MULTIPLE EXON SKIPPING COMPOSITIONS FOR DMD
`
`Atty Docket No. 50450.8090
`
`FIELD OF THE INVENTION
`
`The present invention relates to novel antisense compounds and compositions suitable for
`facilitating exon skipping in the human dystrophin gene. It also provides methods for inducing exon
`skipping using the novel antisense compositions adapted for use in the methodsof the invention.
`
`REFERENCES
`
`Yin et al., (2008) Hum MolGenetSept 10.
`
`Kole, (1997) Acta Biochimica Polonica 44:23 1-238
`
`Sierakowskaet al., (1996) Proc Nat! Acad Sci USA 93:12840-12844
`
`Wilton ef a/., (1999) Neuromusc Disorders 9:330-338
`
`van Deutekom e¢ al., (200!) Human Mol Genet 10:1547-1554
`
`van Deutekomeal., (2007) N Engl J Med 357(26):2677-86
`
`15
`
`Lu e7 al., (2003) Nature Medicine 9:1009-1014
`
`Aartsma-Ruset al., (2004) Am J Hum Genet 74:83-92
`
`Matsuoef al., (1991) J Clin Invest. 87:2127-2131
`
`Takeshima, ef al., (1995) J. Clin. Invest. 95, 515-520
`
`Dunckley et ail., (1997) Nucleosides & Nucleotides. 16, 1665-1668
`
`20
`
`Dunckley er a/., (1998) Human Mol. Genetics, 5, 1083-90
`
`Errington et a/., (2003) J Gen Med 5. 518-527
`
`Mann ef al., (2002) J Gen Med 4, 644- 654
`
`Jearawiriyapaisarn, N., H. M. Moulton,et al. (2008). "Sustained Dystrophin Expression
`
`Induced by Peptide-conjugated Morpholino Oligomers in the Muscles of mdx Mice." Mol Ther.
`
`25
`
`Marshall, N. B., S. K. Oda,et al. (2007). “Arginine-rich cell-penetrating peptidesfacilitate
`
`delivery of antisense oligomers into murine leukocytes and alter pre-mRNAsplicing.” Journal of
`
`Immunological Methods 325(1-2): 114-126.
`
`Wu, B., H. M. Moulton,et al. (2008). “Effective rescue of dystrophin improves cardiac
`
`function in dystrophin-deficient mice by a modified morpholino oligomer." Proc Natl Acad Sci US A
`
`30
`
`105(39): 14814-9.
`
`BACKGROUNDOF THE INVENTION
`
`Antisense technologies are being developed using a range of chemistries to affect gene
`expressionat a variety of different levels (transcription, splicing, stability, translation). Muchofthat
`
`ATL_IMANAGE-5365690 v1
`
`]
`
`

