WO2012143427A1 - Anti polyomavirus compounds - Google Patents

Anti polyomavirus compounds Download PDF

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WO2012143427A1
WO2012143427A1 PCT/EP2012/057134 EP2012057134W WO2012143427A1 WO 2012143427 A1 WO2012143427 A1 WO 2012143427A1 EP 2012057134 W EP2012057134 W EP 2012057134W WO 2012143427 A1 WO2012143427 A1 WO 2012143427A1
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polyomavirus
oligomer
sequence
nucleotide sequence
nucleotides
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French (fr)
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Søren OTTOSEN
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Santaris Pharma A/S
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1131—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against viruses
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    • C12N2310/00—Structure or type of the nucleic acid
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    • C12N2310/11—Antisense
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    • C12N2310/00—Structure or type of the nucleic acid
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/32—Chemical structure of the sugar
    • C12N2310/323—Chemical structure of the sugar modified ring structure
    • C12N2310/3231—Chemical structure of the sugar modified ring structure having an additional ring, e.g. LNA, ENA
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/33—Chemical structure of the base
    • C12N2310/334—Modified C
    • C12N2310/3341—5-Methylcytosine
    • C—CHEMISTRY; METALLURGY
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/34—Spatial arrangement of the modifications
    • C12N2310/341—Gapmers, i.e. of the type ===---===

