Method for reducing dsrna formation during ivt

Abstract

The invention relates to a method for reducing double stranded RNA (dsRNA) formation during in vitro transcription (IVT) of a nucleic acid, comprising performing said in vitro transcription in the presence of one or more compounds selected from a sulfobetaine, a polyamine or a glycoside. More specifically, the invention relates to a method for reducing double stranded RNA formation during in vitro transcription in the presence of said one or more compounds. The invention further relates to RNA obtained by such methods and to the use of said one or more compounds for preventing and / or reducing the formation of double-stranded RNA (dsRNA) in an in vitro transcription reaction.

Description

[0001] METHOD FOR REDUCING dsRNA FORMATION DURING IVT

[0002] FIELD OF THE INVENTION

[0003] The invention relates to a method for reducing double stranded RNA (dsRNA) formation during in vitro transcription (IVT) of a nucleic acid, comprising performing said in vitro transcription in the presence of one or more compound selected from a sulfobetaine, a polyamine or a glycoside. More specifically, the invention relates to a method for reducing double stranded RNA formation during in vitro transcription in the presence of said one or more compounds. The invention further relates to RNA obtained or obtainable by such methods and to the use of said one or more compounds for preventing and / or reducing the formation of double-stranded RNA (dsRNA) in an in vitro transcription reaction.

[0004] BACKGROUND TO THE INVENTION

[0005] The emergence of RNA-based therapeutics demands robust and economical methods to produce RNA with few byproducts from aberrant activity. An in vitro transcription using the bacteriophage T7 RNA polymerase is one such popular method preferably using a purified linear DNA template containing a promoter, ribonucleotide triphosphates (NTPs), a ribonuclease inhibitor, a pyrophosphatase, magnesium ions, an appropriate phage RNA polymerase, and a pH buffer. The exact conditions used in the transcription reaction depend on the amount of RNA needed for a specific application. Methods for RNA in vitro transcription are known in the art (see for example W0200204439).

[0006] One important drawback of a conventional IVT reaction is the presence or generation of certain by-products during the synthesis process, including double-stranded RNA (dsRNA), that trigger cellular immune responses. Therefore, when synthesizing mRNAs for in vivo applications that seek to minimize cellular immune responses, it is preferred to either eliminate these dsRNA contaminants from the mRNA preparations after the IVT or to reduce dsRNA formation during the IVT. Several methods have been described that rely on the removal of the by-products after completion of the IVT reaction, with analytical purification such as chromatography-based purification methods being the most predominant approaches. Although efficient, this approach results in an additional step in the mRNA synthesis workflow, involves specialized instrumentation, is often not compatible with upscaling of the reaction, may reduce RNA yield and impedes the cost effectiveness of the approach. In contrast to post-synthesis purification, preventing dsRNA formation during the IVT reaction is a more cost-efficient approach.

[0007] Piao et al. (2022) investigated in-vitro-transcribing messenger RNA (mRNA) in the presence of a chaotropic agent and achieved production of mRNA with significantly less dsRNA compared with regular IVT. As chaotropic agent, Piao et al used urea and / or formamide. These compounds at higher concentrations were effective in reducing the undesired nucleobase-pairing that caused dsRNA formation, however, at these effective high concentrations, T7 RNA polymerase activity at 40°C was almost completely shut down. In addition, modified IVT conditions using these compounds were effective for a variety of RNA molecules, independent of NTP chemistry or 5' capping method, as significant dsRNA reduction was observed regardless of UTP base modifications. Moderate yield loss was observed when the chaotropic agent concentration approached the upper limit of the working range (1 .2M urea) and little mRNA was yielded when the chaotropic agent concentration exceeded the limit.

[0008] In this paper, Piao et al. further demonstrates urea-containing IVT at higher temperature and found that lower amounts of urea are needed to achieve similar levels of dsRNA with higher mRNA yield. Significantly elevated temperatures may however impose extra economic and technical burdens when applied in industrial-scale mRNA manufacturing.

[0009] Eventually, Piao et al. noticed that not all common chaotropic agents that were tested worked the same on dsRNA reduction. Dimethyl sulfoxide at tested concentrations did not reduce dsRNA, while sodium dodecyl sulfate at low concentrations abolished the IVT. An explanation could be that some chaotropic agents may have poor capabilities to dissociate the undesired nucleobase hybridization or have a stronger tendency to denature enzymes than nucleic acid. In this paper, no compounds were identified which at low (e.g. mM) concentrations enable the reduction of dsRNA without compromising RNA yield.

[0010] The aim of the present invention is therefore to identify compounds which are able to reduce double stranded RNA (dsRNA) formation during in vitro transcription at a concentration which does not denature enzymes used for in vitro transcription and / or do not significantly reduce RNA yield during IVT.

[0011] During the research leading to this invention the inventor surprisingly found that performing in vitro transcription of a nucleic acid in the presence of one or more compounds selected from a sulfobetaine, a polyamine or a glycoside reduced double stranded RNA (dsRNA) formation while maintaining the yield of the RNA production.

[0012] Therefore, the present invention relates to a method for reducing double stranded RNA (dsRNA) formation during in vitro transcription (IVT) of a nucleic acid, comprising performing said in vitro transcription in the presence of one or more compounds selected from a sulfobetaine, a polyamine or a glycoside.

[0013] Thus, the main advantage of performing an IVT reaction in the presence of said one or more compounds is the significant reduction in dsRNA formation during the synthesis process while maintaining a high RNA yield. For example, when an IVT reaction is performed in the presence of a zwitterionic sulfobetaine, an alkyl maltoside, and / or a triamine, dsRNA was reduced significantly.

[0014] Remarkably, performing an IVT reaction in the presence of said one or more compounds in a concentration as low between 0.1 and 1000 mM still achieved the reduction of the formation of dsRNA, whilst chaotropic agents such as urea and formamide require far higher concentrations to effectively reduce reduction of the formation of dsRNA. SUMMARY OF THE INVENTION

[0015] In a first aspect, the present invention provides a method for reducing double stranded RNA (dsRNA) formation during in vitro transcription (IVT) of a nucleic acid, comprising performing said in vitro transcription in the presence of one or more compounds selected from a sulfobetaine, a polyamine or a glycoside.

[0016] In a further embodiment, the in vitro transcription is performed in the presence of a zwitterionic sulfobetaine.

[0017] In another embodiment of the invention, the zwitterionic sulfobetaine is a non-detergent sulfobetaine

[0018] In another embodiment of the non-detergent sulfobetaine comprises an aliphatic group, wherein the aliphatic group comprises between 1 and 18 carbon atoms.

[0019] In a particular embodiment, the method of the present invention is performed in the presence of an non-detergent sulfobetaine, wherein the non-detergent sulfobetaine comprises aliphatic group having a length of between 3 and 10 carbon atoms, preferably between 4 and 9 carbon atoms, more preferably between 5 and 8 carbon atoms, most preferably 6 or 7 carbon atoms.

