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WO2017157950A1 - Oligonucléotides antisens inductibles par la lumière pour application in vivo - Google Patents

Oligonucléotides antisens inductibles par la lumière pour application in vivo Download PDF

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Publication number
WO2017157950A1
WO2017157950A1 PCT/EP2017/056014 EP2017056014W WO2017157950A1 WO 2017157950 A1 WO2017157950 A1 WO 2017157950A1 EP 2017056014 W EP2017056014 W EP 2017056014W WO 2017157950 A1 WO2017157950 A1 WO 2017157950A1
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Prior art keywords
antisense oligonucleotide
antisense
mir
light
irradiation
Prior art date
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PCT/EP2017/056014
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English (en)
Inventor
Stefanie Dimmeler
Alexander Heckel
Tina LUCAS
Florian SCHÄFER
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Johann Wolfgang Goethe-Universität
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Publication of WO2017157950A1 publication Critical patent/WO2017157950A1/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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K41/00—Medicinal preparations obtained by treating materials with wave energy or particle radiation ; Therapies using these preparations
    • A61K41/0042—Photocleavage of drugs in vivo, e.g. cleavage of photolabile linkers in vivo by UV radiation for releasing the pharmacologically-active agent from the administered agent; photothrombosis or photoocclusion
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
    • 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
    • C12N15/111—General methods applicable to biologically active non-coding nucleic acids
    • 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
    • C12N2310/11—Antisense
    • C12N2310/113—Antisense targeting other non-coding nucleic acids, e.g. antagomirs
    • 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
    • C12N2310/30—Chemical structure
    • C12N2310/31—Chemical structure of the backbone
    • C12N2310/315—Phosphorothioates
    • 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
    • C12N2310/30—Chemical structure
    • C12N2310/33—Chemical structure of the base
    • 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
    • C12N2310/30—Chemical structure
    • C12N2310/35—Nature of the modification
    • C12N2310/351—Conjugate
    • C12N2310/3515—Lipophilic moiety, e.g. cholesterol
    • 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
    • C12N2320/00—Applications; Uses
    • C12N2320/50—Methods for regulating/modulating their activity
    • C12N2320/52—Methods for regulating/modulating their activity modulating the physical stability, e.g. GC-content

Definitions

  • the present application provides antisense oligonucleotides for spatially controlled activation of micro RNA silencing via local irradiation after administration of the molecule.
  • the anti- sense design of the invention uses photo labile protecting groups attached to the oligonucleotides which impair antisense binding to its target.
  • the antisense molecules of the invention are useful for in vivo applications and hence will be useful as therapeutics in medicine to activate micro RNA silencing spatially confined, which will result in reduced antisense toxicity.
  • the invention also provides a light inducible miR-92a antisense oligonucleotide for use in aiding tissue generation and wound healing, as well as pharmaceutical compositions comprising the light inducible miR-92a antisense oligonucleotide of the invention.
  • Antisense oligonucleotides or antimiRs are relatively easily taken up by detoxifying organs such as liver or kidney, but uptake in other organs such as muscle or brain tends to be limited. Local delivery or activation may be necessary to augment the biological functions of antimiRs in the target tissue and reduce systemic toxicity.
  • Several targeting strategies have been experimentally used including the linking of miRs or antimiRs to aptamers, cell type-specific delivery by viral vectors, as well as attempts of local delivery by mechanical tools, e.g. catheters.
  • photoactivatable antimiRs were previously developed by attaching photolabile protecting groups (cages) to the nucleobases which temporarily inhibit duplex formation with the target miR, thereby allowing an excellent on/off behavior upon irradiation.
  • photolabile protecting groups cages
  • This invention further relates to the healing of wounds and to antisense agents and techniques for facilitating repair and healing of tissue in humans and animals, especially, but not exclusively, skin or other epithelial tissue that has been damaged by, for example, wounds resulting from accidental injury, surgical operations, other trauma or ischemia e.g. as it occurs in angi- ogenesis impaired diabetic patients.
  • the invention has particular reference to the healing of wounds in humans and other vertebrates.
  • the healing of wounds in tissue such as skin generally involves, at least in adult humans and other mammals, a process of extra cellular matrix (ECM) biosynthesis, turnover and organization which commonly leads to the production of fibrous, connective tissue scars and consequential loss of normal tissue function.
