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WO2025000015A1 - Traitement par oligomère antisens de la perte osseuse - Google Patents

Traitement par oligomère antisens de la perte osseuse Download PDF

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WO2025000015A1
WO2025000015A1 PCT/AU2024/050632 AU2024050632W WO2025000015A1 WO 2025000015 A1 WO2025000015 A1 WO 2025000015A1 AU 2024050632 W AU2024050632 W AU 2024050632W WO 2025000015 A1 WO2025000015 A1 WO 2025000015A1
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antisense
oligomer
sost
protein
antisense oligomer
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Ming Hao Zheng
Jun Yuan
Chi Pang Tai
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Perron Institute for Neurological and Translational Science Ltd
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Perron Institute for Neurological and Translational Science Ltd
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Publication of WO2025000015A1 publication Critical patent/WO2025000015A1/fr
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    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/713—Double-stranded nucleic acids or oligonucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
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    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
    • C07H21/04—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids with deoxyribosyl as saccharide radical
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    • 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
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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
    • C12N2310/30—Chemical structure
    • C12N2310/31—Chemical structure of the backbone
    • C12N2310/314—Phosphoramidates
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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
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/32—Chemical structure of the sugar
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    • C12N2310/3233—Morpholino-type ring
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    • C12N2320/00—Applications; Uses
    • C12N2320/30—Special therapeutic applications
    • C12N2320/33—Alteration of splicing

Definitions

  • Osteoporosis which is characterized by bone loss, is one of the most common disorders affecting ageing population.
  • an antibody against sclerostin named romosozumab (AMG 785, Amgen) has been approved by the U.S. FDA in April 2019 for the treatment of osteoporosis.
  • romosozumab AMG 785, Amgen
  • its clinical outcome showed promising efficacy, adverse effects (including increased cardiovascular and cerebrovascular events) have been commonly reported.
  • the production and purification of antibodies is expensive, which leads to the high cost for monthly injection associated with the use of romosozumab.
  • Sclerostin encoded by SOST gene, is a protein mainly produced in bone.
  • an isolated or purified antisense oligomer for modifying pre-mRNA splicing or mRNA translation in the SOST gene to induce downregulation of the production of functional SOST protein.
  • an isolated or purified antisense oligomer for inducing the production of proteins with retained introns or partial introns, the production of truncated proteins, the production of proteins lacking functional regions, or a reduction in the total amount of protein produced.
  • the antisense oligomer is a phosphorodiamidate morpholino oligomer (PMO) or a 2'-O-methyl phosphorothioate oligomer (2'-OMePS).
  • PMO phosphorodiamidate morpholino oligomer
  • 2'-OMePS 2'-O-methyl phosphorothioate oligomer
  • the antisense oligomer is selected from the group comprising the sequences set forth in Table 1.
  • the antisense oligomer is selected from the list comprising: SEQ ID NO: 1 -11.
  • the antisense oligomer used in the present disclosure is SEQ ID NO: 3-7.
  • the disclosure extends, according to a still further aspect thereof, to cDNA or cloned copies of the antisense oligomer sequences of the disclosure, as well as to vectors containing the antisense oligomer sequences of the disclosure.
  • the disclosure extends further also to cells containing such sequences and/or vectors.
  • [001 1] There is also provided a method for manipulating splicing factor binding in a target protein gene transcript, the method including the step of: providing one or more of the antisense oligomers as described herein and allowing the oligomer(s) to bind to a target nucleic acid site to induce downregulation of the production of functional SOST protein.
  • compositions to treat, prevent or ameliorate the effects of dysregulation of SOST in a subject, the composition comprising: one or more antisense oligomers as described herein; and one or more pharmaceutically acceptable carriers and/or diluents to induce downregulation of the production of functional SOST protein.
  • the bone disease is associated with bone loss in the subject.
  • the bone disease is chosen from: osteoporosis, postmenopausal osteoporosis, osteogenesis imperfecta and bone loss associated with cancer.
  • the subject with bone disease may be a mammal, including a human.
  • a method to treat, prevent or ameliorate the effects of bone disease in a subject comprising the step of: administering to the subject an effective amount of one or more antisense oligomers or pharmaceutical composition comprising one or more antisense oligomers as described herein to induce downregulation of the production of functional SOST protein.
  • kit to treat, prevent or ameliorate the effects of bone disease in a subject comprises at least an antisense oligomer as described herein and combinations or cocktails thereof, packaged in a suitable container, together with instructions for its use.
  • Figure 1 shows the characterization of SOST gene and its gene expression in Saos2 cell line.
  • Fig 1 A SOST expression in Saos 2 cells by Real-time polymerase chain reaction (RT-PCR).
  • Fig 1 B Sequence of mut1 -SOST in human patients (DOI: 10.1086/31881 1 ).
  • Fig 1 C DNA sequence of SOST gene in Saos2 cell line.
  • Figure 2 shows the downregulation of SOST gene expression by ASOs (SEQ ID NO: 1 -11 ) in a dose-dependent manner.
