In vitro transcription method

Abstract

The present disclosure relates generally to methods of producing RNA from a DNA template. More particularly, the method of the present disclosure comprises an in vitro transcription method for producing RNA from close-ended DNA (ceDNA) templates.

Description

[0001] "IN VITRO TRANSCRIPTION METHOD"

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] The present application claims priority from United States Provisional Patent Application No. 63 / 510,969 filed on 29 June 2023, the contents of which is incorporated herein by reference in its entirety.

[0004] TECHNICAL FIELD

[0005] The present disclosure relates generally to methods of producing RNA from a DNA template. More particularly, the method of the present disclosure comprises an in vitro transcription method for producing RNA from close-ended DNA (ceDNA) templates.

[0006] BACKGROUND

[0007] In vitro transcription (IVT) is a biochemical process of mRNA synthesis in a cell-free transcription system in which an RNA polymerase assembles ribonucleotides using DNA as a template. The template typically contains an open reading frame (ORF) that is often, but not exclusively, comprised of the complete coding sequence of a gene, along with 5' and 3' regulatory elements and a poly(A) tail. Therefore, a quality template DNA is a prerequisite for efficient IVT of standard mRNAs or those mRNAs synthesized with modified nucleosides.

[0008] Plasmid DNA is commonly used in IVT methods to produce an RNA of interest. However, conventional bacterial fermentation techniques used to produce plasmid DNA are slow, expensive, limited by a lack of manufacturing capacity and may produce poor DNA quality owing to unstable or difficult sequences. As such, the production of template DNA of sufficient quality and quantity imposes a bottleneck in the process of mRNA synthesis.

[0009] Accordingly, there is a need for improved approaches for producing RNA by IVT to address one or more of these limitations.

[0010] SUMMARY

[0011] In vitro transcription (IVT) is typically performed using a plasmid DNA template. However, the disadvantages associated with the use of plasmid DNA in such reactions has driven the search for improved DNA templates. Close-ended DNA (ceDNA) templates are generated through a cell-free and enzymatically-based process which avoids the selective pressure often associated with plasmid instability, while also delivering rapid and reliable GMP DNA. IVT reactions also generally require less ceDNA material as compared to when using plasmid DNA templates. ceDNA templates can further support the production of difficult sequences, such as those encompassing long polyA tails or those that comprise large (>20kb) genes of interest, thereby allowing greater flexibility in supporting a range of genetic medicines.

[0012] The inventors have surprisingly found that certain parameters, and in particular pH, can play an important role in the yield or efficiency of RNA production from IVT based on a close-ended DNA template.

[0013] In a first aspect, the present disclosure provides a method of producing an RNA molecule by in vitro transcription, said method including the steps of:

[0014] (a) providing a close-ended DNA template encoding the RNA molecule; and

[0015] (b) transcribing the RNA molecule from the close-ended DNA template in a reaction mixture having a pH of between about 6 to about 8.

[0016] Suitably, the reaction mixture has a pH of about 6.5 to about 7.5.

[0017] In one example, the reaction mixture has a pH of about 7.0.

[0018] In certain examples, the close-ended DNA template is formulated in a solution having a pH of about 6 to about 8.

[0019] In other examples, the solution has a pH of about 6.5 to about 7.5.

[0020] According to some examples, the solution has a pH of about 7.0.

[0021] Suitably, the solution comprises a buffer.

[0022] In certain examples, the solution comprises a Tris buffer.

[0023] In other examples, the solution comprises the Tris buffer at a concentration of about 8 mM to about 12 mM.

[0024] Referring to some examples, the solution comprises the Tris buffer at a concentration of about 10 mM.

[0025] In some examples, the close-ended DNA template is or comprises doggybone DNA (dbDNA).

[0026] In certain examples, the close-ended DNA template has been linearized. Suitably, the method further includes the earlier step of linearizing the close-ended DNA template.

[0027] In another example, the RNA molecule is or comprises a non-replicating mRNA molecule or a self-amplifying mRNA molecule.

[0028] Suitably, the RNA molecule encodes an immunogenic protein.

[0029] In a second aspect, the present disclosure provides an isolated RNA molecule produced by the method described herein.

[0030] In a third aspect, the present disclosure provides a pharmaceutical composition comprising the isolated RNA molecule described herein and optionally a pharmaceutically acceptable carrier, diluent or excipient.

[0031] Accordingly, the isolated RNA molecule is contained in or otherwise associated with a lipid-based carrier.

[0032] In some examples, the lipid-based carrier is or comprises a lipid nanoparticle.

[0033] In other examples, the present disclosure provides an isolated RNA molecule or a pharmaceutical composition as described herein for use in: (a) eliciting an immune response; and / or (b) treating a disease, disorder or condition in a subject.

[0034] In a fourth aspect, the present disclosure provides a method of eliciting an immune response in a subject, said method including of step of administering a therapeutically effective amount of the isolated RNA molecule or the pharmaceutical composition as described herein to the subject to thereby elicit the immune response.

[0035] In a fifth aspect, the present disclosure provides a method of preventing and / or treating a disease, disorder or condition in a subject, said method including of step of administering a therapeutically effective amount of the isolated RNA molecule or the pharmaceutical composition as described herein to the subject to thereby prevent and / or treat the disease, disorder or condition.

[0036] In a sixth aspect, the present disclosure provides for the use of the isolated RNA molecule or the pharmaceutical composition as described herein in the manufacture of a medicament for eliciting an immune response in a subject.

[0037] In a seventh aspect, the present disclosure provides for the use of the isolated RNA molecule or the pharmaceutical composition as described herein in the manufacture of a medicament for preventing and / or treating a disease, disorder or condition in a subject. DETAILED DESCRIPTION

[0038] General Techniques and Definitions

[0039] Unless specifically defined otherwise, all technical and scientific terms used herein shall be taken to have the same meaning as commonly understood by one of ordinary skill in the art (e.g. in immunology, molecular biology, immunohistochemistry, biochemistry, genomics and pharmacology).

[0040] The present disclosure is performed without undue experimentation using, unless otherwise indicated, conventional techniques of molecular biology, microbiology, recombinant DNA technology and immunology. Such procedures are described, for example in Sambrook, Fritsch & Maniatis, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratories, New York, Fourth Edition (2012), whole of Vols I, II, and III; DNA Cloning: A Practical Approach, Vols. I and II (D. N. Glover, Second Edition., 1995), IRL Press, Oxford, whole of text; Oligonucleotide Synthesis: A Practical Approach (M. J. Gait, ed, 1984) IRL Press, Oxford, whole of text, and particularly the papers therein by Gait, ppl- 22; Atkinson et al, pp35-81; Sproat et al, pp 83-115; and Wu et al, pp 135-151; 4. Nucleic Acid Hybridization: A Practical Approach (B. D. Hames & S. J. Higgins, eds., 1985) IRL Press, Oxford, whole of text; Immobilized Cells and Enzymes: A Practical Approach (1986) IRL Press, Oxford, whole of text; Perbal, B., A Practical Guide to Molecular Cloning (1984) and Methods In Enzymology (S. Colowick and N. Kaplan, eds., Academic Press, Inc.), whole of series.

[0041] As used herein, the singular forms of “a”, “and” and “the” include plural forms of these words, unless the context clearly dictates otherwise. For example, a reference to “a bacterium” includes a plurality of such bacteria, and a reference to “an antigen” is a reference to one or more antigens.

[0042] The term “and / or”, e.g., “X and / or Y” shall be understood to mean either “X and Y” or “X or Y” and shall be taken to provide explicit support for both meanings or for either meaning.

[0043] Throughout this specification, the word “comprise” or variations such as “comprises” or “comprising” will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

[0044] The term “about” is used herein to mean approximately. When the term “about” is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the recited numerical values. In general, the term “about” is used herein to modify a numerical value above and below the stated value by ± 0.1%, 0.5%, 1.0%, 5.0% or 10%. The extent of such tolerances and variances are well understood by persons skilled in the art. Typically, such tolerances and variances do not compromise the structure, function and / or implementation of the compositions and methods described herein.

[0045] Those skilled in the art will appreciate that the present disclosure is susceptible to variations and modifications other than those specifically described. It is to be understood that the disclosure includes all such variations and modifications. The disclosure also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations of any two or more of said steps or features. Thus, each feature of any particular aspect or embodiment of the present disclosure may be applied mutatis mutandis to any other aspect or embodiment of the present disclosure.

[0046] The present disclosure is not to be limited in scope by the specific embodiments described herein, which are intended for the purpose of exemplification only. Functionally equivalent products, compositions and methods are clearly within the scope of the disclosure, as described herein.

[0047] Throughout this specification, unless specifically stated otherwise or the context requires otherwise, reference to a single step, composition of matter, group of steps or group of compositions of matter shall be taken to encompass one and a plurality (z.e. one or more) of those steps, compositions of matter, groups of steps or group of compositions of matter.

[0048] All computer programs, algorithms, patent and scientific literature referred to herein is incorporated in their entirety herein by reference.

[0049] Method of producing RNA by in vitro transcription

[0050] The present inventors have surprisingly shown that the synthesis of RNA molecules by in vitro transcription of a close-ended DNA template, such as a doggybone DNA template, at a relatively neutral pH improves yield of the synthesized RNA molecules. Accordingly in one form, the present disclosure provides a method of producing an RNA molecule by in vitro transcription, said method including the step of:

[0051] (a) providing a close-ended DNA template encoding the RNA molecule; and

[0052] (b) transcribing the RNA molecule from the close-ended DNA template in a reaction mixture having a pH of between about 6 to about 8.

[0053] In a related form, the present disclosure relates to an isolated RNA molecule produced by the aforementioned method.

