WO2023010070A1 - Procédés et matériaux pour réduire le niveau d'un rna non codant long d'un facteur de transcription activant les lymphocytes b dans les cellules - Google Patents

Procédés et matériaux pour réduire le niveau d'un rna non codant long d'un facteur de transcription activant les lymphocytes b dans les cellules Download PDF

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WO2023010070A1
WO2023010070A1 PCT/US2022/074247 US2022074247W WO2023010070A1 WO 2023010070 A1 WO2023010070 A1 WO 2023010070A1 US 2022074247 W US2022074247 W US 2022074247W WO 2023010070 A1 WO2023010070 A1 WO 2023010070A1
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antisense oligonucleotide
lncbatf
complementary
nucleic acid
sequence
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WO2023010070A8 (fr
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William A. Faubion
Hunter R. GIBBONS
Manuel Bonfim BRAGA NETO
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Mayo Foundation For Medical Education And Research
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    • 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
    • A61K31/712—Nucleic acids or oligonucleotides having modified sugars, i.e. other than ribose or 2'-deoxyribose
    • 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
    • A61K31/7125—Nucleic acids or oligonucleotides having modified internucleoside linkage, i.e. other than 3'-5' phosphodiesters
    • 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
    • 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
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/31—Chemical structure of the backbone
    • C12N2310/315—Phosphorothioates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/32—Chemical structure of the sugar
    • C12N2310/323—Chemical structure of the sugar modified ring structure
    • C12N2310/3231—Chemical structure of the sugar modified ring structure having an additional ring, e.g. LNA, ENA

Definitions

  • This document relates to methods and materials for reducing the level of a long noncoding RNA (LncRNA) of a B cell activating transcription factor (BATF) in cells, including immune cells such as T cells (e.g., CD4 T cells and T-helper 17 (Thl7) cells).
  • LncRNA long noncoding RNA
  • BATF B cell activating transcription factor
  • an antisense oligonucleotide wherein at least a portion of the nucleotide sequence of the antisense oligonucleotide is complementary to the DNA sequence encoding the LncRNA of a BATF or complementary to the RNA sequence of the LncRNA of a BATF, can be used to reduce the level of that LncRNA in a mammal and in some embodiments, can be used to treat an Thl7 inflammatory based disease (e.g., inflammatory bowel disease (IBD), multiple sclerosis, rheumatoid arthritis, and/or cancer).
  • IBD inflammatory bowel disease
  • multiple sclerosis multiple sclerosis
  • rheumatoid arthritis rheumatoid arthritis
  • Thl7 cells are a subset of CD4 T helper cells that produce interleukin- 17 (IL-17) and other inflammatory cytokines such as tumor necrosis factor alpha (TNFa), IL-22, and IL-26. Thl7 cells have been implicated in the pathogenesis of rheumatic diseases, non rheumatic autoimmunity, asthma, and other immune-mediated diseases such as IBD and periodontal disease. See, e.g., Tesmer, et al., Immunol. Rev., 223:87-113 (2008). There is a need for therapies targeting Thl7 cells.
  • IL-17 interleukin- 17
  • TNFa tumor necrosis factor alpha
  • LncBATF LncBATF
  • a LncBATF LncBATF or a LncRNA of BATF.
  • LncBATF was identified in CD4 T cells from patients with Crohn’s disease and patients with ulcerative colitis. T cells from Crohn’s disease patients showed a significantly higher expression of LncBATF compared to healthy controls. As described herein, LncBATF expression in Thl7 cells peaks at day 4 of the seven-day long polarization for effector T cells.
  • Thl7 cells can be targeted by reducing expression of LncBATF, using for example, antisense oligonucleotides, RNA interference, a ribozyme, or other nucleic acid-based method of reducing expression of the non-coding RNA.
  • expression of LncBATF can be reduced, for example, by using an antisense oligonucleotide in which at least a portion of the nucleotide sequence of the antisense oligonucleotide is complementary to the DNA sequence encoding the LncBATF or is complementary to the RNA sequence of the LncBATF.
  • this document features an antisense oligonucleotide for reducing the level of a LncBATF within a cell and a pharmaceutical composition comprising the antisense oligonucleotide.
  • the antisense oligonucleotide can be from about 12 to about 50 nucleotides in length (e.g., about 15 to about 30 nucleotides in length), wherein the antisense oligonucleotide comprises a nucleic acid analog, wherein at least a portion of the nucleotide sequence of the antisense oligonucleotide is (i) complementary to the DNA sequence encoding the LncBATF or (ii) complementary to the RNA sequence of the LncBATF, and wherein the antisense oligonucleotide comprises the ability to reduce the level of the LncBATF in the cell.
