WO2024127317A1 - Traitement d'un dysfonctionnement mitochondrial avec un inhibiteur de miarn-485 - Google Patents

Traitement d'un dysfonctionnement mitochondrial avec un inhibiteur de miarn-485 Download PDF

Info

Publication number
WO2024127317A1
WO2024127317A1 PCT/IB2023/062712 IB2023062712W WO2024127317A1 WO 2024127317 A1 WO2024127317 A1 WO 2024127317A1 IB 2023062712 W IB2023062712 W IB 2023062712W WO 2024127317 A1 WO2024127317 A1 WO 2024127317A1
Authority
WO
WIPO (PCT)
Prior art keywords
seq
aspects
vitamin
inhibitor
fold
Prior art date
Application number
PCT/IB2023/062712
Other languages
English (en)
Inventor
Jin-Hyeob RYU
Han Seok Koh
Young Jin Park
Hannah JANG
Hyun Su Min
Yu Na Lim
Original Assignee
Biorchestra Co., Ltd.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Biorchestra Co., Ltd. filed Critical Biorchestra Co., Ltd.
Publication of WO2024127317A1 publication Critical patent/WO2024127317A1/fr

Links

Definitions

  • the present application is generally directed to the use of a miR-485 inhibitor (e.g., polynucleotide encoding a nucleotide molecule comprising at least one miR-485 binding site) to regulate mitochondrial functions.
  • a miR-485 inhibitor e.g., polynucleotide encoding a nucleotide molecule comprising at least one miR-485 binding site
  • Some aspects of the present disclosure is directed to the use of such miR-485 inhibitors to treat various diseases and disorders associated with mitochondrial dysfunction.
  • Mitochondria are the powerhouse of nearly all cells, converting the energy of food molecules into ATP that powers most cell functions. There is growing evidence that mitochondrial dysfunction is associated with a broad range of human diseases.
  • RNA-485 inhibitor a compound that inhibits an activity of miRNA-485 (miRNA-485 inhibitor).
  • the mitochondrial dysfunction is associated with a disease selected from: a Leigh syndrome (LS), Ataxia telangiectasia (AT), spinocerebellar ataxia (SCA), Danon disease (DD), Autosomal dominant optic atrophy (ADOA), Barth syndrome (BTHS), Charcot-Marie tooth disease, Autosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS), Cockayne syndrome (CS), fabry disease, Fanconi anemia (FA), Vici syndrome (VICIS), Zellweger syndrome (ZS), Werner syndrome (WS), Sengers syndrome, GRACILE syndrome, or a combination thereof.
  • LS Leigh syndrome
  • AT Ataxia telangiectasia
  • SCA spinocerebellar ataxia
  • DD Danon disease
  • ADOA Autosomal dominant optic atrophy
  • BTHS Barth syndrome
  • Charcot-Marie tooth disease Autosomal recessive spastic ataxia of Charlevoix-Saguen
  • Also provided herein is a method of inducing mitophagy in a subject in need thereof, comprising administering to the subject a compound that inhibits an activity of miRNA-485 (miRNA-485 inhibitor).
  • miRNA-485 inhibitor an activity of miRNA-485
  • the level and/or activity of a gene associated with mitophagy is increased in the subject.
  • the level and/or activity of the gene associated with mitophagy is increased by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 100% or more, compared to a reference level and/or activity (e.g., the corresponding level and/or activity in the subject prior to the administration).
  • the gene associated with mitophagy is selected from: PINK1, PARKIN, LC3, LAMP2, or a combination thereof.
  • a method of increasing the level and/or activity of a gene associated with mitophagy in a cell of a subject in need thereof comprising contacting the cell with a compound that inhibits an activity of miRNA-485 (miRNA-485 inhibitor).
  • miRNA-485 inhibitor an activity of miRNA-485
  • the level and/or activity of the gene associated with mitophagy is increased by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 100% or more, compared to a corresponding level and/or activity in a reference cell (e.g., the cell prior to the contacting).
  • the gene associated with mitophagy is selected from: PINK1, PARKIN, LC3, LAMP2, or a combination thereof.
  • a method of increasing one or more mitochondrial function in a cell of a subject in need thereof comprising contacting the cell with a compound that inhibits an activity of miRNA-485 (miRNA-485 inhibitor).
  • the one or more mitochondrial function in the cell is increased by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 100% or more, compared to a reference mitochondrial function (e.g., the corresponding mitochondrial function in the cell prior to the contacting).
  • the one or more mitochondrial function comprises a mitochondrial respiration, glycolysis, mitochondrial biogenesis, mitochondrial fusion, mitochondrial fission, or combinations thereof.
  • Also provided herein is a method of reducing the amount of reactive oxygen species (ROS) produced by a cell of a subject in need thereof, comprising contacting the cell with a compound that inhibits an activity of miRNA-485 (miRNA-485 inhibitor).
  • the amount of ROS produced by the cell is reduced by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or about 100%, compared to the amount of ROS produced by a reference cell (e.g., the cell prior to the contacting).
  • the amount of ROS produced is determined by measuring the level of MitoSOX using flow cytometry.
  • the contacting occurs in vivo in the subject
  • the miRNA-485 inhibitor is administered to the subject prior to the contacting.
  • the contacting occurs ex vivo.
  • a method of reducing the level of reactive oxygen species (ROS) in a subject in need thereof comprising administering to the subject a compound that inhibits an miRNA-485 (miRNA-485 inhibitor).
  • the level of ROS in the subject is reduced by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or about 100%.
  • the subject suffers from a disease selected from: a Leigh syndrome (LS), Ataxia telangiectasia (AT), spinocerebellar ataxia (SCA), Danon disease (DD), Autosomal dominant optic atrophy (ADOA), Barth syndrome (BTHS), Charcot-Marie tooth disease, Autosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS), Cockayne syndrome (CS), fabry disease, Fanconi anemia (FA), Vici syndrome (VICIS), Zellweger syndrome (ZS), Werner syndrome (WS), Sengers syndrome, GRACILE syndrome, or a combination thereof.
  • LS Leigh syndrome
  • AT Ataxia telangiectasia
  • SCA spinocerebellar ataxia
  • DD Danon disease
  • ADOA Autosomal dominant optic atrophy
  • BTHS Barth syndrome
  • Charcot-Marie tooth disease Autosomal recessive spastic ataxia of Charlevoix-Saguenay
  • the miRNA-485 comprises miR485-3p.
  • the miR485-3p comprises 5'- gucauacacggcucuccucucu-3' (SEQ ID NO: 1).
  • the miRNA-485 inhibitor comprises a nucleotide sequence comprising 5'- UGUAUGA-3' (SEQ ID NO: 2) and wherein the miRNA-485 inhibitor comprises about 6 to about 30 nucleotides in length.
  • the miRNA-485 inhibitor comprises at least 1 nucleotide, at least 2 nucleotides, at least 3 nucleotides, at least 4 nucleotides, at least 5 nucleotides, at least 6 nucleotides, at least 7 nucleotides, at least 8 nucleotides, at least 9 nucleotides, at least 10 nucleotides, at least 11 nucleotides, at least 12 nucleotides, at least 13 nucleotides, at least 14 nucleotides, at least 15 nucleotides, at least 16 nucleotides, at least 17 nucleotides, at least 18 nucleotides, at least 19 nucleotides, or at least 20 nucleotides at the 5' of the nucleotide sequence.
  • the miRNA inhibitor comprises at least 1 nucleotide, at least 2 nucleotides, at least 3 nucleotides, at least 4 nucleotides, at least 5 nucleotides, at least 6 nucleotides, at least 7 nucleotides, at least 8 nucleotides, at least 9 nucleotides, at least 10 nucleotides, at least 11 nucleotides, at least 12 nucleotides, at least 13 nucleotides, at least 14 nucleotides, at least 15 nucleotides, at least 16 nucleotides, at least 17 nucleotides, at least 18 nucleotides, at least 19 nucleotides, or at least 20 nucleotides at the 3' of the nucleotide sequence.
  • the miRNA inhibitor has a sequence selected from the group consisting of: 5'-UGUAUGA-3' (SEQ ID NO: 2), 5'-GUGUAUGA-3' (SEQ ID NO: 3), 5'- CGUGUAUGA-3' (SEQ ID NO: 4), 5'-CCGUGUAUGA-3' (SEQ ID NO: 5), 5'- GCCGUGUAUGA-3' (SEQ ID NO: 6), 5'-AGCCGUGUAUGA-3' (SEQ ID NO: 7), 5'- GAGCCGUGUAUGA-3' (SEQ ID NO: 8), 5'-AGAGCCGUGUAUGA-3' (SEQ ID NO: 9), 5'- GAGAGCCGUGUAUGA-3' (SEQ ID NO: 10), 5'-GGAGAGCCGUGUAUGA-3' (SEQ ID NO: 11), 5'-AGGAGAGCCGUGUAUGA-3' (SEQ ID NO: 12), 5'- GAGGAGAGCCGUGUAUGA-3' (SEQ ID NO:
  • the miRNA inhibitor has a sequence selected from the group consisting of: 5'-TGTATGA-3' (SEQ ID NO: 62), 5'-GTGTATGA-3' (SEQ ID NO: 63), 5'-CGTGTATGA-3' (SEQ ID NO: 64), 5'- CCGTGTATGA-3' (SEQ ID NO: 65), 5'-GCCGTGTATGA-3' (SEQ ID NO: 66), 5'- AGCCGTGTATGA-3' (SEQ ID NO: 67), 5'-GAGCCGTGTATGA-3' (SEQ ID NO: 68), 5'- AGAGCCGTGTATGA-3' (SEQ ID NO: 69), 5'-GAGAGCCGTGTATGA-3' (SEQ ID NO: 70), 5'-GGAGAGCCGTGTATGA-3' (SEQ ID NO: 71), 5'-AGGAGAGCCGTGTATGA-3' (SEQ ID NO: 72), 5'-GAGGAGAGCCGTGTATGA-3' (SEQ ID
  • sequence of the miRNA inhibitor is at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% sequence identity to 5'-AGAGAGGAGAGCCGUGUAUGAC-3' (SEQ ID NO: 30) or 5'- AGAGAGGAGAGCCGTGTATGAC-3' (SEQ ID NO: 90).
  • the miRNA inhibitor has a sequence that has at least 90% similarity to 5'- AGAGAGGAGAGCCGUGUAUGAC-3' (SEQ ID NO: 30) or 5'- AGAGAGGAGAGCCGTGTATGAC-3' (SEQ ID NO: 90).
  • the miRNA inhibitor comprises the nucleotide sequence 5'-AGAGAGGAGAGCCGUGUAUGAC-3' (SEQ ID NO: 30) or 5'-AGAGAGGAGAGCCGTGTATGAC-3' (SEQ ID NO: 90) with one substitution or two substitutions.
  • the miRNA inhibitor comprises the nucleotide sequence 5'-AGAGAGGAGAGCCGUGUAUGAC-3' (SEQ ID NO: 30) or 5'- AGAGAGGAGAGCCGTGTATGAC-3' (SEQ ID NO: 90). In some aspects, the miRNA inhibitor comprises the nucleotide sequence 5'- AGAGAGGAGAGCCGUGUAUGAC-3' (SEQ ID NO: 30). [0018] In some aspects, the miRNA inhibitor comprises at least one modified nucleotide.
  • the at least one modified nucleotide comprises a locked nucleic acid (LNA), an unlocked nucleic acid (UNA), an arabino nucleic acid (ABA), a bridged nucleic acid (BNA), a peptide nucleic acid (PNA), or a combination thereof.
  • the miRNA inhibitor comprises a backbone modification.
  • the backbone modification comprises a phosphorodiamidate morpholino oligomer (PMO), a phosphorothioate (PS) modification, or both.
  • PMO phosphorodiamidate morpholino oligomer
  • PS phosphorothioate
  • the miRNA inhibitor is delivered in a delivery agent.
  • the delivery agent comprises a micelle, an exosome, a lipidoid, a liposome, a lipoplex, a lipid nanoparticle, an extracellular vesicle, a synthetic vesicle, a polymeric compound, a peptide, a protein, a cell, a nanoparticle mimic, a nanotube, a conjugate, a viral vector, or combinations thereof.
  • the delivery agent comprises a cationic carrier unit comprising: [WP]-L1-[CC]-L2-[AM] (formula I) or [WP]-L1-[AM]-L2-[CC] (formula II), wherein WP is a water-soluble polymer moiety; CC is a cationic carrier moiety; AM is an adjuvant moiety; and L1 and L2 are independently optional linkers.
  • WP is a water-soluble polymer moiety
  • CC is a cationic carrier moiety
  • AM is an adjuvant moiety;
  • L1 and L2 are independently optional linkers.
  • the cationic carrier unit and the miRNA-485 inhibitor are capable of associating with each other to form a micelle when mixed together.
  • the association is via a covalent bond.
  • the association is via a non-covalent bond.
  • the non-covalent bond comprises an ionic bond.
  • the water-soluble polymer moiety comprises poly(alkylene glycols), poly(oxyethylated polyol), poly(olefinic alcohol), poly(vinylpyrrolidone), poly(hydroxyalkylmethacrylamide), poly(hydroxyalkylmethacrylate), poly(saccharides), poly( ⁇ -hydroxy acid), poly(vinyl alcohol), polyglycerol, polyphosphazene, polyoxazolines (“POZ”) poly(N-acryloylmorpholine), or combinations thereof.
  • the water-soluble polymer moiety comprises polyethylene glycol ("PEG”), polyglycerol, or poly(propylene glycol) (“PPG”). [0022] In some aspects, the water-soluble polymer moiety comprises: , (formula III), wherein n is 1-1000.
  • the n is at least about 110, at least about 111, at least about 112, at least about 113, at least about 114, at least about 115, at least about 116, at least about 117, at least about 118, at least about 119, at least about 120, at least about 121, at least about 122, at least about 123, at least about 124, at least about 125, at least about 126, at least about 127, at least about 128, at least about 129, at least about 130, at least about 131, at least about 132, at least about 133, at least about 134, at least about 135, at least about 136, at least about 137, at least about 138, at least about 139, at least about 140, or at least about 141.
  • the n is about 80 to about 90, about 90 to about 100, about 100 to about 110, about 110 to about 120, about 120 to about 130, about 140 to about 150, or about 150 to about 160.
  • the water-soluble polymer moiety is linear, branched, or dendritic.
  • the cationic carrier moiety comprises one or more basic amino acids.
  • the cationic carrier moiety comprises at least about three, at least about four, at least about five, at least about six, at least about seven, at least about eight, at least about nine, at least about ten, at least about 11, at least about 12, at least about 13, at least about 14, at last about 15, at least about 16, at least about 17, at least about 18, at least about 19, at least about 20, at least about 21, at least about 22, at least about 23, at least about 24, at least about 25, at least about 26, at least about 27, at least about 28, at least about 29, at least about 30, at least about 31, at least about 32, at least about 33, at least about 34, at least about 35, at least about 36, at least about 37, at least about 38, at least about 39, at least about 40, at least about 41, at least about 42, at least about 43, at least about 44, at least about 45, at least about 46, at least about 47, at least about 48, at least about 49, or at least about 50 basic amino acids.
  • the cationic carrier moiety comprises about 30 to about 50 basic amino acids.
  • the basic amino acid comprises arginine, lysine, histidine, or any combination thereof.
  • the cationic carrier moiety comprises about 40 lysine monomers.
  • the adjuvant moiety is capable of modulating an immune response, an inflammatory response, and/or a tissue.
  • the adjuvant moiety comprises an imidazole derivative, an amino acid, a vitamin, or any combination thereof.
  • the adjuvant moiety comprises: , (formula IV), wherein each of G1 and G2 is H, an aromatic ring, or 1-10 alkyl, or G1 and G2 together form an aromatic ring, and wherein n is 1-10.
  • the adjuvant moiety comprises nitroimidazole.
  • the adjuvant moiety comprises metronidazole, tinidazole, nimorazole, dimetridazole, pretomanid, ornidazole, megazol, azanidazole, benznidazole, or any combination thereof.
  • the adjuvant moiety comprises an amino acid.
  • the adjuvant moiety comprises (formula V), wherein wherein each of Z1 and Z2 is H or OH.
  • the adjuvant moiety comprises a vitamin.
  • the vitamin comprises a cyclic ring or cyclic hetero atom ring and a carboxyl group or hydroxyl group.
  • the vitamin comprises: (formula VI), wherein each of Y1 and Y2 is C, N, O, or S, and wherein n is 1 or 2.
  • the vitamin is selected from the group consisting of vitamin A, vitamin B1, vitamin B2, vitamin B3, vitamin B6, vitamin B7, vitamin B9, vitamin B12, vitamin C, vitamin D2, vitamin D3, vitamin E, vitamin M, vitamin H, and any combination thereof.
  • the vitamin is vitamin B3.
  • the adjuvant moiety comprises at least about two, at least about three, at least about four, at least about five, at least about six, at least about seven, at least about eight, at least about nine, at least about ten, at least about 11, at least about 12, at least about 13, at least about 14, at least about 15, at least about 16, at least about 17, at least about 18, at least about 19, or at least about 20 vitamin B3.
  • the adjuvant moiety comprises about 10 vitamin B3.
  • the delivery agent comprises: (i) a water-soluble polymer moiety with about 120 to about 130 PEG units, (ii) a cationic carrier moiety comprising a poly-lysine with about 30 to about 40 lysines, and (iii) an adjuvant moiety with about 5 to about 10 vitamin B3.
  • the cationic carrier unit is capable of protecting the miRNA-485 inhibitor from enzymatic degradation.
  • the delivery agent comprises a cationic carrier unit comprising: [CC]-L1-[CM]-L2-[HM] (Schema I); [CC]-L1-[HM]-L2-[CM] (Schema II); [HM]-L1-[CM]-L2-[CC] (Schema III); [HM]-L1-[CC]-L2-[CM] (Schema IV); [CM]-L1-[CC]-L2-[HM] (Schema V); or [CM]-L1-[HM]-L2-[CC] (Schema VI); wherein CC is a positively charged carrier moiety; CM is a crosslinking moiety; HM is a hydrophobic moiety; and, L1 and L2 are independently optional linkers, and wherein the number of HM is less than 40% relative to [CC] and [CM].
  • the number of HM is less than 39%, less than about 35%, less than about 30%, less than about 25%, less than about 20%, less than about 15%, less than about 10%, less than about 9%, less than about 8%, less than about 7%, less than about 6%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, or about 1% relative to [CC] and [CM].
  • the cationic carrier unit is capable of interacting with the miR-485 inhibitor.
  • the cationic carrier moiety comprises at least about three, at least about four, at least about five, at least about six, at least about seven, at least about eight, at least about nine, at least about ten, at least about 11, at least about 12, at least about 13, at least about 14, at least 15, at least about 16, at least about 17, at least about 18, at least about 19, at least about 20, at least about 21, at least about 22, at least about 23, at least about 24, at least about 25, at least about 26, at least about 27, at least about 28, at least about 29, at least about 30, at least about 31, at least about 32, at least about 33, at least about 34, at least about 35, at least about 36, at least about 37, at least about 38, at least about 39, at least about 40, at least about 41, at least about 42, at least about 43, at least about 44, at least about 45, at least about 46, at least about 47, at least about 48, at least about 49, at least about 50, at least about 51, at least about 52, at least about 53, at least about 54, at least about 55
  • the cationic carrier moiety comprises about 80 amino acids. In some aspects, they amino acids comprise lysines. [0035] In some aspects, the hydrophobic moiety comprises at least about two, at least about three, at least about four, at least about five, at least about six, at least about seven, at least about eight, at least about nine, at least about ten, at least about 11, at least about 12, at least about 13, at least about 14, at least about 15, at least about 16, at least about 17, at least about 18, at least about 19, at least about 20, at least about 21, at least about 22, at least about 23, at least about 24, at least about 25, at least about 26, at least about 27, at least about 28, at least about 29, at least about 30, at least about 31, at least about 32, at least about 33, at least about 34, or at least about 35 amino acids, each linked to a vitamin.
  • the hydrophobic moiety comprises about two vitamin B3, about three vitamin B3, about four vitamin B3, about five vitamin B3, about six vitamin B3, about seven vitamin B3, about eight vitamin B3, about nine vitamin B3, about ten vitamin B3, about 11 vitamin B3, about 12 vitamin B3, about 13 vitamin B3, about 14 vitamin B3, about 15 vitamin B3, about 16 vitamin B3, about 17 vitamin B3, about 18 vitamin B3, about 19 vitamin B3, about 20 vitamin B3, about 21 vitamin B3, about 22 vitamin B3, about 23 vitamin B3, about 24 vitamin B3, about 25 vitamin B3, about 26 vitamin B3, about 27 vitamin B3, about 28 vitamin B3, about 29 vitamin B3, about vitamin B3, about 31 vitamin B3, about 32 vitamin B3, about 33 vitamin B3, about 34 vitamin B3, or about 35 vitamin B3.
  • the cationic carrier moiety comprises about 35 to about 45 lysines, the crosslinking moiety comprises about 5 to about 40 lysine-thiol, and the hydrophobic moiety comprises about 1 to about 10 lysine-vitamin B3. In some aspects, the cationic carrier moiety comprises about 40 lysines, the crosslinking moiety comprises about 35 lysine-thiol, and the hydrophobic moiety comprises about 5 lysine-vitamin B3. [0037] In some aspects, the water-soluble biopolymer moiety comprises about 120 to about 130 PEG units. In some aspects, the water-soluble biopolymer moiety comprises about 114 PEG units.
  • the miRNA inhibitor is administered parenthetically, intramuscularly, subcutaneously, ophthalmic, intravenously, intraperitoneally, intradermally, intraorbitally, intracerebrally, intracranially, intracerebroventricularly, intraspinally, intraventricular, intrathecally, intracistemally, intracapsularly, intratumorally, topically, or any combination thereof.
  • the delivery agent is a micelle.
  • the micelle comprises (i) about 100 to about 200 PEG units, (ii) about 30 to about 40 lysines, each with an amine group, (iii) about 15 to about 20 lysines, each with a thiol group, and (iv) about 30 to about 40 lysines, each linked to vitamin B3.
  • the micelle comprises (i) about 120 to about 130 PEG units, (ii) about 32 lysines, each with an amine group, (iii) about 16 lysines, each with a thiol group, and (iv) about 32 lysines, each linked to vitamin B3.
  • a targeting moiety is further linked to the PEG units.
  • the targeting moiety is a LAT1 targeting ligand. In some aspects, the targeting moiety is phenylalanine.
  • FIGs. 1A-1H show the ability of a miR-485 inhibitor to regulate mitochondrial dysfunction via mitophagy and mitochondrial biogenesis.
  • FIG.1A provides Western blot analysis of MAP1LC3B, SQSTM1/p62, SIRT1, Pink1, Parkin protein expression in primary microglia treated with the following: (i) no treatment ("Con"), (ii) transfected with miR-485 inhibitor (100 nM) alone, (iii) treated with LPS (100 ng/mL) alone, or (iv) treated with LPS (100 ng/mL) and transfected with miR-485 inhibitor (100 nM).
  • FIG.1B provides Western blot analysis of MAP1LC3b, SIRT1, Pink1, Parkin, and VDAC1 protein expression in cytosolic or mitochondrial fractions of primary microglia treated as described in FIG.1A.
  • FIG.1C provides oxygen consumption rate (OCR) analysis in primary microglia treated as described in FIG.1A.
  • FIG. 1D provides comparison of basal respiration (left graph), maximum respiration (center graph), and ATP production (right graph) as determined based on the OCR analysis shown in FIG. 1C.
  • FIG. 1E provides extracellular acidification rate (ECAR) analysis in primary microglia treated as described in FIG. 1A.
  • FIG. 1F provides comparison of glycolysis (left graph), glycolytic capacity (center graph), and glycolytic reserve (right graph) as determined based on the ECAR analysis shown in FIG. 1E.
  • FIG. 1G provides comparison of MitoSOX protein expression (indicator of mitochondrial reactive oxygen species), as measured using flow cytometry in primary microglia treated as described in FIG.1A.
  • the first flow histogram plot (“A") provides an overlay of MitoSOX expression in primary microglia from all the different treatment groups. Unstained cells were used as control.
  • the second flow histogram plot (“B”) provides an overlay of MitoSOX expression in non-treated primary microglia and primary microglia transfected with miR-485 inhibitor alone (i.e., no LPS treatment).
  • the third flow histogram plot (“C”) provides an overlay of MitoSOX expression in non-treated primary microglia and primary microglia treated with LPS alone (i.e., no miR-485 inhibitor).
  • the fourth flow histogram plot (“D") provides an overlay of MitoSOX expression in primary microglia treated with LPS alone and primary microglia treated with LPS and transfected with a miR- 485 inhibitor.
  • FIG.1H shows the results provided in FIG.1G as a bar graph. In each of FIGs. 1A-1H, All data are mean ⁇ SD. *P ⁇ 0.05, **P ⁇ 0.01 and ****P ⁇ 0.0001 compared to the control (Con). ##P ⁇ 0.01 and ####P ⁇ 0.0001 compared to the LPS treated control. [0041] FIGs.
  • FIG.2A provides Western blot analysis of MAP1LC3B, SQSTM1/p62, SIRT1, Pink1, Parkin protein expression in primary microglia treated with the following: (i) no treatment, (ii) treated with A ⁇ oligomers (A ⁇ O) (5 ⁇ M) alone, or (iii) treated with A ⁇ O (5 ⁇ M) and transfected with miR-485 inhibitor (100 nM).
  • the primary microglia were treated either in the presence or absence of bafA1 (inhibitor of autophagosome fusion).
  • FIG. 2B provides Western blot analysis of MAP1LC3b, SIRT1, Pink1, Parkin and VDAC1 protein expression in cytosolic or mitochondrial fractions of primary microglia treated as described in FIG. 2A.
  • FIG. 2C provides Western blot analysis of PGC-1 ⁇ , NRF1, NRF2, TFAM protein expression in primary microglia treated with the following: (i) no treatment, (ii) transfected with miR-485 inhibitor (100 nM) alone, (iii) treated with A ⁇ O (5 ⁇ M) alone, or (iv) treated with A ⁇ O (5 ⁇ M) and transfected with miR-485 inhibitor (100 nM).
  • FIG.2D provides Western blot analysis of DRP1 and p-DRP1 (S616, S637) protein expression in primary microglia treated as described in FIG. 2C.
  • FIG. 2E provides Western blot analysis of MFN1, MFN2, OPA1 protein expression in primary microglia treated as described in FIG. 2C.
  • FIG. 2F provides OCR analysis in primary microglia treated with A ⁇ O (5 ⁇ M) alone (square) or in combination with a miR-485 inhibitor (100 nM) (triangle). Non-treated cells were used as control (circle).
  • FIG. 2G provides comparison of basal respiration (left graph), maximum respiration (center graph), and ATP production (right graph) as determined based on the OCR analysis shown in FIG.2F.
  • FIG.2H provides ECAR analysis of primary microglia treated as described in FIG. 2F.
  • FIG. 2I provides comparison of glycolysis (left graph), glycolytic capacity (center graph), and glycolytic reserve (right graph) as determined based on the ECAR analysis shown in FIG. 2H. All data are mean ⁇ SD. ***P ⁇ 0.001, and ****P ⁇ 0.0001 compared to the control (Con). ##P ⁇ 0.01, ###P ⁇ 0.001 and ####P ⁇ 0.0001 compared to the A ⁇ Os treated control.
  • FIGs. 3A-3D shows the ability of miR-485 inhibitor to regulate microglia dysfunction and thereby improve cognitive decline in A ⁇ O treated mice.
  • FIG. 3A and 3B show relative miR-485-3p expression in acutely isolated astrocytes and microglia, respectively, from mice that were treated with the following: (i) no treatment (i.e., wild-type), (ii) A ⁇ O (2 ⁇ g) alone, or (iii) both A ⁇ O (2 ⁇ g) and miR-485 inhibitor (5 mg/kg).
  • FIG.3C provides OCR analysis in the hippocampal tissue of mice from the different treatment groups as described in FIGs. 3A and 3B.
  • FIG. 3D provides comparison of basal respiration (left graph), maximum respiration (center graph), and ATP production (right graph) as determined based on the OCR analysis shown in FIG.3C.
  • the present disclosure is generally directed to the use of a miR-485 inhibitor to improve and/or prevent mitochondrial dysfunction.
  • the miR-485 inhibitors useful for the present disclosure comprises a nucleotide sequence with at least one miR-485 binding site, and wherein the nucleotide sequence does not encode a protein.
  • such miR-485 inhibitors are capable of improving and/or preventing mitochondrial dysfunction in a variety of manners.
  • a miR-485 inhibitor can promote mitophagy, mitochondrial respiration, glycolysis, mitochondrial biogenesis, mitochondrial dynamics, and/or combinations thereof.
  • ranges recited are understood to be shorthand for all of the values within the range, inclusive of the recited endpoints.
  • a range of 1 to 10 is understood to include any number, combination of numbers, or sub-range from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10.
  • nucleotide sequences are written left to right in 5' to 3' orientation. Nucleotides are referred to herein by their commonly known one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Accordingly, 'a' represents adenine, 'c' represents cytosine, 'g' represents guanine, 't' represents thymine, and 'u' represents uracil. [0052] Amino acid sequences are written left to right in amino to carboxy orientation. Amino acids are referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission.
  • AAV adeno-associated virus
  • AAV includes but is not limited to, AAV type 1, AAV type 2, AAV type 3 (including types 3A and 3B), AAV type 4, AAV type 5, AAV type 6, AAV type 7, AAV type 8, AAV type 9, AAV type 10, AAV type 11, AAV type 12, AAV type 13, AAVrh.74, snake AAV, avian AAV, bovine AAV, canine AAV, equine AAV, ovine AAV, goat AAV, shrimp AAV, those AAV serotypes and clades disclosed by Gao et al. (J. Virol.78:6381 (2004)) and Moris et al.
  • an "AAV” includes a derivative of a known AAV.
  • an “AAV” includes a modified or an artificial AAV.
  • the terms "administration,” “administering,” and grammatical variants thereof refer to introducing a composition, such as a miRNA inhibitor of the present disclosure, into a subject via a pharmaceutically acceptable route.
  • a composition such as a micelle comprising a miRNA inhibitor of the present disclosure
  • introduction of a composition is by any suitable route, including intratumorally, orally, pulmonarily, intranasally, parenterally (intravenously, intra-arterially, intramuscularly, intraperitoneally, or subcutaneously), rectally, intralymphatically, intrathecally, periocularly or topically.
  • Administration includes self-administration and the administration by another.
  • a suitable route of administration allows the composition or the agent to perform its intended function. For example, if a suitable route is intravenous, the composition is administered by introducing the composition or agent into a vein of the subject.
  • the term “associated with” refers to a close relationship between two or more entities or properties.
  • a disease or condition that can be treated with the present disclosure e.g., disease or condition associated with a mitochondrial dysfunction
  • the term “associated with” refers to an increased likelihood that a subject suffers from the disease or condition when the subject exhibits a mitochondrial dysfunction.
  • the mitochondrial dysfunction causes the disease or condition.
  • the mitochondrial dysfunction does not necessarily cause but is correlated with the disease or condition.
  • the term “approximately,” as applied to one or more values of interest refers to a value that is similar to a stated reference value.
  • the term “approximately” refers to a range of values that fall within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than or less than) of the stated reference value unless otherwise stated or otherwise evident from the context (except where such number would exceed 100% of a possible value).
  • abnormal level refers to a level (expression and/or activity) that differs (e.g., increased or decreased) from a reference subject, e.g., who does not suffer from a disease or condition described herein.
  • an abnormal level refers to a level that is increased by at least about 0.1-fold, at least about 0.2-fold, at least about 0.3-fold, at least about 0.4-fold, at least about 0.5-fold, at least about 0.6-fold, at least about 0.7-fold, at least about 0.8-fold, at least about 0.9-fold, at least about 1-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 10-fold, at least about 20-fold, at least about 30-fold, at least about 40-fold, at least about 50-fold, at least about 75-fold, at least about 100-fold, at least about 200-fold, at least about 300-fold, at least about 400-fold, at least about 500-fold, at least about 750-fold, or at least about 1,000-fold or more compared to the corresponding level in a reference subject (e.g., subject
  • an abnormal level refers to a level that is decreased by at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or about 100% compared to the corresponding level in a reference subject (e.g., subject who does not suffer from a disease or condition described herein).
  • Nucleotides or amino acids that are relatively conserved are those that are conserved amongst more related sequences than nucleotides or amino acids appearing elsewhere in the sequences.
  • two or more sequences are said to be "completely conserved” or “identical” if they are 100% identical to one another.
  • two or more sequences are said to be "highly conserved” if they are at least 70% identical, at least 80% identical, at least 90% identical, or at least 95% identical to one another. In some aspects, two or more sequences are said to be “highly conserved” if they are about 70% identical, about 80% identical, about 90% identical, about 95%, about 98%, or about 99% identical to one another. In some aspects, two or more sequences are said to be "conserved” if they are at least 30% identical, at least 40% identical, at least 50% identical, at least 60% identical, at least 70% identical, at least 80% identical, at least 90% identical, or at least 95% identical to one another.