[0054] As used herein, the term “zn vitro transcription” or “IVT” relates to a process wherein RNA molecules are synthesized from a DNA template in a cell-free system (i.e., in vitro). A DNA template, and more particularly a linearized DNA template (e.g., a linearized plasmid DNA template, a linearized ceDNA template, a linearized dbDNA template), can be used as a template for the generation of RNA transcripts. A DNA template for RNA in vitro transcription may be obtained by cloning of a nucleic acid molecule, in particular a cDNA molecule corresponding to the respective RNA to be transcribed in vitro, and introducing it into an appropriate vector for RNA in vitro transcription. Examples of DNA suitable to be used as templates for IVT of RNA comprise plasmid DNA, cDNA or close-ended DNA. For the aspects provided herein, however, the DNA template is or comprises ceDNA, more particularly dbDNA or even more particularly linearized dbDNA.

[0055] The methodology for IVT of mRNA is well-known in the art. (see, e.g., Losick, R., 1972, IVT, Ann Rev Biochem v.41 409-46; Kamakaka, R. T. and Kraus, W. L. 2001. In vitro Transcription. Current Protocols in Cell Biology. 2: 11.6: 11.6.1-11.6.17; Beckert, B. And Masquida, B., (2010) Synthesis of RNA by In vitro Transcription in RNA in Methods in Molecular Biology v. 703 (Neilson, H. Ed), New York, N.Y. Humana Press, 2010; Brunelle, J.L. and Green, R., 2013, Chapter Five - In vitro transcription from plasmid or PCR-amplified DNA, Methods in Enzymology v. 530, 101-114; all of which are incorporated herein by reference). IVT can be performed using a variety of commercially available kits including, but not limited to, RiboMax Large Scale RNA Production System (Promega), MegaScript Transcription kits (Life Technologies), mMESSAGE Transcription kits (Invitrogen), as well as with commercially available reagents including RNA polymerases (e.g., T7 RNA polymerase) and ribonucleotide triphosphates (rNTPs). As used herein, the term “RNA” is an abbreviation for ribonucleic acid. These nucleic acid molecules usually comprise adenosine-monophosphate, uridine-monophosphate, guanosine-monophosphate and cytidine-monophosphate monomers or analogues or modified versions thereof, which are connected to each other along a so-called backbone. The backbone is formed by phosphodiester bonds between the sugar (i.e., ribose) of a first monomer and a phosphate moiety of a second, adjacent monomer. The specific order of the monomers, i.e. the order of the bases linked to the sugar / phosphate-backbone, is called the RNA-sequence. The term “RNA” may refer to a molecule or to a molecule species selected from the group consisting of long-chain RNA, coding RNA, non-coding RNA, single stranded RNA (ssRNA), double stranded RNA (dsRNA), linear RNA (linRNA), circular RNA (circRNA), messenger RNA (mRNA), RNA oligonucleotides, small interfering RNA (siRNA), small hairpin RNA (shRNA), antisense RNA (asRNA), CRISPR / Cas9 guide RNAs, riboswitches, immuno stimulating RNA (isRNA), ribozymes, aptamers, ribosomal RNA (rRNA), transfer RNA (tRNA), viral RNA (vRNA), retroviral RNA or replicon RNA, small nuclear RNA (snRNA), small nucleolar RNA (snoRNA), microRNA (miRNA), circular RNA (circRNA), and a Piwi-interacting RNA (piRNA).

[0056] Suitably, the RNA molecule referred to herein is an mRNA molecule. In particular examples, the RNA molecule is a non-replicating mRNA molecule or a self-amplifying mRNA molecule. According to some examples, the RNA molecule is a non-replicating mRNA molecule. In alternative examples, the RNA molecule is a self-amplifying mRNA molecule.

[0057] As used herein, the term “DNA” is an abbreviation for deoxyribonucleic acid. The component nucleotides of DNA are usually deoxy-adenosine-monophosphate, deoxy- thymidine-monophosphate, deoxy-guanosine-monophosphate and deoxy-cytidine - monophosphate monomers or analogues thereof, which are composed of a sugar moiety (deoxyribose), a base moiety and a phosphate moiety, and polymerize by a characteristic backbone structure. The backbone structure is, typically, formed by phosphodiester bonds between the sugar moiety of the nucleotide (i.e., deoxyribose) of a first monomer and a phosphate moiety of a second, adjacent monomer. The specific order of the monomers, i.e. the order of the bases linked to the sugar / phosphate-backbone, is called the DNA sequence. DNA may be single stranded or double stranded. In the double stranded form, the nucleotides of the first strand typically hybridize with the nucleotides of the second strand, such as by A / T-base-pairing and G / C -base-pairing. Although double stranded DNA comprises two opposing strands in terms of the 5' to 3' direction of the two single strands present in the double strand, it is common to nevertheless refer to a 5' end and a 3' end of the double stranded DNA, namely if the DNA comprises a coding sequence element that introduces a direction of the transcription into the double stranded DNA (and accordingly also a direction of the translation).

[0058] The term “DNA template” refers to a polynucleotide template for an RNA polymerase. Typically a DNA template includes the sequence for a gene of interest operably linked to an RNA polymerase promoter sequence. In particular examples, the DNA template is a double stranded DNA template.

[0059] A DNA template can be prepared for IVT from a number of sources with appropriate techniques which are well-known in the art (see, e.g., Linpinsel, J.L and Conn, G.L., General protocols for preparation of plasmid DNA template; and Bowman, J.C., Azizi, B., Lenz, T.K., Ray, P., and Williams, L.D. in RNA IVT and RNA purification by denaturing PAGE. In: Recombinant and in vitro RNA synthesis. Methods in Molecular Biology, v. 941 Conn G.L. (ed), New York, N.Y. Humana Press, 2012). Suitably, the DNA template comprises a double stranded DNA molecule. Exemplary DNA templates may include plasmid DNA, a PCR product, ceDNA or the like. Referring to the present method, however, the DNA template is suitably a ceDNA template.

[0060] The DNA template typically comprises a suitable RNA polymerase promoter sequence, such as a T7, T3 or SP6 promoter, for IVT at or towards a 5’ end thereof, which is followed by the desired nucleotide sequence, such as a nucleotide sequence encoding a desired mRNA molecule to be prepared, and a 3 ’ terminator for IVT. The desired nucleotide sequence generally includes, in a 5’ to 3’ direction, a 5’UTR, an open reading frame (ORF) encoding a protein of interest and a 3’ UTR. The desired nucleotide sequence or a part thereof (e.g., the ORF) may be codon optimized.

[0061] For the present aspect, the DNA template suitably is or comprises a close-ended or closed linear DNA template that encodes the RNA molecule of interest.

[0062] The terms “close-ended DNA”, “ceDNA”, “closed linear DNA”, “dumbbell DNA”, “doggybone DNA”, “dbDNA”, “clDNA”, “covalently closed linear DNA” (e.g., a linear double stranded covalently closed DNA molecule) and the like, may be used interchangeably herein and typically refer to a linear DNA molecule (e.g., a double stranded linear DNA molecule) that includes at least one covalently closed end, and more particularly two covalently closed ends, such as single stranded hairpin loops or ends, where base pairing between complementary DNA strands is not present. Such hairpin loops suitably join the ends of complementary DNA strands. The hairpin loops may themselves contain complementary sequences, particularly if the hairpin loops comprise part of a protelomerase target sequence. Because they have complementary internal sequences, such DNA molecules can assume a “dumbbell” shape. It is envisaged, however, that the ceDNA described herein may include additional secondary and tertiary nucleic acid structures, as are known in the art (e.g., one or more stem loop structures).

[0063] The ceDNA may be generated from open or closed double stranded or single stranded DNA using conventional molecular biology techniques. For example, ceDNA can be generated by attaching hairpin DNA linkers to one or both ends of an open double- stranded or single stranded DNA molecule, such as under the action of a ligase (see, e.g., US6,451,563 for dsDNA and WO2019101596 for ssDNA). In other examples, ceDNA may be produced by the enzymatic activity of a recombinase or a protelomerase (see, e.g., W02010086626, W02012017210 and WO2016132129 for exemplary methods of producing ceDNA using a protelomerase). As such, the ceDNA template may comprise at least one processing enzyme target sequence, more particularly a recombinase target sequence or a protelomerase target sequence. Typically, a protelomerase target sequence comprises a palindromic sequence (i.e., a double-stranded DNA sequence having two-fold rotational symmetry, also known as an inverted repeat). Suitable protelomerase target sequences are known in the art and are discussed in EP2,391,731, which is incorporated herein by reference. A suitable protelomerase enzyme for the production of ceDNA may be TelN from the Escherichia coli phage N 15.

[0064] In view of the foregoing, the ceDNA template provided herein is suitably an enzymatically-amplified ceDNA template or vector. To this end, the ceDNA template has suitably been produced by a cell-free process and more particularly a bacterial cell-free process. As such, the ceDNA template is suitably free from any bacterial propagation elements and antibiotic resistance elements or markers. Furthermore, the ceDNA template provided herein is suitably not or does not comprise plasmid DNA.

[0065] According to particular examples, the ceDNA template provided herein is a Doggybone™ DNA (dbDNA) template. dbDNA is a minimal, closed linear DNA vector developed by Touchlight Genetics Ltd. dbDNA, which can be rapidly produced, is plasmid- free and is synthesized through an enzymatic process using a DNA polymerase, Phi29, and the protelomerase enzyme, TelN. Such methods may yield a ceDNA template containing only the encoded sequence of interest, a promoter, a poly A tail and telomeric ends.

