Linker sequence potency assay for multiple encoding nucleic acids

Abstract

The present invention provides potency assays for the simultaneous analysis of at least two different nucleic acid sequences, such as RNA and / or DNA sequences, encoding functional sequences, such as antigens or epitopes. The potency determination of the invention may be performed with nucleic acid sequences encoding at least two different functional sequences, including at least two different antigens or epitopes.

Description

[0001] Invention Technology

[0002] This invention provides a potency assay for simultaneously analyzing at least two different nucleic acid sequences (such as RNA and / or DNA sequences) encoding functional sequences (such as antigens or epitopes). The potency assay of this invention can be performed using nucleic acid sequences encoding at least two different functional sequences (including at least two different antigens or epitopes). The nucleic acid sequences analyzed by the method of this invention can be used for downstream clinical applications, such as for inducing an immune response in a subject against two or more antigens or epitopes encoded by the nucleic acid sequence, wherein the immune response can be therapeutic or partially or completely protective. Therefore, the nucleic acid sequences can be used for vaccination. More specifically, the nucleic acid sequences can be used as multivalent T-cell targeted vaccines. Background of the Invention

[0004] In addition to their well-known ability to encode biologically active proteins, nucleic acids such as DNA and RNA possess other remarkable properties that make them attractive therapeutic agents. Nucleic acid-based therapeutic agents are easy to prepare and relatively inexpensive.

[0005] DNA is generally more stable than RNA, but it has some potential safety drawbacks, such as the induction of anti-DNA antibodies and the integration of transgenes into the host genome.

[0006] Using RNA to deliver foreign genetic information into target cells offers an attractive alternative to DNA. Advantages of RNA include transient expression and non-transformation characteristics. RNA does not need to enter the cell nucleus for expression and cannot integrate into the host genome, thus eliminating the risk of tumorigenesis.

[0007] T-cell vaccines are nucleic acid constructs designed to encode highly immunogenic regions or epitopes of a target antigen linked together in a single polypeptide. Multivalent T-cell vaccines can contain multiple nucleic acid constructs. These T-cell vaccines are designed to elicit an immunogenic response through the recognition of epitopes presented by the major histocompatibility complex (MHC) by T cells. In other words, the functional epitope sequence encoded by the T-cell vaccine is expressed and presented by the MHC, thereby eliciting an immunogenic response against the presented (multiple) epitopes. Therefore, the epitopes encoded by T-cell vaccines function in a completely different way than in the more traditional way of evoking an immune response (i.e., by expressing stable folded antigens for antibody recognition).

[0008] Potency testing is used to measure product attributes related to product quality and preparation controls, and is conducted to ensure the characteristics, purity, strength (potency), and stability of the product used throughout all phases of clinical studies. Similarly, potency measurements are used to demonstrate that only batches of product conforming to defined specifications or acceptance criteria are administered throughout all phases of clinical studies and after market approval. Therefore, defining the potency of a biopharmaceutical is a core element during and after product development.

[0009] Potency assays involve the quantitative measurement of certain standards that describe a product’s ability to achieve a defined biological effect. The standards used for measurement should be closely related to the product’s intended biological effect and, ideally, to its clinical purpose. Measuring product potency differs from measuring clinical efficacy. Rather, it is a way to control product quality and provide appropriate release criteria, particularly under GMP. Typically, a separate potency assay must be developed for each product and for each individual product to be administered to a subject. In the rapidly evolving world of nucleic acids, with hundreds of potentially different constructs, and in most cases, no antibodies are available for detection, making it highly beneficial to readily adapt potency assays for new products.

[0010] As part of the drug development process for T-cell vaccines, potency assays must be developed to measure the mechanism of action of T-cell vaccines. However, as mentioned above, T-cell vaccines are not necessarily designed to elicit an immunogenic response by expressing stable folded antigens to be recognized by antibodies. Therefore, the development of potency assays for such vaccines cannot rely on conventional antibody-based methods such as flow cytometry, Western blotting, or ELISA.

[0011] Another consideration in developing such a potency assay is that multivalent T-cell vaccines can encode multiple different epitopes. Therefore, a potency assay for multivalent T-cell vaccines needs to be able to simultaneously determine the expression of several different epitopes. Furthermore, T-cell vaccines can encode de novo epitope sequences. This is because, in some cases, T-cell vaccines are personalized vaccines, with sequences varying from patient to patient. Therefore, it is essential to develop a T-cell vaccine potency assay that can account for the need to assess the potency of de novo or undetermined epitope sequences.

[0012] In short, there is a problem with providing an assay that can be used to determine the potency of an antigen / epitope presented via MHC to elicit an immunogenic response, for which conventional antibody-based methods are useless. There is also a problem with providing an assay that can simultaneously determine the potency of multiple different expressed functional sequences (e.g., epitopes), which may include novel expressed functional sequences. While such an assay can be used to analyze the potency of T-cell vaccines, it can also be used in any situation where any of these limitations apply, such as in any situation where the expression of two or more different analyte sequences is analyzed simultaneously. Invention Overview

[0014] This invention provides a method for the simultaneous analysis of at least two different nucleic acid sequences, the method using adapter sequences that can be proteatically excised from their amino acid sequences. Specifically, when the at least two nucleic acid sequences each encode an amino acid sequence comprising a different expressed functional sequence and a different adapter sequence, each expressed adapter sequence can be excised from its amino acid sequence, and the amount of each adapter sequence can be used as an indicator of the efficacy of its nucleic acid sequence in expressing the functional sequence. Furthermore, since the adapter sequence can be measured and used as an indicator of nucleic acid efficacy, there is no need to directly measure the functional sequence. In other words, each adapter sequence acts as a molecular "barcode" to uniquely identify the expression of its associated functional sequence. For this reason, the method of this invention is also applicable to the analysis of the expression of undetermined, de novo functional sequences (such as variable epitopes). Moreover, it has been observed that adapter sequence expression can be quantified by mass spectrometry, eliminating the need for quantification using antibody-based techniques. Based on these observations, a rapid, cost-effective, reliable, easy-to-use, and interpretable power assay is provided to measure, determine, identify, quantify, confirm, and / or verify the therapeutic potential of at least two different nucleic acids (such as RNA and / or DNA), each encoding at least two different functional sequences.

[0015] This invention encompasses multiple aspects. It should be understood that the various embodiments described herein that are applicable to any one aspect of the invention will generally also apply to all other aspects of the invention.

[0016] In a first aspect, the present invention relates to a method for simultaneously analyzing at least two different nucleic acid sequences, each nucleic acid sequence encoding a different amino acid sequence, wherein each of the at least two different amino acid sequences comprises a different functional sequence and a different adapter sequence, wherein each adapter sequence is proteolytically cleavable from its amino acid sequence, wherein the method comprises the following steps:

[0017] (i) Provide the at least two different nucleic acid sequences;

[0018] (ii) Introducing the at least two different nucleic acid sequences into cells;

[0019] (iii) Expressing at least two different amino acid sequences;

[0020] (iv) Proteolytically cleaved at least two different adapter sequences;

[0021] (v) Determine the amount of each of the excised connector sequences;

[0022] (vi) The amount of each of the excised adapter sequences is used as an indicator of the potency of each of the nucleic acid sequences in expressing each of the functional sequences in a biological system.

[0023] In a second aspect, the present invention relates to a method for analyzing the potency of nucleic acid sequences in expressing functional sequences in a biological system, wherein the method comprises simultaneously analyzing at least two different nucleic acid sequences, each nucleic acid sequence encoding a different amino acid sequence, wherein each of the at least two different amino acid sequences contains a different functional sequence and a different adapter sequence, wherein each adapter sequence is proteolytically cleavable from its amino acid sequence, wherein the method comprises the following steps:

[0024] (i) Provide the at least two different nucleic acid sequences;

[0025] (ii) Introducing the at least two different nucleic acid sequences into cells;

[0026] (iii) Expressing at least two different amino acid sequences;

[0027] (iv) Proteolytically cleaved at least two different adapter sequences;

[0028] (v) Determine the amount of the excised connector sequence.

[0029] In a third aspect, the present invention relates to a kit comprising:

[0030] a) A first nucleic acid sequence comprising an insertion site of a first polynucleotide encoding a functional sequence, wherein the first nucleic acid sequence encodes an amino acid sequence comprising a first adapter sequence, wherein the first adapter sequence is side-mounted at a proteolytic cleavage site such that the first adapter sequence can be cleaved from its amino acid sequence, wherein the sequence of the first adapter sequence is different from any other sequence side-mounted at the same proteolytic cleavage site in its amino acid sequence or in an amino acid sequence encoded by a second nucleic acid sequence; and

[0031] b) A second nucleic acid sequence comprising an insertion site of a second polynucleotide encoding a functional sequence, wherein the second nucleic acid sequence encodes an amino acid sequence comprising a second adapter sequence, wherein the second adapter sequence is side-mounted at a proteolytic cleavage site such that the second adapter sequence can be cleaved from its amino acid sequence, wherein the sequence of the second adapter sequence is different from any other sequence side-mounted at the same proteolytic cleavage site in its amino acid sequence or in the amino acid sequence encoded by the first nucleic acid sequence.

[0032] In a fourth aspect, the present invention relates to a kit comprising:

[0033] a) A first nucleic acid sequence comprising an insertion site for a first polynucleotide encoding a functional sequence, wherein the first nucleic acid sequence encodes an amino acid sequence comprising a first adapter sequence, wherein the first adapter sequence is flanked by a proteolytic cleavage site such that the first adapter sequence can be cleaved from its amino acid sequence; and

[0034] b) A second nucleic acid sequence comprising an insertion site of a second polynucleotide encoding a functional sequence, wherein the second nucleic acid sequence encodes an amino acid sequence comprising a second adapter sequence, wherein the second adapter sequence is flanked by a proteolytic cleavage site such that the second adapter sequence can be cleaved from its amino acid sequence.

[0035] In a fifth aspect, the present invention relates to the use of a kit according to the invention for simultaneously analyzing the efficacy of first and second nucleic acid sequences in expressing first and second functional sequences in a biological system.

[0036] In a sixth aspect, the present invention relates to the use of at least two nucleic acid sequences for simultaneously analyzing the potency of the at least two nucleic acid sequences in expressing at least two different functional sequences in a biological system, wherein each of the at least two nucleic acid sequences encodes an amino acid sequence comprising a different functional sequence and a different adapter sequence, wherein each adapter sequence is 6 to 30 amino acids in length and has the general formula:

[0037] [X] n Y

[0038] in

[0039] X is any amino acid;

[0040] n is an integer from 5 to 29;

[0041] Y is either lysine or arginine;

[0042] In addition, [X] n It may contain the amino acid sequence KP or RP, but it does not contain lysine or arginine.

[0043] Each of these linker sequences is preceded by a lysine or arginine residue at its N-terminus. Brief description of the attached diagram

[0045] Figure 1 A schematic representation of four nucleic acid sequences according to the present invention is shown, each sequence comprising a nucleotide sequence encoding a fixed antigen and a linker sequence capable of being cleaved by proteolytic cleavage. The fixed antigen contains lysine residues (K) and includes portions of the linker sequence. The linker sequence resulting from cleavage is shown below the four nucleic acid sequences.

[0046] Figure 2 A schematic representation of four nucleic acid sequences according to the present invention is shown, each sequence comprising a nucleotide sequence encoding a variable epitope and a linker sequence capable of being cleaved by proteolytic cleavage. A lysine residue (K) is inserted between each functional sequence and the linker sequence. The linker sequence resulting from the cleavage is shown below the four nucleic acid sequences. Invention Details

[0048] Although this disclosure is described in more detail below, it should be understood that this disclosure is not limited to the specific methods, schemes, and reagents described herein, as these can vary. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of this disclosure, which will be defined only by the appended claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0049] The elements of this disclosure will be described in more detail below. These elements are listed together with specific embodiments; however, it should be understood that they can be combined in any way and in any number to create other embodiments. The various examples and preferred embodiments described should not be construed as limiting this disclosure to only the explicitly described embodiments. This description should be understood to support and cover embodiments that combine the explicitly described embodiments with any number of disclosed and / or preferred elements. Furthermore, any permutation and combination of all elements described in this application should be considered as disclosed by the description of this application, unless the context otherwise indicates.

[0050] In a first aspect, the present invention relates to a method for simultaneously analyzing at least two different nucleic acid sequences, each nucleic acid sequence encoding a different amino acid sequence, wherein each of the at least two different amino acid sequences comprises a different functional sequence and a different adapter sequence, wherein each adapter sequence is proteolytically cleavable from its amino acid sequence, wherein the method comprises the following steps:

[0051] (i) Provide the at least two different nucleic acid sequences;

[0052] (ii) Introducing the at least two different nucleic acid sequences into cells;

[0053] (iii) Expressing at least two different amino acid sequences;

[0054] (iv) Proteolytically cleaved at least two different adapter sequences;

[0055] (v) Determine the amount of each of the excised connector sequences;

[0056] (vi) The amount of each of the excised adapter sequences is used as an indicator of the potency of each of the nucleic acid sequences in expressing each of the functional sequences in a biological system.

[0057] At least two different nucleic acid sequences: The method of the present invention is performed on at least two different nucleic acid sequences. Those skilled in the art will understand that the at least two different nucleic acid sequences may be located on the same nucleic acid, or on two or more different nucleic acids. Similarly, when using more than two nucleic acid sequences, one or more nucleic acid sequences may be located on one nucleic acid, and one or more nucleic acid sequences may be located on one or more other nucleic acids.

[0058] In one embodiment, the at least two different nucleic acid sequences are located on a single nucleic acid. In another embodiment, the at least two different nucleic acid sequences are each located on different nucleic acids. It should be understood that there is no strict practical limit to the number of nucleic acid sequences that can be analyzed simultaneously in this invention, as long as the principle of the adapter sequence of this invention is followed; for example, no identical adapter sequences are present within a given assay.

[0059] In one embodiment, the method of the present invention can be used in the following positions: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 6 It can be performed on 0, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, or 10000 different nucleic acid sequences.

[0060] In one embodiment, the at least two different nucleic acid sequences are contained in the same nucleic acid molecule. In another embodiment, the at least two different nucleic acid sequences are contained in more than one nucleic acid molecule. In yet another embodiment, each of the at least two different nucleic acid sequences is contained in a different nucleic acid molecule. In yet another embodiment, at least one of the at least two different nucleic acid sequences is contained in a nucleic acid molecule different from the other of the at least two different nucleic acid sequences.

[0061] The expression of multiple nucleic acid sequences on the same nucleic acid can also be correlated, meaning that each resulting amino acid sequence will have the same or similar expression levels. In this case, the invention may involve the simultaneous analysis of at least two different nucleic acid sequences, including a first nucleic acid sequence contained in a first nucleic acid and a second nucleic acid sequence contained in a second nucleic acid, wherein the first and / or second nucleic acid contains one or more additional nucleic acid sequences. In this embodiment, the additional nucleic acid sequences will be expressed together with the first or second nucleic acid sequence, depending on whether the additional nucleic acid sequences are contained in the first or second nucleic acid, respectively.

[0062] As commonly used herein with reference to certain elements of the invention (e.g., nucleic acids, amino acid sequences, functional sequences, and adapter sequences of the invention), the term "different" means that the element in question is not composed of the same sequence as any other identical element of that aspect of the invention. For example, "comprising at least two different amino acid sequences containing different functional sequences" means that the at least two amino acid sequences are composed of sequences that are different from each other, and that each amino acid sequence contains a functional sequence composed of sequences that are different from the sequences of each other functional sequences contained in the amino acid sequence.

[0063] In one embodiment, the nucleic acid sequence is RNA. In one embodiment, the nucleic acid comprising the nucleic acid sequence of the present invention is RNA. In one embodiment, the nucleic acid sequence is DNA. In one embodiment, the nucleic acid comprising the nucleic acid sequence of the present invention is DNA. In one embodiment, the nucleic acid sequence comprises at least one RNA sequence and at least one DNA sequence. In one embodiment, the nucleic acid comprising the nucleic acid sequence of the present invention comprises at least one RNA polynucleotide and at least one DNA polynucleotide.

[0064] In one embodiment, the at least two different nucleic acid sequences analyzed in the method of the present invention are RNA sequences, DNA sequences, or sequences comprising at least one RNA sequence and at least one DNA sequence. In some embodiments, the nucleic acid is DNA (e.g., one or more DNA sequences), RNA (e.g., one or more RNA sequences), or a mixture of DNA and RNA (e.g., one or more DNA sequences and one or more RNA sequences). In some embodiments, the DNA is present in the form of a vector, for example, a vector comprising DNA encoding an amino acid sequence comprising an amino acid sequence of a biologically active peptide or polypeptide. In some embodiments, the vector is a DNA vector.

[0065] The use of the term "one" nucleic acid sequence in this document should also be understood to apply to at least two nucleic acid sequences in this invention.

[0066] Functional Sequence: The present invention ultimately analyzes the efficacy of nucleic acids in expressing a "functional sequence." In this respect, the functional sequence is an analyte sequence. In one embodiment, the functional sequence is a peptide or polypeptide with therapeutic potential. In one embodiment, the functional sequence is an antigen or epitope. In one embodiment, each functional sequence may contain more than one antigen or epitope sequence. In one embodiment, the functional sequence is an antigen. In one embodiment, the functional sequence is an epitope. In one embodiment, the functional sequence is a T-cell epitope. In one embodiment, the functional sequence is an epitope presented to T cells via the major histocompatibility complex (MHC).

[0067] In one embodiment, the functional sequence is a fixed antigen or a variable epitope. In another embodiment, the functional sequence is a fixed antigen, such as a known tumor antigen. In yet another embodiment, the functional sequence is a variable epitope or a highly variable epitope, such as a patient-specific epitope, a personalized epitope, a de novo epitope sequence, or an undetermined epitope sequence.

[0068] In one embodiment, the functional sequence is a biologically active peptide or polypeptide. In some embodiments, the biologically active peptide or polypeptide is selected from the group consisting of vaccines (e.g., antigens, epitopes), proteins for alternative therapies, antibodies, antibody-like molecules, and cytokines. In some embodiments, the biologically active peptide or polypeptide constitutes a vaccine. In some embodiments, the vaccine is a T-cell vaccine. In some embodiments, the functional sequence of the present invention is a component of a multivalent T-cell vaccine.

[0069] Linker sequence: In this invention, a "linker sequence" is a sequence capable of being proteolytically cleaved from its respective amino acid sequence, the amount of which can be determined and used as an indicator of the effectiveness of the respective nucleic acid sequence in expressing its encoded functional sequence. In one embodiment, the sequence comprising the linker sequence is capable of being proteolytically cleaved from its respective amino acid sequence. In one embodiment, the linker sequence is capable of being proteolytically cleaved from its amino acid sequence as a cleavage sequence comprising the linker sequence.

[0070] It should be understood that the adapter sequence is not necessarily different from the functional sequence of the present invention, and the adapter sequence may actually contain a portion of the functional sequence of the present invention. When the functional sequence of the present invention is a fixed antigen containing a protein hydrolytic cleavage site (see...), Figure 1 This is particularly useful when [the term is missing here]. Therefore, a “linker sequence” is simply a name given to a portion of an amino acid sequence that can be excised (or has been excised) from the amino acid sequence by proteolytic cleavage. This linker sequence may contain a specific sequence included for this purpose (“linker barcode”), and may also contain portions of a functional sequence. When the functional sequence is a variable epitope (see [link missing here]), [the term is missing here]. Figure 2 When the adapter sequence does not contain a portion of the functional sequence, it is particularly useful. In this example case, the adapter sequence consists entirely of a "connector barcode" sequence included for this purpose, and additional amino acid residues can be inserted between the functional sequence and the adapter sequence to create a proteolytic cleavage site (e.g., Figure 2 (in the middle).

[0071] In one embodiment, each adapter sequence of the present invention encodes a polypeptide of 6 to 30 amino acids in length. In a preferred embodiment, each adapter sequence encodes a polypeptide of 8 to 12 amino acids in length.

[0072] In one implementation, each adapter sequence does not encode a functional protein.

[0073] In one embodiment, each adapter sequence is flanked by a proteolytic cleavage site. In one embodiment, "flanked proteolytic cleavage site" means that the amino acid sequence containing the adapter sequence contains a proteolytic cleavage site at the N-terminus (e.g., immediately adjacent to the N-terminus) of the adapter sequence, and the amino acid sequence containing the adapter sequence also contains a proteolytic cleavage site at the C-terminus (e.g., immediately adjacent to the C-terminus) of the adapter sequence. A proteolytic cleavage site should be understood as a position between two amino acid residues in an amino acid sequence that are cleaved by a proteolytic enzyme. In one embodiment, each adapter sequence is flanked by a proteolytic cleavage site, and the adapter sequence itself does not contain any of the same proteolytic cleavage sites. In one embodiment, each adapter sequence does not contain any internal proteolytic cleavage sites identical to any flanking proteolytic cleavage sites. In one embodiment, the amino acid residues within each adapter sequence cannot be proteolytically cleaved by the same proteolytic enzyme that excises the adapter sequence from its amino acid sequence. In one embodiment, each adapter sequence is flanked by a trypsin proteolytic cleavage site, and the adapter sequence itself does not contain any trypsin proteolytic cleavage site. In one embodiment, each linker sequence is side-mounted with a trypsin or Lys-C proteolytic cleavage site, and the linker sequence itself does not contain any trypsin or Lys-C proteolytic cleavage site. It should be understood that different proteolytic enzymes can be used in embodiments of the invention, and therefore the proteolytic cleavage sites can vary depending on the proteolytic enzyme used. A preferred proteolytic enzyme is trypsin, optionally in combination with Lys-C.

[0074] Those skilled in the art should also understand that, depending on the position of the amino acid sequence cleaved by the proteolytic enzyme used in this invention, the residues associated with the proteolytic enzyme cleavage may or may not form a portion of the linker sequence. For example, when a proteolytic enzyme that cleaves the C-terminus of a residue "Z" excises the linker sequence by cleaving at the N-terminus of the linker sequence, the residue "Z" will not form the N-terminus of the linker sequence; instead, the amino acid sequence preceding the N-terminus of the linker sequence will contain the residue "Z". In one embodiment, "before the N-terminus of the linker sequence" means immediately preceding the N-terminus of the linker sequence. As a second example, when a protease that cleaves the N-terminus of a residue "Z" excises the linker sequence by cleaving at the N-terminus of the linker sequence, the residue "Z" will form a portion of the N-terminus of the linker sequence.

[0075] In one embodiment, each adapter sequence is different from the sequences of all other polypeptide sequences that can be proteolytically cleaved in step (iv) of the method according to the invention. In another embodiment, each adapter sequence is different from the sequences of each other different adapter sequences used in the method of the invention. Therefore, in one embodiment, the adapter sequences used in the invention each have different sequences and are unique.

[0076] In one embodiment, the sequence of each adapter sequence is different from the sequence of any other peptide or polypeptide that can be proteolytically cleaved from any functional sequence of the invention. In one embodiment, the sequence of each adapter sequence is different from any other sequence contained in any functional sequence.

[0077] In one embodiment, each adapter sequence is different from the sequence of any other peptide or polypeptide that can be proteatically cleaved from the amino acids encoded by at least two different nucleic acid sequences of the present invention. In one embodiment, each adapter sequence is different from any other sequence encoded by at least two different nucleic acid sequences of the present invention.

[0078] In one embodiment, each adapter sequence is distinct from any other peptide or polypeptide sequence capable of being proteolytically cleaved from the proteome of a cell in which at least two different nucleic acid sequences have been introduced in this invention. In practice, by considering the proteolytic enzymes and cells used, and by applying, for example, computer analysis or in vitro experiments, those skilled in the art can readily determine which sequences can be proteolytically cleaved from the cell's proteome in the method according to the invention.

[0079] In other words, in one embodiment of the invention, each linker sequence is different from any other sequence that can be proteolytically cleaved by the proteolytic enzymes used in the invention. In one embodiment, each linker sequence is different from any other sequence that can be proteolytically cleaved from the amino acids or cellular proteome used in the invention by the proteolytic enzymes used in the invention. In one embodiment, each linker sequence is different from any other sequence that can be proteolytically cleaved from the amino acids or cellular proteome used in the invention by trypsin.

[0080] In one embodiment, the proteolytic resection of step (iv) of the method of the present invention is performed using a proteolytic enzyme or a mixture of proteolytic enzymes. In one embodiment, the proteolytic enzyme is trypsin. In one embodiment, the mixture of proteolytic enzymes comprises trypsin and one or more other proteolytic enzymes selected from the group consisting of Glu-C, Lys-N, Lys-C, Asp-N, and chymotrypsin. In one embodiment, the mixture of proteolytic enzymes comprises trypsin and Lys-C.

[0081] In one embodiment, the proteolytic enzyme used is trypsin, which cleaves the protein to the C-terminus of arginine (R) and lysine (K) residues, unless followed by proline (P). In one embodiment, trypsin is used in conjunction with Lys-C, which cleaves specifically at the C-terminus of K residues, to achieve more efficient and complete digestion. Therefore, in one embodiment, each linker sequence is preceded by a lysine or arginine residue at its N-terminus and contains a lysine or arginine residue at its C-terminus. In one embodiment, each linker sequence is preceded by a K or R residue at its N-terminus and also contains a K or R residue at its C-terminus. In a preferred embodiment, each linker sequence is preceded by a K residue at its N-terminus. In one embodiment, each linker sequence does not contain any other K or R residues. In one embodiment, each linker sequence does not contain any other K or R residues unless followed by a P residue. In one embodiment, each linker sequence does not contain any other K or R residues unless it is part of sequence KP or RP. In one embodiment, the lysine or arginine at the N-terminus of the linker sequence is naturally present within the functional sequence. In another embodiment, a lysine or arginine residue at the N-terminus of the linker sequence has been introduced into the functional sequence via site-directed mutagenesis. In one embodiment, "at the N-terminus of the linker sequence" is understood to mean immediately adjacent to the N-terminus of the linker sequence.

[0082] Following trypsin cleavage, peptides containing a single R or K residue are more readily analyzed by mass spectrometry because the positive charge at the C-terminus favors ionization and consistently produces +2 and +3 peptide charge states. Therefore, in one embodiment, the linker sequence contains only a single R or K residue. In another embodiment, the linker sequence contains only a single K or R residue, wherein the residue is located at its C-terminus. In yet another embodiment, the linker sequence does not contain an internal trypsin-mediated cleavage site.

[0083] In one embodiment, the proteolytic enzyme used in this invention is Lys-C, and its proteolytic cleavage site is at the C-terminus of a lysine (K) residue. In one embodiment, each adapter sequence has K preceding the N-terminus, and each adapter sequence contains K at its C-terminus. In one embodiment, each adapter sequence does not contain an internal Lys-C proteolytic cleavage site.

[0084] In one embodiment, the proteolytic enzyme used in this invention is Glu-C, with its proteolytic cleavage site located at the C-terminus of either an aspartic (D) or glutamic (E) residue. In one embodiment, each adapter sequence is preceded by either D or E at the N-terminus, and the C-terminus of each adapter sequence contains either D or E. In one embodiment, each adapter sequence does not contain an internal Glu-C proteolytic cleavage site.

[0085] In one embodiment, the proteolytic enzyme used in this invention is chymotrypsin, with its proteolytic cleavage site located at the C-terminus of phenylalanine (F), tryptophan (W), and tyrosine (Y) residues. In one embodiment, each linker sequence is preceded by F, W, or Y at the N-terminus, and each linker sequence contains F, W, or Y at its C-terminus. In one embodiment, each linker sequence does not contain an internal chymotrypsin proteolytic cleavage site.

[0086] In one embodiment, a C-terminal "followed by" residue means that the residue immediately following the linker sequence. In another embodiment, a C-terminal "followed by" residue means that the amino acid sequence containing the linker sequence includes the residue immediately following the linker sequence.

[0087] In one embodiment, the proteolytic enzyme used in this invention is Lys-N, with its proteolytic cleavage site located at the N-terminus of a lysine (K) residue. In one embodiment, the N-terminus of each adapter sequence contains K, and the C-terminus of each adapter sequence is followed by K. In one embodiment, each adapter sequence does not contain an internal Lys-N proteolytic cleavage site.

[0088] In one embodiment, the proteolytic enzyme used in this invention is Asp-N, with its proteolytic cleavage site located at the N-terminus of an aspartic (D) or glutamic (E) residue. In one embodiment, the N-terminus of each adapter sequence contains either D or E, and the C-terminus of each adapter sequence is followed by either D or E. In one embodiment, each adapter sequence does not contain an internal Asp-N proteolytic cleavage site.

[0089] It should be understood that, within the context of this invention, proteolytic enzymes can be combined, and in this embodiment, all proteolytic cleavage sites of each proteolytic enzyme included in the combination will be applicable. In one embodiment, the proteolytic enzyme combination comprises trypsin and Lys-C. In another embodiment, the proteolytic enzyme combination consists of trypsin and Lys-C.

[0090] In one embodiment, each adapter sequence is non-immunogenic. In one embodiment, each adapter sequence does not elicit a specific immune response. In one embodiment, each adapter sequence does not elicit an immune response specific to that adapter sequence. In one embodiment, each adapter sequence is immunogenically inert. In one embodiment, each adapter sequence is immunologically cryptic. In one embodiment, each adapter sequence does not stimulate an immune response. In one embodiment, each adapter sequence does not stimulate antibody production. Such embodiments are particularly important when the functional sequence encodes one or more antigens or epitopes. Such embodiments are particularly suitable for non-immunogenicity in animal or human cells, etc.