  • ECM extra cellular matrix
  • wound healing in tissues is generally a reparative process, in contrast to a regenerative process which appears to take place in healing of fetal and embryonic tissue.
  • the outcome of a wound repair process appears to be influenced by a number of different factors, including both intrinsic parameters, e.g. tissue oxygenation, and extrinsic parameters, e.g. wound dressings.
  • intrinsic parameters e.g. tissue oxygenation
  • extrinsic parameters e.g. wound dressings.
  • an antisense oligonucleotide comprising a polynucleotide and at least one photolabile protecting group.
  • the antisense oligonucleotide of the invention in some embodiments further includes a lipophilic moiety.
  • the inventors developed light-activatable antimiRs that efficiently and locally restricted target miR activity in vivo.
  • AntimiRs directed against miR-92a were modified with photolabile protecting groups, so called “cages” and the therapeutic properties of caged antimiR-92a in skin repair was tested.
  • Irradiation (10 min, 385 nm) activated intradermally injected caged anti- miR-92a without significantly affecting miR-92a expression in other organs.
  • Light activation of caged antimiR-92a improved healing in diabetic mice to a similar extent as conventional antimiRs and de-repressed the miR-92a target Sirt-1.
  • the at least one photolabile protecting group is in some embodiments attached to a nucleotide of the polynucleotide, preferably wherein the photolabile protecting group when attached to the antisense oligonucleotide impairs micro RNA (miR)-inhibition of the antisense oligonucleotide.
  • the photo protective group usable in context of the invention is cleavable by irradiation, preferably by a wave length of less than 500nm, more preferably of less than 450nm, even more preferably of 300 to 400 nm, most preferably by 350 to 400nm, most preferably be around 380+/- 5 nm.
  • the applied irradiation will dependent on the caging group used in context of the invention, and how much tissue must be penetrated.
  • caging groups cleavable at the infrared range of light are applied and wherein the wavelength of the used light is between 600nm and 900nm, preferably between 650 and 900nm.
  • Another embodiment pertains to the irradiation of the photo labile protecting groups wherein the exposing step is carried out by "two-photon uncaging" in accordance with known two- photon excitation techniques. See, e.g., U.S. Pat. Nos. 7,049,480; 6,020,591; and 5,034,613.
  • two photons of a suitable wavelength e.g., equal to or greater than 650 or 700 nm
  • the caged oligonucleotide with for example approximately a 100 femtosecond pulse width and an approximately 80 MHz repetition rate, where the photons then double up and remove the caging group.
  • Two photon uncaging can if desired be facilitated through the use of a two-photon sensitizer. This is an important embodiment of the invention, because photons of such wavelengths can be focused more precisely and penetrate tissue more deeply.
  • Suitable photolabile protecting groups for two photon excitation are for example DEACM or /?-dialkylaminonitrobiphenyl (ANBP).
  • Exemplary photolabile protecting groups of the invention may be selected from a caging compound such as orthonitrobenzylic based caging group (l-(4,5-dimethoxy-2-nitrophenyl) diazoethane (DMNPE), [7-(diethylamino)coumarin-4yl]methyl caging group (DEACM), 6- bromo-4-diazomethyl-7-hydroxycoumarin (Bhc-diazo), 2-nitrobenzyl, 4,5 dimethoxy 2- nitrobenzyl, Alpha-carboxy-2-nitrobenzyl, l-(2-nitrophenyl)ethyl, l-(2-nitrophenyl)ethyl ni- troindoline, 4-methoxy 7-nitroindoline, 1-acyl 7-nitroindo lines, l-(2-nitrophenyl)ethyl ethers of 7-hydroxycoumarins, 7-(alkoxy coumarin-4yl)methyl esters, 6,7-(dialkoxy coumarin- 4
  • the photo labile protecting group may be attached at any position in the nucleic acid sequence so long the caging inhibits R A interference. Preferred is the modification of the nu- cleobase of the polynucleotide sequence with photo labile protecting groups.
  • a preferred example provides that the photolabile protecting group is attached to the N 6 of an adenine nucleotide or N 4 of a cytosine nucleotide. These sites are particularly preferred when NPE is used as caging compound. Other possible sites for attaching the photo labile protecting group in the polynucleotide will be dependent on the nature of the caging compound used, as mentioned above.
  • the photo labile protecting group must be attached to the polynucleotide such that the function of the antisense oligonucleotide of the invention as an inhibitor of a targeted micro RNA is impaired in absence of an irradiation signal.