[0066] By way of example, the dbDNA template may be produced by an in vitro cell-free process which comprises (a) contacting a DNA template comprising at least one protelomerase target sequence with at least one DNA polymerase (e.g., Phi29) in the presence of one or more primers under conditions promoting amplification of said template; and (b) contacting amplified DNA (e.g., concatemeric DNA) produced in (a) with at least one protelomerase under conditions promoting production of the dbDNA (see, e.g., the method described in WO2010086626).

[0067] The present inventors have surprisingly found that utilising the ceDNA template when included and / or previously stored in a solution (e.g., a storage solution) which is at a relatively neutral pH (e.g., a pH of about 6.0 to about 8.0) prior to its use in an IVT reaction can increase the efficiency of said reaction and improve RNA production.

[0068] Suitably, the close-ended DNA template is formulated in a solution having a buffer. As used herein, the term “buffer” denotes a weak acid or base used to maintain acidity / alkalinity (pH) of a solution near a chosen value after the addition of another acid or base. Hence, the function of a buffer is to prevent or minimise a rapid or significant change in pH when acids or bases are added to the solution.

[0069] Exemplary buffers that may be used herein include trisaminomethane (Tris) buffers (e.g., Tris HC1), phosphate buffers (e.g., PBS), citrate buffers, glutamate buffers, 4-(2- hydroxyethyl)-l -piperazineethanesulfonic acid (HEPES) and the like. In one example, the solution comprises a Tris buffer, such as Tris HC1. For such examples, the Tris buffer may be included in the solution at a concentration of about 8 mM to about 12 mM (e.g., about 8.0, 8.25, 8.5, 8.75, 9.0, 9.25, 9.5, 9.75, 10.0, 10.25, 10.5, 10.75, 11.0, 11.25, 11.5, 11.75, 12.0 mM or any range therein), more particularly about 8.5 mM to about 11.5 mM, even more particularly about 9 mM to about 11 mM or yet even more particularly about 9.5 mM to about 10.5 mM,. In a further example, the solution containing the ceDNA template comprises a Tris buffer at a concentration of about 10 mM.

[0070] In view of the foregoing, the close-ended DNA template is suitably formulated, such as after production and / or linearization thereof, in a solution, such as a storage solution, having a pH in the range of about 6.0 to about 8.0 (e.g., a pH of about 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0 or any range therein), more particularly about 6.2 to about 7.8, even more particularly about 6.5 to about 7.5, yet even more particularly about 6.6 to about 7.4, still even more particularly about 6.7 to about 7.3, yet still even more particularly about 6.8 to about 7.2 or further more particularly about 6.9 to about 7.1. In a further example, the solution containing the ceDNA template has a pH of about 7.0. In other examples, the solution containing the ceDNA template has a pH of about 6.9. For some examples, the solution containing the ceDNA template has a pH of about 7.1.

[0071] The present method may therefore include the initial or earlier step of adjusting the pH of the solution containing the ceDNA template to a pH in the range of about 6.0 to about 8.0, more particularly about 6.2 to about 7.8, even more particularly about 6.5 to about 7.5, yet even more particularly about 6.6 to about 7.4, still even more particularly about 6.7 to about 7.3, yet still even more particularly about 6.8 to about 7.2 or further more particularly about 6.9 to about 7.1. In particular examples, the present method comprises the step of adjusting the pH of the solution containing the ceDNA template to a pH of about 7. According to some examples, the present method comprises the step of adjusting the pH of the solution containing the ceDNA template to a pH of about 6.9. In other examples, the present method comprises the step of adjusting the pH of the solution containing the ceDNA template to a pH of about 7.1. Such pH adjustment may be achieved by the addition of one or more suitable buffers, such as those provided herein (e.g., Tris HC1). It is further envisaged that adjustment of the pH of the solution containing the ceDNA template may be performed, for example, after production of the ceDNA and more particularly prior to linearization thereof if required. Alternatively or additionally, a pH adjustment or buffering step may be performed after linearization of the ceDNA template so that the solution has a pH in the range provided herein. The DNA template, and more particularly the ceDNA template, may be linearized prior to use in the IVT reaction. Accordingly, the present method may include the earlier or initial step of linearizing the DNA template.

[0072] As used herein and in the context of DNA, the term “linearized” refers to a DNA molecule that comprises at least one free end (e.g., a free 5’ end and / or a free 3’ end) and more particularly two free ends. In particular examples, the linearized ceDNA template includes a free 5’ end and a free 3’ end, which are not linked to each other. Accordingly, the linearized ceDNA template may no longer include hairpin loops at a 5’ end and / or a 3’ end thereof. In this regard, the linearized ceDNA template may be considered or referred to as being “deended”. A linearized ceDNA template in the context of the present disclosure may be obtained by a restriction digest of a ceDNA. It is envisaged that the respective free ends of the linearized ceDNA template may be sticky and / or blunt depending on the restriction enzyme utilised.

[0073] In view of the above, the ceDNA template (e.g., the dbDNA template) may be linearized with a suitable restriction enzyme and optionally isolated or purified before it is subjected to IVT. As used herein, the terms “restriction endonuclease” and “restriction enzyme” refer to a class of enzymes that cleave phosphodiester bonds in both strands of a DNA molecule within specific base sequences. Restriction enzymes recognize specific binding sites, referred to as recognition sequences, on a double-stranded DNA molecule. The sites at which said phosphodiester bonds in the DNA are cleaved by said enzymes are referred to as cleavage sites. Exemplary restriction enzymes include EcoRI, Xbal, PvuII, SapI, Ecil, Bpil, Aarl, Alol, Bael, BbvCI, Ppil, PsrI, BsrDI , BtsI, Earl, BmrI, Bsal, BsmBI, Faul, BbsI, BciVI, BfuAI, BspI, BseRI, Ecil, BtgZI, BpuEI, Bsgl, Mmel, CspCI, Bael, BsaMI, Mval269I, PctI, Bse3DI, BseMI, Bst6I, EamllO4I, Ksp632I, Bfil, Bso31I, BspTNI, BspQI, SapI, Eco31I, Esp3I, Bful, Acc36I, Aarl, Eco57I, Eco57MI, Gsul, Alol, Hin4I, Ppil and PsrI. In particular examples, the ceDNA template has been linearized with SapI, BspQI or an isoschizomer thereof. Similarly, the present method may include the earlier or initial step of linearizing the ceDNA template with SapI, BspQI or an isoschizomer thereof.

[0074] Reagents used in IVT typically comprise: the DNA template (e.g., a linearized ceDNA template) comprising a RNA polymerase promoter sequence; ribonucleotide triphosphates (NTPs) for the four bases (adenine, cytosine, guanine and uracil); optionally, a cap analogue (e.g., m7G(5')ppp(5')G(m7G) or other suitable cap analogues known in the art), a DNA- dependent RNA polymerase capable of binding to the promoter sequence within the DNA template; optionally, a ribonuclease (RNase) inhibitor to inactivate any potentially contaminating RNase; optionally, a pyrophosphatase to degrade pyrophosphate (an inhibitor of RNA synthesis); MgCh, which supplies Mg2+ions as a co-factor for the polymerase; optionally a reaction buffer, such as to maintain a suitable pH value, which can also contain antioxidants (e.g. DTT), and / or polyamines, such as spermidine, at optimal or suitable concentrations.

[0075] Suitably, the reaction mixture includes a reaction buffer. According, to certain examples, the reaction buffer is the same as the buffer included in the solution containing the ceDNA template. It is contemplated, however, that the reaction buffer and the buffer of the solution containing the ceDNA template may differ provided they impart suitable buffering to the reaction mixture. The reaction buffer may be that as hereinbefore described for the solution containing the ceDNA template. In some examples, the reaction buffer comprises a Tris buffer (e.g., Tris HC1). For such examples, the Tris buffer may be included in the reaction mixture at a concentration of about 8 mM to about 12 mM (e.g., about 8.0, 8.25, 8.5, 8.75, 9.0, 9.25, 9.5, 9.75, 10.0, 10.25, 10.5, 10.75, 11.0, 11.25, 11.5, 11.75, 12.0 mM or any range therein), more particularly about 8.5 mM to about 11.5 mM, even more particularly about 9 mM to about 11 mM or yet even more particularly about 9.5 mM to about 10.5 mM. In a further example, the reaction buffer comprises a Tris buffer, such as Tris HC1, at a concentration of about 10 mM.

[0076] In view of the foregoing, the reaction mixture suitably has a pH in the range of about 6.0 to about 8.0 (e.g., a pH of about 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0 or any range therein), more particularly about 6.2 to about 7.8, even more particularly about 6.5 to about 7.5, yet even more particularly about 6.6 to about 7.4, still even more particularly about 6.7 to about 7.3, yet still even more particularly about 6.8 to about 7.2 or further more particularly about 6.9 to about 7.1. In a further example, the reaction mixture for IVT has a pH of about 7.0.

[0077] RNA molecules that are produced by IVT, particularly those for use in vaccines and in other therapeutics, are typically capped in order for the mRNA to be translated. Typically, mature mRNA molecules bear a “cap” structure at their 5 ’-termini, which plays an important role in translation and stability. For example, the 5’ cap plays a pivotal role in mRNA metabolism, and is required to varying degrees for processing and maturation of an RNA transcript in the nucleus, transport of mRNA from the nucleus to the cytoplasm, mRNA stability, and efficient translation of the mRNA to protein. It also helps protect the mRNA from exonuclease degradation such that mRNA lacking a 5’ cap is rapidly degraded, is involved in recognition by the translational initiation factor eIF4E and promotes formation of the translation initiation machinery. In one example, the 5’ cap comprises a 7-methyl guanosine (m7G) that is linked via a 5 ’-5 ’-triphosphate bridge to the 5'-end of the first transcribed nucleotide, resulting in a dinucleotide cap of m7GpppN, where N is any nucleoside (e.g. G, C, A or U) and is the first transcribed nucleotide. This is often referred to as capO. Other examples include capl (m7GpppNmpN) which has additional methylation on the 2'0 position of the first nucleotide and cap2 (m7GpppNmpNm) which has additional methylation on the 2'0 position of both the first and second nucleotides.

