Nucleic acid compositions and methods

Abstract

This invention provides nucleic acid compositions and methods for preparing nucleic acid compositions. Applications of such nucleic acid compositions include their use in therapeutics.

Description

Technical Field

[0001] This invention relates to the technical field of nucleic acid compositions and methods for preparing nucleic acid compositions. Applications of such nucleic acid compositions include their use in therapeutics. Background Technology

[0002] Nucleic acid-based compositions, i.e., compositions containing one or more different nucleic acids, have numerous applications that are being developed and expanded. In particular, compositions containing at least two different nucleic acids (i.e., two nucleic acids with different sequences) are of particular interest. Possible applications of such compositions include mRNA-based vaccines containing multiple different mRNA sequences, each encoding one or more different antigens. Such compositions containing two or more different RNA molecules can be used as vaccines containing / encoding two or more different groups of antigens.

[0003] The production of compositions containing two or more different nucleic acid molecules is typically carried out in two or more separate stages. Traditionally, for each different nucleic acid sequence, there will be a separate production process and reaction vessel. Thus, each different nucleic acid is transcribed from DNA to RNA by, for example, in vitro transcription (IVT) and purified separately. Subsequently, the prepared different nucleic acids are mixed to provide a composition containing two or more different nucleic acids.

[0004] Ideally, particularly for therapeutic applications, compositions containing two or more different nucleic acids should contain a predetermined, known ratio of those two or more different nucleic acids. For example, it might be desirable to provide compositions containing equal amounts or concentrations of different nucleic acids, but it might also be desirable to provide compositions containing, for example, equal amounts or concentrations of first and second nucleic acids and twice the amount or concentration of a third nucleic acid (1:1:2). The specific ratio depends on the application. Therefore, when following the traditional staged approach described above, after the individual production of the nucleic acids, the concentration of each nucleic acid must be determined and carefully adjusted to meet the requirements of the mixed composition—a complex, expensive, and time-consuming process.

[0005] EP3319622B1 discloses a method for preparing compositions comprising nearly equal amounts of different RNA molecules, wherein a reaction mixture comprising nearly equal amounts of corresponding DNA molecules is provided. The illustrated RNA molecules are disclosed to have the same or similar length.

[0006] Marras et al., Nucleic Acids Research 32(9):e72 (2004), disclosed a method for real-time measurement of RNA synthesis of two different RNAs from template DNA molecules of similar length and equal amounts.

[0007] Kerby et al., Appl Biochem Biotechnol. 164(4):497-513 (2011) addressed early transcriptional termination in human H5 influenza RNA synthesis and disclosed an in vitro transcription reaction in which fragments of varying lengths were generated from H5 DNA.

[0008] None of these documents disclose methods for use in single-pot in vitro transcription reactions where the DNA template and / or RNA product have significantly different lengths, and / or methods for improving the production efficiency of multivalent RNA compositions where the desired RNA ratio must be maintained, especially when the desired RNA ratio among all RNA molecules in the multivalent RNA composition is not 1:1. Invention Overview

[0010] While the staged production of nucleic acid compositions containing two or more different nucleic acids is widely used and accepted in the art, the inventors sought to provide an improved method. In particular, the inventors developed a method that takes into account, for example, differences in nucleic acid length and the desired final RNA fraction, and surprisingly found that this method can be used to produce the desired amounts of multiple different nucleic acids in the same IVT reaction. This method was unexpectedly found to be highly advantageous because it was determined to reduce production time by more than three times (when producing compositions containing three different nucleic acids). This includes reducing the number of production reactions and purification steps, and allowing the omission of the final mixing step for a single nucleic acid. Furthermore, the method of the present invention allows the desired proportions of different nucleic acids to be predetermined, easily achieved, and easily adjusted as needed.

[0011] Therefore, in a first aspect, the present invention provides a method for preparing a composition comprising two or more different RNA molecules, wherein each different RNA molecule can be obtained from a different DNA molecule, wherein the method comprises:

[0012] a) Provide a reaction mixture comprising the DNA molecules, wherein each different DNA molecule (X) has a concentration according to formula (i):

[0013] (i)

[0014] in:

[0015] “c(DNAx)” represents the concentration of a given DNA molecule X;

[0016] c(DNA) 总 ")" represents the total concentration of all different DNA molecules;

[0017] "RNA fraction" is the fraction of total RNA molecules in a composition containing RNA molecules that can be obtained from the DNA molecule X;

[0018] "DNAx length fraction" is the base pair (bp) length of the DNA molecule X divided by the total bp length of all different DNA molecules;

[0019] "RNAx length fraction" is the length in bp of the RNA molecule obtainable from the DNA molecule X divided by the total length in bp of all different RNA molecules; and

[0020] b) Obtain the RNA molecule from the DNA molecule in the reaction mixture, thereby preparing the composition.

[0021] In a second aspect, the present invention provides a method for preparing a composition comprising at least a first RNA molecule and a second RNA molecule, wherein the first RNA molecule is obtainable from a first DNA molecule, and the second RNA molecule is obtainable from a second DNA molecule, wherein the method comprises:

[0022] a) Provide a reaction mixture comprising the first DNA molecule and the second DNA molecule; and

[0023] b) Obtain the first RNA molecule and the second RNA molecule from the first DNA molecule and the second DNA molecule, respectively;

[0024] The relative mass of the first RNA molecule is greater than that of the second RNA molecule, and the molar amount of the second RNA molecule obtained from the second DNA molecule is greater than the molar amount of the first RNA molecule obtained from the first DNA molecule, such that the first RNA molecule and the second RNA molecule are obtained in the composition at substantially the same concentration.

[0025] In a third aspect, the present invention provides compositions that are obtained or obtainable by the method of the present invention.

[0026] In a fourth aspect, the present invention provides compositions that are obtained or available by the method of the present invention, which are used as pharmaceuticals.

[0027] In a fifth aspect, the present invention provides a composition comprising two or more different DNA molecules, wherein different RNA molecules can be obtained from each different DNA molecule, wherein each different DNA molecule (X) has a concentration according to formula (i):

[0028] (i)

[0029] in:

[0030] “c(DNAx)” represents the concentration of a given DNA molecule X;

[0031] c(DNA) 总 ")" represents the total concentration of all DNA molecules;

[0032] "RNA fraction" is the fraction of all RNA molecules that can be obtained from a reaction mixture containing RNA molecules that can be obtained from said DNA molecule X;

[0033] "DNAx length fraction" is the base pair (bp) length of the DNA molecule X divided by the total bp length of all different DNA molecules;

[0034] "RNAx length fraction" is the bp length of the RNA molecule that can be obtained from the DNA molecule X divided by the total bp length of all different RNA molecules.

[0035] Brief description of the attached figures

[0036] Figure 1 : A graph showing the dependence of total DNA template concentration on IVT yield in a single-pot reaction calculated to produce three RNA products in a 1:1:1 ratio.

[0037] Figure 2 : A graph showing the dependence of initial ATP / CTP concentrations (ATP and CTP concentrations are initially represented by x-axis values) on the resulting IVT yield in a single-pot reaction containing nucleic acid sequences with high A and C scores.

[0038] Figure 3 The results are derived from the analysis of IVT ratio determination using three different nucleic acids. The method according to the invention utilizes DNA templates encoding RNA product 3 at different concentrations to generate different ratios.

[0039] Figure 4 The results are derived from the analysis of IVT ratio determination using three different nucleic acids. According to the method of the present invention, different concentrations of DNA templates encoding RNA products 1 and 2 are used to generate different ratios. Invention Details

[0041] In a first aspect, the present invention provides a method for preparing a composition comprising two or more different RNA molecules, wherein each different RNA molecule can be obtained from a different DNA molecule, wherein the method comprises:

[0042] a) Provide a reaction mixture comprising the DNA molecules, wherein each different DNA molecule (X) has a concentration according to formula (i):

[0043] (i)

[0044] in:

[0045] “c(DNAx)” represents the concentration of a given DNA molecule X;

[0046] c(DNA) 总 ")" represents the total concentration of all different DNA molecules;

[0047] "RNA fraction" is the fraction of total RNA molecules in a composition containing RNA molecules that can be obtained from the DNA molecule X;

[0048] "DNAx length fraction" is the base pair (bp) length of the DNA molecule X divided by the total bp length of all different DNA molecules;

[0049] "RNAx length fraction" is the length in bp of the RNA molecule obtainable from the DNA molecule X divided by the total length in bp of all different RNA molecules; and

[0050] b) Obtain the RNA molecule from the DNA molecule in the reaction mixture, thereby preparing the composition.

[0051] Therefore, the present invention provides a method for preparing a composition comprising two or more different product nucleic acids (RNAs) from two or more different template nucleic acids (DNAs) in a single reaction mixture. This saves time and cost and is generally more procedurally efficient than prior art methods for producing such compositions. Furthermore, the method of the present invention allows the user to determine the “RNA fraction” of each product nucleic acid (RNA) X to be produced in the resulting composition. This is the proportion of the total resulting product nucleic acids (RNAs) to be composed of each specific product nucleic acid (RNA) X. In other words, this allows those skilled in the art to determine the proportions between the different product nucleic acids (RNAs) produced by the method of the present invention.

[0052] Although the template nucleic acid most commonly comprises DNA in the methods of the present invention, and the product nucleic acid in the methods of the present invention comprises RNA (as described in the first aspect), it should be understood that broader application of the methods and formulas of the present invention (such as those including other types of templates and product nucleic acids) is possible in embodiments. For example, both the template and the product nucleic acid may comprise DNA, and the product nucleic acid may be obtained from the template nucleic acid by DNA replication or non-standard transcription methods. For convenience, the template nucleic acid is generally referred to herein as DNA, and the product nucleic acid as RNA.

[0053] The term "RNA molecule" as used herein refers to a molecule containing RNA polynucleotides. In one embodiment, the RNA molecule contains one or more of a coding sequence, a non-coding RNA (ncRNA) sequence, a microRNA (miRNA) sequence, or a small interfering RNA (siRNA) sequence. The RNA molecule may contain modifications such as conjugation, backbone modifications, base modifications, and sugar modifications. To a certain extent, the RNA molecule may also contain other non-RNA polynucleotides. In one embodiment, the RNA molecule contains at least 100%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 75%, 70%, 65%, 60%, 55%, or 50% RNA polynucleotides.

[0054] The term "DNA molecule" as used herein refers to a molecule containing DNA polynucleotides. In one embodiment, the DNA molecule contains a sequence that can be transcribed into an RNA sequence, said RNA sequence comprising one or more of a non-coding RNA (ncRNA) sequence, a microRNA (miRNA) sequence, or a small interfering RNA (siRNA) sequence. In one embodiment, the DNA molecule also contains one or more non-transcribed DNA, such as a promoter sequence, a PCR template sequence, or a linearized plasmid sequence. The DNA molecule may contain modifications such as conjugation, backbone modifications, base modifications, and sugar modifications. To a certain extent, the DNA molecule may also contain other non-DNA polynucleotides. In one embodiment, the DNA molecule contains at least 100%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 75%, 70%, 65%, 60%, 55%, or 50% DNA polynucleotides.

[0055] This invention relates to a method for preparing a composition comprising two or more different RNA molecules. Generally, in the context of, for example, two or more RNA or DNA molecules, “different” means that the molecules have different nucleotide sequences. In one embodiment, the different RNA molecules also encode different polypeptide sequences. Therefore, it should be understood that references to “different” RNA or DNA “molecules” do not refer to a single molecule itself, but to each different type (sequence) of RNA or DNA molecule present. Thus, in one embodiment, “two or more different RNA molecules” means “two or more different RNA species,” for example, two or more RNA species with different sequences. Thus, in one embodiment, “RNA molecule” means an RNA species. Thus, in one embodiment, “DNA molecule” means a DNA species.

[0056] In one embodiment, each different RNA molecule can be obtained from a different DNA molecule via transcription. Therefore, in one embodiment, the RNA molecule of the present invention can be obtained from two or more different corresponding DNA molecules. In the present invention, the template DNA molecule is the starting material and therefore contains not only the sequence to be transcribed into RNA but also any non-transcribed sequences, including regulatory sequences such as promoter sequences. In one embodiment, the DNA molecule contains a PCR template sequence or a linearized plasmid sequence. Therefore, for considerations involving DNA molecule length, the full length of the DNA molecule, including non-transcribed sequences, should be considered. For considerations involving RNA molecule length, the full length of the RNA molecule to be obtained from the DNA molecule via transcription in the context of the transcription conditions used must be considered.

[0057] In step a) of the method of the present invention, "reaction mixture" should be understood as comprising a single composition in which (two or more) different DNA molecules of the present invention are present. In one embodiment, the reaction mixture is a single composition. In one embodiment, the reaction mixture is contained in a single container. In one embodiment, (two or more) different DNA molecules of the present invention are contained in a single container.

[0058] In this invention, the concentration of each (i.e., every) different DNA molecule in the reaction mixture that contributes to the resulting RNA is determined by formula (i). In this document, “X” is used to refer to a given DNA molecule whose concentration is being calculated, but it should be understood that the calculation is performed sequentially for each different DNA molecule.

[0059] (i)

[0060] “c(DNAx)” represents the concentration of a given DNA molecule X. In this invention, the concentration of each different DNA molecule is expressed as an initial mass concentration, i.e., the mass per unit volume, such as the mass per unit volume of the reaction mixture. The total DNA concentration is also expressed as an initial mass concentration, i.e., the mass per unit volume, such as the mass per unit volume of the reaction mixture.

[0061] c(DNA) 总 ")" represents the total concentration of all different DNA molecules in the reaction mixture. Therefore, the total concentration of DNA to be used in the IVT reaction is predetermined, and the concentration of each different DNA molecule is determined as a relative fraction of that total concentration.

[0062] "RNA fraction" refers to the percentage of total RNA molecules in a composition that contains RNA molecules obtainable from DNA molecule X (i.e., the DNA molecule whose concentration is being calculated). Therefore, the user can predetermine the fraction of each RNA molecule in the composition prepared after the IVT reaction, and thus predetermine the proportion of RNA molecules in the prepared composition. As an example, consider a reaction mixture containing three different DNA molecules, DNA1, DNA2, and DNA3, which encode three different RNA molecules, RNA1, RNA2, and RNA3, respectively. If a composition containing a 1:1:1 ratio of RNA1:RNA2:RNA3 is desired, then the "RNA fraction" when calculating the desired concentration of DNA1 (or DNA2 or DNA3) will be one-third (0.3%). Alternatively, if it is desired to produce a composition comprising RNA1:RNA2:RNA3 in a 2:1:1 ratio, the “RNA fraction” when calculating the concentration of DNA1 will be one-half (0.5). In the latter case, the “RNA fraction” when calculating the desired concentration of DNA2 or DNA3 will be one-quarter (0.25). Therefore, in general, the sum of the RNA fractions for each DNA / corresponding RNA molecule will be 1. In a typical embodiment, each RNA molecule will be available from only one corresponding DNA molecule. However, in embodiments where the RNA molecule can be obtained from more than one different DNA molecule in the reaction mixture, the “RNA fraction” used to determine the concentration of each DNA molecule from which the RNA molecule can be obtained will be further divided by the total number of different DNA molecules from which the RNA molecule can be obtained.

[0063] The "DNAx length fraction" is the base pair (bp) length of the DNA molecule X whose concentration is being determined, divided by the total bp length of all the different DNA molecules. Therefore, the "DNAx length fraction" is a fraction of the total DNA length comprised of the DNA molecules whose concentration is being determined. For example, in a reaction mixture containing three different DNA molecules (DNA1, DNA2, and DNA3, all 1000 bp in length), the "DNAx length fraction" used to determine the desired concentration of DNA1 (or DNA2 or DNA3) would be one-third (0.3 bp). To give another example, in a reaction mixture containing three different DNA molecules (DNA1, 2000 bp in length; DNA2, 1000 bp in length; and DNA3, 1000 bp in length), the "DNA x length fraction" used to determine the desired concentration of DNA1 will be half (0.5). In the latter case, the "DNA x length fraction" used to determine the desired concentration of DNA2 or DNA3 will be one-quarter (0.25). Therefore, in general, the total RNA fraction used for each DNA / corresponding RNA molecule will be 1.

[0064] The "RNAx length fraction" is the ratio of the bp length of the RNA molecules that can be obtained from the DNA molecule X whose concentration is being determined to the total bp length of all the different RNA molecules. Therefore, the "RNAx length fraction" is the fraction of the total RNA length that can be obtained from the DNA molecule X whose concentration is being determined. For example, for a reaction mixture containing three different DNA molecules (DNA1, which yields RNA1 of 1000 bp length; DNA2, which yields RNA2 of 1000 bp length; and DNA3, which yields RNA3 of 1000 bp length), the "RNAx length fraction" used to determine the desired concentration of DNA1 (or DNA2 or DNA3) would be one-third (0.3%). To give another example, for a reaction mixture containing three different DNA molecules (DNA1 yielding RNA1 of 2000 bp, DNA2 yielding RNA2 of 1000 bp, and DNA3 yielding RNA3 of 1000 bp), the "RNA x length fraction" used to determine the desired concentration of DNA1 will be half (0.5). In the latter case, the "DNA x length fraction" used to determine the desired concentration of DNA2 or DNA3 will be one-quarter (0.25). Therefore, in general, the total RNA fraction used for each DNA / corresponding RNA molecule will be 1.

[0065] In the context of this invention, base pair length (bp length) is referred to as the length of a nucleic acid, a concept known to those skilled in the art, referring to the number of consecutive nucleobases that make up a given nucleic acid. This term is essentially interchangeable with nucleotide length (nt length).

[0066] In step a), different DNA molecules are then provided in a reaction mixture containing said DNA molecules. In one embodiment, this involves adding each different DNA molecule to the reaction mixture. In one embodiment, the reaction mixture also contains substances required for transcription to occur, such as RNA polymerase and free RNA nucleotides.

[0067] In one embodiment, step a) involves determining the concentration of each DNA molecule using formula (i), and then providing a reaction mixture containing each DNA molecule at its respective determined concentration.

[0068] In one implementation, different RNA molecules are obtained in a predetermined ratio in step b), and for each different DNA molecule, step a) includes the following steps:

[0069] 1) Determine the length of the DNA molecule;

[0070] 2) Determine the length of the RNA molecule that can be obtained from the DNA molecule;

[0071] 3) Predetermine the ratio between at least two different RNA molecules obtained from the DNA molecule in step b);

[0072] 4) Using formula (i), the length of the DNA molecule, and the length of the available RNA molecule, determine the concentration of DNA molecules for which the RNA molecule will be obtained in step b) at the predetermined ratio; and

[0073] 5) Each DNA molecule is provided in the reaction mixture at the concentration stated therein.

[0074] In embodiments, as those skilled in the art will understand, “determining” the “length” of a DNA or RNA molecule means measuring or calculating the length of the DNA or RNA molecule by the number of base pairs or nucleotides contained therein. In embodiments, as those skilled in the art will understand, determining the concentration of DNA molecules means calculating the concentration of DNA molecules that must be present using formula (i), taking into account both the lengths of the DNA and coding RNA, to obtain different coding RNA molecules in a desired predetermined ratio. It will generally be understood that the predetermined ratio is the proportion of different RNA molecules present in a composition comprising two or more different RNA molecules, the result of the method of the present invention. It will generally be understood that the proportion of different RNA molecules is predetermined, meaning that it is calculated as the desired result before providing the reaction mixture or preparing the composition of the present invention, and used as a factor as described in formula (i).

[0075] In some embodiments, the method of the present invention includes pre-determining the ratio between different RNA molecules to be obtained in step b). This pre-determining step may occur, for example, before or during step a). In one embodiment, the method of the present invention includes pre-determining the ratio between at least two different RNA molecules to be obtained in step b). In one embodiment, the method of the present invention includes pre-determining the ratio between 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more different RNA molecules to be obtained in step b). In one embodiment, the method of the present invention includes pre-determining the ratio between all different RNA molecules to be obtained in step b). In a preferred embodiment, at least one ratio between two different RNA molecules is not 1:1. In a preferred embodiment, at least one ratio between two different RNA molecules is 1:1, and the different RNA molecules in question are (substantially) different in bp length. In a preferred embodiment, at least one ratio between two different RNA molecules is 1:1, and the DNA molecules from which the different RNA molecules in question are obtained are (substantially) different in bp length.

[0076] Subsequently, in step b), different product RNA molecules corresponding to each DNA molecule in the reaction mixture are obtained from the DNA molecules in the reaction mixture. In an embodiment, the RNA molecules are obtained by transcription. In one embodiment, each different RNA molecule is obtained as a fraction of the total RNA obtained, which corresponds to the corresponding “RNA fraction” in formula (i). In one embodiment, each different RNA molecule is obtained as a fraction of the total RNA obtained, which differs from the corresponding “RNA fraction” in formula (i) by up to ±1%, up to ±2%, up to ±3%, up to ±4%, up to ±5%, up to ±6%, up to ±7%, up to ±8%, up to ±9%, up to ±10%, up to ±11%, up to ±12%, up to ±13%, up to ±14%, up to ±15%, up to ±16%, up to ±17%, up to ±18%, up to ±19%, or up to ±20%.

[0077] For a given RNA, the “RNA fraction” in Formula (i) is a predetermined fraction of the total RNA intended to be produced. However, the actual fraction of the total RNA represented by the given RNA (when generated in step b) may deviate from the predetermined value within tolerance. In one embodiment, the tolerance range for the obtained fraction is within ±5%, ±10%, ±15%, or ±20% of the predetermined “RNA fraction” in Formula (i) for each RNA. Furthermore, for a given RNA, the “RNA fraction” in Formula (i) also implies a predetermined concentration of the given RNA relative to the total RNA concentration. Therefore, the actual concentration of the total RNA represented by the given RNA (when generated in step b) may deviate from the predetermined concentration according to the “RNA fraction” in Formula (i) within tolerance. In one embodiment, the tolerance range for the obtained concentration is within ±5%, ±10%, ±15%, or ±20% of the predetermined concentration for each RNA.

[0078] Therefore, in an embodiment where the RNA fraction is 1 / 2, the RNA is obtained at a concentration within ±5%, ±10%, ±15%, or ±20% of the total RNA concentration, for example, 0.5 × ±0.025, ±0.05, ±0.075, or ±0.1 of the total RNA concentration, such as 0.475-0.525 × total RNA concentration, 0.45-0.55 × total RNA concentration, 0.425-0.575 × total RNA concentration, or 0.4-0.6 × total RNA concentration. In an embodiment where the RNA fraction is 1 / 3, the RNA is obtained at a concentration within ±5%, ±10%, ±15%, or ±20% of the total RNA concentration. In an embodiment where the RNA fraction is 1 / 4, the RNA is obtained at a concentration within ±5%, ±10%, ±15%, or ±20% of the total RNA concentration. In embodiments where the RNA fraction is 1 / 5, the RNA is obtained at a concentration within ±5%, ±10%, ±15%, or ±20% of the total RNA concentration. In embodiments where the RNA fraction is 1 / 6, the RNA is obtained at a concentration within ±5%, ±10%, ±15%, or ±20% of the total RNA concentration. In embodiments where the RNA fraction is 5 / 6, the RNA is obtained at a concentration within ±5%, ±10%, ±15%, or ±20% of the total RNA concentration. In embodiments where the RNA fraction is 1 / 7, the RNA is obtained at a concentration within ±5%, ±10%, ±15%, or ±20% of the total RNA concentration. In embodiments where the RNA fraction is 2 / 7, the RNA is obtained at a concentration within ±5%, ±10%, ±15%, or ±20% of the total RNA concentration. In embodiments where the RNA fraction is 3 / 7, the RNA is obtained at a concentration within ±5%, ±10%, ±15%, or ±20% of the total RNA concentration. In embodiments where the RNA fraction is 4 / 7, the RNA is obtained at a concentration within ±5%, ±10%, ±15%, or ±20% of the total RNA concentration. In embodiments where the RNA fraction is 5 / 7, the RNA is obtained at a concentration within ±5%, ±10%, ±15%, or ±20% of the total RNA concentration. In embodiments where the RNA fraction is 6 / 7, the RNA is obtained at a concentration within ±5%, ±10%, ±15%, or ±20% of the total RNA concentration. In embodiments where the RNA fraction is 1 / 8, the RNA is obtained at a concentration within ±5%, ±10%, ±15%, or ±20% of the total RNA concentration.In embodiments where the RNA fraction is 3 / 8, the RNA is obtained at a concentration within ±5%, ±10%, ±15%, or ±20% of the total RNA concentration. In embodiments where the RNA fraction is 5 / 8, the RNA is obtained at a concentration within ±5%, ±10%, ±15%, or ±20% of the total RNA concentration. In embodiments where the RNA fraction is 6 / 8, the RNA is obtained at a concentration within ±5%, ±10%, ±15%, or ±20% of the total RNA concentration. In embodiments where the RNA fraction is 7 / 8, the RNA is obtained at a concentration within ±5%, ±10%, ±15%, or ±20% of the total RNA concentration. In embodiments where the RNA fraction is 1 / 9, the RNA is obtained at a concentration within ±5%, ±10%, ±15%, or ±20% of the total RNA concentration. In embodiments where the RNA fraction is 2 / 9, the RNA is obtained at a concentration within ±5%, ±10%, ±15%, or ±20% of the total RNA concentration. In embodiments where the RNA fraction is 4 / 9, the RNA is obtained at a concentration within ±5%, ±10%, ±15%, or ±20% of the total RNA concentration. In embodiments where the RNA fraction is 5 / 9, the RNA is obtained at a concentration within ±5%, ±10%, ±15%, or ±20% of the total RNA concentration. In embodiments where the RNA fraction is 6 / 9, the RNA is obtained at a concentration within ±5%, ±10%, ±15%, or ±20% of the total RNA concentration. In embodiments where the RNA fraction is 7 / 9, the RNA is obtained at a concentration within ±5%, ±10%, ±15%, or ±20% of the total RNA concentration. In embodiments where the RNA fraction is 8 / 9, the RNA is obtained at a concentration within ±5%, ±10%, ±15%, or ±20% of the total RNA concentration. In embodiments where the RNA fraction is 1 / 10, the RNA is obtained at a concentration within ±5%, ±10%, ±15%, or ±20% of the total RNA concentration. In embodiments where the RNA fraction is 3 / 10, the RNA is obtained at a concentration within ±5%, ±10%, ±15%, or ±20% of the total RNA concentration. In embodiments where the RNA fraction is 4 / 10, the RNA is obtained at a concentration within ±5%, ±10%, ±15%, or ±20% of the total RNA concentration.In embodiments where the RNA fraction is 6 / 10, the RNA is obtained at a concentration within ±5%, ±10%, ±15%, or ±20% of the total RNA concentration. In embodiments where the RNA fraction is 7 / 10, the RNA is obtained at a concentration within ±5%, ±10%, ±15%, or ±20% of the total RNA concentration. In embodiments where the RNA fraction is 8 / 10, the RNA is obtained at a concentration within ±5%, ±10%, ±15%, or ±20% of the total RNA concentration. In embodiments where the RNA fraction is 9 / 10, the RNA is obtained at a concentration within ±5%, ±10%, ±15%, or ±20% of the total RNA concentration. In this regard, it will be understood that the fractions discussed readily correspond to the proportions of different RNA molecules in the composition, for example, a 1:1:2 ratio corresponds to fractions of 1 / 4, 1 / 4, and 1 / 2, respectively.

[0079] In one implementation, each different RNA molecule is obtained as a fraction of the total RNA obtained, which is substantially the same as the corresponding “RNA fraction” in Formula (i). It should be understood that the “corresponding” “RNA fraction” of any given RNA molecule in Formula (i) is the “RNA fraction” in Formula (i) used to determine the initial concentration of DNA molecules from which the given RNA molecule is obtained.

[0080] In one embodiment, the concentration of each RNA molecule obtained in step b) varies only within pharmaceutically acceptable limits of the composition (i.e., the prepared composition). It will be generally understood herein that a pharmaceutically acceptable limit for a given RNA composition is a limit on the amount of RNA molecules present that is necessary to provide the desired therapeutic effect of the composition in vivo and / or avoid excessive toxicity in vivo. It will also be generally understood herein that the pharmaceutically optimal amount of RNA molecules in a given RNA composition will be the amount of RNA molecules that provides optimal therapeutic effect of the composition in vivo and / or minimal excessive toxicity in vivo.

[0081] In one embodiment, the concentration of any given RNA molecule obtained in step b) varies only within 80-120% of the average concentration of all RNA molecules. This embodiment is applicable when each different RNA molecule has the same "RNA fraction" in formula (i). In one embodiment, the concentration of any given RNA molecule obtained in step b) varies only within 80-120% of the total concentration of all RNA molecules multiplied by the corresponding "RNA fraction" of that given RNA molecule in formula (i). This embodiment is applicable when each different RNA molecule has a different or the same "RNA fraction" in formula (i).

[0082] In one embodiment, the composition comprises at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 different RNA molecules.

[0083] In one implementation, RNA molecules are obtained from DNA molecules through transcription.

[0084] In one embodiment, one or more DNA molecules contain a PCR template, or each DNA molecule contains a PCR template.

[0085] In one embodiment, one or more DNA molecules contain a linearized plasmid, or each DNA molecule contains a linearized plasmid.

[0086] In one embodiment, at least one DNA molecule contains a PCR template, and at least one DNA molecule contains a linearized plasmid.

[0087] Each RNA molecule may encode one or more different polypeptides. In one embodiment, each RNA molecule encodes at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 polypeptides. In one embodiment, each polypeptide encoded by the same RNA molecule is identical. In one embodiment, each polypeptide encoded by the same RNA molecule is different. In one embodiment, the polypeptide is an effector polypeptide. In one embodiment, the polypeptide is an antigen.

