WO2021219230A1 - Traitement antiviral - Google Patents

Traitement antiviral Download PDF

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Publication number
WO2021219230A1
WO2021219230A1 PCT/EP2020/062150 EP2020062150W WO2021219230A1 WO 2021219230 A1 WO2021219230 A1 WO 2021219230A1 EP 2020062150 W EP2020062150 W EP 2020062150W WO 2021219230 A1 WO2021219230 A1 WO 2021219230A1
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mirna
ace2
mirnas
mir
inhibitor
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PCT/EP2020/062150
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English (en)
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Paolo Fiorina
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Paolo Fiorina
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Priority to PCT/EP2020/062150 priority Critical patent/WO2021219230A1/fr
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1138—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against receptors or cell surface proteins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/14—Type of nucleic acid interfering N.A.
    • C12N2310/141—MicroRNAs, miRNAs

Definitions

  • the present invention refers to an inhibitor of ACE2 for use in the treatment of a SARS-CoV2 infection, wherein said inhibitor is a miRNA or a mimic thereof or a variant thereof.
  • the present invention also relates to miRNA or a mimic thereof or a variant thereof for medical use and to pharmaceutical compositions thereof.
  • SARS-CoV-2 A newly emerging pathogenic severe respiratory infectious disease has emerged in Wuhan China and has triggered severe pneumonia and acute lung injury.
  • the main compartment of the coronavirus structure is the presence of the transmembrane spike glycoprotein S which promote their entry to the host cells.
  • the distal SI subunit is mainly responsible for its attachment to the host cell receptor as it comprises two receptors membrane domains; domain A (namely S A ) and domain B (namely S B ), the latter has been identified as the main target domain which interacts directly with the angiotensin-converting enzyme 2 (ACE2), established as the main functional receptor for SARS-CoV-2, enabling its efficient entry into susceptible cells [6, 7, 8] ACE2 seems to procure a protective role in acute lung failure as demonstrated in ACE2 KO mice that are protected from SARS-CoV infection; ACE2 downregulation might represent a potential therapy for SARS-CoV2 lethal infection [9, 10] microRNAs (miRNA) are evolving as the main mediators of post-transcriptional control of several proteins encoding genes regulating cell behavior, inflammation and fibrosis [11, 12] Particularly, the specific miRNA network controlling ACE2 during acute lung injury and fibrosis has not been determined yet.
  • ACE2 angiotensin-converting enzyme 2
  • the present invention identifies miRNA-based therapy in the prevention and/or treatment of SARS-COV-2 (or COVTD-19) associated pneumonia.
  • the therapy is in a aerosolized form.
  • COVTD-19 is responsible for a severe acute respiratory syndrome and it has been demonstrated to interact directly with the angiotensin-converting enzyme 2 (ACE2), identified as its main functional receptor, which enables COVID-19 to enter cells.
  • ACE2 angiotensin-converting enzyme 2
  • inventor have now successfully delineated the miRNA network that directly targets ACE2 based on miRnomic profiling of human lung combined with in silico methods for miRNAs- target predictions.
  • the newly identified miRNA targeting allowed to generate miRNA-mimics that modulate ACE2 expression.
  • Administration of the newly identified miRNA-based therapy by decreasing ACE2 expression in lung, attenuate SARS-CoV-2 infection and acute lung injury.
  • the therapy is by aerosol.
  • Inventor are here demonstrating that a newly identified miRNA-based therapy decreases ACE2 expression and mitigate the severity SARS-CoV-2-associated infection (Fig. 1).
  • the invention identifies novel and safe miRNA-based therapeutic approach intended to be formulated as aerosol and delivered through inhalation reducing thus any systemic exposure thereby eliminating any inadvertent possible toxicity and off-target effects.
  • the invention provides an inhibitor of ACE2 for use in the treatment of a SARS- CoV2 infection, wherein said inhibitor is a miRNA or a mimic thereof or a variant thereof.
  • said miRNA or a mimic thereof or a variant thereof comprises any one of SEQ ID No. 1 to SEQ ID No. 62.
  • said miRNA or a mimic thereof or a variant thereof has 70 %, 75 %, 80 %, 85 %, 90 %, 95 %, 99% or 100 % identity with any one of SEQ ID No. 1 to SEQ ID No. 62.
  • the invention also provides a combination of said miRNA or a mimic thereof or a variant thereof.
  • a combination of said miRNA or a mimic thereof or a variant thereof For instance at least one, two, three, four, five or more miRNA can be used, for instance up to 10, 15, 20, 25, 30 or more.
  • said inhibitor is provided within a delivery vehicle, optionally wherein the delivery vehicle is selected from a viral vector, microspheres, liposomes, colloidal gold particles, lipopolysaccharides, polypeptides, polysaccharides, or pegylation of viral vehicles.
  • the delivery vehicle is selected from a viral vector, microspheres, liposomes, colloidal gold particles, lipopolysaccharides, polypeptides, polysaccharides, or pegylation of viral vehicles.
  • the invention also provides a miRNA or a mimic thereof comprising any one of SEQ ID No. 1 to 62 for use in therapy, preferably for use in the treatment of a SARS-CoV2 infection.
  • the invention also provides a pharmaceutical composition comprising at least one inhibitor or miRNA as defined above, and pharmaceutically acceptable excipients and/or diluents for use in the treatment and/or prevention of a SARS-CoV2 infection.
  • the pharmaceutical composition further comprises a further therapeutic agent.
  • the further therapeutic agent is an antiviral agent, an ACE inhibitor or an anti inflammatory agent.
  • the ACE inhibitor is an inhibitor of ACE2.
  • Any known antiviral agent, an ACE inhibitor or an anti-inflammatory agent known in the art may be used.