  • Fig 2A A DNA gel showed that SOST-ASOs (SEQ ID NO: 1 and 3) can greatly reduce the gene expression of SOST, as shown by RT-PCR.
  • Fig 2B Intensity of SOST and ACTS bands were quantified by image J and presented by Graphpad. Relative SOST expression of each sample was compared by normalizing with ACTS expression.
  • Fig 2C NC: transfection reagent.
  • Figure 3 shows that 75-80% transfection efficiency can be achieved by Neon NxT electroporation at 25, 50 and 100 pM.
  • Figure 4 shows downregulation of SOST gene expression by 2’-OMePS modified ASOs (SEQ ID NO: 1 -1 1 ) in a dose-dependent manner (0.2-20pM).
  • a DNA gel showed that SEQ ID NO: 1 and 3), but not SEQ ID NO: 2 can greatly reduce the gene expression of SOST, as shown by RT- PCR.
  • ACTB beta actin housekeeping gene
  • C cell alone with transfection reagent
  • SC standard control oligo
  • Scr scramble ASO.
  • Figure 5 shows reduced SOST protein levels secreted by Saos2 cells after transfection of PMO- modified ASOs (SEQ ID NO: 1 and 3).
  • Fig 5A Ponceau R staining of protein gel showed protein integrity in each sample.
  • Fig 5B Reduction of SOST protein in cells transfected with ASOs (SEQ ID NO: 1 and 3) in a dose dependent manner.
  • Fig 5C cell alone with transfection reagent, SC standard control oligo, Scr: scramble ASO.
  • Figure 6 shows reduced SOST gene expression levels after transfection of ASOs at 5pM.
  • SEQ ID NOs: 3, 4, 6 and 7 showed the highest downregulation of SOSTby RT-PCR.
  • Figure 7 shows the Sanger sequencing analysis of the upper band in the DNA gel shown in figure 4.
  • Exon 1 retained intronic sequence and exon 2 are labelled by underlines of orange and blue colours. The result showed that retention of part of intron 1 ( grey underline) occurred after the treatment of SOST-ASO.
  • Figure 8 shows the in vitro functional analysis of SOST-ASO on human osteoblast cells Saos2 under osteoblastic differentiation medium at concentration of 0, 1 and 10pM.
  • 8a Timeline demonstrating the treatment of SOST-ASO (transfection), and osteoblastic differentiation and osteogenesis analysis at different timepoints.
  • 8b, 8c Alizarin red S staining (ARS) and quantification of Saos2 cells during osteoblastic differentiation. The results showed enhanced ARS which represented higher bone mineralisation (a major procedure of bone formation) following ASO administration.
  • ALP Alkaline phosphatase
  • osteogenic marker genes Osteocalcin OCN, Osteoprotegerin OPG and Runt-related transcription factor 2 RUNX2
  • SOST -ASOs may overcome the concerns related with romosozumab usage.
  • the production cost for oligonucleotides is a lot cheaper than antibody production.
  • antisense oligonucleotides can specially target SOST transcripts in bone, raising the possibility of not affecting SOST involvement in cardiovascular and cerebrovascular systems.
  • SOST-aptamers Some progress has been made in developing SOST-aptamers to target the sclerostin loop 3 region, which is involved in bone formation.
  • aptamer specificity remains unknown due to sequence variations in human Ioop3, and possible post-translational modifications that may intervene in SOST-aptamer binding.
  • unbound aptamer is easily cleared by renal clearance.
  • SOST-ASOs can not only inhibit bone-derived sclerostin production, but also has the advantage of higher stability after delivery into human body.
  • mut1 -SOST A+3T a mutation at the donor splicing site of SOST can cause a splicing shift by using the cryptic site donor located 214bp downstream of the authentic site, leading to partial intron retention which further leads to premature in-frame non-sense codon (DOI: 10.1086/31881 1 ).
  • mut2-SOST A+2692C a mutation near the splicing acceptor site uses the same splice donor and acceptor site, leading to expression of mRNA remaining unaffected.
  • the present disclosure has found that targeting the SOST (mut1 ) region to cause intron retention (instead of exon skipping as ASOs are more commonly used for), results in efficient and sustained splicing modification and protein isoform expression of the SOST protein.
  • the present disclosure provides the ASOs in Table 1 for regulating the expression of functional SOST transcript or/and SOST protein.
  • Particularly preferred sequences are SEQ ID NOs: 3-7, which showed greatest reduction of SOST expression.
  • the antisense oligomer induced exon skipping of the present disclosure need not completely or even substantially ablate the function of the target protein.
  • the intron retention process results in a reduced or compromised functionality of the target protein.
  • the antisense oligomers cover splicing sites in the SOST transcript.
  • the target site may also include some flanking sequences around the splicing sites.
  • the target site is covering the SOST (mut1 ) region.
  • an antisense oligomer of 10 to 50 nucleotides comprising a targeting sequence complementary to a region near or within the splicing sites and/or the polyadenylation site of the target protein pre-mRNA.
