In vitro transcription method

Abstract

The present disclosure generally relates to methods of producing RNA from a DNA template. More specifically, the methods of the present disclosure include an in vitro transcription method for producing RNA from a closed end DNA (ceDNA) template.

Description

[0001] Cross-reference to related applications

[0002] This application claims priority to U.S. Provisional Patent Application No. 63 / 510,969, filed June 29, 2023, the contents of which are incorporated herein by reference in their entirety. Technical Field

[0003] This disclosure generally relates to methods for generating RNA from a DNA template. More specifically, the methods of this disclosure include an in vitro transcription method for generating RNA from a closed-terminal DNA (ceDNA) template. Background Technology

[0004] In vitro transcription (IVT) is a biochemical process for synthesizing mRNA in a cell-free transcription system, in which RNA polymerase uses DNA as a template to assemble ribonucleotides. The template typically contains an open reading frame (ORF), which is usually, but not limited to, the complete coding sequence of a gene, 5' and 3' regulatory elements, and a poly(A) tail. Therefore, high-quality template DNA is a prerequisite for efficient IVT to generate standard mRNA or synthetic mRNAs with modified nucleotides.

[0005] Plasmid DNA is commonly used in IVT methods to generate target RNA. However, conventional bacterial fermentation techniques for producing plasmid DNA are slow, costly, limited in production capacity, and may produce low-quality DNA due to unstable or complex sequences. Therefore, generating sufficient high-quality and abundant template DNA has become a bottleneck in the mRNA synthesis process.

[0006] Therefore, there is a need for improved methods for generating RNA via IVT in order to address one or more of these limitations. Summary of the Invention

[0007] In vitro transcription (IVT) typically uses plasmid DNA templates. However, the drawbacks associated with using plasmid DNA in such reactions have spurred the exploration of improved DNA templates. Closed-terminal DNA (ceDNA) templates are generated via a cell-free, enzymatic method that avoids the selective pressures often associated with plasmid instability, while also rapidly and reliably producing GMP-compliant DNA. IVT reactions generally require less ceDNA material compared to using plasmid DNA templates. ceDNA templates can also support the generation of complex sequences, such as sequences containing long polyadenylated tails or sequences containing large (>20kb) target genes, thus providing greater flexibility in supporting a range of gene therapies.

[0008] The inventors unexpectedly discovered that certain parameters (especially pH) can play an important role in the yield or efficiency of RNA production by IVT based on closed-end DNA templates.

[0009] In a first aspect, this disclosure provides a method for producing RNA molecules via in vitro transcription, the method comprising the following steps:

[0010] (a) Providing a closed-end DNA template encoding the RNA molecule; and

[0011] (b) Transcribe the RNA molecule from the closed-end DNA template in a reaction mixture with a pH of about 6 to about 8.

[0012] Suitable, the pH of the reaction mixture is from about 6.5 to about 7.5.

[0013] In one example, the pH of the reaction mixture was approximately 7.0.

[0014] In some instances, closed-end DNA templates are prepared in solutions with a pH of approximately 6 to approximately 8.

[0015] In other examples, the pH of the solution is approximately 6.5 to approximately 7.5.

[0016] Based on some examples, the pH of the solution is approximately 7.0.

[0017] Suitable, the solution contains a buffer solution.

[0018] In some instances, the solution contains Tris buffer.

[0019] In other examples, the solution contains Tris buffer at a concentration of approximately 8 mM to approximately 12 mM.

[0020] For some examples, the solution contains approximately 10 mM Tris buffer.

[0021] In some instances, the closed-end DNA template is doggybone DNA (dbDNA) or contains doggybone DNA (dbDNA).

[0022] In some instances, the closed-end DNA template has been linearized.

[0023] Suitablely, the method also includes an early step of linearizing the closed-end DNA template.

[0024] In another instance, the RNA molecule is a non-replicating mRNA molecule or a self-amplifying mRNA molecule, or the RNA molecule includes non-replicating mRNA molecules or self-amplifying mRNA molecules.

[0025] Appropriately, RNA molecules encode immunogenic proteins.

[0026] In a second aspect, this disclosure provides isolated RNA molecules produced by the methods described herein.

[0027] In a third aspect, this disclosure provides pharmaceutical compositions comprising the isolated RNA molecules described herein, and optionally comprising a pharmaceutically acceptable carrier, diluent, or excipient.

[0028] Therefore, isolated RNA molecules are contained in lipid-based carriers or otherwise associated with lipid-based carriers.

[0029] In some instances, the lipid-based carrier is a lipid nanoparticle or contains lipid nanoparticles.

[0030] In other instances, this disclosure provides isolated RNA molecules or pharmaceutical compositions as described herein for use in: (a) inducing an immune response; and / or (b) treating a subject’s disease, symptom, or condition.

[0031] In a fourth aspect, this disclosure provides a method for inducing an immune response in a subject, the method comprising the step of administering to the subject a therapeutically effective amount of the isolated RNA molecule or pharmaceutical composition described herein, thereby inducing an immune response.

[0032] In a fifth aspect, this disclosure provides methods for preventing and / or treating a disease, symptom, or condition in a subject, the methods comprising the step of administering a therapeutically effective amount of the isolated RNA molecule or pharmaceutical composition described herein to the subject, thereby preventing and / or treating the disease, symptom, or condition.

[0033] In a sixth aspect, this disclosure provides the use of the isolated RNA molecules or pharmaceutical compositions described herein in the preparation of medicaments for inducing an immune response in a subject.

[0034] In a seventh aspect, this disclosure provides the use of the isolated RNA molecules or pharmaceutical compositions described herein in the preparation of a medicament for the prevention and / or treatment of a disease, symptom, or condition in a subject. Detailed Implementation

[0035] General techniques and definitions

[0036] Unless otherwise specifically defined, all technical and scientific terms used herein should be understood to have the same meaning as commonly understood by one of ordinary skill in the art, such as in the fields of immunology, molecular biology, immunohistochemistry, biochemistry, genomics, and pharmacology.

[0037] This disclosure was made without inappropriate experiments and, unless otherwise stated, it uses conventional techniques of molecular biology, microbiology, recombinant DNA technology, and immunology. These procedures are documented, for example, in the following: Sambrook, Fritsch, and Maniatis, *Molecular Cloning: A Laboratory Manual*, Cold Spring Harbor Laboratory, New York, 4th edition (2012), all contents of Volumes I, II, and III; *DNA Cloning: A Practical Approach*, Volumes I and II (DN Glover, 2nd edition, 1995), IRL Publishing, Oxford, complete book; *Oligonucleotide Synthesis: A Practical Approach* (edited by MJ Gait, 1984), IRL Publishing, Oxford, complete book, especially Gait's edition, pp. 1-22; Atkinson et al., pp. 35-81; Sproat et al., pp. 83-115; and Wu et al., pp. 135-151; 4. *Nucleic Acid Hybridization: A Practical Approach* (edited by BD Hames and SJ Higgins, 1985), IRL Publishing, Oxford, complete book; *Immobilized Cells and Enzymes: A Practical Approach*. Approach (1986), IRL Publishing, Oxford, complete; Perbal, B., Practical Guide to Molecular Cloning (1984) and Methods in Enzymology (edited by S. Colowick and N. Kaplan, Academic Publishing Ltd., complete series).

[0038] As used herein, unless the context clearly indicates otherwise, the singular forms of “a,” “a kind,” and “the” include the plural forms of these words. For example, referring to “a bacterium” includes multiple such bacteria, and referring to “an antigen” refers to one or more antigens.

[0039] The term “and / or”, for example, “X and / or Y”, should be understood to mean “X and Y” or “X or Y”, and should be understood to provide explicit support for both or either of these meanings.

[0040] Throughout this specification, the word “comprising” or variations thereof such as “including” or “containing” shall be understood to mean that the said element, integer or step is included, or that a group of elements, a group of integers or a group of steps is included, but does not exclude any other element, integer or step, or any other group of elements, a group of integers or a group of steps.

[0041] As used herein, the term "about" means approximately. When the term "about" is used with a numerical range, it defines the range by extending the boundaries to both above and below the listed numerical value. Generally, the term "about" herein defines a numerical value as ±0.1%, 0.5%, 1.0%, 5.0%, or 10% above or below the stated value. Such tolerances and variances are well known to those skilled in the art. Typically, such tolerances and variances do not impair the structure, function, and / or implementation of the compositions and methods described herein.

[0042] Those skilled in the art will understand that variations and modifications may exist in this disclosure beyond the specific description. It should be understood that this disclosure includes all such variations and modifications. This disclosure also includes all steps, features, compositions, and compounds, whether individually or collectively mentioned or indicated in this specification, as well as any combination and all combinations of any two or more of the stated steps or features. Therefore, each feature of any particular aspect or embodiment of this disclosure can be applied, with appropriate modifications, to any other aspect or embodiment of this disclosure.

[0043] This disclosure is not limited to the specific embodiments described herein for illustrative purposes only. Functionally equivalent products, compositions, and methods are clearly within the scope of this disclosure, as described herein.

[0044] Throughout this specification, unless otherwise specifically stated or required by the context, references to a single step, substance composition, group of steps, or group of substance compositions shall be understood to cover one or more (i.e., one or more) of such steps, substance compositions, group of steps, or group of substance compositions.

