously replicating vector, i.e., a vector that exists as an extra—chromosomal
entity, the replication of which is independent of chromosomal replication, e.g., a linear or closed
circular plasmid, an extra-chromosomal element, a mini-chromosome, or an artificial chromosome.
The vector can contain any means for assuring self—replication. Alternatively, the vector can be
one which, when introduced into the host cell, is integrated into the genome and replicated
together with the chromosome(s) into which it has been integrated.
“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 lactically or subsequent to the initiation of a pathologic event or contact with
an etiologic agent. Treatment includes any desirable effect on the symptoms or ogy of a
disease or condition associated with the dystrophin protein, as in certain forms of muscular
dystrophy, and may include, for example, minimal changes or improvements in one or more
able markers of the disease or condition being treated. Also included are "prophylactic"
treatments, which can be directed to reducing the rate of progression of the disease or condition
being treated, delaying the onset of that e or condition, or ng the severity of its onset.
"Treatment" or "prophylaxis" does not necessarily indicate complete ation, cure, or
prevention of the disease or condition, or associated ms thereof.
Hence, included are methods of ng muscular dystrophy, such as DMD and BMD, by
administering one or more antisense oligomers of the present invention (e. g., SEQ ID NOs: 1-12,
46, and 47, and variants thereof), optionally as part of a pharmaceutical ation or dosage
form, to a subject in need thereof. Also included are s of inducing exon—skipping in a
subject by stering one or more antisense oligomers, in which the exon is exon 44 from the
dystrophin gene, preferably the human dystrophin gene. A "subject," as used herein, includes any
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animal that exhibits a m, or is at risk for exhibiting a symptom, which can be treated with
an antisense compound of the invention, such as a subject that has or is at risk for having DMD or
BMD, or any of the symptoms associated with these conditions (e.g., muscle fibre loss). Suitable
subjects nts) include laboratory animals (such as mouse, rat, rabbit, or guinea pig), farm
animals, and domestic s or pets (such as a cat or dog). Non-human primates and,
preferably, human patients, are included.
"Alkyl" or "alkylene" both refer to a saturated straight or branched chain arbon
radical containing from 1 to 18 carbons. es include without limitation methyl, ethyl,
propyl, opyl, butyl, iso-butyl, tert-butyl, n—pentyl and n—hexyl. The term "lower alkyl" refers
to an alkyl group, as defined herein, containing between 1 and 8 carbons.
yl" refers to an unsaturated straight or branched chain hydrocarbon radical
containing from 2 to 18 carbons and comprising at least one carbon to carbon double bond.
Examples include without limitation ethenyl, propenyl, iso—propenyl, butenyl, iso—butenyl, tertbutenyl
, n—pentenyl and n—hexenyl. The term "lower alkenyl" refers to an alkenyl group, as
defined herein, containing between 2 and 8 carbons.
"Alkynyl" refers to an unsaturated straight or branched chain hydrocarbon radical
containing from 2 to 18 carbons sing at least one carbon to carbon triple bond. es
include without limitation ethynyl, propynyl, iso—propynyl, butynyl, iso-butynyl, tert-butynyl,
pentynyl and hexynyl. The term "lower alkynyl" refers to an alkynyl group, as defined herein,
containing between 2 and 8 carbons
alkyl" refers to a mono— or poly—cyclic alkyl radical. Examples include without
limitation cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl and cyclooctyl.
"Aryl" refers to a cyclic ic hydrocarbon moiety containing from to 18 carbons
having one or more closed ring(s). es include without limitation phenyl, benzyl, naphthyl,
anthracenyl, phenanthracenyl and biphenyl.
"Aralkyl" refers to a radical of the a RaRb where Ra is an alkylene chain as defined
above and Rb is one or more aryl radicals as defined above, for example, benzyl, diphenylmethyl
and the like.
"Thioalkoxy" refers to a radical of the a —SRc where Rc is an alkyl radical as
defined herein. The term "lower thioalkoxy" refers to an alkoxy group, as defined herein,
containing between 1 and 8 carbons.
"Alkoxy" refers to a radical of the formula —ORda where Rd is an alkyl radical as defined
herein. The term "lower alkoxy" refers to an alkoxy group, as defined herein, containing between
1 and 8 carbons. es of alkoxy groups include, without limitation, methoxy and ethoxy.
"Alkoxyalkyl" refers to an alkyl group substituted with an alkoxy group.
nyl" refers to the C(=O) — radical.
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dynyl" refers to the H2N(C=NH2) —NH— radical.
"Amidinyl" refers to the H2N(C=NH2)CH— radical.
"Amino" refers to the NHZ radical.
"Alkylamino" refers to a radical of the a —NHRd or —NRde where each Rd is,
independently, an alkyl l as defined herein. The term "lower alkylamino" refers to an
alkylamino group, as defined herein, containing between 1 and 8 carbons.
"Heterocycle" means a 5— to 7—membered monocyclic, or 7- to lO-membered ic,
heterocyclic ring which is either saturated, unsaturated, or aromatic, and which contains from 1 to
4 heteroatoms independently selected from nitrogen, oxygen and sulfur, and wherein the nitrogen
and sulfur heteroatoms may be optionally oxidized, and the nitrogen heteroatom may be optionally
quaternized, including bicyclic rings in which any of the above heterocycles are fused to a benzene
ring. The heterocycle may be attached Via any heteroatom or carbon atom. Heterocycles e
heteroaryls as defined below. Thus, in addition to the heteroaryls listed below, heterocycles also
include morpholinyl, pyrrolidinonyl, pyrrolidinyl, piperidinyl, piperizinyl, hydantoinyl,
valerolactamyl, oxiranyl, oxetanyl, tetrahydrofuranyl, ydropyranyl, tetrahydropyridinyl,
tetrahydrothiophenyl, tetrahydrothiopyranyl, tetrahydropyrimidinyl, tetrahydrothiopyranyl, and the
like.
"Heteroaryl" means an aromatic heterocycle ring of 5- to 10 members and having at least
one heteroatom selected from nitrogen, oxygen and , and containing at least 1 carbon atom,
including both mono- and bicyclic ring systems. Representative heteroaryls are pyridyl, furyl,
uranyl, thiophenyl, benzothiophenyl, quinolinyl, pyrrolyl, indolyl, oxazolyl, benzoxazolyl,
imidazolyl, benzimidazolyl, thiazolyl, benzothiazolyl, isoxazolyl, pyrazolyl, isothiazolyl,
pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl, cinnolinyl, phthalazinyl, and olinyl.
The terms "optionally tuted alkyl", "optionally substituted alkenyl", "optionally
substituted alkoxy", "optionally substituted thioalkoxy", "optionally substituted alkyl amino",
"optionally substituted lower alkyl", ”optionally tuted lower alkenyl", "optionally substituted
lower alkoxy”, "optionally tuted lower thioalkoxy", "optionally substituted lower alkyl
amino" and "optionally substituted heterocyclyl" mean that, when tuted, at least one
hydrogen atom is replaced with a substituent. In the case of an oxo substituent (:0) two hydrogen
atoms are replaced. In this regard, substituents include: deuterium, optionally substituted alkyl,
optionally substituted alkenyl, ally substituted alkynyl, optionally substituted aryl, optionally
substituted heterocycle, optionally substituted cycloalkyl, oxo, n, —CN, —ORx, NRny,
NRxC(=O)Ry, NRxSOZRy, —NRxC(=O)NRny, C(=O)Rx, Rx, C(=O)NRny, —SOme
and —SOmNRny, wherein m is 0, 1 or 2, Rx and Ry are the same or different and ndently
hydrogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted
alkynyl, optionally substituted aryl, optionally substituted heterocycle or optionally substituted
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cycloalkyl and each of said optionally substituted alkyl, optionally substituted alkenyl, optionally
substituted alkynyl, optionally substituted aryl, optionally substituted heterocycle and optionally
substituted cycloalkyl substituents may be further substituted with one or more of oxo, halogen, —
CN, —ORX, NRXRy, NRXC(=O)Ry, NRxSOZRy, —NRxC(=O)NRny, C(=O)RX, C(=O)ORX,
RXRy, —SOme and —SOmNRXRy.
An antisense molecule nomenclature system was proposed and published to distinguish
between the different antisense molecules (see Mann et al., (2002) J Gen Med 4, 644-654). This
nomenclature became especially nt when testing several slightly different antisense
molecules, all directed at the same target region, as shown below:
H#A/D(X:y).
The first letter designates the species (e.g. H: human, M: murine, C: canine). "#" designates
target dystrophin exon number. "A/D" indicates acceptor or donor splice site at the beginning and
end of the exon, tively. (X y) represents the annealing coordinates where "-" or "+" indicate
intronic or exonic sequences respectively. For example, A(—6+18) would indicate the last 6 bases
of the intron preceding the target exon and the first 18 bases of the target exon. The closest splice
site would be the acceptor so these coordinates would be preceded with an "A". Describing
annealing nates at the donor splice site could be D(+2-18) where the last 2 exonic bases and
the first 18 ic bases correspond to the annealing site of the antisense molecule. Entirely
exonic annealing coordinates that would be represented by A(+65+85), that is the site between the
65th and 85th tide from the start of that exon.
11. Antisense Oligonucleotides
When antisense molecule(s) are targeted to nucleotide ces ed in splicing of
exons Within pre-mRNA sequences, normal splicing of the exon may be inhibited, causing the
splicing machinery to by-pass the entire ed exon from the mature mRNA. In many genes,
deletion of an entire exon would lead to the production of a non-functional protein through the loss
of important functional domains or the disruption of the reading frame. In some proteins,
however, it is possible to n the protein by deleting one or more exons from within the
protein, without disrupting the reading frame, and without seriously altering the biological activity
of the protein. Typically, such proteins have a structural role and/or possess functional s at
their ends. Duchenne muscular dystrophy arises from mutations that preclude the sis of a
functional dystrophin gene product, typically by disrupting the g frame. Antisense
oligonucleotides that induce exon ng of the region of the dystrophin gene containing the
mutation can allow muscle cells to e a mature mRNA transcript that encodes a functional
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phin protein. The resulting dystrophin protein is not necessarily the "wild-type" form of
dystrophin, but is rather a truncated, yet functional or semi—functional, form of dystrophin. The
present invention describes antisense molecules capable of binding to specified dystrophin pre—
mRNA targets in exon 44, and re—directing processing of that gene.
In particular, the invention relates to isolated nse oligonucleotides of 20 to 50
nucleotides in , including at least 10, 12, 15, 17, 20 or more, consecutive nucleotides
complementary to an exon 44 target region of the phin gene designated as an annealing site
selected from the following: H44A(—O7+17), H44A(—07+20), H44A(-07+22), 8+15),
H44A(-7+15), H44A(-6+15), H44A(-8+17), H44A(—6+17), H44A(+77+101), H44A(+64+91),
H44A(+62+89), H44A(+62+85), H44A(—13+14), H44A(—14+15). Antisense oligonucleotides
specifically hybridize to the annealing site, inducing exon 44 skipping.
The antisense oligonucleotide and the target RNA are complementary to each other when a
sufficient number of corresponding positions in each molecule are occupied by nucleotides which
can hydrogen bond with each other, such that stable and specific g occurs between the
oligonucleotide and the target. Thus, "specifically hybridizable" and "complementary" are terms
which are used to indicate a ient degree of complementarity or precise pairing such that
stable and specific binding occurs between the oligonucleotide and the target. It is understood in
the art that the sequence of an antisense molecule need not be 100% complementary to that of its
target sequence to be specifically hybridizable. An antisense molecule is specifically hybridizable
when binding of the oligonucleotide to the target molecule interferes with the normal function of
the target RNA, and there is a sufficient degree of mentarity to avoid ecific binding
of the antisense oligonucleotide to non-target sequences under conditions in which specific binding
is desired, i.e., under physiological conditions in the case of in vivo assays or therapeutic
treatment, and in the case of in vitro assays, under conditions in which the assays are performed.
The length of an antisense molecule may vary so long as it is capable of binding
selectively to the intended location within the pre-mRNA molecule. The length of such sequences
can be determined in accordance with selection procedures described herein. Generally, the
antisense molecule will be from about 10 nucleotides in length up to about 50 nucleotides in
. It will be appreciated r that any length of nucleotides within this range may be used
in the . Preferably, the length of the antisense molecule is n 10-30 nucleotides in
length.
In one embodiment, oligonucleotides of the invention are 20 to 50 nucleotides in length
and include at least 10, 12, 15, 17, 20 or more, nucleotides of any of SEQ ID NOs: 1-12, 46, and
47. In some ments, thymine bases in SEQ ID NOS: 1—12, 46, and 47 are optionally uracil.
The exon deletion should not lead to a g frame shift in the shortened transcribed
mRNA. Thus, if in a linear sequence of three exons the end of the first exon encodes two of three
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nucleotides in a codon and the next exon is deleted then the third exon in the linear sequence must
start with a single nucleotide that is capable of ting the nucleotide triplet for a codon. If the
third exon does not commence with a single tide there will be a reading frame shift that
would lead to the generation of truncated or a non—functional protein.
It will be appreciated that the codon ements at the end of exons in structural proteins
may not always break at the end of a codon, consequently there may be a need to delete more than
one exon from the NA to ensure in—frame reading of the mRNA. In such circumstances, a
plurality of antisense ucleotides may need to be selected by the method of the invention
wherein each is directed to a different region responsible for inducing splicing in the exons that are
to be deleted.
In some embodiments, the antisense oligonucleotides have the chemical composition of a
naturally occurring nucleic acid le, Le. , the antisense oligonucleotides do not include a
modified or substituted base, sugar, or intersubunit linkage. In a preferred embodiment, the antisense
oligonucleotides of the present invention are non—naturally occurring nucleic acid molecules. For
example, non-naturally occurring nucleic acids can include one or more non-natural base, sugar,
and/or intersubunit linkage, e. g. a base, sugar, and/or linkage that has been modified or substituted
with respect to that found in a lly occurring nucleic acid molecule. Exemplary modifications
are bed below. In some embodiments, non—naturally occurring nucleic acids include more than
one type of modification, 9. g. and base modifications, sugar and e modifications, base
, sugar
and linkage modifications, or base, sugar, and linkage cations. For example, in some
embodiments, the antisense oligonucleotides contain a non—natural (e.g., modified or substituted)
base. In some embodiments, the antisense oligonucleotides contain a non-natural (e. g. modified or
tuted) sugar. In some embodiments, the antisense oligonucleotides contain a non-natural (e. g.
modified or substituted) intersubunit linkage. In some embodiments, the antisense oligonucleotides
contain more than one type of modification or substutution, e.g., a non-natural base and/or a non-
natural sugar, and/or a non-natural intersubuint e.
