Method for treating interferonopathies

Abstract

Methods for interfering or controlling IFN induction, IFN production and / or IFN activity are disclosed, harnessing viral mechanisms for evading or suppressing host anti-viral immune reactions. These methods comprise administration of one or more viral messenger RNAs (mRNAs) coding for proteins or peptides that control or interfere with signal transduction upstream and / or downstream of IFN induction, thereby providing a means for preventing or treating diseases, disorders or conditions associated with increased IFN production, such as cancer, autoimmune diseases, inflammatory diseases and interferonopathies.

Description

[0001] METHOD FOR TREATING INTERFERONOPATHIES

[0002] FIELD OF THE INVENTION

[0003] The present disclosure relates to methods for treating interferonopathies characterized by uncontrolled type I and or type III IFN-dependent immune response, particularly but not exclusively by interfering with the IFN production.

[0004] BACKGROUND

[0005] Interferonopathies are immune system disorders caused by dysregulated type I and type III interferon (IFN) responses, key immune mediators, which are critical, e.g., for antiviral defense. The IFN system operates on a delicate balance, providing protection against infections while avoiding excessive inflammation and immunopathogenesis. To maintain this balance, IFN induction, signaling, and resolution are tightly regulated.

[0006] Type I and type III interferonopathies and related non-monogenic conditions result from disruptions in this regulation. These disorders, often caused by mutations in genes involved in proteasome degradation or RNA- and DNA-sensing pathways, lead to chronic IFN activation or impaired negative regulation. Identifying these conditions is clinically significant, as emerging anti-IFN therapies target the underlying pathogenic mechanisms.

[0007] Type I and III IFNs are key cytokines secreted by host cells upon detecting viral components, such as double-stranded RNA (dsRNA), single-stranded RNA (ssRNA) such as ssRNA with 5' triphosphate, cytosine-guanine dinucleotide (CpG)-capped DNA, viral capsid and / or intermediate proteins (proteins which are generated in infected cells as viral intermediates during). These elements, termed pathogen-associated molecular patterns (PAMP) are recognized by pattern recognition receptors (PRRs), including: Toll-like receptors (TLRs) 3 / 7 / 8 / 9; retinoic acid-inducible gene-1 (RIG-1) like receptors (RLRs) such as RIG-1, melanoma differentiation-associated factor 5 (MDA5), and laboratory of genetics and physiology (LGP2); nucleotide oligomerization domain (NOD)-like receptors (NLRs); and cytosolic DNA sensors.

[0008] Upon PAMP recognition, signaling cascades involving protein conformational changes, ubiquitination, and kinase activation lead to phosphorylation of transcription factors such as IFN- regulatory factors 3, 5 or 7 (IRF3, IRF5 or IRF7, respectfully), and nuclearfactor kappa-light-chain- enhancer of activated B cells (NF-KB). These factors translocate to the nucleus, triggering expression of IFNs and proinflammatory cytokines, shaping a precise and cell-type-specific antiviral response.

[0009] Many viruses have evolved strategies to bypass the IFN response, ensuring their survival and replication. These mechanisms include inhibiting IFN induction, blocking IFN signaling pathways and / or disrupting IFN-mediated antiviral effects

[0010] By targeting innate immune signaling at multiple levels, viruses evade host defenses, allowing continued viral replication and persistence. Understanding these evasion tactics is crucial for developing antiviral therapies and immune-modulating treatments.

[0011] SUMMARY

[0012] The present disclosure is based on a discovery by the present inventor that viral mRNA administered to mice bearing SLE-like disease, dramatically affected the elevated IFN expression in these mice. Specifically, viral mRNA administration substantially reduced INF-p production in the diseased mice.

[0013] The mechanisms viruses utilize to evade the interferon (IFN) system have been adapted and applied, in accordance with the present disclosure, for treating diseases linked to dysregulated or altered IFN expression, production and / or function. Specifically, this disclosure presents strategies for modulating type I and type III IFN production to restore immune balance and improve disease outcomes. These strategies may be used to treat interferonopathies and related conditions.

[0014] Aspects of the present disclosure relate to methods for (i) interfering or controlling IFN-based immune response; (ii) preventing or treating a disease, disorder, syndrome or condition associated with elevated IFN levels; and / or treating interferonopathy. These methods comprise administrating to a subject in need thereof a therapeutically effective amount of one or more viral messenger RNAs (mRNAs) coding for proteins or peptides that interfere with one or more of IFN induction, IFN production and / or IFN activity.

[0015] In the context of the present disclosure, IFN induction is initiation of IFN gene transcription, IFN production is the synthesis and secretion of IFN proteins after induction, and IFN activity is all biological effects of IFN, including antiviral and immune functions.

[0016] In some embodiments, the viral proteins or peptides interfere with IFN-based signaling pathways upstream of INF induction. In some embodiments, interferonopathies treated by a disclosed method are type I or type III IFN interferonopathies.

[0017] Disease, disorder, syndrome or condition treatable by a disclosed method include, but are not limited to, various types of cancer, a neurodegenerative disorder, an inflammatory disease, and / or an autoimmune disease, that may benefit from restoring or remedying altered levels of IFN induction, production and / or activity, for example, altered levels of type I and / or type III IFN.

[0018] Diseases, disorders, syndromes, conditions and / or interferonopathies addressed by the present disclosure include, for example, Aicardi-Goutieres syndrome (AGS); familial chilblain lupus (FCL); spondyloenchondrodysplasia (SPENCD) with immune dysregulation; stimulator of interferon genes (STING)-associated vasculopathy with onset in infancy (SAVI), Japanese autoinflammatory syndrome with lipodystrophy (JASL); ubiquitin-specific peptidase 18 (USP18) deficiency; interferon-stimulated gene 15 (ISG15) deficiency; chronic atypical neutrophilic dermatitis with lipodystrophy; DNAase II deficiency; chronic atypical neutrophilic dermatosis with lipodystrophy and elevated temperature (CANDLE); Nakajo-Nishimura syndrome; X-linked reticulate pigmentary disorder; Singleton-Merten syndrome; joint contractures, muscular atrophy, microcytic anemia; panniculitis-induced lipodystrophy (JMP) syndrome; proteasome-associated autoinflammatory syndrome (PRAAS); systemic lupus erythematosus (SLE); cutaneous lupus erythematosus (CLE), pediatric SLE (pSLE), rheumatoid arthritis (RA), psoriasis, juvenile idiopathic arthritis (JIA), psoriasis, myasthenia gravis (MG), juvenile dermatomyositis (JDM), Sjogren syndrome (SjS); systemic sclerosis (SSc), endometriosis, type I diabetes; dermatomyositis (DM); multiple sclerosis (MS); ataxia telangiectasia; amyotrophic lateral sclerosis (ALS); mendelian susceptibility to mycobacterial disease (MSMD); cytokine storm; bacteriopathy; viropathy; familial systemic lupus erythematosus (FSLE), autosomal dominant immune deregulatory disease (COPA syndrome); a mitochondrial disease; non-alcoholic fatty liver disease (NAFLD); Sjogren's syndrome; nonalcoholic steatohepatitis (NASH); cytokine storm; vasculitides; neurodegenerative diseases such as Parkinson's disease (PD), Lewy body disease (LBD), Huntington's disease (HD) and Alzheimer's disease (AD); and / or traumatic brain injury (TBI).

[0019] The administered viral mRNA may comprise the intact nucleic acid, a derivative thereof, an analog thereof, and / or a biologically active fragment thereof. Exemplary viral proteins that are translated from viral mRNA administered to an infected host and interfere with I FN induction, production and / or activity in the host include, for example, a non-structural (NS) protein; papain-like protease (PLP2)-transmembrane (TM); Z protein; V protein; P protein; C6 protein; E3 protein; virion protein 35 (VP35); nucleocapsid protein (NP); GPC protein; VP24 protein; interferon regulatory factor 1, 2, or 3 (vlRFl / 2 / 3); and / or 3Cprotease protein.

[0020] In some embodiments, the viral protein is a NS protein selected from NS1, NS2, NS3, NS1B, NS2B, NS2B3, NS5, NS5A, NS2A, NS4A, NS4B, NSP1, NSP5, NSP6, NSP7, NSP8, NSP12, NSP13, NSP14, NSP15, NSP16, a biologically active fragment thereof, an analog thereof, a derivative thereof and / or any combination thereof.

[0021] Viral proteins or peptides harnessed in accordance with the present disclosure may interfere with various aspects and pathways of INF induction. For example, they can modulate the function and / or localization of interferon-stimulated gene 15 (ISG15) and / or interfere with the function of a pattern recognition receptor (PRR).

[0022] Specific pathways or host targets that may be affected by the viral proteins or peptides include at least: (i) retinoic acid-inducible gene-l-like receptor (RIG-1) sensing pathway; (ii) ISGylation of RIG-1; (iii) melanoma differentiation-associated gene 5 (MDA5) sensing pathway; (iv) ISGylation of MDA5; (v) interferon regulatory factors (IRFs), preferably, IRF3, IRF5, and IRF7; (vi) cyclic-GMP-AMP synthase (cGAS); (vii) mitochondria antiviral signaling protein (MAVS); (viii) stimulator of IFN genes (STING); (ix) TANK-binding kinase 1 (TBK1) and / or IKB kinase-e (IKKe); (x) NF-KB; and / or (xi) nlrp3 (xii) STATs.

[0023] In another aspect, the present disclosure relates to pharmaceutical compositions and vaccines comprising one or more viral mRNAs coding for any one or more of the abovementions proteins and / or peptides that interfere with IFN induction, IFN production and / or IFN activity. In some embodiments, these viral proteins and peptides interfere with signal transduction upstream and / or downstream of IFN induction for example, signal transduction upstream of IFN induction.

[0024] BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Fig. 1 is schematic (prior art) presentation of exemplary mechanisms of RNA viral antagonism upstream of IFN induction. Solid arrows indicate direct pathway connections. Dashed arrows indicate signal cascade components present in vivo but not shown for space concerns. Asterisks indicate viral antagonism by an unknown mechanism. LGP2, laboratory of genetics and physiology protein 2; MDA5, melanoma differentiation-associated protein 5; TRIF, TIR domain-containing adapter-inducing I FN-fJ; IKK-e, IKB kinase-e; HAV, hepatitis A virus; hCoV-NL63, human coronavirus NL63; PEDV, porcine epidemic diarrhea virus; GTOV, Guanarito virus; JUNV, Junin virus; MAVC, Machupo virus; SABV, Sabia virus; BDV, Borna disease virus; SFTSV, severe fever with thrombocytopenia syndrome virus; JEV, Japanese encephalitis virus; ANDV, Andes virus; SNV, Sin Nombre virus (Taken from Mesev et al., Nature Microbiology, 2019, Vol 4: 914-924).

[0026] DETAILED DESCRIPTION

[0027] Viruses have co-evolved with their hosts to evade, subvert, or directly disrupt the innate immune response, allowing them to replicate efficiently and spread to neighboring cells. A common viral strategy is the disruption of interferon (IFN) signaling, both upstream and downstream of IFN induction. This enables viruses to block IFN signaling pathways, inhibit IFN- stimulated gene (ISG) activity and / or disrupt crosstalk between IFNs and other cellular pathways. By counteracting the host's immune response, viruses reduce the antiviral potency of IFNs, creating a more favorable environment for replication.

[0028] Viruses employ multiple mechanisms to evade IFN-dependent immunity, including concealing viral genomes to avoid immune recognition, and blocking interactions with key host factors involved in IFN induction, manipulating host signaling pathways, e.g., by regulating phosphorylation, ubiquitination, transcription, translation, RNA processing, and protein degradation. Viruses can also employ protein decoys (viral proteins) to mimic or disrupt host immune components.

[0029] Specific viral strategies to suppress IFN responses include, for example:

[0030] (i) Disrupting viral sensors, e.g., inhibiting retinoic acid-inducible gene 1 (RIG-1) receptor or cyclic-GMP-AMP synthase (cGAS), and other immune sensors that detect viral RNA or DNA.

[0031] (ii) Blocking Toll-like receptor (TLR) signaling, thereby preventing IFN activation through TLR pathways. (iii) Inhibiting key IFN signaling proteins such as mitochondria antiviral signaling (MAVS) protein, melanoma differentiation-associated 5 (MDA5), stimulator of IFN genes (STING), TANK- binding kinase 1 (TBK1) and / or IKB kinase-e (IKKe) to suppress IFN production.