[0045] All computer programs, algorithms, patents, and scientific literature cited in this article are incorporated herein by reference in their entirety.

[0046] Methods of producing RNA through in vitro transcription

[0047] The inventors of this invention unexpectedly demonstrated that in vitro transcription of RNA molecules using a closed-terminal DNA template (such as a dog bone-shaped DNA template) at a relatively neutral pH increased the yield of synthesized RNA molecules.

[0048] Therefore, in one form, this disclosure provides a method for producing RNA molecules by in vitro transcription, the method comprising the following steps:

[0049] (a) Provides a DNA template for the closed ends of an RNA molecule; and

[0050] (b) In a reaction mixture with a pH of about 6 to about 8, RNA molecules are transcribed from DNA templates with closed ends.

[0051] In relevant forms, this disclosure relates to isolated RNA molecules produced by the methods described above.

[0052] As used herein, the term "in vitro transcription" or "IVT" refers to the process of synthesizing RNA molecules from a DNA template in a cell-free system (i.e., in vitro). A DNA template, more specifically 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 generating RNA transcripts. A DNA template for in vitro transcription of RNA can be obtained by cloning nucleic acid molecules (particularly cDNA molecules corresponding to the individual RNAs to be transcribed in vitro) and introducing them into a suitable vector for in vitro transcription of RNA. Examples of DNA suitable as templates for RNA IVT include plasmid DNA, cDNA, or closed-terminal DNA. However, for the purposes of this document, the DNA template is or contains ceDNA, more specifically dbDNA, or even more specifically linearized dbDNA.

[0053] In vitro transduction (IVT) of mRNA is well known in the art. (See, for example, Losick, R., 1972, IVT, Ann Rev Biochem v.41 409-46; Kamakaka, RT and Kraus, WL, 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: Humana Press, NY, 2010; Brunelle, JL and Green, R., 2013, Chapter 5 - In vitro transcription from plasmid or PCR-amplified DNA, Methods in Enzymology v.530, 101-114; all of the above references are incorporated herein by reference). IVT can be performed using a variety of commercially available kits and reagents, including but not limited to the RiboMax large-scale RNA production system (Promega), the MegaScript transcription kit (Life Technologies), and the mMESSAGE transcription kit (Invitrogen). The commercially available reagents include RNA polymerases (e.g., T7 RNA polymerase) and ribonucleoside triphosphates (rNTPs).

[0054] As used herein, the term "RNA" is an abbreviation for ribonucleic acid. These nucleic acid molecules typically contain adenosine monophosphate monomers, uridine monophosphate monomers, guanosine monophosphate monomers, and cytidine monophosphate monomers, or their analogues or modified versions, linked together along a so-called backbone. The backbone is formed by phosphodiester bonds between the sugar (i.e., ribose) of the first monomer and the phosphate moiety of the second adjacent monomer. The specific sequence of the monomers (i.e., the sequence of bases attached to the sugar / phosphate backbone) is called the RNA sequence. The term "RNA" can refer to molecules or types of molecules selected from the following groups: long 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 RNA, riboswitch, immune-stimulating RNA (isRNA), ribonuclease, aptamer, 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 Piwi-interacting RNA (piRNA).

[0055] Appropriately, the RNA molecule referred to herein is the mRNA molecule. In certain instances, the RNA molecule is either a non-replicating mRNA molecule or a self-amplifying mRNA molecule. According to some instances, the RNA molecule is a non-replicating mRNA molecule. In alternative instances, the RNA molecule is a self-amplifying mRNA molecule.

[0056] As used herein, the term "DNA" is an abbreviation for deoxyribonucleic acid. The constituent nucleotides of DNA are typically deoxyadenosine monophosphate monomers, deoxythymidine monophosphate monomers, deoxyguanosine monophosphate monomers, and deoxycytidine monophosphate monomers, or their analogues. They consist of a sugar moiety (deoxyribose), a base moiety, and a phosphate moiety, and are polymerized through a characteristic backbone structure. This backbone structure is typically formed by phosphodiester bonds between the sugar moiety (i.e., deoxyribose) of the nucleotide of the first monomer and the phosphate moiety of the second adjacent monomer. The specific sequence of the monomers (i.e., the sequence of bases attached to the sugar / phosphate backbone) is called the DNA sequence. DNA can 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 through A / T base pairing and G / C base pairing, respectively. Although double-stranded DNA contains two single strands in opposite directions (i.e., in terms of the 5' to 3' orientation of the two single strands present in the double strand), the 5' and 3' ends of the double-stranded DNA are usually still referred to if the DNA contains coding sequence elements that introduce the transcriptional orientation (and correspondingly the translational orientation) of the double-stranded DNA.

[0057] The term "DNA template" refers to a polynucleotide template used by RNA polymerase. Typically, a DNA template comprises a target gene sequence that can be operatively linked to the RNA polymerase promoter sequence. In specific instances, the DNA template is a double-stranded DNA template.

[0058] DNA templates for IVT can be prepared from a variety of sources using suitable techniques well known in the art (see, for example, Linpinsel, JL and Conn, GL, General protocols for preparation of plasmid DNA template; and Bowman, JC, Azizi, B., Lenz, TK, Ray, P. and Williams, LD, in RNA IVT and RNA purification by denaturing PAGE, see Recombinant and in vitro RNA synthesis, Methods in Molecular Biology, v. 941, Conn GL (ed.), New York: Humana Press, 2012). Suitably, the DNA template comprises a double-stranded DNA molecule. Exemplary DNA templates may include plasmid DNA, PCR products, ceDNA, etc. However, with reference to the methods of the present invention, the DNA template is suitably a ceDNA template.

[0059] DNA templates typically contain a suitable RNA polymerase promoter sequence for IVT, such as the T7, T3, or SP6 promoter, at or near its 5' end, followed by the desired nucleotide sequence, such as the nucleotide sequence encoding the desired mRNA molecule to be prepared, and a 3' terminator for IVT. The desired nucleotide sequence, in the 5' to 3' direction, typically includes a 5' UTR, an open reading frame (ORF) encoding the target protein, and a 3' UTR. The desired nucleotide sequence or a portion thereof (e.g., the ORF) can be codon-optimized.

[0060] For this purpose, the DNA template is preferably a closed-terminal DNA template or a closed linear DNA template encoding the target RNA molecule, or a closed-terminal DNA template or a closed linear DNA template containing the target RNA molecule.

[0061] The terms “closed-end DNA,” “ceDNA,” “closed linear DNA,” “dumbbell-shaped DNA,” “dog-bone-shaped DNA,” “dbDNA,” “clDNA,” and “covalently closed linear DNA” (e.g., a linear double-stranded covalently closed DNA molecule) are used interchangeably herein and generally refer to a linear DNA molecule (e.g., a double-stranded linear DNA molecule) comprising at least one covalently closed end, and more specifically, a linear DNA molecule comprising two covalently closed ends, such as single-stranded hairpin loops or ends, wherein there is no base pairing between complementary DNA strands. Such hairpin loops are suitable for connecting the ends of complementary DNA strands. The hairpin loop itself may contain a complementary sequence, particularly when the hairpin loop contains a portion of a protelomerase target sequence. Due to their complementary internal sequences, such DNA molecules can exhibit a “dumbbell” shape. However, it is conceivable that the ceDNA described herein may include additional secondary and tertiary nucleic acid structures, such as those known in the art (e.g., one or more stem-loop structures).

[0062] ceDNA can 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 adapters to one or both ends of an open double-stranded or single-stranded DNA molecule, such as in the presence of ligases (see, for example, US6,451,563 on dsDNA and WO2019101596 on ssDNA). In other instances, ceDNA can be generated by the enzymatic activity of recombinases or proteloomerases (see, for example, WO2010086626, WO2012017210, and WO2016132129 for example methods of generating ceDNA using proteloomerases). Therefore, the ceDNA template may contain at least one processing enzyme target sequence, more specifically a recombinase target sequence or a proteloomerase target sequence. Typically, the proteloomerase target sequence contains a palindromic sequence (i.e., a double-stranded DNA sequence with double rotational symmetry, also known as an inverted repeat sequence). Suitable protelomeric target sequences are known in the art and discussed in EP2,391,731, the disclosure of which is incorporated herein by reference. A suitable protelomeric enzyme for generating ceDNA may be TelN from E. coli phage N15.

[0063] In view of the above, the ceDNA template provided herein is preferably an enzymatically amplified ceDNA template or vector. Therefore, the ceDNA template is preferably generated by a cell-free method, more specifically by a bacterial cell-free method. Thus, the ceDNA template is preferably free of any bacterial reproductive elements and antibiotic resistance elements or markers. Furthermore, the ceDNA template provided herein is preferably non-plasmid DNA or does not contain plasmid DNA.

[0064] Based on a specific example, the ceDNA template provided in this article is Doggybone. TM DNA (dbDNA) template. dbDNA is a minimal, closed linear DNA vector developed by Touchlight Genetics Ltd. dbDNA is rapidly generated, plasmid-free, and synthesized via an enzymatic process using DNA polymerase Phi29 and protelomerase TelN. This method produces ceDNA templates containing only the target coding sequence, promoter, polyadenylated tail, and telomere ends.

