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 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

1 MODIFIED AND NON-MODIFIED LONG NON-CODING RNA AND METHODS OF USE THEREOF FIELD

[0001] The present disclosure relates to compositions and uses of modified and unmodified long non-coding RNAs (lncRNA) and in particular to compositions and uses of for delivery of lncRNA complexes such as nanoparticle lncRNA complexes. BACKGROUND

[0002] 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 stability1,2. Conversely, minimal focus has been given to long non-coding RNAs (lncRNA). LncRNAs are a class of nucleic acids involved in gene regulation 3. Over 18,805 lncRNAs catalogued 4.

[0003] 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 lncRNA suppressed this response (5). Similarly, macrophage inflammation-suppressing transcript (MIST) overexpression decreased basal and lipopolysaccharide (LPS)-induced expression of proinflammatory response genes in macrophages(6). MIST serves as a protective lncRNA that perturbs inflammatory phenotype of macrophages(6). Furthermore, diabetes regulated anti-inflammatory RNA (DRAIR) overexpression in monocytes increased anti-inflammatory genes while inhibiting proinflammatory genes(7).

[0004] 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 biologics and small molecule drugs8–11.

[0005] There is limited data on the types of chemical modifications that can impact lncRNA performance. The lncRNAs discovered are mRNA-like lncRNAs 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 / 978-981-10-5203-3.2

[0006] N6- methyladenosine (m6A) and 5-methyl-cytosine (m5C) are two types of commonly found modifications20. M6A is the most abundant modification found in RNA and play important roles in RNA splicing, stability, nuclear export, and translation21. M6A modification can alter RNA structure or recruitment of specific m6A- binding proteins, and thus play a role in gene regulation20,21. 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 of the modified RNA molecules through affecting RNA stability and transcriptional regulation22. In lncRNAs, m5C can augment hydrophobicity of base pairs, promote base stacking, and stabilize base pair interaction22. Pseudouridine (Ψ) modification is also present in lncRNA. The function of Ψ has been found to improve translation of mRNA23; however, the role of this modification for lncRNA remains unknown.

[0007] New modes of targeting disease relevant gene expression levels of for example multiple targets including undruggable targets are desirable. SUMMARY

[0008] Long non-coding ribonucleic acid (lncRNA) 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, lncRNA have not been previously explored as a therapeutic. Here, it is established that lncRNA can be used as a new modality to treat diseases. As examples, four lncRNA from the transcriptome were re-engineered—GAPLINC, MIST DRAIR and PARAIL—and successfully used them to treat acute inflammation in lipopolysaccharide-challenged animals. For each lncRNA, in vitro transcription synthesis and high-performance liquid chromatography purification workflow. As demonstrated herein, lncRNA were tested with various, 5’-cap, 3’ poly(A) tail and chemical base modifications (Ψ-, m1Ψ- and m5C) changes to improve specificity and performance. Additionally, lncRNA was delivered in vivo using a lipid nanoparticle system. Using this pipeline and delivery platform, it is demonstrated that each lncRNA controls inflammation uniquely by specifically regulating different cytokines. This approach and these findings establish this new nucleic acid modality and demonstrates an effective way to re-engineer regulatory lncRNA molecules from the transcriptome for the treatment of disease.

[0009] 5-methyl-cytosine (m5C) is a commonly found RNA modification that can influence RNA stability and transcriptional regulation(21). In lncRNAs, m5C can augment hydrophobicity of base pairs, promote base stacking, and stabilize base pair interaction(21). Pseudouridine (Ψ) modification is another base modification of RNA. The function of Ψ has been found to improve translation of mRNA(22); however, the role and utility of this modification for lncRNA remained unknown until the present.3

[0010] 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(8). The current clinical treatment strategies for systemic inflammatory diseases are predominantly non-RNA biologics and small molecule drugs(9–12). As demonstrated herein, lipid nanoparticles (LNP) can be used for the localized delivery of lncRNAs and treatment of inflammation including sepsis.

[0011] LNPs consist of biocompatible and minimally immunogenic lipids that facilitate delivery across cell membranes, reducing dosage required for effective treatment(13). 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 anti-inflammatory lncRNAs in this LPS-challenged inflammation model, the efficacy of different lncRNAs on the treatment of acute systemic inflammation was demonstrated.

[0012] Localized delivery of lncRNAs can be achieved using for example lipid nanoparticles (LNPs). 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 lncRNA treatment was investigated during the acute phase of systemic inflammation.

[0013] To further expand the therapeutic utility of lncRNA, chemical modifications of this RNA were investigated.

[0014] The space of chemical modifications was investigated for enhancing lncRNA stability and performance and demonstrate the use of lncRNA in vivo for the treatment of 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 lncRNA LNPs . Results revealed that Ψ-modified GAPLINC RNA significantly decreased IL-1β production in vivo.

[0015] Accordingly, an aspect of the disclosure includes a long non-coding (lncRNA)- nanoparticle complex comprising one or more lncRNA molecules and a lipid nanoparticle, and the lncRNA comprises single stranded molecules at a purity of at least 99%.

[0016] An aspect of the disclosure includes a complex comprising a long non-coding (lncRNA) or a DNA encoding a lncRNA and a cell delivery agent wherein the lncRNA lacks a 5’ cap and / or a 3’poly4 A tail, and wherein the lncRNA comprises single stranded molecules at a purity of at least 99%, wherein the lncRNA comprises one or more different lncRNAs.

[0017] Another aspect includes a pharmaceutical composition comprising a complex comprising a long non-coding (lncRNA) or a DNA encoding a lncRNA and a cell delivery agent wherein the lncRNA lacks a 5’ cap and / or a 3’poly A tail, and wherein the lncRNA comprises single stranded molecules at a purity of at least 99%, wherein the lncRNA comprises one or more different lncRNAs.

[0018] In an embodiment, the lncRNA comprises two or more different lncRNAs.

[0019] In another embodiment, the length of one or more of the lncRNA is at least 400 nucleotides, at least 500 nucleotides, at least 1000 nucleotides; and / or up to 1500 nucleotides or up to 2000 nucleotides. For example, the length of the lncRNA can be the active fragment. As another example, the length of the lncRNA can be at least 80% of the naturally occurring lncRNA length. As a further example, the length can comprise additional sequence, for example up to 120% or 125% of the naturally occurring lncRNA length.

[0020] In another embodiment, the lncRNA of the complex or pharmaceutical composition DNA or nucleic acid molecule comprises or is GAPLINC, DRAIR, PARAIL and / or MIST.

[0021] In another embodiment, the lncRNA or DNA of the complex or pharmaceutical composition comprises or is selected from the nucleotide sequence of any one of SEQ ID NOs: 10-21, a sequence with at least 90% sequence identity to any thereof or the DNA encoding the lncRNA is selected from SEQ ID NO: 1, 5 and 6.

[0022] In another embodiment, the lncRNA or DNA of the complex, or the pharmaceutical composition comprises one or more chemically modified nucleotides.

[0023] In another embodiment, the chemical modified nucleotides of the complex or pharmaceutical composition, are: N1-methylpseudouridine (m1Ψ) modification, pseudouridine modification (Ψ), 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),5 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 / or 2'-O-4'-C-Locked-5-methyl-cytidine-triphosphate (2'-O-4'-C-Locked-5-Me-CTP).

[0024] In another embodiment, the lncRNA 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.

[0025] LncRNAs with one or more chemically modified nucleotides (e.g. with one or more of the same residue modified e.g. 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).

[0026] In some embodiments, the lncRNA has similar or decreased inflammasome activity compared to the unmodified molecule.

[0027] 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.

[0028] 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 (MVA), and lentivirus can be used.

[0029] 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 of cholesterol, phosphatidyl choline, optionally 1,2–distearoyl–sn–glycero–3–phosphocholine (DSPC), and / or lipid-PEG, optionally DMG-PEG2000.6

[0030] 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 %.

[0031] In another embodiment, the ionizable lipid of the complex or pharmaceutical composition comprises one or more molecules shown in Fig. 13A or 13I (OC2-K3-E10, C3-K2-E14, SM-102, ALC- 0315, Dlin-MC3-DMA (MC3), βN2 and / or δO3) and / or comprises a tertiary amine.

[0032] In another embodiment, wherein the lncRNA 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.

[0033] In another embodiment, the polydispersity of the complex is below about 0.2.

[0034] 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.

[0035] In another embodiment, the complex comprises a mass ratio of at least 20: 1 of lipid to lncRNA.

[0036] In another embodiment, the complex comprises a mass ratio between about 7:1 to about 20:1 of lipid to lncRNA. The ratio can be any ratio or range between about 7:1to about 20:1 of lipid to lncRNA.

[0037] In another embodiment, the complex comprises a mass ratio of about 20:1 of lipid to lncRNA. The lipid comprises for example, ionizable lipid or other lipid used in the nanoparticle.

[0038] The lipid can for example be ionizable lipid, zwitterionic lipid or cationic lipid depending on the lipid nanoparticle.

[0039] In another embodiment, the lncRNA or DNA of the complex or pharmaceutical composition or nucleic acid, etc is GAPLINC, DRAIR, PARAIL and / or MIST. It is shown herein that the lncRNAs tested inhibited various cytokines and inflammatory mediators for example IL-1β, IFN-γ, IL-6 and / or TNF-α. The lncRNA can also for example be a lncRNA that inhibits or activates the same inflammatory mediators, in response to for example LPS, as GAPLINC, DRAIR, PARAIL and / or MIST.

[0040] In another embodiment, the complex or pharmaceutical composition further comprises optionally one or more pharmaceutically acceptable carrier and / or excipient.

[0041] In another embodiment, the complex or pharmaceutical composition, or the lncRNA or DNA, nucleic acid etc as defined therein is used in the treatment of an acute inflammatory disease or condition or a flare up of a chronic inflammatory disease or condition.7

[0042] In another embodiment, the complex or pharmaceutical composition, or the lncRNA or DNA as defined therein is prepared in a medicament for the treatment of an acute inflammatory disease or condition or flare ups of chronic inflammatory disease or conditions.

[0043] For example, the lncRNA, complexes, nucleic acids that involve elevated levels of cytokines or inflammatory mediators such as IL-1β, IFN-γ, IL-6 and / or TNF-α and / or decreased levels of anti-inflammatory cytokines such as IL-10.

[0044] 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 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-1β, IFN-γ, IL-6 and / or TNF-α and / or decreased levels of IL- 10.

[0045] For example in graft versus host disease, a cytokine storm involving IL-6, Il-1 and TNFa is induced. Accordingly, in one embodiment, the acutely inflammatory condition is graft versus host disease.

[0046] In another embodiment, the vascular disease is or comprises atherosclerosis or atherothrombosis or the CRS is sepsis.

[0047] In another embodiment, the use is in a subject wherein the subject is a human.

[0048] An aspect of the present disclosure includes a nucleic acid molecule comprising or consisting of a long non-coding (lncRNA) wherein the lncRNA lacks a 5’ cap and / or a 3’poly A tail or a DNA molecule encoding said lncRNA, optionally for use in preparing encapsulated lncRNA.

[0049] In an embodiment, the lncRNA of the nucleic acid molecule is chemically modified.

[0050] In another embodiment, the chemical modifications are: N1-methylpseudouridine (m1Ψ) modification, pseudouridine modification (Ψ), 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),8 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 / or 2'-O-4'-C-Locked-5-methyl-cytidine-triphosphate (2'-O-4'-C-Locked-5-Me-CTP).

[0051] An aspect of the present disclosure includes a vector construct comprising a vector backbone (e.g. vector) and the DNA described herein, wherein the vector construct lacks sequence for encoding a 5’ cap and / or a 3’poly A tail.

[0052] In an embodiment, the vector is an expression plasmid or a self-amplified RNA / replicon, optionally for use in an in vitro translation system.

[0053] The vector can be a viral vector such as an adenoviral vector.

[0054] In another embodiment, the replicon structure 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. lncRNA of interest).

[0055] An aspect of the present disclosure includes a kit comprising a complex or pharmaceutical composition, a nucleic acid molecule, a vector described herein and one or more chemically modified residues, and optionally one or more components for in vitro translation or cell delivery agents.

[0056] In an embodiment, the kit includes a cell delivery agent which is a lipid nanoparticle.

[0057] An aspect of the present disclosure includes a method of treating an acute inflammatory disease or flare up of a chronic inflammatory disease or condition, the method comprising administering a complex or pharmaceutical composition, a nucleic acid molecule, or a vector to a subject in need thereof.

[0058] In an embodiment, the acute inflammatory disease is a systemic inflammation, optionally a cytokine release syndrome (CRS).

[0059] In an embodiment, the inflammatory disease is sepsis or graft versus host disease.

[0060] 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, or a vector described herein to a subject.

[0061] In an embodiment, the one or more cytokine comprises IL-1β, TNFα, IFN-γ and / or IL-6.

[0062] In another embodiment, the altering comprises decreasing expression.

[0063] In another embodiment, the one or more cytokine comprises IL-10.

[0064] In another embodiment, the altering comprises increasing IL-10.9

[0065] 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. The inflammatory disease can for example be acute and / or systemic.

[0066] 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.

[0067] Uses of the compositions, nucleic acid molecules, vectors or complexes described herein and the methods described herein are also contemplated.

[0068] 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

[0069] 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:

[0070] 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 lncRNA-based therapeutics. FIG. 1B 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 lncRNAs. FIG. 1D shows LNPs with no cap and tail GAPLINC exhibit smaller sizes than other GAPLINC LNPs. FIG. 1E shows PDI of the GAPLINC LNPs is not significantly different from each other. FIG. 1F shows10 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 GAPLINC without a cap or tail (200 ng) showed the least NF-κB activation compared to other modifications (n=3). Error bars shown are mean ± SEM. * P < 0.05, *** P < 0.001 and **** P < 0.0001.

[0071] FIGs. 2A-2F show exogenously delivered lncRNA attenuates LPS-induced inflammation in vitro and is sensitive to chemical modifications in an exemplary embodiment of the disclosure. FIG. 2A shows NF-κB 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- κB 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. Ψ-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, m1Ψ- and m5C-modified MIST decreases NF-κB activation relative to LPS control. FIG. 2D shows Ψ-modified MIST showed minimal IRF activity compared to other base modifications tested. FIG. 2E shows unmodified and modified DRAIR decreased NF-κB activation compared to LPS control. FIG. 2F shows Ψ- and m1Ψ-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.

[0072] FIGs 3A-3J show exogenous GAPLINC LNPs downregulates proinflammatory cytokines in vivo in an exemplary embodiment of the disclosure. FIG. 3A shows mouse serum collected from C57BL / 6 mice treated with or without 1.0 mg / kg unmodified or modified GAPLINC LNPs and 100 µg LPS for 6 h. Luminex was performed to analyze mouse cytokines, chemokines and growth factor expression. FIG. 3B shows Ψ-modified GAPLINC demonstrated the most significant decrease in IL-1β levels in serum relative to the other unmodified and modified GAPLINC payloads. FIG. 3C shows IFN-γ expression decreased most significantly with Ψ-modified GAPLINC relative to unmodified GAPLINC. FIG. 3D shows unmodified, m1Ψ- and m5C-modified GAPLINC downregulated TNF⍺ levels. FIG. 3R shows m1Ψ-modified GAPLINC decreased IL-6 expression. FIG. 3F shows m1Ψ-modified GAPLINC increased IL-10 expression. FIG. 3G shows formulation optimization of GAPLINC LNPs. Ψ-modified 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 Ψ-modified GAPLINC encapsulation11 efficiency in LNP. FIG. 3I shows a 20:1 formulation that showed significantly more downregulation of IL- 1β expression compared to the 7:1 formulation at 0.10 mg / kg. FIG. 3J shows a treatment of C57BL / 6 mice challenged with 100 µg LPS using a low 0.10 mg / kg dose of Ψ-modified GAPLINC LNPs (20:1) significantly downregulated IL-1β 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. In vivo data represent results from n = 4 to 8 biological replicates. Error bars shown are mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001.

[0073] FIGs 4A-4I show exogenous MIST LNPs downregulates TNF⍺ 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 µg LPS for 6 h. Luminex was used to analyze mouse cytokines, chemokines and growth factor expression. MIST RNA did not change IL-1β expression. FIG. 4F shows m1Ψ-modified MIST upregulated the least IFN-γ compared to the other modifications tested. FIG. 4G shows Ψ-modified MIST showed the most decrease in TNF⍺ 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. 4I shows m1Ψ-modified MIST increased IL-10 expression. A total of three DLS measurements were completed for each LNP shown. In vivo data 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.

[0074] FIGs. 5A-5I show exogenous DRAIR LNPs downregulates TNF⍺ 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 µg LPS for 6 h. Luminex was used to analyze mouse cytokines, chemokines and growth factor expression. DRAIR RNA did not change IL-1β or IFN-γ expression. FIGs. 5G-5H show unmodified and Ψ-, m1Ψ- and m5C-modified DRAIR all decreased TNF⍺ and IL-6 expression. FIG. 5I shows Ψ-modified DRAIR increased IL-10 expression. For nanoparticle characterization, a total of three DLS measurements were completed for each LNP shown. In vivo data represent results from n = 4 to 8 biological replicates. Error bars shown are mean ± SD. * P < 0.05, ** P < 0.01 and **** P < 0.0001.12

[0075] 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 lncRNA. FIG. 6B shows unmodified and m1Ψ-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 cellulosepurified sample. FIG. 6C shows bioanalyzer analysis of cellulose-purified unmodified and m1Ψ-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.

[0076] 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 lncRNA in an exemplary embodiment of the disclosure. FIG. 7A shows HPLC purification workflow to obtain single species of IVTsynthesized lncRNA with minimal dsRNA. FIG. 7B shows bioanalyzer analysis of the reversed-phase HPLC 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 Ψ-modified GAPLINC RNA using different temperatures. Increasing temperature led to fewer peaks which was likely attributed to diminished secondary structure. FIG. 7E shows bioanalyzer of Ψ-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 Ψ-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 lncRNA produced welldefined 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.

