Modified and non-modified long non-coding RNA and methods of use thereof

Abstract

Described herein are long non-coding (lncRNA), DNAs and vectors that encode them, nanoparticle complexes and pharmaceutical compositions comprising the lncRNA. The lncRNA can be encapsulated by a lipid nanoparticle, optionally where the lncRNA lacks a cap and / or tail and wherein the lncRNA comprises one or more lncRNA. Described herein are also methods of treating inflammatory diseases or diseases or conditions associated with a deficiency of a lncRNA, methods of altering expression of one or more cytokine expression in a subject using said long non-coding (lncRNA), DNAs and vectors that encode them, nanoparticle complexes and pharmaceutical compositions.

Description

MODIFIED AND NON-MODIFIED LONG NON-CODING RNA AND METHODS OF USE THEREOF RELATED APPLICATION

[0001] This application claims the benefit of priority to Canadian Patent Application Number 3268456, filed March 20, 2025 and U.S. Provisional Application No. 64 / 001,020, filed March 9, 2026, the contents of each of which is incorporated herein by reference in their entirety.INCORPORTATION OF SEQUENCE LISTING

[0002] A computer readable form ofthe Sequence Listing “P9324914WO01 / 93719423_Sequence Listing” (196,425 bytes) created on March 20. 2026, is herein incorporated by reference.FIELD

[0003] The present disclosure relates to compositions and uses of modified and unmodified long non-coding RNAs (IncRNA) and in particular to compositions and uses thereof, for delivery of IncRNA complexes such as nanoparticle IncRNA complexes.BACKGROUND

[0004] Nucleic acid applications have almost exclusively focused on the delivery of messenger RNA (mRNA), small interfering RNA (siRNA), microRNA (miRNA), and single stranded guide RNA (sgRNA) (FIG. 1A). These RNAs have been well studied, and chemical modifications, which include nucleoside modifications, 5’ caps and 3’ tails, are well known to increase their potency and stability (1-2). Conversely, minimal focus has been given to long non -coding RNAs (IncRNA). LncRNAs are a class of nucleic acids involved in gene regulation (3). Over 18,805 IncRNAs catalogued (4).

[0005] LncRNAs play a role in cell and system functions, such as the innate immune response. For example, the macrophage specific gastric adenocarcinoma predictive long intergenic noncoding RNA (GAPLINC) is downregulated during activation of inflammation (5). Macrophages depleted of GAPLINC showed increased basal expression of inflammatory genes at baseline, and overexpression of this IncRNA suppressed this response (5). Similarly, macrophage inflammation-suppressing transcript (MIST) overexpression decreased basal and lipopolysaccharide (LPS)-induced expression of proinflammatory response genes in macrophages(d). MIST serves as a protective IncRNA that perturbs inflammatory phenotype of macrophages(d). Furthermore, diabetes regulated anti-inflammatory RNA (DRAIR) overexpression in monocytes increased anti-inflammatory genes while inhibiting proinflammatory genes(7).

[0006] Sepsis is currently a significant challenge in intensive care units as it frequently results in multi-organ dysfunction and high morbidity6. This infection-induced systemic inflammatory disorder is estimated to lead to mortality rates of approximately 50% (7). The current clinical treatment strategies for systemic inflammatory diseases are predominantly non-RNA biologies and small molecule drugs (8-11).

[0007] There is limited data on the types of chemical modifications that can impact IncRNA performance. The IncRNAs discovered are mRNA-like IncRNAs that exhibit 5’- capping and 3’ poly(A) tails18 19. See also Table 1.1 of Long Non-Coding RNA Biology, Editor MRS Rao Publisher Springer Singapore (https: / / doi.org / 10.1007 / 978-981-10-5203-3.

[0008] N6- methyladenosine (m6A) and 5 -methyl -cytosine (m5C) are two types of commonly found modifications20. M6A is the most abundant modification found in RNA (21). M6A modification can alter RNA structure or recruitment of specific m6A- binding proteins (20, 21). The addition of a methyl group to the N6- position of adenosine does not disrupt Watson-Crick base pairing but does weaken RNA secondary structure and affect RNA tertiary structure20. M5C modification can affect the fate ofthe modified RNA molecules through affecting RNA stability and transcriptional regulation (22). Pseudouridine (T) modification is also present in IncRNA. The function of has been found to improve translation of mRNA (23); however, the role of this modification for IncRNA remains unknown.

[0009] New modes of targeting disease relevant gene expression levels of for example multiple targets including undruggable targets are desirable.SUMMARY

[0010] Long non-coding ribonucleic acid (IncRNA) molecules make up a significant portion of the transcriptome. LncRNA molecules are bioactive and control cell and system function without protein translation. Normally regarded as a diagnostic marker or drug target, IncRNAs have not been previously explored as a therapeutic. Here, it is established that IncRNA can be used as a new modality to treat diseases. As examples, four IncRNA from the transcriptome were re-engineered — GAPLINC, MIST, DRAIR and PARAIL — were successfully used to treat acute inflammation in lipopolysaccharide -challenged animals. Lor each IncRNA, in vitro transcription synthesis and high-performance liquid chromatography purification was employed. As demonstrated herein, IncRNA were tested with various, 5’-cap, 3’ poly(A) tail and chemical base modifications ( -, m I - and m5C) . IncRNAs which chemically modified sugar backbones, a modification that is generally not useful for mRNA-based applications, were also tested. Using GAPLINC as a model IncRNA, it was demonstrated that 2’OMe-UTP-modified GAPLINC decreased the gene expression of the key inflammatory protein, Illb, in primary human monocytes from donors. Moreover, this 2’ OMe-UTP -modified IncRNA exhibits comparable functional performance as 'P-modified GAPLINCin lipopolysaccharide (LPS) -induced inflammation in primary human monocytes. Additionally, IncRNA was delivered in vivo using a lipid nanoparticle system. Using this pipeline and delivery platform, it is demonstrated that each of the tested IncRNA controls inflammation for example by specifically regulating different cytokines. It is also demonstrated herein that GAPLINC reduced LPS induced inflammation in human monocytes. LncRNAs and particularly stable modified IncRNAs, may be useful for treating diseases associated with mutated IncRNAs or decreased IncRNA expressionThis approach and these findings establish this as a nucleic acid modality and demonstrates an effective way to re-engineer regulatory IncRNA molecules from the transcriptome for the treatment of disease.

[0011] 5 -methyl -cytosine (m5C) is a commonly found RNA modification that can influence RNA stability and transcriptional rcgulation(2 / ). In IncRNAs, m5C can augment hydrophobicity of base pairs, promote base stacking, and stabilize base pair interaction^ / ). Pseudouridine (T) modification is another base modification of RNA. The function of has been found to improve translation of mRNA(22); however, the role and utility of this modification for IncRNA remained unknown until the present.

[0012] Cytokine Release Syndromes include systemic inflammatory conditions such as sepsis. Sepsis is currently a health challenge frequently resulting in multi -organ dysfunction and high morbidity (A). The current clinical treatment strategies for systemic inflammatory diseases are predominantly non-RNA biologies and small molecule drugs (9-12).

[0013] As demonstrated herein, lipid nanoparticles (LNP) can be used for the localized delivery of IncRNAs and treatment of inflammation including sepsis.

[0014] LNPs consist of biocompatible and minimally immunogenic lipids that facilitate delivery across cell membranes, reducing dosage required for effective treatment] / 3). These nanocarriers have been used for example for mRNA loading, controlled and sustained mRNA release, extended stability and lifetime, and targeted delivery (14-17). By administering LNPs encapsulating for example antiinflammatory IncRNAs as in the LPS-challenged inflammation model described in the Examples, the efficacy of different IncRNAs on the treatment of acute systemic inflammation was demonstrated.

[0015] LncRNAs (e.g. modified or non-modified), DNAs encoding IncRNAs, nucleic acid molecules comprising IncRNAs, can also be used to increase expression of IncRNAs that are deficient or absent in a cell or a subject, for example when the naturally occurring IncRNA is truncated, particularly when the IncRNA deficiency or absence is deleterious and causes disease. Examples are truncated IncRNAs associated with disease are provided in Table 6A and deleted IncRNAs associated with disease in Table 6B.

[0016] LncRNAs (e.g. modified or non -modified) can also be used to treat diseases associated with a deficiency (or absence) of a IncRNA.

[0017] Localized delivery of IncRNAs can be achieved using for example LNPs as shown herein. To mimic the course of human sepsis, bacterial component, lipopolysaccharide (LPS), can be exogenously administered to mice17. This leads to the rapid development of systemic inflammatory response, resulting in high levels of proinflammatory cytokines which can be measured in the circulating serum17. By administering LNPs encapsulating GAPLINC in the LPS-induced inflammation model, the efficacy of IncRNA treatment was investigated during the acute phase of systemic inflammation.

[0018] To further expand the therapeutic utility of IncRNA, chemical modifications of this RNA were investigated.

[0019] The space of chemical modifications was investigated for enhancing IncRNA stability and performance and demonstrate the use of IncRNA in vivo for the treatment of for example acute and / or systemic inflammation (FIG. 1A). It was predicted that administering lipid nanoparticles (LNPs) carrying GAPLINC RNA to an LPS-induced inflammation mouse model would lower the production of proinflammatory cytokines. A pipeline was developed to synthesize pure mature forms of unmodified and modified GAPLINC and formulate IncRNA LNPs. Results revealed that 'P-modified GAPLINC RNA significantly decreased IL- 1 [3 production in vivo.

[0020] Accordingly, an aspect of the disclosure includes a long non-coding (IncRNA)-nanoparticle complex comprising one or more IncRNA molecules and a lipid nanoparticle, and the complex or IncRNA comprises single stranded molecules at a purity of at least 99%.

[0021] An aspect of the disclosure includes a complex comprising a long non-coding (IncRNA) or a DNA encoding a IncRNA and a cell delivery agent, wherein the IncRNA lacks a 5’ cap and / or a 3 ’poly A tail, and wherein the complex or IncRNA comprises single stranded molecules at a purity of at least 99%, wherein the IncRNA comprises one or more different IncRNAs.

[0022] Another aspect includes a pharmaceutical composition comprising a complex comprising a long non-coding (IncRNA), a DNA encoding a IncRNA and / or a nucleic acid molecule comprising a IncRNA and a cell delivery agent wherein the IncRNA lacks a 5’ cap and / or a 3 ’poly A tail, and wherein the composition, complex, nucleic acid molecule or IncRNA comprises single stranded molecules at a purity of at least 99%, wherein the IncRNA comprises one or more different IncRNAs.

[0023] In an embodiment, the IncRNA comprises two or more different IncRNAs. For example, the two or more different IncRNAs may comprise two or more different molecules, wherein the difference is different chemical modifications, or presence of absence of a cap or tail of the same IncRNA (e.g.,GAPLINK comprising 2’OMe-UTP chemical modification and GAPLINK with 5 -methyl -cytosine (m5C) modification) and / or wherein the difference is different IncRNAs (e.g. GAPLINK and MIST).

[0024] The IncRNA is in some embodiments, the IncRNA is HPLC purified. In some embodiments, the IncRNA is denatured prior to complexing with the delivery agent.

[0025] In another embodiment, the length of one or more of the IncRNA is at least or about 200 nucleotides, at least or about 400 nucleotides, at least or about 500 nucleotides, at least or about 1000 nucleotides; at least or about 1500 nucleotides, at least or about to 2000 nucleotides or for example up to 3000 nucleotides. For example, the length of the IncRNA can be the active fragment of a IncRNA. The active fragment can be determined in one or more functional assays, including for example in a screening assay as described herein. As another example, the length of the IncRNA can be at least 80% of the naturally occurring IncRNA length. As a further example, the IncRNA can be comprised in a nucleic acid molecule which comprise additional sequence, for example up to 120% or 125% of the naturally occurring IncRNA length.

[0026] In another embodiment, the IncRNA, , DNA encoding the IncRNA or nucleic acid molecule comprises the IncRNA comprises or is or encodes GAPLINC, DRAIR, PARAIL, MIST, MALAT1, NEAT1, HOTAIR, ANRIL, NORAD, PVT1, SNHG1, SNHG3, SNHG5, SNHG7, SNHG12, SNHG14, SNHG15, DNM3OS, CHASERR, H19, MEG3, FENDRR, SOX2OT, LINC00237, LINC-PINT, UCA1, GAS5, MIR17HG, PANDA, LINC00968 and / or LINC01021.

[0027] In another embodiment, the IncRNA, nucleic acid molecule, or DNA of the complex or pharmaceutical composition comprises or is selected from the nucleotide sequence of any one of SEQ ID NOs: 1, 5, 6, 10, 14, 18, 25, 26, or 31-64, or a sequence with at least 80% or at least 90%, or at least 95% sequence identity to any thereof. In an embodiment, the IncRNA, nucleic acid molecule comprises or is selected from 10, 14, 18, 26, or 31-64, or the DNA encoding the IncRNA is selected from SEQ ID NO: 1, 5, 6 and 25 or a sequence with at least 80% or at least 90%, or at least 95% sequence identity to any thereof.

[0028] In an embodiment, the IncRNA is a modified IncRNA.

[0029] In some embodiments, the lincRNA is selected using a screening assay described herein.

[0030] In another embodiment, the IncRNA of the complex, or the pharmaceutical composition comprises one or more chemically modified nucleotides.

[0031] In another embodiment, the chemical modified nucleotides of the IncRNA of the complex, or pharmaceutical composition, are:N1 -methylpseudouridine (m IT) modification,pseudouridine modification (T).5 -methyl -cytosine (m5C) modification,N6-Methyl-adenosine-5'-triphosphate (N6-Me-ATP),2'-fluoro-2'-deoxyuridine-5'-triphosphate (2'-F-dUTP),2'-fluoro-2'-deoxycytidine-5'-triphosphate (2'-F-dCTP),2'-fluoro-2'-deoxyguanosine-5'-triphosphate (2'-F-dGTP),2'-fluoro-2'-Deoxyadenosine-5'-triphosphate (2'-F-dATP),2'-O-methyl-uridine-5'-triphosphate (2'-O-Me-UTP),2'-O-methyl-adenosine-5'-triphosphate (2'-O-Me-ATP),2'-O-methyl-guanosine-5'-triphosphate (2'-O-Me-GTP),2'-O-methyl-cytidine-5'-triphosphate (2'-O-Me-CTP),2'-O-4'-C-Locked-guanosine-triphosphate (2'-O-4'-C-Locked-GTP),2'-O-4'-C-Locked-adenosine-5'-triphosphate (2'-O-4'-C-Locked-ATP), and / or2'-O-4'-C-Locked-5-methyl-cytidine-triphosphate (2'-O-4'-C-Locked-5-Me-CTP).

[0032] In another embodiment, the chemical modification is a base modification.

[0033] In an embodiment, the base modification comprises N 1 -methylpseudouridine (mlT) modification, pseudouridine modification (T). 5 -methyl -cytosine (m5C) modification, and / or N6-Methyl-adenosine-5'-triphosphate (N6-Me-ATP).

[0034] In another embodiment, the chemical modification is a sugar backbone modification.

[0035] In an embodiment, the sugar backbone modification is a 2’0me modification. For example, the sugar modification that may be employed include for example2'-fluoro-2'-deoxyuridine-5'-triphosphate (2'-F-dUTP),2'-fIuoro-2'-deoxycytidine-5'-triphosphate (2'-F-dCTP),2'-fIuoro-2'-deoxyguanosine-5'-triphosphate (2'-F-dGTP),2'-fIuoro-2'-Deoxyadenosine-5'-triphosphate (2'-F-dATP),2'-O-methyl-uridine-5'-triphosphate (2'-O-Me-UTP),2'-O-methyl-adenosine-5'-triphosphate (2'-O-Me-ATP),2'-O-methyl-guanosine-5'-triphosphate (2'-O-Me-GTP),2'-O-methyl-cytidine-5'-triphosphate (2'-O-Me-CTP),2'-O-4'-C-Locked-guanosine-triphosphate (2'-O-4'-C-Locked-GTP),2'-O-4'-C-Locked-adenosine-5'-triphosphate (2'-O-4'-C-Locked-ATP), and / or2'-O-4'-C-Locked-5-methyl-cytidine-triphosphate (2'-O-4'-C-Locked-5-Me-CTP).

[0036] In an embodiment, the sugar backbone modification is selected from 2’0-Me-UTP, 2'-O-Me-ATP, 2'-O-Me-CTP and / or 2'-O-Me-GTP.

[0037] In another embodiment, the IncRNA of the complex or pharmaceutical composition comprises about or at least 10%, about or at least 20%, about or at least 30%, about or at least 40% or about or at least 50% of one or more of A, U, G or C nucleotides replaced by the chemically modified nucleotides, optionally wherein about or at least 50% of A nucleotides are replaced, wherein about or at least 50% of U nucleotides are replaced, wherein about or at least 50% of G nucleotides are replaced, and / or wherein about or at least 50% of C nucleotides are replaced, or optionally wherein 100% of A nucleotides are replaced, wherein 100% of U nucleotides are replaced, wherein 100% of G nucleotides are replaced, and / or wherein 100% of C nucleotides are replaced.

[0038] LncRNAs with one or more chemically modified nucleotides (e.g. with one or more of the same residue modified e.g. one or more cytosine modified residues and / or with one or more different residues modified), can be tested to confirm they do not activate the inflammasome (e.g., do not activate for example Toll-like receptors, or for example do not activate IRF).

[0039] In some embodiments, the IncRNA has similar or decreased inflammasome activity compared to the unmodified molecule.

[0040] In some embodiments, the IncRNA comprising one or more chemically modified residues (e.g. modified IncRNA) comprises similar or greater activity compared to the unmodified IncRNA.

[0041] In another embodiment, the cell delivery agent of the complex or pharmaceutical composition is a lipid nanoparticle, and / or the DNA is comprised in a vector optionally an expression construct lacking sequences for a 5 ’cap and / or polyA tail.

[0042] The lipid of the lipid nanoparticle can for example be ionizable lipid, zwitterionic lipid or cationic lipid depending on the lipid nanoparticle.

[0043] In embodiments using DNA, the cell delivery agent can be a viral vector capable of infecting cells or other delivery agent, for example solid nanoparticles, such as anionic solid nanoparticles, optionally pegylated, in addition to liposomes or other lipid nanoparticles. For example, viral vectors like adenovirus, modified vaccinia ankara (MV A), and lentivirus can be used.

[0044] In another embodiment, the lipid nanoparticle of the complex or pharmaceutical composition is lipofectamine or comprises an ionizable lipid, optionally further comprises one or more ofcholesterol, phosphatidyl choline, optionally l,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), and / or lipid-PEG, optionally DMG-PEG2000.

[0045] In another embodiment, the ionizable lipid of the complex or pharmaceutical composition comprises one or more molecules shown in Fig. 13A or 131 (OC2-K3-E10, C3-K2-E14, SM-102, ALC-0315, Dlin-MC3-DMA (MC3), [3N2 and / or 503), poly beta amino acids (or other polymers) and / or comprises a tertiary amine.

[0046] In another embodiment, the ratio of ionizable lipid, cholesterol, DSPC, and DMG-PEG2000 in the complex or pharmaceutical composition is about 50:38.5: 10: 1.5 mol %.

[0047] In another embodiment, wherein the IncRNA or DNA and the lipid nanoparticle form the complex, the size of the complex is between about 140 nm to about 200 nm or less than 200 nm.

[0048] In another embodiment, the polydispersity of the complex is below about 0.2.

[0049] In another embodiment, the complex can have an overall positive charge, neutral charge or negative charge, for example in water, the complex overall charge can negative, neutral or positive, or about neutral. In some embodiments, the complex overall charge in water is approximately neutral.

[0050] In another embodiment, the complex comprises a mass ratio of at least 20: 1 of lipid to IncRNA.

[0051] In another embodiment, the complex comprises a mass ratio between about 7:1 to about 20:1 of lipid to IncRNA. The ratio can be any ratio or range between about 7:1 to about 20:1 of lipid to IncRNA.

[0052] In another embodiment, the complex comprises a mass ratio of about 20: 1 of lipid to IncRNA. The lipid comprises for example, ionizable lipid or other lipid that can be used in a nanoparticle.

[0053] It is shown herein that the IncRNAs tested inhibited various cytokines and inflammatory mediators for example IL-ip, IFN-y, IL-6 and / or TNF-a. The IncRNA can also for example be a IncRNA that inhibits or activates the same inflammatory mediators, in response to, for example, LPS, as GAPLINC, DRAIR, PARAIL and / or MIST. In addition, truncated IncRNAs have been identified which can cause disturbances or disease. Examples are truncated and absent IncRNAs associated with disease are provided in Table 6 A and 6B. In some embodiments, the IncRNA is or comprises a IncRNA in Table 6 A or B.

[0054] In another embodiment, the IncRNA or DNA of the complex or pharmaceutical composition or nucleic acid molecule, etc is or comprises or encodes GAPLINC, DRAIR, PARAIL, MIST, MALAT1, NEAT1, HOTAIR, ANRIL, NORAD, PVT1, SNHG1, SNHG3, SNHG5, SNHG7, SNHG12,SNHG14, SNHG15, DNM30S, CHASERR, H19, MEG3, FENDRR, S0X20T, LINC00237, LINC-PINT, UCA1, GAS5, MIR17HG, PANDA, LINC00968 and / or LINC0102L

[0055] In another embodiment, the complex or pharmaceutical composition further comprises optionally one or more pharmaceutically acceptable carrier and / or excipient.

[0056] In another embodiment, the complex or pharmaceutical composition, or the IncRNA or DNA, nucleic acid molecule etc as defined therein is used in the treatment diseases and conditions for example of an acute inflammatory disease or condition or a flare up of a chronic inflammatory disease or condition. In an embodiment, the disease or condition is an autoimmune disease. In an embodiment, the disease or condition is rheumatoid arthritis or osteoarthritis.

[0057] In another embodiment, the complex or pharmaceutical composition, or the IncRNA or DNA, nucleic acid molecule etc as defined therein is used in the treatment of a disease or condition associated with a deficiency of a IncRNA. For example, the IncRNA may have reduced expression, reduced function or be completely absent.

[0058] In another embodiment, the complex or pharmaceutical composition, or the IncRNA, nucleic acid molecule or DNA is prepared in a medicament. In some embodiments, the medicament is for the treatment of an acute inflammatory disease or condition or flare ups of chronic inflammatory disease or conditions. In other embodiments, the medicament is for the treatment of a disease or condition associated with a deficiency of a IncRNA

[0059] For example, the IncRNA, complexes, nucleic acid molecules etc can be used for the treatment of conditions and diseases that involve elevated levels of cytokines or inflammatory mediators such as IL-1[3, IFN-y, IL-6 and / or TNF-a and / or decreased levels of anti-inflammatory cytokines such as IL-10.

[0060] The acute inflammatory disease or flare up of a chronic inflammatory disease or condition can involve a cytokine storm and / or be a cytokine release syndrome (CRS) or a vascular disease. For example, the acute inflammatory disease or flare up of a chronic inflammatory disease or condition can be any such condition that involves elevated levels of IL-ip, IFN-y, IL-6 and / or TNF-a and / or decreased levels ofIL-10.

[0061] For example, in graft versus host disease, a cytokine storm involving IL-6, II- 1 and TNFa is induced. Accordingly, in one embodiment, the acutely inflammatory condition is graft versus host disease.

[0062] In another embodiment, the vascular disease is or comprises atherosclerosis or atherothrombosis or the CRS is sepsis.

[0063] In another embodiment, the disease or condition associated with a deficiency of a IncRNA, optionally where the IncRNA that is deficient is selected from Table 6A and / or 6B.

[0064] In another embodiment, the use is in a subject wherein the subject is a human.

[0065] An aspect of the present disclosure includes a nucleic acid molecule comprising or consisting of a long non -coding (IncRNA) wherein the IncRNA lacks a 5’ cap and / or a 3 ’poly A tail or a DNA molecule encoding said IncRNA, optionally for use in preparing encapsulated IncRNA.

[0066] In an embodiment, the nucleic acid molecule or the IncRNA of the nucleic acid molecule is chemically modified (e.g. comprises chemically modified.

[0067] The nucleic acid molecule is for example single stranded RNA, which lacks a 5 ’ cap and / or a 3 ’poly A tail and comprises one or more chemically modified nucleotides.

[0068] In an embodiment, the nucleic acid molecule comprises a modified IncRNA comprising one or more chemically modified nucleotides,wherein the IncRNA is GAPLINC, DRAIR, MIST, PARAIL, MALAT1, NEAT1, HOTAIR, ANRIL, NORAD, PVT1, SNHG1, SNHG3, SNHG5, SNHG7, SNHG12, SNHG14, SNHG15, DNM3OS, CHASERR, H19, MEG3, FENDRR, SOX2OT, LINC00237, LINC-PINT, UCA1, GAS5, MIR17HG, PANDA, LINC00968 or LINC01021;wherein the IncRNA lacks a 5’ cap and / or a 3’ poly-A tail; andwherein the one or more chemically modified nucleotides comprises a base modification and / or a sugar modification selected from:N1 -methylpseudouridine (m IT) modification,pseudouridine modification (T).5 -methyl -cytosine (m5C) modification,N6-Methyl-adenosine-5'-triphosphate (N6-Me-ATP),2'-fhroro-2'-deoxyuridine-5'-triphosphate (2'-F-dUTP),2'-fluoro-2'-deoxycytidine-5'-triphosphate (2'-F-dCTP),2'-fluoro-2'-deoxyguanosine-5'-triphosphate (2'-F-dGTP),2'-fluoro-2'-Deoxyadenosine-5'-triphosphate (2'-F-dATP),2'-O-methyl-uridine-5'-triphosphate (2'-0-Me-UTP),2'-O-methyl-adenosine-5'-triphosphate (2'-0-Me-ATP),2'-O-methyl-guanosine-5'-triphosphate (2'-0-Me-GTP),2'-O-methyl-cytidine-5'-triphosphate (2'-0-Me-CTP),2'-O-4'-C-Locked-guanosine-triphosphate (2'-O-4'-C-Locked-GTP),2'-O-4'-C-Locked-adenosine-5'-triphosphate (2'-O-4'-C-Locked-ATP), and / or2'-O-4'-C-Locked-5-methyl-cytidine-triphosphate (2'-O-4'-C-Locked-5-Me-CTP).

[0069] An aspect of the present disclosure includes a vector construct comprising a vector backbone (e.g. vector) and the DNA described herein (e.g., that encodes the IncRNA), wherein the vector construct lacks sequence for encoding a 5’ cap and / or a 3 ’poly A tail.

[0070] In an embodiment, the vector is an expression plasmid or a self-amplified RNA / replicon, optionally for use in an in vitro translation system.

[0071] The vector can be a viral vector such as an adenoviral vector or lentiviral vector.

[0072] In another embodiment, the self-amplified RNA / replicon is or comprises 5 ’ -RdRp-GOI-3 ’ where RdRp is a polymerase for self-replication and the GOI is a gene of interest (e.g. IncRNA of interest).

[0073] An aspect of the present disclosure includes a kit comprising a complex or pharmaceutical composition, a nucleic acid molecule, a vector or vector construct described herein and one or more chemically modified residues, and optionally one or more components for in vitro translation or cell delivery agents.