Definitions

  • the present invention relates to oligomeric compounds (oligomers), which target DNA viruses in a cell, leading to reduced expression of polyomavirus such as polyomavirus BK.
  • polyomavirus such as polyomavirus BK.
  • Reduction of polyomavirus is beneficial for a range of medical disorders, such as BK virus associated nephropathy.
  • therapeutically effective oligomers By targeting regions of the polyomavirus genome which encodes for multiple transcripts, therapeutically effective oligomers, the risk of the development of viral resistance to the oligomer may be reduced or avoided.
  • Polyomaviruses are DNA-based (double-stranded DNA, -5000 base pairs, circular genome), small (40-50 nanometers in diameter), and icosahedral in shape, and do not have a lipoprotein envelope. Moreover, the genome possess early and late genes, contributing to its complex transcription program. They are potentially oncogenic (tumor-causing); they often persist as latent infections in a host without causing disease, but can lead to significant and damaging infections when activated in, for instance, the kidney, the bladder or the central nervous system. Polyomaviruses may produce tumors in a host of a different species, or a host with an ineffective immune system.
  • BK virus associated nephropathy has been identified as a prevalent cause of kidney graft rejection.
  • the syndrome is caused by the activation of polyomavirus BK (BKV) and subsequent kidney viraemia, leading to damage of the transplanted kidney and eventually to rejection of the graft.
  • BKV polyomavirus BK
  • BKV polyomavirus BK
  • BK virus is involved in as many at 2/3 of all kidney graft losses.
  • BK virus infection may also be a factor in a fraction of haemorrhagic cystitis cases.
  • the BK virus which is a member of the polyomavirus family, carries a ca. 5200 nt double stranded DNA genome (Figure 1).
  • the genome exists as an autologous episome and expresses at least two kinetic classes of transcripts, the early genes and the late genes.
  • the two classes of transcripts are expressed from a divergent promoter and can roughly be divided into the regulatory early genes (Large T antigen and small T antigen) and the structural late genes (VP1 , VP2 and VP3).
  • the major exception from this rule is the so- called Agno protein, which is a primarily regulatory gene, but is expressed from the late gene promoter.
  • the early transcripts encode proteins with multiple functions, including acting as transcriptional activators of the late genes.
  • Each class of genes is expressed from heavily overlapping transcripts, including heavily overlapping reading frames.
  • the expressed genes are transcribed as mRNAs with canonical mammalian 5'-cap structures and 3'
  • DNA viruses Compared to most RNA viruses, DNA viruses generally carry less sequence variation. This holds true for BK virus as well, where a survey of all ca. 15000 possible 14-, 15-, and 16-mers were aligned against the 270 available full length BK virus sequences deposited with Genbank. This alignment identified at least 2000 perfectly conserved target sequences. BK virus also appears to have a high level of conservation to other human polyomaviruses such as the JC virus.
  • the invention provides an oligomer from 10 - 50, such as 10 - 30 nucleotides in length which comprises a contiguous nucleotide sequence (a first region) of a total of from 10 - 30 nucleotides, wherein said contiguous nucleotide sequence (a first region) is at least 80% (e.g., 85%, 90%, 95%, 98%, or 99%) homologous to a region corresponding to the reverse complement of a mammalian polyomavirus such as a polyomavirus BK gene or mRNA, such as any one of SEQ ID NO's: 1-4 mRNA or naturally occurring variant thereof.
  • the oligomer may hybridizes to (i.e. is complementary to) a single stranded nucleic acid molecule having the sequence of a portion of SEQ ID NO: 2 to 4 mRNA.
  • the invention provides an oligomer from 10 - 30 contiguous nucleotides in length, wherein said the contiguous nucleotide sequence of the oligomer is at least 80% (e.g., 85%, 90%, 95%, 98%, or 99% or 100%) homologous to a region corresponding to the
  • the oligomer hybridizes to a single stranded nucleic acid molecule having the sequence of a portion of SEQ ID NO: 2 to 4 mRNA.
  • the invention provides for a conjugate comprising the oligomer according to the invention, and at least one non-nucleotide or non- polynucleotide moiety covalently attached to said oligomer.
  • the invention provides for a pharmaceutical composition
  • a pharmaceutical composition comprising the oligomer or the conjugate according to the invention, and a pharmaceutically acceptable diluent, carrier, salt or adjuvant.
  • the invention provides for the oligomer or the conjugate according to invention, for use as a medicament, such as for the treatment of BK virus associated nephropathy and interstitial nephritis in kidney transplants (Jiang et al., 2009a, Cimbaluk et al. 2009)), ureteral stenosis (often in connection with kidney transplants) (Basara et al. 2010), haemorrhagic cystitis (pediatric; often in connection with bone marrow transplant) (Jiang et al 2009a).
  • the invention provides for the use of an oligomer or the conjugate according to the invention, for the manufacture of a medicament for the treatment of BK virus associated nephropathy and interstitial nephritis in kidney transplants (Jiang et al, 2009a, Cimbaluk et al 2009)), ureteral stenosis (often in connection with kidney transplants) (Basara et a/ 2010), haemorrhagic cystitis (pediatric; often in connection with bone marrow transplant) (Jiang et al 2009a).
  • the invention provides for a method of treating BK virus associated nephropathy and interstitial nephritis in kidney transplants (Jiang et al, 2009a, Cimbaluk et al 2009)), ureteral stenosis (often in connection with kidney transplants) (Basara et a/ 2010), haemorrhagic cystitis (pediatric; often in connection with bone marrow transplant) (Jiang et al 2009a), said method comprising administering an, e.g.
  • an oligomer, a conjugate or a pharmaceutical composition according to the invention to a patient suffering from, or likely to suffer from BK virus associated nephropathy and interstitial nephritis in kidney transplants (Jiang et al, 2009a, Cimbaluk et al 2009)), ureteral stenosis (often in connection with kidney transplants) (Basara et a/ 2010), haemorrhagic cystitis (pediatric; often in connection with bone marrow transplant) (Jiang et al 2009a)(such as a patient suffering from or susceptible to the disease or disorder).
  • the disease or disorder or condition is associated with infection of a patient by polyomavirus BK.
  • the invention provides for a method for the inhibition of polyomavirus such as polyomavirus BK in a cell which is expressing polyomavirus mRNA, said method comprising administering an oligomer, or a conjugate according to the invention to said cell so as to affect the inhibition of polyomavirus such as polyomavirus BK in said cell.
  • the invention provides an oligomer of from 10-50 monomers, which comprises a first region of 10-50 contiguous monomers, wherein the sequence of the first region is at least 80% identical to a region corresponding to a mammalian polyomavirus such as
  • polyomavirus BK gene or to the reverse complement of a target region of a nucleic acid which encodes a mammalian polyomavirus such as polyomavirus BK.
  • the invention further provides a conjugate comprising the oligomer according to the invention, which comprises at least one non-nucleotide or non-polynucleotide moiety ("conjugated moiety") covalently attached to the oligomer of the invention.
  • conjugated moiety at least one non-nucleotide or non-polynucleotide moiety
  • compositions comprising an oligomer or conjugate of the invention, and a pharmaceutically acceptable diluent, carrier, salt or adjuvant.
  • the invention further provides for an oligomer according to the invention, for use in medicine.
  • the invention further provides for the use of the oligomer of the invention for the manufacture of a medicament for the treatment of one or more of the diseases referred to herein, such as a disease selected from the group consisting of BK virus associated nephropathy and interstitial nephritis in kidney transplants (Jiang et al, 2009a, Cimbaluk et al 2009)), ureteral stenosis (often in connection with kidney transplants) (Basara et a/ 2010), haemorrhagic cystitis (pediatric; often in connection with bone marrow transplant) (Jiang et al 2009a).
  • a disease selected from the group consisting of BK virus associated nephropathy and interstitial nephritis in kidney transplants (Jiang et al, 2009a, Cimbaluk et al 2009)
  • ureteral stenosis often in connection with kidney transplants
  • Basara et a/ 2010 haemor
  • the invention further provides for an oligomer according to the invention, for use for the treatment of one or more of the diseases referred to herein, such as a disease selected from the group consisting of BK virus associated nephropathy and interstitial nephritis in kidney transplants (Jiang et al, 2009a, Cimbaluk et al 2009)), ureteral stenosis (often in connection with kidney transplants) (Basara et a/ 2010), haemorrhagic cystitis (pediatric; often in connection with bone marrow transplant) (Jiang et al 2009a).
  • a disease selected from the group consisting of BK virus associated nephropathy and interstitial nephritis in kidney transplants (Jiang et al, 2009a, Cimbaluk et al 2009)
  • ureteral stenosis often in connection with kidney transplants
  • Basara et a/ 2010 haemorrhagic cystit
  • polyomavirus such as polyomavirus BK in cells or tissues
  • methods of down-regulating the expression of polyomavirus comprising contacting said cells or tissues, in vitro or in vivo, with an effective amount of one or more of the oligomers, conjugates or compositions of the invention.
  • the invention provides for a method for treating a disease selected from the group consisting of: BK virus associated nephropathy and interstitial nephritis in kidney transplants (Jiang et al, 2009a, Cimbaluk et a/ 2009)), ureteral stenosis (often in connection with kidney transplants) (Basara et al 2010), haemorrhagic cystitis (pediatric; often in connection with bone marrow transplant) (Jiang et al 2009a), the method comprising administering an effective amount of one or more oligomers, conjugates, or pharmaceutical compositions thereof to an animal in need thereof (such as a patient in need thereof).
  • a disease selected from the group consisting of: BK virus associated nephropathy and interstitial nephritis in kidney transplants (Jiang et al, 2009a, Cimbaluk et a/ 2009)), ureteral stenosis (often in connection with kidney transplants
  • the invention provides for methods of inhibiting (e.g., by down-regulating) the expression of polyomavirus such as polyomavirus BK in a cell or a tissue, the method comprising the step of contacting the cell or tissue, in vitro or in vivo, with an effective amount of one or more oligomers, conjugates, or pharmaceutical compositions thereof, to affect down-regulation of expression of polyomavirus mRNA.
  • polyomavirus such as polyomavirus BK
  • the invention provides for a method for preparing an oligomer for use in inhibiting polyomavirus infection in a cell, said method comprising the steps of i) selecting a target region of a polyomavirus genome (or mRNA encoded by the polyomavirus genome), wherein the target region of the polyomavirus present in at least two, such as two or three independent transcripts expressed by the polyomavirus, and; ii) synthesising an oligomer of up to 30 nucleotides in length which comprises or consists of a contiguous nucleotide sequence of 10 - 30 nucleotides, wherein the contiguous nucleotide sequence is the reverse complement of the target region of the polyomavirus.
  • the invention provides for an oligomer of 10 - 30 nucleotides in length, which comprises or consists of a contiguous nucleotide sequence of 10 - 30 nucleotides, wherein the contiguous nucleotide sequence is the complement of a target region of a polyomavirus genome, wherein the target region of the polyomavirus is present in at least two, such as two or three, independent transcripts expressed by the polyomavirus.
  • the target region of a polyomavirus genome is therefore a region of the polyomavirus genome which is transcribed or a region of a mRNA transcribed from the polyomavirus.
  • the at least two or three independent transcripts may be transcribed from over-lapping open-reading frames present in the polyomavirus genome.
  • the independent transcripts are the results of unique and specific transcription and processing events, although derived, at least partially, from the same genomic sequence.
  • the sequence present in the independent transcripts which is complementary to the contiguous nucleotide sequence of the oligomer may originate from the same genetic loci on the polyomavirus genome.
  • the invention provides for an oligomer of up to 30 nucleotides in length, which comprises or consists of a contiguous nucleotide sequence of 10 - 30 nucleotides, wherein the contiguous nucleotide sequence is the reverse complement of a target region of a polyomavirus genome (or mRNA), wherein the target region of the polyomavirus is present at least two, such as two or three, non-identical transcripts, wherein each non-identical transcript comprises an independent open reading frame.
  • An "independent open reading frame" results in an independently expressed protein, although some non-identical transcripts (also refered to as an independent transcript), in some embodiments, may give rise to proteins with partially conserved protein sequences, but protein products with distinct and different functions.
  • the non-identical transcripts encode for gene-products (typically proteins) with distinct functions.
  • the oligomer may be for use/suitable for use, in inhibiting polyomavirus infection in a cell, such as a mammalian cell or human cell.
  • the polyomavirus infection may be polyomavirus BK infection.
  • FIG 1 circular representation of the genome structure of bk virus.
  • Bk virus expresses at least 6 gene products, from a divergent promoter region located at the replication origin.
  • the early genes are large t antigen (CDS6), which contains an intron, and SMALL T ANTIGEN (CDS6).
  • the late genes are AGNO (CDS 1), VP2 (CDS 2), VP3 (CDS 3) AND VP1 (CDS4).
  • FIG. 2 psiCHECKTM-2 vector.
  • a family of transiently expressed reporters were generated by inserting fragments of the BK virus genome in the MCS in the 3'-UTR of the Renilla luciferase gene of the psiCHECK-2 vector. Oligonucleotide efficacy was assessed by co-transfection of reporter plasmid and oligonucleotides and normalizing the effect on Renilla luciferase (hRluc) to firefly luciferase (hluc+), expressed from a separate promoter on the plasmid. Image borrowed from Promega psiCHECK-2 manual.
  • FIG. 3 Luciferase knockdown of Large T antigen reporter ("VP5") by large T antigen specific oligonucleotides. Renilla luciferase activity was measured 24h after transfection and was normalized to firefly luciferase. Data are expressed as % of mock (100%) and are presented as average ⁇ standard deviation of three independent experiments.
  • VP5 Large T antigen reporter
  • FIG. 4 Luciferase knockdown of Large T antigen/small t antigen reporter ("VP6") by large T/small t antigen specific oligonucleotides. Renilla luciferase activity was measured 24h after transfection and was normalized to firefly luciferase. Data are expressed as % of mock (100%) and are presented as average ⁇ standard deviation of three independent
  • FIG. 5 Luciferase knockdown of Agno protein reporter ("VP6") by Agno protein specific oligonucleotides. Renilla luciferase activity was measured 24h after transfection and was normalized to firefly luciferase. Data are expressed as % of mock (100%) and are presented as average ⁇ standard deviation of three independent experiments.
  • VP6 Agno protein reporter
  • FIG. 6 Luciferase knockdown of VP1 protein reporter ("VP4") by VP1 protein specific oligonucleotides. Renilla luciferase activity was measured 24h after transfection and was normalized to firefly luciferase. Data are expressed as % of mock (100%) and are presented as average ⁇ standard deviation of three independent experiments.
  • FIG. 7 Early (T-antigen) and late (VP1) mRNA expression in infected RPTEC cells after transfection with early-transcript targeting oligonucleotides.
  • Figure 8 Early (T-antigen) and late (VP1) mRNA expression in infected RPTEC cells after transfection with late transcript-targeting oligonucleotides.
  • FIG. 9 Early (T-antigen) and late (VP1) mRNA expression in infected RPTEC cells after gymnosis treatment with early-transcript targeting oligonucleotides. 5911 and 3088 are negative controls, targeting either a host gene (OPN1) or with no target, respectively.
  • OPN1 host gene
  • FIG. 10 Early (T-antigen) and late (VP1) mRNA expression in infected RPTEC cells after gymnosis treatment with late-transcript targeting oligonucleotides. 591 1 and 3088 are negative controls, targeting either a host gene (OPN1) or with no target, respectively. DETAILED DESCRIPTION OF INVENTION
  • oligomeric compounds for use in modulating (e.g. inhibiting) the function of nucleic acid molecules encoding polyomavirus, such as a polyomavirus selected from the group consisting of polyomavirus BK, JC polyomavirus, Kl polyomavirus, WU polyomavirus, Merkel polyomavirus, and Simian virus 40 (SV40).
  • polyomavirus selected from the group consisting of polyomavirus BK, JC polyomavirus, Kl polyomavirus, WU polyomavirus, Merkel polyomavirus, and Simian virus 40 (SV40).
  • polyomavirus selected from the group consisting of polyomavirus BK, JC polyomavirus, Kl polyomavirus, WU polyomavirus, Merkel polyomavirus, and Simian virus 40 (SV40).
  • SV40 Simian virus 40
  • the oligomer of the invention is for use in modulating (e.g. inhibiting) the function of a nucleic acid encoding polyomavirus BK, such as a nucleic acid shown selected from the group consisting of SEQ ID 1 , 2, 3, and 4, and naturally occurring variants of thereof.
  • a nucleic acid encoding polyomavirus BK such as a nucleic acid shown selected from the group consisting of SEQ ID 1 , 2, 3, and 4, and naturally occurring variants of thereof.
  • oligomer in the context of the present invention, refers to a molecule formed by covalent linkage of two or more nucleotides (i.e. an oligonucleotide).
  • a single nucleotide (unit) may also be referred to as a monomer or unit.
  • nucleoside In some embodiments, the terms “nucleoside”, “nucleotide”, “unit” and “monomer” are used interchangeably. It will be recognised that when referring to a sequence of nucleotides or monomers, what is referred to is the sequence of bases, such as A, T, G, C or U.
  • the oligomer consists or comprises of a contiguous nucleotide sequence of from 10 - 50, such as 10 - 30 nucleotides in length.
  • the compound of the invention does not comprise RNA (units). It is preferred that the compound according to the invention is a linear molecule or is synthesised as a linear molecule.
  • the oligomer is a single stranded molecule, and preferably does not comprise short regions of, for example, at least 3, 4 or 5 contiguous nucleotides, which are complementary to equivalent regions within the same oligomer (i.e. duplexes) - in this regards, the oligomer is not (essentially) double stranded. In some embodiments, the oligomer is essentially not double stranded, such as is not a siRNA. In various aspects, the oligomer is essentially not double stranded, such as is not a siRNA.
  • the oligomer of the invention may consist entirely of the contiguous nucleotide region.
  • the oligomer is not substantially self-complementary.
  • the oligomer of the invention is capable of down-regulating (e.g. reducing or removing) expression of the polyomavirus, such as a polyomavirus selected from the group consisting of polyomavirus BK, JC polyomavirus, Kl polyomavirus, WU polyomavirus, Merkel polyomavirus, and Simina virus 40 (SV40).
  • a polyomavirus selected from the group consisting of polyomavirus BK, JC polyomavirus, Kl polyomavirus, WU polyomavirus, Merkel polyomavirus, and Simina virus 40 (SV40).
  • the oligomer of the invention may target/affect the inhibition of polyomavirus BK mRNA, typically in a mammalian such as a human cell, such as a human kidney cell, such as a renal proximal tubular epithelial cell.
  • the oligomers of the invention bind to the target nucleic acid and affect inhibition of expression of at least 10% or 20% compared to the normal expression level, more preferably at least a 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% inhibition compared to the normal expression level (such as the expression level in the absence of the oligomer(s) or conjugate(s)).
  • the normal expression level such as the expression level in the absence of the oligomer(s) or conjugate(s)
  • such modulation is seen when using from 0.04 and 25nM, such as from 0.8 and 20nM concentration of the compound of the invention.
  • the inhibition of expression is less than 100%, such as less than 98% inhibition, less than 95% inhibition, less than 90% inhibition, less than 80% inhibition, such as less than 70% inhibition.
  • Modulation of expression level may be determined by measuring protein levels, e.g. by the methods such as SDS-PAGE followed by western blotting using suitable antibodies raised against the target protein.
  • modulation of expression levels can be determined by measuring levels of mRNA, e.g. by northern blotting or quantitative RT-PCR.
  • the level of down-regulation when using an appropriate dosage is, In some embodiments, typically to a level of from 10-20% the normal levels in the absence of the compound, conjugate or composition of the invention.
  • the cell type may, in some embodiments, be Human Kidney Cells (e.g. in vitro - transfected cells).
  • the oligomer concentration used (e.g. in Human Kidney Cells) may, in some embodiments, be 5nM.
  • the oligomer concentration used may, in some embodiments be 25nM (e.g. in Human Kidney Cells .
  • the oligomer concentration used may, in some embodiments be 1 nM (e.g. in Human Kidney Cells).
  • the concentration of oligomer used to treat the cell is typically performed in an in vitro cell assay, using transfection (Lipofecton), as illustrated in the examples. In the absence of a transfection agent, the oligo concentration required to obtain the down-regulation of the target is typically between 1 and 25 ⁇ , such as 5 ⁇ .
  • the phrase "potent inhibitor” refers to an oligomer with an IC50 of less than 5nM as determined by the lipofectamin transfection assay of example 3. In some embodiments, the IC50 is less than 4nM, such as less than 2nM.
  • the invention therefore provides a method of down-regulating or inhibiting the expression of polyomavirus such as a polyomavirus selected from the group consisting of polyomavirus BK, JC polyomavirus, Kl polyomavirus, WU polyomavirus, Merkel
  • polyomavirus such as a polyomavirus selected from the group consisting of polyomavirus BK, JC polyomavirus, Kl polyomavirus, WU polyomavirus, Merkel
  • the method comprising administering the oligomer or conjugate according to the invention to said cell to down-regulating or inhibiting the expression of polyomavirus such as polyomavirus BK protein and/or mRNA in said cell.
  • the cell is a mammalian cell such as a human cell, such as (e.g. in relation to BKV) a kidney cell, such as a renal proximal tubular epithelial cell
  • the administration may occur, in some embodiments, in vitro.
  • the administration may occur, in some embodiments, in vivo.
  • target nucleic acid refers to the DNA or RNA encoding mammalian polyomavirus such as polyomavirus BK polypeptide mRNA.
  • Polyomavirus such as polyomavirus BK encoding nucleic acids or naturally occurring variants thereof, and RNA nucleic acids derived therefrom, preferably mRNA, such as pre-mRNA, although preferably mature mRNA.
  • the "target nucleic acid” may be a cDNA or a synthetic oligonucleotide derived from the above DNA or RNA nucleic acid targets.
  • the oligomer according to the invention is preferably capable of hybridising to the target nucleic acid.
  • naturally occurring variant thereof refers to variants of the polyomavirus such as polyomavirus BK polypeptide of nucleic acid sequence which exist naturally within the defined taxonomic group.
  • the polyomavirus may, for example, be selected from the following:
  • SEQ ID NO 6 4967 of SEQ ID NO 6 wu possible cancer 440266 NC_009539.1 1003 to 1669, 1670 polyomavirus causing agent Gl: 148724565 to 1821 , 4906 to
  • Simian virus SV40 (non- 10633 NC_001669.1 916 to 1498, 1499 40 (SV40) human viral GI : 9628421 to 1620, 4918 to disease - used SEQ ID NO 84 5163 of SEQ ID NO as a model for 84 polymorviruses
  • the oligomer of the invention may consist or comprise of a contiguous nucleotide sequence of 10 - 30 nucleotides in length, wherein the contiguous nucleotide sequence is at least 80% complementary, such as at least 90% complementary to, such as fully complementary to a target polyomavirus nucleotide sequence.
  • the target polyomavirus sequence may be present in at least one, such as at least two, independent RNA sequences, such as mRNA sequences, which is/are transcribed from at least one region of the polyomavirus genome.
  • the first and second RNA targets may originate from a region of the polyomavirus genome which comprises at least two overlapping and independently expressed transcripts, wherein a first transcript encodes for the first RNA target using a first open reading frame (ORF), and a second transcript encodes for the second RNA target using a second ORF, and wherein both ORFs comprise the target polyomavirus nucleotide sequence or reverse complement thereof.
  • ORF open reading frame
  • both ORFs comprise the target polyomavirus nucleotide sequence or reverse complement thereof.
  • the target polyomavirus nucleotide sequence is present in the two or more ORFs.
  • the oligomer may be targeted to a target sequence selected from the group consisting of SEQ ID NOs 2 - 4.
  • Polyomavirus JC Large T Nucleotides 4771 to 5013 of SEQ ID NO:5 antigen overlap with small T
  • Polyomavirus Kl Large T antigen Nucleotides 4716 to 4967 of SEQ ID NO:6 overlap with small T antigen
  • Polyomavirus WU Large T Nucleotides 4906 to 5157 of SEQ ID NO:7 antigen overlap with small T
  • Simian virus 40 (SV40) VP2 and Nucleotides 916 to 1498 of SEQ ID NO:84 VP3 overlap mRNA sequence
  • Simian virus 40 (SV40) VP2
  • Nucleotides 1499 to 1620 of SEQ ID NO:84 VP3 and VP1 overlap mRNA
  • Simian virus 40 Large T Nucleotides 4918 to 5163 of SEQ ID NO:84 antigen overlap with small T
  • the oligomers may comprise or consist of a contiguous nucleotide sequence which corresponds to the reverse complement of a nucleotide sequence present in SEQ ID NO: 2, 3 or 4.
  • the oligomer may comprise or consist of, or a sequence selected from the group consisting of SEQ ID NOS: 31-53, wherein said oligomer (or contiguous nucleotide portion thereof) may optionally have one, two, or three mismatches against said selected sequence.
  • the oligomer may comprise or consist of a contiguous nucleotide sequence which is fully complementary (perfectly complementary) to the equivalent region of a nucleic acid which encodes a mammalian polyomavirus such as polyomavirus BK (e.g., SEQ ID NO's: 31 - 53 ).
  • the oligomer can comprise or consist of an antisense nucleotide sequence.
  • the oligomer may tolerate e.g 1 , 2, 3, or 4 (or more) mismatches, when hybridising to the target sequence and still sufficiently bind to the target to show the desired effect, i.e. down-regulation of the target.
  • Mismatches may, for example, be compensated by increased length of the oligomer nucleotide sequence and/or an increased number of nucleotide analogues, such as LNA, present within the nucleotide sequence.
  • the contiguous nucleotide sequence comprises no more than 3, such as no more than 2 mismatches when hybridizing to the target sequence, such as to the corresponding region of a nucleic acid which encodes a mammalian polyomavirus such as polyomavirus BK.
  • the contiguous nucleotide sequence comprises no more than a single mismatch when hybridizing to the target sequence, such as the corresponding region of a nucleic acid which encodes a polyomavirus such as polyomavirus BK.
  • the nucleotide sequence of the oligomers of the invention or the contiguous nucleotide sequence is preferably at least 80% homologous to a corresponding region in a sequence selected from the group consisting of SEQ ID NOS: 1 , 5, 6 or 7, such as at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96% homologous, at least 97% homologous, at least 98% homologous, at least 99% homologous, such as 100%
  • the nucleotide sequence of the oligomers of the invention or the contiguous nucleotide sequence is preferably at least 80% homologous to a corresponding region in a sequence selected from the group consisting of SEQ ID NOS: 1 , 3, 2 and 4 such as at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96% homologous, at least 97% homologous, at least 98% homologous, at least 99% homologous, such as 100%
  • the nucleotide sequence of the oligomers of the invention or the contiguous nucleotide sequence is preferably at least 80% homologous to a corresponding region in a sequence selected from the group consisting of SEQ ID NO 5, such as at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96% homologous, at least 97%
  • homologous at least 98% homologous, at least 99% homologous, such as 100%
  • the nucleotide sequence of the oligomers of the invention or the contiguous nucleotide sequence is preferably at least 80% homologous to a corresponding region in a sequence selected from the group consisting of SEQ ID NO 6 such as at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96% homologous, at least 97% homologous, at least 98% homologous, at least 99% homologous, such as 100% homologous (identical). See table 5 for some exemplary target regions of SEQ ID NO 6.
  • the nucleotide sequence of the oligomers of the invention or the contiguous nucleotide sequence is preferably at least 80% homologous to a corresponding region in a sequence selected from the group consisting of SEQ ID NO 7 such as at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96% homologous, at least 97% homologous, at least 98% homologous, at least 99% homologous, such as 100% homologous (identical). See table 5 for some exemplary target regions of SEQ ID NO 7.
  • the nucleotide sequence of the oligomers of the invention or the contiguous nucleotide sequence is preferably at least 80% homologous to a corresponding region in a sequence selected from the group consisting of SEQ ID NO 83 such as at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96% homologous, at least 97%
  • homologous at least 98% homologous, at least 99% homologous, such as 100%
  • the nucleotide sequence of the oligomers of the invention or the contiguous nucleotide sequence is preferably at least 80% homologous to a corresponding region in a sequence selected from the group consisting of SEQ ID NO 84 such as at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96% homologous, at least 97%
  • homologous at least 98% homologous, at least 99% homologous, such as 100%
  • the nucleotide sequence of the oligomers of the invention or the contiguous nucleotide sequence is preferably at least 80% homologous to a corresponding sequence selected from the group consisting of SEQ ID NOS: 54-77, such as at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96% homologous, at least 97% homologous, at least 98% homologous, at least 99% homologous, such as 100% homologous (identical).
  • the nucleotide sequence of the oligomers of the invention or the contiguous nucleotide sequence is preferably at least 80% homologous to the reverse complement of a corresponding sequence present in SEQ ID NO: 31-53, such as at least 85%, at least 90%, at least 91 %, at least 92%at least 93%, at least 94%, at least 95%, at least 96% homologous, at least 97% homologous, at least 98% homologous, at least 99% homologous, such as 100% homologous (identical).
  • the nucleotide sequence of the oligomers of the invention or the contiguous nucleotide sequence is preferably at least 80% complementary to a sub-sequence present in SEQ ID NO: 2, 3 or 4, such as at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96% complementary, at least 97% complementary, at least 98% complementary, at least 99% complementary, such as 100% complementary (perfectly complementary).
  • first region refers to a portion (subsequence) of an oligomer.
  • the 12 monomer sequence SEQ ID No 14 is a subsequence of the 16 monomer sequence SEQ ID No 12 and comprises 12 contiguous monomers of SEQ ID No 12.
  • the oligomer (or contiguous nucleotide portion thereof) is selected from, or comprises, one of the sequences selected from the group consisting of SEQ ID NOS: 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, or a sub-sequence of at least 10 contiguous nucleotides thereof, wherein said oligomer (or contiguous nucleotide portion thereof) may optionally comprise one, two, or three mismatches when compared to the sequence.
  • the sub-sequence may consist of 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, or 29 contiguous nucleotides, such as from 12 -22, such as from 12-18 nucleotides.
  • the sub-sequence is of the same length as the contiguous nucleotide sequence of the oligomer of the invention.
  • the nucleotide sequence of the oligomer may comprise additional 5' or 3' nucleotides, such as, independently, 1 , 2, 3, 4 or 5 additional nucleotides 5' and/or 3', which are non-complementary to the target sequence.
  • the oligomer of the invention may, in some embodiments, comprise a contiguous nucleotide sequence which is flanked 5' and or 3' by additional nucleotides.
  • the additional 5' or 3' nucleotides are naturally occurring nucleotides, such as DNA or RNA.
  • the additional 5' or 3' nucleotides may represent region D as referred to in the context of gapmer oligomers herein.
  • the oligomer according to the invention consists or comprises of a nucleotide sequence according to SEQ ID NO: 2, or a sub-sequence thereof.
  • the oligomer according to the invention consists or comprises of a nucleotide sequence according to SEQ ID NO: 3, or a sub-sequence thereof.
  • the oligomer according to the invention consists or comprises of a nucleotide sequence according to SEQ ID NO: 4, or a sub-sequence thereof.
  • the oligomer according to the invention consists or comprises of a nucleotide sequence according to SEQ ID NO: 31 , or a sub-sequence thereof.
  • the oligomer according to the invention consists or comprises of a nucleotide sequence according to SEQ ID NO: 32, or a sub-sequence thereof.
  • the oligomer according to the invention consists or comprises of a nucleotide sequence according to SEQ ID NO: 33, or a sub-sequence thereof.
  • the oligomer according to the invention consists or comprises of a nucleotide sequence according to SEQ ID NO: 34, or a sub-sequence thereof.
  • the oligomer according to the invention consists or comprises of a nucleotide sequence according to SEQ ID NO: 35, or a sub-sequence thereof.
  • the oligomer according to the invention consists or comprises of a nucleotide sequence according to SEQ ID NO: 36, or a sub-sequence thereof.
  • the oligomer according to the invention consists or comprises of a nucleotide sequence according to SEQ ID NO: 37, or a sub-sequence thereof.
  • the oligomer according to the invention consists or comprises of a nucleotide sequence according to SEQ ID NO: 38, or a sub-sequence thereof.
  • the oligomer according to the invention consists or comprises of a nucleotide sequence according to SEQ ID NO: 39, or a sub-sequence thereof.
  • the oligomer according to the invention consists or comprises of a nucleotide sequence according to SEQ ID NO: 40, or a sub-sequence thereof.