[0020] In another particular embodiment, the method of the present invention is performed in the presence of 3-[Benzyl(dimethyl)azaniumyl]propane-1 -sulfonate (NDSB-256)

[0021] In another embodiment, the method of the present invention is performed in the presence of any of the herein described sulfobetaines, wherein the sulfobetaines is present in said IVT reaction at a concentration between 1 and 1000 mM, preferably between 10 and 500 mM, more preferably, between 100 and 400 mM, most preferably between 200 and 300 mM.

[0022] In a further embodiment, the method of the present invention is performed in the presence of NDSB-256, wherein the NDSB-256 is present in said IVT reaction at a concentration between 1 and 1000 mM, preferably between 10 and 500 mM, more preferably, between 100 and 400 mM, most preferably between 200 and 300 mM.

[0023] In another specific embodiment, the method of the present invention is performed in the presence of NDSB-256, wherein the NDSB-256 is present in said IVT reaction at a concentration of 200 mM or 300 mM.

[0024] In a further embodiment, the in vitro transcription is performed in the presence of a triamine.

[0025] In another embodiment of the invention, the triamine is an aliphatic triamine.

[0026] In another embodiment of the method of the present invention the aliphatic triamine comprises two alkyl chains, and the alkyl chains comprise between 1 and 10 carbon atoms.

[0027] In a particular embodiment, the method of the present invention is performed in the presence of an aliphatic triamine, wherein the aliphatic triamine comprises two alkyl chains having a length of between 1 and 8 carbon atoms, preferably between 2 and 7 carbon atoms, more preferably between 3 and 6 carbon atoms, most preferably 4 or 5 carbon atoms.

[0028] In another particular embodiment, the method of the present invention is performed in the presence of N-(3-aminopropyl)-1 ,4-butaandiamine (spermidine). In another embodiment, the method of the present invention is performed in the presence of any of the herein described polyamines, wherein the polyamine is present in said IVT reaction at a concentration between 0.1 and 200 mM, preferably between 0.5 and 100 mM, more preferably, between 1 and 50 mM, most preferably between 2 and 20 mM.

[0029] In a further embodiment, the method of the present invention is performed in the presence of spermidine, wherein the spermidine is present in said IVT reaction at a concentration between 0.1 and 200 mM, preferably between 0.5 and 100 mM, more preferably, between 1 and 50 mM, even more preferably between 2 and 20 mM, yet more preferably between 7 and 12, most preferably between 10 and 12 mM.

[0030] In another specific embodiment, the method of the present invention is performed in the presence of spermidine, wherein the spermidine is present in said IVT reaction at a concentration of 7 mM or 12 mM.

[0031] In a further embodiment, the in vitro transcription is performed in the presence of an alkyl maltoside.

[0032] In another embodiment of the invention, the alkyl maltoside comprises an alkyl chain comprising between 1 and 18 carbon atoms.

[0033] In another embodiment of the method of the present invention, the alkyl maltoside as described herein comprises an unbranched alkyl chain.

[0034] In a particular embodiment, the method of the present invention is performed in the presence of an alkyl maltoside, wherein the alkyl maltoside comprises an alkyl chain selected from the group consisting of CH3, C2H5, C3H7, C4H9, C5H1 1 , CeHi s, C7H15, CsHi 7, C9H19, C10H21 , C1 1 H23, C12H25, C13H27, C14H29, C15H31 , C16H33, C17H35, and C18H37.

[0035] In another particular embodiment, the method of the present invention is performed in the presence of an alkylmaltoside, wherein the alkyl maltoside is n-Dodecyl-p-D-maltoside or n-Decyl- p-D-maltoside.

[0036] In another embodiment, the method of the present invention is performed in the presence of any of the herein described glycosides, wherein the glycoside is present in said IVT reaction at a concentration between 0.01 and 100 mM, preferably between 0.05 and 10 mM, more preferably, between 0.1 and 5 mM, most preferably between 0.1 and 2 mM.

[0037] In a further embodiment, the method of the present invention is performed in the presence of the n-Dodecyl-p-D-maltoside or n-Decyl-p-D-maltoside at a concentration between 0.01 and 100 mM, preferably between 0.05 and 10 mM, more preferably, between 0.1 1 and 5 mM, most preferably between 0.1 and 2 mM.

[0038] In another specific embodiment, the method of the present invention is performed in the presence of n-Decyl-p-D-maltoside at a concentration of 0.9 mM or 1 .8 mM.

[0039] In yet another specific embodiment, the method of the present invention is performed in the presence of n-Dodecyl-p-D-maltoside being at a concentration of 0.1 mM or 0.2 mM. In a further embodiment, the invention relates to the methods described herein, wherein the one or more compounds selected from sulfobetaine, polyamine or glycoside prevent and / or reduce the formation of double-stranded RNA (dsRNA).

[0040] In another specific embodiment, the invention relates to RNA obtained or obtainable by the methods of the invention.

[0041] In yet another embodiment, the invention relates to the use of one or more compounds selected from a sulfobetaine, a polyamine or a glycoside as defined in the methods disclosed herein for preventing and / or reducing the formation of double-stranded RNA (dsRNA) in an in vitro transcription reaction.

[0042] BRIEF DESCRIPTION OF THE DRAWINGS

[0043] With specific reference now to the figures, it is stressed that the particulars shown are by way of example and for purposes of illustrative discussion of the different embodiments of the present invention only. They are presented in the cause of providing what is believed to be the most useful and readily description of the principles and conceptual aspects of the invention. In this regard no attempt is made to show structural details of the invention in more detail than is necessary for a fundamental understanding of the invention. The description taken with the drawings making apparent to those skilled in the art how the several forms of the invention may be embodied in practice.

[0044] Fig 1 : Impact of NDSB-256 on dsRNA byproduct formation during IVT. Band intensity was normalized relative to the average of Regular IVT’s (WFI control).

[0045] Fig 2: Impact of elevated Spermidine concentrations on dsRNA byproduct formation during IVT. Band intensity was normalized relative to the average of Regular IVT’s (WFI control)

[0046] Fig. 3: Determining optimal concentrations for IVT yield.

[0047] Fig. 4: Determining optimal concentrations for decreasing the formation of dsRNA during IVT.

[0048] Fig. 5: Testing IVT yield for multiple nucleotide sequences.

[0049] Fig. 6 : Transfection of cells using silica-purified mRNA obtained via standard IVT (IVT-Si), silica- and cellulose-ethanol purified mRNA obtained via standard IVT (IVT-Si-Ce) and silica-purified mRNA obtained IVT with additive NDSB-256 (IVT(additive)-Si). (A) 100 ng mRNA per well; (B) 200 ng mRNA per well /

[0050] Fig. 7: Transfection of cells using silica-purified mRNA obtained via standard IVT (IVT-Si), silica- and cellulose-ethanol purified mRNA obtained via standard IVT (IVT-Si-Ce) and silica-purified mRNA obtained IVT with additive spermidine (IVT(additive)-Si). (A) 100 ng mRNA per well; (B) 200 ng mRNA per well /

[0051] Fig. 8 : Transfection of cells using silica-purified mRNA obtained via standard IVT (IVT-Si), silica- and cellulose-ethanol purified mRNA obtained via standard IVT (IVT-Si-Ce) and silica-purified mRNA obtained IVT with additive n-Dodecyl-p-D-maltoside (IVT(additive)-Si). (A) 100 ng mRNA per well; (B) 200 ng mRNA per well / DETAILED DESCRIPTION OF THE INVENTION

[0052] The present invention will now be further described. 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.

