Translation enhancing nucleic acid compounds: aso-conjugated translation-up 1 (act-up1) and uses thereof

Abstract

Disclosed herein are methods and compounds for enhancing gene expression by ACT-UP1 compounds. Such methods and compounds can be used to increase expression of specific genes, many of which are associated with a variety of diseases and disorders.

Description

Cross-references to related applications

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 626,347, filed January 29, 2024; No. 63 / 558,080, filed February 26, 2024; No. 63 / 677,274, filed July 30, 2024; and No. 63 / 727,989, filed December 4, 2024, the entire contents of which are incorporated herein by reference in their entirety.

[0002] By incorporating electronic submission materials The computer-readable nucleotide / amino acid sequence list submitted with this paper is incorporated in full by reference and identified as follows: a text file (1236 KB) named “2025_01_29_Seq_List_Act_Up”, created on January 28, 2025.

[0003] This application references various publications throughout. All publications, gene transcript identifiers, patents, and patent applications mentioned in this specification are incorporated herein by reference to the extent that each individual publication, gene transcript identifier, patent, or patent application is expressly and individually designated as incorporated by reference.

[0004] field Some implementations involve compounds for enhancing gene expression, and methods for using these compounds. Such methods and compounds can be used to increase the expression of specific genes, many of which are associated with a variety of diseases and conditions.

[0005] background Many diseases are caused by insufficient levels of functional proteins that typically play crucial biological roles in ensuring cellular viability. Protein deficiencies can have a variety of causes, including, for example, mutations in the corresponding protein-coding genes, downregulation of gene expression due to alterations in upstream proteins or processes, and others. Increasing the levels of underexpressed disease-related proteins may be a promising approach for treating these diseases.

[0006] Multiple approaches to restore protein expression through different mechanisms have been evaluated. These methods include, but are not limited to: using viruses or LNPs to deliver DNA / mRNA molecules to introduce DNA or RNA expressing wild-type proteins (Samulski, RJ, and N. Muzyczka, 2014 AAV-Mediated Gene Therapy for Research and Therapeutic Purposes. Annu Rev Virol 1:427-451; Wang, J., et al., 2023a Engineered mRNADelivery Systems for Biomedical Applications. Adv Mater: e2308029); and using small activating RNA (saRNA) or oligonucleotides to enhance transcription by targeting promoter regions (Li, LC, 2017, Small RNA-Guided Transcriptional Gene Activation (RNAa) in Mammalian Cells. Adv Exp MedBiol 983: 1-20; Watts, JK, et al., 2010, Effect of chemical modifications on modulation of gene expression by duplex antigene RNAs that are complementary to non-coding transcripts at Gene promoters. Nucleic Acids Res38: 5242-5259; regulating precursor mRNA splicing to generate more stable mRNA isoforms, thereby expressing more proteins (Sergeeva, OV, EY Shcherbinina, N. Shomron and TS Zatsepin, 2022, Modulation of RNA Splicing by Oligonucleotides: Mechanisms of Action and Therapeutic Implications. Nucleic Acid Ther 32: 123-138); using oligonucleotides or small molecules that inhibit mRNA degradation through nonsense-mediated decay (NMD) to improve mRNA stability (Nomakuchi, TT, F. Rigo, I. Aznarez and A.R. Krainer, 2016 Antisense oligonucleotide-directed inhibition of nonsense-mediated mRNA decay. Nat Biotechnol 34: 164-166); using antisense oligonucleotides (ASOs) that target the miRNA itself (antimiR) or target the miRNA binding site in the mRNA sequence to inhibit miRNA function in order to relieve gene expression inhibition (Samad, AFA, and MF Kamaroddin, 2023 Innovative approaches in transforming microRNAs into therapeutic tools. Wiley Interdiscip Rev RNA 14: e1768); antisense oligonucleotides (ASOs) that target repressive elements (e.g., uORF or TIE) in the 5' UTR of mRNA to enhance translation (US Patent 10,822,369; Liang et al., Antisense Oligonucleotides Targeting Translation Inhibitory Elements in 5'UTRs Can Selectively Increase Protein Levels, Nucleic Acids Res, 2017, 45(16): 9528-9546; Liang et al., Translation Efficiency of mRNAs Is Increased by Antisense Oligonucleotides Targeting Upstream Open Reading Frames, Nature Biotechnology, 2016, 34(8):875-880); and, using chimeric oligonucleotide compounds that couple guide RNA (gRNA) to internal ribosome entry sites (IRES) to enhance translation (US Publication US20230090706). Li et al.(Targeting 3' and 5' Untranslated Regions with Antisense Oligonucleotides to Stabilize Frataxin mRNA and Increase Protein Expression, Nucleic Acids Res, 2021, 49(20):11560-11574); Torkzaban et al. (Development of a Tethered mRNAAmplifier to Increase Protein Expression, Biotechnol. J., 2022 Oct, 17(10):e2200214); US Patent 5,916,808; US Patent 9,018,368; US Patent 5,916,808; US Patent 9,297,008; US Publication US20110046200; US Publication US20220204978; US Publication US20190275170; and US Publication US20220127621 describe other examples of methods for upregulating gene expression. .

[0007] Upregulating protein expression is a therapeutic area of ​​interest for disease treatment, and several companies are developing nucleic acid-based therapies to increase protein expression. For example, Sarepta Therapeutics is using phosphoryldiamide morpholino oligomers (PMOs) to promote exon skipping in the treatment of diseases such as Duchenne muscular dystrophy (eteplirsen) (US Publication US20190275072). Ionis developed nusinersen, which modulates alternative splicing to increase protein expression (US Patent 10,436,802). Stoke Therapeutics is developing antisense oligonucleotides (ASOs) to increase protein expression by affecting intron retention through splicing, thereby improving protein levels in haploinsufficiency patients (US Patent 11,096,956).

[0008] These methods offer specific advantages and may be suitable for certain situations. However, each method has its own challenges, limiting its application. For example, efficiently and safely delivering large molecules, including mRNA or DNA, into cells and the nucleus remains challenging (Wang, YS, et al., 2023b mRNA-based vaccines and therapyeutics: an in-depth survey of current and upcoming clinical applications. J Biomed Sci 30: 84). ASOs targeting miRNAs to enhance specific gene expression may lack specificity for the desired target, as each miRNA can regulate the expression of hundreds of genes (Chen, PY, and G. Meister, 2005, microRNA-guided posttranscriptional gene regulation. Biol Chem386: 1205-1218). ASO methods that target other elements (such as 5' UTR uORF, TIE, splice sites, or 3' UTR miRNA binding sites) require the presence of such elements in the target mRNA; therefore, the application of such methods is limited to genes that must have such elements.

[0009] Despite significant progress in the field of oligomeric compound technology, new and efficient methods remain needed to increase protein expression and treat subjects in need, such as those with insufficient protein expression and / or those suffering from the diseases described herein. This paper discloses a novel method, antisense oligonucleotide (ASO)-coupled translation-upregulation 1 (ACT-UP1), which increases protein levels without requiring existing elements in the target mRNA; therefore, theoretically, it can increase protein levels in any gene. Invention Overview Several embodiments described herein relate to the finding that specific modifications to antisense compounds can enhance their effectiveness in regulating gene expression. In some embodiments, antisense compounds enhance gene expression.

[0011] In some embodiments, the present invention relates to antisense compounds, which are antisense oligonucleotide (ASO)-conjugated translation-upregulation 1 (ACT-UP1) compounds. The ACT-UP1 compound comprises an ASO component linked to a protein recruitment sequence (PRS) component, wherein the ASO component hybridizes to a target mRNA, the PRS recruits translation-related proteins, and wherein the ACT-UP1 compound enhances the expression of the target protein. The ACT-UP1 compound may further comprise a conjugate. According to practices of the invention, the ASO component may be directly or indirectly linked to the PRS.

[0012] In some embodiments, the ACT-UP1 compound can be approximately 17 to 45 linked nucleosides in length. The ACT-UP1 compound contains an ASO component linked to a protein recruitment sequence (PRS) component, the ASO component being approximately 12 to 25 linked nucleosides in length, and the PRS component being approximately 5 to 20 linked nucleosides in length. The ASO component hybridizes to the target mRNA, the PRS recruits translation-related proteins, and the ACT-UP1 compound enhances protein expression.

[0013] In some embodiments, the ACT-UP1 compound can be approximately 22 to 35 linked nucleosides. The ACT-UP1 compound contains an ASO component linked to a protein recruitment sequence (PRS) component, the ASO component being 12 to 25 linked nucleosides, the PRS component containing GGACUGGACU (SEQ ID NO: 11) or AAACUAAACU (SEQ ID NO: 13). The ASO component hybridizes to the target mRNA, the PRS recruits translation-related proteins, and the ACT-UP1 compound enhances protein expression.

[0014] In a further aspect, the length of the ACT-UP1 compound can be in the range of about 17 to 45 linked nucleosides, the length of the ASO is about 12 to 25 linked nucleosides, and the length of the PRS is about 5 to 20 linked nucleosides; includes at least one modified sugar, such as a bicyclic sugar, a 2'-O-(2-methoxyethyl) group, a 2'-O-methyl group, and / or a 4'-CH(CH3)-O-2'(cEt) group; includes at least one modified inter-nucleoside link, such as a thiophosphate inter-nucleoside link; and / or contains at least one modified nucleobase, such as 5-methylcytidine.

[0015] Some implementations provide kits and methods for using the compounds disclosed herein, as well as processes for producing these compounds.

[0016] Brief description of the attached diagram Figure 1: Schematic diagram illustrating the potential ACT-UP1 mechanism. ACT-UP1 compounds contain antisense oligonucleotides (ASOs) linked to protein recruitment sequences (PRS). The ASO specifically hybridizes to the target mRNA sequence, bringing the PRS close to the target mRNA. The PRS is a short sequence of linked nucleosides that recruits translation-related proteins (e.g., PABPC1) to the vicinity of the target mRNA, thereby enhancing the translation of the target mRNA.

[0017] Figure 2: Western blot showing Jagged 1 protein levels after transfection of HeLa cells with ACT-UP1 compounds containing different PRS (i.e., ATXL193, ATXL261, and ATXL228). ACT-UP1 compounds containing PRS enhanced Jagged 1 protein expression.

[0018] Figure 3: Western blot showing Jagged 1 protein levels after transfection of HeLa cells with the ACT-UP1 compound. The data show that in the ACT-UP1 compound, the PRS located at the 5' of ASO is more effective in increasing protein levels than the PRS located at the 3' of ASO.

[0019] Figure 4: Western blot showing the level of Jagged 1 protein after transfection of HeLa cells with the ACT-UP1 compound. The data show that the binding site of ASO on the target mRNA affects the ability of the ACT-UP1 compound to increase protein expression. To a certain extent, the closer the binding site is to the stop codon of the transcript, the more protein is expressed.

[0020] Figure 5: Western blot showing the level of Jagged 1 protein after transfecting HeLa cells with ACT-UP1 compounds containing different amounts of phosphate thioester (PS) bonds.

[0021] Figure 6: Western blot showing the Jagged 1 protein level after transfection of HEK293 cells with the ACT-UP1 compound. The data show that the ACT-UP1 compound can increase protein expression in cell lines other than HeLa.

[0022] Figure 7: Western blot showing RAB9 protein levels after transfection of HeLa cells with the ACT-UP1 compound. The data show that the ACT-UP1 compound can increase the protein expression of transcripts of genes other than Jagged 1.

[0023] Figure 8: Western blot showing RNase H1 protein levels after transfection of HeLa cells with the ACT-UP1 compound. The data show that the ACT-UP1 compound can increase the protein expression of gene transcripts other than Jagged 1.

[0024] Figure 9: Western blot showing PBGD protein levels after transfection of HeLa cells with the ACT-UP1 compound. The data show that the ACT-UP1 compound can increase the protein expression of gene transcripts other than Jagged 1.

[0025] Figures 10A-10C: A) Western blot showing PBGD protein levels after transfection of mouse Hepa1-6 cells with ACT-UP1 compounds; and B) bar chart summarizing PBGD protein levels; and C) bar chart summarizing mRNA levels in mouse Hepa1-6 cells transfected with different PBGD ACT-UP1 compounds. The data indicate that ACT-UP1 compounds increased PBGD protein levels but did not significantly increase PBGD mRNA levels.

[0026] Figure 11: Western blot showing FGF21 protein levels after transfection of Hep3B cells with the ACT-UP1 compound. The data indicate that, in addition to Jagged 1, the ACT-UP1 compound can also increase the protein expression of transcripts of other genes in cells other than HeLa.

[0027] Figures 12A-12C: A) Western blot showing FGF21 protein levels after transfection of mouse Hepa1-6 cells with ACT-UP1 compounds; B) Bar chart summarizing FGF21 protein levels; and C) Bar chart summarizing mRNA levels in mouse Hepa1-6 cells transfected with different FGF21 ACT-UP1 compounds. The data indicate that ACT-UP1 compounds increase FGF21 protein levels but do not increase FGF21 mRNA levels.

[0028] Figures 13A-13C: A) Schematic diagram of ACT-UP1 affinity selection assay; B) and C) Western blots showing the recruitment of translation-related proteins by ACT-UP1 compounds.

[0029] Figure 14: Western blot showing the level of Jagged 1 protein after transfecting HeLa cells with ACT-UP1 compounds containing different PRS components.

[0030] Figure 15: Western blot showing the level of Jagged 1 protein in mice after in vivo treatment with the ACT-UP1 compound.

[0031] Figures 16A-16B: A) Western blot showing HNF4A protein levels after transfection of human Hep3B cells with ACT-UP1 compounds; and B) a bar chart summarizing HNF4A protein levels. The data indicate that the use of different ACT-UP1 compounds increased human HNF4A protein levels in Hep3B cells.

[0032] Figures 17A-17B: A) Western blot showing the level of Jagged 1 protein in mice after in vivo treatment with ACT-UP1 compounds with or without GalNAc conjugates; and B) Western blot showing the level of Jagged 1 protein in mice after in vivo treatment with ACT-UP1 compounds with or without (ATXL282) GalNAc conjugates.

[0033] Figure 18: Western blot showing the level of Jagged 1 protein in mice four weeks after in vivo treatment with the ACT-UP1 compound.

[0034] Figure 19: Showing the effect of ACT-UP1 compound on Jag1 + / - Western blot of Jagged 1 protein levels in mice after in vivo treatment.

[0035] Figure 20: Western blot showing the levels of Jagged 1 protein in mice 2 and 3 weeks after administration of the ACT-UP1 compound.

[0036] Figures 21A-21B: Bar charts showing the qRT-PCR results of FGF21 mRNA levels in Hep3B cells treated with different ACT-UP1 compounds for 20 hours (inset A) or 40 hours (inset B).

[0037] Figure 22: A bar chart showing the plasma FGF21 protein levels in mice treated with different ACT-UP1 compounds using ELISA.

[0038] Figures 23A-23B: A) Western blot showing JAG1 protein levels in GM11091 cells 24 hours after transfection with ATXL316 ASO; and B) a bar chart summarizing JAG1 protein levels from the Western blot (with JAG1 protein levels normalized to tubulin). The data indicate that ATXL316 increases JAG1 protein levels in patient GM11091 cells at 24 hours.

[0039] Figures 24A-24B: A) Western blot showing JAG1 protein levels in HeLa cells transfected with 10 nM ATXL316 followed by treatment with 100 µg / ml CHX at different time points (as indicated above the lanes); and B) a plot of JAG1 protein levels from the Western blot, where JAG1 protein levels are normalized to CHX-insensitive protein as detected by Hsp90 antibody. The data indicate that ATXL316 ASO does not affect the stability of JAG1 protein.

[0040] Figures 25A-25C: A) A table listing the sequence, chemical information, and PRS of ACT-UP1 compounds targeting JAG1; B) Western blot showing JAG1 protein levels in HeLa cells transfected with ACT-UP1 compounds; and C) A bar chart summarizing JAG1 protein levels from Western blots (where JAG1 protein is normalized for non-specific proteins). The data indicate that ACT-UP1 compounds with different PRS can increase protein levels at 24 hours.

[0041] Figure 26: A bar chart showing how different bifunctional ASOs can increase FGF21 mRNA levels in human Hep3b cells.

[0042] Figures 27A-27B: Western blot analysis of FGF21 protein levels in human HepG2 cells after transfection with different concentrations of ASO. The data indicate that different bifunctional ACT-UP1 compounds can increase FGF21 protein levels in human HepG2 cells.

[0043] Figures 28A-28B: A) Western blot of HNF4A protein levels in human primary hepatocytes (HPH) treated with the ACT-UP1 compound via free uptake; and B) a bar chart summarizing HNF4A protein levels from the Western blot, with HNF4A protein levels normalized to GAPDH protein. The data indicate that the ACT-UP1 compound increased protein levels at 66 hours.

[0044] Figure 29: Western blot of HNF4A protein levels in mouse Hepa1-6 cells after transfection with different concentrations of ASO. The data indicate that the ACT-UP1 compound increases HNF4A protein in Hepa1-6 mouse cells over a wide dose range.

[0045] Figures 30A-30B: A) Western blot of HNF4A protein levels in mouse liver after treatment with the ACT-UP1 compound; and B) a bar chart summarizing HNF4A protein levels from the Western blot, with HNF4A protein levels normalized to Hsp90 protein. The data indicate that the ACT-UP1 compound can increase HNF4A protein levels in vivo.

[0046] Detailed description of the invention It should be understood that the foregoing general description and the following detailed description are exemplary and explanatory only, and do not constitute a limitation on the claimed invention. In this document, the singular is used to include the plural unless otherwise expressly stated. As used herein, the word “or” means “and / or” unless otherwise stated. Furthermore, the use of the terms “including” and their other forms (e.g., “includes” and “included”) is not limiting. Additionally, unless otherwise expressly stated, terms (e.g., “element” or “component”) cover elements and components comprising one unit as well as elements and components comprising more than one subunit.

[0047] The chapter headings used herein are for organizational purposes only and should not be construed as limiting the subject matter described. All documents or portions thereof cited in this application, including but not limited to patents, patent applications, articles, books, and monographs, are expressly incorporated herein by reference the portions thereof discussed herein, as well as in their entirety.

[0048] definition Unless explicitly defined, the terminology, procedures, and techniques used in relation to analytical chemistry, synthetic organic chemistry, and pharmaceutical chemistry as described herein are well-known and commonly used in the art. Standard techniques can be used for chemical synthesis and analysis. Where permitted, all patents, patent applications, published applications, and other publications cited throughout this disclosure, GENBANK accession numbers and associated sequence information available from databases such as the National Center for Biotechnology Information (NCBI), and other data, are incorporated herein by reference in the sections of the document discussed herein, and in their entirety.

[0049] Unless otherwise stated, the following terms have the following meanings: "2'-O-(2-methoxyethyl)" (also known as 2'-MOE or 2'-O(CH2)2-OCH3) refers to a 2-methoxyethyl modification at the 2' position of the O atom in the furanose ring. Sugars modified with 2'-O-(2-methoxyethyl) are modified sugars.

[0050] "2'-MOE nucleoside" (also known as 2'-O-(2-methoxyethyl) nucleoside) refers to a nucleoside containing a sugar moiety modified with 2'-MOE. "2'-MOE nucleotide" (also known as 2'-O-(2-methoxyethyl) nucleotide) refers to a nucleotide containing a sugar moiety modified with 2'-MOE.

[0051] "2'-O-methyl" (also known as 2'-OCH3 or 2'-OMe) refers to a methyl modification at the 2' position of the O atom in the furanose ring. Sugars modified with 2'-O-methyl are modified sugars.

[0052] "2'-OMe nucleoside" (also known as 2'-O-methyl nucleoside) refers to a nucleoside containing a sugar moiety modified with 2'-OMe. "2'-OMe nucleotide" (also known as 2'-O-methyl nucleotide) refers to a nucleotide containing a sugar moiety modified with 2'-OMe.

[0053] "2'-substituted nucleosides" refers to nucleosides in which the 2'-position of the furanose ring contains a substituent other than H or OH (i.e., modification). In some embodiments, 2'-substituted nucleosides include nucleosides modified with fluorine (2'-F), O-methyl (2'-OMe), O-(2-methoxyethyl) (2'-MOE), or bicyclic sugars. 2'-substituted nucleosides are modified nucleosides.

[0054] "3' target site" refers to the nucleotide in the target nucleic acid that is complementary to the nucleotide at the 3' end of a specific antisense compound.

[0055] "5' target site" refers to the nucleotide in the target nucleic acid that is complementary to the nucleotide at the 5' end of a specific antisense compound.

[0056] "5-Methylcytosine" refers to cytosine modified with a methyl group attached to the 5-position. 5-Methylcytosine is a modified nucleobase and part of a modified nucleoside. "5-Methylcytidine" is the name of a nucleoside when a 5-methyl modified nucleobase is combined with a modified or unmodified sugar.

[0057] As used herein, “about” means within ±7% of a measurable value. For example, if it is stated that “the compound affects at least about 70% mRNA inhibition,” it means that the mRNA level is inhibited in the range of 63% to 77%. As used herein, the term “about” when referring to the amount, dosage, time, temperature, etc., of the compounds or reagents of the invention is intended to cover variations of ±10%, ±5%, ±1%, ±0.5%, or even ±0.1% of the specified amount. For sequence identity, “about” when used as a percentage of sequence identity covers variations of ±5%. Unless otherwise stated, all figures indicating the amount, properties (e.g., reaction conditions), etc., of components used in the specification and claims should in all cases be understood to be modified by the term “about.”

[0058] As used in this article, "mean" can be the mean, mode, or median of a set of measurements.

[0059] As used herein, the singular forms “a,” “an,” and “the” include plural referents unless the context explicitly specifies otherwise. Thus, for example, reference to “a cell” includes multiple such cells; and reference to “the culture” includes one or more cultures, as well as their equivalents known to those skilled in the art.

[0060] “ACT-UP1” or “ASO Coupled Translation - Upregulation 1” refers to an antisense oligonucleotide (ASO) linked to a protein recruitment sequence (PRS). The ASO specifically hybridizes to the target mRNA sequence, bringing the PRS into close proximity to the target mRNA. The PRS is a short sequence of the linked nucleoside that recruits translation regulatory proteins to the vicinity of the target mRNA, thereby enhancing the translation of the target mRNA. As used herein, although the protein recruitment sequence (PRS) contains a short nucleoside linker, it is not an antisense oligonucleotide (ASO) because it does not hybridize to the target nucleic acid (i.e., the protein recruitment sequence is not an antisense sequence of the target nucleic acid). The ASO can be directly linked to the PRS, or they can be linked via a linker. In some preferred embodiments, the PRS is linked to the 5' end of the ASO. In some embodiments, the PRS is linked to the 3' end of the ASO. Linkage = covalent linkage; it should be understood that the ACT-UP1 compound may contain additional sequences besides ASO and PRS. For example, in some implementations, ACT-UP1 includes an ASO, a PRS, and a connector for connecting the ASO and the PRS.

[0061] "ACT-UP1 activity" refers to any detectable or measurable activity attributable to the hybridization of an ACT-UP1 compound with its target nucleic acid. In some embodiments, ACT-UP1 activity is an increase (i.e., upregulation) in the amount of the target nucleic acid and / or the expression of the protein encoded by such target nucleic acid.

[0062] "ACT-UP1 upregulation" or "ACT-UP1 enhancement" means that the target protein level is increased in the presence of ACT-UP1 compounds compared to the target protein level in the absence of ACT-UP1 compounds.

[0063] "Animal" refers to human or non-human animals, including but not limited to mice, rats, rabbits, dogs, cats, pigs, and non-human primates (including but not limited to monkeys and chimpanzees).

[0064] An "antibody" is a molecule characterized by a specific reaction with an antigen in some way, where both antibody and antigen are defined according to each other. An antibody can refer to the complete antibody molecule, or any fragment or region thereof, such as the heavy chain, light chain, or F1 antibody. ab District and F c district.

[0065] "Antisense compound" refers to an oligomeric compound that can hybridize with a target nucleic acid via hydrogen bonding. Examples of antisense compounds include single-stranded and double-stranded compounds, such as antisense oligonucleotides (ASOs), siRNA, shRNA, snoRNA, miRNA, and satellite repeat sequences. It should be understood that antisense compounds include the ACT-UP1 compound, which contains an antisense oligonucleotide (ASO) linked to a protein recruitment sequence (PRS).

[0066] "Antisense oligonucleotide" or "ASO" refers to a single-stranded oligonucleotide having a nucleobase sequence that allows hybridization with a corresponding region or segment of a target nucleic acid. In some embodiments, the ASO comprises one or more modified nucleosides. In some embodiments, the ASO comprises a portion of the ACT-UP1 compound. As used herein, ASO does not refer to the protein recruitment sequence (PRS) in the ACT-UP1 compound.

[0067] "Base complementarity" refers to the ability of a nucleobase in an oligonucleotide to pair (i.e., hybridize) with a corresponding nucleobase in a target nucleic acid, and is regulated by Watson-Crick, Hoogsteen, or reverse Hoogsteen hydrogen binding between the corresponding nucleobases. Base complementarity also refers to canonical base pairing (e.g., A:U, A:T, or C:G) or non-canonical base pairing (e.g., A:G, A:U, G:U, I:U, I:A, or I:C).

[0068] "Bicyclic sugar" refers to a furanose ring modified by bridging two non-homogeneous carbon atoms. Bicyclic sugars are modified sugars.

[0069] "Cap structure" or "terminal cap portion" refers to a chemical modification incorporated into either end of an antisense compound.

[0070] “cEt” or “restricted ethyl” means a bicyclic sugar moiety containing a bridge connecting the 4'-carbon and the 2'-carbon, wherein the bridge has the following formula: 4'-CH(CH3)-O-2'.

[0071] "Restricted ethyl nucleoside" (also known as cEt nucleoside) refers to a nucleoside containing a bicyclic sugar moiety with a 4'-CH(CH3)-O-2' bridge.

[0072] "Chemical modification" refers to the modification of the molecular structure or elements of a naturally occurring molecule. For example, antisense oligonucleotides are composed of linked deoxyribonucleosides (sometimes also referring to DNA nucleotides in this text), therefore, replacing DNA nucleotides with 2'-MOE nucleotides is considered a chemical modification of antisense oligonucleotides. Examples of other chemical modifications can be found below.

[0073] A “chemically distinct region” refers to a region of an antisense compound that is chemically different in some way from another region of the same antisense compound. For example, a region containing a 2'-O-(2-methoxyethyl) nucleotide is chemically different from a region containing a nucleotide lacking the 2'-O-(2-methoxyethyl) modification.

[0074] "Complementarity" refers to the ability of the nucleobases of the first and second nucleic acids to pair.

[0075] "Compliance" refers to an individual's adherence to suggested therapies.

[0076] “Comprise,” “comprises,” and “comprising” will be understood to mean that the step or element, or a group of steps or elements, is included, but does not exclude any other step or element, or a group of steps or elements.

[0077] "Continuous nucleobases" refers to nucleobases that are adjacent to each other.

[0078] "Deoxyribonucleoside" refers to a nucleoside whose sugar moiety has a hydrogen atom at the 2' position. Deoxyribonucleoside is sometimes referred to as DNA nucleoside, "D," or "d" in this document. Deoxyribonucleoside can be modified with any of a variety of substituents and can be linked by covalent bonds other than naturally occurring phosphodiester bonds (e.g., thiophosphates).

[0079] "Deoxyribonucleic acid" refers to a nucleotide that has a hydrogen atom at the 2' position of the sugar portion of the nucleotide. Deoxyribonucleic acid is sometimes referred to as DNA nucleotide, "D," or "d" in this document. Deoxyribonucleic acid can be modified with any of a variety of substituents and can be linked by covalent bonds other than naturally occurring phosphodiester bonds (e.g., thiophosphates).

[0080] A “derivative” of a protein recruitment sequence (PRS) refers to a selected sequence that is then modified to be used as a PRS. For example, GGACU (SEQ ID NO: 8) is an mRNA-derived protein recruitment sequence. 6A methylation target sequence can serve as a basis for preparing GGACU derivatives that are not used as methylation targets and are not present in mRNA (e.g., the GGACU sequence is derivatized for use as a PRS). Derivatives that can be used as PRS components of ACT-UP1 can also be prepared by adding additional nucleobases to the original sequence (e.g., ACGGACUUGGACU, SEQ ID NO: 12), linking repeat sequences of the original sequence together (e.g., GGACUGGACU, SEQ ID NO: 11), linking partially repeat sequences of the original sequence together (GGACUGGAC, SEQ ID NO: 10), or combinations of modifications. In some instances, the derivative contains one or more repeat sequences or partially repeat sequences of a specific sequence. By way of example only, modifications can be the insertion, addition, deletion, or substitution of a specific nucleoside into the sequence (e.g., GAACU (SEQ ID NO: 42), AGACU (SEQ ID NO: 43), or GGACA (SEQ ID NO: 44) are products of substitution of A in GGACU), as well as chemical modifications. For example, in a PRS, the modification includes at least one substitution. In other embodiments, the modification includes at least two substitutions.

[0081] A "bifunctional compound" or "bifunctional ACT-UP1 compound" is a compound designed to have an ACT-UP1 design plus a non-ACT-UP1 secondary function or mechanism. For example, a bifunctional compound may have an ACT-UP1 design (e.g., binding to the 3'UTR of a target transcript and possessing a PRS) and a target sequence rich in AU elements (AREs). Such a bifunctional compound will enhance protein levels by increasing mRNA levels (through blocking ARE binding) and increasing protein translation levels (through ACT-UP1).

[0082] "Efficacy" refers to the ability to produce the desired effect.

[0083] "Expression" encompasses all functions that translate gene-encoded information into structures that exist and function within the cell. These structures include, but are not limited to, the products of transcription and translation.

[0084] "Completely complementary" or "100% complementary" means that every nucleobase in the first nucleic acid is a complementary nucleobase in the second nucleic acid. In some implementations, the first nucleic acid is an antisense compound, and the target nucleic acid is the second nucleic acid.

[0085] "Fully modified" or "fully chemically modified" refers to an antisense compound containing a continuous sequence of nucleosides, in which virtually every nucleoside is chemically modified.

[0086] "Hybridization" refers to the annealing of complementary nucleic acid molecules. In some embodiments, complementary nucleic acid molecules include, but are not limited to, antisense compounds and nucleic acid targets. In some embodiments, complementary nucleic acid molecules include, but are not limited to, antisense oligonucleotides and nucleic acid targets.

[0087] "Closest" means that there are no intermediate elements between the adjacent elements.

[0088] "Individual" refers to a person or non-human animal that chooses to use for treatment or therapy.

[0089] Terms such as “induction,” “inhibition,” “enhancement,” “enhancement,” “increase,” “decrease,” and “strengthening” typically indicate quantitative differences between two states.

[0090] "Inhibit expression or activity" means to reduce or block expression or activity, and does not necessarily mean to completely eliminate expression or activity.

[0091] "Nucleoside linkage" or "linkage" refers to the chemical bond between nucleosides. Here, the 3' position of a nucleoside is connected to the 5' position of the subsequent nucleoside via an inter-nucleoside linkage.

[0092] "Separated" means the state after one or more purification steps, but absolute purity is not required.

