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Title:
TYROSINE KINASE 2 (TYK2) ANTISENSE OLIGONUCLEOTIDES AND THEIR USE
Document Type and Number:
WIPO Patent Application WO/2022/139686
Kind Code:
A1
Abstract:
The present invention relates to antisense-oligonucleotides capable of hybridizing with a specific region of the mRNA encoding Tyrosine Kinase 2 (TYK2) and pharmaceutical compositions comprising same, which can be used to reduce inflammation and/or treat severe coronavirus disease. More specifically, the antisense-oligonucleotides utilize a gapmer design, which are capable of hybridizing with any continuous region within nucleotide position 4214-4233 or nucleotide position 4012-4032 of the mRNA encoding TYK2, and may be further modified with LNA, MOE, cEt and/or 2'-OMe.

Inventors:
PHAN ANH TUAN (SG)
TRAN VAN NHAN (SG)
LIM KAH WAI (SG)
NGUYEN LE TUAN ANH (SG)
Application Number:
PCT/SG2021/050819
Publication Date:
June 30, 2022
Filing Date:
December 22, 2021
Export Citation:
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Assignee:
UNIV NANYANG TECH (SG)
International Classes:
C12N15/113; A61K31/712; A61P31/14; A61P37/06
Other References:
ISHIZAKI MASAYUKI, MUROMOTO RYUTA, AKIMOTO TOSHIHIKO, SEKINE YUICHI, KON SHIGEYUKI, DIWAN MANISH, MAEDA HIROAKI, TOGI SUMIHITO, SH: "Tyk2 is a therapeutic target for psoriasis-like skin inflammation", INTERNATIONAL IMMUNOLOGY, OXFORD UNIVERSITY PRESS, GB, vol. 26, no. 5, 1 May 2014 (2014-05-01), GB , pages 257 - 267, XP055954533, ISSN: 0953-8178, DOI: 10.1093/intimm/dxt062
MINEGISHI Y. ET AL.: "Human Tyrosine Kinase 2 Deficiency Reveals Its Requisite Roles in Multiple Cytokine Signals Involved in Innate and Acquired Immunity", IMMUNITY, vol. 25, no. 5, 9 November 2006 (2006-11-09), pages 745 - 755, XP055573304, [retrieved on 20220218], DOI: 10.1016/J.IMMUNI. 2006.09.00 9
MUROMOTO RYUTA, TAWA KEISUKE, OHGAKIUCHI YUI, SATO AMI, SAINO YUKA, HIRASHIMA KOKI, MINOGUCHI HIROYA, KITAI YUICHI, KASHIWAKURA JU: "IκB-ζ Expression Requires Both TYK2/STAT3 Activity and IL-17–Regulated mRNA Stabilization", IMMUNOHORIZONS, vol. 3, no. 5, 1 May 2019 (2019-05-01), pages 172 - 185, XP055954536, DOI: 10.4049/immunohorizons.1900023
PAIRO-CASTINEIRA E. ET AL.: "Genetic mechanisms of critical illness in COVID-19", NATURE, vol. 591, 11 December 2020 (2020-12-11), pages 92 - 98, XP037386549, [retrieved on 20220218], DOI: 10.1038/S41586-020-03065-Y
Attorney, Agent or Firm:
DOWSING, Bruce John et al. (SG)
Download PDF:
Claims:
32

CLAIMS:

1. Antisense-oligonucleotide consisting of 15 to 20 nucleotides capable of specifically hybridizing with any continuous region within nucleotide position 4204-4233 or nucleotide position 4012-4032 of the mRNA encoding Tyrosine Kinase 2 (TYK2) (SEQ ID NO: 99), wherein the antisense-oligonucleotide has a gapmer structure and phosphate, phosphorothioate and/or phosphorodithioate as internucleotide linkages, and salts and optical isomers of said antisense-oligonucleotide.

2. The antisense-oligonucleotide of claim 1 , wherein the antisense-oligonucleotide consists of 15, 16 or 17 nucleotides.

3. The antisense-oligonucleotide of claim 1 or 2, wherein at least one of the nucleotides at each of the 5’ and 3’ ends are locked nucleic acids (LNAs) and wherein the antisense- oligonucleotide consists of a nucleotide sequence selected from the group comprising the sequence TGAGTTTATTACCAGA (SEQ ID NO: 1), the sequence ATGAGTTTATTACCAG (SEQ ID NO: 2), the sequence TGGTTTCATCCTGGAG (SEQ ID NO: 3), the sequence ATGGTTAGGCTCACGG (SEQ ID NO: 4), the sequence AGATGGTGGAGATGGT (SEQ ID NO: 5), the sequence TTTATTACCAGATGG (SEQ ID NO: 6), the sequence GGTTTCATCCTGGAGC (SEQ ID NO: 7), the sequence TTGGTTTCATCCTGGA (SEQ ID NO: 8), the sequence GAGTTTATTACCAGAT (SEQ ID NO: 9), the sequence GTGGGGTCTCTAGACA (SEQ ID NO: 10), the sequence CAGATGGTGGAGATGG (SEQ ID NO: 11), the sequence GGCCTTGGTACTTCTC (SEQ ID NO: 12), the sequence AGGAGTAAGGCACACG (SEQ ID NO: 13), the sequence GCCTCAAGTTTGGAAG (SEQ ID NO: 14) the sequence CTCTTGGTTTCATCCT (SEQ ID NO: 15), the sequence TTTGGAAGCTGGGGGA (SEQ ID NO: 16), the sequence TCTTGGTTTCATCCTG (SEQ ID NO: 17), the sequence CAAGATCATGGTTAGG (SEQ ID NO: 18), the sequence CCTCAAGATCATGGTT (SEQ ID NO: 19), the sequence ATTACCAGATGGTGGA (SEQ ID NO: 20), the sequence CTTGGTTTCATCCTGG (SEQ ID NO: 21), the sequence TGTTGGGTCCCTCCCT (SEQ ID NO: 22), the sequence GTGGGCCTCAAGTTTG (SEQ ID NO: 23), and the sequence ATGAGTTTATTACCAGA (SEQ ID NO: 24).

4. The antisense-oligonucleotide of any one of claims 1 to 3, wherein the antisense- oligonucleotide consists of a nucleotide sequence selected from the group comprising the sequence T G AGTTT ATT ACCAG A (SEQ ID NO: 25), the sequence

ATGAGTTTATTACCAG (SEQ ID NO: 26), the sequence TGGTTTCATCCTGGAG (SEQ ID NO: 27), the sequence ATGGTTAGGCTCACGG (SEQ ID NO: 28), the sequence AGATGGTGGAGATGGT (SEQ ID NO: 29), the sequence GTTTATTACCAGATGG (SEQ 33

