Linker sequence efficacy assay for multiplex coding nucleic acids

Abstract

The present invention provides potency assays for simultaneously analyzing at least two different nucleic acid sequences, e.g., RNA and / or DNA sequences, encoding functional sequences such as antigens or epitopes. The potency assays of the present invention may be performed using nucleic acid sequences encoding at least two different functional sequences comprising at least two different antigens or epitopes.

Description

[Technical Field]

[0001] The present invention provides potency assays for simultaneously analyzing at least two different nucleic acid sequences (e.g., RNA and / or DNA sequences) encoding functional sequences, such as antigens or epitopes. The potency assays of the present invention may be performed using nucleic acid sequences encoding at least two different functional sequences comprising at least two different antigens or epitopes. Nucleic acid sequences analyzed by the methods of the present invention may be useful in downstream clinical applications for eliciting an immune response against two or more antigens or epitopes encoded by the nucleic acid sequences, e.g., in subjects where the immune response may be therapeutic or partially or fully protective. Thus, the nucleic acid sequences may be useful for vaccination. More particularly, the nucleic acid sequences may be useful as multivalent T cell-targeted vaccines. [Background technology]

[0002] Nucleic acids, such as DNA and RNA, are well known for their ability to encode biologically active proteins, but they also have other attractive properties as therapeutic agents: nucleic acid-based therapeutics are easy to manufacture and relatively inexpensive.

[0003] Generally, DNA is more stable than RNA, but has some potential safety drawbacks, such as the induction of anti-DNA antibodies and integration of the transgene into the host genome.

[0004] The use of RNA to deliver foreign genetic information to target cells offers an attractive alternative to DNA. Advantages of RNA include its transient expression and lack of transforming properties. RNA does not require nuclear infiltration for expression, and it cannot integrate into the host genome, thus eliminating the risk of carcinogenesis.

[0005] T cell vaccines are nucleic acid constructs designed to encode functional sequences that are highly immunogenic regions, or epitopes, of target antigens linked together into a single polypeptide. Multivalent T cell vaccines can contain multiple nucleic acid constructs. These T cell vaccines are designed to generate an immunogenic response through T cell recognition of epitopes presented by the major histocompatibility complex (MHC). In other words, the functional epitope sequences encoded by T cell vaccines are expressed and presented by the MHC, thereby generating an immunogenic response against the presented epitopes. Thus, the epitopes encoded by T cell vaccines function in a manner entirely different from more traditional means of eliciting an immune response, namely, through the expression of stable, folded antigens to be recognized by antibodies.

[0006] Potency testing is used to measure product attributes related to product quality and manufacturing control and is performed to ensure the identity, purity, strength (potency), and stability of the product used in all phases of clinical trials. Similarly, potency measurements are used to demonstrate that only product lots that meet defined specifications or acceptance criteria are administered in all phases of clinical trials and subsequent marketing authorization. Thus, defining biopharmaceutical potency is a central theme in product development and beyond.

[0007] A potency assay involves quantitative measurement of a specific criterion that describes a product's ability to achieve a defined biological effect. The measured criterion should be closely related to the product's intended biological effect and, ideally, to the product's clinical purpose. Measuring product potency is not the same as measuring clinical efficacy. Rather, it is a means of controlling product quality and providing appropriate release standards, especially under GMP. Typically, a separate potency assay must be developed for every product to be administered to a subject. In the rapidly evolving field of nucleic acids, where hundreds of different possible constructs exist and where antibodies for detection are rarely available, potency assays that can be easily adapted to new products would be extremely beneficial.

[0008] As part of the drug development process for T cell vaccines, efficacy 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 generate immunogenic responses by expressing stable, folded antigens that must be recognized by antibodies. Therefore, the development of efficacy assays for such vaccines cannot rely on typical antibody-based approaches such as flow cytometry, Western blot, or ELISA.

[0009] An additional consideration for the development of such efficacy assays is that multivalent T cell vaccines may encode several different epitopes. Therefore, a efficacy assay for a multivalent T cell vaccine must be able to simultaneously determine the expression of several different epitopes. Furthermore, T cell vaccines may encode de novo epitope sequences. This is because, in some cases, T cell vaccines are personalized vaccines with sequences that vary from patient to patient. Therefore, a T cell vaccine efficacy assay must be developed that can take into account the need to evaluate the efficacy of de novo or undetermined epitope sequences.

[0010] Briefly, there is a problem of providing an assay that can be used to determine the potency of antigens / epitopes that generate an immunogenic response by MHC presentation, where typical antibody-based approaches are not useful. There is also a problem of providing an assay that can simultaneously determine the potency of multiple different expressed functional sequences (e.g., epitopes), which may include newly expressed functional sequences. Such an assay would be useful for analyzing the potency of T cell vaccines, but would also be useful in any scenario where any of these limitations apply, such as any scenario in which the expression of two or more different analyte sequences is analyzed simultaneously. Summary of the Invention [Problem to be solved by the invention]

[0011] The present invention provides a method for simultaneously analyzing at least two different nucleic acid sequences using a linker sequence that can be proteolytically excised from the amino acid sequence. In particular, when at least two nucleic acid sequences each encode an amino acid sequence containing a different expressed functional sequence and a different linker sequence, the expressed linker sequence can be excised from the amino acid sequence, and the amount of each linker sequence can be used as an indicator of the efficacy of the nucleic acid sequence expressing the functional sequence. Furthermore, since the linker sequence can be measured and used as an indicator of the efficacy of the nucleic acid, direct measurement of the functional sequence is not required. In other words, each linker sequence acts as a molecular "barcode" for unique identification of the expression of its associated functional sequence. For this reason, the method of the present invention is also useful for analyzing the expression of undetermined de novo functional sequences, such as variable epitopes. Furthermore, it has been observed that the expression of linker sequences can be quantified by mass spectrometry, eliminating the need for antibody-based techniques for quantification. Based on these observations, a rapid, low-cost, reliable, easy-to-use and interpret potency assay is provided to measure, determine, identify, quantify, confirm, and / or validate the therapeutic potential of at least two different nucleic acids (e.g., RNA and / or DNA), each encoding at least two different functional sequences.

[0012] The present invention comprises various aspects, and it will be understood that various embodiments described herein as applicable to any one aspect of the invention are also generally applicable to all other aspects of the invention. [Means for solving the problem]

[0013] In a first aspect, the present invention relates to a method for simultaneously analyzing at least two different nucleic acid sequences, each 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 linker sequence, and each linker sequence is proteolytically cleavable from the amino acid sequence, the method comprising the steps of: (i) providing at least two different nucleic acid sequences; (ii) introducing at least two different nucleic acid sequences into the cell; (iii) expressing at least two different amino acid sequences; (iv) proteolytically excising at least two different linker sequences; (v) determining the amount of each excised linker sequence; (vi) using the amount of each of the excised linker sequences as an indicator of the efficacy of each of the nucleic acid sequences to express each of the functional sequences in a biological system. Includes.

[0014] In a second aspect, the present invention relates to a method for analyzing the efficacy of a nucleic acid sequence to express a functional sequence in a biological system, the method comprising the step of simultaneously analyzing at least two different nucleic acid sequences, each encoding a different amino acid sequence, each of the at least two different amino acid sequences comprising a different functional sequence and a different linker sequence, each linker sequence being proteolytically cleavable from its amino acid sequence, the method comprising the steps of: (i) providing at least two different nucleic acid sequences; (ii) introducing at least two different nucleic acid sequences into the cell; (iii) expressing at least two different amino acid sequences; (iv) proteolytically excising at least two different linker sequences; (v) determining the amount of excised linker sequence Includes.

[0015] In a third aspect, the present invention provides a method for producing a pharmaceutical composition comprising: a) a first nucleic acid sequence comprising an insertion site for a first polynucleotide encoding a functional sequence, the first nucleic acid sequence encoding an amino acid sequence comprising a first linker sequence, the first linker sequence being flanked by proteolytic cleavage sites such that the first linker sequence can be excised from the amino acid sequence, and the sequence of the first linker sequence being different from any other sequence flanking the same proteolytic cleavage site in the 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 for a second polynucleotide encoding a functional sequence, the second nucleic acid sequence encoding an amino acid sequence comprising a second linker sequence, the second linker sequence being flanked by proteolytic cleavage sites that allow the second linker sequence to be excised from the amino acid sequence, and the sequence of the second linker sequence being different from any other sequence flanking the same proteolytic cleavage site in the amino acid sequence or in the amino acid sequence encoded by the first nucleic acid sequence; The present invention relates to a kit comprising:

[0016] In a fourth aspect, the present invention provides a method for producing a composition comprising: a) a first nucleic acid sequence comprising an insertion site for a first polynucleotide encoding a functional sequence, the first nucleic acid sequence encoding an amino acid sequence comprising a first linker sequence, the first linker sequence being flanked by proteolytic cleavage sites so that the first linker sequence can be excised from the amino acid sequence; and b) a second nucleic acid sequence comprising an insertion site for a second polynucleotide encoding a functional sequence, the second nucleic acid sequence encoding an amino acid sequence comprising a second linker sequence, the second linker sequence being flanked by proteolytic cleavage sites so that the second linker sequence can be excised from the amino acid sequence; The present invention relates to a kit comprising:

[0017] 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 to express first and second functional sequences in a biological system.

[0018] In a sixth aspect, the present invention relates to the use of at least two nucleic acid sequences for simultaneously analyzing the efficacy of at least two nucleic acid sequences 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 linker sequence, each linker sequence being between 6 and 30 amino acids in length and having the general formula: [X] n Y where X is any amino acid, n is an integer from 5 to 29, Y is lysine or arginine; Additionally, [X] n may contain the amino acid sequence KP or RP, but otherwise does not contain lysine or arginine, Each linker sequence is preceded at its N-terminus by a lysine or arginine residue. [Brief explanation of the drawings]

[0019] [Figure 1]Schematic diagrams of four nucleic acid sequences according to the present invention are shown. Each nucleic acid sequence contains a nucleotide sequence encoding an immobilized antigen and a linker sequence that can be excised by proteolytic cleavage. The immobilized antigen contains a lysine residue (K) and includes part of the linker sequence. The linker sequence resulting from excision is shown below the four nucleic acid sequences. [Figure 2] Schematic diagrams of four nucleic acid sequences according to the present invention are shown. Each nucleic acid sequence contains a nucleotide sequence encoding a variable epitope and a linker sequence that can be excised by proteolytic cleavage. A lysine residue (K) is inserted between each functional sequence and the linker sequence. The linker sequence resulting from excision is shown below the four nucleic acid sequences. DETAILED DESCRIPTION OF THE INVENTION

[0020] Although the present disclosure is described in more detail below, it should be understood that the disclosure is not limited to the particular methodology, protocols, and reagents described herein, as these may 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 the present disclosure, which is limited 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.

[0021] The elements of the present disclosure are described in more detail below. While these elements are listed with specific embodiments, it should be understood that they may be combined in any manner and in any number to create further embodiments. The various examples and preferred embodiments described should not be construed as limiting the disclosure to only the explicitly described embodiments. The description should be understood to support and encompass embodiments combining the explicitly described embodiments with any number of the disclosed and / or preferred elements. Furthermore, any permutation and combination of all elements described in this application should be considered to be disclosed by the description of this application, unless the context dictates otherwise.

[0022] In a first aspect, the present invention relates to a method for simultaneously analyzing at least two different nucleic acid sequences, each 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 linker sequence, and each linker sequence is proteolytically cleavable from the amino acid sequence, the method comprising the steps of: (i) providing at least two different nucleic acid sequences; (ii) introducing at least two different nucleic acid sequences into the cell; (iii) expressing at least two different amino acid sequences; (iv) proteolytically excising at least two different linker sequences; (v) determining the amount of each excised linker sequence; (vi) using the amount of each of the excised linker sequences as an indicator of the efficacy of each of the nucleic acid sequences to express each of the functional sequences in a biological system. Includes.

[0023] At least two different nucleic acid sequences: The methods of the present invention are carried out on at least two different nucleic acid sequences. It will be understood by those skilled in the art that the at least two different nucleic acid sequences can be present on the same nucleic acid or on two or more different nucleic acids. Similarly, when three or more nucleic acid sequences are used, one or more nucleic acid sequences can be present on one nucleic acid and one or more nucleic acid sequences can be present on one or more additional nucleic acids.

[0024] In one embodiment, at least two different nucleic acid sequences are present on a single nucleic acid.In one embodiment, at least two different nucleic acid sequences are present on different nucleic acids.It can be understood that, as long as the principle of the linker sequence of the present invention is followed, for example, as long as there are no identical linker sequences in a given assay, there is no strict practical limit to the number of nucleic acid sequences that can be analyzed simultaneously in the present invention.

[0025] In one embodiment, the method of the present invention comprises steps 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, 60, 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, 105, 106, 107, 108, 109, 110, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, It can be performed on 2, 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.

[0026] In an embodiment, the at least two different nucleic acid sequences are contained in the same nucleic acid molecule. In an embodiment, the at least two different nucleic acid sequences are contained in two or more nucleic acid molecules. In an embodiment, each of the at least two different nucleic acid sequences is contained in a different nucleic acid molecule. In an embodiment, at least one of the at least two different nucleic acid sequences is contained in a nucleic acid molecule that is different from the other of the at least two nucleic acid sequences.

[0027] It is also possible for the expression of multiple nucleic acid sequences on the same nucleic acid to be linked, i.e., each of the resulting amino acid sequences has the same or similar expression level. In this situation, the present invention may include a step of simultaneously analyzing 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 sequence will be expressed together with the first or second nucleic acid sequence, depending on whether the additional nucleic acid sequence is contained in the first or second nucleic acid, respectively.

[0028] As generally used herein with respect to certain elements of the invention, e.g., nucleic acids, amino acid sequences, functional sequences, and linker sequences of the invention, the term "different" means that the element in question does not consist of the same sequence as any other of the same elements in that aspect of the invention. For example, "at least two different amino acid sequences comprising different functional sequences" means that at least two amino acid sequences consist of sequences that differ from each other, and each amino acid sequence comprises a functional sequence that consists of a sequence that differs from the sequence of each other functional sequence contained in the amino acid sequence.

[0029] 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.

[0030] In one embodiment, the at least two different nucleic acid sequences analyzed in the methods of the invention are RNA sequences, DNA sequences, or comprise at least one RNA sequence and at least one DNA sequence. In some embodiments, the nucleic acids are DNA (e.g., one or more DNAs), RNA (e.g., one or more RNAs), or a mixture of DNA and RNA (e.g., one or more DNAs and one or more RNAs). In some embodiments, the DNA is in the form of a vector, e.g., a vector comprising DNA encoding an amino acid sequence comprising the amino acid sequence of a biologically active peptide or polypeptide. In some embodiments, the vector is a DNA vector.

[0031] References herein defining "one" nucleic acid sequence are understood to also apply to at least two nucleic acid sequences in the present invention.

[0032] Functional Sequence: The present invention ultimately analyzes the efficacy of a nucleic acid in expressing a "functional sequence." In this regard, the functional sequence is a sequence of an analyte. 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 comprise two or more antigen or epitope sequences. 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 by the major histocompatibility complex (MHC).

[0033] In one embodiment, the functional sequence is a fixed antigen or a variable epitope. In one embodiment, the functional sequence is a fixed antigen, such as a known tumor antigen. In another embodiment, the functional sequence is a variable epitope or a highly variable epitope, such as a patient-specific epitope or an individual epitope, or a de novo or undetermined epitope sequence.

[0034] 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 a vaccine (e.g., antigen, epitope), a protein for replacement therapy, an antibody, an antibody-like molecule, and a cytokine. In some embodiments, the biologically active peptide or polypeptide comprises 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.

[0035] Linker sequence: In the present invention, a "linker sequence" is a sequence that can be proteolytically excised from the respective amino acid sequence, and its amount can be determined and used as an indicator of the efficacy of the respective nucleic acid sequence in expressing the encoded functional sequence. In one embodiment, a sequence consisting of the linker sequence can be proteolytically excised from the respective amino acid sequence. In one embodiment, the linker sequence can be proteolytically excised from the amino acid sequence as an excised sequence consisting of the linker sequence.

[0036] It is understood that the linker sequence does not necessarily have to be distinct from the functional sequence of the present invention, and may even comprise a portion of the functional sequence of the present invention. This is particularly useful when the functional sequence of the present invention is a fixed antigen comprising a proteolytic cleavage site (see FIG. 1). Thus, "linker sequence" is merely the name given to a portion of an amino acid sequence that can be (or has been) excised from the amino acid sequence by proteolytic cleavage. This linker sequence may comprise a specific sequence ("linker barcode") included for this purpose, and may further comprise a portion of the functional sequence. When the functional sequence is a variable epitope, it is particularly useful for the linker sequence to not comprise a portion of the functional sequence (see FIG. 2). In this exemplary situation, the linker sequence is generated entirely from the "linker barcode" sequence included for this purpose, and there may be additional amino acid residues inserted between the functional sequence and the linker sequence to create a proteolytic cleavage site between the functional sequence and the linker sequence (as in FIG. 2).

[0037] In one embodiment, each linker sequence of the present invention encodes a polypeptide having a length of 6 to 30 amino acids, hi a preferred embodiment, each linker sequence encodes a polypeptide having a length of 8 to 12 amino acids.

[0038] In one embodiment, each linker sequence does not encode a functional protein.

[0039] In one embodiment, each linker sequence is adjacent to a proteolytic cleavage site. In one embodiment, "adjacent to a proteolytic cleavage site" means that the amino acid sequence comprising the linker sequence comprises a proteolytic cleavage site at the N-terminus, e.g., the very N-terminus, of the linker sequence, and the amino acid sequence comprising the linker sequence further comprises a proteolytic cleavage site at the C-terminus, e.g., the very C-terminus, of the linker sequence. A proteolytic cleavage site is understood to refer to a position in an amino acid sequence between two amino acid residues that are separately cleaved by a proteolytic enzyme. In one embodiment, each linker sequence is adjacent to a proteolytic cleavage site, and the linker sequence itself does not contain any of the same proteolytic cleavage sites. In one embodiment, each linker sequence does not contain any internal proteolytic cleavage site that is the same as any of the adjacent proteolytic cleavage sites. In one embodiment, amino acid residues within each linker sequence are not proteolytically cleaved by the same proteolytic enzyme that excises the linker sequence from its amino acid sequence. In one embodiment, each linker sequence is adjacent to a trypsin proteolytic cleavage site, and the linker sequence itself does not contain any trypsin proteolytic cleavage sites. In one embodiment, each linker sequence is adjacent to a trypsin or Lys-C proteolytic cleavage site, and the linker sequence itself does not contain any trypsin or Lys-C proteolytic cleavage sites. It will be appreciated that different proteases can be used in the practice of the invention, and therefore the proteolytic cleavage sites may vary depending on the protease used. A preferred protease is trypsin, optionally in combination with Lys-C.

[0040] Those skilled in the art will also understand that, depending on the position at which the protease used in the present invention cleaves the amino acid sequence, residues associated with protease cleavage may or may not form part of the linker sequence. For example, if a linker sequence is excised by cleavage at the N-terminus of the linker sequence using a protease that cleaves C-terminally at residue "Z," 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 include residue "Z." In one embodiment, preceding the N-terminus of the linker sequence means preceding the very N-terminus of the linker sequence. As a second example, if a linker sequence is excised by cleavage at the N-terminus of the linker sequence using a protease that cleaves N-terminally at residue "Z," residue "Z" will form part of the N-terminus of the linker sequence.

[0041] In one embodiment, the sequence of each linker 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 one embodiment, the sequence of each linker sequence is different from the sequences of all other different linker sequences used in the method of the invention. Thus, in one embodiment, the linker sequences used in the present invention each have a different sequence and are unique.

[0042] In one embodiment, the sequence of each linker sequence is different from the sequence of any other peptide or polypeptide that can be proteolytically cleaved from any of the functional sequences of the invention, hi one embodiment, the sequence of each linker sequence is different from any other sequence contained in any of the functional sequences.

[0043] In one embodiment, the sequence of each linker sequence is different from the sequence of any other peptide or polypeptide that can be proteolytically cleaved from amino acids encoded by at least two different nucleic acid sequences of the invention. In one embodiment, the sequence of each linker sequence is different from any other sequence encoded by at least two different nucleic acid sequences of the invention.

[0044] In one embodiment, the sequence of each linker sequence is different from the sequence of any other peptide or polypeptide that can be proteolytically excised from the proteome of a cell into which at least two different nucleic acid sequences are introduced according to the present invention. In one embodiment, the sequence of each linker sequence is different from any other sequence in the proteome of a cell into which at least two different nucleic acid sequences are introduced according to the present invention. In practice, a person skilled in the art can easily determine which sequences can be proteolytically excised from the proteome of a cell in a method according to the present invention by identifying the proteolytic enzymes and cells used and applying, for example, in silico analysis or in vitro experiments.

[0045] In other words, in one embodiment of the present invention, each linker sequence is different from any other sequence that can be proteolytically cleaved by the protease used in the present invention. In one embodiment, each linker sequence is different from any other sequence that is proteolytically cleaved by the protease used in the present invention from the amino acid sequence or cellular proteome used in the present invention. In one embodiment, each linker sequence is different from any other sequence that is proteolytically cleaved by trypsin from the amino acid sequence or cellular proteome used in the present invention.

[0046] In one embodiment, the proteolytic excision in step (iv) of the method of the invention is carried out using a proteolytic enzyme or a mixture of proteases. In one embodiment, the proteolytic enzyme is trypsin. In one embodiment, the mixture of proteases comprises trypsin and one or more additional proteases selected from the group consisting of Glu-C, Lys-N, Lys-C, Asp-N, and chymotrypsin. In one embodiment, the mixture of proteases comprises trypsin and Lys-C.

[0047] In one embodiment, the proteolytic enzyme used is trypsin, which cleaves proteins C-terminal to arginine (R) and lysine (K) residues except when followed by proline (P). In one embodiment, trypsin is used in conjunction with Lys-C, which cleaves only C-terminal to K residues, resulting in more efficient and complete digestion. Thus, in one embodiment, each of the linker sequences is preceded at its N-terminus by a lysine or arginine residue, and each of the linker sequences contains a lysine or arginine residue at its C-terminus. In one embodiment, each linker sequence is preceded at its N-terminus by a K or R residue, and each linker sequence further contains a K or R residue at its C-terminus. In a preferred embodiment, each linker sequence is preceded at its N-terminus by a K residue. 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 except when followed by a P residue. In one embodiment, each linker sequence does not contain any other K or R residues except when it is part of the sequence KP or RP. In one embodiment, the lysine or arginine N-terminal to the linker sequence is naturally occurring within the functional sequence. In another embodiment, the lysine or arginine N-terminal to the linker sequence has been introduced into the functional sequence by site-directed mutagenesis. In one embodiment, "N-terminal to the linker sequence" is understood to mean immediately N-terminal to the linker sequence.

[0048] Following excision by trypsin, peptides containing a single R or K are more easily analyzed by mass spectrometry because the positive charge at the C-terminus facilitates ionization, consistently resulting in peptide charge states of +2 and +3. Thus, in one embodiment, the linker sequence contains only a single R or K residue. In one embodiment, the linker sequence consists of only a single K or R residue, and that residue is at its C-terminus. In one embodiment, the linker sequence does not contain an internal trypsin proteolytic cleavage site.

[0049] In one embodiment, the protease used in the invention is Lys-C and the proteolytic cleavage site is C-terminal to a lysine (K) residue. In one embodiment, each linker sequence is preceded at the N-terminus by a K and the C-terminus of each linker sequence comprises a K. In one embodiment, each linker sequence does not contain an internal Lys-C proteolytic cleavage site.

[0050] In one embodiment, the protease used in the invention is Glu-C and the proteolytic cleavage site is C-terminal to an aspartic acid (D) or glutamic acid (E) residue. In one embodiment, each linker sequence is preceded at the N-terminus by D or E and the C-terminus of each linker sequence comprises D or E. In one embodiment, each linker sequence does not contain an internal Glu-C proteolytic cleavage site.

[0051] In one embodiment, the protease used in the present invention is chymotrypsin and the proteolytic cleavage sites are C-terminal to phenylalanine (F), tryptophan (W), and tyrosine (Y) residues. In one embodiment, each linker sequence is preceded at the N-terminus by F, W, or Y, and the C-terminus of each linker sequence comprises F, W, or Y. In one embodiment, each linker sequence does not contain an internal chymotrypsin proteolytic cleavage site.

[0052] In one embodiment, the C-terminus of a linker sequence "followed by" a residue means that the residue immediately follows. In one embodiment, the C-terminus of a linker sequence "followed by" a residue means that the amino acid sequence that includes the linker sequence includes that residue immediately following the linker sequence.

[0053] In one embodiment, the protease used in the invention is Lys-N and the proteolytic cleavage site is N-terminal to a lysine (K) residue. In one embodiment, the N-terminus of each linker sequence comprises a K and the C-terminus of each linker sequence is followed by a K. In one embodiment, each linker sequence does not contain an internal Lys-N proteolytic cleavage site.

[0054] In one embodiment, the protease used in the invention is Asp-N and the proteolytic cleavage site is N-terminal to an aspartic acid (D) or glutamic acid (E) residue. In one embodiment, the N-terminus of each linker sequence comprises D or E and the C-terminus of each linker sequence is followed by D or E. In one embodiment, each linker sequence does not contain an internal Asp-N proteolytic cleavage site.

[0055] It will be understood that proteolytic enzymes can be combined in the context of the present invention, and in this embodiment, all of the proteolytic cleavage sites of the respective proteases included in the combination apply. In one embodiment, the proteolytic enzyme combination comprises trypsin and Lys-C. In one embodiment, the proteolytic enzyme combination consists of trypsin and Lys-C.

[0056] In one embodiment, each of the linker sequences is not immunogenic. In one embodiment, each of the linker sequences does not elicit a specific immune response. In one embodiment, each of the linker sequences does not elicit an immune response specific to that linker sequence. In one embodiment, each of the linker sequences is immunogenically inert. In one embodiment, each of the linker sequences is immunologically masked. In one embodiment, each of the linker sequences does not stimulate an immune response. In one embodiment, each of the linker sequences 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 applicable to non-immunogenicity in animal or human cells, etc.

[0057] In one embodiment, the linker sequences are non-immunogenic in the sense that they do not induce an immune response. In one embodiment, the linker sequences are incapable of inducing an immune response against the linker sequence or cells expressing, containing, and presenting the linker sequence, e.g., human or animal cells. In one embodiment, the linker sequence does not induce an integrated biological response to the antigen, e.g., a cellular immune response, a humoral immune response, or both. In one embodiment, the linker sequence does not induce an immune response, including one or more responses selected from the group consisting of the expression of antibodies against one or more antigens and the proliferation of antigen-specific T lymphocytes, e.g., CD4+ and CD8+ T lymphocytes, e.g., CD8+ T lymphocytes. These can be detected by various in vitro proliferation or cytokine production tests. In one embodiment, the linker sequence does not induce a response by the immune system, e.g., upon administration to a mammal.

[0058] In one embodiment, in each of the nucleic acid sequences encoding the functional sequence and the linker sequence, the nucleotide sequence encoding the functional sequence is "in frame," i.e., in the same reading frame, as the nucleotide sequence encoding the linker sequence. In one embodiment, the nucleotide sequence encoding the functional sequence is separated from the nucleotide sequence encoding the linker sequence by 0 nucleotides or a multiple of 3 nucleotides.

[0059] Some specific linker sequences of the present invention are defined herein. In embodiments, these linker sequences are useful in the present invention using proteolytic enzymes, such as trypsin, that cleave amino acid sequences at the C-terminus of a lysine or arginine residue.

[0060] In one embodiment, the first and second nucleic acid sequences to be analyzed each encode amino acids that make up a linker sequence, the linker sequence being preceded at its N-terminus by a lysine or arginine residue, preferably lysine, and the linker sequence comprising a lysine or arginine residue at its C-terminus.

[0061] In one embodiment, the first nucleic acid sequence to be analyzed encodes an amino acid sequence comprising a first linker sequence, the linker sequence being preceded at its N-terminus by a lysine or arginine residue, preferably lysine, and the first linker sequence comprising a lysine residue at its C-terminus.

[0062] In a further embodiment of this type, the second nucleic acid sequence to be analyzed encodes an amino acid sequence comprising a second linker sequence, the second linker sequence being preceded at its N-terminus by a lysine or arginine residue, preferably lysine, and the second linker sequence comprising an arginine residue at its C-terminus.

[0063] In further embodiments of this type, the first and second linker sequences differ only by a C-terminal lysine residue or a C-terminal arginine residue, respectively.

[0064] In a further embodiment of this type, the third nucleic acid sequence to be analyzed encodes an amino acid sequence comprising a third linker sequence, the third linker sequence being preceded at its N-terminus by a lysine or arginine residue, preferably lysine, and the third linker sequence comprising an arginine residue at its C-terminus.

[0065] In a further embodiment of this type, the fourth nucleic acid sequence being analyzed encodes an amino acid sequence that includes a fourth linker sequence that is preceded at its N-terminus by a lysine or arginine residue, preferably lysine, and that includes an arginine residue at its C-terminus.

[0066] In further embodiments of this type, the first and second linker sequences differ only by a C-terminal lysine residue or a C-terminal arginine residue, respectively, and the third and fourth linker sequences differ only by a C-terminal lysine residue or a C-terminal arginine residue, respectively, but there are further differences between a) the first and second linker sequences and b) the third and fourth linker sequences.