`

`Atty Docket No. 50450.8090
`
`research has focused on the use of antisense compounds to correct or compensate for abnormalor
`disease-associated genes in a myriad ofdifferent conditions. Antisense molecules are able to inhibit
`gene expression with exquisite specificity, and because of this, many research efforts concerning
`
`oligonucleotides as modulators of gene expression have focused oninhibiting the expression of
`targeted genes such as oncogenesorviral genes. The antisense oligonucleotides are directed either
`against RNA (sense strand) or against DNA where they formtriplex structures inhibiting transcription
`
`by RNA polymerase II. To achieve a desired effect of specific gene down-regulation, the
`oligonucleotides generally either promote the decay ofthe targeted mRNAor block translation ofthat
`mRNA, thereby effectively preventing de novo synthesis of the undesirable target protein.
`However, such techniques are not useful where the objectis to up-regulate production of the
`
`10
`
`native protein or compensate for mutations which induce premature termination of translation such as
`
`nonsenseor frame-shifting mutations.
`
`In these cases, the defective gene transcript should not be
`
`subjected to targeted degradation, so the antisense oligonucleotide chemistry should not promote
`
`targetMRNA decay.
`
`In a variety of genetic diseases, the effects of mutations on the eventual expression of a gene
`can be modulated throughaprocessof targeted exon skipping during the splicing process. The
`
`15
`
`splicing processis directed by complex multi-particle machinery that brings adjacent exon-intron
`junctions in pre-mRNAinto close proximity and performs cleavage of phosphodiester bonds at the
`
`ends ofthe introns with their subsequent reformation between exons that are to be spliced together.
`
`20
`
`25
`
`This complex and highly precise process is mediated by sequence motifs in the pre-mRNA thatare
`relatively short semi-conserved RNA segments to which bind the various nuclear splicing factors that
`are then involved in the splicing reactions. By changing the way the splicing machinery reads or
`recognises the motifs involved in pre-mRNA processing, it is possible to create differentially spliced
`mRNA molecules. It has now been recognised that the majority of human genesare alternatively
`spliced during normal gene expression, although the mechanismsinvolved have not been identified.
`In cases where a normally functional protein is prematurely terminated because of mutations |
`therein, a means for restoring some functional protein production through antisense technology has
`
`been shownto be possible through intervention during the splicing processes, and that if exons
`
`30
`
`associated with disease-causing mutations can be specifically deleted from some genes, a shortened
`protein product can sometimes be producedthathassimilar biological propertiesof the native protein
`or has sufficient biological activity to ameliorate the disease caused by mutations associated with the
`
`exon (Sierakowska 1996, Wilton 1999, van Deutekom 2001, Lu 2003, Aartsma-Rus 2004). Kole eal.
`
`(U.S. Patent Nos. 5,627,274; 5,916,808; 5,976,879; and 5,665,593) disclose methods of combating
`
`ATL_IMANAGE-5365690 v1
`
`2
`
`

`

`Atty Docket No. 50450.8090
`
`aberrant splicing using modified antisense oligonucleotides which do not promote decay of the
`
`targeted pre-mRNA.
`
`The process of targeted exon skippingis likely to be particularly useful in long genes where
`
`there are many exons andintrons, where there is redundancyin the genetic constitution of the exonsor
`
`wherea protein is able to function without one or more particular exons(e.g. with the dystrophin gene,
`which consists of 79 exons). Efforts to redirect gene processing for the treatmentof genetic diseases
`associated with truncations caused by mutationsin various genes have focused on the use of antisense
`
`oligonucleotides that either: (1) fully or partially overlap with the elements involvedin the splicing
`
`process; or (2) bind to the pre-mRNAat a position sufficiently close to the element to disrupt the
`binding and function ofthe splicing factors that would normally mediate a particular splicing reaction
`which occursat that element.
`.
`
`Modulation of mutant dystrophin pre-mRNA splicing with antisense oligoribonucleotides has
`
`been reported both in vitro and in vivo.
`In one type of dystrophin mutation reported in Japan, a 52-
`base pair deletion mutation causes exon 19 to be removed with the flanking introns during the splicing
`process (Matsuo efal/., 1991). An invitro minigene splicing system has been used to show that a 31-
`
`mer 2'-O-methy] oligoribonucleotide complementaryto the 5' half of the deleted sequence in
`
`dystrophin Kobe exon 19 inhibited splicing of wild-type pre-mRNA (Takeshimaet al., 1995). The
`same oligonucleotide was used to induce exon skipping fromthe native dystrophin gene transcript in
`
`human cultured lymphoblastoid cells. Dunckley er al., (1997) described in vitro constructs for
`
`analysis of splicing around exon 23 of mutated dystrophin in the md mouse mutant, a model for
`
`muscular dystrophy. Plans to analyse these constructs iv vitro using 2' modified oligonucleotides
`
`targeted to splice sites within and adjacent to mouse dystrophin exon 23 were discussed, though no
`target sites or sequences were given. 2'-O-methyl oligoribonucleotides were subsequently reported to
`correct dystrophin deficiency in myoblasts from the max mouse from this group. An antisense
`
`oligonucleotide targeted to the 31 splice site of murine dystrophin intron 22 wasreported to cause
`skipping of the mutant exon as well as several flanking exons andcreated a novel in-frame dystrophin
`transcript with a novel internal deletion. This mutated dystrophin was expressed in |-2% of antisense
`treated mdx myotubes. Use of other oligonucleotide modifications such as 2'-O- methoxyethy]
`
`phosphodiesters are described by Dunckley er al., (1998).
`Although antisense molecules may provide a tool in the treatment of Duchenne Muscular
`Dystrophy (DMD), attempts to induce exon skipping using antisense molecules have had mixed
`
`success. Successful skipping of dystrophin exon 19 from the dystrophin pre-mRNA wasachieved
`using a variety of antisense molecules directed at the flanking splice sites or motifs within the exon
`involved in exon definition as described by Errington et al., (2003).
`In contrast to the apparent ease of
`
`ATL_IMANAGE-5365690v1
`
`3
`
`10
`
`15
`
`20
`
`25
`
`30
`
`