  • the present disclosure may induce increased degradation of RNA via recruitment of RNase H, wherein the RNase H preferentially binds and degrades RNA bound in duplex to the DNA of the target protein gene.
  • RNase-H recognises the DNA-RNA heteroduplex generated between mRNA and genomic DNA and cleaves the RNA strand.
  • Antisense oligomers designed to serve as substrates for RNase-H are inhibitors of the intermediary metabolism of pre- and spliced mRNAs.
  • antisense oligomer and “antisense compound” and “antisense oligonucleotide” “AON” and “ASO” are used interchangeably and refer to a sequence of cyclic subunits, each bearing a base-pairing moiety, linked by inter-subunit linkages that allow the basepairing moieties to hybridize to a target sequence in a nucleic acid (typically an RNA) by Watson- Crick base pairing, to form a nucleic acid:oligomer heteroduplex within the target sequence.
  • a nucleic acid typically an RNA
  • the cyclic subunits are based on ribose or another pentose sugar or, in a preferred embodiment, a morpholino group (see description of morpholino oligomers below).
  • the oligomer may have exact or near sequence complementarity to the target sequence; variations in sequence near the termini of an oligomer are generally preferable to variations in the interior.
  • pre-RNA and “pre- mRNA” are used interchangeably.
  • isolated is meant material that is substantially or essentially free from components that normally accompany it in its native state.
  • an “isolated polynucleotide” or “isolated oligonucleotide,” as used herein, may refer to a polynucleotide that has been purified or removed from the sequences that flank it in a naturally occurring state, e.g., a DNA fragment that is removed from the sequences that are adjacent to the fragment in the genome.
  • isolated as it relates to cells refers to the purification of cells (e.g., fibroblasts, lymphoblasts) from a source subject (e.g., a subject with bone disease).
  • a source subject e.g., a subject with bone disease.
  • isolated refers to the recovery of mRNA or protein from a source, e.g., cells.
  • an antisense oligomer can be said to be “directed to” or “targeted against” a target sequence with which it hybridizes.
  • the target sequence includes a region including splicing sites and/or the polyadenylation site and surrounding regions.
  • the target sequence is typically a region including an AUG start codon of an mRNA, a Translation Suppressing Oligomer, or splice site of a pre-processed mRNA, a Splice Suppressing Oligomer (SSO).
  • the target sequence for a splice site may include an mRNA sequence having its 5' end 1 to about 25 base pairs downstream of a normal splice acceptor junction in a pre-processed mRNA.
  • a preferred target sequence is any region of a pre-processed mRNA that includes a splice site or is contained entirely within an exon coding sequence or spans a splice acceptor or donor site.
  • An oligomer is more generally said to be "targeted against” a biologically relevant target, such as a protein, virus, or bacteria, when it is targeted against the nucleic acid of the target in the manner described above.
  • sufficient length refers to an antisense oligonucleotide that is complementary to at least 8, more typically 8-30, contiguous nucleobases in a target protein pre- mRNA.
  • an antisense of sufficient length includes at least 8, 9, 10, 1 1 , 12, 13, 14, or 15 contiguous nucleobases in the target protein pre-mRNA.
  • an antisense of sufficient length includes at least 16, 17, 18, 19, 20, 21 , 22, 23, 24, or 25 contiguous nucleobases in the target protein pre-mRNA.
  • An antisense oligonucleotide of sufficient length has at least a minimal number of nucleotides to be capable of specifically hybridizing to exon 2.
  • an oligonucleotide of sufficient length is from about 10 to about 50 nucleotides in length, including oligonucleotides of 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39 and 40 or more nucleotides.
  • an oligonucleotide of sufficient length is from 10 to about 30 nucleotides in length.
  • an oligonucleotide of sufficient length is from 15 to about 25 nucleotides in length.
  • an oligonucleotide of sufficient length is from 20 to 30, or 20 to 50, nucleotides in length.
  • an oligonucleotide of sufficient length is from 22 to 28, 25 to 28, 24 to 29 or 25 to 30 nucleotides in length.
  • the antisense oligomer has sufficient sequence complementarity to a target RNA (i.e., the RNA for which splicing factor binding site selection is modulated) to block a region of a target RNA (e.g., pre-mRNA) in an effective manner.
  • a target RNA e.g., pre-mRNA
  • such blocking of target protein pre-mRNA serves to modulate or modify splicing, either by masking a binding site for a native protein that would otherwise modulate splicing and/or by altering the structure of the targeted RNA.
  • the target RNA is target pre-mRNA (e.g., target protein gene pre-mRNA).
  • An antisense oligomer having a sufficient sequence complementarity to a target RNA sequence to modulate splicing factor binding of the target RNA means that the antisense oligomer has a sequence sufficient to trigger the masking of a binding site for a native protein that would otherwise cause truncation of the target protein and/or alters the three-dimensional structure of the targeted RNA.