  • the portion of the nucleotide sequence of the antisense oligonucleotide is complementary to the DNA sequence encoding the LncBATF. In some cases, the portion of the nucleotide sequence of the antisense oligonucleotide is complementary to the RNA sequence of the LncBATF. In some cases, at least 4, at least 6, or at least 7 of the nucleotides of the antisense oligonucleotide are nucleic acid analogs.
  • the nucleic acid analog comprises one or more of a bridged nucleotide (e.g., a 4’ CH2-O-2’ bridge or an ethylene bridge between the 2’-oxygen and the 4’ carbon of the pentose sugar), a phosphorothioate linkage, or a modification at the 2’ position of the pentose sugar (e.g., a 2’-0-methyl, a 2’-0- methoxy ethyl (MOE), or 2-fluoro modification).
  • a bridged nucleotide e.g., a 4’ CH2-O-2’ bridge or an ethylene bridge between the 2’-oxygen and the 4’ carbon of the pentose sugar
  • a phosphorothioate linkage e.g., a modification at the 2’ position of the pentose sugar (e.g., a 2’-0-methyl, a 2’-0- methoxy ethyl (MOE), or 2-fluoro modification).
  • the oligonucleotide comprises the sequence GCTT GCC AGGCC AT ACGCTT (SEQ ID NO:2), GGAGCCAGTTGCCATGTCCAT (SEQ ID NO:3), or GGCCACACCACAGACTCCCA (SEQ ID NO:4).
  • the antisense oligonucleotide can have the sequence GGAGCCAGTTGCCATGTCCAT (SEQ ID NO:3).
  • This document also features a method of treating a Thl7 inflammatory based disease in a mammal (e.g., a human).
  • the method can include administering an antisense oligonucleotide described herein to a mammal identified as having the Thl7 inflammatory based disease (e.g., inflammatory bowel disease (IBD) such as Crohn’s disease or ulcerative colitis, multiple sclerosis, rheumatoid arthritis, psoriasis, periodontal disease, or cancer).
  • IBD inflammatory bowel disease
  • the antisense oligonucleotide administered to the mammal can be from about 12 to about 50 nucleotides in length (e.g., about 15 to about 30 nucleotides in length), wherein the antisense oligonucleotide comprises a nucleic acid analog, wherein at least a portion of the nucleotide sequence of the antisense oligonucleotide is (i) complementary to the DNA sequence encoding the LncBATF or (ii) complementary to the RNA sequence of the LncBATF, and wherein the antisense oligonucleotide comprises the ability to reduce the level of the LncBATF in the mammal.
  • the portion of the nucleotide sequence of the antisense oligonucleotide is complementary to the DNA sequence encoding the LncBATF. In some cases, the portion of the nucleotide sequence of the antisense oligonucleotide is complementary to the RNA sequence of the LncBATF. In some cases, at least 4, at least 6, or at least 7 of the nucleotides of the antisense oligonucleotide are nucleic acid analogs.
  • the nucleic acid analog comprises one or more of a bridged nucleotide (e.g., a 4’ CH 2 -0-2’ bridge or an ethylene bridge between the 2’-oxygen and the 4’ carbon of the pentose sugar), a phosphorothioate linkage, or a modification at the T position of the pentose sugar (e.g., a 2’-0-methyl, a 2’-0-methoxyethyl (MOE), or 2- fluoro modification).
  • a bridged nucleotide e.g., a 4’ CH 2 -0-2’ bridge or an ethylene bridge between the 2’-oxygen and the 4’ carbon of the pentose sugar
  • a phosphorothioate linkage e.g., a modification at the T position of the pentose sugar (e.g., a 2’-0-methyl, a 2’-0-methoxyethyl (MOE), or 2- fluoro modification).
  • the oligonucleotide comprises the sequence GCTTGCCAGGCCATACGCTT (SEQ ID NO:2), GGAGCCAGTTGCCATGTCCAT (SEQ ID NO:3), or GGCCACACCACAGACTCCCA (SEQ ID NO:4).
  • the antisense oligonucleotide can have the sequence GGAGCCAGTTGCCATGTCCAT (SEQ ID NO:3).
  • FIG. 1 A is the sequence of a human LncBATF (SEQ ID NO: 1).
  • the targets of antisense oligonucleotide (ASO) 1, ASO 2, and ASO 3 are shown in bold, underlined text, with the target of ASO 1 being the first region of highlighted text, the target of ASO 2 being the second region of highlighted text, and the target of ASO 3 being the third region of highlighted text.