`

`Atty Docket No. 50450.8090
`
`exon 19 skipping, the first report of exon 23 skipping in the mdx mouse by Dunckleyeral., (1998)is
`now consideredto be reporting only a naturally occurring revertant transcript or artifact rather than
`any true antisenseactivity. In addition to not consistently generating transcripts missing exon23,
`Dunckleyet al., (1998) did not show any time course of induced exon skippingortitration of antisense
`oligonucleotides to demonstrate dose dependenteffects where the levels of exon skipping
`corresponded with increasing or decreasing amountsofantisense oligonucleotide. Furthermore,this
`
`work could not be replicated by other researchers.
`The first example ofspecific and reproducible exon skipping in the mdx mouse model. was
`reported by Wiltonet al., (1999). By directing an antisense molecule to the donorsplicesite,
`consistentand efficient exon 23 skipping was induced in the dystrophin mRNA within 6 hours of
`
`treatmentof the cultured cells. Wilton ev al., (1999), also describe targeting the acceptor region ofthe
`
`mouse dystrophin pre-mRNA with longer antisense oligonucleotides and being unable to repeat the
`published results of Dunckley ef al., (1998). No exonskipping, either 23 alone or multiple removal of
`several flanking exons, could be reproducibly detected using a selection of antisense oligonucleotides
`
`directed at the acceptorsplice site of intron 22. Whilethe first antisense oligonucleotide directedat
`
`the intron 23 donor splice site induced consistent exon skipping in primary cultured myoblasts, this
`
`compound wasfound to be muchless efficient 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 magnitude (Mannef al., 2002).
`Recent studies have begun to address the challenge of achieving sustained dystrophin
`
`20
`
`expression accompanied by minimal adverseeffects in tissues affected by the absence of dystrophin.
`Intramuscular injection of an antisense oligonucleotide targeted to exon 51 (PROOS51) into the tibialis
`anterior muscle in four patients with DMDresulted in specific skipping of exon 51 without any
`clinically apparent adverse effects (van Deutekom er al., 2007). More recently, studies looking at
`
`systemic delivery of a antisense phosphorodiamidate morpholino oligomer conjugated toacell-
`25
`penetrating peptide (PPMO)targeted to exon 23 in mdx mice produced high and sustained dystrophin
`protein production in skeletal and cardiac muscles without detectable toxicity (Wu et al., 2008;
`Jearawiriypaisarn et al., 2008; Yin et al., 2008).
`
`30
`
`SUMMARYOF THE INVENTION
`
`Accordingto a first aspect, the invention provides antisense molecules capable of binding to a
`
`selected target to induce exon skipping.
`In a further example,it is possible to combine two or more antisense oligonucleotides ofthe
`
`present invention together to induce single or multiple exon skipping.
`
`ATL_IMANAGE-5365690v1
`
`4
`
`