  • the antisense oligonucleotide preferably comprises 1 to 20 photo-labile protecting groups, preferably 2 to 10, more preferably 3 to 8, most preferably about 6. Also the number of protecting groups may vary depending on the length of the polynucleotide sequence, and the photo labile protecting group used.
  • the lipophilic moiety is preferably attached at the 3' end or the 5 ' end of the antisense oligonucleotide of the invention.
  • a lipophilic moiety greatly enhances in vivo applicability of the antisense molecule, however, without impairment of the light inducible function of the antisense oligonucleotide of the invention, which was not obvious.
  • the lipophilic moiety is a fat-soluble moiety capable of intercalating with a phospholipid bi- layer, such as a cell membrane, or delivery vehicle such as a liposome or micelle as disclosed herein.
  • the lipophilic moiety may be conjugated to the hydrocarbon spacer (described below) through an ether or thioether linkage.
  • Exemplary lipophilic moieties include cholesterol and cholesterol derivatives (including cholestenes, cholestanes, and cholestadienes), bile acids (such as cholic acid, deoxycholic acid and dehydrocholic acid), sterols, steroids, or other fat- soluble alcohol or thiol.
  • the lipophilic moiety is cholesterol or a cho- lesterol derivative, or cholic acid or cholic acid derivative as described in U.S. Pat. No. 7,202,227, which is hereby incorporated by reference.
  • Such derivatives include C 1 -C4 alkyl (e.g., methyl) substituted cholesterol or cholic acid structures.
  • the lipophilic moiety is cholesterol or a derivative thereof.
  • the polynucleotide and the lipophilic moiety are separated by a spacer molecule to separate the lipophilic moiety from non- lipophilic, e.g., polar, groups, so as to allow the lipophilic moiety to better interact with, inter alia, membrane phospholipids upon delivery to a patient or upon contact with mammalian cells.
  • the conjugate generally does not have exchangeable protons or other polar groups (e.g., hydroxyl or amide) within the vicinity of the lipophilic moiety.
  • the polynucleotide and the lipophilic moiety are preferably separated by a linker, preferably a hydrocarbon linker.
  • the linker or spacer molecule of the invention may preferably selected from a hydrocarbon linker, such as a linear or cyclic Ci to C 20 , preferably C5 to C I 5, alkyl or alkoxy-alkane, optionally substituted by one or more hetero atoms.
  • a hydrocarbon linker such as a linear or cyclic Ci to C 20 , preferably C5 to C I 5, alkyl or alkoxy-alkane, optionally substituted by one or more hetero atoms.
  • the polynucleotide of the antisense molecule of the invention may have any design known to the skilled artisan for the silencing genes.
  • the polynucleotide may be an antisense oligonucleotide.
  • the antisense oligonucleotide contains at least one chemical modification (e.g. sugar or backbone modification).
  • antisense oligonucleotide may comprise one or more modified nucleotides, such as 2'-0-methyl modifications.
  • antisense oligonucleotides comprise only modified nucleotides, preferably only 2'-0-methyl modifications.
  • antisense oligonucleotides may also comprise one or more phosphorothio- ate linkages resulting in a partial or full phosphorothioate backbone (see below).
  • Antisense oligonucleotides suitable for inhibiting micro RNAs may be about 5 to about 50 nucleotides in length, such as about 10 to about 30 nucleotides in length, or about 20 to about 25 nucleotides in length. In certain embodiments, antisense oligonucleotides are about 8 to about 18 nucleotides in length, and in other embodiments about 12 to about 16 nucleotides in length.
  • the antisense oligonucleotide may comprise a sequence that is at least about 75%, 80%, 85%, 90%), 95%), 96%o, 97%o, 98%, or 99% complementary to a mature miRNA sequence.
  • the antisense oligonucleotides comprise a sequence that is 100% complemen- tary to the mature miRNA sequence.
  • complementary or “base pair” includes or refers only to classic Watson-Crick nucleotide base-pairing.
  • the anti- sense oligonucleotide is an antagomir.
  • An "antagomir” is a single-stranded, optionally chemically-modified, ribonucleotide that is at least partially complementary to a miRNA sequence.
  • the polynucleotide has a sequence designed to mimic a cellular miRNA.
  • the polynucleotide in these embodiments may also comprise one or more modified nucleotides, such as 2'-0-methyl modifications, and one or more phosphorothioate linkages (see below).
