Process

Abstract

The present invention provides a method for determining at least one quality parameter of an RNA sample using reverse transcription and PCR, preferably droplet digital PCR. The quality parameter may be: (a) a quantitative ratio of two or more RNA molecule species in an RNA sample containing RNA molecules of n RNA molecule species; (b) identities of n RNA molecule species in the RNA sample, wherein n is an integer of at least 2; (c) integrity of an RNA sample containing RNA molecules of n RNA molecule species, wherein n is an integer of at least 1; or (d) the potency of a formulated RNA sample comprising the RNA molecule of interest.

Description

Technical Field

[0001] This invention relates to a method for quality control analysis of RNA samples (i.e., RNA mixtures) containing n different RNA molecule species. The method is suitable for quality control of RNA molecule mixtures during or after production. This invention relates to a method for determining at least one quality parameter of an RNA sample using reverse transcription and PCR (preferably droplet digital PCR). This quality parameter may be the quantitative ratio of two or more RNA molecule species in an RNA sample containing n RNA molecule species, or the identity of the n RNA molecule species in the RNA sample, where n is an integer of at least 2. Alternatively, the quality parameter may be the integrity of the RNA sample containing n RNA molecule species, where n is an integer of at least 1. Alternatively, the quality parameter may be the potency of a formulated RNA sample containing the RNA molecule of interest. This method is particularly suitable for analyzing mixtures of encapsulated and / or complexed RNA molecules, especially RNA molecule mixtures formulated with liposomes, lipid nanoparticles, and liposome complexes. Background Technology

[0002] RNA molecules represent a new class of drugs. RNA-based therapies can be used in immunotherapy, gene therapy, and gene vaccination, all of which belong to the most promising and fastest-growing therapeutic areas in modern medicine. RNA-based therapeutic agents can provide highly specific and personalized treatment options for a variety of diseases.

[0003] For certain medical treatments and applications, mixtures of RNA species are required. Examples of such RNA mixture-based therapies may include: using multivalent RNA mixtures that combat multiple pathogen serotypes (e.g., hemagglutinin (HA) from multiple influenza A and B virus serotypes); using RNA mixtures that provide different antigens from the same pathogen (e.g., different antigens from influenza viruses, such as HA, nucleoprotein (NP), neuraminidase (NA), etc.); using RNA mixtures that combat multiple isoforms or variants of cancer antigens (e.g., prostate-specific antigen (PSA) in prostate cancer cases); using RNA mixtures that provide different epitopes of antigens; using RNA mixtures that contain cancer-specific and / or patient-specific cancer antigen mixtures (expressed antigens or mutated antigens); RNA mixtures encoding multiple antibodies (e.g., antibodies targeting different epitopes of one or more proteins), or any other RNA mixture with therapeutic activity (e.g., different isoforms encoding enzymes for molecular therapy, or different therapeutic proteins encoding indications requiring the supplementation of multiple proteins).

[0004] For certain medical indications, RNA molecules in RNA (mixture) samples may be present in the form of a complex, i.e., in the form of at least one RNA-carrier complex. The compounding or encapsulation of RNA in an RNA-carrier complex facilitates successful in vivo delivery. Complex carrier compounds used in the art typically include various types of peptides, polymers, carbohydrates, cholesterol, polyethylene glycol (PEG), lipids, phospholipids, PEGylated lipids, cationic and polycationic compounds, and combinations thereof, as well as other carrier compounds that can assemble into RNA-carrier complexes.

[0005] For RNA-based therapeutics, it is necessary to characterize the presence, integrity, proportions, and quantities (quality control parameters) of the n different components (n different RNA molecule species, whether present in complex or free form) of the drug product and the active pharmaceutical ingredient. Such quality control can be performed during or after RNA sample preparation, and / or during or after RNA sample compounding, and / or as batch release quality control. Because RNA-based therapeutics may consist of multiple RNA species of highly similar size and sequence (e.g., multivalent vaccines composed of multiple similar antigens), standard quality control methods for distinguishing similarly sized RNA species, such as agarose gel electrophoresis or analytical high-performance liquid chromatography (HPLC), are not suitable. An ideal method for quality control of RNA mixtures should be rapid, stable, and cost-effective, capable of characterizing any or all of the following quality control parameters selected from the group consisting of: the presence, quantity, and integrity of at least one RNA molecule species, and the proportions of at least two RNA molecule species in an RNA sample containing n different RNA molecule species. Therefore, there is an urgent need for an RNA quality control analysis method, with particular emphasis on cost-effectiveness, stability, and the ability to distinguish highly similar RNA species in RNA samples containing n different RNA molecule species.

[0006] WO2018 / 211038 describes a method for quality control analysis of RNA samples containing n different RNA molecule species using reverse transcription and polymerase chain reaction (PCR)-based assays, wherein each of the n different RNA molecule species contains one or more synthetically derived coding RNA molecules, where n is an integer at least 1 and in some respects at least 2, thereby determining at least one quality parameter, and wherein the PCR-based assay is digital PCR (dPCR), preferably droplet digital PCR (ddPCR). This assay can be used to determine various quality parameters, including the number of RNA species present, the presence of one or more coding RNA molecules, the integrity of the RNA molecules, and the quantitation ratio among the n RNA species. A disadvantage of the method described in this disclosure is that it requires the introduction of artificial sequences into the RNA sequence, which are purely for analytical purposes and not for RNA function. Furthermore, the analysis of n RNA species requires multiple sets of primers.

[0007] C. Du Cheyne et al., Anal. Biochem. 626 (2021) 114217, describe a 3':5' digital PCR assay for determining the integrity of equine RNA. The assay involves first reverse transcribing RNA into DNA, followed by analysis of the DNA using three probes and three pairs of primers (located at the 3' end, 5' end, and center of the DNA sequence, respectively).

[0008] This method also has a drawback: when analyzing n types of RNA, multiple sets of primers are required. Summary of the Invention

[0009] In a first aspect, the present invention provides a method for determining the quality parameters of an RNA sample containing n types of RNA molecules, wherein n is an integer of at least 2, and the quality parameters are selected from the group consisting of:

[0010] i) The quantitative ratio of two or more RNA molecule types among the n RNA molecule types; and

[0011] ii) The identities of the n RNA molecule species in the RNA sample;

[0012] The method includes the following steps:

[0013] a) Reverse transcribe the n RNA molecules in the RNA sample into cDNA molecules of n DNA molecules; and

[0014] b) The obtained cDNA molecules were subjected to a polymerase chain reaction (PCR)-based assay using a first primer set and a single second primer, wherein...

[0015] The first primer set contains n primer types, each primer type being capable of annealing to a first target region consisting of only one of the n DNA molecule types in the sample; and

[0016] The single second primer can anneal to the second target region of all n types of DNA molecules in the sample.

[0017] The quality parameter can be the quantitative ratio of two or more RNA molecule types out of n RNA molecule types. Therefore, in a second aspect, the present invention provides a method for determining the quantitative ratio of two or more RNA molecule types in an RNA sample containing n RNA molecule types, wherein n is an integer of at least 2, the method comprising the following steps:

[0018] a) Reverse transcribe the n RNA molecules in the RNA sample into cDNA molecules of n DNA molecules; and

[0019] b) The obtained cDNA molecules were subjected to a polymerase chain reaction (PCR)-based assay using a first primer set and a single second primer, wherein...

[0020] The first primer set contains n primer types, each primer type being capable of annealing to a first target region consisting of only one of the n DNA molecule types in the sample; and

[0021] The single second primer can anneal to the second target region of all n types of DNA molecules in the sample.

[0022] The quality parameter can also be the identity of the n RNA molecule species in the RNA sample. Therefore, in a third aspect, the present invention provides a method for determining the identity of the n RNA molecule species in an RNA sample containing n RNA molecule species, where n is an integer of at least 2, the method comprising the following steps:

[0023] a) Reverse transcribe the n RNA molecules in the RNA sample into cDNA molecules of n DNA molecules; and

[0024] b) The obtained cDNA molecules were subjected to a polymerase chain reaction (PCR)-based assay using a first primer set and a single second primer, wherein...

[0025] The first primer set contains n primer types, each primer type being capable of annealing to a first target region consisting of only one of the n DNA molecule types in the sample; and

[0026] The single second primer can anneal to the second target region of all n types of DNA molecules in the sample.

[0027] In a fourth aspect, the present invention provides a method for determining the integrity of an RNA sample containing n types of RNA molecules, wherein n is an integer of at least 1.

[0028] The method includes the following steps:

[0029] a) Reverse transcribe the n RNA molecules in the RNA sample into cDNA molecules of n different DNA types; and

[0030] b) The obtained cDNA molecules were subjected to a polymerase chain reaction (PCR) assay using a first primer set, a single second primer, a third primer set, and a single fourth primer.

[0031] The first primer set contains n primer types, each of which can anneal to the first target region at the 3' end region of the DNA molecule in the sample.

[0032] The single second primer can anneal to the second target region at the 3' end region of all n types of DNA molecules in the sample;

[0033] The third primer set contains n primer types, each of which can anneal to the third target region at the 5' end of a DNA molecule in the sample; and

[0034] The single fourth primer can anneal to the fourth target region at the 5' end region of all n types of DNA molecules in the sample.

[0035] In a fifth aspect, the present invention provides a method for determining the potency of a prepared RNA sample containing an RNA molecule of interest, the method comprising the following steps:

[0036] a) Provide RNA samples isolated from cells that have been transfected with prepared RNA samples;

[0037] b) Reverse transcribe the RNA molecules in the RNA sample into cDNA molecules;

[0038] c) The obtained cDNA molecules are subjected to a polymerase chain reaction (PCR)-based assay using primers 1, 2, 3, and 4, wherein...

[0039] The first primer and the second primer are capable of annealing to the first and second target regions of the cDNA molecule generated from the RNA of interest in the sample, and

[0040] The third and fourth primers are capable of annealing to the first and second target regions of cDNA molecules derived from endogenous RNA in the sample; and

[0041] d) Compare the measured amount of cDNA produced by the RNA molecule of interest with the measured amount of cDNA produced by endogenous RNA.

[0042] In the methods described in the above aspects of this invention, PCR-based assays may use detectable markers. In any of these methods, the detectable marker may be a fluorescent probe. Attached Figure Description

[0043] Figure 1 The percentage of integrity of each RNA that makes up different mixtures of degraded RNA is shown, as measured using ddPCR.

[0044] Figure 2 The copy number (CN) measured by digital droplet polymerase chain reaction (ddPCR) is shown, which measures the power of total RNA of interest (RNA) isolated from Chinese hamster ovary (CHO) cells compared with housekeeping genes. The CHO cells had been previously transfected with four different amounts of formulated RNA of interest.

[0045] Advantages and surprising discoveries

[0046] The inventors have surprisingly discovered that a PCR-based assay can simultaneously measure the quantification ratio of two or more RNA molecule species in a raw RNA sample, the identity of n RNA molecule species in the raw RNA sample, and the integrity of the n RNA molecule species (optionally, these can also be measured in parallel). The PCR-based assay uses a first primer set and a single second primer, wherein the first primer set contains n primer species, each primer species capable of annealing to a first target region of only one of the n DNA molecule species in the sample (i.e., specific to each DNA molecule species in the sample), while the single second primer can anneal to a second target region of all n DNA molecule species in the sample (i.e., universal for all DNA molecule species in the sample). In both respects, this method has advantages over the method described in WO2018 / 211038, particularly in that the experimental system is simplified compared to the method in WO2018 / 211038, given that the two components in each oligonucleotide set (i.e., a universal primer and a universal double-labeled probe) are identical for all RNAs, and only one primer in each set is RNA-specific. This reduces the amount of reagents required, system complexity, and pipetting time compared to assembling two sets of oligonucleotides that are specific to each free RNA.

[0047] The inventors also unexpectedly discovered that the PCR method using four primer sets defined herein has advantages in determining the integrity of n RNA species in a raw RNA sample compared to the methods described in WO2018 / 211038 and Du Cheyne et al. Similar to the identification and ratio determination methods described above, this method has the following advantages compared to the methods described in the two documents: the experimental system is simplified compared to the methods in the two documents: the two components in each oligonucleotide set (i.e., a universal primer and a universal double-labeled probe) are identical for all RNAs, and only one primer in each set is RNA-specific. This reduces the required reagent volume, system complexity, and pipetting time compared to assembling two sets of oligonucleotides each composed of RNA-specific oligonucleotides. This method can also determine the identification and quantification ratio of RNA species in parallel with determining RNA species integrity.

[0048] Finally, the inventors also unexpectedly discovered that, compared with the method described in WO2018 / 211038, the PCR method using the four primer sets defined herein also allows for a simplified determination of the effectiveness of RNA sample preparation.

[0049] definition

[0050] General terminology definition

[0051] 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.

[0052] 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...".

[0053] Unless otherwise stated herein or the context clearly provides otherwise, the terms “a,” “an,” and “the,” as well as similar designations (particularly in the context of the claims) used in the context of describing this disclosure, shall be construed as covering both the singular and the plural.

[0054] Unless otherwise stated herein or the context clearly specifies otherwise, all methods described herein may be performed in any suitable order.

[0055] 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.

[0056] 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.

[0057] 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.

[0058] 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.

[0059] 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.

[0060] All documents cited herein (including all patents, patent applications, scientific publications, manufacturer's instructions, user manuals, etc.), whether mentioned above or below, are incorporated herein by reference in their entirety. Nothing in this document should be construed as an admission that the present invention is not entitled to any prior disclosure based on a priority invention.

[0061] As used in this article, phrases such as “measured amount” or “determined expression” related to amino acid sequences (peptides or polypeptides) or similar phrases refer to the amount or presence of amino acid sequences measured.

[0062] Nucleic acid

[0063] The term "nucleic acid" includes deoxyribonucleic acid (DNA), ribonucleic acid (RNA), combinations thereof, and modified forms thereof. The term also includes genomic DNA, cDNA, mRNA, recombinantly produced molecules, and chemically synthesized molecules.

[0064] In one embodiment, the nucleic acid is DNA. In some embodiments, the nucleic acid is RNA. In some embodiments, the nucleic acid is a mixture of DNA and RNA. 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 gel electrophoresis separation; or (iv) synthesized, for example, by chemical synthesis.

[0065] 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.

[0066] The five standard nucleosides that typically constitute naturally occurring 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.

[0067] 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.

[0068] In one embodiment, the nucleic acid is DNA. Hereinafter, 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 naturally occurring DNA by the addition, deletion, substitution, and / or alteration of one or more nucleotides. Such alterations may refer to the addition of non-nucleotide substances to internal DNA nucleotides or DNA ends. It is also contemplated herein that the nucleotides in the 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 naturally occurring DNA.

[0069] 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).

[0070] DNA can be recombinant DNA, obtained through the cloning of nucleic acids (especially cDNA). cDNA can be obtained through reverse transcription of RNA.

[0071] 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 naturally occurring RNA by the addition, deletion, substitution, and / or alteration of one or more nucleotides. Such alteration 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 naturally occurring nucleotides, and the corresponding RNA containing these altered / modified nucleotides (i.e., altered / modified RNA) may be referred to as analogs of naturally occurring 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).

[0072] “RNA” includes 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 embodiments, “RNA” refers to mRNA. The terms “in vitro transcription” or “IVT” as used herein refer to transcription (i.e., RNA generation) performed under cell-free conditions. That is, IVT does not use living cells / 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)).

[0073] In some embodiments, the nucleic acids of the present invention (such as one, at least two, or all of the nucleic acids of the present invention) are RNA.

[0074] In some implementations, the RNA is single-stranded RNA.

[0075] In some implementations, the RNA is mRNA.

[0076] In some implementations, RNA is produced through in vitro transcription.

[0077] In some implementations, the RNA contains a 5' cap structure.

[0078] In some implementations, the RNA does not contain modified ribonucleotides.

[0079] 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.

[0080] In some embodiments, the nucleic acid of the present invention (such as one, at least two, or all of the nucleic acids of the present invention) is DNA.

[0081] In some implementations, DNA exists in the form of a vector.

[0082] 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.

[0083] In some implementations, the vector is a DNA vector.

[0084] In some embodiments, the nucleic acids of the present invention (such as one, at least two, or all of the nucleic acids of the present invention) comprise a mixture of RNA and DNA.

[0085] In some implementations, the RNA in the mixture is single-stranded RNA.

[0086] In some implementations, the RNA in the mixture is mRNA.

[0087] In some implementations, the RNA in the mixture is produced by in vitro transcription of RNA.

[0088] In some implementations, the RNA in the mixture contains a 5' cap structure.

[0089] In some implementations, the RNA in the mixture does not contain modified ribonucleotides.

[0090] In some embodiments, the RNA in the mixture comprises modified ribonucleotides. In some embodiments, the modified ribonucleotides comprise modified uridines. In some embodiments, the modified uridines comprise N1-methylpseudouridines.

[0091] In some embodiments, the DNA in the mixture is present in the form of a vector. In some embodiments, the vector in the mixture contains DNA encoding an amino acid sequence, said amino acid sequence comprising an amino acid sequence of a biologically active peptide or polypeptide.

[0092] In some implementations, the vector in the mixture is a DNA vector.

[0093] In some embodiments, the nucleic acids (such as RNA and / or DNA) of the present invention may comprise one or at least two or more nucleic acid constructs and be formulated together with a delivery medium.

[0094] In some embodiments, nucleic acids (such as RNA and / or DNA) are formulated together with one or more compounds, said one or more compounds being complexed with said nucleic acids (such as RNA and / or DNA).

[0095] In some implementations, nucleic acids (such as RNA and / or DNA) are formulated into particles.

[0096] In some embodiments, nucleic acids (such as RNA and / or DNA) are formulated into liposome complex particles. In these embodiments, it is preferred that the cell is characterized by having a macropinocytosis-mediated RNA uptake mechanism.

[0097] In some implementations, nucleic acids (such as RNA and / or DNA) are formulated into lipid nanoparticles.

[0098] In some embodiments, nucleic acids (such as RNA and / or DNA) comprise a mixture of different nucleic acids (such as RNA and / or DNA, for example, two or more RNAs, two or more DNAs, or one or more RNAs and one or more DNAs), wherein each nucleic acid (such as RNA and / or DNA) encodes an amino acid sequence comprising an amino acid sequence of a biologically active peptide or polypeptide.

