HIV vaccine

Abstract

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

Description

[0001] Cross-reference to related applications

[0002] This application claims priority to U.S. Provisional Patent Application No. 63 / 459,449, filed April 14, 2023; U.S. Provisional Patent Application No. 63 / 547,796, filed November 8, 2023; and U.S. Provisional Patent Application No. 63 / 549,262, filed February 2, 2024, each of which is incorporated herein by reference in its entirety. Technical Field

[0003] This invention relates to compositions comprising RNA molecules and pharmaceutical formulations comprising such compositions, wherein the RNA molecules comprise an expression cassette encoding an immunogenic peptide, said peptide comprising at least two fragments, wherein each fragment comprises at least one epitope, wherein the epitope is derived from an amino acid sequence encoded by human immunodeficiency virus (HIV). Furthermore, this invention relates to methods for the prevention or treatment of HIV.

[0004] background

[0005] HIV remains a global health threat, with approximately 38 million individuals living with it worldwide (WHO, Globalprogress report on HIV, viral hepatitis and sexually transmitted infections, 2021). Recent advances in successful combination antiretroviral therapy (cART) have significantly reduced the HIV burden. However, a preventative HIV vaccine remains elusive, and cART does not promote complete viral clearance in patients. Therefore, for the more than 21 million people receiving treatment, HIV remains a lifelong chronic condition. Increasing the global applicability of cART, reducing the cost of treatment regimens, and addressing HIV drug resistance are key goals for achieving a “functional cure.”

[0006] Curative treatment is the ultimate goal of HIV therapy and remains a significant challenge in the field. Current curative strategies aim to elicit a strong HIV-specific CD8+ T cell response and / or use bNAb as a therapeutic agent to prevent (re)infection and promote the elimination of infected cells. Key challenges in HIV immunotherapy include i) the development of drug products with broad applicability; ii) the high diversity of circulating HIV strains; iii) reservoir-mediated immune escape within the infected host over time; iv) the presence of immunodominant nonprotective epitopes; and (v) impaired host immune function (Barouch, Nature 2008;455(7213):613-619; Fischer). et al.,Nat Med. 2007;13(1):100-106).

[0007] The rapidly emerging insights into HIV immune control mechanisms, along with the growing spectrum of well-characterized bNAbs, present exciting opportunities for the development of novel pharmaceutical products for HIV prevention and therapeutic treatment.

[0008] Therefore, there remains an urgent need to improve HIV vaccines and / or treatments.

[0009] This invention addresses this need. A particular advantage of this invention is that it provides vaccines that elicit an HIV-specific CD8+ T cell response to control or prevent HIV infection, especially through vaccines that support the generation of CD8+ T cells that support and / or reproduce naturally observed treatment-free viremia control.

[0010] Overview

[0011] One aspect of 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, wherein each fragment comprises at least one epitope, wherein the epitope is derived from an amino acid sequence encoded by human immunodeficiency virus (HIV), and wherein the epitope is contained within a sequence selected from any one of SEQ ID NO: 1 to 148 and 198 to 202 or a variant thereof, or contains a sequence selected from any one of SEQ ID NO: 1 to 148 and 198 to 202 or a variant thereof.

[0012] In the implementation, at least one segment may contain at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 epitopes. In the implementation, an epitope in one segment may be different from epitopes in at least one other segment or all other segments. In the implementation, at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or all of the segments do not contain any epitopes contained in another segment.

[0013] In an implementation, the sequences of the epitopes overlap in at least one segment, and / or the sequences of the epitopes do not overlap in at least one segment.

[0014] In the implementation scheme, the immunogenic peptides comprise 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 the embodiments, 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 those described in SEQ ID NO: 1 to 148 and 198 to 202. In the embodiments, each fragment may contain the same or a different number of epitopes.

[0015] In the implementation scheme, epitopes can be combined within the expression cassette to minimize the length of the RNA molecule, and / or can be ordered within the expression cassette to minimize the HLA-II epitopes of HIV.

[0016] In embodiments, 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. More preferably, at least one or all epitopes may be derived from HIV gp41, gp120, or nef proteins, particularly at least one or all epitopes may be derived from HIV nef protein. In embodiments, the peptide may contain at least one epitope derived from each of HIV gp41, gp120, or nef proteins.

[0017] In an embodiment, at least one amino acid may separate the sequences of non-overlapping epitopes in the peptide and / or fragments, and / or at least one amino acid may separate at least two fragments. Preferably, a linker may separate the sequences of at least two fragments.

[0018] In the implementation scheme, the shared flanking amino acid sequence of the HIV amino acid sequence containing two or more non-overlapping epitopes at 5' and 3' without lateral derived epitopes within one or two or more fragments.

[0019] In the context of this invention, the term "clade" refers to a group of organisms (particularly viruses, such as HIV) that are monophyletic, i.e., composed of a common ancestor on the phylogenetic tree and all its direct descendants. A phylogenetic tree can be constructed, for example, by aligning viral genomes and employing neighbor-joining or maximum parsimony methods known in the art from genome sequences.

[0020] In the implementation, the epitope variant can be an epitope polymorph, wherein the epitope is a shared sequence from at least two different clones of HIV.

[0021] In the context of this invention, the term "polymorphism" refers to a sequence variant of a common sequence in a genome (e.g., from a virus) that differs from the common sequence by at least one different nucleotide. For example, if the sequence atgacc is a common sequence and a "g" is also known to appear at the second position, then aggacc is a polymorphism of the common sequence. Preferably, the polymorphism will have at least 50%, 60%, 70%, 80%, 90%, or 95% sequence identity with the common sequence.

[0022] In the implementation scheme, the variant may differ from the epitope on one, two, three, four, or five amino acids.

[0023] In an embodiment, the composition may further comprise a second RNA molecule, wherein the second RNA molecule comprises a second expression cassette encoding a second immunogenic peptide, the peptide comprising at least two fragments, wherein each fragment comprises at least one epitope, wherein the epitope is derived from an amino acid sequence encoded by human immunodeficiency virus (HIV), and wherein the epitope is contained within a sequence selected from any one of SEQ ID NO: 1 to 148 and 198 to 202 or a variant thereof, or contains a sequence selected from any one of SEQ ID NO: 1 to 148 and 198 to 202 or a variant thereof.

[0024] In one embodiment, the immunogenic peptide contains an epitope from one clade of HIV, and the second immunogenic peptide contains an epitope from another clade of HIV, preferably, one clade is clade B and the other is clade C. In another embodiment, each immunogenic peptide contains the same epitope in both clades. In yet another embodiment, each immunogenic peptide contains a polymorphic epitope from each corresponding clade.

[0025] In the implementation scheme, the epitope may be a T-cell epitope and / or the epitope may be a CD8 minimal epitope.

[0026] In some embodiments, the epitope can be 9 to 21 amino acids long. Preferably, the epitope can be 9 or more amino acids long. Preferably, the epitope can be 10 or more amino acids long. Preferably, the epitope can be 11 or more amino acids long. In some embodiments, the epitope can be 9 to 14 amino acids long. Preferably, the epitope can be 9 to 13 amino acids long. Preferably, the epitope can be 9 to 12 amino acids long. Preferably, the epitope can be 9 to 11 amino acids long. Preferably, the epitope can be 9 or 10 amino acids long. Preferably, the epitope can be 9 amino acids long. In some embodiments, the fragment length can be 9 to 21 amino acids long.

[0027] In the embodiments, the immunogenic peptide may comprise 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 or more fragments, said fragments having a composition selected from SEQ ID NO: The amino acid sequences of 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, 172, 99, 131, 173, 174, 175, 104, 176, 61, 177, 178, 179, 5, 180, 181, 182, 140, 199, 203, and 204.

[0028] In the implementation scheme, the immunogenic peptide may comprise a) SEQ ID NO: 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) SEQ ID NO: The amino acid sequences of 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 the implementation scheme, the immunogenic peptide may comprise a) SEQ ID NO: 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) SEQ ID NO: The amino acid sequences are 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 embodiments, the immunogenic peptide may comprise the amino acid sequences of SEQ ID NO: 183, 185, 187, 189, 205, or 208. The 5' to 3' sequence of fragments in the peptide may be a given sequence or may differ from the given sequence.

[0029] In the implementation scheme, the first immunogenic peptide may comprise a) SEQ ID NO: 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) SEQ ID NO: The amino acid sequence of 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, and the second immunogenic peptide may contain c) SEQ ID NO: The amino acid sequences of 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) SEQ ID NO: 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 comprise the amino acid sequence of SEQ ID NO: 183 or 205, and the second immunogenic peptide comprises the amino acid sequence of SEQ ID NO: 185 or 208; b) the first immunogenic peptide comprises the amino acid sequence of SEQ ID NO: 187, and the second immunogenic peptide may comprise the amino acid sequence of SEQ ID NO: 189. In another embodiment, the first immunogenic peptide comprises the amino acid of SEQ ID: 205, and the second immunogenic peptide comprises the amino acid of SEQ ID NO: 208.

[0030] In the implementation scheme, the RNA molecule may contain the nucleotide sequence of SEQ ID NO: 184, 186, 188, 190, 206, 207, 209, 210 or 211 to 218.

[0031] In the implementation, the epitope can be a non-protective epitope that can cause CD8+ T cell protection collapse. Preferably, protection collapse can be T cell escape, T cell exhaustion, or loss of CD4+ helper T cells.

[0032] In some embodiments, 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 signaling group (MITD).

[0033] In the implementation scheme, the peptide may further include a non-HIV HLA-II helper epitope. Preferably, the helper epitope may be derived from Clostridium tetani (…). Clostridium tetani The P2 and / or P16 amino acid sequences of tetanus toxoid (TT).

[0034] RNA molecules can be linear or circular and / or may contain a 5' cap. Preferably, the 5' cap can be a modified cap, an artificial cap, or a cap analogue.

[0035] In an 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 contain 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 contain 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.

[0036] In an embodiment, the expression cassette may further comprise a poly A structure. Preferably, the poly A structure may be a discontinuous poly A structure. Preferably, the poly A structure may comprise 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.

[0037] In this embodiment, the RNA molecule may be a replicable RNA molecule. Preferably, the replicable RNA molecule may also encode an RNA-dependent RNA polymerase (replicaase) capable of replicating the replicable RNA molecule, or the replicable RNA molecule may not encode an RNA-dependent RNA polymerase (replicaase). Preferably, the composition may also comprise a non-replicable RNA molecule that encodes an RNA-dependent RNA polymerase (replicaase) capable of replicating the replicable RNA molecule.

[0038] In this implementation, the RNA molecule may be non-immunogenic. Preferably, the RNA molecule can be made non-immunogenic by removing the double-stranded RNA.