`

`Atty Docket No. 50450.8090
`
`In another example,it is possible to improve exon skipping of a single or multiple exons by
`
`linking together two or more antisense oligonucleotide molecules. A similar concept has been
`
`described in Aartsma-Rusetal. (2004).
`
`The invention includes, in one general aspect, an antisense oligonucleotide compoundthatis:
`
`(i) composed of morpholino subunits and phosphorus-containing intersubunit linkages joining a
`
`morpholino nitrogen of one subunit to a 5' exocyclic carbon ofan adjacent subunit, (ii) containing
`
`between 10-40 nucleotide bases, preferably 20-35 bases(iii) having a base sequenceeffectiveto
`
`hybridize to at least 12 contiguous bases of a target sequence in dystrophin pre-mRNA andinduce
`
`10
`
`exon skipping.
`The compound may be composed ofphosphorus-containing intersubunit linkages joining a
`morpholino nitrogen of one subunit to a 5' exocyclic carbon of an adjacent subunit. The morpholino
`subunits in the compound maybe joined by phosphorodiamidate linkages, in accordance with the
`structure:
`
`Z—P-X
`
`aOFBi
`
`|
`where Y|=O, Z=O,Pj is a purine or pyrimidine base-pairing moiety effective to bind, by base-
`specific hydrogen bonding, to a base in a polynucleotide, and X is alkyl, alkoxy, thioalkoxy, or alkyl
`
`15
`
`amino e.g., wherein X=NRo, where eachRis 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 numberofpositively charged linkages is between 2 and
`
`20
`
`no more than half of the total numberof linkages. The positively charged linkages may have the
`
`abovestructure in which X is 1-piperazine.
`
`Wherethe antisense compound administeredis effective to target a splice site of preprocessed
`
`human dystrophin, it may have a base sequence complementaryto a target region containing atleast
`12 contiguous bases in preprocessed human dystrophin transcript. Exemplary antisense sequences
`include those identified by SEQ ID NOS: | to 569. Moreparticularly, an antisense sequence of the
`
`25
`
`present invention is contained within:
`
`ATL_IMANAGE-5365690 v1
`
`5
`
`

`

`Atty Docket No. 50450.8090
`
`(a) any of the sequencesidentified by SEQ ID NOS: 1-20, preferably SEQ ID NOS: 1-5 and
`
`16-20, for use in producing skipping of exon 44 in the processing of human dystrophin pre-processed
`
`mRNA;
`
`(b) any of the sequencesidentified by SEQ ID NOS: 21-76, preferably SEQ ID NOS: 21-53,
`and more preferably SEQ ID NOS: 21-25 and 49-53, for use in producing skipping of exon 45in the
`
`processing of human dystrophin pre-processed mRNA,
`
`(c) any of the sequencesidentified by SEQ ID NOS: 77-125, preferably SEQ ID NOS: 77-
`
`105, and more preferably SEQ ID NOS: 77-81 and 101-105, for use in producing skipping of exon 46
`
`in the processing of human dystrophin pre-processed mRNA,comprising
`
`10
`
`(d) any of the sequencesidentified by SEQ ID NOS:'126-169, preferably SEQ ID NOS: 126-
`
`149, and more preferably SEQ ID NOS: 126-130 and 145-149, for use in producing skipping of exon
`
`47 in the processing of human dystrophin pre-processed mRNA;
`
`(e) any of the sequencesidentified by SEQ ID NOS: 170-224, preferably SEQ ID NOS: 170-
`
`201, and more preferably SEQ ID NOS: 170-174 and 197-201, for use in producing skipping of exon
`48 in the processing of human dystrophin pre-processed mRNA;
`(f) any of the sequencesidentified by SEQ ID NOS: 225-266, preferably SEQ ID NOS: 225-
`
`248, and more preferably SEQ ID NOS: 225-229 and 244-248 for use in producing skipping of exon
`
`49 in the processing of human dystrophin pre-processed mRNA;
`
`(g) any of the sequencesidentified by SEQ ID NOS: 267-308, preferably SEQ ID NOS: 267-
`
`20
`
`291, and more preferably SEQ ID NOS: 267-271 and 287-291 for use in producing skipping of exon
`
`50 in the processing of human dystrophin pre-processed mRNA;
`
`(h) any of the sequencesidentified by SEQ ID NOS: 309-371, preferably SEQ ID NOS: 309-
`
`345, and more preferably SEQ ID NOS: 309-313 and 341-345 for use in producing skipping of exon
`51 in the processing of human dystrophin pre-processed mRNA;
`:
`
`25
`
`(i) any of the sequencesidentified by SEQ ID NOS: 372-415, preferably SEQ ID NOS: 372-
`397, and more preferably SEQ ID NOS: 372-376 and 393-397for use in producing skipping of exon
`52 in the processing of human dystrophin pre-processed mRNA;
`.
`(j) any of the sequencesidentified by SEQ ID NOS: 416-475, preferably SEQ ID NOS: 416-
`
`451, and more preferably SEQ ID NOS: 416-420 and 447-451 for use in producing skipping of exon
`
`30
`
`53 in the processing of human dystrophin pre-processed mRNA;
`
`(k) any of the sequencesidentified by SEQ ID NOS: 476-519, preferably SEQ ID NOS: 476-
`
`499, and more preferably SEQ ID NOS: 476-480 and 495-499 for use in producing skipping of exon
`
`54 in the processing of human dystrophin pre-processed mRNA; and
`
`ATL_IMANAGE-5365690 v1
`
`6
`
`

