WO2022109030A1 - Rna-targeting splicing modifiers for treatment of pnpla3-associated conditions and diseases - Google Patents

Rna-targeting splicing modifiers for treatment of pnpla3-associated conditions and diseases Download PDF

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WO2022109030A1
WO2022109030A1 PCT/US2021/059730 US2021059730W WO2022109030A1 WO 2022109030 A1 WO2022109030 A1 WO 2022109030A1 US 2021059730 W US2021059730 W US 2021059730W WO 2022109030 A1 WO2022109030 A1 WO 2022109030A1
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adjacent
rtsm
sequence
independently
exon
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Botao LIU
Amy Larson GORDON
Robert KONCAR
Wei Lu
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Skyhawk Therapeutics, Inc.
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • 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
    • 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
    • C—CHEMISTRY; METALLURGY
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    • 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
    • C12N2310/32—Chemical structure of the sugar
    • C12N2310/323—Chemical structure of the sugar modified ring structure
    • C12N2310/3233—Morpholino-type ring
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2320/00—Applications; Uses
    • C12N2320/30—Special therapeutic applications
    • C12N2320/33—Alteration of splicing
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y301/00—Hydrolases acting on ester bonds (3.1)
    • C12Y301/01—Carboxylic ester hydrolases (3.1.1)
    • C12Y301/01003—Triacylglycerol lipase (3.1.1.3)

Definitions

  • a method of treating a disease or condition in a human subject in need thereof comprising: administering to the subject a therapeutically effective amount of a synthetic Patatin-like phospholipase domain-containing 3 protein (PNPLA3) RNA-targeting splicing modifier (RTSM), thereby treating the disease or condition in the human subject in need thereof;
  • the synthetic RTSM comprises a binding domain that binds to a target region of the PNPLA3 pre-messenger ribonucleic acid (pre-mRNA);
  • the target region comprises an exon-intron junction comprising a target sequence of Formula (I): KGUR, wherein K is G or U; wherein R is A or G; and exon skipping is increased as compared to the PNPLA3 pre-mRNA spliced in the absence of RTSM as demonstrated by an in vitro assay.
  • a synthetic PNPLA3 RTSM that comprises a binding domain that binds to a target region of a PNPLA3 pre-mRNA; wherein: the target region comprises an exon-intron junction comprising a target sequence of Formula (I): KGUR, wherein K is G or U; wherein R is A or G; and exon skipping is increased as compared to when the PNPLA3 pre-mRNA is spliced in the absence of the synthetic RTSM as demonstrated in an in vitro assay.
  • Formula (I) KGUR, wherein K is G or U
  • R is A or G
  • FIG. 1 shows results from RT-PCR analysis of PNPLA3 isoforms present in SNU-886 cells following treatment with antisense oligonucleotides (ASOs).
  • FIG. 2 shows results from RT-PCR analysis of PNPLA3 isoforms present in Hep G2 cells following treatment with antisense oligonucleotides (ASOs).
  • binding domain can comprise a domain or portion of an RTSM which binds to a region or a portion of an PNPLA3 pre-mRNA.
  • the binding can be covalent or non-covalent. Examples of non-covalent binding include binding via hydrogen bonding, Watson-Crick base pairing, wobble-base pairing, and Van der Waals interactions.
  • ASO antisense oligomer
  • oligomer such as a polynucleotide, comprising nucleobases that hybridizes to a target nucleic acid (e.g., a PNPLA3 containing pre-mRNA) sequence by, for example, Watson-Crick base pairing or wobble base pairing (G-U).
  • G-U wobble base pairing
  • complementarity can refer to polynucleotides (e.g., a sequence of nucleotides) related by base-pairing rales.
  • sequence “T-G-A (5 ’-3’) can be complementary to the sequence “T-C-A (5’-3’).
  • Complementarity may’ be “partial,” in which only some of the nucleic acid’s bases are matched according to base pairing rales. Alternatively, there may be “complete” or “total” complementarity between the nucleic acids.
  • base pairing may be contiguous or non-contiguous.
  • the degree of complementarity between nucleic acid strands can impact efficiency and strength of hybridization between nucleic acid strands. While perfect complementarity can be desired, some embodiments can include one or more 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 mismatches with respect to a target RNA.
  • a mismatch can be a mismatch between a base on an RTSM and a base on a target RNA. Variations at any location within the oligomer are included. In certain embodiments, variations in sequence near the termini of an oligomer can be within about 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 nucleotides of the 5 ’ and/or 3’ terminus.
  • a base pairing can be a wobble base pairing.
  • CRISPR Clustered Regularly Interspersed Short Palindromic Repeat
  • CRISPR system CRISPR nuclease system
  • CRISPR nuclease system can include a non-coding RNA molecule (e.g., guide RNA) that binds to DNA or RNA and CRISPR- Associated (Cas) proteins (e.g., Cas9) with, at least some or none, nuclease functionality (e.g., two nuclease domains).
  • Cas CRISPR- Associated
  • exon skipping can refer to a process by which a portion of an exon, an entire exon, or more than one exon are removed from a pre-processed mRNA so that it or they are not present in a mature RNA, such as an mRNA that is translated into a protein. Accordingly, the portion of the protein that can be otherwise encoded by the skipped exon is not present in the expressed form of the protein, and can create a modulated form of the protein.
  • a modulated protein may be functional, less functional or non-functional.
  • a modulated protein may be truncated or subjected to nonsense mediated decay.
  • an exon being skipped can be an aberrant exon from the human PNPLA3 gene which may contain a mutation or other alteration in its sequence that otherwise causes mutated forms of the protein.
  • an exon being skipped can be a wild-type exon.
  • an exon being skipped can be a mutated exon.
  • an exon being skipped can be any' one or more of exons 1-9 of the PNPLA3 gene, such as 1, 2, 3, 4, 5, 6, 7, 8, or 9.
  • sequence identity in the context of two nucleic acid sequences or amino acid sequences of polypeptides can refer to the residues in the two sequences which can be the same when aligned for maximum correspondence over a specified comparison window.
  • a “comparison window”, as used herein, can refer to a segment of at least about: 4, 8, 50, 100, 150, to 200 or more contiguous positions can be compared to a reference sequence of the same number of contiguous positions after the two sequences are aligned optimally.
  • polypeptides herein can be 50%, 60%, 70%, 75%, 80%, 85%, 90%, 98%, 99% or 100% identical to a reference polypeptide, or a fragment thereof, e.g., as measured by BLASTP (or CLUSTAL, or any other available alignment software) using default parameters.
  • nucleic acids can also be described with reference to a starting nucleic acid, e.g., they can be 50%, 60%, 70%, 75%, 80%, 85%, 90%, 98%, 99% or 100% identical to a reference nucleic acid or a fragment thereof, e.g., as measured by BLASTN (or CLUSTAL, or any other available alignment software) using default parameters.
  • BLASTN or CLUSTAL, or any other available alignment software
  • nucleic add or amino acid sequence comprises a sequence that has at least 90% sequence identity or more, at least 95%, at least 98% and at least 99%, compared to a reference sequence using the programs described above, e.g., BLAST, using standard parameters.
  • BLAST for nucleotide sequences
  • W word length
  • E expectation
  • M-5 M-5
  • amino acid sequences the BLAST?
  • Percentage of sequence identity can be determined by comparing two optimally aligned sequences over a comparison w indow ; w'herein the portion of the polynucleotide sequence in the comparison window can comprise additions or deletions (e.g., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences.
  • the percentage can be calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity.
  • the substantial identity exists over a region of the sequences that can be at least about 8, at least, about 14 residues in length, over a region of at least about 20 residues, and in some embodiments, the sequences can be substantially identical over at least about 2.5 residues. In some embodiments, the sequences can be substantially identical over the entire length of the coding regions.
  • nucleobase can generally refer to nitrogen containing compound that is capable of hydrogen bonding with a nucleobase present on a target pre-mRNA .
  • a nucleobase may be any unmodified nucleobase such as adenine, guanine, cytosine, thymine and uracil, or any synthetic or modified nucleobase that is sufficiently similar to an unmodified nucleobase such that it is capable of hydrogen bonding with a nucleobase present on a target pre-mRNA.
  • modified nucleobases include, without limitation, hypoxanthine, xanthine, 7-methylguanine, 5, 6 ⁇ dihydrourac.il, 5-methylcytosine, and 5- hydroxymethoylcytosine.
  • PNPLA3 associated disorder refers to a disease or condition that can be associated with an activity, a reduced activity, an altered activity, and in some cases, mutant activity, of a PNPLA3 protein.
  • the term ‘'targeting domain” can comprise a region or portion of an PNPLA3 pre- mRNA to which a RTSM can bind.
  • the binding can be covalent or non-covalent. Examples of non-covalent binding include binding via hydrogen bonding, Watson-Crick base pairing, wobble-base pairing, and Van derWaals interactions.
  • RNA-Targeting Splicing Modifier RTSM
  • RTSM RNA-Targeting Splicing Modifier
  • the PNPLA3 gene can encode a 481 amino acid Patatin-like phospholipase domain-containing 3 protein (PNPLA3).
  • PNPLA3 481 amino acid Patatin-like phospholipase domain-containing 3 protein
  • Wild-type PNPLA3 can be found in the membranes of lipid droplets, where it can be responsible for the postprandial remodeling of lipid droplets via its triglyceride hydrolase activity.
  • variants such as I148M (rs73840) and S47A have been shown to have reduced catalytic activity.
  • Variants can also resist ubiquitylation-based degradation.
  • variant PNPLA3 proteins can accumulate on the surface of lipid cells and sequester CGI-58, a cofactor required for the activity of adipose triglyceride lipase.
  • pro-fibrogenic cytokines including CCL2 and CCL5. These cytokines can stimulate hepatic stellate cells (HSC)s activation promoting the secretion of collagen 1 , the protein that can be seen as the basis of fibrosis during NALFD/NASH progression.
  • HSC hepatic stellate cells
  • NASH nonalcoholic steatohepatitis
  • RTSMs that can modulate PNPLA3 protein produced in, for example, a cell, organ or subject.
  • a unmodulated PNPLA3 protein can be a reference protein and was identified by homology search and sequences identified by genomic and nucleotide databases, such as ENSEMBL, GenBank, or UniProtKB/Swiss-Prot and in some embodiments comprises a polypeptide sequence of any of the sequences of SEQ ID NO: 9-13.
  • an unmodulated PNPLA3 protein as referred to herein can be a full length, elongated, functional, or wild-type protein or any combination thereof
  • a targeted PNPLA3 gene can encode a PNPLA3 protein.
  • a targeted PNPLA3 gene comprises the sequence as set forth in RefSeq No. NC 000022.11 (disclosed herein as SEQ ID NO: 1 and incorporated by reference).
  • a PNPL.A3 gene comprises one or more gene mutations.
  • the one or more PNPLA3 gene mutations are associated with: lengthening of the protein encoded by the gene; gain-of-function of a protein encoded by the gene; reduced or loss-of-function of a protein encoded by the gene; or an associated disease or a condition thereof.
  • RTSMs disclosed herein can target a PNPLA3 pre-mRNA.
  • a PNPLA3 pre-mRNA can be the precursor PNPLA 3 RNA transcribed from the PNPLA3 gene, but prior to being spliced into a mature RNA.
  • a targeted pre-mRNA comprises a sequence that shares at least about: 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity, to at least about: 4, 5, 6, 7, 10, 15, 20, 25, 50, 75, 100, 150, 200, 250, 300 or more continuous nucleotides of any one of the sequences as set forth SEQ ID NOS: 2-6.
  • RTSMs disclosed herein can modulate PNPLA3 mRNA produced in, tor example, a cell, organ or subject.
  • an unmodulated PNPLA3 mRNA transcript referred to herein can be a reference mRNA and can comprise a sequence as set forth m any of the sequences in SEQ ID NOS: 7-8.
  • an unmodulated PNPLA3 mRNA can be wild-type and in some embodiments can comprise a sequence as set forth in SEQ ID NO: 7.
  • a reference PNPLA mRNA transcript can be a mutant and, in some embodiments, can comprise the sequence as set forth in SEQ ID NO:8.
  • a reference PNPLA3 protein referred to herein can also be mutant and can comprise any one of the sequences in SEQ ID NOS: 10-13.
  • RTSMs disclosed herein can target a target region of a PNPLA3 pre-mRNA.
  • a target region can comprise coding for: protein length; protein stability; protein function; coding specific for RTSM-RNA binding; RTSM-RNA duplex formation; coding which limits off-target binding; coding which recruits one or more splicing complex component; or any combination thereof.
  • RTSMs disclosed herein can increase at least partial exon skipping during splicing of a PNPLA3 pre-mRNA.
  • a targeted pre-mRNA comprises one or more exons and one or more introns.
  • a PNPLA3 pre-mRNA can comprise 9 exons and 8 introns.
  • a targeted pre-mRNA comprises exons 1-9 and introns 1-8.
  • any one of exons 1-9 can comprise, in part or in full, coding for protein length: protein stability; protein function; coding specific for RTSM-RNA binding; RTSM-RNA duplex formation; coding which limits off-target binding; coding which recruits one or more splicing complex component; or any combination thereof.
  • exon 1 can comprise coding for protein length; protein stability; protein function; coding specific tor RTSM-RNA binding; RTSM-RNA duplex formation; coding which limits off-target binding; coding which recruits one or more splicing complex component; or any combination thereof
  • exon 2 can comprise coding for protein length; protein stability; protein function; coding specific for RTSM-RNA binding; RTSM-RNA duplex formation; coding which limits off-target binding; coding which recruits one or more splicing complex component; or any combination thereof
  • exon 3 can comprise coding for protein length; protein stability; protein function; coding specific for RTSM-RNA binding; RTSM-RNA duplex formation; coding which limits off-target binding; coding which recruits one or more splicing complex component; or any combination thereof
  • exon 4 can comprise coding for protein length; protein stability; protein function; coding specific for RTSM- RNA binding; RTSM-RNA duplex formation; coding which limits off-target binding; coding which recruits
  • pre-mRNA exon(s) referred to herein comprises a sequence that shares at least about: 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any one ofthe sequences in Table 2.
  • a pre-mRNA intron(s) referred to herein comprises a sequence that shares at least about: 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any one of the sequences in Table 3.
  • pre-mRNA exon(s), intron(s), or both may comprise one or more nucleotide alterations at one or more positions in any one of the sequences in Tables 2 or 3.
  • Alternative nucleobases can be any one or more of A, C, G or U, or a deletion.
  • a targeted pre-mRNA comprises a target region wherein a target region compri ses an exon-intron junction
  • Exon-intron junctions can be at least 2 nucleic acids in length, at least 3 nucleic acids in length, at least 4 nucleic acids in length, at least 5 nucleic acids in length, at least 6 nucleic acids in length, at least 7 nucleic acids in length, at least 8 nucleic acids in length, at least 9 nucleic acids m length, at least 10 nucleic acids in length, at least 11 nucleic acids in length, at least 12 nucleic acids in length, at least 13 nucleic acids in length, at least 14 nucleic acids or more in length.
  • exon-intron junctions can be about: 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 26, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or more nucleic acids in length.
  • a targeted pre-mRNA comprises a targeted region wherein a targeted region comprises one or more exon-intron junction(s).
  • a target region comprises an exon- intron junction.
  • a target region comprises more than one exon-intron junction.
  • an exon-intron junction can be Exon 1 -Intron 1, Exon2-Intron2, Exon 3 -Intron 3, Exon4- Intrond, Exon5 -Intron 5, Exon6-Intron6, Exon7-Intron7, or Exon8-Intron8, more than one, or any combination thereof.
  • a targeted region comprises a targeted exon or a portion thereof, a targeted intron or a portion thereof, both, more than one of either, or more than one of both, that is targeted by a binding domain of an RTSM herein.
  • a binding domain of an RTSM in some instances can be from about 2. to about 50 nucleobases in length and can bind with about 1 to about 25 nucleobases of an exon to about 1 to about 25 nucleotides of an intron, any number in between, and combinations thereof.
  • Target region can comprise about 1 to about 25 nucleobases of an exon, 1 to about 25 nucleobases of an intron, or any combination thereof.
  • a targeted region comprises a sequence that is at least about 2 to about 50 nucleobases long. In some embodiments, a targeted region comprises a sequence that can be about: 2, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 26, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or more nucleobases in length,
  • a targeted exon can be any one of, or a portion of any one of Exon 1, Exon 2, Exon 3, Exon 4, Exon 5, Exon 6, Exon 7, Exon 8, or Exon 9.
  • a targeted exon can comprise a sequence that shares at least about: 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a sequence of any one of SEQ ID NOS: 14-22.
  • a targeted intron can be any one of, or a portion of any one of Intron 1 , Intron 2, Intron 3, Intron 4, Intron 5, Intron 6, Intron 7, or Intron 8.
  • a targeted intron can comprise a sequence that shares at least about: 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a sequence of any one of SEQ ID NOS: 23-30.
  • an exon -intron junction can comprise a sequence that shares at least about: 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20% or more sequence identity to: SEQ IDS NO: 14 and 23; SEQ IDS NO: 15 and 24; SEQ IDS NO: 16 and 25; SEQ IDS NO: 17 and 26; SEQ IDS NO: 18 and 27; SEQ IDS NO: 19 and 28; SEQ IDS NO: 20 and 29; or SEQ IDS NO: 21 and 30.
  • a targeted mRNA comprises a targeted sequence.
  • an RTSM targets and binds to a targeted sequence of a PNPLA3 pre-mRNA.
  • an exon- intron junction comprises a targeted sequence.
  • a target sequence comprises a sequence that can be at least about 2 to about 50 nucleobases long. In some embodiments, a target sequence comprises a sequence that can be about: 2, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 26, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or more nucleobases in length.
  • a targeted sequence comprises a sequence as set forth in Formula (I) below:
  • a targeted sequence comprises a splice site.
  • an RTSM specifically binds to a splice site.
  • an RTSM specifically hybridizes to a splice site.
  • a splice site comprises a sequence as set forth in Formula (II) below.
  • Formula (I), Formula (II), or both further comprise: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 N(s) wherein each N is independently A, G, C, or U.
  • Formula (I), Formula (II), or both further comprise: N; NN: NNN; NNNN; NNNNN; NNNNNNN; NNNNNNNN; NNNMNNNN; MAWNNNN; NNNNNNNNN; NNNNNNNNNN; NNNNNNNNNNNNN; NNNNNNNNNNNNNN; NNNNNNNNNNNNNNNNN; NNNNNNNNNNNNNNNNNN; or NNNNNNNNNNNNNNNNNNNNN wherein each N is independently A, G, C, or U.
  • an N of a group of: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 contiguous N(s) can be 5’ and adjacent to K or can be 3’ and adjacent to R, wherein each N is independently A, G, C, or U.
  • Formula (I), Formula (II), or both further comprise, a N group of: 1 , 2, 3, or 4 contiguous N(s) that can be 5’ and adjacent to K; a N group of: 1, 2, 3, 4, 5, 6, 7,
  • N(s) 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 contiguous N(s), that can be 3’ and adjacent to R: and combinations thereof, wherein each N is independently A, G, C, or U.
  • Formula (I), Formula (II), or both further comprise: N that is 5 ’ and adjacent to K; NN that is 5’ and adjacent to K; NNN that is 5’ and adjacent to K; NNNN that is 5’ and adjacent to K; NNNNN that is 5’ and adjacent to K; N that is 3’ and adjacent to R; NN that is 3’ and adjacent to R; NNN that is 3’ and adjacent to R; NNNN that is 3’ and adjacent to R; NNNNNNN that is 3’ and adjacent to R; NNNNNNN that is 3’ and adjacent to R; NNNNNNN that is 3’ and adjacent to R; NNNNNNNN that is 3’ and adjacent to R; NNNNNNNNN that is 3’ and adjacent to R; NNNNNNNNN that is 3’ and adjacent to R; NNNNNNNNN that is 3’ and adjacent to R; NNNNNNNNN that is 3’ and adjacent to R; NNNNNNNNN that is 3’ and adjacent to R; NNNNNNNNN that is 3’ and adjacent to R; NNN
  • Formula (I), Formula (II), or both further comprise NNN that is 5’ and adjacent to K and NNNNNN that is 3’ and adjacent to R
  • the sequence of Formula (I), Formula (II), or both may comprise NNNKGL T RNNNNNNNN wherein each N is independently A, G, C or U.
  • Formula (I), Formula (II), or both further comprise NNNNNNN that is 5’ and adjacent to K and NNNNNNNNNNNNNNN that is 3’ and adjacent to R
  • the sequence of Formula (I), Formula (II), or both may comprise NNNNNKGURNNNNNNNNNNN wherein each N is independently A, G, C or U.
  • the sequence set forth in Formula (I), Formula (II), or both further comprising: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 B(s) wherein each B is independently G, C or U; 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 V(s) wherein each V is independently A, G, or C; 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 N wherein each N is independently A, G, C or U; 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 D(s) wherein each D is independently A, G or U; 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 Y(s) wherein each Y is independently C or U; 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 H(s) wherein each H is independently A
  • each K is independently G or U; or any combination thereof.
  • Formula (I), Formula (II), or both further comprise: 1, 2, 3, 4, 5, 6, or 7 B(s) wherein each B is independently G, C or U; 1, 2, or 3 V(s) wherein each V is independently A, G, or C; 1 or 2 D(s) wherein each D is independently A, G or U; 1 , 2, 3, or 4 N(s) wherein each N is independently A, G, C or U; Y wherein Y is C or U; 1 or 2 H(s) wherein each H is independently A, C or U; K wherein K is independently G or U; or any combination thereof.
  • Formula (I), Formula (II), or both further comprises: D, N, V, B or any combination thereof that is 5’ to K, wherein B is G, C or U, wherein V is A, G, or C, wherein I) is A, G or U, and wherein N is A, G, C or U.
  • Formula (I), Formula (II), or both further comprise D that is 5’ and adjacent to K; ND that is 5’ and adjacent to K; VND that is 5’ and adjacent to K; or BAND that is 5’ and adjacent to K; wherein B is G, C or U, wherein B is G, C or U, wherein V is A, G, or C, wherein D is A, G or U, and wherein N is A, G, C or U.
  • Formula (I), Formula (II), or both further comprise: 1 , 2, 3, 4, 5, or 6 B(s) wherein each B is independently G, C or U; 1 or 2 V(s) wherein each V is independently A, G, or C; 1 , 2, or 3, N(s) w herein each N is independently A, G, C or U; 1 or 2 D(s) wherein each D is independently A, G or U; Y wherein Y is C or U; 1 or 2 H(s) wherein each H is independently A, C or U; K wherein K is G or U: or any combination thereof, that is 3‘ and adjacent to R.
  • Formula (I), Formula (II), or both further comprise: H that is 3' and adjacent to R; HV that is 3' and adjacent to R; HVY that is 3' and adjacent to R; HVYH that is 3' and adjacent to R; HVYHB that is 3' and adjacent to R; HVYHBK that is 3‘ and adjacent to R; HVYHBKB that is 3' and adjacent to R; HVYHB KB B that is 3‘ and adjacent to R; HVYHBKB BN that is 3’ and adjacent to R: HVYHBKBBNN that is 3’ and adjacent to R; HAYHBKBBNNB that is 3’ and adjacent to R; HVYHBKBBNNBD that is 3' and adjacent to R; HVYHBKBBNNBDV that is 3' and adjacent to R; HVYHBKBBNNBDVD that is 3' and adjacent to R; HVYHBKBBNNBDVD that is 3' and adjacent to R; HVYHBKBBNNBDVDB that is 3' and adjacent to R; HV
  • Formula (I), Formula (II), or both further comprise: VND that is 5’ and adjacent to K and HVYHBKB that is 3’ and adjacent to R, wherein the sequence of Formula (I), Formula (II), or both, comprise VNDKGURBHVYHBKB wherein each B is independently G, C or U, wherein each V is independently A, G, or C, wherein each D is independently A, G or U, wherein each N is independently A, G, C or U, wherein Y is C or U, wherein each H is independently A, C, or U wherein K is G or U.
  • Formula (I), Formula (II), or both further comprise: BVND that is 5‘ and adjacent to K and HVYHBKBBNNBDVDBBN that is 3’ and adjacent to R, wherein the sequence of Formula (I), Formula (II), or both, comprise B VNDKCilJRflVYIlBKBBNNBDVDBBN wherein each B is independently G, C or U, wherein each V is independently A, G, or C, wherein each D is independently A, G or U, wherein each N is independently A, G, C or U, wherein Y is C or U, wherein each H is independently A, C, or U and wherein K is G or U.
  • a target sequence comprises a sequence that shares at least about: 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any one of SEQ ID NOS: 31-46 of Table 4.
  • Table 4 Exemplary Targeted Sequences
  • a target sequence may comprise one or more nucleotide alterations at one or more positions in any of the sequences in Table 4. Alterations can be made singly or in combination with other alterations at other positions. Alternative nucleobases can be any one or more of A, C, G or U, or a deletion. In some embodiments, alterations can be obtained through computational predictions and alignment, for example, using SPLIGN, ESEMBL, or Blast.
  • the disclosed RTSM herein increases exon skipping of a targeted exon during splicing.
  • the exon to be skipped can be any one of Exon 1, Exon 2, Exon 3, Exon 4, Exon 5, Exon 6, Exon 7, Exon 8, or Exon 9, and any combination thereof.
  • an exon can be skipped when an RTSM can be bound to the target sequence.
  • one or more of Exon 1, Exon 2, Exon 3, Exon 4, Exon 5, Exon 6, Exon 7, Exon 8, Exon 9, and any combination thereof can be skipped during splicing.
  • a target region can comprise a splice site.
  • an exon- intron junction can comprise a splice site.
  • a splice site can be a 5’ splice site.
  • a splice site can be a 3" splice site.
  • an exon-intron junction comprises at least a portion of a target exon, at least a portion of a target intron, both, more than one of either, and more than one ofboth.
  • an exon-intron junction can be located at the 5’ splice site of a target intron, wherein the corresponding targeted exon can be downstream of a target intron.
  • a targeted region can comprise the 5’ splice site of an intron.
  • a targeted region comprises the 5’ splice site of intron I wherein a targeted exon is exon 1.
  • a targeted region comprises the 5’ splice site of intron 2 wherein a targeted exon is exon 2. In some embodiments, a targeted region comprises the 5’ splice site of intron 3 wherein a targeted exon is exon 3. In some embodiments, a targeted region comprises the 5’ splice site of intron 4 wherein a targeted exon is exon 4, In some embodiments, a targeted region comprises the 5’ splice site of intron 5 wherein a targeted exon is exon 5. In some embodiments, a targeted region comprises the 5’ splice site of intron 6 wherein a targeted exon is exon 6.
  • a targeted region comprises the 5’ splice site of intron 7 wherein a targeted exon is exon 7. In some embodiments, a targeted region comprises the 5’ splice site of intron 8 wherein a targeted exon is exon 8.
  • an RTSM binding domain specifically binds to a splice site. In some embodiments, an RTSM binding domain specifically hybridizes to a splice site. In some embodiments, a splice site comprises a sequence as set forth in Formula (II). Various embodiments of Formula (II) can be seen, for example, in any sequence set forth in Table 5.
  • a splice site sequence comprises a sequence that can be at least about 2 to about 50 nucleobases long. In some embodiments, a splice site sequence comprises a sequence that can be about 14 to about 35 nucleobases long. In some embodiments, a splice site sequence comprises a sequence that can be about 14 to about 25 nucleobases long.
  • a splice site sequence comprises a sequence that can be about: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 26, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or more nucleobases in length.
  • a splice site sequence can be about 2 nucleobases in length.
  • a splice site sequence can be about 3 nucleobases in length.
  • a splice site sequence can be about 4 nucleobases in length.
  • a splice site sequence can be about 14 nucleobases in length.
  • a splice site sequence can be about 25 nucleobases in length.
  • a splice site comprises a sequence that shares at least about: 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any one of the sequences in Table 5.
  • a splice site can be a5’ splice site.
  • a 5’ splice site can be the complement of any one of the sequences in SEQ ID NOS: 47-62.
  • a 5’ splice site can be the inverse complement of any one of the sequences in SEQ ID NOS: 47-62.
  • SEQ ID NOS: 47-62 is reproduced in Table 6 below and in some instances, a splice site can be located where the vertical line is located at each sequence.
  • a splice site sequence further comprises one or more alterations at one or more positions on either side of a splice site.
  • an alteration can be seen at -2, -3, -4, -5, -6, -7, -8, -9, -10, -1 1 , -12, -13, -14, -15, -16, -17, -18, -19, -20, -21, -22, -23, -24, -25, -26, -27, -28, -29, -30, -31, -32, -33, -34, -35, -36, -37, -38, -39, -40, -41, -42, -43, -44, -45, -46, -47, -48, -49, -50 or more positions from a splice site in the 3’ to the 5’ direction.
  • an alteration can be seen at +1, +2, +3, +4, +5, +6, +7, +8, +9, + 10, +11, +12, +13, +14, +15, +16, +17, +18, +19, +20, +21, +22, +23, +24, +25, +26, +27, +28, +29, +30, +31, +32, +33, +34, +35, +36, +37, +38, +39, +40, +41, +42, +43, +44, +45, +46, +47, +48, +49, +50 or more positions from a splice site in the 5’ to the 3 ' direction.
  • an alteration can be seen at -25, -24, -23, -22, -21 , -20, -19, -18, -17, -16, -15, -14, -13, -12, -1 1 , -10, -9, -8, - 7. -6, -5, -4, -3. -2. -1 , +1, +2, +3, +4, +5. +6, +7. +8. +9, +10, +11. +12, +13, +14, +15. +16. +17, +18. +19, +20, +21, +22, +23, +24, +25 positions from a splice site.
  • Alterations can be any one of A, C, G or U.
  • alterations can be obtained through computational predictions and alignment, for example, using SPLIGN, ESEMBL, or Blast.
  • RTSM RNA-Targeted Splicing Modifiers
  • An RTSM disclosed herein can be synthetic RNA targeting splicing modifiers that can increase exon skipping during splicing of PNPLA3 pre-mRNA compared to PNPLA3 pre-mRNA spliced in the absence of RTSM.
  • An RTSM disclosed herein can increase the level of modulated PNPLA3 mRNA transcripts as compared to mRNA transcripts processed in the absence of an RTSM,
  • An RTSM disclosed herein can increase the level of modulated PNPLA3 protein production as compared to PNPLA3 produced in the absence of an RTSM.
  • the term “increase” means to induce or to enhance.
  • any incidence of exon skipping or any indication of exon skipping activity resulting from splicing in the presence of RTSM has thereby “increased” exon skipping.
  • an evaluation of an increase in exon skipping can occur in an in vitro assay.
  • comparison of the amount of exon skipping in two otherwise substantially identical systems where one system lacks an RTSM and the other system has an RTSM can determine if exon increases in the presence of the RTSM.
  • an RTSM can be selected from the group consisting of an antibody or a fragment thereof, an aptamer, an antisense oligomer (ASO), or a CRISPR associated protein, and any combination thereof.
  • an RTSM targets and binds to PNPLA3 pre-mRNA.
  • RTSMs wherein RTSM-pre-mRNA binding can prevent recruitment of one or more splicing complex component to the pre-mRNA, decrease the binding affinity of one or more splicing complex component to the pre-mRNA, interfere with splice site signaling, sterically block splicing of the pre-MRNA, or any combination thereof.
  • an RTSM comprises a binding domain that binds to PNPLA3 pre-mRNA.
  • an RTSM comprises a binding domain that binds to a target region of a PNPLA3 pre-mRNA.
  • an RTSM comprises a binding domain that binds to a target sequence of a PNPLA3 pre-mRNA. In some embodiments, an RTSM comprises a binding domain that specifically binds to a splice site sequence of a PNPLA3 pre-mRNA.
  • a binding domain of an RTSM disclosed herein can be from about 2 to about 50 nucleobases in length and can bind with about 1 to about 25 nucleobases of an exon to about 1 to about 25 nucleotides of an intron, any number in between, and combinations thereof.
  • a binding domain of an RTSM comprises a sequence that can be at least about 2 to about 50 nucleobases long.
  • a binding domain of an RTSM comprises a sequence that can be about: 2, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or more nucleobases in length.
  • an RTSM need not to completely bind to all nucleobases in a target sequence and the nucleobases to which it does bind to may be contiguous or noncontiguous.
  • RTSMs may bind over one or more segments of a pre-mRNA transcript, such that intervening or adjacent segments are not involved in the hybridization event (e.g., a loop structure or hairpin structure may be formed).
  • an RTSM binds to noncontiguous nucleobases in a target pre-mRNA transcript.
  • a RTSM may bind to nucleobases in a pre-mRNA transcript that are separated by one or more nucleobase(s) to which an RTSM does not bind.
  • an RTSM can bind to about: 2, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 continuous nucleobases of a target pre-mRNA.
  • an RTSM binding domain comprises a binding sequence.
  • a binding sequence binds to the sequence as set forth in Formula (II) .
  • a binding sequence hybridizes to the sequence as set forth in Formula (II).
  • a binding sequence comprises a sequence as set forth in Formula (III) below:
  • Y is C, T, or U; wherein M is A or C and wherein 5 ’ . . . 3 ’ indicates from the direction.
  • Formula (III) further comprises: I, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 N(s) wherein each N is independently A, G, C, T or U.
  • Formula (I), Formula (II), or both further comprise: N; NN; NNN; NNNN; NNNNNNN; NNNNNNN; NNNNNNNNN; NNNNNNNNNN; NNNNNNNNNNN; NNNNNNNNNNNNNN; NNNNNNNNNNNNN; NNNNNNNNNNNN; NNNNNNNNNNNNN; NNNNNNNNNNNNNN; NNNNNNNNNNNNNN; NNNNNNNNNNNNNNNN; NNNNNNNNNNNNNNNN; or NNNNNNNNNNNNNNNNNNN wherein each N is independently A, G, C, T or U.
  • N group of: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 contiguous N(s) can be 5’ and adjacent to Y or can be 3’ and adjacent to M, wherein each N is independently A, G, C, T or U.
  • Formula (III) further comprises an N group of: 1, 2, 3, or 4 contiguous N(s) that can be 3 ’ and adjacent to M; an N group of: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, I I,
  • N 12, 13, 14, 15, 16, or 17 contiguous N(s), that can be 5’ and adjacent to Y; and combinations thereof, wherein each N is independently A, G, C, T or U.
  • Formula (III) further comprises: N that is 5’ and adjacent to Y; NN that is 5’ and adjacent to Y; NNN that is 5’ and adjacent to Y; NNNN that is 5’ and adjacent to Y; NNNNN that is 5’ and adjacent to Y; NNNNNNN that is 5’ and adjacent to Y; NNNNNN that is 5’ and adjacent to Y; NNNNNNNN that is 5' and adjacent to Y; NNNNNNNNNNN that is 5’ and adjacent to Y; NNNNNNNNNNNNN that is and adjacent to Y; NNNNNNNNNNN that is and adjacent to Y; NNNNNNNNNNNN that is 5’ and adjacent to Y; NNNNNNNNNNNNN that is 5’ and adjacent to Y; NNNNNNNNNNNNNNN that is 5’ and adjacent to Y; NNNNNNNNNNNNNNN that is 5’ and adjacent to Y; NNNNNNNNNNNNNN that is 5’ and adjacent to Y; NNNNNNNNNNNNNN that is
  • Formula (III) further comprises: NNN that is 3 ’ and adjacent to M and NNNNNNNN that is 5’ and adjacent to Y, the sequence of Formula (I), Formula (II), or both, may comprise NNNNNNNNYACMNNN wherein each N is independently A, G, C, T or U.
  • the sequence of Formula (I), Formula (II), or both may comprise NNNNNNNN NNN YACMNNNNNNN wherein each N is independently A, G, C, T or U.
  • the sequence set forth in Formula (III) further comprising: 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 B(s) wherein each B is independently G, C, T or U; 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 V(s) wherein each V is independently A, G, or C; 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, I I, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 N wherein each N is independently A, G, C, T, or U; 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 D(s) wherein each D is independently A, G, T or U; 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 R(s) wherein each R is independently G or A; I, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 H(s) wherein each H
  • each M is independently A or C; or any combination thereof.
