Compositions and methods for increasing protein levels

Abstract

Antisense oligonucleotide strategies, including compositions and methods, for increasing protein levels are provided. Target proteins include but are not limited to tank binding protein kinase 1 (TBK-1 or TBK1), follistatin (FST), and progranulin (PGRN). The compositions and methods described herein are applicable for treatment of neurodegenerative diseases caused by haplo-insufficiency.

Description

[0001] COMPOSITIONS AND METHODS FOR INCREASING PROTEIN LEVELS

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims the benefit under 35 U.S.C. § 119(e) of U.S. Provisional Patent Application Nos. 63 / 726,801 , filed December 2, 2024 and 63 / 781 ,067, filed March 31 , 2025 the entire disclosures of which are incorporated herein by reference.

[0004] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0005] Not applicable.

[0006] FIELD OF THE INVENTION

[0007] The present disclosure generally relates to compositions and methods for increasing a level of one or more target proteins to treat neurodegenerative diseases.

[0008] BACKGROUND OF THE INVENTION

[0009] Genetic haploinsufficiency, in which partial or complete loss of one normal allele is sufficient to cause disease, is the basis of numerous human neurodegenerative diseases. In these disorders, increasing protein levels might potentially slow or cure the disease. The untranslated regions (UTRs) that flank the coding sequence of mRNA are critical regulatory hubs that influence RNA metabolism, including translation and stability. In particular, the 3’UTR encodes numerous regulatory elements that engage trans-acting elements such as RNA binding proteins (RBPs) and microRNAs, many of which repress translation and reduce mRNA stability. Antisense oligonucleotides (ASOs) are DNA sequences that can bind complementarily to target RNA with high affinity and block the binding of certain trans-factors, thereby altering aspects of RNA metabolism. Importantly, ASOs targeting non-coding regions like UTRs can modify stability and translation without interfering directly with the translation machinery. Haploinsufficiency of TANK-binding kinase 1 (TBK-1 ) causes amyotrophic lateral sclerosis and frontotemporal dementia. There is no current way to increase TBK-1 protein in a regulatable manner that can be readily given to humans. In addition, haploinsufficiency of follistatin (FST) can be associated with muscular dystrophies as well as myopathies and ways to increase FST protein in a regulatable manner can provide better therapeutic responses.

[0010] SUMMARY OF THE INVENTION

[0011] Among the various aspects of the present disclosure is the provision of compositions and methods for treating neurodegenerative diseases through targeted modulation or protein expression using 3’UTR-masking compositions.

[0012] Briefly, therefore, the present disclosure is directed to composition and related methods of use of antisense oligonucleotides (ASOs) for the treatment of neurodegenerative disorders.

[0013] The present teachings include a composition comprising a 3’UTR-masking agent, wherein the 3’UTR-masking agent is an antisense oligonucleotide (ASO) comprising a sequence targeting specific regulatory elements of a protein of interest (a target protein). In one aspect, the regulatory element comprises a Pumilio recognition elements and a trans-acting elements.

[0014] The present teachings include a method of selectively increasing expression of a protein of interest in a subject in need thereof, the method comprising: administering to the subject a composition comprising one or more antisense oligonucleotides (ASOs) which specifically bind a 3’UTR of a mRNA encoding the protein of interest such that regulatory elements in the 3’UTR are masked by the ASO(s). In one aspect, the protein of interest is selected from the group consisting of TANK-binding kinase 1 (TBK1 ), progranulin (PGRN), follistatin (FST), and vascular endothelial growth factor (VEGF).

[0015] The present teachings include a method of treating neurodegeneration in a subject in need thereof, the method comprising: administering to the subject a composition comprising one or more antisense oligonucleotides (ASOs) which specifically bind a 3’UTR of a mRNA encoding a protein of interest, such that regulatory elements in the 3’UTR are masked by the ASO(s) and expression of the protein of interest is increased. In one aspect, the protein of interest is selected from the group consisting of TANK-binding kinase 1 (TBK1 ), progranulin (PGRN), follistatin (FST), and vascular endothelial growth factor (VEGF).

[0016] The present teachings include a method of screening for antisense oligonucleotides (ASOs) that increase expression of a protein of interest, the method comprising (i) providing a reporter construct comprising a 3’UTR of a mRNA encoding the protein of interest, operably linked to a reporter gene to a test cell, (ii) contacting the test cell from step a to a candidate ASO that is complementary to a sequence within the 3’UTR and (iii) comparing the level of expression of the reporter gene in the test cell to the level of expression of the reporter gene in a control cell that is not contacted with the candidate ASO or which is contacted with a negative control molecule; wherein increased expression of the reporter gene in the test cell relative to the control cell indicates that the ASO increases expression of the protein of interest and acts as a 3’ UTR masking agent.

[0017] DESCRIPTION OF THE DRAWINGS

[0018] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0019] Those of skill in the art will understand that the drawings, described below, are for illustrative purposes only. The drawings are not intended to limit the scope of the present teachings in any way.

[0020] FIG. 1A, FIG. 1 B, FIG. 1C, and FIG. 1 D depict an antisense oligonucleotide (ASO) screen using 3’UTR reporters to identify ASOs that increase TBK1 protein. The ASO screen was conducted in HEK293, HMC3 microglia, or SH-SY5Y neuroblastoma cells using an EGFP reporter constructs containing the 3’UTR of (FIG. 1A) PGRN or (FIG. 1 B) TBK1. Results are presented as EGFP normalized to mCherry transfection control, relative to scrambled ASO. EGFP protein and TBK1 protein expression was measured in an (FIG. 1C) HEK293 TBK1- inducible (dox) reporter line and (FIG. 1 D) TBK1 + / - induced pluripotent stem cells (iPSCs), respectively, following treatment with scrambled or selected TBK1 -ASOs. EGFP protein and TBK1 protein was quantified by Western blot and normalized to scrambled ASO. (Progranulin ASO:P1 -P10, TBK-1 ASO:T1-T17 each about 20bp).

[0021] FIG. 2A and FIG. 2B depict sequence analysis of TBK1 3’UTR identifying a regulatory hotspot that modifies TBK1 stability or translation. (FIG. 2A) The region of 3’UTR of TBK1 targeted by ASO T2 and T3 contains recognition sites for miR- 200c and Pumilio. (FIG. 2B) Knockdown of miR-200c and treatment with T2 do not alter TBK1 mRNA levels, whereas decreasing Pumilio-homolog- 2 (PIIM2) with siRNA and T3 increases TBK1 mRNA levels.

[0022] FIG. 2C depicts a representative polysome profiling graph of HEK293 cells following treatment with PBS, scramble, T2, or T3. Enrichment of TBK1 mRNA in 40S, 80S, light polysome, or heavy polysome fractions was measured by qPCR and normalized to PBS-40S.

[0023] FIG. 3 depicts Pumilio recognition sites (PREs) in the TBK1 3’UTR that may be universal targets for ASO masking. Use of PRE-targeted ASOs increases expression of an EGFP reporter. Results are shown as EGFP signal normalized to mCherry transfection control, relative to PBS-treated control.

[0024] FIG. 4A and FIG. 4B depict TBK1-upregulating ASOs to restore TBK1 in pre- clinical TBK1 - deficient cell and animal models. (FIG. 4A) Proposed “micro-walk” strategy of tiling ASOs across a regulatory hotspot identified on the 3’UTR of TBK1 . Recognition elements for two likely TBK1 trans-factors miR-200c and Pumilio are marked. (FIG. 4B) Top ASO candidates will be administered to human TBK1 + / - induced pluripotent stem cell (iPSC)-derived neurons, iPSC-derived microglia, and Tbk1 + / - mice (brain and spinal cord). For human cells and mouse tissue, ASO efficacy will be measured by TBK1 protein levels and functional changes in autophagy and innate immune signaling.

[0025] FIG. 5A, FIG. 5B, and FIG. 5C depict ASO 3’UTR masking to increase VEGF expression in the SOD1-G93A mouse model. (FIG. 5A) Representation of the 3’UTR of VEGF with select post-transcriptional regulatory elements annotated. Each depicted element or microRNA site is a potential target for ASO masking. MiRNA sites labeled in red are extensively validated and strong ASO targets. (FIG. 5B) Candidate VEGF-ASOs will be administered by intracerebroventricular injection to SOD1 -G93A mice at phenotypic onset. (FIG. 5C) Primary readouts are survival, motor neuron counts in the spinal cord, and VEGF protein in the brain and spinal cord. Vascular integrity will also be explored. Theoretical survival data shown with VEGF-ASOs compared to a scramble ASO control. FIG. 6A and FIG. 6B depict a strategy to develop a cell-specific catalog of ASO “maskable” 3’UTR elements. (FIG. 6A) A library of barcoded gene reporters that contain 3’UTRs with 4 sites (blue) for any combination of listed cis-regulatory elements specific for neurons and glia or endogenous 3’UTRs will be prepared with a series of ASOs designed to mask these sites. These libraries will be deployed alongside masking ASOs to neurons and glia, which differentially express transfactors. Sequencing of RNA from different polysome fractions will reveal efficiency of ASOs in upregulating translation and mRNA stability of reporter genes. (FIG. 6B) Schematic of translation suppression by cis-elements and ASO effect on stimulating translation and polyribosome association.

[0026] FIG. 7A depicts PRE-containing gene targets with high likelihood for regulation by PUM and includes a set of 2740 transcripts.

[0027] FIG. 7B depicts six disease associated gene transcripts (ITPR1, TBK1, FST, SLC2A1, LDLR, and DEPDC5).

[0028] FIG. 7C depicts results from RNA immunoprecipitation sequencing experiments in HEK293 cell identifying six gene transcripts that are strongly bound by Pumilio proteins PUM1 and PUM2.

[0029] FIG. 7D depicts that PUM proteins negatively regulate expression of selected transcripts as shown by depleting PUM1 and PUM2 using siRNA in HEK293 cells.

[0030] FIG. 8A depicts that PUMs recognize and bind to a highly stereotyped 8 base pair motif that is identifiable within a gene 3’UTR.

[0031] FIG. 8B depicts a strategy that neutralizing Pumilio activity at a single gene target using antisense oligonucleotides (ASOs) provides gene-specific upregulation by augmenting mRNA stability or translation.

[0032] FIG. 8C depicts experimental data showing that ASO treatment results in 1 .5- fold protein upregulation within 24 hours of a fluorescent EGFP reporter that is flanked by a synthetic 3’UTR that contains two instances of a PRE.

[0033] FIG. 8D depicts at least one ASO per gene was able to increase reporter expression by 1.25 to 1.6-fold.

[0034] FIG. 8E depicts the top ASO for each gene can also upregulate endogenous gene mRNA. FIG. 9A depicts a 3’UTR of TBK1 in different species containing 2 PREs that are positioned within 10 base pairs.

[0035] FIG. 9B depicts depleting PUM proteins increases TBK1 protein by 1.7-fold.

[0036] FIG. 9C depicts depleting PUM proteins increased the fraction of TBK1 mRNA remaining after 6 hours after administration of the transcription inhibitor Actinomycin D to HEK293 cells treated with PUMs siRNA.

[0037] FIG. 9D depicts protein expression and mRNA stability with mutagenized 3’UTR luciferase reporter at each PRE site.

[0038] FIG. 9E depicts mutagenesis of each site increased reporter expression and loss of both sites elevated reporter levels by 2.2-fold.

[0039] FIG. 9F depicts the mutagenized reporter achieved greater mRNA stability after incubating with Actinomycin D for 0-8 hours.