To avoid degradation of pre-mRNA during duplex formation with the antisense molecules,
the nse molecules may be adapted to minimize or prevent cleavage by endogenous RNase H.
This property is highly preferred as the treatment of the RNA with the unmethylated
oligonucleotides either intracellularly or in crude extracts that n RNase H leads to
degradation of the pre-mRNA: antisense oligonucleotide duplexes. Any form of modified
antisense molecule that is capable of by—passing or not inducing such ation may be used in
the present method. An example of nse molecules which when ed with RNA are not
cleaved by cellular RNase H is 2'-O-methyl derivatives. 2'—O—methyl—oligoribonucleotides are
very stable in a cellular environment and in animal tissues, and their duplexes with RNA have
higher Tm values than their ribo- or deoxyribo—counterparts. Methylation of the 2' hydroxyribose
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position and the incorporation of a phosphorothioate backbone is a common strategy for producing
molecules that superficially resemble RNA but that are much more ant to nuclease
degradation.
Antisense molecules that do not activate RNase H can be made in accordance with known
techniques (see, e.g., U.S. Pat. No. 5,149,797). Such antisense molecules, which may be
deoxyribonucleotide or ribonucleotide sequences, simply contain any structural modification
which ally hinders or prevents binding of RNase H to a duplex molecule ning the
ucleotide as one member thereof, which structural modification does not substantially hinder
or disrupt duplex formation. Because the portions of the oligonucleotide involved in duplex
formation are substantially different from those portions involved in RNase H binding thereto,
us antisense molecules that do not activate RNase H are ble. For e, such
antisense molecules may be oligonucleotides wherein at least one, or all, of the inter-nucleotide
bridging phosphate residues are modified phosphates, such as methyl phosphonates, methyl
phosphorothioates, phosphoromorpholidates, phosphoropiperazidates and phosphoramidates. For
example, every other one of the internucleotide ng phosphate residues may be modified as
bed. In another non-limiting example, such nse molecules are molecules wherein at
least one, or all, of the tides n a 2' lower alkyl moiety (e. g., C1—C4, linear or branched,
saturated or unsaturated alkyl, such as methyl, ethyl, ethenyl, propyl, l-propenyl, 2-propenyl, and
isopropyl). For example, every other one of the nucleotides may be modified as described.
Specific examples of antisense oligonucleotides useful in this invention include
ucleotides containing modified backbones or non—natural intersubunit linkages.
Oligonucleotides having modified backbones include those that retain a phosphorus atom in the
backbone and those that do not have a phosphorus atom in the backbone. Modified
oligonucleotides that do not have a phosphorus atom in their inter—nucleoside backbone can also be
considered to be oligonucleosides.
In other antisense molecules, both the sugar and the inter-nucleoside linkage, i.e., the
backbone, of the nucleotide units are replaced with novel groups. The base units are ined
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-backbone
of an oligonucleotide is replaced with an amide containing backbone, in ular an
aminoethylglycine ne. The nucleo—bases are retained and are bound directly or indirectly to
aza nitrogen atoms of the amide portion of the backbone.
Modified oligonucleotides may also contain one or more substituted sugar moieties.
Oligonucleotides may also include nucleobase (often referred to in the art simply as
"base") modifications or tutions. Oligonucleotides containing a modified or substituted base
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e oligonucleotides in which one or more purine or pyrimidine bases most commonly found
in nucleic acids are replaced with less common or non—natural bases.
Purine bases se a pyrimidine ring fused to an imidazole ring, as described by the
general formula:
' N“
\ .5
Purine
Adenine and guanine are the two purine nucleobases most commonly found in nucleic acids.
These may be substituted with other naturally—occurring purines, including but not limited to N6-
methyladenine, NZ-methylguanine, hypoxanthine, and 7—methylguanine.
Pyrimidine bases comprise a six—membered pyrimidine ring as described by the general
formula:
dine
Cytosine, uracil, and thymine are the pyrimidine bases most commonly found in c acids.
These may be substituted with other naturally—occurring pyrimidines, including but not limited to
-methylcytosine, 5-hydroxymethylcytosine, pseudouracil, and 4-thiouracil. In one embodiment,
the oligonucleotides described herein contain thymine bases in place of uracil.
Other modified or substituted bases e, but are not limited to, 2,6-diaminopurine, orotic
acid, agmatidine, lysidine, 2-thiopyrimidine (e.g. 2—thiouracil, 2-thiothymine), G-clamp and its
derivatives, 5-substituted pyrimidine (e.g. 5—halouracil, S—propynyluracil, S-propynylcytosine, 5-
aminomethyluracil, oxymethy1uracil, S—aminomethylcytosine, 5—hydroxymethylcytosine,
Super T), 7—deazaguanine, 7—deazaadenine, 7—aza—2,6—diaminopurine, 8-azadeazaguanine, 8-aza-
7—deazaadenine, 7—deaza-2,6-diaminopurine, Super G, Super A, and N4-ethylcytosine, or
derivatives thereof; lopentylguanine (cPent—G), NZ—cyclopentyl—Z—aminopurine (cPent-AP),
and Nz—propyl—Z—aminopurine (Pr—AP), uracil or derivatives thereof; and degenerate or
universal bases, like 2,6-difluorotoluene or absent bases like abasic sites (e.g. 1-deoxyribose, 1,2-
dideoxyribose, l-deoxyO-methylribose; or pyrrolidine derivatives in which the ring oxygen has
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been replaced with nitrogen (azaribose)). es of derivatives of Super A, Super G and Super
T can be found in US. Patent 6,683, 173 (Epoch Biosciences), which is incorporated here entirely
by nce. cPent—G, cPent—AP and Pr—AP were shown to reduce immunostimulatory effects
when incorporated in siRNA (Peacock H. et al. J. Am. Chem. Soc. 2011, 133, 9200). Pseudouracil
is a naturally occuring isomerized version of uracil, with a C-glycoside rather than the regular N-
glycoside as in uridine. Pseudouridine—containing synthetic mRNA may have an improved safety
profile compared to uridine—containing mPvNA (WO 2009127230, incorporated here in its entirety
by reference).
n modified or tuted nucleo—bases are particularly useful for increasing the
binding affinity of the antisense oligonucleotides of the invention. These include 5—substituted
pyrimidines, 6-azapyrimidines and N—2, N—6 and 0—6 tuted purines, ing 2-
aminopropyladenine, 5-propynyluracil and 5-propynylcytosine. 5-methylcytosine substitutions
have been shown to increase nucleic acid duplex stability by 0.6—1.2OC and are presently preferred
base substitutions, even more particularly when combined with 2'-O-methoxyethyl sugar
modifications.
In some embodiments, modified or substituted nucleo—bases are useful for tating
purification of antisense oligonucleotides. For e, in certain embodiments, antisense
oligonucleotides may contain three or more (e.g., 3, 4, 5, 6 or more) consecutive guanine bases. In
certain antisense oligonucleotides, a string of three or more consecutive guanine bases can result in
aggregation of the oligonucleotides, cating purification. In such antisense oligonucleotides,
one or more of the consecutive guanines can be substituted with inosine. The substitution of
inosine for one or more guanines in a string of three or more utive guanine bases can reduce
aggregation of the antisense oligonucleotide, thereby facilitating purification.
In one embodiment, r modification of the antisense oligonucleotides involves
chemically linking to the oligonucleotide one or more moieties or conjugates that enhance the
activity, ar distribution or ar uptake of the oligonucleotide. Such moieties include but
are not limited to lipid moieties such as a cholesterol moiety, cholic acid, a thioether, e. g., hexyl
thiol, a thiocholesterol, an aliphatic chain, e.g., dodecandiol or l residues, a
phospholipid, e.g., di-hexadecyl-rac—glycerol or triethylammonium 1,2-di-O-hexadecy1-rac-
glyceroH-phosphonate, a polyamine or a hylene glycol chain, or tane acetic acid,
a palmityl moiety, or an octadecylamine or hexylamino—carbonyl—oxycholesterol moiety.
It is not necessary for all positions in a given compound to be uniformly modified, and in
fact more than one of the aforementioned modifications may be incorporated in a single compound
or even at a single nucleoside within an oligonucleotide. The present invention also includes
antisense oligonucleotides that are chimeric compounds. "Chimeric" antisense compounds or
"chimeras," in the t of this invention, are antisense molecules, particularly oligonucleotides,
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which contain two or more chemically distinct regions, each made up of at least one r
unit, i.e., a nucleotide in the case of an oligonucleotide compound. These oligonucleotides
lly contain at least one region wherein the oligonucleotide is modified so as to confer upon
the increased resistance to nuclease degradation, increased cellular uptake, and an additional
region for increased g ty for the target nucleic acid.
The antisense molecules used in accordance with this invention may be conveniently and
routinely made through the nown technique of solid phase synthesis. Equipment for such
synthesis is sold by several vendors including, for example, Applied Biosystems (Foster City,
Calif). One method for synthesising oligonucleotides on a modified solid support is described in
US. Pat. No. 4,458,066.
Any other means for such synthesis known in the art may additionally or alternatively be
employed. It is well known to use similar techniques to prepare oligonucleotides such as the
phosphorothioates and alkylated derivatives. In one such ted embodiment, diethyl-
phosphoramidites are used as starting materials and may be synthesized as described by Beaucage,
et al., (1981) Tetrahedron Letters, 22:1859—1862.
The antisense molecules of the invention are synthesised in vitro and do not include
antisense compositions of biological origin. The molecules of the invention may also be mixed,
encapsulated, conjugated or otherwise ated with other molecules, molecule structures or
mixtures of compounds, as for example, liposomes, receptor ed molecules, oral, rectal,
topical or other ations, for assisting in uptake, distribution and/or absorption.
A. Morpholino Oligomers
Exemplary embodiments of the invention relate to morpholino oligomers having
phosphorus-containing backbone es are illustrated in Figs. 1A—1C. In one embodiment, a
phosphorodiamidate—linked morpholino oligomer such as shown in Fig. 1C, which is modified, in
accordance with one aspect of the present invention, to contain positively charged groups at
preferably 10%-50% of its backbone linkages. Morpholino oligomers with uncharged backbone
es, including antisense oligomers, are detailed, for example, in (Summerton and Weller
1997) and in co-owned US. Patent Nos. 5,698,685, 866, 5,142,047, 5,034,506, 5,166,315,
5,185, 444, 5,521,063, 337, 476, 8,299,206 and 7,943,762 all of which are expressly
orated by reference . Morpholino oligomers with modified linkages including charged
linkages can be found in USSN: 13/118,298 (incorporated herein by reference).
Important properties of the morpholino-based subunits include: 1) the ability to be linked
in a oligomeric form by stable, uncharged or positively charged backbone linkages; 2) the ability
to support a nucleotide base (e. g. adenine, cytosine, guanine, thymidine, uracil and inosine) such
that the polymer formed can hybridize with a mentary-base target nucleic acid, including
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target RNA, Tm values above about 45°C in relatively short oligonucleotides (e. g. , 10-15 ;
3) the ability of the oligonucleotide to be actively or passively transported into mammalian cells;
and 4) the ability of the antisense oligonucleotidezRNA heteroduplex to resist RNAse and RNase
H degradation, respectively.
Exemplary backbone structures for antisense oligonucleotides of the claimed subject
matter include the morpholino subunit types shown in Figs. 1D—G, each linked by an uncharged or
positively charged, orus—containing subunit linkage. Fig. 1D shows a phosphorus-
containing linkage which forms the five atom repeating-unit backbone, where the morpholino
rings are linked by a 1-atom phosphoamide linkage. Fig. 1E shows a linkage which produces a 6-
atom repeating-unit ne. In this structure, the atom Y linking the 5' morpholino carbon to
the orus group may be sulfur, nitrogen, carbon or, preferably, oxygen. The X moiety
pendant from the phosphorus may be e, an alkyl or substituted alkyl, an alkoxy or
substituted alkoxy, a thioalkoxy or substituted thioalkoxy, or unsubstituted, monosubstituted, or
disubstituted nitrogen, including cyclic structures, such as morpholines or dines. Alkyl,
alkoxy and thioalkoxy preferably include 1—6 carbon atoms. The Z moieties are sulfur or oxygen,
and are preferably oxygen.
The linkages shown in Figs. 1F and 1G are designed for 7—atom unit—length backbones.
In structure 1F, the X moiety is as in Structure 1E, and the Y moiety may be methylene, sulfur, or,
preferably, oxygen. In Structure 1G, the X and Y es are as in Structure 1E. Particularly
red morpholino oligonucleotides include those composed of lino subunit structures of
the form shown in Fig. 1E, where X=NH2, 2, or 1—piperazine or other charged group, Y=O,
and 2:0.
A substantially uncharged oligonucleotide may be modified, in accordance with an aspect
of the invention, to include charged linkages, e. g., up to about 1 per every 2—5 uncharged linkages,
such as about 4—5 per every 10 uncharged linkages. In certain embodiments, optimal improvement
in antisense activity may be seen when about 25% of the ne es are ic. In
certain embodiments, enhancement may be seen with a small number e.g., 10—20% cationic
linkages, or where the number of cationic linkages are in the range 50-80%, such as about 60%.
Oligomers having any number of cationic es are provided, including fully ic-
linked Oligomers. Preferably, however, the Oligomers are partially charged, having, for e,
%-80%. In preferred embodiments, about 10% to 60%, and preferably 20% to 50% of the
linkages are cationic.
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.
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Also considered are oligomers having blocks of cationic es and blocks of uncharged
es; for example, a central block of ged 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%.
In certain ments, the antisense oligonucleotides can be prepared by stepwise solid-
phase synthesis, employing methods ed in the references cited above, and below with t
to the synthesis of oligonucleotides having a mixture or uncharged and cationic backbone linkages,
and in the Examples herein. In some cases, it may be desirable to add additional chemical
moieties to the antisense compound, e.g., to enhance pharmacokinetics or to facilitate capture or
detection of the compound. Such a moiety may be covalently attached, according to standard
synthetic methods. For example, addition of a polyethylene glycol moiety or other hydrophilic
polymer, e.g., one having 1-100 monomeric subunits, may be useful in enhancing solubility.
A reporter moiety, such as fluorescein or a radiolabeled group, may be attached for
purposes of detection. atively, the reporter label attached to the oligomer may be a ligand,
such as an antigen or biotin, capable of binding a d antibody or streptavidin. In selecting a
moiety for attachment or modification of an antisense oligonucleotide, it is lly of course
desirable to select chemical compounds of groups that are biocompatible and likely to be tolerated
by a subject without undesirable side effects.