[0065] For example, dbDNA templates can be generated in vitro using a cell-free method comprising: (a) contacting a DNA template containing at least one protelomeric target sequence with at least one DNA polymerase (e.g., Phi29) in the presence of one or more primers under conditions that promote the amplification of the template; and (b) contacting the amplified DNA (e.g., multiply DNA) generated in (a) with at least one protelomeric enzyme under conditions that promote the generation of dbDNA (see, for example, the method described in WO2010086626).

[0066] The inventors of this invention have unexpectedly discovered that having the ceDNA template in and / or pre-storing it in a solution (e.g., a storage solution) with a relatively neutral pH (e.g., pH about 6.0 to about 8.0) before using it in an IVT reaction can improve the efficiency of the reaction and enhance RNA yield.

[0067] Closed-end DNA templates are best prepared in solutions containing buffers. As used herein, the term "buffer" refers to a weak acid or weak base used to maintain the pH of a solution near a selected value upon the addition of another acid or base. Therefore, the function of a buffer is to prevent or minimize rapid or significant pH changes when an acid or base is added to a solution.

[0068] Exemplary buffers that may be used in this document include tris(hydroxymethyl)aminomethane (Tris) buffer (e.g., TrisHCl), phosphate buffer (e.g., PBS), citrate buffer, glutamate buffer, 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid (HEPES), etc. In one example, the solution contains a Tris buffer, such as TrisHCl. In such examples, the Tris buffer may be present in solutions with concentrations of about 8 mM to about 12 mM (e.g., about 8.0 mM, 8.25 mM, 8.5 mM, 8.75 mM, 9.0 mM, 9.25 mM, 9.5 mM, 9.75 mM, 10.0 mM, 10.25 mM, 10.5 mM, 10.75 mM, 11.0 mM, 11.25 mM, 11.5 mM, 11.75 mM, 12.0 mM, or any range thereof), more specifically about 8.5 mM to about 11.5 mM, even more specifically about 9 mM to about 11 mM, or even more specifically about 9.5 mM to about 10.5 mM. In a further example, the solution containing the ceDNA template comprises a Tris buffer concentration of about 10 mM.

[0069] In view of the above, closed-end DNA templates, such as those generated and / or linearized, are suitably formulated in solutions with a pH range of about 6.0 to about 8.0 (e.g., pH values ​​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 thereof), such as storage solutions, more specifically with a pH of about 6.2 to about 7.8, even more specifically about 6.5 to about 7.5, even more specifically about 6.6 to about 7.4, still even more specifically about 6.7 to about 7.3, still even more specifically about 6.8 to about 7.2 or further more specifically about 6.9 to about 7.1. In a further example, the pH of the solution containing the ceDNA template is about 7.0. In other instances, the pH of the solution containing the ceDNA template was approximately 6.9. In some instances, the pH of the solution containing the ceDNA template was approximately 7.1.

[0070] Therefore, the method may include an initial or early step of adjusting the pH of a solution containing the ceDNA template to a pH within the following range: about 6.0 to about 8.0, more specifically about 6.2 to about 7.8, even more specifically about 6.5 to about 7.5, even more specifically about 6.6 to about 7.4, still even more specifically about 6.7 to about 7.3, still even more specifically about 6.8 to about 7.2, or further more specifically about 6.9 to about 7.1. In a particular example, the method includes the step of adjusting the pH of a solution containing the ceDNA template to about 7. According to some examples, the method includes the step of adjusting the pH of a solution containing the ceDNA template to about 6.9. In other examples, the method includes the step of adjusting the pH of a solution containing the ceDNA template to about 7.1. Such pH adjustment can be achieved by adding one or more suitable buffers, such as those provided herein (e.g., Tris HCl). Further, it is envisioned that the pH of the solution containing the ceDNA template can be adjusted, for example, after ceDNA production, or more specifically, before linearization if necessary. Alternatively or additionally, a pH adjustment or buffering step can be performed after ceDNA template linearization to bring the solution to a pH value within the range provided herein.

[0071] The DNA template (more specifically, the ceDNA template) can be linearized before being used in an IVT reaction. Therefore, the method may include an early 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 containing at least one free end (e.g., a free 5' end and / or a free 3' end) and more specifically, two free ends. In a particular instance, a linearized ceDNA template comprises a free 5' end and a free 3' end that are not connected to each other. Therefore, a linearized ceDNA template may no longer include hairpin loops at its 5' and / or 3' ends. In this respect, a linearized ceDNA template may be considered or referred to as "determined." In the context of this disclosure, a linearized ceDNA template can be obtained by restriction enzyme digestion of ceDNA. It is envisioned that the individual free ends of the linearized ceDNA template can be sticky ends and / or blunt ends, depending on the restriction endonuclease used.

[0073] Given the above, ceDNA templates (e.g., dbDNA templates) can be linearized with suitable restriction endonucleases and optionally isolated or purified prior to IVT. As used herein, the terms "restriction endonuclease" and "restriction enzyme" refer to a class of enzymes that cleave the phosphodiester bonds in the two strands of a DNA molecule at a specific base sequence. Restriction endonucleases recognize specific binding sites on double-stranded DNA molecules, called recognition sequences. The sites in the DNA cleaved by the enzyme are called cleavage sites. Exemplary restriction endonucleases include EcoRI, XbaI, PvuII, SapI, EciI, BpiI, AarI, AloI, BaeI, BbvCI, PpiI, PsrI, BsrDI, BtsI, EarI, BmrI, BsaI, BsmBI, FauI, BbsI, BciVI, BfuAI, BspI, BseRI, EciI, BtgZI, BpuEI, BsgI, Mmel, CspI, BaeI, BsaMI, Mva1269I, PctI, Bse3DI, BseMI, Bst6I, Eam1104I, Ksp632I, BfiI, Bso31I, BspTNI, BspQI, SapI, Eco31I, Esp3I, BfuI, Acc36I, AarI, Eco57I, Eco57MI, GsuI, AloI, Hin4I, PpiI, and PsrI. In certain instances, the ceDNA template has been linearized with SapI, BspQI, or their isotopes. Similarly, the method may include an early or initial stage of linearizing the ceDNA template with SapI, BspQI, or their isotopes.

[0074] Reagents used for IVT typically include: a DNA template (e.g., a linearized ceDNA template) containing an RNA polymerase promoter sequence; ribonucleoside triphosphates (NTPs) for four bases (adenine, cytosine, guanine, and uracil); and optionally, a cap analogue (e.g., m...). 7 G(5′)ppp(5′)G(m 7 G) or other suitable cap analogues known in the art, capable of binding to a DNA-dependent RNA polymerase within a promoter sequence of a DNA template; optionally, a ribonuclease (RNase) inhibitor that inactivates any potentially contaminating RNase; optionally, a pyrophosphatase that degrades pyrophosphate (an inhibitor of RNA synthesis); MgCl2, which provides Mg 2+ Ions serve as cofactors for polymerases; optionally, the reaction buffer, such as to maintain a suitable pH, may also contain antioxidants (e.g., DTT) and / or polyamines, such as spermidine, at optimal or appropriate concentrations.

[0075] Suitably, the reaction mixture contains a reaction buffer. In some instances, the reaction buffer is the same as the buffer present in the solution containing the ceDNA template. However, it is acceptable that the reaction buffer and the buffer in the solution containing the ceDNA template may differ, as long as they provide adequate buffering for the reaction mixture. The reaction buffer may be the buffer previously described for the solution containing the ceDNA template. In some instances, the reaction buffer contains Tris buffer (e.g., Tris HCl). For such examples, the Tris buffer may be present in the reaction mixture at a concentration of about 8.0 mM to about 12 mM (e.g., about 8.0 mM, 8.25 mM, 8.5 mM, 8.75 mM, 9.0 mM, 9.25 mM, 9.5 mM, 9.75 mM, 10.0 mM, 10.25 mM, 10.5 mM, 10.75 mM, 11.0 mM, 11.25 mM, 11.5 mM, 11.75 mM, 12.0 mM or any range therebetween), more specifically about 8.5 mM to about 11.5 mM, even more specifically about 9 mM to about 11 mM, or even more specifically about 9.5 mM to about 10.5 mM. In further examples, the reaction buffer comprises a Tris buffer, such as Tris HCl, at a concentration of about 10 mM.

[0076] In view of the above, the pH of the reaction mixture is suitably in the range of about 6.0 to about 8.0 (e.g., pH values ​​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 thereof), more specifically about 6.2 to about 7.8, even more specifically about 6.5 to 7.5, even more specifically about 6.6 to about 7.4, still even more specifically about 6.7 to about 7.3, still even more specifically about 6.8 to about 7.2 or further more specifically about 6.9 to about 7.1. In a further example, the pH of the reaction mixture for IVT is about 7.0.