[0053] As used in the specification and the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. By way of example, "a compound" means one compound or more than one compound.

[0054] The terms “comprising”, “comprises” and “comprised of” as used herein are synonymous with “including”, “includes” or “containing”, “contains”, and are inclusive or open-ended and do not exclude additional, non-recited members, elements, or method steps. The terms also encompass “consisting of” and “consisting essentially of”.

[0055] The term "about" or "approximately" as used herein when referring to a measurable value such as a parameter, an amount, a temporal duration, and the like, is meant to encompass variations of + / -10% or less, preferably + / -5% or less, more preferably + / - 1 % or less, and still more preferably + / -0.1 % or less of and from the specified value, insofar such variations are appropriate to perform in the disclosed invention. It is to be understood that the value to which the modifier "about" or "approximately" refers is itself also specifically, and preferably, disclosed.

[0056] The present invention relates to a method to reduce formation of dsRNA during an IVT reaction. The invention further relates to a purified in vitro transcribed RNA composition obtained or obtainable by the methods according to the invention. The inventors of the present invention have unexpectedly found that dsRNA by-product formation can be reduced in an efficient manner by performing the IVT reaction in the presence of one or more compounds selected from a sulfobetaine, a polyamine or a glycoside.

[0057] The main advantage of this method to produce IVT RNAs is a reduction of 60-90% of dsRNA while the yield and integrity of the produced RNA is not compromised. In addition, the described method is compatible with the manufacturing process for different kind of mRNAs including uncapped RNA, CAP-0 and CAP-1 (e.g. Cleancap) capped RNA, nucleoside modified RNA (e.g. including N1 -methyl pseudoruidine), or RNA with and without a polyA tail.

[0058] In the context of the present invention, the terms ‘reducing’ or alternatively ‘to reduce’ are meant to be to ‘lessen’, to ‘decrease’, to ‘minimize’, or to ‘diminish’ the formation of dsRNA.

[0059] Accordingly, where a sample would under normal circumstances contain a particular amount of dsRNA after in vitro transcription, the term ‘reducing’ means that said amount of dsRNA is lower when subjecting said sample to the method of the present invention; in particular when compared to the dsRNA amount obtained in an IVT reaction performed in the absence of said one or more compounds selected from a sulfobetaine, a polyamine or a glycoside. In particular, the amount of dsRNA is preferably reduced by at least 10%, such as at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, when compared to normal circumstances.

[0060] In the context of the present invention, the term ‘the formation’ is meant to be ‘the emergence’, ‘the development’, ‘the origination’, or ‘the generation’ of dsRNA in said in vitro transcription reaction. Specifically, molecules obtained after in vitro transcription typically comprise dsRNA, while we have identified that performing the IVT reaction in the presence of one or more compounds selected from a sulfobetaine, a polyamine or a glycoside results in a reduced formation of such dsRNA.

[0061] In the context of the present invention, the term "RNA" relates to a molecule which comprises ribonucleotide residues and preferably being entirely or substantially composed of ribonucleotide residues. "Ribonucleotide" relates to a nucleotide with a hydroxyl group at the 2'-position of a 0- D- ribofuranosyl group. In particular, the term refers to double stranded RNA, but may also refer to single stranded RNA, isolated RNA such as partially purified RNA, essentially pure RNA, synthetic RNA, recombinantly produced RNA, as well as modified RNA that differs from naturally occurring RNA by the addition, deletion, substitution and / or alteration of one or more nucleotides. Such alterations can include addition of non-nucleotide material, such as to the end(s) of a RNA or internally, for example at one or more nucleotides of the RNA. Nucleotides in RNA molecules can also comprise non-standard nucleotides, such as non-naturally occurring nucleotides or chemically synthesized nucleotides or deoxynucleotides. These altered RNAs can be referred to as analogs or analogs of naturally-occurring RNA.

[0062] According to the present invention, the term "RNA" includes and preferably relates to "mRNA" which means "messenger RNA" and relates to a "transcript" which may be produced using DNA as template and encodes a peptide or protein. mRNA typically comprises a 5’ untranslated region (5’ - UTR), a protein or peptide coding region and a 3’ untranslated region (3’-UTR). mRNA has a limited halftime in cells and in vitro. For the sake of clarity, a mRNA encompasses any coding RNA molecule, which may be translated by a eukaryotic host into a protein.

[0063] The term ‘modified mRNA molecules’ means mRNA molecules that contain one or more modified nucleosides (termed "modified nucleic acids"), which have useful properties such as the lack of a substantial induction of the innate immune response of a cell into which the mRNA is introduced. These modified nucleic acids enhance the efficiency of protein production, intracellular retention of nucleic acids, and viability of contacted cells, as well as possess reduced immunogenicity. An exemplary suitable modified nucleoside may for example by N1 -methyl pseudouridine.

[0064] Preferably, mRNA is produced by in vitro transcription using a nucleic acid template such as a DNA template. In one embodiment of the invention, the RNA is obtained by in vitro transcription. The in vitro transcription methodology is known to the skilled person and may comprise a purified linear DNA template containing a promoter, ribonucleotide triphosphates (NTPs), a buffer system optionally comprising dithiothreitol (DTT) and / or magnesium ions, and an appropriate RNA polymerase such as T7 RNA polymerase. The exact conditions used in the transcription reaction depend on the amount of RNA needed for a specific application. There is a variety of in vitro transcription kits commercially available. In the context of the present invention, the term “double stranded RNA” or “dsRNA” is meant to be any RNA molecule with sufficient internal homology to form significant secondary structures such as hairpins due to hybridization of internal complementary sequences with one another, or a different strand, via Watson-Crick base pairing of nucleotide bases within the complementary sequences. Significant secondary structures generally involve stretches of homology greater than approximately nine bases, but the exact length depends to some extent on context and on whether such secondary structures impart any biological function to the molecule. In particular, molecules obtained after in vitro transcription typically comprise dsRNA with two separate complementary strands and may vary in size for example from 20 nucleotides to 200 nucleotides or even more than 500 nucleotides.

[0065] In the context of the present invention, dsRNA is formed as a by-product identified in IVT reactions which can arise from T7 RNA-dependent RNA polymerase activity. In particular, three main types of by-product in the IVT reaction may result in formation of dsRNA molecules. The first is formed by 3’-extension of the run-off products annealing to complementary sequences in the body of the run-off transcript either in cis (by folding back on the same RNA molecule) or trans (annealing to a second RNA molecule) to form extended duplexes. The second type of dsRNA molecules is formed by hybridization of an antisense RNA molecule to the run-off transcript. The antisense RNA molecules have been reported to be formed in a promoter- and run-off transcript-independent manner. Alternatively, a promoter-independent transcription of full-length anti-sense RNA has also been reported as a novel mechanism of dsRNA generation in T7 RNAPol-driven IVT reaction. A third form of dsRNA results from random pairing of abortive transcripts, either in cis (i.e. within the same molecule) or in trans (between two different molecules). According to the invention, dsRNA encompasses any kind of the described RNA by-products in an IVT reaction.

