WO2025032331A1 - Molécules thérapeutiques - Google Patents

Molécules thérapeutiques Download PDF

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WO2025032331A1
WO2025032331A1 PCT/GB2024/052082 GB2024052082W WO2025032331A1 WO 2025032331 A1 WO2025032331 A1 WO 2025032331A1 GB 2024052082 W GB2024052082 W GB 2024052082W WO 2025032331 A1 WO2025032331 A1 WO 2025032331A1
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antisense oligonucleotide
seq
nucleotides
subject
oligonucleotide according
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Jacqueline VAN DER SPUY
Farah Olivia Toni REZEK
Beatriz SANCHEZ PINTADO
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UCL Business Ltd
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UCL Business Ltd
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/11—Antisense
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/31—Chemical structure of the backbone
    • C12N2310/315—Phosphorothioates
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/32—Chemical structure of the sugar
    • C12N2310/322—2'-R Modification
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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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 ===---===
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/34—Spatial arrangement of the modifications
    • C12N2310/346—Spatial arrangement of the modifications having a combination of backbone and sugar modifications
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2320/00—Applications; Uses
    • C12N2320/30—Special therapeutic applications
    • C12N2320/34—Allele or polymorphism specific uses

Definitions

  • the present invention relates to antisense oligonucleotides for use in the treatment of Doyne Honeycomb Macular Dystrophy
  • Doyne Honeycomb Macular Dystrophy is an autosomal dominant juvenile macular dystrophy for which there is currently no cure or treatments.
  • DHMD is a monogenic disease caused by a dominant variation c.1033C>T, p.(R345W) in the EFEMP1 gene. This dominant variation results in a toxic gain-of-function by the resultant protein, disrupting the function of the retinal pigment epithelial cells in which it is expressed, and ultimately leading to degeneration of the overlying photoreceptor cells in the retinal macular region and loss of central vision.
  • the aim is to develop an RNA-directed therapy that specifically targets the disease-associated allele at the EFEMP1 locus.
  • RNA directed therapies have been developed to treat other diseases. Currently, 15 RNA directed therapies have been approved by the FDA to treat various rare disease, including 1 RNA directed therapy to treat Age-Related Macular Degeneration (Pegaptanib).
  • AON antisense oligonucleotide
  • an antisense oligonucleotide comprising a sequence complementary to at least part of a target nucleic acid sequence, wherein the target sequence comprises SEQ ID NO: 1 or a portion thereof, wherein the antisense oligonucleotide is 100% complementary to position 8 from the 5’ end of SEQ ID NO: 1.
  • the antisense oligonucleotide may selectively reduce expression of a EFEMP1 gene product comprising a single nucleotide point mutation.
  • the antisense oligonucleotide sequence across its entire length may be at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, or 89% complementary upstream and/or downstream of position 8 from the 5’ end of SEQ ID NO: 1 to a target nucleic acid sequence, wherein the target sequence comprises SEQ ID NO: 1 or a portion thereof.
  • the antisense oligonucleotide sequence across its entire length may be at least 90% complementary upstream and/or downstream of position 8 from the 5’ end of SEQ ID NO: 1 to a target nucleic acid sequence wherein the target sequence comprises SEQ ID NO: 1 or a portion thereof.
  • the sequence may be 100% complementary to a target nucleic acid sequence wherein the target sequence comprises SEQ ID NO: 1 or a portion thereof.
  • the antisense oligonucleotide may comprise a sequence complementary to at least nucleotides at positions 2-16 of SEQ ID NO: 1.
  • the antisense oligonucleotide may comprise a sequence complementary to at least nucleotides at positions 4-16 of SEQ ID NO: 1.
  • the antisense oligonucleotide may comprise a sequence complementary to at least nucleotides at positions 6-12 of SEQ ID NO: 1.
  • the antisense oligonucleotide may comprise a sequence complementary to at least nucleotides at positions 6-10 of SEQ ID NO: 1.
  • the antisense oligonucleotide may comprise a sequence complementary to at least nucleotides at positions 7-9 of SEQ ID NO: 1.
  • the antisense oligonucleotide may consist of 10-20 linked nucleotides. In one embodiment the antisense oligonucleotide may consist of 12-18 linked nucleotides.
  • the antisense oligonucleotide may consist of 14-16 linked nucleotides.
  • the antisense oligonucleotide may consist of 16 linked nucleotides.
  • the antisense oligonucleotide may consist of 15 linked nucleotides.
  • the antisense oligonucleotide may consist of 14 linked nucleotides.
  • the antisense oligonucleotide may consist of 10-20 linked nucleotides and comprises at least 8 contiguous nucleotides of any one of SEQ ID Nos: 2, 3, 4, or 5.
  • the antisense oligonucleotide may comprise a contiguous nucleotide sequence of 5’-CTCCCAG-3’.
  • the antisense oligonucleotide may comprise a contiguous nucleotide sequence of 5’-TCCCA-3’.
  • the antisense oligonucleotide may comprise a contiguous nucleotide sequence of 5’-GAC-3.
  • the antisense oligonucleotide may consist of any one of SEQ ID Nos: 2, 3, 4, or 5.
  • the antisense oligonucleotide may consist of SEQ ID No: 2.
  • the antisense oligonucleotide may be modified.
  • the antisense oligonucleotide may be chemically modified.
  • one or more nucleotide(s) may comprise a modified sugar/s and/or sugar substitutes.
  • the modified sugar may be 2’-O-methoxyethyl (MOE), 2'-O-(2-N- methylcarbamoylethyl) (MCE), 2'-O-methylation (OMe), or 2’-Fluoro (2F).
  • the antisense oligonucleotide may comprise a backbone modification, selected from a list comprising phosphorothioate, methyl phosphonate, or methyl phosphorothionate, preferably the antisense oligonucleotide comprises a phosphorothioate backbone.
  • the antisense oligonucleotide may comprise a wing-gap-wing motif.
  • the antisense oligonucleotide may comprise: a gap segment consisting of linked nucleotides; a 5' wing region consisting of linked nucleotides at a 5’ end of the gap segment; and a 3’ wing region consisting of linked nucleotides at a 3’ end of the gap segment; wherein the gap segment is positioned between the 5 ' wing segment and the 3 ' wing segment.
  • the antisense oligonucleotide may consist of a sequence according to any one of SEQ ID Nos: 2, 3, 4, or 5 and wherein the antisense oligonucleotide comprises: a gap segment consisting of linked nucleotides; a 5' wing region consisting of 1-5 linked nucleotides at 5’ end of the gap segment; and a 3’ wing region consisting of 1-5 linked nucleotides at 3’ end of the gap segment; wherein the gap segment is positioned between the 5 ' wing segment and the 3 ' wing segment.
  • the gap segment may comprise a contiguous nucleotide sequence of 5’- CTCCCAG-3’.
  • the gap segment may comprise a contiguous nucleotide sequence of 5’- TCCCA-3’.
  • the gap segment may comprise a contiguous nucleotide sequence of 5’- GAC-3’.
  • the position 6, 7, or 8 of the modified oligonucleotide, as counted from the 5’ terminus of the gap, may align with position 8 from the 5’ end of the target sequence.
  • one or more nucleotides of either or both of the wing regions may comprise a modified sugar or sugar substitute.
  • the modified sugar may be 2’-O-methoxyethyl (MOE), 2'-O-(2-N- methylcarbamoylethyl) (MCE), 2'-O-methylation (OMe), or 2’-Fluoro (2F).
  • MOE 2’-O-methoxyethyl
  • MCE 2'-O-(2-N- methylcarbamoylethyl)
  • OMe 2'-O-methylation
  • 2Fluoro (2F 2’-Fluoro
  • the antisense oligonucleotide may comprise at least one 2’-O-MOE modification.
  • nucleotides of the 5' wing region and/or the 3’ wing region may comprise at least one 2’-O-MOE modification.
  • the 5' wing region may consist of 3 nucleotides comprising 2’-O-MOE modifications and the 3' wing region consists of 3 nucleotides comprising 2’-O-MOE modifications.
  • the antisense oligonucleotide may consist of 10-20 linked nucleotides and comprises at least 8 contiguous nucleotides of any one of SEQ ID Nos: 6, 7, 8, or 9.
