WO2019191109A1 - Inhibiteurs de microarn et leur utilisation dans le traitement de membranes épirétiniennes - Google Patents

Inhibiteurs de microarn et leur utilisation dans le traitement de membranes épirétiniennes Download PDF

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Publication number
WO2019191109A1
WO2019191109A1 PCT/US2019/024090 US2019024090W WO2019191109A1 WO 2019191109 A1 WO2019191109 A1 WO 2019191109A1 US 2019024090 W US2019024090 W US 2019024090W WO 2019191109 A1 WO2019191109 A1 WO 2019191109A1
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mir
inhibitor
nucleotide sequence
seq
eye
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PCT/US2019/024090
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English (en)
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Georgia KAMBOJ
Theodore Leng
Creed STARY
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The Board Of Trustees Of The Leland Stanford Junior University
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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
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0048—Eye, e.g. artificial tears
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • 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

Definitions

  • the present invention pertains generally to compositions and methods for treating epiretinal membranes (ERMs).
  • the invention relates to the use of inhibitors of miR-494 for treatment of epiretinal membranes and eye diseases and conditions associated with epiretinal membrane formation.
  • ERMs are common, with a maximum prevalence of 35% during the eighth decade of life (Mitchell et al. (1997) Ophthalmology 104: 1033-1040). As such, ERMs are the third most common indication for pars plana vitrectomy (Jackson et al. (2013) Eye 27:644-651). Surgery poses a risk of cataract and rhegmatogenous retinal detachment, and recurrence is common (Jackson et al., supra, Rasouli et al. (2012) Can. J.
  • MGCs Muller glial cells at the vitreoretinal interface have been shown to play an important role in the development of ERMs.
  • MGCs are of neuroepithelial origin, and normally located in the retina, but are also found in ERM specimens (Zhao et al. (2013) Retina 33:77-88). Breaks in the internal limiting membrane (ILM) are thought to stimulate MGC end -feet, promoting migration of MGCs through the ILM (Edwards et al. (2016) Exp. Eye Res. 150:44-61). Proliferation along the stiff ILM further stimulates the mechanosentitive MGCs contributing to an“activated” phenotype with greater contractile properties (Bu et al.
  • ERK1/2 extracellular signal-regulated kinases 1 and 2 pathway has been observed to contribute to non-specific activati on of MGCs, while the phosphoinositol-3 kinase (PI3K/Akt) pathway is involved with MGC proliferation (Bringmann et al. (2009) Prog. Retin. Eye Res. 28:423-451).
  • PI3K/Akt phosphoinositol-3 kinase
  • Another cell characteristic frequently found in ERMs is immunoreactivity for alpha-smooth muscle actin (a-SMA) (Zhao et al. (2013) Retina 33:77-88, Bu et al.
  • MGCs have been implicated as the cell type fitting this phenotype. These activated, myofibroblast-like MGCs function in producing extracellular matrix and collagen, which are key features in the transition from a cellular to a contractile, fibrotic ERM. In addition to median o- sti m ul ati on, collagen VI and TGF-b are two factors that have so far been found to act on MGCs to promote myofibroblast-like properties and up-regulate a-SMA (Bu et al. (2014) Retina 34:2317-2335).
  • MicroRNAs are post-transcriptional regulators that provide tissue and cell type specific control of protein expression (Bartel (2009) Cell 136 (2): 215-33; Bartel (2004) Cell 116 (2): 281-97). Consisting of small, noncoding RNA molecules of approximately 21 to 23 nucleotides, microRNAs modulate protein expression by binding to
  • mRNAs complementary or partially complementary target messenger RNAs
  • MicroRNA binding sites are frequently located in the 3 '-untranslated region (UTR) of the target mRNA, but may also be located in coding regions of the mRNA.
  • the 5 '-region of the miRNA, typically nucleotides 2-7 of the miRNA, is termed the "seed sequence" and is particularly important for mRNA repression by miRNA. Because the seed sequence or sequence recognition site is short, generally only 6 base pairs long, mi RNAs tend to have many targets, and each mRNA can be targeted by multiple miRNAs.
  • miRNAs are known to control processes such as development, cell fate, apoptosis, proliferation, differentiation, hematopoiesis, and exocytosis.
  • Dysregulation of miRNA expression is associated with various diseases including many eye disorders, such as myopia, glaucoma, retinoblastoma, age-related macular degeneration, and uveitis (Raghunath et al. (2015) Ophthalmic Res. 53(4): 169- 86, Sundermeier et al. (2012) Cell Mol. Life Sci. 69(16):2739-2750).
  • Previous studies have observed miRNA dysregulation in the vitreous of patients with a number of ocular 7 diseases (Usui-Ouchi et al. (2016) PLoS One H :e0l58043; Russo et al. (2017) PLoS One 12:e0l 74297).
  • the present invention is based, in part, on the discovery that inhibitors of miR- 494 can be used for treatment of epiretinal membranes.
  • the invention includes a method of treating a subject for an eye disease or condition associated with epiretinal membrane formation, the method comprising administering a therapeutically effective amount of an inhibitor of miR-494 to the subject.
  • an effective amount of an inhibitor of miR-494 can be administered to a subject in one or more administrations, applications or dosages.
  • therapeutically effective dose or amount of an inhibitor of miR-494 is intended an amount that, when
  • a therapeutically effective dose or amount of an inhibitor of miR-494 may inhibit Muller glial cell (MGC) migration and/or myofibroblast-like transformation associated with the pathophysiology of epiretinal membrane formation.
  • MMC Muller glial cell
  • the individual undergoing treatment according to the invention exhibits an improvement in one or more symptoms, including such improvements as improved vision, reduced visual distortion, and/or reduced light sensitivity.
  • the inhibitor of miR-494 is administered intraocularly or intravitreally.
  • the inhibitor of miR-494 may be administered locally to the retina or vitreous humor of the subject.
  • the inhibitor of miR-494 is selected from the group consi sting of an antagomir, an antisense oligonucleotide, and an inhibitory RNA (e.g., a small interfering RNA (siRNA) or a short hairpin RNA (shRNA)).
  • an inhibitory RNA e.g., a small interfering RNA (siRNA) or a short hairpin RNA (shRNA)
  • the inhibitor of miR-494 inhibits miR-494-5p or miR-
  • the inhibitor of miR-494 comprises or consists of the nucleotide sequence of SEQ ID NO:2, or a sequence displaying at least about 80-100% sequence identity thereto, including any percent identity within this range, such as 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% sequence identity thereto.
  • the inhibitor of miR-494 comprises at least one locked nucleic acid.
  • the subject is treated therapeutically or prophylactically.
  • the inhibitor of miR-494 is provided by a recombinant polynucleotide comprising a promoter operably linked to a polynucleotide encoding the inhibitor of miR-494.
  • the recombinant polynucl eotide comprises a vector, for example, a nonviral or viral expression vector, such as, but not limited to, an adenovirus, retrovirus (e.g., g-retrovirus and lenti virus), poxvirus, adeno-associated virus, baculovirus, or herpes simplex virus vector.
  • compositions comprising the inhibitor of miR-494 may be administered by any suitable method, including but not limited to, intraocularly, intravitreally, subcutaneously, intra-arterially, or intravenously.
  • a composition is administered locally as eye drops comprising the inhibitor of miR-494 into the retina or as a solution injected into the vitreous humor of the eye of the subject.
  • the invention includes a method for inhibiting formation of an epiretinal membrane in an eye of a subject, the method comprising introducing an effective amount of an inhibitor of miR-494 into the eye.
  • the inhibitor of miR-494 is introduced into the retina or vitreous humor of the eye.