[0067] When deriving a specific linker sequence for use in the present invention, it is understood that the linker sequences are unique to each other and to any other sequences excised by the protease used in the present invention, i.e., functional sequences, amino acids encoded by at least two nucleic acid sequences to be analyzed, and the proteome of the cell used to express the nucleic acid. Thus, the features described herein for these considerations can be logically combined. In one embodiment, each of the linker sequences is unique to any other sequences excised by the protease used in the present invention, i.e., functional sequences, amino acids encoded by at least two nucleic acid sequences to be analyzed, and the proteome of the cell used to express the nucleic acid.

[0068] In one embodiment of the present invention, the linker sequence is preceded at its N-terminus by a lysine residue and can be represented as follows: GGSGGGGSGGR / K. Thus, a portion of the amino acid sequence that makes up the linker sequence can be represented as follows: KΔGGSGGGGSGGR / K (Δ indicates the proteolytic cleavage site). After cleavage, this results in excision of the linker sequence as follows: GGSGGGGSGGR / K.

[0069] Additional sequence: In one embodiment, one or more or all of the at least two amino acid sequences in the method of the present invention comprise a functional sequence and a linker sequence, and the linker sequence is C-terminal to the functional sequence. In one embodiment, the linker sequence comprises the C-terminus of the amino acid sequence. It is therefore understood that a "linker" sequence may be C-terminal to the functional sequence and does not necessarily need to link the functional sequence to any other additional C-terminal sequence.

[0070] In another embodiment, one or more or all of the at least two amino acid sequences further comprise an additional sequence C-terminal to the linker sequence. Thus, in one embodiment, the linker sequence connects the functional sequence to an additional, further C-terminal sequence. In one embodiment, each of the at least two amino acid sequences further comprises a sequence C-terminal to the linker sequence. In one embodiment, the sequence C-terminal to 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 invention, the linker sequence is located C-terminal to the functional sequence and the auxiliary domain sequence is located C-terminal to the linker sequence.

[0071] In one embodiment, the auxiliary domain is a sequence that improves the function of the functional sequence. In one embodiment, where the functional sequence is an antigen or epitope, the auxiliary domain may be a sequence that improves the presentation of the antigen or epitope. In one embodiment, the auxiliary domain is a transport domain. In one embodiment, the auxiliary domain is an MITD domain. Further information regarding the MITD domain can be found in Kreiter et al. (J Immunol 180(1) (2008) 309-318). In an embodiment, each of the at least two nucleic acids in the present invention comprises an auxiliary domain having the same sequence.

[0072] Method Step: In one embodiment, step (ii) of the method of the invention comprises introducing, e.g., transfecting or transducing, at least two different nucleic acids into a cell in vitro. In one embodiment, step (ii) of the method of the invention comprises introducing, e.g., by administration to a subject, at least two different nucleic acid sequences into a cell in vivo.

[0073] Step (iii) of the method of the invention is understood to comprise expressing at least two different amino acid sequences in the cell of step (ii). In one embodiment, step (iii) is defined as attempting to express an amino acid sequence instead.

[0074] In one embodiment, the method of the present invention further comprises a step of lysing cells prior to step (iv). In one embodiment, the method further comprises a step of treating the cell lysate. In one embodiment, the step of treating the cell lysate comprises one or more steps selected from the group consisting of proteolytic enzyme digestion, denaturation, reduction, alkylation, drying, reconstitution, and desalting. In one embodiment, the step of treating the cell lysate comprises proteolytic enzyme treatment of the present invention to excise the linker sequence.

[0075] In an embodiment of step (v) of the method of the present invention, the amount of each excised linker sequence is determined using mass spectrometry. In one embodiment, the amount of each excised linker sequence is determined using liquid chromatography-mass spectrometry (LC-MS). In one embodiment, the amount of each excised linker sequence is determined using target LC-MS.

[0076] In one embodiment, the biological system of the present invention is a biological system present in a human patient.

[0077] In a second aspect, the present invention relates to a method for analyzing the efficacy of a nucleic acid sequence to express a functional sequence in a biological system, the method comprising the step of simultaneously analyzing at least two different nucleic acid sequences, each encoding a different amino acid sequence, each of the at least two different amino acid sequences comprising a different functional sequence and a different linker sequence, each linker sequence being proteolytically cleavable from its amino acid sequence, the method comprising the steps of: (i) providing at least two different nucleic acid sequences; (ii) introducing at least two different nucleic acid sequences into the cell; (iii) expressing at least two different amino acid sequences; (iv) proteolytically excising at least two different linker sequences; (v) determining the amount of excised linker sequence Includes.

[0078] Kits and Use In a third aspect, the present invention provides a method for producing a pharmaceutical composition comprising: a) a first nucleic acid sequence comprising an insertion site for a first polynucleotide encoding a functional sequence, the first nucleic acid sequence encoding an amino acid sequence comprising a first linker sequence, the first linker sequence being flanked by proteolytic cleavage sites such that the first linker sequence can be excised from the amino acid sequence, and the sequence of the first linker sequence being different from any other sequence flanking the same proteolytic cleavage site in the 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 for a second polynucleotide encoding a functional sequence, the second nucleic acid sequence encoding an amino acid sequence comprising a second linker sequence, the second linker sequence being flanked by proteolytic cleavage sites that allow the second linker sequence to be excised from the amino acid sequence, and the sequence of the second linker sequence being different from any other sequence flanking the same proteolytic cleavage site in the amino acid sequence or in the amino acid sequence encoded by the first nucleic acid sequence; The present invention relates to a kit comprising:

[0079] In one embodiment, the insertion site for the functional sequence is a multiple cloning site. In one embodiment, the nucleic acid sequence is a plasmid or vector.

[0080] In one embodiment, the kit of the present invention comprises: c) one or more additional nucleic acid sequences each comprising an insertion site for an additional polynucleotide encoding a functional sequence, wherein each additional nucleic acid sequence encodes an additional amino acid sequence comprising an additional linker sequence, wherein each additional linker sequence is flanked by a proteolytic cleavage site such that each additional linker sequence can be excised from the amino acid sequence, and wherein the sequence of each additional linker sequence differs from any other sequence flanking the same proteolytic cleavage site in the amino acid sequence or in an amino acid sequence encoded by the first nucleic acid sequence, the second nucleic acid sequence, or any other additional nucleic acid sequence.

[0081] In this embodiment, the sequences of the first and second linker sequences are also different from any other sequences flanking the same proteolytic cleavage site in any of the amino acid sequences encoded by the one or more additional nucleic acid sequences.

[0082] In a fourth aspect, the present invention provides a method for producing a composition comprising: a) a first nucleic acid sequence comprising an insertion site for a first polynucleotide encoding a functional sequence, the first nucleic acid sequence encoding an amino acid sequence comprising a first linker sequence, the first linker sequence being flanked by proteolytic cleavage sites so that the first linker sequence can be excised from the amino acid sequence; and b) a second nucleic acid sequence comprising an insertion site for a second polynucleotide encoding a functional sequence, the second nucleic acid sequence encoding an amino acid sequence comprising a second linker sequence, the second linker sequence being flanked by proteolytic cleavage sites so that the second linker sequence can be excised from the amino acid sequence; The present invention relates to a kit comprising:

[0083] In an embodiment of the kit of the present invention, the insertion site contained in each nucleic acid sequence contains a polynucleotide encoding a functional sequence. In one embodiment, the functional sequences are all different.

[0084] 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 to express first and second functional sequences in a biological system.

[0085] In one embodiment, the use of the kit according to the invention is for simultaneously analyzing the efficacy of a first, a second and one or more further nucleic acid sequences to express the first, the second and one or more further functional sequences in a biological system.

[0086] In one embodiment, the invention relates to the use of a kit according to the invention, wherein the insertion sites in each nucleic acid sequence comprise a polynucleotide encoding a functional sequence. In one embodiment, the functional sequences are all different.

[0087] In a sixth aspect, the present invention relates to the use of at least two nucleic acid sequences for simultaneously analyzing the efficacy of at least two nucleic acid sequences 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 linker sequence, each linker sequence being between 6 and 30 amino acids in length and having the general formula: [X] n Y where X is any amino acid, n is an integer from 5 to 29, Y is lysine or arginine; Additionally, [X] n may contain the amino acid sequence KP or RP, but otherwise does not contain lysine or arginine, and each linker sequence is preceded at its N-terminus by a lysine or arginine residue.

[0088] Applications: The present invention has broad application to any method for analyzing at least two different nucleic acid sequences, particularly for analyzing the expression or efficacy of at least two different nucleic acid sequences. In one embodiment, the present invention is a method for analyzing the efficacy of at least two different nucleic acid sequences for expressing at least two different functional sequences. In one embodiment, the present invention is a method for analyzing the efficacy of at least two nucleic acid sequences for expressing at least two functional sequences in a biological system.

[0089] In one embodiment, the biological system is not particularly limited. In one embodiment, the biological system is a cellular 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 vivo in a rodent, such as a mouse. In one embodiment, the biological system is in vivo in a human patient.

[0090] In one embodiment, the invention relates to both in vivo, ex vivo, and in vivo methods and uses. In a different embodiment, the invention relates to in vitro methods and uses. In a different embodiment, the invention relates to ex vivo methods and uses. In a different embodiment, the invention relates to in vivo methods and uses.

[0091] In one embodiment, the present invention is used to analyze multivalent T cell vaccines.

[0092] 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 clinical follow-up study. In one embodiment, the kit of the present invention is used as a companion diagnostic kit.

[0093] General definitions The practice of the present disclosure will employ, unless otherwise indicated, conventional chemical, biochemical, cell biology, immunological, and recombinant DNA techniques which are described in the art.

[0094] Throughout this specification and the claims that follow, unless the context requires otherwise, the word "comprise" and variations such as "comprises" and "comprising" will be understood to imply the inclusion of a stated feature, element, member, integer, or step, or group of features, elements, members, integers, or steps, but not the exclusion of any other feature, element, member, integer, or step, or group of features, elements, members, integers, or steps. The term "consisting essentially of" limits the claim or disclosure to the specified feature, element, member, integer, or step and to features, elements, members, integers, or steps that do not materially affect the basic and novel characteristics of the claim or disclosure. The term "consisting of" limits the claim or disclosure to the specified feature, element, member, integer, or step. The term "comprising" encompasses the term "consisting essentially of," which in turn encompasses the term "consisting of." Thus, wherever it appears in this application, the term "comprising" may be replaced by the term "consisting essentially of" or "consisting of." Similarly, wherever it appears in this application, the term "consisting essentially of" may be replaced by the term "consisting of."

[0095] As used in the context of describing this disclosure (particularly in the context of the claims), the terms "a," "an," and "the" and similar references are to be construed to encompass both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context.

[0096] All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context.

[0097] The use of any examples or exemplary language (e.g., "such as") provided herein is intended merely to better describe the disclosure and does not pose a limitation on the scope of the otherwise claimed disclosure. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the disclosure.

[0098] As used herein, the term "optional" or "optionally" means that the subsequently described event, circumstance, or condition may or may not occur, and that the description includes cases where the event, circumstance, or condition occurs and cases where it does not occur.

[0099] As used herein, "and / or" should be taken 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 taken as a specific disclosure of (i) X, (ii) Y, and (iii) each of X and Y, just as if each were individually set forth herein.

[0100] In the context of the present disclosure, the term "about" indicates an interval of accuracy that a person skilled in the art would understand to still ensure the technical effect of the feature in question. This term typically indicates a deviation from the indicated numerical 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%, e.g., ±0.01%. In some embodiments, "about" indicates a deviation of ±10% from the indicated numerical value. In some embodiments, "about" indicates a deviation of ±5% from the indicated numerical value. In some embodiments, "about" indicates a deviation of ±4% from the indicated numerical value. In some embodiments, "about" indicates a deviation of ±3% from the indicated numerical value. In some embodiments, "about" indicates a deviation of ±2% from the indicated numerical value. In some embodiments, "about" indicates a deviation of ±1% from the indicated numerical value. In some embodiments, "about" indicates a ±0.9% deviation from the indicated numerical value. In some embodiments, "about" indicates a ±0.8% deviation from the indicated numerical value. In some embodiments, "about" indicates a ±0.7% deviation from the indicated numerical value. In some embodiments, "about" indicates a ±0.6% deviation from the indicated numerical value. In some embodiments, "about" indicates a ±0.5% deviation from the indicated numerical value. In some embodiments, "about" indicates a ±0.4% deviation from the indicated numerical value. In some embodiments, "about" indicates a ±0.3% deviation from the indicated numerical value. In some embodiments, "about" indicates a ±0.2% deviation from the indicated numerical value. In some embodiments, "about" indicates a ±0.1% deviation from the indicated numerical value. In some embodiments, "about" indicates a ±0.05% deviation from the indicated numerical value. In some embodiments, "about" indicates a ±0.01% deviation from the indicated numerical value. As will be recognized by those skilled in the art, the specific deviation of the numerical value for a given technical effect will depend on the nature of that technical effect, for example, a natural or biological technical effect may generally have a greater deviation than a man-made or engineered technical effect.

[0101] Recitation of ranges of values ​​herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, and unless otherwise indicated herein, each separate value is incorporated herein as if it were individually referred to herein.

[0102] Each of the documents cited herein (including all patents, patent applications, scientific publications, manufacturer's specifications, instructions, etc.), whether supra or infra, is hereby incorporated by reference in its entirety. Nothing herein should be construed as an admission that the present invention is not entitled to antedate such disclosure by virtue of prior invention.

[0103] Specific Definitions The following provides definitions that apply to all aspects of this disclosure. The following terms have the following meanings unless otherwise indicated. Any terms not defined have their art-recognized meanings.

[0104] The "therapeutic potential" or "potency" of a nucleic acid (e.g., RNA and / or DNA) refers to the therapeutic qualities of the nucleic acid, i.e., the ability of the nucleic acid to provide a 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 expression, particularly strong expression, e.g., expression above a threshold, of a peptide or polypeptide encoded by the nucleic acid, which is indicative of the therapeutic potential of the nucleic acid. In one embodiment, therapeutic potential refers to the ability of a nucleic acid (e.g., RNA and / or DNA) to express a pharmaceutically active peptide or polypeptide in vivo, which exerts its pharmaceutical, e.g., therapeutic, effect.

[0105] In some embodiments, a nucleic acid (e.g., RNA and / or DNA) that exhibits strong expression, e.g., expression above a threshold value, has "sufficient therapeutic potential." The therapeutic potential of a nucleic acid is sufficient if the nucleic acid has the capacity to express an encoded pharmaceutically active peptide or polypeptide in vivo, thereby achieving a meaningful pharmaceutical effect, e.g., a therapeutic effect.

[0106] As used herein, phrases such as "determine the amount" or "determine the expression," or similar phrases in reference to an amino acid sequence (peptide or polypeptide), refer to determining the amount or presence of the amino acid sequence.

[0107] As used herein, terms such as "reduce" or "inhibit" refer to the ability to cause an overall decrease in a level, e.g., by 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 "inhibit" or similar phrases includes complete or essentially complete inhibition, i.e., a reduction to zero or essentially zero.

[0108] As used herein, the term "enhance" refers to the ability to produce an overall increase or enhancement in a level, for example, of at least 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.

[0109] As used herein, "physiological pH" refers to a pH of about 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.

[0110] As used in this disclosure, "% w / v" refers to weight to volume percent, which is a unit of concentration that measures the amount of solute in grams (g) expressed as a percentage of the total volume of the solution in milliliters (mL).

[0111] As used in this disclosure, "wt. %" refers to weight percent, which is a unit of concentration measuring the amount of a substance in grams (g) expressed as a percentage of the total weight of the total composition in grams (g).

[0112] 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.

[0113] As used in this disclosure, "mol % of total lipids" is defined as the ratio of the number of moles of one lipid component to the total number of moles of total lipids multiplied by 100. In this context, in some embodiments, the term "total lipids" includes lipids and lipid-like substances.

[0114] The term "ionic strength" refers to the mathematical relationship between the number of different ionic species in a particular solution and their respective charges. Thus, ionic strength, I, is calculated by the formula:

number

[0115] According to the present disclosure, the term "ionic strength" in some embodiments refers to the presence of monovalent ions. With regard to the presence of divalent ions, particularly divalent cations, their concentration, or the effective concentration (presence of free ions) due to the presence of chelating agents, is sufficiently low in some embodiments to prevent degradation of nucleic acids. In some embodiments, the concentration or effective concentration of divalent ions is below the catalytic level for hydrolyzing phosphodiester bonds between nucleotides, such as RNA nucleotides. In some embodiments, the concentration of free divalent ions is 20 μM or less. In some embodiments, free divalent ions are absent or essentially absent.

[0116] "Osmolality" refers to the concentration of a particular solute expressed as osmoles of solute per kg of solvent.

[0117] The term "lyophilizing" or "freeze-drying" refers to freeze-drying a substance by freezing the substance and then reducing the ambient pressure (e.g., to less than 15 Pa, e.g., less than 10 Pa, less than 5 Pa, or even 1 Pa or less) to cause the freezing medium in the substance to sublimate directly from the solid phase to the gas phase. Thus, the terms "lyophilizing" and "freeze-drying" are used interchangeably herein.

[0118] The term "spray drying" refers to spray drying a substance by mixing a (heated) gas with the fluid to be atomized (atomized) in a vessel (spray dryer), where the solvent from the droplets formed evaporates to produce a dry powder.

[0119] The term "reconstitute" relates to adding a solvent, such as water, to a dry product to return it to a liquid state, such as its original liquid state.

[0120] The term "recombinant" in the context of this disclosure means "produced by genetic engineering." In one embodiment, a "recombinant" in the context of this disclosure is not naturally occurring.

[0121] As used herein, the term "naturally occurring" refers to the fact that an object can be found in nature. For example, a peptide or nucleic acid is naturally occurring if it is present in a living organism (including viruses), can be isolated from a natural source, and has not been intentionally modified by humans in a laboratory. The term "naturally found" means "occurring in nature" and includes known objects as well as 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.

[0122] As used herein, the terms "room temperature" and "ambient temperature" are used interchangeably herein and refer to a temperature of at least about 15°C, e.g., 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 include 15°C, 16°C, 17°C, 18°C, 19°C, 20°C, 21°C, and 22°C. In some embodiments, the temperature is 15°C to about 25°C. In some embodiments, the temperature is 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.

[0123] The term "EDTA" refers to ethylenediaminetetraacetic acid disodium salt. All concentrations are given in terms of EDTA disodium salt.

[0124] The term "cryoprotectant" relates to a substance added to a formulation to protect the active ingredient during the freezing step.

[0125] The term "lyoprotectant" relates to a substance added to a formulation to protect the active ingredient during the drying stage.

[0126] According to the present disclosure, the term "peptide" refers to a substance that includes about 2 or more, about 3 or more, about 4 or more, about 6 or more, about 8 or more, about 10 or more, about 13 or more, about 16 or more, about 20 or more, and up to about 50, about 100, or about 150 consecutive amino acids linked together via peptide bonds. The term "polypeptide" refers to large peptides, particularly peptides having at least about 151 amino acids. Both "peptide" and "polypeptide" are protein molecules.

[0127] The term "biological activity" refers to a response of a biological system to a molecule. Such a biological system may be, for example, a cell or an organism. In some embodiments, such a response is therapeutically or pharmaceutically useful. In some embodiments, biological activity includes pharmaceutical activity.

[0128] The term "biological system," as used herein, refers to any system that interacts or can interact with 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, individual cells, collections of cells such as cell cultures, organs, tissues, and multicellular organisms, such as individuals or subjects, e.g., human patients.

[0129] In some embodiments, a biological system is present in or is an individual or subject, and the biological activity in such a biological system is a therapeutically or pharmaceutically useful activity, i.e., the biological activity results in or contributes to a therapeutically or pharmaceutically useful effect.

[0130] According to various embodiments of the present disclosure, nucleic acids (e.g., RNA and / or DNA) encoding a peptide or polypeptide are taken up or introduced, i.e., transfected or transduced, into a cell, which is present in vitro or in a subject, resulting in expression of the peptide or polypeptide. The cell may, for example, express the encoded peptide or polypeptide intracellularly (e.g., in the cytoplasm and / or nucleus), secrete the encoded peptide or polypeptide, and / or express it on its surface.

[0131] In accordance with the present disclosure, terms such as "expressing nucleic acid" and "encoding nucleic acid" or similar terms are used interchangeably herein to mean, with respect to a particular peptide or polypeptide, that the nucleic acid, when present in an appropriate environment, e.g., a cell, can be expressed to produce said peptide or polypeptide.

[0132] The term "portion" refers to a fraction. With respect to a particular structure such as an amino acid sequence or a protein, the term "portion" may designate a contiguous or non-contiguous fraction of said structure.

[0133] The terms "portion" and "fragment" are used interchangeably herein and refer to a continuous element. For example, a portion of a structure, such as an amino acid sequence or protein, refers to a continuous element of said structure. When used in the context of a composition, the term "portion" refers to a portion of the composition. For example, a portion of a composition can be any portion between 0.1% and 99.9% of the composition (e.g., 0.1%, 0.5%, 1%, 5%, 10%, 50%, 90%, or 99%).

[0134] The term "fragment," in reference to an amino acid sequence (peptide or polypeptide), refers to a portion of the amino acid sequence, i.e., a sequence representing an amino acid sequence truncated at the N-terminus and / or C-terminus. A C-terminally truncated fragment (N-terminal fragment) can be obtained, for example, by translating a truncated open reading frame lacking the 3' end of the open reading frame. An N-terminally truncated fragment (C-terminal fragment) can be obtained, for example, by translating a truncated open reading frame lacking the 5' end of the open reading frame, as long as the truncated open reading frame contains an initiation codon that serves to initiate translation. A fragment of an amino acid sequence contains, 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 can contain, for example, at least 6, in particular 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. Fragments of an amino acid sequence include, for example, sequences of up to 8, in particular up to 10, up to 12, up to 15, up to 20, up to 30 or up to 55 consecutive amino acids of the amino acid sequence.

[0135] "Variant," as used herein and in reference to an amino acid sequence (peptide or polypeptide), refers to an amino acid sequence that differs from a parent amino acid sequence by at least one amino acid (e.g., a different amino acid, or a modification of the same amino acid). The parent amino acid sequence may be a naturally occurring or wild-type (WT) amino acid sequence, or may be a modified version 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, e.g., 1 to about 20 amino acid differences compared to the parent, e.g., 1 to about 10 or 1 to about 5 amino acid differences.

[0136] As used herein, "wild-type" or "WT" or "native" refers to an amino acid sequence found in nature, including allelic variations. A wild-type amino acid sequence, peptide, or polypeptide has an amino acid sequence that has not been intentionally modified.

[0137] 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, splice variants, post-translationally modified variants, conformations, isoforms, allelic variants, species variants, and species homologs, particularly naturally occurring species homologs. The term "variant" particularly includes fragments of an amino acid sequence.

[0138] Amino acid insertion variants include the insertion of a single or two or more amino acids into a specific amino acid sequence. In amino acid sequence variants with insertions, one or more amino acid residues are inserted at specific sites in the amino acid sequence, although random insertion with appropriate screening of the resulting products is also possible. Amino acid addition variants include the fusion of one or more amino acids, e.g., 1, 2, 3, 5, 10, 20, 30, 50, or more amino acids, at the amino and / or carboxy termini. Amino acid deletion variants are characterized by the removal of one or more amino acids from the sequence, e.g., the removal of 1, 2, 3, 5, 10, 20, 30, 50, or more amino acids. The deletion may occur at any position in the protein. Amino acid deletion variants, including deletions at the N-terminus and / or C-terminus of a protein, are also referred to as N-terminal and / or C-terminal truncation variants. Amino acid substitution variants are characterized by the removal of at least one residue in the sequence and the insertion of another residue in its place. Preference is given to modifications at positions in the amino acid sequence that are not conserved between homologous peptides or polypeptides, and / or to substitutions of amino acids with other amino acids having similar properties. In some embodiments, the amino acid changes in the peptide or polypeptide variants are conservative amino acid changes, i.e., substitutions of similarly charged or uncharged amino acids. Conservative amino acid changes involve the substitution of one member of a family of amino acids that are related in their side chains. Naturally occurring amino acids are generally classified into four families: acidic (aspartate, glutamate), 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 jointly classified as aromatic amino acids. In some embodiments, conservative amino acid substitutions are made within the following groups: - glycine, alanine; - valine, isoleucine, leucine; - Aspartic acid, glutamic acid; - Asparagine, glutamine; - serine, threonine; - lysine, arginine; and - Phenylalanine, Tyrosine Contains substitutions in

[0139] In some embodiments, the degree of similarity, e.g., identity, between a given amino acid sequence and an amino acid sequence that is a variant of the 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, i.e., identity, is given over a region of amino acids that is 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 entire length of the reference amino acid sequence. For example, if the reference amino acid sequence consists of 200 amino acids, the degree of similarity, i.e., identity, is provided for, e.g., 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, the degree of similarity, i.e., identity, is provided for the entire length of the reference amino acid sequence. Alignment to determine sequence similarity, e.g., sequence identity, can be performed using tools known in the art, e.g., best sequence alignment, e.g., using Align, with standard settings, preferably EMBOSS::Needle, matrix:Blosum62, gap open 10.0, gap extend 0.5.

[0140] "Sequence similarity" refers to the percentage of amino acids that are identical or represent conservative amino acid substitutions. "Sequence identity" between two amino acid sequences refers to the percentage of amino acids that are identical between the sequences. "Sequence identity" between two nucleic acid sequences refers to the percentage of nucleotides that are identical between the sequences.

[0141] The terms "% identical" and "% identity", or similar terms, are intended to refer to the percentage of nucleotides or amino acids that are identical, particularly in an optimal alignment between the sequences to be compared. Said percentage is purely statistical, and the differences between the two sequences may, but need not, be randomly distributed over the entire length of the sequences to be compared. Comparison of two sequences is usually performed by comparing the sequences after optimal alignment over a segment or "window of comparison" to identify local regions of corresponding sequences. Optimal alignment for comparison may be performed manually, with the aid of the local homology algorithm of Smith and Waterman, 1981, Ads App. Math. 2, 482, with the aid of the local homology algorithm of Neddleman and Wunsch, 1970, J. Mol. Biol. 48, 443, with the aid of the similarity search algorithm of Pearson and Lipman, 1988, Proc. Natl. Acad. Sci. USA 88, 2444, or with the aid of computer programs which use the aforementioned algorithms (GAP, BESTFIT, FASTA, BLAST P, BLAST N, and TFASTA from the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, Wis.). In some embodiments, the percent identity of two sequences is determined using the BLASTN or BLASTP algorithms available at the United States National Center for Biotechnology Information (NCBI) website (e.g., blast.ncbi.nlm.nih.gov / Blast.cgi?PAGE_TYPE=BlastSearch&BLAST_SPEC=blast2seq&LINK_LOC=align2seq).In some embodiments, the algorithm parameters used for the BLASTN algorithm on the NCBI website include (i) setting the expectation threshold to 10, (ii) setting the word size to 28, (iii) setting the maximum match in the query range to 0, (iv) setting the match / mismatch score to 1, -2, (v) setting the gap cost to Linear, and (vi) using a filter for low complexity regions. In some embodiments, the algorithm parameters used for the BLASTP algorithm on the NCBI website include (i) setting the expectation threshold to 10, (ii) setting the word size to 3, (iii) setting the maximum match in the query range to 0, (iv) setting the matrix to BLOSUM62, (v) setting the gap cost to Existence:11 Extension:1, and (vi) a conditional composite score matrix adjustment.

[0142] The percentage identity is obtained by determining the number of corresponding identical positions in the sequences to be compared, dividing this number by the number of positions being compared (e.g., the number of positions in the reference sequence), and multiplying the result by 100.

[0143] In some embodiments, the degree of similarity or identity is given over a region that is at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or about 100% of the entire length of the reference sequence. For example, if the reference nucleic acid sequence consists of 200 nucleotides, the degree of identity is given over 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, contiguous nucleotides. In some embodiments, the degree of similarity or identity is given over the entire length of the reference sequence.

[0144] According to the present disclosure, homologous amino acid sequences exhibit at least 40%, in particular 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.

[0145] The amino acid sequence variants described herein are readily prepared by those skilled in the art, for example, by recombinant DNA manipulation. The manipulation of DNA sequences to prepare peptides or polypeptides with substitutions, additions, insertions, or deletions is described, for example, in Molecular Cloning: A Laboratory Manual, 4 th Edition, M.R. Green and J. Sambrook eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor 2012. Furthermore, the peptide, polypeptide, and amino acid variants described herein can be readily prepared with the aid of known peptide synthesis techniques, for example, by solid phase synthesis and similar methods.

[0146] In some embodiments, a fragment or variant of an amino acid sequence (peptide or polypeptide) is a "functional fragment" or "functional variant." The term "functional fragment" or "functional variant" of an amino acid sequence relates to any fragment or variant that exhibits one or more functional properties that are the same as or similar to those of the original amino acid sequence from which it is derived, i.e., it is functionally equivalent. With respect to an antigen or antigen sequence, one particular function is one or more immunogenic activities exhibited by the original amino acid sequence from which the fragment or variant is derived. As used herein, the term "functional fragment" or "functional variant" particularly refers to a variant molecule or sequence that comprises an amino acid sequence that is altered by one or more amino acids compared to the amino acid sequence of a parent molecule or sequence, yet is still able to fulfill one or more functions of the parent molecule or sequence, e.g., inducing an immune response. In some embodiments, the alterations in the amino acid sequence of the parent molecule or sequence do not significantly affect or change the properties of that molecule or sequence. In different embodiments, the function of the functional fragment or functional variant may be reduced but still significantly present, for example the function of the functional fragment or functional variant may be at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the parent molecule or sequence, however in other embodiments the function of the functional fragment or functional variant may be enhanced compared to the parent molecule or sequence.