  • two or more sequences are said to be "conserved” if they are about 30% identical, about 40% identical, about 50% identical, about 60% identical, about 70% identical, about 80% identical, about 90% identical, about 95% identical, about 98% identical, or about 99% identical to one another.
  • Conservation of sequence can apply to the entire length of a polynucleotide or polypeptide or can apply to a portion, region or feature thereof.
  • the term "derived from,” as used herein, refers to a component that is isolated from or made using a specified molecule or organism, or information (e.g., amino acid or nucleic acid sequence) from the specified molecule or organism.
  • a nucleic acid sequence that is derived from a second nucleic acid sequence can include a nucleotide sequence that is identical or substantially similar to the nucleotide sequence of the second nucleic acid sequence.
  • the derived species can be obtained by, for example, naturally occurring mutagenesis, artificial directed mutagenesis or artificial random mutagenesis.
  • the mutagenesis used to derive nucleotides or polypeptides can be intentionally directed or intentionally random, or a mixture of each.
  • the mutagenesis of a nucleotide or polypeptide to create a different nucleotide or polypeptide derived from the first can be a random event (e.g., caused by polymerase infidelity) and the identification of the derived nucleotide or polypeptide can be made by appropriate screening methods, e.g., as discussed herein.
  • a nucleotide or amino acid sequence that is derived from a second nucleotide or amino acid sequence has a sequence identity of at least about 50%, at least about 51%, at least about 52%, at least about 53%, at least about 54%, at least about 55%, at least about 56%, at least about 57%, at least about 58%, at least about 59%, at least about 60%, at least about 61%, at least about 62%, at least about 63%, at least about 64%, at least about 65%, at least about 66%, at least about 67%, at least about 68%, at least about 69%, at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 8
  • a "coding region” or “coding sequence” is a portion of polynucleotide which consists of codons translatable into amino acids. Although a “stop codon” (TAG, TGA, or TAA) is typically not translated into an amino acid, it can be considered to be part of a coding region, but any flanking sequences, for example promoters, ribosome binding sites, transcriptional terminators, introns, and the like, are not part of a coding region.
  • a coding region typically determined by a start codon at the 5' terminus, encoding the amino terminus of the resultant polypeptide, and a translation stop codon at the 3' terminus, encoding the carboxyl terminus of the resulting polypeptide.
  • the terms "complementary” and “complementarity” refer to two or more oligomers (i.e., each comprising a nucleobase sequence), or between an oligomer and a target gene, that are related with one another by Watson-Crick base-pairing rules.
  • nucleobase sequence "T-G-A (5' ⁇ 3'),” is complementary to the nucleobase sequence "A-C-T (3' ⁇ 5').”
  • Complementarity can be “partial,” in which less than all of the nucleobases of a given nucleobase sequence are matched to the other nucleobase sequence according to base pairing rules.
  • complementarity between a given nucleobase sequence and the other nucleobase sequence can be about 70%, about 75%, about 80%, about 85%, about 90%, or about 95%.
  • the term "complementary” refers to at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% match or complementarity to a target nucleic acid sequence (e.g., miR-485 nucleic acid sequence). Or, there can be “complete” or “perfect” (100%) complementarity between a given nucleobase sequence and the other nucleobase sequence to continue the example. In some aspects, the degree of complementarity between nucleobase sequences has significant effects on the efficiency and strength of hybridization between the sequences.
  • downstream refers to a nucleotide sequence that is located 3' to a reference nucleotide sequence.
  • downstream nucleotide sequences relate to sequences that follow the starting point of transcription.
  • the translation initiation codon of a gene is located downstream of the start site of transcription.
  • carrier refers to an inert substance added to a pharmaceutical composition to further facilitate administration of a compound, e.g., a miRNA inhibitor of the present disclosure.
  • RNA or a polypeptide refers to a process by which a polynucleotide produces a gene product, e.g., RNA or a polypeptide. It includes without limitation transcription of the polynucleotide into micro RNA binding site, small hairpin RNA (shRNA), small interfering RNA (siRNA), or any other RNA product. It includes, without limitation, transcription of the polynucleotide into messenger RNA (mRNA), and the translation of mRNA into a polypeptide. Expression produces a "gene product.”
  • a gene product can be, e.g., a nucleic acid, such as an RNA produced by transcription of a gene.
  • a gene product can be either a nucleic acid, RNA or miRNA produced by the transcription of a gene, or a polypeptide which is translated from a transcript.
  • Gene products described herein further include nucleic acids with post transcriptional modifications, e.g., polyadenylation or splicing, or polypeptides with post translational modifications, e.g., phosphorylation, methylation, glycosylation, the addition of lipids, association with other protein subunits, or proteolytic cleavage.
  • the term "expression” can be used interchangeable with the term “level.”
  • the term “miR-485-3p expression” can be synonymous with the term “miR-485-3p level.”
  • the term “homology” refers to the overall relatedness between polymeric molecules, e.g. between nucleic acid molecules. Generally, the term “homology” implies an evolutionary relationship between two molecules. Thus, two molecules that are homologous will have a common evolutionary ancestor. In the context of the present disclosure, the term homology encompasses both to identity and similarity.
  • polymeric molecules are considered to be "homologous" to one another if at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% of the monomers in the molecule are identical (exactly the same monomer) or are similar (conservative substitutions).
  • the term "homologous” necessarily refers to a comparison between at least two sequences (e.g., polynucleotide sequences).
  • substitutions are conducted at the nucleic acid level, i.e., substituting an amino acid residue with an alternative amino acid residue is conducted by substituting the codon encoding the first amino acid with a codon encoding the second amino acid.
  • identity refers to the overall monomer conservation between polymeric molecules, e.g., between polynucleotide molecules.
  • identity without any additional qualifiers, e.g., polynucleotide A is identical to polynucleotide B, implies the polynucleotide sequences are 100% identical (100% sequence identity).
  • the length of a sequence aligned for comparison purposes is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100% of the length of the reference sequence.
  • the amino acids at corresponding amino acid positions, or bases in the case of polynucleotides are then compared.
  • a position in the first sequence is occupied by the same amino acid or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position.
  • the percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which needs to be introduced for optimal alignment of the two sequences.
  • Suitable software programs that can be used to align different sequences are available from various sources.
  • One suitable program to determine percent sequence identity is bl2seq, part of the BLAST suite of program available from the U.S. government's National Center for Biotechnology Information BLAST web site (blast.ncbi.nlm.nih.gov).
  • Bl2seq performs a comparison between two sequences using either the BLASTN or BLASTP algorithm.
  • BLASTN is used to compare nucleic acid sequences
  • BLASTP is used to compare amino acid sequences.
  • 80.11, 80.12, 80.13, and 80.14 are rounded down to 80.1, while 80.15, 80.16, 80.17, 80.18, and 80.19 are rounded up to 80.2. It also is noted that the length value will always be an integer.
  • sequence alignments can be generated by integrating sequence data with data from heterogeneous sources such as structural data (e.g., crystallographic protein structures), functional data (e.g., location of mutations), or phylogenetic data.
  • a suitable program that integrates heterogeneous data to generate a multiple sequence alignment is T-Coffee, available at worldwideweb.tcoffee.org, and alternatively available, e.g., from the EBI. It will also be appreciated that the final alignment used to calculate percent sequence identity can be curated either automatically or manually.
  • isolating or purifying as used herein is the process of removing, partially removing (e.g., a fraction) of a composition of the present disclosure, e.g., a miRNA inhibitor of the present disclosure from a sample containing contaminants.
  • an isolated composition has no detectable undesired activity or, alternatively, the level or amount of the undesired activity is at or below an acceptable level or amount. In some aspects, an isolated composition has an amount and/or concentration of desired composition of the present disclosure, at or above an acceptable amount and/or concentration and/or activity. In some aspects, the isolated composition is enriched as compared to the starting material from which the composition is obtained.
  • This enrichment can be by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.9%, at least about 99.99%, at least about 99.999%, at least about 99.9999%, or greater than 99.9999% as compared to the starting material.
  • isolated preparations are substantially free of residual biological products.
  • the isolated preparations are 100% free, at least about 99% free, at least about 98% free, at least about 97% free, at least about 96% free, at least about 95% free, at least about 94% free, at least about 93% free, at least about 92% free, at least about 91% free, or at least about 90% free of any contaminating biological matter.
  • Residual biological products can include abiotic materials (including chemicals) or unwanted nucleic acids, proteins, lipids, or metabolites.
  • the term "linked" as used herein refers to a first amino acid sequence or polynucleotide sequence covalently or non-covalently joined to a second amino acid sequence or polynucleotide sequence, respectively.
  • the first amino acid or polynucleotide sequence can be directly joined or juxtaposed to the second amino acid or polynucleotide sequence or alternatively an intervening sequence can covalently join the first sequence to the second sequence.
  • the term "linked" means not only a fusion of a first polynucleotide sequence to a second polynucleotide sequence at the 5'-end or the 3'-end, but also includes insertion of the whole first polynucleotide sequence (or the second polynucleotide sequence) into any two nucleotides in the second polynucleotide sequence (or the first polynucleotide sequence, respectively).
  • the first polynucleotide sequence can be linked to a second polynucleotide sequence by a phosphodiester bond or a linker.
  • the linker can be, e.g., a polynucleotide.
  • a "miRNA inhibitor,” as used herein, refers to a compound that can decrease, alter, and/or modulate miRNA expression, function, and/or activity.
  • the miRNA inhibitor can be a polynucleotide sequence that is at least partially complementary to the target miRNA nucleic acid sequence, such that the miRNA inhibitor hybridizes to the target miRNA sequence.
  • a miRNA inhibitor useful for the present disclosure comprises a nucleotide sequence encoding a nucleotide molecule that is at least partially complementary to a target miR-485 nucleic acid sequence, such that the miRNA inhibitor hybridizes to the miR-485 sequence.
  • a miRNA inhibitor is also referred to herein as a "miR-485 inhibitor.”
  • the hybridization of the miR-485 to the miR-485 sequence decreases, alters, and/or modulates the expression, function, and/or activity of miR-485.
  • miRNA inhibitor can be used interchangeably.
  • miRNA refers to a microRNA molecule found in eukaryotes that is involved in RNA-based gene regulation.
  • the term will be used to refer to the single-stranded RNA molecule processed from a precursor.
  • antisense oligomers can also be used to describe the microRNA molecules of the present disclosure. Names of miRNAs and their sequences related to the present disclosure are provided herein. MicroRNAs recognize and bind to target mRNAs through imperfect base pairing leading to destabilization or translational inhibition of the target mRNA and thereby downregulate target gene expression.
  • miRNA binding site a molecule comprising a sequence complementary to the seed region of the miRNA
  • targeting miRNAs via molecules comprising a miRNA binding site can reduce or inhibit the miRNA-induced translational inhibition leading to an upregulation of the target gene.
  • one or more can occur with respect to the target nucleic acid sequence. Variations at any location within the oligomer are included.
  • antisense oligomers of the disclosure e.g., miR-485 inhibitor
  • antisense oligomers of the disclosure include variations in nucleobase sequence near the termini, variations in the interior, and if present are typically within about 6, 5, 4, 3, 2, or 1 subunits of the 5' and/or 3' terminus.
  • one, two, or three nucleobases can be removed and still provide on-target binding.
  • the terms “modulate,” “modify,” “regulate,” and grammatical variants thereof, generally refer when applied to a specific concentration, level, expression, function or behavior, to the ability to alter, by increasing or decreasing, e.g., directly or indirectly promoting/stimulating/up-regulating or interfering with/inhibiting/down-regulating the specific concentration, level, expression, function or behavior, such as, e.g., to act as an antagonist or agonist.
  • a modulator can increase and/or decrease a certain concentration, level, activity or function relative to a control, or relative to the average level of activity that would generally be expected or relative to a control level of activity.
  • a miRNA inhibitor disclosed herein e.g., a miR-485 inhibitor
  • miRNA inhibitors described herein are capable of regulating mitochondrial function.
  • Nucleic acid refers to the phosphate ester polymeric form of ribonucleosides (adenosine, guanosine, uridine or cytidine; "RNA molecules”) or deoxyribonucleosides (deoxyadenosine, deoxyguanosine, deoxythymidine, or deoxycytidine; "DNA molecules”), or any phosphoester analogs thereof, such as phosphorothioates and thioesters, in either single stranded form, or a double-stranded helix.
  • RNA molecules phosphate ester polymeric form of ribonucleosides
  • deoxyribonucleosides deoxyadenosine, deoxyguanosine, deoxythymidine, or deoxycytidine
  • DNA molecules or any phosphoester analogs thereof, such as phosphorothioates and thioesters, in either single stranded form, or a double-stranded
  • Single stranded nucleic acid sequences refer to single-stranded DNA (ssDNA) or single- stranded RNA (ssRNA). Double stranded DNA-DNA, DNA-RNA and RNA-RNA helices are possible.
  • nucleic acid molecule and in particular DNA or RNA molecule, refers only to the primary and secondary structure of the molecule, and does not limit it to any particular tertiary forms. Thus, this term includes double-stranded DNA found, inter alia, in linear or circular DNA molecules (e.g., restriction fragments), plasmids, supercoiled DNA and chromosomes.
  • a "recombinant DNA molecule” is a DNA molecule that has undergone a molecular biological manipulation.
  • DNA includes, but is not limited to, cDNA, genomic DNA, plasmid DNA, synthetic DNA, and semi-synthetic DNA.
  • a "nucleic acid composition" of the disclosure comprises one or more nucleic acids as described herein.
  • pharmaceutically acceptable carrier encompass any of the agents approved by a regulatory agency of the U.S. Federal government or listed in the U.S. Pharmacopeia for use in animals, including humans, as well as any carrier or diluent that does not cause the production of undesirable physiological effects to a degree that prohibits administration of the composition to a subject and does not abrogate the biological activity and properties of the administered compound. Included are excipients and carriers that are useful in preparing a pharmaceutical composition and are generally safe, non-toxic, and desirable.
  • the term "pharmaceutical composition” refers to one or more of the compounds described herein, such as, e.g., a miRNA inhibitor of the present disclosure, mixed or intermingled with, or suspended in one or more other chemical components, such as pharmaceutically acceptable carriers and excipients.
  • a pharmaceutical composition is to facilitate administration of preparations comprising a miRNA inhibitor of the present disclosure to a subject.
  • polynucleotide refers to polymers of nucleotides of any length, including ribonucleotides, deoxyribonucleotides, analogs thereof, or mixtures thereof.
  • the term refers to the primary structure of the molecule.
  • the term includes triple-, double- and single-stranded deoxyribonucleic acid ("DNA”), as well as triple-, double- and single-stranded ribonucleic acid (“RNA”). It also includes modified, for example by alkylation, and/or by capping, and unmodified forms of the polynucleotide.
  • polynucleotide includes polydeoxyribonucleotides (containing 2-deoxy-D-ribose), polyribonucleotides (containing D-ribose), including tRNA, rRNA, shRNA, siRNA, miRNA and mRNA, whether spliced or unspliced, any other type of polynucleotide which is an N- or C-glycoside of a purine or pyrimidine base, and other polymers containing normucleotidic backbones, for example, polyamide (e.g., peptide nucleic acids "PNAs”) and polymorpholino 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.
  • PNAs peptide nucleic acids
  • a polynucleotide can be, e.g., an oligonucleotide, such as an antisense oligonucleotide.
  • the oligonucleotide is an RNA.
  • the RNA is a synthetic RNA.
  • the synthetic RNA comprises at least one unnatural nucleobase.
  • all nucleobases of a certain class have been replaced with unnatural nucleobases (e.g., all uridines in a polynucleotide disclosed herein can be replaced with an unnatural nucleobase, e.g., 5-methoxyuridine).
  • polypeptide polypeptide
  • peptide protein
  • protein polymers of amino acids of any length.
  • the polymer can comprise modified amino acids.
  • the terms also encompass an amino acid polymer that has been modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component.
  • polypeptides containing one or more analogs of an amino acid including, for example, unnatural amino acids such as homocysteine, ornithine, p-acetylphenylalanine, D-amino acids, and creatine
  • polypeptide refers to proteins, polypeptides, and peptides of any size, structure, or function.
  • Polypeptides include gene products, naturally occurring polypeptides, synthetic polypeptides, homologs, orthologs, paralogs, fragments and other equivalents, variants, and analogs of the foregoing.
  • a polypeptide can be a single polypeptide or can be a multi-molecular complex such as a dimer, trimer or tetramer. They can also comprise single chain or multichain polypeptides. Most commonly disulfide linkages are found in multichain polypeptides.
  • the term polypeptide can also apply to amino acid polymers in which one or more amino acid residues are an artificial chemical analogue of a corresponding naturally occurring amino acid.
  • a "peptide" can be less than or equal to about 50 amino acids long, e.g., about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, or about 50 amino acids long.
  • prevent refers partially or completely delaying onset of an disease, disorder and/or condition; partially or completely delaying onset of one or more symptoms, features, or clinical manifestations of a particular disease, disorder, and/or condition; partially or completely delaying onset of one or more symptoms, features, or manifestations of a particular disease, disorder, and/or condition; partially or completely delaying progression from a particular disease, disorder and/or condition; and/or decreasing the risk of developing pathology associated with the disease, disorder, and/or condition. In some aspects, preventing an outcome is achieved through prophylactic treatment.
  • promoter and “promoter sequence” are interchangeable and refer to a DNA sequence capable of controlling the expression of a coding sequence or functional RNA.
  • a coding sequence is located 3' to a promoter sequence. Promoters can be derived in their entirety from a native gene, or be composed of different elements derived from different promoters found in nature, or even comprise synthetic DNA segments. It is understood by those skilled in the art that different promoters can direct the expression of a gene in different tissues or cell types, or at different stages of development, or in response to different environmental or physiological conditions.
  • Promoters that cause a gene to be expressed in most cell types at most times are commonly referred to as “constitutive promoters.” Promoters that cause a gene to be expressed in a specific cell type are commonly referred to as “cell-specific promoters” or “tissue-specific promoters.” Promoters that cause a gene to be expressed at a specific stage of development or cell differentiation are commonly referred to as “developmentally-specific promoters” or “cell differentiation-specific promoters.” Promoters that are induced and cause a gene to be expressed following exposure or treatment of the cell with an agent, biological molecule, chemical, ligand, light, or the like that induces the promoter are commonly referred to as “inducible promoters” or “regulatable promoters.” It is further recognized that since in most cases the exact boundaries of regulatory sequences have not been completely defined, DNA fragments of different lengths can have identical promoter activity.
  • the promoter sequence is typically bounded at its 3' terminus by the transcription initiation site and extends upstream (5' direction) to include the minimum number of bases or elements necessary to initiate transcription at levels detectable above background. Within the promoter sequence will be found a transcription initiation site (conveniently defined for example, by mapping with nuclease S1), as well as protein binding domains (consensus sequences) responsible for the binding of RNA polymerase.
  • a promoter that can be used with the present disclosure includes a tissue specific promoter.
  • prolactic refers to a therapeutic or course of action used to prevent the onset of a disease or condition, or to prevent or delay a symptom associated with a disease or condition.
  • a “prophylaxis” refers to a measure taken to maintain health and prevent the onset of a disease or condition, or to prevent or delay a symptom associated with a disease or condition.
  • the term “gene regulatory region” or “regulatory region” refers to nucleotide sequences located upstream (5' non-coding sequences), within, or downstream (3' non-coding sequences) of a coding region, and which influence the transcription, RNA processing, stability, or translation of the associated coding region. Regulatory regions can include promoters, translation leader sequences, introns, polyadenylation recognition sequences, RNA processing sites, effector binding sites, or stem-loop structures.
  • a coding region is intended for expression in a eukaryotic cell, a polyadenylation signal and transcription termination sequence will usually be located 3' to the coding sequence.
  • a miR-485 inhibitor disclosed herein e.g., a polynucleotide encoding a RNA comprising one or more miR-485 binding site
  • a coding region for a gene product is associated with one or more regulatory regions in such a way as to place expression of the gene product under the influence or control of the regulatory region(s).
  • a coding region and a promoter are "operably associated” if induction of promoter function results in the transcription of mRNA encoding the gene product encoded by the coding region, and if the nature of the linkage between the promoter and the coding region does not interfere with the ability of the promoter to direct the expression of the gene product or interfere with the ability of the DNA template to be transcribed.
  • Other expression control elements besides a promoter, for example enhancers, operators, repressors, and transcription termination signals, can also be operably associated with a coding region to direct gene product expression.
  • similarity refers to the overall relatedness between polymeric molecules, e.g.
  • polynucleotide molecules e.g. miRNA molecules
  • Calculation of percent similarity of polymeric molecules to one another can be performed in the same manner as a calculation of percent identity, except that calculation of percent similarity takes into account conservative substitutions as is understood in the art. It is understood that percentage of similarity is contingent on the comparison scale used, i.e., whether the nucleic acids are compared, e.g., according to their evolutionary proximity, charge, volume, flexibility, polarity, hydrophobicity, aromaticity, isoelectric point, antigenicity, or combinations thereof.
  • subject refers to any mammalian subject, including without limitation, humans, domestic animals (e.g., dogs, cats and the like), farm animals (e.g., cows, sheep, pigs, horses and the like), and laboratory animals (e.g., monkey, rats, mice, rabbits, guinea pigs and the like) for whom diagnosis, treatment, or therapy is desired, particularly humans.
  • domestic animals e.g., dogs, cats and the like
  • farm animals e.g., cows, sheep, pigs, horses and the like
  • laboratory animals e.g., monkey, rats, mice, rabbits, guinea pigs and the like for whom diagnosis, treatment, or therapy is desired, particularly humans.
  • laboratory animals e.g., monkey, rats, mice, rabbits, guinea pigs and the like
  • the phrase "subject in need thereof” includes subjects, such as mammalian subjects, that would benefit from administration of a miRNA inhibitor of the disclosure (e.g., miR-485 inhibitor), e.g., to increase the expression level of PSD95 protein and/or PSD95 gene; to increase the expression level of synaptophysin protein and/or synaptophysin gene; and/or to decrease the expression level of caspase 3 protein and/or caspase 3 gene.
  • a miRNA inhibitor of the disclosure e.g., miR-485 inhibitor
  • the term "therapeutically effective amount” is the amount of reagent or pharmaceutical compound comprising a miRNA inhibitor of the present disclosure that is sufficient to a produce a desired therapeutic effect, pharmacologic and/or physiologic effect on a subject in need thereof.
  • a therapeutically effective amount can be a "prophylactically effective amount” as prophylaxis can be considered therapy.
  • treat refers to, e.g., the reduction in severity of a disease or condition; the reduction in the duration of a disease course; the amelioration or elimination of one or more symptoms associated with a disease or condition (e.g., diabetes); the provision of beneficial effects to a subject with a disease or condition, without necessarily curing the disease or condition.
  • the term also includes prophylaxis or prevention of a disease or condition or its symptoms thereof.
  • upstream refers to a nucleotide sequence that is located 5' to a reference nucleotide sequence.
  • a "vector” refers to any vehicle for the cloning of and/or transfer of a nucleic acid into a host cell.
  • a vector can be a replicon to which another nucleic acid segment can be attached so as to bring about the replication of the attached segment.
  • a "replicon” refers to any genetic element (e.g., plasmid, phage, cosmid, chromosome, virus) that functions as an autonomous unit of replication in vivo, i.e., capable of replication under its own control.
  • the term “vector” includes both viral and nonviral vehicles for introducing the nucleic acid into a cell in vitro, ex vivo or in vivo.
  • Vectors can be engineered to encode selectable markers or reporters that provide for the selection or identification of cells that have incorporated the vector. Expression of selectable markers or reporters allows identification and/or selection of host cells that incorporate and express other coding regions contained on the vector.
  • selectable marker genes known and used in the art include: genes providing resistance to ampicillin, streptomycin, gentamycin, kanamycin, hygromycin, bialaphos herbicide, sulfonamide, and the like; and genes that are used as phenotypic markers, i.e., anthocyanin regulatory genes, isopentanyl transferase gene, and the like.
  • reporter known and used in the art include: luciferase (Luc), green fluorescent protein (GFP), chloramphenicol acetyltransferase (CAT), ⁇ -galactosidase (LacZ), ⁇ -glucuronidase (Gus), and the like. Selectable markers can also be considered to be reporters.
  • mitochondrial dysfunction refers to an impairment in one or more properties of a mitochondria, such that the mitochondria cannot carry out its normal functions, as compared to a corresponding mitochondria that does not have the impairment (e.g., mitochondria present in cells or tissues of a normal, healthy subject).
  • mitochondrial dysfunction comprises an abnormally low level of at least one mitochondrial activity in one or more mammalian cell types or tissues, where such activity is beneficial to a mammalian cell, tissue, or organism comprising the cell or tissue.
  • mitochondrial dysfunction comprises an abnormally high level of at least one mitochondrial activity in one or more mammalian cell types or tissues, where such activity is detrimental to a mammalian cell, tissue, or organism comprising the cell or tissue.
  • mitochondrial dysfunction comprises an abnormally low number of mitochondria in one or more mammalian cell types or tissues, e.g., relative to a normal level, e.g., a level found in cells or tissues of or obtained from a normal, healthy subject.
  • a normal level e.g., a level found in cells or tissues of or obtained from a normal, healthy subject.
  • each of the mitochondria within a cell exhibits normal function, but the overall number of mitochondria within the cell is reduced, such that the cell is dysfunctional as compared to a corresponding cell with higher number of mitochondria.
  • mitochondrial dysfunction comprises both (i) an abnormally low level of at least one mitochondrial activity in one or more mammalian cell types or tissues, where such activity is beneficial to a mammalian cell, tissue, or organism comprising the cell or tissue; and (ii) an abnormally high level of at least one mitochondrial activity in one or more mammalian cell types or tissues, where such activity is detrimental to a mammalian cell, tissue, or organism comprising the cell or tissue.
  • mitochondria dysfunction comprises (i) an abnormally low level of at least one mitochondrial activity in one or more mammalian cell types or tissues, where such activity is beneficial to a mammalian cell, tissue, or organism comprising the cell or tissue, (ii) an abnormally high level of at least one mitochondrial activity in one or more mammalian cell types or tissues, where such activity is detrimental to a mammalian cell, tissue, or organism comprising the cell or tissue, and (iii) abnormally low number of mitochondria in one or more mammalian cell types or tissues, such that the cell types or tissues exhibit impaired function.
  • mitochondria dysfunction refers to any disease, disorder, or condition which is associated with a mitochondrial dysfunction.
  • the term encompasses any disorder caused at least in part by a defect in amount (e.g., reduced expression), structure, or activity of one or more mitochondrial proteins, protein complexes, or substructures and/or a defect in mitochondrial number.
  • a defect in amount e.g., reduced expression
  • structure e.g., a cell mechanism where cellular components are degraded and recycled.
  • mitochondrial degradation by autophagy is a key mechanism of regulating mitochondrial homeostasis as well as mitochondrial biogenesis. II.
  • Some aspects of the present disclosure are related to the use of miR-485 inhibitors to alleviate and/or prevent a mitochondrial dysfunction.
