HIV vaccine

Abstract

The present invention relates to a composition comprising an RNA molecule, wherein the RNA molecule comprises an expression cassette encoding an immunogenic peptide, the peptide comprising at least two fragments, each fragment comprising at least one epitope, the epitope being derived from an amino acid sequence encoded by human immunodeficiency virus (HIV), and to a pharmaceutical formulation comprising such a composition. Furthermore, the present invention relates to a method for preventing or treating HIV.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority to U.S. Provisional Patent Application No. 63 / 459,449 filed on 14 April 2023, U.S. Provisional Patent Application No. 63 / 547,796 filed on 8 November 2023, and U.S. Provisional Patent Application No. 63 / 549,262 filed on 2 February 2024, all of which are incorporated herein by reference in their entirety.

[0002] The present invention relates to a composition comprising an RNA molecule, wherein the RNA molecule comprises an expression cassette encoding an immunogenic peptide, the peptide comprising at least two fragments, each fragment comprising at least one epitope, the epitope being derived from an amino acid sequence encoded by human immunodeficiency virus (HIV), and to a pharmaceutical formulation comprising such a composition. Furthermore, the present invention relates to a method for preventing or treating HIV. [Background technology]

[0003] HIV remains a global health threat, with approximately 38 million people infected worldwide (WHO, Global progress report on HIV, viral hepatitis and sexually transmitted infections, 2021). Recent advances in the success of combinatorial antiretroviral therapy (cART) have dramatically reduced the HIV burden. However, a preventive HIV vaccine is still not realized, and cART cannot promote complete viral clearance in patients. Therefore, HIV remains a lifelong chronic condition for more than 21 million people who have access to treatment. Increasing the global availability of cART, lowering the cost of treatment regimens, and addressing HIV drug resistance are critical goals for achieving a "functional cure."

[0004] Curative therapy is the ultimate goal of HIV treatment and remains a significant challenge in the HIV field. Current curative strategies aim to induce a strong HIV-specific CD8+ T cell response and / or to use bNAbs as therapeutic agents to prevent (re)infection and promote the elimination of infected cells. Key challenges in HIV immunotherapy include (i) the development of formulations with broad applicability; (ii) the high diversity of circulating HIV strains; (iii) the time-dependent viral repertoire-mediated immune evasion within infected hosts; (iv) the presence of immunodominant unprotective epitopes; and (v) impaired host immune function (Barouch, Nature 2008;455(7213):613-619; Fischer et al., Nat Med. 2007;13(1):100-106).

[0005] Coupled with the expanding spectrum of well-characterized bNAbs, rapidly emerging insights into HIV immune regulatory mechanisms present an excellent opportunity to develop novel formulations for HIV prophylactic and therapeutic treatments. [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Barouch,Nature 2008;455(7213):613-619; [Non-Patent Document 2] Fischer et al., Nat Med.2007;13(1):100-106 [Overview of the project] [Problems that the invention aims to solve]

[0007] Therefore, there remains an urgent need to improve HIV vaccines and / or treatments.

[0008] The present invention addresses such needs. The present invention has a particular advantage in providing a vaccine that induces an HIV-specific CD8+ T cell response for the control or prevention of HIV infection, in particular by providing a vaccine that supports the generation of CD8+ T cells that support and / or replicate naturally observed control of untreated viremia. [Means for solving the problem]

[0009] One aspect provided by this disclosure is a composition comprising an RNA molecule, wherein the RNA molecule comprises an expression cassette encoding an immunogenic peptide, the peptide comprising at least two fragments, each fragment comprising at least one epitope, the epitope being derived from an amino acid sequence encoded by human immunodeficiency virus (HIV), and the epitope being contained in or comprising a sequence selected from any one of SEQ ID NOs: 1-148 and 198-202 or a variant thereof.

[0010] In one embodiment, at least one of the fragments may contain at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 epitopes. In one embodiment, an epitope in one fragment may be different from an epitope in at least one other fragment or all of the other fragments. In one embodiment, at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or all of a fragment may not contain epitopes present in other fragments.

[0011] In one embodiment, the epitope sequences overlap in at least one of the fragments, and / or the epitope sequences do not overlap in at least one of the fragments.

[0012] In one embodiment, the immunogenic peptide comprises a total of at least 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, 35, or 40 fragments. In one embodiment, the immunogenic peptide may contain a total of at least 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, 35, 40, 45, 50, 55, 60, 65, 66, 67, 68, 69, or 70 epitopes from among the epitopes shown in SEQ ID NOs: 1-148 and 198-202. In one embodiment, each fragment may contain the same or different number of epitopes.

[0013] In one embodiment, epitopes can be combined within an expression cassette to minimize the length of the RNA molecule and / or ordered within an expression cassette to minimize the HLA-II epitopes of HIV.

[0014] In one embodiment, the epitopes may be derived from HIV gp41, gp120, p31, p24, nef, p51, protease, tat, rev, vif, vpr, vpx, or vpu proteins. Preferably, at least one or all epitopes may be derived from HIV gp41, gp120, p31, p24, nef, p51, or protease proteins. Preferably, at least one or all epitopes may be derived from HIV gp41, gp120, or nef proteins, and in particular, at least one or all epitopes may be derived from HIV nef protein. In one embodiment, the peptide may contain at least one epitope derived from each of the HIV gp41, gp120, or nef proteins.

[0015] In one embodiment, at least one amino acid can separate the sequence of a non-overlapping epitope in the peptide and / or fragment, and / or at least one amino acid separates at least two fragments. Preferably, the linker can separate the sequences of at least two fragments.

[0016] In one embodiment, two or more non-overlapping epitopes contained within one or more fragments are not flanked at 5' and 3' by the consensus flanking amino acid sequences of the HIV amino acid sequence of the clade from which the epitopes originate.

[0017] In the context of this invention, the term "clade" means a monophyletic group of organisms, i.e., a group of organisms consisting of a common ancestor on a phylogenetic tree and all of its direct descendants, particularly viruses such as HIV. A phylogenetic tree can be constructed from genome sequences, for example, by aligning the viral genomes and using neighbor-joining or parsimonential methods known in the art.

[0018] In one embodiment, a variant of the epitope may be a polymorphism of the epitope, and the epitope is a consensus sequence derived from at least two different clones of HIV.

[0019] In the context of this invention, the term "polymorphism" means a sequence variant of the consensus sequence, for example, a portion of a viral genome, that differs from the consensus sequence by at least one different nucleotide. For example, if the sequence atgacc is the consensus sequence and it is also known to produce a "g" at position 2, then aggacc is a polymorphism of the consensus sequence. Preferably, the polymorphism has at least 50%, 60%, 70%, 80%, 90%, or 95% sequence identity with the consensus sequence.

[0020] In one embodiment, the mutant may have 1, 2, 3, 4, or 5 amino acids that differ from the epitope.

[0021] In one embodiment, the composition may further comprise a second RNA molecule comprising a second expression cassette encoding a second immunogenic peptide, the peptide comprising at least two fragments, each fragment comprising at least one epitope, the epitope being derived from an amino acid sequence encoded by human immunodeficiency virus (HIV), and the epitope being contained within or comprising a sequence selected from any one of sequence codes 1-148 and 198-202 or a variant thereof.

[0022] In one embodiment, the immunogenic peptide contains an epitope derived from HIV of one clade, and the second immunogenic peptide contains an epitope derived from HIV of another clade, preferably one clade being clade B and the other clade being clade C. In one embodiment, each immunogenic peptide contains an epitope that is identical in both clades. In one embodiment, each immunogenic peptide contains a polymorphic epitope derived from its respective clade.

[0023] In one embodiment, the epitope may be a T cell epitope and / or the epitope may be a CD8 minimal epitope.

[0024] In one embodiment, the epitope may be 9 to 21 amino acid long. Preferably, the epitope may be 9 or more amino acid long. Preferably, the epitope may be 10 or more amino acid long. Preferably, the epitope may be 11 or more amino acid long. In some embodiments, the epitope may be 9 to 14 amino acid long. Preferably, the epitope may be 9 to 13 amino acid long. Preferably, the epitope may be 9 to 12 amino acid long. Preferably, the epitope may be 9 to 11 amino acid long. Preferably, the epitope may be 9 or 10 amino acid long. Preferably, the epitope may be 9 amino acid long. In one embodiment, the fragment may be in the range of 9 to 21 amino acid long.

[0025] In one embodiment, the immunogenic peptides are SEQ ID NOs: 60, 103, 98, 149, 2, 150, 142, 86, 1, 141, 151, 152, 130, 153, 154, 155, 156, 157, 158, 4, 159, 160, 161, 162, 35, 28, 163, 164, 165, 166, 87, 167, 85, 125, 29, 168, 169, 57, 3, 170, 171, 17 It may include 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 or more fragments having amino acid sequences selected from the group of amino acid sequences 2, 99, 131, 173, 174, 175, 104, 176, 61, 177, 178, 179, 5, 180, 181, 182, 140, 199, 203 and 204.

[0026] In one embodiment, the immunogenic peptide is a) SEQ ID NOs: 60, 103, 98, 149, 2, 150, 142, 86, 1, 141, 151, 152, 130, 153, 154, 155, 156, 157, 158, 4, 159, 160, 161, 162, 35, 28, 163, 164, 165, 166, 87, 167 and 85, Alternatively, b) may include the amino acid sequences of SEQ ID NOs: 60, 103, 98, 149, 2, 150, 142, 86, 1, 141, 151, 152, 199, 153, 154, 155, 156, 157, 158, 4, 159, 160, 161, 162, 35, 28, 203, 164, 165, 166, 87, 167, and 85. In one embodiment, the immunogenic peptides are a) SEQ ID NOs: 98, 125, 29, 85, 168, 35, 150, 149, 169, 87, 57, 3, 170, 171, 172, 99, 131, 173, 174, 175, 104, 176, 61, 177, 178, 179, 5, 180, 181, 1, 182 and 140, Or b) The amino acid sequences of SEQ ID NOs. 98, 125, 29, 85, 168, 35, 150, 149, 169, 87, 57, 3, 170, 171, 172, 99, 131, 173, 174, 175, 104, 176, 61, 177, 204, 179, 5, 180, 181, 1, 182, and 140 may be included. In one embodiment, the immunogenic peptide may include the amino acid sequences of SEQ ID NOs. 183, 185, 187, 189, 205, or 208. The 5' to 3' order of the fragments in the peptide may be a given order or a different order.

[0027] In one embodiment, the first immunogenic peptide is a) SEQ ID NOs: 60, 103, 98, 149, 2, 150, 142, 86, 1, 141, 151, 152, 130, 153, 154, 155, 156, 157, 158, 4, 159, 160, 161, 162, 35, 28, 163, 164, 165, 166, 87, 167 and 85, or b) Amino acid sequences of SEQ ID NOs: 60, 103, 98, 149, 2, 150, 142, 86, 1, 141, 151, 152, 199, 153, 154, 155, 156, 157, 158, 4, 159, 160, 161, 162, 35, 28, 203, 164, 165, 166, 87, 167 and 85 It may include, and the second immunogenic peptide is c) SEQ ID NOs: 98, 125, 29, 85, 168, 35, 150, 149, 169, 87, 57, 3, 170, 171, 172, 99, 131, 173, 174, 175, 104, 176, 61, 177, 178, 179, 5, 180, 181, 1, 182 and 14 0, or d) may include the amino acid sequences of SEQ ID NOs: 98, 125, 29, 85, 168, 35, 150, 149, 169, 87, 57, 3, 170, 171, 172, 99, 131, 173, 174, 175, 104, 176, 61, 177, 204, 179, 5, 180, 181, 1, 182, and 140. In one embodiment, a) the first immunogenic peptide may include the amino acid sequence of SEQ ID NOs: 183 or 205, and the second immunogenic peptide may include the amino acid sequence of 185 or 208, and b) the first immunogenic peptide may include the amino acid sequence of SEQ ID NOs: 187, and the second immunogenic peptide may include the amino acid sequence of SEQ ID NOs: 189. In one embodiment, the first immunogenic peptide may include the amino acids of SEQ ID NOs: 205, and the second immunogenic peptide may include the amino acids of SEQ ID NOs: 208.

[0028] In one embodiment, the RNA molecule may contain the nucleotide sequences of SEQ ID NOs: 184, 186, 188, 190, 206, 207, 209, 210, or 211-218.

[0029] In one embodiment, the epitope may be a non-protective epitope that can lead to CD8+ T cell protective breakdown. Preferably, the protective breakdown may be T cell escape, T cell exhaustion, or loss of CD4+ helper T cells.

[0030] In one embodiment, the peptide may further comprise a sequence that enhances epitope presentation on the cell surface. Preferably, the cell may be an immune cell. Preferably, the immune cell may be an antigen-presenting cell (APC). In some embodiments, the peptide may further comprise a signal peptide. In some embodiments, the peptide may further comprise an MHC class I transport signal (MITD).

[0031] In one embodiment, the peptide may further comprise a non-HIV HLA-II helper epitope. Preferably, the helper epitope may be a P2 and / or P16 amino acid sequence derived from the tetanus toxoid (TT) of Clostridium tetani.

[0032] RNA molecules may be linear or circular and / or contain a 5' cap. Preferably, the 5' cap may be a modified or artificial cap or cap analogue.

[0033] In one embodiment, the expression cassette may further include a 5' untranslated region (5'UTR) and / or a 3' untranslated region (3'UTR). Preferably, the 5'UTR may include the nucleotide sequence of SEQ ID NO: 71, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity with the nucleotide sequence of SEQ ID NO: 71. Preferably, the 3'UTR may include the nucleotide sequence of SEQ ID NO: 72, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity with the nucleotide sequence of SEQ ID NO: 72.

[0034] In one embodiment, the expression cassette(s) may further include a poly(A) structure. Preferably, the poly(A) structure may be a suspended poly(A) structure. Preferably, the poly(A) structure may include the nucleotide sequence of SEQ ID NO: 73, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity with the nucleotide sequence of SEQ ID NO: 73.

[0035] In one embodiment, the RNA molecule(s) may be a replicable RNA molecule. Preferably, the replicable RNA molecule may further encode an RNA-dependent RNA polymerase (replicase) that can replicate the replicable RNA molecule, or the replicable RNA molecule may not encode an RNA-dependent RNA polymerase (replicase). Preferably, the composition may further contain a non-replicable RNA molecule that encodes an RNA-dependent RNA polymerase (replicase) that can replicate the replicable RNA molecule.

[0036] In one embodiment, the RNA molecule(s) may be non-immunogenic. Preferably, the RNA molecule(s) can be made non-immunogenic by removing double-stranded RNA.

[0037] In one embodiment, the RNA molecule(s) may include nucleotide modifications. Preferably, the modification may be the substitution of one or more U residues with pseudouridine, N1-methylpseudridine, or 5-methyluridine. Preferably, one or more substituted U residues may be N1-methylpseudridine. Preferably, at least 50%, at least 70%, at least 90%, at least 99%, or 100% of the U residues in the RNA molecule may be substituted.

[0038] In one embodiment, RNA molecules(s) can be formulated into a composition containing at least one lipid. Preferably, RNA molecules(s) and at least one lipid can form particles. Preferably, the particles may be lipid nanoparticles (LNPs), lipoplexes (LPXs), or liposomes.

[0039] In one embodiment, the particles may be nanoparticles, where, (i) The number of positive charges in the nanoparticles does not exceed the number of negative charges in the nanoparticles, and / or (ii) The nanoparticles have a net negative charge, and / or (iii) The zeta potential of the nanoparticles is 0 or less. Preferably, the charge ratio of positive charge to negative charge in the nanoparticles can be 1:1 to 1:8, preferably 1:1 to 1:4.

[0040] In one embodiment, at least one lipid may be a cationic lipid. Preferably, the lipid may contain a cationic head group. In some embodiments, the lipid may be a pH-responsive lipid. In some embodiments, at least one lipid may be a PEGylated lipid. In some embodiments, the composition may further contain at least one helper lipid. Preferably, the helper lipid may be a neutral lipid.

[0041] In one embodiment, at least one cationic lipid may include 1,2-di-O-octadecenyl-3-trimethylammoniumpropane (DOTMA), 1,2-dioleyloxy-3-dimethylaminopropane (DODMA), and / or 1,2-dioleoyl-3-trimethylammoniumpropane (DOTAP). In some embodiments, at least one helper lipid may include 1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine (DOPE), cholesterol (Chol), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), and / or 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC).

[0042] In one embodiment, the molar ratio of at least one cationic lipid to at least one helper lipid may be 10:0 to 3:7, preferably 9:1 to 3:7, 4:1 to 1:2, 4:1 to 2:3, 7:3 to 1:1, or 2:1 to 1:1, preferably about 1:1.

[0043] In some embodiments, the nanoparticles may be lipoplexes containing DODMA and DOPE in a molar ratio of 10:0 to 1:9, preferably 8:2 to 3:7, more preferably 7:3 to 5:5, where the charge ratio of positive charge in DODMA to negative charge in RNA is 1.8:2 to 0.8:2, more preferably 1.6:2 to 1:2, even more preferably 1.4:2 to 1.1:2, and even more preferably about 1.2:2.

[0044] In some embodiments, the nanoparticles may be lipoplexes containing DODMA and cholesterol in a molar ratio of 10:0 to 1:9, preferably 8:2 to 3:7, more preferably 7:3 to 5:5, where the charge ratio of positive charge in DODMA to negative charge in RNA is 1.8:2 to 0.8:2, more preferably 1.6:2 to 1:2, even more preferably 1.4:2 to 1.1:2, and even more preferably about 1.2:2, or the nanoparticles may contain DODMA and DSPC in a ratio of 10:0 to 1:9, preferably 8:2 to 3:7, more preferably 7 The lipoplex may contain DODMA:Cholesterol:DOPE:PEGcerC16 in a molar ratio of 3-5:5, with a charge ratio of positive charge in DODMA to negative charge in RNA of 1.8:2-0.8:2, more preferably 1.6:2-1:2, even more preferably 1.4:2-1.1:2, and even more preferably about 1.2:2, or the nanoparticles may be a lipoplex containing DODMA:Cholesterol:DOPE:PEGcerC16 in a molar ratio of 40:48:10:2, or the nanoparticles may contain DODMA and DOPE in a ratio of 10:0-1:9, preferably 8:2-3:7, more preferably The lipoplex may contain DOTMA in a molar ratio of 7:3 to 5:5, with a charge ratio of positive charge in DOTMA to negative charge in RNA of 1.8:2 to 0.8:2, more preferably 1.6:2 to 1:2, even more preferably 1.4:2 to 1.1:2, and even more preferably about 1.2:2, or the nanoparticles may be a lipoplex containing DOTMA and cholesterol in a molar ratio of 10:0 to 1:9, preferably 8:2 to 3:7, more preferably 7:3 to 5:5, with a charge ratio of positive charge in DOTMA to negative charge in RNA of 1.8:2 to 0 The ratio is 0.8:2, more preferably 1.6:2 to 1:2, even more preferably 1.4:2 to 1.1:2, and even more preferably about 1.2:2, or the nanoparticles may be lipoplexes containing DOTAP and DOPE in a molar ratio of 10:0 to 1:9, preferably 8:2 to 3:7, and more preferably 7:3 to 5:5, with a charge ratio of positive charge in DOTMA to negative charge in RNA of 1.8:2 to 0.8:2, more preferably 1.6:2 to 1:2, even more preferably 1.4:2 to 1.1:2, and even more preferably about 1.2:2.

[0045] In one embodiment, the particles may be LNPs that form complexes with and / or encapsulate RNA molecules. In one embodiment, the particles may be vesicles that encapsulate RNA molecules, preferably monolayer liposomes. In some embodiments, the RNA molecule(s) may be formulated into a composition comprising a polyalkyleneimine, preferably a polyalkyleneimine. Preferably, the molar ratio of nitrogen atoms (N) in the polyalkyleneimine to phosphorus atoms (P) in the RNA molecule (N:P ratio) may be 2.0 to 15.0, preferably 6.0 to 12.0.

[0046] In one embodiment, the ionic strength of the composition may be 50 mM or less, preferably the concentration of monovalent cationic ions may be 25 mM or less, and the concentration of divalent cationic ions may be 20 μM or less.

[0047] In one embodiment, the particles formed may be polyplexes.

[0048] In one embodiment, the polyalkyleneimine may include the following general formula (I): [ka] (In the formula, R is H, an acyl group, or a group containing the following general formula (II): [ka] In the formula, R1 is H or a group containing the following general formula (III): [ka] n, m, and l are independently chosen from integers between 2 and 10; and p, q, and r are integers, and the sum of p, q, and r is such that the average molecular weight of the polymer is 1.5 × 10² to 10⁷ Da, preferably 5000 to 10⁵ Da, more preferably 10000 to 40000 Da, more preferably 15000 to 30000 Da, and even more preferably 20000 to 25000 Da).

[0049] In one embodiment, the polyalkyleneimine may include polyethyleneimine and / or polypropyleneimine, preferably polyethyleneimine. In some embodiments, at least 92% of the N atoms in the polyalkyleneimine may be protonable.

[0050] In one embodiment, the composition may be a pharmaceutical composition. Preferably, the composition may further contain a pharmaceutically acceptable carrier or excipient.

[0051] In one embodiment, the composition may be in the form of a dry powder, or the composition may be freeze-dried, or the composition may be frozen. Preferably, the composition may have a temperature of -20°C or lower.

[0052] In one embodiment, the composition may further contain one or more additives, which are optionally selected from the group consisting of buffers, sugars, stabilizers, cryoprotectants, freeze-drying protectants, and chelating agents. Preferably, the buffer may include at least one selected from the group consisting of 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 2-(N-morpholino)ethanesulfonic acid (MES), 3-morpholino-2-hydroxypropanesulfonic acid (MOPSO), acetic acid, acetate buffer and analogues, phosphoric acid and phosphate buffer, and citrate and citrate buffer. Preferably, the sugar may include at least one selected from the group consisting of monosaccharides, disaccharides, trisaccharides, oligosaccharides, and polysaccharides, preferably glucose, trehalose, and saccharose. Preferably, the cryoprotectant may include at least one selected from the group consisting of glycols such as ethylene glycol, propylene glycol, and glycerol. Preferably, the chelating agent may include EDTA.

[0053] In one embodiment, the composition may be a vaccine.

[0054] A further aspect of this disclosure is a pharmaceutical formulation comprising the compositions disclosed herein.

[0055] A further embodiment of this disclosure is a kit comprising one or more RNA molecules as defined herein. Preferably, the RNA molecules may be in the form of a dry powder composition. Preferably, the RNA molecules may be lyophilized. Preferably, the kit may further include instructions for administering the RNA molecules.

[0056] A further aspect of this disclosure is a method for preventing a target HIV infection, which comprises administering a composition disclosed herein to a target.

[0057] Further aspects of the present disclosure are methods for preventing HIV infection in a subject, comprising dissolving a dry powder composition disclosed herein in a suitable liquid pharmaceutical solution to form an administration solution, and administering the administration solution to a subject.

[0058] A further aspect of this disclosure is a method for treating HIV infection in an HIV-positive subject, comprising administering a composition disclosed herein to the subject.

[0059] Further aspects of the present disclosure are methods for treating HIV infection in HIV-positive subjects, comprising dissolving a dry powder composition disclosed herein in a suitable liquid pharmaceutical solution to form an administration solution, and administering the administration solution to a subject.

[0060] In one embodiment of the method disclosed herein, the severity of one or more symptoms of HIV infection can be reduced. In one embodiment, the method may comprise only a single dose of the composition or multiple doses of the composition.

[0061] In one embodiment of the method disclosed herein, the method may further include administering a booster dose of the composition.

[0062] In one embodiment of the methods disclosed herein, administration of the composition may include intradermal, subcutaneous, or intramuscular administration, such as by intradermal, subcutaneous, or intramuscular injection. Preferably, the injection may be by the use of a needle or by the use of a needle-free injection device. In one embodiment of the methods disclosed herein, administration may include administration by intramuscular injection, preferably using a needle.

[0063] Further aspects of this disclosure are compositions described herein for use in a method for preventing or treating a subject HIV infection, the method comprising administering the composition to a subject. In one embodiment, the subject may be HIV-positive.

[0064] A further aspect of this disclosure is the composition described herein for use in the manufacture of a pharmaceutical product for the prevention or treatment of a target HIV infection.

[0065] In one embodiment of the embodiments of this disclosure relating to treatment or prevention, the method may include administering a first composition comprising a first RNA molecule comprising an expression cassette encoding an immunogenic peptide described herein, and the method further includes administering a second composition comprising a second RNA molecule comprising an expression cassette encoding a second immunogenic peptide described herein, wherein the second immunogenic peptide is different from the first immunogenic peptide. The compositions may be administered simultaneously or at different time points by the same or different administration methods. The method may further include further rounds of administering the compositions.

[0066] In one embodiment of the embodiments of this disclosure relating to treatment or prevention, the method may include administering a first RNA molecule and a second RNA molecule to a target, the first RNA molecule being a) SEQ ID NOs: 60, 103, 98, 149, 2, 150, 142, 86, 1, 141, 151, 152, 130, 153, 154, 155, 156, 157, 158, 4, 159, 160, 161, 1 62, 35, 28, 163, 164, 165, 166, 87, 167 and 85, or b) Sequence IDs 60, 103, 98, 149, 2, 150, 142, 86, 1, 141, 151, 152, 199, 153, 154, 155, 156, 157, 158, 4, 159, 160, 161, 162, 35, 28, 203, 164, 165, 166, 87, 167 and 8 The second RNA molecule may contain a nucleotide sequence encoding a first peptide containing an amino acid sequence of 5, and the second RNA molecule may contain SEQ ID NOs: 98, 125, 29, 85, 168, 35, 150, 149, 169, 87, 57, 3, 170, 171, 172, 99, 131, 173, 174, 175, 104, 176, 61, 177, 178, 179, 5, 180, 181, 1, 182 and 140, or d) may include a nucleotide sequence encoding a second peptide containing the amino acid sequences of SEQ ID NOs. 98, 125, 29, 85, 168, 35, 150, 149, 169, 87, 57, 3, 170, 171, 172, 99, 131, 173, 174, 175, 104, 176, 61, 177, 204, 179, 5, 180, 181, 1, 182 and 140. In one embodiment, the first RNA molecule may contain the nucleotide sequence of SEQ ID NO: 184, 211, 212, 206, or 207, and the second RNA molecule may contain the nucleotide sequence of SEQ ID NO: 186, 213, 214, 209, or 210, or b) the first RNA molecule may contain the nucleotide sequence of SEQ ID NO: 188, 215, or 216, and the second RNA molecule may contain the nucleotide sequence of SEQ ID NO: 190, 217, or 218. In one embodiment, the first RNA molecule may contain the nucleotide sequence of SEQ ID NO: 206 or 207, and the second RNA molecule may contain the nucleotide sequence of SEQ ID NO: 209 or 210. In one embodiment, the first and second RNA molecules may be administered at intervals of at least two weeks.In one embodiment, the 5' to 3' order of the amino acid sequences of the first and / or second peptides may be a given order, or the 5' to 3' order of the amino acid sequences of the first and / or second peptides may be different from the given order. [Modes for carrying out the invention]

[0067] The present invention will be described in detail below, but it should be understood that the present invention is not limited to the specific methods, protocols, and reagents described herein, and that these may be modified. Furthermore, it should be understood that the terms used herein are intended solely to describe specific embodiments and are not intended to limit the scope of the present invention, and that the scope of the present invention 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 those skilled in the art.

[0068] Preferably, the terms used herein are defined as those described in "A multilingual glossary of biotechnological terms: (IUPAC Recommendations)", H.G. W. Heuenberger, B. Nagel, and H. Kolbl, Eds., Helvetica Chimica Acta, CH-4010 Basel, Switzerland, (1995).

[0069] The implementation of the present invention will, unless otherwise specified, utilize conventional methods of chemistry, biochemistry, cell biology, immunology, and recombinant DNA techniques as described in the literature of the art (see, for example, Molecular Cloning: A Laboratory Manual, 2nd Edition, J. Sambrook et al. eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor 1989).

[0070] The elements of the present invention are described below. These elements are listed together with specific embodiments, but it should be understood that they may be combined in any way and in any number to create further embodiments. The various examples and preferred embodiments described should not be construed as limiting the invention to only the expressly described embodiments. This description should be understood as disclosing and encompassing embodiments that combine the expressly described embodiments with any number of disclosed elements and / or preferred elements. Furthermore, any rearrangement and combination of all elements described in this application should be considered disclosed by this description unless specifically indicated in the context.

[0071] The term "approximately" means roughly or nearly, and in the context of numerical values ​​or ranges described herein, preferably means + / - 10% of the numerical values ​​or ranges listed or claimed.

[0072] The terms “a,” “an,” and “the” and similar references used in the context describing the present invention (particularly in the context of the claims) should be construed to encompass both singular and plural unless otherwise specifically indicated herein or unless clearly inconsistent with the context. The enumeration of ranges of values ​​herein is intended simply as a way of concisely referring to each distinct value that falls within that range individually. Unless otherwise specifically indicated herein, each individual value is incorporated herein as if it were individually enumerated herein. All methods described herein may be carried out in any suitable order unless otherwise specifically indicated herein or unless clearly inconsistent with the context. The use of any examples or illustrative language provided herein (e.g., “etc.”) is intended simply to better illustrate the present invention and does not impose any limitation on the claimed scope of the present invention. No language herein should be construed to indicate any unclaimed elements essential to the practice of the present invention.

[0073] Unless otherwise specified, the term “including” is used in the context of this document to indicate that there may be additional members in addition to the members of the list introduced by “including”. However, the term “including” is intended to encompass the possibility that there may be no additional members, i.e., for the purposes of this embodiment, “including” should be understood to mean “consisting of”.

[0074] The indication of the relative amount of a component characterized by a collective term is intended to refer to the total amount of all specific variants or members encompassed by the collective term. When a specific component defined by a collective term is identified as existing in a specific relative amount, and this component is further characterized as a specific variant or member encompassed by the collective term, it means that no other variants or members encompassed by the collective term exist additionally such that the total relative amount of the component encompassed by the collective term exceeds the identified relative amount, more preferably, no other variants or members encompassed by the collective term exist at all.

[0075] Throughout this specification, several sources are referenced. Each of the sources referenced herein (including all patents, patent applications, scientific publications, manufacturer specifications, instructions, etc.) is incorporated herein by reference in its entirety, either above or below. Nothing in this specification should be construed as an acknowledgment that the present invention had no prior rights to such disclosures.

[0076] As used herein, terms such as “reduce” or “inhibit” mean the ability to cause an overall reduction of a level of preferably 5% or more, 10% or more, 20% or more, more preferably 50% or more, and most preferably 75% or more. The term “inhibit” or similar phrases include complete or essentially complete inhibition, i.e., a reduction to or essentially zero.

[0077] Terms such as “increase” or “boost” preferably relate to an increase or boost of at least about 10%, preferably at least 20%, preferably at least 30%, more preferably at least 40%, more preferably at least 50%, even more preferably at least 80%, and most preferably at least 100%.

[0078] The term "net charge" refers to the total charge of an object, such as a compound or particle.

[0079] Ions with an overall net positive charge are cations, and ions with an overall net negative charge are anions. Therefore, according to the present invention, anions are ions with more electrons than protons and thus possess a net negative charge, while cations are ions with fewer electrons than protons and thus possess a net positive charge.

[0080] With respect to a given compound or particle, the terms “charged,” “net charge,” “negatively charged,” or “positively charged” refer to the net charge of the given compound or particle when dissolved or suspended in water at pH 7.0.

[0081] The term "nucleic acid" according to the present invention also includes the chemical derivatization of nucleic acids on nucleotide bases, sugars, or phosphates, as well as nucleic acids containing non-natural nucleotides and nucleotide analogs. In some embodiments, the nucleic acid is deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). Generally, a nucleic acid molecule or nucleic acid sequence refers to a nucleic acid which is preferably deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). According to the present invention, nucleic acids include genomic DNA, cDNA, mRNA, viral RNA, recombinantly prepared molecules, and chemically synthesized molecules. According to the present invention, nucleic acids may be in the form of single-stranded or double-stranded linear molecules or covalently ring-bound molecules.

[0082] According to the present invention, "nucleic acid sequence" refers to a sequence of nucleotides in a nucleic acid, such as ribonucleic acid (RNA) or deoxyribonucleic acid (DNA). This term may refer to the entire nucleic acid molecule (such as a single strand of the entire nucleic acid molecule) or a part of it (e.g., a fragment).

[0083] According to the present invention, the terms “RNA” or “RNA molecule” refer to a molecule comprising, and preferably entirely or substantially consisting of, ribonucleotide residues. The term “ribonucleotide” refers to a nucleotide having a hydroxyl group at the 2' position of a β-D-ribofuranosyl group. The term “RNA” includes isolated RNA such as double-stranded RNA, single-stranded RNA, partially or completely purified RNA, essentially pure RNA, synthetic RNA, and recombinant-produced RNA such as modified RNA that differs from naturally occurring RNA by the addition, deletion, substitution and / or alteration of one or more nucleotides. Such alterations may include, for example, the addition of non-nucleotide substances to the terminal(s) or interior of the RNA, for example, at one or more nucleotides of the RNA. Nucleotides in an RNA molecule may also include non-standard nucleotides, such as nucleotides that do not exist in nature or chemically synthesized nucleotides or deoxynucleotides. These modified RNAs may be called analogs, in particular analogs of naturally occurring RNA.

[0084] According to the present invention, RNA can be single-stranded or double-stranded. In some embodiments of the present invention, single-stranded RNA is preferred. The term “single-stranded RNA” generally refers to an RNA molecule that is not associated with a complementary nucleic acid molecule (typically a complementary RNA molecule). Single-stranded RNA may contain self-complementary sequences that allow a portion of the RNA to fold back and form secondary structural motifs, including but not limited to base pairs, stems, stem-loops, and bulges. Single-stranded RNA can exist as a minus [(-)] strand or a plus [(+)] strand. The (+) strand is the strand that contains or codes for genetic information. The genetic information may be, for example, a polynucleotide sequence that codes for a protein. If the (+) strand RNA codes for a protein, the (+) strand can directly function as a template for translation (protein synthesis). The (-) strand is the complementary strand of the (+) strand. In the case of double-stranded RNA, the (+) and (-) strands are two separate RNA molecules, and both of these RNA molecules associate with each other to form double-stranded RNA ("double-stranded RNA").

[0085] The term "stability" of RNA refers to its "half-life." Half-life refers to the time required to remove half of the activity, quantity, or number of molecules. In relation to this invention, the half-life of RNA is an indicator of the stability of the RNA. The half-life of RNA can affect the "duration of expression" of RNA. RNA with a long half-life can be expected to be expressed for a long period of time.

[0086] The term "translation efficiency" refers to the amount of translation products provided by RNA molecules within a specific period of time.

[0087] A “fragment” refers to a sequence that represents a portion of a nucleic acid sequence, i.e., a sequence shortened at the 5' and / or 3' ends. Preferably, a nucleic acid sequence fragment contains at least 80%, preferably at least 90%, 95%, 96%, 97%, 98%, or 99% of nucleotide residues from the nucleic acid sequence. In the present invention, a fragment of an RNA molecule that preserves the stability and / or translation efficiency of the RNA is preferred.

[0088] The term "fragment" in relation to an amino acid sequence (peptide or protein) refers to a sequence representing a portion of the amino acid sequence, i.e., a truncated amino acid sequence at the N-terminus and / or C-terminus. A C-terminally truncated fragment (N-terminal fragment) can be obtained, for example, by translation of 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 translation of a truncated open reading frame lacking the 5' end of the open reading frame, insofar as the truncated open reading frame contains a start codon that helps initiate translation. A fragment of an amino acid sequence includes, for example, at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, and at least 90% of the amino acid residues from the amino acid sequence. When used herein in relation to immunogenic peptides, a fragment is a subpart of a peptide containing one or more epitopes.

[0089] For example, the term “variant” in relation to nucleic acids and amino acid sequences according to the present invention includes any variant, in particular mutants, viral strain variants, splice variants, conformations, isoforms, allele variants, species variants, and species homologs, especially those occurring in nature. Allele variants are associated with normal sequence changes of a gene, and their significance is often unclear. Complete gene sequencing often identifies numerous allele variants for a given gene. With respect to nucleic acid molecules, the term “variant” includes degenerate nucleic acid sequences, and a degenerate nucleic acid according to the present invention is a nucleic acid whose codon sequence differs from that of a reference nucleic acid due to the degeneracy of the genetic code. A species homolog is a nucleic acid or amino acid sequence originating from a different species than that of a given nucleic acid or amino acid sequence. A viral homolog is a nucleic acid or amino acid sequence originating from a different virus than that of a given nucleic acid or amino acid sequence.