[0078] The in vitro transcribed mRNA may be further processed, for example by the addition of a poly(A) tail. The poly(A) tail may be included in the ceDNA template, added via PCR, or added post-transcriptionally by enzymatic polyadenylation. In particular examples, the ceDNA template may include a polyadenylation signal sequence. In further examples, the poly(A) tail is introduced by including a poly(dT) stretch at the end of the ceDNA template. In other examples, the poly(A) tail is added following IVT. In some examples, a 3’ poly(A) tail is added following IVT through the addition of ATP in conjunction with a poly(A) polymerase.

[0079] Following IVT, the DNA template may be removed using any technique known to the person skilled in the art. In certain examples, following IVT, the DNA template is removed by treatment with a DNase. In various examples, the DNase is DNAse I.

[0080] The desired in vitro transcribed RNA may then be purified or isolated from the undesired components of the transcription or associated reactions. Techniques for the isolation of RNA transcripts are well known in the art and include phenol / chloroform extraction or precipitation with either alcohol in the presence of monovalent cations or lithium chloride (LiCl). For example, the RNA molecule can be purified by LiCl precipitation, phenol: chloroform extraction followed by ethanol precipitation, precipitation with ether alcohol in the presence of monovalent cations or by using a spin column based method. In other examples, the RNA molecule is purified using tangential flow filtration (TFF), such as diafiltration. For some examples, the purification step comprises diafiltration into a suitable buffer. According to particular examples, the purification step comprises a spin column based method or column chromatography. Following purification, the RNA molecule can be resuspended in, for example, nuclease-free water or a suitable buffer.

[0081] For the purposes of the present disclosure, by “isolated” or “purified” is meant material, such as protein and nucleic acid molecules, that has been removed from its natural state or otherwise been subjected to human manipulation. Isolated material may be substantially or essentially free from components that normally accompany it in its natural state, or may be manipulated so as to be in an artificial state together with components that normally accompany it in its natural state. Isolated material may be in native, chemical synthetic or recombinant form. A chemically synthesized nucleic acid or protein or one synthesized using IVT / translation is considered “isolated”.

[0082] In one example, the RNA product of the method of IVT provided herein is analysed to assess reaction yield and quality. Various methods for analysing mRNA will be apparent to the skilled person. Such methods may include, but are not limited to, mass spectrometry, gel electrophoresis, liquid chromatography, spectrometry (e.g., Nanodrop) or a combination thereof.

[0083] Non-replicating and self-amplifying mRNA

[0084] According to certain examples, the RNA molecule described herein is or comprises a non-replicating mRNA molecule or a self-amplifying mRNA (sa-mRNA) molecule. As such, the ceDNA template suitably includes a nucleotide sequence that encodes a nonreplicating mRNA molecule or a sa-mRNA molecule.

[0085] For some examples, the RNA molecule referred to herein is a non-replicating mRNA molecule. As their name suggests, non-replicating or non-replicative mRNA molecules are non-self-replicating and typically comprise, in order from 5’ to 3’ : a 5 ’cap structure, a 5’- UTR, a nucleotide sequence encoding a protein of interest, a 3’-UTR and a tailing sequence e.g. a polyadenylation signal or poly-A tail). The non-replicating mRNA may further comprise a translation internal ribosome entry site e.g. Kozak consensus sequence or IRES), a chain terminating nucleotide and / or a stem loop. When contacted with a host cell, non- replicating mRNA is suitably unable to make additional copies of the mRNA encoding a protein of interest.

[0086] In other examples, the RNA molecule provided herein is a sa-mRNA molecule. Selfamplifying mRNA (sa-mRNA; also referred to as self-replicating mRNA), in contrast, is able to amplify copies of the mRNA molecule encoding a protein of interest before transcribing the proteins it codes for, in a host cell. Further, the intracellular replication of sa-mRNA is generally transient and produces a double-stranded RNA (dsRNA) intermediate during replication, which can induce interferon-mediated host-defence mechanisms by triggering pattern recognition receptors. This can result in strong antigen- specific immune responses against the encoded protein of interest. Thus, sa-mRNA vector systems may be particularly suited for vaccine development, as they provide high transient transgene expression and inherent adjuvant effects.

[0087] As used herein, the term “self-amplifying mRNA” or “sa-mRNA” refers to a construct based on an RNA virus that has been engineered to allow expression of heterologous mRNA and proteins. Self-amplifying mRNA can also be referred to as a replicon. Selfamplifying mRNA can amplify in host cells leading to expression of the desired gene product in the host cell. The sa-mRNA of the present disclosure suitably comprises one or more features of a mRNA (e.g., a nucleotide sequence encoding a protein of interest), but further comprises nucleotide sequences encoding non- structural proteins (NSPs), which enable the sa-mRNA to direct its self-amplification. Non-structural proteins can include a viral replicase (or viral polymerase), a viral protease, a viral helicase and optionally other non-structural viral proteins. The skilled person will understand that, in one example, self-amplifying mRNA can be based on the genomic RNA of an RNA virus. The viral RNA is typically positive (+)- stranded so that it can be directly translated after delivery to a cell without the need for intervening replication steps (e.g., reverse transcription). Translation of the viral RNA results in the production of non-structural proteins (NSPs) which combine to form a replicase complex (i.e., an RNA-dependent RNA polymerase). The replicase complex is the component of the sa-mRNA which amplifies the mRNA molecule of interest producing both antisense and sense transcripts, resulting in production of multiple daughter mRNA molecules, and subsequently the encoded protein of interest. For example, the sa-mRNA comprises a viral replicase. According to some examples, the sa-mRNA comprises NSPs derived from (or based on) an alphavirus. Exemplary alphaviruses include, but are not limited to, Venezuelan equine encephalitis virus (VEEV; e.g., Trinidad donkey, TC83CR), Semliki Forest virus (SFV), Sindbis virus (SIN), Ross River virus, Western equine encephalitis virus, Eastern equine encephalitis virus, Chikungunya virus, S.A. AR86 virus, Everglades virus, Mucambo virus, Barmah Forest virus, Middelburg virus, Pixuna virus, O'nyong-nyong virus, Getah virus, Sagiyama virus, Bebaru virus, Mayaro virus, Una virus, Aura virus, Whataroa virus, Banbanki virus, Kyzylagach virus, Highlands J virus, Fort Morgan virus, Ndumu virus, and Buggy Creek virus. The term alphavirus may also include chimeric alphaviruses (e.g., as described by Perri et al., (2003) J. Virol. 77(19): 10394-403) that contain genome sequences from more than one alphavirus. In another example, the self-replicating RNA is derived from or based on a virus other than an alphavirus, for example, a positive-stranded RNA virus. Suitable positive- stranded RNA viruses for use in the present disclosure can include, for example, a picomavirus, a flavivirus, a rubivirus, a pestivirus, a hepacivirus, a calicivirus, or a coronavirus.

[0088] Typically, the sa-mRNA also includes a subgenomic (SG) promoter which, when linked to a nucleotide sequence encoding NSPs and / or a protein of interest, drives the expression of the NSPs and / or the protein of interest. As such, the present disclosure provides in certain examples a sa-mRNA molecule comprising a nucleotide sequence encoding a protein of interest (e.g., an antigen) operably linked to a SG promoter.

[0089] As used herein, the term “subgenomic promoter” or “SG promoter” refers to sequences that constitute a functional element required for the production of subgenomic RNA species. A subgenomic promoter is generally necessary to drive the expression of genes using RNA as the template nucleic acid. The subgenomic promoter can be recognized by an RNA-dependent RNA polymerase, which may be a viral RNA replicase. The promoter itself may be a composite of segments derived from more than one source, naturally occurring or synthetic.

[0090] The RNA molecule, inclusive of non-replicating mRNA and sa-mRNA, of the present disclosure typically comprises a nucleotide or nucleic acid sequence encoding a peptide, polypeptide or protein of interest. By “protein” is meant an amino acid polymer. The amino acids may be natural or non-natural amino acids, D- or L- amino acids as are well understood in the art. A “peptide” is generally considered a protein having no more than fifty (50) amino acids. A “polypeptide” is generally considered a protein having more than fifty (50) amino acids.

[0091] The nucleotide sequence of the RNA molecule may encode any protein known to the person skilled in the art, including any naturally or non-naturally occurring or otherwise modified protein. A protein encoded by the RNA molecule described herein may be of any size and may have any secondary structure or activity. In some examples, a protein encoded by the RNA molecule may have a therapeutic effect when expressed in a cell. In one example, the nucleotide sequence of the RNA molecule encodes an immunogen or an antigen (e.g., a pathogenic antigen). For example, the antigen can induce or elicit an immune response in the subject. According to certain examples, the RNA molecule comprises a nucleotide sequence that encodes an antigen, such as those provided herein, expressed by, derived from or otherwise associated with a pathogen (e.g., a virus, a bacteria, a fungi, a protozoa, etc.) and more particularly an infectious pathogen, such as those described below. In one example, the RNA molecule of the present disclosure comprises a nucleotide sequence that encodes an antigen from a virus. According to various examples, the RNA molecule comprises a nucleotide sequence that encodes an antigen from a respiratory virus, for example, an influenza virus, a parainfluenza virus, a rhinovirus, an avian influenza virus, a coronavirus (e.g., a SARS virus like SARS-CoV-2) or a respiratory syncytial virus.

[0092] Pharmaceutical compositions

[0093] The present disclosure provides a pharmaceutical composition comprising an RNA molecule as described herein, such as a non-replicating mRNA molecule or a sa-mRNA molecule, and optionally one or more pharmaceutically acceptable carriers.