  • the inhibitor of miR-494 is selected from the group consisting of an antagomir, an antisense oligonucleotide, and an inhibitory RNA (e.g., a siRNA or stiRNA).
  • the invention includes a composition for use in the treatment of an eye disease or condition associated with epiretinal membrane formation, the composition comprising an inhibitor of miR-494.
  • compositions may further comprise a pharmaceutically acceptable carrier or excipient.
  • pharmaceutically acceptable carrier can be an aqueous eye drop solution or a solution for injection into the vitreous humor of the eye.
  • the composition comprises an inhibitor of miR-494 selected from the group consisting of an antagomir, an antisense oligonucleotide, and an inhibitory RNA (e.g., siRNA or an shRNA).
  • the inhibitor of miR-494 inhibits miR-494-5p or miR-494-3p.
  • the inhibitor of miR-494 comprises or consists of the nucleotide sequence of SEQ ID NO: 2, or a sequence displaying at least about 80-100% sequence identity thereto, including any percent identity within this range, such as 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92,
  • the inhibitor of miR-494 comprises at least one locked nucleic acid.
  • FIG. 1 shows a cluster plot demonstrating differentially expressed miRs found in ERM specimens versus control vitreous.
  • FIG 2 shows a Box plot demonstrating increased counts of miR-494 in ERM specimens versus control vitreous.
  • microRNA includes a mixture of two or more microRNAs, and the like.
  • microRNA refers to a non-coding single-stranded RNA molecule that is about 19 to about 25 nucleotides in length (including about 19, about 20, about 21, about 22, about 23, about 24, and about 25 nucleotides) that effectively reduces the expression level of target polynucleotides and polypeptides through the RNA interference pathway (i.e., through association with the RISC and subsequent degradation of target mRNA or translational inhibition).
  • microRNA refers to both endogenous miR As that have been found in any organism (e.g., plants, animals) and artificial miRNAs that include single-stranded RNA molecules with sequences of about 19-25 nucleotides in length other than those found in endogenous miRNAs that effectively reduce the expression of target polynucleotides through RNA interference.
  • administering an expression vector, nucleic acid, microRNA, microRNA mimic, or microRNA inhibitor to a cell comprises transducing, transfecting,
  • electroporating, translocating, fusing, phagocytosing, shooting or ballistic methods, etc. i.e., any means by which a nucleic acid can be transported across a cell membrane.
  • derived from is used herein to identify the original source of a molecule but is not meant to limit the method by which the molecule is made which can be, for example, by chemical synthesis or recombinant means.
  • isolated when referring to a polynucleotide, such as a mRNA, microRNA, microRNA mimic, or microRNA inhibitor, is meant that the indicated molecule is present in the substantial absence of other biological macromolecules of the same type.
  • isolated microRNA molecule refers to a polynucleotide molecule, which is
  • RNA nucleotide molecules substantially free of other polynucleotide molecules, e.g., other microRNA molecules that do not target the same RNA nucleotide sequence; however, the molecule may include some additional bases or moieties which do not deleteriously affect the basic
  • substantially purified generally refers to isolation of a substance (compound, polynucleotide, protein, polypeptide, polypeptide composition) such that the substance comprises the majority percent of the sample in which it resides.
  • a substantially purified component comprises 50%, preferably 80%-85%, more preferably 90-95% of the sample.
  • polynucleotide oligonucleotide
  • nucleic acid nucleic acid molecule
  • polynucleotide oligonucleotide
  • nucleic acid molecule a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. This term refers only to the primary structure of the molecule. Thus, the term includes triple-, double- and single-stranded DNA, as well as triple-, double- and single-stranded RNA. It also includes modifications, such as by methylation and/or by capping, and unmodified forms of the polynucleotide.
  • polynucleotide examples include polydeoxyribonucleotides (containing 2-deoxy-D-ribose), polyribonucleotides (containing D-ribose), any other type of polynucleotide which is an N- or C-glycoside of a purine or pyrimidine base, and other polymers containing nonnucleotidic backbones, for example, polyamide (e.g., peptide nucleic acids (PNAs)) and polymorpholino (commercially available from the Anti-Virals, Inc., Corvallis, Oreg., as Neugene) polymers, and other synthetic sequence-specific nucleic acid polymers providing that the polymers contain nucleobases in a configuration which allows for base pairing and base stacking, such as is found in DNA and RNA There is no intended distinction in length between the terms "polyl
  • these terms include, for example, 3'-deoxy-2',5'-DNA, ol igodeoxyrib onuc 1 eoti de N3' P5' phosphoramidates, 2'-0-alkyl-substituted RNA, double- and single-stranded DNA, as well as double- and single- stranded RNA, microRNA, DNA: RNA hybrids, and hybrids between PNAs and DNA or RNA, and also include known types of modifications, for example, labels which are known in the art, methylation, "caps," substitution of one or more of the naturally occurring nucleotides with an analog (e.g., 2-aminoadenosine, 2- thiothymidine, inosine, pyrrolo-pyrimidine, 3 -methyl adenosine, C 5 -propynyl cyti dine,
  • an analog e.g., 2-aminoadenosine, 2- thiothym
  • phosphorothioates, phosphorodithioates, etc. and with positively charged linkages (e.g., aminoalklyphosphoramidates, aminoalkylphosphotriesters), those containing pendant moieties, such as, for example, proteins (including nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.), those with intercalators (e.g., acridine, psoralen, etc.), those containing chelators (e.g., metals, radioactive metals, boron, oxidative metals, etc.), those containing alkyl ators, those with modified linkages (e.g., alpha anomeric nucleic acids, etc.), as well as unmodified forms of the polynucleotide or oligonucleotide.
  • positively charged linkages e.g., aminoalklyphosphoramidates, aminoalkylphosphotriesters
  • pendant moieties such as, for example, proteins (including
  • the term also includes locked nucleic acids (e.g., comprising a ribonucleotide that has a methylene bridge between the 2'-oxygen atom and the 4'-carbon atom).
  • locked nucleic acids e.g., comprising a ribonucleotide that has a methylene bridge between the 2'-oxygen atom and the 4'-carbon atom.
  • label and “detectable label” refer to a molecule capable of detection, including, but not limited to, radioactive isotopes, stable (non-radioactive) heavy isotopes, fluorescers, chemiluminescers, enzymes, enzyme substrates, enzyme cofactors, enzyme inhibitors, chromophores, dyes, metal ions, metal sols, ligands (e.g., biotin or haptens) and the like.
  • fluorescers chemiluminescers
  • enzymes enzyme substrates
  • enzyme cofactors enzyme inhibitors
  • chromophores dyes
  • metal ions metal sols
  • ligands e.g., biotin or haptens
  • labels include, but are not limited to radiolabels (e.g., 3 H, 125 1, 35 S, 14 C, or 32 P), stable (non-radioactive) heavy isotopes (e.g., 13 C or 15 N), phycoerythrin, fluorescein, 7-nitrobenzo-2-oxa-l, 3-diazole (NBD), YPet, CyPet, Cascade blue, allophycocyanin, Alexa dyes (e.g., Alexa 350, Alexa 430, Alexa 488, Alexa 532, Alexa 546, Alexa 555, Alexa 594, Alexa 647, Alexa 660, Alexa 680, and Alexa 750), Atto dyes (e.g., Atto 488, Atto 532, Atto 550, Atto 565, Atto 590, Atto 610, Atto 620, Atto 635, Atto 647, Atto 655, and
  • Enzyme tags are used with their cognate substrate.