Oligonucleotides for use in antisense applications generally range in length from about 10
to about 50 subunits, more preferably about 10 to 30 subunits, and lly 15-25 bases. For
example, an oligonucleotide of the ion having 19-20 subunits, a useful length for an
antisense ucleotide, may ideally have two to ten, e.g., four to eight, cationic linkages, and
the remainder uncharged linkages. An oligonucleotide having 14—15 subunits may ideally have
two to seven, e.g., 3, 4, or 5, cationic linkages and the remainder uncharged linkages. In a
preferred embodiment, the oligonucleotides have 25 to 28 subunits.
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 g moiety may be a purine or pyrimidine found in native
DNA or RNA (e. g. T or U) or an analog, such as hypoxanthine (the base component of
, A, G, C,
the side inosine), 5-methyl cytosine, 2—6—diaminopurine or other modified bases known in
the art and described below.
As noted above, certain embodiments are directed to oligonucleotides comprising novel
intersubunit linkages, including PMO—X oligomers and those having ed terminal groups. In
some embodiments, these ers have higher affinity for DNA and RNA than do the
corresponding fied oligomers and demonstrate improved cell delivery, potency, and/or
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tissue distribution properties compared to ers having other intersubunit linkages. The
ural features and properties of the various linkage types and oligomers are described in more
detail in the following discussion. The synthesis of these and related oligomers is described in co—
owned US. Application No. 13/118,298, which is incorporated by reference in its entirety.
In certain embodiments, the invention provides for an oligonucleotide having a sequence
complementary to the target sequence which is associated with a human disease, and comprises a
sequence of tides having a formula:
Ry/ \Rz H
wherein Nu is a nucleobase;
R1 has the formula
qis0,1,or2;
R2 is selected from the group consisting of en, C1—C5 alkyl, C1—C5 aralkyl, and a
formamidinyl group, and
R3 is selected from the group ting of hydrogen, C1-C10 acyl, C1-C10 aminoacyl, acyl
moiety of a natural or unnatural alpha or beta amino acid, C1—C10 aralkyl, and C1-C10 alkyl, or
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R2 and R3 are joined to form a 5-7 membered ring where the ring may be optionally
tuted with a substituent selected from the group consisting of C1—C10 alkyl, phenyl, halogen,
and C1—C10 aralkyl;
R4 is selected from the group consisting of an electron pair, hydrogen, a C1-C6 alkyl and C1-C6
RX is selected from the group consisting of sarcosinamide, hydroxyl a nucleotide, a cell
penetrating peptide , and piperazinyl;
Ry is selected from the group consisting of hydrogen, a C1—C6 alkyl, a nucleotide a cell
penetrating e moiety, an amino acid, a formamidinyl group, and C1-C6 acyl; and,
R2 is selected from the group consisting of an electron pair, hydrogen, a C1-C6 alkyl, and C1-
C6 acyl pharmaceutically acceptable salts thereof.
Nu may be selected from the group consisting of adenine, guanine, thymine, uracil, cytosine,
and hypoxanthine. More preferably Nu is thymine or uracil.
In preferred ments, the invention provides an oligonucleotide having a sequence of
tides having a formula:
o——P——R1
Ry/ \ H
wherein Nu is a nucleobase;
R1 is selected from the group consisting of R1’ and R1” wherein R1’ is dimethyl- amino
and R1” has the formula
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—N >
\ \,N/R2
R4/ \R3
wherein at least one R1 is R1”;
q is 0, 1, or 2; with the proviso that at least one of R1 is a piperidinyl moiety;
R2 is selected from the group consisting of hydrogen, C1—C5 alkyl, C1—C5 aralkyl, and a
formamidinyl group, and
R3 is selected from the group consisting of hydrogen, C1-C10 acyl, C1-C10 cyl, acyl
moiety of a natural or ral alpha or beta amino acid, C1-C10 l, and C1-C10 alkyl, or
R2 and R3 are joined to form a 5-7 membered ring where the ring may be optionally
substituted With a substituent selected from the group consisting of C1—C10 alkyl, phenyl, halogen,
and C1-C10 aralkyl;
R4 is selected from the group consisting of an electron pair, en, a C1-C6 alkyl and
aralkyl;
Rx is selected from the group consisting of sarcosinamide, hydroxyl a nucleotide, a cell
penetrating peptide moiety, and piperazinyl;
Ry is selected from the group consisting of hydrogen, a C1-C6 alkyl, a nucleotide a cell
penetrating peptide moiety, an amino acid, a formamidinyl group, and C1-C6 acyl; and,
R2 is selected from the group consisting of an electron pair, hydrogen, a C1-C6 alkyl, and C1-
C6 acyl pharmaceutically able salts thereof.
Nu may be selected from the group consisting of adenine, guanine, e, uracil, cytosine,
and hypoxanthine. More preferably Nu is e or uracil.
About 90-50% of the R1 groups are dimethylamino (i.e. Rl’). More, preferably, 90-50% of
the R1 groups are dimethylamino. Most, preferably about 66% of the R1 groups are
dimethylamino.
R1” may be selected from the group consisting of
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«H9H3 N U
CH3 H2N
|® >=NH
wwN N—CH3 mN N\
l CH3
H30>
\ H3C H
H3C /H
>=o mNGN NH2
WNQN m
H O NHZ
’CH3 H2N>=
MGM NH
H WNCH\H
Preferably, at least one tide of the oligonucleotide has the formula:
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N NH2
Rz/ H
wherein Rx, Ry, RZ, and Nu are as stated above. Most preferably, Nu is e or uracil.
Although thymine (T) is the preferred base pairing moiety (Nu or Pi) containing the
chemical modifications described above, any base t known to a person of skill in the art can
be used as the base pairing moiety.
B. Peptide Transporters
The antisense oligonucleotides of the invention may include an oligonucleotide moiety
conjugated to a CPP, preferably an arginine—rich peptide transport moiety effective to enhance
transport of the compound into cells. The transport moiety is preferably attached to a terminus of
the oligomer, as shown, for example, in FIGS 13 and 1C. The peptides have the capability of
inducing cell penetration within 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% of cells of a
given cell culture population, including all integers in n, and allow macromolecular
translocation within multiple tissues in vivo upon systemic stration. In one embodiment, the
cell-penetrating peptide may be an ne—rich peptide transporter. In another ment, the
cell—penetrating peptide may be Penetratin or the Tat peptide. These peptides are well known in
the art and are disclosed, for example, in US Publication No. 2010-0016215 Al, incorporated by
reference in its entirety. A particularly preferred approach to conjugation of peptides to antisense
oligonucleotides can be found in PCT publication W02012/150960, which is incorporated by
reference in its entirety. A preferred embodiment of a peptide conjugated oligonucleotide of the
present invention utilizes glycine as the linker between the CPP and the antisense ucleotide.
For example, a preferred peptide conjugated PMO consists of Ré—G—PMO.
The transport moieties as described above have been shown to greatly enhance cell entry
of attached ers, relative to uptake of the er in the absence of the attached transport
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moiety. Uptake is preferably enhanced at least ten fold, and more preferably twenty fold, relative
to the unconjugated compound.
The use of arginine—rich peptide transporters (i.e., cell—penetrating peptides) are
particularly useful in practicing the present ion. Certain peptide transporters have been
shown to be highly effective at delivery of antisense compounds into primary cells including
muscle cells (Marshall, Oda et al. 2007; iriyapaisarn, Moulton et a1. 2008; Wu, Moulton et
al. 2008). Furthermore, compared to other known peptide transporters such as atin and the
Tat peptide, the peptide transporters described herein, when conjugated to an antisense PMO,
demonstrate an enhanced ability to alter splicing of several gene ripts (Marshall, Oda et al.
2007). Preferred ments of morpholino—peptide transporter conjugates are described in
WO/2012/150960, incorporated herein in its entirety.
Exemplary peptide transporters, excluding linkers are given below in Table 1.
Table 1. Exemplary peptide orters
rTAT RRRQRRKKR 24
R5F2R4 RRRRRFFRRRR 27
(muons2 RARRARRARRARFF
F2 RGRRGRRGRRGRFF
ASequences assigned to SEQ ID NOS do not include the linkage portion (e.g., C, G, P, Ahx, B,
Ath where Ahx and B refer to ohexanoic acid and beta-alanine, tively).
C. Expression Vectors
In one ment, the invention includes expression vectors for expression of the
dystrophin-targeting sequences described herein in cells. Vector delivery systems are capable of
expressing the oligomeric, dystrophin—targeting sequences of the t invention. In one
embodiment, such vectors express a polynucleotide sequence comprising at least 10 consecutive
nucleotides of one or more of SEQ ID NOs: 1-12, 46, and 47. In another embodiment, such
vectors express a polynucleotide sequence comprising one or more of SEQ ID NOs: 1—12, 46, and
47. Expression vectors suitable for gene delivery are known in the art. Such sion vectors
can be modified to express the dystrophin—targeting sequences described herein. Exemplary
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expression vectors include polynucleotide les, preferably DNA molecules, that are d,
for example, from a plasmid, bacteriophage, yeast or virus (e.g., adenovirus, adeno-associated
virus, lentivirus, etc.), into which a polynucleotide can be inserted or cloned. A vector preferably
contains one or more unique restriction sites and can be capable of autonomous ation in a
defined host cell including a target cell or tissue or a progenitor cell or tissue thereof, or be
integrable with the genome of the defined host such that the cloned sequence is reproducible.
Accordingly, the vector can be an autonomously replicating vector, i.e., a vector that exists as an
extra-chromosomal entity, the replication of which is independent of chromosomal replication,
e. g., a linear or closed circular plasmid, an extra—chromosomal element, a hromosome, or an
artificial some. The vector can contain any means for assuring self—replication.
Alternatively, the vector can be one which, when introduced into the host cell, is integrated into
the genome and replicated together with the chromosome(s) into which it has been integrated.
In one embodiment, the expression vectors include a tissue—specific promoter, e.g., a
muscle—specific promoter and/or er, which promotes expression of the oligomeric
dystrophin-targeting ces described herein in particular cells or tissues of interest (6. g. in
). Promoter sequences and expression vectors suitable for sion in muscle cells
include, for e, those described in US 2011/0212529, the entire contents of which are
incorporated herein by reference. Exemplary muscle—specific promoters include a desmin
promoter, a muscle creatine kinase (MCK) promoter, a Pitx3 promoter, a al alpha-actin
promoter, or a troponin I promoter. Use of muscle—specific promoters are further described in, for
example, Talbot et al., Molecular Therapy (2010), 18(3): 601-608; Wang et al., Gene y
(2008), 15(22): 1489-99; and Coulon er al., Journal of Biological Chemistry (2007), 282(45):
33192-33200.
III. Formulations and Modes of Administration
In certain embodiments, the present invention provides formulations or compositions
suitable for the therapeutic delivery of antisense oligomers, as described herein. Hence, in certain
embodiments, the present ion provides pharmaceutically acceptable compositions that
comprise a therapeutically-effective amount of one or more of the oligomers described herein,
ated together with one or more ceutically acceptable carriers (additives) and/or
diluents. While it is possible for an oligomer of the t invention to be administered alone, it
is preferable to administer the compound as a pharmaceutical formulation (composition).
s for the delivery of nucleic acid molecules are described, for example, in Akhtar
et al., 1992, Trends Cell Bio., 2:139; and Delivery Strategies for Antisense Oligonucleotide
Therapeutics, ed. ; Sullivan et al., PCT WO 94/02595. These and other protocols can be
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utilized for the delivery of lly any c acid molecule, including the isolated oligomers of
the present invention.
As detailed below, the pharmaceutical compositions of the present invention may be
specially formulated for administration in solid or liquid form, including those adapted for the
following: (1) oral administration, for e, drenches (aqueous or non-aqueous solutions or
suspensions), tablets, e.g, those targeted for buccal, sublingual, and systemic absorption, boluses,
powders, granules, pastes for application to the ; (2) parenteral administration, for example,
by subcutaneous, intramuscular, intravenous or epidural injection as, for e, a sterile solution
or suspension, or sustained-release formulation; (3) topical application, for example, as a cream,
ointment, or a controlled-release patch or spray applied to the skin; (4) intravaginally or
intrarectally, for example, as a pessary, cream or foam; (5) gually; (6) ly; (7)
transdermally; or (8) nasally.
The phrase "pharmaceutically acceptable" is ed herein to refer to those compounds,
materials, compositions, and/or dosage forms which are, within the scope of sound medical
judgment, suitable for use in contact with the tissues of human beings and animals without
excessive toxicity, irritation, allergic response, or other problem or complication, commensurate
with a able benefit/risk ratio,
The phrase "pharmaceutically—acceptable carrier" as used herein means a
pharmaceutically-acceptable material, composition or vehicle, such as a liquid or solid filler,
diluent, excipient, manufacturing aid (e.g., lubricant, talc magnesium, calcium or zinc stearate, or
steric acid), or solvent encapsulating material, involved in carrying or transporting the subject
compound from one organ, or portion of the body, to another organ, or n of the body. Each
carrier must be "acceptable" in the sense of being compatible with the other ingredients of the
formulation and not injurious to the patient.
Some examples of materials that can serve as pharmaceutically-acceptable carriers
include, t limitation: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as
corn starch and potato starch; (3) ose, and its derivatives, such as sodium carboxymethyl
cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7)
talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil,
cottonseed oil, safflower oil, sesalne oil, olive oil, corn oil and soybean oil; (10) glycols, such as
propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; (12)
, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium
hydroxide and aluminum ide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic
saline; (18) Ringer's solution; (19) ethyl alcohol; (20) pH buffered solutions; (21) polyesters,
polycarbonates and/or hydrides; and (22) other non—toxic compatible substances employed
in pharmaceutical formulations.
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Additional non-limiting es of agents suitable for formulation with the antisense
ers of the instant invention include: PEG conjugated nucleic acids, phospholipid conjugated
nucleic acids, nucleic acids containing lipophilic moieties, phosphorothioates, P—glycoprotein
inhibitors (such as ic P85) which can enhance entry of drugs into various tissues;
biodegradable polymers, such as poly (DL—lactide—coglycolide) microspheres for sustained release
delivery after implantation (Emerich, D F et al., 1999, Cell Transplant, 8, 47—5 8) Alkermes, Inc.
Cambridge, Mass.; and loaded nanoparticles, such as those made of polybutylcyanoacrylate, which
can deliver drugs across the blood brain barrier and can alter neuronal uptake mechanisms (Prog
Neuropsychopharmacol Biol Psychiatry, 23, 941—949, 1999).