[0040] FIG. 10A depicts stable 3’UTR reporter HEK293 cells that express EGFP flanked by the 3’UTR of TBK1 under doxycycline control.

[0041] FIG. 10B depicts TBK1-ASOs dose-dependent mRNA abundance increases in each TBK1 and EGFP.

[0042] FIG. 10C depicts TBK1-ASOs mRNA abundance increases of 1.7-fold for TBK1 and 1 .5-fold for EGFP.

[0043] FIG. 10D depicts ASOs substantially extended mRNA half-life after treating with Actinomycin D for 0-12 hours.

[0044] FIG. 10E depicts mutagenized luciferase reporter with TBK1 ASO.

[0045] FIG. 11 A depicts ASOs targeting FST are fully conserved across species.

[0046] FIG. 11B depicts FST-targeted ASOs strongly upregulated FST by >2.5-fold in fibroblasts.

[0047] FIG. 11C depicts FST-targeted ASOs stabilized FST mRNA in HEK293 cells.

[0048] FIG. 11D depicts FST-ASOs upregulate of Fst mRNA in mouse embryonic fibroblasts.

[0049] FIG. 11E depicts Fst-targeted ASOs increased Fst mRNA by >2-fold in the liver when delivered by intraperitoneal injection at 25mg / kg to C57 naive mice and harvested liver and gastrocnemius muscle after four days.

[0050] FIG. 11 F depicts FST-targeted ASOs can increase Fst mRNA in the central nervous system when delivered by intracerebroventricular stereotaxic injection and harvested cortex and hippocampus after seven days.

[0051] FIG. 12A depicts TBK1 -targeted ASOs can increase TBK UTR reporter expression when delivered in cell lines.

[0052] FIG. 12B depicts FST-targeted ASOs can increase FST UTR reporter expression when delivered in cell lines.

[0053] FIG. 13 depicts FST and TBK-targeted ASOs can increase RNA stability of FST, respectively, in cell lines following block of new transcription with Actinomycin D for 6 hours.

[0054] FIG. 14A depicts that the lead ASO for FST can robustly increase FST mRNA levels in both human and mouse fibroblasts.

[0055] FIG. 14B depicts ASOs for FST can increase FST mRNA levels in the hippocampus.

[0056] FIG. 14C depicts ASOs delivered intracerebroventricularly can increase FST mRNA levels in the frontal cortex.

[0057] FIG. 14D depicts FST-ASOs, administered to a live mouse, can increase FST mRNA levels in the liver.

[0058] FIG. 14E depicts FST-ASOs, administered to a live mouse, also increase the ratio of phosphorylated AKT to total AKT, consistent with the known consequences of increasing FST signaling.

[0059] DETAILED DESCRIPTION OF THE INVENTION

[0060] The present disclosure is based, at least in part, on the discovery that compositions and methods for treating neurodegeneration show success through targeted modulation or protein expression using 3’UTR-masking compositions. The disclosure shows ASOs targeting the 3’UTRs of mRNA are useful in modulating protein expression by, in part, blocking repressive sites. As disclosed herein, antisense oligonucleotides have been designed to selectively increase levels of target proteins. In particular the present disclosure provides the therapeutic strategies to selectively increase protein expression for subjects in need thereof.

[0061] The present disclosure investigated whether ASOs that block the binding sites for repressive trans-factors on 3’UTRs would be a viable strategy to increase protein expression and applied this to progranulin (PGRN), TANK-binding kinase 1 (TBK1 ), and follistatin (FST), in which loss-of-function mutations cause neurodegeneration. Increasing TBK-1 is predicted to be therapeutic. The follistatin (FST) ASO offers an additional method for regulating this pathway (antibodies focused on follistatin / myostatin pathway already exist).

[0062] According to the present disclosure, antisense oligonucleotides were developed that bind to the 3'UTR of the tank binding kinase 1 (TBK-1 ) mRNA and follistatin (FST) mRNA and block binding of negative regulatory elements thus stabilizing TBK-1 mRNA and follistatin (FST) mRNA and increasing TBK-1 and follistatin (FST) protein. Furthermore, for TBK-1 a massively parallel reporter array was used to broadly define regions of the 3' UTR important for mRNA regulation and thus important areas to target for antisense oligo therapeutics.

[0063] One aspect of the present disclosure provides 3’UTR-masking agents. In some embodiments, the 3’UTR-masking agent is antisense oligonucleotide (ASO) specifically targeting regulatory elements in the 3’UTR mRNA. As described herein, an antisense oligonucleotide (ASO) can be useful for the treatment of neurodegenerative diseases. A 3’UTR-masking ASO can be used to specifically increase target protein expression and / or activity (e.g., TKB-1 , PGRN, VEGF, follistatin (FST)). For example, a 3’UTR-masking ASO can mask (through direct binding) regulatory elements such as Pumilio recognition elements, trans-acting elements, thereby altering aspects of RNA metabolism.

[0064] Another aspect of the present disclosure describes the validation of PRE- masking ASOs and their application to several PRE-containing genes that are implicated in haploinsufficiency disorders. Importantly, we validate the therapeutic potential of PRE-masking ASOs by amplifying expression of the haploinsufficiency- related gene TANK-binding kinase 1 (TBK1 ). Loss-of-function mutations in TANK- binding kinase 1 (TBK1 ) result in amyotrophic lateral sclerosis through gene haploinsufficiency. As disclosed herein, the mechanisms of action and specificity of PRE-masking ASOs are established and applied to TBK1 -targeted ASOs that restore TBK1 levels in fibroblasts from individuals with TBK1 haploinsufficiency. Collectively, PRE-masking ASOs introduce a novel antisense strategy to amplify gene expression that can be applied broadly to treat haploinsufficiency-related disorders or to reinforce protective signaling.

[0065] COMPOSITIONS

[0066] In some embodiments, the 3’UTR-masking agent is a locked nucleic acid (LNA) to achieve allele-specific knockdown. In some embodiments, 3’UTR-masking agent is a shRNA or siRNA. A Morpholino, also known as a Morpholino oligomer and as a phosphorodiamidate Morpholino oligomer (PMO), is a type of oligomer molecule (colloquially, an oligo) used in molecular biology to modify gene expression. Its molecular structure can be DNA bases attached to a backbone of methylenemorpholine rings linked through phosphorodiamidate groups. Morpholinos can block access of other molecules to small (~25 base) specific sequences of the base-pairing surfaces of ribonucleic acid (RNA). Morpholinos are used as research tools for reverse genetics by knocking down gene function. Morpholino antisense oligomers can be nucleic acid analogs. The word Morpholino oligos can be referred to as PMO (for phosphorodiamidate morpholino oligomer), especially in medical literature. Vivo- Morpholinos and PPMO can be modified forms of Morpholinos with chemical groups covalently attached to facilitate entry into cells. Gene knockdown can be achieved by preventing cells from making a targeted protein.

[0067] An antisense oligonucleotide of the disclosure may be synthesized using chemical synthesis and enzymatic ligation reactions using procedures known in the art. For example, an oligonucleotide (e.g., an antisense oligonucleotide) may be chemically synthesized using naturally occurring ribonucleotides, deoxyribonucleotides, variously modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex formed between the antisense and sense nucleic acids, or combinations thereof. For example, phosphorothioate derivatives and acridine substituted nucleotides may be used. Other examples of modified nucleotides which may be used to generate an antisense nucleic acid include 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5- iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl) uracil, 5-carboxymethylaminomethyl-2 -thiouridine, 5- carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1 -methylinosine, 2,2- dimethylguanine, 2-methyladenine, 2-methylguanine, 3-m ethylcytosine, 5- methylcytosine, N6-adenine, 7-methylguanine, 5- methylaminomethyluracil, 5- methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine, 5'- methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6- isopentenyladenine, uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, que- osine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5- oxyacetic acid methylester, uracil-5-oxyacetic acid (v), 5-methyl-2- thiouracil, 3-(3-aino- 3-N-2 -carboxypropyl) uracil, (acp3)w, and 2, 6-diam inopurine. Alternatively, the oligonucleotide may be produced biologically using an expression vector into which a nucleic acid has been subcloned in an antisense orientation.

[0068] In certain embodiments, antisense oligonucleotides provided herein may include one or more modifications to a nucleobase, sugar, and / or internucleoside linkage, and as such is a modified oligonucleotide. A modified nucleobase, sugar, or internucleoside linkage may be selected over an unmodified form because of desirable properties such as, for example, enhanced cellular uptake, enhanced affinity for other oligonucleotides or nucleic acid targets, and increased stability in the presence of nucleases. In certain embodiments, a modified nucleoside is a sugar-modified nucleoside. In certain such embodiments, sugar-modified nucleosides may further comprise a natural or modified heterocyclic base moiety or natural or modified internucleoside linkage and may include further modifications independent from the sugar modification. In certain embodiments, a sugar modified nucleoside is a 2’- modified nucleoside, wherein the sugar ring is modified at the 2’ carbon from natural ribose or2’-deoxy-ribose. In certain embodiments, a 2’-modified nucleoside comprises a 2’-substituent group selected from F, O-CH3, and OCH2CH2OCH3. In certain embodiments, a 2’-modified nucleoside has a bicyclic sugar moiety. In certain embodiments, a bicyclic sugar moiety comprises a bridge group between the 2' and the 4' carbon atoms.

[0069] In certain embodiments, a modified oligonucleotide comprises one or more internucleoside modifications. In certain such embodiments, each internucleoside linkage of an oligonucleotide is a modified internucleoside linkage. In certain embodiments, a modified internucleoside linkage comprises a phosphorus atom.

[0070] In certain embodiments, a modified oligonucleotide comprises at least one phosphorothioate internucleoside linkage. In preferred embodiments, each internucleoside linkage of a modified oligonucleotide is a phosphorothioate internucleoside linkage.

[0071] In certain embodiments, a modified oligonucleotide comprises one or more modified nucleobases. In certain embodiments, a modified oligonucleotide comprises one or more 5-methylcytosines. In certain embodiments, each cytosine of a modified oligo-nucleotide comprises a 5-methylcytosine.

[0072] In certain embodiments, a modified nucleobase is selected from 5- hydroxymethyl cytosine, 7-deazaguanine and 7-deazaadenine. In certain embodiments, a modified nucleobase is selected from 7-deazaadenine, 7- deazaguanosine, 2- aminopyridine and 2-pyridone.

[0073] In some embodiments, the antisense molecules of the disclosure may be modified at the base moiety, sugar moiety or phosphate backbone to improve, e.g., the stability, hybridization, or solubility of the molecule. By way of another example, the deoxyribose phosphate backbone of the nucleic acids may be modified to generate peptide nucleic acids (see Hyrup et al. (1996) Bioorganic & Medicinal Chemistry 4(l):5- 23). As used herein, the terms "peptide nucleic acids" or "PNAs" refer to nucleic acid mimics, e.g., DNA mimics, in which the deoxyribose phosphate backbone is replaced by a pseudopeptide backbone and only the four natural nucleobases are retained. The neutral backbone of a PNA has been shown to allow for specific hybridization to DNA and RNA under conditions of low ionic strength. The synthesis of PNA oligomers may be performed using standard solid phase peptide synthesis protocols as described in Hyrup et al. (1996) supra; Perry-O'Keefe et al. (1996) Proc. Natl. Acad. Sci. USA 93:14670-675.