ID NO: 30), the sequence TTTATTACCAGATGG (SEQ ID NO: 31), the sequence GGTTTCATCCTGGAGC (SEQ ID NO: 32), the sequence GTTTATTACCAGATGG (SEQ ID NO: 33), the sequence GTTTATTACCAGATGG (SEQ ID NO: 34), the sequence TGGTTTCATCCTGGAG (SEQ ID NO: 35), the sequence TTGGTTTCATCCTGGA (SEQ ID NO: 36), the sequence G AGTTT ATT ACCAG AT (SEQ ID NO: 37), the sequence GTTTATTACCAGATGG (SEQ ID NO: 38), the sequence GTGGGGTCTCTAGACA (SEQ ID NO: 39), the sequence CAGAT GGTGGAGATGG (SEQ ID NO: 40), the sequence GGCCTTGGTACTTCTC (SEQ ID NO: 41), the sequence AGGAGTAAGGCACACG (SEQ ID NO: 42), the sequence GCCTCAAGTTTGGAAG (SEQ ID NO: 43), the sequence TGGTTTCATCCTGGAG (SEQ ID NO: 44), the sequence CTCTTGGTTTCATCCT (SEQ ID NO: 45), the sequence TTTGGAAGCTGGGGGA (SEQ ID NO: 46), the sequence TGGTTTCATCCTGGAG (SEQ ID NO: 47), the sequence TCTTGGTTTCATCCTG (SEQ ID NO: 48), the sequence CAAGATCATGGTTAGG (SEQ ID NO: 49), the sequence CCTCAAGATCATGGTT (SEQ ID NO: 50), the sequence ATTACCAGATGGTGGA (SEQ ID NO: 51), the sequence TGGTTTCATCCTGGAG (SEQ ID NO: 52), the sequence CTTGGTTTCATCCTGG (SEQ ID NO: 53), the sequence TGTTGGGTCCCTCCCT (SEQ ID NO: 54), the sequence GTGGGCCTCAAGTTTG (SEQ ID NO: 55), the sequence ATGAGTTTATTACCAGA (SEQ ID NO: 56), the sequence ATGAGTTTATTACCAGA (SEQ ID NO: 57), the sequence AT G AGTTT ATT ACCAG A (SEQ ID NO: 58), the sequence ATGAGTTTATTACCAGA (SEQ ID NO: 59), the sequence ATGAGTTTATTACCAGA (SEQ ID NO: 60), the sequence AT G AGTTT ATT ACCAG A (SEQ ID NO: 61), the sequence ATGAGTTTATTACCAGA (SEQ ID NO: 62), the sequence ATGAGTTTATTACCAGA (SEQ ID NO: 63), the sequence ATGAGTTTATTACCAGA (SEQ ID NO: 64), the sequence ATGAGTTTATTACCAGA (SEQ ID NO: 65), the sequence TGAGTTTATTACCAGA (SEQ ID NO: 66), the sequence T G AGTTT ATT ACCAG A (SEQ ID NO: 67), the sequence ATGAGTTTATTACCAG (SEQ ID NO: 68), the sequence ATGAGTTTATTACCAG (SEQ ID NO: 69), the sequence T G AGTTT ATT ACCAG A (SEQ ID NO: 70), the sequence TGAGTTTATTACCAGA (SEQ ID NO: 71), the sequence TGAGTTTATTACCAGA (SEQ ID NO: 72), the sequence AT G AGTTT ATT ACCAG A (SEQ ID NO: 73), the sequence ATGAGTTTATTACCAGA (SEQ ID NO: 74) and the sequence ATGAGTTTATTACCAGA (SEQ ID NO: 75), wherein the LNAs are underlined. The antisense-oligonucleotide of any one of claims 1 to 4, wherein for the outermost 5 nucleotides on each end, 2 to 4 of them are LNA, the rest being DNA. The antisense-oligonucleotide of any one of claims 1 to 5, selected from the group comprising oligonucleotides consisting of the sequence set forth in SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 30, SEQ ID NO: 31 , SEQ ID NO: 32, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 66 and SEQ ID NO: 68. he antisense-oligonucleotide of any one of claims 3 to 6, wherein the LNA are substituted in part or in full by nucleotide analogues including but not limited to 2’-O-methoxyethyl (MOE) and constrained-ethyl (cEt). The antisense-oligonucleotide of any one of claims 1 to 7, further comprising a 2’-OMe nucleotide modification at gap position 2. The antisense-oligonucleotide of claim 8, selected from the group comprising TGAG(mT)TTATTACCAGA (SEQ ID NO: 76), TGGT(mT)TCATCCTGGAG (SEQ ID NO: 77), ATGG(mT)TAGGCTCACGG (SEQ ID NO: 78) and TTGG(mT)TTCATCCTGGA (SEQ ID NO: 79), wherein the LNAs are underlined and wherein “m” represents a 2’-OMe nucleotide modification at gap position 2. The antisense oligonucleotide of claim 9, wherein the antisense oligonucleotide is TGAG(mT)TTATTACCAGA (SEQ ID NO: 76). The antisense oligonucleotide of any one of claims 1 to 10, wherein the antisense oligonucleotide binds with 100% complementarity to the mRNA encoding Tyrosine Kinase 2 and does not bind to any other region in the human transcriptome. A pharmaceutical composition containing an antisense-oligonucleotide of any one of claims 1 to 11 together with at least one pharmaceutically acceptable carrier, excipient, adjuvant, solvent or diluent. The pharmaceutical composition of claim 12, further comprising 4-bromobenzaldehyde /V- (2,6-dimethylphenyl)semicarbazone (EGA). Use of an antisense-oligonucleotide of any one of claims 1 to 11 , or a composition of claim 12 or 13, for the manufacture of a medicament for the treatment of an autoimmune disease, inflammatory disease or severe coronavirus disease. The use according to claim 14, wherein the autoimmune disease is selected from the group comprising rheumatoid arthritis, psoriasis (PSO), systemic lupus erythematosus (SLE), ankylosing spondylitis (AS), ulcerative colitis (UC), Crohn’s disease (CD), type 1 diabetes (T1 D) and multiple sclerosis (MS). The use according to claim 14, wherein the coronavirus disease is COVID- 19. A method of treatment of a subject in need of such treatment comprising administering to the subject an efficacious amount of an antisense-oligonucleotide of any one of claims 1 to 11 or a composition of claim 12 or 13. The method of claim 17, wherein the subject has an autoimmune disease, inflammatory disease or coronavirus disease. The method of claim 18, wherein the autoimmune disease is selected from the group comprising rheumatoid arthritis, psoriasis (PSO), systemic lupus erythematosus (SLE), ankylosing spondylitis (AS), ulcerative colitis (UC), Crohn’s disease (CD), type 1 diabetes (T1 D) and multiple sclerosis (MS). The method of claim 18, wherein the coronavirus disease is COVID-19. A kit comprising an antisense-oligonucleotide of any one of claims 1 to 11 or a composition of claim 12 or 13. Use of 4-bromobenzaldehyde /\/-(2,6-dimethylphenyl)semicarbazone (EGA) as a pharmaceutical composition or research tool to enhance activity of antisense- oligonucleotides.

Description:
TYROSINE KINASE 2 (TYK2) ANTISENSE OLIGONUCLEOTIDES AND THEIR USE

FIELD OF THE INVENTION

The present invention relates to antisense-oligonucleotides capable of hybridizing with a specific region of the mRNA encoding Tyrosine Kinase 2 (TYK2) and pharmaceutical compositions comprising same, which can be used to reduce inflammation and/or treat severe coronavirus disease. More specifically, the antisense-oligonucleotides utilize a gapmer design, which are capable of hybridizing with any continuous region within nucleotide position 4204- 4233 or nucleotide position 4012-4032 of the mRNA encoding TYK2, and may be further modified with LNA, MOE, cEt and/or 2'-OMe.

BACKGROUND OF THE INVENTION

Tyrosine kinase 2 (TYK2) is a member of the Janus kinase (JAK) family of non-receptor tyrosine kinase [O’Shea, J. J. et al., Annu. Rev. Med. 66:311-328 (2015)], together with JAK1 , JAK2, and JAK3. JAK kinases associate with various cytokine receptors at the cytoplasmic side, playing a crucial role as signaling mediators. Upon extracellular cytokine binding of the receptors, the bound JAK proteins phosphorylate each other as well as the receptors, which then serve as anchor points for transcription factors known as signal transducer and activator of transcription (STAT). The STAT members are in turn activated by tyrosine phosphorylation and undergo subsequent dimerization and translocation to the nucleus to propagate downstream gene signaling. Importantly, JAK kinases are critical signal transducers for numerous pro-inflammatory cytokines [Schwartz, D. M. et al., Nat. Rev. Rheumatol. 12:25-36 (2016)] and thus represent compelling pharmacological targets for the treatment of autoimmune and inflammatory diseases [Schwartz, D. M. Y. et al., Nat. Rev. Drug Discov. 16:843-862 (2017)]. As such, intensive efforts have been directed towards the development of small-molecule JAK inhibitors [Clark, J. D. etal., J. Med. Chem. 57:5023-5038 (2014); Bryan, M. C. and Rajapaksa N. S., J. Med. Chem. 61 :9030-9058 (2018)], with the first approval for the treatment of rheumatoid arthritis (RA) by such agents being granted by the U.S. Food and Drug Administration (FDA) in 2012. However, this and other first-generation JAK inhibitors are pan-acting with limited selectivity between different JAK paralogs. Their expansion towards additional autoimmune indications has been hampered by unfavorable safety profiles at higher therapeutic doses, most notably through inhibition of JAK2, a critical signal transducer in hematopoiesis [Neubauer, H. et al., Cell 93:397-409 (1998)]. As such, present drug development efforts focus on the selective targeting of individual JAK members to achieve robust clinical efficacy while minimizing undesirable side effects [Schwartz, D. M. Y. et al., Nat. Rev. Drug Discov. 16:843-862 (2017); Clark, J. D. et al., J. Med. Chem. 57:5023-5038 (2014)]. This presents a significant challenge, owing to the high homology among the various JAK paralogs [Clark, J. D. et a!., J. Med. Chem. 57:5023-5038 (2014)].

Recently, selective TYK2 targeting has emerged as an attractive treatment option for a range of autoimmune diseases. TYK2 plays an essential role in the signaling pathways of multiple cytokines [Minegishi, Y. et al., Immunity 25:745-755 (2006); Ghoreschi, K. A. et al., Immunol. Rev. 228:273-287 (2009); Strobl, B. D. et al. Front. Biosci 16:3214-3232 (2011)], including type l/lll interferon (IFN), interleukin (IL)-12/23, and IL-10. Human genome-wide association studies [Ban, M. et al. Europ. J. Hum. Genet. 17: 1309-1313 (2009); Tao, J. H. et al. Mol Biol Rep 38:4663-4672 (2011); Dendrou, C. A. et al. Sci. Transl. Med. 8:363ra149] revealed that deactivating TYK2 variants could provide protection against a broad range of autoimmune conditions including psoriasis (PSO), RA, systemic lupus erythematosus (SLE), ankylosing spondylitis (AS), ulcerative colitis (UC), Crohn’s disease (CD), type 1 diabetes (T1 D), and multiple sclerosis (MS). Association of TYK2 with these conditions was also supported by findings from other studies [Marroqui, L. et al. Diabetes 64:3808-3817 (2015); Inshaw J. R. J. et al. Nat. Immunol. 19:674-684 (2018); Ishizaki M. et al. Int. Immunol 23:575- 582 (2011); Diogo, D. et al. PLoS One 10:e0122271 (2015); Sigurdsson, S. et al. Am. J. Hum. Genet. 76:528-537 (2005); Cunninghame Graham D. S. et al. PLoS Genet. 7:e1002341 (2011)]. Furthermore, drug agents targeting IFN-a and I L-12/1 L-23 signaling, all of which share TYK2 as a common signaling transducer, have shown clinical efficacy in various autoimmune conditions [Morand, E. F. et al. N. Eng. J. Med. 382:211-221 ; Savage, L. J. et al. Rhematol. Ther. 2:1-16 (2015)].