[0074] In an embodiment, the kit includes a cell delivery agent which is a lipid nanoparticle.

[0075] Another aspect includes a method of increasing a level of a deficient IncRNA in a cell or subject, the method comprising administering the IncRNA, nucleic acid, DNA pharmaceutical composition or a complex described herein.

[0076] In some embodiments, the method is for the treatment of a disease or condition, optionally an acute inflammatory disease or a flare up of a chronic disease or condition or a disease or condition associated with a deficiency of a IncRNA. An aspect of the present disclosure includes a method of treating a disease or condition. In an embodiment, the disease or condition is an acute inflammatory disease or flare up of a chronic inflammatory disease or condition. In an embodiment, the disease or condition is a diseaseor condition associated with a deficiency of a IncRNA. In an embodiment, the method comprises administering a complex or pharmaceutical composition, a nucleic acid molecule, or a vector to a subject in need thereof.

[0077] The inflammatory disease can for example be acute and / or systemic.

[0078] In an embodiment, the acute inflammatory disease is a systemic inflammation, optionally a cytokine release syndrome (CRS).

[0079] In an embodiment, the inflammatory disease is sepsis or graft versus host disease.

[0080] In an embodiment, the disease or condition is an autoimmune disease. For example, the autoimmune disease is rheumatoid arthritis. In an embodiment, the disease or condition is osteoarthritis. Autoimmune diseases, and diseases such as osteoarthritis, increase inflammation, which can exacerbates damage. Anti-inflammatory IncRNAs as demonstrated herein can be used to decrease inflammation.

[0081] In an embodiment, the disease or condition associated with a deficiency of a IncRNA.

[0082] An aspect of the present disclosure includes a method of altering expression of one or more cytokine, optionally proinflammatory cytokine expression in a subject, the method comprising administering a complex, a nucleic acid molecule, a composition or a vector construct described herein to a subject.

[0083] In an embodiment, the one or more cytokine comprises IL-ip, TNFa, IFN-y and / or IL-6.

[0084] In another embodiment, the altering comprises decreasing expression.

[0085] In another embodiment, the one or more cytokine comprises IL-10.

[0086] In another embodiment, the altering comprises increasing IL-10.

[0087] Another aspect of the disclosure includes a method of treating an inflammatory disease, the method comprising administering the compositions, nucleic acid molecules, vector constructs or complexes described herein to a subject in need thereof.

[0088] Another aspect of the disclosure includes a method of altering expression of one or more cytokine, optionally a proinflammatory cytokine, expression in a subject, the method comprising administering the compositions, nucleic acid molecules, vector constructs or complexes described herein to a subject.

[0089] In an embodiment, the administering comprises intravenous administration or subcutaneous administration. In another embodiment, the administering comprises local delivery. Thepharmaceutical compositions, complexes, IncRNAs vector constructs and / or nucleic acid molecules may be formulated for intravenous administration, subcutaneous administration or local delivery. For example, the pharmaceutical compositions, complexes, vector constructs, IncRNAs and / or nucleic acid molecules may be comprised in a sterile vial or syringe.

[0090] Uses of the compositions, nucleic acid molecules, vector constructs, kits and / or complexes described herein and the methods described herein are also contemplated. In some embodiments, the compositions, nucleic acid molecules, vector constructs or complexes described herein are used in the manufacture of a medicament. For example, the medicament can be to treat a disease described herein.

[0091] The preceding section is provided by way of example only and is not intended to be limiting on the scope of the present disclosure and appended claims. Additional objects and advantages associated with the compositions and methods of the present disclosure will be appreciated by one of ordinary skill in the art in light of the instant claims, description, and examples. For example, the various aspects and embodiments of the disclosure may be utilized in numerous combinations, all of which are expressly contemplated by the present description. These additional advantages objects and embodiments are expressly included within the scope of the present disclosure. The publications and other materials used herein to illuminate the background of the disclosure, and in particular cases, to provide additional details respecting the practice, are incorporated by reference, and for convenience are listed in the appended reference section.BRIEF DESCRIPTION OF THE DRAWINGS

[0092] Further objects, features and advantages of the disclosure will become apparent from the following detailed description taken in conjunction with the accompanying figures showing illustrative embodiments of the disclosure, in which:

[0093] FIGs. 1A-1H show LncRNA encapsulated into LNPs can be deployed in vitro to control cell phenotype in an exemplary embodiment of the disclosure. FIG. 1A shows the workflow to design, synthesize, produce and functionally test IncRNA-based therapeutics. FIG. IB shows bioanalyzer analysis of GAPLINC in vitro transcription products reveal a major band at the expected 500 nucleotides (denoted by arrow). FIG. 1C shows the structures of chemical base modifications used to modify IncRNAs. FIG. ID shows LNPs with no cap and tail GAPLINC exhibit smaller sizes than other GAPLINC LNPs. FIG. IE shows PDI of the GAPLINC LNPs is not significantly different from each other. FIG. IF shows encapsulation efficiency varies between the GAPLINC LNPs. FIG. 1G shows the functional testing method of GAPLINC and payload designs. LNP characterization data represent three independent measurements. FIG. 1H shows treatment of RAW-Dual cells with LPS (200 ng / mL) and LNPs containing GAPLINCwithout a cap or tail (200 ng) showed the least NF-KB activation compared to other modifications (n=3). Error bars shown are mean ± SEM. * P < 0.05, *** P < 0.001 and **** P < 0.0001.

[0094] FIGs. 2A-2F show exogenously delivered IncRNA attenuates LPS-induced inflammation in vitro and is sensitive to chemical modifications in an exemplary embodiment of the disclosure. FIG. 2A shows NF-KB activation in RAW-Dual cells after 18 h stimulation with 200 ng / mL LPS and 200 ng of modified or unmodified GAPLINC in lipofectamine. Unmodified and modified GAPLINC attenuates NF-KB activation. FIG. 2B shows IRF activity in RAW-Dual cells after 18 h stimulation with 200 ng unmodified or modified GAPLINC in lipofectamine. The IRF induction of each GAPLINC payload tested is expressed relative to the untreated control. 'P-modified GAPLINC showed minimal IRF activity compared to other base modifications tested. FIG. 2C shows that with the same assay conditions as the GAPLINC RNA in vitro assay, m I - and m5C-modified MIST decreases NF-KB activation relative to LPS control. FIG. 2D shows -modificd MIST showed minimal IRF activity compared to other base modifications tested. FIG. 2E shows unmodified and modified DRAIR decreased NF-KB activation compared to LPS control. FIG. 2F shows - and m IT-modified DRAIR showed minimal IRF activity compared to other base modifications tested. Data shown represent results from n = 3 to 5 technical replicates in three to five independent experiments. Error bars shown are mean ± SEM. * P < 0.05, *** P < 0.001 and **** P < 0.0001.

[0095] FIGs 3A-3M show exogenous GAPLINC LNPs downregulates proinflammatory cytokines in vivo in an exemplary embodiment of the disclosure. FIG. 3 A shows mouse serum collected from C57BL / 6 mice treated with or without 1.0 mg / kg unmodified or modified GAPLINC LNPs and 100 pg LPS for 6 h. Luminex was performed to analyze mouse cytokines, chemokines and growth factor expression. FIG. 3B shows -modificd GAPLINC demonstrated the most significant decrease in IL-1J3 levels in serum relative to the other unmodified and modified GAPLINC payloads. FIG. 3C shows IFN-y expression decreased most significantly with -modificd GAPLINC relative to unmodified GAPLINC. FIG. 3D shows unmodified, m IT- and m5C-modified GAPLINC downregulated TNFa levels. FIG. 3E shows m IT-modificd GAPLINC decreased IL-6 expression. FIG. 3F shows m IT-modificd GAPLINC increased IL-10 expression. FIG. 3G shows formulation optimization of GAPLINC LNPs. -modificd GAPLINC LNPs formulated with 7:1 or 20:1 nanomaterial (e.g., ionizable lipids to RNA mass ratio) to RNA mass ratio showed no significant differences in size, PDI and zeta potential. FIG. 3H shows a formulation with 20: 1 nanomaterial to RNA mass ratio that increased T-modificd GAPLINC encapsulation efficiency in LNP. FIG. 31 shows a 20: 1 formulation that showed significantly more downregulation of IL-1[3 expression compared to the 7: 1 formulation at 0.10 mg / kg. FIG. 3J shows a treatment of C57BL / 6 mice challenged with 100 pg LPS using a low 0.10 mg / kg dose of -modified GAPLINC LNPs (20:1)significantly downregulated IL-1J3 at 6 h compared to LPS control. This effect diminished by 10 h. For nanoparticle characterizations, a total of three DLS measurements were completed for each LNP: ; FIGs.3K-M show quantification of Illb expression in RAW-Dual cells treated with LPS (100 ng / ml) and 'P-modified GAPLINC or Illb siRNA in Lipofectamine (Lipo) as indicated for 3 hours (FIG. 3K), 6 hours (FIG. 3L), or 21 hours (FIG. 3M): FIG 3N shows Illb expression in human PBMC-derived monocytes stimulated with LPS and -modificd GAPLINC in Lipofectamine as indicated at 6 hours; FIG3O shows quantification of IL- ip in C57BL / 6 mice 6 hours after treatment with LPS and 7:1 or 20: 1 GAPLINC LNP formulation at 0.10 mg / kg; FIG. 3P shows IL- 1 in C57BL / 6 mice 6, 10, and 24 hours after challenge with 100 pg of LPS using a low dose (0.10 mg / kg) of 'P-modificd GAPLINC LNPs (20:1). FIG 3Q shows IL-ip in C57BL / 6 mice 6 hours after treatment with LPS with or without 'P-modificd GAPLINC LNPs or LNPs loaded with an irrelevant IncRNA. All in vivo data represent results from n = 4 to 8 biological replicates, and all data points are shown on the graphs. In vitro data represent n = 3 biological replicates with three technical replicates. For FIGs B to F and O to Q, IncRNA was delivered with LNPs made with the OC2-K3-E10 ionizable lipid. For FIGs K to N, RNA was delivered with Lipofectamine. N.D. denotes no detection. Error bars shown are the means ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 by a one-way ANOVA with Tukey’s test.

[0096] FIGs 4A-4I show exogenous MIST LNPs downregulates TNFa in vivo in an exemplary embodiment of the disclosure. Figs. 4A-4C show size, PDI and zeta potential were similar between the LNPs formulated with 20:1 nanomaterial to RNA mass ratio and encapsulating different MIST payloads FIG. 4D shows encapsulation efficiency of LNPs containing unmodified or modified MIST RNA were all above 80%. FIG. 4E shows mouse serum that was collected from C57BL / 6 mice treated with or without 0.10 mg / kg unmodified or modified MIST LNPs and 100 pg LPS for 6 h. Luminex was used to analyze mouse cytokines, chemokines and growth factor expression. MIST RNA did not change IL- 1 [3 expression. FIG. 4F shows ml'P-modified MIST upregulated the least IFN-y compared to the other modifications tested. FIG. 4G shows 'P-modified MIST showed the most decrease in TNFa levels relative to the other unmodified and modified MIST payloads tested. FIG. 4H shows unmodified MIST showed the greatest decrease in IL-6 expression compared to the modified MIST RNAs. FIG. 41 shows ml'P-modified MIST increased IL-10 expression. FIGS. 4J-L show quantification of Tnf expression RAW-Dual cells treated with LPS (100 ng / ml) and 'P-modified MIST or Tnf siRNA in Lipofectamine (Lipo) as indicated at 3 hours (FIG. 4J), 6 hours (FIG. 4K) and 21 hours (FIG.4L). In vivo data represent results from n = 4 to 8 biological replicates. In vitro data represent n = 3 biological replicates with three technical replicates. All data points are shown. For (B) to (D), IncRNA was delivered with LNPs made with the OC2-K3-E10 ionizable lipid. For (J) to (L), RNA was delivered with Lipofectamine. Error bars shown are the means ± SD. *P < 0.05,**P < 0.01, and ****P < 0.0001 by a one-way ANOVA with Tukey’s test. A total of three DLS measurements were completed for each LNP shown..

[0097] FIGs. 5A-5I show exogenous DRAIR LNPs downregulates TNFa and IL-6 in vivo in an exemplary embodiment of the disclosure. FIGs. 5A-5C show size, PDI and zeta potential were similar between the LNPs formulated with 20:1 nanomaterial to RNA mass ratio and encapsulating different DRAIR payloads. FIG. 5D shows encapsulation efficiency of LNPs containing unmodified or modified DRAIR RNA were all above 60%. FIGs 5E-5F show mouse serum that was collected from C57BL / 6 mice treated with or without 0.10 mg / kg unmodified or modified DRAIR LNPs and 100 pg LPS for 6 h. Luminex was used to analyze mouse cytokines, chemokines and growth factor expression. DRAIR RNA did not change IL-1J3 or IFN-y expression. FIGs. 5G-5H show unmodified and -, mlT- and m5C-modified DRAIR all decreased TNFa and IL-6 expression. FIG. 51 shows -modificd DRAIR increased IL- 10 expression. FIGs 5J-L show quantification of 116 expression in RAW-Dual cells treated with LPS (100 ng / ml) and -modificd DRAIR or 116 siRNA in Lipofectamine (Lipo) as indicated at 3 hours (F), 6 hours (G), and 21 hours (H) . In vivo data represent results from n = 4 to 8 biological replicates with all data points shown. In vitro data represent n = 3 biological replicates with three technical replicates, and all data points are shown. For FIGs B to E, IncRNA was delivered with LNPs made with the OC2-K3-E10 ionizable lipid. For FIG J to L, RNA was delivered with Lipofectamine. Error bars shown are the means ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 by a one-way ANOVA with Tukey’s test. For nanoparticle characterization, a total of three DLS measurements were completed for each LNP shown.

[0098] FIGs. 6A-6C show cellulose chromatography removes dsRNA from unmodified and modified GAPLINC RNA in an exemplary embodiment of the disclosure. FIG. 6A shows cellulose purification method for the removal of dsRNA from IVT-synthesized IncRNA. FIG. 6B shows unmodified and ml'P-containing GAPLINC RNA were synthesized and purified using cellulose chromatography. Dot blot (200 ng / dot) analysis using J2 dsRNA-specific mAb reveals a decrease in the intensity of the dot following purification, suggesting the removal of dsRNA in samples. Synthetic double stranded RNA, poly (I:C), was used as a positive control. (-) denotes non-cellulose purified sample. (+) denotes cellulose-purified sample. FIG. 6C shows bioanalyzer analysis of cellulose -purified unmodified and ml'P-modified GAPLINC RNA shows a band around 500 nucleotides (red arrow) corresponding to the size expected for GAPLINC. Additional bands with varying lengths remain following purification.

[0099] FIGs. 7A-7F show reversed-phase HPLC purification of unmodified and modified GAPLINC RNA at 70°C allows for the selection of a single species of IncRNA in an exemplary embodiment of the disclosure . FIG. 7A shows HPLC purification workflow to obtain single species of IVT-synthesized IncRNA with minimal dsRNA. FIG. 7B shows bioanalyzer analysis of the reversed-phaseHPLC purified product (peak 1 elution) show a single band at 500 nucleotides corresponding to the unmodified GAPLINC RNA. FIG. 7C shows unmodified GAPLINC RNA purified at 45°C. Dot blot (200 ng / dot) analysis using J2 dsRNA-specific mAb reveals peak 1 elution fractions show minimal dsRNA compared to peak 2 elution fractions. FIG. 7D shows optimization of reversed-phase HPLC purification of -modificd GAPLINC RNA using different temperatures. Increasing temperature led to fewer peaks which was likely attributed to diminished secondary structure. FIG. 7E shows bioanalyzer of T-modified GAPLINC RNA HPLC purified using different temperatures revealed a single band around 500 nucleotides corresponding to GAPLINC RNA. Increasing purification temperature allowed for better separation of RNA impurities, as additional distinct bands were observed in peak 2 fractions collected in the higher purification temperatures. FIG. 7F shows 'P-modified GAPLINC RNA purified at 45, 60 and 70°C showed minimal presence of dsRNA observed for peak 1 elution fractions relative to peak 2 for all temperatures tested. 70°C was selected to be the best temperature for HPLC purification of the IncRNA produced well-defined peaks, good fraction separation and minimal dsRNA. (-) denotes non-HPLC purified sample. Synthetic double stranded RNA, poly (I:C), was used as a positive control. Red arrow denotes band with expected size for GAPLINC RNA.

[0100] FIGs. 8A-8B show reversed-phase HPLC purification of modified GAPLINC RNA at 70°C allows for the selection of a single species of IncRNA in an exemplary embodiment of the disclosure. FIG. 8A shows bioanalyzer analysis of all the HPLC -purified modified GAPLINC RNA revealed a single band at 500 nucleotides for all peak 1 elution fractions corresponding to the size expected for GAPLINC. FIG. 8B shows dot blot (200 ng / dot) analysis using J2 dsRNA-specific mAb reveals a decrease in dot intensity following HPLC, confirming the removal of dsRNA in samples. Synthetic double stranded RNA, poly (I:C), was used as a positive control. (-) denotes non-HPLC purified sample. Red arrow denotes band with expected size for GAPLINC RNA.

[0101] FIGs. 9A-9E show reversed-phase HPLC traces for unmodified and modified GAPLINC in an exemplary embodiment of the disclosure. A total of 100-500 pg of unmodified, modified or scrambled GAPLINC RNA was purified by reversed-phase (RP)-HPLC. LncRNA was eluted using a linear gradient of buffer B (0.1 M TEAA, 25% acetonitrile, pH 7.0). The gradient spanned 38-65% buffer B over 6-8 column volumes (black line). Absorbance at 260 nm was used to detect RNA species (grey line). Fractions from the two major peaks, denoted as peak 1 and 2, were collected separately, excluding RNA eluted before and after the major peaks.

[0102] FIGs. 10A-10D show reversed-phase HPLC traces for unmodified and modified MIST RNA in an exemplary embodiment of the disclosure. FIGs. 10A-10D show a total of 100-500 pg of unmodified or modified MIST RNA was purified by RP-HPLC in an exemplary embodiment of thedisclosure. IncRNA was eluted using a linear gradient of buffer B (0.1 M TEAA, 25% acetonitrile, pH 7.0). The gradient spanned 38-65% buffer B over 6-8 column volumes (black line). Absorbance at 260 nm was used to detect RNA species (grey line). Fractions from the major peaks, denoted as peak 1 and 2, were collected separately.

[0103] FIGs. 11A-1 ID show reversed-phase HPLC traces for unmodified and modified DRAIR RNA in an exemplary embodiment of the disclosure. A total of 100-500 pg of unmodified or modified DRAIR RNA was purified by RP-HPLC. IncRNA was eluted using a linear gradient of buffer B (0.1 M TEAA, 25% acetonitrile, pH 7.0). The gradient spanned 38-65% buffer B over 6-8 column volumes (grey line). Absorbance at 260 nm was used to detect RNA species (black line). Fractions from the single peak denoted as peak 1 were collected.

[0104] FIGs. 12A-12D show reversed-phase HPLC purification allows forthe selection of a single species of unmodified and modified MIST and DRAIR RNA in an exemplary embodiment of the disclosure. FIG. 12A shows bioanalyzer analysis of all the HPLC-purified unmodified and modified MIST RNA revealed a single band at 1473 nucleotides for all peak 1 elution fractions corresponding to the size expected for MIST. FIG. 12B shows dot blot (200 ng / dot) analysis using J2 dsRNA-specific mAb reveals a decrease in dot intensity following HPLC, confirming the removal of dsRNA in samples. FIG. 12C shows bioanalyzer analysis of all the HPLC-purified unmodified and modified DRAIR RNA revealed a single band at 1432 nucleotides for purified samples corresponding to the size expected for DRAIR. FIG. 12D shows dot blot (200 ng / dot) analysis using J2 dsRNA-specific mAb reveals a decrease in dot intensity following HPLC, confirming the removal of dsRNA in samples. Synthetic double stranded RNA, poly (I:C), was used as a positive control. (-) denotes non-HPLC purified sample. Red arrow denotes band with expected size for the IncRNA.

[0105] FIGs. 13A-13B show ionizable lipids used for delivery of IncRNAs in an exemplary embodiment of the disclosure. FIG. 13A shows the structure of C3-K2-E14 that was used for in vitro GAPLINC RNA delivery experiments. FIG. 13B shows the structure of OC2-K3-E10 that was used for in vivo delivery of GAPLINC RNA delivery experiments. FIGs. 13C-13I show additional ionizable lipids that may be used for delivery of IncRNAs. FIG. 13C shows the structure of C3-K2-E10. FIG. 13D shows the structure of C3-K3-E10. FIG. 13E shows the structure of SM-102. FIG. 13F shows the structure of ALC-0315. FIG. 13G shows the structure of Dlin-MC3-DMA (MC3). FIG. 13H shows the structure of [3N2. FIG.131 shows the structure of 503. FIG. 13J shows co-localization of LNPs formulated with OC2-K3-E10 encapsulating firefly luciferase mRNA in the spleen and liver as revealed by in vivo imaging. C57BL / 6 mice were injected intravenously with 200 pL of PBS (n = 4) or 200 pL of LNP containing 2 pg of firefly luciferase mRNA (n = 3). Data shown is mean ± S.D.

[0106] FIGs. 14A-14D shows unmodified and modified GAPLINC encapsulated in lipofectamine did not induce NF-KB activation and GAPLINC-202 and 205 isoforms did not affect NF-KB and IRF activity. FIG. 14A shows NF-KB activation in RAW-Dual cells after 18 h stimulation with 200 ng / mL LPS and 200 ng of modified or unmodified GAPLINC in lipofectamine. Delivery of modified or unmodified GAPLINC only did not upregulate NF-KB activation. FIG. 14B shows a schematic of GAPLINC isoforms with varying lengths tested. FIG. 14C shows RAW-Dual cells were treated with 200 ng / mL LPS and 200 ng of unmodified GAPLINC-202 or 205 in lipofectamine for 18 h. NF-KB and IRF activity in RAW-Dual cells treated with GAPLINC isoform and LPS did not significantly differ from LPS. The IRF induction of each GAPLINC payload tested is expressed relative to the untreated control. Data shown represent results from n = 3 technical replicates in three independent experiments. Error bars shown are mean ± SEM. * P < 0.05, *** P < 0.001 and **** P < 0.0001.

[0107] FIGs. 15 A- 15D show GAPLINC RNA can be encapsulated into LNPs with different sizes, charges and encapsulation efficiencies. FIGs. 15A-15C show LNPs formulated with 7:1 nanomaterial to RNA mass ratio and encapsulating HPLC-purified scrambled, unmodified or modified GAPLINC RNA have varying sizes, PDI, and zeta potential. FIG. 15D shows the encapsulation efficiency for the LNPs containing GAPLINC with different base modifications. Error bars shown are mean ± SD. A total of three different measurements were completed for each LNP shown.

[0108] FIGs. 16A and 16B show LNPs with unmodified and modified GAPLINC RNA payloads exhibit similar characteristics as determined by DLS and ribogreen assay. FIG. 16A shows similar size and PDI of LNPs formulated with 7:1 nanomaterial to RNA mass ratio and encapsulating unmodified or -modified GAPLINC RNA purified with cellulose or HPLC. FIG. 16B shows similar encapsulation efficiencies for LNPs containing unmodified or 'P-modified GAPLINC RNA purified with cellulose or HPLC. Error bars shown are mean ± SD. A total of three different measurements were completed for each LNP shown.

[0109] FIGs. 17A-17F show GAPLINC LNPs significantly downregulates key inflammatory protein, IL-1J3, in vivo at 1 mg / kg dose and delivery material show minimal stimulation of inflammation in vivo. For FIGs. 17A-17C, mouse serum was collected from C57BL / 6 mice treated simultaneously with or without 1, 3 or 5 mg / kg unmodified GAPLINC LNPs and 100 pg LPS. The unmodified GAPLINC payload was purified using cellulose method. 1 mg / kg LNP significantly downregulated IL- 1 [3 expression. However, IFN-y expression was increased at 1 mg / kg, suggesting payload optimization is required to reduce immunogenicity. LNPs were formulated with 7:1 nanomaterial to RNA mass ratio. For FIGs. 17D-17F, mouse serum was collected from C57BL / 6 mice treated with or without empty LNPs. The amount of delivery material tested was equivalent to the amount of delivery material contained in each LNP dosetested. Delivery material induced minimal inflammation. All heatmaps contain results from Luminex assays quantifying for 10 Mouse cytokines, chemokines and growth factors. Data shown represent results from n = 4 to 12 biological replicates. Error bars shown are mean ± SD. * P < 0.05 and ** P < 0.01.

[0110] FIGs. 18A-18E show the effect of GAPLINC LNPs on expression of cytokines, chemokines and growth factors in vivo. For FIGs. 18A-18E, mouse serum was collected from C57BL / 6 mice treated simultaneously with or without 1.0 mg / kg unmodified or modified GAPLINC LNPs and 100 pg LPS. Expression of GM-CSF, IL-2, IL-12p70, MCP-1 and IL-4, in serum, respectively. LNPs were formulated with 7:1 nanomaterial to RNA mass ratio. Data shown represent results from n = 4 to 8 biological replicates. Error bars shown are mean ± SD. * P < 0.05, ** P < 0.01 and **** P < 0.0001.

[0111] FIGs. 19A-19C show body and organ weights and tissue morphology of -modificd GAPLINC LNP-treated mice are similar to PBS-treated mice in an exemplary embodiment of the disclosure. FIG. 19A shows C57BL / 6 mice were treated with two doses of LNPs formulated with 20:1 nanomaterial to RNA mass ratio and containing -modificd GAPLINC (0.10 mg / kg) on day 1 and 7. Relative body weight changes were similar between PBS-treated and LNP-treated animals during the course of treatment. FIG. 19B shows weights of spleen, kidney and liver for PBS-treated and LNP-treated animals were also similar after 8 days of treatment. FIG. 19C shows hematoxylin and eosin (H&E) stain of tissue slices from the spleen, liver and kidney showed no differences in tissue morphology between PBS-treated and LNP-treated animals following 8 days of treatment. Data shown is mean ± S.D and represents results from n = 4 biological replicates for each treatment group. Black scale bars denote 50 pm. Images are representative of the PBS and LNP groups.

[0112] FIGs. 20A-20E. show the effect of MIST LNPs on expression of cytokines, chemokines and growth factors in vivo in an exemplary embodiment of the disclosure. Mouse serum was collected from C57BL / 6 mice treated simultaneously with or without 0.10 mg / kg unmodified or modified MIST LNPs and 100 pg LPS. FIGs.20A-20E show expression of GM-CSF, IL-2, IL-12p70, MCP-1 and IL-4, in serum, respectively. LNPs were formulated with 20:1 nanomaterial to RNA mass ratio. Data shown represent results from n = 4 to 8 biological replicates. Error bars shown are mean ± SD.