[0039] In the embodiments, the RNA molecule may contain nucleotide modifications. Preferably, the modification may be the substitution of one or more U residues with pseudouridine, N1-methylpseudouridine, or 5-methyluridine. Preferably, the one or more substituted U residues may be N1-methylpseudouridine. 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.

[0040] In this embodiment, the RNA molecule may be formulated in the composition together with at least one lipid. Preferably, the RNA molecule and at least one lipid may form particles. Preferably, the particles may be lipid nanoparticles (LNPs), lipoplexes (LPX), or liposomes.

[0041] In the implementation scheme, the particles can be nanoparticles, wherein: (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 lower. Preferably, the charge ratio of positive to negative charge in the nanoparticles can be from 1:1 to 1:8, more preferably from 1:1 to 1:4.

[0042] In some embodiments, 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 also contain at least one auxiliary lipid. Preferably, the auxiliary lipid may be a neutral lipid.

[0043] In some embodiments, at least one cationic lipid may include 1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA), 1,2-oleyloxy-3-dimethylaminopropane (DODMA), and / or 1,2-dioleyl-3-trimethylammonium propane (DOTAP). In some embodiments, at least one auxiliary lipid may include 1,2-di-(9Z-octadecenoyl)-sn-glycerol-3-phosphate ethanolamine (DOPE), cholesterol (Chol), 1,2-dioleyl-sn-glycerol-3-phosphate choline (DOPC), and / or 1,2-distearate-sn-glycerol-3-phosphate choline (DSPC).

[0044] In the embodiments, the molar ratio of at least one cationic lipid to at least one auxiliary 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.

[0045] In some embodiments, the nanoparticles may be lipoplexes comprising 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 wherein the charge ratio of the positive charge in DODMA to the 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.

[0046] In some embodiments, the nanoparticles may be lipoplexes comprising 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 wherein the charge ratio of the positive charge in DODMA to the 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 be lipoplexes comprising 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 wherein ... The nanoparticles may be DODMA and DSPC in a ratio of 7:3 to 5:5, wherein the charge ratio of the positive charge in DODMA to the 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 be lipoplexes comprising DODMA:cholesterol:DOPE:PEGcerC16 in a molar ratio of 40:48:10:2; or the nanoparticles may be lipoplexes comprising DODMA:cholesterol:DOPE:PEGcerC16 in a molar ratio of 10:0 to 1:9, preferably 8:2 to 3. 7. More preferably, a 7:3 to 5:5 ratio of DOTMA and DOPE, wherein the charge ratio of the positive charge in DOTMA to the 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 be lipoplexes containing a molar ratio of DOTMA to cholesterol of 10:0 to 1:9, preferably 8:2 to 3:7, more preferably 7:3 to 5:5, wherein the charge ratio of the positive charge in DOTMA to the negative charge in RNA is 1. The ratio of positive charge in DOTMA to negative charge in RNA is 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 lipoplexes comprising 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 wherein 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.

[0047] In some embodiments, the particles may be LNPs, which are complexed with and / or encapsulate RNA molecules. In other embodiments, the particles may be vesicles encapsulating RNA molecules, preferably monolayer liposomes. In some embodiments, the RNA molecules may be formulated in a composition comprising polyalkylimide, preferably polyalkylimide. Preferably, the molar ratio (N:P ratio) of the number of nitrogen atoms (N) in the polyalkylimide to the number of phosphorus atoms (P) in the RNA molecule may be 2.0 to 15.0, preferably 6.0 to 12.0.

[0048] In embodiments, the ionic strength of the composition may be 50 mM or lower, preferably wherein the concentration of monovalent cations may be 25 mM or lower and the concentration of divalent cations may be 20 μM or lower.

[0049] In the implementation scheme, the formed particles can be a polyplex.

[0050] In the implementation, the polyalkylene imide may comprise the following general formula (I): , in R is H, an acyl group, or a group containing the following general formula (II): , Where R1 is H or a group containing the following general formula (III): , n, m, and l are independently selected from integers from 2 to 10; and p, q, and r are integers, where the sum of p, q, and r results in an average molecular weight of 1.5 for the polymer. 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.

[0051] In some embodiments, the polyalkylene imide may comprise polyethyleneimine and / or polypropyleneimine, preferably polyethyleneimine. In some embodiments, at least 92% of the nitrogen atoms in the polyalkylene imide may be protonable.

[0052] In this embodiment, the composition may be a pharmaceutical composition. Preferably, the composition may also contain a pharmaceutically acceptable carrier or excipient.

[0053] In embodiments, the composition may be in the form of a dry powder; or the composition may be lyophilized; or the composition may be frozen. Preferably, the temperature of the composition may be -20°C or lower.

[0054] In embodiments, the composition may further comprise one or more additives, wherein the additives are optionally selected from buffering substances, sugars, stabilizers, cryoprotectants, lyophilization protectants, and chelating agents. Preferably, the buffering substance may comprise at least one selected from 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid (HEPES), 2-(N-morpholine) ethanesulfonic acid (MES), 3-morpholine-2-hydroxypropanesulfonic acid (MOPSO), acetic acid, acetate buffers and analogs, phosphate and phosphate buffers, and citric acid and citrate buffers. Preferably, the sugar may comprise at least one selected from monosaccharides, disaccharides, trisaccharides, oligosaccharides, and polysaccharides, preferably at least one selected from glucose, trehalose, and sucrose. Preferably, the cryoprotectant may comprise at least one selected from glycols and glycerol, said glycol being, for example, ethylene glycol or propylene glycol. Preferably, the chelating agent may comprise EDTA.

[0055] In the implementation plan, the composition may be a vaccine.

[0056] Another aspect of this disclosure is pharmaceutical formulations comprising the compositions disclosed herein.

[0057] Another aspect of this disclosure is a kit containing 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 also include instructions for applying the RNA molecules.

[0058] Another aspect of this disclosure is a method for preventing HIV infection in subjects, the method comprising administering to subjects the compositions disclosed herein.

[0059] Another aspect of this disclosure is a method for preventing HIV infection in a subject, the method comprising dissolving the dry powder composition disclosed herein in a suitable liquid pharmaceutical solution to form a solution for application, and applying the solution for application to the subject.

[0060] Another aspect of this disclosure is a method for treating HIV infection in HIV-positive subjects, the method comprising administering the compositions disclosed herein to the subject.

[0061] Another aspect of this disclosure is a method for treating HIV infection in HIV-positive individuals, the method comprising dissolving the dry powder composition disclosed herein in a suitable liquid pharmaceutical solution to form a solution for application, and applying the solution for application to the individual.

[0062] In embodiments of the methods disclosed herein, the severity of one or more symptoms of HIV infection can be reduced. In embodiments, the methods may involve a single application of the composition or may include multiple applications of the composition.

[0063] In embodiments of the methods disclosed herein, the methods may further include administering a stronger dose of the composition.

[0064] In embodiments 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 performed using a needle or by using a needleless injection device. In embodiments of the methods disclosed herein, administration may include intramuscular injection, preferably using a needle for intramuscular injection.

[0065] Another aspect of this disclosure is the use of the compositions described herein in methods for preventing or treating HIV infection in a subject, said methods comprising administering the compositions to said subject. In embodiments, the subject may be HIV-positive.

[0066] Another aspect of this disclosure is the use of the compositions described herein in the preparation of medicaments for the prevention or treatment of HIV infection in subjects.

[0067] In embodiments relating to the treatment or prevention of this disclosure, the method may include administering a first composition comprising a first RNA molecule containing an expression cassette encoding an immunogenic peptide described herein, and wherein the method further includes administering a second composition comprising a second RNA molecule containing an expression cassette encoding a second immunogenic peptide described herein, the second immunogenic peptide being different from the first immunogenic peptide. The compositions may be administered simultaneously or at different time points and via the same or different administration modalities. The method may also include additional rounds of administration of the compositions.

[0068] In 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 subject, wherein the first RNA molecule may comprise a nucleotide sequence encoding a first peptide, the first peptide comprising a) SEQ ID NO: 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) SEQ ID NO: The amino acid sequences of 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; and wherein the second RNA molecule may contain a nucleotide sequence encoding a second peptide, the second peptide comprising c) SEQ ID NO: The amino acid sequences of 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) SEQ ID NO: 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 contains 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 another embodiment, the first RNA molecule contains the nucleotide sequence of SEQ ID NO: 206 or 207, and the second RNA molecule contains the nucleotide sequence of SEQ ID NO: 209 or 210. In yet another embodiment, the first and second RNA molecules may be administered at least two weeks apart.In the implementation, the 5' to 3' sequence of the amino acid sequences in the first and / or second peptides may be a given sequence, or the 5' to 3' sequence of the amino acid sequences in the first and / or second peptides may be different from the given sequence.

[0069] Detailed Explanation

[0070] Although the invention is described in detail below, it should be understood that the invention is not limited to the specific methods, schemes, and reagents described herein, as these can vary. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the invention, which will be defined only by the appended claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0071] Preferably, the terms used herein are defined as described in “A multilingual glossary of biotechnological terms: (IUPAC Recommendations)”, HGW Leuenberger, B. Nagel, and H. Kölbl, Eds., Helvetica Chimica Acta, CH-4010 Basel, Switzerland, (1995).

[0072] Unless otherwise stated, the present invention will be practiced using conventional methods of chemistry, biochemistry, cell biology, immunology, and recombinant DNA technology, methods explained in the literature in this field (see, for example, Molecular Cloning: A Laboratory Manual, 2nd Edition, J. Sambrook). et al. eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor 1989).

[0073] Hereinafter, elements of the invention will be described. These elements are listed together with specific embodiments; however, it should be understood that they can be combined in any manner and in any number to produce additional embodiments. The various examples and preferred embodiments described should not be construed as limiting the invention to the explicitly described embodiments. This description should be understood as disclosing and including embodiments that combine the explicitly described embodiments with any number of disclosed and / or preferred elements. Furthermore, unless the context otherwise requires, any permutation and combination of all elements described in this application should be considered as being disclosed in this specification.

[0074] The term “about” means approximately or close to, and in the context of the numerical values ​​or ranges shown herein, it preferably means + / - 10% of the stated or claimed numerical values ​​or ranges.

[0075] The terms “a,” “an,” and “the,” and similar references used in the context of describing the invention (particularly in the context of the claims) should be interpreted to cover both the singular and plural unless otherwise indicated herein or clearly contradicted by the context. Expressions of numerical ranges herein are intended only as a shorthand method for individually referring to each individual value falling within that range. Each individual value is incorporated into the specification as if it were stated separately herein, unless otherwise stated herein. All methods described herein may be performed in any suitable order unless otherwise indicated herein or clearly contradicted by the context. The use of any and all examples or exemplary language (e.g., “such as”) provided herein is intended only to better illustrate the invention and does not constitute a limitation on the scope of the otherwise claimed invention. The language in the specification should not be construed as indicating any unclaimed element necessary for carrying out the invention.

