RNA production

Abstract

The present disclosure provides techniques for performing in vitro transcription that can generate product RNA preparations with reduced levels of certain contaminants (e.g., aberrant products), particularly double-stranded RNA (dsRNA).

Description

[Technical Field]

[0001] The present invention relates to a method for reducing dsRNA during in vitro transcription by stepwise addition of nucleotides. The present invention further relates to nucleic acids produced by the method of the present invention, and the use of such nucleic acids in methods for treating subjects in need thereof. [Background technology]

[0002] In vitro RNA production is becoming increasingly important in the biotechnology and pharmaceutical industries. Improving manufacturing processes, particularly those capable of large-scale production of high-quality RNA (such as therapeutic-grade RNA), is crucial and valuable. [Overview of the project]

[0003] RNA transcribed in vitro (e.g., by T7 RNA polymerase (RNAP)) may contain abnormal products (at significant levels, etc.). While we do not wish to be bound by theory, it has been proposed that some or all of such products may be generated by the unusual activity of the utilized RNAP. One such abnormal product is dsRNA, which can be particularly problematic given its properties, for example, its ability to induce inflammatory cytokines and / or, above all, its ability to activate immune effector proteins, which can lead to inhibition of protein synthesis. dsRNA is typically a major contaminant of in vitro RNA transcription reactions.

[0004] Generally, abnormal products, particularly dsRNA, are removed from in vitro transcribed RNA preparations by purification, and various purification techniques are available (e.g., LiCl and / or alcohol-based precipitation, size exclusion chromatography and / or ion exchange chromatography, silica matrix purification, ion-pair reversed-phase high-performance liquid chromatography [HPLC], cellulose-based separation, etc.). However, most or all of such purification strategies may be impractical and / or unideal, especially for commercial-scale and / or pharmaceutical-grade production. This is because the desired RNA product is often removed along with abnormal products (and / or other contaminations), resulting in an undesirable and significant loss of the RNA product.

[0005] In particular, this disclosure surprisingly provides the insight that by limiting the amount of UTP or its functional analog when synthesizing RNA by transcription and supplementing the reaction mix with UTP or its functional analog during the transcription process, it is possible to obtain RNA with improved purity, reduced immunogenicity, and good stability.

[0006] Furthermore, this disclosure provides insight that benefits can be gained by first reducing the production of abnormal products (and / or other contaminations). This disclosure provides a technique for performing in vitro transcription that can produce product RNA preparations with reduced levels of specific contamination (e.g., abnormal products), particularly dsRNA. Advantages of the provided technique include, but are not limited to, higher product RNA yield (e.g., less product loss during the process), fewer processing steps (which may contribute to reduced product loss), lower production costs, and shorter production periods, resulting in more efficient manufacturing. Furthermore, this disclosure teaches that the provided improved production technique (e.g., improved transcription reaction conditions) also has various advantages (such as those mentioned above) over improved purification techniques.

[0007] In one embodiment, the present invention relates to a method for producing RNA, comprising transcribing RNA from a DNA template using a reaction mix comprising adenosine triphosphate (ATP), guanosine triphosphate (GTP), cytidine triphosphate (CTP), and uridine triphosphate (UTP), or functional analogs thereof, wherein the starting concentration of UTP or its functional analog is lower than the starting concentration of CTP or its functional analog and / or ATP or its functional analog, the method comprising supplementing the reaction mix with a composition comprising UTP or its functional analog, but substantially free of CTP or its functional analog or ATP or its functional analog, during the transcription reaction.

[0008] In one embodiment, the present invention relates to a method for producing RNA, comprising transcribing RNA from a DNA template using a reaction mix comprising adenosine triphosphate (ATP), guanosine triphosphate (GTP), cytidine triphosphate (CTP), and uridine triphosphate (UTP), or functional analogs thereof, wherein the starting concentration of CTP or its functional analog is equal to the starting concentration of ATP or its functional analog, and the starting concentration of UTP or its functional analog is lower than the starting concentration of CTP or its functional analog or ATP or its functional analog, wherein the method comprises supplementing the reaction mix with UTP or its functional analog during the transcription reaction.

[0009] In one embodiment, the present invention relates to a method for producing a composition containing RNA with reduced double-stranded (ds)RNA content, the method comprising transcribing RNA from a DNA template using a reaction mix comprising adenosine triphosphate (ATP), guanosine triphosphate (GTP), cytidine triphosphate (CTP), and uridine triphosphate (UTP), or functional analogs thereof, wherein the starting concentration of UTP or its functional analog is lower than the starting concentration of CTP or its functional analog and / or ATP or its functional analog, and the method comprises supplementing the reaction mix with a composition comprising UTP or its functional analog, but substantially free of CTP or its functional analog or ATP or its functional analog, during the transcription reaction.

[0010] In one embodiment, the present invention relates to a method for producing a composition containing RNA with reduced double-stranded (ds)RNA content, the method comprising transcribing RNA from a DNA template using a reaction mix comprising adenosine triphosphate (ATP), guanosine triphosphate (GTP), cytidine triphosphate (CTP), and uridine triphosphate (UTP), or functional analogs thereof, wherein the starting concentration of CTP or its functional analog is equal to the starting concentration of ATP or its functional analog, and the starting concentration of UTP or its functional analog is lower than the starting concentration of CTP or its functional analog or ATP or its functional analog, the method comprising supplementing the reaction mix with UTP or its functional analog during the transcription reaction.

[0011] In some embodiments, the double-stranded (ds)RNA content of a composition containing RNA is reduced compared to the dsRNA content of a composition containing RNA transcribed from the same DNA template using equimolar amounts of adenosine triphosphate (ATP), guanosine triphosphate (GTP), cytidine triphosphate (CTP), and uridine triphosphate (UTP), or their functional analogues.

[0012] In some embodiments, the immunogenicity of compositions containing RNA is reduced compared to compositions containing RNA transcribed from the same DNA template using equimolar amounts of adenosine triphosphate (ATP), guanosine triphosphate (GTP), cytidine triphosphate (CTP), and uridine triphosphate (UTP), or their functional analogs.

[0013] In some embodiments, uridine triphosphate (UTP) or a functional analog thereof is present at an initiation concentration that limits the transcription rate.

[0014] In some embodiments, the ratio of the starting concentration of uridine triphosphate (UTP) or its functional analog to the starting concentration of cytidine triphosphate (CTP) or its functional analog, or adenosine triphosphate (ATP) or its functional analog, is approximately 1:1.5 to 1:15.

[0015] In some embodiments, when the concentration of UTP or its functional analog approaches depletion, uridine triphosphate (UTP) or its functional analog is replenished in the reaction mix.

[0016] In some embodiments, the reaction mix is ​​supplemented at least once with uridine triphosphate (UTP) or a functional analog during the transcription reaction.

[0017] In some embodiments, uridine triphosphate (UTP) or a functional analog thereof is continuously replenished in the reaction mix during the transcription process.

[0018] In some embodiments, uridine triphosphate (UTP) or a functional analog thereof is periodically replenished in the reaction mix during the transcription process.

[0019] In some embodiments, the initial ratio of the concentration of uridine triphosphate (UTP) or its functional analog to the concentration of cytidine triphosphate (CTP) or its functional analog, or adenosine triphosphate (ATP) or its functional analog, is maintained or restored by supplementing the reaction mix with uridine triphosphate (UTP) or its functional analog.

[0020] In some embodiments, the reaction mix is ​​replenished with uridine triphosphate (UTP) or a functional analog thereof until the transcription reaction is complete.

[0021] In some embodiments, the starting concentration of guanosine triphosphate (GTP) or its functional analog is lower than the starting concentration of cytidine triphosphate (CTP) or its functional analog, or adenosine triphosphate (ATP) or its functional analog. In some embodiments, it is preferable that guanosine triphosphate (GTP) or its functional analog be present at a starting concentration that limits the transcription rate.

[0022] In some embodiments, the ratio of the starting concentration of guanosine triphosphate (GTP) or its functional analog to the starting concentration of cytidine triphosphate (CTP) or its functional analog, or adenosine triphosphate (ATP) or its functional analog, is approximately 1:1.5 to 1:15.

[0023] In some embodiments, the reaction mix is ​​supplemented with guanosine triphosphate (GTP) or a functional analog thereof during the transcription reaction.

[0024] In some embodiments, when the concentration of GTP or its functional analog approaches depletion, guanosine triphosphate (GTP) or its functional analog is replenished in the reaction mix.

[0025] In some embodiments, the reaction mix is ​​replenished at least once with guanosine triphosphate (GTP) or a functional analog thereof during the transcription reaction.

[0026] In some embodiments, the reaction mix is ​​continuously replenished with guanosine triphosphate (GTP) or a functional analog thereof during the transcription process.

[0027] In some embodiments, the reaction mix is ​​periodically replenished with guanosine triphosphate (GTP) or a functional analog during the transcription process.

[0028] In some embodiments, the initial ratio of the concentration of guanosine triphosphate (GTP) or its functional analog to the concentration of cytidine triphosphate (CTP) or its functional analog, or adenosine triphosphate (ATP) or its functional analog, is maintained or restored by supplementing the reaction mix with guanosine triphosphate (GTP) or its functional analog.

[0029] In some embodiments, the reaction mix is ​​continuously replenished with guanosine triphosphate (GTP) or a functional analog until the transcription reaction is complete.

[0030] In some embodiments, the provided method does not involve supplementing the transcription mix with cytidine triphosphate (CTP) or its functional analog and / or adenosine triphosphate (ATP) or its functional analog during the transcription reaction.

[0031] In some embodiments, the reaction mix includes a starting nucleotide corresponding to the first nucleotide in the RNA molecule.

[0032] In some embodiments, the starting nucleotide is a nucleoside monophosphate, a nucleoside diphosphate, a nucleoside triphosphate, or a dinucleoside triphosphate.

[0033] In some embodiments, the starting nucleotide is a 5' capped or 5' capped analog.

[0034] In some embodiments, the 5' cap or 5' cap analog is G[5']ppp[5']G, m7G[5']ppp[5']G, m3 + G[5']ppp[5']G, m2 7,3’-O G[5']ppp[5']G (3'-ARCA), m2 7,2’-O GpppG (2'-ARCA), m2 7,2’-O Gpp S pG D1 (β-S-ARCA D1), m2 7,2’-O Gpp S pG D2 (β-S-ARCA D2) and m2 7,3’-O Selected from the group consisting of Gppp(m2'-O)ApG (CC413).

[0035] In some embodiments, the 5' cap or 5' cap analog in the reaction mix is present in excess compared to guanosine triphosphate (GTP) or its functional analog.

[0036] In some embodiments, the ratio of the starting concentration of the 5' cap or 5' cap analog to the starting concentration of guanosine triphosphate (GTP) or its functional analog is from about 2:1 to about 20:1.

[0037] In some embodiments, the ratio of the starting concentration of the 5' cap or 5' cap analog to the starting concentration of guanosine triphosphate (GTP) or its functional analog is about 4:1.

[0038] In some embodiments, the reaction mix further comprises RNA polymerase, a buffer, and at least one monovalent or divalent cation.

[0039] In some embodiments, the cation is Li + , Na + , K + , NH 4+ , tris(hydroxymethyl)aminomethane cation, Mg 2+ , Ba 2+ or Mn 2+ is.

[0040] In some embodiments, the RNA polymerase is selected from the group consisting of T7 RNA polymerase, T3 RNA polymerase, and SP6 RNA polymerase.

[0041] In some embodiments, the functional analog of uridine triphosphate (UTP) is selected from the group consisting of pseudo-UTP, N1-methyl pseudo-UTP, 2-thio-UTP, and 4-thio-UTP.

[0042] In some embodiments, the functional analog of guanosine triphosphate (GTP) is selected from the group consisting of 7-deaza-GTP, N1-methyl-GTP, and O6-methyl-GTP.

[0043] In some embodiments, the DNA template encodes one or more of the 5' untranslated region (UTR), 3'UTR, open reading frame, and poly(A) tail.

[0044] In some embodiments, the RNA includes one or more of the following: a 5' untranslated region (UTR), a 3'UTR, an open reading frame, and a poly(A) tail.

[0045] In some embodiments, the RNA encodes at least one peptide or protein.

[0046] In some embodiments, RNA is mRNA.

[0047] In some embodiments, the RNA is self-replicating RNA.

[0048] In some embodiments, the RNA produced by the method of the present invention has 200-20,000 nucleotides, 200-12,000 nucleotides, 200-8,000 nucleotides, 500-5,000 nucleotides, 500-2,500 nucleotides, and in particular 600-2,500 nucleotides or 800-2,000 nucleotides.

[0049] In some embodiments, the pH value of the reaction mix is ​​kept substantially constant throughout the transfer reaction.

[0050] In some embodiments, the progress of the transcription reaction is monitored in real time.

[0051] In some embodiments, this method is carried out using a bioreactor.

[0052] In one aspect, the present invention relates to RNA produced by the method of the present invention.

[0053] In one embodiment, the present invention relates to a composition comprising RNA produced by the method of the present invention.

[0054] In one embodiment, the present invention relates to a method for treating a subject, comprising the steps of (i) obtaining RNA produced by the method of the present invention, or obtaining a composition containing RNA produced by the method of the present invention, and (ii) administering the RNA or the RNA-containing composition to the subject.

[0055] In one embodiment, the present invention relates to a method for producing RNA by in vitro transcription, the method comprising limiting the concentration of UTP or a functional analog thereof during the in vitro transcription reaction.

[0056] In one embodiment, the present invention relates to an in vitro transcription reaction comprising a promoter that directs the transcription of a template to produce a transcript, optionally a poly(A) sequence element; an RNA polymerase that acts on the promoter; and an RNA template comprising adenosine triphosphate (ATP), guanosine triphosphate (GTP), cytidine triphosphate (CTP), and uridine triphosphate (UTP), or functional analogs thereof, wherein the starting concentration of UTP or its functional analog is lower than the concentration of CTP or its functional analog and / or ATP or its functional analog.

[0057] In one embodiment, the present invention relates to a method for treating a subject by administering RNA produced by the method of the present invention, or a composition containing RNA produced by the method of the present invention, to the subject.

[0058] In one embodiment, the present invention relates to a method for reducing the amount of double-stranded RNA resulting from in vitro transcription of RNA, the method comprising in vitro transcription of RNA from a template under transcription conditions in which the amount or concentration of UTP or a functional analog thereof is a limiting amount or concentration in RNA transcription compared to one or more amounts or concentrations of ATP, CTP and / or GTP or their respective analogs.

[0059] In one embodiment, the present invention relates to a method for reducing the amount of double-stranded RNA in a composition comprising RNA resulting from in vitro transcription of RNA, the method comprising in vitro transcription of RNA from a template under transcription conditions in which the amount or concentration of UTP or a functional analog thereof is a limiting amount or concentration in RNA transcription compared to one or more amounts or concentrations of ATP, CTP and / or GTP or their respective analogs.

[0060] In one embodiment, the present invention relates to a method for reducing the immunogenicity of a composition comprising RNA resulting from in vitro transcription of RNA, the method comprising in vitro transcription of RNA from a template under transcription conditions in which the amount or concentration of UTP or a functional analog thereof is a limiting amount or concentration in RNA transcription compared to one or more amounts or concentrations of ATP, CTP and / or GTP or their respective analogs.

[0061] In one embodiment, double-stranded RNA is the result of annealing at least two different RNA molecules to each other, i.e., intermolecular bonding. In another embodiment, double-stranded RNA is the result of intramolecular bonding, i.e., some RNA molecules annealing to themselves, for example, when a transcript folds back. [Brief explanation of the drawing]

[0062] [Figure 1] This section presents exemplary results for dsRNA content produced by an in vitro transduction (IVT) reaction involving the stepwise addition of NTPs to transcribe unmodified RNA. RNA was transcribed in vitro by decreasing (restricting) the starting concentrations of the NTPs shown: G-GTP, A-ATP, U-UTP, and A / U-ATP+UTP. Restricted NTPs were supplied to the IVT reaction stepwise until all NTPs reached the final concentration. All RNAs were co-transcribed and capped using the CC413 capping analog. GTP restriction was used as a control. A. RNA yield was not affected by the type of NTP supplied during the reaction compared to the control. B. RNA integrity was not affected by the type of NTP supplied during the reaction compared to the control. C. dsRNA content increased when ATP was supplied compared to the control, and decreased when UTP was supplied. When both ATP and UTP were supplied, their effects canceled each other out, and the dsRNA content became equivalent to the control (GTP supplied). D. When GTP was not supplied, capping efficiency was reduced compared to the control. [Figure 2]This section presents exemplary results for dsRNA content produced by IVT reactions of unmodified RNA with stepwise addition of UTP or GTP and UTP to restore capping efficiency. RNA was transcribed in vitro by decreasing (restricting) the starting concentrations of the indicated NTPs: G-GTP, U-UTP, and G / U-GTP+UTP. Restricted NTPs were supplied stepwise to the IVT reaction until all NTPs reached the final concentration of NTPs. GTP restriction was used as a control. All RNAs were co-transcribed and capped using a D1-β-S1 ARCA capping analog. A. RNA yield increased when UTP or a combination of UTP and GTP was supplied under these reaction conditions compared to the control. B. The integrity of purified RNA decreased when UTP or GTP and UTP were supplied. When GTP and UTP were supplied in combination, RNA integrity returned to the level of when GTP was supplied to the IVT reaction (control). C. dsRNA content decreased by supplying UTP compared to when control GTP was supplied. When both GTP and UTP were supplied, the dsRNA content decreased, but to a lesser extent than when only UTP was supplied. When D.UTP was supplied alone, the capping efficiency decreased compared to the control, but when GTP and UTP were supplied in combination, the capping efficiency returned to normal. [Figure 3]This paper presents exemplary results of dsRNA content produced by an IVT reaction transcribing RNA-containing N1-methylpseuduridine (m1ΨTP) by stepwise addition of m1ΨTP or m1ΨTP and GTP. RNA was transcribed in vitro by decreasing (restricting) the starting concentrations of the indicated NTPs: G-GTP, m1Ψ-m1ΨTP, and G / m1Ψ-GTP+m1ΨTP. Restricted NTPs were supplied to the IVT reaction stepwise until all NTPs reached the final concentration of NTPs. GTP restriction was used as a control. All RNAs were co-transcribed and capped using a CC413 GAG capping analog. A. RNA yield was not affected by the type of NTP supplied during the reaction compared to the control. B. RNA integrity was reduced when m1ΨTP was supplied compared to the control. When GTP and m1ΨTP were supplied in combination, RNA integrity returned to the level of the control supplied with GTP. C. dsRNA content decreased by supplying m1ΨTP compared to a control supplied with standard GTP. Supplying both GTP and m1ΨTP reduced dsRNA content compared to the response supplied with m1ΨTP alone. D. Capping efficiency decreased when m1ΨTP was supplied compared to the control, but recovered when both GTP and m1ΨTP were supplied. [Figure 4] This shows exemplary results for determining the higher-order structure of RNA products using circular dichroism. [Modes for carrying out the invention]

[0063] A certain definition Certain terms used herein may be understood to be defined as described in "A multilingual glossary of biotechnological terms: (IUPAC Recommendations)," H.G. W. Heuenberger, B. Nagel, and H. Kolbl, Eds., Helvetica Chimica Acta, CH-4010 Basel, Switzerland, (1995).

[0064] Furthermore, the expression of relative amounts of components characterized by a general term typically means referring to the total amount of all specific variations or members included in the general term. If a particular component defined by a general term is specified to exist in a specific relative amount, and this component is further characterized as being a specific variation or member included in the general term, it means that there are no other variations or members included in this general term so that the total relative amount of the components included in the general term exceeds the specified relative amount, more preferably, there are no other variations or members included in this general term at all.

[0065] A: The terms “a,” “an,” “the,” and similar demonstrative pronouns used in the context describing the present invention (particularly in the context of the following claims) should be construed to include both singular and plural unless otherwise indicated herein or unless the context clearly contradicts this. The enumeration of ranges of values ​​herein is merely intended to function as an abbreviation for referring individually to each separate value that falls within that range. Unless otherwise indicated herein, individual values ​​are incorporated herein as if they were individually described herein. All methods described herein may be performed in any preferred order unless otherwise indicated herein or unless the context clearly contradicts this. The use of any and all examples or exemplary language provided herein (e.g., “etc.”) is merely intended to better illustrate the present invention and does not limit the scope of the present invention as otherwise claimed herein. Nothing expressed herein should be construed as indicating that any non-claimed element is essential to the practice of the present invention.

[0066] Approximately or roughly: When used herein in relation to a value, the terms "approximately" or "roughly" refer to a similar value in the context of the reference value described herein. Generally, a person skilled in the art familiar with the context will understand the reasonable degree of difference that "approximately" or "roughly" encompasses in that context. A person skilled in the art will understand that in many embodiments (as understood from the context), the term "approximately" means roughly or nearly, and in the context of a number or range described herein, preferably means + / - 10% of that number or range. For example, in some embodiments, the terms "approximately" or "roughly" may encompass a range of values ​​that fall within the range of 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less of the reference value.

[0067] Administration: As used herein, the term “administration” typically refers to the administration of a composition to a subject or system. Those skilled in the art will recognize the various routes that may be used to administer a subject, e.g., a human, under appropriate circumstances. For example, in some embodiments, administration may be ocular, oral, parenteral, topical, etc. In some specific embodiments, administration may be bronchial (e.g., by bronchial droplet), buccal, percutaneous (e.g., one or more of the following, or including, topical, intradermal, interdermal, transdermal, etc.), enteral, intra-arterial, intradermal, intragastric, intramedullary, intramuscular, intranasal, intraperitoneal, intrathecal, intravenous, intraventricular, intraspecific organ (e.g., intrahepatic), mucosal, nasal, oral, rectal, subcutaneous, sublingual, topical, tracheal (e.g., by intratracheal droplet), vaginal, vitreous, etc. In some embodiments, administration may be intramuscular. In some embodiments, administration may include intermittent (e.g., multiple doses spaced apart in time) and / or periodic (e.g., individual doses spaced apart over a period of time) administration. In some embodiments, administration may include continuous administration (e.g., perfusion) over at least a selected period of time.

[0068] Agent: Generally, as used herein, the term “agent” is used to mean an entity (e.g., lipids, metals, nucleic acids, polypeptides, polysaccharides, small molecules, etc., or complexes, combinations, mixtures or systems thereof [e.g., cells, tissues, organisms]) or a phenomenon (e.g., heat, electric current or electric field, magnetic force or magnetic field, etc.). Under appropriate circumstances, as will be apparent to those skilled in the art, the term may be used to mean an entity that is or contains a cell or organism, or a fraction, extract or component thereof. Or, or further, as will be apparent to the context, the term may be used to mean a natural product in the sense that it is found in nature and / or obtained from nature. In some cases, as will also be apparent to the context, the term may be used to mean one or more entities that are designed, engineered and / or produced by human hands and / or are artificial in the sense that they are not found in nature. In some embodiments, the agent may be available in isolated or pure form, and in some embodiments, the agent may be available in crude form. In some embodiments, potential agents may be provided as aggregates or libraries, for example, that can be screened to identify or analyze the active agents contained therein. In some cases, the term “agent” may mean a compound or entity that is or contains a polymer. In some cases, the term may mean a compound or entity that contains one or more polymeric moieties. In some embodiments, the term “agent” may mean a compound or entity that is not a polymer, and / or a compound or entity that does not substantially contain any polymer, and / or a compound or entity that does not substantially contain one or more specific polymeric moieties. In some embodiments, the term may mean a compound or entity that lacks or substantially contains no polymeric moieties.

[0069] Analog: As used herein, the term “analog” refers to a substance that shares one or more specific structural features, elements, components, or parts with a reference substance. Typically, an “analog” is a substance that exhibits significant structural similarity to a reference substance, for example, having a common core or consensus structure, but is different from the reference substance in a particular distinct way. In some embodiments, an analog is a substance that can be produced from a reference substance, for example, by chemical manipulation of the reference substance. In some embodiments, an analog is a substance that can be produced by carrying out a synthesis process that is substantially similar to the process used to produce the reference substance (for example, having several common steps). In some embodiments, an analog is produced, or can be produced, by carrying out a synthesis process different from the one used to produce the reference substance.

[0070] Antibody Agent: As used herein, the term “antibody agent” refers to an agent that specifically binds to a particular antigen. In some embodiments, the term encompasses any polypeptide or polypeptide complex containing sufficient immunoglobulin structural elements to confer specific binding. Exemplary antibody agents include, but are not limited to, monoclonal or polyclonal antibodies. In some embodiments, an antibody agent may contain one or more constant region sequences characteristic of mouse, rabbit, primate, or human antibodies. In some embodiments, an antibody agent may contain one or more antibody sequence elements that are humanized, primated, chimeric, etc., as are known in the art. In many embodiments, the term “antibody agent” is used to refer to one or more constructs or formats known or developed in the art for utilizing the structural and functional characteristics of an antibody in alternative embodiments.For example, in embodiments, antibody agents used in accordance with this disclosure include, but are not limited to, intact IgA, IgG, IgE, or IgM antibodies; bispecific or polyspecific antibodies (e.g., Zybodies®); antibody fragments, e.g., Fab fragments, Fab' fragments, F(ab')2 fragments, Fd' fragments, Fd fragments, and isolated complementarity-determining regions (CDRs) or sets thereof, single-chain Fv; polypeptide-Fc fusions; single-domain antibodies (e.g., shark single-domain antibodies such as IgNAR or fragments thereof), camelid antibodies, mask antibodies (e.g., Probodies®); and small modular antibodies. The format is selected from ImmunoPharmaceuticals ("SMIPs"); single-stranded diabodies or tandem diabodies (TandAb)); VHH; Anticalins (registered trademark); Nanobodies (registered trademark) minibodies; BiTE (registered trademark); Anrikin repeat proteins or DARPINs (registered trademark); Avimers (registered trademark); DART; TCR-like antibodies; Adnectins (registered trademark); Affilins (registered trademark); Trans-bodies (registered trademark); Affibodies (registered trademark); TrimerX (registered trademark); MicroProteins; Fynomers (registered trademark); Centyrins (registered trademark); and KALBITOR (registered trademark). In some embodiments, antibodies may lack covalent modifications (e.g., glycan binding) that would be present if naturally produced. In some embodiments, the antibody may include covalent modifications (e.g., glycan linkage, payload [e.g., a detectable portion, a therapeutic portion, a catalytic portion, etc.]) or other pendant groups [e.g., polyethylene glycol, etc.]. In many embodiments, the antibody agent is a polypeptide or comprises a polypeptide in which one or more structural elements, recognized by those skilled in the art as complementarity-determining regions (CDRs), are included in the amino acid sequence.In some embodiments, the antibody agent is a polypeptide containing, or comprises, an amino acid sequence containing at least one CDR (e.g., at least one heavy-chain CDR and / or at least one light-chain CDR) that is substantially identical to that found in the reference antibody. In some embodiments, the contained CDR is substantially identical to the reference CDR in that its sequence is identical or it contains 1 to 5 amino acid substitutions when compared to the reference CDR. In some embodiments, the contained CDR is substantially identical to the reference CDR in that it exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the reference CDR. In some embodiments, the contained CDR is substantially identical to the reference CDR in that it exhibits at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the reference CDR. In some embodiments, the included CDR is substantially identical to the reference CDR in that at least one amino acid in the included CDR is deleted, added, or substituted compared to the reference CDR, but the included CDR has an amino acid sequence that is otherwise identical to that of the reference CDR. In some embodiments, the included CDR is substantially identical to the reference CDR in that 1 to 5 amino acids in the included CDR are deleted, added, or substituted compared to the reference CDR, but the included CDR has an amino acid sequence that is otherwise identical to that of the reference CDR. In some embodiments, the included CDR is substantially identical to the reference CDR in that at least one amino acid in the included CDR is substituted compared to the reference CDR, but the included CDR has an amino acid sequence that is otherwise identical to that of the reference CDR. In some embodiments, the included CDR is substantially identical to the reference CDR in that it has an amino acid sequence that is otherwise identical to the reference CDR, although 1 to 5 amino acids in the included CDR are deleted, added, or substituted compared to the reference CDR.In some embodiments, the antibody agent is a polypeptide comprising a structural element whose amino acid sequence is recognized by those skilled in the art as an immunoglobulin variable domain, or comprises a polypeptide comprising a structural element whose amino acid sequence is recognized by those skilled in the art as an immunoglobulin variable domain. In some embodiments, the antibody agent is a polypeptide protein having a binding domain that is homologous to, or largely homologous to, an immunoglobulin binding domain.

[0071] Antibody preparations can be prepared by those skilled in the art using methods known in the art and commercially available services and kits. For example, methods for preparing monoclonal antibodies are well known in the art and include hybridoma technology and phage display technology. Further antibodies suitable for use in this disclosure are described, for example, in the following publications: Antibodies A Laboratory Manual, Second Edition. Edward A. Greenfield. Cold Spring Harbor Laboratory Press (September 30, 2013); Making and Using Antibodies: A Practical Handbook, Second Edition. Eds. Gary C. Howard and Matthew R. Kaser. CRC Press (July 29, 2013); Antibody Engineering: Methods and Protocols, Second Edition (Methods in Molecular Biology). Patrick Chames. Humana Press (August 21, 2012); Monoclonal Antibodies: Methods and Protocols (Methods in Molecular Biology). Eds. Vincent Ossipow and Nicolas Fischer. Humana Press (February 12, 2014); and Human Monoclonal Antibodies: Methods and Protocols(Methods in Molecular Biology).Michael Steinitz.Humana Press(September 30,2013)).

[0072] Antibodies can be produced by standard techniques, for example, by immunization with a suitable polypeptide or part thereof, or by using a phage display library. If polyclonal antibodies are desired, selected mammals (e.g., mice, rabbits, goats, horses, etc.) are immunized with an immunogenic polypeptide having the desired epitope(s) and optionally haptened to another polypeptide. Depending on the host species, various adjuvants may be used to enhance the immunological response. Such adjuvants include, but are not limited to, Freund's gel, mineral gels such as aluminum hydroxide, and surfactants such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanins, and dinitrophenol. Serum from the immunized animals is collected and treated according to known procedures. If the serum containing polyclonal antibodies against the desired epitope also contains antibodies against other antigens, the polyclonal antibodies can be purified by immunoaffinity chromatography or any other method known in the art. Techniques for producing and processing polyclonal antiserums are well known in this field.

[0073] Antigen: As used herein, the term “antigen” means an agent that induces an immune response, and / or (ii) an agent that binds to a T cell receptor (e.g., when presented by an MHC molecule) or an antibody. In some embodiments, the antigen induces a humoral response (e.g., including the production of antigen-specific antibodies). In some embodiments, the antigen induces a cellular response (e.g., the involvement of T cells whose receptors specifically interact with the antigen). In some embodiments, the antigen may or may not bind to an antibody and induce a specific physiological response in an organism. Generally, the antigen is or may include any chemical entity such as, for example, small molecules, nucleic acids, polypeptides, carbohydrates, lipids, or macromolecules (in some embodiments, other than biomolecules [e.g., other than nucleic acids or amino acid macromolecules]). In some embodiments, the antigen is or includes a polypeptide. In some embodiments, the antigen is or includes a glycan. Those skilled in the art will understand that antigens may generally be provided in isolated or pure form, or alternatively in crude form (e.g., together with other materials, such as cell extracts or other relatively crude preparations of antigen-containing sources). In some embodiments, the antigens utilized according to the present invention are provided in crude form. In some embodiments, the antigen is a recombinant antigen.

[0074] Autologous: The term "autologous" is used to describe something that originates from the same source. For example, "autologous cells" refer to cells that originate from the same source. Introducing autologous cells into a target is advantageous because these cells can overcome immunological barriers that would otherwise cause rejection.

[0075] Congenital Cross-Origin: The term "congenital cross-origin" is used to describe things that originate from different individuals of the same species. Two or more individuals are said to be congenital cross-origin if they do not have identical genes at one or more loci.

[0076] Base pairs: As understood in the art, a “base pair” is a structural motif of a secondary structure in which two nucleotide bases are joined to each other via hydrogen bonds between the donor and acceptor sites on the bases. Complementary bases, A:U and G:C, are understood to be able to form stable base pairs via hydrogen bonds between the donor and acceptor sites on the bases. The A:U and G:C base pair is called a Watson-Crick base pair. Weaker base pairs (called wobble base pairs) are formed by bases G and U (G:U). The A:U and G:C base pair can be called a “canonical” base pair. Other base pairs, such as G:U (which occurs fairly often in RNA) and other relatively uncommon base pairs (e.g., A:C, U:U), can be called non-canonical base pairs.

[0077] Batch: As used herein, the terms “batch” or “batch reaction” or similar terms mean at least one separate replenishment event for at least one component (e.g., in some embodiments), in particular at least one such event for UTP or an analog thereof, optionally other components, optionally at least one such event for multiple components replenished in the same separate replenishment event.