[0113] FIGs. 21A-21E show the effect of DRAIR LNPs on expression of cytokines, chemokines and growth factors in vivo in an exemplary embodiment of the disclosure. Mouse serum was collected from C57BL / 6 mice treated simultaneously with or without 0.10 mg / kg unmodified or modified MIST LNPs and 100 pg LPS. FIGs. 21A-21E show expression of GM-CSF, IL-2, IL-12p70, MCP-1 and IL-4, in serum, respectively. LNPs were formulated with 20:1 nanomaterial to RNA mass ratio. Data shown represent results from n = 4 to 8 biological replicates. Error bars shown are mean ± SD.

[0114] FIG. 22 is an alignment of GAPLINC and isoforms 202 and 205.

[0115] FIG. 23 A shows treatment of RAW-Dual cells with LPS (200ng / mL) and LNPs containing PARAIL without a cap or tail (200 ng). FIG. 23B shows IRF activity in RAW-Dual cells after 18 h stimulation with 200 ng unmodified or modified PARAIL.

[0116] FIG. 24 shows sugar-modified GAPLINC can be synthesized using IVT. Bioanalyzer analysis of GAPLINC IVT products synthesized with 2’OMe-UTP shows a major band at 4000 nucleotides, nt, nucleotide.

[0117] FIGs. 25A-C show sugar-modified GAPLINC reduces gene expression of key inflammatory protein in human PBMC-derived monocytes. Quantification oilllb expression in RAW-Dual cells treated with LPS (100 ng / ml), T-modificd GAPLINC and 2’OMe-UTP-modified GAPLINC in Lipofectamine (Lipo) as indicated at (A) 3 hours, (B) 6 hours, and (C) 24 hours.

[0118] FIG. 26 shows reversed-phase HPLC traces for 'P-modified GAPLINC. A total of 400 pg of 'P-modified GAPLINC RNA was purified by reversed-phase (RP)-HPLC. LncRNA was eluted using a linear gradient of buffer B (0.1 M TEAA, 25% acetonitrile, pH 7.0). The gradient spanned 50-65% buffer B over 4.4 column volumes (light grey line). Absorbance at 260 nm was used to detect RNA species (dark grey line). Fractions from the major peak (23.778 min) was collected, excluding RNA eluted before and after the major peaks.

[0119] FIG. 27 shows 'P-modified GAPLINC retains anti-inflammatory activity for 3-6 h. C57BL / 6 mice were injected with 'P-modified GAPLINC LNPs intravenously (0.1 mg / kg) and subsequently challenged with lipopolysaccharide (LPS) intraperitoneally (1 mg / kg) at 0, 3, 6 h post-LNP injection. IL-ip expression was significantly reduced during LPS challenge at 0 and 3 h post-LNP treatment compared to LPS control. .

[0120] FIG. 28 shows T-modified GAPLINC can be delivered using subcutaneous route of administration. C57BL / 6 mice were injected with lipopolysaccharide (LPS) intraperitoneally (5 mg / kg) and T-modified GAPLINC LNPs intravenously or subcutaneously (0.1 mg / kg).DETAILED DESCRIPTION OF VARIOUS EMBODIMENTS

[0121] The following is a detailed description provided to aid those skilled in the art in practicing the present disclosure. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. The terminology used in the description herein is for describing particular embodiments only and is notintended to be limiting of the disclosure. All publications, patent applications, patents, figures and other references mentioned herein are expressly incorporated by reference in their entirety.

[0122] The following non-limiting examples are illustrative of the present application:I. Definitions

[0123] As used herein, the following terms may have meanings ascribed to them below, unless specified otherwise. However, it should be understood that other meanings that are known or understood by those having ordinary skill in the art are also possible, and within the scope of the present disclosure. In the case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

[0124] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range is encompassed within the description. Ranges from any lower limit to any upper limit are contemplated. The upper and lower limits of these smaller ranges which may independently be included in the smaller ranges is also encompassed within the description, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either both of those included limits are also included in the description.

[0125] The term “tail” or “polyA tail” as used herein interchangeably in the specification and in the claims, should be understood to mean a “3 ’polyadenylation (polyA) tail. A “3 ’-polyA tail” refers to a long chain of adenine nucleotides added to the 3 ’-end of a transcription product which provides stability to the RNA molecule and can promote translation.

[0126] The term “cap” or “5’ cap” as used herein in the specification and in the claims, should be understood to mean a guanine nucleoside that is joined via its 5 ’carbon to a triphosphate group.

[0127] The term “cytokine release syndrome” (CRS) as used herein in the specification and in the claims, should be understood to mean a group of clinical syndromes involving activation and dissolution of lymphocytes and release of a large amount of cytokines triggered by infections or therapies with monoclonal antibodies or cytokines. CRS is a type of acute systemic inflammatory disease or condition.

[0128] The term “nucleic acid molecule”, as used herein means two or more covalently linked nucleotides. Unless the context clearly indicates otherwise, the term generally includes, but is not limited to, deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) as well as combinations thereof, which may be single-stranded (ss) or double stranded (ds). The nucleic acids can be any length depending upon the application, for example from 300 bp to 2kb or longer. They can include one or more modified nucleotides.Sequences may be provided using residue nomenclature for DNA or residue nomenclature for RNA. In is to be understood that the sequence in the alternate nucleic acid type is also contemplated. For example, where a DNA sequence is provided, the corresponding RNA is also contemplated herein as the sequence is predictable to a person of skill in the art.

[0129] The term “promoter” or “promoter sequence” generally refers to a regulatory DNA sequence capable of being bound by an RNA polymerase to initiate transcription of a downstream (i.e. 3’) sequence to generate an RNA. Suitable promoters may be derived from any organism and may be bound or recognized by any RNA polymerase. Exemplary promoters include, but are not limited to, a SP6 promoter, a T7 promoter, and a T3 promoter.

[0130] The term “cell-free system” as used herein means a set of reagents that are necessary and sufficient to carry out a specified in vitro biochemical reaction or process e.g. a translation reaction. A cell free system fortranslation can be referred to as an “in vitro translation system”.

[0131] The term “about”, “substantially” and “approximately” as used herein may be used to take into account experimental error and variations that would be expected by a person having ordinary skill in the art. For example, “about” may mean plus or minus 10%, or plus or minus 5%, of the indicated value to which reference is being made. Substantially may for example mean at lest 90%, at least 95% or at least 99%.

[0132] The term “similar” as used herein in the context of a compound inducing an activity, e.g. induction of the inflammasome, refers to a compound that has less than 20% increase compared to the comparator molecule in a same assay.

[0133] As used herein the singular forms "a", "an", and "the" include plural references unless the context clearly dictates otherwise.

[0134] The phrase "and / or" as used herein in the specification and in the claims, should be understood to mean "either or both" of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Multiple elements listed with "and / or" should be construed in the same fashion, i.e., "one or more" of the elements so conjoined. Other elements may optionally be present other than the elements specifically identified by the "and / or" clause, whether related or unrelated to those elements specifically identified.

[0135] As used herein, the phrase "at least one," in reference to a list of one or more elements, should be understood to mean at least one element selected from anyone or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements. This definitionalso allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase "at least one" refers, whether related or unrelated to those elements specifically identified.

[0136] The recitation of numerical ranges by endpoints herein includes all numbers and fractions subsumed within that range (e.g., 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.90, 4, and 5). It is also to be understood that all numbers and fractions thereof are presumed to be modified by the term "about."

[0137] The term "subject" as used herein includes all members of the animal kingdom including mammals, and suitably refers to humans.

[0138] The term "treating" or “treatment” as used herein and as is well understood in the art, means an approach for obtaining beneficial or desired results, including clinical results. Beneficial or desired clinical results can include, but are not limited to, alleviation or amelioration of one or more symptoms or conditions, diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration of the disease state, diminishment of the reoccurrence of disease, and remission (whether partial or total).

[0139] The term "sequence identity" as used herein refers to the percentage of sequence identity between sequences, for example two nucleic acid sequences. To determine the percent identity of two nucleic acid sequences or of two amino acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the sequence of a first amino acid or nucleic acid sequence for optimal alignment with a second amino acid or nucleic acid sequence). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity=number of identical overlapping positions / total number of positions.times.100%). In one embodiment, the two sequences are the same length. The determination of percent identity between two sequences can also be accomplished using a mathematical algorithm. A preferred, non-limiting example of a mathematical algorithm utilized for the comparison of two sequences is the algorithm of Karlin and Altschul, 1990, Proc. Natl. Acad. Sci. U.S.A. 87:2264-2268, modified as in Karlin and Altschul, 1993, Proc. Natl. Acad. Sci. U.S.A. 90:5873-5877. Such an algorithm is incorporated into the NBUAST and XBUAST programs of Altschul et al., 1990, J. Mol. Biol. 215:403. BUAST nucleotide searches can be performed with the NBUAST nucleotide program parameters set, e.g., for score=100, word length=12 to obtain nucleotide sequences homologous to a nucleic acid molecules of the present application. BUAST protein searches can be performed with the XBUAST program parameters set, e.g., to score-50, wordlength=3 to obtain amino acid sequences homologous to a protein molecule described herein. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al., 1997, Nucleic Acids Res. 25:3389-3402. Alternatively, PSI-BLAST can be used to perform an iterated search which detects distant relationships between molecules (Id.). When utilizing BLAST, Gapped BLAST, and PSI-Blast programs, the default parameters of the respective programs (e.g., of XBLAST and NBLAST) can be used (see, e.g., the NCBI website). Another preferred non-limiting example of a mathematical algorithm utilized for the comparison of sequences is the algorithm of Myers and Miller, 1988, CABIOS 4:11-17. Such an algorithm is incorporated in the ALIGN program (version 2.0) which is part of the GCG sequence alignment software package. When utilizing the ALIGN program for comparing amino acid sequences, a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used. The percent identity between two sequences can be determined using techniques similar to those described above, with or without allowing gaps. In calculating percent identity, typically only exact matches are counted.

[0140] The term “IncRNA” as used herein means a class of RNA molecules, typically longer than 200 nucleotides, that do not code for proteins. Naturally occurring IncRNA comprise 5' capping and polyadenylation tails and can function as regulators of gene expression. For example, a IncRNA is a single molecule of a single stranded RNA molecule. The IncRNA can comprise secondary structure and can be human IncRNA, or active fragments thereof. The IncRNAs can be comprised in nucleic acid molecules. The IncRNAs described herein can be comprised of naturally occurring residues or modified residues as discussed herein. As well, for embodiments described herein, the IncRNAs can be capless and / or tailless. It is understood that references and limitations to IncRNA are also applicable to the IncRNA portion of nucleic acid molecules comprising IncRNA and where applicable to DNA encoding the IncRNA. For example, the IncRNA can be a IncRNA or isoform that has a biological activity in disease, or that modify the expression of a gene that has a biological activity in disease.

[0141] The term “dsRNA” as used herein means RNA molecules that comprise at least two strands of RNA that can hybridize partially of fully.

[0142] The phrase “comprises single stranded molecules at a purity of at least 99%” as used herein with respect to a complex, IncRNA, nucleic acid molecule or composition, means that the said complex, IncRNA, nucleic acid or composition has a maximum impurity or contamination with dsRNA of 1%, typically less than 0.5%.

[0143] The term “one or more chemically modified nucleotides” as used herein means that the IncRNA can comprise one or more of the same nucleotide (e.g., only uridine nucleotides) that is modified,or can comprise one or more of different nucleotides modified (e.g. uridine and adenosine nucleotides). The chemical modifications can be sugar modifications and / or base modifications in a same molecule.

[0144] The term “flare up” as used herein refers to a temporary and / or acute worsening of symptoms in a chronic disease. For example, clinical thresholds of key cytokines are elevated. For example, in heathly individuals : IL-lbeta: undetectable or < 1 pg / mL; IL-6: < 5-10 pg / mL, TNF-alpha: < 5-8 pg / mL and IL-10: < 5 pg / mL. In flares ups, these levels may : IL-lbeta: > 5 pg / mL, IL-6: > 10-20 pg / mL, TNF-alpha: > 10 pg / mL and / or IL-10: > 10-20 pg / mL.

[0145] The term “deficient” as used herein with regards to a IncRNA being deficient, refers to a physical molecule deficiency or a functional deficiency, particularly wherein the IncRNA deficiency is deleterious, for example disease causing. The deficiency may be at least 50% of average levels (IncRNA or function) present considered normal (e.g. based on a comparator population) or the IncRNA expression may be absent. When a IncRNA is nonfunctional due to mutation such as truncation, the physical level of the IncRNA may be normal, decreased or increased, and the functional level is decreased by at least 50% of average levels.

[0146] The term "administered" or “administering” as used herein means administration of an amount of compositions, nucleic acid molecules, vector constructs or complexes of the disclosure to a subject or a cell for example in cell culture or in a patient.

[0147] The term “pharmaceutically acceptable” means compatible with the treatment of subjects, for example humans.

[0148] The term “pharmaceutically acceptable carrier” means a non-toxic solvent, dispersant, excipient, adjuvant or other material which is mixed with the active ingredient in order to permit the formation of a pharmaceutical composition, i.e., a dosage form capable of administration to a subject. Pharmaceutically acceptable carriers suitable for use with nucleic acid-based innovations, including RNA molecules formulated with a delivery agent such as a lipid nanoparticle (LNP), may include conventional excipients that ensure stability, biocompatibility, and effective administration. Such carriers can encompass buffered aqueous solutions, isotonic saline, phosphate-buffered saline, tris-based buffers, cryoprotectants, tonicity agents, and stabilizing sugars such as sucrose or trehalose. Additional acceptable components may include polysorbates, polyethylene glycol derivatives, or other formulation excipients that maintain the integrity of the nucleic acid and delivery system during storage and delivery. These carriers are selected to support efficient encapsulation, protect against nuclease degradation, and enable safe in vivo administration without inducing undue toxicity or immunogenicity.

[0149] As used herein in the specification and in the claims, "or" should be understood to have the same meaning as "and / or" as defined above. For example, when separating items in a list, "or" or "and / or" shall be interpreted as being inclusive, i.e., the inclusion of at least one, but also including more than one, of a number or list of elements, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as "only one of or "exactly one of or, when used in the claims, "consisting of will refer to the inclusion of exactly one element of a number or list of elements. In general, the term "or" as used herein shall only be interpreted as indicating exclusive alternatives (i.e., "one or the other but not both") when preceded by terms of exclusivity, such as "either," "one of," "only one of," or "exactly one of."

[0150] As used herein, all transitional phrases such as "comprising," "including," "carrying," "having," "containing," "involving," "holding," "composed of," and the like are to be understood to be open-ended, i.e., to mean including but not limited to. Only the transitional phrases "consisting of’ and "consisting essentially of’ shall be closed or semi-closed transitional phrases, respectively.

[0151] It should also be understood that, in certain methods described herein that include more than one step or act, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are recited unless the context indicates otherwise.

[0152] Further, the definitions and embodiments described in particular sections are intended to be applicable to other embodiments herein described for which they are suitable as would be understood by a person skilled in the art. For example, in the following passages, different aspects are defined in more detail. Each aspect so defined may be combined with any other aspect or aspects unless clearly indicated to the contrary. In particular, any feature indicated as being preferred or advantageous may be combined with any other feature or features indicated as being preferred or advantageous.

[0153] Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present disclosure, examples of methods and materials are now described.II. Products, Compositions, Methods and Uses

[0154] An aspect of the disclosure includes a complex comprising a long non -coding (IncRNA) or a DNA encoding a IncRNA and a cell delivery agent, and wherein the complex or IncRNA comprises single stranded molecules at a purity of at least 99%, wherein the IncRNA comprises one or more different IncRNAs.

[0155] In an embodiment, the IncRNA lacks a 5’ cap and / or a 3’poly A tail. As shown in the Examples, unexpectedly, lacking a cap and / or tail can be beneficial, e.g., for encapsulation and / or activity.

[0156] The inventors have determined, that IncRNAs lacking a cap and / or tail are also therapeutically effective. These IncRNAs which can comprise modifications in U, A, G or C nucleotides (in the bases or the sugar for example) are effective. Modification at all residue types including, A, U, G or C nucleotides were shown to be useful.

[0157] They have also found that these molecules can be effectively delivered to cells with various delivery agents.

[0158] Another aspect of the disclosure includes a long non-coding (IncRNA)-nanoparticle complex comprising a IncRNA or a DNA encoding the IncRNA, or a nucleic acid molecule described herein and a cell delivery agent.

[0159] In an embodiment, the cell delivery agent is a lipid nanoparticle and the IncRNA is encapsulated by a lipid nanoparticle wherein, the IncRNA comprises one or more IncRNA.

[0160] Also provided in another aspect is a nucleic acid molecule comprising the IncRNA.

[0161] In some embodiments, the lipid nanoparticle comprises biocompatible and / or minimally immunogenic lipids and / or comprises a tertiary amine.

[0162] In some embodiments, the IncRNA (e.g. the IncRNA or the IncRNA portion of a nucleic acid molecule) comprises or is selected from GAPLINC, DRAIR, MIST , PARAIL, MALAT1, NEAT1, HOTAIR, ANRIL, NORAD, PVT1, SNHG1, SNHG3, SNHG5, SNHG7, SNHG12, SNHG14, SNHG15, DNM3OS, CHASERR, H19, MEG3, FENDRR, SOX2OT, LINC00237, LINC-PINT, UCA1, GAS5, MIR17HG, PANDA, LINC00968 and / or LINC01021. In some embodiments, the DNA encoding the IncRNA encodes GAPLINC, DRAIR, MIST, PARAIL MALAT1, NEAT1, HOTAIR, ANRIL, NORAD, PVT1, SNHG1, SNHG3, SNHG5, SNHG7, SNHG12, SNHG14, SNHG15, DNM3OS, CHASERR, H19, MEG3, FENDRR, SOX2OT, LINC00237, LINC-PINT, UCA1, GAS5, MIR17HG, PANDA, LINC00968 and / or LINC01021.

[0163] In some embodiments, the IncRNA is an active fragment.

[0164] In some embodiments, the IncRNA or the DNA encoding the IncRNA is or comprises or encodes GAPLINC.

[0165] In some embodiments, the IncRNA or the DNA encoding the IncRNA is or comprises or encodes DRAIR.

[0166] In some embodiments, the IncRNA or the DNA encoding the IncRNA is or comprises or encodes MIST.

[0167] In some embodiments, the IncRNA or the DNA encoding the IncRNA is or comprises or encodes PARAIL.

[0168] In some embodiments, the IncRNA is or the DNA encoding the IncRNA is or comprises or encodes CHASERR.

[0169] The DNA encoding the IncRNA can comprise a promoter sequence such as a T7 promoter for in vitro translation of the IncRNA.

[0170] The nucleic acid molecule comprising the IncRNA can also comprise one or more synthetic nuclease resistant oligonucleotides, optionally 5’ and / or 3’ to the IncRNA.

[0171] The IncRNAs used in the complexes, compositions, kits, nucleic acid molecules etc are purified to remove for example dsRNA or other contaminants, for example by HPLC, such as RP-HPLC.

[0172] As described herein, a linear gradient was used which increased full length RNA isolation. Accordingly, in some embodiments, the IncRNA purification comprises using RP-HPLC and a linear gradient of 38-65% of triethylammonium acetate and acetonitrile buffer, optionally 0.1 M triethylammonium acetate [TEAA, pH 7.0] and 25% [v / v] acetonitrile.

[0173] In some embodiments, IncRNA, DNA encoding the IncRNA or the nucleic acid molecule have 99% purity. In some embodiments, the complex or IncRNA comprises single stranded molecules at a purity of at least 99%. For example, the complex or IncRNA comprises at least 99% single stranded molecules or less than 1% contaminating dsRNA. In some, embodiments, the IncRNA, DNA encoding the IncRNA or the nucleic acid molecule are HPLC purified. In some embodiments, the HPLC is RP-HPLC. The purification can comprise denaturing. Denaturing can be optionally by heating or by chemical methods. The heating or denaturation can be carried out before or during purification. In some embodiments, the purification, optionally HPLC purification, is carried out at a temperature (e.g. a denaturation temperature to disrupt intra and inter IncRNA interactions) of at about or at least 45 °C, at about or at least 50°C, at about or at least 55°C, at about or at least 60°C, at about or at least 65°C or at about or at least 70°C. For example, the purification can be carried out in the range of about 40°C to about 80°C. The temperature can be achieved for example by heating the HPLC column.

[0174] Other purification methods can be used instead of or in combination with HPLC, for example cellulose chromatography purification.

[0175] In some embodiments, the nucleotide sequence of one or more IncRNA molecules is unmodified.

[0176] In some embodiments, the IncRNA is chemically modified or comprises one or more chemically modified nucleotides.

[0177] In some embodiment, the chemical modification is a base modification and / or a sugar modification.

[0178] In some embodiments, the chemically modified IncRNA comprises one or more Nl-methylpseudouridine (m I ) modification, one or more pseudouridine modification ( ). and / or one or more 5 -methyl -cytosine (m5C) modification and / or one or more of N6-Methyl-adenosine-5'-triphosphate (N6-Me-ATP), 2'-fhroro-2'-deoxyuridine-5'-triphosphate (2'-F-dUTP), 2'-fluoro-2'-deoxycytidine-5'-triphosphate (2'-F-dCTP), 2'-fhroro-2'-deoxyguanosine-5'-triphosphate (2'-F-dGTP), 2'-fluoro-2'-Deoxyadenosine-5 '-triphosphate (2'-F-dATP), 2'-O-methyl-uridine-5'-triphosphate (2'-O-Me-UTP), 2'-O-methyl-adenosine-5'-triphosphate (2'-O-Me-ATP), 2'-O-methyl-guanosine-5'-triphosphate (2'-O-Me-GTP), 2'-O-methyl-cytidine-5'-triphosphate (2'-O-Me-CTP), 2'-O-4'-C-Locked-guanosine-triphosphate (2'-O-4'-C-Locked-GTP), 2'-O-4'-C-Locked-adenosine-5'-triphosphate (2'-O-4'-C-Locked-ATP) and 2'-O-4'-C-Locked-5-methyl-cytidine-triphosphate (2'-O-4'-C-Locked-5-Me-CTP).

[0179] For example, one or more 5 -methyl -cytosine (m5C) modification refers to one or more cytidine triphosphate (CTP) in a nucleotide sequence being replaced with 5 -methylcytidine -5 ’-triphosphate (m5C). For example, one or more pseudouridine modification ( ) refers to one or more uridine triphosphate (UTP) in a nucleotide sequence being replaced with pseudouridine -5 ’-triphosphate (T). For example, one or more N 1 -methylpseudouridine (m I ) modification refers to one or more one or more uridine triphosphate (UTP) in a nucleotide sequence being replaced with Nl-methylpseudouridine-5’ -triphosphate (m I ). In some embodiments, one or more pseudouridine modification ( ) refers to all uridine triphosphate (UTP) residues present in a nucleotide sequence being replaced with pseudouridine-5 ’-triphosphate (T). In some embodiments, one or more N 1 -methylpseudouridine (ml ) modification refers to all uridine triphosphate (UTP) residues present in a nucleotide sequence being replaced with N 1 -methylpseudouridine-5 ’-triphosphate (ml ). In some embodiments, one or more 5 -methylcytidine-5 ’ -triphosphate (m5C) modification refers to all cytidine triphosphate (CTP) residues in a nucleotide sequence being replaced with 5 -methylcytidine -5 ’ -triphosphate (m5C) .

[0180] In some embodiments, the chemical modification is a sugar modification. For example, the sugar modification is 2’OMe-UTP, 2'-O-Me-ATP, 2'-O-Me-CTP and / or 2'-O-Me-GTP. Other examplesinclude 2'-F-dUTP, 2'-F-dCTP, 2'-F-dGTP, 2'-F-dATP, 2'-O-4'-C-Locked-GTP, 2'-O-4'-C-Locked-ATP and 2'-O-4’-C-Locked-5-Me-CTP.

[0181] The incorporation of 2’OMe-UTP using T7 RNA polymerase during IVT poses challenges due to the enzyme’s strict substrate specificity for naturally occurring nucleotide triphosphates and difficulty with bulky and rigid substrates(J). A mutant T7 RNA polymerase can be used for synthesis. The successful synthesis of 2’OMe-UTP-modified GAPLINC using the T7 RNA Polymerase Mui is demonstrated herein in model IncRNA GAPLINC. The incorporation of 2’OMe-UTP to GAPLINC which is used a model IncRNA preserves function of GAPLINC to decrease Illb . This sugar modification also maintains functional performance on par with -modificd GAPLINC. -modificd GAPLINC outperforms natural GAPLINC and 2’OMe-UTP -modified GAPLINC has performance that is on-par with -modificd GAPLINC. The sugar modifications are also an improvement over natural GAPLINC.

[0182] In some embodiments, the IncRNA comprises the nucleotide sequence identified in any of Tables 1, 6A or 6B, including any of sequences identified by accession numbers therein. In some embodiments, the IncRNA comprises or is selected from the nucleotide sequence of any one of SEQ ID NOs: 10, 14, 18, 26, or 31-64, or a sequence with at least 80%, at least 90% or at least 95 % sequence identity to any thereof.

[0183] In some embodiments, the IncRNA comprises unmodified GAPLINC (e.g., SEQ ID NO: 10 or 31 or provided by an GAPLINC accession number).

[0184] Sequences with at least 90%, sequence identity can also be used. For example, the IncRNA can comprise or be any of the nucleotide sequences of any one of SEQ ID NOs: 10, 14, 18, 26, or 32-64, , or corresponding DNA sequences or a sequence with at least 85%, or at least 90% sequence identity to any thereof or any of the sequences in described herein. In some embodiments, the DNA encoding the IncRNA is selected from SEQ ID NO: 1, 5, 6 and 25or a sequence with at least 85% or at least 90% sequence identity thereto.

[0185] The sequence identity can be at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical.

[0186] One or more nucleotides can be substituted by different nucleotides (whether chemically modified or not). For example, in some embodiments up to about 9% of the A nucleotides can be substituted. In some embodiments, up to about 10% of the U nucleotides can be substituted. In some embodiments, up to about 12% of the G nucleotides can be substituted. In other embodiments, percent identity is determined using for example BLAST™ alignment or other alignment tool.

[0187] In some embodiments, the IncRNA comprises m5C-modified GAPLINC (e.g., SEQ ID NO: 10, where one or more cytidine triphosphate (CTP) is replaced with 5-methylcytidine-5’-triphosphate). In some embodiments, the one or more IncRNA molecule comprises T -modified GAPLINC (e.g., SEQ ID NO: 10 or a sequence with at least 85% or at least 90% sequence identity thereto where one or more, optionally all, uridine triphosphate (UTP) is replaced with pseudouridine-5’ -triphosphate (T)). In some embodiments, the one or more IncRNA molecule comprises mlT -modified GAPLINC (e.g., SEQ ID NO: 10 or a sequence with at least 85% or at least 90% sequence identity thereto where one or more, optionally all, uridine triphosphate (UTP) is replaced with Nl-methylpseudouridine-5’ -triphosphate (m IT)).

[0188] In some embodiments, the first 19 residues of GAPLINC are not substituted.