[0088] In one embodiment, in step a), the total concentration of all DNA molecules in the reaction mixture is predetermined, preferably wherein the total concentration of DNA molecules is at least 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, or 10 mg / mL. In one embodiment, the total concentration of DNA molecules is 0.05 mg / mL multiplied by the number of different DNA molecules. Thus, in a specific embodiment where two different DNA molecules are present, the total DNA concentration is 0.10 mg / mL. In a specific embodiment where three different DNA molecules are present, the total DNA concentration is 0.15 mg / mL. In a specific embodiment where four different DNA molecules are present, the total DNA concentration is 0.20 mg / mL. In a specific embodiment containing five different DNA molecules, the total DNA concentration is 0.25 mg / mL. In a specific embodiment containing six different DNA molecules, the total DNA concentration is 0.30 mg / mL. In a specific embodiment containing seven different DNA molecules, the total DNA concentration is 0.35 mg / mL. In a specific embodiment containing eight different DNA molecules, the total DNA concentration is 0.40 mg / mL. In a specific embodiment containing nine different DNA molecules, the total DNA concentration is 0.45 mg / mL. In a specific embodiment containing ten different DNA molecules, the total DNA concentration is 0.50 mg / mL. The total DNA concentration in any of these embodiments may vary by ±5%.

[0089] In one embodiment, the method of the present invention further includes:

[0090] c) Determine the amount of each RNA molecule in the composition.

[0091] In one embodiment, determining the amount of each RNA molecule includes determining its concentration, preferably its mass concentration.

[0092] In one embodiment, the method of the present invention further includes determining whether the concentration of one or more RNA molecules in the composition is below or above a reference level, and adjusting the concentration of the corresponding DNA molecules accordingly using a correction factor. In one embodiment, a concentration of RNA molecules less than a pharmaceutically acceptable or pharmaceutically most preferred concentration is considered below a reference level. In one embodiment, a concentration of RNA molecules greater than a pharmaceutically acceptable or pharmaceutically most preferred concentration is considered above a reference level. In one embodiment, a concentration of a given RNA molecule is considered below a reference level when the fraction of a given RNA molecule in the total RNA is less than the corresponding “RNA fraction” in formula (i), such as being less than the corresponding “RNA fraction” in formula (i) by more than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20%. In one implementation, the concentration of a given RNA molecule is considered to be above a reference level when the fraction of the given RNA molecule in the total RNA is greater than the corresponding “RNA fraction” in formula (i), such as being greater than the corresponding “RNA fraction” in formula (i) by more than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20%.

[0093] In one embodiment, the method of the present invention further includes determining that the concentration of one or more RNA molecules in the composition is lower than a reference level, and accordingly increasing the concentration of the corresponding DNA molecule by a correction factor. In one embodiment, increasing the concentration by the correction factor includes multiplying the initial DNA concentration by a value greater than 1. In one embodiment, the method of the present invention further includes determining that the concentration of one or more RNA molecules in the composition is higher than a reference level, and accordingly decreasing the concentration of the corresponding DNA molecule by a correction factor. In one embodiment, decreasing the concentration by the correction factor includes multiplying the initial DNA concentration by a value less than 1 and greater than 0.

[0094] The concentration of RNA molecules can be determined by any means known to those skilled in the art. In one embodiment, the concentration of RNA molecules is determined by UV spectroscopy, for example using a NanoDrop spectrophotometer. In one embodiment, the concentration of RNA molecules is determined by a quantitative method based on fluorescent dyes. In one embodiment, the concentration of RNA molecules is determined by gel electrophoresis and staining intensity analysis. In one embodiment, such methods are used to determine the concentration of each different RNA molecule.

[0095] In this article, references to ATP, CTP, GTP, and TTP concentrations refer to molar concentrations, or the number of moles per unit volume.

[0096] In one embodiment, the reaction mixture contains an ATP concentration of at least 6 mM, preferably at least 9 mM, preferably at least 10 mM, preferably at least 11 mM, preferably at least 12 mM, preferably at least 13 mM, preferably at least 14 mM. In such embodiments, one or more RNA molecules that can be obtained from the DNA molecules in the reaction mixture have a high A fraction, such as an A fraction greater than 25%.

[0097] In one embodiment, the reaction mixture contains a CTP concentration of at least 6 mM, preferably at least 9 mM, preferably at least 10 mM, preferably at least 11 mM, preferably at least 12 mM, preferably at least 13 mM, preferably at least 14 mM. In such embodiments, one or more RNA molecules that can be obtained from the DNA molecules in the reaction mixture have a high C fraction, such as a C fraction greater than 25%.

[0098] In one embodiment, the reaction mixture contains a GTP concentration of at least 6 mM, preferably at least 9 mM, preferably at least 10 mM, preferably at least 11 mM, preferably at least 12 mM, preferably at least 13 mM, preferably at least 14 mM. In such embodiments, one or more RNA molecules that can be obtained from the DNA molecules in the reaction mixture have a high T fraction, such as a T fraction greater than 25%.

[0099] In one embodiment, the reaction mixture contains a TTP concentration of at least 6 mM, preferably at least 9 mM, preferably at least 10 mM, preferably at least 11 mM, preferably at least 12 mM, preferably at least 13 mM, preferably at least 14 mM. In such embodiments, one or more RNA molecules that can be obtained from the DNA molecules in the reaction mixture have a high T fraction, such as a T fraction greater than 25%.

[0100] In one embodiment, the reaction mixture comprises CTP and ATP concentrations of at least 6 mM, preferably at least 9 mM, preferably at least 10 mM, preferably at least 11 mM, preferably at least 12 mM, preferably at least 13 mM, and preferably at least 14 mM, respectively. In such embodiments, the RNA molecule has a high C and A fraction, such as a C and A fraction greater than 25%.

[0101] In one embodiment, the reaction mixture comprises GTP and ATP concentrations of at least 6 mM, preferably at least 9 mM, preferably at least 10 mM, preferably at least 11 mM, preferably at least 12 mM, preferably at least 13 mM, and preferably at least 14 mM, respectively. In such embodiments, one or more RNA molecules that can be obtained from the DNA molecules in the reaction mixture have high G and A fractions, such as greater than 25%.

[0102] In one embodiment, the reaction mixture comprises TTP and ATP concentrations of at least 6 mM, preferably at least 9 mM, preferably at least 10 mM, preferably at least 11 mM, preferably at least 12 mM, preferably at least 13 mM, and preferably at least 14 mM, respectively. In such embodiments, one or more RNA molecules that can be obtained from the DNA molecules in the reaction mixture have high T and ATP fractions, such as greater than 25%.

[0103] In one embodiment, the reaction mixture comprises GTP and CTP concentrations of at least 6 mM, preferably at least 9 mM, preferably at least 10 mM, preferably at least 11 mM, preferably at least 12 mM, preferably at least 13 mM, and preferably at least 14 mM, respectively. In this type of embodiment, one or more RNA molecules that can be obtained from the DNA molecules in the reaction mixture have high G and C fractions, such as greater than 25%.

[0104] In one embodiment, the reaction mixture comprises TTP and CTP concentrations of at least 6 mM, preferably at least 9 mM, preferably at least 10 mM, preferably at least 11 mM, preferably at least 12 mM, preferably at least 13 mM, and preferably at least 14 mM, respectively. In this type of embodiment, one or more RNA molecules that can be obtained from the DNA molecules in the reaction mixture have high C and A fractions, such as T and C fractions greater than 25%.

[0105] In one embodiment, the reaction mixture comprises TTP and GTP concentrations of at least 6 mM, preferably at least 9 mM, preferably at least 10 mM, preferably at least 11 mM, preferably at least 12 mM, preferably at least 13 mM, and preferably at least 14 mM, respectively. In this type of embodiment, one or more RNA molecules that can be obtained from the DNA molecules in the reaction mixture have high T and G fractions, such as greater than 25%.

[0106] In this paper, the A / C / G / T fraction refers to the percentage of nucleobases in a nucleic acid molecule that are A, C, G, and T bases, respectively. Modified bases are still considered as bases of that type; for example, a methyl-C base is still considered a C.

[0107] In one implementation, step b) is performed for at least 105 minutes, at least 180 minutes, or 105 to 180 minutes (including the end value).

[0108] Specific implementation plan and application

[0109] This invention can be particularly used to provide a predetermined proportion of RNA molecules in the prepared composition.

[0110] In one embodiment, the present invention is used to produce a composition comprising two RNA molecules (“RNA1” and “RNA2”). In one embodiment, the method is used to produce a composition comprising RNA1:RNA2 in a ratio of 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, or 1:10.

[0111] In one embodiment, the present invention is used to produce a composition comprising three RNA molecules (“RNA1”, “RNA2”, and “RNA3”). In one implementation, the method is used to produce RNA1:RNA2:RNA3 in ratios of 1:1:1, 1:1:2, 1:1:3, 1:1:4, 1:1:5, 1:1:6, 1:1:7, 1:1:8, 1:1:9, 1:1:10, 1:2:1, 1:2:2, 1:2:3, 1:2:4, 1:2:5, 1:2:6, 1:2:7, 1:2:8, 1:2:9, 1:2:10, 1:3:1, 1:3:2, 1:3:3, 1:3:4, 1:3:5, 1:3:6, 1:3:7, 1:3:8, 1:3:9, 1:3:10, 1:4:1, 1:4:2, 1:4:3, 1:4:4, 1:4:5, 1:4:6, 1:4:7, 1:4:8, 1:4:9, 1:4:10, 1:5:1, 1:5:2, 1:5:3, 1:5:4, 1:5:5, 1:5:6, 1:5:7, 1:5:8, 1:5:9, 1:5:10, 1:6:1, 1:6:2, 1:6:3, 1:6:4, 1:6:5, 1:6:6, 1:6:7, 1:6:8, 1:6:9, 1:6:10, 1:7:1, 1:7:2, 1:7:3, 1:7:4, 1:7:5, 1:7:6, 1:7:7, Compositions of 1:7:8, 1:7:9, 1:7:10, 1:8:1, 1:8:2, 1:8:3, 1:8:4, 1:8:5, 1:8:6, 1:8:7, 1:8:8, 1:8:9, 1:8:10, 1:9:1, 1:9:2, 1:9:3, 1:9:4, 1:9:5, 1:9:6, 1:9:7, 1:9:8, 1:9:9, 1:9:10, 1:10:1, 1:10:2, 1:10:3, 1:10:4, 1:10:5, 1:10:6, 1:10:7, 1:10:8, 1:10:9, or 1:10:10.

[0112] In one embodiment, the present invention is used to produce a composition comprising four RNA molecules (“RNA1”, “RNA2”, “RNA3”, and “RNA4”). In one implementation, the method is used to produce RNA1:RNA2:RNA3:RNA4 in a ratio of 1:1:1:1, 1:1:1:2, 1:1:1:3, 1:1:1:4, 1:1:1:5, 1:1:1:6, 1:1:1:7, 1:1:1:8, 1:1:1:9, 1:1:1:10, 1:1:2:1, 1:1:2:2, 1:1:2:3, 1:1:2:4, 1:1:2:5, 1:1:2:6, 1:1:2:7, 1:1:2:8, 1:1:2:9, 1:1:2:10, 1:1:3:1, 1:1:3:2, 1:1:3:3, 1:1:3:4, 1:1:3:5, 1:1:3:6, 1:1:3:7, 1:1:3:8, 1:1:3:9, 1:1:3:10, 1:1:4:1, 1:1:4:2, 1:1:4:3, 1:1:4:4,1:1:4:5, 1:1:4:6, 1:1:4:7, 1:1:4:8, 1:1:4:9, 1:1:4:10, 1:1:5:1, 1:1:5:2, 1:1:5:3, 1:1:5:4, 1:1:5:5, 1:1:5:6, 1:1:5:7, 1:1:5:8, 1:1:5:9, 1:1:5:10, 1:1:6:1,1:1:6:2, 1:1:6:3, 1:1:6:4, 1:1:6:5, 1:1:6:6, 1:1:6:7, 1:1:6:8, 1:1:6:9, 1:1:6:10, 1:1:7:1, 1:1:7:2, 1:1:7:3, 1:1:7:4, 1:1:7:5, 1:1:7:6, 1:1:7:7, 1:1:7:8, 1:1:7:9, 1:1:7:10, 1:1:8:1, 1:1:8:2, 1:1:8:3, 1:1:8:4, 1:1:8:5, 1:1:8:6, 1:1:8:7, 1:1:8:8, 1:1:8:9, 1:1:8:10, 1:1:9:1, 1:1:9:2, 1:1:9:3, 1:1:9:4, 1:1:9:5, 1:1:9:6, 1:1:9:7, 1:1:9:8, 1:1:9:9, 1:1:9:10, 1:1:10:1, 1:1:10:2, 1:1:10:3, 1:1:10:4, 1:1:10:5, 1:1:10:6, 1:1:10:7, 1:1:10:8, 1:1:10:9,1:1:10:10, 1:2:1:1, 1:2:1:2, 1:2:1:3, 1:2:1:4, 1:2:1:5, 1:2:1:6, 1:2:1:7, 1:2:1:8, 1:2:1:9, 1:2:1:10, 1:2:2:1, 1:2:2:2, 1:2:2:3, 1:2:2:4, 1:2:2:5, 1:2:2:6, 1:2:2:7, 1:2:2:8, 1:2:2:9, 1:2:2:10, 1:2:3:1, 1:2:3:2, 1:2:3:3, 1:2:3:4, 1:2:3:5, 1:2:3:6,1:2:3:7, 1:2:3:8, 1:2:3:9, 1:2:3:10, 1:2:4:1, 1:2:4:2, 1:2:4:3, 1:2:4:4, 1:2:4:5, 1:2:4:6, 1:2:4:7, 1:2:4:8, 1:2:4:9, 1:2:4:10, 1:2:5:1, 1:2:5:2, 1:2:5:3,1:2:5:4, 1:2:5:5, 1:2:5:6, 1:2:5:7, 1:2:5:8, 1:2:5:9, 1:2:5:10, 1:2:6:1, 1:2:6:2, 1:2:6:3, 1:2:6:4, 1:2:6:5, 1:2:6:6, 1:2:6:7, 1:2:6:8, 1:2:6:9, 1:2:6:10,1:2:7:1, 1:2:7:2, 1:2:7:3, 1:2:7:4, 1:2:7:5, 1:2:7:6, 1:2:7:7, 1:2:7:8, 1:2:7:9, 1:2:7:10, 1:2:8:1, 1:2:8:2, 1:2:8:3, 1:2:8:4, 1:2:8:5, 1:2:8:6, 1:2:8:7,1:2:8:8, 1:2:8:9, 1:2:8:10, 1:2:9:1, 1:2:9:2, 1:2:9:3, 1:2:9:4, 1:2:9:5, 1:2:9:6, 1:2:9:7, 1:2:9:8, 1:2:9:9, 1:2:9:10, 1:2:10:1, 1:2:10:2, 1:2:10:3, 1:2:10:4, 1:2:10:5, 1:2:10:6, 1:2:10:7, 1:2:10:8, 1:2:10:9, 1:2:10:10, 1:3:1:1,1:3:1:2, 1:3:1:3, 1:3:1:4, 1:3:1:5, 1:3:1:6, 1:3:1:7, 1:3:1:8,1:3:1:9, 1:3:1:10, 1:3:2:1, 1:3:2:2, 1:3:2:3, 1:3:2:4, 1:3:2:5, 1:3:2:6, 1:3:2:7, 1:3:2:8,1:3:2:9, 1:3:2:10, 1:3:3:1, 1:3:3:2, 1:3:3:3, 1:3:3:4, 1:3:3:5, 1:3:3:6, 1:3:3:7, 1:3:3:8, 1:3:3:9, 1:3:3:10, 1:3:4:1, 1:3:4:2, 1:3:4:3, 1:3:4:4, 1:3:4:5,1:3:4:6, 1:3:4:7, 1:3:4:8, 1:3:4:9, 1:3:4:10, 1:3:5:1, 1:3:5:2, 1:3:5:3, 1:3:5:4, 1:3:5:5, 1:3:5:6, 1:3:5:7, 1:3:5:8, 1:3:5:9, 1:3:5:10, 1:3:6:1, 1:3:6:2,1:3:6:3, 1:3:6:4, 1:3:6:5, 1:3:6:6, 1:3:6:7, 1:3:6:8, 1:3:6:9, 1:3:6:10, 1:3:7:1, 1:3:7:2, 1:3:7:3, 1:3:7:4, 1:3:7:5, 1:3:7:6, 1:3:7:7, 1:3:7:8, 1:3:7:9,1:3:7:10, 1:3:8:1, 1:3:8:2, 1:3:8:3, 1:3:8:4, 1:3:8:5, 1:3:8:6, 1:3:8:7, 1:3:8:8, 1:3:8:9, 1:3:8:10, 1:3:9:1, 1:3:9:2, 1:3:9:3, 1:3:9:4, 1:3:9:5, 1:3:9:6,1:3:9:7, 1:3:9:8, 1:3:9:9, 1:3:9:10, 1:3:10:1, 1:3:10:2, 1:3:10:3, 1:3:10:4,1:3:10:5, 1:3:10:6, 1:3:10:7, 1:3:10:8, 1:3:10:9, 1:3:10:10, 1:4:1:1, 1:4:1:2, 1:4:1:3, 1:4:1:4, 1:4:1:5, 1:4:1:6, 1:4:1:7, 1:4:1:8, 1:4:1:9, 1:4:1:10,1:4:2:1, 1:4:2:2, 1:4:2:3, 1:4:2:4, 1:4:2:5, 1:4:2:6, 1:4:2:7,1:4:2:8, 1:4:2:9, 1:4:2:10, 1:4:3:1, 1:4:3:2, 1:4:3:3, 1:4:3:4, 1:4:3:5, 1:4:3:6, 1:4:3:7,1:4:3:8, 1:4:3:9, 1:4:3:10, 1:4:4:1, 1:4:4:2, 1:4:4:3, 1:4:4:4, 1:4:4:5, 1:4:4:6, 1:4:4:7, 1:4:4:8, 1:4:4:9, 1:4:4:10, 1:4:5:1, 1:4:5:2, 1:4:5:3, 1:4:5:4,1:4:5:5, 1:4:5:6, 1:4:5:7, 1:4:5:8, 1:4:5:9, 1:4:5:10, 1:4:6:1, 1:4:6:2, 1:4:6:3, 1:4:6:4, 1:4:6:5, 1:4:6:6, 1:4:6:7, 1:4:6:8, 1:4:6:9, 1:4:6:10, 1:4:7:1,1:4:7:2, 1:4:7:3, 1:4:7:4, 1:4:7:5, 1:4:7:6, 1:4:7:7, 1:4:7:8, 1:4:7:9, 1:4:7:10, 1:4:8:1, 1:4:8:2, 1:4:8:3, 1:4:8:4, 1:4:8:5, 1:4:8:6, 1:4:8:7, 1:4:8:8,1:4:8:9, 1:4:8:10, 1:4:9:1, 1:4:9:2, 1:4:9:3, 1:4:9:4, 1:4:9:5, 1:4:9:6, 1:4:9:7, 1:4:9:8, 1:4:9:9, 1:4:9:10, 1:4:10:1, 1:4:10:2, 1:4:10:3, 1:4:10:4, 1:4:10:5, 1:4:10:6, 1:4:10:7, 1:4:10:8, 1:4:10:9, 1:4:10:10, 1:5:1:1, 1:5:1:2, 1:5:1:3, 1:5:1:4, 1:5:1:5, 1:5:1:6, 1:5:1:7, 1:5:1:8, 1:5:1:9, 1:5:1:10, 1:5:2:1, 1:5:2:2, 1:5:2:3, 1:5:2:4, 1:5:2:5, 1:5:2:6, 1:5:2:7, 1:5:2:8, 1:5:2:9, 1:5:2:10, 1:5:3:1, 1:5:3:2, 1:5:3:3, 1:5:3:4, 1:5:3:5, 1:5:3:6,1:5:3:7, 1:5:3:8, 1:5:3:9, 1:5:3:10, 1:5:4:1, 1:5:4:2, 1:5:4:3, 1:5:4:4, 1:5:4:5, 1:5:4:6,1:5:4:7, 1:5:4:8, 1:5:4:9, 1:5:4:10, 1:5:5:1, 1:5:5:2, 1:5:5:3, 1:5:5:4, 1:5:5:5, 1:5:5:6, 1:5:5:7, 1:5:5:8, 1:5:5:9, 1:5:5:10, 1:5:6:1, 1:5:6:2, 1:5:6:3,1:5:6:4, 1:5:6:5, 1:5:6:6, 1:5:6:7, 1:5:6:8, 1:5:6:9, 1:5:6:10, 1:5:7:1, 1:5:7:2, 1:5:7:3, 1:5:7:4, 1:5:7:5, 1:5:7:6, 1:5:7:7, 1:5:7:8, 1:5:7:9, 1:5:7:10,1:5:8:1, 1:5:8:2, 1:5:8:3, 1:5:8:4, 1:5:8:5, 1:5:8:6, 1:5:8:7, 1:5:8:8, 1:5:8:9, 1:5:8:10, 1:5:9:1, 1:5:9:2, 1:5:9:3, 1:5:9:4, 1:5:9:5, 1:5:9:6, 1:5:9:7,1:5:9:8, 1:5:9:9, 1:5:9:10, 1:5:10:1, 1:5:10:2, 1:5:10:3, 1:5:10:4, 1:5:10:5,1:5:10:6, 1:5:10:7, 1:5:10:8, 1:5:10:9, 1:5:10:10, 1:6:1:1, 1:6:1:2, 1:6:1:3,1:6:1:4, 1:6:1:5, 1:6:1:6, 1:6:1:7, 1:6:1:8, 1:6:1:9, 1:6:1:10, 1:6:2:1, 1:6:2:2, 1:6:2:3, 1:6:2:4, 1:6:2:5, 1:6:2:6, 1:6:2:7, 1:6:2:8, 1:6:2:9, 1:6:2:10,1:6:3:1, 1:6:3:2, 1:6:3:3, 1:6:3:4, 1:6:3:5, 1:6:3:6, 1:6:3:7, 1:6:3:8, 1:6:3:9, 1:6:3:10, 1:6:4:1, 1:6:4:2, 1:6:4:3, 1:6:4:4, 1:6:4:5,1:6:4:6, 1:6:4:7,1:6:4:8, 1:6:4:9, 1:6:4:10, 1:6:5:1, 1:6:5:2, 1:6:5:3, 1:6:5:4, 1:6:5:5, 1:6:5:6, 1:6:5:7, 1:6:5:8, 1:6:5:9, 1:6:5:10, 1:6:6:1, 1:6:6:2, 1:6:6:3, 1:6:6:4,1:6:6:5, 1:6:6:6, 1:6:6:7, 1:6:6:8, 1:6:6:9, 1:6:6:10, 1:6:7:1, 1:6:7:2, 1:6:7:3, 1:6:7:4, 1:6:7:5, 1:6:7:6, 1:6:7:7, 1:6:7:8, 1:6:7:9, 1:6:7:10, 1:6:8:1,1:6:8:2, 1:6:8:3, 1:6:8:4, 1:6:8:5, 1:6:8:6, 1:6:8:7, 1:6:8:8, 1:6:8:9, 1:6:8:10, 1:6:9:1, 1:6:9:2, 1:6:9:3, 1:6:9:4, 1:6:9:5, 1:6:9:6, 1:6:9:7, 1:6:9:8,1:6:9:9, 1:6:9:10, 1:6:10:1, 1:6:10:2, 1:6:10:3, 1:6:10:4, 1:6:10:5, 1:6:10:6, 1:6:10:7, 1:6:10:8, 1:6:10:9, 1:6:10:10, 1:7:1:1, 1:7:1:2, 1:7:1:3, 1:7:1:4, 1:7:1:5, 1:7:1:6, 1:7:1:7, 1:7:1:8, 1:7:1:9, 1:7:1:10, 1:7:2:1, 1:7:2:2,1:7:2:3, 1:7:2:4, 1:7:2:5, 1:7:2:6, 1:7:2:7, 1:7:2:8, 1:7:2:9, 1:7:2:10, 1:7:3:1, 1:7:3:2, 1:7:3:3, 1:7:3:4, 1:7:3:5, 1:7:3:6, 1:7:3:7, 1:7:3:8, 1:7:3:9,1:7:3:10, 1:7:4:1, 1:7:4:2, 1:7:4:3, 1:7:4:4, 1:7:4:5, 1:7:4:6, 1:7:4:7, 1:7:4:8, 1:7:4:9, 1:7:4:10, 1:7:5:1, 1:7:5:2, 1:7:5:3, 1:7:5:4,1:7:5:5, 1:7:5:6,1:7:5:7, 1:7:5:8, 1:7:5:9, 1:7:5:10, 1:7:6:1, 1:7:6:2, 1:7:6:3, 1:7:6:4, 1:7:6:5, 1:7:6:6, 1:7:6:7, 1:7:6:8, 1:7:6:9, 1:7:6:10, 1:7:7:1, 1:7:7:2, 1:7:7:3,1:7:7:4, 1:7:7:5, 1:7:7:6, 1:7:7:7, 1:7:7:8, 1:7:7:9, 1:7:7:10, 1:7:8:1, 1:7:8:2, 1:7:8:3, 1:7:8:4, 1:7:8:5, 1:7:8:6, 1:7:8:7, 1:7:8:8, 1:7:8:9, 1:7:8:10,1:7:9:1, 1:7:9:2, 1:7:9:3, 1:7:9:4, 1:7:9:5, 1:7:9:6, 1:7:9:7, 1:7:9:8, 1:7:9:9, 1:7:9:10, 1:7:10:1, 1:7:10:2, 1:7:10:3, 1:7:10:4, 1:7:10:5, 1:7:10:6, 1:7:10:7, 1:7:10:8, 1:7:10:9, 1:7:10:10, 1:8:1:1, 1:8:1:2, 1:8:1:3, 1:8:1:4, 1:8:1:5, 1:8:1:6, 1:8:1:7, 1:8:1:8, 1:8:1:9, 1:8:1:10, 1:8:2:1, 1:8:2:2, 1:8:2:3, 1:8:2:4, 1:8:2:5, 1:8:2:6, 1:8:2:7, 1:8:2:8, 1:8:2:9, 1:8:2:10, 1:8:3:1,1:8:3:2, 1:8:3:3, 1:8:3:4, 1:8:3:5, 1:8:3:6, 1:8:3:7, 1:8:3:8, 1:8:3:9, 1:8:3:10, 1:8:4:1, 1:8:4:2, 1:8:4:3, 1:8:4:4, 1:8:4:5, 1:8:4:6, 1:8:4:7, 1:8:4:8,1:8:4:9, 1:8:4:10, 1:8:5:1, 1:8:5:2, 1:8:5:3, 1:8:5:4, 1:8:5:5, 1:8:5:6, 1:8:5:7, 1:8:5:8, 1:8:5:9, 1:8:5:10, 1:8:6:1, 1:8:6:2, 1:8:6:3,1:8:6:4, 1:8:6:5,1:8:6:6, 1:8:6:7, 1:8:6:8, 1:8:6:9, 1:8:6:10, 1:8:7:1, 1:8:7:2, 1:8:7:3, 1:8:7:4, 1:8:7:5, 1:8:7:6, 1:8:7:7, 1:8:7:8, 1:8:7:9, 1:8:7:10, 1:8:8:1, 1:8:8:2,1:8:8:3, 1:8:8:4, 1:8:8:5, 1:8:8:6, 1:8:8:7, 1:8:8:8, 1:8:8:9, 1:8:8:10, 1:8:9:1, 1:8:9:2, 1:8:9:3, 1:8:9:4, 1:8:9:5, 1:8:9:6, 1:8:9:7, 1:8:9:8, 1:8:9:9,1:8:9:10, 1:8:10:1, 1:8:10:2, 1:8:10:3, 1:8:10:4, 1:8:10:5, 1:8:10:6, 1:8:10:7, 1:8:10:8, 1:8:10:9, 1:8:10:10, 1:9:1:1, 1:9:1:2, 1:9:1:3, 1:9:1:4, 1:9:1:5, 1:9:1:6, 1:9:1:7, 1:9:1:8, 1:9:1:9, 1:9:1:10, 1:9:2:1, 1:9:2:2, 1:9:2:3,1:9:2:4, 1:9:2:5, 1:9:2:6, 1:9:2:7, 1:9:2:8, 1:9:2:9, 1:9:2:10, 1:9:3:1, 1:9:3:2, 1:9:3:3, 1:9:3:4, 1:9:3:5, 1:9:3:6, 1:9:3:7, 1:9:3:8, 1:9:3:9, 1:9:3:10,1:9:4:1, 1:9:4:2, 1:9:4:3, 1:9:4:4, 1:9:4:5, 1:9:4:6, 1:9:4:7, 1:9:4:8, 1:9:4:9, 1:9:4:10, 1:9:5:1, 1:9:5:2, 1:9:5:3, 1:9:5:4, 1:9:5:5, 1:9:5:6, 1:9:5:7,1:9:5:8, 1:9:5:9, 1:9:5:10, 1:9:6:1, 1:9:6:2, 1:9:6:3, 1:9:6:4, 1:9:6:5, 1:9:6:6, 1:9:6:7, 1:9:6:8, 1:9:6:9, 1:9:6:10, 1:9:7:1, 1:9:7:2,1:9:7:3, 1:9:7:4,1:9:7:5, 1:9:7:6, 1:9:7:7, 1:9:7:8, 1:9:7:9, 1:9:7:10, 1:9:8:1, 1:9:8:2, 1:9:8:3, 1:9:8:4, 1:9:8:5, 1:9:8:6, 1:9:8:7, 1:9:8:8, 1:9:8:9, 1:9:8:10, 1:9:9:1,1:9:9:2, 1:9:9:3, 1:9:9:4, 1:9:9:5, 1:9:9:6, 1:9:9:7, 1:9:9:8, 1:9:9:9, 1:9:9:10, 1:9:10:1, 1:9:10:2, 1:9:10:3, 1:9:10:4, 1:9:10:5, 1:9:10:6, 1:9:10:7,1:9:10:8, 1:9:10:9, 1:9:10:10, 1:10:1:1, 1:10:1:2, 1:10:1:3, 1:10:1:4, 1:10:1:5, 1:10:1:6, 1:10:1:7, 1:10:1:8, 1:10:1:9, 1:10:1:10, 1:10:2:1, 1:10:2:2,1:10:2:3, 1:10:2:4, 1:10:2:5, 1:10:2:6, 1:10:2:7, 1:10:2:8, 1:10:2:9, 1:10:2:10, 1:10:3:1, 1:10:3:2, 1:10:3:3, 1:10:3:4, 1:10:3:5, 1:10:3:6, 1:10:3:7, 1:10:3:8, 1:10:3:9, 1:10:3:10, 1:10:4:1, 1:10:4:2, 1:10:4:3, 1:10:4:4, 1:10:4:5, 1:10:4:6, 1:10:4:7, 1:10:4:8, 1:10:4:9, 1:10:4:10, 1:10:5:1, 1:10:5:2, 1:10:5:3, 1:10:5:4, 1:10:5:5, 1:10:5:6, 1:10:5:7, 1:10:5:8, 1:10:5:9, 1:10:5:10, 1:10:6:1, 1:10:6:2, 1:10:6:3, 1:10:6:4, 1:10:6:5, 1:10:6:6, 1:10:6:7, 1:10:6:8, 1:10:6:9, 1:10:6:10, 1:10:7:1, 1:10:7:2, 1:10:7:3, 1:10:7:4,1:10:7:5, 1:10:7:6, 1:10:7:7, 1:10:7:8, 1:10:7:9, 1:10:7:10, 1:10:8:1, 1:10:8:2, 1:10:8:3, 1:10:8:4, 1:10:8:5, 1:10:8:6, 1:10:8:7, 1:10:8:8, 1:10:8:9, 1:10:8:10, 1:10:9:1, 1:10:9:2, 1:10:9:3, 1:10:9:4, 1:10:9:5, 1:10:9:6, 1:10:9:7, 1:10:9:8, Compositions of 1:10:9:9, 1:10:9:10, 1:10:10:1, 1:10:10:2, 1:10:10:3, 1:10:10:4, 1:10:10:5, 1:10:10:6, 1:10:10:7, 1:10:10:8, 1:10:10:9, or 1:10:10:10.