[0078] Bonding: As used herein, the term “bonding” is typically understood to mean a non-covalent bond between two or more entities. “Direct” bonding involves physical contact between entities or parts, while indirect bonding involves physical interaction through physical contact with one or more intermediate entities. Bonding between two or more entities can typically be evaluated in any of a variety of contexts, including when the interacting entities or parts are studied alone or in relation to more complex systems (e.g., in a covalent or otherwise related state to a carrier entity, and / or in a biological system or cell).

[0079] Bioreactor: As used herein, the term “bioreactor” refers to the vessel used for in vitro transfer as described herein. A bioreactor can be of any size, as long as it is useful for in vitro transfer. For example, in some embodiments, a bioreactor may be at least 0.5 liters, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50 liters or more, or any capacity in between. Internal conditions of the bioreactor (including, but not limited to, pH and temperature), typically controlled during in vitro transfer. A bioreactor may be constructed of any material suitable for in vitro transfer under the conditions described herein, such as glass, plastic, or metal. Those skilled in the art will recognize and be able to select a suitable bioreactor capacity to use when carrying out in vitro transfer.

[0080] Cap: As used herein, the term “cap” typically refers to a structure comprising, or essentially consisting of, a nucleoside-5'-triphosphate that binds to the 5' end of capless RNA (e.g., capless RNA having a 5'-diphosphate). In some embodiments, the cap is or comprises a guanine nucleotide. In some embodiments, the cap is or comprises a naturally occurring RNA 5' cap, such as, for example, an N7-methylguanosine cap having a structure called “m7G”. In some embodiments, the cap is or comprises a synthetic cap analog that is similar to an RNA cap structure and has the ability to stabilize RNA when attached thereto, for example, but is not limited to, anti-reverse cap analogs (ARCAs) known in the art. Those skilled in the art will understand that methods for ligating a cap to the 5' end of RNA are known in the art. For example, in some embodiments, capped RNA can be obtained by in vitro capping RNA having a 5' triphosphate group or RNA having a 5' diphosphate group with a capping enzyme system (e.g., a vaccinia capping enzyme system or a Saccharomyces cerevisiae capping enzyme system, but not limited to these). Alternatively, capped RNA can be obtained by in vitro transcription (IVT) of a DNA template, where the IVT system includes GTP as well as, for example, capping analogs known in the art. Non-limiting examples of capping analogs include m7GpppG capping analogs or N7-methyl-,2'-O-methyl-GpppG ARCA capping analogs or N7-methyl-,3'-O-methyl-GpppG ARCA capping analogs, or any commercially available capping analog such as CleanCap (Trilink), EZ Cap, etc. In some embodiments, the capping analog is or includes a trinucleotide capping analog.Various capping analogs are described herein, are known in the art, and are commercially available, for example.

[0081] Codon: As understood in the art, the term "codon" refers to a triplet of bases within a coding nucleic acid that specifies the next amino acid to be added during protein synthesis in ribosomes.

[0082] Comparable: As used herein, the term “comparable” means two or more sets of agents, substances, situations, conditions, etc., which do not necessarily have to be identical to one another, but are similar enough to allow comparison between them, such that a person skilled in the art would understand that a reasonable conclusion can be drawn based on observed differences or similarities. In some embodiments, a comparable set of conditions, situations, individuals, or groups is characterized by several substantially identical features and one or a few diverse features. A person skilled in the art will understand the degree of identity required for two or more such sets of agents, substances, situations, conditions, etc., to be considered comparable in any given situation in the context. For example, a person skilled in the art will understand that sets of situations, individuals, or groups are comparable if they are characterized by a sufficient number and type of substantially identical features to ensure a reasonable conclusion that differences in results or observed phenomena obtained under or using different sets of situations, individuals, or groups are due to or exhibit differences in these diverse features.

[0083] Complementary: As used herein, the term “complementary” is used in reference to oligonucleotide hybridization relating to base pairing rules. For example, the sequence “CAGT” is complementary to the sequence “GTCA”. Complementarity can be partial or total. Therefore, any degree of partial complementarity is intended to be within the scope of the term “complementary,” as long as partial complementarity enables oligonucleotide hybridization. Partial complementarity is when one or more nucleic acid bases do not match according to the base pairing rules. Total or complete complementarity between nucleic acids is when each and all nucleic acid bases match with another base according to the base pairing rules. As is understood in the art, percent complementarity refers to the percentage of consecutive residues in a nucleic acid molecule that can form hydrogen bonds (e.g., Watson-Crick base pairing) with a second nucleic acid sequence (e.g., 5, 6, 7, 8, 9, and 10 out of 10 are 50%, 60%, 70%, 80%, 90%, and 100% complementary). "Fully complementary" means that all consecutive residues in the nucleic acid sequence form hydrogen bonds with the same number of consecutive residues in the second nucleic acid sequence. In many embodiments, the degree of complementarity according to the present invention is at least 70%, preferably at least 75%, preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, or most preferably at least 95%, 96%, 97%, 98%, or 99%. In certain embodiments, the degree of complementarity according to the present invention is 100%.

[0084] Comprising: Unless otherwise explicitly specified, the term “comprising” in the context of this document is used to indicate that there may be additional members in addition to the members of the list introduced by “comprising.” However, in certain embodiments of the present invention, the term “comprising” is intended to encompass the possibility that there may be no additional members; that is, for the purposes of this embodiment, “comprising” is understood to mean “consisting of.”

[0085] Decreasing, Lowering, Inhibiting: As understood in the Art, terms such as “decreasing,” “lowering,” or “inhibiting” may be used herein to refer to the ability to cause an overall and / or relative decrease in, for example, an entity, event, frequency, activity, etc. In some embodiments, the decrease may be, for example, 5% or more, 10% or more, 20% or more, 25% or more, 30% or more, 35% or more, 40% or more, 45% or more; in some embodiments, it may be 50% or more, 60% or more, 70% or more; and in some embodiments, it may be 75% or more. In some embodiments, the decrease may be 2 times or more, 3 times or more, 4 times or more, 5 times or more, 6 times or more, 7 times or more, 8 times or more, 9 times or more, 10 times or more, 15 times or more, 20 times or more, 25 times or more, 30 times or more, 40 times or more, 50 times or more, 100 times or more, and so on. In some embodiments, the decrease is evaluated in comparison to an appropriate standard. In some embodiments, the inhibition may be, for example, at an undetectable level, such as zero or essentially zero, or "essentially complete".

[0086] Derivatives: As used herein, the term “derivative” refers to a structural analog of a reference substance. That is, a “derivative” is a substance that exhibits significant structural similarity to a reference substance, for example, having a common core or consensus structure, but differing from the reference substance in a particular distinct way. In some embodiments, a derivative is a substance that can be produced from a reference substance by chemical manipulation. In some embodiments, a derivative is a substance that can be produced by carrying out a synthetic process that is substantially similar to the process that produces the reference substance (e.g., having a common set of steps). For example, in some embodiments, a “derivative” of a nucleic acid residue may be a difference on a nucleotide base, sugar, or phosphate, or may include such differences. In some embodiments, a “derivative” of a nucleic acid may be a nucleic acid containing one or more nucleotides and / or nucleotide analogs that do not exist in nature. In some embodiments, a derivative of a nucleic acid is more stable than an equivalent nucleic acid lacking the relevant derivatization. In some embodiments, the term “derivative” is used to refer to a nucleic acid sequence that is a variant with respect to a particular reference sequence; in some such embodiments, such a derived (i.e., variant) sequence exhibits equivalent or improved stability and / or translation efficiency compared to that parent reference sequence, for example, when substituting such a parent reference sequence in an RNA molecule.

[0087] Detecting: The term “detecting” is used herein to mean a suitable means of determining whether or not the entity of interest is present, or any form of measurement of the entity of interest in a sample. Thus, “detecting” may include determining, measuring, evaluating, or assaying the presence, level, quantity, and / or location of the entity of interest. This includes quantitative and qualitative determinations, measurements, or evaluations, such as semi-quantitative ones. Such determinations, measurements, or evaluations may be relative, for example, if the entity of interest is detected against a control standard, or they may be absolute. Thus, the term “quantify” when used in the context of quantifying the entity of interest refers to absolute or relative quantification. Absolute quantification may be achieved by correlating the detection level of the entity of interest with a known control standard (e.g., by generating a standard curve). Alternatively, relative quantification may be achieved by comparing the detection levels or quantities of two or more different entities of interest, resulting in relative quantification of each of two or more different entities of interest, i.e., one another.

[0088] Determining: Those skilled in the art will understand, upon reading this specification, that the “determining” step may be achieved by utilizing, or by using, any of the various techniques available to those skilled in the art, such as certain techniques explicitly mentioned herein. In some embodiments, determining involves the manipulation of a physical sample. In some embodiments, determining involves the consideration and / or manipulation of data or information, for example, using a computer or other processing unit adapted to perform the relevant analysis. In some embodiments, determining involves receiving relevant information and / or material from a source. In some embodiments, determining involves comparing one or more features of a sample or entity with a comparable reference.

[0089] Dosage Form or Unit Dosage Form: Those skilled in the art will understand that the term “dosage form” may be used to mean a physically independent unit of an active substance (e.g., a therapeutic or diagnostic substance) administered to a subject. Typically, each such unit contains a predetermined amount of the active substance. In some embodiments, such amount is a unit dose (or all thereof) appropriate for administration according to a drug regimen (i.e., a therapeutic drug regimen) that has been established to correlate with a desired or beneficial outcome when administered to a relevant population. Those skilled in the art will understand that the total amount of a therapeutic composition or agent administered to a particular subject may be determined by one or more attending physicians and may include administration in multiple dosage forms.

[0090] Encapsulation: The terms “encapsulation” or “encapsulation” are used herein to mean that at least a portion of a component is enclosed or surrounded by another material or component in the composition. In some embodiments, a component may be completely enclosed or surrounded by another material or component in the composition.

[0091] Excipients: As used herein, the term “excipient” refers to non-therapeutic substances that may be included in a pharmaceutical composition to provide or contribute to, for example, desired properties or effects (e.g., desired consistency, delivery, and / or stabilizing effect). In some embodiments, the term “excipient” is intended to refer to substances that may be present in a pharmaceutical composition and are not active ingredients, such as carriers, binders, lubricants, thickeners, surfactants, preservatives, emulsifiers, buffers, fragrances, or colorants. In some embodiments, suitable pharmaceutical excipients to be added to an LNP composition may include, for example, salts, starch, glucose, lactose, sucrose, gelatin, sodium chloride, glycerol, propylene, glycol, water, ethanol, and the like.

[0092] Code: As used herein, the terms “code” or “to code” refer to sequence information of a first molecule that leads to the production of a second molecule having a defined sequence of nucleotides (e.g., mRNA) or a defined sequence of amino acids. For example, a DNA molecule can code for an RNA molecule (e.g., by a transcription process involving the DNA-dependent RNA polymerase enzyme). An RNA molecule can code for a polypeptide (e.g., by a translation process). Thus, a gene, cDNA, or single-stranded RNA (e.g., mRNA) codes for a polypeptide if the transcription and translation of the mRNA corresponding to that gene produces polypeptides in a cell or other biological system. In some embodiments, the coding region of a single-stranded RNA encoding a target polypeptide agent refers to a coding strand whose nucleotide sequence is identical to the mRNA sequence of such a target polypeptide agent. In some embodiments, the coding region of a single-stranded RNA encoding a target polypeptide agent refers to a non-coding strand of such a target polypeptide agent that can be used as a template for the transcription of a gene or cDNA. As understood in the art, the phrase “nucleic acid encoding a peptide or protein” means that a nucleic acid, when present in a suitable environment, such as intracellular and / or cell-free translation systems, can direct the assembly of amino acids to produce a peptide or protein through the process of translation. In some embodiments, coding can interact with the cellular translation mechanism, thereby yielding a peptide or protein encoded by the translation of such coding RNA.

[0093] Endogenous: As used herein, “endogenous” means any material that originates from or is produced within the organism, cell, tissue, or system in which it is found.

[0094] Exogenous: As used herein, the term “exogenous” means any material that is introduced into or produced outside of an organism, cell, tissue, or system in which it is located.

[0095] Expression: As understood in the Art, the term “expression” is used to refer to the production of a template nucleic acid (typically an RNA template) and / or the polypeptide encoded thereby. Thus, in some embodiments, the term may be used to refer to the production of RNA, polypeptide, RNA and polypeptide; alternatively or additionally, in some embodiments, this may refer to the partial expression of nucleic acids. Furthermore, expression can be transient, stable, or continuous. As used herein, “expression” of a nucleic acid sequence means one or more of the following events: (1) production of an RNA template from a DNA sequence (e.g., by transcription); (2) processing of an RNA transcript (e.g., by splicing, editing, 5' cap formation, and / or 3' end formation); (3) translation of RNA into a polypeptide or protein; and / or (4) post-translational modification of a polypeptide or protein. Those skilled in the art will understand that when the terms “expression” or “translation” are applied to RNA, they typically refer to the process by which ribosomes (e.g., within a cell) read a strand of coding RNA (e.g., messenger RNA) and direct the assembly of the amino acid sequence to produce the encoded peptide or protein.

[0096] Regulatory Sequences: As will be understood by those skilled in the art who read this disclosure, the term “regulatory sequence” as used herein refers to a sequence element whose presence and / or identity influences one or more characteristics of the expression of another sequence. Often, regulatory sequences are nucleic acid sequence elements and often act in cis. Thus, in some embodiments, regulatory sequences may be, for example, promoters, enhancers, repressor elements, loop-forming sites, termination sites, ribosome-binding sequences, translation pause signals, and / or other regulatory elements that can control or regulate, for example, the transcription of a gene and / or the translation of transcribed RNA. In certain embodiments of the present invention, regulatory sequences can be modulated. The exact structure of regulatory sequences present in and / or otherwise linked to a particular expressible construct may vary, for example, depending on the species or cell type of the relevant expression mechanism (e.g., RNA polymerase, splicosome, ribosome, etc.), but in many embodiments, they may include 5' untranscribed sequences and 5' and 3' untranslated sequences, respectively, which are involved in the initiation of transcription and translation. More specifically, in some embodiments, the 5' non-transcriptional expression control sequence may include a promoter region encompassing a promoter sequence for transcriptional control of a functionally linked gene. In some embodiments, the expression control sequence may also include an enhancer sequence or an upstream activator sequence. In many embodiments, the expression control sequence of a DNA molecule may include a 5' non-transcriptional sequence such as a TATA box, a capping sequence, or a CAAT sequence, as well as 5' and 3' untranslated sequences.

[0097] Fed-batch culture process: As used herein, the term “fed-batch culture process” refers to the process of introducing one or more components into a container, such as a bioreactor, at some point after the start of the reaction. In some embodiments, one or more components are introduced by the fed-batch culture process to maintain their concentration at a low level during the reaction. In some embodiments, one or more components are introduced by the fed-batch culture process to replenish those depleted during the reaction.

[0098] Five-prime untranslated region: As used herein, the term “five-prime untranslated region” or “5'UTR” refers to a sequence of an mRNA molecule that begins at the transcription start site and ends one nucleotide (nt) before the start codon (usually AUG) of the RNA coding region.

[0099] Fragment: With respect to nucleic acid sequences, “fragment” refers to a portion of a nucleic acid sequence, for example, a sequence that is smaller than the parent sequence from which the fragment originates (e.g., a nucleic acid sequence shortened at the 5' end and / or 3' end(or more), and / or shortened by the removal of one or more internal residues). In some embodiments, a fragment of a nucleic acid sequence contains at least 80% of the corresponding nucleotide residues from such a parent nucleic acid sequence, or in some embodiments, at least 90%, 95%, 96%, 97%, 98%, or 99%. In many embodiments, a fragment retains one or more characteristics or attributes of its parent sequence. For example, in some embodiments, a fragment of translatable RNA is characterized by stability and / or translation efficiency that is at least reasonably comparable to that of its parent. In some embodiments, a nucleic acid whose sequence represents two or more discontinuous sequences derived from the same parent nucleic acid fused together is considered a fragment of that parent nucleic acid.

[0100] With respect to an amino acid sequence (peptide or protein), the term "fragment" refers to a portion of the amino acid sequence, for example, an amino acid sequence that is shortened at the N-terminus and / or C-terminus, and / or an amino acid sequence in which one or more internal residues are missing. In some embodiments, a C-terminus shortened fragment (N-terminal fragment) can be obtained, for example, by translation of a shortened open reading frame lacking the 3' end of the open reading frame. In some embodiments, an N-terminus shortened fragment (C-terminal fragment) can be obtained, for example, by translation of a shortened open reading frame lacking the 5' end of the open reading frame, insofar as the shortened open reading frame contains a start codon that acts to initiate translation. In many embodiments, a fragment of an amino acid sequence contains, for example, at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, and at least 90% of amino acid residues derived from the amino acid sequence. In many embodiments, a fragment contains at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 or more amino acids, and in many embodiments, a fragment retains one or more characteristics or attributes of its parent sequence. In some embodiments, a polypeptide whose nucleic acid sequence represents two or more discontinuous sequences derived from the same parent polypeptide fused together is considered a fragment of its parent polypeptide.

[0101] Functional: As used herein, a “functional” biomolecule is a biomolecule in a form that exhibits characteristic properties and / or activity. In some embodiments, a biomolecule may have one function (i.e., monofunctional), two functions (i.e., difunctional), or many functions (i.e., polyfunctional).

[0102] Functional Analogue: In certain embodiments, “analog” is a “functional analogue.” The term “functional analogue” refers to an analogue of a substance that contains one or more functions or shares one or more functions with a reference substance. For example, a functional analogue of nucleoside triphosphate (NTP) shares one or more functions with a reference NTP. For example, a functional analogue of GTP shares one or more functions with GTP. For example, a functional analogue of CTP shares one or more functions with CTP. For example, a functional analogue of ATP shares one or more functions with ATP. For example, a functional analogue of UTP shares one or more functions with UTP. In certain embodiments, a functional analogue of NTP is translatable. In certain embodiments, a functional analogue of NTP can be incorporated into a product molecule, such as RNA, instead of replacing, a reference NTP. In certain embodiments, when a functional analogue of NTP is incorporated into an RNA molecule, the RNA molecule becomes translatable, in which the functional analogue functions as the reference NTP during translation. In some embodiments, functional analogs of NTP have additional characteristics not shared with the reference NTP; for example, it is known that by incorporating pseudo-UTP and / or N1-methyl pseudo-UTP instead of UTP, i.e., instead of replacing UTP, RNA with reduced immunogenicity can be obtained compared to RNA transcribed from the same template using unmodified UTP. In some embodiments, functional analogs of UTP can be incorporated into RNA molecules instead of UTP and / or are translatable as, for example, UTP or in place of UTP. Similarly, functional analogs of GTP can be incorporated into RNA molecules instead of GTP and / or are translatable as, for example, GTP or in place of GTP. The same applies to CTP and ATP. In certain embodiments, functional analogs of NTP can be incorporated during the synthesis of the RNA molecule at any predetermined position where each NTP is expected or predicted, depending on the matrix used, such as the DNA on which the RNA is transcribed.

[0103] Functional linkage: As used herein, “functional linkage” or “functionally linked” refers to a linkage within a functional relationship. When a nucleic acid is functionally related to another nucleic acid sequence, that nucleic acid is said to be “functionally linked.” For example, a promoter is functionally linked to a coding sequence if it affects the transcription of that coding sequence. Functionally linked nucleic acids are typically adjacent to each other and may be separated as appropriate by further nucleic acid sequences and, in certain embodiments, transcribed by RNA polymerase to obtain a single RNA molecule (common transcript). In certain embodiments, nucleic acids are functionally linked to expression regulatory sequences that may be homogeneous or heterogeneous with respect to the nucleic acid, in accordance with the present invention.

[0104] Gene: As used herein, the term “gene” refers to a DNA sequence in a chromosome that codes for a product (e.g., an RNA product and / or a polypeptide product). In some embodiments, a gene includes a coding sequence (i.e., a sequence that codes for a particular product); in some embodiments, a gene includes a non-coding sequence. In some specific embodiments, a gene may include both non-coding (e.g., exon) sequences and non-coding (e.g., intron) sequences. In some embodiments, a gene may include one or more regulatory elements that can control or influence one or more aspects of gene expression (e.g., cell type-specific expression, inducible expression, etc.).

[0105] Gene product or expression product: As used herein, the terms “gene product” or “expression product” generally refer to RNA transcribed from a gene (before and / or after processing), or polypeptides encoded by RNA transcribed from a gene (before and / or after modification).

[0106] Heterogeneous: The term “heterogeneous” is used herein to describe any entity relative to a reference substance and to specify that the entity originates from a different source than the reference substance in question and / or is bound to one or more components different from the reference substance in question. For example, introducing cells from one individual into another individual constitutes xenotransplantation. Heterogeneous genes are genes that originate from a source other than the target.

[0107] Homologous: As used herein, the term “homologous” or “homonymy” refers to the overall relationship between polynucleotide molecules (e.g., DNA molecules and / or RNA molecules) and / or polypeptide molecules. In some embodiments, polynucleotide molecules (e.g., DNA molecules and / or RNA molecules) and / or polypeptide molecules are considered “homologous” to one another if their sequences are identical by at least 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%. In some embodiments, polynucleotide molecules (e.g., DNA molecules and / or RNA molecules) and / or polypeptide molecules are considered “homologous” to one another if their sequences are at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% similar (e.g., containing residues with related chemical properties at related positions). For example, as is well known to those skilled in the art, certain amino acids are typically classified as similar to one another as “hydrophobic” or “hydrophilic” amino acids and / or having “polar” or “nonpolar” side chains. Substituting one amino acid for another of the same type can often be considered a “homologous” substitution.

[0108] Host cell: As used herein, refers to a cell into which exogenous material (such as recombinant DNA or other) has been introduced. Those skilled in the art will understand, upon reading this disclosure, that such a term refers not only to a specific target cell but also to the offspring of such a cell. Such offspring may not be identical to the parent cell in practice, because certain modifications may occur in subsequent generations due to either mutation or environmental influences, but they still fall within the scope of the term “host cell” as used herein. In some embodiments, host cells include prokaryotic and eukaryotic cells selected from any living world suitable for expressing exogenous DNA (e.g., recombinant nucleic acid sequences). Examples of cells include prokaryotic and eukaryotic cells (unicellular or multicellular), bacterial cells (e.g., strains such as E. coli, Bacillus spp., and Streptomyces spp.), mycobacterial cells, fungal cells, yeast cells (e.g., S. cerevisiae, S. pombe, P. pastoris, P. methanolica, etc.), plant cells, insect cells (e.g., SF-9, SF-21, baculovirus-infected insect cells, Trichoplusia ni, etc.), non-human animal cells, human cells, or cell fusions such as hybridomas or quadromas. In some embodiments, the host cells are human, monkey, ape, hamster, rat, or mouse cells. In some embodiments, the host cells are eukaryotes. For example, eukaryotic host cells may include: CHO (e.g., CHO Kl, DXB-1 1 CHO, Veggie-CHO), COS (e.g., COS-7), retinal cells, Vero, CV1, kidney (e.g., HEK293, 293 EBNA, MSR 293, MDCK, HaK, BHK), HeLa, HepG2, WI38, MRC 5, Colo205, HB 8065, HL-60, (e.g., BHK21), Jurkat, Daudi, A431 (epidermal), CV-1, U937, 3T3, L cells, C127 cells, SP2 / 0, NS-0, MMT 060562, Sertoli cells, BRL 3 A cells, HT1080 cells, myeloma cells, tumor cells, or cell lines derived from the aforementioned cells.

[0109] Hybridization: A nucleic acid can "hybridize" or "hybridizes" with another nucleic acid if two sequences are complementary to each other. A nucleic acid is "complementary" to another nucleic acid if two sequences can form a stable double helix with each other, for example, if they can hybridize to form a double-stranded molecule. Complementarity can be whole or partial. Those skilled in the art will recognize that the ability of two sequences to hybridize with each other may depend on conditions (e.g., temperature, pH) and / or the presence of other potentially competing sequences. In some embodiments, hybridization is carried out under stringent conditions such that only highly complementary sequences form a stable hybrid. Examples of such stringent conditions are described, for example, in Molecular Cloning: A Laboratory Manual, J. Sambrook et al., Editors, 2nd Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989 or Current Protocols in Molecular Biology, FMAusubel et al., Editors, John Wiley & Sons, Inc., New York. For example, in some embodiments, stringent hybridization may involve incubation of nucleic acids to hybridize with a membrane containing complementary nucleic acids at 65°C in a hybridization buffer (3.5x SSC, 0.02% Ficol, 0.02% polyvinylpyrrolidone, 0.02% bovine serum albumin, 2.5 mM NaH2PO4 (pH 7), 0.5% SDS, 2 mM EDTA). SSC is 0.15 M sodium chloride / 0.15 M sodium citrate, pH 7. After incubation, the membrane to which the DNA has been transferred is washed, for example, at room temperature with 2xSSC, and then washed with 0.1-0.5xSSC / 0.1xSDS at temperatures up to 68°C.

[0110] Identity: As used herein, the term “identity” refers to the overall relationship between polymer molecules, e.g., between nucleic acid molecules (e.g., DNA molecules and / or RNA molecules) and / or between polypeptide molecules. In some embodiments, multiple polymer molecules are considered “substantially identical” to one another if their sequences are identical by at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%. For example, the percentage of identity of the sequences of two nucleic acids or polypeptides can be calculated by aligning the two sequences for the purpose of best comparison (for example, gaps may be introduced in one or both of the first and second sequences for best alignment, and non-identical sequences can be ignored for the purpose of comparison). In certain embodiments, the length of a sequence aligned for comparison purposes is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or substantially 100% of the length of the reference sequence. The nucleotides at the corresponding positions are then compared. The molecules are identical at a given position if the position in the first sequence is occupied by the same residue (e.g., a nucleotide or amino acid) as the corresponding position in the second sequence. The percentage of identity between two sequences is a function of the number of identical positions common to the sequences, taking into account the number of gaps that need to be introduced for optimal alignment of the two sequences and the length of each gap. The comparison of sequences and the determination of the percentage of identity between two sequences can be achieved using mathematical algorithms. For example, the algorithm by Meyers and Miller (CABIOS, 1989, 4:11-17), incorporated into the ALIGN program (version 2.0), can be used to determine the percentage of identity between two nucleotide sequences. In some exemplary embodiments, the comparison of nucleic acid sequences using the ALIGN program utilizes a PAM120 weighted residue table, a gap length penalty 12, and a gap penalty 4.Alternatively, the percentage of identity between two nucleotide sequences can also be determined using the GAP program in the GCG software package, which uses the NWSgapdna.CMP matrix.

[0111] Improvement, Increase, or Decrease: As used herein, these terms or grammatically equivalent comparative terms indicate values ​​relative to a comparable reference measure. For example, in some embodiments, an evaluation value achieved with the agent of interest may be an “improvement” compared to an evaluation value obtained with an equivalent reference agent. Alternatively or additionally, in some embodiments, an evaluation value achieved in the subject or system of interest may be an “improvement” compared to an evaluation value obtained in the same subject or system under different conditions (e.g., before or after an event such as administration of the agent of interest) or in a different equivalent subject (e.g., in the presence of one or more indicators of a particular disease, disorder, or condition of interest, or in an equivalent subject or system different from the subject or system of interest that has been previously exposed to the condition or agent). In some embodiments, comparative terms refer to a statistically significant difference (e.g., an incidence and / or magnitude sufficient to achieve a statistical significance). A person skilled in the art will know, or be able to readily determine, the degree and / or incidence of the difference necessary or sufficient to achieve such a statistically significant difference in a given context.

[0112] Increase, Enhancement: As understood in the Art, terms such as “increase” or “enhancement” may be used herein to refer to an overall and / or relative increase or enhancement in, for example, an entity, event, frequency, or activity. In some embodiments, an increase may be, for example, about at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, 55%, 65%, 70%, 75%, at least 80%, 85%, 90%, 95%, at least 100%, or more. In some embodiments, an increase or enhancement may be twofold, threefold, fourfold, fivefold, sixfold, sevenfold, eightfold, ninefold, tenfold, or more.

[0113] In vitro: As used herein, the term "in vitro" means events that occur in an artificial environment rather than within a multicellular organism, for example, in a test tube or reaction vessel (e.g., a bioreactor), under cell culture conditions, etc.

[0114] In vitro transcription, transcription: As is known in the art, the terms “transcription” and “transcription” relate to the process by which a nucleic acid molecule having a specific nucleic acid sequence (“nucleic acid template”) is read by RNA polymerase, thereby the RNA polymerase synthesizes a complementary single-stranded RNA molecule. During transcription, the genetic information within the nucleic acid template is transcribed. In some embodiments, the nucleic acid template is or contains DNA, but in some embodiments, for example, in the case of transcription from an alphavirus nucleic acid template, the nucleic acid template may be or contain RNA. In some embodiments, the nucleic acid template may not be DNA or RNA and / or may contain one or more residues that are DNA analogs or RNA analogs (e.g., including one or more modifications, e.g., skeletal modifications or base modifications) compared to standard (canonical) DNA or RNA. In many embodiments, the transcribed RNA can be translated into a protein. In many embodiments, as will be apparent from the context, the term “transcription” as used herein refers to “in vitro transcription.” As used herein, the terms “in vitro transcription” or “IVT” refer to the process by which transcription occurs in vitro (i.e., outside of an organism, typically a non-cellular system) to produce a synthetic RNA product; in many embodiments, this disclosure describes IVT for generating RNA products for use in specific applications, such as the production of proteins or polypeptides. In some embodiments, the produced RNA product may be translated in vitro or introduced directly into cells, and in some embodiments, it may be translated. In certain embodiments, the produced RNA product is of sufficient scale and / or quality to be administered to organisms (e.g., as pharmaceutically active RNA), and in some embodiments, to humans. In some embodiments, the RNA product may be selected from, for example, mRNA, antisense RNA molecules, shRNA molecules, long non-coding RNA molecules, ribozymes, aptamers, guide RNA (e.g., for CRISPR), ribosomal RNA, nuclear small RNA, nucleolar small RNA, etc.IVT reactions typically utilize a DNA template (e.g., a linear DNA template), ribonucleotides (e.g., unmodified ribonucleotide triphosphates or modified ribonucleotide triphosphates), and a suitable RNA polymerase, as described and / or utilized herein. In some embodiments, a cloning vector is applied to produce a transcript. In some such embodiments, the cloning vector is called a “transcription vector” (which, according to the present invention, is encompassed by the term “vector”). In some embodiments, the cloning vector may be a plasmid. In some embodiments, the RNA is in vitro transcription RNA (IVT-RNA), which can be obtained by in vitro transcription of a suitable DNA template. Those skilled in the art know of various promoter sequences that can be appropriately used to control transcription by the relevant RNA polymerase. In some embodiments, the DNA template for in vitro transcription can be obtained by cloning a nucleic acid, such as cDNA, and introducing it into a suitable vector for in vitro transcription. In some embodiments, cDNA can be obtained by reverse transcription of RNA.

[0115] In vitro transcription RNA composition: As used herein, the term “in vitro transcription RNA composition” refers to a composition comprising RNA synthesized by in vitro transcription. In some embodiments, such a composition may contain excess in vitro transcription reagents (e.g., ribonucleotides and / or capping agents), nucleic acids or fragments thereof, e.g., DNA templates or fragments thereof, polypeptides or fragments thereof, e.g., recombinant enzymes or host cell proteins or fragments thereof, and / or other impurities. In some embodiments, the in vitro transcription RNA composition may be treated and / or processed in a suitable buffer at a desired concentration before a purification process that ultimately produces an RNA transcript preparation containing the RNA transcript, for formulation and / or further manufacturing and / or processing. For example, in some embodiments, the in vitro transcription RNA composition may be treated to remove or digest DNA templates (e.g., using DNase). In some embodiments, the in vitro transcription RNA composition may be treated to remove or digest polypeptides (e.g., enzymes such as RNA polymerase, RNase inhibitors, etc.) present during the in vitro transcription reaction (e.g., using proteases). Therefore, in some embodiments, the DNA template can be removed or separated from the RNA-containing composition after RNA transcription; those skilled in the art know of various methods, e.g., DNA hydrolysis, that can achieve such removal. In some embodiments, an RNase inhibitor can be added during DNA removal or digestion to protect the RNA from potential degradation. In some embodiments, the in vitro transcribed RNA composition may be treated to remove or digest peptides (e.g., enzymes such as RNA polymerase, RNase inhibitors) present during the in vitro transcription reaction (e.g., using a protease).

[0116] In vivo: The term "in vivo" refers to events that occur within multicellular organisms such as humans and non-human animals.