  • Selected antisense oligomers can be made shorter, e.g., about 12 bases, or longer, e.g., about 50 bases, and include a small number of mismatches, as long as the sequence is sufficiently complementary to effect splicing factor binding modulation upon hybridization to the target sequence, and optionally forms with the RNA antisense oligomer heteroduplex having a Tm of 45°C or greater.
  • the antisense oligomer is selected from the group comprising the sequences set forth in Table 1.
  • the antisense oligomer is selected from the list comprising: SEQ ID NO: 1 -11.
  • the antisense oligomer induced manipulation of protein expression of the present disclosure results in downregulation of the production of functional SOST protein.
  • the degree of complementarity between the target sequence and antisense oligomer is sufficient to form a stable duplex.
  • the region of complementarity of the antisense oligomers with the target RNA sequence may be as short as 8-1 1 bases, but can be 12-15 bases or more, e.g., 10-50 bases, 10-40 bases, 12-30 bases, 12-25 bases, 15-25 bases, 12-20 bases, or 15-20 bases, including all integers in between these ranges.
  • An antisense oligomer of about 16-17 bases is generally long enough to have a unique complementary sequence.
  • a minimum length of complementary bases may be required to achieve the requisite binding Tm, as discussed herein.
  • oligonucleotides as long as 50 bases may be suitable, where at least a minimum number of bases, e.g., 10-12 bases, are complementary to the target sequence.
  • facilitated or active uptake in cells is optimized at oligonucleotide lengths of less than about 30 bases.
  • PMO phosphorodiamidate morpholino oligomer
  • an optimum balance of binding stability and uptake generally occurs at lengths of 18-25 bases.
  • antisense oligomers e.g., PMOs, PMO-X, PNAs, LNAs, 2’-OMe
  • PMOs, PMO-X, PNAs, LNAs, 2’-OMe e.g., PMOs, PMO-X, PNAs, LNAs, 2’-OMe
  • antisense oligomers may be 100% complementary to the target sequence, or may include mismatches, e.g., to accommodate variants, as long as a heteroduplex formed between the antisense oligomer and target sequence is sufficiently stable to withstand the action of cellular nucleases and other modes of degradation which may occur in vivo.
  • certain oligonucleotides may have about or at least about 70% sequence complementarity, e.g., 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence complementarity, between the oligonucleotide and the target sequence.
  • 70% sequence complementarity e.g., 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence complementarity, between the oligonucleotide
  • Mismatches are typically less destabilizing toward the end regions of the hybrid duplex than in the middle.
  • the number of mismatches allowed will depend on the length of the antisense oligomer, the percentage of G:C base pairs in the duplex, and the position of the mismatch(es) in the duplex, according to well understood principles of duplex stability.
  • an antisense oligomer is not necessarily 100% complementary to the target sequence, it is effective to stably and specifically bind to the target sequence, such that splicing of the target pre- mRNA is modulated.
  • the stability of the duplex formed between an antisense oligomer and a target sequence is a function of the binding Tm and the susceptibility of the duplex to cellular enzymatic cleavage.
  • the Tm of an oligonucleotide with respect to complementary-sequence RNA may be measured by conventional methods, such as those described by Hames et aL, Nucleic Acid Hybridization, IRL Press, 1985, pp. 107-108 or as described in Miyada C. G. and Wallace R. B., 1987, Oligonucleotide Hybridization Techniques, Methods Enzymol. Vol. 154 pp. 94-107.
  • antisense oligomers may have a binding Tm, with respect to a complementary- sequence RNA, of greater than body temperature and optionally greater than about 45°C or 50°C. Tm’s in the range 60-80°C or greater are also included.
  • variants include antisense oligomers having about or at least about 70% sequence identity or homology, e.g., 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity or homology, over the entire length of any of SEQ ID NOs: 1 -11.
  • an antisense oligomer capable of binding to a selected target site to modulate or modify splicing in a target protein gene transcript or part thereof.
  • the antisense oligomer may be selected from those provided in Table 1.
  • the antisense oligomer is selected from the list comprising: SEQ ID NO: 1 -1 1.
  • the antisense oligomer induced splicing factor blockage of the present disclosure need not completely or even substantially reduce the amount of target protein produced.
  • the length of an antisense oligomer may vary, as long as it is capable of binding selectively to the intended location within the pre-mRNA molecule.
  • the length of such sequences can be determined in accordance with selection procedures described herein.
  • the antisense oligomer will be from about 10 nucleotides in length, up to about 50 nucleotides in length. It will be appreciated, however, that any length of nucleotides within this range may be used in the method.
  • the length of the antisense oligomer is between 10 and 40, 10 and 35, 15 to 30 nucleotides in length or 20 to 30 nucleotides in length, for example about 25 to 30 nucleotides in length.
  • the oligomer may be 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides in length.
  • an “antisense oligomer”, “AON” or “ASO” refers to a linear sequence of nucleotides, or nucleotide analogues, that allows the nucleobase to hybridize to a target sequence in an RNA by Watson-Crick base pairing, to form an oligonucleotide:RNA heteroduplex within the target sequence.