  • the antisense oligonucleotide may consist of any one of SEQ ID Nos: 6,
  • the antisense oligonucleotide may comprise at least one locked nucleic acid (LNA).
  • LNA locked nucleic acid
  • the gap region may comprise at least one LNA.
  • the LNA may be complementary to position 8 of SEQ ID NO: 1.
  • the antisense oligonucleotide consists of 10-20 linked nucleotides and comprises at least 8 contiguous nucleotides of any one of SEQ I D Nos: 10, 11, 12, or 13. In one embodiment the antisense oligonucleotide may consist of any one of SEQ ID Nos: 10, 11, 12, or 13.
  • the 5' wing region may consist of 3 nucleotides comprising 2’-O-MOE modifications, the 3' wing region may consist of 3 nucleotides comprising 2’-O-MOE modifications, and the gap region may comprise at least one LNA.
  • the antisense oligonucleotide consists of 10-20 linked nucleotides and may comprise at least 8 contiguous nucleotides of any one of SEQ ID Nos: 6, 7, 8, or 9.
  • the antisense oligonucleotide may consist of any one of SEQ ID Nos: 6, 7, 8, or 9.
  • a delivery vehicle comprising a copy of the antisense oligonucleotide according to the invention.
  • a host cell comprising the antisense oligonucleotide according to the invention.
  • an antisense oligonucleotide comprising chemically synthesising an antisense oligonucleotide according to the invention.
  • the method of manufacturing may comprise an additional step of chemically modifying one or more nucleotides.
  • a method of modulating expression of EFEMP1 gene product in a biological system comprising: introducing an antisense oligonucleotide according to the invention or a delivery vehicle according to the invention into the biological system.
  • EFEMP1 gene product may be inhibited.
  • the biological system may be selected from a eukaryotic cell, such as a mammalian cell.
  • the method may be an in vitro or in vivo method.
  • a pharmaceutical composition comprising an antisense oligonucleotide according to the invention or a delivery vehicle according to the invention.
  • the pharmaceutical composition may further comprise a pharmaceutically acceptable carrier, diluent, enhancer or excipient.
  • DHMD Doyne honeycomb macular dystrophy
  • the method of treating, preventing, ameliorating, or slowing progression of DHMD in a subject may selectively reduce expression of a mutant allele.
  • the mutant allele may be a c.1033C>T mutation in the EFEMP1 gene producing a R345W mutant EFEMP1 protein.
  • the method of treating, preventing, ameliorating, or slowing progression of DHMD according to the invention wherein administering the antisense oligonucleotide according to the invention or a delivery vehicle according to the invention or the pharmaceutical composition according to the invention may selectively inhibit expression of R345W mutant EFEMP1 protein expression over wild-type EFEMP1 gene product expression in the subject.
  • the method of treating, preventing, ameliorating, or slowing progression of DHMD in a subject according to the invention wherein the antisense oligonucleotide according to the invention or a delivery vehicle according to the invention or the pharmaceutical composition according to the invention may selectively inhibit expression of R345W mutant EFEMP1 protein by binding to mRNA of the mutant EFEMP1 allele thereby inhibiting R345W mutant EFEMP1 protein expression.
  • the expression of EFEMP1 gene product may be reduced by at least 25- 50% in retinal pigment epithelium in a treated subject compared to untreated subjects.
  • the method of treating, preventing, ameliorating, or slowing progression of DHMD in a subject according to the invention may comprise contacting a cell with the antisense oligonucleotide according to the invention or a delivery vehicle according to the invention or the pharmaceutical composition according to the invention.
  • the cell may be a retinal pigment epithelium (RPE) cell.
  • RPE retinal pigment epithelium
  • the method of treating, preventing, ameliorating, or slowing progression of DHMD in a subject according to the invention wherein the antisense oligonucleotide according to the invention or a delivery vehicle according to the invention or the pharmaceutical composition according to the invention may be administered to the subject intravitreally.
  • the method of treating, preventing, ameliorating, or slowing progression of DHMD in a subject according to the invention wherein the antisense oligonucleotide according to the invention or a delivery vehicle according to the invention or the pharmaceutical composition according to the invention may be administered to the subject by intravitreal injection.
  • the method of treating, preventing, ameliorating, or slowing progression of DHMD in a subject according to the invention wherein the antisense oligonucleotide according to the invention or a delivery vehicle according to the invention or the pharmaceutical composition according to the invention may be administered to the subject by intravitreal injection once every 3 to 12 months.
  • the method of treating, preventing, ameliorating, or slowing progression of DHMD in a subject according to the invention wherein the antisense oligonucleotide according to the invention or a delivery vehicle according to the invention or the pharmaceutical composition according to the invention may be administered to the subject by intravitreal injection once every 3 to 6 months.
  • the method of treating, preventing, ameliorating, or slowing progression of DHMD in a subject according to the invention wherein the antisense oligonucleotide according to the invention or a delivery vehicle according to the invention or the pharmaceutical composition according to the invention may be administered to the subject at a dosage of the antisense oligonucleotide of 1 g -20mg.
  • an antisense oligonucleotide according to the invention or a delivery vehicle according to the invention or the pharmaceutical composition according to the invention for use as a medicament there is provided an antisense oligonucleotide according to the invention or a delivery vehicle according to the invention or the pharmaceutical composition according to the invention for use as a medicament.
  • an antisense oligonucleotide according to the invention or a delivery vehicle according to the invention or the pharmaceutical composition according to the invention for use in treating, preventing, ameliorating, or slowing progression of DHMD in a subject comprising administering to the subject an effective amount of the antisense oligonucleotide.
  • kits comprising an antisense oligonucleotide of any one of the invention or a delivery vehicle according to the invention.
  • Figure 1 Target sequence and ASO design.
  • Upper panel Top line: The genomic DNA (gDNA) sequence (5’-3’) encompassing the mutation of interest (bold underlined, grey fill).
  • Second line The corresponding messenger RNA (mRNA) target sequence (5’-3’) encompassing the mutation of interest (bold underlined, grey fill).
  • Third line The complementary 18 mer ASO1 (SEQ ID NO. 10) and ASO2 sequence (SEQ ID NO. 11) (5’- TCATCCTCCCAGCATTCA-3’) encompassing the mutation of interest (bold underlined, grey fill).
  • Fourth line The complementary 18 mer ASO3 (SEQ ID NO. 12) and ASO4 sequence (SEQ ID NO.
  • genomic DNA reference sequence for ASO1/2 (SEQ ID NO. 15 (5’-TGAATGCTGGGAGGATGA-3’) is located at chr2:55, 871, 081-55, 871 , 098 and for ASO2/3 (SEQ ID NO. 16 (5’-AATGAATGCTGGGAGGAT-3’) at chr:55, 871, 081-55, 871, 100 (IICSC Genome Browser on Human (GRCh38/hg38)).
  • ASO chemistry All ASO (5’-3’) are 18 mer fully substituted phosphorothioate (PS) gapmers, with a gap of 8 deoxynucleotides flanked at the 5’ and 3’ end by five nucleotides with 2’0- methoxyethyl (2’O-MOE) ribose sugar modifications (wings).
  • PS phosphorothioate
  • wings ribose sugar modifications
  • the target nucleotide is flanked by a 5’ and 3’ modified LNA.
  • FIG. 2 In vitro screen of ASO1-4 in a HEK293T heterologous expression system: HEK293T cells were transfected with pEFEMP1(WT)-FLAG3X alone, pEFEMP1(R345W)- mScarlet alone, pEFEMP1(WT)-FLAG3X + pEFEMP1(R345W)-mScarlet, and with control (CTRL) ASO or increasing amounts (25 nM, 50 nM, 100 nM, 200 nM) of ASO1, ASO2, ASO3 and ASO4.
  • CTRL control
  • Figure 3 ASO refinement: Upper box: Summary of ASO1 (18 mer) parameters from which all further ASO were derived, including gDNA reference sequence (5’-3’), mRNA target sequence (5’-3’), complementary 18 mer ASO1 sequence (5’-3’) and chemistry. Target mutation: bold underlined (grey fill).