Among the JAK family members, TYK2 represents a safer target of choice, as JAK1- (perinatal lethal), JAK2- (embryonic lethal), and JAK3- (severe combined immunodeficiency) knockout mice displayed fatal/critical developmental defects [Ghoreschi, K. A. et al., Immunol. Rev. 228:273-287 (2009); Kiu, H. and Nicholson, S. E., Growth Factors 30:88-106 (2012)], whereas TYK2-knockout mice showed relatively milder deficiencies [Minegishi, Y. et al., Immunity 25:745-755 (2006); Kiu, H. and Nicholson, S. E., Growth Factors 30:88-106 (2012); Watford, W. T. and O’Shea, J. J. Immunity 25:695-697 (2006)] (susceptibility to mycobacterial, viral, and fungal infections). There are several TYK2-selective inhibitors undergoing active clinical development [Bryan, M. C. and Rajapaksa N. S., J. Med. Chem. 61 :9030-9058 (2018); He, X. X. et al. Expert Opin. Ther. Pat. 29:137-149 (2019)]. Different strategies were utilized in the design of the inhibitors, including active-site inhibition, simultaneous inhibition of TYK2/JAK1 [Fensome, A. et al. J. Med. Chem. 61 :8597-8612 (2018)], and allosteric binding of pseudokinase domain [Wrobleski, S. T. et al. J. Med. Chem. 62:8973-8995 (2019); Burke, J. R. et al. Sci. Transl. Med. 11 :eaaw1736 (2019)[. Early readouts from the clinical trials are encouraging [Papp, K. etal. N. Engl. J. Med. 379:1313-1321 (2018); Banfield, C. et al., J. Clin. Pharmacol. 58:434-447 (2018)], although longer-term efficacy and safety of these agents are yet to be determined [Winthrop, K. L. Nat. Rev. Rheumatol. 13:234-243 (2017)]. Additional TYK2-targeting agents that utilize different modes of action will expand on the drug development repertoire and are thus highly desirable. RNA therapeutics including antisense oligonucleotides (ASOs) and small-interfering RNAs (siRNAs) represent an emerging drug modality that have seen recent clinical breakthrough successes [Levin, A. A. N. Engl. J. Med. 380: 57-70 (2019)]. These agents operate on a genetic basis through complementary hybridization to their intended RNA targets

In view of the above deficiencies, it is desirable to provide improved inhibitors of TYK2 that may be used in the treatment of autoimmune, inflammatory and/or severe COVID-19 diseases.

SUMMARY OF THE INVENTION

Accordingly, we have developed an improved antisense-oligonucleotide inhibitor and method of inhibiting TYK2 activity to reduce inflammation and/or treat severe coronavirus disease.

According to a first aspect of the invention, there is provided an antisense- oligonucleotide consisting of 15 to 20 nucleotides capable of specifically hybridizing with any continuous region within nucleotide position 4204-4233 or nucleotide position 4012-4032 of the mRNA encoding Tyrosine Kinase 2 (TYK2) (SEQ ID NO: 99), wherein the antisense- oligonucleotide has a gapmer structure and phosphate, phosphorothioate and/or phosphorodithioate as internucleotide linkages, and salts and optical isomers of said antisense-oligonucleotide..

In some embodiments, the antisense-oligonucleotide consists of 15, 16 or 17 nucleotides.

In some embodiments, at least one of the nucleotides at each of the 5’ and 3’ ends are locked nucleic acids (LNAs) and the antisense-oligonucleotide is capable of hybridizing with a region of the mRNA encoding Tyrosine Kinase 2 (TYK2), wherein the antisense- oligonucleotide consists of a nucleotide sequence selected from the group comprising TGAGTTTATTACCAGA (SEQ ID NO: 1), the sequence ATGAGTTTATTACCAG (SEQ ID NO: 2), the sequence TGGTTTCATCCTGGAG (SEQ ID NO: 3), the sequence ATGGTTAGGCTCACGG (SEQ ID NO: 4), the sequence AGATGGTGGAGATGGT (SEQ ID NO: 5), the sequence TTTATTACCAGATGG (SEQ ID NO: 6), the sequence GGTTTCATCCTGGAGC (SEQ ID NO: 7), the sequence TTGGTTTCATCCTGGA (SEQ ID NO: 8), the sequence GAGTTTATTACCAGAT (SEQ ID NO: 9), the sequence GTGGGGTCTCTAGACA (SEQ ID NO: 10), the sequence CAGATGGTGGAGATGG (SEQ ID NO: 11), the sequence GGCCTTGGTACTTCTC (SEQ ID NO: 12), the sequence AGGAGTAAGGCACACG (SEQ ID NO: 13), the sequence GCCTCAAGTTTGGAAG (SEQ ID NO: 14) the sequence CTCTTGGTTTCATCCT (SEQ ID NO: 15), the sequence TTTGGAAGCTGGGGGA (SEQ ID NO: 16), the sequence TCTTGGTTTCATCCTG (SEQ ID NO: 17), the sequence CAAGATCATGGTTAGG (SEQ ID NO: 18), the sequence CCTCAAGATCATGGTT (SEQ ID NO: 19), the sequence ATTACCAGATGGTGGA (SEQ ID NO: 20), the sequence CTTGGTTTCATCCTGG (SEQ ID NO: 21), the sequence TGTTGGGTCCCTCCCT (SEQ ID NO: 22), the sequence GTGGGCCTCAAGTTTG (SEQ ID NO: 23) and the sequence ATGAGTTTATTACCAGA (SEQ ID NO: 24).

In some embodiments, the antisense-oligonucleotide consists of a nucleotide sequence selected from the group comprising T G AGTTT ATT ACCAG A (SEQ ID NO: 25) or the sequence ATGAGTTTATTACCAG (SEQ ID NO: 26), the sequence TGGTTTCATCCTGGAG (SEQ ID NO: 27), the sequence ATGGTTAGGCTCACGG (SEQ ID NO: 28), the sequence AGATGGTGGAGATGGT (SEQ ID NO: 29), the sequence GTTTATTACCAGATGG (SEQ ID NO: 30), the sequence TTTATTACCAGATGG (SEQ ID NO: 31), the sequence GGTTTCATCCTGGAGC (SEQ ID NO: 32), the sequence GTTTATTACCAGATGG (SEQ ID NO: 33), the sequence GTTT ATT ACCAGATGG (SEQ ID NO: 34), the sequence TGGTTTCATCCTGGAG (SEQ ID NO: 35), the sequence TTGGTTTCATCCTGGA (SEQ ID NO: 36), the sequence G AGTTT ATT ACCAG AT (SEQ ID NO: 37), the sequence GTTTATTACCAGATGG (SEQ ID NO: 38), the sequence GTGGGGTCTCTAGACA (SEQ ID NO: 39), the sequence CAGATGGTGGAGATGG (SEQ ID NO: 40), the sequence GGCCTTGGTACTTCTC (SEQ ID NO: 41), the sequence AGGAGTAAGGCACACG (SEQ ID NO: 42), the sequence GCCTCAAGTTTGGAAG (SEQ ID NO: 43), the sequence TGGTTTCATCCTGGAG (SEQ ID NO: 44), the sequence CTCTTGGTTTCATCCT (SEQ ID NO: 45), the sequence TTTGGAAGCTGGGGGA (SEQ ID NO: 46), the sequence TGGTTTCATCCTGGAG (SEQ ID NO: 47), the sequence TCTTGGTTTCATCCTG (SEQ ID NO: 48), the sequence CA AG AT CAT G GTT AG G (SEQ ID NO: 49), the sequence CCTCAAGATCATGGTT (SEQ ID NO: 50), the sequence ATTACCAGATGGTGGA (SEQ ID NO: 51), the sequence TGGTTTCATCCTGGAG (SEQ ID NO: 52), the sequence CTTGGTTTCATCCTGG (SEQ ID NO: 53), the sequence TGTTGGGTCCCTCCCT (SEQ ID NO: 54), the sequence GTGGGCCTCAAGTTTG (SEQ ID NO: 55), the sequence ATGAGTTTATTACCAGA (SEQ ID NO: 56), the sequence ATGAGTTTATTACCAGA (SEQ ID NO: 57), the sequence AT G AGTTT ATT ACCAG A (SEQ ID NO: 58), the sequence ATGAGTTTATTACCAGA (SEQ ID NO: 59), the sequence ATGAGTTTATTACCAGA (SEQ ID NO: 60), the sequence AT G AGTTT ATT ACCAG A (SEQ ID NO: 61), the sequence ATGAGTTTATTACCAGA (SEQ ID NO: 62), the sequence ATGAGTTTATTACCAGA (SEQ ID NO: 63), the sequence AT G AGTTT ATT ACCAG A (SEQ ID NO: 64), the sequence ATGAGTTTATTACCAGA (SEQ ID NO: 65), the sequence TGAGTTTATTACCAGA (SEQ ID NO: 66), the sequence T G AGTTT ATT ACCAG A (SEQ ID NO: 67), the sequence ATGAGTTTATTACCAG (SEQ ID NO: 68), the sequence ATGAGTTTATTACCAG (SEQ ID NO: 69), the sequence T G AGTTT ATT ACCAG A (SEQ ID NO: 70), the sequence TGAGTTTATTACCAGA (SEQ ID NO: 71), the sequence TGAGTTTATTACCAGA (SEQ ID NO: 72), the sequence AT G AGTTT ATT ACCAG A (SEQ ID NO: 73), the sequence ATGAGTTTATTACCAGA (SEQ ID NO: 74) and the sequence ATGAGTTTATTACCAGA (SEQ ID NO: 75), wherein the LNAs are underlined.

In some embodiments, for the outermost 5 nucleotides on each end of said oligonucleotide, 2 to 4 of them are LNA, the rest being DNA. In some embodiments, the antisense-oligonucleotide is selected from the group comprising oligonucleotides consisting of the sequence set forth in SEQ ID NO: 25, SEQ ID

NO: 26, SEQ ID NO: 27, SEQ ID NO: 30, SEQ ID NO: 31 , SEQ ID NO: 32, SEQ ID NO: 56,

SEQ ID NO: 57, SEQ ID NO: 66 and SEQ ID NO: 68.

In some embodiments, the LNA are substituted in part or in full by nucleotide analogues, including but not limited to 2’-O-methoxyethyl (MOE) and constrained-ethyl (cEt), which were shown to produce similar efficacy in other gapmer oligonucleotides [Hong, D. et al. Sci. Transl.

Med. 7:314ea185 (2015); Khvorova, A., et al., Nat. Biotechnol. 35: 238-248 (2017)].

In some embodiments, the antisense-oligonucleotide further comprises a 2’-OMe nucleotide modification at gap position 2.