  • Formula (III) further comprises: M; R; 1 or 2 D(s); 1, 2, or 3 H(s); 1, 2, or 3 B(s); 1, 2, 3, or 4 N(s); 1, 2, 3, 4, 5, 6, or 7 V(s) or any combination thereof, wherein M is A or C, wherein each B is independently G, C, T or U, wherein R is G or A, wherein each D is independently A, G, T or U, wherein each H is independently A, C, T, or U, wherein each N is independently A, G, C, T, or U, and wherein each V is independently A, G. or C.
  • Formula (III) further comprises: R; M; 1 or 2 H(s); 1 or 2 B(s); 1 or 2 D(s): 1, 2, or 3 N(s); I, 2, 3, 4, 5, or 6 V(s); or any combination thereof; that is 5’ and adjacent to Y wherein M is A or C, wherein R is G or A, wherein each D is independently A, G, T or U, wherein each II is independently A, C, T, or U, wherein each N is independently A, G, C, T, or U, and wherein each V is independently A, G, or C.
  • Formula (III) further comprises: H, N, B, V; or any combination thereof that is 3 ’ and adjacent to M; wherein each H is independently A, C, T, or LI, wherein each N is independently A, G, C, T, or U, wherein each B is independently G, C, T or U, and wherein each V is independently A, G, or C.
  • Formula (III) further comprises: D that is 5‘ and adjacent to Y ; BD that is 5' and adjacent to Y; RBD that is 5' and adjacent to ⁇ ; DRBD that is 5' and adjacent to Y; VDRBD that is 5‘ and adjacent to Y; MVDRBD that is 5' and adjacent to Y: VMVDRBD that is 5' and adjacent to Y; VVMVDRBD that is 5' and adjacent to Y; NVVMVDRBD that is 5’ and adjacent to Y; NNVVMVDRBD that is 5' and adjacent to Y; VNNVVMVDRBD that is 5' and adjacent to Y; HVNNVVMVDRBD that is 5' and adjacent to Y; BFIX'TQNVVMVDRBD that is 5' and adjacent to Y; HBI-IVNNVVMVDRBD that is 5' and adjacent to Y; VHBI-RTEWVMVDRBD that is 5’ and adjacent to Y; VA/in3l
  • Formula (III) further comprises VMVDRBD that is 5’ and adjacent to Y, HNB that is 3‘ and adjacent to M, wherein the sequence of Formula (III) comprises VMVDRBDYACM HNB wherein M is independently A or C, wherein each B is independently G, C, T or U, wherein R is G or A, wherein each D is independently A, G, T or U, wherein each H is independently A, C, T, or U, wherein each N is independently A, G, C, T, or U, and wherein each V is independently A, G, or C. In some embodiments.
  • Formula (III) comprises VVHBFIVNNVVMVDRBD that is 5’ and adjacent to B and HNBV that is 3’ and adjacent to Y, wherein the sequence of Formula (III) comprises VVHBHVNNVVMVDRBDYACMHNBV wherein each M is independently A or C, wherein each B is independently G, C, T or U, wherein R is G or A, wherein each D is independently A, G, T or U, wherein each H is independently A, C, T, or LI, wherein each N is independently A, G, C, T, or U, and wherein each V is independently A, G, or C.
  • an RTSM binding domain comprises a sequence about 2 to about 50 nucleobases in length that has at least about: 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence complementarity to any one of the sequences of SEQ ID NOS: 32-46.
  • an RTSM binding domain comprises a sequence about 4 to about 30 nucleobases in length that has at least about: 1 %, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence complementarity to any one of tire sequences of SEQ ID NOS: 32-46.
  • an RTSM binding domain comprises a sequence about 2 to about 50 nucleobases in length that shares at least about: 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any one of the sequences of SEQ ID NOS: 63-94 of Table 6,
  • an RTSM binding domain comprises a sequence about 4 to about 30 nucleobases in length that has at least about: 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any one of the sequences of SEQ ID NOS: 63-94.
  • a binding sequence may comprise one or more nucleotide alterations at one or more positions in any of the sequences in Table 7. Alterations can be made singly or in combmation with other alterations at other positions. Alternative nucleobases can be any one or more of A, C, G, T or U, or a deletion. In some embodiments, alterations can be obtained through computational predictions and alignment, for example, using SPLIGN, ESEMBL, or Blast.
  • a binding sequence comprises a sequence about 2 to about 50 nucleobases in length that has at least about: 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any one of the sequences of SEQ ID NOS: 63-94. In some embodiments, a binding sequence comprises a sequence about 4 to about 30 nucleobases in length that has at least about: 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any one of the sequences of SEQ ID NOS: 63-94.
  • a binding sequence disclosed herein comprises a sequence that can bind or hybridize to 2, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 continuous or non-continuous nucleotides of any one of the sequences of SEQ IDS NOS: 31-46.
  • a binding sequence disclosed herein comprises a sequence that can bind or hybridize to 2, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 continuous or non-continuous nucleotides of any one of the sequences of SEQ IDS NOS: 47-62.
  • An RTSM disclosed herein can increase exon skipping during pre-mRNA splicing as compared to a pre-mRNA spliced in the absence of an RTSM, In some embodiments, exon skipping modulates a PNPLA3 mRNA, a PNPLA3 protein, or both. In some embodiments, modulated exon coding can be determined by modulated mRNA transcription, modulated a PNPLA3 protein translation, or both.
  • modulated exon coding comprises: absence of one or more PNPLA3 gene exon(s) in a PNPLA3 mRNA molecule, a truncated mRNA molecule, absence of one or more PN PLA3 gene exon(s) in a PNPLA3 protein molecule, a non-functional PNPLA3 protein, an at least semi -functional PNPLA3 protein, a truncated PNPLA3 protein molecule, or any combination thereof.
  • RTSM-modulated PNPLA3 mRNA transcript can be encoded by a sequence that excludes a sequence with at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to: SEQ ID NO: 14; SEQ ID NO: 15; SEQ ID NO: 16; SEQ ID NO: 17; SEQ ID NO: 18; SEQ ID NO: 19; SEQ ID NO:20; SEQ ID NO:21 ; SEQ ID NO:22; and any combination thereof.
  • RTSM-modulated PNPLA3-mRNA transcript can be determined by analysis of a PNPLA3 mRNA transcript expressed in the absence of RTSM, or to a wild-type PNPLA3 mRNA, or both, wherein the wild-type PNPLA3 mRNA can be encoded by a sequence with at least: 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence as set forth in SEQ ID NO: 7 or 8.
  • RTSM-modulated PNPLA3 protein expression can be determined by analysis of a PNPLA3 protein translated in the absence of RTSM, or a wild-type PNPLA3 protein, or both, wherein the wild-type PNPLA3 protein can be encoded by a sequence with at least: 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of the sequences as set forth in SEQ ID NOS: 9-13.
  • an RTSM can be an antibody or a fragment thereof specific or selective for a PNPLA3 pre-mRNA. In some embodiments, an RTSM can be an anti -mRNA antibody or a fragment thereof.
  • Antibodies also known as immunoglobulin (Ig), as disclosed herein can be monoclonal or polyclonal antibodies.
  • Die term “monoclonal antibodies,” as used herein, can refer to antibodies that are produced by a single clone of B-cells and bind to the same epitope.
  • polyclonal antibodies can refer to a population of antibodies that are produced by different B-cells and bind to different epitopes of the same antigen. Hie antibodies can be from any animal origin.
  • An antibody can be IgG (including IgGl, IgG2, IgG3, and IgG4), IgA (including IgA1 and IgA2), IgD, IgE, or IgM, and IgY, In some embodiments, the antibody can be whole antibodies, including single-chain whole antibodies.
  • the antibody can be a fragment of an antibody, which can include, but are not limited to, a Fab, a Fab’, a F(ab’)2, a Fd (consisting of VH and CHI), a Fv fragment (consisting of VH and VL), a single-chain variable fragment (scFv), a single-chain antibody, a disulfide-linked variable fragment (dsFv), and fragments comprising either a VL or VH domain.
  • a whole antibody typically consists of tour polypeptides: tw o iden tical copies of a heavy (H) chain polypeptide and two identical copies of a ligh t (L) chain polypeptide.
  • Each of the heavy chains contains one N-terminal variable ( VH) region and three C- terminal constant (CHI, CI 12 and CH3) regions, and each light chain contains one N-terminal variable (VL) region and one C-terminal constant (CL) region.
  • Hie variable regions of each pair of light and heavy chains form the antigen binding site of an antibody.
  • the VH and VL regions have a similar general structure, with each region comprising four framework regions, whose sequences are relatively conserved.
  • the framework regions are connected by three complementarity determining regions (CDRs).
  • the three CDRs form the “hypervariable region” of an antibody, which can be responsible for antigen binding, and can include overlapping or subsets of amino acid residues when compared against each other.
  • the term “CDR” can be a CDR which can be defined based on sequence comparisons.
  • CDRH1, CDRH2 and CDRH3 denote the heavy chain CDRs
  • CDRL1, CDRL2 and CDRL3 denote the light chain CDRs.
  • fragment of an antibody can mean one or more fragments or portions of an antibody that retain the ability to specifically or selectively bind to an antigen.
  • the antibody fragment desirably comprises, for example, one or more CDRs, the variable region (or portions thereof), the constant region (or portions thereof), or any combination thereof.
  • Non-limiting examples of antibody fragments include (1) a Fab fragment, which is a monovalent fragment consisting of the VL, VH, CL, and CHI domains; (2) a F(ab’)2 fragment, which is a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the stalk region; (3) a Fv fragment comprising the VL and VH domains of a single arm of an antibody; (4) a single chain Fv (scFv), which is a monovalent molecule comprising the two domains of the Fv fragment (e.g., VL and VH) joined by a linker which enables the two domains to be synthesized as a single polypeptide chain and (5) a diabody, which is a dimer of polypeptide chains, wherein each polypeptide chain comprises a VH connected to a VL by a peptide linker that is too short to allow pairing between the VH and VL on the same polypeptide chain, thereby driving the pairing between the complementary
  • an RTSM antibody or fragment of described herein comprises a binding region that targets a target region on a PNPLA3 pre-mRNA.
  • an RTSM antibody binding region can be also known as an “antigen recognition domain,” “antigen binding domain,” or “’antigen binding region” which can refer to a portion of an RTSM that specifically binds to a target region.
  • a target region of a pre-mRNA comprising a target sequence can be referred to herein as an antigen .
  • a target sequence is an epitope wherein an RTSM antibody or fragment thereof targets to and binds to a pre-mRNA epitope
  • RTSM antibodies or fragments thereof can be generated by a modified nucleobase-coupling protocol.
  • the antibody can be modified, coupled or conjugated with a nucleic acid probe, such as an antisense oligonucleotide probe, wherein the nucleic acid probe binds to a targeted region of the IFH1 pre-mRNA and increases exon skipping.
  • a nucleic acid probe such as an antisense oligonucleotide probe
  • an RTSM can be an aptamer that binds to a riboswitch on a targeted pre- mRNA
  • an RTSM aptamer can be operably linked to a ligand.
  • an RTSM can be operably linked to a ligand.
  • a ligand or molecule specific to an aptamer it can be helpful to meet some or all of the following criteria. First, it should be able to bind its ligand-binding aptamer with high affinity. Second, ligand-aptamer interaction should not requi re the assistance of any other factor. Third, the ligand-binding site (the aptamer) should be unstructured and only upon binding of ligand should the aptamer undergo a conformational change or rearrangement. Fourth, the ligand-aptamer binding must be preserved under the conditions that support pre-mRNA splicing.
  • the ligand should not affect the splicing of a substrate that does not contain its binding site.
  • the aptamer-ligand comprises a binding region that targets and binds to a target sequence of a pre-mRNA, wherein the aptamer inserts into the strand and the ligand increases exon skipping.
  • a ligand can be tobramycin, neomycin, or theophylline.
  • ASOs Antisense Oligonucleotides
  • an RTSM can be an ASO.
  • an RTSM disclosed herein comprises a binding domain that targets and binds to a target region of a PNPLA3 pre-mRNA
  • a binding domain of an RTSM comprises a sequence that can be about: 2, 4, 5, 6, 7, 8, 9, 10, I I, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 26, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38,
  • an ASO binding domain comprises a sequence about 4 to about 50 nucleobases in length that has at least about: 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence complementarity to any one of the sequences of SEQ ID NOS: 31-46.
  • a binding domain comprises a sequence about 14 to about 30 nucleobases in length that has at least about: 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence complementarity to any one of SEQ ID NOS: 31-46.
  • an ASO-RTSM of the present disclosure comprises a sequence that has at least about 85% sequence complementary to the sequence of SEQ ID NO: 35. In some embodiments, an RTSM of the present disclosure comprises a sequence that has at least about 92% sequence complementary to the sequence of SEQ ID NO: 35. In some embodiments, an RTSM of the present disclosure comprises a sequence that has 100% sequence complementary to the sequence of SEQ ID NO: 35. In some embodiments, an ASO-RTSM of the present disclosure comprises a sequence that has at least about 85% sequence complementary to the sequence of SEQ ID NO: 43, In some embodiments, an RTSM of the present disclosure comprises a sequence that has at least about 92% sequence complementary to the sequence of SEQ ID NO: 43. In some embodiments, an RTSM of the present disclosure comprises a sequence that has 100% sequence complementary to the sequence of SEQ ID NO: 43.
  • An ASO and a DNA or RNA target binding partner can be complementary to each other when a sufficient number of corresponding positions in each molecule are occupied by nucleotides which can hydrogen bond with each other.
  • “specifically hybridizable'’ and “complementary” are terms which can be used to indicate a sufficient degree of complementarity or precise pairing such that stable and specific binding occurs between an ASO and a DNA or RNA target. It can be understood that the sequence of an ASO need not be 100% complementary to that of its target sequence to be specifically hybridi zable.
  • An ASO can be specifically hybridizable when there are sufficient binding interactions between an ASO and DNA or RNA target such that the ASO, at least temporarily, adheres to the specific region which its targeting.
  • Specific binding can occur under physiological conditions, including but not limited to room temperature, in the case of in vivo assays or therapeutic treatment, and in the case of in vitro assays, under conditions in which the assays are performed.
  • the above method may be used to select ASOs.
  • an ASO can have exact sequence complementary to a target sequence or near complementarity (e.g., sufficient complementarity to bind a target sequence and modulating splicing at a splice site).
  • Complementarity the degree to which one polynucleotide is complementary with another
  • the sequence of an antisense oligomer (ASO) need not be 100% complementary’ to that of its target nucleic acid to hybridize.
  • ASOs can comprise at least about: 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence complementarity to a target region within a target nucleic acid sequence to which they are targeted.
  • an ASO in which 18 of 20 nucleobases of the oligomeric compound are complementary to a target region, and would therefore specifically hybridize, would represent 90 percent complementarity'.
  • the remaining non-compiementary nucleobases may be clustered together or interspersed with complementary nucleobases and need not be contiguous to each other or to complementary’ nucleobases.
  • Percent complementarity of an ASO with a region of a target nucleic acid can be determined using BLAST programs (basic local alignment search tools) and PowerBLAST programs.
  • ASOs disclosed herein can be designed so that they bind to a target nucleic acid (e.g., a targeted region of a pre-mRNA transcript) and remain bound under physiological conditions. In some embodiments, binding as described herein can be hybridizing. In some embodiments, if an ASO binds to a site other than the intended (targeted) nucleic acid sequence, it binds to a limited number of sequences that are not a target nucleic acid (to a few sites other than a target nucleic acid).
  • a target nucleic acid e.g., a targeted region of a pre-mRNA transcript
  • Design of an ASO can take into consideration the occurrence of the nucleic acid sequence of a targeted portion of a pre-mRNA transcript or a sufficiently similar nucleic acid sequence in other locations in the genome or cellular pre-mRNA or transcriptome, such that the likelihood that an ASO will bind other sites and cause "off-target” effects is limited .
  • ASOs can bind to a target. pre-mRNA.
  • ASOs can hybridize to a pre-mRNA.
  • ASOs can "specifically hybridize” to or are “specific” to a target nucleic acid or a targeted portion of a pre-mRNA.
  • Such hybridization can occur with a T m (melting temperature) substantially greater than 37°C, at least 50 °C, or between 60 °C to approximately 90 °C.
  • Tm melting temperature
  • the Tm is the temperature at which 50% of a target sequence hybridizes to a complementary' oligonucleotide.
  • an ASO can bind to, hybridize to, or specifically hybridize to a splice site sequence in a target pre-mRNA wherein a splice site sequence comprises a sequence that shares at least about: 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity’ to any' one of the sequences of SEQ ID NOS: 47-62.
  • an ASO RTSM comprises a sequence about 4 to about 50 nucleobases in length that, has at least about: 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity’ to any one of the sequences of SEQ ID NOS:63-94.
  • an ASO comprises a sequence about 14 to about 30 nucleobases in length that has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity’ to any one of the sequences of SEQ ID NOS:63-94.
  • an ASO RTSM of the present disclosure comprises a sequence that shares at least about 85% sequence identity to the sequence of SEQ ID NO: 67. In some embodiments, an ASO RTSM of the present disclosure comprises a sequence that shares at least about 92% sequence identity to the sequence of SEQ ID NO: 67. In some embodiments, an ASO RTSM of the present disclosure comprises a sequence that has 100% sequence identity to the sequence of SEQ ID NO: 67. [0107 j In some embodiments, an ASO RTSM of the present disclosure comprises a sequence that shares at least about 85% sequence identity to the sequence of SEQ ID NO: 75.
  • an ASO RTSM of the present disclosure comprises a sequence that shares at least about 92% sequence identity to the sequence of SEQ ID NO: 75. In some embodiments, an ASO RTSM of the present disclosure comprises a sequence that has 100% sequence identity to the sequence of SEQ ID NO: 75.
  • an ASO RTSM of the present disclosure comprises a sequence that shares at least about 84% sequence identity to the sequence of SEQ ID NO: 83. In some embodiments, an ASO RTSM of the present disclosure comprises a sequence that shares at least about 88% sequence identity to the sequence of SEQ ID NO: 83. In some embodiments, an ASO RTSM of the present disclosure comprises a sequence that shares at least about 84% sequence identity to the sequence of SEQ ID NO: 83. In some embodiments, an ASO RTSM of the present disclosure comprises a sequence that shares at least about 92% sequence identity to the sequence of SEQ ID NO: 83.
  • an ASO RTSM of the present disclosure comprises a sequence that shares at least about 96% sequence identity to the sequence of SEQ ID NO: 83. In some embodiments, an ASO RTSM of the present disclosure comprises a sequence that has 100% sequence identity to the sequence of SEQ ID NO: 83.
  • an ASO RTSM of the present disclosure comprises a sequence that shares at least about 84% sequence identity to the sequence of SEQ ID NO: 91. In some embodiments, an ASO RTSM of the present disclosure comprises a sequence that shares at least about 88% sequence identity to the sequence of SEQ ID NO: 91. In some embodiments, an ASO RTSM of the present disclosure comprises a sequence that shares at least about 84% sequence identity to the sequence of SEQ ID NO: 91 . In some embodiments, an ASO RTSM of the present disclosure comprises a sequence that shares at least about 92% sequence identity’ to the sequence of SEQ ID NO: 91.
  • an ASO RTSM of the present disclosure comprises a sequence that shares at least about 96% sequence identity to the sequence of SEQ ID NO: 91. In some embodiments, an ASO RTSM of the present disclosure comprises a sequence that has 100% sequence identity to the sequence of SEQ ID NO: 91.
  • An ASO disclosed herein can comprise oligonucleotides and any 7 other oligomeric molecule that comprises nucleobases capable of binding to a complementary' nucleobase on a target mRNA, but in some embodiments, an ASO does not comprise a sugar moiety, such as a peptide nucleic acid (PNA).
  • An ASO may comprise naturally-occurring nucleotides, nucleotide analogs, modified nucleotides, or any combination of two or three of the preceding.
  • the term "modified nucleotides" erm include nucleotides with modified or substituted sugar groups and/or having a modified backbone. In some embodiments, all of the nucleotides of an ASO are modified nucleotides.
  • one or more nucleobases of an ASO may be adenine, guanine, cytosine, thymine, uracil, or any synthetic or modified nucleobase that is sufficiently similar to an unmodified nucleobase such that it is capable of hydrogen bonding with a nucleobase present on a target pre-mRNA.
  • modified nucleobases include, without limitation, hypoxanthine, xanthine, 7-methylguanine, 5, 6-dihydrouracil, 5 -methyl cytosine, and 5-hydroxymethoylcytosine.
  • an ASO described herein further comprise a backbone structure that connects the components of an oligomer.
  • backbone structure and “oligomer linkages” may be used interchangeably and can refer to the connection between monomers of an ASO.
  • the backbone comprises a 3'-5' phosphodiester linkage connecting sugar moieties of the oligomer.
  • Tire backbone structure or oligomer linkages of an ASO described herein can include (but are not limited to) phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate, phosphoraniladate, phosphoramidate, and the like.
  • the backbone structure of an ASO does not contain phosphorous but rather contains peptide bonds, for example in a peptide nucleic acid (PNA), or linking groups including carbamate, amides, and linear and cyclic hydrocarbon groups.
  • the backbone modification can be a phosphothioate linkage.
  • the backbone modification can be a phosphoramidate linkage.
  • Any of the ASOs described herein may contain a sugar moiety that comprises ribose or deoxyribose, as present in naturally occurring nucleotides, or a modified sugar moiety or sugar analog, including a morpholine ring.
  • modified sugar moieties include 2' substitutions such as 2'-O-methyl (2'-O-Me), 2' O-methoxyethyl (2'MOE), 2'-O-aminoethyl, 2'F; N3'->P5' phosphoramidate, 2'dimethylaminooxyethoxy, 2'dimethylaminoethoxyethoxy, 2'-guanidinidium, 2'-O- guanidinium ethyl, carbamate modified sugars, and bicyclic modified sugars.
  • the sugar moiety modification can be selected from 2'-O-Me, 2'F, and 2'MOE.
  • the sugar moiety modification can be an extra bridge bond, such as in a locked nucleic acid (LNA).
  • the sugar analog contains a morpholine ring, such as phosphorodiamidate morpholino (PMO).
  • the sugar moiety comprises a ribofuransyl or 2'deoxyribofuransyl modification.
  • the sugar moiety comprises 2'4'-constrained 2'0-methyloxyethyl (cMOE) modifications.
  • the sugar moiety comprises cEt 2', 4' constrained 2'-0 ethyl BNA modifications.
  • the sugar moiety comprises tricycloDNA (tcDNA) modifications.
  • the sugar moiety comprises ethylene nucleic acid (ENA) modifications.
  • the sugar moiety comprises MCE modifications.
  • one or more monomer, or each monomer of an ASO can be modified in the same way, for example each linkage of the backbone of an ASO comprises a phosphorothioate linkage or each ribose sugar moiety comprises a 2'0-methyl modification.
  • Such modifications that are present, on each of the monomer components of an ASO are referred to as "uniform modifications,"
  • a combination of different modifications may be desired, for example, an ASO may comprise a combination of phosphorodiamidate linkages and sugar moieties comprising morpholine rings (morpholines).
  • Combinations of different modifications to an ASO are referred to as “mixed modifications” or “mixed chemistries.”
  • an ASO comprises one or more backbone modifications. In some embodiments, an ASO comprises one or more sugar moiety modification. In some embodiments, an ASO comprises one or more backbone modifications and one or more sugar moiety modifications. In some embodiments, an ASO comprises a 2'MOE modification and a phosphorothioate backbone. In some embodiments, an ASO comprises a phosphorodiamidate morpholino (PMO). In some embodiments, an ASO comprises a peptide nucleic acid (PNA).
  • an ASO comprises a backbone modification comprising a phosphorothioate linkage or a phosphorodiamidate linkage.
  • an ASO comprises a phosphorodiamidate morpholino, a locked nucleic acid, a peptide nucleic acid, a 2'-O-methyl, a 2' -Fluoro, or a 2'-O- methoxyethyl moiety.
  • an ASO comprises at least one modified sugar moiety.
  • each sugar moiety can be a modified sugar moiety.
  • any of the ASOs or any component of an ASO may be independently modified in order to achieve desired properties or activities of the ASO or reduce undesired properties or activities of the ASO.
  • an ASO or one or more components of any ASO may be modified to enhance binding affinity to a target sequence on a pre-mRNA transcript; reduce binding to any non-target sequence; reduce degradation by cellular nucleases (e.g., RNase H); improve uptake of the ASO into a cell and/or into the nucleus of a cell; alter the pharmacokinetics or pharmacodynamics of the ASO; and/or modulate the half-life of the ASO.
  • an ASO can be comprised of one or more 2'-O-(2-methoxyethyl) (MOE) phosphorothioate-modified nucleotides.
  • MOE 2'-O-(2-methoxyethyl)
  • ASOs comprised of such nucleotides can be especially well-suited to the methods disclosed herein; oligomers having such modifications have been shown to have significantly enhanced resistance to nuclease degradation and increased bioavailability, making them suitable, for example, for oral delivery' in some embodiments described herein.
  • ASOs can be synthesized by methods described herein. Alternatively or in addition, ASOs may be obtained from a commercial source. In certain embodiments, an ASO can be prepared by stepwise solid- phase synthesis. In some cases, it may be desirable to add additional chemical moieties to an ASO, e.g., to enhance pharmacokinetics or to facilitate capture or detection of the compound. Such a moiety may be covalently attached, according to standard synthetic methods. For example, addition of a polyethylene glycol moiety or other hydrophilic polymer, e.g., one having 1-100 monomeric subunits, may' be useful in enhancing solubility.
  • a reporter moiety such as fluorescein or a radiolabeled group
  • the reporter label attached to the oligomer may be a ligand, such as an antigen or biotin, capable of binding a labeled antibody or streptavidin.
  • a moiety for attachment or modification of an antisense compound can be generally desirable to select chemical compounds of groups that are biocompatible and likely to be tolerated by a subject without undesirable side effects.
  • RTSMs that can comprise a CRISPR associated protein.
  • An RTSM can be a -Cas system (CC RTSM system) wherein the system comprises a CRISPR associated protein.
  • the system can be designed to target a PNPLA3 pre-mRNA, prevent recruitment of a one or more splicing complex component to a pre-mRNA, decrease the binding affinity of one or more splicing complex component to a pre-mRNA, interfere with splice site signaling, sterically block splicing of a pre-mRNA, and any combination thereof.
  • CC systems that target RNA.
  • CC systems that target RN A and does not rely on consensus protospacer adjacent motif (PAM) for activity, such as Types III and VI, can be used herein. Accordingly, in some embodiments, the CC systems used herein can rely on RNA protospacer flanking sequences (PFS) or PAM sequences. Hence, in some embodiments, a PNPLA3 RNA further comprises a PFS sequence or a PAM sequence.
  • PFS RNA protospacer flanking sequences
  • Subtypes of suitable CC systems disclosed herein can include, but are not limited Type II class 2, Types III-A, III-B, VI-A, VI-A, VI-C, or VI-D.
  • Type II RNA -targeting Cas9 systems can also be used as an RTSM disclosed herein.
  • the CC RTSM system comprises a guide RNA and a Cas nuclease.
  • the guide RNA comprises a crispr RNA (crRNA) and a tracr RNA.
  • the guide RNA comprises a single guide RNA (sgRNA).
  • CC RTSM system can comprise one or more Cas nuclease. Examples of suitable Cas nucleases include, but are not limited to, Csm3, Cmr4, Csm6, Csx1, Csx27, Csx28, a member of a Cas 7 superfamily, or a Cas9, Cas 12, or a Cas 13 effector nuclease.
  • CC Systems that target a PNPLA3 pre-mRNA can be computationally identified through determination of a Cas containing signature genes that express RNAse or RNA targeting activity, and transcribed and processed into a CRISPR gRNA.
  • the gRNA of the CC RTSM system comprises a binding domain that to a target region of a PNPLA3 pre-mRNA.
  • the gRNA or sgRNA binding domain comprises a sequence about 2 to about 50 nucleobases in length that has at least about: 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence complementarity to any one of the sequences of SEQ ID NO:
  • the gRNA or sgRNA binding domain comprises a sequence about 14 to about 30 nucleobases in length that has at least about: 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or
  • the gRNA or sgRNA binding domain comprises a sequence about 4 to about 50 nucleobases in length that has at least about: 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity’ to any one of the sequences of SEQ ID NOS.71-78 and 87-94.
  • the gRNA or sgRNA binding domain comprises a sequence about 14 to about 30 nucleobases in length that has at least about: 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any one of the sequences of SEQ ID NOS.71-78 and 87-94.
  • the gRNA of the CC RTSM system targets a PNPLA3 pre-mRNA of interest and directs the Cas nuclease to a pre-mRNA.
  • the Cas nuclease can be a catalytically dead variant, wherein upon gRNA binding to a target region of a pre-mRNA, the CC RTSM system increases exon skipping wherein the system prevents recruitment of a one or more splicing complex component to a pre-mRNA, decrease the binding affinity of one or more splicing complex component to a pre-mRNA, or sterically block mRNA splicing.
  • the Cas nuclease can be selected and or synthesized to interfere with splice site signaling through RNAse activity' wherein upon gRNA binding to a target region of a pre-mRNA , the the Cas nuclease disrupts splice site signaling sequences of a targeted exon-intron junction thereby inducing exon skipping.
  • any of the RTSMs described herein may be modified in order to achieve desired properties or activities of an RTSM or reduce undesired properties or activities of an RTSM.
  • an RTSM or one or more components of any RTSM may be modified to enhance binding affinity to a target sequence on a pre-mRNA transcript; reduce binding to any non-target sequence; reduce degradation by cellular nucleases (e.g., RNase H); improve uptake of an RTSM into a cell and/or into the nucleus of a cell; alter the pharmacokinetics or pharmacodynamics of the RTSM; and/or modulate the half-life of the RTSM.
  • cellular nucleases e.g., RNase H
  • vector delivery systems that are capable of expressing an RTSM sequences herein, such as vectors that express a polynucleotide sequence comprising any one or more of the sequences shown in Table 7, as described herein.
  • vector or “nucleic acid construct” can be meant to be a polynucleotide molecule, such as a DNA molecule derived, for example, from a plasmid, bacteriophage, yeast or virus, into which a polynucleotide can be inserted or cloned.
  • a vector can contain one or more unique restriction sites and can be capable of autonomous replication in a defined host cell including a target cell or tissue or a progenitor cell or tissue thereof, or be integrated with the genome of the defined host such that the cloned sequence can be reproducible.
  • the vector can be an autonomously replicating vector, e.g., a vector that exists as an extra-chromosomal entity, the replication of which is independent of chromosomal replication, e.g., a linear or closed circular plasmid, an extra-chromosomal element, a mini -chromosome, or an artificial chromosome.
  • the vector can contain any means for assuring self-replication.
  • tire vector can be one which, when introduced into the host cell, is integrated into the genome and replicated together with the chromosome(s) into which it has been integrated.
  • an RTSM of the present disclosure can be conjugated to a cell penetrating peptide.
  • cell penetrating peptide and “CPP” are used interchangeably and can refer to cationic cell penetrating peptides, also called transport peptides, carrier peptides, or peptide transduction domains.
  • the peptides as shown herein, have the capability of inducing cell penetration within 100% of cells of a given cell culture population and allow macromolecular translocation within multiple tissues in vivo upon systemic administrati on .
  • an RTSM can be an ASO wherein an ASO can comprise an oligonucleotide moiety conjugated to a cell penetrating peptide effective to enhance transport of the compound into cells.
  • the oligonucleotide moiety can be an arginine-rich peptide transport moiety effective to enhance transport of the compound into cells.
  • the transport moiety can be attached to a terminus of the oligomer.
  • the peptides have the capability of inducing cell penetration within 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% of cells of a given cell culture population, including all integers in between, and allow macromolecular translocation within multiple tissues in vivo upon systemic administration.
  • the cell-penetrating peptide may be an arginine-rich peptide transporter. In another embodiment, the cell-penetrating peptide may be Penetratm or the Tat peptide.
  • the CPP can be conjugated to an ASO herein and can utilize glycine as the linker between the CPP and the antisense oligonucleotide.
  • a preferred peptide conjugated PMO consists of Re-G-PMO.
  • the transport moieties as described above can enhance cell entry' of attached oligomers, relative to uptake of the oligomer in the absence of the attached transport moiety. Uptake can be enhanced at least ten fold, and at least twenty' fold, relative to the unconjugated compound.
  • arginine-rich peptide transporters e.g., cell-penetrating peptides
  • argine-rich CPPs when conjugated to an antisense PMO, argine-rich CPPs can demonstrate an enhanced ability to alter splicing of several gene transcripts.
  • Exemplary' peptide transporters, excluding linkers, can be seen in Table 8.
  • An RTSM disclosed herein can increase exon skipping during pre-mRNA splicing as compared to a pre-mRNA spliced in the absence of an RTSM herein as determined by an in vitro assay.
  • exon skipping modulates PNPLA3 mRNA transcript production.
  • exon skipping increases production of mRNA transcript excluding coding for one or more exons, a truncated mRNA molecule, or both.
  • RTSM-modulated mRNA expression can be compared to mRNA processed in the absence of RTSM as determined by an in vitro assay.
  • exon skipping modulates PNPLA3 protein production.
  • an increase in exon skipping induces an increase in modulated PNPLA3 protein expression.
  • exon skipping increases production of a non-functional PNPLA3 protein, a semi- functional PNPLA3 protein, a truncated PNPLA3 protein, or any combination thereof.
  • RTSM modulated PNPL.A3 protein production can be compared to PNPLA3 protein production in the absence of RTSM as determined by an in vitro assay.
  • the cell, organ, or subject can be evaluated to determine if appropriate for the methods and compositions described herein. Methods of determining exon skipping, mRNA modulation, protein expression, or PNPLA3 modulation are described, such as in the Examples, herein.
  • qPCR quantitative PCR
  • PCR real-time PCR
  • hot PCR hot PCR
  • expression of one or more genes can be measured by detecting the level of transcripts of the genes.
  • Exon skipping can be measured by detecting the expression of the processed m- RNA.
  • Expression of functional PNPLA3 protein can be measured by detecting the level or length of the protein, or by an assay that measures its biological activity.
  • expression can be measured by- Northern blotting, nuclease protection assays (e.g., RNase protection assays), reverse transcription PCR, quantitative PCR (e.g., real-time PCR such as real-time quantitative reverse transcription PCR), in situ hybridization (e.g., fluorescent in situ hybridization (FISH)), dot-blot analysis, differential display, serial analysis of gene expression, subtractive hybridization, microarrays, nanostrmg, and/or sequencing (e.g., next-generation sequencing)
  • Expression can be measured by protein immunostaining, protein immunoprecipitation, electrophoresis (e.g., SDS-PAGE), Western bloting, bicinchoninic acid assay, spectrophotometry, mass spectrometry, enzyme assays (e.g., enzyme-linked immunosorbent assays), immunohistochemistry-, flow cytometry-, and/or immunocytochemistry-.
  • microscopy can be optical, electron, or scanning probe microscopy.
  • Optical microscopy can comprise use of bright field, oblique illumination, cross-polarized light, dispersion staining, dark field, phase contrast, differential interference contrast, interference reflection microscopy, fluorescence (e.g., when particles, e.g., cells, are immunostained), confocal, single plane illumination microscopy, light sheet fluorescence microscopy, deconvolution, or serial time-encoded amplified microscopy.
  • the disclosed compositions and methods result in a truncated PNPLA3 protein. In some embodiments, the disclosed compositions and methods result in a decrease in the wild- type PNPLA3 protein . In some embodiments, the disclosed compositions and methods result in modulating the splicing of PNPLA3 pre-mRNA. In some embodiments, the disclosed compositions and methods result in an PNPLA3 mRNA lacking exon 5 or portion thereof. In some embodiments, PNPLA3 expression can be modulated compared to a control. A control can be wild-type or non wild-type control. Controls can be positive or negative controls.
  • an RTSM herein increase the level of modulated PNPLA3 mRNA transcripts as compared to mRNA transcripts processed in the absence of RTSM wherein a mRNA transcript modulation comprises a decrease in lull-length PNPLA3 mRNA transcript, an increase in truncated PNPLA3 mRNA transcript, an increase in an PNPLA3 mRNA transcript lacking coding for one or more exons, or any combination thereof.