  • Polynucleotides suitable for mimicking miRNAs may be about 15 to about 50 nucleotides in length, such as about 18 to about 30 nucleotides in length, or about 20 to about 25 nucleotides in length.
  • the synthetic miRNA may comprise a sequence that is at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to a mature miRNA.
  • the polynucleotide comprises a sequence that is 100% identical to the mature miRNA sequence.
  • the polynucleotide may be composed of predominately ribonucleotide units or predominately deoxyribonucleotide units, and may have one or more chemical modification(s).
  • the polynucleotides may be comprised of one or more "conformationally constrained” or bi- cyclic sugar nucleoside modifications (BSN) that confer enhanced thermal stability to complexes formed between the oligonucleotide containing BSN and their complementary mi- croRNA target strand.
  • BSN bi- cyclic sugar nucleoside modifications
  • the polynucleotide includes, for example, from about 1 to 10 locked nucleic acids.
  • LNAs Locked nucleic acids
  • the polynucleotide may comprise peptide nucleic acids (PNAs), which contain a peptide-based backbone rather than a sugar-phosphate backbone.
  • PNAs peptide nucleic acids
  • the polynucleotide contains one or more sugar modifications and/or one or more backbone modifications.
  • Exemplary sugar modifications include 2' and 4' modifications, such as 2'-0-alkyl (e.g.
  • the antisense oligonucleotide of the invention does not comprise a lipophilic moiety.
  • the polynucleotide is a 2'-0-methoxyethyl gapmer.
  • a "gapmer” contains 2'-0-methoxy ethyl-modified ribonucleotides on both 5' and 3' ends with at least ten deoxyribonucleotides in the center. These "gapmers" are capable of triggering RNase- independent degradation mechanisms of R A targets.
  • antisense and sense sequences may be incorporated into shRNAs or other RNA structures containing stem and loop portions, for example. Such sequences are useful for, among other things, mimicking or targeting miRNA function for treatment or amelioration of cardiac hypertrophy, myocardial infarction, heart failure (e.g., congestive heart failure), vascular damage, and/or pathologic cardiac fibrosis, among others.
  • polynucleotide of the invention may be included in the phosphor- backbone linkage between the individual nucleotides of the polynucleotide.
  • at least one phospho linkage between nucleotides is a phosphorothioate linkage.
  • the polynucleotide of the antisense oligonucleotide of the invention may comprise as many phosphorothioate linkages depending on polynucleotide length. Preferred aspects provide 1 to 20 phosphorothioate linkages, preferably 2 to 10, more preferably 3 to 8, most preferably about 6 in the case of the anti miR-92a construct of the invention.
  • the phosphorothioate linkage(s) is (are) preferably located at the 3' and/or 5' ends of polynucleotide, for example wherein at least the two most 3'- and two most 5 '-located nucleotides have a phosphorothioate linkage. Also the number of phosphorothioate linkage at the 3' and 5' ends may vary depending on the length of the polynucleotide.
  • the present invention provides an antisense oligonucleotide which is an anti-micro RNA (miR)-92a antisense oligonucleotide.
  • the antisense oligonucleotides of the invention are preferably for use in medical applications. Envisioned are to supplement with the present invention any treatment of a patient, which comprises a step of administering the antisense oligonucleotide to the patient, and thereafter locally activating the antisense oligonucleotide at the site of treatment using local irradiation. Therefore, the present invention may be used in any medical treatment method that requires a local activation of an antisense oligonucleotide. Although preferred, the present invention shall not be confined to applications in the treatment of the outer body surface of mammals and humans, such as skin or open wounds. The invention may also be used during surgery or within the body cavity, for example by using probes that allow for a local irradiation of tissue in a patient.
  • the invention nevertheless in one embodiment provides the above antisense oligonucleotide for enhancing tissue regeneration, preferably wherein the antisense oligonucleotide is a miR- 92a antisense oligonucleotide.
  • the antisense molecule in this aspect is for example administered to a patient, and subsequently tissue regeneration is enhanced by local irradiation of the target tissue. The local irradiation is started ideally when it is expected that the target area contains a sufficient amount of the administered antisense oligonucleotide.
  • the compound of the invention Upon irradiation, the compound of the invention becomes active by removing the photo labile protecting group and the antisense molecule can induce its therapeutic action, which preferably in context of the invention is the polynucleotides activity as an inhibitor of miR function.