[0113] In one particularly suitable embodiment of the invention, the prepared composition contains at least two different RNA molecules in varying amounts. Therefore, in one particularly suitable embodiment of the invention, the ratio of the different RNA molecules prepared is not 1:1, 1:1:1, 1:1:1:1, 1:1:1:1:1, 1:1:1:1:1:1, 1:1:1:1:1:1:1, 1:1:1:1:1:1:1:1:1:1, 1:1:1:1:1:1:1:1:1:1:1:1:1:1:1:1:1:1:1:1:1:1:1:1:1:1:1:1:1:1:1:1:1:1:1:1:1:1:1:1:1:1:1:1:1:1:1:1:1:1:1:1.

[0114] When the DNA molecules from which RNA molecules are obtained have a large length difference, the present invention is also particularly suitable for providing desired compositions containing two or more RNA molecules.

[0115] In one embodiment, the bp lengths of at least two DNA molecules differ by more than 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 300%, or 400%. In another embodiment, the length of at least one DNA molecule is two, three, or four times that of at least one different DNA molecule.

[0116] Therefore, the present invention is also particularly suitable for providing desired compositions containing two or more RNA molecules when the DNA molecule from which the RNA molecule is derived contains a long non-coding sequence and thus inherently has a high probability of length variation.

[0117] In one embodiment, one or more DNA molecules contain at least 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, or 3000 bp of non-coding sequence. In one embodiment, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the total length of one or more DNA molecules contains non-coding sequence.

[0118] When RNA molecules have significant length differences, the present invention is also particularly suitable for providing desired compositions containing two or more RNA molecules.

[0119] In one embodiment, the bp lengths of at least two RNA molecules differ by more than 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%. In another embodiment, the length of at least one RNA molecule is two, three, or four times that of at least one different RNA molecule.

[0120] In one embodiment, the concentrations of the at least two DNA templates used are not equal. In another embodiment, the concentrations of the at least two DNA templates used differ from the average concentration of the DNA templates by more than 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%.

[0121] Further aspects of the invention

[0122] In a second aspect, the present invention provides a method for preparing a composition comprising at least a first RNA molecule and a second RNA molecule, wherein the first RNA molecule is obtainable from a first DNA molecule and the second RNA molecule is obtainable from a second DNA molecule, wherein the method comprises:

[0123] a) Provide a reaction mixture comprising a first DNA molecule and a second DNA molecule; and

[0124] b) Obtain the first RNA molecule and the second RNA molecule from the first DNA molecule and the second DNA molecule, respectively;

[0125] The first RNA molecule has a larger relative mass than the second RNA molecule, and the molar amount of the second RNA molecule obtained from the second DNA molecule is greater than the molar amount of the first RNA molecule obtained from the first DNA molecule, such that the first RNA molecule and the second RNA molecule are obtained in the composition at substantially the same concentration.

[0126] In an alternative to the second aspect, the present invention provides a method for preparing a composition comprising at least a first RNA molecule and a second RNA molecule, wherein the first RNA molecule is obtainable from a first DNA molecule and the second RNA molecule is obtainable from a second DNA molecule, wherein the method comprises:

[0127] a) Provide a reaction mixture comprising a first DNA molecule and a second DNA molecule; and

[0128] b) Obtain the first RNA molecule and the second RNA molecule from the first DNA molecule and the second DNA molecule, respectively;

[0129] The first RNA molecule has a different relative mass than the second RNA molecule, and the molar amount of the second RNA molecule obtained from the second DNA molecule is different from the molar amount of the first RNA molecule obtained from the first DNA molecule, such that the first RNA molecule and the second RNA molecule are obtained in the composition at concentrations varying according to a predetermined ratio as described herein.

[0130] In an alternative to the second aspect, the present invention provides a method for preparing a composition comprising at least a first RNA molecule and a second RNA molecule, wherein the first RNA molecule is obtainable from a first DNA molecule and the second RNA molecule is obtainable from a second DNA molecule, wherein the method comprises:

[0131] a) Provide a reaction mixture comprising a first DNA molecule and a second DNA molecule; and

[0132] b) Obtain the first RNA molecule and the second RNA molecule from the first DNA molecule and the second DNA molecule, respectively;

[0133] The first DNA molecule has a larger relative mass than the second DNA molecule, and the first DNA molecule is present in the reaction mixture at a mass concentration greater than that of the second DNA molecule, such that the molar amounts of the first DNA molecule and the second DNA molecule are substantially the same, and the first RNA molecule and the second RNA molecule are obtained in the composition at substantially the same molar amounts.

[0134] In an alternative to the second aspect, the present invention provides a method for preparing a composition comprising at least a first RNA molecule and a second RNA molecule, wherein the first RNA molecule is obtainable from a first DNA molecule and the second RNA molecule is obtainable from a second DNA molecule, wherein the method comprises:

[0135] a) Provide a reaction mixture comprising a first DNA molecule and a second DNA molecule; and

[0136] b) Obtain the first RNA molecule and the second RNA molecule from the first DNA molecule and the second DNA molecule, respectively;

[0137] The first DNA molecule has a different relative mass than the second DNA molecule, and the first DNA molecule is present in the reaction mixture at a different mass concentration than the second DNA molecule, such that the molar amounts of the first DNA molecule and the second DNA molecule are varied in a predetermined ratio as described herein, and the first RNA molecule and the second RNA molecule are obtained in the composition at molar amounts varied in a predetermined ratio as described herein.

[0138] "Greater relative mass" refers to the mass of a single RNA molecule itself, which depends in particular on its composition and length.

[0139] In a third aspect, the present invention provides compositions obtained or available by the methods of the present invention. In one embodiment, the composition is a pharmaceutical composition. In one embodiment, the composition comprises a pharmaceutically acceptable carrier, adjuvant, and / or diluent. In one embodiment, the composition is a vaccine composition. In one embodiment, the composition is a multivalent vaccine composition.

[0140] In a fourth aspect, the present invention provides compositions that are obtained or available by the method of the present invention, which are used as pharmaceuticals.

[0141] In a fifth aspect, the present invention provides a composition comprising two or more different DNA molecules, wherein different RNA molecules can be obtained from each different DNA molecule, wherein each different DNA molecule (X) has a concentration according to formula (i):

[0142] (i)

[0143] in:

[0144] “c(DNAx)” represents the concentration of a given DNA molecule X;

[0145] c(DNA) 总 ")" represents the total concentration of all DNA molecules;

[0146] "RNA fraction" is the fraction of all RNA molecules that can be obtained from a reaction mixture containing RNA molecules that can be obtained from said DNA molecule X;

[0147] "DNAx length fraction" is the base pair (bp) length of the DNA molecule X divided by the total bp length of all different DNA molecules;

[0148] "RNAx length fraction" is the bp length of the RNA molecule that can be obtained from the DNA molecule X divided by the total bp length of all different RNA molecules.

[0149] In one embodiment, the composition comprising two or more different DNA molecules is a reaction mixture composition.

[0150] Other definitions and descriptions

[0151] Definition of general terms

[0152] Unless otherwise stated, the practice of this disclosure will employ conventional chemical, biochemical, cell biological, immunological, and recombinant DNA techniques as explained in the literature in this field.

[0153] In this specification and its appended claims, unless the context otherwise requires, the word "comprising" and its variations, such as "including" and "containing," should be understood to imply the inclusion of the stated features, elements, members, integers, or steps, or groups of features, elements, members, integers, or steps, but do not exclude any other features, elements, members, integers, or steps, or groups of features, elements, members, integers, or steps. The term "consistently composed of" limits the scope of the claim or disclosure to the specified features, elements, members, integers, or steps, as well as those features, elements, members, integers, or steps that do not materially affect the essential and novel characteristics of the claim or disclosure. The term "composed of" limits the scope of the claim or disclosure to the specified features, elements, members, integers, or steps. The term "comprising" encompasses the term "consistently composed of," and "consistently composed of" in turn encompasses the term "composed of." Therefore, in this application, whenever the term "comprising" appears, it can be replaced by the terms "consistently composed of" or "composed of." Similarly, in this application, whenever the term "consistently composed of..." appears, it may be replaced with the term "composed of...".

[0154] Unless otherwise stated herein or the context clearly provides otherwise, the terms “a” and “the” as used in the context of describing this disclosure, and similar designations (particularly in the context of the claims), shall be construed as covering both the singular and the plural.

[0155] Unless otherwise stated herein or the context clearly specifies otherwise, all methods described herein may be performed in any suitable order.

[0156] Any or all examples or exemplary language (such as "such as") provided herein are intended only to better illustrate this disclosure and do not constitute a limitation on the scope of protection claimed herein. No language in the specification should be construed as indicating that any unclaimed element is essential to the implementation of this disclosure.

[0157] As used herein, the terms “optional” or “optionally” mean that an event, situation, or condition described below may or may not occur, and the description includes both the possibility that the event, situation, or condition may occur and the possibility that it may not occur.

[0158] When used herein, “and / or” should be understood as a specific disclosure of each of the two specified features or components, regardless of whether the other is included. For example, “X and / or Y” should be understood as a specific disclosure of each of (i) X, (ii) Y, and (iii) X and Y, as if they were each listed separately herein.

[0159] In the context of this disclosure, the term "about" refers to a range of precision understood by those skilled in the art, while ensuring the technical effect of the features in question. This term typically indicates a deviation from the shown value of ±10%, ±5%, ±4%, ±3%, ±2%, ±1%, ±0.9%, ±0.8%, ±0.7%, ±0.6%, ±0.5%, ±0.4%, ±0.3%, ±0.2%, ±0.1%, ±0.05%, for example, ±0.01%. In some embodiments, "about" indicates a deviation from the shown value of ±10%. In some embodiments, "about" indicates a deviation from the shown value of ±5%. In some embodiments, "about" indicates a deviation from the shown value of ±4%. In some embodiments, "about" indicates a deviation from the shown value of ±3%. In some embodiments, "about" indicates a deviation from the shown value of ±2%. In some embodiments, "about" indicates a deviation from the shown value of ±1%. In one embodiment, "about" indicates a deviation from the shown value of ±0.9%. In some embodiments, "about" indicates a deviation from the shown value of ±0.8%. In some embodiments, "about" indicates a deviation from the shown value of ±0.7%. In some embodiments, "about" indicates a deviation from the shown value of ±0.6%. In some embodiments, "about" indicates a deviation from the shown value of ±0.5%. In some embodiments, "about" indicates a deviation from the shown value of ±0.4%. In some embodiments, "about" indicates a deviation from the shown value of ±0.3%. In some embodiments, "about" indicates a deviation from the shown value of ±0.2%. In some embodiments, "about" indicates a deviation from the shown value of ±0.1%. In some embodiments, "about" indicates a deviation from the shown value of ±0.05%. In some embodiments, "about" indicates a deviation from the shown value of ±0.01%. As will be understood by those skilled in the art, for a given technical effect, a particular deviation of such a value will depend on the nature of that technical effect. For example, the deviation of a natural or biotechnological effect may generally be greater than the deviation of a man-made or engineered effect.

[0160] The numerical ranges listed in this document are intended only as a convenient method for individually referring to each specific value falling within that range. Unless otherwise stated herein, each value is included in the specification as if it were listed separately herein.

[0161] All documents cited herein (including all patents, patent applications, scientific publications, manufacturer specifications, instructions, etc.), whether mentioned above or below, are incorporated herein by reference in their entirety. Nothing herein shall be construed as an admission that the invention has lost any rights to any prior art claims.

[0162] As used in this article, phrases such as “measured quantity” or “measured expression”, or similar phrases related to amino acid sequences (peptides or polypeptides), refer to the quantity or presence of amino acid sequences measured.

[0163] As used herein, terms such as “reduction” or “inhibition” refer to the ability to cause an overall decrease in level, for example, about 5% or more, about 10% or more, about 15% or more, about 20% or more, about 25% or more, about 30% or more, about 40% or more, about 50% or more, or about 75% or more. The term “inhibition” or similar phrases include complete or substantially complete inhibition, i.e., a reduction to zero or substantially zero.

[0164] As used in this article, the term “enhancement” refers to the ability to cause an overall increase or enhancement in level, such as at least about 5% or more, about 10% or more, about 15% or more, about 20% or more, about 25% or more, about 30% or more, about 40% or more, about 50% or more, about 75% or more, or about 100% or more.

[0165] As used herein, “physiological pH” refers to a pH of approximately 7.4. In some embodiments, the physiological pH is 7.3 to 7.5. In some embodiments, the physiological pH is 7.35 to 7.45. In some embodiments, the physiological pH is 7.3, 7.35, 7.4, 7.45, or 7.5.

[0166] As used in this disclosure, "% w / v" refers to weight-volume percentage, which is a unit of concentration that measures the mass of solute in grams (g) as a percentage of the total volume of the solution in milliliters (mL).

[0167] As used in this disclosure, "%weight" means weight percentage, which is a unit of concentration, and the amount of substance measured in grams (g) is expressed as a percentage of the total weight of the entire composition in grams (g).

[0168] As used in this disclosure, "mole%" is defined as the ratio of the number of moles of one component to the total number of moles of all components multiplied by 100.

[0169] In the context of this disclosure, the term "recombinant" means "manufactured through genetic engineering." In one embodiment, the "recombinant object" in the context of this disclosure is not naturally occurring.

[0170] As used herein, the term "naturally occurring" refers to the fact that an object can be found in nature. For example, peptides or nucleic acids that exist in organisms (including viruses) and can be isolated from natural sources and have not been intentionally modified by humans in a laboratory are naturally occurring. The term "found in nature" means "existing in nature" and includes both known objects and objects that have not yet been discovered and / or isolated from nature but may be discovered and / or isolated from natural sources in the future.

[0171] According to various embodiments of the invention, the compositions of the invention or the RNA obtained in the invention are taken up or introduced, i.e., transfected or transduced into cells, which may be present in vitro or in a subject, for example resulting in the expression of one or more encoded peptides or polypeptides. The cells may, for example, express the encoded peptides or polypeptides intracellularly (e.g., in the cytoplasm and / or nucleus), may secrete the encoded peptides or polypeptides, and / or may express them on a surface.

[0172] According to this disclosure, terms such as “nucleic acid expression” and “nucleic acid encoding” or similar terms are used interchangeably herein, and a particular peptide or polypeptide means that the nucleic acid, if present in a suitable environment, such as within a cell, can be expressed to produce the peptide or polypeptide.

[0173] The term "portion" refers to a fraction. Regarding a specific structure, such as an amino acid sequence or a protein, the term "portion" can indicate a continuous or discontinuous fraction of that structure.

[0174] The terms “part” and “fragment” are used interchangeably herein and refer to a continuous element. For example, a part of a structure such as an amino acid sequence or a protein refers to a continuous element of that structure. When used in the context of a composition, the term “part” refers to a portion of the composition. For example, a part of a composition can be any portion from 0.1% to 99.9% (e.g., 0.1%, 0.5%, 1%, 5%, 10%, 50%, 90%, or 99%) of the composition.

[0175] A “fragment” of an amino acid sequence (peptide or polypeptide) refers to a portion of an amino acid sequence, specifically a sequence representing a shortened amino acid sequence at the N-terminus and / or C-terminus. A C-terminal shortened fragment (N-terminal fragment) can be obtained, for example, by translating a truncated open reading frame lacking the 3' end. A C-terminal shortened fragment (N-terminal fragment) can be obtained, for example, by translating a truncated open reading frame lacking the 5' end, provided that the truncated open reading frame contains a start codon for initiating translation. A fragment of an amino acid sequence comprises, for example, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the amino acid residues of the amino acid sequence. A fragment of an amino acid sequence comprises, for example, at least 6, particularly at least 8, at least 10, at least 12, at least 15, at least 20, at least 30, at least 50, or at least 100 consecutive amino acids of the amino acid sequence. The amino acid sequence fragment contains, for example, a sequence of up to 8, particularly up to 10, 12, 15, 20, 30, or 55 consecutive amino acids.

[0176] The “sequence identity” between two nucleic acid sequences indicates the percentage of identical nucleotides between the sequences. The terms “%identity” and “% identity” or similar terms are intended to refer, specifically, to the percentage of identical nucleotides or amino acids between the sequences being compared in an optimal alignment. This percentage is purely statistical, and the differences between the two sequences over their entire length can, but do not necessarily, be randomly distributed. Comparison of two sequences is typically performed after optimal alignment, comparing sequences about segments or “comparison windows” to identify local regions of the respective sequences. The best alignment for comparison can be performed manually, or with the local homology algorithm of Smith and Waterman, 1981, Ads App. Math. 2, 482; the local homology algorithm of Needleman and Wunsch, 1970, J. Mol. Biol. 48, 443; the similarity search algorithm of Pearson and Lipman, 1988, Proc. Natl Acad. Sci. USA 88, 2444; or with computer programs that use the algorithms (GAP, BESTFIT, FASTA, BLASTP, BLAST N, and TFASTA in Wisconsin Genetics Software Package, GeneticsComputer Group, 575 Science Drive, Madison, Wis.). In some implementations, the percentage identity of two sequences is determined using the BLASTN or BLASTP algorithm, as available on the National Center for Biotechnology Information (NCBI) website (e.g., at blast.ncbi.nlm.nih.gov / Blast.cgi?PAGE_TYPE=BlastSearch&BLAST_SPEC=blast2seq&LINK_LOC=align2seq). In some implementations, the algorithm parameters used for the BLASTN algorithm on the NCBI website include: (i) an expected threshold of 10; (ii) a word length of 28; (iii) a maximum number of matches within the query range of 0; (iv) a match / mismatch score of 1, -2; (v) a linear void cost; and (vi) the use of a filter with low-complexity regions. In some implementations, the algorithm parameters used for the BLASTP algorithm on the NCBI website include: (i) the expected threshold is set to 10; (ii) the word length is set to 3; (iii) the maximum number of matches in the query range is set to 0; (iv) the matrix is ​​set to BLOSUM62; (v) the empty cost is set to exist: 11 and extend: 1; and (vi) the conditional composition score matrix is ​​adjusted.

[0177] Percentage identity is obtained by determining the number of identical positions corresponding to the sequences to be compared, dividing that number by the number of positions being compared (e.g., the number of positions in the reference sequence), and multiplying the result by 100.

[0178] In some embodiments, the degree of similarity or identity is given for at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or about 100% of the entire length of the reference sequence. For example, if the reference nucleic acid sequence consists of 200 nucleotides, the degree of identity is given for at least about 100, at least about 120, at least about 140, at least about 160, at least about 180, or about 200 nucleotides, and in some embodiments, for consecutive nucleotides. In some embodiments, the degree of similarity or identity is given for the entire length of the reference sequence.

[0179] In some implementations, "isolated" means removed (e.g., purified) from its natural state or from an artificial composition, such as a composition derived from a manufacturing process. For example, nucleic acids, peptides, or polypeptides naturally present in living animals are not "isolated," but the same nucleic acids, peptides, or polypeptides that are partially or completely isolated from their natural coexisting substances are "isolated." Isolated nucleic acids, peptides, or polypeptides may exist in a substantially purified form or may exist in non-natural environments, such as, for example, host cells.

[0180] The term "transfection" refers to the introduction of nucleic acids, particularly RNA, into cells. For the purposes of this disclosure, the term "transfection" also includes the introduction of nucleic acids into or the uptake of nucleic acids by such cells, wherein the cells may be present in a subject, such as a patient, or the cells may be in vitro, such as in vitro from a patient. Thus, according to this disclosure, cells used for transfecting the nucleic acids described herein may be present in vitro or in vivo, for example, the cells may form part of an organ, tissue, and / or a patient's body. According to this disclosure, transfection may be transient or stable. For some applications of transfection, it may be sufficient if the transfected genetic material is expressed only transiently. RNA may be transfected into cells to transiently express the proteins it encodes. Since the nucleic acids introduced during transfection typically do not integrate into the nuclear genome, the exogenous nucleic acids will be diluted or degraded by mitosis. Cells that allow for the amplification of nucleic acid episomes greatly reduce the dilution rate. If it is desired that the transfected nucleic acids actually remain in the genome of the cell and its progeny cells, then stable transfection must occur. Such stable transfection can be achieved, for example, by using a virus-based system or a transposon-based system. Typically, nucleic acids encoding antigens are transiently transfected into cells. RNA can be transfected into cells to transiently express the protein it encodes.

[0181] Cells that can be used for transfection in the methods described herein include, but are not limited to, cells from animal cell lines such as Chinese hamster ovary (CHO), K562, HepG2, HEK293T, RAW, and C2C12 cells. In some embodiments, the cells are CHO, K562, HEK293T, RAW, and C2C12 cells. In some embodiments, the cells are Chinese hamster ovary (CHO) cells.

[0182] As used in this article, “endogenous” means any substance that originates from or is produced within an organism, cell, tissue, or system.

[0183] As used herein, the term "exogenous" means any substance introduced from or produced outside of an organism, cell, tissue, or system.

[0184] As used in this article, the term “expression” is defined as the transcription and / or translation of a specific nucleotide sequence.

[0185] In the context of this disclosure, the term "transcription" refers to the process in which the genetic code in a DNA sequence is transcribed into RNA (specifically mRNA). The RNA can then be translated into peptides or polypeptides.

[0186] Regarding RNA, the terms "expression" or "translation" refer to a process in the cell's ribosomes through which the mRNA chain directs the assembly of amino acid sequences to create peptides or polypeptides.

[0187] Nucleic acid

[0188] The term "nucleic acid" includes deoxyribonucleic acid (DNA), ribonucleic acid (RNA), combinations thereof, and their modified forms. The term includes genomic DNA, cDNA, mRNA, recombinantly produced molecules, and chemically synthesized molecules. Nucleic acids can exist in single-stranded or double-stranded form, and can be linear or covalently closed circular molecules. Nucleic acids can be isolated. According to this disclosure, the term "isolated nucleic acid" means that the nucleic acid is (i) amplified in vitro, for example, by polymerase chain reaction (PCR) for DNA, or by in vitro transcription (e.g., using RNA polymerase) for RNA; (ii) produced by clonal recombination; (iii) purified, for example, by lysis and isolation by gel electrophoresis; or (iv) synthesized, for example, by chemical synthesis.

[0189] The term "nucleoside" (hereinafter referred to as "N") refers to a compound that can be considered as a nucleotide without a phosphate group. Nucleosides are formed by linking a nucleobase to a sugar (such as ribose or deoxyribose), while nucleotides consist of a nucleoside and one or more phosphate groups. Examples of nucleosides include cytidine, uridine, pseudouridine, adenosine, and guanosine.

[0190] The five standard nucleosides that typically constitute natural nucleic acids are uridine, adenosine, thymidine, cytidine, and guanosine. These five nucleosides are usually abbreviated by their single-letter codes U, A, T, C, and G, respectively. However, thymidine is more commonly written as "dT" ("d" stands for "deoxy") because it contains a 2'-deoxyribofuranosylfuranose moiety, rather than the ribofuranosyl ring present in uridine. This is because thymidine is found in deoxyribonucleic acid (DNA), not ribonucleic acid (RNA). Conversely, uridine is found in RNA, not DNA. The other three nucleosides can be found in both RNA and DNA. In RNA, they are represented by A, C, and G, respectively; while in DNA, they are represented by dA, dC, and dG, respectively.

[0191] The modified purine (A or G) or pyrimidine (C, T or U) base moiety is preferably modified with one or more alkyl groups, more preferably with one or more C1-4 alkyl groups, and even more preferably with one or more methyl groups. Specific examples of modified purine or pyrimidine base moiety include N7-alkylguanine, N6-alkyladenine, 5-alkylcytosine, 5-alkyluracil, and N(1)-alkyluracil, such as N7-C1-4 alkylguanine, N6-C1-4 alkyladenine, 5-C1-4 alkylcytosine, 5-C1-4 alkyluracil, and N(1)-C1-4 alkyluracil, preferably with N7-methylguanine, N6-methyladenine, 5-methylcytosine, 5-methyluracil, and N(1)-methyluracil.

[0192] In this document, the term "DNA" refers to a nucleic acid molecule containing deoxyribonucleotide residues. In a preferred embodiment, the DNA contains all or most of the deoxyribonucleotide residues. As used herein, "deoxyribonucleotide" refers to a nucleotide lacking a hydroxyl group at the 2' position of the β-D-furanose group. DNA includes, but is not limited to, double-stranded DNA, single-stranded DNA, isolated DNA (such as partially purified DNA), substantially pure DNA, synthetic DNA, recombinant DNA, and modified DNA that differs from natural DNA by adding, deleting, replacing, and / or altering one or more nucleotides. Such alterations may refer to the addition of non-nucleotide substances to the internal nucleotides or ends of DNA. It is also contemplated herein that the nucleotides in DNA may be non-standard nucleotides, such as chemically synthesized nucleotides or ribonucleotides. For the purposes of this disclosure, these altered DNAs are considered analogs of natural DNA. If the content of deoxyribonucleotide residues in a molecule exceeds 50% (such as 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 96%, at least 97%, at least 98%, at least 99%), then the molecule contains a "majority of deoxyribonucleotide residues," which is calculated based on the total number of nucleotide residues in the molecule. The total number of nucleotide residues in a molecule is the sum of all nucleotide residues (whether these nucleotide residues are standard (i.e., naturally occurring) nucleotide residues or their analogues).

[0193] DNA can be recombinant DNA, obtained through the cloning of nucleic acids (especially cDNA). cDNA can be obtained through reverse transcription of RNA.

[0194] The term "RNA" refers to a nucleic acid molecule containing ribonucleotide residues. In a preferred embodiment, the RNA contains all or most of the ribonucleotide residues. As used herein, "ribonucleotide" refers to a nucleotide having a hydroxyl group at the 2' position of the β-D-furanose group. RNA includes, but is not limited to, double-stranded RNA, single-stranded RNA, isolated RNA (such as partially purified RNA), substantially pure RNA, synthetic RNA, recombinant RNA, and modified RNA that differs from natural RNA by adding, deleting, replacing, and / or altering one or more nucleotides. Such alterations may refer to the addition of non-nucleotide substances to internal RNA nucleotides or RNA ends. It is also contemplated herein that the nucleotides in the RNA may be non-standard nucleotides, such as chemically synthesized nucleotides or deoxyribonucleotides. For the purposes of this disclosure, these altered / modified nucleotides may be referred to as analogs of natural nucleotides, and the corresponding RNA containing these altered / modified nucleotides (i.e., altered / modified RNA) may be referred to as analogs of natural RNA. If the content of ribonucleotide residues in a molecule exceeds 50% (such as 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 96%, at least 97%, at least 98%, at least 99%), then the molecule contains a "majority of ribonucleotide residues," which is calculated based on the total number of nucleotide residues in the molecule. The total number of nucleotide residues in a molecule is the sum of all nucleotide residues (regardless of whether the nucleotide residues are standard (i.e., naturally occurring) nucleotide residues or their analogues).

[0195] "RNA" includes nucleic acids of mRNA, tRNA, ribosomal RNA (rRNA), small nuclear RNA (snRNA), self-amplifying RNA (saRNA), single-stranded RNA (ssRNA), double-stranded RNA (dsRNA), repressive RNA (such as antisense ssRNA, small interfering RNA (siRNA), or microRNA (miRNA)), activating RNA (such as small activating RNA), and immunostimulatory RNA (isRNA). In some implementations, "RNA" refers to mRNA.

[0196] As used herein, the term “in vitro transcription” or “IVT” refers to transcription (i.e., RNA generation) performed under cell-free conditions. That is, IVT does not use living / cultured cells, but rather uses transcription mechanisms extracted from cells (e.g., cell lysates or their isolated components, including RNA polymerases (preferably T7, T3, or SP6 polymerases)).

[0197] In some implementations, the RNA is single-stranded RNA.

[0198] In some implementations, the RNA is mRNA.

[0199] In some implementations, RNA is produced through in vitro transcription.