[0077] RNA molecules produced via IVT (especially those used in vaccines and other therapeutics) are typically capped for mRNA translation. Mature mRNA molecules usually have a "cap" structure at their 5' end, which plays a crucial role in translation and stability. For example, the 5' cap plays a key role in mRNA metabolism and is required to varying degrees for the processing and maturation of RNA transcripts in the cell nucleus, mRNA transport from the nucleus to the cytoplasm, mRNA stability, and efficient mRNA-to-protein translation. It also helps protect mRNA from exonuclease degradation; therefore, mRNA lacking a 5' cap degrades rapidly. Furthermore, it participates in the recognition of the translation initiation factor eIF4E and promotes the formation of translation initiation mechanisms. In one example, the 5' cap contains 7-methylguanosine (m7G), which is linked to the 5' end of the first transcribed nucleotide via a 5'-5' triphosphate bridge, forming the m... 7 The dinucleotide cap of GpppN, where N is any nucleotide (e.g., G, C, A, or U) and is the first nucleotide transcribed. This is often referred to as cap0. Other examples include: cap1 (m 7 GpppNmpN), which has an additional methylation at the 2'O position of the first nucleotide; and cap2 (m 7 GpppNmpNm), which has additional methylation at the 2'O position of both the first and second nucleotides.

[0078] In vitro transcribed mRNA can be further processed, for example, by adding a polyadenylated tail. The polyadenylated tail can be present in the ceDNA template, added via PCR, or added post-transcriptionally via enzymatic polyadenylation. In certain instances, the ceDNA template may include a polyadenylation signal sequence. In further instances, the polyadenylated tail is introduced by including a poly(dT) sequence at the end of the ceDNA template. In other instances, the polyadenylated tail is added after IVT. In some instances, a 3' polyadenylated tail is added after IVT by combining the addition of ATP with poly(A) polymerase.

[0079] Following IVT, the DNA template can be removed using any technique known to those skilled in the art. In some instances, the DNA template is removed after IVT by DNase treatment. In several instances, the DNase is DNase I.

[0080] The desired in vitro transcribed RNA can then be purified or isolated from unwanted components in the transcription or related reactions. Isolation techniques for RNA transcripts are well known in the art and include phenol / chloroform extraction or precipitation with alcohol in the presence of monovalent cations or precipitation with lithium chloride (LiCl). For example, RNA molecules 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 column-based method. In other instances, RNA molecules are purified by tangential flow filtration (TFF), such as percolation. For some instances, the purification step includes percolation into a suitable buffer. Depending on the specific instance, the purification step includes a column-based method or column chromatography. After purification, the RNA molecules can be resuspended in, for example, nuclease-free water or a suitable buffer.

[0081] For the purposes of this disclosure, "isolated" or "purified" means material, such as proteins and nucleic acid molecules, that has been removed from its natural state or otherwise artificially manipulated. Isolated material may be substantially or substantially free of the components normally associated with it in its natural state, or may be manipulated to be in an artificial state together with the components normally associated with it in its natural state. Isolated material may be natural, chemically synthesized, or recombinant. Chemically synthesized nucleic acids or proteins, or nucleic acids or proteins synthesized using IVT / translation, are considered "isolated."

[0082] In one example, the RNA products of the IVT method described herein were analyzed to assess reaction yield and quality. Those skilled in the art will recognize the various methods used for mRNA analysis. These methods may include, but are not limited to, mass spectrometry, gel electrophoresis, liquid chromatography, spectroscopy (e.g., Nanodrop), or combinations thereof.

[0083] Non-replicating mRNA and self-amplifying mRNA

[0084] In some instances, the RNA molecules described herein are non-replicating mRNA molecules or self-amplifying mRNA (sa-mRNA) molecules, or contain non-replicating mRNA molecules or self-amplifying mRNA (sa-mRNA) molecules. Therefore, the ceDNA template is suitable to include a nucleotide sequence encoding a non-replicating mRNA molecule or a sa-mRNA molecule.

[0085] In some instances, the RNA molecules mentioned in this article are non-replicating mRNA molecules. As their name suggests, non-replicating mRNA molecules are not self-replicating and typically consist of, in order from 5' to 3': a 5' cap, a 5'-UTR, a nucleotide sequence encoding the target protein, a 3'-UTR, and a tail sequence (e.g., a polyadenylation signal or polyadenylate tail). Non-replicating mRNAs may also contain translational internal ribosome entry sites (e.g., Kozak concordance sequences or IRES), chain-terminating nucleotides, and / or stem-loops. When in contact with host cells, non-replicating mRNAs are adapted to not produce additional copies of the mRNA encoding the target protein.

[0086] In other instances, the RNA molecules presented herein are sa-mRNA molecules. Self-amplifying mRNAs (sa-mRNAs; also known as self-replicating mRNAs), on the other hand, are capable of first amplifying a copy of the mRNA molecule encoding the target protein in the host cell, and then transcribing the encoded protein. Furthermore, intracellular replication of sa-mRNAs is typically transient, producing a double-stranded RNA (dsRNA) intermediate during replication, which can induce interferon-mediated host defense mechanisms by triggering pattern recognition receptors. This can elicit a strong antigen-specific immune response against the encoded target protein. Therefore, sa-mRNA vector systems are particularly suitable for vaccine development because they provide high transient transgenic expression and an inherent adjuvant effect.

[0087] As used herein, the term "self-amplifying mRNA" or "sa-mRNA" refers to a construct based on an RNA virus engineered to allow the expression of heterologous mRNA and proteins. Self-amplifying mRNA may also be referred to as a replicon. Self-amplifying mRNA can amplify in a host cell, thereby expressing the desired gene product in the host cell. The sa-mRNA of this disclosure suitably includes one or more features of the mRNA (e.g., a nucleotide sequence encoding a target protein), but also includes nucleotide sequences encoding non-structural proteins (NSPs) that enable the sa-mRNA to direct its self-amplification. Non-structural proteins may include viral replicase (or viral polymerase), viral protease, viral helicase, and optionally other non-structural viral proteins. In one instance, those skilled in the art will understand that self-amplifying mRNA may be based on the genomic RNA of an RNA virus. Viral RNA is typically positive (+) strand, so it can be translated directly after delivery to a cell without intermediate replication steps (e.g., reverse transcription). Translation of viral RNA results in the production of non-structural proteins (NSPs), which bind to form a replicase complex (i.e., RNA-dependent RNA polymerase). The replicase complex is a component of sa-mRNA. It amplifies the target mRNA molecule to produce antisense and sense transcripts, thereby generating multiple daughter mRNA molecules, which subsequently produce the target protein encoded. For example, sa-mRNA contains viral replicase.

[0088] According to some examples, sa-mRNA contains NSP derived from (or based on) alphaviruses. Exemplary A viruses include, but are not limited to, Venezuelan equine encephalitis virus (VEEV; e.g., Trinidadian donkey virus, TC83CR), Semliki Forest virus (SFV), Sindbis virus (SIN), Ross River virus, Western equine encephalitis virus, Eastern equine encephalitis virus, Chikungunya virus, SA 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, and Whataroa virus. The term alphavirus can also include chimeric alphaviruses containing genomic sequences from more than one alphavirus (e.g., as described by Perri et al. (2003) J. Virol. 77(19): 10394-403). In another instance, self-replicating RNA is derived from or based on a non-alphavirus, such as a positive-sense RNA virus. Positive-sense RNA viruses suitable for use in this disclosure can include, for example, pituitary viruses, flaviviruses, rubella viruses, plague viruses, hepatitis viruses, caliciviruses, or coronaviruses.

[0089] Typically, sa-mRNA also includes a subgenomic (SG) promoter that drives the expression of NSP and / or the target protein when it is linked to a nucleotide sequence encoding an NSP and / or the target protein. Therefore, this disclosure provides, in some instances, sa-mRNA molecules containing a nucleotide sequence encoding a target protein (e.g., an antigen) operatively linked to an SG promoter.

[0090] As used herein, the term "subgenomic promoter" or "SG promoter" refers to a sequence that constitutes the functional element required to generate subgenomic RNA species. Subgenomic promoters are typically required to drive gene expression that uses RNA as a nucleic acid template. Subgenomic promoters can be recognized by RNA-dependent RNA polymerases, which can be viral RNA replicases. The promoter itself can be a combination of fragments derived from more than one source (natural or synthetic).

[0091] RNA molecules (including non-replicating mRNA and sa-mRNA) of this disclosure typically contain a nucleotide or nucleic acid sequence encoding a target peptide, target polypeptide, or target protein. Here, “protein” refers to a polymer of amino acids. Amino acids can be natural or non-natural, D-amino acids or L-amino acids, as is well known in the art. A “peptide” is generally considered to be a protein having no more than fifty (50) amino acids. A “polypeptide” is generally considered to be a protein having more than fifty (50) amino acids.

[0092] The nucleotide sequence of an RNA molecule can encode any protein known to those skilled in the art, including any naturally occurring or non-naturally occurring protein or a protein otherwise modified. Proteins encoded by the RNA molecules described herein can be of any size and can have any secondary structure or activity. In some instances, proteins encoded by RNA molecules can have therapeutic effects when expressed in cells. In one instance, the nucleotide sequence of an RNA molecule encodes an immunogen or antigen (e.g., a pathogen antigen). For example, an antigen can induce or elicit an immune response in a subject. According to certain instances, the RNA molecule contains a nucleotide sequence encoding an antigen (such as the antigens provided herein) expressed, derived from, or otherwise associated with a pathogen (e.g., a virus, bacteria, fungus, protozoa, etc.), more specifically infectious pathogens, such as those described below. In one instance, the RNA molecule of this disclosure contains a nucleotide sequence encoding an antigen derived from a virus. According to various instances, the RNA molecule contains a nucleotide sequence encoding an antigen derived from a respiratory virus, such as influenza virus, parainfluenza virus, rhinovirus, avian influenza virus, coronavirus (e.g., SARS virus, such as SARS-CoV-2), or respiratory syncytial virus.