[0189] In some embodiments, the IncRNA comprises unmodified MIST (e.g., SEQ ID NO: 14 or 33). In some embodiments, the one or more IncRNA molecule comprises m5C-modified MIST (e.g., SEQ ID NO: 14 or 33 where one or more, optionally all, cytidine triphosphate (CTP) is replaced with 5-methylcytidine-5 ’-triphosphate). In some embodiments, the one or more IncRNA molecule comprises T -modified MIST (e.g., SEQ ID NO: 14 or 33 or a sequence with at least 85% or at least 90% sequence identity thereto where one or more, optionally all, uridine triphosphate (UTP) is replaced with pseudouridine-5 ’-triphosphate (T)). In some embodiments, the one or more IncRNA molecule comprises mlT -modified MIST (e.g., SEQ ID NO: 14 or 33 or a sequence with at least 85% or at least 90% sequence identity thereto where one or more, optionally all, uridine triphosphate (UTP) is replaced with Nl-methylpseudouridine-5 ’-triphosphate (mlT))).

[0190] In some embodiments, the IncRNA comprises unmodified DRAIR (e.g., SEQ ID NO: 18 or 32 or a sequence with at least 85% or at least 90% sequence identity thereto). In some embodiments, the one or more IncRNA molecule comprises m5C-modified DRAIR (e.g., SEQ ID NO: 18 or 32 or a sequence with at least 85% or at least 90% sequence identity thereto where one or more, optionally all, cytidine triphosphate (CTP) is replaced with 5-methylcytidine-5’-triphosphate). In some embodiments, the one or more IncRNA molecule comprises T -modified DRAIR (e.g., SEQ ID NO: 18 or 32 or a sequence with at least 85% or at least 90% sequence identity thereto where one or more, optionally all, uridine triphosphate (UTP) is replaced with pseudouridine-5 ’-triphosphate (T)). In some embodiments, the one or more IncRNA molecule comprises mlT -modified DRAIR (e.g., SEQ ID NO: 18 or 32 or a sequence with at least 85% or at least 90% sequence identity thereto where one or more, optionally all, uridine triphosphate (UTP) is replaced with Nl-methylpseudouridine-5’-triphosphate (m IT)).

[0191] In some embodiments, the IncRNA comprises unmodified PARAIL (e.g., SEQ ID NO: 26 or 34 or a sequence with a least 85% or at least 90% sequence identity thereto). In some embodiments, the one or more IncRNA molecule comprises m5C-modified PARAIL (e.g., SEQ ID NO: 26 or 34 or a sequencewith at least 85% or at least 90% sequence identity thereto where one or more, optionally all, cytidine triphosphate (CTP) is replaced with 5-methylcytidine-5’-triphosphate). In some embodiments, the one or more IncRNA molecule comprises -modified PARAIL (e.g., SEQ ID NO: 26 or 34 or a sequence with at least 85% or at least 90% sequence identity thereto where one or more, optionally all, uridine triphosphate (UTP) is replaced with pseudouridine-5 ’-triphosphate ( )). In some embodiments, the one or more IncRNA molecule comprises mlT -modified PARAIL (e.g., SEQ ID NO: 26 or 34 or a sequence with at least 85% or at least 90% sequence identity thereto where one or more, optionally all, uridine triphosphate (UTP) is replaced with N1 -methylpseudouridine -5 ’-triphosphate (ml'P))). In some embodiments, the one or more IncRNA molecule comprises m6A-modified PARAIL (e.g., SEQ ID NO: 26 or 34 or a sequence with at least 85% or at least 90% sequence identity thereto where one or more, optionally all, adenosine triphosphate (ATP) is replaced withN6-methyl-adenosine-5’-trisphosphate (m6A)).

[0192] The length of the IncRNA can also vary for example by about 20%, For example the sequence can be up to 20% shorter than the naturally occurring IncRNA, or can be up to 20% longer. For example, the sequence can be 5%, 10%, 15% or 20% or up to 20% longer or shorter. The sequence can be added to one end or both.

[0193] In some embodiments, the lipid nanoparticle comprises an ionizable lipid. It can also comprise zwitterionic lipid or cationic lipid.

[0194] In another embodiment, the lipid nanoparticle comprises one or more of cholesterol, a phospholipid and a PEG-lipid. In some embodiments, the nanoparticle comprises an ionizable lipid, cholesterol, DSPC, and DMG-PEG2000 In some embodiments, the nanoparticle comprises a ratio of ionizable lipid, cholesterol, DSPC, and DMG-PEG2000 is about 50:38.5: 10: 1.5 mol %. Other ratios can also be used.

[0195] In some embodiments, the ionizable lipid comprises the ionizable lipids depicted in FIG.13A-I, (OC2-K3-E10, C3-K2-E14, C3-K2-E10, C3-K3-K10, SM-102, ALC-0315, Dlin-MC3-DMA (MC3), [3N2, and 503). In some embodiments, the ionizable lipid comprises D1-L2-C10E. In some embodiments, the ionizable lipid comprises OC2-K3-E10. In some embodiments, the ionizable lipid comprises C3-K2-E14.

[0196] Ionizable lipids that can be used in the lipid nanoparticles, include for example the lipids described in WO2023092242A1 (PCT / CA2022 / 051745) titled “MULTI-MOTIF DENDRONS AND THEIR SUPRAMOLECULAR STRUCTURES AND USES THEREOF”, and WO2025123134A1(PCT / CA2024 / 051647) titled “SUPRAMOLECULAR IONIZABLE LIPID MOLECULES WITH HETERO ATOMIC TUNING FOR NUCLEIC ACID DELIVERY”, each of which are incorporated herein.

[0197] WO2023092242A1 for example discloses OC2-K3-E10 used in the Examples, and WO2025123134A1 discloses among others, [3N2 and 503 also used in the Examples.

[0198] Other nanoparticles can also be used for example those described in EP 3532094 Al, US 8,058,069 B2, WO2016 / 176330 and US 10,195,291 B2, the nanoparticle teachings each of which are incorporated by reference herein.

[0199] In some embodiments, the nanoparticle comprises an ionizable lipid, cholesterol, a phospholipid, and a PEG-lipid.

[0200] In some embodiments, the nanoparticle comprises a PEG-lipid selected from the group consisting of 14:0 PEG2000 PE which is l,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] where 14:0 can be replaced by 18:0 and 2000 can be replaced by 5000, 18:1 PEG2000 PE which is l,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N- [methoxy(polyethylene glycol)-2000] where 2000 can be replaced by 5000, DMG-PEG2000 which is 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000, DSG-PEG2000 which is distearoyl-rac-glycerol-PEG2K, DSPC which is l,2-distearoyl-sn-glycero-3-phosphocholine and combinations thereof.

[0201] In some embodiments, the lipid nanoparticle comprises an ionizable lipid as described in WO2023 / 092242 which is incorporated herein by reference.

[0202] In some embodiments, the lipid nanoparticle comprises an ionizable lipid, cholesterol, DSPC, and DMG-PEG2000. In some embodiments, the nanoparticle comprises a ratio of ionizable lipid, cholesterol, DSPC, and DMG-PEG2000 is about 50:38.5: 10: 1.5 mol %.

[0203] In some embodiments, the lipid nanoparticle comprises an ionizable lipid as described in PCT / CA2024 / 051647 which is incorporated herein by reference.

[0204] In some embodiments, the lipid nanoparticle comprises or is lipofectamine (e.g. the lipid is the lipid in lipofectamine).

[0205] In some embodiments, the complex comprises features as described in Table 2.

[0206] In some embodiments, the size of the complex is between about 140 nm to about 200nm or less than 200 nm. In some embodiments, the size of the complex is between about 144 nm to about 150 nm. In some embodiments, the size of the complex is about 173 nm. In some embodiments, the size of the complex is about 150 nm. In some embodiments, the size of the complex is about 160 nm. In someembodiments, the size of the complex is about 141 nm. In some embodiments, the size of the complex is about 168 nm. In some embodiments, the size of the complex is about 169 nm. In some embodiments, the size of the complex is about 147 nm. In some embodiments, the size of the complex is about 144 nm.

[0207] In some embodiments, the polydispersity of the complex is below about 0.2.

[0208] In some embodiments, the complex is positively charged. In some embodiments, the complex has a charge of about 5 mV to about 32 mV. In some embodiments, the complex has a charge of about 28 mV to about 32 mV. In some embodiments, the complex has a charge of about 24 mV. In some embodiments, the complex has a charge of about 23 mV. In some embodiments, the complex has a charge of about 29 mV. In some embodiments, the complex has a charge of about 25 mV. In some embodiments, the complex has a charge of about 7 mV. In some embodiments, the complex has a charge of about 5 mV.

[0209] In some embodiments, the complex comprises a mass ratio of at least 20:1 of lipid to IncRNA. The lipid can for example be an ionizable lipid, zwitterionic lipid or cationic lipid depending on the lipid nanoparticle. It can be the lipid in lipofectamine.

[0210] In some embodiments, the complex comprises a mass ratio between about 5: 1 to 20: 1 of lipid to IncRNA (or DNA, vector construct or nucleic acid). In some embodiments, the complex comprises a mass ratio between about 7: 1 to 20: 1 of lipid to IncRNA (or DNA, vector construct or nucleic acid). In some embodiments, the complex comprises a mass ratio of about 5: 1 of lipid to IncRNA (or DNA, vector construct or nucleic acid). In some embodiments, the complex comprises a mass ratio of about 6: 1 of lipid to IncRNA (or DNA, vector construct or nucleic acid) . In some embodiments, the complex comprises a mass ratio of about 7: 1 of lipid to IncRNA (or DNA, vector construct or nucleic acid). In some embodiments, the complex comprises a mass ratio of about 20: 1 of lipid to IncRNA (or DNA, vector construct or nucleic acid). In some embodiments, the complex comprises amass ratio of about 8: 1, of about 9:1, of about 10: 1, of about 11:1, of about 12: 1, of about 13:1, of about 14: 1, of about 15:1 of lipid to lncRNA(or DNA, vector construct or nucleic acid).

[0211] In some embodiments, the IncRNA comprises a 5’ cap and / or 3 ’poly A tail. In some embodiments, the IncRNA lacks a 5’ cap and / or 3 ’poly A tail. In some embodiments, the IncRNA lacks a 5’ cap. In some embodiments, the IncRNA lacks 3 ’poly A tail. In some embodiments, the IncRNA lacks a 5’ cap and 3 ’poly A tail.

[0212] A further aspect provides a composition comprising the complex, IncRNA, DNA, nucleic acid, vector construct or any component thereof described herein, and optionally one or more pharmaceutically acceptable carrier and / or excipient, optionally sterile water or buffered saline.

[0213] In certain embodiments, the compositions described herein may be formulated with one or more pharmaceutically acceptable carriers suitable for administration of a nucleic acid molecule, including but not limited to RNA, in combination with a delivery agent such as a lipid nanoparticle (LNP). The pharmaceutically acceptable carriers may comprise any inert or biocompatible substance that facilitates handling, stability, storage, or delivery of the active components, and may include, without limitation, aqueous buffers (e.g., phosphate-buffered saline, Tris-based buffers), isotonic agents, cryoprotectants, stabilizing sugars (e.g., sucrose, trehalose), tonicity-adjusting agents, surfactants, or polymeric excipients. Additional carriers or excipients may be included to maintain nucleic acid integrity, preserve nanoparticle structure, or modulate physicochemical properties of the formulation. The selection of carriers may be made with regard to the intended route of administration, desired pharmacokinetic profde, and acceptable safety and biocompatibility characteristics, and may be varied without departing from the scope of the present disclosure.

[0214] In some embodiments, the composition comprises a concentration of the complex of at least about 30 ng / pL to about 500 ng / pL.

[0215] In some embodiments, the IncRNA of the complex, pharmaceutical composition, nucleic acid, DNA, vector construct, kit etc, is selected using an assay, optionally an assay described herein.

[0216] Another aspect of the disclosure comprises a method for increasing the level of a deficient IncRNA in a cell or subject, the method comprising administering a complex, IncRNA, nucleic acid molecule or DNA described herein to the cell or subject in need thereof. Table 6 A provides a list of IncRNAs that are known to be truncated in disease states and Table 6B provides a list of IncRNAs that are known to be absent in disease states. Any of the IncRNAs listed therein or DNAs encoding them, as well as active fragments thereof can be used in complexes, nucleic acid molecules or methods, uses kits etc described herein.

[0217] As dysregulation of IncRNAs can cause disease, another aspect of the disclosure includes a method of treating a disease or condition, the method comprising administering a complex, IncRNA, nucleic acid molecule for DNA encoding the IncRNA to the cell or subject in need thereof. In one embodiment, the disease or condition is an acute inflammatory disease or condition or a flare up of a chronic inflammatory disease or condition. In another embodiment, the disease or condition is a disease or condition associated with a deficiency of a IncRNA. The deficiency in the IncRNA may be due to mutation (such as a genomic deletion mutant or point mutation) of the IncRNA. In an embodiment, the method comprises administering the IncRNA, a DNA encoding the IncRNA, a nucleic acid molecule, or complex or compositions described herein to a subject in need thereof. In some embodiments, the subject is a human.

[0218] In some embodiments, the inflammatory disease is acute and / or systemic inflammation or a flare up of a chronic inflammatory disease or condition. In some embodiments, the inflammatory disease is sepsis. In some embodiments, the inflammatory disease includes cytokine release syndrome or vascular disease (e.g., atherosclerosis, atherothrombosis).

[0219] In some embodiments, the disease or condition is a disease or condition associated with a deficiency of a IncRNA is a syndromic early-onset neurodevelopmental disorder, developmental delay, growth deficiency. In an embodiment, the disease or condition is neurodegeneration.

[0220] In an embodiment, the disease or condition comprises increased cytokine levels, optionally IL-1[3, TNFa, IFN-y and / or IL-6, for example TNFa.

[0221] In some embodiments, the complex, IncRNA, DNA or pharmaceutical composition is administered in a dosage of up to about 5mg / kg. In some embodiments, the complex, IncRNA, DNA or pharmaceutical composition is administered in a dosage of about O.lmg / kg. In some embodiments, the complex, IncRNA, DNA or pharmaceutical composition is administered in a dosage of about Img / kg. In some embodiments, the complex, IncRNA, DNA or pharmaceutical composition is administered in a dosage of about 3mg / kg. In some embodiments, the complex, IncRNA, DNA or pharmaceutical composition is administered in a dosage of about 5 mg / kg. In some embodiments, the complex, IncRNA, DNA or pharmaceutical composition is administered in a single dose. In some embodiments, the complex, IncRNA, DNA or pharmaceutical composition is administered in a plurality of doses.

[0222] Another aspect of the disclosure includes a method of altering expression of one or more proinflammatory protein, optionally one or more cytokine, optionally a proinflammatory cytokine, expression in a subject, the method comprising administering the complex described or composition described herein to the subject.

[0223] In some embodiments, the one or more cytokine comprises IL-ip, TNFa, IFN-y and / or IL-6. In some embodiments, the altering comprises decreasing expression e.g., as compared to a control.

[0224] In some embodiments, the one or more cytokine comprises IL- 10. In some embodiments, altering comprises increasing IL-10 e.g., as compared to a control.

[0225] In some embodiments, the IncRNA comprises -modified GAPLINC and expression of IL- 1 p is decreased e.g., as compared to a control.

[0226] In some embodiments, the IncRNA comprises unmodified, -, m IT- and / or m5C-modified GAPLINC and expression of IL- i is decreased e.g., as compared to a control.

[0227] In some embodiments, the IncRNA comprises *P-, m IT- and / or m5C-modified GAPLINC and expression of IFN-y is decreased e.g., as compared to a control.

[0228] In some embodiments, the IncRNA comprises unmodified, mlT-, and / or m5C-modified GAPLINC and expression of TNFa is decreased e.g., as compared to a control.

[0229] In some embodiments, the IncRNA comprises mlT-modified GAPLINC and expression of IL-6 is decreased e.g., as compared to a control.

[0230] In some embodiments, the IncRNA comprises ml'P-modified GAPLINC and expression of IL- 10 is increased e.g., as compared to a control.

[0231] In some embodiments, the IncRNA comprises ml'P-modified GAPLINC and expression of MCP-1 is decreased e.g., as compared to a control.

[0232] In some embodiments, the IncRNA comprises 'P-modified GAPLINC and expression of IFN-y is decreased e.g., as compared to a control.

[0233] In some embodiments, the IncRNA comprises unmodified MIST and expression of TNFa and / or IL-6 is decreased e.g., as compared to a control.

[0234] In some embodiments, the IncRNA comprises unmodified, T-, m IT- and / or m5C-modified DRAIR and expression of TNFa and / or IL-6 is decreased e.g., as compared to a control.

[0235] In some embodiments, the IncRNA comprises 'P-modified DRAIR and IL- 10 expression is increased. In some embodiments, the IncRNA comprises ml'P-modified DRAIR and IL- 10 expression is increased e.g., as compared to a control.

[0236] In some embodiments, the IncRNA comprises unmodified, T-, and / or m5C modified MIST and IFN-y expression is increased e.g., as compared to a control.

[0237] In some embodiments, the IncRNA comprises unmodified and / or 'P-modified MIST and TNFa and / or IL-6 expression is decreased e.g., as compared to a control.

[0238] In some embodiments, the IncRNA comprises ml'P-modified MIST and IL- 10 expression is increased e.g., as compared to a control.

[0239] In some embodiments, the control is an unmodified IncRNA (e.g., comprising no chemical modifications). In some embodiments, the control is a IncRNA with a different chemical modification than the IncRNA being compared with the control.

[0240] In some embodiments, the complex, IncRNA, DNA or pharmaceutical composition is administered in a single dose. In some embodiments, the complex, IncRNA, DNA or pharmaceutical composition is administered in a plurality of doses.

[0241] In some embodiments, the complex, IncRNA, DNA or pharmaceutical composition is administered at least about every 10 hours or less. In some embodiments, the complex, IncRNA, DNA or pharmaceutical composition is administered at least about every 9 hours or less. In some embodiments, the complex, IncRNA, DNA or pharmaceutical composition is administered at least about every 8 hours or less. In some embodiments, the complex, IncRNA, DNA or pharmaceutical composition is administered at least about every 7 hours or less. In some embodiments, the complex, IncRNA, DNA or pharmaceutical composition is administered at least about every 6 hours or less. In some embodiments, the complex, IncRNA, DNA or pharmaceutical composition is administered at least about every 5 hours or less. In some embodiments, the complex, IncRNA, DNA or pharmaceutical composition is administered at least about every 4 hours or less. In some embodiments, the complex, IncRNA, DNA or pharmaceutical composition is administered at least about every 3 hours or less. In some embodiments, the complex, IncRNA, DNA or pharmaceutical composition is administered at least about every 2 hours or less. In some embodiments, the complex, IncRNA, DNA or pharmaceutical composition is administered at least about every hour or less. In some embodiments, the complex, IncRNA, DNA or pharmaceutical composition is administered at least about every 6 hours.

[0242] In some embodiments, the complex, IncRNA, DNA or pharmaceutical composition is administered subcutaneously.

[0243] In other embodiments, the complex, IncRNA, DNA or pharmaceutical composition is administered intravenously.

[0244] In some embodiments, the modified IncRNA is a chemically modified IncRNA as described herein. The modified IncRNA can be modified by preparing without a 5’cap and / or 3’poly A tail. In some embodiments, the unmodified IncRNA is not chemically modified (e.g., naturally occurring). In some embodiments, the unmodified IncRNA is an unmodified IncRNA as described herein.

[0245] In some embodiments, the functionality comprises altering levels of expression of one or more proteins in vivo.

[0246] Uses of the complexes described herein and the methods described herein are also contemplated. The IncRNA e.g. modified IncRNA, can be selected for inclusion in the complexes, compositions, nucleic acid molecules, and kits or used to prepare a DNA encoding the IncRNA or used inthe methods and uses described herein, using an assay for identifying active IncRNAs, e.g. modified IncRNAs

[0247] Also provided in another aspect is a screening assay for identifying a modified IncRNA with improved activity, the method comprising:selecting a target IncRNA of interest,preparing a vector construct comprising DNA encoding the IncRNA of interest, wherein the construct is configured to produce the IncRNA of interest lacking a 5 ’cap and / or 3’polyA tail; synthesizing a plurality of IncRNA using one more modified nucleotides in vitro;isolating one or more of the modified IncRNA optionally purifying to a purity of at least 99%; complexing the IncRNA with a delivery agent, optionally a lipid nanoparticle (LNP)testing the one or more isolated modified IncRNA and comparing to a control IncRNA comprising unmodified nucleotides, comprising a 5’cap and / or comprising a 3’polyA tail; and selecting a modified IncRNA with improved activity compared to the control.

[0248] The control IncRNA can for example be an unmodified IncRNA, optionally lacking a 5'cap and / or polyA tail. Alternatively, or as an additional control, can be a scrambled control or an unrelated IncRNA.

[0249] The testing will depend on the activity of the IncRNA for example as shown herein, for IncRNA involved in inflammation, the testing may comprise assessing NF-kB levels, cytokine expression etc, for example as demonstrated in the Examples. In situations where the IncRNA is involved in regulating gene expression as with IncRNA CHASERR, the testing can comprise assessing the expression level of the regulated gene (e.g. CHD2 transcript or protein levels). CHASERR IncRNAs that decrease the increased level of CHD2 seen where CHASERR is truncated, or restore CHD2 levels to normal levels are selected for use, for example for complexing with LNPs.

[0250] In an embodiment, the one or more modified nucleotides are as described herein.

[0251] In an embodiment, the isolating step comprises HPLC or cellulose purification and / or denaturation.

[0252] In an embodiment, wherein the level of dsRNA after the isolating step is less than 0.5% of total RNA, or less than 0.1%.

[0253] In some embodiments, the assay further comprises making the selected modified IncRNA optionally a complex comprising the selected modified IncRNA.

[0254] A selected modified IncRNA can be used in the complexes, compositions, nucleic acid molecules, kits, to prepare DNA encoding the IncRNA, including vector constructs as well as methods and uses described herein

[0255] As used herein in the specification and in the claims, the phrase "at least one," in reference to a list of one or more elements, should be understood to mean at least one element selected from anyone or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements. This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase "at least one" refers, whether related or unrelated to those elements specifically identified.

[0256] It should also be understood that, in certain methods described herein that include more than one step or act, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are recited unless the context indicates otherwise.

[0257] Further, the definitions and embodiments described in particular sections are intended to be applicable to other embodiments herein described for which they are suitable as would be understood by a person skilled in the art. For example, in the following passages, different aspects of the invention are defined in more detail. Each aspect so defined may be combined with any other aspect or aspects unless clearly indicated to the contrary. In particular, any feature indicated as being preferred or advantageous may be combined with any other feature or features indicated as being preferred or advantageous.EXAMPLESExample 1Exogenous GAPLINC LNPs deployed in vitro can control cell phenotype

[0258] As a proof-of-concept IncRNA, unmodified and modified GAPLINC were synthesized using in vitro transcription (IVT). Human and mouse GAPLINC have high sequence homology. The IVT product showed a band of approximately 500 nucleotides by bioanalyzer, which corresponds to the size expected for the unmodified GAPLINC (FIG. 1C). The single band suggested minimal RNA degradation and contaminants. Similarly, there were single bands observed for GAPLINC incorporating m IT. T and m5C modifications. However, additional faint bands at smaller sizes were observed for these modified IncRNAs, suggesting the potential presence of aborted transcripts or other IVT contaminants.

[0259] To remove IVT impurities, cellulose chromatography was utilized (FIG. 6A). This successfully removed dsRNA as suggested by dot blot analysis; however, bioanalyzer revealed additional bands smaller than the expected size of GAPLINC (FIGs. 6B and 6C). Reverse-phase HPLC (RP-HPLC) (FIG. 7A) successfully resolved the different species. The first major peak was a dsRNA-free GAPLINC, and the second major peak was the target RNA with traces of other RNA species and dsRNA (FIGs. 7B and 7C). These results suggest that RP-HPLC removes dsRNA and yields full-length single species ofGAPLINC. Additional optimization of the RP-HPLC method with purification temperature and gradient alterations led to better resolution and purity of GAPLINC (FIGs. 7D-7F). This RP-HPLC protocol developed was adopted for both unmodified and modified IncRNAs (FIGs. 8-12).

[0260] Subsequently, the functional utility of cap and tail modifications for IncRNA was sought to be determined. Three LNP formulations were produced and characterized using the C3-K2-E14 ionizable lipid (FIG. 13A) at a 7: 1 nanomaterial to RNA mass ratio: 1) no 5’ cap or 3’ poly(A) tail; 2) 5’ cap and 3’ poly(A) tail; 3) 5’ cap and no 3’ poly(A) tail. The size of LNPs containing GAPLINC with no cap and tail were smaller compared to the other modifications but all these particles exhibited similar polydispersity index (PDI) (FIGs. IE and F). Encapsulation efficiency was highest overall for LNPs containing GAPLINC with no cap or tail (FIG. IF). Because GAPLINC impacts NF-KB expression, RAW-Dual cells were challenged with LPS and treated with these LNPs for 18 h. Supernatant was collected to assess NF-KB activation using QUANTI-Blue (FIG. 1G). LPS challenge (200 ng / mL) significantly increased activation of NF-KB compared to untreated cells (FIG. 1H). Empty particles did not attenuate NF-KB activation. All LNPs containing GAPLINC significantly decreased NF-KB activation. Notably, LNPs encapsulating GAPLINC with no cap or tail showed the greatest decrease in NF -KB activation relative to other GAPLINC payloads. These findings showed that exogenous IncRNA can alter cell phenotype in vitro and supported the subsequent production of the IncRNA with no cap and tail.Exogenous IncRNA attenuation of NF-KB activation in vitro depends on base modifications

[0261] To screen the functional performance of base -modified IncRNAs, RAW-Dual cells were challenged with LPS (200 ng / mL) and treated for 18 h with 200 ng of unmodified or modified GAPLINC, MIST or DRAIR IncRNA. As expected, the LPS challenge significantly increased NF-KB activation relative to untreated cells (FIGs. 2A, 2C and 2E). For GAPLINC, unmodified and -, ml - and m5C-modified payloads showed significant decreases in NF-KB activation (FIG. 2A). To confirm the effects were caused by the IncRNA and not the delivery system, it was demonstrated that empty LNPs did not induce or reduce NF-KB activation (FIG. 13A). Additionally, interferon regulatory factor (IRF) activity was used as a measurement of immunogenicity of the IncRNAs. IRF activity was minimized with - and m I - modified GAPLINC as they showed no significant differences compared to untreated cells (FIG. 2B). Both unmodified and m5C-modified GAPLINC showed heightened IRF activity. The isoforms GAPLINC 202 and GAPLINC 205 did not lead to the same trends in NF-KB and IRF activities, suggesting that the effect of GAPLINC is isoform specific (FIGs. 14B-14D). For MIST, only mlT- and m5C modifications significantly decreased NF-KB activation compared to untreated LPS challenges (FIG. 2C). However, these payloads increased IRF activity compared to non -challenged untreated cells (FIG. 2D). Unmodified MIST also significantly increased IRF activity. Like GAPLINC, IRF activity was minimized with T modification.For DRAIR, unmodified and -, m I - and m5C-modified payloads significantly attenuated NF-KB activation (FIG. 2E). IRF activity was only minimized with and m I modifications, as the other payloads tested significantly increased IRF activity relative to non-challenged untreated cells (FIG. 2F).GAPLINC LNP performance in vivo is sensitive to IncRNA chemical base modifications

[0262] Upon validating the utility of IncRNAs in vitro, the first model IncRNA, GAPLINC was formulated into LNPs for therapeutic delivery in vivo. LNPs were formulated using D1-L2-C10E ionizable lipid (FIG. 13B), cholesterol, DSPC, and DMG-PEG2000 (50:38.5: 10: 1.5 mol %) mixed with either HPLC-purified unmodified or modified GAPLINC RNA in a microfluidic device as previously dcscribcd(2j. 24). The nanomaterial to RNA mass ratio was 7:1. LNPs formulated with this ionizable lipid were found to target the liver and spleen as determined by intravenously injecting firefly luciferase into C57BL / 6 mice (2 pg; FIG. 13J). The average size of the LNPs with HPLC-purified unmodified GAPLINC RNA was 173 nm (FIG. 15A). Scrambled RNA formed 127 nm LNPs. -modificd GAPLINC LNPs were 150 nm, mlT-modified GAPLINC LNPs were 160 nm and m5C-modified GAPLINC LNPs were 141 nm. The PDI of all particles were below the 0.20 best practice threshold and the particles were positively charged between 28-32 mV (FIGs. 15B-C)(25). Encapsulation efficiency of scrambled GAPLINC was 37%. Unmodified and modified GAPLINC LNPs had encapsulation efficiencies of 49-58% (FIG. 15D). The ranges observed in this characterization data suggested that RNA base modification may impact the size and charge of LNPs formed. Notably, the sizes and PDI of the LNPs made with unmodified and modified IncRNA purified with or without cellulose chromatography and HPLC are similar, suggesting LNP sizes are not impacted by the purification methods used (FIG. 16A). However, unmodified and modified GAPLINC LNPs made with HPLC-purified IncRNA have noticeably lower encapsulation efficiencies compared to no dsRNA and cellulose purification (FIG. 16B).