In some embodiments, the antisense-oligonucleotide is selected from the group comprising:

TGAGmTTTATTACCAGA (SEQ ID NO: 76), TGGTmTTCATCCTGGAG (SEQ ID NO:

77), ATGGmTTAGGCTCACGG (SEQ ID NO: 78) and TTGGmTTTCATCCTGGA (SEQ ID

NO: 79), wherein the LNAs are underlined and wherein “m” represents a 2’-OMe nucleotide modification at gap position 2. More advantageously, the antisense-oligonucleotide is

TGAGmTTTATTACCAGA (SEQ ID NO: 76).

In some embodiments, the antisense oligonucleotide binds with 100% complementarity to the mRNA encoding Tyrosine Kinase 2 and do not bind to any other region in the human transcriptome.

According to a second aspect of the invention, there is provided a pharmaceutical composition containing an antisense-oligonucleotide of the first aspect together with at least one pharmaceutically acceptable carrier, excipient, adjuvant, solvent or diluent and combinations thereof. It has been found that 4-bromobenzaldehyde /V-(2,6-dimethylphenyl)semicarbazone (EGA), which was known to slow down trafficking of oligonucleotides between early endosomes and downstream compartments and exert a negative influence on the activity of endosomal escape agent for oligonucleotides [Juliano, R.L., et al., Nucleic Acids Res. 46: 1601-1613 (2018)], surprisingly significantly enhanced the inhibitory effect of ASOs of the invention by up to 6-fold.

In some embodiments, the pharmaceutical composition further comprises 4- bromobenzaldehyde /\/-(2,6-dimethylphenyl)semicarbazone (EGA).

According to a third aspect of the invention, there is provided a use of an antisense- oligonucleotide of the first or second aspect for the manufacture of a medicament for the treatment of an autoimmune diseases, inflammatory diseases or severe coronavirus disease.

In some embodiments, the autoimmune disease is selected from the group comprising rheumatoid arthritis, psoriasis (PSO), systemic lupus erythematosus (SLE), ankylosing spondylitis (AS), ulcerative colitis (UC), Crohn’s disease (CD), type 1 diabetes (T1 D) and multiple sclerosis (MS).

In some embodiments, the coronavirus disease is COVID-19.

According to a fourth aspect of the invention, there is provided a method of treatment of a subject in need of such treatment comprising administering to the subject an efficacious amount of an antisense-oligonucleotide of the first aspect or a composition of the second aspect.

In some embodiments, the subject has an autoimmune disease, inflammatory disease and/or severe coronavirus disease.

In some embodiments, the autoimmune disease is selected from the group comprising rheumatoid arthritis, psoriasis (PSO), systemic lupus erythematosus (SLE), ankylosing spondylitis (AS), ulcerative colitis (UC), Crohn’s disease (CD), type 1 diabetes (T1 D) and multiple sclerosis (MS). In some embodiments, the coronavirus disease is COVID-19.

According to a fifth aspect of the invention, there is provided a kit comprising an antisense-oligonucleotide or a composition of any aspect of the invention.

To the best of the inventor’s knowledge, this is the first time 4-bromobenzaldehyde /V- (2,6-dimethylphenyl)semicarbazone (EGA) has been used to enhance antisense- oligonucleotide activity.

According to a sixth aspect of the invention, there is provided use of 4- bromobenzaldehyde /\/-(2,6-dimethylphenyl)semicarbazone (EGA) as a pharmaceutical composition or research tool to enhance activity of antisense-oligonucleotides.

In some embodiments the antisense oligonucleotides are selected from the first aspect of the invention.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 shows ASO knockdown of TYK2 mRNA and protein. (A) TYK2 mRNA levels, normalized against GAPDH, of Jurkat cell line after incubation with varying doses of ASOs for 72 h. n = 3, from three independent experiments. IC50 values of the ASOs are shown in the legend. (B) TYK2 mRNA levels, normalized against GAPDH, of different cell lines (Jurkat, K- 562, Karpas-299, MOLT-4, A-431 , and HDLM-2) after incubation with varying doses of ASO- 1 for 72 h. n = 3 for Jurkat, K-562, Karpas-299, and MOLT-4; n = 2 for A-431 and HDLM-2, from at least two independent experiments. The IC50 values across the cell lines are shown in the legend. (C) TYK2 mRNA levels, normalized against GAPDH, of different cell lines (Jurkat, Karpas-299, and MOLT-4) after incubation with 0.5 pM of ASO-1 or the control oligo ASO-ctrl across varying time points from 8 h to 4 days, n = 2, from two independent experiments. Karpas-299 showed a rapid depletion of TYK2 mRNA upon ASO- 1 treatment, whereas MOLT- 4 showed a slower rate of depletion. (D) Western blot showing the dose-dependent knockdown of TYK2 protein in Jurkat cell line after incubation with varying doses of ASO-1 for 72 h. n = 3, from three independent experiments. TYK2 protein levels were not affected by the control oligo ASO-ctrl.

Figure 2 shows selective knockdown of TYK2 protein by ASO- 1. (A) Western blot showing the protein levels of all four JAKs in Jurkat and Karpas-299 cell lines after incubation with varying doses of ASO-1 for 72 h. (B) Western blot showing the protein levels of JAK1 , JAK3, and TYK2 in MOLT-4 and K-562 cell lines after incubation with varying doses of ASO- 1 for 72 h. Experiment was performed on four independent cell lines (n = 4). Potential slight increase of JAK1 and JAK2 were observed in Jurkat, Karpas-299, and K-562 upon TYK2 knockdown. These could have resulted from technical issues from stripping and re-probing (see Materials and Methods), or actual upregulation due to possible compensatory mechanisms following TYK2 depletion.

Figure 3 shows effect of ASO-1 on TYK2, JAK1 , JAK2, and JAK3 expression. (A) Western blot showing the proteins levels of TYK2, JAK1 , JAK2, and JAK3 in Jurkat cell following incubation with 1 pM of ASO-1 for 24 h, 48 h, and 72 h. (B) mRNA levels, normalized against GAPDH, of TYK2, JAK1 , JAK2, and JAK3, in Jurkat cell after incubation with 1 pM of ASO-1, A SO-2, ASOS, ASO-4 or ASOS for 72 h.

Figure 4 shows selective knockdown of TYK2 mRNA by ASO-1. TY 2 mRNA levels, normalized against GAPDH, of different cell lines (Jurkat, K-562, Karpas-299, MOLT-4, A-431 , and HDLM-2) after incubation with 1 pM of ASO-1 for 72 h. n = 2, from two independent experiments. mRNA levels of JAK1/2/3 were largely not affected.

Figure 5 shows dose-dependent inhibition of TYK2-mediated IFN-a signaling by ASO- 1. (A) Western blot showing the protein levels of TYK2, STAT1/3, and phosphorylated STAT1/3 in Jurkat cell, after IFN-a stimulation for 30 mins following incubation with varying doses of ASO-1 for 72 h. (B) Western blot showing the protein levels of STAT1 and phosphorylated STAT1 in Karpas-299 cell, after IFN-a stimulation for 30 mins following incubation with varying doses of ASO-1 for 72 h. Experiment was performed on two independent cell lines (n = 2).

Figure 6A-B shows inhibition of TYK2-mediated IL-12 signaling by ASO-1. (A) Western blot showing the protein levels of STAT1 and phosphorylated STAT1 in HeLa cell after IL-12 stimulation for 1 h following incubation with 500 and 2000 nM of ASO-1 or 2000 nM of the control oligo ASO-ctrl for 72 h. (B) Western blot showing the protein levels of TYK2 and phosphorylated STAT4 in NK-S1 cell, with or without IL-12 stimulation for 1 h following incubation with 10 pM of ASO- 1 or the scrambled control ASO-scrfor 72 h. (C) IFN-y secretion by NK-S1 cells, after baseline subtraction of the respective conditions without IL-12 stimulation, as a percentage of untreated control (UTC) without ASO treatment, as determined at OD450 from ELISA measurement taken following IL-12 stimulation for 12 h following incubation with 10 pM of ASO-1 or the control oligo ASO-ctrl for 72 h.

Figure 7A-B shows dose-dependent inhibition of TYK2-mediated IL-10 signaling by ASO-1. (A) Western blot showing the protein levels of STAT3 and phosphorylated STAT3 in Jurkat cell line, after IL-10 stimulation for 1 h following incubation with varying doses of ASO- 1 for 72 h. (B) Western blot showing the protein levels of STAT3 and phosphorylated STAT3 in K-562 cell line, after IL-10 stimulation for 1 h following incubation with varying doses of ASO- 1 for 72 h. Experiment was performed on two independent cell lines (n = 2). Figure 8 shows ASO-1 treatment has no visible effects on IL-6 signaling. Western blot showing that the protein levels of STAT3 and phosphorylated STAT3 in A-431 cell line following IL-6 stimulation for 1 h were not affected by ASO-1 treatment.

Figure 9 shows TYK2 (ASO-1 and ASO-31) or STAT3 (AZD9150L) mRNA levels, normalized against GAPDH, of KMS-11 cell line after incubation with varying levels of ASOs for 48 h. IC50 values of the ASOs are shown in the legend.

Figure 10 shows Volcano plots of differential gene expression between (A) ASO-1, (B) AZD9150L, and (C) ASO-31 against untreated controls. The target mRNA (TYK2 or STAT3) for each ASO is labeled. Red points, p-value < 0.05; grey points, p-value > 0.05. N = 2 biological replicates.

Figure 11A-B shows that chemical modifications of TYK2 ASOs reduces cytotoxicity. (A) Relative caspase 3/7 activation in HeLa cells induced by TYK2 ASOs and their corresponding counterparts with 2’-OMe at gap position 2. Ref. toxic ASO is derived from Reference 2. (B) TYK2 mRNA levels, normalized against GAPDH, of HDLM-2 cells after incubation with 1 pM of TYK2 ASOs and their corresponding counterparts with 2’-OMe at gap position 2 for 2 days. UTC, untreated control.