  • the miR-485 inhibitors provided herein are capable of regulating various aspects of a mitochondria, such that the mitochondrial dysfunction is alleviated and/or prevented.
  • Non-limiting examples by which miR-485 inhibitors can regulate such aspects of a mitochondria are provided below.
  • Mitophagy As described herein, mitophagy is an important biological process that allows for the selective removal of aged and/or damaged mitochondria. Mitophagy plays a pivotal role in reinstating cellular homeostasis in normal physiology and conditions of stress.
  • a miR-485 inhibitor provided herein is capable of promoting mitophagy.
  • some aspects of the present disclosure are related to methods of inducing mitophagy in a subject in need thereof, comprising administering to the subject a miR- 485 inhibitor.
  • the mitophagy in the subject is increased as compared to a reference subject (e.g., the subject prior to the administration or a corresponding subject that did not receive the administration).
  • the mitophagy in the subject is increased by at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 100%, as compared to the reference subject.
  • the mitophagy in the subject is increased by at least about 1.5-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 11-fold, at least about 12-fold, at least about 13-fold, at least about 14-fold, at least about 15-fold, at least about 20- fold, at least about 25-fold, at least about 30-fold, at least about 35-fold, at least about 40-fold, at least about 45-fold, or at least about 50-fold, as compared to the reference subject.
  • mitophagy can be assessed by measuring the expression and/or activity of one or more genes (or proteins encoded thereof) involved in mitophagy.
  • genes/proteins include: PINK1, Parkin, LC3B, and LAMP2.
  • PINK1 a miR-485 inhibitor provided herein is capable of promoting mitophagy by increasing the expression of PINK1. Unless indicated otherwise, increasing the expression of PINK1 can be at the gene level, at the protein level, or at both the gene level and the protein level.
  • the expression of PINK1 in the subject is increased by at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 100%, as compared to a reference subject (e.g., the subject prior to the administration or a corresponding subject that did not receive the administration).
  • a reference subject e.g., the subject prior to the administration or a corresponding subject that did not receive the administration.
  • the expression of PINK1 in the subject is increased by at least about 1.5-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 11-fold, at least about 12-fold, at least about 13-fold, at least about 14-fold, at least about 15- fold, at least about 20-fold, at least about 25-fold, at least about 30-fold, at least about 35-fold, at least about 40-fold, at least about 45-fold, or at least about 50-fold, as compared to the reference subject.
  • PINK1 is a mitochondrial serine/threonine-protein kinase and plays a role in protecting cells from mitochondrial dysfunction. When mitochondrial membrane potential drops (as in many mitochondrial dysfunction), PINK1 aggregates on the outer mitochondrial membrane to exert its pro-mitophagic function. More specifically, PINK1 activates Parkin which adds ubiquitin moieties on specific mitochondrial outer membrane proteins (e.g., MFN1, MFN2, and FIS1), thus inducing proteasomal degradation, which in turn promotes mitochondrial fission and mitophagy.
  • PTEN-induced kinase 1 is a protein that in humans is encoded by the PINK1 gene.
  • the PINK1 gene is located on chromosome 1 in humans (nucleotides 20,633,458 to 20,651,511 of GenBank Accession Number NC_000001.11, minus strand orientation). Synonyms of the PINK1 gene, and the encoded protein thereof, are known and include "serine/threonine-protein kinase PINK1, mitochondrial,” "PTEN induced putative kinase 1,” “PTEN-induced putative kinase protein 1 ,” “protein kinase BRPK,” “BRPK,” “PARK6.” [0123] There are at least two known isoforms of human PINK1 protein, resulting from alternative splicing.
  • PINK1 isoform 1 (UniProt identifier: Q9BXM7-1-1; also known as PINK1-alpha) consists of 581 amino acids and has been chosen as the canonical sequence (SEQ ID NO: 36).
  • PINK1 isoform 2 (also known as “PINK1-beta”) (UniProt identifier: Q9BXM7- 2) consists of 274amino acids and differs from the canonical sequence as follows: 1-307 (Missing) and 308-320: : LGHGRTLFLVMKN ⁇ MCGSQRPSPLSTS (SEQ ID NO: 37). Table 1 below provides the sequences for the two PINK1 isoforms. Table 1.
  • PINK1 Protein Isoforms Isoform 1 MAVRQALGRGLQLGRALLLRFTGKPGRAYGLGRPGPAAGCVRGERPGWAAGPGAEPRRVG LGLPNRLRFFRQSVAGLAARLQRQFVVRAWGCAGPCGRAVFLAFGLGLIEEKQAESRR (UniProt: AVSACQEIQAIFTQKSKPGPDPLDTRRLQGFRLEEYLIGQSIGKGCSAAVYEATMPTLPQ Q9BXM7- NLEVTKSTGLLPGRGPGTSAPGEGQERAPGAPAFPLAIKMMWNISAGSSSEAILNTMSQE LVPASRVALAGEYGAVTYRKSKRGPKQLAPHPNIIRVLRAFTSSVPLLPGALVDYPDVLP 1) (SEQ ID SRLHPEGLGHGRTLFLVMKNYPCTLRQYLCVNTPSPRLAAMMLLQLLEGVDHLVQQGIAH NO: 36) RDLKSDNILVELDPDGCPWLVIADFGCCLADES
  • a miR-485 inhibitor disclosed herein can increase the expression of PINK1 isoform 1.
  • a miR-485 inhibitor disclosed herein can increase the expression of PINK1 isoform 2.
  • a miR-485 inhibitor disclosed herein can increase the expression of PINK1 isoform 1 and PINK1 isoform 2.
  • PINK1 isoform 1 and isoform 2 are collectively referred to herein as "PINK1.”
  • increasing the expression of Parkin can be at the gene level, at the protein level, or at both the gene level and the protein level. Additionally, increasing the expression of Parkin can result in an increase in the activity of Parkin. Accordingly, unless indicated otherwise, "increasing the expression of Parkin” also comprises increasing the activity of Parkin. [0127]
  • the expression of Parkin in the subject is increased by at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 100%, as compared to a reference subject (e.g., the subject prior to the administration or a corresponding subject that did not receive the administration).
  • a reference subject e.g., the subject prior to the administration or a corresponding subject that did not receive the administration.
  • the expression of Parkin in the subject is increased by at least about 1.5-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 11-fold, at least about 12-fold, at least about 13-fold, at least about 14-fold, at least about 15- fold, at least about 20-fold, at least about 25-fold, at least about 30-fold, at least about 35-fold, at least about 40-fold, at least about 45-fold, or at least about 50-fold, as compared to the reference subject.
  • Parkin is a component of a multiprotein E3 ubiquitin ligase complex, which in turn is part of the ubiquitin-proteasome system that mediates the targeting of proteins for degradation. As described herein, Parkin interacts with PINK1 and plays an important role in the ubiquitination of certain mitochondrial proteins.
  • Parkin is encoded by the PARK2 gene (also referred to herein as the "Parkin" gene).
  • the Parkin gene is located on chromosome 6 in humans (nucleotides 161,347,417to162,727,802, minus strand orientation) of GeneBank Accession Number NC_ 000006.12, minus strand orientation).
  • Synonyms of the Parkin gene, and the encoded protein thereof, are known and include "E3 ubiquitin-protein ligase parkin,” “Parkinson disease (autosomal recessive, juvenile) 2," “PRKN,” “parkinson juvenile disease protein 2,” “parkinson protein 2 E3 ubiquitin protein ligase,” “parkinson protein 2, E3 ubiquitin protein ligase (parkin),” "PDJ,” “AR-JP,” and “LPRS2.” [0130] There are at least eight known isoforms of the human Parkin protein, resulting from alternative splicing.
  • Parkin isoform 1 (UniProt identifier: O60260-1) consists of 465 amino acids and has been chosen as the canonical sequence (SEQ ID NO: 38).
  • Parkin isoform 2 (UniProt identifier: O60260-2; also known as "SV5DEL") consists of 437 amino acids and differs from the canonical sequence as follows: residues 179-206: missing (SEQ ID NO: 39).
  • Parkin isoform 3 (UniProt identifier: O60260-3; SEQ ID NO: 40) consists of 218 amino acids and differs from the canonical sequence as follows: (a) residues 1-79: missing, (b) residues 291-297: AGCPNSL ⁇ VCLLPGM; and (c) residues 298-465: missing.
  • Parkin isoform 4 (UniProt identifier: O60260-4; SEQ ID NO: 41) consists of 274 amino acids and differs from the canonical sequence as follows: residues 1-191: missing.
  • Parkin isoform 5 (UniProt identifier: O60260-5; SEQ ID NO: 42) consists of 387 amino acids and differs from the canonical sequence as follows: (a) residues 290-290: V ⁇ VGTGDTVVLRGALGGFRRGV; (b) residues 362-368: FAFCREC ⁇ YGQRRTK; and (c) residues 369-465: missing.
  • Parkin isoform 6 (UniProt identifier: O60260-6; SEQ ID NO: 43) consists of 316 amino acids and differs from the canonical sequence as follows: residues 58-206: missing.
  • a miR-485 inhibitor disclosed herein can increase the expression of Parkin isoform 1.
  • a miR-485 inhibitor disclosed herein can increase the expression of Parkin isoform 2.
  • a miR-485 inhibitor disclosed herein can increase the expression of Parkin isoform 3.
  • a miR-485 inhibitor disclosed herein can increase the expression of Parkin isoform 4.
  • a miR-485 inhibitor disclosed herein can increase the expression of Parkin isoform 5.
  • a miR-485 inhibitor disclosed herein can increase the expression of Parkin isoform 6.
  • a miR-485 inhibitor disclosed herein can increase the expression of Parkin isoform 7.
  • a miR-485 inhibitor disclosed herein can increase the expression of Parkin isoform 8.
  • a miR-485 inhibitor of the present disclosure can increase the expression of any combination of Parkin isoform 1, Parkin isoform 2, Parkin isoform 3, Parkin isoform 4, Parkin isoform 5, Parkin isoform 6, Parkin isoform 7, and Parkin isoform 8.
  • a miR-485 inhibitor described herein can increase the expression of each of Parkin isoform 1, Parkin isoform 2, Parkin isoform 3, Parkin isoform 4, Parkin isoform 5, Parkin isoform 6, Parkin isoform 7, and Parkin isoform 8.
  • a miR-485 inhibitor disclosed herein increases the expression of Parkin (gene and/or protein) by reducing the expression and/or activity of miR-485-3p.
  • some aspects of the present disclosure are related to methods of increasing the expression of Parkin in a subject in need thereof, comprising administering a miR-485 inhibitor to the subject.
  • Some aspects of the present disclosure are related to methods of treating a disease or disorder associated with an abnormal (e.g., reduced) expression of Parkin in a subject in need thereof.
  • “increasing the expression of LC3B” also comprises increasing the activity of LC3B.
  • Some aspects of the present disclosure are related to methods of inducing mitophagy in a subject in need thereof, comprising administering to the subject a miR-485 inhibitor, wherein after the administration, the expression of LC3B is increased in the subject.
  • the expression of LC3B in the subject is increased by at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 100%, as compared to a reference subject (e.g., the subject prior to the administration or a corresponding subject that did not receive the administration).
  • a reference subject e.g., the subject prior to the administration or a corresponding subject that did not receive the administration.
  • the expression of LC3B in the subject is increased by at least about 1.5-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 11-fold, at least about 12-fold, at least about 13-fold, at least about 14-fold, at least about 15- fold, at least about 20-fold, at least about 25-fold, at least about 30-fold, at least about 35-fold, at least about 40-fold, at least about 45-fold, or at least about 50-fold, as compared to the reference subject.
  • LC3B is a member of the highly conserved ATG8 protein family and plays an important role in mitophagy, e.g., by allowing autophagosomes to recognize target mitochondria (e.g., damaged or dysfunctional) and promoting their degradation.
  • a miR-485 inhibitor provided herein is capable of promoting mitophagy by increasing the expression of Lamp2.
  • increasing the expression of Lamp2 can be at the gene level, at the protein level, or at both the gene level and the protein level. Additionally, increasing the expression of Lamp2 can result in an increase in the activity of Lamp2. Accordingly, unless indicated otherwise, "increasing the expression of Lamp2" also comprises increasing the activity of Lamp2.
  • the expression of NRF1 in the subject is increased by at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 100%, as compared to a reference subject (e.g., the subject prior to the administration or a corresponding subject that did not receive the administration).
  • a reference subject e.g., the subject prior to the administration or a corresponding subject that did not receive the administration.
  • NRF2 isoform 2 (UniProt identifier: Q16236-2; SEQ ID NO: 59) consists of 589 amino acids and differs from the canonical sequence as follows: residues 1-16: missing.
  • NRF2 isoform 3 (UniProt identifier: Q16236-3; SEQ ID NO: 60) consists of 582 amino acids and differs from the canonical sequence as follows: (a) residues 1-16: missing; and (b) residues 135-141: missing. [0164] Table 6 provides the sequences for the three isoforms of the NRF2 protein. Table 6.
  • NRF2 Isoforms Isoform 1 MMDLELPPPGLPSQQDMDLIDILWRQDIDLGVSREVFDFSQRRKEYELEKQKKLEKERQE QLQKEQEKAFFAQLQLDEETGEFLPIQPAQHIQSETSGSANYSQVAHIPKSDALYFDDCM (UniProt: Q QLLAQTFPFVDDNEVSSATFQSLVPDIPGHIESPVFIATNQAQSPETSVAQVAPVDLDGM 16236-1) QQDIEQVWEELLSIPELQCLNIENDKLVETTMVPSPEAKLTEVDNYHFYSSIPSMEKEVG NCSPHFLNAFEDSFSSILSTEDPNQLTVNSLNSDATVNTDFGDEFYSAFIAEPSISNSMP (SEQ ID SPATLSHSLSELLNGPIDVSDLSLCKAFNQNHPESTAEFNDSDSGISLNTSPSVASPEHS NO: 58) VESSSYGDTLLGLSDSEVEELDSAPGSVKQ
  • TFAM is a key activator of mitochondrial transcription as well as a participant in mitochondrial genome replication. TFAM binds mitochondrial promoter DNA to aid transcription of the mitochondrial genome.
  • TFAM isoform 1 (UniProt identifier: Q00059-1; SEQ ID NO: 91) consists of 246 amino acids and has been chosen as the canonical sequence.
  • TFAM isoform 2 (UniProt identifier: Q00059- 2; SEQ ID NO: 92) consists of 214 amino acids and differs from the canonical sequence as follows: residues 148-179: missing. [0172] Table 7 (below) provides the sequences for the two isoforms of TFAM. Table 7.
  • TFAM Isoforms Isoform 1 MAFLRSMWGVLSALGRSGAELCTGCGSRLRSPFSFVYLPRWFSSVLASCPKKPVSSYLRF SKEQLPIFKAQNPDAKTTELIRRIAQRWRELPDSKKKIYQDAYRAEWQVYKEEISRFKEQ (UniProt: Q LTPSQIMSLEKEIMDKHLKRKAMTKKKELTLLGKPKRPRSAYNVYVAERFQEAKGDSPQE 00059-1) KLKTVKENWKNLSDSEKELYIQHAKEDETRYHNEMKSWEEQMIEVGRKDLLRRTIKKQRK YGAEEC (SEQ ID NO: 91) Isoform 2 MAFLRSMWGVLSALGRSGAELCTGCGSRLRSPFSFVYLPRWFSSVLASCPKKPVSSYLRF SKEQLPIFKAQNPDAKTTELIRRIAQRWRELPDSKKKIYQDAYRAEWQVYK
  • a miR-485 inhibitor disclosed herein can increase the expression of TFAM isoform 1.
  • a miR-485 inhibitor disclosed herein can increase the expression of TFAM isoform 2.
  • a miR-485 inhibitor can increase the expression of TFAM isoform 1 and TFAM isoform 2.
  • TFAM refers to any of the above-described isoforms of TFAM.
  • a miR-485 inhibitor disclosed herein increases the expression of TFAM (gene and/or protein) by reducing the expression and/or activity of miR-485-3p.
  • aspects of the present disclosure are related to methods of increasing the expression of NRF2 in a subject in need thereof, comprising administering a miR-485 inhibitor to the subject.
  • Some aspects of the present disclosure are related to methods of treating a disease or disorder associated with an abnormal (e.g., reduced) expression of TFAM in a subject in need thereof.
  • such methods comprise administering a miR-485 inhibitor to the subject, wherein after the administering, the expression of TFAM is increased in the subject.
  • Mitochondrial Fusion and Fission mitochondria are highly versatile and are able to change their shape through a dynamic process of fission (process in which a single entity breaks apart) and fusion (process in which two or more entities join to form a whole). As described herein, the processes of fission and fusion oppose each other and allow the mitochondrial network to constantly remodel itself. As described and demonstrated herein, in some aspects, a miR-485 inhibitor provided herein can regulate the dynamics between mitochondrial fusion and fission that occurs within a cell of a subject. For instance, in some aspects, a miR-485 inhibitor provided herein is capable of reducing mitochondrial fission in a cell of a subject in need thereof.
  • Mitochondrial fission is the process by which mitochondria divide or segregate into two or more separate mitochondrial organelles.
  • mitochondrial fission allows for the creation of new mitochondria.
  • mitochondrial fission contributes to quality control by enabling the removal of damaged mitochondria and can facilitate apoptosis during high levels of cellular stress.
  • a miR-485 inhibitor provided herein is capable of increasing mitochondrial fusion in a cell of a subject in need thereof.
  • Mitochondrial fusion is the process by which two separate mitochondria merge to form a single organelle. Mitochondrial fusion can be particularly useful for cells in times of stress, as it allows functional mitochondria to complement dysfunctional mitochondria by diffusion and sharing of components between organelles.
  • some aspects of the present disclosure are related to methods of reducing mitochondrial fission in a subject in need thereof, comprising administering a miR- 485 inhibitor to the subject.
  • the mitochondrial fission in the subject is reduced by at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or about 100%, as compared to a reference subject (e.g., the subject prior to the administration or a corresponding subject who did not receive the administration).
  • Some of the present disclosure are related to methods of increasing mitochondrial fusion in a subject in need thereof, comprising administering a miR- 485 inhibitor to the subject.
  • the mitochondrial fusion in the subject is increased by at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 100%, as compared to a reference subject (e.g., the subject prior to the administration or a corresponding subject who did not receive the administration).
  • the mitochondrial fusion in the subject is increased by at least about 1.5-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 11-fold, at least about 12-fold, at least about 13-fold, at least about 14-fold, at least about 15-fold, at least about 20-fold, at least about 25-fold, at least about 30-fold, at least about 35-fold, at least about 40-fold, at least about 45-fold, or at least about 50-fold, as compared to the reference subject.
  • a miR-485 inhibitor described herein reduces mitochondrial fission and increases mitochondrial fusion in the subject.
  • any useful method knows in the art can be used.
  • the dynamics between mitochondrial fusion and fission can be observed by assessing the phosphorylation status of the dynamin-related protein 1 (Drp1), which has been shown to promote mitochondrial fission and quality control to maintain cellular homeostasis.
  • Drp1 dynamin-related protein 1
  • the phosphorylation status of amino acid residues S616 and S637 of the Drp1 protein can be assessed.
  • an increase in mitochondrial fission is associated with an increased phosphorylation of residue S616 of the Drp1 protein.
  • an increase in mitochondrial fission is associated with a decreased phosphorylation of residue S637 of the Drp1 protein.
  • an increase in mitochondrial fission is associated with an increased phosphorylation of residue S616 and a decreased phosphorylation of residue S637 of the Drp1 protein.
  • the dynamics between mitochondrial fusion and fission can be assessed based on the expression of a protein involved in either mitochondrial fusion or fission.
  • a miR-485 inhibitor provided herein is capable of regulating the balance between mitochondrial fission and fusion by regulating the expression of the MFN1 protein, which is a mitochondrial outer membrane GTPase and mediates mitochondrial clustering and fusion.
  • a miR-485 inhibitor provided herein is capable of (i) increasing mitochondrial fusion, (ii) reducing mitochondrial fission, or (iii) both (i) and (ii) by increasing the expression of MFN1.
  • increasing the expression of MFN1 can be at the gene level, at the protein level, or at both the gene level and the protein level.
  • increasing the expression of MFN1 can result in an increase in the activity of MFN1.
  • incrementasing the expression of MFN1 also comprises increasing the activity of MFN1.
  • the expression of MFN1 in the subject is increased by at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 100%, as compared to a reference subject (e.g., the subject prior to the administration or a corresponding subject that did not receive the administration).
  • a reference subject e.g., the subject prior to the administration or a corresponding subject that did not receive the administration.
  • the expression of MFN1 in the subject is increased by at least about 1.5-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9- fold, at least about 10-fold, at least about 11-fold, at least about 12-fold, at least about 13-fold, at least about 14-fold, at least about 15-fold, at least about 20-fold, at least about 25-fold, at least about 30-fold, at least about 35-fold, at least about 40-fold, at least about 45-fold, or at least about 50-fold, as compared to the reference subject.
  • MFN1 is a mitochondrial outer membrane GTPase and mediates mitochondrial clustering and fusion. In human, MFN1 is encoded by the MFN1 gene, which is located on chromosome 3 (nucleotides 179,347,709-179,394,936 of GeneBank Accession Number NC_000003.12, plus strand orientation).
  • MFN1 isoform 1 (UniProt identifier: Q8IWA4-1; also known as TG741; SEQ ID NO: 93) consists of 741 amino acids and has been chosen as the canonical sequence.
  • MFN1 isoform 2 (UniProt identifier: Q8IWA4-2; also known as TG370; SEQ ID NO: 94) consists of 370 amino acids and differs from the canonical sequence as follows: (a) residues 367-370: HYSV ⁇ FHVQ; and (b) residues 371-741: missing.
  • MFN1 isoform 3 (UniProt identifier: Q8IWA4-3; SEQ ID NO: 95) consists of 630 amino acids and differs from the canonical sequence as follows: residues 444-554: missing. [0182] Table 8 (below) provides the sequences for the three known isoforms of the MFN1 protein. Table 8.
  • a miR-485 inhibitor disclosed herein can increase the expression of MFN1 isoform 1.
  • a miR-485 inhibitor disclosed herein can increase the expression of MFN1 isoform 2.
  • a miR-485 inhibitor disclosed herein can increase the expression fo MFN1 isoform 3.
  • a miR-485 inhibitor provided herein can increase the expression of MFN1 isoform 1 and MFN1 isoform 2.
  • a miR-485 inhibitor can increase the expression of MFN1 isoform 1 and MFN1 isoform 3.
  • a miR-485 inhibitor can increase the expression of MFN1 isoform 2 and MFN1 isoform 3.
  • a miR-485 inhibitor can increase the expression of MFN1 isoform 1, MFN1 isoform 2, and MFN1 isoform 3.
  • MFN1 refers to any of the above-described isoforms of MFN1.
  • a miR-485 inhibitor disclosed herein increases the expression of MFN1 (gene and/or protein) by reducing the expression and/or activity of miR-485-3p.
  • some aspects of the present disclosure are related to methods of increasing the expression of MFN1 in a subject in need thereof, comprising administering a miR-485 inhibitor to the subject.
  • Some aspects of the present disclosure are related to methods of treating a disease or disorder associated with an abnormal (e.g., reduced) expression of MFN1 in a subject in need thereof.
  • such methods comprise administering a miR-485 inhibitor to the subject, wherein after the administering, the expression of MFN1 is increased in the subject.
  • Mitochondrial Disorder [0185] As will be apparent from the present disclosure, the methods provided herein can be used to treat a wide-range of mitochondrial disorders.
  • a mitochondrial disorder that can be treated with the present disclosure is associated with impaired mitophagy. Accordingly, in some aspects, a mitochondrial disorder that can be treated is associated with abnormal expression of one or more of the following: PINK1, Parkin, LC3B, and LAMP2.
  • a mitochondrial disorder that can be treated is associated with impaired mitochondrial biogenesis.
  • the mitochondrial disorder is associated with abnormal expression of one or more of the following: NRF1, NRF2, and TFAM.
  • a mitochondrial disorder that can be treated with the present disclosure is associated with impaired mitochondrial fission and/or mitochondrial fusion.
  • a mitochondrial disorder that can be treated is associated with abnormal expression of Drp1, MFN1, or both.
  • a mitochondrial disorder that can be treated is impaired with any combination of the following: (i) mitophagy, (ii) mitochondrial biogenesis, and (iii) mitochondrial fission and/or fusion.
  • a mitochondrial disorder that can be treated is impaired with each of the following: (i) mitophagy, (ii) mitochondrial biogenesis, and (iii) mitochondrial fission and/or fusion.
  • mitophagy e.g., mitophagy
  • mitochondrial biogenesis e.g., mitochondrial biogenesis
  • mitochondrial fission and/or fusion e.g., mitochondrial fission and/or fusion.
  • an abnormal expression refers to an expression that is decreased by at least about 1%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 95%, at least about 95%, or about 100% compared to the corresponding level in a reference subject (e.g., subject who does not suffer from a disease or condition described herein).
  • a reference subject e.g., subject who does not suffer from a disease or condition described herein.
  • a disease or disorder that can be treated with the present disclosure is associated with an abnormal expression of PINK1. In some aspects, a disease or disorder that can be treated with the present disclosure is associated with an abnormal expression of Parkin. In some aspects, a disease or disorder that can be treated with the present disclosure is associated with an abnormal expression of LC3B. In some aspects, a disease or disorder that can be treated with the present disclosure is associated with an abnormal expression of LAMP2. In some aspects, a disease or disorder that can be treated with the present disclosure is associated with an abnormal expression of NRF1. In some aspects, a disease or disorder that can be treated with the present disclosure is associated with an abnormal expression of NRF2.
  • a disease or disorder that can be treated with the present disclosure is associated with an abnormal expression of TFAM.
  • a disease or disorder that can be treated with the present disclosure is associated with an abnormal expression of Drp1 (e.g., increased expression of Drp1 with phosphorylation at residue S616 and/or decreased expression of Drp1 with phosphorylation at residue S637).
  • a disease or disorder that can be treated with the present disclosure is associated with an abnormal expression of MFN1.
  • Non-limiting examples of such diseases or disorders include: Leigh syndrome (LS), Ataxia telangiectasia (AT), spinocerebellar ataxia (SCA), Danon disease (DD), Autosomal dominant optic atrophy (ADOA), Barth syndrome (BTHS), Charcot-Marie tooth disease, Autosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS), Cockayne syndrome (CS), fabry disease, Fanconi anemia (FA), Vici syndrome (VICIS), Zellweger syndrome (ZS), Werner syndrome (WS), Sengers syndrome, GRACILE syndrome, or a combination thereof.
  • LS Leigh syndrome
  • AT Ataxia telangiectasia
  • SCA spinocerebellar ataxia
  • DD Danon disease
  • ADOA Autosomal dominant optic atrophy
  • BTHS Barth syndrome
  • Charcot-Marie tooth disease Autosomal recessive spastic ataxia of Charlevoix-Saguenay
  • administering a miR-485 inhibitor can improve one or more symptoms associated with any of the mitochondrial disorder described herein. More specifically, in some aspects, administering a miR-485 inhibitor disclosed herein can improve one or more symptoms associated with impaired mitophagy. In some aspects, administering a miR-485 inhibitor disclosed herein can improve one or more symptoms associated with impaired microbial biogenesis. In some aspects, administering a miR-485 inhibitor disclosed herein can improve one or more symptoms associated with impaired mitochondrial fission and/or fusion.
  • administering a miR-485 inhibitor provided herein can improve one or more symptoms associated with an impairment of any combination of the following: (i) mitophagy, (ii) mitochondrial biogenesis, and (iii) mitochondrial fission and/or fusion. In some aspects, administering a miR-485 inhibitor provided herein can improve one or more symptoms associated with an impairment in each of the following: (i) mitophagy, (ii) mitochondrial biogenesis, and (iii) mitochondrial fission and/or fusion. In some aspects, administering a miR- 485 inhibitor can improve one or more symptoms associated with abnormal expression of any of the following: PINK1, Parkin, LC3B, LAMP2, NRF1, NRF2, TFAM, and MFN1.
  • a miR-485 inhibitor can treat a mitochondrial disorder described herein by improving one or more mitochondrial functions of a cell in a subject. Accordingly, some aspects of the present disclosure are related to methods of increasing one or more mitochondrial functions in a cell of a subject, comprising contacting the cell with a miR-485 inhibitor. In some aspects, the cell is contacted with the miR-485 inhibitor in vivo (e.g., within the subject). In some aspects, the cell is contacted with the miR- 485 inhibitor ex vivo.
  • the one or more mitochondrial functions in the cell is increased by at least about about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 100%, as compared to that of a reference subject (e.g., the subject prior to the administration or a corresponding subject that did not receive the administration).
  • a reference subject e.g., the subject prior to the administration or a corresponding subject that did not receive the administration.
  • the one or more mitochondrial functions in the cell is increased by at least about 1.5-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 11-fold, at least about 12-fold, at least about 13-fold, at least about 14-fold, at least about 15- fold, at least about 20-fold, at least about 25-fold, at least about 30-fold, at least about 35-fold, at least about 40-fold, at least about 45-fold, or at least about 50-fold, as compared to that of the reference subject.
  • mitochondrial functions that can be improved using the present disclosure include: mitochondrial respiration, glycolysis, mitochondrial biogenesis, mitochondrial fission, mitochondrial fusion, or combinations thereof.
  • a miR-485 inhibitor described herein can increase mitochondrial respiration in a cell of a subject in need thereof.
  • a miR-485 inhibitor when administered to a subject in need thereof, can increase mitochondrial respiration in the subject by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 100%, as compared to that of a reference subject (e.g., the subject prior to the administration or a corresponding subject that did not receive the administration).
  • a reference subject e.g., the subject prior to the administration or a corresponding subject that did not receive the administration.
  • the mitochondrial respiration in the subject is increased by at least about 1.5-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 11-fold, at least about 12-fold, at least about 13-fold, at least about 14-fold, at least about 15-fold, at least about 2 0-fold, at least about 25-fold, at least about 30-fold, at least about 35-fold, at least about 40-fold, at least about 45-fold, or at least about 50-fold, as compared to that of the reference subject.
  • an increased mitochondrial respiration can be associated with an increase in each of the following: (i) basal respiration, (ii) maximum respiration, and (iii) ATP production.
  • the basal respiration of the cell is increased by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 100%, as compared to that of a reference (e.g., the cell prior to the contacting or a corresponding cell that was not contacted with the miR-4
  • the basal respiration is increased by at least about 1.5-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9- fold, at least about 10-fold, at least about 11-fold, at least about 12-fold, at least about 13-fold, at least about 14-fold, at least about 15-fold, at least about 20-fold, at least about 25-fold, at least about 30-fold, at least about 35-fold, at least about 40-fold, at least about 45-fold, or at least about 50-fold, as compared to that of the reference.