[0263] For the disease model, systemic inflammation was induced in C57BL / 6 mice by intraperitoneally injecting 100 pg of LPS. These animals were simultaneously treated with 1 mg / kg of scrambled, unmodified or modified GAPLINC LNPs for 6 h followed by serum analysis of proinflammatory and anti-inflammatory protein expression using Luminex (FIG. 3A). The LNP dose was determined through a dose response study using 1, 3, 5 mg / kg of cellulose -purified unmodified GAPLINC LNPs (FIGs. 17A-17C). The delivery material used for this study was confirmed to not elicit changes in the serum compared to PBS control (FIGs. 17D-17F). Unmodified and -, mlT- and m5C-modified GAPLINC LNP significantly attenuated IL- 1 [3 expression compared to LPS and scrambled GAPLINC LNPs (FIG. 3D). Importantly, unmodified and -modificd GAPLINC LNPs had noticeably lower IL-lf> expression compared to the other modifications. Since IL-lf> acts through an NF-KB-dependent pathway, these results are consistent with the in vitro observations (FIG. 2A). Chemically modified basessignificantly lowered IFN-y expression compared to unmodified GAPLINC. Additionally, GAPLINC with the modification was similar to PBS control (FIG. 3C). Unmodified and ml'P- and m5C-modified GAPLINC LNPs downregulated TNFa (FIG. 3D). ml -modified GAPLINC LNPs also decreased IL-6 and increased IL- 10 expression, which was significantly different from the other payloads tested (FIG. 3E-3F). Among other proteins analyzed, GM-CSF, IL-2, IL-12p70 and IL-4 expression were comparable between control and treatment groups (FIG. 18A-18D). Finally, MCP-1 was downregulated with m IT-modified GAPLINC LNPs (FIG. 18E). Results indicate that changes in cytokine expression is sensitive to chemical base modifications of GAPLINC.

[0264] The T-modified GAPLINC LNPs were selected as the lead for further experimentation. In studying the impact of increasing the nanomaterial to RNA mass ratio from 7:1 to 20:1, no significant differences in LNP size, PDI and charge was found (FIG. 3G). However, the 20:1 formulation increased encapsulation of the IncRNA from 53% to 85% (FIG. 3H), thereby meeting best practice criteria(25). Comparing the different formulations and doses of LNPs on IL-1 [3 expression in vivo, the low 0.10 mg / kg dose of T-modified GAPLINC LNPs 20: 1 formulation significantly downregulated IL-1J3 at 6 h compared to LPS (FIG. 31). The effect of this 20: 1 formulation was diminished by 10 h (FIG. 3J). Additional results for a related experiment are shown in Figs. 3O-Q, showing a notable reduction of IL- 1 P expression in vivo at 0.10 mg / kg, a sustained effect with T-modified GAPLINC LNPs for 6 hours in vivo (Fig. 3P) and a IncRNA-specific effect, because IL-1J3 abundance remained high with irrelevant IncRNA controls (Fig.3Q).

[0265] Because of the strong biological effect observed for T-modified GAPLINC LNPs on IL-1 , its mechanism of action was investigated and compare its therapeutic effect to a small interfering RNA (siRNA) that silences Illb mRNA. RAW-Dual cells were treated with LPS (100 ng / ml) and T-modified GAPLINC or Illb siRNA in Lipofectamine for 3, 6, or 21 hours. Gene expression analysis of these cells showed that T-modified GAPLINC transiently decreased Illb expression at 3 and 6 hours, similar to Illb siRNA (Fig. 3, Kto M). Because human and mouse GAPLINC have sequence homology, we examined -modified GAPLINC’s ability to affect Illb expression in primary human cells. The effect seen in mouse cells appeared conserved in human cells, because we observed a decrease in Illb at 6 hours in human peripheral blood mononuclear cell (PBMC)-derived monocytes (Fig. 3N).

[0266] A tolerability study of these IncRNA LNPs was conducted by multiple systemic administrations in C57BL / 6 mice. Weight loss, visually identifiable changes to body condition and gross analysis of organs were used to evaluate host response to LNP treatment. Over 8 days with two LNP treatments (0.10 mg / kg per dose), animals remained active and responsive while maintaining body weights (FIG. 19A). Histopathology analysis of the spleen, liver and kidneys of these animals was conducted to investigate organ toxicity or damage. Results showed no aberrant differences in control and treated animals,suggesting minimal response to materials delivered in vivo (FIGs. 7B-7C). As such, the 20:1 formulation and 0.10 mg / kg dose was used for subsequent IncRNAs tested.^-Modified MIST LNPs serves as a TNFa-specific treatment in vivo

[0267] Next, it was investigated whether MIST, another IncRNA known to perturb macrophage phenotype, would have similar effects as GAPLINC LNPs in vivo after LPS challenge. Unmodified and modified MIST LNPs exhibited similar sizes (144-150 nm) and PDI (<0.20) (FIG. 4A-4B). The charge of unmodified and m5C-modified MIST was 7 mV and *P- and m IT-modified had a charge of 5 mV (FIG. 4C). Using the 20:1 nanomaterial to RNA mass ratio in the formulations, encapsulation efficiencies above 80% for all MIST LNPs was achieved (FIG. 4D). For C57BL / 6 mice challenged with 100 pg LPS and treated for 6 h with 0.10 mg / kg of modified or unmodified MIST LNPs, no changes in IL- 1[3 expression were observed (FIG. 4E). Significant increases in IFN-y expression with unmodified, *P- and m5C-modified MIST LNPs was observed but not m IT-modified MIST LNPs (FIG. 4F). However, unmodified and 'P-modificd MIST LNPs significantly downregulated TNFa (FIG. 4G). Similarly, there were decreases in IL-6 expression with these particles (FIG. 4H). m I 'P-modificd MIST LNPs significantly upregulated IL- 10 levels and this was drastically different compared to the other payloads tested (FIG. 41). Among other proteins analyzed, GM-CSF, IL-2, IL-12p70, MCP-1 and IL-4 expression were comparable between control and treatment groups (FIGs. 20A-20E). The demonstrated function of MIST LNPs in vivo reinforced the GAPLINC result that base modifications influence the performance of exogenous IncRNAs and that the impact of base modifications can change with IncRNA sequence. In contrast to GAPLINC, exogenously delivered 'P-modified MIST LNPs may be a TNFa-specific treatment.Base modification specifies targeting of TNFa, IL-6 and IL-10 by DRAIR in vivo

[0268] IL-6 is another common proinflammatory cytokine upregulated in systemic inflammation(26). Thus, it was investigated whether DRAIR LNPs can act on IL-6. Unmodified and ml'P-modified DRAIR LNPs had similar sizes (168 nm and 169 nm, respectively; FIG. 5A). The sizes of - and m5C-modified DRAIR LNPs were comparable at 147 nm and 144 nm, respectively. PDI of all DRAIR LNPs were below the threshold of 0.20 (FIG. 5B). Similarly, the charge of unmodified and m5C- modified LNPs were comparable at 24 and 23 mV (FIG. 5C), respectively. *P- and ml'P-modified LNPs were akin to each other with a charge of 29 and 25 mV, respectively. DRAIR LNPs exhibited 80% encapsulation for unmodified and 'P-modified, 76% for ml'P-modified, and 60% for m5C- modified. Then, C57BL / 6 mice were challenged with 100 pg LPS and treated with 0.10 mg / kg DRAIR LNPs for 6 h. Results indicate that IL- 1 P and IFN-y expression was not significantly different for all treatments compared to control (FIG. 5E-F). Unmodified and 'P-, ml'P- and m5C-modified DRAIR LNPs significantly downregulated TNFa and IL-6 expression (FIG. 5G-H). Interestingly, 'P-modified DRAIR LNPs significantly upregulated IL-10 levels(FIG. 51). There were no significant differences observed for GM-CSF, IL-2, IL-12p70, MCP-1 and IL-4 expression between control and treatment groups (FIGs. 21A-21E). Thus, results showed that unmodified and -, mlT- and m5C-modified DRAIR can act on TNFa and IL-6. Notably, m IT-modified DRAIR can upregulate the production of the anti-inflammatory IL- 10.

[0269] Unlike coding transcripts, IncRNAs control cell function without translation into protein. For this reason, IncRNA molecules have the incredible potential to become a new class of therapeutics that operate similarly to small molecule drugs. Moreover, unlike small molecule drug discovery campaigns, the function, compatibility, safety and utility of IncRNA have already been established through evolution. Despite their potential, IncRNAs are exclusively used as diagnostic markers or drug targets (27-31). In contrast, this study is the first to report the sourcing and deployment of IncRNA as functional therapeutics.

[0270] LncRNA therapeutics require two important features: the delivery system and pure full-length or active IncRNA transcript with minimal immune system activation. A robust delivery system that can protect the IncRNA from degradation is crucial for successful delivery to desired tissues. LNPs are an established method for mRNA delivery. By adopting LNPs for use with IncRNAs, it was successfully demonstrated that 5 ’-capping and 3’ poly(A) tails are not necessary for the function of the model IncRNA. Functional screening of various unmodified and modified GAPLINC, MIST and DRAIR deployed exogenously in vitro can also control cell phenotype, for example, dependent on the chemical base modifications utilized.

[0271] Base modification of IncRNAs can differentially control cytokine expression in vivo. Due to the labile and immunogenic nature of RNA, unmodified RNA synthesized through IVT may not be feasible for clinical use (32). The incorporation of natural modified nucleotides into transcripts may enhance RNA biological properties. Low dose -modificd GAPLINC LNPs (0.10 mg / kg) targets IL- 1 p, a cytokine regulated by NF-KB, in the LPS-induced systemic inflammation mouse model.

[0272] Other modified IncRNA show different but compelling utility. Low doses of LNP-delivered unmodified MIST downregulated TNFa and IL-6. Additionally, -modificd MIST specifically targeted TNFa. Stapleton et al. implicated MIST in chronic systemic low-grade inflam mation(6). It was shown that loss of MIST led to the upregulation of cytokines such as TNFa and IL-6, shifting macrophages to a proinflammatory phenotype(fi). Thus, it is demonstrated herein that in an acute inflammation, MIST may target TNFa or IL-6 depending on base modifications utilized. Similarly, unmodified and -, m 1 -and m5C-modified DRAIR LNPs drastically downregulated TNFa and IL-6 expression, consistent with a prior study where overexpression of DRAIR attenuated TNF and IL-6 gene expression. Further, it was demonstrated that m I -modificd DRAIR may elevate IL- 10 production. Previously, -modificd RNA was characterized to perform best in suppressing RNA-mediated immune activation(22). Indeed, delivery of -modified GAPLINC may minimize IFN-y expression relative to other base modifications. However, while ml'P-modified MIST had the least activation of IFN-y, there were no differences observed among the different chemically modified DRAIR. Of note, chemical modifications of RNA nucleotides can alter secondary structure of IncRNA and impact fiinction(jj). Given the data, it appears that base modification-induced secondary structure changes alter function.

[0273] The IncRNA LNP modifications described herein included investigating the effects of increasing nanomaterial to RNA mass ratio. A 20:1 formulation was found to significantly improve LNP physical characteristics, IncRNA encapsulation and functional performance at a low dose of 0.10 mg / kg. The single dose effect of these IncRNA LNPs can be sustained for 6 h, demonstrating a utility for treating acute systemic inflammation. In summary, using systemic inflammation as a model disease, four distinct IncRNAs from the human transcriptome were identified and developed IncRNA-based drugs capable of targeting IL-1J3, TNFa and IL-6 in vivo. These IncRNA-based therapies depart from traditional strategies that use recombinant proteins for anti-inflammatory treatments.Materials and MethodsPlasmid construction, PCR amplification and digestion

[0274] To generate DNA template for IVT of the IncRNAs, the sequence of GAPLINC (Gene ID: 100505592), GAPLINC-202 (ENST00000579007.5), GAPLINC-205 (ENST00000700774.1), scrambled GAPLINC, MIST (MIST vl from Stapleton et al. ( ) were individually cloned into a pUCIDT-Kan GoldenGate vector from Integrated DNA Technologies (IDT). The sequence for DRAIR (NR 028417.2) was cloned into pTwist Kan High Copy vector from Twist Bioscience. Additionally, a T7 promoter was inserted before the IncRNA for IVT and an insert for a forward and reverse primer for PCR flank the T7 promoter and IncRNA. The end of the GAPLINC, GAPLINC-202, GAPLINC-205, and MIST gene contains a Nsil enzymatic cut site to allow for the reverse primer to be removed before IVT so that only the IncRNA gene will be present in the IVT amplicon. Similarly, the vector for DRAIR contains a Afel enzymatic cut site. The end of the scrambled GAPLINC sequence has an Apal enzymatic cut site. PCR amplification of plasmid DNA template was completed using the Q5 high-fidelity DNA polymerase (NEB, M0491). The resulting DNA was purified using the Monarch PCR & DNA Cleanup Kit (NEB, T1030L) following the manufacturer’s instructions. After purification, Nsil-HF (NEB R3127), Afel (NEB R0652S) or Apal (NEB R0114S) restriction enzyme were used to remove the reverse primer downstream of the IncRNA gene from the DNA template. For studying the biodistribution of the LNPs, a custom gene for firefly luciferase was obtained from IDT. The vector contained a T7 promoter, a minimal 5' untranslated region, and a 3' untranslated region derived from the mouse alpha globin sequence(40). All sequences used for this study can be found in Table 1.IncRNA IVT

[0275] For the unmodified IncRNA, RNA was synthesized using the HiScribe T7 high yield RNA synthesis kit (NEB, E2040S) following the manufacturer’s instructions. The protocol was altered for the modified IncRNA by using the following nucleotide modifications: cytidine triphosphate (CTP) was replaced with 5 -methylcytidine-5 ’ -triphosphate (m5C) (Hongene, R3-029), and uridine triphosphate (UTP) was replaced with pseudouridine-5 ’-triphosphate (T) (Hongene, R5-022) or Nl-methylpseudouridine-5’-triphosphate (m IT) (Hongene, R5-064). Reactions were incubated at 37°C for up to 20 h. All IVT RNA was purified using the Monarch RNA cleanup kit (NEB, T2050). To determine whether the cap and tail are necessary for GAPLINC function, GAPLINC RNA with cap only or cap & tail was generated. The IVT RNA was capped (10 pg per reaction) using the Vaccinia capping system (NEB, M2080) and mRNA Cap2-O-Methyltransferase (NEB, M0366) at 37°C for 60 min and immediately purified. A poly-A tail was added to the IVT RNA (10 pg per reaction) by using Escherichia coli poly(A) polymerase (5000 U / mL; NEB, M0276) at 37°C for 30 min and immediately purified.Cellulose chromatography of IVT IncRNA

[0276] To purify dsRNA contaminants, a cellulose purification protocol was adapted from Baiersdorfer et al. and Zhong et al. Chromatography buffer containing 10 mM HEPES (pH 7.2), 0.1 mM EDTA, 125 mM NaCl and 16% (v / v) ethanol was prepared. Cellulose was prewashed with chromatography buffer (0.2 g cellulose / mL) by incubating on a multi-purpose tube rotator for 10 min at 80 rpm. 700 pL of cellulose slurry (-0.14 g cellulose) was transferred to 0.45 pm cut-off ultrafiltration spin-columns in 1.5 mb Eppendorf tubes. 100 to 500 pg of IVT IncRNA were loaded to the ultrafiltration spin -columns and incubated on a multi-purpose tube rotator for 30 min at 80 rpm. Samples were centrifuged at 14,000 x g for 60 s and flowthrough containing single stranded IncRNA was collected. The purified IncRNA was recovered by incubating with 0.1 vol 3 M sodium acetate (pH 5.5) and 1 vol isopropanol for 30 min at -20°C. Precipitated IncRNA was pelleted by centrifugation for 15 min at 14,000 x g. The pellet was washed with 500 pL 70% pre-cooled ethanol and then dissolved in 50-100 pL nuclease-free water. Bioanalyzer at the SickKids TCAG Microarray Facility was used to assess the integrity and purity of the purified IncRNA.Reverse-phase HPLC purification of IVT IncRNA

[0277] IncRNA was purified using an Agilent BioHPLC PLRP-S reverse-phase column (lOOOA, 10 pm, 150 mm x 10 mm) packed with poly rigid macroporous styrene / divinylbenzene (PS / DVB) (Agilent, PL1112-9999) connected to an Agilent 1260 Infinity II Bio-Inert liquid chromatography system. IVT IncRNA was purified at either 45, 60 or 70°C with a linear gradient of 38-65% buffer B (0.1 M triethylammonium acetate [TEAA, pH 7.0] and 25% [v / v] acetonitrile) over 6 (unmodified RNA) or 8 (modified RNA) column volumes at a flow rate of 2.4 mL / min. This was followed by a linear gradient of 65-100% buffer B over 0.2 column volumes, a hold at 100% buffer B for 3 column volumes and a lineargradient from 100-38% buffer B over 0.2 column volumes. Elution fractions from major peaks were collected, concentrated, and desalted by multiple centrifugations using Amicon Ultra-15 30 kDa centrifugal filter units (Millipore Sigma, UFC903024), and dilution with nuclease-free water. The purified IncRNA was recovered by incubating with 0.1 vol 3 M sodium acetate (pH 5.5) and 1 vol isopropanol overnight at -20°C. Precipitated IncRNA was pelleted by centrifugation for 15 min at 14,000 x g. The pellet was washed with 500 pL 70% pre-cooled ethanol and then dissolved in 50-100 pL nuclease-free water. Bioanalyzer was used to assess the integrity and purity of the purified IncRNA.Dot Blot analysis of dsRNA

[0278] LncRNA and the positive control synthetic dsRNA (poly (EC) [LMW]; Invivogen, tlrl-picw) was diluted in nuclease-free water to a final concentration of 40 ng / pL. Positively charged Whatman Nytran SuPerCharge nylon membrane (Sigma- Aldrich, WHA 10416230) was placed on a sheet of Whatman GB005 blotting paper (Sigma- Aldrich, WHA10426972). Loading of RNA was performed by pipetting 5 pL (200 ng RNA) of the diluted sample onto the membrane. The sample liquid was drained under the membrane and carried to the underlying blotting paper via capillary forces, allowing the negatively charged RNA to be capture by the positively charged membrane. The air-dried membrane was blocked with 5% (w / v) non-fat dried milk in Tris-buffered saline (TBS) buffer with 2% Tween 20 (TBS-T; Thermo Scientific, AAJ60497K2) on a rocking shaker for 1 h at room temperature. The membrane was washed three times with 35 m TBS-T for 5 min each. To detect dsRNA, the membrane was incubated on a rocking shaker for 2 h at room temperature with J2 anti -dsRNA murine antibody (Novus Biologicals, NBP3-11395-200UG) diluted 1 :5000 in TBS-T containing 1% (w / v) non-fat dried milk. Then the membrane was washed three times with TBS-T for 5 min each. Subsequently, it was incubated with horseradish peroxidase (HRP)-conjugated donkey anti-mouse immunoglobulin G (IgG) (H+L; Jackson ImmunoResearch Laboratories, 715-035-150) diluted at 1: 10,000 in TBS-T containing 1% (w / v) non-fat dried milk on a rocking shaker for 1 h at room temperature. After washing the membrane with 35 mL TBS-T for 15 min, chemiluminescence detection was performed using the SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Scientific, 34094) as per manufacturer’s instructions. The iBright 1500 Imaging System was used to image membrane using the Chemi-Blot setting.Lipid synthesis, formulation and characterization of IncRNA LNPs

[0279] The ionizable lipids, C3-K2-E14 and OC2-K3-E10, were synthesized as previously described by Tilstra et al(23) (FIG. 7A and B). To formulate particles, the ionizable lipid was combined with DMG-PEG2000 and mixed with unmodified or modified IncRNA in a microfluidic device as previously described(23). Lipid components (Mol% ratio: 50% ionizable lipid, 38.5% cholesterol, 10% 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1.5% DMG-PEG2000) was dissolved in ethanol. All formulations were prepared in citrate buffer (45-80 mM) with pH 3 with a total flow rate of 3.5 mL / min.The materials were formulated with a nanomaterial: RNA mass ratio of 7, 9 or 20 Particles were dialyzed 1: 1000 with a Slide -A-Lyzer™ G2 dialysis cassettes (20K MWCO; Thermo Scientific, 87734) against IX PBS to remove ethanol and exchange buffer before sterile filtration through a 0.22 pm polyethersulfone syringe filter. The particles were then concentrated using an Amicon Ultra-0.5 mb 10 kDa centrifugal filter unit (Millipore Sigma, UFC501024) to a concentration of at least 500 ng / pL. All particles were stored at 4°C for up to 1 week prior to use. The size, polydispersity index (PDI) and zeta potential of LNPs were determined using dynamic light scattering (DLS). LNPs were equilibrated at room temperature and diluted in water at 1: 1000 for DLS measurements using the Zetasizer Ultra Red. The target PDI was below 0.2 to ensure consistency of the LNPs. The size of LNPs was expected to be less than 200 nm as larger particles were filtered. The concentration of RNA and encapsulation efficiency of the LNPs was determined using the Quanti-it™ RiboGreen RNA assay kit (Invitrogen, R11490) following the manufacturer’s protocol. All characterization data for formulations used herein can be found in Table 2.Biodistribution of LNPs

[0280] C57BL / 6 mice (The Jackson Laboratory) were administered a 2 pg of firefly luciferase mRNA encapsulated in nanoparticles through tail vein injection. Six hours post injection, mice were administered 3 mg of d-luciferin (Thermo Scientific, 88291), and liver, spleen, kidneys, heart, and lungs were collected for in vivo imaging system (IVIS) detection. IVIS measured fluorescence at an excitation of 535 nm and emission of 600 nm. The total luminescent flux (photons / second) in the organs was determined with the automatic region of interest tool.NF-KB and Interferon Regulatory Factor (IRF) induction assay

[0281] To determine the efficacy of LNPs encapsulating unmodified and modified IncRNA in reducing NF-KB and IRF activation, RAW-Dual (IRF-Lucia / KI-[MIP-2] SEAP) reporter cells acquired from Invivogen were used. These cells were cultured in DMEM with high glucose and pyruvate (Gibco, 11995073) and supplemented with 10% heat inactivated fetal bovine serum (FBS) and 1% penicillinstreptomycin (5000 U / mL). To maintain selective pressure, 100 pg / mL of normocin and 200 pg / mL of zeocin was added into the growth media at every other passage. RAW-Dual cells were seeded at 100,000 cells per well in a 96-well plate. The cells were co-stimulated with 100 ng / mL LPS and 200 ng LNP encapsulating unmodified, modified, scrambled or irrelevant IncRNA or Silencer Select SiRNA (Thermo Fisher Scientific, AM16708; see Table 5) for 3, 6, 18 or 21 h at 37°C with 5% CO2. IncRNA in lipofectamine MessengerMAX transfection reagent (Thermo Scientific, LMRNA003) was used as indicated as per manufacturer’s protocol. Following stimulation, supernatant was collected for SEAP detection of NF-KB induction using QUANTI-Blue and IRF induction using QUANTI-Luc as per manufacturer’s instructions. SEAP levels were measured using the BioTek Synergy Hl microplate reader set at wavelength of 655 nm. Cells were collected for subsequence gene expression analysis.LPS stimulation of human PBMC-derived monocytes and macrophages

[0282] To investigate the efficacy of LNPs encapsulating modified IncRNA in primary human cells, human PBMC-derived monocytes and macrophages from females and males (STEMCELL Technologies, 70034 and 200-0372, respectively) were used. These cells were cultured in Immunocult-SF macrophage medium (STEMCELL Technologies, 10961). To maintain the human PBMC-derived macrophages, macrophage colony-stimulating factor (50 ng / ml) was added to the culture (STEMCELL Technologies, 78057.1). Cells were seeded overnight at 100,000 cells per well in a 96-well plate. The cells were then costimulated with LPS (100 ng / ml) and 200 ng of 'P-modified IncRNA in Lipofectamine MessengerMAX transfection reagent (Thermo Fisher Scientific, LMRNA003) for 3, 6, or 24 hours at 37°C with 5% CO2. The supernatant was subsequently collected for analysis using Luminex. Cells were collected for gene expression analysis.Gene expression analysis by reverse transcription qPCR

[0283] Cells were directly lysed using the TaqMan Cells-to-CT Express Kit (Thermo Fisher Scientific, A57986), and lysates were used directly for reverse transcription using the SuperScript IV VILO Master Mix. Quantitative PCR (qPCR) analysis was completed using the TaqMan Fast Advanced Master Mix and predesigned TaqMan gene expression assays (Applied Biosystems, 4331182) (see Table 5 for assays used). Fast reaction (10 pl) reverse transcription qPCR was carried out using a microamp 96-well Fast Optical reaction plate in a QuantStudio 3 system for 40 cycles (95°C for 1 s and 60°C at 20 s). Relative quantities of each mRNA were determined using the comparative AACT method with GAPDH (glyceraldehyde-3-phosphate dehydrogenase) as the reference gene.IncRNA LNP in vivo testing

[0284] C57BL / 6 wild-type mice were purchased from Charles River Laboratories. To induce systemic inflammatory responses, 100 pg (5 mg / kg) of LPS was administered to the mice intraperitoneally, with control mice receiving PBS injections. Simultaneously, LNPs were administered via intravenous tail vein injection at 0.1, 1, 3 or 5 mg / kg (200 pL / animal). For testing of empty nanoparticle controls, the amount of LNPs injected is equivalent to the amount of delivery material contained in the 1, 3, 5 mg / kg GAPLINC LNP doses. Blood was collected via saphenous vein blood draw 6 h after injections for serum cytokine analysis.Serum Analysis

[0285] To evaluate the efficacy of the IncRNA LNPs on reducing inflammation, mouse serum was collected from C57BL / 6 mice treated with or without LNPs. This study used Luminex xMAP technology for multiplexed quantification of 10 Mouse cytokines, chemokines, and growth factors. The multiplexing analysis was performed using the Luminex™ 200 system (Luminex, Austin, TX, USA) by Eve Technologies Corp. (Calgary, Alberta). Ten markers were simultaneously measured in the samples usingEve Technologies' Mouse Focused 10-Plex Discovery Assay® (MilliporeSigma, Burlington, Massachusetts, USA) according to the manufacturer's protocol. The 10-plex consisted of GM-CSF, IFNy, IL- ip, IL-2, IL-4, IL-6, IL- 10, IL-12p70, MCP-1, and TNFa. Assay sensitivities of these markers ranged from 0.4 to 10.9 pg / mL for the 10-plex assay. For data analysis purposes, out of range below the standard curve readings were designated to 0.001 pg / mL.Animal Weight Maintenance

[0286] Animal weights were recorded prior to and after PBS or pseudouridine-modified GAPLINC LNP (0.1 mg / kg; 200 pL / animal) treatments. Weight change was recorded by dividing initial weight by final weight (i.e., normalizing post sacrifice weight to pretreatment weight).Histology Analysis

[0287] Pseudouridine-modified GAPLINC LNP- and PBS-treated mice were sacrificed by CO2 inhalation and perfused with PBS followed by 4% paraformaldehyde. Spleen, liver, and kidneys were harvested and weighed after seven days of treatment. Organs were post-fixed in 4% paraformaldehyde, cryopreserved in a 30% sucrose gradient and embedded in optimal cutting temperature compound (Fisher Scientific, 23-730-571). 5 pm sections were obtained and stained with Hematoxylin (Sigma-Aldrich, 51275) and Eosin (Sigma-Aldrich, HT110116), mounted with Permount (Fisher Scientific, SP15-100), and visualized on an Axiolmager 2 microscope (Zeiss, Germany).Statistical Analysis

[0288] Statistical analysis was completed using GraphPad Prism 9 and data were represented as mean ± SEM. Comparisons of two groups were calculated using paired two-tailed Student’s t- test. Oneway analysis of variance (ANOVA) and Tukey’s multiple comparison test was used for comparisons of more than two groups.Example 2

[0289] As shown in FIG. 22, GAPLINC 202 and 205 which share overlap with GAPLINC that lack function, lack the first 19 nucleotides of GAPLINC. The first 19 nucleotides may be important for activity. Substitutions are made for example, outside of the first 19 nucleotides, for example so at least 90% sequence identity is maintained. The first 19 nucleotides can comprise chemically modified nucleotides.