[0077] FIGs. 8A-8B show reversed-phase HPLC purification of modified GAPLINC RNA at 70°C allows for the selection of a single species of lncRNA 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.13 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.

[0078] 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 µg 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.

[0079] 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 µg of unmodified or modified MIST RNA was purified by RP-HPLC in an exemplary embodiment of the disclosure. 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 major peaks, denoted as peak 1 and 2, were collected separately.

[0080] FIGs. 11A-11D show reversed-phase HPLC traces for unmodified and modified DRAIR RNA in an exemplary embodiment of the disclosure. A total of 100-500 µg of unmodified or modified DRAIR RNA was purified by 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 (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.

[0081] FIGs. 12A-12D show reversed-phase HPLC purification allows for the 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 intensity14 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 lncRNA.

[0082] FIGs. 13A-13B show ionizable lipids used for delivery of lncRNAs 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 lncRNAs. 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 βN2. FIG. 13I shows the structure of δO3. 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 µL of PBS (n = 4) or 200 µL of LNP containing 2 µg of firefly luciferase mRNA (n = 3). Data shown is mean ± S.D.

[0083] FIGs. 14A-14D shows unmodified and modified GAPLINC encapsulated in lipofectamine did not induce NF-κB activation and GAPLINC-202 and 205 isoforms did not affect NF-κB and IRF activity. FIG. 14A shows NF-κB 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-κB 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-κB 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.

[0084] FIGs. 15A-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.

[0085] 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 and15 PDI of LNPs formulated with 7:1 nanomaterial to RNA mass ratio and encapsulating unmodified or Ψ- modified GAPLINC RNA purified with cellulose or HPLC. FIG. 16B shows similar encapsulation efficiencies for LNPs containing unmodified or Ψ-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.

[0086] FIGs. 17A-17F show GAPLINC LNPs significantly downregulates key inflammatory protein, IL-1β, 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 µg LPS. The unmodified GAPLINC payload was purified using cellulose method. 1 mg / kg LNP significantly downregulated IL-1β expression. However, IFN-γ 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 dose tested. 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.

[0087] 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 µg 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.

[0088] FIGs. 19A-19C show body and organ weights and tissue morphology of Ψ-modified 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 Ψ-modified 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 results16 from n = 4 biological replicates for each treatment group. Black scale bars denote 50 µm. Images are representative of the PBS and LNP groups.

[0089] 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 µg 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.

[0090] 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 µg 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.

[0091] FIG. 22 is an alignment of GAPLINC and isoforms 202 and 205.

[0092] FIG. 23A 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. DETAILED DESCRIPTION OF VARIOUS EMBODIMENTS

[0093] 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 not intended 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.

[0094] The following non-limiting examples are illustrative of the present application: I. Definitions

[0095] 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. In17 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.

[0096] 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.

[0097] 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 the transcription which provides stability to the RNA molecule and can promote translation.

[0098] 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.

[0099] 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.

[00100] 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), 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.

[00101] 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 bound18 or recognized by any RNA polymerase. Exemplary promoters include, but are not limited to, a SP6 promoter, a T7 promoter, and a T3 promoter.

[00102] 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 for translation can be referred to as an “in vitro translation system”.

[00103] 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%.

[00104] As used herein the singular forms "a", "an", and "the" include plural references unless the context clearly dictates otherwise.

[00105] 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.

[00106] 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 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.

[00107] 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."

[00108] The term "subject" as used herein includes all members of the animal kingdom including mammals, and suitably refers to humans.19

[00109] 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).

[00110] 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 NBLAST and XBLAST programs of Altschul et al., 1990, J. Mol. Biol. 215:403. BLAST nucleotide searches can be performed with the NBLAST 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. BLAST protein searches can be performed with the XBLAST program parameters set, e.g., to score-50, word length=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 comparing20 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.

[00111] The term "administered" or “administering” as used herein means administration of a therapeutically effective 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.

[00112] The term “pharmaceutically acceptable” means compatible with the treatment of subjects, for example humans.

[00113] 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.

[00114] 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."

[00115] 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 openended, 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.

[00116] 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.

[00117] 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 to21 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.

[00118] 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 Thereof

[00119] An aspect of the disclosure includes a complex comprising a long non-coding (lncRNA) or a DNA encoding a lncRNA and a cell delivery agent, and wherein the lncRNA comprises single stranded molecules at a purity of at least 99%, wherein the lncRNA comprises one or more different lncRNAs.

[00120] In an embodiment, the lncRNA 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.

[00121] The inventors have determined, that lncRNAs lacking a cap and / or tail are therapeutically effective. These lncRNAs 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, G or C nucleotides were shown to be useful.

[00122] They have also found that these molecules can be effectively delivered to cells with various delivery agents.

[00123] Another aspect of the disclosure includes a long non-coding (lncRNA)-nanoparticle complex comprising a lncRNA and a cell delivery agent.

[00124] In an embodiment, the cell delivery agent is a lipid nanoparticle and the lncRNA is encapsulated by a lipid nanoparticle wherein, the lncRNA comprises one or more lncRNA.

[00125] In some embodiments, the lipid nanoparticle comprises biocompatible and / or minimally immunogenic lipids and / or comprises a tertiary amine.

[00126] In some embodiments, the lncRNA comprises or is selected from GAPLINC, DRAIR, MIST and / or PARAIL.

[00127] The lncRNAs used in the complexes, compositions, kits, nucleic acid molecules etc are purified to remove for example dsRNA or other contaminants, for example by HPLC. 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 at22 a temperature (e.g. a denaturation temperature to disrupt intra and inter lncRNA interactions) of at about or at least 45℃, at about or at least 50℃, at about or at least 55℃, at about or at least 60℃, at about or at least 65℃ or at about or at least 70℃. For example, the purification can be carried out in the range of about 40℃ to about 80℃. The temperature can be achieved for example by heating the HPLC column.

[00128] Other purification methods can be used instead of or in combination with HPLC, for example cellulose chromatography purification.

[00129] In some embodiments, the nucleotide sequence of one or more lncRNA molecules is unmodified.

[00130] In some embodiments, the lncRNA is chemically modified or comprises one or more chemically modified nucleotides.

[00131] In some embodiments, the chemically modified lncRNA comprises one or more N1- methylpseudouridine (m1Ψ) modification, one or more pseudouridine modification (Ψ), 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'-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'-Omethyl-adenosine-5'-triphosphate (2'-O-Me-ATP), 2'-O-methyl-guanosine-5'-triphosphate (2'-O-MeGTP), 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).

[00132] 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 (Ψ) refers to one or more uridine triphosphate (UTP) in a nucleotide sequence being replaced with pseudouridine-5’-triphosphate (Ψ). For example, one or more N1-methylpseudouridine (m1Ψ) modification refers to one or more one or more uridine triphosphate (UTP) in a nucleotide sequence being replaced with N1-methylpseudouridine-5’- triphosphate (m1Ψ). In some embodiments, one or more pseudouridine modification (Ψ) refers to all uridine triphosphate (UTP) residues present in a nucleotide sequence being replaced with pseudouridine- 5’-triphosphate (Ψ). In some embodiments, one or more N1-methylpseudouridine (m1Ψ) modification refers to all uridine triphosphate (UTP) residues present in a nucleotide sequence being replaced with N1- methylpseudouridine-5’-triphosphate (m1Ψ). In some embodiments, one or more 5-methylcytidine-5’-23 triphosphate (m5C) modification refers to all cytidine triphosphate (CTP) residues in a nucleotide sequence being replaced with 5-methylcytidine-5’-triphosphate (m5C).

[00133] In some embodiments, the lncRNA comprises the nucleotide sequence of Table 1. In some embodiments, the lncRNA comprises or is selected from the nucleotide sequence of any one of SEQ ID NOs: 10-21, 26 to 30.

[00134] In some embodiments, the lncRNA comprises unmodified GAPLINC (e.g., SEQ ID NO: 10).

[00135] Sequences with at least 90%, sequence identity can also be used. For example, the lncRNA can comprise or be any of the nucleotide sequences of any one of SEQ ID NOs: 10-21, 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 lncRNA is selected from SEQ ID NO: 1, 5 and 6 or a sequence with at least 85% or at least 90% sequence identity thereto.

[00136] The sequence identity can be at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical.

[00137] 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 BLASTTM alignment or other alignment tool.

[00138] In some embodiments, the lncRNA comprises m5C-modified GAPLINC (e.g., SEQ ID NO: 11). In some embodiments, the one or more lncRNA molecule comprises Ψ -modified GAPLINC (e.g., SEQ ID NO: 12 or a sequence with at least 85% or at least 90% sequence identity thereto). In some embodiments, the one or more lncRNA molecule comprises m1Ψ -modified GAPLINC (e.g., SEQ ID NO: 13 or a sequence with at least 85% or at least 90% sequence identity thereto).

[00139] In some embodiments, the first 19 residues of GAPLINC are not substituted.

[00140] In some embodiments, the lncRNA comprises unmodified MIST (e.g., SEQ ID NO: 14). In some embodiments, the one or more lncRNA molecule comprises m5C-modified MIST (e.g., SEQ ID NO: 15). In some embodiments, the one or more lncRNA molecule comprises Ψ -modified MIST (e.g., SEQ ID NO: 16 or a sequence with at least 85% or at least 90% sequence identity thereto). In some24 embodiments, the one or more lncRNA molecule comprises m1Ψ -modified MIST (e.g., SEQ ID NO: 17 or a sequence with at least 85% or at least 90% sequence identity thereto).

[00141] In some embodiments, the lncRNA comprises unmodified DRAIR (e.g., SEQ ID NO: 18 or a sequence with at least 85% or at least 90% sequence identity thereto). In some embodiments, the one or more lncRNA molecule comprises m5C-modified DRAIR (e.g., SEQ ID NO: 19 or a sequence with at least 85% or at least 90% sequence identity thereto). In some embodiments, the one or more lncRNA molecule comprises Ψ -modified DRAIR (e.g., SEQ ID NO: 20 or a sequence with at least 85% or at least 90% sequence identity thereto). In some embodiments, the one or more lncRNA molecule comprises m1Ψ -modified MIST (e.g., SEQ ID NO: 21 or a sequence with at least 85% or at least 90% sequence identity thereto).

[00142] In some embodiments, the lncRNA comprises unmodified PARAIL (e.g., SEQ ID NO: 26 or a sequence with a least 85% or at least 90% sequence identity thereto). In some embodiments, the one or more lncRNA molecule comprises m5C-modified PARAIL (e.g., SEQ ID NO: 27 or a sequence with at least 85% or at least 90% sequence identity thereto). In some embodiments, the one or more lncRNA molecule comprises Ψ -modified PARAIL (e.g., SEQ ID NO: 28 or a sequence with at least 85% or at least 90% sequence identity thereto). In some embodiments, the one or more lncRNA molecule comprises m1Ψ -modified PARAIL (e.g., SEQ ID NO: 29 or a sequence with at least 85% or at least 90% sequence identity thereto). In some embodiments, the one or more lncRNA molecule comprises m6A-modified PARAIL (e.g., SEQ ID NO: 30 or a sequence with at least 85% or at least 90% sequence identity thereto).

[00143] The length of the lncRNA can also vary for example by about 20%, For example the sequence can be up to 20% shorter than the naturally occurring lncRNA, 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.

[00144] In some embodiments, the lipid nanoparticle comprises an ionizable lipid. It can also comprise zwitterionic lipid or cationic lipid.

[00145] 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.25

[00146] 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), βN2, and δO3). 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. In some embodiments, the ionizable lipid comprises a lipid as described in PCT / CA2024 / 051647 and / or WO 2023 / 092242.

[00147] In some embodiments, the nanoparticle comprises an ionizable lipid, cholesterol, a phospholipid, and a PEG-lipid.

[00148] In some embodiments, the nanoparticle comprises a PEG-lipid selected from the group consisting of 14:0 PEG2000 PE which is 1,2-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 1,2-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-racglycerol-PEG2K, DSPC which is 1,2-distearoyl-sn-glycero-3-phosphocholine and combinations thereof.

[00149] In some embodiments, the lipid nanoparticle comprises an ionizable lipid as described in WO2023 / 092242 which is incorporated herein by reference.

[00150] 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 %.

[00151] In some embodiments, the lipid nanoparticle comprises an ionizable lipid as described in PCT / CA2024 / 051647 which is incorporated herein by reference.

[00152] In some embodiments, the lipid nanoparticle comprises or is lipofectamine (e.g. the lipid is the lipid in lipofectamine).

[00153] In some embodiments, the complex comprise features as described in Table 2.

[00154] 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 150nm. 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 some embodiments, the size of the complex is about 141 nm. In some embodiments, the size of the complex is26 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.

[00155] In some embodiments, the polydispersity of the complex is below about 0.2.

[00156] 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.

[00157] In some embodiments, the complex comprises a mass ratio of at least 20:1 of lipid to lncRNA. 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.

[00158] In some embodiments, the complex comprises a mass ratio between about 5:1 to 20:1 of lipid to lncRNA. In some embodiments, the complex comprises a mass ratio between about 7:1 to 20:1 of lipid to lncRNA. In some embodiments, the complex comprises a mass ratio of about 5: 1 of lipid to lncRNA. In some embodiments, the complex comprises a mass ratio of about 6: 1 of lipid to lncRNA. In some embodiments, the complex comprises a mass ratio of about 7: 1 of lipid to lncRNA. In some embodiments, the complex comprises a mass ratio of about 20: 1 of lipid to lncRNA. In some embodiments, the complex comprises a mass 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.

[00159] In some embodiments, the lncRNA comprises a 5’ cap and / or 3’poly A tail. In some embodiments, the lncRNA lacks a 5’ cap and / or 3’poly A tail. In some embodiments, the lncRNA lacks a 5’ cap. In some embodiments, the lncRNA lacks 3’poly A tail. In some embodiments, the lncRNA lacks a 5’ cap and 3’poly A tail.

[00160] A composition comprising the complex described herein, and optionally one or more pharmaceutically acceptable carrier and / or excipient, optionally sterile water or buffered saline. In some embodiments, the composition comprises a concentration of the complex of at least about 30 ng / µL to about 500 ng / µL.

[00161] Another aspect of the disclosure includes a method of treating acute inflammatory diseases and conditions or flare ups of a chronic inflammatory disease or condition, the method comprising27 administering the lncRNA, DNA the complexes or compositions described herein to a subject in need thereof. In some embodiments, the subject is a human.

[00162] 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).

[00163] In some embodiments, the complex is administered in a dosage of up to about 5mg / kg. In some embodiments, the complex is administered in a dosage of about 0.1mg / kg. In some embodiments, the complex is administered in a dosage of about 1mg / kg. In some embodiments, the complex is administered in a dosage of about 3mg / kg. In some embodiments, the complex is administered in a dosage of about 5 mg / kg. In some embodiments, the complex is administered in a single dose. In some embodiments, the complex is administered in a plurality of doses.

[00164] 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.

[00165] In some embodiments, the one or more cytokine comprises IL-1β, TNFα, IFN-γ and / or IL- 6. In some embodiments, the altering comprises decreasing expression e.g., as compared to a control.

[00166] 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.

[00167] In some embodiments, the lncRNA comprises Ψ-modified GAPLINC and expression of IL-1β is decreased e.g., as compared to a control.

[00168] In some embodiments, the lncRNA comprises unmodified, Ψ-, m1Ψ- and / or m5Cmodified GAPLINC and expression of IL-1β is decreased e.g., as compared to a control.

[00169] In some embodiments, the lncRNA comprises Ψ-, m1Ψ- and / or m5C-modified GAPLINC and expression of IFN-γ is decreased e.g., as compared to a control.

[00170] In some embodiments, the lncRNA comprises unmodified, m1Ψ-, and / or m5Cmodified GAPLINC and expression of TNF⍺ is decreased e.g., as compared to a control.28

[00171] In some embodiments, the lncRNA comprises m1Ψ-modified GAPLINC and expression of IL-6 is decreased e.g., as compared to a control.

[00172] In some embodiments, the lncRNA comprises m1Ψ-modified GAPLINC and expression of IL-10 is increased e.g., as compared to a control.

[00173] In some embodiments, the lncRNA comprises m1Ψ-modified GAPLINC and expression of MCP-1 is decreased e.g., as compared to a control.

[00174] In some embodiments, the lncRNA comprises Ψ-modified GAPLINC and expression of IFN-γ is decreased e.g., as compared to a control.

[00175] In some embodiments, the lncRNA comprises unmodified MIST and expression of TNFα and / or IL-6 is decreased e.g., as compared to a control.

[00176] In some embodiments, the lncRNA comprises unmodified, Ψ-, m1Ψ- and / or m5Cmodified DRAIR and expression of TNFα and / or IL-6 is decreased e.g., as compared to a control.

[00177] In some embodiments, the lncRNA comprises Ψ-modified DRAIR and IL-10 expression is increased. In some embodiments, the lncRNA comprises m1Ψ-modified DRAIR and IL-10 expression is increased e.g., as compared to a control.

[00178] In some embodiments, the lncRNA comprises unmodified, Ψ-, and / or m5C modified MIST and IFN-γ expression is increased e.g., as compared to a control.

[00179] In some embodiments, the lncRNA comprises unmodified and / or Ψ-modified MIST and TNFα and / or IL-6 expression is decreased e.g., as compared to a control.

[00180] In some embodiments, the lncRNA comprises m1Ψ-modified MIST and IL-10 expression is increased e.g., as compared to a control.

[00181] In some embodiments, the control is an unmodified lncRNA (e.g., comprising no chemical modifications). In some embodiments, the control is a lncRNA with a different chemical modification than the lncRNA being compared with the control.

[00182] In some embodiments, the complex is administered in a dosage of up to about 5mg / kg. In some embodiments, the complex is administered in a dosage of about 0.1mg / kg. In some embodiments, the complex is administered in a dosage of about 1mg / kg. In some embodiments, the complex is administered29 in a dosage of about 3mg / kg. In some embodiments, the complex is administered in a dosage of about 5 mg / kg.