[0066] Methods for detecting dsRNA rely essentially on immunological approaches such as immunofluorescence, ELISA, immunoblot as well as antibody-independent methods such as nucleic acid fluorescent in situ hybridization (FISH) or cellulose-based dsRNA isolation have also been used for dsRNA detection. As used herein and as described in the examples, immunological methods such as anti dsRNA J2 antibody immunoblotting, use antibodies as structural probes that specifically recognize the A-helix structure adopted by dsRNA. Commercially available J2 anti-dsRNA lgG2a (and to a lesser extent the lgG2a K1 and IgM K2 mAb or 9D5 mAb) have become the golden standards in dsRNA detection. Furthermore, intact mass spectrometry can be used to quantify the abundance and lengths of different 3’-end-extended dsRNA species.

[0067] As detailed already herein, the invention is based on the identification of sulfobetaines, polyamines and glycosides as an additive to IVT reactions for reducing dsRNA.

[0068] In a particular embodiment, said in vitro reaction can be performed in the additional presence of a pH buffer agent, a cap analog, ribonucleoside triphosphates (NTPs), a nucleic acid template and an RNA polymerase. The in vitro reaction may further be performed in the presence of one or more magnesium salts, one or more of ribonuclease inhibitor, pyrophosphatase, and / or one or more antioxidants. In a first aspect, the present invention provides a method for reducing double stranded RNA (dsRNA) formation during in vitro transcription (IVT) of a nucleic acid, comprising performing said in vitro transcription in the presence of a sulfobetaine.

[0069] In the context of the present invention, sulfobetaines are organic compounds having a positively charged quaternary ammonium and a negatively charged sulfonate group. Additionally, sulfobetaines may have an aliphatic group.

[0070] Due to sulfobetaine having a positively charged group and a negatively charged group, the sulfobetaine is preferably zwitterionic. In the context of the present invention, a zwitterionic sulfobetaine can have surfactant or detergent properties or the zwitterionic sulfobetaine is nondetergent. Sulfobetaine having detergent properties are widely used in cosmetics, protein purification, protein separation, and protein solubilization. Examples of detergent sulfobetaines are Sulfobetaine 8 (SB-8), Sulfobetaine 10 (SB-10), Sulfobetaine 12 (SB-12), Sulfobetaine 14 (SB-14), Sulfobetaine 16 (SB-16).

[0071] Non-detergent sulfobetaines (NDSB) are also zwitterionic and these compounds are known to reduce aggregation and aid in refolding and renaturing of proteins found in bacterial expression systems and in inclusion bodies. Unlike detergents such as CHAPS, NDSBs are nondenaturing, even at higher concentrations and have been shown to increase the yield efficiency of membrane and nuclear proteins, among others.

[0072] Examples of sulfobetaine are NDSB-256, NDSB-195, NDSB-201 , NDSB-21 1 , NDSB-221 .

[0073] In a specific embodiment, said in vitro transcription reaction is performed in the presence of a zwitterionic sulfobetaine. As explained herein, zwitterionic sulfobetaines are organic compounds having a contain a positively charged quaternary ammonium and a negatively charged sulfonate group. Additionally, sulfobetaines may have an aliphatic group.

[0074] In a preferred embodiment, the invention relates to a method for reducing dsRNA formation during IVT of a nucleic acid, comprising performing said in vitro transcription in the presence of any one of NDSB-256, NDSB-195, NDSB-201 , NDSB-21 1 , NDSB-221 .

[0075] The zwitterionic sulfobetaine of the present invention can have any aliphatic group. Therefore, the zwitterionic sulfobetaine of the present invention is in particular a zwitterionic sulfobetaine having an aliphatic group such as a branched or unbranched alkyl chain, a fenyl group. In addition, the aliphatic group may be cyclated to the positively charged quaternary ammonium to make a pyridine or a piperidine group at the position of the positively charged quaternary ammonium. Further, the aliphatic group may additionally comprise a hydroxy compound.

[0076] In a particular embodiment, the invention relates to the method for reducing dsRNA formation during IVT of a nucleic acid, comprising performing said in vitro transcription in the presence of an non-detergent sulfobetaine, wherein the non-detergent sulfobetaine comprises an aliphatic group comprises between 1 and 18 carbon atoms.

[0077] In particular, the invention relates to the methods described herein, wherein the non-detergent sulfobetaine comprises an aliphatic group having a length of between 3 and 10 carbon atoms, preferably between 4 and 9 carbon atoms, more preferably between 5 and 8 carbon atoms, most preferably 6 or 7 carbon atoms. In a further embodiment, the non-detergent sulfobetaine comprises a fenyl group.

[0078] In addition, the invention relates to the methods described herein, wherein the non-detergent sulfobetaine is 3-[Benzyl(dimethyl)azaniumyl]propane-1 -sulfonate (NDSB-256). NDSB-256 is known to prevent protein aggregation and facilitate the renaturation of chemically and thermally denatured proteins.

[0079] Thus, NDSB-256 is can efficiently solubilizes proteins at non-denaturing concentrations. This compounds is therefore know to be useful in preventing protein aggregation. NDSB-256 is used for protein folding, renaturation and crystallization. Also, this compounds is a known protein specific additive for solubility and stability.

[0080] Without being bound by theory, the NDSB-256 can stabilize the RNA produced by for example the method described herein due to its ability to prevent folding and / or formation of hydrogen bonds between different strands of single strand RNA during and after IVT. The conditions leading to dsRNA formation can be prevented due to the stabilization of RNA. The formation of dsRNA is therefore prevented during IVT reactions due to the in the presence of NDSB-256 and its stabilization of single strain RNA.

[0081] In a further embodiment, the invention relates to a method for reducing dsRNA formation during IVT of a nucleic acid, comprising performing said in vitro transcription in the presence of an sulfobetaine, wherein the sulfobetaine is present in said IVT reaction at a concentration between 1 and 1000 mM, preferably between 10 and 500 mM, more preferably, between 100 and 400 mM, most preferably between 200 and 300 mM.

[0082] In another particular embodiment, the invention relates to the methods described herein, wherein the sulfobetaine is NDSB-256 and wherein the NDSB-256 is present in said IVT reaction at a concentration between 1 and 1000 mM, preferably between 10 and 500 mM, more preferably, between 100 and 400 mM, most preferably between 200 and 300 mM.

[0083] In a further specific embodiment of the invention, the NDSB-256 is present in said IVT reaction at a concentration of 200 mM or 300 mM. These moderate concentrations improve overall DNA yield and reduce the formation of dsRNA.