`

`Atty Docket No. 50450.8090
`
`(I) any of the sequencesidentified by SEQ ID NOS: 520-569, preferably SEQ ID NOS: 520-
`546, and more preferably SEQ ID NOS: 520-524 and 542-546 foruse in producing skipping of exon
`
`55 in the processing of human dystrophin pre-processed mRNA;
`
`The compound maybe conjugated to an arginine-rich polypeptide effective to promote uptake
`
`of the compoundinto cells. Exemplary peptides include those identified by SEQ ID NOS: 570 to 578.
`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 ofthe antisense compound. Also in an exemplary
`
`embodiment, the antisense compound is composed of morpholino subunits and phosphorus-containing
`
`intersubunit linkages joining a morpholino nitrogen of one subunit to a 5' exocyclic carbon of an
`
`10
`
`adjacent subunit.
`
`_ In general, the peptide-oligomer conjugate may further comprise a homing peptide whichis
`
`selective for a selected mammalian tissue, i.e. the same tissue being targeted by the cell-penetrating
`
`peptide. The conjugate maybeofthe form cell penetrating peptide - homing peptide - antisense
`
`oligomer, or, more preferably, of the form homing peptide - cell penetrating peptide - antisense
`
`15
`
`oligomer. For example, a peptide conjugate compoundforuse in treating Duchenne muscular
`dystrophy, as described above, can further comprise a homing peptide whichis selective for muscle
`tissue, such as the peptide having the sequence identified as SEQ ID NO: 579, conjugated to the cell-
`penetrating peptide. Exemplary conjugatesofthis type include those represented herein as CP06062-
`MSP-PMO(cell penetrating peptide - homing peptide - antisense oligomer) and as MSP- CP06062-
`
`20.
`
`PMO(homingpeptide - cell penetrating peptide - antisense oligomer) (see SEQ ID NOs: 580-583).
`
`According to a second aspect, the present invention provides antisense molecules selected and
`
`or adapted to aid in the prophylactic or therapeutic treatment of a genetic disorder comprisingat least
`
`an antisense molecule in a formsuitable for delivery to a patient.
`
`According to a third aspect, the invention provides a method for treating a patient suffering
`
`25
`
`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 addresses the use ofpurified and
`isolated antisense oligonucleotides ofthe invention, for the manufacture of a medicamentfor treatment
`of a genetic disease.
`
`30
`
`The invention further provides a method oftreating a condition characterised by Duchenne
`muscular dystrophy, which method comprises administering to a patient in need oftreatment an
`effective amount of an appropriately designed antisense oligonucleotide of the invention, relevant to
`
`the particular genetic lesion in that patient. Further, the invention provides a method for
`
`ATL_IMANAGE-5365690 v1
`
`7
`
`