[00183] In some embodiments, the complex is administered in a single dose. In some embodiments, the complex is administered in a plurality of doses.

[00184] In some embodiments, the complex is administered at least about every 10 hours or less. In some embodiments, the complex is administered at least about every 9 hours or less. In some embodiments, the complex is administered at least about every 8 hours or less. In some embodiments, the complex is administered at least about every 7 hours or less. In some embodiments, the complex is administered at least about every 6 hours or less. In some embodiments, the complex is administered at least about every 5 hours or less. In some embodiments, the complex is administered at least about every 4 hours or less. In some embodiments, the complex is administered at least about every 3 hours or less. In some embodiments, the complex is administered at least about every 2 hours or less. In some embodiments, the complex is administered at least about every hour or less. In some embodiments, the complex is administered at least about every 6 hours.

[00185] In some embodiments, the modified lncRNA is a chemically modified lncRNA as described herein. In some embodiments, the unmodified lncRNA is not chemically modified (e.g., naturally occurring). In some embodiments, the unmodified lncRNA is an unmodified lncRNA as described herein.

[00186] In some embodiments, the functionality comprises altering levels of expression of one or more proteins in vivo.

[00187] Uses of the complexes described herein and the methods described herein are also contemplated.

[00188] 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.

[00189] 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.30

[00190] 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. Examples Example 1 Exogenous GAPLINC LNPs deployed in vitro can control cell phenotype

[00191] As a proof-of-concept lncRNA, unmodified and modified GAPLINC were synthesized using in vitro transcription (IVT). 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 m1Ψ, Ψ and m5C modifications. However, additional faint bands at smaller sizes were observed for these modified lncRNAs, suggesting the potential presence of aborted transcripts or other IVT contaminants.

[00192] 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 of GAPLINC. 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 lncRNAs (FIGs. 8-12).

[00193] Subsequently, the functional utility of cap and tail modifications for lncRNA 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. 1E and F). Encapsulation efficiency was highest overall for LNPs containing GAPLINC with no cap or tail (FIG. 1F). Because GAPLINC impacts NF-κB expression, RAW-Dual cells were31 challenged with LPS and treated with these LNPs for 18 h. Supernatant was collected to assess NF-κB activation using QUANTI-Blue (FIG. 1G). LPS challenge (200 ng / mL) significantly increased activation of NF-κB compared to untreated cells (FIG. 1H). Empty particles did not attenuate NF-κB activation. All LNPs containing GAPLINC significantly decreased NF-κB activation. Notably, LNPs encapsulating GAPLINC with no cap or tail showed the greatest decrease in NF-κB activation relative to other GAPLINC payloads. These findings showed that exogenous lncRNA can alter cell phenotype in vitro and supported the subsequent production of the lncRNA with no cap and tail. Exogenous lncRNA attenuation of NF-κB activation in vitro depends on base modifications

[00194] To screen the functional performance of base-modified lncRNAs, 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 lncRNA. As expected, the LPS challenge significantly increased NF-κB activation relative to untreated cells (FIGs. 2A, 2C and 2E). For GAPLINC, unmodified and Ψ-, m1Ψ- and m5C-modified payloads showed significant decreases in NF-κB activation (FIG. 2A). To confirm the effects were caused by the lncRNA and not the delivery system, it was demonstrated that empty LNPs did not induce or reduce NF-κB activation (FIG. 13A). Additionally, interferon regulatory factor (IRF) activity was used as a measurement of immunogenicity of the lncRNAs. IRF activity was minimized with Ψ- and m1Ψ- 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-κB and IRF activities, suggesting that the effect of GAPLINC is isoform specific (FIGs. 14B-14D). For MIST, only m1Ψ- and m5C modifications significantly decreased NF-κB 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 Ψ modification. For DRAIR, unmodified and Ψ-, m1Ψ- and m5C-modified payloads significantly attenuated NF-κB activation (FIG. 2E). IRF activity was only minimized with Ψ and m1Ψ 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 lncRNA chemical base modifications

[00195] Upon validating the utility of lncRNAs in vitro, the first model lncRNA, 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 HPLCpurified unmodified or modified GAPLINC RNA in a microfluidic device as previously described(23, 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 (232 µg; 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. Ψ-modified GAPLINC LNPs were 150 nm, m1Ψ- 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 lncRNA 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 lncRNA have noticeably lower encapsulation efficiencies compared to no dsRNA and cellulose purification (FIG. 16B).

[00196] For the disease model, systemic inflammation was induced in C57BL / 6 mice by intraperitoneally injecting 100 µg 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 Ψ-, m1Ψ- and m5C-modified GAPLINC LNP significantly attenuated IL-1β expression compared to LPS and scrambled GAPLINC LNPs (FIG. 3D). Importantly, unmodified and Ψ-modified GAPLINC LNPs had noticeably lower IL-1β expression compared to the other modifications. Since IL-1β acts through an NF-κB-dependent pathway, these results are consistent with the in vitro observations (FIG. 2A). Chemically modified bases significantly lowered IFN-γ expression compared to unmodified GAPLINC. Additionally, GAPLINC with the Ψ modification was similar to PBS control (FIG. 3C). Unmodified and m1Ψ- and m5C-modified GAPLINC LNPs downregulated TNF⍺ (FIG. 3D). m1Ψ-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 m1Ψ- modified GAPLINC LNPs (FIG. 18E). Results indicate that changes in cytokine expression is sensitive to chemical base modifications of GAPLINC.

[00197] The Ψ-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 significant33 differences in LNP size, PDI and charge was found (FIG. 3G). However, the 20:1 formulation increased encapsulation of the lncRNA from 53% to 85% (FIG. 3H), thereby meeting best practice criteria(25). Comparing the different formulations and doses of LNPs on IL-1β expression in vivo, the low 0.10 mg / kg dose of Ψ-modified GAPLINC LNPs 20:1 formulation significantly downregulated IL-1β at 6 h compared to LPS (FIG. 3I). The effect of this 20:1 formulation was diminished by 10 h (FIG. 3J). A tolerability study of these lncRNA 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 lncRNAs tested. Ψ-Modified MIST LNPs serves as a TNFα-specific treatment in vivo

[00198] Next, it was investigated whether MIST, another lncRNA 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 Ψ- and m1Ψ-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 µg LPS and treated for 6 h with 0.10 mg / kg of modified or unmodified MIST LNPs, no changes in IL- 1β expression were observed (FIG. 4E). Significant increases in IFN-γ expression with unmodified, Ψ- and m5C-modified MIST LNPs was observed but not m1Ψ-modified MIST LNPs (FIG. 4F). However, unmodified and Ψ-modified MIST LNPs significantly downregulated TNFα (FIG. 4G). Similarly, there were decreases in IL-6 expression with these particles (FIG. 4H). m1Ψ-modified MIST LNPs significantly upregulated IL-10 levels and this was drastically different compared to the other payloads tested (FIG. 4I). 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 lncRNAs and that the impact of base modifications can change with lncRNA sequence. In contrast to GAPLINC, exogenously delivered Ψ-modified MIST LNPs may be a TNFα-specific treatment.34 Base modification specifies targeting of TNFα, IL-6 and IL-10 by DRAIR in vivo

[00199] 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 m1Ψ- modified DRAIR LNPs had similar sizes (168 nm and 169 nm, respectively; FIG. 5A). The sizes of Ψ- 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. Ψ- and m1Ψ-modified LNPs were akin to each other with a charge of 29 and 25 mV, respectively. DRAIR LNPs exhibited 80% encapsulation for unmodified and Ψ-modified, 76% for m1Ψ-modified, and 60% for m5C- modified. Then, C57BL / 6 mice were challenged with 100 µg LPS and treated with 0.10 mg / kg DRAIR LNPs for 6 h. Results indicate that IL-1β and IFN-γ expression was not significantly different for all treatments compared to control (FIG. 5EF). Unmodified and Ψ-, m1Ψ- and m5C-modified DRAIR LNPs significantly downregulated TNFα and IL- 6 expression (FIG. 5G-H). Interestingly, Ψ-modified DRAIR LNPs significantly upregulated IL-10 levels (FIG. 5I). 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 Ψ-, m1Ψ- and m5C-modified DRAIR can act on TNFα and IL-6. Notably, m1Ψ-modified DRAIR can upregulate the production of the anti-inflammatory IL-10.

[00200] Unlike coding transcripts, lncRNAs control cell function without translation into protein. For this reason, lncRNA 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 lncRNA have already been established through evolution. Despite their potential, lncRNAs 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 lncRNA as functional therapeutics.

[00201] LncRNA therapeutics require two important features: the delivery system and pure fulllength or active lncRNA transcript with minimal immune system activation. A robust delivery system that can protect the lncRNA from degradation is crucial for successful delivery to desired tissues. LNPs are an established method for mRNA delivery. By adopting LNPs for use with lncRNAs, it was successfully demonstrated that 5’-capping and 3’ poly(A) tails are not necessary for the function of the model lncRNA. 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.

[00202] Base modification of lncRNAs can differentially control cytokine expression in vivo. Due to the labile and immunogenic nature of RNA, unmodified RNA synthesized through IVT may not be35 feasible for clinical use(32). The incorporation of natural modified nucleotides into transcripts may enhance RNA biological properties. Low dose Ψ-modified GAPLINC LNPs (0.10 mg / kg) targets IL-1β, a cytokine regulated by NF-κB, in the LPS-induced systemic inflammation mouse model.

[00203] Other modified lncRNA show different but compelling utility. Low doses of LNPdelivered unmodified MIST downregulated TNFα and IL-6. Additionally, Ψ-modified MIST specifically targeted TNFα. Stapleton et al. implicated MIST in chronic systemic low-grade inflammation(6). It was shown that loss of MIST led to the upregulation of cytokines such as TNFα and IL-6, shifting macrophages to a proinflammatory phenotype(6). Thus, it is demonstrated herein that in an acute inflammation, MIST may target TNFα or IL-6 depending on base modifications utilized. Similarly, unmodified and Ψ-, m1Ψ- and m5C-modified DRAIR LNPs drastically downregulated TNFα 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 m1Ψ-modified DRAIR may elevate IL-10 production. Previously, Ψ-modified RNA was characterized to perform best in suppressing RNA-mediated immune activation(22). Indeed, delivery of Ψ- modified GAPLINC may minimize IFN-γ expression relative to other base modifications. However, while m1Ψ-modified MIST had the least activation of IFN-γ, there were no differences observed among the different chemically modified DRAIR. Of note, chemical modifications of RNA nucleotides can alter secondary structure of lncRNA and impact function(33). Given the data, it appears that base modificationinduced secondary structure changes alter function.

[00204] The lncRNA 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, lncRNA encapsulation and functional performance at a low dose of 0.10 mg / kg. The single dose effect of these lncRNA 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 lncRNAs from the human transcriptome were identified and developed lncRNA-based drugs capable of targeting IL-1β, TNFα and IL-6 in vivo. These lncRNA-based therapies depart from traditional strategies that use recombinant proteins for anti-inflammatory treatments. Materials and Methods Plasmid construction, PCR amplification and digestion

[00205] To generate DNA template for IVT of the lncRNAs, the sequence of GAPLINC (Gene ID:100505592), GAPLINC-202 (ENST00000579007.5), GAPLINC-205 (ENST00000700774.1), scrambled GAPLINC, MIST (MIST_v1 from Stapleton et al.(6)) were individually cloned into a pUCIDTKan 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 T736 promoter (TAATACGACTCACTATAG) was inserted before the lncRNA for IVT and an insert for a forward and reverse primer for PCR flank the T7 promoter and lncRNA. The end of the GAPLINC, GAPLINC-202, GAPLINC-205, and MIST gene contains a NsiI enzymatic cut site to allow for the reverse primer to be removed before IVT so that only the lncRNA gene will be present in the IVT amplicon. Similarly, the vector for DRAIR contains a AfeI enzymatic cut site. The end of the scrambled GAPLINC sequence has an ApaI 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, NsiI-HF (NEB R3127), AfeI (NEB R0652S) or ApaI (NEB R0114S) restriction enzyme were used to remove the reverse primer downstream of the lncRNA 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. lncRNA IVT

[00206] For the unmodified lncRNA, 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 lncRNA 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 (Ψ) (Hongene, R5-022) or N1-methylpseudouridine-5’- triphosphate (m1Ψ) (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 µg 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 µg 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 lncRNA

[00207] To purify dsRNA contaminants, a cellulose purification protocol was adapted from Baiersdörfer 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 µL of cellulose slurry (~0.14 g cellulose) was transferred to 0.45 µm cut-off ultrafiltration spin-columns in 1.5 mL Eppendorf tubes. 100 to 500 µg of IVT lncRNA were loaded to the ultrafiltration spin-columns and37 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 lncRNA was collected. The purified lncRNA 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 lncRNA was pelleted by centrifugation for 15 min at 14,000 x g. The pellet was washed with 500 µL 70% pre-cooled ethanol and then dissolved in 50-100 µL nuclease-free water. Bioanalyzer at the SickKids TCAG Microarray Facility was used to assess the integrity and purity of the purified lncRNA. Reverse-phase HPLC purification of IVT lncRNA

[00208] lncRNA was purified using an Agilent BioHPLC PLRP-S reverse-phase column (1000Å, 10 µm, 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 lncRNA 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 linear gradient 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 lncRNA was recovered by incubating with 0.1 vol 3 M sodium acetate (pH 5.5) and 1 vol isopropanol overnight at -20°C. Precipitated lncRNA was pelleted by centrifugation for 15 min at 14,000 x g. The pellet was washed with 500 µL 70% pre-cooled ethanol and then dissolved in 50-100 µL nuclease-free water. Bioanalyzer was used to assess the integrity and purity of the purified lncRNA. Dot Blot analysis of dsRNA

[00209] LncRNA and the positive control synthetic dsRNA (poly (I:C) [LMW]; Invivogen, tlrlpicw) was diluted in nuclease-free water to a final concentration of 40 ng / µL. Positively charged Whatman Nytran SuPerCharge nylon membrane (Sigma-Aldrich, WHA10416230) was placed on a sheet of Whatman GB005 blotting paper (Sigma-Aldrich, WHA10426972). Loading of RNA was performed by pipetting 5 µL (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 mL 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, NBP3-11395-38 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 lncRNA LNPs

[00210] The ionizable lipids, C3-K2-E14 and OC2-K3-E10, were synthesized as previously described by Tilstra et al(23) (Supplementary FIG. 7A and B). To formulate particles, the ionizable lipid was combined with DMG-PEG2000 and mixed with unmodified or modified lncRNA 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 1X PBS to remove ethanol and exchange buffer before sterile filtration through a 0.22 µm polyethersulfone syringe filter. The particles were then concentrated using an Amicon Ultra-0.5 mL 10 kDa centrifugal filter unit (Millipore Sigma, UFC501024) to a concentration of at least 500 ng / µL. 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

[00211] C57BL / 6 mice (The Jackson Laboratory) were administered a 2 µg 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 of39 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-κB and Interferon Regulatory Factor (IRF) induction assay

[00212] To determine the efficacy of LNPs encapsulating unmodified and modified lncRNA in reducing NF-κB 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 µg / mL of normocin and 200 µg / 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 or modified lncRNA for 18 h at 37°C with 5% CO2. lncRNA 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-κB induction using QUANTI-Blue and IRF induction using QUANTI-Luc as per manufacturer’s instructions. SEAP levels were measured using the BioTek Synergy H1 microplate reader set at wavelength of 655 nm. lncRNA LNP in vivo testing

[00213] C57BL / 6 wild-type mice were purchased from Charles River Laboratories. To induce systemic inflammatory responses, 100 µg (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 µL / 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

[00214] To evaluate the efficacy of the lncRNA 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 using Eve Technologies' Mouse Focused 10-Plex Discovery Assay® (MilliporeSigma, Burlington, Massachusetts, USA) according to the manufacturer's protocol. The 10-plex consisted of GM-CSF, IFNγ, IL-1β, IL-2, IL-4, IL-6, IL-10, IL-12p70, MCP-1, and TNFα. Assay sensitivities of these markers ranged40 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

[00215] Animal weights were recorded prior to and after PBS or pseudouridine-modified GAPLINC LNP (0.1 mg / kg; 200 µL / animal) treatments. Weight change was recorded by dividing initial weight by final weight (i.e., normalizing post sacrifice weight to pretreatment weight). Histology Analysis

[00216] 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 µm 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 AxioImager 2 microscope (Zeiss, Germany). Statistical Analysis

