WO2022182697A1 - Nouvelle approche à base d'arn pour le traitement du cancer - Google Patents

Nouvelle approche à base d'arn pour le traitement du cancer Download PDF

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Publication number
WO2022182697A1
WO2022182697A1 PCT/US2022/017419 US2022017419W WO2022182697A1 WO 2022182697 A1 WO2022182697 A1 WO 2022182697A1 US 2022017419 W US2022017419 W US 2022017419W WO 2022182697 A1 WO2022182697 A1 WO 2022182697A1
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mettl3
mettl14
hydrochloride
rna
iros
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PCT/US2022/017419
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English (en)
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Yogesh K. GUPTA
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Board Of Regents, The University Of Texas System
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Publication of WO2022182697A1 publication Critical patent/WO2022182697A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1137—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against enzymes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/7115—Nucleic acids or oligonucleotides having modified bases, i.e. other than adenine, guanine, cytosine, uracil or thymine
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/11—Antisense
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y201/00—Transferases transferring one-carbon groups (2.1)
    • C12Y201/01—Methyltransferases (2.1.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y201/00—Transferases transferring one-carbon groups (2.1)
    • C12Y201/01—Methyltransferases (2.1.1)
    • C12Y201/01062—Methyltransferases (2.1.1) mRNA (2'-O-methyladenosine-N6-)-methyltransferase (2.1.1.62)

Definitions

  • AML Acute myeloid leukemia
  • BBM Glioblastoma multiforme
  • iROS inhibitory RNA oligonucleotide scaffolds
  • iROS inhibitory RNA oligonucleotide scaffolds
  • iROS of any preceding aspect, wherein one or more nucleotides are modified.
  • iROS wherein one or more of20 the adenosine bases of the oligonucleotide have been modified to comprise N 6 - methyladenosine (m 6 A), N 6, 2’-O-dimethyladenosine (m 6 Am) and/or wherein one or more the adenosine, uracil, cytidine, and guanine bases of the oligonucleotide have been modified with 2’-O-methyl, 2’-O-methoxyethyl, and/or 2’-Fluoro chemical groups.
  • iROS of any preceding aspect wherein the 5’and 3’-ends have been modified with 25 polyethylene glycol (PEG) and 3’-3’ inverted dU, respectively. Also disclosed herein are iROS of any preceding aspect, wherein one or more uracils have been modified to pseudouridine. 5.
  • iROS of any preceding aspect with alternative chemistries for bridged nucleic acids (BNA) such as: LNA (locked nucleic acid), ENA 30 (ethylene-bridged nucleic acid), PNA (peptide nucleic acid), 2’4’-constrained 2’-O-ethyl (constrained ethyl) BNA (cET), phoshphorodiamidate morphilino oligonucleotide (PMO) and PS (phosphorothioate). 6. Also, disclosed herein are compositions comprising the iROS of any preceding aspect.
  • the oligonucleotide can be encapsulated or comprised in a hydrogel, stable nucleic acid lipid nanoparticles (SNA), spherical nucleic acid, DNA cage, exosome or polymer composition.
  • SNA stable nucleic acid lipid nanoparticles
  • compositions comprising the iROS of any preceding aspect.
  • the oligonucleotide can be chemically conjugated with lipids, cholesterol (to facilitate interactions with lipoproteins), peptides (for cell targeting and/or penetration), aptamer, antibodies, lipoplexes and liposomes, and sugars (for example, N- accetylgalactosamine [GalNAc]).
  • Also disclosed herein are methods of inhibiting the interaction of METTL3 and METTL14 and/or activity of METTL3 or METTL14 comprising contacting METTL3 or METTL14 with the iROS or compositions of any of claims any preceding aspect.
  • methods of inhibiting the interaction of METTL3 and METTL14 and/or activity of METTL3 or METTL14 comprising contacting METTL3 or METTL14 with the iROS as set forth in SEQ ID NO: 4 or SEQ ID NO: 6.
  • cancer and/or metastasis such as, for example, acute myeloid leukemia (AML), glioblastoma (GBM), colorectal cancers, and/or other cancers and diseases that rely upon METTL3, METTL14 enzymatic and/or regulatory activities
  • AML acute myeloid leukemia
  • GBM glioblastoma
  • colorectal cancers and/or other cancers and diseases that rely upon METTL3, METTL14 enzymatic and/or regulatory activities
  • symptoms of said cancer or metastasis in a subject comprising administering to the subject, the iROS or compositions of any preceding aspect.
  • iROS as set forth in SEQ ID NO: 4 or SEQ ID NO: 6.
  • Figures 1A, IB, 1C, ID, IE, IF, 1G, 1H, II, and 1J show structural similarity and biochemical characterization of human METTL3-METTL14 complex.
  • Figure 1A shows crystal structure of EcoP15I-DNA complex; cyan; ModA methyltransferase (MTase), blue; ModB MTase, magenta; Res (ATPase) subunit, grey; DNA duplex, yellow; flipped adenine base.
  • Figure IB shows MTase domains of two Mod subunits (ModA/B) of EcoP15I with target DNA strand.
  • Figure 1C shows an overlay of MTase domains of EcoP15I and METTL3-METTL14 shows high structural similarity within MTase folds.
  • RNA strand here was modeled based on the respective methylating strand in the EcoP15I structure.
  • Figure ID shows the sequence of DNA and RNA oligonucleotides used in this study.
  • Figure IE shows MTase activity of METTL3-METTL14 in the presence of different DNA or RNA substrates. CPM; counts per million. Highest MTase activity was measured with d6T* oligo.
  • Figure IF shows FP-based binding assay for DNA and RNA oligos showing highest affinity for rNEAT2 RNA (green), and lowest affinity for d6T* oligo (red).
  • Figure 1G shows methyltransferase (MTase) activity of METTL3-METTL14 for respective RNA oligo (red), d6T* DNA alone (black), and equimolar mixture of the two (blue).
  • Figure 1H shows dose- dependent inhibition of the m 6 dA activity by rNEAT2 (also referred to herein as AT- 101) and rTCE23 (also referred to herein as AT-201) as measured by a radiometric assay.
  • rNEAT2 also referred to herein as AT- 101
  • rTCE23 also referred to herein as AT-201
  • IC50 concentration of RNA required to achieve 50% inhibition of the m 6 dA activity of METTL3- METTL14.
  • Source data are provided as a Source Data file.
  • Figure 1J shows predicted secondary structures of each oligonucleotides, yellow; RNA strand, black; DNA strand. The values of equilibrium dissociation constants (Kd) for each oligo shown, indicates an inverse relationship between binding affinity and methyltransferase activities.
  • Figures 2A, 2B ,2C, and 2D model of RNA-mediated restriction of m 6 dA activity of METTL3-METTL14.
  • Figure 2A shows the domain architecture of METTL3 and METTL14.
  • LH leader helix
  • NLS nuclear localization signal
  • ZnFl/2 zinc-finger domain 1/2
  • RGG arginine-glycine rich repeats motif.
  • Figure 2B shows FP-based binding assay for DNA and RNA oligos showing highest affinity for rNEAT2 (also referred to herein as AT- 101) RNA (green), and lowest affinity for d6T* oligo (red).
  • Figure 2C shows relative methyltransferase activity of full length METTL3-METTL14, and the truncated form lacking the RGG motif in METTL14 ( [METTL3 -METTL 14 (-RGG)]) in presence of d6T*, rNEAT2, and the equimolar mixture of these two oligos.
  • the results of two groups were analyzed and compared using two-tailed Student’s unpaired T-test (P value ⁇ 0.0001). Details about Student’s T-test are provided in the source data file.
  • FIG. 2D shows METTL3-METTL14 complex can methylate a target adenine (yellow) in a single-stranded DNA region (black). Structured motifs present in ncRNA/mRNAs (yellow) can block the m 6 dA (red sphere) activity by strong shape-dependent binding to METTL3-METTL14.
  • Figures 3A, 3B, 3C, 3D, 3E, and 3F show structure of NEAT2.
  • Figure 3A shows the predicted secondary structure of NEAT2 RNA.
  • Figure 3B shows a 3-D model of rNEAT2-30 as calculated by MC-SYM.
  • Figure 3C shows the predicted secondary structure of TCE23 stem- loop RNA.
  • Figure 3D shows a 3-D model of TCE23 as calculated by MC-SYM.
  • Figure 3E and 3F show chromatogram of final size exclusion chromatography (SEC) step of purification showing METTF3-METTF14 complexes (left, full-length; right, METF3- METTF14 [-RGG]) co-eluted as single homogenous species. Blue; absorbance at 280 nm, Green; absorbance at 260 nm (A260). Fower panels confirm the presence of high purity of METTF3-METTF14 proteins in the SEC peak fractions.
  • SEC final size exclusion chromatography
  • An "increase” can refer to any change that results in a greater amount of a symptom, disease, composition, condition or activity.
  • An increase can be any individual, median, or average increase in a condition, symptom, activity, composition in a statistically significant amount.
  • the increase can be a 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100% increase so long as the increase is statistically significant.
  • a “decrease” can refer to any change that results in a smaller amount of a symptom, disease, composition, condition, or activity.
  • a substance is also understood to decrease the genetic output of a gene when the genetic output of the gene product with the substance is less relative to the output of the gene product without the substance.
  • a decrease can be a change in the symptoms of a disorder such that the symptoms are less than previously observed.
  • a decrease can be any individual, median, or average decrease in a condition, symptom, activity, composition in a statistically significant amount.
  • the decrease can be a 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100% decrease so long as the decrease is statistically significant.