[0074] PNAs of DNAJB6 may be used for therapeutic applications. PNAs of DNAJB6may also be used in the analysis of single base pair mutations in a gene by PNA-directed PCR clamping; as artificial restriction enzymes when used in combination with other enzymes, such as S1 nucleases (Hyrup (1996) supra); or as probes or primers for DNA sequence and hybridization (Hyrup (1996) supra; Perry- O'Keefe et al. (1996) Proc. Natl. Acad. Sci. USA 93: 14670-675). In other embodiments, the oligonucleotides of the invention may include other appended groups such as peptides (e.g., for targeting host cell receptors in vivo), or agents facilitating transport across the cell membrane (see, e.g., Letsinger et al. (1989) Proc. Natl. Acad. Sci. USA 86:6553-6556; Lemaitre et al. (1987) Proc. Natl. Acad. Sci. USA 84:648-652; PCT Publication No. W0 88 / 09810) or the blood-brain barrier (see, e.g., PCT Publication No. WO 89 / 10134). In addition, oligonucleotides may be modified with hybridization-triggered cleavage agents (see, e.g., Krol et al. (1988) Bio / Techniques 6:958-976) or intercalating agents (see, e.g., Zon (1988) Pharm. Res. 5:539-549). To this end, the oligonucleotide may be conjugated to another molecule, e.g., a peptide, hybridization triggered cross-linking agent, transport agent, hybridization-triggered cleavage agent, etc.

[0075] In certain embodiments, an antisense oligonucleotide of the invention is synthesized with a full phosphorothioate backbone with alternating blocks of 2’-MOE and 2’fluoro sugar-modified nucleosides.

[0076] As another example, RNA (e.g., long noncoding RNA (IncRNA)) can be targeted with antisense oligonucleotides (ASOs) as a therapeutic. Processes for making ASOs targeted to RNAs are well known; see e.g. Zhou et al. 2016 Methods Mol Biol. 1402:199-213. Except as otherwise noted herein, therefore, the process of the present disclosure can be carried out in accordance with such processes. Methods of downregulation or silencing genes are known in the art. For example, expressed protein activity can be down-regulated or eliminated using antisense oligonucleotides (ASOs), protein aptamers, nucleotide aptamers, and RNA interference (RNAi) (e.g., small interfering RNAs (siRNA), short hairpin RNA (shRNA), and micro RNAs (miRNA) (see e.g., Rinaldi and Wood (2017) Nature Reviews Neurology 14, describing ASO therapies; Fanning and Symonds (2006) Handb Exp Pharmacol. 173, 289-303G, describing hammerhead ribozymes and small hairpin RNA; Helene, et al. (1992) Ann. N.Y. Acad. Sci. 660, 27-36; Maher (1992) Bioassays 14(12): 807-15, describing targeting deoxyribonucleotide sequences; Lee et al. (2006) Curr Opin Chem Biol. 10, 1 - 8, describing aptamers; Reynolds et al. (2004) Nature Biotechnology 22(3), 326 - 330, describing RNAi; Pushparaj and Melendez (2006) Clinical and Experimental Pharmacology and Physiology 33(5-6), 504-510, describing RNAi; Dillon et al. (2005) Annual Review of Physiology 67, 147-173, describing RNAi; Dykxhoorn and Lieberman (2005) Annual Review of Medicine 56, 401 -423, describing RNAi). RNAi molecules are commercially available from a variety of sources (e.g., Ambion, TX; Sigma Aldrich, MO; Invitrogen). Several siRNA molecule design programs using a variety of algorithms are known to the art (see e.g., Cenix algorithm, Ambion; BLOCK-iT™ RNAi Designer, Invitrogen; siRNA Whitehead Institute Design Tools, Bioinofrmatics & Research Computing). Traits influential in defining optimal siRNA sequences include G / C content at the termini of the siRNAs, Tm of specific internal domains of the siRNA, siRNA length, position of the target sequence within the CDS (coding region), and nucleotide content of the 3' over-hangs.

[0077] One aspect of the invention pertains to isolated nucleic acid molecules that are complementary to the 3’UTR sequence of TBK-1 mRNA, FST mRNA, PGRN mRNA, or VEGF mRNA. A nucleic acid molecule of the present invention, or a complement of any of these nucleotide sequences, may be isolated using standard molecular biology techniques. A nucleic acid of the invention may be amplified using cDNA, mRNA or genomic DNA as a template and appropriate oligonucleotide primers according to standard PCR amplification techniques. The nucleic acid so amplified may be cloned into an appropriate vector and characterized by DNA sequence analysis. A nucleic acid molecule which is complementary to a given nucleotide sequence is one which is sufficiently complementary to the given nucleotide sequence that it can hybridize to the given nucleotide sequence, thereby forming a stable duplex.

[0078] The oligonucleotides typically comprise a region of nucleotide sequence that hybridizes under stringent conditions to at least about 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30 or more consecutive nucleotides of the sense or antisense sequence of 3’UTR sequence of TKB-1 mRNA or 3’UTR sequence of PGRN mRNA, or 3’UTR sequence of VEGF mRNA, or 3’UTR sequence of FST mRNA.

[0079] 3’UTR-masking agents (also referred to herein as "active compounds") of the invention may be incorporated into pharmaceutical compositions suitable for administration. Such compositions typically comprise the agent and a pharmaceutically acceptable carrier. As used herein, the language "pharmaceutically acceptable carrier" is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the compositions is contemplated. Supplementary active compounds may also be incorporated into the compositions.

[0080] The invention includes methods for preparing pharmaceutical compositions for modulating the expression or activity of target peptides. Such methods comprise formulating a pharmaceutically acceptable carrier with a 3’UTR-masking agents which modulates expression or activity of the target protein. Such compositions can further include additional active agents. Thus, the invention further includes methods for preparing a pharmaceutical composition by formulating a pharmaceutically acceptable carrier with an 3’UTR-masking agents and one or more additional active compounds.

[0081] A pharmaceutical composition of the invention may be formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (topical), transmucosal, and rectal administration. Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates, and agents for the adjustment of tonicity such as sodium chloride or dextrose. The pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

[0082] Pharmaceutical compositions suitable for injectable use may include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL (BASF; Parsippany, N.J.), or phosphate buffered saline (PBS). In all cases, a composition may be sterile and may be fluid to the extent that easy syringeability exists. A composition may be stable under the conditions of manufacture and storage and may be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyetheylene glycol, and the like), and suitable mixtures thereof. The proper fluidity may be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion, and by the use of surfactants. Prevention of the action of microorganisms may be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it may be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride, in the composition. Prolonged absorption of the injectable compositions may be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.

[0083] Sterile injectable solutions may be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying, which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile- filtered solution thereof.

[0084] Oral compositions generally may include an inert diluent or an edible carrier. Oral compositions may be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound may be incorporated with excipients and used in the form of tablets, troches, or capsules. Oral compositions may also be prepared using a fluid carrier for use as a mouthwash, wherein the compound in the fluid carrier is applied orally and swished and expectorated or swallowed. Pharmaceutically compatible binding agents and / or adjuvant materials may be included as part of the composition. The tablets, pills, capsules, troches, and the like, may contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose; a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring. For administration by inhalation, the compounds are delivered in the form of an aerosol spray from a pressured container or dispenser which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer.

[0085] Systemic administration may also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and may include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration may be accomplished through the use of nasal sprays or suppositories. For transdermal administration, the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art. The compounds may also be prepared in the form of suppositories (e.g., with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.

[0086] In one embodiment, the active compounds are prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers may be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art. These may be prepared according to methods known to those skilled in the art, for example, as described in U.S. Pat. No. 4,522,811.

[0087] The nucleic acid molecules of the invention may be inserted into vectors and used as gene therapy vectors. Gene therapy vectors may be delivered to a subject by, for example, intravenous injection, local administration (U.S. Pat. No. 5,328,470) or by stereotactic injection (see, e.g., Chen et al. (1994) Proc. Natl. Acad. Sci. USA 91 :3054-3057). The pharmaceutical preparation of the gene therapy vector may include the gene therapy vector in an acceptable diluent, or can comprise a slow release matrix in which the gene delivery vehicle is imbedded.

[0088] The gene therapy vectors of the invention may be either viral or non-viral. Examples of plasmid-based, non-viral vectors are discussed in Huang et al. (1999) Nonviral Vectors for Gene Therapy. A modified plasmid is one example of a non- viral gene delivery system. Peptides, proteins (including antibodies), and oligonucleotides may be stably conjugated to plasmid DNA by methods that do not interfere with the transcriptional activity of the plasmid (Zelphati et al. (2000) BioTechniques 28:304-315). The attachment of proteins and / or oligonucleotides may influence the delivery and trafficking of the plasmid and thus render it a more effective pharmaceutical composition.

[0089] ADMINISTRATION

[0090] Agents and compositions described herein can be administered according to methods described herein in a variety of means known to the art. The agents and composition can be used therapeutically either as exogenous materials or as endogenous materials. Exogenous agents are those produced or manufactured outside of the body and administered to the body. Endogenous agents are those produced or manufactured inside the body by some type of device (biologic or other) for delivery within or to other organs in the body.

[0091] As discussed above, administration can be parenteral, pulmonary, oral, topical, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, ophthalmic, buccal, or rectal administration.

[0092] Agents and compositions described herein can be administered in a variety of methods well-known in the arts. Administration can include, for example, methods involving oral ingestion, direct injection (e.g., systemic or stereotactic), implantation of cells engineered to secrete the factor of interest, drug-releasing biomaterials, polymer matrices, gels, permeable membranes, osmotic systems, multilayer coatings, microparticles, implantable matrix devices, mini-osmotic pumps, implantable pumps, injectable gels and hydrogels, liposomes, micelles (e.g., up to 30 |um), nanospheres (e.g., less than 1 um), microspheres (e.g., 1 -100 um), reservoir devices, a combination of any of the above, or other suitable delivery vehicles to provide the desired release profile in varying proportions. Other methods of control led-release delivery of agents or compositions will be known to the skilled artisan and are within the scope of the present disclosure.

[0093] Delivery systems may include, for example, an infusion pump which may be used to administer the agent or composition in a manner similar to that used for delivering insulin or chemotherapy to specific organs or tumors. Typically, using such a system, an agent or composition can be administered in combination with a biodegradable, biocompatible polymeric implant that releases the agent over a controlled period of time at a selected site. Examples of polymeric materials include polyanhydrides, polyorthoesters, polyglycolic acid, polylactic acid, polyethylene vinyl acetate, and copolymers and combinations thereof. In addition, a controlled release system can be placed in proximity of a therapeutic target, thus requiring only a fraction of a systemic dosage.

[0094] Agents can be encapsulated and administered in a variety of carrier delivery systems. Examples of carrier delivery systems include microspheres, hydrogels, polymeric implants, smart polymeric carriers, and liposomes (see generally, Uchegbu and Schatzlein, eds. (2006) Polymers in Drug Delivery, CRC, ISBN-10: 0849325331 ). Carrier-based systems for molecular or biomolecular agent delivery can: provide for intracellular delivery; tailor biomolecule / agent release rates; increase the proportion of biomolecule that reaches its site of action; improve the transport of the drug to its site of action; allow colocalized deposition with other agents or excipients; improve the stability of the agent in vivcr, prolong the residence time of the agent at its site of action by reducing clearance; decrease the nonspecific delivery of the agent to nontarget tissues; decrease irritation caused by the agent; decrease toxicity due to high initial doses of the agent; alter the immunogenicity of the agent; decrease dosage frequency, improve the taste of the product; or improve the shelf life of the product. THERAPEUTIC METHODS

[0095] Also provided is a process of treating, preventing, or reversing a neurodegenerative disease in a subject in need thereof, the method generally comprises administering a 3’UTR-masking agent composition is as described herein.