  • the increase in modulated mRNA transcript processing can be about: 0.1%, 0.2% 0.3% 0.5%, 0.7%, 0.9%, 1%, 1.01%, 1.5%, 2%, 2.01%, 2.5%, 3%, 3.01%, 3.5%, 4%, 4.01%, 4.5%, 5%, 5.01%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, 10%, 10.5%, 11%, 11.5%, 12%, 12.5%, 13%, 13.5%, 14%, 14.5%, 15%, 15.5%, 16%, 16.5%, 17%, 17.5%, 18%, 18.5%, 19%, 19.5%, 20%, 21 %, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 40%, 45%, 50%, 55%, or 60% or more relative to a non contacted control.
  • the increase in modulated mRNA transcript processing can be about: 65%, 70%, 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%, or 100% relative to a non-contacted control.
  • the increase in modulated PNPLA3 protein production can be about: 0. 1%, 0.2% 0.3% 0.5%, 0.7%, 0.9%, 1%, 1.01 %, 1.5%, 2%, 2.01%, 2.5%, 3%, 3.01%, 3.5%, 4%, 4.01 %, 4.5%, 5%, 5.01%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, 10%, 10.5%, 11%, 11.5%, 12%, 12.5%, 13%, 13.5%, 14%, 14.5%, 15%, 15.5%, 16%, 16.5%, 17%, 17.5%, 18%, 18.5%, 19%, 19.5%, 20%, 21 %, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 40%, 45%, 50%, 55%, or 60% or more relative to a non contacted control.
  • the increase in modulated mRNA transcript processing can be about: 65%, 70%, 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%, or 100% relative to a non -contacted control.
  • Disclosed herein are methods of prophylaxis/treatment of PNPLA3-associated disorders.
  • methods of treatment comprising administering to a subject in need thereof an effective amount of PNPLA3 RTSM, or a pharmaceutical composition comprising the same.
  • the disclosure provides a method for inducing exon skipping to decrease the level of functional PNPLA3 in a subject in need thereof comprising administering to the subject a dose of PNPLA3 RTSM. In some embodiments, the disclosure provides a method for increasing the level of exon modulated PNPLA3 mRN A in a subject in need thereof comprising administering to the subject a dose of PNPLA3 RTSM.
  • treatment with a RTSM of the disclosure increases one or more modulated PNPLA3 mRNA production, decreases full length PNPLA3 mRNA production, decreases full length PNPLA3 mRNA production, decreases functional PNPLA3 production, decreases full-length PNPLA3 protein production, increases truncated PNPLA3 production; decreases, prevents, or delays liver damage; decreases, prevents, or delays steatosis; decreases, prevents, or delays liver fibrosis; decreases, prevents, or delays liver inflammation; decreases, prevents, or delays liver scarring or cirrhosis; decreases, prevents, or delays liver failure; decreases, prevents, or delays liver enlargement; decreases, prevents, or delays elevated transaminases; decreases, prevents, or delays hepatic fat accumulation; decreases PNPLA3 protein accumulation; increases PNPLA3 protein degradation; decreases CGI-58 levels on lipid cells, decreases CCL2 production; decreases CCL5 production,
  • the method disclosed herein can be useful treat subject who is suffering from or is at a risk of developing a PNPLA3-associated disorder.
  • the present disclosure can be useful to treat a subject who is suffering from or at the risk of developing an PNPLA3 associated condition.
  • An PNPLA3 associated condition can be a disorder that can generally characterized by the accumulation of PNPLA3 protein, the dysregulated expression of a PNPLA3 protein, or both.
  • a PNPLA3 protein can comprise a gain-of-function mutation, a loss of function mutation, or a morphological mutation, such as an elongated translated protein, or any combination thereof. Mutants can include the 1148M variant and the S47A variant.
  • a PNPLA3 -associated disorder comprises liver disease, NAFLD, hepatic steatosis, non-alcoholic steatohepatitis (NASH), liver cirrhosis, hepatocellular carcinoma, alcoholic liver disease, alcoholic steatohepatitis (ASH), HCV hepatitis, chronic hepatitis, hereditary hemochromatosis, or primary sclerosing cholangitis.
  • liver disease NAFLD, hepatic steatosis, non-alcoholic steatohepatitis (NASH), liver cirrhosis, hepatocellular carcinoma, alcoholic liver disease, alcoholic steatohepatitis (ASH), HCV hepatitis, chronic hepatitis, hereditary hemochromatosis, or primary sclerosing cholangitis.
  • the method increases truncated PNPLA3 mRNA production by 0.1%, 0.2% 0.3% 0.5%, 0.7%, 0.9%, 1%, 1.01%, 1.5%, 2%, 2.01%, 2.5%, 3%, 3.01%, 3.5%, 4%, 4.01%, 4.5%, 5%, 5.01%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, 10%, 10.5%, 11%, 1 1.5%, 12%, 12.5%, 13%, 13.5%, 14%, 14.5%, 15%, 15.5%, 16%, 16.5%, 17%, 17.5%, 18%, 18.5%, 19%, 19.5%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 40%, 45%, 50%, 55%, or 60% or more relative to a untreated control.
  • the method decreases functional PNPLA3 production by 0. 1 %, 0.2% 0.3% 0.5%, 0.7%, 0.9%, 1%, 1.01%, 1.5%, 2%, 2.01%, 2.5%, 3%, 3.01%, 3.5%, 4%, 4.01%, 4.5%, 5%, 5.01%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, 10%, 10.5%, 1 1%, 11.5%, 12%, 12.5%, 13%, 13.5%, 14%, 14.5%, 15%, 15.5%, 16%, 16.5%, 17%, 17.5%, 18%, 18.5%, 19%, 19.5%, 20%, 21 %, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 40%, 45%, 50%, 55%, or 60% or more relative to a untreated control.
  • the method decreases full-length or elongated PNPLA3 production by 0.1 %, 0.2% 0.3% 0.5%, 0.7%, 0.9%, 1%, 1.01 %, 1.5%, 2%, 2.01%, 2.5%, 3%, 3.01%, 3.5%, 4%, 4.01 %, 4.5%, 5%, 5.01%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, 10%, 10.5%, 11 %, 11 .5%, 12%, 12.5%, 13%, 13.5%, 14%, 14.5%, 15%, 15.5%, 16%, 16.5%, 17%, 17.5%, 18%, 18.5%, 19%, 19.5%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 40%, 45%, 50%, 55%, or 60% or more relative to a untreated control.
  • the method increases truncated PNPLA3 production by 0.1%, 0.2% 0.3% 0.5%, 0.7%, 0.9%, 1%, 1.01%, 1.5%, 2%, 2.01%, 2.5%, 3%, 3.01%, 3.5%, 4%, 4.01%, 4.5%, 5%, 5.01%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, 10%, 10.5%, 1 1%, 11.5%, 12%, 12.5%, 13%, 13.5%, 14%, 14.5%, 15%, 15.5%, 16%, 16.5%, 17%, 17.5%, 18%, 18.5%, 19%, 19.5%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 40%, 45%, 50%, 55%, or 60% or more relative to a untreated control.
  • the method decreases full-length or elongated PNPLA3 production by 0.1%, 0.2% 0.3% 0.5%, 0.7%, 0.9%, 1%, 1.01 %, 1.5%, 2%, 2.01%, 2.5%, 3%, 3.01%, 3.5%, 4%, 4.01 %, 4.5%, 5%, 5.01%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, 10%, 10.5%, 11%, 11.5%, 12%, 12.5%, 13%, 13.5%, 14%, 14.5%, 15%, 15.5%, 16%, 16.5%, 17%, 17.5%, 18%, 18.5%, 19%, 19.5%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 40%, 45%, 50%, 55%, or 60% or more relative to a untreated control.
  • the method increases truncated PNPLA3 production by 0.1%, 0.2% 0.3% 0.5%, 0.7%, 0.9%, 1%, 1.01%, 1.5%, 2%, 2.01%, 2.5%, 3%, 3.01%, 3.5%, 4%, 4.01%, 4.5%, 5%, 5.01%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, 10%, 10.5%, 11%, 11.5%, 12%, 12.5%, 13%, 13.5%, 14%, 14.5%, 15%, 15.5%, 16%, 16.5%, 17%, 17.5%, 18%, 18.5%, 19%, 19.5%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 40%, 45%, 50%, 55%, or 60% or more relative to a untreated control.
  • the method decreases PNPLA3 protein accumulation by 0.1%, 0,2% 0.3% 0.5%, 0.7%, 0.9%, 1%, 1.01%, 1.5%, 2%, 2.01%, 2.5%, 3%, 3.01%, 3.5%, 4%, 4.01%, 4.5%, 5%, 5.01%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, 10%, 10.5%, 11%, 11.5%, 12%, 12.5%, 13%, 13.5%, 14%, 14.5%, 15%, 15.5%, 16%, 16.5%, 17%, 17.5%, 18%, 18.5%, 19%, 19.5%, 20%, 21 %, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 40%, 45%, 50%, 55%, or 60% or more relative to a untreated control.
  • the method increases PNPLA3 protein degradation by 0.1%, 0.2% 0.3% 0.5%, 0.7%, 0.9%, 1%, 1.01%, 1.5%, 2%, 2.01%, 2.5%, 3%, 3.01%, 3.5%, 4%, 4.01%, 4.5%, 5%, 5.01%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, 10%, 10.5%, 1 1%, 11.5%, 12%, 12.5%, 13%, 13.5%, 14%, 14.5%, 15%, 15.5%, 16%, 16.5%, 17%, 17.5%, 18%, 18.5%, 19%, 19.5%, 20%, 21 %, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 40%, 45%, 50%, 55%, or 60% or more relative to a untreated control
  • the method decreases CGI-58 levels on lipid cells by 0.1%, 0.2% 0.3% 0.5%, 0.7%, 0.9%, 1%, 1.01%, 1.5%, 2%, 2.01%, 2.5%, 3%, 3.01%, 3.5%, 4%, 4.01 %, 4.5%, 5%, 5.01 %, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, 10%, 10.5%, 11%, 1 1.5%, 12%, 12.5%, 13%, 13.5%, 14%, 14.5%, 15%, 15.5%, 16%, 16.5%, 17%, 17.5%, 18%, 18.5%, 19%, 19.5%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 40%, 45%, 50%, 55%, or 60% or more relative to a untreated control
  • the method decreases CCL2 production by 0.1 %, 0.2% 0.3% 0.5%, 0.7%, 0.9%, 1%, 1.01%, 1.5%, 2%, 2.01 %, 2.5%, 3%, 3.01%, 3.5%, 4%, 4.01%, 4.5%, 5%, 5.01%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, 10%, 10.5%, 11%, 1 1.5%, 12%, 12.5%, 13%, 13.5%, 14%, 14.5%, 15%, 15.5%, 16%, 16.5%, 17%, 17.5%, 18%, 18.5%, 19%, 19.5%, 2.0%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 40%, 45%, 50%, 55%, or 60% or more relative to a untreated control
  • the method decreases CCL5 production by 0.1%, 0.2% 0.3% 0.5%, 0.7%, 0.9%, 1%, 1.01%, 1.5%, 2%, 2.01%, 2.5%, 3%, 3.01%, 3.5%, 4%, 4.01%, 4.5%, 5%, 5.01%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, 10%, 10.5%, 11%, 11.5%, 12%, 12.5%, 13%, 13.5%, 14%, 14.5%, 15%, 15.5%, 16%, 16.5%, 17%, 17.5%, 18%, 18.5%, 19%, 19.5%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 40%, 45%, 50%, 55%, or 60% or more relative to a untreated control
  • the method decreases activated HSCs by 0.1%, 0,2% 0.3% 0.5%, 0.7%, 0.9%, 1%, 1.01%, 1.5%, 2%, 2.01%, 2.5%, 3%, 3.01%, 3.5%, 4%, 4.01%, 4.5%, 5%, 5.01%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, 10%, 10.5%, 11%, 11.5%, 12%, 12.5%, 13%, 13.5%, 14%, 14.5%, 15%, 15.5%, 16%, 16.5%, 17%, 17.5%, 18%, 18.5%, 19%, 19.5%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 40%, 45%, 50%, 55%, or 60% or more relative to a untreated control
  • the method decreases collagen 1 secretion by 0.1%, 0.2% 0.3% 0.5%, 0.7%, 0.9%, 1%, 1.01%, 1.5%, 2%, 2.01%, 2.5%, 3%, 3.01%, 3.5%, 4%, 4.01%, 4.5%, 5%, 5.01%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, 10%, 10.5%, 11%, 11.5%, 12%, 12.5%, 13%, 13.5%, 14%, 14.5%, 15%, 15.5%, 16%, 16.5%, 17%, 17.5%, 18%, 18.5%, 19%, 19.5%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 40%, 45%, 50%, 55%, or 60% or more relative to a untreated control
  • the method further comprises evaluating the subject prior to RTSM administration to determine whether the subject is suitable for the treatment.
  • evaluating the subject can be determination of mutations of a PNPLA3 gene, increased PNPLA3 protein expression compared to a wild-type control, increased PNPLA3 activity compared to a wild-type control, or any combination thereof.
  • a subject in need thereof expresses PNPLA3 gene OG rs738409 polymorphism.
  • the formulations or preparations herein may be given orally, parenterally, systemically, topically, rectally or intramuscular administration. They can be given in a form suitable for each administration route. For example, they can be administered in tablets or capsule form, by injection, inhalation, eye lotion, ointment, suppository, etc. administration by injection, infusion or inhalation; topical by lotion or ointment; and rectal by suppositories.
  • formulations herein may conveniently be presented in unit dosage form.
  • the amount of active ingredient that can be combined with a carrier material to produce a single dosage form will vary depending upon the host being treated, the particular mode of administration.
  • the amount of active ingredient which can be combined with a carrier material to produce a single dosage form will generally be that amount of the compound which produces a therapeutic effect. Generally, out of one hundred percent, this amount will range from about 0.1 percent to about ninety-nine percent of active ingredient, from about 5 percent to about 70 percent, or from about 10 percent to about 30 percent.
  • the selected dosage level will depend upon a variety of factors including the activity of the particular RTSM herein, the route of administration, the time of administration, the rate of excretion or metabolism of the particular RTSM being employed, the rate and extent of absorption, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular oligomer employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.
  • a suitable daily dose of a compound herein will be that amount of the compound which is the lowest dose effective to produce a therapeutic effect. Such an effective dose will generally depend upon the factors described above. Generally, oral, intravenous, intracerebroventricular, intramuscular and subcutaneous doses of the compounds herein for a patient, when used for the indicated effects, will range from about 0.0001 to about 100 mg per kilogram of body weight per day.
  • an RTSM can be an ASO
  • doses of an ASO herein can be generally administered is from about 0.001 mg/kg to about 1000 mg/kg, wherein mg is mg of RTSM and kg is the body weight, of the subject.
  • mg mg of RTSM
  • kg the body weight
  • preferred doses are from about 0.5 mg to 100 mg/kg.
  • an ASO can be administered at doses of about: 0.001 mg/kg, 0.01 mg/kg, 0.1 mg/kg, 1 mg/kg, 5 mg/kg, 10 mg/kg, 15 mg/kg, 20 mg/kg, 21 mg/kg, 22mg/kg, 23 mg/kg, 24mg/kg, 25 mg/kg, 26 mg/kg, 27 mg/kg, 28 mg/kg, 29 mg/kg, 30 mg/kg, 31 mg/kg, 32 mg/kg, 33 mg/kg, 34 mg/kg, 35 mg/kg, 36 mg/kg, 37 mg/kg, 38 mg/kg, 39 mg/kg, 40 mg/kg, 41 mg/kg, 42 mg/kg, 43 mg/kg, 44 mg/kg, 45 mg/kg, 46 mg/kg, 47 mg/kg, 48 mg/kg, 49 mg/kg 50 mg/kg, 51 mg/kg, 52.
  • mg/kg 53 mg/kg, 54 mg/kg, 55 mg/kg, 56 mg/kg, 57 mg/kg, 58 mg/kg, 59 mg/kg, 60 mg/kg, 65 mg/kg, 70 mg/kg, 75 mg/kg, 80 mg/kg, 85 mg/kg, 90 mg/kg, 95 mg/kg, 100 mg/kg, including all integers in between.
  • the effective daily dose of the active compound may be administered as two, three, four, five, six or more sub-doses administered separately at appropriate intervals throughout the day, optionally, in unit dosage forms.
  • dosing is one administration per day.
  • dosing is one or more administration per every 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 days, or every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 weeks, or every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months, as needed, to maintain the desired expression of a PNPLA3 pre-mRNA and/or a PNPLA3 protein.
  • An RTSM may be administered in continuously or in cycles.
  • an RTSM of the present can be administered, generally at regular intervals (e.g,, daily, weekly, biweekly, monthly, bimonthly).
  • An RTSM may be administered at regular intervals, e.g., daily; once every two days; once every’ three days; once every 3 to 7 days; once every 3 to 10 days; once every 7 to 10 days; once every week; once every two weeks; once monthly.
  • an RTSM may be administered once weekly by intravenous infusion.
  • An RTSM may be administered intermittently over a longer period of time, e.g., for several weeks, months or years.
  • an RTSM may be administered once every' one, two, three, tour, five, six, seven, eight, nine, ten, eleven or twelve months.
  • an RTSM may be administered once every one, two, three, tour or five years.
  • Administration may be followed by, or concurrent with, co-administration with a second agent, for example with an antibiotic, steroid or other therapeutic agent.
  • the treatment regimen may be adjusted (dose, frequency, route, etc.) as indicated, based on the results of immunoassays, other biochemical tests and physiological examination of the subject under treatment.
  • an RTSM of the disclosure may be administered either simultaneously with the other treatment(s), or sequentially in any order and can be temporally spaced up to several days apart.
  • compositions comprising an RTSM described herein and a carrier thereof for administration in a subject.
  • the pharmaceutically acceptable compositions comprise a therapeutically- effective amount of one or more of an RTSM, formulated together with one or more pharmaceutically acceptable: carriers (additives) and/or diluents.
  • an RTSM herein when administered as pharmaceuticals, to humans and animals, they can be given per se or as a pharmaceutical composition containing, for example, 0.1 to 99%, or 10 to 30% of an RTSM in combination with a pharmaceutically acceptable carrier.
  • a pharmaceutical composition of the present disclosure can be delivered, e.g., subcutaneously or intravenously with a standard needle and syringe or a pen delivery device.
  • the injectable preparations may include dosage forms for intravenous, subcutaneous, intracutaneous and intramuscular injections, drip infusions, etc.
  • the injectable preparations may be prepared, e.g., by dissolving, suspending or emulsifying an RTSM herein in a sterile aqueous medium or an oily medium conventionally used for injections.
  • aqueous medium for injections there are, for example, physiological saline, an isotonic solution containing glucose and other auxiliary agents, etc., which may be used in combination with an appropriate solubilizing agent such as an alcohol (e.g., ethanol), a polyalcohol (e.g., propylene glycol, polyethylene glycol), a nonionic surfactant [e.g., polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil)], etc.
  • an alcohol e.g., ethanol
  • a polyalcohol e.g., propylene glycol, polyethylene glycol
  • a nonionic surfactant e.g., polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil
  • oily medium there are employed, eg., sesame oil, soybean oil, etc., which may be used in combination with a solubilizing agent such as benzyl benzoate, benzyl alcohol, etc.
  • a solubilizing agent such as benzyl benzoate, benzyl alcohol, etc.
  • compositions of the present disclosure can be in the form of, tor example, granules, powders, tablets, capsules, syrup, suppositories, injections, emulsions, elixirs, suspensions or solutions.
  • the amount of the aforesaid antibody contained can be about 5 to about 500 mg per dosage form in a unit dose.
  • an RTSM can be contained in about, in about 5 to about. 100 mg, for example for a parental dosage form.
  • an RTSM can be contained in about 10 to about 250 mg for the other dosage forms.
  • oral, buccal, and sublingual administration powders, suspensions, granules, tablets, pills, capsules, gelcaps, and caplets can be used as solid dosage forms. These can be prepared, for example, by mixing the RTSM, with at least one additive such as a starch or other additive.
  • Suitable additives are sucrose, lactose, cellulose sugar, mannitol, maltitol, dextran, starch, agar, alginates, chitins, chitosans, pectins, tragacanth gum, gum arable, gelatins, collagens, casein, albumin, synthetic or semi-synthetic polymers or glycerides.
  • oral dosage forms can contain other ingredients to aid in administration, such as an inactive diluent, or lubricants such as magnesium stearate, or preservatives such as paraben or sorbic acid, or anti-oxidants such as ascorbic acid, tocopherol or cysteine, a disintegrating agent, binders, thickeners, buffers, sweeteners, flavoring agents or perfuming agents. Tablets and pills may be further treated with suitable coating materials known in the art.
  • Liquid dosage forms for oral administration may be in the form of pharmaceutically acceptable emulsions, syrups, elixirs, suspensions, and solutions, which may contain an inactive diluent, such as water.
  • pharmaceutical formulations and medicaments may be prepared as liquid suspensions or aqueous solutions, for example, using a sterile liquid, such as, but not limited to, an oil, water, an alcohol, and combinations of these.
  • pharmaceutical compositions can be prepared in a lyophilized form.
  • the lyophilized preparations can comprise a cryoprotectant known in the art.
  • cryoprotectants as used herein generally includes agents, which provide stability to the protein from freezing-induced stresses.
  • cryoprotectants include polyols such as, for example, mannitol, and include saccharides such as, for example, sucrose, as well as including surfactants such as, for example, polysorbate, poloxamer or polyethylene glycol, and the like. Cryoprotectants also contribute to the tonicity of the formulations. Pharmaceutically suitable surfactants, suspending agents, emulsifying agents, may be added for oral or par- enteral administration.
  • suspensions may include oils.
  • oils include, but are not limited to, peanut oil, sesame oil, cottonseed oil, com oil and olive oil.
  • Suspension preparation may also contain esters of faty acids such as ethyl oleate, isopropyl myristate, fatty acid glycerides and acetylated fatty acid glycerides.
  • Suspension formulations may include alcohols, such as, but not limited to, ethanol, iso- propyl alcohol, hexadecyl alcohol, glycerol and propylene glycol.
  • Ethers such as but not limited to, poly(ethyleneglycoi), petroleum hydrocarbons such as mineral oil and petrolatum: and water may also be used in suspension formulations.
  • the pharmaceutical fonnulations and medicaments may be a spray or aerosol containing an appropriate solvent(s) and optionally other compounds such as, but not limited to, stabilizers, antimicrobial agents, antioxidants, pH modifiers, surfactants, bio- availability modifiers and combinations of these.
  • a propellant for an aerosol Formulation may include compressed air, nitrogen, carbon dioxide, or a hydrocarbon based low boiling solvent.
  • Injectable dosage forms generally include aqueous suspensions or oil suspensions which may be prepared using a suitable dispersant or wetting agent and a suspending agent. Injectable forms may be in solution phase or in the form of a suspension, wdrich can be prepared with a solvent or diluent. Acceptable solvents or vehicles include sterilized water, Ringer’s solution, or an isotonic aqueous saline solution. Alternatively, sterile oils may be employed as solvents or suspending agents. In some embodiments, the oil or fatty acid is non-volatile, including natural or synthetic oils, fatty acids, mono-, di- or tri -glycerides.
  • the pharmaceutical formulation and/ or medicament may be a powder suitable for reconstitution with an appropriate solution as described above.
  • these include, but are not limited to, freeze dried, rotary dried or spray dried powders, amorphous powders, granules, precipitates, or particulates.
  • the formulations may optionally contain stabilizers, pH modifiers, surfactants, bioavailability modifiers and combinations of these.
  • the pharmaceutical formulations and medicaments may be in the form of a suppository, an ointment, an enema, a tablet or a cream for release of compound in the intestines, sigmoid flexure and/or rectum.
  • Rectal suppositories are prepared by mixing one or more compounds herein with acceptable vehicles, for example, cocoa butter or polyethylene glycol, which is present in a solid phase at normal storing temperatures, and present in a liquid phase at those temperatures suitable to release a drug inside the body, such as in the rectum. Oils may also be employed in the preparation of formulations of the soft gelatin type and suppositories.
  • RTSM tetrahydroxymethyl cellulose
  • glycerol glycerol
  • the concentration of an RTSM in these compositions can vary 7 widely, e.g., from less than about 10%, least about 25% to as much as 75% or 90% by weight and wi 11 be selected primarily by fluid volumes, viscosities, etc., in accordance with the particular mode of administration selected.
  • compositions comprising an RTSM described herein can be formulated using one or more physiologically acceptable carriers including excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen.
  • compositions are optionally manufactured such as, by way of example only, by means of mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or compression processes.
  • compositions may also include one or more pH adjusting agents or buffering agents, including acids such as acetic, boric, citric, lactic, phosphoric and hydrochloric acids; bases such as sodium hydroxide, sodium phosphate, sodium borate, sodium citrate, sodium acetate, sodium lactate and tris-hydroxymethylaminomethane; and buffers such as citrate/dextrose, sodium bicarbonate and ammonium chloride.
  • acids such as acetic, boric, citric, lactic, phosphoric and hydrochloric acids
  • bases such as sodium hydroxide, sodium phosphate, sodium borate, sodium citrate, sodium acetate, sodium lactate and tris-hydroxymethylaminomethane
  • buffers such as citrate/dextrose, sodium bicarbonate and ammonium chloride.
  • Such acids, bases and buffers are included in an amount required to maintain pH of the composition in an acceptable range.
  • compositions may also include one or more salts in an amount required to bring osmolality of the composition into
  • Such salts include those having sodium, potassium or ammonium cations and chloride, citrate, ascorbate, borate, phosphate, bicarbonate, sulfate, thiosulfate or bisulfite anions; suitable salts include sodium chloride, potassium chloride, sodium thiosulfate, sodium bisulfite and ammonium sulfate.
  • sustained-release preparations can be used.
  • Suitable examples of sustained- release preparations include semipermeable matrices of solid hydrophobic polymers containing an antibody or antigen binding fragment of the present disclosure, in which the matrices are in the form of shaped articles, e.g., films, or microcapsule.
  • sustained-release matrices include polyesters, hydrogels (for example, poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides (U.S. Patent No.
  • copolymers of L-glutamic acid and y ethyl-L-giutamate non-degradable ethylene-vinyl acetate
  • degradable lactic acid-glycolic acid copolymers such as the LUPRON DEPOTTM (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate)
  • poly-D-(-)-3-hydroxybutyric acid While polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid enable release of molecules for over 100 days, certain hydrogels release proteins for shorter time periods.
  • encapsulated antibodies When encapsulated antibodies remain in the body for a long time, they can denature or aggregate as a result of exposure to moisture at 37°C, resulting in a loss of biological activity and possible changes in immunogenicity. Rational strategies can be devised for stabilization depending on the mechanism involved. For example, if the aggregation mechanism is discovered to be intermolecular S--S bond formation through thiodisulfide interchange, stabilization can be achieved by modifying sulfhydryl residues, lyophilizing from acidic solutions, controlling moisture content, using appropriate additives, and developing specific polymer matrix compositions.
  • the pharmaceutical composition can be delivered in a controlled release system.
  • a pump may be.
  • polymeric materials can be used.
  • a controlled release system can be placed in proximity of the composition's target, thus requiring only a fraction of the systemic dose.
  • an RTSM can be administered with one or more agents capable of promoting penetration of the subject antisense oligonucleotide across the blood-brain barrier.
  • an RTSM can be linked with a viral vector, e.g., to render an RTSM more effective or increase transport across the blood-brain barrier.
  • delivery’ of agents can be by administration of an adenovirus vector to motor neurons in muscle tissue. Delivery of vectors directly to the brain, include but are not limited to the striatum, tire thalamus, the hippocampus, or the substantia nigra.
  • an RTSM can be linked or conjugated with agents that provide desirable pharmaceutical or pharmacodynamic properties.
  • an RTSM can be coupled to a substance that promotes penetration or transport across the blood-brain barrier, e.g., an antibody to the transferrin receptor.
  • osmotic blood brain barrier disruption can be assisted by infusion of sugars, e.g., meso erythritol, xylitol, D(+) galactose, D(+) lactose, D(+) xylose, dulcitol, myo- inositol, L(-) fructose, D(-) mannitol, D(t-) glucose, D(+) arabinose, D(-) arabinose, cellobiose, D(-i-) maltose, D(-f-) raffinose, L(-i-) rhamnose, D(t-) melibiose, D(-) ribose, adonitol, D(+) arabitol,
  • compositions herein may be administered alone or in combination with another therapeutic. Hie additional therapeutic may be administered prior, concurrently or subsequently to the administration of the composition.
  • compositions disclosed herein, comprising an RTSM, described herein can also contain more than one active agent as necessary for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other.
  • the composition can further comprise an anti-inflammatory', a therapeutic protein, a steroid, an analgesic, a non-steroidal anti- inflammatory, a corticosteroid, and combinations thereof.
  • the compositions may be administered in combination with a steroid and/or an antibiotic.
  • Hie steroid may be a glucocorticoid or prednisone.
  • Glucocorticoids such as cortisol control carbohydrate, fat and protein metabolism, and are anti- inflammatory by preventing phospholipid release, decreasing eosinophil action and a number of other mechanisms.
  • Mineralocorticoids such as aldosterone control electrolyte and water levels, mainly by promoting sodium retention in the kidney.
  • Corticosteroids are a class of chemicals that includes steroid hormones naturally produced in the adrenal cortex of vertebrates and analogues of these hormones that are synthesized in laboratories. Corticosteroids are involved in a wide range of physiological processes, including stress response, immune response, and regulation of inflammation, carbohydrate metabolism, protein catabolism, blood electrolyte levels, and behavior.
  • Corticosteroids include Betamethasone, Budesonide, Cortisone, Dexamethasone, Hydrocortisone, Methylprednisolone, Prednisolone, and Prednisone.
  • Such molecules are suitably present in combination in amounts that are effective for the purpose intended.
  • Tire active ingredients of the compositions comprising an antibody or antigen binding fragment thereof described herein can also be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsules and poly-(methylmethacylate) microcapsules, respectively, in colloidal drag delivery systems (for example, liposomes, albumin microspheres, microparticle, microemulsions, nano-particles and nanocapsules) or in macroemulsions.
  • Hie pharmaceutical composition can be also delivered in a vesicle, in particular a liposome.
  • Liposomes include emulsions, foams, micelles, insoluble monolayers, phospholipid dispersions, lamellar layers and the like, and can serve as vehicles to target the M-CSF antibodies to a particular tissue as well as to increase the half life of the composition.
  • Liposomes containing the RTSM, the second active compound or both can be generated by the reverse phase evaporation method with a lipid composition comprising phosphatidylcholine, cholesterol and PEG-derivatized phosphatidylethanolamine (PEG-PE), Liposomes are extruded through filters of defined pore size to yield liposomes with the desired diameter.
  • RTSMs herein can be conjugated to liposomes via a disulfide interchange reaction.
  • the second agent can be optionally contained within the liposome.
  • the second agent may be formulated with the compositions described herein or separately co-administered.
  • a method of decreasing expression of full length PNPLA3 protein comprising contacting a PNPLA3 RNA with a therapeutic agent that binds to a portion of the PNPLA3 RNA, whereby the therapeutic agent causes skipping of an exon in the PNPLA3 RNA that is spliced in the absence of the therapeutic agent.
  • a method of treating a hepatic disease comprising administering a therapeutic agent that binds to a portion of a PNPLA3 RNA to a subject, whereby the therapeutic agent causes skipping of an exon in the PNPLA3 RNA that is spliced in the absence of the therapeutic agent.
  • a pharmaceutical composition comprising a therapeutic agent and a pharmaceutically acceptable excipient, wherein the therapeutic agent binds to a portion of a PNPLA3 RNA.
  • ASO antisense oligomer
  • oligomer such as a polynucleotide, comprising nucleobases that hybridizes to a target nucleic acid (e.g. , a IFIH1 containing pre-mRNA) sequence by Watson-Crick base pairing or wobble base pairing (G-U).
  • the ASO may have exact sequence complementary to tire target sequence or near complementarity (e.g., sufficient complementarity to bind the target sequence and modulating splicing at a splice site).
  • ASOs are designed so that they bind (hybridize) to a target nucleic acid (e.g., a targeted portion of a pre-mRNA transcript) and remain hybridized under physiological conditions. Typically, if they hybridize to a site other than the intended (targeted) nucleic acid sequence, they hybridize to a limited number of sequences that are not a target nucleic acid (to a few sites other than a target nucleic acid).
  • a target nucleic acid e.g., a targeted portion of a pre-mRNA transcript
  • Design of an ASO can take into consideration the occurrence of the nucleic acid sequence of the targeted portion of the pre-mRNA transcript or a sufficiently similar nucleic acid sequence in other locations in the genome or cellular pre- mRNA or transcriptome, such that the likelihood the ASO will bind other sites and cause "off-target” effects is limited.
  • a SOs "specifically hybridize” to or are “specific” to a target nucleic acid or a targeted portion of a pre-mRNA.
  • hybridization occurs with a Tin substantially greater than 37°C, preferably at least 50°C, and typically between 60°C to approximately 90°C
  • Tin is the temperature at which 50% of a target sequence hybridizes to a complementary oligonucleotide.
  • Oligomers such as oligonucleotides, are "complementary" to one another when hybridization occurs in an antiparallel configuration between two single-stranded polynucleotides.
  • a double-stranded polynucleotide can be “complementary” to another polynucleotide, if hybridization can occur between one of the strands of the first polynucleotide and the second.
  • Complementarity (the degree to which one polynucleotide is complementary with another) is quantifiable in terms of the proportion (e.g., the percentage) of bases in opposing strands that are expected to form hydrogen bonds with each other, according to generally accepted base-pairing rales.
  • Tire sequence of an antisense oligomer need not be 100% complementary to that of its target nucleic acid to hybridize.
  • ASOs can comprise at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence complementarity to a target region within the target nucleic acid sequence to which they are targeted.
  • an ASO in which 18 of 20 nucleobases of the oligomeric compound are complementary to a target region, and would therefore specifically hybridize would represent 90 percent complementarity.
  • the remaining non-complementary nucleobases may be clustered together or interspersed with complementary nucleobases and need not be contiguous to each other or to complementary nucleobases.
  • Percent complementarity- 7 of an ASO with a region of a target nucleic acid can be determined routinely using BLAST programs (basic local alignment search tools) and PowerBLAST programs known in the art (Altschul, et al., J. Mol. Biol., 1990, 215, 403- 410; Zhang and Madden, Genome Res., 1997, 7, 649-656).
  • An ASO need not hybridize to all nucleobases in a target sequence and the nucleobases to which it does hybridize may be contiguous or noncontiguous. ASOs may hybridize over one or more segments of a pre-mRNA transcript, such that intervening or adjacent segments are not involved in the hybridization event (e.g., a loop structure or hairpin structure may be formed). In certain embodiments, an ASO hybridizes to noncontiguous nucleobases in a target pre-mRNA transcript. For example, an ASO can hybridize to nucleobases in a pre-mRNA transcript that are separated by one or more nncleobase(s) to which the ASO does not hybridize.
  • the ASOs described herein comprise nucleobases that are complementary to nucleobases present in a targeted portion of a pre-mRNA.
  • ASO embodies oligonucleotides and any other oligomeric molecule that comprises nucleobases capable of hybridizing to a complementary nucleobase on a target mRNA but does not comprise a sugar moiety, such as a peptide nucleic acid (PNA).
  • PNA peptide nucleic acid
  • the ASOs may comprise naturally-occurring nucleotides, nucleotide analogs, modified nucleotides, or any combination of two or three of the preceding.
  • the term "naturally occurring nucleotides" includes deoxyribonucleotides and ribonucleotides.
  • modified nucleotides includes nucleotides with modified or substituted sugar groups and/or having a modified backbone. In some embodiments, all of the nucleotides of the ASO are modified nucleotides.
  • Chemical modifications of ASOs or components of ASOs that are compatible with the methods and compositions described herein will be evident to one of skill in the art and can be found, for example, in U.S. Pat. No. 8,258,109, U.S. Pat. No. 5,656,612, U.S. Patent Publication No. 2012/0190728, and Dias and Stein, Mol. Cancer Ther. 2002, 347-355, herein incorporated by reference in their entirety.
  • nucleobases of an ASO may be any naturally occurring, unmodified nucleobase such as adenine, guanine, cytosine, thymine and uracil, or any synthetic or modified nucleobase that is sufficiently similar to an unmodified nucleobase such that it is capable of hydrogen bonding with a nucleobase present on a target pre-mRNA.
  • modified nucleobases include, without limitation, hypoxanthine, xanthine, 7-methylguanine, 5, 6-dihydrouracil, 5-methylcytosine, and 5- hydroxymethoylcytosine.