[0200] In some implementations, the RNA contains a 5' cap structure.

[0201] In some implementations, the RNA does not contain modified ribonucleotides.

[0202] In some embodiments, the RNA comprises a modified ribonucleotide. In some embodiments, the modified ribonucleotide comprises a modified uridine. In some embodiments, the modified uridine comprises N1-methylpseudouridine.

[0203] In some implementations, DNA exists in the form of a vector.

[0204] In some embodiments, the vector contains DNA encoding an amino acid sequence, said amino acid sequence comprising an amino acid sequence of a biologically active peptide or polypeptide.

[0205] In some implementations, the vector is a DNA vector.

[0206] In some embodiments, the composition of the present invention or the RNA obtained in the present invention (which may contain at least two or more RNA molecules) is formulated together with a delivery vector.

[0207] In some embodiments, the compositions of the present invention or the RNA obtained in the present invention are formulated together with one or more compounds to form a complex with the RNA molecule.

[0208] In some embodiments, the compositions of the present invention or the RNA obtained in the present invention are formulated into particles. In one embodiment, the present invention provides particles comprising the compositions of the present invention. In one embodiment, the present invention provides particles comprising RNA obtained in the present invention. In one embodiment, the present invention provides particles comprising RNA purified from the compositions of the present invention.

[0209] In one embodiment, the composition of the present invention is packaged in particles. In one embodiment, RNA molecules contained in the composition of the present invention are packaged in particles. In one embodiment, RNA molecules obtained in the present invention are packaged in particles. In one embodiment, the particles are lipid complex particles. In one embodiment, the particles are lipid nanoparticles.

[0210] In some embodiments, the compositions of the present invention or the RNA obtained in the present invention are formulated as lipid complex particles. In these embodiments, it is preferred that the cell is characterized by having a macropinocytosis-mediated RNA uptake mechanism.

[0211] In some embodiments, the compositions of the present invention or the RNA obtained in the present invention are formulated as lipid nanoparticles.

[0212] In some embodiments, the compositions of the present invention or the RNA obtained in the present invention comprise at least two RNA molecules, each encoding an amino acid sequence comprising a bioactive peptide or polypeptide.

[0213] In some embodiments, the compositions of the present invention or the RNA obtained in the present invention comprise at least two RNA molecules, each encoding a different amino acid sequence, said amino acid sequence comprising the amino acid sequence of a biologically active peptide or polypeptide.

[0214] In some implementations, different amino acid sequences include different amino acid sequences of bioactive peptides or polypeptides.

[0215] In some implementations, different biologically active peptides or polypeptides contain different antigens.

[0216] In some embodiments, the RNA described herein is a single-stranded RNA that can be translated into a corresponding protein upon entry into a cell (e.g., the cells used in the assays described herein and the recipient cells). In addition to a wild-type or codon-optimized amino acid sequence encoding an amino acid sequence containing a bioactive peptide or polypeptide (e.g., a pharmacologically active peptide or polypeptide, such as an antigen sequence), the RNA may also contain one or more structural elements optimized to maximize RNA stability and translation efficiency (5' cap, 5' UTR, 3' UTR, poly(A) tail). In one embodiment, the RNA contains all of these elements. In one embodiment, β-S-ARCA(D1) (m27,2'-OGppSpG) or m27,3'-OGppp(m12'-O)ApG may be used as a specific capping structure at the 5' end of the RNA drug. As the 5'-UTR sequence, the 5'-UTR sequence of human α-globin mRNA may be used, optionally with an optimized "Kozak sequence" to improve translation efficiency. As a 3'-UTR sequence, a combination of two sequence elements (FI element) derived from N-terminal cleavage enhancer (AES) mRNA (referred to as F) and mitochondrial-encoded 12S ribosomal RNA (referred to as I) can be used, positioned between the coding sequence and the poly(A) tail to ensure higher maximum protein expression levels and longer mRNA presence. These sequences are identified through an in vitro screening process designed to confer RNA stability and enhance total protein expression (see WO 2017 / 060314, which is incorporated herein by reference). Alternatively, the 3'-UTR can be the 3'-UTR sequence of two repeating human β-globin mRNAs. Furthermore, a 110-nucleotide poly(A) tail can be used, consisting of a 30-adenosine residue, followed by a 10-nucleotide linker sequence (random nucleotide), and a sequence segment of an additional 70 adenosine residues. This poly(A) tail sequence is designed to improve RNA stability and translation efficiency.

[0217] An amino acid sequence comprising an amino acid sequence of a biologically active peptide or polypeptide (e.g., a pharmacologically active peptide or polypeptide, such as an antigen sequence) may include other amino acid sequences besides the biologically active peptide or polypeptide amino acid sequence. These other amino acid sequences may support the function or activity of the biologically active peptide or polypeptide. In some embodiments, these other amino acid sequences include amino acid sequences that enhance antigen processing and / or presentation. Alternatively, or additionally, these other amino acid sequences include amino acid sequences that break immune tolerance. Alternatively, or additionally, these other amino acid sequences include amino acid sequences that generate bioluminescence. These other amino acid sequences can be used to determine the amount of amino acid sequences comprising the biologically active peptide or polypeptide amino acid sequence or fragments thereof in the assays described herein. In particular, these other amino acid sequences can be used for quantification by LC-MS / MS analysis.

[0218] The compositions of the present invention and the RNA obtained therein can be complexed with polymers, proteins, and / or lipids (preferably lipids) to generate nucleic acid particles for application. If different combinations of nucleic acids are used, these nucleic acids can be complexed together or separately.

[0219] mRNA

[0220] According to this disclosure, the term "mRNA" refers to "messenger RNA," which is a transcript generated from DNA as a template that can encode a peptide or polypeptide. Typically, mRNA contains a 5'-UTR, a peptide / polypeptide coding region, and a 3'-UTR. In the context of this disclosure, mRNA can be generated from DNA as a template via in vitro transcription (IVT). As mentioned above, in vitro transcription methods are known to those skilled in the art, and various in vitro transcription kits are commercially available.

[0221] mRNA is single-stranded, but it may contain its own complementary sequences, which allow parts of the mRNA sequence to fold and pair with itself to form a double helix structure.

[0222] According to this disclosure, "dsRNA" refers to double-stranded RNA, which is RNA with two partially or completely complementary strands.

[0223] In a preferred embodiment of this disclosure, the mRNA relates to an RNA transcript encoding a peptide or polypeptide.

[0224] In some embodiments, the mRNA encoding the peptide or polypeptide is preferably at least 45 nucleotides in length (such as at least 60, at least 90, at least 100, at least 200, at least 300, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, at least 1000, at least 1500, at least 2000, at least 2500, at least 3000, at least 3500, at least 4000, at least 4500, at least 5000, at least 6000, at least 7000, at least 8000, at least 9000 nucleotides), preferably up to 15000 nucleotides, such as up to 14000, up to 13000, up to 12000, up to 11000, or up to 10000 nucleotides.

[0225] mRNAs known in the art typically comprise a 5' untranslated region (5'-UTR), a peptide / peptide coding region, and a 3' untranslated region (3'-UTR). In some embodiments, mRNA is produced by in vitro transcription or chemical synthesis. In some embodiments, mRNA is produced by in vitro transcription using a DNA template. In vitro transcription methods are known to those skilled in the art; for example, see Molecular Cloning: A Laboratory Manual, 4th Edition, edited by MR Green and J. Sambrook, Cold Spring Harbor Laboratory Press, Cold Spring Harbor 2012. Furthermore, various in vitro transcription kits are commercially available, for example from Thermo Fisher Scientific (such as TranscriptAid). TM Kits from T7 reagent kits, MEGAscript® T7 kits, MAXIscript®, New England BioLabs Inc. (such as HiScribe™ T7 kits, HiScribe™ T7 ARCA mRNA kits), Promega (such as RiboMAX™, HeLaScribe®, Riboprobe® systems), Jena Bioscience (such as SP6 or T7 transcription kits), and Epicentre (such as AmpliScribe™). To provide modified mRNA, appropriate modifying nucleotides, such as modified naturally occurring nucleotides, non-naturally occurring nucleotides, and / or modified non-naturally occurring nucleotides, can be incorporated during synthesis (preferably in vitro transcription), or the mRNA can be modified and / or modified post-transcriptionally.

[0226] In some embodiments, the mRNA is in vitro transcribed mRNA (IVT-RNA), which can be obtained by in vitro transcription of a suitable DNA template. The promoter controlling transcription can be any promoter of any RNA polymerase. Specific examples of RNA polymerases include T7, T3, and SP6 RNA polymerases. Preferably, in vitro transcription is controlled by a T7 or SP6 promoter. The DNA template for in vitro transcription can be obtained by cloning nucleic acids (especially cDNA) and introducing them into a suitable vector for in vitro transcription. cDNA can be obtained by reverse transcription of RNA.

[0227] In some embodiments of this disclosure, the mRNA is a “replicon mRNA” or simply a “replicon,” particularly a “self-replicating mRNA” or “self-amplifying mRNA.” In some embodiments, the replicon or self-replicating mRNA is derived from or contains elements derived from ssRNA viruses, particularly positive-sense ssRNA viruses such as alphaviruses. Alphaviruses are typical representatives of positive-sense RNA viruses. Alphaviruses replicate in the cytoplasm of infected cells (for a review of the alphavirus life cycle, see José et al., Future Microbiol., 2009, vol. 4, pp. 837-856). The total genome length of many alphaviruses is typically between 11,000 and 12,000 nucleotides, and the genomic RNA typically has a 5' cap and a 3' poly(A) tail. The alphavirus genome encodes non-structural proteins (involved in the transcription, modification, and replication of viral RNA, as well as protein modification) and structural proteins (forming viral particles). There are typically two open reading frames (ORFs) in the genome. Four non-structural proteins (nsP1-nsP4) are typically encoded by a first ORF located near the 5' end of the genome, while the structural proteins of alphavirus are encoded by a second ORF located downstream of the first ORF and extending to near the 3' end of the genome. Generally, the first ORF is longer than the second ORF, with a ratio of approximately 2:1. In alphavirus-infected cells, only the nucleic acid sequences encoding non-structural proteins are translated from the genomic RNA, while the genetic information encoding structural proteins is translated from subgenomic transcripts, which are RNA molecules similar to eukaryotic messenger RNA (mRNA) (Gould et al., 2010, Antiviral Res., vol. 87 pp. 111-124). Post-infection, in the early stages of the viral life cycle, the positive-sense genomic RNA acts directly as messenger RNA, translating the open reading frame encoding the non-structural polyprotein (nsP1234). Alphavirus-derived vectors have been proposed for delivering exogenous genetic information to target cells or organisms. In a simplified approach, the open reading frame encoding an alphavirus structural protein is replaced with an open reading frame encoding the target protein. Alphavirus-based trans-replication systems rely on alphavirus nucleotide sequence elements on two separate nucleic acid molecules: one molecule encodes a viral replicase, and the other can be trans-replicated by that replicase (hence the name trans-replication system). Trans-replication requires the simultaneous presence of both nucleic acid molecules in a given host cell. The nucleic acid molecule capable of trans-replication by the replicase must contain certain alphavirus sequence elements so that the alphavirus replicase can recognize them and synthesize RNA.

[0228] In some embodiments of this disclosure, the mRNA includes one or more modifications, for example, to improve its stability and / or translation efficiency and / or reduce immunogenicity and / or cytotoxicity. For example, to improve mRNA expression, its coding region (i.e., the sequence encoding the expressed peptide or polypeptide) may be modified, preferably without altering the sequence of the expressed peptide or polypeptide. Such modifications are described, for example, in WO 2007 / 036366 and PCT / EP2019 / 056502, and include: a 5'-cap structure; extension or truncation of a naturally occurring poly(A) tail; alteration of the 5' and / or 3' untranslated regions (UTRs), such as introducing a UTR unrelated to the RNA coding region; replacement of one or more naturally occurring nucleotides with synthetic nucleotides; and codon optimization (e.g., alteration, preferably increasing the GC content of the RNA).

[0229] In some embodiments, the mRNA contains a 5'-cap structure. In some embodiments, the mRNA does not contain an uncapped 5'-triphosphate. In some embodiments, the mRNA may contain a conventional 5'-cap and / or a 5'-cap analog. The term "conventional 5'-cap" refers to a cap structure located at the 5' end of the mRNA molecule, typically composed of guanosine 5'-triphosphate (Gppp), which is linked to the 5' end of the next nucleotide of the mRNA via its triphosphate portion (i.e., guanosine is linked to the rest of the mRNA via a 5'-5' triphosphate bond). Guanosine may be methylated at the N7 position (forming a cap structure m7Gppp). The term "5'-cap analog" includes 5'-caps based on conventional 5'-caps but modified at the 2' or 3' position of their m7 guanosine structure to prevent the 5'-cap analog from integrating in the reverse direction (such 5'-cap analogs are also called anti-reverse cap analogs (ARCA)). Particularly preferred 5'-cap analogs are analogs having one or more substituents at the bridging oxygen and non-bridging oxygen of the phosphate bridge, such as 5'-cap analogs modified with thiophosphate at the β-phosphate (e.g., m27,2'OG(5')ppSp(5')G (referred to as beta-S-ARCA or β-S-ARCA)), as described in PCT / EP2019 / 056502. The mRNAs with the 5'-cap structure described herein can be obtained by in vitro transcription of a DNA template in the presence of a suitable 5'-cap compound, wherein the 5'-cap structure is integrated into the resulting mRNA strand via co-transcription; alternatively, the mRNA can be generated, for example, by in vitro transcription, and the 5'-cap structure can be ligated to the mRNA post-transcriptionally using a capping enzyme (e.g., a capping enzyme of vaccinia virus).

[0230] In some implementations, the mRNA contains a 5'-cap structure selected from the group consisting of: m27,2'OG(5')ppSp(5')G (especially its D1 diastereomer), m27,3'OG(5')ppp(5')G and m27,3'-OGppp(m12'-O)ApG.

[0231] In some embodiments, the mRNA comprises cap0, cap1, or cap2, preferably cap1 or cap2. According to this disclosure, the term "cap0" refers to the structure "m7GpppN", where N is any nucleotide with an OH moiety at the 2' position. According to this disclosure, the term "cap1" refers to the structure "m7GpppNm", where Nm is any nucleotide with an OCH3 moiety at the 2' position. According to this disclosure, the term "cap2" refers to the structure "m7GpppNmNm", where each Nm is independently any nucleotide with an OCH3 moiety at the 2' position.

[0232] The D1 diastereomer of beta-S-ARCA (β-S-ARCA) has the following structure:

[0233]

[0234] "D1 diastereomer of beta-S-ARCA" or "β-S-ARCA(D1)" refers to the β-S-ARCA diastereomer that elutes before the D2 diastereomer of beta-S-ARCA (β-S-ARCA(D2)) on a high-performance liquid chromatography (HPLC) column, and therefore has a shorter retention time. The HPLC is preferably analytical HPLC. In some embodiments, separation is performed using a Supelcosil LC-18-T RP column, preferably with a specification of 5 μm and a diameter of 4.6 × 250 mm, wherein a flow rate of 1.3 ml / min can be used. In some embodiments, a gradient of methanol in ammonium acetate, for example, a linear gradient of 0-25% methanol in 0.05 M ammonium acetate (pH = 5.9), is used for elution within 15 minutes. Ultraviolet detection (VWD) can be performed at 260 nm, and fluorescence detection (FLD) can be excited at 280 nm and detected at 337 nm.

[0235] The 5'-cap analog m27,3'-OGppp(m12'-O)ApG (also known as m27,3'OG(5')ppp(5')m2'-OApG) is a constituent unit of cap1 and has the following structure:

[0236]

[0237] An exemplary cap0 mRNA containing β-S-ARCA and mRNA has the following structure:

[0238]

[0239] An exemplary cap0 mRNA containing m27,3'OG(5')ppp(5')G and mRNA has the following structure:

[0240]

[0241] An exemplary cap1 mRNA containing m27,3'-OGppp(m12'-O)ApG and mRNA has the following structure:

[0242]

[0243] As used herein, the terms "poly-A tail" or "poly-A sequence" refer to a sequence of continuous or discontinuous adenosine residues typically located at the 3' end of an mRNA molecule. Poly-A tails or poly-A sequences are known to those skilled in the art and may be located after the 3'-UTR of the mRNA described herein. A continuous poly-A tail is characterized by a continuous sequence of adenosine residues. Continuous poly-A tails are common in nature. The mRNAs disclosed herein may have a free 3' poly-A tail that is ligated to the mRNA post-transcriptionally by a template-dependent RNA polymerase, or may be encoded by DNA and transcribed by a template-dependent RNA polymerase to obtain a poly-A tail.

[0244] It has been shown that the poly-A tail of approximately 120 A nucleotides has a beneficial effect on mRNA levels in transfected eukaryotic cells as well as on protein levels translated from open reading frames located upstream (5') of the poly-A tail (Holtkamp et al., 2006, Blood, vol. 108, pp. 4009-4017).

[0245] The poly-A tail can be of any length. In some embodiments, the poly-A tail comprises, substantially consists of, or is composed of at least 20, at least 30, at least 40, at least 80, or at least 100, or at most 500, at most 400, at most 300, at most 200, or at most 150 A nucleotides, particularly comprising about 120 A nucleotides. In this context, "substantially consists of" means that the majority of the nucleotides in the poly-A tail (typically representing at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the total nucleotides in the poly-A tail) are A nucleotides, but the remaining nucleotides are allowed to be other nucleotides besides A nucleotides, such as U nucleotides (uridine monophosphate), G nucleotides (guanosine monophosphate), or C nucleotides (cytidine monophosphate). In this context, "consisting of" means that all nucleotides in the poly-A tail (i.e., representing 100% of the total nucleotides in the poly-A tail) are A nucleotides. The term "A nucleotide" or "A" refers to adenosine monophosphate.

[0246] In some implementations, during RNA transcription (e.g., in the preparation of RNA transcribed in vitro), a poly-A tail is attached to a strand complementary to the coding strand based on a DNA template containing repeating dT nucleotides (deoxythymidines). The DNA sequence encoding the poly-A tail (coding strand) is called a poly(A) box.

[0247] In some embodiments, a poly(A) box present in the DNA coding strand is essentially composed of dA nucleotides but interrupted by random sequences of four nucleotides (dA, dC, dG, and dT). The length of such random sequences can be 5 to 50, 10 to 30, or 10 to 20 nucleotides. Such poly(A) boxes are disclosed in WO 2016 / 005324 A1, which is incorporated herein by reference. Any poly(A) box disclosed in WO 2016 / 005324 A1 can be used in this disclosure. Poly(A) boxes, essentially composed of dA nucleotides but interrupted by random sequences, exhibit the ability to continuously proliferate plasmid DNA in *E. coli* at the DNA level; and at the RNA level, they have beneficial properties supporting RNA stability and translation efficiency, wherein the random sequences are uniformly distributed with the four nucleotides (dA, dC, dG, and dT) and are, for example, 5 to 50 nucleotides in length.

[0248] Therefore, in some embodiments, the poly-A tail contained in the mRNA molecule described herein is essentially composed of A nucleotides, but is interrupted by random sequences of four nucleotides (A, C, G, U). The length of such random sequences can be 5 to 50, 10 to 30, or 10 to 20 nucleotides.

[0249] In some implementations, no nucleotide other than A is located on the 3' flanking side of the poly-A tail, meaning that the 3' end of the poly-A tail is not covered by or immediately followed by a nucleotide other than A.

[0250] In some embodiments, the poly-A tail may comprise at least 20, at least 30, at least 40, at least 80, or at least 100 and up to 500, at most 400, at most 300, at most 200, or at most 150 nucleotides. In some embodiments, the poly-A tail may consist substantially of at least 20, at least 30, at least 40, at least 80, or at least 100 and up to 500, at most 400, at most 300, at most 200, or at most 150 nucleotides. In some embodiments, the poly-A tail may consist of at least 20, at least 30, at least 40, at least 80, or at least 100 and up to 500, at most 400, at most 300, at most 200, or at most 150 nucleotides. In some embodiments, the poly-A tail comprises at least 100 nucleotides. In some embodiments, the poly-A tail contains about 150 nucleotides. In some embodiments, the poly-A tail contains about 120 nucleotides.

[0251] In some embodiments, the mRNA used in this disclosure comprises a 5'-UTR and / or a 3'-UTR. The terms "untranslated region" or "UTR" refer to a region in a DNA molecule that is transcribed but not translated into an amino acid sequence, or a corresponding region in an RNA molecule, such as an mRNA molecule. An untranslated region (UTR) may be located at the 5' end (upstream) of an open reading frame (5'-UTR) and / or the 3' end (downstream) of an open reading frame (3'-UTR). If present, the 5'-UTR is located at the 5' end of a protein-coding region, upstream of the start codon. The 5'-UTR is located downstream of the 5' cap (if present), for example, immediately adjacent to the 5' cap. If present, the 3'-UTR is located at the 3' end of a protein-coding region, downstream of the stop codon, but the term "3'-UTR" typically does not include the poly-A sequence. Therefore, the 3'-UTR is located upstream of the poly-A sequence (if present), for example, immediately adjacent to the poly-A sequence. Integrating a 3'-UTR into the 3' untranslated region of an RNA (preferably mRNA) molecule can improve translation efficiency. Integrating two or more such 3'-UTRs (preferably arranged head-to-tail; see, for example, Holtkamp et al., Blood 108, 4009-4017 (2006)) can produce a synergistic effect. These 3'-UTRs can be homologous or heterologous sequences of the RNA (e.g., mRNA) into which they are introduced. In some embodiments, the 3'-UTR is derived from a globin gene or mRNA, such as the gene or mRNA of α2-globin, α1-globin, or β-globin, for example, β-globin, such as human β-globin. For example, RNA (e.g., mRNA) can be modified by replacing or inserting an existing 3'-UTR with one or more copies (e.g., two copies) of a 3'-UTR derived from a globin gene (such as α2-globin, α1-globin, β-globin, for example, β-globin, such as human β-globin).

[0252] To improve mRNA stability and / or reduce immunogenicity or cytotoxicity, mRNA may contain modified ribonucleotides. For example, in some embodiments, uridine in the mRNA described herein is replaced by a modified nucleoside (partially or entirely, preferably entirely). In some embodiments, the modified nucleoside is a modified uridine.

[0253] In some implementations, the modified uridine that replaces uridine is selected from the group consisting of: pseudouridine (ψ), N1-methylpseudouridine (m1ψ), 5-methyluridine (m5U) and combinations thereof.

[0254] In some embodiments, the modifying nucleoside replacing uridine (partially or entirely, preferably entirely) in the mRNA can be any one or more of the following: 3-methyluridine (m3U), 5-methoxyuridine (mo5U), 5-azauridine, 6-azauridine, 2-thio-5-azauridine, 2-thiouridine (s2U), 4-thiouridine (s4U), 4-thiopseudoruridine, 2-thiopseudoruridine, 5-hydroxyuridine (ho5U), 5-aminoallyluridine, 5-halouridine (e.g., 5-iodouridine or 5-bromouridine), uridine 5-oxyacetic acid (cmo5U), methyl uridine 5-oxyacetate (mcmo5U), and 5-carboxymethyluridine (cm5U). 1-Carboxymethyl pseudouridine, 5-Carboxyhydroxymethyluridine (chm5U), 5-Carboxyhydroxymethyluridine methyl ester (mchm5U), 5-Methoxycarbonylmethyluridine (mcm5U), 5-Methoxycarbonylmethyl-2-thiouridine (mcm5s2U), 5-Aminomethyl-2-thiouridine (nm5s2U), 5-Methylaminomethyluridine (mnm5U), 1-Ethyl pseudouridine, 5-Methylaminomethyl-2-thiouridine (mnm5s2U), 5-Methylaminomethyl-2-selenouridine Uridine (mnm5se2U), 5-carbamoylmethyluridine (ncm5U), 5-carboxymethylaminomethyluridine (cmnm5U), 5-carboxymethylaminomethyl-2-thiouridine (cmnm5s2U), 5-propynyluridine, 1-propynyl pseudouridine, 5-tauronic acid methyluridine (τm5U), 1-tauronic acid methyl pseudouridine, 5-tauronic acid methyl-2-thiouridine (τm5s2U), 1-tauronic acid methyl-4-thiopseudouridine, 5-methyl-2-thiouridine Glycoside (m5s2U), 1-methyl-4-thiopseudouridine (m1s4ψ), 4-thio-1-methylpseudouridine, 3-methylpseudouridine (m3ψ), 2-thio-1-methylpseudouridine, 1-methyl-1-deazonopseudouridine, 2-thio-1-methyl-1-deazonopseudouridine, dihydrouridine (D), dihydrouridine, 5,6-dihydrouridine, 5-methyldihydrouridine (m5D), 2-thiodihydrouridine, 2-thiodihydrouridine, 2-methoxyuridine, 2-methoxy α-Thiouridine, 4-methoxy-2-thiouridine, N1-methyl-2-thiouridine, 3-(3-amino-3-carboxypropyl)uridine (acp3U), 1-methyl-3-(3-amino-3-carboxypropyl)-2-thiouridine (acp3ψ), 5-(isopentenylaminomethyl)uridine (inm5U), 5-(isopentenylaminomethyl)-2-thiouridine (inm5s2U), α-thiouridine, 2′-O-methyluridine (Um), 5,2′-O-dimethyluridine (m5Um), 2′-O-methylpseudouridine (ψm), 2-thio-2′-O-methyluridine (s2Um), 5-methoxycarbonylmethyl-2′-O-methyluridine (mcm5Um), 5-carbamoylmethyl-2′-O-methyluridine (ncm5Um), 5-carboxymethylaminomethyl-2′-O-methyluridine (cmnm5Um), 3,2′-O-dimethyluridine (m3Um), 5-(isopentenylaminomethyl)-2′-O-methyluridine (inm5Um), 1-thiouridine, deoxythymidine, 2′-F-arabinose-uridine, 2′-F-uridine, 2′-OH-arabinose-uridine, 5-(2-methoxycarbonylvinyl)uridine, 5-[3-(1-E-propenylamino)uridine, or any other modified uridine known in the art.

[0255] RNA (preferably mRNA) modified with pseudouridine (partially or completely, preferably completely replacing uridine) is referred to herein as "Ψ-modified RNA," and the term "m1Ψ-modified RNA" refers to RNA (preferably mRNA) containing N(1)-methylpseudouridine (partially or completely, preferably completely replacing uridine). Furthermore, the term "m5U-modified RNA" refers to RNA (preferably mRNA) containing 5-methyluridine (partially or completely, preferably completely replacing uridine). Such Ψ-modified, m1Ψ-modified, or m5U-modified RNAs generally exhibit lower immunogenicity compared to their unmodified forms and are therefore preferred in applications where it is necessary to avoid or minimize the induction of an immune response. In some embodiments, the RNA (preferably mRNA) contains N(1)-methylpseudouridine, which completely replaces uridine.

[0256] The codons of the mRNA used in this disclosure can be further optimized, for example, by increasing the GC content of the RNA and / or replacing rare codons in the cells (or subjects) where the target peptide or polypeptide is to be expressed with common codons that are synonymous in said cells (or subjects). In some embodiments, the amino acid sequence encoded by the mRNA used in this disclosure is encoded by a coding sequence that has undergone codon optimization and / or has an increased G / C content compared to the wild-type coding sequence. This also includes embodiments in which one or more sequence regions of the coding sequence have undergone codon optimization and / or have an increased G / C content compared to the corresponding sequence regions of the wild-type coding sequence. In some embodiments, codon optimization and / or the increase in G / C content preferably does not alter the sequence of the encoded amino acid sequence.

[0257] The term "codon optimization" refers to altering codons in the coding region of a nucleic acid molecule to reflect the typical codon usage habits of the host organism, but preferably without changing the amino acid sequence encoded by the nucleic acid molecule. In the context of this disclosure, codon optimization can be performed on the coding region to achieve optimal expression in subjects treated with the mRNA described herein. Codon optimization is based on the finding that translation efficiency also depends on the frequency of tRNA occurrence in the cell. Therefore, the mRNA sequence can be modified so that codons usable for common tRNAs are inserted into the positions of "rare codons."

[0258] In some implementations, the guanine / cytosine (G / C) content of the coding region of the mRNA described herein is increased compared to the G / C content of the corresponding coding sequence of the wild-type RNA, wherein the amino acid sequence encoded by the mRNA is preferably unmodified compared to the amino acid sequence encoded by the wild-type RNA. This modification of the mRNA sequence is based on the fact that the sequence of any RNA region to be translated is crucial for the efficient translation of that mRNA. Sequences with increased G (guanine) / C (cytosine) content are more stable than sequences with increased A (adenine) / U (uracil) content. Considering that multiple codons encode the same amino acid (i.e., so-called genetic code degeneracy), the codons most favorable for stability (i.e., so-called alternative codon usage) can be determined. Depending on the amino acid to be encoded by the mRNA, there are various ways to modify the mRNA sequence compared to its wild-type sequence. Specifically, codons containing A and / or U nucleotides can be modified by replacing these codons with other codons that encode the same amino acid but do not contain A and / or U, or contain a lower amount of A and / or U nucleotides.

[0259] In various implementation schemes, the G / C content of the mRNA coding region described herein is increased by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 55%, or even more compared to the G / C content of the wild-type RNA coding region.

[0260] The combination of the above modifications, namely the introduction of a 5'-cap structure, the introduction of a poly-A sequence, the demasking of a poly-A sequence, the alteration of the 5'- and / or 3'-UTR (such as the introduction of one or more 3'-UTRs), the replacement of one or more naturally occurring nucleotides with synthetic nucleotides (e.g., replacing cytidine with 5-methylcytidine and / or replacing uridine with pseudouridine (Ψ) or N(1)-methylpseudouridine (m1Ψ) or 5-methyluridine (m5U), and codon optimization, has a synergistic effect on improving the stability and translation efficiency of RNA (preferably mRNA). Therefore, in some embodiments, the mRNA used in this disclosure comprises a combination of at least two, at least three, at least four, or all five modifications described above, namely: (i) introducing a 5'-cap structure; (ii) introducing a poly-A sequence and demasking the poly-A sequence; (iii) altering the 5'- and / or 3'-UTR (such as introducing one or more 3'-UTRs); (iv) replacing one or more naturally occurring nucleotides with synthetic nucleotides (e.g., replacing cytidine with 5-methylcytidine and / or replacing uridine with pseudouridine (Ψ) or N(1)-methylpseudouridine (m1Ψ) or 5-methyluridine (m5U); and (v) codon optimization.