  • the oligomer according to the invention consists or comprises of a nucleotide sequence according to SEQ ID NO: 41 , or a sub-sequence thereof.
  • the oligomer according to the invention consists or comprises of a nucleotide sequence according to SEQ ID NO: 42, or a sub-sequence thereof.
  • the oligomer according to the invention consists or comprises of a nucleotide sequence according to SEQ ID NO: 43, or a sub-sequence thereof. In some embodiments the oligomer according to the invention consists or comprises of a nucleotide sequence according to SEQ ID NO: 44, or a sub-sequence thereof.
  • the oligomer according to the invention consists or comprises of a nucleotide sequence according to SEQ ID NO: 45, or a sub-sequence thereof.
  • the oligomer according to the invention consists or comprises of a nucleotide sequence according to SEQ ID NO: 46, or a sub-sequence thereof.
  • the oligomer according to the invention consists or comprises of a nucleotide sequence according to SEQ ID NO: 47, or a sub-sequence thereof.
  • the oligomer according to the invention consists or comprises of a nucleotide sequence according to SEQ ID NO: 48, or a sub-sequence thereof.
  • the oligomer according to the invention consists or comprises of a nucleotide sequence according to SEQ ID NO: 49, or a sub-sequence thereof.
  • the oligomer according to the invention consists or comprises of a nucleotide sequence according to SEQ ID NO: 50, or a sub-sequence thereof.
  • the oligomer according to the invention consists or comprises of a nucleotide sequence according to SEQ ID NO: 51 , or a sub-sequence thereof.
  • the oligomer according to the invention consists or comprises of a nucleotide sequence according to SEQ ID NO: 52, or a sub-sequence thereof.
  • the oligomer according to the invention consists or comprises of a nucleotide sequence according to SEQ ID NO: 53, or a sub-sequence thereof.
  • Target mRNA 434-449 AAACCTGGACTGGAAC
  • Target mRNA 966-981 TATC AG C AATC AG G C A
  • Target mRNA 1092-1107 TTGTTTGCTACTATTT
  • Target mRNA 1469-1484 AGGAGGTGCTAATCAA
  • Target mRNA 1471-1482 GAGGTGCTAATC
  • Target mRNA 1494-1509 CCTCAATGGATGTTGC
  • Target mRNA 1539-1554 ACACCTGCTCTTGAAG
  • Target mRNA 1583-1598 AAGGAGAGTGTCCAGG
  • Target mRNA 1584-1597 AGGAGAGTGTCCAG
  • Target mRNA 1653-1668 AGGAGGAGTAGAAGTT
  • Target mRNA 2041-2056 TTGGAAATGCAGGGAG
  • Target mRNA 2976-2989 TTCTGAGATAAGTATG
  • Target mRNA 4120-4135 TAAGGGTGTTAATAAG
  • Target mRNA 4121-4134 AAGGGTGTTAATAA
  • Target mRNA 4543-4556 ATGGAACAGAAGAGTG
  • Target mRNA 4906-4919 AGCTCAGAGGTTTGTG
  • Target mRNA 5023-5036 GAATTTCACCCTGACA 54
  • Antisense sequence 434-449 GTTCCAGTCCAGGTTT
  • the oligomer according to the invention consists or comprises of a contiguous nucleotide sequence which is fully complementary to a contiguous nucleotide sequence present in (a sub-sequence of) SEQ ID NO: 79.
  • the oligomer according to the invention consists or comprises of a contiguous nucleotide sequence which is fully complementary to a contiguous nucleotide sequence present in (a sub-sequence of) SEQ ID NO: 80. In some embodiments the oligomer according to the invention consists or comprises of a contiguous nucleotide sequence which is fully complementary to a contiguous nucleotide sequence present in (a sub-sequence of) SEQ ID NO: 81.
  • the oligomer according to the invention consists or comprises of a contiguous nucleotide sequence which is fully homologous to a contiguous nucleotide sequence present in (a sub-sequence of) SEQ ID NO: 82.
  • the degree of “complementarity” is expressed as the percentage identity (or percentage homology) between the sequence of the oligomer (or region thereof) and the sequence of the target region (or the reverse complement of the target region) that best aligns therewith. The percentage is calculated by counting the number of aligned bases that are identical between the 2 sequences, dividing by the total number of contiguous monomers in the oligomer, and multiplying by 100. In such a comparison, if gaps exist, it is preferable that such gaps are merely mismatches rather than areas where the number of monomers within the gap differs between the oligomer of the invention and the target region.
  • nucleotide sequence of the oligomer i.e. the nucleobase or base sequence
  • contiguous nucleotide sequence a first region
  • equivalent contiguous nucleotide sequence of a further sequence selected from either i) a sub-sequence of the reverse complement of the nucleic acid target, such as the mRNA which encodes the polyomavirus proteins, such as the targets listed in table 4 or 5, such as one of the polyomavirus BK proteins, or SEQ ID NO: 2, 3 or 4, and/or ii) the sequence of nucleotides provided herein, such as the group consisting of SEQ ID NOS: 31 to 53, or sub-sequence thereof.
  • Nucleotide analogues are compared directly to their equivalent or corresponding nucleotides.
  • a first sequence which corresponds to a further sequence under i) or ii) typically is identicial to that sequence over the length of the first sequence (such as the contiguous nucleotide sequence) or, as described herein may, in some embodiments, is at least 80% homologous to a corresponding sequence, such as at least 85%, at least 90%, at least 91 %, at least 92%at least 93%, at least 94%, at least 95%, at least 96% homologous, such as 100% homologous (identical).
  • nucleotide analogue and “corresponding nucleotide” are intended to indicate that the nucleotide in the nucleotide analogue and the naturally occurring nucleotide are identical.
  • the "corresponding nucleotide analogue” contains a pentose unit (different from 2-deoxyribose) linked to an adenine.
  • the oligomers may comprise or consist of a contiguous nucleotide sequence of a total of 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29 or 30 contiguous nucleotides in length.
  • the oligomers comprise or consist of a contiguous nucleotide sequence of a total of from 10 - 22, such as 12 - 18, such as 13 - 17 or 12 - 16, or 10-16, such as 10, 11 , 12, 13, 14, 15, 16 contiguous nucleotides in length.
  • the oligomers comprise or consist of a contiguous nucleotide sequence of a total of 10, 11 , 12, 13, or 14 contiguous nucleotides in length.
  • the oligomer according to the invention consists of no more than 22 nucleotides, such as no more than 20 nucleotides, such as no more than 18 nucleotides, such as 15, 16 or 17 nucleotides. In some embodiments the oligomer of the invention comprises less than 20 nucleotides. It should be understood that when a range is given for an oligomer, or contiguous nucleotide sequence length it includes the lower an upper lengths provided in the range, for example from (or between) 10 - 30, includes both 10 and 30.
  • nucleoside analogue and “nucleotide analogue” are used interchangeably.
  • nucleotide refers to a glycoside comprising a sugar moiety, a base moiety and a covalently linked group (linkage group), such as a phosphate or phosphorothioate internucleotide linkage group, and covers both naturally occurring nucleotides, such as DNA or RNA, and non-naturally occurring nucleotides comprising modified sugar and/or base moieties, which are also referred to as “nucleotide analogues" herein.
  • a single nucleotide (unit) may also be referred to as a monomer or nucleic acid unit.
  • nucleoside is commonly used to refer to a glycoside comprising a sugar moiety and a base moiety, and may therefore be used when referring to the nucleotide units, which are covalently linked by the internucleotide linkages between the nucleotides of the oligomer.
  • nucleotide is often used to refer to a nucleic acid monomer or unit, and as such in the context of an oligonucleotide may refer to the base - such as the "nucleotide sequence”, typically refer to the nucleobase sequence (i.e. the presence of the sugar backbone and internucleoside linkages are implicit).
  • nucleotide may refer to a nucleic acid monomer or unit, and as such in the context of an oligonucleotide may refer to the base - such as the "nucleotide sequence", typically refer to the nucleobase sequence (i.e. the presence of the sugar backbone and internucleoside linkages are implicit).
  • nucleoside for example the term “nucleotide” may be used, even when specifiying the presence or nature of the linkages between the nucleosides.
  • the 5' terminal nucleotide of an oligonucleotide does not comprise a 5' internucleotide linkage group, although may or may not comprise a 5' terminal group.
  • Non-naturally occurring nucleotides include nucleotides which have modified sugar moieties, such as bicyclic nucleotides or 2' modified nucleotides, such as 2' substituted nucleotides.
  • Nucleotide analogues are variants of natural nucleotides, such as DNA or RNA nucleotides, by virtue of modifications in the sugar and/or base moieties. Analogues could in principle be merely “silent” or “equivalent” to the natural nucleotides in the context of the oligonucleotide, i.e. have no functional effect on the way the oligonucleotide works to inhibit target gene expression. Such "equivalent” analogues may nevertheless be useful if, for example, they are easier or cheaper to manufacture, or are more stable to storage or manufacturing conditions, or represent a tag or label.
  • the analogues will have a functional effect on the way in which the oligomer works to inhibit expression; for example by producing increased binding affinity to the target and/or increased resistance to intracellular nucleases and/or increased ease of transport into the cell.
  • nucleoside analogues are described by e.g. Freier & Altmann; Nucl. Acid Res., 1997, 25, 4429-4443 and Uhlmann; Curr. Opinion in Drug Development, 2000, 3(2), 293-213, and in Scheme 1 :
  • the oligomer may thus comprise or consist of a simple sequence of natural occurring nucleotides - preferably 2'-deoxynucleotides (referred to here generally as "DNA”), but also possibly ribonucleotides (referred to here generally as "RNA”), or a combination of such naturally occurring nucleotides and one or more non-naturally occurring nucleotides, i.e. nucleotide analogues.
  • nucleotide analogues may suitably enhance the affinity of the oligomer for the target sequence.
  • the oligomer comprises at least 1 nucleoside analogue. In some embodiments the oligomer comprises at least 2 nucleotide analogues. In some embodiments, the oligomer comprises from 3-8 nucleotide analogues, e.g. 6 or 7 nucleotide analogues.
  • At least one of said nucleotide analogues is a locked nucleic acid (LNA); for example at least 3 or at least 4, or at least 5, or at least 6, or at least 7, or 8, of the nucleotide analogues may be LNA.
  • LNA locked nucleic acid
  • all the nucleotides analogues may be LNA.
  • the oligomers of the invention which are defined by that sequence may comprise a corresponding nucleotide analogue in place of one or more of the nucleotides present in said sequence, such as LNA units or other nucleotide analogues, which raise the duplex stability/T m of the oligomer/target duplex (i.e. affinity enhancing nucleotide analogues).
  • any mismatches between the nucleotide sequence of the oligomer and the target sequence are preferably found in regions outside the affinity enhancing nucleotide analogues, such as region B as referred to herein, and/or region D as referred to herein, and/or at the site of non modified such as DNA nucleotides in the oligonucleotide, and/or in regions which are 5' or 3' to the contiguous nucleotide sequence.
  • modification of the nucleotide include modifying the sugar moiety to provide a 2'-substituent group or to produce a bridged (locked nucleic acid) structure which enhances binding affinity and may also provide increased nuclease resistance.
  • a preferred nucleotide analogue is LNA, such as oxy-LNA (such as beta-D-oxy-LNA, and alpha-L-oxy-LNA), and/or amino-LNA (such as beta-D-amino-LNA and alpha-L-amino- LNA) and/or thio-LNA (such as beta-D-thio-LNA and alpha-L-thio-LNA) and/or ENA (such as beta-D-ENA and alpha-L-ENA). Most preferred is beta-D-oxy-LNA.
  • oxy-LNA such as beta-D-oxy-LNA, and alpha-L-oxy-LNA
  • amino-LNA such as beta-D-amino-LNA and alpha-L-amino- LNA
  • thio-LNA such as beta-D-thio-LNA and alpha-L-thio-LNA
  • ENA such as beta-D-ENA and alpha-L-ENA
  • nucleotide analogues present within the oligomer of the invention are independently selected from, for example: 2'-0-alkyl-RNA units, 2'-amino-DNA units, 2'-fluoro-DNA units, LNA units, arabino nucleic acid (ANA) units, 2'-fluoro-ANA units, HNA units, INA (intercalating nucleic acid -Christensen, 2002. Nucl. Acids. Res. 2002 30: 4918-4925, hereby incorporated by reference) units and 2'MOE units.
  • nucleotide analogues are 2'-0-methoxyethyl-RNA (2'MOE), 2'-fluoro-DNA monomers or LNA nucleotide analogues, and as such the oligonucleotide of the invention may comprise nucleotide analogues which are independently selected from these three types of analogue, or may comprise only one type of analogue selected from the three types.
  • at least one of said nucleotide analogues is 2'-MOE- RNA, such as 2, 3, 4, 5, 6, 7, 8, 9 or 10 2'-MOE-RNA nucleotide units.
  • At least one of said nucleotide analogues is 2'-fluoro DNA, such as 2, 3, 4, 5, 6, 7, 8, 9 or 10 2'-fluoro-DNA nucleotide units.
  • the oligomer according to the invention comprises at least one Locked Nucleic Acid (LNA) unit, such as 1 , 2, 3, 4, 5, 6, 7, or 8 LNA units, such as from 3 - 7 or 4 to 8 LNA units, or 3, 4, 5, 6 or 7 LNA units.
  • LNA Locked Nucleic Acid
  • all the nucleotide analogues are LNA.
  • the oligomer may comprise both beta-D-oxy- LNA, and one or more of the following LNA units: thio-LNA, amino-LNA, oxy-LNA, and/or ENA in either the beta-D or alpha-L configurations or combinations thereof.
  • all LNA cytosine units are 5'methyl-Cytosine.
  • the oligomer may comprise both LNA and DNA units.
  • the combined total of LNA and DNA units is 10-25, such as 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, or 25, such as 10 - 24, preferably 10-20, such as 10 - 18, even more preferably 12-16.
  • the nucleotide sequence of the oligomer such as the contiguous nucleotide sequence consists of at least one LNA and the remaining nucleotide units are DNA units.
  • the oligomer comprises only LNA nucleotide analogues and naturally occurring nucleotides (such as RNA or DNA, most preferably DNA nucleotides), optionally with modified internucleotide linkages such as phosphorothioate.
  • nucleobase refers to the base moiety of a nucleotide and covers both naturally occurring a well as non-naturally occurring variants. Thus, “nucleobase” covers not only the known purine and pyrimidine heterocycles but also heterocyclic analogues and tautomeres thereof.
  • nucleobases include, but are not limited to adenine, guanine, cytosine, thymidine, uracil, xanthine, hypoxanthine, 5-methylcytosine, isocytosine, pseudoisocytosine, 5-bromouracil, 5-propynyluracil, 6-aminopurine, 2-aminopurine, inosine, diaminopurine, and 2-chloro-6-aminopurine.
  • At least one of the nucleobases present in the oligomer is a modified nucleobase selected from the group consisting of 5-methylcytosine, isocytosine, pseudoisocytosine, 5-bromouracil, 5-propynyluracil, 6-aminopurine, 2-aminopurine, inosine, diaminopurine, and 2-chloro-6-aminopurine.
  • LNA refers to a bicyclic nucleoside analogue, known as “Locked Nucleic Acid”. It may refer to an LNA monomer, or, when used in the context of an "LNA
  • LNA refers to an oligonucleotide containing one or more such bicyclic nucleotide analogues.
  • LNA nucleotides are characterised by the presence of a linker group (such as a bridge) between C2' and C4' of the ribose sugar ring - for example as shown as the biradical R 4* - R 2* as described below.
  • the LNA used in the oligonucleotide compounds of the invention preferably has the structure of the eneral formula I
  • asymmetric groups may be found in either R or S orientation;
  • X is selected from -0-, -S-, -N(R N* )-, -C(R 6 R 6* )-, such as, in some
  • B is selected from hydrogen, optionally substituted Ci -4 -alkoxy, optionally substituted Ci -4 -alkyl, optionally substituted Ci -4 -acyloxy, nucleobases including naturally occurring and nucleobase analogues, DNA intercalators, photochemically active groups, thermochemically active groups, chelating groups, reporter groups, and ligands; preferably, B is a nucleobase or nucleobase analogue;
  • P designates an internucleotide linkage to an adjacent monomer, or a 5'-terminal group, such internucleotide linkage or 5'-terminal group optionally including the substituent R 5 or equally applicable the substituent R 5* ;
  • P* designates an internucleotide linkage to an adjacent monomer, or a 3'-terminal group
  • each of the substituents R 1* , R 2 , R 3 , R 5 , R 5* , R 6 and R 6* , which are present is independently selected from hydrogen, optionally substituted Ci-12-alkyl, optionally substituted C 2- i2-alkenyl, optionally substituted C 2- i2-alkynyl, hydroxy, Ci-12-alkoxy, C2-12- alkoxyalkyl, C 2- i2-alkenyloxy, carboxy, Ci-12-alkoxycarbonyl, Ci-12-alkylcarbonyl, formyl, aryl, aryloxy-carbonyl, aryloxy, arylcarbonyl, heteroaryl, heteroaryloxy-carbonyl, heteroaryloxy, heteroarylcarbonyl, amino, mono- and di(Ci.
  • R 4* and R 2* together designate a biradical consisting of a groups selected from the group consisting of C(R a R b )-C(R a R b )-, C(R a R b )-0-, C(R a R b )-NR a -, C(R a R b )-S-, and C(R a R b )-C(R a R b )-0-, wherein each R a and R b may optionally be
  • R a and R b may be, optionally independently selected from the group consisting of hydrogen and C i- 6 alkyl, such as methyl, such as hydrogen.
  • R 4* and R 2* together designate the biradical -0-CH(CH 2 OCH 3 )- (2'0-methoxyethyl bicyclic nucleic acid - Seth at al. , 2010, J. Org. Chem) - in either the R- or S- configuration.
  • R 4* and R 2* together designate the biradical -0-CH(CH 2 CH 3 )- (2'0-ethyl bicyclic nucleic acid - Seth at al. , 2010, J. Org. Chem). - in either the R- or S- configuration.
  • R 4* and R 2* together designate the biradical -0-CH(CH 3 )-. - in either the R- or S- configuration. In some embodiments, R 4* and R 2* together designate the biradical -0-CH 2 -0-CH 2 - - (Seth at al. , 2010, J. Org. Chem).
  • R 4* and R 2* together designate the biradical -0-NR-CH 3 - - (Seth at al., 2010, J. Org. Chem) .
  • the LNA units have a structure selected from the following group:
  • R 1* , R 2 , R 3 , R 5 , R 5* are independently selected from the group consisting of hydrogen, halogen, Ci_ 6 alkyl, substituted Ci_ 6 alkyl, C 2-6 alkenyl, substituted C 2-6 alkenyl, C 2-6 alkynyl or substituted C 2-6 alkynyl, Ci -6 alkoxyl, substituted Ci -6 alkoxyl, acyl, substituted acyl, Ci -6 aminoalkyl or substituted Ci -6 aminoalkyl.
  • asymmetric groups may be found in either R or S orientation.
  • R 1* , R 2 , R 3 , R 5 , R 5* are hydrogen.
  • R 1* , R 2 , R 3 are independently selected from the group consisting of hydrogen, halogen, Ci -6 alkyl, substituted Ci -6 alkyl, C 2-6 alkenyl, substituted C 2-6 alkenyl, C 2-6 alkynyl or substituted C 2-6 alkynyl, Ci -6 alkoxyl, substituted Ci -6 alkoxyl, acyl, substituted acyl, Ci -6 aminoalkyl or substituted Ci -6 aminoalkyl.
  • asymmetric groups may be found in either R or S orientation.
  • R 1* , R 2 , R 3 are hydrogen.
  • R 5 or R 5* are hydrogen, where as the other group (R 5 or R 5*
  • R 5 or R 5* is substituted Ci -6 alkyl.
  • each J, and J 2 is, independently H or Ci -6 alkyl.
  • either R 5 or R 5* is methyl, ethyl or methoxymethyl. In some embodiments either R 5 or R 5* is methyl.
  • Such 5' modified bicyclic nucleotides are disclosed in WO 2007/134181 , which is hereby incorporated by reference in its entirety.
  • B is a nucleobase, including nucleobase analogues and naturally occurring nucleobases, such as a purine or pyrimidine, or a substituted purine or substituted pyrimidine, such as a nucleobase referred to herein, such as a nucleobase selected from the group consisting of adenine, cytosine, thymine, adenine, uracil, and/or a modified or substituted nucleobase, such as 5-thiazolo-uracil, 2-thio-uracil, 5-propynyl-uracil, 2'thio-thymine, 5-methyl cytosine, 5-thiozolo-cytosine, 5-propynyl-cytosine, and 2,6- diaminopurine.
  • nucleobase including nucleobase analogues and naturally occurring nucleobases, such as a purine or pyrimidine, or a substituted purine or substituted pyrimidine, such as
  • R 4* and R 2* together designate the biradical C(R a R b )-N(R c )-0-, wherein R a and R b are independently selected from the group consisting of hydrogen, halogen, Ci_ 6 alkyl, substituted Ci_ 6 alkyl, C 2-6 alkenyl, substituted C 2-6 alkenyl, C 2-6 alkynyl or substituted C 2-6 alkynyl, Ci -6 alkoxyl, substituted Ci -6 alkoxyl, acyl, substituted acyl, Ci -6 aminoalkyl or substituted Ci_ 6 aminoalkyl, such as hydrogen, and; wherein R c is selected from the group consisting of hydrogen, halogen, Ci -6 alkyl, substituted Ci -6 alkyl, C 2-6 alkenyl, substituted C 2-6 alkenyl, C 2-6 alkynyl or substituted C 2-6 alkynyl, Ci -6 alkoxyl, substituted Ci_ 6 aminoalky
  • R 4* and R 2* together designate the biradical C(R a R b )-0-C(R c R d ) -0-, wherein R a , R b , R c , and R d are independently selected from the group consisting of hydrogen, halogen, Ci -6 alkyl, substituted Ci -6 alkyl, C 2-6 alkenyl, substituted C 2-6 alkenyl, C 2-6 alkynyl or substituted C 2-6 alkynyl, Ci -6 alkoxyl, substituted Ci -6 alkoxyl, acyl, substituted acyl, Ci-6 aminoalkyl or substituted Ci -6 aminoalkyl, such as hydrogen.
  • Z is Ci -6 alkyl or substituted Ci -6 alkyl. In some embodiments Z is methyl. In some embodiments Z is substituted Ci -6 alkyl. In some embodiments said substituent group is Ci -6 alkoxy. In some embodiments Z is CH 3 OCH 2 -. For all chiral centers, asymmetric groups may be found in either R or S orientation. Such bicyclic nucleotides are disclosed in US 7,399,845 which is hereby incorporated by reference in its entirety.
  • R 1* , R 2 , R 3 , R 5 , R 5* are hydrogen. In some some embodiments, R 1* , R 2 , R 3 * are hydrogen, and one or both of R 5 , R 5* may be other than hydrogen as referred to above and in WO 2007/134181.
  • R 4* and R 2* together designate a biradical which comprise a substituted amino group in the bridge such as consist or comprise of the biradical -CH 2 -N( R c )-, wherein R c is Ci _ i 2 alkyloxy.
  • R 1* , R 2 , R 3 , R 5 , R 5* are independently selected from the group consisting of hydrogen, halogen, Ci -6 alkyl, substituted Ci -6 alkyl, C 2-6 alkenyl, substituted C 2-6 alkenyl, C 2-6 alkynyl or substituted C 2-6 alkynyl, Ci -6 alkoxyl, substituted Ci -6 alkoxyl, acyl, substituted acyl, Ci -6 aminoalkyl or substituted Ci -6 aminoalkyl.
  • R 1* , R 2 , R 3 , R 5 , R 5* are hydrogen. In some embodiments, R 1* , R 2 , R 3 are hydrogen and one or both of R 5 , R 5* may be other than hydrogen as referred to above and in WO 2007/134181.
  • R 4* and R 2* together designate a biradical (bivalent group) C(R a R b )-0-, wherein R a and R b are each independently halogen, C Ci 2 alkyl, substituted C Ci 2 alkyl, C 2 -Ci 2 alkenyl, substituted C 2 -Ci 2 alkenyl, C 2 -Ci 2 alkynyl, substituted C 2 -Ci 2 alkynyl, C Ci 2 alkoxy, substituted C Ci 2 alkoxy, OJi SJi, SOJi, S0 2 Ji, NJiJ 2l N 3 , CN,
  • each J, and J 2 is, independently, H, C1 -C 6 alkyi, substituted C1 -C 6 alkyi, C 2 -C 6 alkenyl, substituted C 2 -C 6 alkenyl, C 2 -C 6 alkynyl, substituted C 2 -C 6 alkynyl, C1 -C 6 aminoalkyl, substituted C1 -C 6 aminoalkyl or a protecting group.
  • Such compounds are disclosed in WO2009006478A, hereby incorporated in its entirety by reference.
  • R 4* and R 2* form the biradical - Q -, wherein Q is
  • R 1* , R 2 , R 3 , R 5 , R 5* are hydrogen.
  • asymmetric groups may be found in either R or S orientation.
  • Such bicyclic nucleotides are disclosed in WO2008/154401 which is hereby incorporated by reference in its entirity.
  • R 1* , R 2 , R 3 , R 5 , R 5* are independently selected from the group consisting of hydrogen, halogen, Ci_ 6 alkyi, substituted Ci -6 alkyi, C 2-6 alkenyl, substituted C 2-6 alkenyl, C 2-6 alkynyl or substituted C 2-6 alkynyl, Ci -6 alkoxyl, substituted Ci -6 alkoxyl, acyl, substituted acyl, Ci -6 aminoalkyl or substituted Ci -6 aminoalkyl.
  • R 1* , R 2 , R 3 , R 5 , R 5* are hydrogen.
  • R 1* , R 2 , R 3 are hydrogen and one or both of R 5 , R 5* may be other than hydrogen as referred to above and in WO 2007/134181 or WO2009/067647 (alpha-L- bicyclic nucleic acids analogs).
  • Y is selected from the group consisting of -0-, -CH 2 0-, -S-, -NH-, N(R e ) and/or - CH 2 -;
  • Z and Z* are independently selected among an internucleotide linkage, R H , a terminal group or a protecting group;
  • B constitutes a natural or non-natural nucleotide base moiety (nucleobase), and
  • R H is selected from hydrogen and Ci -4 -alkyl;
  • R a , R b R c , R d and R e are, optionally independently, selected from the group consisting of hydrogen, optionally substituted Ci_i 2 -alkyl, optionally substituted C 2- i2-alkenyl, optionally substituted C 2- i2-alkynyl, hydroxy, Ci-12-alkoxy, C 2- i2-alkoxyalkyl, C 2- i2-alkenyloxy, carboxy, Ci-12-alkoxycarbonyl,
  • R a , R b R c , R d and R e are, optionally independently, selected from the group consisting of hydrogen and Ci_ 6 alkyl, such as methyl.
  • Ci_ 6 alkyl such as methyl.
  • asymmetric groups may be found in either R or S orientation, for example, two exemplary
  • stereochemical isomers include the beta-D and alpha-L isoforms, which may be illustrated as follows:
  • thio-LNA comprises a locked nucleotide in which Y in the general formula above is selected from S or -CH 2 -S-.
  • Thio-LNA can be in both beta-D and alpha-L- configuration.
  • amino-LNA comprises a locked nucleotide in which Y in the general formula above is selected from -N(H)-, N(R)-, CH 2 -N(H)-, and -CH 2 -N(R)- where R is selected from hydrogen and Ci -4 -alkyl.
  • Amino-LNA can be in both beta-D and alpha-L- configuration.
  • Oxy-LNA comprises a locked nucleotide in which Y in the general formula above represents -0-. Oxy-LNA can be in both beta-D and alpha-L-configuration.
  • ENA comprises a locked nucleotide in which Y in the general formula above is -CH 2 -0- (where the oxygen atom of -CH 2 -0- is attached to the 2'-position relative to the base B).
  • R e is hydrogen or methyl.
  • LNA is selected from beta-D-oxy-LNA, alpha-L-oxy-LNA, beta-D-amino-LNA and beta-D-thio-LNA, in particular beta-D-oxy-LNA.
  • an oligomeric compound may function via non RNase mediated degradation of target mRNA, such as by steric hindrance of translation, or other methods, however, the preferred oligomers of the invention are capable of recruiting an
  • RNase endoribonuclease
  • the oligomer, or contiguous nucleotide sequence comprises of a region of at least 6, such as at least 7 consecutive nucleotide units, such as at least 8 or at least 9 consecutive nucleotide units (residues), including 7, 8, 9, 10, 11 , 12, 13, 14, 15 or 16 consecutive nucleotides, which, when formed in a duplex with the complementary target RNA is capable of recruiting RNase.
  • the contiguous sequence which is capable of recruiting RNAse may be region B as referred to in the context of a gapmer as described herein.
  • the size of the contiguous sequence which is capable of recruiting RNAse, such as region B may be higher, such as 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19 or 20 nucleotide units.
  • EP 1 222 309 provides in vitro methods for determining RNaseH activity, which may be used to determine the ability to recruit RNaseH.
  • a oligomer is deemed capable of recruiting RNase H if, when provided with the complementary RNA target, it has an initial rate, as measured in pmol/l/min, of at least 1 %, such as at least 5%, such as at least 10% or ,more than 20% of the of the initial rate determined using DNA only oligonucleotide, having the same base sequence but containing only DNA monomers, with no 2'
  • an oligomer is deemed essentially incapable of recruiting RNaseH if, when provided with the complementary RNA target, and RNaseH, the RNaseH initial rate, as measured in pmol/l/min, is less than 1 %, such as less than 5%, such as less than 10% or less than 20% of the initial rate determined using the equivalent DNA only oligonucleotide, with no 2' substitutions, with phosphorothioate linkage groups between all nucleotides in the oligonucleotide, using the methodology provided by Example 91 - 95 of EP 1 222 309.
  • an oligomer is deemed capable of recruiting RNaseH if, when provided with the complementary RNA target, and RNaseH, the RNaseH initial rate, as measured in pmol/l/min, is at least 20%, such as at least 40 %, such as at least 60 %, such as at least 80 % of the initial rate determined using the equivalent DNA only oligonucleotide, with no 2' substitutions, with phosphorothioate linkage groups between all nucleotides in the oligonucleotide, using the methodology provided by Example 91 - 95 of EP 1 222 309.
  • the region of the oligomer which forms the consecutive nucleotide units which, when formed in a duplex with the complementary target RNA is capable of recruiting RNase consists of nucleotide units which form a DNA/RNA like duplex with the RNA target - and include both DNA units and LNA units which are in the alpha-L configuration, particularly preferred being alpha-L-oxy LNA.
  • the oligomer of the invention may comprise a nucleotide sequence which comprises both nucleotides and nucleotide analogues, and may be in the form of a gapmer, a headmer or a mixmer.
  • a "headmer” is defined as an oligomer that comprises a region X and a region Y that is contiguous thereto, with the 5'-most monomer of region Y linked to the 3'-most monomer of region X.
  • Region X comprises a contiguous stretch of non-RNase recruiting nucleoside analogues and region Y comprises a contiguous stretch (such as at least 7 contiguous monomers) of DNA monomers or nucleoside analogue monomers recognizable and cleavable by the RNase.
  • a “tailmer” is defined as an oligomer that comprises a region X and a region Y that is contiguous thereto, with the 5'-most monomer of region Y linked to the 3'-most monomer of the region X.
  • Region X comprises a contiguous stretch (such as at least 7 contiguous monomers) of DNA monomers or nucleoside analogue monomers recognizable and cleavable by the RNase, and region X comprises a contiguous stretch of non-RNase recruiting nucleoside analogues.
  • chimeric oligomers consist of an alternating composition of (i) DNA monomers or nucleoside analogue monomers recognizable and cleavable by RNase, and (ii) non-RNase recruiting nucleoside analogue monomers.
  • some nucleoside analogues in addition to enhancing affinity of the oligomer for the target region, some nucleoside analogues also mediate RNase (e.g., RNaseH) binding and cleavage. Since a-L-LNA monomers recruit RNaseH activity to a certain extent, in some embodiments, gap regions (e.g., region B as referred to herein) of oligomers containing a-L- LNA monomers consist of fewer monomers recognizable and cleavable by the RNaseH, and more flexibility in the mixmer construction is introduced.
  • RNase e.g., RNaseH
  • the oligomer of the invention is a gapmer.
  • a gapmer oligomer is an oligomer which comprises a contiguous stretch of nucleotides which is capable of recruiting an RNAse, such as RNAseH, such as a region of at least 6 or 7 DNA nucleotides, referred to herein in as region B (B), wherein region B is flanked both 5' and 3' by regions of affinity enhancing nucleotide analogues, such as from 1 - 6 nucleotide analogues 5' and 3' to the contiguous stretch of nucleotides which is capable of recruiting RNAse - these regions are referred to as regions A (A) and C (C) respectively.
  • the monomers which are capable of recruiting RNAse are selected from the group consisting of DNA monomers, alpha-L-LNA monomers, C4' alkylayted DNA monomers (see PCT/EP2009/050349 and Vester ef a/. , Bioorg. Med. Chem. Lett. 18 (2008) 2296 - 2300, hereby incorporated by reference), and UNA (unlinked nucleic acid) nucleotides (see Flutter ef a/. , Mol. Biosyst. , 2009, 10, 1039 hereby incorporated by reference).
  • UNA is unlocked nucleic acid, typically where the C2 - C3 C-C bond of the ribose has been removed, forming an unlocked "sugar” residue.
  • the gapmer comprises a (poly)nucleotide sequence of formula (5' to 3'), A-B-C, or optionally A-B-C-D or D-A-B-C, wherein; region A (A) (5' region) consists or comprises of at least one nucleotide analogue, such as at least one LNA unit, such as from 1-6 nucleotide analogues, such as LNA units, and; region B (B) consists or comprises of at least five consecutive nucleotides which are capable of recruiting RNAse (when formed in a duplex with a complementary RNA molecule, such as the mRNA target), such as DNA nucleotides, and; region C (C) (3'region) consists or comprises of at least one nucleotide analogue, such as at least one
  • region A consists of 1 , 2, 3, 4, 5 or 6 nucleotide analogues, such as LNA units, such as from 2-5 nucleotide analogues, such as 2-5 LNA units, such as 3 or 4 nucleotide analogues, such as 3 or 4 LNA units; and/or region C consists of 1 , 2, 3, 4, 5 or 6 nucleotide analogues, such as LNA units, such as from 2-5 nucleotide analogues, such as 2-5 LNA units, such as 3 or 4 nucleotide analogues, such as 3 or 4 LNA units.
  • LNA units such as from 2-5 nucleotide analogues, such as 2-5 LNA units, such as 3 or 4 nucleotide analogues, such as 3 or 4 LNA units.
  • B consists or comprises of 5, 6, 7, 8, 9, 10, 11 or 12
  • region B consists or comprises at least one DNA nucleotide unit, such as 1-12 DNA units, preferably from 4-12 DNA units, more preferably from 6-10 DNA units, such as from 7-10 DNA units, most preferably 8, 9 or 10 DNA units.
  • region A consist of 3 or 4 nucleotide analogues, such as LNA
  • region B consists of 7, 8, 9 or 10 DNA units
  • region C consists of 3 or 4 nucleotide analogues, such as LNA.
  • Such designs include (A-B-C) 3-10-3, 3-10-4, 4-10-3, 3-9-3, 3-9-4, 4-9-3, 3-8-3, 3-8-4, 4-8-3, 3-7-3, 3-7-4, 4-7-3, and may further include region D, which may have one or 2 nucleotide units, such as DNA units.
  • WO2008/1 13832 which claims priority from US provisional application 60/977,409 hereby incorporated by reference, refers to 'shortmer' gapmer oligomers.
  • oligomers presented here may be such shortmer gapmers.
  • the oligomer is consisting of a contiguous nucleotide sequence of a total of 10, 1 1 , 12, 13 or 14 nucleotide units, wherein the contiguous nucleotide sequence is of formula (5' - 3'), A-B-C, or optionally A-B-C-D or D-A-B-C, wherein; A consists of 1 , 2 or 3 nucleotide analogue units, such as LNA units; B consists of 7, 8 or 9 contiguous nucleotide units which are capable of recruiting RNAse when formed in a duplex with a complementary RNA molecule (such as a mRNA target); and C consists of 1 , 2 or 3 nucleotide analogue units, such as LNA units.
  • D consists of a single DNA unit.
  • A consists of 1 LNA unit. In some embodiments A consists of 2 LNA units. In some embodiments A consists of 3 LNA units. In some embodiments C consists of 1 LNA unit. In some embodiments C consists of 2 LNA units. In some embodiments C consists of 3 LNA units. In some embodiments B consists of 7 nucleotide units. In some embodiments B consists of 8 nucleotide units. In some embodiments B consists of 9 nucleotide units. . In certain embodiments, region B consists of 10 nucleoside monomers. In certain embodiments, region B comprises 1 - 10 DNA monomers.
  • B comprises of from 1 - 9 DNA units, such as 2, 3, 4, 5, 6, 7 , 8 or 9 DNA units. In some embodiments B consists of DNA units. In some embodiments B comprises of at least one LNA unit which is in the alpha-L configuration, such as 2, 3, 4, 5, 6, 7, 8 or 9 LNA units in the alpha-L-configuration. In some embodiments B comprises of at least one alpha-L-oxy LNA unit or wherein all the LNA units in the alpha-L- configuration are alpha-L- oxy LNA units.