[0032] (iv) Interfering with IFN transcription factors, e.g., inhibiting IRF3, IRF5, and IRF7, which are essential for IFN gene expression.

[0033] (v) Blocking IFN receptors, thereby, preventing host cells from responding to IFNs.

[0034] (vi) Disrupting IFN-induced antiviral activities, for example, by inhibiting IFN-stimulated genes (ISGs) functions to counteract host defenses.

[0035] (vii) Modifying host protein activity: altering post-translational modifications (PTM) such as ubiquitination, SUMOylation, NEDDylation, ISGylation, phosphorylation, and acetylation to manipulate immune responses.

[0036] Therapeutic Applications

[0037] In one aspect, the present disclosure relates to a method for modulating (i.e., inhibiting, arresting, interfering or otherwise attenuating) the IFN-based immune response in patients with conditions linked to excessive or dysregulated IFN activity. The method comprises administering to a subject in need thereof a therapeutically effective amount of one or more viral nucleic acid sequences or peptide-encoding segments thereof that control or interfere with one or more of IFN induction, IFN production and / or IFN activity by mediating IFN signaling pathways and / or targeting specific steps upstream or downstream of IFN induction.

[0038] As used herein, the term "IFN-based immune response" refers to an immune response that is primarily driven by interferons, including the activation, regulation, or modulation of immune cells and signaling pathways through IFN production and downstream signaling. This includes the induction of interferon-stimulated genes (ISGs), activation of natural killer (NK) cells, enhancement of antigen presentation, and modulation of inflammatory cytokine networks. This term specifically refers to immune system activities that are fundamentally dependent on IFNs as key regulators or effectors. "IFN-based immune response" is interchangeable herein with terms "IFN immune activity", "IFN-mediated processes" and "immunomodulatory IFN function".

[0039] The terms "IFN Induction" and "induction of IFN expression", as used herein are interchangeable and refer to the process by which IFN expression is stimulated in response to an inducing agent, such as a viral infection, pathogen-associated molecular patterns (PAMPs), or cytokines. This refers specifically to the initiation of the cellular signaling cascade that leads to IFN gene transcription. These terms refer strictly to the initiation of IFN gene transcription.

[0040] The terms "IFN production" and "IFN synthesis and secretion", as used herein are interchangeable and refer to the synthesis and secretion of interferon proteins by cells following IFN induction. This term encompasses both the transcription and translation of IFN, as well as its subsequent release into the extracellular environment. Although these terms encompass the synthesis and secretion of IFN proteins after induction, sometimes in the context of the present disclosure the terms IFN induction and IFN expression also refer or encompass IFN production.

[0041] The terms "IFN activity" and "functional IFN response" are used interchangeably, referring to all biological effects exerted by interferons, such as antiviral, antiproliferative, or immunomodulatory effects. This term typically refers to measurable functional outcomes of IFN signaling, including gene expression changes or inhibition of viral replication.

[0042] The present disclosure relates to the utilization of viral components known to disrupt, disturb, inhibit, distract, sabotage, obstruct, downregulate or otherwise antagonize or interfere with the normal immune reaction, to control autoinflammation that is associated with interferonopathy. By leveraging viral immune evasion mechanisms, the methods disclosed herein aim to regulate IFN-based immune responses in conditions where IFN signaling contributes to disease pathology.

[0043] As used herein, the term "viral components" encompasses: (i) nonstructural (NS) proteins, namely proteins encoded by a virus that are expressed in host cells during infection but are not incorporated into the viral particle; and (ii) structural components of an infectious virus particle, which are elements that directly or indirectly influence the function or activity of the innate and / or adaptive immune system of an infected host. These components include viral DNA and RNA and viral structural proteins.

[0044] The term "viral structural protein", as used herein, refers to any protein that constitutes a structural component of a mature virus. Such proteins may be integral to the viral capsid, which encases the viral genome, or to the viral envelope, present in enveloped viruses. Non-limiting examples of viral structural proteins that modulate the host immune response include capsid proteins of flaviviruses, which localize to the nucleus during infection and have the potential to alter host gene expression. Another example is the Zika virus (ZIKV) capsid protein, which binds to and inhibits the ubiquitination and activation of RIG-1. For the purposes of this disclosure, the term "viral protein" broadly encompasses both structural and nonstructural viral proteins, as well as peptides derived therefrom.

[0045] In some embodiments, in accordance with a contemplated method described herein, the viral components utilized for therapeutic purposes are viral components that control, downregulate, antagonize or interfere with IFN induction, IFN production and / or IFN-mediated processes.

[0046] The viral components used in accordance with the present disclosure may be genuine components obtained from, produced by, or induced by viruses. Alternatively, or additionally, the viral components may by artificially synthesized or produced by various methods known in the art. The artificial viral components may be similar or identical to genuine or natural viral components. Alternatively, the artificial viral components may be analogs and / or derivatives of natural or genuine viral components.

[0047] Analogous viral components, as referred to herein, are viral components that are comparable, namely, similar, but not necessarily identical, in structure and / or function to natural viral components.

[0048] Derivatives of viral components, as referred to herein, are components based on, or derived from, corresponding natural viral components and featuring or bearing one or more structural modifications. For example, a viral components derivative may comprise an amino acid sequence (e.g., a peptide, protein), or a nucleic acid sequence (e.g., RNA, DNA) derived from a corresponding viral component in which one or more amino acid or nucleic acid, respectively, have been altered (e.g., via chemical modification), replaced, substituted, removed and / or added.

[0049] Biologically active fragments of viral components are further contemplated herein. Such fragments, which may be obtained by truncating corresponding natural components or by synthesis, have similar or identical biological functions as the corresponding intact viral components.

[0050] In some embodiments, fragments, analogs and / or derivatives of a viral components are used, in accordance with a contemplated method, in order to obtain higher or better efficacy and / or specificity or selectivity in controlling or interfering with signal transduction upstream and / or downstream of IFN induction. The term "signal transduction upstream and / or downstream of IFN induction" or the term "upstream and / or downstream molecular pathways regulating IFN expression" and function" are interchangeable and refer to the molecular signaling events that occur before ("upstream") and / or after ("downstream") the initiation of IFN expression.

[0051] "Upstream of IFN induction" refers to the signaling pathways and molecular interactions that lead to the activation of IFN gene transcription. This includes the recognition of stimuli (e.g., viral RNA, cytokines) by pattern recognition receptors (PRRs) such as Toll-like receptors (TLRs) or RIG-l-like receptors (RLRs), and the activation of intracellular signaling cascades (e.g., phosphorylation of I RF3 / IRF7, NF-KB activation).

[0052] Pattern recognition receptors are evolutionarily conserved receptors that detect pathogens and regulate immune responses. They recognize molecular patterns from pathogens, damaged host cells, and foreign molecules, triggering inflammatory and immune reactions to eliminate threats. PRRs can recognize pathogen-associated (PAMPs), damage-associated (DAMPs), microbe-associated (MAMPs), and xenobiotic-associated (XAMPs) molecular patterns.

[0053] PRRs are classified into five categories based on function and structure:

[0054] (1) Toll-like receptors (TLRs) - cell surface receptors that detect self and non-self antigens, regulate cytokine production, and bridge innate and adaptive immunity. Humans possess 10 TLRs (TLR1-10), categorized into cell membrane TLRs (TLR1, 2, 4, 5, 6, 10), which detect external pathogens (In their active signal-transducing form, they exist as dimers: TLR1- TLR2, TLR2-TLR6, and TLR2-TLR4 / 5 / 10), and intracellular TLRs (TLR3, 7, 8, 9), which recognize nucleic acids inside endosomes and lysosomes. These TLRs are expressed within the host cells on the organelle bio-membranes like endoplasmic reticulum (ER), endosomes, and lysosomes;

[0055] (2) AIM2-like receptors (ALRs) - detect cytoplasmic DNA and activate inflammasomes;

[0056] (3) C-type lectin receptors (CLRs) - recognize carbohydrate structures on pathogens;

[0057] (4) retinoic acid-inducible gene (RIG)-l-like receptors (RLRs) - detect viral RNA and trigger antiviral responses. RLRs are key sensors of viral infection, triggering type I interferon production and antiviral responses. RIG-1, a nucleic acid sensor, detects unusual RNAs from both viruses and the host, activating immune defense. While essential for antiviral immunity, uncontrolled RLR activation can contribute to immunopathology; and

[0058] (5) oligomerization domain (NOD)-like receptors (NLRs) - recognize intracellular stress signals and activate immune pathways. NLRs are essential for host defense, but dysregulation is linked to inflammatory and autoimmune diseases, such as Crohn's disease, rheumatoid arthritis, and autoinflammatory syndromes. NLRs comprise inflammasome-forming NLRs such as NLRP1, NLRP3 and NLRC4, signaling NLRs such as NODI, NOD2 (activate N F-KB and MAPK pathways), and regulatory NLRs such as NLRX1 (modulates immune responses and mitochondrial function).

[0059] TLRs signal through two pathways. The MyD88-dependent pathway (used by all TLRs except TLR3) promotes proinflammatory cytokine production. The TIR-domain-containing adapter-inducing IFN (TRIF)-dependent pathway (used by TLR3 and TLR4), induces type I IFN expression.

[0060] Key adaptors in TLR signaling include MyD88, TRIF, Toll-IL-1 receptor (TIR) domaincontaining adaptor protein (TIRAP), and TRIF-related adaptor molecule (TRAM), which regulate immune responses. RIG-1, a nucleic acid sensor, detects viral and host RNA, playing a crucial role in antiviral immunity but can also contribute to immunopathology when dysregulated.

[0061] "Downstream of IFN Induction" refers to the signaling events triggered by IFN once it has been produced and secreted. This includes IFN binding to its receptor, activation of the JAK-STAT signaling pathway, and the transcriptional regulation of interferon-stimulated genes (ISGs), which mediate antiviral, antiproliferative, and immunomodulatory effects.

[0062] Embodiments disclosed herein pertain to applying one or more viral mRNAs as the viral components useful in directly or indirectly controlling, inhibiting, arresting, interfering or otherwise attenuating IFN-based immune response. For example, the be one or more viral mRNA may code for proteins or peptides that control or interfere with signal transduction upstream and / or downstream of IFN induction.

[0063] Many viral proteins are known to interfere with host IFN pathways. Non-limiting examples include: Flavivirus NS2B3; Flebovirus NS; Paramyxovirus C, V; Coronavirus NS2, PLP, YFV NS4B, NS5; Herpes simplex virus (HSV) proteins ICP27m, VP24; West Nile virus (WNV) NS3; Japanese encephalitis virus (JEV) NS5; rotavirus (RotaV) NSP1, NSP3, VP3; Lymphocytic choriomeningitis (LCMV) NP; Poxvirus (POXV) poxins; Phlebo virus (PhleboV) NS3; Hepatitis C virus (HCV) NS4B, NS34A, NS5; respiratory syncytial (RSV) NS1; Epstein-Barr virus (EBV) infectious bronchitis virus (IBV) (IBV) C-terminal domain (CTD) of NS1B; Sendai virus (SendaiV) protein C; Ebola virus (EbolaV) VP35; bluetongue virus (BTV) NS3, MERS CoV M Orf4b; SARS CoV M, Orf9b; SARS CoV-2 NS1, NS13, PLP2; Vaccinia C6; zika virus (ZIKV) NS1, NS2B, NS4A, NS3, NS5; influenza A (IAV) N terminus-truncated NS1 protein (amino acids 79 to 230 from the PR8 virus); dengue virus (DENV) NS2B3, NS3, NS5; Lactate dehydrogenase-elevating virus (LDV) protein Y136; and Human papillomavirus (HPV) E protein.

[0064] In some embodiments, one or more viral mRNAs are utilized for directly or indirectly interfering with or attenuating IFN-based immune response upstream of IFN induction or expression. Exemplary mechanisms for interfering with or controlling the innate immune signaling upstream of IFN induction are shown in Fig. 1 and summarized in Table 1.

[0065] Table 1. Exemplary viral mechanisms for evading IFN induction

[0066] In some embodiments, the viral protein used for targeting the host IFN production is the non-structural protein 1 (NS1), a fragment thereof, an analog thereof or a derivative thereof. In accordance with these embodiments, NS1 is used for interfering with signal transduction upstream of IFN induction. NS1 is a small polypeptide (~26 kDa, 230-237 amino acids) that plays a central role in immune evasion of several viruses. It suppresses IFN signaling, induction, and effector functions through diverse protein-protein interactions, which may differ across hosts and viral strains but ultimately lead to immune suppression.