  • “Inhibit,” “inhibiting,” and “inhibition” mean to decrease an activity, response, condition, disease, or other biological parameter. This can include but is not limited to the complete ablation of the activity, response, condition, or disease. This may also include, for example, a 10% reduction in the activity, response, condition, or disease as compared to the native or control level. Thus, the reduction can be a 10, 20, 30, 40, 50, 60, 70, 80, 90, 100%, or any amount of reduction in between as compared to native or control levels.
  • reducing or other forms of the word, such as “reducing” or “reduction,” is meant lowering of an event or characteristic (e.g. , tumor growth). It is understood that this is typically in relation to some standard or expected value, in other words it is relative, but that it is not always necessary for the standard or relative value to be referred to.
  • reduced tumor growth means reducing the rate of growth of a tumor relative to a standard or a control.
  • prevent or other forms of the word, such as “preventing” or “prevention,” is meant to stop a particular event or characteristic, to stabilize or delay the development or progression of a particular event or characteristic, or to minimize the chances that a particular event or characteristic will occur. Prevent does not require comparison to a control as it is typically more absolute than, for example, reduce. As used herein, something could be reduced but not prevented, but something that is reduced could also be prevented. Likewise, something could be prevented but not reduced, but something that is prevented could also be reduced. It is understood that where reduce or prevent are used, unless specifically indicated otherwise, the use of the other word is also expressly disclosed.
  • the term “subject” refers to any individual who is the target of administration or treatment.
  • the subject can be a vertebrate, for example, a mammal.
  • the subject can be human, non-human primate, bovine, equine, porcine, canine, or feline.
  • the subject can also be a guinea pig, rat, hamster, rabbit, mouse, or mole.
  • the subject can be a human or veterinary patient.
  • patient refers to a subject under the treatment of a clinician, e.g., physician.
  • the term “therapeutically effective” refers to the amount of the composition used is of sufficient quantity to ameliorate one or more causes or symptoms of a disease or disorder. Such amelioration only requires a reduction or alteration, not necessarily elimination.
  • treatment refers to the medical management of a patient with the intent to cure, ameliorate, stabilize, or prevent a disease, pathological condition, or disorder.
  • This term includes active treatment, that is, treatment directed specifically toward the improvement of a disease, pathological condition, or disorder, and also includes causal treatment, that is, treatment directed toward removal of the cause of the associated disease, pathological condition, or disorder.
  • active treatment that is, treatment directed specifically toward the improvement of a disease, pathological condition, or disorder
  • causal treatment that is, treatment directed toward removal of the cause of the associated disease, pathological condition, or disorder.
  • palliative treatment that is, treatment designed for the relief of symptoms rather than the curing of the disease, pathological condition, or disorder
  • preventative treatment that is, treatment directed to minimizing or partially or completely inhibiting the development of the associated disease, pathological condition, or disorder
  • supportive treatment that is, treatment employed to supplement another specific therapy directed toward the improvement of the associated disease, pathological condition, or disorder.
  • Biocompatible generally refers to a material and any metabolites or degradation products thereof that are generally non-toxic to the recipient and do not cause significant adverse effects to the subject.
  • compositions, methods, etc. include the recited elements, but do not exclude others.
  • Consisting essentially of' when used to define compositions and methods shall mean including the recited elements, but excluding other elements of any essential significance to the combination. Thus, a composition consisting essentially of the elements as defined herein would not exclude trace contaminants from the isolation and purification method and pharmaceutically acceptable carriers, such as phosphate buffered saline, preservatives, and the like.
  • Consisting of' shall mean excluding more than trace elements of other ingredients and substantial method steps for administering the compositions provided and/or claimed in this disclosure. Embodiments defined by each of these transition terms are within the scope of this disclosure.
  • control is an alternative subject or sample used in an experiment for comparison purposes.
  • a control can be "positive” or “negative.”
  • Effective amount of an agent refers to a sufficient amount of an agent to provide a desired effect.
  • the amount of agent that is “effective” will vary from subject to subject, depending on many factors such as the age and general condition of the subject, the particular agent or agents, and the like. Thus, it is not always possible to specify a quantified “effective amount.” However, an appropriate “effective amount” in any subject case may be determined by one of ordinary skill in the art using routine experimentation. Also, as used herein, and unless specifically stated otherwise, an “effective amount” of an agent can also refer to an amount covering both therapeutically effective amounts and prophylactically effective amounts. An “effective amount” of an agent necessary to achieve a therapeutic effect may vary according to factors such as the age, sex, and weight of the subject. Dosage regimens can be adjusted to provide the optimum therapeutic response. For example, several divided doses may be administered daily or the dose may be proportionally reduced as indicated by the exigencies of the therapeutic situation.
  • a “pharmaceutically acceptable” component can refer to a component that is not biologically or otherwise undesirable, i.e., the component may be incorporated into a pharmaceutical formulation provided by the disclosure and administered to a subject as described herein without causing significant undesirable biological effects or interacting in a deleterious manner with any of the other components of the formulation in which it is contained.
  • the term When used in reference to administration to a human, the term generally implies the component has met the required standards of toxicological and manufacturing testing or that it is included on the Inactive Ingredient Guide prepared by the U.S. Food and Drug Administration.
  • “Pharmaceutically acceptable carrier” means a carrier or excipient that is useful in preparing a pharmaceutical or therapeutic composition that is generally safe and non-toxic and includes a carrier that is acceptable for veterinary and/or human pharmaceutical or therapeutic use.
  • carrier or “pharmaceutically acceptable carrier” can include, but are not limited to, phosphate buffered saline solution, water, emulsions (such as an oil/water or water/oil emulsion) and/or various types of wetting agents.
  • carrier encompasses, but is not limited to, any excipient, diluent, filler, salt, buffer, stabilizer, solubilizer, lipid, stabilizer, or other material well known in the art for use in pharmaceutical formulations and as described further herein.
  • “Pharmacologically active” (or simply “active”), as in a “pharmacologically active” derivative or analog, can refer to a derivative or analog (e.g., a salt, ester, amide, conjugate, metabolite, isomer, fragment, etc.) having the same type of pharmacological activity as the parent compound and approximately equivalent in degree.
  • “Therapeutic agent” refers to any composition that has a beneficial biological effect. Beneficial biological effects include both therapeutic effects, e.g., treatment of a disorder or other undesirable physiological condition, and prophylactic effects, e.g., prevention of a disorder or other undesirable physiological condition (e.g., a non- immunogenic cancer).
  • the terms also encompass pharmaceutically acceptable, pharmacologically active derivatives of beneficial agents specifically mentioned herein, including, but not limited to, salts, esters, amides, proagents, active metabolites, isomers, fragments, analogs, and the like.
  • therapeutic agent when used, then, or when a particular agent is specifically identified, it is to be understood that the term includes the agent per se as well as pharmaceutically acceptable, pharmacologically active salts, esters, amides, proagents, conjugates, active metabolites, isomers, fragments, analogs, etc.
  • “Therapeutically effective amount” or “therapeutically effective dose” of a composition refers to an amount that is effective to achieve a desired therapeutic result.
  • a desired therapeutic result is the control of type I diabetes.
  • a desired therapeutic result is the control of obesity.
  • Therapeutically effective amounts of a given therapeutic agent will typically vary with respect to factors such as the type and severity of the disorder or disease being treated and the age, gender, and weight of the subject. The term can also refer to an amount of a therapeutic agent, or a rate of delivery of a therapeutic agent (e.g., amount over time), effective to facilitate a desired therapeutic effect, such as pain relief.
  • a desired therapeutic effect will vary according to the condition to be treated, the tolerance of the subject, the agent and/or agent formulation to be administered (e.g., the potency of the therapeutic agent, the concentration of agent in the formulation, and the like), and a variety of other factors that are appreciated by those of ordinary skill in the art.
  • a desired biological or medical response is achieved following administration of multiple dosages of the composition to the subject over a period of days, weeks, or years.
  • iROS AT-101 and/or AT-201
  • AT- 101 and/or AT-201 a particular iROS
  • AT- 101 and/or AT-201 a number of modifications that can be made to a number of molecules including the iROS (AT- 101 and/or AT-201) are discussed
  • specifically contemplated is each and every combination and permutation of iROS (AT-101 and/or AT-201) and the modifications that are possible unless specifically indicated to the contrary.
  • METTL3 methyltransferase like-3
  • AML and GBM cells show great promise for developing a less toxic and more efficacious therapy.
  • METTL3 blocks differentiation of myeloid leukemia cells, and METTL14 inhibits hematopoietic stem/progenitor differentiation and promotes leukemogenesis.
  • METTL3-METTL14 protein complex is believed to change the chemical structure of messenger RNAs - molecules that encode genetic information.
  • AML and GBM cells show elevated levels of METTL3 enzymes, which are essential for their survival, maintenance, and growth.
  • inhibition of METTL3/METTL14 enhances the response to anti-PD- 1 immunotherapy in other cancer types (colorectal cancers and melanoma).
  • RNA modules are expected to serve as potent
  • METTL3-METTL14 antagonists for treatment of most aggressive cancers that rely upon the activity of these enzymes.
  • Recent daunting successes of RNA-based vaccines against SARS-CoV-2 have rejuvenated the area of RNA-based therapeutics.
  • the discovery of the two RNAs as METTL3-METTL14 antagonists has broad utility for treatment of leukemias, brain tumors, colorectal cancers, other cancers and other malignancies that rely upon METTL3, METTL14 enzymatic and/or regulatory activities.
  • the two iROS disclosed herein have higher specificity over a small molecule inhibitor antagonist of METTL3-METTL14 and therefore are more specific, and effective in blocking oncogenic function of these proteins.