  • Middle box 16 mer ASO1A (SEQ ID NO. 6), 15 mer ASO1 B (SEQ ID NO. 7), 15 mer ASO1C (SEQ ID NO.
  • ASO1D 14 mer ASO1D (SEQ ID NO. 9).
  • the mRNA target sequence (5’-3’) for ASO1 is shown at the top.
  • the complementary ASO1A, 1 B, 1C and 1D sequences aligned to the target sequence are shown underneath.
  • Target mutation bold underlined, grey fill.
  • Lower box Summary of ASO1A-1D target sequence (5’-3’) (left) and ASO1A-1 D sequence (5’-3’) (right).
  • the ASO1A-1D sequences (5’-3’) are bold underlined.
  • the ASO chemistry is shown underneath.
  • the target nucleotide (c.1033C>T) in all ASO is a modified LNA (indicated by a +).
  • CTRL chemistry and sequence of the control (CTRL) ASO is shown.
  • Figure 4 In vitro screen of AS01A-1D in a HEK293T heterologous expression system. Levels of EFEMP1 (total), R345W EFEMP1 (MUT) and WT EFEMP1 (WT) measured by qPCR following RNA purification and reverse transcription.
  • HEK293T cells were transfected with pEFEMP1(WT)-FLAG3X + pEFEMP1(R345W)-mScarlet (EFEMP1, black and hatched bars), pEFEMP1(R345W)-mScarlet alone (MUT, light grey bars) or pEFEMP1(WT)-FLAG3X alone (WT, dark grey bars), and with control (CTRL) ASO or increasing amounts (25 nM, 50 nM, 100 nM, 200 nM) of ASO1A (blue), ASO1B (green), ASO1C (red) and ASO1D (yellow).
  • pEFEMP1(R345W)-mScarlet alone MUT, light grey bars
  • FIG. 5 ASO screening in a clinically relevant iPSC-RPE disease model: Immunofluorescent (left) and brightfield (right) images of isogenic control iPSC-RPE (HDR) and patient iPSC-RPE (R345W).
  • CRISPR-Cas9 homology directed repair was used to repair the autosomal dominant heterozygous c.1033C>T mutation in patient-derived iPSC reprogrammed from renal epithelial (RE) cells to create an isogenic control iPSC repaired line.
  • the isogenic control and patient iPSC were differentiated to RPE (protocol based on Michelet et al., 2020).
  • Brightfield microscopy at 72 days of iPSC-RPE differentiation show a typical homogeneous honeycomb cobblestone morphology of isogenic control iPSC-RPE (HDR) cells.
  • patient iPSC-RPE R345W
  • EMT epithelial-mesenchymal transition
  • CTRL control
  • NGS next generation sequencing
  • Enzymatic reactions and purification techniques are performed according to manufacturer's specifications, as commonly accomplished in the art or as described herein.
  • the nomenclatures used in connection with, and the laboratory procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well-known and commonly used in the art. Standard techniques are used for chemical syntheses, chemical analyses, pharmaceutical preparation, formulation, and delivery, and treatment of patients.
  • Nucleotide sequences are presented herein by single strand only, in the 5' to 3' direction, from left to right, unless specifically indicated otherwise. Nucleotides and amino acids are represented herein in the manner recommended by the IUPAC-IUB Biochemical Nomenclature Commission, or (for amino acids) by either the one-letter code, or the three letter code.
  • “enhance” or “increase” refers to an increase in the specified parameter of at least about 1.25-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 8-fold, 10-fold, twelve-fold, or even fifteen-fold.
  • inhibitor or “reduce” or grammatical variations thereof as used herein refers to a decrease or diminishment in the specified level or activity of at least about 15%, 25%, 35%, 40%, 50%, 60%, 75%, 80%, 90%, 95% or more. In particular embodiments, the inhibition or reduction results in little or essentially no detectible activity (at most, an insignificant amount, e.g., less than about 10% or even 5%).
  • nucleic acid As used herein, “nucleic acid,” “nucleotide sequence,” and “polynucleotide” are used interchangeably and encompass both RNA and DNA, including cDNA, genomic DNA, mRNA, synthetic (e.g., chemically synthesized) DNA or RNA and chimeras of RNA and DNA.
  • the term polynucleotide, nucleotide sequence, or nucleic acid refers to a chain of nucleotides without regard to length of the chain.
  • the nucleic acid can be double-stranded or single-stranded. Where single-stranded, the nucleic acid can be a sense strand or an antisense strand.
  • the nucleic acid can be synthesized using oligonucleotide analogs or derivatives (e.g., inosine or phosphorothioate nucleotides). Such oligonucleotides can be used, for example, to prepare nucleic acids that have altered base-pairing abilities or increased resistance to nucleases.
  • the present invention further provides a nucleic acid that is the complement (which can be either a full complement or a partial complement) of a nucleic acid, nucleotide sequence, or polynucleotide of this invention.
  • dsRNA When dsRNA is produced synthetically, less common bases, such as inosine, 5-methylcytosine, 6- methyladenine, hypoxanthine and others can also be used for antisense, dsRNA, and ribozyme pairing.
  • polynucleotides that contain C-5 propyne analogues of uridine and cytidine have been shown to bind RNA with high affinity and to be potent antisense inhibitors of gene expression.
  • Other modifications such as modification to the phosphodiester backbone, or the 2'-hydroxy in the ribose sugar group of the RNA can also be made.
  • an “isolated polynucleotide” is a nucleotide sequence (e.g., DNA or RNA) that is not immediately contiguous with nucleotide sequences with which it is immediately contiguous (one on the 5' end and one on the 3' end) in the naturally occurring genome of the organism from which it is derived.
  • an isolated nucleic acid includes some or all of the 5' non-coding (e.g., promoter) sequences that are immediately contiguous to a coding sequence.
  • the term therefore includes, for example, a recombinant DNA that is incorporated into a vector, into an autonomously replicating plasmid or virus, or into the genomic DNA of a prokaryote or eukaryote, or which exists as a separate molecule (e.g., a cDNA or a genomic DNA fragment produced by PCR or restriction endonuclease treatment), independent of other sequences. It also includes a recombinant DNA that is part of a hybrid nucleic acid encoding an additional polypeptide or peptide sequence.
  • An isolated polynucleotide that includes a gene is not a fragment of a chromosome that includes such gene, but rather includes the coding region and regulatory regions associated with the gene, but no additional genes naturally found on the chromosome.
  • isolated can refer to a nucleic acid, nucleotide sequence or polypeptide that is substantially free of cellular material, viral material, and/or culture medium (when produced by recombinant DNA techniques), or chemical precursors or other chemicals (when chemically synthesized).
  • an “isolated fragment” is a fragment of a nucleic acid, nucleotide sequence or polypeptide that is not naturally occurring as a fragment and would not be found in the natural state. “Isolated” does not mean that the preparation is technically pure (homogeneous), but it is sufficiently pure to provide the polypeptide or nucleic acid in a form in which it can be used for the intended purpose.
  • an “isolated cell” refers to a cell that is separated from other components with which it is normally associated in its natural state.
  • an isolated cell can be a cell in culture medium and/or a cell in a pharmaceutically acceptable carrier of this invention.
  • an isolated cell can be delivered to and/or introduced into a subject.
  • the term “gene” refers to a nucleic acid molecule capable of being used to produce mRNA, antisense RNA, miRNA, and the like. Genes may or may not be capable of being used to produce a functional protein. Genes can include both coding and non-coding regions (e.g., introns, regulatory elements, promoters, enhancers, termination sequences and 5’ and 3’ untranslated regions).
  • a gene may be “isolated” by which is meant a nucleic acid that is substantially or essentially free from components normally found in association with the nucleic acid in its natural state. Such components include other cellular material, culture medium from recombinant production, and/or various chemicals used in chemically synthesizing the nucleic acid.
  • allelic pair is one member of a pair of genes or one member of a series of different forms of a DNA sequences that can exist at a single locus or marker on a specific chromosome.
  • each allelic pair will normally occupy corresponding positions (loci) on a pair of homologous chromosomes, one inherited from the mother and one inherited from the father. If these alleles are identical, the organism or cell is said to be ’homozygous’ for that allele; if they differ, the organism or cell is said to be ’heterozygous’ for that allele.