[0147] An amino acid sequence (peptide or polypeptide) "derived from" a specified amino acid sequence (peptide or polypeptide) refers to the origin of the first amino acid sequence. In some embodiments, an amino acid sequence derived from a particular amino acid sequence has an amino acid sequence that is identical, essentially identical, or homologous to the particular sequence or a fragment thereof. An amino acid sequence derived from a particular amino acid sequence may be a variant of the particular sequence or a fragment thereof. For example, one of skill in the art will understand that antigens suitable for use herein may be altered to differ in sequence from the original naturally occurring or native sequence from which they are derived, while retaining the desired activity of the native sequence.

[0148] In some embodiments, "isolated" means removed (e.g., purified) from a natural state or from a man-made composition, e.g., a composition from a production process. For example, a nucleic acid, peptide, or polypeptide that is naturally present in a living animal is not "isolated," but the same nucleic acid, peptide, or polypeptide that is partially or completely separated from the coexisting materials of its natural state is "isolated." An isolated nucleic acid, peptide, or polypeptide can exist in a substantially purified form and can exist in a non-native environment, such as a host cell.

[0149] The term "transfection" refers to the introduction of nucleic acids, particularly RNA, into cells. For purposes of this disclosure, the term "transfection" also includes the introduction of nucleic acids into or the uptake of nucleic acids by such cells, whether the cells are present in a subject, e.g., a patient, or the cells may be in vitro, e.g., outside the patient. Thus, according to this disclosure, cells for transfection of nucleic acids described herein may be present in vitro or in vivo, e.g., the cells may form part of an organ, tissue, and / or body of a patient. According to this disclosure, transfection may be transient or stable. For some transfection applications, it is sufficient that the transfected genetic material is only transiently expressed. RNA can be transfected into cells to transiently express its encoded protein. Because nucleic acids introduced during the transfection process are not typically integrated into the nuclear genome, the exogenous nucleic acid is diluted or degraded by mitosis. Cells that allow episomal amplification of nucleic acids greatly reduce the rate of dilution. If it is desired that the transfected nucleic acid actually remain in the genome of the cell and its daughter cells, stable transfection must occur. Such stable transfection can be achieved, for example, by using a viral or transposon-based system for transfection. Generally, the nucleic acid encoding the antigen is transiently transfected into the cell. RNA can be transfected into the cell and transiently express its encoded protein.

[0150] Cells useful 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.

[0151] The present disclosure includes peptide or polypeptide analogs. According to the present disclosure, a peptide or polypeptide analog is a modified form of the original peptide or polypeptide from which it is derived, retaining at least one functional property of the peptide or polypeptide. For example, a pharmacologically active analog of a peptide or polypeptide retains at least one pharmacological activity of the original peptide or polypeptide from which it is derived. Such modifications include any chemical modification, including single or multiple substitutions, deletions, and / or additions of any molecules associated with the peptide or polypeptide, such as carbohydrates, lipids, and / or peptides or polypeptides. In some embodiments, a "peptide or polypeptide analog" includes modified forms resulting from glycosylation, acetylation, phosphorylation, amidation, palmitoylation, myristoylation, isoprenylation, lipidation, alkylation, derivatization, introduction of protecting / blocking groups, proteolytic cleavage, or binding to an antibody or another cellular ligand. The term "analog" also extends to all functional chemical equivalents of the peptides and polypeptides.

[0152] As used herein, the terms "linked," "fused," or "fusion" are used interchangeably and refer to the joining together of two or more elements or components or domains.

[0153] As used herein, "endogenous" refers to any material that is from or produced within an organism, cell, tissue, or system.

[0154] As used herein, the term "exogenous" refers to any material introduced from or produced outside an organism, cell, tissue, or system.

[0155] As used herein, the term "expression" is defined as the transcription and / or translation of a particular nucleotide sequence.

[0156] In the context of the present disclosure, the term "transcription" relates to the process by which the genetic code in a DNA sequence is transcribed into RNA (especially mRNA), which can then be translated into peptides or polypeptides.

[0157] With respect to RNA, the terms "expression" or "translation" refer to the process in a cell's ribosomes by which a chain of mRNA directs the assembly of a sequence of amino acids to make a peptide or polypeptide.

[0158] A prodrug of a particular compound described herein is a compound that undergoes chemical conversion under physiological conditions to provide the particular compound upon administration to an individual. Furthermore, a prodrug can be converted to the particular compound by chemical or biochemical methods in an ex vivo environment. For example, a prodrug can be slowly converted to the particular compound when placed in a transdermal patch reservoir with, for example, a suitable enzyme or chemical reagent. Exemplary prodrugs are esters (using alcohol or carboxy groups contained in the particular compound) or amides (using amino or carboxy groups contained in the particular compound) that can be hydrolyzed in vivo. Specifically, any amino group contained in the particular compound that has at least one hydrogen atom can be converted to a prodrug form. Typical N-prodrug forms include carbamates, Mannich bases, enamines, and enaminones.

[0159] In this specification, the structural formula of a compound may represent a particular isomer of said compound, but it should be understood that the present invention includes all isomers, such as structurally occurring geometric isomers, optical isomers based on asymmetric carbons, stereoisomers, tautomers, etc., and isomeric mixtures, and is not limited to the description of the formula.

[0160] "Isomers" are compounds that have the same molecular formula but differ in structure ("structural isomers") or in the geometric (spatial) arrangement of functional groups and / or atoms ("stereoisomers"). "Enantiomers" are a pair of stereoisomers that are non-superimposable mirror images of each other. A "racemic mixture" or "racemate" contains equal amounts of a pair of enantiomers and is designated by the prefix (±). "Diastereomers" are stereoisomers that are non-superimposable and are not mirror images of each other. "Tautomers" are structural isomers of the same chemical substance that spontaneously and reversibly interconvert into each other, even when pure, by the migration of individual atoms or groups of atoms. That is, tautomers are in dynamic chemical equilibrium with each other. An example of a tautomer is keto-enol tautomeric isomer. "Conformers" are stereoisomers that can formally be interconverted only by rotation about a single bond, and in particular include stereoisomers that result in different three-dimensional forms of (hetero)cyclic rings, such as chair, half-chair, boat, and twisted-boat cyclohexane.

[0161] The term "mean diameter" refers to the average hydrodynamic diameter of particles measured by dynamic light scattering (DLS) with data analysis using the so-called cumulant algorithm, which results in a so-called Z dimension with a length dimension. average and the dimensionless polydispersity index (PDI) (Koppel, D., J. Chem. Phys. 57, 1972, pp. 4814-4820, ISO 13321). Here, the "average diameter", "diameter", or "size" of a particle refers to this Z average is used synonymously with the value of

[0162] In some embodiments, the "polydispersity index" may be calculated based on dynamic light scattering measurements by the so-called cumulant analysis mentioned in the definition of "mean diameter." Under certain assumptions, this can be considered as a measure of the size distribution of an ensemble of nanoparticles.

[0163] The "radius of gyration" (herein R) of the particle around the axis of rotationg ) is the radial distance from the axis of rotation of the point at which, if the total mass of a particle were to be condensed, its moment of inertia about a given axis would be the same as its actual mass distribution. Mathematically, R g is the root mean square distance of a particle component from its center of mass or a given axis. For example, at a fixed distance s from the center of mass, i Mass m located at i For a polymer consisting of n mass elements (i=1, 2, 3, . . . , n), R g is the s over all mass elements i 2 is the mass average square root of:

number

[0164] The radius of gyration can be determined experimentally or calculated, for example, using light scattering.

number

number

[0165] The "hydrodynamic radius" of a particle (sometimes called the "Stokes radius" or "Stokes-Einstein radius") is the radius of a hypothetical hard sphere diffusing at the same rate as the particle. The hydrodynamic radius is related to the particle's mobility, taking into account not only its size but also the effects of the solvent. For example, a small, strongly hydrated charged particle may have a larger hydrodynamic radius than a large, weakly hydrated charged particle. This is because the small particle drags many water molecules along as it moves through the solution. Since the actual size of a particle in a solvent cannot be measured directly, the hydrodynamic radius is calculated using the Stokes-Einstein equation:

number

[0166] As used herein, the expression "light scattering" refers to the physical process by which light is caused to deviate from its rectilinear trajectory by one or more paths due to localized inhomogeneities in the medium through which the light passes.

[0167] The term "UV" means ultraviolet and designates the band of the electromagnetic spectrum with wavelengths between 10 nm and 400 nm, i.e., shorter than the wavelengths of visible light but longer than X-rays.

[0168] As used herein, the expression "multi-angle light scattering" or "MALS, multi-angle light scattering" refers to a technique for measuring light scattered by a sample at multiple angles. In this context, "multi-angle" means that the scattered light can be detected at different discrete angles, as measured by, for example, a single detector moving over a range that includes a particular selected angle, or an array of detectors fixed at a particular angular position. In certain embodiments, the light source used in MALS is a laser source (MALLS: multi-angle laser light scattering). Based on the MALS signal of a composition containing particles, and by using an appropriate formalism (e.g., Zimm plot, Berry plot, or Debye plot), the radius of gyration (R g ), and thus it is possible to determine the size of the particles. Preferably, the Zimm plot is calculated using the following formula:

number

number

number

[0169] As used herein, the phrase "dynamic light scattering" or "DLS" refers to a technique for determining particle size and size distribution profiles, particularly with respect to the hydrodynamic radius of the particles. A monochromatic light source, usually a laser, is shone onto a sample through a polarizer. The scattered light then passes through a second polarizer, where the light is detected and the resulting image is projected onto a screen. Particles in solution are illuminated with light, which diffracts the light in all directions. The light diffracted from the particles can interfere constructively (bright areas) or destructively (dark areas). This process is repeated at short time intervals, and the resulting set of spotted patterns is analyzed by an autocorrelator, which compares the light intensity at each point over time.

[0170] As used herein, the phrase "static light scattering" or "SLS" refers to a technique for determining particle size and size distribution profiles, particularly in terms of the particle's radius of gyration and / or its molar mass. A high-intensity monochromatic light, usually a laser, is projected into a solution containing the particles. One or many detectors are used to measure the scattered intensity at one or many angles. The angular dependence is necessary to obtain accurate measurements of both molar mass and size for all macromolecules with a given radius. Therefore, simultaneous measurements at several angles relative to the direction of incident light, known as multi-angle light scattering (MALS) or multi-angle laser light scattering (MALLS), are generally considered the standard practice for static light scattering.

[0171] nucleic acid The term "nucleic acid" includes deoxyribonucleic acid (DNA), ribonucleic acid (RNA), combinations thereof, and modified forms thereof. This term includes genomic DNA, cDNA, mRNA, recombinantly produced molecules, 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 be single-stranded or double-stranded, and can exist as linear or covalently linked circular closed molecules. Nucleic acids can be isolated. The term "isolated nucleic acid," according to the present disclosure, means that the nucleic acid has been (i) amplified in vitro, e.g., by polymerase chain reaction (PCR) of DNA or in vitro transcription of RNA (e.g., using RNA polymerase); (ii) recombinantly produced by cloning; (iii) purified, e.g., by cleavage and separation by gel electrophoresis; or (iv) synthesized, e.g., by chemical synthesis.

[0172] The term "nucleoside" (abbreviated herein as "N") refers to a compound that can be thought of as a nucleotide without the phosphate group. A nucleoside is a nucleic acid base 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.

[0173] The five standard nucleosides that typically make up naturally occurring nucleic acids are uridine, adenosine, thymidine, cytidine, and guanosine. These five nucleosides are commonly abbreviated by their single-letter codes: U, A, T, C, and G, respectively. However, thymidine is more commonly written as "dT" (the "d" stands for "deoxy") because it contains a 2'-deoxyribofuranose moiety rather than the ribofuranose ring found in uridine. This is because thymidine is found in deoxyribonucleic acid (DNA) but not ribonucleic acid (RNA). Conversely, uridine is found in RNA but not DNA. The remaining three nucleosides are found in both RNA and DNA. In RNA, they are represented as A, C, and G, and in DNA, they are represented as dA, dC, and dG.

[0174] The modified purine (A or G) or pyrimidine (C, T, or U) base moiety preferably comprises one or more alkyl groups, more preferably one or more C 1-4 More preferably, the base moiety is modified by one or more alkyl groups, and even more preferably by one or more methyl groups. Particular examples of modified purine or pyrimidine base moieties include N 7 -Alkylguanine, N 6 -alkyladenine, 5-alkylcytosine, 5-alkyluracil, and N(1)-alkyluracil, e.g., N 7 -C 1-4 Alkylguanine, N 6 -C 1-4 Alkyl adenine, 5-C 1-4 Alkylcytosine, 5-C1-4 Alkyluracils and N(1)-C 1-4 Alkyluracil, preferably N 7 -methylguanine, N 6 -methyladenine, 5-methylcytosine, 5-methyluracil, and N(1)-methyluracil.

[0175] Herein, the term "DNA" refers to a nucleic acid molecule containing deoxyribonucleotide residues. In a preferred embodiment, DNA contains all or most deoxyribonucleotide residues. As used herein, "deoxyribonucleotide" refers to a nucleotide lacking a hydroxyl group at the 2' position of the β-D-ribofuranosyl group. DNA includes, without limitation, double-stranded DNA, single-stranded DNA, isolated DNA, e.g., partially purified DNA, essentially pure DNA, synthetic DNA, recombinantly produced DNA, and modified DNA that differs from naturally occurring DNA by the addition, deletion, substitution, and / or modification of one or more nucleotides. Such modifications may refer to internal DNA nucleotides or the addition of non-nucleotide material to the ends of the DNA. It is also contemplated herein that the nucleotides in DNA may be non-standard nucleotides, such as chemically synthesized nucleotides or ribonucleotides. For the purposes of the present disclosure, these modified DNAs are considered analogs of naturally occurring DNA. A molecule contains a "majority of deoxyribonucleotide residues" if the content of deoxyribonucleotide residues in the molecule is greater than 50% (e.g., 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%), based on the total number of nucleotide residues in the molecule. The total number of nucleotide residues in the molecule is the sum of all nucleotide residues (whether the nucleotide residues are standard (i.e., naturally occurring) nucleotide residues or analogs thereof).

[0176] The DNA may be recombinant DNA and may be obtained by cloning a nucleic acid, in particular cDNA, which may be obtained by reverse transcription of RNA.

[0177] The term "RNA" refers to a nucleic acid molecule comprising ribonucleotide residues. In a preferred embodiment, the RNA comprises all or most ribonucleotide residues. As used herein, "ribonucleotide" refers to a nucleotide having a hydroxyl group at the 2' position of a β-D-ribofuranosyl group. RNA includes, without limitation, double-stranded RNA, single-stranded RNA, isolated RNA, e.g., partially purified RNA, essentially pure RNA, synthetic RNA, recombinantly produced RNA, and modified RNA that differs from naturally occurring RNA by the addition, deletion, substitution, and / or modification of one or more nucleotides. Such modifications may refer to the addition of non-nucleotide material to internal RNA nucleotides or to the ends 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 purposes of the present disclosure, these modified / modified nucleotides may be referred to as analogs of naturally occurring nucleotides, and the corresponding RNA comprising such modified / modified nucleotides (i.e., modified / modified RNA) may be referred to as analogs of naturally occurring RNA. A molecule contains a "majority of ribonucleotide residues" if the content of ribonucleotide residues in the molecule is greater than 50% (e.g., 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%) based on the total number of nucleotide residues in the molecule. The total number of nucleotide residues in a molecule is the sum of all nucleotide residues (whether they are standard (i.e., naturally occurring) nucleotide residues or analogs thereof). "RNA" includes mRNA, tRNA, ribosomal RNA (rRNA), small nuclear RNA (snRNA), self-amplifying RNA (saRNA), single-stranded RNA (ssRNA), dsRNA, inhibitory RNA (e.g., antisense ssRNA, small interfering RNA (siRNA), or microRNA (miRNA)), activator RNA (e.g., small activator RNA), and immunostimulatory RNA (isRNA).In some embodiments, "RNA" refers to mRNA.

[0178] As used herein, the term "in vitro transcription" or "IVT" means that transcription (production of RNA) is carried out in a cell-free manner, i.e., IVT does not use live / cultured cells, but rather uses transcription machinery extracted from cells (e.g., a cell lysate or isolated components thereof containing an RNA polymerase (preferably T7, T3, or SP6 polymerase)).

[0179] In some embodiments, the nucleic acids of the invention, eg, one, at least two, or all of the nucleic acids of the invention, are RNA.

[0180] In some embodiments, the RNA is single-stranded RNA.

[0181] In some embodiments, the RNA is mRNA.

[0182] In some embodiments, the RNA is produced by in vitro transcription of RNA.

[0183] In some embodiments, the RNA comprises a 5' cap structure.

[0184] In some embodiments, the RNA does not contain modified ribonucleotides.

[0185] In some embodiments, the RNA comprises modified ribonucleotides. In some embodiments, the modified ribonucleotide comprises a modified uridine. In some embodiments, the modified uridine comprises N1-methylpseudouridine.

[0186] In some embodiments, the nucleic acids of the invention, eg, one, at least two, or all of the nucleic acids of the invention, are DNA.

[0187] In some embodiments, the DNA is in the form of a vector.

[0188] In some embodiments, the vector comprises DNA encoding an amino acid sequence that comprises the amino acid sequence of a biologically active peptide or polypeptide.

[0189] In some embodiments, the vector is a DNA vector.

[0190] In some embodiments, the nucleic acids of the invention, eg, one, at least two, or all of the nucleic acids of the invention, comprise a mixture of RNA and DNA.

[0191] In some embodiments, the RNA in the mixture is single-stranded RNA.

[0192] In some embodiments, the RNA in the mixture is mRNA.

[0193] In some embodiments, the RNA in the mixture is produced by in vitro transcription of RNA.

[0194] In some embodiments, the RNA in the mixture comprises a 5' cap structure.

[0195] In some embodiments, the RNA in the mixture does not contain modified ribonucleotides.

[0196] 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 uridine comprises N1-methylpseudouridine.

[0197] In some embodiments, the DNA in the mixture is in the form of a vector.

[0198] In some embodiments, the vector in the mixture comprises DNA that encodes an amino acid sequence that comprises the amino acid sequence of a biologically active peptide or polypeptide.

[0199] In some embodiments, the vectors in the mixture are DNA vectors.

[0200] In some embodiments, the nucleic acids (eg, RNA and / or DNA) of the invention, which may comprise one or at least two or more nucleic acid constructs, are formulated with a delivery vehicle.

[0201] In some embodiments, the nucleic acid (eg, RNA and / or DNA) is formulated with one or more compounds that complex the nucleic acid (eg, RNA and / or DNA).

[0202] In some embodiments, the nucleic acids (eg, RNA and / or DNA) are formulated as particles.

[0203] In some embodiments, the nucleic acid (e.g., RNA and / or DNA) is formulated as a lipid-complexed particle. In these embodiments, the cells are preferably characterized by a macropinocytosis-mediated RNA uptake mechanism.

[0204] In some embodiments, the nucleic acid (eg, RNA and / or DNA) is formulated as a lipid nanoparticle.

[0205] In some embodiments, the nucleic acids (e.g., RNA and / or DNA) comprise a mixture of different nucleic acids (e.g., RNA and / or DNA, e.g., two or more RNAs, two or more DNAs, or one or more RNAs and one or more DNAs), each nucleic acid (e.g., RNA and / or DNA) encoding an amino acid sequence, including the amino acid sequence of a biologically active peptide or polypeptide.

[0206] In some embodiments, the mixture of different nucleic acids (e.g., RNA and / or DNA, e.g., two or more RNAs, two or more DNAs, or one or more RNAs and one or more DNAs) includes nucleic acids (e.g., RNA and / or DNA, e.g., two or more RNAs, two or more DNAs, or one or more RNAs and one or more DNAs) that encode different amino acid sequences, including the amino acid sequence of a biologically active peptide or polypeptide.

[0207] In some embodiments, the different amino acid sequences comprise amino acid sequences of different biologically active peptides or polypeptides.

[0208] In some embodiments, the different biologically active peptides or polypeptides comprise different antigens.

[0209] In some embodiments, the nucleic acid (e.g., RNA and / or DNA) comprises a mixture of different nucleic acids (e.g., RNA and / or DNA, e.g., two or more RNAs, two or more DNAs, or one or more RNAs and one or more DNAs) that encode amino acid sequences that include amino acid sequences of different antigens.

[0210] In some embodiments, the RNAs described herein are single-stranded RNAs that can be translated into the respective proteins upon entry into cells, such as cells used in the assays described herein and recipient cells. In addition to a wild-type or codon-optimized sequence encoding an amino acid sequence containing the amino acid sequence of a biologically active peptide or polypeptide, e.g., a pharmaceutically active peptide or polypeptide, such as an antigen sequence, the RNA may contain one or more structural elements (5' cap, 5' UTR, 3' UTR, poly(A) tail) optimized to maximize the effectiveness of the RNA in terms of stability and translation efficiency. In one embodiment, the RNA contains all of these elements. In one embodiment, the RNA is a nucleotide sequence encoding beta-S-ARCA(D1)(m2 7,2’-O GppSpG) or m2 7,3’-OGppp(m1 2’-O ) ApG may be used as a specific capping structure at the 5' end of an RNA drug substance. The 5'-UTR sequence of human alpha globin mRNA may be used, optionally with an optimized "Kozak sequence" to increase translation efficiency. The 3'-UTR sequence may be a combination of two sequence elements (FI element) derived from the "amino terminal enhancer of split" (AES) mRNA (called F) and the mitochondrial-encoded 12S ribosomal RNA (called I), located between the coding sequence and the poly(A) tail to ensure higher maximum protein levels and long-term mRNA persistence. These were identified through an ex vivo selection process for sequences that confer RNA stability and increase total protein expression (see WO 2017 / 060314, incorporated herein by reference). Alternatively, the 3'-UTR may be two re-repeated 3'-UTRs of human beta globin mRNA. Additionally, a poly(A) tail may be used that is 110 nucleotides in length and consists of 30 consecutive adenosine residues, followed by a 10 nucleotide linker sequence (of random nucleotides) and an additional 70 adenosine residues, designed to increase RNA stability and translation efficiency.

[0211] An 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 contain amino acid sequences other than the amino acid sequence of the biologically active peptide or polypeptide. Such other amino acid sequences may support the function or activity of the biologically active peptide or polypeptide. In some embodiments, such other amino acid sequences include amino acid sequences that enhance antigen processing and / or presentation. Alternatively or additionally, such other amino acid sequences include amino acid sequences that disrupt immune tolerance. Alternatively or additionally, such other amino acid sequences include amino acid sequences that generate bioluminescence. Such other amino acid sequences may be useful for determining the amount of an amino acid sequence comprising the amino acid sequence of a biologically active peptide or polypeptide or a fragment thereof in the assays described herein. In particular, such other amino acid sequences may be useful for quantitation by LC-MS / MS analysis.

[0212] The nucleic acids described herein (e.g., RNA and / or DNA) may be complexed with polymers, proteins, and / or lipids, preferably lipids, to form nucleic acid-particles for administration. When combinations of different nucleic acids are used, the nucleic acids may be complexed together or individually.

[0213] mRNA According to the present disclosure, the term "mRNA" means "messenger-RNA" and refers to a "transcript" that can be produced by using a DNA template and can encode a peptide or polypeptide. Typically, mRNA comprises a 5'-UTR, a peptide / polypeptide coding region, and a 3'-UTR. In the context of the present disclosure, mRNA can be produced by in vitro transcription (IVT) from a DNA template. As indicated above, in vitro transcription methodologies are known to those skilled in the art, and various in vitro transcription kits are commercially available.

[0214] Although mRNA is single-stranded, it may contain self-complementary sequences that allow part of the mRNA to fold back on itself and pair with itself to form a double helix.

[0215] According to the present disclosure, "dsRNA" means double-stranded RNA, which is RNA having two strands that are partially or completely complementary.

[0216] In a preferred embodiment of the present disclosure, mRNA relates to an RNA transcript that encodes a peptide or polypeptide.

[0217] In some embodiments, the mRNA, preferably encoding a peptide or polypeptide, has a length of at least 45 nucleotides (e.g., 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 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, e.g., up to 14,000, up to 13,000, up to 12,000 nucleotides, up to 11,000 nucleotides, or up to 10,000 nucleotides.

[0218] As is well established in the art, mRNA generally comprises a 5' untranslated region (5'-UTR), a peptide / polypeptide coding region, and a 3' untranslated region (3'-UTR). In some embodiments, mRNA is produced by in vitro transcription or chemical synthesis. In some embodiments, mRNA is produced by in vitro transcription using a DNA template. Methods for in vitro transcription are known to those skilled in the art and are described, for example, in Molecular Cloning: A Laboratory Manual, 4 th Edition, M.R. Green and J. Sambrook eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor 2012. Additionally, various in vitro transcription kits are commercially available, for example, from Thermo Fisher Scientific (e.g., TranscriptAid™ T7 kit, MEGAscript® T7 kit, MAXIscript®), New England BioLabs Inc. (e.g., HiScribe™ T7 kit, HiScribe™ T7 ARCA mRNA kit), Promega (e.g., RiboMAX™, HeLaScribe®, Riboprobe® systems), Jena Bioscience (e.g., SP6 or T7 transcription kits), and Epicentre (e.g., AmpliScribe™). To provide a modified mRNA, the corresponding modified nucleotides, e.g., modified naturally occurring nucleotides, non-naturally occurring nucleotides, and / or modified non-naturally occurring nucleotides, can be introduced during synthesis (preferably in vitro transcription) or can be modified and / or added into the mRNA after transcription.

[0219] In some embodiments, the mRNA is in vitro transcribed mRNA (IVT-RNA), obtained by in vitro transcription of a suitable DNA template. The promoter controlling the transcription may be any promoter for any RNA polymerase. Specific examples of RNA polymerases include T7, T3, and SP6 RNA polymerases. Preferably, the in vitro transcription is controlled by a T7 or SP6 promoter. The DNA template for in vitro transcription can be obtained by cloning a nucleic acid, particularly a cDNA, and introducing it into a suitable vector for in vitro transcription. The cDNA can be obtained by reverse transcription of RNA.

[0220] In some embodiments of the present disclosure, the mRNA is a "replicon mRNA" or simply a "replicon," particularly a "self-replicating mRNA" or "self-amplifying mRNA." In certain embodiments, the replicon or self-replicating mRNA is derived from or contains elements derived from an ssRNA virus, particularly a positive-strand ssRNA virus, such as an alphavirus. Alphaviruses are typical representatives of positive-strand RNA viruses. Alphaviruses replicate in the cytoplasm of infected cells (for a review of the alphavirus life cycle, see Jose 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' poly(A) tail. The genome of an alphavirus encodes nonstructural proteins (involved in viral RNA transcription, modification, and replication, and protein modification) and structural proteins (forming viral particles). Typically, two open reading frames (ORFs) are present in the genome. The four nonstructural proteins (nsP1-nsP4) are co-encoded by the first ORF, which typically begins near the 5' end of the genome, while the structural proteins of alphaviruses are co-encoded by the second ORF, which is found downstream of the first ORF and extends toward the 3' end of the genome. Typically, 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 the nonstructural proteins are translated from the genomic RNA, while the genetic information encoding the structural proteins can be 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).Following infection, i.e., early in the viral life cycle, the (+)-strand genomic RNA acts directly as a messenger RNA for the translation of an open reading frame encoding a nonstructural polyprotein (nsP1234). Alphavirus-derived vectors have been proposed for delivering foreign genetic information to target cells or organisms. In a simple approach, the open reading frame encoding the alphavirus structural proteins is replaced by an open reading frame encoding the desired protein. Alphavirus-based transcriptional replication systems rely on alphavirus nucleotide sequence elements on two separate nucleic acid molecules: one nucleic acid molecule encodes the viral replicase, and the other nucleic acid molecule can be replicated in trans by the replicase (hence the term trans-replication system). Trans-replication requires the presence of both of these nucleic acid molecules in a given host cell. Nucleic acid molecules capable of being replicated in trans by the replicase must contain certain alphavirus sequence elements that allow recognition and RNA synthesis by the alphavirus replicase.

[0221] In some embodiments of the present disclosure, the mRNA contains one or more modifications, for example, to increase its stability, and / or translation efficiency, and / or reduce immunogenicity and / or reduce cytotoxicity. For example, to increase mRNA expression, the mRNA may be modified within the coding region, i.e., the sequence encoding the expressed peptide or polypeptide, preferably without changing the sequence of the expressed peptide or polypeptide. Such modifications are described, for example, in WO 2007 / 036366 and PCT / EP2019 / 056502, and include: 5'-cap structures; extension or shortening of naturally occurring poly(A) tails; alteration of the 5'- and / or 3'-untranslated regions (UTRs), for example, introduction of unrelated UTRs in the coding region of the RNA; replacement of one or more naturally occurring nucleotides with synthetic nucleotides; and codon optimization (e.g., to change, preferably increase, the GC content of the RNA).