Figure 12 shows TYK2 mRNA levels, normalized against GAPDH, of KMS-11 (48 h), K562 (72 h), and Jurkat (72 h) cell lines after incubation with varying levels of ASO- 1, with or without co-formulation with EGA. IC50 values of ASO- 1 are shown in the legend.

Figure 13 shows STAT3 mRNA levels, normalized against GAPDH, of KMS-11 and K562 cell lines after incubation with varying levels of AZD9150L for 48 h, with or without coformulation with EGA. IC50 values of AZD9150L are shown in the legend.

Figure 14 shows MALAT1 RNA levels, normalized against GAPDH, of KMS-11, K562, and MOLM-14 cell lines after incubation with varying levels of MALAT1-ASO for 48 h, with or without co-formulation with EGA. IC50 values of MALAT1-ASO are shown in the legend.

Figure 15 shows the relative enhancement of ASO activity by co-formulation with different doses of EGA. K562 and KMS-11 cell lines were treated with ASO-1 or MALAT1- ASO for 48 h. Relative enhancement in depletion of the target RNA was determined by normalization against the samples treated with no co-formulation of EGA. DETAILED DESCRIPTION OF THE INVENTION

Definitions

Certain terms employed in the specification, examples and appended claims are collected here for convenience.

As used in the specification and the appended claims, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise.

As used herein, the term “comprising” or “including” is to be interpreted as specifying the presence of the stated features, integers, steps or components as referred to, but does not preclude the presence or addition of one or more features, integers, steps or components, or groups thereof. However, in context with the present disclosure, the term “comprising” or “including” also includes “consisting of”. The variations of the word “comprising”, such as “comprise” and “comprises”, and “including”, such as “include” and “includes”, have correspondingly varied meanings.

As used herein, the term “gapmer” is defined as a short DNA antisense oligonucleotide - the gap - with modified RNA-like segments on both sides of the sequence [Khvorova, A., et al., Nat. Biotechnol. 35: 238-248 (2017)]. Gapmers are designed to hybridize to a target piece of RNA and silence the gene through the induction of RNase H cleavage.

As used herein, the term “locked nucleic acid (LNA) is defined as an RNA derivative in which the ribose ring is constrained by a methylene linkage between the 2'-oxygen and the 4'-carbon. This conformation restriction increases binding affinity for complementarity sequences. Further definition of LNA can be found in Obika 1997 [Tetrahedron Lett. 38: 8735-8738] and Koshkin 1998 [Tetrahedron Lett. 54: 3607-3630],

As used herein, the term “severe coronavirus disease” is defined as serious coronavirus disease leading to respiratory failure, septic shock, and/or multiple organ dysfunction (critical illness as defined by COVID- 19 Treatment Guidelines, NIH).

The term "treatment", as used in the context of the invention refers to prophylactic, ameliorating, therapeutic or curative treatment.

The term "subject" is herein defined as vertebrate, particularly mammal, more particularly human. For purposes of research, the subject may particularly be at least one animal model, e.g., a mouse, rat and the like. In particular, for treatment of inflammatory diseases, autoimmune disease and/or severe coronavirus-linked diseases, the subject may be a human. Any discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is not to be taken as an admission that any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to the present disclosure as it existed before the priority date of each of the appended claims.

Bibliographic references mentioned in the present specification are for convenience listed at the end of the examples. The whole content of such bibliographic references are herein incorporated by reference.

EXAMPLES

EXAMPLE 1 : Materials and Methods

ASO design, synthesis, and purification

Unless otherwise specified, 16-nucleotide (nt) fully phosphorothioate [Eckstein, F. Nucleic Acid Ther. 24:374-387 (2014)] (PS)-modified ASOs incorporating locked nucleic acid (LNA) [Obika, S. et al. Tetrahedron Lett. 38:8735-8738 (1997); Koshkin, A. A. et al., Tetrahedron Lett. 54:3607-3630 (1998)] in a 3-10-3 gapmer [Monia, B. P. et al. J. Biol. Chem. 268:14514-14522 (1993)] configuration were designed to be complementary to different segments of TYK2 mRNA (RefSeq ID NM_003331). Several ASOs comprising the sequence of ASO-2 were 17 nucleotides in length after addition of a nucleotide at the 3’ end. All ASOs were synthesized in-house with an ABI 394 DNA/RNA synthesizer on Glen UnySupport (Glen Research) using standard phosphoramidite chemistry. LNA phosphoramidites were purchased from either Exiqon or Sigma-Aldrich, or synthesized from 3’-hydroxyl precursors (Rasayan). Phenylacetyl disulfide (ChemGenes Corporation) was used as the sulfurizing reagent. The ASOs were cleaved from solid support and de-protected with concentrated aqueous ammonia at 55 °C for 16 h, and subsequently purified using Poly-Pak II cartridges (Glen Research) according to the manufacturer’s protocol. The ASOs were desalted using Glen Pak 2.5 desalting column (Glen Research) and dried by lyophilization. The dried ASOs were re-suspended with 10 mM potassium phosphate into 100 pM stock solutions and diluted with cell culture medium before use. All ASOs were characterized using a JEOL SpiralTOF matrix-assisted laser desorption/ionization time-of-f light (MALDI-TOF) mass spectrometer.

Cell culture and reagents

Jurkat acute T-cell leukemia, MOLT-4 acute lymphoblastic leukemia, K-562 chronic myelogenous leukemia, A-431 epidermoid carcinoma, and HeLa adenocarcinoma cells were purchased from American Type Culture Collection (ATCC, USA). Karpas-299 non-Hodgkin’s large cell lymphoma and HDLM-2 Hodgkin lymphoma cells were purchased from Sigma- Aldrich and Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ, Germany), respectively. NK-S1 natural killer/T-cell lymphoma cell was generously provided by Prof. Wee Joo Chng (Cancer Science Institute of Singapore). Suspension cells were typically cultured in RPMI 1640 medium (Thermo Fisher Scientific), while adherent cells were cultured in DMEM medium (Thermo Fisher Scientific), all supplemented with 10-20 % fetal bovine serum (FBS, Thermo Fisher Scientific). NK-S1 cell was cultured in DMEM medium supplemented with 10 % FBS and 10 % equine serum (Sigma-Aldrich). All cells were grown in a 5%-CO2 humidified incubator at 37 °C.

ASO treatment

Cells were seeded on 12-/24-well plates (Corning) and grown for 24 h prior to being treated with ASO. ASOs were diluted with fresh culture media and added directly into treatment wells to attain the desired concentrations, without the use of any transfection reagents. For time-point measurements, cells were harvested at various intervals (8 h, 16 h, 1 day, 2 days, 3 days, and 4 days) post-ASO treatment. For all other experiments, including RNA/protein level measurements and cytokine stimulation assays, cells were harvested 2 or 3 days post-ASO treatment.

Quantitative reverse transcription polymerase chain reaction (RT-qPCR)

Total RNA from each culture well was extracted by successive addition of (i) TRIzol reagent (Thermo Scientific) for cell lysis, (ii) chloroform for phase separation, and (iii) isopropanol for RNA precipitation. The RNA pellet was washed with ethanol and allowed to air dry, and subsequently resuspended in RNase-free water. Quantification of the RNA was performed using Nanodrop 2000 (Thermo Scientific). The RNA was then reverse transcribed with M-MLV reverse transcriptase (Promega) following the manufacturer’s protocol. qPCR of the cDNA was performed using either SYBR Green- or probe-based master mix (Bio-Rad) on a Bio-Rad CFX96 real-time PCR detection system. The 2 -AACT method [Livak, K. J. and Schmittgen T D. Methods 25:402-408 (2001)] was employed to determine expression level of the target genes using GAPDH as a reference.

Primer and probe sequences

The primer pairs used in ASO screening and JAK selectivity measurement are as follows: TYK2: (Forward) 5’-GGA GGA GGG TTC TAG TGG CA-3’ (SEQ ID NO: 83), (Reverse) 5’-ATG TOO CGG AAG TCA CAG AAG-3’ (SEQ ID NO: 84); JAK1: (Forward) 5’-CTT TGC CCT GTA TGA CGA GAA C-3’ (SEQ ID NO: 85), (Reverse) 5’-ACC TCA TCC GGT AGT GGA GC-3’ (SEQ ID NO: 86); JAK2: (Forward) 5’-ATC CAC CCA ACC ATG TCT TCC-3’ (SEQ ID NO: 87), (Reverse) 5’-ATT CCA TGC CGA TAG GCT CTG-3’ (SEQ ID NO: 88); JAK3: (Forward) 5’-CCT GAT CGT GGT CCA GAG AG-3’ (SEQ ID NO: 89), (Reverse) 5’-GCA GGG ATC TTG TGA AAT GTC AT-3’ (SEQ ID NO: 90); GAPDH (reference): (Forward) 5’-CTG GGC TAG ACT GAG CAC C-3’ (SEQ ID NO: 91), (Reverse) 5’-AAG TGG TCG TTG AGG GCA ATG-3’ (SEQ ID NO: 92). The primer pairs and probe sequences used in IC50 measurements are as follows: TYK2: (Forward) 5’-CAG AGT GCC TGA AGG AGT ATA AG-3’ (SEQ ID NO: 93), (Reverse) 5’-TCT GGC TGG AGT CAC AGT-3’ (SEQ ID NO: 94), (Probe) 5’-CGT CAG CAGCTC ATA CAG GGT CAC-3’ (SEQ ID NO: 95); GAPDH (reference): (Forward) 5’-CTG GGC TAG ACT GAG CAC C-3’ (SEQ ID NO: 91), (Reverse) 5’-AAG TGG TCG TTG AGG GCA ATG-3’ (SEQ ID NO: 92), (Probe) 5’-TCT CCT CTG ACT TGA AGA GCG AGA CCC-3’ (SEQ ID NO: 96). All primers and probes were purchased from Integrated DNA Technologies (IDT).