  • Major allele refers to an allele containing the nucleotide present in a statistically significant proportion of individuals in the human population.
  • Minor allele refers to an allele containing the nucleotide present in a relatively small proportion of individuals in the human population.
  • Wild type allele refers to the genotype typically not associated with disease or dysfunction of the gene product.
  • mutant allele refers to one of the pair of genes or DNA sequence existing at a single locus comprising a single point mutation and is the genotype associated with disease or dysfunction of the gene product. For example, a 1033C>T mutation in the EFEMP1 gene which causes DHMD.
  • an antisense oligonucleotide comprising a sequence complementary to at least part of a target nucleic acid sequence, wherein the target sequence comprises SEQ ID NO: 1 or a portion thereof, wherein the antisense oligonucleotide is 100% complementary to position 8 from the 5’ end of SEQ ID NO: 1.
  • “Complementarity” refers to the ability of a nucleic acid to form hydrogen bond(s) with another nucleic acid sequence by either traditional Watson-Crick or other non-traditional types.
  • a percent complementarity indicates the percentage of residues in a nucleic acid molecule which can form hydrogen bonds (e.g., Watson-Crick base pairing) with a second nucleic acid sequence (e.g., 5, 6, 7, 8, 9, 10 out of 10 being 50%, 60%, 70%, 80%, 90% « and 100% complementary).
  • the antisense oligonucleotide can hybridize to a target nucleic acid sequence.
  • the antisense oligonucleotide can hybridize to an EFEMP1 gene product (mRNA) comprising a single nucleotide point mutation.
  • a gene product is the biochemical material, either RNA or protein, resulting from expression of a gene.
  • the antisense oligonucleotide can hybridize to EFEMP1 RNA comprising a single nucleotide point mutation. This single point mutation may result in a R345W mutant EFEMP1 protein.
  • the antisense oligonucleotide can hybridize to mutant EFEMP1 mRNA which would express a R345W mutant EFEMP1 protein. The hybridization may result in a reduction in R345W mutant EFEMP1 protein.
  • the hybridization thus effectively targets the mutant EFEMP1 allele comprising a c.1033C>T mutation and modulates its expression.
  • the antisense oligonucleotide may selectively reduce expression of a EFEMP1 gene product (mRNA) comprising a single nucleotide point mutation. Degradation of this specific mRNA selectively reduces expression of protein comprising the mutation.
  • mRNA EFEMP1 gene product
  • the antisense oligonucleotide sequence across its entire length may be at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, or 89% complementary upstream and/or downstream of position 8 from the 5’ end of SEQ ID NO: 1 to a target nucleic acid sequence, wherein the target sequence comprises SEQ ID NO: 1 or a portion thereof.
  • the antisense oligonucleotide sequence across its entire length may be at least 90% complementary upstream and/or downstream of position 8 from the 5’ end of SEQ ID NO: 1 to a target nucleic acid sequence wherein the target sequence comprises SEQ ID NO: 1 or a portion thereof.
  • the sequence may be 100% complementary to a target nucleic acid sequence wherein the target sequence comprises SEQ ID NO: 1 or a portion thereof.
  • the antisense oligonucleotide may comprise a sequence complementary to at least nucleotides at positions 2-16 of SEQ ID NO: 1. In one embodiment the antisense oligonucleotide may comprise a sequence complementary to at least nucleotides at positions 4-16 of SEQ ID NO: 1. In one embodiment the antisense oligonucleotide may comprise a sequence complementary to at least nucleotides at positions 6-12 of SEQ ID NO: 1.
  • the antisense oligonucleotide may comprise a sequence complementary to at least nucleotides at positions 6-10 of SEQ ID NO: 1. In one embodiment the antisense oligonucleotide may comprise a sequence complementary to at least nucleotides at positions 7-9 of SEQ ID NO: 1.
  • the antisense oligonucleotide may consist of 10-20 linked nucleotides.
  • linked or “linkage” means two entities are bound to one another by any physicochemical means. Any linkage known to those of ordinary skill in the art, covalent or non-covalent, is embraced. Natural linkages, which are those ordinarily found in nature connecting the individual units of a nucleic acid, are most common. The individual units of a nucleic acid may be linked, however, by synthetic or modified linkages.
  • the antisense oligonucleotide may consist of 12-18 linked nucleotides. In one embodiment the antisense oligonucleotide may consist of 14-16 linked nucleotides. In one embodiment the antisense oligonucleotide may consist of 16 linked nucleotides. In one embodiment the antisense oligonucleotide may consist of 15 linked nucleotides. In one embodiment the antisense oligonucleotide may consist of 14 linked nucleotides. In one embodiment the antisense oligonucleotide may consist of 10-20 linked nucleotides and comprises at least 8 contiguous nucleotides of any one of SEQ ID Nos: 2, 3, 4, or 5. Preferably the antisense oligonucleotide is single stranded. The antisense oligonucleotide may be double stranded.
  • the antisense oligonucleotide may comprise a contiguous nucleotide sequence of 5’-CTCCCAG-3’. In one embodiment wherein the antisense oligonucleotide may comprise a contiguous nucleotide sequence of 5’-TCCCA-3’. In one embodiment the antisense oligonucleotide may comprise a contiguous nucleotide sequence of 5’-GAC-3’.
  • the antisense oligonucleotide may consist of any one of SEQ ID Nos: 2, 3, 4, or 5. In one embodiment the antisense oligonucleotide may consist of SEQ ID No: 2.
  • the antisense oligonucleotide may be modified.
  • the antisense oligonucleotide may be chemically modified.
  • the antisense oligonucleotides can be designed and engineered to comprise one or more chemical modifications (e.g. a modified inter-nucleoside linker, a modified nucleoside, or a combination thereof).
  • the antisense oligonucleotide comprises one or more modifications.
  • the modification comprises a modified inter-nucleoside linker, a modified nucleoside, or a combination thereof.
  • the antisense oligonucleotides can comprise one or more nucleotides comprising a modified sugar moiety, wherein the modified sugar moiety is a modification of the sugar moiety when compared to the ribose sugar moiety found in deoxyribose nucleic acid (DNA) and RNA.
  • DNA deoxyribose nucleic acid
  • RNA deoxyribose nucleic acid
  • Numerous nucleotides with modification of the ribose sugar moiety can be utilized, primarily with the aim of improving certain properties of oligonucleotides, such as affinity and/or nuclease resistance. Such modifications include those where the ribose ring structure is modified.
  • HNA hexose ring
  • LNA locked nucleic acids
  • UNA unlinked ribose ring which typically lacks a bond between the C2 and C3 carbons
  • Other sugar modified nucleosides include, for example, bicyclohexose nucleic acids or tricyclic nucleic acids.
  • Modified nucleosides also include nucleosides where the sugar moiety is replaced with a non-sugar moiety, for example in the case of peptide nucleic acids (PNA), or morpholino nucleic acids.
  • Sugar modifications also include modifications made by altering the substituent groups on the ribose ring to groups other than hydrogen, or the 2'-OH group naturally found in DNA and RNA nucleotides. Substituents may, for example be introduced at the 2', 3', 4' or 5' positions.
  • Nucleosides with modified sugar moieties also include 2' modified nucleosides, such as 2' substituted nucleosides. Indeed, much focus has been spent on developing 2' substituted nucleosides, and numerous 2' substituted nucleosides have been found to have beneficial properties when incorporated into oligonucleotides, such as enhanced nucleoside resistance and enhanced affinity.
  • a 2' sugar modified nucleoside is a nucleoside that has a substituent other than H or — OH at the 2' position (2' substituted nucleoside) or comprises a 2' linked biradicle, and includes 2' substituted nucleosides and LNA (2'-4' biradicle bridged) nucleosides.
  • 2' substituted modified nucleosides are 2'-O-alkyl-RNA, 2'-O- methyl-RNA, 2 '-alkoxy -RNA, 2'-O- methoxyethyl-oligos (MOE), 2'-amino-DNA, 2'-Fluoro- RNA, and 2'-F-ANA nucleoside.
  • the antisense oligonucleotide comprises one or more modified sugars. In some embodiments, the antisense oligonucleotide comprises only modified sugars. In certain embodiments, the antisense oligo comprises greater than 10%, 25%, 50%, 75%, or 90% modified sugars. In some embodiments, the modified sugar is a bicyclic sugar. In some embodiments, the modified sugar comprises a 2'-O-methoxyethyl (MOE) group.