[00217] 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

[00218] As shown in FIG. 24, GAPLINC 202 and 205 which share overlap with GAPLINC but lack function, also lack the first 19 nucleotides of GAPLINC. Substitutions are made for example, outside of the first 19 nucleotides. The first 19 nucleotides can comprise chemically modified nucleotides.41 Tables Table 1. Oligonucleotides for GAPLINC DNA template and RNA. Name Sequence (5’ to 3’) GAPLINC DNA Template ACTTGCAGGATCTGACACATCCTCTTGGTTTCCTAAGTCTTATGACTAGCC AATGCCTGAAATAATGAACTCCTCCAAGGCAAGAAATCTGTTTTGAAGCT TCTCTGCGTTCACACACAGCAGCCTGGTTTCCTGGAAGGGCATTTTCCACA TTGTGCGTTATGGATGATCATCCCAGGCATCAGGTGTGAAGCCCTGCATC CACATCCAGGGGCTATCAAATCTCTCTGCAAAAGGAGAAGCTGGACTCAG GCACGTTTACAGTGATGTGTATGCAGGGTATGCACAGATGTGGAAACAGG AACTGATGTGTCCATTACACCACTAGGACAGAGGCCAGAACAATGAAGA AACCAAATACTTGGAAGAGGGTAGAGATAATGAATGGAGTCCAAGAGCC CTGATTGTGCCATAAATGTCCAGATAATTCCATACCTGAGGATTATGTGGT TTGTAAACTTGGCACTTAGAAGAACCAATAAAATCATGTTATAGTTTCAA AAAAAAAAAA (SEQ ID NO: 1) Scrambled GAPLINC DNA Template ACTCTTTTGAGACGACCGGACCGGCTATGTATATCTCTATGGATGGAAAC GTAAGACGATTGCGGTACAGGCTTCCTCTGATTAGGCCATTGTTCGGGTC AATAGCACCCCCGTTGACTTAACTGAGTCTGTTCATGGAAGGGACTTCTA GCCCTGACAGTGTGCGGCAACCACCGAACGGGCCGATATTTAGCTTATGC AAAGTATCAGGTTGGTGATCTTGCTGAGTCCGGAATAGCGCCAGGGTGCA TTCAAGCTATTCCATAAATTTGGTGACCGAATAAAACATTAAACTAAGGG CAGCACCGGTAGTAATGTAATCGGTAAAAAAAACGCACGGGGGAGAGTA ATATTATTAGCATCTCGTCATAAGAAAATAAAGGCAATCCCTAATTATAT ATCGAGTCAATCTAGTTAGTAATAACAAAATACATTAGGGCCAATAACAC AACCCCCAACCACTTAAAGATGGACATTATATCTGAAACCGTCACGAGGG GATCAATATTTG (SEQ ID NO: 2) GAPLINC-202 DNA Template AGCCAATGCCTGAAATAATGAACTCCTCCAAGGCAAGAAATCTGTTTTGA AGCTTCTCTGCGTTCACACACAGCAGCCTGGTTTCCTGGAAGGGCATTTTC CACATTGTGCGTTATGGATGATCATCCCAGGCATCAGGTGTGAAGCCCTG CATCCACATCCAGGGGCTATCAAATCTCTCTGCAAAAGGAGAAGCTGGAC TCAGGCACGTTTACAGTGATGTGTATGCAGTTCTGTGAAGGAAGCTGCAG AAGAAAAAGGTGAAAGAGGTGAGGAAGCTGCAGAAGAAAACCTGGAAG TTAGCAGAGCTTGATCCAGAGGTTTAAGGAAAGAAGCCATCTCCATAACA TAAAAGTGCAAGGTGAAGCAGCAAGTGCTGATGGGGAAGCTGCAGCAAG TCATCCAGAAGATCTTGCTAAGGGTATGCACAGATGTGGAAACAGGAACT GATGTGTCCATTACACCACTAGGACAGAGGCCAGAACAATGAAGAAACC AAATACTTGGAAGAGGGTAGAGATAATGAATGGAGTCCAAGAGCCCTGA TTGTGCCATAAATGTCCAGATAATTCCATACCTGAGGATTATGTGGTTTGT AAACTTGGCACTTAGAAGAACCAATAAAATCATGTTATAGTTTCAAAAAA AAAAAA (SEQ ID NO: 3) GAPLINC-205 DNA Template ATCCTCTTGGTTTCCTAAGTCTTATGACTAGCCAATGCCTGAAATAATGAA CTCCTCCAAGGCAAGAAATCTGTTTTGAAGCTTCTCTGCGTTCACACACAG CAGCCTGGTTTCCTGGAAGGGCATTTTCCACATTGTGCGTTATGGATGATC42 ATCCCAGGCATCAGGTGTGAAGCCCTGCATCCACATCCAGGGGCTATCAA ATCTCTCTGCAAAAGGAGAAGCTGGACTCAGGCACGTTTACAGTGATGTG TATGCAGAGAGAAGGACACATTCTTGGATTGAACCCCAAAGGATGTTGAA CTTAACAAGGATGTCAGAACTAACCCCACCATATTCTGGGACCACTTGCT TGCTAAGAACTAAAAGTCACTTAAACCAGTTAAATAAAAGAGAGTTGATT TTAAAGAAAAAAAAAA (SEQ ID NO: 4) DRAIR DNA Template GACAGTTCCTCCCAGCTCTGACAGCCCGCGGGTGCGGCTGCAGGTGCCGC CTCGGCTTCATTAGCCCAGCTCAGGACAAACTCCAGAAGGGAACAACCCC AGTTCTGGTCCCCTCGGTCACCCCGGGGAAGGACCCAGCCCTAGCTGCTC ACTCTCCGCCCGCCTTCCCGAGAGTGCTGCCCCGACCCCGGGTCCGCCGC CAAGCCGGAGCGCCCGGGGCGTTCGAGGGGGCTTGGCGGCTCCTGTTCCC ACCGCGCACGCCTTACCTGGTGGTGGTTGTACGAGTTCCTCTGCTGCAGG AAGGCGGCTGCGGCAGCCTGGTGCTGCTGCTGCAGCTGCGGGCTGACGGG TGACCTCCGGCTCTGCGGCTGCTGCGGCGCCGCCGCCGGCGGCTGCTGTT GAGGATGATGCCCTCCAGGTCTATCCATTTGCCTAGGAACCAAGAAGTCT TAATAACTGTTTTGGTGATTGGGGCCAGCAGAAACCTTCTTCCTGACATTC TTCTAAGTGACAGGATGTGTGTAGTCTGCTCTCAACTCCCCACTGGTCCAG CTGAGGCTGCCTGAGGAAAGCACTTTTCTCTGAGGTATATTCTTGCCACA GTGGTCACTGATGTGCTTCAAACATGGAAAGAAGAAGTCTCATATGGTCC TACCCCTATAGAAAGAACCACGGTTAACTAATGACTGCTGGATGAAGTTC CTGCTTCCAGCTTCCTGCCAAGACTTCCTTTAATGATGGACTGTAACCTGA AGCTCAAACCCCAAATGACGTTCCTGCTCTGTTGCCTGTACCCTGGATTGC TCACCACCTGAGCCTACCTGCCCTTGTGGGGTTGGAGGTGAGGTGTCACA AAGTCATCAACACAGACTCTGGAAGCAGAGAAGGCTCCTGGAAGGAACT GCTCAGCTGGGGACTACACCTTCTTTTTGTTTTGGTACAGAGAGAGACCA GCTGACTGGTTTTCAGAATGAAAAGAGTCTAGCTGGCAGCCCAAAGAGGT GACTGAGAACTGGCTGTGTAAGAACAGCAGAGCTTCCTGGGAGAAACAG GCTTCATTTTTCCTGCTTTAAAAAAAAAATATGCGAGAACTCCAATGTTCT GGCAAAATTAAGGAAACCTGCAATCAGTTGCTTGGAAAAAAAAACCCAG CAAACCTGTCTGTAGTAAATTACATATTGGGACAAAGCCCCACTGACTCC CGAGGCTGCCTTCCTCCCTGCACCCCACCCTGCTGCCATCCTTAAGGTAAT GTCCTTCAGACTGCACAAGCTGCATTTCAGAGACCTGCAGTCCAAACTTG ACACCTAAGAACTGTGTCCCTTTAGGTAAGTCTCATGGCTTTTTGTCTGAG TTTCATGATATTATTTGTAACATGACCTTAAATCTGTTTCTTCATATTGATA TGAGAAATAAACACACTTTCATCAGC (SEQ ID NO: 5) MIST DNA Template TTGTGGTCACACCAACAGTCTGTCATAAAAGGAGTCTTTGTTCTTCTGTGG AGCTTTCCCAAACTTCTATGTGAATGTCATTCATGCTACTAGTTTGAAGCT CTGAAATCCATCTCTGGGAATTTCCTTTCTTGTATTTTTGTTTTCTTTCAGT AATAGTCTAGATAAAAAAGTCAAGAAGCTAAGAAACAGGATATAAAACA AACAATAACAACAACAAAAAAGTGCCAGGGCTTCAGAGACCTTCACACA TGGGTACCCACAACCTGGTATGGCCATGCACATGGAAGACGGAGAAGAA GGCACTGGCTTTGTGCCTAAGCAAATTCAAGGCTTTACGTAATCATCGGT GACAAAGAAAGAAGACCTCCACTATAAAACAGACATCAATTCTTTAACTG TAGAATAAATGATTCCCCGATCCAGATGTTTGATGTGCAAACAGGGAAAG43 ATGTGATCCCTGCTAGTAAACTGCCTGCTGGGGAAACCATTGTGCAACAT AGAAGCTTCCCTAACTAGGTGACCAACACATCCATTGTCACAAGGCTACC TCTGTCGCTGCCCTCCATGTGAGCTGCTGTGTGTTGAAAACACTGGCAGA AGCAGCCTCCCCAGCAAGTTTTCGCCTGCTGGAGATCTGTCTCTGAGACA AATATAGAGGATGGAGACGTTTGTCGGCTTGTCTGCCATCAAAGACCCAA TTGCTTCCTGCGTTGCAGGAAATGAAGTGCAGCCTCTCAGCCCCGTCCAC CTCCACAAGTGACTGTCAGTGGAGAACAAGCTGAAGCAGTTGGACTTGCT CTGAGTTCCCAGATCTGAGCCAATTCAAAGCTCACAATGTCATTAAACCA CCAGTACTTGGAGGAGAGCTTAAACACTGCTTTGTCCAGTTACTTTCAAA GAGAGTTTCTAGGAAGAGCTATTCCAAATATCAGTCTGTCATTTTCACCAC CGAAGAAATTTTTAAAGTGTGCAGTGCAGGAAGAGACAGGAAGAAGAAG GGACTGGATTGAAAGAGAATGGTCCCTTGTGATCTGGTGACAGAGCCGGA CAGAGCAGATGTCATAAGACTCCAGTTATTTTCCAGCTCTGGTGTGACAG TGGTGTGACCAGCAGCCAGTCATACCACCCTGCCTTTCACAACCTTGAAG AGACACGTCACTCAGCTCTCTAAACCAGTTTGTCAGCTTCATCAGTCCAGC ACACAAGAGCATCTAAGCAGCAGTCAACAGAGCTGAGAGAAATCAAAAA GGGGAAAAAAAACAACAAAAAACTAGGAGAAAGCTGTAATTCTTCTCCT AAGTCAACAGACAATGCAATAGACGTTCACTGACTTTAAAACAGTAAGCA CTGGGGTTTTGAGAAAACAGCTGATGACCTTAAGAATCAAGAAAGCATGT CAGCAATAGACGAAATAGAACCACTGTGTATATGGCATAGAGCAATGTTT CCAATCACCCAAAAAAAAAAAAA (SEQ ID NO: 6) Forward Primer TAGAAACTGGGCTTGTCGAGAC (SEQ ID NO: 7) Reverse Primer TGAATAAAGCCTGAGTAGAGCG (SEQ ID NO: 8) Firefly Luciferase DNA Template TAATACGACTCACTATAAGGGAGACTGCCACCATGGAAGACGCGAAGAA TATCAAGAAGGGACCTGCACCCTTCTATCCACTGGAAGACGGCACTGCTG GAGAGCAGCTCCATAAGGCCATGAAGAGATACGCACTGGTGCCTGGTAC AATCGCATTTACCGACGCTCATATTGAGGTCGATATTACATACGCCGAAT ATTTCGAGATGTCCGTGAGACTCGCTGAGGCAATGAAGCGGTACGGGCTG AATACTAACCACAGGATCGTCGTGTGTTCAGAGAACAGCCTGCAGTTCTT CATGCCTGTGCTGGGTGCTCTGTTCATTGGGGTTGCCGTTGCTCCAGCAAA CGACATTTATAACGAGAGGGAACTGCTTAATAGCATGGGCATTAGCCAGC CGACAGTGGTGTTTGTGAGTAAGAAAGGACTCCAAAAAATTCTGAACGTC CAGAAAAAATTACCTATCATTCAGAAGATCATCATCATGGATAGCAAGAC AGACTACCAGGGCTTTCAGAGCATGTATACCTTTGTGACCAGCCACCTGC CGCCAGGGTTTAACGAGTATGATTTCGTACCCGAAAGCTTCGACCGGGAC AAAACCATCGCATTGATCATGAATTCCTCTGGGTCAACCGGCCTCCCTAA AGGGGTAGCACTGCCACACCGAACTGCGTGCGTGCGCTTTAGCCACGCGA GGGATCCAATTTTTGGAAATCAGATTATCCCCGATACCGCCATACTGTCA GTAGTGCCATTCCACCACGGGTTTGGTATGTTCACCACACTTGGGTATCTA ATCTGTGGATTCAGAGTCGTCCTTATGTACCGCTTTGAAGAGGAATTGTTT CTTCGATCCCTCCAGGACTACAAAATCCAATCCGCCTTATTGGTGCCTACT CTGTTCTCCTTTTTCGCCAAGAGCACGCTGATCGACAAATATGACCTGAGT AACCTCCATGAGATTGCTAGCGGTGGCGCCCCCCTGAGCAAAGAGGTGGG44 CGAGGCAGTTGCTAAGCGCTTCCATCTCCCTGGAATAAGACAGGGATACG GCCTAACAGAGACTACAAGTGCAATCTTAATTACACCCGAAGGCGATGAC AAGCCCGGCGCTGTGGGAAAGGTTGTACCTTTTTTTGAAGCCAAAGTGGT CGACCTCGATACTGGCAAAACGCTAGGCGTCAACCAGCGCGGTGAGCTAT GCGTTAGGGGCCCTATGATCATGAGTGGTTACGTGAACAACCCCGAAGCC ACGAATGCGCTGATCGACAAGGATGGATGGTTGCATTCGGGAGATATCGC TTATTGGGACGAAGACGAGCACTTCTTCATAGTAGACCGACTGAAGAGCC TGATCAAGTACAAGGGATACCAGGTGGCTCCCGCCGAGCTTGAGTCCATC CTCTTGCAACACCCGAATATTTTCGATGCCGGGGTGGCTGGGCTGCCAGA CGATGATGCCGGCGAACTTCCAGCTGCCGTCGTAGTGCTCGAGCACGGGA AGACAATGACCGAAAAGGAGATCGTTGACTACGTGGCCTCACAAGTGAC CACTGCCAAAAAGCTTCGGGGAGGGGTGGTCTTCGTCGATGAGGTCCCCA AAGGCTTGACCGGTAAGCTCGATGCACGGAAGATACGTGAAATTCTGATA AAGGCCAAAAAAGGCGGTAAGATAGCCGTTTGATAAGCTGCCTTCTGCGG GGCTTGCCTTCTGGCCATGCCCTTCTTCTCTCCCTTGCACCTGTACCTCTTG GTCTTTGAATAAAGCCTGAGTAGGAAGAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAATGCATAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAGTACT (SEQ ID NO: 9) Unmodified GAPLINC RNA ACUUGCAGGAUCUGACACAUCCUCUUGGUUUCCUAAGUCUUAUGACUA GCCAAUGCCUGAAAUAAUGAACUCCUCCAAGGCAAGAAAUCUGUUUUG AAGCUUCUCUGCGUUCACACACAGCAGCCUGGUUUCCUGGAAGGGCAU UUUCCACAUUGUGCGUUAUGGAUGAUCAUCCCAGGCAUCAGGUGUGAA GCCCUGCAUCCACAUCCAGGGGCUAUCAAAUCUCUCUGCAAAAGGAGA AGCUGGACUCAGGCACGUUUACAGUGAUGUGUAUGCAGGGUAUGCACA GAUGUGGAAACAGGAACUGAUGUGUCCAUUACACCACUAGGACAGAGG CCAGAACAAUGAAGAAACCAAAUACUUGGAAGAGGGUAGAGAUAAUGA AUGGAGUCCAAGAGCCCUGAUUGUGCCAUAAAUGUCCAGAUAAUUCCA UACCUGAGGAUUAUGUGGUUUGUAAACUUGGCACUUAGAAGAACCAAU AAAAUCAUGUUAUAGUUUCAAAAAAAAAAAA (SEQ ID NO: 10) m5C-modified GAPLINC RNA A5UUG5AGGAU5UGA5A5AU55U5UUGGUUU55UAAGU5UUAUGA5UAG55A AUG55UGAAAUAAUGAA5U55U55AAGG5AAGAAAU5UGUUUUGAAG5UU5 U5UG5GUU5A5A5A5AG5AG55UGGUUU55UGGAAGGG5AUUUU55A5AUUG UG5GUUAUGGAUGAU5AU555AGG5AU5AGGUGUGAAG555UG5AU55A5AU 55AGGGG5UAU5AAAU5U5U5UG5AAAAGGAGAAG5UGGA5U5AGG5A5GU UUA5AGUGAUGUGUAUG5AGGGUAUG5A5AGAUGUGGAAA5AGGAA5UG AUGUGU55AUUA5A55A5UAGGA5AGAGG55AGAA5AAUGAAGAAA55AAA UA5UUGGAAGAGGGUAGAGAUAAUGAAUGGAGU55AAGAG555UGAUUG UG55AUAAAUGU55AGAUAAUU55AUA55UGAGGAUUAUGUGGUUUGUAA A5UUGG5A5UUAGAAGAA55AAUAAAAU5AUGUUAUAGUUU5AAAAAAA AAAAA (SEQ ID NO: 11) Ψ-modified GAPLINC RNA ACPPGCAGGAPCPGACACAPCCPCPPGGPPPCCPAAGPCPPAPGACPAGCCA APGCCPGAAAPAAPGAACPCCPCCAAGGCAAGAAAPCPGPPPPGAAGCPPC PCPGCGPPCACACACAGCAGCCPGGPPPCCPGGAAGGGCAPPPPCCACAPPG PGCGPPAPGGAPGAPCAPCCCAGGCAPCAGGPGPGAAGCCCPGCAPCCACA45 PCCAGGGGCPAPCAAAPCPCPCPGCAAAAGGAGAAGCPGGACPCAGGCAC GPPPACAGPGAPGPGPAPGCAGGGPAPGCACAGAPGPGGAAACAGGAACPG APGPGPCCAPPACACCACPAGGACAGAGGCCAGAACAAPGAAGAAACCAA APACPPGGAAGAGGGPAGAGAPAAPGAAPGGAGPCCAAGAGCCCPGAPPG PGCCAPAAAPGPCCAGAPAAPPCCAPACCPGAGGAPPAPGPGGPPPGPAAAC PPGGCACPPAGAAGAACCAAPAAAAPCAPGPPAPAGPPPCAAAAAAAAAAA A (SEQ ID NO: 12) m1Ψ-modified GAPLINC RNA AC11GCAGGA1C1GACACA1CC1C11GG111CC1AAG1C11A1GAC1AGCCAA1 GCC1GAAA1AA1GAAC1CC1CCAAGGCAAGAAA1C1G1111GAAGC11C1C1G CG11CACACACAGCAGCC1GG111CC1GGAAGGGCA1111CCACA11G1GCG1 1A1GGA1GA1CA1CCCAGGCA1CAGG1G1GAAGCCC1GCA1CCACA1CCAGG GGC1A1CAAA1C1C1C1GCAAAAGGAGAAGC1GGAC1CAGGCACG111ACAG 