[0261] Certain aspects of this disclosure relate to the targeted delivery of the mRNAs disclosed herein to specific cells or tissues. In some embodiments, this disclosure relates to targeting the lymphatic system, particularly secondary lymphatic organs, more specifically the spleen. Targeting the lymphatic system, particularly secondary lymphatic organs, and more specifically, the spleen is preferred if the administered mRNA encodes an antigen or epitope for inducing an immune response. In some embodiments, the target cells are splenic cells. In some embodiments, the target cells are antigen-presenting cells, such as professional antigen-presenting cells in the spleen. In some embodiments, the target cells are dendritic cells in the spleen. The “lymphatic system” is part of the circulatory system and an important component of the immune system, comprising a network of lymphatic vessels that transport lymph fluid. The lymphatic system consists of lymphatic organs, a lymphatic vascular conduction network, and circulating lymph fluid. Primary or central lymphatic organs generate lymphocytes from immature progenitor cells. The thymus and bone marrow constitute primary lymphatic organs. Secondary or peripheral lymphatic organs, including lymph nodes and the spleen, maintain mature immature lymphocytes and initiate adaptive immune responses.

[0262] Lipid-based mRNA delivery systems have an inherent preference for the liver. Hepatic accumulation is due to discontinuities in the hepatic vascular system or lipid metabolism (liposomes conjugated with lipids or cholesterol). In some embodiments, the target organ is the liver, and the target tissue is liver tissue. Delivery to the target tissue is preferred, especially when the presence of mRNA or encoded peptides or polypeptides in the organ or tissue is required, and / or when high levels of the encoded peptides or polypeptides are required, and / or when systemic presence (particularly at significant levels) of the encoded peptides or polypeptides is required or demanded.

[0263] In some embodiments, after administration of the mRNA particles described herein, at least a portion of the mRNA is delivered to target cells or target organs. In some embodiments, at least a portion of the mRNA is delivered to the cytosol of target cells. In some embodiments, the mRNA is mRNA encoding a peptide or polypeptide, and the mRNA is translated by the target cells to produce the peptide or polypeptide. In some embodiments, the target cells are cells in the liver. In some embodiments, the target cells are muscle cells. In some embodiments, the target cells are endothelial cells. In some embodiments, the target cells are tumor cells or cells in the tumor microenvironment. In some embodiments, the target cells are blood cells. In some embodiments, the target cells are cells in lymph nodes. In some embodiments, the target cells are cells in the lungs. In some embodiments, the target cells are blood cells. In some embodiments, the target cells are cells in the skin. In some embodiments, the target cells are spleen cells. In one embodiment, the target cells are antigen-presenting cells, such as professional antigen-presenting cells in the spleen. In some embodiments, the target cells are dendritic cells in the spleen. In some embodiments, the target cells are T cells. In some embodiments, the target cells are B cells. In some embodiments, the target cells are NK cells. In other embodiments, the target cells are monocytes. Therefore, the RNA particles described herein can be used to deliver mRNA to such target cells.

[0264] Pharmaceutically active peptides or polypeptides

[0265] "Encoding" refers to the inherent characteristics of a specific nucleotide sequence in a polynucleotide (such as a gene, cDNA, or mRNA) that serves as a template for the synthesis of other polymers and macromolecules in biological processes. It has a defined nucleotide sequence (i.e., rRNA, tRNA, and mRNA) or a defined amino acid sequence and the biological characteristics it produces. Therefore, if the transcription and translation of the mRNA corresponding to a gene produces a protein in a cell or other biological system, then that gene encodes a protein. Both the coding strand (whose nucleotide sequence is identical to the mRNA sequence and is usually provided in a sequence listing) and the non-coding strand used as a template for gene or cDNA transcription can be referred to as encoding that protein or other products of that gene or cDNA.

[0266] In some embodiments, the compositions of the present invention / RNA obtained in the present invention comprise nucleic acid sequences encoding one or more functional sequences, said functional sequences being peptides or polypeptides, preferably pharmaceutically active peptides or polypeptides.

[0267] In a preferred embodiment, the nucleic acid, such as mRNA, used in this disclosure comprises a nucleic acid sequence encoding a peptide or polypeptide (preferably a pharmaceutically active peptide or polypeptide) and is capable of expressing said peptide or polypeptide, particularly if transferred to a cell or subject. Therefore, in some embodiments, the nucleic acid used in this disclosure comprises a coding region (open reading frame (ORF) encoding a peptide or polypeptide (e.g., encoding a pharmaceutically active peptide or polypeptide). In this respect, an "open reading frame" or "ORF" is a continuous codon sequence from the start codon to the stop codon. Such nucleic acids encoding pharmaceutically active peptides or polypeptides are also referred to herein as "pharmaceutically active nucleic acids." In particular, such mRNAs encoding pharmaceutically active peptides or polypeptides are also referred to herein as "pharmaceutically active mRNAs."

[0268] According to this disclosure, the term "pharmaceutical-active peptide or polypeptide" refers to a peptide or polypeptide that can be used to treat an individual, wherein the expression of the peptide or polypeptide will be beneficial, such as improving disease symptoms. Preferably, the pharmaceutically active peptide or polypeptide has curative or palliative properties and can be administered to improve, alleviate, reduce, reverse, delay the onset of, or reduce the severity of one or more symptoms of a disease. In some embodiments, the pharmaceutically active peptide or polypeptide, when administered to an individual in a therapeutically effective amount, has a positive or beneficial effect on the individual's condition or disease state. The pharmaceutically active peptide or polypeptide may have preventative properties and can be used to delay the onset of a disease or reduce the severity of such a disease. The term "pharmaceutical-active peptide or polypeptide" includes the whole peptide or polypeptide and may also refer to its pharmaceutically active fragment. It may also include pharmaceutically active variants and / or analogs of the peptide or polypeptide.

[0269] Specific examples of pharmaceutically active peptides and polypeptides include, but are not limited to, cytokines, hormones, adhesion molecules, immunoglobulins, immunoactive compounds, growth factors, protease inhibitors, enzymes, receptors, apoptosis regulators, transcription factors, tumor suppressor proteins, structural proteins, reprogramming factors, genome-engineered proteins, and blood proteins.

[0270] The term "cytokine" refers to proteins with a molecular weight of about 5 to 60 kDa that participate in cellular signaling, such as paracrine, endocrine, and / or autocrine signaling. In particular, when released, cytokines influence the behavior of cells surrounding their release site. Examples of cytokines include lymphokines, interleukins, chemokines, interferons, and tumor necrosis factor (TNF). According to this disclosure, cytokines do not include hormones or growth factors. Cytokines differ from hormones in that: (i) they typically act at concentrations that vary much more widely than hormones, and (ii) they are typically produced by a broad range of cells (almost all nucleated cells produce cytokines). Interferons are generally characterized by antiviral, antiproliferative, and immunomodulatory activities. Interferons are proteins that alter and regulate intracellular gene transcription by binding to interferon receptors on the surface of regulated cells, thereby preventing intracellular viral replication. Interferons can be classified into two types. IFN-γ is the only type II interferon; all others are type I interferons. Specific examples of cytokines include erythropoietin (EPO), colony-stimulating factor (CSF), granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), tumor necrosis factor (TNF), bone morphogenetic protein (BMP), interferon α (IFNα), interferon β (IFNβ), interferon γ (IFFγ), interleukin 2 (IL-2), interleukin 4 (IL-4), interleukin 10 (IL-10), interleukin 11 (IL-11), interleukin 12 (IL-12), interleukin 15 (IL-15), and interleukin 21 (IL-21), as well as their variants and derivatives.

[0271] In some embodiments, the pharmaceutically active peptide or polypeptide comprises a substitute protein. In these embodiments, this disclosure provides a method of treating a subject suffering from a condition requiring protein replacement (e.g., a protein deficiency condition), comprising administering to the subject a nucleic acid encoding a substitute protein as described herein. The term "protein replacement" refers to the introduction of a protein (including its functional variants) into a subject lacking such a protein. The term also refers to the introduction of a protein into a subject who would otherwise need or benefit from the provision of protein, such as someone suffering from protein insufficiency. The term "condition characterized by protein deficiency" refers to any condition presenting pathological manifestations caused by the absence or inadequacy of a protein. This term includes protein folding disorders, i.e., conformational disturbances of protein products leading to biological inactivation. Protein insufficiency may involve infectious diseases, immunosuppression, organ failure, glandular problems, radiation sickness, nutritional deficiencies, poisoning, or other environmental or external injuries.

[0272] The term "hormone" refers to a class of signaling molecules produced by glands, where signal transduction typically involves the following steps: (i) synthesis of the hormone in a specific tissue; (ii) storage and secretion; (iii) transport of the hormone to its target; (iv) binding of the hormone to a receptor; (v) signal transmission and amplification; and (vi) degradation of the hormone. Hormones differ from cytokines in that: (1) hormones typically act at relatively small concentrations, and (2) they are typically produced by specific types of cells. In some embodiments, "hormone" is a peptide or polypeptide hormone, such as insulin, vasopressin, prolactin, adrenocorticotropic hormone (ACTH), thyroid hormone, growth hormone (such as human growth hormone or bovine growth hormone), oxytocin, atrial natriuretic peptide (ANP), glucagon, somatostatin, cholecystokinin, gastrin, and leptin.

[0273] The term "adhesion molecule" refers to proteins located on the cell surface that are involved in the binding of the cell to other cells or to the extracellular matrix (ECM). Adhesion molecules are typically transmembrane receptors and can be classified as calcium-independent (e.g., integrins, immunoglobulin superfamily, lymphocyte homing receptors) and calcium-dependent (cadherins and selectins). Specific examples of adhesion molecules are integrins, lymphocyte homing receptors, selectins (e.g., P-selectin), and localizers.

[0274] Integrins also participate in signal transduction. In particular, upon ligand binding, integrins regulate cellular signaling pathways, such as those of transmembrane protein kinases like receptor tyrosine kinases (RTKs). This regulation can lead to cell growth, division, survival or differentiation, or apoptosis. Specific examples of integrins include: α1β1, α2β1, α3β1, α4β1, α5β1, α6β1, α7β1, αLβ2, αMβ2, αIIbβ3, αVβ1, αVβ3, αVβ5, αVβ6, αVβ8, and α6β4.

[0275] The term "immunoglobulin" or "immunoglobulin superfamily" refers to molecules involved in cell recognition, binding, and / or adhesion processes. Molecules belonging to this superfamily share the characteristic that they contain regions called immunoglobulin domains or folds. Members of the immunoglobulin superfamily include antibodies (e.g., IgG), T-cell receptors (TCRs), major histocompatibility complex (MHC) molecules, co-receptors (e.g., CD4, CD8, CD19), antigen receptor helper molecules (e.g., CD-3γ, CD3-δ, CD-3ε, CD79a, CD79b), co-stimulatory or inhibitory molecules (e.g., CD28, CD80, CD86), and others.

[0276] The term "immunoactive compound" refers to any compound that alters the immune response, such as by inducing and / or inhibiting the maturation of immune cells, inducing and / or inhibiting cytokine biosynthesis, and / or by stimulating B cells to produce antibodies. Immunoactive compounds possess potent immunostimulatory activity, including but not limited to antiviral and antitumor activity, and may also downregulate other aspects of the immune response, such as deviating from the TH2 immune response, which is useful for treating a wide range of TH2-mediated diseases. Immunoactive compounds can be used as vaccine adjuvants. Specific examples of immunoactive compounds include interleukins, colony-stimulating factors (CSF), granulocyte colony-stimulating factors (G-CSF), granulocyte-macrophage colony-stimulating factors (GM-CSF), erythropoietin, tumor necrosis factor (TNF), interferons, integrins, localizers, selectins, homing receptors, and antigens, particularly tumor-associated antigens, pathogen-associated antigens (such as bacterial, parasitic, or viral antigens), allergens, and autoantigens. Immunoactive compounds can be vaccine antigens, i.e., antigens administered to a subject to induce an immune response.

[0277] According to this disclosure, "antigen" encompasses any substance that will elicit an immune response and / or any substance capable of directing an immune response or immune mechanism such as a cellular response and / or humoral response. This also includes cases where the antigen is processed into an antigenic peptide and directs an immune response or immune mechanism against one or more antigenic peptides, particularly in the case of presentation in the context of MHC molecules. In particular, "antigen" refers to any substance, such as a peptide or polypeptide, that specifically reacts with an antibody or T lymphocyte (T cell). The term "antigen" may include a molecule comprising at least one epitope, such as a T cell epitope. In some embodiments, an antigen is optionally a molecule that, after processing, induces an immune response that may be specific to the antigen (including cells expressing the antigen). In some embodiments, the antigen is a disease-associated antigen, such as a tumor antigen, viral antigen, or bacterial antigen, or an epitope derived from such antigens.

[0278] The term "autoantigen" or "self-antigen" refers to an antigen originating from a subject's body (i.e., an autoantigen can also be called an "autogenous antigen") that elicits an abnormally strong immune response against that normal part of the body. Such a strong immune response against an autoantigen can be the cause of "autoimmune diseases."

[0279] According to this disclosure, any suitable antigen can be used as a candidate for an immune response, which can be both humoral and cellular immune responses. In the context of some embodiments of this disclosure, the antigen is presented by cells such as antigen-presenting cells in an MHC molecular background, resulting in an immune response against that antigen. The antigen can be a product corresponding to or derived from a naturally occurring antigen. Such naturally occurring antigens can include or may be derived from allergens, viruses, bacteria, fungi, parasites, and other infectious agents and pathogens, or the antigen may also be a tumor antigen. According to this disclosure, the antigen can correspond to a naturally occurring product, such as a viral protein or a portion thereof.

[0280] The term "disease-associated antigen," in its broadest sense, refers to any antigen associated with a disease. A disease-associated antigen is a molecule containing epitopes that stimulate the host's immune system to produce a cellular antigen-specific immune response and / or humoral antibody response against the disease. Disease-associated antigens include pathogen-associated antigens, i.e., antigens associated with microbial infections, typically microbial antigens (such as bacterial or viral antigens), or cancer-associated antigens, typically tumor antigens (such as tumor antigens).

[0281] In some embodiments, the antigen is a tumor antigen, i.e., part of tumor cells, particularly those that are primarily present within the cell or serve as tumor cell surface antigens. In another embodiment, the antigen is a pathogen-associated antigen, i.e., an antigen derived from a pathogen, such as a virus, bacteria, single-celled organism, or parasite, such as viral antigens like viral ribonucleoproteins or capsid proteins. In some embodiments, the antigen is presented by MHC molecules, which leads to the regulation, particularly activation, of immune system cells such as CD4+ and CD8+ lymphocytes, especially through the modulation of T cell receptor activity.

[0282] The term "tumor antigen" refers to a component of cancer cells that can originate from the cytoplasm, cell surface, or cell nucleus. Specifically, it refers to antigens produced within tumor cells or as tumor cell surface antigens. Examples of tumor antigens include carcinoembryonic antigen, alpha-1-fetoprotein, isoferrin and fetal sulfoglycoprotein, alpha-2-H-ferritin and gamma-alpha-fetoprotein, as well as various viral tumor antigens. According to some embodiments of this disclosure, tumor antigens include any antigens that are characteristic of a tumor or cancer in terms of type and / or expression level, and that are characteristic of tumors or cancer cells.

[0283] The term "viral antigen" refers to any viral component that possesses antigenic properties, meaning it can elicit an immune response in an individual. Viral antigens can be viral ribonucleoproteins or envelope proteins.

[0284] The term "bacterial antigen" refers to any bacterial component that possesses antigenic properties, meaning it can elicit an immune response in an individual. Bacterial antigens can originate from the bacterial cell wall or cytoplasmic membrane.

[0285] The term "epitope" refers to an antigenic determinant in a molecule, such as an antigen, that is, a portion or fragment of a molecule recognized by the immune system, for example, by antibodies, T cells, or B cells, particularly when presented in the context of MHC molecules. An epitope of a protein may comprise a continuous or discontinuous portion of the protein, and for example, its length may be about 5 to about 100, about 5 to about 50, about 8 to about 30, or about 10 to about 25 amino acids. For example, the length of an epitope may preferably be 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids. In some embodiments, within the context of this disclosure, the epitope is a T-cell epitope.

[0286] Terms such as “epitope,” “antigen fragment,” “immunogenic peptide,” and “antigen peptide” are used interchangeably herein and may, for example, refer to an incomplete representation of an antigen, such as one capable of evoking an immune response against that antigen or a cell that expresses, contains, and presents that antigen. In some embodiments, these terms refer to an immunogenic portion of an antigen. In some embodiments, it is an antigenic portion that is recognized (i.e., specifically bound) by T cell receptors, particularly when presented in the context of MHC molecules. Certain preferred immunogenic portions bind to class I or class II MHC molecules. The term “epitope” refers to a portion or fragment of a molecule, such as an antigen, that is recognized by the immune system. For example, an epitope can be recognized by T cells, B cells, or antibodies. Epitopes of an antigen may include continuous or discontinuous portions of the antigen and may be about 5 to about 100 amino acids in length, such as about 5 to about 50, about 8 to about 30, or about 8 to about 25 amino acids. For example, the length of an epitope may be 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids. In some embodiments, the length of an epitope is about 10 to about 25 amino acids. The term "eptope" includes T-cell epitopes.

[0287] The term "T-cell epitope" refers to a portion or fragment of a protein that is recognized by T cells when presented in the context of MHC molecules. The term "major histocompatibility complex" and the abbreviation "MHC" encompass both class I and class II MHC molecules and refer to gene complexes present in all vertebrates. MHC proteins or molecules are important for signaling between lymphocytes and antigen-presenting or diseased cells in immune responses, where MHC proteins or molecules bind peptide epitopes and present them to T-cell receptors on T cells for recognition. MHC-encoded proteins are expressed on the cell surface and present T cells with both self-antigens (peptide fragments from the cell itself) and non-self-antigens (e.g., fragments from invading microorganisms). In the case of class I MHC / peptide complexes, the length of the binding peptide is typically from about 8 to about 10 amino acids, although longer or shorter peptides may be effective. In the case of class II MHC / peptide complexes, the length of the binding peptide is typically from about 10 to about 25 amino acids, particularly from about 13 to about 18 amino acids, while longer and shorter peptides may be effective.

[0288] The length of peptide and polypeptide antigens can range from 2 to 100 amino acids, including, for example, 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids. In some embodiments, the peptide can be longer than 50 amino acids. In some embodiments, the peptide can be longer than 100 amino acids.

[0289] A peptide or polypeptide antigen can be any peptide or polypeptide that can induce or enhance the immune system’s ability to produce antibodies and T cell responses against that peptide or polypeptide.

[0290] In some embodiments, the vaccine antigen, i.e., the antigen administered to a subject to induce an immune response, is recognized by immune effector cells. In some embodiments, if the vaccine antigen is recognized by immune effector cells, it is able to induce stimulation, sensitization, and / or expansion of immune effector cells carrying antigen receptors that recognize the vaccine antigen in the presence of appropriate co-stimulatory signals. In the context of embodiments of this disclosure, the vaccine antigen may, for example, be presented or present on the surface of cells such as antigen-presenting cells. In some embodiments, the antigen is presented by diseased cells, such as tumor cells or infected cells. In some embodiments, the antigen receptor is a TCR that binds to an antigen epitope presented in an MHC context. In some embodiments, when expressed by and / or present on T cells, the binding of a TCR to an antigen presented by cells such as antigen-presenting cells results in stimulation, sensitization, and / or expansion of said T cells. In some embodiments, when expressed by and / or present on T cells, the binding of a TCR to an antigen presented on diseased cells results in cell lysis and / or apoptosis of said diseased cells, wherein said T cells release cytotoxic factors, such as perforin and granzymes.

[0291] According to some embodiments, the amino acid sequence that enhances antigen processing and / or presentation is fused directly or via a linker sequence to the antigenic peptide or polypeptide. Therefore, in some embodiments, the nucleic acid (such as RNA and / or DNA) described herein comprises at least one coding region encoding the antigenic peptide or polypeptide and an amino acid sequence that enhances antigen processing and / or presentation.

[0292] Such amino acid sequences that enhance antigen processing and / or presentation are preferably located at the C-terminus of the antigenic peptide or polypeptide and the linker sequence (and optionally at the C-terminus of the amino acid sequence that disrupts immune tolerance), but are not limited thereto. The amino acid sequences that enhance antigen processing and / or presentation as defined herein preferably improve antigen processing and presentation. In one embodiment, the amino acid sequences that enhance antigen processing and / or presentation as defined herein include, but are not limited to, sequences derived from human class I MHC complexes (HLA-B51, haplotypes A2, B27 / B51, Cw2 / Cw3). In addition to improving antigen processing and presentation, such amino acid sequences that enhance antigen processing and / or presentation can also be used to determine the expression of amino acid sequences in the processes described herein.

[0293] Therefore, in a particularly preferred embodiment, the RNA described herein comprises at least one coding region encoding an antigenic peptide or polypeptide and an amino acid sequence that enhances antigen processing and / or presentation, wherein the amino acid sequence that enhances antigen processing and / or presentation is preferably fused to the antigenic peptide or polypeptide, more preferably fused to the C-terminus of the antigenic peptide or polypeptide described herein.

[0294] In addition, the secretory sequence can be fused to the N-terminus of an antigenic peptide or polypeptide.

[0295] The amino acid sequence derived from tetanus toxoid can be used to overcome self-tolerance mechanisms, thereby effectively triggering an immune response against self-antigens by providing T-cell help during sensitization.

[0296] Tetanus toxoid heavy chain is known to contain epitopes that can confound class II MHC alleles and induce CD4+ memory T cells in almost all tetanus-vaccinated individuals. Furthermore, the combination of tetanus toxoid (TT) helper epitopes with tumor-associated antigens (TAAs) is known to improve immunostimulation compared to TAs alone by providing CD4+-mediated T cell helper during sensitization. To reduce the risk of stimulating CD8+ T cells with tetanus sequences, which may compete with the anticipated induction of tumor antigen-specific T cell responses, the complete tetanus toxoid fragment C is not used, as it is known to contain CD8+ T cell epitopes.

[0297] According to some implementation schemes, amino acid sequences that disrupt immune tolerance are fused directly or via linkers to antigenic peptides or polypeptides.

[0298] Such amino acid sequences that disrupt immune tolerance are preferably located at the C-terminus of the antigenic peptide or polypeptide (and optionally at the N-terminus of the amino acid sequence that enhances antigen processing and / or presentation, wherein the amino acid sequence that disrupts immune tolerance and the amino acid sequence that enhances antigen processing and / or presentation may be directly fused or fused via a linker. The amino acid sequence that disrupts immune tolerance as defined herein preferably improves T cell responses. In one embodiment, the amino acid sequence that disrupts immune tolerance as defined herein includes, but is not limited to, sequences derived from tetanus toxoid-derived helper sequences p2 and p16 (P2P16).

[0299] According to some implementation schemes, the bioluminescent amino acid sequence is fused directly or via a linker to an antigenic peptide or polypeptide.

[0300] Such bioluminescent amino acid sequences are preferably located at the C-terminus of the antigenic peptide or polypeptide (and optionally at the N-terminus of an amino acid sequence that enhances antigen processing and / or presentation or an amino acid sequence that disrupts immune tolerance), wherein the bioluminescent amino acid sequence may be directly fused to or fused via a linker with the amino acid sequence that enhances antigen processing and / or presentation or the amino acid sequence that disrupts immune tolerance. Bioluminescent amino acid sequences as defined herein preferably improve the determination of the amount of antigenic peptide or polypeptide. In some embodiments, the bioluminescent amino acid sequences as defined herein generate fluorescence. In some embodiments, the bioluminescent amino acid sequences as defined herein include, but are not limited to, sequences derived from green fluorescent protein (GFP), yellow fluorescent protein (YFP), red fluorescent protein (RFP), blue fluorescent protein (EBFP), cyan fluorescent protein (ECFP), their variants (such as enhanced GFP (EGFP), hyperfolded GFP (sfGFP)), and luciferases.

[0301] The implementation scheme for vaccine RNA is described below, and certain terms used in describing its elements have the following meanings:

[0302] hAg-Kozak: The 5'-UTR sequence of human α-globin mRNA, with an optimized 'Kozak sequence' to increase translation efficiency.

[0303] sec / MITD: A fusion protein tag derived from sequences encoding human class I MHC complexes (HLA-B51, haplotypes A2, B27 / B51, Cw2 / Cw3), which has been shown to improve antigen processing and presentation. Sec corresponds to a 78 bp fragment encoding a secretion signal peptide that guides the translocation of nascent polypeptide chains to the endoplasmic reticulum. MITD corresponds to the transmembrane and cytoplasmic domains of class I MHC molecules, also known as the class I MHC transport domain.

[0304] Antigen: The sequence that encodes the respective antigen / epitope.

[0305] Glycine-Serine Linker (GS): A sequence encoding a linker sequence according to the invention, in one embodiment, which is a glycine-serine linker sequence, a short linker peptide mainly composed of the amino acids glycine (G) and serine (S), commonly used in fusion proteins. In a specific embodiment of the invention, the linker sequence has a lysine residue preceding its N-terminus, and can be represented as follows: GGSGGGGSGGR / K. Therefore, a portion of the amino acid sequence containing the linker sequence can be represented as follows: KΔGGSGGGGSGGR / K (Δ represents a proteolytic cleavage site). Upon cleavage, this results in the excision of the linker sequence as follows: GGSGGGGSGGR / K. In one embodiment, the linker sequence of the invention is a GS linker each containing at least one residue that is not G or S, wherein the amino acid residue forms a proteolytic cleavage site for a proteolytic enzyme.

[0306] P2P16: A sequence that encodes a tetanus toxoid-derived helper epitope to disrupt immune tolerance.

[0307] The FI element: the 3'-UTR is a combination of two sequence elements derived from the "amino-terminal cleavage enhancer" (AES) mRNA (referred to as F) and the mitochondrial-encoded 12S ribosomal RNA (referred to as I). These were identified through an in vitro selection process of sequences that confer RNA stability and enhance total protein expression.

[0308] A30L70: A poly(A) tail of 110 nucleotides in length, consisting of 30 adenosine residues followed by a 10-nucleotide linker sequence and an additional 70 adenosine residues, designed to enhance RNA stability and translation efficiency in dendritic cells.

[0309] In one implementation, the vaccine RNA described herein has the following structure:

[0310] beta-S-ARCA(D1)-hAg-Kozak-sec-GS(1)-antigen-GS(2)-P2P16-GS(3)-MITD-FI-A30L70

[0311] In one implementation, the vaccine antigen described herein has the following structure:

[0312] sec-GS(1)-antigen-GS(2)-P2P16-GS(3)-MITD

[0313] In one implementation, multiple vaccine antigen RNA constructs (nucleic acids) as described herein are contained in a formulation such as a particle (LNP, LPX, PLX, etc.), wherein each vaccine RNA construct contains a different adapter sequence.

[0314] In some embodiments, the antigen receptor is an antibody or B-cell receptor that binds to an epitope of the antigen. In some embodiments, the antibody or B-cell receptor binds to the natural epitope of the antigen.

[0315] The terms "expressed on the cell surface" or "associated with the cell surface" refer to a molecule, such as an antigen, associating with and residing on the cell's plasma membrane, wherein at least a portion of the molecule faces the extracellular space of the cell and is accessible from the outside of the cell, for example, by an antibody located outside the cell. In this context, the portion may be, for example, at least 4, at least 8, at least 12, or at least 20 amino acids. The association can be direct or indirect. For example, the association can occur through one or more transmembrane domains, one or more lipid anchors, or through interaction with any other protein, lipid, sugar, or other structure that may be present on the outer leaflets of the cell's plasma membrane. For example, a molecule associated with the cell surface can be a transmembrane protein having an extracellular portion, or it can be a protein that associates with the cell surface through interaction with another protein that is a transmembrane protein.

[0316] The term "cell surface" or "the surface of the cell" is used in the normal sense in the art and therefore includes the cell exterior that can be approached and bound by proteins and other molecules. If an antigen is located on the surface of the cell and can be approached and bound by antigen-specific antibodies, for example, added to the cell, then the antigen is expressed on the cell surface.

[0317] In the context of this disclosure, the term "extracellular portion" or "extracellular domain" refers to a portion of a molecule, such as a protein, that faces the extracellular space of a cell and is preferably accessible from the outside of the cell, such as an antibody located outside the cell. In some embodiments, the term refers to one or more extracellular loops or domains or fragments thereof.

[0318] The terms “T cell” and “T lymphocyte” are used interchangeably herein and include both helper T cells (CD4+ T cells) and cytotoxic T cells (CTLs, CD8+ T cells), including cytolytic T cells. The term “antigen-specific T cell” or similar terms refer to T cells that recognize antigens targeting T cells, particularly when presented on the surface of antigen-presenting cells or diseased cells such as cancer cells in the context of MHC molecules, and preferably functioning as effectors. A T cell is considered specific to an antigen if it kills a target cell expressing that antigen. T cell specificity can be evaluated using any of a variety of standard techniques, such as in a chromium release assay or a proliferation assay. Alternatively, the synthesis of lymphokines (such as interferon-γ) can be measured.

[0319] The term "target" should refer to a functional factor, such as a cell or tissue, that acts as a target for an immune response, such as a cellular immune response. Targets include cells that present antigens or antigenic epitopes (i.e., peptide fragments derived from antigens). In some embodiments, target cells are cells that express antigens and present said antigens using class I MHC.