  • the maximum respiration of the cell is increased by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 100%, as compared to that of a reference (e.g., the cell prior to the contacting or a corresponding cell that was not contacted with the miR-485 inhibitor).
  • a reference e.g., the cell prior to the contacting or a corresponding cell that was not contacted with the miR-485 inhibitor.
  • the maximum respiration is increased by at least about 1.5-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 11-fold, at least about 12-fold, at least about 13-fold, at least about 14-fold, at least about 15- fold, at least about 20-fold, at least about 25-fold, at least about 30-fold, at least about 35-fold, at least about 40-fold, at least about 45-fold, or at least about 50-fold, as compared to that of the reference.
  • the ATP production by the cell is increased by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 100%, as compared to that of a reference (e.g., the cell prior to the contacting or a corresponding cell that was not contacted with the miR-485 inhibitor).
  • a reference e.g., the cell prior to the contacting or a corresponding cell that was not contacted with the miR-485 inhibitor.
  • an increased glycolysis can be associated with any of the following: (i) glycolytic capacity, (ii) glycolytic reserve, or (iii) both (i) and (ii).
  • the glycolytic capcity of the cell is increased by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 100%, as compared to that of a reference (e.g., the cell prior to the contacting or a corresponding cell that was not contacted with the miR-485
  • the glycolytic capcity is increased by at least about 1.5-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9- fold, at least about 10-fold, at least about 11-fold, at least about 12-fold, at least about 13-fold, at least about 14-fold, at least about 15-fold, at least about 20-fold, at least about 25-fold, at least about 30-fold, at least about 35-fold, at least about 40-fold, at least about 45-fold, or at least about 50-fold, as compared to that of the reference.
  • the glycolytic reserve is increased by at least about 1.5-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 11-fold, at least about 12-fold, at least about 13-fold, at least about 14-fold, at least about 15-fold, at least about 20-fold, at least about 25-fold, at least about 30-fold, at least about 35-fold, at least about 40-fold, at least about 45-fold, or at least about 50-fold, as compared to that of the reference.
  • a miR-485 inhibitor described herein can help regulate the amount of reactive oxygen species (ROS) produced by a cell.
  • ROS reactive oxygen species
  • mitochondria produce some ROS during the process of oxidative phosphorylation (e.g., at the electron transport chain located on the inner mitochondrial membrane).
  • ROS can cause apoptosis/autophagy pathways capable of inducing cell death.
  • a miR-485 inhibitor is capable of reducing and/or preventing ROS overproduction by a mitochondrial of a cell.
  • some aspects of the present disclosure relate to methods of reducing the amount of reactive oxygen species (ROS) produced by a cell of a subject in need thereof, comprising contacting the cell with a miR-485 inhibitor described herein.
  • the amount of ROS produced by the cells is reduced by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or about 100%, compared to the amount of ROS produced by a reference cell (e.g., the cell prior to the contacting or a corresponding cell that is not contacted with the miR-485 inhibitor).
  • the amount of ROS produced by a cell can be assessed using any suitable methods known in the art.
  • the amount of ROS produced by a cell can be assessed by measuring the expression of MitoSOX in the cell, e.g., using flow cytometry.
  • the amount of ROS produced by a cell is directly correlated with the expression of MitoSOX in the cell.
  • the cell is contacted with the miR-485 inhibitor in vivo (e.g., within the subject). In some aspects, the cell is contacted with the miR-485 inhibitor ex vivo.
  • reducing the amount of ROS produced by a cell can be useful in improving one or more properties of a cell.
  • some aspects of the present disclosure is directed to methods of reducing the level of reactive oxygen species (ROS) in a subject in need thereof, comprising administering a miR-485 inhibitor to the subject, wherein the subject suffers from a disease or disorder associated with high level of ROS.
  • ROS reactive oxygen species
  • the disease or disorder associated with high level of ROS comprises any of the mitochondrial disorders described herein.
  • the level of ROS in the subject is reduced by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or about 100%.
  • a miR-485 inhibitor disclosed herein can be administered by any suitable route known in the art.
  • a miR-485 inhibitor is administered parenthetically, intramuscularly, subcutaneously, ophthalmic, intravenously, intraperitoneally, intradermally, intraorbitally, intracerebrally, intracranially, intracerebroventricularly, intraspinally, intraventricular, intrathecally, intracistemally, intracapsularly, intratumorally, or any combination thereof.
  • a miR-485 inhibitor is administered intracerebroventricularly (ICV).
  • a miR-485 inhibitor is administered intravenously.
  • a miR-485 inhibitor of the present disclosure can be used in combination with one or more additional therapeutic agents.
  • the additional therapeutic agent and the miR-485 inhibitor are administered concurrently. In some aspects, the additional therapeutic agent and the miR-485 inhibitor are administered sequentially. [0203] In some aspects, the administration of a miR-485 inhibitor disclosed herein does not result in any adverse effects. In some aspects, miR-485 inhibitors of the present disclosure does not adversely affect body weight when administered to a subject. In some aspects, miR- 485 inhibitors disclosed herein do not result in increased mortality or cause pathological abnormalities when administered to a subject. III. miRNA-485 Inhibitors Useful for the Present Disclosure [0204] Disclosed herein are compounds that can inhibit miR-485 activity (miR-485 inhibitor).
  • a miR-485 inhibitor of the present disclosure comprises a nucleotide sequence encoding a nucleotide molecule that comprises at least one miR-485 binding site, wherein the nucleotide molecule does not encode a protein.
  • the miR-485 binding site is at least partially complementary to the target miRNA nucleic acid sequence (i.e., miR-485), such that the miR-485 inhibitor hybridizes to the miR-485 nucleic acid sequence.
  • the miR-485 binding site of a miR inhibitor disclosed herein has at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence complementarity to the nucleic acid sequence of a miR-485.
  • the miR-485 binding site is fully complementary to the nucleic acid sequence of a miR-485.
  • the miR-485 hairpin precursor can generate both miR-485-5p and miR-485-3p.
  • miR-485" encompasses both miR-485-5p and miR-485- 3p unless specified otherwise.
  • the human mature miR-485-3p has the sequence 5'- GUCAUACACGGCUCUCCUCUCU-3' (SEQ ID NO: 1; miRBase Acc. No. MIMAT0002176).
  • a 5' terminal subsequence of miR-485-3p 5'-UCAUACA-3' (SEQ ID NO: 49) is the seed sequence.
  • the human mature miR-485-5p has the sequence 5'- AGAGGCUGGCCGUGAUGAAUUC-3' (SEQ ID NO: 33; miRBase Acc. No. MIMAT0002175).
  • a 5' terminal subsequence of miR-485-5p 5'-GAGGCUG-3' is the seed sequence.
  • the human mature miR-485-3p has significant sequence similarity to that of other species.
  • the mouse mature miR-485-3p differs from the human mature miR-485-3p by a single amino acid at each of the 5'- and 3'- ends (i.e., has an extra "A” at the 5'-end and missing "C” at the 3'-end).
  • the mouse mature miR-485-3p has the following sequence: 5'-AGUCAUACACGGCUCUCCUCUC-3' (SEQ ID NO: 34; miRBase Acc. No.
  • a miR-485 inhibitor disclosed herein is capable of binding to miR-485-3p and/or miR-485-5p from both human and mouse.
  • the miR-485 binding site is a single-stranded polynucleotide sequence that is complementary (e.g., fully complementary) to a sequence of a miR-485-3p (or a subsequence thereof). In some aspects, the miR-485-3p subsequence comprises the seed sequence.
  • the miR-485 binding site has at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence complementarity to the nucleic acid sequence set forth in SEQ ID NO: 49.
  • the miR-485 binding site is complementary to miR-485-3p except for 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mismatches.
  • the miR-485 binding site is fully complementary to the nucleic acid sequence set forth in SEQ ID NO: 1.
  • the miR-485 binding site is a single-stranded polynucleotide sequence that is complementary (e.g., fully complementary) to a sequence of a miR-485-5p (or a subsequence thereof).
  • the miR-485-5p subsequence comprises the seed sequence.
  • the miR-485 binding site has at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence complementarity to the nucleic acid sequence set forth in SEQ ID NO: 50.
  • the miR-485 binding site is complementary to miR-485-5p except for 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mismatches.
  • the miR-485 binding site is fully complementary to the nucleic acid sequence set forth in SEQ ID NO: 35.
  • the seed region of a miRNA forms a tight duplex with the target mRNA.
  • Most miRNAs imperfectly base-pair with the 3' untranslated region (UTR) of target mRNAs, and the 5' proximal "seed" region of miRNAs provides most of the pairing specificity.
  • UTR 3' untranslated region
  • the 5' proximal "seed" region of miRNAs provides most of the pairing specificity.
  • the first nine miRNA nucleotides (encompassing the seed sequence) provide greater specificity whereas the miRNA ribonucleotides 3' of this region allow for lower sequence specificity and thus tolerate a higher degree of mismatched base pairing, with positions 2-7 being the most important.
  • the miR-485 binding site comprises a subsequence that is fully complementary (i.e., 100% complementary) over the entire length of the seed sequence of miR- 485.
  • miRNA sequences and miRNA binding sequences that can be used in the context of the disclosure include, but are not limited to, all or a portion of those sequences in the sequence listing provided herein, as well as the miRNA precursor sequence, or complement of one or more of these miRNAs.
  • any aspects of the disclosure involving specific miRNAs or miRNA binding sites by name is contemplated also to cover miRNAs or complementary sequences thereof whose sequences are at least about at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to the mature sequence of the specified miRNA
  • miRNA binding sequences of the present disclosure can include additional nucleotides at the 5′, 3′, or both 5′ and 3′ ends of those sequences in the sequence listing provided herein, as long as the modified sequence is still capable of specifically binding to miR-485.
  • miRNA binding sequences of the present disclosure can differ in at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more nucleotides with respect to those sequence in the sequence listing provided, as long as the modified sequence is still capable of specifically binding to miR-485.
  • any methods and compositions discussed herein with respect to miRNA binding molecules or miRNA can be implemented with respect to synthetic miRNAs binding molecules.
  • a miRNA-485 inhibitor of the present disclosure comprises at least 1 nucleotide, at least 2 nucleotides, at least 3 nucleotides, at least 4 nucleotides, at least 5 nucleotides, at least 6 nucleotides, at least 7 nucleotides, at least 8 nucleotides, at least 9 nucleotides, at least 10 nucleotides, at least 11 nucleotides, at least 12 nucleotides, at least 13 nucleotides, at least 14 nucleotides, at least 15 nucleotides, at least 16 nucleotides, at least 17 nucleotides, at least 18 nucleotides, at least 19 nucleotides, or at least 20 nucleotides at the 5' of the nucleotide sequence.
  • a miRNA-485 inhibitor comprises at least 1 nucleotide, at least 2 nucleotides, at least 3 nucleotides, at least 4 nucleotides, at least 5 nucleotides, at least 6 nucleotides, at least 7 nucleotides, at least 8 nucleotides, at least 9 nucleotides, at least 10 nucleotides, at least 11 nucleotides, at least 12 nucleotides, at least 13 nucleotides, at least 14 nucleotides, at least 15 nucleotides, at least 16 nucleotides, at least 17 nucleotides, at least 18 nucleotides, at least 19 nucleotides, or at least 20 nucleotides at the 3' of the nucleotide sequence.
  • a miR-485 inhibitor disclosed herein is about 6 to about 30 nucleotides in length. In some aspects, a miR-485 inhibitor disclosed herein is 7 nucleotides in length. In some aspects, a miR-485 inhibitor disclosed herein is 8 nucleotides in length. In some aspects, a miR-485 inhibitor is 9 nucleotides in length. In some aspects, a miR-485 inhibitor of the present disclosure is 10 nucleotides in length. In some aspects, a miR-485 inhibitor is 11 nucleotides in length. In some aspects, a miR-485 inhibitor is 12 nucleotides in length. In some aspects, a miR-485 inhibitor disclosed herein is 13 nucleotides in length.
  • a miR-485 inhibitor disclosed herein is 14 nucleotides in length. In some aspects, a miR-485 inhibitor disclosed herein is 15 nucleotides in length. In some aspects, a miR-485 inhibitor is 16 nucleotides in length. In some aspects, a miR-485 inhibitor of the present disclosure is 17 nucleotides in length. In some aspects, a miR-485 inhibitor is 18 nucleotides in length. In some aspects, a miR-485 inhibitor is 19 nucleotides in length. In some aspects, a miR-485 inhibitor is 20 nucleotides in length. In some aspects, a miR-485 inhibitor of the present disclosure is 21 nucleotides in length.
  • a miR-485 inhibitor is 22 nucleotides in length.
  • a miR-485 inhibitor disclosed herein comprises a nucleotide sequence that is at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to a sequence selected from SEQ ID NOs: 2 to 30.
  • a miR-485 inhibitor comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 2 to 30, wherein the nucleotide sequence can optionally comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mismatches.
  • a miRNA inhibitor comprises the sequence 5'-UGUAUGA-3' (SEQ ID NO: 2), 5'-GUGUAUGA-3' (SEQ ID NO: 3), 5'-CGUGUAUGA-3' (SEQ ID NO: 4), 5'-CCGUGUAUGA-3' (SEQ ID NO: 5), 5'-GCCGUGUAUGA-3' (SEQ ID NO: 6), 5'- AGCCGUGUAUGA-3' (SEQ ID NO: 7), 5'-GAGCCGUGUAUGA-3' (SEQ ID NO: 8), 5'- AGAGCCGUGUAUGA-3' (SEQ ID NO: 9), 5'-GAGAGCCGUGUAUGA-3' (SEQ ID NO: 10), 5'-GGAGAGCCGUGUAUGA-3' (SEQ ID NO: 11), 5'-AGGAGAGCCGUGUAUGA-3' (SEQ ID NO: 12), 5'-GAGGAGAGCCGUGUAUGA-3' (SEQ ID NO: 13), 5'- AGA
  • the miRNA inhibitor comprises the sequence 5'-UGUAUGAC-3' (SEQ ID NO: 16), 5'-GUGUAUGAC-3' (SEQ ID NO: 17), 5'-CGUGUAUGAC-3' (SEQ ID NO: 18), 5'-CCGUGUAUGAC-3' (SEQ ID NO: 19), 5'-GCCGUGUAUGAC-3' (SEQ ID NO: 20), 5'-AGCCGUGUAUGAC-3' (SEQ ID NO: 21), 5'-GAGCCGUGUAUGAC-3' (SEQ ID NO: 22), 5'-AGAGCCGUGUAUGAC-3' (SEQ ID NO: 23), 5'-GAGAGCCGUGUAUGAC-3' (SEQ ID NO: 24), 5'-GGAGAGCCGUGUAUGAC-3' (SEQ ID NO: 25), 5'- AGGAGAGCCGUGUAUGAC-3' (SEQ ID NO: 26), 5'-GAGGAGAGCCGUGUAUGAC-3' (SEQ ID NO: 27),
  • the miRNA inhibitor has a sequence selected from the group consisting of: 5'-TGTATGA-3' (SEQ ID NO: 62), 5'-GTGTATGA-3' (SEQ ID NO: 63), 5'- CGTGTATGA-3' (SEQ ID NO: 64), 5'-CCGTGTATGA-3' (SEQ ID NO: 65), 5'- GCCGTGTATGA-3' (SEQ ID NO: 66), 5'-AGCCGTGTATGA-3' (SEQ ID NO: 67), 5'- GAGCCGTGTATGA-3' (SEQ ID NO: 68), 5'-AGAGCCGTGTATGA-3' (SEQ ID NO: 69), 5'-GAGAGCCGTGTATGA-3' (SEQ ID NO: 70), 5'-GGAGAGCCGTGTATGA-3' (SEQ ID NO: 71), 5'-AGGAGAGCCGTGTATGA-3' (SEQ ID NO: 72), 5'- GAGGAGAGCCGTGTATGA
  • a miRNA inhibitor disclosed herein comprises a nucleotide sequence that is at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% identical to 5'- AGAGAGGAGAGCCGUGUAUGAC-3' (SEQ ID NO: 30) or 5'- AGAGAGGAGAGCCGTGTATGAC-3' (SEQ ID NO: 90).
  • the miRNA inhibitor comprises a nucleotide sequence that has at least 90% similarity to 5'- AGAGAGGAGAGCCGUGUAUGAC-3' (SEQ ID NO: 30) or 5'- AGAGAGGAGAGCCGTGTATGAC-3' (SEQ ID NO: 90). In some aspects, the miRNA inhibitor comprises the nucleotide sequence 5'- AGAGAGGAGAGCCGUGUAUGAC-3' (SEQ ID NO: 30) or 5'-AGAGAGGAGAGCCGTGTATGAC-3' (SEQ ID NO: 90) with one substitution or two substitutions.
  • the miRNA inhibitor comprises the nucleotide sequence 5'- AGAGAGGAGAGCCGUGUAUGAC-3' (SEQ ID NO: 30) or 5'- AGAGAGGAGAGCCGTGTATGAC-3' (SEQ ID NO: 90). In some aspects, the miRNA inhibitor comprises the nucleotide sequence 5'- AGAGAGGAGAGCCGUGUAUGAC-3' (SEQ ID NO: 30).
  • a miR-485 inhibitor of the present disclosure comprises the sequence disclosed herein, e.g., any one of SEQ ID NOs: 2 to 30, and at least one, at least two, at least three, at least four or at least five additional nucleic acid at the N terminus, at least one, at least two, at least three, at least four, or at least five additional nucleic acid at the C terminus, or both.
  • a miR-485 inhibitor of the present disclosure comprises the sequence disclosed herein, e.g., any one of SEQ ID NOs: 2 to 30, and one additional nucleic acid at the N terminus and/or one additional nucleic acid at the C terminus.
  • a miR-485 inhibitor comprises 5’-AGAGAGGAGAGCCGUGUAUGAC-3' (SEQ ID NO: 30).
  • a miR-485 inhibitor of the present disclosure comprises one miR- 485 binding site.
  • a miR-485 inhibitor disclosed herein comprises at least two miR-485 binding sites.
  • a miR-485 inhibitor comprises three miR-485 binding sites.
  • a miR-485 inhibitor comprises four miR-485 binding sites.
  • a miR-485 inhibitor comprises five miR-485 binding sites.
  • a miR- 485 inhibitor comprises six or more miR-485 binding sites. In some aspects, all the miR-485 binding sites are identical.
  • a miR-485 binding sites are different. In some aspects, at least one of the miR-485 binding sites is different. In some aspects, all the miR-485 binding sites are miR-485-3p binding sites. In some aspects, all the miR-485 binding sites are miR-485-5p binding sites. In some aspects, a miR-485 inhibitor comprises at least one miR- 485-3p binding site and at least one miR-485-5p binding site. 1. Chemically Modified Polynucleotides [0223] In some aspects, a miR-485 inhibitor disclosed herein comprises a polynucleotide which includes at least one chemically modified nucleoside and/or nucleotide.
  • a “nucleoside” refers to a compound containing a sugar molecule (e.g., a pentose or ribose) or a derivative thereof in combination with an organic base (e.g., a purine or pyrimidine) or a derivative thereof (also referred to herein as “nucleobase”).
  • a “nucleotide” refers to a nucleoside including a phosphate group.
  • Modified nucleotides can be synthesized by any useful method, such as, for example, chemically, enzymatically, or recombinantly, to include one or more modified or non-natural nucleosides.
  • Polynucleotides can comprise a region or regions of linked nucleosides. Such regions can have variable backbone linkages. The linkages can be standard phosphodiester linkages, in which case the polynucleotides would comprise regions of nucleotides.
  • the modified polynucleotides disclosed herein can comprise various distinct modifications. In some aspects, the modified polynucleotides contain one, two, or more (optionally different) nucleoside or nucleotide modifications.
  • a modified polynucleotide can exhibit one or more desirable properties, e.g., improved thermal or chemical stability, reduced immunogenicity, reduced degradation, increased binding to the target microRNA, reduced non-specific binding to other microRNA or other molecules, as compared to an unmodified polynucleotide.
  • a polynucleotide of the present disclosure e.g., a miR-485 inhibitor is chemically modified.
  • the terms "chemical modification” or, as appropriate, “chemically modified” refer to modification with respect to adenosine (A), guanosine (G), uridine (U), thymidine (T) or cytidine (C) ribo- or deoxyribonucleosides in one or more of their position, pattern, percent or population, including, but not limited to, its nucleobase, sugar, backbone, or any combination thereof.
  • a polynucleotide of the present disclosure can have a uniform chemical modification of all or any of the same nucleoside type or a population of modifications produced by downward titration of the same starting modification in all or any of the same nucleoside type, or a measured percent of a chemical modification of all any of the same nucleoside type but with random incorporation
  • the polynucleotide of the present disclosure e.g., a miR-485 inhibitor
  • Modified nucleotide base pairing encompasses not only the standard adenine- thymine, adenine-uracil, or guanine-cytosine base pairs, but also base pairs formed between nucleotides and/or modified nucleotides comprising non-standard or modified bases, wherein the arrangement of hydrogen bond donors and hydrogen bond acceptors permits hydrogen bonding between a non-standard base and a standard base or between two complementary non- standard base structures.
  • non-standard base pairing is the base pairing between the modified nucleobase inosine and adenine, cytosine or uracil.
  • TD's of the present disclosure can be administered as RNAs, as DNAs, or as hybrid molecules comprising both RNA and DNA units.
  • the polynucleotide (e.g., a miR-485 inhibitor) includes a combination of at least two (e.g., 2, 3, 4, 5, 6, 7, 8, 8, 10, 11, 12, 13, 14, 15, 16, 17, 18, 18, 20 or more) modified nucleobases.
  • the nucleobases, sugar, backbone linkages, or any combination thereof in a polynucleotide are modified by at least about 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100%.
  • the chemical modification is at nucleobases in a polynucleotide of the present disclosure (e.g., a miR-485 inhibitor).
  • the at least one chemically modified nucleoside is a modified uridine (e.g., pseudouridine ( ⁇ ), 2-thiouridine (s2U), 1- methyl-pseudouridine (m1 ⁇ ), 1-ethyl-pseudouridine (e1 ⁇ ), or 5-methoxy-uridine (mo5U)), a modified cytosine (e.g., 5-methyl-cytidine (m5C)) a modified adenosine (e.g, 1-methyl- adenosine (m1A), N6-methyl-adenosine (m6A), or 2-methyl-adenine (m2A)), a modified guanosine (e.g., 7-methyl-guanosine (m7G) or 1-
  • uridine e.g., pseudouridine (
  • the polynucleotide of the present disclosure is uniformly modified (e.g., fully modified, modified throughout the entire sequence) for a particular modification.
  • a polynucleotide can be uniformly modified with the same type of base modification, e.g., 5-methyl-cytidine (m5C), meaning that all cytosine residues in the polynucleotide sequence are replaced with 5-methyl-cytidine (m5C).
  • m5C 5-methyl-cytidine
  • a polynucleotide can be uniformly modified for any type of nucleoside residue present in the sequence by replacement with a modified nucleoside such as any of those set forth above.
  • the polynucleotide of the present disclosure includes a combination of at least two (e.g., 2, 3, 4 or more) of modified nucleobases.
  • the polynucleotide of the present disclosure can include any useful linkage between the nucleosides.
  • linkages, including backbone modifications, that are useful in the composition of the present disclosure include, but are not limited to the following: 3'-alkylene phosphonates, 3'-amino phosphoramidate, alkene containing backbones, aminoalkylphosphoramidates, aminoalkylphosphotriesters, boranophosphates, -CH2-O-N(CH3)-CH2-, -CH2-N(CH3)-N(CH3)-CH2-, -CH2-NH-CH2-, chiral phosphonates, chiral phosphorothioates, formacetyl and thioformacetyl backbones, methylene (methylimino), methylene formacetyl and thioformacetyl backbones, methyleneimino and methylenehydr
  • the presence of a backbone linkage disclosed above increase the stability and resistance to degradation of a polynucleotide of the present disclosure (i.e., miR- 485 inhibitor).
  • at least about 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% of the backbone linkages in a polynucleotide of the present disclosure (i.e., miR-485 inhibitor) are modified (e.g., all of them are phosphorothioate).
  • a backbone modification that can be included in a polynucleotide of the present disclosure comprises phosphorodiamidate morpholino oligomer (PMO) and/or phosphorothioate (PS) modification.
  • PMO phosphorodiamidate morpholino oligomer
  • PS phosphorothioate
  • sugar modifications [0240] The modified nucleosides and nucleotides which can be incorporated into a polynucleotide of the present disclosure (i.e., miR-485 inhibitor) can be modified on the sugar of the nucleic acid.
  • a miR-485 inhibitor described herein comprises a nucleic acid which comprises at least one nucleoside analog (e.g., a nucleoside with a sugar modification).
  • the sugar modification increases the affinity of the binding of a miR-485 inhibitor to miR-485 nucleic acid sequence.
  • affinity- enhancing nucleoside analogues in the miR-485 inhibitor such as LNA or 2'-substituted sugars, can allow the length and/or the size of the miR-485 inhibitor to be reduced.
  • sugar modifications e.g., LNA
  • RNA includes the sugar group ribose, which is a 5-membered ring having an oxygen.
  • modified nucleotides include replacement of the oxygen in ribose (e.g., with S, Se, or alkylene, such as methylene or ethylene); addition of a double bond (e.g., to replace ribose with cyclopentenyl or cyclohexenyl); ring contraction of ribose (e.g., to form a 4-membered ring of cyclobutane or oxetane); ring expansion of ribose (e.g., to form a 6- or 7-membered ring having an additional carbon or heteroatom, such as for anhydrohexitol, altritol, mannitol, cyclohexanyl, cyclohexenyl, and morpholino that also has a phosphoramidate backbone); multicyclic forms (e.g., tricyclo; and "unlocked" forms, such as glycol nucleic acid (GNA) (e.
  • GAA glyco
  • the sugar group can also contain one or more carbons that possess the opposite stereochemical configuration than that of the corresponding carbon in ribose.
  • a polynucleotide molecule can include nucleotides containing, e.g., arabinose, as the sugar.
  • the 2′ hydroxyl group (OH) of ribose can be modified or replaced with a number of different substituents.
  • substitutions at the 2′-position include, but are not limited to, H, halo, optionally substituted C 1-6 alkyl; optionally substituted C 1-6 alkoxy; optionally substituted C6-10 aryloxy; optionally substituted C3-8 cycloalkyl; optionally substituted C3-8 cycloalkoxy; optionally substituted C 6-10 aryloxy; optionally substituted C 6-10 aryl-C 1-6 alkoxy, optionally substituted C 1-12 (heterocyclyl)oxy; a sugar (e.g., ribose, pentose, or any described herein); a polyethyleneglycol (PEG), -O(CH2CH2O)nCH2CH2OR, where R is H or optionally substituted alkyl, and n is an integer from 0 to 20 (e.g., from 0 to 4, from 0 to 8, from 0 to 10, from 0 to 16, from 1 to 4, from 1 to 8, from 1 to 10, from 1 to 16, from 1 to
  • nucleoside analogues present in a polynucleotide of the present disclosure comprise, e.g., 2'-O-alkyl-RNA units, 2'-OMe-RNA units, 2'-O-alkyl-SNA, 2'-amino-DNA units, 2'-fluoro-DNA units, LNA units, arabino nucleic acid (ANA) units, 2'-fluoro-ANA units, HNA units, INA (intercalating nucleic acid) units, 2'MOE units, or any combination thereof.
  • ANA arabino nucleic acid
  • INA intercalating nucleic acid
  • the LNA is, e.g., oxy-LNA (such as beta- D-oxy-LNA, or alpha-L-oxy-LNA), amino-LNA (such as beta-D-amino-LNA or alpha-L- amino-LNA), thio-LNA (such as beta-D-thio0-LNA or alpha-L-thio-LNA), ENA (such a beta- D-ENA or alpha-L-ENA), or any combination thereof.
  • oxy-LNA such as beta- D-oxy-LNA, or alpha-L-oxy-LNA
  • amino-LNA such as beta-D-amino-LNA or alpha-L- amino-LNA
  • thio-LNA such as beta-D-thio0-LNA or alpha-L-thio-LNA
  • ENA such a beta- D-ENA or alpha-L-ENA
  • nucleoside analogues that can be included in a polynucleotide of the present disclosure comprises a locked nucleic acid (LNA), an unlocked nucleic acid (UNA), an arabino nucleic acid (ABA), a bridged nucleic acid (BNA), and/or a peptide nucleic acid (PNA).
  • LNA locked nucleic acid
  • UNA unlocked nucleic acid
  • ABA arabino nucleic acid
  • BNA bridged nucleic acid
  • PNA peptide nucleic acid
  • nucleoside analog comprises a LNA; 2'-0-alkyl-RNA; 2'-amino-DNA; 2'-fluoro-DNA; arabino nucleic acid (ANA); 2'-fluoro-ANA, hexitol nucleic acid (HNA), intercalating nucleic acid (INA), constrained ethyl nucleoside (cEt), 2'-0-methyl nucleic acid (2'-OMe), 2'-0- methoxyethyl nucleic acid (2'-MOE), or any combination thereof.
  • a polynucleotide of the present disclosure can comprise both modified RNA nucleoside analogues (e.g., LNA) and DNA units.
  • a miR-485 inhibitor is a gapmer. See, e.g., U.S. Pat. Nos. 8,404,649; 8,580,756; 8,163,708; 9,034,837; all of which are herein incorporated by reference in their entireties.
  • a miR-485 inhibitor is a micromir. See U.S. Pat. Appl. Publ. No. US20180201928, which is herein incorporated by reference in its entirety.
  • a polynucleotide of the present disclosure can include modifications to prevent rapid degradation by endo- and exo-nucleases.
  • Modifications include, but are not limited to, for example, (a) end modifications, e.g., 5' end modifications (phosphorylation, dephosphorylation, conjugation, inverted linkages, etc.), 3' end modifications (conjugation, DNA nucleotides, inverted linkages, etc.), (b) base modifications, e.g., replacement with modified bases, stabilizing bases, destabilizing bases, or bases that base pair with an expanded repertoire of partners, or conjugated bases, (c) sugar modifications (e.g., at the 2' position or 4' position) or replacement of the sugar, as well as (d) internucleoside linkage modifications, including modification or replacement of the phosphodiester linkages.
  • end modifications e.g., 5' end modifications (phosphorylation, dephosphorylation, conjugation, inverted linkages, etc.), 3' end modifications (conjugation
  • the miR-485 inhibitors of the present disclosure can be administered, e.g., to a subject suffering from a mitochondrial disorder, using any relevant delivery system known in the art.
  • the delivery system is a vector.
  • the present disclosure provides a vector comprising a miR-485 inhibitor of the present disclosure.
  • the vector is viral vector.
  • the viral vector is an adenoviral vector or an adeno-associated viral vector.
  • the viral vector is an AAV that has a serotype of AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, or any combination thereof.
  • the adenoviral vector is a third generation adenoviral vector.
  • ADEASYTM is by far the most popular method for creating adenoviral vector constructs.
  • the system consists of two types of plasmids: shuttle (or transfer) vectors and adenoviral vectors.
  • the transgene of interest is cloned into the shuttle vector, verified, and linearized with the restriction enzyme PmeI.
  • This construct is then transformed into ADEASIER-1 cells, which are BJ5183 E. coli cells containing PADEASYTM.
  • PADEASYTM is a ⁇ 33Kb adenoviral plasmid containing the adenoviral genes necessary for virus production.
  • the shuttle vector and the adenoviral plasmid have matching left and right homology arms which facilitate homologous recombination of the transgene into the adenoviral plasmid.
  • Recombinant adenoviral plasmids are then verified for size and proper restriction digest patterns to determine that the transgene has been inserted into the adenoviral plasmid, and that other patterns of recombination have not occurred.
  • the recombinant plasmid is linearized with PacI to create a linear dsDNA construct flanked by ITRs.293 or 911 cells are transfected with the linearized construct, and virus can be harvested about 7-10 days later.
  • the viral vector is a retroviral vector, e.g., a lentiviral vector (e.g., a third or fourth generation lentiviral vector). Lentiviral vectors are usually created in a transient transfection system in which a cell line is transfected with three separate plasmid expression systems.
  • the transfer vector plasmid portions of the HIV provirus
  • the packaging plasmid or construct and a plasmid with the heterologous envelop gene (env) of a different virus.
  • the three plasmid components of the vector are put into a packaging cell which is then inserted into the HIV shell.
  • the virus portions of the vector contain insert sequences so that the virus cannot replicate inside the cell system.
  • Current third generation lentiviral vectors encode only three of the nine HIV-1 proteins (Gag, Pol, Rev), which are expressed from separate plasmids to avoid recombination-mediated generation of a replication- competent virus.
  • AAV vector known in the art can be used in the methods disclosed herein.
  • the AAV vector can comprise a known vector or can comprise a variant, fragment, or fusion thereof.