[0117] Isolated: As used herein, the term “isolated” typically refers to a molecule or other entity that substantially contains no other components, such as other cellular material; in some embodiments, the “isolated” entity substantially contains no components that were previously (e.g., when it was first produced) bound to it. In certain embodiments, the term “isolated nucleic acid” as used herein refers to a nucleic acid that is (i) amplified in vitro, e.g., by polymerase chain reaction (PCR), (ii) produced by recombination by cloning, (iii) purified, e.g., by cleavage and gel electrophoresis, or (iv) synthesized, e.g., by chemosynthesis or IVT. In some embodiments, the isolated nucleic acid is a nucleic acid that is available for manipulation by recombination techniques.

[0118] Linked, Fused, Fuse: As used herein, the terms “linked,” “fused,” and “fuse” are interchangeable. These terms refer to the linking of two or more elements, components, or domains, for example, domains from two different proteins or nucleic acid molecules, to one another (e.g., by covalent linking).

[0119] Messenger RNA, mRNA: According to the present invention, the term "mRNA" means "messenger RNA" and relates to a transcript that is typically produced from a template (e.g., a DNA template) and codes for a peptide or protein. Typically, mRNA includes a 5'UTR, a protein coding region, a 3'UTR, and a poly(A) sequence. In some embodiments, mRNA may be produced by in vitro transcription from a DNA template as described herein. In some embodiments, mRNA may be modified, for example, by stabilizing modification and / or capping. In some embodiments, nucleic acids such as RNA, e.g., mRNA, may code for a peptide or protein. Thus, in some embodiments, a transcriptable nucleic acid sequence or its transcript may include an open reading frame (ORF) that codes for a peptide or protein.

[0120] Nanoparticles: As used herein, the term “nanoparticles” refers to particles having a diameter of less than 1,000 nanometers (nm). In some embodiments, nanoparticles have a diameter of less than 300 nm, as defined by the National Science Foundation. In some embodiments, nanoparticles have a diameter of less than 100 nm, as defined by the National Institutes of Health. In some embodiments, nanoparticles have a diameter of less than 80 nm, as defined by the National Institutes of Health. In some embodiments, nanoparticles include one or more containment compartments, which are separated from the bulk solution by a membrane and surround and contain a space or compartment.

[0121] Nucleic Acids / Polynucleotides: As used herein, the term “nucleic acid” refers to a polymer comprising two or more nucleotide or nucleotide analog residues. In some embodiments, a nucleic acid may comprise one or more modified residues or ligatures compared to naturally occurring DNA or RNA residues. For example, in some embodiments, a nucleic acid may have one or more modifications of bases, sugars, or backbone (e.g., phosphates) compared to naturally occurring DNA or RNA residues. In some embodiments, a nucleic acid molecule refers to a nucleic acid that is or contains deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). In some embodiments, a nucleic acid may comprise or contain genomic DNA, cDNA, mRNA, viral RNA, siRNA, miRNA, shRNA, recombinantly prepared, and chemically synthesized molecules, or sequences found in them. In some embodiments, the term “nucleic acid” refers to a polymer comprising at least two residues, or more, e.g., at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, or more. In some embodiments, a nucleic acid is or contains DNA. In some embodiments, the nucleic acid is RNA or contains RNA. In some embodiments, the nucleic acid is peptide nucleic acid (PNA) or contains PNA. In some embodiments, the nucleic acid is single-stranded nucleic acid or contains single-stranded nucleic acid. In some embodiments, the nucleic acid is double-stranded nucleic acid or contains double-stranded nucleic acid. In some embodiments, the nucleic acid contains both single-stranded and double-stranded portions. In some embodiments, the nucleic acid contains a skeleton comprising one or more phosphodiester links. In some embodiments, the nucleic acid contains a skeleton comprising both phosphodiester and non-phosphodiester links. For example, in some embodiments, the nucleic acid may contain a skeleton comprising one or more phosphorothioate or 5'-N-phosphoramidite links and / or one or more peptide bonds, such as in "peptide nucleic acid".In some embodiments, the nucleic acid comprises one or more, or all, of the native residues (e.g., adenine, cytosine, deoxyadenosine, deoxycytidine, deoxyguanosine, deoxythymidine, guanine, thymine, uracil). In some embodiments, the nucleic acid comprises one or more, or all, of the non-native residues. In some embodiments, the non-natural residues include nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyladenosine, 5-methylcytidine, C-5 propynylcytidine, 1-methylpseudridine, C-5 propynyluridine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyluridine, C5-propynylcytidine, C5-methylcytidine, 2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, 6-O-methylguanine, 2-thiocytidine, methylated bases, intercalated bases, and combinations thereof). In some embodiments, non-natural residues include one or more modified sugars (e.g., 2'-fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose) compared to those of natural residues. In some embodiments, the nucleic acid has a nucleotide sequence encoding a functional gene product such as RNA or polypeptide. In some embodiments, the nucleic acid has a nucleotide sequence containing one or more introns. In some embodiments, the nucleic acid may be prepared by isolation from natural sources, enzymatic synthesis (e.g., polymerization based on complementary templates, replication in recombinant cells or systems, e.g., in vivo or in vitro, or by chemosynthesis).In some embodiments, nucleic acids are at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 1 10, 120, 130, 140, 150, 160, 170, 180, 190, 20, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9 The residue length or nucleotide length is 000, 9500, 10,000, 10,500, 11,000, 11,500, 12,000, 12,500, 13,000, 13,500, 14,000, 14,500, 15,000, 15,500, 16,000, 16,500, 17,000, 17,500, 18,000, 18,500, 19,000, 19,500, or 20,000 or more.

[0122] Nucleic acid sequence: According to the present invention, “nucleic acid sequence” refers to a sequence of residues in a nucleic acid, such as ribonucleic acid (RNA) or deoxyribonucleic acid (DNA). In some embodiments, the term is used in reference to the sequence of an entire nucleic acid molecule (e.g., a single strand of the entire nucleic acid molecule); in some embodiments, the term is used to refer to a sequence representing a part thereof (e.g., a fragment).

[0123] Nucleotides: The term "nucleotide" is used herein as is commonly understood in the art and may refer to nucleoside monophosphates, nucleoside diphosphates, and nucleoside triphosphates.

[0124] Pharmaceutical Grade: As used herein, the term “pharmaceutical grade” refers to the standards established by approved national or local pharmacopoeias (e.g., the United States Pharmacopeia and Prescriptions (USP-NF)) for chemical and biological active pharmaceutical ingredients, pharmaceuticals, dosage forms, compound preparations, excipients, medical devices, and dietary supplements.

[0125] Polypeptide: As used herein, the term “polypeptide” typically has the meaning recognized in the art as “a polymer of at least three or more amino acids.” Those skilled in the art will understand that the term “polypeptide” is intended to be sufficiently general to encompass not only polypeptides having the complete sequences described herein, but also polypeptides representing functional, biologically active, or characteristic fragments, parts, or domains (e.g., fragments, parts, or domains retaining at least one activity). In some embodiments, a polypeptide may comprise L-amino acids, D-amino acids, or both, and / or any of the various amino acid modifications or analogs known in the art. Useful modifications include, for example, terminal acetylation, amidation, and methylation. In some embodiments, a polypeptide may comprise native amino acids, non-native amino acids, synthetic amino acids, and combinations thereof (e.g., it may be or may comprise peptide mimetic compounds). In some embodiments, a polypeptide may be or may comprise an enzyme. In some embodiments, a polypeptide may be or may comprise a polypeptide antigen. In some embodiments, a polypeptide may be or may comprise an antibody agent. In some embodiments, the polypeptide may be a cytokine or may contain a cytokine.

[0126] Primary structure: As used herein with respect to nucleic acid molecules, the term “primary structure” refers to the linear sequence of monomer residues.

[0127] Promoter, Promoter Region: The terms “promoter” or “promoter region” refer to a nucleic acid sequence that directs the synthesis of a transcript, such as a coding sequence, by providing a recognition and binding site for, for example, RNA polymerase. In some embodiments, the promoter region may include further recognition or binding sites for further factors involved in regulating gene transcription. In some embodiments, the promoter may control the transcription of a prokaryotic or eukaryotic gene. In some embodiments, the promoter is “inducible” and may initiate transcription in response to an inducer; in some embodiments, the promoter may be “constitutive” if transcription is not regulated by an inducer or a cell-type-specific promoter. In some embodiments, in the absence of an inducer, the inducible promoter may be expressed only to a very low degree or not at all; in the presence of an inducer, typically the promoter is “switched on” or the transcription level increases, mediated by the binding of a particular transcription factor.

[0128] Purity or Purification: As used herein, an agent or entity is “pure” or “purified” if it is substantially free of other components. For example, a preparation containing more than about 90% of a particular agent or entity is typically considered a pure preparation. In some embodiments, the agent or entity is at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% pure in the preparation.

[0129] Ribonucleotides: As used herein, the term “ribonucleotide” encompasses both unmodified and modified ribonucleotides. For example, “unmodified ribonucleotides” include the purine bases adenine (A) and guanine (G), the pyrimidine base cytosine (C), and uracil (U). Modified ribonucleotides (analogs) may include, but are not limited to, one or more modifications such as: (a) terminal modifications, e.g., 5'-terminal modifications (e.g., phosphorylation, dephosphorylation, conjugate linkage, reverse linkage, etc.), 3'-terminal modifications (e.g., conjugate linkage, reverse linkage, etc.), (b) base modifications, e.g., substitutions by modified bases, stabilizing bases, destabilizing bases, or bases that form base pairs with an extended repertoire of a partner, or by conjugate bases, (c) sugar modifications (e.g., at the 2' or 4' position) or sugar substitutions, and (d) nucleoside linkage modifications such as phosphodiester linkage modification or substitution. In some embodiments, the modified ribonucleotide maintains at least one function of the corresponding unmodified ribonucleotide. The term "ribonucleotide" can encompass ribonucleotide triphosphates, such as modified and unmodified ribonucleotide triphosphates.

[0130] Ribonucleic acid (RNA): As used herein, the term “RNA” refers to a polymer of ribonucleotides; the terms “RNA” or “RNA molecule” relate to a molecule containing ribonucleotide residues. In some embodiments, “RNA” is composed entirely or substantially of ribonucleotide residues. As those skilled in the art will know, a standard “ribonucleotide” is a nucleotide having a hydroxyl group at the 2' position of a β-D-ribofuranosyl group. The term “RNA” may include isolated RNA such as double-stranded RNA, single-stranded RNA, partially or completely purified RNA, essentially pure RNA, synthetic RNA, and RNA produced by recombination such as modified RNA, which differs from naturally occurring RNA by the addition, deletion, substitution and / or alteration of one or more nucleotides. In some embodiments, RNA may be modified compared to a reference (e.g., naturally occurring RNA), for example, by the addition of non-nucleotide material to the terminus (or multiple terminus) of the RNA, or internally, for example, to one or more nucleotides of the RNA. In some embodiments, one or more residues or ligatures in an RNA molecule may be non-standard residues or ligatures, such as nucleotides not found in nature, chemically synthesized nucleotides, or deoxynucleotides; in some embodiments, such RNA may be called an analog, e.g., an analog of a naturally occurring RNA. In some embodiments, RNA is single-stranded. In some embodiments, RNA is double-stranded. In some embodiments, RNA contains both single-stranded and double-stranded portions. In some embodiments, RNA may contain a skeletal structure such as those described in the above definition of “nucleic acid / polynucleotide.” RNA may be regulatory RNA (e.g., siRNA, microRNA, etc.) or messenger RNA (mRNA). In some embodiments, RNA is mRNA. In some embodiments where RNA is mRNA, RNA typically contains a poly(A) region at its 3' end.In some embodiments where RNA is mRNA, the RNA typically includes a cap structure at its 5' end, for example, a structure recognized in the art for recognizing mRNA and attaching it to a ribosome to initiate translation. In some embodiments, RNA is synthetic RNA. Examples of synthetic RNA include RNA synthesized in vitro (e.g., by enzymatic synthesis and / or chemical synthesis). In some embodiments, RNA is single-stranded RNA. In some embodiments, single-stranded RNA may include self-complementary elements and / or establish secondary and / or tertiary structures. Those skilled in the art will understand that the term “single-stranded RNA” generally refers to an RNA molecule that is not bound to a complementary nucleic acid molecule (typically, one without a complementary RNA molecule). In some embodiments, single-stranded RNA may include self-complementary sequences that allow a portion of the RNA to fold to form secondary structural motifs, including but not limited to base pairs, stems, stem-loops and / or bulges, known in the art. Those skilled in the art will understand from the context whether, when single-stranded RNA is referred to as "coding," it refers to the coding strand sequence or its complementary strand. In some embodiments, single-stranded RNA may be self-amplifying RNA (also called self-replicating RNA).

[0131] Recombinant: As used herein, the term “recombinant” is intended to mean polypeptides designed, engineered, prepared, expressed, produced, manufactured, and / or isolated by recombinant means, such as polypeptides expressed using recombinant expression vectors transfected into host cells; polypeptides isolated from recombinant combinatorial human polypeptide libraries; polypeptides isolated from animals (e.g., mice, rabbits, sheep, fish, etc.) that are transgenic with respect to genes or genetic components that encode and / or direct the expression of the polypeptide or one or more components, parts, elements, or domains; and / or polypeptides prepared, expressed, produced, or isolated by any other means, including splicing of selected nucleic acid sequence elements or ligation of selected nucleic acid sequence elements, chemical synthesis of selected sequence elements, and / or the production of nucleic acids that encode and / or direct the expression of the polypeptide or one or more components, parts, elements, or domains. In some embodiments, one or more of the sequence elements thus selected are found in nature. In some embodiments, one or more of the selected sequence elements are designed in silico. In some embodiments, one or more of the selected sequence elements are obtained from mutagenesis (e.g., in vivo or in vitro) of known sequence elements from a natural or synthetic source, such as the germline of the source organism of interest (e.g., human, mouse, etc.). In some embodiments, the nucleic acids described herein may be recombinant molecules and / or isolated molecules.

[0132] Reference: As used herein, the term “reference” refers to a standard or control for the subject being compared. For example, in some embodiments, the target agent, animal, individual, population, sample, sequence, or value is compared to the reference or control agent, animal, individual, population, sample, sequence, or value. In some embodiments, the reference or control is tested and / or determined substantially concurrently with the test or determination of the target. In some embodiments, the reference or control is a historical reference or control embodied in a tangible medium, at the discretion of the author. Typically, as will be understood by those skilled in the art, the reference or control is determined or analyzed under conditions or circumstances comparable to the subject being evaluated. Those skilled in the art will understand that there may be sufficient similarity to justify reliance on and / or comparison to a particular reference or control.

[0133] RNA polymerase: As used herein, the term "RNA polymerase" refers to an enzyme that catalyzes polyribonucleotide synthesis by adding ribonucleotide units to a nucleotide chain using DNA or RNA as a template. As is evident from the context, the term refers to either a complete enzyme found in nature, an isolated active catalytic domain or functional domain, or a fragment thereof. In some embodiments, the RNA polymerase enzyme initiates synthesis at a primer or the 3' end of a nucleic acid chain, or at a promoter sequence, and proceeds along the target nucleic acid in the 5' direction, synthesizing a chain complementary to the target nucleic acid until synthesis is complete.

[0134] RNA Transcript Preparations: As used herein, the term “RNA transcript preparation” refers to a preparation comprising an RNA transcript purified from an in vitro transcription RNA composition described herein. In some embodiments, an RNA transcript preparation is a preparation comprising a pharmaceutical-grade RNA transcript. In some embodiments, an RNA transcript preparation is a preparation comprising an RNA transcript, characterized by one or more product quality attributes determined to meet release and / or acceptance criteria (e.g., as described herein). Examples of such product quality attributes, but not limited to, include appearance, RNA length, identity of the drug substance as RNA, RNA integrity, RNA sequence, RNA concentration, pH, osmotic pressure, residual DNA template, residual double-stranded RNA, bacterial endotoxin, bioburden, and combinations thereof.

[0135] Room temperature: As used herein, the term “room temperature” refers to ambient temperature. In some embodiments, room temperature is about 15°C, 16°C, 17°C, 18°C, 19°C, 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, preferably about 18°C ​​to 30°C, for example, about 18°C ​​to 25°C, or about 20°C to 25°C, or about 20 to 30°C, or about 23 to 27°C, or about 25°C.

[0136] Sample: As used herein, the term “sample” typically refers to an aliquot of material obtained from or derived from the source of interest as described herein. In some embodiments, the source of interest is a biological source or an environmental source. In some embodiments, the source of interest is or may include cells or organisms such as microorganisms, plants, or animals (e.g., mice). In some embodiments, the source of interest is or may include biological tissue or body fluids. In some embodiments, body fluids are or may include intracellular fluid, extracellular fluid, intravascular fluid (plasma), interstitial fluid, lymph, and / or cellular fluid. In some embodiments, biological tissue or samples may be obtained, for example, by aspiration, biopsy (e.g., fine needle or tissue biopsy), swab (e.g., oral, nasal, skin, or vaginal swab), scraping, surgery, rinsing, or washing (e.g., bronchoalveolar, tubal, nasal, eye, oral cavity, uterus, vagina, or other rinsing or washing). In some embodiments, the sample is cells obtained from or including cells obtained from the subject. In some embodiments, the sample is a “primary sample” obtained directly from the source of interest by any suitable means. In some embodiments, as is evident from the context, the term “sample” refers to a preparation obtained by processing the primary sample (e.g., removing one or more components and / or adding one or more agents). For example, a “processed sample” may include nucleic acids or proteins extracted from the sample, or obtained by subjecting the primary sample to one or more techniques such as nucleic acid amplification or reverse transcription, isolation and / or purification of specific components.

[0137] Secondary Structure: As understood in the art, the term “secondary structure” is used to refer to the interactions between bases in a nucleic acid molecule. Therefore, secondary structure can be described as a two-dimensional representation of a nucleic acid molecule that reflects base pairs. This term is often used in relation to intramolecular base-pair interactions in single-stranded molecules, such as single-stranded RNA. Indeed, many single-stranded nucleic acid molecules, particularly single-stranded RNA molecules, are characterized by regions of (intramolecular) base pairs. According to the present invention, the term “secondary structure” includes, but is not limited to, structural motifs such as base pairs, stems, stem-loops, bulges, internal loops, and loops such as multi-branched loops. The secondary structure of a nucleic acid molecule can be represented by a two-dimensional figure (planar graph) showing base pairs (for further details on the secondary structure of RNA molecules, see Auber et al., (2006), J. Graph Algorithms Appl., 10:329-351). As described herein, the secondary structure of a particular RNA molecule is relevant in the context of the present invention. The secondary structure of nucleic acid molecules, particularly single-stranded RNA molecules, can be determined by prediction using the RNA secondary structure prediction web server (http: / / rna.urmc.rochester.edu / RNAstructureWeb / Servers / Predict1 / Predict1.html).

[0138] Stability: When the term “stable” is applied to nucleic acids and / or compositions containing nucleic acids (e.g., encapsulated in lipid nanoparticles), it means that such nucleic acids and / or compositions maintain one or more aspects of their characteristics (e.g., physical and / or structural characteristics, function, and / or activity) over a period of time under a specified set of conditions (e.g., pH, temperature, light, relative humidity, etc.). In some embodiments, such stability is maintained for a period of at least about 1 hour; in some embodiments, such stability is maintained for about 5 hours, about 10 hours, about 1 day, about 1 week, about 2 weeks, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 8 months, about 10 months, about 12 months, about 24 months, about 36 months, or longer. In some embodiments, such stability is maintained over periods ranging from about 1 day to about 24 months, about 2 weeks to about 12 months, 2 months to about 5 months, etc. In some embodiments, such stability is maintained under ambient conditions (e.g., room temperature and ambient pressure). In some embodiments, such stability is maintained under physiological conditions (e.g., in vivo, or at about 37°C, e.g., in serum or phosphate-buffered saline). In some embodiments, such stability is maintained under refrigerated storage (e.g., below about 4°C, e.g., -20°C or -70°C). In some embodiments, such stability is maintained when the nucleic acid and / or composition containing it is protected from light (e.g., kept in the dark).

[0139] As an example, in some embodiments, the term “stable” is used in reference to nanoparticle compositions (e.g., lipid nanoparticle compositions). In such embodiments, a stable nanoparticle composition (e.g., a stable nanoparticle composition) and / or its component(s) maintain one or more aspects of its characteristics (e.g., physical and / or structural characteristics, functions, and / or activity) over a period of time under a specified set of conditions. For example, in some embodiments, a stable nanoparticle composition (e.g., a lipid nanoparticle composition) is characterized in that, over a period of time (e.g., as described herein) under a specified set of conditions (e.g., as described herein), the average particle size, particle size distribution, and / or polydispersity of the nanoparticles are substantially maintained (e.g., within 10%) compared to the initial characteristics. In some embodiments, a stable nanoparticle composition (e.g., a lipid nanoparticle composition) is characterized in that, after being maintained over a period of time under a specified set of conditions (e.g., as described herein), no detectable amounts of degradation products (e.g., related to hydrolysis and / or enzymatic digestion) are present.

[0140] RNA Stability: The term “RNA stability” is used herein in reference to the “half-life” of RNA. “Half-life” refers to the time required for half of the activity, quantity, or number of a molecule to disappear. In many embodiments, the half-life of RNA indicates its stability. Those skilled in the art will understand that the half-life of RNA can often affect its “expression period”; typically, RNA with a long half-life expresses for a longer period than RNA with a shorter half-life.

[0141] Stem-loop, hairpin: As used herein, the terms “stem-loop,” “hairpin,” or “hairpin-loop” refer to a specific secondary structure of a nucleic acid molecule, typically a single-stranded nucleic acid molecule, e.g., single-stranded RNA. A specific secondary structure represented by a stem-loop consists of a continuous nucleic acid sequence comprising a stem and a loop (e.g., a terminal loop), also called a hairpin-loop, where the stem is formed by two adjacent, fully or partially complementary sequence elements; these are separated by short sequences (e.g., 3-10 nucleotides) and form the loop of the stem-loop structure. Two adjacent, fully or partially complementary sequences may be defined, for example, as stem-loop elements stem 1 and stem 2. The stem-loop is formed when these two adjacent, fully or partially inversely complementary sequences, e.g., stem-loop elements stem 1 and stem 2, form base pairs with each other. This results in a double-stranded nucleic acid sequence with an unpaired loop at the termination end, formed by a short sequence located between stem-loop elements stem 1 and stem 2. Therefore, a stem-loop consists of two stems (stem 1), one that forms base pairs with the other at the secondary structure level of the nucleic acid molecule, and the other that, at the primary structure level of the nucleic acid molecule, is separated by a short sequence that is not part of stem 1 or stem 2. For illustrative purposes, a two-dimensional representation of a stem-loop is analogous to a lollipop-type structure. The formation of a stem-loop structure involves sequences that can fold back on their own to form a pair of double helixes; these double helixes are formed by stem 1 and stem 2. The stability of the pair of stem-loop elements is typically determined by the nucleotide length of stem 1 that can form base pairs (preferably standard base pairs, more preferably Watson-Crick base pairs) with the nucleotides of stem 2, and the number of nucleotides of stem 2 and the number of nucleotides of stem 1 that cannot form such base pairs (mismatches or bulges). If a given nucleic acid sequence features a stem-loop, then each complementary nucleic acid sequence also typically features a stem-loop. Stem-loops are typically formed by single-stranded RNA molecules.

[0142] Synthetic: As used herein, the term “synthetic” refers to an artificial entity, or an entity created by human intervention, or an entity that is not naturally occurring but produced by synthesis. For example, in some embodiments, synthetic nucleic acid or polynucleotide refers to a nucleic acid molecule that is chemically synthesized, for example, by solid-phase synthesis in some embodiments. In some embodiments, the term “synthetic” refers to an entity that is produced outside of a biological cell. For example, in some embodiments, synthetic nucleic acid or polynucleotide refers to a nucleic acid molecule (e.g., RNA) produced by in vitro transcription using a template.

[0143] Template: As used herein, the terms “template,” “nucleic acid template,” or “template nucleic acid” generally refer to a nucleic acid sequence that can be replicated or transcribed. In some embodiments, the template is DNA. In some embodiments, the DNA template is a linear DNA molecule. In some embodiments, the DNA template is a circular DNA molecule. DNA may be obtained or produced using methods known in the art, such as gene synthesis, recombinant DNA techniques, or a combination thereof. In some embodiments, the DNA template includes a nucleotide sequence encoding a transcribed region of interest (e.g., encoding the RNA described herein) and a promoter sequence recognized by an RNA polymerase selected for use in in vitro transcription, such as the RNA polymerase described herein. In some embodiments, the DNA template encodes one or more elements of a product, such as RNA, e.g., the 5'UTR, 3'UTR, open reading frame (e.g., encoding the peptide or protein of interest, such as an antigen), and poly(A) tail. In some embodiments, the DNA template encodes all elements of the product RNA. In some embodiments, the DNA template does not encode all elements of the product RNA; for example, the DNA template does not encode a poly(A) tail, which can be enzymatically added to the RNA after transcription as described herein.

[0144] Tertiary structure: As used herein, the term “tertiary structure” in relation to nucleic acid molecules refers to the three-dimensional structure of a nucleic acid molecule defined by atomic coordinates.

[0145] Three-Lime Untranslated Region: As used herein, the term “three-lime untranslated region” or “3'UTR” refers to the sequence of the mRNA molecule that begins after the stop codon of the coding region of the open reading frame sequence. In some embodiments, the 3'UTR begins immediately after the stop codon of the coding region of the open reading frame sequence. In other embodiments, the 3'UTR does not begin immediately after the stop codon of the coding region of the open reading frame sequence.

[0146] Threshold Level (e.g., Acceptance Criteria): As used herein, the term “threshold level” refers to a level used as a criterion for obtaining information about the results of a measurement (e.g., the results obtained in an assay) and / or classifying the results of a measurement. For example, in some embodiments, the threshold level means a value measured in an assay that defines a boundary between two subsets of a population (e.g., a batch that meets a quality control criterion versus a batch that does not meet a quality control criterion). Thus, a value greater than or equal to the threshold level defines one subset of the population, and a value less than the threshold level defines the other subset of the population. The threshold level can be determined based on one or more control samples, or in the entire population of control samples. The threshold level can be determined before performing the measurement of interest, simultaneously with the measurement, or after the measurement. In some embodiments, the threshold level may be a range of values.

[0147] Transcription efficiency: The term "transcription efficiency" refers to the amount of transcript produced from a template molecule within a specific period of time.

[0148] Translation efficiency: The term "translation efficiency" refers to the amount of translation products provided by an RNA molecule within a specific period of time.

[0149] Variant: The term “variant,” when used, for example, in relation to nucleic acids and amino acid sequences, includes any variant, particularly mutants, viral strain variants, splice variants, three-dimensional structures, isoforms, allelic variants, species variants, and species homologs, particularly naturally occurring homologs. Allelic variants are related to changes in the normal sequence of a gene, but their significance is often unclear. Complete gene sequencing often identifies numerous allelic variants of a particular gene. In relation to nucleic acid molecules, the term “variant” includes degenerate nucleic acid sequences, and a degenerate nucleic acid according to the present invention is a nucleic acid whose codon sequence differs from a reference nucleic acid due to the degeneracy of the genetic code. A species homolog is a nucleic acid or amino acid sequence that has a different species of origin than a given nucleic acid or amino acid sequence. A viral homolog is a nucleic acid or amino acid sequence that has a different viral origin than a given nucleic acid or amino acid sequence. In some embodiments, a nucleic acid variant may include the deletion, addition, mutation, substitution, and / or insertion of one or more nucleotides compared to a reference nucleic acid. Deletions include the removal of one or more nucleotides from a reference nucleic acid. Addition variants include 5' and / or 3' terminal fusions of one or more nucleotides, such as 1, 2, 3, 5, 10, 20, 30, 50, or more nucleotides. Substitutions involve the removal of at least one nucleotide in a sequence and the insertion of at least one other nucleotide in its place (e.g., transposition and conversion). Mutations may include debasement sites, crosslinking sites, and chemically altered or modified bases. Insertions involve the addition of at least one nucleotide to a reference nucleic acid.

[0150] Vectors: As used herein, the term “vector” refers to a nucleic acid molecule capable of transporting another nucleic acid to which it is linked. Vectors include plasmids, cosmid vectors, phagemids such as lambda phages, viral genomes such as retroviruses, adenoviruses, or baculovirus vectors, artificial chromosome vectors such as bacterial artificial chromosomes (BACs), yeast artificial chromosomes (YACs), or P1 artificial chromosomes (PACs), and their functional portions. One type of vector is a “plasmid,” which refers to a circular double-stranded DNA to which an additional DNA segment can be ligated. Another type of vector is a viral vector, to which an additional DNA segment can be ligated to a viral genome. Certain vectors can self-replicate within the host cell into which they are introduced (e.g., bacterial vectors with a bacterial origin of replication and episomatic mammalian vectors). Other vectors (e.g., non-episomatic mammalian vectors), once introduced into a host cell, can be integrated into the host cell’s genome and thereby replicate with the host genome. Furthermore, certain vectors can induce the expression of the gene to which they are operably linked. Such vectors are referred to herein as “expression vectors.” Expression vectors, including plasmids and viral vectors, generally contain a desired coding sequence and appropriate non-coding sequences necessary for the expression of the operably linked coding sequence in a specific host organism (e.g., bacteria, yeast, plants, insects, or mammals) or in vitro expression system. Cloning vectors are generally used to manipulate and amplify a specific desired DNA fragment, but may lack the functional sequences necessary for the expression of the desired DNA fragment.

[0151] Detailed description of a particular embodiment Specific embodiments of the present invention are described in detail below, but please understand that the present invention is not limited to the specific methodologies, protocols, and reagents described herein, as they may be modified. Please also understand that the technical terms used herein are for the purpose of describing specific embodiments only, and are not intended to limit the scope of the present invention, as it is limited only by the appended claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art.

[0152] In some embodiments, the implementation of the present invention will use conventional methods of chemistry, biochemistry, cell biology, immunology, and recombinant DNA techniques described in the art literature unless otherwise indicated (see, for example, Molecular Cloning: A Laboratory Manual, 2nd Edition, J. Sambrook et al. eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor 1989). In fact, in many embodiments, standard techniques may be used for, for example, the production and / or manipulation of recombinant DNA, oligonucleotide synthesis, tissue culture, and transformation (e.g., electroporation, lipofection). Those skilled in the art will understand, upon reading this disclosure, that enzymatic reactions and purification techniques may be carried out according to the manufacturer's specifications, or as commonly achieved in the art, or as described herein. The techniques and procedures described herein may generally be carried out according to conventional methods well known in the art and / or as described in various general and more specific references cited and discussed throughout this specification. For example, see Green and Sambrook, Molecular Cloning: A Laboratory Manual (4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2012)). This content is incorporated herein by reference for any purpose.

[0153] The following description describes specific elements of the present invention. These elements may be discussed in conjunction with specific embodiments, but it should be understood that these elements may be combined in any way and in any number to create additional embodiments. The various examples and specific embodiments described should not be construed as limiting the invention to only the explicitly described embodiments. This description should be understood as disclosing and encompassing embodiments that combine the explicitly described embodiments with any number of the disclosed and / or preferred elements. Furthermore, any permutation and combination of all elements described in this application should be considered disclosed by this description unless the context indicates otherwise.

[0154] Throughout this specification, several documents are referenced. Each document referenced herein (including all patents, patent applications, scientific publications, manufacturer specifications, instructions, etc.) (whether above or below) is incorporated herein by reference in its entirety. Nothing in this specification should be construed as admitting that the present invention has no prior rights to such disclosures.

[0155] Batch, fed-batch culture This disclosure provides specific techniques for producing RNA molecules, particularly through in vitro transcription (IVT) using “batch” reactions. Those skilled in the art will understand that the terms “batch” or “batch reaction,” or similar terms, are used to refer to reactions such as transcription reactions, in which at least one separate supplementation event occurs for at least one component, at least one NTP, e.g., UTP and / or GTP, or a functional analog thereof, and optionally one or more other components (e.g., components of the reaction mix discussed herein), optionally supplemented in the same separate supplementation event. In some embodiments, supplementing the reaction mix includes supplementing UTP or a functional analog thereof. In some embodiments, supplementing the reaction mix includes supplementing UTP or a functional analog thereof and GTP or a functional analog thereof. In some embodiments, replenishing the reaction mix involves replenishing with ATP or a functional analog thereof and / or CTP or a functional analog thereof and / or one or more salts and / or one or more enzymes, such as polymerases and / or one or more 5' capped nucleotides and / or one or more other components of the reaction mix described herein, such as further components such as transcription buffers, RNase inhibitors, DNA templates, 5' caps and / or 5' capped analogs. In some embodiments, the reaction mix is ​​replenished multiple times during the transcription and / or capping reaction.