[0099] In some embodiments, a mixture of different nucleic acids (such as RNA and / or DNA, for example, two or more RNAs, two or more DNAs, or one or more RNAs and one or more DNAs) comprises nucleic acids (such as RNA and / or DNA, for example, two or more RNAs, two or more DNAs, or one or more RNAs and one or more DNAs) encoding different amino acid sequences, said amino acid sequences comprising the amino acid sequences of biologically active peptides or polypeptides.

[0100] In some implementations, different amino acid sequences include different amino acid sequences of bioactive peptides or polypeptides.

[0101] In some implementations, different biologically active peptides or polypeptides include different antigens.

[0102] In some embodiments, nucleic acids (such as RNA and / or DNA) comprise a mixture of different nucleic acids (such as RNA and / or DNA, for example, two or more RNAs, two or more DNAs, or one or more RNAs and one or more DNAs), said nucleic acid mixture encoding an amino acid sequence containing an amino acid sequence of different antigens.

[0103] 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 (5' cap, 5' UTR, 3' UTR, poly(A) tail) optimized to maximize the RNA's potency for stability and translation efficiency. 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 active ingredient. 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 elements) derived from an 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 selection process targeting sequences that 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 of two repeating human β-globin mRNAs. Furthermore, a poly(A) tail of 110 nucleotides in length can be used, consisting of a 30-adenosine residue, followed by a 10-nucleotide linker sequence (random nucleotide), and a further 70-adenosine residue sequence. This poly(A) tail sequence is designed to improve RNA stability and translation efficiency.

[0104] 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.

[0105] The nucleic acids described herein (such as RNA and / or DNA) 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.

[0106] If the bases forming the first nucleic acid sequence can form base pairs with the bases forming the second nucleic acid sequence via hydrogen bonds, then the two nucleic acid sequences are complementary. Pyrimidines (i.e., cytosine, uracil, thymine, and their analogues) and purines (i.e., adenine, guanine, and their analogues) can form base pairs through hydrogen bonds. For double-stranded formation, the two sequences do not need to be 100% complementary to each other. However, in this invention, the single-stranded nucleic acid molecule serving as the nucleic acid probe is preferably 100% complementary to the target sequence, thereby enabling high-level quality control of in vitro transcribed RNA.

[0107] mRNA

[0108] According to this disclosure, the term "mRNA" refers to "messenger RNA," which relates to a "transcriptum" generated using a DNA template that may 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 a DNA template via in vitro transcription (IVT). As described above, in vitro transcription methods are known to those skilled in the art, and various in vitro transcription kits are commercially available. mRNA is single-stranded but may contain self-complementary sequences, allowing portions of the mRNA sequence to fold and pair with itself to form a double helix structure.

[0109] According to this disclosure, "dsRNA" refers to double-stranded RNA, which is RNA with two partially or completely complementary strands.

[0110] In a preferred embodiment of this disclosure, the mRNA relates to an RNA transcript encoding a peptide or polypeptide.

[0111] 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.

[0112] As is recognized in the art, mRNA typically comprises 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, a variety of in vitro transcription kits are commercially available, for example from Thermo Fisher Scientific (such as TranscriptAid). TMKits from T7 reagent kits (MEGAscript® T7 kit, MAXIscript®), New England BioLabs Inc. (such as HiScribe™ T7 kit, HiScribe™ T7 ARCA mRNA kit), Promega (such as RiboMAX™, HeLaScribe®, Riboprobe® system), 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.

[0113] 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 the promoter of any RNA polymerase. Specific examples of RNA polymerases include T7, T3, and SP6 RNA polymerases. Preferably, in vitro transcription is controlled by the 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.

[0114] In some embodiments of this disclosure, the mRNA is a "replicon mRNA" or simply "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, 4, 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 starting 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. These subgenomic transcripts 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 (OPF) encoding an alphavirus structural protein is replaced by an OPF encoding the target protein. Alphavirus-based trans-replication systems rely on alphavirus nucleotide sequence elements on two separate nucleic acid molecules: one encoding a viral replicase, and the other capable of being 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 that allow the alphavirus replicase to recognize and synthesize RNA.

[0115] 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 WO2019 / 175356, and include the following: a 5'-cap structure; an extension or truncation of a naturally occurring poly(A) tail; alterations to the 5' and / or 3' untranslated regions (UTRs), such as the introduction of 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 an increase in the GC content of the RNA).

[0116] 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 a conventional 5'-cap but modified at the 2' or 3' position of its 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 WO2019 / 175356. The 5'-capped mRNAs described herein can be generated by in vitro transcription of a DNA template in the presence of a suitable 5'-capped compound, wherein the 5'-capped 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'-capped structure can be ligated to the mRNA post-transcriptionally using a capping enzyme (e.g., a capping enzyme of vaccinia virus).

[0117] In some implementations, the mRNA comprises 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.

[0118] 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.

[0119] The D1 diastereomer of beta-S-ARCA (β-S-ARCA) has the following structure:

[0120]

[0121] "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, thus exhibiting 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, 4.6 × 250 mm, where a flow rate of 1.3 ml / min can be used. In some embodiments, a gradient of methanol in ammonium acetate is used, for example, a linear gradient of methanol in 0.05 M ammonium acetate (pH = 5.9) from 0-25% (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.

[0122] The 5'-cap analogue m27,3'-OGppp(m12'-O)ApG (also known as m27,3'OG(5')ppp(5')m2'-OApG) is a constituent unit of cap1, and its structure is as follows:

[0123]

[0124] An exemplary cap0 mRNA containing β-S-ARCA and mRNA has the following structure:

[0125]

[0126] An exemplary cap0 mRNA containing m27,3'OG(5')ppp(5')G and mRNA has the following structure:

[0127]

[0128] An exemplary cap1 mRNA containing m27,3'-OGppp(m12'-O)ApG and mRNA has the following structure:

[0129]

[0130] As used herein, the term "poly-A tail" or "poly-A sequence" refers 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 a poly-A tail encoded by DNA and transcribed by a template-dependent RNA polymerase.

[0131] It has been shown that a 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 an open reading frame present upstream (5') of the poly-A tail (Holtkamp et al., Blood, 2006, 108, 4009-4017).

[0132] The poly-A tail can be of any length. In some embodiments, the poly-A tail comprises, is substantially composed of, or consists of at least 20, at least 30, at least 40, at least 80, or at least 100 and at most 500, at most 400, at most 300, at most 200, or at most 150 A nucleotides, particularly comprising, is substantially composed of, or consists of about 120 A nucleotides. In this context, "substantially composed 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, "composed 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.

[0133] 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.

[0134] In some embodiments, the 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). Such random sequences can be 5 to 50, 10 to 30, or 10 to 20 nucleotides in length. 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 beneficial properties at the DNA level that enable continuous proliferation of plasmid DNA in *E. coli*, while still supporting RNA stability and translation efficiency at the RNA level, wherein the random sequences have a uniform distribution of the four nucleotides (dA, dC, dG, and dT) and a length of, for example, 5 to 50 nucleotides.

[0135] 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.

[0136] 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.

[0137] 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 approximately 150 nucleotides. In other embodiments, the poly-A tail contains approximately 120 nucleotides.

[0138] 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 a 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. Incorporating 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) to which they are introduced. In some embodiments, the 3'-UTRs are derived from globin genes or mRNAs, such as α2-globin, α1-globin, or β-globin (e.g., β-globin, such as human β-globin). For example, RNA (e.g., mRNA) can be modified by replacing 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, e.g., β-globin, such as human β-globin).

[0139] To improve mRNA stability and / or reduce immunogenicity and / or cytotoxicity, mRNA may have 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. In some embodiments, the modified uridine replacing uridine is selected from the group consisting of: pseudouridine (ψ), N1-methylpseudouridine (m1ψ), 5-methyluridine (m5U), and combinations thereof.

[0140] 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), 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- Carboxymethyl aminomethyl-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 (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), di Hydrogenated pseudouridine, 5,6-dihydrouridine, 5-methyldihydrouridine (m5D), 2-thiodihydrouridine, 2-thiodihydropseudouridine, 2-methoxyuridine, 2-methoxy-4-thiouridine, 4-methoxypseudouridine, 4-methoxy-2-thiopseudouridine, N1-methylpseudouridine, 3-(3-amino-3-carboxypropyl)uridine (acp3U), 1-methyl-3-(3-amino-3-carboxypropyl)pseudouridine (acp3ψ), 5-(isopentenylaminomethyl)uridine (inm5U), 5-(isopentenylaminomethyl)-2-thiouridine (inm5s2U), α-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-arasu-uridine, 2′-F-uridine, 2′-OH-arasu-uridine, 5-(2-methoxycarbonylvinyl)uridine, 5-[3-(1-E-propenylamino)uridine, or any other modified uridine known in the art.

[0141] 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.

[0142] 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 codons that are rare 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 been codon-optimized 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 been codon-optimized 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.

[0143] 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 different frequencies 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."

[0144] In some implementations, the guanine / cytosine (G / C) content of the mRNA coding region 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 unchanged 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., the use of so-called alternative codons) can be determined. Depending on the amino acid to be encoded by the mRNA, there are several possibilities for modification of 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 content of A and / or U nucleotides.

[0145] 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.

[0146] The combination of the above modifications, namely incorporation of a 5'-cap structure, incorporation of a poly-A sequence, demasking of a poly-A sequence, alteration of the 5'- and / or 3'-UTR (such as incorporation of one or more 3'-UTRs), replacement of one or more naturally occurring nucleotides with synthetic nucleotides (e.g., replacement of cytidine with 5-methylcytidine and / or replacement of 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) incorporation of a 5'-cap structure; (ii) incorporation of a poly-A sequence, or demasking of the poly-A sequence; (iii) alteration of the 5'- and / or 3'-UTR (such as incorporation of one or more 3'-UTRs); (iv) replacement of one or more naturally occurring nucleotides with synthetic nucleotides (e.g., replacement of cytidine with 5-methylcytidine and / or replacement of uridine with pseudouridine (Ψ) or N(1)-methylpseudouridine (m1Ψ) or 5-methyluridine (m5U); and (v) codon optimization.

[0147] 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, more specifically the spleen, is particularly preferred if the administered mRNA is an mRNA encoding 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.

[0148] 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 with lipid or cholesterol conjugates). 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 the mRNA or the encoded peptide or polypeptide in the organ or tissue is required, and / or when a large amount of the encoded peptide or polypeptide needs to be expressed, and / or when the systemic presence of the encoded peptide or polypeptide (particularly at a significant level) is required or demanded.

[0149] 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.

[0150] RNA molecules

[0151] The term "RNA molecule species" refers to at least one RNA molecule within a group of RNA molecules that are identical in their RNA sequence and / or sequence length. Therefore, all RNA molecules within the same RNA molecule species are encoded by the same template DNA. If the RNA present in a sample is coding RNA, then an RNA species may encode a target peptide or protein or a variant thereof.

[0152] The term "n different RNA molecule species" refers to a set of n RNA molecules whose RNA sequences and / or sequence lengths may differ. Therefore, if an RNA sample contains n different RNA molecule species, and n is 2, then the RNA molecules in the RNA sample belong to either of these two RNA molecule species, meaning they have the same RNA sequence and / or sequence length. In a typical RNA sample containing two different RNA molecule species, one or more RNA molecules of the first RNA molecule species have no difference in RNA sequence and / or sequence length from each other, but differ from one or more RNA molecules of the second RNA molecule species in RNA sequence and / or sequence length. Each RNA molecule species contains at least one RNA molecule, meaning each RNA molecule species contains one or more RNA molecules. Therefore, if an RNA sample contains n different RNA molecule species, then the RNA sample contains at least n RNA molecules (at least one RNA molecule of each RNA molecule species). However, typically, a sample contains a greater number of RNA molecules per RNA molecule species. In this invention, one or more RNA molecules of each RNA molecule species are synthetically derived coding RNA molecule species. If these RNA molecule species are coding RNA molecule species, then each of these n different RNA molecule species preferably (but not necessarily) encodes a target peptide / protein or a variant thereof. In a sample containing n different RNA molecule species analyzed by the method of the present invention, each RNA molecule species may be present in equal, similar, or different amounts, preferably in equal or similar amounts. The number of different RNA molecule species present in the RNA sample is represented by an integer n, which is at least 1, and therefore can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, etc. Preferably, n is an integer of at least 2 or at least 3, or an integer within the following range: 1 to 200, or 2 to 200, more preferably 2 to 150, even more preferably 2 to 100, and most preferably 2 to 50.

[0153] In the method of this invention, the n different RNA molecule types can have similar lengths because the detection and differentiation of these n different RNA molecule types do not depend on length differences. For example, the length difference of the n different RNA molecule types in the sample can not exceed 10% or 8%, preferably not exceed 7% or 6%, more preferably not exceed 5%, and most preferably not exceed 4%.

[0154] Specifically, this method can be used for RNA molecule species that may have similar sequences. For example, sequences of n different RNA molecule species in a sample, or portions of these sequences, may have at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% sequence identity. This sequence identity may be based on regions containing 200, 300, 400, 500, or 600 or more nucleotides in the complete sequence.

[0155] In this invention, the RNA sequences of one or more coding RNA molecules of each of the n different RNA molecule types can be at least 80% identical to each other. This refers to the identity of the entire RNA molecule sequence, not just the identity of the target sequence.

[0156] Integrity of one or more coding RNA molecules of one or more RNA molecule species: The term "integrity" describes the presence of a complete target RNA sequence in an in vitro generated RNA sample. Low integrity can be caused by a variety of factors, including degradation, cleavage, errors or incomplete chemical synthesis, base pairing errors, integration of modified nucleotides or modification of integrated nucleotides, capping deletions or incompleteness, polyadenylation deletions or incompleteness, and incomplete transcription.

[0157] Non-translation area

[0158] The primers used in the method of this invention can target the 3' untranslated region (3'-UTR) or the 5' untranslated region (3'-UTR).

[0159] Generally, the term "3'-UTR" refers to the portion of an RNA molecule located at the 3' end (i.e., "downstream") of the coding sequence and not translated into protein. Typically, the 3'-UTR is the portion of mRNA located between the protein-coding region (coding sequence (CDS)) and the 3' end of the mRNA. In the context of this invention, the term 3'-UTR may also include elements that are not encoded by a template (from which RNA is transcribed) but are added during post-transcriptional maturation of the RNA, such as poly(A) sequences (or poly(A) "tails"). The 3'-UTR of mRNA is not translated into an amino acid sequence. The 3'-UTR sequence is typically encoded by a DNA template and transcribed into the corresponding mRNA during gene expression. In the context of this invention, the 3'-UTR corresponds to the sequence of mature mRNA located between the stop codon of the protein-coding region (preferably immediately adjacent to the 3' end of the stop codon) and the poly(A) sequence of the mRNA.

[0160] 5' Untranslated Region (5'-UTR): The 5'-UTR is generally understood as a specific portion of messenger RNA (mRNA). It is located at the 5' end of the coding sequence of the mRNA. Typically, the 5'-UTR begins at the transcription start site and terminates one nucleotide before the start codon of the coding sequence. The 5'-UTR may contain elements that control gene expression, also known as regulatory elements. For example, such regulatory elements may be ribosome binding sites. The 5'-UTR can be modified post-transcriptionally, for example, by adding a 5' cap structure. In the context of this invention, the term "5'-UTR" generally refers to the sequence in the mRNA located between the 5' cap structure and the start codon. Preferably, the 5'-UTR is a sequence that extends from the nucleotide at the 3' end of the 5' cap structure (preferably, from the nucleotide immediately adjacent to the 3' end of the 5' cap structure) to the nucleotide at the 5' end of the start codon of the coding sequence (preferably, to the nucleotide immediately adjacent to the 5' end of the start codon of the coding sequence).

[0161] RNA sample preparation

[0162] The method of the present invention may include preparing a sample containing RNA. The method may include one or more purification steps.

[0163] As used herein, the term “purification” or “refinement” is to be understood as separating and / or isolating the desired RNA from impurities, intermediates, byproducts and / or reaction components present in a sample, or at least removing impurities, intermediates, byproducts and / or reaction components from a sample containing RNA.

[0164] Non-restrictive examples of unwanted components in RNA-containing samples may include degradation fragments or fragments resulting from premature termination of transcription, or excessively long transcripts generated when plasmids are not fully linearized, and therefore these unwanted components need to be removed. Additionally, some intermediates, such as template DNA (used for in vitro transcription (IVT) of RNA), can be removed from the sample.

[0165] In addition, it may be necessary to remove reaction components from the RNA sample, such as enzymes, proteins, bacterial DNA and RNA, small molecules (such as spermidine), buffer components, etc. Additionally, impurities such as organic solvents, nucleotides, nucleosides, or other small molecules can be separated.

[0166] Reverse transcription

[0167] Each method of the present invention includes the step of reverse transcribing n types of RNA molecules in an RNA sample into cDNA molecules of n types of DNA molecules.

[0168] In this sense, the term "reverse transcription" in the art generally refers to the process of generating complementary DNA (cDNA) from RNA. In this process, RNA (i.e., one or more coding RNA molecules from at least two of the n different RNA molecule species) is typically incubated with reverse transcriptase, deoxyribonucleotides (dNTPs), and at least one suitable primer under conditions sufficient for cDNA synthesis, such as incubation at approximately 37°C to 42°C for 30 minutes to 1 hour.

[0169] Primers used for reverse transcription can be random so that any RNA molecule present in the sample (e.g., one or more coding RNA molecules from all n different RNA molecule species) can be reverse transcribed into cDNA, or they can be target-specific so that only target RNA (e.g., one or more coding RNA molecules from at least two of the n different RNA molecule species) can be reverse transcribed into the corresponding cDNA.

[0170] PCR assay

[0171] The method of the present invention also includes the determination of cDNA molecules generated by reverse transcription of RNA based on polymerase chain reaction (PCR).

[0172] Broadly speaking, the term "PCR-based assay" encompasses any assay that employs a PCR reaction. In one embodiment, the PCR assay is a quantitative polymerase chain reaction (qPCR). In one embodiment, the PCR assay is a digital PCR (dPCR). In one embodiment, the PCR assay is a droplet digital PCR (ddPCR).