  • the AAV vector is selected from the group consisting of AAV type 1 (AAV1), AAV2, AAV3A, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAVrh.74, avian AAV, bovine AAV, canine AAV, equine AAV, goat AAV, primate AAV, non-primate AAV, bovine AAV, shrimp AAV, snake AAV, and any combination thereof.
  • AAV type 1 AAV1
  • AAV2 AAV3A
  • AAV3B AAV4
  • AAV5 AAV6, AAV7, AAV8, AAV9
  • AAV10 AAV11, AAV12, AAV13, AAVrh.74
  • avian AAV bovine AAV
  • bovine AAV canine AAV, equine AAV, goat AAV, primate AAV, non-primate AAV, bovine AAV, shrimp A
  • the AAV vector is derived from an AAV vector selected from the group consisting of AAV1, AAV2, AAV3A, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAVrh.74, avian AAV, bovine AAV, canine AAV, equine AAV, goat AAV, primate AAV, non-primate AAV, ovine AAV, shrimp AAV, snake AAV, and any combination thereof.
  • the AAV vector is a chimeric vector derived from at least two AAV vectors selected from the group consisting of AAV1, AAV2, AAV3A, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAVrh.74, avian AAV, bovine AAV, canine AAV, equine AAV, goat AAV, primate AAV, non-primate AAV, ovine AAV, shrimp AAV, snake AAV, and any combination thereof.
  • the AAV vector comprises regions of at least two different AAV vectors known in the art.
  • the AAV vector comprises an inverted terminal repeat from a first AAV (e.g., AAV1, AAV2, AAV3A, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAVrh.74, avian AAV, bovine AAV, canine AAV, equine AAV, goat AAV, primate AAV, non-primate AAV, ovine AAV, shrimp AAV, snake AAV, or any derivative thereof) and a second inverted terminal repeat from a second AAV (e.g., AAV1, AAV2, AAV3A, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAVrh.74, avian AAV, bovine AAV, canine AAV, equine AAV, goat AAV, primate
  • the AAV vector comprises a portion of an AAV vector selected from the group consisting of AAV1, AAV2, AAV3A, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAVrh.74, avian AAV, bovine AAV, canine AAV, equine AAV, goat AAV, primate AAV, non-primate AAV, ovine AAV, shrimp AAV, snake AAV, and any combination thereof.
  • the AAV vector comprises AAV2.
  • the AAV vector comprises a splice acceptor site.
  • the AAV vector comprises a promoter. Any promoter known in the art can be used in the AAV vector of the present disclosure.
  • the promoter is an RNA Pol III promoter.
  • the RNA Pol III promoter is selected from the group consisting of the U6 promoter, the H1 promoter, the 7SK promoter, the 5S promoter, the adenovirus 2 (Ad2) VAI promoter, and any combination thereof.
  • the promoter is a cytomegalovirus immediate-early gene (CMV) promoter, an EF1a promoter, an SV40 promoter, a PGK1 promoter, a Ubc promoter, a human beta actin promoter, a CAG promoter, a TRE promoter, a UAS promoter, a Ac5 promoter, a polyhedrin promoter, a CaMKIIa promoter, a GAL1 promoter, a GAL10 promoter, a TEF promoter, a GDS promoter, a ADH1 promoter, a CaMV35S promoter, or a Ubi promoter.
  • the promoter comprises the U6 promoter.
  • the AAV vector comprises a constitutively active promoter (constitutive promoter).
  • the constitutive promoter is selected from the group consisting of hypoxanthine phosphoribosyl transferase (HPRT), adenosine deaminase, pyruvate kinase, beta-actin promoter, cytomegalovirus (CMV), simian virus (e.g., SV40), papilloma virus, adenovirus, human immunodeficiency virus (HIV), Rous sarcoma virus, a retrovirus long terminal repeat (LTR), Murine stem cell virus (MSCV) and the thymidine kinase promoter of herpes simplex virus.
  • HPRT hypoxanthine phosphoribosyl transferase
  • CMV cytomegalovirus
  • simian virus e.g., SV40
  • papilloma virus adenovirus
  • the promoter is an inducible promoter.
  • the inducible promoter is a tissue specific promoter.
  • the tissue specific promoter drives transcription of the coding region of the AAV vector in a neuron, a glial cell, or in both a neuron and a glial cell.
  • the AAV vector comprises one or more enhancers.
  • the one or more enhancer are present in the AAV alone or together with a promoter disclosed herein.
  • the AAV vector comprises a 3'UTR poly(A) tail sequence.
  • the 3'UTR poly(A) tail sequence is selected from the group consisting of bGH poly(A), actin poly(A), hemoglobin poly(A), and any combination thereof. In some aspects, the 3'UTR poly(A) tail sequence comprises bGH poly(A). [0261] In some aspects, a polynucleotide of the present disclosure can be administered (e.g., to a subject in need thereof) with a delivery agent.
  • Non-limiting examples of delivery agents that can be used include an exosome, a lipidoid, a liposome, a lipoplex, a lipid nanoparticle, an extracellular vesicle, a synthetic vesicle, a polymeric compound, a peptide, a protein, a cell, a nanoparticle mimic, a nanotube, a micelle, a viral vector, a conjugate, and combinations thereof.
  • the present disclosure also provides a composition comprising a polynucleotide described herein (e.g., miR-485 inhibitor) and a delivery agent.
  • the delivery agent comprises a cationic carrier unit comprising [WP]-L1-[CC]-L2-[AM] (formula I) or [WP]-L1-[AM]-L2-[CC] (formula II) wherein WP is a water-soluble biopolymer moiety; CC is a positively charged carrier moiety; AM is an adjuvant moiety; and, L1 and L2 are independently optional linkers, and wherein when mixed with a nucleic acid at an ionic ratio of about 1:1, the cationic carrier unit forms a micelle.
  • composition comprising a polynucleotide described herein interacts with the cationic carrier unit via an ionic bond.
  • the water-soluble polymer comprises poly(alkylene glycols), poly(oxyethylated polyol), poly(olefinic alcohol), poly(vinylpyrrolidone), poly(hydroxyalkylmethacrylamide), poly(hydroxyalkylmethacrylate), poly(saccharides), poly( ⁇ - hydroxy acid), poly(vinyl alcohol), polyglycerol, polyphosphazene, polyoxazolines ("POZ”) poly(N-acryloylmorpholine), or any combinations thereof.
  • the water-soluble polymer comprises polyethylene glycol (“PEG”), polyglycerol, or poly(propylene glycol) (“PPG").
  • the water-soluble polymer comprises: , (formula III), wherein n is 1-1000.
  • the n is at least about 110, at least about 111, at least about 112, at least about 113, at least about 114, at least about 115, at least about 116, at least about 117, at least about 118, at least about 119, at least about 120, at least about 121, at least about 122, at least about 123, at least about 124, at least about 125, at least about 126, at least about 127, at least about 128, at least about 129, at least about 130, at least about 131, at least about 132, at least about 133, at least about 134, at least about 135, at least about 136, at least about 137, at least about 138, at least about 139, at least about 140, or at least about 141.
  • the n is about 80 to about 90, about 90 to about 100, about 100 to about 110, about 110 to about 120, about 120 to about 130, about 140 to about 150, about 150 to about 160. In some aspects, the n is about 114. [0266] In some aspects, the water-soluble polymer is linear, branched, or dendritic. In some aspects, the cationic carrier moiety comprises one or more basic amino acids.
  • the cationic carrier moiety comprises at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least 11, at least 12, at least 13, at least 14, at last 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 31, at least 32, at least 33, at least 34, at least 35, at least 36, at least 37, at least 38, at least 39, at least 40, at least 41, at least 42, at least 43, at least 44, at least 45, at least 46, at least 47, at least 48, at least 49, at least 50, at least 51, at least 52, at least 53, at least 54, at least 55, at least 56, at least 57, at least 58, at least 59, at least 60, at least 61, at least 62, at least 63, at least 64, at least 65, at least 66,
  • the cationic carrier moiety comprises about 30 to about 50 basic amino acids. In some aspects, the cationic carrier moiety comprises about 60, about 70, about 80, about 90, or about 100 basic amino acids. In some aspects, the cationic carrier moiety comprises about 80 basic amino acids. In some aspects, the basic amino acid comprises arginine, lysine, histidine, or any combination thereof. In some aspects, the cationic carrier moiety comprises about 80 lysine monomers. [0267] In some aspects, the adjuvant moiety is capable of modulating an immune response, an inflammatory response, and/or a tissue microenvironment. In some aspects, the adjuvant moiety comprises an imidazole derivative, an amino acid, a vitamin, or any combination thereof.
  • the adjuvant moiety comprises: wherein each of G1 and G2 is H, an aromatic ring, or 1-10 alkyl, or G1 and G2 together form an aromatic ring, and wherein n is 1-10.
  • the adjuvant moiety comprises nitroimidazole.
  • the adjuvant moiety comprises metronidazole, tinidazole, nimorazole, dimetridazole, pretomanid, ornidazole, megazol, azanidazole, benznidazole, or any combination thereof.
  • the adjuvant moiety comprises an amino acid.
  • the adjuvant moiety comprises (formula V), wherein Ar is wherein each of Z1 and Z2 is H or OH.
  • the adjuvant moiety comprises a vitamin.
  • the vitamin comprises a cyclic ring or cyclic hetero atom ring and a carboxyl group or hydroxyl group.
  • the vitamin comprises: (formula VI), wherein each of Y1 and Y2 is C, N, O, or S, and wherein n is 1 or 2.
  • the vitamin is selected from the group consisting of vitamin A, vitamin B1, vitamin B2, vitamin B3, vitamin B6, vitamin B7, vitamin B9, vitamin B12, vitamin C, vitamin D2, vitamin D3, vitamin E, vitamin M, vitamin H, and any combination thereof.
  • the vitamin is vitamin B3.
  • the adjuvant moiety comprises at least about two, at least about three, at least about four, at least about five, at least about six, at least about seven, at least about eight, at least about nine, at least about ten, at least about 11, at least about 12, at least about 13, at least about 14, at least about 15, at least about 16, at least about 17, at least about 18, at least about 19, at least about 20, at least about 21, at least about 22, at least about 23, at least about 24, at least about 25, at least about 26, at least about 27, at least about 28, at least about 29, at least about 30, at least about 31, at least about 32, at least about 33, at least about 34, at least about 35, at least about 36, at least about 37, at least about 38, at least about 39, at least about 40, at least about 41, at least about 42, at least about 43, at least about 44, at least about 45, at least about 46, at least about 47, at least about 48, at least about 49, or at least about 50 vitamin B3.
  • the adjuvant moiety comprises about 35 vitamin B3.
  • the composition comprises a water-soluble biopolymer moiety with about 100 to about 130 PEG units, a cationic carrier moiety comprising a poly-lysine with about 30 to about 100 lysines (e.g., about 80 lysines), and an adjuvant moiety with about 5 to about 50 vitamin B3 (e.g., about 35 vitamin B3).
  • the composition comprises (i) a water-soluble biopolymer moiety with about 100 to about 200 PEG units, (ii) about 30 to about 100 lysines with an amine group (e.g., about 40 lysines), (iii) about 1 to 20 lysines, each having a thiol group (e.g., about 5 lysines, each with a thiol group), and (iv) about 5 to 50 lysines fused to vitamin B3 (e.g., about 35 lysines, each fused to vitamin B3).
  • an amine group e.g., about 40 lysines
  • a thiol group e.g., about 5 lysines, each with a thiol group
  • vitamin B3 e.g., about 35 lysines, each fused to vitamin B3
  • the composition further comprises a targeting moiety, e.g., a LAT1 targeting ligand, e.g., phenyl alanine, linked to the water soluble polymer.
  • a targeting moiety e.g., a LAT1 targeting ligand, e.g., phenyl alanine
  • the thiol groups in the composition form disulfide bonds.
  • the composition comprises (1) a micelle comprising (i) about 100 to about 200 PEG units (e.g., about 114 units), (ii) about 30 to about 100 lysines with an amine group (e.g., about 40 lysines), (iii) about 3 to 50 lysines, each having a thiol group (e.g., about 35 lysines, each with a thiol group), and (iv) about 2 to 20 lysines fused to vitamin B3 (e.g., about 5 lysines, each fused to vitamin B3), and (2) an isolated polynucleotide described herein (e.g., miR- 485 inhibitor), wherein the isolated polynucleotide is encapsulated within the micelle.
  • a micelle comprising (i) about 100 to about 200 PEG units (e.g., about 114 units), (ii) about 30 to about 100 lysines with an amine group (e.g., about 40
  • the composition further comprises a targeting moiety, e.g., a LAT1 targeting ligand, e.g., phenyl alanine, linked to the PEG units.
  • a targeting moiety e.g., a LAT1 targeting ligand, e.g., phenyl alanine
  • the thiol groups in the micelle form disulfide bonds.
  • the present disclosure also provides a micelle comprising a polynucleotide described herein (e.g., miR-485 inhibitor), wherein the polynucleotide and the delivery agent are associated with each other.
  • the association is a covalent bond, a non-covalent bond, or an ionic bond.
  • the positive charge of the cationic carrier moiety of the cationic carrier unit is sufficient to form a micelle when mixed with the polynucleotide disclosed herein in a solution, wherein the overall ionic ratio of the positive charges of the cationic carrier moiety of the cationic carrier unit and the negative charges of the polynucleotide (or vector comprising the polynucleotide) in the solution is about 1: 1.
  • the cationic carrier unit is capable of protecting the polynucleotide of the present disclosure from enzymatic degradation. See PCT Publication No. WO 2020/261227, which is herein incorporated by reference in its entirety.
  • the polynucleotides disclosed herein are DNA (e.g., a DNA molecule or a combination thereof), RNA (e.g., a RNA molecule or a combination thereof), or any combination thereof.
  • the polynucleotides described herein comprise nucleic acid sequences comprising single stranded or double stranded RNA or DNA (e.g., ssDNA or dsDNA) in genomic or cDNA form, or DNA-RNA hybrids, each of which can include chemically or biochemically modified, non-natural, or derivatized nucleotide bases.
  • nucleic acid sequences can comprise additional sequences useful for promoting expression and/or purification of the encoded polypeptide, including but not limited to polyA sequences, modified Kozak sequences, and sequences encoding epitope tags, export signals, and secretory signals, nuclear localization signals, and plasma membrane localization signals.
  • Carrier Units [0280]
  • a miR-485 inhibitor described herein can be administered (e.g., to a subject suffering from a mitochondrial disorder) using a carrier unit.
  • the carrier units can self-assemble into micelles or be incorporated into micelles.
  • carrier units of the present disclosure comprise a water-soluble biopolymer moiety (e.g., PEG), a charged carrier moiety, a crosslinking moiety, and a hydrophobic moiety.
  • the charged carrier moiety is cationic (e.g., a polylysine).
  • Carrier units of the present disclosure can be used to deliver negatively charged payloads (e.g., polynucleotides disclosed herein).
  • negatively charged payloads (e.g., polynucleotides disclosed herein) that can be delivered by micelles comprises at least about 100, at least about 1000, at least about 2000, at least about 3000, or at least about 4000 nucleotides in length.
  • Carrier units with a cationic charged carrier moiety can be used for the delivery of anionic payloads, e.g., polynucleotides.
  • Carrier units with an anionic charged carrier moiety can be used for the delivery of cationic payloads, e.g., positively charged polynucleotides.
  • the cationic charged carrier moiety and the anionic payloads can electrostatically interact with each other.
  • the resulting carrier unit:payload complex can have a "head” comprising the water- soluble biopolymer moiety and a "tail" comprising the cationic carrier moiety electrostatically bound to the payload.
  • Carrier unit:payload complexes can self-associate, alone or in combination with other molecules, to yield micelles in which the anionic payload (e.g., polynucleotides disclosed herein) is located in the core of the micelle and the water-soluble biopolymer moiety is facing the solvent.
  • the anionic payload e.g., polynucleotides disclosed herein
  • micelles of the present disclosure encompasses not only classic micelles but also small particles, small micelles, micelles, rod-like structures, or polymersomes. Given that polymersomes comprise a luminal space, it is to be understood that all the disclosures related to the "core" of classic micelles are equally applicable to the luminal space in polymersomes comprising carrier units of the present disclosure.
  • the carrier units of the present disclosure can also comprise a targeting moiety (e.g., a targeting ligand) covalently linked to the water-soluble biopolymer moiety via one or more optional linkers.
  • a targeting moiety e.g., a targeting ligand
  • the targeting moiety can be located on the surface of the micelle and can deliver the micelle to a specific target tissue, to a specific cell type, and/or facilitate transport across a physiological barrier (e.g., cell plasma membrane).
  • the micelles of the present disclosure can comprises more than one type of targeting moiety.
  • the carrier units of the present disclosure can also comprise a hydrophobic moiety (HM) covalently linked to the charged cationic carrier moiety.
  • HM hydrophobic moiety
  • the hydrophobic moiety can have, e.g., a therapeutic, a co-therapeutic effect, or positively affect the homeostasis of the target cell or target tissue.
  • the HM comprises one or more amino acids.
  • the HM comprises one or more amino acids linked to a hydrophobic molecule (e.g., a vitamin).
  • the HM comprises one or more lysine residues covalently bound to a hydrophobic molecule (e.g., a vitamin).
  • the anionic payload e.g., miR-485 inhibitor
  • the anionic payload (e.g., miR-485 inhibitor) can be covalently linked to the cationic carrier unit, e.g., a linker such as cleavable linker.
  • a linker such as cleavable linker.
  • Non-limiting examples of various aspects are shown in the present disclosure.
  • the disclosure refers in particular to the use of cationic carrier units, e.g., to deliver anionic payloads such as nucleic acids (e.g., miR-485 inhibitor).
  • the disclosures can be equally applied to the delivery of cationic payloads or to the delivery of neutral payloads by reversing the charges of the carrier moiety and payload (i.e., using an anionic carrier moiety in the carrier unit to deliver a cationic payload), or by using a neutral payload linked to a cationic or anionic adapter that would electrostatically interact with an anionic or cationic carrier moiety, respectively.
  • the present disclosure provides cationic carrier units of Schema I through Schema VI: [CC]-L1-[CM]-L2-[HM] (Schema I); [CC]-L1-[HM]-L2-[CM] (Schema II); [HM]-L1-[CM]-L2-[CC] (Schema III); [HM]-L1-[CC]-L2-[CM] (Schema IV); [CM]-L1-[CC]-L2-[HM] (Schema V); or [CM]-L1-[HM]-L2-[CC] (Schema VI); wherein CC is a cationic carrier moiety, e.g., a polylysine; CM is a crosslinking moiety; HM is a hydrophobic moiety, e.g., vitamin, e.g., vitamin B3; and, L1 and L2 are independently optional linkers.
  • CC is a cationic carrier moiety, e.g.,
  • the cationic carrier unit further comprises a water-soluble polymer (WP).
  • WP water-soluble polymer
  • the water-soluble polymer is attached to [CC], [HM], and/or [CM].
  • the water-soluble polymer is attached to the N terminus of [CC], [HM], or [CM].
  • the water-soluble polymer is attached to the N terminus of [CC].
  • the water-soluble polymer is attached to the C terminus of [CC], [HM], or [CM].
  • the water-soluble polymer is attached to the C terminus of [CC].
  • the cationic carrier unit comprises: [WP]-L3-[CC]-L1-[CM]-L2-[HM] (Schema I’); [WP]-L3-[CC]-L1-[HM]-L2-[CM] (Schema II’); [WP]-L3-[HM]-L1-[CM]-L2-[CC] (Schema III’); [WP]-L3-[HM]-L1-[CC]-L2-[CM] (Schema IV’); [WP]-L3-[CM]-L1-[CC]-L2-[HM] (Schema V’); or [WP]-L3-[CM]-L1-[HM]-L2-[CC] (Schema VI’).
  • the [WP] component can be connected to at least one targeting moiety, i.e., [T] n -[WP]-... wherein n is an integer, e.g., 1, 2 or 3.
  • the carrier unit can comprise the CC, CM, and HM moieties in a linear fashion.
  • the carrier units can comprises the CC, CM, and HM moieties organized in a branched scaffold arrangement, for example, with (i) a polymeric CC moiety comprising positively charged units (e.g., polylysines) and (ii) a CMs (e.g., lysine linked to a crosslinking agent, e.g., lysine-thiol) attached to the N or C terminus of the CC moeity and (iii) a HM (e.g., lysine linked to a hydrophobic agent,e.g., lysine linked to Vitamin B3) attached to the N or C terminus of the CM moiety.
  • a polymeric CC moiety comprising positively charged units (e.g., polylysines) and (ii) a CMs (e.g., lysine linked to a crosslinking agent, e.g., lysine-thiol) attached to the N or C terminus
  • the carrier units can comprises the CC, CM, and HM moieties organized in a branched scaffold arrangement, for example, with (i) a polymeric CC moiety comprising positively charged units (e.g., polylysines) and (ii) a CMs (e.g., lysine linked to a crosslinking agent, e.g., lysine-thiol) attached to the N or C terminus of the CC moeity and (iii) a HM (e.g., lysine linked to a hydrophobic agent, e.g., lysine linked to Vitamin B3) attached to the N or C terminus of the CM moiety.
  • a polymeric CC moiety comprising positively charged units (e.g., polylysines) and (ii) a CMs (e.g., lysine linked to a crosslinking agent, e.g., lysine-thiol) attached to the N or C termin
  • cationic carrier units of the present disclosure are mixed with an anionic payload (e.g., a nucleic acid) at an ionic ratio of about 20:about 1, i.e., the number of negative charges in the anionic payload is about 20 times higher than the number of positive charges in the cationic carrier moiety, to about 20:1, i.e., the number of positive charges in the cationic carrier moiety is about ten times higher than the number of negative charges in the anionic payload
  • the neutralization of negative charges in the anionic payload by positive charges in the cationic carrier moiety mainly via electrostatic interaction leads to the formation of a cationic carrier unit:anionic payload complex having an unaltered hydrophilic portion (comprising the WP moiety) and a substantially more hydrophobic portion (resulting from the association between the cationic carrier moiety plus hydrophobic moiety and the anionic payload).
  • the hydrophobic moiety can contribute its own positive charges to the positive charges of the cationic carrier moiety, which would interact with the negative charges of the anionic payload (e.g., polynucleotides disclosed herein). It is to be understood that references to the interactions (e.g., electrostatic interactions) between a cationic carrier moiety and an anionic payload (e.g., polynucleotides disclosed herein) also encompass interactions between the charges of a cationic carrier moiety plus hydrophobic moiety and the charges of an anionic payload.
  • amphipathic complex results in an amphipathic complex.
  • amphipathic complexes can self-organize, alone or combination with other amphipathic components, into micelles.
  • the resulting micelles comprise the WP moieties facing the solvent (i.e., the WP moieties are facing the external surface of the micelle), whereas the CC and HM moieties as well as the associate payload (e.g., a nucleotide sequence, e.g., RNA, DNA, or any combination thereof) are in the core of the micelle.
  • the cationic carrier unit comprises: (a) a WP moiety, wherein the water-soluble biopolymer is a polyethylene glycol (PEG) of formula III (see below), wherein n is between about 120 to about PEG 130 (e.g., PEG is a PEG5000 or a PEG6000); (b) a CC moiety, wherein the cationic carrier moiety comprises, e.g., about 20 to about 100 lysines (e.g., a linear poly(L-lysine)n wherein n is between about 30 and about 40), a polyethyleneimine (PEI), or chitosan; (c) a CM moiety, wherein the crosslinking moiety comprises about about 10 to about 50 lysines, each of which is linked to a crosslinking agent, e.g., 10-40 lysine-thiol, and (d) an HM moiety, wherein the hydrophobic moiety has about 1 to about PEG (PEG) of formula
  • the cationic carrier unit comprises: (a) a WP moiety, wherein the water-soluble biopolymer is a polyethylene glycol (PEG) of formula III (see below), wherein n is between about 120 to about PEG 130 (e.g., PEG is a PEG5000 or a PEG6000); (b) a CC moiety, wherein the cationic carrier moiety comprises, e.g., about 20 to about 100 lysines (e.g., a linear poly(L-lysine)n wherein n is between about 30 and about 40), a polyethyleneimine (PEI), or chitosan; (c) a CM moiety, wherein the crosslinking moiety comprises about about 10 to about 50 lysines, each of which is linked to a crosslinking agent, e.g., 10-40 lysine-thiol, and (d) an HM moiety, wherein the hydrophobic moiety has about 1 to about PEG (PEG) of formula
  • the cationic carrier unit comprises: (a) a WP moiety, wherein the water-soluble biopolymer is a polyethylene glycol (PEG) of formula III (see below), wherein n is between about 120 to about PEG 130 (e.g., PEG is a PEG5000 or a PEG6000); (b) a CC moiety, wherein the cationic carrier moiety comprises, e.g., about 20 to about 100 lysines (e.g., a linear poly(L-lysine)n wherein n is between about 30 and about 40), a polyethyleneimine (PEI), or chitosan; (c) a CM moiety, wherein the crosslinking moiety comprises about about 10 to about 50 lysines, each of which is linked to a crosslinking agent, e.g., 10-40 lysine-thiol, and (d) an HM moiety, wherein the hydrophobic moiety has about 5 to about PEG (PEG) of formula
  • the cationic carrier unit comprises: (a) a WP moiety, wherein the water-soluble biopolymer is a polyethylene glycol (PEG) of formula III (see below), wherein n is between about 120 to about PEG 130 (e.g., PEG is a PEG5000 or a PEG6000); (b) a CC moiety, wherein the cationic carrier moiety comprises, e.g., about 20 to about 100 lysines (e.g., a linear poly(L-lysine)n, wherein n is between about 30 and about 40, e.g., about 40), a polyethyleneimine (PEI), or chitosan; (c) a CM moiety, wherein the crosslinking moiety comprises about about 10 to about 50 lysines, each of which is linked to a crosslinking agent, e.g., 10-40 lysine-thiol, e.g., 35 lysine-thi
  • the cationic carrier unit further comprises at least one targeting moiety attached to the WP moiety of the cationic carrier unit.
  • the number and/or density of targeting moieties displayed on the surface of the micelle can be modulated by using a specific ratio of cationic carrier units having targeting moieties to cationic carrier units not having targeting moieties.
  • the ratio of cationic carrier units having a targeting moiety to cationic carrier units not having a targeting moiety is at least about 1:5, at least about 1:10, at least about 1:20, at least about 1:30, at least about 1:40, at least about 1:50, at least about 1:60, at least about 1:70, at least about 1:80, at least about 1:90, at least about 1:100, at least about 1:120, at least about 1:140, at least about 1:160, at least about 1:180, at least about 1:200, at least about 1:250, at least about 1:300, at least about 1:350, at least about 1:400, at least about 1:450, at least about 1:500, at least about 1:600, at least about 1:700, at least about 1:800, at least about 1:900, or at least about 1:1000.
  • the cationic carrier unit comprises (i) a targeting moiety (A) which targets the transporter LAT1 (e.g., phenylalanine), (ii) a water soluble polymer which is PEG, (iii) a cationic carrier moiety comprising cationic polymer blocks which are lysine (iv) a crosslinking moiety comprising crosslinking polymer blocks which are lysines linked to crosslinking moieties, and (v) a hydrophobic moiety comprising hydrophobic polymer blocks which are lysines linked to vitamin B3.
  • a targeting moiety A
  • targets the transporter LAT1 e.g., phenylalanine
  • a water soluble polymer which is PEG e.g., a water soluble polymer which is PEG
  • a cationic carrier moiety comprising cationic polymer blocks which are lysine
  • iv a crosslinking moiety comprising crosslinking polymer blocks which are lys
  • a targeting moiety A
  • targets the transporter LAT1 e.g., phenylalanine
  • A which targets the transporter LAT1 (e.g., phenylalanine)
  • the number (percentage) of HM is less than 39%, less than about 35%, less than about 30%, less than about 25%, less than about 20%, less than about 15%, less than about 10%, less than about 9%, less than about 8%, less than about 7%, less than about 6%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, or about 1% relative to [CC] and [CM].
  • the number (percentage) of HM is between about 35% and about 1%, about 35% and about 5%, about 35% and about 10%, about 35% and about 15%, about 35% and about 20%, about 35% and about 25%, about 35% and about 30%, about 30% and about 1%, about 30% and about 5%, about 30% and about 10%, about 30% and about 15%, about 30% and about 20%, about 30% and about 25%, about 25% and about 1%, about 25% and about 5%, about 25% and about 10%, about 25% and about 15%, about 25% and about 20%, about 20% and about 1%, about 20% and about 5%, about 20% and about 10%, about 20% and about 15%, about 15% and about 1%, about 15% and about 5%, about 15% and about 10%, about 10% and about 1%, or about 10% and about 5% relative to [CC] and [CM].
  • the number (percentage) of HM is between about 39% and about 30%, about 30% and about 20%, about 20% and about 10%, about 10% and about 5%, and about 5% and about 1% relative to [CC] and [CM]. In some aspects, the number (percentage) of HM is about 39%, about 30%, about 25%, about 20%, about 15%, about 10%, about 5%, or about 1% relative to [CC] and [CM]. In some aspects, the number of HM is expressed as the percentage of [HM] relative to [CC] and [CM].
  • the vitamin B3 unit are introduced into the side chains of the HM moiety, e.g., by a coupling reaction between NH2 groups in the lysines and COOH groups of vitamin B3, in the presence of suitable conjugation reagents, for example, 1-ethyl-3-(3- dimethylaminopropyl)-carbodiimide (EDC) and N-hydroxy succinimide (NHS).
  • suitable conjugation reagents for example, 1-ethyl-3-(3- dimethylaminopropyl)-carbodiimide (EDC) and N-hydroxy succinimide (NHS).
  • EDC 1-ethyl-3-(3- dimethylaminopropyl)-carbodiimide
  • NHS N-hydroxy succinimide
  • the present disclosure provides complexes comprising a carrier unit (e.g., a cationic carrier unit unit) of the present disclosure non- covalently attached to a payload (e.g., an anionic payload such a nucleotide sequence, e.g., an RNA, DNA, or any combination thereof), wherein the carrier unit and the payload interact electrostatically.
  • a carrier unit e.g., a cationic carrier unit unit
  • a payload e.g., an anionic payload such a nucleotide sequence, e.g., an RNA, DNA, or any combination thereof
  • the present disclosure provides conjugates comprising a carrier unit (e.g., a cationic carrier unit unit) of the present disclosure covalently attached to a payload (e.g., an anionic payload such a nucleotide sequence, e.g., an RNA, DNA, or any combination thereof), wherein the carrier unit and the payload interact electrostatically.
  • a carrier unit e.g., a cationic carrier unit unit
  • a payload e.g., an anionic payload such a nucleotide sequence, e.g., an RNA, DNA, or any combination thereof
  • the carrier unit and the payload can be linked via a cleavable linker.
  • the carrier unit and the payload in addition to interacting electrostatically, can interact covalently (e.g., after electrostatic interaction the carrier unit and the payload can be "locked" via a disulfide bond or a cleavable bond).
  • the cationic carrier unit comprises a water-soluble polymer comprising a PEG with about 100 to about 130 units (e.g., about 114 units), a cationic carrier moiety comprising a polylysine with about 20 to about 60 lysine units, (e.g., about 40 lysines) a crosslinking moiety comprising about 3 to about 40 lysine-thiol units (e.g., about 35 lysines, each with a thiol group), and a hydrophobic moiety comprising about 1 to about 50 lysines linked to a vitamin B3 units (e.g., about 5 lysines, each fused to vitamin B3).
  • a cationic carrier moiety comprising a polylysine with about 20 to about 60 lysine units, (e.g., about 40 lysines)
  • a crosslinking moiety comprising about 3 to about 40 lysine-thiol units (e.g., about 35 lysines,
  • the cationic carrier unit is associated with a negatively charged payload (e.g., a nucleotide sequence, e.g., an RNA, DNA, or any combination thereof), which interacts with the cationic carrier unit via at least one ionic bond (i.e., via electrostatic interaction) with the cationic carrier moiety of the cationic carrier unit.
  • a negatively charged payload e.g., a nucleotide sequence, e.g., an RNA, DNA, or any combination thereof