[0093] In the ACT-UP1 compound, "linked" ASO and PRS components mean that the ASO and PRS components are covalently linked to form the ACT-UP1 compound. It should be understood that the ACT-UP1 compound may contain additional sequences linked to the ASO and / or PRS. For example, in some embodiments, ACT-UP1 comprises ASO and PRS components, as well as a connector for linking the ASO and PRS.

[0094] "Linked deoxynucleosides" refers to nucleic acid bases (A, G, C, T, U) that have been replaced by deoxyribose and linked together by phosphate esters to form nucleotides.

[0095] "Linked nucleosides" refers to adjacent nucleosides (e.g., A, G, C, T, or U) linked together by an internucleotide bond. Examples of linked nucleosides include deoxyribonucleosides (sometimes referred to as DNA nucleosides in this text) or ribonucleosides (sometimes referred to as RNA nucleosides in this text).

[0096] "Connector" refers to a molecule that acts as a spacer between two components (e.g., between the ACT-UP1 compound and the GalNAc conjugate, and / or between PRS and ASO within the ACT-UP1 compound). Examples of connectors include one or more of fatty acids, polyethylene glycol (PEG), or amino acids.

[0097] "Mismatch" or "non-complementary nucleobases" refers to a situation where nucleobases in the first nucleic acid cannot pair with corresponding nucleobases in the second or target nucleic acid through Watson-Crick base pairing (e.g., A:T, A:U, or C:G).

[0098] "Modified nucleoside interbonds" refers to the substitution or any alteration of naturally occurring nucleoside interbonds (i.e., phosphodiester nucleoside interbonds).

[0099] "Modified nucleobases" refers to any base other than adenine, cytosine, guanine, thymidine, or uracil. "Unmodified nucleobases" refers to the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C), and uracil (U). In some embodiments, when a DNA nucleoside replaces an RNA nucleoside, the RNA nucleoside is considered modified. In some embodiments, when an RNA nucleoside replaces a DNA nucleoside, the DNA nucleoside is considered modified.

[0100] "Modified nucleosides" refers to nucleosides that independently possess modified sugar moieties and / or modified nucleobases.

[0101] "Modified nucleotide" refers to a nucleotide that independently has a modified sugar moiety, a modified nucleoside link, and / or a modified nucleobase.

[0102] "Modified oligonucleotide" means an oligonucleotide that contains at least one modified nucleoside link, a modified sugar, and / or a modified nucleobase.

[0103] "Modified sugar" means the substitution and / or any alteration of the natural sugar portion.

[0104] "Part" refers to one of the parts into which something is divided, that is, a portion or component of something. For example, the sugar part of a nucleotide is the sugar component of the nucleotide.

[0105] "Monomer" refers to a single unit of an oligomer. Monomers include, but are not limited to, nucleosides and nucleotides, whether naturally occurring or modified.

[0106] "Modal" refers to the pattern of unmodified and modified nucleosides in an antisense compound. Antisense compounds contain motifs with various modified nucleobases, modified sugars, and / or inter-nucleoside bonds to improve properties such as delivery, stability, specificity, safety, and potency. The motif is independent of the nucleobase sequence of the antisense compound and only identifies the modification pattern.

[0107] "Natural sugar moieties" refers to the sugar moieties that exist in DNA (2'-H) or RNA (2'-OH).

[0108] "Naturally occurring nucleoside-to-nucleotide linkages" refers to 3' to 5' phosphodiester linkages.

[0109] "Non-complementary nucleobases" refer to a pair of nucleobases that do not form hydrogen bonds with each other or otherwise support hybridization.

[0110] "Nucleic acid" refers to a molecule composed of monomeric nucleotides. Nucleic acids include, but are not limited to, ribonucleic acid (RNA, e.g., mRNA), deoxyribonucleic acid (DNA), single-stranded nucleic acids (e.g., antisense oligonucleotides (ASO) and microRNA (miRNA)), and double-stranded nucleic acids (e.g., small interfering RNA (siRNA) and short hairpin RNA (shRNA)).

[0111] "Nucleobase" refers to the heterocyclic portion that can pair with the bases of another nucleic acid.

[0112] "Nucleobase complementarity" refers to the ability of nucleobases to pair with another nucleobase (also known as complementarity). If a nucleobase at a certain position in an oligomer can form a hydrogen bond with a nucleobase at a certain position in a target nucleic acid, the position where the hydrogen bond occurs between the oligomer and the target nucleic acid is considered complementary at that nucleobase pair. For example, in DNA, adenine (A) is complementary to thymine (T); in RNA, adenine (A) is complementary to uracil (U); and in both DNA and RNA, guanine (G) is complementary to cytosine (C). Base pairs, or complementary nucleobases, are typically typical Watson-Crick base pairs (e.g., C:G, A:U, or A:T), but also include atypical base pairs such as Hoogsteen base pairs (e.g., A:G or A:U), wobbling base pairs (e.g., G:U, I:U, I:A, or I:C, where I is hypoxanthine), etc. Nucleobase complementarity facilitates the hybridization of the oligomers described in this paper with their target nucleic acids.

[0113] "Nucleobase sequence" refers to a continuous sequence of nucleobases that is independent of any sugars, linkages, and / or nucleobase modifications.

[0114] "Nucleoside" refers to a nucleobase linked to a sugar.

[0115] "Nucleoside mimics" include those used to replace sugars at one or more positions in an oligomer, or to replace sugars and bases, but not necessarily the linked structures, such as those having morpholino, cyclohexenyl, cyclohexyl, tetrahydropyranoyl, bicyclic, or tricyclic sugar mimics (e.g., non-furanose sugar units). Nucleotide mimics include those used to replace nucleosides at one or more positions in an oligomer (e.g., peptide nucleic acids or morpholino compounds (morpholino compounds linked by -N(H)-C(=O)-O- or other non-phosphodiester bonds)) and the linked structures. Sugar substitutes overlap with the slightly broader term nucleoside mimics, but are intended to refer only to those that replace sugar units (furanose rings). The tetrahydropyran ring provided herein is an exemplary example of a sugar substitute where the furanose group has been replaced by a tetrahydropyran ring system. "Mimetic" refers to a group that substitutes for sugars, nucleobases, and / or the bonds between nucleosides. Typically, analogs are used to replace sugars or sugar-nucleoside linkages, and nucleobases are retained for hybridization with selected targets.

[0116] "Nucleotide" refers to a nucleoside having a linker group (e.g., a phosphate group (P) or a thiophosphate group (PS)) covalently linked to the sugar moiety of the nucleoside. Nucleotides include ribonucleotides and deoxyribonucleotides. Ribonucleotides are the nucleotide units that form the links of RNA. Deoxyribonucleotides are the nucleotide units that form the links of DNA.

[0117] "Off-target effects" refer to unintended or harmful biological effects associated with the regulation of RNA or protein expression of genes other than their intended target nucleic acids.

[0118] "Oligomer" refers to a linked monomeric subunit sequence capable of hybridizing with at least one region of a target nucleic acid via hydrogen bonding. The monomeric subunit can be a modified or unmodified nucleotide or nucleoside. Examples of oligomers include antisense compounds (e.g., antisense oligonucleotides (ASO) or ACT-UP1 compounds containing antisense oligonucleotides).

[0119] As used herein, “oligonucleotide” means a polymer of linked nucleosides, each of which may be independently modified or unmodified. Oligonucleotides may have linking groups other than phosphate groups (e.g., thiophosphate = thiophosphate group) as the linking part between nucleosides.

[0120] "Substantially modified" or "substantially chemically modified" refers to an antisense compound containing a continuous sequence of nucleosides, most (but not all) of which are chemically modified. For example, the compound contains no more than about 5, 4, 3, 2, or 1 unmodified nucleosides.

[0121] "Thiophosphate bond" or "PS" refers to a bond between nucleosides, in which the phosphodiester bond is modified by replacing a non-bridging oxygen atom with a sulfur atom. A thiophosphate bond (also known as thiophosphoric acid) is a modified inter-nucleoside bond.

[0122] A “protein recruitment sequence” or “PRS” is a short sequence of linked nucleosides that attracts and / or binds translation regulatory proteins. By way of example only, a translation regulatory protein may be an RNA-binding protein (RBP) that regulates the translation of mRNA into a protein, as is well known in the art (see Example 12). The PRS is the component of the ACT-UP1 compound that interacts with the RNA-binding protein, while the ASO is the component of the ACT-UP1 compound that hybridizes with the target nucleic acid; the ASO and PRS components of the ACT-UP1 compound together enhance the translation of the mRNA target. Although the protein recruitment sequence contains a short nucleoside linker, it is not an antisense oligonucleotide (ASO) because it does not hybridize with the target nucleic acid (i.e., the protein recruitment sequence is not an antisense sequence of the target nucleic acid). The PRS component of ACT-UP1 may be a sequence derived from a natural sequence. For example, the PRS may be a sequence located in the 3'UTR of the mRNA transcript and derivatized as the trans-acting PRS component of the ACT-UP1 compound. The derivative forming the PRS may optionally be modified with additional nucleobases, nucleobase deletions, nucleobase substitutions, nucleobase repetitions, etc.

[0123] "Partial" refers to a defined number of consecutive (i.e., linked) nucleobases in a nucleic acid. In some embodiments, a partial refers to a defined number of consecutive nucleobases in the target nucleic acid. In some embodiments, a partial is a defined number of consecutive nucleobases in the antisense compound.

[0124] A “region” is defined as a portion of a target nucleic acid that has at least one identifiable structure, function, or feature.

[0125] RNA, or ribonucleic acid, is composed of ribose nucleotides (or ribonucleotides) (nitrogenous bases attached to ribose sugar), linked by phosphodiester bonds to form chains of varying lengths. The nitrogenous bases in RNA are adenine, guanine, cytosine, and uracil. The ribose sugar in RNA is a five-carbon, one-oxygen ring structure.

[0126] "Ribosin" refers to a nucleoside that has a hydroxyl group at the 2' position of the sugar moiety. In this article, ribosins are sometimes referred to as RNA nucleosides, "R", or "r".

[0127] "Ribosonucleotide" refers to a nucleotide that has a hydroxyl group at the 2' position of the sugar portion of the nucleotide. In this article, ribonucleotides are sometimes referred to as RNA nucleotides, "R", or "r".

[0128] A “segment” is defined as a smaller portion or sub-region within a target nucleic acid.

[0129] As used in this article, "site" is defined as a unique nucleobase location within the target nucleic acid.

[0130] "Specific hybridization" refers to an antisense compound (e.g., ASO) that has sufficient complementarity with the target nucleic acid to induce the desired effect, while exhibiting minimal or no effect on non-target nucleic acids under the conditions of desired specific binding (i.e., under physiological conditions in in vivo assays and therapeutic applications). Examples of sufficient complementarity are disclosed herein.

[0131] "Strict hybridization conditions" or "strict conditions" refer to conditions under which an antisense compound will hybridize with its target sequence, but with a very small number of other sequences.

[0132] "Subject" refers to a human or non-human animal selected for treatment or therapy.

[0133] A "target" refers to a protein or nucleic acid sequence (e.g., mRNA) that is intended to be regulated. In some implementations, regulation is an increase in the expression of the target nucleic acid. In other implementations, regulation is a decrease in the expression of the target nucleic acid.

[0134] "Target gene" refers to the gene that encodes the target.

[0135] "Targeting" refers to the process of designing and selecting antisense compounds that will specifically hybridize with the target nucleic acid and induce the desired effect.

[0136] "Target nucleic acid", "target RNA", "target RNA transcript" and "nucleic acid target" all refer to nucleic acids that can be targeted by antisense compounds.

[0137] "Target region" refers to the portion of the target nucleic acid that is targeted by one or more antisense compounds.

[0138] "Target region" refers to the nucleotide sequence of the target nucleic acid targeted by the antisense compound. "5' target site" refers to the 5' terminal nucleotide in the target region. "3' target site" refers to the 3' terminal nucleotide in the target region. In the implementation, the target region is a portion of at least 12 nucleotides (i.e., at least 12 consecutive nucleotides) in the target region targeted by the antisense compound.

[0139] "Therapeutic efficacy" refers to the effectiveness of a therapeutic compound (such as an antisense compound). Therapeutic efficacy can be improved by enhancing the delivery, stability, specificity, safety, and potency of the therapeutic compound.

[0140] "Unmodified" RNA nucleosides refer to purines such as adenine (A) and guanine (G), and pyrimidines such as cytosine (C) and uracil (U). "Unmodified" DNA nucleosides refer to purines such as adenine (A) and guanine (G), and pyrimidines such as thymine (T) and cytosine (C). In some embodiments, when a DNA nucleoside replaces an RNA nucleoside, the unmodified RNA nucleoside is considered modified. In some embodiments, when an RNA nucleoside replaces a DNA nucleoside, the unmodified DNA nucleoside is considered modified.

[0141] "Unmodified nucleosides" refers to nucleosides composed of common, naturally occurring nucleobases and sugar moieties. For example, an unmodified nucleoside used in a DNA sequence is a DNA nucleoside; however, in such a DNA sequence, any other nucleoside (e.g., RNA nucleoside, 2'-OMe, 2'-MOE, or 2'-F) is considered a modified nucleoside.

[0142] "Unmodified nucleotides" refer to nucleotides composed of common, naturally occurring nucleobases, sugar moieties, and internucleotide bonds. For example, an unmodified nucleotide is a DNA nucleotide if used in a DNA sequence; however, in such a DNA sequence, any other nucleotide (e.g., RNA nucleotides, 2'-OMe, 2'-MOE, or 2'-F) is considered a modified nucleotide.

[0143] "Validated target segment" is defined as a portion of at least 8 nucleobases (i.e. 8 consecutive nucleobases) of the target region targeted by the antisense compound.

[0144] "Pterion" refers to multiple nucleosides located at the 3' and / or 5' ends of an antisense oligonucleotide, wherein these nucleosides are modified to endow the antisense oligonucleotide with properties such as enhanced activity, increased affinity for binding to target nucleic acids, and / or resistance to degradation by nucleases in vivo.

[0145] This document discloses antisense compounds that enhance target protein expression. In some embodiments, the antisense compound comprises an antisense oligonucleotide (ASO)-coupled translation-upregulation 1 (ACT-UP1) compound. The ACT-UP1 compound comprises an ASO component linked to a protein recruitment sequence (PRS) component, wherein the ASO component hybridizes to a target mRNA, the PRS recruits translation-related proteins, and wherein the ACT-UP1 compound enhances target protein expression. In some preferred aspects, the PRS is linked to the 5' end of the ASO. In some aspects, the PRS is linked to the 3' end of the ASO.

[0146] In some embodiments, the ACT-UP1 compound comprises approximately 17 to 45, 17 to 44, 17 to 43, 17 to 42, 17 to 41, 17 to 40, 17 to 39, 17 to 38, 17 to 37, 17 to 36, 17 to 35, 17 to 34, 17 to 33, 17 to 32, 17 to 31, 17 to 30, 17 to 29, 17 to 28, 17 to 27, 17 to 26, 17 to 25, 19 to 45, 19 to 40, 19 to 35, 19 to 34, 19 to 33, 19 to 32, and 19 to 31. 19 to 30, 19 to 29, 19 to 28, 19 to 27, 19 to 26, 19 to 25, 22 to 45, 22 to 40, 22 to 35, 22 to 34, 22 to 33, 22 to 32, 22 to 31, 22 to 30, 22 to 29, 22 to 28, 22 to 27, 22 to 26, 22 to 25, 25 to 45, 25 to 40, 25 to 35, 25 to 34, 25 to 33, 25 to 32, 25 to 31, 25 to 30, 25 to 29, 25 to 28, or 25 to 27 connected subunits.

[0147] In some embodiments, the ASO component of the ACT-UP1 compound comprises about 12 to 25, 12 to 24, 12 to 23, 12 to 22, 12 to 21, 12 to 20, 12 to 19, 12 to 18, 12 to 17, 12 to 16, 12 to 15, or 12 to 14 linked nucleosides.

[0148] In some embodiments, the PRS component of the ACT-UP1 compound comprises about 5 to 20, 5 to 19, 5 to 18, 5 to 17, 5 to 16, 5 to 15, 5 to 14, 5 to 13, 5 to 12, 5 to 11, 5 to 10, 5 to 9, 5 to 8, 5 to 7, or 5 to 6 linked nucleosides.

[0149] In some embodiments, the ACT-UP1 compound can be approximately 17 to 45 linked nucleosides. The ACT-UP1 compound may contain an ASO component linked to a protein recruitment sequence (PRS) component, the ASO component being approximately 12 to 25 linked nucleosides, and the PRS component being 5 to 20 linked nucleosides. According to the invention, in some embodiments, the ASO component hybridizes to the target mRNA, the PRS recruits translation-related proteins, thereby the ACT-UP1 compound enhances protein expression. In some preferred aspects, the PRS is linked to the 5' end of the ASO. In some aspects, the PRS is linked to the 3' end of the ASO.

[0150] In some embodiments, the PRS comprises a derivative of the DRACH concordant sequence. In some embodiments, the derivative of the DRACH concordant sequence comprises GGACU (SEQ ID NO: 8), GGAUU (SEQ ID NO: 9), GAACU (SEQ ID NO: 41), AGACU (SEQ ID NO: 42), AAACU (SEQ ID NO: 43), GGACA (SEQ ID NO: 44), AAACA (SEQ ID NO: 154), or a derivative thereof.

[0151] In some embodiments, the PRS comprises a derivative of the RRANN concordant sequence. In some embodiments, the derivative of the RRANN concordant sequence comprises GGACU (SEQ ID NO: 8), GGAUU (SEQ ID NO: 9), GAACU (SEQ ID NO: 41), AGACU (SEQ ID NO: 42), AAACU (SEQ ID NO: 43), or GGACA (SEQ ID NO: 44), or a derivative thereof.

[0152] In some embodiments, the PRS comprises a derivative of the RRAWN concordant sequence. In some embodiments, the derivative of the RRAWN concordant sequence comprises GGACU (SEQ ID NO: 8), GGAUU (SEQ ID NO: 9), GAACU (SEQ ID NO: 41), AGACU (SEQ ID NO: 42), AAACU (SEQ ID NO: 43), or GGACA (SEQ ID NO: 44), or a derivative thereof.

[0153] In some embodiments, the PRS comprises a derivative of the GGACU sequence (SEQ ID NO: 8). In some embodiments, the GGACU derivative is selected from any one of GGACU (SEQ ID NO: 8), GGACUGGAC (SEQ ID NO: 10), GGACUGGACU (SEQ ID NO: 11), GGACUGGACUGGACU (SEQ ID NO: 101), and ACGGACUUGGACU (SEQ ID NO: 12).

[0154] In some implementations, the PRS comprises two to four repeating sequences of the derived sequences GGACU (SEQ ID NO: 8), GGAUU (SEQ ID NO: 9), GAACU (SEQ ID NO: 41), AGACU (SEQ ID NO: 42), AAACU (SEQ ID NO: 43), or GGACA (SEQ ID NO: 44), or a combination of derived sequences (e.g., GGACUGGACU (SEQ ID NO: 11) or AAACUAAACU (SEQ ID NO: 13)).

[0155] In some embodiments, the PRS comprises a derivative of the poly(A) sequence in the 3'UTR of the mRNA. In some embodiments, the derivative of the poly(A) sequence is AAACUAAACU (SEQ ID NO: 13), AAAAAAAAAAAA (SEQ ID NO: 102), or AAAAAAAAAA (SEQ ID NO: 15).

[0156] In some embodiments, the PRS comprises a derivative of the poly(C) sequence in the 3'UTR of the mRNA. In some embodiments, the derivative of the poly(C) sequence is CCCCCCCCCCC (SEQ ID NO: 93).

[0157] In some embodiments, the PRS comprises a derivative of the poly(G) sequence in the 3'UTR of the mRNA. In some embodiments, the derivative of the poly(G) sequence is GGGGGGGGGG (SEQ ID NO: 152).

[0158] In some embodiments, the PRS comprises a derivative of the poly(T) sequence in the 3'UTR of the mRNA. In some embodiments, the derivative of the poly(T) sequence is TTTTTTTTTTT (SEQ ID NO: 153).

[0159] In some embodiments, the PRS comprises a derivative of the poly(U) sequence in the 3'UTR of the mRNA. In some embodiments, the derivative of the poly(U) sequence is UUUUUUUUUU (SEQ ID NO: 94).

[0160] Other examples of PRS are shown in the table in this article.

[0161] In some embodiments, the ACT-UP1 compound has a length of about 22 to 35 linked nucleosides. The ACT-UP1 compound comprises an ASO component linked to a PRS component, the ASO component having a length of about 12 to 25 linked nucleosides, the PRS component comprising GGACUGGACU (SEQ ID NO: 11) or AAACUAAACU (SEQ ID NO: 13). According to the invention, in some embodiments, the ASO component hybridizes to the target mRNA, the PRS recruits translation-related proteins, thereby the ACT-UP1 compound enhances protein expression. In some preferred aspects, the PRS is linked to the 5' end of the ASO. In some aspects, the PRS is linked to the 3' end of the ASO.

[0162] In some embodiments, the ACT-UP1 compound is a trans-acting protein enhancer. In some embodiments, the ACT-UP1 trans-acting protein enhancer targets mRNA transcripts and recruits endogenous translation-related proteins to increase the translation of the target protein.

[0163] In some embodiments, the ACT-UP1 compound targets eukaryotic or prokaryotic mRNA. In some embodiments, the ACT-UP1 compound targets mammalian mRNA, plant mRNA, yeast mRNA, or bacterial mRNA. In some embodiments, the targeted mammalian mRNA encodes any one of the following: Delta / Serrate / Lag-2 (DSL) protein (e.g., JAG1), the RAB family of small GTPases (e.g., RAB9), porphyrin deaminase (PBGD), RNase H family protein (e.g., RNase H1), nuclear transcription factor (e.g., HNF4A), or fibroblast growth factor (e.g., FGF21).

[0164] In some implementations, the ACT-UP1 compound targets a region of approximately 20 to 50, 40 to 70, 60 to 90, 80 to 110, 100 to 130, 120 to 150, 140 to 170, 160 to 190, 180 to 210, 200 to 230, 220 to 250, 240 to 300, or 280 to 500 nucleotides downstream of a stop codon on the target mRNA.

[0165] In some embodiments, the ACT-UP1 compound further comprises a reagent (e.g., a delivery agent, therapeutic agent, or diagnostic agent) to form a conjugated compound. In some embodiments, the conjugating agent (also referred to as the conjugated moiety) may be selected from cholesterol, lipids, carbohydrates, phospholipids, biotin, phenazine, folic acid, phenanthridine, anthraquinone, acridine, fluorescein, rhodamine, coumarin, peptides, antibodies, dyes, and tocopherols. In some embodiments, the conjugating agent is N-acetylgalactosamine (GalNAc). In some embodiments, GalNAc is the GalNAc described in Sharma et al. (2018, Bioconjugate Chem, 29:2478-2488) or the GalNAc described in WO2024137545.

[0166] In some embodiments, the ACT-UP1 compound comprises at least one chemical modification. In some embodiments, the ACT-UP1 compound is partially, substantially, or completely chemically modified. In some embodiments, the ACT-UP1 compound is chemically modified such that the chemical modification is selected from one or more of the following: 2'-O-methyl (2'-OMe), 2'-O-(2-methoxyethyl) (2'-MOE), 2'-fluoro (2'-F), restricted ethyl (cEt), unlocked nucleic acid (UNA), locked nucleic acid (LNA), 2'-MOE-modified T, and / or 5-methylcytosine bases. In some embodiments, the ASO and PRS of the ACT-UP1 compound comprise the same or different chemical modifications. In some embodiments, the PRS chemical modification is 2'-OMe and / or 2'-MOE. In a preferred embodiment, the nucleoside of PRS is modified with 2'-OMe, and the nucleoside of ASO is modified with 2'-MOE. In another preferred embodiment, the PRS nucleoside is modified with 2'-OMe, and the ASO nucleoside comprises cEt, LNA, and / or 2'-MOE modified adenosine (eA), thymidine (eT), uridine (eU), guanosine (eG), cytidine (eC), and 2'-MOE modified 5-methylcytidine (eCm). In embodiments, the PRS comprises ribonucleoside or deoxyribonucleoside. In embodiments, the ASO comprises ribonucleoside or deoxyribonucleoside. In a preferred embodiment, the PRS and ASO comprise modified ribonucleoside, wherein the modification may be the substitution of RNA nucleoside with DNA nucleoside.

[0167] In some embodiments, the ACT-UP1 compound comprises at least one modified internucleotide link. In some embodiments, the at least one modified internucleotide link is a phosphate thioester (PS) link. In one embodiment, the PS link is located / positioned between the first and second nucleosides in the PRS of the ACT-UP1 compound (counted in the 5' to 3' direction). In another embodiment, the PS link is between the second and third nucleosides in the PRS of the ACT-UP1 compound. In embodiments, the PS link is between two or more nucleosides in the ASO of the ACT-UP1 compound. In embodiments, the ASO of the ACT-UP1 compound is substantially modified with an internucleotide PS link. In embodiments, the ASO of the ACT-UP1 compound comprises PS links at the 3' and 5' ends of the ASO, and a phosphate ester (PO) link in the middle of the ASO. In embodiments, the ASO of the ACT-UP1 compound comprises PS bonds between at least two, at least three, at least four, or at least five nucleosides at the 3' end of the ASO, PS bonds between at least two, at least three, at least four, or at least five nucleosides at the 5' end of the ASO, and a phosphate ester (PO) bond in the middle of the ASO. In embodiments, the PS bonds are between each nucleoside in the ASO of the ACT-UP1 compound. In a preferred embodiment, the PS bonds are between the first, second, and third nucleosides of the PRS, and between each nucleoside of the ASO. In some embodiments, the PS bonds are located between nucleosides immediately adjacent to the junction of the PRS and the ASO. In one embodiment, the PS bonds are located between at least two, at least three, at least four, or at least five nucleosides in the PRS immediately adjacent to the ASO. In another embodiment, the PS bonds are located between at least two, at least three, at least four, or at least five nucleosides in the ASO immediately adjacent to the PRS. In another embodiment, the ACT-UP1 compound comprises PS bonds at the following positions: a) between the first, second, and third nucleosides in the PRS; b) between at least 2, 3, 4, or 5 nucleosides immediately adjacent to the ASO in the PRS; c) between at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleosides immediately adjacent to the PRS in the ASO; and / or d) between at least 2, 3, 4, or 5 nucleosides starting from the 3' end of the ASO. In some embodiments, the ACT-UP1 compound includes a linker between the PRS and ASO portions of the ACT-UP1 compound.In another embodiment, the ACT-UP1 compound comprises a linker between the PRS and the ASO, and further comprises PS bonds at the following positions: a) between the first, second, and third nucleosides in the PRS; b) between at least 2, at least 3, at least 4, or at least 5 nucleosides immediately adjacent to the linker in the PRS; c) between at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 nucleosides immediately adjacent to the linker in the ASO; and d) between at least 2, at least 3, at least 4, or at least 5 nucleosides starting from the 3' end of the ASO.

[0168] In some embodiments, the ACT-UP1 compound has a length of about 17 to 45 linked nucleosides, the ASO has a length of about 12 to 25 linked nucleosides, and the PRS has a length of about 5 to 20 linked nucleosides; the ACT-UP1 compound includes at least one modified sugar (e.g., a bicyclic sugar), a 2'-O-(2-methoxyethyl) group (2'-MOE), a 2'-O-methyl group (2'-OMe), and / or a 4'-CH(CH3)-O-2'-restricted ethyl (cEt) group; the ACT-UP1 compound includes at least one modified inter-nucleoside link, such as a phosphate thioester (PS) inter-nucleoside link; and / or the ACT-UP1 compound includes at least one modified nucleobase, such as 5-methylcytidine.

[0169] In some embodiments, the ACT-UP1 compound has a length of about 22 to 35 linked nucleosides. The ACT-UP1 compound comprises an ASO component linked to a PRS component, the ASO component having a length of about 12 to 25 linked nucleosides, the PRS component comprising GGACUGGACU (SEQ ID NO: 11) or AAACUAAACU (SEQ ID NO: 13); the ACT-UP1 compound comprises at least one modified sugar (e.g., a bicyclic sugar), a 2'-O-(2-methoxyethyl) group (2'-MOE), a 2'-O-methyl group (2'-OMe), and / or a 4'-CH(CH3)-O-2'(cEt) group; the ACT-UP1 compound comprises at least one modified inter-nucleoside link, such as a phosphate thioester (PS) inter-nucleoside link; and / or the ACT-UP1 compound comprises at least one modified nucleobase, such as 5-methylcytidine. In addition, in some implementations, the ASO component hybridizes with the target mRNA, and the PRS recruits translation-related proteins, thereby enhancing protein expression with the ACT-UP1 compound.

[0170] In some implementations, the ACT-UP1 compound increases protein expression in cells by at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 150%, 200%, or 300%.

[0171] Some embodiments disclosed herein provide pharmaceutical compositions comprising the ACT-UP1 compound described herein, used alone or in combination with a pharmaceutically acceptable carrier or excipient.

[0172] Certain embodiments disclosed herein provide methods for increasing the translation of target mRNAs in cells, comprising administering an ACT-UP1 compound to cells in an amount sufficient to increase the translation of the target mRNA. The ACT-UP1 compound increases the translation level of the target mRNA by at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 150%, 200%, or 300%.

[0173] Certain embodiments disclosed herein provide a method for increasing the translation of a target mRNA in a subject, comprising administering an ACT-UP1 compound to the subject in an amount sufficient to increase the translation of the target mRNA. The ACT-UP1 compound increases the translation level of the target mRNA by at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 150%, 200%, or 300%.

[0174] Certain embodiments disclosed herein provide a method for treating haploinadenia in a subject, comprising administering an ACT-UP1 compound to the subject in an amount sufficient to treat the subject's haploinadenia. The ACT-UP1 compound increases protein expression by at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 150%, 200%, or 300% to treat the subject's haploinadenia.

[0175] In some implementations, the ACT-UP1 compound is administered to the subject subcutaneously, intrathecally, or intravenously.

[0176] Some embodiments disclosed herein provide a process or method for preparing the ACT-UP1 compound of the present invention, which includes synthesizing oligonucleotides on a solid support using phosphoramide chemicals to obtain the ACT-UP1 compound.

[0177] In one embodiment, a process for preparing the ACT-UP1 compound of the present invention is provided, wherein the process comprises the following steps: a) preparing the compound by sequentially coupling modified and / or unmodified nucleotides and / or linkers onto a conjugated or unmodified solid support via phosphoramidite oligonucleotide synthesis; b) optionally, conjugating the compound onto the solid support via phosphoramidite oligonucleotide synthesis; c) isolating the compound from the solid support and removing the solid support; d) optionally, adding the conjugate after cleavage; and e) optionally, further purifying the compound, optionally using chromatography.