Western blot and cytokine stimulation

Cells were harvested and lysed in mammalian protein extraction reagent (Thermo Scientific) containing protease and phosphatase inhibitor cocktail (Sigma-Aldrich) following the manufacturer’s protocol. Protein samples were quantified by BCA protein assay kit (Thermo Scientific). Approximately 10-30 pg of total protein was loaded and run on 10 % SDS-PAGE with XT MOPS running buffer (Bio-Rad), and subsequently transferred onto a polyvinylidene difluoride (PVDF) membrane (GE Healthcare). The membrane was blocked by Tris-buffered saline-0.05 % Tween 20 (TBST) with 5 % non-fat dry milk (Bio-Rad) for 1 h at room temperature, followed by 5-min wash with TBST 3 times over. The membrane was then probed with primary antibodies against the target proteins (Cell Signaling Technology; TYK2: #9312, JAK1 : #3344, JAK2: #3230, JAK3: #8827, STAT1 : #9172, pSTAT1-Y701 : #7649, STAT3: #4904, pSTAT3-Y705: #9145, and GAPDH: #5174) in the recommended buffers, either at room temperature for 1-2 h or at 4 °C overnight. After washing 3 times, the membrane was incubated with horseradish peroxidase (HRP)-labeled secondary antibodies (Cell Signaling Technology, #7074) at 1 :5,000-1 :2,000 dilution in the blocking buffer at room temperature for 1 h. The membrane was washed 3 times, followed by incubation with HRP substrate according to the manufacturer’s protocol. Finally, the target bands were visualized and captured on an Amersham Imager 680 (GE Healthcare) or Gel ChemiDoc system (BioRad). In cases where multiple JAKs had to be imaged, the order of antibody incubation, stripping, and re-probing were as such: TYK2 followed by JAK2, and JAK3 followed by JAK1. The membrane was stripped using a stripping buffer (100 mM 2-mercaptoethanol, 2 % SDS, 62.5 mM T ris-HCI, pH 6.7) at a temperature of 50 °C for 30 mins with gentle agitation, followed by 6-time washing of 5 mins each by TBST, before proceeding to re-block the membrane and re-probe with antibodies. For cytokine stimulation assays, the cells lines were seeded for 24 h, followed by ASO treatment for a further 3 days before being subjected to stimulation by the respective cytokines, as follows: IFN-a (Genscript) at 30 ng/ml for 30 mins, IL-12 (Sigma-Aldrich) at 20 ng/ml for 1 h, IL-10 (Genscript) at 100 ng/ml for 1 h, and IL-6 (Genscript) at 100 ng/ml for 1 h.

Enzyme-linked immunosorbent assay (ELISA) measurement of IFN-y secretion

IFN-y secreted by NK-S1 cells upon IL-12 stimulation was measured by Quantikine ELISA kit (R&D Systems) following the manufacturer’s protocols. The cells were treated with 10 pM ASO-1 for 3 days before stimulation with 20 ng/ml IL-12 (Sigma-Aldrich) for 12 h. Following stimulation, 100 pl of cell culture was deposited into individual ELISA wells and incubated at room temperature for 2 h to allow IFN-y binding to pre-coated antibodies. The wells were rinsed after removal of media, followed by 2-h incubation with 100 pl HRP-labeled secondary antibodies. Unbound antibodies were washed away before HRP substrate was added to the wells for color development over 30 mins. The reaction was quenched with 0.2 M H2SO4 and OD450 for individual wells were recorded using a pQuant microplate reader (BioTek).

Cytotoxicity measurements of ASOs

Cytotoxicity of ASOs was measured based on their activation of cellular caspase 3/7. 10,000 HeLa cells were seeded in 100 pL DMEM media into 96-well plates for 24 hours before being transfected with 100 nM or 1 pM ASOs using Lipofectamine RNAiMAX (Thermo Fisher) for an additional 24 h. Subsequently, 100 pl Caspase Gio 3/7 Assay (Promega) was added to each well and the plates were kept in the dark for 30 minutes. The resulting luminescence was recorded by a TECAN Infinite M200 plate reader and background subtracted according to the manufacturer’s protocol. Relative caspase activation was calculated as the ratio of luminescence reading between the treated samples and the untreated control (UTC), of which a high value indicates high cytotoxicity of the ASO.

EXAMPLE 2

Potent knockdown of TYK2 mRNA and protein by TYK2-specific ASOs

To identify potent TYK2 ASO candidates, screening was performed on over 200 TYK2- specific ASOs (Table 1) for their potential to induce ribonuclease (RNase) H-mediated mRNA cleavage on Jurkat leukemia cell, a T-lymphocyte model cell line.

Table 1 : Effects of 30 ASOs a ' b targeting TYK2 mRNA (RefSeq ID: NM_003331) in vitro c . a LNA nucleotides are underlined. Phosphorothioate (PS) linkages are represented as asterisks (*). b ln this study, all cytosine residues (C) for both DNA and LNA are 5-methyl cytosines. Note however that these could be interchanged as cytosines. c Jurkat cells were seeded for 24 h followed by treatment with 1 pM of ASOs for 48 h. d TYK2 mRNA level after ASO treatment, normalized against untreated controls

(UTC), using GAPDH mRNA level as a reference. Unless otherwise specified, the ASO configuration comprises 16-nucleotide (nt) fully phosphorothioate [Eckstein, F. Nucleic Acid Ther. 24:374-387 (2014) (PS)-modified gapmer [Monia, B. P. et al. J. Biol. Chem. 268:14514-14522 (1993)] with a middle 10-nt DNA stretch flanked on both ends by 3-nt locked nucleic acid (LNA) segments (i.e. a wing-gap-wing composition of 3-10-3) [Obia, S. et al. Tetrahedron Lett. 38:8735-8738 (1997); Koshkin, A. A. et al., Tetrahedron Lett. 54:3607-3630 (1998)]. The high potency of LNA gapmers enable cellular mRNA knockdown to be achieved in vitro simply by free incubation with ASOs, otherwise termed as free-uptake [Hong, D. et al. Sci. Transl. Med. 7:314ea185 (2015); Ross, S. J. et al. Sci. Transl. Med. 9:eaa5253 (2017)] or gymnosis [Stein, C. A. et al. Nucleic Acids Res. 38:e3 (2010)] in the literature) without the use of any delivery agent. Five selected ASOs (ASO-1-5) demonstrated clear dose-response knockdown of TYK2 mRNA (Figure 1A) with half maximal inhibitory concentration (IC50) values in the range of -90-240 nM following 72 h of ASO treatment. The potency of ASO- 1 was further measured across different cell lines including suspension (Karpas-299 non-Hodgkin lymphoma, MOLT-4 acute lymphoblastic leukemia, K-562 chronic myelogenous leukemia, and HDLM-2 Hodgkin lymphoma) and adherent (A-431 epidermoid carcinoma) cells, giving rise to IC50 values ranging from -13-155 nM (Figure 1 B). Time-course measurement of RNA levels following ASO-1 treatment was performed across different cell lines (Figure 1C); maximal knockdown was generally observed after 48 h and maintained thereafter for at least four days post-treatment with no replacement of media and ASO. In the same experiment, a control oligonucleotide (oligo) comprising the same LNA gapmer configuration (ASO-ctrl) did not lead to reduction in TYK2 mRNA level, thereby confirming the on-target effect of the TYK2-specific ASOs.

TYK2 protein levels in Jurkat cells were measured after treatment with varying concentrations of ASO- 1 for 72 h. Near-complete depletion of TYK2 protein was observed at an ASO concentration of 1 pM (Figure 1 D). At the same dosage, the control oligo ASO-ctrl did not affect the level of TYK2 protein, further validating the on-target effect of the TYK2-specific ASOs. The dose-dependent experiments were repeated on various suspension cell lines and showed a near-complete depletion of TYK2 across these cell lines upon ASO- 1 treatment, even at the lower dosage of 100 nM (Figure 2). Time-course measurements of protein levels following incubation of Jurkat cells with 1 pM of ASO-1 (Figure 3A) showed protein knockdown as soon as 24 h post-treatment, with progressively more knockdown observed at 48 h and 72 h post-treatment. EXAMPLE 3

Selective TYK2 knockdown by ASO-1 with no perturbation on other JAK isoforms

To be an effective therapeutic option, the ASOs should be specific for TYK2 while selective against all other JAK family members. Towards that end, JAK1 , JAK2, and JAK3 protein levels in various cell lines were unaffected by treatment with ASO-1, even at dosages whereby TYK2 protein was completely depleted (Figure 2 and Figure 3A). The TYK2- specificity of ASO-1 was also validated in the context of mRNA, for which only TYK2 mRNA knockdown was observed whereas JAK1 , JAK2, and JAK3 mRNA levels remained largely unchanged when 1 pM of ASO-1 was incubated across different cell lines (Figure 4). High TYK2-selectivity was similarly observed for ASO-2, ASO-3, ASO-4 and ASOS, as JAK1 , JAK2, and JAK3 mRNA levels in Jurkat cells remained largely constant following ASO treatment (Figure 6C).

EXAMPLE 4

ASO-1 inhibition of TYK2-mediated cytokine signaling pathways

The effects of TYK2 knockdown on various TYK2-associated cytokine signaling pathways [Ghoreschi, K., et al. , Immunol. Rev. 228: 273-287 (2009); Strobl, B., D. et al. , Front. Biosci. 16: 3214-3232 (2011)] (i.e. IFN-a, IL-12, IL-10, and IL-6) across different cell lines in vitro were evaluated. For each signaling pathway, the cell lines were pre-treated with ASO-1 for 3 days prior to being subjected to cytokine stimulation. Phosphorylation status of the relevant STAT members was then quantified by western blot of the cell lysate to determine the extent of pathway stimulation (or inhibition by ASO-1).