[0096] Also provided is a process of treating, preventing, or reversing a neuromuscular disease, disorder, or condition in a subject in need of administration of a therapeutically effective amount of a gene-targeting antisense oligonucleotide (ASO) (e.g., designed to reduce or eliminate pathogenic isoform transcripts), so as to reduce or eliminate expression of pathogenic isoforms associated with a neuromuscular disease, disorder, or condition.

[0097] Methods described herein are generally performed on a subject in need thereof. A subject in need of the therapeutic methods described herein can be a subject having, diagnosed with, suspected of having, or at risk for developing a neurodegenerative and / or neuromuscular disease, disorder, or condition. A determination of the need for treatment will typically be assessed by a history, physical exam, or diagnostic tests consistent with the disease or condition at issue. Diagnosis of the various conditions treatable by the methods described herein is within the skill of the art. The subject can be an animal subject, including a mammal, such as horses, cows, dogs, cats, sheep, pigs, mice, rats, monkeys, hamsters, guinea pigs, and humans or chickens. For example, the subject can be a human subject.

[0098] Generally, a safe and effective amount of a gene-targeting ASO is, for example, an amount that would cause the desired therapeutic effect in a subject while minimizing undesired side effects. In various embodiments, an effective amount of a gene-targeting ASO described herein can substantially reduce or inhibit expression of pathogenic isoforms, slow the progress of a neurodegenerative and / or neuromuscular disease, disorder, or condition, or limit the development of a neurodegenerative and / or neuromuscular disease, disorder, or condition.

[0099] When used in the treatments described herein, a therapeutically effective amount of a gene-targeting ASO can be employed in pure form or, where such forms exist, in pharmaceutically acceptable salt form and with or without a pharmaceutically acceptable excipient. For example, the compounds of the present disclosure can be administered, at a reasonable benefit / risk ratio applicable to any medical treatment, in a sufficient amount to reduce or inhibit expression of pathogenic isoforms, slow the progress of a neurodegenerative and / or neuromuscular disease, disorder, or condition, or limit the development of a neurodegenerative and / or neuromuscular disease, disorder, or condition.

[0100] Again, each of the states, diseases, disorders, and conditions, described herein, as well as others, can benefit from compositions and methods described herein. Generally, treating a state, disease, disorder, or condition includes preventing, reversing, or delaying the appearance of clinical symptoms in a mammal that may be afflicted with or predisposed to the state, disease, disorder, or condition but does not yet experience or display clinical or subclinical symptoms thereof. Treating can also include inhibiting the state, disease, disorder, or condition, e.g., arresting or reducing the development of the disease or at least one clinical or subclinical symptom thereof. Furthermore, treating can include relieving the disease, e.g., causing regression of the state, disease, disorder, or condition or at least one of its clinical or subclinical symptoms. A benefit to a subject to be treated can be either statistically significant or at least perceptible to the subject or to a physician.

[0101] Treatment in accord with the methods described herein can be performed prior to, concurrent with, or after conventional treatment modalities for neurodegenerative and / or neuromuscular diseases, disorders, or conditions.

[0102] A gene-targeting ASO can be administered simultaneously or sequentially with another agent, such as an antibiotic, an anti-inflammatory, or another agent. For example, a gene-targeting ASO can be administered simultaneously with another agent, such as an antibiotic or an anti-inflammatory. Simultaneous administration can occur through administration of separate compositions, each containing one or more of a gene-targeting ASO, an antibiotic, an anti-inflammatory, or another agent. Simultaneous administration can occur through administration of one composition containing two or more of a gene-targeting ASO, an antibiotic, an anti- inflammatory, or another agent. A gene-targeting ASO can be administered sequentially with an antibiotic, an anti-inflammatory, or another agent. For example, a gene-targeting ASO can be administered before or after administration of an antibiotic, an anti- inflammatory, or another agent.

[0103] A surgical intervention can be performed simultaneously or sequentially with the administration of an agent, such as an antibiotic, an anti-inflammatory, or another agent. For example, a surgical intervention can be administered simultaneously with another agent, such as an antibiotic or an anti-inflammatory.

[0104] KITS

[0105] Also provided are kits. Such kits can include an agent or composition described herein and, in certain embodiments, instructions for administration. Such kits can facilitate performance of the methods described herein. When supplied as a kit, the different components of the composition can be packaged in separate containers and admixed immediately before use. Packaging of the components separately can, if desired, be presented in a pack or dispenser device which may contain one or more unit dosage forms containing the composition. The pack may, for example, comprise metal or plastic foil such as a blister pack. Such packaging of the components separately can also, in certain instances, permit long-term storage without losing activity of the components.

[0106] In certain embodiments, kits can be supplied with instructional materials. Instructions may be printed on paper or other substrate, and / or may be supplied as an electronic-readable medium or video. Detailed instructions may not be physically associated with the kit; instead, a user may be directed to an Internet web site specified by the manufacturer or distributor of the kit.

[0107] Definitions and methods described herein are provided to better define the present disclosure and to guide those of ordinary skill in the art in the practice of the present disclosure. Unless otherwise noted, terms are to be understood according to conventional usage by those of ordinary skill in the relevant art.

[0108] In some embodiments, numbers expressing quantities of ingredients, properties such as molecular weight, reaction conditions, and so forth, used to describe and claim certain embodiments of the present disclosure are to be understood as being modified in some instances by the term “about.” In some embodiments, the term “about” is used to indicate that a value includes the standard deviation of the mean for the device or method being employed to determine the value. In some embodiments, the numerical parameters set forth in the written description and attached claims are approximations that can vary depending upon the desired properties sought to be obtained by a particular embodiment. In some embodiments, the numerical parameters should be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of some embodiments of the present disclosure are approximations, the numerical values set forth in the specific examples are reported as precisely as practicable. The numerical values presented in some embodiments of the present disclosure may contain certain errors necessarily resulting from the standard deviation found in their respective testing measurements. The recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein. The recitation of discrete values is understood to include ranges between each value.

[0109] In some embodiments, the terms “a” and “an” and “the” and similar references used in the context of describing a particular embodiment (especially in the context of certain of the following claims) can be construed to cover both the singular and the plural, unless specifically noted otherwise. In some embodiments, the term “or” as used herein, including the claims, is used to mean “and / or” unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive.

[0110] The terms “comprise,” “have” and “include” are open-ended linking verbs. Any forms or tenses of one or more of these verbs, such as “comprises,” “comprising,” “has,” “having,” “includes” and “including,” are also open-ended. For example, any method that “comprises,” “has” or “includes” one or more steps is not limited to possessing only those one or more steps and can also cover other unlisted steps. Similarly, any composition or device that “comprises,” “has” or “includes” one or more features is not limited to possessing only those one or more features and can cover other unlisted features.

[0111] As used herein, “administering” is used in its broadest sense to mean contacting a subject with a composition of the invention. As used herein, the term "hybridizes under stringent conditions" is intended to describe conditions for hybridization and washing under which nucleotide sequences at least 60% (65%, 70%, preferably 75%) identical to each other typically remain hybridized to each other. Such stringent conditions are known to those skilled in the art and can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1 -6.3.6. A non-limiting example of stringent hybridization conditions are hybridization in 6x sodium chloride / sodium citrate (SSC) at about 45°C., followed by one or more washes in 0.2.x SSC, 0.1 % SDS at 50-65°C. (e.g., 50°C. or 60°C. or 65°C). Preferably, the isolated nucleic acid molecule of the invention that hybridizes under stringent conditions corresponds to a naturally- occurring nucleic acid molecule. As used herein, a "naturally-occurring" nucleic acid molecule refers to a RNA or DNA molecule having a nucleotide sequence that occurs in a human cell in nature (e.g., encodes a natural protein).

[0112] As used herein, the term "nucleic acid molecule" is intended to include DNA molecules (e.g., cDNA or genomic DNA) and RNA molecules (e.g., mRNA or miRNA) and analogs of the DNA or RNA generated using nucleotide analogs. The nucleic acid molecule may be single-stranded or double-stranded.

[0113] An “isolated nucleic acid molecule” means that the material is removed from its original environment (e.g., the natural environment if it is naturally occurring). For example, a naturally occurring polynucleotide present in a living animal is not isolated, but the same polynucleotide or polypeptide, separated from some or all of the coexisting materials in the natural system, is isolated, even if subsequently reintroduced into the natural system. Such polynucleotides may be part of a vector or other composition and still be isolated in that such vector or composition is not part of its natural environment.

[0114] A miRNA is a small non-coding RNA molecule which functions in transcriptional and post-transcriptional regulation of gene expression. A miRNA functions via base-pairing with complementary sequences within mRNA molecules, usually resulting in gene silencing via translational repression or target degradation. A mature miRNA is processed through a series of steps from a larger primary RNA transcript (pri-miRNA), or from an intron comprising a miRNA (mirtron), to generate a stem loop pre-miRNA structure comprising the miRNA sequence. A pre-miRNA is then cleaved to generate the mature miRNA.

[0115] As used herein, a “pharmaceutical composition” includes a pharmacologically effective amount of a therapeutic agent of the invention and a pharmaceutically acceptable carrier. As used herein, “pharmacologically effective amount,” “therapeutically effective amount” or simply “effective amount” refers to that amount of an agent effective to produce the intended pharmacological, therapeutic or preventive result. For example, if a given clinical treatment is considered effective when there is at least a 15% reduction in a measurable parameter associated with a disease or disorder, a therapeutically effective amount of an agent for the treatment of that disorder or disease is the amount necessary to effect at least a 15% reduction in that parameter.

[0116] All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided with respect to certain embodiments herein is intended merely to better illuminate the present disclosure and does not pose a limitation on the scope of the present disclosure otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the present disclosure.

[0117] Groupings of alternative elements or embodiments of the present disclosure disclosed herein are not to be construed as limitations. Each group member can be referred to and claimed individually or in any combination with other members of the group or other elements found herein. One or more members of a group can be included in, or deleted from, a group for reasons of convenience or patentability. When any such inclusion or deletion occurs, the specification is herein deemed to contain the group as modified thus fulfilling the written description of all Markush groups used in the appended claims.

[0118] All publications, patents, patent applications, and other references cited in this application are incorporated herein by reference in their entirety for all purposes to the same extent as if each individual publication, patent, patent application or other reference was specifically and individually indicated to be incorporated by reference in its entirety for all purposes. Citation of a reference herein shall not be construed as an admission that such is prior art to the present disclosure.

[0119] Having described the present disclosure in detail, it will be apparent that modifications, variations, and equivalent embodiments are possible without departing the scope of the present disclosure defined in the appended claims. Furthermore, it should be appreciated that all examples in the present disclosure are provided as non-limiting examples.

[0120] EXAMPLES

[0121] The following non-limiting examples are provided to further illustrate the present disclosure. It should be appreciated by those of skill in the art that the techniques disclosed in the examples that follow represent approaches the inventors have found function well in the practice of the present disclosure, and thus can be considered to constitute examples of modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments that are disclosed and still obtain a like or similar result without departing from the spirit and scope of the present disclosure.