[0173] As is known to those skilled in the art, polymerase chain reaction (PCR) is a molecular biology technique used to amplify a DNA segment by orders of magnitude, resulting in thousands to millions of copies of a specific DNA sequence. This method relies on thermal cycling, consisting of multiple cycles of repeated heating and cooling of the reaction to allow the DNA to unwind and for enzymatic replication. Primers (short DNA fragments) contain sequences complementary to the target sequence and, together with a thermostable DNA polymerase (such as Taq polymerase), enable selective and repetitive amplification. As the PCR reaction proceeds, the generated DNA itself is used as a template for replication, initiating a chain reaction in which the DNA template is amplified exponentially. DNA polymerases utilize single-stranded DNA as a PCR template and DNA oligonucleotides (also known as DNA primers) to enzymatically assemble a new DNA strand from DNA building blocks (i.e., nucleotides), both of which are essential for initiating DNA synthesis. The vast majority of PCR methods employ thermal cycling, which involves alternating heating and cooling of the PCR sample through a series of predetermined temperature steps. The first step, known as DNA unwinding, involves the physical separation of the two strands of the DNA double helix at high temperatures. The second step involves lowering the temperature, allowing the two DNA strands to become templates for DNA polymerase, thus selectively amplifying the target DNA. The selectivity of PCR stems from the use of primers that are complementary to the DNA region being targeted for amplification under specific thermal cycling conditions.

[0174] RT-qPCR: RT-qPCR assays consist of a first step of reverse transcription and a second step of quantitative PCR, as described above. The reverse transcription and quantitative PCR reactions can be performed separately, such that RNA is reverse transcribed into cDNA in the first step, and the cDNA is transferred to a new reaction mixture for quantitative PCR in the second step. Alternatively, the reverse transcription and quantitative PCR reactions can be performed in one step, such that the reaction mixture contains both components from the reverse transcription reaction and components from the quantitative PCR reaction.

[0175] The PCR process typically involves a series of temperature changes, known as thermal cycling, with each cycle usually consisting of at least two, optionally three, or four independent temperature steps. A single, very high-temperature step (>90°C) usually precedes each cycle, followed by a hold-in step for extension or short-term storage of the final product. The temperature and duration used in each cycle depend on several parameters, including the enzymes used for DNA synthesis, the concentrations of divalent ions and dNTPs in the reaction, and the primer melting temperature. The common steps for most PCR methods are as follows.

[0176] The first step, referred to as the initialization step or activation step, involves heating the reaction chamber to activate the DNA polymerase. In one embodiment, the initialization step is performed at a temperature of 90-100°C. In another embodiment, the initialization step is performed at a temperature of 94-96°C. In another embodiment, the initialization step is performed at a temperature of 95°C. In one embodiment, the initialization step lasts for 1-20 minutes. In another embodiment, the initialization step lasts for 5-15 minutes. In another embodiment, the initialization step lasts for 8-12 minutes. In another embodiment, the initialization step lasts for 10 minutes. In another embodiment, the initialization step is omitted.

[0177] The second step, called denaturation, separates the double strands of nucleic acid, producing two single-stranded DNA molecules.

[0178] In one embodiment, the denaturation step is performed at a temperature of 90-100 °C. In one embodiment, the denaturation step is performed at a temperature of 92-97 °C. In one embodiment, the denaturation step is performed at a temperature of 94 °C. In one embodiment, the denaturation step lasts from 1 second to 1 minute. In one embodiment, the denaturation step lasts from 20 to 40 seconds. In one embodiment, the denaturation step lasts for 30 seconds.

[0179] The third step, called annealing and extension, allows the primers to bind to each single-stranded DNA template, followed by polymerization by DNA polymerase. The temperature of annealing and extension must be low enough for the primers to hybridize with the DNA strands, but high enough to ensure specific hybridization; that is, the primers should only bind to the perfectly complementary portions of the DNA strands and not to any other sites. If the temperature is too low, the primers may not bind completely; if the temperature is too high, the primers may not bind at all. Typical annealing temperatures are about 3-5°C lower than the melting temperature of the primers used. Stable hydrogen bonds can only form between complementary bases when the primer sequence is highly matched to the template sequence. In this step, the polymerase binds to the primer-template hybrid and begins DNA synthesis.

[0180] The temperature for the annealing and extension steps depends on the RNA being studied and the primers used. In one embodiment, the annealing and extension steps are performed at 55-65°C. In another embodiment, the annealing and extension steps are performed at 60-65°C. In another embodiment, the annealing and extension steps are performed at 63°C. In one embodiment, the annealing and extension steps last from 10 seconds to 5 minutes. In another embodiment, the annealing and extension steps last from 20 seconds to 2 minutes. In another embodiment, the annealing and extension steps last from 40 seconds to 80 seconds. In another embodiment, the annealing and extension steps last 60 seconds.

[0181] The denaturation and annealing / extension steps can be repeated the number of times necessary to achieve sufficient DNA amplification. In one embodiment, the denaturation, annealing, and polymerization steps are repeated 10 to 100 times. In one embodiment, the denaturation, annealing, and polymerization steps are repeated 20 to 60 times. In one embodiment, the denaturation, annealing, and polymerization steps are repeated 30 to 50 times. In one embodiment, the denaturation, annealing, and polymerization steps are repeated 35 to 45 times. In one embodiment, the denaturation, annealing, and polymerization steps are repeated 40 times.

[0182] In one embodiment, the PCR procedure includes an enzyme inactivation step performed after cycles of denaturation and annealing / extension. In another embodiment, the enzyme inactivation step is omitted.

[0183] In one embodiment, when the enzyme inactivation step is part of the method of the present invention, the enzyme inactivation step is performed at a temperature of 97-99°C. In another embodiment, the enzyme inactivation step is performed at a temperature of 98°C.

[0184] In the method of the present invention for determining the identity, integrity, and quantification ratio of RNA, in one embodiment, the enzyme inactivation step lasts for 1 to 20 minutes. In one embodiment, the enzyme inactivation step lasts for 5 to 15 minutes. In one embodiment, the enzyme inactivation step lasts for 8 to 12 minutes; in one embodiment, the enzyme inactivation step lasts for 10 minutes.

[0185] In the method for determining RNA potency of the present invention, in one embodiment, the enzyme inactivation step lasts from 10 seconds to 5 minutes. In one embodiment, the enzyme inactivation step lasts from 20 seconds to 2 minutes. In one embodiment, the enzyme inactivation step lasts from 40 seconds to 80 seconds. In one embodiment, the enzyme inactivation step lasts for 60 seconds.

[0186] The temperature and time used in each cycle depend on several parameters, such as the enzymes used to synthesize DNA, the concentrations of divalent ions and deoxyribonucleotides (dNTPs) in the reaction, and the primer binding temperature. The type of quantitative PCR technique used depends on the DNA sequence in the sample, and this technique can use non-specific fluorescent dyes or hybridization probes.

[0187] In one embodiment, the PCR method used in this invention employs a detectable label. A detectable label is a detectable compound that is directly or indirectly attached to another molecule. In the method of this invention, the detectable label is attached to a single-stranded nucleic acid molecule. Methods for attaching labels to nucleic acid molecules are well known to those skilled in the art. Specific, non-limiting examples of labels include fluorescent probes and fluorescent moieties, chromogenic moieties, haptens, affinity tags, and radioisotopes. The label can be detected directly (e.g., by optical methods) or indirectly (e.g., by interaction with one or more subsequently detectable molecules).

[0188] In the method of the present invention, fluorescent probes are preferably used. As those skilled in the art know, a fluorescent probe (also known as a fluorescent label or fluorescent tag) is a molecule linked by a chemical method to assist in the detection of biomolecules such as proteins, antibodies, or amino acids.

[0189] In one embodiment, the fluorescent probe carries one label. In another embodiment, the fluorescent probe carries multiple labels (such as two, three, or four labels).

[0190] The fluorescent probe is labeled with a fluorophore. As is known to those skilled in the art, a fluorophore is a fluorescent substance that can re-emit light upon photoexcitation. The fluorophore selectively binds to specific regions or functional groups on the target molecule and can be linked by chemical or biological methods. Examples of suitable fluorophores known to those skilled in the art include [6-amino-9-[2-carboxy-4-[5-(2,5-dioxopyrrole-1-yl)pentyl-carbamoyl]phenyl]-4,5-disulfonic acid xanthon-3-ylidene]ammonium (Alexa 488), 6-carboxyfluorescein (FAM), and 4-(2,7-difluoro-3-hydroxy-6-oxoxanthon-9-yl)phenyl-1,3-dicarboxylic acid (Oregon). Rhodamine Green, [6-amino-9-(2,5-dicarboxyphenyl)xanthon-3-ylidene]ammonium, 6-[(7-nitro-2,1,3-benzoxadiazol-4-yl)amino]hexanoic acid (NBD-X), Tetrachlorofluorescein (TET), [9-[6-(2,5-dioxopyrrolidone-1-yl)oxy-6-oxohexyl]-8,8-dimethyl-2-oxo-4-(trifluoromethyl)pyrano[3,2-g]quinoline-6-yl]methanesulfonate (Alexa 430), 3-(4,4-difluoro-5-phenyl-3a,4a-diaza-4-boron-s-indan-3-yl)propionic acid (BODIPY) R6G-X), (6-carboxy-4',5'-dichloro-2',7'-dimethoxyfluorescein) (JOE), [2',5,5',6-tetrachloro-6'-(2,2-dimethylpropionyloxy)-7'-[3-[6-[[di(propyl-2-yl)amino]-(2-hydroxyethoxy)phosphino]oxohexylamino]-3-oxopropyl]-4'-methyl-3-oxospiro[2-benzofuran-1,9'-xanthon]-3'-yl] 2,2-dimethylpropionate (Yakima Yellow), 12-[4-(2,5-dioxopyrrolidone-1-yl)oxocarbonylphenyl]-7,8,8,16,16,17-hexamethyl-2-oxa-6,18-diazapentacyclo[11.7.0.0] 3,11 .0 5,9 .0 15,19 [Eicosene-1(20),3,5,9,11,13,15(19)-hepten-4,20-disulfonic acid (Alexa 532), 2'-chloro-7'-phenyl-1,4-dichloro-6-carboxyfluorescein (VIC) and hexachlorofluorescein (HEX). Among them, 5'-hexachlorofluorescein and 6-carboxyfluorescein are preferred.]

[0191] The fluorescent probe is also labeled with a quencher. As is known to those skilled in the art, a quencher is a substance that reduces the fluorescence intensity of a given substance. Examples of quenchers known in the art include 2-[N-(2-hydroxyethyl)-4-[[2-methoxy-5-methyl-4-[(4-methyl-2-nitrophenyl)azo]-phenyl]azo]anilino]ethanol (Black Hole Quencher 1, BHQ 1), ZEN quencher (available from IDT), Iowa Black fluorescein quencher (IBFQ) (available from IDT), 4-N-methyl-N-(4'-nitro-2'-chloroazobenzene-4-yl)-aminobutamido-1-(2-O-dimethoxytriphenylmethyloxymethyl)-pyrrolidine-4-yl-succinyl-long-chain alkylamino-CPG (ECLIPSE quencher), and 5-carboxy-N,N,N',N'-tetramethylrhodamine (TAMRA), and mixtures thereof. Among them, the combination of ZEN and IBFQ and BHQ1 are preferred.

[0192] In one embodiment, the fluorophore is 5'-hexachlorofluorescein, and the quencher is Black Hole Quencher 1. In another embodiment, the fluorophore is 6-carboxyfluorescein, and the quencher is Black Hole Quencher 1. Both the fluorophore and the quencher are commercially available, for example, from Eurofins Genomics.

[0193] The detection of double-stranded nucleic acid molecules generated in the PCR-based assay of this invention depends on a detectable label attached to the nucleic acid probe. For example, if the nucleic acid probe is labeled with a radioactive isotope, double-stranded nucleic acid molecules can be detected by autoradiography. If the nucleic acid probe is labeled with a fluorescent probe, double-stranded nucleic acid molecules can be detected by fluorescence spectroscopy.

[0194] Digital PCR (dPCR)

[0195] In one embodiment, the PCR is digital PCR (dPCR). In one embodiment, the dPCR-based assay uses a detectable label. In one embodiment, the detectable label is a fluorescent probe.

[0196] In digital PCR (dPCR), each PCR reaction is separated into multiple smaller reactions, allowing individual nucleic acid molecules in the sample to be localized and amplified in multiple independent regions. Microplates, capillaries, oil emulsions (droplets), and arrays of miniature reaction chambers with nucleic acid-binding surfaces can all be used to separate RNA samples into multiple small reactions within each reaction vessel. After multiple PCR amplification cycles (typically performed under saturated PCR conditions), the fluorescence signal of the sample is analyzed and detected as a binary reading of "0" (no signal) or "1" (signal present). Utilizing Poisson's law of fractions, the distribution of target molecules in the sample can be accurately approximated, thereby quantifying the target strand in the PCR product. Therefore, dPCR does not rely on the number of amplification cycles to determine the initial sample size, thus avoiding reliance on uncertain exponential data for quantifying target nucleic acids. Therefore, dPCR enables absolute quantification of nucleic acids.

[0197] Suitable commercial systems for performing dPCR include the chip-based QuantStudio™ 3D Digital PCR System (Thermo Fisher, Waltham, MA, USA), Rain Drop Plus™ System (RainDanceTechnologies, Lexington, MA, USA), or QX200™, AutoDG™, and Droplet Digital™ PCR Systems (BioRad Laboratories, Hercules, CA, USA).

[0198] Chip-based digital PCR measures the absolute quantity of nucleic acid molecules distributed in individual reaction wells. Commercially available chip-based dPCR systems include the QuantStudio™ 3D Digital PCR System (Thermo Fisher, Waltham, MA, USA). The PCR reaction is divided into approximately 20,000 individual reaction wells on a chip, each containing or not containing template. The sealed chip is then subjected to PCR amplification. Each reaction well containing template produces a PCR positive signal (positive well), and each reaction well not containing template produces a PCR negative signal (negative well). Template concentration can be quantified using a Poisson distribution algorithm by counting the positive and negative wells.

[0199] Droplet digital PCR (ddPCR)

[0200] In one embodiment, the PCR is droplet digital PCR (ddPCR). In one embodiment, the ddPCR-based assay uses a detectable label. In one embodiment, the detectable label is a fluorescent probe.

[0201] Droplet digital PCR (ddPCR) measures the absolute quantity of nucleic acid molecules by counting them in 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 broken 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.

[0202] Compared to classical relative quantification methods, ddPCR achieves more precise absolute quantification, which is limited by the doubling of sample volume 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 value 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 using a sonic pipette)).

[0203] Quality parameters

[0204] The method according to the invention is applicable to the determination of at least one quality parameter. The term "quality parameter" includes any RNA sample parameter related to the characteristics of the RNA sample, typically obtained for quality control during or after production. Examples of quality parameters include the quantity of one or more coding RNA molecules from at least two RNA molecule species, the integrity of one or more coding RNA molecules from at least two RNA molecule species, and the quantitative ratio between one or more coding RNA molecules from at least two RNA molecule species.

[0205] Quality parameters can be used to determine, for example, whether an RNA sample contains all n required different RNA molecule species, the quantitative ratio between at least two of the n different RNA molecule species, whether the RNA molecules of the n different RNA molecule species are present in their complete form (integrity), and the amount of each RNA molecule species in the RNA sample. By determining at least one quality control parameter, RNA samples can be analyzed and rated according to the regulatory requirements required for drug marketing approval.

[0206] Methods for determining the quantification ratio of RNA molecules

[0207] In one aspect, the quality parameter measured is the quantitative ratio of two or more RNA molecule species in an RNA sample. In this sense, the quantitative ratio can be the ratio between one or more coding RNA molecules of at least two RNA molecule species: the quantitative ratio of one or more coding RNA molecules of at least two RNA molecule species can be determined by measuring the quantity of one or more coding RNA molecules of at least two RNA molecule species and evaluating the quantitative ratio of the two or more quantities. Because this method can measure the quantity of one or more coding RNA molecules of all RNA molecule species, it is possible to determine the quantitative ratio between all RNA molecule species.

[0208] Therefore, in one aspect, the present invention provides a method for determining the quantitative ratio of two or more RNA molecule species in an RNA sample containing n RNA molecule species, wherein n is an integer of at least 2, the method comprising the following steps:

[0209] a) Reverse transcribe n types of RNA molecules in an RNA sample into cDNA molecules of n types of DNA molecules; and

[0210] b) Perform polymerase chain reaction (PCR) assays on the obtained cDNA molecules, wherein the PCR assays use a first primer set and a single second primer, wherein the first primer set contains n primer types, wherein each primer type is annealed to a first target region of only one of the n DNA molecule types in the sample, and the single second primer is annealed to a second target region of all n DNA molecule types in the sample.

[0211] In one embodiment, the first target region is located within the coding sequence of the DNA molecule. In another embodiment, the first target region spans the coding sequence and untranslated region of the DNA molecule.

[0212] In one embodiment, the first target region is located at the 3' end of the DNA molecule. In one embodiment, the first target region is located within the coding sequence of the 3' end of the DNA molecule. In one embodiment, the first target region spans the coding sequence and the 3' untranslated region (as defined herein) of the DNA molecule. In one embodiment, the first target region is located at the 5' end of the DNA molecule. In one embodiment, the first target region is located within the coding sequence of the 5' end of the DNA molecule. In one embodiment, the first target region spans the coding sequence and the 5' untranslated region (as defined herein) of the DNA molecule.

[0213] In one embodiment, the second target region is located within the coding sequence of the DNA molecule. In another embodiment, the second target region spans both the coding sequence and the untranslated region of the DNA molecule. In yet another embodiment, the second target region is located within the untranslated region of the DNA molecule.

[0214] In one embodiment, the second target region is located within the 3' end region of the DNA molecule. In one embodiment, the second target region is located within the coding sequence of the 3' end region of the DNA molecule. In one embodiment, the second target region spans the coding sequence and the 3' untranslated region (as defined herein) of the DNA molecule. In one embodiment, the second target region is located within the coding sequence of the 5' end region of the DNA molecule. In one embodiment, the second target region spans the coding sequence and the 5' untranslated region (as defined herein) of the DNA molecule.