[0076] Unless otherwise expressly stated, the term "comprising" is used in the context of this document to mean that, in addition to the members of the list introduced by "comprising," other members may optionally exist. However, as a particular embodiment of the invention, the term "comprising" is intended to cover the possibility that no other members exist; that is, for the purposes of this embodiment, "comprising" should be understood to mean "consisting of".

[0077] The indication of the relative amount of a component characterized by a generic term refers to the total amount of all specific variants or members covered by the generic term. If a component defined by a generic term is specified to exist in a certain relative amount, and if the component is also characterized as a specific variant or member covered by the generic term, it means that there are no additional variants or members covered by the generic term such that the total relative amount of the components covered by the generic term exceeds the specified relative amount; more preferably, there are no other variants or members covered by the generic term at all.

[0078] Several documents are referenced throughout this specification. Each document referenced herein (including all patents, patent applications, scientific publications, manufacturer's instructions, specifications, etc.), whether mentioned above or below, is incorporated herein by reference in its entirety. Nothing herein should be construed as an admission that the invention does not have rights prior to such disclosure.

[0079] As used herein, terms such as “reduction” or “inhibition” mean that which can cause a reduction in the overall level, preferably 5% or more, 10% or more, 20% or more, more preferably 50% or more, and most preferably 75% or more. The term “inhibition” or similar phrases include complete or substantially complete inhibition, i.e., reduction to zero or substantially to zero.

[0080] Terms such as “increase” or “enhance” preferably refer to an increase or enhancement of 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%.

[0081] The term "net charge" refers to the electrical charge on an entire object (such as a compound or particle).

[0082] Ions with a total net positive charge are cations, while ions with a total net negative charge are anions. Therefore, according to the present invention, anions are ions with more electrons than protons, thus being endowed with a net negative charge; cations are ions with fewer electrons than protons, thus being endowed with a net positive charge.

[0083] 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.

[0084] The term "nucleic acid" according to the invention also includes nucleic acids on nucleotide bases, sugars, or phosphates, and chemically derivatized 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, preferably deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). According to the invention, nucleic acids include genomic DNA, cDNA, mRNA, viral RNA, recombinant-prepared molecules, and chemically synthesized molecules. According to the invention, nucleic acids can be in the form of single-stranded or double-stranded molecules and linear or covalently closed circular molecules.

[0085] According to the present invention, a "nucleic acid sequence" refers to a nucleotide sequence in a nucleic acid (e.g., ribonucleic acid (RNA) or deoxyribonucleic acid (DNA)). This term can refer to a complete nucleic acid molecule (e.g., a single strand of a complete nucleic acid molecule) or a portion thereof (e.g., a fragment).

[0086] According to the present invention, the term "RNA" or "RNA molecule" refers to a molecule comprising ribonucleotide residues and preferably consisting entirely or substantially of ribonucleotide residues. The term "ribonucleotide" refers to a nucleotide having a hydroxyl group at the 2' position of the β-D-furanose group. The term "RNA" includes double-stranded RNA, single-stranded RNA, isolated RNA (e.g., partially or completely purified RNA), substantially pure RNA, synthetic RNA, and recombinant RNA, such as modified RNA, which differs from naturally occurring RNA in that one or more nucleotides are added, deleted, substituted, and / or altered. Such alterations may include the addition of non-nucleotide material, such as adding it to the ends or interior of the RNA, for example, adding it to one or more nucleotides of the RNA. Nucleotides in the RNA molecule may also include non-standard nucleotides, such as non-naturally occurring nucleotides or chemically synthesized nucleotides or deoxynucleotides. These altered RNAs may be referred to as analogs, particularly analogs of naturally occurring RNA.

[0087] According to the present invention, RNA can be single-stranded or double-stranded. In some embodiments of the invention, single-stranded RNA is preferred. The term "single-stranded RNA" generally refers to an RNA molecule that does not associate with a complementary nucleic acid molecule (typically no complementary RNA molecule). Single-stranded RNA may contain self-complementary sequences that allow portions of the RNA to fold back and form secondary structural motifs, including but not limited to base pairs, stems, stem loops, and protrusions. Single-stranded RNA can exist as a negative strand [(-) strand] or a positive strand [(+) strand]. The (+) strand is the strand that contains or encodes genetic information. The genetic information can be, for example, a polynucleotide sequence encoding a protein. When the (+) strand RNA encodes a protein, the (+) strand can be directly used as a template for translation (protein synthesis). The (-) strand is the complement of the (+) strand. In the case of double-stranded RNA, the (+) and (-) strands are two separate RNA molecules, and these two RNA molecules associate with each other to form double-stranded RNA ("double-stranded RNA").

[0088] The term "stability" of RNA is related to its "half-life." Half-life refers to the time required to eliminate half the activity, amount, or number of molecules. In the context of this invention, the half-life of RNA indicates the stability of said RNA. The half-life of RNA may affect the "expression duration" of RNA. RNA with a long half-life can be expected to be expressed over an extended period.

[0089] The term "translation efficiency" refers to the amount of translational product provided by an RNA molecule within a specific time period.

[0090] Regarding nucleic acid sequences, a "fragment" refers to a portion of a nucleic acid sequence, i.e., a sequence representing a shortened nucleic acid sequence at the 5'- and / or 3'- ends. Preferably, the nucleic acid sequence fragment contains at least 80%, more preferably at least 90%, 95%, 96%, 97%, 98%, or 99% of the nucleotide residues from said nucleic acid sequence. In this invention, those fragments that preserve RNA stability and / or translation efficiency of the RNA molecule are preferred.

[0091] Regarding amino acid sequences (peptides or proteins), a "fragment" refers to a portion of an amino acid sequence, specifically a sequence representing a shortened amino acid sequence at the N-terminus and / or C-terminus. A C-terminal shortened fragment (N-terminal fragment) can be obtained, for example, by translating a truncated open reading frame lacking the 3' end. An N-terminal shortened fragment (C-terminal fragment) can be obtained, for example, by translating a truncated open reading frame lacking the 5' end, provided the truncated open reading frame contains a start codon for initiating translation. A fragment of an amino acid sequence comprises, 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%, or at least 90% of the amino acid residues from the amino acid sequence. As used herein with respect to immunogenic peptides, a fragment is a sub-part of a peptide containing one or more epitopes.

[0092] According to the present invention, the term "variant" relating to, for example, nucleic acid and amino acid sequences includes any variant, particularly mutants, viral strain variants, splicing variants, conformations, isotypes, allele variants, species variants, and species homologs, especially those that are naturally occurring. Allele variants involve alterations to the normal sequence of a gene, the significance of which is often unclear. Complete gene sequencing typically identifies many allele variants of a given gene. Regarding nucleic acid molecules, the term "variant" includes degenerate nucleic acid sequences, wherein the degenerate nucleic acid according to the present invention differs from the reference nucleic acid in its codon sequence due to the degeneracy of the genetic code. A species homolog is a nucleic acid or amino acid sequence from a species of a different origin than a given nucleic acid or amino acid sequence. A viral homolog is a nucleic acid or amino acid sequence from a virus of a different origin than a given nucleic acid or amino acid sequence.

[0093] Nucleic acid variants include single or multiple nucleotide deletions, additions, mutations, substitutions, and / or insertions compared to a reference nucleic acid. Deletions include the removal of one or more nucleotides from the reference nucleic acid. Other variants include 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 into its position (e.g., transversion and transition). Mutations include baseless sites, cross-linking sites, and chemically altered or modified bases. Insertions include the addition of at least one nucleotide to the reference nucleic acid.

[0094] According to the present invention, a "nucleotide alteration" can refer to a single or multiple nucleotide deletion, addition, mutation, substitution, and / or insertion compared to a reference nucleic acid. In some embodiments, a "nucleotide alteration" compared to a reference nucleic acid is selected from the deletion, addition, mutation, substitution, and / or insertion of a single nucleotide. According to the present invention, a nucleic acid variant may include one or more nucleotide alterations compared to a reference nucleic acid.

[0095] Variants of a specific nucleic acid sequence preferably have at least one functional property of the specific sequence, and are preferably functionally equivalent to the specific sequence, for example, nucleic acid sequences that exhibit the same or similar properties as the specific nucleic acid sequence.

[0096] 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.

[0097] 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, will be 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%. Preferably, the degree of identity for regions 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 is given. In a preferred embodiment, the degree of identity for the full length of a reference nucleic acid or amino acid sequence is given.

[0098] "Sequence similarity" refers to the percentage of identical or conserved amino acid substitutions. "Sequence identity" between two polypeptide or nucleic acid sequences refers to the percentage of identical amino acids or nucleotides between the sequences.

[0099] The term "%identity" specifically refers to the percentage of identical nucleotides or amino acids in the optimal alignment between two sequences to be compared. This percentage is purely statistical, and the differences between the two sequences can be randomly distributed across the entire length of the sequence. The sequences to be compared may include additions or deletions compared to a reference sequence to obtain the optimal alignment. The comparison of two sequences is typically performed after the optimal alignment by comparing the sequences to segments or "comparison windows" to identify local regions of the respective sequences. The best alignment for comparison can be performed manually or with the aid of the local homology algorithm of Smith and Waterman, 1981, Ads App. Math. 2:482, the local homology algorithm of Needleman and Wunsch, 1970, J. Mol. Biol. 48:443, and the similarity search algorithm of Pearson and Lipman, 1988, Proc. Natl Acad. Sci. USA 85:2444, or with the aid of computer programs using the aforementioned algorithms (GAP, BESTFIT, FASTA, BLAST P, BLAST N and TFASTA in Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, Wis.).

[0100] The percentage of similarity is obtained by determining the number of identical positions corresponding to the sequences to be compared, dividing that number by the number of positions being compared, and then multiplying the result by 100.

[0101] For example, the BLAST program “BLAST 2 sequence”, which is available on the website http: / / www.ncbi.nlm.nih.gov / blast / bl2seq / wblast2.cgi, can be used.