  • the further therapeutic agent is selected from the group consisting of: remdesivir, lisinopril, captopril, benazepril , enalapril, fosinopril, lisinopril, moexipril, perindopril, quinapril, bethamethasone, prednisone, prednisolone , triamcinolone, methylprednisolone, and dexamethasone.
  • the pharmaceutical composition is in the form of a composition for aerosol.
  • the invention further provides a method for the treatment and/or prevention of a S ARS-CoV2 infection in a subject comprising administering the inhibitor or miRNA as defined above or the pharmaceutical composition as defined above to said subject.
  • FIG. 1 miRNA profiling in adult human lung revealed a set of miRNAs predicted to target directly ACE2.
  • A Heatmap delineating a set of 109 miRNAs predicted to target ACE2 ranked by miTG score or prediction score according to Diana-microT-CDS;
  • B Heatmap showing the common miRNAs that were found in Diana-microT-CDS database and TargetScan which are predicted to target ACE2, they are ranked by a scoring provided by TargetScan V7.1 which is calculated as cumulative weighted context++ scores of the miRNAs conserved sites;
  • C Heatmap showing the common miRNAs that were found in the 3 databases Diana-microT- CDS database, TargetScan and miRWalk and ranked following miRWalk scoring determined on the basis of the interactions of miRNA binding sites within the 3’UTR of A( ⁇ 2.
  • FIG. 3 miRNA profiling in murine lung revealed a set of miRNAs predicted to target directly ACE2.
  • A miRNAs network targeting murine ACE2 as determined by several in silico analysis methods for miRNAs-target prediction (Mouse Genome Informatics-MGI, Diana-microT-CDS, TargetScan) showing a total of 177 miRNAs predicted to directly target murine ACE2 gene, the top 5 miRNAs that possess the highest scores were highlighted (mmu- miR-19a-5p, mmu-miR-325-3p, mmu-miR-26b-5p, mmu-miR-26a-5p and mmu-miR-219b- 5p).
  • the selected sets of 5 human and 5 murine relevant miRNAs predicted to target ACE2 gene for an in vitro validation study the first 3 miRNAS in every table possess the highest prediction score as determined by bioinformatic algorithm database (has-miR-3908, has-miR-4773, has- miR-4520-2-3p, and mmu-miR-19a-5p, mmu-miR-6938-3p, mmu-miR-325-3p) and the 2 last miRNAs shown in each table were confirmed in the literature to target ACE2 (hsa-miR-200c- 3p, has-miR-125b-2-3p, and mmu-miR-200c-3p, mmu-miR-125b-2-3p).
  • ACE2-expressing A549 human alveolar epithelial cells and MLE12 murine lung epithelial cells were transfected with the miRNAs mimics shown in (A) and the efficacy of their transfection was confirmed by the detection of FAM labeled miRNA mimic in treated A459 and MLE12 cell lines by fluorescent microscopy, the image is representative of A549 cell line post-transfection with miRNA mimic hsa-miR-3908.
  • C The expression of ACE2 protein was shown as well as the colocalization of ACE2 with FAM reporter of the miRNA mimic as assessed by confocal imaging.
  • RNA comprises "U” (uracil) rather than “T” (thymine) and that depending upon the use of a particular molecule (e.g., vector, delivery etc.) thymidine can be replace with uracil and vice-a- versa in the sequences set forth herein.
  • the disclosure generally provides methods and composition for treating viral infections and more specifically SARS-COV-2 (or COVTD-19) viral infection.
  • the methods and compositions comprise miRNA molecules alone, combined or with other agents that inhibit viral proteins that silence natural anti-viral mechanisms. miRNA and miRNA mimetics
  • MicroRNAs are an abundant class of short endogenous/natural RNAs that act as post-transcriptional regulators of gene expression.
  • MicroRNAs are small RNAs (- 22 nt) that regulate eukaryotic gene expression by binding to specific messenger RNA transcripts, causing the mRNAs to be degraded or causing their translation to be repressed. They are processed from longer (ca 70-80 nt) hairpin-like precursors termed pre-miRNAs by the RNAse III enzyme Dicer.
  • MicroRNAs assemble in ribonucleoprotein complexes termed miRNPs and recognize their target sites by antisense complementarity thereby mediating down- regulation of their target genes.
  • miRNA is used according to its ordinary and plain meaning and refers to a microRNA molecule found in eukaryotes that is involved in RNA-based gene regulation (see, e.g., Carrington et al . , 2003, which is hereby incorporated by reference) .
  • the term can be used to refer to the single-stranded RNA molecule processed from a precursor or in certain instances the precursor itself.
  • miRNA encompasses single-stranded RNA molecules which regulate gene expression.
  • miRNA molecules may be between 10 and 50 nucleotides in length, preferably 15- 40, more preferably 16- 30 and even more preferably 17-25 nucleotides in length. Typically, miRNA molecules may be between 19 and 26 nucleotides in length. MicroRNA molecules are generally 21 to 22 nucleotides in length, though lengths of 19 and up to 23 nucleotides have been reported.
  • the miRNAs are each processed from a longer precursor RNA molecule ("precursor miRNA" or "pre-miRNA”) . Precursor miRNAs are transcribed from non-protein- encoding genes.
  • the precursor miRNAs have two regions of complementarity that enables them to form a stem-loop- or fold-back-like structure, which is cleaved in animals by a ribonuclease Ill-like nuclease enzyme called Dicer.
  • the processed miRNA is typically a portion of the stem.
  • the processed miRNA (also referred to as "mature miRNA") becomes part of a large complex to down-regulate a particular target gene or its gene product.