  • FIG. IB is the genomic sequence from a region of chromosome 14 (75515241-75519816) (SEQ ID NO: 5) that encodes a human LncBATF.
  • FIG. 2 is a graph showing a significant reduction of LncBATF after treatment with 5 mM of antisense oligonucleotide (ASO) 1 (SEQ ID NO:2), ASO 2 (SEQ ID NO:3), or ASO 3 (SEQ ID NO:4).
  • ASO antisense oligonucleotide
  • FIG. 3 contains two graphs showing a significant reduction of RUNX1 RNA (left panel) and RORC RNA (right panel) after treatment with 5 mM of ASO 1 (SEQ ID NO:2), ASO 2 (SEQ ID NO:3), or ASO 3 (SEQ ID NO:4).
  • FIG. 4 contains three plots of the variation in total genes between the different treatment groups (scramble vs combination treatment on the left, scramble vs ASO 2 in the center, and ASO 2 vs combination on the right).
  • a black dot indicates a gene with a variable expression
  • a grey dot indicates a gene that is expressed, but not significantly differentially expressed between the two treatment groups. There were no significantly different genes between ASO 2 and the combination treatment.
  • FIG. 5 is a heat map representing the 500 genes with the biggest differences in expression, including genes that are increased and genes that are decreased following ASO treatment.
  • FIG. 6 is a graph of the level of LncBATF RNA throughout the seven-day long polarization for effector T cells.
  • Thl7 cells are pro-inflammatory, disease inducing cells implicated in the pathogenesis of rheumatic diseases such as psoriasis and rheumatoid arthritis, non rheumatic autoimmunity such as multiple sclerosis, asthma and other allergic diseases such as atopic dermatitis and contact hypersensitivity, and other immune-mediated diseases such as IBD (e.g., Crohn’s disease and ulcerative colitis) and periodontal disease.
  • Thl7 cells have a role in various cancers as the Thl7 cells can promote malignant transformation, angiogenesis, tumor growth, and metastasis based on their pro-inflammatory activities (e.g., cytokine production and/or recruitment of inflammatory cells).
  • Thl7 cells can be targeted by reducing the level of LncBATF present within the cells using, for example, nucleic acid-based methods such as antisense oligonucleotides, RN A interference, CRISPR/Cas (e.g., CRISPR-Cas9, CRISPR- Casl3a), or ribozymes.
  • nucleic acid-based methods such as antisense oligonucleotides, RN A interference, CRISPR/Cas (e.g., CRISPR-Cas9, CRISPR- Casl3a), or ribozymes.
  • the level of LncBATF can be reduced using an antisense oligonucleotide in which at least a portion of the nucleotide sequence of the antisense oligonucleotide is complementary to the DNA encoding the LncBATF (and/or the LncBATF itself) and can bind to that DNA (or the LncBATF) and reduce the level of LncBATF present in the cell.
  • Antisense oligonucleotides typically are at least 8 nucleotides in length (e.g., about 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 10 to 50, 12 to 50, 15 to 20, 18 to 30, 18 to 25, or 20 to 50 nucleotides in length).
  • an antisense oligonucleotide greater than 50 nucleotides in length can be used, including up to the full-length of the LncBATF.
  • An “oligonucleotide” is an oligomer or polymer of ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) or analogs thereof. Nucleic acid analogs can be modified at one or more of the base moiety, sugar moiety, and phosphate backbone to improve, for example, stability, hybridization, or solubility of a nucleic acid.
  • the phosphodiester backbone of an antisense oligonucleotide described herein can be replaced with one or more phosphorothioate, methoylphosphonate, or phosphoroamidate linkages.
  • an antisense oligonucleotide described herein can include one or more nucleotides modified at the 2’ position of the pentose sugar such as 2’-0- methyl, 2’-0-methoxyethyl (MOE), or 2-fluoro compounds.
  • nucleotides modified at the 2’ position of the pentose sugar such as 2’-0- methyl, 2’-0-methoxyethyl (MOE), or 2-fluoro compounds.
  • an antisense oligonucleotide described herein can include 2’ -O-methyl or MOE nucleotides with a phosphodiester backbone or a phosphorothioate backbone, or an antisense oligonucleotide can include 2’-0-methyl or MOE nucleotides with a mix of phosphodiester and phosphorothioate linkages. See, e.g., Scoles, et al. Neurol Genet., 5:e323 (2019).
  • an antisense oligonucleotide described herein can include one or more nucleotides modified to replace the sugar moiety with methylenemorpholine rings and replace the phosophodiester linkage with non-ionic phosphordiamidate linkages (phosphorodiamidate morpholino). See, e.g., Scoles, et al. 2019, supra.