[0102] If two sequences are complementary to each other, the nucleic acid "can hybridize" or "hybridizes" with another nucleic acid. If two sequences can form a stable double helix with each other, the nucleic acid is "complementary" to the other nucleic acid. According to the invention, hybridization is preferably performed under conditions that allow specific hybridization between multiple nucleotides (strict conditions). Strict conditions are described, for example, in *Molecular Cloning: A Laboratory Manual*, J. Sambrook. et al., Editors, 2nd Edition, ColdSpring Harbor Laboratory press, Cold Spring Harbor, New York, 1989 or in Current Protocols in Molecular Biology, FM Ausubel et al. The instructions, in the editors of John Wiley & Sons, Inc., New York, refer, for example, 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). The SSC is 0.15 M sodium chloride / 0.15 M sodium citrate, pH 7. After hybridization, the DNA is transferred to the membrane thereon and washed, for example, at room temperature in 2 × SSC, then washed at temperatures up to 68°C in 0.1–0.5 × SSC / 0.1 × SDS.

[0103] The complementarity percentage represents the percentage of consecutive residues in the nucleic acid molecule that can form hydrogen bonds (e.g., Watson-Crick base pairing) with the 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). "Perfectly complementary" or "fully complementary" means that all consecutive residues in the nucleic acid sequence will form hydrogen bonds with the same number of consecutive residues in the second nucleic acid sequence. Preferably, the complementarity according to the 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 complementarity according to the invention is 100%.

[0104] The term "derivative" includes any chemical derivatization of nucleic acids on the bases of a nucleotide, on a sugar, or on a phosphate group. The term "derivative" also includes nucleic acids containing nucleotides and nucleotide analogs that are not naturally occurring. Preferably, derivatization of nucleic acids increases their stability.

[0105] "A nucleic acid sequence derived from a nucleic acid sequence" refers to a nucleic acid that is a variant of the derived nucleic acid. Preferably, when it replaces a specific sequence in an RNA molecule, the variant sequence relative to the specific sequence maintains RNA stability and / or translation efficiency.

[0106] "nt" is an abbreviation for nucleotide; or for nucleotides, preferably consecutive nucleotides in a nucleic acid molecule.

[0107] According to the present invention, the term "codon" refers to a base triplet that encodes a nucleic acid and specifies which amino acid is added next during the protein synthesis process of the ribosome.

[0108] The terms “transcription” and “transcribing” refer to a process in which a nucleic acid molecule having a specific nucleic acid sequence (“nucleic acid template”) is read by an RNA polymerase, which then produces a single-stranded RNA molecule. During transcription, genetic information in the nucleic acid template is transcribed. The nucleic acid template can be DNA; however, for example, in the case of transcription from an alphaviral nucleic acid template, the template is typically RNA. The transcribed RNA can then be translated into a protein. According to the invention, the term “transcription” includes “in vitro transcription,” which refers to the process of synthesizing RNA, particularly mRNA, in vitro in a cell-free system. Preferably, cloning vectors are used to produce transcripts. These cloning vectors are generally referred to as transcription vectors and are included in the term “vector” according to the invention. Cloning vectors are preferably plasmids. According to the invention, the RNA is preferably in vitro transcribed RNA (IVT-RNA) and can be obtained by in vitro transcription from a suitable DNA template. The promoter used to control transcription can be any promoter of any RNA polymerase. The DNA template for in vitro transcription can be obtained by cloning a nucleic acid (particularly cDNA) and introducing it into a suitable vector for in vitro transcription. cDNA can be obtained through reverse transcription of RNA.

[0109] Single-stranded nucleic acid molecules produced during transcription usually have a nucleic acid sequence that is complementary to the template.

[0110] According to the present invention, the terms "template" or "nucleic acid template" or "template nucleic acid" generally refer to a nucleic acid sequence that can be replicated or transcribed.

[0111] "A nucleic acid sequence transcribed from a nucleic acid sequence" and similar terms refer to a nucleic acid sequence, where appropriate, as part of a complete RNA molecule, which is the transcription product of a template nucleic acid sequence. Typically, the transcribed nucleic acid sequence is a single-stranded RNA molecule.

[0112] According to the present invention, the "3' end" of a nucleic acid refers to the end having a free hydroxyl group. In the diagrammatic representation of double-stranded nucleic acids, especially DNA, the 3' end is always on the right-hand side. According to the present invention, the "5' end" of a nucleic acid refers to the end having a free phosphate group. In the diagrammatic representation of double-stranded nucleic acids, especially DNA, the 5' end is always on the left-hand side.

[0113] 5' end 5'--P-NNNNNNN-OH-3' 3' end

[0114] 3'-HO-NNNNNNN-P--5'

[0115] "Upstream" describes the relative positioning of a first element of a nucleic acid molecule with respect to a second element of the same molecule, where both elements are contained within the same molecule, and where the first element is closer to the 5' end of the molecule than the second element. The second element is then considered "downstream" of the first element. An element located "upstream" of the second element can be synonymously referred to as being located at the "5'" end of the second element. For double-stranded nucleic acid molecules, the indications of "upstream" and "downstream" are given relative to the (+) strand.

[0116] According to the present invention, a "functional link" or "functionally linked" refers to a link within a functional relationship. A nucleic acid is "functionally linked" if it is functionally related to another nucleic acid sequence. For example, if a promoter affects the transcription of a coding sequence, it is functionally linked to said coding sequence. Functionally linked nucleic acids are typically adjacent to each other, separated by other nucleic acid sequences where appropriate, and in a particular embodiment, transcribed by RNA polymerase to yield a single RNA molecule (the common transcript).

[0117] In a particular embodiment, according to the present invention, a nucleic acid is functionally linked to an expression control sequence, which may be homologous or heterologous to the nucleic acid.

[0118] According to the present invention, the term "expression control sequence" includes a promoter, a ribosome-binding sequence, and other control elements that control gene transcription or derivative RNA translation. In specific embodiments of the invention, the expression control sequence can be modulated. The precise structure of the expression control sequence can vary depending on the species or cell type, but generally includes 5'-non-transcriptional and 5'- and 3'-non-translational sequences involved in initiating transcription and translation, respectively. More specifically, the 5'-non-transcriptional expression control sequence includes a promoter region containing a promoter sequence for functionally linking gene transcriptional control. The expression control sequence may also include enhancer sequences or upstream activator sequences. Expression control sequences of DNA molecules typically include 5'-non-transcriptional and 5'- and 3'-non-translational sequences, such as TATA boxes, capping sequences, CAAT sequences, etc. Expression control sequences of alphavirus RNA may include subgenomic promoters and / or one or more conserved sequence elements. The specific expression control sequence according to the present invention is an alphavirus subgenomic promoter, as described herein.

[0119] The nucleic acid sequences specified herein, particularly transcribed and coding nucleic acid sequences, can be combined with any expression control sequence, particularly a promoter, which can be homologous or heterologous to the nucleic acid sequence. The term "homology" means that the nucleic acid sequence is also naturally functionally linked to the expression control sequence, while the term "heterologous" means that the nucleic acid sequence is not naturally functionally linked to the expression control sequence.

[0120] If a transcribed nucleic acid sequence (particularly a nucleic acid sequence encoding a peptide or protein) and an expression control sequence are covalently linked to each other in such a way that the transcription of the transcribed, and particularly the expression of the nucleic acid sequence is under the control of or influenced by the expression control sequence, then they are “functionally” linked to each other. If the nucleic acid sequence is to be translated into a functional peptide or protein, inducing the expression control sequence, which is functionally linked to the coding sequence, results in the transcription of said coding sequence without causing a frameshift in the coding sequence or preventing the coding sequence from being translated into the desired peptide or protein.

[0121] The term "promoter" or "promoter region" refers to a nucleic acid sequence that controls the synthesis of a transcript (e.g., a transcript containing a coding sequence) by providing a recognition and binding site for RNA polymerase. A promoter region may include additional recognition or binding sites for other factors involved in regulating the transcription of the gene. A promoter can control the transcription of prokaryotic or eukaryotic genes. A promoter can be "inducible" and initiate transcription in response to an inducer, or it can be "constitutive" if transcription is not controlled by an inducer. In the absence of an inducer, an inducible promoter is expressed only to a very small degree or not at all. In the presence of an inducer, the gene is "turned on" or the transcription level increases. This is typically mediated by the binding of a specific transcription factor. A specific promoter according to the invention is a subgenomic promoter, for example, a subgenomic promoter of alphavirus as described herein. Other specific promoters are, for example, positive or negative strand promoters of the alphavirus genome.

[0122] The term "core promoter" refers to the nucleic acid sequence contained within a promoter. The core promoter is typically the smallest part of the promoter required for the proper initiation of transcription. A core promoter usually includes a transcription start site and an RNA polymerase binding site.

[0123] "Polymerase" generally refers to a molecular entity capable of catalyzing the synthesis of polymer molecules from monomeric building blocks. "RNA polymerase" is a molecular entity capable of catalyzing the synthesis of RNA molecules from ribonucleotide building blocks. "DNA polymerase" is a molecular entity capable of catalyzing the synthesis of DNA molecules from deoxyribonucleotide building blocks. In the cases of DNA and RNA polymerases, the molecular entity is typically a protein or an assembly or complex of multiple proteins. Typically, DNA polymerases synthesize DNA molecules based on a template nucleic acid, which is usually a DNA molecule. Typically, RNA polymerases synthesize RNA molecules based on a template nucleic acid, which is either a DNA molecule (in this case, the RNA polymerase is a DNA-dependent RNA polymerase, DdRP) or an RNA molecule (in this case, the RNA polymerase is an RNA-dependent RNA polymerase, RdRP).

[0124] RNA-dependent RNA polymerase, or RdRP, is an enzyme that catalyzes the transcription of RNA from an RNA template. In the case of alphavirus RNA-dependent RNA polymerase, the sequential synthesis of the (-) strand complement and (+) strand complementary RNA of the genomic RNA leads to RNA replication. RNA-dependent RNA polymerase is therefore synonymously called "RNA replicase" or simply "replicaase." In nature, RNA-dependent RNA polymerase is typically encoded by all RNA viruses except retroviruses. A typical example of a virus encoding RNA-dependent RNA polymerase is alphavirus.

[0125] According to the present invention, "RNA replication" generally refers to the synthesis of an RNA molecule based on the nucleotide sequence of a given RNA molecule (template RNA molecule). The synthesized RNA molecule may be, for example, identical or complementary to the template RNA molecule. Generally, RNA replication can occur via the synthesis of a DNA intermediate, or it can be directly mediated by RNA-dependent RNA polymerase (RdRP). In the case of alphavirus, RNA replication does not occur via a DNA intermediate, but is mediated by RNA-dependent RNA polymerase (RdRP): the template RNA strand (first RNA strand) (or a portion thereof) serves as a template for the synthesis of a second RNA strand complementary to the first RNA strand or a portion thereof. The second RNA strand (or a portion thereof) may then optionally serve as a template for the synthesis of a third RNA strand complementary to the second RNA strand or a portion thereof. Thus, the third RNA strand is identical to the first RNA strand or a portion thereof. Therefore, RNA-dependent RNA polymerase can directly synthesize the complementary RNA strand of the template and can indirectly synthesize the same RNA strand (via a complementary intermediate strand).