  • MicroRNAs are encoded in the genomes of animals, plants and viruses; these genes are transcribed by RNA polymerase II as part of larger fold-back transcripts (primary miRNAs) , which are processed in the nucleus by Drosha family members to form short stem-loops (pre-miRNAs) , and then exported to the cytoplasm for processing by a Dicer family member to form the mature miRNA.
  • primary miRNAs RNA polymerase II
  • pre-miRNAs short stem-loops
  • animal miRNAs include those that imperfectly base pair with the target, which halts translation (Olsen et al . , 1999; Seggerson et al . , 2002).
  • micro RNA or “miR”, or “miRNA” or the equivalent, means short RNA sequences, which may be between about 15 and 30 nucleotides long (or longer or shorter, preferably 21-23 nucleotides in length) and which may be non-coding.
  • Micro RNAs regulate gene-expression post-transcriptionally by inhibiting mRNA translation.
  • Micro-RNAs are produced as ⁇ 70-nt hairpin RNA precursors and processed to mature miRs by RNase III nucleases Drosha and Dicer. For historical reasons some of the first discovered miRs (inC. elegans) are called “let” (for "lethal”), e.g. miR-let-7a.
  • a miR shall refer to the sequences of the mature miR as well as of any precursors thereof.
  • Host cell miRNA modulating compounds may replicate or mimic the sequence of a host cell miRNA molecule— such compounds are referred to hereinafter as "mimic" miRNA molecules.
  • Mimic miRNA molecules may be exploited as a means of increasing or upregulating the expression, activity and/or function of a particular host cell miRNA.
  • the cell may be contacted or transfected with an miRNA molecule which mimics a host cell miRNA to be up-regulated or over- expressed. In this way, the normal miRNA expression profile of the host cell is supplemented with the mimic miRNA molecule.
  • the mimic miRNA molecules comprise nucleic acid (DNA or RNA) and may themselves be miRNA molecules.
  • miR of the present invention are at least 75, 80, 85, 90, 95, 98, 99 or 100% identical to SEQ ID Nos 1 to 62 provided herein.
  • the general term miR includes all members of the miR family that share at least part of a mature miR sequence.
  • the miR comprises at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 232, 24, 25, 50 nucleotides (including all ranges and integers there between) segment of miR that is at least 75, 80, 85, 90, 95, 98, 99 or 100% identical to SEQ ID NOs: 1 to 62.
  • the miR, miR mimetics or miR variants or miR oligonucleotides for use in accordance with the invention may comprise a variety of sequence and structural modifications, depending on the use and function of the oligonucleotide, as will be described further below.
  • sequence and structural modifications described herein are exemplary only, and the scope of the present invention should not be limited by reference to those modifications, but rather additional modifications known to those skilled in the art may also be employed provided the oligonucleotide retains the desired function or activity.
  • the miR sequence may be modified by the addition of one or more phosphorothioate (for example phosphoromonothioate or phosphorodithioate) linkages between residues in the sequence, or the inclusion of one or morpholine rings into the backbone.
  • phosphorothioate for example phosphoromonothioate or phosphorodithioate
  • Alternative non-phosphate linkages between residues include phosphonate, hydroxlamine, hydroxylhydrazinyl, amide and carbamate linkages, methylphosphonates, phosphorothiolates, phosphoramidates or boron derivatives.
  • the nucleotide residues present in the oligonucleotide may be naturally occurring nucleotides or may be modified nucleotides.
  • Suitable modified nucleotides include 2'-0-methyl nucleotides, 2'-0-flouro nucleotides, 2'-0-methoxyethyl nucleotides, universal nucleobases such as 5-nitro-indole; LNA, UNA, PNA and INA nucleobases, 2'-deoxy-2'-fluoro-arabinonucleic acid (FANA) and arabinonucleic acid (ANA).
  • the ribose sugar moiety that occurs naturally in ribonucleosides may be replaced, for example with a hexose sugar, polycyclic heteroalkyl ring, or cyclohexenyl group.
  • the oligonucleotide sequence may be conjugated to one or more suitable chemical moieties at one or both ends.
  • the oligonucleotide may be conjugated to cholesterol via a suitable linkage such as a hydroxyprolinol linkage at the 3' end.
  • the oligonucleotide may be conjugated to N-acetylgalactosamine (GalNAc).
  • modifications of interest include those that increase the affinity of the oligonucleotide for complementary sequences, i.e. increase the melting temperature of the oligonucleotide base paired to a complementary sequence, or increase the biostability of the oligonucleotide.
  • modifications include 2'-0-flouro, 2'-0-methyl, 2'-0-methoxyethyl groups.
  • LNA, UNA, PNA and INA monomers are also typically employed.
  • affinity increasing modifications are present. If the oligonucleotide is less than 12 or 10 nucleobases in length, it may be composed entirely of affinity increasing units, e.g. LNA monomers, UNA monomers or 2'-0-methyl RNA nucleobases.
  • the fraction of monomers in an oligonucleotide modified at either the base or sugar relatively to the monomers not modified at either the base or sugar may be less than 99%, less than 95%, less than 90%, less than 85%, less than 75%, less than 70%, less than 65%, less than 60%, less than 50%, less than 45%, less than 40%, less than 35%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, less than 5%, less than 1%, more than 99%, more than 95%, more than 90%, more than 85%, more than 75%, more than 70%, more than 65%, more than 60%, more than 50%, more than 45%, more than 40%, more than 35%, more than 30%, more than 25%, more than 20%, more than 15%, more than 10%, and more than 5% or more than 1 %.