[0156] In certain embodiments, a fed-batch culture process is used in a method for producing RNA or RNA-containing compositions. The terms “fed-batch culture process” or “fed-batch culture reaction” or similar terms refer to a process or reaction in which a starting reaction mix (batch reaction product) exists, which is some or all of the components, and in the course of the reaction, one or more components may be replenished in the reaction product. In some embodiments, one or more components, such as UTP or its functional analogues and / or GTP or its functional analogues, are replenished, for example, by a fed-batch culture process, to maintain their concentrations low during the reaction, or to restore their initial concentration ratio to the initial concentration of CTP or its functional analogues and / or ATP or its functional analogues. In some embodiments, one or more components, such as UTP or its functional analogues and / or GTP or its functional analogues, are replenished, for example, by a fed-batch culture process to replenish those that have been depleted during the reaction. “Replenishing the reaction mix” means replenishing the reaction in individual amounts after the reaction has started. However, replenishment in a fed-batch culture reaction is not limited to the replenishment of individual amounts. In some embodiments, replenishment includes replenishment by continuous flow, i.e., continuous replenishment of one or more components of the reaction mix during the transfer and / or capping reaction.

[0157] In some embodiments, the fed-batch culture process involves using a starting reaction mix that contains all the nucleotide triphosphates that are part of the RNA to be synthesized; in other embodiments, the fed-batch culture process involves using a starting reaction mix that does not contain all the nucleotide triphosphates that are part of the RNA to be synthesized.

[0158] In some embodiments, the starting reaction mix includes ATP, GTP, CTP, and UTP, or functional analogs thereof. In some embodiments, the starting reaction mix used is substantially free of ATP or its functional analogs. In some embodiments, the starting reaction mix is ​​substantially free of GTP or its functional analogs. In some embodiments, the starting reaction mix is ​​substantially free of CTP or its functional analogs. In some embodiments, the starting reaction mix is ​​substantially free of UTP or its functional analogs. If the RNA to be synthesized is expected to contain components not present in the starting reaction mix (e.g., according to the template used), it is understood that these components must be supplemented to synthesize the RNA.

[0159] For example, in capping and transcription reactions, the reaction begins when RNA polymerase mediates the formation of a covalent bond between the nucleotide and the capping analog. It will be understood that one difference between capping and transcription reactions and non-capping transcription reactions is the presence of a component that provides a capping structure to the 5' end of the transcript, such as the 5' cap or 5' capping analog described herein.

[0160] This disclosure provides a technique involving a fed-batch culture process in which at least one component of a reaction mix is ​​present in a restricted amount. "Restricted amount" means that the restricted reaction component is present in any amount, e.g., at the starting concentration, such that one or more of the following are restricted: the time until the reaction stops, the amount of product produced by the process until the reaction stops, and the transcription rate. For example, in some embodiments, the amount of the restricting reaction component, e.g., UTP or a functional analog thereof, and / or GTP or a functional analog thereof, restricts one or more of the following: the time until the transcription and / or capping reaction stops, the amount of product produced until the transcription and / or capping reaction stops, e.g., RNA, and / or the rate of the transcription and / or capping reaction, e.g., the rate at which reactants are converted into products.

[0161] In some embodiments, one or more reaction components are added to the reaction continuously, and one or more components, such as one or more limiting components, are added to the reaction periodically by a fed-batch culture process or the like. In certain embodiments, limiting components, such as limiting nucleotides, are replenished to the reaction periodically or intermittently by a fed-batch culture process. The term “periodically” means “occurring at regular intervals” and may also be “regular” meaning “fixed” with respect to characteristics such as time and / or concentration levels in the reaction. “Intermittently” means “occurring at intervals.” “Interval” can refer to both regular and irregular intervals. It will be understood that “intermittent” replenishment of components can also be “periodically.” Furthermore, it will be understood that intermittent introduction or replenishment of components to a reaction means at least once, and “periodically” introduction or replenishment of components means at least twice (to define “regular intervals”). In some embodiments, limiting components, such as UTP or its functional analog and / or GTP or its functional analog, may be replenished during the transcription and / or capping reaction for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100 or more times, at least these times, or up to these times, within any range derived therefrom. In some embodiments, UTP or its functional analog is replenished at least once. In some embodiments, UTP or its functional analog is replenished at least twice. In further embodiments, UTP or its functional analog is introduced into the reaction intermittently or periodically for 3 to 50 times. In some embodiments, such periodic replenishment may be carried out as one or more bolus or batch additions, including, for example, one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen or more bolus or batch additions. In some embodiments, such periodic replenishment may be carried out by a fed-batch culture process.Replenishment may also involve replenishing with a composition comprising UTP or a functional analog thereof, and further components such as a buffer, polymerase, CTP or a functional analog thereof, GTP or a functional analog thereof, ATP or a functional analog thereof, or other components that may be present in the transcription reaction mix described herein. In some embodiments, the composition for replenishment does not essentially contain CTP and / or ATP, or their functional analogs.

[0162] DS RNA contamination As is known in the art, during transcription reactions, such as mRNA synthesis by in vitro transcription (IVT) using T7 RNA polymerase or another RNAP, large amounts of abnormal products, such as double-stranded RNA (dsRNA) (including hybridization of different RNA molecules), are produced due to the non-conventional activity of the enzyme. dsRNA has been shown to induce inflammatory cytokines and activate effector enzymes, leading to inhibition of protein synthesis.

[0163] As described herein, dsRNA contamination in in vitro transcription reactions can be problematic. At least two distinct types of dsRNA contamination have been reported: (i) short dsRNAs in which antisense fragments form base pairs with the target RNA transcript (e.g., mRNA product), and (ii) nearly full-length dsRNAs. Both are generated by promoter-dependent or promoter-independent RNAP (e.g., T7) activity; some may be affected by termination sites within the template.

[0164] While we do not wish to be bound by theory, it should be noted that various mechanisms have been proposed for dsRNA production during in vitro transcription. For example, in some cases, the enzyme that initiated synthesis may terminate before transcription is complete, producing a short RNA transcript (e.g., about 5–11 nt in length), after which transcription of the complementary strand may begin. Alternatively or additionally, RNA folding may lead to transcriptional extension, producing a very long transcript (potentially twice or more in size). Further alternative or additionally, transcription may be brought about by restarting transcription at a specific open template structure, resulting in the production of an antisense strand. Reports also suggest that the termination site of the template may influence dsRNA production.

[0165] This disclosure provides, in particular, a technique for reducing dsRNA in RNA preparations. For example, in some embodiments, the amount of dsRNA present in RNA preparations prepared according to this disclosure is reduced compared to the amount of dsRNA present in RNA preparations prepared using, for example, equimolar amounts of ATP, GTP, CTP, and UTP.

[0166] The level of dsRNA, and therefore the level of its depletion, can be determined using any of the various techniques discussed herein, but are not limited to those described herein. For example, RNA can be spotted onto a membrane such as a nylon membrane, blocked with an appropriate buffer, detected using an antibody-based assay with an antibody specific to dsRNA, such as the J2 antibody (SCICONS English and Scientific Consulting), and then stained with a secondary antibody, anti-mouse HRP antibody (Jackson ImmunoResearch) (see EP18717580.7). Antibody-based detection methods are well known. RNA concentration can also be assessed using UV (e.g., Nanodrop), which may indicate the presence or absence of dsRNA. RNA integrity can be assessed using a Bioanalyzer (Agilent).

[0167] The term "immunogenicity" refers to the ability of a particular substance, especially RNA, to trigger an immune response in the body of an animal, such as a human, e.g., an innate immune response, an adaptive immune response, or both. In other words, immunogenicity is the ability to induce humoral and / or cellular immune responses. Undesirable immunogenicity includes the immune response of an organism to therapeutic substances such as drugs. This reaction can inactivate the therapeutic effect and induce side effects. While not bound by any particular theory, the immunogenicity of many RNA preparations, particularly those produced by conventional in vitro transcription reactions, is thought to be at least partly due to their dsRNA content.

[0168] In some embodiments, the RNA preparations described herein (e.g., RNA preparations produced by the methods provided herein) are significantly less immunogenic than RNA and RNA-containing compositions transcribed from the same DNA template using known methods, such as using equimolar amounts of adenosine triphosphate (ATP), guanosine triphosphate (GTP), cytidine triphosphate (CTP), and uridine triphosphate (UTP), or their functional analogs. In some embodiments, the RNA or RNA-containing compositions of the present invention (i.e., the RNA preparations provided) are at least 5% less immunogenic than RNA or RNA-containing compositions transcribed using equimolar amounts of ATP, GTP, CTP, and UTP, or their functional analogs. In some embodiments, immunogenicity is reduced by at least 10%. In some embodiments, immunogenicity is reduced by at least 20%. In some embodiments, immunogenicity is reduced by at least 30%. In some embodiments, immunogenicity is reduced by at least 40%. In some embodiments, immunogenicity is reduced by at least 50%. In some embodiments, immunogenicity is reduced by at least 60%. In some embodiments, immunogenicity is reduced by at least 70%. In some embodiments, immunogenicity is reduced by at least 80%. In some embodiments, immunogenicity is reduced by at least 90%. In some embodiments, immunogenicity is eliminated or essentially eliminated, i.e., reduced by about 100%.

[0169] In some embodiments, the relative immunogenicity (i.e., the immunogenicity of the provided RNA preparation) of RNA or a composition containing RNA transcribed according to the present invention and RNA or a composition containing RNA transcribed using relevant control methods known in the art, such as using equimolar amounts of ATP, GTP, CTP, and UTP, or functional analogs thereof, can be determined by determining the amount of the provided RNA preparation to produce the same result (e.g., the same amount of protein expression) to the same extent as a given amount of RNA or a composition containing RNA transcribed using, for example, equimolar amounts of ATP, GTP, CTP, and UTP, or functional analogs thereof, using the control method. In one embodiment, the relative immunogenicity of the provided RNA preparation and its respective counterpart transcribed using the relevant control method can be determined by measuring the amount of cytokines (e.g., IL-12, IFN-α, TNF-α, RANTES, MIP-1α or β, IL-6, IFN-β, or IL-8) secreted in response to administration of RNA or a composition containing RNA transcribed according to the present invention, compared to an equivalent amount of RNA or a composition containing RNA transcribed using the control method. For example, when approximately half the amount of cytokine is secreted, RNA transcribed according to the present invention or a composition containing RNA is 50% less immunogenic than RNA transcribed using an appropriate control method or a composition containing RNA.

[0170] "Remarkably low immunogenicity" refers to a detectable reduction in immunogenicity. In one embodiment, this term refers to a reduction that allows an effective amount of RNA or an RNA-containing composition to be administered or repeatedly without eliciting a detectable immune response. In some embodiments, this term refers to a reduction that allows RNA or an RNA-containing composition to be repeatedly administered without inducing an immune response sufficient to detectably reduce the expression of RNA-encoded peptides or proteins, for example, contained in an RNA-containing composition. In some embodiments, the reduction allows RNA or an RNA-containing composition to be repeatedly administered without inducing an immune response sufficient to eliminate the expression of RNA-encoded peptides or proteins.

[0171] As demonstrated herein, the immunogenicity of RNA or RNA-containing compositions (i.e., RNA preparations) can be reduced by transcribing the RNA using the methods of the present invention as described herein. In some embodiments, in such methods, the starting concentration of UTP or its functional analog in the reaction mix used to transcribe RNA from a template is lower than the starting concentration of CTP and / or ATP or their functional analogs. In some embodiments, the method involves transcribing RNA using a starting concentration of UTP or its functional analog lower than the starting concentration of CTP and / or ATP or its functional analog, and replenishing the transcription reaction mix during the transcription reaction with a composition containing UTP or its functional analog, thereby resulting in a reduction in dsRNA formation during transcription compared to a suitable control transcription reaction, such as carrying out the method using equimolar amounts of ATP, GTP, CTP, and UTP or their functional analogs. Thus, in some embodiments, transcribing RNA according to the methods of the present invention results in lower immunogenicity of the RNA compared to RNA transcribed using equimolar amounts of ATP, GTP, CTP, and UTP or their functional analogs, as described herein. In some embodiments, transcription of RNA according to the method of the present invention increases the yield of RNA compared to RNA transcribed using equimolar amounts of ATP, GTP, CTP, and UTP, or their functional analogs, as described herein.

[0172] In vitro transcription reaction As described herein, RNA can be synthesized in vitro. In particular, in vitro transcription allows for the use of cap analogs (e.g., cap analogs that do not exist in nature) that can be added to the in vitro transcription reaction. Those skilled in the art will understand that in many embodiments, if a poly(A) tail is present on the RNA molecule, it is encoded by a complementary sequence on the transcribed template (e.g., by a poly-(dT) sequence in the DNA template). Alternatively or additionally, in some embodiments, capping and / or poly(A) tail addition can be achieved enzymatically after transcription, as is known in the art.

[0173] Those skilled in the art will understand that an in vitro transcription reaction typically comprises: (1) a template (often DNA, typically linear) containing a promoter that directs the transcription of the sequence of interest; (2) ribonucleotide triphosphates (which may be natural compounds or analogs); (3) a buffer system (typically containing magnesium ions); and (4) RNA polymerase. Thus, according to the present invention, an RNA transcription reaction typically comprises: (1) a template (e.g., DNA, typically linear) that may contain a promoter that directs the transcription of the sequence of interest; (2) ribonucleotide triphosphates (natural or functional analogs thereof); (3) a buffer system such as the buffers described herein, which are selected according to the RNA polymerase used, for example; and (4) RNA polymerase. In some embodiments, the transcription reaction mix may further comprise an RNA inhibitor. In some embodiments, the transcription reaction mix may further comprise a pyrophosphatase (e.g., inorganic pyrophosphatase). In some embodiments, the transcription reaction mix may further comprise one or more salts (e.g., monovalent salts and / or divalent salts, e.g., Li + na + , K + NH 4+ Tris(hydroxymethyl)aminomethane cation, Mg 2+ Ba 2+ or Mn 2+The mixture may further contain reducing agents (e.g., dithitreitol, 2-mercaptoethanol, etc.), spermidine, or a combination thereof. In some embodiments, specific reactants are added in a specific order (e.g., pyrophosphatase and polymerase are added last). In some embodiments, the stirring speed is increased after the addition of specific reactants (e.g., pyrophosphatase, polymerase).

[0174] Those skilled in the art will recognize that it is desirable to reduce variability in the in vitro transcription reaction, especially in large-scale cases (e.g., producing more than about 50 μg of RNA per reaction, more commonly more than about 100 μg, or in the range of about 120 μg to about 180 μg per 1 μg of template). For example, in many embodiments, it may be desirable to control the concentration of monovalent or divalent cations and / or the reaction temperature. In some embodiments, the cations in the reaction mix according to the present invention are Li + na + , K + NH 4+ Tris(hydroxymethyl)aminomethane cation, Mg 2+ Ba 2+ or Mn 2+ For example, in many embodiments, Mg 2+ It may be desirable to control the concentration and / or reaction temperature. In some embodiments, Mg 2+ The amount decreases and / or the temperature increases (see, for example, Mu et al., Nuc Acids Res. 10:5239).

[0175] In some embodiments, the Disclosure provides, among other things, techniques for mass-producing pharmaceutical-grade compositions or preparations containing RNA in high-volume batch throughput, for example, at least 10 g of RNA (e.g., at least 15 g RNA, at least 20 g RNA, at least 25 g RNA, at least 30 g RNA, at least 35 g RNA, at least 40 g RNA, at least 45 g RNA, at least 50 g RNA, at least 55 g RNA, at least 60 g RNA, at least 70 g RNA, at least 80 g RNA, at least 90 g RNA, at least 100 g RNA, at least 150 g RNA, at least 200 g RNA, or more). In some embodiments, such methods described herein may be used to produce high-volume batch throughput of about 10 g to about 300 g of RNA, about 10 g to about 200 g of RNA, about 10 g to about 100 g of RNA, about 30 g to about 60 g of RNA, about 50 g to about 300 g of RNA. In some embodiments, such methods described herein are useful for large-scale production to produce high batch throughputs of at least 1 g of RNA per hour, for example, at least 1.5 g of RNA per hour (e.g., at least 2 g of RNA per hour, at least 2.5 g of RNA per hour, at least 3 g of RNA per hour, at least 3.5 g of RNA per hour, at least 4 g of RNA per hour, at least 4.5 g of RNA per hour, at least 5 g of RNA per hour, at least 5.5 g of RNA per hour, at least 6 g of RNA per hour, at least 6.5 g of RNA per hour, at least 7 g of RNA per hour, at least 7.5 g of RNA per hour, at least 8 g of RNA per hour, at least 8.5 g of RNA per hour, at least 9 g of RNA per hour, at least 10 g of RNA per hour or more). In some embodiments, the large-scale production methods described herein can reach a capacity of 15 g to 20 g of RNA per hour (for example, about 17 g per hour).

[0176] Those skilled in the art will understand that the term “polymerase” generally refers to a molecular entity capable of catalyzing the synthesis of polymer molecules from monomer building blocks. “RNA polymerase” is a molecular entity capable of catalyzing the synthesis of RNA molecules from ribonucleotide building blocks. “RNA-dependent RNA polymerase” or “RdRP” is an enzyme that catalyzes the transcription of RNA from an RNA template. “DNA polymerase” is a molecular entity capable of catalyzing the synthesis of DNA molecules from deoxyribonucleotide building blocks. In the case of DNA polymerase and RNA polymerase, the molecular entity is typically a protein, or an aggregate or complex of multiple proteins. Typically, DNA polymerase synthesizes DNA molecules based on a template nucleic acid, which is typically a DNA molecule. Typically, RNA polymerase synthesizes RNA molecules based on a template nucleic acid, which are either DNA molecules (in this case, RNA polymerase is DNA-dependent RNA polymerase, DdRP) or RNA molecules (in this case, RNA polymerase is RNA-dependent RNA polymerase, RdRP).

[0177] Various RNA polymerases suitable for transcription reactions are known in the art, but are not limited to, DNA-dependent RNA polymerases (e.g., T7 RNA polymerase, T3 RNA polymerase, SP6 RNA polymerase, N4 virion RNA polymerase, or their variants or functional domains). Naturally catalyzed RNA-dependent RNA polymerases are typically encoded by all RNA viruses except retroviruses. Alphaviruses are typical representatives of viruses encoding RNA-dependent RNA polymerases. Those skilled in the art will understand that the RNA polymerases used herein may be recombinant RNA polymerases and / or purified RNA polymerases, i.e., they may not be part of a cell extract and may contain other components in addition to the RNA polymerase. In some embodiments, the RNA polymerase useful for commercially-scale transcription is T7 RNA polymerase. In some embodiments, inorganic pyr phosphatase may be added to improve the yield of the transcription reaction (e.g., catalyzed in some embodiments by T7 RNA polymerase).

[0178] In particular, this disclosure establishes that restriction nucleotides can reduce dsRNA formation in in vitro reactions. This disclosure specifically demonstrates that restricting UTPs in in vitro transcription reactions can reduce dsRNA formation and may be particularly useful for the production of transcripts that may contain polyA sequences, such as polyA tails. While not bound by theory, the inventors propose that the observed reduction in dsRNA production may be due to a reduction in reverse transcription (initiated by hybridization with polyA sequences, such as polyA tails).

[0179] Those skilled in the art will understand that as used herein, “UTP restriction” means a restriction at the level of nucleotides that functionally pair with an A residue in the template so that they can be utilized in template synthesis (i.e., in vitro transcription reaction) and incorporated into the resulting chain. Such nucleotides are referred herein as “UTP or its functional analog.” In some embodiments, the functional analog is also typically translatable as “U.”

[0180] Accordingly, in particular, the present disclosure provides specific in vitro transcription techniques in which the concentration of UTP (and / or its functional analog(s)) is restricted, for example, to a lower concentration than that of CTP and / or ATP or their functional analog(s). In some embodiments, UTP is restricted at the start of the reaction. In some embodiments, UTP is restricted throughout the reaction.

[0181] For example, the present disclosure provides an in vitro transcription reaction in which UTP is restricted (e.g., the concentration of UTP and its functional analog is lower than the concentration of one or more other nucleotides (i.e., adenosine triphosphate (ATP) and its functional analog, guanosine triphosphate (GTP) and its functional analog, and cytidine triphosphate (CTP) and its functional analog).

[0182] Furthermore, the Disclosure provides, in particular, a technique in which UTP is restricted in the initial in vitro transcription reaction as described herein. In some embodiments, the Disclosure provides, in particular, a technique in which UTP or a functional analog thereof is replenished over time in the initial in vitro transcription reaction in which UTP is restricted. In some such embodiments, such replenishment is carried out by one or more separate supply events. In some embodiments, such replenishment may be carried out by a continuous process, for example, in some embodiments where the UTP replenishment rate is comparable to the UTP consumption rate during the transcription reaction. In some embodiments, UTP is restricted during replenishment (for example, after such replenishment, UTP or a functional analog thereof is replenished at a concentration such that it is present in the reaction at a concentration lower than one or more of ATP or its functional analog, GTP or its functional analog, and / or CTP or its functional analog, in some embodiments all of them).

[0183] In some embodiments, the in vitro transcription reaction is supplemented with multiple nucleotides (e.g., UTP or its functional analog, and one or more other nucleotides, e.g., ATP or its functional analog, GTP or its functional analog, and / or CTP or its functional analog).

[0184] In some embodiments, both UTP and GTP are restricted during the initial reaction and / or replenishment.

[0185] RNA products This disclosure provides a technology useful for the production of RNA products (i.e., for producing preparations of specific RNA products).

[0186] In certain embodiments of this disclosure, the RNA produced is, for example, messenger RNA (mRNA) associated with an RNA transcript encoding a peptide or protein. As is known in the art, mRNA often comprises a 5' untranslated region (5'UTR), a peptide coding region, and a 3' untranslated region (3'UTR).

[0187] In some embodiments, the RNA product is produced by in vitro transcription. In some embodiments, the mRNA product is produced by in vitro transcription using, for example, a DNA template (where DNA refers to nucleic acids containing deoxyribonucleotides). In some embodiments, RNA synthesis can also be carried out within cells or other systems.

[0188] In some embodiments, the RNA product is in vitro transcription RNA (IVT-RNA), which can be obtained by in vitro transcription of a suitable DNA template.

[0189] In certain embodiments, the DNA template is a linear molecule. In certain embodiments, the DNA template is a cyclic molecule. Generally, DNA can be obtained or produced using methods known in the art, such as gene synthesis, recombinant DNA technology, or a combination thereof. In some embodiments, the DNA template includes a nucleotide sequence encoding a region of interest to be transcribed (for example, encoding RNA such as the RNA described herein) and a promoter sequence recognized by an RNA polymerase selected for use in in vitro transcription. The promoter for controlling transcription may be any promoter of any RNA polymerase. Various RNA polymerases are known in the art, and exemplary polymerases are disclosed herein. Those skilled in the art will understand, by reading this disclosure, that the RNA polymerases used herein may be recombinant RNA polymerases and / or purified RNA polymerases, i.e., they may not be as part of a cell extract and may include other components in addition to the RNA polymerase. Those skilled in the art will recognize the appropriate promoter sequence for a selected RNA polymerase. In some embodiments, the DNA template may include the promoter sequence for a T7 RNA polymerase. DNA templates for in vitro transcription can be obtained by cloning nucleic acids, such as cDNA, and introducing them into a suitable vector for in vitro transcription. Techniques for introducing nucleic acid sequences into vectors, such as cold fusion cloning and others, are well known in this field. cDNA can be obtained by reverse transcription of RNA.

[0190] In some embodiments, the RNA suitable for the techniques described herein is single-stranded RNA (e.g., mRNA as described herein). In some embodiments, the single-stranded RNA is non-coding RNA in that its nucleotide sequence does not contain an open reading frame (or its complement). In some embodiments, the single-stranded RNA has a nucleotide sequence that codes for (or is a complement of the coding sequence of) the polypeptide described herein.

[0191] In some embodiments, the techniques described herein may be particularly useful for synthesizing single-stranded RNA having a length of at least 500 ribonucleotides (for example, at least 600 ribonucleotides, at least 700 ribonucleotides, at least 800 ribonucleotides, at least 900 ribonucleotides, at least 1000 ribonucleotides, at least 1250 ribonucleotides, at least 1500 ribonucleotides, at least 1750 ribonucleotides, at least 2000 ribonucleotides, at least 2500 ribonucleotides, at least 3000 ribonucleotides, at least 3500 ribonucleotides, at least 4000 ribonucleotides, at least 4500 ribonucleotides, at least 5000 ribonucleotides, at least 6000 ribonucleotides, at least 7000 ribonucleotides, at least 8000 ribonucleotides, at least 9000 ribonucleotides, at least 10000 ribonucleotides, at least 11000 ribonucleotides, at least 12000 ribonucleotides, or more). In some embodiments, the techniques described herein may be particularly useful for synthesizing single-stranded RNA having a length of about 1,000 to 5,000 ribonucleotides.

[0192] In some embodiments, the relevant RNA includes a polypeptide coding portion. In some specific embodiments, such a portion may encode an antigen (or its epitope), cytokine, enzyme, etc., or a polypeptide containing them. In some embodiments, the encoded polypeptide may be one or more neoantigens or neoepitopes related to tumors, or may contain them. In some embodiments, the encoded polypeptide may be an antigen (or its epitope) of an infectious agent (e.g., bacteria, fungi, viruses, etc.), or may contain them. In certain embodiments, the encoded polypeptide may be a variant of a wild-type polypeptide.

[0193] This disclosure specifically illustrates the production of RNA encoding viral antigens (or epitopes) (and / or epitopes), for example, coronavirus antigens (or epitopes) and / or epitopes (or epitopes). For example, in some embodiments, this disclosure illustrates the production of single-stranded RNA whose nucleotide sequence encodes a coronavirus polypeptide or a variant thereof. In some embodiments, the single-stranded RNA includes, for example, a nucleotide sequence encoding a pre-fusion coronavirus spike protein, as described in WO2018081318 (the entire contents of which are incorporated herein by reference for the purposes described herein).

[0194] In some embodiments, the single-stranded RNA comprises a nucleotide sequence encoding a SARS-CoV-2 polypeptide (e.g., the spike (S) protein, nucleocapsid (N) protein, envelope (E) protein, and membrane (M) protein) or an immunogenic fragment thereof. In some embodiments, the single-stranded RNA comprises a nucleotide sequence encoding a SARS-CoV-2S polypeptide or an immunogenic fragment thereof (e.g., the receptor-binding domain of the S protein). In some embodiments, such a SARS-CoV-2S polypeptide or its immunogenic fragment may be a mutant protein. In some embodiments, the RNA for use according to this disclosure encodes a SARS-CoV-2 spike protein having K986P and V978P mutations.

[0195] In some embodiments, single-stranded RNA (e.g., mRNA as described herein) may include a secretory signal coding region (e.g., a secretory signal coding region in which the encoded target entity can be secreted by the cell during translation). In some embodiments, such a secretory signal coding region may be a non-human secretory signal or may include a non-human secretory signal. In some embodiments, such a secretory signal coding region may be a human secretory signal or may include a human secretory signal.

[0196] In some embodiments, single-stranded RNA (e.g., mRNA as described herein) may include at least one non-coding sequence element (e.g., to enhance RNA stability and / or translation efficiency). Examples of non-coding sequence elements include, but are not limited to, 3' untranslated regions (UTRs), 5' UTRs, cap structures for co-transcriptional capping of mRNA, polyadenine (PolyA) tails, and any combination thereof.

[0197] UTR (5'UTR and / or 3'UTR): In some embodiments, the single-stranded RNA may include nucleotide sequences encoding the desired 5'UTR and / or 3'UTR. Those skilled in the art will understand that the untranslated region of the mRNA sequence (e.g., the 3'UTR and / or 5'UTR) may contribute to mRNA stability, mRNA localization, and / or translation efficiency. In some embodiments, the untranslated region (UTR) may be located at the 5' (upstream) (5'UTR) and / or the 3' (downstream) (3'UTR) of the open reading frame.

[0198] In some embodiments, single-stranded RNA may include a 5'UTR nucleotide sequence and / or a 3'UTR nucleotide sequence. In some embodiments, such a 5'UTR sequence may be operably ligated to the 3' of a coding sequence (e.g., including one or more coding regions). Additionally or alternatively, in some embodiments, a 3'UTR sequence may be operably ligated to the 5' of a coding sequence (e.g., including one or more coding regions). In some embodiments, the 5'- and / or 3'-untranslated regions may be functionally ligated to an open reading frame so as to associate these regions with the open reading frame in a manner that increases the stability and / or translation efficiency of the RNA including the open reading frame, according to the present invention.

[0199] In some embodiments, the 5'UTR and 3'UTR sequences contained in the single-stranded RNA consist of, or include, naturally occurring or endogenous 5'UTR and 3'UTR sequences relative to the open reading frame of the gene of interest. Alternatively, in some embodiments, the 5' and / or 3'UTR sequences contained in the single-stranded RNA are not endogenous with respect to the coding sequence (e.g., they contain one or more coding regions). In some such embodiments, such 5' and / or 3'UTR sequences may be useful for modifying the stability and / or translation efficiency of the RNA sequence being transcribed. For example, those skilled in the art will understand that AU-rich elements in the 3'UTR sequence can reduce the stability of mRNA. Thus, as will be understood by those skilled in the art, the 3' and / or 5'UTRs can be selected or designed to enhance the stability of the RNA being transcribed, based on the UTR properties well known in the art.

[0200] For example, those skilled in the art will understand that in some embodiments, a nucleotide sequence consisting of, or containing, a Kozak sequence of the open reading frame sequence of the gene or nucleotide sequence of interest can be selectively used as the nucleotide sequence encoding the 5'UTR. As those skilled in the art will understand, Kozak sequences are known to enhance the translation efficiency of some RNA transcripts, but are not necessarily required for all RNAs to enable efficient translation. In some embodiments, single-stranded RNA may include a nucleotide sequence encoding the 5'UTR derived from an RNA virus whose RNA genome is stable in the cell. In some embodiments, for example, various modified ribonucleotides (e.g., as described herein) can be used in the 3' and / or 5'UTR to prevent exonuclease degradation of the transcribed RNA sequence.

[0201] As those skilled in the art will know, the Kozak sequence is the sequence first reported by Kozak (1987), Nucleic Acids Res., 15:8125-8148. Kozak sequences on RNA molecules such as mRNA molecules are recognized by ribosomes as translation initiation sites. Typically, a Kozak sequence contains an AUG start codon, immediately followed by a highly conserved G nucleotide: AUGG. In particular, it has been noted that Kozak sequences can be identified by (gcc)gccRccAUGG as follows: (i) lowercase letters indicate the most common base at a position where the base can change; (ii) uppercase letters indicate a highly conserved base (e.g., "AUGG"); (iii) "R" indicates a purine (adenine or guanine); (iv) the importance of the sequence in parentheses ((gcc)) is uncertain; (v) the underlined AUG base triplet represents the start codon.

[0202] In some embodiments, the RNA produced according to the present invention is (1) 5'UTR, (2) an open reading frame, and / or encoding, for example, the peptide or protein of interest. (3) 3'UTR.

[0203] In some embodiments, such a 5' UTR sequence may be operably concatenated to the 3' of a coding sequence (e.g., including one or more coding regions). Additionally or alternatively, in some embodiments, a 3' UTR sequence may be operably concatenated to the 5' of a coding sequence (e.g., including one or more coding regions).

[0204] In some embodiments, the 5'UTR contained in the single-stranded RNA may originate from human α-globin mRNA bound to the Kozak region.

[0205] In some embodiments, the single-stranded RNA may contain one or more 3'UTRs. For example, in some embodiments, the single-stranded RNA may contain two copies of a 3'UTR derived from globin mRNA, e.g., α2-globin, α1-globin, or β-globin (e.g., human β-globin) mRNA. In some embodiments, two copies of a 3'UTR derived from human β-globin mRNA may be used, for example, in some embodiments, these may be positioned between the coding sequence and the poly(A) tail of the single-stranded RNA to improve protein expression levels and / or long-term persistence of the mRNA. In some embodiments, the 3'UTRs contained in the single-stranded RNA may be one or more (e.g., 1, 2, 3, or more) of the 3'UTR sequences disclosed in WO2017 / 060314 (the entire contents of which are incorporated herein by reference for the purposes described herein). In some embodiments, the 3'-UTR may be a combination of at least two sequence elements (FI elements) derived from “split amino-terminal enhancer” (AES) mRNA (referred to as F) and mitochondrial-encoded 12S ribosomal RNA (referred to as I). These were identified by an ex vivo selection process of sequences that confer RNA stability and increase total protein expression (see WO2017 / 060314 incorporated herein by reference).

[0206] Human betaglobin 3'UTR, particularly two consecutive identical copies of human betaglobin 3'UTR, contributes to high transcriptional stability and translational efficiency (Holtkamp et al., (2006), Blood, 108:4009-4017). Therefore, in some embodiments, the replicase construct according to the present invention contains two consecutive identical copies of human β-globin 3'UTR. Thus, this includes the following in the 5'->3' direction: (a) optionally the 5'UTR; (b) an open reading frame; (c) the 3'UTR, where the 3'UTR contains two consecutive identical copies of human β-globin 3'UTR, its fragment, or a variant of human β-globin 3'UTR or its fragment.