`

`Atty Docket No. 50450.8090
`
`prophylactically treating a patient to prevent or at least minimise Duchene muscular dystrophy,
`
`comprising the step of: administering to the patient an effective amount of an antisense
`oligonucleotide or a pharmaceutical composition comprising one or more of these biological
`
`molecules
`
`The invention also provides kits for treating a genetic disease, which kits comprise at least an
`
`antisense oligonucleotide ofthe present invention, packaged in a suitable container and instructionsfor
`its use.
`.
`These and other objects and features will be more fully understood whenthe following
`detailed description of the invention is read in conjunction with the figures.
`
`10
`
`BRIEF DESCRIPTION OF THE FIGURES
`
`Figure 1 A shows an exemplary morpholino oligomoerstructure with a phosphorodiamidate
`
`linkage;
`
`Figure 1B showsa conjugate of an arginine-rich peptide and an antisense oligomer, in accordance
`
`15
`
`with an embodimentofthe invention;
`
`Figure 1C. shows a conjugate as in Figure 1C, but where the backbone linkages contain one or more
`
`positiviely charged groups; and
`Figs. 1D-1G show various phosphorus-containing linkages in a morpholino oligonucleotide, as
`described below.
`
`20
`
`DETAILED DESCRIPTION OF THE INVENTION
`
`I.
`
`Definitions
`
`The terms below,as used herein, have the following meanings, unless indicated otherwise:
`
`25
`
`The terms“cell penetrating peptide” or “CPP”are used interchangeably and refer to cationic-
`
`cell penetrating peptides, also called transport peptides, carrier peptides, or peptide transduction domains.
`The peptides, as shownherein, have the capability of inducing cell penetration within 100% ofcells ofa
`
`given cell culture population and allow macromoleculartranslocation within multiple tissues in vivo
`
`30
`
`upon systemic administration.
`The terms “antisense oligomer”or “antisense compound”are used interchangeably andrefer
`to a sequence ofcyclic subunits, each bearing a base-pairing moiety,linked by intersubunit linkages
`that allow the base-pairing moieties to hybridize to a target sequence in a nucleic acid (typically an
`
`RNA) by Watson-Crick base pairing, to form a nucleic acid:oligomer heteroduplex within the target
`
`sequence. The cyclic subunits are based on ribose or another pentose sugaror, in a preferred
`
`ATL_IMANAGE-5365690 v1
`
`8
`
`

`

`Atty Docket No. 50450.8090
`
`embodiment, a morpholino group (see description of morpholino oligomers below). The oligomer
`
`may have exact or near sequence complementarity to the target sequence; variations in sequence near
`
`the termini of an oligomerare generally preferable to variations in the interior.
`
`Such an antisense oligomer can be designedto block or inhibit translation of mRNAorto
`inhibit natural pre-mRNAsplice processing, and maybesaid to be “directed to”or “targeted against”
`
`a target sequence with which it hybridizes. The target sequenceis typically a region including an
`AUGstart codon of an mRNA,ora splice site ofa pre-processedmRNA. The target sequencefor a
`splice site may include an mRNAsequencehavingits 5' end | to about 25 base pairs downstream of a
`normalsplice acceptor junction in a preprocessed MRNA.A preferred target sequencefora splice is
`any region of a preprocessed mRNAthatincludesa splicesite or is contained entirely within an exon
`coding sequence or spansa splice acceptor or donorsite. An oligomer is more generally said to be
`“targeted against” a biologically relevanttarget, such as a protein, virus, or bacteria, when it is targeted
`
`against the nucleic acid ofthe 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 structuresare linked together by phosphorus-containing linkages, oneto three atoms long,
`
`preferably two atoms long, and preferably unchargedorcationic, joining the morpholino nitrogen of one
`subunit to a 5° exocyclic carbon ofan adjacent subunit, and (ii) each morpholinoring bears a purine or
`pyrimidine base-pairing moiety effective to bind, by base specific hydrogen bonding, to a base ina
`polynucleotide. See, for example,the structure in Figure 1A, which showsa preferred
`phosphorodiamidate linkage type. Variations can be made to this linkage as long as they do notinterfere
`with bindingor activity. For example, the oxygen attached to phosphorus maybe substituted with sulfur
`(thiophosphorodiamidate). The 5’ oxygen maybe substituted with amino or loweralkyl substituted
`amino. The pendantnitrogen attached to phosphorus maybe unsubstituted, monosubstituted, or
`disubstituted with (optionally substituted) lower alkyl. See also the discussion ofcationic linkages
`below. The purine or pyrimidine basepairing moiety is typically adenine, cytosine, guanine, uracil,
`thymine or inosine. The synthesis, structures, and binding characteristics of morpholino oligomers are
`detailed in U.S. Patent Nos. 5,698,685, 5,217,866, 5,142,047, 5,034,506, 5,166,315, 5,521,063, and
`
`5,506,337, and PCT Appn. No. PCT/US07/11435 (cationic linkages), all of which are incorporated
`
`20
`
`25
`
`30
`
`herein by reference.
`An “amino acid subunit”or “amino acid residue” can refer to an (]-aminoacid residue
`
`(-CO-CHR-NH-) or a (- or other aminoacid residue (e.g—CO-(CH2),CHR-NH-), whereRis a side
`
`chain (which mayinclude hydrogen) andnis | to 6, preferably 1 to 4.
`
`Theterm “naturally occurring amino acid”refers to an aminoacid present in proteins found in
`
`ATL_IMANAGE-5365690 v1
`
`9
`
`