[0067] NS1 proteins are highly expressed in influenza A, B, C, and D viruses (IAV, IBV, ICV, IDV). Despite variations in amino acid sequence, all NS1 proteins target the IFN pathway, though the precise molecular mechanisms may differ among strains. By facilitating viral replication, dampening adaptive immunity, and disrupting host gene expression, NS1 serves as a major factor in virus pathogenesis, particularly of influenza virus.

[0068] In some embodiments, a contemplated method included treatment of a subject in need thereof with viral mRNA coding for NS1, for example, IAV NS1 and / or IBV NS1B, a fragment thereof, an analog thereof and / or a derivative thereof.

[0069] In some embodiments, disrupting signal transduction upstream of IFN induction is achieved by administering mRNAs that encode proteins capable of targeting host nucleic acid sensors or scavengers. These targeted sensors include various pattern recognition receptors (PRRs) such as Toll-like receptors (TLRs), AIM2-like receptors (ALRs), C-type lectin receptors (CLRs), retinoic acid-inducible gene l-(RIG-l)-like receptors (RLRs), and / or oligomerization domain (NOD)-like receptors (NLRs).

[0070] ISGylation is a post-translational modification process in which the ubiquitin-like protein ISG15 (interferon-stimulated gene 15) is covalently conjugated to target proteins. This process is induced by type I interferons (e.g., IFN-a and I FN-fJ) and involves an enzymatic cascade similar to ubiquitination, including El-activating, E2-conjugating, and E3-ligase enzymes. ISGylation modulates antiviral responses by altering stability, localization, or activity of modified proteins for example, by modulating cytokine signaling, or by inhibiting viral replication.

[0071] Interaction with the ubiquitin-proteasome system, sometimes stabilizes proteins rather than marking them for degradation.

[0072] In some embodiments, viral mRNA, its biologically active fragments, analogs, and / or derivatives modulate both RIG-l-mediated signaling and RIG-1 protein levels. Additionally, in some embodiments, viral proteins encoded by the mRNA influence the interferon-induced ISGylation of RIG-L Given this regulatory role, modulation of RIG-1 ISGylation may serve as a potential therapeutic strategy for managing RIG-l-mediated signaling in interferonopathies, particularly in response to endogenous cytosolic RNA.

[0073] In some embodiments, viral mRNA, its biologically active fragments, analogs, and / or derivatives modulate the viral RNA sensor melanoma differentiation-associated gene 5 (MDA5) sensing pathway, for example, by inhibiting ISGylation of MDA5. ISG15 conjugation is essential for antiviral IFN induction mediated by MDA5. ISGylation of the caspase activation and recruitment domains of MDA5 promotes its oligomerization and thereby triggers activation of innate immunity against a range of viruses, including coronaviruses, flaviviruses and picornavi ruses. Overexpression of interferon-induced helicase C domain 1 (IFIH1) gene encoding MDA5 is associated with autoimmune diseases.

[0074] In some embodiments, viral mRNA, its biologically active fragments, analogs, and / or derivatives modulate interferon induction by downregulating, inhibiting, arresting or otherwise disrupting the activity of one or more transcriptional factors, including but not limited to, interferon regulatory factors (IRFs) such as IRF3, IRF5, and IRF7.

[0075] Interferon regulatory factors are a family of transcription factors essential for innate and adaptive immunity, regulating antiviral responses, inflammation, and immune cell differentiation. Among them, IRF3, IRF5, and IRF7 are key to type I interferon (IFN-I) production following pathogen recognition, while IRF9 governs IFN-driven gene expression. IRF4, IRF5, and IRF8 also influence myeloid cell development and inflammatory responses. Dysregulation of IRFs can lead to immune disorders, including autoimmunity.

[0076] The nine IRF family members share a conserved N-terminal DNA-binding domain ("'120 amino acids) that recognizes interferon-stimulated response elements (ISRE). ISRE is a conserved region of about 15bp in all IFN-a / p-stimulated genes promoters. They can form transcriptionally active or repressive complexes with IRFs via homo- or heterodimerization. Given their role in IFN-I regulation, IRFs are implicated in autoimmune diseases such as systemic lupus erythematosus (SLE), where excessive IFN-I contributes to pathology.

[0077] IRF3 and IRF7, the most structurally similar members, are principal IFN inducers. IRF3 is ubiquitously expressed, while IRF7 is lowly expressed except in plasmacytoid dendritic cells (pDCs), where it plays a crucial role in amplifying IFN production. IRF3 activation involves phosphorylation by TRAF-associated NF- KB activator (TANK)-binding kinase 1 (TBK1) and IKB kinase (IKK) epsilon (IKKe), leading to nuclear translocation, co-activator binding (CBP), and IFN- P / IFN-a promoter activation. Various pattern recognition receptors (PRRs) and toll-like receptors (TLRs) initiate IRF3 activation through adaptor proteins (e.g., TRIF, I PS-1, STING) that recruit TBK1 to distinct cellular compartments. In addition to IFN-I, IRF3 regulates cytokines such as CXCL10, RANTES, ISG56, IL-12p35, IL-23, and IL-15, while inhibiting IL-12 and TGF- .

[0078] In some embodiments, a viral protein encoded by an applied mRNA modulates the IRF3 function, for example, by interfering with ISGylation. IRF3 undergoes ISGylation in response to type I interferons and viral infection, a process that can influence its stability and transcriptional activity. Modulation of IRF3 ISGylation by viral proteins may alter IRF3 stability, nuclear translocation, and subsequent IFN promoter activation, thereby affecting type I interferon responses.

[0079] In some embodiments, a viral protein encoded by an applied mRNA mediates or attenuates, e.g., interferes with IRF5 function, for example, by directly or indirectly inhibiting IRF5.

[0080] In some embodiments, a viral protein encoded by an applied mRNA mediates or attenuates, e.g., interferes with IRF7 function, for example, by directly or indirectly inhibiting IRF7. Inhibition of IRF7 and / or IRF5 and / or IRF3 may be effected, e.g., by modulating or blocking IKKE or TBKl - mediated phosphorylation of these IRFs.

[0081] IRF5 and / or IRF7 serve as therapeutic targets for treating or ameliorating systemic lupus erythematosus (SLE). Aberrant IRF5 activation is observed in SLE patients during both active disease and remission, while genetic variants of IRF7 contribute to SLE susceptibility. Additionally, IRF7 is implicated in the pathogenesis of type I diabetes. In autoimmune conditions such as scleroderma and systemic sclerosis (SSc), IRF7 upregulation and activation drive excessive inflammation and fibrosis, further highlighting its role in disease pathology. Inhibiting IRF7 by a viral protein in accordance with the present disclosure may provide an effective means for treating any of these diseases.

[0082] Further exemplary embodiments pertaining to the use of viral proteins for interfering with or controlling IFN-based immune response include:

[0083] (i) use of NS1 for directly modulating the actions of ISGs and / or suppressing host antiviral effector protein kinase R (PKR);

[0084] (ii) use of NS1 protein of Puerto Rico 8 (PR8) influenza A virus or a fragment thereof (truncated PR8 NS1) to suppress the activation of IRF3;

[0085] (iii) use of ZIKV NS1 for interacting with ubiquitin specific peptidase 8 (USP8), for mediating the de-ubiquitination of Caspase 1 and preventing its proteasomal degradation. Enhanced stabilization of Caspase 1 promotes the cleavage of cGAS, which is involved in the initiation of IFN-I induction;

[0086] (iv) use of NS5 of ZIKV and yellow fever virus (YFV) to modulate RIG-1 activity. NS5 suppresses the ubiquitination of RIG-1, followed by reduction of both phosphorylation and nuclear translocation of IRF3, leading to the suppression of IFN induction; (v) use of NS3 / 4A, a major serine protease expressed by HCV, for blocking RIG-1 and / or MDA5. NS3 / 4A cleaves interferon-beta promoter stimulator 1 (IPS-1), an adaptor that triggers RIG-1- and MDA5-mediated type I INF induction;

[0087] (vi) use of NS1B of influenza B virus for inhibiting ISGylation and, subsequently, the generation of ISG15 conjugates;

[0088] (vii) use of NS3 of dengue virus (DENV), alone or in combination with NS2B, for inhibiting IFN induction. NS3 and NS2B form a protease complex that cleaves cGAS (cyclic GIMP-AMP synthase) and STING (stimulator of interferon genes);

[0089] (viii) use of virulence factor C6, encoded by vaccine virus, for interfering with the phosphorylation and activation of IRF3 and IRF7 by binding to TBK-1 / / IKKE;

[0090] (x) use of herpes simplex virus (HSV) VP24 protein for inhibiting the phosphorylation of IRF3 by TBKl / IKKE.

[0091] (xi) use of ebola virus VP35 protein for interfering with the phosphorylation of IRF3 by preventing TBK1- dependent phosphorylation of IRF3;

[0092] (xii) use of 3Cprotease viral protein of enterovirus 71 (EV71) and poliovirus (PV) for inducing RIG-1, IRF7, TRIF, TAB2, TAK1, TAB1, TAB3 and PKR cleavage and degradation;

[0093] (xiii) use of viral proteins of encephalomyocarditis virus (EMCV) for cleaving TANK and disrupt the TANK-TBK1-IKKE-IRF3 complex formation; and / or

[0094] (xv) use of Kaposi's sarcoma-associated herpesvirus (KSHV) viral proteins for blocking host IRFs transactivation. For example, viral interferon regulatory factor 1 (vIRFl) antagonizes both IRF3 and IRF7. vlRF4 antagonizes IRF7.

[0095] In another aspect, the present disclosure relates to a method for preventing or treating a disease, disorder, syndrome or condition associated with elevated IFN levels. This method involves administrating to a subject in need thereof a therapeutically effective amount of one or more viral mRNAs that encode proteins or peptides that are capable of interfering with one or more aspects of IFN induction, IFN production, and / or IFN activity.

[0096] The diseases, disorders, or conditions treatable according to the present disclosure may involve altered IFN production, leading to elevated levels of type I IFN (IFN-I) and / or type III IFN (IFN-III). Dysregulated expression of IFN genes is frequently linked to an increased susceptibility to various IFN-related pathologies, including multiple types of cancer, neurodegenerative disorders, inflammatory diseases, and autoimmune diseases. Exemplary diseases characterized by overproduction of IFN-I include autoimmune diseases with unclear etiology, such as SLE. In these conditions, mutations in genes involved in nucleic acid metabolism contribute to elevated nucleic acid levels, leading to increased cell damage or death. This accumulation of nucleic acids activates pathways involved in interferonopathies, resulting in the overproduction of IFN.

[0097] Patients suffering from diseases such as type 1 diabetes, rheumatoid arthritis, Sjogren's syndrome, dermatomyositis (DM), and multiple sclerosis (MS) often exhibit strong IFN signatures, which reflect elevated levels of interferons in their system. These signatures are indicative of upregulated transcripts (mRNA) produced by different IFN subtypes, suggesting that the immune system is in an active state. This increased IFN production is a hallmark of autoimmune and inflammatory conditions, where the immune system's response is heightened. The specific IFN subtypes involved include type I IFNs (a, p, e, K, and co), which are typically produced in response to viral infections and play a key role in antiviral immune responses, type II IFN (IFN-y), which is crucial for activating immune cells to combat infections and regulate immune functions, and type III IFNs (IFN-X1-4), which are involved in antiviral defense, particularly at mucosal surfaces.

[0098] These elevated IFN signatures reflect the overactivation of the immune system in these diseases, driving pathological immune responses.

[0099] Aberrant IFN-dependent signaling is also implicated in neurodegenerative disorders, some of which are autoimmune in nature, such as ataxia telangiectasia and amyotrophic lateral sclerosis (ALS). Inflammatory diseases, such as mendelian susceptibility to mycobacterial disease (MSMD), bacteriopathies, viropathies, and cytokine storms, as well as injuries (e.g., Traumatic Brain Injury (TBI) in veterans), have also been linked to elevated IFN levels.

[0100] In yet another aspect, the present disclosure relates to a method for preventing or treating interferonopathy, comprising administrating to a subject in need thereof a therapeutically effective amount of one or more viral mRNAs coding for proteins or peptides that interfere with one or more of IFN induction, IFN production and / or IFN activity.