  • Lipids and/or peptides may also be conjugated to the oligonucleotides. Such conjugation may both improve bioavailability and prevent the oligonucleotide from activating RNase H and/or recruiting the RNAi machinery. Conjugation of larger bulkier moieties is typically done at the central part of the oligonucleotide, e.g. at any of the most central 5 monomers. In yet another embodiment, the moiety may be conjugated at the 5'end or the 3'end of the oligonucleotide.
  • One exemplary hydrophobic moiety is a cholesterol moiety that may be conjugated to the oligonucleotide preventing the oligonucleotide from recruiting the RNAi machinery and improving bioavailability of the oligonucleotide.
  • RNAi machinery Different modifications may be placed at different positions within the oligonucleotide to prevent the oligonucleotide from activating RNase H and/or being capable of recruiting the RNAi machinery.
  • phosphorothioate internucleotide linkages may connect the monomers in an oligonucleotide to improve the biostability of the oligonucleotide. All linkages of the oligonucleotide may be phosphorothioate linkages. In another embodiment, the fraction of phosphorothioate linkages may be less than 95%, less than 90%, less than 85%, less than 80%, less than 75%, less than 70%, less than 65%, less than 60%, less than 50%, more than 95%, more than 90%, more than 85%, more than 80%, more than 75%, more than 70%, more than 65%, more than 60% and more than 50%.
  • the oligonucleotide may not comprise any RNA nucleobases. This may assist in preventing the oligonucleotide from being capable of recruiting the RNAi machinery increasing biostability of the oligonucleotide.
  • the oligonucleotide may consist of LNA and DNA nucleobases and these may be connected by phosphorothioate linkages as outlined above.
  • the oligonucleotide does not comprise any DNA nucleobases.
  • the oligonucleotide does not comprise any morpholino and/or LNA nucleobases.
  • the oligonucleotide may comprise a mix of DNA nucleobases and RNA nucleobases to prevent the oligonucleotide from activating RNase H and prevent the oligonucleotide from recruiting the RNAi machinery.
  • DNA and RNA nucleobases may be alternated along the length of the oligonucleotide, or alternatively one or more DNA nucleobases may be located adjacent one another and one or more RNA nucleobases may be located adjacent one another.
  • the oligonucleotide comprises a mix of LNA monomers and 2'-0-methyl RNA nucleobases.
  • LNA and 2'-0-methyl RNA nucleobases may be alternated along the length of the oligonucleotide, or alternatively one or more LNA nucleobases may be located adjacent one another and one or more 2'-0-methyl RNA nucleobases may be located adjacent one another.
  • the number of nucleobases present in an oligonucleotide that increase the affinity of the oligonucleotide for complementary sequences is at least 1 , at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, or at least 22 nucleobases.
  • the number of nucleobases present in a oligonucleotide that increase the affinity of the oligonucleotide for complementary sequences is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, and 22 nucleobases.
  • Synthetic miRNA mimics can assume the regulatory role of natural miRNAs. Like siRNA, double stranded RNA oligonucleotides with minimal chemical modifications are suitable miRNA replacements in vitro and in vivo. Since the synthetic oligonucleotides need to be incorporated into the RNAi machinery, phosphorothioate backbone modifications as well as 2'- alkylated nucleotides are tolerated only up to a small percentage without considerable loss of activity. The lack of making use of the medicinal chemistry toolbox to a higher degree comes at the expense of enzymatic stability and the pharmacokinetic properties. Unformulated dsRNA is degraded in biological fluids within minutes, and thus delivery systems seem to be a prerequisite for successful development of therapeutic miRNA replacements.
  • RNA oligonucleotides Borrowing from siRNA technology, the currently prevailing approaches in preclinical development rely primarily of optimized liposomal formulation (SNALPs, stable nucleic acid lipid particles), viral particles, and cationic polymers such polyethylene imine (PEI).
  • SNALPs stable nucleic acid lipid particles
  • PEI polyethylene imine
  • SNALPs stable nucleic acid lipid particles
  • PEI polyethylene imine
  • These advanced formulations successfully shield RNA oligonucleotides from degrading enzymes, and can deliver their cargo to organs such as liver and kidney and form part of the present invention.
  • the standard design consists of two RNA oligonucleotides with one-two terminal phosphorothioate linkages and several, up to 50 % 2'-methylated nucleosides.
  • the passenger strand which tolerates a higher degree of derivatization, has usually a higher number of these modifications.
  • the sequence of miRNA mimic may be optimized in that it contains mainly a miRNA guide strand which corresponds exactly to one retrieved from the annotation in miRbase and an passenger RNA which consist of 2 LNA-enhanced RNA strands.
  • the mimic may consists of double stranded-RNA of 22 -nt RNAs, generally bound with an argonaute protein to form a silencing complex which is directed by the miRNA guide to the transcript thereby a pairing with the miRNAs seed sites and the complementary sites within the 3’UTR of a target mRNA.
  • the same pharmaceutical formulations encompassing liposomes, polymeric nanoparticles, and viral systems, can be applied to increase stability and enhance the pharmacokinetic behavior of miRNA oligonucleotides.
  • Liposomes mixture of lipids with cationic head groups and helper lipids, including some with polyethylene glycol chains for masking of the surface charge may be used.
  • Polyanionic nucleic acids are electrostatically complexed to the cationic lipid, yielding lipoplexes.
  • lipoplexes include the delivery of miRs with a mixture of DOTMA, cholesterol and a PEG lipid, pre-miR with DDAB, cholesterol and PEG lipids, and the use of solid lipid nanoparticles consisting of DDAB, cholesterol and other components.
  • grafting a targeting molecule on the surface of lipoplexes may constitute a viable strategy for achieving accumulation in certain tissues.
  • Maleimide tethers may be used on lipoplexes made up from protamine, DOTAP, cholesterol and PEG-DPSE.
  • Neutral lipid delivery systems may also be used.