[0215] In one embodiment, the PCR is digital PCR (dPCR). In one embodiment, the dPCR-based assay uses a detectable label. In one embodiment, the detectable label is a fluorescent probe.

[0216] In one embodiment, the PCR is droplet digital PCR (ddPCR). In one embodiment, the ddPCR-based assay uses a detectable label. In one embodiment, the detectable label is a fluorescent probe.

[0217] The identity of RNA molecules

[0218] In one aspect, the quality parameter being measured is the identity of n RNA molecule species in an RNA sample. In this aspect, the present invention provides a method for determining the identity of n RNA molecule species in an RNA sample containing n RNA molecule species, wherein n is an integer of at least 2, the method comprising the following steps:

[0219] a) Reverse transcribe n types of RNA molecules in an RNA sample into cDNA molecules of n types of DNA molecules; and

[0220] b) Perform a polymerase chain reaction (PCR) assay on the obtained cDNA molecules, wherein the PCR assay uses a first primer set and a single second primer, wherein the first primer set contains n primer types, wherein each primer type is annealed to a first target region of only one of the n DNA molecule types in the sample; and the single second primer is annealed to a second target region of all n DNA molecule types in the sample.

[0221] In one embodiment, the first target region is located within the coding sequence of the DNA molecule. In another embodiment, the first target region spans the coding sequence and untranslated region of the DNA molecule.

[0222] In one embodiment, the first target region is located at the 3' end of the DNA molecule. In another embodiment, the first target region is located within the coding sequence of the 3' end of the DNA molecule. In one embodiment, the first target region includes a portion of the coding sequence of the DNA molecule and a portion of the 3' untranslated region (as defined herein). In one embodiment, the first target region is located at the 5' end of the DNA molecule. In another embodiment, the first target region is located within the coding sequence of the 5' end of the DNA molecule. In one embodiment, the first target region includes a portion of the coding sequence of the DNA molecule and a portion of the 5' untranslated region (as defined herein).

[0223] In one embodiment, the second target region is located within the coding sequence of the DNA molecule. In another embodiment, the second target region includes a portion of the coding sequence of the DNA molecule and a portion of the untranslated region. In yet another embodiment, the second target region is located within the untranslated region of the DNA molecule.

[0224] In one embodiment, the second target region is located at the 3' end of the DNA molecule. In another embodiment, the second target region is located within the coding sequence of the 3' end of the DNA molecule. In one embodiment, the second target region includes a portion of the coding sequence of the DNA molecule and a portion of the 3' untranslated region (as defined herein). In one embodiment, the second target region is located at the 5' end of the DNA molecule. In another embodiment, the second target region is located within the coding sequence of the 5' end of the DNA molecule. In one embodiment, the second target region includes a portion of the coding sequence of the DNA molecule and a portion of the 5' untranslated region (as defined herein).

[0225] In one embodiment, the PCR is digital PCR (dPCR). In one embodiment, the dPCR-based assay uses a detectable label. In one embodiment, the detectable label is a fluorescent probe.

[0226] In one embodiment, the PCR is droplet digital PCR (ddPCR). In one embodiment, the ddPCR-based assay uses a detectable label. In one embodiment, the detectable label is a fluorescent probe.

[0227] Integrity of RNA molecules

[0228] In one aspect, the quality parameter being measured is the integrity of n RNA molecule species in an RNA sample (as defined herein). Therefore, in one aspect, the present invention provides a method for determining the integrity of an RNA sample containing n RNA molecule species, wherein n is an integer of at least 1, the method comprising the following steps:

[0229] a) Reverse transcribe the n RNA molecules in the RNA sample into cDNA molecules of n different DNA types; and

[0230] b) The obtained cDNA molecules were subjected to a polymerase chain reaction (PCR) assay using a first primer set, a single second primer, a third primer set, and a single fourth primer.

[0231] The first primer set contains n primer types, each of which can anneal to the first target region at the 3' end region of the DNA molecule in the sample.

[0232] The single second primer can anneal to the second target region at the 3' end region of all n types of DNA molecules in the sample;

[0233] The third primer set contains n primer types, each of which can anneal to the third target region at the 5' end of a DNA molecule in the sample; and

[0234] The single fourth primer can anneal to the fourth target region at the 5' end region of all n types of DNA molecules in the sample.

[0235] In one embodiment, the first target region is located within the coding sequence of the DNA molecule. In another embodiment, the first target region includes a portion of the coding sequence of the DNA molecule and a portion of the untranslated region.

[0236] In one embodiment, the first target region is located at the 3' end of the DNA molecule. In another embodiment, the first target region is located within the coding sequence of the 3' end of the DNA molecule. In one embodiment, the first target region includes a portion of the coding sequence of the DNA molecule and a portion of the 3' untranslated region (as defined herein). In one embodiment, the first target region is located at the 5' end of the DNA molecule. In another embodiment, the first target region is located within the coding sequence of the 5' end of the DNA molecule. In one embodiment, the first target region includes a portion of the coding sequence of the DNA molecule and a portion of the 5' untranslated region (as defined herein).

[0237] In one embodiment, the second target region is located within the coding sequence of the DNA molecule. In another embodiment, the second target region includes a portion of the coding sequence of the DNA molecule and a portion of the untranslated region. In yet another embodiment, the second target region is located within the untranslated region of the DNA molecule.

[0238] In one embodiment, the second target region is located at the 3' end of the DNA molecule. In another embodiment, the second target region is located within the coding sequence of the 3' end of the DNA molecule. In one embodiment, the second target region includes a portion of the coding sequence of the DNA molecule and a portion of the 3' untranslated region (as defined herein). In one embodiment, the second target region is located at the 5' end of the DNA molecule. In another embodiment, the second target region is located within the coding sequence of the 5' end of the DNA molecule. In one embodiment, the second target region includes a portion of the coding sequence of the DNA molecule and a portion of the 5' untranslated region (as defined herein).

[0239] In one embodiment, the third target region is located within the coding sequence of the DNA molecule. In another embodiment, the third target region includes a portion of the coding sequence of the DNA molecule and a portion of the untranslated region.

[0240] In one embodiment, the third target region is located at the 3' end of the DNA molecule. In one embodiment, the third target region is located within the coding sequence of the 3' end of the DNA molecule. In one embodiment, the third target region includes a portion of the coding sequence of the DNA molecule and a portion of the 3' untranslated region (as defined herein). In one embodiment, the third target region is located at the 5' end of the DNA molecule. In one embodiment, the third target region is located within the coding sequence of the 3' end of the DNA molecule. In one embodiment, the third target region includes a portion of the coding sequence of the DNA molecule and a portion of the 5' untranslated region (as defined herein).

[0241] In one embodiment, the fourth target region is located within the coding sequence of the DNA molecule. In another embodiment, the fourth target region includes a portion of the coding sequence of the DNA molecule and a portion of the untranslated region. In yet another embodiment, the fourth target region is located within the untranslated region of the DNA molecule.

[0242] In one embodiment, the fourth target region is located at the 3' end of the DNA molecule. In one embodiment, the fourth target region is located within the coding sequence of the 3' end of the DNA molecule. In one embodiment, the fourth target region includes a portion of the coding sequence of the DNA molecule and a portion of the 3' untranslated region (as defined herein). In one embodiment, the fourth target region is located at the 5' end of the DNA molecule. In one embodiment, the fourth target region is located within the coding sequence of the 5' end of the DNA molecule. In one embodiment, the fourth target region includes a portion of the coding sequence of the DNA molecule and a portion of the 5' untranslated region (as defined herein).

[0243] In one embodiment, the PCR is digital PCR (dPCR). In one embodiment, the dPCR-based assay uses a detectable label. In one embodiment, the detectable label is a fluorescent probe.

[0244] In one embodiment, the PCR is droplet digital PCR (ddPCR). In one embodiment, the ddPCR-based assay uses a detectable label. In one embodiment, the detectable label is a fluorescent probe.

[0245] Efficiency of RNA sample preparation

[0246] In one aspect, the quality parameter being measured is the potency of a formulated RNA sample containing an RNA molecule of interest. In this aspect, the present invention provides a method for determining the potency of a formulated RNA sample containing an RNA molecule of interest, the method comprising the following steps:

[0247] a) Provide RNA samples isolated from cells transfected with prepared RNA samples;

[0248] b) Reverse transcribe the RNA molecules in the RNA sample into cDNA molecules;

[0249] c) Performing a polymerase chain reaction (PCR) assay on the obtained cDNA molecules using a first primer, a second primer, a third primer, and a fourth primer, wherein the first primer and the second primer are annealed to a first target region and a second target region of the cDNA molecule generated from the RNA of interest in the sample, and the third primer and the fourth primer are annealed to the first target region and the second target region of the cDNA molecule derived from endogenous RNA in the sample; and

[0250] d) Compare the measured amount of cDNA produced by the RNA molecule of interest with the measured amount of cDNA produced by endogenous RNA.

[0251] In one embodiment, the first target region is located within the coding sequence of the cDNA molecule. In another embodiment, the first target region includes a portion of the coding sequence of the cDNA molecule and a portion of the untranslated region.

[0252] In one embodiment, the first target region is located at the 3' end of the cDNA molecule. In another embodiment, the first target region is located within the coding sequence of the 3' end of the cDNA molecule. In yet another embodiment, the first target region includes a portion of the coding sequence of the cDNA molecule and a portion of the 3' untranslated region (as defined herein). In one embodiment, the first target region is located at the 5' end of the cDNA molecule. In yet another embodiment, the first target region is located within the coding sequence of the 5' end of the cDNA molecule. In yet another embodiment, the first target region includes a portion of the coding sequence of the cDNA molecule and a portion of the 5' untranslated region (as defined herein).

[0253] In one embodiment, the second target region is located within the coding sequence of the cDNA molecule. In another embodiment, the second target region includes a portion of the coding sequence of the cDNA molecule and a portion of the untranslated region.

[0254] In one embodiment, the second target region is located at the 3' end of the cDNA molecule. In one embodiment, the second target region is located within the coding sequence of the 3' end of the cDNA molecule. In one embodiment, the second target region includes a portion of the coding sequence of the cDNA molecule and a portion of the 3' untranslated region (as defined herein). In one embodiment, the second target region is located at the 5' end of the cDNA molecule. In one embodiment, the second target region is located within the coding sequence of the 5' end of the cDNA molecule. In one embodiment, the second target region includes a portion of the coding sequence of the cDNA molecule and a portion of the 5' untranslated region (as defined herein).

[0255] In one embodiment, the PCR is digital PCR (dPCR). In one embodiment, the dPCR-based assay uses a detectable label. In one embodiment, the detectable label is a fluorescent probe.

[0256] In one embodiment, the PCR is droplet digital PCR (ddPCR). In one embodiment, the ddPCR-based assay uses a detectable label. In one embodiment, the detectable label is a fluorescent probe.

[0257] In one implementation, the endogenous RNA is RNA expressed by housekeeping genes in the sample.

[0258] In one implementation, the endogenous RNA is RNA expressed by the GAPDH gene in the sample.

[0259] In one embodiment, the RNA composition is formulated to contain two or more different RNA molecules of interest, and the PCR-based assay uses two or more different primer pairs, each primer pair being specific to one of the RNA molecules of interest.

[0260] In one embodiment, steps a) through d) are repeated for other RNA samples that have been isolated from cells transfected with a formulated RNA composition containing the RNA molecule of interest to determine the potency level of each formulated RNA composition.

[0261] In one embodiment, the method is implemented to determine the expected potency level of a specific formulated RNA composition containing the RNA of interest, the expected potency level being defined as a reference potency level.

[0262] In one embodiment, the method is performed on other formulated RNA compositions containing RNA of interest and compared to a reference potency level of a specific formulated RNA composition.

[0263] In one implementation, the method further includes the following step z) prior to step a):

[0264] z) Isolate / purify RNA from cells that have been transfected with the RNA sample prepared as described.

[0265] In one implementation, the method further includes the following step y) prior to step z):

[0266] y) Transfect the cells with the prepared RNA composition.

[0267] In one embodiment, cells are transfected with RNA that is complexed with at least one carrier compound, thereby forming at least one RNA-carrier complex.

[0268] RNA vector

[0269] In one embodiment, at least one RNA molecule present in the sample is complexed with at least one carrier compound to form at least one RNA-carrier complex.

[0270] In one embodiment, the at least one carrier compound is a member selected from the group consisting of lipids, phospholipids, polyethylene glycol-modified lipids, cationic and polycationic compounds, and combinations thereof.

[0271] In one embodiment, at least one RNA carrier complex is selected from the group consisting of liposomes, lipid nanoparticles, lipid complexes, and mixtures thereof.

[0272] Particles

[0273] The RNA of the present invention can 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 the complex and the spontaneous formation of nucleic acid particles.

[0274] 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 mediators, RNA nanoparticle encapsulation physically protects RNA from degradation and, depending on specific chemical properties, can aid in cellular uptake and endosome escape.

[0275] In the context of this disclosure, the term "particle" refers to a structural entity formed of molecules or molecular complexes, particularly compounds that form particles. In some embodiments, the particle comprises a coating layer (e.g., one or more layers or one or more 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 a variety of 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.

[0276] "RNA particles" can be used to deliver RNA to target sites of interest (e.g., cells, tissues, organs, etc.). RNA particles can be composed of lipids containing at least one cationic or ionizable cationic lipid or lipid-like substance. While not adhering to any particular theory, it is believed that cationic or ionizable cationic lipids or lipid-like substances combine with RNA to form aggregates, and this aggregation ultimately forms colloidally stable particles.

[0277] The nucleic acid particles (such as RNA particles, DNA particles, or DNA / RNA particles) described herein include formulations based on lipid nanoparticles (LNPs) and formulations based on lipid complexes (LPXs).

[0278] 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.

[0279] 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.

[0280] 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.

[0281] 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).

[0282] 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 the spontaneous formation of 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, nucleic acid (such as RNA and / or DNA, particularly mRNA) liposome complex particles are nanoparticles.

[0283] 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, and they do not form a lamellar (bilayer) phase in water.

[0284] 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.

[0285] 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)).

[0286] 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.

[0287] In some embodiments, the average diameter of the particles described herein (e.g., LNP and LPX) is, in one embodiment, about 50 nm to about 1000 nm, about 50 nm to about 800 nm, about 50 nm to about 700 nm, about 50 nm to about 600 nm, about 50 nm to about 500 nm, about 50 nm to about 450 nm, about 50 nm to about 400 nm, about 50 nm to about 350 nm, about 50 nm to about 300 nm, about 50 nm to about 250 nm, about 50 nm to about 200 nm, about 100 nm to about 1000 nm, about 100 nm to about 800 nm, about 100 nm to about 700 nm, about 100 nm to about 600 nm. nm, approximately 100nm to approximately 500nm, approximately 100nm to approximately 450nm, approximately 100nm to approximately 400nm, approximately 100nm to approximately 350nm, approximately 100nm to approximately 300nm, approximately 100nm to approximately 250nm, approximately 100nm to approximately 200nm, approximately 150nm to approximately 1000nm, approximately 150nm to approximately 800nm, approximately 150nm to approximately 700nm, approximately 150nm to approximately 600nm, approximately 150nm to approximately 500nm, approximately 150nm to approximately 450nm, approximately 150nm to approximately 400nm, approximately 150nm to approximately 350nm, approximately 150nm to approximately 300nm, approximately 150nm to approximately 250nm, approximately 150nm to approximately 200nm, approximately 200nm to approximately 1000nm, approximately 200nm to approximately 800nm, approximately 200nm to approximately 700nm, approximately 200nm to approximately 600nm nm, about 200 nm to about 500 nm, about 200 nm to about 450 nm, about 200 nm to about 400 nm, about 200 nm to about 350 nm, about 200 nm to about 300 nm, or about 200 nm to about 250 nm.

[0288] 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.

[0289] The polydispersity index (PDI) exhibited by the nucleic acid particles (especially mRNA particles) described herein can be less than about 0.5, less than about 0.4, less than about 0.3, less than about 0.2, less than about 0.1, or less than about 0.05. As an example, the polydispersity index exhibited by the nucleic acid particles can be in the range of about 0.01 to about 0.4 or about 0.1 to about 0.3.

[0290] The N / P ratio represents the ratio of the number of nitrogen groups in lipids to the number of phosphate groups in nucleic acids. It is related to the charge ratio, as nitrogen atoms (depending on pH) are generally positively charged, while phosphate groups are negatively charged. In a state of charge equilibrium, the N / P ratio depends on pH. Lipid formulations typically form with N / P ratios greater than 4 up to 12 because positively charged nanoparticles are considered favorable for transfection. In this case, RNA is assumed to be completely bound to the nanoparticles.

[0291] The nucleic acid particles (particularly RNA particles, such as mRNA particles) described herein can be prepared using a variety of methods, which may include obtaining a colloid from at least one cationic or ionizable cationic lipid and mixing the colloid with nucleic acids to obtain nucleic acid particles.

[0292] As used herein, the term "colloid" refers to a type of homogeneous mixture in which dispersed particles do not precipitate. The insoluble particles in the mixture are microscopic, with a particle size between 1 and 1000 nanometers. Such mixtures may be called colloids or colloidal suspensions. Sometimes, the term "colloid" refers only to the particles in the mixture, rather than the entire suspension.

[0293] For the preparation of colloids containing at least one cationic or ionizable cationic lipid, this article applies conventional methods for the preparation of liposome vesicles, with appropriate modifications. The most commonly used methods for the preparation of liposome vesicles have the following basic steps: (i) dissolving the lipid in an organic solvent; (ii) drying the resulting solution; and (iii) hydrating the dried lipid (using various aqueous media).

[0294] In the membrane hydration method, lipids are first dissolved in a suitable organic solvent and dried to form a thin film at the bottom of a flask. The resulting lipid membrane is then hydrated with a suitable aqueous medium to prepare a liposome dispersion. Additionally, an extra particle size reduction step can be added.

[0295] Reverse-phase evaporation is an alternative to membrane hydration for the preparation of liposome vesicles. This method involves forming a water-in-oil emulsion between an aqueous phase and a lipid-containing organic phase. The mixture is then briefly sonicated to homogenize the system. After removing the organic phase under reduced pressure, an emulsion gel is obtained, which is subsequently converted into a liposome suspension.