  • MOE 2'-O-methoxyethyl
  • Modifications to the ribose sugar or nucleobase can also be utilized to increase pharmacodynamic, pharmacokinetic, and biodistribution properties. Nucleoside modifications prevent or reduce degradation by cellular nucleases, thus increasing the pharmacokinetics and bioavailability of the antisense oligonucleotide. Generally, a modified nucleoside includes the introduction of one or more modifications of the sugar moiety or the nucleobase moiety.
  • one or more nucleotide(s) may comprise a modified sugar/s and/or sugar substitutes.
  • the modified sugar may be 2’-O-methoxyethyl (MOE), 2 -O-(2-N- methylcarbamoylethyl) (MCE), 2'-O-methylation (OMe), or 2’-Fluoro (2F).
  • MOE 2’-O-methoxyethyl
  • MCE 2 -O-(2-N- methylcarbamoylethyl)
  • OMe 2'-O-methylation
  • 2Fluoro (2F 2’-Fluoro
  • the antisense oligonucleotide comprises a modified backbone.
  • backbones are provided by morpholino backbones, carbamate backbones, siloxane backbones, sulfide, sulfoxide and sulfone backbones, formacetyl and thioformacetyl backbones, methyleneformacetyl backbones, riboacetyl backbones, alkene containing backbones, sulfamate, sulfonate and sulfonamide backbones, methyleneimino and methylenehydrazino backbones, and amide backbones.
  • the antisense oligonucleotide may comprise a backbone modification, selected from a list comprising phosphorothioate, methyl phosphonate, or methyl phosphorothionate, preferably the antisense oligonucleotide comprises a phosphorothioate backbone.
  • the antisense oligonucleotide may comprise a phosphorothioate backbone.
  • the antisense oligonucleotide may comprise a single-stranded phosphorothioate (PS) backbone.
  • PS phosphorothioate
  • Modification of the inter-nucleoside linker can be utilized to increase pharmacodynamic, pharmacokinetic, and biodistribution properties.
  • inter-nucleoside linker modifications prevent or reduce degradation by cellular nucleases, thus increasing the pharmacokinetics and bioavailability of the antisense oligonucleotide.
  • a modified inter-nucleoside linker includes any linker other than phosphodiester (PO) liners, that covalently couples two nucleosides together.
  • the modified inter-nucleoside linker increases the nuclease resistance of the antisense oligonucleotide compared to a phosphodiester linker.
  • the inter-nucleoside linker includes phosphate groups creating a phosphodiester bond between adjacent nucleosides.
  • Modified inter-nucleoside linkers are particularly useful in stabilizing antisense oligonucleotides for in vivo use and may serve to protect against nuclease cleavage.
  • the phosphorothioate backbone is important for RNAse activity.
  • the antisense oligonucleotide comprises one or more inter- nucleoside linkers modified from the natural phosphodiester to a linker that is for example more resistant to nuclease attack. In some embodiments all of the inter-nucleoside linkers of the antisense oligonucleotide, or contiguous nucleotide sequence thereof, are modified. In some embodiments all of the inter-nucleoside linkers of the antisense oligonucleotide, or contiguous nucleotide sequence thereof, are nuclease resistant inter-nucleoside linkers. In some embodiments the inter-nucleoside linkage comprises sulphur (S), such as a phosphorothioate inter-nucleoside linkage.
  • S sulphur
  • Phosphorothioate inter-nucleoside linkers are particularly useful due to nuclease resistance and improved pharmacokinetics.
  • one or more of the inter-nucleoside linkers of the antisense oligonucleotide, or contiguous nucleotide sequence thereof comprise a phosphorothioate inter-nucleoside linker.
  • all of the inter-nucleoside linkers of the antisense oligonucleotide, or contiguous nucleotide sequence thereof comprise a phosphorothioate inter-nucleoside linker.
  • the antisense oligonucleotide comprises both inter-nucleoside linker modifications and nucleoside modifications.
  • the antisense oligonucleotide may comprise a wing-gap-wing motif. This motif is also known as a “Gapmer”. Gapmers are antisense oligonucleotide structures with RNA-like segments on both sides of a DNA sequence. Gapmers are designed to be complementary to a target RNA and silence the gene by hybridizing to the target sequence and inducing RNase H cleavage.
  • the internal region may be referred to as the "gap" and the external regions may be referred to as the "wings.”
  • the gap may comprise linked DNA nucleotides and the wings may comprise linked RNA nucleotides.
  • the antisense oligonucleotide may comprise a gap segment consisting of linked nucleotides, a 5' wing region consisting of linked nucleotides at a 5’ end of the gap segment, and a 3’ wing region consisting of linked nucleotides at a 3’ end of the gap segment, wherein the gap segment is positioned between the 5 ' wing segment and the 3 ' wing segment.
  • the antisense oligonucleotide may consist of a sequence according to any one of SEQ ID Nos: 2, 3, 4, or 5 and wherein the antisense oligonucleotide comprises a gap segment consisting of linked nucleotides, a 5' wing region consisting of 1-5 linked nucleotides at 5’ end of the gap segment, and a 3’ wing region consisting of 1-5 linked nucleotides at 3’ end of the gap segment, wherein the gap segment is positioned between the 5 ' wing segment and the 3 ' wing segment.
  • the gap segment may comprise a contiguous nucleotide sequence of 5’- CTCCCAG-3’. In one embodiment the gap segment may comprise a contiguous nucleotide sequence of 5’-TCCCA-3’. In one embodiment the gap segment may comprise a contiguous nucleotide sequence of 5’-GAC-3’. In one embodiment the position 6, 7, or 8 of the modified oligonucleotide, as counted from the 5’ terminus of the gap, may align with position 8 from the 5’ end of the target sequence. In one embodiment one or more nucleotides of either or both of the wing regions may comprise a modified sugar or sugar substitute.
  • the modified sugar may be 2’-O-methoxyethyl (MOE), 2'-O-(2-N-methylcarbamoylethyl) (MCE), 2'-O-methylation (OMe), or 2’-Fluoro (2F).
  • MOE 2’-O-methoxyethyl
  • MCE 2'-O-(2-N-methylcarbamoylethyl)
  • OMe 2'-O-methylation
  • 2Fluoro (2F 2’-Fluoro
  • the antisense oligonucleotide may comprise at least one 2’-O-MOE modification.
  • nucleotides of the 5' wing region and/or the 3’ wing region may comprise at least one 2’-O-MOE modification.
  • the 5' wing region may consist of 3 nucleotides comprising 2’-O-MOE modifications and the 3' wing region consists of 3 nucleotides comprising 2’-O-MOE modifications.
  • the antisense oligonucleotide may consist of 10-20 linked nucleotides and comprises at least 8 contiguous nucleotides of any one of SEQ ID Nos: 6, 7, 8, or 9. In one embodiment the antisense oligonucleotide may consist of any one of SEQ ID Nos: 6, 7, 8, or 9.
  • the antisense oligonucleotide may comprise at least one locked nucleic acid (LNA).
  • LNA locked nucleic acid
  • locked nucleic acid or “LNA” means a nucleoside comprising a bicyclic sugar moiety comprising a 4'-CH 2 -0-2'bridge. Locked nucleic acids are described eg. In J. Wengel, Acc. Chem. Res., 120, 5458-5463 (1999 ) or J. Wengel et al., nucleosides & nucleotides, 18(6&7), S. 1365-1370.
  • the gap region may comprise at least one LNA.
  • the LNA may be complementary to position 8 of SEQ ID NO: 1.
  • the antisense oligonucleotide consists of 10-20 linked nucleotides and comprises at least 8 contiguous nucleotides of any one of SEQ ID Nos: 10, 11 , 12, or 13. In one embodiment the antisense oligonucleotide may consist of any one of SEQ ID Nos: 10, 11 , 12, or 13.
  • the 5' wing region may consist of 3 nucleotides comprising 2’-O-MOE modifications, the 3' wing region may consist of 3 nucleotides comprising 2’-O-MOE modifications, and the gap region may comprise at least one LNA.