[0101] At least some of the above-mentioned diseases, disorders, syndromes and / or are interchangeably herein referred to as interferonopathies.

[0102] Interferonopathies are a growing group of monogenic disorders characterized by dysregulated IFN-mediated immune responses due to impaired homeostatic control. Despite variability in clinical severity and presentation, these conditions share common pathogenic mechanisms, primarily involving excessive type I IFN production.

[0103] Monogenic type I interferonopathies are inherited disorders marked by early-onset systemic and organ-specific inflammation driven by constitutive type I IFN activation. Other interferonopathies result from genetic mutations that may be inherited or acquired during a person's lifetime. As research advances, the list of putative monogenic interferonopathies continues to expand with newly identified molecular mechanisms and clinical phenotypes.

[0104] Both type I and type III interferonopathies fall within the autoinflammatory-autoimmune spectrum, where the dominant phenotype depends on the specific IFN signaling pathways involved. However, it is important to note that not all autoinflammatory or autoimmune diseases are classified as interferonopathies.

[0105] While nucleic acid sensing by the innate immune system is essential for host defense against pathogens, it also presents a risk, as viral infections are recognized as potential environmental triggers for autoimmunity.

[0106] Viral infections have been implicated as triggers for autoimmunity through several key mechanisms: molecular mimicry, epitope spreading, and bystander activation. Each of these mechanisms describes how an immune response initially targeting a virus can mistakenly attack the body's own tissues, leading to autoimmune disease.

[0107] Molecular mimicry occurs when viral proteins share structural or sequence similarity with host proteins. The immune system generates antibodies or T cells to fight the virus, but these immune responses cross-react with self-proteins that resemble the viral antigen.

[0108] Viral infection triggers an immune response against one specific viral epitope, but over time, additional self-antigens become targeted (epitope spreading). This happens when tissue damage releases self-proteins, exposing them to the immune system and triggering autoimmunity. For example, in multiple sclerosis (MS), an initial immune response to a viral infection may lead to an immune attack on myelin proteins, causing progressive nerve damage.

[0109] Bystander activation is when a non-specific immune response triggered by viral infection inadvertently activates self-reactive T or B cells. For example, in type 1 diabetes, viral infections such as coxsackievirus B can trigger inflammation in the pancreas, leading to the activation of autoreactive T cells that attack insulin-producing p-cells. All three mechanisms involve the activation of cytosolic nucleic acid sensing pathways, which lead to increased IFN production, a hallmark of interferonopathies.

[0110] Gain-of-function mutations in MDA5 gene (MDA5 / 1 Fl Hl), RIG-1 gene (DDX58), and / or STING gene (TMEM173) further amplify IFN responses, creating a hyperinflammatory state that increases the likelihood of autoimmunity. For example, in Aicardi-Goutieres syndrome, pathogenic IFIH1 mutations enhance MDA5 activity, allowing it to bind more readily to RNA, including self-derived RNA, leading to aberrant IFN production.

[0111] These findings reinforce the connection between dysregulated nucleic acid sensing, interferonopathies, and autoimmunity, further emphasizing the importance of tightly controlled IFN signaling in maintaining immune homeostasis.

[0112] Dysregulated nucleic acid sensing is not only implicated in virus-induced autoimmunity but also plays a central role in genetic interferonopathies. In some cases, mutations in nuclease genes, including TREX1, RNASEH2A, RNASEH2B, and RNASEH2C, impair the degradation of endogenous nucleic acids, leading to chronic activation of the IFN response.

[0113] TREX1 encodes a 3'-5' exonuclease responsible for degrading cytoplasmic DNA fragments, including viral DNA. Its activity prevents immune recognition by sensors like cGAS, thereby suppressing IFN induction. Similarly, RNASEH2A, RNASEH2B, and RNASEH2C encode components of RNAse H2, an enzyme that degrades RNA in RNA:DNA hybrids. Mutations in these genes lead to the accumulation of self-DNA and RNA fragments, mistakenly activating the immune system via cytosolic nucleic acid sensors, resulting in excessive type I IFN production and chronic inflammation.

[0114] The pathological consequences of this dysregulation are exemplified in Aicardi-Goutieres Syndrome (AGS), a severe neurological disorder caused by mutations in RNASEH2A, among other genes. AGS is characterized by progressive microcephaly, psychomotor retardation, intracranial calcifications, and elevated levels of IFN-a in cerebrospinal fluid— clinical features resembling a persistent viral infection despite the absence of an actual pathogen.

[0115] This further highlight how both viral infections and genetic mutations can drive interferonopathies through aberrant activation of nucleic acid sensing pathways, reinforcing the critical role of IFN regulation in preventing autoimmunity and autoinflammation.

[0116] Further type I interferonopathies include, but are not limited to, familial chilblain lupus (FCL); spondyloenchondrodysplasia (SPENCD) with immune dysregulation, STING-associated vasculopathy with onset in infancy (SAVI), Japanese autoinflammatory syndrome with lipodystrophy (JASL); loss of function mutations-related interferonopathies such as ubiquitinspecific peptidase 18 (USP18) deficiency and interferon-stimulated gene 15 (ISG15) deficiency; chronic atypical neutrophilic dermatitis with lipodystrophy; DNAase II deficiency; chronic atypical neutrophilic dermatosis with lipodystrophy and elevated temperature (CANDLE); Nakajo-Nishimura syndrome; X-linked reticulate pigmentary disorder; Singleton-Merten syndrome; joint contractures; muscular atrophy; microcytic anemia; panniculitis-induced lipodystrophy (JMP) syndrome; proteasome-associated autoinflammatory syndrome (PRAAS); familial systemic lupus erythematosus (FSLE), autosomal dominant immune deregulatory disease (COPA syndrome); a mitochondrial disease; non-alcoholic fatty liver disease (NAFLD); and non-alcoholic steatohepatitis (NASH).

[0117] As new mutations are being identified and the inclusion criteria for interferonopathies become more definitive, the above list of interferonopathies may change.

[0118] Drugs inhibiting IFN downstream signaling such as Janus kinase inhibitors, including baricitinib, tofacitinib and ruxolitinib, are currently used for disease control. Reverse transcriptase inhibitors are optional therapeutics for Aicardi-Goutieres syndrome (AGS). Since the discovery of the role of type I and III interferons in the pathogenesis of SLE in the 80's, only one drug was approved, anifrlumab, an antibody which targets the IFN-a / p receptor.

[0119] In accordance with any of the aspects and embodiments of the present disclosure, viral molecular mechanisms are harnessed for treating interferonopathies and diseases, syndromes, disorders and / or conditions associated with altered IFN induction, wherein combinations of several viral mechanisms provide an improved effect with reduced potential side effects.

[0120] The terms "therapy", "treatment", "treating", and "treat" are used interchangeably and refer to: (a) preventing a disease or condition from occurring in someone who may be at risk but has not yet been diagnosed; (b) stopping or slowing the progression of the disease; (c) relieving or improving symptoms of the disease; and (d) curing the disease. These terms also encompass prophylaxis (prevention) as well as treatment for existing conditions.

[0121] In this context, "prevent", "prevention", and "preventing" refer to administering the active agent to someone who has previously experienced the condition (such as an interferonopathy) but is not currently suffering from it. Thus, the terms "treatment", "therapy" and the like include, but are not limited to, changes in the recipient's status. The changes can be either subjective or objective and can relate to features such as symptoms or signs of the disease, disorder, syndrome or condition being treated. For example, if the patient notes relief in at least one symptom of an autoinflammatory disease, then successful treatment has occurred. Similarly, if the clinician notes objective changes, such as by neurologic assessment, then treatment has also been successful. Additionally, preventing further deterioration in the patient's condition is also considered part of effective treatment.

[0122] The term "therapeutically effective amount" as used herein, means the amount or dose of a compound, e.g., one or more viral components as defined herein, that, when administered to a subject for treating a disease, disorder, syndrome or condition, is sufficient to effect such treatment for the disease, disorder, syndrome or condition. The therapeutically effective amount may sometimes be the lowest dose level that yields a therapeutic benefit to patients, on average, or to a given percentage of patients. The 'therapeutically effective amount' can vary depending on the viral component, the disease and its severity, and the age, weight, etc., of the subject to be treated.

[0123] Pharmaceutical compositions and vaccines

[0124] In another aspect, the present disclosure relates to a pharmaceutical composition comprising at least one viral component as defined herein. In some embodiments, the viral component is mRNA that encodes a protein that interferes with IFN induction, production and / or activity.

[0125] In some embodiments, the pharmaceutical composition is a vaccine.

[0126] The term "vaccine," as used herein, refers to a substance designed to alter the host's immune system to help eliminate a disease and create lasting immunity. Vaccines can be both therapeutic and prophylactic. When used prophylactically, vaccines are administered to healthy individuals or those in remission to prevent a disease or its recurrence. Vaccines may include biological preparations such as peptides, antibodies, lymphocytes, or nucleic acids (e.g., mRNA, samRNA, siRNA, circRNA, miRNA), primarily aimed at preventing disease.

[0127] "Vaccination" refers to the process of introducing a vaccine into the body to provide protection against a specific disease or to prevent its recurrence. The term "immunization" is used interchangeably with vaccination and refers to the overall process of becoming protected from a disease through the administration of a vaccine.

[0128] In some embodiments, the vaccine is an mRNA vaccine.

[0129] In some embodiments, the mRNA employed for vaccination encodes a viral protein or peptide as defined herein, a fragment, derivative or analog thereof, that controls or interferes with signal transduction upstream and / or down stream of IFN induction. mRNA vaccines use mRNA's function to prompt the body to make specific proteins or peptides. For example, in COVID-19 vaccines, the viral mRNA administered to the cells promotes production of the spike protein found on the SARS-CoV-2 virus capsid. In the context of the present disclosure, vaccines based on mRNA are means of preventing diseases such as autoimmune diseases and / or interferonopathies as disclosed herein. mRNA is a fragile molecule, so it is "wrapped" in a fat-based coating to protect it. Other ingredients are added to the vaccine to keep it stable and ensure the proper action of the vaccine in the body.

[0130] A contemplated pharmaceutical composition and / or a vaccine may be administered to a subject in need thereof via one or more routes selected from an intradermal, dermal, transdermal, subcutaneous, intranodal, intrasplenic, intranasal, intratracheal, intrathecal, rectal, inhalation, intraocular, intrahepatic, oral, intrauterine, intravaginal, intracerebral, intravascular and / or intramuscular route.

[0131] In some embodiments, a contemplated vaccine may be administered by injection (parenteral administration), by inhalation, by oral or buccal administration.

[0132] The vaccine may be administered at a dose of 0.1-10 mg or 0.02-2.0 mg; at a concentration of 0.1-2 mg / ml, once a week, once every 1-2 weeks, once every 2-4 weeks, once every 1-3 months, once every 3-6 months, once every 6-12 months, or as needed (for example, upon flareup or disease aggravation).

[0133] The terms "comprise", "comprising", "includes", "including", "having" and their conjugates mean "including but not limited to".

[0134] The term "consisting of" means "including and limited to" As used herein, the singular form "a", "an" and "the" include plural references unless the context clearly dictates otherwise. For example, the term "a compound" or "at least one compound" may include a plurality of compounds, including mixtures thereof.

[0135] Whenever a numerical range is indicated herein, it is meant to include any cited numeral (fractional or integral) within the indicated range. The phrases "ranging / ranges between" a first indicate number and a second indicate number and "ranging / ranges from" a first indicate number "to" a second indicate number are used herein interchangeably and are meant to include the first and second indicated numbers and all the fractional and integral numerals therebetween.

[0136] Additional Terminologies

[0137] Interferon stimulated gene factor (ISGF3) - a heterotrimeric transcription factor complex induced by IFN types I and III, consisting of phosphorylated STAT1 and STAT2, and IRF9 . ISGF3 binds to the ISREs.

[0138] 2'3'-Cyclic GMP-AMP (cGAMP) - an intracellular second messenger that is synthesized in response to cytosolic double-stranded DNA and activates the innate immune STING pathway. Guanosine monophosphate (GMP) is a nucleotide derived from guanosine triphosphate (GTP). Adenosine monophosphate (AMP) is a derivative of adenosine triphosphate (ATP). Cyclic GMP (cGMP) and cyclic AMP (cAMP), each alone, act as second messengers, e.g., in activation of intracellular protein kinases.