[0126] According to the present invention, the term "template RNA" refers to RNA that can be transcribed or replicated by RNA-dependent RNA polymerase.

[0127] According to the present invention, the term "gene" refers to a specific nucleic acid sequence responsible for producing one or more cellular products and / or performing one or more intercellular or intracellular functions. More specifically, the term refers to a nucleic acid segment (typically DNA; however, in the case of RNA viruses, RNA) containing nucleic acids encoding specific proteins or functional or structural RNA molecules.

[0128] As used herein, "isolated molecule" is intended to refer to a molecule that is substantially free of other molecules, such as other cellular material. According to the invention, the term "isolated nucleic acid" means that the nucleic acid has been (i) amplified in vitro, for example by polymerase chain reaction (PCR), (ii) produced by clonal recombination, (iii) purified, for example by cleavage and gel electrophoresis, or (iv) synthesized, for example by chemical synthesis. Isolated nucleic acid is a nucleic acid that can be manipulated using recombination techniques.

[0129] The term "vector" is used herein in its most general sense and includes any intermediate vector for nucleic acids, which, for example, enable the nucleic acids to be introduced into prokaryotic and / or eukaryotic host cells and, where appropriate, integrated into the genome. Such vectors preferably replicate and / or are expressed in cells. Vectors include plasmids, phage particles, viral genomes, and portions thereof.

[0130] In the context of this invention, the term "recombinant" means "prepared by genetic engineering." Preferably, in the context of this invention, the "recombinant object," such as recombinant cells, is not naturally occurring.

[0131] As used herein, the term "naturally occurring" refers to the fact that an object can be found in nature. For example, peptides or nucleic acids that exist in organisms (including viruses) and can be isolated from natural sources and have not been intentionally modified in a laboratory are naturally occurring. The term "found in nature" means "existing in nature" and includes both known objects and objects that have not yet been discovered and / or isolated from nature but may be discovered and / or isolated from natural sources in the future.

[0132] According to the present invention, the term "expression" is used in its most general sense and includes the production of RNA and / or proteins. It also includes the partial expression of nucleic acids. Furthermore, expression can be transient or stable. With regard to RNA, the term "expression" or "translation" refers to a process in the ribosomes of a cell by which a strand encoding RNA (e.g., messenger RNA) directs the assembly of an amino acid sequence to prepare a peptide or protein.

[0133] According to the present invention, the term "mRNA" means "messenger RNA" and refers to transcripts that are typically produced using a DNA template and encode peptides or proteins. Typically, mRNA contains a 5'-UTR, a protein-coding region, a 3'-UTR, and a poly(A) sequence. mRNA can be produced from a DNA template by in vitro transcription. In vitro transcription methodologies are known to those skilled in the art. For example, there are various commercially available in vitro transcription kits. According to the present invention, mRNA can be modified by stabilization modifications and capping.

[0134] According to the present invention, the term "poly(A) sequence" or "poly(A) tail" refers to a continuous or discontinuous sequence of adenosine residues typically located at the 3' end of an RNA molecule. A continuous sequence is characterized by a series of adenosine residues. Continuous poly(A) sequences are typical in nature. Although poly(A) sequences are not typically encoded in eukaryotic DNA, but are attached to the free 3' end of RNA post-transcriptionally by template-independent RNA polymerases during eukaryotic transcription within the cell nucleus, the present invention includes poly(A) sequences encoded by DNA.

[0135] According to the present invention, the term "primary structure" in relation to nucleic acid molecules refers to the linear sequence of nucleotide monomers.

[0136] According to the present invention, the term "secondary structure" for nucleic acid molecules refers to a two-dimensional representation of a nucleic acid molecule reflecting base pairing; for example, in the case of single-stranded RNA molecules, particularly intramolecular base pairing. 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, ridges, loops such as inner loops and multi-branched loops. The secondary structure of a nucleic acid molecule can be represented by a two-dimensional diagram (planar diagram) showing base pairing (for further details on the secondary structure of RNA molecules, see Auber). et al. (J. Graph Algorithms Appl. 10:329-351). As described herein, the secondary structures of certain RNA molecules are relevant in the context of this invention.

[0137] According to the present invention, the secondary structure of nucleic acid molecules (especially 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, the "secondary structure" of a nucleic acid molecule specifically refers to the secondary structure determined by the prediction. The MFOLD structure prediction (http: / / unafold.rna.albany.edu / ?q=mfold) can also be used to perform or verify the prediction.

[0138] According to the present invention, a "base pair" is a structural motif of secondary structure in which two nucleotide bases associate with each other through hydrogen bonds between donor and acceptor sites on the bases. Complementary bases A:U and G:C form stable base pairs through hydrogen bonds between donor and acceptor sites on the bases; the A:U and G:C base pairs are called Watson-Crick base pairs. Weaker base pairs (called Wobble base pairs) are formed by the bases G and U (G:U). The base pairs A:U and G:C are called canonical base pairs. Other base pairs, such as G:U (which occurs quite frequently in RNA) and other rare base pairs (e.g., A:C; U:U), are called non-canonical base pairs.

[0139] According to the present invention, "nucleotide pairing" refers to two nucleotides that associate with each other such that their bases form a base pair (ordinary or non-ordinary base pair, preferably ordinary base pair, most preferably Watson-Crick base pair).

[0140] According to the present invention, the terms "stem-loop" or "hairpin" or "hairpin loop" are interchangeable in relation to nucleic acid molecules, typically single-stranded nucleic acid molecules such as single-stranded RNA. A specific secondary structure represented by a stem-loop consists of a continuous nucleic acid sequence comprising a stem and a (terminal) loop (also called a hairpin loop), wherein the stem is formed by two adjacent fully or partially complementary sequence elements; they are separated by a short sequence (e.g., 3-10 nucleotides) to form the loop of the stem-loop structure. Two adjacent fully or partially complementary sequences can be defined, for example, stem-loop elements stem 1 and stem 2. When these two adjacent fully or partially inversely complementary sequences (e.g., stem-loop elements stem 1 and stem 2) form a base pair with each other, a stem-loop is formed, resulting in a double-stranded nucleic acid sequence that contains an unpaired loop at its end formed by a short sequence located between stem-loop elements stem 1 and stem 2. Therefore, the stem-loop comprises two stems (stem 1 and stem 2) that form base pairs with each other at the secondary structure level of the nucleic acid molecule and are separated at the primary structure level by short sequences that are not part of stem 1 or stem 2. For illustration, the two-dimensional representation of the stem-loop resembles a lollipop structure. The formation of the stem-loop structure requires the presence of sequences capable of folding back to form paired double strands; these paired double strands are formed by stem 1 and stem 2. The stability of the paired stem-loop elements is generally determined by the length, the number of stem 1 nucleotides capable of forming base pairs (preferably canonical base pairs, more preferably Watson-Crick base pairs) with nucleotides of stem 2, relative to the number of stem 1 nucleotides capable of forming such base pairs with nucleotides of stem 2 (mismatches or bulges). According to the invention, the optimal loop length is 3-10 nucleotides, more preferably 4 to 7 nucleotides, for example 4, 5, 6, or 7 nucleotides. If a given nucleic acid sequence is characterized by a stem-loop, the corresponding complementary nucleic acid sequence is generally also characterized by a stem-loop. Stem loops are typically formed by single-stranded RNA molecules. For example, several stem loops exist in the 5' replication recognition sequence of the alphavirus genomic RNA.

[0141] According to the present invention, "disruption" or "damage" of a specific secondary structure (e.g., stem-loop) of a nucleic acid molecule means the absence or alteration of that specific secondary structure. Typically, a secondary structure may be disrupted due to a change in at least one nucleotide that is part of the secondary structure. For example, a stem-loop can be disrupted by altering one or more nucleotides forming the stem, making nucleotide pairing impossible.

[0142] According to the present invention, the term "tertiary structure" for nucleic acid molecules refers to the three-dimensional structure of nucleic acid molecules defined by atomic coordinates.

[0143] In some embodiments of this disclosure, the RNA molecule is a "replicon RNA" or "replicon RNA molecule" or simply a "replicon," particularly a "self-replicating RNA" or "self-amplifying RNA" or "replicable RNA molecule." A replicon RNA molecule is RNA that can be replicated by an RNA-dependent RNA polymerase (replicaase) by containing a nucleotide sequence that can be recognized by the replicaase. A replicon does not necessarily encode a replicaase, allowing it to be replicated cis (by an encoded replicaase; also called a "cis replicon") or trans (by a replicaase provided in another manner, such as a separate replicaase encoding a nucleic acid, such as mRNA; also called a "trans replicon").

[0144] In some implementations, the replicon or self-replicating RNA originates from or contains elements derived from ssRNA viruses, particularly positive-sense ssRNA viruses such as alphaviruses. Alphaviruses are typical examples of positive-sense RNA viruses. Alphaviruses replicate in the cytoplasm of infected cells (for a review of the alphavirus life cycle, see José). et al (See Future Microbiol., 2009, vol. 4, pp. 837-856). The total genome length of many alphaviruses is typically 11,000 to 12,000 nucleotides, and the genomic RNA usually has a 5' cap and a 3'-poly (A) tail. The alphavirus genome encodes non-structural proteins (involved in transcription, modification, and replication of viral RNA, as well as protein modification) and structural proteins (forming viral particles). There are typically two open reading frames (ORFs) in the genome. The four non-structural proteins (nsP1-nsP4) are usually encoded by the first ORF, which begins near the 5' end of the genome, while the alphavirus structural proteins are encoded by the second ORF, which is found downstream of the first ORF and extends near the 3' end of the genome. Generally, the first ORF is larger than the second ORF, with a ratio of approximately 2:1. In cells infected with alphavirus, only the nucleic acid sequences encoding non-structural proteins are translated from genomic RNA, while the genetic information encoding structural proteins can be translated from subgenomic transcripts, which are RNA molecules (mRNA) similar to eukaryotic messenger RNA. et al(Reference: ., 2010, Antiviral Res., vol. 87 pp. 111-124). Following infection, in the early stages of the viral life cycle, the (+) strand genomic RNA acts directly as messenger RNA, translating the open reading frame (OPF) encoding the non-structural multiprotein (nsP1234). Alphavirus-derived vectors have been proposed for delivering exogenous genetic information to target cells or organisms. In simpler approaches, the OPF encoding the alphavirus structural protein is replaced by the OPF encoding the target protein. Alphavirus-based trans-replication systems rely on alphavirus nucleotide sequence elements on two separate nucleic acid molecules: one molecule encodes a viral replicase, while the other is capable of being trans-replicated by that replicase (hence the name trans-replication system). Trans-replication requires the presence of both nucleic acid molecules in a given host cell. The nucleic acid molecule capable of trans-replication by the replicase must contain certain alphavirus sequence elements to allow recognition by the alphavirus replicase and RNA synthesis.