[0122] EXAMPLE 1 - 3’ UTR-M ASKING COMPOSITIONS

[0123] The present example provides a screen to identify ASOs that increase protein expression. It was predicted that blocking repressive sites on 3’UTRs of PGRN and TBK1 would in turn increase protein expression. ASOs were designed against the 3’UTR of PGRN (P1-10) and TBK1 (T1 -17). Fluorescence (EGFP) reporter constructs were developed for each 3’UTR and, alongside a transfection control, delivered to human HEK293, microglial (HMC3), and neuroblastoma (SH-SY5Y) cells treated with a single ASO. Fluorescence was measured by plate reader and normalized (FIG. 1A and FIG. 1 B). Two PGRN-ASO and five TBK1 -ASO candidates were identified that increased reporter expression by 1.3x, the threshold for meaningful upregulation. In parallel, an HEK293 cell line was generated with inducible TBK1 (FIG. 1C) and PGRN 3’UTR reporters for further ASO optimization and screening. It was observed that TBK1 -ASO candidates T2 and T3 achieved 2- 3x increases in EGFP protein. With these results, a rapid, generalizable pipeline has been established using a reporter system to identify and screen for ASOs capable of promoting translation. Next, the efficacy of candidate ASOs were tested in induced pluripotent stem cells (iPSCs). Given the initial success of identifying several TBK1 -ASO candidates, the efficiency and specificity of these ASOs in TBK1 -deficient iPSCs to increase endogenous TBK1 protein was tested. Several top TBK1 -ASO candidates achieved the 1 ,3x threshold for upregulation, with some increasing upregulation by 1 ,6x (FIG. 1 D). T2, T3, and others (T4-7) together mask a region spanning ~70 nucleotides, which is presumed to represent a hot-spot for the interaction of multiple ciselements. Sequence analysis predicts at least three cis-elements occur in this region - a seed sequence for miR-200c and two Pumilio recognition elements (PRE) - that could work additively or synergistically to repress TBK1 (FIG. 2A). Pumilio might destabilize TBK1 mRNA at this PRE, as T3 treatment and knockdown of Pumilio increase TBK1 mRNA in a similar fashion. Conversely, T2 treatment or knockdown of miR-200c does not affect TBK1 mRNA levels (FIG. 2B). Polysome profiling of HEK293 cells treated with T2 or T3 revealed trends toward enrichment of TBK1 mRNA in 80S (T2,T3) and polysome fractions (T3) (FIG. 2C). An ASO “micro-walk” across this hot-spot could achieve more efficacious TBK1 -ASOs to advance to pre- clinical testing.

[0124] Here it has been established that upregulating protein expression by 3’UTR- targeting ASOs is feasible. Next, it is tested whether TBK1 -ASOs can influence TBK1 function in TBK1 -iPSCs, iPSC-derived motor neurons and microglia, and in Tbk1 + / - mouse models. Extending beyond TBK1 , it was predicted that ASOs that upregulate neuroprotective genes could provide another test-case and replace current gene therapies in combating neurodegeneration.

[0125] Stereotypically repressive cis-elements, such as PREs, might be potential targets for generalizing the ASO masking approach. A synthetic reporter construct containing PREs, alongside PRE-masking ASOs was prepared and tested. When deployed in HEK293 cells, these ASOs achieved ~1.6x increase in PRE reporter expression (FIG. 3), suggesting that PREs across the genome could be universal targets. It was predicted that this approach can be extending (1 ) to other repressive cis- elements or combinations of elements and (2) across different cell types could achieve a catalog of “maskable” elements applicable to many genes of interest.

[0126] Restoring or upregulating single gene targets could benefit numerous neurodegenerative diseases. Thus, the transformative goal of this disclosure is to develop a bold new approach for safely upregulating protein expression of any gene to ameliorate disease. With genetic haploinsufficiency, partial or complete loss of one normal allele is sufficient to cause disease, but selective restoration of the impacted gene may be curative. Likewise, selective upregulation of protective genes like neurotrophins could counter many central nervous system (CNS) diseases, especially when targeting specific cell types. Viral gene therapies or direct protein delivery are limited by an inability to regulate dosing or to achieve sufficient protein levels within the CNS. The untranslated regions (UTRs) that flank mRNA coding sequences are regulatory hubs for trans-acting elements that determine RNA stability and translation.

[0127] The 3’UTR is preferentially targeted by microRNAs and RNA-binding proteins (RBPs), which can engage stereotyped recognition motifs to influence mRNA translation. The present disclosure provides that masking recognition sites for repressive microRNAs and RBPs increases protein expression for genes affected in neurodegenerative diseases.

[0128] Antisense oligonucleotides (ASOs) are short, synthetic, single- stranded DNA sequences that bind complementarily to target RNA and contain chemically- modified bases that define their mechanisms of action. ASOs are a well- developed and readily translatable technology for manipulating gene expression. In addition, ASO dose can be regulated and safely delivered to the CNS. ASOs have shown enormous therapeutic potential in neurodegenerative conditions via mechanisms of gene knockdown or alternative splicing. Yet, their ability to increase protein expression is relatively unexplored. Using fully-modified, non-mRNA degrading ASOs to increase protein expression of TANK-binding kinase 1 (TBK1 ) as a treatment for amyotrophic lateral sclerosis (ALS) and / or frontotemporal dementia (FTD) by harnessing post-transcriptional regulation at the 3’UTR.

[0129] Addressing this challenge requires an interdisciplinary approach that bridges fundamental mechanisms regulating mRNA translation and RNA therapeutics for neurodegeneration. Rescuing a neurodegenerative haploinsufficiency disease (TBK1 ) and boosting neurotrophin production was examined. Finally, an open- source catalog of motifs responsive to ASO masking generalizes this approach to ~75% of the human genome in a cell-specific manner. The ability to target specific cell populations, such as motor neurons impacted in ALS, carries transformative potential for understanding and treating neurodegenerative disease.

[0130] 3’UTR-masking ASOs that increase TBK1 protein by >1.5x in induced pluripotent stem cells (iPSCs) harboring a TBK1 mutation were developed as discussed above. The ASO screen identified a hot-spot of ~50 nucleotides responsive to ASO masking, likely representing convergence of multiple repressive elements. A fine screening of this region was performed using an ASO “micro-walk” and new iterations produced more efficacious TBK1 -ASOs (FIG. 4A).

[0131] TBK1 has a well-established role in autophagy and innate immunity. ALS- linked mutations in TBK1 disrupt autophagy and reduce survival in TBK1+ / _iPSC motor neurons. Microglia are likely also affected in TBK1 -ALS / FTD; however, the effect of TBK1 deficiency on autophagy or immune signaling in these cells is unknown. Patient-derived iPSC lines were differentiated to motor neurons and microglia and tested if TBK1 -upregulating ASOs restored TBK1 levels and functional deficits, with or without autophagic or immune stressors (FIG. 4B). In parallel, if TBK1 -upregulating ASOs can increase TBK1 protein in the CNS of mice carrying a single Tbk1 allele (Tbk1 + / -) was tested. The top 3 candidate ASOs selected to match mouse Tbk1 sequences (mTbk1 -ASOs), which are highly conserved over this hotspot, and deliver each mTbk1 -ASO by intracerebroventricular injection to Tbk1 + / - mice (FIG. 4B). Effective ASOs were those that achieve a >1.5x increase in TBK1 expression in the cortex and spinal cord and modulate the expression of key genes along autophagy and immune pathways related to TBK1 function.

[0132] Many neurotrophic factors, such as vascular endothelial growth factor (VEGF), can promote neuronal survival, foster regeneration, and influence disease risk. For example, polymorphisms in VEGF that decrease its expression in the brain and spinal fluid increase ALS risk, whereas boosting VEGF by gene transfer, stem cell therapies, or protein delivery promotes survival in pre-clinical ALS models. VEGF gene therapies have entered clinical trials for ALS; however, unlike ASO delivery, these approaches may carry risk for neuro-immune responses, are highly invasive, and cannot be titrated for safety or efficacy.

[0133] Fortunately, VEGF expression is extensively regulated through its 3’UTR with well-characterized repressors, making it a strong candidate for ASO upregulation (FIG. 5A). ASO-mediated VEGF upregulation was predicted to extend survival and promote neuron health in an ALS mouse model.

[0134] The pipeline to design described above was applied to VEGF-ASOs. Briefly, reporter constructs containing the 3’UTR of VEGF were developed alongside ASO libraries that mask well-characterized regulatory elements conserved between human and mouse genomes. This library was screened in multiple human tissue cultures, including glia and neurons, for increased reporter and protein expression. To evaluate ASOs in vivo, the top 3 candidate ASOs were re-designed to match conserved mouse Vegf sequences (mVegf-ASOs), administer these by intracerebroventricular injection to SOD1 -G93A ALS model mice (FIG. 5B), and measure VEGF protein in the cortex and spinal cord using ELISA assays. Whether mVegf-ASOs can extend survival and limit motor neuron loss was tested when delivered at phenotypic onset. Modest (1 ,5-3x) increases in neurotrophins like VEGF achievable with ASOs provide neuroprotection without significant risk. However, because substantially increasing VEGF can induce excessive angiogenesis, blood- brain-bamer integrity in mice was characterized after ASO treatment.

[0135] Regulation of gene expression at the mRNA level is dependent on the network of microRNAs and RBPs expressed in a cell-specific manner. To profile such networks, post-transcriptional regulatory element sequencing (PTRE-seq) was developed, a massively parallel method to catalogue binding efficiency of microRNAs and RBPs to reporter mRNAs and sequence preferences for these interactions (FIG. 6A). These results confirmed complex interactions between microRNA and RBP-binding sites that could be exploited to design cell-specific masking ASOs. the PTRE-seq platform was used to test cell-specific efficiency of ASOs in targeting multiple RBPs and microRNAs in 3’UTR libraries with synthetic or endogenous cis-elements.

[0136] Multiple libraries of reporter and endogenous 3’UTRs with target sites for a defined set of neuron-, astrocyte-, and microglia-specific microRNAs and RBPs were used. The barcoded PTRE-seq libraries will be deployed independently to iPSC- derived neurons and glia, alongside control or cis-element-targeted ASOs. The effect of ASOs on post-transcriptional gene regulation measured by sequencing barcodes specific to each 3’UTR reporter to measure RNA levels and by determining independent translation profiles for mRNAs (FIG. 6B). The mechanism of ASOs will be dependent on either “masking” cell-specific trans-elements or a combination of trans- elements that interact within gene distinct 3’UTRs in cell specific manner.

[0137] This approach allowed deep mechanistic investigation into ASO-mediated effects on mRNA stability and translation when specific trans-factors (microRNA, RBPs) and their cis binding elements are experimentally manipulated. ASOs have neuronal or glial-specific effects on mRNA translation and stability due to different expression levels of trans-factors and their 3’UTR association in those cell types (FIG. 6B). The present disclosure provided 1 ) a generalizable method to increase any protein of interest and 2) complex “barcoded” 3’UTR reporter libraries for mechanistic studies.