  • the term also includes contrast agents such as, but not limited to, MRI contrast agents (e.g., gadodiamide, gadobenic acid, gadopentetic acid, gadoteridol, gadofosveset,
  • gadoversetamide, gadoxetic acid gadoversetamide, gadoxetic acid
  • CT computed tomography contrast agents
  • CT computed tomography
  • Recombinant as used herein to describe a nucleic acid molecule means a polynucleotide of genomic, RNA, miRNA, cDNA, viral, semi synthetic, or synthetic origin which, by virtue of its origin or manipulation is not associated with all or a portion of the polynucleotide with which it is associated in nature.
  • the term "recombinant” as used with respect to a protein or polypeptide means a polypeptide produced by expression of a recombinant polynucleotide. In general, the gene of interest is cloned and then expressed in transformed organisms, as described further below.
  • Recombinant host cells refer to cells which can be, or have been, used as recipients for recombinant vector or other transferred DNA or RNA, and include the original progeny of the original cell which has been transfected.
  • “Operably linked” refers to an arrangement of elements wherein the components so described are configured so as to perform their usual function.
  • a given promoter operably linked to a coding sequence is capable of effecting the expression of the coding sequence when the proper enzymes are present.
  • Expression is meant to include the transcri ption of any one or more of transcri ption of an mRNA, microRNA, microRNA mimic, or microRNA antagonist from a DNA or RNA template and can further include translation of a protein from an mRNA template.
  • the promoter need not be contiguous with the coding sequence, so long as it functions to direct the expression thereof. Thus, for example, intervening untranslated yet transcribed sequences can be present between the promoter sequence and the coding sequence and the promoter sequence can still be considered “operably linked" to the coding sequence.
  • control elements include, but are not limited to, transcription promoters, transcription enhancer elements, transcription termination signals,
  • transfection is used to refer to the uptake of foreign DNA or RNA by a cell.
  • a cell has been "transfected” when exogenous DNA or RNA has been introduced inside the cell membrane.
  • transfection techniques are generally known in the art. See, e.g., Graham et al. (1973) Virology, 52:456, Sambrook et al. (2001)
  • “Pharmaceutically acceptable excipient or carrier” refers to an excipient that may optionally be included in the compositions of the invention and that causes no significant adverse toxicological effects to the patient.
  • “Pharmaceutically acceptable salt” includes, but is not limited to, amino acid salts, salts prepared with inorganic acids, such as chloride, sulfate, phosphate, diphosphate, bromide, and nitrate salts, or salts prepared from the corresponding inorganic acid form of any of the preceding, e.g., hydrochloride, etc., or salts prepared with an organic acid, such as malate, maleate, fumarate, tartrate, succinate, ethyl succinate, citrate, acetate, lactate, methanesulfonate, benzoate, ascorbate, para-toluenesulfonate, palmoate, salicylate and stearate, as well as estolate, gluceptate and lactobionate salts.
  • salts containing pharmaceutically acceptable cations include, but are not limited to, sodium, potassium, calcium, aluminum, lithium, and ammonium (including substituted ammonium).
  • Eye diseases or conditions associated with epiretinal membrane formation include, but are not limited to, cellophane maculopathy, posterior vitreous detachment, retinal tears, retinal detachment, retinal vascular diseases (e.g., diabetic retinopathy and venous occlusive disease), intraocular inflammation, and ocular surgery.
  • treatment refers to the medical management of a patient with the intent to cure, ameliorate, stabilize, or prevent a disease, pathological condition, or disorder.
  • This term includes active treatment, that is, treatment directed specifically toward the improvement of a disease, pathological condition, or disorder, and also includes causal treatment, that is, treatment directed toward removal of the cause of the associated disease, pathological condition, or disorder.
  • this term includes palliative treatment, that is, treatment designed for the relief of symptoms rather than the curing of the disease, pathological condition, or disorder; prophylactic or preventative treatment, that is, treatment directed to minimizing or partially or completely inhibiting the development of the associated disease, pathological condition, or disorder; and supportive treatment, that is, treatment employed to supplement another specific therapy directed toward the improvement of the associated disease, pathological condition, or disorder.
  • an "effective amount" of an inhibitor of miR-494 is an amount sufficient to effect beneficial or desired results, such as an amount that inhibits pathological migration of Muller glial cells through the retina and/or myofibroblast-like transformation of Muller glial cells.
  • An effective amount can be administered in one or more administrations, applications or dosages.
  • a “therapeutically effective dose or amount” of a miRNA, miKNA mimic, or miRNA inhibitor is intended an amount that, when administered as described herein, brings about a positive therapeutic response, such as improved recovery from an eye disease or disorder.
  • the individual undergoing treatment according to the invention exhibits an improvement in one or more symptoms of the eye disease or disorder, including such improvements as improved vision, reduced visual distortion, and/or reduced light sensitivity.
  • a “therapeutically effective dose or amount” of an inhibitor of miR-494 is an amount that, when administered, as described herein, brings about a positive therapeutic response in treatment of an eye disease or condition associated with epiretinal membrane formation, such as preventing formation or reducing growth of an epiretinal membrane.
  • a therapeutically effective dose or amount of an inhibitor of miR-494 may inhibit Muller glial cell (MGC) migration and/or myofibroblast-like transformation associated with the pathophysiology of epiretinal membrane formation.
  • MSC Muller glial cell
  • the exact amount required will vary from subject to subject, depending on the species, age, and general condition of the subject, the severity of the condition being treated, the particular drug or drugs employed, mode of administration, and the like.
  • An appropriate "effective" amount in any individual case may be determined by one of ordinary skill in the art using routine experimentation, based upon the information provided herein.
  • subject any member of the subphylum chordata, including, without limitation, humans and other primates, including non-human primates such as
  • chimpanzees and other apes and monkey species farm animals such as cattle, sheep, pigs, goats and horses; domestic mammals such as dogs and cats; laboratory animals including rodents such as mice, rats and guinea pigs; birds, including domestic, wild and game birds such as chickens, turkeys and other gallinaceous birds, ducks, geese, and the like.
  • the present invention is based on the discovery that inhibitors of miR-494 can be used for treating epiretinal membranes (see Example 1).
  • the present invention pertains generally to compositions containing inhibitors of miR-494 and methods of using such inhibitors of miR-494 for treatment of eye diseases and conditions associated with epiretinal membrane formation.
  • the invention provides a method for treating an epiretinal membrane or an eye disease or condition associated with epiretinal membrane formation by utilizing an inhibitor of miR-494.
  • Eye diseases or conditions associated with epiretinal membrane formation include, but are not limited to, cellophane maculopathy, posterior vitreous detachment, retinal tears or detachment, retinal vascular diseases (e.g., diabetic retinopathy and venous occlusive disease), intraocular inflammation, and ocular surgery.
  • a "therapeutically effective dose or amount" of an inhibitor of miR-494 is an amount that, when administered, as described herein, prevents formation or reduces or slows growth of an epi retinal membrane.
  • one or more symptoms of the eye disease or condition associated with epiretinal membrane formation are ameliorated or eliminated foll owing administration of the inhibitor of miR-494, resulting in improved recovery following treatment.
  • Improved recovery may include, for example, improved vision, reduced visual distortion, and/or reduced light sensitivity.
  • Inhibitors of miR-494 can include antagomirs, antisense oligonucleotides, and inhibitory RNA molecules.
  • inhibition of microRNA function may be achieved by administering antisense oligonucleotides targeting miR-494 that inhibit miR-494-5p and/or miR-494-3p.
  • the antisense oligonucleotides may be ribonucleotides or deoxyribonucleotides.
  • the antisense oligonucleotides have at least one chemical modification.
  • Antisense oligonucleotides may comprise one or more "locked nucleic acids".
  • LNAs Locked nucleic acids
  • the antisense oligonucleotides may comprise peptide nucleic acids (PNAs), which contain a peptide-based backbone rather than a sugar- phosphate backbone.