  • the oligonucleotide for use in enhancing tissue regeneration, for example wound healing comprises an LNA based polynucleotide.
  • the antisense oligonucleotide for use may or may not comprise the lipophilic moiety as mentioned above.
  • a preferred embodiment pertains to the antisense oligonucleotides not comprising a lipophilic moiety.
  • the irradiation is an irradiation with light, preferably light of a wavelength of between 300 and 400nm, preferably 350 to 400nm, more preferably of between 370 and 390 nm, or at a wavelength between 650nm and 900nm, which is particular preferred when a two photon excitation approach is used.
  • the antisense oligonucleotide is preferably for use in wound healing.
  • the antisense oligonucleotide is used to treat a patient with impaired healing capabilities, for example wherein the patient is a diabetes patient.
  • Another aspect of the invention also pertains to a pharmaceutical composition
  • a pharmaceutical composition comprising an antisense oligonucleotide as described herein, together with a pharmaceutically acceptable carrier and/or excipient.
  • the language "pharmaceutically acceptable carrier” is intended to include any and all solvents, solubilizers, fillers, stabilizers, binders, absorbents, bases, buffering agents, lubricants, controlled release vehicles, nanoparticles, liposomes, diluents, emulsifying agents, humectants, lubricants, dispersion media, coatings, antibacterial or antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration.
  • the use of such media and agents for pharmaceutically active substances is well-known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the compositions is contemplated. Supplementary agents can also be incorporated into the compositions.
  • the pharmaceutically acceptable carrier comprises serum albumin.
  • the pharmaceutical composition of the invention is formulated to be compatible with its intended route of administration.
  • routes of administration include parenteral, e.g., intrathecal, intra-arterial, intravenous, intradermal, subcutaneous, oral, transdermal (topical) and transmucosal administration.
  • Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine; propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfate; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide.
  • compositions suitable for injection use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion.
  • suitable carriers include physiological saline, bacteriostatic water, Cremophor ELTM (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS).
  • the injectable composition should be sterile and should be fluid to the extent that easy syringability exists.
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyetheylene glycol, and the like), and suitable mixtures thereof.
  • the proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the requited particle size in the case of dispersion and by the use of surfactants.
  • Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like.
  • isotonic agents for example, sugars, polyalco- hols such as manitol, sorbitol, and sodium chloride in the composition.
  • Prolonged absorption of the inject-able compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.
  • Sterile injectable solutions can be prepared by incorporating the active compound (e.g., a neu- doctorsn) in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization.
  • dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above.
  • the preferred methods of preparation are vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
  • Oral compositions generally include an inert diluent or an edible carrier. They can be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash, wherein the compound in the fluid carrier is applied orally and swished and ex- pectorated or swallowed. Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition.
  • the tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Stertes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.
  • a binder such as microcrystalline cellulose, gum tragacanth or gelatin
  • an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch
  • a lubricant such as magnesium stearate or Stertes
  • a glidant such as colloidal silicon dioxide
  • the compounds are delivered in the form of an aerosol spray from pressured container or dispenser which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer.
  • a suitable propellant e.g., a gas such as carbon dioxide, or a nebulizer.
  • Systemic administration can also be by transmucosal or transdermal means.
  • penetrants appropriate to the barrier to be permeated are used in the formulation.
  • penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives.
  • Transmucosal administration can be accomplished through the use of nasal sprays or suppositories.
  • the pharmaceutical compositions are formulated into ointments, salves, gels, or creams as generally known in the art.
  • the pharmaceutical composition is formulated for sustained or controlled release of the active ingredient.
  • Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art. The materials can also be obtained commercially from e.g. Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) or nanoparticles, including those prepared with poly(dl-lactide-co-glycolide), can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art.
  • Dosage unit form as used herein includes physically discrete units suited as unitary dosages for the subject to be treated; each unit con- taining a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.
  • the specification for the dosage unit forms of the invention are dictated by and directly dependent on the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and the limitations inherent in the art of compounding such an active compound for the treatment of individuals.
  • Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population).
  • the dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50/ED50.
  • Compounds which exhibit large therapeutic indices are preferred. While compounds that exhibit toxic side effects may be used, care should be taken to design a delivery system that targets such compounds to the site of affected tissue in order to minimize potential damage to uninfected cells and, thereby, reduce side effects.