  • the number of nucleotides present in A-B-C are selected from the group consisting of (nucleotide analogue units - region B - nucleotide analogue units): 1-8-1 , 1-8-2, 2-8-1 , 2-8-2, 3-8-3, 2-8-3, 3-8-2, 4-8-1 , 4-8-2, 1-8-4, 2-8-4, or; 1-9-1 , 1-9-2, 2-9-1 , 2-9-2, 2-9-3, 3-9-2, 1-9-3, 3-9-1 , 4-9-1 , 1-9-4, or; 1-10-1 , 1-10-2, 2-10- 1 , 2-10-2, 1-10-3, 3-10-1.
  • the number of nucleotides in A-B-C are selected from the group consisting of: 2-7-1 , 1-7-2, 2-7-2, 3-7-3, 2-7-3, 3-7-2, 3-7-4, and 4-7- 3.
  • each of regions A and C consists of three LNA monomers, and region B consists of 8 or 9 or 10 nucleoside monomers, preferably DNA monomers.
  • both A and C consists of two LNA units each, and B consists of 8 or 9 nucleotide units, preferably DNA units.
  • gapsmer designs include those where regions A and/or C consists of 3, 4, 5 or 6 nucleoside analogues, such as monomers containing a 2'-0-methoxyethyl-ribose sugar (2'-MOE) or monomers containing a 2'-fluoro-deoxyribose sugar, and region B consists of 8, 9, 10, 1 1 or 12 nucleosides, such as DNA monomers, where regions A-B-C have 3-9-3, 3-10-3, 5-10-5 or 4- 12-4 monomers.
  • regions A and/or C consists of 3, 4, 5 or 6 nucleoside analogues, such as monomers containing a 2'-0-methoxyethyl-ribose sugar (2'-MOE) or monomers containing a 2'-fluoro-deoxyribose sugar
  • region B consists of 8, 9, 10, 1 1 or 12 nucleosides, such as DNA monomers, where regions A-B-C have 3-9-3, 3-10-3, 5-10
  • each monomer is linked to the 3' adjacent monomer via a linkage group.
  • linkage groups e.g., each monomer is linked to the 3' adjacent monomer via a linkage group.
  • the 5' monomer at the end of an oligomer does not comprise a 5' linkage group, although it may or may not comprise a 5' terminal group.
  • linkage group or "internucleotide linkage” are intended to mean a group capable of covalently coupling together two nucleotides. Specific and preferred examples include phosphate groups and phosphorothioate groups.
  • nucleotides of the oligomer of the invention or contiguous nucleotides sequence thereof are coupled together via linkage groups.
  • each nucleotide is linked to the 3' adjacent nucleotide via a linkage group.
  • Suitable internucleotide linkages include those listed within WO2007/031091 , for example the internucleotide linkages listed on the first paragraph of page 34 of
  • phosphorothioate or boranophosphate - these two being cleavable by RNase H, also allow that route of antisense inhibition in reducing the expression of the target gene.
  • Suitable sulphur (S) containing internucleotide linkages as provided herein may be preferred.
  • Phosphorothioate internucleotide linkages are also preferred, particularly for the gap region (B) of gapmers.
  • Phosphorothioate linkages may also be used for the flanking regions (A and C, and for linking A or C to D, and within region D, as appropriate).
  • Regions A, B and C may however comprise internucleotide linkages other than phosphorothioate, such as phosphodiester linkages, particularly, for instance when the use of nucleotide analogues protects the internucleotide linkages within regions A and C from endo-nuclease degradation - such as when regions A and C comprise LNA nucleotides.
  • the internucleotide linkages in the oligomer may be phosphodiester, phosphorothioate or boranophosphate so as to allow RNase H cleavage of targeted RNA.
  • Phosphorothioate is preferred, for improved nuclease resistance and other reasons, such as ease of manufacture.
  • nucleotides and/or nucleotide analogues are linked to each other by means of phosphorothioate groups.
  • phosphodiester linkages such as one or two linkages
  • nucleotide analogue units typically in region A and or C
  • all remaining linkage groups are either phosphodiester or phosphorothioate, or a mixture thereof.
  • all the internucleotide linkage groups are phosphorothioate.
  • linkages are phosphorothioate linkages
  • alternative linkages such as those disclosed herein may be used, for example phosphate (phosphodiester) linkages may be used, particularly for linkages between nucleotide analogues, such as LNA, units.
  • phosphate (phosphodiester) linkages may be used, particularly for linkages between nucleotide analogues, such as LNA, units.
  • C residues are annotated as 5'methyl modified cytosine
  • one or more of the Cs present in the oligomer may be unmodified C residues. in some embodimentsin some embodiments
  • the oligomers of the invention may, for example, be selected from the group consisting of: SEQ IDS NO's: 8-30, see also table 2.
  • Table 2 also discloses SEQ ID No: 78 which is the scrambled control compound used.
  • conjugate is intended to indicate a heterogenous molecule formed by the covalent attachment (“conjugation") of the oligomer as described herein to one or more non-nucleotide, or non-polynucleotide moieties.
  • non-nucleotide or non- polynucleotide moieties include macromolecular agents such as proteins, fatty acid chains, sugar residues, glycoproteins, polymers, or combinations thereof.
  • proteins may be antibodies for a target protein.
  • Typical polymers may be polyethylene glycol.
  • the oligomer of the invention may comprise both a polynucleotide region which typically consists of a contiguous sequence of nucleotides, and a further non-nucleotide region.
  • the compound may comprise non-nucleotide
  • components such as a conjugate component.
  • the oligomeric compound is linked to ligands/conjugates, which may be used, e.g. to increase the cellular uptake of oligomeric compounds.
  • ligands/conjugates which may be used, e.g. to increase the cellular uptake of oligomeric compounds.
  • WO2007/031091 provides suitable ligands and conjugates, which are hereby incorporated by reference.
  • the invention also provides for a conjugate comprising the compound according to the invention as herein described, and at least one non-nucleotide or non-polynucleotide moiety covalently attached to said compound.
  • the compound of the invention may also comprise at least one non-nucleotide or non- polynucleotide moiety (e.g. not comprising one or more nucleotides or nucleotide analogues) covalently attached to said compound.
  • Conjugation may enhance the activity, cellular distribution or cellular uptake of the oligomer of the invention.
  • moieties include, but are not limited to, antibodies, polypeptides, lipid moieties such as a cholesterol moiety, cholic acid, a thioether, e.g.
  • a phospholipids e.g., di-hexadecyl-rac-glycerol or triethylammonium 1 ,2-di-o- hexadecyl-rac-glycero-3-h-phospho
  • the oligomers of the invention may also be conjugated to active drug substances, for example, aspirin, ibuprofen, a sulfa drug, an antidiabetic, an antibacterial or an antibiotic.
  • active drug substances for example, aspirin, ibuprofen, a sulfa drug, an antidiabetic, an antibacterial or an antibiotic.
  • the conjugated moiety is a sterol, such as cholesterol.
  • the conjugated moiety comprises or consists of a positively charged polymer, such as a positively charged peptides of, for example from 1 -50, such as 2 - 20 such as 3 - 10 amino acid residues in length, and/or polyalkylene oxide such as polyethylglycol(PEG) or polypropylene glycol - see WO 2008/034123, hereby incorporated by reference.
  • a positively charged polymer such as a positively charged peptides of, for example from 1 -50, such as 2 - 20 such as 3 - 10 amino acid residues in length
  • polyalkylene oxide such as polyethylglycol(PEG) or polypropylene glycol - see WO 2008/034123, hereby incorporated by reference.
  • the positively charged polymer, such as a polyalkylene oxide may be attached to the oligomer of the invention via a linker such as the releasable inker described in WO 2008/034123.
  • conjugate moieties may be used in the conjugates of the invention: 5'- OLIGOM ER -3'
  • activated oligomer refers to an oligomer of the invention that is covalently linked (i.e., functionalized) to at least one functional moiety that permits covalent linkage of the oligomer to one or more conjugated moieties, i.e., moieties that are not themselves nucleic acids or monomers, to form the conjugates herein described.
  • a functional moiety will comprise a chemical group that is capable of covalently bonding to the oligomer via, e.g., a 3'-hydroxyl group or the exocyclic NH 2 group of the adenine base, a spacer that is preferably hydrophilic and a terminal group that is capable of binding to a conjugated moiety (e.g., an amino, sulfhydryl or hydroxyl group).
  • this terminal group is not protected, e.g., is an NH 2 group.
  • the terminal group is protected, for example, by any suitable protecting group such as those described in "Protective Groups in Organic Synthesis" by Theodora W
  • hydroxyl protecting groups include esters such as acetate ester, aralkyl groups such as benzyl, diphenylmethyl, or triphenylmethyl, and tetrahydropyranyl.
  • suitable amino protecting groups include benzyl, alpha-methylbenzyl, diphenylmethyl,
  • the functional moiety is self- cleaving. In other embodiments, the functional moiety is biodegradable. See e.g., U.S. Patent No. 7,087,229, which is incorporated by reference herein in its entirety.
  • oligomers of the invention are functionalized at the 5' end in order to allow covalent attachment of the conjugated moiety to the 5' end of the oligomer.
  • oligomers of the invention can be functionalized at the 3' end.
  • oligomers of the invention can be functionalized along the backbone or on the heterocyclic base moiety.
  • oligomers of the invention can be functionalized at more than one position independently selected from the 5' end, the 3' end, the backbone and the base.
  • activated oligomers of the invention are synthesized by incorporating during the synthesis one or more monomers that is covalently attached to a functional moiety. In other embodiments, activated oligomers of the invention are synthesized with monomers that have not been functionalized, and the oligomer is functionalized upon completion of synthesis.
  • the oligomers are functionalized with a hindered ester containing an aminoalkyi linker, wherein the alkyl portion has the formula (CH 2 ) W , wherein w is an integer ranging from 1 to 10, preferably about 6, wherein the alkyl portion of the alkylamino group can be straight chain or branched chain, and wherein the functional group is attached to the oligomer via an ester group (-O-C(O)- (CH 2 ) W NH).
  • the oligomers are functionalized with a hindered ester containing a (CH 2 ) w -sulfhydryl (SH) linker, wherein w is an integer ranging from 1 to 10, preferably about 6, wherein the alkyl portion of the alkylamino group can be straight chain or branched chain, and wherein the functional group attached to the oligomer via an ester group (-0-C(0)-(CH 2 ) w SH)
  • sulfhydryl-activated oligonucleotides are conjugated with polymer moieties such as polyethylene glycol or peptides (via formation of a disulfide bond).
  • Activated oligomers containing hindered esters as described above can be
  • the oligomers of the invention are functionalized by introducing sulfhydryl, amino or hydroxyl groups into the oligomer by means of a
  • 4,914,210 i.e., a substantially linear reagent having a phosphoramidite at one end linked through a hydrophilic spacer chain to the opposing end which comprises a protected or unprotected sulfhydryl, amino or hydroxyl group.
  • reagents primarily react with hydroxyl groups of the oligomer.
  • activated oligomers have a
  • the activated oligomers have a functionalizing reagent coupled to a 3'- hydroxyl group.
  • the activated oligomers of the invention have a functionalizing reagent coupled to a hydroxyl group on the backbone of the oligomer.
  • the oligomer of the invention is functionalized with more than one of the functionalizing reagents as described in U.S. Patent Nos. 4,962,029 and 4,914,210, incorporated herein by reference in their entirety. Methods of synthesizing such
  • the 5'-terminus of a solid-phase bound oligomer is
  • a dienyl phosphoramidite derivative functionalized with a dienyl phosphoramidite derivative, followed by conjugation of the deprotected oligomer with, e.g., an amino acid or peptide via a Diels-Alder cycloaddition reaction.
  • the incorporation of monomers containing 2'-sugar modifications, such as a 2'-carbamate substituted sugar or a 2'-(0-pentyl-N-phthalimido)- deoxyribose sugar into the oligomer facilitates covalent attachment of conjugated moieties to the sugars of the oligomer.
  • an oligomer with an amino-containing linker at the 2'-position of one or more monomers is prepared using a reagent such as, for example, 5'-dimethoxytrityl-2'-0-(e-phthalimidylaminopentyl)-2'-deoxyadenosine-3'- N,N- diisopropyl-cyanoethoxy phosphoramidite. See, e.g., Manoharan, et al., Tetrahedron Letters, 1991 , 34, 7171.
  • the oligomers of the invention may have amine- containing functional moieties on the nucleobase, including on the N6 purine amino groups, on the exocyclic N2 of guanine, or on the N4 or 5 positions of cytosine.
  • such functionalization may be achieved by using a commercial reagent that is already functionalized in the oligomer synthesis.
  • Some functional moieties are commercially available, for example, heterobifunctional and homobifunctional linking moieties are available from the Pierce Co. (Rockford, III.).
  • Other commercially available linking groups are 5'-Amino-Modifier C6 and 3'-Amino-Modifier reagents, both available from Glen Research Corporation (Sterling, Va.).
  • 5'-Amino-Modifier C6 is also available from ABI (Applied Biosystems Inc., Foster City, Calif.) as Aminolink-2
  • 3'-Amino-Modifier is also available from Clontech Laboratories Inc. (Palo Alto, Calif.).
  • the oligomer of the invention may be used in pharmaceutical formulations and compositions.
  • such compositions comprise a pharmaceutically acceptable diluent, carrier, salt or adjuvant.
  • PCT/DK2006/000512 provides suitable and preferred
  • Suitable dosages, formulations, administration routes, compositions, dosage forms, combinations with other therapeutic agents, pro-drug formulations are also provided in PCT/DK2006/000512 - which are also hereby incorporated by reference.
  • the oligomers of the invention may be utilized as research reagents for, for example, diagnostics, therapeutics and prophylaxis.
  • oligomers may be used to specifically inhibit the synthesis of polyomavirus such as polyomavirus BK protein (typically by degrading or inhibiting the mRNA and thereby prevent protein formation) in cells and experimental animals thereby facilitating functional analysis of the target or an appraisal of its usefulness as a target for therapeutic intervention.
  • polyomavirus such as polyomavirus BK protein
  • the oligomers may be used to detect and quantitate polyomavirus such as polyomavirus BK expression in cell and tissues by northern blotting, in-situ hybridisation or similar techniques.
  • an animal or a human, suspected of having a disease or disorder, which can be treated by modulating the expression of polyomavirus such as polyomavirus BK is treated by administering oligomeric compounds in accordance with this invention.
  • oligomeric compounds in accordance with this invention.
  • methods of treating a mammal, such as treating a human, suspected of having or being prone to a disease or condition, associated with expression of polyomavirus such as polyomavirus BK by administering a therapeutically or prophylactically effective amount of one or more of the oligomers or compositions of the invention.
  • the oligomer, a conjugate or a pharmaceutical composition according to the invention is typically
  • the invention also provides for the use of the compound or conjugate of the invention as described for the manufacture of a medicament for the treatment of a disorder as referred to herein, or for a method of the treatment of as a disorder as referred to herein.
  • the invention also provides for a method for treating a disorder as referred to herein said method comprising administering a compound according to the invention as herein described, and/or a conjugate according to the invention, and/or a pharmaceutical composition according to the invention to a patient in need thereof.
  • the oligomers and other compositions according to the invention can be used for the treatment of conditions associated with over expression or expression of mutated version of the polyomavirus, such as polyomavirus BK.
  • Table 4 provides exemplary medical indications which are associated with certain polyomavirus infections, which may be treated according to some aspects of the present invention.
  • the invention further provides use of a compound of the invention in the manufacture of a medicament for the treatment of a disease, disorder or condition as referred to herein.
  • one aspect of the invention is directed to a method of treating a mammal suffering from or susceptible to conditions associated with abnormal levels of polyomavirus BK, comprising administering to the mammal a therapeutically effective amount of an oligomer targeted to polyomavirus such as polyomavirus BK that comprises one or more LNA units.
  • the oligomer, a conjugate or a pharmaceutical composition according to the invention is typically administered in an effective amount.
  • the disease or disorder may, in some embodiments be associated with a mutation in the polyomavirus such as polyomavirus BK genome or a gene whose protein product is associated with or interacts with polyomavirus BK. Therefore, in some embodiments, the target mRNA is a mutated form of the polyomavirus such as polyomavirus BK sequence.
  • An interesting aspect of the invention is directed to the use of an oligomer (compound) as defined herein or a conjugate as defined herein for the preparation of a medicament for the treatment of a disease, disorder or condition as referred to herein.
  • the methods of the invention are preferably employed for treatment or prophylaxis against diseases caused by abnormal levels of polyomavirus BK.
  • the invention is furthermore directed to a method for treating abnormal levels of polyomavirus BK, said method comprising
  • composition of the invention to a patient in need thereof.
  • the invention also relates to an oligomer, a composition or a conjugate as defined herein for use as a medicament.
  • the invention further relates to use of a compound, composition, or a conjugate as defined herein for the manufacture of a medicament for the treatment of abnormal levels of polyomavirus such as polyomavirus BK or expression of mutant forms of polyomavirus such as polyomavirus BK (such as those associated with one of the diseases referred to herein).
  • polyomavirus such as polyomavirus BK
  • mutant forms of polyomavirus such as those associated with one of the diseases referred to herein.
  • the invention relates to a method of treating a subject suffering from a disease or condition such as those referred to herein.
  • a patient who is in need of treatment is a patient suffering from or likely to suffer from the disease or disorder.
  • treatment refers to both treatment of an existing disease (e.g. a disease or disorder as herein referred to), or prevention of a disease, i.e. prophylaxis. It will therefore be recognised that treatment as referred to herein may, In some embodiments, be prophylactic.
  • the compounds of the invention are for use as a medicament.
  • the compounds of the invention are made having a sequence that is well conserved between one or more polyomavirus forms, such as the sequence may be present in all of polyomavirus BK, polyomavirus JC and SV40, or it may be present in both polyomavirus BK and polyomavirus JC or in polyomavirus BK, and SV40 or in polyomavirus JC and SV40.
  • Aspect 3 In one embodiment according to aspect 2 for use as a medicament for the treatment of polyomavirus BK, polyomavirus JC and SV40, or it may be for treatment of polyomavirus BK and polyomavirus JC or polyomavirus BK, and SV40 or for polyomavirus JC and SV40 infection.
  • the compounds of the invention are for use as a medicament for the treatment of cancer.
  • the compounds of the invention are for use as a medicament for the treatment of progressive multifocal leukoencephalopathy.
  • the oligonucleotide of the invention is targeted to the second exon of Large T antigen.
  • a single stranded oligomer of from 10 - 30 nucleotides in length which comprises a contiguous nucleotide sequence of a total of from 10 - 30 nucleotides, wherein said contiguous nucleotide sequence is at least 80% homologous to a region corresponding to a BKV gene or the reverse complement of an mRNA, such as SEQ ID NO: 2 to 4 or naturally occurring variant thereof.
  • sequence comprises no mismatches or no more than one or two mismatches with the reverse complement of the corresponding region of SEQ ID NO: 31 to 53.
  • sequence of the oligomer consists of the contiguous nucleotide sequence.
  • nucleotide sequence is from 10 - 18 nucleotides in length.
  • nucleotide sequence comprises nucleotide analogues.
  • nucleotide analogues are sugar modified nucleotides, such as sugar modified nucleotides selected from the group consisting of: Locked Nucleic Acid (LNA) units; 2'-0-alkyl-RNA units, 2'-OMe-RNA units, 2'-amino-DNA units, and 2'-fluoro-DNA units.
  • LNA Locked Nucleic Acid
  • nucleotide analogues are LNA.
  • a conjugate comprising the oligomer according to any one of embodiments 1 - 11 , and at least one non-nucleotide or non-polynucleotide moiety covalently attached to said oligomer.
  • a pharmaceutical composition comprising the oligomer according to any one of
  • oligomer according to any one of embodiments 1 - 11 , or the conjugate according to embodiment 12, for use as a medicament such as for the treatment of bk virus associated nephropathy and interstitial nephritis in kidney transplants, ureteral stenosis, haemorrhagic cystitis.
  • a method for the inhibition of polyomavirus such as polyomavirus BK in a cell which is expressing Polyomavirus BK comprising administering an oligomer according to any one of the embodiments 1-10, or a conjugate according to embodiment 1 1 to said cell so as to inhibit polyomavirus such as polyomavirus BK in said cell.
  • oligomers comprising 12 nucleotide sequences of Table 2 are designed as 2-8-2 (LNA-DNA-LNA) oligomers, oligomers comprising 13 nucleotide sequences of Table 2 are designed as 3-8-2, or 2-8-3 (LNA-DNA-LNA) oligomers and oligomers comprising 14 nucleotide sequences of Table 2 are designed as 3-8-3 (LNA-DNA-LNA) oligomers, and the preferred design of the 16 mers are 3-10-3 (LNA-DNA-LNA), wherein the LNAs are independently selected from oxy-LNA, thio-LNA, and amino-LNA, in either of the D- ⁇ and L-a configurations or combinations thereof.
  • Example 2 LNA nucleotides suppresses luciferase reporter-gene expression in primary human kidney cells using a transfection vehicle
  • Reporter gene Reporter constructs were made by inserting fragments of the BK virus early ("VP5", “VP6”) and late ("Agno”, “VP23”, “VP4") into the psiCheck-2 plasmid (Promega) using the Xhol/NotI sites. The early genes are expressed in the antisense orientation to the genomic sequence and thus inserted as the complementary sequence. The late genes are expressed in the sense orientation and are inserted as the direct sequence. The inserts were created by de novo gene synthesis (GeneART). (Table 3 and Figure 2)
  • RPTEC Human renal proximal tubule endothelial cell
  • REGM CC-3190, Lonza
  • the cells were passaged once weekly and seeded at a density of 2500 cells/cm2.
  • Cells were passaged by trypsinization, using the ReagentPack subculture reagents (CC-5034, Lonza)
  • Transfection and reporter assay of the human primary cell RPTEC Suppression of the BK virus-specific reporter gene was performed using the Dual-Luciferase Reporter Assay System (Promega, Cat# E1910) generally as described in Elmen ef a/ 2007 with the following specific alterations: Freshly trypsinized RPTEC cells were plated and left to adhere for 24 hours in 96-well plates at a density of 4000 cells per well.
  • Each reporter construct (figure 2) was mixed with a relevant LNA-oligonucleotide or a scrambled control, using 0.14 ⁇ _ ⁇ ⁇ of Iipofectamine2000 (Invitrogen), 20 ng/well of reporter plasmid and 0.2 to 25 nM of LNA oligonucleotide in 100 ⁇ _ of OptiMEM. Cells were incubated for 4 hours in the presence of the transfection medium, which was then replaced with REGM media. After a further 20 hours, the cells were lysed and assayed according to the Dual-Luciferase Reporter Assay protocol (Promega, Cat# E1910).
  • Oligonucleotides were tested in HeLa cells against reporters carrying their specific target site. The assay was done by co-transfection of the oligonucleotides at concentrations varying from 0.2 to 25 nM along with 10 ng/well of the reporter construct. The luciferase levels were determined 24 hours after transfection. SEQ ID NO: 78, a scrambled oligonucleotide was included as negative control in each experiment.
  • Oligonucleotides targeting the second exon of the VP1 gene were tested against the appropriate reporter "VP5". A potent knockdown could be seen for one of three
  • Oligonucleotides targeting the VP1 open reading frame were tested against their specific target in a luciferase assay (misleadingly named "VP4"). These oligonucleotides include some that target the short region of VP1 that overlap the open reading frame of VP2 and VP3 (SEQ ID NO: 17 and 20). As seen for the previous examples, a robust and dose- dependent knockdown was seen for all oligonucleotides tested. The data is presented in Figure 6.
  • the overall result of the in vitro reporter screening was the identification of multiple effective oligonucleotides against fragments of all the open reading frames in BK virus.
  • Example 3 LNA nucleotides represses BK virus early and late transcripts in in vitro infected primary human kidney cells using a transfection vehicle
  • Propagating the BK virus Gardner strain An aliquot of the BK virus Gardner strain was acquired from ATCC (VR-837). Preparation of BK virus viral stock for infection experiments in Vero cells was done using a modified version of the protocols described in Tremolada et al (2010) and Moriyama et al (2009). In brief, Vero cells were infected an approximate MOI of 0.5. The infection proceeded for approximately 14 days and the cells were mechanically disrupted by repeated freeze-thaw cycles and the virus particles were enzymatically released from the cell debris, using neuraminidase (N2876 Sigma). After removal of debris, the supernatant was stored as viral stock at -80 degrees Celsius. Viral stock was titered on vero cells by immunofluorescence, and titer is expressed as fluorescence forming units (FFU) per ml_ (Tremolada et al).
  • FFU fluorescence forming units
  • Vero cells were propagated using the DMEM AQ (Sigma, D-0819), 10% Fetal bovine serum (Biochrom, vwr) and 25 ⁇ g/mL Gentamycin (Sigma). Cells were passaged 2 or 3 times per week and plated at a density of 10000-30000 cells per cm 2 . Cells were passaged by trypsinization, using phosphate buffered saline (D8537, Sigma) and trypsin (25300054, Invitrogen)
  • RPTEC Human renal proximal tubule endothelial cell (RPTEC) (CC-2553, Lonza) were cultured in media REGM (CC-3190, Lonza). The cells were passaged once weekly and seeded at a density of 2500 cells/cm2. Cells were passaged by trypsinization, using the ReagentPack subculture reagents (CC-5034, Lonza) Transfection of BK virus infected human primary cell RPTEC:
  • Infections were performed on RPTEC cells plated in 12-well plates at 48000 cells per well and allowed 48-72 hours to adhere. The cells were infected with the BK virus, Gardner strain at a multiplicity of infection of 1. The infection was allowed to proceed for 48 hours, before delivery of oligonucleotides by transfection. Transfections were carried out essentially as described by Dean et al. (1994, JBC 269: 16416-16424). In short, cells were preincubated for 7 min. with Lipofectamine in OptiMEM followed by addition of oligonucleotide to a total volume of 1.5 ml_ transfection mix per well. After 4 hours, the transfection mix was removed; cells were washed and grown at 37C for approximately 20 hours in the appropriate growth medium. Cells were then harvested for RNA analysis.
  • Total RNA Isolation Total RNA was isolated using RNeasy mini kit (Qiagen).
  • First strand synthesis was performed using M-MLV Reverse transcriptase essentially as described by manufacturer (Ambion). In brief, 0.5 ⁇ g total RNA of each sample was adjusted to 10.8 ⁇ _ in water. 2 ⁇ _ decamers and 2 ⁇ _ dNTP mix (2.5 mM each) was added. Samples were heated to 70°C for 3 min. and cooled immediately in ice water and added 3.25 ⁇ _ of a mix containing (2 ⁇ _ 10x RT buffer; 1 ⁇ _ M-MLV Reverse Transcriptase; 0.25 ⁇ _ RNAase inhibitor). cDNA is synthesized at 42°C for 60 min followed by a heating inactivation step at 95°C for 10 min and finally cooled to 4°C.
  • Quantitative RT-PCR Viral mRNA levels were determined by quantitative RT-PCR and performed using TaqMan-type probes against either the early or the late viral transcript and normalized to the level of the host cell GAPDH gene.
  • oligonucleotides were tested against wild type, full length virus in an infected cell system in vitro.
  • An in vivo infection system was set up, using wild type BK virus and RPTEC cells. The integrity of the system and the technical skill required for the screening of the oligonucleotides in infected cells was verified. These data were documented and presented at the previous review session and will not be included in this report.
  • RPTEC cells a primary cell line from renal proximal endothelial cells, were infected with the Gardner strain of BK virus at a multiplicity of infection of 1. The infection was allowed to progress for 72 hours before oligonucleotide was delivered by lipid mediated transfection. The oligonucleotides were delivered at concentrations of 1 nM, 5 nM and 25 nM. The cells were harvested 24 hours after transfection. Quantitative PCR was done on to determine the levels of the early and late transcript families.
  • the oligonucleotides targeting the early transcript appeared to have a modest effect on the early transcript (Figure 7).
  • the expression of the late transcripts in some samples appeared in some samples to be more reduced than what might be expected from the modest knock-down of the early, regulatory genes. This effect was most noticeable with SEQ ID NO: 24.
  • the late-transcript-targeting oligonucleotides appear to have a generally robust and dose-dependent effect on their direct target.
  • the one exception to this rule appears to be SEQ ID NO: 11.
  • the viral response to the knockdown of the late gene appears to be the activation of the early genes.
  • the early genes respond by a dramatic increase in expression level. The likely explanation to this response is that the loss of expression in the late genes leads to a feed-back response in the expression of the early genes.
  • the Agno protein has been proposed as an inhibitor of the transactivation function of the large T antigen. Without this inhibition, the large T antigen will continue to act on both the early and late promoters, leading to increased accumulation of the early gene transcripts.
  • the overall result of the transfection of BK virus-directed oligonucleotides is the identification of a several efficient oligonucleotides that target the late transcripts in infected cells. Secondarily, it is also apparent that the knockdown of the late genes will perturb the entire viral gene expression cascade in the infected cells.
  • Example 4 LNA nucleotides represses BK virus early and late transcripts in in vitro infected primary human kidney cells in the absence of a transfection vehicle
  • RPTEC Human renal proximal tubule endothelial cell
  • REGM CC-3190, Lonza
  • the cells were passaged once weekly and seeded at a density of 2500 cells/cm2.
  • Cells were passaged by trypsinization, using the ReagentPack subculture reagents (CC-5034, Lonza)
  • Gymnosis of infected cells were performed on RPTEC cells plated in 12-well plates at 20000 cells per well and allowed 48-72 hours to adhere. The cells were infected with the BK virus, Gardner strain at a multiplicity of infection of 1. The infection was allowed to proceed for 24 hours, before delivery of oligonucleotides by gymnosis. Oligonucleotides were delivered directly to the growth medium, at a concentration of 1 ⁇ , 5 ⁇ or 25 ⁇ . The infected, oligonucleotide treated cells were left to incubate for an additional 144 hours before cells were harvested for RNA analysis.
  • Total RNA Isolation Total RNA was isolated using RNeasy mini kit (Qiagen).
  • First strand synthesis was performed using M-MLV Reverse transcriptase essentially as described by manufacturer (Ambion). In brief, 0.5 ⁇ g total RNA of each sample was adjusted to 10.8 ⁇ _ in water. 2 ⁇ _ decamers and 2 ⁇ _ dNTP mix (2.5 mM each) was added. Samples were heated to 70°C for 3 min. and cooled immediately in ice water and added 3.25 ⁇ _ of a mix containing (2 ⁇ _ 10x RT buffer; 1 ⁇ _ M-MLV Reverse Transcriptase; 0.25 ⁇ RNAase inhibitor). cDNA is synthesized at 42°C for 60 min followed by a heating inactivation step at 95°C for 10 min and finally cooled to 4°C.
  • Quantitative RT-PCR Viral mRNA levels were determined by quantitative RT-PCR and performed using TaqMan-type probes against either the early or the late viral transcript and normalized to the level of the host cell GAPDH gene.
  • the RPTEC cells were infected with BK virus at an MOI of 1 for 48 hours prior to addition of oligonucleotide.
  • the oligonucleotides were added at concentrations of 1 , 5 and 25 ⁇ and left on the cells for an additional 6 days (144h), for a total of 196 hours post infection.
  • the cells were harvested, RNA was isolated and the levels of early and late transcripts were determined by qPCR.
  • Figure 9 shows the effect of treating the infected RPTEC cells with early-transcript targeting oligonucleotides.
  • This observation fits well with what is known about the expression cascade of BK virus, where the loss of the regulatory proteins Large T antigen and small t antigen, would be expected to lead to abolishment of expression of the late, structural genes.
  • oligonucleotides in this experiment appear to be oligonucleotides targeted to the second exon of Large T antigen (SEQ ID NO: 23 and 24), with the oligonucleotides targeting the overlapping open reading frames exerting an intermediate effect (SEQ ID NO: 29 and 30). Oligonucleotides targeting the intron between the two exons of Large T antigen (which also form the 5'-end of the small t antigen open reading frame) appear to have much less activity (SEQ ID NO: 27 and 28). Similarly, the late-transcript targeting oligonucleotides were tested (Figure 10). As seen in transfection, the oligonucleotides generally appear to have a robust effect on their direct target ("VP1"). Most appear to demonstrate a very significant, dose-dependent knockdown of the target. The major exception is SEQ ID NO: 11 , which does show a knockdown, but with an inverse dose response.
  • T-ANTI the level of the early transcripts
  • the longer-term treatment by gymnosis leads comprehensive knockdown of the early transcripts.
  • SEQ ID NO: 1 1 the inverse dose response on the direct target corresponds to a poor response on the indirect target.
  • the most potent oligonucleotides represent target sites throughout the transcript.
  • SEQ ID NO: 12 and SEQ ID NO: 15 target the overlap between VP2 and VP3
  • SEQ ID NO: 17 and SEQ ID NO: 20 target the short overlap between all three major structural proteins (VP1 , VP2 and VP3) ( Figure 10).
  • the overall conclusion of the gymnosis experiment is that efficient knockdown of both classes of transcripts in BK virus can be achieved.
  • the experiments confirm that the gene expression cascade can be perturbed both by knockdown of the regulatory, early transcript, but also by knockdown of the late, predominantly structural genes. The latter observation is unexpected, and strongly suggests that the BK virus is a sensitive target for LNA- oligonucleotide-mediated therapy.