1GA1G1G1A1GCAGGG1A1GCACAGA1G1GGAAACAGGAAC1GA1G1G1CCA 11ACACCAC1AGGACAGAGGCCAGAACAA1GAAGAAACCAAA1AC11GGA AGAGGG1AGAGA1AA1GAA1GGAG1CCAAGAGCCC1GA11G1GCCA1AAA1 G1CCAGA1AA11CCA1ACC1GAGGA11A1G1GG111G1AAAC11GGCAC11AG AAGAACCAA1AAAA1CA1G11A1AG111CAAAAAAAAAAAA (SEQ ID NO: 13) GAPLINC-202 RNA AGCCAAUGCCUGAAAUAAUGAACUCCUCCAAGGCAAGAAAUCUGUUUU GAAGCUUCUCUGCGUUCACACACAGCAGCCUGGUUUCCUGGAAGGGCA UUUUCCACAUUGUGCGUUAUGGAUGAUCAUCCCAGGCAUCAGGUGUGA AGCCCUGCAUCCACAUCCAGGGGCUAUCAAAUCUCUCUGCAAAAGGAG AAGCUGGACUCAGGCACGUUUACAGUGAUGUGUAUGCAGUUCUGUGAA GGAAGCUGCAGAAGAAAAAGGUGAAAGAGGUGAGGAAGCUGCAGAAGA AAACCUGGAAGUUAGCAGAGCUUGAUCCAGAGGUUUAAGGAAAGAAGC CAUCUCCAUAACAUAAAAGUGCAAGGUGAAGCAGCAAGUGCUGAUGGG GAAGCUGCAGCAAGUCAUCCAGAAGAUCUUGCUAAGGGUAUGCACAGA UGUGGAAACAGGAACUGAUGUGUCCAUUACACCACUAGGACAGAGGCC AGAACAAUGAAGAAACCAAAUACUUGGAAGAGGGUAGAGAUAAUGAAU GGAGUCCAAGAGCCCUGAUUGUGCCAUAAAUGUCCAGAUAAUUCCAUA CCUGAGGAUUAUGUGGUUUGUAAACUUGGCACUUAGAAGAACCAAUAA AAUCAUGUUAUAGUUUCAAAAAAAAAAAA (SEQ ID NO: 23) GAPLINC-205 RNA AUCCUCUUGGUUUCCUAAGUCUUAUGACUAGCCAAUGCCUGAAAUAAU GAACUCCUCCAAGGCAAGAAAUCUGUUUUGAAGCUUCUCUGCGUUCAC ACACAGCAGCCUGGUUUCCUGGAAGGGCAUUUUCCACAUUGUGCGUUA UGGAUGAUCAUCCCAGGCAUCAGGUGUGAAGCCCUGCAUCCACAUCCA GGGGCUAUCAAAUCUCUCUGCAAAAGGAGAAGCUGGACUCAGGCACGU UUACAGUGAUGUGUAUGCAGAGAGAAGGACACAUUCUUGGAUUGAACC CCAAAGGAUGUUGAACUUAACAAGGAUGUCAGAACUAACCCCACCAUA UUCUGGGACCACUUGCUUGCUAAGAACUAAAAGUCACUUAAACCAGUU AAAUAAAAGAGAGUUGAUUUUAAAGAAAAAAAAAAA (SEQ ID NO: 24) Unmodified MIST RNA UUGUGGUCACACCAACAGUCUGUCAUAAAAGGAGUCUUUGUUCUUCUG UGGAGCUUUCCCAAACUUCUAUGUGAAUGUCAUUCAUGCUACUAGUUU GAAGCUCUGAAAUCCAUCUCUGGGAAUUUCCUUUCUUGUAUUUUUGUU UUCUUUCAGUAAUAGUCUAGAUAAAAAAGUCAAGAAGCUAAGAAACAG GAUAUAAAACAAACAAUAACAACAACAAAAAAGUGCCAGGGCUUCAGA GACCUUCACACAUGGGUACCCACAACCUGGUAUGGCCAUGCACAUGGA AGACGGAGAAGAAGGCACUGGCUUUGUGCCUAAGCAAAUUCAAGGCUU UACGUAAUCAUCGGUGACAAAGAAAGAAGACCUCCACUAUAAAACAGA46 CAUCAAUUCUUUAACUGUAGAAUAAAUGAUUCCCCGAUCCAGAUGUUU GAUGUGCAAACAGGGAAAGAUGUGAUCCCUGCUAGUAAACUGCCUGCU GGGGAAACCAUUGUGCAACAUAGAAGCUUCCCUAACUAGGUGACCAAC ACAUCCAUUGUCACAAGGCUACCUCUGUCGCUGCCCUCCAUGUGAGCUG CUGUGUGUUGAAAACACUGGCAGAAGCAGCCUCCCCAGCAAGUUUUCG CCUGCUGGAGAUCUGUCUCUGAGACAAAUAUAGAGGAUGGAGACGUUU GUCGGCUUGUCUGCCAUCAAAGACCCAAUUGCUUCCUGCGUUGCAGGA AAUGAAGUGCAGCCUCUCAGCCCCGUCCACCUCCACAAGUGACUGUCAG UGGAGAACAAGCUGAAGCAGUUGGACUUGCUCUGAGUUCCCAGAUCUG AGCCAAUUCAAAGCUCACAAUGUCAUUAAACCACCAGUACUUGGAGGA GAGCUUAAACACUGCUUUGUCCAGUUACUUUCAAAGAGAGUUUCUAGG AAGAGCUAUUCCAAAUAUCAGUCUGUCAUUUUCACCACCGAAGAAAUU UUUAAAGUGUGCAGUGCAGGAAGAGACAGGAAGAAGAAGGGACUGGAU UGAAAGAGAAUGGUCCCUUGUGAUCUGGUGACAGAGCCGGACAGAGCA GAUGUCAUAAGACUCCAGUUAUUUUCCAGCUCUGGUGUGACAGUGGUG UGACCAGCAGCCAGUCAUACCACCCUGCCUUUCACAACCUUGAAGAGAC ACGUCACUCAGCUCUCUAAACCAGUUUGUCAGCUUCAUCAGUCCAGCAC ACAAGAGCAUCUAAGCAGCAGUCAACAGAGCUGAGAGAAAUCAAAAAG GGGAAAAAAAACAACAAAAAACUAGGAGAAAGCUGUAAUUCUUCUCCU AAGUCAACAGACAAUGCAAUAGACGUUCACUGACUUUAAAACAGUAAG CACUGGGGUUUUGAGAAAACAGCUGAUGACCUUAAGAAUCAAGAAAGC AUGUCAGCAAUAGACGAAAUAGAACCACUGUGUAUAUGGCAUAGAGCA AUGUUUCCAAUCACCCAAAAAAAAAAAAA (SEQ ID NO: 14) m5C-modified MIST RNA UUGUGGU5A5A55AA5AGU5UGU5AUAAAAGGAGU5UUUGUU5UU5UGUG GAG5UUU555AAA5UU5UAUGUGAAUGU5AUU5AUG5UA5UAGUUUGAAG5 U5UGAAAU55AU5U5UGGGAAUUU55UUU5UUGUAUUUUUGUUUU5UUU5 AGUAAUAGU5UAGAUAAAAAAGU5AAGAAG5UAAGAAA5AGGAUAUAA AA5AAA5AAUAA5AA5AA5AAAAAAGUG55AGGG5UU5AGAGA55UU5A5A5 AUGGGUA555A5AA55UGGUAUGG55AUG5A5AUGGAAGA5GGAGAAGAAG G5A5UGG5UUUGUG55UAAG5AAAUU5AAGG5UUUA5GUAAU5AU5GGUGA 5AAAGAAAGAAGA55U55A5UAUAAAA5AGA5AU5AAUU5UUUAA5UGUAG AAUAAAUGAUU5555GAU55AGAUGUUUGAUGUG5AAA5AGGGAAAGAUG UGAU555UG5UAGUAAA5UG55UG5UGGGGAAA55AUUGUG5AA5AUAGAA G5UU555UAA5UAGGUGA55AA5A5AU55AUUGU5A5AAGG5UA55U5UGU5G 5UG555U55AUGUGAG5UG5UGUGUGUUGAAAA5A5UGG5AGAAG5AG55U5 555AG5AAGUUUU5G55UG5UGGAGAU5UGU5U5UGAGA5AAAUAUAGAGG AUGGAGA5GUUUGU5GG5UUGU5UG55AU5AAAGA555AAUUG5UU55UG5G UUG5AGGAAAUGAAGUG5AG55U5U5AG5555GU55A55U55A5AAGUGA5UG U5AGUGGAGAA5AAG5UGAAG5AGUUGGA5UUG5U5UGAGUU555AGAU5U GAG55AAUU5AAAG5U5A5AAUGU5AUUAAA55A55AGUA5UUGGAGGAGA G5UUAAA5A5UG5UUUGU55AGUUA5UUU5AAAGAGAGUUU5UAGGAAGA G5UAUU55AAAUAU5AGU5UGU5AUUUU5A55A55GAAGAAAUUUUUAAAG UGUG5AGUG5AGGAAGAGA5AGGAAGAAGAAGGGA5UGGAUUGAAAGA GAAUGGU555UUGUGAU5UGGUGA5AGAG55GGA5AGAG5AGAUGU5AUA AGA5U55AGUUAUUUU55AG5U5UGGUGUGA5AGUGGUGUGA55AG5AG55 AGU5AUA55A555UG55UUU5A5AA55UUGAAGAGA5A5GU5A5U5AG5U5U5 UAAA55AGUUUGU5AG5UU5AU5AGU55AG5A5A5AAGAG5AU5UAAG5AG5 AGU5AA5AGAG5UGAGAGAAAU5AAAAAGGGGAAAAAAAA5AA5AAAAA A5UAGGAGAAAG5UGUAAUU5UU5U55UAAGU5AA5AGA5AAUG5AAUAG A5GUU5A5UGA5UUUAAAA5AGUAAG5A5UGGGGUUUUGAGAAAA5AG5U GAUGA55UUAAGAAU5AAGAAAG5AUGU5AG5AAUAGA5GAAAUAGAA5547 A5UGUGUAUAUGG5AUAGAG5AAUGUUU55AAU5A555AAAAAAAAAAAA A (SEQ ID NO: 15) Ψ-modified MIST RNA PPGPGGPCACACCAACAGPCPGPCAPAAAAGGAGPCPPPGPPCPPCPGPGGA GCPPPCCCAAACPPCPAPGPGAAPGPCAPPCAPGCPACPAGPPPGAAGCPCPG AAAPCCAPCPCPGGGAAPPPCCPPPCPPGPAPPPPPGPPPPCPPPCAGPAAPAG PCPAGAPAAAAAAGPCAAGAAGCPAAGAAACAGGAPAPAAAACAAACAAP AACAACAACAAAAAAGPGCCAGGGCPPCAGAGACCPPCACACAPGGGPAC CCACAACCPGGPAPGGCCAPGCACAPGGAAGACGGAGAAGAAGGCACPGG CPPPGPGCCPAAGCAAAPPCAAGGCPPPACGPAAPCAPCGGPGACAAAGAA AGAAGACCPCCACPAPAAAACAGACAPCAAPPCPPPAACPGPAGAAPAAAP GAPPCCCCGAPCCAGAPGPPPGAPGPGCAAACAGGGAAAGAPGPGAPCCCP GCPAGPAAACPGCCPGCPGGGGAAACCAPPGPGCAACAPAGAAGCPPCCCP AACPAGGPGACCAACACAPCCAPPGPCACAAGGCPACCPCPGPCGCPGCCC PCCAPGPGAGCPGCPGPGPGPPGAAAACACPGGCAGAAGCAGCCPCCCCAG CAAGPPPPCGCCPGCPGGAGAPCPGPCPCPGAGACAAAPAPAGAGGAPGGA GACGPPPGPCGGCPPGPCPGCCAPCAAAGACCCAAPPGCPPCCPGCGPPGCA GGAAAPGAAGPGCAGCCPCPCAGCCCCGPCCACCPCCACAAGPGACPGPCA GPGGAGAACAAGCPGAAGCAGPPGGACPPGCPCPGAGPPCCCAGAPCPGAG CCAAPPCAAAGCPCACAAPGPCAPPAAACCACCAGPACPPGGAGGAGAGCP PAAACACPGCPPPGPCCAGPPACPPPCAAAGAGAGPPPCPAGGAAGAGCPAP PCCAAAPAPCAGPCPGPCAPPPPCACCACCGAAGAAAPPPPPAAAGPGPGCA GPGCAGGAAGAGACAGGAAGAAGAAGGGACPGGAPPGAAAGAGAAPGGP CCCPPGPGAPCPGGPGACAGAGCCGGACAGAGCAGAPGPCAPAAGACPCCA GPPAPPPPCCAGCPCPGGPGPGACAGPGGPGPGACCAGCAGCCAGPCAPACC ACCCPGCCPPPCACAACCPPGAAGAGACACGPCACPCAGCPCPCPAAACCA GPPPGPCAGCPPCAPCAGPCCAGCACACAAGAGCAPCPAAGCAGCAGPCAA CAGAGCPGAGAGAAAPCAAAAAGGGGAAAAAAAACAACAAAAAACPAG GAGAAAGCPGPAAPPCPPCPCCPAAGPCAACAGACAAPGCAAPAGACGPPC ACPGACPPPAAAACAGPAAGCACPGGGGPPPPGAGAAAACAGCPGAPGACC PPAAGAAPCAAGAAAGCAPGPCAGCAAPAGACGAAAPAGAACCACPGPGP APAPGGCAPAGAGCAAPGPPPCCAAPCACCCAAAAAAAAAAAAA (SEQ ID NO: 16) m1Ψ-modified MIST RNA 11G1GG1CACACCAACAG1C1G1CA1AAAAGGAG1C111G11C11C1G1GGAGC 111CCCAAAC11C1A1G1GAA1G1CA11CA1GC1AC1AG111GAAGC1C1GAAA1 CCA1C1C1GGGAA111CC111C11G1A11111G1111C111CAG1AA1AG1C1AGA1 AAAAAAG1CAAGAAGC1AAGAAACAGGA1A1AAAACAAACAA1AACAAC AACAAAAAAG1GCCAGGGC11CAGAGACC11CACACA1GGG1ACCCACAAC C1GG1A1GGCCA1GCACA1GGAAGACGGAGAAGAAGGCAC1GGC111G1GC C1AAGCAAA11CAAGGC111ACG1AA1CA1CGG1GACAAAGAAAGAAGACC 1CCAC1A1AAAACAGACA1CAA11C111AAC1G1AGAA1AAA1GA11CCCCGA 1CCAGA1G111GA1G1GCAAACAGGGAAAGA1G1GA1CCC1GC1AG1AAAC1 GCC1GC1GGGGAAACCA11G1GCAACA1AGAAGC11CCC1AAC1AGG1GACC AACACA1CCA11G1CACAAGGC1ACC1C1G1CGC1GCCC1CCA1G1GAGC1GC 1G1G1G11GAAAACAC1GGCAGAAGCAGCC1CCCCAGCAAG1111CGCC1GC 1GGAGA1C1G1C1C1GAGACAAA1A1AGAGGA1GGAGACG111G1CGGC11G1 C1GCCA1CAAAGACCCAA11GC11CC1GCG11GCAGGAAA1GAAG1GCAGCC 1C1CAGCCCCG1CCACC1CCACAAG1GAC1G1CAG1GGAGAACAAGC1GAA GCAG11GGAC11GC1C1GAG11CCCAGA1C1GAGCCAA11CAAAGC1CACAA1 G1CA11AAACCACCAG1AC11GGAGGAGAGC11AAACAC1GC111G1CCAG11 AC111CAAAGAGAG111C1AGGAAGAGC1A11CCAAA1A1CAG1C1G1CA1111 CACCACCGAAGAAA11111AAAG1G1GCAG1GCAGGAAGAGACAGGAAGA48 AGAAGGGAC1GGA11GAAAGAGAA1GG1CCC11G1GA1C1GG1GACAGAGC CGGACAGAGCAGA1G1CA1AAGAC1CCAG11A1111CCAGC1C1GG1G1GAC AG1GG1G1GACCAGCAGCCAG1CA1ACCACCC1GCC111CACAACC11GAAG AGACACG1CAC1CAGC1C1C1AAACCAG111G1CAGC11CA1CAG1CCAGCAC ACAAGAGCA1C1AAGCAGCAG1CAACAGAGC1GAGAGAAA1CAAAAAGG GGAAAAAAAACAACAAAAAAC1AGGAGAAAGC1G1AA11C11C1CC1AAG1 CAACAGACAA1GCAA1AGACG11CAC1GAC111AAAACAG1AAGCAC1GGG G1111GAGAAAACAGC1GA1GACC11AAGAA1CAAGAAAGCA1G1CAGCAA1 AGACGAAA1AGAACCAC1G1G1A1A1GGCA1AGAGCAA1G111CCAA1CACC CAAAAAAAAAAAAA (SEQ ID NO: 17) Unmodified DRAIR RNA GACAGUUCCUCCCAGCUCUGACAGCCCGCGGGUGCGGCUGCAGGUGCCG CCUCGGCUUCAUUAGCCCAGCUCAGGACAAACUCCAGAAGGGAACAACC CCAGUUCUGGUCCCCUCGGUCACCCCGGGGAAGGACCCAGCCCUAGCUG CUCACUCUCCGCCCGCCUUCCCGAGAGUGCUGCCCCGACCCCGGGUCCG CCGCCAAGCCGGAGCGCCCGGGGCGUUCGAGGGGGCUUGGCGGCUCCUG UUCCCACCGCGCACGCCUUACCUGGUGGUGGUUGUACGAGUUCCUCUGC UGCAGGAAGGCGGCUGCGGCAGCCUGGUGCUGCUGCUGCAGCUGCGGG CUGACGGGUGACCUCCGGCUCUGCGGCUGCUGCGGCGCCGCCGCCGGCG GCUGCUGUUGAGGAUGAUGCCCUCCAGGUCUAUCCAUUUGCCUAGGAA CCAAGAAGUCUUAAUAACUGUUUUGGUGAUUGGGGCCAGCAGAAACCU UCUUCCUGACAUUCUUCUAAGUGACAGGAUGUGUGUAGUCUGCUCUCA ACUCCCCACUGGUCCAGCUGAGGCUGCCUGAGGAAAGCACUUUUCUCU GAGGUAUAUUCUUGCCACAGUGGUCACUGAUGUGCUUCAAACAUGGAA AGAAGAAGUCUCAUAUGGUCCUACCCCUAUAGAAAGAACCACGGUUAA CUAAUGACUGCUGGAUGAAGUUCCUGCUUCCAGCUUCCUGCCAAGACU UCCUUUAAUGAUGGACUGUAACCUGAAGCUCAAACCCCAAAUGACGUU CCUGCUCUGUUGCCUGUACCCUGGAUUGCUCACCACCUGAGCCUACCUG CCCUUGUGGGGUUGGAGGUGAGGUGUCACAAAGUCAUCAACACAGACU CUGGAAGCAGAGAAGGCUCCUGGAAGGAACUGCUCAGCUGGGGACUAC ACCUUCUUUUUGUUUUGGUACAGAGAGAGACCAGCUGACUGGUUUUCA GAAUGAAAAGAGUCUAGCUGGCAGCCCAAAGAGGUGACUGAGAACUGG CUGUGUAAGAACAGCAGAGCUUCCUGGGAGAAACAGGCUUCAUUUUUC CUGCUUUAAAAAAAAAAUAUGCGAGAACUCCAAUGUUCUGGCAAAAUU AAGGAAACCUGCAAUCAGUUGCUUGGAAAAAAAAACCCAGCAAACCUG UCUGUAGUAAAUUACAUAUUGGGACAAAGCCCCACUGACUCCCGAGGC UGCCUUCCUCCCUGCACCCCACCCUGCUGCCAUCCUUAAGGUAAUGUCC UUCAGACUGCACAAGCUGCAUUUCAGAGACCUGCAGUCCAAACUUGAC ACCUAAGAACUGUGUCCCUUUAGGUAAGUCUCAUGGCUUUUUGUCUGA GUUUCAUGAUAUUAUUUGUAACAUGACCUUAAAUCUGUUUCUUCAUAU UGAUAUGAGAAAUAAACACACUUUCAUCAGC (SEQ ID NO: 18) m5C-modified DRAIR RNA GA5AGUU55U555AG5U5UGA5AG555G5GGGUG5GG5UG5AGGUG55G55U5G G5UU5AUUAG555AG5U5AGGA5AAA5U55AGAAGGGAA5AA5555AGUU5U GGU5555U5GGU5A5555GGGGAAGGA555AG555UAG5UG5U5A5U5U55G555 G55UU555GAGAGUG5UG5555GA5555GGGU55G55G55AAG55GGAG5G555G GGG5GUU5GAGGGGG5UUGG5GG5U55UGUU555A55G5G5A5G55UUA55UG GUGGUGGUUGUA5GAGUU55U5UG5UG5AGGAAGG5GG5UG5GG5AG55UG GUG5UG5UG5UG5AG5UG5GGG5UGA5GGGUGA55U55GG5U5UG5GG5UG5 UG5GG5G55G55G55GG5GG5UG5UGUUGAGGAUGAUG555U55AGGU5UAU5 5AUUUG55UAGGAA55AAGAAGU5UUAAUAA5UGUUUUGGUGAUUGGGG5 5AG5AGAAA55UU5UU55UGA5AUU5UU5UAAGUGA5AGGAUGUGUGUAGU 5UG5U5U5AA5U5555A5UGGU55AG5UGAGG5UG55UGAGGAAAG5A5UUUU49 5U5UGAGGUAUAUU5UUG55A5AGUGGU5A5UGAUGUG5UU5AAA5AUGGA AAGAAGAAGU5U5AUAUGGU55UA5555UAUAGAAAGAA55A5GGUUAA5U AAUGA5UG5UGGAUGAAGUU55UG5UU55AG5UU55UG55AAGA5UU55UUU AAUGAUGGA5UGUAA55UGAAG5U5AAA5555AAAUGA5GUU55UG5U5UGU