[0207] In some embodiments, the RNA according to the present invention comprises a 3'UTR that is active to enhance translation efficiency and / or stability, but is not human β-globin 3'UTR, a fragment thereof, or a variant of human β-globin 3'UTR or a fragment thereof.

[0208] Poly-A tail: In some embodiments, single-stranded RNA may include a poly-A tail. The poly-A tail is a nucleotide sequence comprising a series of adenosine nucleotides, which can vary in length (e.g., at least five adenine nucleotides) and may contain up to several hundred adenosine nucleotides. The poly-A tail may be transcribed from a template, e.g., a DNA template, or enzymatically added after a transcription reaction. In some embodiments, the poly-A tail is a nucleotide sequence comprising at least 30 or more adenosine nucleotides, e.g., at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 100, or more adenosine nucleotides. In some embodiments, the poly-A tail is a poly-A homopolymer tail or comprises a poly-A homopolymer tail. In some embodiments, the poly-A tail may comprise one or more modified adenosine nucleosides, including but not limited to cordycepin and 8-azaadenosine. In some embodiments, the poly-A tail may comprise one or more non-adenosine nucleotides. In some embodiments, the poly-A tail may be, or comprise, a disrupted or modified poly-A tail as described in WO2016 / 005324 (the entire contents of which are incorporated herein by reference for the purposes described herein). For example, in some embodiments, the poly-A tail contained in the single-stranded RNA described herein may comprise a modified poly-A sequence comprising, or comprising, a modified poly-A sequence: a linker sequence; a first sequence of at least 20 consecutive A nucleotides, located at 5' of the linker sequence; and a second sequence of at least 20 consecutive A nucleotides, located at 3' of the linker sequence.In some embodiments, the modified polyA sequence may include: a linker sequence comprising at least 10 nucleotides (e.g., U, G, and / or C nucleotides); and a first sequence of at least 30 consecutive A nucleotides, located at 5' of the linker sequence; and a second sequence of at least 70 consecutive A nucleotides, located at 3' of the linker sequence.

[0209] In some embodiments, the poly(A) tail contains, or essentially consists of, or consists of, at least 20, at least 26, at least 40, at least 80, at least 100 and up to 500, up to 400, up to 300, up to 200, up to 150, and especially about 120 A nucleotides. In some embodiments, the poly(A) tail is a nucleotide sequence containing at least 30 or more adenosine nucleotides, for example, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 100, or more adenosine nucleotides. In this context, “essentially consisting of” means that most of the nucleotides in the poly(A)tail, typically at least 50%, and in some embodiments at least 75% of the number of nucleotides in the “poly(A)tail,” are A nucleotides (adenylates), but the remaining nucleotides may be other nucleotides, such as U nucleotides (uridylates), G nucleotides (guanylates), and C nucleotides (cytidylates). In this context, “consisting of” means that all of the nucleotides in the poly(A)tail, i.e., 100% of the number of nucleotides in the poly(A)tail, are A nucleotides. The terms “A nucleotide” or “A” refer to adenylates.

[0210] In fact, a 3' poly(A) tail of approximately 120 A nucleotides has been shown to have beneficial effects on RNA levels in transfected eukaryotic cells and on the levels of proteins translated from the open reading frame located upstream (5') of the 3' poly(A) tail (Holtkamp et al., (2006), Blood, 108:4009-4017).

[0211] According to the present invention, in some embodiments, the 3' poly(A) tail is attached during RNA transcription, i.e., during the preparation of in vitro transcribed RNA, based on a DNA template containing repeating dT nucleotides (deoxythymidylic acid) in a strand complementary to the coding strand. The DNA sequence encoding the poly(A) tail (coding strand) is called the poly(A) cassette.

[0212] 5' Cap: In some embodiments, the RNA product (e.g., single-stranded RNA) prepared as described herein may include a 5' cap that can be incorporated into such single-stranded RNA during transcription or that can be bound to such RNA after transcription. In some embodiments, the RNA may include a 5' cap structure for co-transcriptional capping of mRNA. Examples of cap structures for co-transcriptional capping are known in the art, including, for example, those described in WO2017 / 053297, the entirety of which is incorporated herein by reference for the purposes described herein. In some embodiments, the 5' cap included in the RNA product described herein is or includes a cap1 structure. For example, in some embodiments, the cap1 structure may be or include m7G(5')ppp(5')(2'OMeA)pG, m2 7,3’-O Gppp(m1 2’-O Also known as ApG'.

[0213] In some embodiments, as described herein, the RNA product produced (e.g., single-stranded RNA) may contain at least one modified ribonucleotide, for example, in some embodiments, to increase the stability of such RNA product and / or to reduce the cytotoxicity of such RNA product. For example, in some embodiments, at least one of the A, U, C, and G ribonucleotides of the single-stranded RNA may be substituted with a modified ribonucleotide. For example, in some embodiments, some or all of the cytidine residues present in the single-stranded RNA may be substituted with modified cytidine, in some embodiments the modified cytidine may be, for example, 5-methylcytidine. Alternatively or additionally, in some embodiments, some or all of the uridine residues present in the single-stranded RNA may be substituted with modified uridine, in some embodiments the modified uridine may be, for example, pseudouridine, such as 1-methylpseudridine. In some embodiments, all uridine residues present in the single-stranded RNA are substituted with pseudouridine, for example, 1-methylpseudridine.

[0214] DNA template Those skilled in the art will understand that DNA templates are typically used to direct the synthesis of RNA (e.g., single-stranded RNA). In some embodiments, the DNA template is a linear DNA molecule. In some embodiments, the DNA template is a circular DNA molecule. DNA can be obtained or produced using methods known in the art, such as gene synthesis, recombinant DNA techniques, or combinations thereof. In some embodiments, the DNA template comprises a nucleotide sequence encoding a region of interest to be transcribed (e.g., encoding the RNA described herein) and a promoter sequence recognized by an RNA polymerase selected for use in vitro transcription. Various RNA polymerases are known in the art, such as DNA-dependent RNA polymerases (e.g., T7 RNA polymerase, T3 RNA polymerase, SP6 RNA polymerase, N4 virion RNA polymerase, or variants or functional domains thereof). Those skilled in the art will readily understand that the RNA polymerases used herein may be recombinant RNA polymerases and / or purified RNA polymerases, i.e., they may not be part of a cell extract but may include other components in addition to the RNA polymerase. Those skilled in the art will recognize a suitable promoter sequence for the selected RNA polymerase. In some embodiments, the DNA template may include the promoter sequence of a T7 RNA polymerase.

[0215] In some embodiments, the DNA template includes a nucleotide sequence encoding the RNA described herein (for example, including a nucleotide sequence encoding the antigen of interest, and optionally including one or more nucleotide sequences encoding characteristic elements of the RNA described herein, such as the poly-A tail, 3'UTR, and / or 5'UTR). In some embodiments, such coding sequences may be generated by gene synthesis. In some embodiments, such coding sequences may be inserted into a vector by cold fusion cloning.

[0216] In some embodiments, the DNA template may further include one or more of the following: a suitable restriction endonuclease (e.g., used for linearization), a suitable resistance gene, and / or a recognition sequence for a suitable origin of replication.

[0217] In some embodiments, the DNA template can be amplified, or has been amplified, from plasmid DNA via polymerase chain reaction (PCR). In some embodiments, the plasmid DNA can be obtained, for example, from bacterial cells (e.g., Escherichia coli (E. coli)), and then a plasmid isolation procedure free from endotoxins and animal products may be performed.

[0218] Ribonucleotides Ribonucleotides for use in vitro transcription may comprise at least two (e.g., at least three, at least four, at least five, or at least six) different types of ribonucleotides, each having a different nucleoside. Examples of ribonucleotides for use in vitro transcription include unmodified and / or modified ribonucleotides. “Unmodified ribonucleotides” include the purine bases adenine (A) and guanine (G), and the pyrimidine bases cytosine (C) and uracil (U). In some embodiments, all four types of unmodified ribonucleotides may be used for in vitro transcription.

[0219] In some embodiments, at least one ribonucleotide present in the in vitro transcription is a modified ribonucleotide. Modified ribonucleotides may include, but are not limited to, one or more modifications such as: (a) terminal modifications, e.g., 5' terminal modifications (e.g., phosphorylation, dephosphorylation, conjugate linking, reverse linking, etc.), 3' terminal modifications (e.g., conjugate linking, reverse linking, etc.); (b) base modifications, e.g., substitution by a modified base, stabilizing base, destabilizing base, or base that forms a base pair with an extended repertoire of a partner, or by a conjugate base; (c) sugar modifications (e.g., at the 2' or 4' position) or sugar substitutions; and (d) nucleoside linkage modifications such as phosphodiester linkage modification or substitution. If such modifications interfere with translation to such an extent (e.g., translation is reduced by more than 50% compared to those without modification, e.g., characterized using a rabbit reticulocyte in vitro translation assay), such modified ribonucleotides are undesirable for use in the techniques described herein in some embodiments.

[0220] In some embodiments, one or more modified nucleosides may be used, such as one or more functional analogs of one or more adenosine triphosphate (ATP), guanosine triphosphate (GTP), cytidine triphosphate (CTP), and uridine triphosphate (UTP). When one of the functional analogs of ATP, GTP, CTP, or UTP is used in the method according to the present invention, the resulting RNA molecule will contain one of these functional analogs instead of ATP, GTP, CTP, or UTP, respectively.

[0221] In some embodiments, modified ribonucleotides may have at least one nucleoside ("base") modification or substitution. Various nucleoside modifications or substitutions are known in the art. Those skilled in the art will understand that modified nucleosides include, but are not limited to, synthetic and natural nucleic acid bases such as: for example, inosine, xanthine, hypoxanthine, nubularin, isoguanisine, tubercidine, 2-(halo)adenine, 2-(alkyl)adenine, 2-(propyl)adenine, 2-(amino)adenine, 2-(aminoalkyl)adenine, 2-(aminopropyl)adenine, 2- (Methylthio)-N6-(isopentenyl)adenine, 6-(alkyl)adenine, 6-(methyl)adenine, 7-(deaza)adenine, 8-(alkenyl)adenine, 8-(alkyl)adenine, 8-(alkynyl)adenine, 8-(amino)adenine, 8-(halo)adenine, 8-(hydroxyl)adenine, 8-(thioalkyl)adenine, 8-(thiol)adenine, N6-(isopentyl)adenine, N6-(methyl)adenine, N6,N6-(dimethyl)adenine, 2-(alkyl)guanine, 2-(propyl)guanine, 6-(alkyl)guanine, 6-(methyl)guanine, 7-(alkyl)guanine, 7-(methyl)guanine, 7-(deaza)guanine, 8-(alkyl)guanine, 8-(alkenyl)guanine, 8-(alkynyl)guanine, 8-(amino)guanine, 8-(halo)guanine, 8-(hydroxyl)guanine, 8-(thioalkyl)guanine, 8-(thiol)guanine, N-(methyl)guanine, 2-(thio)cytosine, 3-(deaza)-5-(aza)cytosine Tosine, 3-(alkyl)cytosine, 3-(methyl)cytosine, 5-(alkyl)cytosine, 5-(alkynyl)cytosine, 5-(halo)cytosine, 5-(methyl)cytosine, 5-(propynyl)cytosine, 5-(propynyl)cytosine, 5-(trifluoromethyl)cytosine, 6-(azo)cytosine, N4-(acetyl)cytosine, 3-(3-amino-3 carboxypropyl)uracil, 2-(thio)uracil, 5-(methyl)-2-(thio)uracil, 5-(methylaminomethyl)-2(thio)uracil, 4-(thio)uracil, 5-(methyl)- 4(thio)uracil, 5-(methylaminomethyl)-4(thio)uracil, 5-(methyl)-2,4-(dithio)uracil, 5-(methylaminomethyl)-2,4(dithio)uracil, 5-(2-aminopropyl)uracil, 5-(alkyl)uracil, 5-(alkynyl)uracil, 5-(allylamino)uracil, 5-(aminoallyl)uracil, 5-(aminoalkyl)uracil, 5-(guanidinium alkyl)uracil, 5-(1,3-diazole-1-alkyl)uracil, 5-(cyanoalkyl)uracil, 5-(dialkylamino Lukil)uracil, 5-(dimethylaminoalkyl)uracil, 5-(halo)uracil, 5-(methoxy)uracil, uracil-5-oxyacetic acid, 5-(methoxycarbonylmethyl)-2-(thio)uracil, 5-(methoxycarbonylmethyl)uracil, 5-(propynyl)uracil, 5-(propynyl)uracil, 5-(trifluoromethyl)uracil, 6-(azo)uracil, dihydrouracil, N3-(methyl)uracil, 5-uracil (i.e., pseudouracil), 2-(thio)pseudracil, 4-(thio)pseudracil, 2,4-(dithio)pseudracil, 5-(alkyl)pseudracil, 5-(methyl)pseudracil, 5-(alkyl)-2-(thio)pseudracil, 5-(methyl)-2-(thio)pseudracil, 5-(alkyl)-4(thio)pseudracil, 5-(methyl)-4(thio)pseudracil, 5-(alkyl)-2,4(dithio)pseudracil, 5-(methyl)-2,4(dithio)pseudracil, 1-substituted pseudouracil (e.g., 1-methylpseudridine), C-5 propynyl Uridine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyluridine, 1-substituted-2(thio)pseudracil, 1-substituted-4(thio)pseudracil, 1-substituted-2,4-(dithio)pseudracil, 1-(aminocarbonylethylenyl)pseudracil, 1-(aminocarbonylethylenyl)-2(thio)pseudracil, 1(aminocarbonylethylenyl)-4(thio)pseudracil, 1-(aminocarbonylethylenyl)-2, 4-(dithio)pseudracil, 1-(aminoalkylaminocarbonylethylenyl)-pseudracil, 1(aminoalkylamino-carbonylethylenyl)-2(thio)-pseudracil, 1-(aminoalkylaminocarbonylethylenyl)-4(thio)pseudracil, 1-(aminoalkylaminocarbonylethylenyl)-2,4-(dithio)pseudracil, 1,3-(diaza)-2-(oxo)-phenoxazine-1-yl, 1-(aza)-2-(thio)-3-(aza)-phenoxazine-1 -yl, 1,3-(diaza)-2-(oxo)-phenthiadin-1-yl, 1-(aza)-2-(thio)-3-(aza)-phenthiadin-1-yl, 7-substituted 1,3-(diaza)-2-(oxo)-phenoxazine-1-yl, 7-substituted 1-(aza)-2-(thio)-3-(aza)-phenoxazine-1-yl, 7-substituted 1,3-(diaza)-2-(oxo)-phenthiadin-1-yl, 7-substituted 1-(aza)-2-(thio)-3-(aza)-phenthiadin-1-yl, 7-(aminoalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenoxadin-1-yl,-(aminoalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenoxadin-1-yl,7-(aminoalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenthiadin-1-yl,7-(aminoalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenthiadin-1-yl,7-(guanidiniumalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenoxadin-1-yl,7-(guanidiniumalkylhydroxy) -1-(aza)-2-(thio)-3-(aza)-phenoxazine-1-yl, 7-(guanidinium alkyl-hydroxy)-1,3-(diaza)-2-(oxo)-phenthiadin-1-yl, 7-(guanidinium alkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenthiadin-1-yl, 1,3,5-(triaza)-2,6-(dioxa)-naphthalene, inosine, xanthine, hypoxanthine, nubularin, tubercidine, isoguanisine, inosinyl, 2-aza-inosinyl, 7-deazainosinyl, nitroimidazoly Nitropyrazolyl, nitrobenzimidazolyl, nitroindazolyl, aminoindolyl, pyrrolopyrimidinyl, 3-(methyl)isocarbostyrillyl, 5-(methyl)isocarbostyrillyl, 3-(methyl)-7-(propynyl)isocarbostyrillyl, 7-(aza)indolyl, 6-(methyl)-7-(aza)indolyl, imidizopyridinyl, 9-(methyl)-imidizopyridinyl, pyrrolopyridinyl, isocarbostyrillyl, 7-(propynyl)isocarbostyrillyl, propynyl-7-(aza)indolyl, 2,4,5-(trimethyl)phenyl , 4-(methyl)indolyl, 4,6-(dimethyl)indolyl, phenyl, naphthalenyl, anthracenyl, phenantracenyl, pyrenyl, stilbenyl, tetracerenyl, pentaceryl, difluorotolyl, 4-(fluoro)-6-(methyl)benzimidazole, 4-(methyl)benzimidazole, 6-(azo)thymine, 2-pyridinone, 5-nitroindole, 3-nitropyrrole, 6-(aza)pyrimidine, 2(amino)purine, 2,6-(diamino)purine, 5-substituted pyrimidine, N2-substituted purine, N6-substituted purine, O6-substituted purine, substituted 1,2,4-triazole, pyrrolo-pyrimidine-2-on-3-yl, 6-phenyl-pyrrolo-pyrimidine-2-on-3-yl, para-substituted-6-phenyl-pyrrolo-pyrimidine-2-on-3-yl, ortho-substituted-6-phenyl-pyrrolo-pyrimidine-2-on-3-yl, bis-ortho-substituted-6-phenyl-pyrrolo-pyrimidine-2-on-3-yl, para-(aminoalkylhydroxy)-6-phenyl-pyrrolo-pyrimidine- 2-on-3-yl, ortho-(aminoalkylhydroxy)-6-phenyl-pyrrolo-pyrimidine-2-on-3-yl, bis-ortho-(aminoalkylhydroxy)-6-phenyl-pyrrolo-pyrimidine-2-on-3-yl, pyridopyrimidine-3-yl, 2-oxo-7-amino-pyridopyrimidine-3-yl, 2-oxo-pyridopyrimidine-3-yl, or any O-alkylated or N-alkylated derivative thereof.

[0222] In some embodiments, modified nucleotides utilized in the IVT system and / or methods described herein can disrupt RNA binding to one or more endogenous RNA sensors (e.g., innate immune RNA sensors) of mammals (e.g., humans), such as, but not limited to, Toll-like receptor (TLR) 3, TLR7, TLR8, retinoic acid-inducible gene I (RIG-I), melanoma differentiation-associated gene 5 (MDA5), protein kinase R (PKR), 2'-5' oligoadenylate synthetase (OAS), and the Institute of Genetics and Physiology 2 (LGP2), and combinations thereof. In some embodiments, such modified ribonucleotides may include modifications described in US9,334,328, the contents of which are incorporated herein by reference in their entirety for the purposes described herein. Modified nucleosides are typically preferably translatable within the host cell (e.g., the presence of modified nucleosides does not interfere with the translation of the RNA sequence into its respective protein sequence). The effect of modified nucleotides on translation can be assayed, for example, using a rabbit reticulocyte lysate translation assay, by those skilled in the art.

[0223] In some embodiments, the modified ribonucleotide may include modified nucleoside linkages. Various such modified internucleoside linkages are known in the art; those skilled in the art will understand that non-limiting examples of modified internucleoside linkages that may be used in the art provided herein include phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkyl phosphotriesters, methyl and other alkylphosphonates (such as 3'-alkylene phosphonates and chiral phosphonates), phosphinates, phosphoramidates (such as 3'-aminophosphoramides and aminoalkylphosphoramides), thionophosphoramides, thionoalkyl phosphonates, thionoalkyl phosphotriesters, boranophosphates with normal 3'-5' linkages, their 2'-5' linked analogues, and those with inverted polarity (where pairs of adjacent nucleoside units are linked from 3'-5' to 5'-3' or from 2'-5' to 5'-2'). Various salts, mixed salts, and free acid forms are also included. Modified nucleoside linkages that do not contain phosphorus atoms may have short-chain alkyl or cycloalkyl nucleoside linkages, mixed heteroatoms and alkyl or cycloalkyl nucleoside linkages, or nucleoside linkages formed by one or more short-chain heteroatoms or heterocyclic nucleoside linkages. These include morpholino links (partially formed from the sugar moiety of the nucleoside); siloxane skeletons; sulfides, sulfoxides and sulfone skeletons; formacetyl and thioformacetyl skeletons; methyleneformacetyl and thioformacetyl skeletons; alkene-containing skeletons; sulfamate skeletons; methyleneimino and methylenehydrazino skeletons; sulfonic acid and sulfonamide skeletons; amide skeletons; and nucleotide substitutions having other components with mixed N, O, S, and CH2 constituent parts.

[0224] In some embodiments, a modified ribonucleotide may contain one or more substituted sugar moieties. Various such modified sugar moieties are known in the art. Those skilled in the art will understand that in some embodiments, the sugar moiety of a ribonucleotide may contain one of the following at the 2' position: H (deoxyribose); OH (ribose); F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S-, or N-alkynyl; or O-alkyl-O-alkyl; where alkyl, alkenyl, and alkynyl may be substituted or unsubstituted. In some embodiments, the sugar moiety of the ribonucleotide may be 2'-methoxyethoxy (2'-O-CH2CH2OCH3, also known as 2'-O-(2-methoxyethyl) or 2'-MOE), 2'-dimethylaminooxyethoxy, i.e., the O(CH2)2ON(CH3)2 group, also known as 2'-DMAOE, and 2'-dimethylaminoethoxyethoxy (also known in the art as 2'O-dimethylaminoethoxyethyl or 2'-DMAEOE), i.e., 2'-O-CH2-O-CH2-N(CH2)2;2'-methoxy(2'-OCH3), 2'-aminopropoxy(2'-OCH2CH2CH2NH2), and 2'-fluoro(2'-F). Similar modifications may occur at other positions, e.g., the 3' position of the sugar on the 3'-terminal nucleotide, or at the 5' position of 2'-5' linked nucleotides and 5'-terminal nucleotides.

[0225] In some embodiments, a mixture of ribonucleotides useful for an in vitro transcription reaction may include UTP or a functional analog in combination with at least one or all of ATP or a functional analog thereof, CTP or a functional analog thereof, and GTP or a functional analog thereof. In some embodiments, the functional analog of UTP is or includes N1-methylpseuduridine-5' triphosphate (m1ΨTP). As described herein, the disclosure provides, among other things, a technique in which UTP is restricted in the initial in vitro transcription reaction as described herein (e.g., one or more of ATP or a functional analog thereof, GTP or a functional analog thereof, and / or CTP or a functional analog thereof, in some embodiments at concentrations lower than all of them). In some embodiments, the disclosure provides, among other things, a technique in which UTP or a functional analog thereof is replenished over time in the initial in vitro transcription reaction in which UTP is restricted. In some such embodiments, such replenishment is carried out by one or more separate supply events. In some embodiments, such replenishment may be carried out by a continuous process, for example, in some embodiments where the UTP replenishment rate is comparable to the UTP consumption rate during the transcription reaction. In some embodiments, UTP is restricted during replenishment (for example, after such replenishment, UTP or its functional analog is replenished at concentrations lower than one or more of ATP or its functional analog, GTP or its functional analog, and / or CTP or its functional analog, in some embodiments, all of which are present during the reaction at concentrations lower than all of them).

[0226] In some embodiments, the functional analog of nucleoside triphosphate (NTP) comprises a modified nucleoside. In some embodiments, the modified nucleoside is modified uridine. In certain embodiments, the substitution of uridine with the modified nucleoside is performed by substituting UTP with its functional analog. In some embodiments, the functional analog of UTP is the triphosphate of a modified uridine nucleoside.

[0227] In some embodiments, the modified uridine nucleoside is independently selected from pseudouridine (Ψ), N1-methyl-pseudouridine (m1Ψ), and 5-methyl-uridine (m5U). In some embodiments, the modified nucleoside comprises pseudouridine (Ψ). In some embodiments, the modified nucleoside comprises N1-methyl-pseudouridine (m1Ψ). In some embodiments, the modified nucleoside comprises 5-methyl-uridine (m5U).

[0228] In some embodiments, the RNA may comprise two or more modified uridine nucleosides. In some embodiments, the RNA comprises two or more modified uridine nucleosides independently selected from pseudouridine (Ψ), N1-methyl-pseudouridine (m1Ψ), and 5-methyl-uridine (m5U). In some embodiments, the modified nucleoside comprises pseudouridine (Ψ) and N1-methyl-pseudouridine (m1Ψ). In some embodiments, the modified nucleoside comprises pseudouridine (Ψ) and 5-methyl-uridine (m5U). In some embodiments, the modified nucleoside comprises N1-methyl-pseudouridine (m1Ψ) and 5-methyl-uridine (m5U). In some embodiments, the modified nucleoside comprises pseudouridine (Ψ), N1-methyl-pseudouridine (m1Ψ), and 5-methyl-uridine (m5U).

[0229] In some embodiments, the modified nucleoside is any one or more selected from the group consisting of: 3-methyluridine (m3U), 5-methoxyuridine (mo5U), 5-azauridine, 6-azauridine, 2-thio-5-azauridine, 2-thiouridine (s2U), 4-thiouridine (s4U), 3-methyluridine, 4-thio-pseudridine, 2-thio-pseudridine, 5-hydroxyuridine (ho5U), 5-aminoallyluridine, 5-halouridine (e.g., 5-iodouridine or 5-bro). (Mo-uridine), uridine 5-oxyacetic acid (cmo5U), uridine 5-oxyacetic acid methyl ester (mcmo5U), 5-carboxymethyl-uridine (cm5U), 1-carboxymethyl-pseudridine, 5-carboxyhydroxymethyl-uridine (chm5U), 5-carboxyhydroxymethyl-uridine methyl ester (mchm5U), 5-methoxycarbonylmethyluridine (mcm5U), 5-methoxycarbonylmethyl-2-thio-uridine (mcm5s2U), 5-aminomethyl-2-thio-uridine (nm5s2U), 5- Methylaminomethyluridine (mnm5U), 1-ethyl-pseuduridine, 5-methylaminomethyl-2-thio-uridine (mnm5s2U), 5-methylaminomethyl-2-seleno-uridine (mnm5se2U), 5-carbamoylmethyl-uridine (ncm5U), 5-carboxymethylaminomethyl-uridine (cmnm5U), 5-carboxymethylaminomethyl-2-thio-uridine (cmnm5s2U), 5-propynyl-uridine, 1-propynyl-pseuduridine, 5-taurinomethyl-uridine (τm5U), 1-taurinomethyl Chil-pseuduridine, 5-taurinomethyl-2-thio-uridine (τm5s2U), 1-taurinomethyl-4-thio-pseuduridine), 5-methyl-2-thio-uridine (m5s2U), 1-methyl-4-thio-pseuduridine (m1s4Ψ), 4-thio-1-methyl-pseuduridine, 3-methyl-pseuduridine (m3Ψ), 2-thio-1-methyl-pseuduridine, 1-methyl-1-deaza-pseuduridine, 2-thio-1-methyl-1-deaza-pseuduridine, dihydrouridine (D), dihydropseuduridine, 5,6-Dihydrouridine, 5-methyl-dihydrouridine (m5D), 2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxy-uridine, 2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine, 4-methoxy-2-thio-pseudouridine, N1-methyl-pseudouridine, 3-(3-amino-3)-carboxypropyl)uridine (acp3U), 1-methyl-3-(3-amino-3-carboxypropyl)pseudouridine (acp3Ψ), 5-(isopentenylaminomethyl)uridine (inm5U), 5-(isopentenylaminomethyl)-2-thiouridine (inm5s2U), α-thio-uridine, 2’-O-methyl-uridine (Um), 5,2’-O-dimethyl-uridine (m5Um), 2’-O-methyl-pseudouridine (Ψm), 2-thio-2’-O-methyl-uridine (s2Um), 5-methoxycarbonylmethyl-2’-O-methyl-uridine (mcm5Um), 5-carbamoylmethyl-2’-O-methyl-uridine (ncm5Um), 5-carboxymethylaminomethyl-2’-O-methyl-uridine (cmnm5Um), 3,2’-O-dimethyl-uridine (m3Um), 5-(isopentenylaminomethyl)-2’-O-methyl-uridine (inm5Um), 1-thio-uridine, deoxythymidine, 2’-F-ara-uridine, 2’-F-uridine, 2’-OH-ara-uridine, 5-(2-carbomethoxyvinyl)uridine, 5-[3-(1-E-propenylamino)uridine, or any other modified uridine known in the art.,

[0230] In some embodiments, the functional analog of the NTP comprises a modified nucleoside. In some embodiments, the modified nucleoside is a modified adenosine. In certain embodiments, the substitution of adenosine with the modified nucleoside is effected by substituting ATP with its functional analog. In some embodiments, the functional analog of ATP is the triphosphate of a modified adenosine nucleoside.

[0231] In some embodiments, the modified nucleoside is 2-aminopurine, 2,6-diaminopurine, 7-deaza-adenine, 7-deaza-8-aza-adenine, 7-deaza-2-aminopurine, 7-deaza-8-aza-2-aminopurine, 7-deaza-2,6-diaminopurine, 7-deaza-8-aza-2,6-diaminopurine, 1-methyladenosine, N1-methyladenosine, N6-methyladenosine, N6-isopentenyladenosine, N6-(cis-hydroxyisopentenyl) It is one or more selected from the group consisting of denosine, 2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine, N6-glycinylcarbamoyladenosine, N6-threonylcarbamoyladenosine, 2-methylthio-N6-threonylcarbamoyladenosine, N6,N6-dimethyladenosine, α-thio-adenosine, 8-azido-adenosine, 7-deaza-adenosine, 7-methyladenine, 2-methylthioadenine, and 2-methoxyadenine.

[0232] In some embodiments, the functional analog of NTP comprises a modified nucleoside. In some embodiments, the modified nucleoside is modified guanosine. In certain embodiments, the substitution of guanosine with the modified nucleoside is performed by substituting GTP with its functional analog. In some embodiments, the functional analog of GTP is the triphosphate of the modified guanosine nucleoside.

[0233] In some embodiments, the modified nucleoside is 1-methyl-inosine, waiosine, waibutosine, α-thio-guanosine, 6-methyl-guanosine, 7-deazaguanosine, 7-deaza-8-azaguanosine, 6-thio-guanosine, 6-thio-7-deazaguanosine, 6-thio-7-deaza-8-azaguanosine, 7-methyl-guanosine, 6-thio-7-methyl-guanosine, 7-methyl It is one or more selected from the group consisting of inosine, 6-methoxyguanosine, O6-methylguanosine, N1-methylguanosine, N2-methylguanosine, N2,N2-dimethylguanosine, 8-oxoguanosine, 7-methyl-8-oxoguanosine, 1-methyl-6-thioguanosine, N2-methyl-6-thioguanosine, and N2,N2-dimethyl-6-thioguanosine.

[0234] In some embodiments, the functional analog of NTP comprises a modified nucleoside. In some embodiments, the modified nucleoside is a modified cytidine. In certain embodiments, the substitution of cytidine with the modified nucleoside is performed by substituting CTP with its functional analog. In some embodiments, the functional analog of CTP is the triphosphate of the modified cytidine nucleoside.

[0235] In some embodiments, the modified nucleoside is 5-azacytidine, 6-azacytidine, α-thiocytidine, pseudoisocytidine, 3-methylcytidine, N4-acetylcytidine, 5-formylcytidine, N4-methylcytidine, 5-hydroxymethylcytidine, 1-methylpsudoisocytidine, pyrrolocytidine, pyrrolopsudoisocytidine, 2-thiocytidine, 2-thio-5-methylcytidine, 4-thiopsudoisocytidine, 4-thio-1-methylcytidine It is one or more selected from the group consisting of chylpseudoisocytidine, 4-thio-1-methyl-1-deaza-pseudoisocytidine, 1-methyl-1-deazapseudoisocytidine, zebralin, 5-azazebralin, 5-methyl-zebralin, 5-azazebralin, 2-aza-2-thiozebralin, 2-thiozebralin, 2-methoxycytidine, 2-methoxy-5-methylcytidine, 4-methoxy-pseudoisocytidine, and 4-methoxy-1-methyl-pseudoisocytidine.

[0236] 5' Cap In some embodiments, the RNA produced by the techniques described herein may include a cap at its 5' end.

[0237] The terms “unextended nucleotide” or “starting nucleotide” or similar terms mean a nucleotide that does not have a 5' triphosphate, or a nucleotide that has a modified 5' triphosphate such that the nucleotide can be incorporated only at the 5' end of the transcript and has a 3' hydroxyl group so that it can be extended at the 3' position. The starting nucleotide is the nucleotide corresponding to the first nucleotide of the RNA. In some embodiments, the addition of a starting nucleotide increases the initiation rate of RNA polymerase. In certain embodiments, the starting nucleotide is or comprises a nucleoside monophosphate, a nucleoside diphosphate, a nucleoside triphosphate, or a dinucleoside triphosphate. If the first nucleotide of the RNA is G, the starting nucleotide may be GTP or GMP as described herein, or a functional analog thereof. In some embodiments, the starting nucleotide is a dinucleotide or a trinucleotide. In some embodiments, the starting nucleotide is a nucleoside-5'-triphosphate. In some embodiments, the first nucleotide of the RNA is G, the starting nucleotide is a capped analog of G, and the corresponding ribonucleoside triphosphate is GTP. In some embodiments, the starting nucleotide is a naturally occurring 5' cap or a 5' cap analog such as the cap analogs described herein. In some embodiments, the cap is a guanine nucleotide or comprises a guanine nucleotide. These nucleotides may or may not have capping function. Examples of starting nucleotides include the 5' caps and 5' cap analogs described herein.