The invention also features the use of the composition comprising surface—modified
mes containing poly (ethylene glycol) lipids (PEG—modified, branched and unbranched or
combinations thereof, or long-circulating liposomes or stealth liposomes). Oligomers of the
invention can also comprise covalently attached PEG molecules of various lar weights.
These formulations offer a method for increasing the lation of drugs in target tissues. This
class of drug carriers resists opsonization and elimination by the mononuclear phagocytic system
(MPS or RES), thereby enabling longer blood circulation times and enhanced tissue exposure for
the ulated drug (Lasic et a1. Chem. Rev. 1995, 95, 2601—2627; Ishiwata et al., Chem. Pharm.
Bull. 1995, 43, 1005-1011). Such liposomes have been shown to accumulate selectively in tumors,
presumably by extravasation and e in the neovascularized target tissues (Lasic et al., Science
1995, 267, 1275-1276; Oku et al., 1995, Biochim. Biophys. Acta, 1238, 86—90). The long—
circulating liposomes enhance the pharmacokinetics and pharmacodynamics of DNA and RNA,
particularly ed to conventional cationic liposomes which are known to accumulate in
tissues of the MPS (Liu et al., J. Biol. Chem. 1995, 42, 24864—24870; Choi et al., International
PCT ation No. W0 96/10391; Ansell et al., International PCT Publication No. W0
90; Holland et al., International PCT ation No. W0 96/10392). Long-circulating
mes are also likely to t drugs from nuclease degradation to a greater extent compared
to cationic liposomes, based on their ability to avoid accumulation in metabolically aggressive
MPS tissues such as the liver and spleen.
In a further embodiment, the present invention includes oligomer compositions prepared
for delivery as described in US. Pat. Nos. 6,692,911, 7,163,695 and 7,070,807. In this regard, in
one embodiment, the present invention es an oligomer of the present invention in a
composition comprising copolymers of lysine and ine (HK) (as described in US. Pat. Nos.
7,163,695, 7,070,807, and 6,692,911) either alone or in combination with PEG (e. g., branched or
unbranched PEG or a e of both), in combination with PEG and a ing moiety or any of
the foregoing in combination with a crosslinking agent. In certain embodiments, the present
invention provides antisense oligomers in compositions comprising gluconiC-acid-modified
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polyhistidine or gluconylated-polyhistidine/transferrin-polylysine. One skilled in the art Will also
recognize that amino acids with properties similar to His and Lys may be tuted Within the
composition.
Certain embodiments of the oligomers described herein may contain a basic functional
group, such as amino or alkylamino, and are, thus, capable of g pharmaceutically-acceptable
salts with pharmaceutically-acceptable acids. The term "pharmaceutically—acceptable salts" in this
respect, refers to the relatively non—toxic, inorganic and organic acid addition salts of compounds
of the present invention. These salts can be prepared in situ in the administration vehicle or the
dosage form manufacturing process, or by separately reacting a purified nd of the
invention in its free base form with a suitable c or inorganic acid, and isolating the salt thus
formed during subsequent purification. Representative salts include the hydrobromide,
hydrochloride, sulfate, bisulfate, phosphate, nitrate, acetate, valerate, oleate, palmitate, te,
laurate, benzoate, lactate, phosphate, tosylate, citrate, maleate, fumarate, succinate, tartrate,
napthylate, mesylate, glucoheptonate, lactobionate, and laurylsulphonate salts and the like. (See,
e.g., Berge et al. (1977) aceutical Salts", J. Pharm. Sci. 66:1-19).
The pharmaceutically acceptable salts of the subject oligomers include the conventional
nontoxic salts or quaternary ammonium salts of the compounds, e.g., from non—toxic organic or
inorganic acids. For e, such conventional nontoxic salts include those derived from
inorganic acids such as hydrochloride, hydrobromic, sulfuric, sulfamic, phosphoric, nitric, and the
like; and the salts prepared from organic acids such as acetic, nic, succinic, glycolic, stearic,
lactic, malic, tartaric, citric, ascorbic, palmitic, maleic, hydroxymaleic, phenylacetic, glutamic,
benzoic, salicyclic, sulfanilic, 2-acetoxybenzoic, fumaric, toluenesulfonic, methanesulfonic, ethane
disulfonic, oxalic, isothionic, and the like.
In certain embodiments, the oligomers of the present invention may contain one or more
acidic functional groups and, thus, are capable of forming pharmaceutically-acceptable salts with
pharmaceutically-acceptable bases. The term aceutically-acceptable salts" in these
instances refers to the relatively xic, inorganic and c base addition salts of compounds
of the present invention. These salts can likewise be ed in situ in the administration vehicle
or the dosage form manufacturing process, or by separately reacting the purified compound in its
free acid form with a suitable base, such as the hydroxide, carbonate or bicarbonate of a
pharmaceutically-acceptable metal cation, with ammonia, or with a pharmaceutically—acceptable
organic primary, secondary or ry amine. Representative alkali or alkaline earth salts include
the lithium, sodium, ium, calcium, magnesium, and aluminum salts and the like.
Representative organic amines useful for the formation of base addition salts e ethylamine,
diethylamine, ethylenediamine, ethanolamine, nolamine, piperazine and the like. (See, e. g.,
Berge et al., supra).
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Wetting agents, emulsifiers and lubricants, such as sodium lauryl sulfate and magnesium
stearate, as well as coloring agents, release agents, coating agents, sweetening, flavoring and
perfuming agents, preservatives and antioxidants can also be present in the compositions.
Examples of pharmaceutically—acceptable idants include: (1) water soluble
antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium ate, sodium
metabisulfite, sodium sulfite and the like; (2) oil—soluble antioxidants, such as ascorbyl palmitate,
butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-
tocopherol, and the like; and (3) metal chelating agents, such as citric acid, ethylenediamine
tetraacetic acid (EDTA), sorbitol, ic acid, phosphoric acid, and the like.
Formulations of the present invention include those suitable for oral, nasal, topical
ding buccal and sublingual), rectal, vaginal and/or parenteral administration. The
formulations may conveniently be presented in unit dosage form and may be prepared by any
methods well known in the art of pharmacy. The amount of active ingredient that can be
ed with a carrier material to e a single dosage form will vary depending upon the
host being treated, the particular mode of administration. The amount of active ient which
can be combined with a carrier material to produce a single dosage form will generally be that
amount of the compound which produces a therapeutic effect. Generally, out of one hundred
percent, this amount will range from about 0.1 percent to about ninety-nine t of active
ingredient, preferably from about 5 percent to about 70 percent, most preferably from about 10
percent to about 30 percent.
In certain embodiments, a formulation of the t invention comprises an excipient
selected from cyclodextrins, celluloses, liposomes, micelle forming agents, e.g., bile acids, and
ric carriers, e. g., polyesters and polyanhydrides; and an er of the t invention.
In n embodiments, an aforementioned ation renders orally bioavailable an oligomer of
the present invention.
Methods of preparing these formulations or compositions include the step of bringing into
association an oligomer of the present invention with the carrier and, optionally, one or more
accessory ingredients. In l, the formulations are prepared by uniformly and intimately
bringing into association a compound of the present invention with liquid carriers, or finely
divided solid carriers, or both, and then, if necessary, shaping the product.
Formulations of the invention suitable for oral stration may be in the form of
capsules, s, pills, tablets, lozenges (using a flavored basis, usually sucrose and acacia or
tragacanth), powders, granules, or as a solution or a suspension in an aqueous or non-aqueous
, or as an oil-in-water or water-in—oil liquid emulsion, or as an elixir or syrup, or as pastilles
(using an inert base, such as gelatin and glycerin, or sucrose and acacia) and/or as mouth washes
and the like, each containing a predetermined amount of a compound of the present invention as an
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active ingredient. An oligomer of the present invention may also be administered as a bolus,
electuary or paste.
In solid dosage forms of the invention for oral administration les, tablets, pills,
dragees, powders, es, trouches and the like), the active ingredient may be mixed with one or
more pharmaceutically-acceptable carriers, such as sodium citrate or dicalcium phosphate, and/or
any of the following: (1) fillers or extenders, such as es, lactose, sucrose, glucose, ol,
and/or silicic acid; (2) binders, such as, for example, carboxymethylcellulose, alginates, gelatin,
polyvinyl pyrrolidone, sucrose and/or acacia; (3) humectants, such as glycerol; (4) disintegrating
agents, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain
silicates, and sodium carbonate; (5) solution retarding agents, such as paraffin; (6) absorption
accelerators, such as quaternary ammonium compounds and tants, such as poloxamer and
sodium lauryl sulfate; (7) wetting , such as, for example, cetyl alcohol, glycerol
monostearate, and non-ionic surfactants; (8) ents, such as kaolin and bentonite clay; (9)
lubricants, such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium
lauryl sulfate, zinc stearate, sodium te, stearic acid, and mixtures thereof; (10) coloring
agents; and (11) controlled release agents such as vidone or ethyl cellulose. In the case of
capsules, tablets and pills, the pharmaceutical compositions may also comprise buffering agents.
Solid compositions of a similar type may also be employed as fillers in soft and hard-shelled
gelatin es using such excipients as lactose or milk sugars, as well as high molecular weight
polyethylene s and the like.
A tablet may be made by compression or molding, optionally with one or more accessory
ingredients. Compressed tablets may be prepared using binder (e.g., gelatin or
hydroxypropylmethyl cellulose), lubricant, inert diluent, preservative, disintegrant (for example,
sodium starch glycolate or cross-linked sodium carboxymethyl ose), surface—active or
dispersing agent. Molded tablets may be made by molding in a suitable machine a mixture of the
powdered compound ned with an inert liquid diluent.
The tablets, and other solid dosage forms of the pharmaceutical compositions of the
present invention, such as dragees, capsules, pills and granules, may optionally be scored or
prepared with coatings and shells, such as enteric gs and other coatings well known in the
pharmaceutical-formulating art. They may also be formulated so as to provide slow or controlled
release of the active ingredient therein using, for example, hydroxypropylmethyl cellulose in
g proportions to e the desired release profile, other polymer matrices, liposomes
and/or microspheres. They may be formulated for rapid release, e. g., freeze-dried. They may be
sterilized by, for example, filtration through a bacteria—retaining filter, or by incorporating
sterilizing agents in the form of sterile solid compositions which can be dissolved in sterile water,
or some other sterile injectable medium immediately before use. These compositions may also
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optionally contain opacifying agents and may be of a composition that they release the active
ingredient(s) only, or preferentially, in a certain n of the gastrointestinal tract, optionally, in a
delayed manner. Examples of embedding compositions which can be used include polymeric
substances and waxes. The active ingredient can also be in encapsulated form, if
appropriate, with one or more of the above-described excipients.
Liquid dosage forms for oral administration of the compounds of the invention include
pharmaceutically acceptable emulsions, mulsions, solutions, suspensions, syrups and
elixirs. In addition to the active ingredient, the liquid dosage forms may contain inert diluents
commonly used in the art, such as, for example, water or other solvents, solubilizing agents and
emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol,
benzyl benzoate, propylene glycol, 1,3—butylene glycol, oils (in particular, cottonseed, groundnut,
corn, germ, olive, castor and sesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycols
and fatty acid esters of sorbitan, and mixtures thereof.
Besides inert diluents, the oral compositions can also include adjuvants such as wetting
agents, emulsifying and suspending agents, sweetening, flavoring, coloring, ing and
preservative agents.
Suspensions, in on to the active compounds, may contain suspending agents as, for
example, lated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters,
microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, and
mixtures f.
Formulations for rectal or vaginal administration may be presented as a suppository, which
may be prepared by mixing one or more compounds of the ion with one or more suitable
nonirritating excipients or carriers comprising, for example, cocoa butter, polyethylene glycol, a
suppository wax or a salicylate, and which is solid at room temperature, but liquid at body
temperature and, therefore, will melt in the rectum or vaginal cavity and release the active
compound.
Formulations or dosage forms for the topical or transdermal administration of an oligomer
as provided herein include powders, sprays, nts, pastes, creams, s, gels, solutions,
s and inhalants. The active oligomers may be mixed under sterile conditions with a
pharmaceutically-acceptable carrier, and with any preservatives, buffers, or propellants which may
be required. The ointments, pastes, creams and gels may contain, in on to an active
compound of this invention, excipients, such as animal and vegetable fats, oils, waxes, ins,
starch, anth, cellulose tives, polyethylene glycols, silicones, bentonites, silicic acid,
talc and zinc oxide, or es thereof.
Powders and sprays can contain, in addition to an oligomer of the t invention,
excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicates and polyamide
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powder, or mixtures of these substances. Sprays can additionally contain customary propellants,
such as chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons, such as butane and
propane.
Transdermal patches have the added advantage of providing controlled ry of an
oligomer of the present invention to the body. Such dosage forms can be made by dissolving or
dispersing the oligomer in the proper medium. Absorption enhancers can also be used to increase
the flux of the agent across the skin. The rate of such flux can be controlled by either providing a
rate controlling membrane or dispersing the agent in a polymer matrix or gel, among other
methods known in the art.
Pharmaceutical compositions suitable for parenteral administration may comprise one or
more oligomers of the invention in combination with one or more pharmaceutically-acceptable
e isotonic aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, or sterile
powders which may be reconstituted into sterile injectable solutions or dispersions just prior to
use, which may contain , alcohols, idants, buffers, bacteriostats, solutes which render
the formulation isotonic with the blood of the intended recipient or suspending or thickening
agents. Examples of suitable aqueous and nonaqueous carriers which may be employed in the
pharmaceutical compositions of the invention include water, ethanol, polyols (such as glycerol,
propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils,
such as olive oil, and injectable organic esters, such as ethyl oleate. Proper fluidity can be
maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of
the required particle size in the case of dispersions, and by the use of surfactants.
These compositions may also contain adjuvants such as preservatives, wetting ,
emulsifying agents and dispersing agents. tion of the action of microorganisms upon the
subject oligomers may be ensured by the inclusion of various antibacterial and antifungal agents,
for example, n, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to
include isotonic agents, such as sugars, sodium de, and the like into the compositions. In
addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the
inclusion of agents which delay absorption such as aluminum monostearate and n.
In some cases, in order to g the effect of a drug, it is desirable to slow the absorption
of the drug from aneous or intramuscular injection. This may be accomplished by the use of
a liquid suspension of crystalline or ous material having poor water solubility, among other
methods known in the art. The rate of absorption of the drug then depends upon its rate of
dissolution which, in turn, may depend upon crystal size and crystalline form. atively,
delayed absorption of a parenterally-administered drug form is accomplished by ving or
suspending the drug in an oil vehicle.