  • the antisense oligonucleotide consists of 10-20 linked nucleotides and may comprise at least 8 contiguous nucleotides of any one of SEQ ID Nos: 6, 7, 8, or 9. In one embodiment the antisense oligonucleotide may consist of any one of SEQ ID Nos: 6, 7, 8, or 9.
  • a delivery vehicle comprising a copy of the antisense oligonucleotide according to the invention.
  • the selection of the delivery vehicle may be readily selected by one of skill in the art.
  • the selection of the delivery vehicle is not considered to be a limitation of this invention.
  • suitable delivery vehicles may be a vector, a liposome, a nanoparticle, or a micelle.
  • a host cell comprising the antisense oligonucleotide according to the invention. Also provided is a host cell comprising the delivery vehicle as described above.
  • the host cell may be mammalian, viral, bacterial, a plant or yeast cell.
  • a method of manufacturing an antisense oligonucleotide according to the invention is provided.
  • the method of manufacturing may comprise chemically synthesising an antisense oligonucleotide according to the invention.
  • the method of manufacturing may comprise an additional step of chemically modifying one or more nucleotides.
  • a method of modulating expression of EFEMP1 gene product in a biological system comprising: introducing an antisense oligonucleotide according to the invention or a delivery vehicle according to the invention into the biological system.
  • the antisense oligonucleotide provided herein is useful for targeting nucleic acid expressed from the mutant EFEMP1 allele.
  • the antisense oligonucleotides disclosed herein comprise a sequence complementary to a target nucleic acid sequence wherein the target sequence comprises SEQ ID NO:1 or a portion thereof.
  • the complementary sequence of the antisense oligonucleotide binds and/or hybridizes to a sequence of the mRNA expressed from the mutant EFEMP1 allele comprising SEQ ID NO:1 or a portion thereof.
  • mRNA transcripts for example, mRNA transcripts.
  • the expression of EFEMP1 gene product may be inhibited.
  • the term inhibited is used to indicate a decrease or downregulation of expression or activity.
  • the phrase “inhibit expression” does not necessarily require that the expression of the gene be entirely silenced.
  • the method may result in substantially complete inhibition of expression of the gene or RNA (i.e. 100% inhibition or near 100% of gene expression).
  • the method of the present invention may result in partial, e.g. a slight or moderate reduction in the expression of the target gene or RNA.
  • the method can result in expression of the gene or RNA being inhibited /downregulated by at least 10%, 20%, 30%, 40% or 50% compared to normal or wildtype expression.
  • the method may be an in vitro or in vivo method. In one embodiment the method may be performed in vitro. For example, in a cell, tissue, blood sample or other sample from a human, plant or animal subject. In such embodiments, inhibition of gene expression may be required for research purposes.
  • the method may be performed in vivo.
  • the subject may be a human or animal subject.
  • inhibition of gene expression may result in an altered phenotype in said human or animal subject.
  • inhibition of gene expression, where the gene is disease linked may result in treatment of that disease.
  • the biological system may be selected from a eukaryotic cell, such as a mammalian cell.
  • the biological system may be a cell or plurality of cells, for example a eukaryotic cell/cells, a sample from a subject.
  • the biological system may comprise a retina of a subject.
  • the biological system may comprise retinal pigment epithelium.
  • the subject may be a human or animal subject, for example a subject in which inhibition of gene expression is required.
  • the subject may comprise a synthetic biological system, created from component parts in vitro or created in silico, for example.
  • a pharmaceutical composition comprising an antisense oligonucleotide according to the invention or a delivery vehicle according to the invention.
  • the pharmaceutical composition may further comprise a pharmaceutically acceptable carrier, diluent, enhancer or excipient.
  • the pharmaceutically acceptable carrier or vehicle can be particulate, so that the compositions are, for example, in tablet or powder form.
  • carrier refers to a diluent, adjuvant or excipient, with which a drug antisense oligonucleotide according to the invention is administered.
  • Such pharmaceutical carriers can be liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like.
  • the carriers can be saline, gum acacia, gelatin, starch paste, talc, keratin, colloidal silica, urea, and the like.
  • auxiliary, stabilizing, thickening, lubricating and coloring agents can be used.
  • the antisense oligonucleotide of the present invention or compositions and pharmaceutically acceptable carriers are sterile.
  • Water is a preferred carrier when the drug antisense oligonucleotide according to the invention is administered intravenously.
  • Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions.
  • Suitable pharmaceutical carriers also include excipients such as starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like.
  • excipients such as starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like.
  • the present compositions if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents.
  • the pharmaceutical composition of the invention can be in the form of a liquid, e.g., a solution, emulsion or suspension.
  • the liquid can be useful for delivery by injection (e.g. intravitreal), infusion (e.g., IV infusion) or subcutaneously.
  • the composition When intended for oral administration, the composition is preferably in solid or liquid form, where semi-solid, semi-liquid, suspension and gel forms are included within the forms considered herein as either solid or liquid.
  • the composition can be formulated into a powder, granule, compressed tablet, pill, capsule, chewing gum, wafer or the like form.
  • Such a solid composition typically contains one or more inert diluents.
  • binders such as carboxymethylcellulose, ethyl cellulose, microcrystalline cellulose, or gelatin; excipients such as starch, lactose or dextrins, disintegrating agents such as alginic acid, sodium alginate, corn starch and the like; lubricants such as magnesium stearate; glidants such as colloidal silicon dioxide; sweetening agents such as sucrose or saccharin; a flavoring agent such as peppermint, methyl salicylate or orange flavoring; and a coloring agent.
  • a liquid carrier such as polyethylene glycol, cyclodextrin or a fatty oil.
  • the composition can be in the form of a liquid, e. g. an elixir, syrup, solution, emulsion or suspension.
  • the liquid can be useful for oral administration or for delivery by injection.
  • a composition can comprise one or more of a sweetening agent, preservatives, dye/colorant and flavor enhancer.
  • a surfactant, preservative, wetting agent, dispersing agent, suspending agent, buffer, stabilizer and isotonic agent can also be included.
  • compositions can take the form of one or more dosage units.
  • the pharmaceutical composition of the present invention may be administered orally, topically, by inhalation, insufflation or parenterally.
  • the pharmaceutical composition comprising the antisense oligonucleotide may be administered intravitreally.
  • the pharmaceutical composition comprising the antisense oligonucleotide may be administered by intravitreal injection.
  • the compositions and formulations of said antisense oligonucleotides may be administered topically to the eye. In an embodiment they may be formulated for topical administration to the corneal surface of the eye. Application to the corneal surface may, for example be in the form of eyedrops, a gel, lotion, cream or ocular inserts. In one embodiment, the pharmaceutical composition may be administered topically.
  • the pharmaceutical composition may be administered by a contact lens impregnated with the pharmaceutical composition.
  • the contact lens may be a slow release contact lens.
  • Other administration forms to the eye may include injection into the eye.
  • the amount of the therapeutic that is effective/active in the treatment of a particular disorder or condition will depend on the nature of the disorder or condition and the animal to be treated and can be determined by standard clinical techniques.
  • in vitro or in vivo assays can optionally be employed to help identify optimal dosage ranges.
  • the precise dose to be employed in the compositions will also depend on the route of administration, and the seriousness of the disease or disorder, and should be decided according to the judgment of the practitioner and each subject's circumstances. Factors like age, body weight, sex, diet, time of administration, rate of excretion, condition of the host, drug combinations, reaction sensitivities and severity of the disease shall be taken into account.
  • Toxicity and therapeutic efficacy of the compounds, therapies, combinations and compositions of the invention, administered alone or in combination can be determined by any number of systems or means.
  • the toxicity and therapeutic efficacy can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population).
  • the dose ratio between toxic and therapeutic effects is the therapeutic index (LD50/ ED50).
  • the data obtained from these cell culture assays and animal studies can be used in formulating a range of dosage for use in human.
  • the dosage of such compounds or therapies, alone or in combination lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity.
  • the dosage may vary within this range depending upon the dosage form employed and the route of administration.
  • the pharmaceutical composition may be specifically formulated for delivery of DNA or RNA molecules using non-viral vectors such as exosomes, nanoparticles or liposomes or viral vectors for example retroviral vectors, adenoviral vectors or herpes simplex viral vectors or lipid conjugated.