[0238] In some embodiments, the RNA produced according to the techniques provided herein (i.e., RNA according to the present invention) has a starting nucleotide (starting nucleotide) that is not GTP. In some embodiments, the RNA according to the present invention has a starting nucleotide that competes with GTP or its functional analog with respect to incorporation into RNA. In some embodiments, the starting nucleotide is incorporated into RNA as readily as any other nucleotide. In some embodiments, the starting nucleotide is incorporated into RNA more efficiently than any other nucleotide, and in particular more efficiently than GTP or its functional analog. In some embodiments, the starting nucleotide is incorporated into RNA with less efficiency than any other nucleotide, and in particular less efficiency than GTP or its functional analog. In some embodiments, the starting nucleotide is supplemented during the transcription process. In some embodiments, the starting nucleotide is added to the reaction mix before the start of the transcription reaction.

[0239] In some embodiments, in the reaction mix used according to the present invention, the starting nucleotide corresponding to the first nucleotide of the RNA molecule to be produced is added in excess compared to the fraction of nucleotides that are expected to be found or found at the first position of the RNA molecule. In some embodiments, in the reaction mix, the starting nucleotide corresponding to the first nucleotide of the RNA molecule to be produced is added in excess compared to the fraction of nucleotides that compete for incorporation into the RNA molecule. For example, in some embodiments, the first nucleotide is G, and the 5' cap or 5' cap analog is present in excess of GTP during the initial reaction mix. In some embodiments, the starting nucleotide is added at an initial concentration in the range of about 1–20 mM, 1–17.5 mM, 1–15 mM, 1–12.5 mM, 1–10 mM, 1–7.5 mM, 1–5 mM, or 1 mM–2.5 mM. In some embodiments, the starting nucleotide is added in excess of at least about 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, or more, compared to nucleotides competing for incorporation into RNA. For example, in some embodiments, the starting concentration of the 5' cap or 5' cap analog versus the starting concentration of GTP is about 2:1 to about 20:1, e.g., about 2:1, 3:1, 4:1 in some embodiments, 5:1 in some embodiments, 6:1 in some embodiments, 7:1, 8:1, 9:1, 10:1, or more in some embodiments.

[0240] The terms “5' cap,” “cap,” “5' cap structure,” “cap structure,” “cap nucleotide,” and “5' cap nucleotide” are used synonymously to refer to the dinucleotide found at the 5' end of certain nucleic acids, such as mRNA. A 5' cap is a structure in which (optionally modified) guanosine is attached to the first nucleotide of an mRNA molecule via a 5'-5' triphosphate linkage (or a modified triphosphate linkage in the case of certain cap analogs). In some embodiments, this guanosine is methylated at position 7 (e.g., the naturally occurring m7G cap). The term “conventional 5' cap” refers to the naturally occurring RNA 5' cap, and in some embodiments, to the 7-methylguanosine cap (m7G). The provision of RNA having a 5' cap or a 5' cap analog can be achieved by in vitro transcription in which the 5' cap is simultaneously incorporated into the RNA chain (transcriptional and capping reactions), or by attaching the 5' cap to the RNA after in vitro transcription using a capping enzyme, such as a vaccinia virus-derived capping enzyme or a Saccharomyces cerevisiae capping enzyme system. Alternatively, the capped RNA can be obtained by in vitro transcription (IVT) of a DNA template, where the IVT system includes, for example, GTP, as well as the 5' cap or 5' cap analog known in the art and described herein. Methods for providing RNA having a 5' cap are well known in the art. In capped RNA, the 3' position of the first base of the (capped) RNA molecule is ligated to the 5' position of the next base ("second base") of the RNA molecule via a phosphodiester bond.

[0241] Those skilled in the art will understand that, in some embodiments, adding a 5' cap to RNA (e.g., mRNA) can facilitate the recognition of RNA by ribosomes and its attachment to these ribosomes for the initiation of translation, thereby increasing translation efficiency. Those skilled in the art will also understand that the 5' cap can protect the RNA product from 5' exonuclease-mediated degradation, and thus extend its half-life.

[0242] As described above, in some embodiments, the RNA produced by the techniques described herein may include a cap at its 5' end. In some embodiments, the RNA does not have an uncapped 5'-triphosphate. In some embodiments, the RNA may be modified with a 5' cap analog. In some embodiments, the 5' cap is a synthetic 5' cap analog that is similar to the RNA 5' cap structure and has the ability to stabilize the RNA when attached thereto, including, but not limited to, anti-reverse cap analogs (ARCAs) that are known in the art and described herein. Those skilled in the art will understand that by adding a 5' cap to RNA (e.g., mRNA), the recognition and attachment of the RNA to the ribosome for the initiation of translation may be facilitated, thereby increasing translation efficiency. Those skilled in the art will also understand that the 5' cap may protect the RNA product from 5' exonuclease-mediated degradation and thus extend its half-life. Methods of capping are known in the art; those skilled in the art will recognize that in some embodiments, capping can be performed after in vitro transcription in the presence of a capping system (e.g., an enzyme-based capping system, e.g., a vaccinia virus capping enzyme). In some embodiments, capped RNA can be obtained by in vitro capping RNA having a 5' triphosphate group or RNA having a 5' diphosphate group with a capping enzyme system (e.g., but not limited to, a vaccinia capping enzyme system or a Saccharomyces cerevisiae capping enzyme system). In some embodiments, the capping agent may be introduced into an in vitro transcription reaction mixture (e.g., one described herein) together with a number of ribonucleotides (also known as co-transcription capping) so that the cap is incorporated into the RNA during transcription. In some embodiments, it may be desirable for the RNA to contain a 5' cap, but in some embodiments, the RNA may not have a 5' cap.

[0243] The most frequently used method for producing capped RNA in vitro is to transcribe a DNA template using RNAP, such as bacterial or bacteriophage RNA polymerase, in the presence of all four ribonucleoside triphosphates and a 5' cap or 5' cap analog, e.g., m7G(5')ppp(5')G (also known as m7GpppG). The RNA polymerase initiates transcription by nucleophilic attack of the 3'-OH group of the guanosine moiety of m7GpppG against the α-phosphate of the next template nucleoside triphosphate (pppN), yielding the intermediate m7GpppGpN (where N is the second base of the RNA molecule). The formation of a competing GTP-initiated product, pppGpN, is inhibited by adding an excess of the 5' cap or 5' cap analog compared to GTP, as described herein.

[0244] In some embodiments, a 5' capping agent can be added to the in vitro transcription reaction mixture. In some embodiments, the 5' capping agent may include modified nucleotides, such as modified guanine nucleotides. In some embodiments, the 5' capping agent may include, for example, methyl groups, glyceryl, inverted deoxygenated base deletion moieties (abasic moieties), 4'5' methylene nucleotides, 1-(beta-D-erythrofuranosyl) nucleotides, 4' thionucleotides, carbocyclic nucleotides, 1,5-anhydrohexitol nucleotides, L-nucleotides, alpha-nucleotides, modified base nucleotides, threopentofuranosyl nucleotides, acyclic 3',4'-seconucleotides, acyclic 3,4-dihydroxybutyl nucleotides, acyclic 3,5-dihydroxypentyl nucleotides, 3'-3'-inverted nucleotide moieties, and 3'-3'-inverted base deletion moieties (inverted abasic The 5' capping agent may include a 3'-2'-reverse nucleotide moiety, a 3'-2'-reverse base deletion moiety, a 1,4-butanediol phosphate, a 3'-phosphoramide, a hexyl phosphate, an aminohexyl phosphate, a 3'-phosphate, a 3'-phosphorothioate, a phosphorodithioate, or a crosslinked or uncrosslinked methylphosphonate moiety, inosine, N1-methyl-guanosine, 2'-fluoro-guanosine, 7'-deaza-guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine, or 2-azido-guanosine. In some embodiments, the 5' capping agent may be a dinucleotide capping analog (e.g., m7GpppG capping analog or N7-methyl, 2'-O-methyl-GpppG anti-reverse capping analog (ARCA) capping analog or N7-methyl, 3'-O-methyl-GpppG ARCA capping analog) or may include such an analog. In some embodiments, the 5' capping agent comprises a 5'N7-methyl-3'-O-methylguanosine structure, e.g., CleanCap® Reagents (Trilink BioTechnologies). In some embodiments, the 5' cap is G[5']ppp[5']G, m7G[5']ppp[5']G, m3 2,2,7G[5']ppp[5']G, m2 7,3’-O G[5']ppp[5']G(3'-ARCA), m2 7,2’-O GpppG(2'-ARCA), m2 7,2’-O GppSpG(D1)(β-S-ARCA(D1)), and m2 7,2’-O GppSpG(D2)(β-S-ARCA(D2)) and m2 7,3’-O The 5'-capping agent is or may contain a dinucleotide cap analog such as Gppp(m2'-O)ApG(CC413). In some embodiments, the 5'-capping agent is added in excess to a specific ribonucleotide (plural) (e.g., GTP, ATP, UTP, CTP, or modified forms thereof) to enable the incorporation of the 5'-cap as the initial addition to the RNA transcript. In some embodiments, the 5'-cap used in the present invention is m2 7,3’-O Gppp(m 2’-O This is an ApG 5' cap.

[0245] In the context of this invention, the term "5' cap analog" refers to a molecular structure similar to a conventional 5' cap, but modified in some embodiments to have the ability to stabilize RNA when attached to RNA in vivo and / or intracellularly. The cap analog is not a conventional 5' cap.

[0246] The 5' cap is generally explained as being involved in the efficient translation of mRNA: Generally, in eukaryotes, translation is initiated only at the 5' end of the messenger RNA (mRNA) molecule unless an internal ribosome entry site (IRES) is present. Eukaryotic cells can provide RNA with a 5' cap during transcription in the nucleus: Typically, newly synthesized mRNA is modified with a 5' cap structure, for example, when the transcript reaches 20-30 nucleotides in length. First, the 5' terminal nucleotide pppN (ppp represents triphosphate; N represents any nucleoside) is converted to 5'GpppN in the cell by a capping enzyme having RNA 5'-triphothphatase and guanylyltransferase activity. Subsequently, the GpppN is methylated in the cell by a second enzyme having (guanine-7)-methyltransferase activity to monomethylated m 7 It may form a GpppN cap. In some embodiments, the 5' cap used in the present invention is a natural 5' cap.

[0247] If translation of the protein-coding nucleic acid sequence is desired after introducing each RNA into a host cell or host organism, it is highly preferable that a cap be present on the RNA molecule, especially if translation is desired within the first hour, first two hours, or first three hours after RNA introduction.

[0248] In the present invention, natural 5' capped dinucleotides are typically unmethylated capped dinucleotides (G(5')ppp(5')N; also called GpppN) and methylated capped dinucleotides ((m 7 G(5')ppp(5')N;m 7 Selected from the group consisting of (also called GpppN). 7 GpppN (where N is G) is expressed by the following equation: [ka]

[0249] In certain embodiments of the present invention, the 5' cap is a 5' cap analog. 5' cap analogs were initially reported to facilitate the large-scale synthesis of RNA transcripts by in vitro transcription.

[0250] For RNAs such as mRNA, several 5' cap analogs (synthetic caps) have been generally described, and all of them can be used in connection with the present invention. Ideally, a 5' cap analog associated with higher translation efficiency and / or increased resistance to in vivo degradation and / or increased resistance to in vitro degradation is selected.

[0251] In some embodiments, a 5' cap analog that can be incorporated into the RNA strand only in one direction is used. Pasquinelli et al., (1995), RNA J. 1:957-967 demonstrated that during in vitro transcription, bacteriophage RNA polymerase uses a 7-methylguanosine unit at the start of transcription, such that approximately 40 - 50% of the transcripts with a cap have an inverted cap dinucleotide (i.e., when using m7G, the first reaction product is Gpppm 7 GpN). Compared to RNA with the correct 5' cap, RNA with an inverted 5' cap is not functional with respect to translation from the nucleic acid sequence to protein. Therefore, it is desirable to incorporate the 5' cap in the correct direction, i.e., to obtain RNA having a structure essentially corresponding to m 7 GpppGpN etc. The inverted incorporation of the cap dinucleotide is inhibited by substitution of either the 2'-OH group or the 3'-OH group of the methylated guanosine unit (Stepinski et al., (2001), RNA J., 7:1486-1495; Peng et al., (2002), Org. Lett., 24:161-164). RNA synthesized in the presence of such an "anti-reverse cap analog" or "ARCA" has a conventional 5' cap m 7RNA is translated more efficiently in vitro than RNA transcribed in the presence of GpppG. For this purpose, one cap analog in which the 3'OH group of the methylated guanosine unit is substituted with OCH3 has been described, for example by Holtkamp et al., (2006), Blood, 108:4009-4017 ((7-methyl(3'-O-methyl)GpppG; anti-reverse cap analog (ARCA))). 7-methyl(3'-O-methyl)GpppG (also called 3'-ARCA) is a preferred cap dinucleotide according to the present invention. [ka]

[0252] In some embodiments of the present invention, the RNA of the present invention is inherently resistant to decapping. This is important because, generally, the amount of protein produced from synthetic mRNA introduced into cultured mammalian cells is limited by the natural degradation of the mRNA. One in vivo pathway of mRNA degradation begins with the removal of the mRNA cap. This removal is catalyzed by a heterodimeric pyrophosphatase comprising a regulatory subunit (Dcp1) and a catalytic subunit (Dcp2). The catalytic subunit cleaves between the α-phosphate and β-phosphate groups of the triphosphate crosslink. In the present invention, a cap analog that is resistant to or less susceptible to this type of cleavage may be selected or introduced. A cap analog suitable for this purpose may be selected from capped dinucleotides according to formula (I): [ka] (In the formula, R 1 This is selected from the group consisting of optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted cycloalkyl, optionally substituted heterocyclyl, optionally substituted aryl, and optionally substituted heteroaryl. R 2 and R 3is independently selected from the group consisting of H, halo, OH, and optionally substituted alkoxys, or R 2 and R 3 These combine to form OXO (wherein X is optionally substituted CH2, CH2CH2, CH2CH2CH2, CH2CH(CH3), (Selected from the group consisting of and C(CH3)2) or R 2 R 2 It bonds with the hydrogen atom at the 4' position of the ring to which it is attached, forming -O-CH2- or -CH2-O-. R 5 It is selected from the group consisting of S, Se, and BH3. R 4 and R 6 These are independently selected from the group consisting of O, S, Se, and BH3. n is 1, 2, or 3.

[0253] R 1 , R 2 , R3, R 4 , R 5 , R 6 Specific embodiments are disclosed in WO2011 / 015347A1, which can be selected accordingly in the present invention.

[0254] For example, in some embodiments of the present invention, the 5' cap is a phosphorothioate cap analog or comprises a phosphorothioate cap analog. The phosphorothioate cap analog has one of the three non-bridged oxygen atoms in the triphosphate chain substituted with an oxygen atom, i.e., R of formula (I). 4 , R 5 or R 6One of these is a specific cap analog in which S is present. Phosphothioate cap analogs have been described by Kowalska et al., (2008), RNA, 14:1119-1131 as a solution to the undesirable decapping process and thus as an enhancer of RNA stability in vivo. In particular, stabilization with respect to Dcp2 is achieved by substituting the sulfur atom of the 5' cap beta-phosphate group with an oxygen atom. In a preferred embodiment of the present invention, R of formula (I) 5 S is; R 4 and R 6 It is O.

[0255] In some embodiments of the present invention, the RNA of the present invention comprises a phosphorothioate cap analog in which the phosphorothioate modification of the RNA 5' cap is combined with an “anti-reverse cap analog” (ARCA) modification. Each ARCA-phosphorothioate-cap-analog is described in WO2008 / 157688A2, and all of these can be used in the RNA of the present invention. In this embodiment, R of formula (I) 2 or R 3 At least one of them is not OH, but in some embodiments, R 2 and R 3 One of them is methoxy (OCH3), and R 2 and R 3 Another of these is, in some embodiments, OH. In some embodiments, the oxygen atom is substituted for the sulfur atom of the β-phosphate group (hence the R in formula (I)). 5 S is R 4 and R 6( is O). It is thought that phosphorothioate modification of ARCA ensures that the α, β, and γ phosphorothioate groups are precisely and reliably positioned within the active site of the cap-binding protein in both the translation and decapsulation mechanisms. At least some of these analogs are inherently resistant to pyrophosphatase Dcp1 / Dcp2. Phosphothioate-modified ARCA has been described as having a much higher affinity for eIF4E than the corresponding ARCA lacking the phosphorothioate group.

[0256] In the present invention, each of the 5' capped analogs, namely m2 7,2’-O Gpp s pG is called β-S-ARCA (WO2008 / 157688A2; Kuhn et al., Gene Ther., (2010), 17:961-971). Therefore, in some embodiments of the present invention, the RNA of the present invention is modified with β-S-ARCA (or β-S-ARCA). β-S-ARCA is represented by the following structure: [ka]

[0257] Generally, in cross-linked phosphates, oxygen atoms are replaced by sulfur atoms, generating phosphorothioate diastereomers called D1 and D2 based on the elution pattern in HPLC. In short, "β-S-ARCA D1 diastereomer" or "β-S-ARCA(D1)" or "m2 7,2’-O Gpp S pG(D1) is a diastereomer of β-S-ARCA, and the D2 diastereomer of β-S-ARCA is β-S-ARCA(D2) or m2 7,2’-O Gpp S Compared to pG(D2), it elutes first to the HPLC column and therefore exhibits a shorter retention time. The determination of the stereochemical configuration by HPLC is described in WO2011 / 015347A1.

[0258] In certain embodiments of the present invention, the RNA of the present invention is modified with the β-S-ARCA (D2) diastereomer. The two diastereomers of β-S-ARCA differ in their sensitivity to nuclease. RNA carrying the D2 diastereomer of β-S-ARCA is almost completely resistant to Dcp2 cleavage (only 6% cleavage rate compared to RNA synthesized in the presence of unmodified ARCA 5'-cap), and RNA having a β-S-ARCA (D1) 5'-cap has been shown to exhibit intermediate sensitivity to Dcp2 cleavage (71% cleavage). Furthermore, an increase in stability against Dcp2 cleavage has been shown to correlate with an increase in protein expression in mammalian cells. In particular, RNA carrying the β-S-ARCA (D2) cap has been shown to be translated more efficiently in mammalian cells than RNA having a β-S-ARCA (D1) cap. Thus, in some embodiments of the present invention, the 5'-cap used in the present invention is a 5'-cap analog of formula (I), and the P β atom corresponding to the substituent R in formula (I) 5 characterized by the stereochemical configuration at the P atom containing it. In that embodiment, R 5 of formula (I) is S, and R 4 and R 6 are O. Further, in some embodiments, at least one of R 2 or R 3 of formula (I) is not OH, and / or one of R 2 and R 3 is methoxy (OCH3), and / or the other of R 2 and R 3 is OH; in some embodiments, each of R 2 or R 3 of formula (I) is not OH; in some such embodiments, one of R 2 and R 3 is methoxy (OCH3), and the other of R 2 and R 3 is OH.

[0259] In certain other embodiments of the present invention, the 5' cap used is a β-S-ARCA(D1) diastereomer. This embodiment is particularly suitable for the transfer of capped RNA into immature antigen-presenting cells, such as for vaccination purposes. Each β-S-ARCA(D1) diastereomer has been demonstrated to be particularly suitable for improving RNA stability, improving RNA translation efficiency, extending RNA translation, increasing total RNA protein expression, and / or increasing the immune response to the antigen or antigenic peptide encoded by the RNA when the capped RNA is introduced into immature antigen-presenting cells (Kuhn et al., (2010), Gene Ther., 17:961-971). Accordingly, in some embodiments of the present invention, the RNA of the present invention is modified with the cap analog described in formula (I) and the P of the β-S-ARCA D1 diastereomer β Substituent R in formula (I) corresponding to those present on an atom 5 Characterized by the stereochemical configuration at the P atom containing . Each cap analog and its embodiments are described in WO2011 / 015347A1 and Kuhn et al., (2010), Gene Ther., 17:961-971. Substituent R 5 Any cap analog described in International Publication No. 2011 / 015347A1, in which the stereochemical configuration at the P atom containing corresponds to the stereochemical configuration at the Pβ atom of the D1 diastereomer of β-S-ARCA, can be used in the present invention. In some embodiments, R of formula (I) 5 S is R 4 and R 6 is O. Furthermore, in some embodiments, R of formula (I) 2 or R 3 At least one of them is not OH, and / or R 2 and R 3 One of them is methoxy(OCH3), and / or R 2 and R 3 The other of these is OH; in some embodiments, R of formula (I) 2 or R 3Each of these is not OH; in some such embodiments, R 2 and R 3 One of them is methoxy (OCH3), R 2 and R 3 The other of these is OH. In some embodiments, the 5' cap used in the present invention is m2 7,3’-O Gppp(m1 2’-O )ApG(m2 7,3`O G(5')ppp(5')m 2’-O ApG, m2 7,3’-O Gppp(m 2’-O This is sometimes referred to as ApG, CC413, or CleanCap. In a particularly preferred embodiment, the RNA of the present invention is modified with CleanCap.

[0260] A useful 5' cap in this invention is, but is not limited to, m3 2,2,7 G[5']ppp[5']G, m2 7,2’-O GpppG(2'-ARCA), m 7 Gp3m 2’-O G, m 7 Gp3m 7 G, m2 7,2’-O Gp3G, m2 7,2’-O GpppSG(D1), m2 7,2’-O GpppSG(D2), m2 7,2’-O Gpp S pG(D1), m2 7,2’-O Gpp S pG(D2), m2 7,2’-O Gp S ppG(D1), m2 7,2’-O Gp S ppG(D2) is another example. The 5' cap useful in this invention is also a tetraphosphate derivative of the triphosphate 5' cap analog, for example, m 7 Gp4G(m 7 (A derivative of Gp3G), b 7 Gp4G(m2 7,3’-O (A derivative of Gp3G), b 7 m 3’-O Gp4G(b 7 (A derivative of Gp3G), m2 2,7 Gp4G(e7 (A derivative of Gp3G), m3 2,2,7 Gp4G(m2 2,7 (A derivative of Gp3G), b 7 m 2 Gp4G(m3 2,2,7 (It is a derivative of Gp3G), m 7 Gp4m 7 G(m 7 It may be a derivative of Gp32'dG. Further useful 5' capped analogs are described in US7074596, WO2008 / 016473, WO2008 / 157688, WO2009 / 149253, WO2011 / 015347, and WO2013 / 059475.

[0261] In some embodiments, the 5' cap used in the present invention is a 5' cap structure according to formula (I) in which any one phosphate group is substituted with a boranophosphate group or a phosphoroselenoate group. Such 5' caps have improved stability both in vitro and in vivo. Optionally, each compound has a 2'-O- or 3'-O-alkyl group (in some embodiments, the alkyl is methyl); each cap analog is called BH3-ARCA or Se-ARCA. Compounds particularly suitable for mRNA capping include β-BH3-ARCA and β-Se-ARCA as described in WO2009 / 149253. For these compounds, the D1 diastereomer of β-S-ARCA is P β The substituent R in formula (I) corresponds to the stereochemical configuration at the atom. 5 A stereochemical configuration at the P atom containing is preferred.

[0262] Exemplary in vitro transcription reaction Those skilled in the art will understand the materials and reagents for typical in vitro transcription. In some embodiments, the individual reaction components are thawed before being added to the in vitro transcription reaction mixture. For example, the in vitro transcription reaction mixture typically includes a DNA template (e.g., as described herein), ribonucleotides (e.g., as described herein), RNA polymerase (e.g., DNA-dependent RNA polymerase), and a reaction buffer for a suitablely selected RNA polymerase. In some embodiments, the in vitro transcription reaction mixture may further include an RNase inhibitor. In some embodiments, the in vitro transcription reaction mixture may further include a pyrophosphatase (e.g., inorganic pyrophosphatase). In some embodiments, the in vitro transcription reaction mixture may further include one or more salts (e.g., monovalent and / or divalent salts of Mg2+), a reducing agent (e.g., dithitrateol, 2-mercaptoethanol, etc.), spermidine, or a combination thereof. In some embodiments, specific reaction components are added in a specific order (e.g., pyrophosphatase and polymerase are added last). In some embodiments, the stirring speed is increased after the addition of specific reaction components (e.g., pyrophosphatase, polymerase).

[0263] Various RNA polymerases suitable for in vitro transcription are known in the art, but are not limited to, DNA-dependent RNA polymerases (e.g., T7 RNA polymerase, T3 RNA polymerase, SP6 RNA polymerase, N4 virion RNA polymerase, or their variants or functional domains). Those skilled in the art will understand that the RNA polymerases used herein may be recombinant RNA polymerases and / or purified RNA polymerases, i.e., they may not be part of a cell extract and may include other components in addition to the RNA polymerase. In some embodiments, the RNA polymerase useful for commercial-scale in vitro transcription is T7 RNA polymerase. In some embodiments, inorganic pyr phosphatase may be added to improve the yield of the in vitro transcription reaction (e.g., catalyzed by T7 RNA polymerase in some embodiments).

[0264] In some embodiments, the buffer used for the transcription reaction (transcription buffer) is optimized for the selected RNA polymerase. Typically, the transcription buffer is optimized for the selected RNA polymerase. For example, in some embodiments, the transcription buffer may include Tris-HCl, HEPES, or other suitable buffers. In some embodiments, the transcription buffer may contain 20–60 mM HEPES, 20–60 mM divalent salts (e.g., magnesium salts, e.g., magnesium chloride, magnesium acetate, Li + na + , K + NH 4+ Tris(hydroxymethyl)aminomethane cation, Mg 2+ Ba 2+ or Mn 2+ It may contain, for example, 5-15 mM reducing agents (e.g., dithiothreitol, 2-mercaptoethanol, etc.), and 0.5-3 mM spermidine.

[0265] In some embodiments, the transcription reaction is carried out at a pH of approximately 6, 6.5, 7, 7.5, 8, or 9. In some embodiments, the transcription buffer has a pH of 7–9 (e.g., approximately 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0). In some embodiments, the transcription buffer has a pH of 6–9. In some embodiments, the pH values ​​are approximately 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, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, and 9.0. In some embodiments, the transfer buffer has a pH value of approximately 6 to 8.5. In some embodiments, the buffer has a pH value of approximately 6 to 8.5, approximately 6.5 to 8.0, approximately 7.0 to 7.5, and in some embodiments, approximately 7.5. In some embodiments, the pH value suitable for the transfer reaction may be approximately 7.5 to 8.5. In some embodiments, the pH values ​​are approximately 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, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, and 9.0. In some embodiments, the pH values ​​are approximately 6 to 8.5. In some embodiments, the pH values ​​are 6 to 8.5, 6.5 to 8.0, and 7.0 to 7.5; in some embodiments, the pH value is 7.5.

[0266] In some embodiments, the pH value of the reaction mix is ​​kept essentially constant throughout the transfer reaction, for example, by using a suitable buffer. Suitable buffers for adjusting the pH value include, but are not limited to, NaOH buffer, KOH buffer, or HCl buffer, which are known in the art and described herein.

[0267] In some embodiments, the pH of the reaction mix is ​​kept substantially constant throughout the transcription reaction, for example, by supplementing it with a buffer having a pH similar to or equal to that of the starting reaction mix, and / or, if necessary, by supplementing it with a buffer having a pH different from that of the starting reaction mix.

[0268] In some embodiments, the buffer is selected from the group consisting of 80 mM HEPES / KOH, pH 7.5 and 40 mM Tris / HCl, pH 7.5.

[0269] Exemplary in vitro transcription reaction conditions In some embodiments, the in vitro transcription reaction is carried out for a certain period of time in, for example, a bioreactor as described herein (e.g., selected for a specific in vitro transcription reaction volume as described herein). In some embodiments, this period is at least 20 minutes, for example, at least 25 minutes, at least 30 minutes, at least 40 minutes, at least 55 minutes, at least 60 minutes, at least 75 minutes, at least 90 minutes, at least 105 minutes, at least 120 minutes, at least 135 minutes, at least 150 minutes, at least 165 minutes, or at least 180 minutes. In some embodiments, this period is 20 minutes, 25 minutes, 30 minutes, 35 minutes, 40 minutes, 45 minutes, 50 minutes, 55 minutes, 60 minutes, 65 minutes, 70 minutes, 75 minutes, 80 minutes, 85 minutes, 90 minutes, 95 minutes, 100 minutes, 105 minutes, 110 minutes, 115 minutes, 120 minutes, 125 minutes, 130 minutes, 135 minutes, 140 minutes, 145 minutes, 150 minutes, 155 minutes, 160 minutes, 165 minutes, 170 minutes, 175 minutes, or 180 minutes. In some embodiments, this period is approximately 1.5 to 3 hours. In some embodiments, this period is approximately 25 to 35 minutes.

[0270] In some embodiments, the in vitro transcription reaction is carried out for a certain period of time (e.g., as described herein) at a temperature in which the selected RNA polymerase is functionally active, for example, in a bioreactor as described herein. Typical phage RNA polymerases that carry out in vitro transcription reactions (e.g., T7 polymerase) are usually inactive at high temperatures (e.g., above 45°C), but heat-stable RNA polymerases (e.g., heat-stable variants of T7 RNA polymerase, e.g., those described in US10519431 (the contents of which are incorporated by reference for the purposes described herein)) may show improved stability at high temperatures. In some embodiments, the in vitro transcription is carried out at a temperature of 25°C or higher, for example, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, 41°C, 42°C, 43°C, 44°C, or 45°C. In some embodiments, in vitro transcription is performed at a temperature of approximately 45°C or higher, for example, 46°C, 47°C, 48°C, 49°C, 50°C, 51°C, 52°C, 53°C, 54°C, 55°C or higher.

[0271] In some embodiments, in vitro transfer is carried out, for example, in a bioreactor described herein, at a pH of about 6, 6.5, 7, 7.5, 8, or 9. In some embodiments, a pH suitable for in vitro transfer may be about 7.5 to 8.5.

[0272] In some embodiments, the in vitro transcription reactions carried out according to this disclosure (e.g., in a bioreactor as described herein) may be carried out as continuous feed reactions; in some embodiments, they may be carried out as batch feed reactions. In some embodiments, one or more nucleotides may be added to the in vitro transcription reaction in a stepwise manner (e.g., at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more bolus doses). In some embodiments, the stirring rate is selected so as to achieve a specific mixing time that allows for rapid mixing of the bolus additions in order to ensure optimal availability of the modified nucleotide solution and one or more other nucleotide solutions during RNA synthesis.

[0273] Restriction of reaction components: In certain embodiments, the restricting component is present at a lower starting concentration than the starting concentration of the non-restricting component. In some embodiments, the restricting component is a nucleotide such as ATP, GTP, CTP, UTP, or a functional analog thereof. In some embodiments, the non-restricting component is a nucleotide such as ATP, GTP, CTP, UTP, or a functional analog thereof. The restricting or non-restricting component may be a 5' cap or a 5' cap analog. In some embodiments, the ratio of the restricting component to the non-restricting component, for example, the ratio of UTP or its functional analog to ATP and / or CTP or its functional analog, or the ratio of GTP or its functional analog to ATP and / or CTP or its functional analog, is about 1:1 to about 1:100, for example, 1:1.1 to about 1:80, 1:1.2 to about 1:60, 1:1.3 to about 1:40, in some embodiments 1:1.4 to about 1:30, in some embodiments 1:1.5 to about 1:20, in some embodiments 1:1.5 to about 1:15, 1:1.6 to about 1:10, 1:1.7 to about 1:9, 1:1.8 to about 1:8, 1:1.9 to about 1:7, 1:2 to about 1:6. In some embodiments, the starting concentration of one or more restricting components, such as UTP or its functional analog, or GTP or its functional analog, is 1 / 2, 1 / 3, 1 / 4, 1 / 5, 1 / 6, 1 / 7, 1 / 8, 1 / 9, 1 / 10, 1 / 11, 1 / 12, 1 / 13 compared to the starting concentration of one or more non-restricting components, such as ATP and / or CTP or their functional analogs. These are 1 / 14, 1 / 15, 1 / 16, 1 / 17, 1 / 18, 1 / 19, 1 / 20, 1 / 21, 1 / 22, 1 / 23, 1 / 24, 1 / 25, 1 / 26, 1 / 27, 1 / 28, 1 / 29, 1 / 30, 1 / 31, 1 / 32, 1 / 33, 1 / 34, 1 / 35, 1 / 36, 1 / 37, 1 / 38, 1 / 39, 1 / 40, 1 / 50, 1 / 60, 1 / 70, 1 / 80, 1 / 90, or 1 / 100.