`

`Atty Docket No. 50450.8090
`
`nature. The term “non-natural amino acids” refers to those aminoacids notpresent in proteins foundin
`nature, examples include beta-alanine (B-Ala), 6-aminohexanoic acid (Ahx) and 6-aminopentanoicacid.
`An “effective amount”or“therapeutically effective amount”refers to an amount oftherapeutic
`compound, such as an antisense oligomer, administered to a mammalian subject, either as a single dose
`oras part of a series of doses, whichis effective to produce a desired therapeutic effect. For an antisense
`oligomer, this effect is typically brought aboutby inhibiting translation or natural splice-processing of a
`
`selected target sequence.
`
`“Treatment”of an individual(e.g. a mammal, such as a human)ora cell is any type of
`intervention used in an attemptto alter the natural courseofthe individualor cell. Treatmentincludes,
`butis not limited to, administration of a pharmaceutical composition, and may be performedeither
`
`prophylactically or subsequentto the initiation of a pathologic event or contact with an etiologic agent.
`
`II. Constructing the Antisense Oligonucleotide
`Examples of morpholino oligonucleotides having phosphorus-containing backbonelinkages
`are illustrated in Figs. ]A-1C. Especially preferred is a phosphorodiamidate-linked morpholino
`oligonucleotide such as shownin Fig. 1C, which is modified, in accordance with one aspectof the
`present invention, to contain positively charged groupsat preferably 10%-50% ofits backbone
`linkages. Morpholino oligonucleotides with uncharged backbonelinkages,including antisense
`oligonucleotides, are detailed, for example, in (Summerton and Weller 1997) and in co-owned U.S.
`Patent Nos. 5,698,685, 5,217,866, 5,142,047, 5,034,506, 5,166,315, 5,185, 444, 5,521,063, and
`
`5,506,337, all of which are expressly incorporated by reference herein.
`
`Important properties of the morpholino-based subunits include: 1) the ability to be linked in
`
`a oligomeric form by stable, uncharged orpositively charged backbonelinkages; 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 complementary-basetarget nucleic acid, including target RNA,
`Tm values above about 45°Cin relatively short oligonucleotides (¢.g., 10-15 bases); 3) the ability of
`the oligonucleotide to be actively or passively transported into mammalian cells; and 4) the ability of
`the antisense oligonucleotide:RNA heteroduplex to resist RNAse and RNaseH degradation,
`
`10
`
`15
`
`20
`
`25
`
`respectively.
`Exemplary backbonestructures for antisense oligonucleotides of the claimed subject matter
`
`30
`
`|D-G, each linked by an unchargedorpositively
`include the morpholino subunit types shownin Figs.
`charged, phosphorus-containing subunit linkage. Fig. 1D shows a phosphorus-containing linkage
`which formsthe five atom repeating-unit backbone, where the morpholinoringsare linked by a 1 -
`atom phosphoamidelinkage. Fig. 1E showsa linkage which produces a 6-atom repeating-unit
`
`ATL_IMANAGE-5365690 v1
`
`10
`
`