  • iROS that bind to METTL3 and/or METTL14, inhibit the interaction of METTL3 and METTL14, and or inhibit the activity of METTL3 and/or METTL14.
  • the iROS binds to a METTL3 and/o METTL14 binding site such as, for example, the catalytic (methyltransferase) domains of both METTL3 and METTL14, Leader helix region of METTL3, RGG motif of METTL14 or accessory proteins such as, for example, Wilm’s tumor associated protein (WTAP), Eukaryotic translation initiation factor 3 subunits (eIF3H and others), CBLL1 (HAKAI), VIRMA, ZC3H13, and/or RBM15/15B.
  • WTAP tumor associated protein
  • eIF3H and others Eukaryotic translation initiation factor 3 subunits
  • CBLL1 HKAI
  • VIRMA ZC3H13, and/or RBM15/15B
  • the iROS disclosed herein can be chemically modified, encapsulated or formulated in a protective barrier or time release formulation such as, for example, a hydrogel, exosome, lipid nanocarriers, stable nucleic acid lipid nanoparticles (SNA), spherical nucleic acid, DNA cage, exosome or polymer composition. Additionally, the iROS disclosed herein can be chemically conjugated with lipids, cholesterol (to facilitate interactions with lipoproteins), peptides (for cell targeting and/or penetration), aptamer, antibodies, lipoplexes and liposomes, and sugars (for example, N-accetylgalactosamine [GalNAc]).
  • a protective barrier or time release formulation such as, for example, a hydrogel, exosome, lipid nanocarriers, stable nucleic acid lipid nanoparticles (SNA), spherical nucleic acid, DNA cage, exosome or polymer composition.
  • SNA stable nucleic acid
  • hydrogels disclosed herein can be made using any suitable biodegradable polymer.
  • Polymer refers to a relatively high molecular weight organic compound, natural or synthetic, whose structure can be represented by a repeated small unit, the monomer.
  • Non-limiting examples of polymers include polyethylene, rubber, cellulose. Synthetic polymers are typically formed by addition or condensation polymerization of monomers.
  • copolymer refers to a polymer formed from two or more different repeating units
  • a copolymer can be an alternating copolymer, a random copolymer, a block copolymer, or a graft copolymer. It is also contemplated that, in certain aspects, various block segments of a block copolymer can themselves comprise copolymers.
  • the term “polymer” encompasses all forms of polymers including, but not limited to, natural polymers, synthetic polymers, homopolymers, heteropolymers or copolymers, addition polymers, etc.
  • the hydrogel can comprise a biocompatible polymer (such as, for example, alginate).
  • biocompatible polymers include, but are not limited to polysaccharides such as alginate, chitosan, hyaluronic acid; hydrophilic polypeptides; proteins such as collagen, fibrin, and gelatin; poly(amino acids) such as poly-L-glutamic acid
  • PES polyalkylene glycols and polyalkylene oxides such as polyethylene glycol (PEG), polypropylene glycol (PPG), and poly (ethylene oxide) (PEO); poly (oxy ethylated polyol); poly(olefinic alcohol); polyvinylpyrrolidone); poly(hydroxyalkylmethacrylamide); poly (hydroxy alkylmethacry late); poly(saccharides); poly(hydroxy acids); poly(vinyl alcohol), polyhydroxyacids such as poly(lactic acid), poly (gly colic acid), and poly (lactic acid-co-gly colic acids); polyhydroxyalkanoates such as poly3-hydroxybutyrate or poly4- hydroxybutyrate; polycaprolactones; poly (orthoesters); poly anhydrides; poly(phosphazenes); poly(lactide-co
  • polyesteramides including synthetic and natural polyamides), polypeptides, and poly(amino acids); polyesteramides; polyesters; poly(dioxanones); poly (alky lene alkylates); hydrophobic poly ethers; polyurethanes; polyetheresters; polyacetals; polycyanoacrylates; poly acrylates; polymethylmethacrylates; polysiloxanes; poly (oxy ethylene)/poly(oxypropy lene) copolymers; polyketals; polyphosphates; polyhydroxy valerates; polyalkylene oxalates; polyalkylene succinates; poly(maleic acids), as well as copolymers thereof.
  • Biocompatible polymers can also include polyamides, polycarbonates, poly alky lenes, polyalkylene glycols, polyalkylene oxides, polyalkylene terepthalates, polyvinyl alcohols (PVA), methacrylate PVA(m-PVA), polyvinyl ethers, polyvinyl esters, polyvinyl halides, polyvinylpyrrolidone, polyglycolides, polysiloxanes, polyurethanes and copolymers thereof, alkyl cellulose, hydroxyalkyl celluloses, cellulose ethers, cellulose esters, nitro celluloses, polymers of acrylic and methacrylic esters, methyl cellulose, ethyl cellulose, hydroxypropyl cellulose, hydroxy- propyl methyl cellulose, hydroxybutyl methyl cellulose, cellulose acetate, cellulose propionate, cellulose acetate butyrate, cellulose acetate phthalate, carboxylethyl cellulose, cellulose tri
  • biodegradable polymers include polyesters, poly(ortho esters), poly(ethylene amines), poly(caprolactones), poly(hydroxybutyrates), poly(hydroxyvalerates), poly anhydrides, poly(acrylic acids), polyglycolides, poly (urethanes), polycarbonates, polyphosphate esters, polyphospliazenes, derivatives thereof, linear and branched copolymers and block copolymers (including triblock copolymers) thereof, and blends thereof.
  • the particle contains biocompatible and/or biodegradable polyesters or polyanhydrides such as poly(lactic acid), poly(glycolic acid), and poly(lactic-co-glycolic acid).
  • the particles can contain one more of the following polyesters: homopolymers including glycolic acid units, referred to herein as "PGA", and lactic acid units, such as poly-L-lactic acid, poly-D-lactic acid, poly-D,L-lactic acid, poly-L-lactide, poly-D-lactide, and poly-D,L-lactide5 collectively referred to herein as "PLA”, and caprolactone units, such as poly(e-caprolactone), collectively referred to herein as "PCL”; and copolymers including lactic acid and glycolic acid units, such as various forms of poly(lactic acid-co-glycolic acid) and poly(lactide-co-glycolide) characterized by the ratio of lactic acid:glycolic acid, collectively referred to herein
  • Exemplary polymers also include copolymers of polyethylene glycol (PEG) and the aforementioned polyesters, such as various forms of PLGA-PEG or PLA-PEG copolymers, collectively referred to herein as "PEGylated polymers".
  • PEG polyethylene glycol
  • the PEG region can be covalently associated with polymer to yield "PEGylated polymers" by a cleavable linker.
  • the polymer comprises at least 60, 65, 70, 75, 80, 85, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99 percent acetal pendant groups.
  • the triblock copolymers disclosed herein comprise a core polymer such as, example, polyethylene glycol (PEG), polyvinyl acetate, polyvinyl alcohol, polyvinyl pyrrolidone (PVP), polyethyleneoxide (PEO), poly(vinyl pyrrolidone-co-vinyl acetate), polymethacrylates, polyoxyethylene alkyl ethers, polyoxyethylene castor oils, polycaprolactam, polylactic acid, polyglycolic acid, poly(lactic-glycolic) acid, poly(lactic co- gly colic) acid (PLGA), cellulose derivatives, such as hydroxymethylcellulose, hydroxypropylcellulose and the like.
  • a core polymer such as, example, polyethylene glycol (PEG), polyvinyl acetate, polyvinyl alcohol, polyvinyl pyrrolidone (PVP), polyethyleneoxide (PEO), poly(vinyl pyrrolidone-co-vinyl acetate), polyme
  • compositions comprising iROSs can further comprise one or more immune checkpoint inhibitors and/or chemotherapeutic agents.