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Abstract

The present invention relates to oligomeric compounds (oligomers), which target DNA viruses such as polyomavirus BK in a cell, leading to reduced expression of polyomavirus such as polyomavirus BK. Reduction of polyomavirus such as polyomavirus BK expression is beneficial for a range of medical disorders, such as BK virus associated nephropathy. By targeting regions of the polyomavirus genome which encodes for multiple transcripts, therapeutically effective oligomers, the risk of the development of viral resistance to the oligomer may be reduced or avoided.

Description

ANTI POLYOMAVIRUS COMPOUNDS
FIELD OF INVENTION
The present invention relates to oligomeric compounds (oligomers), which target DNA viruses in a cell, leading to reduced expression of polyomavirus such as polyomavirus BK. Reduction of polyomavirus is beneficial for a range of medical disorders, such as BK virus associated nephropathy. By targeting regions of the polyomavirus genome which encodes for multiple transcripts, therapeutically effective oligomers, the risk of the development of viral resistance to the oligomer may be reduced or avoided.
RELATED APPLICATIONS
This application claims the benefit under 35 U.S.C. § 1 19(e) of U.S. Provisional Application Serial No., 61/476779 filed 19th April 2011 , the disclosure of which is incorporated herein by reference in its entirety.
BACKGROUND
Polyomaviruses are DNA-based (double-stranded DNA, -5000 base pairs, circular genome), small (40-50 nanometers in diameter), and icosahedral in shape, and do not have a lipoprotein envelope. Moreover, the genome possess early and late genes, contributing to its complex transcription program. They are potentially oncogenic (tumor-causing); they often persist as latent infections in a host without causing disease, but can lead to significant and damaging infections when activated in, for instance, the kidney, the bladder or the central nervous system. Polyomaviruses may produce tumors in a host of a different species, or a host with an ineffective immune system.
BK virus associated nephropathy has been identified as a prevalent cause of kidney graft rejection. The syndrome is caused by the activation of polyomavirus BK (BKV) and subsequent kidney viraemia, leading to damage of the transplanted kidney and eventually to rejection of the graft. By some estimates as many as 20% of all kidney transplant recipients experience episodes of BK virus viraemia. It has also been estimated that BK virus is involved in as many at 2/3 of all kidney graft losses.
One of the factors in the emergence of BKVAN is the increased use of
immunosuppressants. The underlying mechanism for this phenomenon is not understood, but the reduction or elimination of immunosuppressants in the treatment of BKVAN is currently the most commonly used response to BKVAN. Other treatments with antivirals and other compounds have proven to be less effective. Monitoring BK levels have become standard, to allow rapid changes in immunosuppressants. The causative agent, polyomavirus BK or BK virus, is widely distributed. Up to 80 percent of all tested subject are seropositive. However, in most individuals, the infection appears to be non-symptomatic. The transmission pathway and complete tropism of the virus is not well understood, but appears slightly more prevalent in young adults, with seropositivity levels decreasing in older adults. In addition to kidney transplants, BKVAN may occur in other immunosuppressed individual, for instance when undergoing
haemopoietic stem cell transplants. BK virus infection may also be a factor in a fraction of haemorrhagic cystitis cases.
The BK virus, which is a member of the polyomavirus family, carries a ca. 5200 nt double stranded DNA genome (Figure 1). The genome exists as an autologous episome and expresses at least two kinetic classes of transcripts, the early genes and the late genes. The two classes of transcripts are expressed from a divergent promoter and can roughly be divided into the regulatory early genes (Large T antigen and small T antigen) and the structural late genes (VP1 , VP2 and VP3). The major exception from this rule is the so- called Agno protein, which is a primarily regulatory gene, but is expressed from the late gene promoter. The early transcripts encode proteins with multiple functions, including acting as transcriptional activators of the late genes. Each class of genes is expressed from heavily overlapping transcripts, including heavily overlapping reading frames. The expressed genes are transcribed as mRNAs with canonical mammalian 5'-cap structures and 3'
polyadenylation.
Compared to most RNA viruses, DNA viruses generally carry less sequence variation. This holds true for BK virus as well, where a survey of all ca. 15000 possible 14-, 15-, and 16-mers were aligned against the 270 available full length BK virus sequences deposited with Genbank. This alignment identified at least 2000 perfectly conserved target sequences. BK virus also appears to have a high level of conservation to other human polyomaviruses such as the JC virus.
SUMMARY OF INVENTION
The invention provides an oligomer from 10 - 50, such as 10 - 30 nucleotides in length which comprises a contiguous nucleotide sequence (a first region) of a total of from 10 - 30 nucleotides, wherein said contiguous nucleotide sequence (a first region) is at least 80% (e.g., 85%, 90%, 95%, 98%, or 99%) homologous to a region corresponding to the reverse complement of a mammalian polyomavirus such as a polyomavirus BK gene or mRNA, such as any one of SEQ ID NO's: 1-4 mRNA or naturally occurring variant thereof. Thus, for example, when targeting BKV, the oligomer may hybridizes to (i.e. is complementary to) a single stranded nucleic acid molecule having the sequence of a portion of SEQ ID NO: 2 to 4 mRNA.
The invention provides an oligomer from 10 - 30 contiguous nucleotides in length, wherein said the contiguous nucleotide sequence of the oligomer is at least 80% (e.g., 85%, 90%, 95%, 98%, or 99% or 100%) homologous to a region corresponding to the
complement of a mammalian polyomavirus such as a polyomavirus BK gene or mRNA, such as any one of SEQ ID NO's: 1-4 mRNA or naturally occurring variant thereof. Thus, for example, the oligomer hybridizes to a single stranded nucleic acid molecule having the sequence of a portion of SEQ ID NO: 2 to 4 mRNA. The invention provides for a conjugate comprising the oligomer according to the invention, and at least one non-nucleotide or non- polynucleotide moiety covalently attached to said oligomer.
The invention provides for a pharmaceutical composition comprising the oligomer or the conjugate according to the invention, and a pharmaceutically acceptable diluent, carrier, salt or adjuvant.
The invention provides for the oligomer or the conjugate according to invention, for use as a medicament, such as for the treatment of BK virus associated nephropathy and interstitial nephritis in kidney transplants (Jiang et al., 2009a, Cimbaluk et al. 2009)), ureteral stenosis (often in connection with kidney transplants) (Basara et al. 2010), haemorrhagic cystitis (pediatric; often in connection with bone marrow transplant) (Jiang et al 2009a).
The invention provides for the use of an oligomer or the conjugate according to the invention, for the manufacture of a medicament for the treatment of BK virus associated nephropathy and interstitial nephritis in kidney transplants (Jiang et al, 2009a, Cimbaluk et al 2009)), ureteral stenosis (often in connection with kidney transplants) (Basara et a/ 2010), haemorrhagic cystitis (pediatric; often in connection with bone marrow transplant) (Jiang et al 2009a).
The invention provides for a method of treating BK virus associated nephropathy and interstitial nephritis in kidney transplants (Jiang et al, 2009a, Cimbaluk et al 2009)), ureteral stenosis (often in connection with kidney transplants) (Basara et a/ 2010), haemorrhagic cystitis (pediatric; often in connection with bone marrow transplant) (Jiang et al 2009a), said method comprising administering an, e.g. effective dose of, an oligomer, a conjugate or a pharmaceutical composition according to the invention, to a patient suffering from, or likely to suffer from BK virus associated nephropathy and interstitial nephritis in kidney transplants (Jiang et al, 2009a, Cimbaluk et al 2009)), ureteral stenosis (often in connection with kidney transplants) (Basara et a/ 2010), haemorrhagic cystitis (pediatric; often in connection with bone marrow transplant) (Jiang et al 2009a)(such as a patient suffering from or susceptible to the disease or disorder).
In one embodiment, the disease or disorder or condition is associated with infection of a patient by polyomavirus BK.
The invention provides for a method for the inhibition of polyomavirus such as polyomavirus BK in a cell which is expressing polyomavirus mRNA, said method comprising administering an oligomer, or a conjugate according to the invention to said cell so as to affect the inhibition of polyomavirus such as polyomavirus BK in said cell.
The invention provides an oligomer of from 10-50 monomers, which comprises a first region of 10-50 contiguous monomers, wherein the sequence of the first region is at least 80% identical to a region corresponding to a mammalian polyomavirus such as
polyomavirus BK gene or to the reverse complement of a target region of a nucleic acid which encodes a mammalian polyomavirus such as polyomavirus BK.
The invention further provides a conjugate comprising the oligomer according to the invention, which comprises at least one non-nucleotide or non-polynucleotide moiety ("conjugated moiety") covalently attached to the oligomer of the invention.
The invention provides for pharmaceutical compositions comprising an oligomer or conjugate of the invention, and a pharmaceutically acceptable diluent, carrier, salt or adjuvant.
The invention further provides for an oligomer according to the invention, for use in medicine.
The invention further provides for the use of the oligomer of the invention for the manufacture of a medicament for the treatment of one or more of the diseases referred to herein, such as a disease selected from the group consisting of BK virus associated nephropathy and interstitial nephritis in kidney transplants (Jiang et al, 2009a, Cimbaluk et al 2009)), ureteral stenosis (often in connection with kidney transplants) (Basara et a/ 2010), haemorrhagic cystitis (pediatric; often in connection with bone marrow transplant) (Jiang et al 2009a).
The invention further provides for an oligomer according to the invention, for use for the treatment of one or more of the diseases referred to herein, such as a disease selected from the group consisting of BK virus associated nephropathy and interstitial nephritis in kidney transplants (Jiang et al, 2009a, Cimbaluk et al 2009)), ureteral stenosis (often in connection with kidney transplants) (Basara et a/ 2010), haemorrhagic cystitis (pediatric; often in connection with bone marrow transplant) (Jiang et al 2009a). Pharmaceutical and other compositions comprising an oligomer of the invention are also provided. Further provided are methods of down-regulating the expression of polyomavirus such as polyomavirus BK in cells or tissues comprising contacting said cells or tissues, in vitro or in vivo, with an effective amount of one or more of the oligomers, conjugates or compositions of the invention.
Also disclosed are methods of treating an animal (a non-human animal or a human) suspected of having, or susceptible to, a disease or condition, associated with expression, or over-expression of polyomavirus such as polyomavirus BK by administering to the non- human animal or human a therapeutically or prophylactically effective amount of one or more of the oligomers, conjugates or pharmaceutical compositions of the invention. Further, methods of using oligomers for the inhibition of expression of polyomavirus BK mRNA, and for treatment of diseases associated with activity of polyomavirus such as polyomavirus BK are provided.
The invention provides for a method for treating a disease selected from the group consisting of: BK virus associated nephropathy and interstitial nephritis in kidney transplants (Jiang et al, 2009a, Cimbaluk et a/ 2009)), ureteral stenosis (often in connection with kidney transplants) (Basara et al 2010), haemorrhagic cystitis (pediatric; often in connection with bone marrow transplant) (Jiang et al 2009a), the method comprising administering an effective amount of one or more oligomers, conjugates, or pharmaceutical compositions thereof to an animal in need thereof (such as a patient in need thereof).
The invention provides for methods of inhibiting (e.g., by down-regulating) the expression of polyomavirus such as polyomavirus BK in a cell or a tissue, the method comprising the step of contacting the cell or tissue, in vitro or in vivo, with an effective amount of one or more oligomers, conjugates, or pharmaceutical compositions thereof, to affect down-regulation of expression of polyomavirus mRNA.
The invention provides for a method for preparing an oligomer for use in inhibiting polyomavirus infection in a cell, said method comprising the steps of i) selecting a target region of a polyomavirus genome (or mRNA encoded by the polyomavirus genome), wherein the target region of the polyomavirus present in at least two, such as two or three independent transcripts expressed by the polyomavirus, and; ii) synthesising an oligomer of up to 30 nucleotides in length which comprises or consists of a contiguous nucleotide sequence of 10 - 30 nucleotides, wherein the contiguous nucleotide sequence is the reverse complement of the target region of the polyomavirus.
The invention provides for an oligomer of 10 - 30 nucleotides in length, which comprises or consists of a contiguous nucleotide sequence of 10 - 30 nucleotides, wherein the contiguous nucleotide sequence is the complement of a target region of a polyomavirus genome, wherein the target region of the polyomavirus is present in at least two, such as two or three, independent transcripts expressed by the polyomavirus. The target region of a polyomavirus genome is therefore a region of the polyomavirus genome which is transcribed or a region of a mRNA transcribed from the polyomavirus.
Suitably the at least two or three independent transcripts may be transcribed from over-lapping open-reading frames present in the polyomavirus genome. The independent transcripts are the results of unique and specific transcription and processing events, although derived, at least partially, from the same genomic sequence. In this respect, the sequence present in the independent transcripts which is complementary to the contiguous nucleotide sequence of the oligomer may originate from the same genetic loci on the polyomavirus genome.
The invention provides for an oligomer of up to 30 nucleotides in length, which comprises or consists of a contiguous nucleotide sequence of 10 - 30 nucleotides, wherein the contiguous nucleotide sequence is the reverse complement of a target region of a polyomavirus genome (or mRNA), wherein the target region of the polyomavirus is present at least two, such as two or three, non-identical transcripts, wherein each non-identical transcript comprises an independent open reading frame. An "independent open reading frame" results in an independently expressed protein, although some non-identical transcripts (also refered to as an independent transcript), in some embodiments, may give rise to proteins with partially conserved protein sequences, but protein products with distinct and different functions.
Typically, the non-identical transcripts encode for gene-products (typically proteins) with distinct functions.
The oligomer may be for use/suitable for use, in inhibiting polyomavirus infection in a cell, such as a mammalian cell or human cell. The polyomavirus infection may be polyomavirus BK infection.
By using a single oligomer which targets more than one independant polyomavirus transcript, it is considered that the occurrence and/or survival (selection) of spontaneous resistance mutations will be greatly diminished or even avoided.
BRIEF DESCRIPTION OF FIGURES
Figure 1 : circular representation of the genome structure of bk virus. Bk virus expresses at least 6 gene products, from a divergent promoter region located at the replication origin. Here indicated by their genebank cds numbers, the early genes are large t antigen (CDS6), which contains an intron, and SMALL T ANTIGEN (CDS6). The late genes are AGNO (CDS 1), VP2 (CDS 2), VP3 (CDS 3) AND VP1 (CDS4).
Figure 2: psiCHECKTM-2 vector. A family of transiently expressed reporters were generated by inserting fragments of the BK virus genome in the MCS in the 3'-UTR of the Renilla luciferase gene of the psiCHECK-2 vector. Oligonucleotide efficacy was assessed by co-transfection of reporter plasmid and oligonucleotides and normalizing the effect on Renilla luciferase (hRluc) to firefly luciferase (hluc+), expressed from a separate promoter on the plasmid. Image borrowed from Promega psiCHECK-2 manual.
Figure 3: Luciferase knockdown of Large T antigen reporter ("VP5") by large T antigen specific oligonucleotides. Renilla luciferase activity was measured 24h after transfection and was normalized to firefly luciferase. Data are expressed as % of mock (100%) and are presented as average ± standard deviation of three independent experiments.
Figure 4: Luciferase knockdown of Large T antigen/small t antigen reporter ("VP6") by large T/small t antigen specific oligonucleotides. Renilla luciferase activity was measured 24h after transfection and was normalized to firefly luciferase. Data are expressed as % of mock (100%) and are presented as average ± standard deviation of three independent
experiments.
Figure 5: Luciferase knockdown of Agno protein reporter ("VP6") by Agno protein specific oligonucleotides. Renilla luciferase activity was measured 24h after transfection and was normalized to firefly luciferase. Data are expressed as % of mock (100%) and are presented as average ± standard deviation of three independent experiments.
Figure 6: Luciferase knockdown of VP1 protein reporter ("VP4") by VP1 protein specific oligonucleotides. Renilla luciferase activity was measured 24h after transfection and was normalized to firefly luciferase. Data are expressed as % of mock (100%) and are presented as average ± standard deviation of three independent experiments.
Figure 7: Early (T-antigen) and late (VP1) mRNA expression in infected RPTEC cells after transfection with early-transcript targeting oligonucleotides.
Figure 8: Early (T-antigen) and late (VP1) mRNA expression in infected RPTEC cells after transfection with late transcript-targeting oligonucleotides.
Figure 9: Early (T-antigen) and late (VP1) mRNA expression in infected RPTEC cells after gymnosis treatment with early-transcript targeting oligonucleotides. 5911 and 3088 are negative controls, targeting either a host gene (OPN1) or with no target, respectively.
Figure 10: Early (T-antigen) and late (VP1) mRNA expression in infected RPTEC cells after gymnosis treatment with late-transcript targeting oligonucleotides. 591 1 and 3088 are negative controls, targeting either a host gene (OPN1) or with no target, respectively. DETAILED DESCRIPTION OF INVENTION
The Oligomer
The present invention employs oligomeric compounds (referred herein as oligomers), for use in modulating (e.g. inhibiting) the function of nucleic acid molecules encoding polyomavirus, such as a polyomavirus selected from the group consisting of polyomavirus BK, JC polyomavirus, Kl polyomavirus, WU polyomavirus, Merkel polyomavirus, and Simian virus 40 (SV40).
In some embodiments, the oligomer of the invention is for use in modulating (e.g. inhibiting) the function of a nucleic acid encoding polyomavirus BK, such as a nucleic acid shown selected from the group consisting of SEQ ID 1 , 2, 3, and 4, and naturally occurring variants of thereof. The term "oligomer" in the context of the present invention, refers to a molecule formed by covalent linkage of two or more nucleotides (i.e. an oligonucleotide). Herein, a single nucleotide (unit) may also be referred to as a monomer or unit. In some embodiments, the terms "nucleoside", "nucleotide", "unit" and "monomer" are used interchangeably. It will be recognised that when referring to a sequence of nucleotides or monomers, what is referred to is the sequence of bases, such as A, T, G, C or U.
The oligomer consists or comprises of a contiguous nucleotide sequence of from 10 - 50, such as 10 - 30 nucleotides in length.
In various embodiments, the compound of the invention does not comprise RNA (units). It is preferred that the compound according to the invention is a linear molecule or is synthesised as a linear molecule. The oligomer is a single stranded molecule, and preferably does not comprise short regions of, for example, at least 3, 4 or 5 contiguous nucleotides, which are complementary to equivalent regions within the same oligomer (i.e. duplexes) - in this regards, the oligomer is not (essentially) double stranded. In some embodiments, the oligomer is essentially not double stranded, such as is not a siRNA. In various
embodiments, the oligomer of the invention may consist entirely of the contiguous nucleotide region. Thus, the oligomer is not substantially self-complementary.
The Target
Suitably the oligomer of the invention is capable of down-regulating (e.g. reducing or removing) expression of the polyomavirus, such as a polyomavirus selected from the group consisting of polyomavirus BK, JC polyomavirus, Kl polyomavirus, WU polyomavirus, Merkel polyomavirus, and Simina virus 40 (SV40).
The oligomer of the invention may target/affect the inhibition of polyomavirus BK mRNA, typically in a mammalian such as a human cell, such as a human kidney cell, such as a renal proximal tubular epithelial cell. In some embodiments, the oligomers of the invention bind to the target nucleic acid and affect inhibition of expression of at least 10% or 20% compared to the normal expression level, more preferably at least a 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% inhibition compared to the normal expression level (such as the expression level in the absence of the oligomer(s) or conjugate(s)). In some
embodiments, such modulation is seen when using from 0.04 and 25nM, such as from 0.8 and 20nM concentration of the compound of the invention. In the same or a different embodiment, the inhibition of expression is less than 100%, such as less than 98% inhibition, less than 95% inhibition, less than 90% inhibition, less than 80% inhibition, such as less than 70% inhibition. Modulation of expression level may be determined by measuring protein levels, e.g. by the methods such as SDS-PAGE followed by western blotting using suitable antibodies raised against the target protein. Alternatively, modulation of expression levels can be determined by measuring levels of mRNA, e.g. by northern blotting or quantitative RT-PCR. When measuring via mRNA levels, the level of down-regulation when using an appropriate dosage, such as from 0.04 and 25nM, such as from 0.8 and 20nM concentration, is, In some embodiments, typically to a level of from 10-20% the normal levels in the absence of the compound, conjugate or composition of the invention.
As illustrated herein the cell type may, in some embodiments, be Human Kidney Cells (e.g. in vitro - transfected cells). The oligomer concentration used (e.g. in Human Kidney Cells) may, in some embodiments, be 5nM. The oligomer concentration used may, in some embodiments be 25nM (e.g. in Human Kidney Cells . The oligomer concentration used may, in some embodiments be 1 nM (e.g. in Human Kidney Cells). It should be noted that the concentration of oligomer used to treat the cell is typically performed in an in vitro cell assay, using transfection (Lipofecton), as illustrated in the examples. In the absence of a transfection agent, the oligo concentration required to obtain the down-regulation of the target is typically between 1 and 25μΜ, such as 5μΜ.
As used herein, the phrase "potent inhibitor" refers to an oligomer with an IC50 of less than 5nM as determined by the lipofectamin transfection assay of example 3. In some embodiments, the IC50 is less than 4nM, such as less than 2nM.
The invention therefore provides a method of down-regulating or inhibiting the expression of polyomavirus such as a polyomavirus selected from the group consisting of polyomavirus BK, JC polyomavirus, Kl polyomavirus, WU polyomavirus, Merkel
polyomavirus, and Simina virus 40, such as polyomavirus BK protein and/or mRNA in a cell which is expressing said polyomavirus and/or mRNA, said method comprising administering the oligomer or conjugate according to the invention to said cell to down-regulating or inhibiting the expression of polyomavirus such as polyomavirus BK protein and/or mRNA in said cell. Suitably the cell is a mammalian cell such as a human cell, such as (e.g. in relation to BKV) a kidney cell, such as a renal proximal tubular epithelial cell The administration may occur, in some embodiments, in vitro. The administration may occur, in some embodiments, in vivo.
The term "target nucleic acid", as used herein refers to the DNA or RNA encoding mammalian polyomavirus such as polyomavirus BK polypeptide mRNA. Polyomavirus such as polyomavirus BK encoding nucleic acids or naturally occurring variants thereof, and RNA nucleic acids derived therefrom, preferably mRNA, such as pre-mRNA, although preferably mature mRNA. In some embodiments, for example when used in research or diagnostics the "target nucleic acid" may be a cDNA or a synthetic oligonucleotide derived from the above DNA or RNA nucleic acid targets. The oligomer according to the invention is preferably capable of hybridising to the target nucleic acid.
The term "naturally occurring variant thereof" refers to variants of the polyomavirus such as polyomavirus BK polypeptide of nucleic acid sequence which exist naturally within the defined taxonomic group.
Polyomaviruses
The polyomavirus may, for example, be selected from the following:
Table 4:
Polyomavirus Therapeutic Taxonomy GENBANK ID Example target indication ID: regions
BK BK virus 10629 NC_001538.1 SEQ ID NO 2 - 4 polyomavirus associated Gl:9627180
nephropathy, BK- virus-associated
hemorrhagic
cystitis
JC causative agent of 10632 NC_001699.1 883 - 1468, 1469 - polyomavirus Progressive Gl:9628642 1560, 4771-5013 of multifocal SEQ ID NO 5 SEQ ID NO 5 leukoencephalopat
hy
Kl possible cancer 423445 NC_009238.1 870 to 1497, 1498 polyomavirus causing agent Gl: 134288556 to 1643, 4716 to
SEQ ID NO 6 4967 of SEQ ID NO 6 wu possible cancer 440266 NC_009539.1 1003 to 1669, 1670 polyomavirus causing agent Gl: 148724565 to 1821 , 4906 to
SEQ ID NO 7 5157 of SEQ ID NO
7
Merkel Likely causative 493803 NC_010277.1 4428 to 4983, 4393 polyomavirus agent in Merkel cell GI : 165973999 to 4427, 196 to 429 carcinomas SEQ ID NO 83 of SEQ ID NO:83
Simian virus SV40 (non- 10633 NC_001669.1 916 to 1498, 1499 40 (SV40) human viral GI : 9628421 to 1620, 4918 to disease - used SEQ ID NO 84 5163 of SEQ ID NO as a model for 84 polymorviruses
in vivo and in
vitro)
Sequences
The oligomer of the invention may consist or comprise of a contiguous nucleotide sequence of 10 - 30 nucleotides in length, wherein the contiguous nucleotide sequence is at least 80% complementary, such as at least 90% complementary to, such as fully complementary to a target polyomavirus nucleotide sequence. The target polyomavirus sequence may be present in at least one, such as at least two, independent RNA sequences, such as mRNA sequences, which is/are transcribed from at least one region of the polyomavirus genome. In the instance that more than one RNA target is target by the oligomer, the first and second RNA targets may originate from a region of the polyomavirus genome which comprises at least two overlapping and independently expressed transcripts, wherein a first transcript encodes for the first RNA target using a first open reading frame (ORF), and a second transcript encodes for the second RNA target using a second ORF, and wherein both ORFs comprise the target polyomavirus nucleotide sequence or reverse complement thereof. In this way the target polyomavirus nucleotide sequence is present in the two or more ORFs. By way of example, with reference to BKV, the oligomer may be targeted to a target sequence selected from the group consisting of SEQ ID NOs 2 - 4.
Table 5: Exemplary target polyomavirus nt target regions: SEQ ID Description Sequences
NO:
1 Polyomavirus BK genomic Genbank accession number: NC_001538 sequence entire nucleotide sequence 1-5153
2 Polyomavirus BK VP2 and VP3 Nucleotides 981 to 1563 of SEQ ID NO: 1 overlap mRNA sequence
3 Polyomavirus BK VP2, VP3 and Nucleotides 1564 to 1679 of SEQ ID NO:1
VP1 overlap mRNA sequence
4 Polyomavirus BK Large T Nucleotides 4635 to 5153 of SEQ ID NO:1 antigen overlap with small T
antigen mRNA sequence
5 Polyomavirus JC genomic Genbank accession number:
sequence NC_001699.1 entire nucleotide sequence
1-5130
6 Polyomavirus Kl genomic Genbank accession number:
sequence NC_009238.1 entire nucleotide sequence
1-5040
7 Polyomavirus WU genomic Genbank accession number:
sequence NC_009539.1 entire nucleotide sequence
1-5229
83 Polyomavirus Merkel genomic Genbank accession number:
sequence NC_010277.1 entire nucleotide sequence 1-5387
84 Simian virus 40 (SV40) genomic Genbank accession number: NC_001669.1 sequence entire nucleotide sequence 1-5243
Polyomavirus JC VP2 and VP3 Nucleotides 883 to 1468 of SEQ ID NO:5 overlap mRNA sequence
Polyomavirus JC VP2, VP3 and Nucleotides 1469 to 1560 of SEQ ID NO:5 VP1 overlap mRNA sequence
Polyomavirus JC Large T Nucleotides 4771 to 5013 of SEQ ID NO:5 antigen overlap with small T
antigen mRNA sequence
Polyomavirus Kl VP2 and VP3 Nucleotides 870 to 1497 of SEQ ID NO:6 overlap mRNA sequence
Polyomavirus Kl VP2, VP3 and Nucleotides 1498 to 1643 of SEQ ID NO:6 VP1 overlap mRNA sequence
Polyomavirus Kl Large T antigen Nucleotides 4716 to 4967 of SEQ ID NO:6 overlap with small T antigen
mRNA sequence
Polyomavirus WU VP2 and VP3 Nucleotides 1003 to 1669 of SEQ ID NO:7 overlap mRNA sequence
Polyomavirus WU VP2, VP3 and Nucleotides 1670 to 1821 of SEQ ID NO:7 VP1 overlap mRNA sequence
Polyomavirus WU Large T Nucleotides 4906 to 5157 of SEQ ID NO:7 antigen overlap with small T
antigen mRNA sequence
Polyomavirus Merkel VP2 and Nucleotides 4428 to 4983 of SEQ ID NO:83 VP3 overlap mRNA sequence
Polyomavirus Merkel VP2, VP3 Nucleotides 4393 to 4427 of SEQ ID NO:83 and VP1 overlap mRNA
sequence
Polyomavirus Merkel Large T Nucleotides 196 to 429 of SEQ ID NO:83 antigen overlap with small T
antigen mRNA sequence
Simian virus 40 (SV40) VP2 and Nucleotides 916 to 1498 of SEQ ID NO:84 VP3 overlap mRNA sequence
Simian virus 40 (SV40) VP2, Nucleotides 1499 to 1620 of SEQ ID NO:84 VP3 and VP1 overlap mRNA
sequence
Simian virus 40 (SV40) Large T Nucleotides 4918 to 5163 of SEQ ID NO:84 antigen overlap with small T
antigen mRNA sequence
The oligomers may comprise or consist of a contiguous nucleotide sequence which corresponds to the reverse complement of a nucleotide sequence present in SEQ ID NO: 2, 3 or 4. Thus, the oligomer may comprise or consist of, or a sequence selected from the group consisting of SEQ ID NOS: 31-53, wherein said oligomer (or contiguous nucleotide portion thereof) may optionally have one, two, or three mismatches against said selected sequence. The oligomer may comprise or consist of a contiguous nucleotide sequence which is fully complementary (perfectly complementary) to the equivalent region of a nucleic acid which encodes a mammalian polyomavirus such as polyomavirus BK (e.g., SEQ ID NO's: 31 - 53 ). Thus, the oligomer can comprise or consist of an antisense nucleotide sequence.
However, in some embodiments, the oligomer may tolerate e.g 1 , 2, 3, or 4 (or more) mismatches, when hybridising to the target sequence and still sufficiently bind to the target to show the desired effect, i.e. down-regulation of the target. Mismatches may, for example, be compensated by increased length of the oligomer nucleotide sequence and/or an increased number of nucleotide analogues, such as LNA, present within the nucleotide sequence.
In some embodiments, the contiguous nucleotide sequence comprises no more than 3, such as no more than 2 mismatches when hybridizing to the target sequence, such as to the corresponding region of a nucleic acid which encodes a mammalian polyomavirus such as polyomavirus BK.
In some embodiments, the contiguous nucleotide sequence comprises no more than a single mismatch when hybridizing to the target sequence, such as the corresponding region of a nucleic acid which encodes a polyomavirus such as polyomavirus BK.
In relation to compounds targeting the polyomavirus BK, the nucleotide sequence of the oligomers of the invention or the contiguous nucleotide sequence is preferably at least 80% homologous to a corresponding region in a sequence selected from the group consisting of SEQ ID NOS: 1 , 5, 6 or 7, such as at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96% homologous, at least 97% homologous, at least 98% homologous, at least 99% homologous, such as 100%
homologous (identical).
In relation to compounds targeting the polyomavirus BK, the nucleotide sequence of the oligomers of the invention or the contiguous nucleotide sequence is preferably at least 80% homologous to a corresponding region in a sequence selected from the group consisting of SEQ ID NOS: 1 , 3, 2 and 4 such as at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96% homologous, at least 97% homologous, at least 98% homologous, at least 99% homologous, such as 100%
homologous (identical).
In relation to compounds targeting the JC polyomavirus, the nucleotide sequence of the oligomers of the invention or the contiguous nucleotide sequence is preferably at least 80% homologous to a corresponding region in a sequence selected from the group consisting of SEQ ID NO 5, such as at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96% homologous, at least 97%
homologous, at least 98% homologous, at least 99% homologous, such as 100%
homologous (identical). See table 5 for some exemplary target regions of SEQ ID NO 5.
In relation to compounds targeting the Kl polyomavirus, the nucleotide sequence of the oligomers of the invention or the contiguous nucleotide sequence is preferably at least 80% homologous to a corresponding region in a sequence selected from the group consisting of SEQ ID NO 6 such as at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96% homologous, at least 97% homologous, at least 98% homologous, at least 99% homologous, such as 100% homologous (identical). See table 5 for some exemplary target regions of SEQ ID NO 6.
In relation to compounds targeting the WU polyomavirus, the nucleotide sequence of the oligomers of the invention or the contiguous nucleotide sequence is preferably at least 80% homologous to a corresponding region in a sequence selected from the group consisting of SEQ ID NO 7 such as at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96% homologous, at least 97% homologous, at least 98% homologous, at least 99% homologous, such as 100% homologous (identical). See table 5 for some exemplary target regions of SEQ ID NO 7.
In relation to compounds targeting the Merkel polyomavirus, the nucleotide sequence of the oligomers of the invention or the contiguous nucleotide sequence is preferably at least 80% homologous to a corresponding region in a sequence selected from the group consisting of SEQ ID NO 83 such as at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96% homologous, at least 97%
homologous, at least 98% homologous, at least 99% homologous, such as 100%
homologous (identical). See table 5 for some exemplary target regions of SEQ ID NO 83.
In relation to compounds targeting the SV40 polyomavirus, the nucleotide sequence of the oligomers of the invention or the contiguous nucleotide sequence is preferably at least 80% homologous to a corresponding region in a sequence selected from the group consisting of SEQ ID NO 84 such as at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96% homologous, at least 97%
homologous, at least 98% homologous, at least 99% homologous, such as 100%
homologous (identical). See table 5 for some exemplary target regions of SEQ ID NO 84.
In relation to compounds targeting the polyomavirus BK, the nucleotide sequence of the oligomers of the invention or the contiguous nucleotide sequence is preferably at least 80% homologous to a corresponding sequence selected from the group consisting of SEQ ID NOS: 54-77, such as at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96% homologous, at least 97% homologous, at least 98% homologous, at least 99% homologous, such as 100% homologous (identical).
In relation to compounds targeting the polyomavirus BK, the nucleotide sequence of the oligomers of the invention or the contiguous nucleotide sequence is preferably at least 80% homologous to the reverse complement of a corresponding sequence present in SEQ ID NO: 31-53, such as at least 85%, at least 90%, at least 91 %, at least 92%at least 93%, at least 94%, at least 95%, at least 96% homologous, at least 97% homologous, at least 98% homologous, at least 99% homologous, such as 100% homologous (identical).
In relation to compounds targeting the polyomavirus BK, the nucleotide sequence of the oligomers of the invention or the contiguous nucleotide sequence is preferably at least 80% complementary to a sub-sequence present in SEQ ID NO: 2, 3 or 4, such as at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96% complementary, at least 97% complementary, at least 98% complementary, at least 99% complementary, such as 100% complementary (perfectly complementary).
In some embodiments, the term "first region" as used herein refers to a portion (subsequence) of an oligomer. For example, the 12 monomer sequence SEQ ID No 14 is a subsequence of the 16 monomer sequence SEQ ID No 12 and comprises 12 contiguous monomers of SEQ ID No 12.
In relation to compounds targeting the polyomavirus BK, in some embodiments the oligomer (or contiguous nucleotide portion thereof) is selected from, or comprises, one of the sequences selected from the group consisting of SEQ ID NOS: 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, or a sub-sequence of at least 10 contiguous nucleotides thereof, wherein said oligomer (or contiguous nucleotide portion thereof) may optionally comprise one, two, or three mismatches when compared to the sequence.
In some embodiments the sub-sequence may consist of 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, or 29 contiguous nucleotides, such as from 12 -22, such as from 12-18 nucleotides. Suitably, in some embodiments, the sub-sequence is of the same length as the contiguous nucleotide sequence of the oligomer of the invention.
However, it is recognised that, in some embodiments the nucleotide sequence of the oligomer may comprise additional 5' or 3' nucleotides, such as, independently, 1 , 2, 3, 4 or 5 additional nucleotides 5' and/or 3', which are non-complementary to the target sequence. In this respect the oligomer of the invention, may, in some embodiments, comprise a contiguous nucleotide sequence which is flanked 5' and or 3' by additional nucleotides. In some embodiments the additional 5' or 3' nucleotides are naturally occurring nucleotides, such as DNA or RNA. In some embodiments, the additional 5' or 3' nucleotides may represent region D as referred to in the context of gapmer oligomers herein.
In some embodiments the oligomer according to the invention consists or comprises of a nucleotide sequence according to SEQ ID NO: 2, or a sub-sequence thereof.
In some embodiments the oligomer according to the invention consists or comprises of a nucleotide sequence according to SEQ ID NO: 3, or a sub-sequence thereof.
In some embodiments the oligomer according to the invention consists or comprises of a nucleotide sequence according to SEQ ID NO: 4, or a sub-sequence thereof.
In some embodiments the oligomer according to the invention consists or comprises of a nucleotide sequence according to SEQ ID NO: 31 , or a sub-sequence thereof.
In some embodiments the oligomer according to the invention consists or comprises of a nucleotide sequence according to SEQ ID NO: 32, or a sub-sequence thereof.
In some embodiments the oligomer according to the invention consists or comprises of a nucleotide sequence according to SEQ ID NO: 33, or a sub-sequence thereof.
In some embodiments the oligomer according to the invention consists or comprises of a nucleotide sequence according to SEQ ID NO: 34, or a sub-sequence thereof.
In some embodiments the oligomer according to the invention consists or comprises of a nucleotide sequence according to SEQ ID NO: 35, or a sub-sequence thereof.
In some embodiments the oligomer according to the invention consists or comprises of a nucleotide sequence according to SEQ ID NO: 36, or a sub-sequence thereof.
In some embodiments the oligomer according to the invention consists or comprises of a nucleotide sequence according to SEQ ID NO: 37, or a sub-sequence thereof.
In some embodiments the oligomer according to the invention consists or comprises of a nucleotide sequence according to SEQ ID NO: 38, or a sub-sequence thereof.
In some embodiments the oligomer according to the invention consists or comprises of a nucleotide sequence according to SEQ ID NO: 39, or a sub-sequence thereof.
In some embodiments the oligomer according to the invention consists or comprises of a nucleotide sequence according to SEQ ID NO: 40, or a sub-sequence thereof.
In some embodiments the oligomer according to the invention consists or comprises of a nucleotide sequence according to SEQ ID NO: 41 , or a sub-sequence thereof.
In some embodiments the oligomer according to the invention consists or comprises of a nucleotide sequence according to SEQ ID NO: 42, or a sub-sequence thereof.
In some embodiments the oligomer according to the invention consists or comprises of a nucleotide sequence according to SEQ ID NO: 43, or a sub-sequence thereof. In some embodiments the oligomer according to the invention consists or comprises of a nucleotide sequence according to SEQ ID NO: 44, or a sub-sequence thereof.
In some embodiments the oligomer according to the invention consists or comprises of a nucleotide sequence according to SEQ ID NO: 45, or a sub-sequence thereof.
In some embodiments the oligomer according to the invention consists or comprises of a nucleotide sequence according to SEQ ID NO: 46, or a sub-sequence thereof.
In some embodiments the oligomer according to the invention consists or comprises of a nucleotide sequence according to SEQ ID NO: 47, or a sub-sequence thereof.
In some embodiments the oligomer according to the invention consists or comprises of a nucleotide sequence according to SEQ ID NO: 48, or a sub-sequence thereof.
In some embodiments the oligomer according to the invention consists or comprises of a nucleotide sequence according to SEQ ID NO: 49, or a sub-sequence thereof.
In some embodiments the oligomer according to the invention consists or comprises of a nucleotide sequence according to SEQ ID NO: 50, or a sub-sequence thereof.
In some embodiments the oligomer according to the invention consists or comprises of a nucleotide sequence according to SEQ ID NO: 51 , or a sub-sequence thereof.
In some embodiments the oligomer according to the invention consists or comprises of a nucleotide sequence according to SEQ ID NO: 52, or a sub-sequence thereof.
In some embodiments the oligomer according to the invention consists or comprises of a nucleotide sequence according to SEQ ID NO: 53, or a sub-sequence thereof.
Table 1 Polyomavirus BK Target regions/sequences and antisense sequences
SEQ ID Description Position in Sequences
NO: NC_001538
1 Polyomavirus Genbank accession number: NC_001538 genomic sequence entire nucleotide sequence 1-5153
2 VP2 and VP3 Nucleotides 981 to 1563 of SEQ ID NO: 1 overlap mRNA
sequence
3 VP2, VP3 and VP1 Nucleotides 1564 to 1679 of SEQ ID NO:1 overlap mRNA
sequence
4 Large T antigen Nucleotides 4635 to 5153 of SEQ ID NO:1 overlap with small T antigen mRNA
sequence
VP2 and VP3 Nucleotides 981 to 1563 of SEQ ID NO: 1 overlap genomic
sequence
VP2, VP3 and VP1 Nucleotides 1564 to 1679 of SEQ ID NO:1 overlap mRNA
sequence
Large T antigen Nucleotides 4635 to 5153 of SEQ ID NO:1 overlap with small T
antigen
Target mRNA 434-449 AAACCTGGACTGGAAC
Target mRNA 662-677 CAGTGTATCTGAGGCT
Target mRNA 966-981 TATC AG C AATC AG G C A
Target mRNA 1092-1107 TTGTTTGCTACTATTT
Target mRNA 1469-1484 AGGAGGTGCTAATCAA
Target mRNA 1470-1483 GGAGGTGCTAATCA
Target mRNA 1471-1482 GAGGTGCTAATC
Target mRNA 1494-1509 CCTCAATGGATGTTGC
Target mRNA 1539-1554 ACACCTGCTCTTGAAG
Target mRNA 1583-1598 AAGGAGAGTGTCCAGG
Target mRNA 1584-1597 AGGAGAGTGTCCAG
Target mRNA 1585-1596 GGAGAGTGTCCA
Target mRNA 1653-1668 AGGAGGAGTAGAAGTT
Target mRNA 2041-2056 TTGGAAATGCAGGGAG
Target mRNA 2525-2540 CTATGTATGGTATGGA
Target mRNA 2976-2989 TTCTGAGATAAGTATG
Target mRNA 4120-4135 TAAGGGTGTTAATAAG
Target mRNA 4121-4134 AAGGGTGTTAATAA
Target mRNA 4122-4133 AGGGTGTTAATA
Target mRNA 4543-4556 ATGGAACAGAAGAGTG
Target mRNA 4906-4919 AGCTCAGAGGTTTGTG
Target mRNA 4988-5001 AATGAAGAGAATGAAT
Target mRNA 5023-5036 GAATTTCACCCTGACA 54 Antisense sequence 434-449 GTTCCAGTCCAGGTTT
55 Antisense sequence 662-677 AGCCTCAGATACACTG
56 Antisense sequence 966-981 TGCCTGATTGCTGATA
57 Antisense sequence 1092-1107 AAATAGTAGCAAACAA