`

`Atty Docket No. 50450.8090
`
`backbone.
`
`In this structure, the atom Y linking the 5' morpholino carbon to the phosphorus group may
`
`be sulfur, nitrogen, carbon or, preferably, oxygen. The X moiety pendant from thephosphorus may be
`
`fluorine, 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 morpholinesor piperidines. Alkyl, alkoxy and thioalkoxy preferably include 1-6 carbon
`atoms. The Z moieties are sulfur or oxygen, and are preferably oxygen.
`.
`The linkages shownin Figs. 1F and 1G are designed for 7-atom unit-length bacbones.
`structure IF, the X moiety is as in Structure IE, and the Y moiety may be methylene,sulfur, or,
`preferably, oxygen. In Structure 1G, the X and Y moieties are as in Structure 1E. Particularly
`preferred morpholino oligonucleotides include those composed of morpholino subunit structures of the
`form shownin Fig. 1E, where X=NH2, N(CH3)2, or 1-piperazine or other charged group, Y=O, and
`
`In
`
`10
`
`Z=0.
`
`Asnoted above,the substantially uncharged oligonucleotide may be modified, in accordance
`with an aspect ofthe invention,to include charged linkages, e.g. up to about | per every 2-5 uncharged
`linkages, such as about 4-5 per every 10 uncharged linkages. Optimal improvementin antisense
`activity may be seen when about 25% of the backbonelinkages are cationic. Suboptimal enhancement
`is typically seen with a small number e.g., 10-20% cationic linkages, and where the numberofcationic
`linkages are in the range 50-80%,andtypically above about 60%, the sequencespecificity of the
`
`antisense binding to its target may be compromisedorlost.
`
`20
`
`Additional experiments conducted in support of the present invention indicate that the
`
`enhancementseen with added cationic backbone charges may, in somecases, be further enhanced by
`
`distributing the bulk of the chargesclose of the "center-region" backbonelinkages of the antisense
`oligonucleotide, e.g., ina 20mer oligonucleotide with 8 cationic backbonelinkages, having atleast
`
`70% of these charged linkages localized in the 10 centermost linkages.
`
`25
`
`The antisense compoundscanbeprepared by stepwise solid-phase synthesis, employing
`
`methodsdetailed in the references cited above, and below with respect to the synthesis of
`
`oligonucleotides having a mixture or uncharged and cationic backbone linkages.
`
`In somecases,it
`
`maybedesirable to add additional chemical moieties to the antisense compound, e.g. to enhance
`pharmacokineticsor to facilitate capture or detection of the compound. Such a moiety may be
`covalently attached, typically to a terminusof the oligomer, according to standard synthetic methods.
`
`30
`
`For example, addition of a polyethyleneglycol moiety or other hydrophilic polymer,e.g., one having
`
`10-100 monomeric subunits, may be useful in enhancing solubility. One or more charged groups,e.g.,
`
`anionic charged groups such as an organic acid, may enhancecell uptake. A reporter moiety, such as
`fluorescein or a radiolabeled group, may be attached for purposes of detection. Alternatively, the
`
`ATL_IMANAGE-5365690 v1
`
`11
`
`

`

`reporter label attached to the oligomer maybealigand, such as an antigen or biotin, capable of
`
`Atty Docket No. 50450.8090
`
`binding a labeled antibodyorstreptavidin. In selecting a moiety for attachment or modification of an
`
`antisense compound,it is generally of course desirable to select chemical compoundsof groupsthat
`are biocompatible and likely to be tolerated by a subject without undesirable side effects.
`Asnoted above,the antisense compoundcan beconstructed to contain a selected number of
`cationic linkages interspersed with uncharged linkages ofthe type described above. The intersubunit
`linkages, both unchargedandcationic, preferably are phosphorus-containing linkages, having the
`structure:
`
`Ww~|——X
`"|
`
`10
`
`where
`
`W is S or O, andis preferably O,
`X = NR'R’or OR®,
`Y =OorNR’,
`
`15
`
`20
`
`25
`
`and each said linkage in the oligomeris selected from:
`(a) uncharged linkage (a), where each of R', R’, R° and R’is independently selected from
`
`hydrogen and loweralkyl;
`(b1) cationic linkage (b1), where X = NR'R? and Y = O, and NR'R’represents an optionally
`substituted piperazino group, such that R'R? = -CHRCHRN(R°)(R‘)CHRCHR-, where
`' each R is independently H or CH,
`R‘ is H, CH3, or an electron pair, and
`R?is selected from H, lower alkyl, e.g. CH3, C(7NH)NH2, Z-L-NHC(=NH)NH)zand
`{C(O)CHR’NH],H, where: Z is C(O)or a direct bond, L is an optional linker up to 18 atomsin
`length, preferably upt to 12 atoms, and more preferably up to 8 atomsin length, having bondsselected _
`from al