[0091] In one embodiment, the adapter sequences are non-immunogenic because they do not elicit an immune response. In one embodiment, the adapter sequences cannot elicit an immune response against the adapter sequence or against cells (such as human or animal cells) that express, contain, and present the adapter sequence. In one embodiment, the adapter sequences do not induce a comprehensive bodily response to an antigen, such as a cellular immune response, a humoral immune response, or both. In one embodiment, the adapter sequences do not elicit an immune response comprising one or more reactions selected from the group consisting of: producing antibodies against one or more antigens and amplifying antigen-specific T lymphocytes (such as CD4+ and CD8+ T lymphocytes, e.g., CD8+ T lymphocytes), which can be detected in various in vitro proliferation or cytokine production assays. In one embodiment, the adapter sequences do not induce an immune system response when administered to, for example, a mammal.

[0092] In one embodiment, in each nucleic acid sequence encoding a functional sequence and a linker sequence, the nucleotide sequence encoding the functional sequence is "in a frame" or in the same reading frame as the nucleotide sequence encoding the linker sequence. In one embodiment, the nucleotide sequence encoding the functional sequence is spaced 0 nucleotides from the nucleotide sequence encoding the linker sequence, or at least a multiple of 3 nucleotides apart.

[0093] Some specific linker sequences of the present invention will now be defined. In embodiments, these linker sequences may be used in conjunction with proteases that cleave the C-terminus of an amino acid sequence containing lysine or arginine residues, such as trypsin.

[0094] In one embodiment, the first and second analyzed nucleic acid sequences each encode an amino acid containing a linker sequence, wherein the linker sequence is preceded by a lysine or arginine residue, preferably lysine, and the linker sequence contains a lysine or arginine residue at its C-terminus.

[0095] In one embodiment, the nucleic acid sequence analyzed first encodes an amino acid sequence containing a first adapter sequence, wherein the adapter sequence is preceded by a lysine or arginine residue, preferably lysine, and the first adapter sequence contains a lysine residue at its C-terminus.

[0096] In another embodiment of this type, the nucleic acid sequence of the second analysis encodes an amino acid sequence containing a second adapter sequence, wherein the second adapter sequence is preceded by a lysine or arginine residue, preferably lysine, and the second adapter sequence contains an arginine residue at its C-terminus.

[0097] In another embodiment of this type, the first and second connector sequences are distinguished only by lysine residues at the C-terminus or arginine residues at the C-terminus, respectively.

[0098] In another embodiment of this type, the nucleic acid sequence of the third analysis encodes an amino acid sequence containing a third adapter sequence, wherein the third adapter sequence is preceded by a lysine or arginine residue, preferably lysine, and the third adapter sequence contains an arginine residue at its C-terminus.

[0099] In another embodiment of this type, the nucleic acid sequence of the fourth analysis encodes an amino acid sequence containing a fourth adapter sequence, wherein the fourth adapter sequence is preceded by a lysine or arginine residue, preferably lysine, and the fourth adapter sequence contains an arginine residue at its C-terminus.

[0100] In another embodiment of this type, the first and second linker sequences are distinguished only by lysine residues or arginine residues at the C-terminus, respectively, and the third and fourth linker sequences are distinguished only by lysine residues or arginine residues at the C-terminus, respectively. However, there are additional differences between a) the first and second linker sequences and b) the third and fourth linker sequences.

[0101] When specific adapter sequences are obtained for use in this invention, it should be understood that these adapter sequences will be unique to each other and to any other sequence that can be cleaved by the proteolytic enzymes used in this invention (i.e., to the functional sequence, the amino acids encoded by the at least two nucleic acid sequences analyzed, and the proteome of the cell expressing the nucleic acid). Therefore, for these considerations, the features mentioned herein can be logically combined. In one embodiment, each adapter sequence is distinct from any other sequence that can be cleaved by the proteolytic enzymes used in this invention, from the functional sequence, the amino acids encoded by the at least two nucleic acid sequences analyzed, and the proteome of the cell expressing the nucleic acid.

[0102] In one embodiment of the invention, the linker sequence is preceded by a lysine residue at its N-terminus and can be represented as follows: GGSGGGGGSGGR / K. Therefore, the portion of the amino acid sequence containing the linker sequence can be represented as follows: KΔGGSGGGGSGGR / K (Δ indicates a protein hydrolytic cleavage site). After cleavage, this results in the removal of the linker sequence as follows: GGSGGGGSGGR / K.

[0103] Additional sequences: In one embodiment, one or more of the at least two amino acid sequences in the method of the present invention comprise a functional sequence and a linker sequence, wherein the linker sequence is located at the C-terminus of the functional sequence. In one embodiment, the linker sequence comprises the C-terminus of the amino acid sequence. Therefore, it should be understood that the "linker" sequence may be located at the C-terminus of the functional sequence, without necessarily linking the functional sequence to any other C-terminal sequence.

[0104] In another embodiment, one or more of the at least two amino acid sequences further comprise an additional sequence located at the C-terminus of the linker sequence. Thus, in one embodiment, the linker sequence does indeed link the functional sequence to an additional, other C-terminal sequence. In one embodiment, each of the at least two amino acid sequences further comprises a sequence located at the C-terminus of the linker sequence. In one embodiment, the sequence located at the C-terminus of the linker sequence is an auxiliary domain sequence. In one embodiment, in each of the at least two amino acid sequences in the method of the present invention, the linker sequence is located at the C-terminus of the functional sequence, and the auxiliary domain sequence is located at the C-terminus of the linker sequence.

[0105] In one embodiment, the auxiliary domain is a sequence that enhances the function of the functional sequence. In embodiments where the functional sequence is an antigen or epitope, the auxiliary domain may be a sequence that enhances the presentation of that antigen or epitope. In one embodiment, the auxiliary domain is a transport domain. In one embodiment, the auxiliary domain is a MITD domain. Further information about MITD domains can be found in Kreiter et al. (J Immunol 180(1)(2008)309-318). In embodiments, each of at least two nucleic acids of the present invention comprises an auxiliary domain having the same sequence.

[0106] Method Steps: In one embodiment, step (ii) of the method of the present invention involves introducing the at least two different nucleic acids in vitro (such as transfection or transduction) into cells. In one embodiment, step (ii) of the method of the present invention involves introducing the at least two different nucleic acid sequences in vivo into cells, such as by administration to a subject.

[0107] Step (iii) of the method of the present invention should be understood to include expressing the at least two different amino acid sequences in the cells of step (ii). In one embodiment, step (iii) is instead defined as attempting to express an amino acid sequence.

[0108] In one embodiment, the method of the present invention further includes lysing cells prior to step (iv). In one embodiment, the method further includes treating the cell lysate. In one embodiment, treating the cell lysate includes one or more steps selected from the group consisting of proteolytic enzyme digestion, denaturation, reduction, alkylation, drying, remodeling, and desalting. In one embodiment, treating the cell lysate includes proteolytic enzyme treatment of the present invention to cleave linker sequences.

[0109] In one embodiment of step (v) of the method of the present invention, mass spectrometry is used to determine the amount of each of the excised adapter sequences. In one embodiment, liquid chromatography-mass spectrometry (LC-MS) is used to determine the amount of each of the excised adapter sequences. In one embodiment, targeted LC-MS is used to determine the amount of each of the excised adapter sequences.

[0110] In one embodiment, the biological system of the present invention is a biological system present in a human patient.

[0111] In a second aspect, the present invention relates to a method for analyzing the potency of nucleic acid sequences in expressing functional sequences in a biological system, wherein the method comprises simultaneously analyzing at least two different nucleic acid sequences, each nucleic acid sequence encoding a different amino acid sequence, wherein each of the at least two different amino acid sequences contains a different functional sequence and a different adapter sequence, wherein each adapter sequence is proteolytically cleavable from its amino acid sequence, wherein the method comprises the following steps:

[0112] (i) Provide the at least two different nucleic acid sequences;

[0113] (ii) Introducing the at least two different nucleic acid sequences into cells;

[0114] (iii) Expressing at least two different amino acid sequences;

[0115] (iv) Proteolytically cleaved at least two different adapter sequences;

[0116] (v) Determine the amount of the excised connector sequence.

[0117] Reagent kit and usage:

[0118] In a third aspect, the present invention relates to a kit comprising:

[0119] a) A first nucleic acid sequence comprising an insertion site of a first polynucleotide encoding a functional sequence, wherein the first nucleic acid sequence encodes an amino acid sequence comprising a first adapter sequence, wherein the first adapter sequence is side-mounted at a proteolytic cleavage site such that the first adapter sequence can be cleaved from its amino acid sequence, wherein the sequence of the first adapter sequence is different from any other sequence side-mounted at the same proteolytic cleavage site in its amino acid sequence or in an amino acid sequence encoded by a second nucleic acid sequence; and

[0120] b) A second nucleic acid sequence comprising an insertion site of a second polynucleotide encoding a functional sequence, wherein the second nucleic acid sequence encodes an amino acid sequence comprising a second adapter sequence, wherein the second adapter sequence is side-mounted at a proteolytic cleavage site such that the second adapter sequence can be cleaved from its amino acid sequence, wherein the sequence of the second adapter sequence is different from any other sequence side-mounted at the same proteolytic cleavage site in its amino acid sequence or in the amino acid sequence encoded by the first nucleic acid sequence.

[0121] In one embodiment, the insertion site for the functional sequence is a multiple cloning site. In another embodiment, the nucleic acid sequence is a plasmid or vector.

[0122] In one embodiment, the kit of the present invention further includes:

[0123] c) one or more additional nucleic acid sequences, each containing an insertion site of an additional polynucleotide encoding a functional sequence, wherein each additional nucleic acid sequence encodes an additional amino acid sequence containing an additional adapter sequence, wherein each additional adapter sequence is side-mounted at a proteolytic cleavage site such that each additional adapter sequence can be cleaved from its amino acid sequence, and wherein the sequence of each additional adapter sequence is different from any other sequence side-mounted at the same proteolytic cleavage site in its amino acid sequence or in an amino acid sequence encoded by a first nucleic acid sequence, a second nucleic acid sequence, or any other additional nucleic acid sequence.

[0124] In this embodiment, the sequences of the first and second adapter sequences are also different from any other sequence that side-joins the same proteolytic cleavage site in any amino acid sequence encoded by one or more other nucleic acid sequences.

[0125] In a fourth aspect, the present invention relates to a kit comprising:

[0126] a) A first nucleic acid sequence comprising an insertion site for a first polynucleotide encoding a functional sequence, wherein the first nucleic acid sequence encodes an amino acid sequence comprising a first adapter sequence, wherein the first adapter sequence is flanked by a proteolytic cleavage site such that the first adapter sequence can be cleaved from its amino acid sequence; and

[0127] b) A second nucleic acid sequence comprising an insertion site of a second polynucleotide encoding a functional sequence, wherein the second nucleic acid sequence encodes an amino acid sequence comprising a second adapter sequence, wherein the second adapter sequence is flanked by a proteolytic cleavage site such that the second adapter sequence can be cleaved from its amino acid sequence.

[0128] In one embodiment of the kit of the present invention, the insertion site contained in each nucleic acid sequence comprises a polynucleotide encoding a functional sequence. In one embodiment, the functional sequences are all different.

[0129] In a fifth aspect, the present invention relates to the use of a kit according to the invention for simultaneously analyzing the efficacy of first and second nucleic acid sequences in expressing first and second functional sequences in a biological system.

[0130] In one embodiment, the kit according to the invention is used for simultaneously analyzing the efficacy of first, second, and one or more additional nucleic acid sequences in expressing first, second, and one or more additional functional sequences in a biological system.

[0131] In one embodiment, the present invention relates to the use of a kit according to the invention, wherein the insertion site in each nucleic acid sequence comprises a polynucleotide encoding a functional sequence. In one embodiment, the functional sequences are all different.

[0132] In a sixth aspect, the present invention relates to the use of at least two nucleic acid sequences for simultaneously analyzing the potency of the at least two nucleic acid sequences in expressing at least two different functional sequences in a biological system, wherein each of the at least two nucleic acid sequences encodes an amino acid sequence comprising a different functional sequence and a different adapter sequence, wherein each adapter sequence is 6 to 30 amino acids in length and has the general formula:

[0133] [X] n Y

[0134] in

[0135] X is any amino acid;

[0136] n is an integer from 5 to 29;

[0137] Y is either lysine or arginine;

[0138] In addition, [X] n It may contain the amino acid sequence KP or RP, but it does not contain lysine or arginine.

[0139] Each of these linker sequences is preceded by a lysine or arginine residue at its N-terminus.

[0140] Applications: This invention can be widely applied to any method for analyzing at least two different nucleic acid sequences, particularly for analyzing the expression or potency of at least two different nucleic acid sequences. In one embodiment, this invention is a method for analyzing the potency of at least two different nucleic acid sequences in expressing at least two different functional sequences. In another embodiment, this invention is a method for analyzing the potency of at least two nucleic acid sequences in expressing at least two functional sequences in a biological system.

[0141] In one embodiment, the biological system is not particularly limited. In one embodiment, the biological system is a cell assay. In another embodiment, the biological system is an ex vivo tissue sample. In one embodiment, the biological system is an ex vivo tissue sample from a rodent such as a mouse. In one embodiment, the biological system is an ex vivo tissue sample from a human. In one embodiment, the biological system is in vivo. In one embodiment, the biological system is in a rodent such as a mouse. In one embodiment, the biological system is in a human patient.

[0142] In one embodiment, the present invention relates to both in vitro, ex vivo, and in vivo methods and uses. In various embodiments, the present invention relates to in vitro methods and uses. In various embodiments, the present invention relates to ex vivo methods and uses. In various embodiments, the present invention relates to in vivo methods and uses.

[0143] In one embodiment, the present invention is used to analyze multivalent T-cell vaccines.

[0144] In one embodiment, the present invention is used in a diagnostic method. In one embodiment, the present invention is used in a companion diagnostic method or in a clinical follow-up study. In one embodiment, the kit of the present invention is used as a companion diagnostic kit.

[0145] Definition of general terms

[0146] Unless otherwise indicated, 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.

[0147] In this specification and the following claims, unless the context otherwise requires, the word “comprise” and its variations such as “comprises” and “comprising” shall be understood to mean comprising the specified features, elements, members, integers or steps, or groups of features, elements, members, integers or steps, but not excluding 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 and features, elements, members, integers or steps that do not substantially affect the basic and new features 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 may be replaced by the terms “consistently composed of” or “composed of”. Similarly, in this application, the term "basically composed of..." can be replaced by the term "composed of..." whenever it appears.

[0148] The terms “a,” “an,” and “the,” as well as similar designations, used in the context of describing this disclosure (especially in the context of the claims) should be interpreted to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by the context.

[0149] All the methods described herein may be performed in any suitable order, unless otherwise indicated herein or otherwise clearly contradicted by the context.

[0150] The use of any and all instances or exemplary language (such as "such as") provided herein is intended only to better illustrate this disclosure and does not limit the scope of any additional claims made herein. No language in the specification should be construed as indicating that any unclaimed element is essential to the practice of this disclosure.

[0151] As used herein, the terms “optional” or “optionally” mean that the event, situation, or condition described below may or may not occur, and the description includes both the scenario in which the event, situation, or condition occurs and the scenario in which the event, situation, or condition does not occur.

[0152] As used herein, “and / or” should be considered as a specific disclosure of each of the two specified features or components (with or without the other). For example, “X and / or Y” should be considered as a specific disclosure of each of (i) X, (ii) Y, and (iii) X and Y, as if each were listed separately herein.

[0153] In the context of this disclosure, the term "about" means, as will be understood by one of ordinary skill, a range of accuracy that still ensures the technical effect of the feature in question. This term typically indicates a deviation from the indicated 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%, and, for example, ±0.01%. In some embodiments, "about" indicates a deviation from the indicated value of ±10%. In some embodiments, "about" indicates a deviation from the indicated value of ±5%. In some embodiments, "about" indicates a deviation from the indicated value of ±4%. In some embodiments, "about" indicates a deviation from the indicated value of ±3%. In some embodiments, "about" indicates a deviation from the indicated value of ±2%. In some embodiments, "about" indicates a deviation from the indicated value of ±1%. In some embodiments, "about" indicates a deviation from the indicated value of ±0.9%. In some embodiments, "about" indicates a deviation of ±0.8% from the indicated value. In some embodiments, "about" indicates a deviation of ±0.7% from the indicated value. In some embodiments, "about" indicates a deviation of ±0.6% from the indicated value. In some embodiments, "about" indicates a deviation of ±0.5% from the indicated value. In some embodiments, "about" indicates a deviation of ±0.4% from the indicated value. In some embodiments, "about" indicates a deviation of ±0.3% from the indicated value. In some embodiments, "about" indicates a deviation of ±0.2% from the indicated value. In some embodiments, "about" indicates a deviation of ±0.1% from the indicated value. In some embodiments, "about" indicates a deviation of ±0.05% from the indicated value. In some embodiments, "about" indicates a deviation of ±0.01% from the indicated value. As will be understood by those skilled in the art, the specific deviation of such a value for a given technical effect will depend on the nature of the technical effect. For example, natural or biotechnological effects can often have a larger deviation than artificial or engineered effects.

[0154] The enumeration of ranges of values ​​in this document is intended only as a shorthand way of referring to each individual value falling within the range. Unless otherwise stated herein, each individual value is included in this specification as if it were enumerated separately.

[0155] Every document cited in this document (including all patents, patent applications, scientific publications, manufacturer specifications, instructions, etc.) is hereby incorporated in its entirety by reference. The content of this document should not be construed as an admission that the present invention is not entitled to any prior art disclosure.

[0156] Specific definition

[0157] Definitions applicable to all aspects of this disclosure are provided below. Unless otherwise indicated, the following terms have the following meanings. Any undefined term has its generally accepted meaning in the art.

[0158] The "therapeutic potential" or "potency" of nucleic acids (such as RNA and / or DNA) refers to the therapeutic quality of the nucleic acid—its ability to provide therapeutic benefit when administered to a subject. In certain embodiments, the therapeutic potential of a nucleic acid can be measured, determined, identified, quantified, confirmed, and / or verified by the expression (particularly strong expression, such as above a threshold) of a peptide or polypeptide encoded by the nucleic acid, said expression indicating the therapeutic potential of the nucleic acid. In one embodiment, therapeutic potential refers to the ability of a nucleic acid (such as RNA and / or DNA) to express a pharmaceutically active peptide or polypeptide in vivo, which exerts its pharmaceutical (e.g., therapeutic) effect.

[0159] In some implementations, nucleic acids (such as RNA and / or DNA) exhibiting strong expression (e.g., expression above a threshold) have “sufficient therapeutic potential.” Therapeutic potential is sufficient if the nucleic acid has the ability to express in vivo a pharmaceutically active peptide or polypeptide that encodes a meaningful drug (e.g., therapeutic) effect.

[0160] As used herein, phrases such as “definite amount” or “definite expression” or similar phrases relating to amino acid sequences (peptides or polypeptides) refer to the amount or presence of a definite amino acid sequence.

[0161] As used herein, terms such as “reduction” or “inhibition” refer to the ability to cause an overall reduction in level, for example, about 5% or more, about 10% or more, about 15% or more, about 20% or more, about 25% or more, about 30% or more, about 40% or more, about 50% or more, or about 75% or more. The term “inhibition” or similar phrases include complete or substantially complete inhibition, i.e., reduction to zero or substantially reduction to zero.

[0162] As used herein, the term “enhancement” refers to the ability to cause an overall increase or enhancement in the level, for example, at least about 5% or more, about 10% or more, about 15% or more, about 20% or more, about 25% or more, about 30% or more, about 40% or more, about 50% or more, about 75% or more, or about 100% or more.

[0163] As used herein, “physiological pH” refers to a pH of approximately 7.4. In some embodiments, the physiological pH is 7.3 to 7.5. In some embodiments, the physiological pH is 7.35 to 7.45. In some embodiments, the physiological pH is 7.3, 7.35, 7.4, 7.45, or 7.5.

[0164] As used in this disclosure, "%w / v" refers to the percentage of weight to volume, which is a unit of concentration that measures the amount of solute in grams (g) and is expressed as a percentage of the total volume of the solution in milliliters (mL).

[0165] As used in this disclosure, “weight%” means weight percentage, which is a unit of concentration that measures the amount of substance in grams (g) and is expressed as a percentage of the total weight of the total composition in grams (g).

[0166] As used in this disclosure, "mol%" is defined as the ratio of the number of moles of one component to the total number of moles of all components multiplied by 100.

[0167] As used in this disclosure, "total lipids (mol%)" is defined as the ratio of the number of moles of one lipid component to the total number of moles of all lipids multiplied by 100. In this context, in some embodiments, the term "total lipids" includes lipids and lipid-like substances.

[0168] The term "ionic strength" refers to the mathematical relationship between the number of different types of ions and their respective charges in a given solution. Therefore, ionic strength I is mathematically expressed by the following formula:

[0169]

[0170] Where c is the molar concentration of a specific ionic substance, and z is the absolute value of its charge. The sum Σ is taken from all the different kinds of ions (i) in the solution.

[0171] According to this disclosure, the term "ionic strength" in some embodiments refers to the presence of monovalent ions. Regarding the presence of divalent ions, particularly divalent cations, their concentration or effective concentration (the presence of free ions) is sufficiently low in some embodiments due to the presence of chelating agents to prevent nucleic acid degradation. In some embodiments, the concentration or effective concentration of divalent ions is below the catalytic level for the hydrolysis of phosphodiester bonds between nucleotides (such as RNA nucleotides). In some embodiments, the concentration of free divalent ions is 20 μM or lower. In some embodiments, free divalent ions are absent or substantially absent.

[0172] "Osmotic pressure" refers to the concentration of a specific solute, expressed as the number of moles of solute per kilogram of solvent.

[0173] The term "lyophilization" or "lyophilization method" refers to the process of freezing a substance by freezing it and then reducing the ambient pressure (e.g., below 15 Pa, such as below 10 Pa, below 5 Pa, or 1 Pa or lower) to allow the freezing medium in the substance to sublimate directly from the solid phase to the gas phase. Therefore, the terms "lyophilization" and "freeze-drying" are used interchangeably herein.

[0174] The term "spray drying" refers to the process of drying a substance by mixing (heated) gas with a fluid that is atomized (sprayed) inside a container (spray dryer), where the solvent from the formed droplets evaporates to produce dry powder.

[0175] The term "reconstruction" refers to adding a solvent, such as water, to a dried product to restore it to a liquid state, such as its original liquid state.

[0176] The term "recombinant" in the context of this disclosure means "made by genetic engineering". In one embodiment, the "recombinant object" in the context of this disclosure is not naturally occurring.

[0177] As used herein, the term "naturally occurring" refers to the fact that an object can be found in nature. For example, peptides or nucleic acids that exist in organisms (including viruses) and can be isolated from natural sources and have not been intentionally modified by humans in a laboratory are naturally occurring. The term "found in nature" means "existing in nature" and includes both known objects and objects that have not yet been discovered and / or isolated from nature but may be discovered and / or isolated from natural sources in the future.

[0178] As used herein, the terms "room temperature" and "ambient temperature" are used interchangeably and refer to a temperature of at least about 15°C, for example, about 15°C to about 35°C, about 15°C to about 30°C, about 15°C to about 25°C, or about 17°C to about 22°C. Such temperatures will include 15°C, 16°C, 17°C, 18°C, 19°C, 20°C, 21°C, and 22°C. In some embodiments, the temperature is about 15°C to about 25°C. In some embodiments, the temperature is about 17°C to about 25°C. In some embodiments, the temperature is about 15°C. In some embodiments, the temperature is about 16°C. In some embodiments, the temperature is about 17°C. In some embodiments, the temperature is about 18°C. In some embodiments, the temperature is about 19°C. In some embodiments, the temperature is about 20°C. In some embodiments, the temperature is about 21°C. In some embodiments, the temperature is about 22°C.

[0179] The term "EDTA" refers to disodium ethylenediaminetetraacetate. All concentrations are given relative to disodium EDTA.

[0180] The term "cryoprotectant" refers to substances added to formulations to protect the active ingredient during the freezing process.

[0181] The term "lyophilization protectant" refers to substances added to formulations to protect the active ingredient during the drying stage.

[0182] According to this disclosure, the term "peptide" refers to a substance comprising about two or more, about three or more, about four or more, about six or more, about eight or more, about ten or more, about thirteen or more, about sixteen or more, about twenty or more, and up to about 50, about 100, or about 150 amino acids linked together by peptide bonds. The term "polypeptide" refers to a large peptide, particularly a peptide having at least about 151 amino acids. Both "peptide" and "polypeptide" are protein molecules.

[0183] The term "bioactivity" refers to the response of a biological system to a molecule. Such a biological system can be, for example, a cell or an organism. In some embodiments, such a response is therapeutically or pharmaceutically useful. In some embodiments, bioactivity includes pharmaceutical activity.

[0184] As used herein, the term “biological system” refers to any system of interacting or potentially interacting biological components whose behavior can be characterized, in whole or in part, by one or more biological processes or mechanisms. Biological systems can include, for example, single cells, cell collections (such as cell cultures), organs, tissues, and multicellular organisms (such as individuals or subjects, e.g., human patients).

[0185] In some implementations, the biological system exists in an individual or subject, and the biological activity in such a biological system is a therapeutically or pharmaceutically useful activity, that is, the biological activity results in or contributes to a therapeutically or pharmaceutically useful effect.

[0186] According to various embodiments of this disclosure, nucleic acids (such as RNA and / or DNA) encoding peptides or polypeptides are absorbed or introduced (i.e., transfected or transduced) into cells, which may be present in vitro or in a subject, resulting in the expression of the peptides or polypeptides. The cells may, for example, express the encoded peptides or polypeptides intracellularly (e.g., in the cytoplasm and / or in the nucleus), secrete the encoded peptides or polypeptides, and / or express them on their surface.

[0187] According to this disclosure, terms such as “nucleic acid expressing…” and “nucleic acid encoding…” or similar terms are used interchangeably herein, and with respect to a particular peptide or polypeptide, it means that a nucleic acid, if present in a suitable environment (e.g., within a cell), can be expressed to produce the peptide or polypeptide.

[0188] The term "portion" refers to a fraction. For a specific structure (such as an amino acid sequence or a protein), the term "portion" can refer to a continuous or discontinuous part of that structure.

[0189] The terms “part” and “fragment” are used interchangeably herein and refer to a continuous element. For example, a part, such as an amino acid sequence or a protein structure, refers to a continuous element of that structure. When used in the context of a composition, the term “part” refers to a portion of the composition. For example, a part of the composition can be any portion from 0.1% to 99.9% (such as 0.1%, 0.5%, 1%, 5%, 10%, 50%, 90%, or 99%) of the composition.

[0190] A “fragment” refers to a portion of an amino acid sequence (peptide or polypeptide), specifically a sequence representing a shortened amino acid sequence at the N-terminus and / or C-terminus. For example, a C-terminal shortened fragment (N-terminal fragment) can be obtained by translating a truncated open reading frame lacking the 3' end. Similarly, a C-terminal shortened fragment (C-terminal fragment) can be obtained by translating a truncated open reading frame lacking the 5' end, provided the truncated open reading frame contains a start codon to initiate translation. A fragment of an amino acid sequence comprises, for example, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the amino acid residues from the amino acid sequence. A fragment of an amino acid sequence comprises, for example, at least 6, particularly at least 8, at least 10, at least 12, at least 15, at least 20, at least 30, at least 50, or at least 100 consecutive amino acids from the amino acid sequence. The amino acid sequence fragment contains, for example, a sequence of up to 8, particularly up to 10, 12, 15, 20, 30, or 55 consecutive amino acids.

[0191] As used herein, and with respect to an amino acid sequence (peptide or polypeptide), a “variant” refers to an amino acid sequence that differs from the parent amino acid sequence due to at least one amino acid (e.g., a different amino acid, or a modification of the same amino acid). The parent amino acid sequence can be a naturally occurring or wild-type (WT) amino acid sequence, or it can be a modified form of a wild-type amino acid sequence. In some embodiments, the variant amino acid sequence has at least one amino acid difference compared to the parent amino acid sequence, for example, a difference of 1 to about 20 amino acids compared to the parent, such as a difference of 1 to about 10 or 1 to about 5 amino acids.

[0192] In this article, "wild-type," "WT," or "natural" refers to amino acid sequences found in nature, including allelic variations. Wild-type amino acid sequences, peptides, or polypeptides have amino acid sequences that have not been intentionally modified.

[0193] For the purposes of this disclosure, a “variant” of an amino acid sequence (peptide or polypeptide) can include amino acid insertion variants, amino acid addition variants, amino acid deletion variants, and / or amino acid substitution variants. The term “variant” includes all mutants, splicing variants, post-translational modification variants, conformations, allotypes, allele variants, species variants, and species homologs, particularly those that are naturally occurring. The term “variant” specifically includes fragments of amino acid sequences.

