(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)
`V
`
`.4121“)3&-\
`team.
`
`\.
`"In?
`
`PCT
`
`(10) International Publication Number
`
`WO 2009/008725 A2
`
`(19) World Intellectual Property Organization
`International Bureau
`
`(43) International Publication Date
`15 January 2009 (15.01.2009)
`
`(51) International Patent Classification:
`A61K 47/48 (2006.01)
`A6IP 25/00 (2006.01)
`A6IP 21/00 (2006.01)
`
`(74) Agent: VAN WESTENBRUGGE, Andries; Nederland—
`sch Octrooibureau, Postbus 29720, NL-2502 LS Den Haag
`(NL).
`
`(21) International Application Number:
`PCT/NL2008/050470
`
`(22) International Filing Date:
`
`11 July 2008 (11.07.2008)
`
`(25) Filing Language:
`(26) Publication Language:
`
`(30) Priority Data:
`07112313.7
`
`English
`English
`
`12 July 2007 (12.07.2007)
`
`EP
`
`(71) Applicant (for all designated States except US): Prosensa
`B.V. [NL/NL]; Wassenaarseweg 72, NL—2333 AL Leiden
`(NL)-
`
`(81) Designated States (unless otherwise indicated, for every
`kind of national protection available): AE, AG, AL, AM,
`AO, AT, AU, AZ, BA, BB, BG, BH, BR, BW, BY, BZ, CA,
`CH, CN, CO, CR, CU, CZ, DE, DK, DM, DO, DZ, EC, EE,
`EG, ES, FI, GB, GD, GE, GH, GM, GT, HN, HR, HU, ID,
`IL, IN, IS, JP, KE, KG, KM, KN, KP, KR, KZ, LA, LC, LK,
`LR, LS, LT, LU, LY, MA, MD, ME, MG, MK, MN, MW,
`MX, MY, MZ, NA, NG, NI, NO, NZ, OM, PG, PH, PL, PT,
`RO, RS, RU, SC, SD, SE, SG, SK, SL, SM, ST, SV, SY, TJ,
`TM, TN, TR, TT, TZ, UA, UG, US, UZ, VC, VN, ZA, ZM,
`ZW.
`
`(72) Inventors; and
`VAN KUIK-
`(75) Inventors/Applicants (for US only):
`ROMEIJN, Petra [NL/NL]; Verlengde Hoogravenseweg
`82, NL—3525 BJ Utrecht (NL). PLATENBURG, Gerard
`Johannes [NL/NL]; Wijngaardenlaan 56, NL-2252 XR
`Voorschoten (NL).
`
`(84) Designated States (unless otherwise indicated, for every
`kind of regional protection available): ARIPO (BW, GH,
`GM, KE, LS, MW, MZ, NA, SD, SL, SZ, TZ, UG, ZM,
`ZW), Eurasian (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM),
`European (AT, BE, BG, CH, CY, CZ, DE, DK, EE, ES, FI,
`FR, GB, GR, HR, HU, IE, IS, IT, LT, LU, LV, MC, MT, NL,
`
`[Continued on next page]
`
`(54) Title: MOLECULES FOR TARGETING COMPOUNDS TO VARIOUS SELECTED ORGANS, TISSUES OR TUMOR
`CELLS
`
`(57) Abstract: The invention provides conjugates,
`comprising an organ, tissue or tumor cell homing
`molecule linked to a moiety. Such a moiety can be,
`for example, an oligonucleotide, small interfen‘ng
`RNA, gene, virus, protein, pharmaceutical or
`detectable agent.
`In addition the
`invention
`provides methods to diagnose or treat neuronal or
`neuromuscular disease, or a pathology of the brain,
`or a tumor of neuronal or neuroectodermal origin,
`by administrating to a subject having or suspected
`of having a pathology a molecule or conjugate that
`homes to, binds to and is taken up by the brain cells
`or neuronal cells, or by the tumor cells of neuronal
`or neuroectodermal origin.
`The invention also
`provides a method of identifying and measun‘ng
`neun‘te growth in neuronal cells.
`
`Fig 1a
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`After 20 minutes
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`Fig 1b
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`After 24 hrs
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`6681
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`WO2009/008725A2|||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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`

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`WO 2009/008725 A2
`
`|||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
`
`NO, PL, PT, RO, SE, SI, SK, TR), OAPI (BF, BJ, CF, CG, — with sequence listing part of description published sepa—
`CI, CM, GA, GN, GQ, GW, ML, MR, NE, SN, TD, TG).
`rately in electronicform and available upon requestfrom
`Published:
`the International Bureau
`— without international search report and to be republished
`upon receipt of that report
`
`