58 Antisense sequence 1469-1484 TTGATTAGCACCTCCT
59 Antisense sequence 1470-1483 TGATTAGCACCTCC
60 Antisense sequence 1471-1482 GATTAGCACCTC
61 Antisense sequence 1494-1509 GCAACATCCATTGAGG
62 Antisense sequence 1539-1554 CTTCAAGAGCAGGTGT
63 Antisense sequence 1583-1598 CCTGGACACTCTCCTT
64 Antisense sequence 1584-1597 CTGGACACTCTCCT
65 Antisense sequence 1585-1596 TGGACACTCTCC
66 Antisense sequence 1653-1668 AACTTCTACTCCTCCT
67 Antisense sequence 2041-2056 CTCCCTGCATTTCCAA
68 Antisense sequence 2525-2540 TCCATACCATACATAG
69 Antisense sequence 2976-2989 ATACATACTTATCTCA
70 Antisense sequence 4120-4135 CTTATTAACACCCTTA
71 Antisense sequence 4121-4134 TTATTAACACCCTT
72 Antisense sequence 4122-4133 TATTAACACCCT
73 Antisense sequence 4543-4556 CACTCTTCTGTTCCAT
74 Antisense sequence 4906-4919 CACAAACCTCTGAGCT
75 Antisense sequence 4988-5001 ATTCATTCTCTTCATT
76 Antisense sequence 5023-5036 TGTCAGGGTGAAATTC
79 "Agno" 388-588 Agno protein
80 "VP23" 624-1679 VP2 and VP3, 5'-end of VP1
81 "VP4" 1564-2652 VP1 , 3'-end of VP2, VP3
82 "VP5" 2722-4566 Exon 2 of large T antigen
(antisense)
In some embodiments the oligomer according to the invention consists or comprises of a contiguous nucleotide sequence which is fully complementary to a contiguous nucleotide sequence present in (a sub-sequence of) SEQ ID NO: 79.
In some embodiments the oligomer according to the invention consists or comprises of a contiguous nucleotide sequence which is fully complementary to a contiguous nucleotide sequence present in (a sub-sequence of) SEQ ID NO: 80. In some embodiments the oligomer according to the invention consists or comprises of a contiguous nucleotide sequence which is fully complementary to a contiguous nucleotide sequence present in (a sub-sequence of) SEQ ID NO: 81.
In some embodiments the oligomer according to the invention consists or comprises of a contiguous nucleotide sequence which is fully homologous to a contiguous nucleotide sequence present in (a sub-sequence of) SEQ ID NO: 82.
In determining the degree of "complementarity" between oligomers of the invention (or regions thereof) and the target region of the nucleic acid which encodes Polyomavirus such as , such as those disclosed herein, the degree of "complementarity" (also, "homology" or "identity") is expressed as the percentage identity (or percentage homology) between the sequence of the oligomer (or region thereof) and the sequence of the target region (or the reverse complement of the target region) that best aligns therewith. The percentage is calculated by counting the number of aligned bases that are identical between the 2 sequences, dividing by the total number of contiguous monomers in the oligomer, and multiplying by 100. In such a comparison, if gaps exist, it is preferable that such gaps are merely mismatches rather than areas where the number of monomers within the gap differs between the oligomer of the invention and the target region.
As used herein, the terms "homologous" and "homology" are interchangeable with the terms "identity" and "identical".
The terms "corresponding to" and "corresponds to" refer to the comparison between the nucleotide sequence of the oligomer (i.e. the nucleobase or base sequence) or contiguous nucleotide sequence (a first region) and the equivalent contiguous nucleotide sequence of a further sequence selected from either i) a sub-sequence of the reverse complement of the nucleic acid target, such as the mRNA which encodes the polyomavirus proteins, such as the targets listed in table 4 or 5, such as one of the polyomavirus BK proteins, or SEQ ID NO: 2, 3 or 4, and/or ii) the sequence of nucleotides provided herein, such as the group consisting of SEQ ID NOS: 31 to 53, or sub-sequence thereof. Nucleotide analogues are compared directly to their equivalent or corresponding nucleotides. A first sequence which corresponds to a further sequence under i) or ii) typically is identicial to that sequence over the length of the first sequence (such as the contiguous nucleotide sequence) or, as described herein may, in some embodiments, is at least 80% homologous to a corresponding sequence, such as at least 85%, at least 90%, at least 91 %, at least 92%at least 93%, at least 94%, at least 95%, at least 96% homologous, such as 100% homologous (identical). The terms "corresponding nucleotide analogue" and "corresponding nucleotide" are intended to indicate that the nucleotide in the nucleotide analogue and the naturally occurring nucleotide are identical. For example, when the 2-deoxyribose unit of the nucleotide is linked to an adenine, the "corresponding nucleotide analogue" contains a pentose unit (different from 2-deoxyribose) linked to an adenine.
The terms "reverse complement", "reverse complementary" and "reverse
complementarity" as used herein are interchangeable with the terms "complement", "complementary" and "complementarity".
Length
The oligomers may comprise or consist of a contiguous nucleotide sequence of a total of 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29 or 30 contiguous nucleotides in length.
In some embodiments, the oligomers comprise or consist of a contiguous nucleotide sequence of a total of from 10 - 22, such as 12 - 18, such as 13 - 17 or 12 - 16, or 10-16, such as 10, 11 , 12, 13, 14, 15, 16 contiguous nucleotides in length.
In some embodiments, the oligomers comprise or consist of a contiguous nucleotide sequence of a total of 10, 11 , 12, 13, or 14 contiguous nucleotides in length.
In some embodiments, the oligomer according to the invention consists of no more than 22 nucleotides, such as no more than 20 nucleotides, such as no more than 18 nucleotides, such as 15, 16 or 17 nucleotides. In some embodiments the oligomer of the invention comprises less than 20 nucleotides. It should be understood that when a range is given for an oligomer, or contiguous nucleotide sequence length it includes the lower an upper lengths provided in the range, for example from (or between) 10 - 30, includes both 10 and 30.
Nucleosides and Nucleoside analogues
In some embodiments, the terms "nucleoside analogue" and "nucleotide analogue" are used interchangeably.
The term "nucleotide" as used herein, refers to a glycoside comprising a sugar moiety, a base moiety and a covalently linked group (linkage group), such as a phosphate or phosphorothioate internucleotide linkage group, and covers both naturally occurring nucleotides, such as DNA or RNA, and non-naturally occurring nucleotides comprising modified sugar and/or base moieties, which are also referred to as "nucleotide analogues" herein. Herein, a single nucleotide (unit) may also be referred to as a monomer or nucleic acid unit. In field of biochemistry, the term "nucleoside" is commonly used to refer to a glycoside comprising a sugar moiety and a base moiety, and may therefore be used when referring to the nucleotide units, which are covalently linked by the internucleotide linkages between the nucleotides of the oligomer. In the field of biotechnology, the term "nucleotide" is often used to refer to a nucleic acid monomer or unit, and as such in the context of an oligonucleotide may refer to the base - such as the "nucleotide sequence", typically refer to the nucleobase sequence (i.e. the presence of the sugar backbone and internucleoside linkages are implicit). Likewise, particularly in the case of oligonucleotides where one or more of the internucleoside linkage groups are modified, the term "nucleotide" may refer to a
"nucleoside" for example the term "nucleotide" may be used, even when specifiying the presence or nature of the linkages between the nucleosides.
As one of ordinary skill in the art would recognise, the 5' terminal nucleotide of an oligonucleotide does not comprise a 5' internucleotide linkage group, although may or may not comprise a 5' terminal group.
Non-naturally occurring nucleotides include nucleotides which have modified sugar moieties, such as bicyclic nucleotides or 2' modified nucleotides, such as 2' substituted nucleotides.
"Nucleotide analogues" are variants of natural nucleotides, such as DNA or RNA nucleotides, by virtue of modifications in the sugar and/or base moieties. Analogues could in principle be merely "silent" or "equivalent" to the natural nucleotides in the context of the oligonucleotide, i.e. have no functional effect on the way the oligonucleotide works to inhibit target gene expression. Such "equivalent" analogues may nevertheless be useful if, for example, they are easier or cheaper to manufacture, or are more stable to storage or manufacturing conditions, or represent a tag or label. Preferably, however, the analogues will have a functional effect on the way in which the oligomer works to inhibit expression; for example by producing increased binding affinity to the target and/or increased resistance to intracellular nucleases and/or increased ease of transport into the cell. Specific examples of nucleoside analogues are described by e.g. Freier & Altmann; Nucl. Acid Res., 1997, 25, 4429-4443 and Uhlmann; Curr. Opinion in Drug Development, 2000, 3(2), 293-213, and in Scheme 1 :
Figure imgf000025_0001
Phosphorthioate 2'-0-Methyl 2'-MOE
2
Figure imgf000025_0002
ate
2'-(3-hydroxy)propyl
Figure imgf000025_0003
Boranophosphates
Scheme 1
The oligomer may thus comprise or consist of a simple sequence of natural occurring nucleotides - preferably 2'-deoxynucleotides (referred to here generally as "DNA"), but also possibly ribonucleotides (referred to here generally as "RNA"), or a combination of such naturally occurring nucleotides and one or more non-naturally occurring nucleotides, i.e. nucleotide analogues. Such nucleotide analogues may suitably enhance the affinity of the oligomer for the target sequence.
Examples of suitable and preferred nucleotide analogues are provided by
WO2007/031091 or are referenced therein.
Incorporation of affinity-enhancing nucleotide analogues in the oligomer, such as LNA or 2'-substituted sugars, can allow the size of the specifically binding oligomer to be reduced, and may also reduce the upper limit to the size of the oligomer before non-specific or aberrant binding takes place. In some embodiments, the oligomer comprises at least 1 nucleoside analogue. In some embodiments the oligomer comprises at least 2 nucleotide analogues. In some embodiments, the oligomer comprises from 3-8 nucleotide analogues, e.g. 6 or 7 nucleotide analogues. In the by far most preferred embodiments, at least one of said nucleotide analogues is a locked nucleic acid (LNA); for example at least 3 or at least 4, or at least 5, or at least 6, or at least 7, or 8, of the nucleotide analogues may be LNA. In some
embodiments all the nucleotides analogues may be LNA.
It will be recognised that when referring to a preferred nucleotide sequence motif or nucleotide sequence, which consists of only nucleotides, the oligomers of the invention which are defined by that sequence may comprise a corresponding nucleotide analogue in place of one or more of the nucleotides present in said sequence, such as LNA units or other nucleotide analogues, which raise the duplex stability/Tm of the oligomer/target duplex (i.e. affinity enhancing nucleotide analogues).
In some embodiments, any mismatches between the nucleotide sequence of the oligomer and the target sequence are preferably found in regions outside the affinity enhancing nucleotide analogues, such as region B as referred to herein, and/or region D as referred to herein, and/or at the site of non modified such as DNA nucleotides in the oligonucleotide, and/or in regions which are 5' or 3' to the contiguous nucleotide sequence.
Examples of such modification of the nucleotide include modifying the sugar moiety to provide a 2'-substituent group or to produce a bridged (locked nucleic acid) structure which enhances binding affinity and may also provide increased nuclease resistance.
A preferred nucleotide analogue is LNA, such as oxy-LNA (such as beta-D-oxy-LNA, and alpha-L-oxy-LNA), and/or amino-LNA (such as beta-D-amino-LNA and alpha-L-amino- LNA) and/or thio-LNA (such as beta-D-thio-LNA and alpha-L-thio-LNA) and/or ENA (such as beta-D-ENA and alpha-L-ENA). Most preferred is beta-D-oxy-LNA.
In some embodiments the nucleotide analogues present within the oligomer of the invention (such as in regions A and C mentioned herein) are independently selected from, for example: 2'-0-alkyl-RNA units, 2'-amino-DNA units, 2'-fluoro-DNA units, LNA units, arabino nucleic acid (ANA) units, 2'-fluoro-ANA units, HNA units, INA (intercalating nucleic acid -Christensen, 2002. Nucl. Acids. Res. 2002 30: 4918-4925, hereby incorporated by reference) units and 2'MOE units. In some embodiments there is only one of the above types of nucleotide analogues present in the oligomer of the invention, or contiguous nucleotide sequence thereof.
In some embodiments the nucleotide analogues are 2'-0-methoxyethyl-RNA (2'MOE), 2'-fluoro-DNA monomers or LNA nucleotide analogues, and as such the oligonucleotide of the invention may comprise nucleotide analogues which are independently selected from these three types of analogue, or may comprise only one type of analogue selected from the three types. In some embodiments at least one of said nucleotide analogues is 2'-MOE- RNA, such as 2, 3, 4, 5, 6, 7, 8, 9 or 10 2'-MOE-RNA nucleotide units. In some
embodiments at least one of said nucleotide analogues is 2'-fluoro DNA, such as 2, 3, 4, 5, 6, 7, 8, 9 or 10 2'-fluoro-DNA nucleotide units.
In some embodiments, the oligomer according to the invention comprises at least one Locked Nucleic Acid (LNA) unit, such as 1 , 2, 3, 4, 5, 6, 7, or 8 LNA units, such as from 3 - 7 or 4 to 8 LNA units, or 3, 4, 5, 6 or 7 LNA units. In some embodiments, all the nucleotide analogues are LNA. In some embodiments, the oligomer may comprise both beta-D-oxy- LNA, and one or more of the following LNA units: thio-LNA, amino-LNA, oxy-LNA, and/or ENA in either the beta-D or alpha-L configurations or combinations thereof. In some embodiments all LNA cytosine units are 5'methyl-Cytosine. In some embodiments of the invention, the oligomer may comprise both LNA and DNA units. Preferably the combined total of LNA and DNA units is 10-25, such as 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, or 25, such as 10 - 24, preferably 10-20, such as 10 - 18, even more preferably 12-16. In some embodiments of the invention, the nucleotide sequence of the oligomer, such as the contiguous nucleotide sequence consists of at least one LNA and the remaining nucleotide units are DNA units. In some embodiments the oligomer comprises only LNA nucleotide analogues and naturally occurring nucleotides (such as RNA or DNA, most preferably DNA nucleotides), optionally with modified internucleotide linkages such as phosphorothioate.
The term "nucleobase" refers to the base moiety of a nucleotide and covers both naturally occurring a well as non-naturally occurring variants. Thus, "nucleobase" covers not only the known purine and pyrimidine heterocycles but also heterocyclic analogues and tautomeres thereof.
Examples of nucleobases include, but are not limited to adenine, guanine, cytosine, thymidine, uracil, xanthine, hypoxanthine, 5-methylcytosine, isocytosine, pseudoisocytosine, 5-bromouracil, 5-propynyluracil, 6-aminopurine, 2-aminopurine, inosine, diaminopurine, and 2-chloro-6-aminopurine.
In some embodiments, at least one of the nucleobases present in the oligomer is a modified nucleobase selected from the group consisting of 5-methylcytosine, isocytosine, pseudoisocytosine, 5-bromouracil, 5-propynyluracil, 6-aminopurine, 2-aminopurine, inosine, diaminopurine, and 2-chloro-6-aminopurine.
LNA The term "LNA" refers to a bicyclic nucleoside analogue, known as "Locked Nucleic Acid". It may refer to an LNA monomer, or, when used in the context of an "LNA
oligonucleotide", LNA refers to an oligonucleotide containing one or more such bicyclic nucleotide analogues. LNA nucleotides are characterised by the presence of a linker group (such as a bridge) between C2' and C4' of the ribose sugar ring - for example as shown as the biradical R4* - R2* as described below.
The LNA used in the oligonucleotide compounds of the invention preferably has the structure of the eneral formula I
Figure imgf000028_0001
Formula 1
wherein for all chiral centers, asymmetric groups may be found in either R or S orientation;
wherein X is selected from -0-, -S-, -N(RN*)-, -C(R6R6*)-, such as, in some
embodiments -0-;
B is selected from hydrogen, optionally substituted Ci-4-alkoxy, optionally substituted Ci-4-alkyl, optionally substituted Ci-4-acyloxy, nucleobases including naturally occurring and nucleobase analogues, DNA intercalators, photochemically active groups, thermochemically active groups, chelating groups, reporter groups, and ligands; preferably, B is a nucleobase or nucleobase analogue;
P designates an internucleotide linkage to an adjacent monomer, or a 5'-terminal group, such internucleotide linkage or 5'-terminal group optionally including the substituent R5 or equally applicable the substituent R5*;
P* designates an internucleotide linkage to an adjacent monomer, or a 3'-terminal group;
R4* and R2* together designate a bivalent linker group consisting of 1 - 4 groups/atoms selected from -C(RaRb)-, -C(Ra)=C(Rb)-, -C(Ra)=N-, -0-, -Si(Ra)2-, -S-, -S02-, -N(Ra)-, and >C=Z, wherein Z is selected from -0-, -S-, and -N(Ra)-, and Ra and Rb each is independently selected from hydrogen, optionally substituted Ci_i2-alkyl, optionally substituted C2-i2-alkenyl, optionally substituted C2-i2-alkynyl, hydroxy, optionally substituted Ci-12-alkoxy, C2-12- alkoxyalkyl, C2-i2-alkenyloxy, carboxy, Ci-12-alkoxycarbonyl, Ci-12-alkylcarbonyl, formyl, aryl, aryloxy-carbonyl, aryloxy, arylcarbonyl, heteroaryl, heteroaryloxy-carbonyl, heteroaryloxy, heteroarylcarbonyl, amino, mono- and di(Ci.6-alkyl)amino, carbamoyl, mono- and di(Ci-6- alkyl)-amino-carbonyl, amino-Ci-6-alkyl-aminocarbonyl, mono- and di(Ci-6-alkyl)amino-Ci-6- alkyl-aminocarbonyl, Ci-6-alkyl-carbonylamino, carbamido, Ci-6-alkanoyloxy, sulphono, Ci-6- alkylsulphonyloxy, nitro, azido, sulphanyl, Ci-6-alkylthio, halogen, DNA intercalators, photochemically active groups, thermochemically active groups, chelating groups, reporter groups, and ligands, where aryl and heteroaryl may be optionally substituted and where two geminal substituents Ra and Rb together may designate optionally substituted methylene (=CH2), wherein for all chiral centers, asymmetric groups may be found in either R or S orientation, and;
each of the substituents R1*, R2, R3, R5, R5*, R6 and R6*, which are present is independently selected from hydrogen, optionally substituted Ci-12-alkyl, optionally substituted C2-i2-alkenyl, optionally substituted C2-i2-alkynyl, hydroxy, Ci-12-alkoxy, C2-12- alkoxyalkyl, C2-i2-alkenyloxy, carboxy, Ci-12-alkoxycarbonyl, Ci-12-alkylcarbonyl, formyl, aryl, aryloxy-carbonyl, aryloxy, arylcarbonyl, heteroaryl, heteroaryloxy-carbonyl, heteroaryloxy, heteroarylcarbonyl, amino, mono- and di(Ci.6-alkyl)amino, carbamoyl, mono- and di(Ci-6- alkyl)-amino-carbonyl, amino-Ci-6-alkyl-aminocarbonyl, mono- and di(Ci-6-alkyl)amino-Ci-6- alkyl-aminocarbonyl, Ci-6-alkyl-carbonylamino, carbamido, Ci-6-alkanoyloxy, sulphono, Ci-6- alkylsulphonyloxy, nitro, azido, sulphanyl, Ci-6-alkylthio, halogen, DNA intercalators, photochemically active groups, thermochemically active groups, chelating groups, reporter groups, and ligands, where aryl and heteroaryl may be optionally substituted, and where two geminal substituents together may designate oxo, thioxo, imino, or optionally substituted methylene; ; wherein RN is selected from hydrogen and Ci-4-alkyl, and where two adjacent (non-geminal) substituents may designate an additional bond resulting in a double bond; and RN*, when present and not involved in a biradical, is selected from hydrogen and Ci-4-alkyl; and basic salts and acid addition salts thereof. For all chiral centers, asymmetric groups may be found in either R or S orientation.
In some embodiments, R4* and R2* together designate a biradical consisting of a groups selected from the group consisting of C(RaRb)-C(RaRb)-, C(RaRb)-0-, C(RaRb)-NRa-, C(RaRb)-S-, and C(RaRb)-C(RaRb)-0-, wherein each Ra and Rb may optionally be
independently selected. In some embodiments, Ra and Rb may be, optionally independently selected from the group consisting of hydrogen and Ci-6alkyl, such as methyl, such as hydrogen. In some embodiments, R4* and R2* together designate the biradical -0-CH(CH2OCH3)- (2'0-methoxyethyl bicyclic nucleic acid - Seth at al. , 2010, J. Org. Chem) - in either the R- or S- configuration.
In some embodiments, R4* and R2* together designate the biradical -0-CH(CH2CH3)- (2'0-ethyl bicyclic nucleic acid - Seth at al. , 2010, J. Org. Chem). - in either the R- or S- configuration.
In some embodiments, R4* and R2* together designate the biradical -0-CH(CH3)-. - in either the R- or S- configuration. In some embodiments, R4* and R2* together designate the biradical -0-CH2-0-CH2- - (Seth at al. , 2010, J. Org. Chem).
In some embodiments, R4* and R2* together designate the biradical -0-NR-CH3- - (Seth at al., 2010, J. Org. Chem) .
In some embodiments, the LNA units have a structure selected from the following group:
Figure imgf000030_0001
In some embodiments, R1*, R2, R3, R5, R5* are independently selected from the group consisting of hydrogen, halogen, Ci_6 alkyl, substituted Ci_6 alkyl, C2-6 alkenyl, substituted C2-6 alkenyl, C2-6 alkynyl or substituted C2-6 alkynyl, Ci-6 alkoxyl, substituted Ci-6 alkoxyl, acyl, substituted acyl, Ci-6 aminoalkyl or substituted Ci-6 aminoalkyl. For all chiral centers, asymmetric groups may be found in either R or S orientation.
In some embodiments, R1*, R2, R3, R5, R5* are hydrogen.
In some embodiments, R1*, R2, R3 are independently selected from the group consisting of hydrogen, halogen, Ci-6 alkyl, substituted Ci-6 alkyl, C2-6 alkenyl, substituted C2-6 alkenyl, C2-6 alkynyl or substituted C2-6 alkynyl, Ci-6 alkoxyl, substituted Ci-6 alkoxyl, acyl, substituted acyl, Ci-6 aminoalkyl or substituted Ci-6 aminoalkyl. For all chiral centers, asymmetric groups may be found in either R or S orientation.
In some embodiments, R1*, R2, R3 are hydrogen.
In some embodiments, R5 and R5* are each independently selected from the group consisting of H, -CH3, -CH2-CH3,- CH2-0-CH3, and -CH=CH2. Suitably in some
embodiments, either R5 or R5* are hydrogen, where as the other group (R5 or R5*
respectively) is selected from the group consisting of Ci-5 alkyl, C2-6 alkenyl, C2-6 alkynyl, substituted Ci_6 alkyl, substituted C2-6 alkenyl, substituted C2-6 alkynyl or substituted acyl (- C(=0)-); wherein each substituted group is mono or poly substituted with substituent groups independently selected from halogen, Ci-6 alkyl, substituted Ci-6 alkyl, C2-6 alkenyl, substituted C2-6 alkenyl, C2-6 alkynyl, substituted C2-6 alkynyl, OJ1 , SJi , NJ^, N3, COOJ1 , CN, 0-C(=0)NJ1J2, N(H)C(=NH)NJ,J2 or N(H)C(=X)N(H)J2 wherein X is O or S; and each J, and J2 is, independently, H, Ci-6 alkyl, substituted Ci-6 alkyl, C2-6 alkenyl, substituted C2-6 alkenyl, C2-6 alkynyl, substituted C2-6 alkynyl, Ci-6 aminoalkyl, substituted Ci-6 aminoalkyl or a protecting group. In some embodiments either R5 or R5* is substituted Ci-6 alkyl. In some embodiments either R5 or R5* is substituted methylene wherein preferred substituent groups include one or more groups independently selected from F, NJ^, N3, CN, OJ1 , SJi , O- C(=0)NJ1J2, N(H)C(=NH)NJ, J2 or N(H)C(0)N(H)J2. In some embodiments each J, and J2 is, independently H or Ci-6 alkyl. In some embodiments either R5 or R5* is methyl, ethyl or methoxymethyl. In some embodiments either R5 or R5* is methyl. In a further embodiment either R5 or R5* is ethylenyl. In some embodiments either R5 or R5* is substituted acyl. In some embodiments either R5 or R5* is C(=0)NJ1J2. For all chiral centers, asymmetric groups may be found in either R or S orientation. Such 5' modified bicyclic nucleotides are disclosed in WO 2007/134181 , which is hereby incorporated by reference in its entirety.
In some embodiments B is a nucleobase, including nucleobase analogues and naturally occurring nucleobases, such as a purine or pyrimidine, or a substituted purine or substituted pyrimidine, such as a nucleobase referred to herein, such as a nucleobase selected from the group consisting of adenine, cytosine, thymine, adenine, uracil, and/or a modified or substituted nucleobase, such as 5-thiazolo-uracil, 2-thio-uracil, 5-propynyl-uracil, 2'thio-thymine, 5-methyl cytosine, 5-thiozolo-cytosine, 5-propynyl-cytosine, and 2,6- diaminopurine.
In some embodiments, R4* and R2* together designate a biradical selected from - C(RaRb)-0-, -C(RaRb)-C(RcRd)-0-, -C(RaRb)-C(RcRd)-C(ReRf)-0-, -C(RaRb)-0-C(RcRd)-, - C(RaRb)-0-C(RcRd)-0-, -C(RaRb)-C(RcRd)-, -C(RaRb)-C(RcRd)-C(ReRf)-, - C(Ra)=C(Rb)-C(RcRd)-, -C(RaRb)-N(Rc)-, -C(RaRb)-C(RcRd)- N(Re)-, -C(RaRb)-N(Rc)-0-, and - C(RaRb)-S-, -C(RaRb)-C(RcRd)-S-, wherein Ra, Rb, Rc, Rd, Re, and Rf each is independently selected from hydrogen, optionally substituted Ci-i2-alkyl, optionally substituted C2-i2-alkenyl, optionally substituted C2-i2-alkynyl, hydroxy, Ci-i2-alkoxy, C2-i2-alkoxyalkyl, C2-i2-alkenyloxy, carboxy, Ci-i2-alkoxycarbonyl, Ci-i2-alkylcarbonyl, formyl, aryl, aryloxy-carbonyl, aryloxy, arylcarbonyl, heteroaryl, heteroaryloxy-carbonyl, heteroaryloxy, heteroarylcarbonyl, amino, mono- and di(Ci-6-alkyl)amino, carbamoyl, mono- and di(Ci-6-alkyl)-amino-carbonyl, amino- Ci-6-alkyl-aminocarbonyl, mono- and di(Ci-6-alkyl)amino-Ci-6-alkyl-aminocarbonyl, Ci-6-alkyl- carbonylamino, carbamido, Ci-6-alkanoyloxy, sulphono, Ci-6-alkylsulphonyloxy, nitro, azido, sulphanyl, Ci-6-alkylthio, halogen, DNA intercalators, photochemically active groups, thermochemically active groups, chelating groups, reporter groups, and ligands, where aryl and heteroaryl may be optionally substituted and where two geminal substituents Ra and Rb together may designate optionally substituted methylene (=CH2). For all chiral centers, asymmetric groups may be found in either R or S orientation.
In a further embodiment R4* and R2* together designate a biradical (bivalent group) selected from -CH2-0-, -CH2-S-, -CH2-NH-, -CH2-N(CH3)-, -CH2-CH2-0-, -CH2-CH(CH3)-, - CH2-CH2-S-, -CH2-CH2-NH-, -CH2-CH2-CH2-, -CH2-CH2-CH2-0-, -CH2-CH2-CH(CH3)-, - CH=CH-CH2-, -CH2-0-CH2-0-, -CH2-NH-0-, -CH2-N(CH3)-0-, -CH2-0-CH2-, -CH(CH3)-0-, and -CH(CH2-0-CH3)-0-, and/or, -CH2-CH2-, and -CH=CH- For all chiral centers, asymmetric groups may be found in either R or S orientation.
In some embodiments, R4* and R2* together designate the biradical C(RaRb)-N(Rc)-0-, wherein Ra and Rb are independently selected from the group consisting of hydrogen, halogen, Ci_6 alkyl, substituted Ci_6 alkyl, C2-6 alkenyl, substituted C2-6 alkenyl, C2-6 alkynyl or substituted C2-6 alkynyl, Ci-6 alkoxyl, substituted Ci-6 alkoxyl, acyl, substituted acyl, Ci-6 aminoalkyl or substituted Ci_6 aminoalkyl, such as hydrogen, and; wherein Rc is selected from the group consisting of hydrogen, halogen, Ci-6 alkyl, substituted Ci-6 alkyl, C2-6 alkenyl, substituted C2-6 alkenyl, C2-6 alkynyl or substituted C2-6 alkynyl, Ci-6 alkoxyl, substituted Ci-6 alkoxyl, acyl, substituted acyl, Ci-6 aminoalkyl or substituted Ci-6 aminoalkyl, such as hydrogen.
In some embodiments, R4* and R2* together designate the biradical C(RaRb)-0-C(RcRd) -0-, wherein Ra, Rb, Rc, and Rd are independently selected from the group consisting of hydrogen, halogen, Ci-6 alkyl, substituted Ci-6 alkyl, C2-6 alkenyl, substituted C2-6 alkenyl, C2-6 alkynyl or substituted C2-6 alkynyl, Ci-6 alkoxyl, substituted Ci-6 alkoxyl, acyl, substituted acyl, Ci-6 aminoalkyl or substituted Ci-6 aminoalkyl, such as hydrogen.
In some embodiments, R4* and R2* form the biradical -CH(Z)-0-, wherein Z is selected from the group consisting of Ci-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, substituted Ci-6 alkyl, substituted C2-6 alkenyl, substituted C2-6 alkynyl, acyl, substituted acyl, substituted amide, thiol or substituted thio; and wherein each of the substituted groups, is, independently, mono or poly substituted with optionally protected substituent groups independently selected from halogen, oxo, hydroxyl, OJi, NJiJ2l SJi, N3, OC(=X)Ji, OC(=X)NJ1J2, NJ3C(=X)NJ1J2 and CN, wherein each J2 and J3 is, independently, H or Ci-6 alkyl, and X is O, S or N^. In some embodiments Z is Ci-6 alkyl or substituted Ci-6 alkyl. In some embodiments Z is methyl. In some embodiments Z is substituted Ci-6 alkyl. In some embodiments said substituent group is Ci-6 alkoxy. In some embodiments Z is CH3OCH2-. For all chiral centers, asymmetric groups may be found in either R or S orientation. Such bicyclic nucleotides are disclosed in US 7,399,845 which is hereby incorporated by reference in its entirety. In some embodiments, R1*, R2, R3, R5, R5* are hydrogen. In some some embodiments, R1*, R2, R3 * are hydrogen, and one or both of R5, R5* may be other than hydrogen as referred to above and in WO 2007/134181.
In some embodiments, R4* and R2* together designate a biradical which comprise a substituted amino group in the bridge such as consist or comprise of the biradical -CH2-N( Rc)-, wherein Rc is Ci _ i2 alkyloxy. In some embodiments R4* and R2* together designate a biradical -Cq3q4-NOR -, wherein q3 and q are independently selected from the group consisting of hydrogen, halogen, Ci_6 alkyl, substituted Ci_6 alkyl, C2-6 alkenyl, substituted C2-6 alkenyl, C2-6 alkynyl or substituted C2-6 alkynyl, Ci-6 alkoxyl, substituted Ci-6 alkoxyl, acyl, substituted acyl, Ci-6 aminoalkyl or substituted Ci-6 aminoalkyl; wherein each substituted group is, independently, mono or poly substituted with substituent groups independently selected from halogen, OJi, SJi, NJiJ2l COOJi, CN, 0-C(=0)NJ1J2, N(H)C(=NH)N or N(H)C(=X=N(H)J2 wherein X is O or S; and each of ^ and J2 is, independently, H, Ci-6 alkyl, C2-6 alkenyl, C2_6 alkynyl, Ci_6 aminoalkyl or a protecting group. For all chiral centers, asymmetric groups may be found in either R or S orientation. Such bicyclic nucleotides are disclosed in WO2008/150729 which is hereby incorporated by reference in its entirity. In some embodiments, R1*, R2, R3, R5, R5* are independently selected from the group consisting of hydrogen, halogen, Ci-6 alkyl, substituted Ci-6 alkyl, C2-6 alkenyl, substituted C2-6 alkenyl, C2-6 alkynyl or substituted C2-6 alkynyl, Ci-6 alkoxyl, substituted Ci-6 alkoxyl, acyl, substituted acyl, Ci-6 aminoalkyl or substituted Ci-6 aminoalkyl. In some embodiments, R1*, R2, R3, R5, R5* are hydrogen. In some embodiments, R1*, R2, R3 are hydrogen and one or both of R5, R5* may be other than hydrogen as referred to above and in WO 2007/134181. In some embodiments R4* and R2* together designate a biradical (bivalent group) C(RaRb)-0-, wherein Ra and Rb are each independently halogen, C Ci2 alkyl, substituted C Ci2 alkyl, C2-Ci2 alkenyl, substituted C2-Ci2 alkenyl, C2-Ci2 alkynyl, substituted C2-Ci2 alkynyl, C Ci2 alkoxy, substituted C Ci2 alkoxy, OJi SJi, SOJi, S02Ji, NJiJ2l N3, CN,
Figure imgf000033_0001
C(=0)NJ1J2,
Figure imgf000033_0002
0-C(=0)NJ1J2, N(H)C(=NH)NJ1J2, N(H)C(=0)NJ1J2 or
N(H)C(=S)NJ1J2; or Ra and Rb together are =C(q3)(q4); q3 and q are each, independently, H, halogen, Ci-Ci2alkyl or substituted C Ci2 alkyl; each substituted group is, independently, mono or poly substituted with substituent groups independently selected from halogen, d- C6 alkyl, substituted Ci-C6 alkyl, C2- C6 alkenyl, substituted C2-C6 alkenyl, C2-C6 alkynyl, substituted C2-C6 alkynyl, OJi, SJi, NJiJ2l N3, CN, C(=0)OJi,
Figure imgf000033_0003
C(=0)Ji, O- C(=0)NJ1J2, N(H)C(=0)NJ1J2 or N(H)C(=S)NJ1J2. and; each J, and J2 is, independently, H, C1 -C6 alkyi, substituted C1 -C6 alkyi, C2-C6 alkenyl, substituted C2-C6 alkenyl, C2-C6 alkynyl, substituted C2-C6 alkynyl, C1 -C6 aminoalkyl, substituted C1 -C6 aminoalkyl or a protecting group. Such compounds are disclosed in WO2009006478A, hereby incorporated in its entirety by reference.
In some embodiments, R4* and R2* form the biradical - Q -, wherein Q is
C(qi)(q2)C(q3)(q4), C(qi)=C(q3), C[=C(qi)(q2)]-C(q3)(q4) or C(qi)(q2)-C[=C(q3)(q4)]; qi , q2, q3, q4 are each independently. H, halogen, Ci_i2 alkyi, substituted Ci_i2 alkyi, C2-i2 alkenyl, substituted Ci_i2 alkoxy, OJi , SJi , SOJi , S02Ji , NJiJ2l N3, CN,
Figure imgf000034_0001
, C(=0)-NJ1J2, C(=0) Ji , -C(=0)NJ1J2, N(H)C(=NH)NJ1J2, N(H)C(=0)NJ1J2 or N(H)C(=S)NJ1J2; each ^ and J2 is, independently, H, Ci-6 alkyi, C2-6 alkenyl, C2-6 alkynyl, Ci-6 aminoalkyl or a protecting group; and, optionally wherein when Q is C(qi)(q2)(q3)(q4) and one of q3 or q4 is CH3 then at least one of the other of q3 or q4 or one of \ and q2 is other than H. In some embodiments, R1*, R2, R3, R5, R5* are hydrogen. For all chiral centers, asymmetric groups may be found in either R or S orientation. Such bicyclic nucleotides are disclosed in WO2008/154401 which is hereby incorporated by reference in its entirity. In some embodiments, R1*, R2, R3, R5, R5* are independently selected from the group consisting of hydrogen, halogen, Ci_6 alkyi, substituted Ci-6 alkyi, C2-6 alkenyl, substituted C2-6 alkenyl, C2-6 alkynyl or substituted C2-6 alkynyl, Ci-6 alkoxyl, substituted Ci-6 alkoxyl, acyl, substituted acyl, Ci-6 aminoalkyl or substituted Ci-6 aminoalkyl. In some embodiments, R1*, R2, R3, R5, R5* are hydrogen. In some embodiments, R1*, R2, R3 are hydrogen and one or both of R5, R5* may be other than hydrogen as referred to above and in WO 2007/134181 or WO2009/067647 (alpha-L- bicyclic nucleic acids analogs).
In some embodiments the LNA used in the oligonucleotide compounds of the invention eneral formula II:
Figure imgf000034_0002
wherein Y is selected from the group consisting of -0-, -CH20-, -S-, -NH-, N(Re) and/or - CH2-; Z and Z* are independently selected among an internucleotide linkage, RH, a terminal group or a protecting group; B constitutes a natural or non-natural nucleotide base moiety (nucleobase), and RH is selected from hydrogen and Ci-4-alkyl; Ra, Rb Rc, Rd and Re are, optionally independently, selected from the group consisting of hydrogen, optionally substituted Ci_i2-alkyl, optionally substituted C2-i2-alkenyl, optionally substituted C2-i2-alkynyl, hydroxy, Ci-12-alkoxy, C2-i2-alkoxyalkyl, C2-i2-alkenyloxy, carboxy, Ci-12-alkoxycarbonyl, C1-12- alkylcarbonyl, formyl, aryl, aryloxy-carbonyl, aryloxy, arylcarbonyl, heteroaryl, heteroaryloxy- carbonyl, heteroaryloxy, heteroarylcarbonyl, amino, mono- and di(Ci-6-alkyl)amino, carbamoyl, mono- and di(Ci-6-alkyl)-amino-carbonyl, amino-Ci-6-alkyl-aminocarbonyl, mono- and di(Ci-6-alkyl)amino-Ci-6-alkyl-aminocarbonyl, Ci-6-alkyl-carbonylamino, carbamido, Ci-6- alkanoyloxy, sulphono, Ci-6-alkylsulphonyloxy, nitro, azido, sulphanyl, Ci-6-alkylthio, halogen, DNA intercalators, photochemically active groups, thermochemically active groups, chelating groups, reporter groups, and ligands, where aryl and heteroaryl may be optionally substituted and where two geminal substituents Ra and Rb together may designate optionally substituted methylene (=CH2); and RH is selected from hydrogen and Ci-4-alkyl. In some embodiments Ra, Rb Rc, Rd and Re are, optionally independently, selected from the group consisting of hydrogen and Ci_6 alkyl, such as methyl. For all chiral centers, asymmetric groups may be found in either R or S orientation, for example, two exemplary
stereochemical isomers include the beta-D and alpha-L isoforms, which may be illustrated as follows:
Figure imgf000035_0001
Specific exemplary LNA units are shown below:
Figure imgf000035_0002
β-D-oxy-LNA
Figure imgf000036_0001
β-D-amino-LNA
The term "thio-LNA" comprises a locked nucleotide in which Y in the general formula above is selected from S or -CH2-S-. Thio-LNA can be in both beta-D and alpha-L- configuration.
The term "amino-LNA" comprises a locked nucleotide in which Y in the general formula above is selected from -N(H)-, N(R)-, CH2-N(H)-, and -CH2-N(R)- where R is selected from hydrogen and Ci-4-alkyl. Amino-LNA can be in both beta-D and alpha-L- configuration.
The term "oxy-LNA" comprises a locked nucleotide in which Y in the general formula above represents -0-. Oxy-LNA can be in both beta-D and alpha-L-configuration.
The term "ENA" comprises a locked nucleotide in which Y in the general formula above is -CH2-0- (where the oxygen atom of -CH2-0- is attached to the 2'-position relative to the base B). Re is hydrogen or methyl.
In some exemplary embodiments LNA is selected from beta-D-oxy-LNA, alpha-L-oxy-LNA, beta-D-amino-LNA and beta-D-thio-LNA, in particular beta-D-oxy-LNA.
RNAse recruitment
It is recognised that an oligomeric compound may function via non RNase mediated degradation of target mRNA, such as by steric hindrance of translation, or other methods, however, the preferred oligomers of the invention are capable of recruiting an
endoribonuclease (RNase), such as RNase H.
It is preferable that the oligomer, or contiguous nucleotide sequence, comprises of a region of at least 6, such as at least 7 consecutive nucleotide units, such as at least 8 or at least 9 consecutive nucleotide units (residues), including 7, 8, 9, 10, 11 , 12, 13, 14, 15 or 16 consecutive nucleotides, which, when formed in a duplex with the complementary target RNA is capable of recruiting RNase. The contiguous sequence which is capable of recruiting RNAse may be region B as referred to in the context of a gapmer as described herein. In some embodiments the size of the contiguous sequence which is capable of recruiting RNAse, such as region B, may be higher, such as 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19 or 20 nucleotide units.
EP 1 222 309 provides in vitro methods for determining RNaseH activity, which may be used to determine the ability to recruit RNaseH. A oligomer is deemed capable of recruiting RNase H if, when provided with the complementary RNA target, it has an initial rate, as measured in pmol/l/min, of at least 1 %, such as at least 5%, such as at least 10% or ,more than 20% of the of the initial rate determined using DNA only oligonucleotide, having the same base sequence but containing only DNA monomers, with no 2'
substitutions, with phosphorothioate linkage groups between all monomers in the
oligonucleotide,, using the methodology provided by Example 91 - 95 of EP 1 222 309.
In some embodiments, an oligomer is deemed essentially incapable of recruiting RNaseH if, when provided with the complementary RNA target, and RNaseH, the RNaseH initial rate, as measured in pmol/l/min, is less than 1 %, such as less than 5%, such as less than 10% or less than 20% of the initial rate determined using the equivalent DNA only oligonucleotide, with no 2' substitutions, with phosphorothioate linkage groups between all nucleotides in the oligonucleotide, using the methodology provided by Example 91 - 95 of EP 1 222 309.
In other embodiments, an oligomer is deemed capable of recruiting RNaseH if, when provided with the complementary RNA target, and RNaseH, the RNaseH initial rate, as measured in pmol/l/min, is at least 20%, such as at least 40 %, such as at least 60 %, such as at least 80 % of the initial rate determined using the equivalent DNA only oligonucleotide, with no 2' substitutions, with phosphorothioate linkage groups between all nucleotides in the oligonucleotide, using the methodology provided by Example 91 - 95 of EP 1 222 309.
Typically the region of the oligomer which forms the consecutive nucleotide units which, when formed in a duplex with the complementary target RNA is capable of recruiting RNase consists of nucleotide units which form a DNA/RNA like duplex with the RNA target - and include both DNA units and LNA units which are in the alpha-L configuration, particularly preferred being alpha-L-oxy LNA.
The oligomer of the invention may comprise a nucleotide sequence which comprises both nucleotides and nucleotide analogues, and may be in the form of a gapmer, a headmer or a mixmer. A "headmer" is defined as an oligomer that comprises a region X and a region Y that is contiguous thereto, with the 5'-most monomer of region Y linked to the 3'-most monomer of region X. Region X comprises a contiguous stretch of non-RNase recruiting nucleoside analogues and region Y comprises a contiguous stretch (such as at least 7 contiguous monomers) of DNA monomers or nucleoside analogue monomers recognizable and cleavable by the RNase.
A "tailmer" is defined as an oligomer that comprises a region X and a region Y that is contiguous thereto, with the 5'-most monomer of region Y linked to the 3'-most monomer of the region X. Region X comprises a contiguous stretch (such as at least 7 contiguous monomers) of DNA monomers or nucleoside analogue monomers recognizable and cleavable by the RNase, and region X comprises a contiguous stretch of non-RNase recruiting nucleoside analogues.
Other "chimeric" oligomers, called "mixmers", consist of an alternating composition of (i) DNA monomers or nucleoside analogue monomers recognizable and cleavable by RNase, and (ii) non-RNase recruiting nucleoside analogue monomers.
In some embodiments, in addition to enhancing affinity of the oligomer for the target region, some nucleoside analogues also mediate RNase (e.g., RNaseH) binding and cleavage. Since a-L-LNA monomers recruit RNaseH activity to a certain extent, in some embodiments, gap regions (e.g., region B as referred to herein) of oligomers containing a-L- LNA monomers consist of fewer monomers recognizable and cleavable by the RNaseH, and more flexibility in the mixmer construction is introduced.
Gapmer Design
Preferably, the oligomer of the invention is a gapmer. A gapmer oligomer is an oligomer which comprises a contiguous stretch of nucleotides which is capable of recruiting an RNAse, such as RNAseH, such as a region of at least 6 or 7 DNA nucleotides, referred to herein in as region B (B), wherein region B is flanked both 5' and 3' by regions of affinity enhancing nucleotide analogues, such as from 1 - 6 nucleotide analogues 5' and 3' to the contiguous stretch of nucleotides which is capable of recruiting RNAse - these regions are referred to as regions A (A) and C (C) respectively.
In some embodiments, the monomers which are capable of recruiting RNAse are selected from the group consisting of DNA monomers, alpha-L-LNA monomers, C4' alkylayted DNA monomers (see PCT/EP2009/050349 and Vester ef a/. , Bioorg. Med. Chem. Lett. 18 (2008) 2296 - 2300, hereby incorporated by reference), and UNA (unlinked nucleic acid) nucleotides (see Flutter ef a/. , Mol. Biosyst. , 2009, 10, 1039 hereby incorporated by reference). UNA is unlocked nucleic acid, typically where the C2 - C3 C-C bond of the ribose has been removed, forming an unlocked "sugar" residue. Preferably the gapmer comprises a (poly)nucleotide sequence of formula (5' to 3'), A-B-C, or optionally A-B-C-D or D-A-B-C, wherein; region A (A) (5' region) consists or comprises of at least one nucleotide analogue, such as at least one LNA unit, such as from 1-6 nucleotide analogues, such as LNA units, and; region B (B) consists or comprises of at least five consecutive nucleotides which are capable of recruiting RNAse (when formed in a duplex with a complementary RNA molecule, such as the mRNA target), such as DNA nucleotides, and; region C (C) (3'region) consists or comprises of at least one nucleotide analogue, such as at least one LNA unit, such as from 1-6 nucleotide analogues, such as LNA units, and; region D (D), when present consists or comprises of 1 , 2 or 3 nucleotide units, such as DNA nucleotides.
In some embodiments, region A consists of 1 , 2, 3, 4, 5 or 6 nucleotide analogues, such as LNA units, such as from 2-5 nucleotide analogues, such as 2-5 LNA units, such as 3 or 4 nucleotide analogues, such as 3 or 4 LNA units; and/or region C consists of 1 , 2, 3, 4, 5 or 6 nucleotide analogues, such as LNA units, such as from 2-5 nucleotide analogues, such as 2-5 LNA units, such as 3 or 4 nucleotide analogues, such as 3 or 4 LNA units.
In some embodiments B consists or comprises of 5, 6, 7, 8, 9, 10, 11 or 12
consecutive nucleotides which are capable of recruiting RNAse, or from 6-10, or from 7-9, such as 8 consecutive nucleotides which are capable of recruiting RNAse. In some embodiments region B consists or comprises at least one DNA nucleotide unit, such as 1-12 DNA units, preferably from 4-12 DNA units, more preferably from 6-10 DNA units, such as from 7-10 DNA units, most preferably 8, 9 or 10 DNA units.
In some embodiments region A consist of 3 or 4 nucleotide analogues, such as LNA, region B consists of 7, 8, 9 or 10 DNA units, and region C consists of 3 or 4 nucleotide analogues, such as LNA. Such designs include (A-B-C) 3-10-3, 3-10-4, 4-10-3, 3-9-3, 3-9-4, 4-9-3, 3-8-3, 3-8-4, 4-8-3, 3-7-3, 3-7-4, 4-7-3, and may further include region D, which may have one or 2 nucleotide units, such as DNA units.
Further gapmer designs are disclosed in WO2004/046160, which is hereby
incorporated by reference. WO2008/1 13832, which claims priority from US provisional application 60/977,409 hereby incorporated by reference, refers to 'shortmer' gapmer oligomers. In some embodiments, oligomers presented here may be such shortmer gapmers.
In some embodiments the oligomer is consisting of a contiguous nucleotide sequence of a total of 10, 1 1 , 12, 13 or 14 nucleotide units, wherein the contiguous nucleotide sequence is of formula (5' - 3'), A-B-C, or optionally A-B-C-D or D-A-B-C, wherein; A consists of 1 , 2 or 3 nucleotide analogue units, such as LNA units; B consists of 7, 8 or 9 contiguous nucleotide units which are capable of recruiting RNAse when formed in a duplex with a complementary RNA molecule (such as a mRNA target); and C consists of 1 , 2 or 3 nucleotide analogue units, such as LNA units. When present, D consists of a single DNA unit.