  • the cyclic subunits may be based on ribose or another pentose sugar or, in certain embodiments, a morpholino group (see description of morpholino oligonucleotides below).
  • PMO phosphoramidate or phosphorodiamidate morpholino oligomer
  • PMO-X PMO-X
  • PPMO thiophosphoramidate morpholinos
  • PNA peptide nucleic acid
  • LNA locked nucleic acid
  • ENA ethylene bridged nucleic acids
  • phosphorothioate oligomer tricyclo-DNA oligomer (tcDNA); tricyclophosphorothioate oligomer; 2’0-Methyl-modified oligomer (2’-OMe); 2’-O-methoxy ethyl (2’-MOE); 2'-O-methyl phosphorothioate oligomer (2'- OMePS); 2’-fluoro, 2’-fluroarabino (FANA); unlocked
  • processing of target protein RNA can be manipulated with specific antisense oligomers. In this way functionally significant decreases in the amount of the target protein can be obtained, thereby reducing the pathology of bone disease.
  • the composition may comprise about 1 nm, 2nm, 3nm, 4nm, 5nm, 6nm, 7nm, 8nm, 9nm, 10nm, 20nm, 50nm, 75nm, 100nm, 150nm, 200nm, 250nm, 300nm, 350nm, 400nm, 450nm, 500nm, 550nm, 600nm, 650nm, 700nm, 750nm, 800nm, 850nm, 900nm, 950nm or 1000nm of each of the desired antisense oligomer(s) of the disclosure.
  • compositions may influence the physical state, stability, rate of in vivo release, and rate of in vivo clearance of the present proteins and derivatives. See, for example, Martin, Remington's Pharmaceutical Sciences, 18th Ed. (1990, Mack Publishing Co., Easton, PA 18042) pages 1435- 1712 that are herein incorporated by reference.
  • the compositions may be prepared in liquid form, or may be in dried powder, such as a lyophilised form.
  • compositions provided according to the present disclosure may be administered by any means known in the art.
  • the pharmaceutical compositions for administration are administered by injection, orally, topically or by the pulmonary or nasal route.
  • the antisense oligomers may be delivered by intravenous, intraarterial, intraperitoneal, intramuscular or subcutaneous routes of administration.
  • the appropriate route may be determined by one of skill in the art, as appropriate to the condition of the subject under treatment.
  • Vascular or extravascular circulation, the blood or lymph system, and the cerebrospinal fluid are some non-limiting sites where the antisense oligomer may be introduced.
  • Direct CNS delivery may be employed, for instance, intracerebral ventricular or intrathecal administration may be used as routes of administration.
  • an injectable, nasal delivery or oral delivery route is preferred.
  • Lipids and liposomes include neutral (e.g. dioleoylphosphatidyl DOPE ethanolamine, dimyristoylphosphatidyl choline DMPC, distearolyphosphatidyl choline) negative (e.g. dimyristoylphosphatidyl glycerol DMPG) and cationic (e.g. dioleoyltetramethylaminopropyl DOTAP and dioleoylphosphatidyl ethanolamine DOTMA).
  • neutral e.g. dioleoylphosphatidyl DOPE ethanolamine, dimyristoylphosphatidyl choline DMPC, distearolyphosphatidyl choline
  • negative e.g. dimyristoylphosphatidyl glycerol DMPG
  • cationic e.g. dioleoyltetramethylaminopropyl DOTAP and dioleoylphosphati
  • compositions and formulations for oral administration include powders or granules, microparticulates, nanoparticulates, suspensions or solutions in water or non-aqueous media, capsules, gel capsules, sachets, tablets or minitablets. Thickeners, flavouring agents, diluents, emulsifiers, dispersing aids or binders may be desirable.
  • Oral formulations are those in which oligomers of the disclosure are administered in conjunction with one or more penetration enhancers surfactants and chelators.
  • Surfactants include fatty acids and/or esters or salts thereof, bile acids and/or salts thereof.
  • Bile acids/salts and fatty acids and their uses are further described in U.S. Pat. No. 6,287,860.
  • the present disclosure provides combinations of penetration enhancers, for example, fatty acids/salts in combination with bile acids/salts.
  • An exemplary combination is the sodium salt of lauric acid, capric acid and UDCA.
  • Further penetration enhancers include polyoxyethylene-9-lauryl ether, polyoxyethylene-20-cetyl ether.
  • Oligomers of the disclosure may be delivered orally, in granular form including sprayed dried particles, or complexed to form micro or nanoparticles. Oligomer complexing agents and their uses are further described in U.S. Pat. No. 6,287,860.
  • Oral formulations for oligomers and their preparation are described in detail in US 6,887,906 and/or US 20030027780.
  • the delivery of a therapeutically useful amount of antisense oligomers may be achieved by methods previously published.
  • intracellular delivery of the antisense oligomer may be via a composition comprising an admixture of the antisense oligomer and an effective amount of a block copolymer.
  • An example of this method is described in US patent application US20040248833.