`

`WO 2009/008725
`
`PCT/NL2008/050470
`
`Molecules for targeting compounds to various selected organs, tissues or tumor cells
`
`FIELD OF THE INVENTION
`
`The present invention is in the field of in vivo targeting and provides molecules that
`
`home to, bind to and are taken up by various organs or tissues or tumor cells.
`
`BACKGROUND OF THE INVENTION
`
`Most therapeutic compounds are delivered to the target organ or tissue through the
`
`circulation. However, in most cases the drug or other treatment will not only target the
`
`diseased organ or tissues, but will also be taken up by other organs and tissues in the
`
`body. This can result in undesirable side effects due to, for example, generalized toxic
`
`effects throughout the patient’s body. Thus, it would be desirable to selectively target
`
`specific organs or tissues, or specific types of tumor cells. In addition, coupling of a
`
`therapeutic compound to a targeting molecule can improve the uptake properties of the
`
`compound into the targeted tissue or cells, resulting in a more effective molecule.
`
`Therefore, coupling to targeting molecules yields compounds that are more effective and
`
`less toxic than the parental compound, see Curnis et al., 2000, Nature Biotechnol. 18,
`
`1185-1190. This can be applied to a wide range of compounds, such as peptides, proteins,
`
`cytostatic agents, antibiotic and antiviral agents.
`
`In the case of neuromuscular diseases such as myotonic dystrophy (MD) or spinal
`
`muscular atrophy (SMA) transport across the blood brain barrier and uptake into the
`
`neuronal cells is mandatory for an effective therapy. Neuron-specific peptides can be
`
`conjugated to, for example, antisense oligonucleotides (AONs) and small
`
`interfering
`
`RNA (siRNA). AONs and siRNAs have high potency to be applied as new classes of
`
`medicines for treatment of specific diseases by blocking undesired gene transcription. In
`
`the field of SMA therapy antisense-induced exon inclusion is gaining attention as a novel
`
`and promising tool
`
`for correction of the translational reading frame of the SMN2
`
`(survival of motor neuron 2) transcript. The aim is to manipulate splicing in such a
`
`manner that the targeted exon will be included (through binding of the AONs to pre—
`
`mRNA). This would allow correction of the translational reading frame, and induction of
`
`the synthesis of a full length SMN protein.
`
`

`

`WO 2009/008725
`
`PCT/NL2008/050470
`
`Several reports have shown the therapeutic potential of the exon inclusion strategy for
`
`restoring fiill
`
`length SMN protein production (Hua et al., 2007, PLoS Biol. 5, e73;
`
`Baughan et al., 2006, Mol. Ther. 14, 54-62). However, the biggest hurdle to overcome is
`
`the poor in vivo neuronal uptake of these AONs and transport across the blood brain
`
`barrier. For other neuronal diseases, or diseases of the brain (e.g. Alzheimer, Parkinson
`
`and the like) the problem is very similar, i.e. poor in vivo uptake of the therapeutic or
`
`diagnostic compounds.
`
`In the case of neuronal or neuro—ectodermal tumors (e.g. neuroblastoma, glioblastoma
`
`and the like), targeting is also of major importance for generating an effective therapy
`
`without side effects. Chemotherapeutic drugs can act both on normal as well as cancerous
`
`tissues, leading to this targeting requirement. For anti—sense oligonucleotide (AON-) or
`
`small interfering (si)RNA-based drugs it is known that pharmacokinetic properties are
`
`unfavourable for the free drug to reach sufficient levels at the site of the tumor, because
`
`the majority is absorbed in the liver and the kidneys. The vehicle delivering the
`
`chemotherapeutic must show sufficient half life time to effectively deliver a therapeutic
`
`agent to the desired cells, also beyond the blood brain barrier.
`
`In light of the above, it is very clear that further improvements in delivery systems are
`
`necessary to achieve specific uptake of agents such as AONs in viva.
`
`SUMMARY OF THE INVENTION
`
`It is an object of the present invention to provide compounds, preferably peptides or
`
`peptidomimetics, that home to an organ or tissue or cell type of interest, especially brain
`
`cells or neuronal cells, or by the tumor cells of neuronal or neuroectodermal origin. By
`
`coupling diagnostic moieties or moieties having a biological activity to such homing
`
`compounds, said moieties are targeted to the specific organs or tissues or cells.
`
`After extensive research,
`
`the present
`
`inventors have identified two peptides that
`
`selectively bind to and are taken up by brain cells, neuronal cells and by tumor cells of
`
`neuronal or neuroectodermal origin. This invention thus fulfills the need of improving the
`
`in vivo uptake of for example (antisense) oligonucleotides, by conjugation of such
`
`oligonucleotides to these specific peptides. The molecules are advantageously useful in
`
`antisense therapy methods for treatment of neuromuscular disease, brain disease or
`
`