[0145] According to the present invention, nucleic acids, such as RNA (e.g., rRNA), can encode peptides or proteins. Therefore, transcribed nucleic acid sequences or their transcripts can contain open reading frames (ORFs) encoding peptides or proteins.

[0146] According to the present invention, the term "nucleic acid encoding a peptide or protein" refers to a nucleic acid, if present in a suitable environment, preferably intracellularly, that can guide the assembly of amino acids to produce a peptide or protein during translation. Preferably, the encoding RNA according to the present invention is capable of interacting with cellular translation mechanisms that allow the encoding RNA to be translated to produce a peptide or protein.

[0147] According to the present invention, the term "peptide" includes oligopeptides and polypeptides, and refers to a substance comprising 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 preferably up to fifty, preferably one hundred or one hundred or preferably one hundred and fifty-five amino acids linked together by peptide bonds. The term "protein" refers to a large peptide, preferably a peptide having at least 151 amino acids, but the terms "peptide" and "protein" are generally used synonymously herein.

[0148] 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 containing bonds such as ester bonds, thioether bonds or disulfide bonds.

[0149] According to the present invention, the terms "initiation codon" and "start codon" are synonymous with the codon (base triplet) of an RNA molecule, which is potentially the first codon translated by the ribosome. Such a codon typically encodes the amino acid methionine in eukaryotes and modified methionine in prokaryotes. The most common start codon in both eukaryotes and prokaryotes is AUG. Unless specifically indicated herein to mean a start codon other than AUG, the terms "initiation codon" and "start codon" in relation to RNA molecules refer to the codon AUG. According to the present invention, the terms "initiation codon" and "start codon" are also used to refer to the corresponding base triplet of deoxyribonucleic acid, i.e., the base triplet encoding the start codon of RNA. If the start codon of messenger RNA is AUG, then the base triplet encoding AUG is ATG. According to the present invention, the terms "initiation codon" and "start codon" preferably refer to a functional start codon or start codon, that is, a start codon or start codon that is used or will be used by the ribosome to initiate translation. AUG codons may exist in RNA molecules that are not used by the ribosome to initiate translation, for example, due to the short distance between the codon and the cap. These codons are not included in the terms "functional start codon" or "start codon".

[0150] The following provides specific and / or preferred variations of various features of the invention. The invention also contemplates embodiments that are particularly preferred, which arise by combining two or more specific and / or preferred variations described for two or more features of the invention.

[0151] "Separated" means altered or removed from its natural state. For example, cells, nucleic acids, or peptides naturally present in living animals are not "separated," but rather are identical cells, partially or completely separated from their natural coexisting substances; nucleic acids or peptides are "separated." Preferably, the separated cells, nucleic acids, or peptides exist in a purified or substantially purified state. The separated cells or cell populations preferably exist in the absence of cells of different cell types; for example, separated T cells exist in the absence of other blood cells such as dendritic cells. Preferably, the separated cells do indeed exist only with genotypical cells of the same cell type.

[0152] The term "syngeneic" is used to describe cells that have the same genetic information as another cell or cell population.

[0153] The term "autologous" is used to describe anything derived from the same object. For example, "autologous transplantation" refers to the transplantation of tissue or organ derived from the same object. Such procedures are advantageous because they overcome immune barriers that would otherwise lead to rejection.

[0154] The term "alien" is used to describe anything that originates from different individuals of the same species. Two or more individuals are considered alliens when the genes at one or more loci are different.

[0155] The term "homogeneous" is used to describe anything derived from individuals or tissues with the same genotype, i.e., identical twins or animals of the same inbred strain, or their tissues.

[0156] The term "heterogeneous" is used to describe something composed of multiple different elements. As an example, transferring bone marrow from one individual to another constitutes a heterologous transplant. Heterogeneous genes are genes derived from sources other than the recipient.

[0157] In the context of this invention, the term "recombinant" means "prepared by genetic engineering." Preferably, in the context of this invention, the "recombinant object," such as recombinant cells, is not naturally occurring.

[0158] As used in this article, the term "naturally occurring" refers to the fact that an object can be found in nature. For example, peptides or nucleic acids that exist in organisms (including viruses) and can be isolated from natural sources and have not been intentionally modified in the laboratory are naturally occurring.

[0159] The term "lentivirus" as used in this article refers to a genus within the family Retroviridae. Lentivirals possess a unique ability among retroviruses to infect non-dividing cells; they can deliver substantial amounts of genetic information into the host cell's DNA, making them one of the most efficient gene delivery vectors. HIV, SIV, and FIV are examples of lentiviruses. Lentiviral vectors provide a means to achieve significant levels of gene transfer in vivo.

[0160] In the context of this invention, the terms "immune effector cells" or "immune reactive cells" refer to cells that perform effector functions during an immune response.

[0161] In the context of this invention, the term "effective function" includes any function mediated by components of the immune system that result in, for example, the killing of diseased cells such as tumor cells, or the inhibition of tumor growth and / or the suppression of tumor development, including the inhibition of tumor spread and metastasis. Preferably, in the context of this invention, an effector function is a T cell-mediated effector function. Such functions include, in the presence of helper T cells (CD4+ T cells), the release of cytokines and / or the activation of CD8+ lymphocytes (CTLs) and / or B cells, and in the presence of CTLs, for example, cell lysis via apoptosis or perforin-mediated cytokine production, the production of cytokines such as IFN-γ and TNF-α, and the specific cytolytic killing of target cells expressing antigens, i.e., cells characterized by antigen expression.

[0162] In one embodiment, "immune effector cells" are capable of binding antigens, such as those presented in the context of MHC (the term MHC includes HLA) on the cell or expressed on the cell surface, and mediating 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 this invention, "immune effector cells" are T cells, preferably CD4+ and / or CD8+ T cells. According to the invention, the term "immune effector cells" also includes cells that can mature into immune cells (e.g., T cells, particularly helper T cells, or cytolytic T cells) upon appropriate stimulation. Immune effector cells include CD34+ hematopoietic stem cells, immature and mature T cells, and immature and mature B cells. When exposed to antigens, T cell precursors differentiate into cytolytic T cells, similar to clonal selection in the immune system.

[0163] Preferably, “immune effector cells” recognize antigens with some degree of specificity, particularly if presented in the context of MHC or present on the surface of diseased cells such as cancer cells. Preferably, this recognition enables the antigen-recognizing cell to respond or react. If the cell is a helper T cell (CD4+ T cell), such responsiveness or reactance may involve the release of cytokines and / or activation of CD8+ lymphocytes (CTLs) and / or B cells. If the cell is a CTL, such responsiveness or reactance may involve, for example, elimination of the cell via apoptosis or perforin-mediated cell lysis, i.e., cells characterized by antigen expression. According to the 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 the antigen. CTL responsiveness can also be determined using artificial reporter assays that precisely indicate CTL responsiveness. Such antigen-recognizing and responsive CTLs are also referred to herein as “antigen-responsive CTLs.”

[0164] "Lymphoid cells" are cells, optionally after appropriate modification, such as after transfer of antigen receptors like TCRs or CARs, capable of generating an immune response, such as a cellular immune response, or precursor cells of such cells, and include lymphocytes, preferably T lymphocytes, lymphoblasts, and plasma cells. Lymphoid cells can be immune effector cells as described herein. Preferred lymphoid cells are T cells that can be modified to express antigen receptors on their cell surface. In one embodiment, the lymphoid cells lack endogenous expression of T cell receptors.

[0165] The terms “T cell” and “T lymphocyte” are used interchangeably herein and include both helper T cells (CD4+ T cells) and cytotoxic T cells (CTL, CD8+ T cells), which include cytolytic T cells. The term “antigen-specific T cell” or similar terms refers to a T cell that recognizes an antigen targeted by the T cell and preferably performs effector functions. A T cell is considered antigen-specific if it kills a target cell expressing an antigen. T cell specificity can be assessed using any of a variety of standard techniques, such as in a chromium release assay or a proliferation assay. Optionally, the synthesis of lymphokines (e.g., interferon-γ) can be measured.

[0166] T cells belong to a group of white blood cells called lymphocytes and play an important role in cell-mediated immunity. They can be distinguished from other lymphocyte types, such as B cells and natural killer cells, by the presence of a special receptor called the T cell receptor (TCR) on their cell surface. The thymus is the main organ responsible for T cell maturation. Several different T cell subsets have been identified, each with distinct functions.

[0167] Helper T cells assist other white blood cells in the immune process, including B cells maturing into plasma cells and activated cytotoxic T cells and macrophages, among other functions. These cells are also called CD4+ T cells because they express the CD4 glycoprotein on their surface. Helper T cells become activated when MHC class II molecules expressed on the surface of antigen-presenting cells (APCs) present peptide antigens to them. Once activated, they rapidly divide and secrete small proteins called cytokines, which regulate or assist active immune responses.

[0168] Cytotoxic T cells destroy virus-infected cells and tumor cells, and are also involved in transplant rejection. These cells are also known as CD8+ T cells because they express the CD8 glycoprotein on their surface. These cells recognize their targets by binding to antigens associated with MHC class I, which are present on the surface of almost every cell in the body.

[0169] Regulatory T cells, or Tregs, are a subset of T cells that regulate the immune system, maintain tolerance to self-antigens, and prevent autoimmune diseases. Tregs are immunosuppressive, typically inhibiting or downregulating the induction and proliferation of effector T cells. Tregs express the biomarkers CD4, FoxP3, and CD25.

[0170] As used herein, the term "naive T cell" refers to a mature T cell that, unlike activated or memory T cells, has not encountered their homologous antigens in the periphery. Naive T cells are typically characterized by surface expression of L-selectin (CD62L), the absence of activation markers CD25, CD44, or CD69, and the absence of the memory CD45RO isotype.

[0171] As used herein, the term "memory T cells" refers to a subset or population of T cells that have previously encountered and responded to their homologous antigens. Upon a second encounter with the antigen, memory T cells can proliferate to produce a faster and stronger immune response than the first time the immune system responded to the antigen. Memory T cells can be CD4+ cells. + or CD8 + And it is usually expressed as CD45RO.

[0172] According to the present invention, the term "T cell" also includes cells that can mature into T cells in response to appropriate stimulation.