  • Chemotherapeutic agents that can be used in the disclosed hydrogel matrixes can comprise any chemotherapeutic known in the art, the including, but not limited to Abemaciclib,
  • Nanoparticle Formulation ABVD, ABVE, ABVE-PC, AC, AC-T, Adcetris (Brentuximab
  • Vedotin Vedotin
  • ADE Ado-Trastuzumab Emtansine
  • Adriamycin Doxorubicin Hydrochloride
  • Chlorambucil Amifostine, Aminolevulinic Acid, Anastrozole, Aprepitant, Aredia
  • Atezolizumab Atezolizumab, Avastin (Bevacizumab), Avelumab, Axitinib, Azacitidine, Bavencio
  • Bexarotene Bexxar (Tositumomab and Iodine I 131 Tositumomab), Bicalutamide, BiCNU
  • Bosutinib Bosutinib
  • Brentuximab Vedotin
  • Brigatinib BuMel
  • Busulfan Busulfex
  • Campath (Alemtuzumab), Camptosar , (Irinotecan Hydrochloride), Capecitabine, CAPOX,
  • Carmubris Carmustine
  • Carmustine Implant Carmustine Implant
  • Casodex Boicalutamide
  • CEM CEM
  • Defitelio (Defibrotide Sodium), Degarelix, Denileukin Diftitox, Denosumab, DepoCyt (Cytarabine Liposome), Dexamethasone, Dexrazoxane Hydrochloride, Dinutuximab,
  • Docetaxel Doxil (Doxorubicin Hydrochloride Liposome), Doxorubicin Hydrochloride,
  • Doxorubicin Hydrochloride Liposome Dox-SL (Doxorubicin Hydrochloride Liposome)
  • Enzalutamide Epirubicin Hydrochloride , EPOCH, Erbitux (Cetuximab), Eribulin Mesylate,
  • Erivedge Vismodegib
  • Erlotinib Hydrochloride Erwinaze (Asparaginase Erwinia chrysanthemi)
  • Ethyol Amifostine
  • Etopophos Etoside Phosphate
  • Etoposide Etoposide
  • Fluorouracil Injection Fluorouracil-Topical, Flutamide, Folex (Methotrexate), Folex PFS
  • FOLFIRINOX FOLFOX
  • Folotyn Pralatrexate
  • FU-LV Fulvestrant
  • Gardasil
  • Gilotrif Afatinib Dimaleate
  • Gleevec Imatinib Mesylate
  • Gliadel Carmustine Implant
  • Gliadel wafer Carmustine Implant
  • Glucarpidase Goserelin
  • Ifex Ifosfamide
  • Ifosfamide Ifosfamide
  • Tfosfamidum Ifosfamide
  • IL-2 Aldesleukin
  • Interleukin-2 Aldesleukin
  • Intron A Recombinant Interferon Alfa-2b
  • Iodine 1 131 Tositumomab and Tositumomab, Ipilimumab, Iressa (Gefitinib), Irinotecan
  • Irinotecan Hydrochloride Irinotecan Hydrochloride Liposome, Istodax (Romidepsin), Ixabepilone, Ixazomib Citrate, Ixempra (Ixabepilone), Jakafi (Ruxolitinib Phosphate), JEB, Jevtana (Cabazitaxel), Kadcyla (Ado-Trastuzumab Emtansine), Keoxifene (Raloxifene Hydrochloride), Kepivance (Palifermin), Keytruda (Pembrolizumab), Kisqali (Ribociclib), Kymriah (Tisagenlecleucel), Kyprolis (Carfilzomib), Lanreotide Acetate, Lapatinib Ditosylate, Lartruvo (Olaratumab), Lenalidomide, Lenvatinib Mesylate, Lenvim
  • Pomalyst (Pomalidomide), Ponatinib Hydrochloride, Portrazza (Necitumumab), Pralatrexate, Prednisone, Procarbazine Hydrochloride , Proleukin (Aldesleukin), Prolia (Denosumab),
  • Promacta (Eltrombopag Olamine), Propranolol Hydrochloride, Provenge (Sipuleucel-T),
  • Rubidomycin (Daunorubicin Hydrochloride), Rubraca (Rucaparib Camsylate), Rucaparib
  • Taxotere Docetaxel
  • Tecentriq a compound that influences the expression of Tecentriq
  • Temodar Temodar
  • Temozolomide Temsirolimus, Thalidomide, Thalomid (Thalidomide), Thioguanine,
  • Tykerb Lapatinib Ditosylate
  • Unituxin Unituximab
  • Uridine Triacetate VAC
  • VAMP Varubi (Rolapitant Hydrochloride), Vectibix (Panitumumab), VelP,
  • Velban (Vinblastine Sulfate), Velcade (Bortezomib), Velsar (Vinblastine Sulfate),
  • Vismodegib Vistogard (Uridine Triacetate), Voraxaze (Glucarpidase), Vorinostat, Votrient
  • Fiposome Fiposome
  • Wellcovorin Flucovorin Calcium
  • Xalkori Crizotinib
  • Xeloda Capecitabine
  • XELIRI XELOX
  • Xgeva Denosumab
  • Xofigo Radium 223 Dichloride
  • Xtandi Enzalutamide
  • Yervoy Ipilimumab
  • Yondelis Trabectedin
  • Zaltrap Ziv-Aflibercept
  • Zarxio Flgrastim
  • Zejula Neraparib Tosylate Monohydrate
  • Zelboraf Vemurafenib
  • Zevalin Ibritumomab Tiuxetan
  • Zinecard Dexrazoxane Hydrochloride
  • Ziv-Aflibercept Zofran (Ondansetron Hydrochloride)
  • Zoladex Goserelin Acetate
  • Zoledronic Acid Zolinza (Vorinostat)
  • Checkpoint inhibitors include, but are not limited to antibodies that block PD-1 (Nivolumab (BMS-936558 or MDX1106), CT-011, MK-3475), PD-L1 (MDX-1105 (BMS-936559), MPDL3280A, MSB0010718C), PD-L2 (rHIgM12B7), CTLA-4 (Ipilimumab (MDX-010), Tremelimumab (CP-675,206)), IDO, B7-H3 (MGA271), B7-H4, TIM3, LAG-3 (BMS-986016).
  • variants of these and other genes and proteins herein disclosed which have at least, 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 percent homology to the stated sequence.
  • the homology can be calculated after aligning the two sequences so that the homology is at its highest level.
  • hybridization typically means a sequence driven interaction between at least two nucleic acid molecules, such as a primer or a probe and a gene.
  • Sequence driven interaction means an interaction that occurs between two nucleotides or nucleotide analogs or nucleotide derivatives in a nucleotide specific manner. For example, G interacting with C or A interacting with T are sequence driven interactions. Typically sequence driven interactions occur on the Watson-Crick face or Hoogsteen face of the nucleotide.
  • the hybridization of two nucleic acids is affected by a number of conditions and parameters known to those of skill in the art. For example, the salt concentrations, pH, and temperature of the reaction all affect whether two nucleic acid molecules will hybridize.
  • selective hybridization conditions can be defined as stringent hybridization conditions.
  • stringency of hybridization is controlled by both temperature and salt concentration of either or both of the hybridization and washing steps.
  • the conditions of hybridization to achieve selective hybridization may involve hybridization in high ionic strength solution (6X SSC or 6X SSPE) at a temperature that is about 12-25°C below the Tm (the melting temperature at which half of the molecules dissociate from their hybridization partners) followed by washing at a combination of temperature and salt concentration chosen so that the washing temperature is about 5°C to 20°C below the Tm.
  • hybridization temperatures are typically higher for DNA-RNA and RNA-RNA hybridizations.
  • the conditions can be used as described above to achieve stringency, or as is known in the art.
  • a preferable stringent hybridization condition for a DNA:DNA hybridization can be at about 68°C (in aqueous solution) in 6X SSC or 6X SSPE followed by washing at 68°C.
  • Stringency of hybridization and washing can be reduced accordingly as the degree of complementarity desired is decreased, and further, depending upon the G-C or A-T richness of any area wherein variability is searched for.
  • stringency of hybridization and washing if desired, can be increased accordingly as homology desired is increased, and further, depending upon the G-C or A-T richness of any area wherein high homology is desired, all as known in the art.
  • selective hybridization conditions would be when at least about, 60, 65, 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 percent of the limiting nucleic acid is bound to the non-limiting nucleic acid.
  • the non-limiting primer is in for example, 10 or 100 or 1000 fold excess.
  • This type of assay can be performed at under conditions where both the limiting and non-limiting primer are for example, 10 fold or 100 fold or 1000 fold below their k d , or where only one of the nucleic acid molecules is 10 fold or 100 fold or 1000 fold or where one or both nucleic acid molecules are above their k d .
  • selective hybridization conditions are when at least about, 60, 65, 70, 71, 72, 73, 74,
  • Preferred conditions also include those suggested by the manufacturer or indicated in the art as being appropriate for the enzyme performing the manipulation.
  • composition or method meets any one of these criteria for determining hybridization either collectively or singly it is a composition or method that is disclosed herein.
  • nucleic acid based there are a variety of molecules disclosed herein that are nucleic acid based, including for example the nucleic acids that encode, for example AT- 101 or AT-201, as well as various functional nucleic acids.
  • the disclosed nucleic acids are made up of for example, nucleotides, nucleotide analogs, or nucleotide substitutes. Non-limiting examples of these and other molecules are discussed herein. It is understood that for example, when a vector is expressed in a cell, that the expressed mRNA will typically be made up of A, C, G, and U.
  • an antisense molecule is introduced into a cell or cell environment through for example exogenous delivery, it is advantagous that the antisense molecule be made up of nucleotide analogs that reduce the degradation of the antisense molecule in the cellular environment.
  • a nucleotide is a molecule that contains a base moiety, a sugar moiety and a phosphate moiety. Nucleotides can be linked together through their phosphate moieties and sugar moieties creating an intemucleoside linkage.
  • the base moiety of a nucleotide can be adenin-9-yl (A), cytosin-l-yl (C), guanin-9-yl (G), uracil-l-yl (U), and thymin-l-yl (T).
  • the sugar moiety of a nucleotide is a ribose or a deoxyribose.
  • the phosphate moiety of a nucleotide is pentavalent phosphate.
  • a non-limiting example of a nucleotide would be 3'- AMP (3'-adenosine monophosphate) or 5'-GMP (5'-guanosine monophosphate).
  • a nucleotide analog is a nucleotide which contains some type of modification to either the base, sugar, or phosphate moieties. Modifications to the base moiety would include natural and synthetic modifications of A, C, G, and T/U as well as different purine or pyrimidine bases, such as uracil-5-yl (.psi.), hypoxanthin-9-yl (I), and 2-aminoadenin-9-yl.
  • a modified base includes but is not limited to 5-methylcytosine (5-me-C), 5 -hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine,
  • 2-thiouracil, 2-thiothymine and 2-thiocytosine 5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo,
  • 5-halo particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines
  • nucleotide analogs such as 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and
  • 0-6 substituted purines including 2-aminopropyladenine, 5-propynyluracil and
  • 5-propynylcytosine can increase the stability of duplex formation.
  • time base modifications can be combined with for example a sugar modification, such as 2'- O-methoxyethyl, to achieve unique properties such as increased duplex stability and/or to prevent enzymatic degradation.
  • iROS such as, for example AT-101 or AT-201
  • one or more nucleotides are modified.
  • iROSs wherein one or more of the adenosine bases of the oligonucleotide have been modified to comprise N 6 - m ethyl ade no sine (m 6 A) and/or wherein one or more the adenosine bases of the oligonucleotide have been modified with 2 ’-0- methyl, 2 ’ - 6>- m eth o x y eth y 1 , and/or 2’-Fluoro chemical groups.