`

`WO 2009/008725
`
`PCT/NL2008/050470
`
`tumors of neuronal or neuro-ectodermal origin, and delivery of a wide variety of
`
`diagnostics or drugs across the blood-brain barrier to brain cells, or to neuronal cells, or
`
`to tumor cells of neuronal or neuroectodermal origin .
`
`Thus the present invention relates to a peptide or peptidomimetic comprising a sequence
`
`or consisting of a sequence selected form the group consisting of THRPPMWSPVWP
`
`(SEQ ID NO: 1) and LPWKPLG (SEQ ID NO: 2).
`
`Also the present
`
`invention concerns conjugates of a peptide or peptidomimetics
`
`comprising a sequence or consisting of a sequence selected form the group consisting of
`
`THRPPMWSPVWP (SEQ ID NO: 1) and LPWKPLG (SEQ ID NO: 2) and a moiety
`
`selected from a biologically active moiety and diagnostic moiety linked thereto.
`
`A conjugate as described above for use as a medicament is an aspect of this invention.
`
`DETAILED DESCRIPTION OF THE INVENTION
`
`The present
`
`invention provides peptides or peptidomimetics for targeting diagnostic
`
`moieties or biologically active moieties to an organ or tissue or cell type of interest,
`
`especially across the blood brain barrier to brain cells, to neuronal cells or to tumor cells
`
`of neuronal or neuroectodermal origin.
`
`A peptide in the context of this invention comprises at least SEQ ID NO:
`
`1 or SEQ ID
`
`NO: 2 identified above.
`
`In one embodiment a peptide in the context of the present
`
`invention comprises a part of SEQ ID NO: 1, said part of SEQ ID NO:
`
`1 being identical
`
`to 11, 10, 9, 8 or 7 amino acids of SEQ ID NO: 1. In one embodiment a peptide in the
`
`context of the present invention comprises or consists of SEQ ID NO: 3-12. In one
`
`embodiment a peptide in the context of the present invention comprises a variant of SEQ
`
`ID NO: 1, said variant comprising one substitution of any amino acid in SEQ ID NO:
`
`1
`
`with any other amino acid or derivative thereof. In one embodiment a peptide in the
`
`context of the present invention comprises or consists of SEQ ID NO: 13-23. The peptide
`
`can be fully constructed of naturally occurring L-amino acids, or can contain one or more
`
`modifications to backbone and/or side chain(s). These modifications can be introduced by
`
`incorporation of amino acid mimetics that show similarity to the natural amino acid. The
`
`group of peptides described above comprising one or more mimetics of amino acids is
`
`

`

`WO 2009/008725
`
`PCT/NL2008/050470
`
`referred to as peptidomimetics. In the context of this invention, mimetics of amino acids
`
`include, but are not limited to, [32- and B3-amino acids, [32,2- [32,3, and [33,3-disubstituted
`
`amino acids, 0L,0L-disubstituted amino acids, statine derivatives of amino acids, D-amino
`
`acids, oc-hydroxyacids, oc-aminonitriles, N-alkylamino acids and the like. In addition, the
`
`C-terminus of the peptide might be carboxylic acid or carboxamide, or other resulting
`
`from incorporation of one of the above mentioned amino acid mimetics. Furthermore, the
`
`peptides described above may contain one or more replacements of native peptide bonds
`
`with groups including, but not limited to, sulfonamide, retroamide, aminooxy-containing
`
`bond, ester, alkylketone, a,a-difluoroketone, a-fluoroketone, peptoid bond (N—alkylated
`
`glycyl
`
`amide bond). Furthermore,
`
`the peptides mentioned above may contain
`
`substitutions in the amino acid side chain (referring to the side chain of the corresponding
`
`natural
`
`amino
`
`acid),
`
`for
`
`instance
`
`4-fluorophenylalanine,
`
`4-hydroxylysine,
`
`3-
`
`aminoproline, 2—nitrotyrosine, N—alkylhistidine or B—branched amino acids or B—branched
`
`amino acid mimetics with chirality at the B-side chain carbon atom opposed to the natural
`
`chirality (e.g. allo-threonine, allo-isoleucine and derivatives). In one other embodiment,
`
`above mentioned group of peptides may contain close structural analogues of amino acid
`
`or amino acids mimetics, for instance omithine instead of lysine, homophenylalanine or
`
`phenylglycine instead of phenylalanine, B-alanine instead of glycine, pyroglutamic acid
`
`instead of glutamic acid, norleucine instead of leucine or the sulfur-oxidized versions of
`
`methionine and/or cysteine. The linear and cyclized forms of the peptides mentioned
`
`above are covered by this patent, as well as their retro,
`
`inverso and/or retroinverso
`
`analogues. To those skilled in the art many more close variations may be known, but the
`
`fact that these are not mentioned here does not limit the scope of this patent. In one
`
`embodiment, a peptide or peptidomimetic according to the present invention is at most 30
`
`amino acids in length, or at least 25 amino acids or 20 amino acids or 19, 18, 17, 16, 15,
`
`14, 13, 12, 11, 10, 9, 8 or 7 amino acids in length.
`
`A biologically active moiety is a compound exerting (directly or indirectly) a biological
`
`function, preferably a therapeutic function, hence is preferably a therapeutically active
`
`compound. A therapeutically active compound can be any compound known in the art
`
`and preferably is a compound that has a therapeutic effect by modulating an intercellular
`
`process. A therapeutically active compound that has a (direct) modulating effect or
`
`(direct) biological
`
`function can be for
`
`instance any protein, enzyme inhibitor,
`
`oligonucleotide, siRNA, gene, or pharmaceutical. Any biologically active compound or
`
`