[0094] By “pharmaceutically-acceptable carrier, diluent or excipient” is meant a solid or liquid filler, diluent or encapsulating substance that may be safely used in systemic administration. Depending upon the particular route of administration, a variety of carriers, well known in the art may be used. These carriers may be selected from a group including sugars, starches, cellulose and its derivatives, malt, gelatine, talc, calcium sulfate, liposomes and other lipid-based carriers, vegetable oils, synthetic oils, polyols, alginic acid, phosphate buffered solutions, emulsifiers, isotonic saline and salts such as mineral acid salts including hydrochlorides, bromides and sulfates, organic acids such as acetates, propionates and malonates and pyrogen-free water. A useful reference describing pharmaceutically acceptable carriers, diluents and excipients is Remington’s Pharmaceutical Sciences (Mack Publishing Co. N.J. USA, 1991), which is incorporated herein by reference.

[0095] Any safe route of administration may be employed for providing a patient with the composition of the present disclosure. For example, oral, rectal, parenteral, sublingual, buccal, intravenous, intra- articular, intra-muscular, intra-dermal, subcutaneous, inhalational, intraocular, intraperitoneal, intracerebroventricular, transdermal and the like may be employed.

[0096] Dosage forms include tablets, dispersions, suspensions, injections, solutions, syrups, troches, capsules, suppositories, aerosols, transdermal patches and the like. These dosage forms may also include injecting or implanting controlled releasing devices designed specifically for this purpose or other forms of implants modified to act additionally in this fashion. Controlled release of the therapeutic agent may be effected by coating the same, for example, with hydrophobic polymers including acrylic resins, waxes, higher aliphatic alcohols, polylactic and polyglycolic acids and certain cellulose derivatives such as hydroxypropylmethyl cellulose. In addition, the controlled release may be effected by using other polymer matrices, liposomes and / or microspheres.

[0097] The above compositions may be administered in a manner compatible with the dosage formulation, and in such amount as is pharmaceutically-effective. The dose administered to a patient, in the context of the present disclosure, should be sufficient to effect a beneficial response in a patient over an appropriate period of time. The quantity of agent(s) to be administered may depend on the subject to be treated inclusive of the age, sex, weight and general health condition thereof, factors that will depend on the judgement of the practitioner.

[0098] Suitably, the isolated RNA molecule of the pharmaceutical composition is contained in, encapsulated by or otherwise associated with (e.g., bound to, absorbed / adsorbed on) a lipid-based carrier, such as a cationic lipid, a lipid nanoparticle, a liposome, a cochleate, a virosome, an immune- stimulating complex, a microparticle, a microsphere, a nanosphere, a unilamellar vesicle, a multilamellar vesicle, an oil-in-water emulsion, a water-in-oil emulsion, an emulsome, and a polycationic peptide, a cationic nano-emulsion or combinations thereof. The lipid-based carrier suitably comprises any lipid or mixture of lipids capable of forming a lipid bilayer structure. These include one or more phospholipids, sterols inclusive of cholesterol, cholesterol-esters and phytosterols, fatty acids and / or triglycerides. Nonlimiting examples of phospholipids include phosphatidylcholine (PC) (lecithin), phosphatidic acid, phosphatidylethanolamine (PE) (cephalin), phosphatidylglycerol (PG), phosphatidylserine (PS), phosphatidylinositol (PI) and sphingomyelin (SM) or natural or synthetic derivatives thereof, as are known in the art.

[0099] According to certain examples, the pharmaceutical composition is formulated as a lipid nanoparticle (LNP) (i.e., the lipid-based carrier is or comprises a lipid nanoparticle). The term “lipid nanoparticle” refers to lipid-based particles having at least one dimension in the order of nanometers (e.g., 1-1,000 nm) which contain one or more lipids (e.g., neutral lipids, anionic lipids, cationic lipids, steroids, polymer-conjugated lipids). Generally, lipid nanoparticles have a single phospholipid outer layer that encapsulates an inner space thereof, which may or may not be aqueous. In some examples, lipid nanoparticles are included in a formulation that can be used to deliver an active agent or therapeutic agent, such as a nucleic acid (e.g., mRNA) to a target site of interest (e.g., cell, tissue, organ, tumor, and the like). Suitably, the active agent or therapeutic agent, such as a nucleic acid, is substantially encapsulated in the lipid portion of the lipid nanoparticle or an inner space enveloped by some or all of the lipid portion of the lipid nanoparticle, thereby protecting it from enzymatic degradation or other undesirable effects induced by the mechanisms of the host organism or cells (e.g., an adverse immune response).

[0100] Formulation of LNPs to be administered may vary according to the route of administration and formulation (e.g., solution, emulsion, capsule) selected. An appropriate pharmaceutical composition comprising an LNP to be administered can be prepared in a physiologically acceptable carrier. For solutions or emulsions, suitable carriers include, aqueous or alcoholic / aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles can include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's or fixed oils. A variety of appropriate aqueous carriers are known to the skilled artisan, including water, buffered water, buffered saline, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol), dextrose solution and glycine. Intravenous vehicles can include various additives, preservatives, or fluid, nutrient or electrolyte replenishers (See, generally, Remington's Pharmaceutical Science, 16th Edition, Mack, Ed. 1980). The compositions can optionally contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions, such as pH adjusting and buffering agents and toxicity adjusting agents, sodium acetate, sodium chloride, potassium chloride, calcium chloride and sodium lactate. The LNPs can be stored in the liquid stage or can be lyophilized for storage and reconstituted in a suitable carrier prior to use according to art-known lyophilization and reconstitution techniques.

[0101] When the LNP composition is or comprises a vaccine composition or an immunogenic composition then the carrier may be water, typically pyrogen-free water, isotonic saline or buffered (aqueous) solutions, such as phosphate or citrate buffered solutions. For injection of an LNP vaccine composition, water or more particularly a buffer, even more particularly an aqueous buffer, may be used, containing a sodium salt, preferably at least about 50 mM of a sodium salt, preferably at least about 0.01 mM of a calcium salt, and optionally a potassium salt, such as at least about 3 mM of a potassium salt. In an example, the sodium, calcium and, optionally, potassium salts may be present as their chloride, iodide, or bromide form or in the form of their hydroxides, carbonates, hydrogen carbonates or sulfates. Non-limiting examples of sodium salts include NaCl, Nal, NaBr, NaiCCE, NaHCCE, NaiSCU. Non-limiting examples of the optional potassium salts include KC1, KI, KBr, K2CO3, KHCO3, K2SO4. Non-limiting examples of calcium salts include CaCh, Cah, CaBn, CaCCh, CaSCU, Ca(OH)2. Furthermore, organic anions of the aforementioned cations may be contained in the buffer. In certain examples, the buffer suitable for injection purposes, may contain salts selected from sodium chloride (NaCl), calcium chloride (CaCh) and optionally potassium chloride (KC1), wherein further anions may be present additional to the chlorides. In one example, the salts in the injection buffer are present in a concentration of at least about 50 mM sodium chloride (NaCl), at least about 3mM potassium chloride (KCI) and at least about O.OlmM calcium chloride (CaCh). The injection buffer may be hypertonic, isotonic or hypotonic with reference to the specific reference medium.

[0102] In some examples, one or more compatible solid or liquid fillers or diluents or encapsulating compounds may be employed which are suitable for administration to a person. Pharmaceutically acceptable carriers, fillers and diluents will have sufficiently high purity and sufficiently low toxicity to make them suitable for administration to a subject. Some examples of compounds which can be used as pharmaceutically acceptable carriers, fillers or constituents thereof are sugars, such as lactose, glucose, trehalose and sucrose; starches, such as corn starch or potato starch; dextrose; cellulose and its derivatives, such as sodium carboxymethylcellulose, ethylcellulose, cellulose acetate; powdered tragacanth; malt; gelatin; tallow; solid glidants, such as stearic acid, magnesium stearate; calcium sulfate; vegetable oils, such as groundnut oil, cottonseed oil, sesame oil, olive oil, com oil and oil from theobroma; polyols, such as polypropylene glycol, glycerol, sorbitol, mannitol and polyethylene glycol; and alginic acid.

[0103] When the pharmaceutical composition, such as one formulated as an LNP, is a vaccine composition it may further comprise one or more pharmaceutically acceptable adjuvants to enhance the immunostimulatory properties of the pharmaceutical composition. The adjuvant may be any compound, which is suitable to support administration and delivery of the pharmaceutical composition and which may initiate or increase an immune response of the innate immune system, (i.e., a non-specific immune response).