[0274] UTP restriction and / or supplementation: In some embodiments, the in vitro transcription reaction includes UTP or its functional analog at a limiting concentration, in combination with at least one or all of ATP or its functional analog, CTP or its functional analog, and optionally GTP or its functional analog. In some embodiments, the functional analog of UTP is or includes N1-methylpseuduridine-5' triphosphate (m1ΨTP). In some embodiments, UTP or its functional analog is present in the in vitro transcription reaction at an initiation concentration that limits the transcription rate. In some embodiments, UTP or its functional analog is present in the in vitro transcription reaction at an initiation concentration lower than the initiation concentration of at least one or all of ATP or its functional analog, CTP or its functional analog, and optionally GTP or its functional analog. In some embodiments, the starting concentration of UTP or its functional analog is at least 30% lower (e.g., at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%) than the starting concentrations of at least one or all of ATP or its functional analog, CTP or its functional analog, and optionally GTP or its functional analog. In some embodiments, the ratio of the starting concentration of UTP or its functional analog to the starting concentrations of ATP or its functional analog, CTP or its functional analog, and optionally at least one or all of GTP or its functional analog is about 1:1.3 or less, for example, 1:1.4; 1:1.5; 1:2; 1:2.5; 1:3; 1:3.5; 1:4; 1:4.5; 1:5; 1:6; 1:7; 1:8; 1:9; 1:10; 1:11; 1:12; 1:13; 1:14; 1:15; 1:16; 1:17; 1:18; 1:19; 1:20 or less.In some embodiments, the ratio of the starting concentration of UTP or its functional analog to the starting concentrations of ATP or its functional analog, CTP or its functional analog, and optionally at least one or all of GTP or its functional analog is about 1:1.3 to about 1:20, or about 1:1.5 to about 1:15, or about 1:5 to about 1:15, or about 1:8 to about 1:12. In some such embodiments, the starting concentrations of ATP or its functional analog, CTP or its functional analog, and optionally GTP or its functional analog may be the same.

[0275] In some embodiments, the in vitro transcription reaction is replenished with UTP or its functional analog at least once during the course of the reaction. In some embodiments, the in vitro transcription reaction is replenished with UTP or its functional analog multiple times (e.g., at least twice, or more, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more times) during the course of the transcription reaction. In some embodiments, replenishment of UTP or its functional analog is performed when its concentration in the reaction mixture is near depletion. In some embodiments, supplementation with UTP or its functional analog is performed when its concentration in the reaction mixture is less than 100 μM, 90 μM, 80 μM, 70 μM, 60 μM, 50 μM, 40 μM, 30 μM, 20 μM, 10 μM, 5 μM, 3 μM, 2 μM, 1 μM, 500 nM, 250 nM, 200 nM, 100 nM, 50 nM, 25 nM, or less. In some embodiments, supplementation with UTP or its functional analog may include supplementing with UTP or its functional analog as a composition optionally containing further reactants as described herein, and supplementing with GTP or its functional analog. In other embodiments, supplementing with UTP does not mean supplementing with other reactants. Similarly, in some embodiments, supplementing with GTP does not mean supplementing with other reactants.

[0276] In some embodiments, replenishment of UTP (or its functional analog) may be carried out continuously during the transcription reaction. For example, in some embodiments, replenishment of UTP (or its functional analog) may be carried out continuously at a rate comparable to its consumption rate (e.g., 10% or less). In some embodiments, replenishment of UTP (or its functional analog) may be carried out at a rate such that, after such replenishment, UTP or its functional analog is present in the reaction at a concentration lower than that of ATP or its functional analog, GTP or its functional analog, and / or CTP or its functional analog, in some embodiments, all of them combined.

[0277] In some embodiments, replenishment of UTP (or its functional analog) may be carried out periodically during the course of the transcription reaction. In some embodiments, replenishment of UTP (or its functional analog) may be carried out periodically such that, after each addition, UTP or its functional analog is present in the reaction at a concentration lower than, in some embodiments, all of, one or more of ATP or its functional analog, GTP or its functional analog, and / or CTP or its functional analog. In some embodiments, such periodic replenishment may be carried out as one or more bolus or batch additions, for example, one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen or more bolus or batch additions. In some embodiments, such periodic replenishment may be carried out by a fed-batch culture process. In some embodiments, supplementation may include supplementing with a composition comprising UTP or a functional analog thereof, and further components such as a buffer, polymerase, CTP or a functional analog thereof, GTP or a functional analog thereof, ATP or a functional analog thereof, or other components that may be present in the transcription reaction mix described herein. In some embodiments, supplementation with UTP or a functional analog thereof does not include supplementation with CTP or a functional analog thereof or ATP or a functional analog thereof.

[0278] In some embodiments, the concentration of UTP or its functional analog added during replenishment is the same as the starting concentration of UTP or its functional analog. In some embodiments, the concentration of UTP or its functional analog added during replenishment is lower than the starting concentration of UTP or its functional analog, for example, by at least 10% (e.g., at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%).

[0279] In some embodiments, the supplementation of UTP (or its functional analog) is carried out at a concentration and / or rate or in a manner such that the ratio of the concentration of UTP or its functional analog to the concentration of ATP or its functional analog, CTP or its functional analog, and optionally at least one or all of GTP or its functional analog (during the reaction) is maintained at substantially the same (e.g., within 10%) as the initial ratio (at the start of the reaction) of the concentration of UTP or its functional analog to the initial concentration of ATP or its functional analog, CTP or its functional analog, and optionally at least one or all of GTP or its functional analog.

[0280] In some embodiments, UTP or its functional analog is replenished until the end of the transcription reaction.

[0281] In some embodiments, UTP or its functional analog is present at an initial concentration of 0.1–2 mM, 0.1–1.5 mM, 0.1–1 mM, 0.5–2 mM, or 1–2 mM during the initial transcription reaction. In some embodiments, UTP or its functional analog is maintained at a concentration of 0.1–2 mM, 0.1–1.5 mM, 0.1–1 mM, 0.5–2 mM, or 1–2 mM throughout the in vitro transcription reaction.

[0282] Optional additional non-UTP restriction and / or supplementation: In some embodiments, at least one non-UTP (or a functional analog thereof) is provided at a restricted concentration (in addition to the restricted UTP or its functional analog) during the initial in vitro transcription reaction (e.g., at the initiation of the in vitro transcription reaction). For example, in some embodiments, at least one of ATP or a functional analog thereof, CTP or a functional analog thereof, or GTP or a functional analog thereof is provided at a restricted concentration (in addition to the restricted UTP or its functional analog) during the initial in vitro transcription reaction (e.g., at the initiation of the in vitro transcription reaction). In some embodiments, GTP or a functional analog thereof is provided at a restricted concentration (in addition to the restricted UTP or its functional analog) during the initial in vitro transcription (e.g., at the initiation of the in vitro transcription reaction).

[0283] In certain embodiments of the present invention, the method involves supplementing a transcription and capping reaction with GTP or a functional analog thereof. This is because, in certain reactions, such as when T7, SP6, or T3 polymerases are used to catalyze the reaction, these compete with the capping analog. However, it will be understood that the present invention is not limited to GTP or a functional analog thereof. Instead, the present invention can be carried out with respect to any reaction involving a capping analog or a nucleotide that competes with a non-extended mononucleotide or dinucleotide, which can be incorporated into the 5' end of the transcript. Thus, any embodiment involving GTP or a functional analog thereof as the competing nucleotide is particularly intended to be carried out with respect to a different nucleotide or nucleotide analog. This method does not depend on whether GTP and / or its functional analogs are used, as long as the analog is incorporated into the transcript extended by the polymerase at a rate similar to that of GTP. As used herein, the term “functional analog of GTP” refers to an extended nucleotide and therefore excludes any capping analogs as defined below.

[0284] In some embodiments, the transcription rate is limited by the starting concentration of GTP or its functional analog. Because GTP competes with 5' cap or 5' cap analogs in certain reactions, such as those using T7, SP6, or T3 polymerases, it is preferable to use a starting concentration of GTP or its functional analog to limit the rate of transcript transcription and / or capping reactions, and to supplement the reaction with GTP or its functional analog. However, it will be understood that the present invention is not limited to GTP or its GTP analogs. In certain embodiments, UTP or its functional analog is present in a starting amount that limits the reaction, and the method according to the present invention involves supplementing the reaction mix with UTP or its functional analog. In certain embodiments, UTP or its functional analog and GTP or its functional analog are present in starting amounts that limit the reaction, and the method according to the present invention involves supplementing the reaction mix with UTP or its functional analog and GTP or its functional analog.

[0285] In some embodiments, GTP or a functional analog is present in the in vitro transcription reaction at an initiation concentration that limits the transcription rate. In some embodiments, GTP or a functional analog is present in the in vitro transcription reaction at an initiation concentration lower than the initiation concentration of at least one or all of ATP or its functional analog, and / or CTP or its functional analog. In some embodiments, the initiation concentration of GTP or a functional analog is at least 30% lower (e.g., at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%) than the initiation concentration of at least one or all of ATP or its functional analog, and / or CTP or its functional analog. In some embodiments, the ratio of the starting concentration of GTP or its functional analog to the starting concentration of ATP or its functional analog, and / or CTP or its functional analog, is about 1:1.3 or less, for example, 1:1.4; 1:1.5; 1:2; 1:2.5; 1:3; 1:3.5; 1:4; 1:4.5; 1:5; 1:6; 1:7; 1:8; 1:9; 1:10; 1:11; 1:12; 1:13; 1:14; 1:15; 1:16; 1:17; 1:18; 1:19; 1:20 or less. In some embodiments, the ratio of the starting concentration of GTP or its functional analog to the starting concentrations of ATP or its functional analog and / or CTP or its functional analog is about 1:1.3 to about 1:20, or about 1:1.5 to about 1:15, or about 1:5 to about 1:15, or about 1:8 to about 1:12. In some such embodiments, the starting concentrations of ATP or its functional analog and / or CTP or its functional analog.

[0286] In some embodiments, the in vitro transcription reaction involves replenishing GTP or its functional analog at least once during the course of the reaction. In some embodiments, the in vitro transcription reaction involves replenishing GTP or its functional analog multiple times (e.g., at least twice, or more, e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen or more times) during the course of the transcription reaction. In some embodiments, the replenishment of GTP or its functional analog occurs when its concentration in the reaction mixture is near depletion. In some embodiments, supplementation with GTP or its functional analog is performed when its concentration in the reaction mixture is less than 100 μM, 90 μM, 80 μM, 70 μM, 60 μM, 50 μM, 40 μM, 30 μM, 20 μM, 10 μM, 5 μM, 3 μM, 2 μM, 1 μM, 500 nM, 250 nM, 200 nM, 100 nM, 50 nM, 25 nM, or less.

[0287] In some embodiments, GTP (or its functional analog) replenishment may be carried out continuously during the transcription reaction. For example, in some embodiments, GTP (or its functional analog) replenishment may be carried out continuously at a rate comparable to its consumption rate (e.g., 10% or less). In some embodiments, GTP (or its functional analog) replenishment may be carried out at a rate such that, after such replenishment, GTP or its functional analog is present in the reaction at a lower concentration than ATP or its functional analog, and / or CTP or its functional analog.

[0288] In some embodiments, GTP (or its functional analog) supplementation may be carried out periodically during the transcription reaction. In some embodiments, GTP (or its functional analog) supplementation may be carried out periodically such that, after each addition, GTP or its functional analog is present in the reaction at a concentration lower than, in some embodiments, all of, ATP or its functional analog and / or CTP or its functional analog. In some embodiments, such periodic supplementation may be carried out as one or more bolus or batch additions, for example, including one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen or more bolus or batch additions. In some embodiments, such periodic supplementation may be carried out by a fed-batch culture process. In some embodiments, supplementation may include supplementing with a composition comprising GTP or a functional analog thereof, and further comprising a buffer, polymerase, CTP or a functional analog thereof, UTP or a functional analog thereof, ATP or a functional analog thereof, or other components that may be present in the transcription reaction mix described herein. In some embodiments, supplementation with GTP or a functional analog thereof does not include supplementation with ATP or a functional analog thereof or CTP or a functional analog thereof.

[0289] In some embodiments, the concentration of GTP or its functional analog added during supplementation is the same as the starting concentration of GTP or its functional analog. In some embodiments, the concentration of GTP or its functional analog added during supplementation is lower than the starting concentration of GTP or its functional analog, for example, by at least 10% (e.g., at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%).

[0290] In some embodiments, GTP (or its functional analog) supplementation is carried out at a concentration and / or rate or in a manner such that the ratio of the concentration of GTP or its functional analog to the concentration of ATP or its functional analog, CTP or its functional analog (during the reaction) is maintained at substantially the same (e.g., within 10%) as the initial ratio of the concentration of GTP or its functional analog to the initial concentration of ATP or its functional analog, CTP or its functional analog (at the start of the reaction).

[0291] In some embodiments, GTP or a functional analog thereof is replenished until the end of the transcription reaction.

[0292] In some embodiments, GTP or its functional analog is present at an initial concentration of 0.1–2 mM, 0.1–1.5 mM, 0.1–1 mM, 0.5–2 mM, or 1–2 mM during the initial transcription reaction. In some embodiments, GTP or its functional analog is maintained at a concentration of 0.1–2 mM, 0.1–1.5 mM, 0.1–1 mM, 0.5–2 mM, or 1–2 mM throughout the in vitro transcription reaction.

[0293] In some embodiments, non-UTP supplementation does not include supplementation with CTP or its functional analog, or ATP or its functional analog.

[0294] In some embodiments in which non-UTP replenishment is carried out, such non-UTP replenishment can be carried out simultaneously with UTP replenishment during the course of the reaction. In some embodiments, non-UTP or its functional analog and UTP or its functional analog can be added to the reaction mixture as a single composition. In some embodiments, non-UTP or its functional analog and UTP or its functional analog can be added to the reaction mixture as separate compositions, for example, at the same or different concentrations, and / or introduced into the reaction mixture at the same or different flow rates. In some embodiments, such non-UTP replenishment and UTP replenishment can be carried out in different ways, for example, one carried out continuously (for example, as described herein) and the other carried out periodically (for example, as described herein).

[0295] In some embodiments, supplementing with nucleotides such as UTP and / or GTP, or their functional analogs, includes supplementing with two or more nucleotides, for example, two or more functional analogs of UTP and / or GTP. For example, in some embodiments, supplementing the reaction mix during a transcription reaction includes supplementing with UTP and pseudo-UTP. In some embodiments, supplementing the reaction mix during a transcription reaction includes supplementing with UTP, pseudo-UTP and GTP. In some embodiments, supplementing the reaction mix during a transcription reaction includes supplementing with pseudo-UTP and / or 1-methyl pseudo-UTP and GTP. In some embodiments, supplementing with two or more nucleotides results in supplementing with an amount of UTP or its functional analog and / or GTP or its functional analog that results in an excess of UTP or its functional analog and / or GTP or its functional analog in the reaction mix. In some embodiments, supplementing with two or more functional analogs of UTP and / or GTP does not result in supplementing with an amount of UTP or its functional analog and / or GTP or its functional analog that would result in an excess of UTP or its functional analog and / or GTP or its functional analog in the reaction mix.

[0296] In some embodiments, methods for increasing the yield of capped RNA transcripts and / or decreasing dsRNA include incubating components for transcription and capping reactions under conditions that promote transcription polymerization, wherein the concentration of the 5' capping analog is maintained during the reaction at a ratio of approximately 1:1 to approximately 50:1 relative to the concentration of the competing nucleotide component by multiple doses of the competing nucleotide component. In certain embodiments, the competing nucleotide is GTP or a functional analog thereof. In reactions involving T7, T3, or SP6 RNA polymerases, the competing nucleotide is typically GTP or a functional analog thereof. Any embodiment involving the use of GTP or a functional analog thereof is particularly intended to be substituted at the +1 position with another nucleotide triphosphate or a functional analog thereof when using an RNA polymerase that uses a particular nucleotide. The present invention also relates to a method for increasing the yield of capped transcripts and / or reducing dsRNA in in vitro transcription and capping reactions, the method comprising incubating the reaction components under conditions that enable transcription, wherein the concentration of GTP or its functional analog during the reaction is maintained at a concentration of about 0.2 mM to about 2.0 mM for at least 30 minutes during the reaction, and the concentration of other nucleotides is at least about 0.2 mM.

[0297] Furthermore, the present invention relates to a method for producing RNA having a non-elongated nucleotide at its 5' end, comprising introducing a nucleotide that competes with the non-elongated nucleotide by a fed-batch culture process into a transcription reaction involving RNA polymerase and the non-elongated nucleotide. In certain embodiments, the non-elongated nucleotide is not a functional cap analog. Any embodiment discussed with respect to GTP or a GTP analog can be carried out with respect to another nucleotide, insofar as that nucleotide competes with the non-elongated nucleotide at its 5' end, and vice versa, and so on. Furthermore, it will be understood that any embodiment discussed with respect to a 5' cap or a 5' cap analog can be carried out with respect to a non-elongated nucleotide that can only be added to the 5' end of a transcript, and vice versa.

[0298] In certain embodiments, the reaction may be supplemented with a 5' cap or a 5' cap analog during the transfer and / or capping reaction. In certain embodiments, the reaction is not supplemented with a 5' cap or a 5' cap analog during the transfer and / or capping reaction.

[0299] In some embodiments, one of the components supplemented to the reaction by, for example, a fed-batch culture process is a nucleotide. In some cases, multiple nucleotides are introduced by the fed-batch culture process. For example, both UTP and GTP nucleotides or their functional analogs may be supplemented by the fed-batch culture process, or UTP and its functional analogs may be supplemented by the fed-batch culture process, and / or GTP and its functional analogs may be supplemented by the fed-batch culture process. In further embodiments, all nucleotides are supplemented by the fed-batch culture process. One or more of the nucleotides in this reaction may be modified nucleotides, such as the functional analogs of nucleoside triphosphates described herein. Uncapped nucleotides, although modified, can still be functional because they can be incorporated at the 3' end of the polymerized transcript. That is, these uncapped nucleotides have a 5' triphosphate and are therefore elongable.

[0300] In some embodiments, a programmable pump may be used for replenishment. In some embodiments, a programmable syringe pump may be used to automatically perform, for example, the stepwise addition of one or more reaction components. Alternatively or additionally, in some embodiments, a monitor (e.g., a sensor) may be used to detect the level(s) of one or more components; in some such embodiments, the monitor may automatically communicate with the pump so that, for example, if a decrease in the amount of such component(s) is detected, additional supply may be released.

[0301] In some embodiments, after RNA transcription, the DNA template can be removed or separated from the in vitro transcribed RNA composition, for example, by methods known in the art, such as DNA hydrolysis.

[0302] In some embodiments, RNase inhibitors may be added during DNA removal or digestion to protect RNA from potential degradation. In some embodiments, chelating agents may be added to the DNase-treated transcription mixture to form complexes with divalent ions that may be added during the in vitro transcription reaction. Exemplary chelating agents may be ethylenediaminetetraacetic acid (EDTA) or contain ethylenediaminetetraacetic acid (EDTA). In some embodiments, the temperature may change by at least 1°C (e.g., at least 2°C, 3°C, 4°C, 5°C, 6°C, 7°C, 8°C, 9°C, 10°C or higher) upon addition of the chelating agent.

[0303] bioreactor In some embodiments, the transcription reaction is carried out in (i.e., using) a bioreactor as described herein.

[0304] As described herein, in some embodiments, the transfer reaction is carried out at a pH of about 6, 6.5, 7, 7.5, 8, or 9. In some embodiments, a pH value suitable for the transfer reaction may be about 7.5 to 8.5. In some embodiments, the pH values ​​are 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, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0. In some embodiments, the pH value is about 6 to 8.5. In some embodiments, the pH values ​​are 6 to 8.5, 6.5 to 8.0, 7.0 to 7.5; in some embodiments, the pH value is 7.5. In some embodiments, the pH value of the reaction mix is ​​kept essentially constant throughout the transfer reaction, for example, by using a suitable buffer. Suitable buffers for adjusting the pH value include, but are not limited to, NaOH buffer, KOH buffer, or HCl buffer, which are known in the art and described herein. In some embodiments, the buffer has a pH value as described herein, for example, 6–8.5, 6.5–8.0, 7.0–7.5, or 7.5. In some embodiments, the buffer is selected from the group consisting of 80 mM HEPES / KOH, pH 7.5 and 40 mM Tris / HCl, pH 7.5. In some embodiments, maintaining a constant pH value and / or monitoring the pH value throughout the transfer reaction is done in a bioreactor, i.e., using a bioreactor.

[0305] In some embodiments, the progress of the transfer reaction is monitored in real time. In some embodiments, monitoring of the progress of the transfer reaction is achieved using sensors, such as a bioreactor, including a bioreactor equipped with a UV flow cell for UV260 / 280nm measurement.

[0306] As used herein, the terms “bioreactor” or “transcription reactor” refer to a container, such as a chamber, test tube, or column, in which a transcription reaction is carried out under specific conditions as described herein. Transcription bioreactors are known in the art (see WO1995 / 08626 and EP3155129). Bioreactors are typically configured such that reaction components are delivered to a reactor core by a supply line, and RNA products are removed by passing through an ultrafiltration membrane to an outlet stream (EP3155129 and van de Merbel, (1999), J. Chromatogr. A 856(1-2):55-82). A bioreactor useful in the method of the present invention may comprise a reaction module for carrying out the transcription reaction, a capture module for transiently capturing the transcribed RNA molecules, and a control module for controlling the supply of components of the reaction mix to the reaction module, wherein the reaction module may comprise a filtration membrane for separating nucleotides from the reaction mix, and the control of the supply of components of the reaction mix by the control module may be based on the measured concentration of the separated nucleotides. The bioreactor may be thermoregulated to precisely maintain a specific temperature, such as the temperature of the transfer reaction described herein, for example, typically 4°C to 40°C. The bioreactor may be equipped with inlet feet and outlet ports. The bioreactor may be capable of stirring the reaction mix during the transfer reaction, for example, at a variable stirring speed. The stirring may be continuous or discontinuous, such as at intervals.

[0307] The bioreactor for use in accordance with the present invention may be of any size, as long as it is useful for the transfer. For example, in some embodiments, the bioreactor may be at least 0.2 liters or more, e.g., 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50 liters or more, or any volume in between. The internal conditions of the bioreactor (including, but not limited to, pH and temperature), typically controlled during the transfer reaction described herein. The bioreactor may be constructed of any material suitable for in vitro transfer under the conditions described herein, such as glass, plastic, or metal.

[0308] In some embodiments, the bioreactor may be equipped with a pump for replenishing the reaction mix. In some embodiments, a programmable pump may be used for replenishment. In some embodiments, a programmable syringe pump may be used to automatically perform, for example, the stepwise addition of one or more reaction components. Alternatively or additionally, in some embodiments, a monitor (e.g., a sensor) may be used to detect the level(s) of one or more components; in some such embodiments, the monitor may automatically communicate with the pump so that, for example, if a decrease in the amount of such component(s) is detected, additional feed may be released.

[0309] use In many embodiments, the RNA products produced by the methods described herein have a lower amount of dsRNA contamination compared to RNA products produced by processes in which UTP is limited during the in vitro transcription process. In some embodiments, such RNA products have low levels of dsRNA contamination that do not require a purification process to remove dsRNA contamination. In some embodiments, such RNA (e.g., mRNA) is for therapeutic use. In some embodiments, such RNA (e.g., mRNA) is a pharmaceutical-grade product.

[0310] In some embodiments, one or more RNAs (e.g., single-stranded RNAs) can be formulated with lipid nanoparticles or complexed with liposomes to produce pharmaceutical-grade compositions or preparations containing RNA-LNPs or RNA-lipoplexes. In some embodiments, the lipid nanoparticles are lipid nanoparticles comprising one or more cationic or ionizable lipids, such as those known in the art. In some embodiments, the lipid nanoparticles may comprise at least one cationic or ionizable lipid, at least one polymer-conjugated lipid, and at least one helper lipid (e.g., at least one neutral lipid such as a phospholipid and / or sterol).

[0311] In some embodiments, such RNA products described herein can be administered to subjects that require them (e.g., subjects that would benefit from the expression of the encoded polypeptide, e.g., as a substitution or to stimulate or enhance an immune response), typically via incorporation into LNPs in some embodiments.

[0312] Treatment of conditions and vaccination: In many embodiments, the RNA products provided (e.g., preparations produced via the use of the processes described herein) are used for the treatment of conditions and / or vaccination.

[0313] The terms “immunization” or “vaccination” generally refer to the process of treating a subject for therapeutic or preventive reasons. Treatment, in particular preventive treatment, is preferably a treatment aimed at inducing or enhancing an immune response in a subject to, for example, one or more antigens, or includes such treatment. According to the present invention, where it is desirable to induce or enhance an immune response by using the RNA described herein, the immune response may be induced or enhanced by the RNA. In some embodiments, the present invention preferably provides a preventive treatment which is a vaccination of a subject or includes such treatment. One embodiment of the present invention in which the RNA of the present invention encodes a pharmaceutically active peptide or protein which is an immunologically active compound or antigen as the protein of interest is particularly useful for vaccination. When the purpose is to treat a condition, the RNA of the present invention preferably encodes a peptide or protein or nucleic acid (e.g., therapeutic nucleic acids such as siRNA, shRNA, miRNA, etc.) which can treat or is sufficient to treat the condition.

[0314] The terms “subject” and “individual” are used interchangeably and relate to mammals. For example, mammals in the context of this invention include humans, non-human primates, livestock such as dogs, cats, sheep, cattle, goats, pigs, and horses, laboratory animals such as mice, rats, rabbits, and guinea pigs, and domesticated animals such as zoo animals. The term “animal” as used herein also includes humans. The term “subject” may also include patients, i.e., animals with a disease, preferably humans.

[0315] The agents (e.g., RNA products) and compositions described herein are preferably administered in an effective dose. “Effective dose” means an amount that, alone or in combination with additional doses, achieves the desired response or effect. In the case of treating a particular disease or condition, the desired response preferably relates to inhibiting the course of the disease. This includes slowing the progression of the disease, in particular interrupting or reversing its progression. The desired response in the treatment of a disease or condition may also be to delay or prevent the onset of the disease or condition.

[0316] The effective dose of the agents or compositions described herein will depend on the condition being treated, the severity of the disease, the individual parameters of the patient (age, physiological state, body size and weight, duration of treatment, type of concomitant therapy (if any), specific route of administration), and similar factors. Therefore, the dose administered of the agents described herein may depend on some of these parameters. If the patient's response is insufficient with the initial dose, a higher dose (or an effective higher dose achieved by a different, more localized route of administration) may be used.

[0317] kit In particular, the present invention provides a kit comprising one or more components useful for producing RNA according to the present invention, and / or a kit comprising RNA produced by the method of the present invention. In some embodiments, the kit may include, for example, pharmaceutically acceptable excipients, diluents, carriers, etc. In some embodiments, the kit comprises a preparation of the RNA product produced as described herein, and one or more pharmaceutically acceptable excipients, diluents, carriers, etc. In some embodiments, the kit provided comprises one or more devices for administration (e.g., syringes, vials, or IV bags), or components thereof. In some embodiments, the provided kit includes one or more devices for dilution.

[0318] In some embodiments, various components of a kit exist as separate entities. For example, a kit containing two or more nucleic acid molecules (e.g., two or more RNA products as described herein) may contain them in separate containers. Alternatively or additionally, a kit may contain one or more nucleic acid molecules in one or more containers, and also contain one or more other components (e.g., buffers, carriers, diluents, excipients, etc.) in one or more containers separate from any containers containing nucleic acids.

[0319] In some embodiments, the separate containers may be open or sealed. In some embodiments, some or all of the containers are sealed.

[0320] In some embodiments, any container containing RNA, or components combined with RNA (e.g., buffers, carriers, diluents, excipients, etc.), is either RNAse-free or essentially RNAse-free.

[0321] In some embodiments, the kit of the present invention comprises RNA for inoculating cells and / or for administration to human or animal subjects. In some embodiments, the RNA preparation is frozen. In some embodiments, the RNA preparation is dried. In some embodiments, the RNA preparation comprises lipids (e.g., LNPs).

[0322] The kit according to the present invention optionally includes a label or other form of information element, such as an electronic data carrier. The label or information element preferably includes instructions, such as printed written instructions, or optionally instructions in a printable electronic format. The instructions may refer to at least one preferred way of using the kit.

[0323] RNA preparations and compositions As described herein, this disclosure provides various preparations of RNA products and / or other compositions comprising RNA (e.g., RNA produced as described herein).

[0324] Accordingly, this disclosure provides comparative compositions that contain less double-stranded RNA compared to compositions obtained by the method of the present invention, for example, compositions that cannot be obtained by the method of the present invention.

[0325] In some embodiments, the provided composition is purified; in some embodiments, the provided composition does not need to be purified. If further purification is not performed, the RNA-containing composition may further contain other chemical substances and molecules, such as components of a transcription mix used for RNA transcription, DNA template molecules, enzymes, salts, NTPs, and the like.

[0326] In some embodiments, the RNA-containing composition according to the present invention is pure enough to be used in subsequent processes without further purification. In some embodiments, the RNA-containing composition according to the present invention is pure enough to be used for administration to cells and / or subjects requiring it. In some embodiments, the RNA-containing composition according to the present invention needs to be purified after the transcription reaction before being used in further processes. In some embodiments, the RNA-containing composition according to the present invention needs to be purified after the transcription reaction before being used for administration to cells or subjects requiring it.

[0327] In some embodiments, the amount of dsRNA produced by the method of the present invention is reduced compared to the amount of dsRNA produced by a method using equimolar amounts of ATP, CTP, GTP, UTP, or functional analogs thereof. In some embodiments, the amount of dsRNA produced by the method of the present invention is reduced by at least 10%, for example, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, preferably at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 80%, at least 90%, at least 95%, or 100% compared to the amount of dsRNA produced by a method using equimolar amounts of ATP, CTP, GTP, UTP, or functional analogs thereof. Preferably, the amount of dsRNA produced by the method of the present invention is reduced by at least 40%, at least 41%, at least 42%, at least 43%, at least 44%, at least 45%, at least 46%, at least 57%, at least 48%, at least 49%, at least 60%, at least 51%, at least 52%, at least 53%, at least 54%, at least 55%, at least 56%, preferably at least 57%, at least 58%, at least 59%, at least 60%, or more, compared to the amount of dsRNA produced by a method using equimolar amounts of ATP, CTP, GTP, UTP, or their functional analogs.

[0328] In some embodiments, the yield of RNA produced by the method of the present invention is increased compared to the yield of RNA produced by a method using equimolar amounts of ATP, CTP, GTP, UTP, or functional analogs thereof. In some embodiments, the yield of RNA produced by the method of the present invention is increased by at least 10%, for example, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, preferably at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 80%, at least 90%, at least 95%, or 100% compared to the yield of RNA produced by a method using equimolar amounts of ATP, CTP, GTP, UTP, or functional analogs thereof. Preferably, the yield of RNA produced by the method of the present invention is increased by at least 40%, at least 41%, at least 42%, at least 43%, at least 44%, at least 45%, at least 46%, at least 57%, at least 48%, at least 49%, at least 60%, at least 51%, at least 52%, at least 53%, at least 54%, at least 55%, at least 56%, preferably at least 57%, at least 58%, at least 59%, at least 60%, or more, compared to the yield of RNA produced by a method using equimolar amounts of ATP, CTP, GTP, UTP, or functional analogs thereof.

[0329] In some embodiments, one or more RNAs (e.g., single-stranded RNAs) can be formulated with lipid nanoparticles or complexed with liposomes to produce pharmaceutical-grade compositions or preparations containing RNA-LNPs or RNA-lipoplexes. In some embodiments, the lipid nanoparticles are lipid nanoparticles comprising one or more cationic or ionizable lipids, such as those known in the art. In some embodiments, the lipid nanoparticles may comprise at least one cationic or ionizable lipid, at least one polymer-conjugated lipid, and at least one helper lipid (e.g., at least one neutral lipid such as a phospholipid and / or sterol).

[0330] Pharmaceutical composition In some embodiments, the RNA of the present invention, for example, the RNA produced by the method of the present invention, may exist in the form of a pharmaceutical composition. A pharmaceutical composition according to the present invention may comprise at least one RNA molecule according to the present invention. A pharmaceutical composition according to the present invention may further comprise one or more pharmaceutically acceptable diluents, excipients, carriers and / or vehicles. The selection of pharmaceutically acceptable carriers, vehicles, excipients or diluents is not particularly limited. Any suitable pharmaceutically acceptable carrier, vehicle, excipient or diluent known in the art may be used.