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Inj ectable depot forms may be made by forming microencapsule matrices of the subject
oligomers in biodegradable rs such as polylactide—polyglycolide. Depending on the ratio of
oligomer to polymer, and the nature of the particular polymer employed, the rate of oligomer
release can be controlled. Examples of other biodegradable polymers include poly(orthoesters)
and poly(anhydrides). Depot injectable formulations may also prepared by entrapping the drug in
liposomes or microemulsions that are compatible with body tissues.
When the oligomers of the present invention are administered as pharmaceuticals, to
humans and animals, they can be given per se or as a pharmaceutical composition containing, for
example, 0.1 to 99% (more preferably, 10 to 30%) of active ingredient in combination with a
pharmaceutically able carrier.
As noted above, the formulations or preparations of the present invention may be given
orally, parenterally, topically, or rectally. They are typically given in forms suitable for each
administration route. For e, they are administered in tablets or capsule form, by injection,
inhalation, eye lotion, ointment, suppository, etc. stration by injection, on or
inhalation; topical by lotion or ointment; and rectal by itories.
The phrases "parenteral administration" and "administered parenterally" as used herein
means modes of administration other than enteral and l administration, usually by ion,
and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular,
intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular,
intraarticular, subcapsular, subarachnoid, pinal and intrasternal injection and infusion.
The phrases "systemic administration,n n stered systemically, n nperipheral
administration" and "administered peripherally" as used herein mean the administration of a
compound, drug or other material other than directly into the central nervous system, such that it
enters the patient‘s system and, thus, is subject to lism and other like ses, for
example, subcutaneous administration.
Regardless of the route of administration selected, the oligomers of the present invention,
which may be used in a suitable hydrated form, and/or the pharmaceutical itions of the
present invention, may be formulated into pharmaceutically—acceptable dosage forms by
conventional methods known to those of skill in the art. Actual dosage levels of the active
ients in the pharmaceutical compositions of this invention may be varied so as to obtain an
amount of the active ient which is effective to e the d therapeutic response for a
particular patient, composition, and mode of administration, without being unacceptably toxic to
the patient.
The selected dosage level will depend upon a variety of factors including the activity of
the particular er of the present invention employed, or the ester, salt or amide thereof, the
route of administration, the time of administration, the rate of excretion or metabolism of the
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particular oligomer being employed, the rate and extent of absorption, the duration of the
treatment, other drugs, compounds and/or materials used in combination with the particular
oligomer employed, the age, sex, weight, condition, l health and prior medical history of the
patient being treated, and like factors well known in the medical arts.
A physician or veterinarian having ordinary skill in the art can readily determine and
prescribe the effective amount of the pharmaceutical ition required. For example, the
physician or veterinarian could start doses of the compounds of the invention employed in the
pharmaceutical composition at levels lower than that required in order to achieve the desired
eutic effect and gradually increase the dosage until the desired effect is achieved. In general,
a suitable daily dose of a compound of the invention will be that amount of the compound which is
the lowest dose effective to produce a therapeutic effect. Such an effective dose will lly
depend upon the factors bed above. Generally, oral, intravenous, intracerebroventricular and
subcutaneous doses of the compounds of this invention for a patient, when used for the indicated
s, will range from about 0.0001 to about 100 mg per kilogram of body weight per day.
If desired, the effective daily dose of the active compound may be administered as two,
three, four, five, six or more ses administered separately at appropriate intervals throughout
the day, optionally, in unit dosage forms. In certain situations, dosing is one administration per
day. In certain embodiments, dosing is one or more administration per every 2, 3, 4, 5, 6, 7, 8, 9,
,11,12,13,14 days, or every 1, 2, 3, 4, 5, 6, 7, 8, 9,10,11,12 weeks, or every 1, 2, 3, 4, 5, 6, 7,
8, 9, 10, 11, 12 months, as needed, to maintain the desired expression of a onal dystrophin
protein.
Nucleic acid molecules can be administered to cells by a variety of methods known to
those familiar to the art, ing, but not cted to, encapsulation in liposomes, by
iontophoresis, or by incorporation into other es, such as hydrogels, cyclodextrins,
biodegradable psules, and bioadhesive microspheres, as described herein and known in the
art. In certain embodiments, microemulsification technology may be utilized to improve
bioavailability of lipophilic (water insoluble) pharmaceutical agents. Examples include Trimetrine
(Dordunoo, S. K., et al., Drug Development and Industrial Pharmacy, 17(12), 1685-1713, 1991
and REV 5901 (Sheen, P. C., et al., J Pharm Sci 80(7), 712-714, 1991). Among other benefits,
microemulsification provides enhanced bioavailability by preferentially directing absorption to the
lymphatic system d of the circulatory system, which thereby bypasses the liver, and prevents
destruction of the compounds in the hepatobiliary circulation.
In one aspect of invention, the ations contain micelles formed from an er as
provided herein and at least one amphiphilic carrier, in which the micelles have an average
diameter of less than about 100 nm. More preferred ments provide micelles having an
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e diameter less than about 50 nm, and even more preferred embodiments e micelles
having an average diameter less than about 30 nm, or even less than about 20 nm.
While all suitable amphiphilic carriers are contemplated, the presently preferred carriers
are generally those that have Generally—Recognized—as—Safe (GRAS) status, and that can both
solubilize the compound of the t invention and microemulsify it at a later stage when the
solution comes into a contact with a complex water phase (such as one found in human gastro-
intestinal . y, amphiphilic ingredients that satisfy these requirements have HLB
(hydrophilic to lipophilic balance) values of 2—20, and their structures n straight chain
aliphatic radicals in the range of C-6 to C-20. Examples are polyethylene-glycolized fatty
glycerides and polyethylene glycols.
Examples of hilic carriers include ted and monounsaturated
polyethyleneglycolyzed fatty acid glycerides, such as those obtained from fully or partially
hydrogenated various vegetable oils. Such oils may advantageously consist of tri—, di—, and monofatty
acid glycerides and di— and mono—polyethyleneglycol esters of the corresponding fatty acids,
with a particularly preferred fatty acid composition including capric acid 4-10, capric acid 3-9,
lauric acid 40-50, myristic acid 14-24, palmitic acid 4—14 and stearic acid 5-15%. Another useful
class of amphiphilic carriers includes partially esterified sorbitan and/or sorbitol, with saturated or
mono-unsaturated fatty acids (SPAN—series) or corresponding ethoxylated analogs (TWEEN-
series).
Commercially available amphiphilic carriers may be ularly useful, including
Gelucire—series, Labrafil, Labrasol, or Lauroglycol (all ctured and distributed by Gattefosse
ation, Saint Priest, ), PEG-mono—oleate, PEG-di-oleate, PEG-mono-laurate and di-
laurate, Lecithin, Polysorbate 80, etc (produced and distributed by a number of ies in USA
and worldwide).
In certain embodiments, the ry may occur by use of liposomes, nanocapsules,
microparticles, microspheres, lipid particles, vesicles, and the like, for the introduction of the
compositions of the present invention into suitable host cells. In particular, the compositions of
the present invention may be formulated for delivery either encapsulated in a lipid particle, a
liposome, a vesicle, a nanosphere, a nanoparticle or the like. The formulation and use of such
delivery es can be carried out using known and conventional techniques.
Hydrophilic polymers suitable for use in the t invention are those which are readily
water-soluble, can be covalently attached to a vesicle—forming lipid, and which are tolerated in
vivo without toxic effects (i.e., are biocompatible). Suitable polymers include polyethylene glycol
(PEG), polylactic (also termed polylactide), polyglycolic acid (also termed ycolide), a
polylactic—polyglycolic acid copolymer, and polyvinyl alcohol. In n embodiments, polymers
have a molecular weight of from about 100 or 120 daltons up to about 5,000 or 10,000 daltons, or
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from about 300 daltons to about 5,000 daltons. In other embodiments, the polymer is
polyethyleneglycol having a molecular weight of from about 100 to about 5,000 daltons, or having
a molecular weight of from about 300 to about 5,000 daltons. In certain embodiments, the
polymer is polyethyleneglycol of 750 daltons (PEG(750)). Polymers may also be defined by the
number of monomers therein; a preferred embodiment of the present invention utilizes polymers of
at least about three monomers, such PEG polymers consisting of three monomers (approximately
150 daltons).
Other hydrophilic polymers which may be suitable for use in the present invention include
polyvinylpyrrolidone, thoxazoline, polyethyloxazoline, droxypropyl
methacrylamide, polymethacrylamide, polydimethylacrylamide, and derivatized celluloses such as
hydroxymethylcellulose or hydroxyethylcellulose.
In certain embodiments, a formulation of the present invention comprises a biocompatible
polymer selected from the group consisting of polyamides, polycarbonates, polyalkylenes,
polymers of acrylic and methacrylic esters, polyvinyl polymers, polyglycolides, polysiloxanes,
polyurethanes and co-polymers f, celluloses, opylene, hylenes, yrene,
polymers of lactic acid and ic acid, polyanhydrides, poly(ortho)esters, poly(butic acid),
poly(valeric acid), poly(lactide—co—caprolactone), polysaccharides, proteins, aluronic acids,
polycyanoacrylates, and blends, mixtures, or copolymers thereof.
Cyclodextrins are cyclic oligosaccharides, consisting of 6, 7 or 8 glucose units, ated
by the Greek letter a, B, or y, respectively. The glucose units are linked by a—1,4—glucosidic bonds.
As a consequence of the chair conformation of the sugar units, all secondary hydroxyl groups (at
C-2, C-3) are located on one side of the ring, while all the primary hydroxyl groups at C-6 are
situated on the other side. As a result, the al faces are hydrophilic, making the cyclodextrins
water-soluble. In contrast, the cavities of the cyclodextrins are hydrophobic, since they are lined
by the hydrogen of atoms C—3 and C—5, and by ether—like oxygens. These es allow
complexation with a variety of relatively hydrophobic compounds, including, for instance, d
compounds such as 17a-estradiol (see, e.g., van Uden et al. Plant Cell Tiss. Org. Cult. -113
(1994)). The complexation takes place by Van der Waals interactions and by hydrogen bond
formation. For a general review of the chemistry of cyclodextrins, see, Wenz, Agnew. Chem. Int.
Ed. Engl., 332803-822 .
The physico-chemical properties of the cyclodextrin derivatives depend strongly on the
kind and the degree of substitution. For example, their solubility in water ranges from insoluble
(e.g., triacetyl-beta-cyclodextrin) to 147% soluble (w/v) (Gbeta-cyclodextrin). In on, they
are soluble in many organic solvents. The ties of the cyclodextrins enable the control over
solubility of various formulation components by increasing or decreasing their solubility.
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Numerous cyclodextrins and methods for their preparation have been described. For
e, Parmeter (I), et al. (US. Pat. No. 3,453,259) and Gramera, et al. (US. Pat. No.
3,459,731) described electroneutral cyclodextrins. Other tives include cyclodextrins With
cationic properties [Parmeter (II), US. Pat. No. 257], insoluble crosslinked cyclodextrins
(Solms, US. Pat. No. 3,420,788), and cyclodextrins with anionic properties ter (111), US.
Pat. No. 3,426,011]. Among the cyclodextrin tives with anionic properties, carboxylic acids,
phosphorous acids, inous acids, phosphonic acids, phosphoric acids, thiophosphonic acids,
thiosulphinic acids, and sulfonic acids have been appended to the parent cyclodextrin [see,
Parmeter (III), supra]. Furthermore, sulfoalkyl ether cyclodextrin derivatives have been described
by , et al. (US. Pat. No. 5,134,127).
Liposomes consist of at least one lipid r ne enclosing an aqueous internal
compartment. Liposomes may be characterized by membrane type and by size. Small unilamellar
vesicles (SUVs) have a single membrane and lly range between 0.02 and 0.05 um in
diameter; large unilamellar vesicles (LUVS) are typically larger than 0.05 um. Oligolamellar large
vesicles and multilamellar vesicles have multiple, usually concentric, membrane layers and are
typically larger than 0.1 um. Liposomes with several nonconcentric membranes, i.e., several
smaller vesicles contained Within a larger vesicle, are termed multivesicular vesicles.
One aspect of the present invention relates to formulations comprising liposomes
containing an oligomer of the present invention, where the liposome membrane is formulated to
provide a liposome with increased carrying capacity. atively or in addition, the compound
of the present invention may be contained within, or adsorbed onto, the liposome bilayer of the
liposome. An oligomer of the present invention may be aggregated With a lipid surfactant and
carried Within the liposome's al space; in these cases, the liposome membrane is formulated
to resist the disruptive effects of the active agent—surfactant ate.
According to one embodiment of the t invention, the lipid bilayer of a liposome
contains lipids derivatized with polyethylene glycol (PEG), such that the PEG chains extend from
the inner surface of the lipid bilayer into the interior space ulated by the liposome, and
extend from the exterior of the lipid bilayer into the surrounding environment.
Active agents contained Within liposomes of the present invention are in solubilized form.
Aggregates of surfactant and active agent (such as emulsions or micelles containing the active
agent of st) may be entrapped within the interior space of liposomes according to the present
invention. A surfactant acts to disperse and solubilize the active agent, and may be selected from
any suitable aliphatic, cycloaliphatic or aromatic surfactant, including but not limited to
biocompatible lysophosphatidylcholines (LPGs) of varying chain lengths (for example, from about
C14 to about C20). Polymer—derivatized lipids such as PEG—lipids may also be utilized for micelle
formation as they Will act to inhibit micelle/membrane fusion, and as the on of a polymer to
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surfactant molecules ses the CMC of the tant and aids in e ion. Preferred
are surfactants with CMOs in the micromolar range; higher CMC surfactants may be ed to
prepare micelles entrapped within liposomes of the t invention.
Liposomes according to the present invention may be prepared by any of a variety of
techniques that are known in the art. See, e.g., US. Pat. No. 4,235,871; Published PCT
applications W0 96/14057; New RRC, Liposomes: A cal approach, IRL Press, Oxford
(1990), pages 33—104; Lasic DD, Liposomes from physics to applications, Elsevier Science
Publishers BV, Amsterdam, 1993. For example, liposomes of the present invention may be
prepared by diffusing a lipid derivatized with a hydrophilic polymer into preformed liposomes,
such as by exposing preformed liposomes to micelles composed of grafted polymers, at lipid
concentrations corresponding to the final mole percent of derivatized lipid which is desired in the
liposome. Liposomes containing a hydrophilic polymer can also be formed by homogenization,
lipid-field hydration, or extrusion techniques, as are known in the art.