  • the cationic polymer polyethylene imine (PEI) may be used.
  • PEI can also be used as carrier system for targeted delivery by attaching specific ligands to the polymer.
  • the rabies peptide RVG was attached to PEI for transport to and across the blood-brain barrier.
  • Poly(lactic-co-glycolic acid) may also be used.
  • PLGA needs to be coated or functionalized for efficient oligonucleotide delivery, but affords long-term dissociation from the carrier for a prolonged effect.
  • Coating with cationic peptides nona-arginine or penetratin may be used.
  • oligonucleotide cargo is noncovalently entrapped in the silica matrix, and dissociates upon hydrolysis of the matrix.
  • Conjugation of lipids or receptor-binding molecules directly to the nucleic acid is a promising way to increase cellular uptake of miRNAs.
  • By attaching cholesterol to the 3 '-end of the passenger strand an accumulation in target tissue can be achieved.
  • the asialoglycoprotein receptor ligand N-acetylgalactosamine may also be used.
  • Circulating miRNAs are found in body fluids (plasma, saliva, etc.) and are exchanged between cells despite the abundance of nucleases throughout the body. Natural shielding of endogenous miRNAs is afforded through extracellular vesicles, called exosomes. They are small membrane vesicles (up to 100 nm), and are produced by many cell types, including epithelial, dendritic, and immune cells. Thus exosomes can be used to encapsulate and deliver synthetic or endogenously expressed miRNAs in vivo.
  • the oligonucleotide cargo can be introduced by transfection of corresponding plasmid into exosome-producing cells, or synthetic oligonucleotides can be inferred through electroporation of the mature exosomes.
  • transfection of exosome- producing HEK293 cells with synthetic let-7 may be employed to produce miRNA containing exosomes.
  • Exosomes are similar to liposomes in terms of consisting of bilayered phospholipids, but the biogenesis of exosomes ensures their biocompatibility and low toxicity. It also significantly complicates pharmaceutical development, production and safety profiling (immunogenicity, and potential biological impurities).
  • Bacteriophages may also be used to develop virus-like particles for oligonucleotide and drug delivery.
  • compositions comprising any of the nucleic acid compounds described above in association with a pharmaceutically acceptable excipient, carrier or diluent. Such compositions may find application in, for example, the treatment of viral infections and/or diseases and/or conditions caused or contributed to by, viruses.
  • the pharmaceutical compositions provided by the disclosure are formulated as sterile pharmaceutical compositions.
  • Suitable excipients, carriers or diluents may include, for example, water, saline, phosphate buffered saline, dextrose, glycerol, ethanol, ion exchangers, alumina, aluminium stearate, lecithin, serum proteins, such as serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water salts or electrolytes, such as protamine sulphate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycon, sodium carboxymethylcellulose, polyacrylates , waxes, polyethylene- polypropylene- block polymers, polyethylene glycol and wool fat and the like, or combinations thereof.
  • Said pharmaceutical formulation may be formulated, for example, in a form suitable for aerosol administration.
  • the invention is also directed to the formulations comprising the miRNA of the invention in the form of powders for inhalation.
  • the administration of pharmacologically active ingredients by inhalation to the airways is a widely used technique especially for the treatment of reversible airway inflammation.
  • the technique is also used for the administration of active agents having systemic action, which are absorbed via the lungs, into the bloodstream.
  • Some of the most widely used systems for the administration of drugs to the airways are the dry powder inhalers (DPIs).
  • Drugs intended for inhalation as dry powders by means of DPIs should be used in the form of particles of few microns ([mu]m) particle size.
  • Micronised particles generally considered "respirable” are those with a particle size comprised from 0.5 to 10 micron, preferably 0.5 to 5 micron, as they are able of penetrating into the lower airways, i.e. the bronchiolar and alveolar sites, which are the site of action for the pulmonary drugs and where absorption takes place for the systemic drugs. Larger particles are mostly deposited in the oropharyngeal cavity so they cannot reach said sites, whereas the smaller ones are exhaled.
  • the desirable particle sizes are generally achieved by grinding or so-called micronisation of the active agent.
  • several documents deal with the physico-chemical characteristics of micronised active ingredients for inhalation in particular in terms of particle size (US 2004002510, WO 03/90715, WO 03/24396, WO 02/85326, WO 98/52544, EP 680752, WO 98/17676 and WO 95/01324 incorporated by reference).
  • Powders for inhalation may be formulated by mixing the micronised drug with a carrier (generally a physiologically acceptable material, commonly lactose or mannitol, preferably [alpha] -lactose monohydrate) consisting of coarser particles.
  • a carrier generally a physiologically acceptable material, commonly lactose or mannitol, preferably [alpha] -lactose monohydrate
  • subject is intended to encompass a singular "subject” and plural “subjects” and includes, but is not limited to humans; primates such as apes, monkeys, orangutans, and chimpanzees; canids such as dogs and wolves; felids such as cats, lions, and tigers; equids such as horses, donkeys, and zebras; food animals such as cows, pigs, and sheep; ungulates such as deer and giraffes; rodents such as mice, rats, hamsters and guinea pigs; and bears .
  • inhibiting includes any measurable decrease or complete inhibition to achieve a desired result. For example, there may be a decrease of 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or more, or any range derivable therein, reduction of activity compared to normal.
  • a subject following administering of a miR of the invention, a subject may experience a reduction in severity or duration of one or more viral infection, preferably COVTD-19 symptoms.
  • the term "effective amount”, as used herein, refers to the amount that is safe and sufficient to treat, lesson the likelihood of, or delay the progress of a viral infection.