  • PNAs peptide nucleic acids
  • the antisense oligonucleotides may contain one or more chemical modifications, including, but are not limited to, sugar modifications, such as 2'-0-alkyl (e.g. 2'-0-methyl, 2'-0-methoxyethyl), 2'-fluoro, and 4' thio modifications, and backbone modifications, such as one or more phosphorothi oate, morpholino, or
  • suitable antisense oligonucleotides are 2'-0-methoxyethyl "gapmers" which contain 2’-0-methoxyethyl-modified ribonucleotides on both 5' and 3' ends with at least ten deoxyribonucleotides in the center. These "gapmers” are capable of triggering RNase H-dependent degradation mechanisms of RNA targets.
  • Other modifications of antisense oligonucleotides to enhance stability and improve efficacy such as those described in U.S. Pat. No. 6,838,283, which is herein incorporated by reference in its entirety, are known in the art and are suitable for use in the methods of the invention.
  • Antisense oligonucleotides useful for inhibiting the activity of microRNAs, are typically about 19 to about 25 nucleotides in length. Antisense oligonucleotides may comprise a sequence that is at least partially complementary to a mature miRNA sequence, e.g , at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% complementary to a target sequence of miR-494. In some
  • the antisense oligonucleotide may be substantially complementary to the target sequence of miR-494, that is at least about 95%, 96%, 97%, 98%, or 99% complementary to a target polynucleotide sequence.
  • the antisense oligonucleotide comprises a sequence that is 100% complementary to the target sequence of miR-494.
  • the inhibitor of miR-494 is a chemi call y-modi fi ed antisense oligonucleotide.
  • Antisense oligonucleotides may comprise a sequence that is substantially complementary to a precursor miRNA sequence (pre-miRNA).
  • pre-miRNA precursor miRNA sequence
  • the antisense oligonucleotide comprises a sequence that is substantially complementary to a sequence located outside the stem -loop region of the pre-miRNA sequence.
  • the antisense oligonucleotides are antagomirs.
  • Antagomirs are single- stranded, chemically-modified ribonucleotides that are at least partially complementary to the miRNA sequence.
  • Antagomirs may comprise one or more modified nucleotides, such as 2'-0-methyl -sugar modifications. In some embodiments, antagomirs comprise only modified nucleotides.
  • Antagomirs may also comprise one or more phosphorothioate linkages resulting in a partial or full
  • antagomir may be linked to a cholesterol or other moiety at its 3' end.
  • Antagomirs suitable for inhibiting miRNAs may be about 15 to about 50 nucleotides in length, more preferably about 18 to about 30 nucleotides in length, and most preferably about 20 to about 25 nucleotides in length. "Partially complementary” refers to a sequence that is at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%
  • the antagomirs may be at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% complementary to a target sequence of miR-494.
  • the antagomir may be substantially complementary to a mature miRNA sequence, that is at least about 95%, 96%, 97%, 98%, or 99% complementary to a target sequence of miR-494.
  • the antagomirs are 100% complementary to the target sequence of miR- 494.
  • the inhibitor of miR-494 is an inhibitory RNA molecule having a double stranded region that is at least partially identical and partially
  • the inhibitory RNA molecule may be a double-stranded, small interfering RNA (siRNA) or a short hairpin RNA molecule (shRNA) comprising a stem -loop structure.
  • the double-stranded regions of the inhibitory RNA molecule may comprise a sequence that is at least partially identical and partially complementary, e.g., about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical and complementary, to the mature miRNA sequence.
  • the double-stranded regions of the inhibitory RNA comprise a sequence that is at least substantially identical and substantially complementary to a target sequence of miR-494.
  • Substantially identical and substantially complementary refers to a sequence that is at least about 95%, 96%, 97%, 98%, or 99% identical and complementary to a target polynucleotide sequence.
  • the double- stranded regions of the inhibitory RNA molecule may contain 100% identity and complementarity to the target miRNA sequence.
  • the inhibitors of miR-494 are inhibitory RNA molecul es, such as ribozymes, siRNAs, or shRNAs.
  • an inhibitor of miR-494 is an inhibitory RNA molecule comprising a double-stranded region, wherein the double- stranded region comprises a sequence having 100% identity and complementarity to miR-494.
  • inhibitors of miR-494 are inhibitory RNA molecules which comprise a double-stranded region, wherein said double-stranded region comprises a sequence of at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity and compl em entari ty to a target sequence of miR-494.
  • inhibitors of miR-494 may target a sequence of miR-494, such as a target sequence within the human miR-494 (SEQ ID NO: 1).
  • inhibitors of miR-494 are antagomirs comprising a sequence that is perfectly complementary to a target sequence of the human miR-494.
  • an inhibitor of the human miR-494 is an antisense oligonucleotide, an antagomir, or siRNA comprising a sequence complementary or substantially
  • the inhibitor of miR-494 comprises or consists of the nucleotide sequence of SEQ ID NO:2, or a sequence displaying at least about 80-100% sequence identity thereto, including any percent identity within this range, such as 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% sequence identity thereto.
  • the inhibitor of miR-494 is expressed in vivo from a vector.
  • a "vector” is a composition of matter which can be used to deliver a nucleic acid of interest to the interior of a cell. Numerous vectors are known in the art including, but not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses. Thus, the term “vector” includes an autonomously replicating plasmid or a virus. Examples of viral vectors include, but are not limited to, adenoviral vectors, adeno-associated virus vectors, retroviral vectors, lenti viral vectors, and the like.
  • an expression construct can be replicated in a living cell, or it can be made synthetically.
  • expression construct an expression vector
  • vector an expression vector
  • an expression vector for expressing an inhibitor of miR-494 comprises a promoter "operably linked” to a polynucleotide encoding the inhibitor of miR-494.
  • the phrase "operably linked” or “under transcriptional control” as used herein means that the promoter is in the correct location and orientation in relation to a polynucleotide to control the initiation of transcription by RNA polymerase and expression of the polynucleotide.
  • the nucl eic acid encoding a polynucleotide of interest is under transcriptional control of a promoter.
  • a "promoter” refers to a DNA sequence recognized by the synthetic machinery of the cell, or introduced synthetic machinery, required to initiate the specific transcription of a gene.
  • the term promoter will be used here to refer to a group of transcriptional control modules that are clustered around the initiation site for RNA polymerase I, II, or III.
  • Typical promoters for mammalian cell expression include the SV40 early promoter, a CMV promoter such as the CMV immediate early promoter (see, U.S. Patent Nos.
  • mice mammary tumor virus LTR promoter the mouse mammary tumor virus LTR promoter
  • Ad MLP adenovirus major late promoter
  • herpes simplex virus promoter among others.
  • Other nonviral promoters such as a promoter derived from the murine metallothionein gene, will also find use for mammalian expression.
  • promoters can be obtained from commercially available plasmids, using techniques well known in the art. See, e g., Sambrook et al., supra. Enhancer elements may be used in association with the promoter to increase expression levels of the constructs.
  • Examples include the SV40 early gene enhancer, as described in Dijkema et al., EMBO J (1985) 4:761, the enhancer/promoter derived from the long terminal repeat (LTR) of the Rous Sarcoma Virus, as described in Gorman et al., Proc. Natl. Acad. Sci. USA (1982) 79:6777 and elements derived from human CMV, as described in Boshart et al., Cell (1985) 41 :521. such as elements included in the CMV intron A sequence.
  • LTR long terminal repeat
  • transcription termi nator/poly adenyl ati on signals will also be present in the expression construct.
  • sequences include, but are not limited to, those derived from SV4Q, as described in Sambrook et al., supra, as well as a bovine growth hormone terminator sequence (see, e.g., U.S. Patent No. 5,122,458).