[0320] "Antigen processing" refers to the degradation of an antigen into processed products, which are fragments of the antigen (e.g., peptides degraded into peptides); and one or more of these fragments (e.g., by binding) associating with MHC molecules for presentation by cells such as antigen-presenting cells to specific T cells.

[0321] "Antigen-responsive CTL" refers to CD8+ T cells that respond to antigens or peptides derived from antigens presented on the surface of antigen-presenting cells by class I MHC.

[0322] According to this disclosure, CTL responsiveness can include sustained calcium flux, cell division, production of cytokines such as IFN-γ and TNF-α, upregulation of activation markers such as CD44 and CD69, and specific cytolytic killing of target cells expressing tumor antigens. CTL responsiveness can also be determined using artificial reporter genes that accurately indicate CTL responsiveness.

[0323] As used herein, “activation” or “stimulation” refers to a cellular state in which cells have been adequately stimulated to induce detectable cell proliferation, such as immune effector cells like T cells. Activation can also be associated with the initiation of signaling pathways, induced cytokine production, and detectable effector function. The term “activated immune effector cells” specifically refers to immune effector cells undergoing cell division.

[0324] The term "sensitization" refers to the process in which immune effector cells, such as T cells, come into contact with a specific antigen for the first time, leading to their differentiation into effector cells, such as effector T cells.

[0325] The term "amplification" refers to the process in which a specific entity is multiplied. In some embodiments, the term is used in the context of an immune response, where immune effector cells are stimulated by an antigen, proliferate, and specific immune effector cells that recognize said antigen are amplified. In some embodiments, amplification leads to the differentiation of immune effector cells.

[0326] The terms “immune response” and “immune reaction” are used interchangeably in their conventional sense herein, referring to a comprehensive bodily response to an antigen, which may refer to a cellular immune response, a humoral immune response, or both. According to this disclosure, with respect to an acting factor such as an antigen, cell, or tissue, the terms “immune response to” or “immune response against” refer to an immune response against that acting factor, such as a cellular response. An immune response may include one or more reactions selected from the group consisting of: the production of antibodies against one or more antigens and the expansion of antigen-specific T lymphocytes such as CD4+ and CD8+ T lymphocytes (e.g., CD8+ T lymphocytes), which can be detected in various in vitro proliferation or cytokine production assays.

[0327] In the context of this disclosure, the terms "inducing an immune response" and "initiating an immune response," and similar terms, refer to the induction of an immune response, such as the induction of a cellular immune response, a humoral immune response, or both. An immune response can be protective / prophylactic / preventive and / or therapeutic. An immune response can be against any immunogen or antigen or antigenic peptide, such as against tumor-associated antigens or pathogen-associated antigens (e.g., antigens of viruses such as influenza viruses (A, B, or C), CMV, or RSV). In this context, "inducing" can mean that no immune response against a specific antigen or pathogen exists prior to induction, but it can also mean that a certain level of immune response exists prior to induction and that the immune response is enhanced after induction. Therefore, "inducing an immune response" in this context also includes "enhancing an immune response." In some embodiments, after inducing an immune response in an individual, the individual is protected from developing a disease such as an infectious disease or cancer, or the disease condition is improved by inducing an immune response.

[0328] The terms “cellular immune response,” “cellular response,” “cell-mediated immunity,” or similar terms refer to cellular responses that include those against cells characterized by expressing antigens and / or presenting antigens using class I or class II MHC. Cellular responses involve cells called T cells or T lymphocytes, which act as “helpers” or “killers.” Helper T cells (also known as CD4+ T cells) play a central role in regulating the immune response, while killer cells (also known as cytotoxic T cells, cytolytic T cells, CD8+ T cells, or CTLs) kill cells such as diseased cells.

[0329] The term "humoral immune response" refers to the process in a living organism of producing antibodies in response to acting factors and organisms, ultimately neutralizing and / or eliminating these acting factors and organisms. The specificity of the antibody response is mediated by T and / or B cells through binding to membrane-associated receptors with a single specific antigen. Upon binding to the appropriate antigen and receiving various other activation signals, B lymphocytes divide, producing memory B cells and antibody-secreting plasma cell clones, each producing antibodies that recognize the same antigenic epitopes recognized by their antigen receptors. Memory B lymphocytes remain dormant until they are subsequently activated by their specific antigens. These lymphocytes provide the cellular basis of memory and the corresponding escalation of the antibody response upon re-exposure to a particular antigen.

[0330] As used herein, the term "antibody" refers to an immunoglobulin molecule that specifically binds to an epitope on an antigen. Specifically, the term "antibody" refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. The term "antibody" includes monoclonal antibodies, recombinant antibodies, human antibodies, humanized antibodies, chimeric antibodies, and combinations thereof. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL). The variable and constant regions are also referred to herein as variable domains and constant domains, respectively. The VH and VL regions can be further subdivided into hypervariable regions, called complementarity-determining regions (CDRs), interspersed with more conserved regions, called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The CDRs of VH are called HCDR1, HCDR2, and HCDR3, and the CDRs of VL are called LCDR1, LCDR2, and LCDR3. The variable regions of the heavy and light chains contain binding domains that interact with the antigen. The constant regions of an antibody include a heavy chain constant region (CH) and a light chain constant region (CL), where CH can be further subdivided into constant domain CH1, a hinge region, and constant domains CH2 and CH3 (arranged in the following order from the amino terminus to the carboxyl terminus: CH1, CH2, CH3). The constant regions of antibodies can mediate the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. Antibodies can be complete immunoglobulins derived from natural or recombinant sources and can be the immunologically active portion of a complete immunoglobulin. Antibodies are typically tetramers of immunoglobulin molecules. Antibodies can exist in a variety of forms, including, for example, polyclonal antibodies, monoclonal antibodies, Fv, Fab, and F(ab)2, as well as single-chain antibodies and humanized antibodies.

[0331] The term "immunoglobulin" refers to proteins in the immunoglobulin superfamily, such as antigen receptors like antibodies or B cell receptors (BCRs). Immunoglobulins are characterized by their structural domains, or immunoglobulin domains, which exhibit characteristic immunoglobulin (Ig) folds. The term encompasses both membrane-bound immunoglobulins and soluble immunoglobulins. Membrane-bound immunoglobulins, also known as surface immunoglobulins or membrane immunoglobulins, are typically part of the BCR. Soluble immunoglobulins are commonly referred to as antibodies. Immunoglobulins typically consist of several chains, typically two identical heavy chains and two identical light chains linked by disulfide bonds. These chains are primarily composed of immunoglobulin domains, such as the VL (variable light chain) domain, CL (constant light chain) domain, VH (variable heavy chain) domain, and CH (constant heavy chain) domains CH1, CH2, CH3, and CH4. Five types of mammalian immunoglobulin heavy chains exist: α, δ, ε, γ, and μ, which constitute different classes of antibodies: IgA, IgD, IgE, IgG, and IgM. In contrast to the heavy chains of soluble immunoglobulins, the heavy chains of membrane or surface immunoglobulins contain a transmembrane domain and a short cytoplasmic domain at their carboxyl terminus. In mammals, two types of light chains exist: λ and κ. Immunoglobulin chains contain variable and constant regions. The constant region is largely conserved across different isotypes of immunoglobulins, while the variable region is highly diverse and responsible for antigen recognition.

[0332] The terms “immunization” and “immunity” describe the process of treating an individual for therapeutic or preventative reasons and involve the administration of one or more immunogens or antigens or derivatives thereof to the individual, particularly in the form of RNA (especially mRNA) encoding them, as described herein, and the stimulation of an immune response against said one or more immunogens or antigens or cells characterized by presenting said one or more immunogens or antigens.

[0333] "Cells characterized by antigen presentation," "antigen-presenting cells," or "MHC molecules that present antigens on the surface of antigen-presenting cells," or similar expressions, refer to cells that present antigens or antigenic peptides directly or after processing in the context of MHC molecules such as class I MHC and / or class II MHC molecules, such as diseased cells, particularly tumor cells or infected cells, or antigen-presenting cells. In some embodiments, the MHC molecule is a class I MHC molecule.

[0334] The term "allergen" refers to an antigen that originates outside a subject (i.e., an allergen can also be called a "heterologous antigen") and elicits an abnormally strong immune response in the subject, in which the subject's immune system fights against a perceived threat that would otherwise be harmless to the subject. An "allergic reaction" is a disease caused by such a strong immune response to an allergen. Allergens are typically antigens that can stimulate a type I hypersensitivity response in atopic individuals through an immunoglobulin E (IgE) response. Specific examples of allergens include allergens derived from peanut proteins (e.g., Ara h 2.02), ovalbumin, grass pollen proteins (e.g., Phl p 5), and house dust mite proteins (e.g., Der p 2).

[0335] The term "growth factor" refers to molecules that can stimulate cell growth, proliferation, healing, and / or cell differentiation. Typically, growth factors act as signaling molecules between cells. The term "growth factor" includes specific cytokines and hormones that bind to specific receptors on the surface of their target cells. Examples of growth factors include bone morphogenetic protein (BMP), fibroblast growth factor (EGF), vascular endothelial growth factor (VEGF), such as VEGFA, epidermal growth factor (EGF), insulin-like growth factor, hepatocyte glycosides, macrophage colony-stimulating factor, granulocyte colony-stimulating factor, granulocyte-macrophage colony-stimulating factor, neuromodulatory proteins, neurotrophic factors (e.g., brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF)), placental growth factor (PGF), platelet-derived growth factor (PDGF), renin-angiotensin II receptor agonists (RNLS) (anti-apoptotic survival factor), T-cell growth factor (TCGF), thrombopoietin (TPO), transforming growth factors (transforming growth factor α (TGF-α), transforming growth factor β (TGF-β)), and tumor necrosis factor-α (TNF-α). In some embodiments, "growth factor" is a peptide or polypeptide growth factor.

[0336] The term "protease inhibitor" refers to molecules that inhibit the function of proteases, particularly peptides or polypeptides. Protease inhibitors can be classified by the protease they inhibit (e.g., aspartic protease inhibitors) or by their mechanism of action (e.g., suicide inhibitors, such as serine protease inhibitors). Specific examples of protease inhibitors include serine protease inhibitors, such as α1-antitrypsin, aprotinin, and ubenimex.

[0337] The term "enzyme" refers to a large molecular biological catalyst that accelerates a chemical reaction. Like any catalyst, an enzyme is not consumed in the reaction it catalyzes and does not alter the equilibrium of said reaction. Unlike many other catalysts, enzymes are more specific. In some embodiments, the enzyme is essential for the homeostasis of a subject; for example, any dysfunction of the enzyme (especially reduced activity or decreased production that may be caused by mutation, deletion, or other factors) can lead to disease. Examples of enzymes include herpes simplex virus type 1 thymidine kinase (HSV1-TK), hexosaminease, phenylalanine hydroxylase, pseudocholinesterase, and lactase.

[0338] The term "receptor" refers to a protein molecule that receives signals (particularly chemical signals called ligands) from outside the cell. The binding of a signal (e.g., a ligand) to a receptor elicits some type of cellular response, such as the activation of intracellular kinases. Receptors include transmembrane receptors (such as ion channel-coupled (ionotropic) receptors, G protein-coupled (metabolic) receptors, and enzyme-coupled receptors) and intracellular receptors (such as cytoplasmic receptors and nuclear receptors). Specific examples of receptors include steroid hormone receptors, growth factor receptors, and peptide receptors (i.e., receptors whose ligands are peptides), such as P-selectin glycoprotein ligand-1 (PSGL-1). The term "growth factor receptor" refers to a receptor that binds to growth factors.

[0339] The term "apoptotic regulators" refers to molecules, particularly peptides or polypeptides, that regulate apoptosis, either activating or inhibiting it. Apoptotic regulators can be divided into two main categories: those regulating mitochondrial function and those regulating caspases. The first category includes proteins that maintain mitochondrial integrity by preventing loss of mitochondrial membrane potential and / or the release of pro-apoptotic proteins such as cytochrome C into the cytoplasm (e.g., BCL-2, BCL-xL). This category also includes pro-apoptotic proteins that promote cytochrome C release (e.g., BAX, BAK, BIM). The second category includes proteins that inhibit caspase activation, such as apoptotic proteins (e.g., XIAP) or FLIP inhibitors.

[0340] The term "transcription factor" refers to proteins that regulate the rate at which genetic information is transcribed from DNA to messenger RNA, particularly by binding to specific DNA sequences. Transcription factors can regulate cell division, cell growth, and cell death throughout life; cell migration and organization during embryonic development; and / or responses to signals from outside the cell, such as hormones. Transcription factors contain at least one DNA-binding domain that binds to a specific DNA sequence, typically adjacent to the gene regulated by the transcription factor. Specific examples of transcription factors include MECP2, FOXP2, FOXP3, the STAT protein family, and the HOX protein family.

[0341] The term "tumor suppressor protein" refers to molecules, particularly peptides or polypeptides, that protect cells from stopping at a step in the pathway to cancer. Tumor suppressor proteins (usually encoded by corresponding tumor suppressor genes) exhibit attenuation or inhibition of cell cycle regulation and / or promote apoptosis. Their functions can be one or more of the following: inhibiting genes essential for cell cycle continuity; coupling the cell cycle to DNA damage (cell division should not occur as long as damaged DNA is present in the cell); initiating apoptosis if the damaged DNA cannot be repaired; metastasis inhibition (e.g., preventing tumor cell spread, blocking loss of contact inhibition, inhibiting metastasis); and DNA repair. Specific examples of tumor suppressor proteins include p53 protein, phosphatase and tensin homolog (PTEN), SWI / SNF (SWItch / sucrose non-fermentable complex), von Hippel-Lindau tumor suppressor protein (pVHL), adenomatous polyposis protein (APC), CD95 protein, tumor suppressor gene 5 protein (ST5), tumor suppressor gene 14 protein (ST14), and Yippee-like 3 protein (YPEL3).

[0342] The term "structural protein" refers to proteins that impart stiffness and rigidity to otherwise fluid biological components. Most structural proteins are fibrous (such as collagen and elastin), but they can also be globular (such as actin and tubulin). Typically, globular proteins are soluble as monomers but aggregate to form long fibers, which can, for example, form the cytoskeleton. Other structural proteins are motor proteins (such as myosin, kinesin, and dynein) and surfactant proteins. Specific examples of structural proteins include collagen, surfactant protein A, surfactant protein B, surfactant protein C, surfactant protein D, elastin, tubulin, actin, and myosin.

[0343] The term "reprogramming factor" or "reprogramming transcription factor" refers to molecules, particularly peptides or polypeptides, that, when expressed in somatic cells, optionally in conjunction with other agents such as other reprogramming factors, cause said somatic cells to be reprogrammed or dedifferentiated into cells with stem cell characteristics (especially pluripotency). Specific examples of reprogramming factors include OCT4, SOX2, c-MYC, KLF4, LIN28, and NANOG.

[0344] The term "genome-engineered protein" refers to proteins that can be inserted, deleted, or replaced in the DNA of a subject's genome. Specific examples of genome-engineered proteins include a wide range of nucleases, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly spaced short palindromic repeats-related protein 9 (CRISPR-Cas9).

[0345] The term "blood proteins" refers to peptides or polypeptides present in the plasma of a subject, particularly a healthy subject. Blood proteins have a variety of functions, such as transport (e.g., albumin, transferrin), enzymatic activity (e.g., thrombin or ceruloplasmin), blood clotting (e.g., fibrinogen), defense against pathogens (e.g., complement components and immunoglobulins), and protease inhibitors (e.g., α1-antitrypsin). Specific examples of blood proteins include thrombin, serum albumin, factor VII, factor VIII, insulin, factor IX, factor X, tissue plasminogen activator, protein C, von Willebrand factor, antithrombin III, glucocerebrosidase, erythropoietin, granulocyte colony-stimulating factor (G-CSF), modified factor VIII, and anticoagulants.

[0346] Therefore, in some embodiments, the pharmaceutically active peptide or polypeptide is (i) a cytokine, preferably selected from the group consisting of erythropoietin (EPO), interleukin-4 (IL-2), and interleukin-10 (IL-11), more preferably EPO; (ii) an adhesion molecule, particularly an integrin; (iii) an immunoglobulin, particularly an antibody; (iv) an immunologically active compound, particularly an antigen; (v) a hormone, particularly vasopressin, insulin, or growth hormone; (vi) a growth factor, particularly VEGFA; (vii) a protease inhibitor, particularly α1-antitrypsin; (viii) an enzyme, preferably selected from the group consisting of herpes simplex virus type 1 thymidine kinase (HSV1-TK), hexosaminease, phenylalanine hydroxylase, pseudocholinesterase, trypsin, and lactase; (ix) a receptor, particularly a growth factor receptor; (x) an apoptosis regulator, particularly BAX; (xi) a transcription factor, particularly FOXP3; (xii) Tumor suppressor proteins, particularly p53; (xiii) structural proteins, particularly surfactant protein B; (xiv) reprogramming factors, such as those selected from the group consisting of OCT4, SOX2, c-MYC, KLF4, LIN28, and NANOG; (xv) genome-engineered proteins, particularly clustered regularly spaced short palindromic repeats-CRISPR-associated protein 9 (CRISPR-Cas9); and (xvi) blood proteins, particularly fibrinogen.

[0347] In some embodiments, the pharmaceutically active peptide or polypeptide contains one or more antigens or one or more epitopes, i.e., administration of the peptide or polypeptide to a subject elicits an immune response in the subject against the one or more antigens or one or more epitopes, which may be therapeutic or partially or completely protective.

[0348] In some implementations, nucleic acids such as mRNA encode at least one epitope.

[0349] In some implementations, the epitope is derived from a tumor antigen. The tumor antigen can be a “standard” antigen, which is generally known to be expressed in various cancers. The tumor antigen can also be a “neoantigen,” which is specific to an individual tumor and has not previously been recognized by the immune system. Neoantigens or neoepitaxes can be generated by amino acid changes resulting from one or more cancer-specific mutations in the cancer cell genome. Examples of tumor antigens include, but are not limited to, p53, ART-4, BAGE, β-catenin / m, Bcr-abL, CAMEL, CAP-1, CASP-8, CDC27 / m, CDK4 / m, CEA, cell surface proteins of the tight junction protein family such as CLAUDIN-6, CLAUDIN-18.2 and CLAUDIN-12, c-MYC, CT, Cyp-B, DAM, ELF2M, ETV6-AML1, G250, GAGE, GnT-V, and Gap. 100, HAGE, HER-2 / neu, HPV-E7, HPV-E6, HAST-2, hTERT (or hTRT), LAGE, LDLR / FUT, MAGE-A, preferably MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A5, MAGE-A6, MAGE-A7, MAGE-A8, MAGE-A9, MAGE-A10, MAGE-A11 or MAGE-A12, MAGE-B, MAGE-C, MART-1 / Melan-A, MC1R, myosin White / m, MUC1, MUM-1, MUM-2, MUM-3, NA88-A, NF1, NY-ESO-1, NY-BR-1, p190 minor BCR-abL, Pml / RARa, PRAME, protease 3, PSA, PSM, RAGE, RU1 or RU2, SAGE, SART-1 or SART-3, SCGB3A2, SCP1, SCP2, SCP3, SSX, survival protein, TEL / AML1, TPI / m, TRP-1, TRP-2, TRP-2 / INT2, TPTE, WT and WT-1.

[0350] Cancer mutations vary from person to person. Therefore, cancer mutations encoding novel epitopes (neo-epitopes) represent attractive targets for vaccine composition and immunotherapy development. The efficacy of tumor immunotherapy depends on the selection of cancer-specific antigens and epitopes capable of inducing a potent immune response in the host. RNA can be used to deliver patient-specific tumor epitopes to patients. Dendritic cells (DCs) residing in the spleen represent antigen-presenting cells of particular interest for RNA expression of immunogenic epitopes or antigens such as tumor epitopes. The use of multiple epitopes has been shown to enhance the therapeutic efficacy of tumor vaccine compositions. Rapid sequencing of the tumor mutation genome can provide personalized vaccines with multiple epitopes, which can be encoded by mRNA as described herein, for example, as a single polypeptide, wherein the epitopes are optionally separated by a linker. In some embodiments of this disclosure, the mRNA encodes at least one epitope, at least two epitopes, at least three epitopes, at least four epitopes, at least five epitopes, at least six epitopes, at least seven epitopes, at least eight epitopes, at least nine epitopes, or at least ten epitopes. Exemplary implementations include mRNAs encoding at least five epitopes (referred to as "pentatopes") and mRNAs encoding at least ten epitopes (referred to as "decatopes").

[0351] In some embodiments, the antigen or epitope is derived from pathogen-associated antigens, particularly viral antigens. In some embodiments, the antigen or epitope is derived from the SARS-CoV-2 S protein, its immunogenic variants, or immunogenic fragments of the SARS-CoV-2 S protein or its immunogenic variants. Therefore, in some embodiments, the mRNA used in this disclosure encodes an amino acid sequence comprising the SARS-CoV-2 S protein, its immunogenic variants, or immunogenic fragments of the SARS-CoV-2 S protein or its immunogenic variants.

[0352] In some embodiments of this disclosure, the antigen (such as a tumor antigen or a vaccine antigen) is preferably administered as a single-stranded, 5'-capped mRNA, which is translated into the corresponding protein upon entering the cells of the subject to which the RNA is administered. Preferably, the RNA contains structural elements (5' cap, 5' UTR, 3' UTR, poly(A) sequence) optimized for maximum RNA efficiency in terms of stability and translation efficiency.

[0353] In some embodiments, beta-S-ARCA (D1) is used as a specific cap structure at the 5' end of the mRNA. In some embodiments, m27,3'-OGppp (m12'-O)ApG is used as a specific cap structure at the 5' end of the mRNA. In some embodiments, the 5'-UTR sequence is derived from human α-globin mRNA and optionally has an optimized 'Kozak sequence' to increase translation efficiency. In some embodiments, a combination of two sequence elements (FI element) derived from 'amino-terminal cleavage enhancer' (AES) mRNA (referred to as F) and mitochondrial-encoded 12S ribosomal RNA (referred to as I) is placed between the coding sequence and the poly(A) sequence to ensure higher maximum protein levels and extended mRNA durability. In some embodiments, two repeating 3'-UTRs derived from human β-globin mRNA are placed between the coding sequence and the poly(A) sequence to ensure higher maximum protein levels and extended mRNA durability. In some implementations, a 110-nucleotide poly(A) sequence is used, comprising a 30-nucleotide segment of adenosine residues, followed by a 10-nucleotide linker sequence and an additional 70 adenosine residues. This poly(A) sequence is designed to enhance RNA stability and translation efficiency.

[0354] In some embodiments, mRNA encoding an antigen (such as a tumor antigen or vaccine antigen) is expressed in the cells of the treated subject to provide the antigen. In some embodiments, the mRNA is transiently expressed in the subject's cells. In some embodiments, the mRNA is transcribed in vitro. In some embodiments, the antigen is expressed on the cell surface. In some embodiments, the antigen is expressed and presented in an MHC context. In some embodiments, the antigen is expressed into the extracellular space, i.e., the antigen is secreted.

[0355] Antigen molecules or their processed products, such as fragments thereof, can bind to antigen receptors such as BCRs or TCRs carried by immune effector cells, or bind to antibodies.

[0356] According to this disclosure, peptides and polypeptide antigens are provided to a subject by administering mRNA encoding peptide and polypeptide antigens, wherein the antigen is a vaccine antigen, preferably inducing an immune response, such as a humoral and / or cellular immune response in a subject provided with the peptide or polypeptide antigen. The immune response is preferably against a target antigen. Therefore, the vaccine antigen may comprise a target antigen, a variant thereof, or a fragment thereof. In some embodiments, such fragments or variants are immunologically equivalent to the target antigen. In the context of this disclosure, the terms "antigen fragment" or "antigen variant" refer to an agent that induces an immune response targeting that antigen, i.e., the target antigen. Therefore, a vaccine antigen may correspond to or may comprise a target antigen, may correspond to or may comprise a fragment of the target antigen, or may correspond to or may comprise an antigen homologous to the target antigen or a fragment thereof. Therefore, according to this disclosure, a vaccine antigen may comprise an immunogenic fragment of the target antigen or an amino acid sequence homologous to an immunogenic fragment of the target antigen. The "immunogenic fragment of the antigen" according to this disclosure preferably relates to an antigen fragment capable of inducing an immune response against the target antigen. The vaccine antigen may be a recombinant antigen.

[0357] The term "immunologic equivalence" refers to immunologically equivalent molecules, such as immunologically equivalent amino acid sequences, exhibiting the same or substantially the same immunological properties and / or exerting the same or substantially the same immunological effects, for example, regarding the type of immunological effect. In the context of this disclosure, the term "immunologic equivalence" is preferably used with respect to the immunological effects or properties of an antigen or antigen variant used for immunization. For example, an amino acid sequence is immunologically equivalent to a reference amino acid sequence if, when exposed to the immune system of a subject, it induces an immune response having a specificity for reacting with a reference amino acid sequence.

[0358] In some embodiments, the mRNA used in this disclosure is non-immunogenic. According to this disclosure, RNA encoding an immunostimulant can be administered to provide an adjuvant effect. The RNA encoding the immunostimulant can be a standard RNA or a non-immunogenic RNA.

[0359] As used herein, the term "non-immunogenic RNA" (such as "non-immunogenic mRNA") refers to RNA that, upon administration, for example, to mammals, does not induce an immune system response, or induces a response that is weaker than that induced by the same RNA differing only in that it has not undergone modifications and treatments that render the non-immunogenic RNA non-immunogenic, i.e., a weaker response than that induced by standard RNA (stdRNA). In some embodiments, non-immunogenic RNA, also referred to herein as modified RNA (modRNA), is made non-immunogenic by incorporating a modifying nucleotide that inhibits RNA-mediated activation of innate immune receptors into the RNA and / or removing double-stranded RNA (dsRNA).

[0360] To render non-immunogenic RNA (especially mRNA) non-immunogenic by incorporating a modified nucleoside, any modified nucleoside can be used, as long as it reduces or inhibits the immunogenicity of the RNA. Particularly preferred are modified nucleosides that inhibit RNA-mediated activation of innate immune receptors. In some embodiments, the modified nucleoside involves replacing one or more uridines with a nucleoside containing a modified nucleobase. In some embodiments, the modified nucleobase is a modified uracil. In some embodiments, the nucleoside comprising the modified nucleobase is 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), 4-thiopseudouridine, 2-thiopseudouridine, 5-hydroxyuridine (ho5U), 5-aminoallyluridine, 5-halouridine (e.g., 5-iodouridine or 5-bromouridine), uridine 5-oxyacetic acid (cmo5U), methyl uridine 5-oxyacetate (mcmo5U), and 5-carboxymethyluridine (cm5U). 1-Carboxymethyl pseudouridine, 5-carboxyhydroxymethyluridine (chm5U), 5-carboxyhydroxymethyluridine methyl ester (mchm5U), 5-methoxycarbonylmethyluridine (mcm5U), 5-methoxycarbonylmethyl-2-thiouridine (mcm5s2U), 5-aminomethyl-2-thiouridine (nm5s2U), 5-methylaminomethyluridine (mnm5U), 1-ethyl pseudouridine, 5-methylaminomethyl-2-thiouridine (mnm5s2U), 5-methylaminomethyl-2-selenouridine (mnm5se2U), 5-carbamoylmethyluridine (ncm5U), 5-carboxymethylaminomethyluridine (cmnm5U), 5-carboxymethylaminomethyl -2-Thiouridine (cmnm5s2U), 5-propynyluridine, 1-propynyl pseudouridine, 5-Taurine methyluridine (τm5U), 1-Taurine methyl pseudouridine, 5-Taurine methyl-2-thiouridine (τm5s2U), 1-Taurine methyl-4-thiopseudouridine, 5-Methyl-2-thiouridine (m5s2U), 1-Methyl-4-thiopseudouridine (m1s4ψ), 4-Thio-1-methylpseudouridine, 3-Methylpseudouridine (m3ψ), 2-Thio-1-methylpseudouridine, 1-Methyl-1-deazinopseudouridine, 2-Thio-1-methyl-1-deazinopseudouridine, dihydrouridine (D), dihydropseudouridine, 5,6-Dihydrouridine, 5-Methyldihydrouridine (m5D), 2-Thiodihydrouridine, 2-Thiodihydropseuuridine, 2-Methoxyuridine, 2-Methoxy-4-Thiouridine, 4-Methoxypseuuridine, 4-Methoxy-2-Thiopseuuridine, N1-Methylpseuuridine, 3-(3-amino-3-carboxypropyl)uridine (acp3U), 1-Methyl-3-(3-amino-3-carboxypropyl)pseuuridine (acp3ψ), 5-(isopentenylaminomethyl)uridine (inm5U), 5-(isopentenylaminomethyl)-2-thiouridine (inm5s2U), α-Thiouridine, 2′-O-methyluridine (Um), 5,2′-O-dimethyluridine (m5Um), 2′-O-methylpseuuridine (ψm) 2-Thio-2′-O-methyluridine (s2Um), 5-methoxycarbonylmethyl-2′-O-methyluridine (mcm5Um), 5-carbamoylmethyl-2′-O-methyluridine (ncm5Um), 5-carboxymethylaminomethyl-2′-O-methyluridine (cmnm5Um), 3,2′-O-dimethyluridine (m3Um), 5-(isopentenylaminomethyl)-2′-O-methyluridine (inm5Um), 1-thiouridine, deoxythymidine, 2′-F-arabinose-uridine, 2′-F-uridine, 2′-OH-arabinose-uridine, 5-(2-methoxycarbonylvinyl)uridine, 5-[3-(1-E-propenylamino)uridine. In some embodiments, the nucleoside containing the modified nucleobase is pseudouridine (ψ), N1-methylpseudouridine (m1ψ), or 5-methyluridine (m5U), particularly N1-methylpseudouridine.