[0138] EXAMPLE 2 - PUMILIO PROTEINS REGULATE MANY GENE TARGETS RELATED TO DISEASE

[0139] Pumilio proteins are well-characterized repressors of mRNA stability and translation, suggesting Pumilio proteins as targets for gene upregulation. First, existing data-sets were mined to examine genes potentially regulated by Pumilio proteins. Pumilio proteins commonly bind at Pumilio recognition elements (PREs) that are found in the 3’UTR of about 40% of genes. In the subset of genes expressed in HEK293 cells, ~3350 transcripts are bound by PUMs. The union of “bound genes” and PRE-containing genes represents targets with high likelihood for regulation by PUM and includes a set of 2740 transcripts (FIG. 7A). This set of genes is enriched with ~193 haploinsufficiency-associated genes (annotated by OMIM) and ~682 other disease-associated genes (annotated by Orphanet). To examine this potential regulation, six disease associated gene transcripts were selected (ITPR1, TBK1, FST, SLC2A1, LDLR, and DEPDC5) as potential targets that are strongly bound by Pumilio proteins PUM1 and PUM2 by RNA immunoprecipitation sequencing in HEK293 cells (FIGS. 7B and FIG. 7C). To test whether PUM proteins negatively regulate expression of these selected transcripts, PUM1 and PUM2 was depleted using siRNA in HEK293 cells. Indeed, PUM depletion increased mRNA of each gene target by >1.5-fold (FIG. 7D). Therefore, Pumilio depletion might represent a nonspecific strategy to upregulate many genes simultaneously. EXAMPLE 3 - MASKING PRES ON 3’UTRS UPREGULATES DISEASE-ASSOCIATED PROTEIN TARGETS

[0140] Selective gene upregulation has been an elusive but attractive therapeutic goal. PLIMs recognize and bind at a highly stereotyped 8 base pair motif (UGUAHAWW) that is readily identifiable within a gene 3’UTR (FIG. 8A). It was proposed that neutralizing Pumilio activity at a single gene target using antisense oligonucleotides (ASOs) could provide gene-specific upregulation by augmenting mRNA stability or translation (FIG. 8B).

[0141] To provide a proof-of-principle for PRE-masking as a strategy for protein upregulation, a fluorescent and luciferase-based reporter systems was utilized. First a fluorescent EGFP reporter was developed that is flanked by a synthetic 3’UTR that contains two instances of a PRE (FIG. 8C) and designed reporter-specific ASOs that target individual PRE segments. Transfecting an empty vector (PBS), non-targeting control (NTC) ASO, or a reporter-specific PRE-targeting ASO revealed that ASOs can amplify fluorescence. Protein upregulation by 1.5-fold occurred within 24 hours of ASO treatment and was sustained through 72 hours of incubation (FIG. 8C).

[0142] To demonstrate that this effect is not restricted to EGFP mRNA, dual luciferase reporter systems were implemented to extend this approach to 3’UTRs from endogenous genes that contain PREs that are potentially regulated by PUMs. The initial focus centered on the six disease-associated transcripts that the previous experiments demonstrated are repressed by PUM proteins (ITPR1, TBK1, FST, SLC2A1, LDLR, and DEPDC5), and a transcript-specific ASOs for each gene were developed. The results indicated at least one ASO per gene that was able to increase reporter expression by between 1 .25 to 1 .6-fold (FIGS. 8D, 12A, 12B, and 13). Further experiments demonstrated using the top ASO for each gene can also upregulate endogenous gene mRNA (FIG. 8E). To demonstrate the mechanism of action, specificity, and applicability of PRE-masking ASOs, further experimentation focused on the regulation of TANK-binding kinase 1 (TBK1).

[0143] EXAMPLE 4 - PUMILIO PROTEINS REPRESS TBK1 LEVELS THROUGH MRNA DESTABILIZATION

[0144] Amyotrophic lateral sclerosis is a neurodegenerative disease that may result from haploinsufficiency of TBK1. The 3’UTR of TBK1 contains 2 PREs that are positioned within 10 base pairs (FIG. 9A), and PUM proteins strongly bind TBK1 mRNA (FIG. 7C). To evaluate how Pumilio proteins repress TBK1 expression, PUM proteins were depleted by siRNA in HEK293 cells. In addition to mRNA upregulation (FIG. 7D), it was found that depleting PUM proteins increases TBK1 protein by 1 .7- fold (FIG. 9B). It was suspected this might occur through mRNA destabilization. To test this possibility, the transcription inhibitor Actinomycin D was administered for 0 or 6 hours to HEK293 cells treated with PUMs siRNA. The results show that depleting PUM proteins increased the fraction of TBK1 mRNA remaining after 6 hours, suggesting that PUM proteins de-stabilize TBK1 mRNA (FIG. 9C). To test whether this repression is indeed occurring through PREs on the 3’UTR of TBK1 , the 3’UTR luciferase reporter was mutagenized at each PRE site and protein expression and mRNA stability were examined (FIG. 9D). Mutagenesis of each site increased reporter expression and loss of both sites elevated reporter levels by 2.2- fold (FIG. 9E). In addition, the mutagenized reporter achiever greater mRNA stability after incubating with Actinomycin D for 0-8 hours (FIG. 9F). Finally, depleting PUM proteins increased luciferase expression.

[0145] EXAMPLE 5 - PRE-MASKING ASOS INCREASE TBK1 LEVELS THROUGH MRNA DE-STABILIZATION

[0146] It was suspected that ASOs targeting the PREs on TBK1 mRNA operate through eliminating Pumilio activity. To test this possibility, stable 3’UTR reporter HEK293 cells were developed that express EGFP flanked by the 3’UTR of TBK1 under doxycycline control (FIG. 10A). First, experiments examined whether TBK1 - ASOs can increase mRNA abundance of TBK1 and EGFP. Indeed, the experiments resulted in dose-dependent increases in each, achieving >1.8-fold increase in TBK1 and 1.6-fold in EGFP at the highest dose tested (FIG. 10B). Examination of TBK1 or EGFP protein in these cells revealed increases of each to about 1 .7-fold for TBK1 and 1.5-fold for EGFP (FIG. 10C). To examine whether ASOs similarly stabilize target mRNA, experiments investigated TBK1 and EGFP mRNA after ASO treatment. ASOs substantially extended mRNA half-life after treating with Actinomycin D for 0-12 hours (FIGS. 10D and 13). Finally, treating the mutagenized luciferase reporter with TBK1 ASO did not affect reporter levels, suggesting that ASOs are specific to the 3’UTR PRE sites (FIG. 10E). Thus, these results demonstrate by using two reporter systems and endogenous TBK1 itself that ASOs operate through targeting the 3’UTR of TBK1 to increase mRNA and protein expression.

[0147] EXAMPLE 6 - ASOs TARGETING PRES ARE ACTIVE IN IN VIVO

[0148] Further studies evaluated whether PRE-targeting ASOs can increase protein abundance in the mouse. While the PREs in TBK1 are conserved across species, the surrounding and intervening sequences are highly different, making the ASOs inactive against the mouse TBK1 sequence. Of those examined, FST mRNA is strongly repressed by PUM proteins (FIG. 7D), and ASOs targeting FST are fully conserved across species; therefore, ASOs targeting FST were selected for in vivo testing (FIG. 11 A).

[0149] FST is abundantly expressed in human fibroblasts, so initial studies tested if FST-targeted ASOs could increase FST mRNA in vitro. ASOs strongly upregulated FST by >2.5-fold in fibroblasts (FIG. 11 B) and stabilized FST mRNA in HEK293 cells (FIG. 11C). To examine whether ASOs are also active against mouse Fst, FST- ASOs were delivered to mouse embryonic fibroblasts and achieved upregulation of Fst mRNA (FIG. 11D). Candidate ASOs were then optimized, based on ASO F4, to advance to in vivo study. To examine improved candidates in the mouse, FST- targeted ASOs were delivered by intraperitoneal injection at 25mg / kg to C57 naive mice and harvested liver and gastrocnemius muscle after four days (FIG. 11 E). FST- targeted ASOs were then evaluated to determine whether FST-targeted ASOs can increase Fst mRNA in the central nervous system. ASO candidates were delivered by intracerebroventricular stereotaxic injection and harvested cortex and hippocampus after seven days (FIG. 11 F).

[0150] EXAMPLE 7 - ASOPE AGAINST FOLLISTATIN, ENHANCING MUSCLE PRODUCTION

[0151] An experiment to confirm that F4 can increase FST mRNA from the genome in mouse and human fibroblasts was designed. This RT-qPCR confirmed F4 robustly increased FST mRNA levels (FIGS. 14A-14E). Because the PUM target sequence is highly conserved, the same ASO worked in both mouse and humans.

[0152] EXAMPLE 8 - MATERIALS AND METHODS

[0153] Cell culture Human embryonic kidney 293 (HEK293) cells were obtained from the American Type Culture Collection (CRL-1573; ATCC, Manassas, VA, USA). HeLa cells were obtained from the American Type Culture Collection (CCL2; ATCC, Manassas, VA, USA). HEK293 and HeLa cells were maintained in DMEM and 10% FBS, containing antibiotics, unless described otherwise.

[0154] Stable Flp-ln T-REx-HEK293 cells expressing an inducible EGFP TBK1 3'UTR reporter were generated using the Flp-ln T-REx system (Invitrogen). The EGFP coding sequence followed by the TBK1 3'UTR was cloned into the pcDNA5 / FRT / TO vector. Flp-ln T-REx-HEK293 host cells were co-transfected with the pcDNA5 / FRT / TO-EGFP-3'UTR construct and pOG44 Flp recombinase expression plasmid at a 1 :9 ratio using Lipofectamine 3000 (Invitrogen) according to the manufacturer's protocol. Forty-eight hours post-transfection, cells were selected with 100 pg / mL hygromycin B for two weeks. Individual colonies were isolated, expanded, and screened for tetracycline-inducible EGFP expression. EGFP expression was induced with 1 pg / mL tetracycline for 24 hours and verified by fluorescence microscopy and flow cytometry. Positive clones were maintained in DMEM supplemented with 10% tetracycline-free FBS, 100 pg / mL hygromycin B, and 15 pg / mL blasticidin. Induction of EGFP in cell culture was performed by adding doxycycline to final concentration of 1 ug / mL in the media without replacement. iPSC-derived iNeurons with a doxycycline-inducible NGN2 promoter were cultured and differentiated using a modified protocol. On day 0, cells were seeded onto a Matrigel-coated (Coming) plate into Stemflex media (Thermo Fisher Scientific) without antibiotics, supplemented with 10 pM ROCK inhibitor Y-27632 (Tocris). Media was changed daily after the first day. On day 1 , the culture was switched to N2B27 media (1 :1 mixture of DMEM / F12 and Neurobasal media supplemented with 1 % N2, 2% B27, 1 % GlutaMAX, and 0.1 % P-mercaptoethanol, all from Thermo Fisher Scientific). Cells were maintained in N2B27 media with daily changes until day 5. On day 5, cells were dissociated using Accutase (Stemcell Technologies) and re-plated onto 12-well plates coated with poly-d-lysine (Sigma) in N2B27 media containing 10 pM Y-27632. Neuronal differentiation was induced by adding 2 pg / mL doxycycline to the culture media. On day 8 (DIV8), cells were transfected using Lipofectamine RNAiMAX (Invitrogen) according to the manufacturer's protocol. Media was changed 24 hours post-transfection. Cells were maintained until DIVI O, at which point they were collected for downstream analyses. Throughout the differentiation process, cells were kept at 37°C in a humidified incubator with 5% CO2.