UG55UGUA555UGGAUUG5U5A55A55UGAG55UA55UG555UUGUGGGGUUG GAGGUGAGGUGU5A5AAAGU5AU5AA5A5AGA5U5UGGAAG5AGAGAAGG 5U55UGGAAGGAA5UG5U5AG5UGGGGA5UA5A55UU5UUUUUGUUUUGGU A5AGAGAGAGA55AG5UGA5UGGUUUU5AGAAUGAAAAGAGU5UAG5UGG 5AG555AAAGAGGUGA5UGAGAA5UGG5UGUGUAAGAA5AG5AGAG5UU55 UGGGAGAAA5AGG5UU5AUUUUU55UG5UUUAAAAAAAAAAUAUG5GAG AA5U55AAUGUU5UGG5AAAAUUAAGGAAA55UG5AAU5AGUUG5UUGGA AAAAAAAA555AG5AAA55UGU5UGUAGUAAAUUA5AUAUUGGGA5AAAG 5555A5UGA5U555GAGG5UG55UU55U555UG5A5555A555UG5UG55AU55UU AAGGUAAUGU55UU5AGA5UG5A5AAG5UG5AUUU5AGAGA55UG5AGU55A AA5UUGA5A55UAAGAA5UGUGU555UUUAGGUAAGU5U5AUGG5UUUUUG U5UGAGUUU5AUGAUAUUAUUUGUAA5AUGA55UUAAAU5UGUUU5UU5A UAUUGAUAUGAGAAAUAAA5A5A5UUU5AU5AG5 (SEQ ID NO: 19) Ψ-modified DRAIR RNA GACAGPPCCPCCCAGCPCPGACAGCCCGCGGGPGCGGCPGCAGGPGCCGCC PCGGCPPCAPPAGCCCAGCPCAGGACAAACPCCAGAAGGGAACAACCCCA GPPCPGGPCCCCPCGGPCACCCCGGGGAAGGACCCAGCCCPAGCPGCPCAC PCPCCGCCCGCCPPCCCGAGAGPGCPGCCCCGACCCCGGGPCCGCCGCCAA GCCGGAGCGCCCGGGGCGPPCGAGGGGGCPPGGCGGCPCCPGPPCCCACC GCGCACGCCPPACCPGGPGGPGGPPGPACGAGPPCCPCPGCPGCAGGAAGG CGGCPGCGGCAGCCPGGPGCPGCPGCPGCAGCPGCGGGCPGACGGGPGACC PCCGGCPCPGCGGCPGCPGCGGCGCCGCCGCCGGCGGCPGCPGPPGAGGAP GAPGCCCPCCAGGPCPAPCCAPPPGCCPAGGAACCAAGAAGPCPPAAPAAC PGPPPPGGPGAPPGGGGCCAGCAGAAACCPPCPPCCPGACAPPCPPCPAAGP GACAGGAPGPGPGPAGPCPGCPCPCAACPCCCCACPGGPCCAGCPGAGGCP GCCPGAGGAAAGCACPPPPCPCPGAGGPAPAPPCPPGCCACAGPGGPCACPG APGPGCPPCAAACAPGGAAAGAAGAAGPCPCAPAPGGPCCPACCCCPAPAG AAAGAACCACGGPPAACPAAPGACPGCPGGAPGAAGPPCCPGCPPCCAGCP PCCPGCCAAGACPPCCPPPAAPGAPGGACPGPAACCPGAAGCPCAAACCCC AAAPGACGPPCCPGCPCPGPPGCCPGPACCCPGGAPPGCPCACCACCPGAGC CPACCPGCCCPPGPGGGGPPGGAGGPGAGGPGPCACAAAGPCAPCAACACA GACPCPGGAAGCAGAGAAGGCPCCPGGAAGGAACPGCPCAGCPGGGGACP ACACCPPCPPPPPGPPPPGGPACAGAGAGAGACCAGCPGACPGGPPPPCAGA APGAAAAGAGPCPAGCPGGCAGCCCAAAGAGGPGACPGAGAACPGGCPGP GPAAGAACAGCAGAGCPPCCPGGGAGAAACAGGCPPCAPPPPPCCPGCPPP AAAAAAAAAAPAPGCGAGAACPCCAAPGPPCPGGCAAAAPPAAGGAAACC PGCAAPCAGPPGCPPGGAAAAAAAAACCCAGCAAACCPGPCPGPAGPAAAP PACAPAPPGGGACAAAGCCCCACPGACPCCCGAGGCPGCCPPCCPCCCPGC ACCCCACCCPGCPGCCAPCCPPAAGGPAAPGPCCPPCAGACPGCACAAGCP GCAPPPCAGAGACCPGCAGPCCAAACPPGACACCPAAGAACPGPGPCCCPPP AGGPAAGPCPCAPGGCPPPPPGPCPGAGPPPCAPGAPAPPAPPPGPAACAPGA CCPPAAAPCPGPPPCPPCAPAPPGAPAPGAGAAAPAAACACACPPPCAPCAG C (SEQ ID NO: 20) m1Ψ-modified DRAIR RNA GACAG11CC1CCCAGC1C1GACAGCCCGCGGG1GCGGC1GCAGG1GCCGCC1 CGGC11CA11AGCCCAGC1CAGGACAAAC1CCAGAAGGGAACAACCCCAG1 1C1GG1CCCC1CGG1CACCCCGGGGAAGGACCCAGCCC1AGC1GC1CAC1C1 CCGCCCGCC11CCCGAGAG1GC1GCCCCGACCCCGGG1CCGCCGCCAAGCC GGAGCGCCCGGGGCG11CGAGGGGGC11GGCGGC1CC1G11CCCACCGCGC50 ACGCC11ACC1GG1GG1GG11G1ACGAG11CC1C1GC1GCAGGAAGGCGGC1G CGGCAGCC1GG1GC1GC1GC1GCAGC1GCGGGC1GACGGG1GACC1CCGGC1 C1GCGGC1GC1GCGGCGCCGCCGCCGGCGGC1GC1G11GAGGA1GA1GCCC1 CCAGG1C1A1CCA111GCC1AGGAACCAAGAAG1C11AA1AAC1G1111GG1G A11GGGGCCAGCAGAAACC11C11CC1GACA11C11C1AAG1GACAGGA1G1G 1G1AG1C1GC1C1CAAC1CCCCAC1GG1CCAGC1GAGGC1GCC1GAGGAAAG CAC1111C1C1GAGG1A1A11C11GCCACAG1GG1CAC1GA1G1GC11CAAACA 1GGAAAGAAGAAG1C1CA1A1GG1CC1ACCCC1A1AGAAAGAACCACGG11 AAC1AA1GAC1GC1GGA1GAAG11CC1GC11CCAGC11CC1GCCAAGAC11CC 111AA1GA1GGAC1G1AACC1GAAGC1CAAACCCCAAA1GACG11CC1GC1C1 G11GCC1G1ACCC1GGA11GC1CACCACC1GAGCC1ACC1GCCC11G1GGGG11 GGAGG1GAGG1G1CACAAAG1CA1CAACACAGAC1C1GGAAGCAGAGAAG GC1CC1GGAAGGAAC1GC1CAGC1GGGGAC1ACACC11C11111G1111GG1AC AGAGAGAGACCAGC1GAC1GG1111CAGAA1GAAAAGAG1C1AGC1GGCAG CCCAAAGAGG1GAC1GAGAAC1GGC1G1G1AAGAACAGCAGAGC11CC1GG GAGAAACAGGC11CA11111CC1GC111AAAAAAAAAA1A1GCGAGAAC1CC AA1G11C1GGCAAAA11AAGGAAACC1GCAA1CAG11GC11GGAAAAAAAA ACCCAGCAAACC1G1C1G1AG1AAA11ACA1A11GGGACAAAGCCCCAC1GA C1CCCGAGGC1GCC11CC1CCC1GCACCCCACCC1GC1GCCA1CC11AAGG1A A1G1CC11CAGAC1GCACAAGC1GCA111CAGAGACC1GCAG1CCAAAC11G ACACC1AAGAAC1G1G1CCC111AGG1AAG1C1CA1GGC11111G1C1GAG111C A1GA1A11A111G1AACA1GACC11AAA1C1G111C11CA1A11GA1A1GAGAAA 1AAACACAC111CA1CAGC (SEQ ID NO: 21) Firefly Luciferase RNA AGGGAGACUGCCACCAUGGAAGACGCGAAGAAUAUCAAGAAGGGACCU GCACCCUUCUAUCCACUGGAAGACGGCACUGCUGGAGAGCAGCUCCAU AAGGCCAUGAAGAGAUACGCACUGGUGCCUGGUACAAUCGCAUUUACC GACGCUCAUAUUGAGGUCGAUAUUACAUACGCCGAAUAUUUCGAGAUG UCCGUGAGACUCGCUGAGGCAAUGAAGCGGUACGGGCUGAAUACUAAC CACAGGAUCGUCGUGUGUUCAGAGAACAGCCUGCAGUUCUUCAUGCCU GUGCUGGGUGCUCUGUUCAUUGGGGUUGCCGUUGCUCCAGCAAACGAC AUUUAUAACGAGAGGGAACUGCUUAAUAGCAUGGGCAUUAGCCAGCCG ACAGUGGUGUUUGUGAGUAAGAAAGGACUCCAAAAAAUUCUGAACGUC CAGAAAAAAUUACCUAUCAUUCAGAAGAUCAUCAUCAUGGAUAGCAAG ACAGACUACCAGGGCUUUCAGAGCAUGUAUACCUUUGUGACCAGCCAC CUGCCGCCAGGGUUUAACGAGUAUGAUUUCGUACCCGAAAGCUUCGAC CGGGACAAAACCAUCGCAUUGAUCAUGAAUUCCUCUGGGUCAACCGGC CUCCCUAAAGGGGUAGCACUGCCACACCGAACUGCGUGCGUGCGCUUU AGCCACGCGAGGGAUCCAAUUUUUGGAAAUCAGAUUAUCCCCGAUACC GCCAUACUGUCAGUAGUGCCAUUCCACCACGGGUUUGGUAUGUUCACC ACACUUGGGUAUCUAAUCUGUGGAUUCAGAGUCGUCCUUAUGUACCGC UUUGAAGAGGAAUUGUUUCUUCGAUCCCUCCAGGACUACAAAAUCCAA UCCGCCUUAUUGGUGCCUACUCUGUUCUCCUUUUUCGCCAAGAGCACGC UGAUCGACAAAUAUGACCUGAGUAACCUCCAUGAGAUUGCUAGCGGUG GCGCCCCCCUGAGCAAAGAGGUGGGCGAGGCAGUUGCUAAGCGCUUCC AUCUCCCUGGAAUAAGACAGGGAUACGGCCUAACAGAGACUACAAGUG CAAUCUUAAUUACACCCGAAGGCGAUGACAAGCCCGGCGCUGUGGGAA AGGUUGUACCUUUUUUUGAAGCCAAAGUGGUCGACCUCGAUACUGGCA AAACGCUAGGCGUCAACCAGCGCGGUGAGCUAUGCGUUAGGGGCCCUA UGAUCAUGAGUGGUUACGUGAACAACCCCGAAGCCACGAAUGCGCUGA UCGACAAGGAUGGAUGGUUGCAUUCGGGAGAUAUCGCUUAUUGGGACG AAGACGAGCACUUCUUCAUAGUAGACCGACUGAAGAGCCUGAUCAAGU51 ACAAGGGAUACCAGGUGGCUCCCGCCGAGCUUGAGUCCAUCCUCUUGC AACACCCGAAUAUUUUCGAUGCCGGGGUGGCUGGGCUGCCAGACGAUG AUGCCGGCGAACUUCCAGCUGCCGUCGUAGUGCUCGAGCACGGGAAGA CAAUGACCGAAAAGGAGAUCGUUGACUACGUGGCCUCACAAGUGACCA CUGCCAAAAAGCUUCGGGGAGGGGUGGUCUUCGUCGAUGAGGUCCCCA AAGGCUUGACCGGUAAGCUCGAUGCACGGAAGAUACGUGAAAUUCUGA UAAAGGCCAAAAAAGGCGGUAAGAUAGCCGUUUGAUAAGCUGCCUUCU GCGGGGCUUGCCUUCUGGCCAUGCCCUUCUUCUCUCCCUUGCACCUGUA CCUCUUGGUCUUUGAAUAAAGCCUGAGUAGGAAGAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA UGCAUAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAGU (SEQ ID NO: 22) PARAIL DNA Template ACAAGCCGGGTGGACATCGCTGAGGCTTGGAAAAGATTTCCTCCGGGGA AAATCCCTCCGGAGTTCTCCAGTCTGGTGTCAGAGAGGAATACTAAAGAA GAAGGAGAAATTCAAGGTACAAGAGAGGCAAAGAATGTTGCTCCTACAA AGGAACTCTCCTGACAGAAGTCCACAGAGGACAGATGGATGGGGATGAT TTCCACACTAGTTCCTAACTTTAAATAAAACCAAGCCTGCAGCGTAGTTTC CTGAGTCCTAATGGAAGGATTTGATGAAGACATCCCATTTAGGAATCTGC ACTGTTCTCCCTGGGCCACAGCTATGCCTGCTTCCCTGGAGGCCTGCTGCC ATCCAGGACAACGAGATGGAGATGTGTTAAAACTCAGAAGTCCTGTTCTA GAGTGACTCTCTTACTACACAGAGCATCCCTTGCCATTGAAGAGGTAAAA CTTAATTCCCGGCAGGCAGCCCAATGTGGGAGCGAGCGTCAGCCATGCTG CGGAAGGCAGAATAAAGTGTCTTCCTGGGAGGAGCAATGTCCAAAATAT GGTCTTTCTACTGGTGAAAGGTGGATATTGGAATCACCTACAGTTGATAG CGGCCAGAAGAAAGACACCCAAGCAACTGCCGCAGGCCAGTCAGCCCTA TGGAAGCTGGAAGAAAGATACCCAAGTGCCCGGCCATACTGACCCTGAA AGAAAGACATACAAACACCTGCCTCTGGAATCACTGTTATTTGAATGTGT GCTGGGGATATGGAAAAGACAAGAGTACATGGTGGAGAGGTGGAAGAGA AAATGAAGCCAAGGTGTTTGTATATTGTAGAGAAAGGTAGAACTAGAGA TGGTAGTGAGGAACAGGGAGATATGAGGACGCCATAGTGAGTTCCTACC CTGGGATGCCACTAAGGGCCATGTTTGCATCCATGGTCCTGTTGCAGCTG GGGTCGTTGCTGATGTCTTGGCCCGAGTTACCATCAAAGACCAAGTGGAT GTCCCATGGTCTAGGTTGCCACTGGAAAACGTGTGGATATCTGAGGGTCA TGCTGCTTAGGGAAGACTTGCTCATCTTCGTGGCCTGCCCTGTCACATGAG GCCATAGGGACATTTGGGCCCAGACTAGTTCAATGGGCTATGTCTTGGTC CTTGACTTCATGGCAGCTGAGGTCTGTGTTGATGTCCCAGTCTCATGAATT ACCAAAGGCCATGCAGATATCCCTAGTCTGGAATGCTGCCTGAGTACTAT GCTGAGCTGGAGTGTGTGGGGTGATCAGCCCCAAAGGTGTGACTGGGGG AGAGCTGGCCCGGGGCCCTGCCTCTTATCTGCCCCACAACAGTGGCAGAG ATAGCTAGGGCAGTACCTCCCAGGGCACAGAAGAGCTGATGGGCTAACT AGCTCAATTACCTGCGCAGATCTAGGGCAGTGAGTTGGCCCACTCCAACA TAGTTCTATCCCAACTATAAACTGCTGGGGCATGTGAAAGGGCTGGTTTT GCAGATCCAAAGCAGCAGGATCTCCATGGCATGGGGCTGAAACAGAATA TCCAAGCCGTGTCCCGGTGAGGATCCAGTATATATAGTATAGCAGAAGCC AGAGACCTTGAAACAGACCATGACTTATTGCAAGGAACATTTCCAAGTGA AAATATATGGACAAAAGGGTGTACCATGCGACACAACAGCTTCCAGAGC52 AAGATTTTTTTTTTTTTAATTCTGTCTCCTTTTCTTTTCTTTTGTTGGGGGAG GTTGTAAGGGCAGAGGGCAGAGAACAGATATGAAGGGATGGGGAAATGA GTGAAATTGGGGTATGCGATGTAAAACACACAAAATAATCAATAAAAAT TTAAAAAACA (SEQ ID NO: 25) Unmodified PARAIL RNA ACAAGCCGGGUGGACAUCGCUGAGGCUUGGAAAAGAUUUCCUCCGGGG AAAAUCCCUCCGGAGUUCUCCAGUCUGGUGUCAGAGAGGAAUACUAAA GAAGAAGGAGAAAUUCAAGGUACAAGAGAGGCAAAGAAUGUUGCUCCU ACAAAGGAACUCUCCUGACAGAAGUCCACAGAGGACAGAUGGAUGGGG AUGAUUUCCACACUAGUUCCUAACUUUAAAUAAAACCAAGCCUGCAGC GUAGUUUCCUGAGUCCUAAUGGAAGGAUUUGAUGAAGACAUCCCAUUU AGGAAUCUGCACUGUUCUCCCUGGGCCACAGCUAUGCCUGCUUCCCUGG AGGCCUGCUGCCAUCCAGGACAACGAGAUGGAGAUGUGUUAAAACUCA GAAGUCCUGUUCUAGAGUGACUCUCUUACUACACAGAGCAUCCCUUGC CAUUGAAGAGGUAAAACUUAAUUCCCGGCAGGCAGCCCAAUGUGGGAG CGAGCGUCAGCCAUGCUGCGGAAGGCAGAAUAAAGUGUCUUCCUGGGA GGAGCAAUGUCCAAAAUAUGGUCUUUCUACUGGUGAAAGGUGGAUAUU GGAAUCACCUACAGUUGAUAGCGGCCAGAAGAAAGACACCCAAGCAAC UGCCGCAGGCCAGUCAGCCCUAUGGAAGCUGGAAGAAAGAUACCCAAG UGCCCGGCCAUACUGACCCUGAAAGAAAGACAUACAAACACCUGCCUCU GGAAUCACUGUUAUUUGAAUGUGUGCUGGGGAUAUGGAAAAGACAAGA GUACAUGGUGGAGAGGUGGAAGAGAAAAUGAAGCCAAGGUGUUUGUA UAUUGUAGAGAAAGGUAGAACUAGAGAUGGUAGUGAGGAACAGGGAG AUAUGAGGACGCCAUAGUGAGUUCCUACCCUGGGAUGCCACUAAGGGC CAUGUUUGCAUCCAUGGUCCUGUUGCAGCUGGGGUCGUUGCUGAUGUC UUGGCCCGAGUUACCAUCAAAGACCAAGUGGAUGUCCCAUGGUCUAGG UUGCCACUGGAAAACGUGUGGAUAUCUGAGGGUCAUGCUGCUUAGGGA AGACUUGCUCAUCUUCGUGGCCUGCCCUGUCACAUGAGGCCAUAGGGA CAUUUGGGCCCAGACUAGUUCAAUGGGCUAUGUCUUGGUCCUUGACUU CAUGGCAGCUGAGGUCUGUGUUGAUGUCCCAGUCUCAUGAAUUACCAA AGGCCAUGCAGAUAUCCCUAGUCUGGAAUGCUGCCUGAGUACUAUGCU GAGCUGGAGUGUGUGGGGUGAUCAGCCCCAAAGGUGUGACUGGGGGAG AGCUGGCCCGGGGCCCUGCCUCUUAUCUGCCCCACAACAGUGGCAGAGA UAGCUAGGGCAGUACCUCCCAGGGCACAGAAGAGCUGAUGGGCUAACU AGCUCAAUUACCUGCGCAGAUCUAGGGCAGUGAGUUGGCCCACUCCAA CAUAGUUCUAUCCCAACUAUAAACUGCUGGGGCAUGUGAAAGGGCUGG UUUUGCAGAUCCAAAGCAGCAGGAUCUCCAUGGCAUGGGGCUGAAACA GAAUAUCCAAGCCGUGUCCCGGUGAGGAUCCAGUAUAUAUAGUAUAGC AGAAGCCAGAGACCUUGAAACAGACCAUGACUUAUUGCAAGGAACAUU53 UCCAAGUGAAAAUAUAUGGACAAAAGGGUGUACCAUGCGACACAACAG CUUCCAGAGCAAGAUUUUUUUUUUUUUAAUUCUGUCUCCUUUUCUUUU CUUUUGUUGGGGGAGGUUGUAAGGGCAGAGGGCAGAGAACAGAUAUGA AGGGAUGGGGAAAUGAGUGAAAUUGGGGUAUGCGAUGUAAAACACACA AAAUAAUCAAUAAAAAUUUAAAAAACA (SEQ ID NO: 26) m5C-modified PARAIL RNA A5AAG55GGGUGGA5AU5G5UGAGG5UUGGAAAAGAUUU55U55GGGGAAA AU555U55GGAGUU5U55AGU5UGGUGU5AGAGAGGAAUA5UAAAGAAGAA GGAGAAAUU5AAGGUA5AAGAGAGG5AAAGAAUGUUG5U55UA5AAAGG AA5U5U55UGA5AGAAGU55A5AGAGGA5AGAUGGAUGGGGAUGAUUU55A 5A5UAGUU55UAA5UUUAAAUAAAA55AAG55UG5AG5GUAGUUU55UGAG U55UAAUGGAAGGAUUUGAUGAAGA5AU555AUUUAGGAAU5UG5A5UGU U5U555UGGG55A5AG5UAUG55UG5UU555UGGAGG55UG5UG55AU55AGGA 