[0093] Pharmaceutical Composition

[0094] This disclosure provides pharmaceutical compositions comprising RNA molecules described herein (such as non-replicating mRNA molecules or sa-mRNA molecules), and optionally comprising one or more pharmaceutically acceptable carriers.

[0095] "Pharmaceutically acceptable carriers, diluents, or excipients" refers to solid or liquid fillers, diluents, or encapsulating substances that are safe for systemic administration. Depending on the specific route of administration, a variety of carriers known in the art may be used. These carriers may be selected from the group consisting of: sugars, starches, cellulose and their derivatives, malt, gelatin, talc, calcium sulfate, liposomes and other lipid-based carriers, vegetable oils, synthetic oils, polyols, alginic acid, phosphate buffers, emulsifiers, isotonic saline and salts (such as inorganic 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., NJ, USA, 1991), the contents of which are incorporated herein by reference.

[0096] The compositions disclosed herein can be administered to patients via any safe route of administration. For example, they can be administered orally, rectally, parenterally, sublingually, sublingually, intravenously, intra-articularly, intramuscularly, intradermally, subcutaneously, by inhalation, intraocularly, intraperitoneally, intravenously, or percutaneously.

[0097] Dosage forms include tablets, dispersants, suspensions, injections, solutions, syrups, lozenges, capsules, suppositories, aerosols, transdermal patches, etc. These dosage forms may also include injectable or implantable controlled-release devices specifically designed for this purpose, or other forms of modified implants that additionally function in this manner. Controlled release of therapeutic agents can be achieved by coating them, for example, using hydrophobic polymers, including acrylic resins, waxes, higher fatty alcohols, polylactic acid and polyglycolic acid, and certain cellulose derivatives (such as hydroxypropyl methylcellulose). Alternatively, controlled release can be achieved using other polymer matrices, liposomes, and / or microspheres.

[0098] The above compositions can be administered in a dosage form compatible manner and in a pharmaceutically effective amount. In the context of this disclosure, the dose administered to the patient should be sufficient to produce a beneficial response in the patient within an appropriate timeframe. The amount of one or more agents to be administered may depend on the subject being treated, including factors such as their age, sex, weight, and overall health condition, which will depend on the physician's judgment.

[0099] Suitablely, the isolated RNA molecules in the pharmaceutical composition are contained in, encapsulated in, or otherwise associated (e.g., bound, absorbed / adsorbed) with a lipid-based carrier (such as cationic lipids, lipid nanoparticles, liposomes, spirochetes, virions, immunostimulatory complexes, microparticles, microspheres, nanospheres, monolayer vesicles, multilayer vesicles, oil-in-water emulsions, water-in-oil emulsions, emulsions, polycationic peptides, cationic nanoemulsions, or combinations thereof).

[0100] Lipid-based carriers are suitable to comprise any lipid or mixture of lipids capable of forming a lipid bilayer. These include one or more phospholipids, sterols (including cholesterol, cholesterol esters, and phytosterols), fatty acids, and / or triglycerides. Non-limiting examples of phospholipids include phosphatidylcholine (PC) (lecithin), phosphatidic acid, phosphatidylethanolamine (PE) (cephalin), phosphatidylglycerol (PG), phosphatidylserine (PS), phosphatidylinositol (PI), and sphingomyelin (SM), or their natural or synthetic derivatives, as known in the art.

[0101] In some instances, pharmaceutical compositions are formulated as lipid nanoparticles (LNPs) (i.e., the lipid-based carrier is a lipid nanoparticle or contains lipid nanoparticles). The term "lipid nanoparticle" refers to lipid-based particles having at least one dimension at the nanometer scale (e.g., 1-1000 nm) containing one or more lipids (e.g., neutral lipids, anionic lipids, cationic lipids, steroids, polymer-conjugated lipids). Typically, lipid nanoparticles have a monolayer of phospholipid encapsulating their internal space, which may or may not be aqueous. In some instances, lipid nanoparticles are present in formulations that can be used to deliver active agents or therapeutic agents (such as nucleic acids, e.g., mRNA) to target sites (e.g., cells, tissues, organs, tumors, etc.). Suitably, the active agent or therapeutic agent (such as nucleic acids) is substantially encapsulated within the lipid portion of the lipid nanoparticle, or encapsulated within an internal space wholly or partially surrounded by the lipid portion of the lipid nanoparticle, thereby protecting the active agent or therapeutic agent from enzymatic degradation or other adverse effects caused by mechanisms of the host organism or cells (e.g., adverse immune responses).

[0102] The formulation of the LNP to be administered may vary depending on the route of administration and the chosen formulation (e.g., solution, emulsion, capsule). Suitable pharmaceutical compositions containing LNPs to be administered can be prepared in physiologically acceptable carriers. For solutions or emulsions, suitable carriers include aqueous solutions or alcohol / aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral carriers may include sodium chloride solution, Ringer's dextran, dextran and sodium chloride, lactated Ringer's solution, or non-volatile oils. Various suitable aqueous carriers are known to those skilled in the art, including water, buffered water, buffered saline, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol), dextran solution, and glycine. Intravenous carriers may include various additives, preservatives, or fluids, nutrients, or electrolyte supplements (see generally Remington's Pharmaceutical Science, 16th edition, Mack ed., 1980). These compositions may optionally contain pharmaceutically acceptable excipients that approximate physiological conditions, such as pH adjusters and buffers, as well as toxicity modifiers, sodium acetate, sodium chloride, potassium chloride, calcium chloride, and sodium lactate. LNPs may be stored in a liquid state or lyophilized and reconstituted in a suitable carrier prior to use using lyophilization and reconstitution techniques known in the art.

[0103] When the LNP composition is or contains a vaccine composition or an immunogenic composition, the carrier can be water (typically pyrogen-free water), isotonic saline, or a buffered (aqueous) solution (such as a phosphate buffer or a citrate buffer). For injection of the LNP vaccine composition, water, or more specifically a buffer solution, or even more specifically an aqueous buffer solution, can be used, containing: a sodium salt, preferably at least about 50 mM; a calcium salt, preferably at least about 0.01 mM; and optionally a potassium salt, such as at least about 3 mM. In one example, the sodium, calcium, and optionally potassium salts may be present in the form of their chloride, iodide, or bromide, or in the form of their hydroxide, carbonate, bicarbonate, or sulfate. Non-limiting examples of sodium salts include NaCl, NaI, NaBr, Na₂CO₃, NaHCO₃, and Na₂SO₄. Non-limiting examples of optional potassium salts include KCl, KI, KBr, K₂CO₃, KHCO₃, and K₂SO₄. Non-limiting examples of calcium salts include CaCl2, CaI2, CaBr2, CaCO3, CaSO4, and Ca(OH)2. Furthermore, organic anions of the above cations may be included in the buffer solution. In some cases, buffer solutions suitable for injection purposes may contain salts selected from sodium chloride (NaCl), calcium chloride (CaCl2), and optionally potassium chloride (KCl), wherein anions other than chlorides may be present. In one example, the salts in the injection buffer are present at a concentration of at least about 50 mM sodium chloride (NaCl), at least about 3 mM potassium chloride (KCl), and at least about 0.01 mM calcium chloride (CaCl2). The injection buffer may be hypertonic, isotonic, or hypotonic relative to a particular reference medium.

[0104] In some instances, one or more compatible solid or liquid fillers, diluents, or encapsulating compounds suitable for human administration may be used. Pharmaceutically acceptable carriers, fillers, and diluents will have sufficiently high purity and sufficiently low toxicity to be suitable for administration to subjects. Some examples of compounds that may be used as pharmaceutically acceptable carriers, fillers, or components 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 carboxymethyl cellulose, ethyl cellulose, and cellulose acetate; powdered tragacanth; malt; gelatin; animal fats; solid glidants, such as stearic acid and magnesium stearate; calcium sulfate; vegetable oils, such as peanut oil, cottonseed oil, sesame oil, olive oil, corn oil, and cocoa butter; polyols, such as polypropylene glycol, glycerin, sorbitol, mannitol, and polyethylene glycol; and alginic acid.

[0105] When the pharmaceutical composition (such as a composition formulated as 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 suitable for supporting the administration and delivery of the pharmaceutical composition and capable of initiating or enhancing an immune response of the innate immune system (i.e., a nonspecific immune response).