[0173] Most T cells possess a T cell receptor (TCR) that exists as a complex of several proteins. The actual T cell receptor consists of two separate polypeptide chains, produced by independent T cell receptor α and β (TCRα and TCRβ) genes, and are referred to as α- and β-TCR chains. γδ T cells (γδ T cells) represent a small subgroup of T cells that possess 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 much smaller than αβ T cells (2% of total T cells).

[0174] All T cells originate from hematopoietic stem cells in the bone marrow. Hematopoietic progenitor cells derived from hematopoietic stem cells reside in the thymus and proliferate through cell division to produce a large number of immature thymocytes. The earliest thymocytes express neither CD4 nor CD8, and are therefore classified as double-negative (CD4+). - CD8 - ) cells. As they progress through their development, they transform into double-positive thymocytes (CD4+). + CD8 + Finally, it matures into a single positive (CD4) positivity. + CD8 - or CD4 - CD8 + Thymocytes are then released from the thymus into the peripheral tissues.

[0175] T cells can typically be prepared in vitro or ex vivo using standard procedures. For example, commercially available cell isolation systems can be used to isolate T cells from the bone marrow, peripheral blood, or a portion of the bone marrow or peripheral blood of mammals (e.g., patients). Optionally, T cells can be derived from related or unrelated humans, non-human animals, cell lines, or cultures. Samples containing T cells can be, for example, peripheral blood mononuclear cells (PBMCs).

[0176] The term "epitope" refers to a portion or fragment of a molecule, such as an antigen, that is recognized by the immune system. For example, an epitope can be recognized by T cells, B cells, or antibodies. An epitope of an antigen may include continuous or discontinuous portions of the antigen, and its length can be from about 5 to about 100 amino acids, for example, from about 5 to about 50, more preferably from about 8 to about 30, and most preferably from about 10 to about 25 amino acids. For example, the length of an epitope can preferably be 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids. In one embodiment, the epitope length is from about 10 to about 25 amino acids. The term "epitope" includes T-cell epitopes.

[0177] The term "T-cell epitope" refers to a portion or fragment of a protein that is recognized by T cells when presented in the context of MHC molecules. The terms "major histocompatibility complex" and the abbreviation "MHC" encompass MHC class I and MHC class II molecules and refer to gene complexes present in all vertebrates. MHC proteins or molecules are important for signaling between lymphocytes and antigen-presenting or diseased cells in immune responses, where MHC proteins or molecules bind peptide epitopes and present them for recognition by T-cell receptors on T cells. MHC-encoded proteins are expressed on the cell surface and present T cells with both self-antigens (peptide fragments from the cell itself) and non-self-antigens (e.g., fragments from invading microorganisms). In the case of class I MHC / peptide complexes, the binding peptide is typically about 8 to about 10 amino acids long, although longer or shorter peptides may be effective. In the case of class II MHC / peptide complexes, the binding peptide is typically about 10 to about 25 amino acids long, particularly about 13 to about 18 amino acids long, while longer and shorter peptides may be effective.

[0178] The medical preparations described herein, particularly kit products, may include instruction materials or instructions. As used herein, "instruction materials" or "instructions" include publications, recordings, diagrams, or any other medium of expression that can be used to convey the usefulness of the compositions and methods of the present invention. Instruction materials for kit products of the present invention may, for example, be affixed to or shipped with a container containing the composition of the present invention. Alternatively, the instruction materials may be shipped separately from the container, with the intention that the instruction materials and the composition be used synergistically by the recipient.

[0179] The following provides specific and / or preferred variations of various features of the invention. The invention also contemplates embodiments that are particularly preferred, which arise by combining two or more specific and / or preferred variations described for two or more features of the invention.

[0180] Nucleic acid

[0181] As used herein, the terms "polynucleotide" or "nucleic acid" are intended to include DNA and RNA, such as genomic DNA, cDNA, mRNA, recombinant and chemically synthesized molecules. Nucleic acids can be single-stranded or double-stranded. RNA includes in vitro transcribed RNA (IVT RNA) or synthetic RNA. According to the invention, it is preferable to isolate polynucleotides.

[0182] Nucleic acids can be contained in vectors. As used herein, the term "vector" includes any vector known to those skilled in the art, including plasmid vectors, granular vectors, bacteriophage vectors (e.g., λ phage), viral vectors (e.g., retroviral, adenoviral, or baculoviral vectors), or artificial chromosome vectors (e.g., bacterial artificial chromosomes (BAC), yeast artificial chromosomes (YAC), or P1 artificial chromosomes (PAC)). Vectors include expression vectors and cloning vectors. Expression vectors include plasmids and viral vectors and typically contain the desired coding sequence and appropriate DNA sequences necessary for expressing the operable linked coding sequence in a specific host organism (e.g., bacteria, yeast, plants, insects, or mammals) or in an in vitro expression system. Cloning vectors are typically used for engineering and amplifying a desired DNA fragment and may lack the functional sequences required for expressing the desired DNA fragment.

[0183] In some implementations, at least one or all of the first, second, third, and fourth RNA molecules are modified RNA molecules.

[0184] Modified RNA

[0185] In some embodiments, the RNA or RNA molecule described herein is modified RNA. In some embodiments, the modified RNA contains at least one functional analog of A, C, G and / or U.

[0186] In the implementation scheme, the RNA described herein may have modified nucleotide / nucleoside / backbone modifications. The term "RNA modification" as used herein may refer to chemical modifications that include backbone modifications as well as sugar or base modifications.

[0187] In this document, modified RNA molecules as defined herein may contain nucleotide analogs / modifications, such as backbone modifications, sugar modifications, or base modifications. A backbone modification relevant to this disclosure is a modification in which the phosphate group in the nucleotide backbone of an RNA molecule as defined herein is chemically modified. A sugar modification relevant to this disclosure is a chemical modification of the sugar in the nucleotide backbone of an RNA molecule as defined herein. Furthermore, a base modification relevant to this disclosure is a chemical modification of the base portion of the nucleotide in an RNA molecule. In this document, the nucleotide analog or modification is preferably selected from nucleotide analogs suitable for transcription and / or translation.

[0188] Sugar modification: Modified nucleosides and nucleotides that can be incorporated into the sugar moiety of RNA molecules described herein can be modified. For example, the 2' hydroxyl group (OH) can be modified or replaced with a number of different "oxygen" or "deoxy" substituents. Examples of "oxygen"-2' hydroxyl group modifications include, but are not limited to, alkoxy or aryloxy groups (-OR, e.g., R = H, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl, or sugar); polyethylene glycol (PEG), -O(CH2CH2O)nCH2CH2OR; locked nucleic acids (LNA), where the 2' hydroxyl group is linked to the 4' carbon of the same ribose, for example, via a methylene bridge; and amino groups (-O-amino, where the amino group, e.g., NRR, can be alkylamino, dialkylamino, heterocyclic, arylamino, diarylamino, heteroarylamino, or diheteroarylamino, ethylenediamine, polyamino) or aminoalkoxy groups. "Deoxy" modifications include hydrogen, amino groups (e.g., NH₂; alkylamino, dialkylamino, heterocyclic, arylamino, diarylamino, heteroarylamino, diheteroarylamino, or amino acids); or the amino group can be attached to a sugar via a linker containing one or more C, N, and O atoms. The sugar group may also contain one or more carbons having a stereochemical configuration opposite to the corresponding carbon in ribose. Therefore, modified RNA molecules can include nucleotides containing, for example, arabinose as a sugar.

[0189] Backbone Modification: The phosphate backbone can also be modified in modified nucleosides and nucleotides, which can be incorporated into modified RNA molecules as described herein. The phosphate groups of the backbone can be modified by replacing one or more oxygen atoms with different substituents. Furthermore, modified nucleosides and nucleotides can include complete replacement of unmodified phosphate ester moieties with modified phosphate esters as described herein. Examples of modified phosphate ester groups include, but are not limited to, thiophosphates, selenophosphates, borano phosphates, borano phosphate esters, hydrophosphonates, aminophosphates, alkyl or aryl phosphonates, and phosphate triesters. Dithiophosphates have two non-linked oxygen atoms replaced by sulfur. Phosphate linkers can also be modified by replacing the linked oxygen atoms with nitrogen (bridged aminophosphate), sulfur (bridged thiophosphate), and carbon (bridged methylene-phosphonate).

[0190] Base modification: Modified nucleosides and nucleotides, which can be incorporated into RNA molecules modified as described herein, can be further modified at the nucleobase moiety. Examples of nucleobases 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 at the major groove face. In some embodiments, major groove chemical modifications may include amino groups, thiol groups, alkyl groups, or halogen groups.

[0191] In specific embodiments of this disclosure, the nucleotide analog / modification is selected from base modifications, preferably from 2-amino-6-chloropurine nucleotide-5'-triphosphate, 2-aminopurine-ribonucleoside-5'-triphosphate, 2-aminoadenosine-5'-triphosphate, 2'-amino-2'-deoxy-cytidine-triphosphate, 2-thiocytidine-5'-triphosphate, 2-thiouridine-5'-triphosphate, 2'-fluorothymidine-5'-triphosphate, etc. 2'-O-methylinosine-5'-triphosphate, 4-thiouridine-5'-triphosphate, 5-aminoallylcytidine-5'-triphosphate, 5-aminoallylcytidine-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 - Triphosphate, 5-iodouridine-5'-triphosphate, 5-iodo-2'-deoxyuridine-5'-triphosphate, 5-methylcytidine-5'-triphosphate, 5-methyluridine-5'-triphosphate, 5-propynyl-2'-deoxycytidine-5'-triphosphate, 5-propynyl-2'-deoxyuridine-5'-triphosphate, 6-azacytidine-5'-triphosphate, 6-azauridine-5'-triphosphate, 6-chloropurine nucleoside-5'-triphosphate, 7-deazo-adenosine-5'-triphosphate, 7 -Denitroguanosine-5'-triphosphate, 8-azaadenosine-5'-triphosphate, 8-azidoadenosine-5'-triphosphate, benzimidazole-ribonucleoside-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, flavin-5'-triphosphate. Particularly preferred are nucleotides that can be given base modifications, selected from nucleotides with base modifications consisting of 5-methylcytidine-5'-triphosphate, 7-denitroguanosine-5'-triphosphate, 5-bromocytidine-5'-triphosphate, and pseudouridine-5'-triphosphate.In some embodiments, the modified nucleosides include pyridine-4-ketoribonucleotide, 5-aza-uridine, 2-thio-5-aza-uridine, 2-thiouridine, 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxyuridine, 3-methyluridine, 5-carboxymethyluridine, 1-carboxymethyl-pseudouridine, 5-propynyl-uridine, 1-propynyl-pseudouridine, 5-taurate methyluridine, 1-taurate methyl-pseudouridine, 5-taurate methyl-2-thiouridine, 1-taurate methyl -4-Thio-uridine, 5-methyl-uridine, 1-methyl-pseudouridine, 4-thio-1-methyl-pseudouridine, 2-thio-1-methyl-pseudouridine, 1-methyl-1-denitro-pseudouridine, 2-thio-1-methyl-1-denitro-pseudouridine, dihydrouridine, dihydro-pseudouridine, 2-thio-dihydrouridine, 2-methoxy-uridine, 2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine, and 4-methoxy-2-thio-pseudouridine. In a preferred embodiment, the functional analogue replacing uridine is N1-methyl-pseudouridine (m1Ψ).