  • Tire ASOs described herein also comprise a backbone structure that connects the components of an oligomer.
  • backbone structure and “oligomer linkages” may be used interchangeably and refer to the connection between monomers of the ASO.
  • the backbone comprises a 3 ’-5 ’ phosphodiester linkage connecting sugar moieties of the oligomer.
  • the backbone structure or oligomer linkages of the ASOs described herein may include (but are not limited to) phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate, phosphoramladate, phosphoramidate, and the like. See, e.g., LaPlanche, et al., Nucleic Acids Res. 14:9081 (1986); Stec, et al., J. Am. Chem. Soc. 106:6077 (1984), Stem, et al.. Nucleic Acids Res.
  • the backbone structure of the ASO does not contain phosphorous but rather contains peptide bonds, for example in a peptide nucleic acid (PN A), or finking groups including carbamate, amides, and linear and cyclic hydrocarbon groups.
  • PN A peptide nucleic acid
  • the backbone modification is a phosphothioate linkage. In some embodiments, the backbone modification is a phosphoramidate linkage. [02021 Any of the ASOs described herein may contain a sugar moiety that comprises ribose or deoxyribose, as present in naturally occurring nucleotides, or a modified sugar moiety or sugar analog, including a morpholine ring.
  • Non-limiting examples of modified sugar moieties include 2' substitutions such as 2'-O-methyl (2'-O-Me), 2‘-O-methoxyethyl (2'MOE), 2'-O-aminoethyl, 2'F; N3'->P5' phosphoramidate, 2’dimethylaminooxyethoxy, 2’dimethylaminoethoxyethoxy, 2'-guanidinidium, 2 -0- guanidinium ethyl, carbamate modified sugars, and bicyclic modified sugars.
  • the sugar moiety modification is selected from 2'-0-Me, 2'F, and 2'MOE.
  • the sugar moiety modification is an extra bridge bond, such as in a locked nucleic acid (LNA).
  • the sugar analog contains a morpholine ring, such as phosphorodiamidate morpholino (PMO).
  • the sugar moiety comprises a ribofuransyl or 2‘deoxyribofuransyl modification.
  • the sugar moiety’ comprises 2'4 '-constrained 2'O-methyloxyethyl (cMOE) modifications.
  • the sugar moiety comprises cEt 2’, 4' constrained 2'-0 ethyl BNA modifications.
  • the sugar moiety comprises tricycloDNA (tcDNA) modifications.
  • the sugar moiety comprises ethylene nucleic acid (ENA) modifications.
  • the sugar moiety comprises MCE modifications. Modifications are known in the art and described in the literature, e.g, by Jarver, et al, 2014, "A Chemical View of Oligonucleotides for Exon Skipping and Related Drug Applications,” Nucleic Acid Therapeutics 24(1): 37-47, incorporated by reference for this purpose herein.
  • each monomer of the ASO is modified in the same way, for example each linkage of the backbone of the ASO comprises a phosphorothioate linkage or each ribose sugar moiety comprises a 2'0-methyI modification.
  • Such modifications that are present on each of the monomer components of an ASO are referred to as “uniform modifications.”
  • a combination of different modifications may be desired, for example, an ASO may comprise a combination of phosphorodiamidate linkages and sugar moieties comprising morpholine rings (morpholinos).
  • Combinations of different modifications to an ASO are referred to as “mixed modifications” or “mixed chemistries.”
  • the ASO comprises one or more backbone modifications. In some embodiments, the ASO comprises one or more sugar moiety modification. In some embodiments, the ASO comprises one or more backbone modifications and one or more sugar moiety modifications. In some embodiments, the ASO comprises a 2'MOE modification and a phosphorothioate backbone. In some embodiments, the ASO comprises a phosphorodiamidate morpholino (PMO). In some embodiments, the ASO comprises a peptide nucleic acid (PNA).
  • PMO phosphorodiamidate morpholino
  • any of the ASOs or any component of an ASO may be modified in order to achieve desired properties or activities of the ASO or reduce undesired properties or activities of the ASO.
  • an ASO or one or more components of any ASO may be modified to enhance binding affinity’ to a target sequence on a pre-mRNA transcript; reduce binding to any non-target sequence; reduce degradation by cellular nucleases (i.e, RNase H); improve uptake of the ASO into a cell and/or into the nucleus of a cell; alter the pharmacokinetics or pharmacodynamics of the ASO; and/or modulate the half-life of the ASO.
  • the ASOs are comprised of 2'-O-(2-methoxyethyl) (MOE) phosphorothioate-modified nucleotides.
  • MOE 2'-O-(2-methoxyethyl)
  • ASOs comprised of such nucleotides are especially well-suited to the methods disclosed herein; oligomers having such modifications have been shown to have significantly enhanced resistance to nuclease degradation and increased bioavailability, making them suitable, for example, for oral delivery in some embodiments described herein. See e.g, Geary, et al., J Pharmacol Exp Ther. 2001 ; 296(3):890-7; Geary, et al, J Pharmacol Exp Ther. 2001; 296(3):898-904.
  • compositions and methods useful for treating hepatic diseases may be obtained from a commercial source.
  • these compositions and methods result in a truncated PNPLA3 protein.
  • these compositions and methods result in a decrease in the wild-type PNPLA3 protein.
  • the compositions and methods result in modulating the splicing of PNPLA3 RNA,
  • the compositions and methods result in an PNPLA3 RN A lacking exon 5.
  • a method of decreasing expression of foil length PNPLA3 protein comprising contacting a PNPLA3 RNA with a therapeutic agent that binds to a portion of the PNPLA3 RNA, whereby the therapeutic agent causes skipping of an exon in the PNPLA3 RNA that is spliced in the absence of the therapeutic agent.
  • the therapeutic agent causes skipping of exon 5 in the PNPLA3 RNA.
  • the therapeutic agent binds to a 5" splice site sequence in the PNPLA3 RNA.
  • the 5’ splice site sequence is in Intron 5 of the PNPLA3 RNA.
  • the 5’ splice site comprises a sequence with at least 95% sequence identity to SEQ ID No: 43.
  • the therapeutic agent is an antisense oligonucleotide (ASO).
  • the ASO comprises a sequence that is at least about 80% identity 7 to SEQ ID NO: 83, In some embodiments, the ASO comprises a sequence that is at least about 90% identity to SEQ ID NO: 83.
  • hepatic disease comprising administering a therapeutic agent that binds to a portion of a PNPLA3 RNA to a subject, whereby the therapeutic agent causes skipping of an exon in the PNPLA3 RNA that is spliced in the absence of the therapeutic agent.
  • the hepatic disease is nonalcoholic steatohepatitis (NASH).
  • a pharmaceutical composition comprising a therapeutic agent and a pharmaceutically acceptable excipient, wherein the therapeutic agent binds to a portion of a
  • the therapeutic agent is an antisense oligomer (ASO) and wherein the ASO comprises a sequence that is at least about 80%, 85%, 90%, 95%, 97%, or 100% identity to any one of SEQ ID NO: 83.
  • ASO antisense oligomer
  • the ASO comprises a backbone modification comprising a phosphorothioate linkage or a phosphorodiamidate linkage.
  • the ASO comprises a phosphorodiamidate morpholino, a locked nucleic acid, a peptide nucleic acid, a 2'-O-methyl, a 2'-Fluoro, or a 2'-O- niethoxyethyl moiety.
  • the ASO comprises at least one modified sugar moiety. In some embodiments, each sugar moiety is a modified sugar moiety.
  • the ASO consists of from 8 to 50 nucleobases, 8 to 40 nucleobases, 8 to 35 nucleobases, 8 to 30 nucleobases, 8 to 25 nucleobases, 8 to 20 nucleobases, 8 to 15 nucleobases, 9 to 50 nucleobases, 9 to 40 nucleobases, 9 to 35 nucleobases, 9 to 30 nucleobases, 9 to 25 nucleobases, 9 to 20 nucleobases, 9 to 15 nucleobases, 10 to 50 nucleobases, 10 to 40 nucleobases, 10 to 35 nucleobases, 10 to 30 nucleobases, 10 to 25 nucleobases, 10 to 20 nucleobases, 10 to 15 nucleobases, 1 1 to 50 nucleobases, 11 to 40 nucleobases, 11 to 35 nucleobases, 11 to 30 nucleobases, 11 to 25 nucleobases, 11 to 20 nucleobases,
  • the ASO is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, complementary 7 to the targeted portion of the PNPLA3 mRNA encoding the PNPLA3 protein.
  • the method further comprises assessing PNPLA3 mRNA or PNPLA3 protein expression.
  • the cells are ex vivo.
  • the therapeutic agent is administered to the subject by mtravitreal injection, intrathecal injection, intraperitoneal injection, subcutaneous injection, intravenous injection, subretinal injection, intracerebro ventricular injection, intramuscular injection, topical application, or implantation.
  • the therapeutic agent is administered with one or more agents capable of promoting penetration of the subject antisense oligonucleotide across the blood-brain barrier by any method known in the art.
  • the therapeutic agent is linked with a viral vector, e.g., to render the therapeutic agent more effective or increase transport across the blood-brain barrier.
  • the therapeutic agent is linked or conjugated with agents that provide desirable pharmaceutical or pharmacodynamic properties.
  • the therapeutic agent is coupled to a substance, known in the art to promote penetration or transport across the blood-brain barrier, e.g., an antibody to the transferrin receptor.
  • osmotic blood brain barrier disruption is assisted by infusion of sugars, e.g., meso erythritol, xylitol, D(+) galactose, D(+) lactose, D(+) xylose, dulcitol, myo-inositol, L(-) fructose, D(-) mannitol, D(+) glucose, D(+) arabinose, D(-) arabinose, cellobiose, D(+) maltose, D(-t-) raffinose, L(-i-) rhamnose, D(t-) melibiose, D(-) ribose, adonitol, D(+) arabitol, L(-) arabitol, D(t-) fucose, L(-) fucose, D(-) lyxose, L(t-) lyxo
  • sugars
  • the therapeutic agent is encapsulated in glucose-coated polymeric nanocarriers, such as those described in Min et al. "‘Systemic Brain Delivery' of Antisense Oligonucleotides across the Blood-Brain Barner with a Glucose-Coated Polymeric Nanocarrier,” Angew. Chem. Int. Ed. 2020, 59, 8173-8180, incorporated herein by reference.
  • a method of decreasing expression of full length PNPLA3 protein comprising contacting a PNPLA3 RNA with a therapeutic agent that binds to a portion of the PNPLA3 RNA, whereby the therapeutic agent causes skipping of an exon in the PNPLA3 RNA that is spliced in the absence of the therapeutic agent.
  • the therapeutic agent causes skipping of exon 5 in the PNPLA3 RNA.
  • the therapeutic agent binds to a 5’ splice site sequence in the PNPLA3 RNA.
  • the 5’ splice site sequence is in Intron 5 of the PNPLA3 RNA.
  • the 5’ splice site comprises a sequence with at least 95% sequence identity to SEQ ID No: 43.
  • the therapeutic agent is an antisense oligonucleotide (ASO).
  • ASO antisense oligonucleotide
  • the ASO comprises a sequence that is at least about 80% identity to SEQ ID NO: 83.
  • the ASO comprises a sequence that is at least about 90% identity to SEQ ID NO: 83.
  • disclosed herein is a method of treating a hepatic disease comprising administering a therapeutic agent that binds to a portion of a PNPLA3 RNA to a subject, whereby the therapeutic agent causes skipping of an exon in tire PNPLA3 RNA that is spliced in the absence of the therapeutic agent,
  • the therapeutic agent causes skipping of exon 5 in the PNPLA3 RNA.
  • the therapeutic agent binds to a 5’ splice site sequence in the PNPLA3 RNA.
  • the 5’ splice site sequence is in Intron 5 of the PNPLA3 RNA.
  • the 5’ splice site comprises a sequence with at least 95% sequence identity to SEQ ID No: 43.
  • the therapeutic agent is an antisense oligonucleotide (ASO).
  • ASO antisense oligonucleotide
  • the ASO comprises a sequence that is at least about 80% identity to SEQ ID NO: 83.
  • the ASO comprises a sequence that is at least about 90% identity to SEQ ID NO: 83.
  • hepatic diseases is NASH.
  • a pharmaceutical composition comprising a therapeutic agent and a pharmaceutically acceptable excipient, wherein the therapeutic agent binds to a portion of a PNPLA3 RNA.
  • the therapeutic agent causes skipping of an exon in the PNPLA3 RN A that is spliced in the absence of the therapeutic agent.
  • the therapeutic agent causes skipping of exon 5 in the PNPLA3 RNA.
  • the therapeutic agent binds to a 5’ splice site sequence in the PNPLA3 RNA.
  • the therapeutic agent binds to the 5 ’ splice site sequence is in Intron 5 of the PNPLA3 RNA
  • the 5’ splice site comprises a sequence with at least 95% sequence identity to SEQ ID No: 43.
  • the therapeutic agent is an antisense oligonucleotide (ASO).
  • ASO antisense oligonucleotide
  • the ASO comprises a sequence that is at least about 80% identity to SEQ ID NO: 83.
  • the ASO comprises a sequence that is at least about 90% identity to SEQ ID NO: 83.
  • nucleobases corresponding to the abbreviations in various nucleobase sequences disclosed herein can be found in Table 9A below.
  • Table 9A Nudeobase Abbreviations
  • amino acids corresponding to the abbreviations in various polypeptide sequences disclosed herein can be found, for example, in Table 9B below.
  • ASOs described herein can be synthesized using synthetic techniques, using methods described herein or combinations of both. Alternatively, ASOs are available commercially from various sources, including Integrated DNA Technologies (IDT), Coralville, Iowa and Gene Tools, LLC.
  • Example 1 PNPLA3 splicing modulation in hepatocellular carcinoma cells
  • SNL-886 is an adult hepatocellular carcinoma cell line, and it contains wild type PNPLA3.
  • SN U- 886 cells (KCLB 00886) were plated at a concentration of 125,000 cells/well in a 12-well plate and were cultured using RPMI 1640 (Gibco, Cat # A10491-01) supplemented with 1% Penicillin-Streptomycin (Gibco, #15140163), and 10% Fetal Bovine Seram (Gibco, Cat# A38401-01) for 24 hours.
  • Hep G2 is an adolescent hepatocellular carcinoma cell line, and it carries the rs738409 variant in PNPLA3.
  • Hep G2 cells ATCC® HB8065
  • EMEM with L-Glutamine ATCC, Cat #30-2003
  • MEM Non-Essential Amino Acids Gibco, #11140076
  • Penicillin-Streptomycin Gibco, #15140163
  • Fetal Bovine Seram Gibco, Cat# A38401-01
  • ASOs with morpholino-modified backbones were transfected individually into each well using Endo-Porter (Gene-Tools, LLC at 6ul/ml). Each morpholino was at a 500uM stock concentration and was added to the wells to achieve final concentrations of 5uM (10 pl/well) and 10 pM (20 pL/well) for control and PNPLA3 morpholinos.
  • NTC is a non-targeting control morpholino
  • PNPLA3 E5-I5 morphRK (labeled as 5’SS in FIGS. 1 and 2) is a morpholino that targets PNPLA3 at the Exon5-Intron5 boundary'.
  • Tire sequences of the ASOs used are set forth below in Table 10.
  • RNA samples were harvested after 48hrs. Six hours before harvest, samples were treated with either 100 pg/mL. cycloheximide (Sigma C4859) or 0.1%DMSO (Tocris Bioscience 3176/25mL).
  • RNA was prepared from each sample, using the RNeasy plus mini kits (Qiagen # 74134). RNA was quantitated on a ThermoScientific NanodropTM 8000 Spectrophotometer, and cDNA was synthesized with lug total RNA using SuperScriptTM III First-Strand Synthesis SuperMix (Invitrogen 11752-050). RT-PCR reactions were performed using Invitrogen PlatinumTM SuperFiTM DNA Polymerase (Invitrogen 12351050), with the following primers:
  • Example 2 In Vivo Activity of an PNPLA3-RTSM on PNPLA3 pre-mRNA and PNPLA3 protein expression
  • mice of 5-6 weeks age can be injected with exemplary murine PNPLA3-RTSMs herein at 5 mg/kg, or PBS, subcutaneously once a day for three days.
  • a PNPLA3-RTSM control according to Example 1 can be administered to the control group using the same regime.
  • blood can be collected and PNPLA3 mRNA and PNPLA3 protein can be evaluated.
  • RT-PCR evaluation of the PNPLA3-RTSM treated mRNA can depict that the mRNA product contains exons 4 and 6, exon 5 having been skipped.
  • BCA assays and SDS-PAGE gel probed with anti-PNPLA3 antibodies can demonstrate that the PNPLA3-RTSMs of the present disclosure decreased the level of functional PNPLA3 protein compared to the control .
  • Example 3 Splicing modification of the PNPLA3 pre-mRNA will prevent the progression of or treat NASH
  • mice predisposed to NASH C57BL/6NTac mice conditioned on a modified Amylin liver NASH (AMLN) diet can be obtained.
  • Diet can be # D091003 lOi (source Research Diets) which contains 40 kcal% fat, 20 kcal% fructose and 2% cholesterol and is an irradiated diet.
  • C57BL/6NTac males are put on this diet at 6 weeks of age and housed at reduced density.
  • Control males are housed in the same location, also at reduced density, and are fed NIH-31M diet. Control males can also be generated using a low-fat purified diet.
  • Mice can be split into two treatment groups, treatment with or without the RTSM herein.
  • NASH B6 mice become obese, get fatty liver and develop liver inflammation and fibrosis after 26+ weeks on diet, with inter-animal variability observed for development of the inflammation and fibrosis phenotype. Observations can include: significantly elevated ALT levels compared to controls, robust steatosis, hepatic inflammation including the presence of hepatic crown-like structures, activated stellate cells, and development of consistent fibrosis by 26 w eeks on diet.
  • RTSM treatment groups can be injected with exemplary murine PNPLA3-RTSMs herein at 5 mg/kg, or PBS, subcutaneously, once a day for up to 90 days.
  • RTSM treated NASH mice exhibit decreased ALT levels, steatosis, hepatic inflammation, and fibrosis compared to the untreated controls.

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Abstract

Provided herein are methods and compositions for decreasing the expression of a protein, and for treating a subject in need thereof, e.g., a subject with excess protein expression or a subject having an associate disease described herein.

Description

RNA-TARGETING SPLICING MODIFIERS FOR TREATMENT OF PNPLA3- ASSOCIATED CONDITIONS AND DISEASES RELATED APPLICATIONS [0001] This application claims priority to U.S. Provisional Patent Application No. 63/114,871, filed November 17, 2020 the contents of which is entirely incorporated herein by reference for all purposes. SUMMARY [0002] Disclosed herein is a method of treating a disease or condition in a human subject in need thereof, the method comprising: administering to the subject a therapeutically effective amount of a synthetic Patatin-like phospholipase domain-containing 3 protein (PNPLA3) RNA-targeting splicing modifier (RTSM), thereby treating the disease or condition in the human subject in need thereof; wherein: the synthetic RTSM comprises a binding domain that binds to a target region of the PNPLA3 pre-messenger ribonucleic acid (pre-mRNA); the target region comprises an exon-intron junction comprising a target sequence of Formula (I): KGUR, wherein K is G or U; wherein R is A or G; and exon skipping is increased as compared to the PNPLA3 pre-mRNA spliced in the absence of RTSM as demonstrated by an in vitro assay. [0003] Disclosed herein is a synthetic PNPLA3 RTSM that comprises a binding domain that binds to a target region of a PNPLA3 pre-mRNA; wherein: the target region comprises an exon-intron junction comprising a target sequence of Formula (I): KGUR, wherein K is G or U; wherein R is A or G; and exon skipping is increased as compared to when the PNPLA3 pre-mRNA is spliced in the absence of the synthetic RTSM as demonstrated in an in vitro assay. INCORPORATION BY REFERENCE [0004] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. BRIEF DESCRIPTION OF THE DRAWINGS [0005] FIG. 1 shows results from RT-PCR analysis of PNPLA3 isoforms present in SNU-886 cells following treatment with antisense oligonucleotides (ASOs). [0006] FIG. 2 shows results from RT-PCR analysis of PNPLA3 isoforms present in Hep G2 cells following treatment with antisense oligonucleotides (ASOs). DETAILED DESCRIPTION [0007] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which embodiments herein belongs. Any methods, devices and materials similar or equivalent to those described herein can be used in the practice of embodiments herein. The following definitions are provided to facilitate understanding of certain terms used frequently herein and are not meant to limit the scope of the present disclosure. Definitions
[0008] The term “binding domain” as used herein can comprise a domain or portion of an RTSM which binds to a region or a portion of an PNPLA3 pre-mRNA. The binding can be covalent or non-covalent. Examples of non-covalent binding include binding via hydrogen bonding, Watson-Crick base pairing, wobble-base pairing, and Van der Waals interactions.
[0009] As used herein, the terms "ASO" and "antisense oligomer" are used interchangeably and can refer to an oligomer such as a polynucleotide, comprising nucleobases that hybridizes to a target nucleic acid (e.g., a PNPLA3 containing pre-mRNA) sequence by, for example, Watson-Crick base pairing or wobble base pairing (G-U).
[0010] The terms “complementary'” and “complementarity” can refer to polynucleotides (e.g., a sequence of nucleotides) related by base-pairing rales. For example, the sequence “T-G-A (5 ’-3’),” can be complementary to the sequence “T-C-A (5’-3’).” Complementarity may’ be “partial,” in which only some of the nucleic acid’s bases are matched according to base pairing rales. Alternatively, there may be “complete” or “total” complementarity between the nucleic acids. Furthermore, base pairing may be contiguous or non-contiguous. The degree of complementarity between nucleic acid strands can impact efficiency and strength of hybridization between nucleic acid strands. While perfect complementarity can be desired, some embodiments can include one or more 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 mismatches with respect to a target RNA. A mismatch can be a mismatch between a base on an RTSM and a base on a target RNA. Variations at any location within the oligomer are included. In certain embodiments, variations in sequence near the termini of an oligomer can be within about 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 nucleotides of the 5 ’ and/or 3’ terminus. In some embodiments, a base pairing can be a wobble base pairing.
[0011] A “CRISPR” (Clustered Regularly Interspersed Short Palindromic Repeat) “CRISPR system,” or “CRISPR nuclease system” and their grammatical equivalents can include a non-coding RNA molecule (e.g., guide RNA) that binds to DNA or RNA and CRISPR- Associated (Cas) proteins (e.g., Cas9) with, at least some or none, nuclease functionality (e.g., two nuclease domains).
|0012] The term “exon skipping” can refer to a process by which a portion of an exon, an entire exon, or more than one exon are removed from a pre-processed mRNA so that it or they are not present in a mature RNA, such as an mRNA that is translated into a protein. Accordingly, the portion of the protein that can be otherwise encoded by the skipped exon is not present in the expressed form of the protein, and can create a modulated form of the protein. In some embodiments, a modulated protein may be functional, less functional or non-functional. In some embodiments, a modulated protein may be truncated or subjected to nonsense mediated decay. In certain embodiments, an exon being skipped can be an aberrant exon from the human PNPLA3 gene which may contain a mutation or other alteration in its sequence that otherwise causes mutated forms of the protein. In certain embodiments, an exon being skipped can be a wild-type exon. In certain embodiments, an exon being skipped can be a mutated exon. In some embodiments, an exon being skipped can be any' one or more of exons 1-9 of the PNPLA3 gene, such as 1, 2, 3, 4, 5, 6, 7, 8, or 9.
[0013] The terms “identical” and its grammatical equivalents as used herein or “sequence identity” in the context of two nucleic acid sequences or amino acid sequences of polypeptides can refer to the residues in the two sequences which can be the same when aligned for maximum correspondence over a specified comparison window. A “comparison window”, as used herein, can refer to a segment of at least about: 4, 8, 50, 100, 150, to 200 or more contiguous positions can be compared to a reference sequence of the same number of contiguous positions after the two sequences are aligned optimally. Methods of sequences for comparison can be conducted by7 the local homology' algorithm or by7 computerized implementations of these algorithms including, but not limited to CLUSTAL, GAP, BESTFIT, BLAST, FASTA, and TFASTA. Alignment can be also often performed by inspection and manual alignment. In one class of embodiments, the polypeptides herein can be 50%, 60%, 70%, 75%, 80%, 85%, 90%, 98%, 99% or 100% identical to a reference polypeptide, or a fragment thereof, e.g., as measured by BLASTP (or CLUSTAL, or any other available alignment software) using default parameters. Similarly, nucleic acids can also be described with reference to a starting nucleic acid, e.g., they can be 50%, 60%, 70%, 75%, 80%, 85%, 90%, 98%, 99% or 100% identical to a reference nucleic acid or a fragment thereof, e.g., as measured by BLASTN (or CLUSTAL, or any other available alignment software) using default parameters. When one molecule can be said to have certain percentage of sequence identity with a larger molecule, can mean that when the two molecules are optimally aligned, said percentage of residues in the smaller molecule finds a match residue in the larger molecule in accordance with the order by which the two molecules are optimally aligned. The term “substantially identical” and its grammatical equivalents as applied to nucleic acid or amino acid sequences can mean that a nucleic add or amino acid sequence comprises a sequence that has at least 90% sequence identity or more, at least 95%, at least 98% and at least 99%, compared to a reference sequence using the programs described above, e.g., BLAST, using standard parameters. For example, the BLASTN program (for nucleotide sequences) uses as defaults a word length (W) of 11, an expectation (E) of 10, M-5, N=-4, and a comparison of both strands. In another example, for amino acid sequences, the BLAST? program uses as defaults a word length (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix. Percentage of sequence identity can be determined by comparing two optimally aligned sequences over a comparison w indow ; w'herein the portion of the polynucleotide sequence in the comparison window can comprise additions or deletions (e.g., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage can be calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity. In some embodiments, the substantial identity exists over a region of the sequences that can be at least about 8, at least, about 14 residues in length, over a region of at least about 20 residues, and in some embodiments, the sequences can be substantially identical over at least about 2.5 residues. In some embodiments, the sequences can be substantially identical over the entire length of the coding regions.
100141 The term “nucleobase” can generally refer to nitrogen containing compound that is capable of hydrogen bonding with a nucleobase present on a target pre-mRNA . For example, a nucleobase may be any unmodified nucleobase such as adenine, guanine, cytosine, thymine and uracil, or any synthetic or modified nucleobase that is sufficiently similar to an unmodified nucleobase such that it is capable of hydrogen bonding with a nucleobase present on a target pre-mRNA. Examples of modified nucleobases include, without limitation, hypoxanthine, xanthine, 7-methylguanine, 5, 6~dihydrourac.il, 5-methylcytosine, and 5- hydroxymethoylcytosine.
[0015] The term “PNPLA3 associated disorder” as used herein refers to a disease or condition that can be associated with an activity, a reduced activity, an altered activity, and in some cases, mutant activity, of a PNPLA3 protein.
[0016i As used herein, the term ‘'targeting domain” can comprise a region or portion of an PNPLA3 pre- mRNA to which a RTSM can bind. The binding can be covalent or non-covalent. Examples of non-covalent binding include binding via hydrogen bonding, Watson-Crick base pairing, wobble-base pairing, and Van derWaals interactions.
Overview
[0017] Some embodiments here are based, at least in part, on compelling evidence of a therapeutic effect of an RNA-Targeting Splicing Modifier (RTSM). Specifically, such embodiments arise out of the novel finding that treatment with an exon skipping RTSM disclosed herein can produce decreased levels of a PNPLA3 protein. [0018] Without being bound by theory, the PNPLA3 gene can encode a 481 amino acid Patatin-like phospholipase domain-containing 3 protein (PNPLA3). Variants of PNPLA3 have been associated with increased hepatic fat levels and with hepatic inflammation in a genome-wide association study. Wild-type PNPLA3 can be found in the membranes of lipid droplets, where it can be responsible for the postprandial remodeling of lipid droplets via its triglyceride hydrolase activity. However, variants, such as I148M (rs73840) and S47A have been shown to have reduced catalytic activity. Variants can also resist ubiquitylation-based degradation. Accordingly, variant PNPLA3 proteins can accumulate on the surface of lipid cells and sequester CGI-58, a cofactor required for the activity of adipose triglyceride lipase. Furthermore, while some variants exhibit reduced catalytic activity, some variants can also promote the production of pro-fibrogenic cytokines, including CCL2 and CCL5. These cytokines can stimulate hepatic stellate cells (HSC)s activation promoting the secretion of collagen 1 , the protein that can be seen as the basis of fibrosis during NALFD/NASH progression.
[0019] Decreasing the abundance or activity of this protein could improve the outcome of liver steatosis, liver inflammation and liver fibrosis, and be a useful treatment of nonalcoholic steatohepatitis (NASH). Hence, in certain embodiments, the methods described herein can be useful to increase exon skipping in PNPLA3 pre-mRNA to decrease active PNPLA3 levels.
Patatin-Iike phospholipase domain-containing 3 (PNPLA3) gene and protein
[0020 ] Disclosed herein are RTSMs that can modulate PNPLA3 protein produced in, for example, a cell, organ or subject. A unmodulated PNPLA3 protein can be a reference protein and was identified by homology search and sequences identified by genomic and nucleotide databases, such as ENSEMBL, GenBank, or UniProtKB/Swiss-Prot and in some embodiments comprises a polypeptide sequence of any of the sequences of SEQ ID NO: 9-13. In some embodiments, an unmodulated PNPLA3 protein as referred to herein can be a full length, elongated, functional, or wild-type protein or any combination thereof
[0021[ It was identified that the PNPLA3 gene can encode a PNPLA3 protein. In one embodiment, a targeted PNPLA3 gene comprises the sequence as set forth in RefSeq No. NC 000022.11 (disclosed herein as SEQ ID NO: 1 and incorporated by reference).
[0022] In some embodiments, a PNPL.A3 gene comprises one or more gene mutations. In some embodiments, the one or more PNPLA3 gene mutations are associated with: lengthening of the protein encoded by the gene; gain-of-function of a protein encoded by the gene; reduced or loss-of-function of a protein encoded by the gene; or an associated disease or a condition thereof.
[0023] RTSMs disclosed herein can target a PNPLA3 pre-mRNA. A PNPLA3 pre-mRNA can be the precursor PNPLA 3 RNA transcribed from the PNPLA3 gene, but prior to being spliced into a mature RNA. In some embodiments, a targeted pre-mRNA comprises a sequence that shares at least about: 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity, to at least about: 4, 5, 6, 7, 10, 15, 20, 25, 50, 75, 100, 150, 200, 250, 300 or more continuous nucleotides of any one of the sequences as set forth SEQ ID NOS: 2-6.
[00241 RTSMs disclosed herein can modulate PNPLA3 mRNA produced in, tor example, a cell, organ or subject. In some embodiments, an unmodulated PNPLA3 mRNA transcript referred to herein can be a reference mRNA and can comprise a sequence as set forth m any of the sequences in SEQ ID NOS: 7-8. In some embodiments, an unmodulated PNPLA3 mRNA can be wild-type and in some embodiments can comprise a sequence as set forth in SEQ ID NO: 7. In some embodiments, a reference PNPLA mRNA transcript can be a mutant and, in some embodiments, can comprise the sequence as set forth in SEQ ID NO:8. In some embodiments, a reference PNPLA3 protein referred to herein can also be mutant and can comprise any one of the sequences in SEQ ID NOS: 10-13. [0025] In some embodiments, RTSMs disclosed herein can target a target region of a PNPLA3 pre-mRNA. In some embodiments, a target region can comprise coding for: protein length; protein stability; protein function; coding specific for RTSM-RNA binding; RTSM-RNA duplex formation; coding which limits off-target binding; coding which recruits one or more splicing complex component; or any combination thereof.
[ 0026 i In some embodiments, upon binding to a target region, RTSMs disclosed herein can increase at least partial exon skipping during splicing of a PNPLA3 pre-mRNA. Accordingly, in some embodiments a targeted pre-mRNA comprises one or more exons and one or more introns. A PNPLA3 pre-mRNA can comprise 9 exons and 8 introns. Accordingly, in some embodiments a targeted pre-mRNA comprises exons 1-9 and introns 1-8. In some embodiments, any one of exons 1-9 can comprise, in part or in full, coding for protein length: protein stability; protein function; coding specific for RTSM-RNA binding; RTSM-RNA duplex formation; coding which limits off-target binding; coding which recruits one or more splicing complex component; or any combination thereof. For example, exon 1 can comprise coding for protein length; protein stability; protein function; coding specific tor RTSM-RNA binding; RTSM-RNA duplex formation; coding which limits off-target binding; coding which recruits one or more splicing complex component; or any combination thereof; exon 2 can comprise coding for protein length; protein stability; protein function; coding specific for RTSM-RNA binding; RTSM-RNA duplex formation; coding which limits off-target binding; coding which recruits one or more splicing complex component; or any combination thereof; exon 3 can comprise coding for protein length; protein stability; protein function; coding specific for RTSM-RNA binding; RTSM-RNA duplex formation; coding which limits off-target binding; coding which recruits one or more splicing complex component; or any combination thereof; exon 4 can comprise coding for protein length; protein stability; protein function; coding specific for RTSM- RNA binding; RTSM-RNA duplex formation; coding which limits off-target binding; coding which recruits one or more splicing complex component; or any combination thereof; exon 5 can comprise coding for protein length; protein stability; protein function; coding specific for RTSM-RNA binding; RTSM-RNA duplex formation; coding which limits off-target binding; coding which recruits one or more splicing complex component; or any combination thereof; exon 6 can comprise coding for protein length; protein stability; protein function; coding specific for RTSM-RNA binding; RTSM-RNA duplex formation; coding which limits off-target binding; coding which recruits one or more splicing complex component; or any combination thereof; exon 7 can comprise coding for protein length; protein stability; protein function; coding specific for RTSM-RNA binding; RTSM-RNA duplex formation; coding which limits off-target binding; coding which recruits one or more splicing complex component; or any combination thereof; exon 8 can comprise coding for protein length; protein stability; protein function; coding specific for RTSM- RNA binding; RTSM-RNA duplex formation; coding which limits off-target binding; coding which recruits one or more splicing complex component; or any combination thereof; and exon 9 can comprise coding tor protein length; protein stability; protein function; coding specific for RTSM-RNA binding; RTSM-RNA duplex formation; coding which limits off-target binding; coding which recruits one or more splicing complex component; or any combination thereof.
[0027] In some embodiments, pre-mRNA exon(s) referred to herein comprises a sequence that shares at least about: 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any one ofthe sequences in Table 2. In some embodiments, a pre-mRNA intron(s) referred to herein comprises a sequence that shares at least about: 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any one of the sequences in Table 3.
[0028] In some embodiments, pre-mRNA exon(s), intron(s), or both, may comprise one or more nucleotide alterations at one or more positions in any one of the sequences in Tables 2 or 3. Alternative nucleobases can be any one or more of A, C, G or U, or a deletion. [0029] When an intron is contiguous with the 3’ end of an exon, the exon and intron can correspond for splicing purposes and create an exon-intron junction. In some embodiments, a targeted pre-mRNA comprises a target region wherein a target region compri ses an exon-intron junction , Exon-intron junctions can be at least 2 nucleic acids in length, at least 3 nucleic acids in length, at least 4 nucleic acids in length, at least 5 nucleic acids in length, at least 6 nucleic acids in length, at least 7 nucleic acids in length, at least 8 nucleic acids in length, at least 9 nucleic acids m length, at least 10 nucleic acids in length, at least 11 nucleic acids in length, at least 12 nucleic acids in length, at least 13 nucleic acids in length, at least 14 nucleic acids or more in length. For example, exon-intron junctions can be about: 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 26, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or more nucleic acids in length.
[0030 ] In some embodiments, a targeted pre-mRNA comprises a targeted region wherein a targeted region comprises one or more exon-intron junction(s). In some embodiments, a target region comprises an exon- intron junction. In some embodiments, a target region comprises more than one exon-intron junction. In some embodiments, an exon-intron junction can be Exon 1 -Intron 1, Exon2-Intron2, Exon 3 -Intron 3, Exon4- Intrond, Exon5 -Intron 5, Exon6-Intron6, Exon7-Intron7, or Exon8-Intron8, more than one, or any combination thereof.