[0361] In some embodiments, replacing one or more uridines with a nucleoside containing a modified nucleobase includes replacing at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 10%, at least 25%, at least 50%, at least 75%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of the uridine.

[0362] During mRNA synthesis via in vitro transcription (IVT) using T7 RNA polymerase, significant amounts of aberrant products, including double-stranded RNA (dsRNA), are generated due to the enzyme's unconventional activity. dsRNA induces inflammatory cytokines and activates effector enzymes, leading to inhibition of protein synthesis. dsRNA can be removed from RNA such as IVT RNA, for example, by using ion-pair reversed-phase HPLC with a non-porous or porous C-18 polystyrene-divinylbenzene (PS-DVB) matrix. Alternatively, an enzyme-based method can be used, employing *E. coli* RNase III, which specifically hydrolyzes dsRNA but not ssRNA, thereby eliminating dsRNA contaminants from the IVT RNA preparation. Furthermore, dsRNA can be separated from ssRNA by using a cellulose material. In some embodiments, the RNA preparation is contacted with a cellulose material, and ssRNA is separated from the cellulose material under conditions that allow dsRNA to bind to the cellulose material but do not allow ssRNA to bind to the cellulose material. Suitable methods for providing ssRNA are disclosed, for example, in WO 2017 / 182524.

[0363] As used herein, the term "removal" or "elimination" refers to the characteristic of the separation of a first substance group, such as non-immunogenic RNA, from the vicinity of a second substance group, such as dsRNA, wherein the first substance group is not necessarily devoid of the second substance, and the second substance group is not necessarily devoid of the first substance. However, the characteristic is that the first substance group with the second substance group removed has a measurably lower content of the second substance compared to an unseparated mixture of the first and second substances.

[0364] In some embodiments, removing dsRNA (especially mRNA) from non-immunogenic RNA includes removing dsRNA such that less than 10%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, less than 0.5%, less than 0.3%, or less than 0.1% of the RNA in the non-immunogenic RNA composition is dsRNA. In some embodiments, the non-immunogenic RNA (especially mRNA) contains no or substantially no dsRNA. In some embodiments, the non-immunogenic RNA (especially mRNA) composition comprises a purified preparation of single-stranded nucleoside-modified RNA. For example, in some embodiments, the purified preparation of single-stranded nucleoside-modified RNA (especially mRNA) is substantially free of double-stranded RNA (dsRNA). In some embodiments, the purified preparation contains at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% single-stranded nucleoside-modified RNA relative to all other nucleic acid molecules (DNA, dsRNA, etc.).

[0365] In some embodiments, the translation of non-immunogenic RNA (especially mRNA) in cells is more efficient than that of standard RNA having the same sequence. In some embodiments, translation is enhanced by 2-fold relative to its unmodified counterpart. In some embodiments, translation is enhanced by 3-fold. In some embodiments, translation is enhanced by 4-fold. In some embodiments, translation is enhanced by 5-fold. In some embodiments, translation is enhanced by 6-fold. In some embodiments, translation is enhanced by 7-fold. In some embodiments, translation is enhanced by 8-fold. In some embodiments, translation is enhanced by 9-fold. In some embodiments, translation is enhanced by 10-fold. In some embodiments, translation is enhanced by 15-fold. In some embodiments, translation is enhanced by 20-fold. In some embodiments, translation is enhanced by 50-fold. In some embodiments, translation is enhanced by 50-fold. In some embodiments, translation is enhanced by 1000-fold. In some embodiments, translation is enhanced by 2000-fold. In some embodiments, the fold increase is 10-1000-fold. In some embodiments, the fold increase is 10-100-fold. In some embodiments, the multiplier is 10-200 times. In some embodiments, the multiplier is 10-300 times. In some embodiments, the multiplier is 10-500 times. In some embodiments, the multiplier is 20-1000 times. In some embodiments, the multiplier is 30-1000 times. In some embodiments, the multiplier is 50-1000 times. In some embodiments, the multiplier is 100-1000 times. In some embodiments, the multiplier is 200-1000 times. In some embodiments, the translation enhances any other significant amount or range of amounts.

[0366] In some embodiments, non-immunogenic RNA (particularly mRNA) exhibits significantly lower innate immunogenicity than standard RNA having the same sequence. In some embodiments, non-immunogenic RNA (particularly mRNA) exhibits an innate immune response that is 2-fold lower than its unmodified counterpart. In some embodiments, innate immunogenicity is reduced by 3-fold. In some embodiments, innate immunogenicity is reduced by 4-fold. In some embodiments, innate immunogenicity is reduced by 5-fold. In some embodiments, innate immunogenicity is reduced by 6-fold. In some embodiments, innate immunogenicity is reduced by 7-fold. In some embodiments, innate immunogenicity is reduced by 8-fold. In some embodiments, innate immunogenicity is reduced by 9-fold. In some embodiments, innate immunogenicity is reduced by 10-fold. In some embodiments, innate immunogenicity is reduced by 15-fold. In some embodiments, innate immunogenicity is reduced by 20-fold. In some embodiments, innate immunogenicity is reduced by 50-fold. In some embodiments, innate immunogenicity is reduced by 100-fold. In some embodiments, innate immunogenicity is reduced by 200-fold. In some embodiments, innate immunogenicity is reduced by 500-fold. In some implementations, innate immunogenicity is reduced by 1000-fold. In some implementations, innate immunogenicity is reduced by 2000-fold.

[0367] The term "exhibiting significantly lower innate immunogenicity" refers to a detectable reduction in innate immunogenicity. In some embodiments, the term refers to a reduction such that an effective amount of non-immunogenic RNA (especially mRNA) can be administered without triggering a detectable innate immune response. In some embodiments, the term refers to a reduction such that non-immunogenic RNA (especially mRNA) can be repeatedly administered without triggering an innate immune response sufficient to detectably reduce the protein encoded by said non-immunogenic RNA. In some embodiments, the reduction is such that non-immunogenic RNA (especially mRNA) can be repeatedly administered without triggering an innate immune response sufficient to eliminate the detectable protein encoded by said non-immunogenic RNA.

[0368] Immunogenicity is the ability of a foreign substance, such as RNA, to elicit an immune response in a human or other animal. The innate immune system is a relatively nonspecific and immediate component of the immune system. It is one of the two main components of the vertebrate immune system, the other being the adaptive immune system.

[0369] Particles

[0370] The RNA-containing compositions of the present invention, or the RNA obtained in the present invention, may be present in particles comprising: (i) RNA; and (ii) at least one cationic or ionizable cationic compound, such as a polymer or lipid that forms a complex with RNA. Electrostatic interactions between positively charged molecules (such as polymers and lipids) and negatively charged nucleic acids participate in particle formation. This leads to the formation of complexes and the spontaneous formation of nucleic acid particles containing RNA.

[0371] Previous studies have described different types of RNA-containing particles suitable for delivering RNA in particulate form (e.g., see Kaczmarek, JC et al., 2017, Genome Medicine 9, 60). For non-viral RNA delivery vectors, the nanoparticle encapsulation of RNA physically protects the RNA from degradation and, depending on its specific chemical properties, can facilitate cellular uptake and endosome escape.

[0372] In the context of this disclosure, the term "particle" refers to a structural entity formed of molecules or molecular complexes, particularly particulate compounds. In some embodiments, the particle comprises a coating layer (e.g., one or more layers or sheets) consisting of one or more types of amphiphilic substances (e.g., amphiphilic lipids). In this context, the expression "amphiphilic substance" means that the substance is both hydrophilic and lipophilic. The coating layer may also comprise other substances (e.g., other lipids), which need not be amphiphilic. Thus, the particle can be a monolayer or multilayer structure, wherein the substances constituting one or more layers comprise one or more types of amphiphilic substances (particularly selected from the group consisting of various amphiphilic lipids), optionally combined with other substances (e.g., other lipids), which need not be amphiphilic. In some embodiments, the term "particle" refers to micrometer-scale or nanometer-scale structures, such as dense structures of the micrometer or nanometer size. According to this disclosure, the term "particle" includes nanoparticles.

[0373] "RNA particles" can be used to deliver RNA to target sites (such as cells, tissues, organs, etc.). RNA particles can be composed of lipids containing at least one cationic or ionizable cationic lipid or lipid-like substance. Although not bound by any particular theory, it is believed that cationic or ionizable cationic lipids or lipid-like substances bind to RNA to form aggregates, and this aggregation ultimately forms colloidally stable particles.

[0374] Nucleic acid particles (such as RNA particles) include formulations based on lipid nanoparticles (LNPs) and lipid complexes (LPXs).

[0375] Typically, liposome complexes (LPX) are obtained by mixing two aqueous phases: a phase containing nucleic acids (such as RNA and / or DNA) and a phase containing lipid dispersions. In some embodiments, the lipid phase contains liposomes.

[0376] In some embodiments, liposomes are self-enclosed monolayer or multilayer vesicle particles, wherein the layers comprise lipid bilayers and the enclosed cavities contain an aqueous phase. A prerequisite for using liposomes to form nanoparticles is that the desired lipids in the mixture are capable of forming a sheet-like (bilayer) phase in the applied aqueous environment.

[0377] In some embodiments, liposomes comprise a monolayer or multilayer phospholipid bilayer encapsulating an aqueous core (also referred to herein as an aqueous cavity). They can be prepared from materials having polar head (hydrophilic) groups and nonpolar tail (hydrophobic) groups. In some embodiments, the cationic lipids used to formulate liposomes intended for nucleic acid delivery are amphiphilic, consisting of a positively charged (cationic) amine head group linked to a hydrocarbon chain or cholesterol derivative via glycerol.

[0378] In some embodiments, the liposome complex is a formulation based on multilayered liposomes, formed by electrostatic interactions between cationic liposomes and nucleic acids (such as RNA and / or DNA). In some embodiments, the formed liposome complex has a unique internal molecular arrangement due to a shift in the liposome structure towards a compact nucleic acid-liposome complex (such as an RNA-liposome complex and / or a DNA-liposome complex). In some embodiments, these formulations are characterized by low encapsulation rates and incomplete encapsulation of nucleic acids (such as RNA).

[0379] In some embodiments, LPX particles comprise amphiphilic lipids, particularly cationic or ionizable cationic amphiphilic lipids, and nucleic acids (such as RNA and / or DNA, particularly mRNA), as described herein. In some embodiments, electrostatic interactions between positively charged liposomes (composed of one or more amphiphilic lipids, particularly cationic or ionizable cationic amphiphilic lipids) and negatively charged nucleic acids (particularly mRNA) lead to the formation of a complex and spontaneously form nucleic acid liposome complex particles. Positively charged liposomes are typically synthesized using cationic or ionizable cationic amphiphilic lipids (such as DOTMA and / or DODMA) and other lipids (such as DOPE). In some embodiments, the nucleic acid (such as RNA and / or DNA, particularly mRNA) liposome complex particle is a nanoparticle.

[0380] Typically, lipid nanoparticles (LNPs) can be obtained by directly mixing nucleic acids (such as RNA and / or DNA) in an aqueous phase with lipids in a phase containing an organic solvent (such as ethanol). In this case, lipids or lipid mixtures can be used for particle formation without forming a sheet-like (bilayer) phase in water.

[0381] In some embodiments, the LNP comprises or is composed of cationic / ionizable lipids and accessory lipids (such as phospholipids, cholesterol, and / or polyethylene glycol (PEG) lipids). In some embodiments, in the nucleic acid LNPs described herein (such as RNA LNPs, e.g., mRNA LNPs), nucleic acids (such as RNA, e.g., mRNA) are bound to ionizable lipids occupying the central core of the LNP. In some embodiments, PEG lipids co-form the surface of the LNP with phospholipids. In some embodiments, the surface comprises a bilayer. In some embodiments, charged and uncharged forms of cholesterol and ionizable lipids may be distributed throughout the LNP.

[0382] In some embodiments, nucleic acids (such as RNA and / or DNA, e.g., mRNA) may be non-covalently associated with the particle, as described herein. In some embodiments, nucleic acids (such as RNA and / or DNA, particularly mRNA) may adhere to the outer surface of the particle (surface nucleic acids (such as surface RNA, particularly surface mRNA)) and / or may be contained within the particle (encapsulated nucleic acids (such as encapsulated RNA, particularly encapsulated mRNA)).

[0383] In some embodiments, the size (e.g., diameter) of the particles described herein (e.g., LNP and LPX) is in the range of about 10 to about 2000 nm, such as at least about 15 nm (e.g., at least about 20 nm, at least about 25 nm, at least about 30 nm, at least about 35 nm, at least about 40 nm, at least about 45 nm, at least about 50 nm, at least about 55 nm, at least about 60 nm, at least about 65 nm, at least about 70 nm, at least about 75 nm, at least about 80 nm, at least about 85 nm, at least about 90 nm, at least about 95 nm, or at least about 100 nm) and / or at most 1900 nm (e.g., at most about 1900 nm, at most about 1800 nm, at most about 1700 nm, at most about 1600 nm, at most about 1500 nm, at most about 1400 nm, at most about 1300 nm, at most about 1200 nm, at most about 1100 nm). nm, up to about 1000 nm, up to about 950 nm, up to about 900 nm, up to about 850 nm, up to about 800 nm, up to about 750 nm, up to about 700 nm, up to about 650 nm, up to about 600 nm, up to about 550 nm or up to about 500 nm), such as in the range of about 20 to about 1500 nm, such as about 30 to about 1200 nm, about 40 to about 1100 nm, about 50 to about 1000 nm, about 60 to about 900 nm, about 70 to 800 nm, about 80 to 700 nm, about 90 to 600 nm, or about 50 to 500 nm, or about 100 to 500 nm, such as in the range of 10 to 1000 nm, 15 to 500 nm, 20 to 450 nm, 25 to 400 nm, 30 to 350 nm, 40 to 300 nm. The range is within the range of nm, 50 to 250 nm, 60 to 200 nm, or 70 to 150 nm.

[0384] In some embodiments, the particles described herein are nanoparticles. The term "nanoparticle" refers to nanoscale particles comprising the nucleic acids (particularly mRNA) described herein and at least one cationic or ionizable cationic lipid, wherein all three external dimensions of the particle are at the nanoscale, i.e., at least about 1 nm and less than about 1000 nm. Preferably, the size of the particle is its diameter.

[0385] Pharmaceutical compositions containing nucleic acid particles

[0386] In some embodiments, the compositions of the present invention (such as those prepared by the methods of the present invention) are pharmaceutical compositions. In one embodiment, the composition may comprise salts, buffers, or other components as further described below. In the most preferred embodiment, the composition comprises two or more different RNA molecules.

[0387] In some embodiments, the salt used in the compositions described herein comprises sodium chloride. While not wishing to be bound by theory, sodium chloride functions as an ion osmotic pressure regulator for pretreating nucleic acids (such as RNA and / or DNA) prior to mixing with lipids. In some embodiments, the compositions described herein may comprise alternative organic or inorganic salts. Alternative salts include, but are not limited to, potassium chloride, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, potassium acetate, potassium bicarbonate, potassium sulfate, disodium hydrogen phosphate, sodium dihydrogen phosphate, sodium acetate, sodium bicarbonate, sodium sulfate, lithium chloride, magnesium chloride, magnesium phosphate, calcium chloride, and sodium salts of ethylenediaminetetraacetic acid (EDTA).

[0388] Typically, compositions used for storing nucleic acid particles (such as those used for freezing nucleic acid particles) contain low concentrations of sodium chloride or have low ionic strength. In some embodiments, the concentration of sodium chloride is from 0 mM to about 50 mM, from 0 mM to about 40 mM, or from about 10 mM to about 50 mM.

[0389] According to this disclosure, the nucleic acid particle compositions described herein have a pH value suitable for the stability of nucleic acid particles, and particularly suitable for the stability of nucleic acids. It is not desirable to be bound by theory; a buffer system is used to maintain the pH value of the particle compositions described herein during the manufacture, storage, and use of the compositions. In some embodiments of this disclosure, the buffer system may include a solvent (particularly water, such as deionized water, particularly water for injection) and a buffering substance. The buffering substance may be selected from 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid (HEPES), 2-amino-2-(hydroxymethyl)propane-1,3-diol (Tris), acetate, and histidine. A preferred buffering substance is HEPES.

[0390] The compositions described herein may also contain cryoprotectants and / or surfactants as stabilizers to avoid significant loss of product quality, particularly to avoid significant loss of nucleic acid (especially mRNA) activity during storage, freezing and / or lyophilization, such as reducing or preventing aggregation, particle disintegration, nucleic acid (especially mRNA) degradation and / or other types of damage.

[0391] In one implementation, the cryoprotectant is a carbohydrate. As used herein, the term "carbohydrate" refers to and encompasses monosaccharides, disaccharides, trisaccharides, oligosaccharides, and polysaccharides.

[0392] In one embodiment, the cryoprotectant is a monosaccharide. As used herein, the term "monosaccharide" refers to a single carbohydrate unit (e.g., a simple sugar) that cannot be hydrolyzed into simpler carbohydrate units. Exemplary monosaccharide cryoprotectants include glucose, fructose, galactose, xylose, ribose, etc.

[0393] In one embodiment, the cryoprotectant is a disaccharide. As used herein, the term "disaccharide" refers to a compound or chemical moiety consisting of two monosaccharide units linked by a glycosidic bond (e.g., a 1-4 or 1-6 glycosidic bond). Disaccharides can be hydrolyzed into two monosaccharides. Exemplary disaccharide cryoprotectants include sucrose, trehalose, lactose, maltose, etc.

[0394] The term "trisaccharide" refers to three sugar molecules linked together to form a single molecule. Examples of trisaccharides include raffinose and pinotriose.

[0395] In one embodiment, the cryoprotectant is an oligosaccharide. As used herein, the term "oligosaccharide" refers to a compound or chemical moiety consisting of 3 to 15 (e.g., 3 to 10) monosaccharide units linked by glycosidic bonds (e.g., by 1-4 or 1-6 glycosidic bonds) to form a linear, branched, or cyclic structure. Exemplary oligosaccharide cryoprotectants include cyclodextrin, raffinose, melitriose, maltotriose, stachyose, acarbose, etc. Oligosaccharides can be oxidized or reduced.

[0396] In one embodiment, the cryoprotectant is a cyclic oligosaccharide. As used herein, the term "cyclic oligosaccharide" refers to a compound or chemical motif consisting of 3 to 15 (such as 6, 7, 8, 9, or 10) monosaccharide units linked by glycosidic bonds (e.g., by 1-4 or 1-6 glycosidic bonds) to form a cyclic structure. Exemplary cyclic oligosaccharide cryoprotectants include cyclic oligosaccharides that are discrete compounds, such as α-cyclodextrin, β-cyclodextrin, or γ-cyclodextrin.

[0397] Other exemplary cyclic oligosaccharide cryoprotectants include compounds that contain a cyclodextrin moiety within a larger molecular structure, such as a polymer containing a cyclic oligosaccharide moiety. Cyclic oligosaccharides can be oxidized or reduced, for example, oxidized to a dicarbonyl compound form. As used herein, the term "cyclodextrin moiety" refers to a cyclodextrin (e.g., α, β, or γ cyclodextrin) radical that is incorporated into or constitutes part of a larger molecular structure, such as a polymer. A cyclodextrin moiety can be directly or optionally linked to one or more other moieties. A cyclodextrin moiety can be oxidized or reduced, for example, oxidized to a dicarbonyl compound form.

[0398] Carbohydrate cryoprotectants, such as cyclic oligosaccharide cryoprotectants, can be derivatized carbohydrates. For example, in one embodiment, the cryoprotectant is a derivatized cyclic oligosaccharide, such as a derivatized cyclodextrin, such as 2-hydroxypropyl-β-cyclodextrin, such as a partially etherified cyclodextrin (e.g., partially etherified β-cyclodextrin).

[0399] An exemplary cryoprotectant is a polysaccharide. As used herein, the term "polysaccharide" refers to a compound or chemical motif consisting of at least 16 monosaccharide units linked by glycosidic bonds (e.g., by 1-4 or 1-6 glycosidic bonds) forming a linear, branched, or cyclic structure, including polymers in which a polysaccharide is part of its main chain structure. In the main chain, the polysaccharide can be linear or cyclic. Exemplary polysaccharide cryoprotectants include glycogen, amylase, cellulose, dextran, maltodextrin, etc.

[0400] In some embodiments, the nucleic acid particle composition may contain sucrose. Not wishing to be theoretically constrained, the role of sucrose is to promote cryoprotection of the composition, thereby preventing the aggregation of nucleic acid (especially mRNA) particles and maintaining the chemical and physical stability of the composition. In some embodiments, the nucleic acid particle composition may contain alternative cryoprotectants to sucrose. Alternative stabilizers include, but are not limited to, trehalose and glucose. In one specific embodiment, an alternative stabilizer to sucrose is trehalose or a mixture of sucrose and trehalose.

[0401] Preferred cryoprotectants are selected from the group consisting of sucrose, trehalose, glucose, and combinations thereof, such as a combination of sucrose and trehalose. In a preferred embodiment, the cryoprotectant is sucrose.

[0402] Some embodiments of this disclosure consider the use of chelating agents in the nucleic acid compositions described herein. A chelating agent is a compound capable of forming at least two coordinated covalent bonds with a metal ion, thereby generating a stable, water-soluble complex. Undesirably, chelating agents reduce the concentration of free divalent ions that might otherwise induce accelerated degradation of the nucleic acids described herein. Examples of suitable chelating agents include, but are not limited to, ethylenediaminetetraacetic acid (EDTA), EDTA salts, deferoxamine B, deferoxamine, sodium diethyldithiocarbamate, penicillamine, calcium valerate, sodium valerate, dimercaptosuccinic acid, trientine, hypozoxytriacetic acid, trans-diaminocyclohexanetetraacetic acid (DCTA), diethylenetriaminepentaacetic acid (DTPA), and bis(aminoethyl)ethylene glycol ether-N,N,N',N'-tetraacetic acid. In some embodiments, the chelating agent is EDTA or an EDTA salt. In one exemplary embodiment, the chelating agent is EDTA disodium dihydrate. In one embodiment, the molar concentration of EDTA is from about 0.05 mM to about 5 mM, from about 0.1 mM to about 2.5 mM, or from about 0.25 mM to about 1 mM.

[0403] In an alternative implementation, the nucleic acid particle composition described herein does not contain a chelating agent.

[0404] Compositions containing the nucleic acids described herein, optionally formulated into particles, may be used as or for the preparation of pharmaceutical compositions or drugs for therapeutic or preventative treatment.

[0405] The term "pharmaceutical composition" refers to a composition comprising a therapeutically effective ingredient, preferably comprising a pharmaceutically acceptable carrier, diluent, and / or excipient. The pharmaceutical composition can be used to treat, prevent, or reduce the severity of a disease by administration to a subject.

[0406] The pharmaceutical compositions disclosed herein may comprise one or more adjuvants, or may be used in combination with one or more adjuvants. The term "adjuvant" refers to a compound that prolongs, enhances, or accelerates an immune response. Adjuvants comprise a heterogeneous group of compounds, such as oil emulsions (e.g., Freund's adjuvant), mineral compounds (such as alum), bacterial products (such as pertussis toxin), or immunostimulatory complexes. Examples of adjuvants include, but are not limited to, LPS, GP96, CpG oligodeoxyribonucleotides, growth factors, and cytokines such as monocytokines, lymphokines, interleukins, and chemokines. Chemokines may be IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12, INFα, INF-γ, GM-CSF, and LT-α. Other known adjuvants are aluminum hydroxide, Freund's adjuvant, or oil formulations such as Montanide® ISA51. Other adjuvants suitable for use in this disclosure include lipopeptides, such as Pam3Cys, and lipophilic ingredients, such as saponins, trehalose-6,6-dibenzyl ester (TDB), monophospholipid A (MPL), monosaccharide glycerol (MMG), or glucopyranosyl lipid adjuvant (GLA).

[0407] The pharmaceutical compositions disclosed herein may be in a storable form (e.g., frozen or lyophilized / freeze-dried form) or an "ready-to-use form" (i.e., a form that can be immediately administered to a subject, e.g., without any treatment, such as dilution). Therefore, the storable form of the pharmaceutical composition must be processed or converted into a ready-to-use or administerable form before administration. For example, a frozen pharmaceutical composition must be thawed, or a lyophilized pharmaceutical composition must be reconstituted, for example using a suitable solvent (e.g., deionized water, such as water for injection) or a liquid (e.g., an aqueous solution).

[0408] The pharmaceutical compositions according to this disclosure are generally administered in a “pharmaceuticalally effective amount” and a “pharmaceuticalally acceptable formulation”.

[0409] The term "pharmaceutical acceptable" means that a substance is non-toxic and does not interact with the active ingredient in a pharmaceutical composition.

[0410] The term "pharmaceutical effective amount" refers to the amount, alone or in combination with subsequent doses, that achieves the desired response or effect. In some embodiments related to the treatment of a specific disease, the desired response may be related to inhibiting disease progression. This includes slowing disease progression, and in some embodiments, interrupting or reversing disease progression. In disease treatment, the desired response may also be the delay or prevention of the onset of the disease or condition. The effective amount of the pharmaceutical composition described herein depends on the condition being treated, the severity of the disease, the patient's individual parameters (including age, physical condition, body size, and weight), the duration of treatment, the type of concomitant therapy (if any), the specific route of administration, and similar factors. Therefore, the dosage of the pharmaceutical composition described herein may depend on several of the parameters mentioned above. If the initial dose is insufficient to elicit a response in the patient, a higher dose may be used (or a higher effective dose may be achieved through a different, more localized route of administration).

[0411] The pharmaceutical compositions disclosed herein may comprise buffers, preservatives, and optionally other therapeutic agents. In some embodiments, the pharmaceutical compositions disclosed herein comprise one or more pharmaceutically acceptable carriers, diluents, and / or excipients.

[0412] Preservatives suitable for use in the pharmaceutical compositions disclosed herein include, but are not limited to, benzalkonium chloride, chlorobutanol, p-hydroxybenzoate and thimerosal.

[0413] As used herein, the term "excipient" refers to a substance that may be present in the pharmaceutical compositions of this disclosure but is not an active ingredient. Examples of excipients include, but are not limited to, carriers, binders, diluents, lubricants, thickeners, surfactants, preservatives, stabilizers, emulsifiers, buffers, flavoring agents, or coloring agents.

[0414] The term "diluent" refers to an agent used for dilution and / or thinning. Furthermore, the term "diluent" also includes any one or more fluids, liquids, or solid suspensions and / or mixtures. Suitable examples of diluents include ethanol, glycerol, and water.

[0415] The term "carrier" refers to a component, which may be natural, synthetic, organic, or inorganic, to which the active component is combined to facilitate, enhance, or achieve administration of the pharmaceutical composition. Carriers as used herein may be one or more compatible solid or liquid fillers, diluents, or encapsulating substances suitable for administration to a subject. Suitable carriers include, but are not limited to, sterile water, Ringer's solution, lactated Ringer's solution, sterile sodium chloride solution, isotonic saline, polyalkylene glycols, hydrogenated naphthalene, and especially biocompatible lactic acid polymers, lactic acid / glycolic acid copolymers, or polyoxyethylene / polyoxypropylene copolymers. In some embodiments, the pharmaceutical compositions of this disclosure comprise isotonic saline.

[0416] Pharmaceutically acceptable carriers, excipients, or diluents for therapeutic use are well known in the pharmaceutical industry, as described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (A. RGennaro edit. 1985).

[0417] The drug carrier, excipient, or diluent can be selected based on the intended route of administration and standard pharmaceutical practice.

[0418] Route of administration of the pharmaceutical composition

[0419] In some embodiments, the pharmaceutical compositions described herein can be administered intravenously, intra-arterially, subcutaneously, intradermally, through the skin, intralymphaticly, intramuscularly, intratumorally, or peritumorally. In some embodiments, the pharmaceutical compositions are formulated for local or systemic administration. Systemic administration may include enteral administration (involving absorption via the gastrointestinal tract) or parenteral administration. As used herein, “parenteral administration” means administration by any means other than the gastrointestinal tract, such as intravenous injection. In some embodiments, the pharmaceutical compositions are formulated for systemic administration. In some embodiments, systemic administration is performed via intravenous administration.

[0420] Use of pharmaceutical compositions

[0421] Compositions containing the RNA of the present invention / RNA molecules obtained in the present invention (optionally formulated as granules) can be used to treat or prevent a variety of diseases, particularly those in which providing a subject with a peptide or polypeptide produces a therapeutic or preventative effect. For example, providing a viral antigen or epitope can be used to treat viral diseases caused by said virus. Providing a tumor antigen or epitope can be used to treat cancerous diseases in which cancer cells express said tumor antigen. Providing a functional protein or enzyme can be used to treat genetic diseases characterized by abnormally functioning proteins, such as lysosomal storage diseases (e.g., mucopolysaccharidosis) or factor deficiencies. Providing cytokines or cytokine fusion proteins can be used to modulate the tumor microenvironment.

[0422] The term "disease" (also referred to as "symptom" in this text) refers to an abnormal condition affecting an individual's body. Disease is generally understood as a medical condition associated with specific symptoms and signs. Diseases can be caused by factors originating from external sources, such as infectious diseases, or by internal dysfunctions, such as autoimmune diseases. In humans, the term "disease" is used more broadly, generally referring to any condition that causes pain, dysfunction, suffering, social problems, or death to the affected individual, or similar problems to those in contact with that individual. In this broad sense, it sometimes includes injury, disability, symptom, syndrome, infection, isolated symptoms, abnormal behavior, and atypical variations in structure and function, while in other contexts and for other purposes, these may be considered different categories. Diseases often affect not only an individual physically but also emotionally, as suffering from and coexisting with many diseases can alter a person's outlook on life and personality.