[0194] Amino acid insertion variants involve the insertion of one, two, or more amino acids into a specific amino acid sequence. In the case of amino acid sequence variants with insertions, one or more amino acid residues are inserted into a specific site in the amino acid sequence, although random insertions are also possible while the resulting product is appropriately screened. Amino acid addition variants involve the fusion of the amino and / or carboxyl ends of one or more amino acids (such as 1, 2, 3, 5, 10, 20, 30, 50, or more amino acids). Amino acid deletion variants are characterized by the removal of one or more amino acids from the sequence, such as the removal of 1, 2, 3, 5, 10, 20, 30, 50, or more amino acids. Deletions can occur at any position in the protein. Amino acid deletion variants containing deletions at the N-terminus and / or C-terminus of a protein are also called N-terminal and / or C-terminal truncation variants. Amino acid substitution variants are characterized by the removal of at least one residue from the sequence and the insertion of another residue into its position. Preferably, the modification occurs at a non-conserved position between homologous peptides or polypeptides in the amino acid sequence, and / or the amino acid is replaced with another amino acid having similar properties. In some embodiments, amino acid changes in peptide and polypeptide variants are conserved amino acid changes, i.e., substitutions of similar charged or uncharged amino acids. Conserved amino acid changes involve the substitution of one of the related amino acid families in its side chain. Naturally occurring amino acids are generally classified into four families: acidic (aspartic acid, glutamic acid), basic (lysine, arginine, histidine), nonpolar (alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), and uncharged polar (glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine) amino acids. Phenylalanine, tryptophan, and tyrosine are sometimes collectively classified as aromatic amino acids. In some embodiments, conserved amino acid substitutions include substitutions within the following groups:

[0195] - Glycine, alanine;

[0196] -Valine, Isoleucine, Leucine;

[0197] -Aspartic acid, glutamic acid;

[0198] -Asparagine, glutamine;

[0199] -Serine, Threonine;

[0200] -Lysine, arginine; and

[0201] - Phenylalanine, tyrosine.

[0202] In some embodiments, the degree of similarity, such as the identity between a given amino acid sequence and an amino acid sequence that is a variant of said given amino acid sequence, will be at least about 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. In some embodiments, the degree of similarity or identity is given for at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or about 100% of the amino acid region of the entire length of the reference amino acid sequence. For example, if the reference amino acid sequence consists of 200 amino acids, a degree of similarity or identity is given for, for example, at least about 20, at least about 40, at least about 60, at least about 80, at least about 100, at least about 120, at least about 140, at least about 160, at least about 180, or about 200 amino acids (in some embodiments, consecutive amino acids). In some embodiments, a degree of similarity or identity is given for the entire length of the reference amino acid sequence. Alignments used to determine sequence similarity (such as sequence identity) can be performed using tools known in the art, such as using optimal sequence alignment, for example using Align, using standard settings, preferably EMBOSS::needle, Matrix:Blosum62, Gap Open 10.0, or Gap Extend 0.5.

[0203] "Sequence similarity" indicates the percentage of identical or conserved amino acid substitutions. "Sequence identity" between two amino acid sequences indicates the percentage of identical amino acids between the sequences. "Sequence identity" between two nucleic acid sequences indicates the percentage of identical nucleotides between the sequences.

[0204] The terms “% identical” and “% identity” or similar terms are intended to specifically refer to the percentage of identical nucleotides or amino acids in an optimal alignment between sequences to be compared. This percentage is purely statistical, and the differences between the two sequences may, but are not necessarily, randomly distributed across the entire length of the sequences to be compared. The comparison of two sequences is typically performed by comparing the sequences relative to a fragment or “comparison window” after optimal alignment to determine local regions of the respective sequences. The best alignment for comparison can be performed manually, or by means of the local homology algorithm of Smith and Waterman, 1981, AdsApp.Math.2, 482; the local homology algorithm of Neddleman and Wunsch, 1970, J.Mol.Biol.48, 443; the similarity search algorithm of Pearson and Lipman, 1988, Proc.Natl Acad.Sci.USA 88, 2444; or by means of computer programs using the aforementioned algorithms (GAP, BESTFIT, FASTA, BLAST P, BLAST N, and TFASTA, in the Wisconsin Genetics software package, Genetics Computer Group, 575 ScienceDrive, Madison, Wis.). In some implementations, the BLASTN or BLASTP algorithm is used to determine the percentage of identity between two sequences, as can be obtained from the National Center for Biotechnology Information (NCBI) website (e.g., at blast.ncbi.nlm.nih.gov / Blast.cgi?PAGE_TYPE=BlastSearch&BLAST_SPEC=blast2seq&LINK_LOC=align2seq). In some implementations, the algorithm parameters used for the BLASTN algorithm on the NCBI website include: (i) an expected threshold of 10; (ii) a word length of 28; (iii) a maximum number of matches within the query range of 0; (iv) match / mismatch scores of 1 and -2; (v) a gap penalty of linearity; and (vi) the use of a filter with low-complexity regions. In some implementations, the algorithm parameters for the BLASTP algorithm used on the NCBI website include: (i) the expected threshold is set to 10; (ii) the word length is set to 3; (iii) the maximum number of matches in the query range is set to 0; (iv) the matrix is ​​set to BLOSUM62; (v) the gap penalty is set to exist: 11 and extend: 1; and (vi) the conditional composition score matrix is ​​adjusted.

[0205] The identity percentage is obtained by determining the number of identical positions corresponding to the sequences to be compared, dividing that number by the number of positions being compared (e.g., the number of positions in the reference sequence), and multiplying the result by 100.

[0206] In some embodiments, a degree of similarity or identity is given for regions comprising at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or about 100% of the entire length of the reference sequence. For example, if the reference nucleic acid sequence consists of 200 nucleotides, a degree of identity is given for at least about 100, at least about 120, at least about 140, at least about 160, at least about 180, or about 200 nucleotides (in some embodiments, consecutive nucleotides). In some embodiments, a degree of similarity or identity is given for the entire length of the reference sequence.

[0207] According to this disclosure, the homologous amino acid sequences exhibit at least 40%, particularly at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, and, for example, at least 95%, at least 98%, or at least 99% amino acid residue identity.

[0208] The amino acid sequence variants described herein can be readily prepared by a technician, for example, through recombinant DNA manipulation. DNA sequence manipulations for preparing peptides or polypeptides with substitutions, additions, insertions, or deletions are described in detail, for example, in *Molecular Cloning: A Laboratory Manual*, 4th edition, eds. M. Green and J. Sambrook, ColdSpring Harbor Laboratory Press, ColdSpring Harbor 2012. Furthermore, the peptides, polypeptides, and amino acid variants described herein can be readily prepared using known peptide synthesis techniques, such as, for example, solid-phase synthesis and similar methods.

[0209] In some embodiments, fragments or variants of an amino acid sequence (peptide or polypeptide) are “functional fragments” or “functional variants.” The term “functional fragment” or “functional variant” of an amino acid sequence refers to any fragment or variant that exhibits one or more functional properties that are the same as or similar to the functional properties of the amino acid sequence from which it originates; that is, it is functionally equivalent. For an antigen or antigen sequence, a particular function is one or more immunogenic activities exhibited by the amino acid sequence from which the fragment or variant originates. As used herein, the terms “functional fragment” or “functional variant” specifically refer to a variant molecule or sequence that comprises an amino acid sequence that has one or more amino acids altered compared to the amino acid sequence of a parent molecule or sequence, and that still performs one or more functions of the parent molecule or sequence, such as inducing an immune response. In some embodiments, modifications to the amino acid sequence of the parent molecule or sequence do not significantly affect or alter the characteristics of said molecule or sequence. In various embodiments, the function of a functional fragment or functional variant may be reduced but still significantly present; for example, the function of a functional fragment or functional variant may be at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of that of the parent molecule or sequence. However, in other implementations, the function of the functional fragment or functional variant can be enhanced compared to the parent molecule or sequence.

[0210] The amino acid sequence (peptide or polypeptide) “derived from” a specified amino acid sequence (peptide or polypeptide) refers to the source of the first amino acid sequence. In some embodiments, the amino acid sequence derived from a specific amino acid sequence has the same, substantially the same, or homologous amino acid sequence as that specific sequence or a fragment thereof. The amino acid sequence derived from a specific amino acid sequence can be a variant of that specific sequence or a fragment thereof. For example, those skilled in the art will understand that antigens applicable herein can be modified such that their sequences differ from those of their naturally occurring or native sequences from which they are derived, while retaining the desired activity of the native sequence.

[0211] In some implementations, "isolated" means removed (e.g., purified) from a natural state or from an artificial composition (such as a composition derived from a preparation process). For example, nucleic acids, peptides, or polypeptides naturally present in living organisms are not "isolated," but the same nucleic acids, peptides, or polypeptides partially or completely isolated from their natural coexisting substances are "isolated." Isolated nucleic acids, peptides, or polypeptides may exist in a substantially purified form or may exist in non-natural environments (such as, for example, host cells).

[0212] The term "transfection" refers to the introduction of nucleic acids (particularly RNA) into cells. For the purposes of this disclosure, the term "transfection" also includes the introduction of nucleic acids into cells or the uptake of nucleic acids by such cells, which may be present in a subject (e.g., a patient) or in vitro (e.g., outside the patient). Thus, according to this disclosure, cells used for transfecting the nucleic acids described herein may be present in vitro or in vivo, for example, cells that may form organs, tissues, and / or parts of a patient's body. According to this disclosure, transfection can be transient or stable. For some applications of transfection, it is sufficient if the transfected genetic material is expressed only transiently. RNA can be transfected into cells to transiently express the proteins it encodes. Since the nucleic acids introduced during transfection typically do not integrate into the nuclear genome, the foreign nucleic acids will be diluted or degraded by mitosis. Cells that allow free amplification of nucleic acids greatly reduce the dilution rate. If it is desired that the transfected nucleic acids are truly retained in the genome of the cell and its daughter cells, stable transfection must occur. Such stable transfection can be achieved, for example, by using virus-based systems or transposon-based systems for transfection. Typically, nucleic acids encoding antigens are transiently transfected into cells. RNA can be transfected into cells to transiently express the proteins it encodes.

[0213] Cells that can be used for transfection in the methods described herein include, but are not limited to, cells derived from animal cell lines, such as Chinese hamster ovary (CHO), K562, HepG2, HEK293T, RAW, and C2C12 cells. In some embodiments, the cells are CHO, K562, HEK293T, RAW, and C2C12 cells. In some embodiments, the cells are Chinese hamster ovary (CHO) cells.

[0214] This disclosure includes analogs of peptides or polypeptides. According to this disclosure, an analog of a peptide or polypeptide is a modified form of the peptide or polypeptide derived from it and having at least one functional property of the peptide or polypeptide. For example, a pharmacologically active analog of a peptide or polypeptide has at least one pharmacological activity of the peptide or polypeptide from which the analog is derived. Such modifications include any chemical modifications and include single or multiple substitutions, deletions, and / or additions of any molecule associated with the peptide or polypeptide (such as carbohydrates, lipids, and / or peptides or polypeptides). In some embodiments, an “analog” of a peptide or polypeptide includes those modified forms resulting from glycosylation, acetylation, phosphorylation, amidation, palmitoylation, myristylation, isopreneation, esterification, alkylation, derivatization, introduction of protecting / blocking groups, proteolytic cleavage, or binding to an antibody or another cellular ligand. The term “analog” is also extended to all functional chemical equivalents of the peptides and polypeptides.

[0215] The terms “connected,” “integrated,” or “integrated” as used herein are used interchangeably. These terms refer to two or more elements, components, or domains connected together.

[0216] As used in this article, "endogenous" means any substance that originates from or is produced within an organism, cell, tissue, or system.

[0217] As used herein, the term "exogenous" means any substance introduced from or produced outside an organism, cell, tissue, or system.

[0218] As used in this article, the term “expression” is defined as the transcription and / or translation of a specific nucleotide sequence.

[0219] In the context of this disclosure, the term "transcription" refers to the process in which the genetic code in a DNA sequence is transcribed into RNA (especially mRNA). Subsequently, the RNA can be translated into peptides or polypeptides.

[0220] For RNA, the terms “expression” or “translation” refer to a process in the cell’s ribosomes through which the mRNA chain directs the assembly of amino acid sequences to form peptides or polypeptides.

[0221] The prodrugs of the specific compounds described herein are those compounds that undergo chemical transformation under physiological conditions to provide the specific compound when administered to an individual. Furthermore, prodrugs can be converted into the specific compound in an in vitro environment by chemical or biochemical methods. For example, when placed in a transdermal patch reservoir containing suitable enzymes or chemical reagents, a prodrug can be slowly converted into the specific compound. Exemplary prodrugs are esters (using the alcohol or carboxyl group contained in the specific compound) or amides (using the amino or carboxyl group contained in the specific compound) that can be hydrolyzed in vivo. Specifically, any amino group contained in the specific compound and carrying at least one hydrogen atom can be converted into a prodrug form. Common N-prodrug forms include carbamates, Mannich bases, enamines, and enamine ketones.

[0222] In this specification, the structural formula of a compound may represent a certain isomer of the compound. However, it should be understood that the invention includes all structural isomers, such as geometric isomers, optical isomers based on asymmetric carbon, stereoisomers, tautomers, and mixtures of isomers, and is not limited to the description of the formula.

[0223] "Isomers" are compounds with the same molecular formula but different structures ("structural isomers") or different geometric (spatial) orientations of functional groups and / or atoms ("stereoisomers"). "Enantiomers" are a pair of stereoisomers that are non-overlapping mirror images of each other. A "racemic mixture" or "racemate" contains an equal amount of a pair of enantiomers and is indicated by the prefix (±). "Diabeta-isomers" are non-overlapping stereoisomers that are not mirror images of each other. "Tautomers" are structural isomers of the same chemical substance that, even when pure, spontaneously and reversibly interconvert to each other due to the migration of individual atoms or groups; that is, tautomers are in dynamic chemical equilibrium with each other. An example of a tautomer is the ketone-enol-tautomer. "Conformation isomers" are stereoisomers that can interconvert by rotating around a formal single bond, and include stereoisomers that result in different three-dimensional forms of (hetero)cycles, such as chair, half-chair, boat and twist boat shapes of cyclohexane.

[0224] The term "average diameter" refers to the average hydrodynamic diameter of a particle, as measured by dynamic light scattering (DLS), where data analysis is performed using a so-called cumulant algorithm that provides a so-called Z-axis with a length dimension. 平均 The dimensionless polydispersity index (PDI) is used as the result (Koppel, D., J. Chem. Phys. 57, 1972, pp. 4814-4820, ISO 13321). Here, the particle's "mean diameter," "diameter," or "size" is related to Z. 平均 This value is used synonymously.

[0225] In some implementations, the “polydispersity index” can be calculated based on dynamic light scattering measurements via so-called cumulative analysis, as mentioned in the definition of “average diameter.” Under certain prerequisites, it can be considered a measure of the size distribution of an aggregate of nanoparticles.

[0226] The particle's "radius of gyration" around the axis of rotation (abbreviated as R in this article) g R is the radial distance from the axis of rotation such that, if we assume the entire mass of the particle is concentrated at that point, its moment of inertia about a given axis will be the same as its actual mass distribution. Mathematically, R... g This is the root mean square distance between the particle component and its centroid or a given axis. For example, for a particle component located at a fixed distance s from its centroid... i The mass at point m i A macromolecule composed of mass elements (i = 1, 2, 3, ..., n), R g It is the s of all quality elements i 2 The square root of the average mass value can be calculated as follows:

[0227]

[0228] The radius of gyration can be determined experimentally or calculated, for example, by using light scattering. Especially for small scattering vectors... The structure function S is defined as follows:

[0229]

[0230] Where N is the number of components (Guinier's Law).

[0231] The "hydrodynamic radius" of a particle (sometimes called the "Stokes radius" or "Stokes-Einstein radius") is the radius of a hypothetical hard sphere that diffuses at the same rate as the particle. The hydrodynamic radius relates to the particle's mobility and takes into account not only its size but also the effects of the solvent. For example, a smaller, more hydrated charged particle can have a larger hydrodynamic radius than a larger, less hydrated charged particle. This is because a smaller particle drags a greater number of water molecules along with it as it moves through the solution. Since the actual size of a particle in a solvent cannot be directly measured, the hydrodynamic radius can be defined by the Stokes-Einstein equations:

[0232]

[0233] Where k B η is the Boltzmann constant; T is the temperature; η is the viscosity of the solvent; and D is the diffusion coefficient. The diffusion coefficient can be determined experimentally, for example, by using dynamic light scattering (DLS). Therefore, one procedure for determining the hydrodynamic radius of a particle or particle group (such as the hydrodynamic radius of particles contained in a sample or control composition as disclosed herein, or the hydrodynamic radius of a particle peak obtained by field-flow fractionation of such a sample or control composition) is to measure the DLS signal of said particle or particle group (such as the DLS signal of particles contained in a sample or control composition as disclosed herein, or the DLS signal of a particle peak obtained by field-flow fractionation of such a sample or control composition).

[0234] As used in this article, "light scattering" refers to the physical process in which light is forced to deviate from one or more straight paths due to local inhomogeneities in the medium through which it passes.

[0235] The term "UV" refers to ultraviolet light and specifically to the electromagnetic spectrum with wavelengths ranging from 10 nm to 400 nm (shorter than visible light but longer than X-rays).

[0236] As used herein, the terms "multi-angle light scattering" or "MALS" refer to techniques for measuring light scattered from a sample at multiple angles. "Multi-angle" in this respect means that the scattered light can be detected at different discrete angles, for example, by means of a single detector that moves within a range including a selected specific angle or by an array of detectors fixed at a specific angular location. In some embodiments, the light source used in MALS is a laser source (MALLS: Multi-Angle Laser Scattering). Based on the MALS signal of the composition containing particles and by using an appropriate form (e.g., Zimm diagram, Berry diagram, or Debye diagram), the radius of gyration (R) can be determined. g This allows for the determination of the particle size. Preferably, the Zim diagram is a graphical representation using the following equation:

[0237]

[0238] Where c is the mass concentration of particles in the solvent (g / mL); A2 is the second inertial force coefficient (mol·mL / g). 2 P(θ) is a shape factor related to the dependence of scattered light intensity on angle; R θ It is a super Rayleigh ratio (cm) -1 And K* is equal to 4π. 2 η o (dn / dc) 2 λ0 -4 N A -1 The optical constants, where η o λ is the refractive index of the solvent at the incident radiation (vacuum) wavelength, λ0 is the incident radiation (vacuum) wavelength (nm), and N A It is Avogadro's constant (mol) -1 ), and dn / dc is the differential refractive index increment (mL / g) (see, for example, Buchholz et al. (Electrophoresis 22(2001), 4118-4128); BHZimm (J. Chem. Phys. 13(1945), 141; P. Debye (J. Appl. Phys. 15(1944): 338; and W. Burchard (Anal. Chem. 75(2003), 4279-4291). Preferably, the Berry plot is calculated by the following terms:

[0239]

[0240] Among them, c and R θ And K* as defined above. Preferably, the Debye diagram is calculated using the following terms:

[0241]

[0242] Among them, c and R θ And K* are defined as above.

[0243] As used herein, “dynamic light scattering” or “DLS” refers to a technique for determining the size and size distribution profile of particles, particularly regarding their hydrodynamic radius. A monochromatic light source (typically a laser) is emitted through a polarizer and enters the sample. The scattered light then passes through a second polarizer, where it is detected, and the resulting image is projected onto a screen. Particles in the solution are struck by the light, diffracting it in all directions. The diffracted light from the particles can exhibit constructive interference (bright areas) or destructive interference (dark areas). This process is repeated at short time intervals, and the resulting speckle pattern set is analyzed by an autocorrelator, which compares the light intensity at each point over time.

[0244] As used herein, "static light scattering" or "SLS" refers to a technique for determining the size and size distribution of particles, particularly regarding their radius of gyration and / or molar mass. High-intensity monochromatic light (typically a laser) is emitted into a solution containing the particles. One or more detectors are used to measure the scattering intensity at one or more angles. Angle dependence is required to obtain accurate measurements of both the molar mass and size of macromolecules of all radii. Therefore, simultaneous measurements at several angles relative to the direction of incident light (referred to as multi-angle light scattering (MALS) or multi-angle laser scattering (MALLS)) are generally considered the standard implementation of static light scattering.

[0245] Nucleic acid

[0246] The term "nucleic acid" includes deoxyribonucleic acid (DNA), ribonucleic acid (RNA), combinations thereof, and modified forms thereof. The term includes genomic DNA, cDNA, mRNA, recombinant-derived and chemically synthesized molecules. In some embodiments, 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. In some embodiments, the nucleic acid is DNA. Nucleic acids can exist as single-stranded or double-stranded, linear or covalently circularly closed molecules. Nucleic acids can be isolated. According to this disclosure, the term "isolated nucleic acid" refers to nucleic acids that are (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 cleavage and separation by gel electrophoresis, or (iv) synthesized, for example by chemical synthesis.

[0247] The term "nucleoside" (abbreviated as "N" in this document) refers to compounds that can be considered as nucleotides without a phosphate group. A nucleoside is a nucleobase linked to a sugar (e.g., ribose or deoxyribose), while a nucleotide consists of a nucleoside and one or more phosphate groups. Examples of nucleosides include cytidine, uridine, pseudouridine, adenosine, and guanosine.

[0248] 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'-deoxyfuranose portion, unlike the furanose found 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 as A, C, and G, while in DNA, they are represented as dA, dC, and dG.

[0249] The modified purine (A or G) or pyrimidine (C, T or U) base moiety is preferably modified by one or more alkyl groups, more preferably by one or more C groups. 1-4 Alkyl group modification, or even more preferably modification with one or more methyl groups. Specific examples of modified purine or pyrimidine base moieties include N... 7 -alkyl-guanine, N 6 -alkyl-adenine, 5-alkyl-cytosine, 5-alkyl-uracil and N(1)-alkyl-uracil, such as N 7 -C 1-4 Alkyl-guanine, N 6 -C 1-4 Alkyl-adenine, 5-C 1-4 Alkyl-cytosine, 5-C 1-4 Alkyl-uracil and N(1)-C 1-4 Alkyl-uracil, preferably N 7 -Methyl-guanine, N 6 -Methyl-adenine, 5-methyl-cytosine, 5-methyl-uracil and N(1)-methyl-uracil.

[0250] In this document, the term "DNA" refers to a nucleic acid molecule containing deoxyribonucleotide residues. In a preferred embodiment, the DNA contains all or most of the deoxyribonucleotide residues. As used herein, "deoxyribonucleotide" refers to a nucleotide lacking a hydroxyl group at the 2'-position of the β-D-furanose ribosyl group. DNA encompasses, 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 target internal DNA nucleotides or add non-nucleotide substances to the ends(s) of the DNA. 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, such altered DNA is considered an analogue of naturally occurring DNA. A molecule is considered to contain "major deoxyribonucleotide residues" if the content of deoxyribonucleotide residues in the molecule is based on the total number of nucleotide residues in the molecule exceeding 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%). The total number of nucleotide residues in the 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).

[0251] DNA can be recombinant DNA and can be obtained by cloning nucleic acids (especially cDNA). cDNA can be obtained through reverse transcription of RNA.

[0252] 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 ribosyl group. RNA encompasses, 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 alterations may target internal RNA nucleotides or add non-nucleotide substances to the ends(s) of the RNA. It is also contemplated herein that the nucleotides in the RNA may be non-standard nucleotides, such as chemically synthesized nucleotides or deoxynucleotides. For the purposes of this disclosure, these altered / modified nucleotides may be referred to as analogs of naturally occurring nucleotides, and corresponding RNA containing such altered / modified nucleotides (i.e., altered / modified RNA) may be referred to as analogs of naturally occurring RNA. A molecule is considered to contain "major ribonucleotide residues" if the content of ribonucleotide residues in the molecule is based on the total number of nucleotide residues in the molecule exceeding 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%). The total number of nucleotide residues in the 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).

[0253] "RNA" includes mRNA, tRNA, ribosomal RNA (rRNA), small nuclear RNA (snRNA), self-amplified RNA (saRNA), single-stranded RNA (ssRNA), dsRNA, repressive RNA (such as antisense ssRNA, small interfering RNA (siRNA), or microRNA (miRNA)), activating RNA (such as small activating RNA), and immunostimulatory RNA (isRNA). In some implementations, "RNA" refers to mRNA.

[0254] As used herein, the term "in vitro transcription" or "IVT" refers to transcription (i.e., RNA generation) performed in a cell-free manner. That is, IVT does not use living / cultured cells, but rather uses transcription machinery extracted from cells (e.g., cell lysates or isolated components thereof, including RNA polymerases (preferably T7, T3, or SP6 polymerases)).

[0255] 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.

[0256] In some implementations, the RNA is a single-stranded RNA.

[0257] In some implementations, the RNA is mRNA.

[0258] In some implementations, the RNA is generated by in vitro transcription of RNA.

[0259] In some implementations, the RNA includes a 5' cap structure.

[0260] In some implementations, the RNA does not contain modified ribonucleotides.

[0261] 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-methyl-pseudouridine.

[0262] 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 DNA.

[0263] In some implementations, the DNA is present in the form of a vector.

[0264] In some embodiments, the vector comprises DNA encoding an amino acid sequence, which includes the amino acid sequence of a biologically active peptide or polypeptide.

[0265] In some implementations, the vector is a DNA vector.

[0266] 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.

[0267] In some embodiments, the RNA in the mixture is single-stranded RNA.

[0268] In some embodiments, the RNA in the mixture is mRNA.

[0269] In some implementations, the RNA in the mixture is generated by in vitro transcription of RNA.

[0270] In some implementations, the RNA in the mixture contains a 5' cap structure.

[0271] In some embodiments, the RNA in the mixture does not contain modified ribonucleotides.

[0272] 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-methyl-pseudouridine.

[0273] In some implementations, the DNA in the mixture is present in the form of a carrier.

[0274] In some embodiments, the carrier in the mixture comprises DNA encoding an amino acid sequence, which includes the amino acid sequence of a biologically active peptide or polypeptide.

[0275] In some embodiments, the carrier in the mixture is a DNA carrier.

[0276] In some embodiments, the nucleic acids of the present invention (such as RNA and / or DNA, which may contain one or at least two or more nucleic acid constructs) are formulated together with a delivery medium.

[0277] In some embodiments, the nucleic acid (such as RNA and / or DNA) is formulated together with one or more compounds that are complexed with the nucleic acid (such as RNA and / or DNA).

[0278] In some implementations, the nucleic acids (such as RNA and / or DNA) are formulated as particles.

[0279] In some embodiments, the nucleic acids (such as RNA and / or DNA) are formulated as lipoplex particles. Preferably, in these embodiments, the cells are characterized by a large pinocytosis-mediated RNA uptake mechanism.

[0280] In some implementations, the nucleic acids (such as RNA and / or DNA) are formulated as lipid nanoparticles.

[0281] In some embodiments, the nucleic acid (such as RNA and / or DNA) comprises 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.

[0282] In some embodiments, the 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.

[0283] In some embodiments, the different amino acid sequences comprise different amino acid sequences of bioactive peptides or polypeptides.

[0284] In some embodiments, the different biologically active peptides or polypeptides include different antigens.

[0285] In some embodiments, the nucleic acid (such as RNA and / or DNA) comprises 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) encoding amino acid sequences, the amino acid sequences comprising amino acid sequences of different antigens.

[0286] In some embodiments, the RNA described herein is a single-stranded RNA that can be translated into a corresponding protein upon entering a cell (e.g., the cells used in the assays described herein and the cells of the receptor). In addition to a wild-type or codon-optimized sequence encoding an amino acid sequence (which contains an amino acid sequence of a bioactive peptide or polypeptide (e.g., a pharmaceutically 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, multiple (A) tail) optimized for maximum efficiency in terms of RNA stability and translation efficiency. In one embodiment, the RNA contains all of these elements. In one embodiment, β-S-ARCA(D1)(m2 7,2'-0 GppSpG) or m2 7,3’-0 Gppp(m1 2’-0ApG can be used as a specific capping structure at the 5' end of RNA drug substances. As a 5'-UTR sequence, the 5'-UTR sequence of human α-globin mRNA can 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 "split N-terminal enhancer" (AES) mRNA (referred to as F) and mitochondrial-encoded 12S ribosomal RNA (referred to as I) can be used, placed between the coding sequence and the multiple (A) tail to ensure higher maximum protein levels and extended persistence of the mRNA. For sequences that confer RNA stability and enhance total protein expression, these are determined by an in vitro selection process (see WO 2017 / 060314, incorporated herein by reference). Alternatively, the 3'-UTR can be two repeating 3'-UTRs of human β-globin mRNA. Alternatively, a multi(A) tail with a length of 110 nucleotides can be used, consisting of a 30-adenosine residue segment, a subsequent 10-nucleotide (random nucleotide) linker sequence, and an additional 70 adenosine residues. This multi(A) tail sequence is designed to enhance RNA stability and translation efficiency.

[0287] The amino acid sequence comprising the amino acid sequence of a biologically active peptide or polypeptide (e.g., a pharmaceutically active peptide or polypeptide such as an antigen sequence) may include amino acid sequences other than the amino acid sequence of the biologically active peptide or polypeptide. Such additional amino acid sequences may support the function or activity of the biologically active peptide or polypeptide. In some embodiments, such additional amino acid sequences comprise amino acid sequences that enhance antigen processing and / or presentation. Or, additionally, such additional amino acid sequences comprise amino acid sequences that disrupt immune tolerance. Or, additionally, such additional amino acid sequences comprise amino acid sequences that generate bioluminescence. Such additional amino acid sequences can be used to determine the amount of amino acid sequences comprising the amino acid sequence of a biologically active peptide or polypeptide or a fragment thereof in the assays described herein. In particular, such additional amino acid sequences can be used for quantification by LC-MS / MS analysis.