Figure imgf000009_0001
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Figure imgf000019_0002
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Figure imgf000021_0001
Figure imgf000022_0001
Figure imgf000023_0001
Figure imgf000024_0001
Figure imgf000025_0001
[0031] In some embodiments, a targeted region comprises a targeted exon or a portion thereof, a targeted intron or a portion thereof, both, more than one of either, or more than one of both, that is targeted by a binding domain of an RTSM herein. A binding domain of an RTSM in some instances can be from about 2. to about 50 nucleobases in length and can bind with about 1 to about 25 nucleobases of an exon to about 1 to about 25 nucleotides of an intron, any number in between, and combinations thereof. Target region can comprise about 1 to about 25 nucleobases of an exon, 1 to about 25 nucleobases of an intron, or any combination thereof. In some embodiments, a targeted region comprises a sequence that is at least about 2 to about 50 nucleobases long. In some embodiments, a targeted region comprises a sequence that can be about: 2, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 26, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or more nucleobases in length,
[0032] In some embodiments, a targeted exon can be any one of, or a portion of any one of Exon 1, Exon 2, Exon 3, Exon 4, Exon 5, Exon 6, Exon 7, Exon 8, or Exon 9. A targeted exon can comprise a sequence that shares at least about: 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a sequence of any one of SEQ ID NOS: 14-22.
[0033] In some embodiments, a targeted intron can be any one of, or a portion of any one of Intron 1 , Intron 2, Intron 3, Intron 4, Intron 5, Intron 6, Intron 7, or Intron 8. A targeted intron can comprise a sequence that shares at least about: 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a sequence of any one of SEQ ID NOS: 23-30.
[0034] In some embodiments, an exon -intron junction can comprise a sequence that shares at least about: 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20% or more sequence identity to: SEQ IDS NO: 14 and 23; SEQ IDS NO: 15 and 24; SEQ IDS NO: 16 and 25; SEQ IDS NO: 17 and 26; SEQ IDS NO: 18 and 27; SEQ IDS NO: 19 and 28; SEQ IDS NO: 20 and 29; or SEQ IDS NO: 21 and 30.
[0035] In some embodiments, a targeted mRNA comprises a targeted sequence. In some embodiments, an RTSM targets and binds to a targeted sequence of a PNPLA3 pre-mRNA. In some embodiments, an exon- intron junction comprises a targeted sequence.
[0036] In some embodiments, a target sequence comprises a sequence that can be at least about 2 to about 50 nucleobases long. In some embodiments, a target sequence comprises a sequence that can be about: 2, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 26, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or more nucleobases in length.
[0037] In some embodiments, a targeted sequence comprises a sequence as set forth in Formula (I) below:
Formula (I): Exemplary Targeted Sequence
5’ ... KGl. R ... 3’ wherein K is G or U; wherein R is A or G; and wherein 5 ’ ...3’ indicates from the 5’
Figure imgf000026_0001
3’ direction.
[0038] In some embodiments, a targeted sequence comprises a splice site. In some embodiments, an RTSM specifically binds to a splice site. In some embodiments, an RTSM specifically hybridizes to a splice site. In some embodiments, a splice site comprises a sequence as set forth in Formula (II) below.
Formula (II): Exemplary Splice Site
5’ ... K | GUR ... 3’ wherein K is G or U; wherein R is A or G; wherein | ... ” indicates the splice site; and wherein 5 ’ . . . 3 ’ indicates from the 5
Figure imgf000026_0002
3 ’ direction. [0039] In some embodiments, the sequence set forth in Formula (I), Formula (II), or both wherein: K is G; K is U; R is A; or R is G; and any combination thereof. In some embodiments, the sequence set forth in Formula (I), Formula (II), or both wherein: K is G and R is G; K is G and R is A; or K is U and R is A.
[0040] In some embodiments, Formula (I), Formula (II), or both, further comprise: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 N(s) wherein each N is independently A, G, C, or U. In some embodiments, Formula (I), Formula (II), or both, further comprise: N; NN: NNN; NNNN; NNNNN; NNNNNN; NNNNNNN; NNNNNNNN; NNNMNNNN; MAWNNNN; NNNNNNNNNNN; NNNNNNNNNNNN; NNNNNNNNNNNNN; NNNNNNNNNNNNNN; NNNNNNNNNNNNNNN; NNNNNNNNNNNNNNNN; or NNNNNNNNNNNNNNNNN wherein each N is independently A, G, C, or U.
[0041] In some embodiments, an N of a group of: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 contiguous N(s) can be 5’ and adjacent to K or can be 3’ and adjacent to R, wherein each N is independently A, G, C, or U. In some embodiments, Formula (I), Formula (II), or both, further comprise, a N group of: 1 , 2, 3, or 4 contiguous N(s) that can be 5’ and adjacent to K; a N group of: 1, 2, 3, 4, 5, 6, 7,
8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 contiguous N(s), that can be 3’ and adjacent to R: and combinations thereof, wherein each N is independently A, G, C, or U.
[0042] In some embodiments, Formula (I), Formula (II), or both, further comprise: N that is 5 ’ and adjacent to K; NN that is 5’ and adjacent to K; NNN that is 5’ and adjacent to K; NNNN that is 5’ and adjacent to K; NNNNN that is 5’ and adjacent to K; N that is 3’ and adjacent to R; NN that is 3’ and adjacent to R; NNN that is 3’ and adjacent to R; NNNN that is 3’ and adjacent to R; NNNNN that is 3’ and adjacent to R; NNNNNN that is 3’ and adjacent to R; NNNNNNN that is 3’ and adjacent to R; NNNNNNNN that is 3’ and adjacent to R; NNNNNNNNN that is 3‘ and adjacent to R; NNNNNNNNNN that is 3’ and adjacent to R; NNNNNNNNNNN that is 3’ and adjacent to R; NNNNNNNNNNNN that is 3’ and adjacent to R; NNNNNNNNNNNNN that is 3’ and adjacent to R; NNNNNNNNNNNNNN that is 3’ and adjacent to R; NNNNNNNNNNNNNNN that is 3’ and adjacent to R; NNNNNNNNNNNNNN^ that is 3’ and adjacent to R; NNNNNNNNNNNNNNNNN that is 3’ and adjacent to R; more than one of the foregoing; and combinations thereof; and any combination thereof, wherein each N is independently A, G, C or U.
[0043] For example, wherein Formula (I), Formula (II), or both, further comprise NNN that is 5’ and adjacent to K and NNNNNNNN that is 3’ and adjacent to R, the sequence of Formula (I), Formula (II), or both, may comprise NNNKGLTRNNNNNNNN wherein each N is independently A, G, C or U. For example, wherein Formula (I), Formula (II), or both, further comprise NNNNNNN that is 5’ and adjacent to K and NNNNNNNNNNN that is 3’ and adjacent to R, the sequence of Formula (I), Formula (II), or both, may comprise NNNNNNNKGURNNNNNNNNNNN wherein each N is independently A, G, C or U.
[0044] In some embodiments, the sequence set forth in Formula (I), Formula (II), or both, further comprising: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 B(s) wherein each B is independently G, C or U; 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 V(s) wherein each V is independently A, G, or C; 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 N wherein each N is independently A, G, C or U; 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 D(s) wherein each D is independently A, G or U; 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 Y(s) wherein each Y is independently C or U; 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 H(s) wherein each H is independently A, C or U; 1, 2, 3, 4, 5, 6, 7, 8,
9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 K(s) wherein each K is independently G or U; or any combination thereof.
[0045] In some embodiments, Formula (I), Formula (II), or both, further comprise: 1, 2, 3, 4, 5, 6, or 7 B(s) wherein each B is independently G, C or U; 1, 2, or 3 V(s) wherein each V is independently A, G, or C; 1 or 2 D(s) wherein each D is independently A, G or U; 1 , 2, 3, or 4 N(s) wherein each N is independently A, G, C or U; Y wherein Y is C or U; 1 or 2 H(s) wherein each H is independently A, C or U; K wherein K is independently G or U; or any combination thereof.
[0046J In some embodiments, Formula (I), Formula (II), or both, further comprises: D, N, V, B or any combination thereof that is 5’ to K, wherein B is G, C or U, wherein V is A, G, or C, wherein I) is A, G or U, and wherein N is A, G, C or U.
[0047] In some embodiments. Formula (I), Formula (II), or both, further comprise D that is 5’ and adjacent to K; ND that is 5’ and adjacent to K; VND that is 5’ and adjacent to K; or BAND that is 5’ and adjacent to K; wherein B is G, C or U, wherein B is G, C or U, wherein V is A, G, or C, wherein D is A, G or U, and wherein N is A, G, C or U.
[0048] In some embodiments. Formula (I), Formula (II), or both, further comprise: 1 , 2, 3, 4, 5, or 6 B(s) wherein each B is independently G, C or U; 1 or 2 V(s) wherein each V is independently A, G, or C; 1 , 2, or 3, N(s) w herein each N is independently A, G, C or U; 1 or 2 D(s) wherein each D is independently A, G or U; Y wherein Y is C or U; 1 or 2 H(s) wherein each H is independently A, C or U; K wherein K is G or U: or any combination thereof, that is 3‘ and adjacent to R.
[0049] In some embodiments, Formula (I), Formula (II), or both, further comprise: H that is 3' and adjacent to R; HV that is 3' and adjacent to R; HVY that is 3' and adjacent to R; HVYH that is 3' and adjacent to R; HVYHB that is 3' and adjacent to R; HVYHBK that is 3‘ and adjacent to R; HVYHBKB that is 3' and adjacent to R; HVYHB KB B that is 3‘ and adjacent to R; HVYHBKB BN that is 3' and adjacent to R: HVYHBKBBNN that is 3’ and adjacent to R; HAYHBKBBNNB that is 3’ and adjacent to R; HVYHBKBBNNBD that is 3' and adjacent to R; HVYHBKBBNNBDV that is 3' and adjacent to R; HVYHBKBBNNBDVD that is 3' and adjacent to R; HVYHB KBBNNBDVDB that is 3' and adjacent to R; HVYHBKBBNNBDVDBB that is 3' and adjacent to R; or HVYHBKBBNNBDVDBBN, that is 3' and adjacent to R; wherein each B is independently G, C or U, wherein each V is independently A, G, or C, wherein each D is independently A, G or U, wherein each N is independently A, G, C or U, wherein Y is C or U, wherein each H is independently A, C, or U and wherein K is G or U.
[0050] In some embodiments, Formula (I), Formula (II), or both, further comprise: VND that is 5’ and adjacent to K and HVYHBKB that is 3’ and adjacent to R, wherein the sequence of Formula (I), Formula (II), or both, comprise VNDKGURBHVYHBKB wherein each B is independently G, C or U, wherein each V is independently A, G, or C, wherein each D is independently A, G or U, wherein each N is independently A, G, C or U, wherein Y is C or U, wherein each H is independently A, C, or U wherein K is G or U.
[0051] In some embodiments, Formula (I), Formula (II), or both, further comprise: BVND that is 5‘ and adjacent to K and HVYHBKBBNNBDVDBBN that is 3’ and adjacent to R, wherein the sequence of Formula (I), Formula (II), or both, comprise B VNDKCilJRflVYIlBKBBNNBDVDBBN wherein each B is independently G, C or U, wherein each V is independently A, G, or C, wherein each D is independently A, G or U, wherein each N is independently A, G, C or U, wherein Y is C or U, wherein each H is independently A, C, or U and wherein K is G or U.
[0052] Various embodiments of Formula (I) can be seen, for example, in any of the sequences of Table 4. Various embodiments of Formula (II) can be seen, for example, in any of the sequences of Table 5.
[0053] In some embodiments, a target sequence comprises a sequence that shares at least about: 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any one of SEQ ID NOS: 31-46 of Table 4. Table 4: Exemplary Targeted Sequences
Figure imgf000029_0001
[0054] In some embodiments, a target sequence may comprise one or more nucleotide alterations at one or more positions in any of the sequences in Table 4. Alterations can be made singly or in combination with other alterations at other positions. Alternative nucleobases can be any one or more of A, C, G or U, or a deletion. In some embodiments, alterations can be obtained through computational predictions and alignment, for example, using SPLIGN, ESEMBL, or Blast.
[0055] In some embodiments, the disclosed RTSM herein increases exon skipping of a targeted exon during splicing. In some embodiments, the exon to be skipped can be any one of Exon 1, Exon 2, Exon 3, Exon 4, Exon 5, Exon 6, Exon 7, Exon 8, or Exon 9, and any combination thereof. In some embodiments, an exon can be skipped when an RTSM can be bound to the target sequence. In some embodiments when an RTSM is bound to the target sequence, one or more of Exon 1, Exon 2, Exon 3, Exon 4, Exon 5, Exon 6, Exon 7, Exon 8, Exon 9, and any combination thereof, can be skipped during splicing.
[0056] In some embodiments, a target region can comprise a splice site. In some embodiments, an exon- intron junction can comprise a splice site. In certain embodiments, a splice site can be a 5’ splice site. In other embodiments, a splice site can be a 3" splice site.
[0057] In some embodiments, an exon-intron junction comprises at least a portion of a target exon, at least a portion of a target intron, both, more than one of either, and more than one ofboth. In some embodiments, an exon-intron junction can be located at the 5’ splice site of a target intron, wherein the corresponding targeted exon can be downstream of a target intron. In some embodiments, a targeted region can comprise the 5’ splice site of an intron. In some embodiments, a targeted region comprises the 5’ splice site of intron I wherein a targeted exon is exon 1. In some embodiments, a targeted region comprises the 5’ splice site of intron 2 wherein a targeted exon is exon 2. In some embodiments, a targeted region comprises the 5’ splice site of intron 3 wherein a targeted exon is exon 3. In some embodiments, a targeted region comprises the 5’ splice site of intron 4 wherein a targeted exon is exon 4, In some embodiments, a targeted region comprises the 5’ splice site of intron 5 wherein a targeted exon is exon 5. In some embodiments, a targeted region comprises the 5’ splice site of intron 6 wherein a targeted exon is exon 6. In some embodiments, a targeted region comprises the 5’ splice site of intron 7 wherein a targeted exon is exon 7. In some embodiments, a targeted region comprises the 5’ splice site of intron 8 wherein a targeted exon is exon 8.
[0058] In some embodiments, an RTSM binding domain specifically binds to a splice site. In some embodiments, an RTSM binding domain specifically hybridizes to a splice site. In some embodiments, a splice site comprises a sequence as set forth in Formula (II). Various embodiments of Formula (II) can be seen, for example, in any sequence set forth in Table 5.
[0059] In some embodiments, a splice site sequence comprises a sequence that can be at least about 2 to about 50 nucleobases long. In some embodiments, a splice site sequence comprises a sequence that can be about 14 to about 35 nucleobases long. In some embodiments, a splice site sequence comprises a sequence that can be about 14 to about 25 nucleobases long. In some embodiments, a splice site sequence comprises a sequence that can be about: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 26, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or more nucleobases in length. In some embodiments, a splice site sequence can be about 2 nucleobases in length. In some embodiments, a splice site sequence can be about 3 nucleobases in length. In some embodiments, a splice site sequence can be about 4 nucleobases in length. In some embodiments, a splice site sequence can be about 14 nucleobases in length. In some embodiments a splice site sequence can be about 25 nucleobases in length.
[0060] In some embodiments, a splice site comprises a sequence that shares at least about: 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any one of the sequences in Table 5. In some embodiments, a splice site can be a5’ splice site. In some embodiments, a 5’ splice site can be the complement of any one of the sequences in SEQ ID NOS: 47-62. In some embodiments, a 5’ splice site can be the inverse complement of any one of the sequences in SEQ ID NOS: 47-62.
Table 5: Exemplary Splice Site Sequences
Figure imgf000030_0001
[0061] SEQ ID NOS: 47-62 is reproduced in Table 6 below and in some instances, a splice site can be located where the vertical line is located at each sequence.
Table 6: Representative Splice Site Sequences
Figure imgf000031_0001
[0062] In some embodiments, a splice site sequence further comprises one or more alterations at one or more positions on either side of a splice site. For example, an alteration can be seen at -2, -3, -4, -5, -6, -7, -8, -9, -10, -1 1 , -12, -13, -14, -15, -16, -17, -18, -19, -20, -21, -22, -23, -24, -25, -26, -27, -28, -29, -30, -31, -32, -33, -34, -35, -36, -37, -38, -39, -40, -41, -42, -43, -44, -45, -46, -47, -48, -49, -50 or more positions from a splice site in the 3’ to the 5’ direction. In a further example, an alteration can be seen at +1, +2, +3, +4, +5, +6, +7, +8, +9, + 10, +11, +12, +13, +14, +15, +16, +17, +18, +19, +20, +21, +22, +23, +24, +25, +26, +27, +28, +29, +30, +31, +32, +33, +34, +35, +36, +37, +38, +39, +40, +41, +42, +43, +44, +45, +46, +47, +48, +49, +50 or more positions from a splice site in the 5’ to the 3 ' direction. In another example, an alteration can be seen at -25, -24, -23, -22, -21 , -20, -19, -18, -17, -16, -15, -14, -13, -12, -1 1 , -10, -9, -8, - 7. -6, -5, -4, -3. -2. -1 , +1, +2, +3, +4, +5. +6, +7. +8. +9, +10, +11. +12, +13, +14, +15. +16. +17, +18. +19, +20, +21, +22, +23, +24, +25 positions from a splice site. Alterations can be any one of A, C, G or U. In some embodiments, alterations can be obtained through computational predictions and alignment, for example, using SPLIGN, ESEMBL, or Blast.
RNA-Targeted Splicing Modifiers (RTSM)
[0063] An RTSM disclosed herein can be synthetic RNA targeting splicing modifiers that can increase exon skipping during splicing of PNPLA3 pre-mRNA compared to PNPLA3 pre-mRNA spliced in the absence of RTSM. An RTSM disclosed herein can increase the level of modulated PNPLA3 mRNA transcripts as compared to mRNA transcripts processed in the absence of an RTSM, An RTSM disclosed herein can increase the level of modulated PNPLA3 protein production as compared to PNPLA3 produced in the absence of an RTSM. [0064] As used herein, the term “increase” means to induce or to enhance. For example, if no exon skipping occurred in the absence of RTSM, then any incidence of exon skipping or any indication of exon skipping activity resulting from splicing in the presence of RTSM has thereby “increased” exon skipping. In some instances, an evaluation of an increase in exon skipping can occur in an in vitro assay. In some instances, comparison of the amount of exon skipping in two otherwise substantially identical systems where one system lacks an RTSM and the other system has an RTSM can determine if exon increases in the presence of the RTSM.
[0065] In some embodiments, an RTSM can be selected from the group consisting of an antibody or a fragment thereof, an aptamer, an antisense oligomer (ASO), or a CRISPR associated protein, and any combination thereof.
100661 In some embodiments, an RTSM targets and binds to PNPLA3 pre-mRNA. Disclosed herein are RTSMs, wherein RTSM-pre-mRNA binding can prevent recruitment of one or more splicing complex component to the pre-mRNA, decrease the binding affinity of one or more splicing complex component to the pre-mRNA, interfere with splice site signaling, sterically block splicing of the pre-MRNA, or any combination thereof. In some embodiments, an RTSM comprises a binding domain that binds to PNPLA3 pre-mRNA. In some embodiments, an RTSM comprises a binding domain that binds to a target region of a PNPLA3 pre-mRNA. In some embodiments, an RTSM comprises a binding domain that binds to a target sequence of a PNPLA3 pre-mRNA. In some embodiments, an RTSM comprises a binding domain that specifically binds to a splice site sequence of a PNPLA3 pre-mRNA.
[0067] In some embodiments, a binding domain of an RTSM disclosed herein can be from about 2 to about 50 nucleobases in length and can bind with about 1 to about 25 nucleobases of an exon to about 1 to about 25 nucleotides of an intron, any number in between, and combinations thereof. In some embodiments, a binding domain of an RTSM comprises a sequence that can be at least about 2 to about 50 nucleobases long. In some embodiments, a binding domain of an RTSM comprises a sequence that can be about: 2, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or more nucleobases in length.
[0068] In some embodiments, an RTSM need not to completely bind to all nucleobases in a target sequence and the nucleobases to which it does bind to may be contiguous or noncontiguous. RTSMs may bind over one or more segments of a pre-mRNA transcript, such that intervening or adjacent segments are not involved in the hybridization event (e.g., a loop structure or hairpin structure may be formed). In certain embodiments, an RTSM binds to noncontiguous nucleobases in a target pre-mRNA transcript. For example, a RTSM may bind to nucleobases in a pre-mRNA transcript that are separated by one or more nucleobase(s) to which an RTSM does not bind. In some embodiments, an RTSM can bind to about: 2, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 continuous nucleobases of a target pre-mRNA.
[0069] In some embodiments, an RTSM binding domain comprises a binding sequence. In some embodiments, a binding sequence binds to the sequence as set forth in Formula (II) . In some embodiments, a binding sequence hybridizes to the sequence as set forth in Formula (II). In some embodiments, a binding sequence comprises a sequence as set forth in Formula (III) below:
Formula (III): Exemplary Binding Sequence
5’ ... YAC.M... 3’ wherein Y is C, T, or U; wherein M is A or C and wherein 5 ’ . . . 3 ’ indicates from the direction.
Figure imgf000032_0001
[0070] In some embodiments, the sequence set forth in Formula (III) wherein: Y is C; Y is T; Y is U; M is A; M is C; and any combination thereof. In some embodiments, the sequence set forth in Formula (III) wherein: Y is C and M is C; Y is T and M is A; Y is U and M is A; Y is Y and M is C; or Y is U and M is C.
[0071] In some embodiments, Formula (III) further comprises: I, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 N(s) wherein each N is independently A, G, C, T or U. In some embodiments, Formula (I), Formula (II), or both, further comprise: N; NN; NNN; NNNN; NNNNN; NNNNNN; NNNNNNN; NNNNNNNN; NNNNNNNNN; NNNNNNNNNN; NNNNNNNNNNN; NNNNNNNNNNNN; NNNNNNNNNNNNN; NNNNNNNNNNNNNN; NNNNNNNNNNNNNNN; NNNNNNNNNNNNNNNN; or NNNNNNNNNNNNNNNNN wherein each N is independently A, G, C, T or U.
[0072] In some embodiments, wherein an N group of: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 contiguous N(s) can be 5’ and adjacent to Y or can be 3’ and adjacent to M, wherein each N is independently A, G, C, T or U. In some embodiments, Formula (III) further comprises an N group of: 1, 2, 3, or 4 contiguous N(s) that can be 3 ’ and adjacent to M; an N group of: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, I I,
12, 13, 14, 15, 16, or 17 contiguous N(s), that can be 5’ and adjacent to Y; and combinations thereof, wherein each N is independently A, G, C, T or U.
[0073] In some embodiments, Formula (III) further comprises: N that is 5’ and adjacent to Y; NN that is 5’ and adjacent to Y; NNN that is 5’ and adjacent to Y; NNNN that is 5’ and adjacent to Y; NNNNN that is 5’ and adjacent to Y; NNNNNN that is 5’ and adjacent to Y; NNNNNNN that is 5’ and adjacent to Y; NNNNNNNN that is 5' and adjacent to Y; NNNNNNNNN that is 5' and adjacent to Y; NNNNNNNNNN that is 5’ and adjacent to Y; NNNNNNNNNNN that is and adjacent to Y; NNNNNNNNNNNN that is 5’ and adjacent to Y; NNNNNNNNNNNNN that is 5’ and adjacent to Y; NNNNNNNNNNNNNN that is 5’ and adjacent to Y; NNNNNNN^^ that is 5’ and adjacent to Y; NNNNNNNNs^^ that is 5’ and adjacent to Y; NNNNNNNNN that is 5’ and adjacent to Y; N that is 3’ and adjacent to M; NN that is 3’ and adjacent to M; NNN that is 3’ and adjacent to M; NNNN that is 3’ and adjacent to M; NNNNN that is 3’ and adjacent to M; and any combination thereof wherein each N is independently A, G, C, I' or U.
[0074] For example. Formula (III) further comprises: NNN that is 3 ’ and adjacent to M and NNNNNNNN that is 5’ and adjacent to Y, the sequence of Formula (I), Formula (II), or both, may comprise NNNNNNNNYACMNNN wherein each N is independently A, G, C, T or U. For example, wherein Formula (I), Formula (II), or both, further comprise NNNNNNN that is 3’ and adjacent to M and NNNNNNNNNNN that is 5’ and adjacent to Y, the sequence of Formula (I), Formula (II), or both, may comprise NNNNNNNN NNN YACMNNNNNNN wherein each N is independently A, G, C, T or U.
[0075] In some embodiments, the sequence set forth in Formula (III) further comprising: 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 B(s) wherein each B is independently G, C, T or U; 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 V(s) wherein each V is independently A, G, or C; 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, I I, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 N wherein each N is independently A, G, C, T, or U; 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 D(s) wherein each D is independently A, G, T or U; 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 R(s) wherein each R is independently G or A; I, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 H(s) wherein each H is independently A, C, T, or U; 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,
13, 14, 15, 16, 17, 18, 19, 20, or 21 M(s) wherein each M is independently A or C; or any combination thereof.
[0076] In some embodiments, Formula (III) further comprises: M; R; 1 or 2 D(s); 1, 2, or 3 H(s); 1, 2, or 3 B(s); 1, 2, 3, or 4 N(s); 1, 2, 3, 4, 5, 6, or 7 V(s) or any combination thereof, wherein M is A or C, wherein each B is independently G, C, T or U, wherein R is G or A, wherein each D is independently A, G, T or U, wherein each H is independently A, C, T, or U, wherein each N is independently A, G, C, T, or U, and wherein each V is independently A, G. or C.
[0077] In some embodiments, Formula (III) further comprises: R; M; 1 or 2 H(s); 1 or 2 B(s); 1 or 2 D(s): 1, 2, or 3 N(s); I, 2, 3, 4, 5, or 6 V(s); or any combination thereof; that is 5’ and adjacent to Y wherein M is A or C, wherein R is G or A, wherein each D is independently A, G, T or U, wherein each II is independently A, C, T, or U, wherein each N is independently A, G, C, T, or U, and wherein each V is independently A, G, or C.
[0078] In some embodiments, Formula (III) further comprises: H, N, B, V; or any combination thereof that is 3 ’ and adjacent to M; wherein each H is independently A, C, T, or LI, wherein each N is independently A, G, C, T, or U, wherein each B is independently G, C, T or U, and wherein each V is independently A, G, or C.
[0079] In some embodiments. Formula (III) further comprises: D that is 5‘ and adjacent to Y ; BD that is 5' and adjacent to Y; RBD that is 5' and adjacent to ¥; DRBD that is 5' and adjacent to Y; VDRBD that is 5‘ and adjacent to Y; MVDRBD that is 5' and adjacent to Y: VMVDRBD that is 5' and adjacent to Y; VVMVDRBD that is 5' and adjacent to Y; NVVMVDRBD that is 5’ and adjacent to Y; NNVVMVDRBD that is 5' and adjacent to Y; VNNVVMVDRBD that is 5' and adjacent to Y; HVNNVVMVDRBD that is 5' and adjacent to Y; BFIX'TQNVVMVDRBD that is 5' and adjacent to Y; HBI-IVNNVVMVDRBD that is 5' and adjacent to Y; VHBI-RTEWVMVDRBD that is 5’ and adjacent to Y; VA/in3lTVT4NVVMVDRBD that is 5* and adjacent to Y; NVVHBHVNNVVMVDRBD that is 5' and adjacent to Y; H that is 3' and adjacent to M; HN that is 3' and adjacent to M; HNB that is 3‘ and adjacent to M; HNBV that is 3' and adjacent to M; or any combination thereof, wherein M is A or C, wherein each B is independently G, C, T or U, wherein R is G or A, wherein each D is independently A, G, I' or U, wherein each H is independently A, C, T, or U, wherein each N is independently A, G, C, T, or U, and wherein each V is independently A, G, or C.
[0080] In some embodiments, Formula (III) further comprises VMVDRBD that is 5’ and adjacent to Y, HNB that is 3‘ and adjacent to M, wherein the sequence of Formula (III) comprises VMVDRBDYACM HNB wherein M is independently A or C, wherein each B is independently G, C, T or U, wherein R is G or A, wherein each D is independently A, G, T or U, wherein each H is independently A, C, T, or U, wherein each N is independently A, G, C, T, or U, and wherein each V is independently A, G, or C. In some embodiments. Formula (III) comprises VVHBFIVNNVVMVDRBD that is 5’ and adjacent to B and HNBV that is 3’ and adjacent to Y, wherein the sequence of Formula (III) comprises VVHBHVNNVVMVDRBDYACMHNBV wherein each M is independently A or C, wherein each B is independently G, C, T or U, wherein R is G or A, wherein each D is independently A, G, T or U, wherein each H is independently A, C, T, or LI, wherein each N is independently A, G, C, T, or U, and wherein each V is independently A, G, or C.
[0081] Various embodiments of Formula (III) can be seen in SEQ ID NOS: 63-94 as set forth in Table 7 below. Various embodiments of Formula (III) can be modified as described in the methods herein.
[0082] In some embodiments, an RTSM binding domain comprises a sequence about 2 to about 50 nucleobases in length that has at least about: 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence complementarity to any one of the sequences of SEQ ID NOS: 32-46. In some embodiments, an RTSM binding domain comprises a sequence about 4 to about 30 nucleobases in length that has at least about: 1 %, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence complementarity to any one of tire sequences of SEQ ID NOS: 32-46.
[0083] In some embodiments, an RTSM binding domain comprises a sequence about 2 to about 50 nucleobases in length that shares at least about: 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any one of the sequences of SEQ ID NOS: 63-94 of Table 6, In some embodiments, an RTSM binding domain comprises a sequence about 4 to about 30 nucleobases in length that has at least about: 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any one of the sequences of SEQ ID NOS: 63-94.
Table 7: Exemplary Binding Sequences
Figure imgf000035_0001
[0084] In some embodiments, a binding sequence may comprise one or more nucleotide alterations at one or more positions in any of the sequences in Table 7. Alterations can be made singly or in combmation with other alterations at other positions. Alternative nucleobases can be any one or more of A, C, G, T or U, or a deletion. In some embodiments, alterations can be obtained through computational predictions and alignment, for example, using SPLIGN, ESEMBL, or Blast.
[0085] In some embodiments, a binding sequence comprises a sequence about 2 to about 50 nucleobases in length that has at least about: 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any one of the sequences of SEQ ID NOS: 63-94. In some embodiments, a binding sequence comprises a sequence about 4 to about 30 nucleobases in length that has at least about: 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any one of the sequences of SEQ ID NOS: 63-94.
[0086 [ In some embodiments, a binding sequence disclosed herein comprises a sequence that can bind or hybridize to 2, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 continuous or non-continuous nucleotides of any one of the sequences of SEQ IDS NOS: 31-46.
[00871 In some embodiments, a binding sequence disclosed herein comprises a sequence that can bind or hybridize to 2, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 continuous or non-continuous nucleotides of any one of the sequences of SEQ IDS NOS: 47-62.
[0088] An RTSM disclosed herein can increase exon skipping during pre-mRNA splicing as compared to a pre-mRNA spliced in the absence of an RTSM, In some embodiments, exon skipping modulates a PNPLA3 mRNA, a PNPLA3 protein, or both. In some embodiments, modulated exon coding can be determined by modulated mRNA transcription, modulated a PNPLA3 protein translation, or both. In some embodiments, modulated exon coding comprises: absence of one or more PNPLA3 gene exon(s) in a PNPLA3 mRNA molecule, a truncated mRNA molecule, absence of one or more PN PLA3 gene exon(s) in a PNPLA3 protein molecule, a non-functional PNPLA3 protein, an at least semi -functional PNPLA3 protein, a truncated PNPLA3 protein molecule, or any combination thereof.
[0089[ In some embodiments, RTSM-modulated PNPLA3 mRNA transcript can be encoded by a sequence that excludes a sequence with at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to: SEQ ID NO: 14; SEQ ID NO: 15; SEQ ID NO: 16; SEQ ID NO: 17; SEQ ID NO: 18; SEQ ID NO: 19; SEQ ID NO:20; SEQ ID NO:21 ; SEQ ID NO:22; and any combination thereof.
[00901 In some embodiments, wherein RTSM-modulated PNPLA3-mRNA transcript can be determined by analysis of a PNPLA3 mRNA transcript expressed in the absence of RTSM, or to a wild-type PNPLA3 mRNA, or both, wherein the wild-type PNPLA3 mRNA can be encoded by a sequence with at least: 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence as set forth in SEQ ID NO: 7 or 8.
[0091] In some embodiments wherein RTSM-modulated PNPLA3 protein expression can be determined by analysis of a PNPLA3 protein translated in the absence of RTSM, or a wild-type PNPLA3 protein, or both, wherein the wild-type PNPLA3 protein can be encoded by a sequence with at least: 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of the sequences as set forth in SEQ ID NOS: 9-13.
Antibodies
[0092[ In some embodiments, an RTSM can be an antibody or a fragment thereof specific or selective for a PNPLA3 pre-mRNA. In some embodiments, an RTSM can be an anti -mRNA antibody or a fragment thereof. Antibodies, also known as immunoglobulin (Ig), as disclosed herein can be monoclonal or polyclonal antibodies. "Die term “monoclonal antibodies,” as used herein, can refer to antibodies that are produced by a single clone of B-cells and bind to the same epitope. In contrast, “polyclonal antibodies” can refer to a population of antibodies that are produced by different B-cells and bind to different epitopes of the same antigen. Hie antibodies can be from any animal origin. An antibody can be IgG (including IgGl, IgG2, IgG3, and IgG4), IgA (including IgA1 and IgA2), IgD, IgE, or IgM, and IgY, In some embodiments, the antibody can be whole antibodies, including single-chain whole antibodies. In some embodiments, the antibody can be a fragment of an antibody, which can include, but are not limited to, a Fab, a Fab’, a F(ab’)2, a Fd (consisting of VH and CHI), a Fv fragment (consisting of VH and VL), a single-chain variable fragment (scFv), a single-chain antibody, a disulfide-linked variable fragment (dsFv), and fragments comprising either a VL or VH domain. A whole antibody typically consists of tour polypeptides: tw o iden tical copies of a heavy (H) chain polypeptide and two identical copies of a ligh t (L) chain polypeptide. Each of the heavy chains contains one N-terminal variable ( VH) region and three C- terminal constant (CHI, CI 12 and CH3) regions, and each light chain contains one N-terminal variable (VL) region and one C-terminal constant (CL) region. Hie variable regions of each pair of light and heavy chains form the antigen binding site of an antibody. The VH and VL regions have a similar general structure, with each region comprising four framework regions, whose sequences are relatively conserved. The framework regions are connected by three complementarity determining regions (CDRs). The three CDRs, known as CDR1 , CDR2, and CDR3, form the “hypervariable region” of an antibody, which can be responsible for antigen binding, and can include overlapping or subsets of amino acid residues when compared against each other. In one embodiment, the term “CDR” can be a CDR which can be defined based on sequence comparisons. CDRH1, CDRH2 and CDRH3 denote the heavy chain CDRs, and CDRL1, CDRL2 and CDRL3 denote the light chain CDRs.
[0093] The terms “fragment of an antibody,” “antibody fragment,” “functional fragment of an antibody,” “antigen-binding portion” or its grammatical equivalents are used interchangeably herein and can mean one or more fragments or portions of an antibody that retain the ability to specifically or selectively bind to an antigen. The antibody fragment desirably comprises, for example, one or more CDRs, the variable region (or portions thereof), the constant region (or portions thereof), or any combination thereof. Non-limiting examples of antibody fragments include (1) a Fab fragment, which is a monovalent fragment consisting of the VL, VH, CL, and CHI domains; (2) a F(ab’)2 fragment, which is a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the stalk region; (3) a Fv fragment comprising the VL and VH domains of a single arm of an antibody; (4) a single chain Fv (scFv), which is a monovalent molecule comprising the two domains of the Fv fragment (e.g., VL and VH) joined by a linker which enables the two domains to be synthesized as a single polypeptide chain and (5) a diabody, which is a dimer of polypeptide chains, wherein each polypeptide chain comprises a VH connected to a VL by a peptide linker that is too short to allow pairing between the VH and VL on the same polypeptide chain, thereby driving the pairing between the complementary domains on different VH-VL polypeptide chains to generate a dimeric molecule having two functional antigen binding sites.