  • an antisense oligonucleotide described herein can contain one or more bridged nucleotides.
  • an antisense oligonucleotide described herein can include one or more methylene bridge connections between the 2’-oxygen and the 4’ carbon of a pentose sugar (4’ CH 2 -0-2’ bridge).
  • An antisense oligonucleotide containing one or more methylene bridges can be referred to as a locked nucleic acid (LNA).
  • LNA locked nucleic acid
  • a LNA also can include one or more MOE modifications.
  • a LNA described herein contains a phosphodiester backbone.
  • a LNA described herein contains a phosphorothioate backbone. In some embodiments, a LNA described herein contains a mixture of a phosphodiester and phosphorothioate linkages. See, e.g., Soler-Bistue, et al, Molecules, 24(12):2297 (2019).
  • an antisense oligonucleotide described herein can include one more ethylene bridge connections between the 2’ -oxygen and the 4’ carbon of the pentose sugar or one or more aminoethylene bridged nucleic acids between the 2’-oxygen and the 4’ carbon of the pentose sugar.
  • An antisense oligonucleotide described herein containing one or more ethylene bridges can be referred to as an ethylene bridged nucleic acid (ENA). See, e.g., Soler-Bistue, et al, 2019, supra.
  • any appropriate method can be used to make an antisense oligonucleotide described herein (e.g., an ASO, a LNA, and/or ENA).
  • methods for synthesizing antisense oligonucleotides include solid phase synthesis techniques. Equipment for such synthesis is commercially available from several vendors including, for example, Applied Biosystems (Foster City, CA). See, e.g., International PCT Patent Application Publication No. W09914226 for synthesis of LNA.
  • expression vectors that contain a regulatory element that directs production of an antisense oligonucleotide can be used to produce an antisense oligonucleotide.
  • Antisense oligonucleotides described herein can bind to the DNA encoding a LncBATF and/or to the LncBATF itself that was transcribed from such DNA, under physiological conditions (i.e., physiological pH and ionic strength).
  • physiological conditions i.e., physiological pH and ionic strength
  • the sequence of an antisense oligonucleotide need not be 100% complementary to that of its target nucleic acid in order to hybridize under physiological conditions.
  • an antisense oligonucleotide can hybridize under physiological conditions to DNA encoding an LncBATF and/or to the LncBATF itself in a manner that reduces the level of the LncBATF present within a cell, with non-specific binding to non-target sequences being minimal.
  • an antisense oligonucleotide described herein includes a contiguous sequence of between 12 and 50 nucleotides that is at least 85% complementary (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% complementary) with a region of the LncBATF and/or of the DNA encoding the LncBATF.
  • a representative human LncBATF sequence is set forth in FIG. 1 A (SEQ ID NO:l).
  • the genomic sequence from chromosome 14 (75515241-75519816) is set forth in FIG. IB (SEQ ID NO:5).
  • Target sites for LncBATF antisense oligonucleotides can include, for example, an exon, a splice site, regions lacking secondary structure, and regions left unbound by proteins or other nucleic acids. Further criteria that can be applied to the design of antisense oligonucleotides include, for example, the lack of predicted secondary structure of a potential antisense oligonucleotide, an appropriate G and C nucleotide content (e.g., about 50%), and the absence of sequence motifs such as single nucleotide repeats (e.g., GGGG runs).
  • the effectiveness of antisense oligonucleotides at reducing the level of LncBATF within cells can be evaluated by measuring levels of the LncBATF or a downstream target (e.g., a transcription factor such as RUNX or RORC) via RNA sequencing or Northern blotting, or by detecting RUNX or RORC polypeptides.
  • a downstream target e.g., a transcription factor such as RUNX or RORC
  • an antisense oligonucleotide described herein can include one of the following sequences: GCTTGCCAGGCCATACGCTT (SEQ ID NO:2), GGAGCCAGTTGCCATGTCCAT (SEQ ID NO:3), and GGCCACACCACAGACTCCCA (SEQ ID NO:4).
  • the targets of each of SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4 in the LncBATF are shown in FIG. 1 A.
  • an antisense oligonucleotide can be designed to include a sequence that targets a region containing the second exon of LncBATF and is in proximity to a splice site, that includes GCCA, that has a 50 ⁇ 10% (e.g., 50 ⁇ 9%, 50 ⁇ 8%, 50 ⁇ 7%, 50 ⁇ 6%, 50 ⁇ 5%, 50 ⁇ 4%, 50 ⁇ 3%, 50 ⁇ 2%, or 50 ⁇ 1%) GC content, and/or lacks four contiguous identical bases.