[0222] 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 analog. The term "conventional 5' cap" refers to a cap structure found on the 5' end of an mRNA molecule, generally consisting of a guanosine 5'-triphosphate (Gppp) connected via its triphosphate moiety to the 5' end of the next nucleotide of the mRNA (i.e., the guanosine is connected to the remainder of the mRNA via a 5'-to-5' triphosphate linkage). The guanosine is attached at position N 7 may be methylated in the cap structure m 7 The term "5' cap analog" is based on the traditional 5' cap, but is modified to avoid incorporation of the 5' cap analog in the reverse orientation. 7These include 5' caps modified at the 2' or 3' position of the guanosine structure (such 5' cap analogs are also referred to as anti-reverse cap analogs (ARCA)). Particularly preferred 5' cap analogs include those described in PCT / EP2019 / 056502 that have one or more substitutions at the bridging and non-bridging oxygens in the phosphate bridge, such as phosphorothioate-modified 5' cap analogs (e.g., m2 7,2’O G(5')ppSp(5')G (referred to as beta-S-ARCA or β-S-ARCA). Providing mRNA with a 5' cap structure as described herein may be achieved by in vitro transcription of a DNA template in the presence of the corresponding 5' cap compound, with the 5' cap structure incorporated into the resulting mRNA strand by co-transcription, or by generating mRNA, for example, by in vitro transcription, and then post-transcriptionally attaching the 5' cap structure to the mRNA using a capping enzyme, for example, vaccinia viri capping enzyme.

[0223] In some embodiments, the mRNA is m2 7,2’O G(5')ppSp(5')G (especially its D1 diastereomer), m2 7,3’O G(5')ppp(5')G, and m2 7,3’-O Gppp(m1 2’-O ) ApG.

[0224] In some embodiments, the mRNA comprises cap 0, cap 1, or cap 2, preferably cap 1 or cap 2. According to the present disclosure, the term "cap 0" refers to the structure "m 7 GpppN" where N is any nucleoside having an OH moiety at the 2' position. According to the present disclosure, the term "Cap 1" refers to the structure "m 7 GpppNm, where Nm is any nucleoside having an OCH3 moiety at the 2' position. According to the present disclosure, the term "cap2" refers to the structure "m 7GpppNmNm" where each Nm is independently any nucleoside having an OCH3 moiety at the 2' position.

[0225] The D1 diastereomer of beta-S-ARCA (β-S-ARCA) has the following structure: [ka] It has.

[0226] The "D1 diastereomer of beta-S-ARCA" or "beta-S-ARCA(D1)" is the diastereomer of beta-S-ARCA that elutes first on an HPLC column compared to the D2 diastereomer of beta-S-ARCA (beta-S-ARCA(D2)) and therefore exhibits a shorter retention time. The HPLC is preferably analytical. In some embodiments, a Supelcosil LC-18-T RP column, preferably 5 μm in size and 4.6 × 250 mm in format, is used for the separation, and a flow rate of 1.3 ml / min can be applied. In some embodiments, a gradient of methanol in ammonium acetate is used, e.g., a linear gradient of 0 to 25% methanol in 0.05 M ammonium acetate, pH 5.9, within 15 minutes. UV detection (UV-detection) can be performed at 260 nm, and fluorescence detection (FLD) can be performed with excitation at 280 nm and detection at 337 nm.

[0227] m2, the 5' cap analogue that is a component of cap1 7,3’-O Gppp(m1 2’-O )ApG(m2 7,3’O G(5')ppp(5')m 2’-O ApG) has the following structure: [ka] It has.

[0228] An exemplary cap 0 mRNA containing β-S-ARCA and mRNA has the following structure: [ka] It has.

[0229] m2 7,3’O An exemplary cap 0 mRNA containing G(5')ppp(5')G and mRNA has the following structure: [ka] It has.

[0230] m2 7,3’-O Gppp(m1 2’-O ) An exemplary cap 1 mRNA containing ApG and mRNA has the following structure: [ka] It has.

[0231] As used herein, the term "poly A tail" or "poly A sequence" refers to an uninterrupted or interrupted sequence of adenylate residues typically located at the 3' end of an mRNA molecule. Poly A tails or poly A sequences are known to those skilled in the art and may follow the 3'UTR of the mRNAs described herein. An uninterrupted poly A tail is characterized by consecutive adenylate residues. Uninterrupted poly A tails are typical in nature. The mRNAs disclosed herein may have a poly A tail attached to the free 3' end of the mRNA after transcription by a non-template-dependent RNA polymerase, or a poly A tail encoded by DNA and transcribed by a template-dependent RNA polymerase.

[0232] It has been demonstrated that poly(A) tails of approximately 120 nucleotides have a beneficial effect on the levels of mRNA 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).

[0233] The poly-A tail can be of any length. In some embodiments, the poly-A tail comprises, consists essentially of, or consists of at least 20, at least 30, at least 40, at least 80, or at least 100, and up to 500, 400, 300, 200, or 150 A nucleotides, particularly 120 A nucleotides. In this context, "consisting essentially of" means that the majority of nucleotides in the poly-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 poly-A tail, are A nucleotides, although the remaining nucleotides may be nucleotides other than A nucleotides, such as U nucleotides (uridylate), G nucleotides (guanylate), or C nucleotides (cytidylate). In this context, "consisting of" means that all nucleotides in the poly-A tail, i.e., 100% of the number of nucleotides in the poly-A tail, are A nucleotides. The term "A nucleotide" or "A" refers to adenylate.

[0234] In some embodiments, the poly(A) tail is attached during transcription of the RNA, e.g., during preparation of the in vitro transcribed RNA, based on a DNA template containing repeated dT nucleotides (deoxythymidylate) in the strand complementary to the coding strand. The DNA sequence encoding the poly(A) tail (coding strand) is referred to as a poly(A) cassette.

[0235] In some embodiments, the poly(A) cassette present in the coding strand of DNA consists essentially of dA nucleotides but is interrupted by random sequences of the four nucleotides (dA, dC, dG, and dT). Such random sequences may be 5 to 50, 10 to 30, or 10 to 20 nucleotides in length. Such cassettes are disclosed in WO 2016 / 005324 A1, which is incorporated herein by reference. Any poly(A) cassette disclosed in WO 2016 / 005324 A1 may be used in the present disclosure. Poly(A) cassettes consisting essentially of dA nucleotides but interrupted by random sequences with an equal distribution of the four nucleotides (dA, dC, dG, dT), e.g., 5 to 50 nucleotides in length, are included that exhibit sustained propagation of plasmid DNA in E. coli at the DNA level, yet are associated with beneficial properties that support RNA stability and translation efficiency at the RNA level. Thus, in some embodiments, the poly-A tails included in the mRNA molecules described herein consist essentially of A nucleotides, but are interrupted by random sequences of four nucleotides (A, C, G, U). Such random sequences can be 5-50, 10-30, or 10-20 nucleotides in length.

[0236] In some embodiments, no nucleotides other than A nucleotides are adjacent to the poly A tail at its 3' end, i.e., the poly A tail is not masked or followed at its 3' end by a nucleotide other than A.

[0237] In some embodiments, the polyA tail may comprise at least 20, at least 30, at least 40, at least 80, or at least 100, and up to 500, 400, 300, 200, or 150 nucleotides. In some embodiments, the polyA tail may consist essentially of at least 20, at least 30, at least 40, at least 80, or at least 100, and up to 500, 400, 300, 200, or 150 nucleotides. In some embodiments, the polyA tail may consist of at least 20, at least 30, at least 40, at least 80, or at least 100, and up to 500, 400, 300, 200, or 150 nucleotides. In some embodiments, the polyA tail comprises at least 100 nucleotides. In some embodiments, the polyA tail comprises about 150 nucleotides. In some embodiments, the polyA tail comprises about 120 nucleotides.

[0238] In some embodiments, the mRNA used in the present disclosure includes a 5' UTR and / or a 3' UTR. The term "untranslated region" or "UTR" refers to a region in a DNA molecule that is transcribed but not translated into an amino acid sequence, or a corresponding region in an RNA molecule, such as an mRNA molecule. An untranslated region (UTR) can be located 5' (upstream) of an open reading frame (5' UTR) and / or 3' (downstream) of an open reading frame (3' UTR). When present, the 5' UTR is located at the 5' end, i.e., upstream of the start codon of the protein-coding region. The 5' UTR is downstream of the 5' cap (if present), e.g., directly adjacent to the 5' cap. When present, the 3' UTR is located at the 3' end, i.e., downstream of the stop codon of the protein-coding region, although the term "3' UTR" generally does not include a polyA sequence. Thus, the 3' UTR is upstream of the polyA sequence (if present), e.g., directly adjacent to the polyA sequence. The incorporation of a 3' UTR into the 3' untranslated region of an RNA (preferably an mRNA) molecule can result in improved translation efficiency. Synergistic effects can be achieved by incorporating two or more such 3' UTRs (preferably arranged in a head-to-tail orientation; see, e.g., Holtkamp et al., Blood 108, 4009-4017 (2006)). The 3' UTR may be autologous or heterologous to the RNA (e.g., mRNA) into which it is introduced. In certain embodiments, the 3' UTR is derived from a globin gene or mRNA, such as alpha2-globin, alpha1-globin, or beta globin, e.g., beta globin, e.g., human beta globin. For example, an RNA (e.g., an mRNA) may be modified by replacing or inserting one or more, e.g., two, copies of a 3' UTR from a globin gene, such as alpha2-globin, alpha1-globin, or beta globin, e.g., beta globin, e.g., human beta globin.

[0239] The mRNA may have modified ribonucleotides to increase its stability, and / or reduce immunogenicity, and / or reduce cytotoxicity. For example, in some embodiments, uridines in the mRNAs described herein are replaced (partially or completely, preferably completely) by modified nucleosides. In some embodiments, the modified nucleoside is a modified uridine.

[0240] In some embodiments, the modified uridine replacing the uridine is selected from the group consisting of pseudouridine (Ψ), N1-methyl-pseudouridine (mΨ), 5-methyl-uridine (m5U), and combinations thereof.

[0241] In some embodiments, modified nucleosides that replace (partially or completely, preferably completely) uridine in an mRNA include 3-methyluridine (m3U, 3-methyl-uridine), 5-methoxyuridine (mo5U, 5-methoxy-uridine), 5-azauridine, 6-azauridine, 2-thio-5-azauridine, 2-thiouridine (s2U, 2-thio-uridine), 4-thiouridine (s4U, 4-thio-uridine), 4-thiopseudouridine, 2-thiopseudouridine, 5-hydroxyuridine (ho5U, 5-hydroxy-uridine), 5-aminoallyluridine, 5-halouridine (e.g., 5-iodouridine or 5-bromouridine), uridine 5-oxyacetic acid (cmo5U, uridine 5-oxyacetic acid methyl ester) (m ... ester), 5-carboxymethyluridine (cm5U, 5-carboxymethyl-uridine), 1-carboxymethylpseudouridine, 5-carboxyhydroxymethyluridine (chm5U, 5-carboxyhydroxymethyl-uridine), 5-carboxyhydroxymethyluridine methyl ester (mchm5U, 5-carboxyhydroxymethyl-uridine methyl ester), 5-methoxycarbonylmethyluridine (mcm5U, 5-methoxycarbonylmethyl-uridine), 5-methoxycarbonylmethyl-2-thiouridine (mcm5s2U, 5-methoxycarbonylmethyl-2-thio-uridine), 5-aminomethyl-2-thiouridine (nm5s2U, 5-aminomethyl-2-thio-uridine), 5-methylaminomethyluridine (mnm5U, 5-methylaminomethyl-uridine), 1-ethylpseudouridine, 5-methylaminomethyl-2-thiouridine (mnm5s2U, 5-methylaminomethyl-2-thio-uridine), 5-methylaminomethyl-2-selenouridine (mnm5se2U,5-methylaminomethyl-2-seleno-uridine), 5-carbamoylmethyluridine (ncm5U, 5-carbamoylmethyl-uridine), 5-carboxymethylaminomethyluridine (cmnm5U, 5-carboxymethylaminomethyl-uridine), 5-carboxymethylaminomethyl-2-thiouridine (cmnm5s2U, 5-carboxymethylaminomethyl-2-thiouridine), 5-propynyluridine, 1-propynyl Lupseudouridine, 5-taurinomethyluridine (τm5U, 5-taurinomethyl-uridine), 1-taurinomethylpseudouridine, 5-taurinomethyl-2-thiouridine (τm5s2U, 5-taurinomethyl-2-thio-uridine), 1-taurinomethyl-4-thiopseudouridine, 5-methyl-2-thiouridine (m5s2U, 5-methyl-2-thio-uridine), 1-methyl-4-thiopseudouridine (m1s4Ψ, 1-methyl-4-thio-pseudouridine) idine), 4-thio-1-methylpseudouridine, 3-methylpseudouridine (m3Ψ, 3-methyl-pseudouridine), 2-thio-1-methylpseudouridine, 1-methyl-1-deazapseudouridine, 2-thio-1-methyl-1-deazapseudouridine, dihydrouridine (D, dihydrouridine), dihydropseudouridine, 5,6-dihydrouridine, 5-methyldihydrouridine (m5D, 5-methyl-dihydrouridine), 2-thiodihydrouridine, 2-thiodihydrouridine pseudouridine, 2-methoxyuridine, 2-methoxy-4-thiouridine, 4-methoxypseudouridine, 4-methoxy-2-thiopseudouridine, N1-methylpseudouridine, 3-(3-amino-3-carboxypropyl)uridine (acp3U), 1-methyl-3-(3-amino-3-carboxypropyl)pseudouridine (acp3Ψ),5-(isopentenylaminomethyl)uridine (inm5U, 5-(isopentenylaminomethyl)uridine), 5-(isopentenylaminomethyl)-2-thiouridine (inm5s2U, 5-(isopentenylaminomethyl)-2-thiouridine), α-thiouridine, 2'-O-methyluridine (Um, 2'-O-methyl-uridine), 5,2'-O-dimethyluridine (m5Um, 5,2'-O-dimethyl-uridine), 2'-O-methylpseudouridine (Ψm, 2'-O-methyl-pseudouridine), 2-thio-2'-O-methyluridine (s2Um, 2-thio-2'-O-methyl-uridine), 5-methoxycarbonylmethyl-2'-O-methyluridine (mcm5Um, 5-methoxycarbonylmethyl-2'-O-methyl-uridine), 5-carbamoylmethyl The modified uridine may be one or more of 2'-O-methyluridine (ncm5Um, 5-carbamoylmethyl-2'-O-methyl-uridine), 5-carboxymethylaminomethyl-2'-O-methyluridine (cmnm5Um, 5-carboxymethylaminomethyl-2'-O-methyl-uridine), 3,2'-O-dimethyluridine (m3Um, 3,2'-O-dimethyl-uridine), 5-(isopentenylaminomethyl)-2'-O-methyluridine (inm5Um, 5-(isopentenylaminomethyl)-2'-O-methyl-uridine), 1-thiouridine, deoxythymidine, 2'-F-ara-uridine, 2'-F-uridine, 2'-OH-ara-uridine, 5-(2-carbomethoxyvinyl)uridine, 5-[3-(1-E-propenylamino)uridine]uridine, or any other modified uridine known in the art.

[0242] RNA (preferably mRNA) modified with pseudouridine (partially or completely, preferably completely replacing uridine) is referred to herein as "Ψ-modified," and the term "mΨ-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 mΨ- or m5U-modified RNAs typically exhibit reduced immunogenicity compared to their unmodified forms and are therefore preferred in applications where induction of an immune response is to be avoided or minimized. In some embodiments, the RNA (preferably mRNA) contains N(1)-methylpseudouridine, completely replacing uridine.

[0243] The codons of the mRNA used in the present disclosure may be further optimized, for example, to increase the GC content of the RNA and / or to replace codons that are rare in a cell (or subject) in which the desired peptide or polypeptide is expressed with codons that are of higher frequency in said cell (or subject). In some embodiments, the amino acid sequence encoded by the mRNA used in the present disclosure is encoded by a coding sequence that has been codon-optimized and / or whose G / C content has been increased compared to a wild-type coding sequence. This includes embodiments in which one or more sequence regions of the coding sequence have been codon-optimized and / or have an increased G / C content compared to the corresponding sequence region of a wild-type coding sequence. In some embodiments, the codon optimization and / or increased G / C content preferably does not change the sequence of the encoded amino acid sequence.

[0244] The term "codon optimization" refers to changing the codons in the coding region of a nucleic acid molecule to reflect the typical codon usage of the host organism, preferably without changing the amino acid sequence encoded by the nucleic acid molecule.In the context of the present disclosure, the coding region may be codon-optimized for optimal expression in the subject to be treated with the mRNA described herein.Codon optimization is based on the discovery that translation efficiency is also determined by the different frequencies of occurrence of tRNA in cells.Therefore, the sequence of mRNA may be modified so that codons that are available for frequently occurring tRNAs are inserted instead of "rare codons".

[0245] 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 the wild-type RNA, and the amino acid sequence encoded by the mRNA is preferably unaltered 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 efficient translation of that mRNA. Sequences with an increased G (guanosine) / C (cytosine) content are more stable than sequences with an increased A (adenosine) / U (uracil) content. In view of the fact that several codons encode the same amino acid (the so-called degeneracy of the genetic code), it is possible to determine the codon most favorable for stability (the so-called alternative codon usage). Depending on the amino acid to be encoded by the mRNA, there are various possibilities for modifying the mRNA sequence compared to its wild-type sequence. In particular, codons containing A and / or U nucleotides can be modified by replacing these codons with other codons that encode the same amino acids but that do not contain A and / or U or that contain fewer A and / or U nucleotides.

[0246] In various embodiments, the G / C content of the coding regions of the mRNAs described herein is increased by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 55%, or more compared to the G / C content of the coding regions of the wild-type RNA.

[0247] The combination of the above modifications, i.e., incorporation of a 5' cap structure, incorporation of a polyA sequence, demasking of a polyA sequence, alteration of the 5'- and / or 3'-UTR (e.g., incorporation of one or more 3'UTRs), replacement of one or more naturally occurring nucleotides with synthetic nucleotides (e.g., replacement of cytidine with 5-methylcytidine, and / or uridine with pseudouridine (Ψ) or N(1)-methylpseudouridine (mΨ) or 5-methyluridine (m5U)), and codon optimization, has a synergistic effect on increasing RNA (preferably mRNA) stability and translation efficiency. Thus, in some embodiments, the mRNA used in the present disclosure includes a combination of at least two, at least three, at least four, or all five of the above-mentioned modifications: (i) incorporation of a 5' cap structure; (ii) incorporation of a polyA sequence, demasking of a polyA sequence; (iii) alteration of the 5'- and / or 3'-UTR (e.g., 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 uridine with pseudouridine (Ψ) or N(1)-methylpseudouridine (mΨ) or 5-methyluridine (m5U)); and (v) codon optimization.

[0248] Some aspects of the present disclosure include targeted delivery of the mRNA disclosed herein to certain cells or tissues. In some embodiments, the present disclosure includes targeting the lymphatic system, particularly secondary lymphoid organs, more specifically the spleen. Targeting the lymphatic system, particularly secondary lymphoid organs, more specifically the spleen, is particularly preferred when the administered mRNA encodes an antigen or epitope for inducing an immune response. In some embodiments, the target cell is a splenocyte. In some embodiments, the target cell is an antigen-presenting cell, such as a professional antigen-presenting cell in the spleen. In some embodiments, the target cell is a dendritic cell in the spleen. The "lymphatic system" is part of the circulatory system and is an important part of the immune system, including a network of lymphatic vessels that transport lymph. The lymphatic system consists of lymphoid organs, a conducting network of lymphatic vessels, and circulating lymph. Primary or central lymphoid organs generate lymphocytes from immature progenitor cells. The thymus and bone marrow constitute primary lymphoid organs. Secondary or peripheral lymphoid organs, including lymph nodes and the spleen, maintain mature and immature lymphocytes and initiate adaptive immune responses.

[0249] Lipid-based mRNA delivery systems have an inherent preference for the liver. Accumulation in the liver is caused by the discontinuous nature of the hepatic vasculature or lipid metabolism (liposomes and lipid or cholesterol conjugates). In some embodiments, the target organ is the liver and the target tissue is hepatic tissue. Delivery to such target tissue is particularly preferred when the presence of the mRNA or the encoded peptide or polypeptide in this organ or tissue is desired, and / or when abundant expression of the encoded peptide or polypeptide is desired, and / or when a particularly large systemic presence of the encoded peptide or polypeptide is desired or required.

[0250] In some embodiments, after administration of the mRNA particles described herein, at least a portion of the mRNA is delivered to a target cell or target organ. In some embodiments, at least a portion of the mRNA is delivered to the cytoplasm of the target cell. In some embodiments, the mRNA encodes a peptide or polypeptide, and the mRNA is translated by the target cell to produce the peptide or polypeptide. In some embodiments, the target cell is a cell in the liver. In some embodiments, the target cell is a muscle cell. In some embodiments, the target cell is an endothelial cell. In some embodiments, the target cell is a tumor cell or a cell in the tumor microenvironment. In some embodiments, the target cell is a blood cell. In some embodiments, the target cell is a cell in a lymph node. In some embodiments, the target cell is a cell in the lung. In some embodiments, the target cell is a blood cell. In some embodiments, the target cell is a cell in the skin. In some embodiments, the target cell is a splenocyte. In some embodiments, the target cell is an antigen-presenting cell, such as a professional antigen-presenting cell in the spleen. In some embodiments, the target cell is a dendritic cell in the spleen. In some embodiments, the target cell is a T cell. In some embodiments, the target cell is a B cell. In some embodiments, the target cell is a NK cell. In some embodiments, the target cell is a monocyte. Thus, the RNA particles described herein can be used to deliver mRNA to such target cells.

[0251] Pharmaceutically active peptides or polypeptides "Encoding" refers to the inherent property of a particular sequence of nucleotides in a polynucleotide, e.g., a gene, cDNA, or mRNA, to serve as a template for the synthesis of other polymers and macromolecules in biological processes, having either a defined sequence of nucleotides (i.e., rRNA, tRNA, and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom. Thus, a gene encodes a protein if transcription and translation of its corresponding mRNA produces the protein in a cell or other biological system. Both the coding strand, whose nucleotide sequence is identical to the mRNA sequence and usually provided in a sequence listing, and the non-coding strand, which is used as a template for transcription of the gene or cDNA, can be said to encode the protein or other product of that gene or cDNA.

[0252] In some embodiments, nucleic acids such as mRNAs used in the present disclosure comprise nucleic acid sequences that encode one or more functional sequences, which may be peptides or polypeptides, preferably pharmaceutically active peptides or polypeptides.

[0253] In a preferred embodiment, a nucleic acid such as an mRNA used in the present 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 when transferred to a cell or a subject. Thus, in some embodiments, a nucleic acid used in the present disclosure comprises a coding region (open reading frame (ORF)) encoding a peptide or polypeptide, e.g., a pharmaceutically active peptide or polypeptide. In this regard, an "open reading frame" or "ORF" is a stretch of consecutive codons beginning with an initiation codon and ending with a stop codon. Such a nucleic acid encoding a pharmaceutically active peptide or polypeptide is also referred to herein as a "pharmaceutically active nucleic acid." In particular, such an mRNA encoding a pharmaceutically active peptide or polypeptide is also referred to herein as a "pharmaceutically active mRNA."

[0254] According to the present disclosure, the term "pharmaceutically active peptide or polypeptide" refers to a peptide or polypeptide that can be used to treat an individual where expression of the peptide or polypeptide is beneficial, for example, in ameliorating disease symptoms. Preferably, a pharmaceutically active peptide or polypeptide has therapeutic or palliative properties and may be administered to improve, alleviate, relieve, reverse, delay the onset, or reduce the severity of one or more symptoms of a disease. In some embodiments, a pharmaceutically active peptide or polypeptide, when administered to an individual in a therapeutically effective amount, has a beneficial or advantageous effect on the individual's condition or disease state. A pharmaceutically active peptide or polypeptide has prophylactic properties and may be used to delay the onset or reduce the severity of a disease. The term "pharmaceutically active peptide or polypeptide" includes whole proteins or polypeptides and can also refer to pharmaceutically active fragments thereof. It can also include pharmaceutically active analogs of peptides or polypeptides.

[0255] 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-modifying proteins, and blood proteins.

[0256] The term "cytokine" refers to a protein having a molecular weight of approximately 5 to 60 kDa that contributes to cell signaling (e.g., paracrine, endocrine, and / or autocrine signaling). Specifically, upon release, cytokines affect the behavior of cells surrounding their release site. Examples of cytokines include lymphokines, interleukins, chemokines, interferons, and tumor necrosis factors (TNFs). According to the present disclosure, cytokines do not include hormones or growth factors. Cytokines differ from hormones in that (i) they typically act at more variable concentrations than hormones and (ii) they are generally produced by a wide range of cells (almost all nucleated cells can produce cytokines). Interferons are generally characterized by antiviral, antiproliferative, and immunomodulatory activities. Interferons are proteins that alter and regulate gene transcription in cells by binding to interferon receptors on the surface of the regulated cells, thereby preventing viral replication within the cells. Interferons can be classified into two types: IFN-gamma is the only type II interferon; all others are type I interferons.Specific examples of cytokines include erythropoietin (EPO), colony-stimulating factors (CSFs), granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), tumor necrosis factors (TNFs), bone morphogenetic proteins (BMPs), interferon alpha (IFNα), interferon beta (IFNβ), interferon gamma (INFγ), interleukin 2 (IL-2), and erythrocyte-associated cytokines (IL-1, IL-2). 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), and variants and derivatives thereof.

[0257] In some embodiments, the pharmaceutically active peptide or polypeptide comprises a replacement protein. In these embodiments, the present disclosure provides a method of treating a subject having a disorder requiring protein replacement (e.g., a protein deficiency disorder), comprising administering to the subject a nucleic acid described herein encoding the replacement protein. The term "protein replacement" refers to the introduction of a protein (including a functional variant thereof) to a subject having a deficiency of such protein. The term also refers to the introduction of a protein to a subject who otherwise requires or would benefit from the provision of a protein, e.g., a subject suffering from a protein deficiency. The term "disorder characterized by protein deficiency" refers to any disorder exhibiting pathology caused by the absence or insufficient amount of a protein. This term encompasses protein folding or conformational disorders that result in a biologically inactive protein product. Protein deficiency may be involved in infectious disease, immunosuppression, organ failure, glandular disease, radiation damage, malnutrition, poisoning, or other environmental or external causes of pathology.

[0258] The term "hormone" refers to a class of signaling molecules produced by glands, and signal transduction typically involves the following steps: (i) synthesis of the hormone in a specific tissue, (ii) storage and secretion, (iii) transport of the hormone to its target, (iv) binding of the hormone by a receptor, (v) signal transmission and amplification, and (vi) destruction of the hormone. Hormones differ from cytokines in that (1) hormones typically act at concentrations with low fluctuations and (2) are generally produced by specific cell types. In some embodiments, a "hormone" is a peptide or polypeptide hormone, such as insulin, vasopressin, prolactin, adrenocorticotropic hormone (ACTH), thyroid hormone, growth hormone (e.g., human growth hormone or bovine somatotropin), oxytocin, atrial natriuretic peptide (ANP), glucagon, somatostatin, cholecystokinin, gastrin, and leptin.

[0259] The term "adhesion molecule" refers to a protein located on the surface of a cell and involved in binding of the cell to other cells or to the extracellular matrix (ECM). Adhesion molecules are typically transmembrane receptors and can be classified as calcium-independent (e.g., integrins, immunoglobulin superfamily, lymphocyte homing receptors) and calcium-dependent (cadherins and selectins). Specific examples of adhesion molecules include integrins, lymphocyte homing receptors, selectins (e.g., P-selectin), and addressins.

[0260] Integrins are also involved in signal transduction. In particular, upon ligand binding, integrins regulate cellular signaling pathways, e.g., transmembrane protein kinase pathways such as receptor tyrosine kinases (RTKs). Such regulation can result in cell proliferation, division, survival, or 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 These include β8, and α6β4.

[0261] The term "immunoglobulin" or "immunoglobulin superfamily" refers to molecules involved in the processes of cell recognition, binding, and / or adhesion. Molecules belonging to this superfamily share the characteristic that they 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 accessory molecules (e.g., CD3-γ, CD3-δ, CD3-ε, CD79a, CD79b), costimulatory or inhibitory molecules (e.g., CD28, CD80, CD86), and others.

[0262] The term "immunologically active compound" relates to any compound that alters the immune response, for example, by inducing and / or suppressing immune cell maturation, inducing and / or suppressing cytokine biosynthesis, and / or altering humoral immunity by stimulating antibody production by B cells. Immunologically active compounds have potent immunostimulatory activity, including but not limited to antiviral and antitumor activity, and can downregulate other aspects of the immune response, for example, decoupling the immune response from a TH2 immune response. This is useful for the treatment of a wide range of TH2-mediated diseases. Immunologically active compounds may be useful as vaccine adjuvants. Specific examples of immunologically active compounds include interleukins, colony-stimulating factors (CSFs), granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), erythropoietin, tumor necrosis factor (TNF), interferons, integrins, addressins, selectins, homing receptors, and antigens, particularly tumor-associated antigens, pathogen-associated antigens (e.g., bacterial, parasitic, or viral antigens), allergens, and autoantigens. The immunologically active compound may be a vaccine antigen, i.e., an antigen whose inoculation into a subject induces an immune response.