[0094] In one embodiment, an RTSM antibody or fragment of described herein comprises a binding region that targets a target region on a PNPLA3 pre-mRNA. In some embodiments, an RTSM antibody binding region can be also known as an “antigen recognition domain,” “antigen binding domain,” or “’antigen binding region” which can refer to a portion of an RTSM that specifically binds to a target region. In some embodiments, a target region of a pre-mRNA comprising a target sequence can be referred to herein as an antigen . In some embodiments, wherein a target region of a pre-mRNA is an antigen, a target sequence is an epitope wherein an RTSM antibody or fragment thereof targets to and binds to a pre-mRNA epitope,
[0095] In some embodiments, RTSM antibodies or fragments thereof can be generated by a modified nucleobase-coupling protocol. In such embodiments, the antibody can be modified, coupled or conjugated with a nucleic acid probe, such as an antisense oligonucleotide probe, wherein the nucleic acid probe binds to a targeted region of the IFH1 pre-mRNA and increases exon skipping. Aptamers
[0096] In some embodiments an RTSM can be an aptamer that binds to a riboswitch on a targeted pre- mRNA In some embodiments, an RTSM aptamer can be operably linked to a ligand.
[0097] In some embodiments, an RTSM can be operably linked to a ligand. For modulating mRNA splicing, a ligand or molecule specific to an aptamer it can be helpful to meet some or all of the following criteria. First, it should be able to bind its ligand-binding aptamer with high affinity. Second, ligand-aptamer interaction should not requi re the assistance of any other factor. Third, the ligand-binding site (the aptamer) should be unstructured and only upon binding of ligand should the aptamer undergo a conformational change or rearrangement. Fourth, the ligand-aptamer binding must be preserved under the conditions that support pre-mRNA splicing. Finally, the ligand should not affect the splicing of a substrate that does not contain its binding site. When an RTSM can be an aptamer-ligand system, the aptamer-ligand comprises a binding region that targets and binds to a target sequence of a pre-mRNA, wherein the aptamer inserts into the strand and the ligand increases exon skipping. In some embodiments, a ligand can be tobramycin, neomycin, or theophylline.
Antisense Oligonucleotides (ASOs)
[0098] In some embodiments, an RTSM can be an ASO. In some embodiments, an RTSM disclosed herein comprises a binding domain that targets and binds to a target region of a PNPLA3 pre-mRNA In some embodiments, a binding domain of an RTSM comprises a sequence that can be about: 2, 4, 5, 6, 7, 8, 9, 10, I I, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 26, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38,
39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or more nucleobases in length.
[0099] In some embodiments, an ASO binding domain comprises a sequence about 4 to about 50 nucleobases in length that has at least about: 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence complementarity to any one of the sequences of SEQ ID NOS: 31-46. In some embodiments, a binding domain comprises a sequence about 14 to about 30 nucleobases in length that has at least about: 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence complementarity to any one of SEQ ID NOS: 31-46.
[0100] In some embodiments, an ASO-RTSM of the present disclosure comprises a sequence that has at least about 85% sequence complementary to the sequence of SEQ ID NO: 35. In some embodiments, an RTSM of the present disclosure comprises a sequence that has at least about 92% sequence complementary to the sequence of SEQ ID NO: 35. In some embodiments, an RTSM of the present disclosure comprises a sequence that has 100% sequence complementary to the sequence of SEQ ID NO: 35. In some embodiments, an ASO-RTSM of the present disclosure comprises a sequence that has at least about 85% sequence complementary to the sequence of SEQ ID NO: 43, In some embodiments, an RTSM of the present disclosure comprises a sequence that has at least about 92% sequence complementary to the sequence of SEQ ID NO: 43. In some embodiments, an RTSM of the present disclosure comprises a sequence that has 100% sequence complementary to the sequence of SEQ ID NO: 43.
[0101] An ASO and a DNA or RNA target binding partner can be complementary to each other when a sufficient number of corresponding positions in each molecule are occupied by nucleotides which can hydrogen bond with each other. Thus in some embodiments, “specifically hybridizable'’ and “complementary” are terms which can be used to indicate a sufficient degree of complementarity or precise pairing such that stable and specific binding occurs between an ASO and a DNA or RNA target. It can be understood that the sequence of an ASO need not be 100% complementary to that of its target sequence to be specifically hybridi zable. An ASO can be specifically hybridizable when there are sufficient binding interactions between an ASO and DNA or RNA target such that the ASO, at least temporarily, adheres to the specific region which its targeting. Specific binding can occur under physiological conditions, including but not limited to room temperature, in the case of in vivo assays or therapeutic treatment, and in the case of in vitro assays, under conditions in which the assays are performed. In some embodiments, the above method may be used to select ASOs.
[0102] In some embodiments, an ASO can have exact sequence complementary to a target sequence or near complementarity (e.g., sufficient complementarity to bind a target sequence and modulating splicing at a splice site). Complementarity (the degree to which one polynucleotide is complementary with another) is quantifiable in terms of the proportion (e.g., the percentage) of bases in opposing strands that are expected to form hydrogen bonds with each other, according to generally accepted base-pairing rules. The sequence of an antisense oligomer (ASO) need not be 100% complementary’ to that of its target nucleic acid to hybridize. In certain embodiments, ASOs can comprise at least about: 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence complementarity to a target region within a target nucleic acid sequence to which they are targeted. For example, an ASO in which 18 of 20 nucleobases of the oligomeric compound are complementary to a target region, and would therefore specifically hybridize, would represent 90 percent complementarity'. In this example, the remaining non-compiementary nucleobases may be clustered together or interspersed with complementary nucleobases and need not be contiguous to each other or to complementary’ nucleobases. Percent complementarity of an ASO with a region of a target nucleic acid can be determined using BLAST programs (basic local alignment search tools) and PowerBLAST programs.
[0103] ASOs disclosed herein can be designed so that they bind to a target nucleic acid (e.g., a targeted region of a pre-mRNA transcript) and remain bound under physiological conditions. In some embodiments, binding as described herein can be hybridizing. In some embodiments, if an ASO binds to a site other than the intended (targeted) nucleic acid sequence, it binds to a limited number of sequences that are not a target nucleic acid (to a few sites other than a target nucleic acid). Design of an ASO can take into consideration the occurrence of the nucleic acid sequence of a targeted portion of a pre-mRNA transcript or a sufficiently similar nucleic acid sequence in other locations in the genome or cellular pre-mRNA or transcriptome, such that the likelihood that an ASO will bind other sites and cause "off-target" effects is limited .
[0104] In certain embodiments, ASOs can bind to a target. pre-mRNA. In certain embodiments, ASOs can hybridize to a pre-mRNA. In some instances, ASOs can "specifically hybridize" to or are "specific" to a target nucleic acid or a targeted portion of a pre-mRNA. Such hybridization can occur with a Tm (melting temperature) substantially greater than 37°C, at least 50 °C, or between 60 °C to approximately 90 °C. Such hybridization can correspond to stringent hybridization conditions. At a given ionic strength and pH, the Tm is the temperature at which 50% of a target sequence hybridizes to a complementary' oligonucleotide. In some embodiments, an ASO can bind to, hybridize to, or specifically hybridize to a splice site sequence in a target pre-mRNA wherein a splice site sequence comprises a sequence that shares at least about: 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity’ to any' one of the sequences of SEQ ID NOS: 47-62.
[0105] In some embodiments, an ASO RTSM comprises a sequence about 4 to about 50 nucleobases in length that, has at least about: 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity’ to any one of the sequences of SEQ ID NOS:63-94. In some embodiments, an ASO comprises a sequence about 14 to about 30 nucleobases in length that has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity’ to any one of the sequences of SEQ ID NOS:63-94.
[0106] In some embodiments, an ASO RTSM of the present disclosure comprises a sequence that shares at least about 85% sequence identity to the sequence of SEQ ID NO: 67. In some embodiments, an ASO RTSM of the present disclosure comprises a sequence that shares at least about 92% sequence identity to the sequence of SEQ ID NO: 67. In some embodiments, an ASO RTSM of the present disclosure comprises a sequence that has 100% sequence identity to the sequence of SEQ ID NO: 67. [0107 j In some embodiments, an ASO RTSM of the present disclosure comprises a sequence that shares at least about 85% sequence identity to the sequence of SEQ ID NO: 75. In some embodiments, an ASO RTSM of the present disclosure comprises a sequence that shares at least about 92% sequence identity to the sequence of SEQ ID NO: 75. In some embodiments, an ASO RTSM of the present disclosure comprises a sequence that has 100% sequence identity to the sequence of SEQ ID NO: 75.
[0108] In some embodiments, an ASO RTSM of the present disclosure comprises a sequence that shares at least about 84% sequence identity to the sequence of SEQ ID NO: 83. In some embodiments, an ASO RTSM of the present disclosure comprises a sequence that shares at least about 88% sequence identity to the sequence of SEQ ID NO: 83. In some embodiments, an ASO RTSM of the present disclosure comprises a sequence that shares at least about 84% sequence identity to the sequence of SEQ ID NO: 83. In some embodiments, an ASO RTSM of the present disclosure comprises a sequence that shares at least about 92% sequence identity to the sequence of SEQ ID NO: 83. In some embodiments, an ASO RTSM of the present disclosure comprises a sequence that shares at least about 96% sequence identity to the sequence of SEQ ID NO: 83. In some embodiments, an ASO RTSM of the present disclosure comprises a sequence that has 100% sequence identity to the sequence of SEQ ID NO: 83.
[01091 In some embodiments, an ASO RTSM of the present disclosure comprises a sequence that shares at least about 84% sequence identity to the sequence of SEQ ID NO: 91. In some embodiments, an ASO RTSM of the present disclosure comprises a sequence that shares at least about 88% sequence identity to the sequence of SEQ ID NO: 91. In some embodiments, an ASO RTSM of the present disclosure comprises a sequence that shares at least about 84% sequence identity to the sequence of SEQ ID NO: 91 . In some embodiments, an ASO RTSM of the present disclosure comprises a sequence that shares at least about 92% sequence identity’ to the sequence of SEQ ID NO: 91. In some embodiments, an ASO RTSM of the present disclosure comprises a sequence that shares at least about 96% sequence identity to the sequence of SEQ ID NO: 91. In some embodiments, an ASO RTSM of the present disclosure comprises a sequence that has 100% sequence identity to the sequence of SEQ ID NO: 91.
[0110] An ASO disclosed herein can comprise oligonucleotides and any7 other oligomeric molecule that comprises nucleobases capable of binding to a complementary' nucleobase on a target mRNA, but in some embodiments, an ASO does not comprise a sugar moiety, such as a peptide nucleic acid (PNA). An ASO may comprise naturally-occurring nucleotides, nucleotide analogs, modified nucleotides, or any combination of two or three of the preceding. The term "modified nucleotides" erm include nucleotides with modified or substituted sugar groups and/or having a modified backbone. In some embodiments, all of the nucleotides of an ASO are modified nucleotides.
[0111] In some embodiments, one or more nucleobases of an ASO may be adenine, guanine, cytosine, thymine, uracil, or any synthetic or modified nucleobase that is sufficiently similar to an unmodified nucleobase such that it is capable of hydrogen bonding with a nucleobase present on a target pre-mRNA. Examples of modified nucleobases include, without limitation, hypoxanthine, xanthine, 7-methylguanine, 5, 6-dihydrouracil, 5 -methyl cytosine, and 5-hydroxymethoylcytosine.
[0l I 2[ In some embodiments, an ASO described herein further comprise a backbone structure that connects the components of an oligomer. The term "backbone structure" and "oligomer linkages" may be used interchangeably and can refer to the connection between monomers of an ASO. In naturally occurring oligonucleotides, the backbone comprises a 3'-5' phosphodiester linkage connecting sugar moieties of the oligomer. Tire backbone structure or oligomer linkages of an ASO described herein can include (but are not limited to) phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate, phosphoraniladate, phosphoramidate, and the like. In some embodiments, the backbone structure of an ASO does not contain phosphorous but rather contains peptide bonds, for example in a peptide nucleic acid (PNA), or linking groups including carbamate, amides, and linear and cyclic hydrocarbon groups. In some embodiments, the backbone modification can be a phosphothioate linkage. In some embodiments, the backbone modification can be a phosphoramidate linkage.
[0113] Any of the ASOs described herein may contain a sugar moiety that comprises ribose or deoxyribose, as present in naturally occurring nucleotides, or a modified sugar moiety or sugar analog, including a morpholine ring. Mon-limiting examples of modified sugar moieties include 2' substitutions such as 2'-O-methyl (2'-O-Me), 2' O-methoxyethyl (2'MOE), 2'-O-aminoethyl, 2'F; N3'->P5' phosphoramidate, 2'dimethylaminooxyethoxy, 2'dimethylaminoethoxyethoxy, 2'-guanidinidium, 2'-O- guanidinium ethyl, carbamate modified sugars, and bicyclic modified sugars. In some embodiments, the sugar moiety modification can be selected from 2'-O-Me, 2'F, and 2'MOE. In some embodiments, the sugar moiety modification can be an extra bridge bond, such as in a locked nucleic acid (LNA). In some embodiments the sugar analog contains a morpholine ring, such as phosphorodiamidate morpholino (PMO). In some embodiments, the sugar moiety comprises a ribofuransyl or 2'deoxyribofuransyl modification. In some embodiments, the sugar moiety comprises 2'4'-constrained 2'0-methyloxyethyl (cMOE) modifications. In some embodiments, the sugar moiety comprises cEt 2', 4' constrained 2'-0 ethyl BNA modifications. In some embodiments, the sugar moiety comprises tricycloDNA (tcDNA) modifications. In some embodiments, the sugar moiety comprises ethylene nucleic acid (ENA) modifications. In some embodiments, the sugar moiety comprises MCE modifications.
[Oil 4] In some embodiments, one or more monomer, or each monomer of an ASO can be modified in the same way, for example each linkage of the backbone of an ASO comprises a phosphorothioate linkage or each ribose sugar moiety comprises a 2'0-methyl modification. Such modifications that are present, on each of the monomer components of an ASO are referred to as "uniform modifications," In some examples, a combination of different modifications may be desired, for example, an ASO may comprise a combination of phosphorodiamidate linkages and sugar moieties comprising morpholine rings (morpholines). Combinations of different modifications to an ASO are referred to as "mixed modifications" or "mixed chemistries."
[0115] In some embodiments, an ASO comprises one or more backbone modifications. In some embodiments, an ASO comprises one or more sugar moiety modification. In some embodiments, an ASO comprises one or more backbone modifications and one or more sugar moiety modifications. In some embodiments, an ASO comprises a 2'MOE modification and a phosphorothioate backbone. In some embodiments, an ASO comprises a phosphorodiamidate morpholino (PMO). In some embodiments, an ASO comprises a peptide nucleic acid (PNA).
[0116] In some embodiments, an ASO comprises a backbone modification comprising a phosphorothioate linkage or a phosphorodiamidate linkage. In some embodiments, an ASO comprises a phosphorodiamidate morpholino, a locked nucleic acid, a peptide nucleic acid, a 2'-O-methyl, a 2' -Fluoro, or a 2'-O- methoxyethyl moiety. In some embodiments, an ASO comprises at least one modified sugar moiety. In some embodiments, each sugar moiety can be a modified sugar moiety.
[0117] Any of the ASOs or any component of an ASO (e.g., a nucleobase, sugar moiety, backbone) described herein may be independently modified in order to achieve desired properties or activities of the ASO or reduce undesired properties or activities of the ASO. For example, an ASO or one or more components of any ASO may be modified to enhance binding affinity to a target sequence on a pre-mRNA transcript; reduce binding to any non-target sequence; reduce degradation by cellular nucleases (e.g., RNase H); improve uptake of the ASO into a cell and/or into the nucleus of a cell; alter the pharmacokinetics or pharmacodynamics of the ASO; and/or modulate the half-life of the ASO.
[0118] In some embodiments, an ASO can be comprised of one or more 2'-O-(2-methoxyethyl) (MOE) phosphorothioate-modified nucleotides. ASOs comprised of such nucleotides can be especially well-suited to the methods disclosed herein; oligomers having such modifications have been shown to have significantly enhanced resistance to nuclease degradation and increased bioavailability, making them suitable, for example, for oral delivery' in some embodiments described herein.
[0119] ASOs can be synthesized by methods described herein. Alternatively or in addition, ASOs may be obtained from a commercial source. In certain embodiments, an ASO can be prepared by stepwise solid- phase synthesis. In some cases, it may be desirable to add additional chemical moieties to an ASO, e.g., to enhance pharmacokinetics or to facilitate capture or detection of the compound. Such a moiety may be covalently attached, according to standard synthetic methods. For example, addition of a polyethylene glycol moiety or other hydrophilic polymer, e.g., one having 1-100 monomeric subunits, may' be useful in enhancing solubility.
[0120] Further a reporter moiety, such as fluorescein or a radiolabeled group, may be attached for purposes of detection. Alternatively, the reporter label attached to the oligomer may be a ligand, such as an antigen or biotin, capable of binding a labeled antibody or streptavidin. In selecting a moiety for attachment or modification of an antisense compound, can be generally desirable to select chemical compounds of groups that are biocompatible and likely to be tolerated by a subject without undesirable side effects.
Figure imgf000042_0003
[0121] Disclosed herein are RTSMs that can comprise a CRISPR associated protein. An RTSM can be a
Figure imgf000042_0001
-Cas system (CC RTSM system) wherein the system comprises a CRISPR associated protein. The
Figure imgf000042_0002
system can be designed to target a PNPLA3 pre-mRNA, prevent recruitment of a one or more splicing complex component to a pre-mRNA, decrease the binding affinity of one or more splicing complex component to a pre-mRNA, interfere with splice site signaling, sterically block splicing of a pre-mRNA, and any combination thereof. Envisioned herein are CC systems that target RNA. While any suitable CC system may be used for the purposes of the disclosure herein, in some embodiments, systems that target RN A and does not rely on consensus protospacer adjacent motif (PAM) for activity, such as Types III and VI, can be used herein. Accordingly, in some embodiments, the CC systems used herein can rely on RNA protospacer flanking sequences (PFS) or PAM sequences. Hence, in some embodiments, a PNPLA3 RNA further comprises a PFS sequence or a PAM sequence.
[0122] Subtypes of suitable CC systems disclosed herein can include, but are not limited Type II class 2, Types III-A, III-B, VI-A, VI-A, VI-C, or VI-D. In other embodiments, Type II RNA -targeting Cas9 systems can also be used as an RTSM disclosed herein.
|0123] In some embodiments, the CC RTSM system comprises a guide RNA and a Cas nuclease. In one embodiment, the guide RNA comprises a crispr RNA (crRNA) and a tracr RNA. In another embodiment, the guide RNA comprises a single guide RNA (sgRNA). In some embodiments, CC RTSM system can comprise one or more Cas nuclease. Examples of suitable Cas nucleases include, but are not limited to, Csm3, Cmr4, Csm6, Csx1, Csx27, Csx28, a member of a Cas 7 superfamily, or a Cas9, Cas 12, or a Cas 13 effector nuclease.
[0124] CC Systems that target a PNPLA3 pre-mRNA can be computationally identified through determination of a Cas containing signature genes that express RNAse or RNA targeting activity, and transcribed and processed into a CRISPR gRNA.
[0125] In some embodiments, the gRNA of the CC RTSM system comprises a binding domain that to a target region of a PNPLA3 pre-mRNA. In one embodiment, the gRNA or sgRNA binding domain comprises a sequence about 2 to about 50 nucleobases in length that has at least about: 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence complementarity to any one of the sequences of SEQ ID
NOS: 31-46. In one embodiment, the gRNA or sgRNA binding domain comprises a sequence about 14 to about 30 nucleobases in length that has at least about: 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or
100% sequence complementarity to any one of the sequences of SEQ ID NOS: 31-46. [0126] In some embodiments, the gRNA or sgRNA binding domain comprises a sequence about 4 to about 50 nucleobases in length that has at least about: 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity’ to any one of the sequences of SEQ ID NOS.71-78 and 87-94. In some embodiments, the gRNA or sgRNA binding domain comprises a sequence about 14 to about 30 nucleobases in length that has at least about: 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any one of the sequences of SEQ ID NOS.71-78 and 87-94.
10127] In some embodiments, the gRNA of the CC RTSM system targets a PNPLA3 pre-mRNA of interest and directs the Cas nuclease to a pre-mRNA. According to some embodiments, the Cas nuclease can be a catalytically dead variant, wherein upon gRNA binding to a target region of a pre-mRNA, the CC RTSM system increases exon skipping wherein the system prevents recruitment of a one or more splicing complex component to a pre-mRNA, decrease the binding affinity of one or more splicing complex component to a pre-mRNA, or sterically block mRNA splicing.
[0128] In other embodiments, the Cas nuclease can be selected and or synthesized to interfere with splice site signaling through RNAse activity' wherein upon gRNA binding to a target region of a pre-mRNA , the the Cas nuclease disrupts splice site signaling sequences of a targeted exon-intron junction thereby inducing exon skipping.
Modifications
JOI 29] Any of the RTSMs described herein may be modified in order to achieve desired properties or activities of an RTSM or reduce undesired properties or activities of an RTSM. For example, an RTSM or one or more components of any RTSM may be modified to enhance binding affinity to a target sequence on a pre-mRNA transcript; reduce binding to any non-target sequence; reduce degradation by cellular nucleases (e.g., RNase H); improve uptake of an RTSM into a cell and/or into the nucleus of a cell; alter the pharmacokinetics or pharmacodynamics of the RTSM; and/or modulate the half-life of the RTSM.
[ 0130 J Also included herein are vector delivery systems that are capable of expressing an RTSM sequences herein, such as vectors that express a polynucleotide sequence comprising any one or more of the sequences shown in Table 7, as described herein. By “vector” or “nucleic acid construct” can be meant to be a polynucleotide molecule, such as a DNA molecule derived, for example, from a plasmid, bacteriophage, yeast or virus, into which a polynucleotide can be inserted or cloned. A vector can contain one or more unique restriction sites and can be capable of autonomous replication in a defined host cell including a target cell or tissue or a progenitor cell or tissue thereof, or be integrated with the genome of the defined host such that the cloned sequence can be reproducible. Accordingly, the vector can be an autonomously replicating vector, e.g., a vector that exists as an extra-chromosomal entity, the replication of which is independent of chromosomal replication, e.g., a linear or closed circular plasmid, an extra-chromosomal element, a mini -chromosome, or an artificial chromosome. The vector can contain any means for assuring self-replication. Alternatively, tire vector can be one which, when introduced into the host cell, is integrated into the genome and replicated together with the chromosome(s) into which it has been integrated.
[0131] For example, an RTSM of the present disclosure can be conjugated to a cell penetrating peptide. The term “cell penetrating peptide” and “CPP” are used interchangeably and can refer to cationic cell penetrating peptides, also called transport peptides, carrier peptides, or peptide transduction domains. The peptides, as shown herein, have the capability of inducing cell penetration within 100% of cells of a given cell culture population and allow macromolecular translocation within multiple tissues in vivo upon systemic administrati on .
10132] In one embodiment, an RTSM can be an ASO wherein an ASO can comprise an oligonucleotide moiety conjugated to a cell penetrating peptide effective to enhance transport of the compound into cells. In some embodiment, the oligonucleotide moiety can be an arginine-rich peptide transport moiety effective to enhance transport of the compound into cells. The transport moiety can be attached to a terminus of the oligomer. The peptides have the capability of inducing cell penetration within 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% of cells of a given cell culture population, including all integers in between, and allow macromolecular translocation within multiple tissues in vivo upon systemic administration. In one embodiment, the cell-penetrating peptide may be an arginine-rich peptide transporter. In another embodiment, the cell-penetrating peptide may be Penetratm or the Tat peptide. In one embodiment, the CPP can be conjugated to an ASO herein and can utilize glycine as the linker between the CPP and the antisense oligonucleotide. For example, a preferred peptide conjugated PMO consists of Re-G-PMO.
[0133] The transport moieties as described above can enhance cell entry' of attached oligomers, relative to uptake of the oligomer in the absence of the attached transport moiety. Uptake can be enhanced at least ten fold, and at least twenty' fold, relative to the unconjugated compound.
[0134] The use of arginine-rich peptide transporters (e.g., cell-penetrating peptides) can be used herein. For example, when conjugated to an antisense PMO, argine-rich CPPs can demonstrate an enhanced ability to alter splicing of several gene transcripts. Exemplary' peptide transporters, excluding linkers, can be seen in Table 8.
Table 8: Exemplary Peptide transporters
Figure imgf000044_0001
Assays
[0135] An RTSM disclosed herein can increase exon skipping during pre-mRNA splicing as compared to a pre-mRNA spliced in the absence of an RTSM herein as determined by an in vitro assay. In some embodiments, exon skipping modulates PNPLA3 mRNA transcript production. In some embodiments, exon skipping increases production of mRNA transcript excluding coding for one or more exons, a truncated mRNA molecule, or both. In some embodiments, RTSM-modulated mRNA expression can be compared to mRNA processed in the absence of RTSM as determined by an in vitro assay.
[0136] In some embodiments, exon skipping modulates PNPLA3 protein production. In certain embodiments, an increase in exon skipping induces an increase in modulated PNPLA3 protein expression. In some embodiments, exon skipping increases production of a non-functional PNPLA3 protein, a semi- functional PNPLA3 protein, a truncated PNPLA3 protein, or any combination thereof. In some embodiments, RTSM modulated PNPL.A3 protein production can be compared to PNPLA3 protein production in the absence of RTSM as determined by an in vitro assay.
[0137] In some embodiments, the cell, organ, or subject can be evaluated to determine if appropriate for the methods and compositions described herein. Methods of determining exon skipping, mRNA modulation, protein expression, or PNPLA3 modulation are described, such as in the Examples, herein.
[0138] Other non-limiting assays to determine gene expression, exon skipping and PNPLA3 expression include quantitative PCR (qPCR), including but not limited to PCR, real-time PCR (e.g., Sybr-green), and/or hot PCR. In some cases, expression of one or more genes can be measured by detecting the level of transcripts of the genes. Exon skipping can be measured by detecting the expression of the processed m- RNA. Expression of functional PNPLA3 protein can be measured by detecting the level or length of the protein, or by an assay that measures its biological activity. For example, expression can be measured by- Northern blotting, nuclease protection assays (e.g., RNase protection assays), reverse transcription PCR, quantitative PCR (e.g., real-time PCR such as real-time quantitative reverse transcription PCR), in situ hybridization (e.g., fluorescent in situ hybridization (FISH)), dot-blot analysis, differential display, serial analysis of gene expression, subtractive hybridization, microarrays, nanostrmg, and/or sequencing (e.g., next-generation sequencing) Expression can be measured by protein immunostaining, protein immunoprecipitation, electrophoresis (e.g., SDS-PAGE), Western bloting, bicinchoninic acid assay, spectrophotometry, mass spectrometry, enzyme assays (e.g., enzyme-linked immunosorbent assays), immunohistochemistry-, flow cytometry-, and/or immunocytochemistry-. Expression of one or more genes can also be measured by microscopy. The microscopy can be optical, electron, or scanning probe microscopy. Optical microscopy can comprise use of bright field, oblique illumination, cross-polarized light, dispersion staining, dark field, phase contrast, differential interference contrast, interference reflection microscopy, fluorescence (e.g., when particles, e.g., cells, are immunostained), confocal, single plane illumination microscopy, light sheet fluorescence microscopy, deconvolution, or serial time-encoded amplified microscopy.
Methods
[0139] Disclosed herein, among other disclosures, are methods of exon skipping, modulating PNPLA3 pre-mRNA expression, modulating PNPLA3 protein expression, and prophylaxis/treatment of: PNPLA3- mediated conditions, liver disorder, liver disease, and any combination thereof.
[0140] In some embodiments, the disclosed compositions and methods result in a truncated PNPLA3 protein. In some embodiments, the disclosed compositions and methods result in a decrease in the wild- type PNPLA3 protein . In some embodiments, the disclosed compositions and methods result in modulating the splicing of PNPLA3 pre-mRNA. In some embodiments, the disclosed compositions and methods result in an PNPLA3 mRNA lacking exon 5 or portion thereof. In some embodiments, PNPLA3 expression can be modulated compared to a control. A control can be wild-type or non wild-type control. Controls can be positive or negative controls.
Methods of modulating PNPLA3 expression
[0141] Disclosed herein are methods to increase exon skipping of an PNPLA3 mRNA comprising contacting a PNPLA3 pre-mRNA with an RTSM as disclosed herein, and allowing modulated splicing to occur. In some embodiments, an RTSM herein increase the level of modulated PNPLA3 mRNA transcripts as compared to mRNA transcripts processed in the absence of RTSM wherein a mRNA transcript modulation comprises a decrease in lull-length PNPLA3 mRNA transcript, an increase in truncated PNPLA3 mRNA transcript, an increase in an PNPLA3 mRNA transcript lacking coding for one or more exons, or any combination thereof.
[0142] In some embodiments, the increase in modulated mRNA transcript processing can be about: 0.1%, 0.2% 0.3% 0.5%, 0.7%, 0.9%, 1%, 1.01%, 1.5%, 2%, 2.01%, 2.5%, 3%, 3.01%, 3.5%, 4%, 4.01%, 4.5%, 5%, 5.01%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, 10%, 10.5%, 11%, 11.5%, 12%, 12.5%, 13%, 13.5%, 14%, 14.5%, 15%, 15.5%, 16%, 16.5%, 17%, 17.5%, 18%, 18.5%, 19%, 19.5%, 20%, 21 %, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 40%, 45%, 50%, 55%, or 60% or more relative to a non contacted control. In certain embodiments, the increase in modulated mRNA transcript processing can be about: 65%, 70%, 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%, or 100% relative to a non-contacted control.
[0143] In some embodiments, the increase in modulated PNPLA3 protein production can be about: 0. 1%, 0.2% 0.3% 0.5%, 0.7%, 0.9%, 1%, 1.01 %, 1.5%, 2%, 2.01%, 2.5%, 3%, 3.01%, 3.5%, 4%, 4.01 %, 4.5%, 5%, 5.01%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, 10%, 10.5%, 11%, 11.5%, 12%, 12.5%, 13%, 13.5%, 14%, 14.5%, 15%, 15.5%, 16%, 16.5%, 17%, 17.5%, 18%, 18.5%, 19%, 19.5%, 20%, 21 %, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 40%, 45%, 50%, 55%, or 60% or more relative to a non contacted control. In certain embodiments, the increase in modulated mRNA transcript processing can be about: 65%, 70%, 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%, or 100% relative to a non -contacted control.
Methods of Treating Diseases and Conditions
[0144] Disclosed herein are methods of prophylaxis/treatment of PNPLA3-associated disorders. Disclosed herein are methods of treatment comprising administering to a subject in need thereof an effective amount of PNPLA3 RTSM, or a pharmaceutical composition comprising the same.
[0145] In some embodiments, the disclosure provides a method for inducing exon skipping to decrease the level of functional PNPLA3 in a subject in need thereof comprising administering to the subject a dose of PNPLA3 RTSM. In some embodiments, the disclosure provides a method for increasing the level of exon modulated PNPLA3 mRN A in a subject in need thereof comprising administering to the subject a dose of PNPLA3 RTSM. In some embodiments, treatment with a RTSM of the disclosure increases one or more modulated PNPLA3 mRNA production, decreases full length PNPLA3 mRNA production, decreases full length PNPLA3 mRNA production, decreases functional PNPLA3 production, decreases full-length PNPLA3 protein production, increases truncated PNPLA3 production; decreases, prevents, or delays liver damage; decreases, prevents, or delays steatosis; decreases, prevents, or delays liver fibrosis; decreases, prevents, or delays liver inflammation; decreases, prevents, or delays liver scarring or cirrhosis; decreases, prevents, or delays liver failure; decreases, prevents, or delays liver enlargement; decreases, prevents, or delays elevated transaminases; decreases, prevents, or delays hepatic fat accumulation; decreases PNPLA3 protein accumulation; increases PNPLA3 protein degradation; decreases CGI-58 levels on lipid cells, decreases CCL2 production; decreases CCL5 production, decreases activated HSCs, decreases collagen 1 secretion; or any combination thereof to be expected in the absence of treatment with a RTSM.
[0146] In one embodiment, the method disclosed herein can be useful treat subject who is suffering from or is at a risk of developing a PNPLA3-associated disorder. In one embodiment, the present disclosure can be useful to treat a subject who is suffering from or at the risk of developing an PNPLA3 associated condition. An PNPLA3 associated condition can be a disorder that can generally characterized by the accumulation of PNPLA3 protein, the dysregulated expression of a PNPLA3 protein, or both. In some embodiments, a PNPLA3 protein can comprise a gain-of-function mutation, a loss of function mutation, or a morphological mutation, such as an elongated translated protein, or any combination thereof. Mutants can include the 1148M variant and the S47A variant. In some embodiments, a PNPLA3 -associated disorder comprises liver disease, NAFLD, hepatic steatosis, non-alcoholic steatohepatitis (NASH), liver cirrhosis, hepatocellular carcinoma, alcoholic liver disease, alcoholic steatohepatitis (ASH), HCV hepatitis, chronic hepatitis, hereditary hemochromatosis, or primary sclerosing cholangitis.
[0147] In some embodiments, the method increases truncated PNPLA3 mRNA production by 0.1%, 0.2% 0.3% 0.5%, 0.7%, 0.9%, 1%, 1.01%, 1.5%, 2%, 2.01%, 2.5%, 3%, 3.01%, 3.5%, 4%, 4.01%, 4.5%, 5%, 5.01%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, 10%, 10.5%, 11%, 1 1.5%, 12%, 12.5%, 13%, 13.5%, 14%, 14.5%, 15%, 15.5%, 16%, 16.5%, 17%, 17.5%, 18%, 18.5%, 19%, 19.5%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 40%, 45%, 50%, 55%, or 60% or more relative to a untreated control.
[0148] In certain embodiments, the method decreases functional PNPLA3 production by 0. 1 %, 0.2% 0.3% 0.5%, 0.7%, 0.9%, 1%, 1.01%, 1.5%, 2%, 2.01%, 2.5%, 3%, 3.01%, 3.5%, 4%, 4.01%, 4.5%, 5%, 5.01%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, 10%, 10.5%, 1 1%, 11.5%, 12%, 12.5%, 13%, 13.5%, 14%, 14.5%, 15%, 15.5%, 16%, 16.5%, 17%, 17.5%, 18%, 18.5%, 19%, 19.5%, 20%, 21 %, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 40%, 45%, 50%, 55%, or 60% or more relative to a untreated control.
[0149] In certain embodiments, the method decreases full-length or elongated PNPLA3 production by 0.1 %, 0.2% 0.3% 0.5%, 0.7%, 0.9%, 1%, 1.01 %, 1.5%, 2%, 2.01%, 2.5%, 3%, 3.01%, 3.5%, 4%, 4.01 %, 4.5%, 5%, 5.01%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, 10%, 10.5%, 11 %, 11 .5%, 12%, 12.5%, 13%, 13.5%, 14%, 14.5%, 15%, 15.5%, 16%, 16.5%, 17%, 17.5%, 18%, 18.5%, 19%, 19.5%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 40%, 45%, 50%, 55%, or 60% or more relative to a untreated control.
[0150] In certahi embodiments, the method increases truncated PNPLA3 production by 0.1%, 0.2% 0.3% 0.5%, 0.7%, 0.9%, 1%, 1.01%, 1.5%, 2%, 2.01%, 2.5%, 3%, 3.01%, 3.5%, 4%, 4.01%, 4.5%, 5%, 5.01%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, 10%, 10.5%, 1 1%, 11.5%, 12%, 12.5%, 13%, 13.5%, 14%, 14.5%, 15%, 15.5%, 16%, 16.5%, 17%, 17.5%, 18%, 18.5%, 19%, 19.5%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 40%, 45%, 50%, 55%, or 60% or more relative to a untreated control.
[0151] In certain embodiments, the method decreases full-length or elongated PNPLA3 production by 0.1%, 0.2% 0.3% 0.5%, 0.7%, 0.9%, 1%, 1.01 %, 1.5%, 2%, 2.01%, 2.5%, 3%, 3.01%, 3.5%, 4%, 4.01 %, 4.5%, 5%, 5.01%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, 10%, 10.5%, 11%, 11.5%, 12%, 12.5%, 13%, 13.5%, 14%, 14.5%, 15%, 15.5%, 16%, 16.5%, 17%, 17.5%, 18%, 18.5%, 19%, 19.5%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 40%, 45%, 50%, 55%, or 60% or more relative to a untreated control.