  • the data obtained from the cell culture assays and animal studies can be used in formulating a range of dosage for use in humans.
  • the dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity.
  • the dosage may vary within this range depending upon the dosage form employed and the route of administration utilized.
  • the therapeutically effective dose can be estimated initially from cell culture assays.
  • a dose may be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture.
  • IC50 i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms
  • Such information can be used to more accurately determine useful doses in humans.
  • the pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration.
  • RNA was isolated after 48 h of injection. n 4 different animals/group. Data are expressed as means ⁇ SEM. **p ⁇ 0.01, *p ⁇ 0.05.
  • FIG. 2 Caged antimiR-92a treatment with subsequent light activation accelerates and improves wound healing in db/db mice.
  • A Macroscopic quantification of the wound size over time post wounding. Different groups as indicated.
  • B Representative photographs of the quantified wound area at different time points post injury. Morphometric quantification of (C) the amount of granulation tissue, as a measure for wound quality; (D) the length of reepithelializa- tion, as a measure for the wound size and (E) the distance between the ends of panniculus carnosus, as a measure for wound contraction.
  • FIG. 3 Inducible miR-92a inhibition increases angiogenesis and the expression of the miR-92a target Sirt-1 in wound tissue.
  • A Immunohistochemical quantification of the CD31 positive stained area within the granulation tissue and
  • C Morphometric quantification of the Sirt-1 + area normalized to the Hoechst + area within the granulation tissue 11 days post injury.
  • FIG. 5 Repetitive caged antimiR-92a injection with subsequent light induction successfully downregulates miR-92a levels in cutaneous wounds with no obvious toxic side effects in excretory organs.
  • FIG. 6 Inducible miR-92a inhibition slightly increases ITGA5 protein levels in wound tissue.
  • A Morphometricquantification of immunohistochemically stained ITGA5 area within wounds upon caged antimiRtreatment as indicated and
  • NPE-protected 2'-OMe RNA phosphoramidite precursors and antimiRs without cholesterol were prepared (6).
  • Solid phase synthesis of cholesterol-modified antimiRs was performed in 14 ⁇ scale on an automated DNA/RNA synthesizer (Akta Oligopilot plus) in DMTr-Off mode.
  • 3 ' -Cholesteryl-TEG CPG was purchased from GlenResearch, 2 ' -OMe RNA amidites were supplied by LinkTech.
  • the oligonucleotides were synthesized using a modified synthesis protocol with six minutes recycling time and a Cap-Ox-Cap or Cap-Thio-Cap protocol.
  • the caged antimiR was prepurified on an Akta Purifier system with a 12 mL Source 15RPC column (flow: 4 mL/min) using a two-step linear gradient with 0.1 M triethylammonium acetate (pH 7) in water (buffer A) and 0.1 M triethylammonium acetate in 90 % acetonitrile (buffer B).
  • Step 1 0-20% B in 1 column volume
  • Step 1 0-20% B in 9 minutes
  • Step 2 20-90% B in 82.7 minutes
  • Non-caged antimiR-92a and caged antimiR-Ctrl were purified by RP-HPLC on the YoungLin system in one step.
  • All product-containing fractions were concentrated, analyzed by LC-MS (Bruker micrOTOF-Q) and the purest fractions were pooled and de- salted via NAP25 columns (GE Healthcare). Oligonucleotides were then lyophilized and dissolved in PBS buffer (80 ng ⁇ L).
  • Non-caged antimiR-92a 5 ' - CsAsGGCCGGGACAAGUGCAsAsUsAs-Chol ⁇ (SEQ ID NO: 1)
  • Caged antimiR-92a S'-CsAsGGCCGGGACAAGUGCAsAsUsAs-Chol ⁇
  • Non-caged antimiR-92a 7940.8 Da (expected: 7939.7 Da)
  • mice were treated with light-inducible antimiRs by i.d. injection into the wound margin (2 ⁇ g in 25 ⁇ ). Again after 10 min, wounds were irradiated for 10 min.
  • the wound healing kinetics was documented photographically on days 1, 4, 7 and 11 post wounding and on day 11 mice were euthanized. Wounds were bisected in caudocranial direction, and the tissue was either fixed overnight in 4% paraformaldehyde for paraffin embedding or mixed with QIAzol (Qiagen) before RNA isolation.