`

`WO 2009/008725
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`PCT/NL2008/050470
`
`therapeutically active compound can be used as long as it can be linked to or can be made
`
`suitable to be linked to a peptide or peptidomimetic according to the present invention.
`
`The biologically active compound or therapeutically active compound so becomes the
`
`moiety in the compound according to the present invention. The skilled person will be
`
`able to identify suitable biologically active or therapeutically active compounds. In one
`
`embodiment the biologically active compound is a cytostatic, e.g. anti-cancer compound,
`
`such as anthracyclines (including daunorubicin, doxorubicin, epirubicin,
`
`idarubicin,
`
`mitoxantrone),
`
`alkylating
`
`agents
`
`(including
`
`cisplatin,
`
`carboplatin,
`
`oxaliplatin,
`
`chlorambucil, busulfan, melphalan, mechloethamine, cyclophosphamide, iphosphamide),
`
`anti-metabolites
`
`(including azathioprine, mercaptopurine), plant
`
`alkaloids
`
`and/or
`
`terpenoids (including vinca alkaloids and taxanes such as vincristine, vinblastine,
`
`vinorelbine, vindesine podophyllotoxin, etoposide, paclitaxel, docetaxel),
`
`tenoposide),
`
`topoisomerase inhibitors (including campthotecins irinotecan,
`
`topotecan, amsacrine),
`
`dactinomycin, dacarbazine, gemcitabine, temozolamide, mAbs (including trastuzumab,
`
`cetuximab, bevacizumab, rituximab).
`
`In one embodiment the biologically active compound or therapeutically active compound
`
`is a compound comprising or consisting of nucleic acids or analogues thereof. Such
`
`compounds can be considered to exert (indirectly) a biological function, preferably a
`
`therapeutic function, by modulating the genetic machinery within a cell, in particular on
`
`the level of production of proteins. The nucleic acid may be a DNA, RNA or analogues
`
`thereof, such as compounds comprising 2’-0-alkyl or 2’-0-alkenyl (allyl) or 2’-0-
`
`alkynyl nucleotides, e.g. 2’-0-methyl-, 2’-0-methoxyethyl-
`
`(MOE) and 2’-0-allyl-
`
`nucleotides, locked nucleic acids (LNAs), peptide nucleic acids (PNAs), ethylene bridged
`
`nucleic acids (ENAs), phosphorothioate modified nucleotides, e.g. 2’-0-methoxyethyl
`
`phosphorothioate RNA nucleotides or 2’-0-methyl phosphorothioate RNA nucleotides,
`
`morpholino based nucleotides and combinations thereof etc. The compound comprising
`
`or consisting of nucleic acids or analogues thereof may also comprise a mixture of
`
`various nucleic acids or its analogues. It may e.g. be a chimera of a mixture of 2’0-
`
`methyl RNA and RNA, of DNA and LNA, etcetera. It may also be a gapmer, e.g. having
`
`tenninal 2’O-methyl RNA nucleotides and inner DNA nucleotides. The compound may
`
`be a gene, plasmid, a polynucleotide or oligonucleotide, small interfering RNA and the
`
`like. The compound may be single stranded or double stranded.
`
`