  • SEQ ID NO: 3 An example of such a sequence is set forth in SEQ ID NO: 3 as it targets a region that contains the second exon of LncBATF, and is in proximity to a splice site, it includes a GCCA sequence, it has near 50% GC content, and it lacks four contiguous identical bases.
  • antisense oligonucleotides can contain one or more nucleic acid analogs (e.g., LNA and/or phosphorothioate linkages).
  • the antisense oligonucleotide can be a mixmer, in which the LNA residues and other residues are interspersed in different configurations throughout the sequence. See, e.g. Soler- Bistue, etal.
  • the antisense oligonucleotide can be a gapmer, in which the LNA residues are on the ends of the oligonucleotide. See, e.g., Soler-Bistue, etal 2019, supra.
  • an antisense oligonucleotide provided herein can include SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4 with the first one to four outermost bases being LNA (denoted by the + in the below sequence) and the backbone can have phosphorothioate linkages (denoted by the * in the below sequence).
  • an antisense oligonucleotide provided herein can have the following sequence and modifications to the sugar and phosphate backbone: G+C*+T*+T*G*C*C*A*G*G*C*C*A*T*A*C*G*+C*+T*+T, G+G*+A*+G*C*C*A*G*T*T*G*C*C*A*T*G*T*+C*+C*+A*+T, or G+G*+C*+C*A*C*A*C*C*A*C*A*G*A*C*T*C*+C*+A.
  • an antisense oligonucleotide described herein can be conjugated to a sugar such as N-acetylgalactosamine, a cell surface receptor ligand, a drug, a hormone, a lipid, a polymer (e.g., a cationic polymer), a polypeptide, a toxin, a vitamin, or a viral polypeptide.
  • a sugar such as N-acetylgalactosamine, a cell surface receptor ligand, a drug, a hormone, a lipid, a polymer (e.g., a cationic polymer), a polypeptide, a toxin, a vitamin, or a viral polypeptide.
  • an antisense oligonucleotide described herein can be formulated as a pharmaceutical composition for administration to a mammal (e.g. a human).
  • a pharmaceutical composition provided herein can include a pharmaceutically acceptable carrier such as a buffer, a salt, a surfactant, a sugar, a tonicity modifier, or combinations thereof.
  • an antisense oligonucleotide described herein can be conjugated to a lipid (e.g., cholesterol), polypeptides (e.g., cell-penetrating polypeptides), aptamers, antibodies, sugars (e.g., N- acetylgalactosamine), cationic polymers (e.g., polyethyleneimine), exosomes, or nanoparticles to improve, for example, intracellular uptake, reduce clearance, or target particular tissues as, for example, described in Roberts etal. , Nat. Reviews Drug Discovery , 19:673-694 (2020)
  • lipid e.g., cholesterol
  • polypeptides e.g., cell-penetrating polypeptides
  • aptamers e.g., antibodies, sugars (e.g., N- acetylgalactosamine), cationic polymers (e.g., polyethyleneimine), exosomes, or nanoparticles to improve, for example
  • a pharmaceutical composition provided herein can be formulated to be a liquid that includes from about 1 mg to about 500 mg (e.g., from about 1 mg to about 500 mg, from about 10 mg to about 500 mg, from about 50 mg to about 500 mg, from about 100 mg to about 500 mg, from about 0.5 mg to about 250 mg, from about 0.5 mg to about 150 mg, from about 0.5 mg to about 100 mg, from about 0.5 mg to about 50 mg, from about 1 mg to about 300 mg, from about 2 mg to about 200 mg, from about 10 mg to about 300 mg, from about 25 mg to about 300 mg, from about 50 mg to about 150 mg, or from about 150 mg to about 300 mg) of an antisense oligonucleotide described herein per mL.
  • a pharmaceutical composition provided herein can be formulated to be a liquid that includes from about 1 mg to about 500 mg (e.g., from about 1 mg to about 500 mg, from about 10 mg to about 500 mg, from about 50 mg to about 500 mg, from about 100 mg to about 500 mg,
  • a pharmaceutical composition provided herein can be formulated to be a solid or semi-solid that includes from about 0.5 mg to about 500 mg (e.g., from about 1 mg to about 500 mg, from about 10 mg to about 500 mg, from about 50 mg to about 500 mg, from about 100 mg to about 500 mg, from about 0.5 mg to about 250 mg, from about 0.5 mg to about 150 mg, from about 0.5 mg to about 100 mg, from about 0.5 mg to about 50 mg, from about 1 mg to about 300 mg, from about 10 mg to about 300 mg, from about 25 mg to about 300 mg, from about 50 mg to about 150 mg, or from about 150 mg to about 300 mg) of an antisense oligonucleotide described herein.