[0263] An "antigen" according to the present disclosure encompasses any substance that elicits an immune response and / or any substance against which an immune response or mechanism, such as a cellular and / or humoral response, is directed. This also includes situations where an antigen is processed into antigenic peptides, particularly when presented in the context of MHC molecules, and an immune response or mechanism is directed against one or more antigenic peptides. In particular, "antigen" relates 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 molecules that contain at least one epitope, e.g., a T cell epitope. In some embodiments, an antigen is a molecule that elicits an immune response, possibly after processing, that may be specific for the antigen (including cells expressing the antigen). In some embodiments, the antigen is a disease-associated antigen, such as a tumor antigen, a viral antigen, or a bacterial antigen, or an epitope derived from these antigens.

[0264] The term "autoantigen" (or "self-antigen") refers to an antigen that originates within a subject's body (i.e., an autoantigen may also be referred to as an "autologous antigen") and that produces an abnormally vigorous immune response against this normal part of the body. Such a vigorous immune response against an autoantigen can be the cause of an "autoimmune disease."

[0265] According to the present disclosure, any suitable antigen that is a candidate for an immune response may be used, and the immune response may be a humoral or cellular immune response. In the context of some embodiments of the present disclosure, the antigen is presented by cells, for example, by antigen-presenting cells in the context of MHC molecules, which results in an immune response to the antigen. The antigen may correspond to or be a product derived from a naturally occurring antigen. Such naturally occurring antigens may include or be derived from allergens, viruses, bacteria, fungi, parasites, and other infectious agents and pathogens, and the antigen may also be a tumor antigen. According to the present disclosure, the antigen may correspond to a naturally occurring product, for example, a viral protein or portion thereof.

[0266] The term "disease-associated antigen" is used in its broadest sense and 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 elicit a cellular, antigen-specific immune response and / or a humoral antibody response against the disease. Disease-associated antigens include pathogen-associated antigens, i.e., antigens associated with microbial infections, typically microbial antigens (e.g., bacterial or viral antigens), or antigens associated with cancer, typically tumors, e.g., tumor antigens.

[0267] In some embodiments, the antigen is a tumor antigen, i.e., an antigen that occurs primarily intracellularly or as a surface antigen on tumor cells. In another embodiment, the antigen is a pathogen-associated antigen, i.e., an antigen derived from a pathogen, e.g., a virus, bacterium, single-cell organism, or parasite, e.g., a viral antigen, e.g., a viral ribonucleoprotein or coat protein. In some embodiments, the antigen may be presented by MHC molecules, which result in modulation, particularly through modulation of T cell receptor activity, particularly activation of cells of the immune system, e.g., CD4+ and CD8+ lymphocytes.

[0268] The term "tumor antigen" refers to a component of a cancer cell, which may originate from the cytoplasm, cell surface, or cell nucleus. In particular, it refers to an antigen produced intracellularly or as a surface antigen on a tumor cell. For example, tumor antigens include carcinoembryonic antigen, alpha 1-fetoprotein, isoferritin, and fetal sulfoglycoprotein, alpha 2-H-ferroprotein, and gamma-fetoprotein, as well as various viral tumor antigens. According to some embodiments of the present disclosure, tumor antigens include any antigen that is characteristic of a tumor or cancer, and tumor or cancer cells associated with the type and / or expression level.

[0269] The term "viral antigen" refers to any viral component that has antigenic properties, i.e., is capable of eliciting an immune response in an individual. A viral antigen may be a viral ribonucleoprotein or an envelope protein.

[0270] The term "bacterial antigen" refers to any bacterial component that has antigenic properties, i.e., is capable of eliciting an immune response in an individual. Bacterial antigens can be derived from the bacterial cell wall or cytoplasmic membrane.

[0271] The term "epitope" refers to an antigenic determinant within a molecule, e.g., an antigen, i.e., 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 an MHC molecule. An epitope of a protein may comprise a contiguous or non-contiguous portion of the protein and may be, e.g., about 5 to about 100, about 5 to about 50, about 8 to about 30, or about 10 to about 25 amino acids in length; e.g., an epitope may be preferably 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids in length. In some embodiments, an epitope in the context of the present disclosure is a T cell epitope.

[0272] Terms such as "epitope," "fragment of an antigen," "immunogenic peptide," and "antigenic peptide" are used interchangeably herein and may refer, for example, to an antigen or an incomplete representation of an antigen that is capable of eliciting an immune response against a cell that expresses or contains and presents the antigen. In some embodiments, the term refers to an immunogenic portion of an antigen. In some embodiments, this is the part of the antigen that is recognized (i.e., specifically bound) by a T cell receptor, particularly when presented in the context of an MHC molecule. Certain preferred immunogenic portions bind to MHC class I or class 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 can be recognized by a T cell, a B cell, or an antibody. An epitope of an antigen includes a contiguous or discontinuous portion of the antigen and may be about 5 to about 100, e.g., about 5 to about 50, about 8 to about 30, or about 8 to about 25 amino acids in length; for example, 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 length. In some embodiments, an epitope is about 10 to about 25 amino acids in length. The term "epitope" includes T-cell epitopes.

[0273] 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 an MHC molecule. The terms "major histocompatibility complex" and the abbreviation "MHC" refer to a complex of genes present in all vertebrates, including MHC class I and MHC class II molecules. MHC proteins or molecules are important for signaling between lymphocytes and antigen-presenting cells or diseased cells in the immune response; they bind peptide epitopes and present them for recognition by T cell receptors on T cells. Proteins encoded by MHC are expressed on the surface of cells and present both self-antigens (peptide fragments derived from the cell itself) and non-self-antigens (e.g., fragments of invading microorganisms) to T cells. In the case of class I MHC / peptide complexes, the binding peptides are typically about 8 to about 10 amino acids in length, although longer or shorter peptides can also be effective. In the case of class II MHC / peptide complexes, the binding peptides are typically about 10 to about 25 amino acids in length, particularly about 13 to about 18 amino acids in length, although longer or shorter peptides may also be effective.

[0274] Peptide and polypeptide antigens are between 2 and 100 amino acids in length, including lengths of 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids. In some embodiments, peptides may be greater than 50 amino acids. In some embodiments, peptides may be greater than 100 amino acids.

[0275] A peptide or polypeptide antigen may be any peptide or polypeptide that is capable of inducing or increasing the ability of the immune system to mount antibody and T cell responses against that peptide or polypeptide.

[0276] In some embodiments, vaccine antigens, i.e., antigens whose inoculation into a subject induces an immune response, are recognized by immune effector cells. In some embodiments, once recognized by immune effector cells, vaccine antigens can, in the presence of appropriate costimulatory signals, induce stimulation, priming, and / or proliferation of immune effector cells bearing antigen receptors that recognize the vaccine antigen. In the context of embodiments of the present disclosure, vaccine antigens may be presented or present, for example, on the surface of a cell, e.g., an antigen-presenting cell. In some embodiments, the antigen is presented by a diseased cell (e.g., a tumor cell or an infected cell). In some embodiments, the antigen receptor is a TCR that binds to an epitope of an antigen presented in the context of an MHC. In some embodiments, binding of a TCR, when expressed by and / or present on a T cell, to an antigen presented by a cell, such as an antigen-presenting cell, results in stimulation, priming, and / or proliferation of the T cell. In some embodiments, binding of a TCR, when expressed by and / or present on a T cell, to an antigen presented on the diseased cell results in cytolysis and / or apoptosis of the diseased cell, and the T cell releases cytokine factors, such as perforin and granzymes.

[0277] In some embodiments, the antigen processing and / or presentation enhancing amino acid sequence is fused to the antigenic peptide or polypeptide, either directly or via a linker sequence. Thus, in some embodiments, the nucleic acids (e.g., RNA and / or DNA) described herein comprise at least one coding region that encodes an antigenic peptide or polypeptide and an amino acid sequence that enhances antigen processing and / or presentation.

[0278] Such amino acid sequences that enhance antigen processing and / or presentation are preferably, but not limited to, located C-terminal to the antigenic peptide or polypeptide and linker sequence (and may be located C-terminal to the amino acid sequence that breaks immune tolerance). 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 the human MHC class I complex (HLA-B51, haplotype A2, B27 / B51, Cw2 / Cw3). In addition to improving antigen processing and presentation, such amino acid sequences that enhance antigen processing and / or presentation may also be used to determine the expression of the amino acid sequences in the processes described herein.

[0279] Thus, in a particularly preferred embodiment, the RNA described herein comprises at least one coding region encoding an antigenic peptide or polypeptide and an amino acid sequence that enhances antigen processing and / or presentation, wherein said amino acid sequence that enhances antigen processing and / or presentation is preferably fused to the antigenic peptide or polypeptide, more preferably to the C-terminus of the antigenic peptide or polypeptide described herein.

[0280] Additionally, the secretory sequence may be fused to the N-terminus of the antigenic peptide or polypeptide.

[0281] To efficiently mount an immune response against self-antigens and overcome self-tolerance mechanisms by providing help to T cells during priming, an amino acid sequence derived from tetanus toxoid of Clostridium tetani may be employed.

[0282] The heavy chain of tetanus toxoid contains epitopes that can bind promiscuously to MHC class II alleles, and induces CD4 expression in almost all tetanus-vaccinated individuals. +It is known that tetanus toxoid (TT) helper epitopes in combination with tumor-associated antigens induce memory T cells. Furthermore, the combination of tetanus toxoid (TT) helper epitopes with tumor-associated antigens induces CD4+ T cells during priming. + It is known that tumor-associated antigens improve immune stimulation by boosting CD8 T cells compared to the application of tumor-associated antigens alone. + To reduce the risk of stimulating T cells, CD8 + The whole fragment C of tetanus toxoid, which is known to contain T cell epitopes, is not used.

[0283] In some embodiments, the immune tolerance-breaking amino acid sequence is fused, either directly or via a linker, to the antigenic peptide or polypeptide.

[0284] Such tolerance-breaking amino acid sequences are preferably located at the C-terminus of the antigenic peptide or polypeptide (and optionally N-terminus of the antigen processing and / or presentation enhancing amino acid sequence), and the tolerance-breaking amino acid sequence and the antigen processing and / or presentation enhancing amino acid sequence may be fused directly or via a linker. The tolerance-breaking amino acid sequences defined herein preferably improve T cell responses. In one embodiment, tolerance-breaking amino acid sequences defined herein include, but are not limited to, sequences derived from the helper sequences p2 and p16 (P2P16) from tetanus toxoid.

[0285] In some embodiments, the bioluminescence producing amino acid sequence is fused, either directly or via a linker, to the antigenic peptide or polypeptide.

[0286] Such a bioluminescence-producing amino acid sequence is preferably located at the C-terminus of the antigenic peptide or polypeptide (and optionally at the N-terminus of the (i) antigen processing and / or presentation enhancing amino acid sequence or (ii) immune tolerance disrupting amino acid sequence), and the bioluminescence-producing amino acid sequence and the (i) antigen processing and / or presentation enhancing amino acid sequence or the (ii) immune tolerance disrupting amino acid sequence may be fused directly or via a linker. The bioluminescence-producing amino acid sequence defined herein preferably improves the determination of the quantity of the antigenic peptide or polypeptide. In some embodiments, the bioluminescence-producing amino acid sequence defined herein fluoresces. In some embodiments, bioluminescence-producing 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), variants thereof (e.g., enhanced GFP (EGFP), superfolder GFP (sfGFP), and luciferase.

[0287] Vaccine RNA embodiments are described below, and certain terms used in describing these elements have the following meanings. hAg-Kozak: 5'UTR sequence of human alpha globin mRNA with an optimized "Kozak sequence" to increase translation efficiency.

[0288] sec / MITD: A fusion protein tag derived from sequences encoding the human MHC class I complex (HLA-B51, haplotype A2, B27 / B51, Cw2 / Cw3). It has been shown to improve antigen processing and presentation. Sec corresponds to a 78-bp fragment encoding a secretory signal peptide, which guides translocation of the nascent polypeptide chain into the endoplasmic reticulum. MITD corresponds to the transmembrane and cytoplasmic domains of the MHC class I molecule and is also referred to as the MHC class I transport domain.

[0289] Antigen: The sequence encoding the respective antigen / epitope.

[0290] Glycine-serine linker (GS): A sequence encoding a linker sequence according to the present invention, which in one embodiment is a glycine-serine linker sequence, a short linker peptide consisting mainly of the amino acids glycine (G) and serine (S), commonly used for fusion proteins. In a particular embodiment of the present invention, the linker sequence is preceded by a lysine residue at its N-terminus, and the sequence can be represented as follows: GGSGGGGSGGR / K. Thus, a portion of the amino acid sequence constituting the linker sequence can be represented as follows: KΔGGSGGGGSGGR / K (Δ indicates the proteolytic cleavage site). After cleavage, this results in the excision of the linker sequence as follows: GGSGGGGSGGR / K. In one embodiment, the linker sequence of the present invention is a GS linker containing at least one residue that is not G or S, respectively, and this amino acid residue forms the proteolytic cleavage site for a protease.

[0291] P2P16: A sequence encoding a helper epitope derived from tetanus toxoid for breaking immune tolerance.

[0292] The FI element:3'UTR is a combination of two sequence elements derived from the "amino-terminal cleavage enhancer" (AES) mRNA (termed F) and the mitochondrially encoded 12S ribosomal RNA (termed I). These were identified by an ex vivo selection process for sequences that confer RNA stability and increase total protein expression.

[0293] A30L70: 110 nucleotides in length, consisting of 30 consecutive adenosine residues, a 10 nucleotide linker sequence, and a poly(A) tail of 70 more adenosine residues, designed to increase RNA stability and translation efficiency in dendritic cells.

[0294] In one embodiment, the vaccine RNA described herein has the structure: Beta-S-ARCA(D1)-hAg-Kozak-sec-GS(1)-Antigen-GS(2)-P2P16-GS(3)-MITD-FI-A30L70 It has.

[0295] In one embodiment, the vaccine antigen described herein has the structure: sec-GS(1)-antigen-GS(2)-P2P16-GS(3)-MITD It has.

[0296] In one embodiment, there are multiple vaccine antigen RNA constructs (nucleic acids) as described herein contained in one formulation, e.g., one particle (LNP, LPX, PLX, etc.), and each vaccine RNA construct contains a different linker sequence.

[0297] 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 a natural epitope of the antigen.

[0298] The terms "expressed on the cell surface" or "associated with the cell surface" mean that a molecule, such as an antigen, is associated with and located at the plasma membrane of a cell, with at least a portion of the molecule facing the extracellular space of the cell, e.g., an antibody located on the exterior of the cell being accessible from the exterior of the cell. In this context, a portion can be, for example, at least 4, at least 8, at least 12, or at least 20 amino acids. The association can be direct or indirect. For example, the association can be through interaction with one or more transmembrane domains, one or more lipid anchors, or any other protein, lipid, sugar, or other structure that can be found on the outer leaflet of the plasma membrane of a cell. For example, a molecule associated with the surface of a cell can be a transmembrane protein having an extracellular portion, or a protein that associates with the surface of a cell by interacting with another protein that is a transmembrane protein.

[0299] "Cell surface" or "surface of a cell" is used according to its ordinary meaning in the art and thus includes the exterior of a cell that is accessible for binding by proteins and other molecules. An antigen is expressed on the surface of a cell if it is located on the surface of the cell and is accessible for binding, for example, by an antigen-specific antibody that is added to the cell.

[0300] The term "extracellular portion" or "exodomain" in the context of the present disclosure refers to a part of a molecule, such as a protein, that faces the extracellular space of a cell and is preferably accessible from outside the cell, e.g., by a binding molecule, such as an antibody, that is located on the outside of the cell. In some embodiments, the term refers to one or more extracellular loops or domains or fragments thereof.

[0301] The terms "T cell" and "T lymphocyte" are used interchangeably herein and include T helper cells (CD4+ T cells) and cytotoxic T cells (CTL, cytotoxic T cells, CD8+ T cells), including cytolytic T cells. The term "antigen-specific T cell" and similar terms refer to a T cell that recognizes the antigen targeted by the T cell in association with an MHC molecule, particularly when presented on the surface of an antigen-presenting cell or a diseased cell, such as a cancer cell, and preferably exerts T cell effector function. A T cell is considered specific for an antigen if it kills a target cell expressing the antigen. T cell specificity may be assessed using any of a variety of standard techniques, e.g., chromium elution assays or proliferation assays. Alternatively, the synthesis of lymphokines (e.g., interferon-γ) can be measured.

[0302] The term "target" is intended to mean an agent, such as a cell or tissue, that is the target for an immune response, such as a cellular immune response. Targets include cells that present an antigen or an antigen epitope, i.e., a peptide fragment derived from an antigen. In some embodiments, target cells are cells that express an antigen and present said antigen in conjunction with class I MHC.

[0303] "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 peptides) and the association (e.g., via binding) of one or more of these fragments with an MHC molecule for presentation to a specific T cell by a cell, e.g., an antigen-presenting cell.

[0304] "Antigen-responsive CTL" refers to a CD8 CTL that responds to an antigen or a peptide derived from the antigen. + It refers to T cells, which are presented with class I MHC on the surface of antigen-presenting cells.

[0305] According to the present disclosure, CTL responsiveness can include sustained calcium flux, cell division, production of cytokines such as IFN-γ and TNF-α, upregulation of activation markers such as CD44 and CD69, and specific cytolytic killing of target cells expressing tumor antigens. CTL responsiveness may also be determined using artificial reporters that accurately represent CTL responsiveness.

[0306] "Activation" or "stimulation," as used herein, refers to the state of a cell, e.g., an immune effector cell, such as a T cell, that has been stimulated sufficiently to induce detectable cell proliferation. Activation can also be associated with the initiation of signal transduction pathways, the induction of cytokine production, and detectable effector function. The term "activated immune effector cell" refers, inter alia, to an immune effector cell that is undergoing cell division.

[0307] The term "priming" refers to the process by which an immune effector cell, such as a T cell, first contacts its specific antigen, causing it to differentiate into an effector cell, such as an effector T cell.

[0308] The term "proliferation" refers to the process by which a specific entity increases. In some embodiments, the term is used in reference to an immune response in which immune effector cells are stimulated by an antigen, causing proliferation and amplification of specific immune effector cells that recognize the antigen. In some embodiments, proliferation results in differentiation of immune effector cells.

[0309] The terms "immune response" and "immune reaction" are used interchangeably herein in their conventional sense to refer to the body's integrated response to an antigen and may refer to a cellular immune response, a humoral immune response, or both. According to the present disclosure, the terms "immune response to" or "immune response against" in reference to an agent such as an antigen, cell, or tissue refers to an immune response, such as a cellular response, directed against that agent. An immune response includes the development of antibodies against one or more antigens and the production of antigen-specific T lymphocytes, e.g., CD4 + and CD8 + T lymphocytes, e.g., CD8 +The present invention may include one or more responses selected from the group consisting of proliferation of T lymphocytes, which may be detected in various in vitro proliferation or cytokine production tests.

[0310] The terms "induction of an immune response" and "eliciting an immune response," and similar terms in the context of the present disclosure, refer to the induction of an immune response, e.g., the induction of a cellular immune response, a humoral immune response, or both. The immune response may be protective / preventative / prophylactic and / or therapeutic. The immune response may be directed against any immunogen or antigen or antigenic peptide, such as a tumor-associated antigen or a pathogen-associated antigen (e.g., an antigen of a virus (e.g., influenza virus (A, B, or C), CMV, or RSV)). "Induction" in this context can mean that there was no immune response against a particular antigen or pathogen before induction, but it can also mean that there was some level of immune response against a particular antigen or pathogen before induction, and that after induction, the immune response is enhanced. Thus, "induction of an immune response" in this context also includes "enhancing an immune response." In some embodiments, after induction of an immune response in an individual, the individual is protected from developing a disease, such as an infectious disease or a cancer disease, or the disease state is ameliorated by the induction of an immune response.

[0311] The terms "cellular immune response," "cell-mediated response," "cell-mediated immunity," or similar terms are meant to include cell-directed responses characterized by expression of an antigen and / or presentation of an antigen with class I or class II MHC. Cellular responses involve cells called T cells or T lymphocytes that act as "helpers" or "killers." Helper T cells (CD4 + T cells (also named T cells) play a central role by regulating the immune response, and killer cells (cytotoxic T cells, cytolytic T cells, CD8 + T cells, or CTLs, kill cells such as diseased cells.

[0312] The term "humoral immune response" refers to the process in an organism in which antibodies are produced in response to agents and organisms, ultimately neutralizing and / or eliminating them. The specificity of the antibody response is mediated by T and / or B cells through membrane-associated receptors that bind to a single specific antigen. Following binding of the appropriate antigen and reception of various other activation signals, B lymphocytes divide, producing memory B cells and antibody-secreting plasma cell clones, each of which produces antibodies that recognize the same antigenic epitope as that recognized by its antigen receptor. Memory B lymphocytes remain quiescent until subsequently activated by their specific antigen. These lymphocytes provide the cellular basis of memory, resulting in an enhanced antibody response upon re-exposure to a specific antigen.

[0313] As used herein, the term "antibody" refers to an immunoglobulin molecule capable of specifically binding to an epitope on an antigen. In particular, the term "antibody" refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds. The term "antibody" includes monoclonal antibodies, recombinant antibodies, human antibodies, humanized antibodies, chimeric antibodies, and any combination of the above. Each heavy chain consists of a heavy chain variable region (VH, heavy chain variable region) and a heavy chain constant region (CH, heavy chain constant region). Each light chain consists of a light chain variable region (VL, light chain variable region) and a light chain constant region (CL, light chain constant region). 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 regions of hypervariability, termed complementarity determining regions (CDRs), interspersed with more conserved regions, termed framework regions (FRs). Each VH and VL consists of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The CDRs of VH are termed HCDR1, HCDR2, and HCDR3, and the CDRs of VL are termed LCDR1, LCDR2, and LCDR3. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant region of an antibody comprises a heavy chain constant region (CH) and a light chain constant region (CL), which can be further subdivided into a constant domain CH1, a hinge region, and constant domains CH2 and CH3 (arranged from the amino terminus to the carboxy terminus in the following order: CH1, CH2, CH3). The constant regions of the antibody can mediate the binding of the immunoglobulin 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 intact immunoglobulins derived from natural or recombinant sources, or immunologically active portions of intact immunoglobulins. 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.

[0314] The term "immunoglobulin" relates to proteins of the immunoglobulin superfamily, such as antigen receptors, e.g., antibodies or B cell receptors (BCRs). Immunoglobulins are characterized by structural domains, i.e., immunoglobulin domains, with a characteristic immunoglobulin (Ig) fold. The term encompasses membrane-bound immunoglobulins as well as soluble immunoglobulins. Membrane-bound immunoglobulins are also named surface or membrane immunoglobulins, and are generally part of the BCR. Soluble immunoglobulins are generally named antibodies. Immunoglobulins generally contain several chains, typically two identical heavy chains and two identical light chains linked via disulfide bonds. These chains are primarily composed of immunoglobulin domains, e.g., 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 H4. There are five types of mammalian immunoglobulin heavy chains, namely α, δ, ε, γ, and μ, which correspond to the different classes of antibodies, namely 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 carboxy termini. In mammals, there are two types of light chains, namely lambda and kappa. Immunoglobulin chains contain a variable region and a constant region. The constant region is essentially conserved among immunoglobulins of different isotypes, while the variable portion is highly variable and is responsible for antigen recognition.

[0315] The terms "vaccination" and "immunization" describe the process of treating an individual for therapeutic or prophylactic reasons and relate to the procedure of administering to an individual one or more of the immunogens or antigens described herein or derivatives thereof, particularly in the form of RNA (especially mRNA) encoding them, to stimulate an immune response against said one or more immunogens or antigens or against cells characterized by the presentation of said one or more immunogens or antigens.

[0316] By "cells characterized by antigen presentation" or "cells presenting antigen" or "MHC molecules presenting antigen on the surface of antigen-presenting cells" or similar expressions is meant diseased cells, particularly tumor cells or infected cells, or cells such as antigen-presenting cells that present antigens or antigenic peptides, either directly or after processing, in association with MHC molecules, e.g., MHC class I and / or MHC class II molecules. In some embodiments, the MHC molecules are MHC class I molecules.

[0317] The term "allergen" refers to a type of antigen that originates from outside the subject's body (i.e., allergens are also referred to as "xenoantigens") and causes the subject's immune system to produce an unusually vigorous immune response to fight off a perceived threat that would otherwise be harmless to the subject. "Allergy" is a disease caused by such a vigorous immune response to allergens. Allergens are typically antigens that can stimulate a type I hypersensitivity reaction via an immunoglobulin E (IgE) response in atopic individuals. Specific examples of allergens include allergens derived from peanut proteins (e.g., Ara h 2.02), ovalbumin, grass pollen proteins (e.g., Phl p 5), and house dust mite proteins (e.g., Der p 2).

[0318] The term "growth factor" refers to a molecule 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 proteins (BMPs), fibroblast growth factors (FGFs), vascular endothelial growth factors (VEGFs), e.g., VEGFA, epidermal growth factor (EGF), insulin-like growth factors, ephrins, macrophage colony-stimulating factor, granulocyte colony-stimulating factor, granulocyte-macrophage colony-stimulating factor, neuregulins, neurotrophins (e.g., brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF)), placental growth factor (PGF), platelet-derived growth factor (PDGF), renalase (RNLS) (an anti-apoptotic survival factor), T-cell growth factor (TCGF), and erythrocyte-associated growth factor (YAG). Growth factors include transforming growth factor (transforming growth factor alpha (TGF-α)), transforming growth factor beta (TGF-β), and tumor necrosis factor alpha (TNF-α). In some embodiments, a "growth factor" is a peptide or polypeptide growth factor.

[0319] The term "protease inhibitor" refers to a molecule, particularly a peptide or polypeptide, that inhibits the function of a protease. 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 serpins). Specific examples of protease inhibitors include serpins, such as alpha-1-antitrypsin, aprotinin, and bestatin.

[0320] The term "enzyme" refers to a macromolecular biological catalyst that facilitates chemical reactions. Like any catalyst, enzymes are not consumed in the reactions they catalyze and do not alter the equilibrium of the reaction. Unlike many other catalysts, enzymes are more specific. In some embodiments, enzymes are important to the homeostasis of a subject; for example, any malfunction of an enzyme (particularly a decrease in activity, which can be caused by either mutation, deletion, or reduced production) can result in disease. Examples of enzymes include herpes simplex virus type 1 thymidine kinase (HSV1-TK), hexosaminidase, phenylalanine hydroxylase, pseudocholinesterase, and lactase.

[0321] The term "receptor" refers to a protein molecule that receives a signal (e.g., a chemical signal called a ligand) from outside the cell. Binding of a signal (e.g., a ligand) to a receptor triggers a certain cellular response, such as the intracellular activation of a kinase. Receptors include transmembrane receptors (e.g., ion channel-linked (ionotropic) receptors, G protein-linked (metabotropic) receptors, and enzyme-linked receptors) and intracellular receptors (e.g., cytoplasmic receptors and nuclear receptors). Specific examples of receptors include steroid hormone receptors, growth factor receptors, and peptide receptors (i.e., receptors whose ligands are peptides), such as P-selectin glycoprotein ligand-1 (PSGL-1). The term "growth factor receptor" refers to a receptor that binds to a growth factor.

[0322] The term "apoptosis-controlling agent" refers to molecules, particularly peptides or polypeptides, that control apoptosis, i.e., activate or inhibit apoptosis. Apoptosis-controlling agents can be divided into two broad classes: those that regulate mitochondrial function and those that regulate caspases. The first class includes proteins (BCL-2, BCL-xL) that act to preserve mitochondrial integrity by preventing loss of mitochondrial membrane potential and / or release of pro-apoptotic proteins, such as cytochrome c, into the cytosol. Also included in this first class are pro-apoptotic proteins (e.g., BAX, BAK, BIM) that promote the release of cytochrome c. The second class includes proteins such as inhibitors of apoptosis proteins (e.g., XIAP) or FLIP, which block caspase activation.

[0323] 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 in response to extracellular signals such as hormones. Transcription factors contain at least one DNA-binding domain that binds to specific DNA sequences that are usually adjacent to the genes regulated by the transcription factor. Specific examples of transcription factors include MECP2, FOXP2, FOXP3, the STAT protein family, and the HOX protein family.

[0324] The term "tumor suppressor protein" relates to molecules, particularly peptides or polypeptides, that protect cells from one step on the path to cancer. Tumor suppressor proteins (usually encoded by corresponding tumor suppressor genes) exert a dampening or suppressing effect on cell cycle control and / or promote apoptosis. Their functions may be one or more of the following: suppression of genes essential for the continuation of the cell cycle; linking the cell cycle to DNA damage (cell division cannot occur as long as damaged DNA is present in the cell); initiation of apoptosis if damaged DNA cannot be repaired; inhibition of metastasis (e.g., preventing tumor cells from dispersing, blocking 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 (SWItch / Sucrose Non-Fermentable), von Hippel-Lindau tumor suppressor (pVHL), adenomatous polyposis coli (APC), CD95, suppression of tumorigenicity 5 (ST5), suppression of tumorigenicity 14 (ST14), and Yippee-like 3 (YPEL3).

[0325] The term "structural protein" refers to a protein that adds rigidity and stiffness to an otherwise fluid biological component. Structural proteins are often fibrous (e.g., collagen and elastin), but can also be globular (e.g., actin and tubulin). Globular proteins are usually soluble as monomers but polymerize to form long fibers that can, for example, constitute the cytoskeleton. Other structural proteins are motor proteins (e.g., myosin, kinesin, and dynein) that can generate mechanical force, 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.