[0288] The nucleic acids (such as RNA and / or DNA) described herein 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, the nucleic acids can be complexed together or individually.

[0289] mRNA

[0290] According to this disclosure, the term "mRNA" refers to "messenger RNA" and relates to a "transcription" that can be generated using a DNA template and 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 mentioned above, in vitro transcription methods are known to those skilled in the art, and various in vitro transcription kits are commercially available.

[0291] mRNA is single-stranded, but it can contain self-complementary sequences that allow the mRNA to partially fold and pair with itself to form a double helix.

[0292] According to this disclosure, "dsRNA" refers to double-stranded RNA, and is RNA having two partially or completely complementary strands.

[0293] In a preferred embodiment of this disclosure, the mRNA relates to an RNA transcript encoding a peptide or polypeptide.

[0294] In some embodiments, the mRNA encoding a peptide or polypeptide preferably has at least 45 nucleotides (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 1,000, at least 1,500, at least 2,000, at least 2,500, at least 3,000, at least...). The length can be 3,500, at least 4,000, at least 4,500, at least 5,000, at least 6,000, at least 7,000, at least 8,000, at least 9,000 nucleotides, preferably up to 15,000 (such as up to 14,000, up to 13,000, up to 12,000, up to 11,000, or up to 10,000 nucleotides).

[0295] As is known in the art, mRNA typically contains a 5' untranslated region (5'-UTR), a peptide / peptide coding region, and a 3' untranslated region (3'-UTR). In some embodiments, the mRNA is prepared by in vitro transcription or chemical synthesis. In some embodiments, the mRNA is prepared by in vitro transcription using a DNA template. In vitro transcription methods are known to those skilled in the art; see, for example, Molecular Cloning: A Laboratory Manual, 4th Edition, eds. MR. Green and J. Sambrook, ColdSpring Harbor Laboratory Press, ColdSpring Harbor 2012. Furthermore, a variety of in vitro transcription kits are commercially available, such as those from Thermo Fisher Scientific (e.g., TranscriptAid). TM T7 reagent kit T7 reagent kit ), New England BioLabs Inc. (such as HiScribe) TM T7 reagent kit, HiScribe TM T7 ARCA mRNA kit), Promega (such as RiboMAX) TM , systems), Jena Bioscience (such as SP6 or T7 transcription kits), and Epicentre (such as AmpliScribe) TM To provide modified mRNA, the corresponding modified 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 modification can be achieved in the mRNA after transcription and / or the modification can be added to the mRNA.

[0296] In some embodiments, the mRNA is in vitro transcribed mRNA (IVT-RNA) and can be obtained by in vitro transcription from a suitable DNA template. The promoter used to control transcription can be any promoter of any RNA polymerase. Specific examples of RNA polymerases are T7, T3, and SP6 RNA polymerases. Preferably, in vitro transcription is controlled by a T7 or SP6 promoter. A 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.

[0297] In some embodiments of this disclosure, mRNA is referred to as "replicon mRNA" or simply "replicon," particularly "self-replicating mRNA" or "self-amplifying mRNA." In some embodiments, the replicon or self-replicating mRNA is derived from or contains elements derived from ssRNA viruses (particularly positive-sense ssRNA viruses such as alphaviruses). Alphaviruses are typical representatives of positive-sense RNA viruses. Alphaviruses replicate in the cytoplasm of infected cells (for a review of the alphavirus life cycle, see José et al., Future Microbiol., 2009, Vol. 4, pp. 837-856). The total genome length of many alphaviruses typically ranges from 11,000 to 12,000 nucleotides, and the genomic RNA typically has a 5' cap and a 3' multi-(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 originating near the 5' end of the genome, while alphavirus structural proteins are encoded by a second ORF located downstream of the first ORF and extending near the 3' end of the genome. Generally, the first ORF is larger 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 genomic RNA, while the genetic information encoding structural proteins is translated from subgenomic transcripts, which are RNA molecules similar to eukaryotic messenger RNA (mRNA; Gould et al., 2010, Antiviral Res., Vol. 87, pp. 111-124). Post-infection, in the early stages of the viral life cycle, the (+) strand genomic RNA directly acts as messenger RNA for translating the open reading frame encoding the non-structural polyprotein (nsP1234). Alphavirus-derived vectors have been proposed for delivering foreign genetic information to target cells or organisms. In a simplified approach, the open reading frame encoding an alphavirus structural protein is replaced by an open reading frame encoding the target protein. Alphavirus-based trans-replication systems rely on alphavirus nucleotide sequence elements on two separate nucleic acid molecules: one molecule encodes a viral replicase, and the other is capable of being trans-replicated by that replicase (hence the name trans-replication system). Trans-replication requires the 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 to allow for recognition by the alphavirus replicase and RNA synthesis.

[0298] In some embodiments of this disclosure, the mRNA contains one or more modifications, for example, to increase its stability and / or increase translation efficiency and / or reduce immunogenicity and / or reduce cytotoxicity. For example, to increase mRNA expression, it may be modified within the coding region (i.e., the sequence encoding the expressed peptide or polypeptide), preferably without altering the sequence of the expressed peptide or polypeptide. Such modifications are described, for example, in WO 2007 / 036366 and PCT / EP2019 / 056502, and include the following: 5'-cap structure; extension or truncation of naturally occurring multiple (A) tails; alteration of the 5'- and / or 3'-untranslated regions (UTRs), such as introducing a UTR unrelated to the coding region of the RNA; replacement of one or more naturally occurring nucleotides with synthetic nucleotides; and codon optimization (e.g., alteration, preferably increasing, the GC content of the RNA).

[0299] In some embodiments, the mRNA comprises a 5'-cap structure. In some embodiments, the mRNA does not have an uncapped 5'-triphosphate. In some embodiments, the mRNA may comprise a conventional 5'-cap and / or a 5'-cap analogue. The term "conventional 5'-cap" refers to a cap structure present at the 5' end of the mRNA molecule and is 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 remainder of the mRNA via a 5'-to-5' triphosphate ester bond). Guanosine can be present at position N. 7 The site is methylated (producing a cap structure m) 7 (Gppp). The term "5'-cap analogue" includes those based on the conventional 5'-cap but in m 7 A 5'-cap modified at the 2'- or 3'-position of the guanosine structure prevents the 5'-cap analog from integrating in reverse orientation (such 5'-cap analogs are also called anti-reverse-cap analogs (ARCA)). Particularly preferred 5'-cap analogs are those with one or more substitutions at the bridging and non-bridging oxygen sites in the phosphate bridge, such as 5'-cap analogs modified with thiophosphate at the β-phosphate site (e.g., m2). 7,2'0 G(5')ppSp(5')G (referred to as β(beta)-S-ARCA or β-S-ARCA)), as described in PCT / EP2019 / 056502. The provision of mRNA having a 5'-cap structure as described herein can be achieved by in vitro transcription of a DNA template in the presence of a suitable 5'-cap compound, wherein the 5'-cap structure is co-transcribed into the resulting mRNA chain, or the mRNA can be generated, for example, by in vitro transcription, and the 5'-cap structure can be ligated to the mRNA post-transcriptionally using a capping enzyme (e.g., a capping enzyme of vaccinia virus).

[0300] In some implementations, the mRNA comprises m2-selected molecules. 7,2'0 G(5')ppSp(5')G (especially its D1 diastereomer), m2 7,3'0 G(5')ppp(5')G and m2 7,3'-0 Gppp(m1 2'-0 The 5'-cap structure of the group composed of ApG.

[0301] In some embodiments, the mRNA comprises cap 0, cap 1, or cap 2, preferably cap 1 or cap 2. According to this disclosure, the term "cap 0" refers to the structure "m..." 7 "GpppN", where N is any nucleoside carrying an OH moiety at position 2'. According to this disclosure, the term "cap 1" refers to the structure "m 7 "GpppNm", where Nm is any nucleotide carrying the OCH3 moiety at position 2'. According to this disclosure, the term "cap 2" refers to the structure "m". 7 "GpppNmNm", where each Nm is independently any nucleoside carrying the OCH3 moiety at position 2'.

[0302] The D1 diastereomer of β(beta)-S-ARCA (β-S-ARCA) has the following structure:

[0303]

[0304] "β-S-ARCA D1 diastereomer" or "β-S-ARCA(D1)" is a diastereomer of β-S-ARCA that elutes first on the HPLC column compared to the D2 diastereomer of β-S-ARCA (β-S-ARCA(D2)) and therefore exhibits a shorter retention time. The HPLC is preferably analytical HPLC. In some embodiments, a Supelcosil LC-18-T RP column (preferably in the form of 5 μm, 4.6 x 250 mm) is used for separation, thereby allowing a flow rate of 1.3 ml / min. In some embodiments, a methanol gradient in ammonium acetate is used, for example, a linear gradient of 0-25% methanol in 0.05 M ammonium acetate at pH = 5.9 over 15 min. UV detection (VWD) can be performed at 260 nm, and fluorescence detection (FLD) can be performed, wherein excitation is performed at 280 nm and detection is performed at 337 nm.

[0305] 5'-Cap Analog m2 7,3'-0 Gppp(m1 2'-0 ApG (also known as m2) 7,3'0 G(5')ppp(5')m2'-0 ApG (which is the building block of cap 1) has the following structure:

[0306]

[0307] An exemplary capped 0 mRNA containing β-S-ARCA and mRNA has the following structure:

[0308]

[0309] An example includes m2 7,3'0 G(5')ppp(5')G and the cap of mRNA 0 mRNA have the following structure:

[0310]

[0311] An example includes m2 7,3'-0 Gppp(m1 2'-0 The cap of ApG and mRNA has the following structure:

[0312]

[0313] As used herein, the terms "multiple A tail" or "multiple A sequence" refer to a continuous or discontinuous sequence of adenosine residues typically located at the 3' end of an mRNA molecule. Multiple A tails or multiple A sequences are known to those skilled in the art and may follow the 3'-UTR in the mRNAs described herein. A continuous multiple A tail is characterized by a continuous sequence of adenosine residues. Continuous multiple A tails are common in nature. The mRNAs disclosed herein may have a free 3' multiple A tail that is ligated to the mRNA post-transcriptionally by a template-independent RNA polymerase, or a multiple A tail encoded by DNA and transcribed by a template-dependent RNA polymerase.

[0314] It has been shown that the poly-A tail of approximately 120 A nucleotides has a beneficial effect on mRNA levels in transfected eukaryotic cells as well as on the levels of proteins translated from open reading frames located upstream (5') of the poly-A tail (Holtkamp et al., 2006, Blood, Vol. 108, pp. 4009-4017).

[0315] The multi-A tail can be of any length. In some embodiments, the multi-A tail comprises at least 20, at least 30, at least 40, at least 80, or at least 100, and up to 500, up to 400, up to 300, up to 200, or up to 150 A nucleotides (and particularly about 120 A nucleotides), substantially composed of or consisting of. In this context, "substantially composed of" means that the majority of the nucleotides in the multi-A tail, typically 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 number of nucleotides in the multi-A tail, are A nucleotides, but the remaining nucleotides are allowed to be nucleotides other than 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 multi-A tail (i.e., 100% of the number of nucleotides in the multi-A tail) are A nucleotides. The term "A nucleotide" or "A" refers to adenosine monophosphate.

[0316] In some implementations, during RNA transcription (e.g., in the preparation of in vitro transcribed RNA), a multi-A tail is attached to a DNA template containing repeating dT nucleotides (deoxythymidines) in a strand complementary to the coding strand. The DNA sequence encoding the multi-A tail (coding strand) is called a multi(A) box.

[0317] In some embodiments, the multi(A) box present in the coding strand of DNA consists primarily of dA nucleotides but is interrupted by random sequences of four nucleotides (dA, dC, dG, and dT). The length of such random sequences can be 5 to 50, 10 to 30, or 10 to 20 nucleotides. Such boxes are disclosed in WO 2016 / 005324 A1, which is hereby incorporated by reference. Any multi(A) box disclosed in WO2016 / 005324 A1 can be used in this disclosure. Covering such multi(A) boxes, which consist primarily of dA nucleotides but are interrupted by random sequences having a uniform distribution of the four nucleotides (dA, dC, dG, and dT) and a length of, for example, 5 to 50 nucleotides, demonstrates at the DNA level the continuous proliferation of plasmid DNA in *E. coli* and remains associated with beneficial properties supporting RNA stability and translation efficiency at the RNA level. Therefore, in some embodiments, the multi-A tail contained in the mRNA molecule described herein is primarily composed of A nucleotides, but is interrupted by random sequences of four nucleotides (A, C, G, U). Such random sequences can be 5 to 50, 10 to 30, or 10 to 20 nucleotides in length.

[0318] In some implementations, the multi-A tail is not side-attached to any nucleotide other than A at its 3' end; that is, the multi-A tail is not covered or followed by any nucleotide other than A at its 3' end.

[0319] In some embodiments, the multi-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 multi-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 multi-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 multi-A tail comprises at least 100 nucleotides. In some embodiments, the multi-A tail comprises about 150 nucleotides. In some embodiments, the multi-A tail comprises about 120 nucleotides.

[0320] 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 to a corresponding region in an RNA molecule (such as an mRNA molecule). An untranslated region (UTR) may be present at the 5' (upstream) end of an open reading frame (5'-UTR) and / or the 3' (downstream) end of an open reading frame (3'-UTR). If present, the 5'-UTR is located upstream of the 5' end of the start codon in a protein-coding region. The 5'-UTR is located downstream of the 5'-cap (if present), for example, directly adjacent to the 5'-cap. If present, the 3'-UTR is located downstream of the 3' end of the stop codon in a protein-coding region, but the term "3'-UTR" typically does not include a multi-A sequence. Therefore, the 3'-UTR is located upstream of the multi-A sequence (if present), for example, directly adjacent to the multi-A sequence. Incorporating a 3'-UTR into the 3'-untranslated region of an RNA (preferably mRNA) molecule can lead to enhanced translation efficiency. A synergistic effect can be achieved by incorporating two or more such 3'-UTRs (preferably arranged head-to-tail; see, for example, Holtkamp et al., Blood 108, 4009-4017 (2006)). The 3'-UTRs can be autologous or heterologous with the RNA (e.g., mRNA) in which they are incorporated. In some embodiments, the 3'-UTR is derived from a globin gene or mRNA, such as α2-globin, α1-globin, or β-globin (e.g., β-globin, e.g., human β-globin). For example, RNA (e.g., mRNA) can be modified by replacing or inserting one or more (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, e.g., human β-globin).

[0321] mRNA may have modified ribonucleotides to increase its stability and / or reduce its immunogenicity and / or cytotoxicity. For example, in some embodiments, uridine in the mRNA described herein is replaced (partially or completely, preferably completely) with a modified nucleoside. In some embodiments, the modified nucleoside is a modified uridine.

[0322] In some embodiments, the uridine that replaces the modified uridine is selected from the group consisting of pseudouridine (ψ), N1-methyl-pseudouridine (m1ψ), 5-methyl-uridine (m5U) and combinations thereof.

[0323] In some embodiments, the nucleoside that replaces (partially or completely, preferably completely) uridine in the mRNA can be one or more of the following: 3-methyluridine (m3U), 5-methoxyuridine (mo5U), 5-aza-uridine, 6-aza-uridine, 2-thio-5-aza-uridine, 2-thio-uridine (s2U), 4-thio-uridine (s4U), 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxyuridine (ho5U), 5-aminoallyl-uridine, 5-halo-uridine (e.g., 5-iodo-uridine or 5-bromo-uridine), uridine 5-oxyacetic acid (cmo5U), methyl uridine 5-oxyacetate (mcmo5U), 5-carboxymethyl uridine... 5-Carboxymethyl-uridine (cm5U), 1-Carboxymethyl-pseudouridine, 5-Carboxyhydroxymethyl-uridine (chm5U), 5-Carboxyhydroxymethyl-uridine methyl ester (mchm5U), 5-Methoxycarbonylmethyl-uridine (mcm5U), 5-Methoxycarbonylmethyl-2-thio-uridine (mcm5s2U), 5-Aminomethyl-2-thio-uridine (nm5s2U), 5-Methylaminomethyl-uridine (mnm5U), 1-Ethyl-pseudouridine, 5-Methylaminomethyl-2-thio-uridine (mnm5s2U), 5-Methylaminomethyl-2-seleno-uridine (mnm5se2U), 5-Carbamoylmethyl-uridine (ncm5U), 5-Carboxymethylaminomethyl-uridine (c mnm5U), 5-carboxymethylaminomethyl-2-thio-uridine (cmnm5s2U), 5-propynyl-uridine, 1-propynyl-pseudouridine, 5-tauronic acid methyl-uridine (τm5U), 1-tauronic acid methyl-2-thio-uridine (τm5s2U), 1-tauronic acid methyl-4-thio-pseudouridine), 5-methyl-2-thio-uridine (m5s2U), 1-methyl-4-thio-pseudouridine (m1s4ψ), 4-thio-1-methyl-pseudouridine, 3-methyl-pseudouridine (m3ψ), 2-thio-1-methyl-pseudouridine, 1-methyl-1-deazo-pseudouridine, 2-thio-1-methyl-1-deazo-pseudouridine, dihydrouridine Glycoside (D), dihydro-dihydrouridine, 5,6-dihydrouridine, 5-methyl-dihydrouridine (m5D), 2-thio-dihydrouridine, 2-thio-dihydro-dihydrouridine, 2-methoxy-uridine, 2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine, 4-methoxy-2-thio-pseudouridine, N1-methyl-pseudouridine, 3-(3-amino-3-carboxypropyl)uridine (acp3U), 1-methyl-3-(3-amino-3-carboxypropyl)pseudouridine (acp3ψ), 5-(isopentenylaminomethyl)uridine (inm5U), 5-(isopentenylaminomethyl)-2-thio-uridine (inm5s2U), α-thio-uridine, 2′-O-methyl-uridine (Um), 5,2′-O-dimethyluridine (m5Um), 2′-O-methyl-pseudouridine (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.

[0324] RNA (preferably mRNA) modified with pseudouridine (partially or completely, preferably completely replacing uridine) is referred to herein as "Ψ-modified", and the term "m1Ψ-modified" means that the RNA (preferably mRNA) contains N(1)-methylpseudouridine (partially or completely, preferably completely replacing uridine). Furthermore, the term "m5U-modified" means that the RNA (preferably mRNA) contains 5-methyluridine (partially or completely, preferably completely replacing uridine). Such Ψ- or m1Ψ- or m5U-modified RNA generally exhibits reduced immunogenicity compared to its unmodified form and is therefore preferred in applications where the induction of an immune response should be avoided or minimized. In some embodiments, the RNA (preferably mRNA) contains N(1)-methylpseudouridine that completely replaces uridine.

[0325] The codons of the mRNA used in this disclosure may be further optimized, for example, by increasing the GC content of the RNA and / or replacing rare codons in the cells (or subject) where the target peptide or polypeptide is expressed with common codons that are synonymous with those in the cells (or subject). 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 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 increased G / C content compared to the corresponding sequence regions of the wild-type coding sequence. In some embodiments, codon optimization and / or increased G / C content preferably do not alter the sequence of the encoded amino acid sequence.

[0326] The term "codon optimization" refers to altering the codons in the coding region of a nucleic acid molecule to reflect the host organism's usual codon selection, preferably without changing the amino acid sequence encoded by the nucleic acid molecule. Within the context of this disclosure, codon optimization can be performed on coding regions to achieve optimal expression in subjects treated with the mRNA described herein. Codon optimization is based on the finding that translation efficiency is also determined by the varying frequencies of tRNA occurrence in cells. Therefore, the mRNA sequence can be modified so that frequently occurring tRNAs can be used to insert their codons into "rare codon" positions.

[0327] In some embodiments, the guanosine / cytosine (G / C) content of the coding region of the mRNA described herein is increased compared to the G / C content of the corresponding coding sequence of wild-type RNA, wherein the amino acid sequence encoded by the mRNA is preferably unmodified compared to the amino acid sequence encoded by the wild-type RNA. This modification of the mRNA sequence is based on the fact that the sequence of any RNA region to be translated is important for the efficient translation of that mRNA. Sequences with increased G (guanosine) / C (cytosine) content are more stable than sequences with increased A (adenosine) / U (uracil) content. Given the fact that multiple codons encode one and the same amino acid (so-called degeneracy of the genetic code), the codons most favorable for stability can be determined (so-called alternative codon selection). Depending on the amino acid encoded by the mRNA, there are several possibilities for modification of the mRNA sequence compared to its wild-type sequence. In particular, these codons can be modified by replacing codons containing A and / or U nucleotides 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.

[0328] In various implementations, the G / C content of the coding region of the mRNA 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 coding region of wild-type RNA.

[0329] The above modifications, namely the incorporation of a 5'-cap structure, the incorporation of a multi-A sequence, the exposure of a multi-A sequence, the alteration of the 5'- and / or 3'-UTR (such as incorporating one or more 3'-UTRs), the replacement of one or more naturally occurring nucleotides with synthetic nucleotides (e.g., replacing cytidine with 5-methylcytidine, and / or replacing uridine with pseudouridine (Ψ) or N(1)-methylpseudouridine (m1Ψ) or 5-methyluridine (m5U), and codon optimization, have a synergistic effect on improving the stability and translation efficiency of RNA (preferably mRNA). Therefore, in some embodiments, the mRNA used in this disclosure contains at least two, at least three, at least four, or all five of the above modifications, namely (i) incorporation of a 5'-cap structure, (ii) incorporation of a multi-A sequence to expose the multi-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.

[0330] Some aspects of this disclosure relate to the targeted delivery of the mRNAs disclosed herein to certain 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 part of the immune system, comprising a network of lymphatic vessels carrying lymph. The lymphatic system consists of lymphatic organs, a network of lymphatic vessels, and circulating lymph. 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 (which include lymph nodes and the spleen) maintain mature naïve lymphocytes and initiate adaptive immune responses.

[0331] Lipid-based mRNA delivery systems have an inherent preference for the liver. Hepatic accumulation is caused by discontinuities in the hepatic vascular system or lipid metabolism (liposomes and lipid or cholesterol conjugates). In some embodiments, the target organ is the liver and the target tissue is liver tissue. Delivery to such a target tissue is preferred, particularly if the presence of mRNA or encoded peptides or polypeptides in that organ or tissue is desired and / or if the expression of large amounts of encoded peptides or polypeptides is desired and / or if systemic presence of encoded peptides or polypeptides (especially significant amounts) is desired or required.

[0332] 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 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. In some embodiments, the target cells are T cells. In some embodiments, the target cell is a B cell. In some embodiments, the target cell is an NK cell. In some embodiments, the target cell is a monocyte. Therefore, the RNA particles described herein can be used to deliver mRNA to such target cells.

[0333] Pharmaceutically active peptides or polypeptides

[0334] "Encoding" refers to the inherent properties of a specific nucleotide sequence in a polynucleotide (such as a gene, cDNA, or mRNA) to serve as a template for other polymers and macromolecules in synthetic biology processes, which have defined nucleotide sequences (i.e., rRNA, tRNA, and mRNA) or defined amino acid sequences, and the resulting biological properties. Therefore, if the transcription and translation of the mRNA corresponding to a gene produces a protein in a cell or other biological system, then that gene encodes a protein. Both the coding strand (whose nucleotide sequence is identical to the mRNA sequence and is usually provided in a sequence listing) and the non-coding strand (which serves as a template for gene or cDNA transcription) can be referred to as encoding the protein or other product of that gene or cDNA.

[0335] In some embodiments, the nucleic acids (such as mRNA) used in this disclosure comprise nucleic acid sequences encoding one or more functional sequences, which may be peptides or polypeptides, preferably pharmaceutically active peptides or polypeptides.

[0336] In a preferred embodiment, the nucleic acid (such as mRNA) used in this disclosure comprises a nucleic acid sequence encoding a peptide or polypeptide (preferably a pharmaceutically active peptide or polypeptide) and is capable of expressing said peptide or polypeptide, particularly if transferred to cells or a subject. Therefore, in some embodiments, the nucleic acid used in this disclosure contains a coding region (open reading frame (ORF)) encoding a peptide or polypeptide (e.g., encoding a pharmaceutically active peptide or polypeptide). In this respect, an "open reading frame" or "ORF" is a continuous codon segment that begins with a start codon and ends with a stop codon. Such nucleic acids encoding pharmaceutically active peptides or polypeptides are also referred to herein as "pharmaceutically active nucleic acids." In particular, such mRNAs encoding pharmaceutically active peptides or polypeptides are also referred to herein as "pharmaceutically active mRNAs."

[0337] According to this disclosure, the term "pharmaceutically active peptide or polypeptide" refers to a peptide or polypeptide that can be used to treat an individual, wherein the expression of the peptide or polypeptide would be beneficial, for example, in improving the symptoms of a disease. Preferably, the pharmaceutically active peptide or polypeptide has curative or palliative properties and can be administered to improve, alleviate, reduce, reverse one or more symptoms of a disease, delay its onset, or reduce its severity. In some embodiments, when administered to an individual in a therapeutically effective amount, the pharmaceutically active peptide or polypeptide has a positive or beneficial effect on the individual's condition or disease state. Pharmacologically active peptides or polypeptides may have preventative properties and can be used to delay the onset of a disease or reduce the severity of such a disease. The term "pharmaceutically active peptide or polypeptide" includes the whole peptide or polypeptide and may also refer to its pharmaceutically active fragment. It may also include pharmaceutically active variants and / or analogs of the peptide or polypeptide.

[0338] Specific examples of pharmaceutically active peptides and polypeptides include, but are not limited to, cytokines, hormones, adhesion molecules, immunoglobulins, immunologically active compounds, growth factors, protease inhibitors, enzymes, receptors, apoptosis regulators, transcription factors, tumor suppressor proteins, structural proteins, reprogramming factors, genome-engineered proteins, and blood proteins.

[0339] The term "cytokine" refers to proteins with a molecular weight of approximately 5 to 60 kDa that participate in cellular signaling (e.g., paracrine, endocrine, and / or autocrine signaling). Specifically, when released, cytokines influence the behavior of cells surrounding their release site. Examples of cytokines include lymphokines, interleukins, chemokines, interferons, and tumor necrosis factor (TNF). According to this disclosure, cytokines do not include hormones or growth factors. Cytokines differ from hormones in that: (i) they typically act at much more variable concentrations than hormones; and (ii) they are generally produced by a wide range of cells (almost all nucleated cells produce cytokines). Interferons are generally characterized by antiviral, antiproliferative, and immunomodulatory activities. Interferons are proteins that prevent viral replication within cells by altering and regulating intracellular gene transcription through binding to interferon receptors on the surface of regulated cells. Interferons can be classified into two types. IFN-γ is the only type II interferon; all other interferons are type I interferons. Specific examples of cytokines include erythropoietin (EPO), colony-stimulating factor (CSF), granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), tumor necrosis factor (TNF), bone morphogenetic protein (BMP), interferon α (IFNα), interferon β (IFNβ), interferon γ (IFFγ), interleukin 2 (IL-2), interleukin 4 (IL-4), interleukin 10 (IL-10), interleukin 11 (IL-11), interleukin 12 (IL-12), interleukin 15 (IL-15), and interleukin 21 (IL-21), as well as their variants and derivatives.

[0340] In some embodiments, the pharmaceutically active peptide or polypeptide comprises a substitute protein. In these embodiments, this disclosure provides a method for treating a subject suffering from a disorder requiring protein replacement (e.g., a protein deficiency disorder), comprising administering to said subject a nucleic acid encoding a substitute protein as described herein. The term “protein replacement” refers to the introduction of a protein (including its functional variants) into a subject having a deficiency of such a protein. The term also refers to the introduction of a protein into a subject who otherwise needs or benefits from the provision of protein, such as a subject suffering from protein insufficiency. The term “disorder characterized by protein deficiency” refers to any disorder manifesting as a pathological condition due to the absence or inadequacy of a protein. This term encompasses protein folding disorders, i.e., conformational disorders, that produce biologically inactive protein products. Protein insufficiency can be involved in infectious diseases, immunosuppression, organ failure, glandular problems, radiation sickness, nutritional deficiencies, poisoning, or other environmental or external injuries.

[0341] The term "hormone" refers to a class of signaling molecules produced by glands, where signaling typically involves the following steps: (i) synthesis of the hormone in a specific tissue; (ii) storage and secretion; (iii) delivery of the hormone to its target; (iv) receptor binding of the hormone; (v) signal transmission and amplification; and (vi) breakdown of the hormone. Hormones differ from cytokines in that: (1) hormones typically act at relatively fixed concentrations; and (2) they are typically produced by specific types of cells. In some embodiments, "hormone" is a peptide or polypeptide hormone, such as insulin, vasopressin, prolactin, adrenocorticotropic hormone (ACTH), thyroid hormone, growth hormone (such as human growth hormone or bovine growth hormone), oxytocin, atrial natriuretic peptide (ANP), glucagon, somatostatin, cholecystokinin, gastrin, and leptin.