[0084] In a further aspect, the present invention provides a method for reducing double stranded RNA (dsRNA) formation during in vitro transcription (IVT) of a nucleic acid, comprising performing said in vitro transcription in the presence of a polyamine.

[0085] In the context of the present invention, polyamines are organic compounds having more than one aminogroup. Polyamines can be cyclic and / or comprise at least two aliphatic or alkyl chains.

[0086] An example of a polyamine is triamine. In the context of the present invention, triamine is a biogenic polyamine which has three amino groups and at least two aliphatic or alkyl chains.

[0087] Triamines are able to interact with ribonucleic acid and changes their properties. For instance, it is known that polyamines can stabilize supercoiled and folded DNA. The amount of polyamines or triamines in the cell would be stoichiometric with the amount of ribonucleic acids. Polyamines occur in cell nucleus and ribosomes and may play a vital role in the synthesis of nucleic acids and proteins and development of the cell. Examples of polyamines are putrescine, cadaverine, spermidine, spermine, thermospermine, caldopentamine, caldohexamine, tris(3-aminopropyl)amine, and tetrakis (3-aminopropyl ammonium).

[0088] In a specific embodiment, said in vitro transcription reaction is performed in the presence of a triamine. As explained herein, triamines are compounds having three amino groups and an aliphatic or alkyl chain differing in chain length, unsaturation and position of the cis-double bond.

[0089] In a preferred embodiment, the invention relates to a method for reducing dsRNA formation during IVT of a nucleic acid, comprising performing said in vitro transcription in the presence of any one of putrescine, cadaverine, spermidine, spermine, thermospermine, caldopentamine, caldohexamine, tris(3-aminopropyl)amine, and tetrakis (3-aminopropyl ammonium).

[0090] The triamine of the present invention can have any aliphatic chain length. Therefore, the triamine of the present invention is in particular an aliphatic triamine.

[0091] In a particular embodiment, the invention relates to the method for reducing dsRNA formation during IVT of a nucleic acid, comprising performing said in vitro transcription in the presence of an aliphatic triamine, wherein the aliphatic triamine comprises two alkyl chains, and wherein the alkyl chains comprise between 1 and 10 carbon atoms.

[0092] In particular, the invention relates to the herein defined method, wherein aliphatic triamine comprises two alkyl chains having a length of between 1 and 8 carbon atoms, preferably between 2 and 7 carbon atoms, more preferably between 3 and 6 carbon atoms, most preferably 4 or 5 carbon atoms.

[0093] In a further embodiment, the aliphatic triamine comprises two unbranched alkyl chains.

[0094] In addition, the invention relates to the herein defined method, wherein the aliphatic triamine is N-(3-aminopropyl)-1 ,4-butaandiamine (spermidine). Spermidine can also be defined as monoaminopropylputrescine, 1 ,5,10-triazadecaan.

[0095] Spermidine is usually considered a critical component for in vitro transcription reactions. Spermidine is a polyamine that potentially interacts with inhibitory polyanions and has been shown to enable the polymerase to dissociate from the DNA template and initiate new DNA chain synthesis. In addition, spermidine has been reported to prevent the inhibition of RNA synthesis by exogenous RNA and to improve the overall efficiency of T7 RNAP transcription in vitro.

[0096] Without being bound by theory, the polyamine may stabilize the RNA produced by the method of the invention due to its ability to interact with ribonucleic acids. The conditions leading to dsRNA formation can be prevented due to the stabilization of RNA. The formation of dsRNA in the presence of polyamine may therefore be prevented by stabilization of the single strand RNA produced by IVT reactions.

[0097] In a further embodiment, the invention relates to a method for reducing dsRNA formation during IVT of a nucleic acid, comprising performing said in vitro transcription in the presence of an aliphatic triamine, wherein the aliphatic triamine is present in said IVT reaction at a concentration between 0.1 and 200 mM, preferably between 0.5 and 100 mM, more preferably, between 1 and 50 mM, most preferably between 2 and 20 mM.. In another particular embodiment, the invention relates to the herein defined method, wherein the aliphatic triamine is spermidine and wherein the spermidine is present in said IVT reaction at a concentration between 0.1 and 200 mM, preferably between 0.5 and 100 mM, more preferably, between 1 and 50 mM, even more preferably between 2 and 20 mM, yet more preferably between 7 and 12, most preferably between 10 and 12 mM.

[0098] In a further specific embodiment of the invention, the spermidine is present in said IVT reaction at a concentration of 7 mM or 12 mM. These moderate concentrations improve overall DNA yield and reduce the formation of dsRNA.

[0099] In a further aspect, the present invention provides a method for reducing double stranded RNA (dsRNA) formation during in vitro transcription (IVT) of a nucleic acid, comprising performing said in vitro transcription in the presence of a glycoside.

[0100] In the context of the present invention, a glycoside is a molecule in which a sugar is bound to another functional group via a glycosidic bond. In other words, a glycoside is any molecule in which a sugar group is bonded through its anomeric carbon to another group via a glycosidic bond.

[0101] Without being bound by theory the dsRNA may be reduced by the presence of a glycoside for example due to the chaotropic capabilities of glycosides to dissociate the nucleobase hybridization and / or a stronger tendency to denature nucleic acids than enzymes.

[0102] In a specific embodiment, said in vitro transcription reaction is performed in the presence of an alkyl maltoside.

[0103] A maltoside is a glycoside with maltose as the glycone (sugar) functional group. An alkyl maltoside contains one or more hydrophobic alkyl chains as the aglycone and is therefore a sugar- based nonionic surfactant. Alkyl maltosides are known as a class of detergents. Variation in the alkyl chain confers a range of detergent properties including Critical Micelle Concentration (CMC) and solubility. The present invention therefore relates to performing the herein defined method in the presence of sugar-based nonionic surfactants, in particular in the presence of an alkyl p-D-maltoside.

[0104] In a particular embodiment, the invention relates to a method for reducing dsRNA formation during IVT of a nucleic acid, comprising performing said in vitro transcription in the presence of alkyl maltoside having an alkyl chain comprising between 1 and 18 carbon atoms. The alkyl chain of the alkyl maltoside in particular comprises between 3 and 17 carbon atoms, more in particular between 5 and 16 carbon atoms, even more in particular between 7 and 14 carbon atoms, most in particular between 10 and 12 carbon atoms.

[0105] In a further embodiment, the alkyl maltoside comprises an unbrached alkyl chain.

[0106] The invention further relates to the method as defined herein, wherein the alkyl maltoside comprises an alkyl chain selected from the group consisting of CH3, C2H5, C3H7, C4H9, C5H11 , CeHis, C7H15, CsHi7, C9H19, C10H21 , C11H23, C12H25, C13H27, C14H29, C15H31 , C16H33, C17H35, and C18H37.