5AA5GAGAUGGAGAUGUGUUAAAA5U5AGAAGU55UGUU5UAGAGUGA5U 5U5UUA5UA5A5AGAG5AU555UUG55AUUGAAGAGGUAAAA5UUAAUU555 GG5AGG5AG555AAUGUGGGAG5GAG5GU5AG55AUG5UG5GGAAGG5AGA AUAAAGUGU5UU55UGGGAGGAG5AAUGU55AAAAUAUGGU5UUU5UA5U GGUGAAAGGUGGAUAUUGGAAU5A55UA5AGUUGAUAG5GG55AGAAGA AAGA5A555AAG5AA5UG55G5AGG55AGU5AG555UAUGGAAG5UGGAAGA AAGAUA555AAGUG555GG55AUA5UGA555UGAAAGAAAGA5AUA5AAA5A 55UG55U5UGGAAU5A5UGUUAUUUGAAUGUGUG5UGGGGAUAUGGAAAA GA5AAGAGUA5AUGGUGGAGAGGUGGAAGAGAAAAUGAAG55AAGGUG UUUGUAUAUUGUAGAGAAAGGUAGAA5UAGAGAUGGUAGUGAGGAA5A GGGAGAUAUGAGGA5G55AUAGUGAGUU55UA555UGGGAUG55A5UAAGG G55AUGUUUG5AU55AUGGU55UGUUG5AG5UGGGGU5GUUG5UGAUGU5U UGG555GAGUUA55AU5AAAGA55AAGUGGAUGU555AUGGU5UAGGUUG55 A5UGGAAAA5GUGUGGAUAU5UGAGGGU5AUG5UG5UUAGGGAAGA5UU G5U5AU5UU5GUGG55UG555UGU5A5AUGAGG55AUAGGGA5AUUUGGG55 5AGA5UAGUU5AAUGGG5UAUGU5UUGGU55UUGA5UU5AUGG5AG5UGAG GU5UGUGUUGAUGU555AGU5U5AUGAAUUA55AAAGG55AUG5AGAUAU5 55UAGU5UGGAAUG5UG55UGAGUA5UAUG5UGAG5UGGAGUGUGUGGGG UGAU5AG5555AAAGGUGUGA5UGGGGGAGAG5UGG555GGGG555UG55U5 UUAU5UG5555A5AA5AGUGG5AGAGAUAG5UAGGG5AGUA55U555AGGG5 A5AGAAGAG5UGAUGGG5UAA5UAG5U5AAUUA55UG5G5AGAU5UAGGG5 AGUGAGUUGG555A5U55AA5AUAGUU5UAU555AA5UAUAAA5UG5UGGGG 5AUGUGAAAGGG5UGGUUUUG5AGAU55AAAG5AG5AGGAU5U55AUGG5A UGGGG5UGAAA5AGAAUAU55AAG55GUGU555GGUGAGGAU55AGUAUAU AUAGUAUAG5AGAAG55AGAGA55UUGAAA5AGA55AUGA5UUAUUG5AA GGAA5AUUU55AAGUGAAAAUAUAUGGA5AAAAGGGUGUA55AUG5GA5A 5AA5AG5UU55AGAG5AAGAUUUUUUUUUUUUUAAUU5UGU5U55UUUU5U UUU5UUUUGUUGGGGGAGGUUGUAAGGG5AGAGGG5AGAGAA5AGAUA UGAAGGGAUGGGGAAAUGAGUGAAAUUGGGGUAUG5GAUGUAAAA5A5 A5AAAAUAAU5AAUAAAAAUUUAAAAAA5A (SEQ ID NO: 27)54 Ψ-modified PARAIL RNA ACAAGCCGGGPGGACAPCGCPGAGGCPPGGAAAAGAPPPCCPCCGGGGAA AAPCCCPCCGGAGPPCPCCAGPCPGGPGPCAGAGAGGAAPACPAAAGAAGA AGGAGAAAPPCAAGGPACAAGAGAGGCAAAGAAPGPPGCPCCPACAAAGG AACPCPCCPGACAGAAGPCCACAGAGGACAGAPGGAPGGGGAPGAPPPCC ACACPAGPPCCPAACPPPAAAPAAAACCAAGCCPGCAGCGPAGPPPCCPGA GPCCPAAPGGAAGGAPPPGAPGAAGACAPCCCAPPPAGGAAPCPGCACPGPP CPCCCPGGGCCACAGCPAPGCCPGCPPCCCPGGAGGCCPGCPGCCAPCCAG GACAACGAGAPGGAGAPGPGPPAAAACPCAGAAGPCCPGPPCPAGAGPGAC PCPCPPACPACACAGAGCAPCCCPPGCCAPPGAAGAGGPAAAACPPAAPPCC CGGCAGGCAGCCCAAPGPGGGAGCGAGCGPCAGCCAPGCPGCGGAAGGC AGAAPAAAGPGPCPPCCPGGGAGGAGCAAPGPCCAAAAPAPGGPCPPPCPA CPGGPGAAAGGPGGAPAPPGGAAPCACCPACAGPPGAPAGCGGCCAGAAG AAAGACACCCAAGCAACPGCCGCAGGCCAGPCAGCCCPAPGGAAGCPGGA AGAAAGAPACCCAAGPGCCCGGCCAPACPGACCCPGAAAGAAAGACAPAC AAACACCPGCCPCPGGAAPCACPGPPAPPPGAAPGPGPGCPGGGGAPAPGGA AAAGACAAGAGPACAPGGPGGAGAGGPGGAAGAGAAAAPGAAGCCAAGG PGPPPGPAPAPPGPAGAGAAAGGPAGAACPAGAGAPGGPAGPGAGGAACAG GGAGAPAPGAGGACGCCAPAGPGAGPPCCPACCCPGGGAPGCCACPAAGG GCCAPGPPPGCAPCCAPGGPCCPGPPGCAGCPGGGGPCGPPGCPGAPGPCPP GGCCCGAGPPACCAPCAAAGACCAAGPGGAPGPCCCAPGGPCPAGGPPGCC ACPGGAAAACGPGPGGAPAPCPGAGGGPCAPGCPGCPPAGGGAAGACPPGC PCAPCPPCGPGGCCPGCCCPGPCACAPGAGGCCAPAGGGACAPPPGGGCCC AGACPAGPPCAAPGGGCPAPGPCPPGGPCCPPGACPPCAPGGCAGCPGAGGP CPGPGPPGAPGPCCCAGPCPCAPGAAPPACCAAAGGCCAPGCAGAPAPCCCP AGPCPGGAAPGCPGCCPGAGPACPAPGCPGAGCPGGAGPGPGPGGGGPGAP CAGCCCCAAAGGPGPGACPGGGGGAGAGCPGGCCCGGGGCCCPGCCPCPP APCPGCCCCACAACAGPGGCAGAGAPAGCPAGGGCAGPACCPCCCAGGGC ACAGAAGAGCPGAPGGGCPAACPAGCPCAAPPACCPGCGCAGAPCPAGGG CAGPGAGPPGGCCCACPCCAACAPAGPPCPAPCCCAACPAPAAACPGCPGG GGCAPGPGAAAGGGCPGGPPPPGCAGAPCCAAAGCAGCAGGAPCPCCAPGG CAPGGGGCPGAAACAGAAPAPCCAAGCCGPGPCCCGGPGAGGAPCCAGPA PAPAPAGPAPAGCAGAAGCCAGAGACCPPGAAACAGACCAPGACPPAPPGC AAGGAACAPPPCCAAGPGAAAAPAPAPGGACAAAAGGGPGPACCAPGCGA CACAACAGCPPCCAGAGCAAGAPPPPPPPPPPPPPAAPPCPGPCPCCPPPPCPP PPCPPPPGPPGGGGGAGGPPGPAAGGGCAGAGGGCAGAGAACAGAPAPGAA GGGAPGGGGAAAPGAGPGAAAPPGGGGPAPGCGAPGPAAAACACACAAAA PAAPCAAPAAAAAPPPAAAAAACA (SEQ ID NO: 28) m1Ψ-modified PARAIL RNA ACAAGCCGGG1GGACA1CGC1GAGGC11GGAAAAGA111CC1CCGGGGAAA A1CCC1CCGGAG11C1CCAG1C1GG1G1CAGAGAGGAA1AC1AAAGAAGAA GGAGAAA11CAAGG1ACAAGAGAGGCAAAGAA1G11GC1CC1ACAAAGGA AC1C1CC1GACAGAAG1CCACAGAGGACAGA1GGA1GGGGA1GA111CCAC AC1AG11CC1AAC111AAA1AAAACCAAGCC1GCAGCG1AG111CC1GAG1CC 1AA1GGAAGGA111GA1GAAGACA1CCCA111AGGAA1C1GCAC1G11C1CCC 1GGGCCACAGC1A1GCC1GC11CCC1GGAGGCC1GC1GCCA1CCAGGACAAC GAGA1GGAGA1G1G11AAAAC1CAGAAG1CC1G11C1AGAG1GAC1C1C11AC 1ACACAGAGCA1CCC11GCCA11GAAGAGG1AAAAC11AA11CCCGGCAGGC AGCCCAA1G1GGGAGCGAGCG1CAGCCA1GC1GCGGAAGGCAGAA1AAAG 1G1C11CC1GGGAGGAGCAA1G1CCAAAA1A1GG1C111C1AC1GG1GAAAGG 1GGA1A11GGAA1CACC1ACAG11GA1AGCGGCCAGAAGAAAGACACCCAA55 GCAAC1GCCGCAGGCCAG1CAGCCC1A1GGAAGC1GGAAGAAAGA1ACCC AAG1GCCCGGCCA1AC1GACCC1GAAAGAAAGACA1ACAAACACC1GCC1C 1GGAA1CAC1G11A111GAA1G1G1GC1GGGGA1A1GGAAAAGACAAGAG1A CA1GG1GGAGAGG1GGAAGAGAAAA1GAAGCCAAGG1G111G1A1A11G1A GAGAAAGG1AGAAC1AGAGA1GG1AG1GAGGAACAGGGAGA1A1GAGGAC GCCA1AG1GAG11CC1ACCC1GGGA1GCCAC1AAGGGCCA1G111GCA1CCA1 GG1CC1G11GCAGC1GGGG1CG11GC1GA1G1C11GGCCCGAG11ACCA1CAA AGACCAAG1GGA1G1CCCA1GG1C1AGG11GCCAC1GGAAAACG1G1GGA1A 1C1GAGGG1CA1GC1GC11AGGGAAGAC11GC1CA1C11CG1GGCC1GCCC1G1 CACA1GAGGCCA1AGGGACA111GGGCCCAGAC1AG11CAA1GGGC1A1G1C 11GG1CC11GAC11CA1GGCAGC1GAGG1C1G1G11GA1G1CCCAG1C1CA1GA A11ACCAAAGGCCA1GCAGA1A1CCC1AG1C1GGAA1GC1GCC1GAG1AC1A1 GC1GAGC1GGAG1G1G1GGGG1GA1CAGCCCCAAAGG1G1GAC1GGGGGAG AGC1GGCCCGGGGCCC1GCC1C11A1C1GCCCCACAACAG1GGCAGAGA1A GC1AGGGCAG1ACC1CCCAGGGCACAGAAGAGC1GA1GGGC1AAC1AGC1C AA11ACC1GCGCAGA1C1AGGGCAG1GAG11GGCCCAC1CCAACA1AG11C1 A1CCCAAC1A1AAAC1GC1GGGGCA1G1GAAAGGGC1GG1111GCAGA1CCA AAGCAGCAGGA1C1CCA1GGCA1GGGGC1GAAACAGAA1A1CCAAGCCG1G 1CCCGG1GAGGA1CCAG1A1A1A1AG1A1AGCAGAAGCCAGAGACC11GAA ACAGACCA1GAC11A11GCAAGGAACA111CCAAG1GAAAA1A1A1GGACAA AAGGG1G1ACCA1GCGACACAACAGC11CCAGAGCAAGA1111111111111AA 11C1G1C1CC1111C1111C1111G11GGGGGAGG11G1AAGGGCAGAGGGCAGA GAACAGA1A1GAAGGGA1GGGGAAA1GAG1GAAA11GGGG1A1GCGA1G1A AAACACACAAAA1AA1CAA1AAAAA111AAAAAACA (SEQ ID NO: 29) m6A-modified PARAIL RNA 6C66GCCGGGUGG6C6UCGCUG6GGCUUGG6666G6UUUCCUCCGGGG6666U CCCUCCGG6GUUCUCC6GUCUGGUGUC6G6G6GG66U6CU666G66G66GG6G 666UUC66GGU6C66G6G6GGC666G66UGUUGCUCCU6C666GG66CUCUCCUG 6C6G66GUCC6C6G6GG6C6G6UGG6UGGGG6UG6UUUCC6C6CU6GUUCCU66 CUUU666U6666CC66GCCUGC6GCGU6GUUUCCUG6GUCCU66UGG66GG6U UUG6UG66G6C6UCCC6UUU6GG66UCUGC6CUGUUCUCCCUGGGCC6C6GC U6UGCCUGCUUCCCUGG6GGCCUGCUGCC6UCC6GG6C66CG6G6UGG6G6U GUGUU6666CUC6G66GUCCUGUUCU6G6GUG6CUCUCUU6CU6C6C6G6GC6 UCCCUUGCC6UUG66G6GGU6666CUU66UUCCCGGC6GGC6GCCC66UGUGG G6GCG6GCGUC6GCC6UGCUGCGG66GGC6G66U666GUGUCUUCCUGGG6G G6GC66UGUCC6666U6UGGUCUUUCU6CUGGUG666GGUGG6U6UUGG66UC 6CCU6C6GUUG6U6GCGGCC6G66G666G6C6CCC66GC66CUGCCGC6GGCC6 GUC6GCCCU6UGG66GCUGG66G666G6U6CCC66GUGCCCGGCC6U6CUG6C CCUG666G666G6C6U6C666C6CCUGCCUCUGG66UC6CUGUU6UUUG66UGU GUGCUGGGG6U6UGG6666G6C66G6GU6C6UGGUGG6G6GGUGG66G6G6666 UG66GCC66GGUGUUUGU6U6UUGU6G6G666GGU6G66CU6G6G6UGGU6GU G6GG66C6GGG6G6U6UG6GG6CGCC6U6GUG6GUUCCU6CCCUGGG6UGCC6 CU66GGGCC6UGUUUGC6UCC6UGGUCCUGUUGC6GCUGGGGUCGUUGCU G6UGUCUUGGCCCG6GUU6CC6UC666G6CC66GUGG6UGUCCC6UGGUCU6 GGUUGCC6CUGG6666CGUGUGG6U6UCUG6GGGUC6UGCUGCUU6GGG66G 6CUUGCUC6UCUUCGUGGCCUGCCCUGUC6C6UG6GGCC6U6GGG6C6UUU GGGCCC6G6CU6GUUC66UGGGCU6UGUCUUGGUCCUUG6CUUC6UGGC6G CUG6GGUCUGUGUUG6UGUCCC6GUCUC6UG66UU6CC666GGCC6UGC6G6 U6UCCCU6GUCUGG66UGCUGCCUG6GU6CU6UGCUG6GCUGG6GUGUGUG GGGUG6UC6GCCCC666GGUGUG6CUGGGGG6G6GCUGGCCCGGGGCCCUG CCUCUU6UCUGCCCC6C66C6GUGGC6G6G6U6GCU6GGGC6GU6CCUCCC6G GGC6C6G66G6GCUG6UGGGCU66CU6GCUC66UU6CCUGCGC6G6UCU6GGG56 C6GUG6GUUGGCCC6CUCC66C6U6GUUCU6UCCC66CU6U666CUGCUGGGG C6UGUG666GGGCUGGUUUUGC6G6UCC666GC6GC6GG6UCUCC6UGGC6U GGGGCUG666C6G66U6UCC66GCCGUGUCCCGGUG6GG6UCC6GU6U6U6U6 GU6U6GC6G66GCC6G6G6CCUUG666C6G6CC6UG6CUU6UUGC66GG66C6UU UCC66GUG6666U6U6UGG6C6666GGGUGU6CC6UGCG6C6C66C6GCUUCC6G 6GC66G6UUUUUUUUUUUUU66UUCUGUCUCCUUUUCUUUUCUUUUGUUG GGGG6GGUUGU66GGGC6G6GGGC6G6G66C6G6U6UG66GGG6UGGGG666U G6GUG666UUGGGGU6UGCG6UGU6666C6C6C6666U66UC66U66666UUU6666 66C6 (SEQ ID NO: 30) “P” identifies residues where uridine triphosphate (UTP) was replaced with pseudouridine-5’-triphosphate (Ψ). “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 N1- methylpseudouridine-5’-triphosphate (m1Ψ). “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 ID Ionizable Lipid RNA Type Total Lipids / RNA (wt / wt) N / P Mean by Intensity (nm) PDI EE (%) JP01 C3-K2- E14 - - - 81 0.18 - JP02 C3-K2- E14 GAPLINC Unmodified 9.0 5.01 91 0.16 87 JP03 C3-K2- E14 GAPLINC Unmodified cap + tail 9.0 5.23 230 0.14 64 JP04 C3-K2- E14 GAPLINC Unmodified cap only 9.0 5.21 234 0.12 58 JP05 C3-K2- E14 GAPLINC m1Ψ 9.0 5.21 129 0.19 76 JP07 C3-K2- E14 GAPLINC m1Ψ 20.0 11.58 199 0.13 65 JP09 OC2-K3- E10 GAPLINC Ψ 7.70 5.01 141 0.15 97 JP10 OC2-K3- E10 GAPLINC Ψ (cellulose) 7.70 5.01 159 0.12 6157 JP11 OC2-K3- E10 GAPLINC Unmodified (cellulose) 7.70 5.01 192 0.11 87 JP12 OC2-K3- E10 GAPLINC Unmodified (cellulose) 7.70 5.01 145 0.06 95 JP13 OC2-K3- E10 GAPLINC Ψ (cellulose) 7.70 5.01 152 0.08 89 JP14 OC2-K3- E10 GAPLINC Unmodified (cellulose) 7.70 5.05 187 0.08 84 JP15 OC2-K3- E10 GAPLINC Unmodified (cellulose) 7.70 5.05 195 0.09 88 JP16 OC2-K3- E10 - 7.70 5.05 175 0.08 - JP17 OC2-K3- E10 GAPLINC Unmodified (cellulose) 7.70 5.05 161 0.10 87 JP18 OC2-K3- E10 GAPLINC Unmodified (HPLC) 7.70 5.05 173 0.13 54 JP19 OC2-K3- E10 GAPLINC m5C (HPLC) 7.30 5.02 141 0.11 49 JP20 OC2-K3- E10 GAPLINC Ψ (HPLC) 7.40 5.03 150 0.16 58 JP21 OC2-K3- E10 GAPLINC Scrambled (HPLC) 7.70 5.02 127 0.07 37 JP22 OC2-K3- E10 GAPLINC m1Ψ (HPLC) 7.70 5.06 160 0.11 54 JP24 OC2-K3- E10 MIST Unmodified (HPLC) 7.70 5.03 135 0.07 44 JP25 OC2-K3- E10 DRAIR Unmodified (HPLC) 7.70 5.02 161 0.10 61 JP26 OC2-K3- E10 MIST Ψ (HPLC) 7.30 5.00 124 0.06 6258 JP27 OC2-K3- E10 DRAIR Ψ (HPLC) 7.40 5.06 140 0.07 42 JP29 OC2-K3- E10 GAPLINC Ψ (HPLC) 7.30 5.02 177 0.12 53 JP30 OC2-K3- E10 GAPLINC Ψ (HPLC) 20.0 13.76 164 0.09 85 JP31 OC2-K3- E10 MIST m1Ψ (HPLC) 20.0 13.20 156 0.07 88 JP32 OC2-K3- E10 MIST m5C (HPLC) 20.0 13.79 147 0.09 80 JP34 OC2-K3- E10 MIST Unmodified (HPLC) 20.0 13.06 148 0.07 80 JP35 OC2-K3- E10 MIST Ψ (HPLC) 20.0 13.71 146 0.07 85 JP36 OC2-K3- E10 DRAIR Unmodified (HPLC) 20.0 13.03 168 0.09 82 JP37 OC2-K3- E10 DRAIR Ψ (HPLC) 20.0 13.68 147 0.08 87 JP38 OC2-K3- E10 DRAIR m1Ψ (HPLC) 20.0 13.17 169 0.05 76 JP39 OC2-K3- E10 DRAIR m5C (HPLC) 20.0 13.94 144 0.06 60 JP41 OC2-K3- E10 GAPLINC Ψ (HPLC) 20.0 13.76 151 0.07 89 Note: N / P stands for nitrogen to phosphate. 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Claims