  • 5'- UTR sequences can be placed adjacent to the coding sequence in order to enhance expression of the same.
  • IJTRs which include an Internal Ribosome Entry Site (IRES) present in the leader sequences of picomaviruses such as the encephalomyocarditis virus (EMCV) UTR (Jang et al. J. Virol. (1989)
  • picomavirus UTR sequences that will also find use in the present invention include the polio leader sequence and hepatitis A virus leader and the hepatitis C IRES.
  • the cells containing nucleic acid constructs of the present invention may be identified in vitro or in vivo by including a marker in the expression construct. Such markers would confer an identifiable change to the cell permitting easy identification of cells containing the expression construct.
  • a drug selection arker aids in cloning and in the selection of transformants, for example, genes that confer resistance to neomycin, puromycin, hygromycin, DHFR, GPT, zeocin and histidinol are useful selectable markers.
  • enzymes such as herpes simplex virus thymidine kinase (tk) or
  • chloramphenicol acetyltransferase may be employed.
  • Fluorescent markers e.g., GFP, EGFP, Dronpa, mCherry, mOrange, mPlum, Venus, YPet, phycoerythrin
  • immunologic markers can also be employed.
  • the selectable marker employed is not believed to be important, so long as it is capable of being expressed simultaneously with the nucleic acid encoding a gene product. Further examples of selectable markers are well known to one of skill in the art.
  • the expression construct comprises a virus or engineered construct derived from a viral genome.
  • viral based systems have been developed for gene transfer into mammalian cells. These include adenoviruses, retroviruses (g-retroviruses and lenti viruses), poxviruses, adeno-associated viruses, baculoviruses, and herpes simplex viruses (see e.g., Wamock et al. (2011) Methods Mol. Biol. 737: 1-25; Walther et al. (2000) Drugs 60(2):249-27l; and Lundstrom (2003) Trends Biotechnol.
  • retroviruses provide a convenient platform for gene delivery systems. Selected sequences can be inserted into a vector and packaged in retroviral particles using techniques known in the art. The recombinant virus can then be isolated and delivered to cells of the subject either in vivo or ex vivo.
  • retroviral systems have been described (U.S. Pat. No. 5,219,740; Miller and Rosman (1989) BioTechniques 7:980-990; Miller, A. D. (1990) Human Gene Therapy 1 :5-14; Scarpa et al. (1991) Virology 180:849-852; Burns et al. (1993) Proc. Natl. Acad. Sci. USA
  • Lenti viruses are a class of retroviruses that are particularly useful for delivering polynucleotides to mammalian cells because they are able to infect both dividing and nondividing cells (see e.g., Lois et al (2002) Science 295:868-872; Durand et al. (2011) Viruses 3(2): 132-159; herein incorporated by reference).
  • adenovirus vectors have also been described. Unlike retroviruses which integrate into the host genome, adenoviruses persist extrachromosomally thus minimizing the risks associated with insertional mutagenesis (Haj -Ahmad and Graham, J. Virol. (1986) 57:267-274; Bett et al., J. Virol. (1993) 67:5911-5921; Mittereder et al., Human Gene Therapy (1994) 5:717-729; Seth et al., J. Virol. (1994) 68:933-940; Barr et al., Gene Therapy (1994) 1 :51-58; Berkner, K. L.
  • AAV vector systems have been developed for gene delivery.
  • AAV vectors can be readily constructed using techniques well known in the art. See, e.g., U.S. Pat. Nos. 5,173,414 and 5,139,941; International Publication Nos. WO 92/01070
  • Another vector system useful for delivering the polynucleotides of the present invention is the enterically administered recombinant poxvirus vaccines described by Small, Jr., P. A., et al. (U.S. Pat. No. 5,676,950, issued Oct. 14, 1997, herein incorporated by reference).
  • vaccinia virus recombinants expressing a nucleic acid molecule of interest can be constructed as follows. The DNA encoding the particular nucleic acid sequence is first inserted into an appropriate vector so that it is adjacent to a vaccinia promoter and flanking vaccinia DNA sequences, such as the sequence encoding thymidine kinase (TK). This vector is then used to transfect cells which are simultaneously infected with vaccinia.
  • TK thymidine kinase
  • Homologous recombination serves to insert the vaccinia promoter plus the gene encoding the sequences of interest into the viral genome.
  • the resulting TK-recombinant can be selected by culturing the cells in the presence of 5-bromodeoxyuridine and picking viral plaques resistant thereto.
  • avipoxvi ruses such as the fowlpox and canarypox viruses
  • canarypox viruses can also be used to deliver the nucleic acid molecules of interest.
  • the use of an avipox vector is particularly desirable in human and other mammalian species since members of the avipox genus can only productively replicate in susceptible avian species and therefore are not infective in mammalian cells.
  • Methods for producing recombinant avipoxviruses are known in the art and employ genetic recombination, as described above with respect to the production of vaccinia viruses. See, e.g., WO 91/12882; WO 89/03429; and WO 92/03545.
  • Molecular conjugate vectors such as the adenovirus chimeric vectors described in Michael et ah, J. Biol. Chem. (1993) 268:6866-6869 and Wagner et al., Proc. Natl. Acad. Sci. USA (1992) 89:6099-6103, can also be used for gene delivery.
  • Alphavirus genus such as, but not limited to, vectors derived from the Sindbis virus (SIN), Semliki Forest virus (SFV), and Venezuelan Equine Encephalitis virus (VEE), will also find use as viral vectors for delivering the Sindbis virus (SIN), Semliki Forest virus (SFV), and Venezuelan Equine Encephalitis virus (VEE), will also find use as viral vectors for delivering the Sindbis virus (SIN), Semliki Forest virus (SFV), and Venezuelan Equine Encephalitis virus (VEE), will also find use as viral vectors for delivering the Sindbis virus (SIN), Semliki Forest virus (SFV), and Venezuelan Equine Encephalitis virus (VEE), will also find use as viral vectors for delivering the Sindbis virus (SIN), Semliki Forest virus (SFV), and Venezuelan Equine Encephalitis virus (VEE), will also find use as viral vectors for delivering the Sindbis virus (SIN), Semliki Forest virus (SFV), and Venezuelan
  • chimeric alphavirus vectors comprised of sequences derived from Sindbis virus and Venezuelan equine encephalitis virus. See, e.g., Perri et al. (2003) J. Virol. 77: 10394-10403 and International Publication Nos. WO 02/099035, WO 02/080982, WO 01/81609, and WO 00/61772; herein incorporated by reference in their entireties.
  • a vaccinia-based infection/transfection system can be conveniently used to provide for inducible, transient expression of the polynucleotides of interest (e.g., encoding an inhibitor of miR-494) in a host cell.
  • cells are first infected in vitro with a vaccinia virus recombinant that encodes the bacteriophage T7 RNA polymerase.
  • This polymerase displays extraordinar specificity in that it only transcribes templates bearing T7 promoters.
  • cells are transfected with the polynucleotide of interest, driven by a T7 promoter.
  • the polymerase expressed in the cytoplasm from the vaccinia virus recombinant transcribes the transfected DNA into RNA.
  • RNA RNA-binding protein
  • Elroy-Stein and Moss Proc. Natl. Acad. Sci. USA (1990) 87:6743-6747; Fuerst et al., Proc. Natl. Acad. Sci. USA (1986) 83:8122-8126.
  • amplification system can be used that will lead to high level expression following introducti on into host cells.