`

`WO 2009/008725
`
`PCT/NL2008/050470
`
`In one embodiment a diagnostic moiety is linked to the peptides or peptidomimetics
`
`according to the present invention. The diagnostic moiety may be for in vivo or in vitro
`
`diagnostic purposes. Commonly used imaging labels, radio labels or fluorescent labels
`
`such as Cy3, Cy5, Cy5.5 and the like or green fluorescent protein (GFP) or other
`
`diagnostic proteins, possibly via recombinant expression, may be used as diagnostic
`
`moieties.
`
`In order to prepare the conjugates according to the present invention, coupling of the
`
`biologically active moiety or diagnostic moiety to the peptides or peptidomimetics
`
`according to the present invention occurs via known methods to couple compounds to
`
`amino acids or peptides. A common method is to link a moiety to a free amino group or
`
`free hydroxyl group or free carboxylic acid group or free thiol group in a peptide or
`
`peptidomimetic. Common conjugation methods include thiol/maleimide coupling, amide
`
`or ester bond formation, or heterogeneous diSulfide formation. The skilled person is well
`
`aware of standard chemistry that can be used to bring about the required coupling. The
`
`biologically active moiety or diagnostic moiety may be coupled directly to a peptide or
`
`peptidomimetic or may be coupled via a spacer or linker molecule. It is not necessary that
`
`the biologically active or diagnostic moiety is covalently linked to the peptide or
`
`peptidomimetic of the invention. It may also be conjugated Via electrostatic interactions.
`
`In one embodiment the present invention also relates to a molecule comprising a peptide
`
`or peptidomimetic according to the invention and a linking part, which is not a peptide,
`
`for linking the molecule to a biologically active moiety or a diagnostic moiety. The
`
`linking part
`
`for example may be a (poly)cationic group that complexes with a
`
`biologically active poly- or oligonucleotide. Such a (poly)cationic group may be a
`
`spermine or polyethyleneimine, polyethylene glycol, poly-L-lysine and the like.
`
`As mentioned in one embodiment the peptide or peptidomimetic according to the present
`
`invention is
`
`linked to a biologically active moiety. For example the peptide or
`
`peptidomimetic can be linked to a biologically active or therapeutic peptide and in one
`
`embodiment can even be part of the peptide or peptidomimetic basic structure. For
`
`example the amino- or carboxy-terminus of a therapeutic peptide can be extended with a
`
`sequence comprising or consisting of the peptides described above. It is to be understood
`
`that such a peptide extended with a peptide or peptidomimetic according to the invention
`
`

`

`WO 2009/008725
`
`PCT/NL2008/050470
`
`is encompassed by a conjugate according to the present invention. The preparation of
`
`such peptides can be achieved via standard amino acid or peptide coupling procedures.
`
`In one embodiment the peptide or peptidomimetic according to the present invention is
`
`combined with a nuclear localisation signal
`
`(NLS). In one embodiment a conjugate
`
`according to the present invention is combined with a NLS. In the context of the present
`
`invention the NLS functions to direct the present conjugates, e.g. the biologically active
`
`moiety or a diagnostic moiety,
`
`into a cell nucleus, presumably via its recognition by
`
`cytosolic nuclear
`
`transport
`
`receptors. The NLS may be part of the peptide or
`
`peptidomimetic according to the present invention, e.g. the amino- or carboxy-terminus
`
`of a NLS can be extended with a sequence comprising or consisting of the peptides
`
`described above. Also a NLS may be coupled at a different position than that of the
`
`peptide or peptidomimetic according to the present invention to a biologically active
`
`moiety or a diagnostic moiety. NLS sequences are known in the art. Typically a NLS
`
`signal consists of or comprises (a few) short sequences of positively charged lysines
`
`and/or arginines, for example a NLS consist of or comprises (K)KKR(K), (K)KRS(K),
`
`(K)(S)RK(R)(K). Known NLS are PKKKRKV, GKKRSKV, KSRKRKL.
`
`In one
`
`embodiment
`
`the peptide or peptidomimetic according to the present
`
`invention is
`
`combined with a NLS selected from the group consisting of SEQ ID NO: 24-39.
`
`In one embodiment a conjugate according to the invention wherein the biologically active
`
`moiety is a protein or polypeptide and wherein the peptide or peptidomimetic is
`
`comprised in the protein or polypeptide backbone is prepared by recombinant expression
`
`of the peptide or peptidomimetic together with the biologically active protein. Preferably
`
`a DNA construct is prepared such that the peptide or peptidomimetic according to the
`
`invention is expressed at a terminus of the biologically active peptide, preferably at the
`
`C-terminus of the biologically active peptide. Such preparation of DNA constructs by
`
`recombinant DNA methodology and expression in a suitable host is common practice to
`
`the skilled person.
`
`Thus in one embodiment the present conjugate is a fusion protein of a peptide according
`
`to the present invention, e.g. a peptide of SEQ ID NO:
`
`1 or SEQ ID NO: 2, with a
`
`therapeutically active protein, e.g. antibody, or a diagnostic (e.g. fluorescent) protein or
`
`both, optionally also comprising a NLS. Such a fusion protein can be prepared by
`
`expression of the appropriate DNA construct.
`
`