[0178] Certain embodiments disclosed herein provide an ACT-UP1 compound for enhancing JAG1 expression in cells, wherein the compound comprises any of the antisense oligomeric sequences targeting JAG1 in Table 52D. Certain embodiments disclosed herein provide an ACT-UP1 compound for enhancing JAG1 expression in cells, wherein the compound comprises any of the modified antisense oligomeric sequences targeting JAG1 in Table 52C. In a preferred embodiment, the compound comprises the sequence of ATXL316 (SEQ ID NO: 111), or the chemical information and sequence of ATXL316 (SEQ ID NO: 36). In another preferred embodiment, the compound comprises the chemical structure of ATXL316:

[0179] In one embodiment, the pharmaceutical composition comprises an antisense oligomeric compound for enhancing JAG1 expression in cells, used alone or in combination with a pharmaceutically acceptable carrier and / or excipient. In a preferred embodiment, the pharmaceutical composition comprises ATXL316, used alone or in combination with a pharmaceutically acceptable carrier and / or excipient.

[0180] Some embodiments provide a process or method for enhancing JAG1 mRNA expression in cells, comprising administering to cells an antisense oligomer compound for increasing JAG1 translation, or a pharmaceutical composition comprising an antisense oligomer compound for increasing JAG1 translation, in an amount sufficient to increase JAG1 mRNA translation. In one embodiment, the antisense oligomer compound or the pharmaceutical composition thereof increases JAG1 protein translation by at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 150%, 200%, 250%, or 300%. In a preferred embodiment, the antisense oligomer compound for increasing JAG1 translation is ATXL316.

[0181] Some embodiments provide a method for treating Alagille syndrome in a subject, comprising administering an antisense oligomer or a pharmaceutical composition thereof to the subject to increase JAG1 expression to an amount sufficient to increase JAG1 levels in the subject. In a preferred embodiment, the compound or pharmaceutical composition comprises ATXL316. In some embodiments, the compound or pharmaceutical composition is administered to the subject subcutaneously, intrathecally, or intravenously.

[0182] Some embodiments provide a process for preparing ATXL316, wherein the process includes the following steps: a) preparing the compound by sequentially coupling modified and / or unmodified nucleotides and / or linkers onto a conjugated or unmodified solid support via phosphoramidite oligonucleotide synthesis; b) optionally, coupling the conjugated portion to the compound onto the solid support via phosphoramidite oligonucleotide synthesis; c) isolating the compound from the solid support and removing the solid support; and d) optionally, adding the conjugate after cleavage; and / or e) optionally, further purifying the compound, optionally using chromatography.

[0183] Certain embodiments disclosed herein provide an ACT-UP1 compound for enhancing FGF21 expression in cells, wherein the compound comprises any of the antisense oligomeric sequences targeting FGF21 in Table 52D. Certain embodiments disclosed herein provide an ACT-UP1 compound for enhancing FGF21 expression in cells, wherein the compound comprises any of the modified antisense oligomeric sequences targeting FGF21 in Table 52C. In a preferred embodiment, the compound comprises the sequence of ATXL506 (SEQ ID NO: 136), or the chemical information and sequence of ATXL506 (SEQ ID NO: 81). In another preferred embodiment, the compound comprises the chemical structure of ATXL506:

[0184] In one embodiment, the pharmaceutical composition comprises an antisense oligomeric compound for enhancing FGF21 expression in cells, used alone or in combination with a pharmaceutically acceptable carrier and / or excipient. In a preferred embodiment, the pharmaceutical composition comprises ATXL506, used alone or in combination with a pharmaceutically acceptable carrier and / or excipient.

[0185] Some embodiments provide a process or method for enhancing FGF21 mRNA expression in cells, comprising administering to cells an antisense oligomer compound for increasing FGF21 translation, or a pharmaceutical composition comprising an antisense oligomer compound for increasing FGF21 translation, in an amount sufficient to increase FGF21 mRNA translation. In one embodiment, the antisense oligomer compound or the pharmaceutical composition thereof increases FGF21 protein translation by at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 150%, 200%, 250%, or 300%. In a preferred embodiment, the antisense oligomer compound for increasing FGF21 translation is ATXL506.

[0186] Some embodiments provide a method for treating an inflammatory disease, condition, or symptom in a subject, comprising administering an antisense oligomer or a pharmaceutical composition thereof to the subject to an amount sufficient to increase FGF21 in the subject. In some embodiments, the inflammatory disease, condition, or symptom is a metabolic disease, condition, or symptom, a cardiovascular disease, condition, or symptom, and / or dyslipidemia. In some embodiments, dyslipidemia is hyperlipidemia, hypercholesterolemia, and / or hypertriglyceridemia. In some embodiments, hypertriglyceridemia is severe hypertriglyceridemia (SHTG). In some embodiments, the metabolic disease, condition, or symptom is impaired glucose tolerance (e.g., peripheral glucose intolerance), steatosis, obesity, diabetes, nonalcoholic fatty liver disease (NAFLD), and / or metabolic dysfunction-associated steatohepatitis (MASH) (formerly known as nonalcoholic-associated steatohepatitis (NASH)).

[0187] In a preferred embodiment, the compound or pharmaceutical composition comprises ATXL506. In some embodiments, the compound or pharmaceutical composition is administered subcutaneously, intrathecally, or intravenously to the subject.

[0188] Some embodiments provide a process for preparing ATXL506, wherein the process includes the following steps: a) preparing the compound by sequentially coupling modified and / or unmodified nucleotides and / or linkers onto a conjugated or unmodified solid support via phosphoramidite oligonucleotide synthesis; b) optionally, coupling the conjugated portion to the compound onto the solid support via phosphoramidite oligonucleotide synthesis; c) isolating the compound from the solid support and removing the solid support; and d) optionally, adding the conjugate after cleavage; and / or e) optionally, further purifying the compound, optionally using chromatography.

[0189] Certain embodiments disclosed herein provide an ACT-UP1 compound for enhancing HNF4A expression in cells, wherein the compound comprises any of the antisense oligomeric sequences targeting HNF4A in Table 52D. Certain embodiments disclosed herein provide an ACT-UP1 compound for enhancing HNF4A expression in cells, wherein the compound comprises any of the modified antisense oligomeric sequences targeting HNF4A in Table 52C. In a preferred embodiment, the compound comprises the sequence of ATXL546 (SEQ ID NO: 150), or the chemical information and sequence of ATXL546 (SEQ ID NO: 91). In another preferred embodiment, the compound comprises the chemical structure of ATXL546:

[0190] In a preferred embodiment, the compound comprises the sequence of ATXL547 (SEQ ID NO: 151), or the chemical information and sequence of ATXL547 (SEQ ID NO: 92). In another preferred embodiment, the compound comprises the chemical structure of ATXL547:

[0191] In one embodiment, the pharmaceutical composition comprises an antisense oligomeric compound for enhancing HNF4A expression in cells, used alone or in combination with a pharmaceutically acceptable carrier and / or excipient. In a preferred embodiment, the pharmaceutical composition comprises ATXL546, used alone or in combination with a pharmaceutically acceptable carrier and / or excipient. In a preferred embodiment, the pharmaceutical composition comprises ATXL547, used alone or in combination with a pharmaceutically acceptable carrier and / or excipient.

[0192] Some embodiments provide a process or method for enhancing HNF4A mRNA expression in cells, comprising administering to cells an antisense oligomer compound for increasing HNF4A translation, or a pharmaceutical composition comprising an antisense oligomer compound for increasing HNF4A translation, in an amount sufficient to increase HNF4A mRNA translation. In one embodiment, the antisense oligomer compound or the pharmaceutical composition thereof increases HNF4A protein translation by at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 150%, 200%, 250%, or 300%. In a preferred embodiment, the antisense oligomer compound for increasing HNF4A translation is ATXL546. In a preferred embodiment, the antisense oligomer compound for increasing HNF4A translation is ATXL547.

[0193] Certain embodiments provide methods for treating a subject with fibrosis, cirrhosis, cancer, and / or diabetes, comprising administering an antisense oligomer compound or a pharmaceutical composition thereof to the subject to an amount sufficient to increase HNF4A in the subject. In some embodiments, the fibrosis is liver fibrosis. In some embodiments, the cancer is hepatocellular carcinoma (HCC). In some embodiments, the diabetes is juvenile-onset diabetes (MODY1). Certain embodiments provide methods for treating a subject with liver disease, comprising administering an antisense oligomer compound or a pharmaceutical composition thereof to the subject to an amount sufficient to increase HNF4A in the subject. In a preferred embodiment, the compound or pharmaceutical composition comprises ATXL546. In another preferred embodiment, the compound or pharmaceutical composition comprises ATXL547. In some embodiments, the antisense oligomer compound or pharmaceutical composition is administered to the subject subcutaneously, intrathecally, or intravenously.

[0194] Some embodiments provide a process for preparing ATXL546 or ATXL547, wherein the process includes the following steps: a) preparing the compound by sequentially coupling modified and / or unmodified nucleotides and / or linkers onto a conjugated or unmodified solid support via phosphoramidite oligonucleotide synthesis; b) optionally, conjugating the compound onto the solid support via phosphoramidite oligonucleotide synthesis; c) isolating the compound from the solid support and removing the solid support; and d) optionally, adding the conjugate after cleavage; and / or e) optionally, further purifying the compound, optionally using chromatography.

[0195] Implementation Scheme 1 provides an antisense oligonucleotide (ASO)-conjugated translation-upregulation 1 (ACT-UP1) compound, wherein the ACT-UP1 compound comprises an ASO component linked to a protein recruitment sequence (PRS) component, wherein the ASO component is capable of hybridizing with a target mRNA, wherein the PRS is capable of recruiting translation-related proteins, and wherein the ACT-UP1 compound is capable of enhancing protein expression. In one embodiment, the ASO component binds directly or indirectly to the PRS.

[0196] Implementation Scheme 2 provides the ACT-UP1 compound of Implementation Scheme 1, wherein the length of the PRS is 5 to 20, 5 to 19, 5 to 18, 5 to 17, 5 to 16, 5 to 15, 5 to 14, 5 to 13, 5 to 12, 5 to 11, 5 to 10, 5 to 9, 5 to 8, 5 to 7, or 5 to 6 linked nucleosides.

[0197] Implementation Scheme 3 provides the ACT-UP1 compound of Implementation Scheme 1, wherein PRS is a derivative of the DRACH concordant sequence, or a sequence comprising (a) the DRACH concordant sequence and (b) one or more repeating or partially repeating sequences comprising the DRACH concordant sequence, wherein D is adenine (A), guanine (G), or thymine (T); R is adenine (A) or guanine (G); A is adenine (A); C is cytosine (C); and H is adenine (A), cytosine (C), or uracil (U). In one embodiment, the DRACH concordant sequence is GGACU (SEQ ID NO: 8), GAACU (SEQ ID NO: 41), AGACU (SEQ ID NO: 42), AAACU (SEQ ID NO: 43), GGACA (SEQ ID NO: 44), or AAACA (SEQ ID NO: 154), or a derivative thereof.

[0198] Embodiment 4 provides the ACT-UP1 compound of Embodiment 1, wherein PRS is an RRANN concordant sequence, or a sequence comprising (a) the RRANN concordant sequence and (b) one or more repeating or partially repeating sequences comprising the RRANN concordant sequence, wherein R is adenine (A) or guanine (G); and N is adenine (A), guanine (G), cytosine (C), thymine (T), or uracil (U). In one embodiment, the RRANN concordant sequence is GGACU (SEQ ID NO: 8), GGAUU (SEQ ID NO: 9), GAACU (SEQ ID NO: 41), AGACU (SEQ ID NO: 42), AAACU (SEQ ID NO: 43), GGACA (SEQ ID NO: 44), AAACA (SEQ ID NO: 154), or a derivative thereof. In some embodiments, the N at the 3' end of the RRANN concordant sequence is T.

[0199] Embodiment 5 provides the ACT-UP1 compound of Embodiment 1, wherein PRS is the RRAWN concordant sequence, or an additional sequence comprising (a) the RRAWN concordant sequence and (b) one or more repeating or partially repeating sequences comprising the RRANN concordant sequence, wherein R is adenine (A) or guanine (G); W is adenine (A) or cytosine (C); and N is adenine (A), guanine (G), cytosine (C), thymine (T), or uracil (U). In one embodiment, the DRACH concordant sequence is GGACU (SEQ ID NO: 8), GAACU (SEQ ID NO: 41), AGACU (SEQ ID NO: 42), AAACU (SEQ ID NO: 43), GGACA (SEQ ID NO: 44), AAACA (SEQ ID NO: 154), or a derivative thereof. In some embodiments, N in the RRAWN concordant sequence is T.

[0200] Implementation Scheme 6 provides the ACT-UP1 compound of Implementation Scheme 3, 4 or 5, wherein GGACU or a derivative thereof is selected from one of GGACU (SEQ ID NO: 8), GGACUGGAC (SEQ ID NO: 10), GGACUGGACU (SEQ ID NO: 11) and ACGGACUUGGACU (SEQ ID NO: 12).

[0201] Embodiment 7 provides the ACT-UP1 compound of Embodiment 1, wherein the PRS is a derivative of a poly(A), poly(C), poly(G), poly(T), or poly(U) sequence in the 3'UTR of the mRNA. Embodiment 7 further provides a PRS having the sequence GUGUGUGUGU (SEQ ID NO: 76) or CUCUCUCUCU (SEQ ID NO: 75).

[0202] Implementation Scheme 8 provides the ACT-UP1 compound of Implementation Scheme 7, wherein the poly(A) tail derivatives are AAACUAAACU (SEQ ID NO: 13), AAAAAAAAA (SEQ ID NO: 15), AAACAAAACA (SEQ ID NO: 99), and AAAAAAAAAAAA (SEQ ID NO: 102); the poly(C) sequence is CCCCCCCCCCC (SEQ ID NO: 93); and the poly(U) sequence is UUUUUUUUUU (SEQ ID NO: 94).

[0203] Implementation Scheme 9 provides the ACT-UP1 compound of Implementation Scheme 1, wherein PRS supports the binding of RNA-binding proteins and their associated complexes. In one embodiment, the RNA-binding protein and its associated complexes are translation-related proteins. In another embodiment, the translation-related protein is selected from the group consisting of PABPC1, YTHDF1, ALKBH5, METTL3, and METTL14, and combinations thereof.

[0204] Implementation Scheme 10 provides the ACT-UP1 compound of Implementation Scheme 1, wherein the compound comprises 17 to 45, 17 to 44, 17 to 43, 17 to 42, 17 to 41, 17 to 40, 17 to 39, 17 to 38, 17 to 37, 17 to 36, 17 to 35, 17 to 34, 17 to 33, 17 to 32, 17 to 31, 17 to 30, 17 to 29, 17 to 28, 17 to 27, 17 to 26, 17 to 25, 19 to 45, 19 to 40, 19 to 35, 19 to 34, 19 to 33, 19 to 32, 19 1 to 31, 19 to 30, 19 to 29, 19 to 28, 19 to 27, 19 to 26, 19 to 25, 22 to 45, 22 to 40, 22 to 35, 22 to 34, 22 to 33, 22 to 32, 22 to 31, 22 to 30, 22 to 29 22 to 28, 22 to 27, 22 to 26, 22 to 25, 25 to 45, 25 to 40, 25 to 35, 25 to 34, 25 to 33, 25 to 32, 25 to 31, 25 to 30, 25 to 29, 25 to 28, or 25 to 27 connected subunits.

[0205] Implementation scheme 11 provides the ACT-UP1 compound of implementation scheme 1, wherein the length of the ASO is 12 to 25, 12 to 24, 12 to 23, 12 to 22, 12 to 21, 12 to 20, 12 to 19, 12 to 18, 12 to 17, 12 to 16, 12 to 15, or 12 to 14 linked nucleosides.

[0206] Implementation Scheme 12 provides the ACT-UP1 compound of Implementation Scheme 1, wherein ASO binds to a sequence in the 3' UTR of the target mRNA. In one embodiment, ASO binds to a sequence between a stop codon and a polyadenylation signal in the 3'-UTR of the target mRNA. In one embodiment, the polyadenylation signal is AAUAAA.

[0207] Implementation Scheme 13 provides the ACT-UP1 compound of Implementation Scheme 1, wherein the ACT-UP1 compound comprises a bifunctional ASO. This bifunctional compound can enhance protein expression through two mechanisms of action: (1) ACT-UP1 recruits translating proteins to increase protein expression, and (2) blocks cis-elements in target mRNA to stabilize mRNA or prevent its degradation. In one embodiment, the cis-element is an AU-rich element (ARE) of the mRNA transcript. In one embodiment, the bifunctional ACT-UP1 compound binds to the 3'UTR of the target transcript and comprises an AU-rich element (ARE) targeting sequence, wherein the ARE targeting sequence of the ACT-UP1 compound blocks the binding of cellular proteins to the transcript ARE and inhibits target mRNA degradation. In one embodiment, inhibition of target mRNA degradation increases target mRNA and protein expression. In one embodiment, the bifunctional ASO can increase FGF21 mRNA levels and FGF21 protein expression. In one embodiment, the bifunctionality of the ACT-UP1 compound has an additive or synergistic effect in increasing the protein expression of the target gene.

[0208] Implementation scheme 14 provides the ACT-UP1 compound of implementation scheme 1, wherein the ASO sequence is not the reverse complementary sequence of the PRS sequence.

[0209] Implementation 15 provides the ACT-UP1 compound of Implementation 1, wherein PRS is not an internal ribosome entry site (IRES) sequence.

[0210] Implementation Scheme 16 provides the ACT-UP1 compound of Implementation Scheme 1, wherein the ACT-UP1 compound is a trans-acting protein enhancer. In one embodiment, the trans-acting protein enhancer recruits translation-related proteins to the target mRNA. In one embodiment, the translation-related proteins are selected from the group consisting of PABPC1, YTHDF1, ALKBH5, METTLE3, and METTL14, and combinations thereof.

[0211] Implementation scheme 17 provides the ACT-UP1 compound of implementation scheme 1, wherein the target mRNA is mammalian mRNA, plant mRNA, yeast mRNA or bacterial mRNA.

[0212] Implementation scheme 18 provides the ACT-UP1 compound of implementation scheme 17, wherein the target mRNA is mammalian JAG1, RAB9, PBGD, RNase H1, HNF4A or FGF21.

[0213] Implementation Scheme 19 provides the ACT-UP1 compound of Implementation Scheme 1, wherein the ACT-UP1 compound targets a region of approximately 20 to 50, 40 to 70, 60 to 90, 80 to 110, 100 to 130, 120 to 150, 140 to 170, 160 to 190, 180 to 210, 200 to 230, 220 to 250, 240 to 300, or 280 to 500 nucleotides downstream of a stop codon on mRNA.

[0214] Embodiment 20 provides the ACT-UP1 compound of Embodiment 1, further comprising a conjugate. In one embodiment, the conjugate may be selected from cholesterol, lipids, carbohydrates, phospholipids, biotin, phenazine, folic acid, phenanthridine, anthraquinone, acridine, fluorescein, rhodamine, coumarin, peptides, antibodies, dyes, and tocopherols. In one embodiment, the conjugate is a compound containing N-acetylgalactosamine (GalNAc).

[0215] Embodiment 21 provides the ACT-UP1 compound of Embodiment 1, wherein the compound comprises at least one chemical modification. In one embodiment, the compound is fully chemically modified. In one embodiment, ASO and PRS comprise the same chemical modification. In one embodiment, ASO and PRS comprise different chemical modifications. In one embodiment, the chemical modification may be selected from 2'-O-methyl (2'-OMe), 2'-O-(2-methoxyethyl) (2'-MOE), 2'-fluoro (2'-F), restricted ethyl (cEt), non-locked nucleic acid (UNA), locked nucleic acid (LNA), 2'-MOE-modified T, and / or 5-methylcytosine bases. In one embodiment, the PRS chemical modification is 2'-O-methyl (2'-OMe). In one embodiment, the ASO chemical modification is cEt and / or 2'-O-MOE-modified 5-methylcytidine (eCm).

[0216] Embodiment 22 provides the ACT-UP1 compound of Embodiment 1, wherein the compound comprises at least one modified internucleotide link. In one embodiment, the at least one modified internucleotide link is a phosphate thioester (PS) link.

[0217] Implementation 23 provides the ACT-UP1 compound of Implementation 1, which comprises 17 to 45 linked nucleosides, wherein the ACT-UP1 compound comprises an ASO component linked to a protein recruitment sequence (PRS) component, the ASO component comprising 12 to 25 linked nucleosides, and the PRS component comprising 5 to 20 linked nucleosides.

[0218] Implementation 24 provides the ACT-UP1 compound of Implementation 1, which comprises 22 to 35 linked nucleosides, wherein the ACT-UP1 compound comprises an ASO component linked to a protein recruitment sequence (PRS) component, the ASO component comprising 12 to 25 linked nucleosides, the PRS component comprising the sequence GGACUGGACU (SEQ ID NO: 11) or the sequence AAACUAAACU (SEQ ID NO: 13).

[0219] Implementation 25 provides any of the aforementioned implementations of the ACT-UP1 compound, wherein the compound increases protein expression by at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 150%, 200%, 250%, or 300%.

[0220] Embodiment 26 provides a pharmaceutical composition comprising any of the ACT-UP1 compounds of the foregoing embodiments, used alone or in combination with a pharmaceutically acceptable carrier and / or excipients.

[0221] Implementation Scheme 27 provides a method for increasing the translation of target mRNA in cells, comprising administering to cells an ACT-UP1 compound of any one of Implementation Schemes 1 to 25, or a pharmaceutical composition of Implementation Scheme 26, in an amount sufficient to increase the translation of target mRNA. In one embodiment, the ACT-UP1 compound or pharmaceutical composition may be administered to a subject subcutaneously, intrathecally, or intravenously.

[0222] Embodiment 28 provides a method for treating a subject with haploinadenia, comprising administering to the subject the ACT-UP1 compound of any one of claims 1 to 25, or the pharmaceutical composition of claim 26, in an amount sufficient to treat the subject's haploinadenia. In one embodiment, the ACT-UP1 compound or pharmaceutical composition may be administered to the subject subcutaneously, intrathecally, or intravenously.

[0223] Implementation Scheme 29 provides a method for preparing the compound of any of the foregoing embodiments, wherein the method comprises the following steps: a) The compound was prepared by sequentially coupling modified and / or unmodified nucleotides and / or linkers onto a conjugated or unmodified solid support via phosphoramide oligonucleotide synthesis. b) Optionally, a compound whose conjugated portion is coupled to a solid support by means of phosphoramidoid oligonucleotide synthesis; c) Separate the compound from the solid support and remove the solid support; and d) Optionally, add conjugates after cutting.

[0224] e) Optionally, the compound may be further purified, optionally using chromatography.

[0225] Antisense compounds Antisense compounds include antisense oligonucleotides (ASOs) and compounds containing ASOs, such as ASO-conjugated translation-upregulation 1 (ACT-UP1) compounds. An antisense compound is "antense" to the target nucleic acid, meaning it can hybridize with the target nucleic acid via hydrogen bonding.

[0226] In some embodiments, the antisense oligonucleotide comprises a nucleobase sequence that, when written in a 5' to 3' orientation, contains the inverse complementary sequence of the target segment of the target nucleic acid it targets. In some such embodiments, the antisense oligonucleotide has a nucleobase sequence that, when written in a 5' to 3' orientation, contains the inverse complementary sequence of the target segment of the target nucleic acid it targets.

[0227] In some embodiments, the antisense compound comprises an ACT-UP1 compound, which in turn comprises an antisense oligonucleotide (ASO) component and a protein recruitment sequence (PRS) component.

[0228] In some embodiments, the antisense compound is about 17 to 45 subunits in length. In some embodiments, the antisense compound comprises an ACT-UP1 compound, which in turn comprises an antisense oligonucleotide of about 12 to 25 subunits in length and a protein recruitment sequence of about 5 to 20 subunits in length.

[0229] In some embodiments, the antisense compound is about 17 to 45 subunits in length. In some embodiments, the antisense compound comprises an ACT-UP1 compound, which in turn comprises an antisense oligonucleotide of about 12 to 25 subunits in length and a protein recruitment sequence of about 5 to 19 subunits in length.

[0230] In some embodiments, the antisense compound is about 17 to 45 subunits in length. In some embodiments, the antisense compound comprises an ACT-UP1 compound, which in turn comprises an antisense oligonucleotide of about 12 to 25 subunits in length and a protein recruitment sequence of about 5 to 18 subunits in length.

[0231] In some embodiments, the antisense compound is about 17 to 45 subunits in length. In some embodiments, the antisense compound comprises an ACT-UP1 compound, which in turn comprises an antisense oligonucleotide of about 12 to 25 subunits in length and a protein recruitment sequence of about 5 to 17 subunits in length.

[0232] In some embodiments, the antisense compound is about 17 to 45 subunits in length. In some embodiments, the antisense compound comprises an ACT-UP1 compound, which in turn comprises an antisense oligonucleotide of about 12 to 25 subunits in length and a protein recruitment sequence of about 5 to 16 subunits in length.

[0233] In some embodiments, the antisense compound is about 17 to 45 subunits in length. In some embodiments, the antisense compound comprises an ACT-UP1 compound, which in turn comprises an antisense oligonucleotide of about 12 to 25 subunits in length and a protein recruitment sequence of about 5 to 15 subunits in length.

[0234] In some embodiments, the antisense compound is about 17 to 45 subunits in length. In some embodiments, the antisense compound comprises an ACT-UP1 compound, which in turn comprises an antisense oligonucleotide of about 12 to 25 subunits in length and a protein recruitment sequence of about 5 to 14 subunits in length.

[0235] In some embodiments, the antisense compound is about 17 to 45 subunits in length. In some embodiments, the antisense compound comprises an ACT-UP1 compound, which in turn comprises an antisense oligonucleotide of about 12 to 25 subunits in length and a protein recruitment sequence of about 5 to 13 subunits in length.

[0236] In some embodiments, the antisense compound is about 17 to 45 subunits in length. In some embodiments, the antisense compound comprises an ACT-UP1 compound, which in turn comprises an antisense oligonucleotide of about 12 to 25 subunits in length and a protein recruitment sequence of about 5 to 12 subunits in length.

[0237] In some embodiments, the antisense compound is about 17 to 45 subunits in length. In some embodiments, the antisense compound comprises an ACT-UP1 compound, which in turn comprises an antisense oligonucleotide of about 12 to 25 subunits in length and a protein recruitment sequence of about 5 to 11 subunits in length.

[0238] In some embodiments, the antisense compound is about 17 to 45 subunits in length. In some embodiments, the antisense compound comprises an ACT-UP1 compound, which in turn comprises an antisense oligonucleotide of about 12 to 25 subunits in length and a protein recruitment sequence of about 5 to 10 subunits in length.

[0239] In some embodiments, the antisense compound is about 17 to 45 subunits in length. In some embodiments, the antisense compound comprises an ACT-UP1 compound, which in turn comprises an antisense oligonucleotide of about 12 to 25 subunits in length and a protein recruitment sequence of about 5 to 9 subunits in length.

[0240] In some embodiments, the antisense compound is about 17 to 45 subunits in length. In some embodiments, the antisense compound comprises an ACT-UP1 compound, which in turn comprises an antisense oligonucleotide of about 12 to 25 subunits in length and a protein recruitment sequence of about 5 to 8 subunits in length.

[0241] In some embodiments, the antisense compound is about 17 to 45 subunits in length. In some embodiments, the antisense compound comprises an ACT-UP1 compound, which in turn comprises an antisense oligonucleotide of about 12 to 25 subunits in length and a protein recruitment sequence of about 5 to 7 subunits in length.

[0242] In some embodiments, the antisense compound is about 17 to 45 subunits in length. In some embodiments, the antisense compound comprises an ACT-UP1 compound, which in turn comprises an antisense oligonucleotide of about 12 to 25 subunits in length and a protein recruitment sequence of about 5 or 6 subunits in length.

[0243] In other embodiments, the antisense compound has about 17 to 45, 17 to 44, 17 to 43, 17 to 42, 17 to 41, 17 to 40, 17 to 39, 17 to 38, 17 to 37, 17 to 36, 17 to 35, 17 to 34, 17 to 33, 17 to 32, 17 to 31, 17 to 30, 17 to 29, 17 to 28, 17 to 27, 17 to 26, 17 to 25, 19 to 45, 19 to 40, 19 to 35, 19 to 34, 19 to 33, 19 to 32, 19 to 31, 19 1 to 30, 19 to 29, 19 to 28, 19 to 27, 19 to 26, 19 to 25, 22 to 45, 22 to 40, 22 to 35, 22 to 34, 22 to 33, 22 to 32, 22 to 31, 22 to 30, 22 to 29, 22 to 28, 22 to 27, 22 to 26, 22 to 25, 25 to 45, 25 to 40, 25 to 35, 25 to 34, 25 to 33, 25 to 32, 25 to 31, 25 to 30, 25 to 29, 25 to 28, or 25 to 27 connected subunits. In some such embodiments, the length of the antisense compound is about 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 or 45 connected subunits, or a range defined by any two of the above values.

[0244] In some embodiments, the antisense oligonucleotide (ASO) has a length of about 12 to 25, 12 to 24, 12 to 23, 12 to 22, 12 to 21, 12 to 20, 12 to 19, 12 to 18, 12 to 17, 12 to 16, 12 to 15, or 12 to 14 linked subunits. In some embodiments, the antisense oligonucleotide has a length of about 13 to 25, 13 to 24, 13 to 23, 13 to 22, 13 to 21, 13 to 20, 13 to 19, 13 to 18, 13 to 17, 13 to 16, 13 to 15, or 13 to 14 linked subunits. In some embodiments, the antisense oligonucleotide has a length of about 14 to 25, 14 to 24, 14 to 23, 14 to 22, 14 to 21, 14 to 20, 14 to 19, 14 to 18, 14 to 17, 14 to 16, or 14 to 15 linked subunits. In some embodiments, the antisense oligonucleotide has a length of about 15 to 25, 15 to 24, 15 to 23, 15 to 22, 15 to 21, 15 to 20, 15 to 19, 15 to 18, 15 to 17, or 15 to 16 linked subunits. In some embodiments, the antisense oligonucleotide has a length of about 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 linked subunits, or a range defined by any two of the above values.

[0245] In some embodiments, the length of the protein recruitment sequence (PRS) is about 5 to 20, 5 to 19, 5 to 18, 5 to 17, 5 to 16, 5 to 15, 5 to 14, 5 to 13, 5 to 12, 5 to 11, 5 to 10, 5 to 9, 5 to 8, 5 to 7, or 5 to 6 linked subunits. In some embodiments, the length of the protein recruitment sequence is about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 linked subunits, or a range defined by any two of the above values.

[0246] The length of the antisense compound (e.g., an antisense oligonucleotide) may be increased or decreased, and / or base mismatches with the target may be introduced without eliminating activity (U.S. Patent 7,772,203, incorporated herein by reference). For example, non-canonical base pairs (e.g., A:G, A:C, G:U, I:U, I:A, or I:C) may be introduced into the antisense oligonucleotide without eliminating activity. In some embodiments, antisense oligonucleotides are designed to have one or more non-canonical base pairs (i.e., mismatches) to enhance the activity of the antisense compound.