[0155] Plasmid constructs and mutagenesis

[0156] The 3'UTRs of interest (TBK1, DEPDC5, ITPR1, LDLR, FST, SLC2A1, WAC) were amplified from human genomic DNA. The PCR reactions were performed using Q5 High-Fidelity DNA Polymerase (New England Biolabs) according to the manufacturer's instructions. The pMirGlo vector (Promega) were digested with Xhol and Notl restriction enzymes (New England Biolabs) for 1 hour at 37°C. The digested vector was dephosphorylated using Antarctic Phosphatase (New England Biolabs) to prevent self-ligation. The PCR products were purified using the Monarch PCR cleanup kit (NEB). The In-Fusion cloning reaction was set up using the InFusion Snap Assembly Master Mix (Takara Bio) with a 3:1 molar ratio of insert to vector. The reaction was incubated at 50°C for 15 minutes, then placed on ice. The ligation products were transformed into competent E. coli DH5ct cells (Invitrogen) using heat shock at 42°C for 30 seconds and according to manufacturer instructions. Transformed bacteria were plated on LB agar containing 100 pg / mL ampicillin and incubated overnight at 37°C. Individual colonies were screened by colony PCR, and positive clones were confirmed by Sanger sequencing (Genewiz).

[0157] Mutations in the PRE sites of TBK1 were introduced using site-directed mutagenesis. Mutagenic primers were designed with Quickchange software. PCR amplification was performed using Phusion High-Fidelity DNA Polymerase (Thermo Fisher Scientific) with 5 ng of template DNA, 0.5 pM of each primer, 200 pM dNTPs, and 1X Phusion HF Buffer in a 50 pL reaction volume. Thermal cycling conditions were: initial denaturation at 98°C for 30 seconds; 19 cycles of 98°C for 10 seconds, 65°C for 30 seconds, and 72°C for 8 minutes; followed by a hold at 4°C. The PCR product was treated with 1 pL Dpnl (New England Biolabs) at 37°C for 1 hour to digest the parental DNA template. NEB 5-alpha Competent E. coli cells (New England Biolabs) were transformed with 5 pL of the Dpnl-treated PCR product using heat-shock. Transformants were selected on LB agar plates containing appropriate antibiotics. Positive clones were identified by colony PCR and confirmed by Sanger sequencing (Genewiz). Dual luciferase assays

[0158] Firefly and Renilla luciferase activities were quantified using the Dual-Glo® Luciferase Assay System (Promega) following the manufacturer’s protocol. Cells were seeded in 96-well plates and transfected after 24 hours with the experimental Firefly / Renilla dual luciferase reporter construct using Lipofectamine 3000 (Thermo Fisher Scientific). After 48 hours, cells were lysed in 50 pl Dual-Glo® reagent, incubated for 10 minutes at room temperature, and firefly luciferase activity was measured using a luminometer (Agilent, BioTek Synergy H1 ). Subsequently, 50 pl of Dual-Glo® Stop & Gio reagent was added to quench firefly luciferase activity and activate Renilla luciferase, followed by a 30-minute incubation and measurement. Results were normalized to Renilla luciferase activity to control for transfection efficiency and cell viability. All experiments were repeated with at least three independent biological replicates. Empty vector and mock-transfected cells served as controls.

[0159] Drug treatments

[0160] For transcriptional inhibition, cells were treated with Actinomycin D (ActD, Sigma-Aldrich A9415) at a final concentration of 5 pg / mL for 0-12 hours. At each time point, cells were harvested for RNA extraction and subsequent analysis. The 0-hour time point served as the baseline for normalization. For autophagy inhibition, cells were treated with Bafilomycin A1 (BafA, Sigma-Aldrich B1793) at a final concentration of 100 nM for 4 hours. Control cells were treated with an equivalent volume of DMSO (vehicle).

[0161] A SO designs

[0162] All ASOs were designed as indicated and ordered from Integrated DNA Technologies or GeneTools (only morpholinos). All were fully modified 15-25 base pair sequences consisting of 2’-methoxyethyl or locked nucleic acid bases. Backbones used were phosphorothioate or morpholino.

[0163] A SO and siRNA treatments in cells

[0164] Treatments using ASOs or siRNA were performed using reverse transfection unless otherwise indicated. HEK293 cells were plated in DMEM at 75% confluency at the time of transfection. ASO or siRNA was complexed with RNAiMAX transfection reagent (Invitrogen) in Opti-MEM Reduced Serum Medium according to manufacturer recommendations, with final ASO or siRNA concentrations ranging from 10-250 nM in culture medium. The RNAiMAX:ASO complexes were incubated at room temperature for 20 minutes before being added dropwise to cells. Transfected cultures were maintained for 48-72 hours at 37°C with 5% CO2, without media replacement. Non-targeting ASO controls and lipofectamine only (PBS) cells serving as negative controls. Cells were harvested for downstream analysis (qPCR, western blot, or functional assays) at experimental endpoints.

[0165] Immunoblotting

[0166] Cells were washed once with ice-cold PBS and lysed in RIPA buffer (50 mM Tris-HCI pH 7.4, 150 mM NaCI, 1 % NP-40, 0.5% sodium deoxycholate, 0.1 % SDS) supplemented with fresh EDTA-free protease inhibitor cocktail (Roche Diagnostics). Lysates were sonicated for 5 minutes at 4°C, followed by centrifugation at 21 ,000 x g for 15-20 minutes at 4°C to pellet insoluble debris. Supernatants were quantified using the Pierce BCA Protein Assay Kit according to the manufacturer’s protocol, with absorbance measured at 562 nm after a 30-m inute incubation at 37°C. Protein concentrations were normalized against a bovine serum albumin (BSA) standard curve. For electrophoresis, 20 pg of protein per sample was mixed with Laemmli buffer, denatured at 95°C for 10 minutes, and resolved on a 4%-20% gradient polyacrylamide gel (Bio-Rad) using Tris-glycine-SDS running buffer. Proteins were transferred to PVDF membranes using a transfer system at constant voltage (400 mA, 1 hour) for downstream immunoblotting. Membranes were incubated in primary antibodies overnight, then 1 hour incubation in secondary antibody. Membranes were visualized using Clarity ECL reagent (Biorad, 1705061 ). Primary antibodies used for blotting: VCL at 1 :4000 (Sigma, V9131 ), TBK1 at 1 :1000 (Abeam, ab40676), pAKT at 1 :1000 (Cell Signaling Technologies, 9271 S), AKT at 1 :1000 (Cell Signaling Technologies, 9272), EGFP at 1 :1000 (Invitrogen MA1 -952), p62 at 1 :3000 (Abeam, ab56416), and LC3b at 1 :1000 (Cell Signaling Technologies, 83506). Secondary antibodies used: anti-Rabbit IgG HRP (Sigma, GENA934) and anti-Mouse IgG HRP (Sigma, GENA931 ). mRNA quantification

[0167] RNA was isolated from cell, liver, or muscle tissue using the RNeasy Mini Kit (Qiagen) following the manufacturer's instructions. Cells or tissue were lysed with 300 pL RLT buffer (Qiagen) in the plate or in Eppendorf tubes. If using cells, the plate was shaken for 10 minutes at room temperature. The sample was then mixed with 1.5 volumes of 100% ethanol and transferred to an RNeasy column for RNA purification with DNase treatment according to the kit protocol. cDNA synthesis was carried out using the High-Capacity cDNA Reverse Transcription Kit (Invitrogen). Quantitative PCR was performed on the QuantStudio 12K Flex Real-Time PCR System using Power SYBR Green Power PCR Master Mix (Thermo Fisher Scientific). Gene expression was quantified using the AACt method, using GAPDH and ACTB (human) or Gapdh and Actb (mouse) as reference genes.

[0168] RNA-seq analysis was performed on samples sequenced using an Illumina NovaSeq X Plus platform. Raw data processing, including basecalling and demultiplexing, was conducted using Illumina's DRAGEN and BCLconvert (v4.2.4) software. Reads were aligned to the Ensembl release 101 primary assembly using STAR (v2.7.9a1 ), with gene counts determined by Subread:featureCount (v2.0.32) and isoform quantification by Salmon (v1.5.2). Sequencing quality was assessed using RSeQC (v4.0). Data normalization and differential expression analysis were performed using EdgeR and Limma R / B ioconductor packages, with TMM normalization and voomWithQualityWeights transformation. Only genes containing Pumilio recognition elements or that are bound by Pumilio were compared. Benjamini-Hochberg FDR-adjusted p-values < 0.05 based on this subsetted genome were considered significantly differentially expressed.

[0169] Animals

[0170] C57BL / 6J mice (The Jackson Laboratory, stock no. 000664) were bred and housed at Washington University in St. Louis. Mice were provided with unlimited food and water and kept on a standard 12-hour light / dark cycle. Animal use was conducted in accordance with the National Institutes of Health (NIH) guidelines for animal research, under protocols approved by the Institutional Animal Care and Use Committee of Washington University in St. Louis.

[0171] A SO intraperitoneal injection

[0172] All experiments were performed using male C57BL / 6J mice. Mice received a single intraperitoneal (IP) injection of saline, non-targeting control (NTC) ASO, or an FST-targeting ASO at a dose of 25 mg / kg prepared in sterile saline. Four days after IP injection, mice were perfused with cold PBS and euthanized. The liver and the right and left gastrocnemius muscles were rapidly dissected. Tissues were immediately flash-frozen in liquid nitrogen and subsequently stored at -80°C until further processing and analysis.

[0173] Statistical analysis

[0174] For all experiments, the primary comparison was between NTC-ASO control versus any targeting ASOs that were tested. Comparisons between two groups were conducted using student’s t-test. Multiple comparisons were conducted using ANOVA with Dunnett’s post-hoc test, such as in ASO screening experiments. Measurements of mRNA stability (factors: condition, hours incubated in actinomycin D) and TBK1 -ASO reporter specificity (factors: ASO treatment, each reporter) were analyzed with Two-way ANOVA with Dunnett’s post-hoc test where appropriate. Significance is defined as follows: *p < 0.05, **p < 0.01 , ***p < 0.001 , ****p < 0.0001 , and ns, no statistical significance (P > 0.05). All data were analyzed with GraphPad Prism (version 10, USA).

[0175] MPRA Library Design

[0176] MPRA elements were designed using the region spanning chr12:6450138- 64502114 in GRCh38.p14. Using a custom Python script, the region was tiled by selecting 180nt with a 5nt step until all 5nt steps were exhausted resulting in 111 unique sequences. From those unmodified tiles, the middle 20nt (from position 80 to 100) were either removed or swapped for a null sequence (Plassmeyer et al., 2023) resulting in 111 new sequences with lengths of 160nt and 180nt, respectively. For plasmid library cloning, Kpnl and Nhel sites were appended to the 5’ and 3’ ends of each sequence, respectively, with primer binding sites flanking each restriction site. To ensure all elements were the same length as per synthesis requirements, a 20nt filler was placed between the 5’ primer and the 5’ restriction site in the swap elements. The resulting length of each designed element was 240nt elements.

[0177] MPRA Library Cloning

[0178] The designed 3’UTR library was synthesized by Twist Bioscience via oligo array synthesis. The oligo pool was amplified using NEBNext Ultra II Q5 Master Mix for 12 cycles with 5ng of ssDNA input for 2 technical replicate reactions. PCR products were purified with a 1 : 1 ratio of Mag-Bind TotalPure NGS beads to sample. Purified PCR products were digested with Kpnl and Nhel followed by a 1 :1 bead purification. Partial Illumina sequencing adapters were ligated onto purified digestions, which was followed by a double-sided bead purification (0.8x / 1.2x). Primers with a unique P7 index and a universal P5 index were used to amplify and index the ligation products, which was followed by a double-sided bead purification (0.8x / 1 ,2x). Sequencing libraries were submitted to the Genome Technology Access Center (GTAC) at Washington University School of Medicine with an average read depth of 12.5 million reads using a NovaSeq (Illumina). Upon confirmation of successful enrichment of the oligo pool, residual digested oligos described above were ligated into pJD599 in the 3’UTR region downstream of a tdTomato coding region (Selmanovic et al., n.d.). The cloned library was transformed into NEB 10- beta Electrocom petent E. coli (NEB C3020K), and plasmid library was purified using a Maxi-prep. Plasmid DNA library elements were amplified using primers that flank the 3’UTRs (oligo table), and element distribution was determined by Illumina sequencing as described above.