In another exemplary formulation procedure, the active agent is first sed by
sonication in a lysophosphatidylcholine or other low CMC surfactant (including polymer grafted
lipids) that y solubilizes hydrophobic molecules. The resulting micellar suspension of active
agent is then used to rehydrate a dried lipid sample that contains a suitable mole percent of
polymer-grafted lipid, or cholesterol. The lipid and active agent suspension is then formed into
liposomes using extrusion techniques as are known in the art, and the resulting liposomes
separated from the unencapsulated solution by standard column separation.
In one aspect of the present invention, the liposomes are prepared to have substantially
homogeneous sizes in a ed size range. One effective sizing method involves ing an
aqueous suspension of the liposomes h a series of polycarbonate membranes having a
selected uniform pore size; the pore size of the ne will correspond roughly with the largest
sizes of liposomes produced by extrusion through that membrane. See e. g., US. Pat. No.
4,737,323 (Apr. 12, 1988). In certain embodiments, reagents such as DharmaFECT® and
Lipofectamine® may be ed to introduce polynucleotides or proteins into cells.
The release characteristics of a formulation of the present invention depend on the
encapsulating material, the concentration of encapsulated drug, and the presence of release
modifiers. For example, release can be manipulated to be pH dependent, for example, using a pH
sensitive coating that releases only at a low pH, as in the stomach, or a higher pH, as in the
ine. An enteric coating can be used to prevent release from occurring until after passage
through the stomach. Multiple coatings or mixtures of cyanamide encapsulated in different
materials can be used to obtain an initial release in the stomach, followed by later release in the
intestine. Release can also be manipulated by inclusion of salts or pore forming , which can
increase water uptake or release of drug by ion from the capsule. Excipients which modify
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the solubility of the drug can also be used to control the release rate. Agents which enhance
degradation of the matrix or release from the matrix can also be incorporated. They can be added
to the drug, added as a separate phase (i.e., as particulates), or can be co—dissolved in the polymer
phase depending on the compound. In most cases the amount should be between 0.1 and thirty
percent (w/w polymer). Types of ation ers include inorganic salts such as
ammonium sulfate and ammonium chloride, organic acids such as citric acid, benzoic acid, and
ascorbic acid, inorganic bases such as sodium carbonate, potassium carbonate, calcium carbonate,
Zinc carbonate, and zinc hydroxide, and organic bases such as protamine sulfate, spermine,
choline, ethanolamine, diethanolamine, and triethanolamine and tants such as Tween® and
Pluronic®. Pore forming agents which add tructure to the matrices (i.e., water soluble
compounds such as inorganic salts and ) are added as particulates. The range is typically
n one and thirty percent (w/w polymer).
Uptake can also be manipulated by altering residence time of the particles in the gut. This
can be achieved, for e, by g the particle with, or selecting as the encapsulating
material, a mucosal adhesive polymer. Examples include most polymers with free carboxyl
, such as chitosan, celluloses, and ally rylates (as used herein, polyacrylates
refers to polymers including acrylate groups and modified acrylate groups such as cyanoacrylates
and methacrylates).
An oligomer may be formulated to be contained within, or, adapted to release by a surgical
or medical device or implant. In certain aspects, an implant may be coated or ise treated
with an oligomer. For example, hydrogels, or other polymers, such as patible and/or
biodegradable polymers, may be used to coat an implant with the compositions of the present
invention (i.e., the composition may be adapted for use with a medical device by using a hydrogel
or other polymer). Polymers and copolymers for coating medical devices with an agent are well—
known in the art. Examples of implants include, but are not limited to, stents, drug-eluting stents,
sutures, prosthesis, vascular catheters, dialysis catheters, vascular grafts, prosthetic heart valves,
cardiac pacemakers, implantable cardioverter defibrillators, IV needles, devices for bone setting
and formation, such as pins, screws, plates, and other devices, and artificial tissue matrices for
wound healing.
In addition to the methods provided herein, the oligomers for use according to the
invention may be formulated for administration in any convenient way for use in human or
veterinary medicine, by analogy with other pharmaceuticals. The nse oligomers and their
corresponding formulations may be administered alone or in combination with other therapeutic
strategies in the treatment of ar dystrophy, such as myoblast transplantation, stem cell
therapies, administration of aminoglycoside antibiotics, proteasome inhibitors, and ulation
therapies (e.g., upregulation of utrophin, an autosomal paralogue of dystrophin).
AVN-013BPC
The routes of stration described are intended only as a guide since a d
practitioner will be able to determine readily the m route of administration and any dosage
for any particular animal and condition. Multiple approaches for introducing functional new
genetic material into cells, both in Vitro and in vivo have been attempted (Friedmann (1989)
Science, 75-1280). These approaches include integration of the gene to be expressed into
ed retroviruses (Friedmann (1989) supra; Rosenberg (1991) Cancer ch 51(18), suppl.:
5074S—5079S); integration into non—retrovirus vectors (e.g., adeno-associated viral vectors)
(Rosenfeld, et al. (1992) Cell, 68:143—155; Rosenfeld, et al. (1991) Science, 252:431-434); or
delivery of a transgene linked to a heterologous promoter—enhancer element via liposomes
(Friedmann (1989), supra; Brigham, et al. (1989) Am. J. Med. Sci., 298:278—281; Nabel, et al.
(1990) Science, 249:1285—1288; Hazinski, et al. (1991) Am. J. Resp. Cell Molec. Biol., 4:206-209;
and Wang and Huang (1987) Proc. Natl. Acad. Sci. (USA), 84:7851-7855); coupled to ligand-
specific, cation-based transport systems (Wu and Wu (1988) J. Biol. Chem., 263: 14621-14624) or
the use of naked DNA, expression vectors (Nabel et al. , supra); Wolff et al. (1990) Science,
247: 468). Direct injection of transgenes into tissue produces only localized expression
(Rosenfeld (1992) supra); Rosenfeld et al. (1991) supra; Brigham et al. (1989) supra; Nabel (1990)
supra; and Hazinski et al. (1991) supra). The Brigham et al. group (Am. J. Med. Sci. (1989)
298:278-281 and Clinical Research (1991) 39 (abstract)) have reported in vivo transfection only of
lungs of mice following either intravenous or intratracheal administration of a DNA liposome
complex. An example of a review article of human gene y procedures is: Anderson, Science
(1992) 256:808—813.
IV. Kits
The invention also provides kits for treatment of a patient with a genetic disease which kit
comprises at least an antisense molecule (e.g., an antisense oligomer set forth in SEQ ID NOs: 1-
12, 46, and 47), packaged in a suitable container, together with instructions for its use. The kits
may also contain eral reagents such as buffers, stabilizers, etc. Those of ordinary skill in the
field should appreciate that ations of the above method has wide ation for identifying
antisense molecules suitable for use in the treatment of many other diseases.
V. Examples
Although the foregoing invention has been described in some detail by way of illustration
and example for purposes of clarity of understanding, it will be readily apparent to one of ordinary
skill in the art in light of the teachings of this invention that n changes and modifications may
be made thereto without departing from the spirit or scope of the appended . The following
AVN-013BPC
examples are provided by way of ration only and not by way of limitation. Those of skill in
the art will readily recognize a variety of noncritical parameters that could be changed or modified
to yield essentially similar results.
Materials and Methods
Cells and Tissue Culture Treatment Conditions
Human Rhabdomyosarcoma cells (ATCC, CCL-136; RD cells) were seeded into tissue
culture-treated T75 flasks (Nunc) at 1.5 x 106 cells/flask in 24 mL of warmed DMEM with L-
Glutamine (HyClone), 10% fetal bovine serum, and 1% Penicillin—Streptomycin antibiotic solution
(CelGro); after 24 hours, media was aspirated, cells were washed once in warmed PBS, and fresh
media was added. Cells were grown to 80% confluence in a 37°C incubator at 5.0% C02 and
ted using trypsin. Lyophilized phosphorodiamidate morpholino oligomers (PMOs) were re-
suspended at imately 0.5 to 2.0 mM in nuclease—free water; to verify molarity, PMO
solutions were ed using a NanoDrop 2000 spectrophotometer (Thermo Scientific). PMOs
were delivered to RD cells using nucleoporation according to the manufacturer’s instructions and
the SG kit ). PMOs were tested at various concentrations as indicated (e.g., 2.5, 5, 10, 12.5,
and 25 micromolar). Cells were incubated for 24 hours post nucleoporation at approximately 2
- 3 X 105 cells per well of a 12 or 24-well plate (n=2 or 3) and then subjected to RNA extraction as
described below.
Primary human myoblasts were cultured in Skeletal Muscle Cell Growth Media
(PromoCell) using standard techniques. Nucleoporation of the PMOs at various concentrations
was done as described for RD cells above. Cells were then plated in triplicate wells of a 12-well
plate in PromoCell growth media and allowed to incubate for 24 hours before RNA extraction as
described below.
RNA Extraction and PCR Amplification
RNA was extracted from eated cells (RD cells or primary human myoblasts) using
the n 96 well RNA isolation kit from GE care and subjected to either nested or
unnested RT-PCR using standard techniques and the following primer pairs. Outer primers:
d 5’-CAATGCTCCTGACCTCTGTGC —3’ (SEQ ID NO: 40), reverse 5’—
GCTCTTTTCCAGGTTCAAGTGG —3’ (SEQ ID NO: 41); inner primers: forward 5’-
AACAAAGCTCAGGTCG -3’ (SEQ ID NO: 42), reverse 5’-
GCAATGTTATCTGCTTCCTCCAACC —3’ (SEQ ID NO: 43). In some cases, unnested PCR was
performed using the inner primers. Exon skipping was measured by gel electrophoresis or by
AVN-013BPC
using the Caliper LabChip bioanalyzer and the % exon skipping (i.e., band intensity of the exon-
skipped product relative to the full length PCR product) was graphed as shown in FIGS 3 to 6.
Pre aration of Mor holino Subunits PMO and PMO with Modified Intersubunit
Linkages
B O
o 1. NalO4, MeoH (aq) HO
2- B4O7
3. Borane-triethylamine
4. Methanolic acid (p-TsOH /\
HO OH or HCI) H H
1 2
3 R2 i
IF! \ /p
N/ \
1_ 1 Z / Cl ,N /\Cl
R3 CI
4a 4b
X B
R2 X B
0 \ || 0
N Lo N—P—o
R1—L1 Z | / |
Cl R3 CI
T T
PG PG
5a 5b
Scheme 1: General synthetic route to PMO and ed—PMO Subunits
Referring to Reaction Scheme 1, wherein B represents a base pairing moiety and
PG represents a protecting group, the morpholino subunits may be prepared from the
corresponding ribinucleoside (1) as shown. The morpholino subunit (2) may be optionally
ted by reaction with a suitable protecting group precursor, for example trityl chloride. The
AVN-013BPC
3’ protecting group is generally removed during solid—state oligomer synthesis as described in
more detail below. The base pairing moiety may be suitably protected for solid-phase oligomer
sis. Suitable protecting groups include benzoyl for adenine and cytosine, acetyl for
guanine, and pivaloyloxymethyl for hypoxanthine (I). The yloxymethyl group can be
introduced onto the N1 position of the hypoxanthine heterocyclic base. Although an ected
hypoxanthine subunit, may be employed, yields in activation reactions are far superior when the
base is protected. Other suitable protecting groups include those disclosed in U.S. Patent No.
8,076,476, which is hereby incorporated by reference in its entirety.
Reaction of 3 with the activated phosphorous compound 4a or 4b results in
morpholino subunits having the desired linkage moiety (Sa or 5b). It should be noted that the R1
and/or L1 moieties may also be installed on the heterocyclic ring Z after ion of the P-O bond
or even after the subunit has been incorporated into an oligomer.
Compounds of structure 421 or 4b can be ed using any number of methods
known to those of skill in the art, including those bed in the examples. Coupling with the
morpholino moiety then proceeds as outlined above.
Compounds of structure 5a or 5b can be used in solid-phase oligomer synthesis
for ation of oligomers comprising the intersubunit linkages. Such methods are well known
in the art. Briefly, a compound of ure 5a or 5b may be modified at the 5’ end to contain a
linker to a solid support. Once supported, the protecting group of 5a or 5b (e. g., trityl at 3’—end))
is removed and the free amine is reacted with an activated phosphorous moiety of a second
compound of structure 5a or 5b (or analogue thereof). This sequence is repeated until the desired
length oligo is obtained. The ting group in the terminal 3’ end may either be removed or left
on if a 3’ modification is desired. The oligo can be removed from the solid support using any
number of methods, or example treatment with a base to cleave the e to the solid support.
The preparation of morpholino oligomers in general and specific morpholino
oligomers of the invention are described in more detail in the Examples.
Preparation of Morpholino Oligomers
The preparation of the compounds of the invention are performed using the following
protocol:
AVN-013BPC
Preparation of trityl zine phenyl ate 35 ( and 2B): To a
cooled suspension of compound 11 in dichloromethane (6 mL/g 11) was added a solution of
potassium carbonate (3.2 eq) in water (4 mL/g potassium carbonate). To this two-phase mixture
was slowly added a solution of phenyl chloroformate (1.03 eq) in dichloromethane (2 g/g phenyl
chloroformate). The reaction mixture was warmed to 20 °C. Upon reaction completion (1-2 hr),
the layers were separated. The c layer was washed with water, and dried over anhydrous
ium carbonate. The product 35 was isolated by crystallization from acetonitrile.
Preparation of carbamate alcohol 36: Sodium hydride (1.2 eq) was suspended in 1-
methyl—2—pyrrolidinone (32 mL/g sodium hydride). To this suspension were added triethylene
glycol (10.0 eq) and compound 35 (1.0 eq). The resulting slurry was heated to 95 °C. Upon
reaction completion (1-2 hr), the mixture was cooled to 20 0C. To this mixture was added 30%
dichloromethane/methyl tert—butyl ether (vzv) and water. The product-containing organic layer
was washed successively with aqueous NaOH, aqueous succinic acid, and saturated aqueous
sodium chloride. The product 36 was isolated by crystallization from dichloromethane/methyl
utyl ether/heptane.
Preparation of Tail acid 37: To a solution of compound 36 in tetrahydrofuran (7
mL/g 36) was added succinic ide (2.0 eq) and DMAP (0.5 eq). The mixture was heated to
50 OC. Upon reaction tion (5 hr), the mixture was cooled to 20 °C and adjusted to pH 8.5
with aqueous NaHCO3. Methyl tert-butyl ether was added, and the product was extracted into the
aqueous layer. Dichloromethane was added, and the mixture was ed to pH 3 with aqueous
citric acid. The product-containing c layer was washed with a mixture of pH=3 citrate
buffer and saturated aqueous sodium chloride. This romethane solution of 37 was used
without isolation in the preparation of compound 38.