[0139] Stimulator of Interferon Genes (STING) is an ER-bound protein that regulates host defense gene transcription upon detecting cytosolic DNA, such as cyclic dinucleotides (CDNs) from infections. It is expressed in various epithelial, endothelial, and immune cells. STING activation is triggered by cGAS, which synthesizes cGAMP in response to cytosolic DNA. This leads to TBKl-mediated phosphorylation of IRF3, promoting cytokine production, immune activation, and inflammation.

[0140] Interferons (IFNs) are cytokines crucial for antiviral defense, immune regulation, and inflammation. IFNs induce hundreds of IFN-stimulated genes (ISGs) to inhibit viral replication and modulate immune responses. They are classified into:

[0141] (i) Type I IFNs (IFN-a, IFN- , etc.), rapid response, produced by most cells; (ii) Type II IFN (IFN-y), regulates adaptive immunity, produced mainly by NK and T cells; and

[0142] (iii) Type III IFNs (IFN-X1-4), which act similarly to type I IFNs but target specific cells.

[0143] IFNs bind specific receptors (IFNAR1 / 2, I FN LR1 / IL10R2, IFNGR1 / 2), activating JAK-STAT signaling, which leads to STAT phosphorylation, nuclear translocation, and IFN-stimulated gene (ISG) expression. Non-canonical pathways, such as unphosphorylated ISGF3 and CrkL:pSTAT5, ensure prolonged antiviral protection, while alternative pathways (MARK, PI3K, NF-KB) fine-tune responses.

[0144] IFNs influence immune cells directly via receptor activation or indirectly by inducing chemokines, cytokines (e.g., IL-15), and antigen-presenting cell (APC) activity. They regulate monocytes, macrophages, and dendritic cells (DCs), supporting differentiation, antigen presentation, and cytokine production.

[0145] IFN induction and production

[0146] IFNs are produced in response to microbial components (viral RNA, bacterial endotoxins, CpG motifs) through pattern recognition receptors (PRRs), including cytoplasmic helicases and Toll-like receptors (TLRs). Detection of viral nucleic acids triggers signaling cascades involving ubiquitin modification, protein aggregation, and kinase activation, leading to transcription factor (TF) phosphorylation. Key phosphorylated TFs (IRF3, IRF7, NF-KB) translocate to the nucleus to drive IFN gene expression.

[0147] IFN-P (and some IFN-X subtypes) requires a multiprotein enhanceosome (a highly ordered protein complex that assembles on an enhancer region of DNA to regulate gene transcription) that remodels chromatin for gene activation. Type I and III IFNs are regulated similarly but differ in IRF isoform usage, allowing context-dependent gene expression. Negative regulation maintains low IFN levels in the absence of stimuli, involving chromatin repression and transcriptional repressors like IRF2, which competes with activators (IRF3, IRF7, I RF1, IRF5) and may degrade upon viral infection.

[0148] Type I IFN (IFN- ) induction is driven by a coordinated binding of TFs: IRF3 & IRF7 (bind PRDI / III); ATF-2 / c-Jun (AP-1) (binds PRDIV); and NF-KB (p50 / RelA) (binds PRDII).

[0149] NF-KB, IRF3, and IRF7 are critical, non-redundant regulators of IFN-P expression during viral infections. Type III IFNs (IFN-X1-3) rely on IRF and NF-KB binding sites for transcription. IFN-Xi is regulated by IRF3 and NF-KB, while I FN-X2 / 3 depend on IRF7. Some cell types exhibit independent regulation of IFN-X genes, unlike type I IFNs, allowing distinct antiviral responses.

[0150] Cytoplasmic RLR Signaling and IFN Induction

[0151] The retinoic acid-inducible gene l-like receptors (RLRs)-RIG-l, MDA5, and LGP2, act as primary sensors of RNA virus infection by detecting viral RNA structures. RIG-1 recognizes short double-stranded and 5'-phosphorylated RNA, while MDA5 detects long double-stranded RNA. LGP2 plays an auxiliary role in RNA recognition. Upon binding viral RNA, RIG-1 and MDA5 undergo a conformational change, exposing their N-terminal caspase activation and recruitment domains (CARDs), which interact with MAVS (also known as I PS-1, VISA, or Cardif) at the mitochondria.

[0152] RIG-1 activation requires Lys63-linked polyubiquitin chains, leading to the formation of MAVS aggregates on the mitochondrial membrane. These aggregates recruit TRAF2, TRAF3, and TRAF6, which act as ubiquitin ligases and are essential for activating key signaling cascades, including: MAPK -> AP-1 activation; IKK complex -> NF-KB activation; and TBKl / IKKe -> IRF3 and IRF7 activation.

[0153] The translocation of IRF3, IRF7, and NF-KB to the nucleus initiates IFN gene expression. Notably, ubiquitin modifications serve as a common regulatory mechanism for antiviral signaling.

[0154] Additional modulators fine-tune RLR signaling. One such regulator, STING, is an ER- localized transmembrane protein that plays a key role in RIG-l-mediated but not MDA5- mediated signaling, allowing diversification of signal transduction. Other helicases like DDX3 and DDX60 assist in IFN production but are also exploited by viruses for immune evasion.

[0155] Toll-like Receptor (TLR) Signaling and IFN Production

[0156] TLRs, expressed primarily in innate immune cells, serve as crucial viral sensors. In plasmacytoid dendritic cells (pDCs), which are specialized for high IFN production, TLR7 and TLR9 detect single-stranded RNA and CpG DNA, respectively. Their activation recruits MyD88, which further interacts with IRAKI, IRAK4, TRAF3, and TRAF6, leading to IRF7 phosphorylation. PIN1- dependent isomerization and IKKa may also contribute to IRF7 activation. In contrast, TLR3, which senses extracellular and endosomal double-stranded RNA, utilizes TRIF (TICAM-l) as an adapter. This pathway recruits TBK1 and IKKe, as well as NAP1 and TRAF proteins, to activate IRF3. RIP1 and TRAF6 contribute to NF-KB and AP-1 activation, although the role of TRAF6 may be cell-type dependent.

[0157] IFN Signaling and Antiviral Responses

[0158] Once type I (IFN-a / P) and type III (IFN-X) IFNs are induced, they are secreted by infected or activated cells and bind their respective receptors, initiating JAK-STAT signaling. This leads to the formation of ISGF3 (STAT1, STAT2, and IRF9), which drives the expression of interferon- stimulated genes (ISGs) responsible for antiviral, immunostimulatory, and antiproliferative effects.

[0159] Both type I and III IFNs activate ISGF3, though they differ in regulation and signaling output, influencing viral evasion strategies and host / tissue tropism. Viruses such as influenza, WNV, YFV, DENV, HSV, and hepatitis viruses actively antagonize type I IFN signaling to facilitate replication. Flaviviruses (e.g., ZIKV, JEV, WNV, DENV) are particularly adept at suppressing IFN responses in human cells, underscoring the critical role of IFN-mediated immunity in viral defense.

[0160] Various embodiments and aspects as delineated hereinabove and as claimed in the claims section below find experimental support in the following examples.

[0161] EXAMPLES

[0162] Reference is now made to the following examples, which together with the above descriptions illustrate some embodiments in a non-limiting fashion.

[0163] Material and Methods

[0164] (i) Materials

[0165] The following materials, nucleic acids and kits were used: Table 2. Materials

[0166] (ii) Cells

[0167] Human primary keratinocytes. IFNs are produced not only by infiltrating immune cells but also by resident skin cells, with increased baseline IFN production priming for inflammatory cell activation, immune response amplification, and development of skin lesions. Human primary keratinocytes were obtained from Reprocell (USA) and expanded for long-term culture using keratinocyte Growth Medium 3 (Reprocell).

[0168] RAW 264.7 cell line. A murine macrophage-like cell line widely used in immunology, inflammation, and cancer research. These cells behave similarly to primary macrophages, making them useful for studying immune responses, particularly in inflammation and infection. RAW 264.7 cells can engulf and digest pathogens or apoptotic cells, mimicking real macrophage functions. In response to lipopolysaccharide (LPS) or poly(l:C) stimulation, they produce inflammatory cytokines (e.g., TNF-a, IL-6, IFN-P) and nitric oxide (NO), playing a role in antimicrobial defense. These cells express TLR3, TLR4, TLR7, and TLR9, allowing them to detect viral RNA. Since RAW 264.7 cells are responsive to pathogen-associated molecular patterns (PAMPs), they are commonly used to study host-pathogen interactions, including viral infections like influenza and coronaviruses.

[0169] RAW 264.7 cells (purchased from ATCC) were transfected with viral mRNA for assessing transfection efficiency and effects thereof on cytokine production, particularly IFN-p.

[0170] (Hi) Receptor agonists / activators

[0171] Poly(deoxyadenylic-deoxythymidylic) acid sodium salt (poly(dA:dT). Poly(dA:dT), is a synthetic double-stranded polynucleotide composed of repeating deoxyadenylic (dA) and deoxythymidylic (dT) sequences. It mimics AT-rich regions of DNA and has immunostimulatory properties, as it is recognized as foreign DNA by intracellular immune receptors. When introduced into cells, it can activate the innate immune response, particularly through the cytosolic DNA-sensing pathway. Poly(dA:dT) is sensed by cyclic GMP-AMP synthase (cGAS) in the cytoplasm, and this leads to the production of cyclic GMP-AMP (cGAMP), which activates STING (Stimulator of Interferon Genes). STING activation triggers the production of type I interferons (IFN-a / P) and pro-inflammatory cytokines, leading to an antiviral and immune response.

[0172] Though mainly sensed in the cytoplasm, poly(dA:dT) can also interact with TLR9, a receptor that detects unmethylated CpG DNA in endosomes. This can lead to additional immune activation pathways, particularly in plasmacytoid dendritic cells.

[0173] Poly(dA:dT) does not directly bind to or activate RIG-1, but it can lead to secondary activation of RIG-1 through an indirect mechanism. RIG-1 is a cytosolic sensor that primarily detects short double-stranded RNA (dsRNA) with 5'-triphosphate ends, not DNA. However, if poly(dA:dT) can be transcribed into dsRNA by RNA polymerase III, such dsRNA contains 5'- triphosphate ends, making it a ligand for RIG-1. This indirect sensing by RIG-1 leads to the production of type I interferons. The cGAS-STING pathway is the dominant response, while RIG- I activation can be a secondary effect.

[0174] To achieve stimulation of cGAS and other cytosolic pattern recognition receptors (PRRs), Poly(dA:dT) is delivered into the cytoplasm, for example by using a transfection agent, such as LyoVec™. Polyinosinic-polycytidylic acid (poly(l:C)). Poly(l:C) is a synthetic double-stranded RNA (dsRNA) analog that mimics viral dsRNA, which is a common molecular pattern associated with viral infections. Poly( I :C) is composed of two complementary strands: polyinosinic acid (poly(l)), a strand made up of repeating inosine (I) nucleotides, and polycytidylic acid (poly(C)), a complementary strand made up of repeating cytidine (C) nucleotides. These two strands form a stable, double-stranded helical structure, mimicking the dsRNA of viruses.

[0175] Poly(l:C) functions as a pathogen-associated molecular pattern (PAMP) that activates the innate immune system by stimulating PRRs, particularly Toll-like receptor 3 (TLR3) and RIG-I-I ike receptors (RLRs).

[0176] Poly(l:C)'s receptor TLR3, is located in endosomes (mostly in dendritic cells, macrophages, and epithelial cells). Poly( I :C) is taken up into endosomes and binds to TLR3. TLR3 recruits TRIF (TIR-domain-containing adapter-inducing interferon-P), which activates IRF3 (Interferon Regulatory Factor 3) leading to type I interferon (IFN-a / P) production.

[0177] Poly(l:C) is sensed in the cytoplasm of fibroblasts, epithelial cells, and immune cells by MDA5 (Melanoma Differentiation-Associated Gene 5) preferentially and by RIG-1 (to a lesser extent). MDA5 and RIG-1 activate MAVS (Mitochondrial Antiviral Signaling Protein), which, in turn, triggers IRF3 and IRF7, leading to type I interferons (IFN-a / P) production and further leading to NF-KB activation, thereby inducing inflammatory cytokines. These pathways enhance antiviral defenses and immune activation.