  • a T7 RNA polymerase promoter preceding the coding region for T7 RNA polymerase can be engineered. Translation of RNA derived from this template will generate T7 RNA polymerase which in turn will transcribe more template. Concomitantly, there will be a cDNA whose expression is under the control of the T7 promoter. Thus, some of the T7 RNA polymerase generated from translation of the amplification template RN A will lead to transcription of the desired gene. Because some T7 RNA polymerase is required to initiate the amplification, T7 RNA polymerase can be introduced into cells along with the tempi ate(s) to prime the transcription reaction.
  • the polymerase can be introduced as a protein or on a plasmid encoding the RNA polymerase.
  • T7 systems and their use for transforming cells see, e.g., International Publication No. WO 94/26911; Studier and Moffatt, J. Mol. Biol. (1986) 189:113-130; Deng and Wolff, Gene (1994) 143:245-249; Gao et al., Biochem. Biophys. Res. Commun. (1994) 200: 1201-1206; Gao and Huang, Nuc. Acids Res. (1993) 21 :2867-2872; Chen et al., Nuc. Acids Res. (1994) 22:2114-2120; and U.S. Pat. No. 5,135,855.
  • the expression construct In order to effect expression of sense or antisense gene constructs, the expression construct must be delivered into a cell. This delivery may be accomplished in vitro, as in laboratory procedures for transforming cells lines, or in vivo or ex vivo, as in the treatment of certain disease states.
  • One mechanism for delivery is via viral infection where the expression construct is en cap si dated in an infectious viral particle.
  • the nucleic acid encoding the gene of interest may be positioned and expressed at different sites.
  • the nucleic acid encoding the gene may be stably integrated into the genome of the cell. This integration may be in the cognate location and orientation via homologous recombination (gene replacement) or it may be integrated in a random, non specific location (gene augmentation).
  • the nucleic acid may be stably maintained in the cell as a separate, episomal segment of DN A. Such nucleic acid segments or "episomes" encode sequences sufficient to permit maintenance and replication independent of or in synchronization with the host cell cycle. Flow the expression construct is delivered to a cell and where in the cell the nucleic acid remains is dependent on the type of expression construct employed.
  • the expression construct may simply consist of naked recombinant DNA or plasmids. Transfer of the construct may be performed by any of the methods mentioned above which physically or chemically permeabilize the cell membrane. This is particularly applicable for transfer in vitro but it may be applied to in vivo use as well.
  • Dubensky et al. Proc. Natl. Acad. Sci. USA (1984) 81 :7529-7533
  • Benvenisty and Neshif Proc. Natl. Acad. Sci.
  • a naked DNA expression construct may be transferred into cells by particle bombardment.
  • This method depends on the ability to accelerate DNA-coated microprojectiles to a high velocity allowing them to pierce cell membranes and enter cells without killing them (Klein et al. (1987) Nature 327:70-73).
  • Several devices for accelerating small particles have been developed.
  • One such device relies on a high voltage discharge to generate an electrical current, which in turn provides the motive force (Yang et al. (1990) Proc. Natl. Acad. Sci. USA 87:9568-9572).
  • the microprojectiles may consist of biologically inert substances, such as tungsten or gold beads.
  • the expression construct may be delivered using liposomes.
  • Liposomes are vesicular structures characterized by a phospholipid bilayer membrane and an inner aqueous medium. Multilamellar liposomes have multiple lipid layers separated by aqueous medium. They form spontaneously when phospholipids are suspended in an excess of aqueous solution. The lipid components undergo self rearrangement before the formation of closed structures and entrap water and dissolved solutes between the lipid bilayers (Ghosh and Bachhawat (1991) Liver Diseases,
  • the liposome may be complexed with a hemagglutinating virus (HVJ). This has been shown to facilitate fusion with the cell membrane and promote cell entry of liposome-encapsulated DNA (Kaneda et al. (1989) Science 243:375-378).
  • HVJ hemagglutinating virus
  • the liposome may be complexed or employed in conjunction with nuclear non-histone chromosomal proteins (HMG-I) (Kato et al. (1991) J. Biol. Chem. 266(6):336l-3364).
  • HMG-I nuclear non-histone chromosomal proteins
  • the liposome may be complexed or employed in conjunction with both HVJ and HMG-I.
  • receptor-mediated delivery vehicles which can be employed to deliver a nucleic acid encoding a particular gene into cells. These take advantage of the selective uptake of macromolecules by receptor-mediated endocytosis in almost all eukaryotic cells. Because of the cell type-specific di stribution of vari ous receptors, the delivery' can be highly specific (Wu and Wu (1993) Adv. Drug Delivery' Rev. 12:159-167).
  • Receptor-m edi ated gene targeting vehicles generally consist of two components: a cell receptor-specific ligand and a DNA-binding agent.
  • ligands have been used for receptor-mediated gene transfer. The most extensively characterized ligands are asialoorosomucoid (ASOR) and transferrin (see, e.g., Wu and Wu (1987), supra, Wagner et al. (1990) Proc. Natl. Acad. Sci. USA 87(9):34l0-3414).
  • ASOR asialoorosomucoid
  • transferrin see, e.g., Wu and Wu (1987), supra, Wagner et al. (1990) Proc. Natl. Acad. Sci. USA 87(9):34l0-3414.
  • neoglycoprotein which recognizes the same receptor as ASOR, has been used as a gene delivery vehicle (Ferkol et al. (1993) FASEB J.
  • EGF epidermal growth factor
  • the delivery vehicle may comprise a ligand and a liposome.
  • a ligand for example, Nicolau et al. (Methods Enzymol. (1987) 149: 157-176) employed lactosyl-ceramide, a galactose-terminal asialganglioside, incorporated into liposomes and observed an increase in the uptake of the insulin gene by hepatocytes.
  • a nucleic acid encoding a particular gene also may be specifically delivered into a cell type by any number of receptor-ligand systems with or without liposomes.
  • epidermal growth factor EGF
  • EGF epidermal growth factor
  • Mannose can be used to target the mannose receptor on liver cells.
  • antibodies to CDS (CLL), CD22 (lymphoma), CD25 (T-cell leukemia) and MAA (melanoma) can similarly be used as targeting moieties.
  • an oligonucleotide may be administered in combination with a cationic lipid.
  • cationic lipids include, but are not limited to, lipofectin, DOTM A, DOPE, and DOTAP.
  • Other disclosures also discuss different lipid or liposomal formulations including nanoparticles and methods of administration; these include, but are not limited to, U.S. Patent Publication 20030203865, 20020150626, 20030032615, and
  • gene transfer may more easily be performed under ex vivo conditions.
  • Ex vivo gene therapy refers to the isolation of cells from an animal, the delivery of a nucleic acid into the cells in vitro, and then the return of the modified cells back into an animal. This may involve the surgical removal of tissue/organs from an animal or the primary culture of cells and tissues.
  • a miRNA, miRNA mimic, or miR A inhibitor may comprise a detectable label in order to facilitate detection of binding to a target nucleic acid.
  • Detectable labels suitable for use in the present invention include any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical, or chemical means.
  • Useful labels in the present invention include biotin or other
  • streptavidin-binding proteins for staining with labeled streptavidin conjugate, magnetic beads (e.g., Dynabeads), fluorescent dyes (e.g., phycoerythrin, YPet, fluorescein, Texas Red, rhodamine, green fluorescent protein, and the like, see, e.g., Molecular Probes, Eugene, OR, USA), radiolabels (e.g., 3 ⁇ 4, 125 1, 35 S, 14 C, or 32 P), enzymes (e.g., horse radish peroxidase, alkaline phosphatase and others commonly used in an ELISA), and colorimetric labels such as colloidal gold (e.g., gold particles in the 40-80 nm diameter size range scatter green light with high efficiency) or colored glass or plastic (e.g., polystyrene, polypropylene, latex, etc.) beads.