  • 0.5 mg to about 500 mg e.g., from about 1 mg to about 500 mg, from about 10 mg to about 500 mg, from about 50 mg to about 500 mg, from about 100 mg to about 500 mg, from about 0.5 mg to about 250 mg, from about 0.5 mg to about 150 mg, from about 0.5 mg to about
  • a pharmaceutical composition provided herein can be in any appropriate form.
  • a pharmaceutical composition provided herein can designed to be a liquid, a semi-solid, or a solid.
  • a pharmaceutical composition provided herein can be a liquid solution (e.g., an injectable and/or infusible solution), a dispersion, a suspension, a tablet, a pill, a powder, a microemulsion, a liposome, or a suppository.
  • a pharmaceutical composition provided herein can be lyophilized.
  • a pharmaceutical composition provided herein e.g., a pharmaceutical composition that includes one or more antisense oligonucleotides described herein can be formulated with a carrier or coating designed to protect against rapid release.
  • a pharmaceutical composition described herein can be formulated as a controlled release formulation or as a regulated release formulation as described elsewhere (U.S. Patent Application Publication Nos. 2019/0241667; 2019/0233522; and 2019/0233498).
  • compositions e.g., a pharmaceutical composition described herein
  • a composition e.g., a pharmaceutical composition described herein
  • a composition containing one or more antisense oligonucleotides can be administered to a mammal (e.g., a human) identified as having a Thl7 inflammatory based disease to treat that mammal.
  • Thl7 inflammatory based disease can be treated using a composition (e.g., a pharmaceutical composition provided herein) containing one or more antisense oligonucleotides described herein.
  • a mammal e.g., a human
  • a composition e.g., a pharmaceutical composition
  • a composition e.g., a pharmaceutical composition
  • Thl7 inflammatory based diseases examples include, without limitation, inflammatory bowel disease (IBD) such as Crohn’s disease or ulcerative colitis, multiple sclerosis, rheumatoid arthritis, psoriasis, periodontal disease, or cancer (e.g., ovarian cancer, pancreatic cancer, bladder cancer, colon cancer, prostate cancer, or lymphoma).
  • IBD inflammatory bowel disease
  • cancer e.g., ovarian cancer, pancreatic cancer, bladder cancer, colon cancer, prostate cancer, or lymphoma.
  • a mammal having a Thl7 inflammatory based disease can be administered a composition (e.g., a pharmaceutical composition) containing one or more antisense oligonucleotides described herein to treat that mammal (e.g., to reduce one or more symptoms of the Thl7 inflammatory based disease such as reduced inflammatory immune response, reduced cytokine production, reduced tumor size, decreased gastrointestinal symptoms such as diarrhea or abdominal pain, or decreased joint pain).
  • a composition e.g., a pharmaceutical composition
  • one or more antisense oligonucleotides described herein to treat that mammal (e.g., to reduce one or more symptoms of the Thl7 inflammatory based disease such as reduced inflammatory immune response, reduced cytokine production, reduced tumor size, decreased gastrointestinal symptoms such as diarrhea or abdominal pain, or decreased joint pain).
  • compositions e.g., a pharmaceutical composition described herein
  • a composition e.g., a pharmaceutical composition described herein
  • a composition containing one or more antisense oligonucleotides described herein
  • a mammal e.g., a human
  • a composition e.g., a pharmaceutical composition provided herein
  • a composition containing one or more antisense oligonucleotides described herein can be administered to a mammal (e.g. a human) to reduce the inflammatory immune response within the mammal and/or to decrease other symptoms of the particular Thl7 inflammatory based disease such as joint pain or gastrointestinal distress.
  • a composition provided herein e.g., a pharmaceutical composition containing one or more antisense oligonucleotides described herein
  • a mammal e.g., a human
  • intravenously e.g., via an intravenous injection or infusion
  • subcutaneously e.g., via a subcutaneous injection
  • intraperitoneally e.g., via an intraperitoneal injection
  • intramuscularly e.g., via intramuscular injection.
  • the route and/or mode of administration of a composition can be adjusted for the mammal being treated.
  • an effective amount of a composition containing one or more antisense oligonucleotides described herein can be an amount that reduces the symptoms associated with a Thl7 inflammatory based disease within a mammal without producing significant toxicity to the mammal.