  • the effective amount can thus cure or result in amelioration of the symptoms of the viral infection, slow the course of disease progression resulting from viral infection, slow or inhibit a symptom of a viral infection (e.g. flu symptoms), slow or inhibit the establishment of secondary symptoms of a viral infection or inhibit the development of a secondary symptom of a viral infection.
  • the effective amount for the treatment of the viral infection depends on the type of viral infection to be treated, the severity of the symptoms, the subject being treated, the age and general condition of the subject, the mode of administration and so forth. Thus, it is not possible or prudent to specify an exact "effective amount”.
  • the effective amount is a "therapeutically effective amount” for the alleviation of the symptoms of the disease mediated by the viral (e.g., COVTD-19) infection or condition being treated.
  • the effective amount is a "prophylactically effective amount” for prophylaxis of the symptoms of the disease or condition being prevented.
  • compositions of the disclosure can be administered by any means that produces contact of the active agent with the agent's site of action. They can be administered by any conventional means available for use in conjunction with pharmaceuticals, either as individual therapeutic agents or in a combination of therapeutic agents. They can be administered alone, but typically are administered with a pharmaceutical carrier selected on the basis of the chosen route of administration and standard pharmaceutical practice.
  • the compounds can, for example, be administered orally, transmucosally, parenterally (including subcutaneous injections, intravenous, intramuscular, intrasternal injection or infusion techniques) , by inhalation spray, or rectally, in the form of a unit dosage of a pharmaceutical composition containing an effective amount of the compound and conventional non-toxic pharmaceutically- acceptable carriers, adjuvants and vehicles.
  • injectable solutions can be prepared according to methods known in the art wherein the carrier comprises a saline solution, a glucose solution or a solution containing a mixture of saline and glucose. Further description of methods suitable for use in preparing pharmaceutical compositions of the disclosure and of ingredients suitable for use in said compositions is provided in Remington's Pharmaceutical Sciences, 18th edition, edited by A. R.
  • the methods of treatment according to the disclosure ameliorate one or more symptoms in a subject associated with the viral infection.
  • the symptoms associated with the infection can include, but are not limited to, reduction in CD4+ T cell numbers, pain (peripheral neuropathy); fever, cough, and other cold/flu symptoms; night sweats; diarrhea, nausea, and other indigestion symptoms; lymph swelling or other immunological symptoms; weight loss and loss of appetite; Candida in the mouth; secondary bacterial and/or viral infections; elevated liver enzymes; reduction in central nervous system and brain function; depression; overall reduced immunity; AIDS-related complications (ARC) , including, but not limited to, progressive generalization lymphadenia (PGL) , Kaposi's sarcoma, Pneumocystis carinii pneumonia, cataplectic purpura thrombocytopenica; neurological syndromes, including, but not limited to, dementia complications, encephalopathy, disseminated sclerosis ortropical paraplegia; as well as anti influenza antibody-positive and influenza-positive syndrome including that in silent
  • Treatment and “treating” as used herein refer to administration or application of a therapeutic agent to a subject or performance of a procedure or modality on a subject for the purpose of obtaining a therapeutic benefit of a disease or health- related condition.
  • a subject or patient e.g., a mammal, such as a human
  • a treatment comprising administration of a compound or composition of the disclosure.
  • therapeutic benefit or “therapeutically effective” as used throughout this application refers to anything that promotes or enhances the well-being of the subject with respect to the medical treatment of a condition.
  • a therapeutically effective amount of a miRNA of the disclosure may be an amount sufficient to treat or prevent the COVTD-19 infection.
  • compositions, carriers, diluents and reagents are used interchangeably and represent that the materials are capable of administration to or upon a mammal without the production of undesirable physiological effects such as nausea, dizziness, gastric upset and the like.
  • Each carrier must also be “acceptable” in the sense of being compatible with the other ingredients of the formulation.
  • a pharmaceutically acceptable carrier will not promote the raising of an immune response to an agent with which it is admixed, unless so desired.
  • the preparation of a pharmacological composition that contains active ingredients dissolved or dispersed therein is well understood in the art and need not be limited based on formulation.
  • the pharmaceutical formulation contains a compound of the disclosure in combination with one or more pharmaceutically acceptable ingredients.
  • the carrier can be in the form of a solid, semi-solid or liquid diluent, cream or a capsule.
  • Such compositions are prepared as injectable either as liquid solutions or suspensions, however, solid forms suitable for solution, or suspensions, in liquid prior to use can also be prepared.
  • the preparation can also be emulsified or presented as a liposome composition.
  • the active ingredient can be mixed with excipients which are pharmaceutically acceptable and compatible with the active ingredient and in amounts suitable for use in the therapeutic methods described herein. Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol or the like and combinations thereof.
  • compositions can contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents and the like which enhance the effectiveness of the active ingredient.
  • auxiliary substances such as wetting or emulsifying agents, pH buffering agents and the like which enhance the effectiveness of the active ingredient.
  • the therapeutic composition of the disclosure can include pharmaceutically acceptable salts of the components therein.
  • Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the polypeptide) that are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, tartaric, mandelic and the like.
  • Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine, procaine and the like.
  • Physiologically tolerable carriers are well known in the art.
  • Exemplary liquid carriers are sterile aqueous solutions that contain no materials in addition to the active ingredients and water, or contain a buffer such as sodium phosphate at physiological pH value, physiological saline or both, such as phosphate-buffered saline.
  • aqueous carriers can contain more than one buffer salt, as well as salts such as sodium and potassium chlorides, dextrose, polyethylene glycol and other solutes.
  • Liquid compositions can also contain liquid phases in addition to and to the exclusion of water.
  • glycerin examples include glycerin, vegetable oils such as cottonseed oil, and water-oil emulsions.