[0106] Such adjuvants may be selected from any adjuvant known to the art and suitable for the specific properties of the vaccine composition or immunogenic composition (i.e., for inducing an appropriate immune response in mammals). In some instances, adjuvants may be selected from the group consisting of: MF59® (squalene-water emulsion), TDM, MDP, muramyl dipeptide, pluronics, alum solution, aluminum hydroxide, ADJUMER™ (polyphosphazene); aluminum phosphate gel; dextran from algae; algammulin; aluminum hydroxide gel (alum); aluminum hydroxide gel with high protein adsorption; low viscosity aluminum hydroxide gel; AF or SPT (emulsion of squalane (5%), Tween 80 (0.2%), pluronics L121 (1.25%), and phosphate-buffered saline (pH 7.4)); AVRIDINE™ (propylene diamine); BAY R1005™ ((N-(2-deoxy-2-L-leucylamino-bD-glucopyranosyl)-N-octadecyl-dodecanoyl-amide hydroacetate)); CALCITRIOL™ (l-α,25-dihydroxyvitamin D3); calcium phosphate gel; CAP™ (calcium phosphate nanoparticles); cholera toxin, cholera toxin-Al-protein-AD-fragment fusion protein, cholera toxin B subunit; CRL 1005 (block copolymer P1205); liposomes containing cytokines; DDA (dimethyl dioctadecyl ammonium bromide); DHEA (dehydroepiandrosterone); DMPC (dimyristoyl phosphatidylcholine); DMPG (dimyristoyl phosphatidylglycerol); DOC / alum complex (sodium deoxycholate); Freund's complete adjuvant; Freund's incomplete adjuvant; gamma inulin; Gerbu adjuvant (a mixture of the following: i) N-acetylglucosamine-(bl-4)-N-acetylmurayl-L-propionyl-D-glutamine (GMDP), ii) dimethyl dioctadecyl ammonium chloride (DDA), iii) zinc-L-proline complex (ZnPro-8); GM-CSF); GMDP (N-acetylglucosamine-(bl-4)-N-acetylmurayl-L-propionyl-D-isoglutamine) Amide); Imiquimod (l-(2-methylpropyl)-1H-imidazo[4,5-c]quinoline-4-amine); ImmTher™ (N-acetylglucosamine-N-acetylmurayl-L-Ala-D-isoglut-L-Ala-dipalmitate); DRV (immunoliposomes prepared from dehydrated-rehydrated vesicles); Interferon-γ; Interleukin-1β; Interleukin-2; Interleukin-7; Interleukin-12; ISCMS™;ISCOPREP 7.0.3™; Liposomes; LOXORIBINE™ (7-allyl-8-oxoguanosine); LT oral adjuvant (E. coli unstable enterotoxin-protoxin); Microspheres and microparticles of any composition; MONTANIDE ISA 51™ (purified incomplete Freund's adjuvant); MONTANIDE ISA 720™ (Metabolizable Oil Adjuvant); MPL™ (3-Q-Deacylated-4'-Monophosphoryllipid A); MTP-PE and MTP-PE Liposomes ((N-acetyl-L-alanyl-D-isoglutamyl-L-alanine-2-(1,2-dipalmitoyl-sn-glycerol-3-(hydroxyphosphoryloxy))-acetamide, monosodium salt); MURAMETIDE™ (Nac-Mur-L-Ala-D-Gln-OCH3); MURAPALMITINE™ and D-MURAPALMITINE™ (Nac-Mur-L-Thr-D-isoGln-sn-glycerol-dipalmitoyl); NAGO (Neuraminidase-galactose oxidase); Nanospheres or nanoparticles of any composition; NISV (Nonionic Surfactant Vesicles); PLEURAN™ (β-glucan); PLGA, PGA, and PLA (Homopolymers and copolymers of lactic and glycolic acids; Microspheres / nanospheres); PLURONIC L121™; PMMA (polymethyl methacrylate); PODDS™ (protein-like microspheres); polycarbamate derivatives; poly-rA:poly-rU (polyadenylate-polyuridine complex); polysorbate 80 (Tween 80); protein spirochetes (Avanti Polar Lipids, Inc., Alabaster, AL); STIMULON™ (QS-21); Quil-A (Quil-A saponins); S-28463 (4-amino-otec-dimethyl-2-ethoxymethyl-1H-imidazo[4,5c]quinoline-l-ethanol); SAF-1™ (“Syntex adjuvant formulation”); Sendai protein liposomes and lipid matrices containing Sendai virus; Span-85 (sorbitol trioleate); Spector (Marcol 52, Span Emulsions of 85 and Tween 85); squalene or Robane® (2,6,10,15,19,23-hexamethyltetracosahexane and 2,6,10,15,19,23-hexamethyl-2,6,10,14,18,22-tetracosahexane); stearoyl tyrosine (octadecyl tyrosine hydrochloride);Theramid® (N-acetylglucosamine-N-acetylmurayl-L-Ala-D-isoglutamate-L-Ala-dipalmitoylpropionamide); Theronyl-MDP (Termurtide™ or [thrl]-MDP; N-acetylmurayl-L-threonyl-D-isoglutamine); Ty particles (Ty-VLP or virus-like particles); Walter-Reed liposomes (liposomes containing lipid A adsorbed onto aluminum hydroxide), and lipopeptides, including Pam3Cys, particularly aluminum salts such as Adju-phos, Alhydrogel, and Rehydragel; emulsions, including CFA, SAF, IFA, MF59, Provax, TiterMax, Montanide, and Vaxfectin; copolymers, including Optivax (CRL1005), L121, and Poloaxmer4010; liposomes, including Stealth; spirochetes, including BIORAL; and plant-derived adjuvants, including QS21 and Quil. A. Iscomatrix, ISCOM; suitable adjuvants for co-stimulation, including lycopene; biopolymers, including PLG, PMM, inulin; adjuvants of microbial origin, including Romurtide, DETOX, MPL, CWS, mannose, CpG polynucleotide sequence, CpG7909, human TLR 1-10 ligands, mouse TLR 1-13 ligands, ISS-1018, IC31, imidazoquinolines, Ampligen, Ribi529, IMOxine, IRIV, VLP, cholera toxin, heat-labile toxin, Pam3Cys, flagellin, GPI anchoring protein, LNFPIII / Lewis X, antimicrobial peptides, UC-1V150, RSV fusion protein, cdiGMP; and adjuvants suitable as antagonists, including CGRP neuropeptide. In some instances, the adjuvant is a squalene emulsion or an emulsion containing squalene. In a specific instance, the adjuvant is MF-59 or an emulsion containing MF-59.

[0107] The concentration of RNA molecules in a pharmaceutical composition can vary and will be selected based on fluid volume, viscosity, body weight, type of mRNA molecules (non-replicating vs. self-amplifying), and other considerations according to a specific route of administration. Whether in a single dose or as part of a series of doses, the concentration of RNA molecules in the pharmaceutical composition is suitable for the prevention or treatment of disease, symptom, or condition. The amount used may vary depending on the individual's health status, physical condition, age, and taxonomy (e.g., non-human primates, primates, etc.), the individual's immune system's responsiveness to the encoded antigenic protein or peptide, the condition being treated, and other relevant factors.

[0108] Treatment

[0109] The RNA molecules and pharmaceutical compositions disclosed herein can be used for treatment, such as in methods of inducing an immune response; and / or in methods of treating a subject’s disease, symptom, or condition.

[0110] Therefore, in one form, this disclosure provides a method for inducing an immune response in a subject, the method comprising the step of administering to the subject a therapeutically effective amount of the isolated RNA molecule disclosed herein or the pharmaceutical composition disclosed herein, thereby inducing an immune response.

[0111] In related forms, this disclosure relates to the use of the isolated RNA molecules or pharmaceutical compositions disclosed herein in the preparation of a medicament for inducing an immune response in a subject.

[0112] In relation to the various aspects described herein, the terms “subject,” “patient,” and “individual” are used interchangeably, including but not limited to mammals, including humans, performing animals (such as horses, camels, greyhounds), livestock (such as cattle, sheep, and horses), and companion animals (such as cats and dogs). Fittingly, a subject is a human being.

[0113] "Initiating an immune response" means causing or stimulating the production or activity of one or more elements of the immune system, including the cellular immune system, the humoral immune system (i.e., antibodies), and / or the innate 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 (including plasmacytoid dendritic cells), cytokines, and / or chemokines. Non-limiting examples of cytokines include pro-inflammatory cytokines such as TNF-α, IL-2, IL-6, IL-8, IL-17A, and IL-1 (e.g., IL-1β). A non-limiting example of a chemokine is the neutrophil chemokine IL-8. In some instances, the immune response induced by the vaccine composition described herein is protective.

[0114] As used herein, the terms “immunization,” “vaccination,” and “vaccine” refer to methods and / or compositions that elicit a protective immune response against a pathogen, such as an infectious pathogen, thereby at least partially preventing or minimizing subsequent infection or exposure to that pathogen or an associated serotype, subtype, pathogenic strain, or variant.

[0115] In another form, this disclosure provides a method for preventing and / or treating a disease, symptom, or condition in a subject, the method comprising the step of administering to the subject a therapeutically effective amount of the isolated RNA molecule disclosed herein or the pharmaceutical composition disclosed herein, thereby preventing and / or treating the disease, symptom, or condition.

[0116] In a further related form, this disclosure relates to the use of the isolated RNA molecules or pharmaceutical compositions disclosed herein in the preparation of a medicament for the prevention and / or treatment of a disease, symptom, or condition in a subject.