[0090] Nucleic acid variants include single or multiple nucleotide deletions, additions, mutations, substitutions, and / or insertions compared to a reference nucleic acid. A deletion involves the removal of one or more nucleotides from the reference nucleic acid. Addition variants involve 5' and / or 3' terminal fusions of one or more nucleotides, e.g., 1, 2, 3, 5, 10, 20, 30, 50 or more nucleotides. In the case of substitution, at least one nucleotide in the sequence is removed and at least one other nucleotide is inserted in its place (e.g., transversion and transition). Mutations include debasement sites, crosslinking sites, and chemically altered or modified bases. Insertions involve the addition of at least one nucleotide to the reference nucleic acid.

[0091] According to the present invention, a “nucleotide change” may refer to one or more nucleotide deletions, additions, mutations, substitutions, and / or insertions compared to a reference nucleic acid. In some embodiments, a “nucleotide change” is selected from the group consisting of a single nucleotide deletion, a single nucleotide addition, a single nucleotide mutation, a single nucleotide substitution, and / or a single nucleotide insertion compared to a reference nucleic acid. According to the present invention, a nucleic acid variant may comprise one or more nucleotide changes compared to a reference nucleic acid.

[0092] A variant of a specific nucleic acid sequence preferably has at least one functional characteristic of the specific sequence, and preferably is functionally equivalent to the specific sequence, for example, a nucleic acid sequence that exhibits the same or similar characteristics as the specific nucleic acid sequence.

[0093] As described below, some embodiments of the present invention are characterized, in particular, by nucleic acid sequences homologous to other nucleic acid sequences. These homologous sequences are variants of other nucleic acid sequences.

[0094] Preferably, the degree of identity between a given nucleic acid sequence and a nucleic acid sequence that is a variant of the given nucleic acid sequence, or between a given amino acid sequence and an amino acid sequence that is a variant of the given amino acid sequence, is at least 70%, preferably at least 75%, preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, or most preferably at least 95%, 96%, 97%, 98%, or 99%. The degree of identity is preferably given over a region of at least about 30, at least about 50, at least about 70, at least about 90, at least about 100, at least about 150, at least about 200, at least about 250, at least about 300, or at least about 400 nucleotides. In a preferred embodiment, the degree of identity is given over the entire length of the reference nucleic acid sequence or the reference amino acid sequence.

[0095] "Sequence similarity" indicates the percentage of amino acids that are identical or represent conserved amino acid substitutions. "Sequence identity" between two polypeptide or nucleic acid sequences indicates the percentage of amino acids or nucleotides that are identical between the sequences.

[0096] The term "% identical" is intended to refer specifically to the percentage of nucleotides or amino acids that are identical in the optimal alignment between two sequences being compared, wherein the percentage is purely statistical, the differences between the two sequences may be randomly distributed over the entire length of the sequences, and the sequences being compared may contain additions or deletions compared to a reference sequence in order to obtain the optimal alignment between the two sequences. The comparison of two sequences is typically performed by comparing the sequences with respect to a segment or "comparison window" after optimal alignment in order to identify local regions of the corresponding sequences. Optimal alignment for comparison can be performed manually, or using the local homology algorithm by Smith and Waterman, 1981, Ads App.Math.2:482, the local homology algorithm by Needleman and Wunsch, 1970, J.Mol.Biol.48:443, and the similarity search algorithm by Pearson and Lipman, 1988, Proc.Natl Acad.Sci.USA 85:2444, or with the assistance of computer programs using the said algorithms (GAP, BESTFIT, FASTA, BLAST P, BLAST N, and TFASTA from Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, Wis.).

[0097] The identity percentage is obtained by determining the number of identical positions in the sequences being compared, dividing this number by the number of positions being compared, and multiplying the result by 100.

[0098] For example, you could use the BLAST program "BLAST 2 sequences," which is available on the website http: / / www.ncbi.nlm.nih.gov / blast / bl2seq / wblast2.cgi.

[0099] Nucleic acids can "hybridize" or "hybridize" with another nucleic acid if two sequences are complementary to each other. Nucleic acids are "complementary" to another nucleic acid if two sequences can form a stable double helix with each other. According to the present invention, hybridization is preferably carried out under conditions that allow for specific hybridization between polynucleotides (stringent conditions). Stringent conditions are described, for example, in Molecular Cloning: A Laboratory Manual, J. Sambrook et al., Editors, 2nd Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989 or Current Protocols in Molecular Biology, FMAusubel et al., Editors, John Wiley & Sons, Inc., New York, and refer to hybridization at 65°C in hybridization buffer (3.5 × SSC, 0.02% Ficoll, 0.02% polyvinylpyrrolidone, 0.02% bovine serum albumin, 2.5 mM NaH2PO4 (pH 7), 0.5% SDS, 2 mM EDTA). SSC is 0.15 M sodium chloride / 0.15 M sodium citrate, pH 7. After hybridization, the DNA-transcribed membrane is washed, for example, in 2×SSC at room temperature, and then washed again in 0.1–0.5×SSC / 0.1×SDS at temperatures up to 68°C.

[0100] The complementarity percentage indicates the proportion of consecutive residues in a nucleic acid molecule that can form hydrogen bonds (e.g., Watson-Crick base pairs) with a second nucleic acid sequence (e.g., 5, 6, 7, 8, 9, and 10 out of 10 are 50%, 60%, 70%, 80%, 90%, and 100% complementary). "Fully complementary" or "completely complementary" means that all consecutive residues in the nucleic acid sequence form hydrogen bonds with the same number of consecutive residues in the second nucleic acid sequence. Preferably, the degree of complementarity according to the present invention is at least 70%, preferably at least 75%, preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, or most preferably at least 95%, 96%, 97%, 98%, or 99%. Most preferably, the degree of complementarity according to the present invention is 100%.

[0101] The term “derivative” includes any chemical derivatization of nucleic acids on a nucleotide base, sugar, or phosphate. The term “derivative” also includes nucleic acids containing nucleotides and nucleotide analogs that do not exist in nature. Preferably, the derivatization of nucleic acids increases their stability.

[0102] A "nucleic acid sequence derived from a nucleic acid sequence" refers to a nucleic acid that is a variant of the nucleic acid from which it is derived. Preferably, when substituting a specific sequence within an RNA molecule, the variant sequence for that specific sequence maintains the stability and / or translation efficiency of the RNA.

[0103] "nt" is an abbreviation for one nucleotide, or for multiple nucleotides, preferably a sequence of nucleotides in a nucleic acid molecule.

[0104] According to the present invention, the term "codon" refers to a base triplet in a coding nucleic acid that specifies which amino acid will be added next during protein synthesis in a ribosome.

[0105] The terms “transcription” and “to transcribe” relate to the process by which a nucleic acid molecule having a specific nucleic acid sequence (“nucleic acid template”) is read by an RNA polymerase, resulting in the RNA polymerase producing a single-stranded RNA molecule. During transcription, the genetic information in the nucleic acid template is transcribed. The nucleic acid template may be DNA; however, in the case of transcription from, for example, an alphavirus nucleic acid template, the template is typically RNA. The transcribed RNA can then be translated into a protein. According to this invention, the term “transcription” includes “in vitro transcription,” and the term “in vitro transcription” relates to the process by which RNA, particularly mRNA, is synthesized in vitro in a cell-free system. Preferably, a cloning vector is applied to the production of a transcript. These cloning vectors are generally called transcription vectors and, according to this invention, are encompassed in the term “vector.” The cloning vector is preferably a plasmid. According to this invention, the RNA is preferably in vitro transcription RNA (IVT-RNA), which can be obtained by in vitro transcription of a suitable DNA template. The promoter for controlling transcription can be any promoter for any RNA polymerase. DNA templates for in vitro transcription can be obtained by cloning nucleic acids, particularly cDNA, and introducing them into a suitable vector for in vitro transcription. cDNA can be obtained by reverse transcription of RNA.

[0106] Single-stranded nucleic acid molecules produced during transcription typically have a nucleic acid sequence that is complementary to the template sequence.

[0107] According to the present invention, the terms “template,” “nucleic acid template,” or “template nucleic acid” generally refer to a nucleic acid sequence that can be replicated or transcribed.

[0108] The terms "nucleotide sequence transcribed from a nucleotide sequence" and similar terms refer, where appropriate, to a nucleotide sequence as part of a complete RNA molecule that is the transcript of a template nucleotide sequence. Typically, the transcribed nucleotide sequence is a single-stranded RNA molecule.

[0109] According to this invention, the "3' end of a nucleic acid" refers to the end containing a free hydroxyl group. In a schematic diagram of a double-stranded nucleic acid, particularly DNA, the 3' end is always on the right side. According to this invention, the "5' end of a nucleic acid" refers to the end containing a free phosphate group. In a schematic diagram of a double-stranded nucleic acid, particularly DNA, the 5' end is always on the left side. 5' end 5'--P-NNNNNNN-OH-3' 3' end 3'-HO-NNNNNNN-P--5'

[0110] "Upstream" refers to the relative position of the first element of a nucleic acid molecule to its second element, where both elements are contained within the same nucleic acid molecule, and the first element is located closer to the 5' end of the nucleic acid molecule than the second element. In this case, the second element is said to be "downstream" of the first element of the nucleic acid molecule. An element located "upstream" of the second element can synonymously be said to be located on the "5' side" of the second element. For double-stranded nucleic acid molecules, designations such as "upstream" and "downstream" are given with respect to the "+" strand.

[0111] According to the present invention, "functional linkage" or "functionally linked" refers to linkage within a functional relationship. A nucleic acid is "functionally linked" if it is functionally related to another nucleic acid sequence. For example, a promoter is functionally linked to a coding sequence if it affects the transcription of that coding sequence. Functionally linked nucleic acids are typically adjacent to each other but, where appropriate, are separated by further nucleic acid sequences, which in certain embodiments are transcribed by RNA polymerase to give a single RNA molecule (common transcript).

[0112] In certain embodiments, according to the present invention, nucleic acids are functionally linked to expression regulatory sequences that may be homogeneous or heterogeneous with respect to the nucleic acid.

[0113] The term “expression regulatory sequence” according to the present invention includes promoters, ribosome-binding sequences, and other regulatory elements that control the transcription of a gene or the translation of derived RNA. In certain embodiments of the present invention, expression regulatory sequences can be modulated. The exact structure of an expression regulatory sequence may vary depending on the species or cell type, but typically includes a 5' untranscribed sequence and 5' and 3' untranslated sequences that are involved in the initiation of transcription and translation, respectively. More specifically, the 5' untranscribed expression regulatory sequence includes a promoter region that contains a promoter sequence for the transcriptional control of a functionally linked gene. Expression regulatory sequences may also include enhancer sequences or upstream activation sequences. Expression regulatory sequences of DNA molecules typically include 5' untranscribed sequences such as TATA boxes, capping sequences, and CAAT sequences, as well as 5' and 3' untranslated sequences. Expression regulatory sequences of alphaviral RNA may include subgenome promoters and / or one or more conserved sequence elements. A particular expression regulatory sequence according to the present invention is an alphaviral subgenome promoter, as described herein.

[0114] Nucleic acid sequences identified herein, in particular transcriptionable coding nucleic acid sequences, may be combined with any regulatory expression sequences, in particular promoters, which may be homogeneous or heterogeneous. The term “homologous” refers to the fact that the nucleic acid sequence is functionally linked to the regulatory expression sequence in nature, while the term “heterogeneous” refers to the fact that the nucleic acid sequence is not functionally linked to the regulatory expression sequence in nature.

[0115] A transcriptionable nucleic acid sequence, particularly a nucleic acid sequence encoding a peptide or protein, and an expression regulatory sequence are "functionally" linked to each other if they are covalently linked to each other such that the transcription or expression of the transcriptionable, particularly the coding nucleic acid sequence, is under the control or influence of the expression regulatory sequence. When a nucleic acid sequence is translated into a functional peptide or protein, induction of an expression regulatory sequence functionally linked to the coding sequence results in the transcription of the coding sequence without causing a frameshift of the coding sequence or making it impossible for the coding sequence to be translated into the desired peptide or protein.

[0116] The terms “promoter” or “promoter region” refer to a nucleic acid sequence that controls the synthesis of a transcript, such as a coding sequence, by providing a recognition and binding site for RNA polymerase. A promoter region may include further recognition or binding sites for further factors involved in regulating the transcription of the gene. Promoters can control the transcription of prokaryotic or eukaryotic genes. Promoters can be “inducible,” meaning they can initiate transcription in response to an inducer, or they can be “constitutive,” meaning transcription is not controlled by an inducer. Inducible promoters are expressed only slightly or not at all in the absence of an inducer. In the presence of an inducer, the gene is “switched on,” or the level of transcription increases. This is usually mediated by the binding of a specific transcription factor. A specific promoter according to the present invention is, as described herein, for example, an alphavirus subgenome promoter. Other specific promoters are, for example, alphavirus genome positive-strand or negative-strand promoters.

[0117] The term "core promoter" refers to the nucleic acid sequence contained within a promoter. Typically, the core promoter is the smallest portion of the promoter necessary to properly initiate transcription. The core promoter typically includes the transcription initiation site and the RNA polymerase binding site.

[0118] "Polymerase" generally refers to a molecular entity that can catalyze the synthesis of polymer molecules from monomer building blocks. "RNA polymerase" is a molecular entity that can catalyze the synthesis of RNA molecules from ribonucleotide building blocks. "DNA polymerase" is a molecular entity that can catalyze the synthesis of DNA molecules from deoxyribonucleotide building blocks. In the case of DNA polymerase and RNA polymerase, the molecular entity is typically a protein or an aggregate or complex of multiple proteins. Typically, DNA polymerase synthesizes DNA molecules based on a template nucleic acid, which is typically a DNA molecule. Typically, RNA polymerase synthesizes RNA molecules based on a template nucleic acid, which is either a DNA molecule (in which case RNA polymerase is DNA-dependent RNA polymerase, DdRP) or an RNA molecule (in which case RNA polymerase is RNA-dependent RNA polymerase, RdRP).

[0119] RNA-dependent RNA polymerase, or RdRP, is an enzyme that catalyzes the transcription of RNA from an RNA template. In the case of alphaviral RNA-dependent RNA polymerase, RNA replication is brought about by the sequential synthesis of the (-) strand complement and (+) strand genomic RNA of the genomic RNA. Therefore, RNA-dependent RNA polymerase is synonymously called "RNA replicase" or simply "replicase." In nature, RNA-dependent RNA polymerase is typically encoded by all RNA viruses except retroviruses. Alphaviruses are a typical example of viruses that encode RNA-dependent RNA polymerase.

[0120] According to the present invention, "RNA replication" generally refers to an RNA molecule synthesized based on the nucleotide sequence of a given RNA molecule (template RNA molecule). The synthesized RNA molecule may be identical or complementary to the template RNA molecule, for example. Generally, RNA replication can occur via the synthesis of DNA intermediates or directly by RNA-dependent RNA replication mediated by RNA-dependent RNA polymerase (RdRP). In the case of alphaviruses, RNA replication does not occur via DNA intermediates but is mediated by RNA-dependent RNA polymerase (RdRP): the template RNA strand (first RNA strand) - or a part thereof - acts as a template for the synthesis of a second RNA strand complementary to the first RNA strand or a part thereof. The second RNA strand - or a part thereof - then optionally acts as a template for the synthesis of a third RNA strand complementary to the second RNA strand or a part thereof. Thus, the third RNA strand is identical to the first RNA strand or a part thereof. Therefore, RNA-dependent RNA polymerases can directly synthesize complementary RNA strands of a template, and can also indirectly synthesize identical RNA strands (via complementary intermediate strands).

[0121] According to the present invention, the term "template RNA" refers to RNA that can be transcribed or replicated by RNA-dependent RNA polymerase.

[0122] According to the present invention, the term “gene” refers to a specific nucleic acid sequence responsible for the production of one or more cell products and / or the achievement of one or more intercellular or intracellular functions. More specifically, the term refers to a nucleic acid portion (typically DNA; however, RNA in the case of RNA viruses) that comprises nucleic acids encoding a specific protein or functional or structural RNA molecule.

[0123] As used herein, “isolated molecule” is intended to mean a molecule that substantially does not contain other molecules, such as other cellular material. According to the present invention, the term “isolated nucleic acid” means a nucleic acid that has been (i) amplified in vitro, for example by polymerase chain reaction (PCR), (ii) recombinantly produced by cloning, (iii) purified, for example by cleavage and gel electrophoresis, or (iv) synthesized, for example by chemical synthesis. An isolated nucleic acid is a nucleic acid that is available for manipulation by recombinant techniques.

[0124] The term “vector” is used herein in its most common sense and includes, for example, any intermediate vehicle for nucleic acids that enables the introduction of nucleic acids into prokaryotic and / or eukaryotic host cells and, where appropriate, their integration into the genome. Such vectors are preferably replicated and / or expressed within cells. Vectors include plasmids, phagemids, viral genomes, and fractions thereof.

[0125] In the context of this invention, the term "recombinant" means "produced through genetic manipulation." Preferably, the "recombinant object," such as recombinant cells in the context of this invention, does not exist in nature.

[0126] As used herein, the term “naturally occurring” refers to the fact that an object can be found in nature. For example, peptides or nucleic acids that are present in living organisms (including viruses), can be isolated from natural sources, and have not been intentionally modified by humans in a laboratory are naturally occurring. The term “found in nature” means “naturally occurring” 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.

[0127] According to the present invention, the term “expression” is used in its most common sense and includes the production of RNA and / or proteins. This term also includes the partial expression of nucleic acids. Furthermore, expression can be transient or stable. With respect to RNA, the terms “expression” or “translation” refer to the process in the ribosomes of a cell in which a chain of coding RNA (e.g., messenger RNA) directs the assembly of an amino acid sequence to produce a peptide or protein.

[0128] According to the present invention, the term "mRNA" means "messenger RNA" and refers to a transcript that codes for a peptide or protein, typically produced by using a DNA template. Typically, mRNA includes a 5'-UTR, a protein-coding region, a 3'-UTR, and a poly(A) sequence. mRNA can be produced by in vitro transcription from a DNA template. In vitro transcription methods are known to those skilled in the art. For example, various in vitro transcription kits are commercially available. According to the present invention, mRNA can be modified by stabilization modifications and capping.

[0129] According to the present invention, the terms “poly(A) sequence” or “poly(A) tail” typically refer to a continuous or discontinuous sequence of adenylate residues located at the 3' end of an RNA molecule. Continuous sequences are characterized by consecutive adenylate residues. In nature, continuous poly(A) sequences are typical. Poly(A) sequences are not usually encoded by eukaryotic DNA, but during eukaryotic transcription in the cell nucleus, they bind to the free 3' end of RNA by post-transcriptional template-independent RNA polymerase, and the present invention encompasses poly(A) sequences encoded by DNA.

[0130] According to the present invention, the term "primary structure" with respect to nucleic acid molecules refers to the linear sequence of nucleotide monomers.

[0131] According to the present invention, the term “secondary structure” in relation to nucleic acid molecules refers to a two-dimensional representation of the nucleic acid molecule that reflects base pairing, for example, intramolecular base pairing in the case of single-stranded RNA molecules. Although each RNA molecule has only a single polynucleotide chain, the molecule is typically characterized by regions of (intramolecular) base pairs. According to the present invention, the term “secondary structure” includes structural motifs including, but not limited to, base pairs, stems, stem-loops, bulges, internal loops, and loops such as multibranched loops. The secondary structure of a nucleic acid molecule can be represented by a two-dimensional drawing (planar graph) showing base pairing (for further details on the secondary structure of RNA molecules, see Auber et al., 2006; J. Graph Algorithms Appl. 10:329-351). As described herein, the secondary structure of a particular RNA molecule is relevant in the context of the present invention.

[0132] According to the present invention, the secondary structure of nucleic acid molecules, particularly single-stranded RNA molecules, is determined by prediction using a web server for RNA secondary structure prediction (http: / / rna.urmc.rochester.edu / RNAstructureWeb / Servers / Predict1 / Predict1.html). Preferably, according to the present invention, "secondary structure" with respect to nucleic acid molecules specifically refers to the secondary structure determined by the aforementioned prediction. The prediction may also be performed or verified using MFOLD structure prediction (http: / / unafold.rna.albany.edu / ?q=mfold).

[0133] According to this invention, a "base pair" is a structural motif of a secondary structure in which two nucleotide bases associate with each other via hydrogen bonds between donor and acceptor sites on the bases. Complementary bases A:U and G:C form stable base pairs via hydrogen bonds between donor and acceptor sites on the bases; A:U and G:C base pairs are called Watson-Crick base pairs. Weaker base pairs (called wobble base pairs) are formed by bases G and U (G:U). Base pairs A:U and G:C are called canonical base pairs. Other base pairs such as G:U (which occur fairly frequently in RNA) and other rare base pairs (e.g., A:C;U:U) are called non-canonical base pairs.

[0134] According to the present invention, "nucleotide pairing" refers to two nucleotides that associate with each other such that the bases of the two nucleotides form a base pair (a canonical or non-canonical base pair, preferably a canonical base pair, most preferably a Watson-Crick base pair).

[0135] According to the present invention, the terms “stem-loop,” “hairpin,” or “hairpin-loop” are all interchangeable in reference to a specific secondary structure of a nucleic acid molecule, typically a single-stranded nucleic acid molecule such as single-stranded RNA. The specific secondary structure represented by a stem-loop consists of a contiguous nucleic acid sequence containing a stem and a (terminal) loop, also called a hairpin-loop, where the stem is formed by two adjacent, fully or partially complementary sequence elements, separated by a short sequence (e.g., 3-10 nucleotides) that forms the loop of the stem-loop structure. The two adjacent, fully or partially complementary sequences can be defined, for example, as stem 1 and stem 2 of the stem-loop element. The stem-loop is formed when these two adjacent, fully or partially inversely complementary sequences, for example, stem 1 and stem 2 of the stem-loop element, form base pairs with each other, resulting in a double-stranded nucleic acid sequence containing an unpaired loop at its terminal, formed by a short sequence located between stem 1 and stem 2 of the stem-loop element. Therefore, a stem-loop consists of two stems (stem 1 and stem 2), which, at the level of the secondary structure of the nucleic acid molecule, form base pairs with each other, and at the level of the primary structure of the nucleic acid molecule, are separated by a short sequence that is not part of stem 1 or stem 2. For illustrative purposes, a two-dimensional representation of a stem-loop is analogous to a lollipop-shaped structure. The formation of a stem-loop structure requires the presence of a sequence that can fold back and form a paired double helix; the paired double helix is ​​formed by stem 1 and stem 2. The stability of the paired stem-loop element is typically determined by its length, i.e., the number of nucleotides in stem 1 that can form base pairs (preferably canonical base pairs, more preferably Watson-Crick base pairs) with the nucleotides in stem 2, and the number of nucleotides in stem 1 that cannot form such base pairs with the nucleotides in stem 2 (mismatches or bulges). According to the present invention, the optimal loop length is 3 to 10 nucleotides, more preferably 4 to 7 nucleotides, e.g., 4, 5, 6, or 7 nucleotides.When a given nucleic acid sequence is characterized by a stem-loop, its complementary nucleic acid sequences are also typically characterized by stem-loops. Stem-loops are typically formed by single-stranded RNA molecules. For example, the 5' replication recognition sequence of alphaviral genomic RNA contains several stem-loops.

[0136] According to the present invention, "disruption" or "disruption" of a specific secondary structure (e.g., stem-loop) of a nucleic acid molecule means that the specific secondary structure is absent or altered. Typically, a secondary structure can be disrupted as a result of a change in at least one nucleotide that is part of the secondary structure. For example, a stem-loop can be disrupted by a change in one or more nucleotides that form the stem, resulting in nucleotide pairing being impossible.

[0137] According to the present invention, the term "tertiary structure" in relation to nucleic acid molecules refers to the three-dimensional structure of a nucleic acid molecule as defined by atomic coordinates.

[0138] In some embodiments of this disclosure, the RNA molecule is a “replicon RNA” or “replicon RNA molecule” or simply “replicon,” in particular “self-replicating RNA” or “self-amplifying RNA” or “replicable RNA molecule.” A replicon RNA molecule is RNA that can be replicated by RNA-dependent RNA polymerase (replicase) by containing a nucleotide sequence that can be recognized by a replicase so that the RNA may be replicated. Since a replicon does not necessarily encode a replicase, a replicon can be replicated in cis (by an encoded replicase; also called a “cis-replicon”) or trans (by a replicase provided in another way, a separate replicase that encodes a nucleic acid such as mRNA; also called a “trans-replicon”).

[0139] In certain embodiments, the replicon or self-replicating RNA is derived from or contains elements derived from ssRNA viruses, particularly positive-strand ssRNA viruses such as alphaviruses. Alphaviruses are typical representatives of positive-strand RNA viruses. Alphaviruses replicate in the cytoplasm of infected cells (see Jose et al., Future Microbiol., 2009, vol. 4, pp. 837-856 for a review of the alphavirus life cycle). The whole genome length of many alphaviruses is typically in the range of 11,000–12,000 nucleotides, and the genomic RNA typically has a 5' cap and a 3' poly(A) tail. The alphavirus genome encodes non-structural proteins (involved in the transcription, modification, and replication of viral RNA, as well as protein modification) and structural proteins (forming the viral particle). Typically, there are two open reading frames (ORFs) in the genome. The four non-structural proteins (nsP1-nsP4) are typically encoded together by a first ORF (Order of Reference Frame) beginning near the 5' end of the genome, while the alphaviral structural proteins are found downstream of the first ORF and are encoded together by a second ORF extending near the 3' end of the genome. Typically, the first ORF is larger than the second ORF, with a ratio of approximately 2:1. In cells infected with alphaviruses, only the nucleic acid sequences encoding non-structural proteins are translated from the genomic RNA, while the genetic information encoding structural proteins is translatable from subgenomic transcripts, which are RNA molecules similar to eukaryotic messenger RNA (mRNA; Gould et al., 2010, Antiviral Res., vol.87, pp.111-124). After infection, i.e., in the early stages of the viral life cycle, the (+) strand genomic RNA acts directly like messenger RNA for the translation of the open reading frame encoding the non-structural polyprotein (nsP1234). Alphavirus-derived vectors have been proposed for delivering foreign genetic information to target cells or target organisms.A simple approach involves replacing the open reading frame encoding the alphaviral structural protein with the open reading frame encoding the protein of interest. An alphaviral-based trans replication system relies on alphaviral nucleotide sequence elements on two distinct nucleic acid molecules: one encoding a viral replicase, and the other being able to be replicated in trans by the replicase (hence the name trans replication system). Trans replication requires the presence of both of these nucleic acid molecules in a given host cell. The nucleic acid molecule that can be replicated in trans by the replicase must contain specific alphaviral sequence elements that enable recognition of the alphaviral replicase and RNA synthesis.

[0140] According to the present invention, nucleic acids such as RNA, for example rRNA, can encode peptides or proteins. Therefore, a transcriptable nucleic acid sequence or its transcript may include an open reading frame (ORF) that encodes a peptide or protein.

[0141] According to the present invention, the term "nucleic acid encoding a peptide or protein" means that, when present in a suitable environment, preferably within a cell, the nucleic acid can direct the assembly of amino acids to produce a peptide or protein during the translation process. Preferably, the coding RNA according to the present invention can interact with the cellular translation mechanism that enables the translation of the coding RNA to produce a peptide or protein.

[0142] According to the present invention, the term "peptide" includes oligopeptides and polypeptides and refers to a substance containing two or more, preferably three or more, preferably four or more, preferably six or more, preferably eight or more, preferably ten or more, preferably thirteen or more, preferably sixteen or more, preferably twenty or more, and up to a maximum preferably fifty, preferably 100 or more, consecutive amino acids linked to one another via peptide bonds. The term "protein" refers to a large peptide, preferably a peptide having at least 151 amino acids, however, the terms "peptide" and "protein" are generally used as synonyms herein.

[0143] According to the present invention, the terms "peptide" and "protein" include substances that contain not only amino acid components but also non-amino acid components such as sugars and phosphate structures, and also include substances that contain bonds such as esters, thioethers, or disulfide bonds.

[0144] According to the present invention, the terms “start codon” and “start codon” are synonymous with a codon (base triplet) of an RNA molecule that may be the first codon translated by the ribosome. Such codons typically encode the amino acid methionine in eukaryotes and modified methionine in prokaryotes. The most common start codon in eukaryotes and prokaryotes is AUG. Unless otherwise specifically stated herein, the terms “start codon” and “start codon” refer to the codon AUG with respect to RNA molecules. According to the present invention, the terms “start codon” and “start codon” are also used to refer to the corresponding base triplet of deoxyribonucleic acid, i.e., the base triplet that encodes the start codon of RNA. If the start codon of messenger RNA is AUG, then the base triplet that encodes AUG is ATG. According to the present invention, the terms “start codon” and “start codon” preferably refer to a functional start codon or start codon, i.e., a start codon or start codon that is or will be used as a codon by the ribosome to initiate translation. For example, AUG codons may exist in RNA molecules that are not used by ribosomes to initiate translation because the distance from the codon to the cap is too short. These codons are not included in the term functional start codon or departure codon.

[0145] The following provides specific and / or preferred variations of the individual features of the present invention. The present invention also intends, as a particularly preferred embodiment, to be an embodiment generated by combining two or more specific and / or preferred variations described for two or more features of the present invention.

[0146] "Isolated" means modified or removed from its natural state. For example, cells, nucleic acids, or peptides that are naturally present in a living animal are not "isolated," but the same cells, nucleic acids, or peptides that have been partially or completely separated from their natural coexisting substances are "isolated." Preferably, isolated cells, nucleic acids, or peptides exist in a purified or substantially purified state. Isolated cells or cell populations preferably exist without cells of different cell types; for example, isolated T cells exist without other blood cells such as dendritic cells. Preferably, isolated cells exist only with isogenic cells of the same cell type.

[0147] The term "isogenetic" is used to describe cells that have the same genetic information as another cell or cell population.

[0148] The term "autologous" is used to describe something that originates from the same source. For example, "autotransplantation" refers to the transplantation of tissue or organs from the same source. Such a procedure is advantageous because it overcomes immunological barriers that would otherwise lead to rejection.

[0149] The term "homogenetic" is used to describe things that originate from different individuals of the same species. Two or more individuals are said to be homogeneous if they do not have identical genes at one or more gene loci.

[0150] The term "related" is used to describe individuals or tissues that have the same genotype, i.e., identical twins or animals of the same inbred lineage, or tissues derived from them.

[0151] The term "xenotransplant" is used to describe something consisting of multiple different elements. For example, transferring bone marrow from one individual to another constitutes xenotransplantation. Xenogenes are genes that originate from a source other than the target organism.

[0152] In the context of this invention, the term "recombinant" means "produced through genetic manipulation." Preferably, "recombinant objects," such as recombinant cells, in the context of this invention do not exist in nature.

[0153] As used herein, the term “naturally occurring” refers to the fact that a substance can be found in nature. For example, a peptide or nucleic acid that is present in living organisms (including viruses), can be isolated from a natural source, and has not been intentionally modified by humans in a laboratory is considered naturally occurring.

[0154] As used herein, "lentivirus" refers to a genus of the family Retroviridae. Lentiviruses are unique among retroviruses in that they can infect non-dividing cells; they are one of the most efficient methods of gene delivery vectors because they can deliver a significant amount of genetic information to the host cell's DNA. HIV, SIV, and FIV are all examples of lentiviruses. Vectors derived from lentiviruses provide a means of achieving significant levels of gene transfer in vivo.

[0155] In relation to the present invention, the terms "immune effector cells" or "immunoreactive cells" refer to cells that exert effector functions during an immune response.

[0156] In the context of the present invention, the term "effector function" includes any function mediated by components of the immune system that result in inhibition of tumor growth and / or tumor development, including, for example, the death of diseased cells such as tumor cells, or the inhibition of tumor dissemination and metastasis. Preferably, the effector function in the context of the present invention is a T cell-mediated effector function. Such a function is a helper T cell (CD4 + In the case of T cells, cytokine release and / or CD8 +This includes activation of lymphocytes (CTLs) and / or B cells, and in the case of CTLs, elimination of cells, i.e., cells characterized by antigen expression, via apoptosis or perforin-mediated cytolysis, production of cytokines such as IFN-γ and TNF-α, and specific cytolytic death of target cells expressing the antigen.

[0157] In one embodiment, "immune effector cells" can bind to antigens, such as antigens presented in relation to MHC (the term MHC includes HLA) on cells or expressed on the surface of cells, and mediate an immune response. For example, immune effector cells include T cells (cytotoxic T cells, helper T cells, tumor-infiltrating T cells), B cells, natural killer cells, neutrophils, macrophages, and dendritic cells. Preferably, in the context of the present invention, "immune effector cells" are T cells, preferably CD4 + and / or CD8 + These are T cells. According to the present invention, the term “immune effector cells” also includes cells that can mature into immune cells (such as T cells, particularly T helper cells, or cytolytic T cells) upon appropriate stimulation. Immune effector cells are CD34 + This includes hematopoietic stem cells, immature and mature T cells, and immature and mature B cells. The differentiation of T cell precursors into cytolytic T cells is analogous to clonal selection in the immune system when exposed to antigens.

[0158] Preferably, “immune effector cells” recognize antigens with some degree of specificity, particularly when presented in relation to MHC or present on the surface of disease cells such as cancer cells. Preferably, such recognition allows the antigen-recognizing cells to be responsive or reactive. + If it is a T cell, such responsiveness or reactivity is due to the release of cytokines and / or CD8 +It may involve the activation of lymphocytes (CTL) and / or B cells. When the cell is a CTL, such responsiveness or reactivity may involve the elimination of cells, i.e., cells expressing an antigen, for example, via apoptosis or perforin-mediated cytolysis. According to the present invention, CTL responsiveness may include sustained calcium flux, cell division, production of cytokines such as IFN-γ and TNF-α, upregulation of activation markers such as CD44 and CD69, and specific cytolytic killing of target cells expressing an antigen. CTL responsiveness can also be determined using an artificial reporter that accurately indicates CTL responsiveness. Such CTLs that recognize an antigen and are responsive or reactive are also referred to herein as "antigen-responsive CTLs."

[0159] A "lymphoid cell" is a cell that can generate an immune response, such as a cellular immune response, or a progenitor cell of such a cell, optionally after appropriate modification, such as transfer of an antigen receptor such as a TCR or CAR, and includes lymphocytes, preferably T lymphocytes, lymphoblasts, and plasma cells. A lymphoid cell can be an immune effector cell as described herein. Preferred lymphoid cells are T cells that can be modified to express an antigen receptor on the cell surface. In one embodiment, the lymphoid cell lacks endogenous expression of the T cell receptor.

[0160] The terms "T cell" and "T lymphocyte" are used interchangeably herein and include cytotoxic T cells (CTL, CD8 + T cells) consisting of T helper cells (CD4 + T cells) and cytolytic T cells. The term "antigen-specific T cell" or a similar term refers to a T cell that recognizes an antigen targeted by the T cell and preferably exhibits the effector function of the T cell. A T cell is considered antigen-specific when the cell kills a target cell expressing the antigen. T cell specificity can be evaluated using any of a variety of standard techniques, for example, in a chromium release assay or a proliferation assay. Alternatively, synthesis of a lymphokine (such as interferon γ) can be measured.

[0161] T cells belong to the group of white blood cells known as lymphocytes and play a central role in cellular immunity. They can be distinguished from other lymphocyte types, such as B cells and natural killer cells, by the presence of special receptors on their cell surface called T cell receptors (TCRs). The thymus is the main organ involved in the maturation of T cells. Several different subsets of T cells have been discovered, each with a different function.

[0162] Among its many functions, T helper cells assist other leukocytes in immunological processes, including the maturation of B cells into plasma cells and the activation of cytotoxic T cells and macrophages. These cells express the CD4 glycoprotein on their surface, hence the name CD4. + They are also known as T cells. Helper T cells are activated when presented with peptide antigens by MHC class II molecules expressed on the surface of antigen-presenting cells (APCs). Once activated, they rapidly divide and secrete small proteins called cytokines that regulate or assist the active immune response.

[0163] Cytotoxic T cells destroy virus-infected cells and tumor cells and are also involved in transplant rejection. These cells express the CD8 glycoprotein on their surface, therefore CD8 + They are also known as T cells. These cells recognize their targets by binding to antigens associated with MHC class I, which are present on the surface of almost all cells in the body.

[0164] Regulatory T cells, or Tregs, are a subpopulation of T cells that modulate the immune system, maintain tolerance to autoantigens, and prevent autoimmune diseases. Tregs are immunosuppressive and generally suppress or downregulate the induction and proliferation of effector T cells. Tregs express the biomarkers CD4, FoxP3, and CD25.