[0247] Antisense oligonucleotide motif A motif refers to a modification pattern of an antisense oligonucleotide. Various motifs have been described in the art and are incorporated herein by reference (e.g., U.S. Patent 11,203,755; U.S. Patent 10,870,849; European Patent 1,532,248; U.S. Patent 11,406,716; U.S. Patent 10,668,170; U.S. Patent 9,796,974; U.S. Patent 8,754,201; U.S. Patent 10,837,013; U.S. Patent 7,732,593; U.S. Patent 7,015,315; U.S. Patent 7,750,144; U.S. Patent 8,420,799; U.S. Patent 8,809,516; U.S. Patent 8,796,436). U.S. Patents 8,859,749, 9,708,615, 10,233,448, 10,273,477, 10,612,024, 10,612,027, 10,669,544, 11,401,517, 9,260,471, 9,970,005, 11,193,126, 8,604,183, 9,150,605, 9,708,610, USSN 2020 / 0031862, and USSN 2016 / 0272970.

[0248] In some embodiments, the antisense oligonucleotides disclosed herein have chemically modified subunits that are arranged in motifs or patterns (i.e., chemically modified motifs / patterns) to impart beneficial properties to the antisense oligonucleotides, including but not limited to: enhancing activity to improve potency; increasing binding affinity to increase specificity for target nucleic acids, thereby limiting off-target effects and improving safety; or enhancing resistance to degradation by nucleases in vivo, thereby improving stability and durability.

[0249] Target mRNA and related gene expression Several implementation schemes involve methods for upregulating target mRNA and / or upregulating target gene expression using ACT-UP1 compounds.

[0250] In some embodiments, the target mRNA is a transcript of JAG1. In some embodiments, the JAG1 mRNA sequence is SEQ ID NO: 1 (GENBANK accession number NM_000214.3), or a sequence containing at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with it.

[0251] In some embodiments, the target mRNA is a transcript of RAB9A. In some embodiments, the RAB9A mRNA sequence is SEQ ID NO: 2 (GENBANK accession number NM_004251.5), or contains a sequence with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity. In some embodiments, the target mRNA is a transcript of RNase H1. In some embodiments, the RNase H1 mRNA sequence is SEQ ID NO: 3 (GENBANK accession number NM_001286834.3), or contains a sequence with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity.

[0252] In some embodiments, the target mRNA is a transcript of PBGD. In some embodiments, the PBGD mRNA sequence is SEQ ID NO: 4 (GENBANK accession number NM_000190.4), or a sequence containing at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with it.

[0253] In some embodiments, the target mRNA is a transcript of FGF21. In some embodiments, the FGF21 mRNA sequence is SEQ ID NO: 5 (GENBANK accession number NM_019113.4), or contains a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with it.

[0254] In some embodiments, the target mRNA is a transcript of HNF4A. In some embodiments, the HNF4A mRNA sequence is SEQ ID NO: 45 (GENBANK accession number NM_178849.3), or contains a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with it.

[0255] In some embodiments, the target mRNA is a transcript of klotho (KL). In some embodiments, the KL mRNA sequence is SEQ ID NO: 46 (GENBANK accession number NM_004795.4), or a sequence containing at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with it.

[0256] In some embodiments, the target mRNA is a transcript of OPA1. In some embodiments, the OPA1 mRNA sequence is SEQ ID NO: 47 (GENBANK accession number NM_015560.3), or contains a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with it.

[0257] In some embodiments, the target mRNA is a transcript of PKD1. In some embodiments, the PKD1 mRNA sequence is SEQ ID NO: 48 (GENBANK accession number NM_001009944.3), or a sequence containing at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with it.

[0258] In some embodiments, the target mRNA is a transcript of PKD2. In some embodiments, the PKD2 mRNA sequence is SEQ ID NO: 49 (GENBANK accession number NM_000297.4), or contains a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with it.

[0259] In some embodiments, the target mRNA is a transcript of HBB. In some embodiments, the HBB mRNA sequence is SEQ ID NO: 50 (GENBANK accession number NM_000518.5), or a sequence containing at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with it.

[0260] In some embodiments, the target mRNA is a transcript of GRN. In some embodiments, the GRN mRNA sequence is SEQ ID NO: 51 (GENBANK accession number NM_002087.4), or a sequence containing at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with it.

[0261] In some embodiments, the target mRNA is a transcript of SCN1A. In some embodiments, the SCN1A mRNA sequence is SEQ ID NO: 52 (GENBANK accession number NM_001165963.4), or contains a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with it.

[0262] In some embodiments, the ACT-UP1 compound of the present invention targets a sequence of no more than about 500 nucleotides, about 300 nucleotides, about 280 nucleotides, about 250 nucleotides, about 240 nucleotides, about 210 nucleotides, about 200 nucleotides, about 190 nucleotides, about 180 nucleotides, about 170 nucleotides, about 160 nucleotides, about 150 nucleotides, about 140 nucleotides, about 130 nucleotides, about 120 nucleotides, about 110 nucleotides, about 100 nucleotides, about 95 nucleotides, about 90 nucleotides, about 80 nucleotides, about 70 nucleotides, about 60 nucleotides, about 50 nucleotides, or about 40 nucleotides downstream of a stop codon on an mRNA transcript. In some embodiments, the ACT-UP1 compound targets a region on the mRNA transcript downstream of a stop codon consisting of approximately 20 to approximately 50 nucleotides, approximately 40 to approximately 70 nucleotides, approximately 60 to approximately 90 nucleotides, approximately 80 to approximately 110 nucleotides, approximately 100 to approximately 130 nucleotides, approximately 120 to approximately 150 nucleotides, approximately 140 to approximately 170 nucleotides, approximately 160 to approximately 190 nucleotides, approximately 70 to approximately 240 nucleotides, or approximately 180 to approximately 210 nucleotides. In some embodiments, the ACT-UP1 compound targets a sequence on the mRNA transcript downstream of a stop codon consisting of approximately 50, approximately 70, approximately 80, approximately 90, approximately 100, approximately 150, approximately 160, or approximately 190 nucleotides. In some implementations, the ACT-UP1 compound targets a region on the mRNA transcript downstream of a stop codon for approximately 20 to 50, 40 to 70, 60 to 90, 80 to 110, 100 to 130, 120 to 150, 140 to 170, 160 to 190, 180 to 210, 200 to 230, 220 to 250, 240 to 300, or 280 to 500 nucleotides. In some embodiments, the target region on the mRNA is approximately 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides long. In some embodiments, the target region on the mRNA is located in the 3' UTR. In some embodiments, the target region on the mRNA is the 3' UTR, rather than a poly(A) tail.

[0263] In some preferred embodiments of the invention, the ACT-UP1 compound of the invention targets a sequence on the mRNA transcript not more than about 240 nucleotides, about 210 nucleotides, about 200 nucleotides, about 190 nucleotides, about 180 nucleotides, about 170 nucleotides, about 160 nucleotides, about 150 nucleotides, about 140 nucleotides, about 130 nucleotides, about 120 nucleotides, about 110 nucleotides, about 100 nucleotides, about 95 nucleotides, about 90 nucleotides, about 80 nucleotides, about 70 nucleotides, about 60 nucleotides, about 50 nucleotides, or about 40 nucleotides downstream of a stop codon. In a preferred embodiment, the target sequence is located about 50 to about 240 nucleotides downstream of a stop codon on the mRNA transcript. In another preferred embodiment, the target sequence is located about 70 to about 240 nucleotides downstream of a stop codon on the mRNA transcript. In yet another preferred embodiment, the target sequence is located approximately 95 to approximately 240 nucleotides downstream of the stop codon on the mRNA transcript. In a further preferred embodiment, the target sequence is located approximately 140 to approximately 240 nucleotides downstream of the stop codon on the mRNA transcript.

[0264] In some embodiments, the target mRNA is present in eukaryotic or prokaryotic cells. In some embodiments, the target protein is expressed in eukaryotic or prokaryotic cells. In some embodiments, the eukaryotic or prokaryotic cells are mammalian cells, plant cells, yeast cells, or bacterial cells. In some embodiments, the mammalian cells include cells derived from rodent mammals (e.g., mice and hamsters) and lagomorph mammals (e.g., rabbits), carnivorous mammals (including felines (cats) and canines (dogs)), even-toed ungulates (including bovines (cattle) and suidae (pigs)), or perissodactyl mammals (including equines (horses)). In some aspects, these mammals belong to primates, ceboids, or simoids (monkeys), or to apes (i.e., humans and apes). In a preferred aspect, the mammalian cells are human cells. In some embodiments, the cells are in the form of cultured cell lines. In some embodiments, the cell lines are primary cell lines.

[0265] Hybridization In some embodiments, hybridization occurs between the antisense compound disclosed herein and the mRNA. The most common hybridization mechanism involves hydrogen bonding between complementary bases of nucleic acid molecules (e.g., Watson-Crick, Hoogsteen, or reverse Hoogsteen hydrogen bonding).

[0266] In Watson-Crick canonical base pairing, adenine (A) is complementary to thymine (T) in DNA; adenine (A) is complementary to uracil (U) in RNA; and guanine (G) is complementary to cytosine (C) in both DNA and RNA. Base pairs or complementary nucleobases are typically Watson-Crick base pairs (e.g., C:G, A:U, or A:T), but non-canonical base pairs, such as Hoogsteen base pairs (e.g., A:G or A:U), wobbling base pairs (e.g., G:U, I:U, I:A, or I:C, where I is hypoxanthine), are also permitted during hybridization of antisense compounds with target nucleic acids or target regions. Wobbling base pairs in RNAi reagents have been previously described (see, for example, U.S. Patent 7,732,593 and U.S. Patent 7,750,144).

[0267] Nucleobase complementarity facilitates hybridization of the antisense compounds described herein with their target nucleic acids, where stronger pairing (e.g., more base pairs and / or stronger hydrogen bonds) results in stronger hybridization between the antisense compound and the target. Hybridization can occur under various conditions. Strict conditions are sequence-dependent and determined by the properties and composition of the antisense compound to be hybridized.

[0268] Methods for determining whether a sequence can specifically hybridize with a target nucleic acid are well known in the art. In some embodiments, the antisense compounds provided herein can specifically hybridize with the target mRNA with little to no off-target binding.

[0269] Complementarity When a sufficient number of nucleobases in an antisense oligonucleotide can bind hydrogen to the corresponding nucleobases in the target nucleic acid, thereby producing the desired effect (e.g., ACT-UP1 upregulates the target nucleic acid, such as mRNA), the antisense compound containing the antisense oligonucleotide is "complementary" to the target nucleic acid, is a "complementary" to the target nucleic acid, or has "complementarity" with the target nucleic acid.

[0270] Non-complementary nucleotide bases between antisense oligonucleotides and mRNA are tolerable, provided that the antisense oligonucleotide can still specifically hybridize with the target nucleic acid. Furthermore, antisense oligonucleotides can hybridize with one or more segments of mRNA, such that intermediate or adjacent segments do not participate in the hybridization event (e.g., loop structures, mismatches, or hairpin structures).

[0271] In some embodiments, for an antisense compound containing an antisense oligonucleotide, the antisense oligonucleotide moiety has at least about 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% complementarity with the mRNA, target region, target segment, or a specific portion thereof. The percentage of complementarity between the antisense oligonucleotide and the target nucleic acid can be determined using conventional methods.

[0272] For example, in an antisense compound, approximately 18 of the 20 nucleotides of the antisense oligonucleotide are complementary to the target region and thus hybridize specifically with the target, representing 90% complementarity. In this example, the remaining non-complementary nucleotides of the antisense oligonucleotide can be clustered or interspersed with complementary nucleotides, and do not need to be adjacent to each other or to complementary nucleotides. Therefore, an antisense oligonucleotide of 18 nucleotides in length with four non-complementary nucleotides (flanked by two regions completely complementary to the target nucleic acid) will have an overall complementarity of 77.8% with the target nucleic acid, thus falling within the scope of this invention. The percentage of complementarity between the antisense oligonucleotide and the target nucleic acid region can be routinely determined using the BLAST (Basic Local Alignment Search) and PowerBLAST programs known in the art (Altschul). et al ., J.Mol.Biol ., 1990, 215, 403 410; Zhang and Madden, Genome Res., 1997, 7, 649656. For example, the percentage of homology, sequence identity, or complementarity can be determined using the Gap program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, Madison Wis.) with default settings, which uses the algorithm of Smith and Waterman (Adv. Appl. Math., 1981, 2, 482 489).

[0273] In some embodiments, the antisense oligonucleotides or specific portions thereof provided herein are completely complementary (i.e., 100% complementary) to the target nucleic acid or specific portions thereof. For example, the antisense oligonucleotide may be completely complementary to mRNA, or a target region, or a target segment, or its target sequence. As used herein, “completely complementary” means that each nucleobase of the antisense oligonucleotide is capable of precise base pairing with the corresponding nucleobase of the target nucleic acid. For example, if a 20-nucleobase portion of the target nucleic acid is completely complementary to the antisense oligonucleotide, then the 20-nucleobase antisense oligonucleotide is completely complementary to a 400-nucleobase-long target sequence. Complete complementarity can also be used to refer to specific portions of the first and / or second nucleic acids. For example, a 20-nucleobase portion of a 30-nucleobase antisense oligonucleotide may be “completely complementary” to a 400-nucleobase-long target sequence. If the target sequence has a corresponding 20-base portion, where each nucleotide is complementary to the 20-base portion of the antisense oligonucleotide, then the 20-base portion of the 30-base oligonucleotide is perfectly complementary to the target sequence. Simultaneously, the entire 30-base antisense oligonucleotide may or may not be perfectly complementary to the target sequence, depending on whether the remaining 10 nucleotides of the antisense oligonucleotide are also complementary to the target sequence.

[0274] The non-complementary nucleobases can be located at the 5' or 3' end of the antisense oligonucleotide. Alternatively, one or more non-complementary nucleobases can be located inside the antisense oligonucleotide. When two or more non-complementary nucleobases are present, they can be continuous (i.e., linked) or discontinuous. In one embodiment, the non-complementary nucleobases are located on the wing of the nicked antisense oligonucleotide.

[0275] In some implementations, antisense oligonucleotides of about 14, 15, 16, 17, 18, 19 or 20 nucleotides in length relative to the target nucleic acid (e.g., mRNA or a specific portion thereof) contain no more than 4, no more than 3, no more than 2 or no more than one (1) non-complementary nucleotides.

[0276] In some implementations, antisense oligonucleotides with a length of about 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides relative to the target nucleic acid (e.g., mRNA or a specific portion thereof) contain no more than 6, 5, 4, 3, 2, or 1 non-complementary nucleotides.

[0277] The provided antisense oligonucleotides also include those that are partially complementary to the target nucleic acid. As used herein, "partial" refers to a defined number of consecutive (i.e., linked) nucleotides within a region or segment of the target nucleic acid. "Partial" can also refer to a defined number of consecutive nucleotides in the antisense oligonucleotide. In some embodiments, the antisense oligonucleotide is complementary to at least 10 nucleotides of the target segment. In some embodiments, the antisense oligonucleotide is complementary to at least 11 nucleotides of the target segment. In some embodiments, the antisense oligonucleotide is complementary to at least 12 nucleotides of the target segment. In some embodiments, the antisense oligonucleotide is complementary to at least 13 nucleotides of the target segment. In some embodiments, the antisense oligonucleotide is complementary to at least 14 nucleotides of the target segment. In some embodiments, the antisense oligonucleotide is complementary to at least 15 nucleotides of the target segment. Antisense oligonucleotides were also envisioned that are complementary to at least about 12, 13, 14, 15, 16, 17, 18, 19, 20 or more nucleobase portions of the target segment (or a range defined by any two of these values).

[0278] identity The antisense oligonucleotides provided herein may also have a defined percentage of identity with a specific nucleotide sequence, SEQ ID NO, or a compound or portion thereof represented by a specific identifier. As used herein, an antisense oligonucleotide is considered identical to the sequence disclosed herein if it has the same nucleobase pairing ability. For example, in a disclosed DNA sequence, RNA containing uracil instead of thymidine would be considered identical to that DNA sequence because both uracil and thymidine pair with adenine. Truncated and extended versions of the antisense oligonucleotides described herein, as well as oligonucleotides with different bases relative to the antisense oligonucleotides provided herein, are also contemplated. Dissimilar bases may be adjacent to each other or scattered throughout the antisense oligonucleotide. The percentage of identity of the antisense oligonucleotide is calculated based on the number of bases with the same base pairing ability relative to the compared sequence.

[0279] In some embodiments, the antisense oligonucleotide or a portion thereof has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with one or more of the antisense oligonucleotides or SEQ IDNOs or portions thereof disclosed herein.

[0280] In some embodiments, a portion of the antisense oligonucleotide is compared to an equal-length portion of the target nucleic acid. In some embodiments, approximately 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides are compared to an equal-length portion of the target nucleic acid.

[0281] Chemical modification Nucleosides are base-sugar combinations. The nucleobase (also called the base) portion of a nucleoside is typically a heterocyclic base portion. A nucleotide is a nucleoside that further comprises a covalently linked (e.g., a phosphate ester group, or a chemically modified linker as described below) linking to the sugar portion of the nucleoside. Oligonucleotides are formed by the covalent linking of adjacent nucleotides to each other to form linear polymeric oligonucleotides. Within the oligonucleotide structure, the linking groups are generally referred to as the internucleotide links that form the oligonucleotide.

[0282] Antisense compounds include antisense oligonucleotides (ASOs) and compounds containing ASOs, such as ASO-coupled translation-upregulation 1 (ACT-UP1) compounds.

[0283] Modification of antisense compounds encompasses substitution or alteration of nucleobases, internucleotide bonds, or sugar moieties. Modified antisense compounds are generally preferred over their native or unmodified forms because they possess desired properties, such as improved delivery (e.g., increased cellular uptake), enhanced specificity or affinity for nucleic acid targets, improved stability in the presence of nucleases, enhanced safety (e.g., fewer side effects after administration of the compound to a subject), or increased efficacy (e.g., increased activity).

[0284] Nucleoside interbond modification Naturally occurring internucleotide bonds in RNA and DNA are 3' to 5' phosphodiester linkages. For nucleosides including pentose furans, the phosphate ester group can be linked to the 2', 3', or 5' hydroxyl portion of the sugar. Oligomers with one or more modified (i.e., non-naturally occurring) internucleotide bonds are generally preferred over oligomers with naturally occurring internucleotide bonds because they offer desired properties, such as enhanced cellular uptake, increased affinity for target nucleic acids, reduced toxicity, increased stability and persistence, reduced degradation, and other desired characteristics of oligomers. Modified internucleotide bonds and their advantages are well known in the art (Crooke, ST, et al., 2021a Antisense technology: an overview and prospectus. Nat Rev Drug Discov 20: 427-453; Crooke, ST, et al., 2021b Antisense technology: A review. J Biol Chem 296: 100416).

[0285] Oligomeric compounds with modified nucleoside internucleotide bonds include those retaining phosphorus atoms and those without phosphorus atoms. Typical phosphorus-containing nucleoside internucleotide bonds include, but are not limited to, phosphate diesters, phosphate triesters, methylphosphonates (e.g., 5'-methylphosphonate (5'-MP)), phosphoramide esters, thiophosphate esters (e.g., dithiophosphate Rp isomers (PS, Rp), dithiophosphate Rp isomers (PS, Sp), or 5'-thiophosphate (5'-PS)), methoxypropylphosphonates, and phosphate-containing (… S 5'-5'-C-methyl and 5'-(E)-vinylphosphonate. In some respects, the nucleoside-to-nucleotide (N-nucleotide) bonds can be replaced by peptide-nucleotide (PNA) bonds.

[0286] In some embodiments, the oligomeric compound targeting nucleic acids comprises one or more modified internucleotide links. In some embodiments, the modified internucleotide links are phosphate thioester (PS) links. In some embodiments, one or more internucleotide links in the oligomeric compound are phosphate thioester internucleotide links. In some embodiments, each internucleotide link in the oligomeric compound is a phosphate thioester internucleotide link. In some embodiments, each internucleotide link in the oligonucleotide is a phosphate thioester internucleotide link.

[0287] Sugar modification The oligomeric compounds described herein may contain one or more nucleosides in which the sugar groups have been modified. Such sugar-modified nucleosides can endow oligomeric compounds with desired characteristics, such as increased stability, increased persistence (e.g., prolonged half-life), increased binding affinity, reduced off-target effects, reduced immunogenicity, reduced toxicity, increased potency, or other beneficial biological properties. Sugar modification and its advantages are known in the art (Faria, M., and H. Ulrich, 2008 Sugarboost: when ribose modifications improve oligonucleotide performance. CurrOpin Mol Ther 10: 168-175; Crooke, ST, et al., 2021b Antisense technology: A review. J Biol Chem 296: 100416; Egli, M., and M. Manoharan, 2023 Chemistry, structure and function of approved oligonucleotide therapeutics. Nucleic Acids Res 51: 2529-2573).

[0288] In some embodiments, the nucleoside comprises a chemically modified ribofuran ring moiety. Examples of chemically modified ribofuran rings may include, but are not limited to, the addition of substituents (e.g., 5' sugar modification or 2' sugar modification); bridging non-isositu ring atoms to form bicyclic nucleic acids (BNAs); and replacing the ribose epoxide atom with S, N(R), or C(R1)(R)2 (where R=H, C1-C). 12 Alkyl groups or protecting groups); nucleoside analogs; and combinations thereof.

[0289] Examples of chemically modified sugars include 2'-F-5'-methyl-substituted nucleosides (see, for example, PCT Publication WO2008101157 for other disclosed 5',2'-disubstituted nucleosides), ribose epoxide atoms replaced with S, further substituted at the 2'-position (see, for example, US Publication US20050130923), or, optionally, 5'-substituted BNA (see, PCT Publication WO2007134181, wherein LNA is substituted with, for example, a 5'-methyl or 5'-vinyl group).

[0290] 2'-Modified sugars refer to furanoses modified at the 2' position. 2'-Modified nucleosides refer to nucleosides containing a sugar with a 2'-position modification of the furanose ring. In some embodiments, such modifications include substituents selected from: halides, including but not limited to substituted and unsubstituted alkoxy groups, substituted and unsubstituted thioalkyl groups, substituted and unsubstituted aminoalkyl groups, substituted and unsubstituted alkyl groups, substituted and unsubstituted allyl groups, and substituted and unsubstituted alkynyl groups. In some embodiments, the 2' modification is selected from substituents including but not limited to: O[(CH2)] n O] m CH3, O(CH2) n NH2, O(CH2) n CH3, O(CH2) n ONH2, OCH2C(=O)N(H)CH3 and O(CH2) n ON[(CH2) n [CH3]2, where n and m are 1 to approximately 10. Other 2'-substituent groups may also be selected from: C1-C 12Alkyl, substituted alkyl, alkenyl, alkynyl, aryl, aralkyl, O-alkaneary or O-aralkyl, SH, SCH3, OCN, Cl, Br, CN, CF3, OCF3, SOCH3, SO2CH3, ONO2, NO2, N3, NH2, heterocyclic alkyl, heterocyclic aryl, aminoalkylamino, polyalkylamino, substituted silyl, RNA cleaving group, reporter group, intercalator, group used to improve pharmacokinetic properties and group used to improve the pharmacodynamic properties of oligomeric compounds, and other substituents with similar properties.

[0291] Other examples of nucleosides with modified sugar moieties include, but are not limited to, nucleosides comprising: 5'-vinyl, 5'-methyl (R or S), 2'-F-5'-methyl, 4'-S, 2'-deoxy-2'-fluoro (2'-F), 2'-OCH3 (2'-O-methyl, 2'-OMe), 2'-O(CH2)2OCH3 (2'-O-(2-methoxyethyl), 2'-O-MOE, 2'-MOE), 2'-O-methyl-4-pyridine, phosphoryldiamine morpholino (PMO), tricyclic DNA (tcDNA), 2'-arabino-fluorine, 2'-O-benzyl, glycol nucleic acid (GNA), and / or nonlocked nucleic acid (UNA) substituents. The substituent at the 2' position may also be selected from allyl, amino, azide, thio, O-allyl, O-Cl-C 10 Alkyl, OCF3, O(CH2)2SCH3, O(CH2)2-ON(Rm)(Rn) and O-CH2-C(=O)-N(Rm)(Rn), wherein each Rm and Rn is independently H, or substituted or unsubstituted C1-C 10 Alkyl. 2'-OMe, 2'-OCH3, or 2'-O-methyl each refer to a nucleoside containing a sugar with a -OCH3 group at the 2' position of the sugar ring. 2'-F refers to a sugar containing a fluorine group at the 2' position. 2'-O-(2-methoxyethyl), 2'-O-MOE, or 2'-MOE each refer to a nucleoside containing a sugar with a -O(CH2)2OCH3 group at the 2' position of the sugar ring.

[0292] BNA refers to a modified nucleoside comprising a bicyclic sugar moiety, wherein a bridging link connecting two carbon atoms on the sugar ring connects the 2' carbon atom on the sugar ring to another carbon atom. Examples of bicyclic nucleosides include, but are not limited to, nucleosides comprising a bridging link between 4' and 2' ribose ring atoms, such as nucleosides in locked nucleic acids (LNAs). In some embodiments, the oligomeric compounds provided herein comprise one or more bicyclic nucleosides, wherein the bridging link comprises a 4' to a 2' bicyclic nucleoside. Campbell and Wengel (Chem SocRev, 2011, 40(12):5680-9) have described LNAs and UNAs, which are incorporated herein by reference.

[0293] In some embodiments, the oligomeric compound comprises one or more nucleotides having a modified sugar moiety. In some embodiments, the modified sugar moiety is 2'-MOE. In some embodiments, the modified sugar moiety has a 2'-OMe modification. In some embodiments, the modified sugar moiety has a 2'-F modification. In some embodiments, the modified sugar moiety is cEt.

[0294] Nucleobase modification ACT-UP1 compounds may also include nucleobase (often simply referred to in the art as "base") modifications or substitutions. As used herein, the term "natural" nucleobase includes purine bases adenine (A) and guanine (G), and pyrimidine bases thymine (T), cytosine (C), and uracil (U). Modified nucleobases include other synthetic and natural nucleobases, such as replacing deoxyribonucleosides with ribonucleosides in DNA oligomers (e.g., replacing T with U in RNA chains), replacing ribonucleosides with deoxyribonucleosides in RNA oligomers (e.g., replacing U with T in RNA chains), and universally paired nucleosides, such as 3-formylindole and / or 5-nitroindole (TS Zatsepin, DA Stetsenko, MJ Gait and TSOretskaya, Bioconjugate Chemistry, 16, 471-489, 2005; A. Okamoto, K.Tainaka and I. Saito, Tetrahedron Lett., 43, 4581-4583, 2002; D. Loakes, NucleicAcids Research, 29, 2437-2447, 2001; FH Martin, MM Castro, F. Aboul-elaand I. Tinoco, Jr, Nucleic Acids Research, 13, 8927-8938, 1985; SC Case-Green, EM Southern, Nucleic Acids Research, 22, 131-136, 1994; R. Eritja, DM Horowitz, PA Walker, JP Ziehler-Martin, MS Boosalis, MF Goodman, K. Itakura and BE Kaplan, Nucleic Acids Research, 14, 8135-8153, 1986; D.Picken and V. Gault, Nucleosides, Nucleotides and Nucleic Acids, 16, 937-939, 1997) The modified nucleobases may further include deoxythymidine (dT), 5-methylcytosine (also known as 5-me-C or mC), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymidine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyluracil and cytosine, 6-azauracil, cytosine, and cytosine. Pyrimidines and thymines, 5-uracil (pseudouracil), 4-thiouracil, 8-halogenated, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxy and other 8-substituted adenines and guanines, 5-halogenated (especially 5-bromo), 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deadenine and 7-deadenine, and 3-deadenine and 3-deadenine. Other nucleobases include those disclosed in U.S. Patent 3,687,808; those disclosed in *Modified Nucleosides in Biochemistry, Biotechnology and Medicine*, Herdewijn, P. ed. Wiley-VCH, 2008; those disclosed in *The Concise Encyclopedia Of Polymer Science and Engineering*, pages 858-859, Kroschwitz, JL cd. John Wiley & Sons, 1990; those disclosed in *Englisch et al.*, *Angewandte Chemie*, International Edition, 1991, 30, 613; and those disclosed in *Sanghvi, Y S.*, Chapter 15, dsRNA Research and Applications, pages 289-302, Crooke, ST and Lebleu, B., Ed., CRC Press, 1993. Some of these nucleobases are particularly useful for enhancing the binding affinity of the oligomeric compounds characterized by the features described in this invention. These include 5-substituted pyrimidines, 6-azapyrimidines, and N-2, N-6, and O-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil, and 5-propynylcytosine. 5-methylcytosine substitution has been shown to increase the stability of nucleic acid duplexes by 0.6–1.2 °C.(Sanghvi, YS, Crooke, ST and Lebleu, B., Eds., dsRNA Research and Applications, CRCPress, Boca Raton, 1993, pp. 276-278), and is an exemplary base substitution, even more specifically in its combination with 2'-O-methoxyethyl sugar modification. Representative U.S. patents teaching the preparation of certain of the above-described modified nucleosides and other modified nucleosides include, but are not limited to, U.S. patent numbers 3,687,808; 4,845,205; 5,130,30; 5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469; and 5,594,121. 5,596,091; 5,614,617; 5,681,941; 5,750,692; 6,015,886; 6,147,200; 6,166,197; 6,222,025; 6,235,887; 6,380,368; 6,528,640; 6,639,062; 6,617,438; 7,045,610; 7,427,672; and 7,495,088, the entire contents of which are hereby incorporated by reference.

[0295] Oligomeric compound delivery system Oligomeric compounds need to enter target cells to become active. Various methods have been used to deliver oligomeric compounds into target cells, including viral delivery vectors, lipid-based delivery, polymer-based delivery, and conjugate-based delivery (Paunovska et al., Drug Delivery Systems for RNA Therapeutics, 2022, Nature Reviews Genetics, 23(5):265-280; Chen et al., 2022, Molecular Therapy, Nucleic Acids, 29:150-160).

[0296] Lipid-based particles can form specific structures, such as micelles, liposomes, and lipid nanoparticles (LPNs), to deliver oligomeric compounds into cells. To form these particles, LPNs can include one or more of the following: cationic or ionizable lipids (e.g., DLin-MC3-DMA, SM-102, or ALC-0315), cholesterol, accessory lipids, 1,2-distearate-sn-glycerol-3-phosphocholine (DSPC), poly(ethylene glycol) (PEG) modified lipids (e.g., PEG-2000-C-DMG, PEG-2000-DMG, or ALC-0159), C12-200, cKK-E12, etc. Different lipid combinations can be formulated to influence the delivery of oligomeric compounds to different cell types. In one instance, the therapeutic siRNA patisiran was formulated in cationic ionizable lipids DLin-MC3-DMA, cholesterol, polar phospholipids DSPC, and PEG-2000-C-DMG for delivery to hepatocytes.

[0297] Polymer-based particles are also used in oligomeric compound delivery systems. Such polymers include poly(lactic-co-glycolic acid) (PLGA), polyethyleneimine (PEI), poly(l-lysine) (PLL), poly(β-amino ester) (PBAE), dendritic polymers (e.g., poly(amidoamine) (PAMAM) or PLL), and other polymers or polymers modified thereof. The polymer composition can be varied to suit the desired properties of the oligomeric compound delivery.