[0107] In addition, the invention relates to a method as defined herein, wherein the alkyl maltoside is n-Dodecyl-p-D-maltoside or n-Decyl-p-D-maltoside. Synonyms for n-Decyl-p-D-maltoside are n- Decyl-b-D-maltopyranoside, Decyl-p-maltopyranoside, Decyl-p-maltoside, Decylmaltoside, decyl 4- O-a-D-glucopyranosyl-p-D-glucopyranoside or DM. Synonyms for Dodecyl-p-D-maltoside are n- Dodecyl-p-D-maltopyranoside, Lauryl maltoside, Dodecyl 4-O-a-D-glucopyranosyl-p-D- glucopyranoside or DDM.

[0108] In a further embodiment, the invention relates to a method for reducing dsRNA formation during IVT of a nucleic acid, comprising performing said in vitro transcription in the presence of a glycoside, wherein the glycoside is present in said IVT reaction at a concentration between 0.01 and 100 mM, preferably between 0.05 and 10 mM, more preferably, between 0.1 and 5 mM, most preferably between 0.1 and 2 mM.

[0109] In another particular embodiment, the invention relates to the herein defined method, wherein the alkyl maltoside is n-Dodecyl-p-D-maltoside or n-Decyl-p-D-maltoside and wherein the n-Dodecyl- p-D-maltoside or n-Decyl-p-D-maltoside is present in said IVT reaction at a concentration between 0.01 and 100 mM, preferably between 0.05 and 10 mM, more preferably, between 0.1 and 5 mM, most preferably between 0.1 and 5 mM.

[0110] In a specific embodiment of the invention the n-Decyl-p-D-maltoside can be present in said IVT reaction at a concentration of 0.9 mM or 1 .8 mM.

[0111] In a further specific embodiment of the invention, the n-Dodecyl-p-D-maltoside is present in said IVT reaction at a concentration of 0.1 mM or 0.2 mM.

[0112] In a specific embodiment, the integrity and / or yield of RNA after the IVT reaction using the one or more compounds as defined herein, is not compromised or preferably increased when compared to normal circumstances. The integrity of the RNA can be measured by any suitable means such as by capillary electrophoresis peak profiles, which may be obtained on a bioanalyzer. Specifically, no significant degradation was observed in the experiments performed herein and peak profiles were nearly identical for IVT reactions in the absence of a sulfobetaine, polyamine or glycoside.

[0113] The invention further relates to RNA obtained or obtainable by the method of the invention.

[0114] In a further aspect, the invention relates to the use of one or more compounds selected from a sulfobetaine, a polyamine or a glycoside as defined herein for preventing and / or reducing the formation of dsRNA in an in vitro transcription reaction.

[0115] In particular, the invention relates to the use of zwitterionic sulfobetaine as defined herein for preventing and / or reducing the formation of dsRNA in an in vitro transcription reaction.

[0116] In a more specific embodiment, the invention relates to the use of an non-detergent sulfobetaine, in particular N-[Benzyl(dimethyl)azaniumyl]propane-1 -sulfonate (NDSB-256) for preventing and / or reducing the formation of dsRNA in an in vitro transcription reaction.

[0117] Furthermore, the invention relates to the method described herein, wherein the [Benzyl(dimethyl)azaniumyl]propane-1 -sulfonate (NDSB-256) prevents and / or reduces the formation of dsRNA.

[0118] In particular, the invention relates to the use of a aliphatic polyamine as defined herein for preventing and / or reducing the formation of dsRNA in an in vitro transcription reaction.

[0119] In a more specific embodiment, the invention relates to the use of an aliphatic triamine, in particular N-(3-aminopropyl)-1 ,4-butaandiamine (sperimdine) for preventing and / or reducing the formation of dsRNA in an in vitro transcription reaction. Furthermore, the invention relates to the herein defined method, wherein the N-(3- aminopropyl)-1 ,4-butaandiamine prevents and / or reduces the formation of dsRNA.

[0120] In a further aspect, the invention relates to the use of a glycoside as defined herein for preventing and / or reducing the formation of double-stranded RNA (dsRNA) in an in vitro transcription reaction.

[0121] Furthermore, the invention relates to the method as disclosed herein, wherein the glycoside prevents and / or reduces the formation of double-stranded RNA (dsRNA).

[0122] In a particular embodiment, RNA in said in vitro transcription reaction may be capped or uncapped RNA, modified or unmodified RNA, or RNA with or without poly(A) tail, or any combination thereof. A mature mRNA ready for efficient translation by the ribosome contains two major modifications: a 5' cap structure and a poly(A) tail. Moreover, performing the IVT reaction in the presence of one or more compounds selected from a sulfobetaine, a polyamine or a glycoside is not compromised towards short or long RNA molecules, i.e. it works equally well on small and long templates.

[0123] According to the invention, an RNA molecule, such as a messenger RNA (or mRNA), comprises the following types: uncapped unmodified RNA without poly(A)tail, uncapped unmodified RNA with poly(A)tail, uncapped modified RNA without poly(A)tail, uncapped modified RNA with poly(A)tail, capped unmodified RNA without poly(A)tail, capped unmodified RNA with poly(A)tail, capped modified RNA without poly(A)tail, capped modified RNA with poly(A)tail.

[0124] In the context of the present invention, the term “capped RNA” is to be understood as an RNA molecule of which the 5' end is linked to a guanosine or a modified guanosine, preferably a 7- methylguanosine (N7-methyl guanosine or m7G), connected to a 5' to 5' triphosphate linkage or analogue. "Capping" of the RNA structure plays a crucial role in a variety of cellular processes which include translation initiation, splicing, intracellular transport and turnover. In vitro synthesis of capped mRNAs is performed by bacteriophage RNA polymerase (T7, SP6 or T3)-mediated in vitro transcription that co-transcriptionally incorporate cap analogues at the 5'-end of the transcripts. Alternatively, post-transcriptional enzymatic capping may also be used to add a 5’CAP to the IVT produced RNA molecules.

[0125] As used herein, “cap analogues” are caps which are biologically equivalent to a 7- methylguanosine (m7G), and comprise traditional analogues such as G(5’)ppp(5’)G, m7G(5’)ppp(5’)G or m2,2,7G(5’)ppp(5’)G, but also Anti-Reverse Cap Analog (ARCA) 3'-O-Me- m7G(5')ppp(5')G, Unmethylated Cap Analog G(5')ppp(5')G, Methylated Cap Analog for A+1 sites m7G(5')ppp(5')A; Unmethylated Cap Analog for A+1 sites G(5')ppp(5')A. Anti-Reverse Cap Analog (ARCA) is a modified cap analogue in which the 3' OH group (closer to m7G) is replaced with - OCH3 (i.e. methylated) preventing RNA elongation by phosphodiester bond formation at this position. This modification forces ARCA incorporation in the correct orientation and subsequently results in a translatable mRNA population.

[0126] In some preferred embodiments, the mRNA used in the methods of the present invention has a 5’ cap structure with a so-called CAP-1 structure (such as CleanCap®), meaning that the 2' hydroxyl of the ribose in the penultimate nucleotide with respect to the cap nucleotide is methylated, such as illustrated below:

[0127] In the context of the present invention, the term “uncapped RNA” is to be understood as any RNA molecule that does not comprise a cap as defined in the definition “capped RNA”. Thus, in a particular embodiment, “uncapped mRNA” may refer to an mRNA of which the 5' end is not linked to a 7-methylguanosine, through a 5' to 5' triphosphate linkage, or an analogue as previously defined.