[0290] Fig. 22 more particularly shows that the the length of GAPLINC is 511 nts, GAPLINC-202 is 653 nts, GAPLINC-205 is 420 nts. GAPLINC-202 exon 1 aligns with position 48-108 on GAPLINC exon 1. GAPLINC-202 exon 2 aligns with position 109-278 on GAPLINC 2. GAPLINC-202 exon 3 does not align with any position on GAPLINC. GAPLINC-202 exon 4 aligns with position 279 to 501 on GAPLINC exon 3. GAPLINC-205 exon 1 aligns with position 19-108 on GAPLINC exon 1. GAPLINC-205 exon 2 aligns with position 109-278 on GAPLINC 2. GAPLINC-205 exon 3 does not align with any position on GAPLINC.Example 3Modification of the sugars on IncRNA

[0291] The results show that incorporating 2’OMe-UTP in IncRNA can reduce Illb gene expression following 6 and 24 h of LPS stimulation in human PBMC-derived monocytes to the same degree as other base modified versions of GAPLINC, such as those which incorporates base modifications. As demonstrated herein 'P-modified GAPLINC outperform natural, unmodified GAPLINC, the sugar modifications are also useful performance -enhancing method.MethodsPlasmid construction

[0292] To generate a DNA template of the IncRNA for IVT, GAPLINC (Gene ID: 100505592) was cloned into a pVAXl pro vector from GenScript. A T7 promoter was inserted before the IncRNA for IVT. The end of the GAPLINC sequence was flanked by a Nsil enzymatic cut site at the 3’ end of the sequence to allow for linearization of the plasmid. All sequences used for this study can be found in Table 4.IncRNA IVT

[0293] For the base modified IncRNA, RNA was synthesized using the HiScribe T7 high yield RNA synthesis kit (NEB, E2040S) following the manufacturer’s instructions. Uridine 5 ’ -triphosphate was replaced with (Hongene, R5-022). Reactions were incubated at 37°C for up to 21 hours. For the sugar modified IncRNA, RNA was synthesized using T7 RNA Polymerase Mui (Hongene, 25KU), 10 mM 2'0-Me-UTP (Hongene, R5-016), 10 mM ATP (Hongene, R1331), 10 mM GTP (Hongene, R2331), 10 mM CTP (Hongene, R3331), and IVT buffer containing 200 mM HEPES (pH 7.3), 5.5 mM MgCL, 2 mM spermidine, 40 mM DTT, 0.01% Triton, and 1.5 mM MnCL. Reactions were incubated for up to 21 hours at 37°C. To remove DNA template, reactions were incubated with DNase I (NEB, M0303S) according to the manufacturer’s instructions. All IVT RNA was purified using the Monarch RNA Cleanup Kit (NEB, T2050L).LPS stimulation of human PBMC-derived monocytes

[0294] To investigate the utility of base and sugar modified IncRNA in primary human cells, human PBMC-derived monocytes from females and males (STEMCELL Technologies, 70034) were used. Cells were cultured in Immunocult-SF macrophage medium (STEMCELL Technologies, 10961). Cells were seeded overnight at 100,000 cells per well in a 96-well plate. The cells were then costimulated withLPS (100 ng / mL) and 200 ng of T-modificd IncRNA and 2'0-Me-UTP -modified IncRNA in Lipofectamine MessengerMAX transfection reagent (Thermo Fisher Scientific, LMRNA003) for 3, 6, 24 h at 37°C with 5% CO2. Cells were collected for gene expression analysis.Gene expression analysis by reverse transcription quantitative polymerase chain reaction (RT-qPCR)

[0295] Cells were directly lysed using the TaqMan Cells-to-CT Express Kit (Thermo Fisher Scientific, A57986) and lysates were used directly for reverse transcription using the SuperScript IV VILO Master Mix. Gene expression analysis was completed using the TaqMan Fast Advanced Master Mix and predesigned TaqMan gene expression assays (Applied Biosystems, 4331182) (see Table 5 for assays used). Fast reaction (10 pL) RT-qPCR was carried out using a microamp 96-well Fast Optical reaction plate in a QuantStudio 3 system for 40 cycles (95 °C for 1 s and 60°C for 20 s). Relative quantities of each mRNA were calculated using the comparative AACT method with GAPDH (glyceraldehyde-3-phosphate dehydrogenase) as the reference gene.Statistical Analysis

[0296] Statistical analysis was completed using GraphPad Prism 10, and data were represented as the means ± S.D. A one-way analysis of variance (ANOVA) and Tukey’s multiple comparison test were used for comparisons of more than two groups.ResultsSugar modified IncRNA can be synthesized using in vitro transcription (IVT)

[0297] As a proof-of-concept, we sought to synthesize 2’OMe-UTP modified GAPLINC using IVT. The use of canonical T7 RNA polymerase led to zero yield, suggesting the need for a mutant T7 RNA polymerase that will overcome the potential steric hindrance posed by the incorporation of 2’OMe-UTP. In utilizing the T7 RNA Polymerase Mui from Hongene, we were able to achieve synthesis of 2’OMe-UTP-modified GAPLINC (Fig. 24). Bioanalyzer revealed a distinct dominant band at 4000 nucleotides. Incorporation of 2’OMe-UTP may change the RNA secondary structure, charge density and RNA interaction with the polymer matrix, which can lead to slower electrophoretic mobility. Therefore, although we expected GAPLINC to be around 500 nucleotides, it is not surprising that the bioanalyzer results reveal an apparently larger fragment.

[0298] To determine the effects of sugar-modified IncRNA compared to base-modified IncRNA, we challenged human PBMC-derived monocytes with LPS (100 ng / ml) and treated the cells for 3, 6, and 24 h with 165 ng of 2’OMe-UTP- or -modificd GAPLINC delivered using Lipofectamine. Results of gene expression analysis show that there were no changes in Illb at 3 h compared to LPS control. At 6 h, therewas a significant reduction of Illb for both - and 2’OMe-UTP-modified GAPLINC relative to LPS. This decrease in Illb was sustained at 24 h (Fig. 25).

[0299] In both timepoints, - and 2’OMe-UTP-modified GAPLINC had similar effects in lowering Illb, suggesting that 2’0Me modification maintains functional performance of GAPLINC.Example 4Exogenous delivery of IncRNA LNPs

[0300] It was shown that exogenously delivered -modificd GAPLINC retains anti-inflammatory effects of IL- ip for 3-6 h. This demonstrates that the LNPs administered can suppress the early rise of cytokines and potentially limit downstream inflammatory propagation observed in acute inflammatory diseases.Subcutaneous injection of ^-modified GAPLINC LNPs

[0301] The results show that -modificd GAPLINC LNPs can be subcutaneously injected to lower IL-1 expression. These results were also comparable to the intravenous route of administration, as no substantial differences were observed between the two modes of injections tested. S.C. LNP delivery offers several advantages over intravenous route of administration. There can be greater convenience and patient accessibility using subcutaneous injections(56).MethodsPlasmid construction

[0302] To generate a DNA template of the IncRNA for IVT, GAPLINC (Gene ID: 100505592) was cloned into a pVAXl pro vector from GenScript. A T7 promoter was inserted before the IncRNA for IVT. The end of the GAPLINC sequence was flanked by a Nsil enzymatic cut site at the 3’ end of the sequence to allow for linearization of the plasmid. SEQ ID NO: 1 and SEQ ID NO:2 were used for this study.LncRNA IVT

[0303] For the base modified IncRNA, RNA was synthesized using the HiScribe T7 high yield RNA synthesis kit (NEB, E2040S) following the manufacturer’s instructions. Uridine 5 ’ -triphosphate was replaced with (Hongene, R5-022). Reactions were incubated at 37°C for up to 21 hours. For the sugar modified IncRNA, RNA was synthesized using T7 RNA Polymerase Mui (Hongene, 25KU), 10 mM 2'0-Me-UTP (Hongene, R5-016), 10 mM ATP (Hongene, R1331), 10 mM GTP (Hongene, R2331), 10 mM CTP (Hongene, R3331), and IVT buffer containing 200 mM HEPES (pH 7.3), 5.5 mM MgCL, 2 mM spermidine, 40 mM DTT, 0.01% Triton, and 1.5 mM MnCL. Reactions were incubated for up to 21 hoursat 37°C. To remove DNA template, reactions were incubated with DNase I (NEB, M0303S) according to the manufacturer’s instructions. All IVT RNA was purified using the Monarch RNA Cleanup Kit (NEB, T2050L).RP-HPLC purification of IVT IncRNA

[0304] IncRNA was purified using an Agilent BioHPLC PLRP-S reverse-phase column (1000 A, 10 pm, 150 mm by 10 mm) packed with polyrigid macroporous styrene / divinylbenzene (Agilent, PL1112-9999) connected to an Agilent 1260 Infinity II Bio-Inert liquid chromatography system. IVT IncRNA was purified at 70°C with a linear gradient of 38-50% buffer B [0.1 M triethylammonium acetate (pH 7.0) and 25% (v / v) acetonitrile] over 0.2 column volumes at a flow rate of 1.2 ml / min. Then a linear gradient from 50-65% buffer B over 4.4 column volumes at a flow rate of 2.4 ml / min. This was followed by a linear gradient from 65-100% buffer B over 0.2 column volumes then 100-38% over 0.2 column volumes. A hold at 38% buffer B was completed over 1.2 column volumes. Elution fractions from major peaks were collected, concentrated and desalted by multiple centrifugations using Amicon Ultra-15 30-kDa centrifugal filter units (Millipore Sigma, UFC903024) and dilution with nuclease -free water. The purified IncRNA was recovered by incubating with 0.1 vol 3 M sodium acetate (pH 5.5) and 1 vol isopropanol overnight at -20°C. Precipitated IncRNA was pelleted by centrifugation for 15 min at 14,000g. The pellet was washed with 500 pl of 70% precooled ethanol and then dissolved with 50 pl of nuclease-free water.Lipid synthesis, formulation, and characterization of IncRNA LNPs

[0305] The ionizable lipid, OC2-K3-E10, was synthesized as previously described by Tilstra et al. (53, 27, 59). To formulate particles, the ionizable lipid was combined with DMG-PEG2000 (l,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000) and mixed with unmodified or modified IncRNA in a microfluidic device as previously described (53, 27, 59). Lipid components (mol % ratio: 50% ionizable lipid, 38.5% cholesterol, 10% l,2-distearoyl-sn-glycero-3-phosphocholine, and 1.5% DMG-PEG2000) were dissolved in ethanol. All formulations were prepared in citrate buffer (70 mM) with pH 3 at a total flow rate of 3.5 ml / min. The materials were formulated with a nanomaterial: RNA mass ratio of 20. Particles were dialyzed 1:1000 with Slide-A-Lyzer G2 dialysis cassettes (20,000 molecular weight cutoff; Thermo Fisher Scientific, 87734) against 1 x PBS to remove ethanol and exchange buffer before sterile filtration through a 0.22-pm polyethersulfone syringe filter. All particles were stored at 4°C for up to 1 week before use. The size, PDI, and zeta potential of LNPs were determined using dynamic light scattering. LNPs were equilibrated at room temperature and diluted in water at 1:1000 for dynamic light scattering measurements using the Zetasizer Ultra Red. The target PDI was below 0.2 to ensure the consistency of the LNPs. The size of LNPs was expected to be less than 200 nm as larger particles werefiltered. The concentration of RNA and the EE of the LNPs were determined using the Quanti-it RiboGreen RNA assay kit (Invitrogen, R11490) following the manufacturer’s protocol.LncRNA in vivo testing

[0306] C57BL / 6 wild-type female mice (aged 9-10 weeks) were purchased from Charles River Laboratories. Systemic inflammation was induced in the mice using 100 (5 mg / kg) that was administered intraperitoneally. Simultaneously, LNPs were administered via EV. or S.C. injection at 0.1 mg / kg (200pl per animal). Blood was collected via saphenous vein blood draw 6 hours after injections for serum cytokine analysis. For duration of effect study, LNPs were administered using intravenous tail vein injections at 0.1 mg / kg (200pl per animal). Then, after 3 and 6 hours, 20 pg (1 mg / kg) of LPS was administered to the animals intraperitoneally. Blood was collected via saphenous vein blood draw 4 hours after LPS administration for serum cytokine analysis.Serum Analysis

[0307] To evaluate the efficacy of the IncRNA LNPs on reducing LPS-induced inflammation, mouse serum was collected from C57BL / 6 mice. This study used Luminex® xMAP® technology to quantitatively and simultaneously detect fourteen mouse cytokines, chemokines and growth factors. The multiplexing analysis was performed by Eve Technologies Corporation (Calgary, Alberta, Canada) using the Luminex® 200™ system (Luminex Corporation / DiaSorin, Saluggia, Italy) with Bio-Plex Manager™ software (Bio-Rad Laboratories Inc., Hercules, California, USA). Fourteen markers were measured in the samples using the Eve Technologies' Mouse Cytokine / Chemokine Focused 14-Plex Discovery Assay® Array (MDF14) as per the manufacturer’s instructions for use (MILLIPLEX® Mouse Cytokine / Chemokine Magnetic Bead Panel Cat. #MCYT1-19OK, Millipore Sigma, Burlington, Massachusetts, USA). The 14-plex consisted of GM-CSF, IFNy, IL- 1 , IL-2, IL-4, IL-5, IL-6, IL- 10, IL-12p70, IL- 13, IL-17A, IL-17F, MCP-1 / CCL2, and TNFa. Assay sensitivities of these markers range from 0.52 - 6.61 pg / mL. Individual analyte sensitivity values are available in the MilliporeSigma MILLIPLEX® protocol.Statistical Analysis

[0308] Statistical analysis was completed using GraphPad Prism 10, and data were represented as the means ± S.D. A one-way analysis of variance (ANOVA) and Tukey’s multiple comparison test were used for comparisons of more than two groups.ResultsExogenously delivered E-modified GAPLINC retains anti-inflammatory activity for 3-6 h

[0309] In the previous Examples it was shown that a high dose of LPS (5 mg / kg) in C57BL / 6 mice can induce a rapid spike in inflammatory cytokines in 6 hours, allowing us to evaluate the efficacy of -modified GAPLINC LNPs in attenuation of LPS-induced inflammation. Since this model only allowed toevaluate how -modificd GAPLINC LNPs can suppress a strong acute inflammatory response at a single time point, the inventors wanted to next evaluate how long the anti-inflammatory effect of 'P-modified GAPLINC LNPs persists.

[0310] LNPs were formulated with a nanomaterial -to-RNA mass ratio of 20:1 using OC2-K3-E10. The average size of the LNPS with 'P-modified GAPLINC was 115 nm with a polydispersity index (PDI) was below the 0.20 best practice threshold (6). The particles were 6 mV. The encapsulation efficiency (EE) was 86%. All LNPs were formulated using HPLC-purified 'P-modified GAPLINC. Trace of purification can be found in Fig. 26.

[0311] To evaluate the duration of effect, 'P-modified GAPLINC LNPs were administered using I.V. injection at 0.1 mg / kg, then LPS (1 mg / kg) was either administered intraperitoneally at 0, 3, or 6 hours post-LNP treatment. Serum samples were collected 4 hours post-LPS injection. By using a lower LPS dose, we produce a moderate and more sustained inflammatory response, enabling us to monitor the expression of inflammatory cytokines in a wider dynamic window. Results revealed that IL- 1 [3 expression was significantly higher at 6 hours compared to 0 and 3 hours post-LNP treatment (Fig. 27). Therefore, results suggest that the 'P-modified GAPLINC LNPs likely retains anti-inflammatory protective effects between 3-6 hours.Subcutaneous injection of P-modified GAPLINC achieves similar efficacy as intravenous route of administration