  • amount of an active agent used in the disclosure that will be effective in the treatment of a particular disorder or condition will depend on the nature of the disorder or condition and can be determined by standard clinical techniques.
  • pharmaceutically acceptable carrier or diluent means a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, involved in carrying or transporting the subject agents from one organ, or portion of the body, to another organ, or portion of the body.
  • 3 miRNAs predictions databases were employed: Diana-micoT-CDS, TargetScan and miRwalk with the criterion of ranking of the miRNAs based on their primary prediction score which following the algorithm used is mainly built based on the number of binding sites to the 3’UTR of the mRNA, the site type, the distance from the 3’UTR end, high AU content also the presence of 2 miRNA features TA and SPS (TA) lower target-site abundance within the transcriptome and stronger predicted seed-pairing stability (SPS)).
  • TA TA
  • SPS seed-pairing stability
  • Droplet digital PCR was performed using a Bio-Rad QX100 Droplet Digital PCR system (Bio-Rad, Hercules, CA). Reactions were performed in appropriate volumes using 10 m ⁇ ddPCR 2x Master Mix, 1 m ⁇ 20X Primer and TaqMan Probe Mix, 5 m ⁇ Nuclease free water, and 4 m ⁇ reverse transcriptase product. Tables 3, 4 and 5 contains the list and sequences of all primers used. Sample was loaded into a droplet generator cartridge. 20 m ⁇ of sample preparation was then transferred into the cartridge’s middle wells, taking caution to avoid bubbles. 70 m ⁇ of oil were added into lower wells, and the cartridge containing the samples was placed into the droplet generator to generate individual droplets.
  • ddPCR Droplet digital PCR
  • Human epithelial cell line A549 and murine epithelial cell line MLE12 were purchased from the American Type Culture Collection (respectively ATCC® CCL185TM (A549) and ATCC ® CRF-2110TM(MFE12); Manassas, VA, USA) .
  • A549 Cells were cultured in Kaighn's Modification of Ham's F-12 Medium (F-12K Medium, ATCC ® 30-2004TM, Manassas, VA, USA) supplemented with 10% heat-inactivated fetal calf serum (Gibco, Thermo Fisher Scientific), 100 units/ml penicillin, 100 pg/ml streptomycin, and 2 mM F-glutamine as recommended by the manufacturer.
  • murine MFE12 cells were cultured in DMEM:F-12 Medium (ATCC ® 30-2006TM) supplemented with 2% heat- inactivated fetal calf serum (Gibco, Thermo Fisher Scientific), 100 units/ml penicillin, 100 pg/ml streptomycin, and 2 mM F-glutamine as recommended by the manufacturer. Cells in logarithmic phase were used for the further analysis.
  • Cell lysates for western blot analysis were obtained in RIPA buffer (50 mmol/1 Tris-HCl, pH 8.0, 1% Triton-x, 0.5% sodium deoxycholate, 0.1% SDS, 150 mmol/1 sodium chloride) with protease inhibitor cocktail (Roche). Extract from cells homogenate for Elisa were prepared as follows: A549 cells and MLE12 cells were homogenized in 500 m ⁇ of chilled IX PBS, after incubation and centrifugation, supernatant were collected and used for Elisa assay as recommended by the manufacturer (ELISA kit ACE2 Human (E-EL-H0281 96T, Elabscience) and ELISA kit ACE2 Murine (MBS24565, MyBioSource).
  • RIPA buffer 50 mmol/1 Tris-HCl, pH 8.0, 1% Triton-x, 0.5% sodium deoxycholate, 0.1% SDS, 150 mmol/1 sodium chloride
  • Extract from cells homogenate for Elisa were prepared as follows: A
  • Table 1 list of miRNA mimics and their sequences Targeted
  • ACE2 protein expression was quantified in extracts from lysates of A549 and MFE12 cell lines using respectively the EFISA kit ACE2 Human (E-EF-H0281 96T, Elabscience) and the EFISA kit ACE2 Murine (MBS24565, MyBioSource) following manufacturer’s instructions. Briefly, all extract from samples and serial standard dilutions prepared from an ACE2 stock standard solution were distributed into the appropriate wells of the Elisa microplate, then antibody cocktail containing the capture and detector ACE2 antibodies was distributed to all the samples and the standard wells. After an incubation followed by 3 washes-decanting cycles with the appropriate wash buffer, a development solution was added to every well and then incubated. After incubation a stop solution was applied to every well and the plate was processed with an Elisa reader MULTISKAN GO (Thermo, Scientific) recording the OD at 450 nm and the protein concentration was calculated accordingly.
  • Elisa reader MULTISKAN GO Therm
  • Protein concentration in A549 and MLE12 cell lysates was measured. Fifteen micrograms of total proteins were electrophoresed on 8-16% gradient SDS-PAGE gels and blotted onto PVDF membrane (Bio-Rad, Hercules, CA, USA). Blots were then stained with Ponceau S.
  • Membranes were blocked for 1 h in 5% non-fat dry milk in TBST (Tris [10 mmol/1], NaCl [150mmol/l]), 0.1% Tween-20, 5% non-fat dry milk, pH 7.4 at 25°C) and then incubated for 12 h with a polyclonal rabbit ACE2 antibody ((MBS 150723), MYBioSource, USA) diluted 1:500 or with anti-rabbit GAPDH antibody (218S, Cell Signaling Technology, Danvers, MA) diluted at 1 : 1000 in TBS-5% milk at 4°C, washed four times with TBS-0.1% Tween-20.