[0326] The term "reprogramming factor" or "reprogramming transcription factor" relates to a molecule, in particular a peptide or polypeptide, which when expressed in a somatic cell, optionally together with further agents such as further reprogramming factors, leads to the reprogramming or dedifferentiation of said somatic cell into a cell with stem cell characteristics, in particular pluripotency. Particular examples of reprogramming factors include OCT4, SOX2, c-MYC, KLF4, LIN28, and NANOG.

[0327] The term "genetically engineered protein" refers to a protein that can insert, delete, or replace DNA in the genome of a subject.Specific examples of genetically engineered proteins include meganucleases, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly spaced short palindromic repeat-CRISPR-associated protein 9 (CRISPR-Cas9).

[0328] The term "blood protein" refers to a peptide or polypeptide 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), enzymatic activity (e.g., thrombin or ceruloplasmin), clotting (e.g., fibrinogen), defense against pathogens (e.g., complement components and immunoglobulins), protease inhibitors (e.g., alpha-1-antitrypsin), and others. 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.

[0329] Thus, in some embodiments, the pharmaceutically active peptide or polypeptide is selected from the group consisting of: (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 immunoactive compound, particularly an antigen; (v) a hormone, particularly vasopressin, insulin, or growth hormone; (vi) a growth factor, particularly VEGFA; (vii) a protease inhibitor, particularly alpha 1-antitrypsin; (viii) a protease inhibitor, preferably herpes simplex virus type 1 thymidine kinase (HSV1-TK), hexosaminidase, phenylalanine ... (xii) tumor suppressor proteins, particularly p53; (xiii) structural proteins, particularly surfactant protein B; (xiv) reprogramming factors, such as those selected from the group consisting of OCT4, SOX2, c-MYC, KLF4, LIN28, and NANOG; (xv) genome engineering proteins, particularly clustered regularly interspaced short inverted palindromic nucleotide sequences-CRISPR-associated protein 9 (CRISPR-Cas9); and (xvi) blood proteins, particularly fibrinogen.

[0330] In some embodiments, the pharmaceutically active peptide or polypeptide comprises one or more antigens or one or more epitopes, i.e., administration of the peptide or polypeptide to a subject elicits a therapeutic or partially or fully protective immune response in the subject against the one or more antigens or one or more epitopes.

[0331] In some embodiments, the nucleic acid, such as an mRNA, encodes at least one epitope.

[0332] In some embodiments, the epitope is derived from a tumor antigen. The tumor antigen may be a "standard" antigen commonly known to be expressed in various cancers. The tumor antigen may also be a "neoantigen" that is specific to an individual's tumor and has not previously been recognized by the immune system. The neoantigen or neoepitope may result from one or more cancer-specific mutations in the genome of cancer cells that result in amino acid changes. Examples of tumor antigens include p53, ART-4, BAGE, beta-catenin / m, Bcr-abL, CAMEL, CAP-1, CASP-8, CDC27 / m, CDK4 / m, CEA, cell surface proteins of the claudin family, e.g., CLAUDIN-6, CLAUDIN-18.2, and CLAUDIN-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, MAGE-A, preferably MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A5, MAGE-A6, MAGE-A7, MAGE-A8, MAGE-A9, MAGE-A10, MAGE-A11, or MAGE-A12, MAGE-B, MAGE-C, MART-1 / Melan-A, MC1R, myosin / m, MUC1, M These include, but are not limited to, UM-1, MUM-2, MUM-3, NA88-A, NF1, NY-ESO-1, NY-BR-1, p190 minor BCR-abL, Pml / RARa, PRAME, proteinase 3, PSA, PSM, RAGE, RU1 or RU2, SAGE, SART-1 or SART-3, SCGB3A2, SCP1, SCP2, SCP3, SSX, SURVIVIN, TEL / AML1, TPI / m, TRP-1, TRP-2, TRP-2 / INT2, TPTE, WT, and WT-1.

[0333] Cancer mutations vary from individual to individual. Therefore, cancer mutations encoding novel epitopes (neoepitopes) represent attractive targets for the development of vaccine compositions and immunotherapeutics. The effectiveness of tumor immunotherapy depends on the selection of cancer-specific antigens and epitopes that can induce a strong immune response in the host. RNA can be used to deliver patient-specific tumor epitopes to patients. Dendritic cells (DCs) present in the spleen represent particularly interesting antigen-presenting cells for RNA expression of immunogenic epitopes or antigens, such as tumor epitopes. The use of multiple epitopes has been shown to increase the therapeutic efficacy of tumor vaccine compositions. Rapid sequencing of the tumor mutagenesis can provide multiple epitopes for personalized vaccines, which can be encoded by the mRNA described herein, for example, as a single polypeptide in which the epitopes may be separated by a linker. In some embodiments of the present disclosure, the mRNA encodes at least 1 epitope, at least 2 epitopes, at least 3 epitopes, at least 4 epitopes, at least 5 epitopes, at least 6 epitopes, at least 7 epitopes, at least 8 epitopes, at least 9 epitopes, or at least 10 epitopes. Exemplary embodiments include mRNAs encoding at least 5 epitopes (designated "pentatopes") and mRNAs encoding at least 10 epitopes (designated "decatopes").

[0334] In some embodiments, the antigen or epitope is derived from a pathogen-associated antigen, particularly a viral antigen. In some embodiments, the antigen or epitope is derived from the SARS-CoV-2 S protein, an immunogenic variant thereof, or an immunogenic fragment of the SARS-CoV-2 S protein or its immunogenic variant. Thus, in some embodiments, the mRNA used in the present disclosure encodes an amino acid sequence comprising the SARS-CoV-2 S protein, an immunogenic variant thereof, or an immunogenic fragment of the SARS-CoV-2 S protein or its immunogenic variant.

[0335] In some embodiments of the present disclosure, antigens (e.g., tumor antigens or vaccine antigens) are preferably administered as single-stranded, 5'-capped mRNA that is translated into the respective protein upon entry into the cells of the subject receiving the RNA. Preferably, the RNA contains structural elements (5' cap, 5' UTR, 3' UTR, poly(A) sequence) optimized to maximize the effectiveness of the RNA with respect to stability and translation efficiency.

[0336] In some embodiments, beta-S-ARCA (D1) is utilized as the specific capping structure at the 5' end of the mRNA. In some embodiments, m2 is utilized as the specific capping structure at the 5' end of the mRNA. 7,3’-O Gppp(m1 2’-O ) ApG is utilized. In some embodiments, the 5' UTR sequence is derived from human alpha globin mRNA and may have an optimized "Kozak sequence" to increase translation efficiency. In some embodiments, a combination of two sequence elements (FI element) derived from the "amino-terminal cleavage enhancer" (AES) mRNA (termed F) and the mitochondrially encoded 12S ribosomal RNA (termed I) is placed between the coding sequence and the poly(A) sequence to ensure higher maximum protein levels and long-term mRNA persistence. In some embodiments, two re-repeated 3' UTRs derived from human beta globin mRNA are placed between the coding sequence and the poly(A) sequence to ensure higher maximum protein levels and long-term mRNA persistence. In some embodiments, a poly(A) sequence 110 nucleotides in length and consisting of 30 consecutive adenosine residues, followed by a 10-nucleotide linker sequence and an additional 70 adenosine residues, is used. This poly(A) sequence was designed to increase RNA stability and translation efficiency.

[0337] In some embodiments, mRNA encoding an antigen (e.g., a tumor antigen or a vaccine antigen) is expressed in cells of a subject treated 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, expression of the antigen occurs at the cell surface. In some embodiments, the antigen is expressed and presented in association with MHC. In some embodiments, expression of the antigen is directed to the extracellular space, i.e., the antigen is secreted.

[0338] An antigen molecule or its processing product, such as a fragment thereof, can bind to an antigen receptor, such as a BCR or TCR, or an antibody carried by an immune effector cell.

[0339] Peptide and polypeptide antigens provided to a subject according to the present disclosure by administering mRNA encoding the peptide and polypeptide antigen preferably result in the induction of an immune response, e.g., a humoral and / or cellular immune response, in the subject provided with the peptide or polypeptide antigen, when the antigen is a vaccine antigen. The immune response is preferably directed against a target antigen. Thus, a vaccine antigen may comprise the 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 the present disclosure, the term "antigen fragment" or "antigen variant" refers to an agent that results in the induction of an immune response targeted to the antigen, i.e., the target antigen. Thus, a vaccine antigen may correspond to or comprise the target antigen, a fragment of the target antigen, or an antigen that is homologous to the target antigen or a fragment thereof. That is, according to the present disclosure, a vaccine antigen may comprise an immunogenic fragment of the target antigen or an amino acid sequence that is homologous to an immunogenic fragment of the target antigen. An "immunogenic fragment of an antigen" according to the present disclosure preferably relates to a fragment of an antigen that is capable of inducing an immune response against the target antigen. The vaccine antigen may be a recombinant antigen.

[0340] The term "immunologically equivalent" means that immunologically equivalent molecules, e.g., immunologically equivalent amino acid sequences, exhibit the same or essentially the same immunological properties and / or exert the same or essentially the same immunological effect, e.g., with respect to the type of immunological effect. In the context of the present disclosure, the term "immunologically equivalent" is preferably used with reference to the immunological effect or properties of an antigen or antigen variant used for immunization. For example, an amino acid sequence is immunologically equivalent to a reference amino acid sequence if, when exposed to a subject's immune system, the amino acid sequence induces an immune response with specificity that reacts with the reference amino acid sequence.

[0341] In some embodiments, the mRNA used in the present disclosure is non-immunogenic. RNA encoding an immunostimulatory agent may be administered in accordance with the present disclosure to provide an adjuvant effect. The RNA encoding the immunostimulatory agent may be standard RNA or non-immunogenic RNA.

[0342] As used herein, the term "non-immunogenic RNA" (e.g., "non-immunogenic mRNA") refers to RNA that, e.g., upon administration to a mammal, does not induce a response by the immune system or that induces a weaker response than that induced by the same RNA, i.e., standard RNA (stdRNA), except that the RNA has not been subjected to the modifications and processes that render it non-immunogenic. In certain embodiments, non-immunogenic RNA, also designated herein as modified RNA (modRNA), is made non-immunogenic by incorporating modified nucleosides into the RNA and / or by eliminating double-stranded RNA (dsRNA) that inhibit RNA-mediated activation of specific immune receptors.

[0343] To render non-immunogenic RNA (particularly mRNA) non-immunogenic by incorporating a modified nucleoside, any modified nucleoside may be used as long as it reduces or suppresses the immunogenicity of the RNA. Particularly preferred are modified nucleosides that suppress RNA-mediated activation of specific immune receptors. In some embodiments, the modified nucleoside comprises the replacement of one or more uridines with a nucleoside comprising a modified nucleobase. In some embodiments, the modified nucleobase is a modified uracil. In some embodiments, the nucleoside comprising a modified nucleobase is a 3-methyluridine (m ). 3 U), 5-methoxyuridine (mo 5 U), 5-azauridine, 6-azauridine, 2-thio-5-azauridine, 2-thiouridine (s 2 U), 4-thiouridine (s 4 U), 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxyuridine (ho 5 U), 5-aminoallyl uridine, 5-halouridine (e.g., 5-iodouridine or 5-bromouridine), uridine 5-oxyacetic acid (cmo 5 U), uridine 5-hydroxyacetic acid methyl ester (mcmo 5 U), 5-carboxymethyluridine (cm 5 U), 1-carboxymethylpseudouridine, 5-carboxyhydroxymethyluridine (chm 5 U), 5-carboxyhydroxymethyluridine methyl ester (mchm 5 U), 5-methoxycarbonylmethyluridine (mcm 5 U), 5-methoxycarbonylmethyl-2-thiouridine (mcm 5 s 2 U), 5-aminomethyl-2-thiouridine (nm 5 s 2 U), 5-methylaminomethyluridine (mnm 5 U), 1-ethylpseudouridine, 5-methylaminomethyl-2-thiouridine (mnm 5 s 2 U), 5-methylaminomethyl-2-selenouridine (mnm5 se 2 U), 5-carbamoylmethyluridine (ncm 5 U), 5-carboxymethylaminomethyluridine (cmnm 5 U), 5-carboxymethylaminomethyl-2-thiouridine (cmnm 5 s 2 U), 5-propynyluridine, 1-propynylpseudouridine, 5-taurinomethyluridine (τm 5 U), 1-taurinomethylpseudouridine, 5-taurinomethyl-2-thiouridine (τm5s2U), 1-taurinomethyl-4-thiopseudouridine, 5-methyl-2-thiouridine (m 5 s 2 U), 1-methyl-4-thiopseudouridine (m 1 s 4 Ψ), 4-thio-1-methylpseudouridine, 3-methylpseudouridine (m 3 Ψ), 2-thio-1-methylpseudouridine, 1-methyl-1-deazapseudouridine, 2-thio-1-methyl-1-deazapseudouridine, dihydrouridine (D), dihydropseudouridine, 5,6-dihydrouridine, 5-methyldihydrouridine (m 5 D), 2-thiodihydrouridine, 2-thiodihydropseudouridine, 2-methoxyuridine, 2-methoxy-4-thiouridine, 4-methoxypseudouridine, 4-methoxy-2-thiopseudouridine, N1-methylpseudouridine, 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-thiouridine (inm 5 s 2 U), α-thiouridine, 2'-O-methyluridine (Um), 5,2'-O-dimethyluridine (m 5 Um), 2'-O-methylpseudouridine (Ψm), 2-thio-2'-O-methyluridine (s 2Um), 5-methoxycarbonylmethyl-2'-O-methyluridine (mcm 5 Um), 5-carbamoylmethyl-2'-O-methyluridine (ncm 5 Um), 5-carboxymethylaminomethyl-2'-O-methyluridine (cmnm 5 Um), 3,2'-O-dimethyluridine (m 3 Um), 5-(isopentenylaminomethyl)-2'-O-methyluridine (inm 5 Um), 1-thiouridine, deoxythymidine, 2'-F-ara-uridine, 2'-F-uridine, 2'-OH-ara-uridine, 5-(2-carbomethoxyvinyl)uridine, and 5-[3-(1-E-propenylamino)uridine. In certain embodiments, the nucleoside comprising a modified nucleobase is pseudouridine (Ψ), N1-methylpseudouridine (m1Ψ), or 5-methyluridine (m5U), particularly N1-methylpseudouridine.

[0344] In some embodiments, the replacement of one or more uridines with nucleosides comprising modified nucleobases comprises replacement of 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 uridines.

[0345] During the synthesis of mRNA by in vitro transcription (IVT) using T7 RNA polymerase, significant amounts of aberrant products, including double-stranded RNA (dsRNA), are produced by the activity of atypical enzymes. dsRNA induces inflammatory cytokines and activates effector enzymes, leading to the inhibition of protein synthesis. dsRNA can be removed from RNA, such as IVT RNA, by ion-pair reverse-phase HPLC, for example, using a nonporous or porous C-18 polystyrene-divinylbenzene (PS-DVB) matrix. Alternatively, an enzyme-based method can be used, using Escherichia coli (E. coli) RNase III, which specifically hydrolyzes dsRNA but does not degrade ssRNA, thereby eliminating dsRNA contaminants from IVT RNA preparations. Furthermore, dsRNA can be separated from ssRNA by using cellulosic materials. In some embodiments, the RNA preparation is contacted with cellulosic material, and ssRNA is separated from the cellulosic material under conditions that allow binding of dsRNA to the cellulosic material but not ssRNA to the cellulosic material. Suitable methods for providing ssRNA are disclosed, for example, in WO 2017 / 182524.

[0346] As used herein, "removing" or "removal" refers to the characteristic of a population of a first substance, e.g., non-immunogenic RNA, being separated from the vicinity of a population of a second substance, e.g., dsRNA, where the population of the first substance is not necessarily devoid of the second substance, and the population of the second substance is not necessarily devoid of the first substance. However, a population of the first substance characterized by the removal of the population of the second substance will have a somewhat reduced content of the second substance compared to an unseparated mixture of the first and second substances.

[0347] In some embodiments, removing dsRNA (particularly 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 (particularly mRNA) is free of or essentially free of dsRNA. In some embodiments, the non-immunogenic RNA (particularly mRNA) composition comprises a purified preparation of single-stranded nucleoside-modified RNA. For example, in some embodiments, a purified preparation of single-stranded nucleoside-modified RNA (particularly mRNA) is substantially free of double-stranded RNA (dsRNA). In some embodiments, a purified preparation 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.).

[0348] In some embodiments, non-immunogenic RNA (especially mRNA) is translated more efficiently in cells than standard RNA with the same sequence. In some embodiments, translation is enhanced by a factor of 2 relative to its unmodified counterpart. In some embodiments, translation is enhanced by a factor of 3. In some embodiments, translation is enhanced by a factor of 4. In some embodiments, translation is enhanced by a factor of 5. In some embodiments, translation is enhanced by a factor of 6. In some embodiments, translation is enhanced by a factor of 7. In some embodiments, translation is enhanced by a factor of 8. In some embodiments, translation is enhanced by a factor of 9. In some embodiments, translation is enhanced by a factor of 10. In some embodiments, translation is enhanced by a factor of 15. In some embodiments, translation is enhanced by a factor of 20. In some embodiments, translation is enhanced by a factor of 50. In some embodiments, translation is enhanced by a factor of 100. In some embodiments, translation is enhanced by a factor of 200. In some embodiments, translation is enhanced by a factor of 500. In some embodiments, translation is enhanced by a factor of 1000-fold. In some embodiments, translation is enhanced by a factor of 2000-fold. In some embodiments, the factor is 10-1000-fold. In some embodiments, the factor is 10-1000-fold. In some embodiments, the factor is 10-200-fold. In some embodiments, the factor is 10-300-fold. In some embodiments, the factor is 10-500-fold. In some embodiments, the factor is 20-1000-fold. In some embodiments, the factor is 30-1000-fold. In some embodiments, the factor is 50-1000-fold. In some embodiments, the factor is 100-1000-fold. In some embodiments, the factor is 200-1000-fold. In some embodiments, translation is enhanced by any other significant amount or range of amounts.

[0349] In some embodiments, non-immunogenic RNA (especially mRNA) exhibits significantly lower inherent immunogenicity than standard RNA having the same sequence. In some embodiments, non-immunogenic RNA (especially mRNA) exhibits an inherent immune response that is at least two-fold lower than its unmodified counterpart. In some embodiments, the inherent immunogenicity is reduced by three-fold. In some embodiments, the inherent immunogenicity is reduced by four-fold. In some embodiments, the inherent immunogenicity is reduced by five-fold. In some embodiments, the inherent immunogenicity is reduced by six-fold. In some embodiments, the inherent immunogenicity is reduced by seven-fold. In some embodiments, the inherent immunogenicity is reduced by eight-fold. In some embodiments, the inherent immunogenicity is reduced by nine-fold. In some embodiments, the inherent immunogenicity is reduced by ten-fold. In some embodiments, the inherent immunogenicity is reduced by fifteen-fold. In some embodiments, the inherent immunogenicity is reduced by twenty-fold. In some embodiments, the inherent immunogenicity is reduced by fifty-fold. In some embodiments, the inherent immunogenicity is reduced by 100-fold. In some embodiments, the inherent immunogenicity is reduced by 200-fold. In some embodiments, the inherent immunogenicity is reduced by 500-fold. In some embodiments, the inherent immunogenicity is reduced by 1000-fold. In some embodiments, the inherent immunogenicity is reduced by 2000-fold.

[0350] The term "exhibiting significantly reduced intrinsic immunogenicity" refers to a detectable reduction in intrinsic immunogenicity. In some embodiments, this term refers to a reduction such that an effective amount of non-immunogenic RNA (especially mRNA) can be administered without eliciting a detectable intrinsic immune response. In some embodiments, this term refers to a reduction such that the non-immunogenic RNA (especially mRNA) can be repeatedly administered without eliciting a sufficient intrinsic immune response to detectably reduce production of the protein encoded by the non-immunogenic RNA. In some embodiments, the reduction is such that the non-immunogenic RNA (especially mRNA) can be repeatedly administered without eliciting a sufficient intrinsic immune response to eliminate detectable production of the protein encoded by the non-immunogenic RNA.

[0351] "Immunogenicity" is the ability of a foreign substance, such as RNA, to elicit an immune response in humans or other animals. The innate immune system is a relatively nonspecific and rapid component of the immune system. It is one of the two major components of the vertebrate immune system, along with the adaptive immune system.

[0352] particle The nucleic acids described herein (e.g., RNA and / or DNA, particularly mRNA) may be present in particles comprising (i) the nucleic acid and (ii) at least one cationic or cationic ionizable compound, such as a polymer or lipid, complexed with the nucleic acid. Electrostatic interactions between positively charged molecules, such as polymers and lipids, and negatively charged nucleic acids are responsible for particle formation. This results in complexation and spontaneous formation of nucleic acid particles.

[0353] Various types of RNA-containing particles have already been described as suitable for the delivery of RNA in particulate form (see, for example, Kaczmarek, JC et al., 2017, Genome Medicine 9, 60). For non-viral RNA delivery vehicles, nanoparticle encapsulation of RNA can physically protect the RNA from degradation and, depending on the specific chemistry, aid in cellular uptake and endosomal escape.

[0354] In the context of the present disclosure, the term "particle" refers to a structural entity formed by molecules or molecular complexes, particularly particle-forming compounds. In some embodiments, a particle comprises an envelope (e.g., one or more layers or lamellae) composed of one or more types of amphiphilic substances (e.g., amphiphilic lipids). In this context, the expression "amphiphilic substance" means that the substance has both hydrophilic and lipophilic properties. The envelope may contain additional substances (e.g., additional lipids) that may not be amphiphilic. Thus, the particle may have a monolayer or multilayer structure, and the substances constituting one or more layers or lamellae comprise one or more types of amphiphilic substances (e.g., selected from the group consisting of amphiphilic lipids), which may be combined with additional substances (e.g., additional lipids) that may not be amphiphilic. In some embodiments, the term "particle" refers to a micro- or nano-sized structure, such as a micro- or nano-sized compact structure. According to the present disclosure, the term "particle" includes nanoparticles.

[0355] "RNA particles" can be used to deliver RNA to a desired target site (e.g., a cell, tissue, organ, etc.). The RNA particles may be formed from lipids, including at least one cationic or cationic ionizable lipid or lipid-like substance. Without intending to be bound by any theory, it is believed that the cationic or cationic ionizable lipid or lipid-like substance combines with the RNA to form aggregates, which aggregates result in colloidally stable particles.

[0356] The nucleic acid particles (eg, RNA particles, DNA particles, or DNA / RNA particles) described herein include lipid nanoparticle (LNP)-based and lipoplex (LPX)-based formulations.

[0357] Generally, lipoplexes (LPX) are obtained by mixing two aqueous phases: one containing nucleic acids (e.g., RNA and / or DNA) and one containing a lipid dispersion. In some embodiments, the lipid phase comprises liposomes.

[0358] In some embodiments, liposomes are self-sealing unilamellar or multilamellar vesicular particles, the lamellae of which comprise a lipid bilayer and the encapsulated lumen of which comprises an aqueous phase. A prerequisite for using liposomes to form nanoparticles is that the lipids in the required mixture can form a lamellar (bilayer) phase in the aqueous environment to which they are applied.

[0359] In some embodiments, liposomes comprise a single or multiple phospholipid bilayer enclosing an aqueous core (also referred to herein as an aqueous lumen). They may be prepared from materials with polar head (hydrophilic) groups and nonpolar tail (hydrophobic) groups. In some embodiments, cationic lipids employed in formulating liposomes designed for nucleic acid delivery are amphiphilic in nature and consist of a positively charged (cationic) amine head group linked via glycerol to a hydrocarbon chain or cholesterol derivative.

[0360] In some embodiments, lipoplexes are multilamellar liposome-based formulations that form upon electrostatic interaction between cationic liposomes and nucleic acids (e.g., RNA and / or DNA). In some embodiments, the formed lipoplexes have a distinct internal arrangement of molecules that results from the conversion of the liposomal structure to a packed nucleic acid-lipoplex (e.g., RNA and / or DNA-lipoplex). In some embodiments, these formulations are characterized by poor encapsulation of nucleic acids (e.g., RNA) and incomplete entrapment of nucleic acids (e.g., RNA).

[0361] In some embodiments, LPX particles comprise amphiphilic lipids, particularly cationic or cationic ionizable amphiphilic lipids, and nucleic acids (e.g., RNA and / or DNA, particularly mRNA), as described herein. In some embodiments, electrostatic interactions between positively charged liposomes (made from one or more amphiphilic lipids, particularly cationic or cationic ionizable amphiphilic lipids) and negatively charged nucleic acids (e.g., mRNA) result in complexation and spontaneous formation of nucleic acid lipoplex particles. Positively charged liposomes may generally be synthesized using cationic or cationic ionizable amphiphilic lipids, such as DOTMA and / or DODMA, and additional lipids, such as DOPE. In some embodiments, nucleic acid (e.g., RNA and / or DNA, particularly mRNA) lipoplex particles are nanoparticles.

[0362] Generally, lipid nanoparticles (LNPs) are obtained by directly mixing nucleic acids (e.g., 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 that do not form a lamellar (bilayer) phase in water can be used for particle formation.

[0363] In some embodiments, LNPs comprise or consist of cationic / ionizable lipids and helper lipids, such as phospholipids, cholesterol, and / or polyethylene glycol (PEG) lipids. In some embodiments, in nucleic acid LNPs (e.g., RNA LNPs, e.g., mRNA LNPs) described herein, nucleic acids (e.g., RNA, e.g., mRNA) are bound to ionizable lipids that occupy the central core of the LNP. In some embodiments, PEG lipids, along with phospholipids, form the surface of the LNP. In some embodiments, the surface comprises a bilayer. In some embodiments, charged and uncharged forms of cholesterol and ionizable lipids can be distributed throughout the LNP.

[0364] In some embodiments, nucleic acids (e.g., RNA and / or DNA, e.g., mRNA) may be non-covalently associated with the particles described herein. In embodiments, the nucleic acids (e.g., RNA and / or DNA, particularly mRNA) may be attached to the outer surface of the particle (surface nucleic acids (e.g., surface RNA, particularly surface mRNA)) and / or may be contained within the particle (encapsulated nucleic acids (e.g., encapsulated RNA, particularly encapsulated mRNA)).

[0365] In some embodiments, the particles described herein (e.g., LNP and LPX) have a diameter of about 10 to about 2000 nm, e.g., 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). m, at most about 1100 nm, at most about 1000 nm, at most about 950 nm, at most about 900 nm, at most about 850 nm, at most about 800 nm, at most about 750 nm, at most about 700 nm, at most about 650 nm, at most about 600 nm, at most about 550 nm, or at most about 500 nm), for example, from about 20 to about 1500 nm, for example, from about 30 to about 1200 nm, from about 40 to about 1100 nm, They have a size (e.g., diameter) in the range of 50 to about 1000 nm, about 60 to about 900 nm, about 70 to 800 nm, about 80 to 700 nm, about 90 to 600 nm, or about 50 to 500 nm, or about 100 to 500 nm, for example, in the range of 10 to 1000 nm, 15 to 500 nm, 20 to 450 nm, 25 to 400 nm, 30 to 350 nm, 40 to 300 nm, 50 to 250 nm, 60 to 200 nm, or 70 to 150 nm.

[0366] In some embodiments, the particles described herein (e.g., LNP and LPX) may have a particle size 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, or about 50 nm to about 600 nm. nm, about 50nm to about 250nm, about 50nm to about 200nm, about 100nm to about 1000nm, about 100nm to about 800nm, about 100nm to about 700nm, about 100nm to about 60 0nm, about 100nm to about 500nm, about 100nm to about 450nm, about 100nm to about 400nm, about 100nm to about 350nm, about 100nm to about 300nm, about 100nm to about 2 50nm, about 100nm to about 200nm, about 150nm to about 1000nm, about 150nm to about 800nm, about 150nm to about 700nm, about 150nm to about 600nm, about 150nm ~about 500nm, about 150nm to about 450nm, about 150nm to about 400nm, about 150nm to about 350nm, about 150nm to about 300nm, about 150nm to about 250nm, about 150 The average diameter ranges from about 200 nm to about 1000 nm, from about 200 nm to about 800 nm, from about 200 nm to about 700 nm, from about 200 nm to about 600 nm, from about 200 nm to about 500 nm, from about 200 nm to about 450 nm, from about 200 nm to about 400 nm, from about 200 nm to about 350 nm, from about 200 nm to about 300 nm, or from about 200 nm to about 250 nm.

[0367] In some embodiments, the particle described herein is nanoparticle.The term " nanoparticle " refers to the nano-sized particle that comprises nucleic acid (particularly mRNA) described herein and at least one cationic or cationic ionizable lipid, and all three external dimensions of the particle are nanoscale, i.e. at least about 1 nm and less than about 1000 nm.Preferably, the size of the particle is its diameter.

[0368] The nucleic acid particles (particularly mRNA particles) described herein can exhibit a polydispersity index (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. By way of example, the nucleic acid particles can exhibit a polydispersity index ranging from about 0.01 to about 0.4, or from about 0.1 to about 0.3.

[0369] The N / P ratio gives the ratio of the number of nitrogen groups in the lipid to the number of phosphate groups in the nucleic acid. This correlates with the charge ratio, since nitrogen atoms are usually positively charged (depending on the pH) and phosphate groups are negatively charged. The N / P ratio is pH dependent when charge balance exists. Because positively charged nanoparticles are thought to be preferable for transfection, lipid formulations are often formed with an N / P ratio greater than 4 and up to 12. In that case, the RNA is thought to be fully bound to the nanoparticles.