Preparation of 38: To the solution of nd 37 was added N—hydroxy
norbornene—2,3—dicarboxylic acid imide (HONB) (1.02 eq), 4-dimethylaminopyridine (DMAP)
(0.34 eq), and then 1-(3-dimethylaminopropyl)—N'—ethylcarbodiimide hydrochloride (EDC) (1.1
eq). The mixture was heated to 55 0C. Upon reaction completion (4—5 hr), the mixture was cooled
to 20 °C and washed successively with 1:1 0.2 M citric acid/brine and brine. The dichloromethane
solution underwent solvent exchange to acetone and then to N,N—dimethylformamide, and the
product was ed by precipitation from acetone/ N,N—dimethylformamide into saturated
AVN-013BPC
aqueous sodium chloride. The crude product was reslurried l times in water to remove
al N,N-dimethylformamide and salts.
Introduction of the activated “Tail” onto the anchor-loaded resin was performed
in dimethyl imidazolidinone (DMI) by the procedure used for incorporation of the subunits during
solid phase synthesis.
Preparation of the Solid Support for Synthesis of Morpholino Oligomers: This
procedure was performed in a silanized, jacketed peptide vessel (ChemGlass, NJ, USA) with a
coarse ty (40-60 pm) glass frit, overhead stirrer, and 3-way Teflon stopcock to allow N2 to
bubble up through the frit or a vacuum extraction.
The resin treatment/wash steps in the following procedure consist of two basic
operations: resin fluidization or stirrer bed reactor and solvent/solution extraction. For resin
fluidization, the stopcock was positioned to allow N2 flow up through the frit and the specified
resin treatment/wash was added to the reactor and allowed to te and completely wet the
resin. Mixing was then d and the resin slurry mixed for the specified time. For
solvent/solution extraction, mixing and N2 flow were stopped and the vacuum pump was started
and then the stopcock was positioned to allow evacuation of resin treatment/wash to waste. All
resin treatment/wash volumes were 15 mL/g of resin unless noted otherwise.
To aminomethylpolystyrene resin (100-200 mesh; ~1.0 mmol/g load based on
nitrogen substitution; 75 g, 1 eq, Polymer Labs, UK, part #1464—X799) in a silanized, jacketed
e vessel was added 1—methyl—2—pyrrolidinone (NMP; 20 ml/g resin) and the resin was
allowed to swell with mixing for 1-2 hr. Following evacuation of the swell solvent, the resin was
washed with dichloromethane (2 x 1—2 min), 5% diisopropylethylamine in 25%
isopropanol/dichloromethane (2 x 3-4 min) and dichloromethane (2 x 1-2 min). After evacuation
of the final wash, the resin was treated with a on of disulfide anchor 34 in 1—methyl
pyrrolidinone (0.17 M; 15 mL/g resin, ~2.5 eq) and the resin/reagent mixture was heated at 45 0C
for 60 hr. On reaction tion, heating was discontinued and the anchor solution was
evacuated and the resin washed with 1—methyl—2—pyrrolidinone (4 x 3—4 min) and romethane
(6 x 1-2 min). The resin was treated with a solution of 10% (v/v) diethyl dicarbonate in
dichloromethane (16 mL/g; 2 x 5-6 min) and then washed with dichloromethane (6 x 1-2 min).
The resin 39 (see was dried under a N2 stream for 1—3 hr and then under vacuum to
constant weight (i 2%). Yield: 110-150% of the original resin weight.
AVN-013BPC
Determination of the Loading of Aminomethylpolystyrene—disulfide resin: The
loading of the resin (number of ially available reactive sites) is determined by a
spectrometric assay for the number of triphenylmethyl (trityl) groups per gram of resin.
A known weight of dried resin (25 i 3 mg) is transferred to a silanized 25 ml
volumetric flask and ~5 mL of 2% (V/V) trifluoroacetic acid in romethane is added. The
contents are mixed by gentle swirling and then allowed to stand for 30 min. The volume is
brought up to 25 mL with additional 2% (v/v) roacetic acid in dichloromethane and the
contents thoroughly mixed. Using a positive displacement pipette, an aliquot of the -
ning solution (500 uL) is transferred to a 10 mL volumetric flask and the volume brought up
to 10 mL with methanesulfonic acid.
The trityl cation content in the final solution is measured by UV absorbance at
431.7 nm and the resin loading calculated in trityl groups per gram resin (umol/g) using the
appropriate volumes, dilutions, extinction coefficient (8: 41 umol-lcm—l) and resin weight. The
assay is performed in triplicate and an average loading calculated.
The resin loading procedure in this example will provide resin with a loading of
imately 500 umol/g. A loading of 300—400 in umol/g was obtained if the ide anchor
incorporation step is performed for 24 hr at room temperature.
Tail loading: Using the same setup and volumes as for the preparation of
aminomethylpolystyrene-disulfide resin, the Tail can be introduced into solid support. The anchor
loaded resin was first deprotected under acidic condition and the ing material neutralized
before coupling. For the ng step, a solution of 38 (0.2 M) in DMI containing 4-
ethylmorpholine (NEM, 0.4 M) was used instead of the disulfide anchor solution. After 2 hr at 45
0C, the resin 39 was washed twice with 5% diisopropylethylamine in 25%
isopropanol/dichloromethane and once with DCM. To the resin was added a solution of benzoic
anhydride (0.4 M) and NEM (0.4 M). After 25 min, the reactor jacket was cooled to room
temperature, and the resin washed twice with 5% diisopropylethylamine in 25%
isopropanol/dichloromethane and eight times with DCM. The resin 40 was filtered and dried
under high vacuum. The loading for resin 40 is defined to be the g of the original
aminomethylpolystyrene-disulfide resin 39 used in the Tail loading.
Solid Phase Synthesis: lino Oligomers were prepared on a Gilson AMS-
422 Automated Peptide Synthesizer in 2 mL Gilson polypropylene reaction columns (Part #
AVN-013BPC
3980270). An aluminum block with channels for water flow was placed around the columns as
they sat on the synthesizer. The AMS—422 will alternatively add reagent/wash solutions, hold for a
ied time, and evacuate the columns using vacuum.
For oligomers in the range up to about 25 subunits in length,
aminomethylpolystyrene-disulfide resin with loading near 500 umol/g of resin is preferred. For
larger oligomers, aminomethylpolystyrene—disulfide resin with loading of 300-400 umol/g of resin
is red. If a molecule with 5’—Tail is desired, resin that has been loaded with Tail is chosen
with the same loading guidelines.
The ing t solutions were prepared:
ylation Solution: 10% Cyanoacetic Acid (w/V) in 4:1
dichloromethane/acetonitrile; Neutralization Solution: 5% Diisopropylethylamine in 3:1
dichloromethane/isopropanol; ng Solution: 0.18 M (or 0.24 M for oligomers having grown
longer than 20 subunits) activated Morpholino Subunit of the desired base and linkage type and 0.4
M N ethylmorpholine, in 1,3-dimethylimidazolidinone. Dichloromethane (DCM) was used as a
transitional wash separating the different t solution washes.
On the synthesizer, with the block set to 42 0C, to each column containing 30 mg
of aminomethylpolystyrene—disulfide resin (or Tail resin) was added 2 mL of 1-methyl
pyrrolidinone and allowed to sit at room temperature for 30 min. After washing with 2 times 2 mL
of dichloromethane, the following synthesis cycle was employed:
Step Volume Delivery Hold time
Detritylation 1.5 mL Manifold 15 s
Detritylation 1.5 mL Manifold 15 seconds
ylation 1.5 mL Manifold 15 seconds
ylation 1.5 mL Manifold 15 seconds
Detritylation 1.5 mL Manifold 15 seconds
Detritylation 1.5 mL Manifold 15 seconds
Detritylation 1.5 mL Manifold 15 seconds
DCM 1.5 mL Manifold 30 seconds
Neutralization 1.5 mL Manifold 30 seconds
Neutralization 1.5 mL Manifold 30 seconds
Neutralization 1.5 mL Manifold 30 seconds
AVN-013BPC
Neutralization 1.5 mL Manifold 30 seconds
Neutralization 1.5 mL Manifold 30 seconds
Neutralization 1.5 mL Manifold 30 seconds
DCM 1.5 mL Manifold 30 seconds
ng 350—500uL Syringe 40 minutes
DCM 1.5 mL Manifold 30 seconds
Neutralization 1.5 mL ld 30 seconds
Neutralization 1.5 mL Manifold 30 seconds
DCM 1.5 mL Manifold 30 seconds
DCM 1.5 mL Manifold 30 seconds
DCM 1.5 mL Manifold 30 seconds
The sequences of the individual oligomers were programmed into the synthesizer
so that each column receives the proper coupling solution (A,C,G,T,I) in the proper sequence.
When the oligomer in a column had completed incorporation of its final t, the column was
removed from the block and a final cycle performed manually with a coupling solution comprised
of oxytriphenylmethyl de (0.32 M in DMI) containing 0.89 M 4-ethylmorpholine.
Cleavage from the resin and removal of bases and backbone protecting groups:
After methoxytritylation, the resin was washed 8 times with 2 mL 1—methyl—2—pyrrolidinone. One
mL of a cleavage solution consisting of 0.1 M 1,4—dithiothreitol (DTT) and 0.73 M triethylamine
in l-methylpyrrolidinone was added, the column capped, and allowed to sit at room temperature
for 30 min. After that time, the solution was drained into a 12 mL Wheaton vial. The greatly
shrunken resin was washed twice with 300 uL of cleavage solution. To the solution was added 4.0
mL conc. aqueous ammonia (stored at —20 0C), the vial capped tightly (with Teflon lined screw
cap), and the mixture swirled to mix the solution. The vial was placed in a 45 OC oven for 16-24
hr to effect cleavage of base and ne protecting .
Crude product purification: The vialed ammonolysis solution was removed from
the oven and allowed to cool to room temperature. The solution was diluted with 20 mL of 0.28%
aqueous ammonia and passed h a 2.5x10 cm column containing Macroprep HQ resin
(BioRad). A salt gradient (A: 0.28% a with B: 1 M sodium chloride in 0.28% ammonia;
AVN-013BPC
0-100% B in 60 min) was used to elute the methoxytrityl ning peak. The combined fractions
were pooled and further processed depending on the desired product.
Demethoxytritylation of Morpholino Oligomers: The pooled fractions from the
Macroprep purification were treated with 1 M H3PO4 to lower the pH to 2.5. After initial mixing,
the s sat at room temperature for 4 min, at which time they are neutralized to pH 10-11 with
2.8% a/water. The products were purified by solid phase extraction (SPE).
SPE column packing and conditioning: Amberchrome CG-300M (Rohm and
Haas; Philadelphia, PA) (3 mL) is packed into 20 mL fritted columns (BioRad Econo-Pac
Chromatography s (732—1011)) and the resin rinsed with 3 mL of the following: 0.28%
NH4OH/80% acetonitrile; 0.5M NaOH/20%ethanol; water; 50 mM H3PO4/80% acetonitrile;
water; 0.5 NaOH/20% ethanol; water; 0.28% NH4OH.
SPE cation: The solution from the demethoxytritylation was loaded onto the
column and the resin rinsed three times with 3—6 mL 0.28% aqueous ammonia. A Wheaton Vial
(12 mL) was placed under the column and the product eluted by two washes with 2 mL of 45%
acetonitrile in 0.28% aqueous ammonia.
Product isolation: The solutions were frozen in dry ice and the Vials placed in a
freeze dryer to produce a fluffy white powder. The samples were ved in water, filtered
through a 0.22 micron filter (Pall Life Sciences, sc 25 mm syringe filter, with a 0.2 micron
HT Tuffryn membrane) using a syringe and the Optical Density (OD) was measured on a UV
spectrophotometer to determine the OD units of oligomer present, as well as dispense sample for
analysis. The solutions were then placed back in Wheaton Vials for lyophilization.
Analysis of Morpholino ers by MALDI: TOF mass spectrometry
was used to determine the composition of fractions in purifications as well as provide evidence for
identity (molecular weight) of the Oligomers. Samples were run following dilution with solution
of 3,5—dimethoxy—4—hydroxycinnamic acid (sinapinic acid), trihydoxyacetophenone (THAP)
or alpha-cyanohydoxycinnarnic acid (HCCA) as matrices.
AVN-013BPC
Using the protocol described in Example 1, the following PMO was sized, NG0391
H44A(-8+15), SEQ ID NO: 4 (5’-GAT CTG TCA AAT CGC CTG CAG GT-3’) and used in
Examples 8 and 9.
Elfireak A Ereak B Bre:ak C
: i ' NMe2
' NIVIe2 A u/NMez A F"/
O O O A ./ ,P a
Y \/\O/\/ \/\OH O;P\o O’ \O O \O
N G
T o G o
T N N
/NMez “Flip I ,0 A
(DA/(PK /AP<
O M N/P‘o
MezN/ 0 MezN 0 e2
KEOJ/G Kon/C KEOD/C KEOD/G
T T T N
1’0 (O A ’O
A ’0
, P P,\ P/\
M 2N’P\o MezN \O MeZN/ O MezN O
C T
of Yr" Yr Yr
N N
T T Al 0 H
A ‘0 “P”O . 3
P\ Me N’ ‘o MezN o
MezN O 2
K6)” A T [ 3 ]
03’ 03’
N T
A to
A Lo f :0
M 2N, \O MezN O
Me N/F'\O2 KEOD/C OJ/A Kfioj/G
N N
/\ [1,0 A 60 :P30
MeZN’ \O MezN’P‘o M 2N O
Oj/T KEO‘J/T KEQD/C
N N, N
A ,0 O A
A [’0
-P{ E P
MezN o MezN/P o MSZN/ ‘o
’6 K103” *6)”
N N N
éreak A Break B Brea'k C
NH2 NH2 0 o
N NH
N \N NH
A: (NI I
_
J C= I NAG G: <’ | T—m U—_ l
/ /N NANHg
| rTl’go
A The stereochemistry of the phosphorous center is not defined
AVN-013BPC
Example 3
Using the ol described in Example 1, the following PMO was synthesized, NG0392
H44A(-7+15), SEQ ID NO: 5 (5’- GAT CTG TCA AAT CGC CTG CAG G-3’) and used as
described in Examples 8 and 9.