[0296] The term "ethanol injection technique" refers to the process of rapidly injecting an ethanol solution containing lipids into an aqueous solution through a needle. This operation disperses the lipids throughout the solution and promotes the formation of lipid structures, such as lipid vesicles (e.g., liposomes). Typically, the nucleic acid (such as RNA and / or DNA, particularly mRNA) liposome complex particles described herein can be obtained by adding nucleic acids (such as RNA and / or DNA, particularly mRNA) to a colloidal liposome dispersion. In some embodiments, the ethanol injection technique is used to form such a colloidal liposome dispersion by injecting an ethanol solution containing lipids (such as cationic or ionizable cationic lipids, such as DOTMA and / or DODMA, and other lipids) into an aqueous solution under stirring. In some embodiments, the nucleic acid (such as RNA and / or DNA, particularly mRNA) liposome complex particles described herein are obtained without an extrusion step.

[0297] The term "extrusion" or "extrusion" refers to the production of particles with a fixed cross-sectional profile. Specifically, it refers to the reduction of particle size, achieved by forcing particles through a filter with a specific pore size.

[0298] According to this disclosure, other methods characterized by the absence of organic solvents can also be used to prepare colloids.

[0299] In some embodiments, the LNP comprises four components: ionizable cationic lipids, neutral lipids (such as phospholipids), steroids (such as cholesterol), and polymer-conjugated lipids. In some embodiments, the LNP can be prepared by rapidly mixing lipids dissolved in ethanol with nucleic acids (such as RNA and / or DNA) in an aqueous buffer. While the nucleic acid (such as RNA and / or DNA) particles described herein may contain polymer-conjugated lipids (such as PEG lipids), nucleic acid (such as RNA and / or DNA) particles that do not contain polymer-conjugated lipids (such as PEG lipids) are also provided herein.

[0300] In some embodiments, the method for preparing the LNP comprising nucleic acids (such as RNA and / or DNA) and at least one cationic or ionizable cationic lipid described herein is as follows: (a) preparing a nucleic acid (such as RNA and / or DNA) solution containing water and a buffer system; (b) preparing an ethanol solution comprising a cationic or ionizable cationic lipid and (if present) one or more other lipids; and (c) mixing the nucleic acid (such as RNA and / or DNA) solution prepared in step (a) with the ethanol solution prepared in step (b) to prepare a formulation comprising the LNP. Following step (c), one or more steps selected from dilution and filtration (such as tangential flow filtration) may be performed.

[0301] In some embodiments, the method for preparing the LNP comprising nucleic acids (such as RNA and / or DNA) and at least one cationic or ionizable cationic lipid described herein is as follows: (a') preparing a liposome or colloidal formulation of the cationic or ionizable cationic lipid and (if present) one or more other lipids in an aqueous phase; (b') preparing a nucleic acid (such as RNA and / or DNA) solution containing water and a buffer system; and (c') mixing the liposome or colloidal formulation prepared in step (a') with the nucleic acid (such as RNA and / or DNA) solution prepared in step (b'). Following step (c'), one or more steps selected from dilution and filtration (such as tangential flow filtration) may be performed.

[0302] This disclosure describes particles comprising nucleic acids (such as RNA and / or DNA, particularly mRNA) and at least one cationic or ionizable cationic lipid, said cationic or ionizable cationic lipid associating with nucleic acids (such as RNA and / or DNA) to form nucleic acid (such as RNA and / or DNA) particles, and describes compositions comprising such particles. The nucleic acid (such as RNA and / or DNA) particles may contain nucleic acids (such as RNA and / or DNA) complexed with the particles in different forms through non-covalent interactions. The particles described herein are not viral particles, and in particular, are not infectious viral particles, i.e., they cannot virally infect cells.

[0303] Suitable cationic lipids or ionizable cationic lipids refer to those lipids that form nucleic acid particles, and they fall within the scope of the terms "particle-forming components" or "granulating agents." The terms "particle-forming components" or "granulating agents" refer to any component capable of associating with nucleic acids to form nucleic acid particles. Such components include any component that can become part of a nucleic acid particle.

[0304] In some implementations, nucleic acid particles (such as RNA and / or DNA particles, especially mRNA particles) contain more than one type of nucleic acid (such as RNA and / or DNA) molecules, wherein the molecular parameters of the nucleic acid (such as RNA and / or DNA) molecules may be similar or different from each other, for example, in terms of molar mass or basic structural elements, such as molecular structure, capping (RNA only), coding regions or other features.

[0305] In granule formulations, each type of nucleic acid (such as RNA and / or DNA) can be formulated into a separate granule formulation. In this case, each separate granule formulation will contain one type of nucleic acid (such as RNA and / or DNA). These separate granule formulations can exist as independent entities, for example, in separate containers. Such formulations can be obtained by providing each type of nucleic acid (such as RNA and / or DNA) separately (usually in the form of a solution containing nucleic acids (such as RNA and / or DNA)) with a granulating agent to form granules. Each granule will contain only the specific type of nucleic acid (such as RNA and / or DNA) provided when forming the granule (separate granule formulation). In some embodiments, a composition (such as a pharmaceutical composition) contains more than one separate granule formulation. The corresponding pharmaceutical composition is referred to as a mixed granule formulation. According to the invention, a mixed granule formulation can be obtained by the steps of forming separate granule formulations separately and then mixing these separate granule formulations. Through the mixing step, a formulation containing a mixed population of particles containing nucleic acids (such as RNA and / or DNA) is obtained. Multiple separate granule populations can be placed together in a container containing a mixed population of multiple separate granule formulations. Alternatively, all nucleic acid types (such as RNA and / or DNA) in the pharmaceutical composition can be formulated together into a compound granule formulation. Such formulations can be obtained by providing a compound formulation (typically a compound solution) of all nucleic acid types (such as RNA and / or DNA) with a granulating agent to form granules. Unlike mixed granule formulations, compound granule formulations typically contain granules containing more than one type of nucleic acid (such as RNA and / or DNA). In compound granule compositions, different nucleic acid types (such as RNA and / or DNA) are typically present together in a single granule.

[0306] polymer

[0307] Polymers are frequently used as materials for nanoparticle-based delivery due to their high chemical flexibility. Typically, cationic polymers are used to aggregate negatively charged nucleic acids into nanoparticles via electrostatic interactions. These positively charged groups are usually composed of amines, whose protonation state changes within a pH range of 5.5 to 7.5, which is thought to lead to ionic imbalance and consequently endosome disruption. Polymers such as poly-L-lysine, polyamidoamine, protamine, and polyethyleneimine, as well as natural polymers such as chitosan, have been used for nucleic acid delivery and are suitable for use as the cationic polymers described herein. Furthermore, some researchers have synthesized polymers specifically for nucleic acid delivery. In particular, poly(β-amino esters), due to their ease of synthesis and biodegradability, have been widely used in nucleic acid delivery. Such synthetic polymers are also suitable for use as the cationic polymers described herein.

[0308] As used herein, “polymer” has its usual meaning, referring to a molecular structure containing one or more repeating units (monomers) linked by covalent bonds. These repeating units may be identical, or in some cases, more than one type of repeating unit may be present in a polymer. In some cases, polymers are of biological origin, i.e., biopolymers, such as proteins. In other cases, polymers may also contain other parts, such as targeting moieties.

[0309] A polymer is called a "polymer" if it contains more than one type of repeating unit. It should be understood that the polymer used herein can be a copolymer. The repeating units forming a copolymer can be arranged in any manner. For example, the repeating units can be arranged in a random order, an alternating order, or as a "block" copolymer, i.e., comprising one or more regions each containing a first repeating unit (e.g., a first block) and one or more regions each containing a second repeating unit (e.g., a second block), and so on. Block copolymers can have two (diblock copolymers), three (triblock copolymers), or more different blocks.

[0310] In some embodiments, the polymer is biocompatible. A biocompatible polymer is a polymer that typically does not cause significant cell death at moderate concentrations. In some embodiments, the biocompatible polymer is biodegradable, meaning that the polymer is capable of chemical and / or biodegradation within a physiological environment, such as in vivo. In some embodiments, the polymer may be protamine sulfate or polyalkylene imide.

[0311] The term "protamine" refers to any of a variety of strongly basic, relatively low molecular weight, arginine-rich proteins that specifically associate with DNA, replacing somatic histones in sperm cells of many animals (such as fish). More specifically, "protamine" refers to the strongly basic, water-soluble protein found in fish sperm that does not coagulate upon heating and primarily produces arginine upon hydrolysis. Purified forms of protamine are used in long-acting insulin preparations to neutralize the anticoagulant effect of heparin.

[0312] According to this disclosure, the term "protamine" as used herein refers to any protamine amino acid sequence, including fragments thereof, and multimeric forms of said amino acid sequence or fragments thereof, obtained or derived from natural or biological sources, as well as (synthetic) polypeptides that are artificial and specifically designed for a particular purpose and cannot be isolated from natural or biological sources.

[0313] In one embodiment, the polyalkylene imide comprises polyethyleneimine and / or polypropyleneimine, preferably polyethyleneimine. A preferred polyalkylene imide is polyethyleneimine (PEI). The average molecular weight of PEI is preferably 0.75 × 10⁻⁶. 2Up to 10 7 Da, preferably 1000 to 10 5 Da, more preferably 10,000 to 40,000 Da, more preferably 15,000 to 30,000 Da, and even more preferably 20,000 to 25,000 Da.

[0314] According to this disclosure, linear polyalkylene imides, such as linear polyethyleneimine (PEI), are preferred.

[0315] The cationic polymers (including polycationic polymers) considered for use herein include any cationic polymer capable of binding nucleic acids via electrostatic interactions. In one embodiment, the cationic polymers considered for use herein include any cationic polymer capable of associating with nucleic acids, for example, by forming a complex with the nucleic acid or forming vesicles therein that encapsulate or encapsulate the nucleic acid.

[0316] The particles described herein may also contain polymers other than cationic polymers, namely non-cationic polymers and / or anionic polymers. In this document, anionic polymers and neutral polymers are collectively referred to as non-cationic polymers.

[0317] lipids

[0318] The terms “lipid” and “lipid-like substances” are broadly defined herein to refer to molecules containing one or more hydrophobic moieties or groups and optionally one or more hydrophilic moieties or groups. Molecules containing both hydrophobic and hydrophilic moieties are also often referred to as amphiphilic molecules. Lipids are generally insoluble or slightly soluble in water, but soluble in many organic solvents. In an aqueous environment, amphiphilicity allows lipid molecules to self-assemble into ordered structures and different phases. When present in an aqueous environment in vesicles, multilayer / monolayer liposomes, or membranes, one phase consists of a lipid bilayer. Hydrophobicity can be imparted by the introduction of nonpolar groups, including but not limited to long-chain saturated and unsaturated aliphatic hydrocarbon groups, and such groups substituted with one or more aromatic, cyclic aliphatic, or heterocyclic groups. Hydrophilic groups can include polar groups and / or charged groups, including carbohydrate, phosphate, carboxyl, sulfate, amino, thiol, nitro, hydroxyl, and other similar groups.

[0319] As used herein, the term "hydrophobic" refers to any molecule, part, or group that is substantially immiscible or insoluble in aqueous solutions. The term "hydrophobic group" includes hydrocarbon compounds containing at least six carbon atoms. Hydrophobic groups may have functional groups (e.g., ethers, esters, halogens, etc.) and atoms other than carbon and hydrogen, provided that the group satisfies the condition of being substantially immiscible or insoluble in aqueous solutions.

[0320] The term "hydrocarbon" as defined herein includes alkyl, alkenyl, or alkynyl groups. It should be understood that one or more hydrogen atoms in an alkyl, alkenyl, or alkynyl group may be substituted with other atoms (e.g., halogen, oxygen, or sulfur). Unless otherwise stated, a hydrocarbon group may also include cyclic (alkyl, alkenyl, or alkynyl) groups or aryl groups, provided that the overall polarity of the hydrocarbon remains relatively nonpolar.

[0321] The term "alkyl" refers to a saturated straight-chain or branched monovalent hydrocarbon moiety that can have 6 to 30 carbon atoms, typically 6 to 20, and commonly 6 to 18. Exemplary nonpolar alkyl groups include, but are not limited to, hexyl, decyl, dodecyl, tetradecyl, hexadecyl, octadecyl, etc.

[0322] The term "alkenyl" refers to a straight-chain or branched monovalent hydrocarbon moiety having at least one carbon-carbon double bond, wherein the total number of carbon atoms can be 6 to 30, usually 6 to 20, and often 6 to 18.

[0323] The term "alkynyl" refers to a straight-chain or branched monovalent hydrocarbon moiety having at least one carbon-carbon triple bond, wherein the total number of carbon atoms can be 6 to 30, typically 6 to 20, and often 6 to 18. The alkynyl group may optionally have one or more carbon-carbon double bonds.

[0324] As used herein, the term "amphiphilic" refers to a molecule that simultaneously possesses both a polar and a nonpolar portion. Typically, amphiphilic compounds have a polar head connected to a long hydrophobic tail. In some embodiments, the polar portion is soluble in water, while the nonpolar portion is insoluble in water. Furthermore, the polar portion may carry a positive or negative charge. Alternatively, the polar portion may carry both a positive and a negative charge and be an amphoteric ion or an inner salt. For the purposes of this disclosure, the amphiphilic compound may be, but is not limited to, one or more natural or non-natural lipid and lipid-like compounds.

[0325] The terms "lipid-like substances," "lipid-like compounds," or "lipid-like molecules" refer to substances that are structurally and / or functionally related to lipids but can not be strictly considered lipids, particularly amphiphilic substances. For example, the term includes compounds capable of forming amphiphilic layers when present in an aqueous environment in vesicles, multilayer / monolayer liposomes, or membranes, including surfactants or synthetic compounds possessing both hydrophilic and hydrophobic portions. Generally, the term refers to molecules comprising hydrophilic and hydrophobic portions with different structural organization, whose structures may be similar to or dissimilar to lipids. Examples of lipid-like compounds capable of spontaneously integrating into cell membranes include functional lipid constructs such as synthetic functional-spacer-lipid constructs (FSL), synthetic functional-spacer-sterol constructs (FSS), and artificial amphiphilic molecules. Lipids are typically cylindrical. The area occupied by the two alkyl chains is similar to that occupied by the polar head groups. Lipid monomers have low solubility and tend to aggregate to form water-insoluble planar bilayer structures. Conventional surfactant monomers are typically conical. Hydrophilic head groups tend to occupy more molecular space than linear alkyl chains. In some embodiments, surfactants tend to aggregate to form water-soluble spherical or elliptical micelles. While lipids also have the same basic structure as surfactants (polar hydrophilic head groups and nonpolar hydrophobic tails), lipids differ from surfactants in the shape of the monomers, the type of aggregates formed in solution, and the concentration range required for aggregation. Unless otherwise stated herein or explicitly required by context, the term "lipid" as used herein should be interpreted to encompass both lipids and lipid-like substances.

[0326] Lipids are generally classified into eight major categories: fatty acids, glycerides, glycerophospholipids, sphingolipids, glycolipids, polyketides (formed by the condensation of ketoacyl subunits), sterol lipids, and isopentenol lipids (formed by the condensation of isopentenyl subunits). Although the term "lipid" is sometimes used synonymously with fat, fat is a subclass of lipid called triglycerides. Lipids also encompass molecules such as fatty acids and their derivatives (including triglycerides, diglycerides, monoglycerides, and phospholipids) as well as sterol compounds, i.e., sterol-containing metabolites, such as cholesterol or its derivatives. Examples of cholesterol derivatives include, but are not limited to, cholesterolanol, cholesterol ketone, cholesterol sterol, coprostinol, cholesterol-2'-hydroxyethyl ether, cholesterol-4'-hydroxybutyl ether, tocopherol and its derivatives and mixtures.

[0327] Fatty acids, or fatty acid residues, are a diverse group of molecules composed of hydrocarbon chains terminated by carboxylic acid groups. This arrangement gives the molecule a polar hydrophilic end and a nonpolar hydrophobic end, making it insoluble in water. The carbon chains are typically 4 to 24 carbon atoms long, can be saturated or unsaturated, and can be linked with functional groups containing oxygen, halogens, nitrogen, and sulfur. If a fatty acid contains double bonds, it may exist as cis or trans geometric isomers, which significantly affect the molecular configuration. Cis double bonds cause the fatty acid chain to bend, and the more double bonds in the chain, the more pronounced this bending effect. Other major lipid types in the fatty acid class include fatty acid esters and fatty acid amides.

[0328] Glycerides consist of monosubstituted, disubstituted, and trisubstituted glycerols, the most well-known of which are fatty acid triesters of glycerol, called triglycerides. Sometimes, the term "triacylglycerol" is synonymous with "triglyceride." In these compounds, the three hydroxyl groups of glycerol are each esterified, typically by different fatty acids. Another subclass of glycerides is represented by glycosylglycerol, characterized by the presence of one or more sugar residues linked to glycerol via glycosidic bonds.

[0329] Glycerophospholipids are amphiphilic molecules (containing both hydrophobic and hydrophilic regions) with a glycerol core linked to two fatty acid-derived "tails" via ester bonds and to a "head" group via phosphate ester bonds. Examples of glycerophospholipids (commonly referred to as phospholipids, although sphingomyelins are also classified as phospholipids) are phosphatidylcholine (also known as PC, GPCho, or lecithin), phosphatidylethanolamine (PE or GPEtn), and phosphatidylserine (PS or GPSer).

[0330] Sphingolipids are a class of complex compounds sharing a common structural feature: a sphingosine base skeleton. The main sphingosine base in mammals is commonly referred to as sphingosine. Ceramides (N-acylsphingosine bases) are a major subclass of sphingosine base derivatives, consisting of fatty acids linked by amide bonds. These fatty acids are typically saturated or monounsaturated, with carbon chain lengths ranging from 16 to 26 carbon atoms. The main phosphospholipid in mammals is sphingomyelin (ceramide phosphocholine), while insects primarily contain ceramide phosphoethanolamine, and fungi contain phytoceramide phosphoinositol and a mannose-containing head group. Glycosphingolipids are a diverse family of molecules composed of one or more sugar residues linked to a sphingosine base via glycosidic bonds. Examples of these substances range from simple to complex glycosphingolipids, such as cerebrosides and gangliosides.

[0331] Sterol lipids, such as cholesterol and its derivatives, or tocopherol and its derivatives, along with glycerophospholipids and sphingomyelin, are important components of membrane lipids.