[0128] In the context of the present invention, the term “modified RNA” is to be understood as an RNA molecule which contains at least one modified nucleotide, nucleoside or base, such as a modified purine or a modified pyrimidine. A modified nucleoside or base can be any nucleoside or base that is not A, U, C or G (respectively Adenosine, Uridine, Cytidine or Guanosine for nucleosides; and Adenine, Uracil, Cytosine or Guanine when referring solely to the sugar moiety).

[0129] In the context of the present invention, the term “unmodified RNA” is to be understood as any RNA molecule that does not comprise a modification as defined in the definition “modified RNA”.

[0130] As used herein, the term “poly(A) tail” is to be understood as a moiety comprising multiple adenosine monophosphates and is well known in the art. A poly(A) tail is generally produced during a step called polyadenylation that is one of the post-translation modifications which generally occur during the production of mature messenger RNAs; such poly(A) tail contributes to the stability and the half-life of said mRNAs, and can be of variable length. In particular, a poly(A) tail may be equal or longer than 10 adenosine nucleotides, which includes equal or longer than 20 adenosine nucleotides, which includes equal or longer than 100 adenosine nucleotides, and for example about 120 adenosine nucleotides.

[0131] In the context of the present invention, the term “without poly(A) tail” is to be understood as any RNA molecule that does not comprise a poly(A) tail as described in the definition “poly(A) tail”.

[0132] In the sense of the invention, the terms “modified and unmodified” are considered distinctly from “capped and uncapped”, as the latter specifically relates to the base at the 5'-end of a RNA molecule, and also distinctly from “with poly(A)tail and without poly(A)tail”. EXAMPLES

[0133] The invention will now be illustrated by means of the following examples. It will be understood by those skilled in the art that various changes and modifications can be easily made without departing from the technical spirit or essential features of the present invention. Therefore, it is to be understood that the below-described examples are illustrative in all aspects and do not limit the scope of the invention in any way.

[0134] EXAMPLE 1 - Sulfobetaine

[0135] Screening of compounds for reduction of dsRNA formation during IVT

[0136] An IVT reaction was performed in a 96 well plate with the following conditions:

[0137] Lin pDNA template muCD70

[0138] Test transcription 0.1 ml

[0139] 4h reaction

[0140] Purification with magnetic beds

[0141] The IVT reaction was performed in the presence of the following components:

[0142] Table 1. Components of the IVT reaction

[0143] The WFI was (partially) replaced by additives in order to perform the IVT reaction with the desired concentration of additives. The additive NDSB-256 was tested in 200 mM and 300 mM, WFI was used as a control sample. After completing the IVT reaction, dsRNA formation was measured by gel electrophoresis (Figure 1 ). The graph in Figure 1 demonstrates the decrease in dsRNA formation when increasing the concentration of NDSB-256 to 200 mM and 300 mM as an additive in IVT reactions as compared to the WFI control.

[0144] Results dsRNA content was significantly decreased in comparison with the WFI (water for injection) control in the presence of NDSB-256. The bands measured are the band at dsRNA.

[0145] Table 2. Results screening compounds

[0146] EXAMPLE 2 - Polyamine

[0147] Screening of compounds for reduction of dsRNA formation during IVT

[0148] An IVT reaction was performed in a 96 well plate with the following conditions:

[0149] Lin pDNA template muCD70

[0150] Test transcription 0.1 ml

[0151] 4h reaction

[0152] Purification with magnetic beds

[0153] The IVT reaction was performed in the presence of the following components:

[0154] Table 3. Components of the IVT reaction

[0155] The WFI was (partially) replaced by additives in order to perform the IVT reaction with the desired concentration of additives. The additive spermidine was tested in 7 mM and 12 mM. After completing the IVT reaction, dsRNA formation was measured by gel electrophoresis (Figure 2). The graph in Figure 2 demonstrates the decrease in dsRNA formation when increasing the concentration of spermidine to 7 mM and 12 mM as an additive in IVT reactions, as compared to the WFI control.

[0156] Results dsRNA content was decreased in comparison with the WFI (water for injection) control in the presence of spermidine. The bands measured are the band at dsRNA.

[0157] Table 4. Results screening compounds

[0158] EXAMPLE 3 - Glycoside

[0159] Screening of compounds for reduction of dsRNA formation during IVT

[0160] An IVT reaction was performed in a 96 well plate with the following conditions:

[0161] Lin pDNA template muCD70

[0162] Test transcription 0.1 ml

[0163] 4h reaction

[0164] 10 pl of Detergent added to the IVT reaction from the Detergent screen 96 well-plate Purification with magnetic beads

[0165] The IVT reaction was performed in the presence of the following components:

[0166] Table 5. Components of the IVT reaction

[0167] Results dsRNA content was decreased in comparison with the WFI (water for injection) control in the presence of the below compounds in the below concentrations:

[0168] Table 6. Results screening compounds EXAMPLE 4 - Testing various glycoside concentrations

[0169] Determining optimal concentration of compounds in the IVT reaction.

[0170] Different concentrations of the glycoside compounds were tested with the screening conditions and components as demonstrated in Example 3 (Figure 3 and 4). For n-Decyl-0-D- maltoside (DM) the following concentrations were screened: 1 .8 mM and 0.9 mM. For n-Dodecyl-0- D-maltoside (DDM) the following concentrations were screened 0.4 mM, 0.2 mM and 0.1 mM.

[0171] Figure 3 demonstrates that IVT yield was optimal when performing the IVT reaction in the presence of 0.9 mM n-Decyl-p-D-maltoside (DM) and / or when performing the IVT reaction in the presence of 0.1 mM n-Dodecyl-p-D-maltoside (DDM).

[0172] Figure 4 demonstrates that the relative dsRNA content was optimal when performing the IVT reaction in the presence of 1 .8 mM n-Decyl-p-D-maltoside (DM) and / or when performing the IVT reaction in the presence of 0.2 mM n-Dodecyl-p-D-maltoside (DDM).

[0173] EXAMPLE 5 - Glycoside

[0174] Testing IVT of different nucleotide sequences.

[0175] Multiple nucleotide sequence candidates were transcribed with the screening conditions and components as demonstrated in Example 3 (Figure 5). The candidates are TLR4 (1136nt), Flue (2039nt), Spike (4202nt).

[0176] Figure 5 shows that performing the IVT reaction in the presence of n-Decyl-p-D-maltoside (DM) in a concentration of 1 .8 mM and n-Dodecyl-p-D-maltoside (DDM) in a concentration of 0.2 mM are capable of yielding very comparable amounts of RNA regardless of the sequence of the RNA.

[0177] EXAMPLE 6 - Sulfobetaine, polyamine and glycoside

[0178] N1 -methyl pseudouridine modified mRNA encoding eGFP was produced using standard in vitro transcription and using a modified in vitro transcription reaction containing 200mM NDSB-256, 12mM spermidine or 0.2mM n-Dodecyl-p-D-maltoside respectively.