[0165] As used herein, the term “naive T cell” refers to a mature T cell that has never encountered its alloantigen in the periphery, unlike activated T cells or memory T cells. Naive T cells are generally characterized by surface expression of L-selectin (CD62L), absence of the activation markers CD25, CD44, or CD69, and absence of the memory CD45RO isoform.

[0166] As used herein, the term “memory T cells” refers to a subgroup or subpopulation of T cells that have previously encountered and responded to their allogeneic antigens. Upon a second encounter with the antigen, memory T cells can proliferate to initiate a faster and more powerful immune response than the first time the immune system responded to the antigen. Memory T cells are CD4 + or CD8 + It can be one of the following, and usually expresses CD45RO.

[0167] According to the present invention, the term "T cell" also includes cells that can mature into T cells upon appropriate stimulation.

[0168] Most T cells possess a T cell receptor (TCR), which exists as a complex of several proteins. The actual T cell receptor is produced from independent T cell receptor alpha and beta (TCRα and TCRβ) genes and consists of two distinct peptide chains called the α-TCR chain and the β-TCR chain. γδ T cells (gamma delta T cells) are a small subset of T cells that have a different T cell receptor (TCR) on their surface. However, in γδ T cells, the TCR consists of one γ chain and one δ chain. This group of T cells is far less numerous than αβ T cells (2% of all T cells).

[0169] All T cells originate from hematopoietic stem cells in the bone marrow. Hematopoietic progenitor cells derived from hematopoietic stem cells reside in the thymus, where they expand through cell division to form a large population of immature thymocytes. The earliest thymocytes do not express CD4 or CD8, and are therefore double-negative (CD4). - CD8 -They are classified as ) cells. As development progresses, they become double-positive thymocytes (CD4 + CD8 + ) and ultimately single positive (CD4 + CD8 - or CD4 - CD8 + They mature into thymocytes and are then released from the thymus into peripheral tissues.

[0170] T cells can generally be prepared in vitro or ex vivo using standard procedures. For example, T cells can be isolated from mammals, such as a patient's bone marrow, peripheral blood, or a portion of bone marrow or peripheral blood, using commercially available cell isolation systems. Alternatively, T cells may originate from related or unrelated human, non-human animals, cell lines, or cultures. A sample containing T cells may, for example, be peripheral blood mononuclear cells (PBMCs).

[0171] The term “epitope” refers to a portion or fragment of a molecule, such as an antigen, that is recognized by the immune system. For example, an epitope may be recognized by T cells, B cells, or antibodies. An antigen epitope may consist of a continuous or discontinuous portion of the antigen and may be about 5 to about 100, for example, about 5 to about 50, more preferably about 8 to about 30, and most preferably about 10 to about 25 amino acid lengths. For example, an epitope may preferably be 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acid lengths. In one embodiment, the epitope is about 10 to about 25 amino acid lengths. The term “epitope” includes T cell epitopes.

[0172] The term "T cell epitope" refers to a portion or fragment of a protein that is recognized by T cells when presented in association with an MHC molecule. The term "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 disease cells in immune responses, and MHC proteins or molecules bind to 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 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 bound peptide is typically about 8 to 10 amino acids long, but longer or shorter peptides may be effective. For class II MHC / peptide complexes, the bound peptide is typically about 10 to 25 amino acids long, and especially about 13 to 18 amino acids long, although longer and shorter peptides may also be effective.

[0173] The pharmaceutical preparations, in particular kits, described herein may include explanatory materials or instructions. Where used herein, “explanatory materials” or “instructions” includes publications, records, figures, or any other medium of expression that can be used to convey the usefulness of the compositions and methods of the present invention. The explanatory materials for a kit of the present invention may, for example, be affixed to the container containing the compositions of the present invention, or shipped together with the container containing the compositions. Alternatively, the explanatory materials may be shipped separately from the container, with the intention that the explanatory materials and the compositions be used in conjunction by the recipient.

[0174] The following provides specific and / or preferred variations of the individual features of the present invention. The present invention also intends, as a particularly preferred embodiment, to be an embodiment generated by combining two or more specific and / or preferred variations described for two or more features of the present invention.

[0175] nucleic acid As used herein, the terms “polynucleotide” or “nucleic acid” are intended to include DNA and RNA, such as genomic DNA, cDNA, mRNA, recombinantly produced molecules, and chemically synthesized molecules. Nucleic acids may be single-stranded or double-stranded. RNA includes in vitro transcribed RNA (IVT RNA) or synthetic RNA. According to the present invention, polynucleotides are preferably isolated.

[0176] Nucleic acids may be included in vectors. As used herein, the term “vector” includes any vector known to those skilled in the art, including plasmid vectors, cosmid vectors, phage vectors such as lambda phages, retroviral vectors, viral vectors such as adenovirus vectors or baculovirus vectors, or artificial chromosome vectors such as bacterial artificial chromosomes (BACs), yeast artificial chromosomes (YACs), or P1 artificial chromosomes (PACs). The vectors include expression vectors and cloning vectors. Expression vectors include plasmids and viral vectors and generally contain a desired coding sequence and appropriate DNA sequences necessary for the expression of an operablely linked coding sequence in a particular host organism (e.g., bacteria, yeast, plants, insects, or mammals) or in an in vitro expression system. Cloning vectors are generally used to manipulate and amplify a particular desired DNA fragment and may lack the functional sequences necessary for the expression of the desired DNA fragment.

[0177] In some embodiments, at least one or all of the first, second, third, and fourth RNA molecules are modified RNA molecules.

[0178] modified RNA In some embodiments, the RNA or RNA molecule described herein is modified RNA. In some embodiments, the modified RNA comprises at least one functional analogue of A, C, G, and / or U.

[0179] In one embodiment, the RNA described herein may have modified nucleotides / nucleosides / skeletal modifications. As used herein, the term “RNA modification” may refer to skeletal modifications as well as chemical modifications, including sugar modifications or base modifications.

[0180] In this regard, the modified RNA molecules as defined herein may include nucleotide analogs / modifications, such as skeletal modifications, sugar modifications, or base modifications. Skeletal modifications as defined herein are modifications in which the phosphate of the nucleotide backbone in the RNA molecule as defined herein is chemically modified. Sugar modifications as defined herein are chemical modifications of the sugars in the nucleotides of the RNA molecule as defined herein. Furthermore, base modifications as defined herein are chemical modifications of the base portion of the nucleotides of the RNA molecule. In this regard, nucleotide analogs or modifications are preferably selected from nucleotide analogs applicable to transcription and / or translation.

[0181] Sugar Modification: Modified nucleosides and nucleotides that can be incorporated into the modified RNA molecules described herein can be modified with sugar moieties. For example, the 2' hydroxyl group (OH) can be modified or substituted with several different "oxy" or "deoxy" substituents. Examples of "oxy"-2' hydroxyl group modifications include, but are not limited to, alkoxy or aryloxy (-OR, e.g., R=H, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl, or sugar); polyethylene glycol (PEG), -O(CH2CH2O)nCH2CH2OR; "locked" nucleic acids (LNA) in which the 2' hydroxyl is linked to the 4' carbon of the same ribose sugar, for example by a methylene crosslink; and amino groups (-O-amino, where the amino group, e.g., NRR, can be alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, or diheteroarylamino, ethylenediamine, polyamino) or aminoalkoxy. "Deoxy" modifications include hydrogen, amino (e.g., NH2; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, diheteroarylamino, or amino acid), or the amino group can be attached to a sugar via a linker, the linker containing one or more atoms C, N, and O. The sugar group can also contain one or more carbons having a stereochemistry opposite to that of the corresponding carbon in ribose. Thus, a modified RNA molecule can contain, for example, a nucleotide containing arabinose as the sugar.

[0182] Skeletal Modification: The phosphate skeleton may be further modified in modified nucleosides and nucleotides and may be incorporated into the modified RNA molecules described herein. The phosphate group of the skeleton can be modified by substituting one or more oxygen atoms with different substituents. Furthermore, modified nucleosides and nucleotides may include complete substitution of the unmodified phosphate moiety with the modified phosphate described herein. Examples of modified phosphate groups include, but are not limited to, phosphorothioates, phosphoroselenates, boranophosphates, boranophosphate esters, hydrogen phosphonates, phosphoramidates, alkyl or aryl phosphonates, and phosphotryesters. In phosphorothioates, both unbound oxygen atoms are substituted with sulfur. The phosphate linker can also be modified by substituting the bound oxygen with nitrogen (bridged phosphoramidate), sulfur (bridged phosphorothioate), and carbon (bridged methylenephosphonate).

[0183] Base Modification: Modified nucleosides and nucleotides that can be incorporated into the modified RNA molecules described herein can be further modified at the nucleic acid base portion. Examples of nucleic acid bases found in RNA include, but are not limited to, adenine, guanine, cytosine, and uracil. For example, the nucleosides and nucleotides described herein can be chemically modified on the main groove surface. In some embodiments, the main groove chemical modification may include an amino group, a thiol group, an alkyl group, or a halo group.

[0184] In certain embodiments of this disclosure, the nucleotide analogs / modifications are preferably 2-amino-6-chloropurine riboside-5'-triphosphate, 2-aminopurine riboside-5'-triphosphate, 2-aminoadenosine-5'-triphosphate, 2'-amino-2'-deoxycytidine-triphosphate, 2-thiocytidine-5'-triphosphate, 2-thiouridine-5'-triphosphate, 2'-fluorothymidine-5'-triphosphate, 2'-0-methylinosine-5'-triphosphate, 4-thiouridine-5' - Triphosphate, 5-aminoallylcytidine-5'-triphosphate, 5-aminoallyluridine-5'-triphosphate, 5-bromocytidine-5'-triphosphate, 5-bromouridine-5'-triphosphate, 5-bromo-2'-deoxycytidine-5'-triphosphate, 5-bromo-2'-deoxyuridine-5'-triphosphate, 5-iodocytidine-5'-triphosphate, 5-iodo-2'-deoxycytidine-5'-triphosphate, 5-iodouridine-5'-triphosphate, 5-iodo-2'-deoxyuridine Zin-5'-triphosphate, 5-methylcytidine-5'-triphosphate, 5-methyluridine-5'-triphosphate, 5-propynyl-2'-deoxycytidine-5'-triphosphate, 5-propynyl-2'-deoxyuridine-5'-triphosphate, 6-azacitidine-5'-triphosphate, 6-azauridine-5'-triphosphate, 6-chloropurine riboside-5'-triphosphate, 7-deazaadenosine-5'-triphosphate, 7-deazaguanosine-5'-triphosphate, 8-azacidenosine-5'-triphosphate The base modification is selected from 8-azidoadenosine-5'-triphosphate, benzimidazole-riboside-5'-triphosphate, N1-methyladenosine-5'-triphosphate, N1-methylguanosine-5'-triphosphate, N6-methyladenosine-5'-triphosphate, O6-methylguanosine-5'-triphosphate, N6-methylguanosine-5'-triphosphate, pseudouridine-5'-triphosphate, or puromycin-5'-triphosphate, xanthosine-5'-triphosphate. Nucleotides for base modification are particularly preferred, selected from the group of base-modified nucleotides consisting of 5-methylcytidine-5'-triphosphate, 7-deazaguanosine-5'-triphosphate, 5-bromocytidine-5'-triphosphate, and pseudouridine-5'-triphosphate.In some embodiments, the modified nucleosides include pyridine-4-onribonucleoside, 5-azauridine, 2-thio-5-azauridine, 2-thiouridine, 4-thio-pseudridine, 2-thio-pseudridine, 5-hydroxyuridine, 3-methyluridine, 5-carboxymethyluridine, 1-carboxymethyl-pseudridine, 5-propynyluridine, 1-propynyl-pseudridine, 5-taurinomethyluridine, 1-taurinomethyl-pseudridine, 5-taurinomethyl-2-thiouridine, and l-taurinomethyl These include -4-thiouridine, 5-methyluridine, 1-methylpsuduridine, 4-thio-1-methylpsuduridine, 2-thio-1-methylpsuduridine, 1-methyl-1-deazapsuduridine, 2-thio-1-methyl-1-deazapsuduridine, dihydrouridine, dihydropsuduridine, 2-thio-dihydrouridine, 2-thio-dihydropsuduridine, 2-methoxyuridine, 2-methoxy-4-thiouridine, 4-methoxypsuduridine, and 4-methoxy-2-thiopsuduridine. In preferred embodiments, a functional analogue substituting uridine is N1-methylpsuduridine (m1Ψ).

[0185] In some embodiments, the modified nucleosides include 5-aza-cytidine, pseudoisocytidine, 3-methylcytidine, N4-acetylcytidine, 5-formylcytidine, N4-methylcytidine, 5-hydroxymethylcytidine, 1-methyl-pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine, 2-thiocytidine, 2-thio-5-methylcytidine, 4-thio-pseudoisocytidine, 4-thio-1-methyl- This includes pseudoisocytidine, 4-thio-1-methyl-1-deaza-pseudoisocytidine, 1-methyl-1-deaza-pseudoisocytidine, zebralin, 5-aza-zebralin, 5-methyl-zebralin, 5-aza-2-thio-zebralin, 2-thio-zebralin, 2-methoxy-cytidine, 2-methoxy-5-methyl-cytidine, 4-methoxy-pseudoisocytidine, and 4-methoxy-l-methyl-pseudoisocytidine.

[0186] In other embodiments, the modified nucleosides include 2-aminopurine, 2,6-diaminopurine, 7-deaza-adenine, 7-deaza-8-aza-adenine, 7-deaza-2-aminopurine, 7-deaza-8-aza-2-aminopurine, 7-deaza-2,6-diaminopurine, 7-deaza-8-aza-2,6-diaminopurine, 1-methyladenosine, N6-methyladenosine, N6-isopentenyladenosine, N6 -(cis-hydroxyisopentenyl)adenosine, 2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine, N6-glycinylcarbamoyladenosine, N6-threonylcarbamoyladenosine, 2-methylthio-N6-threonylcarbamoyladenosine, N6,N6-dimethyladenosine, 7-methyladenine, 2-methylthio-adenine, and 2-methoxyadenine. In other embodiments, the modified nucleosides include inosine, 1-methylinosine, waiosine, waibutosine, 7-deaza-guanosine, 7-deaza-8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deaza-8-aza-guanosine, 6-thio-7-deaza-8-aza-guanosine, 7-methylguanosine, 6-thio-7-methylguanosine, 7-methylinosine, 6-methoxyguanosine, 1-methylguanosine, N2-methylguanosine, N2,N2-dimethylguanosine, 8-oxo-guanosine, 7-methyl-8-oxo-guanosine, l-methyl-6-thioguanosine, N2-methyl-6-thioguanosine, and N2,N2-dimethyl-6-thioguanosine.

[0187] In some embodiments, the nucleotide can be modified on the main groove surface, which may include substituting a hydrogen atom on C-5 of uracil with a methyl group or a halo group. In certain embodiments, the modified nucleoside is 5'-0-(l-thiophosphate)-adenosine, 5'-0-(1-thiophosphate)-cytidine, 5'-0-(l-thiophosphate)-guanosine, 5'-0-(l-thiophosphate)-uridine, or 5'-0-(l-thiophosphate)-pseudridine.

[0188] In further embodiments, the modified RNA is 6-aza-cytidine, 2-thio-cytidine, α-thio-cytidine, pseudo-iso-cytidine, 5-aminoallyl-uridine, 5-iodo-uridine, Nl-methyl-pseudouridine, 5,6-dihydrouridine, α-thio-uridine, 4-thio-uridine, 6-aza-uridine, 5-hydroxy-uridine, deoxythymidine, 5-methyluridine, pyrrolo-cytidine, inosine, α - May include nucleoside modifications selected from thio-guanosine, 6-methyl-guanosine, 5-methyl-cytidine, 8-oxo-guanosine, 7-deaza-guanosine, Nl-methyl-adenosine, 2-amino-6-chloropurine, N6-methyl-2-aminopurine, pseudo-isocytidine, 6-chloropurine, N6-methyl-adenosine, α-thio-adenosine, 8-azido-adenosine, and 7-deaza-adenosine.

[0189] In certain preferred embodiments, the RNA comprises a modified nucleoside instead of at least one (e.g., all) uridines.

[0190] As used herein, the term "uracil" refers to one of the nucleic acid bases that may be present in RNA. The structure of uracil is as follows: [ka]

[0191] As used herein, the term "uridine" refers to one of the nucleosides that can be present in RNA. The structure of uridine is as follows: [ka]

[0192] UTP (uridine 5'-triphosphate) has the following structure. [ka]

[0193] Pseudo-UTP (pseudouridine 5'-triphosphate) has the following structure. [ka]

[0194] "Pseudouridine" is an example of a modified nucleoside, an isomer of uridine, in which uracil is bonded to the pentose ring via a carbon-carbon bond instead of a nitrogen-carbon glycosidic bond.

[0195] Another exemplary modified nucleoside is N1-methylpseudridine (m1Ψ), which has the following structure: [ka]

[0196] N1-methyl-pseudo-UTP has the following structure. [ka]

[0197] Another exemplary modified nucleoside is 5-methyluridine (m5U), which has the following structure: [ka]

[0198] In certain preferred embodiments, one or more uridines in the RNA described herein are replaced with modified nucleosides. In some embodiments, the modified nucleosides are modified uridines.

[0199] In certain preferred embodiments, the RNA comprises a modified nucleoside in place of at least one uridine. In some embodiments, the RNA comprises a modified nucleoside in place of each uridine.

[0200] In certain preferred embodiments, the modified nucleoside is independently selected from pseudouridine (ψ), N1-methyl-pseudridine (m1ψ), and 5-methyl-uridine (m5U). In some embodiments, the modified nucleoside includes pseudouridine (ψ). In some embodiments, the modified nucleoside includes N1-methyl-pseudridine (m1ψ). In some embodiments, the modified nucleoside includes 5-methyl-uridine (m5U). In some embodiments, the RNA may contain two or more modified nucleosides, which are independently selected from pseudouridine (ψ), N1-methyl-pseudridine (m1ψ), and 5-methyl-uridine (m5U). In some embodiments, the modified nucleoside includes pseudouridine (ψ) and N1-methyl-pseudridine (m1ψ). In some embodiments, the modified nucleoside comprises pseudouridine (ψ) and 5-methyluridine (m5U). In some embodiments, the modified nucleoside comprises N1-methyl-pseudridine (m1ψ) and 5-methyluridine (m5U). In some embodiments, the modified nucleoside comprises pseudouridine (ψ), N1-methyl-pseudridine (m1ψ), and 5-methyluridine (m5U).

[0201] In certain preferred embodiments, the modified nucleoside that replaces one or more, for example, all, uridines in the RNA is 3-methyluridine (m 3 U), 5-methoxyuridine (mo 5 U), 5-aza-uridine, 6-aza-uridine, 2-thio-5-aza-uridine, 2-thiouridine(s) 2 U), 4-Thiouridine(s) 4 U), 4-thiopseudolidine, 2-thiopseudolidine, 5-hydroxyuridine (ho 5 U), 5-aminoalliuluridine, 5-halouridine (e.g., 5-iodouridine or 5-bromoruridine), uridine 5-oxyacetic acid (cmo 5 U), Uridine 5-oxyacetate methyl ester (mcmo 5U), 5-carboxymethyluridine (cm 5 U), 1-carboxymethyl-pseudridine, 5-carboxyhydroxymethyl-uridine (chm 5 U), 5-carboxyhydroxymethyl-uridinemethyl 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-ethyl-pseudolidine, 5-methylaminomethyl-2-thiouridine (mnm 5 s 2 U), 5-methylaminomethyl-2-selenouridine (mnm 5 se 2 U), 5-Carbamoylmethyluridine (ncm 5 U), 5-carboxymethylaminomethyluridine (cmnm 5 U), 5-carboxymethylaminomethyl-2-thiouridine (cmnm 5 s 2 U), 5-propinyl-uridine, 1-propinyl-pseudolidine, 5-taurinomethyl-uridine (τm 5 U), 1-taurinomethyl-pseuduridine, 5-taurinomethyl-2-thiouridine (τm5s2U), 1-taurinomethyl-4-thiopseuduridine), 5-methyl-2-thiouridine (m 5 s 2 U), 1-methyl-4-thiopsuduridine (m 1 s 4 Ψ), 4-thio-1-methylpseuduridine, 3-methylpseuduridine (m 3 Ψ), 2-thio-1-methyl-pseuduridine, 1-methyl-1-deaza-pseuduridine, 2-thio-1-methyl-1-deaza-pseuduridine, dihydrouridine(D), dihydropseuduridine, 5,6-dihydrouridine, 5-methyl-dihydrouridine(m5 D) 2-Thiodihydrouridine, 2-Thiodihydropsuduridine, 2-Methoxyuridine, 2-Methoxy-4-Thiouridine, 4-Methoxypsuduridine, 4-Methoxy-2-Thiopsuduridine, N1-Methylpsuduridine, 3-(3-amino-3-carboxypropyl)uridine (acp 3 U), 1-methyl-3-(3-amino-3-carboxypropyl)pseudridine (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-methylpsuduridine (Ψm), 2-thio-2'-O-methyluridine (s 2 Um), 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 It may be one or more of the following modified uridines known in the art: Um), 1-thiouridine, deoxythymidine, 2'-F-alaruridine, 2'-F-uridine, 2'-OH-alaruridine, 5-(2-carbomethoxyvinyl)uridine, 5-[3-(1-E-propenylamino)uridine, or any other modified uridine known in the art.

[0202] In one embodiment, the RNA contains other modified nucleosides or further modified nucleosides, such as modified cytidines, including those described above. For example, in one embodiment, in the RNA, 5-methylcytidine partially or completely, preferably completely, replaces cytidine. In one embodiment, the RNA contains 5-methylcytidine and one or more selected from pseudouridine (ψ), N1-methyl-pseudridine (m1ψ), and 5-methyl-uridine (m5U). In one embodiment, the RNA contains 5-methylcytidine and N1-methyl-pseudridine (m1ψ). In some embodiments, the RNA contains 5-methylcytidine instead of each cytidine and N1-methyl-pseudridine (m1ψ) instead of each uridine.

[0203] cap In some embodiments, the RNA or RNA molecule described herein may optionally include a 5' cap, a 5' UTR, a coding sequence, a 3' UTR, and / or a poly(A) tail. In some embodiments, the coding sequence or open reading frame may be optimized with respect to codon usage frequency.

[0204] "RNA containing a 5' cap," "RNA with a 5' cap," "RNA modified with a 5' cap," or "capped RNA" refers to RNA containing a 5' cap. For example, providing a 5' cap to RNA can be achieved by in vitro transcription of a DNA template in the presence of the 5' cap, the 5' cap being co-transcribed into the resulting RNA strand, or the RNA can be produced, for example, by in vitro transcription, and the 5' cap can be attached to the RNA post-transcriptionally using a capping enzyme, such as the capping enzyme of vaccinia virus. In capped RNA, the 3' position of the first base of the (capped) RNA molecule is ligated to the 5' position of the next base ("second base") of the RNA molecule via a phosphodiester bond.

[0205] In the present disclosure, naturally occurring caps typically consist of non-methylated cap dinucleotides (G(5’)ppp(5’)N; also called GpppN) and methylated cap dinucleotides ((m 7 G(5’)ppp(5’)N; m 7 GpppN) and are selected from the group consisting of. m 7 GpppN (where N is G) is represented by the following formula.

Chemical Formula

[0206] The capped RNA of the present disclosure can be prepared in vitro and thus does not rely on the capping mechanism in host cells. Cotranscriptional capping functions by transcribing a DNA template in vitro with any of bacterial or bacteriophage nucleic acid polymerases in the presence of all four ribonucleoside triphosphates or their functional analogs and a capping reagent, such as m 7 G(5’)ppp(5’)G (m 7 GpppG). The nucleic acid polymerase initiates transcription by a nucleophilic attack of the 3’-OH of the guanosine moiety of m 7 GpppG on the α-phosphate of the next template ribonucleoside triphosphate (pppN), resulting in an intermediate m 7 GpppGpN (where N is the second base of the RNA molecule).

[0207] In a preferred embodiment of the present disclosure, the RNA molecule contains a 5’ cap analog. Cap analogs have been previously described as first promoting the large-scale synthesis of RNA transcripts by in vitro transcription.

[0208] For messenger RNA, several cap analogs (also called synthetic caps) have been previously generally described and all of them can be used in the context of the present disclosure. Ideally, cap analogs associated with higher translation efficiency and / or increased resistance to in vivo degradation and / or increased resistance to in vitro degradation are selected.

[0209] Preferably, a cap analog that can be incorporated into the RNA strand only in one orientation is used. Pasquinelli et al. (1995, RNA J. 1:957-967) showed that during in vitro transcription, bacteriophage RNA polymerase uses a 7-methylguanosine unit for the initiation of transcription, such that about 40-50% of the transcripts with a cap have the cap dinucleotide in the reverse orientation (i.e., the initial reaction product is Gpppm 7 GpN). Compared to RNA with the correct cap, RNA with an inverted cap is not functional with respect to the translation of the nucleic acid sequence into protein. Thus, it is desirable to incorporate the cap in the correct orientation, i.e., to obtain RNA having a structure essentially corresponding to m 7 GpppGpN, etc. The reverse incorporation of the cap dinucleotide has been shown to be inhibited by substitution of either the 2'-OH or 3'-OH group of the methylated guanosine unit (Stepinski et al., 2001, RNA J. 7:1486-1495; Peng et al., 2002, Org. Lett. 24:161-164). RNA synthesized in the presence of such "anti-reverse cap analogs" is translated more efficiently than RNA transcribed in vitro in the presence of the conventional 5' cap m 7 GpppG. For this purpose, one cap analog in which the 3'OH group of the methylated guanosine unit is replaced by OCH3 has been described, for example, by Holtkamp et al., 2006, Blood 108:4009-4017 (7-methyl(3'-O-methyl)GpppG; anti-reverse cap analog (ARCA)). ARCA is an appropriate cap dinucleotide according to the present disclosure.

Chemical formula

[0210] In one embodiment, the cap has the effect that RNA having such a cap is inherently less susceptible to decapping. This is important because, generally, the amount of protein produced from synthetic mRNA introduced into cultured mammalian cells is limited by the spontaneous degradation of mRNA. One in vivo pathway of mRNA degradation begins with the removal of an mRNA cap. This removal is catalyzed by a heterodimeric pyrophosphatase comprising a regulatory subunit (Dcp1) and a catalytic subunit (Dcp2). The catalytic subunit cleaves between the α-phosphate and β-phosphate groups of a triphosphate crosslink. In this disclosure, caps that are resistant to or less resistant to this type of cleavage can be selected. Suitable cap analogs for this purpose can be selected from capped dinucleotides following formula (I): [ka] (In the formula, R 1 This is selected from the group consisting of optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted cycloalkyl, optionally substituted heterocyclyl, optionally substituted aryl, and optionally substituted heteroaryl. R 2 and R 3 is independently selected from the group consisting of H, halo, OH, and optionally substituted alkoxy, or R 2 and R 3 They combine to form OXO (where X is selected from the group consisting of CH2, CH2CH2, CH2CH2CH2, CH2CH(CH3), and C(CH3)2, which may be substituted), or R 2 R 2 It bonds with the hydrogen atom at the 4' position of the ring to which it is bonded, forming -O-CH2- or -CH2-O-. R 5 It is selected from the group consisting of S, Se, and BH3. R 4 and R 6 (It is independently selected from the group consisting of O, S, Se, and BH3). n is 1, 2, or 3.

[0211] R 1 , R 2 , R3, R 4 , R 5 , R 6 Preferred embodiments of R, R, R3, R, R are disclosed in International Publication No. WO 2011 / 015347 A1 and can be selected accordingly in the present disclosure.

[0212] For example, in one embodiment, the RNA molecule of the present disclosure comprises a phosphorothioate cap analog. The phosphorothioate cap analog is one in which one of the three non-bridging O atoms in the triphosphate chain is replaced by an S atom, i.e., R, R, or R of formula (I) is a specific cap analog in which one of them is S. The phosphorothioate cap analog is described by J. Kowalska et al., 2008, RNA, 14: 1119-1131 as a solution to the unwanted decapping process and thus a solution for enhancing the stability of RNA in vivo. In particular, substitution of the sulfur atom in the β-phosphate group of the 5' cap with an oxygen atom results in stabilization against Dcp2. In a preferred embodiment thereof in the present disclosure, R in formula (I) is S, and R and R are O. 4 , R 5 or R 6 is a specific cap analog in which one of them is S. The phosphorothioate cap analog is described by J. Kowalska et al., 2008, RNA, 14: 1119-1131 as a solution to the unwanted decapping process and thus a solution for enhancing the stability of RNA in vivo. In particular, substitution of the sulfur atom in the β-phosphate group of the 5' cap with an oxygen atom results in stabilization against Dcp2. In a preferred embodiment thereof in the present disclosure, R in formula (I) is S, and R and R are O. 5 is S, and R 4 and R 6 are O.

[0213] In a further embodiment, the RNA of the present disclosure comprises a phosphorothioate cap analog in which the phosphorothioate modification of the RNA 5' cap is combined with an "anti-reverse cap analog" (ARCA) modification. Each ARCA-phosphorothioate cap analog is described in International Publication No. WO 2008 / 157688 A2, and all of them can be used for the RNA of the present disclosure. In that embodiment, at least one of R or R in formula (I) is not OH, preferably R and R 2 or R 3 in formula (I) is not OH, preferably R and R 2 and R 3One of them is methoxy(OCH3), and R 2 and R 3 The other is preferably OH. In a preferred embodiment, the sulfur atom in the β-phosphate group is substituted with an oxygen atom (hence R in formula (I)). 5 S is R 4 and R 6 (is O). The phosphorothioate modification of ARCA is thought to ensure that the α, β, and γ phosphorothioate groups are precisely positioned within the active site of the cap-binding protein in both the translation and decapping mechanisms. At least some of these analogs are inherently resistant to pyrophosphatase Dcp1 / Dcp2. Phosphothioate-modified ARCA has been described as having a much higher affinity for eIF4E than the corresponding ARCA lacking the phosphorothioate group.

[0214] In this disclosure, each of the caps that is particularly preferred, namely m 2’ 7,2’-O Gpp s pG is called β-S-ARCA (International Publication No. 2008 / 157688A2; Kuhn et al., 2010, Gene Ther. 17:961-971). Therefore, in one embodiment of the present disclosure, the RNA of the present disclosure is modified with β-S-ARCA. β-S-ARCA is represented by the following structure. [ka]

[0215] Generally, replacing an oxygen atom with a sulfur atom in a crosslinked phosphate yields phosphorothioate diastereomas called D1 and D2, based on their elution patterns in HPLC. In short, the D1 diastereoma of β-S-ARCA, or "β-S-ARCA(D1)", is a diastereoma of β-S-ARCA that elutes first on the HPLC column and therefore exhibits a shorter retention time compared to the D2 diastereoma of β-S-ARCA (β-S-ARCA(D2)). The determination of the stereochemistry by HPLC is described in International Publication No. 2011 / 015347A1.

[0216] In a first particularly preferred embodiment of the present disclosure, the RNA of the present disclosure is modified with the β-S-ARCA(D2) diastereoma. The two diastereomas of β-S-ARCA differ in their sensitivity to nucleases. RNA carrying the β-S-ARCA D2 diastereoma is shown to be almost completely resistant to Dcp2 cleavage (only 6% cleavage compared to RNA synthesized in the presence of an unmodified ARCA 5' cap), while RNA with the β-S-ARCA(D1) 5' cap exhibits moderate sensitivity to Dcp2 cleavage (71% cleavage). The increased stability to Dcp2 cleavage has further been shown to correlate with increased protein expression in mammalian cells. In particular, RNA with the β-S-ARCA(D2) cap has been shown to be translated more efficiently in mammalian cells than RNA with the β-S-ARCA(D1) cap. Therefore, in one embodiment of the present disclosure, the RNA of the present disclosure is modified with the β-S-ARCA D2 diastereoma P β The substituent R in formula (I) corresponds to the stereochemical configuration at the atom. 5 It is modified with a cap analog according to formula (I), characterized by the stereochemical configuration of the P atom containing R 5 S is R 4 and R 6 is O. Furthermore, R in equation (I) 2 or R 3 At least one of them is preferably not OH, but preferably R 2and R 3 One of them is methoxy(OCH3), and R 2 and R 3 The other is preferably OH.

[0217] In a second particularly preferred embodiment, the RNA of the Disclosure is modified with the β-S-ARCA(D1) diastereoma. This embodiment is particularly suitable for the transfer of capped RNA into immature antigen-presenting cells, such as for vaccination purposes. Each β-S-ARCA(D1) diastereoma has been demonstrated to enhance RNA stability, improve RNA translation efficiency, prolong RNA translation, increase total RNA protein expression, and / or enhance the immune response to the antigen or antigenic peptide encoded by the RNA, upon transfer of the capped RNA into immature antigen-presenting cells (Kuhn et al., 2010, Gene Ther. 17:961-971). Therefore, in an alternative embodiment of the Disclosure, the RNA of the Disclosure is modified with the P of the β-S-ARCA(D1) diastereoma. β The substituent R in formula (I) corresponds to the stereochemical configuration at the atom. 5 Modified with a cap analogue following formula (I), characterized by a stereochemical configuration at the P atom containing . Each cap analogue and its embodiments are described in International Publication No. 2011 / 015347A1 and Kuhn et al., 2010, Gene Ther. 17:961-971. Substituent R 5 The stereochemical configuration of the P atom containing this is a β-S-ARCA D1 diastereoma of P β Any cap analogue described in International Publication No. 2011 / 015347A1, corresponding to the stereochemical configuration of an atom, may be used in this disclosure. Preferably, R in formula (I) 5 S is R 4 and R 6 is O. Furthermore, R in equation (I) 2 or R 3 At least one of them is preferably not OH, but preferably R 2 and R 3 One of them is methoxy(OCH3), and R2 and R 3 The other is preferably OH.

[0218] In one embodiment, the RNA of the present disclosure is modified with a 5' cap structure according to formula (I), in which any one phosphate group is substituted with a boranophosphate group or a phosphoselenoate group. Such caps have increased stability both in vitro and in vivo. Optionally, each compound has a 2'-O- or 3'-O-alkyl group (the alkyl is preferably methyl); each cap analog is called BH3-ARCA or Se-ARCA. Compounds particularly suitable for capping mRNA include β-BH3-ARCA and β-Se-ARCA, described in International Publication No. 2009 / 149253A2. For these compounds, the P1 diastereoma of β-S-ARCA is used. β The substituent R in formula (I) corresponds to the stereochemical configuration at the atom. 5 A stereochemical configuration at the P atom containing is preferred.

[0219] In some embodiments, the RNA may have caps that may be appropriate in the context of this disclosure, for example, cap 0 (methylation of the first nucleic acid base, e.g., m7 GpppN), Cap 1 ( m7 Further methylation of the ribose of the adjacent nucleotide of GpppN), cap 2 ( m7 Further methylation of the ribose of the second nucleotide downstream of GpppN), cap 3 ( m7 Further methylation of the ribose of the third nucleotide downstream of GpppN), cap 4 ( m7 This includes further methylation of the ribose of the fourth nucleotide downstream of GpppN), ARCA (anti-reverse cap analogues), modified ARCA (e.g., phosphorothioate-modified ARCA, e.g., β-S-ARCA), inosine, N1-methyl-guanosine, 2'-fluoro-guanosine, 7-deaza-guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine, and 2-azido-guanosine.

[0220] In some embodiments, the RNA comprises a cap that is cap-0 (also referred to herein as "cap 0"), cap-1 (also referred to herein as "cap 1"), or cap-2 (also referred to herein as "cap 2"). See, for example, FIG. 1 of Ramanathan A et al. and FIG. 1 of Decroly E et al.

[0221] In some embodiments, cap 0 comprises a guanosine nucleoside methylated at the 7-position of guanine ( m7 G). In some embodiments, cap 0 is linked to the RNA via a 5'-5' triphosphate bridge and is referred to herein as m7 Gppp or m7 G(5')ppp(5') as well.

[0222] In some embodiments, cap 1 comprises a guanosine nucleoside methylated at the 7-position of guanine ( m7 G or 7m G) and a first nucleotide methylated at the 2'O position in the RNA ( 2’OMe N1 or N12'OMe or N1 2’OMe ). In some embodiments, cap 1 is linked to the RNA via a 5'-5' triphosphate bridge. In some embodiments, cap 1 can be represented as m7 Gppp(N1 2’OMe ) or m7 G(5')ppp(5')(N1 2’OMe ) or 7m G(5')ppp(5')N1 2’-OMe ). In some embodiments, N1 is selected from A, C, G, or U. In some embodiments, N1 is A. In some embodiments, N1 is C. In some embodiments, N1 is G. In some embodiments, N1 is U.