[0331] The term “carrier” refers to an organic or inorganic component of natural or non-natural (synthetic) nature to which an active ingredient is combined in order to facilitate, enhance, or enable its application. According to the present invention, the term “carrier” also includes one or more suitable solid or liquid fillers, diluents, or encapsulating materials that are suitable for administration to a patient.

[0332] Possible carriers for parenteral administration include, for example, sterile water, glucose solution, Ringer's solution, Ringer's lactate solution, sterile sodium chloride solution, polyalkylene glycol, hydrogenated naphthalene, and especially biocompatible lactide polymers, lactide / glycolide copolymers, or polyoxyethylene / polyoxypropylene copolymers.

[0333] The pharmaceutical compositions described herein may be administered via any conventional parenteral route, such as by injection or infusion. Administration is preferably parenteral, for example, intravenous, intra-arterial, subcutaneous, lymph node, intradermal, or intramuscular.

[0334] In some embodiments of the present invention, the pharmaceutical composition may further contain a solvent, such as an aqueous solvent or any solvent that allows for the preservation of RNA integrity. In some embodiments, the pharmaceutical composition is an aqueous solution containing RNA. The aqueous solution may optionally contain a solute, such as a salt.

[0335] In some embodiments of the present invention, the pharmaceutical composition is in the form of a lyophilized composition. The lyophilized composition can be obtained by lyophilizing each aqueous composition.

[0336] Compositions suitable for parenteral administration typically contain a sterile aqueous or non-aqueous preparation of the active compound and are preferably isotonic with respect to the recipient's blood. Examples of suitable carriers and solvents include Ringer's solution and isotonic sodium chloride solution. Furthermore, sterile fixative oil is usually used as the solution or suspension medium.

[0337] In some embodiments, the pharmaceutical composition comprises at least one cationic entity. Generally, cationic lipids, cationic polymers, and other positively charged substances can form complexes with negatively charged nucleic acids. The RNA according to the present invention can be stabilized by complex formation with a cationic compound, preferably a polycationic compound such as a cationic or polycationic peptide or protein. In some embodiments, the pharmaceutical composition according to the present invention comprises at least one cationic molecule selected from the group consisting of protamine, polyethyleneimine, poly-L-lysine, poly-L-arginine, histone, or cationic lipid.

[0338] In some embodiments, cationic lipids useful according to the present invention are cationic amphiphilic molecules, for example, molecules comprising at least one hydrophilic moiety and a lipophilic moiety. In some embodiments, cationic lipids may be monocationic or polycationic. Cationic lipids typically have a lipophilic moiety such as a sterol, acyl, or diacyl chain and have a net positive charge as a whole. The head group of the lipid typically retains a positive charge. Cationic lipids preferably have a positive charge of 1 to 10 valencies, more preferably 1 to 3 valencies, and even more preferably 1 valency. Examples of cationic lipids, but not limited to these, include 1,2-di-O-octadecenyl-3-trimethylammoniumpropane (DOTMA), dimethyldioctadecylammonium (DDAB); 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP); 1,2-dioleoyl-3-dimethylammonium-propane (DODAP); 1,2-diacyloxy-3-dimethylammoniumpropane; 1,2-dialkyloxy-3-dimethylammoniumpropane; dioctadecyldimethylammonium chloride (DODAC), 1,2-dimyristoyloxypropyl-1,3-dimethylhydroxyethylammonium (DMRIE), and 2,3-dioleoyloxy-N-[2(sperminecarboxamide)ethyl]-N,N-dimethyl-1-propanumtrifluoroacetic acid (DOSPA). Cationic lipids also include lipids having a tertiary amine group, such as 1,2-dilinoleyloxy-N,N-dimethyl-3-aminopropane (DLinDMA). Cationic lipids are suitable for formulating RNA in the lipid formulations described herein, such as liposomes, emulsions, and lipoplexes. Typically, the positive charge is contributed by at least one cationic lipid, and the negative charge is contributed by the RNA. In some embodiments, the pharmaceutical composition includes at least one helper lipid in addition to the cationic lipids. The helper lipid may be a neutral lipid or anionic lipid. The helper lipid may be a natural lipid such as a phospholipid, or an analog of a natural lipid, or a completely synthetic lipid with no similarity to a natural lipid, or a lipid-like molecule.When a pharmaceutical composition contains both cationic lipids and helper lipids, the molar ratio of cationic lipids to neutral lipids can be appropriately determined considering factors such as the stability of the formulation.

[0339] In some embodiments, the pharmaceutical composition according to the present invention comprises protamine. In some embodiments, protamine may be useful as a cationic carrier. The term “protamine” refers to any of a variety of relatively low molecular weight strongly basic proteins that are arginine-rich and have been found to bind to DNA in place of somatic histones in the sperm cells of animals such as fish. In particular, the term “protamine” refers to a strongly basic, water-soluble, heat-independent protein found in fish sperm that contains multiple arginine monomers. According to the present invention, the term “protamine” as used herein means any protamine amino acid sequence obtained or derived from natural or biological sources, including its fragments and amino acid sequences or the polymeric forms of those fragments. Furthermore, the term includes (synthetic) polypeptides that are artificial, specifically designed for a particular purpose, and cannot be isolated from natural or biological sources.

[0340] In some embodiments, the compositions provided by the present invention may comprise one or more adjuvants. Adjuvants may be added to vaccines to stimulate an immune system response; typically, adjuvants themselves do not provide immunity. Examples of adjuvants, but not limited to, include: inorganic compounds (e.g., alum, aluminum hydroxide, aluminum phosphate, calcium hydroxide phosphate); mineral oils (e.g., paraffin oil); cytokines (e.g., IL-1, IL-2, IL-12); immunostimulatory polynucleotides (such as RNA or DNA, e.g., CpG-containing oligonucleotides); saponins (e.g., plant saponins derived from quillaya, soybean, and senega); oil emulsions or liposomes; polyoxyethylene ethers and polyoxyethylene ester formulations; polyphosphazenes (PCPP); muramyl peptides; imidazoquinolone compounds; thiosemicarbazone compounds; Flt3 ligands (WO2010 / 066418A1); or other adjuvants known to those skilled in the art. A preferred adjuvant for RNA administration according to the present invention is the Flt3 ligand (WO2010 / 066418A1). When the Flt3 ligand and the RNA encoding the antigen are administered together, a significant increase in antigen-specific CD8+ T cells may be observed.

[0341] In some embodiments, the pharmaceutical compositions provided by the present invention may be buffered (for example, with acetate buffer, citrate buffer, succinate buffer, Tris buffer, or phosphate buffer).

[0342] host cell In some embodiments, a pharmaceutical (or other) composition is appropriately formulated for introduction into cells; cells to which one or more RNA molecules can be inoculated, i.e., administered, may be called “host cells.” As used herein, the term “host cells” refers to any cells that can be transformed or transfected with exogenous RNA molecules. In many embodiments, the term “cell” refers to an intact cell, i.e., a cell with an intact membrane that does not release normal intracellular components such as enzymes, organelles, or genetic material. In many embodiments, an intact cell is a viable cell, i.e., a living cell capable of performing its normal metabolic functions. According to the present invention, the term “host cells” includes prokaryotic cells (e.g., E. coli) or eukaryotic cells (e.g., human and animal cells, plant cells, yeast cells, and insect cells). Examples of cells include prokaryotes and eukaryotes (unicellular or multicellular), bacterial cells (e.g., strains such as E. coli, Bacillus spp., and Streptomyces spp.), mycobacterial cells, fungal cells, yeast cells (e.g., S. cerevisiae, S. pombe, P. pastoris, P. methanolica, etc.), plant cells, insect cells (e.g., SF-9, SF-21, baculovirus-infected insect cells, Trichoplusia ni, etc.), non-human animal cells, human cells, or cell fusions such as hybridomas or quadromas. In some embodiments, the host cells are human, monkey, ape, hamster, rat, or mouse cells. In some embodiments, the host cells are eukaryotes.For example, eukaryotic host cells may include: CHO (e.g., CHO Kl, DXB-1 1 CHO, Veggie-CHO), COS (e.g., COS-7), retinal cells, Vero, CV1, kidney (e.g., HEK293, 293 EBNA, MSR 293, MDCK, HaK, BHK), HeLa, HepG2, WI38, MRC 5, Colo205, HB 8065, HL-60, (e.g., BHK21), Jurkat, Daudi, A431 (epidermal), CV-1, U937, 3T3, L cells, C127 cells, SP2 / 0, NS-0, MMT 060562, Sertoli cells, BRL 3 A cells, HT1080 cells, myeloma cells, tumor cells, or cell lines derived from the aforementioned cells. Of particular interest are mammalian cells, such as those from humans, mice, hamsters, pigs, livestock, e.g., horses, cattle, sheep, and goats, as well as primates. The cells may be derived from numerous tissue types and include primary cells and cell lines. Specific examples include keratinocytes, peripheral blood leukocytes, bone marrow stem cells, and embryonic stem cells. In some embodiments, the host cells are antigen-presenting cells, particularly dendritic cells, monocytes, or macrophages. In some embodiments, the compositions provided herein (e.g., pharmaceutical compositions) deliver nucleic acids (e.g., RNA) to host cells, thereby enabling the nucleic acids to exist in single or multiple copies within the host cells, and in some embodiments, to be expressed within the host cells.

[0343] In some embodiments, the host cell may be a prokaryotic cell; in some embodiments, the host cell may be a eukaryotic cell.

[0344] In some embodiments, prokaryotic cells are used here for, for example, DNA propagation according to the present invention, and eukaryotic cells are preferred here for, for example, the expression of open reading frames of replicons.

[0345] Specific Embodiments

[0346] Specific embodiments provided by this disclosure are described below: 1. A method for producing RNA, comprising transcribing RNA from a DNA template using a reaction mix comprising adenosine triphosphate (ATP), guanosine triphosphate (GTP), cytidine triphosphate (CTP), and uridine triphosphate (UTP), or functional analogs thereof, wherein the starting concentration of UTP or its functional analog is lower than the starting concentration of CTP or its functional analog and / or ATP or its functional analog, and the method comprises supplementing the reaction mix during the transcription reaction with a composition comprising UTP or its functional analog and substantially free of CTP or its functional analog or ATP or its functional analog.

[0347] 2. A method for producing RNA, comprising transcribing RNA from a DNA template using a reaction mix comprising adenosine triphosphate (ATP), guanosine triphosphate (GTP), cytidine triphosphate (CTP), and uridine triphosphate (UTP), or functional analogs thereof, wherein the starting concentration of CTP or its functional analog is equal to the starting concentration of ATP or its functional analog, and the starting concentration of UTP or its functional analog is lower than the starting concentration of CTP or its functional analog or ATP or its functional analog, and the method comprises supplementing the reaction mix with UTP or its functional analog during the course of the transcription reaction.

[0348] 3. A method for producing a composition containing RNA having a reduced double-stranded (ds)RNA content, wherein the method comprises transcribing RNA from a DNA template using a reaction mix comprising adenosine triphosphate (ATP), guanosine triphosphate (GTP), cytidine triphosphate (CTP), and uridine triphosphate (UTP), or functional analogs thereof, wherein the starting concentration of UTP or its functional analog is lower than the starting concentration of CTP or its functional analog and / or ATP or its functional analog, and the method comprises supplementing the reaction mix with a composition containing UTP or its functional analog, but substantially free of CTP or its functional analog or ATP or its functional analog, during the transcription reaction.

[0349] 4. A method for producing a composition containing RNA having a reduced double-stranded (ds)RNA content, wherein the method comprises transcribing RNA from a DNA template using a reaction mix comprising adenosine triphosphate (ATP), guanosine triphosphate (GTP), cytidine triphosphate (CTP), and uridine triphosphate (UTP), or functional analogs thereof, wherein the starting concentration of CTP or its functional analog is equal to the starting concentration of ATP or its functional analog, and the starting concentration of UTP or its functional analog is lower than the starting concentration of CTP or its functional analog or ATP or its functional analog, and the method comprises supplementing the reaction mix with UTP or its functional analog during the transcription reaction.

[0350] 5. The method according to Embodiment 3 or 4, wherein the double-stranded (ds)RNA content of the RNA-containing composition is reduced compared to the dsRNA content of a composition containing RNA transcribed from the same DNA template using equimolar amounts of adenosine triphosphate (ATP), guanosine triphosphate (GTP), cytidine triphosphate (CTP), and uridine triphosphate (UTP), or functional analogs thereof.

[0351] 6. The method according to any one of Embodiments 3 to 5, wherein the immunogenicity of the RNA-containing composition is reduced compared to the immunogenicity of a composition containing RNA transcribed from the same DNA template using equimolar amounts of adenosine triphosphate (ATP), guanosine triphosphate (GTP), cytidine triphosphate (CTP), and uridine triphosphate (UTP), or functional analogs thereof.

[0352] 7. The method according to any one of Embodiments 1 to 6, wherein uridine triphosphate (UTP) or a functional analog thereof is present at an initiation concentration that limits the transcription rate.

[0353] 8. The method according to any one of Embodiments 1 to 7, wherein the ratio of the initial concentration of uridine triphosphate (UTP) or its functional analog to the initial concentration of cytidine triphosphate (CTP) or its functional analog, or adenosine triphosphate (ATP) or its functional analog, is approximately 1:1.5 to 1:15.

[0354] 9. The method according to any one of Embodiments 1 to 8, wherein when the concentration of UTP or its functional analog approaches depletion, uridine triphosphate (UTP) or its functional analog is replenished in the reaction mix.

[0355] 10. The method according to any one of Embodiments 1 to 9, wherein during the transcription reaction, the reaction mix is ​​supplemented at least once with uridine triphosphate (UTP) or a functional analog thereof.

[0356] 11. The method according to any one of Embodiments 1 to 10, wherein uridine triphosphate (UTP) or a functional analog thereof is continuously replenished in the reaction mix during the transcription reaction.

[0357] 12. The method according to any one of Embodiments 1 to 10, wherein uridine triphosphate (UTP) or a functional analog thereof is periodically replenished in the reaction mix during the transcription reaction.

[0358] 13. The method according to any one of Embodiments 1 to 12, wherein the initial ratio of the concentration of UTP or its functional analog to the concentration of cytidine triphosphate (CTP) or its functional analog, or adenosine triphosphate (ATP) or its functional analog, is maintained or restored by supplementing the reaction mix with uridine triphosphate (UTP) or its functional analog.

[0359] 14. The method according to any one of Embodiments 1 to 13, wherein the reaction mix is ​​replenished with uridine triphosphate (UTP) or a functional analog thereof until the transcription reaction is completed.

[0360] 15. The method according to any one of Embodiments 1 to 14, wherein the initial concentration of guanosine triphosphate (GTP) or its functional analog is lower than the initial concentration of cytidine triphosphate (CTP) or its functional analog, or adenosine triphosphate (ATP) or its functional analog.

[0361] 16. The method according to Embodiment 15, wherein guanosine triphosphate (GTP) or a functional analog thereof is present at an initiation concentration that limits the transfer rate.

[0362] 17. The method according to Embodiment 15 or 16, wherein the ratio of the initial concentration of guanosine triphosphate (GTP) or its functional analog to the initial concentration of cytidine triphosphate (CTP) or its functional analog, or adenosine triphosphate (ATP) or its functional analog, is approximately 1:1.5 to 1:15.

[0363] 18. The method according to any one of embodiments 15 to 17, wherein guanosine triphosphate (GTP) or a functional analog thereof is added to the reaction mix during the transcription reaction.

[0364] 19. The method according to Embodiment 18, wherein guanosine triphosphate (GTP) or its functional analog is replenished in the reaction mix when the concentration of GTP or its functional analog approaches depletion.

[0365] 20. The method according to any one of embodiments 15 to 19, wherein the reaction mix is ​​supplemented at least once with guanosine triphosphate (GTP) or a functional analog thereof during the transcription reaction.

[0366] 21. The method according to any one of Embodiments 15 to 20, wherein guanosine triphosphate (GTP) or a functional analog thereof is continuously replenished in the reaction mix during the transcription reaction.

[0367] 22. The method according to any one of Embodiments 15 to 20, wherein guanosine triphosphate (GTP) or a functional analog thereof is periodically replenished in the reaction mix during the transcription reaction.

[0368] 23. The method according to any one of Embodiments 15 to 22, wherein the initial ratio of the concentration of guanosine triphosphate (GTP) or its functional analog to the concentration of cytidine triphosphate (CTP) or its functional analog, or adenosine triphosphate (ATP) or its functional analog, is maintained or restored by supplementing the reaction mix with guanosine triphosphate (GTP) or its functional analog.

[0369] 24. The method according to any one of embodiments 15 to 23, wherein the reaction mix is ​​replenished with guanosine triphosphate (GTP) or a functional analog thereof until the transcription reaction is completed.

[0370] 25. The method according to any one of Embodiments 1 to 24, wherein the method does not involve supplementing the transcription mix with cytidine triphosphate (CTP) or a functional analog thereof and / or adenosine triphosphate (ATP) or a functional analog thereof during the transcription reaction.

[0371] 26. The method according to any one of Embodiments 1 to 25, wherein the reaction mix comprises a starting nucleotide corresponding to the first nucleotide in the RNA molecule.

[0372] 27. The method according to Embodiment 26, wherein the starting nucleotide is a nucleoside monophosphate, a nucleoside diphosphate, a nucleoside triphosphate, or a dinucleoside triphosphate.

[0373] 28. The method according to Embodiment 26 or 27, wherein the starting nucleotide is a 5' cap or a 5' cap analog.

[0374] 29. The 5' capped analog is G[5']ppp[5']G, m7G[5']ppp[5']G, m3 2,2,7 G[5']ppp[5']G, m2 7,3’-O G[5']ppp[5']G(3'-ARCA), m2 7,2’-O GpppG(2'-ARCA), m2 7,2’-O Gpp S pG D1(β-S-ARCA D1), m2 7,2’-O Gpp S pG D2 (β-S-ARCA D2) and m2 7,3’-O Gppp(m 2’-O The method according to Embodiment 28, selected from the group consisting of ApG(CC413).

[0375] 30. The method according to Embodiment 28 or 29, wherein the 5' cap or 5' cap analog in the reaction mix is ​​present in excess compared to guanosine triphosphate (GTP) or its functional analog.

[0376] The method according to Embodiment 30, wherein the ratio of the starting concentration of a 31.5' cap or 5' cap analog to the starting concentration of guanosine triphosphate (GTP) or its functional analog is about 2:1 to about 20:1.

[0377] The method according to Embodiment 31, wherein the ratio of the initial concentration of the 32.5' cap or 5' cap analog to the initial concentration of guanosine triphosphate (GTP) or its functional analog is approximately 4:1.

[0378] 33. The method according to any one of Embodiments 1 to 32, wherein the reaction mix further comprises RNA polymerase, a buffer, and at least one monovalent or divalent cation.

[0379] 34. The cation is Li + na + , K + NH 4+ Tris(hydroxymethyl)aminomethane cation, Mg 2+ Ba 2+ or Mn 2+ The method according to embodiment 33.

[0380] 35. The method according to embodiment 33 or 34, wherein the RNA polymerase is selected from the group consisting of T7 RNA polymerase, T3 RNA polymerase, and SP6 RNA polymerase.

[0381] 36. The method according to any one of Embodiments 1 to 35, wherein the functional analog of uridine triphosphate (UTP) is selected from the group consisting of pseudo-UTP, N1-methyl pseudo-UTP, 2-thio-UTP, and 4-thio-UTP.

[0382] 37. The method according to any one of Embodiments 1 to 36, wherein the functional analog of guanosine triphosphate (GTP) is selected from the group consisting of 7-deaza-GTP, N1-methyl-GTP, and O6-methyl-GTP.

[0383] 38. The method according to any one of Embodiments 1 to 37, wherein the DNA template encodes one or more of the 5' untranslated region (UTR), 3'UTR, open reading frame, and poly(A) tail.

[0384] 39. The method according to any one of Embodiments 1 to 38, wherein the RNA comprises one or more of the following: a 5' untranslated region (UTR), a 3'UTR, an open reading frame, and a poly(A) tail.

[0385] 40. The method according to Embodiment 39, wherein the RNA encodes at least one peptide or protein.

[0386] 41. The method according to any one of Embodiments 1 to 40, wherein the RNA is mRNA.

[0387] 42. The method according to any one of Embodiments 1 to 41, wherein the pH value of the reaction mix is ​​kept substantially constant throughout the process of the transfer reaction.

[0388] 43. The method according to any one of Embodiments 1 to 42, wherein the progress of the transcription reaction is monitored in real time.

[0389] 44. The method according to any one of Embodiments 1 to 43, carried out using a bioreactor.

[0390] 45. RNA produced by the method described in any one of Embodiments 1 to 44.

[0391] 46. ​​A composition comprising RNA produced by the method described in any one of Embodiments 3 to 44.

[0392] 47. A method of treating the subject, (i) the step of obtaining RNA produced by any one of Embodiments 1 to 44, or the step of obtaining a composition comprising RNA produced by any one of Embodiments 3 to 44, (ii) The method comprising the step of administering the RNA or the composition containing the RNA to the subject.

[0393] 48. A method of treating a subject by administering to the subject a composition containing the RNA described in Embodiment 45 or the RNA described in Embodiment 46.

[0394] 49. Improvements have been made to the method of producing RNA by in vitro transcription. The method comprising limiting the concentration of UTP or a functional analog thereof during the in vitro transcription reaction.

[0395] 50. An in vitro transcription reaction comprising limiting the concentration of UTP or a functional analog thereof in the in vitro transcription reaction. An in vitro transcription reaction comprising an RNA template containing a promoter that directs the transcription of a template for producing a transcript having poly(A) sequence elements, RNA polymerase acting on the promoter; The in vitro transcription reaction comprises adenosine triphosphate (ATP), guanosine triphosphate (GTP), cytidine triphosphate (CTP), and uridine triphosphate (UTP), or functional analogs thereof, wherein the starting concentration of UTP or its functional analog is lower than the concentration of CTP or its functional analog and / or ATP or its functional analog. [Examples]

[0396] The present invention will be described in detail and illustrated by figures and examples, which are for illustrative purposes only and not intended to limit the invention. Those skilled in the art will also be able to access further embodiments included in the present invention as described and illustrated by the examples.

[0397] Example 1: Exemplary IVT reactions that restrict UTP or UTP and ATP in transcription reactions can reduce the generation of dsRNA content. This embodiment demonstrates an exemplary fed-batch culture procedure for improving capping efficiency and / or manipulating the double-stranded RNA (dsRNA) content generated during in vitro transcription (IVT) reactions. In some embodiments, dsRNA is produced by reverse transcription (e.g., 3' to 5' direction). In some embodiments, this effect is minimized by restricting the NTP required for transcription starting at the 3' end. This embodiment specifically demonstrates that restricting UTP in in vitro transcription reactions can reduce dsRNA formation, which may be particularly useful for the production of transcripts that may contain polyA sequences, such as polyA tails. While not bound by theory, the inventors propose that the observed reduction in dsRNA production may be due to a reduction in reverse transcription (e.g., initiated during hybridization with polyA sequences, such as polyA tails).

[0398] To test the effect of limiting NTPs during IVT, ATP(A), UTP(U), and a combination of ATP and UTP (A / U) were supplied to the reaction at specific time intervals. A standard GTP(G) supply was used as a control.

[0399] In some embodiments, the starting concentrations of G, U, A, or A / U were reduced to 20% of their respective starting concentrations (e.g., typical starting concentrations used in IVT) and supplied in four additions during the transcription reaction until the final concentration was reached.

[0400] Exemplary IVT is m2 for DNA template, cotranscription capping. 7,3’-O Gppp(m 2’-O The procedure was carried out in the presence of an ApG(CC413) cap analog and nucleoside triphosphates (GTP, ATP, UTP, CTP). The IVT reaction was performed for 150 minutes using magnesium acetate buffer containing dithiothreitol and spermidine in the presence of T7 RNA polymerase, an RNAse inhibitor (Ribolock), and an inorganic pyrophosphatase.

[0401] In some embodiments, after IVT, the DNA template was removed (e.g., via DNAse digestion), and the RNA was purified (e.g., using magnetic beads for immobilization (Berensmeier, S. Magnetic particles for the separation and purification of nucleic acids. Appl. Microbiol. Biotechnol. 73, 495-504; 10.1007 / s00253-006-0675-0 (2006)). In some embodiments, the RNA was eluted in H2O, for ex...

Claims

1. 1. A method for producing RNA comprising transcribing RNA from a DNA template using a reaction mix comprising adenosine triphosphate (ATP), guanosine triphosphate (GTP), cytidine triphosphate (CTP) and uridine triphosphate (UTP), or functional analogs thereof, wherein an initial concentration of UTP or its functional analog is lower than an initial concentration of CTP or its functional analog and / or ATP or its functional analog, the method comprising supplementing the reaction mix during the course of the transcription reaction with a composition comprising UTP or its functional analog and substantially free of CTP or its functional analog or ATP or its functional analog.

2. The method described in claim 1, wherein the initial concentration of guanosine triphosphate (CTP) or a functional analog thereof is equal to the initial concentration of adenosine triphosphate (ATP) or a functional analog thereof.

3. 2. The method of claim 1, wherein the double-stranded (ds)RNA content of the composition comprising RNA is reduced compared to the dsRNA content of a composition comprising RNA transcribed from the same DNA template using equimolar amounts of adenosine triphosphate (ATP), guanosine triphosphate (GTP), cytidine triphosphate (CTP) and uridine triphosphate (UTP), or functional analogs thereof.

4. 2. The method of claim 1, wherein the immunogenicity of the composition comprising RNA is reduced compared to the immunogenicity of a composition comprising RNA transcribed from the same DNA template using equimolar amounts of adenosine triphosphate (ATP), guanosine triphosphate (GTP), cytidine triphosphate (CTP) and uridine triphosphate (UTP), or functional analogs thereof.

5. 2. The method of claim 1, wherein the ratio of the starting concentration of uridine triphosphate (UTP) or a functional analogue thereof to the starting concentration of cytidine triphosphate (CTP) or a functional analogue thereof or adenosine triphosphate (ATP) or a functional analogue thereof is about 1:1.5 to 1:

15.

6. 2. The method of claim 1, wherein the reaction mix is ​​replenished with uridine triphosphate (UTP) or a functional analog thereof when the concentration of UTP or a functional analog thereof approaches depletion.

7. 2. The method of claim 1, wherein the reaction mix is ​​supplemented with uridine triphosphate (UTP) or a functional analog thereof at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100 or more times during the course of the transcription reaction.

8. 10. The method of claim 1, wherein the reaction mix is ​​continuously replenished with uridine triphosphate (UTP) or a functional analog thereof during the course of the transcription reaction.

9. 2. The method of claim 1, wherein the reaction mix is ​​periodically replenished with uridine triphosphate (UTP) or a functional analog thereof during the course of the transcription reaction.

10. 2. The method of claim 1, wherein the initial ratio of the concentration of uridine triphosphate (UTP) or a functional analog thereof to the concentration of cytidine triphosphate (CTP) or a functional analog thereof or adenosine triphosphate (ATP) or a functional analog thereof is maintained or restored by supplementing the reaction mix with UTP or a functional analog thereof.

11. 2. The method of claim 1, wherein the initial concentration of guanosine triphosphate (GTP) or a functional analog thereof is lower than the initial concentration of cytidine triphosphate (CTP) or a functional analog thereof or adenosine triphosphate (ATP) or a functional analog thereof.

12. 12. The method of claim 11, wherein the ratio of the starting concentration of guanosine triphosphate (GTP) or a functional analogue thereof to the starting concentration of cytidine triphosphate (CTP) or a functional analogue thereof or adenosine triphosphate (ATP) or a functional analogue thereof is about 1:1.5 to 1:

15.

13. 12. The method of claim 11, wherein the reaction mix is ​​supplemented with guanosine triphosphate (GTP) or a functional analog thereof during the course of the transcription reaction.

14. 14. The method of claim 13, wherein the reaction mix is ​​replenished with guanosine triphosphate (GTP) or a functional analog thereof when the concentration of GTP or a functional analog thereof approaches depletion.

15. 12. The method of claim 11, wherein the reaction mix is ​​replenished with guanosine triphosphate (GTP) or a functional analog thereof at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100 or more times during the course of the transcription reaction.

16. 12. The method of claim 11, wherein the reaction mix is ​​continuously replenished with guanosine triphosphate (GTP) or a functional analog thereof during the course of the transcription reaction.

17. 12. The method of claim 11, wherein the reaction mix is ​​periodically replenished with guanosine triphosphate (GTP) or a functional analog thereof during the course of the transcription reaction.

18. 12. The method of claim 11, wherein the initial ratio of the concentration of guanosine triphosphate (GTP) or a functional analog thereof to the concentration of cytidine triphosphate (CTP) or a functional analog thereof or adenosine triphosphate (ATP) or a functional analog thereof is maintained or restored by supplementing the reaction mix with GTP or a functional analog thereof.

19. 12. The method of claim 11, wherein the reaction mix is ​​supplemented with guanosine triphosphate (GTP) or a functional analog thereof until the transcription reaction is terminated.

20. 2. The method of claim 1, wherein the method does not include supplementing a transcription mix with cytidine triphosphate (CTP) or a functional analog thereof and / or adenosine triphosphate (ATP) or a functional analog thereof during the course of the transcription reaction.

21. The method of claim 1 , wherein the reaction mix comprises a starting nucleotide corresponding to the first nucleotide in the RNA molecule.

22. 22. The method of claim 21, wherein the starting nucleotide is a nucleoside monophosphate, a nucleoside diphosphate, a nucleoside triphosphate, or a dinucleoside triphosphate.

23. 22. The method of claim 21, wherein the starting nucleotide is a 5' cap or a 5' cap analog.

24. The 5' cap analog is G[5']ppp[5']G, m ７ G[5']ppp[5']G,m ３ ２，２，７ G[5']ppp[5']G,m ２ ７，３’－Ｏ G[5']ppp[5']G (3'-ARCA), m ２ ７，２’－Ｏ GpppG (2'-ARCA), m ２ ７，２’－Ｏ GppspG D1 (β-S-ARCA D1), m ２ ７，２’－Ｏ GppspG D2 (β-S-ARCA D2) and m ２ ７，３’－Ｏ Gppp(m ２’－Ｏ 24. The method of claim 23, wherein said polypeptide is selected from the group consisting of ApG(CC413).

25. 24. The method of claim 23, wherein the 5' cap or 5' cap analog in the reaction mix is ​​present in excess compared to guanosine triphosphate (GTP) or a functional analog thereof.

26. 26. The method of claim 25, wherein the ratio of the starting concentration of 5' cap or 5' cap analog to the starting concentration of guanosine triphosphate (GTP) or a functional analog thereof is from about 2:1 to about 20:

1.

27. 27. The method of claim 26, wherein the ratio of the starting concentration of 5' cap or 5' cap analog to the starting concentration of guanosine triphosphate (GTP) or a functional analog thereof is about 4:

1.

28. 2. The method of claim 1, wherein the functional analog of uridine triphosphate (UTP) is selected from the group consisting of pseudo-UTP, N1-methyl pseudo-UTP, 2-thio-UTP, and 4-thio-UTP.

29. 2. The method of claim 1, wherein the functional analog of guanosine triphosphate (GTP) is selected from the group consisting of 7-deaza-GTP, N1-methyl-GTP, and O6-methyl-GTP.

30. 2. The method of claim 1, wherein the DNA template encodes one or more of a 5' untranslated region (UTR), a 3' UTR, an open reading frame, and a poly(A) tail.

31. 2. The method of claim 1, wherein the RNA comprises one or more of a 5' untranslated region (UTR), a 3' UTR, an open reading frame, and a poly(A) tail.

32. 32. The method of claim 31 , wherein the RNA encodes at least one peptide or protein.

33. The method of claim 1 , wherein the pH value of the reaction mix is ​​kept substantially constant during the course of the transcription reaction.

34. The method of claim 1 , wherein the progress of the transcription reaction is monitored in real time.

35. 10. The method of claim 1, carried out using a bioreactor.

36. A composition comprising RNA produced by the method of claim 1.

37. 1. A method of treating a subject, comprising: (i) obtaining RNA produced by the method of claim 1; (ii) administering to the subject the RNA or a composition comprising the RNA.

38. A method of treating a subject by administering to the subject a composition comprising the RNA described in claim 36.

39. A method for producing RNA by in vitro transcription comprising: The method comprising limiting the concentration of UTP or a functional analogue thereof in the in vitro transcription reaction.

40. An in vitro transcription reaction comprising: an RNA template comprising a promoter that directs transcription of the template to produce a transcript having a polyA sequence element; an RNA polymerase acting on the promoter; adenosine triphosphate (ATP), guanosine triphosphate (GTP), cytidine triphosphate (CTP) and uridine triphosphate (UTP), or functional analogs thereof, wherein the initial concentration of UTP or its functional analog is lower than the concentration of CTP or its functional analog and / or ATP or its functional analog.

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