  • an effective amount of an antisense oligonucleotide described herein can be from about 0.001 mg/kg to about 100 mg/kg (e.g., from about 0.001 mg/kg to about 90 mg/kg, from about 0.001 mg/kg to about 80 mg/kg, from about 0.001 mg/kg to about 70 mg/kg, from about 0.001 mg/kg to about 60 mg/kg, from about 0.001 mg/kg to about 50 mg/kg, from about 0.001 mg/kg to about 40 mg/kg, from about 0.001 mg/kg to about 30 mg/kg, from about 0.005 mg/kg to about 100 mg/kg, from about 0.01 mg/kg to about 100 mg/kg, from about 0.05 mg/kg to about 100 mg/kg, from about 0.1 mg/kg to about 100 mg/kg, from about 0.5 mg/kg to about 100 mg/kg, from about 1 mg/kg to about 100 mg/kg, from about 5 mg/kg to about 100 mg/kg, from about 0.01 mg/kg to about
  • the effective amount can remain constant or can be adjusted as a sliding scale or variable dose depending on the mammal’s response to treatment.
  • Various factors can influence the actual effective amount used for a particular application. For example, the severity of the Thl7 inflammatory based disease when treating a mammal having such a disease, the route of administration, the age and general health condition of the mammal, excipient usage, the possibility of co-usage with other therapeutic or prophylactic treatments such as use of other agents (e.g., anti-inflammatory drugs), and the judgment of the treating physician may require an increase or decrease in the actual effective amount of a composition provided herein (e.g., a pharmaceutical composition containing one or more antisense oligonucleotides described herein) that is administered.
  • a composition provided herein e.g., a pharmaceutical composition containing one or more antisense oligonucleotides described herein
  • an effective frequency of administration of a composition containing one or more antisense oligonucleotides described herein can be a frequency that reduces the symptoms associated with a Thl7 inflammatory based disease without producing significant toxicity to the mammal.
  • an effective frequency of administration of a composition containing one or more antisense oligonucleotides described herein can be a frequency that reduces the symptoms associated with a Thl7 inflammatory based disease of a mammal as compared to a control mammal having comparable Thl7 inflammatory based disease and not treated with the composition.
  • an effective frequency of administration of a pharmaceutical composition described herein can be from about twice daily to about three times a year (e.g., from about every other day to about once a month, from about once daily to about once a month, from about twice daily to about once a week, or from once daily to about once a week).
  • the frequency of administration of a pharmaceutical composition described herein such as a pharmaceutical composition containing one or more antisense oligonucleotides described herein can remain constant or can be variable during the duration of treatment. Various factors can influence the actual effective frequency used for a particular application.
  • the severity of the Thl7 inflammatory based disease when treating a mammal having such a disease may require an increase or decrease in the actual effective frequency of administration of a composition provided herein (e.g., a pharmaceutical composition containing one or more antisense oligonucleotides described herein).
  • a composition provided herein e.g., a pharmaceutical composition containing one or more antisense oligonucleotides described herein.
  • an effective duration of administration of a composition containing one or more antisense oligonucleotides described herein can be a duration that reduces the symptoms associated with a Thl7 inflammatory based disease of a mammal as compared to a control mammal having comparable Thl7 inflammatory based disease and not treated with the composition.
  • an effective duration of administration of a composition containing one or more antisense oligonucleotides described herein can be a duration that reduces the symptoms associated with a Thl7 inflammatory based disease of a mammal having such a disease as compared to a control mammal having comparable Thl7 inflammatory based disease and not treated with the composition.
  • an effective duration of administration of a pharmaceutical composition provided herein such as a pharmaceutical composition containing one or more antisense oligonucleotides described herein can vary from a single time point of administration to several weeks to several months (e.g., 4 to 12 weeks). Multiple factors can influence the actual effective duration used for a particular application.
  • the severity of the Thl7 inflammatory based disease, the route of administration, the age and general health condition of the mammal, excipient usage, the possibility of co-usage with other therapeutic or prophylactic treatments such as use of other agents (e.g., anti-inflammatory agents), and the judgment of the treating physician may require an increase or decrease in the actual effective duration of administration of a composition provided herein (e.g., a pharmaceutical composition containing one or more antisense oligonucleotides described herein).
  • LncBATF a long noncoding RNA, is transcribed from a region upstream of the gene encoding BATF.
  • the sequence of the LncBATF is shown in FIG. 1 A.
  • LncBATF was initially identified in RNA sequencing data from CD4 + T cells isolated from the lamina intestinal of Crohn’s disease and ulcerative colitis patients. T cells from Crohn’s patients showed a significantly higher level of LncBATF as compared to healthy controls. Thl7 cells expressed the highest level of LncBATF when compared to other T cell subsets.
  • ASO 3 G+G*+C*+C*A*C*A*C*C*A*G*A*C*T*C*+C*+A Naive T cells from a healthy donor were plated according to a standard Thl7 polarization protocol.