  • iROSs wherein one or more uracils have been modified to pseudouridine.
  • Nucleotide analogs can also include modifications of the sugar moiety. Modifications to the sugar moiety would include natural modifications of the ribose and deoxy ribose as well as synthetic modifications. Sugar modifications include but are not limited to the following modifications at the 2' position: OH; F; 0-, S-, or N-alkyl; 0-, S-, or N-alkenyl; 0-, S- or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl may be substituted or unsubstituted Ci to Cio, alkyl or C2 to C10 alkenyl and alkynyl.
  • 2' sugar modiifcations also include but are not limited to -0[(CH2) n 0] m CH3, -0(CH2) n OCH3, - 0(CH 2 ) radical NH 2 , -0(CH 2 )n CH 3 , -0(CH 2 )n -ONH 2 , and -0(CH 2 )n0N[(CH 2 )n CH 3 )] 2 , where n and m are from 1 to about 10.
  • Ci Cio lower alkyl
  • substituted lower alkyl alkaryl, aralkyl, O-alkaryl or O-aralkyl
  • SH SCH3
  • sugars Similar modifications may also be made at other positions on the sugar, particularly the 3' position of the sugar on the 3' terminal nucleotide or in 2'-5' linked oligonucleotides and the 5' position of 5' terminal nucleotide. Modified sugars would also include those that contain modifications at the bridging ring oxygen, such as CH2 and S. Nucleotide sugar analogs may also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar.
  • Nucleotide analogs can also be modified at the phosphate moiety.
  • Modified phosphate moieties include but are not limited to those that can be modified so that the linkage between two nucleotides contains a phosphorothioate, chiral phosphorothioate, phosphorodithioate, phosphotriester, aminoalkylphosphotriester, methyl and other alkyl phosphonates including 3'-alkylene phosphonate and chiral phosphonates, phosphinates, phosphoramidates including 3'-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates.
  • these phosphate or modified phosphate linkage between two nucleotides can be through a 3'-5' linkage or a 2'-5' linkage, and the linkage can contain inverted polarity such as 3'-5' to 5'-3' or 2'-5' to 5'-2'.
  • Various salts, mixed salts and free acid forms are also included.
  • nucleotide analogs need only contain a single modification, but may also contain multiple modifications within one of the moieties or between different moieties.
  • the iROS can comprise alternative chemistries for bridged nucleic acids (BNA) such as: LNA (locked nucleic acid), ENA (ethylene-bridged nucleic acid), PNA (peptide nucleic acid), 2’ 4’ -constrained 2’-0-ethyl (constrained ethyl) BNA (cET), phoshphorodiamidate morphilino oligonucleotide (PMO) and PS (phosphorothioate).
  • BNA bridged nucleic acids
  • Nucleotide substitutes are molecules having similar functional properties to nucleotides, but which do not contain a phosphate moiety, such as peptide nucleic acid (PNA). Nucleotide substitutes are molecules that will recognize nucleic acids in a Watson- Crick or Hoogsteen manner, but which are linked together through a moiety other than a phosphate moiety. Nucleotide substitutes are able to conform to a double helix type structure when interacting with the appropriate target nucleic acid.
  • PNA peptide nucleic acid
  • Nucleotide substitutes are nucleotides or nucleotide analogs that have had the phosphate moiety and/or sugar moieties replaced. Nucleotide substitutes do not contain a standard phosphorus atom. Substitutes for the phosphate can be for example, short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl intemucleoside linkages, or one or more short chain heteroatomic or heterocyclic intemucleoside linkages.
  • morpholino linkages formed in part from the sugar portion of a nucleoside
  • siloxane backbones sulfide, sulfoxide and sulfone backbones ;formacetyl and thioformacetyl backbones; methylene formacetyl and thioformacetyl backbones; alkene containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, O, S and CPE component parts.
  • conjugates include but are not limited to lipid moieties such as a cholesterol moiety (Letsinger et ak, Proc. Natl. Acad. Sci. USA, 1989, 86, 6553-6556), cholic acid (Manoharan et ak, Bioorg. Med. Chem. Let., 1994, 4,
  • a thioether e.g., hexyl-S-tritylthiol (Manoharan et ak, Ann. N.Y. Acad. Sci.,
  • a phospholipid e.g., di-hexadecyl-rac-glycerol or triethylammonium 1,2-di- O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et ak, Tetrahedron Lett., 1995, 36, 3651-3654; Shea et ak, Nucl.
  • Acids Res., 1990, 18, 3777-3783 a polyamine or a polyethylene glycol chain (Manoharan et ak, Nucleosides & Nucleotides, 1995, 14, 969-973), or adamantane acetic acid (Manoharan et ak, Tetrahedron Lett., 1995, 36, 3651-3654), a palmityl moiety (Mishra et ak, Biochim. Biophys. Acta, 1995, 1264, 229-237), or an octadecylamine or hexylamino-carbonyl-oxy cholesterol moiety (Crooke et ak, J. Pharmacol. Exp. Ther., 1996, 277, 923-937.
  • a polyamine or a polyethylene glycol chain Manoharan et ak, Nucleosides & Nucleotides, 1995, 14, 969-973
  • adamantane acetic acid
  • a Watson-Crick interaction is at least one interaction with the Watson-Crick face of a nucleotide, nucleotide analog, or nucleotide substitute.
  • the Watson-Crick face of a nucleotide, nucleotide analog, or nucleotide substitute includes the C2, Nl, and C6 positions of a purine-based nucleotide, nucleotide analog, or nucleotide substitute and the C2, N3, C4 positions of a pyrimidine-based nucleotide, nucleotide analog, or nucleotide substitute.
  • a Hoogsteen interaction is the interaction that takes place on the Hoogsteen face of a nucleotide or nucleotide analog, which is exposed in the major groove of duplex DNA.
  • the Hoogsteen face includes the N7 position and reactive groups (NH2 or O) at the C6 position of purine nucleotides.
  • the disclosed nucleic acids can be in the form of naked DNA or RNA, or the nucleic acids can be in a vector for delivering the nucleic acids to the cells, whereby the antibody-encoding
  • the vector can be a commercially available preparation, such as an adenovirus vector (Quantum Biotechnologies, Inc. (Laval, Quebec, Canada). Delivery of the nucleic acid or vector to cells can be via a variety of mechanisms. As one example, delivery can be via a liposome, using commercially available liposome preparations such as LIPOFECTIN, LIPOFECT AMINE (GIBCO-BRL, Inc., Gaithersburg, MD), SUPERFECT (Qiagen, Inc. Hilden, Germany) and TRANSFECTAM (Promega Biotec, Inc., Madison, WI), as well as other liposomes developed according to procedures standard in the art.
  • the disclosed nucleic acid or vector can be delivered in vivo by electroporation, the technology for which is available from Genetronics, Inc. (San Diego,
  • vector delivery can be via a viral system, such as a retroviral vector system which can package a recombinant retroviral genome (see e.g., Pastan et al., Proc. Natl. Acad. Sci. U.S.A. 85:4486, 1988; Miller et al., Mol. Cell. Biol. 6:2895, 1986).
  • the recombinant retrovirus can then be used to infect and thereby deliver to the infected cells nucleic acid encoding a broadly neutralizing antibody (or active fragment thereof).
  • the exact method of introducing the altered nucleic acid into mammalian cells is, of course, not limited to the use of retroviral vectors.
  • compositions and methods can be used in conjunction with any of these or other commonly used gene transfer methods.
  • the dosage for administration of adenovirus to humans can range from about 10 7 to 10 9 plaque forming units (pfu) per injection but can be as high as 10 12 pfu per injection (Crystal, Hum. Gene Ther. 8:985-1001, 1997; Alvarez and Curiel,
  • a subject can receive a single injection, or, if additional injections are necessary, they can be repeated at six-month intervals (or other appropriate time intervals, as determined by the skilled practitioner) for an indefinite period and/or until the efficacy of the treatment has been established.
  • Parenteral administration of the nucleic acid or vector, if used, is generally characterized by injection. Injectables can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution of suspension in liquid prior to injection, or as emulsions. A more recently revised approach for parenteral administration involves use of a slow release or sustained release system such that a constant dosage is maintained.
  • suitable formulations and various routes of administration of therapeutic compounds see, e.g., Remington: The Science and Practice of Pharmacy (19th ed.) ed. A.R. Gennaro, Mack Publishing Company, Easton, PA 1995.
  • compositions can also be administered in vivo in a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable is meant a material that is not biologically or otherwise undesirable, i.e., the material may be administered to a subject, along with the nucleic acid or vector, without causing any undesirable biological effects or interacting in a deleterious manner with any of the other components of the pharmaceutical composition in which it is contained.
  • the carrier would naturally be selected to minimize any degradation of the active ingredient and to minimize any adverse side effects in the subject, as would be well known to one of skill in the art.
  • compositions may be administered orally, parenterally (e.g., intravenously), by intramuscular injection, by intraperitoneal injection, transdermally, extracorporeally, topically or the like, including topical intranasal administration or administration by inhalant ⁇
  • topical intranasal administration means delivery of the compositions into the nose and nasal passages through one or both of the nares and can comprise delivery by a spraying mechanism or droplet mechanism, or through aerosolization of the nucleic acid or vector.
  • Administration of the compositions by inhalant can be through the nose or mouth via delivery by a spraying or droplet mechanism. Delivery can also be directly to any area of the respiratory system (e.g., lungs) via intubation.