[0117] As used herein, “treatment,” “curing,” or “management” means a therapeutic intervention that at least partially improves, eliminates, or alleviates the symptoms or pathological signs of a disease, symptom, or condition (such as an infectious disease, symptom, or condition (e.g., a viral infection)) after it has begun to develop. Treatment need not be absolutely perfect to be beneficial to the subject. Such beneficial effects can be determined using any method or standard known to a person skilled in the art.

[0118] As used herein, “prevention,” “avoidance,” or “treatment” refers to a process of action initiated before infection or exposure to a pathogen (e.g., a virus) or its molecular components and / or before the onset of symptoms or pathological signs of a disease, condition, or illness, in order to prevent infection and / or alleviate symptoms or pathological signs. It should be understood that such prevention need not be absolutely perfect to be beneficial to the subject. “Prophylactic” treatment refers to treatment administered to subjects who do not exhibit signs of a disease, condition, or illness, or who exhibit only early signs, with the aim of reducing the risk of the development of symptoms or pathological signs of a disease, condition, or illness.

[0119] As used herein, the terms “disease,” “symptom,” or “condition” refer to an interruption or interference with the normal physiological or biological functions of a subject, and are not limited to any specific condition. Diseases, symptoms, or conditions that can be treated with the RNA molecules or pharmaceutical compositions of this disclosure include, but are not limited to, rare diseases, infectious diseases, cancers and proliferative diseases, genetic diseases, autoimmune diseases, diabetes, neurodegenerative diseases, cardiovascular and renal vascular diseases, and metabolic diseases.

[0120] Suitablely, a disease, symptom, or condition is an infectious disease, symptom, or condition (e.g., an infection), that is, a disease, symptom, or condition caused or mediated at least in part by an infectious agent or infectious pathogen (such as those described herein). More specifically, a disease, symptom, or condition can be a viral disease, symptom, or condition. Thus, in some instances, a disease, symptom, or condition is caused or mediated at least in part by a respiratory virus such as influenza virus, parainfluenza virus (e.g., parainfluenza virus type 3; PIV3), rhinovirus, avian influenza virus, coronavirus (e.g., SARS virus, such as SARS-CoV-1 and SARS-CoV-2), metapneumovirus (e.g., human metapneumovirus; hMPV), or respiratory syncytial virus (RSV). More specifically, a disease, symptom, or condition may be caused or mediated at least in part by SARS-CoV-2, influenza A virus, influenza B virus, RSV, PIV3, or hMPV. According to various instances, a disease, symptom, or condition is caused or mediated at least in part by an influenza virus.

[0121] A “therapeutic effective dose” is at least the minimum concentration required to achieve a measurable improvement in a particular disease, condition, or illness. Therapeutic effective doses as used herein can vary depending on factors such as the patient’s disease state, age, sex, weight, and the ability of the RNA molecules of this disclosure to elicit a desired response (such as an immune response) in an individual. Arapeutic effective doses also refer to the amount by which any toxic or harmful effects of the RNA molecule are outweighed by its beneficial therapeutic effects. Therapeutic effective doses can vary depending on the disease, condition, or illness to be treated, the factors to be modified, and the weight, age, ethnic background, sex, health and / or physical condition of the subject being treated, and other relevant factors. Typically, therapeutic effective doses fall within a relatively broad range (e.g., a “dosage” range) that can be determined by medical personnel through routine testing and experimentation. Therefore, this term should not be construed as limiting this disclosure to a specific amount, such as the weight or number of RNA molecules. Therapeutic effective doses can be administered as a single dose or in repeated doses during treatment.

[0122] The RNA molecules described herein can be delivered as naked RNA (e.g., simply as an aqueous solution of RNA). In other instances, to enhance cell entry and subsequent intercellular effects, the RNA molecules can be co-administered with lipid-based carriers, such as liposomes or lipid nanoparticles as described herein.

[0123] The RNA molecules or pharmaceutical compositions disclosed herein can be administered to a subject in the form of one or more dose units, wherein, for example, a tablet or injectable liquid volume may be a single dose unit. In multi-dose formulations requiring a series of two or more doses, the RNA molecules or pharmaceutical compositions are administered over a predetermined time span. Such time spans may be one week, two weeks, three weeks, four weeks, five weeks, six weeks, seven weeks, eight weeks, nine weeks, ten weeks, eleven weeks, or even one year. Practical methods for preparing such dose forms are known or readily apparent to those skilled in the art (see, for example, Remington: The Science and Practice of Pharmacy, 20th edition, Philadelphia College of Pharmacy and Science, 2000).

[0124] antigen protein

[0125] Consider the RNA molecules described herein, which include nucleotide sequences encoding antigenic or immunogenic proteins or fragments, variants, or derivatives thereof. Antigenic proteins can be pathogen antigens, tumor antigens, allergen antigens, or autoimmune autoantigens. These pathogen antigens can originate from pathogenic microorganisms, particularly bacteria, viruses, fungi, or protozoa, and these antigens elicit an immune response in mammalian subjects, such as humans. Pathogen antigens can be surface antigens (e.g., proteins or fragments thereof) located on the surface of, for example, viruses, bacteria, fungi, or protozoa.

[0126] Pathogen antigens may include antigens derived from one or more of the following pathogens: *Acinetobacter baumannii*, *Anaplasma* genus, *Anaplasma phagocytophilum*, *Ancylostoma braziliense*, *Ancylostoma duodenale*, *Arcanobacterium haemolyticum*, *Ascaris lumbricoides*, *Aspergillus*, *Astroviridae*, *Babesia*, *Bacillus anthracis*, *Bacillus cereus*, *Bartonella henselae*, BK virus, *Blastocysts hominis*, *Blastomyces dermatitidis*, and *Bordetella pertussis*. *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* genus, *Candida* spp., *Chlamydia trachomatis*, *Chlamydophila pneumoniae*, *Chlamydophila psittaci* psittaci), QD prions, Clonorchis sinensis, Clostridium botulinum, Clostridium difficile, Clostridium perfringensClostridium perfringens, Clostridium spp., Clostridium tetani, Coccidioides spp., Coronavirus, Corynebacterium diphtheriae, Coxiella burnetii, Crimean-Congo hemorrhagic fever virus, Cryptococcus neoformans, Cryptosporidium, Cytomegalovirus (CMV), Dengue virus (DEN-1, DEN-2, DEN-3 and DEN-4), Dinuclear ferruginosa, Ebola virus (EBOV), Echinococcus, Ehrlichiachaffeensis, Ehrlichia *E. ewingii*, *Ehrlichia*, *Entamoeba histolytica*, *Enterococcus*, *Enterovirus*, *Enteroviruses* (mainly Coxsackie A virus and Enterovirus 71 (EV71)), *Epidermophyton spp*, *EBV*, *Escherichia coli* O157:H7, O111 and O104:H4, *Fasciola hepatica* and *Fasciola gigantica*, FFI prions, *Filarioidea* superfamily, flaviviruses, *Francisella tularensis*, *Fusobacterium*, *Geotrichum candidum*, *Giardia intestinalis*, *Gnathostoma* species. spp), GSS prions, Guanarito virus, Haemophilus ducreyi, Haemophilus influenzae, Helicobacter pylori, Henipavirus (Hendra virus)Hepatitis A virus, Nipah virus, Hepatitis B virus (HBV), Hepatitis C virus (HCV), Hepatitis D virus, Hepatitis E virus, Herpes simplex virus types 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 Metapneumovirus (hMPV), Human Papillomavirus (HPV), Human Parainfluenza Virus (HPIV), Japanese Encephalitis Virus, JC Virus, Junin Virus, Kingella kingae, Klebsiella granulomatis, Kurukinum virus, Lassa virus, Legionella pneumophila. * *Pneumophila*, *Leishmania*, *Leptospira*, *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., Onchocercavolvulus, Orientia tsutsugamushi, Orthomyxoviridae (influenza), Paracoccidioides brasiliensis, Paragonimus spp, Paragonimus westermani, Parvovirus B19, Pasteurella, Plasmodium, Pneumocystis jirovecii, Poliovirus, Rabies virus, Respiratory syncytial virus (RSV), Rhinovirus, Rickettsia akari, Rickettsia, Rickettsia prowazekii, Rickettsia rickettsii, Rickettsia typhi, Rift Valley fever virus Virus, rotavirus, rubella virus, Sabiavirus, Salmonella, Sarcoptes scabiei, SARS coronavirus, Schistosoma, Shigella, Sin Nombre virus, Hantavirus, Sporothrix schenckii, Staphylococcus, Streptococcus, Streptococcus agalactiae, Streptococcus pneumoniae, Streptococcus pyogenes, Strongyloides stercoralis, Taenia, Taenia solium, tick-borne encephalitis virus (TBEV), Toxocara canis * *Toxocara cati*, *Toxoplasma gondii*, *Treponema pallidum*, *Trichinella spiralis*, *Trichomonas vaginalis*, and *Trichophyton* species.The following bacteria are listed: Trichomonas vaginalis (spp.), Trichomonas trichomonas, Trypanosoma brucei, Trypanosoma cruzi, Ureaplasma urealyticum, Varicella virus (VZV), Smallpox or smallpox, vCJD prions, 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.