  • fluorescent dyes e.g., phycoerythrin, YP
  • MRI contrast agents e.g., gadodi amide, gadobenic acid, gadopentetic acid, gadoteridol, gadofosveset, gadoversetamide, gadoxetic acid
  • CT contrast agents e.g., Diatrizoic acid, Metrizoic acid, lodamide, Iotalamic acid, Ioxitalamic acid, Ioglicic acid, Acetrizoic acid, Iocarmic acid, Methiodal, Diodone, Metrizamide, Iohexol, loxaglic acid, Iopamidol, Iopromide, Iotrolan, Ioversol, lopentol, Iodixanol, Iomeprol, Iobitridol, Ioxilan, Iodoxamic acid, Iotroxic acid, Ioglycamic acid, Adipiodone,
  • MRI magnetic resonance imaging
  • CT computed tomography
  • Iobenzamic acid, Iopanoic acid, Iocetamic acid, Sodium iopodate, Tyropanoic acid, Calcium iopodate) are useful as labels in medical imaging.
  • Patents teaching the use of such labels include U.S. Pat. Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345;
  • compositions comprising one or more inhibitors of miR-494 and a pharmaceutically acceptable carrier.
  • pharmaceutical compositions will be prepared in a form appropriate for the intended application. Generally, this will entail preparing compositions that are essentially free of pyrogens, as well as other impurities that could be harmful to humans or animals.
  • Colloidal dispersion systems such as macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes, may be used as delivery vehicles for the agonists or inhibitors of microRNA function described herein.
  • Commercially available fat emulsions that are suitable for delivering the nucleic acids of the invention to tissues, such as cardiac muscle tissue and smooth muscle tissue, include Intralipid, Liposyn, Liposyn II, Liposyn III, Nutrilipid, and other similar lipid emulsions.
  • a preferred colloidal system for use as a delivery vehicle in vivo is a liposome (i.e., an artificial membrane vesicle). The preparation and use of such systems is well known in the art. Exemplary
  • Aqueous compositions of the present invention comprise an effective amount of the delivery' vehicle, di ssolved or dispersed in a pharmaceutically acceptable carrier or aqueous medium.
  • pharmaceutically acceptable or “pharmacologically acceptable” refers to molecular entities and compositions that do not produce adverse, allergic, or other untoward reactions when administered to an animal or a human.
  • pharmaceutically acceptable carrier includes solvents, buffers, solutions, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like acceptable for use in formulating pharmaceuticals, such as pharmaceuticals suitable for administration to humans.
  • 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 ingredients of the present invention, its use in therapeutic compositions is contemplated. Supplementary active ingredients also can be
  • compositions incorporated into the compositions, provided they do not inactivate the nucleic acids of the compositions.
  • compositions for use in the invention will comprise a therapeutically effective amount of a miRNA, miRNA mimic, or miRNA inhibitor.
  • a therapeutically effective dose or amount of a miRNA, miRNA mimic, or miRNA inhibitor is intended an amount that, when administered as described herein, brings about a positive therapeutic response, such as improved recovery from an eye disease or disorder.
  • the individual undergoing treatment according to the invention exhibits an improvement in one or more symptoms of the eye disease or disorder, including such improvements as improved vision, reduced visual distortion, and/or reduced light sensitivity.
  • a “therapeutically effective dose or amount” of an inhibitor of miR-494 is an amount that, when administered, as described herein, brings about a positive therapeutic response, such as preventing formation or reducing growth of an epiretinal membrane.
  • an inhibitor of miR-494 is an amount sufficient to effect beneficial or desired results, such as an amount that inhibits pathological migration of Muller glial cells through the retina and/or myofibroblast-like transformation of Muller glial cells.
  • An effective amount can be administered in one or more administrations, applications or dosages.
  • an inhibitor of miR-494 is administered prophylactically, e.g., to prevent or slow growth of an epiretinal membrane.
  • prophylactic uses will be of particular value for subjects who have an eye disease or condition likely to lead to development of an epiretinal membrane or who are otherwise at risk of forming an epiretinal membrane.
  • compositions can be administered as eye drops comprising the miRNA, miRNA mimic, or miRNA inhibitor into the eye of the subject.
  • compositions may also be administered parenterally, e.g., by injection intraocularly, intravitreally, subcutaneously, intra-arterially, or intravenously.
  • Additional formulations suitable for other modes of administration include oral and pulmonary formulations, suppositories, and transderm al formulations, aerosol, intranasal, and sustained release formulations.
  • Dosage treatment may be a single dose schedule or a multiple dose schedule.
  • the exact amount necessary will vary depending on the desired response; the subject being treated; the age and general condition of the individual to be treated; the severity of the condition being treated; the mode of administration, among other factors.
  • An appropriate effective amount can be readily determined by one of skill in the art.
  • a "therapeutically effective amount” will fall in a relatively broad range that can be determined through routine trials using in vitro and in vivo models known in the art.
  • the pharmaceutical forms suitable for injectable use or catheter delivery include, for example, sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions.
  • these preparations are sterile and fluid to the extent that easy injectability exists. Preparations should be stable under the conditions of manufacture and storage and should be preserved against the contaminating acti on of microorganisms, such as bacteria and fungi .
  • Appropriate solvents or dispersion media may contain, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils.
  • the proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • the prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like.
  • isotonic agents for example, sugars or sodium chloride.
  • Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.
  • Sterile injectable solutions may be prepared by incorporating the active compounds in an appropriate amount into a solvent along with any other ingredients (for example as enumerated above) as desired, followed by filtered sterilization.
  • dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the desired other ingredients, e.g., as enumerated above.
  • the preferred methods of preparation include vacuum - drying and freeze-drying techniques which yield a powder of the active ingredient(s) plus any additional desired ingredient from a previously sterile-filtered solution thereof.
  • compositions of the present invention generally may be formulated in a neutral or salt form.
  • Pharmaceutically-acceptable salts include, for example, acid addition salts (formed with the free amino groups of the protein) derived from inorganic acids (e.g., hydrochloric or phosphoric acids, or from organic acids (e.g., acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups of the protein can also be derived from inorganic bases (e.g., sodium, potassium, ammonium, calcium, or ferric hydroxides) or from organic bases (e.g., isopropylamine, trimethylamine, histidine, procaine and the like).
  • inorganic acids e.g., hydrochloric or phosphoric acids, or from organic acids (e.g., acetic, oxalic, tartaric, mandelic, and the like.
  • Salts formed with the free carboxyl groups of the protein can also be
  • solutions are preferably administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective.
  • the formulations may easily be admini stered in a variety of dosage forms such as injectable solutions, drug release capsules and the like.
  • the solution generally is suitably buffered and the liquid diluent first rendered isotonic for example with sufficient saline or glucose.
  • aqueous solutions may be used, for example, for intravenous, intramuscular, subcutaneous and intraperitoneal administration.
  • sterile aqueous media are employed as is known to those of skill in the art, particularly in light of the present disclosure.
  • a single dose may be dissolved in 1 ml of isotonic NaCl solution and either added to 1000 ml of hypodermocly sis fluid or injected at the proposed site of infusion, (see for example, "Remington's Pharmaceutical Sciences” 15th Edition, pages 1035-1038 and 1570-1580).
  • Some variation in dosage will necessarily occur depending on the condition of the subject being treated.
  • the person responsible for administration will, in any event, determine the appropriate dose for the individual subject.
  • preparations should meet sterility, pyrogenicity, and general safety and purity standards as required by FDA Office of Biologies standards.
  • compositions described herein may be included in a kit.
  • at least one inhibitor of miR-494 or a recombinant polynucleotide encoding it may be included in a kit.
  • the kit may also include one or more transfection reagents to facilitate delivery of polynucleotides to cells.