62 CLAIMS 1. A complex comprising a long non-coding (lncRNA) or a DNA encoding a lncRNA and a cell delivery agent wherein the lncRNA lacks a 5’ cap and / or a 3’poly A tail, and wherein the lncRNA comprises single stranded molecules at a purity of at least 99%, wherein the lncRNA comprises one or more different lncRNAs.

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 lncRNA comprises two or more different lncRNAs and / or wherein the lncRNA was purified, optionally by HPLC or cellulose chromatography and / or using a method comprising a denaturation step, optionally a heating step or chemical denaturation step, for example at heating a denaturation temperature.

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 lncRNA or DNA is at least 400 nucleotides, at least 500 nucleotides, at least 1000 nucleotides; and / or up to 1500 nucleotides or up to 2000 nucleotides.

5. The complex or pharmaceutical composition of any one of claims 1 to 4 wherein the lncRNA or DNA comprises or is GAPLINC, DRAIR, MIST and / or PARAIL.

6. The complex or pharmaceutical composition of any one of claims 1 to 5, wherein the lncRNA comprises or is selected from the nucleotide sequence of any one of SEQ ID NOs: 10-21 or 26- 30, a sequence with at least 90% sequence identity to any thereof or the DNA encoding the lncRNA is selected from SEQ ID NO: 1, 5 6 and 25.

7. The complex of any one of claims 1 to 6, or the pharmaceutical composition of any one of claims 2 to 4, wherein the lncRNA 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: N1-methylpseudouridine (m1Ψ) modification, pseudouridine modification (Ψ), 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),63 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 / or 2'-O-4'-C-Locked-5-methyl-cytidine-triphosphate (2'-O-4'-C-Locked-5-Me-CTP).

9. The complex or pharmaceutical composition of 7 or 8, wherein the lncRNA comprises about or at about or 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,.

10. The complex or pharmaceutical composition of any one of claims 1 to 9, wherein the cell delivery agent is a lipid nanoparticle, 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 an expression plasmid, a viral vector, optionally an adenoviral viral vector, or a self-amplified 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 1,2–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), βN2 and / or δO3) and / or comprises a tertiary amine.

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

15. The complex or pharmaceutical composition of any one of claims 11 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 an adenoviral vector or the DNA is complexed with the cell64 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 15, wherein the complex comprises a mass ratio of at least 15:1, 16:1, 17:1, 18:1, 19:1 or 20: 1 of lipid to lncRNA.

18. The complex or pharmaceutical composition of any one of claims 10 to15, wherein the complex comprises a mass ratio between about 7:1 to about 20:1 of lipid to lncRNA.

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 lncRNA.

20. The complex or pharmaceutical composition of any one of claims 1 to 4, wherein the lncRNA is an anti inflammatory lncRNA optionally wherein the lncRNA comprises or is GAPLINC, DRAIR, MIST and / or PARAIL.

21. The complex or pharmaceutical composition of any one of claims 1 to 20 further comprising optionally one or more pharmaceutically acceptable carrier and / or excipient.

22. The complex or pharmaceutical composition of any one of claims 1 to 21, or the lncRNA or DNA as defined therein for use in the treatment of an acute inflammatory disease or condition or a flare up of a chronic inflammatory disease or condition.

23. Use of the complex or pharmaceutical composition according to any one of claims 1-21, or the lncRNA or DNA as defined therein in the treatment of an acute inflammatory disease or condition or a flare up of a chronic inflammatory disease or condition.

24. Use of the complex or pharmaceutical composition according to any one of claims 1-21, or the lncRNA or DNA as defined therein in the preparation of a medicament for the treatment of an acute inflammatory disease or condition or flare up of a chronic inflammatory disease or condition.

25. The complex or pharmaceutical composition or use of any one of claims 22 to 24, wherein the acute inflammatory disease is a cytokine release syndrome (CRS) or vascular disease.

26. The complex or pharmaceutical composition or use of claim 25, wherein the vascular disease is or comprises atherosclerosis or atherothrombosis or the CRS is sepsis or graft versus host disease.

27. The complex or pharmaceutical composition or use of claim according to any one of claims 21- 26, wherein the subject is a human.

28. A nucleic acid molecule comprising or consisting of a long non-coding (lncRNA) wherein the lncRNA lacks a 5’ cap and / or a 3’poly A tail or a DNA molecule encoding said lncRNA, optionally for use in preparing encapsulated lncRNA optionally for use in a method or use described herein.65 29. The nucleic acid molecule of claim 28, wherein the lncRNA has any one of the modifications as defined in any of claims 7 to 8 optionally for use in a method or use described herein.

30. A vector construct comprising a vector backbone and the DNA of claim 29, wherein the vector construct lacks sequence for encoding a 5’ cap and / or a 3’poly A tail, optionally as described in any one of claims 1 to 6, optionally for use in a method or use described herein.

31. The vector construct of claim 30 wherein the vector is an expression plasmid or replicon / self amplified RNA , adenoviral (for DNA) or other DNA delivery vector, optionally for use in an in vitro translation system, or a method or use described herein.

32. A kit comprising the complex or pharmaceutical composition of any one of claims 1 to 21, the nucleic acid molecule of claim 28 or 29, the vector of claim 30 or 31 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.

33. The kit of claim 32 wherein the cell delivery agent is a lipid nanoparticle, optionally for use in a method or use described herein.

34. A method of treating an acute inflammatory disease or a flare up of a chronic disease or condition, the method comprising administering the complex or pharmaceutical composition of any one of claims 1 to 21 the nucleic acid molecule of claim 28 or 29, the vector of claim 30 or 31 to a subject in need thereof.

35. The method of claim 34, wherein the acute inflammatory disease is a systemic inflammation, optionally a cytokine release syndrome.

36. The method of claim 23, wherein the inflammatory disease is sepsis or graft versus host disease.

37. A method of altering expression of one or more cytokine, optionally proinflammatory cytokine expression in a subject, the method comprising administering the complex of any one of claims 1 to 21 or the nucleic acid molecule of claim 28 or 29, the vector of claim 30 or 31 to the subject.

38. The method of claim 37, wherein the one or more cytokine comprises IL-1β, TNFα, IFN-γ and / or IL-6.

39. The method of claim 36 or 37, wherein the altering comprises decreasing expression.

40. The method of claim 38, wherein the one or more cytokine comprises IL-10.

41. The method of claim 40, wherein the altering comprises increasing IL-10.