[0298] The oligomeric compounds disclosed herein can be covalently linked to one or more moieties or conjugates, which enhance the activity, cellular distribution, or cellular uptake of the resulting compounds. Conjugates may include cholesterol, lipids, carbohydrates, phospholipids, biotin, phenazine, folic acid, phenanthridine, anthraquinones, acridine, fluorescein, rhodamine, coumarin, peptides, antibodies, dyes, tocopherols (Nishina et al., 2008, Molecular Therapy, 16(4):734-740), etc. Conjugate-based delivery can actively deliver oligomeric compounds to specific cell types.

[0299] In this example, N-acetylgalactosamine (GalNAc) is conjugated with an oligomer and delivered into hepatocytes. Various GalNAc conjugates are available in several publications, all of which are incorporated herein by reference: Sharma et al., 2018, Bioconjugate Chem, 29:2478-2488; Nair et al., J. Am. Chem. Soc.2014, 136(49):16958 16961; Keam, 2022, Drugs, 82:1419-1425; US Patent 10,087,208; Prakash et al., 2014, Nucleic Acids Res, 42(13):8796-807; Debacker et al., 2020, Molecular Therapy, 28(8):1759-1771; US ​​Patent 11,110,174; US Patent 9,796,756; US Patent 9,181,549; US Patent 10,344,275; US Patent 10,570,169; US Patent 9,506,030; US Patent 7,582,744; and WO2024137545.

[0300] Oligomeric compound synthesis Oligomeric compounds are designed, synthesized, and prepared using methods known in the art.

[0301] Solid-phase synthesis of oligonucleotides was performed using standard phosphoramidite chemistry on a MerMade™ 48x synthesizer (BioAutomation, LGC, Biosearch Technologies, Hoddesdon, UK), which can prepare up to 48 oligonucleotides in 1 µmol or 5 µmol increments per run. Phosphoramidite synthesis of oligonucleotides on solid supports is well known in the art (e.g., Beaucage and Caruthers, 1981, Tetrahedron Letters, 22(20): 1859-1862; Roy and Caruthers, 2013, Molecules, 18:14268-14284; and Roy and Caruthers et al., 2021, Nature Communications, 12:2760). The solid support was a porous glass (500–1400 Å) loaded with a universal linker or with a 3'-GalNAc conjugate (AM Chemicals, Vista, CA, USA; Primetech ALC, Minsk, Belarus; Gene Link, Elmsford, NY, USA; or any GalNAc conjugate disclosed herein) or a universal solid support (AM Chemicals, Vista, CA, USA). Auxiliary reagents and standard 2'-cyanoethylphosphoramide monomers (2'-fluoronucleoside, 2'-O-methylnucleoside, RNA nucleoside, DNA nucleoside) were obtained from multiple sources (Hongene Biotech, Shanghai, China; Sigma-Aldrich, St. Louis, MO, USA; Glen Research, Sterling, VA, USA; ThermoFisher Scientific, Waltham, MA, USA; LGC Biosearch Technologies, Hoddesdon, UK). The phosphoramidite mixture was prepared in anhydrous acetonitrile or 30% DMF / acetonitrile and coupled using 0.25M 4,5-dicyanimidazole (DCI) (Sigma-Aldrich, St. Louis, MO, USA) for coupling times ranging from 120 to 360 seconds. Standard phosphodiester linkage was achieved using a mixture of 0.02M iodine in tetrahydrofuran (THF), pyridine, and water.Phosphophosphate linkages were generated using 0.05 M sulfiding reagent II (3-((dimethylaminomethylene)amino)-3H-1,2,4-dithiazolyl-3-thione, DDTT) (40:60, pyridine / acetonitrile) (LGC Biosearch Technologies, Hoddesdon, UK) with an oxidation time of 6 min. All sequences were synthesized after removing the dimethoxytriphenylmethyl (DMT) protecting group.

[0302] After solid-phase synthesis, oligonucleotides were cleaved from the solid support, and base-unstable groups were removed by incubation in ammonium hydroxide at 55°C for 6 hours. The ammonium hydroxide was then removed to dryness using a centrifugal vacuum concentrator at room temperature. For sequences containing native ribonucleotides (2'-OH) protected with tert-butyldimethylsilyl (TBDMS), a second deprotection was performed using triethylamine;trihydrofluoride (TEA:3HF). 100 µL LDMSO and 125 µL TEA:3HF were added to each TBDMS-protected oligonucleotide, and the mixture was incubated at 65°C for 2.5 hours. After incubation, 25 µL of 3M sodium acetate was added to the solution, followed by precipitation in butanol at -20°C for 30 minutes. The turbid solution was centrifuged to form a cake, and the supernatant was carefully decanted using a pipette. A standard precipitation process was then performed using 75% ethanol:water, followed by 100% ethanol as the supernatant. The oligonucleotide cake was dried in a centrifugal vacuum concentrator for 30 minutes.

[0303] After precipitation with 3M sodium acetate, desalting was performed without HPLC purification, followed by chromatography on G25 Sephadex. ® Elution was performed using a column (Sigma-Aldrich, St. Louis, MO, USA). Purification of the oligonucleotides was achieved by anion exchange chromatography on a Gilson GX271 preparative HPLC system (Middleton, WI, USA) using BioWorks Q40 resin (Uppsala, Sweden). Final desalting was performed using Sephadex. ® G25 column. Purity analysis of all oligonucleotides was performed by ion-pair reversed-phase HPLC on an Agilent 1200 analytical HPLC system (Santa Clara, CA, USA), and intact mass analysis was performed by negative ion mass spectrometry on an Agilent 6130 single quadrupole mass spectrometer (Santa Clara, CA, USA). Finally, analysis was performed by UV / Vis on a Tecan Infinite column. ® A260 quantitative analysis was performed using an M Plex microplate reader (Zurich, Switzerland).

[0304] In vitro testing of oligomers This article describes a method for treating cells with oligomeric compounds, such as the ACT-UP1 compound.

[0305] When cells reach approximately 60-80% confluence during culture, they can be treated with oligomeric compounds.

[0306] Reagents commonly used to introduce oligomeric compounds into cultured cells include the cationic lipid transfection reagent Oligofectamine. TM 2000 or Lipofectamine TM 2000 (ThermoFisher Scientific, Waltham, MA). In one example, the oligomeric compound can be combined with Oligofectamine. TM Mix 2000 in OPTI-MEM 1 (ThermoFisher Scientific, Waltham, MA) to achieve the desired final concentration of the oligomer, which can range from 0.001 nM to 300 nM in the culture medium. The transfection procedure should be performed according to the manufacturer's recommended protocol.

[0307] Another technique used to introduce oligomeric compounds into cultured cells includes electroporation.

[0308] Oligomeric compounds conjugated to GalNAc can be introduced into cells by incubating the conjugated compound with the cells without the use of transfection reagents, a process referred to here as “free uptake.” Oligomeric GalNAc conjugates are transported to desialyl glycoprotein receptor (ASGR) positive cells (e.g., hepatocytes) via endocytosis.

[0309] Cells were treated with oligomeric compounds using conventional methods. Cells were collected 4–144 hours after oligomeric compound treatment, at which time the mRNA levels of the target nucleic acid (collected at 4–144 hours) or the protein levels (extracted at 24–96 hours) were measured using methods known in the art and described herein. Typically, the treatment was repeated multiple times, and the data are presented as the mean (e.g., standard deviation) of the repeated treatments.

[0310] The concentration of the oligomer used varies depending on the cell line and target. Methods for determining the optimal oligomer concentration for a specific target in a particular cell line are well known in the art. Typically, cells are treated with the oligomer in a dose-dependent manner to calculate the half-maximum inhibitory concentration (IC50). In the case of Oligofectamine... TM 2000 or Lipofectamine TMWhen transfecting with 2000, oligomers are typically used at concentrations ranging from 0.001 nM to 300 nM. When transfecting using electroporation or free uptake, oligomers are used at higher concentrations (ranging from 7.5 nM to 20,000 nM).

[0311] In vivo testing of oligomers The oligomeric compounds of the present invention (e.g., the ACT-UP1 compound) are tested in animals to assess their ability to regulate target protein expression and produce phenotypic changes (e.g., changes in one or more markers affected by the target nucleic acid). Furthermore, phenotypic changes may also result in the reduction of diseases, conditions, illnesses, or symptoms associated with the target nucleic acid. Testing can be performed in normal animals or experimental disease models. For animal administration, the oligomeric compounds are formulated in a pharmaceutically acceptable diluent (e.g., phosphate-buffered saline (PBS)). Administration includes parenteral routes, such as intraperitoneal, intravenous, and subcutaneous administration. Dosage and frequency of administration are calculated based on various factors, such as the route of administration and animal body weight. In one embodiment, after treatment with the oligomeric compounds of the present invention for a period of time, RNA encoding the target nucleic acid is isolated from liver tissue, and changes in target nucleic acid expression are measured. Changes in protein levels expressed by the target nucleic acid can also be measured.

[0312] RNA isolation RNA analysis can be performed on total cellular RNA or poly(A)+ mRNA. RNA isolation methods are well known in the art. RNA preparation is performed using methods well known in the art, such as using TRIZOL reagents (Thermo Fisher Scientific, Waltham, MA), the Qiagen RNeasy kit (Qiagen, Hilden, Germany), or an AcroPrep Advance 96-well filter plate with Qiagen's RLT, RW1, and RPE buffers (Pall Corporation, Port Washington, New York). RNA extraction procedures are performed according to the manufacturer's recommended protocol.

[0313] protein separation Protein analysis can be performed on whole-cell extracts or tissue lysates. Methods for preparing cell extracts or tissue lysates are well known in the art. Cellular proteins are prepared using methods well known in the art, such as RIPA buffer (ThermoFisher Scientific, Waltham, MA) or other suitable buffers. Tissue lysates are prepared in RIPA buffer using a tissue homogenizer. Protein levels can be analyzed using Western blotting, ELISA, or other methods.

[0314] Compositions and methods for preparing pharmaceutical compositions The oligomeric compounds of the present invention (such as the ACT-UP1 compound described herein) can be combined with pharmaceutically acceptable active or inert substances (such as diluents, excipients, or carriers) for the preparation of pharmaceutical compositions or formulations.

[0315] The composition and method used to formulate the pharmaceutical composition depend on several criteria, including but not limited to route of administration, disease severity, or dose to be administered.

[0316] In some embodiments, the drug carrier or excipient is a pharmaceutically acceptable solvent, suspension, or any other pharmacologically inert solvent for delivering one or more oligomeric compounds to animals. Excipients can be liquid or solid and can be selected in conjunction with the planned administration method to provide the desired volume, consistency, etc., after mixing with the nucleic acid and other components in a given drug composition. Typical drug carriers include, but are not limited to, binders (e.g., pregelatinized corn starch, polyvinylpyrrolidone, and / or hydroxypropyl methylcellulose); fillers (e.g., lactose and other sugars, microcrystalline cellulose, pectin, gelatin, calcium sulfate, ethyl cellulose, polyacrylates, and / or dicalcium phosphate); lubricants (e.g., magnesium stearate, talc, silica, colloidal silica, stearic acid, metal stearates, hydrogenated vegetable oils, corn starch, polyethylene glycol, sodium benzoate, and / or sodium acetate); disintegrants (e.g., starch and / or sodium starch-hydroxyacetate); and wetting agents (e.g., sodium dodecyl sulfate).

[0317] The compositions of the present invention can also be formulated using pharmaceutically acceptable organic or inorganic excipients that do not react adversely with nucleic acid compounds and are suitable for parenteral or non-parenteral administration. Suitable pharmaceutically acceptable carriers include, but are not limited to, water, salt solutions, alcohols, polyethylene glycol, gelatin, lactose, amylose, magnesium stearate, talc, silica, viscous paraffin, hydroxymethyl cellulose, polyvinylpyrrolidone, etc. Pharmaceutically acceptable diluents include phosphate-buffered saline (PBS). PBS is a suitable diluent for compositions intended for parenteral delivery. Therefore, in one embodiment, the methods described herein employ a pharmaceutical composition comprising an oligomeric compound and a pharmaceutically acceptable diluent. In some embodiments, the pharmaceutically acceptable diluent is PBS. In some embodiments, the oligomeric compound is the ACT-UP1 compound.

[0318] Pharmaceutical compositions comprising oligomeric compounds (such as the ACT-UP1 compound) may encompass any pharmaceutically acceptable salt, ester, or salt of such esters, which, when administered to animals (including humans), can (directly or indirectly) provide a biologically active metabolite or residues thereof. Therefore, this disclosure also relates, for example, to pharmaceutically acceptable salts of oligomeric compounds, prodrugs, pharmaceutically acceptable salts of such prodrugs, and other bioequivalents. Suitable pharmaceutically acceptable salts include, but are not limited to, sodium and potassium salts.

[0319] In some embodiments, the pharmaceutical composition is prepared for administration by injection (e.g., intravenous, subcutaneous, and / or intramuscular). In some such embodiments, the pharmaceutical composition comprises a carrier and is formulated in an aqueous solution (e.g., water or a physiologically compatible buffer, such as Hanks' solution, Ringer's solution, or physiological saline buffer (e.g., PBS)). In some embodiments, other components are included (e.g., components that contribute to solubility or act as preservatives). In some embodiments, the suspension for injection is prepared using a suitable liquid carrier, suspension, etc. Some pharmaceutical compositions for injection are provided in unit dose form (e.g., ampoules or multi-dose containers).

[0320] dose For the purposes of this disclosure, the amount or dose of the active agent (i.e., the oligomeric compound of the present invention) applied should be sufficient, for example, to regulate the expression of the target protein in an animal. In animals (e.g., humans), the dose will be determined by the efficacy of the particular active agent, the condition of the animal, and the weight of the animal to be treated.

[0321] Various methods for determining the dosage are known in the art.

[0322] The dosage of the active agent disclosed herein will also be determined by the presence, nature, and extent of any adverse side effects that may occur with the administration of the specific active agent disclosed herein. Typically, the attending physician will consider a variety of factors, such as age, weight, overall health, diet, sex, the active agent of this disclosure to be administered, route of administration, and severity of the condition being treated, to determine the dosage of the active agent of this disclosure for each individual patient.

[0323] Dosage In some embodiments, the pharmaceutical composition is administered according to a dosing regimen (e.g., dose, dosing frequency, and duration), wherein the dosing regimen can be selected to achieve a desired effect. The desired effect may be, for example, a reduction in the target nucleic acid, or prevention, reduction, relief, or slowing of the progression of a disease, condition, or symptom associated with the target nucleic acid. In some embodiments, variables of the dosing regimen are adjusted to achieve a desired concentration of the pharmaceutical composition in the subject. The term "concentration of the pharmaceutical composition" used with respect to the dosing regimen may refer to the oligomeric compound or active ingredient of the pharmaceutical composition. For example, in some embodiments, the dose and dosing frequency are adjusted to provide a tissue or plasma concentration of the pharmaceutical composition sufficient to achieve the desired effect.

[0324] Dosage depends on the severity and responsiveness of the disease state being treated, and the course of treatment may last from several days to several months, or until a cure or remission of the disease state is achieved. Dosage also depends on drug potency and metabolism. In some embodiments, the dose is from about 0.01 μg to 50 mg per kg body weight, from 0.01 μg to 100 mg per kg body weight, or within the range of about 0.001 mg to 1000 mg, and may be administered once or more daily, weekly, monthly, quarterly, or annually, or even once every 2 to 20 years. After successful treatment, patients are expected to receive maintenance therapy to prevent relapse of the disease state, wherein the oligomeric compound is administered at a maintenance dose ranging from about 0.01 μg to 100 mg per kg body weight, once or more daily, once or more weekly, once or more monthly, once or more quarterly, once or more annually, up to once every 20 years, or within the range of about 0.001 mg to 1000 mg. In some implementation schemes, it is expected that the oligomeric compound will be applied at most once daily, once weekly, once monthly, once quarterly, once annually, once every two years, once every three years, once every four years, once every five years, once every ten years, up to once every twenty years.

[0325] In some embodiments, the dosage range is between any of the following: about 1 mg - 1500 mg, 100 mg - 1400 mg, 100 mg - 1300 mg, 100 mg - 1200 mg, 100 mg - 1100 mg, 100 mg - 1000 mg, 100 mg - 900 mg, 200 mg - 800 mg, 300 mg - 700 mg, 400 mg - 600 mg, 100 mg - 400 mg, 200 mg - 500 mg, 300 mg - 600 mg, and 400 mg - 700 mg. In some embodiments, the dosage is about 100 mg, 150 mg, 200 mg, 250 mg, 300 mg, 350 mg, 400 mg, 450 mg, 500 mg, 550 mg, 600 mg, 650 mg, 700 mg, 800 mg, 850 mg, 900 mg, 950 mg, 1000 mg, 1050 mg, 1100 mg, 1150 mg, 1200 mg, 1250 mg, 1300 mg, 1350 mg, 1400 mg, 1450 mg, or 1500 mg.

[0326] In some embodiments, the oligomeric compound is administered twice annually at any dose of about 150 mg, 200 mg, 300 mg, 400 mg, 500 mg, 600 mg, 700 mg, 800 mg, or 900 mg. In some embodiments, the oligomeric compound is administered quarterly at a dose of about 100 mg, 150 mg, 200 mg, 300 mg, 400 mg, 500 mg, 600 mg, 700 mg, 800 mg, or 900 mg. In some embodiments, the oligomeric compound is administered monthly or every two months at a dose of about 50 mg, 100 mg, 150 mg, 200 mg, 300 mg, 400 mg, 500 mg, 600 mg, 700 mg, 800 mg, or 900 mg. In some implementations, the oligomeric compound is administered weekly or every two weeks at doses of approximately 10 mg, 15 mg, 20 mg, 25 mg, 50 mg, 100 mg, 150 mg, 200 mg, 300 mg, 400 mg, 500 mg, 600 mg, 700 mg, 800 mg, or 900 mg.

[0327] application The oligomeric compounds (e.g., ACT-UP1) or pharmaceutical compositions of the present invention can be administered via a variety of routes, depending on whether local or systemic treatment is desired and the site of treatment. Administration can be oral, inhaled, or parenteral.

[0328] In some embodiments, the compounds and compositions described herein are administered parenterally. Parenterical administration includes intravenous, intra-arterial, subcutaneous, intraperitoneal, or intramuscular injection or infusion; or intracranial administration, such as intrathecal or intraventricular administration. In some embodiments, parenterical administration is performed by infusion. Infusion can be chronic or continuous, or short-term or intermittent. In some embodiments, the infused pharmaceutical formulation is delivered via a pump.

[0329] In some embodiments, parenteral administration is performed by injection. Injection can be delivered using a syringe or pump. In some embodiments, the injection is a bolus. In some embodiments, the injection is administered directly to a tissue or organ.

[0330] In some embodiments, formulations for parenteral, intrathecal, or intraventricular administration may include sterile aqueous solutions and may also contain buffers, diluents, and other suitable additives, such as, but not limited to, penetration enhancers, carrier compounds, and other pharmaceutically acceptable carriers or excipients.

[0331] In some embodiments, formulations for oral administration of compounds or compositions may include, but are not limited to, drug carriers, excipients, powders or granules, microparticles, nanoparticles, suspensions or solutions in aqueous or non-aqueous media, capsules, gel capsules, pouch formulations, tablets, or microtablets. Thickeners, flavoring agents, diluents, emulsifiers, dispersants, or binders may be desired. In some embodiments, oral formulations are formulations in which the compounds provided herein are administered in combination with one or more penetration enhancers, surfactants, and chelating agents.

[0332] The reagent kit of the present invention According to another aspect of the invention, a kit is provided. The kit according to the invention includes packaging containing any composition of the invention or an oligomeric compound of the invention. In various aspects, the kit contains any composition of the invention as a unit dose. For purposes herein, "unit dose" refers to a discrete amount dispersed in a suitable carrier.

[0333] The phrase “packaging” means any container containing the compositions provided herein. In a preferred embodiment, the packaging may be a box or an outer packaging. Packaging materials used for packaging pharmaceutical products are well known to those skilled in the art. Examples of pharmaceutical packaging materials include, but are not limited to, blister packs, bottles, tubes, inhalers, pumps, bags, vials, containers, syringes (including pre-filled syringes), bottles, and any packaging materials suitable for the selected formulation and the intended method of administration and treatment.

[0334] The kit may also contain items not included inside the packaging but attached to the outside of the packaging, such as pipettes.

[0335] The kit may optionally contain instructions for administering the compositions of the present invention to subjects with conditions requiring treatment. The kit may also contain instructions regarding the regulatory-approved use of the components of the compositions described herein (e.g., the U.S. Food and Drug Administration). The kit may optionally contain a label or instruction manual for use with the compositions of the present invention. The packaging and / or any instruction manual itself may have been approved by a regulatory agency. These kits may include components packaged in solid or liquid phase form (e.g., provided buffer solutions). These kits may also include buffer solutions for preparing solutions to carry out the relevant methods, and pipettes for transferring liquids from one container to another.

[0336] The kit may also optionally contain one or more other compositions for use in the combination therapy described herein. In some embodiments, the packaging is a container for any mode of administration, such as intravitreal delivery, intraocular delivery, intratumoral delivery, peritumoral delivery, intraperitoneal delivery, intrathecal delivery, intramuscular delivery, subcutaneous delivery, intravenous delivery, intra-arterial delivery, intraventricular delivery, intrasternal delivery, intracranial delivery, or intradermal delivery.

[0337] How to use This invention provides a method for enhancing the expression of a target protein in a subject, comprising administering an effective amount of the oligomeric compound of the invention or the pharmaceutical composition of the invention, thereby increasing the expression of the target protein in the subject. In some embodiments, the oligomeric compound is an ACT-UP1 compound. In some preferred aspects, in the ACT-UP1 compound, PRS is linked to the 5' end of ASO. In some aspects, PRS is linked to the 3' end of ASO.

[0338] This invention provides a method for enhancing the expression of a target protein in eukaryotic or prokaryotic cells, comprising administering an effective amount of the oligomeric compound of the invention or the pharmaceutical composition of the invention, thereby increasing the expression of the target protein in a subject. In some embodiments, the oligomeric compound is an ACT-UP1 compound. In some embodiments, the eukaryotic or prokaryotic cells are mammalian cells, plant cells, yeast cells, or bacterial cells.

[0339] In some embodiments, methods for enhancing the expression of target genes in cells include administering an oligomeric compound that targets mRNA transcripts to the cells. In some embodiments, the oligomeric compound is the ACT-UP1 compound. In some preferred aspects, in the ACT-UP1 compound, PRS is linked to the 5' end of ASO. In some aspects, PRS is linked to the 3' end of ASO.

[0340] In some embodiments, the oligomeric compound of the present invention increases protein expression by at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%. In one embodiment, the oligomeric compound is an ACT-UP1 compound.

[0341] In some embodiments, a method for enhancing Jagged 1 (JAG1) gene expression in cells is provided, comprising administering to cells an oligomeric compound that targets the human JAG1 mRNA transcript (GenBank ID: NM_000214.3, SEQ ID NO: 1). In some embodiments, the oligomeric compound is an ACT-UP1 compound that targets JAG1. In some embodiments, enhancing JAG1 gene expression in cells is used to treat a subject suffering from a JAG1-related disease. In some embodiments, JAG1-related disease is associated with decreased JAG1 protein levels. In a preferred embodiment, the oligomeric compound is an ACT-UP1 compound that targets JAG1.

[0342] In some embodiments, a method for enhancing RAB9A gene expression in cells is provided, comprising administering to cells an oligomeric compound that targets the human RAB9A mRNA transcript (GenBank ID: NM_004251.5, SEQ ID NO: 2). In some embodiments, the oligomeric compound is an ACT-UP1 compound that targets RAB9A. In some embodiments, enhancing RAB9A gene expression in cells is used to treat a subject suffering from RAB9-related disease. In some embodiments, RAB9A-related disease is associated with decreased RAB9A protein levels. In a preferred embodiment, the oligomeric compound is an ACT-UP1 compound that targets RAB9A.

[0343] In some embodiments, a method for enhancing RNase H1 gene expression in cells is provided, comprising administering an oligomeric compound that targets the human RNase H1 mRNA transcript (GenBank ID: NM_001286834.3, SEQ ID NO: 3) to cells. In some embodiments, enhancing RNase H1 gene expression in cells is used to treat a subject suffering from RNase H1-related disease. In some embodiments, RNase H1-related disease is associated with decreased RNase H1 protein levels. In a preferred embodiment, the oligomeric compound is an ACT-UP1 compound that targets RNase H1.

[0344] In some embodiments, a method for enhancing PBGD gene expression in cells is provided, comprising administering to cells an oligomeric compound that targets the human PBGD (also known as HMBS) mRNA transcript (GenBank ID: NM_000190.4, SEQ ID NO: 4). In some embodiments, a method for enhancing PBGD gene expression in cells is provided, comprising administering to cells an oligomeric compound that targets mouse PBGD mRNA (GenBank ID: NM_013551.2, SEQ ID NO: 6). In some embodiments, enhancing PBGD gene expression in cells is used to treat a subject suffering from PBGD-related disease. In some embodiments, PBGD-related disease is associated with decreased PBGD protein levels. In a preferred embodiment, the oligomeric compound is an ACT-UP1 compound that targets PBGD.

[0345] In some embodiments, a method for enhancing FGF21 gene expression in cells is provided, comprising administering to cells an oligomeric compound that targets the human FGF21 mRNA transcript (GenBank ID: NM_019113.4, SEQ ID NO: 5). In some embodiments, a method for enhancing FGF21 gene expression in cells is provided, comprising administering to cells an oligomeric compound that targets mouse FGF21 mRNA (GenBank ID: NM_020013.4, SEQ ID NO: 7). In some embodiments, enhancing FGF21 gene expression in cells is used to treat a subject suffering from an FGF21-related disease. In some embodiments, the FGF21-related disease is associated with reduced levels of FGF21 protein production and / or secretion. In a preferred embodiment, the oligomeric compound is an ACT-UP1 compound that targets FGF21.

[0346] In some embodiments, methods for enhancing HNF4α (HNF4A) gene expression in cells include administering an oligomeric compound that targets the HNF4α mRNA transcript (GenBank ID: NM_178849.3, SEQ ID NO: 45) to cells. In some embodiments, enhancing HNF4α gene expression in cells is used to treat a subject with HNF4α-related disease. In some embodiments, HNF4α-related disease is associated with decreased HNF4α protein levels. In a preferred embodiment, the oligomeric compound is an ACT-UP1 compound that targets HNF4α.

[0347] In some embodiments, a method for enhancing klotho (KL) gene expression in cells includes administering an oligomeric compound that targets the mRNA transcript (GenBank ID: NM_004795.4, SEQ ID NO: 46) to cells. In some embodiments, enhancing KL gene expression in cells treats a subject with a KL-related disease. In some embodiments, the KL-related disease is associated with decreased klotho protein levels. In a preferred embodiment, the oligomeric compound is an ACT-UP1 compound that targets KL.

[0348] In some embodiments, a method for enhancing OPA1 gene expression in cells includes administering an oligomeric compound that targets the OPA1 mRNA transcript (GenBank ID: NM_015560.3, SEQ ID NO: 47) to cells. In some embodiments, enhancing OPA1 gene expression in cells is used to treat a subject suffering from an OPA1-related disease. In some embodiments, OPA1-related disease is associated with decreased OPA1 protein levels. In a preferred embodiment, the oligomeric compound is an ACT-UP1 compound that targets OPA1.

[0349] In some embodiments, a method for enhancing PKD1 gene expression in cells includes administering an oligomeric compound that targets the PKD1 mRNA transcript (GenBank ID: NM_001009944.3, SEQ ID NO: 48) to cells. In some embodiments, enhancing PKD1 gene expression in cells is used to treat a subject with PKD1-related disease. In some embodiments, PKD1-related disease is associated with decreased PKD1 protein levels. In a preferred embodiment, the oligomeric compound is an ACT-UP1 compound that targets PKD1.

[0350] In some embodiments, a method for enhancing PKD2 gene expression in cells includes administering an oligomeric compound that targets the PKD2 mRNA transcript (GenBank ID: NM_000297.4, SEQ ID NO: 49) to cells. In some embodiments, enhancing PKD2 gene expression in cells is used to treat a subject with PKD2-related disease. In some embodiments, PKD2-related disease is associated with decreased PKD2 protein levels. In a preferred embodiment, the oligomeric compound is an ACT-UP1 compound that targets PKD2.

[0351] In some embodiments, methods for enhancing HBB gene expression in cells include administering an oligomeric compound that targets the HBB mRNA transcript (GenBank ID: NM_000518.5, SEQ ID NO: 50) to cells. In some embodiments, enhancing HBB gene expression in cells is used to treat a subject with HBB-related disease. In some embodiments, HBB-related disease is associated with decreased HBB protein levels. In a preferred embodiment, the oligomeric compound is an ACT-UP1 compound that targets HBB.

[0352] In some embodiments, methods for enhancing GRN gene expression in cells include administering an oligomeric compound that targets the GRN mRNA transcript (GenBank ID: NM_002087.4, SEQ ID NO: 51) to cells. In some embodiments, enhancing GRN gene expression in cells is used to treat a subject with a GRN-related disease. In a preferred embodiment, the oligomeric compound is an ACT-UP1 compound that targets GRN.

[0353] In some embodiments, a method for enhancing SCN1A gene expression in cells includes administering an oligomeric compound that targets the SCN1Am RNA transcript (GenBank ID: NM_001165963.4, SEQ ID NO: 52) to cells. In some embodiments, enhancing SCN1A gene expression in cells is used to treat a subject suffering from an SCN1A-related disease. In some embodiments, the SCN1A-related disease is associated with decreased SCN1A protein levels. In a preferred embodiment, the oligomeric compound is an ACT-UP1 compound that targets SCN1A.

[0354] In some embodiments of this disclosure, the subject is a mammal, including but not limited to rodents (e.g., mice and hamsters) and lagomorphs (e.g., rabbits), carnivores (including felines (cats) and canines (dogs)), even-toed ungulates (including bovines (cattle) and suidae (pigs)), or perissodactyls (including equines (horses)). In some respects, these mammals belong to primates, ceboids, or simoids (monkeys), or to apes (i.e., humans and apes). In a preferred aspect, the mammal is a human.

[0355] In vivo testing of the ACT-UP1 compound In some embodiments of this disclosure, the ACT-UP1 compound can be tested in subjects to assess its ability to enhance protein expression. In some embodiments, the ACT-UP1 compound can be tested in subjects to assess its ability to increase mRNA translation, thereby increasing protein production and / or producing phenotypic changes associated with that protein. In some embodiments, the ACT-UP1 compound can be tested in subjects to assess its ability to treat protein-related diseases. Testing can be performed in normal subjects or experimental disease models. For administration to subjects, the ACT-UP1 compound is formulated in a pharmaceutically acceptable diluent (e.g., phosphate-buffered saline). Administration includes parenteral routes, such as intraperitoneal, intravenous, and subcutaneous. The calculation of the ACT-UP1 dose and frequency of administration depends on various factors, such as the route of administration and the subject's weight. In one embodiment, after treatment with the ACT-UP1 compound for a period of time, the protein is isolated from tissue, and changes in protein expression are measured.

[0356] In vitro assay for identifying ACT-UP1 compounds that enhance protein expression In some embodiments of this disclosure, an in vitro assay is provided for identifying ACT-UP1 compounds that enhance target protein expression.