`

`WO 2009/008725
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`PCT/NL2008/050470
`
`In one embodiment the present invention concerns the use of a conjugate according to
`
`present invention for the preparation of a medicament for targeting a biological active
`
`moiety or a diagnostic moiety across the blood brain barrier to brain cells, to neuronal
`
`cells or to tumor cells of neuronal or neuroectodermal origin. In one embodiment the
`
`medicament is for the treatment of a brain disorder. In one embodiment the medicament
`
`is for the treatment of a neuronal or neuromuscular disease. In one embodiment the
`
`medicament is for the treatment of a tumor of neuronal or neuroectodermal origin.
`
`Examples of brain disorders
`
`are
`
`those
`
`involving a neurodegeneration and/or
`
`neuroinflammation event such as Stroke, Alzheimer's Disease, Parkinson's Disease and
`
`Multiple Sclerosis. Also therapy of (CNS—)disorders that benefit from neurotrophic
`
`factors, including GDNF, BDNF, EPO (erythropoietin) and anti-inflammatory antibodies
`
`(e.g. Enbrel® and Remicade®) are encompassed by the present invention.
`
`Also therapy of disorders that benefit from enzyme replacement therapies to treat the
`
`neurological
`
`component of
`
`inherited
`
`lysosomal
`
`storage
`
`diseases
`
`(Cerezyme®,
`
`AldurazymeTM, Farbrazyme®) and Pompe disease (Myozyme®) are encompassed by the
`
`present invention.
`
`Also therapy of tumors that metastasize to the brain are encompassed by the present
`
`invention, for example those tumors that can be treated with therapeutic anti-cancer
`
`antibodies (e.g. Rituxan®, Herceptin® and ErbituxTM) and anti-cancer compounds (e.g.
`
`GleevecTM and IressaTM)
`
`Examples of neuronal or neuromuscular diseases are myotonic dystrophy (MD) or spinal
`
`muscular atrophy (SMA), DNA repeat diseases, such as, but not
`
`limited to: coding
`
`regions repeat diseases having a polyglutamine (CAG) repeat: Huntington’s disease, Haw
`
`River syndrome, Kennedy’s disease/spinobulbar muscular atrophy, spino-cerebellar
`
`ataxia, or diseases having polyalanine (GCG) repeats such as: infantile spasm syndrome,
`
`deidocranial dysplasia, blepharophimosis/ptosis/epicanthus invensus syndrome, hand-
`
`foot-genital
`
`syndrome,
`
`synpolydactyly,
`
`oculopharyngeal muscular
`
`dystrophy,
`
`holoprosencephaly. Diseases with repeats in non-coding regions of genes to be treated
`
`according to the invention comprise the trinucleotide repeat disorders (mostly CTG
`
`and/or CAG repeats): myotonic dystrophy type
`
`1, myotonic dystrophy type 2,
`
`Friedreich’s ataxia, spino-cerebellar ataxia, autism. Furthermore, the present conjugates
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`can be applied for therapy of fragile site associated repeat disorder comprising various
`
`fragile X-syndromes, Jacobsen syndrome and other unstable repetitive element disorders
`
`such as myoclonus epilepsy,
`
`facioscapulohumeral dystrophy and certain forms of
`
`diabetes mellitus type 2.
`
`Examples of tumors of neuronal or neuroectodermal origin include all neoplasms of the
`
`CNS and PNS,
`
`such as, but not
`
`limited to, neuroblastoma, medulloblastoma,
`
`glioblastoma,
`
`oligodendroglioma,
`
`oligoastrocytoma,
`
`astrocytoma,
`
`neurofibroma,
`
`ependymoma, MPNST (malignant peripheral nerve sheath tumors), ganglioneuroma or
`
`Schwannoma. Also of neuroectodermal origin are tumours such as rhabdomyosarcoma,
`
`retinoblastoma, small cell lung carcinoma, adrenal pheochromocytoma, primitive PNET
`
`(peripheral neuroectodermal
`
`tumor), Ewing’s
`
`sarcoma
`
`and melanoma.
`
`In one
`
`embodiment the medicament is for the treatment of neuroblastoma, medulloblastoma,
`
`glioblastoma,
`
`oligodendroglioma,
`
`oligoastrocytoma,
`
`astrocytoma,
`
`neurofibroma,
`
`ependymoma, MPNST (malignant peripheral nerve sheath tumors), ganglioneuroma,