  • Other methods of delivery of antisense oligomers to the nucleus are described in Mann CJ et al. (2001) Proc, Natl. Acad. Science, 98(1 ) 42-47, and in Gebski et al. (2003) Human Molecular Genetics, 12(15): 1801 -181 1.
  • a method for introducing a nucleic acid molecule into a cell by way of an expression vector either as naked DNA or complexed to lipid carriers, is described in US 6,806,084.
  • colloidal dispersion systems include macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes or liposome formulations. These colloidal dispersion systems can be used in the manufacture of therapeutic pharmaceutical compositions.
  • Liposomes are artificial membrane vesicles, which are useful as delivery vehicles in vitro and in vivo. These formulations may have net cationic, anionic, or neutral charge characteristics and have useful characteristics for in vitro, in vivo and ex vivo delivery methods. It has been shown that large unilamellar vesicles can encapsulate a substantial percentage of an aqueous buffer containing large macromolecules. RNA and DNA can be encapsulated within the aqueous interior and be delivered to cells in a biologically active form (Fraley, et al., Trends Biochem. Sci. 6:77, 1981).
  • liposomes depend on pH, ionic strength, and the presence of divalent cations.
  • Cationic liposomes are positively charged liposomes which are believed to interact with negatively charged DNA molecules to form a stable complex. Liposomes that are pH-sensitive or negatively charged are believed to entrap DNA rather than complex with it. Both cationic and noncationic liposomes have been used to deliver DNA to cells.
  • the antisense oligomer may also be combined with other pharmaceutically acceptable carriers or diluents to produce a pharmaceutical composition.
  • Suitable carriers and diluents include isotonic saline solutions, for example phosphate-buffered saline.
  • the composition may be formulated for parenteral, intramuscular, intravenous, subcutaneous, intraocular, oral, or transdermal administration.
  • the antisense oligomers of the disclosure encompass any pharmaceutically acceptable salts, esters, or salts of such esters, or any other compound which, upon administration to an animal including a human, is capable of providing (directly or indirectly) the biologically active metabolite or residue thereof. Accordingly, as an example, the disclosure is also drawn to prodrugs and pharmaceutically acceptable salts of the compounds of the disclosure, pharmaceutically acceptable salts of such pro-drugs, and other bioequivalents.
  • pharmaceutically acceptable salts refers to physiologically and pharmaceutically acceptable salts of the compounds of the disclosure: i.e. salts that retain the desired biological activity of the parent compound and do not impart undesired toxicological effects thereto.
  • salts formed with cations such as sodium, potassium, ammonium, magnesium, calcium, polyamines such as spermine and spermidine, etc.
  • acid addition salts formed with inorganic acids for example hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid and the like
  • salts formed with organic acids such as, for example, acetic acid, oxalic acid, tartaric acid, succinic acid, maleic acid, fumaric acid, gluconic acid, citric acid, malic acid, ascorbic acid, benzoic acid, tannic acid, palmitic acid, alginic acid, polyglutamic acid, naphthalenesulfonic acid, methanesulfonic acid, p-toluenesulfonic acid, naphthalenedisulfonic acid, polygalacturonic acid, and the like
  • compositions of the present disclosure may be administered in a number of ways depending upon whether local or systemic treatment is desired and upon the area to be treated. Administration may be topical (including ophthalmic and mucous membranes, as well as rectal delivery), pulmonary, e.g., by inhalation or insufflation of powders or aerosols (including by nebulizer, intratracheal, intranasal, epidermal and transdermal), oral or parenteral. Parenteral administration includes intravenous, intra-arterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; or intracranial, e.g., intrathecal or intraventricular, administration.
  • Oligomers with at least one 2'-0-methoxyethyl modification are believed to be particularly useful for oral administration.
  • the antisense oligomer is delivered via the subcutaneous or intravenous route.
  • the pharmaceutical formulations of the present disclosure may be prepared according to conventional techniques well known in the pharmaceutical industry. Such techniques include the step of bringing into association the active ingredients with the pharmaceutical carrier(s) or excipient(s). In general, the formulations are prepared by uniformly and intimately bringing into association the active ingredients with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.
  • the antisense oligomer may be administered at regular intervals for a short time period, e.g., daily for two weeks or less. However, in some cases the oligomer is administered intermittently over a longer period of time. Administration may be followed by, or concurrent with, administration of an antibiotic or other therapeutic treatment.
  • the treatment regimen may be adjusted (dose, frequency, route, etc.) as indicated, based on the results of immunoassays, other biochemical tests and physiological examination of the subject under treatment.
  • T reatment includes, but is not limited to, administration of a pharmaceutical composition, and may be performed either prophylactically or subsequent to the initiation of a pathologic event or contact with an etiologic agent.
  • Treatment includes any desirable effect on the symptoms or pathology of bone disease, and may include, for example, minimal changes or improvements in one or more measurable markers of the bone disease being treated.