[0423] In the context of this invention, the terms "treatment," "being treated," or "therapeutic intervention" refer to the management and care of a subject in order to combat a condition such as a disease. This term is intended to include all treatment methods for a given condition suffered by the subject, such as administering compounds with effective therapeutic effects to relieve symptoms or complications, slow the progression of the disease, symptom, or condition, reduce or eliminate symptoms and complications, and / or cure or eliminate the disease, symptom, or condition, as well as preventing the condition, wherein prevention is to be understood as the management and care of an individual in order to combat a disease, condition, or symptom, including the administration of active compounds to prevent the occurrence of symptoms or complications.

[0424] The term "therapeutic treatment" refers to any treatment that improves an individual's health and / or prolongs (increases) lifespan. Such treatment may eliminate an individual's disease, stop or delay the development of an individual's disease, inhibit or slow the progression of an individual's disease, reduce the frequency or severity of an individual's symptoms, and / or reduce the recurrence rate of a disease in an individual who currently has or has previously had a certain disease.

[0425] The term “preventive treatment” or “preventive therapy” refers to any treatment designed to prevent an individual from developing a disease. As used herein, the terms “preventive treatment” or “preventive therapy” are used interchangeably.

[0426] As used herein, the terms “individual” and “subject” are used interchangeably. They refer to humans or other mammals (e.g., mice, rats, rabbits, dogs, cats, cattle, pigs, sheep, horses, or primates), or any other non-mammal, including birds (chickens), fish, or any other animal species that may have or be susceptible to a disease (e.g., cancer, infectious disease), but may or may not have the disease, or may require preventative interventions (such as vaccination), or may require interventions (such as protein replacement). In many embodiments, “individual” is a human being. Unless otherwise stated, the terms “individual” and “subject” do not indicate a specific age and therefore encompass adults, the elderly, children, and newborns. In some embodiments of this disclosure, “individual” or “subject” is a “patient.”

[0427] The term "patient" refers to an individual or subject receiving treatment, especially an individual or subject who is ill.

[0428] The compositions of the present invention / RNA obtained in the present invention can be administered to a subject to deliver nucleic acids into the subject's cells.

[0429] The compositions of the present invention / RNA obtained in the present invention can be administered to a subject to deliver a therapeutic or preventative peptide or polypeptide (e.g., a pharmacologically active peptide or polypeptide) to the subject, wherein the nucleic acid encodes a therapeutic or preventative peptide or polypeptide.

[0430] The compositions of the present invention / RNA obtained in the present invention can be administered to a subject for the treatment or prevention of a disease in the subject, wherein delivery of nucleic acids to the subject's cells is beneficial for the treatment or prevention of the disease.

[0431] The compositions of the present invention / RNA obtained in the present invention can be administered to a subject to treat or prevent a disease in the subject, wherein the RNA encodes a therapeutic or preventive peptide or polypeptide, and wherein delivery of the therapeutic or preventive peptide or polypeptide to the subject is beneficial to the treatment or prevention of the disease.

[0432] In some embodiments, RNA is present in the composition described herein.

[0433] In some implementations, RNA is administered in pharmaceutically effective amounts.

[0434] In some implementations, the subjects are mammals. In some implementations, the mammals are humans.

[0435] In some embodiments of this disclosure, the aim is to induce an immune response by providing a vaccine.

[0436] Those skilled in the art will understand that one of the principles of immunotherapy and vaccination is based on the fact that an immune protective response against a disease is generated by immunizing a subject with an antigen or epitope that is immunologically relevant to the disease to be treated. Therefore, the RNA described herein can be used to induce or enhance an immune response. The RNA described herein can therefore be used for the preventive and / or therapeutic treatment of diseases involving antigens or epitopes.

[0437] In some embodiments of this disclosure, the aim is to treat cancer through vaccination.

[0438] In some embodiments of this disclosure, the aim is to provide protection against infectious diseases through vaccination.

[0439] In some embodiments of this disclosure, the aim is to provide secretory therapeutic proteins, such as antibodies, bispecific antibodies, cytokines, cytokine fusion proteins, and enzymes, to subjects, particularly those in need.

[0440] In some embodiments of this disclosure, the aim is to provide subjects, particularly those in need, with protein replacement therapy, such as the production of erythropoietin, factor VII, von Willebrand factor, β-galactosidase, and α-N-acetylglucosidase.

[0441] In some embodiments of this disclosure, the aim is to modulate / reprogram immune cells in the blood.

[0442] In some embodiments of this disclosure, the aim is to provide subjects, particularly those in need, with one or more cytokines or cytokine fusion proteins that modulate the tumor microenvironment.

[0443] In some embodiments of this disclosure, the aim is to provide subjects, particularly those in need, with one or more cytokines or cytokine fusion proteins that have antitumor activity.

[0444] The documents and studies cited and referenced in this document are not intended to acknowledge any prior art. All statements regarding the contents of these documents are based on information available to the applicant and do not constitute any acknowledgment of the accuracy of those contents.

[0445] This specification (including the following examples) is intended to enable those skilled in the art to make and use various embodiments. The descriptions of specific apparatuses, techniques, and applications are provided by way of example only. Those skilled in the art can readily make various modifications to the embodiments described herein, and the general principles defined herein can be applied to other embodiments and applications without departing from the spirit and scope of the various embodiments. Therefore, the various embodiments are not intended to be limited to the embodiments described and shown herein, but should have the scope consistent with the claims.

[0446] Digital droplet PCR

[0447] In some embodiments, the present invention includes determining the amount of each RNA molecule in the composition.

[0448] Two or more different RNA molecules obtained by the methods and compositions of the present invention can be quantitatively determined by digital droplet polymerase chain reaction (ddPCR).

[0449] Specifically, this can be achieved as follows: First, reverse transcription is performed on the composition (“RNA sample” composition) ultimately obtained in this invention, which contains two or more different RNA molecules. This results in a composition containing n different cDNA (DNA) molecules (n = the number of different RNA molecules initially present in the composition). Then, a polymerase chain reaction (PCR)-based assay is performed using a first primer set and a single second primer, wherein the first primer set contains n primer species, each primer species being annealed to a first target region of only one of the n different DNA molecules in the sample (i.e., specific to each different DNA molecule in the sample), and the single second primer being annealed to a second target region of all n DNA molecules in the sample (i.e., shared for all DNA molecules in the sample). PCR is performed using these primers. The different DNA molecules produced by the PCR-based assay can be tandemly measured and quantified as substitutes for the RNA molecules initially present in the original RNA sample (this can be done using ddPCR, more details below). Therefore, such assays can measure the quantitative proportions of two or more RNA molecules in the original RNA sample in parallel.

[0450] Droplet digital PCR (ddPCR) measures the absolute quantity of nucleic acid molecules by counting the dispensing regions of discrete, volume-defined water-in-oil droplets. Commercially available ddPCR systems include the Droplet Digital™ PCR system (BioRad Laboratories, Hercules, CA, USA) or the Rain Drop Plus™ system (RainDance Technologies, Lexington, MA, USA). The PCR reaction is split into approximately 20,000 droplets, some containing and some not containing the template, resulting in PCR-positive and negative droplets. The template concentration is quantified by counting these droplets using a Poisson distribution algorithm. These droplets are generated by a droplet generator.

[0451] Compared to classical relative quantification methods, ddPCR achieves more precise absolute quantification, which is limited by the doubling of sample size in each cycle. ddPCR offers several advantages over traditional methods (e.g., qPCR) because it eliminates the need for a standard curve, is more robust (even suboptimal primer pairs that lead to false positives or false negatives eventually yield the correct concentration values ​​due to the Poisson distribution algorithm), and enables precise (diagnostic) quantification (standard qPCR has a resolution of 0.5 cycles (±50%), while ddPCR has a resolution of 10% (1.5% accuracy can be achieved using a sonic pipette)).

[0452] Therefore, in one embodiment, the method of the present invention further includes the step of determining the quantitative proportion of two or more RNA molecules present in the RNA sample composition obtained in the present invention. In one embodiment, this additional step includes the following sub-steps:

[0453] a) Reverse transcription of two or more (n) different RNA molecules in an RNA sample composition into cDNA molecules of n different DNA molecules; and

[0454] b) Perform a PCR-based assay on the obtained cDNA molecules using a first primer set and a single second primer, wherein the first primer set contains n primer species, each primer species being annealed to a first target region of only one of the n different DNA molecules in the sample, and the single second primer being annealed to a second target region of all n different DNA molecules in the sample; optionally, the PCR-based assay is ddPCR.

[0455] Example

[0456] Example 1: Generation of a 1:1:1 RNA composition from a PCR template

[0457] A composition containing three different RNA molecules in a 1:1:1 (by mass) ratio was produced using PCR template DNA starting material. This included IVT followed by tangential flow filtration (TFF), purification only, and sterile filtration. To achieve the desired ratio, it was determined that shorter RNA molecules required more frequent transcription (higher molar amounts of RNA molecules) to achieve the same mass (or mass concentration) as longer RNA molecules. Furthermore, determining the length of the DNA template was crucial. The DNA template is always longer than its corresponding RNA product because the DNA molecule must contain at least a promoter sequence; in this embodiment, a PCR template was included. Therefore, formula (i) was developed to take all these parameters into account:

[0458] (i)

[0459] The RNA fraction was determined based on a desired 1:1:1 ratio of the three RNA molecules (RNA1-1, RNA1-2, and RNA1-3). The total amount of DNA used was determined by titration to be 0.03 mg / mL. Based on this desired ratio, the total DNA concentration, and the known lengths of the DNA and RNA molecules, the initial concentration of each DNA template to be used was determined using Formula (i). The calculated values ​​are shown in Table 1.

[0460] Table 1 Calculate the PCR template DNA concentration of the 1:1:1 composition.

[0461]

[0462] In this particular embodiment, the very similar DNA and RNA molecule lengths, along with the same RNA fraction, resulted in similar concentrations of each DNA template molecule in the reaction mixture. Using the calculated DNA concentrations, IVT was performed, completing the entire process development pathway. The resulting RNA molecule proportions were within acceptable limits.

[0463] Example 2: Changing the parameters of a single-pot IVT reaction

[0464] As described in Example 1, PCR template DNA molecules were also used for single-pot IVT reactions in the following experiments. The parameters of the novel single-pot IVT reaction were varied, and the effects of these parameters were determined.

[0465] Example 2.1 Total DNA Concentration

[0466] In this experiment, the total amount of DNA (PCR template) was titrated, and the yield data were analyzed. The results are listed in Table 2 below.

[0467] Table 2 Effect of total DNA concentration on IVT yield

[0468]

[0469] IVT production increases with increasing DNA template concentration, such as Figure 1 As shown, the yield increase plateaued when the DNA concentration was between 0.03 and 0.05 mg / mL. For further experiments, the DNA template concentration was set at 0.03 mg / mL because the yield was high, but the DNA template concentration was low enough to reduce costs and the amount of protein that needed to be separated from the reaction was small.

[0470] Example 2.2 ATP / CTP concentration

[0471] The RNA sequences generated in the single-pot IVT reaction all exhibited high C and A content. Therefore, the effect of adjusting the initial concentrations of CTP and ATP was investigated. ATP and CTP were titrated from 9 mM to 12.6 mM. The results are listed in Table 3 below.

[0472] Table 3 Effects of ATP / CTP concentration on IVT production and RNA ratio

[0473]

[0474] Higher levels of ATP and CTP were determined to have a positive impact on integrity, yield, and dsRNA content. IVT yield was particularly significantly affected. Data have been plotted on [website / platform name - missing in original text]. Figure 2 IVT production is influenced by the additional ATP and CTP in the initial reaction. IVT production increases with increasing NTP levels (9 mM ATP / CTP as the initial condition), and plateaus with approximately 11.7 mM ATP / CTP.

[0475] Furthermore, the proportions of the three RNAs in the mixture were determined by digital droplet PCR (ddPCR). In this embodiment, where a 1:1:1 ratio was desired, each RNA theoretically accounted for 33%. The specification range was set at 20% of the theoretical value, which is an acceptable range of 33% ± 6%. The data showed that the proportions were correct for all ten reaction conditions and were independent of the initial CTP / ATP concentration (Table 3).

[0476] Additional titration experiments were performed to further characterize the concentration range. The tested ATP / CTP concentration range was 11.7 mM to 13.5 mM. The results are listed in Table 4 below.

[0477] Table 4 Results of the effect of ATP / CTP concentration on IVT yield and integrity

[0478]

[0479] As shown in Table 4, the integrity of IVT production increases with increasing ATP and CTP levels. Based on these data, a decrease in dsRNA slightly improves capping efficiency. For this type of reaction, ATP and CTP concentrations are considered optimal at 12.2 mM.

[0480] Other parameters

[0481] With the increase of ATP and CTP, it is speculated that the reaction requires more Mg. 2+ Cations, because they generate more PPi, cause more of these ions to precipitate out of the solution. To further investigate Mg... 2+ The effect of cations, titrating extra Mg 2+ However, adding more Mg 2+ The ratio of cations has no effect.

[0482] Example 3: Production of a 1:1:1 RNA composition from a linearized plasmid

[0483] Certain DNA sequences may be difficult to clone into a suitable PCR template form due to, for example, high CG content and high sequence repetitions, which can lead to problems with PCR template generation and specificity issues with analytical primers. Therefore, it may be advantageous to start a single-pot IVT reaction containing less refined forms of DNA, such as linearized plasmid DNA.

[0484] Unlike PCR DNA templates, when using linearized plasmid DNA to generate RNA molecules according to the present invention, the non-transcribed DNA differences between one DNA template and another are more significant. However, the robust and adaptable method developed by the inventors using formula (i) is able to account for this weighted variation.

[0485] Then, a single-pot IVT reaction was performed using linearized plasmids as DNA templates to provide a composition containing RNA1, RNA2, and RNA3 in a 1:1:1 ratio. The required concentrations of each linearized plasmid DNA template were calculated as shown in Table 5 below.

[0486] Table 5 Calculate the linearized plasmid DNA concentration of the 1:1:1 composition.

[0487]

[0488] Based on the determined desired DNA concentration, the sequence was then successfully transcribed in a single-pot reaction using linearized plasmid DNA.

[0489] Due to the high C content of RNA molecules, further investigation was conducted to determine whether the IVT reaction conditions could be optimized by including additional CTP at the start of the reaction. The results are shown in Table 6 below.

[0490] Table 6 The effect of CTP concentration on yield

[0491]

[0492] As shown in the table above, transcription yield increases with increasing CTP levels.

[0493] Example 4: Changing the proportion of obtained RNA molecules

[0494] A significant advantage of performing single-pot IVT reactions is the reduced time required. However, single-pot IVT reactions following formula (i) also benefit from improved flexibility. If the desired proportion of RNA molecules within a composition changes (e.g., during preclinical and clinical trials), single-pot IVT production can be easily adjusted to accommodate this change according to formula (i). To investigate the feasibility of this flexibility, different proportions were tested, i.e., by adding two, four, and eight times the amount of DNA template (for one template) to achieve different RNA fractions. The results showed that these changes in the DNA proportion input successfully corresponded to providing the correct proportion of RNA molecules in the resulting RNA composition after the single-pot IVT reaction.

[0495] The RNA fraction was initially set at 1:1:1 (RNA1:RNA2:RNA3) to obtain the corresponding mass concentration of RNA in the resulting composition. The potential benefits of obtaining, for example, a higher mass concentration of RNA3 for certain applications were then considered. Therefore, the flexibility of varying the RNA ratio was explored for subsequent applications. Different single-pot IVT experiments were performed, varying the mass concentration of the DNA template used to transcribe RNA3, and the RNA ratio in the resulting composition was measured. As a baseline, at least 0.01 μg / μL of each DNA template was used; for example, for a 1:1:4 ratio, 0.01 μg / μL of RNA1 and RNA2 DNA templates and 0.04 μg / μL of RNA3 DNA template were used. Data from the RNA3 DNA template titration were shown in... Figure 3 middle.

[0496] The detected RNA ratios were comparable to theoretical values. Therefore, it was determined that the RNA ratios could be rapidly adjusted by changing the concentration of the input DNA template used in a single-pot IVT reaction.

[0497] These experiments involved large amounts of DNA that needed to be removed by digestion with DNase I. Therefore, the inventors investigated whether a smaller amount of DNA could be used to reduce costs and the potential negative impact on dsRNA. In a second experiment, the total DNA template concentration of a 1:1:4 mixture was titrated. The results are listed in Table 7 below.

[0498] Table 7 Effect of total DNA concentration on yield

[0499]

[0500] As shown in the table above, IVT yield increases with increasing total DNA template concentration, but plateaus at a DNA concentration of 0.04 μg / μL. Further increases in DNA template concentration result in a slight increase in dsRNA content. The RNA ratio is unaffected by the total DNA template concentration; therefore, the optimal DNA concentration for this 1:1:4 single-pot IVT reaction is considered to be at least 0.04 μg / μL.

[0501] Furthermore, to obtain a complete dataset on the flexible alteration of RNA ratios in the method of this invention, the ratios of RNA1 and RNA2 were also varied. The data are shown in Figure 16, demonstrating the possibility of flexibly generating customized mixtures of three different RNA molecules.

[0502] Example 5: Production of a 1:1:1:1 RNA composition from a linearized plasmid

[0503] Compositions containing four different RNA molecules encoding eight different antigens were produced using the same method (each RNA molecule encoding two antigens). The task was to optimize the reaction for a 1:1:1:1 ratio. Linearized plasmid DNA molecules were used as starting materials. Linearized plasmid DNA molecules are longer than equivalent PCR templates because they contain, for example, selection marker genes and origins of replication. This means that non-transcribed DNA is a more significant factor in this experiment. Furthermore, the length of the RNA molecules obtained in this experiment was variable, with the shortest RNA being 1283 nt and the longest being 3002 nt. Using formula (i), the values ​​shown in Table 8 below were obtained.

[0504] Table 8 Calculate the linearized plasmid DNA concentration of the 1:1:1:1 composition.

[0505]

[0506] Single-pot IVT reactions were performed using the calculated concentrations, and the proportion of RNA produced was within acceptable limits.

[0507] Further investigation confirmed that the composition of the modified RNA molecules used, the composition of the buffer solution, the NTP composition, and the concentration of the capping reagent had no effect on the proportion of RNA molecules obtained.

[0508] Example 6: Determining the quantitative ratio of at least two different RNA molecules in the same RNA sample composition using ddPCR

[0509] This experiment further details the use of ddPCR to determine the quantitative ratio of (at least two) different RNA molecules present in the RNA sample composition finally obtained in this invention.

[0510] Materials and Methods:

[0511] cDNA synthesis

[0512] Complementary DNA synthesis was performed using the SuperScript IV First-Strand Synthesis Kit (Invitrogen) according to the manufacturer's protocol. The RNA mixture sample was diluted to 5 ng / µL. For a single reaction, 5 ng of RNA mixture, 1 µL of 10 µM cDNA primers, and 1 µL of 10 mM dNTPs were mixed, and the volume was adjusted to 13.5 µL with H2O. The cDNA primers were annealed at the 3' UTR to partially cover the poly(A) tail. Typically, a master mixture containing the RNA sample, primers, dNTPs, and water was prepared and aliquoted into four tubes for triplet measurements, with one negative control included. The RNA was denatured at 80°C for 5 min, rapidly cooled on ice for at least 1 min, and then 4 µL of 5× SuperScript IV buffer, 1 µL of 40 U / µL RNase inhibitor, 1 µL of 0.1 M DTT, and 0.5 µL of 200 U / µL SuperScript IV reverse transcriptase were added. For the negative control, 0.5 µL of water was added instead of reverse transcriptase. Incubate the samples in a PCR cycler using the following program: 55°C for 10 minutes, 80°C for 10 minutes, and store at 4°C. Then, add 0.5 µL of RNase H (2 U / µL) and incubate the samples at 37°C for 20 minutes. Store cDNA samples in low DNA binding tubes at -20°C or process directly for ddPCR.

[0513] Droplet digital PCR

[0514] Droplet digital PCR was performed on the QX200 / C1000 system (Bio-Rad) according to the manufacturer's instructions. cDNA was always freshly diluted to a concentration of approximately 1000 CN / µL. For a single reaction, 5.5 µL of cDNA, 11 µL of 2× ddPCRSuperMix (Bio-Rad), 0.25 µM of double-labeled HEX-BHQ1 probe, 0.9 µM of RNA-specific forward primer, and 0.9 µM of universal reverse primer were mixed to a final volume of 22 µL. Each sample was performed in triplicate, and a negative control was included for each sample. After droplet formation, the samples were incubated in a C1000 thermal cycler (Bio-Rad) and the following thermal cycling procedure was performed:

[0515] Activation step 1): 95°C for 600 seconds.

[0516] Denaturation step 2): 30 seconds at 94°C.

[0517] Annealing and extension step 3): 63°C for 60 seconds.

[0518] Enzyme inactivation step 4): 98°C for 600 seconds.

[0519] Steps 2 and 3 were repeated 40 times. After PCR was completed, the fluorescence of the droplets was read using a QX200 droplet reader (Bio-Rad).

[0520] Data Analysis

[0521] Data were analyzed using QuantaSoft version 1.7.4.0917 (Bio-Rad). QuantaSoft can usually automatically distinguish and define positive and negative groups. In rare cases where automatic distinction is not possible, thresholds are set manually. The software automatically calculates CN / µL. To calculate the RNA proportion, CN / µL is converted to mass / volume concentration (g / µL) using the following formula:

[0522]

[0523] Where MW is the molecular weight of RNA. The proportion (expressed as a percentage) is then calculated using the following formula:

[0524]

[0525] To calculate the proportions of RNA B, C, or D, the concentration of the corresponding RNA must be given in the molecule.

[0526] result:

[0527] To verify the accuracy of the RNA proportioning method, several solutions containing different RNAs mixed in varying proportions were measured. To assess the accuracy of the method, the "recovery rate %" was calculated using the following formula, which indicates the closeness of the measured value to the theoretical value:

[0528]

[0529] Recovery rates ranged from 89.3% to 119.4%. The ratio of the four RNAs was calculated as the ratio of the amount of one RNA to the amounts of the other three RNAs in the solution; that is, if the amount of one RNA is low, the amounts of the other three RNAs will increase in a complementary manner. Therefore, the same difference between the measured and theoretical values ​​will result in different recovery rate % values. To more comprehensively assess the accuracy of the method, the “delta” value was also evaluated, where delta is the mathematical distance between the measured value of two RNAs in a mixture and the target value. The maximum delta value for all tested RNA mixtures was 3, which further confirms the accuracy of the method.

[0530] Table 9 ddPCR was used to measure the quantitative ratio between at least two different RNA molecules present in the same composition.

[0531]

[0532] The data in Table 9 report the mean and standard deviation (SD) of three replicate experiments, as well as the recovery rate (mean of measured values ​​÷ theoretical value × 100) and delta value (mathematical distance between measured and theoretical values). Compare the "Theoretical RNA Proportion" column with the "Mean" column to verify whether the measured proportion reflects the theoretical value with minimal deviation.

[0533] Therefore, ddPCR can be used to verify and determine the quantitative ratio of at least two different RNA molecules present in the RNA sample composition of the present invention.

[0534] discuss

[0535] The single-pot IVT process is robust and scalable. Production consists of one day of IVT and the next day of purification. In contrast, traditional staged production of compositions containing three different RNA molecules results in a total production time of at least seven days, plus the need to mix the RNA molecules in the correct proportions at the end. Therefore, a single-pot IVT process was developed to maintain low production time and cost by producing three or more RNAs in the correct proportions in a single-pot reaction by mixing DNA templates. Surprisingly, this process, which occurs in a single reaction mixture, has been found to successfully deliver good yields of RNA molecules in the desired proportions.

[0536] It was also demonstrated that the RNA ratio can be flexibly altered using a single-pot IVT method by adjusting the DNA template concentration according to formula (i). This process showed that the total DNA concentration could be reduced to only 0.04 mg / mL, while successfully producing a composition containing three RNA molecules in the desired ratio.

Claims

1. A method for preparing a composition comprising two or more different RNA molecules, wherein each different RNA molecule can be obtained from a different DNA molecule, wherein the method comprises: a) Provide a reaction mixture comprising the DNA molecules, wherein each different DNA molecule (X) has a concentration according to formula (i): (i) in: "c(DNAx)" represents the concentration of a given DNA molecule X; c(DNA) 总 ")" represents the total concentration of all different DNA molecules; "RNA fraction" is the fraction of total RNA molecules in a composition containing RNA molecules that can be obtained from the DNA molecule X; "DNAx length fraction" is the base pair (bp) length of the DNA molecule X divided by the total bp length of all different DNA molecules; "RNAx length fraction" is the length in bp of the RNA molecule obtainable from the DNA molecule X divided by the total length in bp of all different RNA molecules; and b) Obtain the RNA molecule from the DNA molecule in the reaction mixture, thereby preparing the composition.

2. The method of claim 1, wherein step a) comprises determining the concentration of each DNA molecule using formula (i), and then providing a reaction mixture comprising each DNA molecule at its respective determined concentration.

3. The method according to claim 1 or 2, wherein different RNA molecules are obtained in a predetermined proportion in step b), and step a) comprises performing the following steps for each different DNA molecule: 1) Determine the length of the DNA molecule; 2) Determine the length of the RNA molecule that can be obtained from the DNA molecule; 3) Predetermine the ratio between at least two different RNA molecules obtained from the DNA molecule in step b); 4) Using formula (i), the length of the DNA molecule, and the length of the available RNA molecule, determine the concentration of DNA molecules for which the RNA molecule will be obtained in step b) at the predetermined ratio; and 5) Each DNA molecule is provided in the reaction mixture at the concentration stated therein.

4. The method according to any one of claims 1 to 3, wherein the RNA molecules are obtained in step b) at a predetermined ratio, wherein the ratio between at least two types of RNA molecules is not 1:

1.

5. The method according to any one of claims 1 to 4, wherein the base pair length difference between at least two DNA molecules exceeds 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 300%, or 400%.

6. The method according to any one of claims 1 to 5, wherein the base pair length difference between at least two RNA molecules exceeds 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%.

7. The method according to any one of claims 1 to 6, wherein the concentrations of at least two DNA molecules differ from the average concentration of the DNA molecules by more than 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%.

8. The method according to any one of claims 1 to 7, wherein the composition comprises at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 different RNA molecules.

9. The method according to any one of claims 1 to 8, wherein the RNA molecule is obtained from the DNA molecule by transcription.

10. The method according to any one of claims 1 to 9, wherein one or more DNA molecules contain a PCR template, or each DNA molecule contains a PCR template.

11. The method according to any one of claims 1 to 10, wherein one or more DNA molecules contain a linearized plasmid, or each DNA molecule contains a linearized plasmid.

12. The method according to any one of claims 1 to 11, wherein at least one DNA molecule contains a PCR template and at least one DNA molecule contains a linearized plasmid.

13. The method according to any one of claims 1 to 12, wherein each RNA molecule encodes at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 polypeptides.

14. The method according to any one of claims 1 to 13, wherein in step a), the total concentration of all DNA molecules in the reaction mixture is predetermined, preferably, wherein the total concentration of the DNA molecules is at least 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, or 10 mg / mL.

15. The method according to any one of claims 1 to 14, wherein the method further comprises: c) Determine the amount of each RNA molecule in the composition.

16. The method according to any one of claims 1 to 15, wherein the reaction mixture contains CTP and ATP at concentrations of at least 6 mM, preferably at least 9 mM, preferably at least 10 mM, preferably at least 11 mM, preferably at least 12 mM, preferably at least 13 mM, and preferably at least 14 mM.

17. The method according to any one of claims 1 to 16, wherein performing step b) lasts for at least 105 minutes, at least 180 minutes, or between 105 minutes and 180 minutes, including 105 minutes and 180 minutes.

18. A method for preparing a composition comprising at least a first RNA molecule and a second RNA molecule, wherein the first RNA molecule is obtainable from a first DNA molecule, and the second RNA molecule is obtainable from a second DNA molecule, wherein the method comprises: a) Provide a reaction mixture comprising the first DNA molecule and the second DNA molecule; and b) Obtain the first RNA molecule and the second RNA molecule from the first DNA molecule and the second DNA molecule, respectively; The relative mass of the first RNA molecule is greater than that of the second RNA molecule, and the molar amount of the second RNA molecule obtained from the second DNA molecule is greater than the molar amount of the first RNA molecule obtained from the first DNA molecule, such that the first RNA molecule and the second RNA molecule are obtained in the composition at substantially the same concentration.

19. A composition that is obtained or can be obtained by the method according to any one of claims 1 to 18.

20. The composition according to claim 19, used as a medicine.

21. A composition comprising two or more different DNA molecules, wherein different RNA molecules can be obtained from each different DNA molecule, wherein each different DNA molecule (X) has a concentration according to formula (i): (i) in: "c(DNAx)" represents the concentration of a given DNA molecule X; c(DNA) 总 ")" represents the total concentration of all DNA molecules; "RNA fraction" is the fraction of all RNA molecules that can be obtained from a reaction mixture containing RNA molecules that can be obtained from said DNA molecule X; "DNAx length fraction" is the base pair (bp) length of the DNA molecule X divided by the total bp length of all different DNA molecules; "RNAx length fraction" is the bp length of the RNA molecule that can be obtained from the DNA molecule X divided by the total bp length of all different RNA molecules.

22. A method for preparing a composition comprising two or more different RNA molecules, wherein each different RNA molecule can be obtained from a different DNA molecule, wherein the method comprises: a) Determine the concentration of each DNA molecule in the reaction mixture using formula (i), and then provide a reaction mixture containing each DNA molecule at its respective determined concentration: (i) in: "c(DNAx)" represents the concentration of a given DNA molecule X; c(DNA) 总 ")" represents the total concentration of all different DNA molecules; "RNA fraction" is the fraction of total RNA molecules in a composition containing RNA molecules that can be obtained from the DNA molecule X; "DNAx length fraction" is the base pair (bp) length of the DNA molecule X divided by the total bp length of all different DNA molecules; "RNAx length fraction" is the length in bp of the RNA molecule obtainable from the DNA molecule X divided by the total length in bp of all different RNA molecules; and b) Obtain the RNA molecule from the DNA molecule in the reaction mixture, thereby preparing the composition.

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