[0357] In one instance, the mRNA was chosen as the target for upregulating its protein expression. Cultured cells (e.g., HeLa, Hepa1-6, or HEK293) were seeded and allowed to grow for one day to ~70% confluence. Then, Lipofectamine was used. TM Cells were transfected with the 2000 transfection reagent (ThermoFisher Scientific, Waltham, MA) at a final concentration of approximately 7.5 nM or 15 nM of the ACT-UP1 compound of interest, or blank transfection was performed as a control. Twenty-four (24) hours post-transfection, cells were collected and lysed, and proteins were extracted using RIPA lysis extraction buffer (ThermoFisher Scientific, Waltham, MA). Target protein levels were determined by Western blotting or chemiluminescence assay using a target protein-specific antibody and, optionally, a secondary antibody conjugated to alkaline phosphatase or horseradish peroxidase. Western blot images were quantified using ImageJ (an open-source software for processing and analyzing scientific images), and results were quantified as a percentage of protein levels relative to blank transfected cells after normalization to the loaded control protein (e.g., GAPDH or nucleolin (NCL)). Since the control protein was not targeted by the ACT-UP1 compound tested in the assay, its levels were unaffected, demonstrating the specificity of the ACT-UP1 compound in regulating target protein levels.

[0358] Advantages of the present invention Protein expression involves many biological steps, such as RNA transcription, precursor mRNA splicing, mRNA stability, translation, and protein stability. Difficulty in any of these steps can lead to reduced protein expression or the production of nonfunctional proteins; for example, missense or nonsense mutations can result in haplo-inadequate dosing.

[0359] The use of antisense compounds in therapeutics is becoming increasingly sophisticated, with several commercially available antisense compounds currently available for treating a variety of diseases (Crooke, ST, et al., 2021c, Antisense drug discovery and development technology considered in a pharmacological context. BiochemPharmacol 189: 114196). As disclosed herein, antisense oligonucleotide (ASO)-coupled translation-upregulation 1 (also known as ACT-UP1) is a novel approach to enhancing protein expression. As also disclosed herein, novel types of antisense compounds, ACT-UP1 compounds, have been developed for this approach, enhancing protein levels without requiring existing repressive elements in the target mRNA. This paper demonstrates that these ACT-UP1 compounds enhance protein levels in a variety of genes.

[0360] The ACT-UP1 compound contains an antisense oligonucleotide (ASO) linked to a protein recruitment sequence (PRS). Without being bound by any particular theory, the ASO component of the ACT-UP1 compound specifically hybridizes to the mRNA sequence of interest, bringing the PRS component of the ACT-UP1 compound to a position immediately adjacent to the target mRNA. The PRS component then directs translation regulatory proteins to the target mRNA, promoting the interaction between translation-related proteins and the mRNA of interest, thereby increasing the translation of the targeted mRNA.

[0361] PRS are short, single-stranded sequences of linked nucleosides that can interact with and / or bind to regulatory proteins. Single-stranded RNA can bind more proteins than double-stranded RNA (Liang, XH, et al., 2015, Identification and characterization of intracellular proteins that bind oligonucleotides with phosphorothioate linkages. Nucleic Acids Res 43: 2927-2945). Therefore, this study aimed to assess the protein recruitment capacity of single-stranded sequences as PRS components of ACT-UP1.

[0362] m 6 RNA methylation plays an important role in gene expression regulation (He and He, m) 6 A RNAMethylation: From Mechanisms to Therapeutic Potential, EMBO, 2021 Feb 1, 40(3):e105977), in which m 6 A specific protein involved in RNA methylation (such as the RNA methyltransferase METTL3) enhances mRNA translation by interacting with cellular translation initiation mechanisms (Lin et al., METTL3 Promotes Translation in Human Cancer Cells, Mol Cell., 2016, 62(3):335-345; Choe et al., mRNA Circularization by METTL3-eIF3h Enhances Translation and Promotes Oncogenesis). 6 Among the many components of the A-methyltransferase complex, METTL3 and METTL14 form a heterodimer to mediate the conversion of adenine (A) to N in mRNA. 6 -Methyl-adenosine (m 6 The transformation of A). The METTL3-METTL14 complex recognizes the concordant sequence DRACH (D is adenine (A), guanine (G), or thymine (T); R is adenine (A) or guanine (G); A is adenine (A); C is cytosine (C); H is adenine (A), cytosine (C), or uracil (U)) (He and He, EMBO, 2021 Feb1, 40(3):e105977). Examples of DRACH include, but are not limited to, the sequence GGACU (SEQ ID NO: 8), which can mediate N 6 -Methyl-adenosine (m 6 A) Transformation (Liu et al., A METTL3-METTL14 Complex Mediates MammalianNuclear RNA N 6-Adenosine Methylation, Nat Chem Biol, 2014, 10(2):93-95). Sequences derived from the GGACU sequence in mRNA (e.g., GGACU (SEQ ID NO: 8), GGAUU (SEQ ID NO: 9), GGACUGGAC (SEQ ID NO: 10), GGACUGGACU (SEQ ID NO: 11), ACGGACUUGGACU (SEQ ID NO: 12), and GGACUGGACUGGACU (SEQ ID NO: 101)) were designed as PRS components in the ACT-UP1 compound and tested in the examples below.

[0363] For the PRS component of ACT-UP1, other sequences derived from the DRACH concordant sequence are also envisioned (e.g., AAACAAAACA (SEQ ID NO: 99)). Additionally, PRS components derived from the RRANN concordant sequence (R is adenine or guanine; A is adenine; and N is adenine, guanine, cytosine, thymine, or uracil) or the RRAWN concordant sequence (R is adenine or guanine; A is adenine; W is adenine or cytosine; N is adenine, guanine, cytosine, thymine, or uracil) in the mRNA are also envisioned. PRS sequences derived from DRACH, RRANN, or RRAWN shared sequences include, but are not limited to, any of the following sequences: five to 20 linked nucleosides containing sequences or combinations thereof of GGACU (SEQ ID NO: 8), GGAUU (SEQ ID NO: 9), GAACU (SEQ ID NO: 41), AGACU (SEQ ID NO: 42), AAACU (SEQ ID NO: 43), GGACA (SEQ ID NO: 44), AAACA (SEQ ID NO: 154). For example, a PRS may contain two to four repeating sequences of the sequence elements GGACU, GAACU, AGACU, AAACU, or GGACA, or combinations thereof.

[0364] The 3'UTR of mRNA plays a crucial role in regulating translation through interactions with miRNAs or proteins (Hong, D., and S. Jeong, 2023, 3'UTR Diversity: Expanding Repertoire of RNAAlterations in Human mRNAs. Mol Cells 46: 48-56). For example, poly(A)-binding proteins (PABPs) that interact with the 3' UTR poly(A) tail are known to be essential for translation. Sequences derived from the poly(A) tail in mRNA (e.g., AAACUAAACU (SEQ ID NO: 13), AAAAAAAA (poly(A)8) (SEQ ID NO: 14), AAAAAAAAAA (poly(A)8) (SEQ ID NO: 14)) are also relevant. 10 ) (SEQ ID NO: 15), AAACAAACA (SEQ ID NO: 99) or AAAAAAAAAAAA (poly(A) 12 (SEQ ID NO: 102) was designed as the PRS component in the ACT-UP1 compound and tested in the examples below.

[0365] While some methylation targets and poly(A) tails have been shown to be associated with translation-related proteins, as discussed above, the idea of ​​using these RNA sequences outside their native mRNA environment to attract and recruit translation-related proteins is novel and inventive. In other words, naturally occurring cis elements in mRNA (e.g., the DRACH concordant sequence, GGACU (SEQ ID NO: 8), and / or the poly(A) tail) are converted into trans elements (i.e., PRS) for use in ACT-UP1 compounds, which can be administered to subjects to regulate gene expression. These PRS are further transformed to produce the novel PRS disclosed herein by chemically modifying their sequences to stabilize them, adding nucleotides to the sequences, and / or altering nucleotide sequences to enhance their protein recruitment function.

[0366] Surprisingly, ACT-UP1 compounds conjugated with short PRS and ASO effectively enhanced protein expression. As illustrated in the examples below, ACT-UP1 compounds effectively upregulated the expression of multiple target genes, achieving ACT-UP1-mediated protein upregulation both in vitro and in vivo. Therefore, the ACT-UP1 approach has the potential to significantly increase the number of targetable genes in subjects requiring enhanced protein expression (e.g., haplo-underdose patients) and expand potential therapeutic applications to a wider range of diseases.

[0367] The ACT-UP1 method and compounds have several advantages: As antisense compounds, ACT-UP1 compounds possess the mature delivery, chemical information, and stability of antisense compounds. Tolerance to various chemical modifications that support antisense compound specificity and activity allows for longer durations of action and lower dosing frequencies.

[0368] The ACT-UP1 compound contains a short PRS element. The short length of the PRS element results in a short ACT-UP1 compound, making its synthesis simpler and shorter, and requiring less material. Short ACT-UP1 compounds are more easily absorbed into cells or tissues, which is an important prerequisite for the safe and effective application of therapeutic agents.

[0369] In theory, ACT-UP1 compounds can be used to upregulate any protein of interest because the ASO element of ACT-UP1 compounds can be programmed to target any mRNA transcript, and the PRS element does not depend on the target to enhance protein expression.

[0370] ACT-UP1 compounds do not require co-delivery of exogenous proteins. Instead, when administered to subjects, ACT-UP1 compounds function independently to enhance target protein expression by recruiting endogenous translated proteins to the target mRNA. Therefore, ACT-UP1 compounds can be relatively readily used as therapeutic agents.

[0371] ACT-UP1 compounds primarily work by increasing translation without affecting the stability of the target mRNA, and may be a better treatment option for patients with haplo-insufficient doses, where the restoration of the target protein to normal levels does not exceed twice or approximately twice the protein levels observed in haplo-insufficient patients, thus avoiding excessive overexpression of the target protein.

[0372] ACT-UP1 compounds contain modular components, suitable for adding additional elements to the compound. For example, when significantly high protein expression levels are desired, ACT-UP1 compounds can be incorporated into sequences targeting AREs to further stabilize the target mRNA. Due to PRS, the homeostatic level of mRNA is higher and the translation of any single target mRNA is increased, as shown below. The bifunctional ACT-UP1 compound targeting FGF21 mRNA achieves the significantly high protein levels shown.

[0373] ACT-UP1 compounds allow for the design of ACT-UP1 compounds, enabling flexible modulation or fine-tuning of the protein expression level of target mRNAs in cells by selecting the location of the ACT-UP1 binding site in the target mRNA, modifications to the ACT-UP1 sequence, the length of the PRS, the type of the PRS, the position of the PRS relative to the ASO, the presence of existing elements (e.g., ARE target sequences), and the dosage of the ACT-UP1 compound.

[0374] Example Non-restricted public information and incorporated by reference While certain compounds, compositions, and methods described herein have been specifically described according to certain embodiments, the following examples are for illustrative purposes only and are not intended to limit the scope of the compounds described herein. Each reference cited in this application is incorporated herein by reference in its entirety.

[0375] The following applies to all modified sequences disclosed in this article.

[0376] The markings preceding or following each nucleoside indicate the type of chemical modification performed on that nucleoside (if any). If no markings precede or follow the letter representing the nucleoside, the nucleoside is a deoxyribonucleoside. The markings for chemical modifications of compounds can be as follows: The "(5p)" before nucleoside refers to 5'-phosphate. The "r" before nucleoside refers to ribonucleoside (ribonucleoside that has replaced deoxyribonucleoside). The "d" before (or without a label before or after) a nucleoside indicates a deoxynucleoside. The "f" before nucleoside refers to 2'-fluoro(2'-F) sugar modification. The "m" before nucleoside refers to 2'-OCH3 (also known as 2'-O-methyl, 2'-OMe) modification. The "e" before nucleoside indicates a 2'-O(CH2)2OCH3 modification (also known as 2'-O-(2-methoxyethyl), 2'-O-MOE, or 2'-MOE). “eCm” refers to 5-methylcytidine modified with 2'-O-MOE. “ "Refers to thiophosphate (PS) bonds that have been replaced by substituted phosphate (PO) bonds" The "gna" preceding nucleoside refers to ethylene glycol-modified nucleic acid. The "L" after nucleoside refers to the locked nucleic acid (LNA) modification of the nucleoside. “GL-GalNAc” is the GalNAc moiety described by Sharma et al. (2018, Bioconjugate Chem, 29:2478-2488). “AN-GalNAc” is the GalNAc part described in WO2024137545.

[0377] If more than one sequence is disclosed in a row of the table, the SEQ ID NO applies to the modified sequence (“Sequence + Chemical Information”). Bold and underlined sequences indicate the PRS of the ACT-UP1 compound.

[0378] Example 1: ACT-UP1 compound increases Jagged 1 protein levels Alagille syndrome (ALGS) is a rare genetic disorder primarily caused by haploinous deficiency mutations in the JAG1 gene (i.e., one allele of JAG1 remains wild-type, while the other allele contains a mutation that disrupts the normal production and / or function of the Jagged 1 protein) (Kohut, TJ, et al., 2021, Alagille Syndrome: AFocused Review on Clinical Features, Genetics, and Treatment. Semin Liver Dis41:525-537). These mutations result in insufficient levels of functional Jagged 1 protein, leading to impaired development of the hepatic bile ducts. This bile duct damage causes bile acids to accumulate in the liver and blood, resulting in liver and other tissue damage. Therefore, restoring functional Jagged 1 protein expression holds promise as a therapeutic option for ALGS.

[0379] An ACT-UP1 compound targeting human JAG1 mRNA (GenBank NM_000214.3; SEQ ID NO: 1) was designed to evaluate whether it could increase JAG1 expression. The ACT-UP1 compound contains two elements coupled together: (1) an antisense oligonucleotide targeting JAG1 mRNA (JAG1 ASO); and (2) a protein recruitment sequence (PRS).

[0380] The ASO element in the ACT-UP1 compound was designed to bind to the 3'UTR of JAG1 mRNA located approximately 160 nucleotides downstream of the stop codon.

[0381] The PRS elements in the ACT-UP1 compound were designed as single-stranded sequences containing a GGACU sequence (SEQ IDNO: 8), which commonly appears as an m6A modification site in mRNA (Linder, B., et al., 2015, Single-nucleotide-resolution mapping of m6A and m6Am throughout the transcriptome. Nat Methods 12: 767-772), or an incomplete repeat sequence containing the GGACU sequence, namely GGACUGGAC (SEQ IDNO: 10). The GGACU sequence can recruit cellular proteins to modify adenosine nucleotides and can regulate translation (Meyer, KD, 2019, m(6)A-mediated translation regulation. Biochim Biophys Acta GeneRegul Mech 1862: 301-309). The PRS elements in the ACT-UP1 compound are shown in Table 1 as bolded and underlined sequences.

[0382] The compound ATXL228, which contains only JAG1 ASO, targets the same JAG1 mRNA sequence as the ACT-UP1 compound and was used as a control.

[0383] To protect the compounds from nuclease degradation, these compounds are modified with 2'-O-methyl (also known as 2'-OMe), as shown in Table 1, with the modified nucleotides prefixed with "m".

[0384] Table 1. Sequences and chemical information of compounds

[0385] In vitro assay HeLa cells were seeded and allowed to grow to ~70% confluence for one day, then Lipofectamine was used. TMTransfection was performed using 2000 transfection reagent (ThermoFisher Scientific, Waltham, MA) with compounds listed in Table 1 at final concentrations of 7.5 nM or 15 nM, or blank transfection as a control. Twenty-four (24) hours post-transfection, cells were collected and lysed, and proteins were extracted using RIPA lysis extraction buffer (ThermoFisher Scientific, Waltham, MA). Jagged 1 protein levels were determined by Western blotting using a Jagged 1-specific antibody (ab109536, from Abcam, Waltham, MA). Western blot results are shown in Figure 2. Western blot images were quantified using ImageJ, and the results are shown in Table 2 as percentages of protein levels relative to blank transfected cells after normalization to the sample control protein GAPDH. GAPDH was not targeted by the assayed compounds, and GAPDH levels were unaffected, indicating that these compounds are specific in regulating target protein levels.

[0386] Table 2. Jagged 1 protein levels in cells treated with different compounds

[0387] The results showed that the ACT-UP1 compounds ATXL193 and ATXL261 increased the level of the targeted Jagged 1 protein. Furthermore, increasing the length of the PRS (ATXL193) increased Jagged 1 protein expression more significantly than the shorter PRS (ATXL261). On the other hand, the control JAG1ASO ATXL228, which lacked PRS but had the same mRNA binding sequence as both ACT-UP1 compounds, did not increase Jagged 1 protein levels, indicating the importance of the presence of PRS in upregulating JAG1 expression.

[0388] Example 2. The position of the protein recruitment sequence (PRS) affects activity. The tested ACT-UP1 compounds contained a protein recruitment sequence (PRS) at the 5' end. To determine whether ACT-UP1 compounds with a 3' PRS could also enhance protein levels, the ACT-UP1 compound ATXL384 was designed and synthesized. ATXL384 has the same mRNA-binding domain as ATXL193 previously described in Example 1 above and Table 3 below, but the PRS is located at the 3' end of the ACT-UP1 compound instead of the 5' end of JAG1 ASO. The tested PRS are shown in bold and underlined sequences in Table 3. The sequence and chemical information of ATXL384 are listed in Table 3.

[0389] Table 3. Sequence and chemical information of ATXL193 and ATXL384

[0390] In vitro assay HeLa cells were seeded and allowed to grow to ~70% confluence for one day, then Lipofectamine was used. TM Transfection was performed using 2000 transfection reagent (ThermoFisher Scientific, Waltham, MA) with compounds listed in Table 3 at final concentrations of 7.5 nM or 15 nM, or blank transfection as a control. Twenty-four (24) hours post-transfection, cells were collected and lysed, and proteins were extracted using RIPA lysis extraction buffer (ThermoFisher Scientific, Waltham, MA). Jagged 1 protein levels were determined by Western blotting using a Jagged 1-specific antibody (ab109536, from Abcam, Waltham, MA). Western blot results are shown in Figure 3. Western blots were quantified using ImageJ, and the results are shown in Table 4 as percentages of protein levels relative to blank transfected cells after normalization to the sample control protein GAPDH. GAPDH was not targeted by the assayed compounds, and GAPDH levels were unaffected, indicating that these compounds are specific in regulating target protein levels.

[0391] Table 4. Jagged 1 protein levels in HeLa cells

[0392] The results showed that the ACT-UP1 compound ATXL384 with 3' PRS failed to significantly increase the level of Jagged 1 protein, while under the same experimental conditions, the ACT-UP1 compound ATXL193 containing 5' PRS successfully increased the level of Jagged 1 protein again, indicating the importance of the position of PRS in ACT-UP1 compounds.

[0393] Example 3. The ASO binding site in mRNA can affect ACT-UP1 activity. To determine whether the binding position of ASO within the 3' UTR of the target mRNA affects the protein-enhancing activity of ACT-UP1 compounds, ACT-UP1 compounds were designed to bind to approximately 50 (ATXL257), 100 (ATXL258), 500 (ATXL259), and 1000 (ATXL260) nucleotides downstream of the stop codon in JAG1 mRNA. As shown in Table 5, these ACT-UP1 compounds contain the same PRS (bold and underlined sequence) at the 5' end of the JAG1 ASO.

[0394] To protect the compounds from nuclease degradation, these compounds were modified with 2'-O-methyl (also known as 2'-OMe), as shown in Table 5, with the modified nucleosides prefixed with "m". To further enhance the stability of the compounds, two phosphate thioesters (PS) were introduced immediately adjacent to the first two nucleosides, and are indicated in Table 5 with "m". "express.

[0395] Table 5. Sequence and chemical information of JAG1 ACT-UP1 compounds targeting different positions within the 3' UTR of JAG1 mRNA

[0396] In vitro assay HeLa cells were seeded and allowed to grow to ~70% confluence for one day, then Lipofectamine was used. TM Transfection was performed using 2000 transfection reagent (ThermoFisher Scientific, Waltham, MA) with compounds listed in Table 5 at final concentrations of 7.5 nM or 15 nM, or blank transfection as a control. Twenty-four (24) hours post-transfection, cells were collected and lysed, and proteins were extracted using RIPA lysis extraction buffer (ThermoFisher Scientific, Waltham, MA). Jagged 1 protein levels were determined by Western blotting using a Jagged 1-specific antibody (ab109536, from Abcam, Waltham, MA). Western blot results are shown in Figure 4. Western blots were quantified using ImageJ, and the results are shown in Table 6 as percentages of protein levels relative to blank transfected cells after normalization to the sample control protein GAPDH. GAPDH was not targeted by the assayed compounds, and GAPDH levels were unaffected, indicating that these compounds are specific in regulating target protein levels.

[0397] Table 6. Jagged 1 protein levels in cells treated with ACT-UP1 compounds targeting different positions within the 3' UTR of JAG1 mRNA

[0398] The results showed that ACT-UP1 compounds targeting different positions within the 3' UTR exhibited varying activities in enhancing protein levels. ASOs binding to positions closer to the stop codon showed better activity. These results further suggest that positions closer to the stop codon may be preferentially targeted.

[0399] Example 4. ACT-UP1 compound containing PS and 2'-MOE modification For antisense oligonucleotides (ASOs) previously described in the art, phosphate thioester (PS) backbone modification is typically used to increase nuclease resistance, enhance cellular uptake, and improve ASO drug performance. Therefore, the ACT-UP1 compound is designed to have a PS backbone modification.

[0400] Different amounts of PS backbone modifications were introduced into ACT-UP1 compounds similar to ATXL193 (as previously described), generating alternative ACT-UP1 compounds ATXL234 and ATXL262. Furthermore, since 2'-O-(2-methoxyethyl) (also known as 2'-O-MOE, 2'-MOE, or MOE) modification has been successfully used in ASO drugs with excellent safety benefits (Crooke, ST, et al., 2021a, Antisense technology: an overview and prospectus. Nat Rev DrugDiscov 20: 427-453), 2'-MOE modification was introduced into the ASO moiety of the ACT-UP1 compounds, where the ASO hybridizes to the target JAG1 mRNA sequence. To avoid the potential impact of the large size of the 2'-MOE on protein-protein recruitment sequence (PRS) binding, the PRS was modified with 2'-OMe. The sequence and chemical information of these ACT-UP1 compounds are listed in Table 7. In Table 7, PS is indicated by "…". The symbols indicate that 2'-OMe is represented by "m", 2'-MOE by "e", and 5-methylcytidine modified by 2'-MOE is represented by "eCm".

[0401] As shown in Table 7, the ACT-UP1 compound contains the same PRS (bold and underlined sequence) at the 5' end of JAG1 ASO.

[0402] Table 7. Sequence and chemical information of Jagged 1 ACT-UP1 compounds with different PS numbers

[0403] In vitro assay HeLa cells were seeded and allowed to grow to ~70% confluence for one day, then Lipofectamine was used. TM Transfection was performed using 2000 transfection reagent (ThermoFisher Scientific, Waltham, MA) with compounds listed in Table 7 at final concentrations of 7.5 nM or 15 nM, or blank transfection as a control. Twenty-four (24) hours post-transfection, cells were collected and lysed, and proteins were extracted using RIPA lysis extraction buffer (ThermoFisher Scientific, Waltham, MA). Jagged 1 protein levels were determined by Western blotting using a Jagged 1-specific antibody (ab109536, from Abcam, Waltham, MA). Western blot results are shown in Figure 5. Western blots were quantified using ImageJ, and the results are shown in Table 8 as percentages of protein levels relative to blank transfected cells after normalization to the sample control protein GAPDH. GAPDH was not targeted by the compounds being measured, and GAPDH levels were unaffected, indicating that these compounds are specific in regulating target protein levels.

[0404] Table 8. Jagged 1 protein levels in cells treated with ACT-UP1 compounds of varying amounts of PS

[0405] The results showed that ACT-UP1 compounds with multiple PS and 2'-MOE modifications could also increase Jagged 1 protein levels, and that 14 or 16 PS modifications in ACT-UP1 compounds did not show a significant difference in ACT-UP1 activity.

[0406] Example 5: The ACT-UP1 compound can increase the level of Jagged 1 protein in different cell types. The effects of the aforementioned ACT-UP1 compound ATXL193 were also evaluated in HEK293 cells. Lipofectamine was used. TMCells were transfected with ATXL193 at different concentrations using 2000 transfection reagent (ThermoFisher Scientific, Waltham, MA), or blank transfection was performed as a control. Twenty-four (24) hours post-transfection, cells were collected and lysed, and proteins were extracted using RIPA lysis extraction buffer (ThermoFisher Scientific, Waltham, MA). Jagged1 protein levels were determined by Western blotting using a Jagged1-specific antibody (ab109536, from Abcam, Waltham, MA). Western blot results are shown in Figure 6. Western blots were quantified using ImageJ, and the results are shown in Table 9, representing the percentage of protein levels relative to blank transfected cells after normalization to the sample control protein GAPDH. GAPDH was not targeted by the compounds being measured, and GAPDH levels were unaffected, suggesting that these compounds are specific in regulating target protein levels.

[0407] Table 9. Jagged 1 protein levels in HEK293 cells treated with ATXL193

[0408] The results showed that the use of the ACT-UP1 compound also increased the level of Jagged 1 protein in HEK293 cells, indicating that this upregulated phenotype is not unique to a specific cell type.

[0409] Example 6. The use of the ACT-UP1 compound can increase Rab9 protein levels. To determine whether the ACT-UP1 compound could enhance the levels of other proteins besides Jagged 1, it was engineered to target RAB9, a protein encoding the endosome lysosomal system. The ACT-UP1 compound was engineered to bind to an 18-nucleotide region of RAB9 mRNA approximately 90 nt downstream of the stop codon of human RAB9A mRNA (GenBank ID: NM_004251.5, SEQ ID NO: 2). To determine whether altering the length and position of the ACT-UP1 PRS affected protein level enhancement, two additional nucleosides were added to the 5' end of the PRS, and a nucleoside was inserted between the two GGACU sequences. Sequence and chemical information are shown in Table 10. In Table 10, PS is indicated by "...". The symbol “” indicates that 2'-OMe is represented by “m”. As shown in Table 10, the PRS of the ACT-UP1 compound is the bolded and underlined sequence at the 5' end of RAB9 ASO.

[0410] Table 10. Sequence and chemical information of ATXL230

[0411] In vitro assay HeLa cells were seeded and allowed to grow to ~70% confluence for one day, then Lipofectamine was used. TM Transfection was performed using 2000 transfection reagent (ThermoFisher Scientific, Waltham, MA) with final concentrations of 5 nM, 10 nM, 20 nM, and 40 nM of the ACT-UP1 compound ATXL230, or as a blank control. Twenty-four (24) hours post-transfection, cells were collected and lysed, and proteins were extracted using RIPA lysis extraction buffer (ThermoFisher Scientific, Waltham, MA). RAB9 protein levels were determined by Western blotting using a RAB9-specific antibody (ab2810, from Abcam, Waltham, MA). Western blot results are shown in Figure 7. Western blots were quantified using ImageJ, and the results are shown in Table 11 as percentages of protein levels relative to blank transfected cells after normalization to the sample control protein GAPDH. GAPDH was not targeted by the assayed compounds, and GAPDH levels were unaffected, suggesting that these compounds are specific in regulating target protein levels.

[0412] Table 11. Rab9 protein levels in HeLa cells treated with ATXL230

[0413] The results showed that although the ACT-UP1 compound contained PRS (13 nt) that had been further elongated by additional nucleotides, it was also able to increase Rab9 protein.

[0414] Example 7. The use of the ACT-UP1 compound can increase RNase H1 protein. RNase H1 protein is an endonuclease involved in R-loop solving and genome integrity (Cerritelli, SM, and RJ Crouch, 2019 RNases H: Multiple roles in maintaining genome integrity. DNA Repair (Amst) 84: 102742). Increasing the level of this protein has potential benefits for the treatment of myelodysplastic syndromes (Chen, L., et al., 2018, The Augmented R-Loop Is a Unifying Mechanism for Myelodysplastic Syndromes Induced by High-Risk Splicing Factor Mutations. Mol Cell 69: 412-425 e416). The ACT-UP1 compound ATXL231 was designed to bind to the 3' UTR of human RNase H1, where 18 nucleotides hybridize with RNase H mRNA approximately 80 nucleotides downstream of the stop codon of human RNase H mRNA (GenBank ID: NM_001286834.3, SEQ ID NO: 3). ATXL231 targeting RNase H1 was designed to have the same PRS as previously described for ATXL230 targeting RAB9. The sequence and chemical information of ATXL231 are shown in Table 12. In Table 12, PRS are indicated by "…". The symbol “” indicates that 2'-OMe is represented by “m”. As shown in Table 12, the PRS of the ACT-UP1 compound is the bolded and underlined sequence at the 5' end of RNase H1 ASO.

[0415] Table 12. Sequence and chemical information of ATXL231

[0416] In vitro assay HeLa cells were seeded and allowed to grow to ~70% confluence for one day, then Lipofectamine was used. TMCells were transfected with ACT-UP1 compound ATXL231 at final concentrations of 5 nM, 10 nM, 20 nM, and 40 nM using 2000 transfection reagent (ThermoFisher Scientific, Waltham, MA), or blank transfection was performed as a control. Twenty-four (24) hours post-transfection, cells were collected and lysed, and proteins were extracted using RIPA lysis extraction buffer (ThermoFisher Scientific, Waltham, MA). RNase H1 protein levels were determined by Western blotting using an RNase H1-specific antibody (15606-1-AP, from Proteintech). ® (Rosemont, IL). Western blot results are shown in Figure 8. Western blots were quantified using ImageJ, and the results are shown in Table 13, representing the percentage of protein levels relative to blank transfected cells after normalization for the loading control protein GAPDH. GAPDH was not targeted by the assayed compounds, and GAPDH levels were unaffected, indicating that these compounds are specific in regulating target protein levels.

[0417] Table 13. RNase H1 protein levels in HeLa cells treated with ATXL231

[0418] The results showed that the use of ACT-UP1 compounds significantly increased the level of RNase H1 protein, indicating that the technology can increase different target proteins, and that active ACT-UP1 compounds can contain different lengths or sequences.

[0419] Example 8. The use of the ACT-UP1 compound can increase PBGD protein. Acute intermittent porphyria (AIP) is a severe genetic disorder caused by a haploinufficient mutation in the bilirubinogen deaminase gene (PBGD, also known as hydroxymethylcholine synthase (HMBS)). PBGD is a protein required for the heme biosynthesis pathway. Insufficient PBGD levels in subjects can lead to the accumulation of heme synthesis intermediates. Therefore, increasing PBGD levels in subjects could potentially restore normal heme biosynthesis and treat the disease.

[0420] The ACT-UP1 compound ATXL243 was designed to bind to the 3' UTR of PBGD, located at a conserved sequence approximately 190 nucleotides downstream of the stop codon in human PBGD mRNA (GenBank ID: NM_000190.4, SEQ ID NO: 4) between human, monkey, and mouse PBGD mRNAs. The sequence and chemical information of ATXL243 are shown in Table 14. In Table 14, PS is indicated by "..." “” indicates that 2'-OMe is represented by “m”. As shown in Table 14, the PRS of the ACT-UP1 compound is the bolded and underlined sequence at the 5' end of PBGD ASO.