  • prophylactic treatments which can be directed to reducing the rate of progression of the bone disease being treated, delaying the onset of the bone disease, or reducing the severity of its onset. “Treatment” or “prophylaxis” does not necessarily indicate complete eradication, cure, or prevention of the disease or condition, or associated symptoms thereof.
  • a "subject,” as used herein, includes any animal that exhibits a symptom, or is at risk for exhibiting a symptom, which can be treated with an antisense compound of the disclosure, or any of the symptoms associated with the condition (e.g. bone disease).
  • Suitable subjects include laboratory animals (such as mouse, rat, rabbit, or guinea pig), farm animals, and domestic animals or pets (such as a cat or dog).
  • Non-human primates, such as human subjects, are included.
  • the efficacy of an in vivo administered antisense oligomers of the disclosure may be determined from biological samples (tissue, blood, urine etc.) taken from a subject prior to, during and subsequent to administration of the antisense oligomer.
  • Assays of such samples include (1 ) monitoring the presence or absence of heteroduplex formation with target and nontarget sequences, using procedures known to those skilled in the art, e.g., an electrophoretic gel mobility assay; (2) monitoring the amount of a mutant RNA in relation to a reference normal RNA or protein as determined by standard techniques such as RT-PCR, Northern blotting, ELISA or Western blotting.
  • one or more antisense oligomers as described herein for use in an antisense oligomer-based therapy is provided.
  • the therapy is for bone disease.
  • the antisense oligomer may be selected from the group consisting of any one or more of SEQ ID NOs: 1 -11 , and combinations or cocktails thereof. This includes sequences which can hybridise to such sequences under stringent hybridisation conditions, sequences complementary thereto, sequences containing modified bases, modified backbones, and functional truncations or extensions thereof which possess or modulate pre-RNA processing activity in a target protein gene transcript.
  • the antisense oligomer induced manipulation of protein expression of the present disclosure results in downregulation of the production of functional SOST protein.
  • the disclosure extends also to a combination of two or more antisense oligomers capable of binding to a selected target to modify splicing of a target protein gene transcript.
  • the combination may be a cocktail of two or more antisense oligomers, a construct comprising two or more or two or more antisense oligomers joined together for use in an antisense oligomer-based therapy.
  • the disclosure provides a method to treat, prevent or ameliorate the effects of bone disease, comprising the step of: administering to the subject an effective amount of one or more antisense oligomers or pharmaceutical composition comprising one or more antisense oligomers as described herein to induce downregulation of the production of functional SOST protein.
  • the therapy may be used to develop non-functional, extended, truncated or nonsense target proteins, or to reduce target protein expression.
  • the decrease in levels of target protein may be achieved by decreasing the amount of normal native SOST RNA through intron retention.
  • the present disclosure may induce increased degradation of RNA via recruitment of RNase H, wherein the RNase H preferentially binds and degrades RNA bound in duplex to the DNA of the target protein gene.
  • the reduction in target protein may lead to a reduction in the quantity, duration or severity of the symptoms of bone disease.
  • the disclosure also provides for the use of purified and isolated antisense oligomers as described herein, for the manufacture of a medicament for treatment of bone disease.
  • the antisense oligomer used in the present disclosure is chosen from SEQ ID NO: 1-11.
  • the antisense oligomer induced manipulation of protein expression of the present disclosure results in downregulation of the production of functional SOST protein.
  • the disclosure extends, according to a still further aspect thereof, to cDNA or cloned copies of the antisense oligomer sequences of the disclosure, as well as to vectors containing the antisense oligomer sequences of the disclosure.
  • the disclosure extends further also to cells containing such sequences and/or vectors.
  • kits to treat, prevent or ameliorate bone disease in a subject which kit comprises at least an isolated or purified antisense oligomer for modifying pre-mRNA splicing or mRNA translation in a target protein gene transcript or part thereof, packaged in a suitable container, together with instructions for its use.
  • kits will contain at least one antisense oligomer as described herein or as shown in Table 1 or SEQ ID NOs: 1 -1 1 , or a cocktail of the same, as described herein.
  • the kits may also contain peripheral reagents such as buffers, stabilizers, etc.
  • kit to treat, prevent or ameliorate bone disease in a subject comprises at least an antisense oligomer described herein as SEQ ID NOs: 1 - 1 1 , the antisense oligomers of Table 1 and combinations or cocktails thereof, packaged in a suitable container, together with instructions for its use.
  • kit to treat, prevent or ameliorate bone disease in a subject comprises at least an antisense oligomer selected from the group consisting of any one or more of SEQ ID NOs: 1 -1 1 , and combinations or cocktails thereof, packaged in a suitable container, together with instructions for its use.
  • kits may also be provided in dried or lyophilized forms.
  • reconstitution generally is by the addition of a suitable solvent.
  • the kit can additionally contain a suitable solvent for reconstitution of the lyophilized components.
  • Individual components of the kit may be packaged in separate containers. Irrespective of the number or type of containers, the kits of the disclosure also may comprise, or be packaged with, an instrument for assisting with the injection/administration or placement of the ultimate complex composition within the body of an animal.