  • ionic bond i.e., via electrostatic interaction
  • water-soluble biopolymer refers to a biocompatible, biologically inert, non-immunogenic, non-toxic, and hydrophilic polymer, e.g., PEG.
  • the water-soluble polymer comprises poly(alkylene glycols), poly(oxyethylated polyol), poly(olefinic alcohol), poly(vinylpyrrolidone), poly(hydroxyalkylmethacrylamide), poly(hydroxyalkylmethacrylate), poly(saccharides), poly( ⁇ - hydroxy acid), poly(vinyl alcohol), polyglycerol, polyphosphazene, polyoxazolines (“POZ”) poly(N-acryloylmorpholine), or any combinations thereof.
  • POZ polyoxazolines
  • the water-soluble biopolymer is linear, branched, or dendritic.
  • the water-soluble biopolymer comprises polyethylene glycol ("PEG"), polyglycerol (“PG”), or poly(propylene glycol) (“PPG”).
  • PPG is less toxic than PEG, so many biological products are now produced in PPG instead of PEG.
  • the water-soluble biopolymer comprises a PEG characterized by a formula R 3 -(O-CH2-CH2)n- or R 3 -(0-CH2-CH2)n-O- with R 3 being hydrogen, methyl or ethyl and n having a value from 2 to 200.
  • the PEG has the formula (Formula III) wherein n is 1 to 1000.
  • the n of the PEG has a value of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103,
  • n is at least about 10, at least about 20, at least about 30, at least about 40, at least about 50, at least about 60, at least about 70, at least about 80, at least about 90, at least about 100, at least about 110, at least 120, at least about 130, at least about 140, at least about 150, at least about 160, at least about 170, at least about 180, at least about 190, at least about 200, at least about 210, at least about 220, at least about 230, at least about 240, at least about 250, at least about 260, at least about 270, at least about 280, at least about 290, at least about 300, at least about 310, at least about 320, at least about 330, at least about 340, at least about 350, at least about 360, at least about 370, at least about 380, at least about 390, at least about 400, at least about 410, at least about 420, at least about 430, at least about 440, at least about 450, at least about
  • n is between about 50 and about 100, between about 100 and about 150, between about 150 and about 200, between about 200 and about 250, between about 250 and about 300, between about 300 and about 350, between about 350 and about 400, between about 400 and about 450, between about 450 and about 500, between about 500 and about 550, between about 550 and about 600, between about 600 and about 650, between about 650 and about 700, between about 700 and about 750, between about 750 and about 800, between about 800 and about 850, between about 850 and about 900, between about 900 and about 950, or between about 950 and about 1000.
  • n is at least about 80, at least about 81, at least about 82, at least about 83, at least about 84, at least about 85, at least about 86, at least about 87, at least about 88, at least about 89, at least about 90, at least about 91, at least about 92, at least about 93, at least about 94, at least about 95, at least about 96, at least about 97, at least about 98, at least about 99, at least about 100, at least about 101, at least about 102, at least about 103, at least about 104, at least about 105, at least about 106, at least about 107, at least about 108, at least about 109, at least 110, at least about 111, at least about 112, at least about 113, at least about 114, at least about 115, at least about 116, at least about 117, at least about 118, at least about 119, at least about 120, at least about 121, at least about
  • n is about 80 to about 90, about 90 to about 100, about 100 to about 110, about 110 to about 120, about 120 to about 130, about 130 to about 140, about 140 to about 150, about 150 to about 160, about 85 to about 95, about 95 to about 105, about 105 to about 115, about 115 to about 125, about 125 to about 135, about 135 to about 145, about 145 to about 155, about 155 to about 165, about 80 to about 100, about 100 to about 120, about 120 to about 140, about 140 to about 160, about 85 to about 105, about 105 to about 125, about 125 to about 145, or about 145 to about 165. [0320] In some aspects, n is about 100 to about 150.
  • n is about 100 to about 140. In some aspects, n is about 100 to about 130. In some aspects, n is about 110 to about 150. In some aspects, n is about 110 to about 140. In some aspects, n is about 110 to about 130. In some aspects, n is about 110 to about 120. In some aspects, n is about 120 to about 150. In some aspects, n is about 120 to about 140. In some aspects, n is about 120 to about 130. In some aspects, n is about 130 to about 150. In some aspects, n is about 130 to about 140. In some aspects, n is about 114. [0321] Thus, is some aspects, the PEG is a branched PEG.
  • Branched PEGs have three to ten PEG chains emanating from a central core group.
  • the PEG moiety is a monodisperse polyethylene glycol.
  • a monodisperse polyethylene glycol is a PEG that has a single, defined chain length and molecular weight. mdPEGs are typically generated by separation from the polymerization mixture by chromatography.
  • a monodisperse PEG moiety is assigned the abbreviation mdPEG.
  • the PEG is a Star PEG. Star PEGs have 10 to 100 PEG chains emanating from a central core group.
  • the PEG is a Comb PEGs.
  • Comb PEGs have multiple PEG chains normally grafted onto a polymer backbone.
  • the PEG has a molar mass between about 1000 g/mol and about 2000 g/mol, between about 2000 g/mol and about 3000 g/mol, between about 3000 g/mol to about 4000 g/mol, between about 4000 g/mol and about 5000 g/mol, between about 5000 g/mol and about 6000 g/mol, between about 6000 g/mol and about 7000 g/mol, or between 7000 g/mol and about 8000 g/mol.
  • the PEG is PEG100, PEG200, PEG300, PEG400, PEG500, PEG600, PEG 700, PEG 800, PEG 900, PEG 1000, PEG 1100, PEG 1200, PEG 1300, PEG 1400, PEG 1500, PEG 1600, PEG 1700, PEG1800, PEG1900, PEG2000, PEG2100, PEG2200, PEG2300, PEG2400, PEG2500, PEG1600, PEG1700, PEG1800, PEG1900, PEG2000, PEG2100, PEG2200, PEG2300, PEG2400, PEG2500, PEG2600, PEG2700, PEG2800, PEG2900, PEG 3000, PEG 3100, PEG 3200 , PEG 3300 , PEG 3400 , PEG 3500 , PEG 3600 , PEG 3700 , PEG 3800 , PEG 3900 , PEG 4000
  • the PEG is PEG5000. In some aspects, the PEG is PEG6000. In some aspects, the PEG is PEG4000. [0325] In some aspects, the PEG is monodisperse, e.g., mPEG 100 , mPEG 200, mPEG 300, mPEG400, mPEG500, mPEG600, mPEG700, mPEG800, mPEG900, mPEG1000, mPEG1100, mPEG1200, mPEG1300, mPEG1400, mPEG1500, mPEG1600, mPEG1700, mPEG1800, mPEG1900, mPEG2000, mPEG2100, mPEG 2200, mPEG 2300, mPEG 2400, mPEG 2500, mPEG 1600, mPEG 1700, mPEG 1800, mPEG 1900, mPEG 2000,
  • the mPEG is mPEG5000. In some aspects, the mPEG is mPEG6000. In some aspects, the mPEG is mPEG4000.
  • the water-soluble biopolymer moiety is a polyglycerol (PG) described by the formula ((R3—O—(CH2—CHOH—CH2O)n—) with R3 being hydrogen, methyl or ethyl, and n having a value from 3 to 200.
  • PG polyglycerol
  • the water-soluble biopolymer moiety is a branched polyglycerol described by the formula (R 3 —O—(CH 2 —CHOR 5 —CH 2 —O) n —) with R 5 being hydrogen or a linear glycerol chain described by the formula (R 3 —O—(CH 2 — CHOH—CH2—O)n—) and R 3 being hydrogen, methyl or ethyl.
  • the water-soluble biopolymer moiety is a hyperbranched polyglycerol described by the formula (R 3 —O—(CH 2 — CHOR 5 —CH 2 —O) n —) with R 5 being hydrogen or a glycerol chain described by the formula (R 3 — O—(CH2—CHOR 6 —CH2—O)n—), with R 6 being hydrogen or a glycerol chain described by the formula (R 3 —O—(CH2—CHOR 7 —CH2—O)n—), with R 7 being hydrogen or a linear glycerol chain described by the formula (R 3 —O—(CH 2 —CHOH—CH 2 —O) n —) and R 3 being hydrogen, methyl or ethyl.
  • the PG has a molar mass between about 1000 g/mol and about 2000 g/mol, between about 2000 g/mol and about 3000 g/mol, between about 3000 g/mol to about 4000 g/mol, between about 4000 g/mol and about 5000 g/mol, between about 5000 g/mol and about 6000 g/mol, between about 6000 g/mol and about 7000 g/mol, or between 7000 g/mol and about 8000 g/mol.
  • the PG is PG100, PG200, PG300, PG400, PG500, PG600, PG700, PG800, PG 900, PG 1000, PG 1100, PG 1200, PG 1300, PG 1400, PG 1500, PG 1600, PG 1700, PG 1800, PG 1900, PG 2000, PG 2100, PG2200, PG2300, PG2400, PG2500, PG1600, PG1700, PG1800, PG1900, PG2000, PG2100, PG2200, PG2300, PG2400, PG2500, PG2600, PG2700, PG2800, PG2900, PG3000, PG3100, PG3200, PG3300, PG3400, PG3500, PG3600, PG3700, PG 3800 , PG 3900 , PG 4000 , PG 4100, PG 4000 , PG
  • the PG is PG 5000 . In some aspects, the PG is PG 6000 . In some aspects, the PG is PG 4000 . [0329] In some aspects, the PG is monodisperse, e.g., mPG100, mPG200, mPG300, mPG400, mPG 500, mPG 600, mPG 700, mPG 800, mPG 900, mPG 1000, mPG 1100, mPG 1200, mPG 1300, mPG 1400, mPG 1500, mPG 1600, mPG 1700, mPG 1800, mPG 1900, mPG 2000, mPG 2100, mPG 2200, mPG 2300, mPG 2400, mPG 2500, mPG1600, mPG1700, mPG1800, mPG1900, mPG2000, mPG2100, mPG2200, mPG2300, mPG2400, m
  • the water-soluble biopolymer comprises poly(propylene glycol) ("PPG").
  • PPG is characterized by the following formula, with n having a value from 1 to 1000.
  • the n of the PPG has a value of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89,
  • n of the PPG is at least about 10, at least about 20, at least about 30, at least about 40, at least about 50, at least about 60, at least about 70, at least about 80, at least about 90, at least about 100, at least about 110, at least 120, at least about 130, at least about 140, at least about 150, at least about 160, at least about 170, at least about 180, at least about 190, at least about 200, at least about 210, at least about 220, at least about 230, at least about 240, at least about 250, at least about 260, at least about 270, at least about 280, at least about 290, at least about 300, at least about 310, at least about 320, at least about 330, at least about 340, at least about 350, at least about 360, at least about 370, at least about 380, at least about 390, at least about 400, at least about 410, at least about 420, at least about 430, at least about 440, at least about
  • the n of the PPG is between about 50 and about 100, between about 100 and about 150, between about 150 and about 200, between about 200 and about 250, between about 250 and about 300, between about 300 and about 350, between about 350 and about 400, between about 400 and about 450, between about 450 and about 500, between about 500 and about 550, between about 550 and about 600, between about 600 and about 650, between about 650 and about 700, between about 700 and about 750, between about 750 and about 800, between about 800 and about 850, between about 850 and about 900, between about 900 and about 950, or between about 950 and about 1000.
  • the n of the PPG is at least about 80, at least about 81, at least about 82, at least about 83, at least about 84, at least about 85, at least about 86, at least about 87, at least about 88, at least about 89, at least about 90, at least about 91, at least about 92, at least about 93, at least about 94, at least about 95, at least about 96, at least about 97, at least about 98, at least about 99, at least about 100, at least about 101, at least about 102, at least about 103, at least about 104, at least about 105, at least about 106, at least about 107, at least about 108, at least about 109, at least 110, at least about 111, at least about 112, at least about 113, at least about 114, at least about 115, at least about 116, at least about 117, at least about 118, at least about 119, at least about 120, at least about
  • the n of the PPG is about 80 to about 90, about 90 to about 100, about 100 to about 110, about 110 to about 120, about 120 to about 130, about 130 to about 140, about 140 to about 150, about 150 to about 160, about 85 to about 95, about 95 to about 105, about 105 to about 115, about 115 to about 125, about 125 to about 135, about 135 to about 145, about 145 to about 155, about 155 to about 165, about 80 to about 100, about 100 to about 120, about 120 to about 140, about 140 to about 160, about 85 to about 105, about 105 to about 125, about 125 to about 145, or about 145 to about 165.
  • the PPG is a branched PPG. Branched PPGs have three to ten PPG chains emanating from a central core group.
  • the PPG moiety is a monodisperse polyethylene glycol.
  • a monodisperse polyethylene glycol mdPPG
  • mdPEG monodisperse polyethylene glycol
  • mdPEG monodisperse polyethylene glycol
  • a monodisperse PPG moiety is assigned the abbreviation mdPPG.
  • the PPG is a Star PPG.
  • Star PPGs have 10 to 100 PPG chains emanating from a central core group.
  • the PPG is a Comb PPGs.
  • Comb PPGs have multiple PPG chains normally grafted onto a polymer backbone.
  • the PPG has a molar mass between about 1000 g/mol and about 2000 g/mol, between about 2000 g/mol and about 3000 g/mol, between about 3000 g/mol to about 4000 g/mol, between about 4000 g/mol and about 5000 g/mol, between about 5000 g/mol and about 6000 g/mol, between about 6000 g/mol and about 7000 g/mol, or between 7000 g/mol and about 8000 g/mol.
  • the PPG is PPG 100 , PPG 200, PPG 300, PPG 400, PPG 500, PPG 600, PPG 700, PPG800, PPG900, PPG1000, PPG1100, PPG1200, PPG1300, PPG1400, PPG1500, PPG1600, PPG1700, PPG1800, PPG 1900, PPG 2000, PPG 2100, PPG 2200, PPG 2300, PPG 2400, PPG 2500, PPG 1600, PPG 1700, PPG 1800, PPG 1900, PPG 2000, PPG 2100, PPG 2200, PPG 2300, PPG 2400, PPG 2500, PPG 2600, PPG 2700, PPG 2800, PPG 2900, PPG 3000, PPG3100, PPG3200, PPG3300, PPG3400, PPG3500, PPG3600, PPG3700, PPG3800, PPG3900, PPG4000, PPG 4100, PPG 4200
  • the PPG is PPG 5000 . In some aspects, the PPG is PPG 6000 . In some aspects, the PPG is PPG 4000 . [0340] In some aspects, the PPG is monodisperse, e.g., mPPG100, mPPG200, mPPG300, mPPG400, mPPG500, mPPG600, mPPG700, mPPG800, mPPG900, mPPG1000, mPPG1100, mPPG1200, mPPG 1300, mPPG 1400, mPPG 1500, mPPG 1600, mPPG 1700, mPPG 1800, mPPG 1900, mPPG 2000, mPPG 2100, mPPG 2200, mPPG 2300, mPPG 2400, mPPG 2500, mPPG 1600, mPPG 1700, mPPG 1800, mPPG 1900, mPPG 2000, m
  • the mPPG is mPPG5000. In some aspects, the mPPG is mPPG6000. In some aspects, the mPPG is mPPG 4000 .
  • Cationic carrier [0341] In some aspects, the cationic carrier units of the present disclosure comprise at least one cationic carrier moiety.
  • the term "cationic carrier" refers to a moiety or portion of a cationic carrier unit of the present disclosure comprising a plurality of positive charges that can interact and bind electrostatically an anionic payload (or an anionic carrier attached to a payload).
  • the number of positive charges or positively charged groups on the cationic carrier is similar to the number of negative charges or negatively charged groups on the anionic payload (or an anionic carrier attached to a payload).
  • the cationic carrier comprises a biopolymer, e.g., a peptide (e.g., a polylysine).
  • the cationic carrier comprises one or more basic amino acids (e.g., lysine, arginine, histidine, or a combination thereof).
  • the cationic carrier comprises at least about three, at least about four, at least about five, at least about six, at least about seven, at least about eight, at least about nine, at least about ten, at least about 11, at least about 12, at least about 13, at least about 14, at least about 15, at least about 16, at least about 17, at least about 18, at least about 19, at least about 20, at least about 21, at least about 22, at least about 23, at least about 24, at least about 25, at least about 26, at least about 27, at least about 28, at least about 29, at least about 30, at least about 31, at least about 32, at least about 33, at least about 34, at least about 35, at least about 36, at least about 37, at least about 38, at least about 39, at least about 40, at least about 41, at least about 42, at least about 43, at least about 44, at least about 45, at least about 46, at least about 47, at least about 48, at least about 49, at least about 50, at least about 51, at least about 52, at least about 53, at least about 54, at least about 55,
  • the cationic carrier moiety comprises about 60, about 70, about 80, about 90, or about 100 basic amino acids. In some aspects, the cationic carrier moiety comprises about 80 basic amino acids. [0343] In some aspects, the cationic carrier unit comprises at least about 40 basic amino acids, e.g., lysines. In some aspects, the cationic carrier unit comprises at least about 45 basic amino acids, e.g., lysines. In some aspects, the cationic carrier unit comprises at least about 50 basic amino acids, e.g., lysines. In some aspects, the cationic carrier unit comprises at least about 55 basic amino acids, e.g., lysines.
  • the cationic carrier unit comprises at least about 60 basic amino acids, e.g., lysines. In some aspects, the cationic carrier unit comprises at least about 65 basic amino acids, e.g., lysines. In some aspects, the cationic carrier unit comprises at least about 70 basic amino acids, e.g., lysines. In some aspects, the cationic carrier unit comprises at least about 75 basic amino acids, e.g., lysines. In some aspects, the cationic carrier unit comprises at least about 80 basic amino acids, e.g., lysines.
  • the cationic carrier unit comprises about 30 to about 1000, about 30 to about 900, about 30 to about 800, about 30 to about 700, about 30 to about 600, about 30 to about 500, about 30 to about 400, about 30 to about 300, about 30 to about 200, about 30 to about 100, about 40 to about 1000, about 40 to about 900, about 40 to about 800, about 40 to about 700, about 40 to about 600, about 40 to about 500, about 40 to about 400, about 40 to about 300, about 40 to about 200, or about 40 to about 100 basic amino acids, e.g., lysines.
  • basic amino acids e.g., lysines.
  • the basic amino acids e.g., lysines
  • the basic amino acids are not modified such that they possess –NH3+(e.g., positive charge).
  • the cationic carrier unit comprises about 30 to about 100, about 30 to about 90, about 30 to about 80, about 30 to about 70, about 30 to about 60, about 30 to about 50, about 30 to about 40, about 40 to about 100, about 40 to about 90, about 40 to about 80, about 40 to about 70, about 40 to about 60, about 70 to about 80, about 75 to about 85, about 65 to about 75, about 65 to about 80, about 60 to about 85, or about 40 to about 500 basic amino acids, e.g., lysines.
  • the cationic carrier unit comprises about 100 to about 1000, about 100 to about 900, about 100 to about 800, about 100 to about 700, about 100 to about 600, about 100 to about 500, about 100 to about 400, about 100 to about 300, about 100 to about 200, about 200 to about 1000, about 200 to about 900, about 200 to about 800, about 200 to about 700, about 200 to about 600, about 200 to about 500, about 200 to about 400, about 200 to about 300, about 300 to about 1000, about 300 to about 900, about 300 to about 800, about 300 to about 700, about 300 to about 600, about 300 to about 500, about 300 to about 400, about 400 to about 1000, about 400 to about 900, about 400 to about 800, about 400 to about 700, about 400 to about 600, about 400 to about 500, about 500 to about 1000, about 500 to about 600, about 600 to about 1000, about 600 to about 900, about 600 to about 800, about 600 to about 700, about 400 to about 600, about 400 to about 500, about 500 to about 1000, about 500 to about 600, about 600
  • the number of basic amino acids can be adjusted based on the length of the anionic payload. For example, an anionic payload with a longer sequence can be paired with higher number of basic amino acids, e.g., lysines.
  • the number of basic amino acids, e.g., lysines, in the cationic carrier unit can be calculated so that the molar ratio of protonated amine in polymer to phosphate in an anionic payload, e.g., mRNA (N/P ratio) is at least about 1, at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 11, at least about 12, at least about 13, at least about 14, at least about 15, at least about 16, at least about 17, at least about 18, at least about 19, or at least about 20.
  • N/P ratio molar ratio of protonated amine in polymer to phosphate in an anionic payload
  • the molar ratio of protonated amine in polymer to phosphate in an anionic payload, e.g., mRNA is between about 1 to about 20, about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, or about 20.
  • the number of basic amino acids, e.g., lysines, in the cationic carrier unit is calculated so that the molar ratio of protonated amine in polymer to phosphate in an anionic payload, e.g., mRNA (N/P ratio) is about 1 to about 10, e.g., about 3 to about 4, about 4 to about 5, about 5 to about 6, about 6 to about 7, or about 7 to about 8.
  • the number of basic amino acids, e.g., lysines, in the cationic carrier unit is calculated so that the molar ratio of protonated amine in polymer to phosphate in an anionic payload, e.g., mRNA (N/P ratio) is about 1 to about 2.
  • the number of basic amino acids, e.g., lysines, in the cationic carrier unit is calculated so that the molar ratio of protonated amine in polymer to phosphate in an anionic payload, e.g., mRNA (N/P ratio) is about 3 to about 4.
  • the number of basic amino acids, e.g., lysines, in the cationic carrier unit is calculated so that the molar ratio of protonated amine in polymer to phosphate in an anionic payload, e.g., mRNA (N/P ratio) is about 2 to about 3.
  • the number of basic amino acids, e.g., lysines, in the cationic carrier unit is calculated so that the molar ratio of protonated amine in polymer to phosphate in an anionic payload, e.g., mRNA (N/P ratio) is about 4 to about 5. In some aspects, the number of basic amino acids, e.g., lysines, in the cationic carrier unit is calculated so that the molar ratio of protonated amine in polymer to phosphate in an anionic payload, e.g., mRNA (N/P ratio) is about 5 to about 6.
  • the number of basic amino acids, e.g., lysines, in the cationic carrier unit is calculated so that the molar ratio of protonated amine in polymer to phosphate in an anionic payload, e.g., mRNA (N/P ratio) is about 6 to about 7.
  • the number of basic amino acids, e.g., lysines, in the cationic carrier unit is calculated so that the molar ratio of protonated amine in polymer to phosphate in an anionic payload, e.g., mRNA (N/P ratio) is about 7 to about 8.
  • the number of basic amino acids, e.g., lysines, in the cationic carrier unit is calculated so that the molar ratio of protonated amine in polymer to phosphate in an anionic payload, e.g., mRNA (N/P ratio) is about 8 to about 9.
  • the number of basic amino acids, e.g., lysines, in the cationic carrier unit is calculated so that the molar ratio of protonated amine in polymer to phosphate in an anionic payload, e.g., mRNA (N/P ratio) is about 9 to about 10.
  • a role of the cationic carrier moiety is to neutralize negative charges on the payload (e.g., negative changes in the phosphate backbone of an mRNA) via electrostatic interaction, in some aspects (e.g., when the payload is a nucleic acid such as an antimir), the length of the cationic carrier, number of positively charged groups on the cationic carrier, and distribution and orientation of charges present on the cationic carrier will depend on the length and charge distribution on the payload molecule.
  • the cationic carrier comprises between about 5 and about 10, between about 10 and about 15, between about 15 and about 20, between about 20 and about 25, between about 25 and about 30, between about 30 and about 35, between about 35 and about 40, between about 40 and about 45, between about 45 and about 50, between about 50 and about 55, between about 55 and about 60, between about 60 and about 65, between about and about 70, between about 70 and about 75, or between about 75 and about 80 basic amino acids.
  • the positively charged carrier comprises between 30 and about 50 basic amino acids.
  • the positively charged carrier comprises between 70 and about 80 basic amino acids.
  • the basic amino acid comprises arginine, lysine, histidine, or any combination thereof.
  • the basic amino acid is a D-amino acid. In some aspects, the basic amino acid is an L-amino acid. In some aspects, the positively charged carrier comprises D- amino acids and L-amino acids. In some aspects, the basic amino acid comprises at least one unnatural amino acid or a derivative thereof.
  • the basic amino acid is arginine, lysine, histidine, L-4-aminomethyl-phenylalanine, L-4-guanidine-phenylalanine, L-4- aminomethyl-N-isopropyl-phenylalanine, L-3-pyridyl-alanine, L-trans-4- aminomethylcyclohexyl-alanine, L-4-piperidinyl-alanine, L-4-aminocyclohexyl-alanine, 4- guanidinobutyric acid, L-2-amino-3-guanidinopropionic acid, DL-5-hydroxylysine, pyrrolysine, 5-hydroxy-L-lysine, methyllysine, hypusine, or any combination thereof.
  • the positively charged carrier comprises about 40 lysines. In a particular aspect, the positively charged carrier comprises about 50 lysines. In a particular aspect, the positively charged carrier comprises about 60 lysines. In a particular aspect, the positively charged carrier comprises about 70 lysines. In a particular aspect, the positively charged carrier comprises about 80 lysines.
  • the cationic carrier comprises a polymer or copolymer comprising at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least 11, at least 12, at least 13, at least 14, at last 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 31, at least 32, at least 33, at least 34, at least 35, at least 36, at least 37, at least 38, at least 39, at least 40, at least 41, at least 42, at least 43, at least 44, at least 45, at least 46, at least 47, at least 48, at least 49, at least 50, at least 51, at least 52, at least 53, at least 54, at least 55, at least 56, at least 57, at least 58, at least 59, at least 60, at least 61, at least 62, at least 63,
  • the cationic carrier comprises a polymer or copolymer comprising between about 5 and about 10 cationic groups, between about 10 and about 15 cationic groups, between about 15 and about 20 cationic groups, between about 20 and about 25 cationic groups, between about 25 and about 30 cationic groups, between about 30 and about 35 cationic groups, between about 35 and about 40 cationic groups, between about 40 and about 45 cationic groups, between about 45 and about 50 cationic groups, between about 50 and about 55 cationic groups, between about 55 and about 60 cationic groups, between about 60 and about 65 cationic groups, between about 65 and about 70 cationic groups, between about 70 and about 75 cationic groups, or between about 45 and about 50 cationic groups (e.g., amino groups).
  • amino groups e.g., amino groups
  • the cationic carrier comprises a polymer or copolymer comprising between 30 and about 50 cationic groups (e.g., amino groups). In some specific aspects, the cationic carrier comprises a polymer or copolymer comprising between 70 and about 80 cationic groups (e.g., amino groups). In some aspects, the polymer or copolymer is an acrylate, a polyalcohol, or a polysaccharide. [0352] In some aspects, the cationic carrier moiety binds to a single payload molecule. In some aspects, a cationic carrier moiety can bind to multiple payload molecules, which can be identical or different.
  • the positive charges of the cationic carrier moiety and negative charges of a nucleic acid payload are at an ionic ratio of about 20:1, about 19:1, about 18:1, about 17:1, about 16:1, about 15:1, about 14:1, about 13:1, about 12:1, about 11:1, about 10:1, about 9:1, about 8:1 about 7:1, about 6:1 about 5:1, about 4:1, about 3:1, about 2:1, or about 1:1.
  • the negative charges of a nucleic acid payload and the positive charges of the cationic carrier moiety are at an ionic ratio of about 20:1, about 19:1, about 18:1, about 17:1, about 16:1, about 15:1, about 14:1, about 13:1, about 12:1, about 11:1, about 10:1, about 9:1, about 8:1 about 7:1, about 6:1 about 5:1, about 4:1, about 3:1, about 2:1, or about 1:1.
  • the anionic payload comprises a nucleotide sequence having about 10 to about 1000 (e.g., about 100 to about 1000) in length, wherein the N/P ratio of the the cationic carrier moiety and the anionic payload is about 2 to about 10, e.g., about 2 to about 9, about 2 to about 8, about 2 to about 7, about 2 to about 6, about 2 to about 5, about 2 to about 4, about 2 to about 3, e.g., e.g., about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, or about 10.
  • an N/P ratio of the cationic carrier moiety and the anionic payload of about 10 to about 1000 nucleotides in length is between about 1 and about 10, e.g., about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, or about 10.
  • the anionic payload comprises a nucleotide sequence having about 1000 to about 2000 in length, wherein the N/P ratio of the cationic carrier moiety and the anionic payload is about 3 to about 12, e.g., about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, or about 12.
  • the N/P ratio of the cationic carrier moiety and the anionic payload is between about 4 and about 7, e.g., about 4, about 5, about 6, or about 7.
  • the anionic payload comprises a nucleotide sequence having about 2000 to about 3000 in length, wherein the N/P ratio of the cationic carrier moiety and the anionic payload is about 3 to about 16, e.g., about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16.
  • the N/P ratio of the cationic carrier moiety and the anionic payload is between about 6 and about 9, e.g., about 6, about 7, about 8, or about 9.
  • the anionic payload comprises a nucleotide sequence having about 3000 to about 4000 in length, wherein the N/P ratio of the cationic carrier moiety and the anionic payload is about 3 to about 20, e.g., about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, or about 20. In some aspects, wherein the N/P ratio of the cationic carrier moiety and the anionic payload is between about 7 and about 10, e.g., about 7, about 8, about 9, or about 10. [0358] In some aspects, the cationic carrier moiety has a free terminus wherein the end group is a reactive group.
  • the cationic carrier moiety has a free terminus (e.g., the C-terminus in a poly-lysine cationic carrier moiety) wherein the end group is an amino (-NH 2 ) group. In some aspects, the cationic carrier moiety has a free terminus wherein the end group is an sulfhydryl group. In some apects, the reactive group of the cationic carrier moiety is attached to a hydrophobic moiety, e.g., a vitamin B3 hydrophobic moiety.
  • Crosslinking Moiety [0359] In some aspects, the cationic carrier units of the present disclosure comprise at least one crosslinking moiety.
  • crosslinking moiety refers to a moiety or portion of a polymer block comprising a plurality of agents that are capable of forming crosslinks.
  • the number of agents that are capable of forming crosslinks comprises an amino acid with a side chain of a crosslinking agent.
  • the CM comprises a biopolymer, e.g., a peptide (e.g., a polylysine) linked to a crosslinking agent.
  • the crosslinking moiety comprises one or more amino acids (e.g., lysine, arginine, histidine, or a combination thereof).
  • the crosslinking moiety comprises at least about three, at least about four, at least about five, at least about six, at least about seven, at least about eight, at least about nine, at least about ten, at least about 11, at least about 12, at least about 13, at least about 14, at least about 15, at least about 16, at least about 17, at least about 18, at least about 19, at least about 20, at least about 21, at least about 22, at least about 23, at least about 24, at least about 25, at least about 26, at least about 27, at least about 28, at least about 29, at least about 30, at least about 31, at least about 32, at least about 33, at least about 34, at least about 35, at least about 36, at least about 37, at least about 38, at least about 39, at least about 40, at least about 41, at least about 42, at least about 43, at least about 44, at least about 45, at least about 46, at least about 47, at least about 48, at least about 49, or at least about 50 amino acids, e.g., lysines, arginines, or combinations thereof, each
  • the crosslinking moiety comprises at least about 10 amino acids, e.g., lysines, each of which is linked to a crosslinking agent. In some aspects, the crosslinking moiety comprises at least about 11 amino acids, e.g., lysines, each of which is linked to a crosslinking agent. In some aspects, the crosslinking moiety comprises at least about 12 amino acids, e.g., lysines, each of which is linked to a crosslinking agent. In some aspects, the crosslinking moiety comprises at least about 13 amino acids, e.g., lysines, each of which is linked to a crosslinking agent.