  • the kit comprises a
  • composition comprising an inhibitor of miR-494, wherein the
  • the pharmaceutical composition is suitable for delivery to the eye.
  • the pharmaceutically composition can be an aqueous eye drop solution or a solution for injection into the vitreous humor of the eye.
  • the components of the kit may be packaged either in aqueous media or in lyophilized form.
  • the container means of the kits will generally include at least one vial, test tube, flask, bottle, syringe or other container means, into which a component may be placed, and preferably, suitably aliquoted.
  • the container can be configured to administer a solution in a drop wise manner.
  • kits of the present invention also will typically contain a means for containing the nucleic acids, and any other reagent containers in close confinement for commercial sale. Such containers may include injection or blow-molded plastic containers into which the desired vials are retained.
  • the liquid solution is an aqueous solution, with a sterile aqueous solution being particularly preferred.
  • the components of the kit may be provided as dried powder(s).
  • the powder can be reconstituted by the addition of a suitable solvent. It is envisioned that the solvent may also be provided in another container means.
  • the container means will generally include at least one vial, test tube, flask, bottle, syringe and/or other container means, into which the nucleic acid formulations are placed, preferably, suitably allocated.
  • the kits may also comprise a second container means for containing a sterile, pharmaceutically acceptable buffer and/or other diluent.
  • kits may also include components that preserve or maintain the microRNA inhibitors/polynucleotides or that protect against their degradation. Such components may be RNAse-free or protect against RNAses.
  • Such kits generally will comprise, in suitable means, distinct containers for each individual reagent or solution.
  • kits will also include instructions for employing the kit components as well the use of any other reagent not included in the kit. Instructions may include variations that can be implemented.
  • a kit may also include utensils or devices for administering the mi NA inhibitor by various administration routes, such as parenteral or catheter administration or coated stent.
  • ECM Epiretinal membrane formation involves the migrati on of Muller glial cells (MGCs) through damaged internal limiting membrane, followed by the transition of MGCs to a myofibroblast-like state, similar to the endothelial to
  • EMT mesenchymal transition
  • ERM tissue was collected under IRB approval and with informed consent during routine vitrectomy from patients with ERM undergoing membrane peel.
  • Control vitreous was collected from patients undergoing vitrectomy for primary symptomatic idiopathic floaters without ERM.
  • a Nano String nCounter assay was used to quantify (by solution hybridization without amplification) and compare miRs expressed in ER tissue with control vitreous.
  • Raw counts for each miR were normalized and analyzed using n Solver analysis software to obtain differential expression analysis of 800 miRs.
  • MiRs are small, noncoding RNAs that modulate gene expression by acting as regulators of their target messenger RNAs (mRNAs).
  • mRNAs target messenger RNAs
  • 12 Miravirsen (a miR- 122 inhibitor) has been successfully used in the treatment of patients with Hepatiti s C virus in a phase 2 clinical trial, suggesting that identification of critical miRs is an important step in the development of new treatments for known diseases.
  • Previous studies have observed miR dysregulation in the vitreous of patients with a number of ocular disease.
  • 13 Russo et al. 14 assessed miRs found circulating in the vitreous of patients with ERMs compared with controls, and found that the top 3 differentially expressed miRs included miR- 19b, miR- 24 and miR-l42-3p. The presence of these miRs is thought to reflect those secreted into the vitreous by cells involved in the development of ERMs.
  • miR-494 has been associated with an increase in a-SMA and reduction of the epithelial marker E-cadherin, consistent with induction of EMT in tubular epithelial cells. 16
  • the role of miR-494 in the transition to a proliferating and migrating a-SMA producing cell type associated with fibrosis represents a critical knowledge gap with respect to MGCs, and is the focus of this work. We hypothesize that targeted miR-494 inhibition is a novel therapeutic strategy for prevention of MGC migration, proliferation and a-SMA mediated contraction within a newly formed ERM.
  • MIO-M1 Muller glia cell line
  • MIO-M1 cells are treated with TGF-b! for 48 hours to promote an activated phenotype, namely expression of a-SMA and enhanced myofibroblast-like properties 9 .
  • Cellular miR-494 expression is measured using RT-qPCR pre- and post-MGC activation. Expression of a-SMA as an indication of contractile potential can be studied with immun ocytol ogy . 9
  • Exosomes are membrane bound vesicles secreted by cells and distributed through body fluids, including vitreous. Exosomal nucleic acids (mainly RNAs and miRs) can be released into recipient cells and alter cell function and gene expression. 18 MIO-M1 exosomes will be isolated from cells prior to, during, and after, TGF-bI treatment. Exosomal RNA can be isolated and miRs are compared with those found circulating in the vitreous of patients with ERM. Direct treatment of MIO-M1 cells with exosomes isolated following TGF-pl treatment is also performed. Expression of a-SMA can be measured by western blot following exosomal transfection, and compared with control cells.
  • RNAs and miRs mainly RNAs and miRs
  • MIO-M1 cells are transfected with a miR-494 mimic, inhibitor or negative control and expression of a-SMA is measured by western blot and immunocytology.
  • MIO-M1 cells are transfected with a miR-494 inhibitor or negative control and transferred to a matrigel-coated invasion chamber in serum-free DMEM.
  • DMEM containing 10 % FBS are used as a chemoattractant.
  • Successfully migrating cells are fixed and counted by a blinded observer.
  • Zhao F Gandorfer A, Haritoglou C, Scheler R, Schaumberger MM, Kampik A, et al. Epiretinal cell proliferation in macular pucker and vitreomacular traction syndrome: analysis of flat-mounted internal limiting membrane specimens.
  • MicroRNA-494 promotes cyclosporine-induced nephrotoxicity and epithelial to mesenchymal transition by inhibiting PTEN. Am J Transplant 2015;15: 1682-91.

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Abstract

L'invention concerne des compositions et des procédés de traitement de membranes épirétiniennes. En particulier, l'invention concerne l'utilisation d'inhibiteurs de miR-494 pour le traitement de membranes épirétiniennes.
PCT/US2019/024090 2018-03-28 2019-03-26 Inhibiteurs de microarn et leur utilisation dans le traitement de membranes épirétiniennes WO2019191109A1 (fr)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20170082611A1 (en) * 2015-09-21 2017-03-23 Regenerative Research Foundation Methods for Inhibiting Epithelial to Mesenchymal Transition by Inhibition of FOXS1
WO2017152149A1 (fr) * 2016-03-03 2017-09-08 University Of Massachusetts Adn double hélice linéaire à extrémité fermée pour transfert de gène non viral

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20170082611A1 (en) * 2015-09-21 2017-03-23 Regenerative Research Foundation Methods for Inhibiting Epithelial to Mesenchymal Transition by Inhibition of FOXS1
WO2017152149A1 (fr) * 2016-03-03 2017-09-08 University Of Massachusetts Adn double hélice linéaire à extrémité fermée pour transfert de gène non viral

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
KAIDONIS ET AL.: "Micro-RNAs in the pathogenesis of epiretinal membrane (ERM) formation", INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE, vol. 59, no. 9, 1 July 2018 (2018-07-01), pages 5263. *
RUSSO ET AL.: "miRNAs in the vitreous humor of patients affected by idiopathic epiretinal membrane and macular hole", PLOS ONE, vol. 12, no. 3, 22 March 2017 (2017-03-22), pages 1 - 13, XP055638993 *
YUAN ET AL.: "MicroRNA-494 promotes cyclosporine-induced nephrotoxicity and epithelial to mesenchymal transition by inhibiting PTEN", AM J TRANSPLANT, vol. 15, no. 6, June 2015 (2015-06-01), pages 1682 - 1691, XP055638999 *

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