[0223] In some embodiments, m7 G(5')ppp(5')(N1 2’OMe) Cap 1 contains a second nucleotide N2 which is A, G, C or U near the cap at the +2 position. In some embodiments, such cap 1 is ( m7 G(5')ppp(5')(N1 2’OMe It is represented as )pN2). In some embodiments, N2 is A. In some embodiments, N2 is C. In some embodiments, N2 is G. In some embodiments, N2 is U.

[0224] In some embodiments, the cap 1 is m7 G(5')ppp(5')(A1 2’OMe )pG2 (where A1 is the A proximal to the cap at position +1, and G2 is the G proximal to the cap at position +2) or containing it, having the following structure: [ka]

[0225] In some embodiments, the cap 1 is m7 G(5')ppp(5')(A1 2’OMe )pU2 (where A1 is the proximal A of the +1 cap and U2 is the proximal U of the +2 cap) or including it, having the following structure: [ka]

[0226] In some embodiments, the cap 1 is m7 G(5')ppp(5')(G1 2’OMe )pG2 (where G1 is the cap-proximal G at position +1 and G2 is the cap-proximal G at position +2) or containing it, having the following structure: [ka]

[0227] In some embodiments, the cap 1 contains guanine ( m7G) comprises a guanosine nucleoside methylated at position 7 and one or more further modifications, such as methylation at ribose, and a first nucleotide with 2'O methylation in RNA. In some embodiments, cap 1 comprises a guanosine nucleoside methylated at position 7 of guanine and 3'O methylation at ribose (m7G3'OMe or 7m G 3’OMe ), and the first nucleotide (N1) in RNA that has been 2'O methylated. 2’OMe ) includes. In some embodiments, cap 1 is linked to RNA via a 5'-5' triphosphate crosslink, which herein refers to (m7G3'OMe)ppp(2'OMeN1) or ( m7 G 3’OMe )(5')ppp(5')( 2’OMe Also called N1). In some embodiments, N1 is selected from A, C, G, or U. In some embodiments, N1 is A. In some embodiments, N1 is C. In some embodiments, N1 is G. In some embodiments, N1 is U.

[0228] In some embodiments, ( m7 G 3’OMe )(5')ppp(5')(N1 2’OMe ) Cap 1 is the second nucleotide, i.e., the nucleotide proximal to the cap at position 2, and contains N2 selected from A, G, C, or U. m7 G 3’OMe )(5')ppp(5')(N1 2’OMe )pN2). In some embodiments, N2 is A. In some embodiments, N2 is C. In some embodiments, N2 is G. In some embodiments, N2 is U.

[0229] In some embodiments, cap 1 is ( m7 G 3’OMe )(5')ppp(5')(A1 2’OMe )pG2 (where A1 is the A proximal to the cap at position +1, and G2 is the G proximal to the cap at position +2) or containing it, having the following structure: [ka]

[0230] In some embodiments, cap 1 is ( m7 G 3’OMe )(5')ppp(5')(G1 2’OMe )pG2 (where G1 is the cap-proximal G at position +1 and G2 is the cap-proximal G at position +2) or containing it, having the following structure: [ka]

[0231] In some embodiments, the second nucleotide in cap 1 may include one or more modifications, such as methylation. In some embodiments, cap 1 containing the second nucleotide including 2'O methylation is a cap 2 structure.

[0232] In some embodiments, the RNA polynucleotide containing cap 1 has increased translation efficiency, increased translation rate and / or increased expression of the encoded payload compared to a suitable reference comparator. In some embodiments, ( m7 G 3’OMe )(5')ppp(5')(A1 2’OMe RNA polynucleotides containing cap 1 having pG2 (where A1 is the nucleotide near the cap at position +1 and G2 is the nucleotide near the cap at position +2) are ( m7 G 3’OMe )(5')ppp(5')(G1 2’OMe Compared to RNA polynucleotides containing cap 1 with pG2 (where G1 is the nucleotide proximal to the cap at position 1 and G2 is the nucleotide proximal to the cap at position 2), the translation efficiency is increased. In some embodiments, the increase in translation efficiency is evaluated upon administration of the RNA polynucleotide to cells or organisms.

[0233] In some embodiments, the cap analog used in RNA polynucleotides is m7 G 3’OMe Gppp(m1 2’-OMe )ApG(m2 7,3’-OMe G(5')ppp(5')m 2’-OMe ApG or ( m7 G 3’OMe )(5')ppp(5')(A 2’OMe It is also called pG, and it has the following structure. [ka]

[0234] The following are RNA and m2 7,3`OMe G(5')ppp(5')m 2’-OMe This is an example of cap 1 RNA containing ApG. [ka]

[0235] The following is another example of cap 1 RNA. [ka]

[0236] UTR The term "untranslated region" or "UTR" refers to a region within a DNA molecule that is transcribed but not translated into an amino acid sequence, or the corresponding region within an RNA molecule such as an mRNA molecule. Untranslated regions (UTRs) can be located on the 5' side (upstream) (5'-UTR) and / or the 3' side (downstream) (3'-UTR) of an open reading frame.

[0237] The 3'-UTR, if present, is located at the 3' end of a gene, downstream of the stop codon in the protein-coding region; however, the term "3'-UTR" preferably does not include the poly(A) tail. Therefore, the 3'-UTR is upstream of the poly(A) tail (if present), for example, directly adjacent to the poly(A) tail.

[0238] The 5'-UTR, if present, is located at the 5' end of a gene, upstream of the start codon of the protein-coding region. The 5'-UTR is downstream of the 5' cap, for example, directly adjacent to the 5' cap.

[0239] The 5' and / or 3' untranslated regions can be functionally linked to the open reading frame, according to this disclosure, such that these regions associate with the open reading frame in a manner that enhances the stability and / or translation efficiency of the RNA containing the open reading frame.

[0240] In some embodiments, the RNA molecule according to this disclosure comprises a 5'-UTR and / or a 3'-UTR.

[0241] UTRs are involved in RNA stability and translation efficiency. In addition to the structural modifications relating to the 5' cap and / or 3' poly(A) tail described herein, both can be improved by selecting specific 5' and / or 3' untranslated regions (UTRs). Sequence elements within UTRs are generally understood to affect translation efficiency (primarily the 5'-UTR) and RNA stability (primarily the 3'-UTR). The presence of active 5'-UTRs is preferable to enhance RNA translation efficiency and / or stability. The presence of active 3'-UTRs is preferable, independently or further, to enhance the translation efficiency and / or stability of the RNA molecule.

[0242] With respect to a first nucleic acid sequence (e.g., UTR), the terms “active for increasing translation efficiency” and / or “active for increasing stability” mean that the first nucleic acid sequence can modify the translation efficiency and / or stability of the second nucleic acid sequence in a transcript common to the second nucleic acid sequence, such that the translation efficiency and / or stability of the second nucleic acid sequence is increased compared to the translation efficiency and / or stability of the second nucleic acid sequence in the absence of the first nucleic acid sequence.

[0243] The 5'-UTR according to this disclosure may include any combination of multiple nucleic acid sequences, optionally separated by a linker. The 3'-UTR according to this disclosure may include any combination of multiple nucleic acid sequences, optionally separated by a linker.

[0244] The term "linker" as used in this disclosure refers to a nucleic acid sequence that is added between two nucleic acid sequences in order to concatenate them. There are no particular limitations on the linker sequence.

[0245] The 3'-UTR typically has a length of 200–2000 nucleotides, e.g., 500–1500 nucleotides. The 3' untranslated region of immunoglobulin mRNA is relatively short (less than about 300 nucleotides), while that of other genes is relatively long. For example, the 3' untranslated region of tPA is about 800 nucleotides long, that of factor VIII is about 1800 nucleotides long, and that of erythropoietin is about 560 nucleotides long. The 3' untranslated region of mammalian mRNA typically has a homologous region known as the AAUAAA hexanucleotide sequence. This sequence is likely a poly(A) attachment signal and is often located 10–30 bases upstream of the poly(A) attachment site. The 3'-untranslated region may contain one or more reverse repeat sequences that can fold to give a stem-loop structure that interacts with proteins known to act as a barrier against exoribonucleases or enhance RNA stability (e.g., RNA-binding proteins).

[0246] Human β-globin 3'-UTR, particularly two consecutive identical copies of human β-globin 3'-UTR, contribute to high transcript stability and translational efficiency (Holtkamp et al., 2006, Blood 108:4009-4017). Therefore, in one embodiment, the RNA molecule according to this disclosure comprises two consecutive identical copies of human β-globin 3'-UTR. Thus, it comprises in the 5'→3' direction: (a) optionally 5'-UTR; (b) open reading frame; and (c) 3'-UTR, wherein the 3'-UTR comprises two consecutive identical copies of human β-globin 3'-UTR, a fragment thereof, or a variant or fragment of human β-globin 3'-UTR.

[0247] In one embodiment, the RNA molecule according to the Disclosure comprises human β-globin 3'-UTR, a fragment thereof, or a 3'-UTR that is not a variant or fragment of human β-globin 3'-UTR, but is active to enhance translation efficiency and / or stability.

[0248] In one embodiment, the RNA molecule according to this disclosure contains an active 5'-UTR to enhance translation efficiency and / or stability.

[0249] Poly(A) array In some embodiments, the RNA molecule according to this disclosure includes a 3'-poly(A) sequence.

[0250] According to this disclosure, in one embodiment, the poly(A) sequence contains, or essentially consists of, or consists of, at least 20, preferably at least 26, preferably at least 40, preferably at least 80, preferably at least 100, and preferably up to 500, preferably up to 400, preferably up to 300, preferably up to 200, and especially up to 150 A nucleotides, particularly about 120 A nucleotides. In this context, “essentially consists of” means that most of the nucleotides in the poly(A) sequence, typically at least 50%, preferably at least 75%, of the number of nucleotides in the “poly(A) sequence” are A nucleotides (adenylate), but the remaining nucleotides may be other nucleotides such as U nucleotides (uridylate), G nucleotides (guanylate), C nucleotides (cytidylate), etc. In this regard, “consists of” means that all nucleotides in the poly(A) sequence, i.e., 100% of the number of nucleotides in the poly(A) sequence are A nucleotides. The term "A nucleotide" or "A" refers to adenylic acid.

[0251] In fact, it has been demonstrated that a 3' poly(A) sequence of approximately 120 A nucleotides has beneficial effects on RNA levels in transfected eukaryotic cells, as well as on the levels of proteins translated from the open reading frame located upstream (5' side) of the 3' poly(A) sequence (Holtkamp et al., 2006, Blood, vol.108, pp.4009-4017).

[0252] This disclosure provides a 3' poly(A) sequence that is added during RNA transcription, i.e., during the preparation of in vitro transcription RNA, based on a DNA template containing repeating dT nucleotides (deoxythymidylate) in a strand complementary to the coding strand. The DNA sequence encoding the poly(A) sequence (coding strand) is called a poly(A) cassette.

[0253] In preferred embodiments of this disclosure, the 3' poly(A) cassette present in the coding strand of DNA is essentially composed of dA nucleotides but interrupted by a random sequence having an equal distribution of four nucleotides (dA, dC, dG, dT). Such a random sequence may be 5 to 50 nucleotides long, preferably 10 to 30 nucleotides long, and more preferably 10 to 20 nucleotides long. Such a cassette is disclosed in International Publication 2016 / 005004A1. Any poly(A) cassette disclosed in International Publication 2016 / 005004A1 may be used in this disclosure. A poly(A) cassette, essentially composed of dA nucleotides but having an equal distribution of four nucleotides (dA, dC, dG, dT) and interrupted by a random sequence having, for example, a length of 5 to 50 nucleotides, is still associated with beneficial properties at the DNA level, in terms of sustained proliferation of plasmid DNA in Escherichia coli (E. coli), and at the RNA level, in terms of supporting RNA stability and translation efficiency.

[0254] Accordingly, in some embodiments of this disclosure, the 3' poly(A) sequence contained in the RNA molecule described herein is essentially composed of A nucleotides but is interrupted by a random sequence having an equal distribution of four nucleotides (A, C, G, U). Such a random sequence may be 5 to 50 nucleotides long, preferably 10 to 30 nucleotides long, and more preferably 10 to 20 nucleotides long.

[0255] Codon usage frequency Generally, the degeneracy of the genetic code allows for the substitution of certain codons (base triplets encoding amino acids) present in an RNA sequence with other codons (base triplets) while maintaining the same coding ability (so that the substituting codon codes for the same amino acid as the codon being substituted). In some embodiments of this disclosure, at least one codon in an open reading frame contained in an RNA molecule is different from each codon in each open reading frame of the species from which the open reading frame originates. In those embodiments, the coding sequence of the open reading frame is said to be "adapted" or "modified". The coding sequence of an open reading frame contained in an RNA molecule can be adapted.

[0256] For example, when fitting the coding sequence of an open reading frame, frequently used codons can be selected: International Publication 2009 / 024567A1 describes the fitting of coding sequences of nucleic acid molecules, including the substitution of rare codons with more frequently used codons. Since codon usage frequency depends on the host cell or host organism, this type of fitting is suitable for adapting nucleic acid sequences to expression in a particular host cell or host organism. Generally speaking, more frequently used codons are typically translated more efficiently in the host cell or host organism, but fitting all codons in the open reading frame is not always necessary.

[0257] For example, when fitting the coding sequence of an open reading frame, the content of G (guanylic acid) and C (cytidylic acid) residues can be modified by selecting codons with the highest GC-rich content for each amino acid. RNA molecules with GC-rich open reading frames have been reported to potentially reduce immune activation and improve RNA translation and half-life (Thess et al., 2015, Mol.Ther.23, 1457-1465).

[0258] particle To overcome barriers to safe and effective nucleic acid delivery, nucleic acids may be administered with one or more delivery vehicles that protect the nucleic acids from degradation, maximize delivery to on-target cells, and minimize exposure to off-target cells. Such nucleic acid delivery vehicles may complex or encapsulate nucleic acids and may contain a variety of materials, including polymers and lipids. In some embodiments, such nucleic acid delivery vehicles may form particles having nucleic acids, preferably RNA.

[0259] The RNA, particularly mRNA, described herein may be present in particles comprising (i) RNA and (ii) at least one cationic compound or cationic ionizable compound, such as a polymer or lipid, that complexes the RNA. Electrostatic interactions between positively charged molecules, such as polymers and lipids, and negatively charged RNA are involved in particle formation. This results in the complexation and spontaneous formation of nucleic acid particles, particularly RNA particles.

[0260] In some embodiments, the compositions described herein contain one or more RNA molecules in the particles.

[0261] Various types of nucleic acid-containing particles have been previously described as suitable for the delivery of RNA in particle form (see, for example, Kaczmarek, JCet al., 2017, Genome Medicine 9, 60). In the case of nonviral RNA delivery vehicles, encapsulation of nucleic acids in nanoparticles can physically protect the nucleic acids from degradation and, depending on the specific chemistry, can assist in cellular uptake and endosomal extrusion.

[0262] In the context of this disclosure, the term “particle” refers to a structured entity formed by a molecule or molecular complex, in particular by a particle-forming compound. In some embodiments, the particle contains an envelope (e.g., one or more layers or lamellae) made from one or more 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 also contain further substances (e.g., further lipids) that do not need to be amphiphilic. Thus, the particle may be a monolamellar or multilamellar structure in which the substances constituting one or more layers or lamellae optionally contain one or more amphiphilic substances (in particular, selected from the group consisting of amphiphilic lipids) in combination with further substances (e.g., further lipids) that do not need to be amphiphilic. In some embodiments, the term “particle” refers to a micro-sized or nano-sized structure, e.g., a micro-sized or nano-sized compact structure. According to this disclosure, the term “particle” includes nanoparticles.

[0263] DNA and / or RNA can be delivered to target sites of interest (e.g., cells, tissues, organs) using "DNA particles," "RNA particles," or "DNA and RNA particles." DNA and / or RNA particles may be formed from lipids containing at least one cationic lipid or cationic ionizable lipid. While not intended to be bound by any theory, it is thought that cationic lipids or cationic ionizable lipids form aggregates with nucleic acids, and these aggregates result in colloidally stable particles.

[0264] The RNA particles described herein include lipid nanoparticle (LNP)-based and lipoplex (LPX)-based formulations.

[0265] The lipoplex (LPX) described herein can be obtained by mixing two aqueous phases, namely a phase containing RNA and a phase containing a lipid dispersion. In some embodiments, the lipid phase contains liposomes.

[0266] In some embodiments, liposomes are self-closed monolayer or multilayer vesicular particles, where the lamellae contain a lipid bilayer and the enclosed lumen contains an aqueous phase. A requirement for using liposomes for nanoparticle formation is that the lipids in the mixture can, if necessary, form a lamellar (bilayer) phase in the applied aqueous environment.

[0267] In some embodiments, liposomes comprise a monolayer or multilayer phospholipid bilayer surrounding an aqueous core (also referred herein as an aqueous lumen). They can be prepared from materials having polar head (hydrophilic) groups and nonpolar tail (hydrophobic) groups. In some embodiments, cationic lipids used to formulate liposomes designed for RNA delivery are inherently amphiphilic and consist of positively charged (cationic) amine head groups linked to hydrocarbon chains or cholesterol derivatives via glycerol.

[0268] In some embodiments, lipoplexes are multilayer liposome-based formulations formed during electrostatic interactions between cationic liposomes and nucleic acids. In some embodiments, the formed lipoplexes have different internal molecular arrangements resulting from the conversion of liposome structures to compact RNA-lipoplexes.

[0269] In some embodiments, LPX particles comprise amphiphilic lipids, particularly cationic amphiphilic lipids or cationic ionizable amphiphilic lipids, and RNA (particularly mRNA) as described herein. In some embodiments, electrostatic interactions between positively charged liposomes (made from one or more amphiphilic lipids, particularly cationic amphiphilic lipids or cationic ionizable amphiphilic lipids) and negatively charged RNA (particularly mRNA) result in the complexation and spontaneous formation of RNA lipoplex particles. Positively charged liposomes can generally be synthesized using cationic amphiphilic lipids or cationic ionizable amphiphilic lipids, e.g., DOTMA and / or DODMA, and optionally further lipids, e.g., DOPE or DSPC.

[0270] Generally, lipid nanoparticles (LNPs) can typically be obtained from the direct mixing of RNA 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.

[0271] In some embodiments, the LNP comprises or consists of cationic lipids / cationically ionizable lipids and helper lipids, such as phospholipids, cholesterol, and / or polymer conjugate lipids (e.g., polyethylene glycol (PEG) lipids). In some embodiments, in the RNA LNP described herein, RNA (particularly mRNA) is bound to a cationically ionizable lipid occupying the central core of the LNP. In some embodiments, polymer conjugate lipids, together with phospholipids, form the surface of the LNP. In some embodiments, cholesterol and cationically ionizable lipids in charged and uncharged forms may be distributed throughout the LNP.

[0272] In some embodiments, the RNA (e.g., mRNA) described herein may non-covalently associate with the particles described herein. In embodiments, the RNA (especially mRNA) may be attached to the outer surface of the particle (surface RNA (especially surface mRNA)) and / or contained within the particle (inclusion RNA (especially inclusion mRNA)).

[0273] In some embodiments, the particles described herein (e.g., LNPs and LPXs) are in the range of about 10 to about 2000 nm, for example, 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 up to about 1900 nm (e.g., up to about 1800 nm, up to about 1700 nm, up to about 1600 nm, up to about 1500 nm, up to about 1400 nm, up to about 1300 nm, up to about 1200 nm, up to about 1 100nm, up to approximately 1000nm, up to approximately 950nm, up to approximately 900nm, up to approximately 850nm, up to approximately 800nm, up to approximately 750nm, up to approximately 700nm, up to approximately 650nm, up to approximately 600nm, up to approximately 550nm, or up to approximately 500nm), for example, in the range of approximately 20 to approximately 1500nm, for example, approximately 30 to approximately 1200nm, approximately 40 to approximately 1100nm, approximately 50 to approximately 1000nm, approximately 60 to approximately The particles have sizes (such as diameter) in the range of 900 nm, about 70 to about 800 nm, about 80 to about 700 nm, about 90 to about 600 nm, or about 50 to about 500 nm or about 100 to about 500 nm, for example, 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, 70 to 150 nm, or 80 to 150 nm. In some embodiments, the particles described herein (e.g., LNPs and LPXs) have sizes (such as diameter) in the range of about 40 nm to about 200 nm, for example, about 50 nm to about 180 nm, about 60 nm to about 160 nm, about 80 nm to about 150 nm, or about 80 nm to about 120 nm.

[0274] In some embodiments, the particles described herein (e.g., LNP and LPX) are, in some embodiments, approximately 50 nm to approximately 1000 nm, approximately 50 nm to approximately 800 nm, approximately 50 nm to approximately 700 nm, approximately 50 nm to approximately 600 nm, approximately 50 nm to approximately 500 nm, approximately 50 nm to approximately 450 nm, approximately 50 nm to approximately 400 nm, approximately 50 nm to approximately 350 nm, and approximately 50 nm to approximately 300 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 600nm, 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 250nm, Approximately 100nm to approximately 200nm, approximately 150nm to approximately 1000nm, approximately 150nm to approximately 800nm, approximately 150nm to approximately 700nm, approximately 150nm to approximately 600nm, approximately 150nm to approximately 500nm , 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 150nm to about 200nm , have an average diameter in the range of approximately 200 nm to approximately 1000 nm, approximately 200 nm to approximately 800 nm, approximately 200 nm to approximately 700 nm, approximately 200 nm to approximately 600 nm, approximately 200 nm to approximately 500 nm, approximately 200 nm to approximately 450 nm, approximately 200 nm to approximately 400 nm, approximately 200 nm to approximately 350 nm, approximately 200 nm to approximately 300 nm, approximately 200 nm to approximately 250 nm, or approximately 80 nm to approximately 150 nm. In some embodiments, the particles described herein (e.g., LNPs and LPXs) have an average diameter in the range of approximately 40 nm to approximately 200 nm, for example, approximately 50 nm to approximately 180 nm, approximately 60 nm to approximately 160 nm, approximately 80 nm to approximately 150 nm, or approximately 80 nm to approximately 120 nm.

[0275] RNA particles (especially mRNA particles) described herein may exhibit polydispersity index (PDI) less than about 0.5, less than about 0.4, less than about 0.3, less than about 0.2, less than about 0.1, or less than about 0.05. For example, RNA particles may exhibit polydispersity index in the range of about 0.01 to about 0.4 or about 0.1 to about 0.3.

[0276] 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. Since nitrogen atoms are typically positively charged (pH-dependent) and phosphate groups are negatively charged, this correlates with the charge ratio. The N / P ratio, when charge equilibrium exists, is pH-dependent. Because positively charged nanoparticles may be favorable for transfection, lipid formulations can be formed with N / P ratios greater than 4 and up to 12. In this case, the RNA is considered to be completely bound to the nanoparticles.

[0277] This disclosure describes compositions comprising RNA (particularly mRNA) and at least one cationic lipid or cationic ionizable lipid that associates with the RNA to form RNA particles, and formulations comprising such particles. The RNA particles may include RNA complexed in various forms by non-covalent interactions with the particles. The particles described herein are not viral particles, in particular infectious viral particles; that is, they cannot virally infect cells.

[0278] Suitable cationic lipids or cationic ionizable lipids that form RNA particles are included in the term “particle-forming components” or “particle-forming agents.” The term “particle-forming components” or “particle-forming agents” refers to any component that associates with RNA to form RNA particles. Such components include any component that may be part of an RNA particle.

[0279] In some embodiments, RNA particles (particularly mRNA particles) comprise two or more RNA molecules, and the molecular parameters of the RNA molecules may be similar or different from one another, such as molar mass or basic structural elements such as molecular structure, capping, coding region, or other features.

[0280] In particulate formulations, each RNA species can be formulated separately as an individual particulate formulation. In this case, each individual particulate formulation contains one RNA species. Each individual particulate formulation may exist as a distinct entity, for example, in a separate container. Such formulations can be obtained by providing each RNA species separately (typically in the form of RNA-containing solutions) together with a particle-forming agent, thereby enabling particle formation. Each particle contains only the specific RNA species provided when the particle is formed (individual particulate formulation). In some embodiments, a composition, such as a pharmaceutical composition, comprises two or more individual particulate formulations. Each pharmaceutical composition is called a mixed particulate formulation. A mixed particulate formulation according to this disclosure can be obtained by forming the individual particulate formulations separately and then mixing the individual particulate formulations. The mixing step can yield a formulation containing a mixed population of RNA-containing particles. The individual particle populations may exist together in a single container containing the mixed population of individual particulate formulations. Alternatively, all RNA species of a pharmaceutical composition can be formulated together as a combined particulate formulation. Such formulations can be obtained by providing a combined formulation (typically a combined solution) of all RNA species together with a particle-forming agent, thereby enabling particle formation. In contrast to mixed particle formulations, combination particle formulations typically contain particles with two or more RNA species. In combination particle compositions, different RNA species typically coexist within a single particle.

[0281] polymer Polymers are commonly used materials for nanoparticle-based delivery due to their high degree of chemical flexibility. Typically, cationic polymers are used to electrostatically condense negatively charged RNA into particles, particularly nanoparticles. These positively charged groups often consist of amines that change their protonation state in the pH range of 5.5–7.5, which is thought to lead to ionic imbalances resulting in endosomal rupture. Polymers such as poly-L-lysine, polyamidoamines, protamines, and polyethyleneimines, 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-aminoesters) have become widely used in nucleic acid delivery, particularly due to their ease of synthesis and biodegradability. Such synthetic polymers are also suitable as cationic polymers herein.

[0282] As used herein, “polymer” is given its usual meaning, namely a molecular structure comprising one or more repeating units (monomers) linked by covalent bonds. All repeating units may be identical, or, in some cases, two or more different types of repeating units may be present within the polymer. In some cases, the polymer is of biological origin, i.e., a biopolymer such as a protein. In some cases, further parts, such as a targeted moiety, may also be present within the polymer.

[0283] A polymer is said to be a "copolymer" if it contains two or more types of repeating units. It should be understood that the polymers used herein may be copolymers. The repeating units forming a copolymer can be arranged in any way. For example, the repeating units may be arranged in a random order, an alternating order, or as a "block" copolymer, i.e., such that each of the following includes one or more regions containing a first repeating unit (e.g., a first block) and one or more regions containing a second repeating unit (e.g., a second block). A block copolymer may have two (diblock copolymer), three (triblock copolymer), or more distinct blocks.

[0284] In certain embodiments, the polymer is biocompatible. Biocompatible polymers are typically polymers that do not cause significant cell death at moderate concentrations. In certain embodiments, the biocompatible polymer is biodegradable, meaning the polymer can be chemically and / or biologically degraded in a physiological environment, such as inside the body.

[0285] In certain embodiments, the polymer may be a protamine or a polyalkyleneimine.

[0286] The term "protamine" refers to any of several relatively low molecular weight strongly basic proteins that are rich in arginine and found in the sperm cells of various animals (such as fish), particularly associating with DNA in place of somatic histones. Specifically, the term "protamine" refers to proteins found in fish sperm that are strongly basic, water-soluble, do not coagulate with heat, and primarily produce arginine upon hydrolysis. In purified form, they are used in long-acting insulin formulations and to neutralize the anticoagulant effect of heparin.

[0287] As used herein, the term “protamine” is intended to include any protamine amino acid sequence, fragment thereof, and polymeric forms of such amino acid sequence or fragment thereof, as well as artificial, specifically designed for a particular purpose, unisolated (synthesized) polypeptides from natural or biological sources.

[0288] In one embodiment, the polyalkyleneimine includes polyethyleneimine and / or polypropyleneimine, preferably polyethyleneimine. The preferred polyalkyleneimine is polyethyleneimine (PEI). The average molecular weight of PEI is preferably 0.75 × 10² to 10⁷ Da, preferably 1000 to 10⁵ Da, more preferably 10000 to 40000 Da, more preferably 15000 to 30000 Da, and even more preferably 20000 to 25000 Da.

[0289] According to this disclosure, linear polyalkyleneimines such as linear polyethyleneimine (PEI) are preferred.

[0290] The cationic polymers intended for use herein (including polycationic polymers) include any cationic polymers that can electrostatically bind to nucleic acids. In one embodiment, the cationic polymers intended for use herein include any cationic polymer to which nucleic acids can associate, for example, by forming a complex with nucleic acids or by forming vesicles in which nucleic acids are encapsulated or enclosed.

[0291] The particles described herein may also include polymers other than cationic polymers, namely non-cationic polymers and / or anionic polymers. Collectively, anionic polymers and neutral polymers are referred to herein as non-cationic polymers.

[0292] 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 both hydrophobic and hydrophilic moieties are also often referred to as amphiphilic substances. Lipids are typically insoluble or sparingly soluble in water, but soluble in many organic solvents. In aqueous environments, their amphiphilic nature allows the molecules to self-assemble into organized structures and various phases. One of these phases consists of a lipid bilayer, if they exist in vesicles, multilayer / monolayer liposomes, or membranes in aqueous environments. Hydrophobicity can be conferred by the presence of long-chain saturated and unsaturated aliphatic hydrocarbon groups, as well as nonpolar groups, including but not limited to those substituted with one or more aromatic, alicyclic, or heterocyclic groups. Hydrophilic groups may include polar and / or charged groups, including carbohydrates, phosphate groups, carboxylic acid groups, sulfate groups, amino groups, sulfhydryl groups, nitro groups, hydroxyl groups, and other similar groups.

[0293] As used herein, the term “hydrophobic” refers to any molecule, part, or group that is substantially immiscible or insoluble in aqueous solution. The term hydrophobic group includes hydrocarbons having at least six carbon atoms. Monovalent radicals of hydrocarbons are referred to herein as hydrocarbyls. Hydrophobic groups may have functional groups (e.g., ethers, esters, halides, etc.) as well as atoms other than carbon and hydrogen, insofar as they satisfy the condition of being substantially immiscible or insoluble in aqueous solution.

[0294] The term "hydrocarbon" includes acyclic, e.g., linear or branched hydrocarbyl groups, e.g., alkyl, alkenyl, or alkynyl groups as defined herein. It should be understood that one or more hydrogen atoms in an alkyl, alkenyl, or alkynyl group may be substituted with other atoms, e.g., halogen, oxygen, or sulfur. Unless otherwise specified, hydrocarbon groups may also include cyclic (alkyl, alkenyl, or alkynyl) groups or aryl groups, as long as the overall polarity of the hydrocarbon remains relatively nonpolar.

[0295] The term "alkyl" refers to a saturated linear or branched monovalent hydrocarbon moiety that may have 1 to 30, typically 1 to 20, and often 6 to 18 carbon atoms. Examples of nonpolar alkyl groups include, but are not limited to, methyl, ethyl, propyl, isopropyl, hexyl, decyl, dodecyl, tetradecyl, hexadecyl, and octadecyl.

[0296] The term "alkenyl" refers to a linear or branched monovalent hydrocarbon moiety having at least one carbon-carbon double bond, with a total carbon atom count of 6 to 30, typically 6 to 20, and often 6 to 18. Generally, the maximum number of carbon-carbon double bonds in an alkenyl group can be equal to an integer calculated by dividing the number of carbon atoms in the alkenyl group by 2, rounding down the result of the division to the smaller integer if the number of carbon atoms in the alkenyl group is odd. For example, for an alkenyl group with 9 carbon atoms, the maximum number of carbon-carbon double bonds is 4. Preferably, an alkenyl group has 1 to 6 (e.g., 1 to 4), i.e., 1, 2, 3, 4, 5, or 6 carbon-carbon double bonds.

[0297] The term "alkynyl" refers to a linear or branched monovalent hydrocarbon moiety having at least one carbon-carbon triple bond, with a total carbon atom count of 6 to 30, typically 6 to 20, and often 6 to 18. An alkynyl group may have one or more carbon-carbon double bonds. Generally, the maximum number of carbon-carbon triple bonds in an alkynyl group can be equal to an integer calculated by dividing the number of carbon atoms in the alkynyl group by 2, rounding down the result of the division to the smaller integer if the number of carbon atoms in the alkynyl group is odd. For example, for an alkynyl group with 9 carbon atoms, the maximum number of carbon-carbon triple bonds is 4. Preferably, an alkynyl group has 1 to 6 (e.g., 1 to 4), i.e., 1, 2, 3, 4, 5, or 6, more preferably 1 or 2 carbon-carbon triple bonds.

[0298] The term "alkylene" refers to a saturated linear or branched divalent hydrocarbon moiety that may have 1 to 30, typically 2 to 20, and often 4 to 12 carbon atoms. Exemplary nonpolar alkylene groups include, but are not limited to, methylene, ethylene, trimethylene, hexamethylene, decamethylene, dodecamethylene, tetradecamethylene, hexadecamethylene, and octadecamethylene.

[0299] The term "alkenylene" refers to a linear or branched divalent hydrocarbon moiety having at least one carbon-carbon double bond, with a total carbon atom count of 2 to 30, typically 2 to 20, and often 4 to 12. Generally, the maximum number of carbon-carbon double bonds in an alkenylene group can be equal to an integer calculated by dividing the number of carbon atoms in the alkenylene group by 2, rounding down the result of the division to the smaller integer if the number of carbon atoms in the alkenylene group is odd. For example, for an alkenylene group with 9 carbon atoms, the maximum number of carbon-carbon double bonds is 4. Preferably, an alkenylene group has 1 to 6 (e.g., 1 to 4), i.e., 1, 2, 3, 4, 5, or 6 carbon-carbon double bonds.

[0300] The term "cycloalkyl" preferably refers to a cyclic non-aromatic version of "alkyl" and "alkenyl" having 3 to 14 carbon atoms, for example 3 to 12 or 3 to 10 carbon atoms, i.e., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 carbon atoms (for example 3, 4, 5, 6, 7, 8, 9 or 10 carbon atoms), more preferably 3 to 7 carbon atoms. Examples of cycloalkyl groups include cyclopropyl, cyclopropenyl, cyclobutyl, cyclobutenyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cycloheptyl, cycloheptenyl, cyclooctyl, cyclooctenyl, cyclononyl, cyclononenyl, cyclodecyl, cyclodecenyl, and adamantyl. Cycloalkyl groups may consist of one ring (monocyclic), two rings (bicyclic), or three or more rings (polycyclic).

[0301] The term "aryl" refers to a monoradical of an aromatic cyclic hydrocarbon. Preferably, the aryl group contains 3 to 14 carbon atoms (e.g., 5, 6, 7, 8, 9, or 10) that can be arranged in one ring (e.g., phenyl) or two or more fused rings (e.g., naphthyl). Exemplary aryl groups include cyclopropenilium, cyclopentadienyl, phenyl, indenyl, naphthyl, azlenyl, fluorenyl, anthryl, and phenanthryl. Preferably, "aryl" refers to a monocyclic ring containing 6 carbon atoms or an aromatic bicyclic ring system containing 10 carbon atoms. Preferred examples are phenyl and naphthyl. The term aryl does not include fullerenes.

[0302] In the context of hydrocarbons, the term "aromatic" means that the entire molecule must be aromatic. For example, when a monocyclic aryl is hydrogenated (partially or completely), the resulting hydrogenated cyclic structure is classified as a cycloalkyl for the purposes of this disclosure. Similarly, when a bicyclic or polycyclic aryl (such as naphthyl) is hydrogenated, the resulting hydrogenated bicyclic or polycyclic structure (such as 1,2-dihydronaphthyl) is classified as a cycloalkyl for the purposes of this disclosure (even if one ring remains aromatic, as in 1,2-dihydronaphthyl).

[0303] As used herein, the term “amphiphilic” refers to a molecule having both a polar and a nonpolar moiety. Often, amphiphilic compounds have a polar head attached to a long hydrophobic tail. In some embodiments, the polar moiety is soluble in water, while the nonpolar moiety is insoluble in water. Furthermore, the polar moiety may have either a formal positive charge or a formal negative charge. Alternatively, the polar moiety may have both a formal positive and a formal negative charge, and may be a zwitterion or an intramolecular salt. For the purposes of this disclosure, amphiphilic compounds may be, but are not limited to, one or more natural or non-natural lipids and lipid-like compounds.