[0342] The term "adhesion molecule" refers to proteins located on the cell surface and involved in cell binding to other cells or to the extracellular matrix (ECM). Adhesion molecules are typically transmembrane receptors and can be classified as calcium-independent (e.g., integrins, immunoglobulin superfamily, lymphocyte homing receptors) and calcium-dependent (cadherins and selectins). Specific examples of adhesion molecules include integrins, lymphocyte homing receptors, selectins (e.g., P-selectin), and addressins.

[0343] Integrins also participate in signal transduction. Specifically, upon ligand binding, integrins regulate cellular signaling pathways, such as those of transmembrane protein kinases like receptor tyrosine kinases (RTKs). Such regulation can lead to cell growth, division, survival, differentiation, or apoptosis. Specific examples of integrins include: α1β1, α2β1, α3β1, α4β1, α5β1, α6β1, α7β1, α... L β2, α M β2, α IIb β3, α V β1, α V β3, α V β5, α V β6, α V β8 and α6β4.

[0344] The term "immunoglobulin" or "immunoglobulin superfamily" refers to molecules involved in cell recognition, binding, and / or adhesion. Molecules belonging to this superfamily share the following characteristics: they all contain regions known as immunoglobulin domains or folds. Members of the immunoglobulin superfamily include antibodies (e.g., IgG), T-cell receptors (TCRs), major histocompatibility complex (MHC) molecules, co-receptors (e.g., CD4, CD8, CD19), antigen receptor co-molecules (e.g., CD-3γ, CD3-δ, CD-3ε, CD79a, CD79b), co-stimulatory or inhibitory molecules (e.g., CD28, CD80, CD86), and others.

[0345] The term "immunologicly active compound" refers to any compound that alters the immune response, such as by inducing and / or inhibiting the maturation of immune cells, inducing and / or inhibiting cytokine biosynthesis, and / or by stimulating B cells to produce antibodies. Immunologicly active compounds possess potent immunostimulatory activity, including, but not limited to, antiviral and antitumor activity, and can also downregulate other aspects of the immune response, such as shifting the immune response from a TH2 immune response, which is useful for treating a wide range of TH2-mediated diseases. Immunologicly active compounds can be used as vaccine adjuvants. Specific examples of immunologically active compounds include interleukins, colony-stimulating factor (CSF), granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), erythropoietin, tumor necrosis factor (TNF), interferon, integrins, addressins, selectins, homing receptors, and antigens, particularly tumor-associated antigens, pathogen-associated antigens (such as bacterial, parasitic, or viral antigens), allergens, and autoantigens. Immunologicly active compounds can be vaccine antigens, i.e., antigens that induce an immune response when administered to a subject.

[0346] According to this disclosure, "antigen" encompasses any substance that will elicit an immune response and / or any substance targeted by an immune response or immune mechanism (such as a cellular response and / or humoral response). This also includes cases where the antigen is processed into an antigenic peptide and the immune response or immune mechanism targets one or more antigenic peptides, particularly if presented in the context of MHC molecules. In particular, "antigen" refers to any substance, such as a peptide or polypeptide, that specifically reacts with an antibody or T lymphocyte (T cell). The term "antigen" can include a molecule containing at least one epitope (such as a T cell epitope). In some embodiments, an antigen is a molecule that optionally induces an immune response after processing, which can be specific to the antigen (including cells expressing the antigen). In some embodiments, the antigen is a disease-associated antigen, such as a tumor antigen, viral antigen, or bacterial antigen, or an epitope derived from such an antigen.

[0347] The term "autoantigen" or "autologous antigen" refers to an antigen that originates within a subject's body (i.e., an autoantigen, also known as an "autologous antigen") and elicits an abnormally strong immune response against that normal part of the body. Such a strong immune response against an autoantigen can be the cause of "autoimmune diseases."

[0348] According to this disclosure, any suitable antigen can be used as a candidate for an immune response, which can be both humoral and cellular immune responses. In the context of some embodiments of this disclosure, the antigen is presented by cells (such as antigen-presenting cells) in an MHC molecular background, resulting in an immune response against the antigen. The antigen can be a product corresponding to or derived from a naturally occurring antigen. Such naturally occurring antigens can include or may be derived from allergens, viruses, bacteria, fungi, parasites, and other infectious agents and pathogens, or the antigen may be a tumor antigen. According to this disclosure, the antigen can correspond to a naturally occurring product, such as a viral protein or a portion thereof.

[0349] The term "disease-associated antigen," used in its broadest sense, refers to any antigen associated with a disease. A disease-associated antigen is a molecule containing an epitope that stimulates the host's immune system to produce a cellular antigen-specific immune response and / or humoral antibody response against the disease. Disease-associated antigens include pathogen-associated antigens, i.e., antigens associated with microbial infections, typically microbial antigens (such as bacterial or viral antigens), or antigens associated with cancer (typically tumors), such as tumor antigens.

[0350] In some embodiments, the antigen is a tumor antigen, i.e., a part of tumor cells, particularly an antigen that is primarily present intracellularly or serves as a surface antigen of tumor cells. In another embodiment, the antigen is a pathogen-associated antigen, i.e., an antigen derived from a pathogen, such as an antigen derived from a virus, bacteria, single-celled organism, or parasite, such as viral antigens like viral ribonucleoproteins or capsid proteins. In some embodiments, the antigen is to be presented by MHC molecules that lead to the regulation, particularly activation, of cells of the immune system (such as CD4+ and CD8+ lymphocytes), especially by modulating the activity of T-cell receptors.

[0351] The term "tumor antigen" refers to components of cancer cells that can originate from the cytoplasm, cell surface, or cell nucleus. Specifically, it refers to antigens produced intracellularly or as surface antigens on tumor cells. Examples of tumor antigens include carcinoembryonic antigen, α1-fetoprotein, isoferrin and fetal sulfoglycoprotein, α2-H-ferritin and γ-fetoprotein, and various viral tumor antigens. According to some embodiments of this disclosure, tumor antigens include any antigens that characterize a tumor or cancer, and tumor or cancer cells, in terms of type and / or expression level.

[0352] The term "viral antigen" refers to any viral component that possesses antigenic properties (i.e., the ability to elicit an immune response in an individual). Viral antigens can be viral ribonucleoproteins or envelope proteins.

[0353] The term "bacterial antigen" refers to any bacterial component that possesses antigenic properties (i.e., the ability to elicit an immune response in an individual). Bacterial antigens can originate from the cell wall or cytoplasmic membrane of bacteria.

[0354] The term "epitope" refers to an antigenic determinant in a molecule (such as an antigen), that is, a portion or fragment of a molecule that is recognized by the immune system (e.g., by antibodies, T cells, or B cells, particularly when presented in the context of MHC molecules). An epitope of a protein may comprise a continuous or discontinuous portion of the protein, and its length may be, for example, about 5 to about 100, about 5 to about 50, about 8 to about 30, or about 10 to about 25 amino acids. For example, the length of an epitope may preferably be 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids. In some embodiments, the epitope in the context of this disclosure is a T-cell epitope.

[0355] Terms such as “epitope,” “antigen fragment,” “immunogenic peptide,” and “antigen peptide” are used interchangeably herein and may, for example, refer to an incomplete representation of an antigen that, for example, is capable of evoking an immune response against the antigen or against cells that express, contain, and present the antigen. In some embodiments, the term refers to an immunogenic portion of an antigen. In some embodiments, it is a portion of the antigen that is recognized (i.e., specifically bound) by T cell receptors, particularly if presented in the context of MHC molecules. Certain preferred immunogenic portions bind to MHC class I or II molecules. The term “epitope” refers to a portion or fragment of a molecule (such as an antigen) that is recognized by the immune system. For example, an epitope may be recognized by T cells, B cells, or antibodies. Epitopes of an antigen may include continuous or discontinuous portions of the antigen, and their length may be from about 5 to about 100, such as from about 5 to about 50, from about 8 to about 30, or from about 8 to about 25 amino acids. For example, the length of an epitope may be 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids. In some embodiments, the length of an epitope is from about 10 to about 25 amino acids. The term "epitaph" includes T-cell epitopes.

[0356] The term "T-cell epitope" refers to a portion or fragment of a protein that is recognized by T cells when presented in the context of MHC molecules. The term "major histocompatibility complex" and the abbreviation "MHC" encompasses MHC class I and MHC class II molecules and refers to gene complexes present in all vertebrates. MHC proteins or molecules play a crucial role in signaling between lymphocytes and antigen-presenting cells or diseased cells in immune responses, where MHC proteins or molecules bind peptide epitopes and present them for recognition by T-cell receptors on T cells. MHC-encoded proteins are expressed on the cell surface and present both self-antigens (peptide fragments from the cell itself) and non-self-antigens (e.g., fragments from invading microorganisms) to T cells. In the case of class I MHC / peptide complexes, the binding peptide is typically about 8 to about 10 amino acids long, but longer or shorter peptides can be effective. In the case of class II MHC / peptide complexes, the binding peptide is typically about 10 to about 25 amino acids long, and particularly about 13 to about 18 amino acids long, while longer and shorter peptides can be effective.

[0357] The length of peptide and polypeptide antigens can be from 2 to 100 amino acids, including, for example, 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids. In some embodiments, the peptide can be longer than 50 amino acids. In some embodiments, the peptide can be longer than 100 amino acids.

[0358] A peptide or polypeptide antigen can be any peptide or polypeptide that can induce or increase the immune system’s ability to produce antibodies and T-cell responses against the peptide or polypeptide.

[0359] In some embodiments, the vaccine antigen, i.e., the antigen that, when administered to a subject, induces an immune response, is recognized by immune effector cells. In some embodiments, if recognized by immune effector cells, the vaccine antigen can induce stimulation, initiation, and / or expansion of immune effector cells carrying antigen receptors that recognize the vaccine antigen in the presence of appropriate co-stimulatory signals. In the context of embodiments of this disclosure, the vaccine antigen may, for example, be presented or present on the surface of cells (such as antigen-presenting cells). In some embodiments, the antigen is presented by diseased cells (such as tumor cells or infected cells). In some embodiments, the antigen receptor is a TCR that binds to an epitope of the antigen presented in the context of MHC. In some embodiments, when expressed by and / or present on T cells, the binding of a TCR to an antigen presented by a cell (such as an antigen-presenting cell) leads to the stimulation, initiation, and / or expansion of the T cell. In some implementations, when expressed by and / or present on T cells, the binding of TCR to antigens presented on diseased cells leads to cell lysis and / or apoptosis of the diseased cells, wherein the T cells release cytotoxic factors, such as perforin and granzyme.

[0360] According to some embodiments, the amino acid sequence that enhances antigen processing and / or presentation is fused to the antigenic peptide or polypeptide directly or via a linker sequence. Therefore, in some embodiments, the nucleic acids (such as RNA and / or DNA) described herein contain at least one coding region encoding the antigenic peptide or polypeptide, as well as the amino acid sequence that enhances antigen processing and / or presentation.

[0361] Such amino acid sequences that enhance antigen processing and / or presentation are preferably located at the C-terminus of the antigenic peptide or polypeptide and the linker sequence (and optionally at the C-terminus of the amino acid sequence that disrupts immune tolerance), but are not limited thereto. Amino acid sequences that enhance antigen processing and / or presentation as defined herein preferably improve antigen processing and presentation. In one embodiment, amino acid sequences that enhance antigen processing and / or presentation as defined herein include, but are not limited to, sequences derived from human MHC class I complexes (HLA-B51, monomers A2, B27 / B51, Cw2 / Cw3). In addition to improving antigen processing and presentation, such amino acid sequences that enhance antigen processing and / or presentation can also be used to determine the expression of amino acid sequences in the processes described herein.

[0362] Therefore, in a particularly preferred embodiment, the RNA described herein comprises at least one coding region encoding an antigenic peptide or polypeptide and an amino acid sequence that enhances antigen processing and / or presentation, said amino acid sequence being preferably fused to the antigenic peptide or polypeptide, more preferably fused to the C-terminus of the antigenic peptide or polypeptide as described herein.

[0363] In addition, the secreted sequence can fuse with the N-terminus of an antigenic peptide or polypeptide.

[0364] The amino acid sequence of tetanus toxoid derived from Clostridium tetani can be used to overcome self-tolerance mechanisms in order to effectively generate an immune response to autoantigens by providing T cell assistance during initiation.

[0365] Tetanus toxoid heavy chain is known to contain epitopes that can bind indiscriminately to MHC class II alleles and induce CD4 in almost all individuals vaccinated against tetanus. + Memory T cells. Furthermore, it is known that the combination of tetanus toxoid (TT) helper epitopes and tumor-associated antigens can provide CD4+ during initiation, compared to administration of tumor-associated antigens alone. + Mediated T cells help improve immune stimulation. This is to reduce the stimulation of CD8 cells with tetanus sequences. + The risk to T cells (the sequence may compete with the expected induction of tumor antigen-specific T cell responses), the entire fragment C of tetanus toxoid is not used because it is known to contain CD8. + T-cell epitopes.

[0366] According to some implementation schemes, amino acid sequences that disrupt immune tolerance are fused directly or through linkers to antigenic peptides or polypeptides.

[0367] Such amino acid sequences that disrupt immune tolerance are preferably located at the C-terminus of the antigenic peptide or polypeptide (and optionally at the N-terminus of the amino acid sequence that enhances antigen processing and / or presentation), wherein the amino acid sequence disrupting immune tolerance may be directly fused to or fused via a linker with the amino acid sequence that enhances antigen processing and / or presentation. The amino acid sequence disrupting immune tolerance as defined herein preferably improves T cell responses. In one embodiment, the amino acid sequence disrupting immune tolerance as defined herein includes, but is not limited to, sequences derived from tetanus toxoid-derived helper sequences p2 and p16 (P2P16).

[0368] According to some implementation schemes, the bioluminescent amino acid sequence is fused directly or through a linker to an antigenic peptide or polypeptide.

[0369] Such bioluminescent amino acid sequences are preferably located at the C-terminus of the antigenic peptide or polypeptide (and optionally at the N-terminus of (i) an amino acid sequence that enhances antigen processing and / or presentation or (ii) an amino acid sequence that disrupts immune tolerance), wherein the bioluminescent amino acid sequence and (i) the amino acid sequence that enhances antigen processing and / or presentation or (ii) the amino acid sequence that disrupts immune tolerance can be directly fused or fused via a linker. Bioluminescent amino acid sequences as defined herein preferably improve the determination of the amount of antigenic peptide or polypeptide. In some embodiments, bioluminescent amino acid sequences as defined herein generate fluorescence. In some embodiments, bioluminescent amino acid sequences as defined herein include, but are not limited to, sequences derived from green fluorescent protein (GFP), yellow fluorescent protein (YFP), red fluorescent protein (RFP), blue fluorescent protein (EBFP), cyan fluorescent protein (ECFP), their variants (such as enhanced GFP (EGFP), superfolded GFP (sfGFP), and luciferase sequences.

[0370] The implementation scheme for vaccine RNA is described below, and certain terms used in describing its components have the following meanings:

[0371] hAg-Kozak: The 5'-UTR sequence of human α-globin mRNA with an optimized "Kozak sequence" to improve translation efficiency.

[0372] sec / MITD: A fusion protein tag derived from the sequence encoding the human MHC class I complex (HLA-B51, monomers A2, B27 / B51, Cw2 / Cw3), which has been shown to improve antigen processing and presentation. Sec corresponds to a 78 bp fragment encoding a secretion signal peptide that guides the translocation of nascent polypeptide chains into the endoplasmic reticulum. MITD corresponds to the transmembrane and cytoplasmic domain of MHC class I molecules, also known as the MHC class I transport domain.

[0373] Antigen: The sequence that encodes the corresponding antigen / epitope.

[0374] Glycine-Serine Linker (GS): A sequence encoding a linker sequence according to the invention, in one embodiment, which is a glycine-serine linker sequence—a short linker peptide consisting primarily of the amino acids glycine (G) and serine (S), as commonly used in fusion proteins. In a specific embodiment of the invention, the linker sequence is preceded by a lysine residue at its N-terminus and can be represented as follows: GGSGGGGSGGR / K. Thus, the portion of the amino acid sequence containing the linker sequence can be represented as follows: KΔGGSGGGGSGGR / K (Δ indicates a proteolytic cleavage site). After cleavage, this results in the linker sequence being excised as follows: GGSGGGGSGGR / K. In one embodiment, the linker sequence of the invention is a GS linker, each containing at least one non-G or S residue, wherein said amino acid residue forms a proteolytic cleavage site for a proteolytic enzyme.

[0375] P2P16: The sequence encoding a tetanus toxoid-derived helper epitope to disrupt immune tolerance.

[0376] The FI element: The 3'-UTR is a combination of two sequence elements derived from the "split amino-terminal enhancer" (AES) mRNA (called F) and the mitochondrial-encoded 12S ribosomal RNA (called I). These are determined through an in vitro selection process to obtain sequences that confer RNA stability and enhance total protein expression.

[0377] A30L70: A multi-(A) tail measuring 110 nucleotides in length, consisting of a 30-adenosine residue segment, followed by a 10-nucleotide linker sequence and an additional 70 adenosine residues, designed to enhance RNA stability and translation efficiency in dendritic cells.

[0378] In one implementation, the vaccine RNA described herein has the following structure:

[0379] β-S-ARCA(D1)-hAg-Kozak-sec-GS(1)-antigen-GS(2)-P2P16-GS(3)-MITD-FI-A30L70.

[0380] In one implementation, the vaccine antigen described herein has the following structure:

[0381] sec-GS(1)-antigen-GS(2)-P2P16-GS(3)-MITD.

[0382] In one implementation, a formulation (such as a particle (LNP, LPX, PLX, etc.)) contains multiple vaccine antigen RNA constructs (nucleic acids) as described herein, wherein each vaccine RNA construct contains a different adapter sequence.

[0383] In some embodiments, the antigen receptor is an antibody or B-cell receptor that binds to an epitope in the antigen. In some embodiments, the antibody or B-cell receptor binds to the natural epitope of the antigen.

[0384] The terms "expressed on the cell surface" or "associated with the cell surface" refer to a molecule, such as an antigen, associating with and residing on the cell's plasma membrane, wherein at least a portion of the molecule faces the extracellular space of the cell and is accessible from the outside of the cell, for example, through an antibody located outside the cell. In this context, the portion may be, for example, at least 4, at least 8, at least 12, or at least 20 amino acids. The association may be direct or indirect. For example, the association may be through one or more transmembrane domains, one or more lipid anchors, or through interaction with any other protein, lipid, carbohydrate, or other structure that can be found on the outer lobules of the cell membrane. For example, a molecule associated with the cell surface may be a transmembrane protein having an extracellular portion, or it may be a protein that associates with the cell surface through interaction with another protein that is a transmembrane protein.

[0385] The term "cell surface" or "the surface of the cell" is used according to its normal meaning in the art and therefore includes the cell exterior that can be bound by proteins and other molecules. If an antigen is located on the cell surface and can be bound by antigen-specific antibodies, for example, added to the cell, then the antigen is expressed on the cell surface.

[0386] In the context of this disclosure, the term "extracellular portion" or "exodomain" refers to a portion of a molecule (such as a protein) that faces the extracellular space of the cell and is preferably accessible from the outside of the cell, for example, by binding molecules (such as antibodies) located outside the cell. In some embodiments, the term refers to one or more extracellular loops or domains or fragments thereof.

[0387] The terms “T cell” and “T lymphocyte” are used interchangeably herein and include both helper T cells (CD4+ T cells) and cytotoxic T cells (CTL, CD8+ T cells), including cytolytic T cells. The term “antigen-specific T cell” or similar terms refer to T cells that recognize antigens targeted by T cells (particularly when presented on the surface of antigen-presenting cells or diseased cells, such as cancer cells, in the context of MHC molecules) and preferably perform effector functions. A T cell is considered antigen-specific if it kills a target cell expressing an antigen. T cell specificity can be evaluated using any of a variety of standard techniques, such as chromium release assays or proliferation assays. Alternatively, the synthesis of lymphokines (such as interferon-γ) can be measured.

[0388] The term "target" should refer to a factor (such as a cell or tissue) that serves as a target for an immune response (such as a cellular immune response). Targets include cells that present antigens or antigenic epitopes (i.e., peptide fragments derived from antigens). In some embodiments, the target cells are cells that express antigens and present them using class I MHC.

[0389] "Antigen processing" refers to the degradation of an antigen into processing products that are fragments of the antigen (e.g., degradation of a polypeptide into a peptide) and the association (e.g., by binding) of one or more of these fragments with an MHC molecule for presentation by a cell (such as an antigen-presenting cell) to a specific T cell.

[0390] "Antigen-responsive CTL" refers to CD8+ cells that are responsive to an antigen or a peptide derived from that antigen. + T cells, where the antigen is presented on the surface of antigen-presenting cells along with class I MHC.

[0391] According to this disclosure, CTL responsiveness may include sustained calcium flux, cell division, production of cytokines (such as IFN-γ and TNF-α), upregulation of activation markers (such as CD44 and CD69), and specific cytolytic killing of target cells expressing tumor antigens. Artificial reporter molecules that accurately indicate CTL responsiveness may also be used to determine CTL responsiveness.

[0392] As used herein, “activation” or “stimulation” refers to the state of cells (such as immune effector cells, such as T cells) that have been adequately stimulated to induce detectable cell proliferation. Activation can also be associated with the initiation of signaling pathways, induced cytokine production, and detectable effector function. The term “activated immune effector cells” specifically refers to immune effector cells undergoing cell division.

[0393] The term "initiation" refers to the process in which immune effector cells (such as T cells) have their first contact with their specific antigens and are induced to differentiate into effector cells (such as effector T cells).

[0394] The term "amplification" refers to the process in which a specific entity multiplies. In some embodiments, the term is used in the context of an immune response, in which immune effector cells are stimulated by an antigen, proliferate, and specific immune effector cells that recognize said antigen are amplified. In some embodiments, amplification leads to the differentiation of immune effector cells.

[0395] The terms “immune response” and “immune reaction” are used interchangeably in their conventional sense herein and refer to a comprehensive bodily response to an antigen, and may refer to a cellular immune response, a humoral immune response, or both. According to this disclosure, the terms “immune response to” or “immune response against” relating to factors (such as antigens, cells, or tissues) refer to an immune response, such as a cellular response against said factor. An immune response may include one or more reactions selected from the group consisting of: producing antibodies against one or more antigens and amplifying antigen-specific T lymphocytes, such as CD4+. + and CD8 + T lymphocytes, such as CD8 + T lymphocytes can be detected in various in vitro proliferation or cytokine production assays.

[0396] The terms “inducing an immune response” and “initiating an immune response,” and similar terms, in the context of this disclosure, refer to inducing an immune response, such as inducing a cellular immune response, a humoral immune response, or both. An immune response can be protective / preventive / prophylactic and / or therapeutic. An immune response can be against any immunogen or antigen or antigenic peptide, such as against tumor-associated antigens or pathogen-associated antigens (e.g., viral antigens such as influenza virus (A, B, or C), CMV, or RSV). “Inducing” in this context can mean that there is no immune response against a specific antigen or pathogen prior to induction, but it can also mean that a certain level of immune response against a specific antigen or pathogen exists prior to induction, and that the immune response is enhanced after induction. Therefore, “inducing an immune response” in this context also includes “enhancing an immune response.” In some embodiments, after inducing an immune response in an individual, the individual is protected from disease (such as an infectious disease or cancerous disease) or the disease condition is improved by inducing an immune response.

[0397] The terms "cellular immune response," "cellular response," "cell-mediated immunity," or similar terms are intended to encompass cellular responses against cells characterized by expressing antigens and / or presenting antigens using class I or class II MHC. Cellular responses involve cells called T cells or T lymphocytes, which act as "helper factors" or "killer factors." Helper T cells (also known as CD4+) + T cells play a central role in regulating the immune response, and killer cells (also known as cytotoxic T cells, cytolytic T cells, CD8+ cells) also play a crucial role. + T cells (or CTLs) kill cells such as diseased cells.

[0398] The term "humoral immune response" refers to a process in a living organism in which antibodies are produced in response to factors and organisms, ultimately neutralizing and / or eliminating those factors and organisms. The specificity of the antibody response is mediated by T and / or B cells via membrane-associated receptors that bind to a single specific antigen. Upon binding to an appropriate antigen and receiving various other activation signals, B lymphocytes divide, producing memory B cells and antibody-secreting plasma cell clones, each producing antibodies that recognize the same antigenic epitopes as their antigen receptors. Memory B lymphocytes remain dormant until they are subsequently activated by their specific antigens. These lymphocytes provide the cellular basis of memory and the escalation of the antibody response upon re-exposure to the specific antigen.

[0399] As used herein, the term "antibody" refers to an immunoglobulin molecule capable of specifically binding to epitopes on an antigen. Specifically, the term "antibody" refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains linked together by disulfide bonds. The term "antibody" includes monoclonal antibodies, recombinant antibodies, human antibodies, humanized antibodies, chimeric antibodies, and any combination thereof. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL). The variable and constant regions are also referred to herein as variable domains and constant domains, respectively. The VH and VL regions can be further subdivided into hypervariable regions, called complementarity-determining regions (CDRs), interspersed with more conserved regions, called framework regions (FRs). Each VH and VL consists of 3 CDRs and 4 FRs, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The CDRs of VH are called HCDR1, HCDR2, and HCDR3, and the CDRs of VL are called LCDR1, LCDR2, and LCDR3. The variable regions of the heavy and light chains contain binding domains that interact with the antigen. The constant regions of an antibody include a heavy chain constant region (CH) and a light chain constant region (CL), where CH can be further subdivided into constant domain CH1, a hinge region, and constant domains CH2 and CH3 (arranged in the following order from the amino terminus to the carboxyl terminus: CH1, CH2, CH3). The constant regions of an antibody can mediate the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. Antibodies can be complete immunoglobulins derived from natural or recombinant sources, and can be the immunoactive portion of a complete immunoglobulin. Antibodies are typically tetramers of immunoglobulin molecules. Antibodies can exist in a variety of forms, including, for example, polyclonal antibodies, monoclonal antibodies, Fv, Fab, and F(ab)2, as well as single-chain antibodies and humanized antibodies.

[0400] The term "immunoglobulin" refers to proteins in the immunoglobulin superfamily, such as antigen receptors, antibodies, or B cell receptors (BCRs). Immunoglobulins are characterized by domains that exhibit a characteristic immunoglobulin (Ig) fold, known as immunoglobulin domains. The term encompasses both membrane-bound and soluble immunoglobulins. Membrane-bound immunoglobulins, also called surface immunoglobulins or membrane immunoglobulins, are typically part of the BCR. Soluble immunoglobulins are often referred to as antibodies. Immunoglobulins typically consist of several chains, usually two identical heavy chains and two identical light chains linked by disulfide bonds. These chains are primarily composed of immunoglobulin domains, such as V... L (Variable light chain) domain, C L (Constant light chain) domain, V H (Variable Heavy Chain) domain and C H (Constant Heavy Chain) Domain C H 1. C H 2. C H 3 and C H 4. There are five types of mammalian immunoglobulin heavy chains: α, δ, ε, γ, and μ, corresponding to different classes of antibodies: IgA, IgD, IgE, IgG, and IgM. In contrast to the heavy chains of soluble immunoglobulins, the heavy chains of membrane or surface immunoglobulins contain a transmembrane domain and a short cytoplasmic domain at their carboxyl terminus. There are two types of light chains in mammals: λ and κ. Immunoglobulin chains contain variable and constant regions. The constant region is largely conserved across different isotypes of immunoglobulins, while the variable portion is highly diverse and responsible for antigen recognition.

[0401] The terms “vaccination” and “immunization” describe the process of treating an individual for therapeutic or preventative reasons and involve procedures such as administering to an individual one or more immunogens or antigens or derivatives thereof as described herein, particularly in the form of RNA (especially mRNA) encoding them, and stimulating an immune response against said one or more immunogens or antigens or cells characterized by presenting said one or more immunogens or antigens.

[0402] The phrase "characterized by antigen-presenting cells," "antigen-presenting cells," or "MHC molecules presenting antigens on the surface of antigen-presenting cells," or similar expressions, refer to cells, such as diseased cells (particularly tumor cells or infected cells), or antigen-presenting cells, that directly or after processing antigens or antigenic peptides in the presence of MHC molecules (such as MHC class I and / or MHC class II molecules). In some embodiments, the MHC molecules are MHC class I molecules.

[0403] The term "allergen" refers to an antigen originating outside the body of a subject (i.e., an allergen can also be called a "heterologous antigen") that elicits an abnormally strong immune response, in which the subject's immune system resists a perceived threat that would otherwise be harmless to the subject. An "allergic reaction" is a disease caused by such a strong immune response to an allergen. Allergens are typically antigens that can stimulate a type I hypersensitivity response in atopic individuals through an immunoglobulin E (IgE) response. Specific examples of allergens include allergens derived from peanut protein (e.g., Ara h 2.02), ovalbumin, grass pollen protein (e.g., Phlp5), and house dust mite protein (e.g., Der p 2).