  • compositions required will vary from subject to subject, depending on the species, age, weight and general condition of the subject, the severity of the allergic disorder being treated, the particular nucleic acid or vector used, its mode of administration and the like. Thus, it is not possible to specify an exact amount for every composition. However, an appropriate amount can be determined by one of ordinary skill in the art using only routine experimentation given the teachings herein.
  • Parenteral administration of the composition is generally characterized by injection.
  • Injectables can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution of suspension in liquid prior to injection, or as emulsions.
  • a more recently revised approach for parenteral administration involves use of a slow release or sustained release system such that a constant dosage is maintained. See, e.g., U.S. Patent No. 3,610,795, which is incorporated by reference herein.
  • the materials may be in solution, suspension (for example, incorporated into microparticles, liposomes, or cells). These may be targeted to a particular cell type via antibodies, receptors, or receptor ligands.
  • the following references are examples of the use of this technology to target specific proteins to tumor tissue (Senter, et al., Bioconjugate Chem., 2:447-451, (1991); Bagshawe, K.D., Br. J. Cancer, 60:275-281, (1989); Bagshawe, et al., Br. J. Cancer, 58:700-703, (1988); Senter, et al., Bioconjugate Chem., 4:3-9, (1993); Battelli, et al., Cancer Immunol.
  • Vehicles such as "stealth” and other antibody conjugated liposomes (including lipid mediated drug targeting to colonic carcinoma), receptor mediated targeting of DNA through cell specific ligands, lymphocyte directed tumor targeting, and highly specific therapeutic retroviral targeting of murine glioma cells in vivo.
  • receptors are involved in pathways of endocytosis, either constitutive or ligand induced. These receptors cluster in clathrin-coated pits, enter the cell via clathrin-coated vesicles, pass through an acidified endosome in which the receptors are sorted, and then either recycle to the cell surface, become stored intracellularly, or are degraded in lysosomes.
  • the internalization pathways serve a variety of functions, such as nutrient uptake, removal of activated proteins, clearance of macromolecules, opportunistic entry of viruses and toxins, dissociation and degradation of ligand, and receptor- level regulation. Many receptors follow more than one intracellular pathway, depending on the cell type, receptor concentration, type of ligand, ligand valency, and ligand concentration. Molecular and cellular mechanisms of receptor-mediated endocytosis have been reviewed (Brown and Greene, DNA and Cell Biology 10:6, 399-409 (1991)). a) Pharmaceutically Acceptable Carriers
  • compositions, including antibodies, can be used therapeutically in combination with a pharmaceutically acceptable carrier.
  • a pharmaceutically acceptable carrier Suitable carriers and their formulations are described in Remington: The Science and Practice of Pharmacy (19th ed.) ed. A.R. Gennaro, Mack Publishing Company, Easton, PA 1995.
  • an appropriate amount of a pharmaceutically acceptable salt is used in the formulation to render the formulation isotonic.
  • the pharmaceutically acceptable carrier include, but are not limited to, saline, Ringer's solution and dextrose solution.
  • the pH of the solution is preferably from about 5 to about 8, and more preferably from about 7 to about 7.5.
  • Further carriers include sustained release preparations such as semipermeable matrices of solid hydrophobic polymers containing the antibody, in which matrices are in the form of shaped articles, e.g., films, liposomes or microparticles. It will be apparent to those persons skilled in the art that certain carriers may be more preferable depending upon, for instance, the route of administration and concentration of composition being administered.
  • compositions can be administered intramuscularly or subcutaneously. Other compounds will be administered according to standard procedures used by those skilled in the art.
  • compositions may include carriers, thickeners, diluents, buffers, preservatives, surface active agents and the like in addition to the molecule of choice.
  • Pharmaceutical compositions may also include one or more active ingredients such as antimicrobial agents, antiinflammatory agents, anesthetics, and the like.
  • the pharmaceutical composition may be administered in a number of ways depending on whether local or systemic treatment is desired, and on the area to be treated. Administration may be topically (including ophthalmically, vaginally, rectally, intranasally), orally, by inhalation, or parenterally, for example by intravenous drip, subcutaneous, intraperitoneal or intramuscular injection.
  • the disclosed antibodies can be administered intravenously, intraperitoneally, intramuscularly, subcutaneously, intracavity, or transdermally.
  • Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions.
  • non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate.
  • Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media.
  • Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils.
  • Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Preservatives and other additives may also be present such as, for example, antimicrobials, antioxidants, chelating agents, and inert gases and the like.
  • Formulations for topical administration may include ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders.
  • Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable.
  • compositions for oral administration include powders or granules, suspensions or solutions in water or non-aqueous media, capsules, sachets, or tablets. Thickeners, flavorings, diluents, emulsifiers, dispersing aids or binders may be desirable.
  • compositions may potentially be administered as a pharmaceutically acceptable acid- or base- addition salt, formed by reaction with inorganic acids such as hydrochloric acid, hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, and phosphoric acid, and organic acids such as formic acid, acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid, malonic acid, succinic acid, maleic acid, and fumaric acid, or by reaction with an inorganic base such as sodium hydroxide, ammonium hydroxide, potassium hydroxide, and organic bases such as mono-, di-, trialkyl and aryl amines and substituted ethanolamines.
  • inorganic acids such as hydrochloric acid, hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, and phosphoric acid
  • organic acids such as formic acid, acetic acid, propionic acid, glyco
  • Effective dosages and schedules for administering the compositions may be determined empirically, and making such determinations is within the skill in the art.
  • the dosage ranges for the administration of the compositions are those large enough to produce the desired effect in which the symptoms of the disorder are affected.
  • the dosage should not be so large as to cause adverse side effects, such as unwanted cross-reactions, anaphylactic reactions, and the like.
  • the dosage will vary with the age, condition, sex and extent of the disease in the patient, route of administration, or whether other drugs are included in the regimen, and can be determined by one of skill in the art.
  • the dosage can be adjusted by the individual physician in the event of any counterindications. Dosage can vary, and can be administered in one or more dose administrations daily, for one or several days.
  • a typical daily dosage of the antibody used alone might range from about 1 pg/kg to up to 100 mg/kg of body weight or more per day, depending on the factors mentioned above.
  • Also disclosed herein are methods of inhibiting the interaction of METTL3 and METTL14 and/or activity of METTL3 or METTL14 comprising contacting METTL3 or METTL14 with the iROS or compositions disclosed herein.
  • methods of inhibiting the interaction of METTL3 and METTL14 and/or activity of METTL3 or METTL14 comprising contacting METTL3 or METTL14 with the iROS as set forth in SEQ ID NO: 4 or SEQ ID NO: 6.
  • the disclosed iROS can be used to treat any disease where uncontrolled cellular proliferation occurs such as cancers.
  • methods of treating, inhibiting, decreasing, reducing, ameliorating and/or preventing cancer and/or metastasis such as, for example, acute myeloid leukemia (AML), glioblastoma (GBM), colorectal cancers, and/or other cancers that rely upon METTL3, METTL14 enzymatic and/or regulatory activities
  • AML acute myeloid leukemia
  • GBM glioblastoma
  • colorectal cancers and/or other cancers that rely upon METTL3, METTL14 enzymatic and/or regulatory activities
  • symptoms of said cancer or metastasis in a subject comprising administering to the subject, the iROS or compositions disclosed herein.
  • cancer such as, for example, acute myeloid leukemia (AML), glioblastoma (GBM), colorectal cancers, and/or other cancers that rely upon METTL3, METTL14 enzymatic and/or regulatory activities
  • AML acute myeloid leukemia
  • GBM glioblastoma
  • colorectal cancers and/or other cancers that rely upon METTL3, METTL14 enzymatic and/or regulatory activities
  • metastasis or symptoms of said cancer or metastasis in a subject comprising administering to the subject the iROS as set forth in SEQ ID NO: 4 or SEQ ID NO: 6.
  • the disclosed oligonucleotides can be used to treat, inhibit, reduce, decrease, ameliorate, and/or prevent any disease where uncontrolled cellular proliferation occurs such as cancers or symptoms associated with said cancers.
  • a representative but non- limiting list of different types of cancers is as follows: lymphoma, B cell lymphoma, T cell lymphoma, mycosis fungoides, Hodgkin’s Disease, myeloid leukemia, bladder cancer, brain cancer, nervous system cancer, head and neck cancer, squamous cell carcinoma of head and neck, lung cancers such as small cell lung cancer and non-small cell lung cancer, neuroblastoma/glioblastoma, ovarian cancer, skin cancer, liver cancer, melanoma, squamous cell carcinomas of the mouth, throat, larynx, and lung, cervical cancer, cervical carcinoma, breast cancer, and epithelial cancer, renal cancer, genitourinary cancer, pulmonary cancer, esophageal carcinoma, head and neck
  • Compounds disclosed herein may also be used for the treatment of precancer conditions such as cervical and anal dysplasias, other dysplasias, severe dysplasias, hyperplasias, atypical hyperplasias, and neoplasias.
  • the disclosed iROS (such as, for example AT-101 and AT-201) and compositions comprising said iROS used in the disclosed methods of treating, preventing, inhibiting, and/or reducing a cancer or metastasis can further comprise one or more immune blockade inhibitors and/or chemotherapeutic agents.