  • non-viral vectors such as exosomes, nanoparticles or liposomes or viral vectors for example retroviral vectors, adenoviral vectors or herpes simplex viral vectors or lipid conjugated.
  • Methods of delivery of the antisense oligonucleotide, pharmaceutical composition, or vehicle include injection of naked antisense oligonucleotide, physical delivery such as electroporation, gene gun, sonoporation, magnetofection, hydrodynamic delivery, and chemical methods to enhance delivery such as inorganic nanoparticles and cell-penetrating peptides.
  • the antisense oligonucleotide may be administered as a bare molecule in a carrier.
  • the carrier may be a liquid.
  • the antisense oligonucleotide may be conjugated to another moiety to aid its delivery.
  • the moiety may be selected from a small molecule, for example a chemical, nanoparticle, small molecule, liposome or extracellular vesicle.
  • the antisense oligonucleotide of the invention may be complexed with membrane disruptive agents and/or a cationic lipid or helper lipid molecule.
  • the pharmaceutical composition may further comprise a further DHMD therapy.
  • a pharmaceutically effective dose is that dose required to prevent, inhibit the occurrence, or treat (alleviate a symptom to some extent, preferably all of the symptoms) a disease state.
  • the pharmaceutically effective dose generally depends on the type of disease, the composition used, the route of administration, the type of mammal being treated, the physical characteristics of the specific mammal under consideration, concurrent medication, and other factors that those skilled in the medical arts will recognize.
  • an amount of 1pg -30mg may be administered.
  • An amount of 1pg -30mg may be administered by intravitreal injection.
  • the pharmaceutical composition may be administered once every 3 to 12 months.
  • the pharmaceutical composition may be administered once every 3 to 6 months.
  • DHMD Doyne honeycomb macular dystrophy
  • the method of treating, preventing, ameliorating, or slowing progression of DHMD in a subject may selectively reduce expression of a mutant allele.
  • the mutant allele may be a c.1033C>T mutation in the EFEMP1 gene producing a R345W mutant EFEMP1 protein.
  • administering the antisense oligonucleotide may improve, preserve, or prevent worsening of visual function, visual field, retinal function, retinal pigment epithelium, function, electroretinogram (ERG) response, or visual acuity compared to an untreated subject.
  • the inventors have demonstrated that antisense oligonucleotide reduced c.1033C>T, p.R345W mutant allele expression in a heterologous HEK in vitro model and iPSC-retinal pigment epithelial cells derived from a molecularly confirmed c.1033C>T, p.R345W Doyne Honeycomb Macular Dystrophy patient.
  • Administering the antisense oligonucleotide may improve retinal structure and/or function and this can be measured by techniques known in the art such as ERG, OCT, microperimetry, FST, adaptive optics, pupillometry.
  • Administering the antisense oligonucleotide may inhibit, prevent, or delay progression of retinal pigment epithelial cell loss or deterioration of the retina outer nuclear layer in a subject compared to an untreated subject.
  • the method of treating, preventing, ameliorating, or slowing progression of DHMD according to the invention wherein administering the antisense oligonucleotide according to the invention or a delivery vehicle according to the invention or the pharmaceutical composition according to the invention may selectively inhibit expression of R345W mutant EFEMP1 protein expression over wild-type EFEMP1 gene product expression in the subject.
  • the method of treating, preventing, ameliorating, or slowing progression of DHMD in a subject according to the invention wherein the antisense oligonucleotide according to the invention or a delivery vehicle according to the invention or the pharmaceutical composition according to the invention may selectively inhibit expression of R345W mutant EFEMP1 protein by binding to mRNA of the EFEMP1 gene thereby inhibiting R345W mutant EFEMP1 protein expression.
  • the expression of EFEMP1 gene product may be reduced by at least 25- 50% in retinal pigment epithelium in a treated subject compared to untreated subjects.
  • the method of treating, preventing, ameliorating, or slowing progression of DHMD in a subject according to the invention may comprise contacting a cell with the antisense oligonucleotide according to the invention or a delivery vehicle according to the invention or the pharmaceutical composition according to the invention.
  • the cell may be a retinal pigment epithelium (RPE) cell.
  • the method of treating, preventing, ameliorating, or slowing progression of DHMD in a subject according to the invention wherein the antisense oligonucleotide according to the invention or a delivery vehicle according to the invention or the pharmaceutical composition according to the invention may be administered to the subject intravitreally.
  • the method of treating, preventing, ameliorating, or slowing progression of DHMD in a subject according to the invention wherein the antisense oligonucleotide according to the invention or a delivery vehicle according to the invention or the pharmaceutical composition according to the invention may be administered to the subject by intravitreal injection.
  • the method of treating, preventing, ameliorating, or slowing progression of DHMD in a subject according to the invention wherein the antisense oligonucleotide according to the invention or a delivery vehicle according to the invention or the pharmaceutical composition according to the invention may be administered to the subject by intravitreal injection once every 3 to 12 months.
  • the antisense oligonucleotide or pharmaceutical composition comprising the antisense oligonucleotide may be administered by intravitreal injection at regular intervals.
  • the method of treating, preventing, ameliorating, or slowing progression of DHMD in a subject according to the invention wherein the antisense oligonucleotide according to the invention or a delivery vehicle according to the invention or the pharmaceutical composition according to the invention may be administered to the subject by intravitreal injection once every 3 to 6 months.
  • the antisense oligonucleotide or pharmaceutical composition comprising the antisense oligonucleotide may be administered by intravitreal injection at regular intervals.
  • the antisense oligonucleotide or pharmaceutical composition comprising the antisense oligonucleotide may be administered at a dosage determined by the skilled person. Determination of the dosage may be based on techniques well known in the art and may vary dependent on the subject, desired effect, frequency of administration and safety. In one embodiment the method of treating, preventing, ameliorating, or slowing progression of DHMD in a subject according to the invention, wherein the antisense oligonucleotide according to the invention or a delivery vehicle according to the invention or the pharmaceutical composition according to the invention may be administered to the subject at a dosage of the antisense oligonucleotide of 1 pg - 30mg.
  • the antisense oligonucleotide or pharmaceutical composition comprising the antisense oligonucleotide may be administered initially in a loading dose and subsequently as a maintenance dose.
  • the loading dose may comprise a greater concentration of the antisense oligonucleotide than a subsequent maintenance dose of the antisense oligonucleotide.
  • an antisense oligonucleotide according to the invention or a delivery vehicle according to the invention or the pharmaceutical composition according to the invention for use as a medicament there is provided an antisense oligonucleotide according to the invention or a delivery vehicle according to the invention or the pharmaceutical composition according to the invention for use as a medicament.
  • an antisense oligonucleotide according to the invention or a delivery vehicle according to the invention or the pharmaceutical composition according to the invention for use in treating, preventing, ameliorating, or slowing progression of DHMD in a subject comprising administering to the subject an effective amount of the antisense oligonucleotide.
  • the antisense oligonucleotide may be administered together with a second therapy.
  • the present invention therefore also provides a combination therapy comprising the administration of the antisense oligonucleotide and another active ingredient such as, for example, another DHMD therapy.
  • Other therapies may include cell stress modifiers (inhibitors and activators) or neuroprotective strategies.
  • the therapies may be administered at the same time (for example in the same medicament) or at a different time (for example in a different medicament).
  • the therapies may be provided as different medicaments administered sequentially.
  • the antisense oligonucleotide may be administered prior to or after the other DHMD therapeutic.
  • kits comprising an antisense oligonucleotide of any one of the invention or a delivery vehicle according to the invention.
  • the kit comprises instructions for use.
  • ASO antisense oligonucleotides
  • EGF EGF containing fibulin extracellular matrix protein 1 (EFEMP1, OMIM 601548) gene located at 2p16.1 (genomic coordinates (GRCh38): 2:55,865,967-55,923,782 (NCBI)).
  • ASO target sequences were selected based on in silico prediction of target RNA secondary structure and accessibility, and optimal in silica properties of the complementary ASO (%GC, TM, G, maxG, secondary structure formation and stability).