[0179] All generated mRNA was purified using silica membrane spin columns. Part of the material produced using standard IVT was subsequently further purified using cellulose-ethanol purification in order to remove the dsRNA present.

[0180] The three generated mRNA constructs were then used to transfect A549 reporter cells which act as a reporter for activation of the innate immune system. Transfections were performed at both 100 and 200ng mRNA per well.

[0181] As indicated in figure 6 (NDSB-256), figure 7 (spermidine) and figure 8 (n-Dodecyl-p-D-maltoside), transfection of silica purified mRNA generated using standard IVT results in a strong signal in the reporter cells. As expected, for material where dsRNA is removed using cellulose-ethanol purification, the resulting signal is much reduced. Interestingly, the signal generated by mRNA that was generated using IVT containing an increased concentration of NDSB-256, spermidine or n- Dodecyl-p-D-maltoside is comparable to that of the mRNA generated using standard IVT and subjected to cellulose-ethanol purification. These data indicate that by adding an increased concentration of NDSB-256, spermidine or n-Dodecyl-p-D-maltoside, the formation of mRNA byproducts leading to activation of the innate immune system (most likely dsRNA) can be significantly reduced. REFERENCES

[0182] Piao et al., 2022 - dsRNA reduction by chaotropic agents during in vitro transcription of messenger RNA - Molecular Therapy Nucleic Acids; Vol. 29 Sep, 2022.

Claims

CLAIMS1 . Method for reducing double stranded RNA (dsRNA) formation during in vitro transcription (IVT) of a nucleic acid, said method comprising performing said in vitro transcription in the presence of one or more compounds selected from a sulfobetaine, a polyamine or a glycoside.

2. Method as claimed in claim 1 , wherein the sulfobetaine is a zwitterionic sulfobetaine.

3. Method as claimed in claim 2, wherein the zwitterionic sulfobetaine is a non-detergent sulfobetaine.

4. Method as claimed in claim 3, wherein the non-detergent sulfobetaine comprises an aliphatic group, wherein the aliphatic group comprises between 1 and 18 carbon atoms.

5. Method as claimed in any one of claims 3 or 4, wherein the non-detergent sulfobetaine comprises an aliphatic group having a length of between 3 and 10 carbon atoms, preferably between 4 and 9 carbon atoms, more preferably between 5 and 8 carbon atoms, most preferably 6 or 7 carbon atoms.

6. Method as claimed in any one of claims 3-5, wherein the non-detergent sulfobetaine is 3- [Benzyl(dimethyl)azaniumyl]propane-1 -sulfonate (NDSB-256).

7. Method as claimed in any one of claims 1 -6, wherein the sulfobetaine is present in said IVT reaction at a concentration between 1 and 1000 mM, preferably between 10 and 500 mM, more preferably, between 100 and 400 mM, most preferably between 200 and 300 mM.

8. Method as claimed in claim 6, wherein NDSB-256 is present in said IVT reaction at a concentration between 1 and 1000 mM, preferably between 10 and 500 mM, more preferably, between 100 and 400 mM, most preferably between 200 and 300 mM9. Method as claimed in any one of claims 6 or 8, wherein NDSB-256 is present in said IVT reaction at a concentration of 200 mM or 300 mM.

10. Method as claimed in claim 1 , wherein the polyamine is a triamine.1 1 . Method as claimed in claim 10, wherein the triamine is an aliphatic triamine.

12. Method as claimed in claim 1 1 , wherein the aliphatic triamine comprises two alkyl chains, and wherein the alkyl chains comprise between 1 and 10 carbon atoms.

13. Method as claimed in any one of claims 1 1 or 12, wherein the aliphatic triamine comprises two alkyl chains having a length of between 1 and 8 carbon atoms, preferably between 2 and 7 carbon atoms, more preferably between 3 and 6 carbon atoms, most preferably 4 or 5 carbon atoms.

14. Method as claimed in any one of claims 1 1 -13, wherein the aliphatic triamine is N-(3- aminopropyl)-1 ,4-butaandiamine (spermidine).

15. Method as claimed in any one of claims 10-14, wherein the aliphatic triamine is present in said IVT reaction at a concentration between 0.1 and 200 mM, preferably between 0.5 and 100 mM, more preferably, between 1 and 50 mM, most preferably between 2 and 20 mM.

16. Method as claimed in claim 14, wherein spermidine is present in said IVT reaction at a concentration between 0.1 and 200 mM, preferably between 0.5 and 100 mM, more preferably, between 1 and 50 mM, even more preferably between 2 and 20 mM, yet more preferably between 7 and 12, most preferably between 10 and 12 mM.

17. Method as claimed in any one of claims 14 or 16, wherein spermidine is present in said IVT reaction at a concentration of 7 mM or 12 mM.

18. Method as claimed in claim 1 , wherein the glycoside is an alkyl maltoside.

19. Method as claimed in claim 18, wherein the alkyl maltoside comprises an alkyl chain comprising between 1 and 18 carbon atoms.

20. Method as claimed in claim 18 or 19, wherein the alkyl maltoside comprises an unbranched alkyl chain.21 . Method as claimed in any one of claims 18-20, wherein the alkyl maltoside comprises an alkyl chain selected from the group consisting of CH3, C2H5, C3H7, C4H9, C5H11 , CeHis, C7H15, CsHi7, C9H19, C10H21 , C11H23, C12H25, C13H27, C14H29, C15H31 , C16H33, C17H35, and C18H37.

22. Method as claimed in any one of claims 18-21 , wherein the alkyl maltoside is n-Dodecyl- p-D-maltoside or n-Decyl-p-D-maltoside.

23. Method as claimed in any one of claims 1 or 18-22, wherein the glycoside is present in said IVT reaction at a concentration between 0.1 and 100 mM, preferably between 0.05 and 10 mM, more preferably, between 0.1 and 5 mM, most preferably between 0.1 and 2 mM.

24. Method as claimed in claim 22, wherein the n-Dodecyl-p-D-maltoside or n-Decyl-0-D- maltoside is present in said IVT reaction at a concentration between 0.1 and 100 mM, preferably between 0.05 and 10 mM, more preferably, between 0.1 and 5 mM, most preferably between 0.1 and 2 mM.

25. Method as claimed in claim 22, wherein the n-Decyl-p-D-maltoside is present in said IVT reaction at a concentration of 0.9 mM or 1 .8 mM.

26. Method as claimed in any one of claims 22 or 23, wherein the n-Dodecyl-p-D-maltoside is present in said IVT reaction at a concentration of 0.1 mM or 0.2 mM.

27. Method as claimed in any one of claims 1 -26, wherein the one or more compounds prevent and / or reduce the formation of double-stranded RNA (dsRNA).

28. Use of one or more compounds as defined in any one of claims 1 -26 for preventing and / or reducing the formation of double-stranded RNA (dsRNA) in an in vitro transcription reaction.

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