[0104] Such an adjuvant may be selected from any adjuvant known to a skilled person and suitable for the particular nature of the vaccine or immunogenic composition (i.e., for the induction of a suitable immune response in a mammal). In certain examples, the adjuvant may be selected from the group consisting of: MF59® (squalene-water emulsion), TDM, MDP, muramyl dipeptide, pluronics, alum solution, aluminium hydroxide, ADJUMER™ (polyphosphazene); aluminium phosphate gel; glucans from algae; algammulin; aluminium hydroxide gel (alum); highly protein-adsorbing aluminium hydroxide gel; low viscosity aluminium hydroxide gel; AF or SPT (emulsion of squalane (5%), Tween 80 (0.2%), Pluronic L121 (1.25%), phosphate-buffered saline, pH 7.4); AVRIDINE™ (propanediamine); BAY R1005™ ((N-(2-deoxy-2-L-leucylamino-b-D-glucopyranosyl)-N-octadecyl-dodecanoyl- amide hydroacetate); CALCITRIOL™ (l-alpha,25-dihydroxy-vitamin D3); calcium phosphate gel; CAP™ (calcium phosphate nanoparticles); cholera holotoxin, cholera-toxin- Al-protein-A-D-fragment fusion protein, sub-unit B of the cholera toxin; CRL 1005 (block copolymer P1205); cytokine-containing liposomes; DDA (dimethyldioctadecylammonium bromide); DHEA (dehydroepiandrosterone); DMPC (dimyristoylphosphatidylcholine); DMPG (dimyristoylphosphatidylglycerol); DOC / alum complex (deoxycholic acid sodium salt); Freund's complete adjuvant; Freund's incomplete adjuvant; gamma inulin; Gerbu adjuvant (mixture of: i) N-acetylglucosaminyl-(Pl-4)-N-acetylmuramyl-L-alanyl-D- glutamine (GMDP), ii) dimethyldioctadecylammonium chloride (DDA), iii) zinc-L-proline salt complex (ZnPro-8); GM-CSF); GMDP (N-acetylglucosaminyl-(bl-4)-N-acetylmuramyl- L-alanyl-D-isoglutamine); imiquimod (l-(2-methypropyl)-lH-imidazo[4,5-c]quinoline-4- amine); ImmTher™ (N-acetylglucosaminyl-N-acetylmuramyl-L-Ala-D-isoGlu-L- Alaglycerol dipalmitate); DRVs (immunoliposomes prepared from dehydration-rehydration vesicles); interferon-gamma; interleukin-lbeta; interleukin-2; interleukin-7; interleukin- 12; ISCOMS™; ISCOPREP 7.0.3.™; liposomes; LOXORIBINE™ (7-allyl-8-oxoguanosine); LT oral adjuvant (E.coli labile enterotoxin-protoxin); microspheres and microparticles of any composition;; MONTANIDE ISA 51™ (purified incomplete Freund's adjuvant); MONTANIDE ISA 720™ (metabolisable oil adjuvant); MPL™ (3-Q-desacyl-4'- monophosphoryl lipid A); MTP-PE and MTP-PE liposomes ((N-acetyl-L-alanyl-D- isoglutaminyl-L-alanine-2-(l,2-dipalmitoyl-sn-glycero-3-(hydroxyphosphoryloxy))- ethylamide, monosodium salt); MURAMETIDE™ (Nac-Mur-L-Ala-D-Gln-OCH3); MURAPALMITINE™ and D-MURAPALMITINE™ (Nac-Mur-L-Thr-D-isoGIn-sn- glyceroldipalmitoyl); NAGO (neuraminidase-galactose oxidase); nanospheres or nanoparticles of any composition; NISVs (non-ionic surfactant vesicles); PLEURAN™ (P- glucan); PLGA, PGA and PLA (homo- and co-polymers of lactic acid and glycolic acid; microspheres / nanospheres); PLURONIC L121™; PMMA (polymethyl methacrylate); PODDS™ (proteinoid microspheres); polyethylene carbamate derivatives; poly-rA: poly-rU (polyadenylic acid-polyuridylic acid complex); polysorbate 80 (Tween 80); protein cochleates (Avanti Polar Lipids, Inc., Alabaster, AL); STIMULON™ (QS-21); Quil-A (Quil-A saponin); S-28463 (4-amino-otec-dimethyl-2-ethoxymethyl-lH-imidazo[4,5 c]quinoline-l-ethanol); SAF-1™ ("Syntex adjuvant formulation"); Sendai proteoliposomes and Sendai-containing lipid matrices; Span-85 (sorbitan trioleate); Speed (emulsion of Marcol 52, Span 85 and Tween 85); squalene or Robane® (2,6,10,15,19,23-hexamethyltetracosan and 2,6,10,15,19,23-hexamethyl-2,6,10,14,18,22-tetracosahexane); stearyltyrosine

[0105] (octadecyltyrosine hydrochloride); Theramid® (N-acetylglucosaminyl-N-acetylmuramyl-L- Ala-D-isoGlu-L-Ala-dipalmitoxypropylamide); Theronyl-MDP (Termurtide™ or [thrl]- MDP; N-acetylmuramyl-L-threonyl-D-isoglutamine); Ty particles (Ty-VLPs or virus-like particles); Walter-Reed liposomes (liposomes containing lipid A adsorbed on aluminium hydroxide), and lipopeptides, including Pam3Cys, in particular aluminium salts, such as Adju- phos, Alhydrogel, Rehydragel; emulsions, including CFA, SAF, IFA, MF59, Provax, TiterMax, Montanide, Vaxfectin; copolymers, including Optivax (CRL1005), L121, Poloaxmer4010), etc.; liposomes, including Stealth, cochleates, including BIORAL; plant derived adjuvants, including QS21, Quil A, Iscomatrix, ISCOM; adjuvants suitable for costimulation including Tomatine, biopolymers, including PLG, PMM, Inulin; microbe derived adjuvants, including Romurtide, DETOX, MPL, CWS, Mannose, CpG polynucleotide sequences, CpG7909, ligands of human TLR 1-10, ligands of murine TLR 1-13, ISS-1018, IC31, Imidazoquinolines, Ampligen, Ribi529, IMOxine, IRIVs, VLPs, cholera toxin, heat- labile toxin, Pam3Cys, Flagellin, GPI anchor, LNFPIII / Lcwis X, antimicrobial peptides, UC- 1V150, RSV fusion protein, cdiGMP; and adjuvants suitable as antagonists including CGRP neuropeptide. For certain examples, the adjuvant is or comprises a squalene-water emulsion. In particular examples, the adjuvant is or comprises MF-59.

[0106] The concentration of the RNA molecule in the pharmaceutical composition can vary and will be selected based on fluid volumes, viscosities, body weight, type of mRNA molecule (non-replicating vs self-amplifying) and other considerations in accordance with the particular mode of administration. The concentration of the RNA molecule in the pharmaceutical composition will suitably be effective for prevention or treatment of a disease, disorder or condition, either in a single dose or as part of a series of doses. The amount may vary depending upon the health, physical condition, age and taxonomic group of the individual to be treated (e.g. non-human primate, primate, etc.), the capacity of the individual's immune system to react to the encoded antigenic protein or peptide, the condition to be treated and other relevant factors.

[0107] Methods of treatment

[0108] The RNA molecules and pharmaceutical compositions disclosed herein may be for use in therapy, such as in methods of: eliciting an immune response; and / or treating a disease, disorder or condition in a subject.

[0109] Accordingly, in one form the present disclosure provides a method of eliciting an immune response in a subject, said method including of step of administering a therapeutically effective amount of the isolated RNA molecule disclosed herein or the pharmaceutical composition disclosed herein to the subject to thereby elicit the immune response.

[0110] In a related form, the present disclosure relates to the use of the isolated RNA molecule disclosed herein or the pharmaceutical composition disclosed herein in the manufacture of a medicament for eliciting an immune response in a subject.

[0111] With respect to the aspects described herein, the terms “subject”, “patient” and “individual” are used interchangeably and include, but are not limited to, mammals, inclusive of humans, performance animals (such as horses, camels, greyhounds), livestock (such as cows, sheep, horses) and companion animals (such as cats and dogs). Suitably, the subject is a human.

[0112] By “elicit an immune response” is meant generate or stimulate the production or activity of one or more elements of the immune system inclusive of the cellular immune system, humoral immune system (i.e., antibodies) and / or the native immune system. Suitably, the immune response described herein includes one or more elements of the immune system, such as T lymphocytes, B lymphocytes, antibodies, neutrophils, dendritic cells inclusive of plasmacytoid dendritic cells, cytokines and / or chemokines. Non-limiting examples of cytokines include pro-inflammatory cytokines such as TNF-a, IL-2, IL-6, IL-8, IL-17A and IL-1 (e.g., IL-ip). A non-limiting example of a chemokine is the neutrophil chemo -attractant IL-8. In certain examples, the immune response that is elicited by the vaccine compositions described herein is protective.

[0113] As generally used herein, the terms “immunize”, “vaccinate” and “vaccine” refer to methods and / or compositions that elicit a protective immune response against a pathogen, such as an infectious pathogen, whereby subsequent infection or exposure to the pathogen, or a related serotype, subtype, strain or variant, is at least partly prevented or minimized.

[0114] In another form, the present disclosure provides a method of preventing and / or treating a disease, disorder or condition in a subject, said method including of step of administering a therapeutically effective amount of the isolated RNA molecule disclosed herein or the pharmaceutical composition disclosed herein to the subject to thereby prevent and / or treat the disease, disorder or condition.

[0115] In a further related form, the present disclosure relates to the use of the isolated RNA molecule disclosed herein or the pharmaceutical composition disclosed herein in the manufacture of a medicament for preventing and / or treating a disease, disorder or condition in a subject.

[0116] As used herein, “treating”, “treat” or “treatment” refers to a therapeutic intervention that at least partly ameliorates, eliminates or reduces a symptom or pathological sign of a disease, disorder or condition, such as an infectious disease, disorder or condition (e.g., a viral infection), after it has begun to develop. Treatment need not be absolute to be beneficial to the subject. The beneficial effect can be determined using any methods or standards known to the ordinarily skilled artisan.

[0117] As used herein, “preventing”, “prevent” or “prevention” refers to a course of action initiated prior to infection by, or exposure to, a pathogen (e.g., a virus) or molecular components thereof and / or before the onset of a symptom or pathological sign of the disease, disorder or condition, so as to prevent infection and / or reduce the symptom or pathological sign. It is to be understood that such preventing need not be absolute to be beneficial to a subject. A “prophylactic” treatment is a treatment administered to a subject who does not exhibit signs of the disease, disorder or condition, or exhibits only early signs for the purpose of decreasing the risk of developing a symptom or pathological sign of the disease, disorder or condition.