[0178] Cyclic guanosine monophosphate-adenosine monophosphate (cyclic GMP-AMP, cGAMP). cGAMP is a cyclic dinucleotide (CDN) that acts as a second messenger in the innate immune system. It plays a crucial role in detecting cytosolic DNA and triggering an antiviral immune response. There are two types of cGAMP: 2'3'-cGAMP (canonical cGAMP), produced by mammals via cGAS (cyclic GMP-AMP synthase) and most common in immunity, and 3'3'-cGAMP (bacterial cGAMP), found in some bacteria as part of their own signaling pathways. cGAMP is synthesized by cGAS, a cytosolic PRR that detects double-stranded DNA (dsDNA) in the cytoplasm. When cGAS binds to DNA, it becomes enzymatically active and catalyzes the formation of cGAMP from GTP (Guanosine triphosphate) and ATP (Adenosine triphosphate). cGAMP binds to STING, a transmembrane protein located on the endoplasmic reticulum (ER). Upon binding, STING undergoes a conformational change, allowing it to Translocate to the Golgi apparatus, recruit and activate TBK1 (TANK-binding kinase 1) and phosphorylate IRF3 (Interferon Regulatory Factor 3).

[0179] Phosphorylated IRF3 translocates to the nucleus, where it induces the expression of type I Interferons and pro-inflammatory cytokines like IL-6 and TNF-a. This response helps protect the host against viral, bacterial, and tumorigenic threats. cGAMP is a critical molecule in the innate immune system that bridges DNA sensing to interferon production via the cGAS-STING pathway. It plays essential roles in antiviral defense, inflammation, and cancer immunotherapy.

[0180] G3-ended Y-form Short DNA (G3-YSD). G3-YSD is a synthetic 26-nucleotide DNA sequence designed to activate cGAS. Derived from the HIV-1 RNA genome, G3-YSD forms a Y- shaped structure due to its palindromic sequence flanked by unpaired guanosine trimers (G3). These guanosine overhangs are critical for cGAS recognition and activation. Upon binding to G3- YSD, cGAS becomes activated and catalyzes the synthesis of cyclic GMP-AMP (cGAMP) from ATP and GTP. The produced cGAMP acts as a second messenger, binding to and activating the STING which, in turn, initiates downstream signaling cascades, leading to the production of type I interferons and other cytokines (see above).

[0181] (iv) FLAG-Tag ELISA

[0182] FLAG expression of mRNA refers to the process where an mRNA molecule containing a FLAG-tag sequence is translated into a FLAG-tagged protein in a cell. The FLAG tag (DYKDDDDK) is a small peptide sequence added to the coding region of a mRNA. This sequence is engineered at either the N-terminal (beginning) or C-terminal (end) of the protein. When the mRNA is transfected into cells, it is translated into a protein that carries the FLAG tag at the designated position.

[0183] The FLAG-tagged protein can be detected and quantified using FLAG-specific antibodies in FLAG-Tag enzyme-linked immunosorbent assay (ELISA).

[0184] The kit employed in this assay utilizes a Competitive-ELISA detection method to measure FLAG-Tag concentration. The microplate included in the kit is precoated with the FLAG-Tag peptide. This serves as a reference antigen that will compete with the sample for antibody binding. In the competition step, the test sample (which contains FLAG-Tag) or known FLAG-Tag standards are added to the wells. A biotin-labeled FLAG-Tag is also added to each well. These two forms of FLAG-Tag (sample-derived vs. biotin-labeled) compete for binding sites on a FLAG- Tag-specific antibody in the well. The plate is incubated, allowing the FLAG-Tag molecules (sample-derived or biotin-labeled) to bind to the antibodies.

[0185] After incubation, unbound components are washed away, and a streptavidin-HRP (Horseradish Peroxidase) conjugate (SABC) is added. Since biotin binds strongly to streptavidin, the HRP-Streptavidin complex specifically binds to any biotin-labeled FLAG-Tag molecules that remain attached to the antibody. Excess unbound streptavidin-HRP is washed away, ensuring that only wells containing biotin-labeled FLAG-Tag retain HRP activity. If a sample contains a high concentration of FLAG-Tag, it outcompetes the biotin-labeled FLAG-Tag, meaning fewer biotin- labeled molecules remain for streptavidin-HRP binding.

[0186] Following another wash step, a 3,3',5,5'-Tetramethylbenzidine (TMB)) substrate solution is added. HRP catalyzes the TMB reaction, producing a blue color that turns yellow upon the addition of a stop solution (usually an acidic solution like sulfuric acid). The intensity of the yellow color corresponds to the amount of HRP-bound biotin-labeled FLAG-Tag. The optical density (OD) is measured at 450 nm using a microplate reader. The sample's OD450 value is compared to a standard curve generated using known FLAG-Tag concentrations. Since this is a competitive ELISA, the FLAG-Tag concentration in the sample is inversely proportional to the OD450 value: higher FLAG-Tag levels lead to lower OD readings.

[0187] This method provides precise quantification of the FLAG-tagged protein in the sample.

[0188] FLUC (Firefly Luciferase, encoded by the luc gene) is an enzyme from the firefly (Photinus pyralis) that catalyzes a bioluminescent reaction, producing light (luminescence). It is commonly used as a reporter gene in biological research. mRNA encoding FLUC is transfected into cells and serves as control in transfection experiments disclosed herein. The transfected cells express FLUC, producing luciferase enzyme. Upon addition of a substrate (D-luciferin), luciferase catalyzes the reaction, emitting light. Luminescence is detected using a luminometer, providing a quantitative readout. FLUC may also be Flag tagged and serve as control in FLAG-Tag ELISA.

[0189] (v) IFN production / expression measurement

[0190] IFN expression was quantified by human or mouse specific IFN-ai, IFN-012, or IFN-b ELISA kits (according to manufacturer's instructions. (vi) Viral mRNA

[0191] Viral ribonucleotide sequences were custom made and, optionally, modified to improve their stability and transfection efficiency.

[0192] The coding sequences for influenza A NS1 (KR700628.1); a similar sequence codon- optimized with an F9A mutation (Yu et al., 2022); influenza B NS1 (AF492479.1); Zika virus PRVABC-59 NS1 (KX377337.1) and Vaccina virus C6 protein (MT227314.1) were subcloned into the multiple cloning site of pcDNA under the control of a T7 promoter. All sequences included an in-frame 3 x FLAG C-terminal tag and were flanked by 5' _UTR (TEV) and 3' _UTR (F-l) as described (Stadler et al., 2017). The plasmid was modified to contain two stretches of 49 nucleotide poly A sequences separated by a linker. The obtained plasmid was linearized and was used as a template for in vitro transcription using the MEGAscript™ T7 Transcription Kit (Invitrogen) according to the manufacturer instructions. The RNA sequences were capped during the in vitro transcription reaction using a 7mGpppA Cap Analog (NEB).

[0193] (vii) In vitro transient cell transfection

[0194] Transient transfection involves the temporary alteration of genetic material within specific cell lines. This process enables the manipulation of gene expression without permanently modifying the host genome. The technique introduces exogenous genetic material into mammalian cells, e.g., a plasmid DNA, mRNA, miRNA or siRNA. In transient transfection, the introduced nucleic acid exists in the cell only for a limited period of time and is not integrated into the genome. As a result, transiently transfected genetic material is not passed from generation to generation during cell division, and it can be lost by environmental factors or diluted out during cell division. The high copy number of the transfected genetic material leads to substantial levels of expressed proteins within the temporary period that it exists in the cell.

[0195] One method to achieve transient transfection involves cationic polymers or lipid-based carriers; these vectors effectively deliver genetic cargo into cells.

[0196] A transient influence on gene expression was assessed by transient transfection of primary human keratinocytes by a chemical transfection method using a cationic lipid-based transfection reagent. Specifically, lyophilized lipid-based transfection reagent LyoVec™ (InVivoGen), belonging to the family of phosphonolipids was employed for in vitro transfection. The major constituent of LyoVec™ is the phosphonolipid DTCPTA, which is coupled to DiPPE, a neutral lipid that helps destabilizing membrane bilayers and increases the in vitro transfection efficiency of LyoVec™. LyoVec™ is often used as a nucleic acid complexing agent to facilitate the cellular entry of mRNAs, such as RIG-1 or STING ligands (e.g., poly(dA:dT)), and TLR7 / 8 ligands (e.g., ssPolyU5). This complexation step is crucial for inducing an effective response to the nucleic acids by intracellular pathogen recognition receptors.

[0197] (viii) Stabilization and amplification

[0198] For further stabilizing the therapeutic mRNA molecule, modified nucleotides such as mltp, m5C, 5moU are used.

[0199] (ix) Transfection confirmation

[0200] Transfection confirmation was done using fluorescent nucleic acid stains such as green fluorescent protein (GFP). Qualitative assessment of transfection efficiency was done using a fluorescent microscope.

[0201] (x) Spleen cells isolation

[0202] Spleens harvested from mice were each is placed in one well of a 24-well plate containing 0.5 ml cold PBS and kept on ice.

[0203] A 100 pm cell strainer was placed over a 1.7 ml Eppendorf tube. The spleen was crushed using the plunger end of a syringe and transferred to the tube through the cell strainer. The cells were washed through the strainer with 1 ml PBS. Then, the cells were centrifuged at 700 x g for 5 minutes at 4°C; the supernatant was discarded.

[0204] One ml Gibco™ ACK Lysing Buffer was added to cell pellet and incubated for 5 minutes at room temperature. The cell suspension was then centrifuged at 700 x g for 5 minutes at 4°C, and the supernatant was discarded. The pellet was suspended in 1 ml cold PBS and centrifuged at 700 x g for 5 minutes at 4°C (followed by discarding the supernatant). Thereafter, the pellet was resuspended in 200 pl cold PBS and cells were counted.

[0205] (xi) Cells and tissue lysis

[0206] Whole blood was collected via venipuncture into EDTA-treated collection tubes to prevent coagulation. An amount of 0.1 ml of the EDTA-treated whole blood was transferred into a tube containing 1 mL of Gibco™ ACK Lysing Buffer and left at room temperature for 3 - 5 minutes to lyse red blood cells.

[0207] The crude white blood cells (WBCs) suspension was centrifuged at 300 x g for 5 minutes at room temperature. The supernatant was carefully removed, leaving approximately 50 pL of residual liquid containing white blood cells. Cold PBS (5 mL) were added to the cell pellet and the suspension was centrifuged at 300 x g for 5 minutes at 2 - 8°C to wash the cells.

[0208] For cell Lysis, the pellet was resuspended in cell lysis buffer and incubated for 30 minutes to ensure complete lysis.

[0209] Finally, the lysate was processed by centrifuging at 10,000 x g for 10 minutes in a precooled microcentrifuge to separate cellular debris. The resulting pellet was collected and stored at -80°C for further analysis.

[0210] EXAMPLE 1

[0211] Preparation of viral mRNA for transfection

[0212] The following coding nucleotide sequences were subcloned into the multiple cloning sites of pcDNA, under the control of a T7 promoter, as described in the Materials and Methods section:

[0213] (i) influenza A NS1 (KR700628.1; SEQ ID NO:1);

[0214] (ii) a similar sequence codon-optimized with an F9A mutation (Yu et al., 2022) (SEQ. ID NO:2);

[0215] (iii) influenza B NS1 (AF492479.1; SEQ ID NO:3);

[0216] (iv) Vaccina virus C6 protein (MT227314.1; SEQ ID NO:4); and

[0217] (v) Zika virus PRVABC-59 NS1 (KX377337.1; SEQ ID NO ).

[0218] EXMPLE 2

[0219] IFN-p production in viral mRNA-transfected primary human keratinocytes

[0220] The effect of purified viral mRNA sequences, known to evade the IFN-dependent antiviral immune response, on the secretion of IFN- was assessed in human primary epidermal keratinocytes, following activation with one or more of following receptor agonist / activators: STING activator (2,3-cGAMP), RIG-1 indirect activator (poly(dA:dT)), cGAS activator (G3-YSD) and TLR3 activator (poly( l:C)). Primary human keratinocytes were isolated and sub-cultured in serum-free and bovine pituitary extract (BPE)-free Keratinocyte Growth Medium 3. The cells were seeded in 48-well plates, 5 - 7.5 x 103cells / well in 200 pL medium and incubated in a cell culture incubator.