[0421] Table 14. Sequence and chemical information of ATXL243 targeting PBGD mRNA

[0422] In vitro assay HeLa cells were seeded and allowed to grow to ~70% confluence for one day, then Lipofectamine was used. TM Transfection was performed using 2000 transfection reagent (ThermoFisher Scientific, Waltham, MA) with final concentrations of 5 nM, 10 nM, 20 nM, 40 nM, and 80 nM of the ACT-UP1 compound ATXL231, or as a blank transfection control. Twenty-four (24) hours post-transfection, cells were collected and lysed, and proteins were extracted using RIPA lysis extraction buffer (ThermoFisher Scientific, Waltham, MA). PBGD protein levels were determined by Western blotting using a PBGD-specific antibody (ab129092, from Abcam, Waltham, MA). Western blot results are shown in Figure 9. Western blots were quantified using ImageJ, and the results are shown in Table 15 as percentages of protein levels relative to blank transfected cells after normalization for the sample control protein nucleolin (NCL). NCL was not targeted by the assayed compounds, and NCL levels were unaffected, suggesting that these compounds are specific in regulating target protein levels.

[0423] Table 15. PBGD protein levels in HeLa cells transfected with ATXL243

[0424] The results showed that the ACT-UP1 compound could also increase the level of PBGD protein, further demonstrating that the ACT-UP1 upregulation method described in this paper can be applied to upregulate the expression of different target nucleic acids.

[0425] Example 9: The ACT-UP1 compound can increase PBGD protein in mouse Hepa1-6 cells. To determine whether ACT-UP1-mediated increases in PBGD protein in human HeLa cells could also be observed in different species, ACT-UP1 compounds were engineered to target mouse PBGD mRNA.

[0426] The ACT-UP1 compound ATXL319 was designed to target approximately 150 nt downstream of the stop codon of mouse PBGD mRNA (GenBank ID: NM-013551.2, SEQ ID NO: 6). To enhance binding affinity, ATXL319 contains three locked nucleic acid (LNA) modified nucleotides at its 3' end. ATXL320 and ATXL321 were designed to target a site 5 nucleotides upstream of the ATXL319 target site, but with different lengths of ASO hybridization regions. The sequences and chemical information of the compounds are listed in Table 16. In Table 16, PS is indicated by "..." The text indicates that 2'-OMe is preceded by "m" before the modified nucleoside, and LNA is followed by "L" after the modified nucleoside. [mCL] specifically represents LNA 5-methylcytidine 5'-thiophosphate. As shown in Table 16, the PRS of the ACT-UP1 compounds is the bolded and underlined sequence at the 5' end of PBGD ASO. Each compound further comprises a GalNAc conjugate, as described in WO2024137545, and is designated as "AN-GalNAc".

[0427] Table 16. Sequence and chemical information of ACT-UP1 compounds targeting mouse PGBD

[0428] In vitro assays – protein assessment Hepa1-6 cells were inoculated and allowed to grow to ~70% confluence for one day, then Lipofectamine was used. TMTransfection was performed using 2000 transfection reagent (ThermoFisher Scientific, Waltham, MA) with ACT-UP1 compound at final concentrations of 7.5 nM or 15 nM, or as a control. Twenty-four (24) hours post-transfection, cells were collected and lysed, and proteins were extracted using RIPA lysis extraction buffer (ThermoFisher Scientific, Waltham, MA). PBGD protein levels were determined by Western blotting using a PBGD-specific antibody (ab129092, from Abcam, Waltham, MA). Western blotting results are shown in Figure 10A, and a plot of the results is shown in Figure 10B. Western blotting was quantified using ImageJ, and the results are shown in Table 17 as a percentage of protein levels relative to blank transfected cells after normalization for the sample control protein NCL. NCL was not targeted by the assayed compounds, and NCL levels were unaffected, indicating that these compounds are specific in regulating target protein levels.

[0429] Table 17. PBGD protein levels in mouse Hepa1-6 cells transfected with ACT-UP1 compound.

[0430] Overall, all three ACT-UP1 compounds increased protein levels, with higher doses showing greater protein increases. Although the different compounds may have varying degrees of effect on increasing PBGD protein levels, the results demonstrate that the use of ACT-UP1 compounds can increase PBGD protein levels in mouse cells, suggesting that the ACT-UP1 approach described herein is not species-specific.

[0431] In vitro assay – mRNA assessment To determine whether the ACT-UP1 compound, which showed an increase in protein levels, also affected PBGD mRNA levels, total RNA was collected from cells as described above, and PBGD mRNA levels were determined using quantitative real-time PCR (qRT-PCR) with a mouse PBGD-specific TaqMan primer and probe set (Mm01143545_m1, from ThermoFisher Scientific, Waltham, MA). qRT-PCR was performed using AgPath-ID™ one-step RT-PCR reagents on a QS3 real-time PCR system (ThermoFisher Scientific, Waltham, MA). The target PBGD mRNA levels detected in the qRT-PCR assay were compared with those detected in aliquots of the corresponding RNA samples using Ribogreen.TM Total RNA levels were normalized as measured by ThermoFisher Scientific, Waltham, MA. mRNA levels are shown in Figure 10C and Table 18.

[0432] Table 18. PBGD mRNA levels in cells treated with various ACT-UP1 compounds

[0433] The results showed that the ACT-UP1 compound increased PBGD protein levels but did not significantly affect PBGD mRNA levels. This indicates that the ACT-UP1 compound can increase protein levels by enhancing translation rather than by increasing mRNA levels.

[0434] Example 10. The use of the ACT-UP1 compound can increase FGF21 protein. Fibroblast growth factor 21 (FGF21) is a hormone that regulates important metabolic pathways, including energy balance and glucose and lipid homeostasis. Administration of FGF21 to rodents or non-human primates has resulted in considerable pharmacological benefits against a range of obesity-related metabolic complications, including reduced fat mass and relief of hyperglycemia, insulin resistance, dyslipidemia, cardiovascular disease, and non-alcoholic steatohepatitis (NASH). Currently, FGF21 and its analogues are being tested in clinical trials. However, due to the relatively short half-life of synthetic FGF21 and potential immune responses, increasing endogenous FGF21 protein expression is attractive for the treatment of chronic metabolic diseases.

[0435] The ACT-UP1 compound ATXL251 was designed to bind to the 3'UTR of human FGF21 mRNA (GenBank ID: NM_019113.4, SEQ ID NO: 5) approximately 90 nucleotides downstream of the termination codon. The sequence and chemical information of ATXL251 are listed in Table 19. In Table 19, PS is indicated by "..." The symbols “” indicate that 2'-OMe is indicated by “m” before the modified nucleoside (e.g., mG), 2'-MOE is indicated by “e” before the modified nucleoside (e.g., eG), and 5-methylcytidine modified by 2'-MOE is indicated by “eCm”. As shown in Table 19, the PRS of ATXL251 is the bolded and underlined sequence at the 5' end of FGF21 ASO.

[0436] Table 19. Sequence and chemical information of ATXL251

[0437] In vitro assay Based on the Protein Atlas database (www.proteinatlas.com), Hep3B cells express relatively high levels of FGF21. Hep3B cells were seeded and allowed to grow for one day until ~70% confluence was achieved, and then Lipofectamine was used. TM Transfection was performed using ACT-UP1 compound at final concentrations of 5 nM, 10 nM, 20 nM, 40 nM, or 80 nM using 2000 transfection reagent (ThermoFisher Scientific, Waltham, MA), or as a blank transfection control. Twenty-four (24) hours post-transfection, cells were collected and lysed, and proteins were extracted using RIPA lysis extraction buffer (ThermoFisher Scientific, Waltham, MA). FGF21 protein levels were determined by Western blotting using an FGF21-specific antibody (ab171941, from Abcam, Waltham, MA). Western blot results are shown in Figure 11. Western blots were quantified using ImageJ, and the results are shown in Table 20 as percentages of protein levels relative to blank transfected cells after normalization for the sample control protein NCL. NCL was not targeted by the assayed compounds, and NCL levels were unaffected, indicating that these compounds are specific in regulating target protein levels.

[0438] Table 20. FGF21 protein levels in Hep3B cells transfected with ATXL251

[0439] The results showed that ATXL251 could increase the level of FGF21 protein, which further confirms that the ACT-UP1 method described in this paper can be applied to increase the protein levels of different genes.

[0440] Example 11. The use of the ACT-UP1 compound increased FGF21 protein in mouse cells. To determine whether ACT-UP1-mediated increase in FGF21 protein in human HeLa cells could also be observed in different species, ACT-UP1 compounds were engineered to target the 3'UTR of mouse FGF21 mRNA.

[0441] The ACT-UP1 compound was designed to target approximately 70 nt downstream of the stop codon of mouse FGF21 mRNA (GenBank ID: NM-020013.4, SEQ ID NO: 7) (ATXL318). This sequence is conserved among humans, monkeys, and mice. Additionally, ATXL317, derived from ATXL251 but possessing a GalNAc conjugate (as described in WO2024137545), was synthesized. ATXL317 exhibits two mismatches with mouse FGF21 mRNA. The sequence and chemical information of the compounds are listed in Table 21. In Table 21, PS is indicated by "..." The designation “” indicates that 2’-OMe is indicated by “m” before the modified nucleoside (e.g., mG), 2’-MOE is indicated by “e” before the modified nucleoside (e.g., eG), and 5-methylcytidine modified with 2’-MOE is indicated by “eCm”. In Table 21, the PRS of the ACT-UP1 compounds are shown as a bolded and underlined sequence at the 5’ end of the FGF21 ASO. Each compound further comprises a GalNAc conjugate, as described in WO2024137545, and is designated as AN-GalNAc.

[0442] Table 21. Sequence and chemical information of ACT-UP1 compounds targeting mouse FGF21 mRNA

[0443] In vitro assays – protein assessment Hepa1-6 cells were inoculated and allowed to grow to ~70% confluence for one day, then Lipofectamine was used. TM Transfection was performed using 2000 transfection reagent (ThermoFisher Scientific, Waltham, MA) with ACT-UP1 compound at final concentrations of 7.5 nM or 15 nM, or as a control. Twenty-four (24) hours post-transfection, cells were collected and lysed, and proteins were extracted using RIPA lysis extraction buffer (ThermoFisher Scientific, Waltham, MA). FGF21 protein levels were determined by Western blotting using an FGF21-specific antibody (ab171941, from Abcam, Waltham, MA). Western blotting results are shown in Figure 12A, and a plot of the results is shown in Figure 12B. Western blotting was quantified using ImageJ, and the results are shown in Table 22 as percentages of protein levels relative to blank transfected cells after normalization for the sample control protein NCL. NCL was not targeted by the assayed compounds, and NCL levels were unaffected, indicating that these compounds are specific in regulating target protein levels.

[0444] Table 22. FGF21 protein levels in mouse Hepa1-6 cells transfected with ACT-UP1 compound.

[0445] Overall, all evaluated ACT-UP1 compounds increased FGF21 protein levels, with higher doses showing greater protein increase compared to lower doses. Consistent with the results observed in the Jagged 1 ASO example above, the FGF21 ACT-UP1 compound ATXL318 (hybridizing closer to the stop codon) appeared to increase FGF21 protein expression more than ATXL317 (hybridizing further away from the stop codon and having two mismatches). In conclusion, these results demonstrate that the use of ACT-UP1 compounds can increase FGF21 protein levels in mouse cells, further indicating that the ACT-UP1 approach described herein is not species-specific.

[0446] In vitro assay – mRNA assessment To determine whether the ACT-UP1 compound, which showed an increase in protein levels, also affected FGF21 mRNA levels, total RNA was collected from cells as described above, and FGF21 mRNA levels were determined using quantitative real-time PCR (qRT-PCR) with a mouse FGF21-specific TaqMan primer and probe set (Mm07297622_g1, from ThermoFisher Scientific, Waltham, MA). qRT-PCR was performed using the AgPath-ID™ one-step RT-PCR reagent in a QS3 real-time PCR system (ThermoFisher Scientific, Waltham, MA). The target FGF21 mRNA levels detected in the qRT-PCR assay were compared with those detected in aliquots of RNA samples using Ribogreen. TM Total RNA levels were normalized based on measurements taken at ThermoFisher Scientific, Waltham, MA. mRNA levels are shown in Figure 12C and Table 23.

[0447] Table 23. FGF21 mRNA levels in Hepa1-6 cells treated with ACT-UP1 compound

[0448] The results showed that the ACT-UP1 compound increased FGF21 protein levels but not FGF21 mRNA levels. This indicates that the ACT-UP1 compound can increase protein levels by enhancing translation rather than by increasing mRNA levels.

[0449] Example 12. ACT-UP1 can recruit cellular proteins that may be involved in translation. “ASO-coupled translation-upregulation 1” or “ACT-UP1” refers to an antisense oligonucleotide (ASO) linked to a protein recruitment sequence (PRS).

[0450] ASO specifically hybridizes with the target mRNA sequence, bringing PRS to a location immediately adjacent to the target mRNA. As shown in the previous examples, it targets specific nucleic acids and can increase the expression of specific target proteins.

[0451] PRS is a short sequence of linked nucleosides. It is not an antisense oligonucleotide (ASO) because it does not hybridize with the target nucleic acid (i.e., the protein recruitment sequence is neither an antisense sequence of the target nucleic acid nor does it pair with bases specific to the target nucleic acid sequence).

[0452] Unbound by any particular theory, it is believed that the PRS element of the ACT-UP1 compound draws translation regulatory proteins to the target mRNA that is adjacent to the ASO element of the ACT-UP1 compound, thereby increasing the translation of the targeted mRNA.

[0453] To evaluate this hypothesis, a 17-nucleotide oligonucleotide (ATXL263) was designed and synthesized. ATXL263 contains a 5' biotin and a 3' UTR sequence derived from JAG1 mRNA (SEQ ID NO: 1). The sequence and chemical information of ATXL263 are shown in Table 24. In Table 24, 2'-OMe is indicated by "m".

[0454] Table 24. Sequence and chemical information of ATXL263

[0455] ATXL263 can form a 17-base-pair-long double strand with the previously described compounds ATXL228, ATXL261, and ATXL193 (see Table 1). Additionally, as shown in Table 1, ATXL228 has no PRS component and is only ASO. ATXL261 is an example of an ACT-UP1 compound with a PRS length of 5 nucleosides, and ATXL193 is an ACT-UP1 compound with a PRS length of 9 nucleosides.

[0456] ATXL263 was added to solutions of the other three compounds in 1× phosphate buffered saline, and these solutions were heated at 94°C for 4 min in a modular heating apparatus to form 30 μM double strands. The heating module containing the solutions was then removed from the modular heating apparatus and allowed to cool to room temperature gradually over 1 hour.

[0457] Neutroavidin resin (Thermofisher Scientific, Waltham, MA) was pre-washed with W100 buffer (50 mM Tris-HCl (pH 7.5), 100 mM NaCl, 5 mM EDTA, and 0.1% NP40) and then incubated with 30 μM of duplexes. These duplexes were then attached to Neutroavidin beads (see Figure 13A for a schematic diagram). After washing twice with W100 buffer to remove excess duplexes, the resin was blocked for 1 hour with blocking buffer (W100 containing 10 mg / mL BAS, 1.2 mg / mL glycogen, and 0.2 mg / mL transfer RNA). Then, after washing twice with W100, 2.5 mg of total protein prepared from HeLa cells was incubated with the duplex-coated beads for 2 hours. The protein associated with the duplex was washed and eluted from the beads by boiling in 2X SDS loading buffer (ThermoFisher Scientific, Waltham, MA).

[0458] The isolated proteins were analyzed on SDS-PAGE, and the presence of several proteins that may regulate translation was assessed: PABPC1 and m6A recognition proteins (e.g., Mettl3, YTHDF1, and ALKBH5).

[0459] PABPC1 It has recently been shown that by guiding the RNA-Cas13-PABPC1 fusion system, the fusion protein can be recruited to the 3'UTR of the target mRNA to increase the level of the target protein (Torkzaban et al., Biotechnol. J., 2022 Oct, 17(10):e2200214). PABPC1 is an abundant cytoplasmic poly(A)-binding protein that is essential for translation (Lemay, JF, et al., 2010 Crossing the borders: poly(A)-binding proteins working on both sides of the fence. RNA Biol 7: 291-295). Therefore, by using a specific PABPC1 antibody (10970-1-AP, from Proteintech...).® Western blot analysis (Rosemont, IL, USA) was used to evaluate the co-segregation of PABPC1 and ACT-UP1 compounds. The results shown in Figure 13B indicate that the significant co-segregation between PABPC1 and ACT-UP1 compounds occurred in a PRS length-dependent manner; higher levels of PABPC1 protein were found to co-segregate with ATXL193, an ACT-UP1 compound with a longer PRS, compared to ATXL261, which has a shorter PRS. This is consistent with previous findings that RNA length can affect the binding affinity of PABPC1 (Sachs, AB, et al., 1987, A single domain of yeast poly(A)-binding protein is necessary and sufficient for RNA binding and cell viability. Mol Cell Biol 7: 3268-3276).

[0460] As a control, another RNA-binding protein, HuR (antibody ab200342, from Abcam, Cambridge, UK), did not show significant PRS-dependent binding to these compounds (Figure 13B). These results suggest that ACT-UP1 compounds can recruit the RNA-binding protein PABPC1 through their PRS components, which can enhance the translation of target mRNAs.

[0461] In addition, the binding of a few proteins involved in the m6A modification pathway to the ACT-UP1 compound was evaluated. m6A is a nucleotide modification present in many mRNAs, and it can regulate translation by binding to m6A recognition proteins (He, PC, and C. He, 2021, m(6)A RNA methylation: from mechanisms to therapeutic potential. EMBO J 40: e105977; Meyer, KD, 2019, m(6)A-mediated translation regulation. Biochim Biophys Acta Gene Regul Mech 1862: 301-309). The proteins evaluated included: Mettl3 (antibody ab195352, from Abcam, Waltham, MA), an m6A writer protein; YTHDF1 (antibody ab252346, from Abcam, Waltham, MA), an m6A reader protein; and ALKBH5 (antibody ab195377, from Abcam, Waltham, MA), an m6A eraser protein (Huang, H., et al., 2020, The Biogenesis and Precise Control of RNA m(6)A Methylation. Trends Genet 36: 44-52). Western blot results are shown in Figure 13C.

[0462] Data indicate that ACT-UP1 compounds significantly recruit YTHDF1 and ALKBH5, and recruit Mettl3 protein to a lesser extent. As a control, ASO ATXL228 without PRS binds very weakly to these proteins. Similar to PABPC1, these proteins also bind more strongly to ACT-UP1 compounds with longer PRS (ATXL193) compared to ACT-UP1 compounds with shorter PRS (ATXL261). This protein binding trend is consistent with the observation that ATXL193 caused a greater increase in Jagged 1 protein compared to ATXL261, while ATXL228 (without PRS) did not increase this protein level (see Example 1 above). These combined results suggest that ACT-UP1 compounds can recruit cellular RNA-binding proteins, including Mettl3, YTHDF1, and ALKBH5, to target mRNAs, thereby enhancing translation.

[0463] Example 13. Different PRS can increase target protein levels. The protein recruitment sequence (PRS) described above is derived from the consortium sequence GGACU (SEQ ID NO: 8), which is present in mRNAs that preferentially undergo m6A modification (Linder, B., et al., 2015, Single-nucleotide-resolution mapping of m6A and m6Am throughout the transcriptome. Nat Methods 12: 767-772). The aforementioned example demonstrates that PABPC1 can be recruited to the GGACU-derived PRS.

[0464] Next, we evaluated the ability of other sequences to act as protein recruitment sequences (PRS) in the ACT-UP1 compound.

[0465] ACT-UP1 compounds were designed based on the Jagged 1-targeting compound ATXL234, using the same ASO sequence but with different PRS. The PRS components of the ACT-UP1 compounds were designed as derivatives of the poly(A) sequence because PABP proteins have been shown to preferentially bind to poly(A) sequences (Lemay, JF, et al., 2010 Crossing the borders: poly(A)-binding proteins working on both sides of the fence. RNABiol 7: 291-295). The sequences and chemical modifications of these compounds are shown in Table 25. As shown in Table 25, PS is used to define… The symbols “m” indicate that 2'-OMe is indicated by “m” before the modified nucleoside, and 2'-MOE is indicated by “e” before the modified nucleoside (e.g., eG), and 5-methylcytidine modified with 2'-MOE is indicated by “eCm”. In Table 25, the PRS of the ACT-UP1 compounds are shown as the bolded and underlined sequence at the 5' end of JAG1 ASO. Each newly designed compound further comprises a GalNAc conjugate, as described in WO2024137545, and is designated as AN-GalNAc. ATXL316 has the same sequence and sequence modifications as ATXL234, but additionally has an attached GalNAc conjugate. ATXL234 is as previously described above.

[0466] Table 25. Sequence and chemical information of ACT-UP1 compounds containing different PRS

[0467] In vitro assay HeLa cells were seeded and allowed to grow to ~70% confluence for one day, then Lipofectamine was used. TM Transfection was performed using 2000 transfection reagent (ThermoFisher Scientific, Waltham, MA) with ACT-UP1 compounds at final concentrations of 7.5 nM and 15 nM, or as a control (blank transfection). Twenty-four (24) hours post-transfection, cells were collected and lysed, and proteins were extracted using RIPA lysis extraction buffer (ThermoFisher Scientific, Waltham, MA). Jagged 1 protein levels were determined by Western blotting using a Jagged 1-specific antibody (ab109536, from Abcam, Waltham, MA). Western blot results are shown in Figure 14. Western blots were quantified using ImageJ, and the results are shown in Table 26 as percentages of protein levels relative to blank transfected cells after normalization for the sample control protein NCL. NCL was not targeted by the assayed compounds, and NCL levels were unaffected, indicating that these compounds are specific in regulating target protein levels.

[0468] Table 26. Jagged 1 protein levels in HeLa cells transfected with ACT-UP1 compounds containing different PRS.

[0469] The results showed that other short linker nucleoside sequences (e.g., AAACUAAACU or AAAAAAAAAA) could also function as PRS in ACT-UP1 compounds and enhance the level of the target Jagged 1 protein. However, reducing the Poly(A) length to 8 nt appeared to eliminate the PRS function in ACT-UP1 compounds. The data suggest that PRS length is a significant factor influencing the activity of ACT-UP1 compounds, likely because it affects the recruitment of important proteins that regulate translation, including PABPC1. In conclusion, these results indicate that different PRS sequences can be employed to enhance protein levels.

[0470] Example 14. The use of the ACT-UP1 compound increased Jagged 1 protein in mouse liver. To determine whether ACT-UP1 compounds that enhance protein levels in vitro can also enhance protein levels in vivo, a novel ACT-UP1 compound, ATXL246, was designed and synthesized. ATXL246 is derived from the ACT-UP1 compound ATXL234, but three nucleotides near the 3' end of the ASO region were removed to determine whether shorter base pairs could still enhance protein levels. Furthermore, ATXL246 contains a GalNAc conjugate (as described in WO2024137545) and is designated as AN-GalNAc to facilitate hepatic delivery. The sequence and chemical information of ASO ATXL246 are listed in Table 27. In Table 27, PS is indicated by " The designation “” indicates that 2’-OMe is indicated by “m” before the modified nucleoside (e.g., mG), 2’-MOE is indicated by “e” before the modified nucleoside (e.g., eG), and 5-methylcytidine modified with 2’-MOE is indicated by “eCm”. ATXL316 was previously described in Table 25 above. AN-GalNAc is the GalNAc conjugate described in WO2024137545.

[0471] Table 27. Sequence and chemical information of ATXL246

[0472] Three male Balb / C mice aged 7 to 8 weeks in each group received subcutaneous injections of ATXL246 or ATXL316 at doses of 0.5 mg / kg or 3 mg / kg. Phosphate-buffered saline (PBRS) was used as a control. Ninety-six hours after administration, the mice were given a second dose, and after another 96 hours, they were sacrificed and their organs were collected for analysis. Total liver protein was prepared from the mice and analyzed using Western blotting. Jagged 1 protein was detected using an antibody (ab109536, from Abcam, Waltham, MA). The Western blotting results are shown in Figure 15. Western blots were quantified using Image J, and the results are shown in Table 28, representing the percentage of protein levels in PBS-treated control mice after normalization for the sample control protein α-tubulin.

[0473] Table 28. Jagged 1 protein levels in mice treated with ATXL246 and ATXL316

[0474] The results showed that the ACT-UP1 compounds ATXL246 and ATXL316 could increase the level of Jagged 1 protein in animals, consistent with the results observed in vitro.

[0475] Example 15. The use of the ACT-UP1 compound can increase HNF4A protein in cells. HNF4A is a transcription factor important for metabolic pathways in the liver. Protein levels tend to decrease with the progression of metabolic diseases (Baciu et al., 2017, PLoS ONE 12(12): e0189223; Lu et al., 2022, Lipids Health Dis., 21(1):46). Increasing HNF4A protein levels in the liver via mRNA delivery or saRNA has resulted in beneficial effects on disease phenotypes, including NASH and obesity (Yang et al., 2021, J Hepatol., 75(6):1420-1433; Huang et al., 2020, Mol Ther Nucleic Acids, 19:361-370). To determine whether the ACT-UP1 mechanism could increase HNF4A protein levels, ACT-UP1 compounds were engineered to target the 3'UTR of human HNF4A mRNA.

[0476] Four ACT-UP1 compounds were designed to target human HNF4A mRNA (GenBank ID: NM_178849.3, SEQ ID NO: 45) downstream of the stop codon for approximately 70 nt (ATXL394), 95 nt (ATXL395), 140 nt (ATXL396), and 240 nt (ATXL397). These sequences were conserved between humans and monkeys. ATXL395 was also conserved in mice. The sequences and chemical information of the compounds are listed in Table 29. As shown in Table 29, PS is indicated by " The 'm' indicates that 2'-OMe is preceded by 'm' before the modified nucleoside. In Table 29, the PRS of the ACT-UP1 compound is shown as the bolded and underlined sequence at the 5' end of HNF4A ASO.

[0477] Table 29. Sequence and chemical information of ACT-UP1 compounds targeting human HNF4A mRNA

[0478] In vitro assays – protein assessment Hep3B cells were seeded and allowed to grow to ~70% confluence for one day, then Lipofectamine was used. TMTransfection was performed using 2000 transfection reagent (ThermoFisher Scientific, Waltham, MA) with ACT-UP1 compound at final concentrations of 7.5 nM or 15 nM, or as a control (blank transfection). Twenty-four (24) hours post-transfection, cells were collected and lysed, and proteins were extracted using RIPA lysis extraction buffer (ThermoFisher Scientific, Waltham, MA). HNF4A protein levels were determined by Western blotting using an HNF4A-specific antibody (ab181604, from Abcam, Waltham, MA). Western blotting results are shown in Figure 16A, and a bar chart of the quantified results is shown in Figure 16B. Western blotting was quantified using ImageJ, and the results are shown in Table 30, representing the percentage of protein levels relative to the mean of four blank transfected cell samples after normalization to the sample control protein NCL. NCL was not targeted by the assayed compounds, and NCL levels were unaffected, indicating that these compounds are specific in regulating target protein levels.

[0479] Table 30. HNF4A protein levels in human Hep3B cells transfected with ACT-UP1 compound

[0480] Overall, the evaluated ACT-UP1 compounds increased HNF4A protein levels. In conclusion, the results demonstrate that the use of ACT-UP1 compounds can increase HNF4A protein levels in human cells, further indicating that the ACT-UP1 method described herein can be applied to various genes.

[0481] Example 16. GalNAc-conjugated ACT-UP1 compounds showed better activity in increasing JAG1 protein levels. To determine whether ACT-UP1 compounds without the GalNAc conjugate could also enhance protein levels, several ACT-UP1 compounds were designed and evaluated. Compound ATXL398 has the same sequence and sequence modifications as the previously described ATXL246, but lacks the GalNAc conjugate of ATXL246. Compound ATXL282 targets a different position on the 3' UTR of Jagged 1 mRNA than ATXL398. Compound ATXL283 has the same sequence and sequence modifications as ATXL282, but lacks the GalNAc conjugate. Sequence and chemical information for these compounds is listed in Table 31. The GalNAc conjugate is designated AN-GalNAc and described in WO2024137545.

[0482] Table 31. Sequence and chemical information of ASO

[0483] Three male Balb / C mice aged 7 to 8 weeks in each group received subcutaneous injections of 0.5 mg / kg or 3 mg / kg of the GalNAc-conjugated compounds ATXL246 or ATXL316. Additionally, the corresponding non-GalNAc-conjugated counterparts ATXL398 and ATXL234 were administered at 15 mg / kg and 50 mg / kg, respectively. The non-GalNAc-conjugated compounds were administered at higher concentrations because they entered cells less efficiently than the GalNAc-conjugated compounds.

[0484] In another study, ATXL282 was administered at 0.5 mg / kg or 2.5 mg / kg, while the ATXL283 compound was administered at 25 mg / kg. Phosphate-buffered saline (PBS) was injected as a control in each case. Mice were given a second dose 96 hours after initial administration, and then sacrificed and their organs were collected for analysis after another 96 hours. Total liver protein was prepared from the mice and analyzed using Western blotting. Jagged 1 protein was detected using an antibody (ab109536, from Abcam, Cambridge, UK). Western blotting results are shown in Figures 17A and 17B. Western blots were quantified using Image J, and the results are shown in Tables 32A–32B, representing the percentage of protein levels in PBS-treated control mice after normalization against the sampled control protein NCL (ab22758, from Abcam, Cambridge, UK).

[0485] Table 32A. Jagged 1 protein levels in mouse livers treated with different compounds

[0486] Table 32B. Jagged 1 protein levels in mouse livers treated with different compounds

[0487] The results showed that ACT-UP1 compounds without GalNAc conjugation could also increase protein levels, while compounds with GalNAc conjugation could increase protein levels even at low doses, which is consistent with the observation that GalNAc conjugation helps hepatocytes take up compounds.

[0488] Example 17. The ACT-UP1 compound increased JAG1 protein levels in mice 4 weeks after administration. To determine whether the activity of the ACT-UP1 compound was persistent in mice after administration, three 7- to 8-week-old male Balb / c mice in each cohort received subcutaneous injections of 0.5 mg / kg or 3 mg / kg of GalNAc-conjugated ATXL316. A second dose was administered 96 hours later. Mice were sacrificed 4 weeks after the initial administration, and organs were collected for analysis. Total liver protein was prepared from mice and analyzed using Western blotting. Jagged 1 protein was detected using an antibody (ab109536, from Abcam, Cambridge, UK). Western blot results are shown in Figure 18. Quantification of the Western blot was performed using Image J, and the results are shown in Table 33, representing the percentage of protein levels in PBS-treated mice after normalization to the sample control protein NCL (ab22758, from Abcam, Cambridge, UK).

[0489] Table 33. Jagged 1 protein levels in mice treated with ATXL316

[0490] The results showed that the ACT-UP1 compound was able to increase Jagged 1 protein levels even 4 weeks after administration, indicating that it has a durable effect.