  • Chemotherapeutic agents that can be used in the disclosed hydrogel matrixes can comprise any chemotherapeutic known in the art, the including, but not limited to Abemaciclib, Abiraterone Acetate, Abitrexate (Methotrexate), Abraxane (Paclitaxel Albumin- stabilized Nanoparticle Formulation), ABVD, ABVE, ABVE-PC, AC, AC-T, Adcetris (Brentuximab Vedotin), ADE, Ado-Trastuzumab Emtansine, Adriamycin (Doxorubicin Hydrochloride), Afatinib Dimaleate, Afinitor (Everolimus), Akynzeo (Netupitant and Palonosetron Hydrochloride), Aldara (Imiquimod), Aldesleukin, Alecensa (Alectinib), Alectinib, Alemtuzumab, Alimta (Pemetrexed Disodium), Aliqo
  • Vaccine Cetuximab, CEV, Chlorambucil, CHLORAMBUCIL-PREDNISONE, CHOP, Cisplatin, Cladribine, Clafen (Cyclophosphamide), Clofarabine, Clofarex (Clofarabine), Clolar (Clofarabine), CMF, Cobimetinib, Cometriq (Cabozantinib-S-Malate), Copanlisib Hydrochloride, COPDAC, COPP, COPP-ABV, Cosmegen (Dactinomycin), Cotellic (Cobimetinib), Crizotinib, CVP, Cyclophosphamide, Cyfos (Ifosfamide), Cyramza (Ramucirumab), Cytarabine, Cytarabine Liposome, Cytosar-U (Cytarabine), Cytoxan (Cyclophosphamide), Dabrafenib, dacarbazine, Dacogen (
  • Etoposide Phosphate Evacet (Doxorubicin Hydrochloride Liposome), Everolimus, Evista , (Raloxifene Hydrochloride), Evomela (Melphalan Hydrochloride), Exemestane, 5-FU (Fluorouracil Injection), 5-FU (Fluorouracil-Topical), Fareston (Toremifene), Farydak (Panobinostat), Faslodex (Fulvestrant), EEC, Femara (Letrozole), Filgrastim, Fludara (Fludarabine Phosphate), Fludarabine Phosphate, Fluoroplex (Fluorouracil-Topical), Fluorouracil Injection, Fluorouracil-Topical, Flutamide, Folex (Methotrexate), Folex PFS (Methotrexate), FOLFIRI, FOLFIRI-B E V ACIZUM AB , FOLFIRI-
  • Ifex Ifosfamide
  • Ifosfamide Ifosfamide
  • Ifosfamidum Ifosfamide
  • IL-2 Aldesleukin
  • Interleukin-2 Aldesleukin
  • Intron A Recombinant Interferon Alfa-2b
  • Iodine 1 131 Tositumomab and Tositumomab, Ipilimumab, Iressa (Gefitinib), Irinotecan
  • Kymriah (Tisagenlecleucel), Kyprolis (Carfilzomib), Lanreotide Acetate, Lapatinib
  • Leustatin (Cladribine), Levulan (Aminolevulinic Acid), Linfolizin (Chlorambucil), LipoDox
  • Lupron (Leuprolide Acetate), Lupron Depot (Leuprolide Acetate), Lupron
  • Megestrol Acetate Mekinist (Trametinib), Melphalan, Melphalan Hydrochloride,
  • Methotrexate LPL Metallocate
  • Methylnaltrexone Bromide Mexate (Methotrexate)
  • Mexate-AQ Metalhotrexate
  • Midostaurin Mitomycin C
  • Mitoxantrone Hydrochloride
  • Mitozytrex (Mitomycin C), MOPP, Mozobil (Plerixafor), Mustargen (Mechlorethamine
  • Nanoparticle Lormulation Navelbine (Vinorelbine Tartrate), Necitumumab, Nelarabine,
  • Neosar (Cyclophosphamide), Neratinib Maleate, Nerlynx (Neratinib Maleate), Netupitant and
  • Niraparib Tosylate Monohydrate Nivolumab
  • Nolvadex Teamoxifen Citrate
  • Olaratumab Omacetaxine Mepesuccinate, Oncaspar (Pegaspargase), Ondansetron Hydrochloride, Onivyde (Irinotecan Hydrochloride Liposome), Ontak (Denileukin Diftitox), Opdivo (Nivolumab), OPPA, Osimertinib, Oxaliplatin, Paclitaxel, Paclitaxel Albumin- stabilized Nanoparticle Formulation, PAD, Palbociclib, Palifermin, Palonosetron Hydrochloride, Palonosetron Hydrochloride and Netupitant, Pamidronate Disodium, Panitumumab, Panobinostat, Paraplat (Carboplatin), Paraplatin (Carboplatin), Pazopanib Hydrochloride, PCV, PEB, Pegaspargase, Pegfilgrastim, Peginterferon Alfa- 2b, PEG-Intron (Peginterferon Alfa-2b), Pe
  • Steritalc Talc
  • Stivarga Regorafenib
  • Sunitinib Malate Sutent (Sunitinib Malate)
  • Sylatron Peginterferon Alfa-2b
  • Sylvant Siltuximab
  • Synribo Omacetaxine Mepesuccinate
  • Tabloid Thioguanine
  • TAC Tafinlar
  • Tagrisso Osimertinib
  • Talc Talimogene Laherparepvec
  • Tamoxifen Citrate Tarabine PFS (Cytarabine), Tarceva (Erlotinib Hydrochloride), Targretin (Bexarotene), Tasigna (Nilotinib), Taxol (Paclitaxel), Taxotere (Docetaxel), Tecentriq , (Atezolizumab), Temodar (Temozolomide),
  • Temozolomide Temsirolimus, Thalidomide, Thalomid (Thalidomide), Thioguanine, Thiotepa, Tisagenlecleucel, Tolak (Fluorouracil— Topical), Topotecan Hydrochloride, Toremifene, Torisel (Temsirolimus), Tositumomab and Iodine 1 131 Tositumomab, Totect (Dexrazoxane Hydrochloride), TPF, Trabectedin, Trametinib, Trastuzumab, Treanda
  • Checkpoint inhibitors include, but are not limited to antibodies that block PD-1 (Nivolumab (BMS-936558 or MDX1106), CT-011, MK-3475), PD-L1 (MDX-1105 (BMS-936559), MPDL3280A, MSB0010718C), PD-L2 (rHIgM12B7), CTLA-4 (Ipilimumab (MDX-010), Tremelimumab (CP-675,206)), IDO, B7-H3 (MGA271), B7-H4, TIM3, LAG-3 (BMS-986016).
  • N 6 methyl adenos i ne is considered a major covalent modification of the adenosine (A) base in coding and noncoding (nc) RNAs. It is linked to diverse physiologic processes, including - but not limited to - RNA turnover, stem cell differentiation, oncogenic translation, and DNA damage repair. In human cells, most m 6 A in cellular RNAs is installed by METTL3-METTL14 methyltransferases, both of which belong to b-class of S-adenosyl methionine (SAM)-dependent methyltransferases ( N 6 -MTases).
  • SAM S-adenosyl methionine
  • m 6 dA MTases High structural similarity of the MTase cores of human METTL3-METTL14 and N 6 -deoxyadenosi ne DNA methyltransferases (m 6 dA MTases), especially those from Type III restriction-modification (R-M) systems in bacteria (e.g. Mod A/B dimer of EcoP15I), hints that these enzymes originated from a common ancestor (fig. 1 a-c).
  • R-M Type III restriction-modification
  • RNAs e.g., nascent pre-mRNA (CA-RNA), promoter- associated RNA, enhancer RNA, and repeat RNA elements.
  • METTL3-METTL14 m 6 A writers can be recruited to chromatin by modified histone tails (e.g. H3K36me3) or transcription machinery.
  • modified histone tails e.g. H3K36me3
  • MALAT1 chromatin associated XIST and NEAT2
  • a role of m 6 A in DNA damage response and repair has also been suggested.
  • METTL3 localizes at sites of ds DNA breaks and installs the m 6 A on DNA-damage-associated RNAs.
  • m 6 dA levels in mammalian DNA are extremely low (0.006 to 0.01%) and originate from RNA catabolism rather than SAM-dependent MTase reactions, fundamental questions about substrate(s) specificity and mechanisms of action of METTL3-METTL14 have emerged.
  • RNA substrates of varied lengths (5 to 30 nucleotides) with at least one canonical m 6 A signature motif (RRACH) except for an RNA oligo (rPal-Top+bat) in which two RRACH sites were arranged in a palindromic fashion (fig. Id).
  • RRACH canonical m 6 A signature motif
  • d6T (or 6T, as referred to by Woodcock et al) does not truly represent a single- stranded DNA (ssDNA) substrate.
  • d6T* a new DNA oligo in which two nucleotides, a cytidine and a guanine at -2 and -3 positions (outside of the core RRACH motif), were replaced by two thymine nts to disrupt base pairing and palindrome formation without affecting the core RRACH motif required for m 6 dA installation (fig. Id).
  • the d6T* represents a true ssDNA substrate, and when METTL3-METTL14 indeed prefers a ssDNA for methylation, it shows higher activity for d6T* over d6T.
  • methyltransferase activity of METTL3-METTL14 increased 2-fold (compared to d6T, and >12-fold compared to r6T) (fig. le).
  • METTL3-METTL14 showed no activity in the presence of a perfect duplex version of d6T DNA (d6T-ds).
  • mRNAs to IncRNA XIST , NEAT2 or MALATl
  • NEAT2 IncRNA in regulated transcription can relocate a large subset of transcription units across cellular compartments from polycomb bodies (PcGs) to interchromatin granules (ICGs).
  • PcGs polycomb bodies
  • ICGs interchromatin granules
  • RNA As a negative control, we included a noncognate RNA, rTCE23 (also referred to herein as AT-201) which forms a perfect stem-loop structure, but lacks a m 6 A signature motif (fig. Id, S. fig. lb). We observed no methyltransferase activity for AT-201 RNA, as expected: but surprisingly, AT-101 also exhibited negligible methyltransferase activity compared to ssDNA d6T* (fig. le).