  • RNA from murine skin, liver or kidney was isolated using the miRNeasy Mini Kit (Qiagen) according to the manufactures protocol.
  • miR-92a detection 10 ng RNA was reverse transcribed and quantified by TaqMan real-time PCR using the TaqMan microRNA Assay (Applied Biosystems). RNU6 was used as the endogenous control. A StepOnePlus device was used for detection (Applied Biosystems). Measurement for toxicity of light-inducible antimiRs
  • Plasma levels for the transaminases AST and ALT as well as for AP were quantified. Creatinine and urea plasma levels were determined to proof kidney functionality. Measurements occurred in accordance to standard procedures in the central laboratory of the university hospital Frankfurt am Main, Germany.
  • Immunohisto chemical stainings were performed on 4 ⁇ paraffin sections. The tissue was rehydrated and demasked with citrate buffer (pH 6). Before antibody incubation, sections were blocked with 1% BSA / 2% normal goat serum (Dako) in PBS and then incubated with the primary antibody over night at 4°C. Monoclonal primary antibodies used: rat a mouse CD31 (1 :30, Dianova), rabbit a mouse ITGA5 (1 : 100, Abeam) and rabbit a mouse Sirtl (1 : 100, Millipore).
  • Bound primary antibody was detected by incubation with Alexa Fluor 488- or Alexa Fluor 555-conjugated (Invitrogen) secondary antibodies (1 h, room temperature), followed by counterstaining with Hoechst 33342 (Anaspec. Inc.).
  • the macroscopic wound area was quantified based on photographs taken at various time points post injury and was calculated as the percentage of the initial wound area with a diameter of 6 mm. For scaling a ruler was placed next to the wound.
  • the extent of granulation tissue formation was determined on 4 ⁇ haematoxy- lin and eosin (H&E) stained paraffin tissue sections from the central portion of the wound using a Zeiss microscope equipped with a bright field filter (Axio Observer.Zl, Zeiss). Additionally, the distance between the ends of panniculus carnosus was determined as a measure of wound contraction and the length of the re-epithelialized area was determined as a measure for the microscopic wound size.
  • Example 1 Efficient and locally restricted miR-92a downregulation in murine skin upon treatment with light-inducible antimiR-92a
  • the inventors established an ex vivo skin explant culture model to define suitable wavelengths, light dose and concentration of applied caged antimiRs.
  • the inventors used isolated skin tissue of adult male mice and measured light intensity behind a skin barrier by testing LEDs of different wavelengths and currents. These findings were compared to light intensities without the skin barrier. Although longer wavelengths show, as expected, a better penetration depth, as shown by less intensity attenuation, 385 nm light efficiently enters murine skin. Since wavelengths of this range can effectively remove photolabile protecting groups, but are not expected to induce direct photo damage as compared to shorter wavelengths of the ultraviolet spectrum, light of 385 nm was used for the subsequent experiments.
  • Caged antimiR- 92a developed in earlier studies, was injected intradermally into murine skin explants (1 ⁇ g diluted in 50 ⁇ PBS). After irradiation for 10 min (385 nm, 300 mA) and subsequent cultivation for 48 h, levels of free miR-92a were analyzed by quantitative real-time PCR. In comparison to the samples treated with non- irradiated caged antimiR-92a miR-92a levels were significantly down-regulated upon light induction (Figure 1A). The efficiency of miR-92a inhibition by light-induced caged antimiRs was similar as compared to conventional constitutive ly active antimiRs, which were used as positive control (Figure 1A).
  • the inventors synthesized antimiRs with a slightly changed architecture,in accordance with first antagomiR studies by Krutzfeldt et al. The inventors attached a cholesterol moiety on the 3 '-end of the antimiRs for better in vivo delivery.
  • AntimiRs were composed of 2 -OMe modified nucleotides and contained in total six phosphorothioates at the 3'- and 5 '-end to allow stability against endo- and exonucleases.
  • the inventors attached six l-(2-nitrophenyl)ethyl (NPE) photo labile protecting groups to the N 6 of A and N 4 of C nucleotides ( Figure 4) respectively. Additionally, the inventors synthesized a non-caged antimiR-92a as positive control and a caged antimiR negative control that does not affect miR-92a levels.