[0304] The terms “lipid-like substance,” “lipid-like compound,” or “lipid-like molecule” refer to substances that are structurally and / or functionally related to lipids but cannot be considered lipids in the strict sense, particularly amphiphilic substances. For example, this term includes compounds that can form amphiphilic layers when present in vesicles, multilayer / monolayer liposomes, or membranes in an aqueous environment, and includes surfactants or synthetic compounds having both hydrophilic and hydrophobic parts. Generally speaking, this term includes molecules that have hydrophilic and hydrophobic parts with different structural arrangements, which may or may not be similar to the structure of lipids. Examples of lipid-like compounds that can be spontaneously incorporated 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 containing two long alkyl chains and a polar head group are generally cylindrical. The area occupied by the two alkyl chains is similar to the area occupied by the polar head group. Such lipids have low solubility as monomers and tend to aggregate into planar bilayers that are insoluble in water. Conventional surfactant monomers, which consist of only one linear alkyl chain and a hydrophilic head group, are generally conical. The hydrophilic head group tends to occupy more molecular space than the linear alkyl chain. In some embodiments, surfactants tend to aggregate into spherical or elliptical micelles that are water-soluble. Lipids also have the same general structure as surfactants—a polar hydrophilic head group and a nonpolar hydrophobic tail—but lipids differ from surfactants in monomer shape, the type of aggregates formed in solution, and the concentration range required for aggregation. As used herein, the term “lipid” should be interpreted to encompass both lipids and lipid-like substances unless otherwise specifically indicated herein or unless it is clearly inconsistent with the context.

[0305] 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 prenolipids (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 encompass molecules such as fatty acids and their derivatives (including triglycerides, diglycerides, monoglycerides, and phospholipids), as well as steroids, i.e., sterol-containing metabolites such as cholesterol or its derivatives. Examples of cholesterol derivatives include, but are not limited to, cholestanol, cholestanone, cholestenone, coprostanol, cholesteryl-2'-hydroxyethyl ether, cholesteryl-4'-hydroxybutyl ether, tocopherol and their derivatives, and mixtures thereof.

[0306] Fatty acids, or fatty acid residues, are a diverse group of molecules consisting of hydrocarbon chains with carboxylic acid groups at their ends; this arrangement confers a polar, hydrophilic end and a water-insoluble, nonpolar, hydrophobic end to the molecule. Typically 4–24 carbon chains, they may be saturated or unsaturated and bonded to functional groups including oxygen, halogens, nitrogen, and sulfur. If a fatty acid contains a double bond, it can be either cis or trans geometric isomerized, which significantly affects the molecule's stereochemistry. A cis double bond bends the fatty acid chain, an effect that occurs when multiple cis double bonds combine in the chain. Other major lipid classes in the fatty acid category are fatty acid esters and fatty acid amides.

[0307] Glycerolipids are composed of monosubstituted, disubstituted, and trisubstituted glycerols, the most well known being fatty acid triesters of glycerol called triglycerides. The term "triacylglycerol" is sometimes used synonymously with "triglyceride." In these compounds, each of the three hydroxyl groups of glycerol is typically esterified by a different fatty acid. 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 bonds.

[0308] Glycerophospholipids are amphiphilic molecules (containing both hydrophobic and hydrophilic regions) that contain a glycerol core, which is bonded to two fatty acid-derived "tails" by ester bonds and to a single "head" group by a phosphate ester bond. Examples of glycerophospholipids commonly referred to as phospholipids (although sphingomyelin is also classified as a phospholipid) include phosphatidylcholine (also known as PC, GPCho, or lecithin), phosphatidylethanolamine (PE or GPEtn), and phosphatidylserine (PS or GPSer).

[0309] Sphingolipids are a complex family of compounds that share a common structural feature: a sphingoid base skeleton. The major sphingoid base in mammals is generally called sphingosine. Ceramides (N-acyl-sphingoid bases) are a major subclass of sphingoid base derivatives containing amide-linked fatty acids. These fatty acids are typically saturated or monounsaturated, with chain lengths of 16–26 carbon atoms. The major sphingophospholipid in mammals is sphingomyelin (ceramidephosphocholine), while insects primarily contain ceramidephosphoethanolamine, and fungi have phytoceramidephosphoinositol and mannose-containing head groups. Sphingoglycolipids are a diverse family of molecules composed of one or more sugar residues linked to a sphingoid base via glycosidic bonds. Examples include simple and complex sphingoglycolipids such as cerebrosides and gangliosides.

[0310] Sterol lipids, such as cholesterol and its derivatives, or tocopherol and its derivatives, are important components of membrane lipids, along with glycerophospholipids and sphingomyelin.

[0311] Saccharolipids are compounds in which fatty acids are directly linked to a sugar backbone, forming a structure compatible with the membrane bilayer. In saccharolipids, monosaccharides replace the glycerol backbone found in glycerolipids and glycerophospholipids. The best-known saccharolipid is the acylated glucosamine precursor of lipid A component of the lipopolysaccharide in Gram-negative bacteria. A typical lipid A molecule is a glucosamine disaccharide derivatized by as many as seven fatty acyl chains. The minimum lipopolysaccharide required for growth in E. coli is Kdo2-lipid A, a hexaacylated disaccharide of glucosamine glycosylated by two 3-deoxy-D-mann-octurosone (Kdo) residues.

[0312] Polyketides are synthesized by polymerization of acetyl and propionyl subunits using classical enzymes, as well as repeating and multimodular enzymes that share mechanistic features with fatty acid synthases. They exhibit great structural diversity, encompassing numerous secondary metabolites and natural products from animal, plant, bacterial, fungal, and marine sources. Many polyketides are cyclic molecules whose backbone is often further modified by glycosylation, methylation, hydroxylation, oxidation, or other processes.

[0313] According to this disclosure, lipids and lipid-like substances may be cationic, anionic, or neutral. Neutral lipids or lipid-like substances exist in uncharged or neutral zwitterionic forms at a selected pH.

[0314] Cationic lipids / Cationic ionizable lipids In some embodiments, the RNA compositions and formulations and nucleic acid particles described herein include at least one cationic lipid or cationic ionizable lipid as a particle-forming agent. The cationic lipid or cationic ionizable lipid intended for use herein includes any cationic lipid or cationic ionizable lipid (including lipid-like substances) that can electrostatically bind to nucleic acids. In some embodiments, the cationic lipid or cationic ionizable lipid intended for use herein can associate with nucleic acids, for example, by forming a complex with the nucleic acid or by forming a vesicle in which the nucleic acid is encapsulated or enclosed.

[0315] 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. Generally, cationic lipids have a lipophilic moiety such as a sterol, acyl chain, diacyl chain or more, and the lipid head group is typically positively charged.

[0316] In some embodiments, cationic lipids have a net positive charge only at specific pH levels, particularly acidic pH levels, but at different pH levels, preferably higher pH levels such as physiological pH, they preferably have no net positive charge, and preferably are chargeless, i.e., neutral. This ionizable behavior is thought to enhance efficacy by facilitating endosomal escape and reducing toxicity compared to particles that remain cationic at physiological pH.

[0317] As used herein, “cationic ionizable lipid” refers to a lipid or lipid-like substance that is either positively charged or neutral, i.e., not permanently cationic. Therefore, depending on the pH of the composition in which the cationic ionizable lipid is dissolved, the cationic ionizable lipid may be either positively charged or neutral. For the purposes of this disclosure, cationic ionizable lipids are encompassed within the term “cationic lipid” unless inconsistent with the context.

[0318] In some embodiments, the cationic lipid or cationic ionizable lipid includes a head group comprising at least one nitrogen atom (N) that is positively charged or protonable, for example, under physiological conditions.

[0319] Examples of cationic lipids or cationic ionizable lipids include N,N-dimethyl-2,3-dioleyloxypropylamine (DODMA), 1,2-dioleoyl-3-trimethylammoniumpropane (DOTAP); 1,2-di-O-octadecenyl-3-trimethylammoniumpropane (DOTMA), 3-(N-(N',N'-dimethylaminoethane)-carbamoyl)cholesterol (DC-Chol), dimethyldioctadecylammonium (DDAB); 1,2-dioleoyl-3-dimethylammonium-propane (DODA P); 1,2-Diacyloxy-3-dimethylammoniumpropane; 1,2-Dialkyloxy-3-dimethylammoniumpropane; Dioctadecyldimethylammonium chloride (DODAC), 1,2-Distearyloxy-N,N-dimethyl-3-aminopropane (DSDMA), 2,3-Di(tetradecoxy)propyl-(2-hydroxyethyl)-dimethylazanium (DMRIE), 1,2-Dimiristoyl-sn-glycero-3-ethylphosphocholine (DMEPC), l,2-Dimiristoyl-3-trimethylammoniumpropane (DMTAP), 1,2-dioleyloxypropyl-3-dimethyl-hydroxyethylammonium bromide (DORIE), and 2,3-dioleoyloxy-N-[2(sperminecarboxamide)ethyl]-N,N-dimethyl-l-propanamium trifluoroacetate (DOSPA), 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 1,2-dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA), dioctadecylamideglycylspermine (DOGS), 3-dimethylamino -2-(cholest-5-ene-3-beta-oxybutane-4-oxy)-1-(cis,cis-9,12-octadecadieneoxy)propane (CLinDMA), 2-[5'-(cholest-5-ene-3-beta-oxy)-3'-oxapentoxy)-3-dimethyl-1-(cis,cis-9',12'-octadecadieneoxy)propane (CpLinDMA), N,N-dimethyl-3,4-dioleyloxybenzylamine (DMOBA), 1,2-N,N'-dioleylcarbamyl-3-dimethylaminopropane (DOcarbDAP), 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-dimethylaminoethyl-[1,3]-Dioxolane (DLin-K-XTC2-D MA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA), heptatriaconta-6,9,28,31-tetraen-19-yl-4-(dimethylamino)butanoate (DLin-MC3-DMA), N-(2-hydroxyethyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)-1-propanaminonium bromide (DMRIE), (±)-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(dodecyloxy)-1-propanaminium bromide (GAP-DLRIE), (±)-N-(3-aminopropyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)-1-propanaminium bromide (GAP-DMRIE), N-(2-aminoethyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)-1-propanami Nium bromide (βAE-DMRIE), N-(4-carboxybenzyl)-N,N-dimethyl-2,3-bis(oleoyloxy)propane-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)propane-1-amine (octyl-CLinDMA), 1,2-dimyristoyl-3-dimethylammoniumpropane (DMDAP), 1,2-Dipalmitoyl-3-dimethylammonium propane (DPDAP), N1-(2-((1S)-1-[(3-aminopropyl)amino]-4-[di(3-amino-propyl)amino]butylcarboxamide)ethyl]-3,4-di[oleyloxy]-benzamide (MVL5), 1,2-Dioleoyl-sn-glycero-3-ethylphosphocholine (DOEPC), 2,3-Bis(dodecyloxy)-N-(2-hydroxyethyl (Tyl)-N,N-dimethylpropane-1-amonium bromide (DLRIE), N-(2-aminoethyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)propane-1-amonium bromide (DMORIE), di((Z)-non-2-en-1-yl)8,8'-((((2(dimethylamino)ethyl)thio)carbonyl)azandiyl)dioctanoate (ATX), N,N-dimethyl-2,3-bis(dodecyloxy (C)propan-1-amine (DLDMA), N,N-dimethyl-2,3-bis(tetradecyloxy)propan-1-amine (DMDMA), di((Z)-non-2-en-1-yl)-9-((4-(dimethylaminobutanoyl)oxy)heptadecanedioate (L319), N-dodecyl-3-((2-dodecylcarbamoyl-ethyl)-{2-[(2-dodecylcarbamoyl-ethyl)-2-{(2-dodecylcarbamoyl Examples include, but are not limited to, ethyl)-[2-(2-dodecylcarbamoyl-ethylamino)-ethyl]-amino}-ethylamino)propionamide (Lipidoid 98N12-5) and 1-(2-[bis(2-hydroxydodecyl)amino]ethyl-(2-[4-[2-[bis(2-hydroxydodecyl)amino]ethyl]piperazine-1-yl]ethyl]amino]dodecane-2-ol (Lipidoid C12-200).

[0320] In some embodiments, the cationic lipid or cationic ionizable lipid is DOTMA. In some embodiments, the cationic lipid or cationic ionizable lipid is DODMA.

[0321] DOTMA is a cationic lipid having a quaternary amine head group. The structure of DOTMA can be represented as follows: [ka]

[0322] DODMA is an ionizable cationic lipid having a tertiary amine head group. The structure of DODMA can be represented as follows: [ka]

[0323] In some embodiments, cationic lipids or cationic ionizable lipids may constitute about 10 mol% to about 95 mol%, about 20 mol% to about 95 mol%, about 20 mol% to about 90 mol%, about 30 mol% to about 90 mol%, about 40 mol% to about 90 mol%, or about 40 mol% to about 80 mol% of the total lipids present in the particles.

[0324] Further lipids The RNA compositions and formulations and RNA particles described herein may also contain lipids other than cationic lipids 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). Collectively, anionic lipids and neutral lipids or lipid-like substances are referred to herein as non-cationic lipids. By optimizing the formulation of RNA particles by adding other hydrophobic moieties such as cholesterol and lipids in addition to cationic lipids or cationic ionizable lipids, particle stability and nucleic acid delivery effectiveness can be enhanced.

[0325] One or more additional lipids may or may not affect the overall charge of the RNA particles. In some embodiments, one or more additional lipids are noncationic lipids or lipid-like substances. Noncationic 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.

[0326] In some embodiments, the RNA compositions and formulations and RNA particles described herein include cationic lipids or cationic ionizable lipids and one or more further lipids.

[0327] While we do not wish to be bound by theory, the amount of cationic lipids or cationically ionizable lipids compared to the amount of one or more additional lipids may affect important RNA particle properties such as RNA charge, particle size, stability, tissue selectivity, and biological activity. Therefore, in some embodiments, the molar ratio of cationic lipids or cationically ionizable lipids 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.

[0328] In some embodiments, one or more additional lipids contained in the RNA compositions and formulations and RNA particles described herein include one or more neutral lipids, steroids, and combinations thereof.

[0329] In some embodiments, one or more further lipids include neutral lipids that are phospholipids. In some embodiments, the phospholipids are selected from the group consisting of phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidic acid, phosphatidylserine, and sphingomyelin. Specific phospholipids that can be used include, but are not limited to, phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidic acid, phosphatidylserine, or sphingomyelin.Such phospholipids include, in particular, diacylphosphatidylcholine, such as distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dimyristoylphosphatidylcholine (DMPC), dipentadecanoylphosphatidylcholine, dilauroylphosphatidylcholine, dipalmitoylphosphatidylcholine (DPPC), diarachidoylphosphatidylcholine (DAPC), and dibehenoylphosphatidylcholine (D BPC), ditricosanoylphosphatidylcholine (DTPC), dilignoceroylphatidylcholine (DLPC), palmitoyloleoylphosphatidylcholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 diether PC), 1-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC) and phosphatidylethanolamine, especially diacylphosphatidylethanolamine, such as dioleoylphosphatidylethanolamine (DOPE), distearoylphosphatidylethanolamine (DSPE), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphatidylethanolamine (DMPE), dilauroylphosphatidylethanolamine (DLPE), and diphytanoylphosphatidylethanolamine (D Examples include PyPE), 1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphocholine (DOPG), 1,2-dipalmitoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (DPPG), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), N-palmitoyl-D-erythro-sphingosylphosphorylcholine (SM), and further, phosphatidylethanolamine lipids having different hydrophobic chains. In some embodiments, the neutral lipid is selected from the group consisting of DSPC, DOPC, DMPC, DPPC, POPC, DOPE, DOPG, DPPG, POPE, DPPE, DMPE, DSPE, and SM.In some embodiments, the neutral lipid is selected from the group consisting of DSPC, DPPC, DMPC, DOPC, POPC, DOPE, and SM. In some embodiments, the neutral lipid is DSPC. In some embodiments, the neutral lipid is DOPE.

[0330] In some embodiments, the further lipids include one of the following: (1) phospholipids, (2) cholesterol or its derivatives, or (3) mixtures of phospholipids and cholesterol or its derivatives. Examples of cholesterol derivatives include, but are not limited to, cholestanol, cholestanone, cholestenone, coprostanol, cholesteryl-2'-hydroxyethyl ether, cholesteryl-4'-hydroxybutyl ether, tocopherol and its derivatives, and mixtures thereof.

[0331] Accordingly, in some embodiments, the RNA compositions and formulations and RNA particles described herein include (1) cationic or cationic ionizable lipids and phospholipids such as DSPC or DOPE, or (2) cationic or cationic ionizable lipids and phospholipids such as DSPC or DOPE, and cholesterol.

[0332] In some embodiments, the RNA particles described herein (particularly those containing mRNA) include (1) DOTMA and DOPE, (2) DOTMA, DOPE and cholesterol, (3) DODMA and DOPE, or (4) DODMA, DOPE and cholesterol.

[0333] DSPC is a neutral phospholipid. The structure of DSPC can be represented as follows: [ka]

[0334] DOPE is a neutral phospholipid. The structure of DOPE can be represented as follows: [ka]

[0335] The structure of cholesterol can be represented as follows: [ka]

[0336] In some embodiments, the RNA compositions and formulations and RNA particles described herein do not contain polymer conjugate lipids such as pegylated lipids. The term "pegylated lipid" refers to a molecule that includes both a lipid moiety and a polyethylene glycol moiety. Pegylated lipids are known in the art.

[0337] In some embodiments, additional lipids (e.g., one or more phospholipids and / or cholesterol) may constitute about 0 mol% to about 90 mol%, about 0 mol% to about 80 mol%, about 2 mol% to about 80 mol%, about 5 mol% to about 80 mol%, about 5 mol% to about 60 mol%, about 5 mol% to about 50 mol%, about 7.5 mol% to about 50 mol%, or about 10 mol% to about 40 mol% of the total lipids present in the particles. In some embodiments, additional lipids (e.g., one or more phospholipids and / or cholesterol) may constitute about 10 mol%, about 15 mol%, or about 20 mol% of the total lipids present in the particles.

[0338] In some embodiments, the further lipids include a mixture of (i) phospholipids such as DOPE and (ii) cholesterol or a derivative thereof. In some embodiments, the molar ratio of phospholipids such as DOPE to cholesterol or a derivative thereof is about 9:0 to about 1:10, about 2:1 to about 1:4, about 1:1 to about 1:4, or about 1:1 to about 1:3.

[0339] Polymer-conjugated lipids In some embodiments, the RNA compositions and formulations and RNA particles described herein may contain at least one polymer-conjugate lipid. A polymer-conjugate lipid is typically a molecule comprising a lipid moiety and a polymer moiety conjugated thereto. In some embodiments, the polymer-conjugate lipid is a PEG-conjugate lipid, also referred herein as a pegylated lipid or PEG lipid. The term "pegylated lipid" refers to a molecule comprising both a lipid moiety and a polyethylene glycol moiety. Pegylated lipids are known in the art. In some embodiments, the polymer-conjugate lipid is a polysarcosine-conjugate lipid, also referred herein as a sarcosinylated lipid or pSar lipid. The term "sarcosinylated lipid" refers to a molecule comprising both a lipid moiety and a polysarcosine moiety.

[0340] In some embodiments, polymer-conjugated lipids are designed to sterically stabilize lipid particles by forming a protective hydrophilic layer that shields the hydrophobic lipid layer. In some embodiments, polymer-conjugated lipids can reduce the association of such lipid particles with serum proteins and / or the resulting uptake by the reticuloendothelial system when such lipid particles are administered in vivo.

[0341] Polyethylene glycol (PEG) conjugate lipids In some embodiments, the RNA compositions / formulations and RNA particles described herein contain PEG-conjugated lipids.

[0342] In some embodiments, PEG-conjugated lipids (PEG-conjugated lipids) are lipids having the following general formula structure: [ka] (In the formula, each of R12 and R13 is independently a linear or branched alkyl or alkenyl chain containing 10 to 30 carbon atoms, where the alkyl / alkenyl chain may be interrupted by one or more ester bonds, and w has an average value in the range of 30 to 60.) or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof.

[0343] In some embodiments of this formula, each of R12 and R13 is independently a linear alkyl chain containing 10 to 18 carbon atoms, preferably 12 to 16 carbon atoms.

[0344] In some embodiments of this formula, R12 and R13 are identical. In some embodiments, each of R12 and R13 is a linear alkyl chain containing 12 carbon atoms. In some embodiments, each of R12 and R13 is a linear alkyl chain containing 14 carbon atoms. In some embodiments, each of R12 and R13 is a linear alkyl chain containing 16 carbon atoms.

[0345] In some embodiments of this formula, R12 and R13 are different. In some embodiments, one of R12 and R13 is a linear alkyl chain containing 12 carbon atoms, and the other of R12 and R13 is a linear alkyl chain containing 14 carbon atoms.

[0346] In some embodiments of this formula, w has an average value in the range of 40 to 50, for example, an average value of 45.

[0347] In some embodiments of this formula, w is in a range such that the PEG portion of the PEGylated lipid has an average molecular weight of about 400 to about 6000 g / mol, for example, about 1000 to about 5000 g / mol, about 1500 to about 4000 g / mol, or about 2000 to about 3000 g / mol. In some embodiments, each of R12 and R13 is a linear alkyl chain containing 14 carbon atoms, and w has an average value of 45.

[0348] Various PEG conjugate lipids are known in the art and include, but are not limited to, pegylated diacylglycerols (PEG-DAG), such as 1-(monomethoxy-polyethylene glycol)-2,3-dimyristoylglycerol (PEG-DMG), pegylated phosphatidylethanolamine (PEG-PE), PEG-succinate diacylglycerol (PEG-S-DAG), such as 4-O-(2',3'-di(tetradecanoyloxy)propyl-1-O-(ω-methoxy(polyethoxy)ethyl)butanediate (PEG-S-DMG), pegylated ceramide (PEG-cer), or PEG dialkoxypropyl carbamates, such as ω-methoxy(polyethoxy)ethyl-N-(2,3-di(tetradecanoxy)propyl)carbamate or 2,3-di(tetradecanoxy)propyl-N-(ω-methoxy(polyethoxy)ethyl)carbamate.

[0349] In some embodiments, the PEG conjugate lipid (PEG-conjugated lipid) is or comprises 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide. In some embodiments, the PEG-conjugated lipid has the following structure: [ka]

[0350] In some embodiments, the PEG conjugate lipid (PEGylated lipid) is, for example, DMG-PEG 2000 having the following structure. [ka]

[0351] In some embodiments, the PEG conjugate lipid (PEG-conjugated lipid) has the following structure: [ka] (wherein n has an average value in the range of 30 to 60, for example, an average value of about 50). In one embodiment, the PEG conjugate lipid (PEG-2000-C-DMA) is preferably PEG2000-C-DMA, which refers to 3-N-[(ω-methoxypoly(ethylene glycol)2000)carbamoyl]-1,2-dimyristyloxypropylamine (MPEG-(2kDa)-C-DMA) or methoxy-polyethylene glycol-2,3-bis(tetradecyloxy)propylcarbamate (2000).

[0352] In some embodiments, the RNA compositions / formulations described herein may comprise one or more PEG-conjugate lipids or PEGylated lipids described in International Publication No. 2017 / 075531 and International Publication No. 2018 / 081480, the entire contents of which are incorporated herein by reference for the purposes described herein.

[0353] In some embodiments, the pegylated lipids constitute about 1 mol% to about 10 mol%, preferably about 1 mol% to about 5 mol%, and more preferably about 1 mol% to about 2.5 mol%, of the total lipids present in the RNA composition / formulation and RNA particles described herein.

[0354] Embodiment of lipoplex particles In some embodiments of this disclosure, the RNA described herein may be present in RNA lipoplex particles.

[0355] A lipoplex (LPX) is generally an electrostatic complex formed by mixing pre-formed cationic lipid liposomes with anionic nucleic acids. The formed lipoplex has a different internal molecular arrangement resulting from the conversion from a liposome structure to a compact RNA-lipoplex.

[0356] In certain embodiments, the RNA lipoplex particles contain both cationic lipids and further lipids. In exemplary embodiments, the cationic lipid is DOTMA and the further lipid is DOPE.

[0357] In some embodiments, the molar ratio of at least one cationic lipid to at least one further 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 at least one cationic lipid to at least one further lipid is about 2:1.

[0358] The RNA 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 nm to about 700 nm, about 400 nm to about 600 nm, about 300 nm to about 500 nm, or about 350 nm to about 400 nm. In certain embodiments, RNA lipoplex particles have an average diameter of approximately 200 nm, approximately 225 nm, approximately 250 nm, approximately 275 nm, approximately 300 nm, approximately 325 nm, approximately 350 nm, approximately 375 nm, approximately 400 nm, approximately 425 nm, approximately 450 nm, approximately 475 nm, approximately 500 nm, approximately 525 nm, approximately 550 nm, approximately 575 nm, approximately 600 nm, approximately 625 nm, approximately 650 nm, approximately 675 nm, approximately 700 nm, approximately 725 nm, approximately 750 nm, approximately 775 nm, approximately 800 nm, approximately 825 nm, approximately 850 nm, approximately 875 nm, approximately 900 nm, approximately 925 nm, approximately 950 nm, approximately 975 nm, or approximately 1000 nm. In some embodiments, RNA lipoplex particles have an average diameter in the range of approximately 250 nm to approximately 700 nm. In some embodiments, the RNA lipoplex particles have an average diameter in the range of about 300 nm to about 500 nm. In exemplary embodiments, the RNA lipoplex particles have an average diameter of about 400 nm.

[0359] The RNA lipoplex particles and compositions comprising RNA lipoplex particles described herein are useful for the delivery of RNA to target tissues after parenteral administration, particularly after intravenous administration.

[0360] RNA lipoplex particles targeting the spleen are described in International Publication No. 2013 / 143683, incorporated herein by reference. It has been found that RNA lipoplex particles having a net negative charge can selectively target splenic tissue or splenic cells, such as antigen-presenting cells, particularly dendritic cells. Thus, RNA accumulation and / or RNA expression occur in the spleen after administration of RNA lipoplex particles. Therefore, the RNA lipoplex particles of this disclosure can be used to express RNA in the spleen. In one embodiment, RNA accumulation and / or RNA expression in the lungs and / or liver does not occur, or does not occur essentially, after administration of RNA lipoplex particles. In some embodiments, RNA accumulation and / or RNA expression occurs in antigen-presenting cells, such as professional antigen-presenting cells of the spleen, after administration of DNA and / or RNA lipoplex particles. Therefore, the RNA lipoplex particles of this disclosure can be used to target RNA, such as RNA encoding an antigen or at least one epitope, to the lymphoid system, particularly secondary lymphoid organs, more specifically the spleen. Targeting the lymphoid system, particularly secondary lymphoid organs, more specifically the spleen, is particularly preferred when the administered RNA is RNA encoding a vaccine antigen. In some embodiments, the target cells are spleen cells. In some embodiments, the target cells are antigen-presenting cells, such as professional antigen-presenting cells in the spleen. In some embodiments, the target cells are dendritic cells in the spleen.

[0361] The charge of the RNA lipoplex particles of this disclosure is the sum of the charges present in at least one cationic lipid and the charges present in the RNA. The charge ratio is the ratio of the positive charges present in at least one cationic lipid to the negative charges present in the RNA. The charge ratio of the positive charges present in at least one cationic lipid to the negative charges present in the RNA is calculated by the following formula: Charge ratio = [(concentration of cationic lipid (mol)) * (total number of positive charges in cationic lipid)] / [(concentration of RNA (mol)) * (total number of negative charges in RNA)]. The concentration of RNA and the amount of at least one cationic lipid can be determined by a person skilled in the art using routine methods.

[0362] In some embodiments, at physiological pH, the charge ratio of positive to negative charges in RNA lipoplex particles is approximately 1.6:2 to approximately 1:2, or approximately 1.6:2 to approximately 1.1:2. In specific embodiments, the charge ratio of positive to negative charges in RNA lipoplex particles at physiological pH is approximately 1.6:2.0, approximately 1.5:2.0, approximately 1.4:2.0, approximately 1.3:2.0, approximately 1.2:2.0, approximately 1.1:2.0, or approximately 1:2.0.

[0363] Embodiments of Lipid Nanoparticles (LNPs) In some embodiments, the RNA described herein exists in the form of lipid nanoparticles (LNPs). LNPs typically comprise four components: a cationic ionizable lipid, a neutral lipid such as a phospholipid, a steroid such as cholesterol, and a polymer conjugate lipid such as a PEG lipid. LNPs can be prepared by mixing lipids dissolved in ethanol with RNA in an aqueous buffer.

[0364] In some embodiments, in the RNA LNPs described herein, the RNA is bound to a cationic ionizable lipid that occupies the central core of the LNP. Polymer conjugate lipids, together with phospholipids, form the surface of the LNP. In some embodiments, cholesterol and cationic ionizable lipids may be distributed throughout the LNP.

[0365] In some embodiments, the LNP comprises one or more cationic ionizable lipids and one or more stabilizing lipids. The stabilizing lipids include neutral lipids and polymer conjugate lipids.

[0366] In some embodiments, the LNPs include cationic ionizable lipids, neutral lipids, steroids, polymer-conjugated lipids, and RNA encapsulated within or associated with lipid nanoparticles.

[0367] In some embodiments, the LNP contains 35-65 mol%, 40-60 mol%, 40-55 mol%, 45-55 mol%, or 45-50 mol% of cationic ionizable lipids.

[0368] In some embodiments, neutral lipids are present at concentrations ranging from 5–15 mol%, 7–13 mol%, or 9–11 mol%.

[0369] In some embodiments, the steroid is present at concentrations ranging from 30–50 mol%, 30–45 mol%, 35–45 mol%, or 35–43 mol%.

[0370] In some embodiments, the LNP contains 1-10 mol%, 1-5 mol%, or 1-2.5 mol% of polymer-conjugated lipids.

[0371] In some embodiments, the LNP comprises 45–55 mol% cationic ionizable lipids; 5–15 mol% neutral lipids; 30–45 mol% steroids; 1–5 mol% polymer conjugate lipids; and RNA encapsulated within or associated with lipid nanoparticles.

[0372] In some embodiments, the mole percentage is determined based on the total moles of lipids present in the lipid nanoparticles. In some embodiments, the mole percentage is determined based on the total moles of cationic ionizable lipids, neutral lipids, steroids, and polymer conjugate lipids present in the lipid nanoparticles.

[0373] In some embodiments, the neutral lipid is selected from the group consisting of DSPC, DPPC, DMPC, DOPC, POPC, DOPE, DOPG, DPPG, POPE, DPPE, DMPE, DSPE, and SM. In some embodiments, the neutral lipid is DSPC.

[0374] In some embodiments, the steroid is cholesterol.

[0375] In some embodiments, the polymer conjugate lipid is a pegylated lipid, such as the pegylated lipid described above.

[0376] In some embodiments, the cationic ionizable lipid component of the LNP has the structure of formula (III): [ka] (In the formula, One of L1 or L2 is -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O)x-, -SS-, -C(=O)S-, SC(=O)-, -NRaC(=O)-, -C(=O)NRa-, NRaC(=O)NRa-, -OC(=O)NRa- or -NRaC(=O)O-, and the other of L1 or L2 is -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O)x-, -SS-, -C(=O)S-, SC(=O)-, -NRaC(=O)-, -C(=O)NRa-, NRaC(=O)NRa-, -OC(=O)NRa- or -NRaC(=O)O- or a direct bond; G1 or G2 is independently an unsubstituted C1-C12 alkylene or a C1-C12 alkenylene; G3 consists of C1-C24 alkylene, C1-C24 alkenylene, C3-C8 cycloalkylene, and C3-C8 cycloalkenylene; Ra is either H or a C1-C12 alkyl group; R1 and R2 are independently a C6-C24 alkyl or a C6-C24 alkenyl; R3 is H, OR5, CN, -C(=O)OR4, -OC(=O)R4, or -NR5C(=O)R4; R4 is a C1-C12 alkyl group; R5 is H or C1-C6 alkyl; and x is 0, 1, or 2. or it has a pharmaceutically acceptable salt, tautomer, prodrug or stereoisomer thereof.

[0377] In some of the aforementioned embodiments of formula (III), the lipid has one of the following structures: (IIIA) or (IIIB): [ka] (Here, A is a 3- to 8-membered cycloalkyl or cycloalkylene ring; R6 is independently H, OH, or C1-C24 alkyl in each instance; n is an integer in the range of 1 to 15.

[0378] In some of the aforementioned embodiments of formula (III), the lipid has structure (IIIA), and in other embodiments, the lipid has structure (IIIB).

[0379] In other embodiments of formula (III), the lipid has one of the following structures: (IIIC) or (IIID): [ka] (Here, y and z are independent integers in the range of 1 to 12.)

[0380] In any of the embodiments of formula (III) described above, either L1 or L2 is -O(C=O)-. For example, in some embodiments, each of L1 and L2 is -O(C=O)-. In any of the several different embodiments described above, L1 and L2 are independently -(C=O)O- or -O(C=O)-. For example, in some embodiments, each of L1 and L2 is -(C=O)O-.

[0381] In several different embodiments of formula (III), the lipid has one of the following structures: (IIIE) or (IIIF). [ka]

[0382] In some of the aforementioned embodiments of formula (III), the lipid has one of the following structures: (IIIG), (IIIH), (IIII), or (IIIJ). [ka]

[0383] In some of the aforementioned embodiments of formula (III), n is an integer in the range of 2 to 12, for example, 2 to 8 or 2 to 4. For example, in some embodiments, n is 3, 4, 5 or 6. In some embodiments, n is 3. In some embodiments, n is 4. In some embodiments, n is 5. In some embodiments, n is 6.

[0384] In some other embodiments of the aforementioned embodiments of formula (III), y and z are each independently integers in the range of 2 to 10. For example, in some embodiments, y and z are each independently integers in the range of 4 to 9 or 4 to 6.

[0385] In some of the aforementioned embodiments of formula (III), R6 is H. In other embodiments of the aforementioned embodiments, R6 is a C1-C24 alkyl group. In other embodiments, R6 is an OH group.

[0386] In some embodiments of formula (III), G3 is unsubstituted. In other embodiments, G3 is substituted. In various different embodiments, G3 is a linear C1-C24 alkylene or a linear C1-C24 alkenylene.

[0387] In some other aforementioned embodiments of formula (III), R1 or R2, or both, are C6-C24 alkenyls. For example, in some embodiments, R1 and R2 each independently have the following structure: [ka] (Here, R7a and R7b are, at each occurrence, independently H or C1-C12 alkyl; and a is an integer between 2 and 12. Here, R7a, R7b, and a are selected such that R1 and R2 each independently contain 6 to 20 carbon atoms. For example, in some embodiments, a is an integer in the range of 5 to 9 or 8 to 12.

[0388] In some of the aforementioned embodiments of formula (III), at least one occurrence of R7a is H. For example, in some embodiments, R7a is H at each occurrence. In other different embodiments of the aforementioned embodiments, at least one occurrence of R7b is a C1-C8 alkyl. For example, in some embodiments, the C1-C8 alkyl is methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-hexyl, or n-octyl.

[0389] In a different embodiment of formula (III), R1 or R2, or both, have one of the following structures: [ka]

[0390] In some of the aforementioned embodiments of formula (III), R3 is OH, CN, -C(=O)OR4, -OC(=O)R4, or -NHC(=O)R4. In some embodiments, R4 is methyl or ethyl.

[0391] In various different embodiments, the cationic lipid of formula (III) has one of the structures shown in the following table.

[0392] A representative compound of formula (III). [ka]

[0393] [ka]

[0394] [ka]

[0395] [ka]

[0396] [ka]

[0397] [ka]

[0398] [ka]

[0399] Further representative cationic ionizable lipids are as follows: [ka]

[0400] [ka]

[0401] In some embodiments, the RNA described herein is formulated into an LNP composition comprising cationic ionizable lipids, such as the cationic ionizable lipids described above, neutral lipids, steroids, and polymer conjugate lipids.

[0402] In some embodiments, the RNA described herein is formulated into an LNP composition comprising a cationic ionizable lipid of formula III, a neutral lipid, a steroid, and a polymer conjugate lipid.

[0403] In some embodiments, the RNA described herein is formulated into an LNP composition comprising cationic ionizable lipids, neutral lipids, steroids, and polymer conjugate lipids as shown in the table above.

[0404] In some embodiments, the RNA described herein is formulated into an LNP composition comprising 3D-P-DMA, neutral lipids, steroids, and polymer-conjugated lipids.

[0405] In some embodiments, the RNA described herein is formulated into an LNP composition comprising ALC-0366, a neutral lipid, a steroid, and a polymer conjugate lipid.

[0406] In some embodiments, the RNA described herein is formulated into an LNP composition comprising ALC-0315, a neutral lipid, a steroid, and a polymer conjugate lipid.

[0407] In some embodiments, the neutral lipid is DSPC. In some embodiments, the steroid is cholesterol. In some embodiments, the polymer conjugate lipid is a pegylated lipid, such as DMG-PEG 2000, PEG2000-C-DMA, or ALC-0159.

[0408] In some embodiments, the RNA described herein is formulated into an LNP composition comprising cationic ionizable lipids, such as the cationic ionizable lipids described above, neutral lipids, steroids, and pegylated lipids.