[0221] Four days after cell seeding, the keratinocytes were transiently transfected with one of the mRNAs disclosed in Table 3, using the lyophilized lipid-based transfection reagent LyoVec™, as described in the Materials and Methods section. The nucleic acids used for in vitro transfection were in their naked form, namely, carrier-free. These mRNAs were not complexed with proteins, lipids, or other protective carriers. Furthermore, these viral mRNA are natural viral nucleic acid that are expressed / translated in infected human host but not necessarily in non-human species.

[0222] Table 3. Nucleic acid used for transient transfection of keratinocytes

[0223] The medium was changed 24 and 48 hours after transfection. Transfection efficiency was qualitatively evaluated using a fluorescent microscope. Three days following transfection, the keratinocytes were activated for a period of 16-20 h with one or more of the activators: 2,3-cGAMP, poly(dA:dT), G3-YSD and / or poly(l:C), in 0.2 mL medium (the activators are described in the Materials and Methods section). On the next day, supernatants were collected and the amount of IFN-p was quantified using the IFN- ELISA kit. The following control groups were used: (1) keratinocytes not transfected with mRNA and not activated with receptors agonists (non-transfected and non-activated); (2) keratinocytes transfected with non-relevant coding mRNA sequence (CT) or mRNA encoding GFP, and activated with each one of the receptors agonists; For the evaluation of mRNA transfection efficiency, keratinocytes were transfected with mRNA encoding for green fluorescent protein (GFP), which emits bright green fluorescence (peak at ~509 nm) when exposed to blue or ultraviolet light. The results, expressed as IFN-p expression, are depicted in Tables 4-6.

[0224] The effect of transfection with viral mRNA coding for IVA-NS1 protein on the production of IFN- in human primary keratinocytes is summarized in Table 4:

[0225] Table 4. IFN-p expression (pg / ml) in keratinocytes transfected with viral mRNA encoding for IVA-NS1 (500 ng / ml), following activation with TLR3 and cGAS agonists.

[0226] As seen in Table 4, mRNA encoding for IVA-NS1 significantly inhibited the expression of IFN-p in human primary keratinocytes activated by the TLR3 activator (poly(l:C), and cGAS activator (G3-YSD).

[0227] The effect of transfection with naked viral mRNA coding for VV-C6 protein on the expression of IFN-p in human primary keratinocytes activated with various agonists is summarized in Table 5:

[0228] Table 5. IFN-p expression (pg / ml) in keratinocytes transfected with mRNA encoding for VV-C6 (250 ng / ml) and activated with various agonists, and the expression of IFN- relative to non-transfected keratinocytes (%) As seen in Table 5, mRNA coding for the viral protein VV-C6 significantly inhibited the expression of IFN-p in keratinocytes activated by the TLR3 activator poly(l:C) and the RIG-1 indirect activator poly(dA:dT).

[0229] The effect on expression of IFN-p in human primary keratinocytes transfected with various naked mRNAs and then activated by the cGAS-STING and RIG-1 activator poly(dA:dT) is summarized in Table 6. Values are expressed as % of control (cells transfected with GFP-coding mRNA).

[0230] Table 6. The change (% reduction) in IFN-p expression in keratinocytes transfected with various viral mRNAs (50 ng / ml) and activated with poly(dA:dT) As seen in Tables 4-6, and under the experimental conditions used activation of RIG- 1 / cGAS-STING by poly(dA:dT) and TLR3 by poly(l:C) had a significant effect on IFN-p secretion by human primary keratinocytes, whereas the activators of cGAS (G3-YSD) and STING (2' 3' cGAMP) had a lesser or no effect.

[0231] Since poly(dA:dT) activated the STING pathway, via activation of cyclic GMP-AMP synthase (cGAS) in the cytoplasm, leading to the production of cyclic GMP-AMP (cGAMP), the results show that cGAS-STING pathway is highly inhibited or blocked by the viral mRNA resulting is reduced IFN- expression. G3-YSD-activated cGAS was also inhibited by a specific viral RNA (see table 5), supporting the findings that inhibiting the cGAS-STING pathway has direct and substantial influence on IFN-p production.

[0232] With respect to viral mechanism for evading the IFN-induced immunity, it is shown herein that mRNA coding for NS1 of IVA, IVB and ZIKV and protein C6 of VV reduced the secretion of IFN-p following activation of RIG-l / cGAS-STING (poly(dA:dT)) and TLR3 / RIG1-MDA5 (poly(l:C)). Furthermore, reduction in IFN-p production was sequence dependent: while a dose of 50 ng / ml of mRNA encoding for VV-C6 protein significantly reduced the secretion of IFN-p induced by RIGl / cGAS STING and TLR3 / RIG1-MDA5 activation / stimulation, viral mRNA encoding for IVB- NS1 and ZIKA-NS1 proteins provided a milder reduction, whereas mRNA encoding the IVA-NS1 had a relatively low effect.

[0233] The order of reactivity or efficiency of blocking the RIGl / cGAS-STING pathway in human keratinocytes by viral mRNAs, in response to activation with poly(dA:dT) is: VV-C6 > IVB-NS1 = ZIKA-NS1 > IVA-NS1.

[0234] EXAMPLE 3

[0235] Induction of IFN-P production in RAW 264.7 cells transfected with viral mRNA

[0236] Macrophage RAW 264.7 cells were seeded at 25,000 per well (96 well plate). On the next day, 200 ng / ml naked (lipid nanoparticle-free) flag-tagged viral mRNA was transfected, and the transfected cells were incubated for 24 h. Thereafter, the cells were stimulated for 24 h by the addition of poly(l:C) (50 pg / ml). The supernatant was collected and cells were frozen at -80°C.

[0237] For assessing transfection efficacy and INF-p production, FLAG-Tag ELISA was employed on cell lysates using FLUC as control, as described in the Materials and Methods section.

[0238] The following naked flag-tagged viral mRNA were transfected: Table 7. Flag-tagged viral mRNA transfected into RAW 264.7 cells

[0239] The amount of viral proteins detected (as quantified by FLAG-tag ELISA, in pg / ml) and amounts of INF-p (pg / ml) produced are summarized in Table 8:

[0240] Table 8. Amounts of viral proteins and INF-P in RAW 264.7 cells transfected with viral mRNA (200 mg / ml) and stimulated with poly(l:C)

[0241] These results demonstrate that transfection with the indicated viral mRNA was highly effective in inhibiting IFN-p production in response to activation of PRRs, particularly TLR3.

[0242] EXAMPLE 4

[0243] Expression of transfected viral mRNA and IFN-p in naive mice

[0244] The effects of viral mRNA transfection on viral protein (NS1 and C6) expression and IFN production were evaluated in female C57BI / 6 mice. Viral mRNA was intramuscular (IM) administered to the mice at a dose of 10 pg / mouse. The viral mRNA employed was lipid nanoparticle (LNP)-encapsulated FLAG-encoding mRNA, for brevity designated herein as "LNP- flag mRNA". LNPs are lipid-based carriers used to protect and deliver mRNA into cells, particularly for in vivo delivery. The LNPs used were Ionizable lipids in combination with a PEG shielding lipid and a helper lipid (DSPC). Particle size ranged between 100-125 nm. Four groups of 3 mice each were tested. Each group was treated with one of the following viral LNP-flag mRNA: LNP-FlaglNB3 encoding the viral protein IVB-NS1 (SED ID NO: 6); LNP- FlaglNB4 encoding the viral protein VV-C6 (SED ID NO: 8) LNP-FlaglNB5 the viral protein ZV-NS1 (SED ID NO: 7); or LNP-FLUC encoding luciferase.

[0245] Bleeding of the mice was performed prior to treatment and then again, 3 days after viral nucleic acid administration.

[0246] Three days after treatment, the mice were euthanized. White blood cells (WBCs) from whole blood, and cells from spleen and iliac lymph nodes were extracted and lysed for subsequent Flag-Tag ELISA. INF-p production was determined in plasma using IFN-p ELISA kit as described in the Materials and Methods section.

[0247] The amounts of viral proteins (pg) per 105WBCs, spleen cells and iliac lymph nodes (LN), are disclosed in Table 9.

[0248] Table 9. Amount of viral protein (pg) per 105cells measured 3 days after viral mRNA administration

[0249] Significant presence of viral proteins was found in WBCs, spleen and lymph nodes, with distribution and extent of expression differing among the mRNA sequences tested. Significant amounts of IVB-NS1 and ZV-NS1 were found in WBCs and LN. All three viral proteins were expressed in the spleen.

[0250] The amounts of IFN-p production as measured in the blood (pg / ml) prior to (baseline), and 3 days after viral mRNA administration are disclosed in Table 10. Table 10. Amounts of IFN-p production (pg / ml): baseline (re-treatment), and 3 days after viral mRNA administration

[0251] The expression of IFN- was elevated following viral mRNA administration and viral protein expression, with similar IFN-p levels measure in the control mice (FLUC) and the INB4- administered mice, followed by a milder elevation following INB5 administration, and the lower elevation seen for INB3 administration. This suggests the following immunogenicity of INB3 <<INB5<INB4 for the viral mRNAs tested.

[0252] EXAMPLE 5

[0253] Establishment of mice models of systemic lupus and psoriasis-like diseases associated with interferonopathy

[0254] (i) Systemic lupus erythematosus (SLE) model

[0255] Systemic lupus erythematosus (SLE)-like disease was induced with a single intraperitoneal (IP) injection of 0.5 mg pristane to 8-week aged C57BL6 female mice. To assess dysregulated type I interferon (IFN) response (interferonopathy) in the model, IFN-p production was quantified by ELISA analysis of blood samples taken from the mice at the day of pristane injection (day 0) and then 1, 6, 12 and 26 weeks after SLE-like disease induction. The results (average of 8 mice) are summarized in Table 11. The severity of the disease was evaluated by histopathological analysis.

[0256] Table 11. IFN-p production (pg / ml) in SLE-like disease mouse model The increase in IFN-p production over time indicates the successful establishment of the SLE mice model.

[0257] (ii) Imiquimod (IMQ)-induced psoriasis-like mouse model

[0258] The Imiquimod (IMQ)-induced psoriasis-like mouse model is a widely used experimental model for studying psoriasis, an autoimmune skin disorder characterized by keratinocyte hyperproliferation, immune activation, and chronic inflammation. It mimics key aspects of human psoriasis, particularly the I L-23 / 1 L-17 immune axis, making it a valuable tool for studying disease mechanisms and testing potential treatments.

[0259] Eight-week old female C57BI and Balb / C mice (4 mice of each strain) were shaved and depilated on their back using a Mini Clipper razor and commercially available depilatory cream (Veet®, Reckitt Benckiser Group, Slough, England) working on back skin area of 2x3 cm. The psoriasis-like disease was induced by applying 5% Imiquimod (Aldara™) cream topically to the shaved back skin of the mice. The application was repeated daily for 6 days (days 0 - 5), 62.5 mg / day, to induce psoriasis-like skin lesions.

[0260] IMQ. is a TLR7 / TLR8 agonist, meaning it triggers innate immune responses similar to viral infections. This leads to activation of plasmacytoid dendritic cells (pDCs) and keratinocytes, which release various cytokines (IL-23, TNF-a, IL-6 and IFN-y) and chemokines which recruit immune cells to the skin. The mice developed psoriasis-like lesions featuring scaly, red, thickened skin similar to human psoriasis.

[0261] The disease was evaluated by histopathological analysis. The skin showed acanthosis (epidermal thickening), parakeratosis (retained nuclei in the stratum corneum) and Infiltration of neutrophils, T cells, and dendritic cells (results not shown).

[0262] To verify the interferonopathy involvement, blood samples were taken on the day of disease induction (day 0) and then on day 1, 5 and 9 post psoriasis-like disease induction and subjected to type I IFNs analysis using dedicated mouse ELISA kits. The results (average of 8 mice) are summarized in Table 12. Table 12. Type 1 IFNs production in Imiquimod (IMQ)-induced psoriasis-like disease mice model

[0263] The significant increase in IFN-p production and, to a lesser extent, IFN-ai production, on day 1 post disease induction demonstrated substantive interferonopathy involvement. However, this effect vanished by day 5.

[0264] "Humanized" mice are mice engrafted with human plasmacytoid dendritic cells (pDCs), peripheral mononuclear cells (PBMCs) or CD34+hematopoietic stem cells (HSCs).