  • an instrument may be an inhalant, syringe, pipette, forceps, measured spoon, eye dropper or any such medically approved delivery vehicle.
  • the liquid solution can be an aqueous solution, for example a sterile aqueous solution.
  • the expression construct may be formulated into a pharmaceutically acceptable syringeable composition.
  • the container means may itself be an inhalant, syringe, pipette, eye dropper, or other such like apparatus, from which the formulation may be applied to an affected area of the animal, such as the skin, injected into an animal, or even applied to and mixed with the other components of the kit.
  • antisense oligomers of the present disclosure may also be used in conjunction with alternative therapies, such as drug therapies.
  • the present disclosure therefore provides a method of treating, preventing or ameliorating the effects of bone disease, wherein the antisense oligomers of the present disclosure and administered sequentially or concurrently with another alternative therapy associated with treating, preventing or ameliorating bone disease.
  • the alternative therapy may be chosen from the list comprising: bisphosphonates (alendronate, risedronate, ibandronate, zoledronic acid); denosumab; romosozumab; teriparatide and abaloparatide; raloxifene; calcitonin; calcium supplementation; dietary modifications; regular weight bearing exercise.
  • the transfection efficiency of ASOs was tested using a negative control PMO- modified oligo with 3’ fluorescein (CCTCTTACCTCAGTTACAATTTATA [SEQ ID NO: 16], Gene Tools) at 25pM, 50 pM and 100 pM by Neon NxT electroporation (ThermoFisher) (Pulse voltage 1200V, Pulse Width 40ms and Pulse number 1 ) and found that 75-80% of transfection efficiency can be achieved (Figure 3).
  • SEQ ID NOs: 3, 4, 6, 7 showed highest downregulation of SOST at 5pM (Figure 6). They are microwalked (Table 1 ), highlighting that the overlapping region among these ASOs is the most susceptible to PMO steric hindrance. Furthermore, the switching of the usage of splicing site to the cryptic donor site is evidenced by the presence of additional upper DNA bands (marked by the upper black arrow in Figure 6). This shift will result in a frameshift non-sense mutation (DOI: 10.1086/318811 ).
  • Example 2
  • Sequencing data supports the intron retention after treatment of SOST-ASO
  • osteogenic marker genes Osteocalcin OCN, Osteoprotegerin OPG and Runt-related transcription factor 2 RUNX2
  • SOST-ASO No.13
  • Figure 8e quantitative polymerase chain reaction

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Abstract

L'invention concerne un oligomère antisens isolé ou purifié et des combinaisons et des cocktails pour modifier l'épissage de pré-ARNm ou la traduction d'ARNm dans le transcrit de gène SOST ou une partie de celui-ci pour induire une régulation à la baisse de la production de protéine SOST fonctionnelle.
PCT/AU2024/050632 2023-06-27 2024-06-18 Traitement par oligomère antisens de la perte osseuse Pending WO2025000015A1 (fr)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003073991A2 (fr) * 2002-03-01 2003-09-12 Celltech R & D, Inc. Procedes destines a accroitre ou a reduire la densite osseuse
WO2009039175A2 (fr) * 2007-09-17 2009-03-26 Amgen Inc. Procédé pour inhiber la résorption osseuse

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003073991A2 (fr) * 2002-03-01 2003-09-12 Celltech R & D, Inc. Procedes destines a accroitre ou a reduire la densite osseuse
WO2009039175A2 (fr) * 2007-09-17 2009-03-26 Amgen Inc. Procédé pour inhiber la résorption osseuse

Non-Patent Citations (4)

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Title
AARTSMA-RUS, A ET AL.: "Antisense-mediated exon skipping: a versatile tool with therapeutic and research applications", RNA, vol. 13, no. 10, 2007, pages 1609 - 1624, XP009144451 *
HAVENS, M.A. ET AL.: "Splice-switching antisense oligonucleotides as therapeutic drugs.", NUCLEIC ACIDS RESEARCH, vol. 44, no. 14, 2016, pages 6549 - 6563, XP093196279, DOI: 10.1093/nar/gkw533 *
MCNALLY, E.M. ET AL.: "Welcome to the splice age: antisense oligonucleotide- mediated exon skipping gains wider applicability", THE JOURNAL OF CLINICAL INVESTIGATION, vol. 126, no. 4, 2016, pages 1236 - 1238, XP055704166, DOI: 10.1172/JCI86799 *
MORA‐RAIMUNDO PATRICIA, LOZANO DANIEL, BENITO MANUEL, MULERO FRANCISCA, MANZANO MIGUEL, VALLET‐REGÍ MARÍA: "Osteoporosis Remission and New Bone Formation with Mesoporous Silica Nanoparticles", ADVANCED SCIENCE, JOHN WILEY & SONS, INC, GERMANY, vol. 8, no. 16, 1 August 2021 (2021-08-01), Germany, XP093258078, ISSN: 2198-3844, DOI: 10.1002/advs.202101107 *

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