[0409] In some embodiments, the RNA described herein is formulated into an LNP composition comprising a cationic ionizable lipid of formula III, a neutral lipid, a steroid, and a pegylated lipid.

[0410] In some embodiments, the RNA described herein is formulated into an LNP composition comprising cationic ionizable lipids, neutral lipids, steroids, and pegylated lipids as shown in the table above.

[0411] In some embodiments, the RNA described herein is formulated into an LNP composition comprising 3D-P-DMA, neutral lipids, steroids, and pegylated lipids.

[0412] In some embodiments, the RNA described herein is formulated into an LNP composition comprising ALC-0366, a neutral lipid, a steroid, and a pegylated lipid.

[0413] In some embodiments, the RNA described herein is formulated into an LNP composition comprising ALC-0315, a neutral lipid, a steroid, and a pegylated lipid.

[0414] In some embodiments, the neutral lipid is DSPC. In some embodiments, the steroid is cholesterol. In some embodiments, the pegylated lipid is DMG-PEG 2000, PEG2000-C-DMA, or ALC-0159.

[0415] In some embodiments, the RNA described herein is formulated into an LNP composition comprising cationic ionizable lipids, such as the cationic ionizable lipids described above, DSPC, cholesterol, and pegylated lipids.

[0416] In some embodiments, the RNA described herein is formulated into an LNP composition comprising a cationic ionizable lipid of formula III, DSPC, cholesterol, and pegylated lipids.

[0417] In some embodiments, the RNA described herein is formulated into an LNP composition comprising the cationic ionizable lipids, DSPCs, cholesterol, and pegylated lipids shown in the table above.

[0418] In some embodiments, the RNA described herein is formulated into an LNP composition comprising 3D-P-DMA, DSPC, cholesterol, and pegylated lipids.

[0419] In some embodiments, the RNA described herein is formulated into an LNP composition comprising ALC-0366, DSPC, cholesterol, and pegylated lipids.

[0420] In some embodiments, the RNA described herein is formulated into an LNP composition comprising ALC-0315, DSPC, cholesterol, and pegylated lipids.

[0421] In some embodiments, the pegylated lipid is DMG-PEG 2000, PEG2000-C-DMA, or ALC-0159.

[0422] In some embodiments, the RNA described herein is formulated into an LNP composition comprising a cationic ionizable lipid, such as the cationic ionizable lipid described above, DSPC, cholesterol, and DMG-PEG 2000.

[0423] In some embodiments, the RNA described herein is formulated into an LNP composition comprising a cationic ionizable lipid of formula III, DSPC, cholesterol, and DMG-PEG 2000.

[0424] In some embodiments, the RNA described herein is formulated into an LNP composition comprising the cationic ionizable lipids, DSPC, cholesterol, and DMG-PEG 2000 shown in the table above.

[0425] In some embodiments, the RNA described herein is formulated into an LNP composition comprising 3D-P-DMA, DSPC, cholesterol, and DMG-PEG 2000.

[0426] In some embodiments, the RNA described herein is formulated into an LNP composition comprising ALC-0366, DSPC, cholesterol, and DMG-PEG 2000.

[0427] In some embodiments, the RNA described herein is formulated into an LNP composition comprising ALC-0315, DSPC, cholesterol, and DMG-PEG 2000.

[0428] In some embodiments, the RNA described herein is formulated into an LNP composition comprising a cationic ionizable lipid, such as the cationic ionizable lipid described above, DSPC, cholesterol, and PEG2000-C-DMA.

[0429] In some embodiments, the RNA described herein is formulated into an LNP composition comprising a cationic ionizable lipid of formula III, DSPC, cholesterol, and PEG2000-C-DMA.

[0430] In some embodiments, the RNA described herein is formulated into an LNP composition comprising the cationic ionizable lipids, DSPC, cholesterol, and PEG2000-C-DMA shown in the table above.

[0431] In some embodiments, the RNA described herein is formulated into an LNP composition comprising 3D-P-DMA, DSPC, cholesterol, and PEG2000-C-DMA.

[0432] In some embodiments, the RNA described herein is formulated into an LNP composition comprising ALC-0366, DSPC, cholesterol, and PEG2000-C-DMA.

[0433] In some embodiments, the RNA described herein is formulated into an LNP composition comprising ALC-0315, DSPC, cholesterol, and PEG2000-C-DMA.

[0434] In some embodiments, the RNA described herein is formulated into an LNP composition comprising a cationic ionizable lipid, such as the cationic ionizable lipid described above, DSPC, cholesterol, and ALC-0159.

[0435] In some embodiments, the RNA described herein is formulated into an LNP composition comprising a cationic ionizable lipid of formula III, DSPC, cholesterol, and ALC-0159.

[0436] In some embodiments, the RNA described herein is formulated into an LNP composition comprising the cationic ionizable lipids, DSPC, cholesterol, and ALC-0159 shown in the table above.

[0437] In some embodiments, the RNA described herein is formulated into an LNP composition comprising 3D-P-DMA, DSPC, cholesterol, and ALC-0159.

[0438] In some embodiments, the RNA described herein is formulated into an LNP composition comprising ALC-0366, DSPC, cholesterol, and ALC-0159.

[0439] In some embodiments, the RNA described herein is formulated into an LNP composition comprising ALC-0315, DSPC, cholesterol, and ALC-0159.

[0440] 3D-P-DMA:(6Z,16Z)-12-((Z)-deca-4-en-1-yl)docosa-6,16-dien-11-yl5-(dimethylamino)pentanoate [ka]

[0441] ALC-0366: ((3-hydroxypropyl)azandiyl)bis(nonane-9,1-diyl)bis(2-butyloctanoate) [ka]

[0442] ALC-0315: ((4-hydroxybutyl)azandiyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate) / 6-[N-6-(2-hexyldecanoyloxy)hexyl-N-(4-hydroxybutyl)amino]hexyl2-hexyldecanoate [ka]

[0443] DMG-PEG 2000: [ka] PEG2000-C-DMA:3-N-[(ω-methoxypoly(ethylene glycol)2000)carbamoyl]-1,2-dimyristyloxypropylamine (MPEG-(2kDa)-C-DMA or methoxy-polyethylene glycol-2,3-bis(tetradecyloxy)propylcarbamate(2000)) (Here, n has an average value in the range of 30 to 60, for example, an average value of approximately 50). [ka]

[0444] ALC-0159:2-[(Polyethylene Glycol)-2000]-N,N-Ditetradecylacetamide / 2-[2-(ω-Methoxy(Polyethylene Glycol 2000)Ethoxy]-N,N-Ditetradecylacetamide [ka]

[0445] DSPC:1,2-distearoyl-sn-glycero-3-phosphocholine [ka]

[0446] cholesterol: [ka]

[0447] The N / P value is preferably at least about 4. In some embodiments, the N / P value is in the range of 4-20, 4-12, 4-10, 4-8, or 5-7. In some embodiments, the N / P value is about 6.

[0448] The term “stealth” is used herein to describe the ability of the particles described herein to be undetectable by the immune system of the host to which they are administered, and subsequently isolated and / or degraded, or barely detectable, and subsequently isolated and / or degraded, and / or detected, and later isolated and / or degraded.

[0449] Macrophages constitute one of the most important components of the immune system and play a dominant role in removing foreign particles, including liposomes and other colloidal particles, from the bloodstream. At the molecular level, particle clearance occurs in two steps: opsonization, which involves the deposition of serum proteins (or "opsonins") on the surface of the particles, followed by recognition and capture of the opsonized particles by macrophages.

[0450] Modification of the particle surface with chains of hydrophilic and flexible polymers, such as poly(ethylene glycol) type polymers, provides steric protection by preventing opsonins from reaching the particle surface.

[0451] In some embodiments, the amphiphilic derivatives of the polymers used herein have a hydrophobic group (e.g., a lipid) as specified herein. In some embodiments, the amphiphilic derivatives of the polymers used herein have a phospholipid as the hydrophobic group (e.g., a lipid), such as a biodegradable phospholipid like phosphatidylethanolamine. In some embodiments, the phospholipid is selected from the group consisting of DSPE (distearoylphosphatidylethanolamine), DPPE (dipalmitoylphosphatidylethanolamine), DOPE (dioleoylphosphatidylethanolamine), and POPE (palmitoyloleylphosphatidylethanolamine), and mixtures thereof. In some embodiments, as the phospholipid, DSPE would be used for its stability quality in the particles described herein. Furthermore, as the hydrophobic group (e.g., a lipid), a compound having at least one alkyl chain that provides hydrophobic fixation to the particles described herein may be used.

[0452] In some embodiments, the polymers for use herein are selected from the group consisting of poly(ethylene glycol) (PEG), polysarcosine (pSar) (poly(N-methylglycine), polyoxazoline (POX), polyoxazine (POZ), and poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA) (including their derivatives).

[0453] In some embodiments, the polymer is designed to sterically stabilize the particles by forming a protective hydrophilic layer. In some embodiments, the polymer can reduce the association of the particles with serum proteins and / or the resulting uptake by the reticuloendothelial system when such particles are administered in vivo.

[0454] In some embodiments, PEG is a linear or branched polymer which may be substituted with ethylene glycol or ethylene oxide. In some embodiments, PEG is unsubstituted. In some embodiments, PEG is substituted with, for example, one or more alkyl, alkoxy, acyl, hydroxy, or aryl groups. In some embodiments, PEG has a molecular weight of about 130 to about 50,000, in another embodiment about 150 to about 30,000, in another embodiment about 150 to about 20,000, in another embodiment about 150 to about 15,000, in another embodiment about 150 to about 10,000, in another embodiment about 150 to about 6,000, in another embodiment about 150 to about 5,000, in another embodiment about 150 to about 4,000, in another embodiment about 150 to about 3,000, in another embodiment about 300 to about 3,000, in another embodiment about 1,000 to about 3,000, and in yet another embodiment about 1,500 to about 2,500.

[0455] In some embodiments, the PEG portion of the amphiphilic derivative of the polymer has a molecular weight of 1000 or more. In some embodiments, the PEG portion of the amphiphilic derivative of the polymer contains 10 or more units of the formula (O-CH2-CH2)n. In some embodiments, PEG contains 20 to 200 ethylene oxide units, for example, about 45 ethylene oxide units.

[0456] In some embodiments, PEG includes "PEG2k," also known as "PEG 2000," which has an average molecular weight of about 2000 daltons.

[0457] In some embodiments, DSPE-PEG2000, DSPE-PEG3000, and DSPE-PEG5000 are used as amphiphilic derivatives of the polymer.

[0458] In some embodiments, pSar contains 2 to 200 sarcosine units, e.g., 5 to 100 sarcosine units, 10 to 50 sarcosine units, 15 to 40 sarcosine units, e.g., about 23 sarcosine units.

[0459] In some embodiments, pSar includes the structure of the following general formula: [ka] (In the formula, s is the number of sarcosine units).

[0460] In some embodiments, the POX and / or POZ polymers are 2-200, 2-190, 2-180, 2-170, 2-160, 2-150, 2-140, 2-130, 2-120, 2-110, 2-100, 2-90, 2-80, 2-70, 5-200, 5-190, 5-180, 5-170, 5-160, 5-150, 5-140, 5-1 Contains 30, 5-120, 5-110, 5-100, 5-90, 5-80, 5-70, 10-200, 10-190, 10-180, 10-170, 10-160, 10-150, 10-140, 10-130, 10-120, 10-110, 10-100, 10-90, 10-80, or 10-70 POX and / or POZ repeating units.

[0461] In some embodiments, the POX and / or POZ polymers include the following general formula: [ka] (wherein a formula a is an integer between 1 and 2; R11 is an alkyl, particularly a C1-3 alkyl such as methyl, ethyl, isopropyl, or n-propyl, which is independently selected for each repeating unit; and m refers to the number of POX and / or POZ repeating units).

[0462] In some embodiments, the POX and / or POZ polymer is a polymer of POX and comprises repeating units of the following general formula. [ka]

[0463] In some embodiments, the POX and / or POZ polymer is a polymer of POZ and comprises repeating units of the following general formula. [ka]

[0464] In any of the above embodiments of the formula, m (i.e., the number of repeating units in the polymer) is preferably 2 to 190, for example 2 to 180, 2 to 170, 2 to 160, 2 to 150, 2 to 140, 2 to 130, 2 to 120, 2 to 110, 2 to 100, 2 to 90, 2 to 80, 2 to 70, 5 to 200, 5 to 190, 5 to 180, 5 to 170, 5 to 160 , 5-150, 5-140, 5-130, 5-120, 5-110, 5-100, 5-90, 5-80, 5-70, 10-200, 10-190, 10-180, 10-170, 10-160, 10-150, 10-140, 10-130, 10-120, 10-110, 10-100, 10-90, 10-80, or 10-70. In certain embodiments, m is 2-180, e.g., 4-160, 6-140, 8-120, or 10-100, e.g., 20-80, 30-70, or 40-50.

[0465] In some embodiments, the POX and / or POZ polymer is a copolymer comprising repeating units of the following general formula: [ka] (In the formula, the number of repeating units shown to the left of the copolymer is between 1 and 199; the number of repeating units in the formula to the right of the copolymer is between 1 and 199; and the sum of the number of repeating units in the formula to the left of the copolymer and the number of repeating units in the formula to the right is between 2 and 200).

[0466] In some embodiments of the oxazolinylated and / or oxazinylated hydrophobic moiety (e.g., lipids), the number of repeating units in the left-hand equation of the copolymer is 1 to 179, e.g., 1 to 159, 1 to 139, 1 to 119, or 1 to 99; the number of repeating units in the right-hand equation of the copolymer is 1 to 179, e.g., 1 to 159, 1 to 139, 1 to 119, or 1 to 99; and the sum of the number of repeating units in the left-hand and right-hand equations of the copolymer is 2 to 180, e.g., 4 to 160, 6 to 140, 8 to 120, or 10 to 100, e.g., 20 to 80, 30 to 70, or 40 to 50.

[0467] In some of the embodiments described above, R11 may be the same alkyl group at each occurrence (i.e., in each repeating unit) (for example, R11 may be methyl in each repeating unit). In some alternative embodiments, R11 in at least one repeating unit may be different from R11 in another repeating unit (for example, for at least one repeating unit, R11 is one specific alkyl group (e.g., ethyl), and for at least one different repeating unit, R11 is a different specific alkyl group (e.g., methyl)). For example, each R11 may be selected from two different alkyl groups (e.g., methyl and ethyl), and not all R11s are the same alkyl group.

[0468] In any of the embodiments described above, R11 is preferably methyl or ethyl, more preferably methyl. Thus, in some embodiments, each R11 is either methyl or each R11 is ethyl. In some alternative embodiments, R11 is independently selected from methyl and ethyl for each repeating unit, and in at least one repeating unit, R11 is methyl and in at least one repeating unit, R11 is ethyl.

[0469] In some embodiments, the polymer comprises poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA) or poly-2-(2-(2-methylaminoethoxy)ethoxy)acetic acid (pMAEEA), or a derivative thereof.

[0470] In some embodiments, the polymer includes the following general formula: [ka] (In the formula, X2 and X1 together are optionally substituted amides, optionally substituted thioamides, or esters; Y is -CH2-, -(CH2)2-, or -(CH2)3-; z is 2 to 24; and n is between 1 and 100.

[0471] In some embodiments, (i) If X1 is -C(O)-, then X2 is -NR1-; (ii) If X1 is -NR1-, then X2 is -C(O)-; (iii) If X1 is -C(S)-, then X2 is -NR1-; (iv) If X1 is -NR1-, then X2 is -C(S)-; (v) If X1 is -C(O)-, then X2 is -O-; or (vi) If X1 is -O-, then X2 is -C(O)-; Here, R1 is hydrogen or a C1-8 alkyl group.

[0472] In some embodiments, X1 is -C(O)- and X2 is -NR1-, where R1 is hydrogen or C1-8 alkyl. In some embodiments, X1 is -C(O)- and X2 is -NR1-, where R1 is hydrogen or methyl. In some embodiments, X1 is -C(O)- and X2 is -NR1-, where R1 is hydrogen.

[0473] In some embodiments, Y is -CH2- or -(CH2)2-.

[0474] In some embodiments, the polymer includes the following general formula: [ka] (In the formula, R1 is hydrogen or a C1-8 alkyl group; z is 2 to 24; and n is between 1 and 100.

[0475] In some embodiments of the above formula, z is between 2 and 10. In some embodiments, z is between 2 and 7. In some embodiments, z is between 2 and 5. In some embodiments, z is 2 or 3. In some embodiments, z is 2.

[0476] In some embodiments, the polymer includes the following general formula: [ka] (In the formula, R1 is hydrogen or a C1-8 alkyl group; and n is between 1 and 100.

[0477] In some embodiments of the above formula, R1 is hydrogen or methyl. In some embodiments, R1 is hydrogen.

[0478] In some embodiments, the polymer includes the following general formula: [ka] (In the formula, n is between 1 and 100.

[0479] In some embodiments of the above formula, n is between 5 and 50. In some embodiments, n is between 5 and 25. In some embodiments, n is between 7 and 14. In some embodiments, n is between 10 and 25. In some embodiments, n is between 14 and 17. In some embodiments, n is 8 or 14.

[0480] In some embodiments, the molar ratio of the amphiphilic derivative of the polymer incorporated into the particles is 0.5 to 20 mol%, preferably 1 to 10 mol%, of the lipid molecules constituting the particles.

[0481] Pharmaceutical composition The particles or compositions described herein may be administered as pharmaceutical compositions or pharmaceuticals, or in any suitable form of pharmaceutical composition. In some embodiments, the pharmaceutical compositions are for use in therapeutic or preventive measures, such as in the treatment or prevention of cancer or infectious diseases, particularly antigen-related diseases such as HIV infection.

[0482] The term "pharmaceutical composition" relates to a composition comprising a therapeutically effective agent, preferably together with a pharmaceutically acceptable carrier, diluent, and / or excipient. The pharmaceutical composition is useful for treating, preventing, or reducing the severity of a disease by administering the pharmaceutical composition to a subject.

[0483] The pharmaceutical compositions of this disclosure may contain one or more adjuvants, or may be administered together with one or more adjuvants. In some embodiments, the pharmaceutical compositions do not contain adjuvants. The term “adjuvant” relates to compounds that prolong, enhance, or accelerate an immune response. Adjuvants include a heterogeneous group of compounds such as oil emulsions (e.g., Freund’s adjuvants), inorganic compounds (e.g., alum), bacterial products (e.g., Bordetella pertussis toxin), or immune stimuli complexes. Examples of adjuvants, but not limited to, include LPS, GP96, CpG oligodeoxynucleotides, growth factors, and cytokines, such as monokines, lymphokines, interleukins, and chemokines. Chemokines may be IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12, INFa, INF-γ, GM-CSF, and LT-a. Further known adjuvants include aluminum hydroxide, Freund's adjuvant, or oils such as Montanide® ISA51. Other suitable adjuvants for use in this disclosure include lipopeptides such as Pam3Cys, as well as lipophilic components such as saponins, trehalose-6,6-dibehenate (TDB), monophosphoryl lipid A (MPL), monomycoloylglycerol (MMG), or glucopyranosyllipid adjuvants (GLA).

[0484] The pharmaceutical compositions of this disclosure may be in a storable form (e.g., frozen or freeze-dried form) or a "ready-to-use form" (i.e., a form that can be immediately administered to a subject without any processing such as dilution). Therefore, before administration of the storable form of a pharmaceutical composition, this storable form must be processed or converted to a ready-to-use or administerable form. For example, a frozen pharmaceutical composition must be thawed or a freeze-dried pharmaceutical composition must be reconstituted by using a suitable solvent (e.g., deionized water such as water for injection) or a liquid (e.g., an aqueous solution).

[0485] The pharmaceutical compositions described herein are generally applied in terms of "pharmaceutically effective amounts" and "pharmaceutically acceptable formulations."

[0486] The term "pharmaceutically acceptable" refers to a non-toxic substance that does not interact with the action of the active ingredient in a pharmaceutical composition.

[0487] The term "pharmaceutical effective dose" refers to the amount that achieves the desired response or effect, either alone or in combination with additional doses. In some embodiments relating to the treatment of a particular disease, the desired response may relate to inhibiting the course of the disease. This includes slowing the progression of the disease, and in some embodiments, interrupting or reversing the progression of the disease. The desired response in the treatment of a disease may also be the delay or prevention of the onset of the disease or condition or its symptoms. The effective dose of the pharmaceutical composition described herein depends on the individual parameters of the patient, including the condition being treated, the severity of the disease, age, physiological state, size and weight, the duration of treatment, the type of accompanying treatment (if any), the specific route of administration, and similar factors. Therefore, the dose administered of the pharmaceutical composition described herein may depend on a variety of such parameters. If the response in the patient is insufficient with the initial dose, a higher dose (or an effectively higher dose achieved by a different, more localized route of administration) may be used.

[0488] The pharmaceutical compositions of this disclosure may include buffers, preservatives, and optionally other therapeutic agents. In some embodiments, the pharmaceutical compositions of this disclosure include one or more pharmaceutically acceptable carriers, diluents, and / or excipients.

[0489] Suitable preservatives for use in the pharmaceutical compositions of this disclosure include, but are not limited to, benzalkonium chloride, chlorobutanol, parabens, and thimerosal.

[0490] As used herein, the term “excipient” refers to a substance that may be present in the pharmaceutical compositions of this disclosure but is not an active ingredient. Examples of excipients include, but are not limited to, carriers, binders, diluents, lubricants, thickeners, surfactants, preservatives, stabilizers, emulsifiers, buffers, flavoring agents, or colorants.

[0491] The term “diluent” refers to a substance used to dilute and / or reduce an agent. Furthermore, the term “diluent” includes one or more fluids, liquids, or solid suspensions and / or mixtures. Examples of suitable diluents include ethanol, glycerol, and water.

[0492] The term "carrier" refers to a component that may be natural, synthetic, organic, or inorganic, to which the active ingredient is combined to facilitate, enhance, or enable the administration of the pharmaceutical composition. As used herein, carriers may be one or more suitable solid or liquid fillers, diluents, or encapsulants suitable for administration to a target. Suitable carriers include, but are not limited to, sterile water, Ringer's solution, Ringer's lactate, sterile sodium chloride solution, isotonic saline, polyalkylene glycol, naphthalene hydrogenate, and, in particular, biocompatible lactide polymers, lactide / glycolide copolymers, or polyoxyethylene / polyoxypropylene copolymers. In some embodiments, the pharmaceutical compositions of this disclosure include isotonic saline.

[0493] Pharmacopoeia-acceptable carriers, excipients, or diluents for therapeutic use are well known in the pharmaceutical field and are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (AR Gennaro edit. 1985).

[0494] The pharmaceutical carrier, excipient, or diluent may be selected in relation to the intended route of administration and standard pharmaceutical practices.

[0495] Treatment method The agents, compositions, and methods described herein can be used to treat subjects having diseases, such as HIV infection. The agents, compositions, and methods described herein can be used in the therapeutic or prophylactic treatment of various diseases. In some embodiments, the agents, compositions, and methods described herein are useful in the prophylactic and / or therapeutic treatment of antigen-related diseases.

[0496] Such antigens can serve as targets for immune effector cells that express antigen receptors. For example, if the antigen is derived from a virus, the drugs, compositions, and methods may be useful in treating viral diseases caused by the virus. If the antigen is a tumor antigen, the drugs, compositions, and methods may be useful in treating cancerous diseases in which cancer cells express the tumor antigen.

[0497] The term “disease” refers to an abnormal condition that affects an individual’s body. A disease is often interpreted as a medical condition associated with specific symptoms and signs. Diseases can be caused by factors originating from external sources, such as infections, or by internal dysfunctions, such as autoimmune diseases. In humans, “disease” is often used more broadly to refer to a condition that causes pain, dysfunction, distress, social problems, or death in the affected individual, or similar problems in those in contact with the individual. In this broader sense, disease may sometimes include injury, physical disability, impairment, syndrome, infection, isolated symptoms, deviant behavior, and atypical changes in structure and function, although in other contexts and for other purposes these may be considered distinct categories. Many diseases, and living with them, can alter one’s outlook on life and personality, so diseases usually affect individuals not only physically but also emotionally.

[0498] The term "infectious disease" refers to any disease caused by microbial factors that can be transmitted from individual to individual or from organism to organism (e.g., the common cold). Infectious diseases are well known in the art and include, for example, viral diseases, bacterial diseases, or parasitic diseases caused by viruses, bacteria, and parasites, respectively. In this regard, infectious diseases may include, for example, hepatitis, sexually transmitted infections (e.g., chlamydia or gonorrhea), tuberculosis, HIV / acquired immunodeficiency syndrome (AIDS), diphtheria, hepatitis B, hepatitis C, cholera, severe acute respiratory syndrome (SARS), avian influenza, and influenza.

[0499] In this context, the terms “treatment,” “to treat,” or “therapeutic intervention” refer to the management and care of an individual aimed at combating a condition such as a disease or disorder. The term is intended to include all forms of treatment for a given condition in which an individual is afflicted, such as the administration of therapeutic compounds to alleviate symptoms or complications, to slow the progression of a disease, disorder or condition, to alleviate or reduce symptoms and complications, and / or to cure or eliminate a disease, disorder or condition, and to prevent a condition. Prevention should be understood as the management and care of an individual aimed at combating a disease, condition or disorder, and includes the administration of active compounds to prevent the onset of symptoms or complications.

[0500] The term "therapeutic treatment" refers to any treatment that improves an individual's health and / or extends (increases) their lifespan. Treatment may eliminate a disease in an individual, halt or delay the progression of a disease in an individual, inhibit or delay the progression of a disease in an individual, reduce the frequency or severity of symptoms in an individual, and / or reduce recurrence in an individual who currently has or has previously had the disease.

[0501] The terms “prophylactic treatment” or “preventive treatment” refer to any treatment intended to prevent the development of disease in an individual. The terms “prophylactic treatment” and “preventive treatment” are used interchangeably herein.

[0502] The terms “individual” and “subject” are used interchangeably herein. They refer to a human or other mammal (e.g., mouse, rat, rabbit, dog, cat, cattle, pig, sheep, horse, or primate) that is susceptible to, but may or may not have, a disease or disorder (e.g., cancer). In many embodiments, the individual is a human. Unless otherwise specified, the terms “individual” and “subject” do not indicate a specific age and therefore encompass adults, the elderly, children, and newborns. In embodiments of this disclosure, the “individual” or “subject” is a “patient.”

[0503] The term "patient" means an individual or subject for treatment, in particular an affected individual or subject.

[0504] The terms “antigen-related disease,” “antigen-expressing cell-related disease,” or similar terms refer to any disease related to an antigen, such as a disease characterized by the presence of an antigen. Antigen-related diseases may be infectious diseases, or cancerous diseases or simply cancer. As mentioned above, the antigen may be a disease-related antigen such as a tumor-associated antigen, a viral antigen, or a bacterial antigen. In some embodiments, the antigen-related disease is preferably a disease involving cells that express the antigen on their cell surface.

[0505] The term “infectious disease” refers to any disease caused by a microbial factor (e.g., HIV) that can be transmitted from individual to individual or from organism to organism. Infectious diseases are well known in the art and include, for example, viral diseases, bacterial diseases, or parasitic diseases caused by viruses, bacteria, and parasites, respectively. In this regard, infectious diseases may include, for example, hepatitis, sexually transmitted infections (e.g., chlamydia or gonorrhea), tuberculosis, HIV / acquired immunodeficiency syndrome (AIDS), diphtheria, hepatitis B, hepatitis C, cholera, severe acute respiratory syndrome (SARS), avian influenza, and influenza.

[0506] The methods and agents described herein are particularly useful for the treatment of HIV infection.

[0507] The terms "cell-mediated immunity," "cellular immunity," "cellular immune response," or similar terms are intended to include cellular responses to cells characterized by antigen expression, particularly antigen presentation by class I or class II MHC. The cellular response relates to cells called T cells or T lymphocytes that act as either "helpers" or "killers." Helper T cells (also called CD4+ T cells) play a central role by modulating the immune response, while killer cells (also called cytotoxic T cells, cytolytic T cells, CD8+ T cells, or CTLs) kill disease cells such as cancer cells and prevent the production of further disease cells.

[0508] The term "antigen-presenting cell" (APC) refers to one of the various cells that can display, acquire, and / or present at least one antigen or antigen fragment on (or on) its cell surface. Antigen-presenting cells can be distinguished into professional antigen-presenting cells and non-professional antigen-presenting cells.

[0509] The term "professional antigen-presenting cells" refers to antigen-presenting cells that constitutively express major histocompatibility complex class II (MHC class II) molecules necessary for interaction with naive T cells. When T cells interact with the MHC class II molecular complex on the membrane of antigen-presenting cells, the antigen-presenting cells produce costimulatory molecules that induce T cell activation. Professional antigen-presenting cells include dendritic cells and macrophages.

[0510] The term "non-professional antigen-presenting cells" refers to antigen-presenting cells that do not constitutively express MHC class II molecules but express them in response to stimulation by certain cytokines, such as interferon-gamma. Examples of non-professional antigen-presenting cells include fibroblasts, thymic epithelial cells, thyroid epithelial cells, glial cells, pancreatic beta cells, or vascular endothelial cells.

[0511] "Antigen processing" refers to the breakdown of an antigen into processing products, which are fragments of the antigen (e.g., breakdown of proteins into peptides), and the association (e.g., by binding) of an MHC molecule with one or more of these fragments for presentation to specific T cells by cells such as antigen-presenting cells.

[0512] In some embodiments, the compositions, particles, or pharmaceutical compositions described herein may be administered intravenously, intra-arterially, subcutaneously, intradermally, transdermally, intra-lymph nodely, intramuscularly, intratumorally, or peritumorally. In some embodiments, the compositions, particles, or pharmaceutical compositions described herein may be administered intramuscularly. In some embodiments, the compositions, particles, or pharmaceutical compositions are formulated for topical or systemic administration. Systemic administration may include enteral administration, including absorption through the gastrointestinal tract, or parenteral administration. As used herein, “parenteral administration” refers to administration by any method other than through the gastrointestinal tract, such as intravenous injection. In some embodiments, the compositions, particles, or pharmaceutical compositions are formulated for systemic administration. In some embodiments, systemic administration is by intravenous administration. In some embodiments, the compositions, particles, or pharmaceutical compositions are formulated for intramuscular administration.

[0513] The present invention will be described in detail and illustrated by the drawings and examples, which are for illustrative purposes only and are not intended to limit it. Further embodiments similarly included in the present invention will be accessible to those skilled in the art through the description and examples.

[0514] [Table 1-1]

[0515] [Table 1-2]

[0516] [Table 1-3]

[0517] [Table 1-4]

[0518] [Table 2-1]

[0519] [Table 2-2]

[0520] [Table 2-3]

[0521] [Table 2-4]

[0522] [Table 2-5]

[0523] [Table 2-6]

[0524] [Table 2-7]

[0525] [Table 2-8]

[0526] [Table 2-9]

[0527] [Table 2-10]

[0528] [Table 2-11]

[0529] [Table 2-12] [Brief explanation of the drawing]