  • RNA oligos with varied length (5-nt to 22-nt), and sequence (fig. Id). We also varied the position of the RRACH motif, and the flanking sequences in these oligos. All oligos comprise one RRACH motif except for the Pal-Top+bot; where two RRACH sites were arranged in a palindromic fashion within each strand of a 10-mer RNA duplex.
  • probe 5 ssRNA
  • c2-flip, 22-nt 5-nt shorter version
  • RNA oligos The other three RNA oligos, FoxMl-pl (13-nt with one RRACH at 3’-end), c-Myc-p5 (8-nt), and c-Myc-p6 (11-nt; RRACH at 5’-end) were derived from the FOXM1, and MYC genes because of their involvement in m 6 A-mediated processes in glioblastoma and acute myeloid leukemia.
  • a fluorescein moiety at the 5 ’-end of these oligos to facilitate the quantitative measurement of their binding affinities (equilibrium dissociation constant or Kd) to METTL3-METTL14 (see methods section for details).
  • RNA oligos with high propensity toward forming secondary structures showed negligible methylation by METTL3-METTL14.
  • the other RNA oligos were more likely to remain linear: although they showed some activity, they could not be methylated with as much efficiency as the ssDNA substrate, d6T*.
  • IC 50 216 nM
  • IC 50 887 nM
  • METTL3-METTL14 were to methylate ssDNA (m 6 dA) in vivo, and the evidence to date does not support its direct enzymatic origin, then what could be the significance of tight RNA binding for structured RNA oligos (AT-101 and AT-201)?
  • structural motifs in ncRNA or mRNAs can be recruitment platforms for METTL3-METTL14 in a specific context. But how would the RNA binding activity then affect DNA m 6 dA activity?
  • METTL3-METTL14 we measured METTL3-METTL14’s methyltransferase activity in the presence of an equimolar mixture of d6T* DNA and each RNA oligo.
  • RNAs with propensity to form secondary structures and highest binding affinity to METTL3-METTL14 AT-101, AT-201 attenuated methyltransferase activity to negligible levels (fig. lg).
  • DNA methyltransferase activity (m 6 dA) of METTL3-METTL14 is restricted by shape-specific RNA recognition (fig. 1).
  • RGG motif in METTL3-METTL14 should diminish RNA binding and subsequently compromise the ability of RNA to attenuate DNA methylation. Consistently, we observed 2- 10-fold decrease in RNA binding affinity by METTL3-METTL14 (-RGG) (fig. 2b). Of note, the preferentially binding (albeit 2- 10-fold lower affinity) of the enzyme devoid of RGG to structured RNA oligos indicates that the code for shape specific RNA recognition resides in other parts of the enzyme and the RGG motif strengthens this shape-specific RNA recognition (fig. 2b).
  • RNA shape-dependent recognition of RNA by METTL3- METTL14, and new N 6 -deoxy adenosine methylation (m 6 dA) activity for ssDNA, which is restricted by shape-dependent recognition of structured regions in nc RNAs and chromatin- associated nascent pre-mRNAs (CA-RNA) (fig. 2d).
  • the structured elements in RNA can also facilitate recruitment of METTL3-METTL14 to chromatin and/or keep the writer complex engaged during co-transcriptional occurrence of m 6 A.
  • Such a topologic arrangement also can be important to protect the single- stranded regions of DNA exposed during transcription, DNA recombination, damage and repair, and R-loop metabolism from m 6 dA methylation by METTL3-METTL14. These are all processes in which m 6 A plays important roles and unrestricted installation of m 6 dA on DNA could otherwise be deleterious to genome integrity. b) METHODS
  • the coding sequence of full length human METTL3 (NCBI reference sequence GI: 3330131) was used in this study. The gene was cloned into a plasmid suitable for expression of proteins in insect cells with an N-terminal poly histidine tag followed by a tobacco etch virus (TEV) protease site. This plasmid (5TEY (METTL3) was a kind gift from Dr. Cheryl Arrowsmith.
  • the coding sequence of full length human METTL14 (NCBI reference number GI: 172045930) was cloned into the pFastBacl vector between BamHI and Notl restriction enzyme sites. For expression of recombinant proteins, we used ExpiSf Expression System (Thermo Fisher).
  • the viral DNA bacmids for each gene were prepared from individual plasmids transformed into MAX EfficiencyTM DHlOBacTM competent cells (Thermo Fisher). We confirmed the identity of the inserted genes by PCR amplification and DNA sequencing. The recombinant P0 virus for each gene were generated in ExpiSf9 insect cells following the manufacturer’ s recommendations (Thermo Fisher). The amount of each vims needed for infection has been optimized for maximal production of the two proteins. Infected cells were grown in ExpiSf CD medium (Thermo Fisher) at 27 °C on an orbital shaker (speed 125 rpm) in a controlled (non-humidified, air regulated) environment.
  • Cells were harvested at 72 hours post-infection by spinning at 300 X g for 5 minutes, washed with phosphate buffered saline (PBS) twice, and resuspended in a buffer A (0.025 M Tris-HCl pH 8.0, 0.5 M NaCl, 0.005 M Imidazole, 0.1 mM TCEP, 10% [(v/v)] glycerol) supplemented with 0.5% (v/v) Igepal, 2 mini-protease inhibitor cocktail tablets (Roche), DNase I, and lysozyme.
  • PBS phosphate buffered saline
  • the protein complex was eluted as a single homogenous species in a final buffer containing 0.02 M Tris-HCl pH 8.0, 0.15 M NaCl, 0.1 mM TCEP.
  • the purified protein complex was concentrated to 3-5 mg/mL and used immediately, or flash frozen in liquid nitrogen and stored at -80 °C.
  • the METTL3-METT14 (-RGG) complex lacks the c-terminus RGG repeats motif (amino acids 400 - 457) of METTL14. It was expressed and purified using same method as the full length METTL3-METTL14 complex.
  • RNA and DNA oligonucleotides oligonucleotides used in this study were synthesized, purified by HPLC, and received in deprotected (for RNAs) and desalted form from Dharmacon and Integrated DNA Technologies, respectively. Oligos were dissolved in IX buffer containing 0.01 M Tris-HCl pH 8.0, 0.05 M NaCl. To prepare double- stranded oligos, the two strands were mixed in equimolar amounts and annealing was done by heating the mixture to 95 °C for 2 minutes followed by gradual cooling to 25 °C over 45 minutes in a thermocycler (Eppendorf).
  • RNA/DNA probes were crosslinked to membrane by exposure to ultraviolet light (254 nm) for 2 minutes.
  • Source data are provided as a Source Data file. The results of two groups in fig. 2c were derived from three independent experiments and were analyzed using two- tailed Student’s T-test (unpaired).
  • Each reaction was carried out in a 10 pL mixture containing 50 mM Tris-HCl, pH 7.5, 5 mM NaCl, 1 mM DTT, 200 pM SAM, 10 pM each of substrate RNA/DNA probe, and 1 pM of METTL3-METTL14 enzyme complex.
  • the reactions were incubated at 37°C for 1 hour, followed by incubation with 0.8 units of proteinase K (New England Biolabs) at
  • the buffer corrected values were used to calculate the equilibrium dissociation constant (K d ) for protein-DNA/RNA binding using a simple 1:1 specific binding model.
  • Source data are provided as a Source Data file.
  • Vu, L. P., Pickering B. F., Cheng, Y., Zaccara, S., Nguyen, D., Minuesa, G., Chou, T., Chow, A., Saletore, Y., MacKay, M., Schulman, J., Famulare, C., Patel, M., Klimek, V. M., Garrett-Bakelman, F. E., Melnick, A., Carroll, M., Mason, C. E., Jaffrey, S. R., and Kharas, M. G.
  • the N6-methyladenosine (m6A)-forming enzyme METTL3 controls myeloid differentiation of normal hematopoietic and leukemia cells, Nat Med. Vu, L.P. et al.
  • the N6-methyladenosine (m6A)-forming enzyme METTL3 controls myeloid differentiation of normal hematopoietic and leukemia cells. Nat Med (2017).
  • Woodcock, C.B. et al. Human MettL3-MettL14 complex is a sequence- specific DNA adenine methyltransferase active on single-strand and unpaired DNA in vitro. Cell Discov 5, 63 (2019).
  • RNA m6A methylation regulates the ultraviolet-induced DNA damage response. Nature 543, 573-576 (2017). Yang, L. et al. ncRNA- and Pc2 methylation-dependent gene relocation between nuclear structures mediates gene activation programs. Cell 147, 773-88 (2011). Zhang, C. et al. METTL3 and N6-Methyladenosine Promote Homologous Recombination- Mediated Repair of DSBs by Modulating DNA-RNA Hybrid Accumulation. Mol Cell 79, 425-442 e7 (2020).
  • SEQ ID NO: 3 nucleic acid sequence for Full length human rNEAT2 RNA from where the rNEAT2 oligo (AT-101) was derived.
  • SEQ ID NO: 4 nucleic acid sequence for AT-101 (or rNEAT2): nucleotide 4927-4956
  • SEQ ID NO: 5 Full length sequence of drosophila melanogaster rTCE23 RNA from where the rTCE23 oligo (AT-201) was derived. The highlighted sequence represents AT-201.
  • SEQ ID NO: 6 nucleic acid sequence for AT-201 (rTCE23):

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Abstract

L'invention concerne des iROS qui se lient à METTL3 ou METTL14 et leurs procédés d'utilisation pour le traitement du cancer.
PCT/US2022/017419 2021-02-23 2022-02-23 Nouvelle approche à base d'arn pour le traitement du cancer WO2022182697A1 (fr)

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