[0152] In certain embodiments, the method increases truncated PNPLA3 production by 0.1%, 0.2% 0.3% 0.5%, 0.7%, 0.9%, 1%, 1.01%, 1.5%, 2%, 2.01%, 2.5%, 3%, 3.01%, 3.5%, 4%, 4.01%, 4.5%, 5%, 5.01%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, 10%, 10.5%, 11%, 11.5%, 12%, 12.5%, 13%, 13.5%, 14%, 14.5%, 15%, 15.5%, 16%, 16.5%, 17%, 17.5%, 18%, 18.5%, 19%, 19.5%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 40%, 45%, 50%, 55%, or 60% or more relative to a untreated control.
[0153] In certain embodiments, the method decreases PNPLA3 protein accumulation by 0.1%, 0,2% 0.3% 0.5%, 0.7%, 0.9%, 1%, 1.01%, 1.5%, 2%, 2.01%, 2.5%, 3%, 3.01%, 3.5%, 4%, 4.01%, 4.5%, 5%, 5.01%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, 10%, 10.5%, 11%, 11.5%, 12%, 12.5%, 13%, 13.5%, 14%, 14.5%, 15%, 15.5%, 16%, 16.5%, 17%, 17.5%, 18%, 18.5%, 19%, 19.5%, 20%, 21 %, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 40%, 45%, 50%, 55%, or 60% or more relative to a untreated control. [0154] In some embodiments, the method increases PNPLA3 protein degradation by 0.1%, 0.2% 0.3% 0.5%, 0.7%, 0.9%, 1%, 1.01%, 1.5%, 2%, 2.01%, 2.5%, 3%, 3.01%, 3.5%, 4%, 4.01%, 4.5%, 5%, 5.01%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, 10%, 10.5%, 1 1%, 11.5%, 12%, 12.5%, 13%, 13.5%, 14%, 14.5%, 15%, 15.5%, 16%, 16.5%, 17%, 17.5%, 18%, 18.5%, 19%, 19.5%, 20%, 21 %, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 40%, 45%, 50%, 55%, or 60% or more relative to a untreated control
[0155] In some embodiments, the method decreases CGI-58 levels on lipid cells by 0.1%, 0.2% 0.3% 0.5%, 0.7%, 0.9%, 1%, 1.01%, 1.5%, 2%, 2.01%, 2.5%, 3%, 3.01%, 3.5%, 4%, 4.01 %, 4.5%, 5%, 5.01 %, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, 10%, 10.5%, 11%, 1 1.5%, 12%, 12.5%, 13%, 13.5%, 14%, 14.5%, 15%, 15.5%, 16%, 16.5%, 17%, 17.5%, 18%, 18.5%, 19%, 19.5%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 40%, 45%, 50%, 55%, or 60% or more relative to a untreated control
[0156] In some embodiments, the method decreases CCL2 production by 0.1 %, 0.2% 0.3% 0.5%, 0.7%, 0.9%, 1%, 1.01%, 1.5%, 2%, 2.01 %, 2.5%, 3%, 3.01%, 3.5%, 4%, 4.01%, 4.5%, 5%, 5.01%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, 10%, 10.5%, 11%, 1 1.5%, 12%, 12.5%, 13%, 13.5%, 14%, 14.5%, 15%, 15.5%, 16%, 16.5%, 17%, 17.5%, 18%, 18.5%, 19%, 19.5%, 2.0%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 40%, 45%, 50%, 55%, or 60% or more relative to a untreated control
[0157] In some embodiments, the method decreases CCL5 production by 0.1%, 0.2% 0.3% 0.5%, 0.7%, 0.9%, 1%, 1.01%, 1.5%, 2%, 2.01%, 2.5%, 3%, 3.01%, 3.5%, 4%, 4.01%, 4.5%, 5%, 5.01%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, 10%, 10.5%, 11%, 11.5%, 12%, 12.5%, 13%, 13.5%, 14%, 14.5%, 15%, 15.5%, 16%, 16.5%, 17%, 17.5%, 18%, 18.5%, 19%, 19.5%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 40%, 45%, 50%, 55%, or 60% or more relative to a untreated control
[0158] In some embodiments, the method decreases activated HSCs by 0.1%, 0,2% 0.3% 0.5%, 0.7%, 0.9%, 1%, 1.01%, 1.5%, 2%, 2.01%, 2.5%, 3%, 3.01%, 3.5%, 4%, 4.01%, 4.5%, 5%, 5.01%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, 10%, 10.5%, 11%, 11.5%, 12%, 12.5%, 13%, 13.5%, 14%, 14.5%, 15%, 15.5%, 16%, 16.5%, 17%, 17.5%, 18%, 18.5%, 19%, 19.5%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 40%, 45%, 50%, 55%, or 60% or more relative to a untreated control
[0159] In some embodiments, the method decreases collagen 1 secretion by 0.1%, 0.2% 0.3% 0.5%, 0.7%, 0.9%, 1%, 1.01%, 1.5%, 2%, 2.01%, 2.5%, 3%, 3.01%, 3.5%, 4%, 4.01%, 4.5%, 5%, 5.01%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, 10%, 10.5%, 11%, 11.5%, 12%, 12.5%, 13%, 13.5%, 14%, 14.5%, 15%, 15.5%, 16%, 16.5%, 17%, 17.5%, 18%, 18.5%, 19%, 19.5%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 40%, 45%, 50%, 55%, or 60% or more relative to a untreated control
[0160] In some embodiments, the method further comprises evaluating the subject prior to RTSM administration to determine whether the subject is suitable for the treatment. In some embodiments, evaluating the subject can be determination of mutations of a PNPLA3 gene, increased PNPLA3 protein expression compared to a wild-type control, increased PNPLA3 activity compared to a wild-type control, or any combination thereof. In some embodiments, a subject in need thereof expresses PNPLA3 gene OG rs738409 polymorphism.
Methods of Administration
[0161] As disclosed in further detail below, the formulations or preparations herein may be given orally, parenterally, systemically, topically, rectally or intramuscular administration. They can be given in a form suitable for each administration route. For example, they can be administered in tablets or capsule form, by injection, inhalation, eye lotion, ointment, suppository, etc. administration by injection, infusion or inhalation; topical by lotion or ointment; and rectal by suppositories.
[0162] Regardless of the route of administration selected, formulations herein may conveniently be presented in unit dosage form. The amount of active ingredient that can be combined with a carrier material to produce a single dosage form will vary depending upon the host being treated, the particular mode of administration. The amount of active ingredient which can be combined with a carrier material to produce a single dosage form will generally be that amount of the compound which produces a therapeutic effect. Generally, out of one hundred percent, this amount will range from about 0.1 percent to about ninety-nine percent of active ingredient, from about 5 percent to about 70 percent, or from about 10 percent to about 30 percent.
101631 The selected dosage level will depend upon a variety of factors including the activity of the particular RTSM herein, the route of administration, the time of administration, the rate of excretion or metabolism of the particular RTSM being employed, the rate and extent of absorption, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular oligomer employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.
[0164] In general, a suitable daily dose of a compound herein will be that amount of the compound which is the lowest dose effective to produce a therapeutic effect. Such an effective dose will generally depend upon the factors described above. Generally, oral, intravenous, intracerebroventricular, intramuscular and subcutaneous doses of the compounds herein for a patient, when used for the indicated effects, will range from about 0.0001 to about 100 mg per kilogram of body weight per day.
[0165] In some embodiments wherein an RTSM can be an ASO, doses of an ASO herein can be generally administered is from about 0.001 mg/kg to about 1000 mg/kg, wherein mg is mg of RTSM and kg is the body weight, of the subject. For example, 0.001 mg/kg to about 1 mg/kg, 1 -20 mg/kg, 20-40 mg/kg, 40- 60mg.kg, 60-80 mg/kg, or 80-100 mg/kg. For i.v. administration, preferred doses are from about 0.5 mg to 100 mg/kg. In some embodiments, an ASO can be administered at doses of about: 0.001 mg/kg, 0.01 mg/kg, 0.1 mg/kg, 1 mg/kg, 5 mg/kg, 10 mg/kg, 15 mg/kg, 20 mg/kg, 21 mg/kg, 22mg/kg, 23 mg/kg, 24mg/kg, 25 mg/kg, 26 mg/kg, 27 mg/kg, 28 mg/kg, 29 mg/kg, 30 mg/kg, 31 mg/kg, 32 mg/kg, 33 mg/kg, 34 mg/kg, 35 mg/kg, 36 mg/kg, 37 mg/kg, 38 mg/kg, 39 mg/kg, 40 mg/kg, 41 mg/kg, 42 mg/kg, 43 mg/kg, 44 mg/kg, 45 mg/kg, 46 mg/kg, 47 mg/kg, 48 mg/kg, 49 mg/kg 50 mg/kg, 51 mg/kg, 52. mg/kg, 53 mg/kg, 54 mg/kg, 55 mg/kg, 56 mg/kg, 57 mg/kg, 58 mg/kg, 59 mg/kg, 60 mg/kg, 65 mg/kg, 70 mg/kg, 75 mg/kg, 80 mg/kg, 85 mg/kg, 90 mg/kg, 95 mg/kg, 100 mg/kg, including all integers in between.
[0166] If desired, the effective daily dose of the active compound may be administered as two, three, four, five, six or more sub-doses administered separately at appropriate intervals throughout the day, optionally, in unit dosage forms. In certain situations, dosing is one administration per day. In certain embodiments, dosing is one or more administration per every 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 days, or every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 weeks, or every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months, as needed, to maintain the desired expression of a PNPLA3 pre-mRNA and/or a PNPLA3 protein. An RTSM may be administered in continuously or in cycles.
[0167] In some embodiments, an RTSM of the present can be administered, generally at regular intervals (e.g,, daily, weekly, biweekly, monthly, bimonthly). An RTSM may be administered at regular intervals, e.g., daily; once every two days; once every’ three days; once every 3 to 7 days; once every 3 to 10 days; once every 7 to 10 days; once every week; once every two weeks; once monthly. For example, an RTSM may be administered once weekly by intravenous infusion. An RTSM may be administered intermittently over a longer period of time, e.g., for several weeks, months or years. For example, an RTSM may be administered once every' one, two, three, tour, five, six, seven, eight, nine, ten, eleven or twelve months. In addition, an RTSM may be administered once every one, two, three, tour or five years. Administration may be followed by, or concurrent with, co-administration with a second agent, for example with an antibiotic, steroid or other therapeutic agent. The treatment regimen may be adjusted (dose, frequency, route, etc.) as indicated, based on the results of immunoassays, other biochemical tests and physiological examination of the subject under treatment.
[01681 When co-administered with one or more other therapies, an RTSM of the disclosure may be administered either simultaneously with the other treatment(s), or sequentially in any order and can be temporally spaced up to several days apart.
Pharmaceutical Compositions and Dosage Forms
[0169] Disclosed herein, in certain embodiments, are pharmaceutical compositions comprising an RTSM described herein and a carrier thereof for administration in a subject.
[0170] In certain embodiments, the pharmaceutically acceptable compositions comprise a therapeutically- effective amount of one or more of an RTSM, formulated together with one or more pharmaceutically acceptable: carriers (additives) and/or diluents. In some embodiments, when an RTSM herein are administered as pharmaceuticals, to humans and animals, they can be given per se or as a pharmaceutical composition containing, for example, 0.1 to 99%, or 10 to 30% of an RTSM in combination with a pharmaceutically acceptable carrier.
[0171] A pharmaceutical composition of the present disclosure can be delivered, e.g., subcutaneously or intravenously with a standard needle and syringe or a pen delivery device. The injectable preparations may include dosage forms for intravenous, subcutaneous, intracutaneous and intramuscular injections, drip infusions, etc. The injectable preparations may be prepared, e.g., by dissolving, suspending or emulsifying an RTSM herein in a sterile aqueous medium or an oily medium conventionally used for injections. As the aqueous medium for injections, there are, for example, physiological saline, an isotonic solution containing glucose and other auxiliary agents, etc., which may be used in combination with an appropriate solubilizing agent such as an alcohol (e.g., ethanol), a polyalcohol (e.g., propylene glycol, polyethylene glycol), a nonionic surfactant [e.g., polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil)], etc. As the oily medium, there are employed, eg., sesame oil, soybean oil, etc., which may be used in combination with a solubilizing agent such as benzyl benzoate, benzyl alcohol, etc. The injection thus prepared can be filled m an appropriate ampoule.
[0172] Compositions of the present disclosure can be in the form of, tor example, granules, powders, tablets, capsules, syrup, suppositories, injections, emulsions, elixirs, suspensions or solutions. The amount of the aforesaid antibody contained can be about 5 to about 500 mg per dosage form in a unit dose. In one embodiment, an RTSM can be contained in about, in about 5 to about. 100 mg, for example for a parental dosage form. In other embodiments, an RTSM can be contained in about 10 to about 250 mg for the other dosage forms.
[0173] For example, oral, buccal, and sublingual administration, powders, suspensions, granules, tablets, pills, capsules, gelcaps, and caplets can be used as solid dosage forms. These can be prepared, for example, by mixing the RTSM, with at least one additive such as a starch or other additive. Suitable additives are sucrose, lactose, cellulose sugar, mannitol, maltitol, dextran, starch, agar, alginates, chitins, chitosans, pectins, tragacanth gum, gum arable, gelatins, collagens, casein, albumin, synthetic or semi-synthetic polymers or glycerides. Optionally, oral dosage forms can contain other ingredients to aid in administration, such as an inactive diluent, or lubricants such as magnesium stearate, or preservatives such as paraben or sorbic acid, or anti-oxidants such as ascorbic acid, tocopherol or cysteine, a disintegrating agent, binders, thickeners, buffers, sweeteners, flavoring agents or perfuming agents. Tablets and pills may be further treated with suitable coating materials known in the art. [01741 Liquid dosage forms for oral administration may be in the form of pharmaceutically acceptable emulsions, syrups, elixirs, suspensions, and solutions, which may contain an inactive diluent, such as water. In some embodiments, pharmaceutical formulations and medicaments may be prepared as liquid suspensions or aqueous solutions, for example, using a sterile liquid, such as, but not limited to, an oil, water, an alcohol, and combinations of these. In some embodiments, pharmaceutical compositions can be prepared in a lyophilized form. The lyophilized preparations can comprise a cryoprotectant known in the art. The term “cryoprotectants” as used herein generally includes agents, which provide stability to the protein from freezing-induced stresses. Examples of cryoprotectants include polyols such as, for example, mannitol, and include saccharides such as, for example, sucrose, as well as including surfactants such as, for example, polysorbate, poloxamer or polyethylene glycol, and the like. Cryoprotectants also contribute to the tonicity of the formulations. Pharmaceutically suitable surfactants, suspending agents, emulsifying agents, may be added for oral or par- enteral administration.
[01751 As noted above, suspensions may include oils. Such oils include, but are not limited to, peanut oil, sesame oil, cottonseed oil, com oil and olive oil. Suspension preparation may also contain esters of faty acids such as ethyl oleate, isopropyl myristate, fatty acid glycerides and acetylated fatty acid glycerides. Suspension formulations may include alcohols, such as, but not limited to, ethanol, iso- propyl alcohol, hexadecyl alcohol, glycerol and propylene glycol. Ethers, such as but not limited to, poly(ethyleneglycoi), petroleum hydrocarbons such as mineral oil and petrolatum: and water may also be used in suspension formulations.
[0176] For nasal administration, the pharmaceutical fonnulations and medicaments may be a spray or aerosol containing an appropriate solvent(s) and optionally other compounds such as, but not limited to, stabilizers, antimicrobial agents, antioxidants, pH modifiers, surfactants, bio- availability modifiers and combinations of these. A propellant for an aerosol Formulation may include compressed air, nitrogen, carbon dioxide, or a hydrocarbon based low boiling solvent.
[0177] Injectable dosage forms generally include aqueous suspensions or oil suspensions which may be prepared using a suitable dispersant or wetting agent and a suspending agent. Injectable forms may be in solution phase or in the form of a suspension, wdrich can be prepared with a solvent or diluent. Acceptable solvents or vehicles include sterilized water, Ringer’s solution, or an isotonic aqueous saline solution. Alternatively, sterile oils may be employed as solvents or suspending agents. In some embodiments, the oil or fatty acid is non-volatile, including natural or synthetic oils, fatty acids, mono-, di- or tri -glycerides.
[0178] For injection, the pharmaceutical formulation and/ or medicament may be a powder suitable for reconstitution with an appropriate solution as described above. Examples of these include, but are not limited to, freeze dried, rotary dried or spray dried powders, amorphous powders, granules, precipitates, or particulates. For injection, the formulations may optionally contain stabilizers, pH modifiers, surfactants, bioavailability modifiers and combinations of these.
[0179] For rectal administration, the pharmaceutical formulations and medicaments may be in the form of a suppository, an ointment, an enema, a tablet or a cream for release of compound in the intestines, sigmoid flexure and/or rectum. Rectal suppositories are prepared by mixing one or more compounds herein with acceptable vehicles, for example, cocoa butter or polyethylene glycol, which is present in a solid phase at normal storing temperatures, and present in a liquid phase at those temperatures suitable to release a drug inside the body, such as in the rectum. Oils may also be employed in the preparation of formulations of the soft gelatin type and suppositories. Water, saline, aqueous dextrose and related sugar solutions, and glycerols may be employed in the preparation of suspension formulations which may also contain suspending agents such as pectins, carbomers, methyl cellulose, hydroxypropyl cellulose or carboxymethyl cellulose, as well as buffers and preservatives. [01801 The concentration of an RTSM in these compositions can vary7 widely, e.g., from less than about 10%, least about 25% to as much as 75% or 90% by weight and wi 11 be selected primarily by fluid volumes, viscosities, etc., in accordance with the particular mode of administration selected.
[0181] In some embodiments, pharmaceutical compositions comprising an RTSM described herein can be formulated using one or more physiologically acceptable carriers including excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen.
[0182] Pharmaceutical compositions are optionally manufactured such as, by way of example only, by means of mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or compression processes.
[0183] In certain embodiments, compositions may also include one or more pH adjusting agents or buffering agents, including acids such as acetic, boric, citric, lactic, phosphoric and hydrochloric acids; bases such as sodium hydroxide, sodium phosphate, sodium borate, sodium citrate, sodium acetate, sodium lactate and tris-hydroxymethylaminomethane; and buffers such as citrate/dextrose, sodium bicarbonate and ammonium chloride. Such acids, bases and buffers are included in an amount required to maintain pH of the composition in an acceptable range. In other embodiments, compositions may also include one or more salts in an amount required to bring osmolality of the composition into an acceptable range. Such salts include those having sodium, potassium or ammonium cations and chloride, citrate, ascorbate, borate, phosphate, bicarbonate, sulfate, thiosulfate or bisulfite anions; suitable salts include sodium chloride, potassium chloride, sodium thiosulfate, sodium bisulfite and ammonium sulfate.
[0184] In some embodiments, sustained-release preparations can be used. Suitable examples of sustained- release preparations include semipermeable matrices of solid hydrophobic polymers containing an antibody or antigen binding fragment of the present disclosure, in which the matrices are in the form of shaped articles, e.g., films, or microcapsule. Examples of sustained-release matrices include polyesters, hydrogels (for example, poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides (U.S. Patent No. 3,773,919), copolymers of L-glutamic acid and y ethyl-L-giutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as the LUPRON DEPOT™ (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly-D-(-)-3-hydroxybutyric acid. While polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid enable release of molecules for over 100 days, certain hydrogels release proteins for shorter time periods. When encapsulated antibodies remain in the body for a long time, they can denature or aggregate as a result of exposure to moisture at 37°C, resulting in a loss of biological activity and possible changes in immunogenicity. Rational strategies can be devised for stabilization depending on the mechanism involved. For example, if the aggregation mechanism is discovered to be intermolecular S--S bond formation through thiodisulfide interchange, stabilization can be achieved by modifying sulfhydryl residues, lyophilizing from acidic solutions, controlling moisture content, using appropriate additives, and developing specific polymer matrix compositions. In certain situations, the pharmaceutical composition can be delivered in a controlled release system. In one embodiment, a pump may be. In another embodiment, polymeric materials can be used. In yet another embodiment, a controlled release system can be placed in proximity of the composition's target, thus requiring only a fraction of the systemic dose.
[0185] In some embodiments, an RTSM can be administered with one or more agents capable of promoting penetration of the subject antisense oligonucleotide across the blood-brain barrier. In some embodiments, an RTSM can be linked with a viral vector, e.g., to render an RTSM more effective or increase transport across the blood-brain barrier. For example, delivery’ of agents can be by administration of an adenovirus vector to motor neurons in muscle tissue. Delivery of vectors directly to the brain, include but are not limited to the striatum, tire thalamus, the hippocampus, or the substantia nigra. [0186] In embodiments, an RTSM can be linked or conjugated with agents that provide desirable pharmaceutical or pharmacodynamic properties. In some embodiments, an RTSM can be coupled to a substance that promotes penetration or transport across the blood-brain barrier, e.g., an antibody to the transferrin receptor. In some embodiments, osmotic blood brain barrier disruption can be assisted by infusion of sugars, e.g., meso erythritol, xylitol, D(+) galactose, D(+) lactose, D(+) xylose, dulcitol, myo- inositol, L(-) fructose, D(-) mannitol, D(t-) glucose, D(+) arabinose, D(-) arabinose, cellobiose, D(-i-) maltose, D(-f-) raffinose, L(-i-) rhamnose, D(t-) melibiose, D(-) ribose, adonitol, D(+) arabitol, L(-) arabitol, D(+) fucose, L(-) fucose, D(-) lyxose, L(+) lyxose, and L(-) lyxose, or amino acids, e.g., glutamine, lysine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glycine, histidine, leucine, methionine, phenylalanine, proline, serine, threonine, tyrosine, valine, and taurine. In some embodiments, the composition can be encapsulated in glucose-coated polymeric nanocarriers.
Second Agent
[0187] The compositions herein may be administered alone or in combination with another therapeutic. Hie additional therapeutic may be administered prior, concurrently or subsequently to the administration of the composition.
[0188] The compositions disclosed herein, comprising an RTSM, described herein, can also contain more than one active agent as necessary for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other. For example, the composition can further comprise an anti-inflammatory', a therapeutic protein, a steroid, an analgesic, a non-steroidal anti- inflammatory, a corticosteroid, and combinations thereof. For example, the compositions may be administered in combination with a steroid and/or an antibiotic. Hie steroid may be a glucocorticoid or prednisone. Glucocorticoids such as cortisol control carbohydrate, fat and protein metabolism, and are anti- inflammatory by preventing phospholipid release, decreasing eosinophil action and a number of other mechanisms. Mineralocorticoids such as aldosterone control electrolyte and water levels, mainly by promoting sodium retention in the kidney. Corticosteroids are a class of chemicals that includes steroid hormones naturally produced in the adrenal cortex of vertebrates and analogues of these hormones that are synthesized in laboratories. Corticosteroids are involved in a wide range of physiological processes, including stress response, immune response, and regulation of inflammation, carbohydrate metabolism, protein catabolism, blood electrolyte levels, and behavior. Corticosteroids include Betamethasone, Budesonide, Cortisone, Dexamethasone, Hydrocortisone, Methylprednisolone, Prednisolone, and Prednisone.
[0189] Such molecules are suitably present in combination in amounts that are effective for the purpose intended. Tire active ingredients of the compositions comprising an antibody or antigen binding fragment thereof described herein can also be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsules and poly-(methylmethacylate) microcapsules, respectively, in colloidal drag delivery systems (for example, liposomes, albumin microspheres, microparticle, microemulsions, nano-particles and nanocapsules) or in macroemulsions. Hie pharmaceutical composition can be also delivered in a vesicle, in particular a liposome. Liposomes include emulsions, foams, micelles, insoluble monolayers, phospholipid dispersions, lamellar layers and the like, and can serve as vehicles to target the M-CSF antibodies to a particular tissue as well as to increase the half life of the composition.
[0190] Liposomes containing the RTSM, the second active compound or both can be generated by the reverse phase evaporation method with a lipid composition comprising phosphatidylcholine, cholesterol and PEG-derivatized phosphatidylethanolamine (PEG-PE), Liposomes are extruded through filters of defined pore size to yield liposomes with the desired diameter. RTSMs herein can be conjugated to liposomes via a disulfide interchange reaction. The second agent, can be optionally contained within the liposome.
[0191] In some embodiments, the second agent may be formulated with the compositions described herein or separately co-administered.
Further Embodiments
[0192] In some embodiments, described herein, is a method of decreasing expression of full length PNPLA3 protein comprising contacting a PNPLA3 RNA with a therapeutic agent that binds to a portion of the PNPLA3 RNA, whereby the therapeutic agent causes skipping of an exon in the PNPLA3 RNA that is spliced in the absence of the therapeutic agent.
[0193] In some embodiments, described herein, is a method of treating a hepatic disease comprising administering a therapeutic agent that binds to a portion of a PNPLA3 RNA to a subject, whereby the therapeutic agent causes skipping of an exon in the PNPLA3 RNA that is spliced in the absence of the therapeutic agent.
[0194] In some embodiments, described herein, is a pharmaceutical composition comprising a therapeutic agent and a pharmaceutically acceptable excipient, wherein the therapeutic agent binds to a portion of a PNPLA3 RNA.
[0195] As used herein, the terms "ASO" and "antisense oligomer" are used interchangeably and refer to an oligomer such as a polynucleotide, comprising nucleobases that hybridizes to a target nucleic acid (e.g. , a IFIH1 containing pre-mRNA) sequence by Watson-Crick base pairing or wobble base pairing (G-U). The ASO may have exact sequence complementary to tire target sequence or near complementarity (e.g., sufficient complementarity to bind the target sequence and modulating splicing at a splice site). ASOs are designed so that they bind (hybridize) to a target nucleic acid (e.g., a targeted portion of a pre-mRNA transcript) and remain hybridized under physiological conditions. Typically, if they hybridize to a site other than the intended (targeted) nucleic acid sequence, they hybridize to a limited number of sequences that are not a target nucleic acid (to a few sites other than a target nucleic acid). Design of an ASO can take into consideration the occurrence of the nucleic acid sequence of the targeted portion of the pre-mRNA transcript or a sufficiently similar nucleic acid sequence in other locations in the genome or cellular pre- mRNA or transcriptome, such that the likelihood the ASO will bind other sites and cause "off-target” effects is limited.
[0196] In some embodiments, A SOs "specifically hybridize" to or are "specific" to a target nucleic acid or a targeted portion of a pre-mRNA. Typically such hybridization occurs with a Tin substantially greater than 37°C, preferably at least 50°C, and typically between 60°C to approximately 90°C Such hybridization preferably corresponds to stringent hybridization conditions. At a given ionic strength and pH, the Tin is the temperature at which 50% of a target sequence hybridizes to a complementary oligonucleotide.
[0197] Oligomers, such as oligonucleotides, are "complementary" to one another when hybridization occurs in an antiparallel configuration between two single-stranded polynucleotides. A double-stranded polynucleotide can be "complementary" to another polynucleotide, if hybridization can occur between one of the strands of the first polynucleotide and the second. Complementarity (the degree to which one polynucleotide is complementary with another) is quantifiable in terms of the proportion (e.g., the percentage) of bases in opposing strands that are expected to form hydrogen bonds with each other, according to generally accepted base-pairing rales. Tire sequence of an antisense oligomer (ASO) need not be 100% complementary to that of its target nucleic acid to hybridize. In certain embodiments, ASOs can comprise at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence complementarity to a target region within the target nucleic acid sequence to which they are targeted. For example, an ASO in which 18 of 20 nucleobases of the oligomeric compound are complementary to a target region, and would therefore specifically hybridize, would represent 90 percent complementarity. In this example, the remaining non-complementary nucleobases may be clustered together or interspersed with complementary nucleobases and need not be contiguous to each other or to complementary nucleobases. Percent complementarity-7 of an ASO with a region of a target nucleic acid can be determined routinely using BLAST programs (basic local alignment search tools) and PowerBLAST programs known in the art (Altschul, et al., J. Mol. Biol., 1990, 215, 403- 410; Zhang and Madden, Genome Res., 1997, 7, 649-656).
[0198] An ASO need not hybridize to all nucleobases in a target sequence and the nucleobases to which it does hybridize may be contiguous or noncontiguous. ASOs may hybridize over one or more segments of a pre-mRNA transcript, such that intervening or adjacent segments are not involved in the hybridization event (e.g., a loop structure or hairpin structure may be formed). In certain embodiments, an ASO hybridizes to noncontiguous nucleobases in a target pre-mRNA transcript. For example, an ASO can hybridize to nucleobases in a pre-mRNA transcript that are separated by one or more nncleobase(s) to which the ASO does not hybridize.
[0199] The ASOs described herein comprise nucleobases that are complementary to nucleobases present in a targeted portion of a pre-mRNA. lire term ASO embodies oligonucleotides and any other oligomeric molecule that comprises nucleobases capable of hybridizing to a complementary nucleobase on a target mRNA but does not comprise a sugar moiety, such as a peptide nucleic acid (PNA). The ASOs may comprise naturally-occurring nucleotides, nucleotide analogs, modified nucleotides, or any combination of two or three of the preceding. The term "naturally occurring nucleotides" includes deoxyribonucleotides and ribonucleotides. The term "modified nucleotides" includes nucleotides with modified or substituted sugar groups and/or having a modified backbone. In some embodiments, all of the nucleotides of the ASO are modified nucleotides. Chemical modifications of ASOs or components of ASOs that are compatible with the methods and compositions described herein will be evident to one of skill in the art and can be found, for example, in U.S. Pat. No. 8,258,109, U.S. Pat. No. 5,656,612, U.S. Patent Publication No. 2012/0190728, and Dias and Stein, Mol. Cancer Ther. 2002, 347-355, herein incorporated by reference in their entirety.
[02001 One or more nucleobases of an ASO may be any naturally occurring, unmodified nucleobase such as adenine, guanine, cytosine, thymine and uracil, or any synthetic or modified nucleobase that is sufficiently similar to an unmodified nucleobase such that it is capable of hydrogen bonding with a nucleobase present on a target pre-mRNA. Examples of modified nucleobases include, without limitation, hypoxanthine, xanthine, 7-methylguanine, 5, 6-dihydrouracil, 5-methylcytosine, and 5- hydroxymethoylcytosine.
[0201] Tire ASOs described herein also comprise a backbone structure that connects the components of an oligomer. The term "backbone structure" and "oligomer linkages" may be used interchangeably and refer to the connection between monomers of the ASO. In naturally occurring oligonucleotides, the backbone comprises a 3 ’-5 ’ phosphodiester linkage connecting sugar moieties of the oligomer. The backbone structure or oligomer linkages of the ASOs described herein may include (but are not limited to) phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate, phosphoramladate, phosphoramidate, and the like. See, e.g., LaPlanche, et al., Nucleic Acids Res. 14:9081 (1986); Stec, et al., J. Am. Chem. Soc. 106:6077 (1984), Stem, et al.. Nucleic Acids Res. 16:3209 (1988), Zon, et al., Anti-Cancer Drug Design 6:539 (1991); Zon, et al., Oligonucleotides and Analogues: A Practical Approach, pp. 87-108 (F. Eckstein, Ed., Oxford University Press, Oxford England (1991)); Stec, et al., U.S. Pat. No. 5,151,510; Uhlmann and Peyman, Chemical Review's 90:543 (1990). In some embodiments, the backbone structure of the ASO does not contain phosphorous but rather contains peptide bonds, for example in a peptide nucleic acid (PN A), or finking groups including carbamate, amides, and linear and cyclic hydrocarbon groups. In some embodiments, the backbone modification is a phosphothioate linkage. In some embodiments, the backbone modification is a phosphoramidate linkage. [02021 Any of the ASOs described herein may contain a sugar moiety that comprises ribose or deoxyribose, as present in naturally occurring nucleotides, or a modified sugar moiety or sugar analog, including a morpholine ring. Non-limiting examples of modified sugar moieties include 2' substitutions such as 2'-O-methyl (2'-O-Me), 2‘-O-methoxyethyl (2'MOE), 2'-O-aminoethyl, 2'F; N3'->P5' phosphoramidate, 2’dimethylaminooxyethoxy, 2’dimethylaminoethoxyethoxy, 2'-guanidinidium, 2 -0- guanidinium ethyl, carbamate modified sugars, and bicyclic modified sugars. In some embodiments, the sugar moiety modification is selected from 2'-0-Me, 2'F, and 2'MOE. In some embodiments, the sugar moiety modification is an extra bridge bond, such as in a locked nucleic acid (LNA). In some embodiments the sugar analog contains a morpholine ring, such as phosphorodiamidate morpholino (PMO). In some embodiments, the sugar moiety comprises a ribofuransyl or 2‘deoxyribofuransyl modification. In some embodiments, the sugar moiety’ comprises 2'4 '-constrained 2'O-methyloxyethyl (cMOE) modifications. In some embodiments, the sugar moiety comprises cEt 2’, 4' constrained 2'-0 ethyl BNA modifications. In some embodiments, the sugar moiety comprises tricycloDNA (tcDNA) modifications. In some embodiments, the sugar moiety comprises ethylene nucleic acid (ENA) modifications. In some embodiments, the sugar moiety comprises MCE modifications. Modifications are known in the art and described in the literature, e.g, by Jarver, et al, 2014, "A Chemical View of Oligonucleotides for Exon Skipping and Related Drug Applications," Nucleic Acid Therapeutics 24(1): 37-47, incorporated by reference for this purpose herein.
[0203] In some embodiments, each monomer of the ASO is modified in the same way, for example each linkage of the backbone of the ASO comprises a phosphorothioate linkage or each ribose sugar moiety comprises a 2'0-methyI modification. Such modifications that are present on each of the monomer components of an ASO are referred to as "uniform modifications." In some examples, a combination of different modifications may be desired, for example, an ASO may comprise a combination of phosphorodiamidate linkages and sugar moieties comprising morpholine rings (morpholinos). Combinations of different modifications to an ASO are referred to as "mixed modifications” or "mixed chemistries."
[02041 In some embodiments, the ASO comprises one or more backbone modifications. In some embodiments, the ASO comprises one or more sugar moiety modification. In some embodiments, the ASO comprises one or more backbone modifications and one or more sugar moiety modifications. In some embodiments, the ASO comprises a 2'MOE modification and a phosphorothioate backbone. In some embodiments, the ASO comprises a phosphorodiamidate morpholino (PMO). In some embodiments, the ASO comprises a peptide nucleic acid (PNA). Any of the ASOs or any component of an ASO (e.g, a nucleobase, sugar moiety, backbone) described herein may be modified in order to achieve desired properties or activities of the ASO or reduce undesired properties or activities of the ASO. For example, an ASO or one or more components of any ASO may be modified to enhance binding affinity’ to a target sequence on a pre-mRNA transcript; reduce binding to any non-target sequence; reduce degradation by cellular nucleases (i.e, RNase H); improve uptake of the ASO into a cell and/or into the nucleus of a cell; alter the pharmacokinetics or pharmacodynamics of the ASO; and/or modulate the half-life of the ASO.
[0205] In some embodiments, the ASOs are comprised of 2'-O-(2-methoxyethyl) (MOE) phosphorothioate-modified nucleotides. ASOs comprised of such nucleotides are especially well-suited to the methods disclosed herein; oligomers having such modifications have been shown to have significantly enhanced resistance to nuclease degradation and increased bioavailability, making them suitable, for example, for oral delivery in some embodiments described herein. See e.g, Geary, et al., J Pharmacol Exp Ther. 2001 ; 296(3):890-7; Geary, et al, J Pharmacol Exp Ther. 2001; 296(3):898-904.
[0206] Methods of synthesizing ASOs will be known to one of skill in the art. Alternatively or in addition,
ASOs may be obtained from a commercial source. [0207] Described, herein are compositions and methods useful for treating hepatic diseases. In some embodiments, these compositions and methods result in a truncated PNPLA3 protein. In some embodiments, these compositions and methods result in a decrease in the wild-type PNPLA3 protein. In some embodiments, the compositions and methods result in modulating the splicing of PNPLA3 RNA, In some embodiments, the compositions and methods result in an PNPLA3 RN A lacking exon 5.