  • NPE l-(2-nitrophenyl)ethyl
  • Example 2 Inducible miR-92a inhibition improves skin repair in healing impaired diabetic mice
  • Cutaneous wound healing is a highly orchestrated physiological process involving the interplay of keratinocytes, fibroblasts, endothelial cells, extracellular matrix remodeling and the immune system (13). Inflammation, proliferation and remodeling, the three different healing phases, that overlap in time and space, are regulated by several cytokines and growth factors, but also by various miRs (14). The formation of new blood vessels by proliferation and migration of endothelial cells (a process termed angiogenesis) promotes the wound healing response (15, 16). Although the therapeutic effects of miR-92a inhibition on wound healing has not been reported, the inventors hypothesized that the known pro-angiogenic activity of anti- miR-92a may accelerate the healing response.
  • mice were used diabetic mice as a relevant model of impaired wound healing.
  • Non-healing, chronic wounds are a particularly common problem in diabetes.
  • Db/db mice are deficient for leptin receptor activity by carrying a homozygous point mutation in the gene for this receptor and are a well-established model to test therapeutic improvements for chronic wound healing (17).
  • To study skin repair with or without functional miR-92a in diabetic mice 13 weeks old mice were wounded by using 6 mm punch biopsies and wounds were treated with caged antimiRs (2 ⁇ g diluted in 25 ⁇ PBS) at days 0, 4 and 7. Knockdown efficiency was quantified via qPCR 1 1 days post injury. Light-activated caged antimiR-92a therapy led to a marked downregulation of miR-92a levels whereas no effect was seen in control groups (Figure 5A).
  • liver transaminases AST (aspartate aminotransferase) and ALT (alanine aminotransferase) were measured as biomarkers for liver injury. Furthermore, creatinine and urea plasma levels were determined to control for kidney functionality. For all used biomarkers no significant upregulation was detected in comparison to untreated db/db mice ( Figure 5B-F), indicating that caged antimiRs and the released photolysis side product had no obvious toxic short-term effects on organs, such as liver and kidney.
  • Validated pro-angiogenic miR-92a targets are the integrin subunit a5 (ITGA5), that mediates cell-matrix interactions and cell migration (18, 19) via binding to fibronectin, and the class III histone deacetylase sirtuin (Sirt)-l (10).
  • ITGA5 integrin subunit a5
  • Sirt class III histone deacetylase sirtuin
  • Sirt-1 was shown to promote corneal epithelialization (22) and improved keratinocyte proliferation during skin repair (23).
  • a strong upregulation of Sirt-1 upon miR-92a inhibition in the epithelium covering the wound area was observed (Figure 3D), probably leading to the thick and hyperproliferative epithelium in miR-92a inhibited wounds and thereby promoting an accelerated healing response ( Figure 2).
  • the second analyzed miR- 92a target ITGA5 is regulated in a transient manner after skin injury.
  • ITGA5 While an increase of ITGA5 during the early phase of the wound healing response is contributing to keratinocyte migration over the fibronectin-rich matrix, ITGA5 is down-regulated during later phases where a collagen-rich matrix replaces the fibronectin matrix.
  • the inventors did not see a significant difference of ITGA5 after antimiR-92a treatment at the end of the experiment, the inventors cannot exclude a derepression of ITGA5 by antimiR-92a treatment during early wound healing phases, probably mediating an improved keratinocyte migration and finally forcing wound closure.
  • the invention shows that caged antimiRs are effectively activated by light in non-transparent living tissue.

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Abstract

La présente invention concerne des oligonucléotides antisens pour l'activation spatialement régulée du silençage de micro-ARN par irradiation locale après l'administration de la molécule. La conception anti-sens de l'invention utilise des groupes protecteurs photolabiles fixés aux oligonucléotides qui affectent la liaison antisens à leur cible. Les molécules antisens de l'invention sont utiles pour des applications in vivo et seront donc utiles en tant qu'agents thérapeutiques en médecine pour activer un silençage de micro-ARN confiné spatialement, ce qui entraînera une toxicité antisens réduite. L'invention concerne également un oligonucléotide antisens de miR-92 inductible par la lumière destiné à être utilisé pour aider la génération de tissu et la cicatrisation des plaies, ainsi que des compositions pharmaceutiques comprenant l'oligonucléotide antisens de miR-92 inductible par la lumière de l'invention.
PCT/EP2017/056014 2016-03-15 2017-03-14 Oligonucléotides antisens inductibles par la lumière pour application in vivo WO2017157950A1 (fr)

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