In some embodiments A consists of 1 LNA unit. In some embodiments A consists of 2 LNA units. In some embodiments A consists of 3 LNA units. In some embodiments C consists of 1 LNA unit. In some embodiments C consists of 2 LNA units. In some embodiments C consists of 3 LNA units. In some embodiments B consists of 7 nucleotide units. In some embodiments B consists of 8 nucleotide units. In some embodiments B consists of 9 nucleotide units. . In certain embodiments, region B consists of 10 nucleoside monomers. In certain embodiments, region B comprises 1 - 10 DNA monomers. In some embodiments B comprises of from 1 - 9 DNA units, such as 2, 3, 4, 5, 6, 7 , 8 or 9 DNA units. In some embodiments B consists of DNA units. In some embodiments B comprises of at least one LNA unit which is in the alpha-L configuration, such as 2, 3, 4, 5, 6, 7, 8 or 9 LNA units in the alpha-L-configuration. In some embodiments B comprises of at least one alpha-L-oxy LNA unit or wherein all the LNA units in the alpha-L- configuration are alpha-L- oxy LNA units. In some embodiments the number of nucleotides present in A-B-C are selected from the group consisting of (nucleotide analogue units - region B - nucleotide analogue units): 1-8-1 , 1-8-2, 2-8-1 , 2-8-2, 3-8-3, 2-8-3, 3-8-2, 4-8-1 , 4-8-2, 1-8-4, 2-8-4, or; 1-9-1 , 1-9-2, 2-9-1 , 2-9-2, 2-9-3, 3-9-2, 1-9-3, 3-9-1 , 4-9-1 , 1-9-4, or; 1-10-1 , 1-10-2, 2-10- 1 , 2-10-2, 1-10-3, 3-10-1. In some embodiments the number of nucleotides in A-B-C are selected from the group consisting of: 2-7-1 , 1-7-2, 2-7-2, 3-7-3, 2-7-3, 3-7-2, 3-7-4, and 4-7- 3. In certain embodiments, each of regions A and C consists of three LNA monomers, and region B consists of 8 or 9 or 10 nucleoside monomers, preferably DNA monomers. In some embodiments both A and C consists of two LNA units each, and B consists of 8 or 9 nucleotide units, preferably DNA units. In various embodiments, other gapmer designs include those where regions A and/or C consists of 3, 4, 5 or 6 nucleoside analogues, such as monomers containing a 2'-0-methoxyethyl-ribose sugar (2'-MOE) or monomers containing a 2'-fluoro-deoxyribose sugar, and region B consists of 8, 9, 10, 1 1 or 12 nucleosides, such as DNA monomers, where regions A-B-C have 3-9-3, 3-10-3, 5-10-5 or 4- 12-4 monomers. Further gapmer designs are disclosed in WO 2007/14651 1A2, hereby incorporated by reference.
Internucleotide Linkages
The monomers of the oligomers described herein are coupled together via linkage groups. Suitably, each monomer is linked to the 3' adjacent monomer via a linkage group. The person having ordinary skill in the art would understand that, in the context of the present invention, the 5' monomer at the end of an oligomer does not comprise a 5' linkage group, although it may or may not comprise a 5' terminal group.
The terms "linkage group" or "internucleotide linkage" are intended to mean a group capable of covalently coupling together two nucleotides. Specific and preferred examples include phosphate groups and phosphorothioate groups.
The nucleotides of the oligomer of the invention or contiguous nucleotides sequence thereof are coupled together via linkage groups. Suitably each nucleotide is linked to the 3' adjacent nucleotide via a linkage group.
Suitable internucleotide linkages include those listed within WO2007/031091 , for example the internucleotide linkages listed on the first paragraph of page 34 of
WO2007/031091 (hereby incorporated by reference).
It is, in some embodiments, preferred to modify the internucleotide linkage from its normal phosphodiester to one that is more resistant to nuclease attack, such as
phosphorothioate or boranophosphate - these two, being cleavable by RNase H, also allow that route of antisense inhibition in reducing the expression of the target gene.
Suitable sulphur (S) containing internucleotide linkages as provided herein may be preferred. Phosphorothioate internucleotide linkages are also preferred, particularly for the gap region (B) of gapmers. Phosphorothioate linkages may also be used for the flanking regions (A and C, and for linking A or C to D, and within region D, as appropriate).
Regions A, B and C, may however comprise internucleotide linkages other than phosphorothioate, such as phosphodiester linkages, particularly, for instance when the use of nucleotide analogues protects the internucleotide linkages within regions A and C from endo-nuclease degradation - such as when regions A and C comprise LNA nucleotides.
The internucleotide linkages in the oligomer may be phosphodiester, phosphorothioate or boranophosphate so as to allow RNase H cleavage of targeted RNA. Phosphorothioate is preferred, for improved nuclease resistance and other reasons, such as ease of manufacture.
In one aspect of the oligomer of the invention, the nucleotides and/or nucleotide analogues are linked to each other by means of phosphorothioate groups.
It is recognised that the inclusion of phosphodiester linkages, such as one or two linkages, into an otherwise phosphorothioate oligomer, particularly between or adjacent to nucleotide analogue units (typically in region A and or C) can modify the bioavailability and/or bio-distribution of an oligomer - see WO2008/053314, hereby incorporated by reference. In some embodiments, such as the embodiments referred to above, where suitable and not specifically indicated, all remaining linkage groups are either phosphodiester or phosphorothioate, or a mixture thereof.
In some embodiments all the internucleotide linkage groups are phosphorothioate.
When referring to specific gapmer oligonucleotide sequences, such as those provided herein it will be understood that, in various embodiments, when the linkages are phosphorothioate linkages, alternative linkages, such as those disclosed herein may be used, for example phosphate (phosphodiester) linkages may be used, particularly for linkages between nucleotide analogues, such as LNA, units. Likewise, when referring to specific gapmer oligonucleotide sequences, such as those provided herein, when the C residues are annotated as 5'methyl modified cytosine, in various embodiments, one or more of the Cs present in the oligomer may be unmodified C residues. in some embodimentsin some embodiments
Oligome c Compounds
The oligomers of the invention may, for example, be selected from the group consisting of: SEQ IDS NO's: 8-30, see also table 2. Table 2 also discloses SEQ ID No: 78 which is the scrambled control compound used.
Conjugates
In the context the term "conjugate" is intended to indicate a heterogenous molecule formed by the covalent attachment ("conjugation") of the oligomer as described herein to one or more non-nucleotide, or non-polynucleotide moieties. Examples of non-nucleotide or non- polynucleotide moieties include macromolecular agents such as proteins, fatty acid chains, sugar residues, glycoproteins, polymers, or combinations thereof. Typically proteins may be antibodies for a target protein. Typical polymers may be polyethylene glycol.
Therefore, in various embodiments, the oligomer of the invention may comprise both a polynucleotide region which typically consists of a contiguous sequence of nucleotides, and a further non-nucleotide region. When referring to the oligomer of the invention consisting of a contiguous nucleotide sequence, the compound may comprise non-nucleotide
components, such as a conjugate component.
In various embodiments of the invention the oligomeric compound is linked to ligands/conjugates, which may be used, e.g. to increase the cellular uptake of oligomeric compounds. WO2007/031091 provides suitable ligands and conjugates, which are hereby incorporated by reference. The invention also provides for a conjugate comprising the compound according to the invention as herein described, and at least one non-nucleotide or non-polynucleotide moiety covalently attached to said compound. Therefore, in various embodiments where the compound of the invention consists of a specified nucleic acid or nucleotide sequence, as herein disclosed, the compound may also comprise at least one non-nucleotide or non- polynucleotide moiety (e.g. not comprising one or more nucleotides or nucleotide analogues) covalently attached to said compound.
Conjugation (to a conjugate moiety) may enhance the activity, cellular distribution or cellular uptake of the oligomer of the invention. Such moieties include, but are not limited to, antibodies, polypeptides, lipid moieties such as a cholesterol moiety, cholic acid, a thioether, e.g. Hexyl-s-tritylthiol, a thiocholesterol, an aliphatic chain, e.g., dodecandiol or undecyl residues, a phospholipids, e.g., di-hexadecyl-rac-glycerol or triethylammonium 1 ,2-di-o- hexadecyl-rac-glycero-3-h-phosphonate, a polyamine or a polyethylene glycol chain, an adamantane acetic acid, a palmityl moiety, an octadecylamine or hexylamino-carbonyl- oxycholesterol moiety.
The oligomers of the invention may also be conjugated to active drug substances, for example, aspirin, ibuprofen, a sulfa drug, an antidiabetic, an antibacterial or an antibiotic.
In certain embodiments the conjugated moiety is a sterol, such as cholesterol.
In various embodiments, the conjugated moiety comprises or consists of a positively charged polymer, such as a positively charged peptides of, for example from 1 -50, such as 2 - 20 such as 3 - 10 amino acid residues in length, and/or polyalkylene oxide such as polyethylglycol(PEG) or polypropylene glycol - see WO 2008/034123, hereby incorporated by reference. Suitably the positively charged polymer, such as a polyalkylene oxide may be attached to the oligomer of the invention via a linker such as the releasable inker described in WO 2008/034123.
By way of example, the following conjugate moieties may be used in the conjugates of the invention: 5'- OLIGOM ER -3'
Figure imgf000043_0001
5'- OLIGOMER -3'
Figure imgf000043_0002
Activated oligomers
The term "activated oligomer," as used herein, refers to an oligomer of the invention that is covalently linked (i.e., functionalized) to at least one functional moiety that permits covalent linkage of the oligomer to one or more conjugated moieties, i.e., moieties that are not themselves nucleic acids or monomers, to form the conjugates herein described.
Typically, a functional moiety will comprise a chemical group that is capable of covalently bonding to the oligomer via, e.g., a 3'-hydroxyl group or the exocyclic NH2 group of the adenine base, a spacer that is preferably hydrophilic and a terminal group that is capable of binding to a conjugated moiety (e.g., an amino, sulfhydryl or hydroxyl group). In some embodiments, this terminal group is not protected, e.g., is an NH2 group. In other embodiments, the terminal group is protected, for example, by any suitable protecting group such as those described in "Protective Groups in Organic Synthesis" by Theodora W
Greene and Peter G M Wuts, 3rd edition (John Wiley & Sons, 1999). Examples of suitable hydroxyl protecting groups include esters such as acetate ester, aralkyl groups such as benzyl, diphenylmethyl, or triphenylmethyl, and tetrahydropyranyl. Examples of suitable amino protecting groups include benzyl, alpha-methylbenzyl, diphenylmethyl,
triphenylmethyl, benzyloxycarbonyl, tert-butoxycarbonyl, and acyl groups such as trichloroacetyl or trifluoroacetyl. In some embodiments, the functional moiety is self- cleaving. In other embodiments, the functional moiety is biodegradable. See e.g., U.S. Patent No. 7,087,229, which is incorporated by reference herein in its entirety.
In some embodiments, oligomers of the invention are functionalized at the 5' end in order to allow covalent attachment of the conjugated moiety to the 5' end of the oligomer. In other embodiments, oligomers of the invention can be functionalized at the 3' end. In still other embodiments, oligomers of the invention can be functionalized along the backbone or on the heterocyclic base moiety. In yet other embodiments, oligomers of the invention can be functionalized at more than one position independently selected from the 5' end, the 3' end, the backbone and the base.
In some embodiments, activated oligomers of the invention are synthesized by incorporating during the synthesis one or more monomers that is covalently attached to a functional moiety. In other embodiments, activated oligomers of the invention are synthesized with monomers that have not been functionalized, and the oligomer is functionalized upon completion of synthesis. In some embodiments, the oligomers are functionalized with a hindered ester containing an aminoalkyi linker, wherein the alkyl portion has the formula (CH2)W, wherein w is an integer ranging from 1 to 10, preferably about 6, wherein the alkyl portion of the alkylamino group can be straight chain or branched chain, and wherein the functional group is attached to the oligomer via an ester group (-O-C(O)- (CH2)WNH).
In other embodiments, the oligomers are functionalized with a hindered ester containing a (CH2)w-sulfhydryl (SH) linker, wherein w is an integer ranging from 1 to 10, preferably about 6, wherein the alkyl portion of the alkylamino group can be straight chain or branched chain, and wherein the functional group attached to the oligomer via an ester group (-0-C(0)-(CH2)wSH)
In some embodiments, sulfhydryl-activated oligonucleotides are conjugated with polymer moieties such as polyethylene glycol or peptides (via formation of a disulfide bond).
Activated oligomers containing hindered esters as described above can be
synthesized by any method known in the art, and in particular by methods disclosed in PCT Publication No. WO 2008/034122 and the examples therein, which are incorporated herein by reference in its entirety.
In still other embodiments, the oligomers of the invention are functionalized by introducing sulfhydryl, amino or hydroxyl groups into the oligomer by means of a
functionalizing reagent substantially as described in U.S. Patent Nos. 4,962,029 and
4,914,210, i.e., a substantially linear reagent having a phosphoramidite at one end linked through a hydrophilic spacer chain to the opposing end which comprises a protected or unprotected sulfhydryl, amino or hydroxyl group. Such reagents primarily react with hydroxyl groups of the oligomer. In some embodiments, such activated oligomers have a
functionalizing reagent coupled to a 5'-hydroxyl group of the oligomer. In other
embodiments, the activated oligomers have a functionalizing reagent coupled to a 3'- hydroxyl group. In still other embodiments, the activated oligomers of the invention have a functionalizing reagent coupled to a hydroxyl group on the backbone of the oligomer. In yet further embodiments, the oligomer of the invention is functionalized with more than one of the functionalizing reagents as described in U.S. Patent Nos. 4,962,029 and 4,914,210, incorporated herein by reference in their entirety. Methods of synthesizing such
functionalizing reagents and incorporating them into monomers or oligomers are disclosed in U.S. Patent Nos. 4,962,029 and 4,914,210.
In some embodiments, the 5'-terminus of a solid-phase bound oligomer is
functionalized with a dienyl phosphoramidite derivative, followed by conjugation of the deprotected oligomer with, e.g., an amino acid or peptide via a Diels-Alder cycloaddition reaction.
In various embodiments, the incorporation of monomers containing 2'-sugar modifications, such as a 2'-carbamate substituted sugar or a 2'-(0-pentyl-N-phthalimido)- deoxyribose sugar into the oligomer facilitates covalent attachment of conjugated moieties to the sugars of the oligomer. In other embodiments, an oligomer with an amino-containing linker at the 2'-position of one or more monomers is prepared using a reagent such as, for example, 5'-dimethoxytrityl-2'-0-(e-phthalimidylaminopentyl)-2'-deoxyadenosine-3'- N,N- diisopropyl-cyanoethoxy phosphoramidite. See, e.g., Manoharan, et al., Tetrahedron Letters, 1991 , 34, 7171.
In still further embodiments, the oligomers of the invention may have amine- containing functional moieties on the nucleobase, including on the N6 purine amino groups, on the exocyclic N2 of guanine, or on the N4 or 5 positions of cytosine. In various embodiments, such functionalization may be achieved by using a commercial reagent that is already functionalized in the oligomer synthesis.
Some functional moieties are commercially available, for example, heterobifunctional and homobifunctional linking moieties are available from the Pierce Co. (Rockford, III.). Other commercially available linking groups are 5'-Amino-Modifier C6 and 3'-Amino-Modifier reagents, both available from Glen Research Corporation (Sterling, Va.). 5'-Amino-Modifier C6 is also available from ABI (Applied Biosystems Inc., Foster City, Calif.) as Aminolink-2, and 3'-Amino-Modifier is also available from Clontech Laboratories Inc. (Palo Alto, Calif.).
Compositions
The oligomer of the invention may be used in pharmaceutical formulations and compositions. Suitably, such compositions comprise a pharmaceutically acceptable diluent, carrier, salt or adjuvant. PCT/DK2006/000512 provides suitable and preferred
pharmaceutically acceptable diluent, carrier and adjuvants - which are hereby incorporated by reference. Suitable dosages, formulations, administration routes, compositions, dosage forms, combinations with other therapeutic agents, pro-drug formulations are also provided in PCT/DK2006/000512 - which are also hereby incorporated by reference.
Applications
The oligomers of the invention may be utilized as research reagents for, for example, diagnostics, therapeutics and prophylaxis.
In research, such oligomers may be used to specifically inhibit the synthesis of polyomavirus such as polyomavirus BK protein (typically by degrading or inhibiting the mRNA and thereby prevent protein formation) in cells and experimental animals thereby facilitating functional analysis of the target or an appraisal of its usefulness as a target for therapeutic intervention. In diagnostics the oligomers may be used to detect and quantitate polyomavirus such as polyomavirus BK expression in cell and tissues by northern blotting, in-situ hybridisation or similar techniques.
For therapeutics, an animal or a human, suspected of having a disease or disorder, which can be treated by modulating the expression of polyomavirus such as polyomavirus BK is treated by administering oligomeric compounds in accordance with this invention. Further provided are methods of treating a mammal, such as treating a human, suspected of having or being prone to a disease or condition, associated with expression of polyomavirus such as polyomavirus BK by administering a therapeutically or prophylactically effective amount of one or more of the oligomers or compositions of the invention. The oligomer, a conjugate or a pharmaceutical composition according to the invention is typically
administered in an effective amount.
The invention also provides for the use of the compound or conjugate of the invention as described for the manufacture of a medicament for the treatment of a disorder as referred to herein, or for a method of the treatment of as a disorder as referred to herein.
The invention also provides for a method for treating a disorder as referred to herein said method comprising administering a compound according to the invention as herein described, and/or a conjugate according to the invention, and/or a pharmaceutical composition according to the invention to a patient in need thereof.
Medical Indications
The oligomers and other compositions according to the invention can be used for the treatment of conditions associated with over expression or expression of mutated version of the polyomavirus, such as polyomavirus BK. Table 4 provides exemplary medical indications which are associated with certain polyomavirus infections, which may be treated according to some aspects of the present invention.
The invention further provides use of a compound of the invention in the manufacture of a medicament for the treatment of a disease, disorder or condition as referred to herein.
Generally stated, one aspect of the invention is directed to a method of treating a mammal suffering from or susceptible to conditions associated with abnormal levels of polyomavirus BK, comprising administering to the mammal a therapeutically effective amount of an oligomer targeted to polyomavirus such as polyomavirus BK that comprises one or more LNA units. The oligomer, a conjugate or a pharmaceutical composition according to the invention is typically administered in an effective amount.
The disease or disorder, as referred to herein, may, in some embodiments be associated with a mutation in the polyomavirus such as polyomavirus BK genome or a gene whose protein product is associated with or interacts with polyomavirus BK. Therefore, in some embodiments, the target mRNA is a mutated form of the polyomavirus such as polyomavirus BK sequence.
An interesting aspect of the invention is directed to the use of an oligomer (compound) as defined herein or a conjugate as defined herein for the preparation of a medicament for the treatment of a disease, disorder or condition as referred to herein.
The methods of the invention are preferably employed for treatment or prophylaxis against diseases caused by abnormal levels of polyomavirus BK.
Alternatively stated, In some embodiments, the invention is furthermore directed to a method for treating abnormal levels of polyomavirus BK, said method comprising
administering an oligomer of the invention, or a conjugate of the invention or a
pharmaceutical composition of the invention to a patient in need thereof.
The invention also relates to an oligomer, a composition or a conjugate as defined herein for use as a medicament.
The invention further relates to use of a compound, composition, or a conjugate as defined herein for the manufacture of a medicament for the treatment of abnormal levels of polyomavirus such as polyomavirus BK or expression of mutant forms of polyomavirus such as polyomavirus BK ( such as those associated with one of the diseases referred to herein).
Moreover, the invention relates to a method of treating a subject suffering from a disease or condition such as those referred to herein.
A patient who is in need of treatment is a patient suffering from or likely to suffer from the disease or disorder.
In some embodiments, the term 'treatment' as used herein refers to both treatment of an existing disease (e.g. a disease or disorder as herein referred to), or prevention of a disease, i.e. prophylaxis. It will therefore be recognised that treatment as referred to herein may, In some embodiments, be prophylactic.
ASPECTS
The following aspects of the present invention may be used in combination with the other embodiments described herein.
Aspect 1. In one embodiment, the compounds of the invention are for use as a medicament.
Aspect 2. In one embodiment, the compounds of the invention are made having a sequence that is well conserved between one or more polyomavirus forms, such as the sequence may be present in all of polyomavirus BK, polyomavirus JC and SV40, or it may be present in both polyomavirus BK and polyomavirus JC or in polyomavirus BK, and SV40 or in polyomavirus JC and SV40.
Aspect 3 In one embodiment according to aspect 2 for use as a medicament for the treatment of polyomavirus BK, polyomavirus JC and SV40, or it may be for treatment of polyomavirus BK and polyomavirus JC or polyomavirus BK, and SV40 or for polyomavirus JC and SV40 infection.
Aspect 4. In one embodiment, the compounds of the invention are for use as a medicament for the treatment of cancer.
Aspect 5. In one embodiment the compounds of the invention are for use as a medicament for the treatment of progressive multifocal leukoencephalopathy.
Aspect 6. In one embodiment, the oligonucleotide of the invention is targeted to the second exon of Large T antigen.
FURTHER EMBODIMENTS
The following further emboidments of the present invention may be used in combination with the other embodiments described herein
1. A single stranded oligomer of from 10 - 30 nucleotides in length which comprises a contiguous nucleotide sequence of a total of from 10 - 30 nucleotides, wherein said contiguous nucleotide sequence is at least 80% homologous to a region corresponding to a BKV gene or the reverse complement of an mRNA, such as SEQ ID NO: 2 to 4 or naturally occurring variant thereof.
2. The oligomer according to embodiment 1 , wherein the contiguous nucleotide sequence is at least 80% homologous to a region corresponding to any of SEQ ID NO: 31 to 53.
3. The oligomer according to embodiment 1 or 2, wherein the contiguous nucleotide
sequence comprises no mismatches or no more than one or two mismatches with the reverse complement of the corresponding region of SEQ ID NO: 31 to 53.
4. The oligomer according to any one of embodiments 1 - 3, wherein the nucleotide
sequence of the oligomer consists of the contiguous nucleotide sequence.
5. The oligomer according to any one of embodiments 1 - 4, wherein the contiguous
nucleotide sequence is from 10 - 18 nucleotides in length.
6. The oligomer according to any one of embodiments 1 - 5, wherein the contiguous
nucleotide sequence comprises nucleotide analogues.
7. The oligomer according to embodiment 6, wherein the nucleotide analogues are sugar modified nucleotides, such as sugar modified nucleotides selected from the group consisting of: Locked Nucleic Acid (LNA) units; 2'-0-alkyl-RNA units, 2'-OMe-RNA units, 2'-amino-DNA units, and 2'-fluoro-DNA units. 8. The oligomer according to embodiment 6, wherein the nucleotide analogues are LNA.
9. The oligomer according to any one of embodiments 6 - 8 which is a gapmer.
10. The oligomer according to any one of embodiments 1 - 9, which inhibits the expression of polyomavirus such as polyomavirus bk gene or mrna in a cell which is expressing polyomavirus such as polyomavirus bk gene or mrna.
1 1. The oligomer according to any one of embodiments 1-10, wherein the oligomer consist of or comprises any one of SEQ ID NO's: 8 to 30.
12. A conjugate comprising the oligomer according to any one of embodiments 1 - 11 , and at least one non-nucleotide or non-polynucleotide moiety covalently attached to said oligomer.
13. A pharmaceutical composition comprising the oligomer according to any one of
embodiments 1 - 11 , or the conjugate according to embodiment 12, and a
pharmaceutically acceptable diluent, carrier, salt or adjuvant.
14. The oligomer according to any one of embodiments 1 - 11 , or the conjugate according to embodiment 12, for use as a medicament, such as for the treatment of bk virus associated nephropathy and interstitial nephritis in kidney transplants, ureteral stenosis, haemorrhagic cystitis.
15. The use of an oligomer according to any one of the embodiments 1-1 1 , or a conjugate as defined in embodiment 12, for the manufacture of a medicament for the treatment of BK virus associated nephropathy and interstitial nephritis in kidney transplants, ureteral stenosis, haemorrhagic cystitis.
16. A method of treating BK virus associated nephropathy and interstitial nephritis in kidney transplants, ureteral stenosis, haemorrhagic cystitis, said method comprising
administering an effective amount of an oligomer according to any one of the
embodiments 1-10, or a conjugate according to embodiment 11 , or a pharmaceutical composition according to embodiment 12, to a patient suffering from, or likely to suffer from bk virus associated nephropathy and interstitial nephritis in kidney transplants, ureteral stenosis, haemorrhagic cystitis.
17. A method for the inhibition of polyomavirus such as polyomavirus BK in a cell which is expressing Polyomavirus BK, said method comprising administering an oligomer according to any one of the embodiments 1-10, or a conjugate according to embodiment 1 1 to said cell so as to inhibit polyomavirus such as polyomavirus BK in said cell.
EXAMPLES LNA monomer and oligonucleotide synthesis were performed using the methodology referred to in Examples 1 and 2 of PCT/EP2007/060703.
The stability of LNA oligonucleotides in human or rat plasma is performed using the methodology referred to in Example 4 of PCT/EP2007/060703
The above mentioned examples of PCT/EP2007/060703 are hereby specifically incorporated by reference.
Example 1: Design of the oligonucleotide
In a specific preferred design of the oligonucleotides of the invention, oligomers comprising 12 nucleotide sequences of Table 2 are designed as 2-8-2 (LNA-DNA-LNA) oligomers, oligomers comprising 13 nucleotide sequences of Table 2 are designed as 3-8-2, or 2-8-3 (LNA-DNA-LNA) oligomers and oligomers comprising 14 nucleotide sequences of Table 2 are designed as 3-8-3 (LNA-DNA-LNA) oligomers, and the preferred design of the 16 mers are 3-10-3 (LNA-DNA-LNA), wherein the LNAs are independently selected from oxy-LNA, thio-LNA, and amino-LNA, in either of the D-β and L-a configurations or combinations thereof.
Table 2
In Table 2, bold capital letters indicate LNA, s indicate phosphothioate bond, m indicate methyl C, O indicate oxy LNA.
SEQ ID NO Target Sequence
8 BKV °T OT O T OT OTO
5 -Qs' V V ¾¾¾¾¾¾¾¾¾¾Τ8" Ts' T' -3
9 BKV
b -Ag- tjg- us- cstscsasgsastsascsas I S J -._>
10 BKV I-J T* o_ om_ o , , , . OT o . o
1 1 BKV
5J-Ag- As- As- tsasgstsasgscsasasas Cs m As m A- -3'
12 BKV ci -r oT o_ o , , , iri- omr oTo
ΰ " ' s' ' s "s" ^Vs^Ss^s^' s us' us' ' ' ~ό
13 BKV ci -r o_ o . o, , T om« omr o
ΰ " ' s' As Vs^Q's0^3^0^ s s s
14 BKV o . oT o, T om« o
ΰ 1 As 1 s rsas9scsascscs 1 s us' "
15 BKV r, _ om_ o . o , . o _ o_o ,
5J-GS- C3- As- ascsastscscsaststsgsAg- Gs" G- -3'
16 BKV R, IT1 _ OT OT O T 0_ OTO ,
5 - cs' V V ¾ s s¾¾¾Ts' Gs' T' "3
17 BKV cs nip omr oT o , , m oT oTo„,
¾ " us' us' 1 s' QsSs^^s^s^s^ us' ' s ~ά
Figure imgf000052_0001
Example 2: LNA nucleotides suppresses luciferase reporter-gene expression in primary human kidney cells using a transfection vehicle
Reporter gene: Reporter constructs were made by inserting fragments of the BK virus early ("VP5", "VP6") and late ("Agno", "VP23", "VP4") into the psiCheck-2 plasmid (Promega) using the Xhol/NotI sites. The early genes are expressed in the antisense orientation to the genomic sequence and thus inserted as the complementary sequence. The late genes are expressed in the sense orientation and are inserted as the direct sequence. The inserts were created by de novo gene synthesis (GeneART). (Table 3 and Figure 2)
Table 3
Figure imgf000053_0001
Propagating the cell line: Human renal proximal tubule endothelial cell (RPTEC) (CC-2553, Lonza) were cultured in media REGM (CC-3190, Lonza). The cells were passaged once weekly and seeded at a density of 2500 cells/cm2. Cells were passaged by trypsinization, using the ReagentPack subculture reagents (CC-5034, Lonza)
Transfection and reporter assay of the human primary cell RPTEC: Suppression of the BK virus-specific reporter gene was performed using the Dual-Luciferase Reporter Assay System (Promega, Cat# E1910) generally as described in Elmen ef a/ 2007 with the following specific alterations: Freshly trypsinized RPTEC cells were plated and left to adhere for 24 hours in 96-well plates at a density of 4000 cells per well. Each reporter construct (figure 2) was mixed with a relevant LNA-oligonucleotide or a scrambled control, using 0.14 μΙ_Λ βΙΙ of Iipofectamine2000 (Invitrogen), 20 ng/well of reporter plasmid and 0.2 to 25 nM of LNA oligonucleotide in 100 μΙ_ of OptiMEM. Cells were incubated for 4 hours in the presence of the transfection medium, which was then replaced with REGM media. After a further 20 hours, the cells were lysed and assayed according to the Dual-Luciferase Reporter Assay protocol (Promega, Cat# E1910).
Oligonucleotides were tested in HeLa cells against reporters carrying their specific target site. The assay was done by co-transfection of the oligonucleotides at concentrations varying from 0.2 to 25 nM along with 10 ng/well of the reporter construct. The luciferase levels were determined 24 hours after transfection. SEQ ID NO: 78, a scrambled oligonucleotide was included as negative control in each experiment.
Oligonucleotides targeting the second exon of the VP1 gene were tested against the appropriate reporter "VP5". A potent knockdown could be seen for one of three
oligonucleotides (SEQ ID NO: 23), whereas a less pronounced knockdown was seen for the remaining two oligonucleotides (SEQ ID NO: 24 and 27) targeting this reporter construct. The data is presented in figure 3.
Oligonucleotides targeting the overlapping open reading frame between the large T and small t antigens were tested against their specific reporter ("VP6"). For all three oligonucleotides, a robust and dose dependent knockdown was seen. The data is presented in Figure 4.
A single oligonucleotide (SEQ ID NO: 8) targeting the Agno protein ORF was tested against its specific target in the luciferase assay ("Agno"). A very robust and dose-dependent response was seen for this oligonucleotide (Figure 5).
Oligonucleotides targeting the VP1 open reading frame were tested against their specific target in a luciferase assay (misleadingly named "VP4"). These oligonucleotides include some that target the short region of VP1 that overlap the open reading frame of VP2 and VP3 (SEQ ID NO: 17 and 20). As seen for the previous examples, a robust and dose- dependent knockdown was seen for all oligonucleotides tested. The data is presented in Figure 6.
The overall result of the in vitro reporter screening was the identification of multiple effective oligonucleotides against fragments of all the open reading frames in BK virus.
Example 3: LNA nucleotides represses BK virus early and late transcripts in in vitro infected primary human kidney cells using a transfection vehicle
Propagating the BK virus Gardner strain: An aliquot of the BK virus Gardner strain was acquired from ATCC (VR-837). Preparation of BK virus viral stock for infection experiments in Vero cells was done using a modified version of the protocols described in Tremolada et al (2010) and Moriyama et al (2009). In brief, Vero cells were infected an approximate MOI of 0.5. The infection proceeded for approximately 14 days and the cells were mechanically disrupted by repeated freeze-thaw cycles and the virus particles were enzymatically released from the cell debris, using neuraminidase (N2876 Sigma). After removal of debris, the supernatant was stored as viral stock at -80 degrees Celsius. Viral stock was titered on vero cells by immunofluorescence, and titer is expressed as fluorescence forming units (FFU) per ml_ (Tremolada et al).
Propagating Vero cells: Vero cells were propagated using the DMEM AQ (Sigma, D-0819), 10% Fetal bovine serum (Biochrom, vwr) and 25 μg/mL Gentamycin (Sigma). Cells were passaged 2 or 3 times per week and plated at a density of 10000-30000 cells per cm2. Cells were passaged by trypsinization, using phosphate buffered saline (D8537, Sigma) and trypsin (25300054, Invitrogen)
Propagating the RPTEC cell line: Human renal proximal tubule endothelial cell (RPTEC) (CC-2553, Lonza) were cultured in media REGM (CC-3190, Lonza). The cells were passaged once weekly and seeded at a density of 2500 cells/cm2. Cells were passaged by trypsinization, using the ReagentPack subculture reagents (CC-5034, Lonza) Transfection of BK virus infected human primary cell RPTEC:
Infections were performed on RPTEC cells plated in 12-well plates at 48000 cells per well and allowed 48-72 hours to adhere. The cells were infected with the BK virus, Gardner strain at a multiplicity of infection of 1. The infection was allowed to proceed for 48 hours, before delivery of oligonucleotides by transfection. Transfections were carried out essentially as described by Dean et al. (1994, JBC 269: 16416-16424). In short, cells were preincubated for 7 min. with Lipofectamine in OptiMEM followed by addition of oligonucleotide to a total volume of 1.5 ml_ transfection mix per well. After 4 hours, the transfection mix was removed; cells were washed and grown at 37C for approximately 20 hours in the appropriate growth medium. Cells were then harvested for RNA analysis.
Total RNA Isolation. Total RNA was isolated using RNeasy mini kit (Qiagen).
First strand synthesis. First strand synthesis was performed using M-MLV Reverse transcriptase essentially as described by manufacturer (Ambion). In brief, 0.5 μg total RNA of each sample was adjusted to 10.8 μΙ_ in water. 2 μΙ_ decamers and 2 μΙ_ dNTP mix (2.5 mM each) was added. Samples were heated to 70°C for 3 min. and cooled immediately in ice water and added 3.25 μΙ_ of a mix containing (2 μΙ_ 10x RT buffer; 1 μΙ_ M-MLV Reverse Transcriptase; 0.25 μΙ_ RNAase inhibitor). cDNA is synthesized at 42°C for 60 min followed by a heating inactivation step at 95°C for 10 min and finally cooled to 4°C.
Quantitative RT-PCR: Viral mRNA levels were determined by quantitative RT-PCR and performed using TaqMan-type probes against either the early or the late viral transcript and normalized to the level of the host cell GAPDH gene.
To determine the efficacy of the oligonucleotides in an infected cell system, they were tested against wild type, full length virus in an infected cell system in vitro. An in vivo infection system was set up, using wild type BK virus and RPTEC cells. The integrity of the system and the technical skill required for the screening of the oligonucleotides in infected cells was verified. These data were documented and presented at the previous review session and will not be included in this report.
In brief, RPTEC cells, a primary cell line from renal proximal endothelial cells, were infected with the Gardner strain of BK virus at a multiplicity of infection of 1. The infection was allowed to progress for 72 hours before oligonucleotide was delivered by lipid mediated transfection. The oligonucleotides were delivered at concentrations of 1 nM, 5 nM and 25 nM. The cells were harvested 24 hours after transfection. Quantitative PCR was done on to determine the levels of the early and late transcript families.
The effect of the transfected oligonucleotides on the BK virus gene expression in the infected cells differed in part from what was seen in the reporter assay. In particular, the oligonucleotides targeting the early transcript appeared to have a modest effect on the early transcript (Figure 7). In fact, only a single oligonucleotide appeared to generate a better- than-50% knockdown and that only at the highest oligonucleotide concentration. However, even though the reduction of the early transcripts were modest, the expression of the late transcripts in some samples appeared in some samples to be more reduced than what might be expected from the modest knock-down of the early, regulatory genes. This effect was most noticeable with SEQ ID NO: 24.
When examining the effect of the late-transcript targeting oligonucleotides on both the early and late transcripts (Figure 8), a significantly altered image was seen. Unlike the first class of oligonucleotides, the late-transcript-targeting oligonucleotides appear to have a generally robust and dose-dependent effect on their direct target. The one exception to this rule appears to be SEQ ID NO: 11. Remarkably, the viral response to the knockdown of the late gene appears to be the activation of the early genes. For almost all late-transcript targeting oligonucleotides, the early genes respond by a dramatic increase in expression level. The likely explanation to this response is that the loss of expression in the late genes leads to a feed-back response in the expression of the early genes. Some evidence exists to support this model, in particular the Agno protein has been proposed as an inhibitor of the transactivation function of the large T antigen. Without this inhibition, the large T antigen will continue to act on both the early and late promoters, leading to increased accumulation of the early gene transcripts. The overall result of the transfection of BK virus-directed oligonucleotides is the identification of a several efficient oligonucleotides that target the late transcripts in infected cells. Secondarily, it is also apparent that the knockdown of the late genes will perturb the entire viral gene expression cascade in the infected cells.
Example 4: LNA nucleotides represses BK virus early and late transcripts in in vitro infected primary human kidney cells in the absence of a transfection vehicle
Propagating the cell line: Human renal proximal tubule endothelial cell (RPTEC) (CC-2553, Lonza) were cultured in media REGM (CC-3190, Lonza). The cells were passaged once weekly and seeded at a density of 2500 cells/cm2. Cells were passaged by trypsinization, using the ReagentPack subculture reagents (CC-5034, Lonza)
Gymnosis of infected cells. Infections were performed on RPTEC cells plated in 12-well plates at 20000 cells per well and allowed 48-72 hours to adhere. The cells were infected with the BK virus, Gardner strain at a multiplicity of infection of 1. The infection was allowed to proceed for 24 hours, before delivery of oligonucleotides by gymnosis. Oligonucleotides were delivered directly to the growth medium, at a concentration of 1 μΜ, 5 μΜ or 25 μΜ. The infected, oligonucleotide treated cells were left to incubate for an additional 144 hours before cells were harvested for RNA analysis.
Total RNA Isolation. Total RNA was isolated using RNeasy mini kit (Qiagen).
First strand synthesis. First strand synthesis was performed using M-MLV Reverse transcriptase essentially as described by manufacturer (Ambion). In brief, 0.5 μg total RNA of each sample was adjusted to 10.8 μΙ_ in water. 2 μΙ_ decamers and 2 μΙ_ dNTP mix (2.5 mM each) was added. Samples were heated to 70°C for 3 min. and cooled immediately in ice water and added 3.25 μΙ_ of a mix containing (2 μΙ_ 10x RT buffer; 1 μΙ_ M-MLV Reverse Transcriptase; 0.25 μί RNAase inhibitor). cDNA is synthesized at 42°C for 60 min followed by a heating inactivation step at 95°C for 10 min and finally cooled to 4°C.
Quantitative RT-PCR: Viral mRNA levels were determined by quantitative RT-PCR and performed using TaqMan-type probes against either the early or the late viral transcript and normalized to the level of the host cell GAPDH gene.
In addition to the transfection screen of the library of BK virus directed oligonucleotides, data was acquired for the treatment of BK virus infected RPTEC cells using the methodology described above.
Here, the RPTEC cells were infected with BK virus at an MOI of 1 for 48 hours prior to addition of oligonucleotide. The oligonucleotides were added at concentrations of 1 , 5 and 25 μΜ and left on the cells for an additional 6 days (144h), for a total of 196 hours post infection. The cells were harvested, RNA was isolated and the levels of early and late transcripts were determined by qPCR.
Figure 9 shows the effect of treating the infected RPTEC cells with early-transcript targeting oligonucleotides. The oligonucleotides that were designed to act directly on the early transcript, do in fact lead to knockdown of the viral transcripts in infection. This knockdown leads to a corresponding knockdown in the late genes. This observation fits well with what is known about the expression cascade of BK virus, where the loss of the regulatory proteins Large T antigen and small t antigen, would be expected to lead to abolishment of expression of the late, structural genes. The most potent oligonucleotides in this experiment appear to be oligonucleotides targeted to the second exon of Large T antigen (SEQ ID NO: 23 and 24), with the oligonucleotides targeting the overlapping open reading frames exerting an intermediate effect (SEQ ID NO: 29 and 30). Oligonucleotides targeting the intron between the two exons of Large T antigen (which also form the 5'-end of the small t antigen open reading frame) appear to have much less activity (SEQ ID NO: 27 and 28). Similarly, the late-transcript targeting oligonucleotides were tested (Figure 10). As seen in transfection, the oligonucleotides generally appear to have a robust effect on their direct target ("VP1"). Most appear to demonstrate a very significant, dose-dependent knockdown of the target. The major exception is SEQ ID NO: 11 , which does show a knockdown, but with an inverse dose response.
As in the transfection experiment, an indirect effect can also be seen on the level of the early transcripts (T-ANTI). However, unlike transfection, the longer-term treatment by gymnosis leads comprehensive knockdown of the early transcripts. Like the effect on the direct target, this appears to be a dose dependent response and demonstrate kinetics depending on the strength of the direct activity of the oligonucleotide. This includes SEQ ID NO: 1 1 , where the inverse dose response on the direct target corresponds to a poor response on the indirect target. For both the direct and the indirect effect, the most potent oligonucleotides represent target sites throughout the transcript. Of these, SEQ ID NO: 12 and SEQ ID NO: 15 target the overlap between VP2 and VP3, whereas SEQ ID NO: 17 and SEQ ID NO: 20 target the short overlap between all three major structural proteins (VP1 , VP2 and VP3) (Figure 10). The overall conclusion of the gymnosis experiment is that efficient knockdown of both classes of transcripts in BK virus can be achieved. The experiments confirm that the gene expression cascade can be perturbed both by knockdown of the regulatory, early transcript, but also by knockdown of the late, predominantly structural genes. The latter observation is unexpected, and strongly suggests that the BK virus is a sensitive target for LNA- oligonucleotide-mediated therapy.