[0118] As used herein, the terms “disease”, “disorder” or “condition” refers to a disruption of or interference with a normal physiological or biological function of a subject, and is not to be limited to any specific condition. Diseases, disorders, and / or conditions which may be treated by the RNA molecules or pharmaceutical compositions of the present disclosure may include, but are not limited to, rare diseases, infectious diseases, cancer and proliferative diseases, genetic diseases, autoimmune diseases, diabetes, neurodegenerative diseases, cardio- and reno-vascular diseases, and metabolic diseases.

[0119] Suitably, the disease, disorder or condition is an infectious disease, disorder or condition (e.g., an infection), that is, one that is at least partly caused or mediated by an infectious agent or pathogen, such as those described herein. More particularly, the disease, disorder or condition can be a viral disease, disorder or condition. As such, in some examples, the disease, disorder or condition is at least partly caused or mediated by a respiratory virus, such an influenza virus, a parainfluenza virus (e.g., parainfluenza virus type 3; PIV3), a rhinovirus, an avian influenza virus, a coronavirus (e.g., a SARS virus, such as SARS-CoV-1 and SARS-CoV-2), a me tapneumo virus (e.g., human metapneumovirus; hMPV) or a respiratory syncytial virus (RSV). More particularly, the disease, disorder or condition can be at least partly caused or mediated by SARS-CoV-2, an influenza A virus, an influenza B virus, RSV, PIV3 or hMPV. According to various examples, the disease, disorder or condition is at least partly caused or mediated by an influenza virus.

[0120] A “therapeutically effective amount” is at least the minimum concentration required to effect a measurable improvement of a particular disease, disorder or condition. A therapeutically effective amount herein may vary according to factors such as the disease state, age, sex, and weight of the patient, and the ability of the RNA molecule of the present disclosure to elicit a desired response, such as an immune response, in the individual. A therapeutically effective amount is also one in which any toxic or detrimental effects of the RNA molecule are outweighed by the therapeutically beneficial effects thereof. The therapeutically effective amount may vary according to the disease, disorder or condition to be treated or factor to be altered and also according to the weight, age, racial background, sex, health and / or physical condition and other factors relevant to the subject being treated. Typically, the therapeutically effective amount will fall within a relatively broad range (e.g., a “dosage” range) that can be determined through routine trial and experimentation by a medical practitioner. Accordingly, this term is not to be construed to limit the disclosure to a specific quantity, such as weight or number of RNA molecules. The therapeutically effective amount can be administered in a single dose or in a dose repeated once or several times over a treatment period.

[0121] The RNA molecule described herein can be delivered as naked RNA (e.g., merely as an aqueous solution of RNA). In other examples, and to enhance entry into cells and subsequent intercellular effects, the RNA molecule is administered in combination with a lipid-based carrier, such as a liposome or lipid nanoparticle, as described herein.

[0122] The RNA molecule or pharmaceutical composition of the present disclosure may be administered to a subject in the form of one or more dosage units, where for example, a tablet or injectable liquid volume may be a single dosage unit. In a multi-dosage form, requiring a series of two or more doses, the RNA molecule or pharmaceutical composition is administered within a pre-defined timespan. Such a timespan may be a week, two weeks, three weeks, four weeks, five weeks, six weeks, seven weeks, eight weeks, nine weeks, ten weeks, eleven weeks up until one year. Actual methods of preparing such dosage forms are known, or will be apparent, to those skilled in this art (see, e.g., Remington: The Science and Practice of Pharmacy, 20th Edition, Philadelphia College of Pharmacy and Science, 2000).

[0123] Antigenic proteins

[0124] It is contemplated that the RNA molecules described herein include a nucleotide sequence encoding an antigenic or immunogenic protein, or a fragment, variant or derivative thereof. The antigenic protein may be a pathogen antigen, a tumour antigen, an allergenic antigen or an autoimmune self-antigen. Such pathogen antigens may be those derived from pathogenic organisms, in particular bacterial, viral, fungal or protozoal pathogenic organisms, which evoke an immunological reaction in a mammalian subject, such as a human. Pathogen antigens may be surface antigens, for example proteins or fragments thereof, located at the surface of, for example, the virus, bacteria, fungus or protozoa.

[0125] Pathogen antigens may include those derived from one or more of: Acinetobacter baumannii, Anaplasma genus, Anaplasma phagocytophilum, Ancylostoma braziliense, Ancylostoma duodenale, Arcanobacterium haemolyticum, Ascaris lumbricoides, Aspergillus genus, Astroviridae family, Babesia genus, Bacillus anthracis, Bacillus cereus, Bartonella henselae, BK virus, Blastocysts hominis, Blastomyces dermatitidis, Bordetella pertussis, Borrelia burgdorferi, Borrelia genus, Borrelia spp, Brucella genus, Brugia malayi, Bunyaviridae family, Burkholderia cepacia and other Burkholderia species, Burkholderia mallei, Burkholderia pseudomallei, Caliciviridae family, Campylobacter genus, Candida albicans, Candida spp, Chlamydia trachomatis, Chlamydophila pneumoniae, Chlamydophila psittaci, QD prion, Clonorchis sinensis, Clostridium botulinum, Clostridium difficile, Clostridium perfringens, Clostridium perfringens, Clostridium spp, Clostridium tetani, Coccidioides spp, coronaviruses, Corynebacterium diphtheriae, Coxiella burnetii, Crimean- Congo hemorrhagic fever virus, Cryptococcus neoformans, Cryptosporidium genus, Cytomegalovirus (CMV), Dengue viruses (DEN-1, DEN-2, DEN-3 and DEN-4), Dientamoeba fragilis, Ebolavirus (EBOV), Echinococcus genus, Ehrlichia chaffeensis, Ehrlichia ewingii, Ehrlichia genus, Entamoeba histolytica, Enterococcus genus, Enterovirus genus, Enteroviruses, mainly Coxsackie A virus and Enterovirus 71 (EV71), Epidermophyton spp, Epstein-Barr Virus (EBV), Escherichia coli O157:H7, 0111 and O104:H4, Fasciola hepatica and Fasciola gigantica, FFI prion, Filarioidea superfamily, Flaviviruses, Francisella tularensis, Fusobacterium genus, Geotrichum candidum, Giardia intestinalis, Gnathostoma spp, GSS prion, Guanarito virus, Haemophilus ducreyi, Haemophilus influenzae, Helicobacter pylori, Henipavirus (Hendra virus, Nipah virus), Hepatitis A Virus, Hepatitis B Virus (HBV), Hepatitis C Virus (HCV), Hepatitis D Virus, Hepatitis E Virus, Herpes simplex virus 1 and 2 (HSV-1 and HSV-2), Histoplasma capsulatum, HIV (Human immunodeficiency virus), Hortaea werneckii, Human bocavirus (HBoV), Human herpesvirus 6 (HHV-6) and Human herpesvirus 7 (HHV-7), Human metapneumo virus (hMPV), Human papillomavirus (HPV), Human parainfluenza viruses (HPIV), Japanese encephalitis virus, JC virus, Junin virus, Kingella kingae, Klebsiella granulomatis, Kuru prion, Lassa virus, Legionella pneumophila, Leishmania genus, Leptospira genus, Listeria monocytogenes, Lymphocytic choriomeningitis virus (LCMV), Machupo virus, Malassezia spp, Marburg virus, Measles virus, Metagonimus yokagawai, Microsporidia phylum, Molluscum contagiosum virus (MCV), Mumps virus, Mycobacterium leprae and Mycobacterium lepromatosis, Mycobacterium tuberculosis, Mycobacterium ulcerans, Mycoplasma pneumoniae, Naegleria fowleri, Necator americanus, Neisseria gonorrhoeae, Neisseria meningitidis, Nocardia asteroides, Nocardia spp, Onchocerca volvulus, Orientia tsutsugamushi, Orthomyxoviridae family (Influenza), Paracoccidioides brasiliensis, Paragonimus spp, Paragonimus westermani, Parvovirus B 19, Pasteurella genus, Plasmodium genus, Pneumocystis jirovecii, Poliovirus, Rabies virus, Respiratory syncytial virus (RSV), Rhinovirus, rhinoviruses, Rickettsia akari, Rickettsia genus, Rickettsia prowazekii, Rickettsia rickettsii, Rickettsia typhi, Rift Valley fever virus, Rotavirus, Rubella virus, Sabia virus, Salmonella genus, Sarcoptes scabiei, SARS coronavirus, Schistosoma genus, Shigella genus, Sin Nombre virus, Hantavirus, Sporothrix schenckii, Staphylococcus genus, Streptococcus genus, Streptococcus agalactiae, Streptococcus pneumoniae, Streptococcus pyogenes, Strongyloides stercoralis, Taenia genus, Taenia solium, Tick-bome encephalitis virus (TBEV), Toxocara canis, Toxocara cati, Toxoplasma gondii, Treponema pallidum, Trichinella spiralis, Trichomonas vaginalis, Trichophyton spp, Trichuris trichiura, Trypanosoma brucei, Trypanosoma cruzi, Ureaplasma urealyticum, Varicella zoster virus (VZV), Varicella zoster virus (VZV), Variola major or Variola minor, vCJD prion, Venezuelan equine encephalitis virus, Vibrio cholerae, West Nile virus, Western equine encephalitis virus, Wuchereria bancrofti, Yellow fever virus, Yersinia enterocolitica, Yersinia pestis, and Yersinia pseudotuberculosis.