[0370] The nucleic acid particles described herein (particularly RNA particles such as mRNA particles) can be prepared using a wide variety of methods, which may include obtaining a colloid from at least one cationic or cationic ionizable lipid and mixing the colloid with nucleic acid to obtain the nucleic acid particles.

[0371] As used herein, the term "colloid" refers to a type of homogeneous mixture in which dispersed particles do not settle. The insoluble particles in the mixture are microscopic and have particle sizes between 1 and 1000 nm. The mixture may be referred to as a colloid or a colloidal suspension. The term "colloid" may refer only to the particles in the mixture and not to the suspension as a whole.

[0372] For the preparation of colloids containing at least one cationic or cationic-ionizable lipid, suitably adapted methods conventionally used to prepare liposomal vesicles are applicable herein. The most commonly used methods for preparing liposomal vesicles share the following basic steps: (i) dissolving lipids in an organic solvent, (ii) drying the resulting solution, and (iii) hydrating the dried lipids (using various aqueous media).

[0373] In the film hydration method, lipids are first dissolved in a suitable organic solvent and dried to obtain a thin film on the bottom of a flask. The resulting lipid film is hydrated with a suitable aqueous medium to produce a liposome dispersion. Additional miniaturization steps may also be included.

[0374] Reverse phase evaporation is an alternative method to film hydration for preparing liposomal vesicles and involves the formation of a water-in-oil emulsion between an aqueous phase and an organic phase containing lipids. To homogenize the system, mild sonication of the mixture is necessary. Removal of the organic phase under reduced pressure results in a milky gel, which subsequently becomes a liposomal suspension.

[0375] 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, e.g., lipid vesicles such as liposomes. Generally, the nucleic acid (e.g., RNA and / or DNA, particularly mRNA) lipoplex particles described herein are obtained by adding nucleic acid (e.g., RNA and / or DNA, particularly mRNA) to a colloidal liposome dispersion. Using the ethanol injection technique, such colloidal liposome dispersions are formed, in some embodiments, as follows: An ethanol solution containing lipids, e.g., cationic or cationic ionizable lipids such as DOTMA and / or DODMA, and additional lipids, is injected into an aqueous solution under stirring. In some embodiments, the nucleic acid (e.g., RNA and / or DNA, particularly mRNA) lipoplex particles described herein are obtained without an extrusion step.

[0376] The term "extrusion" or "extrusion" refers to the creation of particles having a fixed cross-sectional shape. In particular, it refers to the reduction in size of particles so that they can be passed through a filter with defined pores.

[0377] Other methods that feature no organic solvents may also be used in accordance with the present disclosure to prepare colloids.

[0378] In some embodiments, LNPs comprise four components: an ionizable cationic lipid, a neutral lipid such as a phospholipid, a steroid such as cholesterol, and a polymer-conjugated lipid. In some embodiments, LNPs may be prepared by rapidly mixing lipids dissolved in ethanol with nucleic acid (e.g., RNA and / or DNA) in an aqueous buffer. While the nucleic acid (e.g., RNA and / or DNA) particles described herein may comprise a polymer-conjugated lipid such as a PEG-lipid, nucleic acid (e.g., RNA and / or DNA) particles that do not comprise a polymer-conjugated lipid such as a PEG-lipid are also provided herein.

[0379] In some embodiments, LNPs comprising nucleic acids (e.g., RNA and / or DNA) and at least one cationic or cationic ionizable lipid described herein are prepared by the following steps: (a) preparing a nucleic acid (e.g., RNA and / or DNA) solution comprising water and a buffer system; (b) preparing an ethanol solution comprising cationic or cationic ionizable lipids and, if present, one or more additional lipids; and (c) mixing the nucleic acid (e.g., RNA and / or DNA) solution prepared in (a) with the ethanol solution prepared in (b), thereby preparing a formulation comprising the LNP. Step (c) may be followed by one or more additional steps selected from dilution and filtration, e.g., tangential flow filtration.

[0380] In some embodiments, LNPs comprising nucleic acid (e.g., RNA and / or DNA) and at least one cationic or cationic ionizable lipid described herein are prepared by (a') preparing a liposomal or colloidal preparation of the cationic or cationic ionizable lipid and, if present, one or more additional lipids in an aqueous phase, (b') preparing a nucleic acid (e.g., RNA and / or DNA) solution comprising water and a buffer system, and (c') mixing the liposomal or colloidal preparation prepared in (a') with the nucleic acid (e.g., RNA and / or DNA) solution prepared in (b'). Step (c') may be followed by one or more additional steps selected from dilution and filtration, e.g., tangential flow filtration.

[0381] The present disclosure describes particles comprising nucleic acid (e.g., RNA and / or DNA, particularly mRNA) and at least one cationic or cationic ionizable lipid that associates with the nucleic acid (e.g., RNA and / or DNA) to form nucleic acid (e.g., RNA and / or DNA) particles, as well as compositions comprising such particles. The nucleic acid (e.g., RNA and / or DNA) particles may comprise nucleic acid (e.g., RNA and / or DNA) complexed with the particles in various forms through non-covalent interactions. The particles described herein are not viral particles, particularly infectious viral particles. That is, the particles cannot infect cells via viruses.

[0382] Suitable cationic or cationic ionizable lipids are those lipids that form nucleic acid particles and are encompassed 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 acid to form a nucleic acid particle. Such components include any component that may be part of a nucleic acid particle.

[0383] In some embodiments, nucleic acid particles (e.g., RNA and / or DNA particles, particularly mRNA particles) comprise two or more nucleic acid (e.g., RNA and / or DNA) molecules, which may be similar or different from each other in terms of molecular parameters such as molar mass or basic structural elements such as molecular architecture, capping (for RNA only), coding regions, or other features.

[0384] In a particle formulation, each nucleic acid (e.g., RNA and / or DNA) species can be formulated separately as an individual particle formulation. In that case, each individual particle formulation will contain one nucleic acid (e.g., RNA and / or DNA) species. The individual particle formulations may be present as separate entities, for example, in separate containers. Such formulations are obtained by providing each nucleic acid (e.g., RNA and / or DNA) species separately (typically in the form of a nucleic acid (e.g., RNA and / or DNA)-containing solution) with a particle-forming agent, thereby forming particles. Each particle will contain only the specific nucleic acid (e.g., RNA and / or DNA) species provided when the particle is formed (individual particle formulation). In some embodiments, a composition, such as a pharmaceutical composition, contains two or more individual particle formulations. Each pharmaceutical composition is referred to as a mixed particle formulation. A mixed particle formulation according to the present invention can be obtained by separately forming individual particle formulations and then mixing the individual particle formulations. The mixing step results in a formulation containing a mixed population of nucleic acid (e.g., RNA and / or DNA)-containing particles. The individual particle populations may be present together in one container containing a mixed population of individual particle preparations. Alternatively, all nucleic acid (e.g., RNA and / or DNA) species of a pharmaceutical composition can be formulated together as a combined particle preparation. Such a preparation can be obtained by providing a combined preparation (typically a combined solution) of all nucleic acid (e.g., RNA and / or DNA) species and a particle-forming agent, thereby forming particles. In contrast to a mixed particle preparation, a combined particle preparation will typically contain particles containing two or more nucleic acid (e.g., RNA and / or DNA) species. In a combined particle composition, different nucleic acid (e.g., RNA and / or DNA) species typically exist together in a single particle.

[0385] polymer Given their high degree of chemical flexibility, polymers are commonly used materials for nanoparticle-based delivery. Cationic polymers are typically used to electrostatically condense negatively charged nucleic acids into nanoparticles. These positively charged groups often consist of amines that change protonation state in the pH range of 5.5 to 7.5, which is thought to lead to ionic imbalances that result in endosomal disruption. Polymers such as poly-L-lysine, polyamidoamine, protamine, and polyethyleneimine, as well as naturally occurring polymers such as chitosan, have all been applied to nucleic acid delivery and are suitable as cationic polymers herein. Furthermore, some researchers have synthesized polymers specifically for nucleic acid delivery. Poly(β-amino esters), in particular, have become widely used in nucleic acid delivery due to their ease of synthesis and biodegradability. Such synthetic polymers are also suitable as cationic polymers herein.

[0386] As used herein, "polymer" is given its ordinary meaning, i.e., a molecular structure comprising one or more repeating units (monomers) connected by covalent bonds. The repeating units may all be identical, or in some cases, more than one type of repeating unit may be present in the polymer. In some cases, the polymer is of biological origin, i.e., a biopolymer such as a protein. In some cases, additional moieties, such as targeting moieties, may be present in the polymer.

[0387] If two or more types of repeating units are present in a polymer, the polymer is referred to as a "copolymer." It should be understood that the polymers employed herein may be copolymers. The repeating units forming a copolymer can be arranged in any manner. For example, the repeating units can be arranged in a random order, an alternating order, or as a "block" copolymer in which one or more regions each contain a first repeating unit (e.g., a first block) and one or more regions each contain a second repeating unit (e.g., a second block). Block copolymers can have two (diblock copolymers), three (triblock copolymers), or a greater number of distinct repeating units.

[0388] In certain embodiments, the polymer is biocompatible. Biocompatible polymers are typically polymers that do not cause significant cell death at reasonable concentrations. In certain embodiments, the biocompatible polymer is biodegradable. That is, the polymer can be chemically and / or biologically degraded in a physiological environment, e.g., in vivo.

[0389] In certain embodiments, the polymer may be protamine or a polyalkyleneimine.

[0390] The term "protamine" refers to any of a variety of relatively low molecular weight, strongly basic proteins that are rich in arginine and are found in the sperm cells of various animals (such as fish) in place of somatic histones, particularly in association with DNA. In particular, the term "protamine" refers to a protein found in fish sperm cells that is strongly basic, water-soluble, heat-resistant, and hydrolyzes to yield primarily arginine. In purified form, they are used in long-acting formulations of insulin and to neutralize the anticoagulant effect of heparin.

[0391] In accordance with the present disclosure, the term "protamine" as used herein is meant to include any protamine amino acid sequence obtained or derived from a natural or biological source, including fragments thereof and multimeric forms of said amino acid sequence or fragments thereof, as well as polypeptides that are man-made and specifically designed for a particular purpose and cannot be isolated from a natural or biological source (synthetic).

[0392] In one embodiment, the polyalkyleneimine comprises polyethyleneimine and / or polypropyleneimine, preferably polyethyleneimine. A preferred polyalkyleneimine is polyethyleneimine (PEI). The average molecular weight of PEI is preferably 0.75×10 2 ~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.

[0393] Preferred according to the present disclosure are linear polyalkyleneimines, such as linear polyethyleneimine (PEI).

[0394] Cationic polymers (including polycationic polymers) contemplated for use herein include any cationic polymer that can electrostatically bind to nucleic acids. In one embodiment, cationic polymers contemplated for use herein include any cationic polymer with which nucleic acids can associate, for example, by forming a complex with the nucleic acid or by forming a vesicle in which the nucleic acid is entrapped or encapsulated.

[0395] The particles described herein may include polymers other than cationic polymers, i.e., non-cationic polymers and / or anionic polymers. Anionic and neutral polymers are collectively referred to herein as non-cationic polymers.

[0396] lipids The terms "lipid" and "lipid-like substance" are broadly defined herein as molecules containing one or more hydrophobic moieties or groups and, optionally, one or more hydrophilic moieties or groups. Molecules containing hydrophobic and hydrophilic moieties are often referred to as amphiphiles. Lipids are typically insoluble or poorly soluble in water, but are soluble in many organic solvents. In aqueous environments, their amphiphilic nature allows them to self-assemble into organized structures and distinct phases. One of these phases consists of a lipid bilayer, as it exists in vesicles, multilamellar / unilamellar liposomes, or membranes in aqueous environments. Hydrophobicity can be imparted by the inclusion of nonpolar groups, including, but not limited to, long-chain saturated and unsaturated aliphatic hydrocarbon groups, and such groups substituted with one or more aromatic, cycloaliphatic, or heterocyclic groups. Hydrophilic groups may contain polar and / or charged groups, including carbohydrate, phosphate, carboxylic acid, sulfate, amino, sulfhydryl, nitro, hydroxyl, and other similar groups.

[0397] As used herein, the term "hydrophobic" refers to any molecule, moiety, or group that is substantially immiscible or insoluble in aqueous solution. The term "hydrophobic group" includes hydrocarbons having at least six carbon atoms. Hydrophobic groups may contain functional groups (e.g., ether, ester, halide, etc.) and atoms other than carbon and hydrogen, provided that the group is substantially immiscible or insoluble in aqueous solution.

[0398] The term "hydrocarbon" includes alkyl, alkenyl, or alkynyl as defined herein. It is recognized that one or more of the hydrogens in the alkyl, alkenyl, or alkynyl may be replaced with other elements, such as halogen, oxygen, or sulfur. Unless otherwise stated, the hydrocarbon group may include cyclic (alkyl, alkenyl, or alkynyl) groups or aryl groups, provided that the overall polarity of the hydrocarbon remains relatively nonpolar.

[0399] The term "alkyl" refers to a saturated, straight-chain or branched, monovalent hydrocarbon moiety that may have 6 to 30, typically 6 to 20, and often 6 to 18 carbon atoms. Exemplary nonpolar alkyl groups include, but are not limited to, hexyl, decyl, dodecyl, tetradecyl, hexadecyl, octadecyl, and the like.

[0400] The term "alkenyl" refers to a straight-chain or branched monovalent hydrocarbon moiety having at least one carbon-carbon double bond, which may contain from 6 to 30, typically from 6 to 20, and often from 6 to 18 total carbon atoms.

[0401] The term "alkynyl" refers to a straight-chain or branched monovalent hydrocarbon moiety having at least one carbon-carbon triple bond, in which the total carbon atoms can be 6 to 30, typically 6 to 20, and often 6 to 18. An alkynyl group may have one or more carbon-carbon double bonds.

[0402] As used herein, the term "amphiphilic" refers to a molecule having both polar and non-polar portions. Often, amphiphilic compounds have a polar head attached to a long hydrophobic tail. In some embodiments, the polar portion is water-soluble, while the non-polar portion is water-insoluble. Furthermore, the polar portion may have a formal positive or negative charge. Alternatively, the polar portion may have both a formal positive and a formal negative charge and may be a zwitterion or inner salt. For purposes of this disclosure, an amphiphilic compound may be one or more of, but is not limited to, natural or non-natural lipids and lipid-like compounds.

[0403] The terms "lipid-like substance," "lipid-like compound," or "lipid-like molecule" refer to substances structurally and / or functionally related to lipids but not strictly considered lipids, particularly amphiphiles. For example, the term includes compounds capable of forming amphiphilic layers due to their presence in vesicles, multilamellar / unilamellar liposomes, or membranes in an aqueous environment, including surfactants or synthetic compounds containing both hydrophilic and hydrophobic moieties. Generally speaking, the term refers to molecules containing hydrophilic and hydrophobic moieties with a structural organization that may or may not resemble that of lipids. Examples of lipid-like compounds capable of spontaneously incorporating into cell membranes include functional lipid constructs, such as synthetic function-spacer-lipid constructs (FSLs), synthetic function-spacer-sterol constructs (FSSs), and artificial amphiphilic molecules. Lipids are generally 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 water-insoluble planar bilayers. Conventional surfactant monomers generally have a conical shape. 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 ellipsoidal micelles. While lipids have the same general structure as surfactants (polar hydrophilic head groups and nonpolar hydrophobic tails), lipids differ from surfactants in the shape of the monomer, the type of aggregates they form 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 context.

[0404] Generally, lipids can be divided into eight categories: fatty acids, glycerolipids, glycerophospholipids, sphingolipids, saccharolipids, polyketides (derived from the condensation of ketoacyl subunits), sterol lipids, and prenol lipids (derived from the condensation of isoprene subunits). The term "lipid" is sometimes used as a synonym for fat, but fat is a subgroup of lipids called triglycerides. Lipids also include molecules such as fatty acids and their derivatives (including tri-, di-, and monoglycerides and phospholipids) and steroids, i.e., sterol-containing metabolites, such as cholesterol or its derivatives. Examples of cholesterol derivatives include, but are not limited to, cholesterol, cholestanone, coprostanol, cholesteryl-2'-hydroxyethyl ether, cholesteryl-4'-hydroxybutyl ether, tocopherol, and their derivatives, and mixtures thereof.

[0405] Fatty acids or fatty acid residues are a diverse group of molecules consisting of a hydrocarbon chain terminating in a carboxylic acid group. This configuration gives the molecule a polar, hydrophilic end and a water-insoluble, nonpolar, hydrophobic end. The carbon chain, typically 4 to 24 carbons in length, can be saturated or unsaturated and can be bonded to functional groups including oxygen, halogens, nitrogen, and sulfur. When fatty acids contain double bonds, they can be cis or trans geometric isomers, which significantly affect the configuration of the molecule. Cis double bonds cause the fatty acid chain to bend, an effect that is combined with more double bonds in the chain. Other major lipid classes in the fatty acid category are fatty acid esters and fatty acid amides.

[0406] Glycerolipids consist of mono-, di-, and tri-substituted glycerols, the most well-known of which are 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 with different fatty acids. A further subclass of glycerolipids is represented by glycosylglycerols, which are characterized by the presence of one or more sugar residues attached to glycerol via glycosidic linkages.

[0407] Glycerophospholipids are amphipathic molecules (containing both hydrophobic and hydrophilic regions) that contain a glycerol core linked by ester linkages to two fatty acid-derived "tails" and a phosphate ester linkage to a single "head" group. Examples of glycerophospholipids, commonly referred to as phospholipids (although sphingomyelin is also classified as a phospholipid), include phosphatidylcholine (PC, GPCho, also known as phosphatidylcholine or lecithin), phosphatidylethanolamine (PE or GPEtn, phsophatidylethanolamine), and phosphatidylserine (PS or GPSer, phosphatidylserine).

[0408] Sphingolipids are a complex family of compounds that share a common structural feature: a sphingoid base backbone. The major sphingoid base in mammals is commonly referred to as sphingosine. Ceramides (N-acylsphingoid bases) are a major subclass of sphingoid base derivatives with amide-linked fatty acids. The fatty acids typically have chain lengths of 16 to 26 carbon atoms and are saturated or monounsaturated. The major phosphosphingolipid in mammals is sphingomyelin (ceramide phosphocholine), while insects primarily contain ceramide phosphoethanolamine, and fungi have plant ceramides with phosphoinositol- and mannose-containing head groups. Glycosphingolipids are a diverse family of molecules consisting 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.

[0409] Sterol lipids, such as cholesterol and its derivatives, or tocopherol and its derivatives, are important constituents of membrane lipids, together with glycerophospholipids and sphingomyelins.

[0410] Saccharolipids describe compounds in which fatty acids are directly linked to a sugar backbone to form structures compatible with membrane bilayers. In saccharolipids, monosaccharide substitution of the glycerol backbone gives rise to glycerolipids and glycerophospholipids. The best-known saccharolipid is the acylated glucosamine precursor of the lipid A component of the lipopolysaccharide of Gram-negative bacteria. A typical lipid A molecule is a disaccharide of glucosamine, which is derivatized with as many as seven fatty acyl chains. The minimal lipopolysaccharide required for growth in Escherichia coli is Kdo2-lipid A, a hexaacylated disaccharide of glucosamine glycosylated with two 3-deoxy-D-manno-octulosonic acid (Kdo) residues.

[0411] Polyketides are synthesized by the polymerization of acetyl and propionyl subunits by iterative and multimodular enzymes that share mechanistic features with classical enzymes and fatty acid synthases. They are found in numerous secondary metabolites and natural products from animals, plants, bacteria, fungi, and aquatic 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.

[0412] According to the present disclosure, lipids and lipid-like substances can be cationic, anionic, or neutral. Neutral lipids or lipid-like substances exist in an uncharged or neutral zwitterionic form at a selected pH.

[0413] Cationic / cationic ionizable lipids The nucleic acid particles (e.g., RNA and / or DNA particles) described herein comprise at least one cationic or cationic ionizable lipid as a particle-forming agent. The cationic or cationic ionizable lipids intended for use herein include any cationic or cationic ionizable lipid (including lipid-like substances) that can electrostatically bind to nucleic acids. In some embodiments, the cationic or cationic ionizable lipids intended for use herein can associate with nucleic acids, for example, by forming a complex with nucleic acids or by forming vesicles in which the nucleic acids are enclosed or encapsulated.

[0414] As used herein, "cationic lipid" refers to a lipid or lipid-like substance that has a net positive charge. Cationic lipids bind to negatively charged nucleic acids through electrostatic interactions. Cationic lipids generally have a lipophilic moiety, such as a sterol, an acyl chain, a diacyl or higher acyl chain, and the lipid head group typically carries a positive charge.

[0415] In some embodiments, cationic lipid has net positive charge only at certain pH, particularly acidic pH, while at different pH, preferably high pH such as physiological pH, it preferably does not have net positive charge, preferably has no charge, that is, it is neutral.This ionization behavior is thought to increase efficacy by helping endosomal escape and reducing toxicity, compared with particles that remain cationic at physiological pH.

[0416] As used herein, "cationic ionizable lipid" refers to a lipid or lipid-like substance that has a net positive charge or is neutral, i.e., is not permanently cationic. Thus, depending on the pH of the composition in which the cationic ionizable lipid is dissolved, the cationic ionizable lipid is either positively charged or neutral. For the purposes of this disclosure, cationic ionizable lipids are encompassed by the term "cationic lipid" unless the context dictates otherwise.

[0417] In some embodiments, the cationic or cationic-ionizable lipid comprises a head group that includes at least one nitrogen atom (N) that is positively charged or capable of being protonated, e.g., under physiological conditions.

[0418] Examples of cationic or cationic ionizable lipids include, but are not limited to, N,N-dimethyl-2,3-dioleyloxypropylamine (DODMA), 1,2-dioleoyl-3-trimethylammonium propane (DOTAP), 1,2-dioleoyl-3-trimethylammonium propane (DOTMA), 1,2-di-O-octadecen ...AP), 1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA), 1,2-di-O-octadecenyl-3-trimethylammonium propane propane), 3-(N-(N',N'-dimethylaminoethane)-carbamoyl)cholesterol (DC-Chol, 3-(N-(N',N'-dimethylaminoethane)-carbamoyl)cholesterol), dimethyldioctadecylammonium (DDAB, dimethyldioctadecylammonium); 1,2-dioleoyl-3-dimethylammonium propane (DODAP, 1,2-dioleoyl-3-dimethylammonium propane); 1,2-diacyloxy-3-dimethylammonium propane; 1,2-dialkyloxy-3-dimethylammonium propane; dioctadecyldimethylammonium chloride (DODAC, dioctadecyldimethyl ammonium chloride), 1,2-distearyloxy-N,N-dimethyl-3-aminopropane (DSDMA), 2,3-di(tetradecoxy)propyl-(2-hydroxyethyl)dimethylazanium (DMRIE), 1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine (DMEPC), l,2-dimyristoyl-3-trimethylammonium propane (DMTAP), 1,2-Dioleyloxypropyl-3-dimethyl-hydroxyethyl ammonium bromide (DORIE) and 2,3-dioleoyloxy-N-[2(sperminecarboxamide)ethyl]-N,N-dimethyl-1-propanamium trifluoroacetate (DOSPA) trifluoroacetate), 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 1,2-dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA), dioctadecylamidoglycylspermine (DOGS) spermine), 3-dimethylamino-2-(cholest-5-en-3-beta-oxybutan-4-oxy)-1-(cis,cis-9,12-octadecadienoxy)propane (CLinDMA), 2-[5'-(cholest-5-en-3-beta-oxy)-3'-oxapentoxy]-3-dimethyl-1-(cis,cis-9',12' -octadecadienoxy)propane (CpLinDMA, 2-[5'-(cholest-5-en-3-beta-oxy)-3'-oxapentoxy)-3-dimethyl-1-(cis,cis-9',12'-octadecadienoxy)propane), N,N-dimethyl-3,4-dioleyloxybenzylamine (DMOBA, N,N-dimethyl-3,4-dioleyloxybenzylamine), 1,2-N,N'-dioleylcarbamyl-3-dimethylaminopropane (DOcarbDAP, 1,2-N,N'-dioleylcarbamyl-3-dimethylaminopropane), 2,3-Dilinoleoyloxy-N,N-dimethylpropylamine (DLinDAP), 1,2-N,N'-Dilinoleylcarbamyl-3-dimethylaminopropane (DLincarbDAP) ), 1,2-Dilinoleoylcarbamyl-3-dimethylaminopropane (DLinCDAP), 2,2-Dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), 2,2-Dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane Xolane (DLin-K-XTC2-DMA, 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA, 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane), heptatriaconta-6,9,28,31-tetraen-19-yl-4-(dimethylaminoethyl) (amino)butanoate (DLin-MC3-DMA, heptatriaconta-6,9,28,31-tetraen-19-yl-4-(dimethylamino)butanoate), N-(2-Hydroxyethyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)-1-propanaminium bromide (DMRIE, N-(2-Hydroxyethyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)-1-propanaminium bromide), (±)-N-(3-aminopropyl)-N,N-dimethyl-2,3-Bis(cis-9-tetradecenyloxy)-1-propanaminium bromide (GAP-DMORIE, (±)-N-(3-aminopropyl)-N,N-dimethyl-2,3-bis(cis-9-tetradecenyloxy)-1-propanaminium bromide), (±)-N-(3-aminopropyl)-N,N-dimethyl-2,3-bis(dodecyloxy)-1-propanaminium bromide (GAP-DLRIE, (±)-N-(3-aminopropyl)-N,N-dimethyl-2,3-bis(dodecyloxy)-1-propanaminium bromide), (±)-N-(3-aminopropyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)-1-propanaminium bromide (GAP-DMRIE, (±)-N-(3-aminopropyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)-1-propanaminium bromide), N-(2-aminoethyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)-1-propanaminium bromide (βAE-DMRIE, N-(2-Aminoethyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)-1-propanaminium bromide), N-(4-carboxybenzyl)-N,N-dimethyl-2,3-bis(oleoyloxy)propan-1-aminium (DOBAQ), 2-({8-[(3β)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z) -octadeca-9,12-dien-1-yloxy]propan-1-amine (Octyl-CLinDMA, 2-({8-[(3β)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-1-amine), 1,2-dimyristoyl-3-dimethylammonium propane (DMDAP, 1,2-dimyristoyl-3-dimethylammonium-propane), 1,2-dipalmitoyl-3-dimethylammonium-propane (DPDAP, 1,2-dipalmitoyl-3-dimethylammonium-propane), N1-[2-((1S)-1-[(3-aminopropyl)amino]-4-[di(3-aminopropyl)amino]butylcarboxamido)ethyl]-3,4-di[oleyloxy]benzamide (MVL5, N1-[2-((1S)-1-[(3-aminopropyl)amino]-4-[di(3-aminopropyl) amino]butylcarboxamido)ethyl]-3,4-di[oleyloxy]-benzamide), 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine (DOEPC, 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine), 2,3-bis(dodecyloxy)-N-(2-hydroxyethyl)-N,N-dimethylpropan-1-amonium bromide (DLRIE, 2,3-bis(dodecyloxy)-N-(2-hydroxyethyl)-N,N-dimethylpropan-1-amonium bromide) bromide), N-(2-aminoethyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)propan-1-aminium bromide (DMORIE), di((Z)-non-2-en-1-yl)8,8'-((((2(dimethylamino)ethyl)thio)carbonyl)azanediyl)dioctanoate (ATX, di((Z)-non-2-en-1-yl) 8,8'-((((2(dimethylamino)ethyl)thio)carbonyl)azanediyl)dioctanoate), N,N-dimethyl-2,3-bis(dodecyloxy)propan-1-amine (DLDMA, N,N-dimethyl-2,3-bis(dodecyloxy)propan-1-amine), N,N-dimethyl-2,3-bis(tetradecyloxy)propan-1-amine (DMDMA, N,N-dimethyl-2,3-bis(tetradecyloxy)propan-1-amine), di((Z)-non-2-en-1-yl)-9-((4-(dimethylaminobutanoyl)oxy)heptadecanedioate (L319), N-dodecyl-3-((2-dodecylcarbamoylethyl)-{2-[(2-dodecylcarbamoylethyl)-2-{(2-dodecylcarbamoylethyl)-[2-(2-dodecylcarbamoylethylamino)ethyl]amino}ethylamino)propionamide (Lipidoid 98N), 12 -5), 1-[2-[bis(2-hydroxydodecyl)amino]ethyl-[2-[4-[2-[bis(2-hydroxydodecyl)amino]ethyl]piperazin-1-yl]ethyl]amino]dodecan-2-ol (lipidoid C12-200).

[0419] In some embodiments, the cationic or cationic ionizable lipid is DOTMA. In some embodiments, the cationic or cationic ionizable lipid is DODMA.

[0420] DOTMA is a cationic lipid with a quaternary amine head group. The structure of DOTMA is represented as follows: [ka] DODMA is an ionizable cationic lipid with a tertiary amine head group. The structure of DODMA is represented as follows: [ka]

[0421] In some embodiments, the cationic or cationic ionizable lipid may comprise from about 10 mol% to about 95 mol%, from about 20 mol% to about 95 mol%, from about 20 mol% to about 90 mol%, from about 30 mol% to about 90 mol%, from about 40 mol% to about 90 mol%, or from about 40 mol% to about 80 mol% of the total lipid present in the particle.