[02081 In some embodiments, disclosed herein is a method of decreasing expression of foil length PNPLA3 protein comprising contacting a PNPLA3 RNA with a therapeutic agent that binds to a portion of the PNPLA3 RNA, whereby the therapeutic agent causes skipping of an exon in the PNPLA3 RNA that is spliced in the absence of the therapeutic agent.
[0209] In some embodiments, the therapeutic agent causes skipping of exon 5 in the PNPLA3 RNA. In some embodiments, the therapeutic agent binds to a 5" splice site sequence in the PNPLA3 RNA. In some embodiments, the 5’ splice site sequence is in Intron 5 of the PNPLA3 RNA. In some embodiments, the 5’ splice site comprises a sequence with at least 95% sequence identity to SEQ ID No: 43. In some embodiments, the therapeutic agent is an antisense oligonucleotide (ASO). In some embodiments, the ASO comprises a sequence that is at least about 80% identity7 to SEQ ID NO: 83, In some embodiments, the ASO comprises a sequence that is at least about 90% identity to SEQ ID NO: 83.
[0210] In some embodiments, disclosed herein is a method of treating a hepatic disease comprising administering a therapeutic agent that binds to a portion of a PNPLA3 RNA to a subject, whereby the therapeutic agent causes skipping of an exon in the PNPLA3 RNA that is spliced in the absence of the therapeutic agent. In some embodiments, the hepatic disease is nonalcoholic steatohepatitis (NASH).
[0211] In some embodiments, disclosed herein is a pharmaceutical composition comprising a therapeutic agent and a pharmaceutically acceptable excipient, wherein the therapeutic agent binds to a portion of a
PNPLA3 RNA,
[0212] In some embodiments, the therapeutic agent is an antisense oligomer (ASO) and wherein the ASO comprises a sequence that is at least about 80%, 85%, 90%, 95%, 97%, or 100% identity to any one of SEQ ID NO: 83.
[0213] In some embodiments, the ASO comprises a backbone modification comprising a phosphorothioate linkage or a phosphorodiamidate linkage. In some embodiments, the ASO comprises a phosphorodiamidate morpholino, a locked nucleic acid, a peptide nucleic acid, a 2'-O-methyl, a 2'-Fluoro, or a 2'-O- niethoxyethyl moiety. In some embodiments, the ASO comprises at least one modified sugar moiety. In some embodiments, each sugar moiety is a modified sugar moiety. In some embodiments, the ASO consists of from 8 to 50 nucleobases, 8 to 40 nucleobases, 8 to 35 nucleobases, 8 to 30 nucleobases, 8 to 25 nucleobases, 8 to 20 nucleobases, 8 to 15 nucleobases, 9 to 50 nucleobases, 9 to 40 nucleobases, 9 to 35 nucleobases, 9 to 30 nucleobases, 9 to 25 nucleobases, 9 to 20 nucleobases, 9 to 15 nucleobases, 10 to 50 nucleobases, 10 to 40 nucleobases, 10 to 35 nucleobases, 10 to 30 nucleobases, 10 to 25 nucleobases, 10 to 20 nucleobases, 10 to 15 nucleobases, 1 1 to 50 nucleobases, 11 to 40 nucleobases, 11 to 35 nucleobases, 11 to 30 nucleobases, 11 to 25 nucleobases, 11 to 20 nucleobases, 11 to 15 nucleobases, 12. to 50 nucleobases, 12 to 40 nucleobases, 12 to 35 nucleobases, 12 to 30 nucleobases, 12 to 25 nucleobases, 12 to 20 nucleobases, or 12 to 15 nucleobases. In some embodiments, the ASO is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, complementary7 to the targeted portion of the PNPLA3 mRNA encoding the PNPLA3 protein. In some embodiments, the method further comprises assessing PNPLA3 mRNA or PNPLA3 protein expression. In some embodiments, the cells are ex vivo.
[0214] In some embodiments, the therapeutic agent is administered to the subject by mtravitreal injection, intrathecal injection, intraperitoneal injection, subcutaneous injection, intravenous injection, subretinal injection, intracerebro ventricular injection, intramuscular injection, topical application, or implantation. [0215] In some embodiments, the therapeutic agent is administered with one or more agents capable of promoting penetration of the subject antisense oligonucleotide across the blood-brain barrier by any method known in the art. In some embodiments, the therapeutic agent is linked with a viral vector, e.g., to render the therapeutic agent more effective or increase transport across the blood-brain barrier. For example, delivery' of agents by administration of an adenovirus vector to motor neurons in muscle tissue is described in U.S. Pat. No. 6,632,427, "Adenoviral-vector-mediated gene transfer into medullary motor neurons," incorporated herein by reference. Delivery' of vectors directly to the brain, e.g., the striatum, the thalamus, the hippocampus, orthe substantia nigra, is described, e.g., in U.S. Pat. No. 6,756,523, "Adenovirus vectors for the transfer of foreign genes into cells of the central nervous system particularly’ in brain," incorporated herein by reference.
[0216] In embodiments, the therapeutic agent is linked or conjugated with agents that provide desirable pharmaceutical or pharmacodynamic properties. In some embodiments, the therapeutic agent is coupled to a substance, known in the art to promote penetration or transport across the blood-brain barrier, e.g., an antibody to the transferrin receptor. In some embodiments, osmotic blood brain barrier disruption is assisted by infusion of sugars, e.g., meso erythritol, xylitol, D(+) galactose, D(+) lactose, D(+) xylose, dulcitol, myo-inositol, L(-) fructose, D(-) mannitol, D(+) glucose, D(+) arabinose, D(-) arabinose, cellobiose, D(+) maltose, D(-t-) raffinose, L(-i-) rhamnose, D(t-) melibiose, D(-) ribose, adonitol, D(+) arabitol, L(-) arabitol, D(t-) fucose, L(-) fucose, D(-) lyxose, L(t-) lyxose, and L(-) lyxose, or amino acids, e.g., glutamine, lysine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glycine, histidine, leucine, methionine, phenylalanine, proline, serine, threonine, tyrosine, valine, and taurine. Methods and materials for enhancing blood brain barrier penetration are described, e.g., in U.S. Pat. No. 9,193,969, "Compositions and methods for selective delivery’ of oligonucleotide molecules to specific neuron types," U.S. Pat. No. 4,866,042, "Method for the delivery’ of genetic material across the blood brain barrier," U.S. Pat. No. 6,294,520, "Material for passage through the blood-brain barrier," and U.S. Pat. No. 6,936,589, "Parenteral delivery systems," each incorporated herein by reference.
[0217] In some embodiments, the therapeutic agent is encapsulated in glucose-coated polymeric nanocarriers, such as those described in Min et al. "‘Systemic Brain Delivery' of Antisense Oligonucleotides across the Blood-Brain Barner with a Glucose-Coated Polymeric Nanocarrier,” Angew. Chem. Int. Ed. 2020, 59, 8173-8180, incorporated herein by reference.
[0218] In some embodiments, disclosed herein is a method of decreasing expression of full length PNPLA3 protein comprising contacting a PNPLA3 RNA with a therapeutic agent that binds to a portion of the PNPLA3 RNA, whereby the therapeutic agent causes skipping of an exon in the PNPLA3 RNA that is spliced in the absence of the therapeutic agent.
[0219] In some embodiments, wherein the therapeutic agent causes skipping of exon 5 in the PNPLA3 RNA.
[0220] In some embodiments, wherein the therapeutic agent binds to a 5’ splice site sequence in the PNPLA3 RNA.
[0221] In some embodiments, wherein the 5’ splice site sequence is in Intron 5 of the PNPLA3 RNA.
[0222] In some embodiments, wherein the 5’ splice site comprises a sequence with at least 95% sequence identity to SEQ ID No: 43.
[0223] In some embodiments, wherein the therapeutic agent is an antisense oligonucleotide (ASO).
[0224] In some embodiments, wherein the ASO comprises a sequence that is at least about 80% identity to SEQ ID NO: 83.
[0225] In some embodiments, wherein the ASO comprises a sequence that is at least about 90% identity to SEQ ID NO: 83. [0226] In some embodiments, disclosed herein is a method of treating a hepatic disease comprising administering a therapeutic agent that binds to a portion of a PNPLA3 RNA to a subject, whereby the therapeutic agent causes skipping of an exon in tire PNPLA3 RNA that is spliced in the absence of the therapeutic agent,
[0227] In some embodiments, wherein the therapeutic agent causes skipping of exon 5 in the PNPLA3 RNA.
[0228] In some embodiments, wherein the therapeutic agent binds to a 5’ splice site sequence in the PNPLA3 RNA.
[0229] In some embodiments, wherein the 5’ splice site sequence is in Intron 5 of the PNPLA3 RNA.
[0230] In some embodiments, wherein the 5’ splice site comprises a sequence with at least 95% sequence identity to SEQ ID No: 43.
[0231] In some embodiments, wherein the therapeutic agent is an antisense oligonucleotide (ASO).
[0232] In some embodiments, wherein the ASO comprises a sequence that is at least about 80% identity to SEQ ID NO: 83.
[0233] In some embodiments, wherein the ASO comprises a sequence that is at least about 90% identity to SEQ ID NO: 83.
[0234] In some embodiments, wherein the hepatic diseases is NASH.
[0235] In some embodiments, disclosed herein is a pharmaceutical composition comprising a therapeutic agent and a pharmaceutically acceptable excipient, wherein the therapeutic agent binds to a portion of a PNPLA3 RNA.
[0236] In some embodiments, wherein the therapeutic agent causes skipping of an exon in the PNPLA3 RN A that is spliced in the absence of the therapeutic agent.
[0237] In some embodiments, wherein the therapeutic agent causes skipping of exon 5 in the PNPLA3 RNA.
[0238] In some embodiments, wherein the therapeutic agent binds to a 5’ splice site sequence in the PNPLA3 RNA.
[0239] In some embodiments, wherein the therapeutic agent binds to the 5 ’ splice site sequence is in Intron 5 of the PNPLA3 RNA
[0240] In some embodiments, wherein the 5’ splice site comprises a sequence with at least 95% sequence identity to SEQ ID No: 43.
[0241] In some embodiments, wherein the therapeutic agent, is an antisense oligonucleotide (ASO).
[0242] In some embodiments, wherein the ASO comprises a sequence that is at least about 80% identity to SEQ ID NO: 83.
[0243] In some embodiments, wherein the ASO comprises a sequence that is at least about 90% identity to SEQ ID NO: 83.
[0244] In some embodiments, nucleobases corresponding to the abbreviations in various nucleobase sequences disclosed herein can be found in Table 9A below. Table 9A: Nudeobase Abbreviations
Figure imgf000060_0001
[0245] In some embodiments, amino acids corresponding to the abbreviations in various polypeptide sequences disclosed herein can be found, for example, in Table 9B below.
Table 9B: Amino Acid Abbreviations
Figure imgf000060_0002
Figure imgf000061_0001
EXAMPI.ES
J 0246] These examples are provided for illustrative purposes only and not to limit the scope of the claims provided herein. ASOs described herein can be synthesized using synthetic techniques, using methods described herein or combinations of both. Alternatively, ASOs are available commercially from various sources, including Integrated DNA Technologies (IDT), Coralville, Iowa and Gene Tools, LLC.
Example 1: PNPLA3 splicing modulation in hepatocellular carcinoma cells
[0247] SNL-886 is an adult hepatocellular carcinoma cell line, and it contains wild type PNPLA3. SN U- 886 cells (KCLB 00886) were plated at a concentration of 125,000 cells/well in a 12-well plate and were cultured using RPMI 1640 (Gibco, Cat # A10491-01) supplemented with 1% Penicillin-Streptomycin (Gibco, #15140163), and 10% Fetal Bovine Seram (Gibco, Cat# A38401-01) for 24 hours.
[0248 [ Hep G2 is an adolescent hepatocellular carcinoma cell line, and it carries the rs738409 variant in PNPLA3. Hep G2 cells (ATCC® HB8065) were plated at a concentration of 125,000 cells/well in a 12- well plate and were cultured using EMEM with L-Glutamine (ATCC, Cat #30-2003), supplemented with 1% MEM Non-Essential Amino Acids (Gibco, #11140076) to 1%, Penicillin-Streptomycin (Gibco, #15140163), and 10% Fetal Bovine Seram (Gibco, Cat# A38401-01) for 24 hours.
[0249] ASOs with morpholino-modified backbones were transfected individually into each well using Endo-Porter (Gene-Tools, LLC at 6ul/ml). Each morpholino was at a 500uM stock concentration and was added to the wells to achieve final concentrations of 5uM (10 pl/well) and 10 pM (20 pL/well) for control and PNPLA3 morpholinos. NTC is a non-targeting control morpholino and PNPLA3 E5-I5 morphRK (labeled as 5’SS in FIGS. 1 and 2) is a morpholino that targets PNPLA3 at the Exon5-Intron5 boundary'. Tire sequences of the ASOs used are set forth below in Table 10.
Table 10. ASO sequences
Figure imgf000062_0001
[0250] Samples were harvested after 48hrs. Six hours before harvest, samples were treated with either 100 pg/mL. cycloheximide (Sigma C4859) or 0.1%DMSO (Tocris Bioscience 3176/25mL). RNA was prepared from each sample, using the RNeasy plus mini kits (Qiagen # 74134). RNA was quantitated on a ThermoScientific Nanodrop™ 8000 Spectrophotometer, and cDNA was synthesized with lug total RNA using SuperScript™ III First-Strand Synthesis SuperMix (Invitrogen 11752-050). RT-PCR reactions were performed using Invitrogen Platinum™ SuperFi™ DNA Polymerase (Invitrogen 12351050), with the following primers:
Table 11. Primers
Figure imgf000062_0002
[0251] Afterthe initial denaturation step (98°C, 30s), 32 cycles were performed (98°C 10s, 63°C 10s, 72°C 30s), followed by a final extension step (72°C 5mm). Ihe PCR products were run on a 2% agarose gel (FIGS. 1 and 2).
Example 2: In Vivo Activity of an PNPLA3-RTSM on PNPLA3 pre-mRNA and PNPLA3 protein expression
[02521 Female C57BL/6 mice of 5-6 weeks age (N=3/group) can be injected with exemplary murine PNPLA3-RTSMs herein at 5 mg/kg, or PBS, subcutaneously once a day for three days. A PNPLA3-RTSM control according to Example 1 can be administered to the control group using the same regime. After 3 days, blood can be collected and PNPLA3 mRNA and PNPLA3 protein can be evaluated. RT-PCR evaluation of the PNPLA3-RTSM treated mRNA can depict that the mRNA product contains exons 4 and 6, exon 5 having been skipped. BCA assays and SDS-PAGE gel probed with anti-PNPLA3 antibodies can demonstrate that the PNPLA3-RTSMs of the present disclosure decreased the level of functional PNPLA3 protein compared to the control .
Example 3: Splicing modification of the PNPLA3 pre-mRNA will prevent the progression of or treat NASH
[0253] Mice predisposed to NASH (C57BL/6NTac mice) conditioned on a modified Amylin liver NASH (AMLN) diet can be obtained. Diet can be # D091003 lOi (source Research Diets) which contains 40 kcal% fat, 20 kcal% fructose and 2% cholesterol and is an irradiated diet. C57BL/6NTac males are put on this diet at 6 weeks of age and housed at reduced density. Control males are housed in the same location, also at reduced density, and are fed NIH-31M diet. Control males can also be generated using a low-fat purified diet. Mice can be split into two treatment groups, treatment with or without the RTSM herein.
[0254] NASH B6 mice become obese, get fatty liver and develop liver inflammation and fibrosis after 26+ weeks on diet, with inter-animal variability observed for development of the inflammation and fibrosis phenotype. Observations can include: significantly elevated ALT levels compared to controls, robust steatosis, hepatic inflammation including the presence of hepatic crown-like structures, activated stellate cells, and development of consistent fibrosis by 26 w eeks on diet.
[0255] RTSM treatment groups can be injected with exemplary murine PNPLA3-RTSMs herein at 5 mg/kg, or PBS, subcutaneously, once a day for up to 90 days.
[0256] The data can depict that RTSM treated NASH mice exhibit decreased ALT levels, steatosis, hepatic inflammation, and fibrosis compared to the untreated controls.

Claims

CLAIMS What is claimed is:
1. A method of treating a disease or condition in a human subject in need thereof, the method comprising: a) administering to the subject a therapeutically effective amount of a synthetic Patat in-like phospholipase domain-containing 3 protein (PNPLA3) RNA-targeting splicing modifier (RTSM), thereby treating the disease or condition in the human subject in need thereof; wherein: the synthetic RTSM comprises a binding domain that binds to a target region of the PNPLA3 pre-messenger ribonucleic acid (pre-m RNA); the target region comprises an exon-intron junction comprising a target sequence of Formula (I):
KGUR, wherein K is G or U; wherein R is A or G; and exon skipping is increased as compared to the PNPLA3 pre-mRNA spliced m the absence of RTSM as demonstrated by an m vitro assay.
2. The method of claim 1, wherein increasing exon skipping comprises modulation of expression PNPLA3 mRNA, PNPLA3 protein or both comprising: a decrease in full-length PNPLA3 mRNA production, an increase m truncated PNPLA3 mRNA production, an increase in PNPLA3 mRNA production wherein the transcript lacks coding for one or more exons, decrease in active PNPLA3 expression, a decrease in full-length or elongated PNPLA3 protein production, increase in truncated PNPLA3 production, or any combination thereof.
3. The method of claim 1, wherein the exon-intron junction comprises a sequence of Formula (I) wherein K is G and R is G; K is G and R is A; or K is U and R is A.
4. lire method of claim 1, wherein Formula (I) further comprises: 1 or 2 D(s) wherein each D is independently A, G or U; optionally 1, 2, or 3 V(s) wherein each V is independently A, G, or C; optionally 1, 2, 3, 4, 5, 6, or 7 B(s) wherein each B is independently G, C or U; optionally 1 , 2, 3, or 4 N(s) wherein each N is independently A, G, C, or U; optionally Y wherein ¥ is C, or U; optionally 1 or 2 H(s) wherein each H is independently A, C, or U; optionally K wherein each K is independently G, or L! ; or any combination thereof.
5. Tire method of claim 1, wherein Formula (I) further comprises H that is 3' and adjacent to R; HV that is 3‘ and adjacent to R; HVY that is 3' and adjacent to R; HVYH that is 3' and adjacent to R; HVYHB that is 3' and adjacent to R; HVYHBK that is 3’ and adjacent to R; HVYHBKB that is 3' and adjacent to R; HVYHBKBB that is 3' and adjacent to R; HVYHBKBBN that is 3' and adjacent to R;
HVYHB KBBNN that is 3' and adjacent to R; HVYHBKBBNNB that is 3' and adjacent to R; HVYHBKBBNNBD that is 3' and adjacent to R; HVYHBKBBNNBDV that is 3' and adjacent to R; HVYHB KBBNNBDVD that is 3' and adjacent to R; HVYHB KB BNNBDVDB that is 3' and adjacent to R; HVYHBKBBNNBDVDBB that is 3‘ and adjacent to R; or HVYHBKBBNNBDVDBBN that is 3' and adjacent to R; or any combination thereof, wherein each D is independently A, G or U, wherein each V is independently A, G, or C, wherein each B is independently G, C or U, w herein each N is independently A, G, C, or U, wherein Y is C, or U, wherein each H is independently A, C, or U, and wherein each K is independently G, or U.
6. The method of claim 5, w herein Formula (I) comprises the VND and the HVYHBKB or the BVND and the HVYHBKBBNNBDVDBBN wherein each V is independently A, G, or C, wherein each B is independently G, C or U, wherein each D is independently A, G or U, wherein each N is independently A, G, C, or U, wherein ¥ is C, or U, wherein each H is independently A, C, or U, and wherein each K is independently G, or U. . The method of claim 1, w herein the target sequence comprises a sequence about 2 to about 50 nucleobases in length with at least about: 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence set forth in Formula (I) or to any one of the sequences of SEQ. ID NO: 31-46. . Tire method of claim 1, wherein the synthetic RTSM binding domain comprises a sequence of Formula (III):
YACM, wherein Y is C, T, or U; wherein M is A or C. . The method of claim 8, wherein the binding domain comprises a sequence of Formula (III) wherein ¥ is C and M is C; Y is T and M is A; Y is U and M is A; Y is Y and M is C; or Y is U and M is C.0. lire method of claim 8, wherein Formula (III) further comprises: 1 or 2 D(s) wherein each D is independently G, A, T or U; optionally: M wherein M is A or C; optionally R wherein R is G or A: optionally 1, 2, or 3 H(s) wherein each H is individually A, C, T, or U; optionally 1 , 2, or 3 B(s) wherein each B is independently G, C, T or U; optionally 1, 2, 3, or 4 N(s) wherein each N is independently A, G, C, T, or U; optionally 1 , 2, 3, 4, 5, 6, or 7 V(s) wherein each V is independently A, G, or C; or any combination thereof. 1 . Tire method of claim 8, wherein Formula (III) further comprises D that is 5' and adjacent to Y; BD that is 5‘ and adjacent to Y; RBD that is 5* and adjacent to Y; DRBD that is 5' and adjacent to Y; VDRBD that is 5’ and adjacent to Y; MVDRBD that is 5' and adjacent to Y; VMVDRBD that is 5’ and adjacent to Y ; VVMVDRBD that is 5' and adjacent to Y; NWMVDRBD that is 5' and adjacent to Y; NNVVMVDRBD that is 5' and adjacent to Y: VNNWMVDRBD that is 5' and adjacent to Y; HVNNVVMVDRBD that is 5' and adjacent to Y ; BHVNNVVMVDRBD that is 5' and adjacent to Y ; HBHVNNVA^MVDRBD that is 5' and adjacent to Y; VIIBHVNNVVMVDRBD that is 5' and adjacent to Y; VVHBHVNNVVM VDRBD that is 5' and adjacent to Y ;
NV VHBHVNNV VMVDRBD that is 5' and adjacent to Y: H that is 3' and adjacent to M; HN that is 3' and adjacent to M; HNB that is 3' and adjacent to M; HNBV that is 3! and adjacent to M; or any combination thereof, or any combination thereof, wherein M is A or C, wherein each R is independently G or A, wherein each D is independently A, G, T or U, wherein each H is A, C, T or U, wherein each B is independently G, C, T or U; wherein each N is independently A, G, C, T, or U, and each V is independently A, G, or C: or any combination thereof. . The method of claim 1 1 , wherein Formula (III) comprises the VMVDRBD and the HNB or the
VVHBHVNNVVMVDRBD and the HNBV, wherein M is A or C, wherein each R is independently G or A, wherein each D is independently A, G, T or U, wherein each H is A, C, T or U, wherein each B is independently G, C, T or U; wherein each N is independently A, G, C, T, or U, and each V is independently A, G, or C; or any combination thereof. 3. The method of claim 8, wherein the binding domain comprises a binding sequence comprising a sequence about 2 to about 50 nucleobases in length that has at least about: 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence complementarity to the sequence set forth in Formula (I) orto any one of the sequences of SEQ ID NOS: 31-46. . Hie method of claim 8, wherein the binding domain comprises a binding sequence comprising a sequence about 2 to about 50 nucleobases in length that has at least about: 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence set forth in Formula (III) or to any one of the sequences of SEQ ID NOS: 63-94. 5. The method of claim 1, further comprising testing the subject prior to administration of the synthetic RTSM to determine whether the subject is suitable for the treatment. The method of claim 1 , wherein the disease or condition is a PNPL A3 -associated disorder. The method of claim 1, wherein the disease or condition is liver disease. The method of claim 17. wherein liver disease is characterized by hepatic fat levels and with hepatic inflammation. The method of claim 1, wherein the disease or condition is a liver disease; optionally selected from the group consisting of Nonalcoholic fatty’ liver disease (NAFLD), hepatic steatosis, non-alcoholic steatohepatitis (NASH), liver cirrhosis, hepatocellular carcinoma, alcoholic liver disease, alcoholic steatohepatitis (ASH), HCV hepatitis, chronic hepatitis, hereditary hemochromatosis, or primary sclerosing cholangitis. The method of claim 19, wherein the disease is associated with PNPLA3 rs738409 encoding I148M mutant. The method of claim 1, wherein the disease or condition is characterized by liver damage, steatosis, liver fibrosis, liver inflammation, liver scarring or cirrhosis, liver failure, liver enlargement, elevated transaminases, or hepatic fat accumulation. The method of claim 21, wherein the PNPLA3 associated disease comprises NASH or NAFLD. The method of claim 1, wherein a therapeutically effective amount is about 0.001 mg, 'leg to about 1000 mg/kg, wherein mg is mg of the RTSM and kg is the body weight of the subject. The method of claim 1, wherein the in vitro assay comprises a reverse transcription polymerase chain reaction (RT-PCR), Western blot analysis, bicinchoninic acid (BCA) assay or immunohistochemical detection . The method of claim 1, wherein the RTSM is administered as a pharmaceutical composition which also comprises a pharmaceutically acceptable: excipient, diluent, or carrier. Tire method of claim 25, wherein the pharmaceutical composition is in unit dose form. The method of claim 1, wherein the RTSM is a polynucleotide comprising from about 4 to about 30 nucleotides. The method of claim 1, wherein the RTSM is administered once every- 7 to 10 days for at least about 90 days. The method of claim 1, further comprising administering a second therapy wherein the second therapy is administered consecutively or concurrently to administration of the RTSM. The method of claim 1, wherein the exon-intron junction is located at the 5’ splice site of the target intron and wherein the target exon is upstream of the exon-intron junction. The method of claim 30, wherein the exon-intron junction is selected from the group consisting of Exon2-Intron2, Exon3-Intron3, Exon4-Intron4, Exon5 -IntronS, Exon6-Intron6, Exon7-Intron7, or Exon8-Intron8. The method of claim 31, wherein the targeted exon is exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, or exon 8. The method of claim 1, wherein the binding sequence binds at a splice site sequence, wherein the splice site sequence comprises a sequence about 4 to about 25 nucleobases in length and is located at the 5’ splice site of the target intron. The method of claim 33, wherein the splice site comprises a splice site sequence about 2 to about 50 nucleobases long with at least about: 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any one of the sequences in SEQ, ID NOS: 47-62. The method of claim 1, wherein the synthetic RTSM is selected from the group consisting of an antibody or a fragment thereof, an aptamer, an antisense oligomer (ASO), or CRISPR associated proteins. The method of claim 35, wherein the synthetic RTSM is a synthetic ASO comprising one or more nucleobases, one or more sugar moieties, a backbone, modifications thereof, more than one of the foregoing, and combinations thereof. The method of claim 36, wherein the synthetic RTSM-ASO comprises backbone modifications comprising a phosphorothioate linkage; a sugar moiety comprising a 2'0-methyl modification; more than one of each; and in combination thereof. The method of claim 37, wherein the synthetic ASO comprises one or more 2'-O-(2-methoxyethyl) (MOE) phosphorothioate-modified nucleotides. The method of claim 1, wherein expression of exon 5 by an PNPLA3 mRNA or by an PNPLA3 protein is skipped in the presence of the RTSM as confirmed by an in vitro assay. A synthetic PNPLA3 RTSM that comprises a) a binding domain that binds to a target region of a PNPLA3 pre-mRNA; wherein: the target region comprises an exon-intron junction comprising a target sequence of Formula (I):
KGUR, wherein K is G or U; wherein R is A or G; and exon skipping is increased as compared to when the PNPLA3 pre-mRNA is spliced in the absence of the synthetic RTSM as demonstrated in an in vitro assay. The synthetic RTSM of claim 40, wherein the exon-intron junction compri ses a sequence of Formula (1) wherein K is G and R is G; K is G and R is A; K is T and R is A; or K is U and R is A. The synthetic RTSM of claim 40, wherein Formula (I) further comprises: 1 or 2 D(s) wherein each D is independently A, G or U; optionally 1, 2, or 3 V(s) wherein each V is independently A, G, or C; optionally 1, 2, 3, 4, 5, 6, or 7 B(s) wherein each B is independently G, C or U; optionally 1, 2, 3, or 4 N(s) wherein each N is independently A, G, C, T, or U; optionally Y wherein Y is C, T, or U; optionally 1 or 2 H(s) wherein each H is independently A, C, T, or U; optionally K wherein each K is independently G, T, or U; or any combination thereof. The synthetic RTSM of claim 40, wherein Formula (I) further comprises H that is 3! and adjacent to R; HV that is 3' and adjacent to R; HVY that is 3' and adjacent to R; HVYH that is 3' and adjacent to R; HVYIIB that is 3' and adjacent to R; HVYHBK that is 3' and adjacent to R; HVYHBKB that is 3' and adjacent to R; HVYHBKBB that is 3‘ and adjacent to R; HVYHB KBBN that is 3' and adjacent to R; HVYHBKBBNN that is 3' and adjacent to R; H VYHBK BBNN B that is 3! and adjacent to R; HVYHB KBBNNBD that is 3' and adjacent to R; HVYHB KBBNNBDV that is 3' and adjacent to R; HVYHBKBBNNBDVD that is 3' and adjacent to R; HVYHBKBBNNBDVDB that is 3' and adjacent to R; HVYHBKBBNNBDVDBB that is 3' and adjacent to R; or HVYHB KBBNNBDVDBBN that is 3' and adjacent to R; or any combination thereof herein each D is independently A, G or U, wherein each V is independently A, G, or C, wherein each B is independently G, C or U, wherein each N is independently A, G, C, T, or U, wherein Y is C, T, or U, wherein each H is independently A, C, T, or U, and wherein each K is independently G, T, or U. The synthetic RTSM of claim 43, wherein Formula (I) comprises the VND and the HVYHBKB or the BVND and the HVYHBKBBNNBDVDBBN herein each D is independently A, G or U, wherein each V is independently A, G, or C, wherein each B is independently G, C or U, wherein each N is independently A, G, C, T, or U, wherein Y is C, T, or U, wherein each H is independently A, C, T, or U, and wherein each K is independently G, T, or L! . The synthetic RTSM of claim 40, wherein the exon-intron junction is located at the 5’ splice site of the target intron and wherein the target exon is upstream of the exon-intron junction. The synthetic RTSM of claim 45, wherein the exon -intron junction is selected from the group consisting of Exon2.-Intron2, Exon 3 -Intron 3, Exon4-Intron4, Exon5-lntron5, Exon6-Intron6, Exon7- Intron7, or Exon8-lntron8 Tire synthetic RTSM of claim 46, wherein the targeted exon is exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9 or any combination thereof. The synthetic RTSM of claim 45, wherein the target sequence comprises a sequence about 2 to about 50 nucleobases long with at least about: 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence set forth in Formula (I) or to any one of the sequences of SEQ ID NOS: 31-46. ’ Tire synthetic RTSM of claim 40, wherein the RTSM binding domain comprises a sequence of Formula (III):
YACM, wherein Y is C, T, or U; wherein M is A or C. The synthetic RTSM of claim 40, wherein the RTSM binding domain comprises a sequence of Formula (III) wherein Y is C and M is C; Y is T and M is A; Y is LI and M is A; Y is Y and M is C; or Y is U and M is C. The synthetic RTSM of claim 40, wherein Formula (III) further comprises: M wherein M is A or C; optionally R wherein R is A, G, T or U: optionally 1 or 2 D(s) wherein each D is independently G or A; optionally 1, 2, or 3 H(s): optionally 1, 2, or 3 B(s) wherein each B is independently G, C, T or U; optionally 1, 2, 3, or 4 N(s) wherein each N is independently A, G, C, T, or U; optionally 1, 2, 3, 4, 5, 6, or 7 V(s) wherein each V is independently A, G, or C; or any combination thereof. The synthetic RTSM of claim 40, wherein Formula (III) further comprises: D that is 5' and adjacent to Y ; BD that is 5' and adjacent to Y ; RBD that is 5' and adjacent to Y ; DRBD that is 5' and adjacent to Y; VDRBD that is 5' and adjacent to Y ; MVDRBD that is 5' and adjacent to Y ; VMVDRBD that is 5' and adjacent to Y; WMVDRBD that is 5' and adjacent to Y; NWMVDRBD that is 5' and adjacent to Y; NN WMVDRBD that is 5' and adjacent to Y; VNN WMVDRBD that is 5' and adjacent to Y; HVNNVVMVDRBD that is 5' and adjacent to Y; BHVNNVVMVDRBD that is 5' and adjacent to Y; HBHVNN WMVDRBD that is 5' and adjacent to Y; VHBHVNNVVMVDRBD that is 5' and adjacent to Y ; VVHBHVNNVVMVDRBD that is 5' and adjacent to Y;
NVVTTBT-RTVNVVMVDRBD that is 5' and adjacent to Y; H that is 3' and adjacent to M; HN that is 3' and adjacent to M; HNB that is 3‘ and adjacent to M; HNBV that is 3' and adjacent to M; or any combination thereof wherein M is A or C, wherein R is A, G, T or L!; wherein each D is independently G or A, wherein each H is A, C, T or U, wherein each B is independently G, C, T or U,; wherein each N is independently A, G, C, T, or U, and each V is independently A, G, or C; or any combination thereof. The synthetic RTSM of claim 52, wherein Formula (III) comprises the VMVDRBD and the HNB or the VVHBHVNNVVMVDRBD and the HNBV wherein M is A or C, wherein R is A, G, T or U; wherein each D is independently G or A, wherein each H is A, C, T or U, wherein each B is independently G, C, T or U,; wherein each N is independently A, G, C, T, or U, and each V is independently A, G, or C; or any combination thereof. The synthetic RTSM of claim 50 w herein the RTSM binding domain comprises a binding sequence comprising a sequence about 2 to about 50 nucleobases in length that has at least about: 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence complementarity to the sequence set forth in Formula (I) or to any one of the sequences of SEQ ID NOS: 31 -46, The synthetic RTSM of claim 50 wherein the RTSM binding domain comprises a binding sequence comprising a sequence about 2 to about 50 nucleobases in length that has at least about: 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence set forth in Formula (III) or to any one of tire sequences of SEQ ID NOS: 63-94. The synthetic RTSM of claim 50 wherein the target sequence comprises a sequence that is about 14 to about 25 nucleobases in length that has at least about: 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NOS:35 or 43. The synthetic RTSM of claim 50, wherein the binding sequence comprises a sequence that is about 14 to about 25 nucleobases in length that has at least about: 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence complementarity to SEQ ID NOS: 35 or 43. The synthetic RTSM of claim 50, wherein the binding sequence comprises a sequence that is about 14 to about 25 nucleobases in length that has at least about: 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity SEQ ID NOS:67, 75, 83, or 91. The synthetic RTSM of claim 50 wherein the binding sequence binds at a splice site sequence, wherein the splice site sequence comprises a sequence about 4 to about 25 nucleobases in length and is located at the 5 ’ splice site of the target intron. The synthetic RTSM of claim 50 wherein the RTSM is a polynucleotide comprising from about 4 to about 30 nucleotides. Tire synthetic RTSM of claim 50 wherein the synthetic RTSM is selected from the group consisting of an antibody or a fragment thereof, an aptamer, an antisense oligomer (ASO), or a CRISPR associated protein. Tire synthetic RTSM of claim 40, wherein the synthetic RTSM is a synthetic ASO comprising one or more nucleobases, one or more sugar moieties, a backbone, modifications thereof, more than one of the foregoing, and combinations thereof. Tire synthetic RTSM of claim 62, wherein the synthetic RTSM- ASO comprises backbone modifications comprising a phosphorothioate linkage; a sugar moiety comprising a 2'0-methyl modification; more than one of each; and in combination thereof. Tire synthetic RTSM of claim 63, wherein the synthetic ASO comprises one or more 2'-O-(2- methoxyethyl) (MOE) phosphorothioate-modified nucleotides The synthetic RTSM of claim 40, wherein the in vitro assay comprises RT-PCR, BCA Assay, Western blot analysis, or immunohistochemical detection, or any combination thereof. The synthetic RTSM of claim 40, wherein expression of exon 5 by an PNPLA3 mRNA or by an PNPLA3 protein is skipped in the presence of the RTSM as confirmed by an in vitro assay. The synthetic RTSM of claim 40, wherein the RTSM is administered as a pharmaceutical composition which also comprises a pharmaceutically acceptable: excipient, diluent, or carrier. The synthetic RTSM of claim 67, wherein the pharmaceutical composition is in unit dose form. The synthetic RTSM of claim 67, wherein the composition further comprises a second active agent comprising an anti-inflammatory, a therapeutic protein, a steroid, an analgesic, a non-steroidal anti- inflammatory, a corticosteroid, and any combination thereof.
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