[0179] Cell Culture and Transfection

[0180] HEK293 cells for each condition were grown in T-75 flasks in a medium consisting of 0.22uM vacuum-filtered EMEM with 10% Fetal Bovine Serum and 1 % Pen / Strep. For transfection, cells were plated at a density of 2x10A6 cells in 10cm culture dishes using reverse transfection with 2ug plasmid delivered by Lipofectamine 3000. The MPRA library was individually transfected across n=6 replicate plates. 24 hours post transfection, medium was replaced with antibiotic containing medium. Forty-eight hours after transfection, cells were collected using Trizol reagent and stored in 1.5mL microcentrifuge tubes at -80°C until all samples were collected for RNA extraction (see below).

[0181] RNA Extraction and purification

[0182] RNA was extracted and purified from Trizol lysates using isopropanol precipitation followed by Zymo Clean and Concentrator - 25 kit (Zymo, CA USA). Once samples had thawed after removal from -80°C, 20% volume of chloroform (200pL) was added to each sample and hand-shaken for 30 seconds. After a 5- minute incubation at RT, samples were centrifuged at 12,000g for 15 minutes at 4°C to allow phase separation of the samples. After phase separation, 450pL of aqueous phase was transferred to a clean microcentrifuge tube and mixed with an equal volume of 100% isopropanol. After a 5-minute incubation with isopropanol, samples were centrifuged at 20,000g for 20 minutes at 4°C to pellet the RNA. After pelleting RNA, the supernatant was removed, and the pellet was washed with 1 mL of 100% ethanol followed by centrifugation at 20,000g for 20 minutes at 4°C. The ethanol supernatant was then removed and the pellet allowed to dry. Once dry, the pellet was resuspended and DNase treated in a 50pL reaction mixture Turbo DNA-free kit (Ambion #AM1907, Austin, TX, USA) to removal residual plasmid DNA. DNase treated RNA samples were then applied to the kit per manufacturer protocol.

[0183] Illumina Library Preparation of MPRA from Cultured Cells

[0184] 5pg of RNA from each sample was used to prepare targeted cDNA sequencing libraries using a reverse transcription (RT) primer distal to the 3’UTR tiled sequence. cDNA products were split in half with one half undergoing PCR amplification of prespliced (intron) mRNA with a forward primer that binds the intron within the reporter gene and a reverse primer flanked by the 3’UTR and RT primer and the other half with a forward primer that spans the exon-exon junction of the spliced reporter (exon). Both intron and exon PCRs were purified using a 0.4x / 1 ,2x magnetic bead purification and subsequently digested with Nhel (New England Biolabs) and Kpnl (New England Biolabs). Digested amplicons were isolated using a 0.8x / 1.2x bead purification wherein Illumina P7 and P5 adapters were ligated. Ligation products were isolated using a 0.8x / 1.2x bead purification and amplified with flanking primers that added the i7 and i5 index sequences. Sequencing depth averaged ~59 million reads for each sample. No reverse transcriptase controls were simultaneously performed for a subset of replicates that did not produce sequencing libraries indicating that all libraries were generated only RNA. Input plasmid DNA was sequenced following the intron PCR method described above.

[0185] MPRA Data Analysis

[0186] Paired end reads were merged using Pandaseq (v2.11 ) with default parameters. Merged reads were analyzed using a custom Python counting script that searches for exact matches of designed 3’UTR tiling elements. All downstream analysis was performed using a custom R script. Raw counts were normalized as counts per million (CPM). These normalized counts were used to calculate expression as the log2(RNA / DNA). To find potential ASO targets, positionally identical tiles with the middle 20nt removed or swapped were compared to the unmodified tiles, and statistical analysis was performed using a Wilcoxon Rank Sum Test.

[0187] Dual luciferase assays

[0188] Firefly and Renilla luciferase activities were quantified using the Dual-Glo® Luciferase Assay System (Promega) following the manufacturer’s protocol. Cells were seeded in 96-well plates and transfected after 24 hours with the experimental firefly / Renilla bicistronic luciferase reporter construct using Lipofectamine 3000 (Thermo Fisher Scientific). After 48 hours, cells were lysed in 50 pl Dual-Glo® reagent, incubated for 10 minutes at room temperature, and firefly luciferase activity was measured using a luminometer (Agilent, BioTek Synergy H1 ). Subsequently, 50 pl of Dual-Glo® Stop & Gio reagent was added to quench firefly luciferase activity and activate Renilla luciferase, followed by a 30-m inute incubation and measurement. Results were normalized to Renilla luciferase activity to control for transfection efficiency and cell viability. All experiments were repeated with at least three independent biological replicates. Empty vector and mock-transfected cells served as controls. qRT-PCR mRNA quantification

[0189] RNA was isolated from cell, brain, liver, or muscle tissue using the RNeasy Mini Kit (Qiagen) following the manufacturer's instructions. Cells or tissue were lysed with 300 pL QIAzol RLT buffer (Qiagen) in the plate or in Eppendorf tubes. If using cells, the plate was shaken for 10 minutes at room temperature. The sample was the mixed with 1.5 volumes of 100% ethanol and transferred to an RNeasy column for RNA purification according to the kit protocol. cDNA synthesis was carried out using the High-Capacity cDNA Reverse Transcription Kit (Invitrogen). Quantitative PCR was performed on the QuantStudio 12K Flex Real-Time PCR System using Power SYBR Green Power PCR Master Mix (Thermo Fisher Scientific). Gene expression was quantified using the AACt method, with GAPDH and ACTB as reference genes. Luciferase constructs

[0190] The 3'UTRs of interest were amplified from human genomic DNA. The PCR reactions were performed using Q5 High-Fidelity DNA Polymerase (New England Biolabs) according to the manufacturer's instructions. The pMirGlo vector (Promega) were digested with Xhol and Notl restriction enzymes (New England Biolabs) for 1 hour at 37°C. The digested vector was dephosphorylated using Antarctic Phosphatase (New England Biolabs) to prevent self-ligation. The PCR products were purified using the Monarch PCR cleanup kit (NEB). The In-Fusion cloning reaction was set up using the In-Fusion Snap Assembly Master Mix (Takara Bio) with a 3:1 molar ratio of insert to vector. The reaction was incubated at 50°C for 15 minutes, then placed on ice. The ligation products were transformed into competent E. coli DH5a cells (Invitrogen) using heat shock at 42°C for 30 seconds and according to manufacturer instructions. Transformed bacteria were plated on LB agar containing 100 pg / mL ampicillin and incubated overnight at 37°C. Individual colonies were screened by colony PCR, and positive clones were confirmed by Sanger sequencing (Genewiz).

[0191] A SO sequences

[0192] Table 1 : ASO sequences

Claims

ClaimsWhat is claimed is:

1. A composition comprising a 3’UTR-masking agent, wherein the 3’UTR- masking agent is an antisense oligonucleotide (ASO) comprising a sequence targeting specific regulatory elements of a protein of interest.

2. The composition of claim 1 , wherein the regulatory elements are selected from a Pumilio recognition element, a trans-acting element, or a combination thereof.

3. The composition of claim 1 , wherein the protein of interest is selected from the group consisting of follistatin (FST), TANK-binding kinase 1 (TBK1 ), progranulin (PGRN), and vascular endothelial growth factor (VEGF).

4. The composition of claim 1 , wherein the ASO targets FST and comprises the sequence of F1 , F2, F3, F4, F5, F6, F7, F4.1 , F4.2, F4.3, F4.4, F4.5, F4.6, F4.7, or F4.8.

5. The composition of claim 1 , wherein the ASO targets TBK1 and comprises the sequence of T1 , T2, T3, T4, T5, T6, T7, T8, T9, T10, T11 , T12, T13, T14, T15, T16, or T17.

6. A method of selectively increasing expression of a protein of interest in a subject in need thereof, the method comprising: administering to the subject a composition comprising one or more antisense oligonucleotides (ASOs) which specifically bind a 3’UTR of an mRNA encoding the protein of interest such that regulatory elements in the 3’UTR are masked by the ASO(s).

7. The method of claim 6, wherein the regulatory elements are selected from a Pumilio recognition element, a trans-acting element, or a combination thereof.

8. The method of claim 6, wherein the protein of interest is selected from the group consisting of follistatin (FST), TANK-binding kinase 1 (TBK1 ), progranulin (PGRN), and vascular endothelial growth factor (VEGF).

9. The method of claim 6, wherein the one or more ASOs target FST and comprises the sequence of F1 , F2, F3, F4, F5, F6, F7, F4.1 , F4.2, F4.3, F4.4, F4.5, F4.6, F4.7, or F4.8.

10. The method of claim 6, wherein the one or more ASOs target TBK1 and comprises the sequence of T1 , T2, T3, T4, T5, T6, T7, T8, T9, T10, T11 , T12, T13, T14, T15, T16, or T17.

11. A method of treating neurodegeneration in a subject in need thereof, the method comprising: administering to the subject a composition comprising one or more antisense oligonucleotides (ASOs) which specifically bind a 3’UTR of a mRNA encoding a protein of interest, such that regulatory elements in the 3’UTR are masked by the ASO(s) and expression of the protein of interest is increased.

12. The method of claim 11 , wherein the regulatory elements are selected from a Pumilio recognition element, a trans-acting element, or a combination thereof.

13. The method of claim 11 , wherein the protein of interest is selected from the group consisting of follistatin (FST), TANK-binding kinase 1 (TBK1 ), progranulin (PGRN), and vascular endothelial growth factor (VEGF).

14. The method of claim 11 , wherein the one or more ASOs target FST and comprises the sequence of F1 , F2, F3, F4, F5, F6, F7, F4.1 , F4.2, F4.3, F4.4, F4.5, F4.6, F4.7, or F4.8.

15. The method of claim 11 , wherein the one or more ASOs target TBK1 and comprises the sequence of T1 , T2, T3, T4, T5, T6, T7, T8, T9, T10, T11 , T12, T13, T14, T15, T16, or T17.

16. A method of screening for antisense oligonucleotides (ASOs) that increase expression of a protein of interest, the method comprising: providing a reporter construct comprising a 3’UTR of a mRNA encoding the protein of interest, operably linked to a reporter gene to a test cell; contacting the test cell from step a with a candidate ASO which is complementary to a sequence within the 3’UTR; and comparing the level of expression of the reporter gene in the test cell to the level of expression of the reporter gene in a control cell which is not contacted with the candidate ASO or which is contacted with a negative control molecule; wherein increased expression of the reporter gene in the test cell relative to the control cell indicates that the ASO increases expression of the protein of interest and acts as a 3’ UTR masking agent.

17. The method of claim 16, wherein the protein of interest is selected from the group consisting of follistatin (FST), TANK-binding kinase 1 (TBK1 ), progranulin (PGRN), and vascular endothelial growth factor (VEGF).

18. The method of claim 16, wherein the reporter gene is EGFP.

19. The method of claim 16, wherein the test cell is selected from the group consisting of human HEK293, microglial (HMC3), and neuroblastoma (SH- SY5Y).

20. The method of claim 16, wherein the ASO acts as a 3’ UTR masking agent when the expression of the protein of interest is increased by a factor of 1 .3.

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