[0422] Additional fats The particles described herein may contain lipids (including lipid-like substances) other than cationic or cationic ionizable lipids (collectively referred to herein as cationic lipids), i.e., non-cationic lipids (including non-cationic or non-cationic ionizable lipids or lipid-like substances). Anionic and neutral lipids or lipid-like substances are collectively referred to herein as non-cationic lipids. Optimizing the formulation of nucleic acid particles by adding other hydrophobic moieties, such as cholesterol and lipids, in addition to cationic or cationic ionizable lipids can enhance particle stability and nucleic acid delivery efficacy.

[0423] The one or more additional lipids may or may not affect the overall charge of the nucleic acid particle. In some embodiments, the one or more additional lipids are non-cationic lipids or lipid-like substances. Non-cationic lipids may include, for example, one or more anionic lipids and / or neutral lipids. As used herein, "anionic lipid" refers to any lipid that is negatively charged at a selected pH. As used herein, "neutral lipid" refers to any of several lipid species that exist in an uncharged or neutral zwitterionic form at a selected pH.

[0424] In some embodiments, the nucleic acid particles described herein (particularly those containing mRNA) comprise a cationic or cationic ionizable lipid and one or more additional lipids.

[0425] Without wishing to be bound by theory, the amount of cationic or cationic ionizable lipid, relative to the amount of one or more additional lipids, can affect important characteristics of the nucleic acid particles, such as charge, particle size, stability, tissue selectivity, and nucleic acid bioactivity. Thus, in some embodiments, the molar ratio of cationic or cationic ionizable lipid to one or more additional 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.

[0426] In some embodiments, the one or more additional lipids included in the nucleic acid particles described herein (particularly in particles comprising mRNA) include one or more of the following: neutral lipids, steroids, and combinations thereof.

[0427] In some embodiments, the one or more additional lipids comprise a neutral lipid that is a phospholipid. In some embodiments, the phospholipid is 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 include, in particular, diacylphosphatidylcholines, such as distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dimyristoylphosphatidylcholine (DMPC), dipentadecanoylphosphatidylcholine, dilauroylphosphatidylcholine, dipalmitoylphosphatidylcholine (DPPC), dipentadecanoylphosphatidylcholine, dilauroylphosphatidylcholine, dipalmitoylphosphatidylcholine (DPPC), diacylphosphatidylcholine, ... Arachidoylphosphatidylcholine (DAPC, diarachidoylphosphatidylcholine), dibehenoylphosphatidylcholine (DBPC, dibehenoylphosphatidylcholine), ditricosanoylphosphatidylcholine (DTPC, ditricosanoylphosphatidylcholine), dilignoceroylphosphatidylcholine (DLPC, dilignoceroylphosphatidylcholine), palmitoyloleoylphosphatidylcholine (POPC, palmitoyloleoyl-phosphatidylcholine), 1,2-Di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 diether PC), 1-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC, 1-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC) and phosphatidylethanolamines, in particular diacylphosphatidylethanolamines, such as dioleoylphosphatidylethanolamine (DOPE), distearoylphosphatidylethanolamine (DSPE), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphatidylethanolamine (DMPE), lethanolamine), dilauroylphosphatidylethanolamine (DLPE), diphytanoylphosphatidylethanolamine (DPyPE), 1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphocholine (DOPG), 1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phospho-(1'-rac-glycerol) (DPPG, 1,2-dipalmitoyl-sn-glycero-3-phospho-(1'-rac-glycerol)), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine), N-palmitoyl-D-erythro-sphingosylphosphorylcholine (SM, N-palmitoyl-D-erythro-sphingosylphosphorylcholine), and additional phosphatidylethanolamine lipids with various 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 selected from the group consisting of DSPC, DPPC, DMPC, DOPC, POPC, DOPE, and SM. In some embodiments, the neutral lipid is DOPE. ,

[0428] In some embodiments, the additional lipid comprises one of the following: (1) a phospholipid, (2) cholesterol or a derivative thereof, or (3) a mixture of a phospholipid and cholesterol or a derivative thereof. Examples of cholesterol derivatives include, but are not limited to, cholestanol, cholestanone, cholestenone, coprostanol, cholesteryl-2'-hydroxyethyl ether, cholesteryl-4'-hydroxybutyl ether, tocopherol, and derivatives thereof, and mixtures thereof.

[0429] Thus, in some embodiments, the nucleic acid particles described herein (particularly particles comprising mRNA) comprise (1) a cationic or cationic ionizable lipid and a phospholipid, such as DOPE, or (2) a cationic or cationic ionizable lipid and a phospholipid, such as DOPE, and cholesterol.

[0430] In some embodiments, the nucleic acid particles described herein (particularly particles comprising mRNA) comprise (1) DOTMA and DOPE, (2) DOTMA, DOPE, and cholesterol, (3) DODMA and DOPE, or (4) DODMA, DOPE, and cholesterol.

[0431] DOPE is a neutral phospholipid. The structure of DOPE is shown below. [ka]

[0432] The structure of cholesterol is shown below: [ka]

[0433] In some embodiments, the particles described herein do not include polymer-conjugated lipids, such as pegylated lipids. The term "pegylated lipid" refers to a molecule that includes both a lipid portion and a polyethylene glycol portion. Pegylated lipids are known in the art.

[0434] 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 particle. 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 particle.

[0435] In some embodiments, the additional lipid comprises a mixture of (i) a phospholipid, such as DOPE, and (ii) cholesterol or a derivative thereof, in which the molar ratio of the phospholipid, 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.

[0436] Polymer-conjugated lipids In some embodiments, particle may comprise at least one polymer-conjugated lipid.Polymer-conjugated lipid is typically a molecule comprising a lipid portion and a polymer portion conjugated thereto.In some embodiments, polymer-conjugated lipid is PEG-conjugated lipid, also referred to herein as PEG-conjugated lipid or PEG-lipid.

[0437] In some embodiments, the polymer-conjugated lipids are designed to sterically stabilize the lipid particles by forming a protective hydrophilic layer that shields the hydrophobic lipid layer, hi some embodiments, the polymer-conjugated lipids can reduce association with serum proteins and / or the resulting uptake by the reticuloendothelial system when such lipid particles are administered in vivo.

[0438] Various PEG-conjugated lipids are known in the art, including, but not limited to, pegylated diacylglycerols (PEG-DAGs), such as 1-(monomethoxypolyethyleneglycol)-2,3-dimyristoylglycerol (PEG-DMG), pegylated phosphatidylethanolamine (PEG-PE), PEG succinate diacylglycerols (PEG-S-DAGs), such as 4-O-(2'-,3'-di(tetradecanoyloxy)propyl) 1-O-(ω-methoxy(polyethoxy)ethyl)butanedioate (PEG-S-DMG), 4-O-(2'-,3'-di(tetradecanoyloxy)propyl) but ... PEG-ceramide, or PEG dialkoxypropyl carbamate, such as ω-methoxy(polyethoxy)ethyl-N-(2,3-di(tetradecanoyloxy)propyl)carbamate or 2,3-di(tetradecanoyloxy)propyl-N-(ω-methoxy(polyethoxy)ethyl)carbamate, and the like.

[0439] In some embodiments, the particles may include one or more PEG-conjugated or pegylated lipids 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.

[0440] Lipoplex particles In some embodiments of the present disclosure, the nucleic acids (eg, RNA and / or DNA) described herein may be present in nucleic acid lipoplex particles (eg, RNA and / or DNA lipoplex particles).

[0441] Lipoplexes (LPXs) are generally electrostatic complexes formed by mixing preformed cationic lipid liposomes with anionic nucleic acids (e.g., RNA and / or DNA). The formed lipoplexes have a well-defined internal arrangement of molecules resulting from the conversion of the liposomal structure into dense nucleic acid lipoplexes (e.g., RNA and / or DNA lipoplexes). These formulations are generally characterized by poor encapsulation and incomplete nucleic acid entrapment.

[0442] In certain embodiments, nucleic acid lipoplex particles (e.g., RNA and / or DNA lipoplex particles) comprise both a cationic lipid and an additional lipid. In an exemplary embodiment, the cationic lipid is DOTMA and the additional lipid is DOPE.

[0443] In some embodiments, the molar ratio of the at least one cationic lipid to the at least one additional 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 certain 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 the at least one cationic lipid to the at least one additional lipid is about 2:1.

[0444] The nucleic acid lipoplex particles (e.g., RNA and / or DNA lipoplex particles) described herein, in some embodiments, have an average diameter in the range of about 200 nm to about 1000 nm, about 200 nm to about 800 nm, about 250 to about 700 nm, about 400 to about 600 nm, about 300 nm to about 500 nm, or about 350 nm to about 400 nm. In certain embodiments, the RNA lipoplex 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 lipoplex particles (e.g., RNA and / or DNA lipoplex particles) have an average diameter in the range of about 250 nm to about 700 nm. In another embodiment, the nucleic acid lipoplex particles (e.g., RNA and / or DNA lipoplex particles) have an average diameter in the range of about 300 nm to about 500 nm. In an exemplary embodiment, the nucleic acid lipoplex particles (e.g., RNA and / or DNA lipoplex particles) have an average diameter of about 400 nm.

[0445] The nucleic acid lipoplex particles (e.g., RNA and / or DNA lipoplex particles) and compositions comprising nucleic acid lipoplex particles (e.g., RNA and / or DNA lipoplex particles) described herein are useful for delivering nucleic acids (e.g., RNA and / or DNA) to target tissues following parenteral administration, particularly following intravenous administration.

[0446] RNA lipoplex particles targeted to the spleen are described in International Publication No. WO 2013 / 143683, which is incorporated herein by reference. It has been discovered that RNA lipoplex particles having a net negative charge can be used to preferentially target spleen tissue or splenocytes, such as antigen-presenting cells, particularly dendritic cells. Thus, after administration of the RNA lipoplex particles, RNA accumulation and / or RNA expression occurs in the spleen. Thus, the nucleic acid (e.g., RNA and / or DNA) lipoplex particles of the present disclosure may be used to express nucleic acids (e.g., RNA and / or DNA) in the spleen. In embodiments, after administration of the nucleic acid (e.g., RNA and / or DNA) lipoplex particles, no or essentially no nucleic acid (e.g., RNA) accumulation and / or nucleic acid (e.g., RNA) expression occurs in the lung and / or liver. In one embodiment, administration of the nucleic acid (e.g., RNA and / or DNA) lipoplex particles results in the accumulation of the nucleic acid (e.g., RNA) and / or expression of the nucleic acid (e.g., RNA) in antigen-presenting cells, such as professional antigen-presenting cells in the spleen. Thus, the nucleic acid (e.g., RNA and / or DNA) lipoplex particles of the present disclosure may be used to express a nucleic acid (e.g., RNA and / or DNA), for example, a nucleic acid (e.g., RNA and / or DNA) encoding an antigen or at least one epitope, in such antigen-presenting cells. In one embodiment, the antigen-presenting cells are dendritic cells and / or macrophages.

[0447] The charge of the nucleic acid (e.g., RNA and / or DNA) lipoplex particles of the present disclosure is the sum of the charge present on at least one cationic lipid and the charge present on the nucleic acid (e.g., RNA). The charge ratio is the ratio of the positive charge present on at least one cationic lipid to the negative charge present on the nucleic acid (e.g., RNA). The charge ratio of the positive charge present on at least one cationic lipid to the negative charge present on the nucleic acid (e.g., RNA) is calculated by the following formula: Charge ratio = [(cationic lipid concentration (molar)) x (total number of positive charges in the cationic lipid)] / [(nucleic acid (e.g., RNA) concentration (molar)) x (total number of negative charges in the nucleic acid (e.g., RNA)]. The concentration of the nucleic acid (e.g., RNA) and the amount of the at least one cationic lipid can be determined by a person skilled in the art using routine methods.

[0448] In one embodiment, the charge ratio of positive to negative charges of nucleic acid (e.g., RNA and / or DNA) lipoplex particles at physiological pH is about 1.6:2 to about 1:2, or about 1.6:2 to about 1.1:2. In certain embodiments, the charge ratio of positive to negative charges of nucleic acid (e.g., RNA and / or DNA) lipoplex particles at physiological pH 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.

[0449] Lipid nanoparticles (LNPs) In some embodiments, the nucleic acids (e.g., RNA and / or DNA) described herein are present in the form of lipid nanoparticles (LNPs). LNPs can include any lipid capable of forming particles to which one or more nucleic acid molecules are bound or in which one or more nucleic acid molecules are encapsulated.

[0450] LNPs typically contain four components: an ionizable cationic lipid, a neutral lipid such as a phospholipid, a steroid such as cholesterol, and a polymer-conjugated lipid such as a PEG-lipid. LNPs may be prepared by mixing lipids dissolved in ethanol with nucleic acid in an aqueous buffer.

[0451] In some embodiments, in the nucleic acid (e.g., RNA and / or DNA) LNPs described herein, the nucleic acid (e.g., RNA and / or DNA, particularly mRNA) is bound to an ionizable lipid that occupies the central core of the LNP. PEG lipids, along with phospholipids, form the surface of the LNP. In some embodiments, the surface comprises a bilayer. In some embodiments, cholesterol and charged and uncharged forms of ionizable lipids can be distributed throughout the LNP.

[0452] In some embodiments, the LNPs comprise one or more cationic lipids and one or more stabilizing lipids, including neutral lipids and pegylated lipids.

[0453] In some embodiments, the LNPs comprise cationic lipids, neutral lipids, steroids, polymer-conjugated lipids, and nucleic acids (e.g., RNA and / or DNA) encapsulated within or associated with the lipid nanoparticles.

[0454] In some embodiments, LNPs comprise 40-55 mole percent, 40-50 mole percent, 41-50 mole percent, 42-50 mole percent, 43-50 mole percent, 44-50 mole percent, 45-50 mole percent, 46-50 mole percent, or 46-49 mole percent.

[0455] In some embodiments, the neutral lipid is present at a concentration ranging from 5 to 15 mole percent, from 7 to 13 mole percent, or from 9 to 11 mole percent.

[0456] In some embodiments, the steroid is present at a concentration ranging from 30 to 50 mole percent, 35 to 45 mole percent, or 38 to 43 mole percent.

[0457] In some embodiments, the LNP comprises between 1 and 10 mole percent, between 1 and 5 mole percent, or between 1 and 2.5 mole percent of polymer-conjugated lipid.

[0458] In some embodiments, the LNPs comprise 45-50 mole percent cationic lipids, 5-15 mole percent neutral lipids, 35-45 mole percent steroids, 1-5 mole percent polymer-conjugated lipids, and nucleic acids (e.g., RNA and / or DNA) encapsulated within or associated with the lipid nanoparticles.

[0459] In some embodiments, the mole percent is determined based on the total moles of lipids present in the lipid nanoparticle, hi some embodiments, the mole percent is determined based on the total moles of cationic lipids, neutral lipids, steroids, and polymer-conjugated lipids present in the lipid nanoparticle.

[0460] 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 selected from the group consisting of DSPC, DPPC, DMPC, DOPC, POPC, DOPE, and SM. In some embodiments, the neutral lipid is DSPC.

[0461] In some embodiments, the steroid is cholesterol.

[0462] In some embodiments, the polymer-conjugated lipid is a pegylated lipid. In some embodiments, the pegylated lipid has the following structure: [ka] or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof, and R 12 and R 13 are each independently a linear or branched, saturated or unsaturated alkyl chain containing 10 to 30 carbon atoms, the alkyl chain optionally being interrupted by one or more ester bonds, and w has an average value in the range of 30 to 60. In some embodiments, R 12 and R13 are each independently a linear 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 is each independently a linear saturated alkyl chain containing about 14 carbon atoms, and w has an average value of about 45.

[0463] In some embodiments, the pegylated lipid is or comprises 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide.

[0464] In some embodiments, the cationic lipid component of the LNP has the structure of formula (III): [ka] or a pharmaceutically acceptable salt, tautomer, prodrug, or stereoisomer thereof, wherein L 1 or L 2 One of the 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 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- or a direct bond, G1 and G 2 are each independently unsubstituted C1-C 12 Alkylene or C1-C 12 is alkenylene, G 3 is C1-C 24 Alkylene, C1-C 24 alkenylene, C3-C8 cycloalkylene, C3-C8 cycloalkenylene; R a is H or C1-C 12 is alkyl, R 1 and R 2 are independently C6-C 24 Alkyl or C6-C 24 is alkenyl, R 3 is H, OR 5 , CN, -C(=O)OR 4 , -OC(=O)R 4 or -NR 5 C(=O)R 4 and R 4 is C1-C 12 is alkyl, R 5 is H or C1-C6 alkyl, x is 0, 1, or 2.

[0465] In some of the above embodiments of formula (III), the lipid has the following structure (IIIA) or (IIIB): [ka] (IIIA) (IIIB) It has one of the following. A is a 3- to 8-membered cycloalkyl or cycloalkylene ring; R 6 is independently H, OH, or C1-C 24 is alkyl, n is an integer ranging from 1 to 15.

[0466] In some of the above embodiments of formula (III), the lipid has structure (IIIA), and in other embodiments, the lipid has structure (IIIB).

[0467] In other embodiments of formula (III), the lipid has the following structure (IIIC) or (IIID): [ka] (IIIC) (IIID) It has one of the following. y and z are each independently an integer ranging from 1 to 12.

[0468] In any of the above embodiments of formula (III), L 1 and L 2 One of the groups is —O(C═O)—. For example, in some embodiments, L 1 and L 2 Each of is —O(C═O)—. In some different embodiments of any of the above, L 1 and L 2 are each independently -(C=O)O- or -O(C=O)-. For example, in some embodiments, L 1 and L 2 Each of is —(C═O)O—.

[0469] In some different embodiments of formula (III), the lipid has the following structure (IIIE) or (IIIF): [ka] (IIIE) (IIIF) It has one of the following.

[0470] In some of the above embodiments of formula (III), the lipid has the following structure (IIIG), (IIIH), (IIII), or (IIIJ): [ka] (IIIG) (IIIH) JPEG2026508330000025.jpg38170(IIII) (IIIJ) It has one of the following.

[0471] In some of the above embodiments of Formula (III), n is an integer ranging from 2 to 12, such as from 2 to 8 or from 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.

[0472] In some other of the above embodiments of Formula (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.

[0473] In some of the above embodiments of formula (III), R 6 is H. In other embodiments of the invention, R 6 is C1-C 24 In other embodiments, R 6 is OH.

[0474] In some embodiments of Formula (III), G 3 is unsubstituted. In other embodiments, G is substituted. In various different embodiments, G 3 is a linear C1-C 24 Alkylene or linear C1-C 24 It is alkenylene.

[0475] In some other of the above embodiments of formula (III), R 1 or R 2 Or both are C6-C 24 For example, in some embodiments, R 1 and R 2 each independently have the following structure: [ka] It has. R 7a and R 7b Each occurrence of is independently H or C1-C 12 alkyl, and a is an integer from 2 to 12; R 7a , R 7b , and a are R 1 and R 2 is each independently selected to contain from 6 to 20 carbon atoms. For example, in some embodiments, a is an integer ranging from 5 to 9 or from 8 to 12.

[0476] In some of the above embodiments of formula (III), R 7a At least one occurrence of is H. For example, in some embodiments, R 7a is H at each occurrence. In another different embodiment of the above, R 7b is C1-C8 alkyl. For example, in some embodiments, C1-C8 alkyl is methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-hexyl, or n-octyl.

[0477] In different embodiments of formula (III), R 1 or R 2 or both have the following structure: [ka] It has one of the following.

[0478] In some of the above embodiments of formula (III), R 3 OH, CN, -C(=O)OR 4 , -OC(=O)R 4 , or -NHC(=O)R 4 In some embodiments, R 4 is methyl or ethyl.

[0479] In various different embodiments, the cationic lipid of formula (III) has one of the structures shown in the table below.

[0480] Representative compounds of formula (III) [Table 1] JPEG2026508330000029.jpg244170 JPEG2026508330000030.jpg235170 JPEG2026508330000031.jpg228170 JPEG2026508330000032.jpg251170 JPEG2026508330000033.jpg228170 JPEG2026508330000034.jpg138170

[0481] A variety of 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 targeted to specific cell types (e.g., hepatocytes). In some embodiments, the neutral lipid can be or include a phospholipid or a derivative thereof (e.g., 1,2-distearoyl-sn-glycero-3-phosphocholine (DPSC)) and / or cholesterol. In some embodiments, the polymer-conjugated lipid can be a PEG-conjugated lipid (e.g., 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide or a derivative thereof).

[0482] In some embodiments, the LNP comprises a lipid of Formula (III), a nucleic acid (e.g., RNA and / or DNA), a neutral lipid, a steroid, and a pegylated lipid. In some embodiments, the neutral lipid is DSPC. In some embodiments, the steroid is cholesterol. In some embodiments, the pegylated lipid is ALC-0159. ALC-0159: [ka]

[0483] In some embodiments, the cationic lipid is present in the LNP in an amount of about 45 to about 50 mole percent. In some embodiments, the neutral lipid is present in the LNP in an amount of about 5 to about 15 mole percent. In some embodiments, the steroid is present in the LNP in an amount of about 35 to about 45 mole percent. In some embodiments, the pegylated lipid is present in the LNP in an amount of about 1 to about 5 mole percent.

[0484] In some embodiments, the LNPs comprise cationic lipid in an amount of about 45 to about 50 mole percent, DSPC in an amount of about 5 to about 15 mole percent, cholesterol in an amount of about 35 to about 45 mole percent, and ALC-0159 in an amount of about 1 to about 5 mole percent.

[0485] 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.

[0486] Measuring nucleic acid expression in transfected cells To quantify an amino acid sequence expressed in cells transfected with a nucleic acid encoding the amino acid sequence, one or more peptides of the sequence encoded by the nucleic acid, such as a unique MITD (MHC class I trafficking domain) peptide or a specific fragment of a bioluminescent peptide, for example, present at the C-terminus of the encoded amino acid sequence, can be quantified from whole cell lysates using LC-MS / MS analysis.

[0487] cell In some embodiments, the cells of the present invention mimic the nucleic acid uptake mechanism (e.g., 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 macropinocytosis-mediated RNA uptake mechanism. In these embodiments, the nucleic acids (e.g., RNA and / or DNA) are preferably formulated as lipoplex particles. In some embodiments, the cells are characterized by an endosomal-mediated RNA uptake mechanism. In these embodiments, the nucleic acids (e.g., RNA and / or DNA) are preferably formulated as lipid nanoparticles. In some embodiments, the cells are cells derived from an animal cell line, particularly cells that take up nucleic acid products, such as RNA-LPX, DNA-LPX, or RNA-LNP, using the same mechanism as recipient cells (i.e., recipient target cells, e.g., dendritic cells (DCs)) and are suitable for routine testing in a QC environment (e.g., a GMP QC environment). In some embodiments, the cell is a Chinese hamster ovary (CHO) cell. In some embodiments, the cell is selected from K562, HepG2, HEK293T, RAW, and C2C12 cells, such as K562, HEK293T, RAW, and C2C12 cells.

[0488] Cell lysis In some embodiments, the methods described herein comprise a step of lysing the cells prior to determining the amount of an amino acid sequence comprising the amino acid sequence of a biologically active peptide or polypeptide or fragment thereof.

[0489] In some embodiments, the methods described herein further comprise the step of treating the cell lysate prior to determining the amount of an amino acid sequence comprising the amino acid sequence of a biologically active peptide or polypeptide or fragment thereof.

[0490] In some embodiments, the step of processing the cell lysate includes one or more selected from denaturation, reduction, proteolytic enzyme digestion according to the present invention (e.g., digestion with trypsin, Glu-C, LysN, Lys-C, Asp-N chymotrypsin, or a mixture of any two or more of these proteases), alkylation, drying, reconstitution, and desalting, e.g., after trypsin digestion, alkylation, and desalting.

[0491] Any suitable method for lysing cells may be used in the assays described herein. In some embodiments, a buffer such as a Tris / HCl buffer having a pH of about 7.5 (e.g., adjusted with HCl) and containing a mild zwitterionic detergent, such as CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate) and / or CHAPSO (3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate), is used as the cell lysis buffer. The cell lysis buffer may further contain a chelating agent, such as EDTA, and / or one or more protease inhibitors.

[0492] The table below shows an example of a cell lysis buffer preparation and final component concentrations. [Table 2]

[0493] Quantification of expression products Any method suitable for quantifying peptides and polypeptides may be used in the assays described herein. In preferred embodiments, antibody-free methods are used. In preferred embodiments, 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.

[0494] In some embodiments, the amount of an amino acid sequence comprising a functional sequence, eg, an amino acid sequence of a biologically active peptide or polypeptide or fragment thereof, is determined using mass spectroscopy.

[0495] In some embodiments, the amount of an amino acid sequence comprising a functional sequence, eg, an amino acid sequence of a biologically active peptide or polypeptide or fragment thereof, is determined using liquid chromatography-mass spectrometry (LC-MS).

[0496] In some embodiments, the amount of an amino acid sequence comprising a functional sequence, eg, an amino acid sequence of a biologically active peptide or polypeptide or fragment thereof, is determined using targeted LC-MS.

[0497] In some embodiments, the amount of an amino acid sequence comprising a functional sequence, e.g., an amino acid sequence of a biologically active peptide or polypeptide or fragment thereof, is determined using one or more amino acid sequences expressed by the cell as a reference for quantification.

[0498] In some embodiments, the one or more amino acid sequences expressed by the cell include one or more amino acid sequences of housekeeping proteins.

[0499] In some embodiments, the efficacy of a nucleic acid (eg, RNA and / or DNA) to induce biological activity in a biological system includes the therapeutic efficacy of the nucleic acid (eg, RNA and / or DNA).

[0500] In some embodiments, the nucleic acid (e.g., RNA and...

Claims

1. 1. A method for simultaneously analyzing at least two different nucleic acid sequences, each 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 linker sequence, each linker sequence being capable of being proteolytically cleaved from the amino acid sequence, the method comprising the steps of: (i) providing said at least two different nucleic acid sequences; (ii) introducing the at least two different nucleic acid sequences into a cell; (iii) expressing the at least two different amino acid sequences; (iv) proteolytically excising said at least two different linker sequences; (v) determining the amount of each of the excised linker sequences; (vi) using the amount of each of the excised linker sequences as an indicator of the efficacy of each of the nucleic acid sequences to express each of the functional sequences in a biological system;

2. 10. 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 2, wherein the functional sequence is an epitope.

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

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

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

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

8. The method of any one of claims 1 to 7, wherein each of the linker sequences is preceded at its N-terminus by a lysine or arginine residue 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 N-terminal to the linker sequence is naturally occurring within the functional sequence.

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

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

12. The method according to any one of claims 1 to 11, wherein the proteolytic excision in step (iv) is carried out using a proteolytic enzyme or a mixture of proteases.

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

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

15. The method according to any one of claims 1 to 14, wherein the amount of each of the excised linker sequences is determined using mass spectrometry.

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

17. The method of any one of claims 1 to 16, wherein the amount of each of the excised linker sequences is determined using targeted LC-MS.

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 C-terminal to the linker sequence.

19. 19. The method of claim 18, wherein the sequence C-terminal to the linker sequence is an auxiliary domain sequence.

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

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

22. 22. The method of claim 21, further comprising processing the cell lysate.

23. 23. The method of claim 22, wherein the step of processing the cell lysate comprises one or more steps selected from the group consisting of protease digestion, denaturation, reduction, alkylation, drying, reconstitution, 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. 1. A method for analyzing the efficacy of a nucleic acid sequence to express a functional sequence in a biological system, comprising the step of simultaneously analyzing at least two different nucleic acid sequences, each encoding a different amino acid sequence, each of the at least two different amino acid sequences comprising a different functional sequence and a different linker sequence, each linker sequence being capable of being proteolytically cleaved from the amino acid sequence, the method comprising the steps of: (i) providing said at least two different nucleic acid sequences; (ii) introducing the at least two different nucleic acid sequences into a cell; (iii) expressing the at least two different amino acid sequences; (iv) proteolytically excising said at least two different linker sequences; (v) determining the amount of the excised linker sequence;

26. a) a first nucleic acid sequence comprising an insertion site for a first polynucleotide encoding a functional sequence, said first nucleic acid sequence encoding an amino acid sequence comprising a first linker sequence, said first linker sequence being flanked by proteolytic cleavage sites such that said first linker sequence can be excised from said amino acid sequence, and the sequence of said first linker sequence being different from any other sequence flanking the same proteolytic cleavage site in said 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 for a second polynucleotide encoding a functional sequence, said second nucleic acid sequence encoding an amino acid sequence comprising a second linker sequence, said second linker sequence being flanked by proteolytic cleavage sites so that said second linker sequence can be excised from said amino acid sequence, and the sequence of said second linker sequence being different from any other sequence flanking the same proteolytic cleavage site in said amino acid sequence or in the amino acid sequence encoded by said first nucleic acid sequence; Kit including:

27. 27. Use of the kit of claim 26 for simultaneously analyzing the efficacy of first and second nucleic acid sequences expressing first and second functional sequences in a biological system.

28. 1. Use of at least two nucleic acid sequences for simultaneously analyzing the efficacy of at least two nucleic acid sequences 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 linker sequence, each linker sequence being 6 to 30 amino acids in length and having the general formula: [X] ｎ Y where X is any amino acid, n is an integer from 5 to 29; Y is lysine or arginine; Furthermore, [X] ｎ may contain the amino acid sequence KP or RP, but otherwise does not contain lysine or arginine, The above use, wherein each linker sequence is preceded at its N-terminus by a lysine or arginine residue.

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