Type I IFNs (IFN-a/IFN-P) signal through binding to the interferon-a/p receptor (IFNAR), which is mediated by TYK2 and JAK1 [Silvennoinen, O., et al., Nature 366: 583-585 (1993)], and eventually lead to the activation of several STAT members including STAT1/3 [Velichko, S., et al., J. Biol. Chem. 277: 35635-35641 (2002); Platanias, L. C. Nat. Rev. Immunol. 5: 375-386 (2005); van Boxel-Dezaire, A. H., et al., Immunity 25: 361-372 (2006)]. Following ASO-1 treatment for 3 days, Jurkat and Karpas-299 cells were stimulated with 30 ng/ml of IFN-a for 30 min. Western blot of cell lysate showed that STAT1 and/or STAT3 phosphorylation within both cell lines was inhibited by ASO- 1 in a dose-dependent manner (Figure 5) while the total amounts of non-phosphorylated STAT1 and STAT3 remained unchanged. Our results indicated the dependence of type I IFN signaling on TYK2 protein, consistent with previous findings from human TYK2 deficiency [Minegishi, Y., et al., Immunity 25: 745-755 (2006)] and TYK2-knockout mice [Karaghiosoff, M., H. et al., Immunity 13: 549- 560 (2000); Shimoda, K., et al., Immunity 13: 561-571 (2000)], albeit in the mice knockouts, TYK2 protein was found to play a more limited role in type I IFN signaling.

IL-12 signaling occurs through its binding to the IL-12 receptor, a heterodimeric complex consisting of I L12R|31 and IL12R 2. Upon IL-12 binding to the receptor, the bound TYK2 and JAK2 are activated and phosphorylate STAT1/3/4 [Bacon, C. M., et al., Proc. Natl. Acad. Sci. USA 92: 7307-7311 (1995); Jacobson, N. G., et al., J. Exp. Med. 181 : 1755-1762 (1995)], thereby leading to downstream gene regulation events, among which include interferon y (IFN-y) production. After ASO-1 treatment, HeLa adenocarcinoma cells were stimulated with 100 ng/ml of IL-12 for 1 h. Western blot of the cell lysate showed that phosphorylated STAT 1 levels following IL-12 stimulation were reduced in cells that were pretreated with ASO-1 for 3 days, as compared to the controls (Figure 6A). After ASO treatment for 3 days, NK-S1 natural killer/T-cell lymphoma cells were stimulated with 20 ng/ml of IL-12 for 1 h. Western blot of the cell lysate showed that phosphorylated STAT4 levels following IL- 12 stimulation were reduced in cells that were pre-treated with ASO-1, as compared to the controls (Figure 6B). Effects of TYK2 knockdown were also evident from IFN-y quantification through enzyme-linked immunosorbent assay (ELISA) measurement, wherein IFN-y secretion (stimulated over a duration of 12 h with 20 ng/ml of IL-12) was drastically reduced in NK-S1 cells pre-treated with ASO-1, as compared to non-treated controls and cells pre-treated with ASO-ctrl (SEQ ID NO: 61) (Figure 6C). Our results indicate an essential role of TYK2 protein in mediating IL- 12 signal transduction, as has been reported previously from studies in humans [Minegishi, Y., et al., Immunity 25: 745-755 (2006); Kreins, A. Y., et al., J. Exp. Med. 212: 1641-1662 (2015)] and mice [Karaghiosoff, M., H. et al., Immunity 13: 549-560 (2000); Shimoda, K., et al., Immunity 13: 561-571 (2000)].

IL-10 signaling occurs through a heterotetrameric receptor complex comprising two each of IL10R1 and IL10R2, which is mediated by TYK2 and JAK1 , subsequently leading to STAT3 phosphorylation. Jurkat and K-562 cells were stimulated with 100 ng/ml of IL-10 for 1 hour following pre-treatment with ASO- 1 for 3 days. Western blot of the cell lysate showed that STAT3 phosphorylation was inhibited in a dose-dependent manner (Figure 7), in agreement with TYK2 protein depletion, whereas normal STAT3 protein levels were not affected. Our results suggest at least a similar level of involvement from TYK2 in IL- 10 signaling as compared to JAK1 , which contrasts with results from previous studies reporting a dominant role of JAK1 over TYK2 in IL-10 signal transduction [Shimoda, K., et al., Immunity 13: 561-571 (2000); Rodig, S. J., et al., Cell 93: 373-383 (1998); Sohn, S. J., et al., J. Immunol. 191 : 2205-2216 (2013)]. IL-6 signaling is mediated through its receptor complex, which is composed of gp130 and IL-6Ra. Upon IL-6 binding, the associated JAK1 , JAK2, and TYK2 are activated, thereby leading to phosphorylation of STAT3 protein [Minegishi, Y., et al., Immunity 25: 745-755 (2006); Ghoreschi, K., et al., Immunol. Rev. 228: 273-287 (2009)]. Here we measured the levels of phosphorylated STAT3 in A-431 cells after IL-6 (100 ng/ml) stimulation for 1 h following pre-treatment with ASO-1 for 3 days. In this case, TYK2 depletion with ASO-1 did not lead to any visible changes on both phosphorylated and normal STAT3 (Figure 8), even at the highest dose tested (2 pM), suggesting minimal impact of TYK2 knockdown on IL-6 signaling through STAT3. Our results are consistent with multiple reports showing a dominant role of JAK1 over JAK2 and TYK2 in mediating IL-6 signal transduction [Parganas, E., et al., Cell 93: 385-395 (1998)].

EXAMPLE 5

Comparison of ASO-1 against benchmark ASOs.

We have shown that ASO- 1 led to highly potent knockdown of TYK2 mRNA and protein (Figs. 1, 2 and 4). Here its potency was compared against two ASOs, AZD9150L (LNA variant of a benchmark ASO gapmer targeting STAT3 mRNA [Hong, D., et al. Sci. Transl. Med., 7: 314ra185 (2015)] in Phase II clinical trial; Table 2) and ASO-31 (another potent TYK2 ASO, Table 2).

Table 2. Additional ASOs a b used in this study a. LNA nucleotides are underlined. Phosphorothioate (PS) linkages are represented as asterisks (*). b. In this study, all cytosine residues (C) for both DNA and LNA are 5-methyl cytosines. Note however that these could be interchanged as cytosines. The IC50 values for ASO-1 in KMS-11 myeloma cell line following 72 h of treatment was 12.9 nM (Fig. 9), which was superior in comparison to those of both AZD9150L (39.8 nM) and ASO-31 (42.3 nM).

We have also shown that ASO- 1 is specific for TYK2 mRNA and protein while selective against all other JAK paralogs (Figs. 2 and 4). This was further supported by RNA-seq data of Jurkat cell line following treatment by the three ASOs individually (Fig. 10), for which the volcano plot of differential gene expression between A SO- 7-treated samples and untreated controls showed the highest knockdown of its target mRNA with the strongest statistical significance. Thus, the specificity of ASO- 1 for its target mRNA was higher than that of AZD9150L, as well as ASO-31, which showed broader effects across the transcriptome. Hence these results supported the highly targeted action of ASO- 1 with minimal off-target activity, a unique combination of properties that are critical for effective therapeutics.

EXAMPLE 6

Modifications to chemical configuration of TYK2 ASOs

Chemical configurations including gapmer design exert considerable effects on activity of ASOs. Here we tested a series of ASOs with further modifications beyond the gapmer configuration (Table 3); for the outermost 5 nucleotides on each end, at least 2 to 4 are LNA with the rest being DNA, while keeping a DNA gap in the middle that is at least 9-nucleotide long. Together with ASO- 1, ASO-32 and ASO-33 (both 17-nt long) as well as ASO-42 and ASO-44 (both 16-nt long) exhibited the most potent activity.

Table 3. Effects of ASOs a ' b targeting TYK2 mRNA (RefSeq ID: NM_003331) in vitro c a. LNA nucleotides are underlined. Phosphorothioate (PS) linkages are represented as asterisks (*). b. In this study, all cytosine residues (C) for both DNA and LNA are 5-methyl cytosines. Note however that these could be interchanged as cytosines. c. Jurkat cells were seeded for 24 h followed by treatment with 1 pM of ASOs for 48 h. d. TYK2 mRNA level after ASO treatment, normalized against untreated controls (UTC), using GAPDH mRNA level as a reference.

EXAMPLE 7

Cytotoxicity of TYK2 ASOs can be reduced by modification

The cytotoxicity of the ASOs was determined by the extent to which they activate the caspase 3 and caspase 7 enzymes in living cells [Dieckmann, A., et al., Mol. Ther. Nucleic Acids 10: 45-54 (2018); Shen, W., et al., Nat. Biotechnol. 37: 640-650 (2019)], which was shown to be a good predictor of their in vivo toxicity. Briefly, 10,000 HeLa cells were seeded in 100 pL DMEM media into 96-well plates for 24 hours before being transfected with 100 nM or 1 pM ASOs using Lipofectamine RNAiMAX (Thermo Fisher) for an additional 24 h. Subsequently, 100 pL Caspase Gio® 3/7 Assay (Promega) was added to each well and the plates were kept in the dark for 30 minutes. The resulting luminescence was recorded by a TECAN Infinite M200 plate reader and background subtracted according to the manufacturer’s protocol. The relative caspase activation was calculated as the ratio of luminescence reading between the treated samples and the untreated control (UTC), of which a high value indicates high cytotoxicity of the ASO.