`
`Schwannoma, rhabdomyosarcoma, retinoblastoma, small cell
`
`lung carcinoma, adrenal
`
`pheochromocytoma, primitive PNET (peripheral neuroectodermal
`
`tumor), Ewing’s
`
`sarcoma and melanoma.
`
`In one embodiment
`
`the biologically active moiety is an oligonucleotide that
`
`is
`
`complementary to and/or capable of hybridizing to a repetitive stretch selected from the
`
`group consisting of (CAG)n, (GCG)n, (CUG)n, (CGG)n and (CCUG)n, wherein n is
`
`selected from 1 — 50, preferably 2 - 20. The integer n is selected such that in a preferred
`
`embodiment
`
`the oligonucleotide comprises at
`
`least
`
`10
`
`to about 50 consecutive
`
`nucleotides complementary to a repetitive element, more preferably 12 to 45 nucleotides,
`
`even more preferably 12 to 30, and most preferably 12 to 25 nucleotides complementary
`
`to a repetitive stretch.
`
`The use of an oligonucleotide that is complementary to a polyglutamine (CAG)n tract in
`
`a transcript is particularly useful for the diagnosis, treatment and/or prevention of the
`
`human disorders Huntington’s disease, several forms of spino-cerebellar ataxia or Haw
`
`River syndrome, caused by repeat expansions in the HD, HDL2/JPH3, SBMA/AR,
`
`SCAl/ATXI, SCA2/ATX2, SCA3/ATX3, SCA6/CACNAIA, SCA7, SCA17 or DRPLA
`
`human genes.
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`The use of an oligonucleotide that is complementary to a polyalanine (GCG)n tract in a
`
`transcript
`
`is particularly useful for the diagnosis,
`
`treatment and/or prevention of the
`
`human disorders:
`
`infantile spasm syndrome, deidocranial dysplasia, blepharophimosis,
`
`hand—foot—genital disease, synpolydactyly, oculopharyngeal muscular dystrophy and/or
`
`holoprosencephaly, which are caused by repeat expansions in the ARX, CBFAI , FOXLZ,
`
`HOXA13, HOXD13, OPDM/PABPZ, TCFBRl or ZIC2 human genes.
`
`The use of an oligonucleotide complementary to a (CUG)n repeat in a transcript
`
`is
`
`particularly useful for the diagnosis, treatment and/or prevention of the human genetic
`
`disorder myotonic dystrophy type 1, spino-cerebellar ataxia, caused by repeat expansions
`
`in the DMl/DMPK or SCA8 human genes respectively.
`
`The use of an oligonucleotide complementary to a (CCUG)n repeat in a transcript is
`
`particularly useful for the diagnosis, treatment and/or prevention of the human genetic
`
`disorder myotonic dystrophy type 2, caused by repeat expansions in the DMZ/ZNF9
`
`gene.
`
`The use of an oligonucleotide complementary to a (CGG)n repeat in a transcript
`
`is
`
`particularly useful for the diagnosis, treatment and/or prevention of human fragile X
`
`syndromes, caused by repeat expansion in the FRAXA/FMRI, FRAXE/FMRZ and
`
`FRAXF/FAM] 1A genes.
`
`The use of an oligonucleotide complementary to a (CGG)n repeat in a transcript is
`
`particularly useful for the diagnosis, treatment and/or prevention of the human genetic
`
`disorder Jacobsen syndrome, caused by repeat expansion in the FRAl 1B/CBL2 gene.
`
`In one embodiment the biologically active moiety in the peptide or peptidomimetic
`
`according to the present invention is an antisense oligonucleotide or siRNA comprising a
`
`sequence from the table below.
`
`In one embodiment
`
`the peptide or peptidomimetic
`
`conjugates according to the present invention are for the modulation of (expression oi) a
`
`target gene and/or protein selected from the table 1 below and/or for therapy, in particular
`
`treatment of a disease selected from the table below.
`
`
`
`Antisense oligonucleotide sequence
`
`Target gene Disease
`/ rotein
`
`
`
`
`
`5’ CTTC TC GGAGCTGAGTGTC T neuroectodermal tumors
`
`10
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`
`
`neuroectodermal tumors
`DCL
`5 ’-CTTCTCGGAGCTGAGCGTCT
`
`
`5 ’-GCUGGGCAGGCCAUUCACAC
`
`DCL
`
`neuroectodermal tumors
`
`5’-GCUCGGCAGGCCGUUCACCC
`