[0404] The term "growth factor" refers to molecules that can stimulate cell growth, proliferation, healing, and / or cell differentiation. Typically, growth factors act as signaling molecules between cells. The term "growth factor" includes specific cytokines and hormones that bind to specific receptors on the surface of their target cells. Examples of growth factors include bone morphogenetic protein (BMP), fibroblast growth factor (FGF), vascular endothelial growth factor (VEGF) (such as VEGFA), epidermal growth factor (EGF), insulin-like growth factor, hepatocyte glycosides, macrophage colony-stimulating factor, granulocyte colony-stimulating factor, granulocyte-macrophage colony-stimulating factor, neuromodulatory proteins, neurotrophic factors (e.g., brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF)), placental growth factor (PGF), platelet-derived growth factor (PDGF), renal enzyme (RNLS) (an anti-apoptotic survival factor), T-cell growth factor (TCGF), thrombopoietin (TPO), transforming growth factors (transforming growth factor α (TGF-α), transforming growth factor β (TGF-β)), and tumor necrosis factor-α (TNF-α). In some embodiments, "growth factor" is a peptide or polypeptide growth factor.

[0405] The term "protease inhibitor" refers to molecules, particularly peptides or polypeptides, that inhibit the function of proteases. Protease inhibitors can be classified by the protease they inhibit (e.g., aspartic protease inhibitors) or by their mechanism of action (e.g., suicide inhibitors, such as serine protease inhibitors). Specific examples of protease inhibitors include serine protease inhibitors, such as α1-antitrypsin, protease inhibitory peptides, and phenbutatin.

[0406] The term "enzyme" refers to a large molecular biological catalyst that accelerates a chemical reaction. Like any catalyst, an enzyme is not consumed in the reaction it catalyzes and does not alter the equilibrium of said reaction. Unlike many other catalysts, enzymes are more specific. In some implementations, enzymes are essential for the homeostasis of a subject; for example, any dysfunction of an enzyme (particularly reduced activity that can be caused by mutation, deletion, or decreased production) can lead to disease. Examples of enzymes include herpes simplex virus type 1 thymidine kinase (HSV1-TK), aminohexosidase, phenylalanine hydroxylase, pseudocholinesterase, and lactase.

[0407] The term "receptor" refers to a protein molecule that receives signals (particularly chemical signals called ligands) from outside the cell. The binding of the signal (e.g., the ligand) to the receptor elicits a cellular response, such as intracellular activation of a kinase. Receptors include transmembrane receptors (such as ion channel-coupled (ionotropic) receptors, G protein-coupled (metabolic) receptors, and enzyme-coupled receptors) and intracellular receptors (such as cytoplasmic and nuclear receptors). Specific examples of receptors include steroid hormone receptors, growth factor receptors, and peptide receptors (i.e., receptors whose ligands are peptides), such as P-selectin glycoprotein ligand-1 (PSGL-1). The term "growth factor receptor" refers to a receptor that binds to growth factors.

[0408] The term "apoptosis regulator" refers to molecules, particularly peptides or polypeptides, that regulate apoptosis (i.e., activate or inhibit apoptosis). Apoptosis regulators can be divided into two main categories: regulators of mitochondrial function and regulators of caspases. The first category includes proteins (e.g., BCL-2, BCL-xL) that maintain mitochondrial integrity by preventing loss of mitochondrial membrane potential and / or the release of pro-apoptotic proteins (such as cytochrome C) into the cytosol. Pro-apoptotic proteins that promote the release of cytochrome C (e.g., BAX, BAK, BIM) also belong to this first category. The second category includes proteins such as inhibitors of apoptotic proteins (e.g., XIAP) or FLIPs, which block the activation of caspases.

[0409] The term "transcription factor" refers to proteins that regulate the rate of transcription of genetic information from DNA to messenger RNA, particularly by binding to specific DNA sequences. Transcription factors can regulate cell division, cell growth, and cell death throughout life; cell migration and organization during embryonic development; and / or responses to signals from outside the cell, such as hormones. Transcription factors contain at least one DNA-binding domain that binds to a specific DNA sequence, typically adjacent to the gene that the transcription factor regulates. Specific examples of transcription factors include MECP2, FOXP2, FOXP3, the STAT protein family, and the HOX protein family.

[0410] The term "tumor suppressor protein" refers to molecules, particularly peptides or polypeptides, that protect cells from taking a step on the path to cancer. Tumor suppressor proteins (typically encoded by corresponding tumor suppressor genes) exhibit attenuating or inhibiting effects on cell cycle regulation and / or promote apoptosis. Their functions can include one or more of the following: inhibiting genes essential for cell cycle persistence; coupling the cell cycle to DNA damage (cell division will not occur as long as damaged DNA is present in the cell); initiating apoptosis if the damaged DNA cannot be repaired; metastasis inhibition (e.g., preventing tumor cell spread, preventing loss of contact inhibition, and inhibiting metastasis); and DNA repair. Specific examples of tumor suppressor proteins include p53, phosphatase and tensin homolog (PTEN), SWI / SNF (convertible / non-fermentable sucrose), von Hippel-Lindau tumor suppressor protein (pVHL), adenomatous polyposis protein (APC), CD95, tumorigenesis suppressor 5 (ST5), tumorigenesis suppressor 14 (ST14), and Yippee-like 3 (YPEL3).

[0411] The term "structural protein" refers to proteins that impart stiffness and rigidity to biological components that are otherwise fluid. Most structural proteins are fibrous (such as collagen and elastin), but they can also be globular (such as actin and tubulin). Typically, globular proteins are soluble as monomers but aggregate to form long filaments, which can, for example, constitute the cytoskeleton. Other structural proteins include motor proteins (such as myosin, kinesin, and dynein) and surfactant proteins. Specific examples of structural proteins include collagen, surfactant protein A, surfactant protein B, surfactant protein C, surfactant protein D, elastin, tubulin, actin, and myosin.

[0412] The term "reprogramming factor" or "reprogramming transcription factor" refers to molecules, particularly peptides or polypeptides, that, when optionally expressed in somatic cells along with other factors (such as additional reprogramming factors), cause said somatic cells to be reprogrammed or dedifferentiated into cells with stem cell characteristics (especially pluripotency). Specific examples of reprogramming factors include OCT4, SOX2, c-MYC, KLF4, LIN28, and NANOG.

[0413] The term "genome-engineered protein" refers to proteins that can be inserted, deleted, or replaced in a subject's genome. Specific examples of genome-engineered proteins include megnucleases, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regular-spaced short palindromic repeats-CRISPR-associated protein 9 (CRISPR-Cas9).

[0414] The term "blood proteins" refers to peptides or polypeptides present in the plasma of a subject, particularly in the plasma of a healthy subject. Blood proteins have a variety of functions, such as transport (e.g., albumin, transferrin), enzyme activity (e.g., thrombin or ceruloplasmin), blood clotting (e.g., fibrinogen), defense against pathogens (e.g., complement components and immunoglobulins), and protease inhibitors (e.g., α1-antitrypsin). Specific examples of blood proteins include thrombin, serum albumin, factor VII, factor VIII, insulin, factor IX, factor X, tissue plasminogen activator, protein C, von Willebrand factor, antithrombin III, glucocerebrosidase, erythropoietin, granulocyte colony-stimulating factor (G-CSF), modified factor VIII, and anticoagulants.

[0415] Therefore, in some embodiments, the pharmaceutically active peptide or polypeptide is (i) a cytokine, preferably selected from the group consisting of erythropoietin (EPO), interleukin-4 (IL-2) and interleukin-10 (IL-11), more preferably EPO; (ii) an adhesion molecule, particularly an integrin; (iii) an immunoglobulin, particularly an antibody; (iv) an immunologically active compound, particularly an antigen; (v) a hormone, particularly angiotensin, insulin or growth hormone; (vi) a growth factor, particularly VEGFA; (vii) a protease inhibitor, particularly α1-antitrypsin; (viii) an enzyme, preferably selected from herpes simplex virus type 1 thymidine kinase (HSV1-TK), aminohexosidase, phenylalanine... The group consisting of amino acid hydroxylases, pseudocholinesterases, pancreatic enzymes, and lactases; (ix) receptors, particularly growth factor receptors; (x) apoptosis regulators, particularly BAX; (xi) transcription factors, particularly FOXP3; (xii) tumor suppressor proteins, particularly p53; (xiii) structural proteins, particularly surfactant protein B; (xiv) reprogramming factors, such as the group consisting of OCT4, SOX2, c-MYC, KLF4, LIN28, and NANOG; (xv) genome-engineered proteins, particularly clustered regular-spaced short palindromic repeats-CRISPR-associated protein 9 (CRISPR-Cas9); and (xvi) blood proteins, particularly fibrinogen.

[0416] In some embodiments, the pharmaceutically active peptide or polypeptide contains one or more antigens or one or more epitopes, i.e., administration of the peptide or polypeptide to a subject elicits an immune response in the subject against the one or more antigens or one or more epitopes, which may be therapeutic or partially or completely protective.

[0417] In some implementations, nucleic acids (such as mRNA) encode at least one epitope.

[0418] In some implementations, the epitope is derived from a tumor antigen. A tumor antigen can be a “standard” antigen, generally known to be expressed in various cancers. A tumor antigen can also be a “neoantigen,” specific to an individual’s tumor and not previously recognized by the immune system. Neoantigens or novel epitopes can arise from one or more cancer-specific mutations in the cancer cell genome that cause amino acid changes. Examples of tumor antigens include, but are not limited to, p53, ART-4, BAGE, β-catenin / m, Bcr-abLCAMEL, CAP-1, CASP-8, CDC27 / m, CDK4 / m, CEA, cell surface proteins of the sealing protein family (such as sealing protein-6, sealing protein-18.2, and sealing protein-12), c-MYC, CT, Cyp-B, DAM, ELF2M, ETV6-AML1, G250, GAGE, GnT-V, Gap 100, HAGE, HER-2 / neu, HPV-E7, HPV-E6, HAST-2, hTERT (or hTRT), LAGE, LDLR / FUT, and MAGE-A (preferably MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A5, MAGE-A6, MAGE-A7, MAGE-A8, MAGE-A9, MAGE-A). 10, MAGE-A 11 or MAGE-A12), MAGE-B, MAGE-C, MART-1 / Melan-A, MC1R, myosin / m, MUC1, MUM-1, MUM-2, MUM-3, NA88-A, NF1, NY-ESO-1, NY-BR-1, pl90 minor BCR-abL, Pml / RARa, PRAME, protease 3, PSA, PSM, RAGE, RU1 or RU2, SAGE, SART-1 or SART-3, SCGB3A2, SCP1, SCP2, SCP3, SSX, survival protein, TEL / AML1, TPI / m, TRP-1, TRP-2, TRP-2 / INT2, TPTE, WT and WT-1.

[0419] Cancer mutations vary from person to person. Therefore, cancer mutations encoding novel epitopes (novel epitopes) represent attractive targets for the development of vaccine compositions and immunotherapies. The efficacy of tumor immunotherapies depends on the selection of cancer-specific antigens and epitopes capable of inducing a potent immune response within the host. RNA can be used to deliver patient-specific tumor epitopes to patients. Dendritic cells (DCs) located in the spleen represent antigen-presenting cells with particular interest in the RNA expression of immunogenic epitopes or antigens, such as tumor epitopes. The use of multiple epitopes has been shown to enhance the therapeutic efficacy of tumor vaccine compositions. Rapid sequencing of the tumor mutant genome can provide personalized vaccines with multiple epitopes, which can be encoded by mRNA as described herein, for example, as a single polypeptide, wherein the epitopes are optionally separated by linkers. In some embodiments of this disclosure, the mRNA encodes at least one epitope, at least two epitopes, at least three epitopes, at least four epitopes, at least five epitopes, at least six epitopes, at least seven epitopes, at least eight epitopes, at least nine epitopes, or at least ten epitopes. Exemplary implementations include mRNAs encoding at least five epitopes (referred to as "pentatopes") and mRNAs encoding at least ten epitopes (referred to as "decatopes").

[0420] In some embodiments, the antigen or epitope is derived from pathogen-associated antigens, particularly viral antigens. In some embodiments, the antigen or epitope is derived from the SARS-CoV-2S protein, its immunogenic variants, or immunogenic fragments of the SARS-CoV-2S protein or its immunogenic variants. Therefore, in some embodiments, the mRNA used in this disclosure encodes an amino acid sequence comprising the SARS-CoV-2S protein, its immunogenic variants, or immunogenic fragments of the SARS-CoV-2S protein or its immunogenic variants.

[0421] In some embodiments of this disclosure, the antigen (such as a tumor antigen or a vaccine antigen) is preferably administered as a single-stranded, 5'-capped mRNA, which is translated into the corresponding protein upon entering the cells of the subject to which the RNA is administered. Preferably, the RNA contains structural elements (5' cap, 5' UTR, 3' UTR, multiple (A) sequences) optimized for maximum efficiency in terms of RNA stability and translation.

[0422] In some implementations, β-S-ARCA(D1) is used as a specific capping structure at the 5' end of the mRNA. In some implementations, m2 7,3’-0 Gppp(m1 2’-0ApG is used as a specific capping structure at the 5' end of mRNA. In some embodiments, the 5'-UTR sequence is derived from human α-globin mRNA and optionally has an optimized "Kozak sequence" to improve translation efficiency. In some embodiments, a combination of two sequence elements (FI elements) derived from "split N-terminal enhancer" (AES) mRNA (referred to as F) and mitochondrial-encoded 12S ribosomal RNA (referred to as I) is placed between the coding sequence and the multiple (A) sequence to ensure higher maximum protein levels and extended durability of mRNA. In some embodiments, two repeating 3'-UTRs derived from human β-globin mRNA are placed between the coding sequence and the multiple (A) sequence to ensure higher maximum protein levels and extended durability of mRNA. In some embodiments, a multiple (A) sequence of 110 nucleotides in length is used, consisting of a 30-adenosine residue segment, a subsequent 10-nucleotide linker sequence, and an additional 70 adenosine residues. This multiple (A) sequence is designed to enhance RNA stability and translation efficiency.

[0423] In some embodiments, mRNA encoding an antigen (such as a tumor antigen or vaccine antigen) is expressed in the cells of the treated subject to provide the antigen. In some embodiments, the mRNA is transiently expressed in the subject's cells. In some embodiments, the mRNA is transcribed in vitro. In some embodiments, the antigen is expressed at the cell surface. In some embodiments, the antigen is expressed and presented in the MHC context. In some embodiments, the antigen is expressed into the extracellular space, i.e., the antigen is secreted.

[0424] Antigen molecules or their processed products (such as fragments thereof) can bind to antigen receptors (such as BCRs or TCRs) carried by immune effector cells, or to antibodies.

[0425] According to this disclosure, peptide and polypeptide antigens (wherein the antigen is a vaccine antigen) provided to a subject via administration of mRNA encoding peptide and polypeptide antigens preferably induce an immune response, such as a humoral and / or cellular immune response, in the subject to which the peptide or polypeptide antigen was provided. The immune response is preferably directed against a target antigen. Therefore, a vaccine antigen may comprise a target antigen, a variant thereof, or a fragment thereof. In some embodiments, such a fragment or variant is immunologically equivalent to the target antigen. In the context of this disclosure, the terms "antigen fragment" or "antigen variant" refer to an agent that induces an immune response targeting the antigen, i.e., the target antigen. Therefore, a vaccine antigen may correspond to or may comprise a target antigen, may correspond to or may comprise a fragment of the target antigen, or may correspond to or may comprise an antigen homologous to the target antigen or a fragment thereof. Therefore, according to this disclosure, a vaccine antigen may comprise an immunogenic fragment of the target antigen or an amino acid sequence homologous to an immunogenic fragment of the target antigen. The "immunogenic fragment of the antigen" according to this disclosure preferably refers to an antigen fragment capable of inducing an immune response against the target antigen. The vaccine antigen may be a recombinant antigen.

[0426] The term "immunologically equivalent" means that immunologically equivalent molecules (such as immunologically equivalent amino acid sequences) exhibit the same or substantially the same immunological properties and / or exert the same or substantially the same immunological effects, for example, in terms of the type of immunological effect. In the context of this disclosure, the term "immunologically equivalent" is preferably used in reference to the immunological effects or properties of an antigen or antigen variant used for immunization. For example, if an amino acid sequence induces an immune response with specificity to a reference amino acid sequence when exposed to the immune system of a subject, then said amino acid sequence is immunologically equivalent to the reference amino acid sequence.

[0427] In some embodiments, the mRNA used in this disclosure is non-immunogenic. RNA encoding an immunostimulant can be administered according to this disclosure to provide adjuvant action. The RNA encoding the immunostimulant can be standard RNA or non-immunogenic RNA.

[0428] As used herein, the term "non-immunogenic RNA" (such as "non-immunogenic mRNA") refers to RNA that, when administered to, for example, mammals, does not induce an immune system response, or induces a weaker response than that induced by the same RNA (distinguished only by not being modified or treated to make non-immunogenic RNA non-immunogenic) (i.e., induced by standard RNA (stdRNA)). In some embodiments, non-immunogenic RNA (also referred to herein as modified RNA (modRNA)) is made non-immunogenic by incorporating a modified nucleoside that inhibits RNA-mediated activation of innate immune receptors into the RNA and / or removing double-stranded RNA (dsRNA).

[0429] To render non-immunogenic RNA (especially mRNA) non-immunogenic by incorporating a modified nucleoside, any modified nucleoside can be used, as long as it reduces or inhibits the immunogenicity of the RNA. Particularly preferred are modified nucleosides that inhibit RNA-mediated activation of innate immune receptors. In some embodiments, the modified nucleoside comprises replacing one or more uridines with a nucleoside containing a modified nucleobase. In some embodiments, the modified nucleobase is a modified uracil. In some embodiments, the nucleoside containing the modified nucleobase is selected from the group consisting of: 3-methyl-uridine (mRNA) 3 U), 5-methoxyuridine (mo) 5 U), 5-aza-uridine, 6-aza-uridine, 2-thio-5-aza-uridine, 2-thio-uridine (s 2 U), 4-thiouridine (s) 4 U), 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxy-uridine (ho) 5 U), 5-aminoallyl-uridine, 5-halo-uridine (e.g., 5-iodo-uridine or 5-bromo-uridine), uridine 5-oxyacetic acid (cmo 5 U), uridine 5-oxyacetic acid methyl ester (mcmo) 5 U), 5-carboxymethyluridine (cm) 5 U), 1-carboxymethyl-pseudouridine, 5-carboxyhydroxymethyl-uridine (chm) 5 U), 5-carboxyhydroxymethyl-uridine methyl ester (mchm) 5 U), 5-methoxycarbonylmethyl-uridine (mcm) 5 U), 5-methoxycarbonylmethyl-2-thio-uridine (mcm) 5 s 2 U), 5-aminomethyl-2-thio-uridine (nm) 5 s 2 U), 5-methylaminomethyluridine (mnm) 5U), 1-ethyl-pseudouridine, 5-methylaminomethyl-2-thio-uridine (mnm) 5 s 2 U), 5-methylaminomethyl-2-seleno-uridine (mnm) 5 se 2 U), 5-carbamoylmethyluridine (ncm) 5 U), 5-Carboxymethylaminomethyluridine (cmnm) 5 U), 5-Carboxymethylaminomethyl-2-thio-uridine (cmnm) 5 s 2 U), 5-propynyl-uridine, 1-propynyl-pseudouridine, 5-tauric acid methyl-uridine (τm) 5 U), 1-Taurate methyl-pseudouridine, 5-Taurate methyl-2-thio-uridine (τm5s2U), 1-Taurate methyl-4-thio-pseudouridine, 5-methyl-2-thio-uridine (m 5 s 2 U), 1-methyl-4-thio-pseuuridine (m 1 s 4 ψ), 4-thio-1-methyl-pseuuridine, 3-methyl-pseuuridine (m 3 ψ), 2-thio-1-methyl-pseudouridine, 1-methyl-1-denitro-pseudouridine, 2-thio-1-methyl-1-denitro-pseudouridine, dihydrouridine (D), dihydrouridine, 5,6-dihydrouridine, 5-methyl-dihydrouridine (m) 5 D) 2-Thio-dihydrouridine, 2-Thio-dihydropseudouridine, 2-methoxy-uridine, 2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine, 4-methoxy-2-thio-pseudouridine, N1-methyl-pseudouridine, 3-(3-amino-3-carboxypropyl)uridine (acp) 3 U), 1-methyl-3-(3-amino-3-carboxypropyl)pseudouridine (acp) 3 ψ), 5-(isopentenylaminomethyl)uridine (inm) 5 U), 5-(isopentenylaminomethyl)-2-thio-uridine (inm 5 s 2 U), α-thio-uridine, 2′-O-methyl-uridine (Um), 5,2′-O-dimethyl-uridine (m) 5 Um), 2′-O-methyl-pseuuridine (ψm), 2-thio-2′-O-methyl-uridine (s) 2 Um), 5-methoxycarbonylmethyl-2′-O-methyl-uridine (mcm) 5 Um), 5-carbamoylmethyl-2′-O-methyluridine (ncm) 5Um), 5-carboxymethylaminomethyl-2′-O-methyl-uridine (cmnm) 5 Um), 3,2′-O-dimethyluridine (m) 3 Um), 5-(isopentenylaminomethyl)-2′-O-methyl-uridine (inm) 5 Um), 1-thio-uridine, deoxythymidine, 2′-F-arasu-uridine, 2′-F-uridine, 2′-OH-arasu-uridine, 5-(2-methoxycarbonylvinyl)uridine and 5-[3-(1-E-propenylamino)uridine. In some embodiments, the nucleoside containing the modified nucleobase is pseudouridine (ψ), N1-methyl-pseudouridine (m1ψ) or 5-methyl-uridine (m5U), especially N1-methyl-pseudouridine.

[0430] In some embodiments, replacing one or more uridines with a nucleoside containing a modified nucleobase includes replacing at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 10%, at least 25%, at least 50%, at least 75%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of the uridine.

[0431] During the synthesis of mRNA via in vitro transcription (IVT) using T7 RNA polymerase, significant amounts of aberrant products, including double-stranded RNA (dsRNA), are generated due to the enzyme's unconventional activity. dsRNA induces inflammatory cytokines and activates effector enzymes, leading to inhibition of protein synthesis. dsRNA can be removed from RNA (such as IVT RNA), for example, using a non-porous or porous C-18 polystyrene-divinylbenzene (PS-DVB) matrix by ion-pair reversed-phase HPLC. Alternatively, an enzyme-based method can be used, employing *E. coli* RNase III, which specifically hydrolyzes dsRNA but not ssRNA, to eliminate dsRNA contaminants from the IVT RNA formulation. Furthermore, dsRNA can be separated from ssRNA by using a cellulose material. In some embodiments, the RNA formulation is contacted with a cellulose material, and ssRNA is separated from the cellulose material under conditions that allow dsRNA to bind to the cellulose material but do not allow ssRNA to bind to the cellulose material. Suitable methods for providing ssRNA are disclosed, for example, in WO 2017 / 182524.

[0432] As used herein, “removal” or “elimination” refers to the feature of separating a population of first substances (such as non-immunogenic RNA) from a population of second substances (such as dsRNA), wherein the population of first substances is not necessarily devoid of the second substance, and the population of second substances is not necessarily devoid of the first substance. However, a population of first substances characterized by the removal of the second substance population has a measurably lower content of the second substance compared to a mixture of unseparated first and second substances.

[0433] In some embodiments, removing dsRNA (especially mRNA) from non-immunogenic RNA includes removing dsRNA such that less than 10%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, less than 0.5%, less than 0.3%, or less than 0.1% of the RNA in the non-immunogenic RNA composition is dsRNA. In some embodiments, the non-immunogenic RNA (especially mRNA) contains no or substantially no dsRNA. In some embodiments, the non-immunogenic RNA (especially mRNA) composition comprises a purified formulation of single-stranded nucleoside-modified RNA. For example, in some embodiments, the purified formulation of single-stranded nucleoside-modified RNA (especially mRNA) is substantially free of double-stranded RNA (dsRNA). In some implementations, the purified formulation is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% single-stranded nucleoside-modified RNA, relative to all other nucleic acid molecules (DNA, dsRNA, etc.).

[0434] In some embodiments, the non-immunogenic RNA (especially mRNA) is translated more efficiently in cells than standard RNA having the same sequence. In some embodiments, translation is enhanced by a 2-fold increase relative to its unmodified counterpart. In some embodiments, translation is enhanced by a 3-fold increase. In some embodiments, translation is enhanced by a 4-fold increase. In some embodiments, translation is enhanced by a 5-fold increase. In some embodiments, translation is enhanced by a 6-fold increase. In some embodiments, translation is enhanced by a 7-fold increase. In some embodiments, translation is enhanced by an 8-fold increase. In some embodiments, translation is enhanced by a 9-fold increase. In some embodiments, translation is enhanced by a 10-fold increase. In some embodiments, translation is enhanced by a 15-fold increase. In some embodiments, translation is enhanced by a 20-fold increase. In some embodiments, translation is enhanced by a 50-fold increase. In some embodiments, translation is enhanced by a 50-fold increase. In some embodiments, translation is enhanced by a 1000-fold increase. In some embodiments, the translation is enhanced by a factor of 2000. In some embodiments, the factor is 10-1000. In some embodiments, the factor is 10-100. In some embodiments, the factor is 10-200. In some embodiments, the factor is 10-300. In some embodiments, the factor is 10-500. In some embodiments, the factor is 20-1000. In some embodiments, the factor is 30-1000. In some embodiments, the factor is 50-1000. In some embodiments, the factor is 100-1000. In some embodiments, the factor is 200-1000. In some embodiments, the translation is enhanced by any other significant amount or range.

[0435] In some embodiments, the non-immunogenic RNA (especially mRNA) exhibits significantly lower innate immunogenicity than standard RNA having the same sequence. In some embodiments, the non-immunogenic RNA (especially mRNA) exhibits a 2-fold lower innate immune response than its unmodified counterpart. In some embodiments, innate immunogenicity is reduced by a 3-fold. In some embodiments, innate immunogenicity is reduced by a 4-fold. In some embodiments, innate immunogenicity is reduced by a 5-fold. In some embodiments, innate immunogenicity is reduced by a 6-fold. In some embodiments, innate immunogenicity is reduced by a 7-fold. In some embodiments, innate immunogenicity is reduced by an 8-fold. In some embodiments, innate immunogenicity is reduced by a 9-fold. In some embodiments, innate immunogenicity is reduced by a 10-fold. In some embodiments, innate immunogenicity is reduced by a 15-fold. In some embodiments, innate immunogenicity is reduced by a 20-fold. In some embodiments, innate immunogenicity is reduced by a 50-fold. In some embodiments, innate immunogenicity is reduced by a 100-fold. In some embodiments, innate immunogenicity is reduced by a 200-fold. In some embodiments, innate immunogenicity is reduced by a 500-fold. In some embodiments, innate immunogenicity is reduced by a 1000-fold. In some embodiments, innate immunogenicity is reduced by a 2000-fold.

[0436] The term "exhibiting significantly lower innate immunogenicity" refers to a detectable reduction in innate immunogenicity. In some embodiments, the term refers to a reduction such that an effective amount of non-immunogenic RNA (especially mRNA) can be administered without triggering a detectable innate immune response. In some embodiments, the term refers to a reduction such that repeated administration of non-immunogenic RNA (especially mRNA) can be performed without evoking an innate immune response sufficient to detectably reduce the production of proteins encoded by the non-immunogenic RNA. In some embodiments, the reduction allows repeated administration of non-immunogenic RNA (especially mRNA) without evoking an innate immune response sufficient to eliminate the detectable production of proteins encoded by the non-immunogenic RNA.

[0437] "Immunogenicity" is the ability of a foreign substance (such as RNA) to elicit an immune response in a human or other animal. The innate immune system is a relatively nonspecific and direct component of the immune system. It is one of the two main components of the vertebrate immune system, the other being the adaptive immune system.

[0438] Particles

[0439] Nucleic acids (such as RNA and / or DNA, especially mRNA) described herein can be present in particles containing (i) nucleic acids and (ii) at least one cationic or cationically ionizable compound (such as polymers or lipids complexed with nucleic acids). Electrostatic interactions between positively charged molecules (such as polymers and lipids) and negatively charged nucleic acids are involved in particle formation. This leads to the complexation and spontaneous formation of nucleic acid particles.

[0440] Different types of RNA-containing particles have been previously described for use in delivering RNA in particulate form (see, for example, Kaczmarek, JC et al., 2017, Genome Medicine 9, 60). For non-viral RNA delivery mediators, the encapsulation of RNA in nanoparticles physically protects the RNA from degradation and, depending on the specific chemical properties, can facilitate cellular uptake and endosome escape.

[0441] 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 particles contain a coating (e.g., one or more layers or thin layers) made 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 may also contain other substances that are not necessarily amphiphilic (e.g., other lipids). Thus, the particles can be monolayer or multilayer structures, wherein the substances constituting one or more layers or thin layers include one or more types of amphiphilic substances (particularly selected from the group consisting of amphiphilic lipids), optionally combined with other substances that are not necessarily amphiphilic (e.g., other lipids). In some embodiments, the term "particle" refers to micron or nanometer-sized structures, such as micron or nanometer-sized compact structures. According to this disclosure, the term "particle" includes nanoparticles.

[0442] “RNA particles” can be used to deliver RNA to a target site (e.g., cells, tissues, organs, etc.). RNA particles can be formed from lipids or lipid-like substances containing at least one cation or a cation-ionizable lipid. Without intending to be bound by any theory, it is believed that cations or cation-ionizable lipids or lipid-like substances combine with RNA to form aggregates, and that such aggregates produce colloidally stable particles.