Ereak A I?reak B Bre:ak C
oYowow \/\OH Ogé<ONMe2 ng<:Me2 Ogé<ZMe2
EN] [5‘ Yr Yrs Yre
T N
N N
OA‘P<:M92 AFLEO /“p20 MEZNLPES
ofWe) {0L40 more\—§O Ho.—.\—<O
N N
A ,,o FLO /P30
MezN’ ‘o MezN/ \0 MezN O
‘5’“ ‘6’)” KEY
T T T,o
A ,o " $0 AP”
P’ ,P\ Me N’ ‘o
MezN/ \o MezN o 2
RE? T
Oj/T KEOj/A
T To
A $0 40 CP"
/P\O MezN’P\O MeZN \O
MezN
of ofG of
N N N
A LO A $0
MezN’P o< 4o
MezN’P‘o ,R
MezN o
01/ ‘6? Y)”
N N’ N
" $0 A ¢O "P’ro
MegN'P\O /P‘o MezN/ \
NH2 NH2 0 O
A=’f\NN \ c=fk N
\N NH
G=<’| T—mU—I_ 5
<N I N/J _
III/go NAO
/N NANHZ
A The stereochemistry of the orous center is not defined
AVN-013BPC
Example 4
Using the protocol described in Example 1, the following PMO was synthesized, NG0393
H44A(-6+15), SEQ ID NO: 6 (5’- GAT CTG TCA AAT CGC CTG CAG -3’) and used as
described in Examples 8 and 9.
Ereak A I‘la‘reak B Bre:ak C
: : ' NMe2
- NMe2 A i/NMez A “3/
o o O A '/ ,P N,
Y \/\o/\/ \/\OH O¢P\O o' \o o \o
N G
T o G o
T N
N N
5P<NMezI All/’0 lip/,0
O \
MeZN \0 Me2N O [3 I
K10)” K63” ‘63”C
N N T
A ”’0 £20 ,Pi’g
MezN’ ‘o MezN/ 0 MezN
of Yr" Yr
” T T,o
A"0 “ ”’0 3P:
/P< ‘o Me2N o
MezN O
A T
Oj/T o j/
N N
'10 l go A T,o
A I
;P<o MezN’ \o MezN’RO
Me N2
oj/C Oj/A oj/G
N N
A T,O A
’ O ¢O
KO ”
MezN’P\o ,R
MezN O
Oj/T KEO‘J/T KEOD/C
N N’ N
A '0 N
A éo
,FK’ p
MeZN O MeZN/F’\o MezN/ \O
(5/6 Rog/C K103”
N N N
éreak A Break B Brea'k c
0 o
NH2 NH2
N NH
N \N NH 5
A=<’| C=| G=<’| T—mU—_ _ |
J A
/N III/go /N NANHZ [I] O
A The stereochemistry of the phosphorous center is not defined
AVN-013BPC
Example 5
Using the protocol described in Example 1, the ing PMO was synthesized, NG0394
H44A(-8+17), SEQ ID NO: 7 (5’- CAG ATC TGT CAA ATC GCC TGC AGG T -3’) and used as
described in Examples 8 and 9.
ll3reak A Il3reak B Bre:ak C
. . .
: NMe2 :/NMe2 . /NMe2
o/\/Ov\oH 02P<' ,P
O,,P\O O’ \O
N KC 1/ RE 1/ C
E j T O T O
I O
I KC 1/
T N N
NW2 | l A T
/ O ,0
A‘P A ’
" ’“O P” P
O \0
Me N/P\0 MeQN/ ‘0 MezN/ \O
KEOJ/C 2 Kon/G KEOD/C Ktoj/A
T N T N
A ,0 Lo
, P‘O
P’ \ AP‘O
MezN’ \O M62N \O MezN/ O MezN/ O
G G
orA Kon/T Kfioj/ KEOD/
N T T N
A. o A 40
P Arno
’1 A
\ aO P ’ \ Me N O P\
Me2N/ \o MeZN o 2
C M92N/
, Lo
A .,o /,0 ’,‘P'
P‘o MezN’P\O M92” \0 A P30
MezN
A C MezN/
OJ’A 01’ O
N N N
I “LO ,Pi’oI\ C 30
MSQN O MeQN’ \O MegN O
Oj/T KEOD/T
N Kon/A
N~ N
A 90 l\ ¢O Ap’ro
Me2N’ ‘0 P‘o
Me2N Me N/ \O2
‘3” Y)” YO)”
l‘.‘ N N
I I 'l
éreak A Break B Break C
NH2 NH2 0 O
“1*“ e ”
”H 5
A= <N’ IN’J C= | Ill/k0 G= <IN’ | T—m U—- - I
N/’J\NH2 “*0
A The stereochemistry of the phosphorous center is not defined
3BPC
Example 6
Using the protocol described in Example 1, the following PMO was synthesized, NG—13—0008
H44A(-7+17), SEQ ID NO: 1 (5’- CAG ATC TGT CAA ATC GCC TGC AGG-S’) and used as
described in Examples 8 and 9.
Break A ll3reak B Brelak C
: : :
' - NMe2
. NMez A . /NMe2
0 O o A ./ fi/
Y \/\O/\/ \/\OH ,P
O,,P\O 0’ \O OI, \o
N o C
T o T
T N N
€P<NM62 Allfio AFLO A ,,o
O Me2N/ ‘0 MezN/ O MezN O
KEOj/C KKOJ/G KEOD/C Kfioj/A
N N
‘ ¢0 T¢0 I\ T '130 ¢0
A /P\O
MezN’ \O MeZN 0 MezN MezN FKO
o A
Nj/ Ktoj/T KEOJ/G KEOj/e
N N
Algo T
ALO AP¢O A O
P] ’P‘ M 2N’ ‘o P», M 2N
meZN \O O ‘o
KEOJ/G c c MezN
01/ ]/ KEOD/G
T T T o N
,0 A (’0 AP”
/AP<0 MeZN’P\O MezN’ ‘o
MezN
A [ 3 ]
O Oj/A Oj/C
N N
ALO A
, o ,,o
MeZN’RO a P
\o MezN’ \o
Oj/T KEO‘J/A Kfioj/T
T N’ N
A ‘0 A
A ,o ,,o
,P ’ P\
MeZN \O \0 0
MezN MezN/
OTC Yr" *6)”
N N N
I I
éreak A Break B Brea'k C
NH2 NH2 0 O
A-< N
I c—fiN w NH
- a - A - . a WU:
/N N O
'i' O N NH2
/ |
A The stereochemistry of the phosphorous center is not defined
AVN-013BPC
Example 7
Using the ol described in Example 1, the following PMO was synthesized, NG0395
H44A(-6+17), SEQ ID NO: 8 (5’- CAG ATC TGT CAA ATC GCC TGC AG -3’) and used as
described in Examples 8 and 9.
T N
NMe T |
/ 2 ’0 ’/o
A [,0
\O P p’ /P\
O O
MezN O \0
MezN MezN
K10 ‘63” KEY: RE)”A C
I ,,0 ,o A ,o
A 40
MezN/P\O K P,
MezN/ MezN/ ‘ O
MezN’ ‘o
o A
Mr Kon/T KEOJ/G Kfioj/G
N T u
A LO A'L/(O 1‘on
/P<O MezN’ ‘0 M92N 0
MezN
REC Oj/C C I 3 ]
73/ T To
A $0 ‘0 CP”
/P\O MezN’ \O MeZN \O
° of 01’C
N N N
A LO A
’ o [,0
¢ ,P\
M 2N’P\O MezN’P\O MezN O
Oj/T KEOj/A Ktoj/T
T N’ N
I\ ,O "
A ”/0
,P< ”wO P
M82N O MezN/P\O MezN/ \O
Y Yr" Yr
N N 'y'
E'sreak A Break B Break C
0 o
NH2 NH2
N NH
NH 5
A=/(/NNIKNIN’J c=|(KN G=’| T—mU—l_ _
til/g0 (IN N/)\NH2 T/go
A The chemistry of the phosphorous center is not defined
AVN-013BPC
Example 8
Exon 44 skipping
A series of antisense oligomers that target human dystrophin exon 44 were designed and
synthesized as follows:
Description Seguence SEQ ID NO
H44A(-07+17) CAGATCTGTCAAATCGCCTGCAGG
H44A(-07+20) CAACAGATCTGTCAAATCGCCTGCAGG
CTCAACAGATCTGTCAAATCGCCTGCAGG
GATCTGTCAAATCGCCTGCAGGT
-CAAATCGCCTGCAGG
TCAAATCGCCTGCAG
H44A( 8+17) CAGA-C-G1CAAATCGCCTGCAGGT
CAGATCTGTCAAATCGCCTGCAG
G1G1C--1CTGAGAAACTGTTCAGC
GAGAAACTGTTCAGCTTCTGTTAGCCAC
GAAACTGTTCAGCTTCTGTTAGCCACTG
CTGTTCAGCTTCTGTTAGCCACTG
13+14) ATCTGTCAAATCGCCTGCAGGTAAAAG
H44A(-14+15) TCAAATCGCCTGCAGGTAAAAGC
Selected antisense oligomers shown above were evaluated for exon skipping efficacy by
treating RD cells at the s indicated concentrations. In these experiments, published
antisense oligomers corresponding to H44A(—06+14) and H44A(+85+104) (US 8,232,384; SEQ ID
NOs: 167 and 165, respectively) and H44A(—06+20), H44A(—09+17), H44A(+59+85) and
H44A(+65+90) (WO2011/057350; SEQ ID NOS: 68, 220, 54 and 10, respectively) were used as
ative oligomers. As shown in oligomer H44A(-07+17) (SEQ ID NO: 1) was
highly effective at inducing exon 44 skipping in RD cells compared to known sequences. As
shown in H44A(+62+89) (SEQ ID NO: 11) was highly effective in inducing exon 44
skipping in cultured RD cells compared to other highly active antisense oligonucleotides known in
the art.
Example 9
AVN-013BPC
Exon 44 skipping in primary human myoblasts
Based on the above results described in e 8, additional oligomers were designed
and tested in primary human myoblasts. Additional sequences known in the art were included in
the analysis (H44A(-10+15) and 20+5); SEQ ID NOS: 44 and 45, tively) as
comparators to the newly designed oligomers. These sequences are disclosed as SEQ ID NOS: 4
and 2 in PCT publication WO/2010/048586. As shown in FIGS. 5-6, a range of oligomers nested
within a target region defined as H44A(—07+17) all have the ability to induce exon 44 skipping at
relatively high levels compared to sequences known in the art. Preferred oligomers are shown
above in examples 2 — 7 (SEQ ID NOs: 4, 5, 6, 7, 1 and 8, respectively).
*********************
All publications and patent applications cited in this specification are herein incorporated
by reference as if each individual publication or patent application were specifically and
dually ted to be incorporated by reference.
AVN-013BPC
SEQUENCE G
Description Mm
H44A(-07+17) CAGATCTGTCAAATCGCCTGCAGG
07+20) CAACAGATCTGTCAAATCGCCTGCAGG
H44A(-O7+22) CTCAACAGATCTGTCAAATCGCCTGCAGG
H44A(—8+15) GAT CTG TCA AAT CGC CTG CAG GT
H44A(-7+15) GAT CTG TCA AAT CGC CTG CAG G
H44A(-6+15) GAT CTG TCA AAT CGC CTG CAG
H44A(—8+17) CAG ATC TGT CAA ATC GCC TGC AGG T
H44A(-6+17) CAG ATC TGT CAA ATC GCC TGC AG
H44A(+77+101) GTGTCTTTCTGAGAAACTGTTCAGC
H44A(+64+91) GAGAAACTGTTCAGCTTCTGTTAGCCAC
H44A(+62+89) GAAACTGTTCAGCTTCTGTTAGCCACTG
H44A(+62+85) CTGTTCAGCTTCTGTTAGCCACTG
06+ 14) ATCTGTCAAATCGCCTGCAG
H44A(+85+104) TTTGTGTCTTTCTGAGAAAC
H44A(+6 1 +84) - G - - CAGC - i CTGTTAGCCACTGA
H44A(-10+15) AAATCGCCTGCAGGTAA
H44A(+64+88)
H44A(+79+103)
H44A(-06+20) CTGTCAAATCGCCTGCAG
H44A(-09+17)
H44A(+59+85)
H44A(+59+89)
H44A(+65+90)
rTAT RRRQRRKKR
RKKRRQRRR
RRRRRRRRRFF
RRRRRFFRRRR
RRRR
{Ki-{R
' RRRRR
' RRRRRR
-{ {Bi-{RR
-{ {REL-{RR
(RX)s {XRXRXRXRXRXRX
(RAth)4; (P007) 'RAhXRRAhXRRAhXRRAhXR
; (CP04057) {AhX {RAhXRRAhXRRAhXRRAhXR
(RAthRBRh; {Ahx {RBRRAhXRRBR
(CPO6062)
AVN-013BPC
(RAR)4F2 RARRARRARRARFF 38
(RGR)4F2 RGRRGRFF 39
Pr imer CAATGCTCCTGACCTCTGTGC 4 O
Pr imer GCTCTTTTCCAGGTTCAAGTGG
Pr imer GTCTACAACAAAGCTCAGGTCG 4 2
Pr imer GCAATGTTATCTGCTTCCTCCAACC
H44A(-10+15) GA-C -G-CAAAL‘CGCCTGCAGGTAA
H44A(-20+5) -CGCC - GCAGGTAAAAGCATATGG
H44A(-13+14) CTGTCAAATCGCCTGCAGGTAAAAG
H44A(-14+15) - c - G - CAAATCGCCTGCAGGTAAAAGC
AVN-013BPC
REFERENCES
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378(9791): 595—605.
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placebo—controlled, dose—escalation, proof—of—concept study." Lancet Neurol 8(10): 918-
Lu, Q. L., C. J. Mann, et al. . "Functional amounts of dystrophin produced by skipping the
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skipping in the mdx mouse model of muscular dystrophy." J Gene Med 4(6): .
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transcript in lymphoblastoid cells by transfecting an antisense oligodeoxynucleotide
AVN-013BPC
complementary to an exon recognition sequence." Biochem Biophys Res Commun 226(2):
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, P., R. Kole, et al. (2007). Splice switching oligomers for the TNF superfamily receptors
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Claims (4)
1. An nse oligonucleotide of 22 bases comprising the base sequence GAT CTG TCA AAT CGC CTG CAG G (SEQ ID NO: 5), in which thymine bases are optionally uracil bases, n the antisense oligonucleotide is ated to an arginine-rich peptide; or a pharmaceutically acceptable salt thereof.
2. The antisense oligonucleotide of claim 1, wherein the antisense oligonucleotide is chemically linked to a polyethylene glycol molecule and conjugated to an arginine rich peptide.
3. The antisense oligonucleotide of claim 1 or 2, wherein the arginine-rich peptide consists of a sequence selected from SEQ ID NOS: 24-39.
4. The antisense oligonucleotide of any one of claims 1-3, wherein the antisense oligonucleotide has the following structure:
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