[0491] Example 18. The ACT-UP1 compound increased JAG1 protein levels in JAG1 heterozygous mice. The upregulation of JAG1 by the ACT-UP1 compound was evaluated using a mouse model carrying JAG1 heterozygous deletion (JAG+ / -) (pr139410, The Jackson Laboratory, Bar Harbor, Maine, USA).

[0492] The compounds evaluated in the JAG+ / - model were two newly designed control compounds (ATXL245 and ATXL233) without PRS elements, and the previously described ACT-UP1 compound ATXL246. ATXL245 does not target JAG1. ATXL233 targets the 5' UTR of the JAG1 mRNA transcript. Sequence and chemical information of the new compounds are listed in Table 34. GL-GalNAc is the GalNAc conjugate described by Sharma et al. (2018, Bioconjugate Chem, 29:2478-2488). AN-GalNAc is the GalNAc conjugate described in WO2024137545.

[0493] Table 34. Sequence and chemical information of ASO

[0494] Male JAG1+ / - mice aged 6-8 weeks were administered 3 mg / kg of the compound twice, 96 hours apart. Mice were sacrificed 96 hours after the last administration, and organs were collected for analysis. Total liver protein was prepared and analyzed using Western blotting. JAG1 protein was detected using an antibody (ab109536, from Abcam, Cambridge, UK). The Western blotting results are shown in Figure 19. Western blotting was quantified using Image J, and the results are shown in Table 35, representing the percentage of protein levels in PBS-treated mice after normalization against the sample control protein GAPDH (G8795, from Sigma-Aldrich, St. Louis, MO, USA).

[0495] Table 35. Jag1 + / - Jagged 1 protein levels in mice

[0496] The results showed that the ACT-UP1 compound ATXL246 significantly increased JAG1 protein levels in JAG1+ / - mice. Furthermore, another compound, ATXL233, which targets the uORF region within the 5' UTR of JAG1 mRNA, also significantly increased protein levels. However, the control compound ATXL245, which does not target JAG1, did not show a statistically significant increase in JAG1 protein.

[0497] Example 19. ACT-UP1 compounds with different amounts of PS bonds increased JAG1 protein levels in animals. The aforementioned ACT-UP1 compounds (e.g., ATXL316) contain a PRS with two PS-linked nucleosides at its 5' end to protect it from degradation, and also contain eight 2'-modified nucleosides linked to a phosphodiester (PO) backbone to reduce nonspecific protein binding. To determine whether the amount of PS bonds in ACT-UP1 compounds could improve protein binding, two new compounds were designed based on the ATXL316 sequence, containing additional PS bonds between the PRS nucleosides. Table 36 lists the new compounds with different amounts of PS bonds in their PRS (bolded and underlined in the table). AN-GalNAc is a GalNAc conjugate described in WO2024137545.

[0498] Table 36. Sequence and chemical information of Jag1 ACT-UP1 compounds

[0499] In vivo assay These new compounds, along with ATXL316, were tested in animals to evaluate their activity and duration. Three male Balb / C mice aged 7 to 8 weeks in each group received a subcutaneous injection of 3 mg / kg of the GalNAc-conjugated compound. Phosphate-buffered saline (PBS) was administered as a control. Ninety-six hours after administration, mice were given a second dose, and sacrificed at week 2 or 3 following the second dose, with organs collected for analysis. Total liver protein was prepared from the mice and analyzed using Western blot. Jagged1 protein was detected using an antibody (70109T, from Cell Signaling Technology, Danvers, MA, USA). Western blot results are shown in Figure 20. Western blot quantification was performed using Image J, and the results are shown in Table 37 as percentages of protein levels relative to the saline-treated control mice after normalization against the sample control protein GAPDH (G8795, from Sigma-Aldrich, St. Louis, MO, USA).

[0500] Table 37. JAG1 protein levels in the livers of mice treated with ACT-UP1 compound

[0501] The results showed that ACT-UP1 compounds with different amounts of PS could also increase the level of the target JAG1 protein, and a significant increase in ATXL316 could still be observed 3 weeks after compound administration.

[0502] Example 20. Bifunctional ACT-UP1 compound increases FGF21 mRNA levels in vitro. To further enhance FGF21 levels, a bifunctional ACT-UP1 compound, ATXL464, was designed. Unbound by any particular theory, this bifunctional compound functions through two mechanisms: (1) ACT-UP1 recruits translational proteins to increase protein expression; and (2) it inhibits mRNA degradation by blocking the binding of cellular proteins to AU-rich elements (AREs) in transcripts. As a comparison with the bifunctional ACT-UP1 compound, four other compounds were designed to act solely through the ARE mechanism and target FGF21 mRNA AREs: ATXL460-ATXL463. ATXL464 has the same sequence and chemical information as ATXL461, plus PRS, thus it is a bifunctional compound targeting both ACT-UP1 and AREs. The sequence and chemical information of the compounds are listed in Table 38. PS: “ The symbols “” indicate that 2'-OMe is indicated by “m” before the modified nucleoside, LNA by “L” after the modified nucleoside, 2'-MOE by “e” before the modified nucleoside, and 5-methylcytidine modified by 2'-MOE is indicated by “eCm”. In the table, PRS is shown in bold and underlined.

[0503] Table 38. Sequence and chemical information of FGF21 compounds

[0504] In vitro assay Hep3B cells were seeded and allowed to grow to ~70% confluence for one day, then Lipofectamine was used. TM Transfection was performed using the 2000 transfection reagent (ThermoFisher Scientific, Waltham, MA) with five different concentrations of the compound, or blank transfection as a control. At 20 or 40 hours post-transfection, cells were collected and total RNA was prepared using the Qiagen RNeasy kit (Qiagen, Hilden, Germany). FGF21 mRNA levels were measured using quantitative real-time PCR (qRT-PCR) with a human FGF21-specific TaqMan primer and probe set (AssayID: Hs00173927_m1, ThermoFisher Scientific, Waltham, MA). qRT-PCR was performed using the AgPath-ID™ one-step RT-PCR reagent on a QS3 real-time PCR system (ThermoFisher Scientific, Waltham, MA). The FGF21 mRNA level detected in the qRT-PCR assay was compared with the level detected in aliquots of the corresponding RNA sample using Ribogreen. TM Total RNA levels were normalized using measurements taken at ThermoFisherScientific, Waltham, MA. mRNA levels are shown in Figure 21 and Table 39.

[0505] Table 39. FGF21 mRNA levels in Hep3B cells after transfection

[0506] The results showed that all three compounds targeting ARE alone increased FGF21 mRNA levels in a dose-dependent manner at two different time points. The bifunctional ARE-targeting compound plus ACT-UP1 (ATXL464) significantly increased FGF21 mRNA levels more than the ARE-targeting compound alone (ATXL461), especially at high doses.

[0507] Example 21. A bifunctional FGF21 ACT-UP1 compound increased FGF21 protein levels in mouse plasma. To determine whether bifunctional FGF21 ACT-UP1 compounds could increase plasma FGF21 protein levels, novel compounds ATXL482 and ATXL499 were designed based on the sequences of previously described compounds ATXL461 and ATXL464, respectively, but with phosphate thioester bonds in their ARE-binding regions. Furthermore, these compounds were conjugated with GalNAc to facilitate delivery to hepatocytes. The sequences and chemical information of these compounds are listed in Table 40. AN-GalNAc is the GalNAc conjugate described in WO2024137545. The bolded and underlined sequences are PRS.

[0508] Table 40. Sequence and chemical information of FGF21 ASO

[0509] To determine the activity of the novel FGF21 bifunctional ACT-UP1 compound in mice, three 7- to 8-week-old male Balb / C mice in each group were subcutaneously injected with 1 mg / kg, 3 mg / kg, 9 mg / kg, or 75 mg / kg of the GalNAc-conjugated compounds ATXL482 and ATXL499. A second dose was administered 72 hours later. Mice were sacrificed 72 hours after the second dose, and blood samples were collected from the mice using tubes coated with lithium heparin (as an anticoagulant). Plasma was prepared by centrifuging the blood samples at 2000 × g for 10 min at 4°C. The mice were then analyzed using a mouse FGF21-specific ELISA kit (KE10042, Proteintech). ® Plasma FGF21 protein levels were measured in mice at Rosemont, IL, USA. FGF21 protein levels relative to saline control mice are shown in Figure 22 and Table 41.

[0510] Table 41. Relative plasma FGF21 protein levels in mice treated with ASO

[0511] The results showed that, compared with the saline-treated group, the bifunctional ACT-UP1 compound ATXL499 induced a greater increase in protein than the ARE-targeting compound ATXL482, with an increase of about 4-fold at 9 mg / kg and more than 10-fold at 75 mg / kg.

[0512] Example 22. The ACT-UP1 compound can increase the level of Jagged 1 protein in fibroblasts derived from ALGS patients. To evaluate whether the ACT-UP1 compound could increase target proteins in the context of disease, the effect of the aforementioned ACT-UP1 compound ATXL316 was also evaluated in GM11091 cells. This cell line was derived from ALGS patients and contained a heterozygous mutation in the JAG1 gene (Brooks BM, et al., 2021, Stem Cell Res: 54:102447).

[0513] GM11091 cells were grown to approximately 70% confluence and then treated with Lipofectamine. TM Transfection was performed using 2000 transfection reagent (ThermoFisher Scientific, Waltham, MA) with different concentrations of ATXL316, or blank transfection as a control. Twenty-four (24) hours post-transfection, cells were collected and lysed, and proteins were extracted using RIPA lysis extraction buffer (ThermoFisher Scientific, Waltham, MA). Jagged 1 protein levels were determined by Western blotting using a JAG1-specific antibody (ab109536, from Abcam, Waltham, MA). Western blot results are shown in Figure 23. Western blots were quantified using Image J, and the results are shown in Table 42 as percentages of protein levels relative to blank transfected cells after normalization against the control protein α-tubulin (ab7291, from Abcam, Waltham, MA).

[0514] Table 42. JAG1 protein levels in GM11091 cells treated with ATXL316

[0515] The results showed that the use of the ATXL316 ACT-UP1 compound also increased the level of Jagged1 protein in GM11091 patient cells, indicating that this upregulation method can increase the level of JAG1 protein in patients.

[0516] Example 23. The ACT-UP1 compound ATXL316 can increase Jagged 1 protein levels but does not improve protein stability. As demonstrated in previous examples, ATXL316 can increase JAG1 protein levels in cells. Without being bound by any particular theory, the increase in JAG1 protein levels could be due to increased JAG1 translation. However, the increase in JAG1 protein levels could also be a result of increased JAG1 protein stability. To assess this possibility, the stability of JAG1 protein in HeLa cells treated with and without ATXL316 was evaluated using cycloheximide (CHX), a potent translation inhibitor that terminates the synthesis of new protein products.

[0517] Seed HeLa cells and allowed to grow to ~70% confluence for one day, then performed blank transfection or Lipofectamine. TM Transfection was performed using 20 nMATXL316 with 2000 transfection reagent (ThermoFisher Scientific, Waltham, MA). Twelve (12) hours post-transfection, cells were treated with 100 μg / mL CHX for 0, 4, 8, or 12 hours to terminate translation. Cells were then collected and lysed, and proteins were extracted using RIPA lysis extraction buffer (ThermoFisher Scientific, Waltham, MA). JAG1 protein levels were determined by Western blotting using a JAG1-specific antibody (ab109536, from Abcam, Waltham, MA), as shown in Figure 24A. JAG1 protein levels in the Western blot were quantified using ImageJ, and the results are shown in Table 43 as a percentage of the protein level in the control cells at time point 0, after normalization to the protein level in the control cells detected by Hsp90 antibody (ProteinTech, Rosemont, IL, USA; catalog number 13171-1-AP).

[0518] Table 43. JAG1 protein levels in HeLa cells after cyclohexylimide treatment

[0519] The results showed that ATXL316 increased JAG1 protein levels by approximately 63% before CHX treatment (time point 0). The degradation rate of JAG1 protein was comparable in cells treated with ATXL316 or in the control group, indicating that ATXL316 did not affect JAG1 protein stability. CHX treatment reduced protein levels over time by inhibiting JAG1 oxidative synthesis, reflecting the stability of the existing protein. These results suggest that the ACT-UP1 compound ATXL316 increases protein translation.

[0520] Example 24. Different PRS of ACT-UP1 compounds targeting Jagged 1 can increase protein levels. In Example 13, it was shown that several different protein recruitment sequences (PRS) in the ACT-UP1 compounds could enhance target protein levels. To evaluate the effect of additional ACT-UP1 PRS on their ability to enhance protein levels, 10 ACT-UP1 compounds with the same JAG1 mRNA binding sequence as ATXL316 but different PRS (bolded and underlined in the table), as shown in Figure 25A and Table 44A, were designed. These ACT-UP1 compounds were evaluated in HeLa cells.

[0521] HeLa cells were seeded and allowed to grow to ~70% confluence for one day, then Lipofectamine was used. TM Transfection was performed using 2000 transfection reagent (ThermoFisher Scientific, Waltham, MA) with ACT-UP1 compound at final concentrations of 7.5 nM and 15 nM, or as a control (blank transfection). Twenty-four (24) hours post-transfection, cells were collected and lysed, and proteins were extracted using RIPA lysis extraction buffer (ThermoFisher Scientific, Waltham, MA). JAG1 protein levels were determined by Western blotting using a JAG1-specific antibody (ab109536, from Abcam, Waltham, MA). Western blotting results are shown in Figure 25B. Western blotting was quantified using ImageJ, and the results are shown in Table 44B and Figure 25C, representing the percentage of protein levels relative to blank transfected cells after normalization for non-specific protein bands detected by the same antibody.

[0522] Table 44A. Sequence and chemical information of ASOs with novel PRS

[0523] Table 44B. JAG1 protein levels in HeLa cells transfected with ACT-UP1 compounds containing different PRS.

[0524] These results, combined with those of Example 13, show that the PRS in many tests can increase JAG1 protein levels to varying degrees, indicating that the ACT-UP1 PRS region can tolerate sequence variations in terms of enhancing protein levels.

[0525] Example 25. Different bifunctional ACT-UP1 compounds targeting FGF21 mRNA can increase FGF21 mRNA levels in Hep3B cells. To evaluate the effects of different chemical information and sequences on increasing FGF21 mRNA levels, several compounds with different chemical information, sequences, and / or PRS were designed around the bifunctional ACT-UP1 compound ATXL499 (previously described in Example 21). Examples of these compounds are listed in Table 45.

[0526] Table 45. Sequence and chemical information of ACT-UP1 compounds targeting FGF21

[0527] In vitro determination of FGF21 mRNA levels in Hep3B cells Hep3B (human liver cancer) cells were inoculated and allowed to grow to ~70% confluence for one day, then Lipofectamine was used. TMTransfection was performed using the 2000 transfection reagent (ThermoFisher Scientific, Waltham, MA) with five different concentrations of the compound, or blank transfection as a control. Forty-eight hours post-transfection, cells were collected, and total RNA was prepared using the Qiagen RNeasy Mini kit (Qiagen, Hilden, Germany). Quantitative real-time PCR (qRT-PCR) was used to determine FGF21 mRNA levels using a human FGF21-specific TaqMan primer and probe set (Assay ID: Hs00173927_m1, ThermoFisher Scientific, Waltham, MA). qRT-PCR was performed using the AgPath-ID™ one-step RT-PCR reagent on a QS3 real-time PCR system (ThermoFisher Scientific, Waltham, MA). The FGF21 mRNA levels detected by qRT-PCR were compared with those detected in aliquots of the corresponding RNA sample using Ribogreen. TM Total RNA levels were normalized based on measurements taken at ThermoFisher Scientific, Waltham, MA. mRNA levels are shown in Figure 26 and Table 46.

[0528] Table 46. FGF21 mRNA levels in Hep3B cells transfected with different compounds

[0529] The results showed that these compounds with different chemical information, sequences, and / or PRS were able to increase FGF21 mRNA levels in Hep3B cells, especially those with longer PRS (e.g., GGACUGGACU). The compounds containing shorter PRS (ATXL523, ATXL524, and ATXL525) appeared to be less active than those with longer PRS.

[0530] In vitro determination of FGF21 mRNA levels in HepG2 cells HepG2 cells derived from the liver and expressing the FGF21 protein were inoculated and allowed to grow for one day to ~70% confluence, then Lipofectamine was used. TMTransfection was performed using 2000 transfection reagent (ThermoFisher Scientific, Waltham, MA) with compounds listed in Table 45 at final concentrations of 7.5 nM and 15 nM, or blank transfection as a control. Twenty-four (24) hours post-transfection, cells were collected and lysed, and proteins were extracted using RIPA lysis extraction buffer (ThermoFisher Scientific, Waltham, MA). FGF21 protein levels were determined by Western blotting using an FGF21-specific antibody (ab171941, from Abcam, Waltham, MA). Western blotting results for 7.5 nM and 15 nM samples are shown in insets A and B of Figure 27, respectively. Western blot images were quantified using ImageJ, normalized against the loading control protein GAPDH, and the percentage of protein levels relative to blank transfected cells was calculated, as shown in Table 47.

[0531] Table 47. FGF21 protein levels in HepG2 cells transfected with 7.5 nM compound

[0532] The results showed that these different ACT-UP1 compounds could increase FGF21 protein levels in HepG2 cells, although compounds with short PRS (e.g., ATXL525) showed lower activity than those with longer PRS, especially at higher compound concentrations.

[0533] Example 26. The use of the newly designed ACT-UP1 compound increased HNF4A protein in human primary hepatocytes. To further evaluate the activity of ACT-UP1 compounds in increasing HNF4A protein levels, two novel compounds were designed around the previously described ATXL395 (see Example 15). These two compounds have different sequences and chemical information but share the same PRS. These novel compounds contain a phosphate thioester (PS) backbone and a 2'-MOE sugar modification in the mRNA-binding region to improve in vivo stability, as well as a GalNAc conjugate for delivery to hepatocytes. These compounds are listed in Table 48, with the PRS of the ACT-UP1 compounds shown as the bolded and underlined sequence at the 5' end of the HNF4A antisense oligonucleotide sequence.

[0534] Table 48. Sequence and chemical information of novel ACT-UP1 compounds targeting human HNF4A mRNA

[0535] In vitro assays – protein assessment These two GalNAc-conjugated compounds were evaluated in human primary hepatocytes (HPH) via free uptake, i.e., the compounds were incubated with the cells in the absence of transfection reagents and entered the cells via endocytosis through the interaction of the GalNAc conjugates with the ASGR receptor.

[0536] HPH cells were grown to ~70% confluence, and then the compound was delivered into the cells at final concentrations of 5 µM and 25 µM in OptiCulture medium (XenoTech, KansasCity, KS, USA, K8300). Cells were incubated in OptiCulture medium with either the compound or water for 66 h, collected, and lysed. Protein was then extracted using RIPA lysis extraction buffer (ThermoFisher Scientific, Waltham, MA). HNF4A protein levels were determined by Western blot using an HNF4A-specific antibody (ab181604, from Abcam, Waltham, MA). The Western blot results are shown in Figure 28A, and the bar chart of the quantitative results is shown in Figure 28B. Western blots were quantified using ImageJ, and the results are shown in Table 49, representing the percentage of protein levels of the loading control protein GAPDH, after normalization relative to the mean of four blank control (PBS) cell samples, as detected using a GAPDH-specific antibody (G8795, from Sigma-Aldrich, St. Louis, MO, USA).

[0537] Table 49. HNF4A protein levels in HPH incubated with the ACT-UP1 compound

[0538] The results showed that the GalNAc-conjugated compound derived from ATXL395 could increase the HNF4A protein level in human primary hepatocytes.

[0539] Example 27. Dose-response study of ACT-UP1 compound ATXL547 in mouse cells. To evaluate the dose-response relationship, the activity of ATXL547 (previously described in Example 26) was tested by transfection into mouse Hepa1-6 cells. Cells were seeded and allowed to grow for one day to ~70% confluence, and then treated with Lipofectamine. TMTransfection was performed using ATXL547 transfection reagent 2000 (ThermoFisher Scientific, Waltham, MA) at final concentrations of 5 nM, 10 nM, 20 nM, 40 nM, and 80 nM, or as a control. Twenty-four (24) hours post-transfection, cells were collected and lysed, and proteins were extracted using RIPA lysis extraction buffer (ThermoFisher Scientific, Waltham, MA). HNF4A protein levels were determined by Western blotting using an HNF4A-specific antibody (R&D Systems, Minneapolis, MN, USA; catalog number PP-H1415-0C). Western blot results are shown in Figure 29. Western blots were quantified using ImageJ, and the results are shown in Table 50, representing the percentage of protein levels relative to the mean of four blank transfected cell samples after normalization of the loading control protein detected by the Hsp90 antibody (ProteinTech, Rosemont, IL, USA; catalog number 13171-1-AP).

[0540] Table 50. HNF4A protein levels in mouse Hepa1-6 cells transfected with ATXL547

[0541] The results showed that ATXL547 could increase HNF4A protein levels over a wide dose range of 5 nM to 80 nM, and the protein levels remained controllable without extremely high overexpression.

[0542] Example 28. The ACT-UP1 compound increases HNF4A protein levels in vivo. To evaluate the activity of the HNF4A ACT-UP1 compound in vivo, mice were treated with the compound and HNF4A protein levels were measured.

[0543] Three male Balb / c mice aged 7 to 8 weeks in each group received subcutaneous injections of 2.0 mg / kg, 8.0 mg / kg, 16.0 mg / kg, or 30.0 mg / kg of GalNAc-conjugated ATXL546 or ATXL547. Phosphate-buffered saline (PBS) was administered as a control. Ninety-six hours after administration, the mice were given a second dose at the same dosage. Four days after the second administration, the mice were sacrificed, and their organs were collected for analysis. Total liver protein was prepared and analyzed using Western blotting. HNF4A protein was detected using an antibody (R&D Systems, Minneapolis, MN, USA; catalog number PP-H1415-0C). The Western blot results are shown in Figure 30. Western blots were quantified using Image J, and the results are shown in Table 51, representing the percentage of protein levels in the sample control protein Hsp90, as detected using the antibody (ProteinTech, Rosemont, IL, USA; catalog number 13171-1-AP), relative to the PBS-treated control mice.

[0544] Table 51. HNF4A protein levels in mice treated with ACT-UP1 compound

[0545] The results showed that the ACT-UP1 compound could increase HNF4A protein levels in vivo, consistent with the results observed in vitro.

[0546] Table 52A: Sequence Listing of GenBank Sequences

[0547]

[0548]

[0549]

[0550]

[0551]

[0552]

[0553]

[0554]

[0555]

[0556]

[0557]

[0558]

[0559]

[0560]

[0561]

[0562]

[0563]

[0564]

[0565]

[0566]

[0567]

[0568]

[0569] Table 52B: PRS Sequence List

[0570] Table 52C: Sequence listing and chemical information of compound sequences

[0571] Table 52D: Sequence listing of compounds (no chemical information)

Claims

1. An antisense oligonucleotide (ASO)-conjugated translation-upregulation 1 (ACT-UP1) compound, wherein the ACT-UP1 compound comprises an ASO component linked to a protein recruitment sequence (PRS) component, wherein the ASO component is capable of hybridizing with a target mRNA, wherein the PRS is capable of recruiting translation-related proteins, and wherein the ACT-UP1 compound is capable of enhancing protein expression.

2. The ACT-UP1 compound according to claim 1, wherein the length of the PRS is 5 to 20, 5 to 19, 5 to 18, 5 to 17, 5 to 16, 5 to 15, 5 to 14, 5 to 13, 5 to 12, 5 to 11, 5 to 10, 5 to 9, 5 to 8, 5 to 7, or 5 to 6 linked nucleosides.

3. The ACT-UP1 compound according to claim 1, wherein the PRS is a derivative of the DRACH concordant sequence, or a sequence comprising (a) the DRACH concordant sequence and (b) an additional sequence comprising one or more repeating sequences or partially repeating sequences of the DRACH concordant sequence.

4. The ACT-UP1 compound according to claim 3, wherein the DRACH concordant sequence is GGACU (SEQ ID NO: 8) or a derivative thereof.

5. The ACT-UP1 compound according to claim 1, wherein the PRS is a derivative of the GGACU sequence (SEQ ID NO: 8).

6. The ACT-UP1 compound according to claim 4 or 5, wherein the GGACU or its derivative is selected from one of GGACU (SEQ ID NO: 8), GGACUGGAC (SEQ ID NO: 10), GGACUGGACU (SEQ ID NO: 11), and ACGGACUUGGACU (SEQ ID NO: 12).

7. The ACT-UP1 compound according to claim 1, wherein the PRS is a derivative of a poly(A), poly(C), poly(T), or poly(U) sequence in the 3'UTR of the mRNA.

8. The ACT-UP1 compound according to claim 7, wherein the poly(A) tail derivative is AAACUAAACU (SEQ ID NO: 13), AAAAAAAAAA (SEQ ID NO: 15), AAACAAAACA (SEQ ID NO: 99), AAAAAAAAAAAA (SEQ ID NO: 102); the poly(C) sequence is CCCCCCCCCCC (SEQ ID NO: 93); and the poly(U) sequence is UUUUUUUUUU (SEQ ID NO: 94).

9. The ACT-UP1 compound according to claim 1, wherein the compound comprises 17 to 45, 17 to 44, 17 to 43, 17 to 42, 17 to 41, 17 to 40, 17 to 39, 17 to 38, 17 to 37, 17 to 36, 17 to 35, 17 to 34, 17 to 33, 17 to 32, 17 to 31, 17 to 30, 17 to 29, 17 to 28, 17 to 27, 17 to 26, 17 to 25, 19 to 45, 19 to 40, 19 to 35, 19 to 34, 19 to 33, 19 to 32, or 19. Up to 31, 19 to 30, 19 to 29, 19 to 28, 19 to 27, 19 to 26, 19 to 25, 22 to 45, 22 to 40, 22 to 35, 22 to 34, 22 to 33, 22 to 32, 22 to 31, 22 to 30, 22 to 29 22 to 28, 22 to 27, 22 to 26, 22 to 25, 25 to 45, 25 to 40, 25 to 35, 25 to 34, 25 to 33, 25 to 32, 25 to 31, 25 to 30, 25 to 29, 25 to 28, or 25 to 27 connected subunits.

10. The ACT-UP1 compound according to claim 1, wherein the length of the ASO is 12 to 25, 12 to 24, 12 to 23, 12 to 22, 12 to 21, 12 to 20, 12 to 19, 12 to 18, 12 to 17, 12 to 16, 12 to 15, or 12 to 14 linked nucleosides.

11. The ACT-UP1 compound according to claim 1, wherein the ACT-UP1 compound is a trans-acting protein enhancer.

12. The ACT-UP1 compound according to claim 1, wherein the target mRNA is mammalian mRNA, plant mRNA, yeast mRNA, or bacterial mRNA.

13. The ACT-UP1 compound of claim 12, wherein the ACT-UP1 compound targets a region downstream of a stop codon on the mRNA of about 20 to 50, 40 to 70, 60 to 90, 80 to 110, 100 to 130, 120 to 150, 140 to 170, 160 to 190, 180 to 210, 200 to 230, 220 to 250, 240 to 300, or 280 to 500 nucleotides.

14. The ACT-UP1 compound of claim 12, wherein the target mRNA is JAG1, RAB9, PBGD, RNase H1, HNF4A, or FGF21.

15. The ACT-UP1 compound according to claim 1, further comprising a conjugate.

16. The ACT-UP1 compound of claim 15, wherein the conjugate may be selected from cholesterol, lipids, carbohydrates, phospholipids, biotin, phenazine, folic acid, phenanthridine, anthraquinone, acridine, fluorescein, rhodamine, coumarin, peptides, antibodies, dyes, and tocopherols.

17. The ACT-UP1 compound according to claim 15, wherein the conjugate is a compound containing N-acetylgalactosamine (GalNAc).

18. The ACT-UP1 compound according to claim 1, wherein the compound comprises at least one chemical modification.

19. The ACT-UP1 compound according to claim 18, wherein the compound is fully chemically modified.

20. The ACT-UP1 compound of claim 18, wherein the ASO and PRS contain the same chemical modifications.

21. The ACT-UP1 compound according to claim 18, wherein the ASO and PRS contain different chemical modifications.

22. The ACT-UP1 compound according to claim 21, wherein the PRS chemical modification is 2'-O-methyl (2'-OMe).

23. The ACT-UP1 compound according to any one of claims 18 to 21, wherein the chemical modification may be selected from 2'-O-methyl (2'-OMe), 2'-O-(2-methoxyethyl) (2'-MOE), 2'-fluorine (2'-F), restricted ethyl (cEt), non-locked nucleic acid (UNA), locked nucleic acid (LNA), 2'-MOE-modified T and / or 5-methylcytosine bases.

24. The ACT-UP1 compound according to claim 1, wherein the compound comprises at least one modified nucleoside interlinking bond.

25. The ACT-UP1 compound according to claim 24, wherein the at least one modified nucleoside inter-linkage is a phosphate thioester nucleoside inter-linkage (PS).

26. The ACT-UP1 compound according to any one of claims 1 to 25, comprising 17 to 45 linked nucleosides, wherein the ACT-UP1 compound comprises an ASO component linked to a protein recruitment sequence (PRS) component, the ASO component comprising 12 to 25 linked nucleosides, and the PRS component comprising 5 to 20 linked nucleosides.

27. The ACT-UP1 compound according to any one of claims 1 to 26, comprising 22 to 35 linked nucleosides, wherein the ACT-UP1 compound comprises an ASO component linked to a protein recruitment sequence (PRS) component, the ASO component comprising 12 to 25 linked nucleosides, the PRS component comprising the sequence GGACUGGACU (SEQ ID NO: 11) or the sequence AAACUAAACU (SEQ ID NO: 13).

28. The ACT-UP1 according to any one of claims 1 to 27, wherein the compound increases protein expression by at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 150%, 200%, 250%, or 300%.

29. A pharmaceutical composition comprising any of the ACT-UP1 compounds of the preceding claims, used alone or in combination with a pharmaceutically acceptable carrier and / or excipient.

30. A method for increasing the translation of a target mRNA in a cell, comprising administering to the cell the ACT-UP1 compound of any one of claims 1 to 28 or the pharmaceutical composition of claim 29 in an amount sufficient to increase the translation of the target mRNA.

31. A method for treating a subject with haplo-inadequacy syndrome, comprising administering to the subject the ACT-UP1 compound of any one of claims 1 to 28 or the pharmaceutical composition of claim 29 in an amount sufficient to treat the subject's haplo-inadequacy syndrome.

32. The method of claim 30 or 31, wherein the ACT-UP1 compound or pharmaceutical composition may be administered subcutaneously, intravenously or intrathecally to the subject.

33. A method for preparing the compound according to any one of claims 1-9, wherein the method comprises the following steps: a. The compound is prepared by sequentially coupling modified and / or unmodified nucleotides and / or linkers onto a conjugated or unmodified solid support via phosphoramide oligonucleotide synthesis; b. Optionally, a compound to which the conjugated portion is coupled to the solid support by means of phosphoramidite oligonucleotide synthesis; c. Separate the compound from the solid support and remove the solid support; as well as d. Optionally, add a conjugate after cutting; e. Optionally, the compound may be further purified, optionally using chromatography.

34. The ACT-UP1 compound according to claim 1, wherein the ASO component is directly or indirectly connected to the PRS.

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