[0332] Glycolipids are compounds in which fatty acids are directly attached to a sugar backbone, forming a structure compatible with the membrane bilayer. In glycolipids, monosaccharides replace the glycerol backbone in glycerides and glycerophospholipids. The most common glycolipid is the acylated glucosamine precursor of the lipid A component of lipopolysaccharides in Gram-negative bacteria. The typical lipid A molecule is a glucosamine disaccharide, which is derivatized to have up to seven fatty-acyl chains. The smallest lipopolysaccharide required for the growth of *E. coli* is Kdo2-lipopolysaccharide A, a hexaacylated glucosamine disaccharide glycosylated by two 3-deoxy-D-mannooctulose (Kdo) residues.

[0333] Polyketides are synthesized via the polymerization of acetyl and propionyl subunits using classical enzymes, as well as iterative and modular enzymes with mechanisms similar to fatty acid synthases. They comprise a wide range of secondary metabolites and natural products from animal, plant, bacterial, fungal, and marine organisms, exhibiting remarkable structural diversity. Many polyketides are cyclic molecules, and their backbones are often further modified through glycosylation, methylation, hydroxylation, oxidation, or other processes.

[0334] According to this disclosure, lipids and lipid-like substances can be cationic, anionic, or neutral. Neutral lipids or lipid-like substances exist as uncharged or neutral zwitterionic forms at a selected pH value.

[0335] Cationic / ionizable cationic lipids

[0336] The nucleic acid particles (such as RNA and / or DNA particles) described herein contain at least one cationic or ionizable cationic lipid as a granulating agent. The cationic or ionizable cationic lipids considered herein include any cationic or ionizable cationic lipid (including lipid-like substances) capable of electrostatically binding nucleic acids. In some embodiments, the cationic or ionizable cationic lipids considered herein may associate with nucleic acids, for example, by forming a complex with nucleic acid or forming vesicles that encapsulate or block nucleic acids.

[0337] As used herein, “cationic lipids” refers to lipids or lipid-like substances that carry a net positive charge. Cationic lipids bind to negatively charged nucleic acids through electrostatic interactions. Typically, cationic lipids have a lipophilic moiety, such as a sterol, acyl chain, diacyl chain, or multiple acyl chains, and the head group of the lipid is usually positively charged.

[0338] In some embodiments, cationic lipids carry a net positive charge only at specific pH values ​​(especially acidic pH values), while at different pH values ​​(preferably higher pH values, such as physiological pH values), they preferably do not carry a net positive charge, and are preferably uncharged, i.e., neutral. This ionization behavior is believed to enhance therapeutic efficacy compared to particles that remain cationic at physiological pH values, as it facilitates endosome escape and reduces toxicity.

[0339] As used herein, "ionizable cationic lipid" refers to a lipid or lipid-like substance having a net positive charge or being neutral (i.e., not permanently cationic). Therefore, depending on the pH of the composition in which the ionizable cationic lipid is dissolved, the ionizable cationic lipid is either positively charged or neutral. For the purposes of this disclosure, unless otherwise specified, the term "cationic lipid" is used to refer to ionizable cationic lipids.

[0340] In some embodiments, the cationic or ionizable cationic lipid includes a head group comprising at least one positively charged or protonable nitrogen atom (N), for example, positively charged or protonable under physiological conditions.

[0341] Examples of cationic or ionizable cationic lipids include, but are not limited to: N,N-dimethyl-2,3-dioleoyloxypropylamine (DODMA), 1,2-dioleoyl-3-trimethylammonium propane (DOTAP), 1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA), 3-(N-(N′,N′-dimethylaminoethane)-carbamoyl)cholesterol (DC-Chol), dimethylbis(octadecyl)ammonium (DDAB); 1,2-dioleoyl-3-dimethylammonium propane (DODAP); 1,2-diacyloxy-3-dimethylammonium propane; 1,2-dialkoxy-3-dimethylammonium propane; bis(octadecyl)dimethylammonium chloride (DODAC), 1,2-distearateoxy-N,N-dimethyl-3-aminopropane (DSDMA), 2,3-bis(tetradecoxy)propyl-(2-hydroxyethyl)-dimethylammonium (DMRI). E), 1,2-dimyristoyl-sn-glycerol-3-ethylphosphocholine (DMEPC), 1,2-dimyristoyl-3-trimethylammonium propane (DMTAP), 1,2-dioleoyloxypropyl-3-dimethyl-hydroxyethylammonium bromide (DORIE), and 2,3-dioleoyloxy-N-[2-(sperminecarbamate)ethyl]-N,N-dimethyl-1-propylamine trifluoroacetate (DOSPA), 1,2-dilinoleoyloxy-N,N-dimethylaminopropane (DLinDMA), 1,2-dilinoleoyloxy-N,N-dimethylaminopropane (DLenDMA), and bis(octadecylamidoglycylspermine)spermine (DOGS). 3-Dimethylamino-2-(cholest-5-en-3-β-oxobut-4-oxy)-1-(cis,cis-9,12-octadecadienoxy)propane (CLinDMA), 2-[5′-(cholest-5-en-3-β-oxy)-3′-oxaproloxy)-3-dimethyl-1-(cis,cis-9′,12′-octadecadienoxy)propane (CpLinDMA), N,N-dimethyl-3,4-dioleoyloxybenzylamine (DMOBA), 1,2-N,N′-dioleoylcarbamoyl-3-dimethylaminopropane (DOcarbDAP), 2,3-dilinoleoyloxy-N,N-dimethylpropylamine (DLi) nDAP), 1,2-N,N′-dilinoleylcarbamoyl-3-dimethylaminopropane (DLincarbDAP), 1,2-dilinoleylcarbamoyl-3-dimethylaminopropane (DLinCDAP), 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-K-XTC2-DMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA), heptadecane-6,9,28,31-Tetraen-19-yl-4-(dimethylamino)butyrate (DLin-MC3-DMA), N-(2-hydroxyethyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)-1-propylamine bromide (DMRIE), (±)-N-(3-aminopropyl)-N,N-dimethyl-2,3-bis(cis-9-tetradecenyloxy)-1-propylamine bromide (GAP-DMORIE), (±)-N-(3-aminopropyl)-N,N-dimethyl-2,3-bis(dodecyloxy)-1-propylamine bromide (GAP-DL) RIE), (±)-N-(3-aminopropyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)-1-propanamine bromide (GAP-DMRIE), N-(2-aminoethyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)-1-propanamine bromide (βAE-DMRIE), N-(4-carboxybenzyl)-N,N-dimethyl-2,3-bis(oleoyloxy)prop-1-amine (DOBAQ), 2-({8-[(3β)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3- [(9Z,12Z)-Octadeca-9,12-dien-1-yloxy]prop-1-amine (octyl-CLinDMA), 1,2-dimyristoyl-3-dimethylammonium propane (DMDAP), 1,2-dipalmitoyl-3-dimethylammonium propane (DPDAP), N1-[2-((1S)-1-[(3-aminopropyl)amino]-4-[di(3-amino-propyl)amino]butylcarbamoyl)ethyl]-3,4-di[oleoyloxy]benzamide (MVL5), 1,2-dioleoyl-sn-glycerol-3-ethylphosphocholine (DOEPC), 2,3-bis(dodecyloxy)-N-(2-hydroxyethyl)-N,N-dimethylpropane-1-ammonium bromide (DLRIE), N-(2-aminoethyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)propane-1-ammonium bromide (D MORIE), di((Z)-non-2-en-1-yl)8,8'-((((2(dimethylamino)ethyl)thio)carbonyl)azadiyl)dioctanoate (ATX), N,N-dimethyl-2,3-bis(dodecyloxy)prop-1-amine (DLDMA), N,N-dimethyl-2,3-bis(tetradecyloxy)prop-1-amine (DMDMA), di((Z)-non-2-en-1-yl)-9-((4-(dimethylaminobutyryl)oxy)heptadecanoate (L319), N-dodecyl-3-((2-dodecylcarbamoyl-ethyl)-{2-[(2-dodecylcarbamoyl-ethyl)-2-{(2-dodecylcarbamoyl-ethyl)-[2-(2-dodecylcarbamoyl-ethylamino)ethyl]amino}-ethylamino)propionamide (lipidoid 98N, 12-5), 1-[2-[bis(2-hydroxydodecyl)amino]ethyl-[2-[4-[2-[bis(2-hydroxydodecyl)amino]ethyl]piperazin-1-yl]ethyl]amino]dodecane-2-ol (lipidoid C12-200).

[0342] In some embodiments, the cationic or ionizable cationic lipid is DOTMA. In some embodiments, the cationic or ionizable cationic lipid is DODMA.

[0343] DOTMA is a cationic lipid with a quaternary ammonium head group. The structure of DOTMA can be represented as follows:

[0344]

[0345] DODMA is an ionizable cationic lipid with a tertiary amine head group. The structure of DODMA can be represented as follows:

[0346]

[0347] In some embodiments, cationic or ionizable cationic lipids may account for about 10 mol% to about 95 mol%, about 20 mol% to about 95 mol%, about 20 mol% to about 90 mol%, about 30 mol% to about 90 mol%, about 40 mol% to about 90 mol%, or about 40 mol% to about 80 mol% of the total lipids present in the particles.

[0348] Other lipids

[0349] The particles described herein may also contain lipids (including lipid-like substances) other than cationic or ionizable cationic lipids (collectively referred to herein as cationic lipids), i.e., non-cationic lipids (including non-cationic or non-ionizable cationic lipids or lipid-like substances). Anionic and neutral lipids or lipid-like substances are collectively referred to herein as non-cationic lipids. In addition to cationic or ionizable cationic lipids, optimizing the formulation of nucleic acid particles by adding other hydrophobic components (such as cholesterol and lipids) can improve particle stability and nucleic acid delivery efficiency.

[0350] One or more other lipids may or may not affect the overall charge of the nucleic acid particles. In some embodiments, one or more other lipids are non-cationic lipids or lipid-like substances. Non-cationic lipids may include, for example, one or more anionic lipids and / or neutral lipids. As used herein, "anionic lipid" means any lipid that carries a negative charge at a selected pH. As used herein, "neutral lipid" means any of a variety of lipid species that exist at a selected pH in an uncharged or neutral zwitterionic form.

[0351] In some embodiments, the nucleic acid particles described herein (particularly those containing mRNA) comprise cationic or ionizable cationic lipids and one or more other lipids.

[0352] While not wishing to be bound by theory, the molar ratio of cationic or ionizable cationic lipids to one or more other lipids can influence important nucleic acid particle properties, such as nucleic acid charge, particle size, stability, tissue selectivity, and bioactivity. Therefore, in some embodiments, the molar ratio of cationic or ionizable cationic lipids to one or more other lipids is about 10:0 to about 1:9, about 4:1 to about 1:2, about 4:1 to about 1:1, about 3:1 to about 1:1, or about 3:1 to about 2:1.

[0353] In some implementations, the nucleic acid particles (particularly those containing mRNA) described herein contain one or more other lipids, including one or more of the following: neutral lipids, steroids, and combinations thereof.

[0354] In some embodiments, one or more other lipids include neutral lipids, which are phospholipids. In some embodiments, the phospholipids are selected from the group consisting of phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidic acid, phosphatidylserine, and sphingomyelin. Specific phospholipids that may be used include, but are not limited to, phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidic acid, phosphatidylserine, or sphingomyelin. These phospholipids particularly include diacylphosphatidylcholine, such as distearylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dimyristoylphosphatidylcholine (DMPC), bispentadecanylphosphatidylcholine, dilauroylphosphatidylcholine, dipalmitoylphosphatidylcholine (DPPC), arachidoylphosphatidylcholine (DAPC), dibenzylphosphatidylcholine (DBPC), bis(t- ... PC) and phosphatidylethanolamines, especially diacylphosphatidylethanolamines, such as dioleoylphosphatidylethanolamine (DOPE), distearylphosphatidylethanolamine (DSPE), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphatidylethanolamine (DMPE), dilauroylphosphatidylethanolamine (DLPE), diphyranoylphosphatidylethanolamine (DPyPE), 1,2-di-(9Z-octadecenoyl)-sn-glycerol-3-phosphate choline (DOPG), 1,2-dipalmitoyl-sn-glycerol-3-phosphate-(1′-rac-glycerol) (DPPG), 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphate ethanolamine (POPE), N-palmitoyl-D-erythrosine-sphingosine phosphatidylcholine (SM) and other phosphatidylethanolamine lipids with different hydrophobic chains. In some embodiments, the neutral lipid is selected from the group consisting of DSPC, DOPC, DMPC, DPPC, POPC, DOPE, DOPG, DPPG, POPE, DPPE, DMPE, DSPE, and SM. In some embodiments, the neutral lipid is DOPE.

[0355] In one embodiment, the other lipid comprises one of the following: (1) phospholipids; (2) cholesterol or a derivative thereof; or (3) a mixture of phospholipids and cholesterol or a derivative thereof. Examples of cholesterol derivatives include, but are not limited to, cholesterol, cholesterol ketones, coprolitols, cholesterol-2'-hydroxyethyl ether, cholesterol-4'-hydroxybutyl ether, tocopherol and its derivatives, and mixtures thereof.

[0356] Therefore, in some embodiments, the nucleic acid particles described herein (particularly particles containing mRNA) include: (1) cationic or ionizable cationic lipids and phospholipids, such as DOPE; or (2) cationic or ionizable cationic lipids and phospholipids, such as DOPE and cholesterol.

[0357] In one embodiment, the nucleic acid particles described herein (particularly particles containing mRNA) include: (1) DOTMA and DOPE, (2) DOTMA, DOPE and cholesterol, (3) DODMA and DOPE, or (4) DODMA, DOPE and cholesterol.

[0358] DOPE is a neutral phospholipid. The structure of DOPE can be represented as follows:

[0359]

[0360] The structure of cholesterol can be represented as follows:

[0361]

[0362] In some embodiments, the particles described herein do not contain polymer-conjugated lipids, such as polyethylene glycol-modified lipids. The term "polyethylene glycol-modified lipid" refers to a molecule comprising a lipid portion and a polyethylene glycol portion. Polyethylene glycol-modified lipids are known in the art.

[0363] In some embodiments, other lipids (e.g., one or more phospholipids and / or cholesterol) may comprise about 0 mol% to about 90 mol%, about 0 mol% to about 80 mol%, about 2 mol% to about 80 mol%, about 5 mol% to about 80 mol%, about 5 mol% to about 60 mol%, about 5 mol% to about 50 mol%, about 7.5 mol% to about 50 mol%, or about 10 mol% to about 40 mol% of the total lipids present in the particles. In some embodiments, other lipids (e.g., one or more phospholipids and / or cholesterol) may comprise about 10 mol%, about 15 mol%, or about 20 mol% of the total lipids present in the particles.

[0364] In some embodiments, the other lipids comprise a mixture of: (i) phospholipids, such as DOPE; and (ii) cholesterol or a derivative thereof. In some embodiments, the molar ratio of phospholipids (such as DOPE) to cholesterol or a derivative thereof is about 9:0 to about 1:10, about 2:1 to about 1:4, about 1:1 to about 1:4, or about 1:1 to about 1:3.

[0365] Polymer-conjugated lipids

[0366] In some embodiments, the particles may comprise at least one polymer-conjugated lipid. A polymer-conjugated lipid is typically a molecule comprising a lipid moiety and a polymer moiety conjugated thereto. In some embodiments, the polymer-conjugated lipid is a PEG-conjugated lipid, also referred to herein as a polyethylene glycol-modified lipid or PEG-lipid.

[0367] In some embodiments, polymer-conjugated lipids are designed to spatially stabilize lipid particles by forming a protective hydrophilic layer that shields the hydrophobic lipid layer. In some embodiments, when such lipid particles are administered in vivo, the polymer-conjugated lipids can reduce their association with serum proteins and / or the resulting uptake by the reticuloendothelial system.

[0368] Various PEG-conjugated lipids are known in the art, including but not limited to: polyethylene glycol diacylglycerol (PEG-DAG), such as 1-(monomethoxy-polyethylene glycol)-2,3-dimyristoylglycerol (PEG-DMG); polyethylene glycol phosphatidylethanolamine (PEG-PE); polyethylene glycol succinate diacylglycerol (PEG-S-DAG), such as 4-O-(2',3'-di(tetradecanoyloxy)propyl-1-O-(ω-methoxy(polyethoxy)ethyl)succinate (PEG-S-DMG); polyethylene glycol ceramide (PEG-cer) or polyethylene glycol dialkoxypropyl carbamate, such as ω-methoxy(polyethoxy)ethyl-N-(2,3-di(tetradecanoyloxy)propyl)carbamate or 2,3-di(tetradecanoyloxy)propyl-N-(ω-methoxy(polyethoxy)ethyl)carbamate, etc.

[0369] In some embodiments, the particles may comprise one or more PEG-conjugated lipids or polyethylene glycol-modified lipids, as described in WO 2017 / 075531 and WO 2018 / 081480, the entire contents of each of which are incorporated herein by reference for the purposes described herein.

[0370] Liposome complex particles

[0371] In some embodiments of this disclosure, the nucleic acids (such as RNA and / or DNA) described herein may be present in nucleic acid liposome complex particles (such as RNA and / or DNA liposome complex particles).

[0372] Liposome complexes (LPX) are electrostatic complexes typically formed by mixing pre-formed cationic liposomes with anionic nucleic acids such as RNA and / or DNA. Due to the transformation of the liposome structure into a dense nucleic acid-liposome complex (such as an RNA- and / or DNA-liposome complex), the resulting liposome complexes exhibit a unique internal molecular arrangement. These formulations are typically characterized by low nucleic acid encapsulation efficiency and incomplete nucleic acid embedding.

[0373] In some embodiments, nucleic acid liposome complex particles (such as RNA and / or DNA liposome complex particles) comprise cationic lipids and other lipids. In an exemplary embodiment, the cationic lipid is DOTMA, and the other lipids are DOPE.

[0374] In some embodiments, the molar ratio of at least one cationic lipid to at least one other lipid is about 10:0 to about 1:9, about 4:1 to about 1:2, or about 3:1 to about 1:1. In specific embodiments, the molar ratio may be about 3:1, about 2.75:1, about 2.5:1, about 2.25:1, about 2:1, about 1.75:1, about 1.5:1, about 1.25:1, or about 1:1. In an exemplary embodiment, the molar ratio of at least one cationic lipid to at least one other lipid is about 2:1.