[0443] The nucleic acid particles described in this article (e.g., RNA particles, DNA particles, or DNA / RNA particles) include formulations based on lipid nanoparticles (LNPs) and lipid complexes (LPXs).

[0444] Generally, lipid complexes (LPX) can be 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 comprises liposomes.

[0445] In some embodiments, liposomes are self-enclosed monolayer or multilayer vesicle particles, wherein the thin layer comprises a lipid bilayer and the encapsulated cavity contains an aqueous phase. A prerequisite for using liposomes to form nanoparticles is that the desired lipids in the mixture are capable of forming a layered (bilayer) phase in the aqueous environment of the application.

[0446] In some embodiments, liposomes comprise a single or multiple 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 and consist of positively charged (cationic) amine head groups linked by glycerol to a hydrocarbon chain or cholesterol derivative.

[0447] In some embodiments, the lipid complex is a multilayered liposome-based formulation that forms upon electrostatic interaction between cationic liposomes and nucleic acids (such as RNA and / or DNA). In some embodiments, the formed lipid complex exhibits a different internal molecular arrangement due to a transformation from a liposome structure to a compact nucleic acid-lipid complex (such as an RNA- and / or DNA-lipid complex). In some embodiments, these formulations are characterized by poor encapsulation and incomplete embedding of nucleic acids (such as RNA).

[0448] In some embodiments, LPX particles comprise amphiphilic lipids (particularly cationic or cationically ionizable amphiphilic lipids) and nucleic acids (such as RNA and / or DNA, especially mRNA) as described herein. In some embodiments, electrostatic interactions between positively charged liposomes (made from one or more amphiphilic lipids, particularly cationic or cationically ionizable amphiphilic lipids) and negatively charged nucleic acids (especially mRNA) lead to the recombination and spontaneous formation of nucleic acid-liposome complex particles. Positively charged liposomes can typically be synthesized using cationic or cationically ionizable amphiphilic lipids (such as DOTMA and / or DODMA) and other lipids (such as DOPE). In some embodiments, the nucleic acid (such as RNA and / or DNA, especially mRNA)-liposome complex particles are nanoparticles.

[0449] Generally, 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 to form particles that do not form a layered (bilayer) phase in water.

[0450] 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, together with phospholipids, form the surface of the LNP. In some embodiments, the surface comprises a bilayer. In some embodiments, cholesterol and ionizable lipids, in both charged and uncharged forms, may be distributed throughout the LNP.

[0451] In some embodiments, nucleic acids (such as RNA and / or DNA, e.g., mRNA) may non-covalently associate with particles as described herein. In embodiments, the nucleic acids (such as RNA and / or DNA, especially mRNA) may adhere to the outer surface of the particle (surface nucleic acids (such as surface RNA, especially surface mRNA)) and / or may be contained within the particle (encapsulated nucleic acids (such as encapsulated RNA, especially encapsulated mRNA)).

[0452] In some embodiments, the particles described herein (e.g., LNP and LPX) have dimensions (such as diameter) 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). Up to approximately 1000nm, up to approximately 950nm, up to approximately 900nm, up to approximately 850nm, up to approximately 800nm, up to approximately 750nm, up to approximately 700nm, up to approximately 650nm, up to approximately 600nm, up to approximately 550nm, or up to approximately 500nm), such as in the range of approximately 20 to approximately 1500nm, such as approximately 30 to approximately 1200nm, approximately 40 to approximately 1100nm, approximately 50 to approximately 1000nm, etc. 0nm, approximately 60 to approximately 900nm, approximately 70 to 800nm, approximately 80 to 700nm, approximately 90 to 600nm, or approximately 50 to 500nm, or approximately 100 to 500nm, such as in the range of 10 to 1000nm, 15 to 500nm, 20 to 450nm, 25 to 400nm, 30 to 350nm, 40 to 300nm, 50 to 250nm, 60 to 200nm, or 70 to 150nm.

[0453] In some embodiments, the particles described herein (e.g., LNP and LPX) have a range in some embodiments of 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, about 100 nm to about 500 nm, about 100 nm to about 450 nm, about 100 nm to about 400 nm, about 100 nm to about 350 nm, about 100 nm to about 300 nm, about 100 nm to about 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 Average diameter of 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, approximately 200nm to approximately 500nm, approximately 200nm to approximately 450nm, approximately 200nm to approximately 400nm, approximately 200nm to approximately 350nm, approximately 200nm to approximately 300nm, or approximately 200nm to approximately 250nm.

[0454] In some embodiments, the particles described herein are nanoparticles. The term "nanoparticle" refers to nanoscale particles comprising nucleic acids (especially mRNA) as described herein and at least one cation or cationically ionizable lipids, wherein all three external dimensions of the particle are in the nanoscale, i.e., at least about 1 nm and less than about 1000 nm. Preferably, the size of the particle is its diameter.

[0455] The nucleic acid particles (especially mRNA particles) described herein can exhibit polydispersity indices (PDI) of 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. For example, nucleic acid particles can exhibit polydispersity indices in the range of about 0.01 to about 0.4 or about 0.1 to about 0.3.

[0456] The N / P ratio gives 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 because nitrogen atoms (depending on pH) are generally positively charged, while phosphate groups are negatively charged. In the presence of charge balance, the N / P ratio depends on pH. Lipid formulations are typically formed with N / P ratios greater than 4, up to 12, because positively charged nanoparticles are considered favorable for transfection. In this case, RNA is considered to bind completely to the nanoparticles.

[0457] A wide range of methods can be used to prepare the nucleic acid particles (especially RNA particles, such as mRNA particles) described herein, which may include obtaining a colloid from at least one cationic or cationically ionizable lipid and mixing the colloid with nucleic acids to obtain nucleic acid particles.

[0458] As used herein, the term "colloid" refers to a class of homogeneous mixtures in which dispersed particles do not precipitate. The insoluble particles in the mixture are microscopic, with a particle size between 1 and 1000 nanometers. This mixture may be referred to as a colloid or a colloidal suspension. Sometimes, the term "colloid" refers only to the particles in the mixture, rather than the entire suspension.

[0459] For the preparation of colloids containing at least one cation or cationically ionizable lipids, conventional methods for preparing liposome vesicles, appropriately adapted, can be used in this paper. The most commonly used methods for preparing liposome vesicles generally involve the following basic stages: (i) dissolving the lipids in an organic solvent, (ii) drying the resulting solution, and (iii) hydrating the dried lipids (using various aqueous media).

[0460] In the thin-film 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 obtained lipid film is then hydrated using a suitable aqueous medium to produce a liposome dispersion. Additionally, a further size reduction step may be included.

[0461] Reverse-phase evaporation is an alternative method for thin-film hydration in the preparation of liposome vesicles, involving the formation of 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. Removal of the organic phase under reduced pressure yields an emulsion gel, which subsequently transforms into a liposome suspension.

[0462] The term "ethanol injection technique" refers to a process in which an ethanol solution containing lipids is rapidly injected through a needle into an aqueous solution. This action disperses the lipids throughout the solution and promotes the formation of lipid structures, such as lipid vesicles, like liposomes. Typically, the nucleic acid (such as RNA and / or DNA, especially mRNA) lipid complex particles described herein can be obtained by adding nucleic acids (such as RNA and / or DNA, especially mRNA) to colloidal liposome dispersions. In some embodiments, the ethanol injection technique is used to form such colloidal liposome dispersions as follows: an ethanol solution containing lipids (such as cationic or cationically ionizable lipids (such as DOTMA and / or DODMA) and other lipids) is injected into an aqueous solution under stirring. In some embodiments, the nucleic acid (such as RNA and / or DNA, especially mRNA) lipid complex particles described herein are obtained without an extrusion step.

[0463] The term "extrusion" or "extruded" refers to producing particles with a fixed cross-sectional profile. In particular, it refers to reducing the particle size, thereby forcing the particles through a filter with defined pores.

[0464] According to this disclosure, other methods that are characterized by not containing organic solvents can also be used to prepare colloids.

[0465] 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. Although 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.

[0466] In some embodiments, the LNP described herein, comprising nucleic acids (such as RNA and / or DNA) and at least one cation or a cationicly ionizable lipid, is prepared by: (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 cationicly ionizable lipid and (if present) one or more other lipids; and (c) mixing the nucleic acid (such as RNA and / or DNA) solution prepared in (a) with the ethanol solution prepared in (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.

[0467] In some embodiments, the LNP described herein, comprising nucleic acids (such as RNA and / or DNA) and at least one cation or cationic ionizable lipid, is prepared by: (a') preparing a colloidal formulation of liposomes or cationic or cationic ionizable lipids and (if present) one or more other lipids in an aqueous phase; and (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 (a') with the nucleic acid (such as RNA and / or DNA) solution prepared in (b'). Following step (c'), one or more steps selected from dilution and filtration (such as tangential flow filtration) may be performed.

[0468] This disclosure describes particles comprising nucleic acids (such as RNA and / or DNA, especially mRNA) and at least one cation or cationic ionizable lipid, and compositions comprising such particles, said lipid associating with nucleic acids (such as RNA and / or DNA) to form nucleic acid (such as RNA and / or DNA) particles. Nucleic acid (such as RNA and / or DNA) particles may contain nucleic acids (such as RNA and / or DNA) complexed with the particle in different forms through non-covalent interactions. The particles described herein are not viral particles, particularly infectious viral particles, i.e., they cannot virally infect cells.

[0469] Suitable cationic or cationically ionizable lipids are lipids that form nucleic acid particles and are included by the terms "particle-forming component" or "particle-forming agent." The terms "particle-forming component" or "particle-forming agent" refer to any component that associates with nucleic acids to form nucleic acid particles. Such components include any component that can serve as part of a nucleic acid particle.

[0470] 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) molecule, wherein the molecular parameters of the nucleic acid (such as RNA and / or DNA) molecules may be similar to or different from each other, such as in terms of molar mass or basic structural elements (such as molecular structure, capping (RNA only), coding regions or other features).

[0471] In granule formulations, each type of nucleic acid (such as RNA and / or DNA) can be formulated separately as a separate granule formulation. In this case, each separate granule formulation will contain one type of nucleic acid (such as RNA and / or DNA). The separate granule formulations can exist as separate 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 (typically each in the form of a solution containing nucleic acid (such as RNA and / or DNA)) together with a granule forming agent, thereby allowing granule formation. Each granule will specifically contain a specific type of nucleic acid (such as RNA and / or DNA) provided during granule formation (a separate granule formulation). In some embodiments, a composition (such as a pharmaceutical composition) comprises more than one separate granule formulation. The individual pharmaceutical compositions are referred to as a mixed granule formulation. The mixed granule formulation according to the invention can be obtained by the steps of separately forming separate granule formulations and then mixing the separate granule formulations. Through the mixing step, a formulation comprising a mixed population of granules containing nucleic acids (such as RNA and / or DNA) is obtained. The separate granule populations can be together in a single container, comprising a mixed population of separate granule formulations. Alternatively, all nucleic acid types (such as RNA and / or DNA) of the pharmaceutical composition can be formulated together as a combined particle formulation. Such a formulation can be obtained by providing a combined formulation (typically a combination solution) of all nucleic acid types (such as RNA and / or DNA) together with a particle-forming agent, thereby allowing particle formation. In contrast to mixed particle formulations, combined particle formulations typically contain particles containing more than one type of nucleic acid (such as RNA and / or DNA). In combined particle compositions, different nucleic acid types (such as RNA and / or DNA) are typically present together within a single particle.

[0472] polymer

[0473] Polymers are commonly used materials for nanoparticle-based delivery due to their high chemical flexibility. Typically, cationic polymers are used to electrostatically aggregate negatively charged nucleic acids into nanoparticles. These positively charged groups are usually composed of amines, which alter their protonation state in a pH range of 5.5 to 7.5, which is thought to lead to ionic imbalance, resulting in endosome disruption. Polymers such as poly-L-lysine, polyamidoamine, protamine, and polyethyleneimine, as well as naturally occurring polymers such as chitosan, have been used for nucleic acid delivery and are suitable as cationic polymers in this paper. Furthermore, some researchers have synthesized polymers specifically for nucleic acid delivery. In particular, poly(β-amino esters) have been widely used in nucleic acid delivery due to their ease of synthesis and biodegradability. Such synthetic polymers are also suitable as cationic polymers in this paper.

[0474] As used herein, “polymer” is given its usual meaning, referring to a molecular structure comprising one or more repeating units (monomers) linked by covalent bonds. The repeating units may be all identical, or in some cases, more than one type of repeating unit may be present within a polymer. In some cases, polymers are biologically derived, i.e., biopolymers such as proteins. In some cases, additional components may be present in the polymer, such as targeting moieties.

[0475] 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, in 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), etc. Block copolymers can have two (diblock copolymers), three (triblock copolymers), or more different blocks.

[0476] In some embodiments, the polymer is biocompatible. A biocompatible polymer is one that does not typically cause significant cell death at moderate concentrations. In some embodiments, the biocompatible polymer is biodegradable, meaning that the polymer is capable of chemically and / or biologically degrading within physiological environments, such as in vivo.

[0477] In some implementations, the polymer may be protamine or polyalkylene imide.

[0478] The term "protamine" refers to any of a variety of relatively low-molecular-weight, strongly basic proteins rich in arginine, found particularly in the sperm cells of many animals (such as fish) to associate with DNA, replacing somatic cell histones. Specifically, the term "protamine" refers to strongly basic proteins found in fish sperm that are soluble in water, do not coagulate upon heating, and primarily produce arginine upon hydrolysis. They are used in purified forms in long-acting insulin preparations and to neutralize the anticoagulant effect of heparin.

[0479] According to this disclosure, the term "protamine" as used herein is intended to include any protamine amino acid sequence obtained or derived from natural or biological sources, including fragments thereof and polymeric forms of said amino acid sequence or fragments thereof, as well as artificial and (synthetic) polypeptides specifically designed for a particular purpose and which cannot be isolated from natural or biological sources.

[0480] 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. 2 Up 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.

[0481] According to this disclosure, linear polyalkylene imides, such as linear polyethyleneimine (PEI), are preferred.

[0482] The cationic polymers (including polycationic polymers) considered for use herein include any cationic polymer capable of electrostatically binding nucleic acids. In one embodiment, the cationic polymers considered for use herein include any cationic polymer with which nucleic acids can associate, for example, by forming a complex with the nucleic acid or forming vesicles therein encapsulating or encapsulating the nucleic acid.

[0483] The particles described herein may also contain polymers other than cationic polymers, namely non-cationic polymers and / or anionic polymers. Anionic and neutral polymers are collectively referred to herein as non-cationic polymers.

[0484] lipids

[0485] The terms “lipid” and “lipid-like substance” are broadly defined herein as molecules comprising one or more hydrophobic moieties or groups and optionally one or more hydrophilic moieties or groups. Molecules comprising both hydrophobic and hydrophilic moieties are also commonly referred to as amphiphiles. Lipids are generally insoluble or sparingly soluble in water, but soluble in many organic solvents. In an aqueous environment, the amphiphilic nature allows molecules to self-assemble into organized structures and different phases. One of these phases consists of lipid bilayers, as they are present in vesicles, multilayer / monolayer liposomes, or membranes in an aqueous environment. Hydrophobicity can be imparted by comprising 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 and / or charged groups and include carbohydrate, phosphate, carboxylic acid, sulfate, amino, thiol, nitro, hydroxyl, and other groups.

[0486] As used herein, the term "hydrophobic" means any molecule, part, or group that is substantially immiscible or insoluble in aqueous solutions. The term "hydrophobic group" includes hydrocarbons having at least six carbon atoms. A hydrophobic group may have functional groups (e.g., ethers, esters, halides, etc.) and atoms other than carbon and hydrogen, provided that the group satisfies the condition of being substantially immiscible or insoluble in aqueous solutions.

[0487] The term "hydrocarbon" includes alkyl, alkenyl, or alkynyl groups as defined herein. 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, hydrocarbon groups may also include cyclic (alkyl, alkenyl, or alkynyl) groups or aryl groups, provided that the overall polarity of the hydrocarbon remains relatively nonpolar.

[0488] The term "alkyl" refers to a saturated straight-chain or branched monovalent hydrocarbon moiety that may have six to thirty, typically six to twenty, and often six to eighteen carbon atoms. Exemplary nonpolar alkyl groups include, but are not limited to, hexyl, decyl, dodecyl, tetradecyl, hexadecyl, octadecyl, etc.

[0489] 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, typically 6 to 20, and often 6 to 18.

[0490] 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.

[0491] As used herein, the term "amphiphilic" refers to a molecule having both a polar and a nonpolar moiety. Typically, amphiphilic compounds have a polar head attached to a long hydrophobic tail. In some embodiments, the polar moiety is soluble in water, while the nonpolar moiety is insoluble in water. Furthermore, the polar moiety may have a positive or negative charge. Alternatively, the polar moiety may have both a positive and a negative charge and be an amphoteric ion or an inner salt. For the purposes of this disclosure, amphiphilic compounds may be, but are not limited to, one or more natural or non-natural lipids and lipid-like compounds.

[0492] 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 cannot be strictly considered lipids, particularly amphiphilic substances. For example, the term includes compounds capable of forming amphiphilic layers (when they are present in vesicles, multilayer / monolayer liposomes, or membranes in an aqueous environment) and includes surfactants or synthetic compounds having both hydrophilic and hydrophobic portions. Generally, the term refers to molecules containing hydrophilic and hydrophobic portions with different structural organization, which may be similar to or dissimilar to the structural organization of 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 the area occupied by the polar head group. Lipids have low solubility as monomers and tend to aggregate into planar bilayers that are insoluble in water. 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 into water-soluble spherical or elliptical micelles. While lipids also possess the same general structure as surfactants—a polar hydrophilic head group and a nonpolar hydrophobic tail—lipids differ from surfactants in monomer shape, the type of aggregates formed in solution, and the concentration range required for aggregation. As used herein, the term "lipid" should be interpreted to encompass both lipids and lipid-like substances, unless otherwise indicated herein or clearly contradicted by the context.

[0493] Lipids are generally classified into eight categories: fatty acids, glycerides, glycerophospholipids, sphingolipids, glycolipids, polyketides (derived from the condensation of ketoacyl subunits), sterol lipids, and prenol lipids (derived from the condensation of isoprene subunits). Although the term "lipid" is sometimes used synonymously with fat, fat is a subgroup of lipids called triglycerides. Lipids also encompass molecules such as fatty acids and their derivatives (including triglycerides, diglycerides, monoglycerides, and phospholipids) as well as steroids (i.e., sterol-containing metabolites, such as cholesterol or its derivatives). Examples of cholesterol derivatives include, but are not limited to, cholesterol alcohols, cholesterol ketones, cholesterol ketones, coprosterol, cholesterol-2'-hydroxyethyl ether, cholesterol-4'-hydroxybutyl ether, tocopherol and its derivatives, and mixtures thereof.

[0494] Fatty acids, or fatty acid residues, are diverse molecular groups composed of hydrocarbon chains terminated by carboxylic acid groups; this arrangement endows the molecule with a polar hydrophilic end and a water-insoluble nonpolar hydrophobic end. The carbon chains, typically 4 to 24 carbons long, can be saturated or unsaturated and can be linked to functional groups containing oxygen, halogens, nitrogen, and sulfur. If fatty acids contain double bonds, they can exhibit cis or trans geometric isomers, which significantly affect the molecular configuration. Cis double bonds cause the fatty acid chain to bend, an effect exacerbated by the presence of more double bonds in the chain. Other major lipid classes within the fatty acid category are fatty esters and fatty amides.

[0495] Glycerides consist of mono-, di-, and tri-substituted glycerols, the most well-known being fatty acid triesters of glycerol, called triglycerides. The term "triacylglycerol" is sometimes used synonymously with "triglyceride." In these compounds, the three hydroxyl groups of glycerol are typically esterified 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.

[0496] 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 sphingomyelin is also classified as a phospholipid) are phosphatidylcholine (also known as PC, GPCho, or lecithin), phosphatidylethanolamine (PE or GPEtn), and phosphatidylserine (PS or GPSer).

[0497] Sphingolipids are a complex family of compounds sharing a common structural feature—a sphingoid base backbone. The dominant sphingoid base in mammals is generally referred to as sphingosine. Ceramides (N-acyl-sphingoid bases) are the major subclass of sphingoid base derivatives of fatty acids with amide linkages. These fatty acids are typically saturated or monounsaturated, with 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 possess 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 sphingoid base via glycosidic bonds. Examples of these include simple and complex glycosphingolipids such as cerebrosides and gangliosides.

[0498] Steroid lipids, such as cholesterol and its derivatives, or tocopherol and its derivatives, are important components of membrane lipids, along with glycerophospholipids and sphingomyelins.

[0499] Glycolipids are compounds in which fatty acids are directly linked to the sugar backbone to form a structure compatible with the membrane bilayer. In glycolipids, monosaccharides replace the glycerol backbone present in glycerides and glycerophospholipids. The most familiar glycolipid is the acylated glucosamine precursor of the lipid A component of lipopolysaccharides in Gram-negative bacteria. The typical lipid A molecule is a disaccharide of glucosamine, which is derivatized by up to seven fatty acyl chains. The smallest lipopolysaccharide required for the growth of *E. coli* is Kdo2-lipid A, a hexaacylated disaccharide of glucosamine glycosylated by two 3-deoxy-D-manno-octulose (Kdo) residues.

[0500] Polyketides are synthesized by the polymerization of acetyl and propionyl subunits using classical enzymes and iterative and modular enzymes that share mechanical features with fatty acid synthases. They comprise a large number of secondary metabolites and natural products from animal, plant, bacterial, fungal, and marine sources, and exhibit great structural diversity. Many polyketides are cyclic molecules, and their backbones are often further modified by glycosylation, methylation, hydroxylation, oxidation, or other processes.

[0501] 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.

[0502] Cation / Cationally Ionizable Lipids

[0503] The nucleic acid particles (e.g., RNA and / or DNA particles) described herein contain at least one cationic or cationically ionizable lipid as a particle-forming agent. The cationic or cationically ionizable lipids considered for use herein include any cationic or cationically ionizable lipid (including lipid-like substances) capable of electrostatically binding nucleic acids. In some embodiments, the cationic or cationically ionizable lipids considered for use herein can associate with nucleic acids, for example, by forming a complex with the nucleic acid or forming vesicles therein encapsulating or encapsulating the nucleic acid.

[0504] 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. Generally, cationic lipids have a lipophilic moiety, such as a sterol, acyl chain, diacyl, or more acyl chains, and the head group of the lipid usually carries a positive charge.

[0505] In some embodiments, the cationic lipid carries a net positive charge only at a specific pH (particularly acidic pH), while it preferably does not carry a net positive charge at different pH levels (preferably higher pH levels, such as physiological pH), and is preferably uncharged, i.e., it is neutral. This ionizable behavior is thought to enhance efficacy by facilitating endosome escape and reducing toxicity compared to particles that remain cationic at physiological pH.

[0506] As used herein, "cationically ionizable lipid" refers to lipids or lipid-like substances that are either net positively charged or neutral (i.e., non-permanently cations). Therefore, cationically ionizable lipids can be positively charged or neutral, depending on the pH of the composition in which they are dissolved. For the purposes of this disclosure, unless contradicted, the term "cationically ionizable lipid" encompasses cationically ionizable lipids.

[0507] In some embodiments, the cationic or cationically ionizable lipid comprises a head group containing at least one positively charged or protonable nitrogen atom (N).

[0508] Examples of cationic or cationically ionizable lipids include, but are not limited to, N,N-dimethyl-2,3-diolenyloxypropylamine (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-Distearyloxy-N,N-dimethyl-3-aminopropane (DSDMA), 2,3-di(tetradecoxy)propyl-(2-hydroxyethyl)-dimethylazonium (DMRIE), 1,2-dimyristoyl-sn-glycerol-3-ethylphosphocholine (DMEPC), 1,2-dimyristoyl-3-trimethylammonium propane (DMTAP), 1,2-diolenoyloxypropyl-3-dimethyl-hydroxyethylammonium bromide (DORIE), 2,3-diolenoyloxy-N-[2-(sperminecarbamoyl)ethyl]-N,N-dimethyl-1-trifluoroacetic acid propane (DOSPA), 1,2-dilinolenoyloxy-N,N-dimethylaminopropane (DLinDMA), 1 2-Dilinylamino-N,N-dimethylaminopropane (DLenDMA), bis(octadecylamidoglycylsperyl)sperylamine (DOGS), 3-dimethylamino-2-(cholesterol-5-en-3-β-oxybutane-4-oxy)-1-(cis,cis-9,12-octadecadienoxy)propane (CLinDMA), 2-[5′-(cholesterol-5-en-3-β-oxy)-3′-oxaproloxy)-3-dimethyl-1-(cis,cis-9′,12′-octadecadienoxy)propane (CpLinDMA), N,N-dimethyl-3,4-dioleylaminobenzylamine (DMOBA), 1,2-N,N′-dioleylcarbamoyl-3-dimethyl Aminopropane (DOcarbDAP), 2,3-dilinoleyloxy-N,N-dimethylpropylamine (DLinDAP), 1,2-N,N′-dilinoleenylcarbamoyl-3-dimethylaminopropane (DLincarbDAP), 1,2-dilinoleylcarbamoyl-3-dimethylaminopropane (DLinCDAP), 2,2-dilinoleenyl-4-dimethylaminomethyl-[1,3]-dioxacyclopentane (DLin-K-DMA), 2,2-dilinoleenyl-4-dimethylaminoethyl-[1,3]-dioxacyclopentane (DLin-K-XTC2-DMA), 2,2-dilinoleenyl-4-(2-dimethylaminoethyl)-[1,3]-Dioxacyclopentane (DLin-KC2-DMA), heptadecyl-6,9,28,31-tetraen-19-yl-4-(dimethylamino)butyrate (DLin-MC3-DMA), N-(2-hydroxyethyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)-1-propanium bromide (DMRIE), (±)-N-(3-aminopropyl)-N,N-dimethyl-2,3-bis(cis-9-tetradecenyloxy)-1-propanium bromide (GAP-DMORIE), (±)-N-(3-aminopropyl)-N,N-dimethyl-2,3-bis(cis-9-tetradecenyloxy)-1-propanium bromide (GAP-DMORIE), (±)-N-(3-aminopropyl)-N,N-dimethyl-2,3 -bis(dodecyloxy)-1-propanediamine bromide (GAP-DLRIE), (±)-N-(3-aminopropyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)-1-propanediamine bromide (GAP-DMRIE), N-(2-aminoethyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)-1-propanediamine bromide (βAE-DMRIE), N-(4-carboxybenzyl)-N,N-dimethyl-2,3-bis(oleoyloxy)propane-1-ammonium (DOBAQ), 2-({8-[(3β)-cholesterol-5-en-3-yloxy]octyl}oxy)- N,N-Dimethyl-3-[(9Z,12Z)-octadec-9,12-dien-1-yloxy]propane-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-aminopropyl)amino]butylcarbamoyl)ethyl]-3,4-di[oleenoyloxy]-benzamide (MVL5), 1,2-dioleoyl-sn-glycerol-3-ethyl 2,3-Di(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 (DMORIE), di((Z)-non-2-en-1-yl)8,8'-((((2(dimethylamino)ethyl)thio)carbonyl)azanyl)dioctanoate (ATX), N,N-dimethyl-2,3-bis(dodecyloxy)propane-1-amine (DLDMA), N,N-dimethyl-2,3-Bis(tetradecoxy)propane-1-amine (DMDMA), di((Z)-non-2-en-1-yl)-9-((4-(dimethylaminobutyryl)oxy)heptadecanoic acid ester (L319), N-dodecyl-3-((2-dodecylcarbamoyl-ethyl)-{2-[(2-dodecylcarbamoyl-ethyl)-2-{(2-dodecylcarbamoyl-ethyl)-[2-(2-dodecylcarbamoyl-ethylamino)-ethyl]-amino}-ethylamino)propionamide (lipid 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 (lipid C12-200).

[0509] In some embodiments, the cationic or cationically ionizable lipid is DOTMA. In some embodiments, the cationic or cationically ionizable lipid is DODMA.

[0510] DOTMA is a cationic lipid with a quaternary ammonium head group. The structure of DOTMA can be represented as follows:

[0511]

[0512] DODMA is an ionizable cationic lipid with a tertiary amine head group. The structure of DODMA can be represented as follows:

[0513]

[0514] In some embodiments, the cationic or cationically ionizable lipids may comprise 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.

[0515] Other lipids

[0516] The particles described herein may also contain lipids (including lipid-like substances) other than cationic or cationically ionizable lipids (collectively referred to herein as cationic lipids), i.e., non-cationic lipids (including non-cationic or non-cationically ionizable 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 cationically ionizable lipids, optimizing the formulation of nucleic acid particles by adding other hydrophobic moieties (such as cholesterol and lipids) can enhance particle stability and nucleic acid delivery efficiency.

[0517] One or more additional lipids may or may not affect the total charge of the nucleic acid particles. In some embodiments, the one or more additional lipids are noncationic lipids or lipid-like substances. Noncationic lipids may comprise, 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.

[0518] In some embodiments, the nucleic acid particles described herein (especially those containing mRNA) comprise cationic or cationically ionizable lipids and one or more other lipids.