  • the crosslinking moiety comprises at least about 14 amino acids, e.g., lysines, each of which is linked to a crosslinking agent. In some aspects, the crosslinking moiety comprises at least about 15 amino acids, e.g., lysines, each of which is linked to a crosslinking agent. In some aspects, the crosslinking moiety comprises at least about 16 amino acids, e.g., lysines, each of which is linked to a crosslinking agent. In some aspects, the crosslinking moiety comprises at least about 17 amino acids, e.g., lysines, each of which is linked to a crosslinking agent.
  • the crosslinking moiety comprises at least about 18 amino acids, e.g., lysines, each of which is linked to a crosslinking agent. In some aspects, the crosslinking moiety comprises at least about 19 amino acids, e.g., lysines, each of which is linked to a crosslinking agent. In some aspects, the crosslinking moiety comprises at least about 20 amino acids, e.g., lysines, each of which is linked to a crosslinking agent. [0362] In some aspects, a crosslinking agent is a thiol. In some aspects, a crosslinking agent is a thiol derivative.
  • the cationic carrier units of the present disclosure comprise at least one hydrophobic moiety.
  • hydrophobic moiety refers to a molecular entity that can, e.g., (i) complement the therapeutic or prophylactic activity of the payload, (ii) modulate the therapeutic or prophylactic activity of the payload, (iii) function as a therapeutic and/or prophylactic agent in the target tissue or target cells, (iv) facilitate the transport of the cationic carrier unit across a physiological barrier, e.g., the BBB and/or the plasma membrane, (v) improve the homeostasis of the target tissue or target cell, (vi) contribute positively charges groups to the cationic carried moiety, or (vii) any combination thereof.
  • the hydrophobic moiety is capable of modulating, e.g., an immune response, an inflammatory response, or a tissue microenvironment.
  • a hydrophobic moiety capable of modulating an immune response can comprise, e.g., tyrosine or dopamine. Tyrosine can be transformed into L-DOPA, and then be converted to dopamine via 2-step enzymatic reaction. Normally, dopamine levels are low in the Parkinson’s disease patients. Therefore, in some aspects, tyrosine is a hydrophobic moiety in cationic carrier units used for the treatment of Parkinson’s disease.
  • cationic carrier units of the present disclosure used for the treatment of disease or conditions related to low serotonin levels comprise tryptophan as a hydrophobic moiety.
  • a hydrophobic moiety can modulate a tumor microenvironment in a subject with a tumor, for example, by inhibiting or reducing hypoxia in the tumor microenvironment.
  • the hydrophobic moiety comprises, e.g., an amino acid linked to an imidazole derivative, a vitamin, or any combination thereof.
  • the hydrophobic moiety comprises an amino acid (e.g., lysine) linked to an imidazole derivative comprising: (Formula VI), wherein each of G 1 and G 2 is independently H, an aromatic ring, or 1-10 alkyl, or G 1 and G 2 together form an aromatic ring, and wherein n is 1-10.
  • the hydrophobic moiety comprises an amino acid (e.g., lysine) linked to nitroimidazole. Nitroimidazoles function as antibiotics. Nitroheterocycles in nitroimidazoles can be reductively activated in hypoxic cells, and then undergo redox recycling or decompose to cytotoxic products.
  • the hydrophobic moiety comprises an amino acid (e.g., lysine) linked to metronidazole, tinidazole, nimorazole, dimetridazole, pretomanid, ornidazole, megazol, azanidazole, benznidazole, nitroimidazole, or any combination thereof.
  • the hydrophobic moiety comprises (Formula VII), wherein Ar is wherein each of Z1 and Z2 is H or OH.
  • the hydrophobic moiety is capable of inhibiting or reducing an inflammatory response.
  • the hydrophobic moiety is an amino acid (e.g., lysine) linked to a vitamin.
  • the vitamin comprises a cyclic ring or cyclic hetero atom ring and a carboxyl group or hydroxyl group.
  • the vitamin comprises: (Formula VIII), wherein each of Y1 and Y2 is C, N, O, or S, and wherein n is 1 or 2.
  • the vitamin is selected from the group consisting of vitamin A (retinol), vitamin B1 (Thiamine Chloride), vitamin B2 (Riboflavin), vitamin B3 (Niacinamide), vitamin B6 (Pyridoxal), vitamin B7 (Biotin), vitamin B9 (Folic acid), vitamin B12 (Cobalamin), vitamin C (Ascorbic acid), vitamin D2, vitamin D3, vitamin E (Tocopherol), vitamin M, vitamin H, a derivative thereof, and any combination thereof.
  • the vitamin is vitamin B3 (also known as niacin or nicotinic acid).
  • the hydrophobic moiety comprises at least about one, at least about two, at least about three, at least about four, at least about five, at least about six, at least about seven, at least about eight, at least about nine, at least about ten, at least about 11, at least about 12, at least about 13, at least about 14, at least about 15, at least about 16, at least about 17, at least about 18, at least about 19, or at least about 20 amino acids (e.g., lysines), each of which is linked to vitamin B3.
  • the hydrophobic moiety comprises about 1 amino acid (e.g., lysine), each of which is linked to vitamin B3.
  • the hydrophobic moiety comprises about 2 amino acids (e.g., lysines), each of which is linked to vitamin B3. In some aspects, the hydrophobic moiety comprises about 3 amino acids (e.g., lysines), each of which is linked to vitamin B3. In some aspects, the hydrophobic moiety comprises about 4 amino acids (e.g., lysines), each of which is linked to vitamin B3. In some aspects, the hydrophobic moiety comprises about 5 amino acids (e.g., lysines), each of which is linked to vitamin B3. In some aspects, the hydrophobic moiety comprises about 6 amino acids (e.g., lysines), each of which is linked to vitamin B3.
  • the hydrophobic moiety comprises about 2 amino acids (e.g., lysines), each of which is linked to vitamin B3. In some aspects, the hydrophobic moiety comprises about 3 amino acids (e.g., lysines), each of which is linked to vitamin B3. In some aspects, the hydrophobic moiety comprises about 4 amino acids
  • the hydrophobic moiety comprises about 7 amino acids (e.g., lysines), each of which is linked to vitamin B3. In some aspects, the hydrophobic moiety comprises about 8 amino acids (e.g., lysines), each of which is linked to vitamin B3. In some aspects, the hydrophobic moiety comprises about 9 amino acids (e.g., lysines), each of which is linked to vitamin B3. In some aspects, the hydrophobic moiety comprises about 10 amino acids (e.g., lysines), each of which is linked to vitamin B3. In some aspects, the hydrophobic moiety comprises about 11 amino acids (e.g., lysines), each of which is linked to vitamin B3.
  • the hydrophobic moiety comprises about 12 amino acids (e.g., lysines), each of which is linked to vitamin B3. In some aspects, the hydrophobic moiety comprises about 13 amino acids (e.g., lysines), each of which is linked to vitamin B3. In some aspects, the hydrophobic moiety comprises about 14 amino acids (e.g., lysines), each of which is linked to vitamin B3. In some aspects, the hydrophobic moiety comprises about 15 amino acids (e.g., lysines), each of which is linked to vitamin B3. In some aspects, the hydrophobic moiety comprises about 16 amino acids (e.g., lysines), each of which is linked to vitamin B3.
  • the hydrophobic moiety comprises about 12 amino acids (e.g., lysines), each of which is linked to vitamin B3. In some aspects, the hydrophobic moiety comprises about 13 amino acids (e.g., lysines), each of which is linked to vitamin B3. In some aspects, the hydrophobic moiety comprises about 14 amino acids
  • the hydrophobic moiety comprises about 17 amino acids (e.g., lysines), each of which is linked to vitamin B3. In some aspects, the hydrophobic moiety comprises about 18 amino acids (e.g., lysines), each of which is linked to vitamin B3. In some aspects, the hydrophobic moiety comprises about 19 amino acids (e.g., lysines), each of which is linked to vitamin B3. In some aspects, the hydrophobic moiety comprises about 20 amino acids (e.g., lysines), each of which is linked to vitamin B3.
  • the hydrophobic moiety comprises from about 1 to about 10 amino acids (e.g., lysines), each of which is linked to vitamin B3, about 5 to about 10 amino acids (e.g., lysines), each of which is linked to vitamin B3, about 10 to about 15 amino acids (e.g., lysines), each of which is linked to vitamin B3, about 15 to about 20 amino acids (e.g., lysines), each of which is linked to vitamin B3, about 1 to about 20 vitamin amino acids (e.g., lysines), each of which is linked to B3, about 1 to about 15 vitamin amino acids (e.g., lysines), each of which is linked to B3, about 1 to about 10 amino acids (e.g., lysines), each of which is linked to vitamin B3, about 1 to about 5 amino acids (e.g., lysines), each of which is linked to vitamin B3.
  • amino acids e.g., lysines
  • lysines amino acids
  • lysines amino acids
  • Niacin is a precursor of the coenzymes nicotinamide adenine dinucleotide (NAD) and nicotinamide adenine dinucleotide phosphate (NADP) in vivo.
  • NAD converts to NADP by phosphorylation in the presence of the enzyme NAD+ kinase.
  • NADP and NAD are coenzymes for many dehydrogenases, participating in many hydrogen transfer processes.
  • NAD is important in catabolism of fat, carbohydrate, protein, and alcohol, as well as cell signaling and DNA repair, and NADP mostly in anabolism reactions such as fatty acid and cholesterol synthesis.
  • Niacin produces marked anti-inflammatory effects in a variety of tissues – including the brain, gastrointestinal tract, skin, and vascular tissue – through the activation of NIACR1. Niacin has been shown to attenuate neuroinflammation and can have efficacy in treating neuroimmune disorders such as multiple sclerosis and Parkinson's disease. See Offermanns & Schwaninger (2015) Trends in Molecular Medicine 21:245-266; Chai et al (2013) Current Atherosclerosis Reports 15:325; Graff et al.
  • the cationic carrier unit comprises a targeting moiety, which is linked to the water-soluble polymer optionally via a linker.
  • targeting moiety refers to a biorecognition molecule that binds to a specific biological substance or site.
  • the targeting moiety is specific for a certain target molecule (e.g., a ligand targeting a receptor, or an antibody targeting a surface protein), tissue (e.g., a molecule that would preferentially carry the micelle to a specific organ or tissue, e.g., liver, brain, or endothelium), or facilitate transport through a physiological barrier (e.g., a peptide or other molecule that can facilitate transport across the brain blood barrier or plasma membrane).
  • a certain target molecule e.g., a ligand targeting a receptor, or an antibody targeting a surface protein
  • tissue e.g., a molecule that would preferentially carry the micelle to a specific organ or tissue, e.g., liver, brain, or endothelium
  • a physiological barrier e.g., a peptide or other molecule that can facilitate transport across the brain blood barrier or plasma membrane.
  • a targeting moiety can be coupled to a cationic carrier unit, and therefore, to the external surface of a micelle, whereas the micelle has the payload entrapped within its core.
  • the targeting moiety is a targeting moiety capable of targeting the micelle of the present disclosure to a tissue.
  • the tissue is liver, brain, kidney, lung, ovary, pancreas, thyroid, breast, stomach, or any combination thereof.
  • the tissue is cancer tissue, e.g., liver cancer, brain cancer, kidney cancer, lung cancer, ovary cancer, pancreas cancer, thyroid cancer, breast cancer, stomach cancer, or any combination thereof.
  • the tissue is liver.
  • the targeting moiety targeting liver is cholesterol.
  • the targeting moiety targeting liver is a ligand that binds an asialoglycoprotein receptor targeting moiety.
  • the asialoglycoprotein receptor targeting moiety comprises a GalNAc cluster.
  • the GalNAc cluster is a monovalent, divalent, trivalent, or tetravalent GalNAc cluster.
  • the tissue is pancreas.
  • the targeting moiety targeting pancreas comprises a ligand targeting ⁇ v ⁇ 3 integrin receptors on pancreatic cells.
  • the targeting moiety comprises an arginylglycylaspartic acid (RGD) peptide sequence (L- Arginyl-Glycyl-L-Aspartic acid; Arg-Gly-Asp).
  • RGD arginylglycylaspartic acid
  • the tissue is a tissue in the central nervous system, e.g., neural tissue.
  • the targeting moiety targeting the central nervous system is capable being transported by Large-neutral Amino Acid Transporter 1 (LAT1).
  • LAT1 Large-neutral Amino Acid Transporter 1
  • LAT1 (SLC7A5) is a transporter for both the uptake of large neutral amino acids and a number of pharmaceutical drugs. LAT1 can transport drugs such as L-dopa or gabapentin.
  • a targeting moiety comprises glucose, e.g., D-glucose, which can bind to Glucose transporter 1 (or GLUT1) and cross BBB.
  • GLUT1 also known as solute carrier family 2, facilitated glucose transporter member 1 (SLC2A1), is a uniporter protein that in humans is encoded by the SLC2A1 gene. GLUT1 facilitates the transport of glucose across the plasma membranes of mammalian cells.
  • a targeting moiety comprises galactose, e.g., D-galactose, which can bind to GLUT1 transporter to cross BBB.
  • a targeting moiety comprises glutamic acid, which can bind to acetylcholinesterase inhibitor (AChEI) and/or EAATs inhibitors and cross BBB.
  • Acetylcholinesterase is the enzyme that is the primary member of the cholinesterase enzyme family.
  • an acetylcholinesterase inhibitor is the inhibitor that inhibits acetylcholinesterase from breaking down acetylcholine into choline and acetate, thereby increasing both the level and duration of action of the neurotransmitter acetylcholine in the central nervous system, autonomic ganglia and neuromuscular junctions, which are rich in acetylcholine receptors.
  • Acetylcholinesterase inhibitors are one of two types of cholinesterase inhibitors; the other being butyryl-cholinesterase inhibitors.
  • the tissue targeted by a targeting moiety is a skeletal muscle.
  • the targeting moiety targeting skeletal muscle is capable being transported by Large- neutral Amino Acid Transporter 1 (LAT1).
  • LAT1 Large- neutral Amino Acid Transporter 1
  • LAT1 is consistently expressed at high levels in brain microvessel endothelial cells. Being a solute carrier located primarily in the BBB, targeting the micelles of the present disclosure to LAT1 allows delivery through the BBB.
  • the targeting moiety targeting a micelle of the present disclosure to the LAT1 transporter is an amino acid, e.g., a branched-chain or aromatic amino acid.
  • the amino acid is valine, leucine, and/or isoleucine.
  • the amino acid is tryptophan and/or tyrosine. In some aspects, the amino acid is tryptophan. In some aspects, the amino acid is tyrosine.
  • the targeting moiety is a LAT1 ligand selected from tryptophan, tyrosine, phenylalanine, tryptophan, methionine, thyroxine, melphalan, L-DOPA, gabapentin, 3,5- I-diiodotyrosine, 3-iodo-I-tyrosine, fenclonine, acivicin, leucine, BCH, methionine, histidine, valine, or any combination thereof.
  • a ligand functions as a type of targeting moiety defined as a selectively bindable material that has a selective (or specific), affinity for another substance.
  • the ligand is recognized and bound by a usually, but not necessarily, larger specific binding body or "binding partner,” or "receptor.”
  • binding partner or “receptor.”
  • ligands suitable for targeting are antigens, haptens, biotin, biotin derivatives, lectins, galactosamine and fucosylamine moieties, receptors, substrates, coenzymes and cofactors among others.
  • Other substances that can function as ligands for targeting a micelle of the present disclosure are vitamins (i.e.
  • the targeting moiety comprises a protein or protein fragment (e.g., hormones, toxins), and synthetic or natural polypeptides with cell affinity.
  • Ligands also include various substances with selective affinity for ligators that are produced through recombinant DNA, genetic and molecular engineering. Except when stated otherwise, ligands of the instant disclosure also include ligands as defined in U.S. Pat. No. 3,817,837, which is herein incorporated by reference in its entirety.
  • a ligator functions as a type of targeting moiety defined for this disclosure as a specific binding body or "partner" or “receptor,” that is usually, but not necessarily, larger than the ligand it can bind to. For the purposes of this disclosure, it can be a specific substance or material or chemical or “reactant” that is capable of selective affinity binding with a specific ligand.
  • a ligator can be a protein such as an antibody, a nonprotein binding body, or a "specific reactor.”
  • a ligator includes an antibody, which is defined to include all classes of antibodies, monoclonal antibodies, chimeric antibodies, Fab fractions, fragments and derivatives thereof.
  • antibody encompasses an immunoglobulin whether natural or partly or wholly synthetically produced, and fragments thereof. The term also covers any protein having a binding domain that is homologous to an immunoglobulin binding domain. “Antibody” further includes a polypeptide comprising a framework region from an immunoglobulin gene or fragments thereof that specifically binds and recognizes an antigen.
  • antibody is meant to include whole antibodies, polyclonal, monoclonal and recombinant antibodies, fragments thereof, and further includes single-chain antibodies, humanized antibodies, murine antibodies, chimeric, mouse-human, mouse-primate, primate-human monoclonal antibodies, anti-idiotype antibodies, antibody fragments, such as, e.g., scFv, scFab, (scFab)2, (scFv)2, Fab, Fab', and F(ab')2, F(ab1)2, Fv, dAb, and Fd fragments, diabodies, and antibody-related polypeptides.
  • Antibody includes bispecific antibodies and multispecific antibodies so long as they exhibit the desired biological activity or function.
  • the targeting moiety is an antibody or a molecule comprising an antigen binding fragment thereof.
  • the antibody is a nanobody.
  • the antibody is an ADC.
  • the terms "antibody-drug conjugate” and “ADC” are used interchangeably and refer to an antibody linked, e.g., covalently, to a therapeutic agent (sometimes referred to herein as agent, drug, or active pharmaceutical ingredient) or agents.
  • the targeting moiety is an antibody-drug conjugate. [0397] Under some conditions, the instant disclosure is also applicable to using other substances as ligators.
  • ligators suitable for targeting include naturally occurring receptors, any hemagglutinins and cell membrane and nuclear derivatives that bind specifically to hormones, vitamins, drugs, antibiotics, cancer markers, genetic markers, viruses, and histocompatibility markers.
  • Another group of ligators includes any RNA and DNA binding substances such as polyethylenimine (PEI) and polypeptides or proteins such as histones and protamines.
  • Other ligators also include enzymes, especially cell surface enzymes such as neuraminidases, plasma proteins, avidins, streptavidins, chalones, cavitands, thyroglobulin, intrinsic factor, globulins, chelators, surfactants, organometallic substances, staphylococcal protein A, protein G, ribosomes, bacteriophages, cytochromes, lectins, resins, and organic polymers.
  • Targeting moieties also include various substances such as any proteins, protein fragments or polypeptides with affinity for the surface of any cells, tissues or microorganisms that are produced through recombinant DNA, genetic and molecular engineering.
  • the targeting moiety directs a micelle of the present disclosure to a specific tissue (i.e., liver tissue or brain tissue), to a specific type of cell (e.g., cancer cells), or to a physiological compartment or physiological barrier (e.g., the BBB).
  • a cationic carrier unit disclosed herein can comprise one or more linkers.
  • the term "linker” refers to a peptide or polypeptide sequence (e.g., a synthetic peptide or polypeptide sequence), or a non-peptide linker for which its main function is to connect two moieties in a cationic carrier unit disclosed herein.
  • cationic carrier units of the present disclosure can comprise at least one linker connecting a tissue-specific targeting moiety (TM) with a water soluble polymer (WS), at least one linker connecting a water-soluble biopolymer (WP) with cationic carrier (CC) or a hydrophobic moiety (HM) or a crosslinking moiety (CM), at least one linker connecting a cationic carrier (CC) with a hydrophobic moiety (HM), or any combination thereof.
  • two or more linkers can be linked in tandem.
  • each of the linkers can be the same or different.
  • linkers provide flexibility to the cationic carrier unit.
  • Linkers are not typically cleaved; however, in some aspects, such cleavage can be desirable. Accordingly, in some aspects a linker can comprise one or more protease-cleavable sites, which can be located within the sequence of the linker or flanking the linker at either end of the linker sequence. [0402] In one aspect, the linker is a peptide linker.
  • the peptide linker can comprise at least about two, at least about three, at least about four, at least about five, at least about 10, at least about 15, at least about 20, at least about 25, at least about 30, at least about 35, at least about 40, at least about 45, at least about 50, at least about 55, at least about 60, at least about 65, at least about 70, at least about 75, at least about 80, at least about 85, at least about 90, at least about 95, or at least about 100 amino acids.
  • the peptide linker can comprise at least about 110, at least about 120, at least about 130, at least about 140, at least about 150, at least about 160, at least about 170, at least about 180, at least about 190, or at least about 200 amino acids.
  • the peptide linker can comprise at least about 200, at least about 250, at least about 300, at least about 350, at least about 400, at least about 450, at least about 500, at least 550, at least about 600, at least about 650, at least about 700, at least about 750, at least about 800, at least about 850, at least about 900, at least about 950, or at least about 1,000 amino acids.
  • the peptide linker can comprise between 1 and about 5 amino acids, between 1 and about 10 amino acids, between 1 and about 20 amino acids, between about 10 and about 50 amino acids, between about 50 and about 100 amino acids, between about 100 and about 200 amino acids, between about 200 and about 300 amino acids, between about 300 and about 400 amino acids, between about 400 and about 500 amino acids, between about 500 and about 600 amino acids, between about 600 and about 700 amino acids, between about 700 and about 800 amino acids, between about 800 and about 900 amino acids, or between about 900 and about 1000 amino acids.
  • Examples of peptide linkers are well known in the art. In some aspects, the linker is a glycine/serine linker.
  • the peptide linker is glycine/serine linker according to the formula [(Gly)n-Ser]m where n is any integer from 1 to 100 and m is any integer from 1 to 100.
  • the glycine/serine linker is according to the formula [(Gly)x-Sery]z (SEQ ID NO: 1) wherein x in an integer from 1 to 4, y is 0 or 1, and z is an integers from 1 to 50.
  • the peptide linker comprises the sequence Gn, where n can be an integer from 1 to 100 (SEQ ID NO: 32).
  • the sequence of the peptide linker is GGGG (SEQ ID NO: 2).
  • the peptide linker can comprise the sequence (GlyAla)n (SEQ ID NO: 3), wherein n is an integer between 1 and 100. In some aspects, the peptide linker can comprise the sequence (GlyGlySer)n (SEQ ID NO: 4), wherein n is an integer between 1 and 100. [0408] In some aspects, the peptide linker comprises the sequence (GGGS)n (SEQ ID NO: 5). In still some aspects, the peptide linker comprises the sequence (GGS)n(GGGGS)n (SEQ ID NO: 6). In these instances, n can be an integer from 1-100.
  • n can be an integer from one to 20, i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20.
  • linkers include, but are not limited to, GGG, SGGSGGS (SEQ ID NO: 7), GGSGGSGGSGGSGGG (SEQ ID NO: 8), GGSGGSGGGGSGGGGS (SEQ ID NO: 9), GGSGGSGGSGGSGGSGGS (SEQ ID NO: 10), or GGGGSGGGGSGGGGS (SEQ ID NO: 11).
  • the linker is a poly-G sequence (GGGG)n (SEQ ID NO: 12), where n can be an integer from 1-100.
  • the peptide linker is synthetic, i.e., non-naturally occurring.
  • a peptide linker includes peptides (or polypeptides) (e.g., natural or non-naturally occurring peptides) which comprise an amino acid sequence that links or genetically fuses a first linear sequence of amino acids to a second linear sequence of amino acids to which it is not naturally linked or genetically fused in nature.
  • the peptide linker can comprise non-naturally occurring polypeptides which are modified forms of naturally occurring polypeptides (e.g., comprising a mutation such as an addition, substitution or deletion).
  • the peptide linker can comprise non-naturally occurring amino acids.
  • the peptide linker can comprise naturally occurring amino acids occurring in a linear sequence that does not occur in nature. In still some aspects, the peptide linker can comprise a naturally occurring polypeptide sequence. [0411] In some aspects, the linker comprises a non-peptide linker. In some aspects, the linker consists of a non-peptide linker.
  • the non-peptide linker can be, e.g., maleimido caproyl (MC), maleimido propanoyl (MP), methoxyl polyethyleneglycol (MPEG), succinimidyl 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (SMCC), m-maleimidobenzoyl- N-hydroxysuccinimide ester (MBS), succinimidyl 4-(p-maleimidophenyl)butyrate (SMPB), N- succinimidyl(4-iodoacetyl)aminobenzonate (SIAB), succinimidyl 6-[3-(2-pyridyldithio)- propionamide]hexanoate (LC-SPDP), 4-succinimidyloxycarbonyl-alpha-methyl-alpha-(2- pyridyldithio)toluene (SMPT), etc.
  • MC maleimido caproy
  • Linkers can be introduced into polypeptide sequences using techniques known in the art (e.g., chemical conjugation, recombinant techniques, or peptide synthesis). Modifications can be confirmed by DNA sequence analysis.
  • the linkers can be introduced using recombinant techniques.
  • the linkers can be introduced using solid phase peptide synthesis.
  • a cationic carrier unit disclosed herein can contain simultaneously one or more linkers that have been introduced using recombinant techniques and one or more linkers that have been introduced using solid phase peptide synthesis or methods of chemical conjugation known in the art.
  • the linker comprises a cleavage site.
  • the present disclosure also provides pharmaceutical compositions comprising a miR-485 inhibitor disclosed herein (e.g., a polynucleotide or a vector comprising the miR-485 inhibitor) that are suitable for administration to a subject.
  • the pharmaceutical compositions generally comprise a miR-485 inhibitor described herein (e.g., a polynucleotide or a vector) and a pharmaceutically-acceptable excipient or carrier in a form suitable for administration to a subject.
  • Pharmaceutically acceptable excipients or carriers are determined in part by the particular composition being administered, as well as by the particular method used to administer the composition.
  • kits or products of manufacture comprising a miRNA inhibitor of the present disclosure (e.g., a polynucleotide, vector, or pharmaceutical composition disclosed herein) and optionally instructions for use, e.g., instructions for use according to the methods disclosed herein.
  • a miRNA inhibitor of the present disclosure e.g., a polynucleotide, vector, or pharmaceutical composition disclosed herein
  • optionally instructions for use e.g., instructions for use according to the methods disclosed herein.
  • the kit or product of manufacture comprises a miR-485 inhibitor (e.g., vector, e.g., an AAV vector, a polynucleotide, or a pharmaceutical composition of the present disclosure) in one or more containers.
  • the kit or product of manufacture comprises miR-485 inhibitor (e.g., a vector, e.g., an AAV vector, a polynucleotide, or a pharmaceutical composition of the present disclosure) and a brochure.
  • miR-485 inhibitors disclosed herein can be readily incorporated into one of the established kit formats which are well known in the art.
  • the following examples are offered by way of illustration and not by way of limitation. Examples Example 1: In Vitro Analysis of Effect on LPS-Mediated Mitochondrial Dysfunction [0417] LPS (toll-like receptor 4 ligand) has been previously shown to repress autophagy and impair the phagocytic capability of microglia. Jin et al., Front Aging Neurosci 10: 378 (Nov. 20, 2018).
  • the miR-485 inhibitor can have an effect on metabolic reprogramming by regulating both OXPHOS and glycolysis. Induction of metabolic shift from OXPHOS to glycolysis by inflammatory stimuli has been shown to impair ATP production, increasing the production of reactive oxygen species (ROS), and inducing impaired mitochondrial fission and fragmentation in microglial chronic activation.
  • ROS reactive oxygen species
  • the miR-485 inhibitor reduced ROS overproduction through the regulation of metabolic reprogramming by regulating both OXPHOS and glycolysis in LPS- treated microglia.
  • Example 2 In Vitro Analysis of Effect on A ⁇ O-Mediated Mitochondrial Dysfunction [0424] Next, the effect of the miR-485 inhibitors described herein on mitochondrial metabolism was assessed. Amyloid beta (A ⁇ ) peptides are major constituents of the senile plaques associated with some neurodegenerative diseases. A ⁇ peptides have also been shown to be associated with multiple diverse mitochondrial dysfunctions accompanying such diseases (e.g., Alzheimer's disease). Mossman et al., Cell Metab 20(4): 662-9 (Oct. 7, 2014).
  • a ⁇ peptides are capable of sensitizing the mitochondrial permeability transition pore to ca2+ resulting in mitochondrial dysfunction, and impaired mitochondria often release mitochondrial deoxynucleic acid (mtDNA), which activates NLRP3 inflammasome by potassium outflow or calcium influx.
  • mtDNA mitochondrial deoxynucleic acid
  • Example 1 the expression of different proteins thought to be important in autophagy regulation was assessed (e.g., MAP1LC3B, SQSTM1/p62, SIRT1, Pink1, and Parkin) by Western blot analysis.
  • MAP1LC3B the expression of different proteins thought to be important in autophagy regulation was assessed (e.g., MAP1LC3B, SQSTM1/p62, SIRT1, Pink1, and Parkin) by Western blot analysis.
  • FIG. 2A similar to LPS, treating primary microglia with A ⁇ Os resulted in noticeably reduced expression of many of the proteins involved in mitophagy (e.g., LAMP2, Pink1, and Parkin). Treatment with A ⁇ Os also resulted in increased expression of MAP1LC3B.
  • treating primary microglia with a miR-485 inhibitor also had an effect on mitochondrial biogenesis.
  • treatment of primary microglia with A ⁇ Os resulted in decreased expression of the proteins associated with the PGC-1 ⁇ -NRF1/NRF2-TFAM pathway (a known mitochondrial biogenesis signaling pathway).
  • PGC-1 ⁇ -NRF1/NRF2-TFAM pathway a known mitochondrial biogenesis signaling pathway.
  • PGC-1 ⁇ -NRF1/NRF2-TFAM pathway a known mitochondrial biogenesis signaling pathway.
  • Further treating the primary microglia with a miR-485 inhibitor resulted in comparable expression of the proteins tested (i.e., PGC- 1 ⁇ , NRF1, NRF2, and TFAM) to those of the control non-A ⁇ O treated primary microglia.
  • a ⁇ O- treated primary microglia exhibited decreased phosphorylation of the pS616 site of Drp1 (i.e., decreased fission) and increased phosphorylation of the pS637 site (i.e., increased fusion).
  • the ability of a miR-485 inhibitor to promote mitochondrial fusion was further confirmed by the increased expression of mitofusin-1 (MFN1) protein in primary microglia treated with both the A ⁇ O and miR-485-inhibitor, as compared to those cells treated with the A ⁇ O alone (see FIG. 2E).
  • mice were treated with one of the following: (i) no treatment ("Con"), (ii) A ⁇ Os (2 ⁇ g) alone, or (iii) A ⁇ Os (2 ⁇ g) and a miR-485 inhibitor (5 mg/kg).
  • the A ⁇ O and miR-485 inhibitor were administered via intracerebroventricular (ICV) injection.
  • ICV intracerebroventricular
  • the miR-485 inhibitor treated mice also exhibited improved ATP turnover and increased basal and maximal respiration within the hippocampus, as based on OCR analysis (see FIGs.3C and 3D).
  • the above results confirm that the miR-485 inhibitors provided herein can improve impaired mitochondrial function in vivo.
  • *** [0433] It is to be appreciated that the Detailed Description section, and not the Summary and Abstract sections, is intended to be used to interpret the claims. The Summary and Abstract sections can set forth one or more but not all exemplary aspects of the present disclosure as contemplated by the inventor(s), and thus, are not intended to limit the present disclosure and the appended claims in any way.