  • ASOs were transfected using LIPOFECTAMINE® RNAiMax (Thermofisher) on day 3 of T cell polarization, and RNA was isolated on day 4.
  • LIPOFECTAMINE® RNAiMax Thermofisher
  • RNA sequencing of these knockdown samples was completed, including the single treatment of ASO 2 and the combination treatment including all three ASOs.
  • a scramble control was compared to the two different treatments.
  • the plots in FIG. 4 show the variation in total genes between the different treatment groups.
  • a red dot indicates a gene with a variable expression, while a grey dot indicates a gene that is expressed, but not significantly differentially expressed between the two treatment groups. There were no significantly different genes between the ASO 2 and combination treatment groups.
  • FIG. 5 contains a heat map representing the 500 genes with the biggest differences in expression, including genes that were increased and genes that were decreased following ASO treatment. There was a stark separation between the ASO treated samples and the scramble controls.
  • RNA sequencing data also was used to understand which pathways were most disrupted within the treated cells (single treatment with AS02 and the combination of all three ASOs). Based on IPA canonical pathway analysis, the standard T cell developmental pathways were most inhibited within the Thl7 cells after ASO treatment as compared with the scramble control. There was no significant transcriptional difference between the two treatments.
  • Example 2 Time dependent changes in LncBATF expression in Thl7 cells Naive CD4+ T cells were isolated from total peripheral blood mononuclear cells
  • PBMCs peripheral blood mononuclear cells
  • RNA was extracted using the Qiagen MIRNAEASY® kit according to manufacturer’s instructions. RNA was then converted to cDNA using the GOSCRIPTTM Reverse Transcriptase system (Promega) according to manufacturer’s instructions. Converted cDNAthen was evaluated using quantitative PCR using LncBATF specific primers (these were the same primers used in every evaluation of the lncRNA). LncBATF transcript levels were identified relative to the housekeeping gene HPRT, and then evaluated according to the baseline expression of LncBATF in a nonpolarized ThO cell (FIG. 6). LncBATF RNA levels peaked at day 4.

Abstract

La présente invention concerne des procédés et des matériaux permettant de réduire le taux d'un ARN non codant long d'un facteur de transcription activant les lymphocytes B (LncBATF) dans une cellule.
PCT/US2022/074247 2021-07-28 2022-07-28 Procédés et matériaux pour réduire le niveau d'un rna non codant long d'un facteur de transcription activant les lymphocytes b dans les cellules WO2023010070A1 (fr)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20160115218A1 (en) * 2010-04-30 2016-04-28 Medical Diagnostic Laboratories, Llc Anti-Sense Oligonucleotides Targeted Against Exon 9 of IL-23R-alpha Gene and Method of Using Same to Induce Exon Skipping and to Treat Inflammatory Bowel Diseases
US20170016004A1 (en) * 2015-05-29 2017-01-19 Dan R. Littman DDX5 AND ASSOCIATED NON-CODING RNAs AND MODULATION OF TH17 EFFECTOR FUNCTION
WO2017211999A1 (fr) * 2016-06-08 2017-12-14 Aalborg Universitet Oligonucléotides antisens pour la modulation de longs arn non codants

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US20160115218A1 (en) * 2010-04-30 2016-04-28 Medical Diagnostic Laboratories, Llc Anti-Sense Oligonucleotides Targeted Against Exon 9 of IL-23R-alpha Gene and Method of Using Same to Induce Exon Skipping and to Treat Inflammatory Bowel Diseases
US20170016004A1 (en) * 2015-05-29 2017-01-19 Dan R. Littman DDX5 AND ASSOCIATED NON-CODING RNAs AND MODULATION OF TH17 EFFECTOR FUNCTION
WO2017211999A1 (fr) * 2016-06-08 2017-12-14 Aalborg Universitet Oligonucléotides antisens pour la modulation de longs arn non codants

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BRAGA-NETO MANUEL B, GABALLA JOSEPH M, BAMIDELE ADEBOWALE O, SARMENTO OLGA F, SVINGEN PHYLLIS, GONZALEZ MICHELLE, RAMOS GUILHERME : "Deregulation of Long Intergenic Non-coding RNAs in CD4+ T Cells of Lamina Propria in Crohn’s Disease Through Transcriptome Profiling", JOURNAL OF CROHN'S AND COLITIS, ELSEVIER BV, NL, vol. 14, no. 1, 1 January 2020 (2020-01-01), NL , pages 96 - 109, XP093031120, ISSN: 1873-9946, DOI: 10.1093/ecco-jcc/jjz109 *

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