[0519] It is not desirable to be bound by theory, but the amount of cationic or cationically ionizable lipids relative to one or more other lipids can affect important nucleic acid particle characteristics, such as nucleic acid charge, particle size, stability, tissue selectivity, and biological activity. Therefore, in some embodiments, the molar ratio of cationic or cationically ionizable 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.

[0520] In some implementations, one or more additional lipids contained in the nucleic acid particles described herein (especially those containing mRNA) include one or more of the following: neutral lipids, steroids, and combinations thereof.

[0521] In some embodiments, the one or more additional lipids comprise neutral lipids as phospholipids. In some embodiments, the phospholipids are selected from the group consisting of phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidic acid, phosphatidylserine, and sphingomyelin. Specific phospholipids that can be used include, but are not limited to, phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidic acid, phosphatidylserine, or sphingomyelin. Such phospholipids particularly include diacylphosphatidylcholine, such as distearatelphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dimyristoylphosphatidylcholine (DMPC), bispentadecanylphosphatidylcholine, dilaurylphosphatidylcholine, dipalmitoylphosphatidylcholine (DPPC), diarachidonicylphosphatidylcholine (DAPC), disamylphosphatidylcholine (DBPC), and ditrisaccharoylphosphatidylcholine. DTPC, DLPC, POPC, 1,2-di-O-octadecenyl-sn-glycerol-3-phosphate choline (18:0 diether PC), OChemsPC, 1-oleoyl-2-cholesterolyl-sn-glycerol-3-phosphate choline (C16Lyso) PC), and phosphatidylethanolamines, especially diacylphosphatidylethanolamines, such as dioleoylphosphatidylethanolamine (DOPE), distearate-phosphatidylethanolamine (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′-racemic-glycerol) (DPPG), 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphate ethanolamine (POPE), N-palmitoyl-D-erythrosphoylphosphatidylcholine (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.

[0522] In some embodiments, the additional lipids include one of: (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 alcohols, cholesterol ketones, cholesterol ketones, coprosterol, cholesterol-2'-hydroxyethyl ether, cholesterol-4'-hydroxybutyl ether, tocopherol and its derivatives, and mixtures thereof.

[0523] Therefore, in some embodiments, the nucleic acid particles described herein (especially those containing mRNA) comprise (1) cationic or cationically ionizable lipids and phospholipids such as DOPE or (2) cationic or cationically ionizable lipids and phospholipids such as DOPE and cholesterol.

[0524] In some implementations, the nucleic acid particles described herein (especially those containing mRNA) contain (1) DOTMA and DOPE, (2) DOTMA, DOPE and cholesterol, (3) DODMA and DOPE, or (4) DODMA, DOPE and cholesterol.

[0525] DOPE is a neutral phospholipid. The structure of DOPE can be represented as follows:

[0526]

[0527] The structure of cholesterol can be represented as follows:

[0528]

[0529] 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 both a lipid moiety and a polyethylene glycol moiety. Polyethylene glycol-modified lipids are known in the art.

[0530] In some embodiments, the additional 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, the additional lipids (e.g., one or more phospholipids and / or cholesterol) comprise about 10 mol%, about 15 mol%, or about 20 mol% of the total lipids present in the particles.

[0531] In some embodiments, the additional 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.

[0532] Polymer-conjugated lipids

[0533] In some embodiments, the particles may comprise at least one polymer-conjugated lipid. The 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.

[0534] In some embodiments, the polymer-conjugated lipids are designed to spatially stabilize the lipid particles by forming a protective hydrophilic layer that shields the hydrophobic lipid layer. In some embodiments, when the polymer-conjugated 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.

[0535] Various PEG-conjugated lipids are known in the art, and include, but are not limited to, polyethylene glycol-modified diacylglycerols (PEG-DAG) such as 1-(monomethoxy-polyethylene glycol)-2,3-dimyristoylglycerol (PEG-DMG), polyethylene glycol-modified phosphatidylethanolamine (PEG-PE), PEG succinate diacylglycerol esters (PEG-S-DAG) such as 4-O-(2',3'-di(tetradecanoyloxy)propyl-1-O-(ω-methoxy(polyethoxy)ethyl)butane ester (PEG-S-DMG), polyethylene glycol-modified ceramides (PEG-cer), or PEG dialkoxypropyl carbamates 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.

[0536] In some embodiments, the particles may contain 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.

[0537] lipid complex particles

[0538] In some embodiments of this disclosure, the nucleic acids (such as RNA and / or DNA) described herein may be present in nucleic acid lipid complex particles (such as RNA and / or DNA lipid complex particles).

[0539] Lipid complexes (LPX) are electrostatic complexes typically formed by mixing pre-formed cationic liposomes with anionic nucleic acids (such as RNA and / or DNA). The resulting lipid complexes have a unique intramolecular arrangement resulting from the transformation of the liposome structure into a tight nucleic acid-lipid complex (such as RNA- and / or DNA-lipid complexes). These formulations are generally characterized by poor encapsulation and incomplete embedding of nucleic acids.

[0540] In some embodiments, nucleic acid lipid complex particles (such as RNA and / or DNA lipid complex particles) comprise both cationic lipids and other lipids. In an exemplary embodiment, the cationic lipid is DOTMA and the other lipid is DOPE.

[0541] 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.

[0542] The nucleic acid lipid complex particles (such as RNA and / or DNA lipid complex particles) described herein have an average diameter ranging from 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 some embodiments. In a specific embodiment, the RNA lipid complex particles have an average diameter of 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 nucleic acid lipid complex particles (such as RNA and / or DNA lipid complex particles) have an average diameter ranging from about 250 nm to about 700 nm. In another embodiment, the nucleic acid lipid complex particles (such as RNA and / or DNA lipid complex particles) have an average diameter ranging from about 300 nm to about 500 nm. In an exemplary embodiment, the nucleic acid lipid complex particles (such as RNA and / or DNA lipid complex particles) have an average diameter of about 400 nm.

[0543] The nucleic acid lipid complex particles (such as RNA and / or DNA lipid complex particles) and compositions containing nucleic acid lipid complex particles (such as RNA and / or DNA lipid complex particles) described herein can be used to deliver nucleic acids (such as RNA and / or DNA) to target tissues after parenteral administration, particularly after intravenous administration.

[0544] RNA-lipid complex particles targeting the spleen are described in WO 2013 / 143683, which is incorporated herein by reference. It has been found that RNA-lipid 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, RNA accumulation and / or RNA expression occur in the spleen after administration of the RNA-lipid complex particles. Therefore, nucleic acid (such as RNA and / or DNA) lipid complex particles of this disclosure can be used to express nucleic acids (such as RNA and / or DNA) in the spleen. In one embodiment, after administration of the nucleic acid (such as RNA and / or DNA) lipid complex particles, no or substantially no nucleic acid (such as RNA) accumulation and / or nucleic acid (such as RNA) expression occurs in the lungs and / or liver. In one embodiment, after administration of the nucleic acid (such as RNA and / or DNA) lipid complex particles, nucleic acid (such as RNA) accumulation and / or nucleic acid (such as RNA) expression occurs in antigen-presenting cells (such as professional antigen-presenting cells) in the spleen. Therefore, the nucleic acid (such as RNA and / or DNA) lipid 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 such antigen-presenting cells. In one embodiment, the antigen-presenting cells are dendritic cells and / or macrophages.

[0545] The charge of the nucleic acid (such as RNA and / or DNA) lipid complex particles of this disclosure 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 (moles)) * (Total positive charge in cationic lipids)] / [(Nucleic acid (such as RNA) concentration (moles)) * (Total negative charge in nucleic acids (such as RNA))]. The concentration of the nucleic acid (such as RNA) and the amount of at least one cationic lipid can be determined by those skilled in the art using conventional methods.

[0546] In one embodiment, at physiological pH, the charge ratio of positive to negative charge in nucleic acid (such as RNA and / or DNA) lipid 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, at physiological pH, the charge ratio of positive to negative charge in nucleic acid (such as RNA and / or DNA) lipid 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.

[0547] Lipid nanoparticles (LNP)

[0548] In some embodiments, the nucleic acids (such as RNA and / or DNA) described herein are present in the form of lipid nanoparticles (LNPs). LNPs may contain any lipids capable of forming particles, with one or more nucleic acid molecules linked to the particles, or with one or more nucleic acid molecules encapsulated within the particles.

[0549] 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.

[0550] In some embodiments, in the nucleic acid (such as RNA and / or DNA) LNPs described herein, the nucleic acids (such as RNA and / or DNA, especially mRNA) are bound to ionizable lipids occupying the central core of the LNP. PEG lipids, together with phospholipids, form the surface of the LNP. In some embodiments, the surface comprises a bilayer. In some embodiments, cholesterol and ionizable lipids in charged and uncharged forms may be distributed throughout the LNP.

[0551] 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.

[0552] In some embodiments, the LNP comprises cationic lipids, neutral lipids, steroids, polymer-conjugated lipids; and nucleic acids (such as RNA and / or DNA) encapsulated within or associated with lipid nanoparticles.

[0553] In some embodiments, the 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%.

[0554] In some embodiments, the neutral lipids are present at concentrations ranging from 5 to 15 mol%, 7 to 13 mol%, or 9 to 11 mol%.

[0555] In some embodiments, the steroid is present at a concentration ranging from 30 to 50 mol%, 35 to 45 mol%, or 38 to 43 mol%.

[0556] 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.

[0557] 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.

[0558] In some embodiments, the mole % is determined based on the total number of moles of lipids present in the lipid nanoparticles. In some embodiments, the mole % is determined based on the total number of moles of cationic lipids, neutral lipids, steroids, and polymer-conjugated lipids present in the lipid nanoparticles.

[0559] 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.

[0560] In some implementations, the steroid is cholesterol.

[0561] 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:

[0562] Or its pharmaceutically acceptable salts, tautomers or stereoisomers, wherein:

[0563] 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; and w has an average value ranging from 30 to 60. In some embodiments, R 12 and R 13 Each is independently a straight-chain saturated alkyl chain containing 12 to 16 carbon atoms. In some embodiments, w has an average value ranging from 40 to 55. In some embodiments, the average w is about 45. In some embodiments, R 12 and R 13 Each is independently a straight-chain saturated alkyl chain containing about 14 carbon atoms, and w has an average value of about 45.

[0564] In some embodiments, the PEGylated lipid is or includes 2-[(PEG)-2000]-N,N-bistetradecylacetamide.

[0565] In some embodiments, the cationic lipid component of LNP has the structure of formula (III):

[0566]

[0567] Or a pharmaceutically acceptable salt, tautomer, prodrug, or stereoisomer thereof, wherein:

[0568] 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;

[0569] G 1 and G 2 Each is an independent, unreplaced C1-C 12 Alkylene or C1-C 12 alkenyl;

[0570] G 3 It is C1-C 24 Alkylene, C1-C 24 C3-C8 cycloalkylene, C3-C8 cycloalkylene;

[0571] R a Is it H or C1-C? 12 alkyl;

[0572] R 1 and R 2 Each is independently C6-C 24 Alkyl or C6-C 24 alkenyl;

[0573] R 3 Is it H or OR? 5 CN, -C(=O)OR 4 -OC(=O)R 4 or -NR 5 C(=O)R 4 ;

[0574] R 4 It is C1-C 12 alkyl;

[0575] R 5 It is an H or C1-C6 alkyl group; and

[0576] x is 0, 1, or 2.

[0577] In some of the foregoing embodiments of formula (III), the lipid has one of the following structures (IIIA) or (IIIB):

[0578]

[0579] in:

[0580] A is a 3- to 8-membered cycloalkyl or cyclohexane ring;

[0581] R 6 Each time it appears, it is independently H, OH, or Cl-C. 24 alkyl;

[0582] n is an integer ranging from 1 to 15.

[0583] In some of the foregoing embodiments of formula (III), the lipid has structure (IIIA), and in other embodiments, the lipid has structure (IIIB).

[0584] In other embodiments of formula (III), the lipid has one of the following structures (IIIC) or (IIID):

[0585]

[0586] Where y and z are each an independent integer ranging from 1 to 12.

[0587] In any of the aforementioned embodiments of formula (III), L 1 or L 2One of them is -O (C = O)-. For example, in some implementations, L 1 and L 2 One is -O(C=O)-. In some different implementations of any of the foregoing, 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-.

[0588] In some different embodiments of formula (III), the lipid has one of the following structures (IIIE) or (IIIF):

[0589] In some of the foregoing embodiments of formula (III), the lipid has one of the following structures: (IIIG), (IIIH), (IIII), or (IIIJ):

[0590]

[0591] In some of the foregoing embodiments of formula (III), n is an integer ranging 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.

[0592] In some other of the foregoing embodiments of equation (III), y and z are each independently an integer ranging from 2 to 10. For example, in some embodiments, y and z are each independently an integer ranging from 4 to 9 or from 4 to 6.

[0593] In some of the aforementioned embodiments of formula (III), R 6 It is H. In other of the aforementioned embodiments, R 6 It is C1-C 24 Alkyl group. In other embodiments, R 6 It is OH.

[0594] In some implementations of formula (III), G 3 It is not replaced. In other implementations, G3 is replaced. In various different implementations, G... 3 It is a straight-chain C1-C 24 Alkylene or straight-chain C1-C 24 Alkenyl group.

[0595] In some other of the foregoing embodiments of formula (III), R 1 Or R2 Or both are C6-C 24 Alkenyl. For example, in some embodiments, R 1 and R 2 Each has the following structure independently:

[0596]

[0597] in:

[0598] R 7a and R 7b Each occurrence is independently H or C1-C 12 Alkyl; and

[0599] a is an integer from 2 to 12.

[0600] Where R 7a R 7b 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 ranging from 5 to 9 or from 8 to 12.

[0601] In some of the aforementioned embodiments of formula (III), R 7a The at least one occurrence of is H. For example, in some implementations, R 7a It is H each time it appears. In other different of the aforementioned embodiments, R... 7b The presence of at least one of the elements 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.

[0602] In different implementations of formula (III), R 1 or R 2 Or both have one of the following structures:

[0603]

[0604] In some of the aforementioned embodiments of formula (III), R 3 It is OH, CN, -C(=O)OR 4 -OC(=O)R 4 or -NHC(=O)R 4 In some implementations, R 4 It is methyl or ethyl.

[0605] In various different embodiments, the cationic lipid of formula (III) has one of the structures listed in the table below.

[0606] Representative compounds of formula (III).

[0607]

[0608]

[0609]

[0610]

[0611]

[0612]

[0613]

[0614] 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 include 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).

[0615] In some embodiments, the 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.

[0616] ALC-0159:

[0617]

[0618] In some embodiments, the cationic lipid is present in the LNP at an amount of about 45 to about 50 mol%. In some embodiments, the neutral lipid is present in the LNP at an amount of about 5 to about 15 mol%. In some embodiments, the steroid is present in the LNP at an amount of about 35 to about 45 mol%. In some embodiments, the PEGylated lipid is present in the LNP at an amount of about 1 to about 5 mol%.

[0619] In some embodiments, the LNP comprises about 45 to about 50 mol% of cationic lipids, about 5 to about 15 mol% of DSPC, about 35 to about 45 mol% of cholesterol, and about 1 to about 5 mol% of ALC-0159.

[0620] 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.

[0621] Measuring nucleic acid expression in transfected cells

[0622] To quantify the amino acid sequence expressed in cells transfected with nucleic acids encoding amino acid sequences, LC-MS / MS analysis can be used to quantify one or more peptides encoding nucleic acid sequences from total cell lysates, such as unique MITD (MHCI class transport domain) peptides or specific fragments of bioluminescent peptides, for example, present at the C-terminus of the encoded amino acid sequence.

[0623] cell

[0624] In some embodiments, the cells of the present invention mimic the nucleic acid uptake mechanism (such as RNA and / or DNA uptake mechanism) of a biological system. In some embodiments, the biological system is present in a human patient. In some embodiments, the biological system comprises antigen-presenting cells, preferably dendritic cells. In some embodiments, the dendritic cells comprise immature dendritic cells. In some embodiments, the cells are characterized by a large pinocytosis-mediated RNA uptake mechanism. In these embodiments, nucleic acids (such as RNA and / or DNA) are preferably formulated as lipid complex particles. In some embodiments, the cells are characterized by an endosome-mediated RNA uptake mechanism. In these embodiments, nucleic acids (such as RNA and / or DNA) are preferably formulated as lipid nanoparticles. In some embodiments, the cells are cells derived from animal cell lines, particularly using the same mechanism as receptor cells (i.e., the receptor's target cells, which will take up nucleic acid products, such as dendritic cells (DCs)) and suitable for routine testing in a QC environment (such as a GMP QC environment) (which takes up nucleic acid products, e.g., RNA-LPX, DNA-LPX, or RNA-LNP). In some embodiments, the cells are Chinese hamster ovary (CHO) cells. In some embodiments, the cells are selected from K562, HepG2, HEK293T, RAW, and C2C12 cells, such as those selected from K562, HEK293T, RAW, and C2C12 cells.

[0625] Cell lysis

[0626] In some embodiments, the method described herein includes lysing cells before determining the amount of an amino acid sequence comprising an amino acid sequence of a bioactive peptide or polypeptide or a fragment thereof.

[0627] In some embodiments, the methods described herein further include treating cell lysates prior to determining the amount of an amino acid sequence comprising an amino acid sequence of a bioactive peptide or polypeptide or a fragment thereof.

[0628] In some embodiments, processing cell lysates includes one or more of the following: denaturation, reduction, digestion with a proteolytic enzyme according to the invention (such as digestion using trypsin, Glu-C, LysN, Lys-C, Asp-N chymotrypsin, or any two or more of these proteolytic enzymes), alkylation, drying, reconstitution, and desalting, such as trypsin digestion, alkylation, and desalting.

[0629] Any method suitable for lysing cells can be used in the assays described herein. In some embodiments, buffers such as Tris / HCl buffers are used as lysis buffers, having, for example, a pH of about 7.5 (e.g., adjusted with HCl), and containing detergents such as mild zwitterionic detergents, for example, CHAPS (3-[(3-cholamidopropyl)dimethylammonium]-1-propanesulfonate) and / or CHAPSO (3-[(3-cholamidopropyl)dimethylammonium]-2-hydroxy-1-propanesulfonate). The lysis buffer may also contain chelating agents such as EDTA and / or one or more protease inhibitors.

[0630] The table below shows examples of lysis buffer formulations and final component concentrations.

[0631] Components quantity Final concentration Ultrapure water 9180μL - 200mM Tris / HCl, pH 7.5 500μL 10mM 5M NaCl 300μL 150mM 0.5M EDTA 20 μL 1mM CHAPS 100mg 1% (w / v) Protease inhibitor mixture 1 piece -

[0632] Quantification of expression products

[0633] Any method suitable for quantifying peptides and polypeptides can be used in the assays described herein. In a preferred embodiment, an antibody-free method is used. In a preferred embodiment, mass spectrometry is used. In some embodiments, liquid chromatography-tandem mass spectrometry (LC-MS / MS) is used. In some embodiments, targeted LC-MS is used.

[0634] In some implementations, mass spectrometry is used to determine the amount of amino acid sequence containing a functional sequence, such as a bioactive peptide or polypeptide or fragment thereof.

[0635] In some embodiments, liquid chromatography-mass spectrometry (LC-MS) is used to determine the amount of amino acid sequence containing a functional sequence, such as a bioactive peptide or polypeptide or fragment thereof.

[0636] In some implementations, targeted LC-MS is used to determine the amount of amino acid sequence containing a functional sequence, such as a bioactive peptide or polypeptide or fragment thereof.

[0637] In some embodiments, one or more amino acid sequences expressed by cells are used as a quantitative reference to determine the amount of amino acid sequence containing a functional sequence (such as a bioactive peptide or polypeptide or fragment thereof).

[0638] In some implementations, one or more amino acid sequences expressed by cells contain one or more amino acid sequences of housekeeping proteins.

[0639] In some implementations, the potency of nucleic acids (such as RNA and / or DNA) in inducing biological activity in biological systems includes the therapeutic potency of nucleic acids (such as RNA and / or DNA).

[0640] In some embodiments, the nucleic acid (such as RNA and / or DNA) has sufficient potency to induce biological activity, such as therapeutic potency, in a biological system if the amount of an amino acid sequence containing a bioactive peptide or polypeptide or a fragment thereof is above a predetermined cutoff value.

[0641] In some embodiments, if the amount of an amino acid sequence containing a bioactive peptide or polypeptide or fragment thereof is below a predetermined cutoff value, the nucleic acid (such as RNA and / or DNA) does not have sufficient potency to induce bioactivity, such as therapeutic potency, in a biological system.

[0642] In some implementations, a predetermined cutoff value is determined using nucleic acids (such as RNA and / or DNA) known to have acceptable potency in inducing biological activity (such as therapeutic potency) in biological systems.

[0643] In some implementations, the nucleic acid (such as RNA and / or DNA) used to determine the predetermined cutoff value and the nucleic acid (such as RNA and / or DNA) to be analyzed have the same chemical composition.

[0644] In some implementations, the methods described herein are used to analyze different batches of the same nucleic acids (such as RNA and / or DNA).

[0645] In some implementations, nucleic acids (such as RNA and / or DNA) or batches of nucleic acids (such as RNA and / or DNA) that have sufficient potency to induce biological activity (such as therapeutic potency) in a biological system are used or will be used for treatment, and / or nucleic acids (such as RNA and / or DNA) or batches of nucleic acids (such as RNA and / or DNA) that do not have sufficient potency to induce biological activity (such as therapeutic potency) in a biological system are not used or will not be used for treatment.

[0646] In some implementations, the potency of nucleic acids (such as RNA and / or DNA) in inducing biological activity in a biological system (such as the therapeutic potency of nucleic acids (such as RNA and / or DNA)) reflects the quality of nucleic acids (such as RNA and / or DNA), such as therapeutic quality.

[0647] In some implementations, the quality of nucleic acids (such as RNA and / or DNA) reflects whether and / or the extent to which they have been exposed to harmful conditions.

[0648] In some implementations, the harmful conditions include heat.

[0649] Using a living cell system instead of a cell-free system (such as reticulocyte lysate) offers the advantage that the power assays presented herein can indicate whether the efficacy of nucleic acids (such as RNA and / or DNA) in inducing biological activity in a biological system (such as the therapeutic efficacy of nucleic acids) reflects one or more parameters of the nucleic acid (or a formulation / composition containing nucleic acids, such as RNA-LPX, etc.), which is not provided by power assays based on cell-free systems.

[0650] Therefore, in some embodiments, the efficacy of nucleic acids (such as RNA and / or DNA) in inducing biological activity in a biological system (such as the therapeutic efficacy of nucleic acids (such as RNA and / or DNA)) reflects one or more parameters selected from the group consisting of particle parameters, formulation / composition parameters, and nucleic acid (such as RNA and / or DNA) parameters.

[0651] In some embodiments, particle parameters include size, surface charge, lipid mass (e.g., degradation), particle structure (e.g., laminarity), and intracellular nucleic acid (such as RNA and / or DNA) release, N / P ratio, concentration of free nucleic acids (such as RNA and / or DNA), and concentration of accessible nucleic acids (such as RNA and / or DNA).

[0652] In some implementations, formulation / composition parameters include osmotic p...

Claims

1. A method for simultaneously analyzing at least two different nucleic acid sequences, each nucleic acid sequence encoding a different amino acid sequence, wherein each of the at least two different amino acid sequences comprises a different functional sequence and a different adapter sequence, wherein each adapter sequence is proteolytically cleavable from its amino acid sequence, wherein the method comprises the following steps: (i) Provide the at least two different nucleic acid sequences; (ii) Introducing the at least two different nucleic acid sequences into cells; (iii) Expressing at least two different amino acid sequences; (iv) Proteolytically cleaved at least two different adapter sequences; (v) Determine the amount of each of the excised connector sequences; (vi) The amount of each of the excised adapter sequences is used as an indicator of the potency of each of the nucleic acid sequences in expressing each of the functional sequences in a biological system.

2. The method of claim 1, wherein the biological system is a biological system present in a human patient.

3. The method of claim 1 or claim 2, wherein the functional sequence is a tabletop.

4. The method of any one of claims 1 to 3, wherein the functional sequence is a T-cell epitope.

5. The method of any one of claims 1 to 4, wherein the functional sequence is an epitope presented to T cells via the major histocompatibility complex (MHC).

6. The method of any one of claims 1 to 5, wherein the functional sequence is a fixed antigen.

7. The method of any one of claims 1 to 5, wherein the functional sequence is a variable tabletop.

8. The method of any one of claims 1 to 7, wherein each of the linker sequences is preceded by a lysine or arginine residue at its N-terminus and contains a lysine or arginine residue at its C-terminus.

9. The method of any one of claims 1 to 8, wherein the lysine or arginine at the N-terminus of the linker sequence is naturally present within the functional sequence.

10. The method of any one of claims 1 to 8, wherein the lysine or arginine at the N-terminus of the linker sequence has been introduced into the functional sequence by site-directed mutagenesis.

11. The method of any one of claims 1 to 10, wherein each of the adapter sequences is different from all other polypeptide sequences that can be hydrolyzed in step (iv).

12. The method of any one of claims 1 to 11, wherein the proteolytic excision of step (iv) is performed using a proteolytic enzyme or a mixture of proteolytic enzymes.

13. The method of claim 12, wherein the proteolytic enzyme is trypsin.

14. The method of claim 12, wherein the mixture of proteolytic enzymes comprises trypsin and one or more other proteases selected from the group consisting of Glu-C, Lys-N, Lys-C, Asp-N and chymotrypsin.

15. The method of any one of claims 1 to 14, wherein mass spectrometry is used to determine the amount of each of the excised connector sequences.

16. The method of any one of claims 1 to 15, wherein liquid chromatography-mass spectrometry (LC-MS) is used to determine the amount of each of the excised connector sequences.

17. The method of any one of claims 1 to 16, wherein targeted LC-MS is used to determine the amount of each of the excised connector sequences.

18. The method of any one of claims 1 to 17, wherein each of the at least two amino acid sequences further comprises a sequence at the C-terminus of the linker sequence.

19. The method of claim 18, wherein the sequence at the C end of the connector sequence is an auxiliary structural domain sequence.

20. The method of claim 19, wherein, In each of the at least two amino acid sequences, the linker sequence is located at the C-terminus of the functional sequence, and the auxiliary domain sequence is located at the C-terminus of the linker sequence.

21. The method of any one of claims 1 to 20, wherein the method comprises lysing the cells prior to step (iv).

22. The method of claim 21, wherein the method further comprises treating cell lysates.

23. The method of claim 22, wherein processing the cell lysate comprises one or more steps selected from the group consisting of protease digestion, denaturation, reduction, alkylation, drying, remodeling, and desalting.

24. The method of any one of claims 1 to 23, wherein the at least two different nucleic acid sequences are RNA sequences, DNA sequences, or comprise at least one RNA sequence and at least one DNA sequence.

25. A method for analyzing the potency of nucleic acid sequences in expressing functional sequences in a biological system, wherein the method comprises simultaneously analyzing at least two different nucleic acid sequences, each nucleic acid sequence encoding a different amino acid sequence, wherein each of the at least two different amino acid sequences contains a different functional sequence and a different adapter sequence, wherein each adapter sequence is proteolytically cleavable from its amino acid sequence, wherein the method comprises the following steps: (i) Provide the at least two different nucleic acid sequences; (ii) Introducing the at least two different nucleic acid sequences into cells; (iii) Expressing at least two different amino acid sequences; (iv) Proteolytically cleaved at least two different adapter sequences; (v) Determine the amount of the excised connector sequence.

26. A reagent kit comprising: a) A first nucleic acid sequence comprising an insertion site of a first polynucleotide encoding a functional sequence, wherein the first nucleic acid sequence encodes an amino acid sequence comprising a first adapter sequence, wherein the first adapter sequence is side-mounted at a proteolytic cleavage site such that the first adapter sequence can be cleaved from its amino acid sequence, wherein the sequence of the first adapter sequence is different from any other sequence side-mounted at the same proteolytic cleavage site in its amino acid sequence or in an amino acid sequence encoded by a second nucleic acid sequence; and b) A second nucleic acid sequence comprising an insertion site of a second polynucleotide encoding a functional sequence, wherein the second nucleic acid sequence encodes an amino acid sequence comprising a second adapter sequence, wherein the second adapter sequence is side-mounted at a proteolytic cleavage site such that the second adapter sequence can be cleaved from its amino acid sequence, wherein the sequence of the second adapter sequence is different from any other sequence side-mounted at the same proteolytic cleavage site in its amino acid sequence or in the amino acid sequence encoded by the first nucleic acid sequence.

27. The kit according to claim 26 is used for simultaneously analyzing the efficacy of first and second nucleic acid sequences in expressing first and second functional sequences in a biological system.

28. Use of at least two nucleic acid sequences for simultaneously analyzing the potency of said at least two nucleic acid sequences in expressing at least two different functional sequences in a biological system, wherein each of said at least two nucleic acid sequences encodes an amino acid sequence comprising a different functional sequence and a different adapter sequence, wherein each adapter sequence is 6 to 30 amino acids in length and has the general formula: [X] n AND in X is any amino acid; n is an integer from 5 to 29; Y is either lysine or arginine; In addition, [X] n It may contain the amino acid sequence KP or RP, but it does not contain lysine or arginine. Each of these linker sequences is preceded by a lysine or arginine residue at its N-terminus.

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