[0127] In some instances, the relevant antigenic or immunogenic proteins may originate from pathogens selected from: Severe Acute Respiratory Syndrome (SARS), SARS-CoV-1 and SARS-CoV-2, influenza viruses (including influenza A and influenza B), respiratory syncytial virus (RSV), herpes simplex virus (HSV), human papillomavirus (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 (such as parainfluenza virus type 3 or PIV3), human metapneumovirus (hMPV), and yellow fever virus. More specifically, the antigen may be derived from SARS-CoV-2, influenza A and influenza B viruses, RSV, PIV3, hMPV, Hendra virus, or CMV. In some instances, the antigenic protein or immunogenic protein is derived from SARS-CoV-2, influenza A virus, influenza B virus, RSV, PIV3, or hMPV.

[0128] In some instances, the antigenic protein or immunogenic protein is derived from the influenza virus. In such instances, the mRNA may have a nucleotide sequence encoding at least one antigenic protein derived from the hemagglutinin (HA), neuraminidase (NA), nucleoprotein (NP), matrix protein 1 (M1), 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 PB1, PB1-F2, or polymerase basic protein 2 (PB2) of the influenza virus, or fragments, derivatives, or variants thereof. In some instances, the nucleotide sequence encodes at least one antigenic protein derived from the hemagglutinin (HA) and / or neuraminidase (NA) of the influenza virus, or fragments, derivatives, or variants thereof. The HA and / or NA may be independently derived from influenza A virus or influenza B virus, or fragments, variants, or derivatives thereof (e.g., chimeric versions).

[0129] In some instances, the antigenic protein or immunogenic protein is derived from coronaviruses such as SARS-CoV-1 or SARS-CoV-2. In these cases, the mRNA may have a nucleotide sequence encoding at least one antigenic protein derived from the spike (S) protein or fragments, derivatives, or variants thereof.

[0130] Those skilled in the art will understand that various changes and / or modifications can be made to the above embodiments without departing from the broad general scope of this disclosure. Therefore, the embodiments are to be considered illustrative rather than restrictive in all respects.

[0131] Item-by-item list of the implementation plan

[0132] 1. A method for producing RNA molecules via in vitro transcription, the method comprising the following steps:

[0133] (a) Providing a closed-end DNA template encoding the RNA molecule; and

[0134] (b) Transcribe the RNA molecule from the closed-end DNA template in a reaction mixture with a pH of about 6 to about 8.

[0135] 2. The method according to embodiment 1, wherein the pH of the reaction mixture is about 6.5 to about 7.5.

[0136] 3. The method according to embodiment 1 or embodiment 2, wherein the pH of the reaction mixture is about 7.0.

[0137] 4. The method according to any one of the foregoing embodiments, wherein the closed-end DNA template is prepared in a solution with a pH of about 6 to about 8.

[0138] 5. The method according to embodiment 4, wherein the pH of the solution is from about 6.5 to about 7.5.

[0139] 6. The method according to embodiment 4 or embodiment 5, wherein the pH value of the solution is about 7.0.

[0140] 7. The method according to any one of the foregoing embodiments, wherein the solution comprises a buffer solution.

[0141] 8. The method according to embodiment 7, wherein the solution comprises Tris buffer.

[0142] 9. The method according to embodiment 8, wherein the solution comprises a Tris buffer solution with a concentration of about 8 mM to about 12 mM.

[0143] 10. The method according to embodiment 8 or embodiment 9, wherein the solution contains a Tris buffer solution with a concentration of about 10 mM.

[0144] 11. The method according to any one of the foregoing embodiments, wherein the closed-end DNA template is dog bone-type DNA (dbDNA) or contains dog bone-type DNA (dbDNA).

[0145] 12. The method according to any one of the foregoing embodiments, wherein the closed-end DNA template has been linearized.

[0146] 13. The method according to any one of the foregoing embodiments, wherein the method further comprises an early step of linearizing the closed-end DNA template.

[0147] 14. The method according to any one of the foregoing embodiments, wherein the RNA molecule is a non-replicating mRNA molecule or a self-amplifying mRNA molecule, or the RNA molecule includes a non-replicating mRNA molecule or a self-amplifying mRNA molecule.

[0148] 15. The method according to any one of the foregoing embodiments, wherein the RNA molecule encodes an immunogenic protein.

[0149] 16. An isolated RNA molecule produced by the method of any one of embodiments 1 to 15.

[0150] 17. A pharmaceutical composition comprising the isolated RNA molecule as described in embodiment 16 and optionally comprising a pharmaceutically acceptable carrier, diluent, or excipient.

[0151] 18. The pharmaceutical composition according to embodiment 17, wherein the isolated RNA molecule is contained in or otherwise associated with a lipid-based carrier.

[0152] 19. The pharmaceutical composition according to embodiment 18, wherein the lipid-based carrier is a lipid nanoparticle or comprises lipid nanoparticles.

[0153] 20. The isolated RNA molecule according to embodiment 16 or the pharmaceutical composition according to any one of embodiments 17 to 19, for use in: (a) inducing an immune response; and / or (b) treating a subject’s disease, symptom or condition.

[0154] 21. A method for inducing an immune response in a subject, the method comprising the step of administering to the subject a therapeutically effective amount of an isolated RNA molecule according to embodiment 16 or embodiment 20, or a pharmaceutical composition according to any one of embodiments 17 to 20, thereby inducing the immune response.

[0155] 22. A method for preventing and / or treating a disease, symptom, or condition of a subject, the method comprising the step of administering to the subject a therapeutically effective amount of an isolated RNA molecule according to embodiment 16 or embodiment 20, or a pharmaceutical composition according to any one of embodiments 17 to 20, thereby preventing and / or treating the disease, symptom, or condition.

[0156] 23. Use of the isolated RNA molecule according to embodiment 16 or embodiment 20, or the pharmaceutical composition according to any one of embodiments 17 to 20, in the preparation of a medicament for inducing an immune response in a subject.

[0157] 24. Use of the isolated RNA molecule according to embodiment 16 or claim 20, or the pharmaceutical composition according to any one of embodiments 17 to 20, in the preparation of a medicament for the prevention and / or treatment of a disease, symptom, or condition of a subject.

Claims

1. A method for producing RNA molecules via in vitro transcription, the method comprising the following steps: (a) Provide a closed-terminal DNA template encoding the RNA molecule; and (b) Transcribe the RNA molecule from the closed-end DNA template in a reaction mixture with a pH of about 6 to about 8.

2. The method of claim 1, wherein the pH of the reaction mixture is about 6.5 to about 7.

5.

3. The method according to claim 1, wherein the pH of the reaction mixture is about 7.

0.

4. The method of claim 1, wherein the closed-end DNA template is prepared in a solution with a pH of about 6 to about 8.

5. The method of claim 4, wherein the pH of the solution is about 6.5 to about 7.

5.

6. The method of claim 4, wherein the pH of the solution is about 7.

0.

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

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

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

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

11. The method of claim 1, wherein the closed-end DNA template is dog bone DNA (dbDNA) or contains dog bone DNA (dbDNA).

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

13. The method of claim 1, wherein the method further comprises an early step of linearizing a closed-end DNA template.

14. The method according to claim 1, wherein the RNA molecule is a non-replicating mRNA molecule or a self-amplifying mRNA molecule, or the RNA molecule includes 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 in vitro transcription, the method comprising the steps of: (a) Provide a closed-terminal DNA template encoding the RNA molecule; and (b) Transcribe the RNA molecule from the closed-end DNA template in a reaction mixture with a pH of about 6 to about 8.

17. A pharmaceutical composition comprising the isolated RNA molecule according to claim 16, and optionally comprising 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 a lipid nanoparticle or comprises lipid nanoparticles.

20. The isolated RNA molecule according to claim 16, used for: (a) inducing an immune response; and / or (b) treating a subject's disease, ailment, or condition.

21. The pharmaceutical composition according to claim 17, used for: (a) inducing an immune response; and / or (b) treating a subject's disease, ailment, or condition.

22. A method for inducing an immune response in a subject, the method comprising administering to the subject a therapeutically effective amount of the isolated RNA molecule according to claim 16, thereby inducing the immune response.

23. A method for inducing an immune response in a subject, the method comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition according to claim 17, thereby inducing the immune response.

24. A method for preventing and / or treating a disease, symptom, or condition of a subject, the method comprising administering to the subject a therapeutically effective amount of the isolated RNA molecule according to claim 16, thereby preventing and / or treating the disease, symptom, or condition.

25. A method for preventing and / or treating a disease, symptom, or condition of a subject, the method comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition according to claim 17, thereby preventing and / or treating the disease, symptom, or condition.

26. Use of the isolated RNA molecule according to claim 16 in the preparation of a medicament for inducing an immune response in a subject.

27. Use of the pharmaceutical composition according to claim 17 in the preparation of a medicament for inducing an immune response in a subject.

28. Use of the isolated RNA molecule according to claim 16 in the preparation of a medicament for the prevention and / or treatment of a subject’s disease, symptom or condition.

29. Use of the pharmaceutical composition according to claim 17 in the preparation of a medicament for the prevention and / or treatment of a disease, symptom or condition of a subject.