Claims

1. An oligomer of 10 - 30 nucleotides in length, which comprises or consists of a
contiguous nucleotide sequence of 10 - 30 nucleotides, wherein the contiguous nucleotide sequence is the complement of a target region of a polyomavirus genome, or a polyomavirus transcript such as a polyomavirus mRNA, wherein the target region of the polyomavirus is present in at least two, such as two or three, independent transcripts expressed by the polyomavirus genome, wherein the at least two
independent transcripts, such as two or three, are transcribed from over-lapping open- reading frames present in the polyomavirus genome.
2. The oligomer according to claim 1 , wherein said contiguous nucleotide sequence is complementary to a sequence selected from the group consisting of: SEQ ID NO s 1 , 3, 2, 4, 5, 6, 7, 83 and 84.
3. The oligomer according to claim 1 or 2, wherein the contiguous nucleotide sequence is present in the complement of a sequence selected from SEQ ID NO 31 to 53.
4. The oligomer according to any one of claims 1 - 3, wherein the nucleotide sequence of the oligomer consists of the contiguous nucleotide sequence.
5. The oligomer according to any one of claims 1 - 4, wherein the contiguous nucleotide sequence is from 10 - 18 nucleotides in length.
6. The oligomer according to any one of claims 1 - 5, wherein the contiguous nucleotide sequence comprises nucleotide analogues.
7. The oligomer according to claim 6, wherein the nucleotide analogues are sugar modified nucleotides, such as sugar modified nucleotides selected from the group consisting of: Locked Nucleic Acid (LNA) units; 2'-0-alkyl-RNA units, 2'-OMe-RNA units, 2'-amino- DNA units, and 2'-fluoro-DNA units.
8. The oligomer according to claim 6, wherein the nucleotide analogues are LNA.
9. The oligomer according to any one of claims 6 - 8 which is a gapmer.
10. The oligomer according to any one of claims 1 - 9, which inhibits the expression of polyomavirus, such as polyomavirus BK gene or mRNA in a cell which is expressing polyomavirus, such as polyomavirus BK gene or mRNA.
1 1. The oligomer according to any one of claims 1-10, wherein the oligomer consist of or comprises any one of SEQ ID NO's: 8 to 30.
12. A conjugate comprising the oligomer according to any one of claims 1 - 1 1 , and at least one non-nucleotide or non-polynucleotide moiety covalently attached to said oligomer.
13. A pharmaceutical composition comprising the oligomer according to any one of claims 1 - 11 , or the conjugate according to claim 12, and a pharmaceutically acceptable diluent, carrier, salt or adjuvant.
14. The oligomer according to any one of claims 1 - 11 , or the conjugate according to claim 12, for use as a medicament, such as for the treatment of a disease or disorder selected from the group consisting of BK virus associated nephropathy, interstitial nephritis in kidney transplants, ureteral stenosis and haemorrhagic cyctitis..
15. The use of an oligomer according to any one of the claims 1-1 1 , or a conjugate as
defined in claim 12, for the manufacture of a medicament for the treatment of a disease or disorder associated with polyomavirus infection, such as a disease or disorder selected from the group consisting of BK virus associated nephropathy, BK virus associated interstitial nephritis in kidney transplants, BK virus associated ureteral stenosis and BK virus associated haemorrhagic cystitis.
16. A method of treating of a disease or disorder associated with polyomavirus infection, such as a disease or disorder selected from the group consisting of BK virus associated nephropathy, BK virus associated interstitial nephritis in kidney transplants, BK virus associated ureteral stenosis and BK virus associated haemorrhagic cystitis, said method comprising administering an effective amount of an oligomer according to any one of the claims 1-10, or a conjugate according to claim 1 1 , or a pharmaceutical composition according to claim 12, to a patient suffering from, or likely to suffer from said disease or disorder.
17. A method for the inhibition of polyomavirus, such as polyomavirus BK in a cell which is expressing said polyomavirus, said method comprising administering an oligomer according to any one of the claims 1-10, or a conjugate according to claim 11 to said cell so as to inhibit said polyomavirus in said cell.
18. A method for preparing an oligomer for use in inhibiting polyomavirus infection in a cell, said method comprising the steps of i) selecting a target region of a polyomavirus genome, wherein the target region of the polyomavirus present in at least two, such as two or three independent transcripts expressed by the polyomavirus, and; ii)
synthesising an oligomer of up to 30 nucleotides in length which comprises or consists of a contiguous nucleotide sequence of 10 - 30 nucleotides, wherein the contiguous nucleotide sequence is the reverse complement of the target region of the polyomavirus.
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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2015042466A3 (en) * 2013-09-19 2015-08-27 The United States Of America, As Represented By The Secretary, Department Of Health & Human Services Compositions and methods for inhibiting jc virus (jcv)
WO2018146110A1 (en) * 2017-02-08 2018-08-16 Universitaet Des Saarlandes Assay system for bk polyomavirus and uses thereof
WO2019168402A1 (en) 2018-03-02 2019-09-06 Academisch Ziekenhuis Leiden H.O.D.N. Lumc Inhibition of polyomavirus replication
WO2023214373A3 (en) * 2022-05-06 2024-01-11 Academisch Ziekenhuis Leiden H.O.D.N. Lumc Oligonucleotide

Citations (24)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4914210A (en) 1987-10-02 1990-04-03 Cetus Corporation Oligonucleotide functionalizing reagents
US4962029A (en) 1987-10-02 1990-10-09 Cetus Corporation Covalent oligonucleotide-horseradish peroxidase conjugate
WO2000055344A1 (en) * 1999-03-17 2000-09-21 The Government Of The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Adenoviral vector expressing a sv40 t antigen antisense rna
EP1222309A1 (en) 1999-09-30 2002-07-17 Isis Pharmaceuticals, Inc. Human rnase h and oligonucleotide compositions thereof
WO2004046160A2 (en) 2002-11-18 2004-06-03 Santaris Pharma A/S Amino-lna, thio-lna and alpha-l-oxy-ln
WO2005018534A2 (en) * 2003-05-16 2005-03-03 Rosetta Inpharmatics, Llc Methods and compositions for rna interference
WO2005112636A2 (en) * 2004-05-12 2005-12-01 Kamel Khalili COMPOSITIONS AND METHODS FOR siRNA INHIBITION OF PRIMATE POLYOMAVIRUS GENES
US7087229B2 (en) 2003-05-30 2006-08-08 Enzon Pharmaceuticals, Inc. Releasable polymeric conjugates based on aliphatic biodegradable linkers
WO2007031091A2 (en) 2005-09-15 2007-03-22 Santaris Pharma A/S Rna antagonist compounds for the modulation of p21 ras expression
WO2007127919A2 (en) * 2006-04-28 2007-11-08 Alnylam Pharmaceuticals, Inc. Compositions and methods for inhibiting expression of a gene from the jc virus
WO2007134181A2 (en) 2006-05-11 2007-11-22 Isis Pharmaceuticals, Inc. 5'-modified bicyclic nucleic acid analogs
WO2007146511A2 (en) 2006-05-05 2007-12-21 Isis Pharmaceuticals, Inc. Compounds and methods for modulating gene expression
WO2008034123A2 (en) 2006-09-15 2008-03-20 Enzon Pharmaceuticals, Inc. Polymeric conjugates containing positively-charged moieties
WO2008034122A2 (en) 2006-09-15 2008-03-20 Enzon Pharmaceuticals, Inc. Hindered ester-based biodegradable linkers for oligonucleotide delivery
WO2008053314A2 (en) 2006-10-30 2008-05-08 Nokia Corporation Method, apparatus and system providing operator controlled mobility for user equipment
US7399845B2 (en) 2006-01-27 2008-07-15 Isis Pharmaceuticals, Inc. 6-modified bicyclic nucleic acid analogs
WO2008094516A2 (en) * 2007-01-29 2008-08-07 City Of Hope Multi-targeting short interfering rnas
WO2008113832A2 (en) 2007-03-22 2008-09-25 Santaris Pharma A/S SHORT RNA ANTAGONIST COMPOUNDS FOR THE MODULATION OF TARGET mRNA
WO2008150729A2 (en) 2007-05-30 2008-12-11 Isis Pharmaceuticals, Inc. N-substituted-aminomethylene bridged bicyclic nucleic acid analogs
WO2008154401A2 (en) 2007-06-08 2008-12-18 Isis Pharmaceuticals, Inc. Carbocyclic bicyclic nucleic acid analogs
WO2009006478A2 (en) 2007-07-05 2009-01-08 Isis Pharmaceuticals, Inc. 6-disubstituted bicyclic nucleic acid analogs
WO2009067647A1 (en) 2007-11-21 2009-05-28 Isis Pharmaceuticals, Inc. Carbocyclic alpha-l-bicyclic nucleic acid analogs
WO2009083790A2 (en) * 2007-12-28 2009-07-09 Qiagen Sciences, Inc 'apoptosis inducing positive control for expression modulating experiments'
WO2009105212A2 (en) * 2008-02-20 2009-08-27 Metic Immunogenetic Consultant, Inc Detection of polyomavirus

Patent Citations (24)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4962029A (en) 1987-10-02 1990-10-09 Cetus Corporation Covalent oligonucleotide-horseradish peroxidase conjugate
US4914210A (en) 1987-10-02 1990-04-03 Cetus Corporation Oligonucleotide functionalizing reagents
WO2000055344A1 (en) * 1999-03-17 2000-09-21 The Government Of The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Adenoviral vector expressing a sv40 t antigen antisense rna
EP1222309A1 (en) 1999-09-30 2002-07-17 Isis Pharmaceuticals, Inc. Human rnase h and oligonucleotide compositions thereof
WO2004046160A2 (en) 2002-11-18 2004-06-03 Santaris Pharma A/S Amino-lna, thio-lna and alpha-l-oxy-ln
WO2005018534A2 (en) * 2003-05-16 2005-03-03 Rosetta Inpharmatics, Llc Methods and compositions for rna interference
US7087229B2 (en) 2003-05-30 2006-08-08 Enzon Pharmaceuticals, Inc. Releasable polymeric conjugates based on aliphatic biodegradable linkers
WO2005112636A2 (en) * 2004-05-12 2005-12-01 Kamel Khalili COMPOSITIONS AND METHODS FOR siRNA INHIBITION OF PRIMATE POLYOMAVIRUS GENES
WO2007031091A2 (en) 2005-09-15 2007-03-22 Santaris Pharma A/S Rna antagonist compounds for the modulation of p21 ras expression
US7399845B2 (en) 2006-01-27 2008-07-15 Isis Pharmaceuticals, Inc. 6-modified bicyclic nucleic acid analogs
WO2007127919A2 (en) * 2006-04-28 2007-11-08 Alnylam Pharmaceuticals, Inc. Compositions and methods for inhibiting expression of a gene from the jc virus
WO2007146511A2 (en) 2006-05-05 2007-12-21 Isis Pharmaceuticals, Inc. Compounds and methods for modulating gene expression
WO2007134181A2 (en) 2006-05-11 2007-11-22 Isis Pharmaceuticals, Inc. 5'-modified bicyclic nucleic acid analogs
WO2008034123A2 (en) 2006-09-15 2008-03-20 Enzon Pharmaceuticals, Inc. Polymeric conjugates containing positively-charged moieties
WO2008034122A2 (en) 2006-09-15 2008-03-20 Enzon Pharmaceuticals, Inc. Hindered ester-based biodegradable linkers for oligonucleotide delivery
WO2008053314A2 (en) 2006-10-30 2008-05-08 Nokia Corporation Method, apparatus and system providing operator controlled mobility for user equipment
WO2008094516A2 (en) * 2007-01-29 2008-08-07 City Of Hope Multi-targeting short interfering rnas
WO2008113832A2 (en) 2007-03-22 2008-09-25 Santaris Pharma A/S SHORT RNA ANTAGONIST COMPOUNDS FOR THE MODULATION OF TARGET mRNA
WO2008150729A2 (en) 2007-05-30 2008-12-11 Isis Pharmaceuticals, Inc. N-substituted-aminomethylene bridged bicyclic nucleic acid analogs
WO2008154401A2 (en) 2007-06-08 2008-12-18 Isis Pharmaceuticals, Inc. Carbocyclic bicyclic nucleic acid analogs
WO2009006478A2 (en) 2007-07-05 2009-01-08 Isis Pharmaceuticals, Inc. 6-disubstituted bicyclic nucleic acid analogs
WO2009067647A1 (en) 2007-11-21 2009-05-28 Isis Pharmaceuticals, Inc. Carbocyclic alpha-l-bicyclic nucleic acid analogs
WO2009083790A2 (en) * 2007-12-28 2009-07-09 Qiagen Sciences, Inc 'apoptosis inducing positive control for expression modulating experiments'
WO2009105212A2 (en) * 2008-02-20 2009-08-27 Metic Immunogenetic Consultant, Inc Detection of polyomavirus

Non-Patent Citations (18)

* Cited by examiner, † Cited by third party
Title
CHRISTENSEN, NUCL. ACIDS. RES., vol. 30, 2002, pages 4918 - 4925
DEAN ET AL., JBC, vol. 269, 1994, pages 16416 - 16424
FLUITER ET AL., MOL. BIOSYST., vol. 10, 2009, pages 1039
FORTIN MARYLÃNE ET AL: "A multi-target antisense approach against PDE4 and PDE7 reduces smoke-induced lung inflammation in mice", RESPIRATORY RESEARCH, BIOMED CENTRAL LTD., LONDON, GB, vol. 10, no. 1, 20 May 2009 (2009-05-20), pages 39, XP021055519, ISSN: 1465-9921, DOI: 10.1186/1465-9921-10-39 *
FREIER; ALTMANN, NUCL. ACID RES., vol. 25, 1997, pages 4429 - 4443
GRUNWELLER A ET AL: "Locked nucleic acid oligonucleotides: The next generation of antisense agents?", BIODRUGS: CLINICAL IMMUNOTHERAPEUTICS, BIOPHARMACEUTICALS AND GENE THERAPY, ADIS INTERNATIONAL, FR, vol. 21, no. 4, 1 January 2007 (2007-01-01), pages 235 - 243, XP009125276, ISSN: 1173-8804 *
HINTON T M ET AL: "Inhibition of chicken anaemia virus replication using multiple short-hairpin RNAs", ANTIVIRAL RESEARCH, ELSEVIER BV, NL, vol. 80, no. 2, 1 November 2008 (2008-11-01), pages 143 - 149, XP025506389, ISSN: 0166-3542, [retrieved on 20080618], DOI: 10.1016/J.ANTIVIRAL.2008.05.009 *
JACKSON A L ET AL: "Widespread siRNA off-target transcript silencing mediated by seed region sequence complementarity", RNA, COLD SPRING HARBOR LABORATORY PRESS, US, vol. 12, no. 7, 1 July 2006 (2006-07-01), pages 1179 - 1187, XP002569696, ISSN: 1355-8382, [retrieved on 20060508], DOI: 10.1261/RNA.25706 *
JOSEPHSON M A ET AL: "TREATMENT OF RENAL ALLOGRAFT POLYOMA BK VIRUS INFECTION WITH LEFLUNOMIDE", TRANSPLANTATION, WILLIAMS AND WILKINS, BALTIMORE US, vol. 81, no. 5, 15 March 2006 (2006-03-15), pages 704 - 710, XP009074619, ISSN: 0041-1337, DOI: 10.1097/01.TP.0000181149.76113.50 *
MANOHARAN ET AL., TETRAHEDRON LETTERS, vol. 34, 1991, pages 7171
ORBA Y ET AL: "Inhibition of Virus Production in JC Virus-Infected Cells by Postinfection RNA Interference", JOURNAL OF VIROLOGY, THE AMERICAN SOCIETY FOR MICROBIOLOGY, US, vol. 78, no. 13, 1 July 2004 (2004-07-01), pages 7270 - 7273, XP002997068, ISSN: 0022-538X, DOI: 10.1128/JVI.78.13.7270-7273.2004 *
PETTENAZZO E ET AL: "3'-5' L-capped-antisense oligodeoxynucleotides targeted at the SV40 T-antigen gene: Pharmacological and biological properties", MICROBIOLOGICA, XX, IT, vol. 19, no. 4, 1 January 1996 (1996-01-01), pages 273 - 284, XP008152954, ISSN: 0391-5352 *
RADHAKRISHAN S ET AL: "Intracellular approach for blocking JC virus gene expression by using RNA interference during viral infection", JOURNAL OF VIROLOGY, THE AMERICAN SOCIETY FOR MICROBIOLOGY, US, vol. 78, no. 13, 1 January 2004 (2004-01-01), pages 7264 - 7269, XP002997069, ISSN: 0022-538X, DOI: 10.1128/JVI.78.13.7264-7269.2004 *
S. SEI ET AL: "Identification of a Key Target Sequence To Block Human Immunodeficiency Virus Type 1 Replication within the gag-pol Transframe Domain", JOURNAL OF VIROLOGY, vol. 74, no. 10, 15 May 2000 (2000-05-15), pages 4621 - 4633, XP055030068, ISSN: 0022-538X, DOI: 10.1128/JVI.74.10.4621-4633.2000 *
THEODORA W GREENE; PETER G M WUTS: "Protective Groups in Organic Synthesis, 3rd edition", 1999, JOHN WILEY & SONS
UHLMANN, CURR. OPINION IN DRUG DEVELOPMENT, vol. 3, no. 2, 2000, pages 293 - 213
VESTER ET AL., BIOORG. MED. CHEM. LETT., vol. 18, 2008, pages 2296 - 2300
WESTERMANN P ET AL: "INHIBITION OF EXPRESSION OF SV40 VIRUS LARGE T-ANTIGEN BY ANTISENSEOLIGODEOXYRIBONUCLEOTIDES", BIOMEDICA BIOCHIMICA ACTA, AKADEMIE VERLAG, BERLIN, DE, vol. 48, no. 1, 1 January 1989 (1989-01-01), pages 85 - 93, XP000886838, ISSN: 0232-766X *

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* Cited by examiner, † Cited by third party
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US10036020B2 (en) 2013-09-19 2018-07-31 The United States Of America, As Represented By The Secretary, Department Of Health & Human Services Compositions and methods for inhibiting JC virus (JCV)
WO2018146110A1 (en) * 2017-02-08 2018-08-16 Universitaet Des Saarlandes Assay system for bk polyomavirus and uses thereof
WO2019168402A1 (en) 2018-03-02 2019-09-06 Academisch Ziekenhuis Leiden H.O.D.N. Lumc Inhibition of polyomavirus replication
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US11661602B2 (en) 2018-03-02 2023-05-30 Academisch Ziekenhuis Leiden H.O.D.N. Lumc Inhibition of polyomavirus replication
WO2023214373A3 (en) * 2022-05-06 2024-01-11 Academisch Ziekenhuis Leiden H.O.D.N. Lumc Oligonucleotide

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