ASO-1 exhibits an excellent safety profile, eliciting a low caspase activation close to basal level in HeLa cells even at a high dose of 1 pM with transfection (Fig. 11A). On the other hand, ASO-3, ASO-4, and ASO-12 led to high relative caspase activation levels (up to -6-10 times that of UTC) at a lower dose of 100 nM with transfection (Fig. 11A). Caspase activation by a toxic ASO [Shen, W., et al., Nat. Biotechnol. 37: 640-650 (2019)] (100 nM with transfection) was measured for comparison. It has been reported that the cytotoxicity of PS-ASO gapmers could be reduced by chemical modifications [Shen, W., et al., Nat. Biotechnol. 37: 640-650 (2019)], specifically through incorporation of 2’-O-methyl (2’-OMe) at gap position 2, i.e. position 5 of 3-10-3 gapmers. Accordingly, corresponding modifications to ASO-1 , ASO-3, ASO-4, and ASO-12 (Table 4) reduced caspase activation in HeLa cells back to basal levels while still retaining their TYK2 mRNA knockdown activity (Fig. 11 B).

Table 4. TYK2 ASOs and their corresponding counterparts with 2’-OMe at gap position 2. a a LNA nucleotides are underlined. 2’-OMe nucleotides are labeled with the letter “m”. Phosphorothioate (PS) linkages are represented as asterisks (*).

EXAMPLE 8

A new formulation to enhance cellular activity of ASOs

Different formulation and vehicle agents have been explored towards the enhancement of cellular uptake and activity of ASOs. The small molecule 4- bromobenzaldehyde /\/-(2,6-dimethylphenyl)semicarbazone (named EGA) was shown to be a selective inhibitor of endosomal trafficking pathways exploited by toxins and viruses [Gillespie, E.J., et al. Proc Natl Acad Sci U SA, 110: E4904-4912 (2013)]. A later publication also showed that EGA slowed down trafficking of oligonucleotides between early endosomes and downstream compartments, and that it exerts negative influence on the activity of endosomal escape agent for oligonucleotides [Juliano, R.L., et al., Nucleic Acids Res., 46: 1601-1613 (2018)] and thus would be non-desirable.

Unexpectedly, contrary to the reported inhibitory effects of EGA on oligonucleotide trafficking, we co-formulated EGA with ASO-1 across multiple cell lines and observed up to more than 6-fold enhancement in potency based on IC50 values (Figure 12). The same enhancement effects were also observed for AZD9150L (targeting STAT3 mRNA) and an additional ASO (MALAT1-ASO targeting MALAT1 IncRNA) on various cell lines (Figures 13 and 14), indicating the effects to be generally applicable across different ASOs and cell types. Thus, co-formulation with EGA can be used to enhance the activity of ASOs towards inducing RNase H-mediated degradation of target RNA. Dose-dependent treatment of ASO- 1 and MALAT1-ASO on K562 and KMS-11 showed that co-formulation with doses of EGA up to 10 pM led to around 3-fold improvement in ASO activity (Figure 15).

Summary

JAK proteins, acting as critical signaling mediators of many pro-inflammatory cytokines, represent attractive drug targets for the treatment of autoimmune and inflammatory diseases. Mounting evidence from human genetics studies and clinical findings of several drug agents against TYK2-dependent cytokine signaling pathways (e.g. IFN-a and IL-12/IL-23), combined with the less-severe phenotype of TYK2-knockouts as compared to the other JAK paralogs, indicate TYK2 as an excellent target for the treatment of autoimmune diseases with an optimal balance between efficacy and safety. Additionally, high TYK2 expression has been linked to severe COVID-19 from a recent GWAS of critically ill COVID- 19 patients [Pairo-Castineira, E., et al., Nature 591 : 92-98 (2021)], pointing to its selective targeting as a potential treatment for these severe cases. Among small-molecule inhibitors of TYK2 that have been advanced to clinical testing, nearly all were designed to block or to compete with adenosine triphosphate (ATP) binding to the catalytic domain, also known as the Janus Homology 1 (JH1) domain, where selectivity is especially challenging to achieve given the high homology among the JAK isoforms. A small-molecule inhibitor utilizing an alternative approach of allosteric inhibition through selective binding to the pseudokinase (JH2) domain has shown a remarkable selectivity for TYK2 against all other JAK isoforms, and early clinical results of the inhibitor against PSO [Papp, K., K. et al., N. Engl. J. Med. 379: 1313-1321 (2018)] look promising. Despite its high selectivity for TYK2, the inhibitor still binds to JAK1 (JH2 domain) and BMPR2 kinase with high affinity [Wrobleski, S. T., et al., J. Med. Chem. 62: 8973-8995 (2019)]. Longer-term efficacy and safety of this and other TYK2 inhibitors remain to be determined.

In the case of the TYK2 ASO gapmers of the invention, RNase H is recruited upon ASO hybridization to the sequence target, thereby resulting in specific cleavage and subsequent degradation of TYK2 mRNA, and eventually depletion of TYK2 protein. Fully PS- modified LNA gapmers were utilized in the design of the TYK2 ASOs, which exhibit IC50 values matching those of recent ASO candidates with a similar chemical configuration that have been advanced to clinical testing [Hong, D., et al., Sci. Transl. Med. 7: 314ra185 (2015); Ross, S. J., et al., Sci. Transl. Med. 9: eaal5253 (2017)]. Sequence alignments of ASO-1-ASO-5 against the human genome transcript database found no matching sequences with significant overlap with ASO-1, ASO-2, and ASO-4, supporting their specificity for TYK2 mRNA. Experimentally, we have demonstrated their selectivity against the other JAK isoforms, as the mRNA and protein levels of JAK1/2/3 were not affected by TYK2 ASO treatment.

To achieve targeted delivery of ASOs in vivo, intensive efforts have been driven towards direct chemical conjugation of ASO with cell type-/organ-selective ligands [Dowdy, S. F. Nat. Biotechnol. 35: 222-229 (2017)]. Tremendous success has been observed for their targeted delivery to liver hepatocytes upon conjugation with multiple copies of the simple carbohydrate /V-acetylgalactosamine [Prakash, T. P., et al. , Nucleic Acids Res. 42: 8796-8807 (2014)] (GalNAc). Conjugation of TYK2 ASO with ligands that are specific for immune cell/tissue subtypes thus holds great promise for its targeted delivery to address particular autoimmune diseases.

Beyond TYK2 mRNA and protein level measurements, we observed dose-dependent reduction of IFN-a signaling with TYK2 ASO treatment, as measured by pSTAT1/3 levels. Antibodies against IFN-a and IFNAR have been evaluated for the treatment of SLE, with the latter showing clinical efficacy in a phase 3 trial [Morand, E. F., et al., N. Engl. J. Med. 382: 211-221 (2019)]. Our results are consistent with the essential role of TYK2 in IFN-a signaling, supporting further development of the TYK2 ASOs towards treatment/application on autoimmune diseases such as SLE where elevated IFN-a signaling was identified as a major driver of pathology [Crow, M. K., J. Immunol. 192: 5459-5468 (2014)]. On a similar note, we showed that ASO-mediated TYK2 knockdown led to reduction of IL-12 signaling as measured by pSTATI levels. This observation is consistent with TYK2 being an important signaling mediator of the I L-12/1 L-23 pathway, for which a monoclonal antibody against the common p40 subunit of I L-12/1 L-23 was approved for the treatment of PSO, PSA, CD, and UC [Sands, B. E., et al., N. Engl. J. Med. 381 : 1201-1214 (2019)]. We have shown that IL-10 signaling was inhibited upon TYK2 depletion through ASO treatment, as measured by pSTAT3 levels. Unlike IFN-a and IL-12/IL-23, IL-10 is generally regarded as an anti-inflammatory cytokine [Schwartz, D. M., et al., Nat. Rev. Rheumatol. 12: 25-36 (2016)], although its overproduction has been raised as a predisposing factor for SLE [Beebe, A. M., etal., Cytokine Growth Factor Rev. 13: 403-412 (2002)]. In this case, our results show that TYK2 exerts an essential role as a signaling mediator for IL-10, which contrasts against previous reports on the dominant role of JAK1 over TYK2 in IL-10 signal transduction [Shimoda, K., et al., Immunity 13: 561-571 (2000); Rodig, S. J., et al., Cell 93 373-383 (1998); Sohn, S. J., et al., J. Immunol. 191 : 2205- 2216 (2013)]. On the other hand, TYK2 depletion showed no effects on IL-6 signaling through pSTAT3, suggesting a limited role for TYK2 in signal transduction of the IL-6 pathway. This is consistent with reports of the dominant role of JAK1 in mediating IL-6 signal transduction [Shimoda, K., et al., Immunity 13: 561-571 (2000); Kreins, A. Y., et al., J Exp Med 212:1641- 1662 (2015); Rodig, S. J., et al., Cell 93: 373-383 (1998); Sohn, S. J., et al., J. Immunol. 191 : 2205-2216 (2013); Paragas, E., et al., Cell 93:385-395 (1998)]. We further measured the cytotoxicity of the TYK2 ASOs through cellular caspase 3/7 activation [Linnane, E., et al., Nucleic Acids Res. 47: 4375-4392 (2019)]; Dowdy, S. F., Nat. Biotechnol. 35: 222-229 (2017)], which was shown to be a good predictor of their in vivo toxicity. ASO-1 exhibited an excellent safety profile, while cytotoxicity of the other ASOs could be largely eliminated through judicious modifications [Dowdy, S. F., Nat. Biotechnol. 35: 222-229 (2017)]. Aside from autoimmune diseases, TYK2 was also implicated in the pathology of various cancers including T-cell acute lymphoblastic leukemia [Sanda, T., et al., Cancer Discov. 3: 564-577 (2013)] (T-ALL), anaplastic large cell lymphoma [Prutsch, N., et al., Leukemia 33: 696-709 (2019)] (ALCL), and nerve sheath tumors [Qin, W., et al., Cancer Med. 8: 5232-5241 (2019)].

The TYK2 ASOs of the invention have potential as therapeutic agents for the treatment of autoimmune diseases, inflammatory diseases and severe COVID-19.

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