[0530] [Figure 1] Figure 1 shows the string representation of two RNA molecules. The first RNA molecule (string 1) encodes an immunogenic peptide containing 33 fragments, and the second RNA molecule (string 2) encodes an immunogenic peptide containing 32 fragments. Each pair of fragments is separated by up to four cleavage-promoting amino acids. The length of the immunogenic peptide is approximately 550 amino acids. [Figure 2]Figure 2 shows the string representation of two RNA molecules. The first RNA molecule (string 1) encodes an immunogenic peptide containing 33 fragments, and the second RNA molecule (string 2) encodes an immunogenic peptide containing 32 fragments. Each pair of fragments is separated by a 10-amino acid non-immunogenic linker adjacent to cleavage-promoting amino acids (up to 4). The length of the immunogenic peptide is approximately 950 amino acids. [Figure 3] Amino acid changes between RNA strings 1 and 2 and RNA strings 1.1 and 2.1. [Figure 4A] In vitro expression of mRNA-encoded polypeptides from two mRNA strings (string 1 no GS, string 2 no GS, and string 2 GS) with or without a flexible glycine-serine (GS) linker. For each string, three different nucleotide sequences were generated (nt1, nt2, and nt3), where nt1 corresponds to the wild-type sequence, and nt2 and nt3 represent two different codon optimization methods. Each string was transcribed in vitro in the presence of either unmodified (uRNA) or methylpseudridine (modRNA). For string 2 GS, only nt3 was tested. Each mRNA string, i.e., uRNA / modRNA, nt1-3, strings 1-2, GS / no GS, was separately transfected into human cell lines in or without the proteasome inhibitor MG132. The relative abundance of each peptide is shown as follows: (A) for two trypsin peptides common to each string tested with modRNA; (B) for trypsin peptides specific to string 1 (no GS, modRNA, and uRNA); and (C) for trypsin peptides specific to string 2 (GS and no GS, modRNA, and uRNA). [Figure 4D]The trypsin peptides YPLTFGWCFK and IYSYFPSVISK, common to all tested strings, are located in the central and C-terminal regions of the polypeptides encoded by mRNA string 1 (no GS) (D), mRNA string 2 (no GS), and GS (E). The locations of the string-specific trypsin peptide TSTLQEQIGWAR (D), tested for string 1 (no GS), and the string-specific trypsin peptide QNYTPGPGVR (E), tested for string 2 (GS) and (no GS), are also shown. [Figure 5A] In vitro processing and presentation of HLA-I alleles from mRNA-encoded polypeptides from two modRNA strings (string 1 and string 2) were evaluated by immunopeptidomics. Each string was transfected into the A375 cell line encoding a single HLA class I allele, and HIV-specific epitopes in string 1(AB) were detected by targeted mass spectrometry. The detected epitope locations are annotated below string 1(A). [Figure 5B] Detailed information related to each detected epitope, such as the HLA class-I allele, the location of the fragment ends, and the non-HIV junction sequence (end), as well as sequence matches for string 1, are shown in panel (B), and the corresponding information for string 2 is shown in panel (D). Legend: Epitope sequence perfectly matches 2021 consensus B=ConB; Epitope sequence perfectly matches 2021 consensus C=ConC; Epitope sequence perfectly matches consensus B and C=ConB / C; Epitope does not perfectly match either consensus B or C = polymorphism. [Figure 5C]In vitro processing and presentation of HLA-I alleles from mRNA-encoded polypeptides from two modRNA strings (string 1 and string 2) were evaluated by immunopeptidomics. Each string was transfected into the A375 cell line encoding a single HLA class I allele, and the HIV-specific epitope in string 2 (CD) could be detected by targeted mass spectrometry. The detected epitope location is annotated below string 2 (C). [Figure 5D] Panel (D) shows detailed information related to each detected epitope, including the HLA class-I allele, the location of the fragment ends, and the non-HIV junction sequence (end), as well as the corresponding information for String 2. Legend: Epitope sequence perfectly matches 2021 consensus B=ConB; Epitope sequence perfectly matches 2021 consensus C=ConC; Epitope sequence perfectly matches consensus B and C=ConB / C; Epitope does not perfectly match either consensus B or C = polymorphism. [Figure 6]TCR engagement from processed and presented epitopes from different mRNA strings in the K562 cell line. Jurkat cell lines expressing a single TCR were co-incubated with monoHLA-I allele K562 cells, and downstream signaling of the expressed TCR was measured as a log2 factor change signal of luciferase. K562 was pulsed (PP) with peptides encoded by the tested strings as a positive control controlling TCR activity against the tested specific epitopes, or transfected with mRNA encoding polypeptides (striped bars). Two different mRNA strings (string 1 and string 2) were tested with different nucleotide optimizations (nt2 and nt3). PHA-L was used as a positive control for TCR activity. % unique epitopes AK11, DA9, +DA9+, KF11, and IW9 were tested and reacted with their cognitive TCRs. The processing and presentation of the precise epitopes KF11 and IW9 were investigated from different mRNA strings. The polymorphic version of DA9, shown as +DA9+ and encoded by the string 2 version, was cross-reactive with the non-cognitive DA9-specific TCR. AK11 was tested using A*11:01, EL9 using A*26:01, KF11 using B*57:01, DA9 using B*14:01, and IW9 was presented by both the cognitive HLA-I allele B*57:03 and the non-cognitive B*57:01 allele, shown as +B*57:01+ for both string 1 and string 2. Legend: AK11:ACQGVGGPGHK;DA9:DRFYKTLRA;+DA9+:DRFFKTLRA;KF11:KAFSPEVIPMF;IW9:ISPRTLNAW [Figure 7A]TCR engagement from processed and presented epitopes in iDCs generated from two donors. Jurkat cell lines expressing a single TCR were co-incubated with iDCs, and expressed TCR downstream signaling was measured as a log2 factor change signal of luciferase. The iDCs were pulsed with peptides encoded by the tested strings. To control TCR activity, pulsed iDCs were either pre-co-transfected with mRNA encoding an HLA class I allele (PP) or, without co-transfection, expressed only donor-derived endogenous HLA-I to control potential endogenous HLA-I mediated background presentation (PP+eHLA). iDCs were co-transfected with polypeptide-encoding mRNA and cognitive HLA class I alleles (striped bars). Two different mRNA strings (string 1 and string 2) were tested with different nucleotide optimizations (nt2 and nt3). PHA-L was used as a positive control for TCR activity. For donor 1(A) and donor 2(B), six (AK11, DA9, +DA9+, KF11, EL9, and IW9) and five (AK11, DA9, +DA9+, EL9, and IW9) unique epitopes were tested and reacted with their cognitive TCRs. Processing and presentation of epitope IW9 were examined from two different mRNA strings (string 1 and string 2). The polymorphic version of DA9, shown as +DA9+ and encoded by the string 2 version, was cross-reactive with the non-cognitive DA9-specific TCR. AK11 was tested using A*11:01, EL9 was tested using A*26:01, KF11 was tested using B*57:01, DA9 was tested using B*14:01, and IW9 was presented with both the cognitive HLA-I allele B*57:03 and the noncognitive B*57:01 allele, which was shown as +B*57:01+ for both string 1 and string 2.Legend: AK11:ACQGVGGPGHK;DA9:DRFYKTLRA;+DA9+:DRFFKTLRA;KF11:KAFSPEVIPMF;IW9:ISPRTLNAW;EL9:EVIPMFSAL. [Figure 7B] For donor 1(A) and donor 2(B), six (AK11, DA9, +DA9+, KF11, EL9, and IW9) and five (AK11, DA9, +DA9+, EL9, and IW9) unique epitopes were tested and reacted with their cognitive TCRs. Processing and presentation of epitope IW9 were examined from two different mRNA strings (string 1 and string 2). The polymorphic version of DA9, shown as +DA9+ and encoded by the string 2 version, was cross-reactive with the non-cognitive DA9-specific TCR. AK11 was tested using A*11:01, EL9 was tested using A*26:01, KF11 was tested using B*57:01, DA9 was tested using B*14:01, and IW9 was presented with both the cognitive HLA-I allele B*57:03 and the noncognitive B*57:01 allele, which was shown as +B*57:01+ for both string 1 and string 2. Legend: AK11:ACQGVGGPGHK;DA9:DRFYKTLRA;+DA9+:DRFFKTLRA;KF11:KAFSPEVIPMF;IW9:ISPRTLNAW;EL9:EVIPMFSAL [Figure 8A]CD8+ and CD4+ T cell responses to two different mRNA strings (string 1 and string 2): Elispot data of IFN-γ production by isolated CD8+ T cells and isolated CD4+ T cells stimulated with a peptide pool. T cells were isolated from HLA-A*02:01 CB6F1 transgenic mice and stimulated with a peptide pool for 18 hours on Elispot plates coated with anti-IFN-γ antibody. CD8+ T cells from groups immunized with string 1 (A) or string 2 (B) (n=5 mice / group) were incubated with an 11-mer peptide pool covering vaccine fragments containing at least one known / predicted HIV-1 epitope (prctrk<1%) derived from A*02:01 (white box-black circle) or H2b / H2d (striped box-white square) or neither (diamond box-white triangle). [Figure 8B] CD8+ and CD4+ T cell responses to two different mRNA strings (string 1 and string 2): Elispot data of IFN-γ production by isolated CD8+ T cells and isolated CD4+ T cells stimulated with a peptide pool. T cells were isolated from HLA-A*02:01 CB6F1 transgenic mice and stimulated with a peptide pool for 18 hours on Elispot plates coated with anti-IFN-γ antibody. CD8+ T cells from groups immunized with string 1 (A) or string 2 (B) (n=5 mice / group) were incubated with an 11-mer peptide pool covering vaccine fragments containing at least one known / predicted HIV-1 epitope (prctrk<1%) derived from A*02:01 (white box-black circle) or H2b / H2d (striped box-white square) or neither (diamond box-white triangle). [Figure 8C]CD8+ T cells from groups immunized with string 1 (C) or string 2 (D) were incubated with a heterologous minimal A*02:01 (white box - black circle) or H2b / H2b (striped box - white square) HIV epitope pool, i.e., minimal HIV epitopes (8-11mers) containing at least one polymorphism compared to the immunization string sequence. Pools I / K tested in the string 1 immunization group and G / H tested in the string 2 immunization group represent exact sequence matches found only in string 2 and string 1, respectively. [Figure 8D] CD8+ T cells from groups immunized with string 1 (C) or string 2 (D) were incubated with a heterologous minimal A*02:01 (white box - black circle) or H2b / H2b (striped box - white square) HIV epitope pool, i.e., minimal HIV epitopes (8-11mers) containing at least one polymorphism compared to the immunization string sequence. Pools I / K tested in the string 1 immunization group and G / H tested in the string 2 immunization group represent exact sequence matches found only in string 2 and string 1, respectively. [Figure 8E] Isolated CD4+ T cells and CD8+ T cells (E) were incubated with a pool of 15-mer peptides encompassing the entire tetanus toxin. [Figure 8F] Isolated CFD8+ T cells and CD4+ T cells from the indicated immune groups were tested using an AH-1-independent 15-mer peptide pool negative control (F). [Figure 8G] Isolated CFD8+ T cells and CD4+ T cells from the indicated immune groups were tested using concanavalin A-positive control (G). [Figure 8H] Panel (H) shows an overview of the experimental setup. [Figure 9A]CD8+ and CD4+ T cell responses to two different mRNA strings (string 1 and string 2): Elispot data of IFN-γ production by isolated CD8+ T cells and isolated CD4+ T cells stimulated with a peptide pool. T cells were isolated from HLA-A*02:01 CB6F1 transgenic mice and stimulated with a peptide pool for 18 hours on Elispot plates coated with anti-IFN-γ antibody. CD8+ T cells were immunized with either a single mRNA, string 1 (10 μg) or string 2 (10 μg), or co-immunized with both mRNA strings 1 and 2 in a 1:1 ratio (10 μg + 10 μg; 5 μg + 5 μg; 2.5 μg + 2.5 μg) (n=5 mice / group). Animals immunized with string 1 (A,C) alone or in combination, or with string 2 (B,D) alone or in combination, were incubated with 11-mer peptide pools covering vaccine fragments containing at least one known / predicted HIV epitope (prctrk < 1%) from A*02:01 (white box - black circle) or H2b / H2d (striped box - white square) or neither (diamond box - white triangle), pools A, B, C and D, E, F, respectively. CD8+ T cells from groups immunized with string 1 (A) or string 2 (B) were also incubated with minimal A*02:01 (white box - black circle) or H2b / H2b (striped box - white square) HIV epitope pools (8-11mers) exclusively encoded by either string 1 (pools G and H) or string 2 (pools I / K). [Figure 9B]CD8+ and CD4+ T cell responses to two different mRNA strings (string 1 and string 2): Elispot data of IFN-γ production by isolated CD8+ T cells and isolated CD4+ T cells stimulated with a peptide pool. T cells were isolated from HLA-A*02:01 CB6F1 transgenic mice and stimulated with a peptide pool for 18 hours on Elispot plates coated with anti-IFN-γ antibody. CD8+ T cells were immunized with either a single mRNA, string 1 (10 μg) or string 2 (10 μg), or co-immunized with both mRNA strings 1 and 2 in a 1:1 ratio (10 μg + 10 μg; 5 μg + 5 μg; 2.5 μg + 2.5 μg) (n=5 mice / group). Animals immunized with string 1 (A,C) alone or in combination, or with string 2 (B,D) alone or in combination, were incubated with 11-mer peptide pools covering vaccine fragments containing at least one known / predicted HIV epitope (prctrk < 1%) from A*02:01 (white box - black circle) or H2b / H2d (striped box - white square) or neither (diamond box - white triangle), pools A, B, C and D, E, F, respectively. CD8+ T cells from groups immunized with string 1 (A) or string 2 (B) were also incubated with minimal A*02:01 (white box - black circle) or H2b / H2b (striped box - white square) HIV epitope pools (8-11mers) exclusively encoded by either string 1 (pools G and H) or string 2 (pools I / K). [Figure 9C] Similarly, CD4+ T cells from groups immunized with string 1(C) were incubated with a minimum A*02:01 (white box-black circle) or H2b / H2b (striped box-white square) HIV epitope pool (8-11 units). [Figure 9D] Similarly, CD4+ T cells from groups immunized with string 2(D) were incubated with a minimum A*02:01 (white box-black circle) or H2b / H2b (striped box-white square) HIV epitope pool (8-11 units). [Figure 9E] Isolated CD4+ T cells and CD8+ T cells (E) were incubated with a pool of 15-mer peptides encompassing the entire tetanus toxin. [Figure 9F] Isolated CD8+ and CD4+ T cells from the indicated immune groups were tested using an AH-1-independent 15-mer peptide pool negative control (F). Quantification of IFNg spot-forming units per 100,000 cells for each peptide pool was performed. [Figure 9G] Isolated CD8+ and CD4+ T cells from the indicated immune groups were tested using a concanavalin A-positive control (G). IFNg spot-forming units per 100,000 cells were quantified for each peptide pool. [Figure 9H] Panel (H) shows an overview of the experimental setup. [Figure 10A] TCR engagement from epitopes processed from different mRNA strings in the K562 cell line: Jurkat cell lines expressing a single TCR were co-incubated with the monoHLA-I allele K562, and the expressed TCR downstream signaling was measured as a log2 factor change signal of luciferase. K562 was either pulsed (PP) with peptides encoded by the tested strings as a positive control for controlling TCR activity against the specific epitopes tested, or transfected with mRNA strings encoding polypeptides. PHA-L was used as a positive control for TCR activity. Five unique epitopes were tested and reacted with their cognitive TCRs. Four different mRNA strings with different amino acid sequences were tested with two nucleotide sequence optimizations (nt2 and nt3), each. String 1 and its derivative, string 1.1 (four point mutations), were tested in K562 cells by transfecting the cells with 3 μg(A) of methylpseudridine mRNA. All of the displayed epitopes were perfectly matched in the sequences of these mRNA strings. [Figure 10B]String 1 and its derivative, string 1.1 (four point mutations), were tested in K562 cells by transfecting the cells with 0.3 μg (B) of methylpseudridine mRNA. All indicated epitopes were perfectly matched in the sequences of these mRNA strings. [Figure 10C] String 2 and its derivative, string 2.1 (a single point mutation), were tested using K562 cells transfected with 3 μg (C) of methylpseudridine mRNA. AK11 was tested using A*11:01, EL9 using A*26:01, KF11 using B*57:01, DA9 using B*14:01, and IW9 using the HLA-I allele B*57:03. Epitopes marked with an asterisk (*) contain at least one polymorphism compared to the sequences of these mRNA strings. [Figure 10D] String 2 and its derivative, string 2.1 (a single point mutation), were tested using K562 cells transfected with 0.3 μg (D) of methylpseudridine mRNA. AK11 was tested using A*11:01, EL9 using A*26:01, KF11 using B*57:01, DA9 using B*14:01, and IW9 using the HLA-I allele B*57:03. Epitopes marked with an asterisk (*) contain at least one polymorphism compared to the sequences of these mRNA strings. [Figure 11A]TCR engagement from presented epitopes processed from different mRNA strings in K562 cell lines generated from two donors: Effector Jurkat cell lines expressing a single TCR were co-incubated with donor-derived target monoHLA-I allele K562 or HLA-I transfected iDCs, and expressed TCR downstream signaling was measured as a log2 factor change signal of luciferase. Target cells were transfected with either pulsed peptides encoded by the tested strings as a positive control to regulate TCR activity against the tested specific epitopes (PP), or with mRNA strings encoding polypeptides. Target iDCs were also tested with pulsed peptides without HLA allele co-transfection to control background presentation by donor-derived endogenous HLA-I (PP 10 μM + eHLA). PHA-L was used as a positive control for TCR activity. Three unique epitopes were tested and reacted with their cognitive TCRs. Four different mRNA strings with different amino acid sequences were tested with two nucleotide sequence optimizations (nt2 and nt3). Strings 1 and 2 were tested by transfecting target K562 cells (A) or target iDCs (B,C) from two donors with 3 μg of methylpseudridine mRNA. Both the tested TL9 and RI8 epitopes were encoded by strings 1 and 2, respectively, with complete sequence matching. In parallel, the epitope TW10 encoded by string 2 contained a single mutant polymorphism but was in complete agreement with string 1. TL9 was tested using the HLA allele B*39:10, EI8 was tested using the HLA allele B*52:01, and TW10 was tested using the HLA alleles B*57:01, B*57:03, and B*58:01. [Figure 11B]Strings 1 and 2 were tested by transfecting target K562 cells (A) or target iDCs (B,C) derived from two donors with 3 μg of methylpseudridine mRNA. Both the tested TL9 and RI8 epitopes were encoded by strings 1 and 2, exhibiting complete sequence matching. In parallel, the epitope TW10 encoded by string 2 contained a single mutant polymorphism but was in complete agreement with string 1. TL9 was tested using the HLA allele B*39:10, EI8 was tested using the HLA allele B*52:01, and TW10 was tested using the HLA alleles B*57:01, B*57:03, and B*58:01. [Figure 11C] Strings 1 and 2 were tested by transfecting target K562 cells (A) or target iDCs (B,C) derived from two donors with 3 μg of methylpseudridine mRNA. Both the tested TL9 and RI8 epitopes were encoded by strings 1 and 2, exhibiting complete sequence matching. In parallel, the epitope TW10 encoded by string 2 contained a single mutant polymorphism but was in complete agreement with string 1. TL9 was tested using the HLA allele B*39:10, EI8 was tested using the HLA allele B*52:01, and TW10 was tested using the HLA alleles B*57:01, B*57:03, and B*58:01. [Figure 12A]CD8+ and CD4+ T cell responses to two different mRNA string (string 1 and string 2) combinations mediated by intramuscular lipid nanoparticle (LNP) nanocarriers: Elispot data of IFN-γ production from CD8+ T cell-depleted and CD4+ T cell-depleted splenocytes stimulated with a peptide pool. Splenocytes were isolated from HLA-A*02:01 CB6F1 transgenic mice and stimulated with a peptide pool for 18 hours on Elispot plates coated with anti-IFN-γ antibody. CD4+ T cell-depleted and CD8+ T cell-depleted splenocytes were prepared from animal populations co-immunized with a 1:1 ratio (2.5 μg + 2.5 μg) single LNP formulation mRNA string 1 + string 2 combination (n=5 mice / group) or from animal populations administered with saline placebo. mRNA combinations were tested using string 1 + string 2 as either unmodified (uRNA) or methylpseudridine (modRNA). Animals were immunized with a single intramuscular injection, and splenocytes were collected 7 days after injection. CD4+ T cell-depleted splenocytes from animals immunized with a string 1 + string 2 modRNA or uRNA combination (Figure 12A) were incubated with an 11-mer peptide pool covering vaccine fragments containing at least one known / predicted HIV epitope (prctrk < 1%) derived from A*02:01 (white box - black circle), H2b / H2d (striped box - white square), or neither (diamond box - white triangle). [Figure 12B] CD8+ T cell-depleted splenocytes from animals immunized with a combination of string 1 + string 2 modRNA or uRNA (Figure 12B) were incubated with an 11-mer peptide pool covering vaccine fragments containing at least one known / predicted HIV epitope (prctrk < 1%) derived from A*02:01 (white box - black circle), H2b / H2d (striped box - white square), or neither (diamond box - white triangle). [Figure 12C]CD4+ T cell-depleted splenocytes and CD8+ T cell-depleted splenocytes were incubated with a 15-mer peptide pool encompassing the entire tetanus toxin (Figure 12C). Background IFN-γ signals from CD4+ T cell-depleted splenocytes and CD8+ T cell-depleted splenocytes incubated with a DMSO peptide diluent control are shown as the median signal measured across all groups tested in panel (A) or (B)—"DMSO median." Quantification of IFNg spot-forming units per 10e6 cells for each peptide pool. [Figure 12D] Figure 12D shows a summary of the experiment. [Examples]

[0531] Example 1 HLA-I-restricted epitopes were selected for improved coverage and escape mutation constraints. These panels of CD8+ T cell epitopes were assembled into multi-epitope cassettes to generate vaccines, as described below.

[0532] A list of highly networked epitopes on CD8+ T cells, providing broad coverage based on HLA class I allele prevalence, has been accumulated and is being used to design a rational set of RNA vaccines. Using the highly networked scoring approach described in Gaiha et al., Structural topology defines protective CD8+ T cell epitopes in the HIV proteome. Science 364, 480-484 (2019), we constructed an initial list of target epitopes based on potentially high thresholds for mutations and using publicly available data demonstrating immunogenicity in humans. We evaluated their binding using an internal pMHC prediction tool and performed pMHC binding assays for epitope-MHC pairs that could not be internally checked. The sequences of each epitope within HIV clade B and C consensus 2021 were identified and their conservation was assessed. For all amino acids in epitopes where patient sequence conservation was less than 75% (according to the Los Alamos HIV database), the inventors considered adding polymorphic epitopes to achieve coverage across the majority of patients. The epitopes were systematized and processed into fragments distributed on two different RNAs. The fragments were separated by auxiliary residues using two linker approaches to produce two sets of two RNAs.

[0533] Each epitope was fitted to an HLA class I allele to which they could bind, and their amino acid sequences were defined in the context of HIV clade B and C consensus, and screened for the most frequent polymorphisms within the clade; see Table 1 below.

[0534] Epitopes were selected from the "A" and "Silver" CTL epitope lists in the Los Alamos HIV database (LADB) (https: / / www.hiv.lanl.gov / content / immunology / tables / tables.html). Epitopes with expected high mutational constraints were selected. After multiple selection rounds, 70 epitopes were listed at the top, with network scores exceeding the selection threshold and possessing literature-defined, proven human immunogenicity and their HLA class-I binding partners.

[0535] [Table 3-1]

[0536] [Table 3-2]

[0537] [Table 3-3]

[0538] Epitope-allelic pairs reported in LADB were collected across numerous studies for further evaluation, and the inventors identified corresponding sequences in the clade B and clade C 2021 consensus sequences from LADB. Where an LADB epitope sequence differed from clade B / C, it was discarded, and only the consensus sequence was retained. To further validate the publicly available information regarding epitope / HLA interactions, the inventors used peptide-MHC binding prediction tools. Their analysis revealed that approximately 40% of the published epitope-allelic pairs were predicted as non-binding pairs. Of these pairs, the inventors aimed to confirm the binding of epitopes where the HLA class I partner allele was observed at a >2% prevalence in either the Caucasian European population or the South African population.

[0539] pMHC stability assays were used to confirm binding. Due to the unavailability of MHC monomers for some alleles, not all desired epitope-allele pairs could be tested. Of the available alleles, 18 epitope / allele pairs were confirmed and retained as binding pairs, while 37 epitope / allele pairs were unbinding pairs and excluded from vaccine design. Epitope / allele pairs that could not be confirmed for binding were still retained in the vaccine but were not used in coverage calculations.

[0540] The selected epitopes are structurally important and should be concentrated in conserved regions of the HIV proteome. To confirm this and ensure that relevant polymorphisms are not lost, the inventors determined what percentage of patient sequences within clades B and C matched the consensus by comparing sequence data (Gag / Pol / Env / Nef) from LADB for the target protein. Each amino acid within each epitope with less than 75% match was added to the most common polymorphic version of the epitope. A total of 17 polymorphic fragments were added to the vaccine.

[0541] Overlapping epitopes were combined and fused into peptide fragments to reduce the number of linkers required and decrease the overall size of the RNA cassette. The fragments were designed to maximize the removal of expected HIV-specific class II core epitopes to limit the undesirable induction of HIV-specific CD4 responses and to minimize the length of the coding sequence.

[0542] The inventors combined the epitopes and fragments into 26 fragments (52 fragments in total) where the epitopes are represented by clade B and clade C consensus sequences, and distributed them between two coding RNA molecules to which 17 polymorphic fragments were added. Four of the fragments containing polymorphisms were matched to the same clade B / C consensus sequence. To reduce redundancy and limit the final RNA template size, consensus fragments from clade C were replaced with their polymorphic versions. The vaccine is bivalent and contains two RNAs: one RNA matches the "clade B" consensus (26 fragments), and the second RNA matches the "clade C" consensus (26 fragments + 4 polymorphic fragments). The inventors distributed the remaining 13 polymorphic fragments across both RNA strings, distributing 7 to the "clade B RNA string" and 6 to the "clade C RNA string." In our final design, as shown in Figures 1 and 2, RNA string 1 ("clade B string") comprises 33 fragments with a cumulative length of 411 amino acids, and string 2 ("clade C string") comprises 32 fragments with a total length of 405 amino acids.

[0543] String 1 ("Clade B string") (SEQ ID NOs. 183 and 187) contains the following 33 fragments, each containing 1, 2, 3, 4, 5, 6, and 7 of the above epitopes derived from the pol, gag, env, and nef proteins: DCKTILKAL, GERIVDIIA, IVTDSQYAL, AEQASQEVKNWM, SFNCGGEFF, GHQAAMQMLKETI, KQNPEIVIY, TAFTIPSI, KLTPLCVTL, TAFTIPSV, QNYTPG PGIRYPLTFGWCFKL, FKRKGGIGGY, NTQGYYPDW, SALSEGATPQDLNTMLNT, LVGPTPVNIIGRNLL, VKVVEEKAFSPEVIPMFSAL, KQNPDIVIYQYMDDL, FRD YVDRFYKTLRA, AEQASQDVKNWM, RAIEAQQHL, VRMYSPTSILDI, PIQKETWEAWWTEYW, HTQGYFPDWQNYT, EIYKRWIILGLNK, GLNKIVRMY, TSTLQEQIGW, PIQKEIWETWWTDYW, GQMVHQPLSPRTLNAW, YTPGPGTRYPLTFGW, GQMVHQAISPRTLNAWVKVV, TPGIRYQYNVL, ACQGVGGPGHKARVL, and GIPHPAGLK.

[0544] String 2 ("Clade C string") (SEQ ID NOs: 185 and 189) contains the following 32 fragments, each containing one, two, three, four, five, six, and seven of the above epitopes derived from the pol, gag, env, and nef proteins: IVTDSQYAL, AEQATQEVKNWM, TSTLQEQIAW, GIPHPAGLK, VRMYSPVSILDI, GLNKIVRMY, GHQAAMQMLKETI, AKNPEIVIY, GQMVHQALSPRTLNAWVKV I, TPGIRYQYNVL, AEQATQDVKNWM, SFNCRGEFF, YTPGPGVRFPLTFGWCF, AQNPEIVIYQYMDDL, QNYTPGPGVRYPLTFGWCFKL, FKRKGGIGGY, NTQGYF PDW, HTQGFFPDWQNYT, PIQKETWETWWTDYW, GQMVHQPISPRTLNAW, GERIIDIIA, LIGPTPVNIIGRNML, DCKTILRAL, VKVIEEKAFSPEVIPMFTAL, PI QKEIWETWWTEYW, FRDYVDRFFKTLRA, RAIEAQQHM, TALSEGATPQDLNTMLNT, DIYKRWIILGLNK, KLTPLCVTL, ACQGVGGPSHKARVL, and TAFTIPST.

[0545] String 1.1 ("Clade B string") (SEQ ID NO: 205) contains the following 33 fragments, each containing 1, 2, 3, 4, 5, 6, and 7 of the above epitopes derived from the pol, gag, env, and nef proteins: DCKTILKAL, GERIVDIIA, IVTDSQYAL, AEQASQEVKNWM, SFNCGGEFF, GHQAAMQMLKETI, KQNPEIVIY, TAFTIPSI, KLTPLCVTL, TAFTIPSV, QNYTPG PGIRYPLTFGWCFKL, FKRKGGIGGY, NTQGFFPDW, SALSEGATPQDLNTMLNT, LVGPTPVNIIGRNLL, VKVVEEKAFSPEVIPMFSAL, KQNPDIVIYQYMDDL, FRD YVDRFYKTLRA, AEQASQDVKNWM, RAIEAQQHL, VRMYSPTSILDI, PIQKETWEAWWTEYW, HTQGYFPDWQNYT, EIYKRWIILGLNK, GLNKIVRMY, TSTLQEQIGW, PIQKETWEIWWTDYW, GQMVHQPLSPRTLNAW, YTPGPGTRYPLTFGW, GQMVHQAISPRTLNAWVKVV, TPGIRYQYNVL, ACQGVGGPGHKARVL, and GIPHPAGLK.

[0546] String 2.1 ("Clade C string") (SEQ ID NO: 208) contains the following 32 fragments, each containing one, two, three, four, five, six, and seven of the above epitopes derived from the pol, gag, env, and nef proteins: IVTDSQYAL, AEQATQEVKNWM, TSTLQEQIAW, GIPHPAGLK, VRMYSPVSILDI, GLNKIVRMY, GHQAAMQMLKETI, AKNPEIVIY, GQMVHQALSPRTLNAWVKV I, TPGIRYQYNVL, AEQATQDVKNWM, SFNCRGEFF, YTPGPGVRFPLTFGWCF, AQNPEIVIYQYMDDL, QNYTPGPGVRYPLTFGWCFKL, FKRKGGIGGY, NTQGYF PDW, HTQGFFPDWQNYT, PIQKETWETWWTDYW, GQMVHQPISPRTLNAW, GERIIDIIA, LIGPTPVNIIGRNML, DCKTILRAL, VKVIEEKAFSPEVIPMFTAL, PI QKETWETWWTEYW, FRDYVDRFFKTLRA, RAIEAQQHM, TALSEGATPQDLNTMLNT, DIYKRWIILGLNK, KLTPLCVTL, ACQGVGGPSHKARVL, and TAFTIPST.

[0547] The inventors added further non-human sequence domains enriched with universal non-HIV class II epitopes to each string to induce CD4+ T cells that can help activate CD8+ T cells.

[0548] Two strategies were used to link the fragments within each string: 1) Direct addition of up to four cleavage-promoting amino acids between each pair of fragments, determined by an internal algorithm. In this strategy, the final string length was approximately 550 amino acids (Figure 1). 2) Flexible non-immunogenic linkers (10 amino acids) adjacent to the cleavage-promoting amino acids (up to 4) between each pair of fragments. In this strategy, the final string length was approximately 950 amino acids (Figure 2). mRNA strings 1 and 2 differ from mRNA strings 1.1 and 2.1 in that they show very little amino acid change, as shown in Figure 3.

[0549] The cleavage-promoting auxiliary residues were selected based on an algorithm using one additional parameter: the selected residues must not coincide with any native adjacent residues present in either the consensus B sequence or the consensus C sequence. This prevents the extension of our fragments to non-network residues, thus avoiding the addition of undesirable non-networked HIV class I epitopes or auxiliary HIV class II epitopes.

[0550] The fragments were randomly assigned to each RNA in a different order for string 1 and string 2 to improve downstream RNA analysis and junctional epitope repetition.

[0551] Example 2: Mass Spectrometry Proteomics Analysis In short, 5 x 10 6Each HEK293T cell was transfected with 1 μg of mRNA encoding strings 1 and 2 and MessengerMax Lipofectamine Reagent. Cells were incubated for 24 hours in or without 1 μM of the proteasome inhibitor MG132. 24 hours after transfection, cells were harvested, washed with 1 mL of PBS, rapidly frozen, and stored until analysis. The frozen cell pellet was thawed and then lysed in cold lysis buffer. Approximately 100 μg of protein from each sample was normalized to a concentration of 1 μg / μL in lysis buffer. The lysates were diluted to reduce the urea content in the lysis buffer to 1 M. Trypsin / Lys-C was added at a ratio of 1 μg per 50 μg of total protein, and the samples were incubated overnight. Synthetic trypsin peptides were labeled with heavy isotopes and injected for targeted MS / MS analysis. Data analysis was performed using Skyline-daily software. Retention times and peptide fragments were identified by matching with spiked heavy isotope-labeled synthetic peptides. The relative abundance of each peptide was calculated by measuring the area under the curve (AUC) of the top 10 most abundant fragment ions. Load normalization was calculated using a panel of peptides derived from housekeeping proteins with constant intracellular abundance. The AUC of each housekeeping peptide was normalized to the average AUC of the entire sample, and the median of each average AUC was calculated for the entire panel of housekeeping proteins. This median is the loading normalization factor for each sample. Relative abundance was calculated by dividing the AUC of each peptide derived from each mRNA string by the loading normalization factor. The AUC of each target peptide was normalized to the average AUC of the entire sample.

[0552] Figure 4 shows the in vitro expression of mRNA-encoded polypeptides from two mRNA strings (string 1 no GS, string 2 no GS, and string 2 GS) with and without a flexible glycine-serine (GS) linker. For each string, three different nucleotide sequences were generated (nt1, nt2, and nt3), where nt1 corresponds to the wild-type sequence and nt2 and nt3 represent two different codon optimization methods. Each string was transcribed in vitro in the presence of either unmodified (uRNA) or methylpseudridine (modRNA). For string 2 GS, only nt3 was tested. Each mRNA string, i.e., uRNA / modRNA, nt1-3, strings 1-2, GS / no GS, was separately transfected into human cell lines in or without the proteasome inhibitor MG132. The relative abundance of each peptide is shown (A) for two trypsin peptides common to each string tested with modRNA. (B) The relative abundance of each peptide is shown for trypsin peptides specific to string 1 no-GS, modRNA, and uRNA. (C) The relative abundance of each peptide is shown for trypsin peptides specific to string 2 GS and non-GS, modRNA and uRNA. The trypsin peptides YPLTFGWCFK and IYSYFPSVISK, common to all tested strings, are located in the central and C-terminal regions of polypeptides encoded by mRNA string 1 non-GS (D), mRNA string 2 non-GS and GS (E). The positions of the string-specific trypsin peptide TSTLQEQIGWAR(D), tested for string 1 without GS, and the string-specific trypsin peptide QNYTPGPGVR(E), tested for string 2 with and without GS, are also shown.

[0553] The experiment as a whole demonstrates that higher in vitro expression can be achieved using modRNA compared to uRNA. Detection of the IYSYFPSVISK trypsin peptide indicates that different strings and their codon-optimized versions were translated to the C-terminal region of the encoded polypeptide. Codon optimization appears to influence the level of translation from different strings and is sequence-dependent. The presence of the proteasome inhibitor MG132 resulted in an increase in detectable polypeptides, indicating that the translated polypeptides target the proteasome degradation pathway required for epitope processing.

[0554] Example 3: Immunopeptidemics A375 cells were used for transfection, either by manipulating them to stably express the target BAP-tagged allele or by overexpressing the target allele intracellularly. 5 × 10⁷ manipulated cells were transfected with 1 μg of mRNA encoding modRNA strings 1 and 2 and Messenger Max Lipofectamine reagent, and then harvested. Transfected cells were lysed and clarified before processing. For BAP-tagged cell lines, the clarified lysates were biotinylated with biotin, ATP, and BirA, and then incubated with NeutrAvidin beads to obtain affinity-enriched biotinylated HLA-peptide complexes. For overexpression cell lines, pan-class-I antibody was loaded onto Sepharose beads and incubated with the clarified lysates to isolate all HLA-peptide complexes. The peptides were washed, eluted from the antibody-bound HLA complexes, and isolated by molecular weight filtration. The isolated peptides were then labeled with TMTzero, reduced using TCEP, alkylated with iodoacetamide (IAA), desalted, and analyzed by nLC-MS / MS. Samples were resuspended in 3% acetonitrile, 5% formic acid, with 150 femtomoles of each TMT-131C-labeled heavy isotope-labeled synthetic peptide added per injection. Peptides were chromatographically separated using a Vanquish Neo uHPLC with an Aurora Ultimate packed emitter column, heated at 60°C during separation. Peptides were eluted into an Orbitrap Ascend Tribrid mass spectrometer with a Nanospray Flex ion source. Data were acquired using internal standard-triggered parallel reaction monitoring. A fast, low-resolution precursor scan was used to explore m / z values ​​in the inclusion list associated with the internal standard for the TMT-131C-labeled heavy isotope-labeled synthetic peptide. If m / z values ​​were observed from the inclusion list, a fast, low-resolution tandem mass spectrometry (MS / MS) survey scan was performed to monitor characteristic fragment ions associated with the peptide.If five or more monitored ions were observed, a second MS / MS scan was performed with a mass offset equal to the difference between the TMT-131C-labeled heavy isotope-labeled synthetic peptide and the TMTzero-labeled target HLA peptide.

[0555] Data analysis was performed using Skyline-daily software. Retention times and peptide fragments were identified by matching with spiked heavy isotope-labeled synthetic peptides. The relative abundance of each peptide was calculated by measuring the AUC of the top 10 most abundant fragment ions.

[0556] Figure 5 shows the in vitro processing and presentation of HLA-I alleles from mRNA-encoded polypeptides from two modRNA strings (string 1 and string 2) evaluated by immunopeptidomics. Each string was transfected into the A375 cell line encoding a single HLA class I allele, and HIV-specific epitopes in string 1 (AB) and string 2 (CD) were detected by targeted mass spectrometry. The detected epitope locations are annotated below string 1 (A) and string 2 (C). Detailed information related to each detected epitope, such as the HLA class-I allele, the location of the fragment ends, and the non-HIV junction sequence (end), as well as sequence matches for string 1, are shown in panel (B) and the corresponding information for string 2 is shown in panel (D). Legend: Epitope sequence perfectly matches 2021 consensus B=ConB; Epitope sequence perfectly matches 2021 consensus C=ConC; Epitope sequence perfectly matches consensus B and C=ConB / C; Epitope does not perfectly match either consensus B or C = polymorphism.

[0557] These datasets confirm the processing and presentation of epitopes from mRNA-encoded polypeptides to human MHC class I alleles. Detection of epitopes located at the ends of fragments by pull-down confirms polypeptide cleavage between fragments in non-HIV junction regions.

[0558] Example 4: T cell activation assay using K562 NFAT-TCR / CD3 effector cells were purchased from Promega as cryopreserved cells. These Jurkat T cells express luciferase driven by the NFAT response element (NFAT-RE) as a reporter. The endogenous TCR and β2M genes were removed in Jurkat reporter cells by CRISPR-Casp9 mediated knockout. The α and β chains of the CD8 coreceptor were stably inserted into Jurkat reporter cells via transposons. Two mRNAs encoding the α and β chains of the TCR clone were co-electroporated into reporter NFAT-luciferase cells. After transfection, 2 × 10⁴ Jurkat cells were co-cultured with K562 cells in a 2:1 ratio in a 384-well plate containing 25 μL of medium (RPMI1640 + 10% non-thermally inactivated FBS) / well. Prior to co-culture, K562 cells were transfected with mRNA encoding an HIV-derived polypeptide (string 1 or string 2) and mRNA encoding an HLA class I allele. As a positive control for the specificity of the TCRs used, K562 cells transfected only with mRNA encoding HLA-I pulsed with a minimal HIV-epitope peptide target were co-cultured with each TCR-encoded mRNA-transfected Jurkat reporter cells. Furthermore, TCR expression and downstream signaling were confirmed using stimulation with 2 μg / ml phytohemagglutinin-L (PHA-L). Transient expression of transfected HLA class-I was verified by flow cytometry after staining with HLA-A or HLA-B specific antibodies. After 16 hours, an equal volume (15 μL) of luciferin (Bio-Glo, Promega) was added to each well, and luciferase activity was measured using a luminescence plate reader. Luminescence signals measured in different wells corresponded to the level of TCR-mediated activation in Jurkat cells. For each TCR, the log2 factor change in luminescence compared to an "effector-only control" was calculated, and the 2x change cutoff was used to determine the specific TCR.

[0559] Figure 6 shows TCR engagement from epitopes processed from different mRNA strings and presented in the K562 cell line. Jurkat cell lines expressing a single TCR were co-incubated with monoHLA-I allele K562 cells, and the expressed TCR downstream signaling was measured as a log2 factor change signal of luciferase. K562 was pulsed (PP) with peptides encoded by the tested strings as a positive control to control TCR activity against the tested specific epitopes, or transfected with mRNA encoding polypeptides (striped bars). Two different mRNA strings (string 1 and string 2) were tested with different nucleotide optimizations (nt2 and nt3). PHA-L was used as a positive control for TCR activity. % unique epitopes AK11, DA9, +DA9+, KF11, and IW9 were tested and reacted with their cognitive TCRs. The processing and presentation of the precise epitopes KF11 and IW9 were investigated from different mRNA strings. The polymorphic version of DA9, shown as +DA9+ and encoded by the string 2 version, was cross-reactive with the non-cognitive DA9-specific TCR. Similarly, IW9 was presented by both the cognitive HLA-I allele B*57:03 and the non-cognitive B*57:01 allele, shown as +B*57:01+ for both string 1 and string 2. Legend: AK11: ACQGVGGPGHK; DA9: DRFYKTLRA; +DA9+: DRFFKTLRA; KF11: KAFSPEVIPMF; IW9: ISPRTLNAW.