[0126] According to certain examples, relevant antigens or immunogenic proteins may be derived from the pathogens selected from: Severe Acute Respiratory Syndrome (SARS), Severe Acute Respiratory Syndrome Coronavirus and Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-1 and SARS-CoV-2), Influenza virus (inclusive of influenza A and B), respiratory syncytial virus (RSV), Herpes simplex virus (HSV), human Papilloma virus (HPV), Human immunodeficiency virus (HIV), Plasmodium, Staphylococcus aureus, Dengue virus, Chlamydia trachomatis, Cytomegalovirus (CMV), Hepatitis B virus (HBV), Mycobacterium tuberculosis, Rabies virus, Hendra virus, Parainfluenza virus (e.g., Parainfluenza virus type 3 or PIV3), human Metapneumo virus (hMPV) and Yellow Fever Virus. More particularly, the antigen may be derived from SARS-CoV-2, an influenza A virus, an influenza B virus, RSV, PIV3, hMPV, Hendra virus or CMV. In some examples, the antigen or immunogenic protein is derived from SARS-CoV-2, an influenza A virus, an influenza B virus, RSV, PIV3 or hMPV.

[0127] According to certain examples, the antigen or immunogenic protein is derived from an influenza virus. In such examples, the mRNA may have a nucleotide sequence encoding at least one antigenic protein derived from hemagglutinin (HA), neuraminidase (NA), nucleoprotein (NP), matrix protein 1 (Ml), matrix protein 2 (M2), non-structural protein 1 (NS1), non-structural protein 2 (NS2), nuclear export protein (NEP), polymerase acidic protein (PA), polymerase basic protein PB 1, PB 1-F2, or polymerase basic protein 2 (PB2) of an influenza virus or a fragment, derivative or variant thereof. For some examples, the nucleotide sequence encodes at least one antigenic protein derived from hemagglutinin (HA) and / or neuraminidase (NA) of an influenza virus or a fragment, derivative or variant thereof. The HA and / or NA may, independently, be derived from an influenza A virus or an influenza B virus or a fragment, variant or derivative (e.g., chimeric versions) of either.

[0128] For other examples, the antigen or immunogenic protein is from a coronavirus, such as a SARS-CoV-1 or SARS-CoV-2. In such instances, the mRNA may have a nucleotide sequence encoding at least one antigenic protein derived from the Spike (S) protein or a fragment, derivative or variant thereof. It will be appreciated by persons skilled in the art that numerous variations and / or modifications may be made to the above-described embodiments, without departing from the broad general scope of the present disclosure. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive.

[0129] ITEMIZED LISTING OF EMBODIMENTS

[0130] 1. A method of producing an RNA molecule by in vitro transcription, said method including the steps of:

[0131] (a) providing a close-ended DNA template encoding the RNA molecule; and

[0132] (b) transcribing the RNA molecule from the close-ended DNA template in a reaction mixture having a pH of between about 6 to about 8.

[0133] 2. The method of claim 1, wherein the reaction mixture has a pH of about 6.5 to about 7.5.

[0134] 3. The method of claim 1 or claim 2, wherein the reaction mixture has a pH of about 7.0.

[0135] 4. The method of any one of the preceding claims, wherein the close-ended DNA template is formulated in a solution having a pH of about 6 to about 8.

[0136] 5. The method of claim 4, wherein the solution has a pH of about 6.5 to about 7.5.

[0137] 6. The method of claim 4 or claim 5, wherein the solution has a pH of about 7.0.

[0138] 7. The method of any one of the preceding claims, wherein the solution comprises a buffer.

[0139] 8. The method of claim 7, wherein the solution comprises a Tris buffer. 9. The method of claim 8, wherein the solution comprises the Tris buffer at a concentration of about 8 mM to about 12 mM.

[0140] 10. The method of claim 8 or claim 9, wherein the solution comprises the Tris buffer at a concentration of about 10 mM.

[0141] 11. The method of any one of the preceding claims, wherein the close-ended DNA template is or comprises doggybone DNA (dbDNA).

[0142] 12. The method of any one of the preceding claims, wherein the close-ended DNA template has been linearized.

[0143] 13. The method of any one of the preceding claims, wherein the method further includes the earlier step of linearizing the close-ended DNA template.

[0144] 14. The method of any one of the preceding claims, wherein the RNA molecule is or comprises a non-replicating mRNA molecule or a self-amplifying mRNA molecule.

[0145] 15. The method of any one of the preceding claims, wherein the RNA molecule encodes an immunogenic protein.

[0146] 16. An isolated RNA molecule produced by the method of any one of claims 1 to 15.

[0147] 17. A pharmaceutical composition comprising the isolated RNA molecule of claim 16 and optionally a pharmaceutically acceptable carrier, diluent or excipient.

[0148] 18. The pharmaceutical composition of claim 17, wherein the isolated RNA molecule is contained in or otherwise associated with a lipid-based carrier.

[0149] 19. The pharmaceutical composition of claim 18, wherein the lipid-based carrier is or comprises a lipid nanoparticle. 20. The isolated RNA molecule of claim 16 or the pharmaceutical composition of any one of claims 17 to 19 for use in: (a) eliciting an immune response; and / or (b) treating a disease, disorder or condition in a subject.

[0150] 21. A method of eliciting an immune response in a subject, said method including of step of administering a therapeutically effective amount of the isolated RNA molecule of claim 16 or claim 20 or the pharmaceutical composition of any one of claims 17 to 20 to the subject to thereby elicit the immune response.

[0151] 22. A method of preventing and / or treating a disease, disorder or condition in a subject, said method including of step of administering a therapeutically effective amount of the isolated RNA molecule of claim 16 or claim 20 or the pharmaceutical composition of any one of claims 17 to 20 to the subject to thereby prevent and / or treat the disease, disorder or condition.

[0152] 23. Use of the isolated RNA molecule of claim 16 or claim 20 or the pharmaceutical composition of any one of claims 17 to 20 in the manufacture of a medicament for eliciting an immune response in a subject.

[0153] 24. Use of the isolated RNA molecule of claim 16 or claim 20 or the pharmaceutical composition of any one of claims 17 to 20 in the manufacture of a medicament for preventing and / or treating a disease, disorder or condition in a subject.

Claims

CLAIMS:

1. A method of producing an RNA molecule by in vitro transcription, said method including the steps of:(a) providing a close-ended DNA template encoding the RNA molecule; and(b) transcribing the RNA molecule from the close-ended DNA template in a reaction mixture having a pH of between about 6 to about 8.

2. The method of claim 1, wherein the reaction mixture has a pH of about 6.5 to about 7.5.

3. The method of claim 1, wherein the reaction mixture has a pH of about 7.0.

4. The method of claim 1, wherein the close-ended DNA template is formulated in a solution having a pH of about 6 to about 8.

5. The method of claim 4, wherein the solution has a pH of about 6.5 to about 7.5.

6. The method of claim 4, wherein the solution has a pH of about 7.0.

7. The method of claim 1, wherein the solution comprises a buffer.

8. The method of claim 7, wherein the solution comprises a Tris buffer.

9. The method of claim 8, wherein the solution comprises the Tris buffer at a concentration of about 8 mM to about 12 mM.

10. The method of claim 8, wherein the solution comprises the Tris buffer at a concentration of about 10 mM.

11. The method of claim 1, wherein the close-ended DNA template is or comprises doggybone DNA (dbDNA).

12. The method of claim 1, wherein the close-ended DNA template has been linearized.

13. The method of claim 1, wherein the method further includes the earlier step of linearizing the close-ended DNA template.

14. The method of claim 1, wherein the RNA molecule is or comprises a non-replicating mRNA molecule or a self-amplifying mRNA molecule.

15. The method of claim 1, wherein the RNA molecule encodes an immunogenic protein.

16. An isolated RNA molecule produced by a method of in vitro transcription, said method including the steps of:(a) providing a close-ended DNA template encoding the RNA molecule; and(b) transcribing the RNA molecule from the close-ended DNA template in a reaction mixture having a pH of between about 6 to about 8..

17. A pharmaceutical composition comprising the isolated RNA molecule of claim 16 and optionally a pharmaceutically acceptable carrier, diluent or excipient.

18. The pharmaceutical composition of claim 17, wherein the isolated RNA molecule is contained in or otherwise associated with a lipid-based carrier.

19. The pharmaceutical composition of claim 18, wherein the lipid-based carrier is or comprises a lipid nanoparticle.

20. The isolated RNA molecule of claim 16 for use in: (a) eliciting an immune response; and / or (b) treating a disease, disorder or condition in a subject.

21. The pharmaceutical composition of claim 17 for use in: (a) eliciting an immune response; and / or (b) treating a disease, disorder or condition in a subject.

22. A method of eliciting an immune response in a subject, said method including of step of administering a therapeutically effective amount of the isolated RNA molecule of claim 16 to the subject to thereby elicit the immune response.

23. A method of eliciting an immune response in a subject, said method including of step of administering a therapeutically effective amount of the pharmaceutical composition of claim 17 to the subject to thereby elicit the immune response.

24. A method of preventing and / or treating a disease, disorder or condition in a subject, said method including of step of administering a therapeutically effective amount of the isolated RNA molecule of claim 16 to the subject to thereby prevent and / or treat the disease, disorder or condition.

25. A method of preventing and / or treating a disease, disorder or condition in a subject, said method including of step of administering a therapeutically effective amount of the pharmaceutical composition of claim 17 to the subject to thereby prevent and / or treat the disease, disorder or condition.

26. Use of the isolated RNA molecule of claim 16 in the manufacture of a medicament for eliciting an immune response in a subject.

27. Use of the pharmaceutical composition of claim 17 in the manufacture of a medicament for eliciting an immune response in a subject.

28. Use of the isolated RNA molecule of claim 16 in the manufacture of a medicament for preventing and / or treating a disease, disorder or condition in a subject.

29. Use of the pharmaceutical composition of claim 17 in the manufacture of a medicament for preventing and / or treating a disease, disorder or condition in a subject.