[0375] In some embodiments, the average diameter of the nucleic acid liposome complex particles (such as RNA and / or DNA liposome complex particles) is about 200 nm to about 1000 nm, about 200 nm to about 800 nm, about 250 nm to about 700 nm, about 400 nm to about 600 nm, about 300 nm to about 500 nm, or about 350 nm to about 400 nm. In specific embodiments, the average diameter of the RNA liposome complex particles is about 200 nm, about 225 nm, about 250 nm, about 275 nm, about 300 nm, about 325 nm, about 350 nm, about 375 nm, about 400 nm, about 425 nm, about 450 nm, about 475 nm, about 500 nm, about 525 nm, about 550 nm, about 575 nm, about 600 nm, about 625 nm, about 650 nm, about 700 nm, about 725 nm, about 750 nm, about 775 nm, about 800 nm, about 825 nm, about 850 nm, about 875 nm, about 900 nm, about 925 nm, about 950 nm, about 975 nm, or about 1000 nm. In one embodiment, the average diameter of the nucleic acid liposome complex particles (such as RNA and / or DNA liposome complex particles) is about 250 nm to about 700 nm. In another embodiment, the nucleic acid liposome complex particles (such as RNA and / or DNA liposome complex particles) have an average diameter of about 300 nm to about 500 nm. In an exemplary embodiment, the nucleic acid liposome complex particles (such as RNA and / or DNA liposome complex particles) have an average diameter of about 400 nm.

[0376] The nucleic acid liposome complex particles (such as RNA and / or DNA liposome complex particles) described herein and compositions containing nucleic acid liposome complex particles (such as RNA and / or DNA liposome complex particles) can be used to deliver nucleic acids (such as RNA and / or DNA) to target tissues after parenteral administration, particularly after intravenous administration.

[0377] WO 2013 / 143683 describes RNA liposome complex particles targeting the spleen, which is incorporated herein by reference. It has been found that RNA liposome complex particles with a net negative charge can be used to preferentially target spleen tissue or spleen cells, such as antigen-presenting cells, particularly dendritic cells. Therefore, following administration of the RNA liposome complex particles, RNA accumulation and / or RNA expression occur in the spleen. Therefore, the nucleic acid (such as RNA and / or DNA) liposome complex particles of this disclosure can be used to express nucleic acids (such as RNA and / or DNA) in the spleen. In one embodiment, following administration of the nucleic acid (such as RNA and / or DNA) liposome complex particles, no or minimal accumulation and / or expression of nucleic acids (such as RNA) occurs in the lungs and / or liver. In one embodiment, following administration of the nucleic acid (such as RNA and / or DNA) liposome complex particles, accumulation and / or expression of nucleic acids (such as RNA) occur in antigen-presenting cells (such as specialized antigen-presenting cells in the spleen). Therefore, the nucleic acid (such as RNA and / or DNA) liposome complex particles of this disclosure can be used to express nucleic acids (such as RNA and / or DNA), for example, nucleic acids (such as RNA and / or DNA) encoding antigens or at least one epitope, in these antigen-presenting cells. In one embodiment, the antigen-presenting cells are dendritic cells and / or macrophages.

[0378] The charge of the nucleic acid (such as RNA and / or DNA) liposome complex particles disclosed herein is the sum of the charges present in at least one cationic lipid and the charges present in the nucleic acid (such as RNA). The charge ratio is the ratio of the positive charge present in at least one cationic lipid to the negative charge present in the nucleic acid (such as RNA). The charge ratio of the positive charge present in at least one cationic lipid to the negative charge present in the nucleic acid (such as RNA) is calculated by the following equation: Charge ratio = [(Cationic lipid concentration (mol))] [Total positive charge in cationic lipids] / [Concentration of nucleic acids (such as RNA) (mol)] (Total negative charge in nucleic acids (such as RNA)). The concentration of nucleic acids (such as RNA) and the amount of at least one cationic lipid can be determined by those skilled in the art using conventional methods.

[0379] In one embodiment, under physiological pH conditions, the charge ratio of positive to negative charges in the nucleic acid (such as RNA and / or DNA) liposome complex particles is about 1.6:2 to about 1:2, or about 1.6:2 to about 1.1:2. In a specific embodiment, under physiological pH conditions, the charge ratio of positive to negative charges in the nucleic acid (such as RNA and / or DNA) liposome complex particles is about 1.6:2.0, about 1.5:2.0, about 1.4:2.0, about 1.3:2.0, about 1.2:2.0, about 1.1:2.0, or about 1:2.0.

[0380] Lipid nanoparticles (LNP)

[0381] In some embodiments, the nucleic acids described herein (such as RNA and / or DNA) are present in the form of lipid nanoparticles (LNPs). LNPs may comprise any lipid capable of forming particles to which one or more nucleic acid molecules are attached, or encapsulated within the particles.

[0382] LNPs typically consist of four components: ionizable cationic lipids, neutral lipids (such as phospholipids), steroids (such as cholesterol), and polymer-conjugated lipids (such as PEG-lipids). LNPs can be prepared by mixing lipids dissolved in ethanol with nucleic acids in an aqueous buffer.

[0383] In some embodiments, in the nucleic acid (such as RNA and / or DNA) LNP described herein, the nucleic acid (such as RNA and / or DNA, particularly mRNA) is bound to an ionizable lipid occupying the central nucleus of the LNP. PEG lipids, together with phospholipids, form the surface of the LNP. In some embodiments, this surface comprises a bilayer structure. In some embodiments, charged and uncharged forms of cholesterol and ionizable lipids may be distributed throughout the LNP.

[0384] In some embodiments, the LNP comprises one or more cationic lipids, and one or more stable lipids. Stable lipids include neutral lipids and polyethylene glycol-modified lipids.

[0385] In some implementations, LNPs comprise: cationic lipids, neutral lipids, steroids, polymer-conjugated lipids; and nucleic acids (such as RNA and / or DNA) encapsulated within or associated with lipid nanoparticles.

[0386] In some implementations, LNP comprises 40 to 55 mol%, 40 to 50 mol%, 41 to 50 mol%, 42 to 50 mol%, 43 to 50 mol%, 44 to 50 mol%, 45 to 50 mol%, 46 to 50 mol%, or 46 to 49 mol%.

[0387] In some implementations, the concentration of neutral lipids ranges from 5 to 15 mol%, 7 to 13 mol%, or 9 to 11 mol%.

[0388] In some implementations, the concentration of steroids ranges from 30 to 50 mol%, 35 to 45 mol%, or 38 to 43 mol%.

[0389] In some embodiments, the LNP comprises 1 to 10 mol%, 1 to 5 mol%, or 1 to 2.5 mol% of a polymer-conjugated lipid.

[0390] In some embodiments, the LNP comprises: 45 to 50 mol% cationic lipids; 5 to 15 mol% neutral lipids; 35 to 45 mol% steroids; 1 to 5 mol% polymer-conjugated lipids; and nucleic acids (such as RNA and / or DNA) encapsulated within or associated with lipid nanoparticles.

[0391] In some embodiments, the molar percentage is determined based on the total number of moles of lipids present in the lipid nanoparticles. In some embodiments, the molar percentage is determined based on the total number of moles of cationic lipids, neutral lipids, steroids, and polymer-conjugated lipids present in the lipid nanoparticles.

[0392] In some embodiments, the neutral lipid is selected from the group consisting of DSPC, DPPC, DMPC, DOPC, POPC, DOPE, DOPG, DPPG, POPE, DPPE, DMPE, DSPE, and SM. In some embodiments, the neutral lipid is DSPC.

[0393] In some implementations, the steroid is cholesterol.

[0394] In some embodiments, the polymer-conjugated lipid is a polyethylene glycol-modified lipid. In some embodiments, the polyethylene glycol-modified lipid has the following structure:

[0395]

[0396] Or its pharmaceutically acceptable salts, tautomers or stereoisomers, wherein:

[0397] R 12 and R 13 Each is independently a straight-chain or branched saturated or unsaturated alkyl chain containing 10 to 30 carbon atoms, wherein the alkyl chain is optionally interrupted by one or more ester bonds; the average value of w ranges from 30 to 60. In some embodiments, R 12 and R 13Each is independently a straight-chain saturated alkyl chain containing 12 to 16 carbon atoms. In some embodiments, the average value of w ranges from 40 to 55. In some embodiments, the average value of w is about 45. In some embodiments, R 12 and R 13 Each is an independent straight-chain saturated alkyl chain containing about 14 carbon atoms, and the average value of w is about 45.

[0398] In some embodiments, the PEGylated lipid is or contains 2-[(PEG)-2000]-N,N-bistetradecylacetamide.

[0399] In some embodiments, the cationic lipid component of LNP has the structure of formula (III):

[0400]

[0401] Or a pharmaceutically acceptable salt, tautomer, prodrug, or stereoisomer thereof, wherein:

[0402] L 1 or L 2 One of them is –O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O)-. x -, -SS-, -C(=O)S-, SC(=O)-, -NR a C(=O)-、-C(=O)NR a -、NR a C(=O)NR a -、-OC(=O)NR a -or-NR a C(=O)O-, and L 1 or L 2 The other two are –O(C=O)-, -(C=O)O-, -C(=O)-, -O-, and -S(O). x -, -SS-, -C(=O)S-, SC(=O)-, -NR a C(=O)-、-C(=O)NR a -、NR a C(=O)NR a -、-OC(=O)NR a -or-NR a C(=O)O- or direct bond;

[0403] G 1 and G 2 Each is an unsubstituted C1-C 12 Alkylene or C1-C 12 alkenyl;

[0404] G 3 It is C1 -C 24 Alkylene, C1-C 24 alkenyl, C3-C8 cycloalkylene, C3-C8 cycloalkenyl;

[0405] R a Is it H or C1-C? 12 alkyl;

[0406] R 1 and R 2 Each is independently C6-C 24 Alkyl or C6-C 24 alkenyl;

[0407] R 3 Is it H or OR? 5 CN, -C(=O)OR 4 -OC(=O)R 4 Or –NR 5 C(=O)R 4 ;

[0408] R 4 It is C1-C 12 alkyl;

[0409] R 5 It is an H or C1-C6 alkyl group;

[0410] x is 0, 1, or 2.

[0411] In some of the above embodiments of formula (III), the lipid has one of the following structures (IIIA) or (IIIB):

[0412]

[0413] in:

[0414] A is a 3- to 8-membered cycloalkyl or cycloalkylene ring;

[0415] R 6 Each time it appears, it is independently H, OH, or Cl-C. 24 alkyl;

[0416] n is an integer from 1 to 15.

[0417] In some of the above embodiments of formula (III), the lipid has structure (IIIA), and in other embodiments, the lipid has structure (IIIB).

[0418] In other embodiments of formula (III), the lipid has one of the following structures (IIIC) or (IIID):

[0419]

[0420] Where y and z are each an independent integer from 1 to 12.

[0421] In any of the above embodiments of formula (III), L 1 or L 2 One of them is -O (C=O). For example, in some implementations, L 1 and L 2 Each of them is -O (C=O)-. In some different implementations of any of the above general formulas, L 1 and L 2 Each is independently -(C=O)O- or -O(C=O)-. For example, in some implementations, L 1 and L 2 Each of them is -(C=O)O-.

[0422] In some different embodiments of formula (III), the lipid has one of the following structures (IIIE) or (IIIF):

[0423]

[0424] In some of the above embodiments of formula (III), the lipid has one of the following structures: (IIIG), (IIIH), (IIII), or (IIIJ):

[0425]

[0426] In some of the above embodiments of equation (III), n is an integer from 2 to 12, such as 2 to 8 or 2 to 4. For example, in some embodiments, n is 3, 4, 5, or 6. In some embodiments, n is 3. In some embodiments, n is 4. In some embodiments, n is 5. In some embodiments, n is 6.

[0427] In some other embodiments of equation (III) described above, y and z are each independently integers from 2 to 10. For example, in some embodiments, y and z are each independently integers from 4 to 9 or from 4 to 6.

[0428] In some of the above-described embodiments of formula (III), R 6 For H. In other of the above embodiments, R 6 For C1 - C 24 Alkyl group. In other embodiments, R 6 It is OH.

[0429] In some implementations of formula (III), G 3It has not been replaced. In other implementations, G 3 Replaced. In various different implementation schemes, G 3 It is linear C1 - C 24 Alkylene or linear C1-C 24 Alkenyl group.

[0430] In some other embodiments of formula (III) described above, R 1 or R 2 Or both are C6-C 24 Alkenyl. For example, in some embodiments, R 1 and R 2 Each of them independently has the following structure:

[0431]

[0432] in:

[0433] R 7a and R 7b Each occurrence is independently H or Cl-C 12 Alkyl groups; and

[0434] a is an integer from 2 to 12.

[0435] Among them, R 7a R 7b a and a are each chosen such that R 1 and R 2 Each contains 6 to 20 carbon atoms independently. For example, in some embodiments, a is an integer from 5 to 9 or from 8 to 12.

[0436] In some of the above-described embodiments of formula (III), R 7a H appears at least once. For example, in some implementations, R 7a Each occurrence is H. In other different embodiments of the above general formula, R... 7b The presence of at least one of the compounds is a C1-C8 alkyl group. For example, in some embodiments, the C1-C8 alkyl group is methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-hexyl, or n-octyl.

[0437] In different implementations of formula (III), R 1 or R 2 Or both have one of the following structures:

[0438]

[0439] In some of the above-described embodiments of formula (III), R 3 For OH, CN, -C(=O)OR4 -、-OC(=O)R 4 Or –NHC(=O)R 4 In some implementations, R 4 It can be methyl or ethyl.

[0440] In various implementations, the cationic lipid of formula (III) has one of the structures listed in the table below.

[0441] Representative compounds of formula (III).

[0442]

[0443]

[0444]

[0445]

[0446]

[0447]

[0448]

[0449] Various lipids (including, for example, cationic lipids, neutral lipids, and polymer-conjugated lipids) are known in the art and can be used herein to form lipid nanoparticles, such as lipid nanoparticles targeting specific cell types (e.g., hepatocytes). In some embodiments, neutral lipids may be or comprise phospholipids or derivatives thereof (e.g., 1,2-distearate-sn-glycerol-3-phosphocholine (DPSC)) and / or cholesterol. In some embodiments, polymer-conjugated lipids may be PEG-conjugated lipids (e.g., 2-[(polyethylene glycol)-2000]-N,N-bistetradecylacetamide or derivatives thereof).

[0450] In one embodiment, LNP comprises lipids of formula (III), nucleic acids (such as RNA and / or DNA), neutral lipids, steroids, and PEGylated lipids. In some embodiments, the neutral lipid is DSPC. In some embodiments, the steroid is cholesterol. In some embodiments, the PEGylated lipid is ALC-0159, with the structure shown below.

[0451]

[0452] In one embodiment, the cationic lipid content in the LNP is about 45 to about 50 mol%. In some embodiments, the neutral lipid content in the LNP is about 5 to about 15 mol%. In some embodiments, the steroid content in the LNP is about 35 to about 45 mol%. In some embodiments, the PEGylated lipid content in the LNP is about 1 to about 5 mol%.

[0453] In some embodiments, the LNP comprises: cationic lipids in a concentration of about 45 to about 50 mol%, DSPC in a concentration of about 5 to about 15 mol%, cholesterol in a concentration of about 35 to about 45 mol%, and ALC-0159 in a concentration of about 1 to about 5 mol%.

[0454] The N / P value is preferably at least about 4. In some embodiments, the N / P value ranges from 4 to 20, 4 to 12, 4 to 10, 4 to 8, or 5 to 7. In some embodiments, the N / P value is about 6.

[0455] Pharmaceutical compositions containing nucleic acid particles

[0456] In some embodiments, the composition comprising the nucleic acid molecules of the present invention (such as nucleic acid molecules prepared by the method of the present invention) is a pharmaceutical composition. In one embodiment, the composition may comprise the salt, buffer, or other components further described below. In the most preferred embodiment, the composition comprises two or more different RNA molecules.

[0457] 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).

[0458] Typically, compositions for storing nucleic acid (such as RNA and / or DNA) particles (such as those for freezing nucleic acid (such as RNA and / or DNA) 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.

[0459] According to this disclosure, the nucleic acid (such as RNA and / or DNA) particle compositions described herein have a pH value suitable for the stability of nucleic acid (such as RNA and / or DNA) particles, and particularly suitable for the stability of nucleic acids (such as RNA and / or DNA). 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.

[0460] 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 (such as RNA and / or DNA, especially mRNA) activity during storage, freezing and / or lyophilization, such as reducing or preventing aggregation, particle disintegration, nucleic acid (such as RNA and / or DNA, especially mRNA) degradation and / or other types of damage.

[0461] In one implementation, the cryoprotectant is a carbohydrate. As used herein, the term "carbohydrate" refers to and encompasses monosaccharides, disaccharides, trisaccharides, oligosaccharides, and polysaccharides.

[0462] 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.

[0463] 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 together 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.

[0464] The term "trisaccharide" refers to three sugar molecules linked together to form a single molecule. Examples of trisaccharides include raffinose and pinotriose.

[0465] 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 together 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.

[0466] 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 monosaccharide units (such as 6, 7, 8, 9, or 10) linked together 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.

[0467] 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.

[0468] 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).

[0469] 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 together 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 backbone structure. In the backbone, the polysaccharide can be linear or cyclic. Exemplary polysaccharide cryoprotectants include glycogen, amylase, cellulose, dextran, maltodextrin, etc.

[0470] In some embodiments, the nucleic acid (such as RNA and / or DNA) 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 (such as RNA and / or DNA, particularly mRNA) particles and maintaining the chemical and physical stability of the composition. In some embodiments, the nucleic acid (such as RNA and / or DNA) particle composition may contain alternative cryoprotectants to sucrose. Alternative stabilizers include, but are not limited to, trehalose and glucose. In one specific embodiment, the alternative stabilizer to sucrose is trehalose or a mixture of sucrose and trehalose.

[0471] 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.