[0560] These datasets confirm that epitopes can be generated by enzymatically processing polypeptides encoded in all mRNA strings, and that these epitopes are then presented on their cognitive HLA class I alleles. The epitope:HLA-I complex can induce TCR downstream signaling in vitro. These data also show the effect of nucleotide sequence optimization on the magnitude of the TCR-mediated response.

[0561] Example 5: T cell activation assay using iDCs Cells were used and prepared as described in Example 4. After transfection, 2 × 10⁴ Jurkat cells were co-cultured with immature dendritic cell (iDC) cells in a 2:1 ratio in a 384-well plate containing 25 μL of medium (RPMI1640 + 10% non-thermally inactivated FBS) / well. Prior to co-culture, iDCs were prepared from donor PBMCs. Briefly, CD14+ monocytes were actively isolated from human PBMCs and cultured for 5 days in RPMI16407 / 5% pooled human serum (PHS) / 1% sodium pyruvate / 0.5% penicillin-streptomycin with 1000 U / mL IL-4 and 1600 U / mL GM-CSF to produce iDCs. Next, iDCs were transfected with mRNA encoding HIV-derived polypeptides (strings 1 and 2) and mRNA encoding HLA class I alleles, and then co-incubated with Jurkat reporter cells. As a positive control for the specificity of the TCRs used, iDCs transfected with only HLA-I encoding mRNA and pulsed with minimal HIV-epitope peptide targets were co-cultured with Jurkat reporter cells transfected with mRNA encoding each TCR. A control containing iDCs pulsed with peptide only was included to evaluate the effect of endogenous HLA-I alleles from PBMC donors. Furthermore, TCR expression and downstream signaling were confirmed using stimulation with 2 μg / ml phytohemagglutinin-L (PHA-L). After 16 hours, an equal volume (15 μL) of luciferin (Bio-Glo, Promega) was added to each well, and luciferase activity was measured using a luminescence plate reader. Luminescence signals measured in different wells corresponded to the level of TCR-mediated activation in Jurkat cells. For each TCR, the log2 factor change in luminescence compared to an "effector-only control" was calculated, and the 2x change cutoff was used to determine the specific TCR.

[0562] Figure 7 shows TCR engagement from processed and presented epitopes in iDCs generated from two donors using different mRNA strings. Jurkat cell lines expressing a single TCR were co-incubated with iDCs, and the expressed TCR downstream signaling was measured as a log2 factor change signal of luciferase. The iDCs were pulsed with peptides encoded by the tested strings. To control TCR activity, pulsed iDCs were either pre-co-transfected with mRNA encoding an HLA class I allele (PP) or, without co-transfection, expressed only donor-derived endogenous HLA-I to control potential endogenous HLA-I mediated background presentation (PP+eHLA). iDCs were co-transfected with polypeptide-encoding mRNA and cognitive HLA class I alleles (striped bars). Two different mRNA strings (string 1 and string 2) were tested with different nucleotide optimizations (nt2 and nt3). PHA-L was used as a positive control for TCR activity. For donor 1(A) and donor 2(B), six unique epitopes (AK11, DA9, +DA9+, KF11, EL9, and IW9) and five unique epitopes (AK11, DA9, +DA9+, EL9, and IW9) were tested and reacted with their cognitive TCRs. The processing and presentation of epitope IW9 were investigated from two different mRNA strings (string 1 and string 2). The polymorphic version of DA9, encoded by the string 2 version and shown as +DA9+, was cross-reactive with the non-cognitive DA9-specific TCR. Similarly, IW9 was presented by both the cognitive HLA-I allele B*57:03 and the non-cognitive B*57:01 allele, shown as +B*57:01+ for both string 1 and string 2. Legend: AK11: ACQGVGGPGHK; DA9: DRFYKTLRA; +DA9+: DRFFKTLRA; KF11: KAFSPEVIPMF; IW9: ISPRTLNAW; EL9: EVIPMFSAL.

[0563] These datasets demonstrate that polypeptides encoded in all mRNA strings can be enzymatically processed to generate epitopes, which are then presented on their cognitive HLA class I alleles in human iDCs derived from PBMCs. The epitope:HLA-I complex can induce TCR downstream signaling in vitro. These data also show the effect of nucleotide sequence optimization on the magnitude of the TCR-mediated response.

[0564] Example 6 Immunogenicity studies will be conducted in BALB-C (wild-type) mice and HLA knock-in mice using the RNA molecules shown in Figures 1 and 2. For this purpose, compositions containing lipids for intramuscular (im) or intravenous (iv) delivery will be combined with the RNA molecules. Mouse vaccination

[0565] Wild-type mice: Wild-type mice are vaccinated either in vitro with a lipid nanoparticle formulation or in vitro with a lipoplex formulation. Wild-type mice are inoculated with vaccine, i.e., co-formulated 2-4 RNA or monoformulated 2-4 RNA, at up to three doses (low, medium, high) according to different prime / boost regimens - i.e., time between injections and number of boosts. Vaccine-induced CD8+ T cells are quantified from splenocytes and whole blood via peptide pool ELISPOT at euthanasia. Drainage lymph nodes are collected for immunostaining of relevant markers. Cytokine levels in the blood are measured by multiplex ELISA during the course of the experiment. The amount of HIV-specific CD8+ T cells is compared across regimens, dosing, formulations (if applicable), and RNA platforms (if applicable) to define the optimal vaccination schedule for both injection routes.

[0566] HLA knock-in (HLA KI) mice: HLA KI mice are vaccinated either im with a lipid nanoparticle formulation or iv with a lipoplex formulation. The HLA panel is designed to cover relevant HLA-I alleles, i.e., those that are very common in the population or related to negative / positive outcomes during the course of HIV infection. HLA KI mice are vaccinated for each RNA platform / lipid formulation according to the optimal regimen defined for wild-type mice. Vaccine-induced CD8+ T cells are quantified from splenocytes and whole blood via ELISPOT with specific peptides against the HLA-Is under consideration. Drainage lymph nodes are collected for immunostaining of relevant markers. These experiments confirm the proper presentation and immunogenicity of the selected epitopes.

[0567] Example 7 Sample preparation and immunization mRNAs generated by in vitro transcription were tested for endotoxin content and formulated in lipoplex lipid nanocarriers. Each mRNA was formulated individually at one of the following concentrations: 0.1, 0.2, or 0.25 mg / mL, 24 hours prior to injection, and kept at 4°C. On the day of immunization, the particles were lightly resuspended, diluted in PBS where applicable, and / or co-mixed to a final injection volume of 100 μL. CB6F1-Tg(HLA-A*0201 / H2-Kb)A*0201(CB6F1) mice were immunized by tail vein injection (29G needle) on d0 (prime), d7 (boost), and d14 (boost). Six hours after immunization, blood samples were collected from each animal and stored at -80°C. On d21, one week after the last boost, the animals were sacrificially killed, and the spleen was immediately collected to isolate CD4+ and CD8+ T cells. The animals were tracked for health monitoring throughout the entire experiment, including until their death.

[0568] T cell isolation and ELISpot Freshly harvested splenocytes were subjected to negative CD4- or CD8- isolation kits (Miltenyi biotec catalog numbers 130-104-454 and 130-104-075). Briefly, 10 e7 cells were incubated with a biotin antibody cocktail for 5 minutes at 4°C. The antibody / cell mixture was then incubated with anti-biotin microbeads (2 μl / 10 e6 cells) for 10 minutes at 4°C. Unlabeled CD4+ or CD8+ T cells were separated from labeled cells via magnetic separation by collecting the flow-through of the cell suspension. Cells were immediately used for the IFNg-ELISpot assay. 5 × 10 e4 BMDCs derived from CB6F1 were pulsed with 2–6 μg / mL peptide test or control at 37°C for 2 hours, and then co-incubated overnight with 1 × 10 e5 isolated T cells on an anti-IFNg coated plate (Mabtech 321-4HPW-2). Spots were detected using a biotinylated anti-mouse IFNg detection antibody, followed by incubation with streptavidin-HRP. CD8+ and CD4+ T cell responses to two different mRNA strings (string 1 and string 2)

[0569] Elispot data on IFN-γ production by isolated CD8+ T cells and isolated CD4+ T cells stimulated with a peptide pool. T cells were isolated from HLA-A*02:01 CB6F1 transgenic mice and stimulated with a peptide pool for 18 hours on Elispot plates coated with anti-IFN-γ antibody. CD8+ T cells from groups immunized with string 1 (Figure 8A) or string 2 (Figure 8B) (n=5 mice / group) were incubated with an 11-mer peptide pool covering vaccine fragments containing at least one known / predicted HIV-1 epitope (prctrk<1%) derived from A*02:01 (white box-black circle), H2b / H2d (striped box-white square), or neither (diamond box-white triangle). CD8+ T cells from groups immunized with String 1 (Figure 8C) or String 2 (Figure 8D) were incubated with heterologous minimal A*02:01 (white box - black circle) or H2b / H2b (striped box - white square) HIV epitope pools, i.e., minimal HIV epitopes (8-11 mers) containing at least one polymorphism compared to the immune string sequence. Pools I / K tested in the String ...

Claims

1. A composition comprising an RNA molecule, wherein the RNA molecule comprises an expression cassette encoding an immunogenic peptide, the peptide comprises at least two fragments, each fragment comprising at least one epitope, the epitope being derived from an amino acid sequence encoded by human immunodeficiency virus (HIV), and the epitope being contained in or comprising a sequence selected from any one of SEQ ID NOs: 1-148 and 198-202 or a variant thereof.

2. The composition according to claim 1, wherein at least one of the fragments comprises at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 epitopes.

3. The composition according to claim 1 or 2, wherein the epitope in one fragment is different from the epitopes in at least one other fragment or all of the fragments.

4. The composition according to any one of claims 1 to 3, wherein at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or all of the fragments are free from epitopes contained in other fragments.

5. The composition according to any one of claims 1 to 4, wherein the arrangement of the epitopes overlaps in at least one of the fragments.

6. The composition according to any one of claims 1 to 5, wherein the arrangement of the epitopes does not overlap in at least one of the fragments.

7. The composition according to any one of claims 1 to 6, wherein the peptide comprises in total at least 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, 35, or 40 fragments.

8. The composition according to any one of claims 1 to 7, wherein the peptide comprises in total at least 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, 35, 40, 45, 50, 55, 60, 65, 66, 67, 68, 69, or 70 epitopes from among the epitopes shown in SEQ ID NOs: 1 to 148 and 198 to 202.

9. The composition according to any one of claims 1 to 8, wherein each fragment contains the same or different number of epitopes.

10. The composition according to any one of claims 1 to 9, wherein the epitopes are combined within the expression cassette in such a way that the length of the RNA molecule is minimized.

11. The composition according to any one of claims 1 to 10, wherein the epitope is derived from the HIV proteins gp41, gp120, p31, p24, nef, p51, protease, tat, rev, vif, vpr, vpx, or vpu.

12. The composition according to any one of claims 1 to 11, wherein the peptide comprises at least one epitope derived from each of the HIV gp41, gp120, or nef proteins.

13. The composition according to any one of claims 1 to 12, wherein at least one amino acid separates the sequence of the non-overlapping epitope, and / or at least one amino acid separates at least two fragments.

14. The composition according to any one of claims 1 to 13, wherein two or more non-overlapping epitopes contained within one or more fragments are not adjacent at 5' and 3' by the consensus adjacent amino acid sequences of the HIV amino acid sequence of the clade from which the epitopes originate.

15. The composition according to any one of claims 1 to 14, wherein the linker separates at least two fragments.

16. The composition according to any one of claims 1 to 15, wherein the variant of the epitope is a polymorphism of the epitope, the epitope is a consensus sequence derived from at least two different clones of HIV, and / or the variant differs from the epitope by one, two, three, four, or five amino acids.

17. The composition according to any one of claims 1 to 16, further comprising a second RNA molecule, the second RNA molecule comprising a second expression cassette encoding a second immunogenic peptide, the peptide comprising at least two fragments, each fragment comprising at least one epitope, the epitope being derived from an amino acid sequence encoded by human immunodeficiency virus (HIV), and the epitope being contained in or comprising a sequence selected from any one of SEQ ID NOs: 1 to 148 and 198 to 202 or a variant thereof.

18. The composition according to claim 17, wherein at least one of the fragments of the second immunogenic peptide comprises at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 epitopes.

19. The composition according to claim 17 or 18, wherein the epitope in one fragment of the second immunogenic peptide is different from the epitopes in at least one other fragment of the second immunogenic peptide or in all other fragments of the second immunogenic peptide.

20. The composition according to any one of claims 17 to 19, wherein at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or all of the fragment of the second immunogenic peptide does not contain an epitope contained in another fragment of the second immunogenic peptide.

21. The composition according to any one of claims 17 to 20, wherein the sequence of the epitope overlaps in at least one of the fragments of the second immunogenic peptide.

22. The composition according to any one of claims 17 to 21, wherein the sequence of the epitope does not overlap in at least one of the fragments of the second immunogenic peptide.

23. The composition according to any one of claims 17 to 22, wherein the second immunogenic peptide comprises in total at least 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, 35, or 40 fragments.

24. The composition according to any one of claims 17 to 23, wherein the second immunogenic peptide comprises in total at least 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, 35, 40, 45, 50, 55, 60, 65, 66, 67, 68, 69, or 70 epitopes from among the epitopes shown in SEQ ID NOs: 1 to 148 and 198 to 202.

25. The composition according to any one of claims 17 to 24, wherein each fragment of the second immunogenic peptide comprises the same or different number of epitopes.

26. The composition according to any one of claims 17 to 25, wherein the epitope is combined within the expression cassette of the second immunogenic RNA molecule such that the length of the RNA molecule is minimized.

27. The composition according to any one of claims 17 to 26, wherein the epitope is derived from the HIV proteins gp41, gp120, p31, p24, nef, p51, protease, tat, rev, vif, vpr, vpx, or vpu.

28. The composition according to any one of claims 17 to 27, wherein the second immunogenic peptide comprises at least one epitope derived from each of the HIV gp41, gp120, or nef proteins.

29. The composition according to any one of claims 17 to 28, wherein two or more non-overlapping epitopes contained in one fragment of the second immunogenic peptide or two or more fragments of the second immunogenic peptide are not adjacent at 5' and 3' by the consensus adjacent amino acid sequences of the HIV amino acid sequence of the clade from which the epitopes originate.

30. The composition according to any one of claims 17 to 29, wherein at least one amino acid isolates the sequence of the non-overlapping epitope, and / or at least one amino acid isolates at least two fragments of the second immunogenic peptide.

31. The composition according to any one of claims 17 to 30, wherein the linker separates at least two fragments of the second immunogenic peptide.

32. The composition according to any one of claims 17 to 31, wherein the variant of the epitope is a polymorphism of the epitope, the epitope is a consensus sequence derived from at least two different clones of HIV, and / or the variant differs from the epitope by one, two, three, four, or five amino acids.

33. The composition according to any one of claims 1 to 32, wherein the immunogenic peptide comprises an epitope derived from HIV of one clade, and the second immunogenic peptide comprises an epitope derived from HIV of another clade.

34. The composition according to claim 33, wherein the one clade is clade B, and the other clade is clade C.

35. The composition according to claim 33 or 34, wherein each immunogenic peptide comprises an epitope that is identical in both clades.

36. The composition according to any one of claims 33 to 35, wherein each immunogenic peptide contains a polymorphic epitope derived from its respective clade.

37. The composition according to any one of claims 1 to 36, wherein the epitope is a T cell epitope.

38. The composition according to any one of claims 1 to 37, wherein the epitope is the CD8 minimum epitope.

39. The composition according to any one of claims 1 to 38, wherein the epitope has a length of 9 to 21 amino acids.

40. The composition according to any one of claims 1 to 39, wherein the epitope has a length of 9 amino acids or more.

41. The composition according to any one of claims 1 to 40, wherein the epitope has a length of 10 amino acids or more.

42. The composition according to any one of claims 1 to 41, wherein the epitope has a length of 11 amino acids or more.

43. The composition according to any one of claims 1 to 42, wherein the epitope has a length of 9 to 14 amino acids.

44. The composition according to any one of claims 1 to 43, wherein the epitope has a length of 9 to 13 amino acids.

45. The composition according to any one of claims 1 to 44, wherein the epitope has a length of 9 to 12 amino acids.

46. The composition according to any one of claims 1 to 45, wherein the epitope has a length of 9 to 11 amino acids.

47. The composition according to any one of claims 1 to 46, wherein the epitope has a length of 9 or 10 amino acids.

48. The composition according to any one of claims 1 to 47, wherein the epitope has a length of 9 amino acids.

49. The composition according to any one of claims 1 to 48, wherein the fragment is in the range of 9 to 21 amino acid lengths.

50. The aforementioned peptide a) Sequence numbers 60, 103, 98, 149, 2, 150, 142, 86, 1, 141, 151, 152, 130, 153, 154, 155, 156, 157, 158, 4, 159, 160, 161, 162, 35, 28, 163, 164, 165, 166, 87, 167 and 85, or b) Sequence numbers 60, 103, 98, 149, 2, 150, 142, 86, 1, 141, 151, 152, 199, 153, 154, 155, 156, 157, 158, 4, 159, 160, 161, 162, 35, 28, 203, 164, 165, 166, 87, 167 and 85 A composition according to any one of claims 1 to 49, comprising the amino acid sequence.

51. The aforementioned peptide a) Sequence numbers 98, 125, 29, 85, 168, 35, 150, 149, 169, 87, 57, 3, 170, 171, 172, 99, 131, 173, 174, 175, 104, 176, 61, 177, 178, 179, 5, 180, 181, 1, 182 and 140, or b) Sequence numbers 98, 125, 29, 85, 168, 35, 150, 149, 169, 87, 57, 3, 170, 171, 172, 99, 131, 173, 174, 175, 104, 176, 61, 177, 204, 179, 5, 180, 181, 1, 182 and 140 A composition according to any one of claims 1 to 49, comprising the amino acid sequence.

52. The composition according to any one of claims 1 to 49, wherein the peptide comprises the amino acid sequence of SEQ ID NO: 183, 185, 187, 189, 205, or 208.

53. The first peptide described above, a) Sequence numbers 60, 103, 98, 149, 2, 150, 142, 86, 1, 141, 151, 152, 130, 153, 154, 155, 156, 157, 158, 4, 159, 160, 161, 162, 35, 28, 163, 164, 165, 166, 87, 167 and 85, or b) Sequence numbers 60, 103, 98, 149, 2, 150, 142, 86, 1, 141, 151, 152, 199, 153, 154, 155, 156, 157, 158, 4, 159, 160, 161, 162, 35, 28, 203, 164, 165, 166, 87, 167 and 85 It contains the amino acid sequence, The second peptide described above, c) Sequence numbers 98, 125, 29, 85, 168, 35, 150, 149, 169, 87, 57, 3, 170, 171, 172, 99, 131, 173, 174, 175, 104, 176, 61, 177, 178, 179, 5, 180, 181, 1, 182 and 140, or d) Sequence numbers 98, 125, 29, 85, 168, 35, 150, 149, 169, 87, 57, 3, 170, 171, 172, 99, 131, 173, 174, 175, 104, 176, 61, 177, 204, 179, 5, 180, 181, 1, 182 and 140 The amino acid sequence includes, The composition according to any one of claims 17 to 49.

54. a) The first peptide contains the amino acid sequence of SEQ ID NO: 183 or 205, and the second peptide contains the amino acid sequence of 185 or 208, or b) The first peptide comprises the amino acid sequence of SEQ ID NO: 187, and the second peptide comprises the amino acid sequence of SEQ ID NO: 189, Optionally, the first peptide contains the amino acid of SEQ ID NO: 205, and the second peptide contains the amino acid of SEQ ID NO:

208. The composition according to any one of claims 17 to 49.

55. The composition according to any one of claims 1 to 54, wherein the RNA molecule comprises the nucleotide sequence of SEQ ID NOs: 184, 186, 188, 190, 206, 207, 209, 210, or 211-218.

56. The composition according to any one of claims 1 to 55, wherein the epitope is a non-protective epitope that can cause CD8+ T cell protective breakdown.

57. The composition according to claim 56, wherein the protective breakdown is T cell escape or T cell exhaustion or loss of CD4+ helper T cells.

58. The composition according to any one of claims 1 to 57, wherein the peptide further comprises a sequence that enhances epitope presentation on the surface of cells.

59. The composition according to claim 58, wherein the cells are immune cells.

60. The composition according to claim 59, wherein the immune cells are antigen-presenting cells (APCs).

61. The composition according to any one of claims 1 to 60, further comprising the peptide signal peptide.

62. The composition according to any one of claims 1 to 61, wherein the peptide further comprises an MHC class I transport signal (MITD).

63. The composition according to any one of claims 1 to 62, wherein the peptide further comprises a non-HIV HLA-II helper epitope.

64. The composition according to claim 63, wherein the helper epitope is a P2 and / or P16 amino acid sequence derived from tetanus toxoid (TT) of Clostridium tetanus.

65. The composition according to any one of claims 1 to 64, wherein the RNA molecule is linear or cyclic.

66. The composition according to any one of claims 1 to 65, wherein the RNA molecule includes a 5' cap.

67. The composition according to claim 66, wherein the 5' cap is a modified or artificial cap or a cap analogue.

68. The composition according to any one of claims 1 to 67, wherein the expression cassette further comprises a 5' untranslated region (5'UTR) and / or a 3' untranslated region (3'UTR).

69. The composition according to claim 68, wherein the 5'UTR comprises the nucleotide sequence of SEQ ID NO: 195, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity with the nucleotide sequence of SEQ ID NO:

195.

70. The composition according to claim 69, wherein the 3'UTR comprises the nucleotide sequence of SEQ ID NO: 196, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity with the nucleotide sequence of SEQ ID NO:

196.

71. The composition according to any one of claims 1 to 70, wherein the expression cassette further comprises a poly(A) structure.

72. The composition according to claim 71, wherein the poly-A structure is an interrupted poly-A structure.

73. The composition according to claim 72, wherein the polyA structure comprises the nucleotide sequence of Sequence ID No. 197, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity with the nucleotide sequence of Sequence ID No.

197.

74. The composition according to any one of claims 1 to 73, wherein the RNA molecule is a replicable RNA molecule.

75. The composition according to claim 74, wherein the replicable RNA molecule further encodes an RNA-dependent RNA polymerase (replicase) capable of replicating the replicable RNA molecule.

76. The composition according to claim 74, wherein the replicable RNA molecule does not encode RNA-dependent RNA polymerase (replicase).

77. The composition according to any one of claims 74 to 76, further comprising a non-replicable RNA molecule encoding an RNA-dependent RNA polymerase (replicase) capable of replicating the replicatable RNA molecule.

78. The composition according to any one of claims 1 to 77, wherein the RNA molecule is non-immunogenic.

79. The composition according to claim 78, wherein the RNA molecule is made non-immunogenic by the removal of double-stranded RNA.

80. The composition according to any one of claims 1 to 79, wherein the RNA molecule includes nucleotide modifications.

81. The composition according to claim 80, wherein the modification is the substitution of one or more U residues with pseudouridine, N1-methylpseudridine, or 5-methyluridine.

82. The composition according to claim 81, wherein one or more of the substituted U residues are N1-methylpsuduridine.

83. The composition according to claim 80 or 81, wherein at least 50%, at least 70%, at least 90%, at least 99%, or 100% of the U residues in the RNA molecule are substituted.

84. The composition according to any one of claims 1 to 83, wherein the RNA molecule is formulated into a composition comprising at least one lipid.

85. The composition according to claim 84, wherein the RNA molecule and at least one lipid form particles.

86. The composition according to claim 85, wherein the particles are lipid nanoparticles (LNPs), lipoplexes (LPXs), or liposomes.

87. The aforementioned particles are nanoparticles, (i) The number of positive charges in the nanoparticles does not exceed the number of negative charges in the nanoparticles, and / or (ii) The nanoparticles have a net negative charge, and / or (iii) The zeta potential of the nanoparticles is 0 or less. The composition according to claim 85 or 86.

88. The composition according to claim 87, wherein the charge ratio of positive charge to negative charge in the nanoparticles is 1:1 to 1:8, preferably 1:1 to 1:

4.

89. The composition according to any one of claims 84 to 88, wherein the at least one lipid is a cationic lipid.

90. The composition according to claim 89, wherein the lipid comprises a cationic head group.

91. The composition according to claims 84 to 90, wherein the lipid is a pH-responsive lipid.

92. The composition according to any one of claims 84 to 91, wherein the at least one lipid is a PEG-modified lipid.

93. The composition according to any one of claims 84 to 92, further comprising at least one helper lipid.

94. The composition according to claim 93, wherein the helper lipid is a neutral lipid.

95. The composition according to any one of claims 89 or 90, wherein the at least one cationic lipid comprises 1,2-di-O-octadecenyl-3-trimethylammoniumpropane (DOTMA), 1,2-dioleyloxy-3-dimethylaminopropane (DODMA), and / or 1,2-dioleoyl-3-trimethylammoniumpropane (DOTAP).

96. The composition according to any one of claims 93 or 94, wherein the at least one helper lipid comprises 1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine (DOPE), cholesterol (Chol), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), and / or 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC).

97. The composition according to claim 93 or 94, wherein the molar ratio of the at least one cationic lipid to the at least one helper lipid is 10:0 to 3:7, preferably 9:1 to 3:7, 4:1 to 1:2, 4:1 to 2:3, 7:3 to 1:1, or 2:1 to 1:1, preferably about 1:

1.

98. The composition according to any one of claims 87 to 97, wherein the nanoparticles are a lipoplex containing DODMA and DOPE in a molar ratio of 10:0 to 1:9, preferably 8:2 to 3:7, more preferably 7:3 to 5:5, and the charge ratio of positive charge in DODMA to negative charge in RNA is 1.8:2 to 0.8:2, more preferably 1.6:2 to 1:2, even more preferably 1.4:2 to 1.1:2, and even more preferably about 1.2:

2.

99. The composition according to any one of claims 87 to 97, wherein the nanoparticles are a lipoplex containing DODMA and cholesterol in a molar ratio of 10:0 to 1:9, preferably 8:2 to 3:7, more preferably 7:3 to 5:5, and the charge ratio of positive charge in DODMA to negative charge in RNA is 1.8:2 to 0.8:2, more preferably 1.6:2 to 1:2, even more preferably 1.4:2 to 1.1:2, and even more preferably about 1.2:

2.

100. The composition according to any one of claims 87 to 97, wherein the nanoparticles are a lipoplex containing DODMA and DSPC in a molar ratio of 10:0 to 1:9, preferably 8:2 to 3:7, more preferably 7:3 to 5:5, and the charge ratio of positive charge in DODMA to negative charge in RNA is 1.8:2 to 0.8:2, more preferably 1.6:2 to 1:2, even more preferably 1.4:2 to 1.1:2, and even more preferably about 1.2:

2.

101. The composition according to any one of claims 87 to 97, wherein the nanoparticles are a lipoplex containing DODMA:cholesterol:DOPE:PEGcerC16 in a molar ratio of 40:48:10:

2.

102. The composition according to any one of claims 87 to 97, wherein the nanoparticles are a lipoplex containing DOTMA and DOPE in a molar ratio of 10:0 to 1:9, preferably 8:2 to 3:7, more preferably 7:3 to 5:5, and the charge ratio of positive charge in DOTMA to negative charge in RNA is 1.8:2 to 0.8:2, more preferably 1.6:2 to 1:2, even more preferably 1.4:2 to 1.1:2, and even more preferably about 1.2:

2.

103. The composition according to any one of claims 87 to 97, wherein the nanoparticles are a lipoplex containing DOTMA and cholesterol in a molar ratio of 10:0 to 1:9, preferably 8:2 to 3:7, more preferably 7:3 to 5:5, and the charge ratio of positive charge in DOTMA to negative charge in RNA is 1.8:2 to 0.8:2, more preferably 1.6:2 to 1:2, even more preferably 1.4:2 to 1.1:2, and even more preferably about 1.2:

2.

104. The composition according to any one of claims 87 to 97, wherein the nanoparticles are a lipoplex containing DOTAP and DOPE in a molar ratio of 10:0 to 1:9, preferably 8:2 to 3:7, more preferably 7:3 to 5:5, and the charge ratio of positive charge in DOTMA to negative charge in RNA is 1.8:2 to 0.8:2, more preferably 1.6:2 to 1:2, even more preferably 1.4:2 to 1.1:2, and even more preferably about 1.2:

2.

105. The composition according to claim 85 or 86, wherein the particles are LNPs that form a complex with the RNA molecule and / or encapsulate the RNA molecule.

106. The composition according to claim 85 or 86, wherein the particles are vesicles that encapsulate the RNA molecule, preferably monolayer liposomes.

107. The composition according to any one of claims 1 to 84, wherein the RNA molecule(s) is formulated into a composition containing a polyalkyleneimine, preferably a polyalkyleneimine.

108. The composition according to claim 107, wherein the molar ratio (N:P ratio) of the number of nitrogen atoms (N) in the polyalkylene imine to the number of phosphorus atoms (P) in the RNA molecule is 2.0 to 15.0, preferably 6.0 to 12.

0.

109. The composition according to claim 107 or 108, wherein the ionic strength of the composition is 50 mM or less, preferably the concentration of monovalent cationic ions is 25 mM or less, and the concentration of divalent cationic ions is 20 μM or less.

110. The composition according to claims 107 to 109, wherein the particles formed are polyplex.

111. The aforementioned polyalkyleneimine is given by the following general formula (I): 【Chemistry 1】 (In the formula, R is H, an acyl group, or the following general formula (II): 【Chemistry 2】 It is a group that includes, In the formula, R1 is H or the following general formula (III): 【Transformation 3】 It is a group that includes, n, m, and l are independently selected from integers between 2 and 10; and (where p, q, and r are integers, and the sum of p, q, and r is such that the average molecular weight of the polymer is 1.5 × 10² to 10⁷ Da, preferably 5000 to 10⁵ Da, more preferably 10000 to 40000 Da, more preferably 15000 to 30000 Da, and even more preferably 20000 to 25000 Da.) A composition according to any one of claims 107 to 110, comprising:

112. The composition according to any one of claims 107 to 111, wherein the polyalkyleneimine comprises polyethyleneimine and / or polypropyleneimine, preferably polyethyleneimine.

113. The composition according to any one of claims 107 to 112, wherein at least 92% of the N atoms in the polyalkyleneimine are protonable.

114. A pharmaceutical composition, as described in any one of claims 1 to 113.

115. The composition according to claim 114, further comprising a pharmaceutically acceptable carrier or excipient.

116. The composition according to any one of claims 1 to 115, in the form of a dried powder.

117. A composition according to any one of claims 1 to 115, which is freeze-dried.

118. A composition according to any one of claims 1 to 115, which is frozen.

119. The composition according to claim 118, having a temperature of -20°C or lower.

120. The composition according to any one of claims 1 to 119, further comprising one or more additives, wherein the additives are optionally selected from the group consisting of buffering substances, sugars, stabilizers, freeze-protecting agents, freeze-drying protective agents, and chelating agents.

121. The composition according to claim 120, wherein the buffering substance comprises at least one selected from the group consisting of 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 2-(N-morpholino)ethanesulfonic acid (MES), 3-morpholino-2-hydroxypropanesulfonic acid (MOPSO), acetic acid, acetate buffer and analogs, phosphoric acid and phosphate buffer, and citrate and citrate buffer.

122. The composition according to claim 120 or 121, wherein the sugar comprises at least one selected from the group consisting of monosaccharides, disaccharides, trisaccharides, oligosaccharides, and polysaccharides, preferably from glucose, trehalose, and saccharose.

123. The composition according to any one of claims 120 to 122, wherein the cryoprotectant comprises at least one selected from the group consisting of glycols such as ethylene glycol, propylene glycol, and glycerol.

124. The composition according to any one of claims 120 to 123, wherein the chelating agent comprises EDTA.

125. A vaccine, the composition according to any one of claims 1 to 124.

126. A pharmaceutical preparation comprising the composition according to any one of claims 1 to 125.

127. A kit comprising one or more RNA molecules as defined in any one of claims 1 to 58.

128. The kit according to claim 127, wherein the RNA molecule is in the form of a dried powder composition.

129. The kit according to claim 127 or 128, wherein the RNA molecule is freeze-dried.

130. The kit according to any one of claims 127 to 129, further comprising instructions for administering the RNA molecule.

131. A method for preventing a target HIV infection, comprising administering a composition according to any one of claims 1 to 125 to the target.

132. A method for preventing a target HIV infection, comprising dissolving the composition described in 116 in a suitable liquid pharmaceutical solution to form an administration solution, and administering the administration solution to the target.

133. A method for treating HIV infection in an HIV-positive subject, comprising administering a composition according to any one of claims 1 to 125 to the subject.

134. A method for treating HIV infection in an HIV-positive subject, comprising dissolving the composition described in 116 in a suitable liquid pharmaceutical solution to form an administration solution, and administering the administration solution to the subject.

135. The method according to any one of claims 131 to 134, wherein the severity of one or more symptoms of the HIV infection is reduced.

136. The method according to any one of claims 131 to 135, comprising only a single dose of the composition.

137. The method according to any one of claims 131 to 135, comprising administering the composition multiple times.

138. The method according to any one of claims 131 to 137, further comprising administering a booster dose of the composition.

139. The method according to any one of claims 131 to 138, wherein the administration of the composition includes intradermal, subcutaneous, or intramuscular administration by intradermal, subcutaneous, or intramuscular injection.

140. The method according to claim 139, wherein the injection is performed using a needle or using a needle-free injection device.

141. The method according to any one of claims 131 to 139, wherein administration preferably includes administration by intramuscular injection using a needle.

142. A composition according to any one of claims 1 to 125 for use in a method for preventing or treating a target HIV infection, wherein the method comprises administering the composition to the target.

143. The composition for use according to claim 142, wherein the subject is HIV-positive.

144. A composition according to any one of claims 1 to 125, for use in the manufacture of a pharmaceutical product for the prevention or treatment of a target HIV infection.

145. A method for treating or preventing HIV infection, comprising administering a first RNA molecule and a second RNA molecule as targets, The first RNA molecule comprises a nucleotide sequence encoding a first peptide, and the first peptide is a) Sequence numbers 60, 103, 98, 149, 2, 150, 142, 86, 1, 141, 151, 152, 130, 153, 154, 155, 156, 157, 158, 4, 159, 160, 161, 162, 35, 28, 163, 164, 165, 166, 87, 167 and 85, or b) Sequence numbers 60, 103, 98, 149, 2, 150, 142, 86, 1, 141, 151, 152, 199, 153, 154, 155, 156, 157, 158, 4, 159, 160, 161, 162, 35, 28, 203, 164, 165, 166, 87, 167 and 85 It includes the amino acid sequence, and The second RNA molecule comprises a nucleotide sequence encoding a second peptide, and the second peptide is c) Sequence numbers 98, 125, 29, 85, 168, 35, 150, 149, 169, 87, 57, 3, 170, 171, 172, 99, 131, 173, 174, 175, 104, 176, 61, 177, 178, 179, 5, 180, 181, 1, 182 and 140, or d) Sequence numbers 98, 125, 29, 85, 168, 35, 150, 149, 169, 87, 57, 3, 170, 171, 172, 99, 131, 173, 174, 175, 104, 176, 61, 177, 204, 179, 5, 180, 181, 1, 182 and 140 A method comprising the amino acid sequence.

146. a) The first RNA molecule contains the nucleotide sequence of SEQ ID NO: 184, 211, 212, 206, or 207, and the second RNA molecule contains the nucleotide sequence of SEQ ID NO: 186, 213, 214, 209, or 210, or b) The first RNA molecule contains the nucleotide sequence of SEQ ID NO: 188, 215, or 216, and the second RNA molecule contains the nucleotide sequence of SEQ ID NO: 190, 217, or 218. The method according to claim 145.

147. The method according to claim 146, wherein the first RNA molecule comprises the nucleotide sequence of SEQ ID NO: 206 or 207, and the second RNA molecule comprises the nucleotide sequence of SEQ ID NO: 209 or 210.

148. The method according to claims 145 to 147, wherein the first and second RNA molecules are administered at an interval of at least two weeks.

149. The method according to any one of claims 145 to 148, wherein the order of the amino acids from 5' to 3' of the first and / or second peptide is a given order, or the order of the amino acids from 5' to 3' of the first and / or second peptide is different from a given order.

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