`neuroectodermal tumors
`DCL
`5 ’-CUUCUCGGAGCUGAGUGUCU
`
`
`5 ’-CUUCUCGGAGCUGAGCGUCU
`
`5 ’-CAAGAAGACGGCUCACUCCTT
`
`3 ’-TTGUUCUUCUGCCGAGUGAGG
`
`DCL
`
`DCL
`
`neuroectodermal tumors
`
`neuroectodermal tumors
`
`5’-CAAGAAAACGGCUCAUUCCTT
`
`DCL
`
`neuroectodermal tumors
`
`3 ’-TTGUUCUUUUGCCGAGUAAGG
`
`
`5 ’-GAAAGCCAAGAAGGUUCGATT
`
`DCL
`
`neuroectodermal tumors
`
`3 ’-TTCUUUCGGUUCUUC CAAGCT
`
`5’-GAAGGCCAAGAAAGUUCGUTT
`
`D
`
`CL
`
`neuroectodermal tumors
`
`3 ’-TTCUUCCGGUUCUUUCAAGCA
`
`5=<CAG>n<n=2—12>
`
`
`5’-(CAG)n (n = 2 — 12)
`Spinocerebellar ataxia 8
`
`5’-(CCG)n (n = 2 — l2)
`
`5’-(CGG)n (n = 2 — l2)
`
`5’-(UUC)n (n = 2 — 12)
`
`Fragile X syndrome
`
`Fragile XE
`
`Friedreich’s ataxia
`
`
`5’-(CUG)n (n = 2 — 12)
`AR
`Spinal muscular atrophy
`
`Dentatorubralpallidolusian
`DRPL4
`5’-(CUG)n (n = 2 — 12)
`
`atrophy
`
`5’-(CUG)n (n = 2 — l2)
`
`SCAl , 2, 3,
`7
`
`Spinocerebellar ataxia type
`1,2,3,7
`
`5’-(CUG)n (n = 2 7 12)
`
`CACNAIA Spinocerebellar ataxia type 6
`
`5’—(CGCG4CG4)n (n = 2 — l2)
`
`CSTB
`
`Progressive myoclonus
`
`
`
`
`
`5'-AUUCACUUUCAUAAUGCUGG Spinal muscular atrophy SMN2
`
`
`
`
`5’-TTTTTGATTTTGTCT
`SMN2
`Spinal muscular atrophy
`
`5’-ATTTAAGGAATGTGA
`
`SMN2
`
`Spinal muscular atrophy
`
`5'-CCGTCGCCCTTCAGCACGCA-3’
`SOD
`amyotrophic lateral sclerosis
`
`(ALS)
`
`5'-GTCGCCCTTCAGCACGCACA-3’
`SOD
`amyotrophic lateral sclerosis
`
`(ALS)
`
`amyotrophic lateral sclerosis
`SOD
`5'-CTACAGTTTAGCAGGACAG-3’
`(ALS)
`
`
`
`5’-TCTCTATTGCACATTCCAAG Huntington’s disease Huntington
`
`
`
`ll
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`WO 2009/008725
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`
`
`(IT15)
`
`5’-TGATCAGATCTTGAATGTGA
`Huntington
`Huntington’s disease
`
`(IT15)
`
`5’-GTAATCAGGCCTGCACCATG
`Huntington
`Huntington’s disease
`(IT15)
`
`
`5’-AAGCAATCCATGGACTGAAG
`Huntington
`Huntington’s disease
`(IT15)
`
`
`5’-CTGCTGCTGTTGCTGCTGCT
`Huntington
`Huntington’s disease
`
`(IT] 5)
`
`Huntington’s disease
`Huntington
`5’-C GCCTGCACCATGTTCCTCA
`(IT15)
`
`A person skilled in the art will readily recognise that variations on the sequences in the
`
`table above are possible while retaining complementarity to the target sequence. Uracile
`
`and thymidine nucleotides are interexchangable while retaining complementarity to the
`
`target sequence. The key is that the oligonucleotide should be able to bind with sufficient
`
`efficiency to the intended target sequence. Similarly, inosine (i.e. a nucleotide containing
`
`a base able to form a wobble base pair) could replace nucleotides while retaining
`
`complementarity. In other embodiments containing (triplet) nucleotide repeat sequences,
`
`it is readily recognised that for effective binding complementarity, the oligonucleotide
`
`can start and finish with any nucleotide of the repeat sequence and the oligonucleotide
`
`does not require to be an exact multiple of the repeat sequence. As an illustrative
`
`example, (CUG)n in the table above, could also be represented amongst others by
`
`(UGC)n or (CTG)n or (CIG)n or (CUG)nCU or (CUG)n(CTG)m(CUG)p (with n, m, p
`
`being integers), etcetera.
`
`One embodiment of the invention is the targeting of a virus or viral particle to cells. In a
`
`conjugate according to the invention the Virus or Viral particle is the biologically active
`
`moiety. In one embodiment the peptide or peptidomimetic according to the invention is
`
`linked to the Viral biologically active moiety by including the DNA/RNA sequence of the
`
`peptide or peptidomimetic in the genome of a virus such that
`
`the peptide or
`
`peptidomimetic is expressed at
`
`the outer surface of the virus or viral particle. The
`
`recombinant methodology to bring such expression about is well known to the skilled
`
`person. The peptide or peptidomimetic thus targets the virus or Viral particle to specific
`
`cells/tissue. This is of par