[0472] Some embodiments of this disclosure contemplate the use of chelating agents in the nucleic acid (such as RNA and / or DNA) compositions described herein. A chelating agent is a compound capable of forming at least two 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 nucleic acids (such as RNA and / or DNA) in this disclosure. 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.

[0473] In alternative embodiments, the nucleic acid (such as RNA and / or DNA) particle compositions described herein do not contain chelating agents.

[0474] 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.

[0475] 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.

[0476] 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, INFa, INF-γ, GM-CSF, and LT-a. 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).

[0477] 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, frozen pharmaceutical compositions must be thawed, or lyophilized pharmaceutical compositions must be reconstituted, e.g., using a suitable solvent (e.g., deionized water, such as water for injection) or a liquid (e.g., an aqueous solution).

[0478] The pharmaceutical compositions according to this disclosure are generally administered in a “pharmaceuticalally effective amount” and a “pharmaceuticalally acceptable formulation”.

[0479] The term "pharmaceutical acceptable" means that a substance is non-toxic and does not interact with the active ingredient in a pharmaceutical composition.

[0480] The term "pharmaceutical effective dose" refers to the amount that, alone or in combination with subsequent doses, 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 dose 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).

[0481] 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.

[0482] Preservatives suitable for use in the pharmaceutical compositions disclosed herein include, but are not limited to, benzalkonium chloride, chlorobutanol, p-hydroxybenzoate and thimerosal.

[0483] 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.

[0484] The term "diluent" refers to a reagent used for dilution and / or thinning. Furthermore, the term "diluent" also includes any one or more fluids, liquids, or solid suspensions and / or mixing media. Suitable examples of diluents include ethanol, glycerol, and water.

[0485] 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.

[0486] Pharmaceutically acceptable carriers, excipients, or diluents for therapeutic use are well known in the pharmaceutical industry, and are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (edited by A. RGennaro, 1985).

[0487] The drug carrier, excipient, or diluent can be selected based on the intended route of administration and standard pharmaceutical practice.

[0488] Route of administration of the pharmaceutical composition

[0489] 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.

[0490] Use of pharmaceutical compositions

[0491] Compositions containing the nucleic acids described herein (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 (where 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.

[0492] 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 use of "disease" is broader, 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.

[0493] 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.

[0494] 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.

[0495] 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.

[0496] 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, older adults, children, and newborns. In some embodiments of this disclosure, “individual” or “subject” is a “patient.”

[0497] The term "patient" refers to an individual or subject receiving treatment, especially an individual or subject who is ill.

[0498] According to the assay described herein, effective nucleic acids, particularly RNA, can be administered to a subject to deliver nucleic acids into the subject's cells.

[0499] According to the assay described herein, a potent nucleic acid, particularly RNA, can be administered to a subject to deliver a therapeutic or preventative peptide or polypeptide (e.g., a pharmacologically active peptide or polypeptide), wherein the nucleic acid encodes a therapeutic or preventative peptide or polypeptide.

[0500] According to the assay described herein, effective nucleic acids, particularly RNA, can be administered to subjects for the treatment or prevention of diseases, wherein delivery of nucleic acids to the subject's cells is beneficial for the treatment or prevention of the disease.

[0501] According to the assay described herein, a potent nucleic acid, particularly RNA, can be administered to a subject to treat or prevent a disease in the subject, wherein the nucleic acid encodes a therapeutic or preventive peptide or polypeptide, and wherein delivery of the therapeutic or preventive peptide or polypeptide to the subject is beneficial for treating or preventing the disease.

[0502] In some implementations, nucleic acids are present in the compositions described herein.

[0503] In some implementation schemes, nucleic acids are administered in pharmaceutically effective amounts.

[0504] In some implementations, the subjects are mammals. In some implementations, the mammals are humans.

[0505] In some embodiments of this disclosure, the aim is to induce an immune response by providing a vaccine.

[0506] 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 nucleic acids described herein can be used to induce or enhance an immune response. The nucleic acids described herein can therefore be used for the preventive and / or therapeutic treatment of diseases involving antigens or epitopes.

[0507] In some embodiments of this disclosure, the aim is to treat cancer through vaccination.

[0508] In some embodiments of this disclosure, the aim is to provide protection against infectious diseases through vaccination.

[0509] 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.

[0510] 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.

[0511] In some embodiments of this disclosure, the aim is to modulate / reprogram immune cells in the blood.

[0512] 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 regulate the tumor microenvironment.

[0513] 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.

[0514] 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.

[0515] 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 a scope consistent with the claims.

[0516] Example

[0517] Example 1: RNA Identity

[0518] Materials and Methods:

[0519] cDNA synthesis

[0520] 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 primer, and 1 µL of 10 mM dNTP were mixed, and the volume was adjusted to 13.5 µL with water (H2O). The cDNA primer was 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. Three replicate measurements were performed on each sample, and a negative control was included. The RNA was denatured at 80°C for 5 minutes, rapidly cooled on ice for at least 1 minute, 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.

[0521] Droplet digital PCR

[0522] 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 (Eurofins Genomics), 0.9 µM of RNA-specific forward primer (Eurofins Genomics), and 0.9 µM of universal reverse primer were mixed to a final volume of 22 µL. Each sample was measured 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:

[0523] Activation step 1): 95℃ for 600 seconds.

[0524] Denaturation step 2): 94℃ for 30 seconds.

[0525] Annealing and extension step 3): 63℃ for 60 seconds.

[0526] Enzyme inactivation step 4): 98℃ for 600 seconds.

[0527] 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).

[0528] Data Analysis

[0529] Data were analyzed using QuantaSoft version 1.7.4.0917 (Bio-Rad). QuantaSoft can usually automatically distinguish and define positive and negative populations. In rare cases where automatic distinction is not possible, thresholds are set manually. The software automatically calculates CN / µL. For RNA identification, samples must show a minimum of 300 CN / µL, and negative controls must show a maximum of 10 CN / µL.

[0530] result:

[0531] To verify that this method could correctly identify RNA, each oligonucleotide group was tested for each individual RNA that made up the sample RNA mixture. When the corresponding oligonucleotide group was used to analyze the 3' end region, ddPCR only produced positive droplets when the RNA-specific forward primer was combined with the target RNA (e.g., when RNA A was amplified using the RNA A forward primer). In all other forward primer / RNA combinations, only negative droplets were observed.

[0532] Table 1

[0533]

[0534] The data in Table 1 report the CN / µL values ​​(CN = copy number) of each RNA in four RNA mixtures tested using forward primers specific to each RNA (e.g., primer pair A is specific to RNA A). Each sample was measured in triplicate. Bold numbers in the table indicate high CN / µL values, and normal font numbers indicate low CN / µL values.

[0535] Example 2: RNA ratio

[0536] Materials and Methods:

[0537] cDNA synthesis

[0538] 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 primer, and 1 µL of 10 mM dNTP were mixed, and the volume was adjusted to 13.5 µL with water (H2O). The cDNA primer was 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. Three replicate measurements were performed on each sample, and a negative control was included. The RNA was denatured at 80°C for 5 minutes, rapidly cooled on ice for at least 1 minute, 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.

[0539] Droplet digital PCR

[0540] 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 measured 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:

[0541] Activation step 1): 95℃ for 600 seconds.

[0542] Denaturation step 2): 94℃ for 30 seconds.

[0543] Annealing and extension step 3): 63℃ for 60 seconds.

[0544] Enzyme inactivation step 4): 98℃ for 600 seconds.

[0545] 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).

[0546] Data Analysis

[0547] Data were analyzed using QuantaSoft version 1.7.4.0917 (Bio-Rad). QuantaSoft can usually automatically distinguish and define positive and negative populations. In rare cases where automatic distinction is not possible, thresholds are set manually. The software automatically calculates CN / µL. To calculate the RNA ratio, CN / µL is converted to mass / volume concentration (g / µL) using the following formula:

[0548]

[0549] Where MW is the molecular weight of RNA. The ratio (expressed as a percentage (%)) is then calculated using the following formula:

[0550]

[0551] To calculate the ratio of RNA B, C, or D, the concentration of the corresponding RNA must be given in the molecule of the formula.

[0552] result:

[0553] To verify the accuracy of the RNA ratio measurement method, several solutions containing RNA mixed in different ratios 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:

[0554]

[0555] 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.

[0556] Table 2

[0557]

[0558] The data in Table 2 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). The "Theoretical RNA Ratio" column is compared with the "Mean" column to verify that the measured ratio reflects the theoretical value with minimal bias.

[0559] Example 3: RNA Integrity

[0560] Materials and Methods:

[0561] cDNA synthesis

[0562] 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 primer, and 1 µL of 10 mM dNTP were mixed, and the volume was adjusted to 13.5 µL with water (H2O). The cDNA primer was 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. Three replicate measurements were performed on each sample, and a negative control was included. The RNA was denatured at 80°C for 5 minutes, rapidly cooled on ice for at least 1 minute, 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.

[0563] Droplet digital PCR

[0564] Droplet digital PCR was performed on a 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 primer, and 0.9 µM of universal reverse primer were mixed to a final volume of 22 µL. The reaction mixture contained two oligonucleotide sets, one targeting the 5' end of the RNA and the other targeting the 3' end. The 5' end oligonucleotide set consisted of one universal forward primer, one RNA-specific reverse primer, and one universal FAM-BHQ1 double-labeled probe. The 3' end oligonucleotide set consisted of one RNA-specific forward primer, one universal reverse primer, and one universal HEX-BHQ1 double-labeled probe. Each sample was measured in triplicate, and a negative control was included for each sample. After oil droplets are generated, the sample is incubated in a C1000 thermal cycler (Bio-Rad) and the following thermal cycling procedure is performed:

[0565] Activation step 1): 95℃ for 600 seconds.

[0566] Denaturation step 2): 94℃ for 30 seconds.

[0567] Annealing and extension step 3): 63℃ for 60 seconds.

[0568] Enzyme inactivation step 4): 98℃ for 600 seconds.

[0569] 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).

[0570] Data Analysis

[0571] Data were analyzed using QuantaSoft version 1.7.4.0917 (Bio-Rad). QuantaSoft can usually automatically distinguish and define positive and negative populations. However, due to the complex mixture of RNA templates and primers / probes, negative, single-positive, and double-positive populations are not always clearly distinguishable, and the software fails to automatically identify them. Therefore, populations must be manually defined by visualizing the data on a two-dimensional (2D) amplitude plot. Once these four populations were identified, the software automatically calculated the CN / µL values ​​for HEX-positive and FAM-positive samples for each sample. In addition, linkage was calculated. Linkage was originally defined to analyze the proximity of two sequences that are physically linked on the same DNA segment (JF Regan et al., PLOS ONE, 2015, 10(3), e0118270). Since the 5' and 3' ends of intact RNA are physically linked, this value can also be used to study RNA integrity. Linkage describes the copy number (CN) / droplet concentration of 5' and 3' end double-positive droplets after normalizing the probability of random co-localization of single-positive and double-positive templates. To determine the proportion of intact RNA, linkage was automatically calculated and converted to CN / µL by ddPCR software, divided by the total number of 3' end (HEX) CN / µL, yielding the linkage percentage. This value is correlated with RNA integrity.

[0572]

[0573] result:

[0574] To verify the accuracy of this RNA integrity detection method, each RNA group was individually incubated at 90°C for different times to degrade it to different integrity levels. RNAs at the same degradation time points were then mixed and measured using ddPCR. This method successfully detected the decrease in RNA integrity over time for all RNA groups.

[0575] Figure 1 The results show that the integrity of individual RNAs constituting RNA mixtures with different degrees of degradation was measured using ddPCR. The longer the RNA was degraded at 90°C, the lower the integrity measured by ddPCR.

[0576] To calculate RNA integrity, the 3' end CN / µL information is quantified. This is the same information required to assess RNA ratio and RNA identity. Therefore, the method proposed in this paper can measure RNA identity, RNA ratio, and RNA integrity in parallel.

[0577] Furthermore, the experimental system is simplified because the two components in each oligonucleotide set (i.e., a universal primer and a universal double-labeled probe) are identical for all RNAs, and only one primer in each set is RNA-specific. This reduces the required reagent volume, system complexity, and pipetting time compared to assembling two sets of RNA-specific oligonucleotides.

[0578] Example 4: RNA efficacy

[0579] Method description:

[0580] Total ribonucleic acid (RNA) was isolated from Chinese hamster ovary (CHO) cells previously transfected with varying amounts of formulated RNA of interest. Total RNA was isolated using a commercially available kit (RNeasy Micro kit, Qiagen) and reverse transcribed into complementary deoxyribonucleic acid (cDNA) using oligo(dT) primers (Oligo(dT)20, part of the Invitrogen SuperScript IV kit).

[0581] The cDNA was diluted and analyzed by digital droplet polymerase chain reaction (ddPCR) using two sets of specific primers and fluorescently labeled probes targeting the RNA of interest and housekeeping RNA. The probes were labeled with hexachlorofluorocarbon (HEX) and 6-carboxyfluorocarbon (FAM) and carried Black Hole quencher 1 (BHQ1). The PCR cycling program consisted of initial activation at 95°C for 600 seconds, followed by 40 cycles, each consisting of denaturation at 94°C for 30 seconds and annealing / extension at 61°C for 60 seconds. A final enzyme inactivation treatment was then performed at 98°C for 60 seconds.

[0582] Figure 2The copy number (CN) of RNA of interest and housekeeping gene in total RNA isolated from Chinese hamster ovary (CHO) cells, measured by digital droplet polymerase chain reaction (ddPCR), is shown. These cells had previously been transfected with four different formulations of RNA of interest.

[0583] All publications mentioned in the foregoing specification are incorporated herein by reference. Various modifications and variations to the methods and systems described herein will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in conjunction with specific preferred embodiments, it should be understood that the claimed invention should not be unduly limited to these specific embodiments. Indeed, it will be apparent to those skilled in the fields of chemistry, biochemistry, molecular biology, biotechnology, or related fields that various modifications to the embodiments of the invention are intended to be included within the scope of the following claims.

Claims

1. A method for determining quality parameters of an RNA sample containing n types of RNA molecules, wherein n is an integer of at least 2, and the quality parameters are selected from the group consisting of: i) The quantitative ratio of two or more RNA molecule types among the n RNA molecule types; and ii) The identities of the n RNA molecule species in the RNA sample; The method includes the following steps: a) Reverse transcribe the n types of RNA molecules in the RNA sample into cDNA molecules of n types of DNA molecules; as well as b) The obtained cDNA molecules were subjected to a polymerase chain reaction (PCR)-based assay using a first primer set and a single second primer, wherein... The first primer set contains n primer types, each of which can anneal to a first target region consisting of only one of the n DNA molecule types in the sample. The single second primer can anneal to the second target region of all n types of DNA molecules in the sample.

2. The method according to claim 1, wherein the quality parameter is the quantitative ratio of two or more RNA molecule types among the n RNA molecule types.

3. The method according to claim 1, wherein the quality parameter is the identity of the n RNA molecule species in the RNA sample.

4. A method for determining the integrity of an RNA sample containing n different types of RNA molecules, where n is an integer at least 1. The method includes the following steps: a) Reverse transcribe the n RNA molecules in the RNA sample into cDNA molecules of n DNA molecule types; as well as b) The obtained cDNA molecules were subjected to a polymerase chain reaction (PCR) assay using a first primer set, a single second primer, a third primer set, and a single fourth primer. The first primer set contains n primer types, each of which can anneal to the first target region at the 3' end region of the DNA molecule in the sample. The single second primer can anneal to the second target region at the 3' end region of all n types of DNA molecules in the sample; The third primer set contains n primer types, each of which can anneal to the third target region at the 5' end of a DNA molecule in the sample; and The single fourth primer can anneal to the fourth target region at the 5' end region of all n types of DNA molecules in the sample.

5. A method for determining the potency of a prepared RNA sample containing an RNA molecule of interest, the method comprising the following steps: a) Provide RNA samples isolated from cells transfected with prepared RNA samples; b) Reverse transcribe the RNA molecules in the RNA sample into cDNA molecules; c) The obtained cDNA molecules are subjected to a polymerase chain reaction (PCR)-based assay using primers 1, 2, 3, and 4, wherein... The first primer and the second primer are capable of annealing to the first and second target regions of the cDNA molecule generated from the RNA of interest in the sample, and The third and fourth primers are capable of annealing to the first and second target regions of cDNA molecules derived from endogenous RNA in the sample. as well as d) Compare the measured amount of cDNA produced by the RNA molecule of interest with the measured amount of cDNA produced by endogenous RNA.

6. The method according to claim 5, wherein the endogenous RNA is RNA expressed by a housekeeping gene in the sample, preferably, wherein the endogenous RNA is RNA expressed by a GAPDH gene in the sample.

7. The method according to claim 5 or 6, wherein the RNA composition is prepared comprising two or more different RNA molecules of interest, and the PCR-based assay uses two or more different primer pairs, each primer pair being specific to one of the said RNA molecules of interest.

8. The method according to any one of claims 5 to 7, wherein steps a) to d) are repeated for other RNA samples isolated from cells transfected with a formulated RNA composition containing the RNA molecule of interest to determine the potency level of each formulated RNA composition.

9. The method according to any one of claims 5 to 8, wherein the method is carried out to determine the expected potency level of a specific formulated RNA composition containing the RNA of interest, the expected potency level being defined as a reference potency level.

10. The method of claim 9, wherein the method is performed on other formulated RNA compositions containing the RNA of interest and compared with a reference potency level of the particular formulated RNA composition.

11. The method according to any one of claims 6 to 10, further comprising step z) prior to step a): z) Isolate / purify RNA from cells transfected with the RNA sample prepared as described, and Optionally, it also includes the following step y) prior to step z): y) Transfect the cells with the prepared RNA composition.

12. The method according to any of the preceding claims, wherein the PCR-based assay in step b) is digital PCR (dPCR), preferably wherein the PCR-based assay in step b) is droplet digital PCR (ddPCR).

13. The method according to any of the preceding claims, wherein the PCR-based assay uses a detectable label, preferably wherein the detectable label is a fluorescent probe.

14. The method according to any one of the preceding claims, wherein at least one RNA in the RNA molecules present in the sample is complexed with at least one carrier compound to form at least one RNA-carrier complex.

15. The method of claim 14, wherein cells are transfected with RNA in combination with at least one carrier compound to form at least one RNA-carrier complex.

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