[0312] To test GAPLINC in vivo using subcutaneous as the administration route, systemic inflammation in C57BL / 6 mice were induced using lipopolysaccharide (LPS) injected intraperitoneally (5 mg / kg). These animals were simultaneously injected intravenously and subcutaneously with LNPs containing 'P-modified GAPLINC (0.1 mg / kg). Results showed that there were no substantial differences in IL- 1 P expression between intravenous and subcutaneous routes of administration (Fig. 28). Thus, it was shown that GAPLINC LNPs may be administered subcutaneously to attenuate LPS-induced inflammation in mice.TablesTable 1. Oligonucleotides for GAPLINC DNA template and RNA.Name and Sequence (5’ to 3’) and / or SEQ ID NOAccessionGAPLINC ACTTGCAGGATCTGACACATCCTCTTGGTTTCCTAAGTCTTATGACTAGCCAATGCCTGAAAT DNA Template AATGAACTCCTCCAAGGCAAGAAATCTGTTTTGAAGCTTCTCTGCGTTCACACACAGCAGCCT GGTTTCCTGGAAGGGCATTTTCCACATTGTGCGTTATGGATGATCATCCCAGGCATCAGGTGT GAAGCCCTGCATCCACATCCAGGGGCTATCAAATCTCTCTGCAAAAGGAGAAGCTGGACTCAG GCACGTTTACAGT GAT GT GTAT GCAGGGTAT GCACAGAT GT GGAAACAGGAACT GAT GT GT CC ATTACACCACTAGGACAGAGGCCAGAACAATGAAGAAACCAAATACTTGGAAGAGGGTAGAGA TAATGAATGGAGTCCAAGAGCCCTGATTGTGCCATAAATGTCCAGATAATTCCATACCTGAGG ATTATGTGGTTTGTAAACTTGGCACTTAGAAGAACCAATAAAATCATGTTATAGTTTCAAAAA AAAAAAA ( SEQ ID NO : 1 )Scrambled ( SEQ ID NO : 2 )GAPLINC DNA TemplateGAPLINC-202 ( SEQ ID NO : 3 )DNA TemplateGAPLINC-205 ( SEQ ID NO : 4 )DNA TemplateDRAIR DNA GACAGTTCCTCCCAGCTCTGACAGCCCGCGGGTGCGGCTGCAGGTGCCGCCTCGGCTTCATTATemplate GCCCAGCTCAGGACAAACTCCAGAAGGGAACAACCCCAGTTCTGGTCCCCTCGGTCACCCCGG GGAAGGACCCAGCCCTAGCTGCTCACTCTCCGCCCGCCTTCCCGAGAGTGCTGCCCCGACCCC GGGTCCGCCGCCAAGCCGGAGCGCCCGGGGCGTTCGAGGGGGCTTGGCGGCTCCTGTTCCCAC CGCGCACGCCTTACCTGGTGGTGGTTGTACGAGTTCCTCTGCTGCAGGAAGGCGGCTGCGGCA GCCTGGTGCTGCTGCTGCAGCTGCGGGCTGACGGGTGACCTCCGGCTCTGCGGCTGCTGCGGC GCCGCCGCCGGCGGCTGCTGTTGAGGATGATGCCCTCCAGGTCTATCCATTTGCCTAGGAACC AAGAAGTCTTAATAACTGTTTTGGTGATTGGGGCCAGCAGAAACCTTCTTCCTGACATTCTTC TAAGTGACAGGATGTGTGTAGTCTGCTCTCAACTCCCCACTGGTCCAGCTGAGGCTGCCTGAG GAAAGCACTTTTCTCTGAGGTATATTCTTGCCACAGTGGTCACTGATGTGCTTCAAACATGGA AAGAAGAAGTCTCATATGGTCCTACCCCTATAGAAAGAACCACGGTTAACTAATGACTGCTGG ATGAAGTTCCTGCTTCCAGCTTCCTGCCAAGACTTCCTTTAATGATGGACTGTAACCTGAAGC TCAAACCCCAAATGACGTTCCTGCTCTGTTGCCTGTACCCTGGATTGCTCACCACCTGAGCCT ACCTGCCCTTGTGGGGTTGGAGGTGAGGTGTCACAAAGTCATCAACACAGACTCTGGAAGCAG AGAAGGCTCCTGGAAGGAACTGCTCAGCTGGGGACTACACCTTCTTTTTGTTTTGGTACAGAG AGAGACCAGCTGACTGGTTTTCAGAATGAAAAGAGTCTAGCTGGCAGCCCAAAGAGGTGACTG AGAACTGGCTGTGTAAGAACAGCAGAGCTTCCTGGGAGAAACAGGCTTCATTTTTCCTGCTTT AAAAAAAAAATATGCGAGAACTCCAATGTTCTGGCAAAATTAAGGAAACCTGCAATCAGTTGC TTGGAAAAAAAAACCCAGCAAACCTGTCTGTAGTAAATTACATATTGGGACAAAGCCCCACTG ACTCCCGAGGCTGCCTTCCTCCCTGCACCCCACCCTGCTGCCATCCTTAAGGTAATGTCCTTC AGACTGCACAAGCTGCATTTCAGAGACCTGCAGTCCAAACTTGACACCTAAGAACTGTGTCCC TTTAGGTAAGTCTCATGGCTTTTTGTCTGAGTTTCATGATATTATTTGTAACATGACCTTAAA TCTGTTTCTTCATATTGATATGAGAAATAAACACACTTTCATCAGC ( SEQ ID NO : 5 ) MIST DNA TTGTGGTCACACCAACAGTCTGTCATAAAAGGAGTCTTTGTTCTTCTGTGGAGCTTTCCCAAATemplate CTTCTATGTGAATGTCATTCATGCTACTAGTTTGAAGCTCTGAAATCCATCTCTGGGAATTTC CTTTCTTGTATTTTTGTTTTCTTTCAGTAATAGTCTAGATAAAAAAGTCAAGAAGCTAAGAAA CAGGATATAAAACAAACAATAACAACAACAAAAAAGTGCCAGGGCTTCAGAGACCTTCACACA TGGGTACCCACAACCTGGTATGGCCATGCACATGGAAGACGGAGAAGAAGGCACTGGCTTTGT GCCTAAGCAAATTCAAGGCTTTACGTAATCATCGGTGACAAAGAAAGAAGACCTCCACTATAA AAC AGAC AT C AAT TCTTTAACT GT AGAAT AAAT GAT T C C C C GAT C C AGAT GT T T GAT GT G C AA ACAGGGAAAGATGTGATCCCTGCTAGTAAACTGCCTGCTGGGGAAACCATTGTGCAACATAGAAGCTTCCCTAACTAGGTGACCAACACATCCATTGTCACAAGGCTACCTCTGTCGCTGCCCTCC ATGTGAGCTGCTGTGTGTTGAAAACACTGGCAGAAGCAGCCTCCCCAGCAAGTTTTCGCCTGC TGGAGATCTGTCTCTGAGACAAATATAGAGGATGGAGACGTTTGTCGGCTTGTCTGCCATCAA AGACCCAATTGCTTCCTGCGTTGCAGGAAATGAAGTGCAGCCTCTCAGCCCCGTCCACCTCCA CAAGTGACTGTCAGTGGAGAACAAGCTGAAGCAGTTGGACTTGCTCTGAGTTCCCAGATCTGA GCCAATTCAAAGCTCACAATGTCATTAAACCACCAGTACTTGGAGGAGAGCTTAAACACTGCT TTGTCCAGTTACTTTCAAAGAGAGTTTCTAGGAAGAGCTATTCCAAATATCAGTCTGTCATTT TCACCACCGAAGAAATTTTTAAAGTGTGCAGTGCAGGAAGAGACAGGAAGAAGAAGGGACTGG ATTGAAAGAGAATGGTCCCTTGTGATCTGGTGACAGAGCCGGACAGAGCAGATGTCATAAGAC TCCAGTTATTTTCCAGCTCTGGTGTGACAGTGGTGTGACCAGCAGCCAGTCATACCACCCTGC CTTTCACAACCTTGAAGAGACACGTCACTCAGCTCTCTAAACCAGTTTGTCAGCTTCATCAGT CCAGCACACAAGAGCAT CTAAGCAGCAGT CAACAGAGCT GAGAGAAAT CAAAAAGGGGAAAAA AAACAACAAAAAACTAGGAGAAAGCT GTAATT CTT CT CCTAAGT CAACAGACAAT GCAATAGA CGTTCACTGACTTTAAAACAGTAAGCACTGGGGTTTTGAGAAAACAGCTGATGACCTTAAGAA T CAAGAAAGCAT GT CAGCAATAGACGAAATAGAACCACT GT GTATAT GGCATAGAGCAAT GTT TCCAATCACCCAAAAAAAAAAAAA ( SEQ ID NO : 6 )Forward TAGAAACTGGGCTTGTCGAGAC (SEQ ID NO : 7 )PrimerReverse Primer TGAATAAAGCCTGAGTAGAGCG (SEQ ID NO : 8 )Firefly (SEQ ID NO: 9)LuciferaseDNA TemplateUnmodified ACUUGCAGGAUCUGACACAUCCUCUUGGUUUCCUAAGUCUUAUGACUAGCCAAUGCC GAPLINC UGAAAUAAUGAACUCCUCCAAGGCAAGAAAUCUGUUUUGAAGCUUCUCUGCGUUCACRNAACACAGCAGCCUGGUUUCCUGGAAGGGCAUUUUCCACAUUGUGCGUUAUGGAUGAUC AUCCCAGGCAUCAGGUGUGAAGCCCUGCAUCCACAUCCAGGGGCUAUCAAAUCUCUC UGCAAAAGGAGAAGCUGGACUCAGGCACGUUUACAGUGAUGUGUAUGCAGGGUAUGC ACAGAUGUGGAAACAGGAACUGAUGUGUCCAUUACACCACUAGGACAGAGGCCAGAA CAAUGAAGAAACCAAAUACUUGGAAGAGGGUAGAGAUAAUGAAUGGAGUCCAAGAGC CCUGAUUGUGCCAUAAAUGUCCAGAUAAUUCCAUACCUGAGGAUUAUGUGGUUUGUA AACUUGGCACUUAGAAGAACCAAUAAAAUCAUGUUAUAGUUUCAAAAAAAAAAAA ( S EQ ID NO : 10 )m5C-modified (SEQ ID NO: 10 where cytidine triphosphate (CTP) was replaced with 5- GAPLINC methylcytidine -5 ’-triphosphate (m5C))RNA'P-modified (SEQ ID NO: 10 where uridine triphosphate (UTP) was replaced with pseudouridine - GAPLINC 5 ’-triphosphate ( ))RNAml'P-modified (SEQ ID NO: 10 where uridine triphosphate (UTP) was replaced with Nl-GAPLINC methylpseudouridine-5 ’-triphosphate (m IT))RNA GAPLINC-202 (SEQ ID NO: 23)RNA GAPLINC-205 (SEQ ID NO: 24)RNAUnmodified UUGUGGUCACACCAACAGUCUGUCAUAAAAGGAGUCUUUGUUCUUCUGUGGAGCUUUCCCAAA MIST RNA CUUCUAUGUGAAUGUCAUUCAUGCUACUAGUUUGAAGCUCUGAAAUCCAUCUCUGGGAAUUUCCUUUCUUGUAUUUUUGUUUUCUUUCAGUAAUAGUCUAGAUAAAAAAGUCAAGAAGCUAAGAAA CAGGAUAUAAAACAAACAAUAACAACAACAAAAAAGUGCCAGGGCUUCAGAGACCUUCACACA UGGGUACCCACAACCUGGUAUGGCCAUGCACAUGGAAGACGGAGAAGAAGGCACUGGCUUUGU GCCUAAGCAAAUUCAAGGCUUUACGUAAUCAUCGGUGACAAAGAAAGAAGACCUCCACUAUAA AACAGACAU CAAUU CUUUAACU GUAGAAUAAAU GAUU C C C C GAU C CAGAU GUUU GAU GU GCAA ACAGGGAAAGAUGUGAUCCCUGCUAGUAAACUGCCUGCUGGGGAAACCAUUGUGCAACAUAGA AGCUUCCCUAACUAGGUGACCAACACAUCCAUUGUCACAAGGCUACCUCUGUCGCUGCCCUCC AUGUGAGCUGCUGUGUGUUGAAAACACUGGCAGAAGCAGCCUCCCCAGCAAGUUUUCGCCUGC UGGAGAUCUGUCUCUGAGACAAAUAUAGAGGAUGGAGACGUUUGUCGGCUUGUCUGCCAUCAA AGACCCAAUUGCUUCCUGCGUUGCAGGAAAUGAAGUGCAGCCUCUCAGCCCCGUCCACCUCCA CAAGUGACUGUCAGUGGAGAACAAGCUGAAGCAGUUGGACUUGCUCUGAGUUCCCAGAUCUGA GCCAAUUCAAAGCUCACAAUGUCAUUAAACCACCAGUACUUGGAGGAGAGCUUAAACACUGCU UU GU C CAGUUACUUU CAAAGAGAGUUU CUAGGAAGAGCUAUU C CAAAUAU CAGU CU GU CAUUU UCACCACCGAAGAAAUUUUUAAAGUGUGCAGUGCAGGAAGAGACAGGAAGAAGAAGGGACUGG AUUGAAAGAGAAUGGUCCCUUGUGAUCUGGUGACAGAGCCGGACAGAGCAGAUGUCAUAAGAC UCCAGUUAUUUUCCAGCUCUGGUGUGACAGUGGUGUGACCAGCAGCCAGUCAUACCACCCUGC CUUU CACAAC CUU GAAGAGACAC GU CACU CAGCU CU CUAAAC CAGUUU GU CAGCUU CAU CAGU CCAGCACACAAGAGCAUCUAAGCAGCAGUCAACAGAGCUGAGAGAAAUCAAAAAGGGGAAAAA AAACAACAAAAAACUAGGAGAAAGCU GUAAUU CUU CU C CUAAGU CAACAGACAAU GCAAUAGA CGUUCACUGACUUUAAAACAGUAAGCACUGGGGUUUUGAGAAAACAGCUGAUGACCUUAAGAA UCAAGAAAGCAUGUCAGCAAUAGACGAAAUAGAACCACUGUGUAUAUGGCAUAGAGCAAUGUU U C C AAU C AC C C AAAAAAAAAAAAA( SEQ ID NO : 14 )m5C-modified (SEQ ID NO: 14 where cytidine triphosphate (CTP) was replaced with 5-MIST RNA methylcytidine -5 ’-triphosphate (m5C))T-modified (SEQ ID NO: 14 where uridine triphosphate (UTP) was replaced withMIST RNA pseudouridine-5’ -triphosphate ( ))mlT-modified (SEQ ID NO: 14 where uridine triphosphate (UTP) was replaced with Nl- MIST RNA methylpseudouridine-5 ’-triphosphate (m I ))Unmodified GACAGUUCCUCCCAGCUCUGACAGCCCGCGGGUGCGGCUGCAGGUGCCGCCUCGGCUUCAUUA DRAIR RNA GCCCAGCUCAGGACAAACUCCAGAAGGGAACAACCCCAGUUCUGGUCCCCUCGGUCACCCCGG GGAAGGACCCAGCCCUAGCUGCUCACUCUCCGCCCGCCUUCCCGAGAGUGCUGCCCCGACCCC GGGUCCGCCGCCAAGCCGGAGCGCCCGGGGCGUUCGAGGGGGCUUGGCGGCUCCUGUUCCCAC CGCGCACGCCUUACCUGGUGGUGGUUGUACGAGUUCCUCUGCUGCAGGAAGGCGGCUGCGGCA GCCUGGUGCUGCUGCUGCAGCUGCGGGCUGACGGGUGACCUCCGGCUCUGCGGCUGCUGCGGC GCCGCCGCCGGCGGCUGCUGUUGAGGAUGAUGCCCUCCAGGUCUAUCCAUUUGCCUAGGAACC AAGAAGUCUUAAUAACUGUUUUGGUGAUUGGGGCCAGCAGAAACCUUCUUCCUGACAUUCUUC UAAGUGACAGGAUGUGUGUAGUCUGCUCUCAACUCCCCACUGGUCCAGCUGAGGCUGCCUGAG GAAAGCACUUUUCUCUGAGGUAUAUUCUUGCCACAGUGGUCACUGAUGUGCUUCAAACAUGGA AAGAAGAAGU CU CAUAU GGU C CUAC C C CUAUAGAAAGAAC CAC GGUUAACUAAU GACU GCU GG AUGAAGUUCCUGCUUCCAGCUUCCUGCCAAGACUUCCUUUAAUGAUGGACUGUAACCUGAAGC UCAAACCCCAAAUGACGUUCCUGCUCUGUUGCCUGUACCCUGGAUUGCUCACCACCUGAGCCU ACCUGCCCUUGUGGGGUUGGAGGUGAGGUGUCACAAAGUCAUCAACACAGACUCUGGAAGCAG AGAAGGCUCCUGGAAGGAACUGCUCAGCUGGGGACUACACCUUCUUUUUGUUUUGGUACAGAG AGAGACCAGCUGACUGGUUUUCAGAAUGAAAAGAGUCUAGCUGGCAGCCCAAAGAGGUGACUG AGAACUGGCUGUGUAAGAACAGCAGAGCUUCCUGGGAGAAACAGGCUUCAUUUUUCCUGCUUU AAAAAAAAAAUAUGCGAGAACUCCAAUGUUCUGGCAAAAUUAAGGAAACCUGCAAUCAGUUGC UUGGAAAAAAAAACCCAGCAAACCUGUCUGUAGUAAAUUACAUAUUGGGACAAAGCCCCACUG ACUCCCGAGGCUGCCUUCCUCCCUGCACCCCACCCUGCUGCCAUCCUUAAGGUAAUGUCCUUC AGACU GCACAAGCU GCAUUU CAGAGAC CU GCAGU C CAAACUU GACAC CUAAGAACU GU GU C C CUUUAGGUAAGU CU CAU GGCUUUUU GU CU GAGUUU CAU GAUAUUAUUU GUAACAU GAC CUUAAA UCUGUUUCUUCAUAUUGAUAUGAGAAAUAAACACACUUUCAUCAGC ( SEQ ID NO : 18 ) m5C-modified (SEQ ID NO: 18 where cytidine triphosphate (CTP) was replaced with 5-DRAIR RNA methylcytidine -5 ’-triphosphate (m5C))'P-modified (SEQ ID NO: 18 where uridine triphosphate (UTP) was replaced withDRAIR RNA pseudouridine-5’ -triphosphate ( ))ml -modified (SEQ ID NO: 18 where uridine triphosphate (UTP) was replaced with N1 -DRAIR RNA methylpseudouridine-5 ’-triphosphate (m IT))Firefly (SEQ ID NO: 22)LuciferaseRNA PARATL DNA ACAAGCCGGGTGGACATCGCTGAGGCTTGGAAAAGATTTCCTCCGGGGAAAATCCCTCCGGAGTemplate TT CT CCAGT CT GGT GT CAGAGAGGAATACTAAAGAAGAAGGAGAAATT CAAGGTACAAGAGAG GCAAAGAAT GTT GGT CCTACAAAGGAACT CT GGT GACAGAAGT CCACAGAGGACAGAT GGAT G GGGATGATTTCCACACTAGTTCCTAACTTTAAATAAAACCAAGCCTGCAGCGTAGTTTCCTGA GTCCTAATGGAAGGATTTGATGAAGACATCCCATTTAGGAATCTGCACTGTTCTCCCTGGGCC ACAGCTATGCCTGCTTCCCTGGAGGCCTGCTGCCATCCAGGACAACGAGATGGAGATGTGTTA AAACTCAGAAGTCCTGTTCTAGAGTGACTCTCTTACTACACAGAGCATCCCTTGCCATTGAAG AGGTAAAACTTAATTCCCGGCAGGCAGCCCAATGTGGGAGCGAGCGTCAGCCATGCTGCGGAA GGCAGAATAAAGTGTCTTCCTGGGAGGAGCAATGTCCAAAATATGGTCTTTCTACTGGTGAAA GGTGGATATTGGAATCACCTACAGTTGATAGCGGCCAGAAGAAAGACACCCAAGCAACTGCCG CAGGCCAGTCAGCCCTATGGAAGCTGGAAGAAAGATACCCAAGTGCCCGGCCATACTGACCCT GAAAGAAAGACATACAAACACCTGCCTCTGGAATCACTGTTATTTGAATGTGTGCTGGGGATA T GGAAAAGACAAGAGTACAT GGT GGAGAGGT GGAAGAGAAAAT GAAGCCAAGGT GTTT GTATA TTGTAGAGAAAGGTAGAACTAGAGATGGTAGTGAGGAACAGGGAGATATGAGGACGCCATAGT GAGTTCCTACCCTGGGATGCCACTAAGGGCCATGTTTGCATCCATGGTCCTGTTGCAGCTGGG GTCGTTGCTGATGTCTTGGCCCGAGTTACCATCAAAGACCAAGTGGATGTCCCATGGTCTAGG TTGCCACTGGAAAACGTGTGGATATCTGAGGGTCATGCTGCTTAGGGAAGACTTGCTCATCTT CGTGGCCTGCCCTGTCACATGAGGCCATAGGGACATTTGGGCCCAGACTAGTTCAATGGGCTA TGTCTTGGTCCTTGACTTCATGGCAGCTGAGGTCTGTGTTGATGTCCCAGTCTCATGAATTAC CAAAGGCCATGCAGATATCCCTAGTCTGGAATGCTGCCTGAGTACTATGCTGAGCTGGAGTGT GTGGGGTGATCAGCCCCAAAGGTGTGACTGGGGGAGAGCTGGCCCGGGGCCCTGCCTCTTATC TGCCCCACAACAGTGGCAGAGATAGCTAGGGCAGTACCTCCCAGGGCACAGAAGAGCTGATGG GCTAACTAGCTCAATTACCTGCGCAGATCTAGGGCAGTGAGTTGGCCCACTCCAACATAGTTC TATCCCAACTATAAACTGCTGGGGCATGTGAAAGGGCTGGTTTTGCAGATCCAAAGCAGCAGG ATCTCCATGGCATGGGGCTGAAACAGAATATCCAAGCCGTGTCCCGGTGAGGATCCAGTATAT ATAGTATAGCAGAAGCCAGAGACCTTGAAACAGACCATGACTTATTGCAAGGAACATTTCCAA GTGAAAATATATGGACAAAAGGGTGTACCATGCGACACAACAGCTTCCAGAGCAAGATTTTTT TTTTTTTAATTCTGTCTCCTTTTCTTTTCTTTTGTTGGGGGAGGTTGTAAGGGCAGAGGGCAG AGAACAGATAT GAAGGGAT GGGGAAAT GAGT GAAATT GGGGTAT GCGAT GTAAAACACACAAA ATAATCAATAAAAATTTAAAAAACA ( SEQ ID NO : 25 )Unmodified ACAAGCCGGGUGGACAUCGCUGAGGCUUGGAAAAGAUUUCCUCCGGGGAAAAUCCCUCCGGAG PARAIL RNA uu cu c CAGU CU GGU GU CAGAGAGGAAUACUAAAGAAGAAGGAGAAAUU CAAGGUACAAGAGAG GCAAAGAAUGUUGCUCCUACAAAGGAACUCUCCUGACAGAAGUCCACAGAGGACAGAUGGAUG GGGAUGAUUUCCACACUAGUUCCUAACUUUAAAUAAAACCAAGCCUGCAGCGUAGUUUCCUGA GUCCUAAUGGAAGGAUUUGAUGAAGACAUCCCAUUUAGGAAUCUGCACUGUUCUCCCUGGGCC ACAGCUAUGCCUGCUUCCCUGGAGGCCUGCUGCCAUCCAGGACAACGAGAUGGAGAUGUGUUA AAACU CAGAAGU C CU GUU CUAGAGU GACU CU CUUACUACACAGAGCAU C C CUU GC CAUU GAAG AGGUAAAACUUAAUUCCCGGCAGGCAGCCCAAUGUGGGAGCGAGCGUCAGCCAUGCUGCGGAA GGCAGAAUAAAGUGUCUUCCUGGGAGGAGCAAUGUCCAAAAUAUGGUCUUUCUACUGGUGAAA GGUGGAUAUUGGAAUCACCUACAGUUGAUAGCGGCCAGAAGAAAGACACCCAAGCAACUGCCG CAGGCCAGUCAGCCCUAUGGAAGCUGGAAGAAAGAUACCCAAGUGCCCGGCCAUACUGACCCU GAAAGAAAGACAUACAAACACCUGCCUCUGGAAUCACUGUUAUUUGAAUGUGUGCUGGGGAUA UGGAAAAGACAAGAGUACAUGGUGGAGAGGUGGAAGAGAAAAUGAAGCCAAGGUGUUUGUAUA UUGUAGAGAAAGGUAGAACUAGAGAUGGUAGUGAGGAACAGGGAGAUAUGAGGACGCCAUAGU GAGUUCCUACCCUGGGAUGCCACUAAGGGCCAUGUUUGCAUCCAUGGUCCUGUUGCAGCUGGG GUCGUUGCUGAUGUCUUGGCCCGAGUUACCAUCAAAGACCAAGUGGAUGUCCCAUGGUCUAGG UUGCCACUGGAAAACGUGUGGAUAUCUGAGGGUCAUGCUGCUUAGGGAAGACUUGCUCAUCUU CGUGGCCUGCCCUGUCACAUGAGGCCAUAGGGACAUUUGGGCCCAGACUAGUUCAAUGGGCUA UGUCUUGGUCCUUGACUUCAUGGCAGCUGAGGUCUGUGUUGAUGUCCCAGUCUCAUGAAUUAC CAAAGGCCAUGCAGAUAUCCCUAGUCUGGAAUGCUGCCUGAGUACUAUGCUGAGCUGGAGUGU GUGGGGUGAUCAGCCCCAAAGGUGUGACUGGGGGAGAGCUGGCCCGGGGCCCUGCCUCUUAUC UGCCCCACAACAGUGGCAGAGAUAGCUAGGGCAGUACCUCCCAGGGCACAGAAGAGCUGAUGG GCUAACUAGCUCAAUUACCUGCGCAGAUCUAGGGCAGUGAGUUGGCCCACUCCAACAUAGUUC UAUCCCAACUAUAAACUGCUGGGGCAUGUGAAAGGGCUGGUUUUGCAGAUCCAAAGCAGCAGG AUCUCCAUGGCAUGGGGCUGAAACAGAAUAUCCAAGCCGUGUCCCGGUGAGGAUCCAGUAUAU AUAGUAUAGCAGAAGC CAGAGAC CUU GAAACAGAC CAU GACUUAUU GCAAGGAACAUUU C CAA GUGAAAAUAUAUGGACAAAAGGGUGUACCAUGCGACACAACAGCUUCCAGAGCAAGAUUUUUU UUUUUUUAAUUCUGUCUCCUUUUCUUUUCUUUUGUUGGGGGAGGUUGUAAGGGCAGAGGGCAG AGAACAGAUAUGAAGGGAUGGGGAAAUGAGUGAAAUUGGGGUAUGCGAUGUAAAACACACAAA AUAAUCAAUAAAAAUUUAAAAAACA ( SEQ ID NO : 26 )m5C-modified (SEQ ID NO: 26 where cytidine triphosphate (CTP) was replaced with 5-PARAIL RNA methylcytidine -5 ’-triphosphate (m5C))T-modified (SEQ ID NO: 26 where uridine triphosphate (UTP) was replaced with PARAIL RNA pseudouridine-5’ -triphosphate ( ))mlT-modified (SEQ ID NO: 26 where uridine triphosphate (UTP) was replaced with Nl- PARAIL RNA methylpseudouridine-5 ’-triphosphate (m IT))m6A-modified (SEQ ID NO: 26 where adenosine triphosphate (ATP) was replaced with N6-methyl- PARAIL RNA adenosine-5 ’-trisphosphate (m6A))Human NR_110428.1NR_110429.1GAPLINC SEQ ID NO: 10Human DRAIR NR 038857.1 GGUUCCGCCCUGGGUGGGGAGAAGCCCUGCUGCACCACUCGGGCUUCCCAGAGCGGGGCUGGC CCGCGGGGGAAACUGAGGCCCGGGACCCCGACCGGCGCCUGCACUGCGCUGGUAGGAGUGCUG UCACCGACGUCGUAGAGCCGCCGAGGGUGUUUCUGUAGGGUAAACAUGAAGCCUCGGCUAUAU U C CAGGCAUUUU CACUU GGAAACAUUUUACAUAAAGUAUUU GAAGGU CUAGAUAAAGAAUAAC AGAGUGAUCCAUACUACAUGAUGAGGAGCUGUCCAAACACCAUGCAGAUGCUCCUAGGAUUCU UAAACUAGCCAAAAAUAAUUCCAGGCAUUGUCAGAUAUAGGAUAAGUACAUGAAGACAGCAUU AGUCGCAGUAGCGAAGACACGGAAACAACCUAAGUGUUCACCAACAGAUGAAUGGAUGAAGCA AAUGUAGCAUAUAUACACAGUGGAAUACUAUUCAGCCUUUAAAAAGAAGGAAAUCUGGCUAAC UGCAACAACAUGGAUGAACCUGGAGGAUAUUAUAUAAGUGAAAUAAGCCAGGCAUGGAAAGUU UUUGUAGAGAUGAAGUCUUGCUGUGUUGCCCAGGCUGGUCUCCAACUCCUGUACUCAAGCUAG CUUCCUGCUUUGGCCUCCCAAACUGCUGGAAUUAAGUUGUCUGCCCUAUUGCUCAACAAGAUA AAAUGUCUGGGCUGGUGGCUGGAGGCUGGAGGAAGAAUCUGGAAAGAGAGAGAGACUGUGGAA AAGGCAUAGAAAGACCUUUGAACUAGCAGAACAUAUUUCUUUGAGGGACUUAGGAACUUCAUU CUGGGCAAUCUUCCCAAAAGGUACACAGCUCUGGACGUACCAACACUCUAUUUAAAAACCAGU AAGGUAGAGUGGAAGGCAGAGCUGAGUGAAACUGAAACCUGGUAUUUGCUGGAGGUUUCUUUC UGGAAAGCUGACAGCACACCUCUGACCCUCUCCAAAAGGUCUGAUAUGAAAUAUUGCGACAAA GUUGGAAUCAACUGGCAGGCAUUUCAGUAGAUGUGCUAAUAGCAUGCAACAAUGGGAAGGCGC CAAAACUGAACUGAGGUGGUGAAAUGAGGUAGAGAUAAGCAAGUACCGCAAAAAAGGAGGCAA GAGAAAUGGCAAAACUGGGGUGGUUCAAAGAAGGAAACAGGGUUUGAUGCCAAUGGUUAUUGU CUGGCUUAUUCGAGUAGUUUUUAAAGGGAGAGGAAGGGAGAGGUGUGUCGCUACCUGCUGGUA CUGUGGUGUGCCUUUAUUGCGUUUGGACAUCCAAGCAGACCUAUUUAUUUGUUCCCUAAGCAA AUAUUUAUU GAAUAU GUAUUAU GU C CUAGGCACU GU GUAU CAGAAAUAACU GCAUAUAU GAGC CUUGGAGGGUGAUUGUGGUUGAUAUGCAUUGACAUGGGUGAAAGGGAAGAGACAUAUUUUCAA CUUUGUAUUUUUCAACACAUCCAGCUGCGUGCUUUCAGCCCAGCAGGUGCUCAAAAUAGGUUU ACUGAGGUGAAAACCAAUUCUUGCCUCUAAAGGGUGUUCCAUGGUCUCACAGGAAUUAGGAAG GAGU GAG GAG GAU CUUU AGU AAAU AAAC AAU GU G GU GAU AG C AU AAAAAAU AC AAAAAAAAAA AAAAAA_ (SEQ ID NO: 32) _MIST NM_052964 . 4XM_011513775 . 3XM_054348851 . 1XM_017007684 . 2XM_054348852 . 1 GGCUGCAUUUCACAGGAAACCAAGUCUAAAACGGACCUAUCAGGAGGUUUUCUGCUGAAGGGC ACUGCUUAGCAUCGAGAAGAAUUCAACCCACCGCCUUACUAAUUUCCAGUGCCCCAAGGUCUC UGCACUGCCGCCCCUCCUCACAGGAGACGGACACCUCAGCCUAGAUCCCUUGGUGCUCUCCAC GCUGUUCAGGCUGAAUUGAAGAGCCCUCUUACCCGCCAGGUGCCAAGAACUAUGAACAGGCAG GGCAAUAGAAAGACAACUAAAGAAGGAU C CAAC GAUUU GAAAUU C CAGAACUU CAGU CU GC CA AAAAACAGGUCAUGGCCUCGCAUCAAUAGUGCCACAGGCCAGUACCAGAGGAUGAACAAGCCU CUUCUAGACUGGGAAAGAAACUUUGCUGCAGUCCUGGAUGGAGCAAAAGGCCACAGUGAUGAU GACUAUGAUGACCCUGAGCUUCGGAUGGAAGAGACAUGGCAGUCGAUUAAAAUUUUACCAGCC CGGCCUAUAAAGGAAUCUGAAUAUGCAGAUACACACUAUUUCAAGGUUGCAAUGGACACUCCC CUUCCGUUAGACACCAGGACCUCUAUCUCCAUUGGACAGCCGACCUGGAACACACAGACGAGG UUGGAAAGAGUGGACAAACCCAUUUCCAGGGACGUCAGAAGCCAAAACAUUAAAGGAGAUGCA U C C GUAAGAAAGAACAAGAUU C CUUUAC CAC CU C CU C GGC CU CU CAUAACACUU C C GAAGAAG UACCAACCCUUGCCCCCUGAGCCGGAGAGCAGCAGGCCACCUUUAUCUCAGAGACACACCUUU CCAGAAGUCCAGGGAAUGCCCAGUCAGAUAAGCUUAAGGGACUUAAGUGAGGUCCUUGAAGCA GAAAAAGUU C CU CAUAAC CAGAGGAAGC CU GAAU CAACU CAU CU GUUAGAAAAC CAAAAUACU CAAGAGAUU C CACUU GC CAUUAGCAGUU CUU CAUU CAC GACAAGCAAC CACAGU GU GCAAAAC AGAGAUCAUAGAGGAGGCAUGCAGCCCUGUUCUCCUCAGAGAUGCCAGCCUCCAGCCAGCUGC AGCCCUCACGAAAAUAUACUGCCCUAUAAAUACACAAGCUGGAGACCACCUUUCCCCAAAAGG UCUGAUAGAAAGGAUGUCCAGCACAAUGAAUGGUACAUUGGAGAAUACAGCCGCCAGGCAGUG GAAGAGGCAUUCAUGAAGGAGAACAAGGAUGGUAGUUUCUUGGUCCGAGAUUGUUCCACAAAA U C CAAGGAAGAGC C CUAU GUUUUGGCUGU GUUUUAU GAGAACAAAGU CUACAAU GUAAAAAU CCGCUUCCUGGAGAGGAAUCAGCAGUUUGCCCUGGGGACAGGACUCAGAGGAGAUGAGAAGUUU GAUU CAGUAGAAGACAU GAU C GAACACUACAAGAAUUUU C C CAUUAUACUAAUU GAU GGGAAA GAUAAAACUGGGGUCCACAGGAAACAGUGUCACCUCACUCAGCCACUCCCUCUCACCAGACAC CUCUUGCCUCUGUAGCCUGGUCUUUGUGUUAUCUUUGGUUUACUGGAUUCAGCGCUUCCAUUG UUUUCAUUGAUUUCAAAAGUUUAUUUUCUGUGCCUUCAAGGGACAACUUUUUUAACUUUGGAG AAAAGAAAAACACU CUAUAACAGAGAGU GGAAAAU CACU GAG GGUUUU GAAAGUU CAAAC GAG AGAGAAAAUAUUUAUAACAU GCAAAAAAUAAAAACAUUU CUAGUAACU GGC CACU GGAAAAUA AAUAAAAAUAAAAACUAAAAAAAAAGAAAAAAAAAAAAAAAAAAAAAAAA SEQ ID NO : 33PARAIL NR_125822 . 1GGACGGCAAGGCGGGGAGAGGGGCAGCCCGCUCGCGGCCCUCCCGCCUGGCUCUCGCCUCGGC CUGCGGCCGGGAUCCUCCGCCCGGGUCGCGCAGCGGAGGCCGAGGGCUGGGACGGGCGGCUGG GAAGUGGGAAAGGGAGCUCCUUUUGUCUUCUCUUCCCAUCCCCCAGGUUUGGGAAAGUUUUCC UUUUGGGAAAGCCCCUCUCUUCAGCCUGCUGGCGGGUGUCGGAUUGGUUAGCGACCACCGGCC CCCGCCUCCCCCAACAGGAUUCUCCUUUGGGCCACAGGUCUUCCGGAAGUUGCCAAACCCCGG UAGCGGCAGCGUCCGGCUGGGGGCUUUAUUAGGGGGCACCGGGGUCUGCUUUCCGACUCCCUU CCGACUCCGCGCCUGCGCAGGAAGCGAUUUGGGGAGUGUUGUGAAUUCCGAGGGCUCCACACU UAAGAGGUU GUACACU CAC CU GU CUAC CU GGACU C CAAUUU C CAUAUUU C CAGC CACUU GAGG ACUGAGAGGUGGAUGAUAAACCCUGUCAUAGUGGAGCAAGUUCAGGUGUUUAACCGCUGUUAU GGGGGAUCUGCCUUUUCUCCCUCUUCUUUCUUACUUCCAUAAGUAUGUAUGUGCAGAGAUUGA AAAAUAAC CUU GAGAGAGU C CAU C CAU CUAACU C CAGUU CAUU GGAAAUUU CUGCUUCUUUGA GACUGAGGUUAUAUUCCACUUCUUCUGACUACACCACCACCAUCAAUCUCCUUCUCUGAACUC UUGGAUCAUUACUUCAACCUGAAACAACUCAUCAGGCAGAAUAGUUGCCUACUGCCCCAACCC CACCAAAUGUUCCAGCACCUCUGAUAUUACCUCACAUGGCAAGAGGGCUUUGAAGAUGUGAUU GAGUCAAGGAUCUUGAAUGGGGAGAUUAUCCUGAAUUAUUCAGCUGGACCCAGUGUAAUCACA AAGGUUUUUCAAAGAUGGAAGAGAGGCAGAAGAGCCAGAAAAGGAUAUGUGACAAUGGAGUCA GUGUCAGAGUUGGAGUGACGUGAUGUGAGAAGGAUUCAACCUGCUGUUUCUGGCUUUGAGGAU GAAAGAAGGGGCCAUGAGCCAAGCAAUACAGGUGGCUUCUUUCAAGAAGUUGGAAAAGUGGUA AUAUUUUAUGUUAUUUUGGUUUGAUUUUUGUUUGUGGCAUAUGGCUCUGCUGUCCUGUGACUC UAAGUAGAAGAGAAAAUAGU GC CAAUAAUU CUU CAU GGAGAU GCAC CU CU CU CAAGU CUU GAA CAAUGUUUGAGAUUUCCCCAGGCCACCAUCACUGGAUUUGGCUAUUUGACUUGCUGUAGUCAA UGGAAUGUGAGUGAAAAGUGACUCUGUACCAGUGGGGAGGCAAAGCCUUAAAAGACCCAGCAA AUUUCUUCCAGCCCUUUUGUGCUUCUGCCAAAAGAAUACUGUGAGCAAUCAGUGCCCUUAAGC CUGGGCCUCAGAACGAGAGAUAUGGAGCAAACCUGAACCUGAACAGCAGCCUACAAUCAAGCC AGGAUGACCCUGCAAAGCCCAGCCAACGAUAGCAAAGCCACAGCAGACUUGUAGACCAAUGAG CAUGAAAUAAAUUGCAUUGAAAGCUA SEQ ID NO : 34The sequences can presented such that“P” identifies residues where uridine triphosphate (UTP) was replaced with pseudouridine-5’ -triphosphate ( ): “5” identifies residues where cytidine triphosphate (CTP) was replaced with 5 -methylcytidine-5 ’ -triphosphate (m5C); “1” identifies residues where uridine triphosphate (UTP) was replaced with N 1 -methylpseudouridine-5 ’ -triphosphate (m I ) and / or “6” identifies residues where adenosine triphosphate (ATP) was replaced with N6-methyl-adenosine-5’-trisphosphate (m6A).Table 2. Characterization Summary of LNPs formulated in this study.Formulation Ionizable RNA Type Total N / P Mean by PDI EE (%) ID Lipid Lipids / RNA Intensity(wt / wt) (nm)JP01 C3-K2- 81 0.18E14JP02 C3-K2- GAPLINC 9.0 5.01 91 0.16 87 E14 UnmodifiedJP03 C3-K2- GAPLINC 9.0 5.23 230 0.14 64 E14 Unmodifiedcap + tailJP04 C3-K2- GAPLINC 9.0 5.21 234 0.12 58 E14 Unmodifiedcap onlyJP05 C3-K2- GAPLINC 9.0 5.21 129 0.19 76 E14 mlTJP07 C3-K2- GAPLINC 20.0 11.58 199 0.13 65 E14 mlTJP09 OC2-K3- GAPLINC T 7.70 5.01 141 0.15 97 E10JP10 OC2-K3- GAPLINC T 7.70 5.01 159 0.12 61 E10 (cellulose)JP11 OC2-K3- GAPLINC 7.70 5.01 192 0.11 87 E10 Unmodified(cellulose)JP12 OC2-K3- GAPLINC 7.70 5.01 145 0.06 95 E10 Unmodified(cellulose)JP13 OC2-K3- GAPLINC T 7.70 5.01 152 0.08 89 E10 (cellulose)JP14 OC2-K3- GAPLINC 7.70 5.05 187 0.08 84 E10 Unmodified(cellulose)JP15 OC2-K3- GAPLINC 7.70 5.05 195 0.09 88 E10 Unmodified(cellulose)JP16 OC2-K3- - 7.70 5.05 175 0.08E10JP17 OC2-K3- GAPLINC 7.70 5.05 161 0.10 87 E10 Unmodified(cellulose)JP18 OC2-K3- GAPLINC 7.70 5.05 173 0.13 54 E10 Unmodified(HPLC)JP19 OC2-K3- GAPLINC 7.30 5.02 141 0.11 49 E10 m5C (HPLC)JP20 OC2-K3- GAPLINCT 7.40 5.03 150 0.16 58 E10 (HPLC)JP21 OC2-K3- GAPLINC 7.70 5.02 127 0.07 37E10 Scrambled(HPLC)JP22 OC2-K3- GAPLINC 7.70 5.06 160 0.11 54E10 mlTJP38 OC2-K3- DRAIR 20.0 13.17 169 0.05 76E10 mlT(HPLC)JP39 OC2-K3- DRAIR m5C 20.0 13.94 144 0.06 60E10 (HPLC)JP41 OC2-K3- GAPLINCT 20.0 13.76 151 0.07 89E10 (HPLC)Note: N / P stands for nitrogen to phosphate.Table 3. siRNAs used for RAW-Cell treatmentsGene Organism siRNA ID116 Mouse 159540Tnf Mouse 188364Illb Mouse 158282Table 4. Oligonucleotides for GAPLINC DNA template and RNA.Name Sequence (5’ to 3’)GAPLINC DNA (SEQ ID NO: 1)Template-modificd (SEQ ID NO: 10 where cytidine triphosphate (CTP) was replaced with 5- GAPLINC RNA methylcytidine -5 ’-triphosphate (m5C).)2’OMe-UTP- (SEQ ID NO: 10 where uridine triphosphate (UTP) was fully replaced with 2'-O- modified methyl -uridine-5 '-triphosphate.)GAPLINC RNATable 5. TaqMan Gene Expression Assays used for RT-qPCR Gene Expression Analysis Gene Organism TaqMan Gene Expression Assay ID116 Mouse Mm00446190_mlTnf Mouse Mm00443258_mlIllb Mouse Mm00434228_mlGapdh Mouse Mm999999l5_glIllb Human Hs01555410_mlTnf Human Hs00174128_ml116 Human Hs00174131_mlGapdh Human Hs02786624_g 1Table 6A Disease Associated Truncated IncRNAsTable 6B Disease Associated Deleted IncRNAsREFERENCES1. Kariko, K., Buckstein, M., Ni, H. & Weissman, D. Suppression of RNA Recognition by Toll-like Receptors:The Impact of Nucleoside Modification and the Evolutionary Origin of RNA. 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Claims