  • the reference gDNA sequence for ASO1 and ASO2 SEQ ID NO. 15
  • ASO3 and ASO4 SEQ ID NO.
  • ASO sequences are as follows: ASO1 (SEQ ID NO 10) and 2 (SEQ ID NO. 11): (5’TCATCCTCCCAGCATTCA3’) ASO 3 (SEQ ID NO. 12) and 4 (SEQ ID NO.
  • ASO chemistry for all four 18 mer ASO is a ‘gapmer’ configuration with a fully substitutes phosphorothioate (PS) backbone, and a gap of 8 deoxynucleotides (dDNA bases) flanked on either side by 5 nucleotides with 2’0- methoxyethyl (2’O-MOE) ribose sugar modifications.
  • the target nucleotide c.1033C>T
  • ASO3 and ASO4 it is located at position 4 of the ‘gap’.
  • the target nucleotide itself is a linked nucleic acid (LNA).
  • ASO2 and ASO4 the target nucleotide is flanked on the 5’ and 3’ side by an LNA.
  • HEK293T cells were transfected with pEFEMP1(WT)-FLAG3X alone, pEFEMP1(R345W)- mScarlet alone, pEFEMP1(WT)-FLAG3X + pEFEMP1(R345W)-mScarlet, and with control (CTRL) ASO or increasing amounts (25 nM, 50 nM, 100 nM, 200 nM) of ASO1, ASO2, ASO3 and ASO4.
  • CTRL control
  • EFEMP1(WT) common EFEMP1 primer and FLAG-specific primer
  • EFEMP1(R345W) common EFEMP1 primer and mScarlet-specific primer
  • EFEMP1 EFEMPI-specific primer pair
  • ASO refinement ( Figure 3) ASO1 (18 mer) was selected as the lead ASO for further refinement of the ASO design to improve specificity, stability and efficiency.
  • ASO1A is a 16 mer ASO
  • ASO1B and C are 15 mer ASO
  • ASO1D is a 14 mer ASO.
  • All ASO (1A-1D) are fully substituted phosphorothioate (PS) gapmers in which the target nucleotide (c.1033C>T) is a modified LNA.
  • PS phosphorothioate
  • the wings flanking the gap are comprised of three 2’O-MOE ribose modified nucleotides (shortened from the previous 5 nucleotide wings in ASO1).
  • the gap in ASO1A is 10 deoxynucleotides in length, 9 deoxynucleotides in ASO1B and 1C, and 8 deoxynucleotides in the shortest 14 mer ASO1 D.
  • the position of the target nucleotide LNA within the gap shifts according to the alignment of the gapmer configuration to the target sequence, and is located at position 7, 8, 6 and 6 of the gap respectively for ASO1A, 1B, 10 and 1D.
  • HEK293T cells were transfected with pEFEMP1(WT)-FLAG3X alone, pEFEMP1(R345W)- mScarlet alone or pEFEMP1(WT)-FLAG3X + pEFEMP1(R345W)-mScarlet, and with control (CTRL) ASO or increasing amounts (25 nM, 50 nM, 100 nM, 200 nM) of ASO1A, ASO1B, ASO1C or ASO1D.
  • CTRL control
  • Reverse transcriptase quantitative PCR (qPCR) of purified RNA was conducted using primer pairs specific for EFEMP1(WT), EFEMP1(R345W) or both (EFEMP1) and the results compared to cells transfected with plasmid only (NT; non-treated) or treated with control ASO (CTRL ASO). Whilst all ASO were effective at reducing EFEMP1 levels in comparison to untreated cells (NT) and cells treated with control (CTRL) ASO, a concentration-dependent reduction was seen for ASO1A, 1C and 1D, and preferential reduction of EFEMP1(R345W) was evident for all ASO (1A-1 D) ( Figure 4).
  • Renal epithelial (RE) cells were retrieved from a molecularly confirmed c.1033C>T, p.R345W Doyne Honeycomb Macular Dystrophy patient.
  • the RE cells were reprogrammed to induced pluripotent stem cells (iPSC) via ectopic expression of the ‘Yamanaka factors’ and the pluripotency and trilineage differentiation potential of the iPSC characterised by immunocytochemistry (ICC) of pluripotency markers (TRA-1-60, OCT4, NANOG, SSEA-4) and RT-qPCR of markers for trilineage potential following directed differentiation, respectively.
  • ICC immunocytochemistry
  • pluripotency markers TRA-1-60, OCT4, NANOG, SSEA-4
  • HDR CRISPR-Cas9 homology directed repair
  • RNP EFEMPI-specific crisprRNA/tracrRNA/ribonucleotide protein
  • ssODN repair template was employed to correct the c.1033C>T variation in the patient iPSC line, thereby generating an isogenic corrected control.
  • the patient and corrected iPSC were differentiated to retinal pigment epithelial cells (RPE), using a modified protocol based on Michelet et al., 2020. Significant morphological disruption of the iPSC-RPE cells from the patient compared to the isogenic control were evident (data not shown).
  • the isogenic control cells formed a uniform honeycomb monolayer of cuboidal shaped epithelial cells characterised by ZO-1 tight junctions and specific markers of RPE differentiation (e.g. MERTK) ( Figure 5).
  • the patient-derived iPSC-RPE formed a heterogeneous population of mixed morphology epithelial and non-epithelial cells indicative of upregulation of epithelial- mesenchymal transition (EMT), a clinically relevant phenotype.
  • EMT epithelial- mesenchymal transition
  • Reverse transcriptase qPCR of purified RNA confirmed the significant downregulation of RPE markers (BEST1, MERTK, RPE65 and PMEL) in the patient iPSC-RPE compared to the isogenic control, and the significant upregulation of EMT markers (ZEB1 , TGFB1, SNAI2).
  • the transfection of iPSC- RPE with 5’ 6-FAM conjugated ASO confirmed successful uptake of ASO into the iPSC-RPE ( Figure 5).
  • CT control ASO
  • ASO1A, 1B, 1C and 1D 200 nM
  • the target (spanning EFEMP1 c.1033C>T) was amplified and sequenced (paired-end reads) by Illumina MiSeq next generation sequencing (NGS) (read depth -150,000).
  • NGS next generation sequencing
  • the target locus was also amplified and quantified from patient renal epithelial cells (gDNA and cDNA).

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Abstract

L'invention concerne un oligonucléotide antisens comprenant une séquence complémentaire d'au moins une partie d'une séquence d'acide nucléique cible, la séquence cible comprenant SEQ ID NO : 1 ou une partie de celle-ci. L'invention concerne également des procédés de traitement, de prévention, de réduction ou de ralentissement de la progression de la dystrophie maculaire en nid d'abeille de Doyne (DHMD), ainsi que des utilisations médicales connexes des oligonucléotides antisens.
PCT/GB2024/052082 2023-08-08 2024-08-07 Molécules thérapeutiques Pending WO2025032331A1 (fr)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2013173637A1 (fr) * 2012-05-16 2013-11-21 Rana Therapeutics, Inc. Compositions et méthodes pour moduler l'expression génique
WO2019183630A2 (fr) * 2018-03-23 2019-09-26 The Trustees Of Columbia University In The City Of New York Édition de gènes pour maladies autosomiques dominantes
WO2023154964A1 (fr) * 2022-02-14 2023-08-17 Alloy Therapeutics, Inc. Procédés et compositions pour cibler efemp1

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2013173637A1 (fr) * 2012-05-16 2013-11-21 Rana Therapeutics, Inc. Compositions et méthodes pour moduler l'expression génique
WO2019183630A2 (fr) * 2018-03-23 2019-09-26 The Trustees Of Columbia University In The City Of New York Édition de gènes pour maladies autosomiques dominantes
WO2023154964A1 (fr) * 2022-02-14 2023-08-17 Alloy Therapeutics, Inc. Procédés et compositions pour cibler efemp1

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
GREENSAMBROOK ET AL.: "Molecular Cloning: A Laboratory Manual", 2012, COLD SPRING HARBOR LABORATORY PRESS
IN J. WENGEL, ACC. CHEM. RES., vol. 120, 1999, pages 5458 - 5463
J. WENGEL ET AL., NUCLEOSIDES & NUCLEOTIDES, vol. 18, no. 6-7, pages 1365 - 1370

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