  • a polyclonal rabbit ACE2 antibody ((MBS 150723), MYBioSource, USA) diluted 1:500 or with anti-rabbit GAPDH antibody (218S, Cell Signaling Technology, Danvers, MA) diluted at 1 : 1000 in TBS-5% milk at 4°C, washed four times with TBS-0.1% Tween-20
  • Detection was performed using an anti -rabbit IgG HRP linked-secondary antibody diluted 1 : 2000 (7074S, Cell Signaling Technology, Danvers, MA) in TBS-5% milk for 1 h, and finally washed with TBS- 0.1% Tween-20.
  • the resulting bands were visualized using Clarity Max western ECL substrate (Bio-Rad, Hercules, CA, USA) on a Uvitec Mini HD9 (Cleaver Scientific, Rugby, Warwickshire, UK) image documentation system.
  • Clarity Max western ECL substrate Bio-Rad, Hercules, CA, USA
  • Uvitec Mini HD9 Cleaver Scientific, Rugby, Warwickshire, UK
  • Flow cytometry was performed to analyze ACE2 surface expression on miRNA mimic-treated A549- cells.
  • Rabbit polyclonal PE-conjugated ACE2 antibody (orb486003, Biorbyt, FCC USA) was used to stain the cells. Background staining was determined using PE labelled Rabbit IgG antibody (orb490756, Biorbyt LLC, USA), nonreactive isotype-matched control antibody with gates positioned to exclude 99% of non-reactive cells.
  • Cells were subjected to FACS analysis and were run on a FACSCelestaTM (Becton Dickinson). Data were analyzed using FlowJo software version 10 (Treestar).
  • A549 and MLE12 cells were collected and washed in 1 ml of BD staining buffer (BD Biosciences). Cells were staining with PE-conjugated anti-ACE2 monoclonal antibody (Santa Cruz Biotechnology, sc-390851). After a fixation and washing step, cells were stained with DAPI (1:5000) (DAPI 1.5mM solution in H2O [Molecular Probes, Thermo Fisher, Waltham, MA]), then mounted with FluorSave (FluorSaveTM Reagent [Calbiochem, Merck KGaA, Germany]) mounting media into SuperfrostTM Plus glass slides and imaged using a confocal microscope (Leica TCS SP2 Laser Scanning Confocal).
  • angiotensin-Aldosterone System the Angiotensin converting enzyme 2 (ACE2) as illustrated in Figure 1.
  • ACE2 Angiotensin converting enzyme 2
  • the set of 109 miRNAs predicted to target ACE2 were ranked by miTG score or prediction score according to Diana-microT-CDS (Fig. 2A), or as ranked by TargetScan which provided prediction scoring as cumulative weighted context++ scores of the miRNAs conserved sites (Fig. 2B).
  • the Ace2 targeting miRNA were also ranked by using the miRWalk algorithm where the scoring was ranked following interactions of miRNA binding sites within the 3’UTR of ACE2 (Fig. 2C).
  • Inventor’s digital-PCR analysis confirmed the expression of the selected miRNAs at different levels, particularly hsa-miR-4270, hsa-miR- 216b, hsa-miR-3529-3p and hsa-miR-362-5p appeared to be highly expressed in human lung (Fig. 2D).
  • miRNA profiling in murine lung revealed a set of miRNAs predicted to target directly ACE2.
  • inventor used several in silico analysis methods for miRNAs-target prediction (Mouse Genome Informatics- MGI, Diana-microT-CDS, TargetScan).
  • a total of 177 miRNAs predicted to directly target ACE2 were identified (Fig. 3A), with 5 top miRNAs strongly being predicted to target ACE2 (mmu-miR-19a-5p, mmu-miR-325-3p, mmu-miR-26b-5p, mmu-miR-26a-5p and mmu-miR- 219b-5p).
  • all miRNA discovered are ranked by their relative expression (X-axis) and by prediction score as calculated by Diana-microT-CDS algorithm (Y- axis) (Fig. 3B).
  • inventor performed a digital-PCR analysis screening for the expression in murine pulmonary tissues of these top 5 miRNAs candidates predicted to have ACE2 as target (Fig. 3C and 3D).
  • our digital-PCR analysis confirmed the expression of the selected miRNAs in murine lung.
  • miRNA targeting decreases ACE 2 in human and murine lung cell lines.
  • a miRNA mimic is of ⁇ 22-nt RNAs which consist of a guide RNA strand and its complementary passenger RNA strand that function to mediate post- translational gene repression, Fig. 4A).
  • Inventor tested one set of miRNAs not previously known to modify ACE2 expression has-miR-3908, has-miR-4773, has-miR-4520-2-3p, mmu- miR-19a-5p, mmu-miR-6938-3p, mmu-miR-325-3p
  • one set of miRNAs known directly target ACE2 hsa-miR-200c-3p, has-miR-125b-2-3p, mmu-miR-200c-3p, mmu-miR-125b-2- 3p.
  • Tortorici M.A., Walls, A.C., Lang, Y., Wang, C., Li, Z., Koerhuis, D., Boons, G.J., Bosch, B.J., Rey, F.A., de Groot, R. T, and Veesler, D. (2019). Structural basis for human coronavirus attachment to sialic acid receptors. Nat. Struct. Mol. Biol. 26, 481-489.
  • Angiotensin-converting enzyme 2 is a functional receptor for the SARS coronavirus. Nature 426, 450-454. 9. Keiji Kuba, Yumiko Imai, Shuan Rao, et al. A crucial role of angiotensin converting enzyme

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Abstract

La présente invention concerne un inhibiteur de ACE2 destiné à être utilisé dans le traitement d'une infection par le SARS-CoV2, ledit inhibiteur étant un miARN ou un analogue de celui-ci ou un variant de celui-ci. La présente invention concerne également un miARN ou un analogue de celui-ci ou un variant de celui-ci à usage médical et des compositions pharmaceutiques associées.
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