CLAIMS1. A complex comprising a long non-coding (IncRNA), a nucleic acid molecule comprising a IncRNA or a DNA encoding a IncRNA, and a cell delivery agent, wherein the IncRNA lacks a 5 ’ cap and / or a 3 ’poly A tail, and wherein the complex, nucleic acid molecule or IncRNA comprises single stranded molecules at a purity of at least 99%, wherein the IncRNA comprises one or more different IncRNAs.

2. A pharmaceutical composition comprising the complex of claim 1.

3. The complex of claim 1 or the pharmaceutical composition of claim 2, wherein the IncRNA comprises two or more different IncRNAs and / or wherein the IncRNA or nucleic acid molecule was purified, optionally by HPLC or cellulose chromatography and / or using a method comprising a denaturation step, optionally a heating denaturation step or chemical denaturation step.

4. The complex of any one of claims 1 to 3, or the pharmaceutical composition of any one of claims 2 to 3, wherein the length of one or more of the IncRNA or nucleic acid molecule is at least or about 200 nucleotides, at least or about 400 nucleotides, at least or about 500 nucleotides, at least or about 1000 nucleotides; at least or about 1500 nucleotides, at least or about 2000 nucleotides and optionally up to 3000 nucleotides.

5. The complex or pharmaceutical composition of any one of claims 1 to 4 wherein the IncRNA, nucleic acid molecule comprises or is or the DNA encodes GAPLINC, DRAIR, MIST, PARAIL, MALAT1, NEAT1, HOTAIR, ANRIL, NORAD, PVT1, SNHG1, SNHG3, SNHG5, SNHG7, SNHG12, SNHG14, SNHG15, DNM3OS, CHASERR, H19, MEG3, FENDRR, SOX2OT, LINC00237, LINC-PINT, UCA1, GAS5, MIR17HG, PANDA, LINC00968 and / or LINC01021.

6. The complex or pharmaceutical composition of any one of claims 1 to 5, wherein the IncRNA or nucleic acid molecule comprises or is selected from the nucleotide sequence of any one of SEQ ID NOs: 10, 14, 18, 26, or 31-64, a sequence with at least 80% or at least 90% sequence identity to any thereof or the DNA encodes the IncRNA, optionally selected from SEQ ID NO: 1, 5 6 and 25 or a sequence with at least 90% sequence identity to any thereof.

7. The complex of any one of claims 1 to 6, or the pharmaceutical composition of any one of claims 2 to 6, wherein the IncRNA comprises one or more chemically modified nucleotides.

8. The complex or pharmaceutical composition of claim 7, wherein the one or more chemically modified nucleotides are a base chemical modification and / or a sugar chemical modification selected from:N1 -methylpseudouridine (m IT) modification,pseudouridine modification (T).5 -methyl -cytosine (m5C) modification,N6-Methyl-adenosine-5'-triphosphate (N6-Me-ATP),2'-fhroro-2'-deoxyuridine-5'-triphosphate (2'-F-dUTP),2'-fluoro-2'-deoxycytidine-5'-triphosphate (2'-F-dCTP),2'-fluoro-2'-deoxyguanosine-5'-triphosphate (2'-F-dGTP),2'-fluoro-2'-Deoxyadenosine-5'-triphosphate (2'-F-dATP),2'-O-methyl-uridine-5'-triphosphate (2'-O-Me-UTP),2'-O-methyl-adenosine-5'-triphosphate (2'-O-Me-ATP),2'-O-methyl-guanosine-5'-triphosphate (2'-O-Me-GTP),2'-O-methyl-cytidine-5'-triphosphate (2'-O-Me-CTP),2'-O-4'-C-Locked-guanosine-triphosphate (2'-O-4'-C-Locked-GTP),2'-O-4'-C-Locked-adenosine-5'-triphosphate (2'-O-4'-C-Locked-ATP), and / or2'-O-4'-C-Locked-5-methyl-cytidine-triphosphate (2'-O-4'-C-Locked-5-Me-CTP).

9. The complex or pharmaceutical composition of claim 7 or 8, wherein the IncRNA comprises about or at least 10%, about or at least 20%, about or at least 30%, about or at least 40% or about or at least 50% chemically modified nucleotides, wherein the chemically modified nucleotides replace one or more of A, U, G, or C nucleotides, optionally wherein about or at least 50% of A nucleotides are replaced, wherein about or at least 50% of U nucleotides are replaced, wherein about or at least 50% of G nucleotides are replaced, and / or wherein about or at least 50% of C nucleotides are replaced, or optionally wherein 100% of A nucleotides are replaced, wherein 100% of U nucleotides are replaced, wherein 100% of G nucleotides are replaced, and / or wherein 100% of C nucleotides are replaced.

10. The complex or pharmaceutical composition of any one of claims 1 to 9, wherein the cell delivery agent is a lipid nanoparticle, and / or the DNA is comprised in a vector construct, optionally a vector expression construct lacking sequences for a 5 ’cap and / or polyA tail or the vector is anexpression plasmid, a viral vector, optionally an adenovirus or lentivirus viral vector, or a selfamplified RNA / replicon, optionally lacking sequences for a 5 ’cap and / or polyA tail.

11. The complex or pharmaceutical composition of claim 10, wherein the lipid nanoparticle is or comprises lipofectamine or comprises an ionizable lipid, and optionally one or more of cholesterol, phosphatidyl choline, optionally l,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), and a PEG-lipid optionally DMG-PEG2000.

12. The complex or pharmaceutical composition of claim 11, wherein the ratio of ionizable lipid, cholesterol, DSPC, and DMG-PEG2000 is about 50:38.5: 10: 1.5 mol %.

13. The complex or pharmaceutical composition of claim 11 or 12, wherein the ionizable lipid is or comprises one or more molecules shown in Fig. 13A-I (OC2-K3-E10, C3-K2-E14, SM-102, ALC-0315, Dlin-MC3-DMA (MC3), PN2 and / or 503) and / or comprises a tertiary amine.

14. The complex or pharmaceutical composition of any one of claims 10 to 13, wherein the IncRNA and the lipid nanoparticle form the complex, wherein the size of the complex is between about 140 nm to about 200 run or less than 200 nm.

15. The complex or pharmaceutical composition of any one of claims 10 to 14, wherein the polydispersity of the complex is below about 0.2.

16. The complex or pharmaceutical composition of any one of claims 1-15, comprising DNA, wherein the DNA is comprised in a vector, optionally an adenoviral vector or lentiviral vector, and / or the DNA is complexed with the cell delivery agent, optionally wherein the cell delivery agent is or comprises anionic solid nanoparticles, optionally pegylated.

17. The complex or pharmaceutical composition of any one of claims 10 to 16, wherein the complex comprises a mass ratio between about 5: 1 to 20: 1 of lipid to IncRNA, DNA, vector construct or nucleic acid molecule or a mass ratio of at least 5 : 1, 7:1, 10:1, 15:1, 16:1, 17:1, 18:1, 19:1 or 20: 1 of lipid to IncRNA, DNA, vector construct or nucleic acid molecule.

18. The complex or pharmaceutical composition of any one of claims 10 to 15, wherein the complex comprises a mass ratio between about 7: 1 to about 20: 1 of lipid to IncRNA.

19. The complex or pharmaceutical composition of any one of claims 10 to 15, wherein the complex comprises a mass ratio of about 20: 1 of lipid to IncRNA.

20. The complex or pharmaceutical composition of any one of claims 1 to 19, wherein the IncRNA is an anti inflammatory IncRNA optionally wherein the IncRNA comprises or is GAPLINC, DRAIR, MIST, PARAIL, MALAT1, NEAT1, HOTAIR, ANRIL, NORAD, PVT1, SNHG1, SNHG3, SNHG5, SNHG7, SNHG12, SNHG14, SNHG15, DNM3OS, CHASERR, H19, MEG3, FENDRR, SOX2OT, LINC00237, LINC-PINT, UCA1, GAS5, MIR17HG, PANDA, LINC00968 and / or LINC01021.

21. The complex or pharmaceutical composition of any one of claims 1 to 20, wherein the IncRNA is purified comprising a purification step comprising HPLC and / or cellulose chromatography purification.

22. The complex or pharmaceutical composition of claim 21, wherein the purification step comprises heating or is performed, at about or at least 45°C, at about or at least 50°C, at about or at least 55 °C, at about or at least 60°C, at about or at least 65 °C or at about or at least 70°C or the HPLC purification comprises a linear gradient.

23. The complex or pharmaceutical composition of any one of claims 1 to 22 further comprising one or more pharmaceutically acceptable carrier and / or excipient.

24. The complex or pharmaceutical composition of any one of claims 1 to 23, or the IncRNA, nucleic acid molecule or DNA as defined therein for use in increasing the level of IncRNA, optionally a deficient IncRNA in a cell or subject, for use as a medicament, for use in the treatment of a disease or condition optionally an acute inflammatory disease or condition or a flare up of a chronic inflammatory disease or condition or for the treatment of a disease or condition associated with a deficiency of a IncRNA.

25. Use of the complex comprising a long non-coding (IncRNA), a nucleic acid molecule comprising a IncRNA or a DNA encoding a IncRNA, and a cell delivery agent or a pharmaceuticalcomposition comprising any of the foregoing, optionally the complex or pharmaceutical composition according to any one of claims 1 -24, or the IncRNA or DNA as defined therein for increasing the level of a IncRNA, optionally a deficient IncRNA, in a cell or subject, or in the treatment of a disease or condition, optionally an acute inflammatory disease or condition or a flare up of a chronic inflammatory disease or condition or a disease or condition associated with a deficiency of a IncRNA.

26. Use of the complex comprising a long non-coding (IncRNA), a nucleic acid molecule comprising a IncRNA or a DNA encoding a IncRNA, and a cell delivery agent or a pharmaceutical composition comprising any of the foregoing, optionally the complex or pharmaceutical composition according to any one of claims 1 -24, or the IncRNA or DNA as defined therein in the preparation of a medicament for increasing the level of a deficient IncRNA in a cell or subject, or the treatment of a disease or condition, optionally an acute inflammatory disease or condition or flare up of a chronic inflammatory disease or condition or a disease or condition associated with a deficiency of a IncRNA.

27. The complex or pharmaceutical composition or use of any one of claims 24 to 26, wherein the acute inflammatory disease is a cytokine release syndrome (CRS) or vascular disease.

28. The complex or pharmaceutical composition or use of claim 27, wherein the vascular disease is or comprises atherosclerosis or atherothrombosis or the CRS is sepsis or graft versus host disease.

29. The complex or pharmaceutical composition or use of claim according to any one of claims 24- 28, wherein the subject is a human.

30. The complex or pharmaceutical composition for use of any one of claim 22 to 29, wherein the disease or condition is associated with a deficiency of a IncRNA.

31. The complex or pharmaceutical composition for use of any one of claim 22 to 30, wherein the deficiency of a IncRNA is a IncRNA selected from Table 6A or Table 6B and / or wherein the IncRNA comprises or consists of any of SEQ ID Nos 35-64.

32. A nucleic acid molecule comprising or consisting of a long non-coding (IncRNA) wherein the IncRNA lacks a 5 ’ cap and / or a 3 ’poly A tail or a DNA molecule encoding said IncRNA, optionally for use in preparing encapsulated IncRNA optionally for use in a screening assay, forincreasing the level of a deficient IncRNA in a cell or subject, and / or in the treatment of a disease or condition, optionally an acute inflammatory disease or condition or a flare up of a chronic inflammatory disease or condition or a disease or condition associated with a deficiency of a IncRNA.

33. The nucleic acid molecule of claim 32, wherein the IncRNA has any one of the modifications as defined in any one of claims 7 to 9 optionally for use in a screening assay, for increasing the level of a deficient IncRNA in a cell or subject, and / or in the treatment of a disease or condition, optionally an acute inflammatory disease or condition or a flare up of a chronic inflammatory disease or condition or a disease or condition associated with a deficiency of a IncRNA.

34. The nucleic acid molecule of any one of claim 32 or 33 comprising a modified IncRNA comprising one or more chemically modified nucleotides,wherein the IncRNA is GAPLINC, DRAIR, MIST, PARAIL, MALAT1, NEAT1, HOTAIR, ANRIL, NORAD, PVT1, SNHG1, SNHG3, SNHG5, SNHG7, SNHG12, SNHG14, SNHG15, DNM30S, CHASERR, H19, MEG3, FENDRR, SOX2OT, LINC00237, LINC-PINT, UCA1, GAS5, MIR17HG, PANDA, LINC00968 or LINC01021;wherein the IncRNA lacks a 5’ cap and / or a 3’ poly-A tail; andwherein the one or more chemically modified nucleotides selected from:N1 -methylpseudouridine (m IT) modification,pseudouridine modification (T).5 -methyl -cytosine (m5C) modification,N6-Methyl-adenosine-5'-triphosphate (N6-Me-ATP),2'-fhroro-2'-deoxyuridine-5'-triphosphate (2'-F-dUTP),2'-fluoro-2'-deoxycytidine-5'-triphosphate (2'-F-dCTP),2'-fluoro-2'-deoxyguanosine-5'-triphosphate (2'-F-dGTP),2'-fluoro-2'-Deoxyadenosine-5'-triphosphate (2'-F-dATP),2'-O-methyl-uridine-5'-triphosphate (2'-0-Me-UTP),2'-O-methyl-adenosine-5'-triphosphate (2'-0-Me-ATP),2'-O-methyl-guanosine-5'-triphosphate (2'-0-Me-GTP),2'-O-methyl-cytidine-5'-triphosphate (2'-0-Me-CTP),2'-O-4'-C-Locked-guanosine-triphosphate (2'-O-4'-C-Locked-GTP),2'-O-4'-C-Locked-adenosine-5'-triphosphate (2'-O-4'-C-Locked-ATP), and / or 2'-O-4'-C-Locked-5-methyl-cytidine-triphosphate (2'-O-4'-C-Locked-5-Me-CTP).

35. A vector construct comprising a vector backbone and a DNA encoding the IncRNA of as defined in any one of claims 1 to 31, optionally wherein the vector construct lacks sequence for encoding a 5 ’ cap and / or a 3 ’poly A tail, optionally wherein the IncRNA is as defined in any one of claims 1 to 34, optionally for use in a screening assay, for increasing the level of a deficient IncRNA in a cell or subject, and / or in the treatment of a disease or condition, optionally an acute inflammatory disease or condition or a flare up of a chronic inflammatory disease or condition or a disease or condition associated with a deficiency of a IncRNA.

36. The vector construct of claim 35 wherein the vector is an expression plasmid or replicon / self amplified RNA, adenoviral or lentiviral vector (for DNA) or other DNA delivery vector, optionally for use in an in vitro translation system, or in a screening assay, for increasing the level of a deficient IncRNA in a cell or subject, and / or in the treatment of a disease or condition, optionally an acute inflammatory disease or condition or a flare up of a chronic inflammatory disease or condition or a disease or condition associated with a deficiency of a IncRNA.

37. A kit comprising the complex or pharmaceutical composition of any one of claims 1 to 31, the nucleic acid molecule of claim 32 to 34, the vector of claim 35 or 36 and one or more chemically modified residues, optionally as defined in claim 8, and optionally one or more components for in vitro translation or cell delivery agents.

38. The kit of claim 37 wherein the cell delivery agent is a lipid nanoparticle, optionally for use in a in a screening assay, for increasing the level of a deficient IncRNA in a cell or subject, and / or in the treatment of a disease or condition, optionally an acute inflammatory disease or condition or a flare up of a chronic inflammatory disease or condition or a disease or condition associated with a deficiency of a IncRNA.

39. A method of increasing a level of a deficient IncRNA in a cell or subject, the method comprising administering the pharmaceutical composition or the complex of any one of claims 1 to 31, the nucleic acid of any one of claims 32 to 34 or the vector of claim 35 or 36.

40. The method of claim 39 for the treatment of a disease or condition, optionally an acute inflammatory disease or a flare up of a chronic disease or condition or a disease or condition associated with a deficiency of a IncRNA, an autoimmune disease and / or osteoarthritis.

41. The method of claim 40, wherein the acute inflammatory disease is a systemic inflammation, optionally a cytokine release syndrome or wherein the disease or condition associated with a deficiency of a IncRNA is a syndromic early-onset neurodevelopmental disorder, developmental delay, growth deficiency or the disease or condition is a neurodegenerative disease or wherein the autoimmune disease is rheumatoid arthritis.

42. The method of claim 40, wherein the inflammatory disease is sepsis or graft versus host disease.

43. The method of any one of claims 39 to 44 or the uses defined in any one of claims 25 to 31, wherein the administering is intravenous or subcutaneous or formulated therefore.

44. The method of any one of claims 39 to 44 wherein the method is for altering expression of one or more cytokine, optionally proinflammatory cytokine expression.in a cell or in a subject.

45. The method of claim 44, wherein the one or more cytokine comprises IL-ip, TNFa, IFN-y and / or IL-6.

46. The method of claim 44 or 45, wherein the altering comprises decreasing expression.

47. The method of claim 44, wherein the one or more cytokine comprises IL- 10.

48. The method of claim 46, wherein the altering comprises increasing IL-10.

49. A screening assay for identifying a modified IncRNA with improved activity, the methodcomprising:a. selecting a target IncRNA of interest,b. preparing a vector construct comprising DNA encoding the IncRNA of interest, wherein the construct is configured to produce the IncRNA of interest lacking a 5 ’cap and / or 3’polyA tail;c. synthesizing a plurality of IncRNA using one more modified nucleotides in vitro; d. isolating one or more of the modified IncRNA at a purity of at least 99%;e. complexing the IncRNA with a delivery agent, optionally a lipid nanoparticle (LNP) f. testing the one or more isolated modified IncRNA and comparing to a control IncRNA comprising unmodified nucleotides, comprising a 5’cap and / or comprising a 3’polyA tail; andg. selecting a modified IncRNA with improved activity compared to the control.

50. The method of claim 49, wherein the isolating step comprises HPLC or cellulose purification and / or denaturation.

51. The method of claim 50, wherein the level of dsRNA after the isolating step is less than 0.5% of total RNA, or less than 0.1%.

52. The method of any one of claims 49 to 51, wherein the selected modified IncRNA or an active fragment thereof is the IncRNA of the complex, pharmaceutical composition, nucleic acid molecule, method, use or kit of any one of claims 1 to 48.

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