Landscapes

  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

La présente invention concerne l'utilisation d'un inhibiteur de miR-485 pour soulager et/ou prévenir une fonction mitochondriale. Selon certains aspects, un inhibiteur de miR-485 décrit dans la présente invention peut être utilisé pour réguler la mitophagie, la biogenèse mitochondriale, la fission mitochondriale et/ou la fusion mitochondriale. La présente invention concerne également l'utilisation d'un inhibiteur de miR-485 pour traiter un trouble mitochondrial.
PCT/IB2023/062712 2022-12-14 2023-12-14 Traitement d'un dysfonctionnement mitochondrial avec un inhibiteur de miarn-485 WO2024127317A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US202263387455P 2022-12-14 2022-12-14
US63/387,455 2022-12-14

Publications (1)

Publication Number Publication Date
WO2024127317A1 true WO2024127317A1 (fr) 2024-06-20

Family

ID=91485283

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/IB2023/062712 WO2024127317A1 (fr) 2022-12-14 2023-12-14 Traitement d'un dysfonctionnement mitochondrial avec un inhibiteur de miarn-485

Country Status (1)

Country Link
WO (1) WO2024127317A1 (fr)

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20190142289A (ko) * 2019-12-12 2019-12-26 주식회사 바이오오케스트라 miR-485-3p를 이용한 알츠하이머병 진단 방법
WO2021015683A1 (fr) * 2019-07-23 2021-01-28 Lakto Hayvancilik Teknoloji̇leri̇ Sanayi̇ Ve Ti̇caret Li̇mi̇ted Şi̇rketi̇ Système de gestion de troupeau et capteur de suivi

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2021015683A1 (fr) * 2019-07-23 2021-01-28 Lakto Hayvancilik Teknoloji̇leri̇ Sanayi̇ Ve Ti̇caret Li̇mi̇ted Şi̇rketi̇ Système de gestion de troupeau et capteur de suivi
KR20190142289A (ko) * 2019-12-12 2019-12-26 주식회사 바이오오케스트라 miR-485-3p를 이용한 알츠하이머병 진단 방법

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
PETERM.J. QUINN;PAULAI. MOREIRA;ANTóNIOFRANCISCO AMBRóSIO;C.HENRIQUE ALVES: "PINK1/PARKIN signalling in neurodegeneration and neuroinflammation", ACTA NEUROPATHOLOGICA COMMUNICATIONS, BIOMED CENTRAL LTD, LONDON, UK, vol. 8, no. 1, 9 November 2020 (2020-11-09), London, UK , pages 1 - 20, XP021283380, DOI: 10.1186/s40478-020-01062-w *
ST-PIERRE JULIE, DRORI STAVIT, ULDRY MARC, SILVAGGI JESSICA M., RHEE JAMES, JÄGER SIBYLLE, HANDSCHIN CHRISTOPH, ZHENG KANGNI, LIN : "Suppression of Reactive Oxygen Species and Neurodegeneration by the PGC-1 Transcriptional Coactivators", CELL, ELSEVIER, AMSTERDAM NL, vol. 127, no. 2, 1 October 2006 (2006-10-01), Amsterdam NL , pages 397 - 408, XP093181450, ISSN: 0092-8674, DOI: 10.1016/j.cell.2006.09.024 *
YU LING, LI HAITING, LIU WENHU, ZHANG LIGONG, TIAN QUN, LI HAIRONG, LI MIN: "Retracted: MiR‐485‐3p serves as a biomarker and therapeutic target of Alzheimer's disease via regulating neuronal cell viability and neuroinflammation by targeting AKT3", MOLECULAR GENETICS & GENOMIC MEDICINE, vol. 9, no. 1, 1 January 2021 (2021-01-01), pages e1548, XP093063010, ISSN: 2324-9269, DOI: 10.1002/mgg3.1548 *

Similar Documents

Publication Publication Date Title
US20230304014A1 (en) Mirna-485 inhibitor for huntington's disease
WO2024127317A1 (fr) Traitement d'un dysfonctionnement mitochondrial avec un inhibiteur de miarn-485
US20230099372A1 (en) Use of mirna-485 inhibitors for treating tauopathy
US20230119699A1 (en) Diagnostic methods using sirt1 expression
WO2023079499A1 (fr) Utilisation d'inhibiteurs de mir-485 pour traiter des maladies ou des pathologies associées au vieillissement
US20230121720A1 (en) Diagnostic methods using pcg-1a expression
WO2022168007A1 (fr) Utilisation d'inhibiteurs de miarn-485 pour traiter des maladies ou des troubles associés à une expression de nlrp3 anormale
US20230131083A1 (en) Use of mirna-485 inhibitors for treating amyotrophic lateral sclerosis (als)
US20240117350A1 (en) Use of mirna-485 inhibitor to regulate psd95, synaptophysin, and caspase-3 expression
US20240209035A1 (en) Polynucleotides capable of enhanced protein expression and uses thereof
US20230126157A1 (en) Mirna-485 inhibitor for gene upregulation
WO2023089506A1 (fr) Procédés sans amplification pour la mesure de miarn
WO2022264038A1 (fr) Utilisation d'inhibiteurs de miarn-485 pour le traitement de maladies ou de troubles liés aux inflammasomes
CN116963785A (zh) miRNA-485抑制剂用于治疗与异常NLRP3表达相关的疾病或病症的用途
WO2022003609A1 (fr) Procédés de diagnostic snp