[0265] SLE and psoriasis disease models are established, and interferonopathy involvement is verified in these humanized mice according to the procedures described above.

[0266] EXAMPLE 6

[0267] The effect of viral mRNA on IFN-P expression in SLE mice model

[0268] The systemic lupus erythematosus (SLE)-like disease model of Example 5 was used for assessing the efficacy of vaccination against the disease with viral mRNA. C57BI female mice were injected with IP administration of 0.5 ml pristane. Six months later, blood was collected. This bleeding time point is designated herein as "day 0" or "baseline". The baseline IFN-p was quantified using mouse IFN-p ELISA.

[0269] Three groups of female C57BI / 6 mice (Groups 1-3) were treated by intramuscular administration of one or more of the following viral LNP-flag mRNAs (10 pg / mouse): LNP- FlaglNB3 encoding the viral protein IVB-NS1 (SED ID NO: 6); LNP-FlaglNB5 encoding the viral protein ZV-NS1 (SED ID NO: 7); and / or LNP-FlaglNB4 encoding the viral protein VV-C6 (SED ID NO: 8); or LNP-FLUC encoding luciferase. The treatment regimen consisted of one or two sessions of IM injection (0.1 ml / mouse) of LNP-flag mRNA, the first one on day 0, and the second 2 weeks later (i.e., day 13), as follows: Group 1 (3 mice) was treated IM with a mix of 3 types of LNP-flag mRNAs comprising 1:1:1 relative amounts of LNP-FlaglNB3, LNP-FlaglNB4, and LNP-FlaglNB5, herein designated "MIX". This group was administered with MIX only once (i.e., on "day 0");

[0270] Group 2 (3 mice) served as control group and treated IM with LNP-FLUC, herein designated "FLUC", once (on day 0); and

[0271] Group 3 (2 mice) was treated with LNP-FlaglNB3, herein designated "INB3" twice: LNP- encapsulated viral mRNA was IM injected on study day 0 and then again 2 weeks later (study day 13).

[0272] After viral mRNA injection to the mice, blood was collected at four time points: prior to treatment (Day 0), days 3 and 13, and 30 days following the first treatment, at termination of study.

[0273] The amount of IFN-p was analyzed using mouse IFN-p ELISA kit and compared to the baseline (day 0) IFN-p amount. The expression of LNP-FlagINB, namely, the amount of proteins translated therefrom, was determined in WBCs, spleen and draining lymph nodes by Flag-tag ELISA as described in the Material and Methods section.

[0274] The results are summarized in Tables 13-16.

[0275] Table 13. Relative amounts of plasma IFN- (% of baseline) at various time points following LNP-FlagINB IM administration to female C57BI / 6 mice Table 14. LNP-FlagINB protein expression (pg / ml) in WBCs at various time points following LNP-FlagINB IM administration to female C57BI / 6 mice

[0276] Flag-1 NB3 expression was maintained in WBCs for 3 days.

[0277] 5 Table 15. LNP-FlagINB protein production (pg / ml) in the spleen and iliac lymph nodes

[0278] (LN) 13 and 30 days following LNP-FlagINB IM administration to female C57BI / 6 mice

[0279] Pristane-induced SLE in C57BL / 6 female mice leads to increased levels of Type I IFNs in the blood. The present results demonstrate that mRNA of influenza B virus encoding for NS1 o protein (LNP-INB3), and a mixture of viral mRNAs encoding for NS1 proteins of 2 different viruses and C6 protein (MIX), all reduced the levels of IFN-p in the blood.

[0280] The second injection of LNP-INB3, 2 weeks after the first injection, further reduced the levels of blood IFN-p.

[0281] Overall, the effect on IFN-p secretion / production was maintained for at least 30 days 5 (MIX) and 14 days (INB3). The effect correlated with NS1 protein amounts detected in at least the spleen and LNs.

[0282] The effect of treatment on the weight of the spleen and kidneys relative to control (FLUC group) is summarized in Table 16. Table 16. The relative weight (compared to body weight, %) of the spleen and kidneys of female C57BI / 6 mice, 30 days following LNP-FlagINB IM administration

[0283] Pristane-induced SLE in C57BL / 6 female mice leads to splenomegaly as a result of immune cell activation and proliferation (especially B and T cells), myeloid cell expansion, and increased immune complex deposition. The spleen's weight increases as it becomes a central site for immune response in the context of autoimmune disease, making it a useful indicator of disease progression and immune activation in this model. As demonstrated herein, LNP-INB3 and MIX reduced spleen weight compared to untreated control (LNP-FLUC).

[0284] Pristane induced SLE in C57BL / 6 female mice typically further results in an increase in kidney weight due to the inflammatory processes such as edema, immune cell infiltration, and the accumulation of extracellular matrix proteins and subsequent renal damage associated with lupus nephritis.

[0285] LNP-INB3 and LNP-INB MIX administration reduced the kidneys weight as compared to untreated control (LNP-FLUC).

[0286] Under the experimental conditions employed it may be concluded that LNP-INBs reduce the severity of pristane-induced SLE.

[0287] EXAMPLE 7

[0288] Human IFN production in lupus disease mouse model in response to intranodal delivery of viral mRNA

[0289] Viral constructs (e.g., viral mRNA) are directly administered intranodal. For intranodal delivery of mRNA, mice are anesthetized with ketamine (70 mg / kg; Ceva) and xylazine (10 mg / kg; Bayer). The inguinal lymph node is surgically exposed and injected with 10 pg viral mRNA in a total volume of 10-15 pl.

Claims

WHAT IS CLAIMED IS:

1. A method for interfering with or controlling IFN-based immune response in a subject in need thereof, comprising administrating to the subject a therapeutically effective amount of one or more viral messenger RNAs (mRNAs) coding for proteins or peptides that interfere with one or more of I FN induction, I F N production and / or IFN activity, thereby interfering with or controlling the IFN-based immune response in the subject.

2. A method for preventing or treating a disease, disorder, syndrome or condition associated with elevated IFN levels, comprising administrating to a subject in need thereof a therapeutically effective amount of one or more viral messenger RNAs (mRNAs) coding for proteins or peptides that interfere with one or more of IFN induction, IFN production and / or IFN activity, thereby preventing or treating the disease, disorder, syndrome or condition in the subject.

3. A method for treating interferonopathy, comprising administrating to a subject in need thereof a therapeutically effective amount of one or more viral messenger RNAs (mRNAs) coding for proteins or peptides that interfere with one or more of IFN induction, IFN production and / or IFN activity, thereby treating interferonopathy in the subject.

4. The method of any one of claims 1 to 3, wherein IFN induction is initiation of IFN gene transcription, IFN production is the synthesis and secretion of IFN proteins after induction, and IFN activity is all biological effects of IFN, including antiviral and immune functions.

5. The method of claim 1 or 4, wherein the viral proteins or peptides interfere with or control signaling pathways upstream of INF induction.

6. The method of any one of claims 2 to 5, wherein the disease, disorder, syndrome or condition is cancer, a neurodegenerative disorder, an inflammatory disease, and / or an autoimmune disease, that may benefit from remedying altered levels of IFN induction, IFN production and / or IFN activity.

7. The method of claim 6, wherein the altered levels of IFN induction, production and / or activity are of type I and / or type III IFN, optionally IFN-p.

8. The method of any one of claims 3 to 5, wherein the interferonopathy is type I or type III IFN interferonopathy.

9. The method of any one of any one of claims 2 to 8, wherein the disease, disorder, syndrome, condition and / or interferonopathy is selected from: Aicardi-Goutieres syndrome (AGS); familial chilblain lupus (FCL); spondyloenchondrodysplasia (SPENCD) with immune dysregulation; stimulator of interferon genes (STING)-associated vasculopathy with onset in infancy (SAVI), Japanese autoinflammatory syndrome with lipodystrophy (JASL); ubiquitinspecific peptidase 18 (USP18) deficiency; interferon-stimulated gene 15 (ISG15) deficiency; chronic atypical neutrophilic dermatitis with lipodystrophy; DNAase II deficiency; chronic atypical neutrophilic dermatosis with lipodystrophy and elevated temperature (CANDLE); Nakajo-Nishimura syndrome; X-linked reticulate pigmentary disorder; Singleton-Merten syndrome; joint contractures, muscular atrophy, microcytic anemia; panniculitis-induced lipodystrophy (JMP) syndrome; proteasome-associated autoinflammatory syndrome (PRAAS); systemic lupus erythematosus (SLE); cutaneous lupus erythematosus (CLE), pediatric SLE (pSLE), rheumatoid arthritis (RA), psoriasis, juvenile idiopathic arthritis (JIA), psoriasis, myasthenia gravis (MG), juvenile dermatomyositis (JDM), Sjogren syndrome (SjS); systemic sclerosis (SSc), endometriosis, type I diabetes; dermatomyositis (DM); multiple sclerosis (MS); ataxia telangiectasia; amyotrophic lateral sclerosis (ALS); mendelian susceptibility to mycobacterial disease (MSMD); cytokine storm; bacteriopathy; viropathy; familial systemic lupus erythematosus (FSLE), autosomal dominant immune deregulatory disease (COPA syndrome); a mitochondrial disease; non-alcoholic fatty liver disease (NAFLD); Sjogren's syndrome; non-alcoholic steatohepatitis (NASH); cytokine storm; vasculitides; Parkinson's disease (PD); Lewy body disease (LBD); Huntington's disease (HD); Alzheimer's disease (AD); and traumatic brain injury (TBI).

10. The method of any one of claims 1 to 9, wherein the viral mRNA comprises the intact nucleic acid, a derivative thereof, an analog thereof, a biologically active fragment thereof and any combination thereof.

11. The method of any one of claims 1 to 10, wherein the viral mRNAs code for at least one viral protein selected from: a non-structural (NS) protein; papain-like protease (PLP2)- transmembrane (TM); Z protein; V protein; P protein; C6 protein; E3 protein; virion protein 35 (VP35); nucleocapsid protein (NP); GPC protein; VP24 protein; interferon regulatory factor 1, 2 or 3 (vlRFl / 2 / 3); and / or 3Cprotease protein.

12. The method of claim 11, wherein the viral protein is a non-structural (NS) protein selected from NS1, NS2, NS3, NS1B, NS2B, NS2B3, NS5, NS5A, NS2A, NS4A, NS4B, NSP1, NSP5, NSP6, NSP7, NSP8, NSP12, NSP13, NSP14, NSP15, NSP16, a biologically active fragment thereof, an analog thereof, a derivative thereof and / or any combination thereof.

13. The method of claim 11 or 12, wherein the viral proteins or peptides interfere with the function of a pattern recognition receptor (PRR).

14. The method of any one of claims 1 to 13, wherein the viral proteins or peptides affect at least one of: (i) retinoic acid-inducible gene-l-like receptor (RIG-1) sensing pathway; (ii) ISGylation of RIG-1; (iii) melanoma differentiation-associated gene 5 (MDA5) sensing pathway; (iv) ISGylation of MDA5; (v) interferon regulatory factors (IRFs), preferably, IRF3, IRF5, and IRF7; (vi) cyclic-GMP-AMP synthase (cGAS); (vii) mitochondria antiviral signaling protein (MAVS); (viii) stimulator of IFN genes (STING); (ix) TANK-binding kinase 1 (TBK1) and / or IKB kinase-e (IKKe); (x) NF-KB; and / or (xi) nlrp3 (xii) STATs.

15. The method of any one of claims 1 to 14, wherein the one or more viral mRNAs are administered to the subject by at least one route selected from the group consisting of intradermal, dermal, transdermal, subcutaneous, intranodal, intrasplenic, intranasal, intratracheal, intrathecal, rectal, inhalation, intraocular, intrahepatic, oral, intrauterine, intravaginal, intracerebral, intravascular and / or intramuscular route.

16. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and one or more viral mRNAs coding for proteins or peptides that interfere with interfere with IFN induction, IFN production and / or IFN activity.

17. A vaccine comprising one or more viral mRNAs coding for proteins or peptides that interfere with IFN induction, INF production and / or IFN activity.

18. The pharmaceutical composition of claim 16 or the vaccine of claim 17, wherein proteins or peptides interfere with signal transduction upstream and / or downstream of IFN induction, preferably, signal transduction upstream of IFN induction, optionally, IFN- induction.

19. The pharmaceutical composition or the vaccine of claim 18, wherein the protein or peptide are as defined in any one of claims 11 to 14.

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