[0192] In some embodiments, the modified nucleosides include 5-aza-cytidine, pseudocytidine, 3-methylcytidine, N4-acetylcytidine, 5-formylcytidine, N4-methylcytidine, 5-hydroxymethylcytidine, 1-methyl-pseudocytidine, pyrrolocytidine, pyrrolo-pseudocytidine, 2-thiocytidine, 2-thio-5-methylcytidine, 4-thio-pseudocytidine, 4-thio-1-methyl-pseudocytidine, 4-thio-1-methyl-pseudocytidine, etc. 1-Methyl-1-deazo-pseudoisocytidine, 1-methyl-1-deazo-pseudoisocytidine, zebularine, 5-aza-zebularine, 5-methyl-zebularine, 5-aza-2-thio-zebularine, 2-thio-zebularine, 2-methoxy-cytidine, 2-methoxy-5-methyl-cytidine, 4-methoxy-pseudoisocytidine, and 4-methoxy-1-methyl-pseudoisocytidine.

[0193] In other embodiments, the modified nucleosides include 2-aminopurine, 2,6-diaminopurine, 7-deadenine, 7-deadenine-8-azaadenine, 7-deadenine-2-aminopurine, 7-deadenine-8-azaa-2-aminopurine, 7-deadenine-2,6-diaminopurine, 7-deadenine-8-aza-2,6-diaminopurine, 1-methyladenosine, N6-methyladenosine, N... 6-Isopentenyl adenosine, N6-(cis-hydroxyisopentenyl) adenosine, 2-methylthio-N6-(cis-hydroxyisopentenyl) adenosine, N6-glycylcarbamoyl adenosine, N6-threonylcarbamoyl adenosine, 2-methylthio-N6-threonylcarbamoyl adenosine, N6,N6-dimethyl adenosine, 7-methyl adenosine, 2-methylthio-adenosine, and 2-methoxy-adenosine. In other embodiments, the modified nucleosides include inosine, 1-methyl-inosine, wyosine, wyosine, 7-deazoguanosine, 7-deazo-8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deazo-guanosine, 6-thio-7-deazo-8-aza-guanosine, 7-methyl-guanosine, 6-thio-7-methyl-guanosine, 7-methyl-guanosine, 6-thio-7-methyl-guanosine, 7-methyl-inosine, 6-methoxy-guanosine, 1-methyl-guanosine, N2-methyl-guanosine, N2,N2-dimethyl-guanosine, 8-oxo-guanosine, 7-methyl-8-oxo-guanosine, 1-methyl-6-thio-guanosine, N2-methyl-6-thio-guanosine, and N2,N2-dimethyl-6-thio-guanosine.

[0194] In some embodiments, the nucleotide may be modified on the main groove face and may include replacing the hydrogen at C-5 of uracil with a methyl or halogen group. In specific embodiments, the modified nucleoside is 5'-O-(1-thiophosphate)-adenosine, 5'-O-(1-thiophosphate)-cytidine, 5'-O-(1-thiophosphate)-guanosine, 5'-O-(1-thiophosphate)-uridine, or 5'-O-(1-thiophosphate)-pseuuridine.

[0195] In another embodiment, the modified RNA may comprise a subset selected from 6-aza-cytidine, 2-thio-cytidine, α-thio-cytidine, pseudo-isocytidine, 5-aminoallyl-uridine, 5-iodo-uridine, N1-methyl-pseudo-uridine, 5,6-dihydrouridine, α-thio-uridine, 4-thio-uridine, 6-aza-uridine, 5-hydroxy-uridine, deoxy-thymidine, 5-methyl-uridine, pyridoxine, etc. Nucleoside modification of troponin, inosine, α-thioguanosine, 6-methylguanosine, 5-methylcytidine, 8-oxoguanosine, 7-deazoguanosine, N1-methyladenosine, 2-amino-6-chloropurine, N6-methyl-2-aminopurine, pseudo-isocytidine, 6-chloropurine, N6-methyladenosine, α-thioadenosine, 8-azido-adenosine, and 7-deazo-adenosine.

[0196] In some preferred embodiments, the RNA comprises a modified nucleoside that replaces at least one (e.g., each) uridine.

[0197] The term "uracil" as used in this article describes one of the nucleobases that may be present in RNA nucleic acids. The structure of uracil is: .

[0198] The term "uridine" as used in this article describes one of the nucleosides that may exist in RNA. The structure of uridine is: .

[0199] UTP (uridine 5'-triphosphate) has the following structure: .

[0200] Pseudo-UTP (pseudouridine 5'-triphosphate) has the following structure: .

[0201] “Pseudouridine” is an example of a modified nucleoside that is an isomer of uridine in which uracil is linked to a pentose ring via a carbon-carbon bond instead of a nitrogen-carbon glycosidic bond.

[0202] Another exemplary modified nucleoside is N1-methyl-pseuuridine (m1Ψ), which has the following structure: .

[0203] N1-Methyl-pseudo-UTP has the following structure: .

[0204] Another exemplary modified nucleoside is 5-methyluridine (m5U), which has the following structure: .

[0205] In some preferred embodiments, one or more uridines in the RNA described herein are replaced by modified nucleosides. In some embodiments, the modified nucleosides are modified uridines.

[0206] In some preferred embodiments, the RNA comprises a modified nucleoside replacing at least one uridine. In some embodiments, the RNA comprises a modified nucleoside replacing each uridine.

[0207] In some preferred embodiments, the modified nucleoside is independently selected from pseudouridine (ψ), N1-methyl-pseudouridine (m1ψ), and 5-methyl-uridine (m5U). In some embodiments, the modified nucleoside includes pseudouridine (ψ). In some embodiments, the modified nucleoside includes N1-methyl-pseudouridine (m1ψ). In some embodiments, the modified nucleoside comprises 5-methyl-uridine (m5U). In some embodiments, the RNA may contain more than one type of modified nucleoside, and the modified nucleoside is independently selected from pseudouridine (ψ), N1-methyl-pseudouridine (m1ψ), and 5-methyl-uridine (m5U). In some embodiments, the modified nucleoside includes pseudouridine (ψ) and N1-methyl-pseudouridine (m1ψ). In some embodiments, the modified nucleoside includes pseudouridine (ψ) and 5-methyl-uridine (m5U). In some embodiments, the modified nucleoside comprises N1-methyl-pseudouridine (m1ψ) and 5-methyl-uridine (m5U). In some embodiments, the modified nucleosides include pseudouridine (ψ), N1-methyl-pseudouridine (m1ψ), and 5-methyl-uridine (m5U).

[0208] In some preferred embodiments, the modifying nucleoside replacing one or more (e.g., all) uridines in the RNA can be any one or more of the following: 3-methyl-uridine (m 3 U), 5-methoxyuridine (mo) 5 U), 5-aza-uridine, 6-aza-uridine, 2-thio-5-aza-uridine, 2-thio-uridine (s 2 U), 4-thiouridine (s) 4 U), 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxy-uridine (ho) 5 U), 5-aminoallyl-uridine, 5-halo-uridine (e.g., 5-iodo-uridine or 5-bromo-uridine), uridine 5-oxyacetic acid (cmo 5 U), uridine 5-oxyacetic acid methyl ester (mcmo) 5 U), 5-carboxymethyluridine (cm) 5 U), 1-carboxymethyl-pseudouridine, 5-carboxyhydroxymethyl-uridine (chm) 5U), 5-carboxyhydroxymethyl-uridine methyl ester (mchm) 5 U), 5-methoxycarbonylmethyluridine (mcm) 5 U), 5-methoxycarbonylmethyl-2-thio-uridine (mcm) 5 s 2 U), 5-aminomethyl-2-thio-uridine (nm) 5 s 2 U), 5-methylaminomethyluridine (mnm) 5 U), 1-ethyl-pseudouridine, 5-methylaminomethyl-2-thio-uridine (mnm) 5 s 2 U), 5-methylaminomethyl-2-seleno-uridine (mnm) 5 se 2 U), 5-carbamoylmethyluridine (ncm) 5 U), 5-Carboxymethylaminomethyluridine (cmnm) 5 U), 5-Carboxymethylaminomethyl-2-thio-uridine (cmnm) 5 s 2 U), 5-propynyl-uridine, 1-propynyl-pseudouridine, 5-tauric acid methyl-uridine (τm) 5 U), 1-Taurate methyl-pseudouridine, 5-Taurate methyl-2-thio-uridine (τm5s2U), 1-Taurate methyl-4-thio-pseudouridine, 5-methyl-2-thio-uridine (m 5 s 2 U), 1-methyl-4-thio-pseuuridine (m 1 s 4 ψ), 4-thio-1-methyl-pseuuridine, 3-methyl-pseuuridine (m 3 ψ), 2-thio-1-methyl-pseudouridine, 1-methyl-1-denitro-pseudouridine, 2-thio-1-methyl-denitro-pseudouridine, dihydrouridine (D), dihydrouridine, 5,6-dihydrouridine, 5-methyl-dihydrouridine (m) 5 D) 2-Thio-dihydrouridine, 2-Thio-dihydropseudouridine, 2-methoxy-uridine, 2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine, 4-methoxy-2-thio-pseudouridine, N1-methyl-pseudouridine, 3-(3-amino-3-carboxypropyl)uridine (acp) 3 U), 1-methyl-3-(3-amino-3-carboxypropyl)pseudouridine (acp) 3 ψ), 5-(isopentenylaminomethyl)uridine (inm) 5 U), 5-(isopentenylaminomethyl)-5-thio-uridine (inm) 5 s 2U), α-thiouridine, 2'-O-methyluridine (Um), 5,2'-O-dimethyluridine (m) 5 Um), 2'-O-methyl-pseudouridine (ψm), 2-thio-2'-O-methyluridine (s) 2 Um), 5-methoxycarbonylmethyl-2'-O-methyl-uridine (mcm) 5 Um), 5-carbamoylmethyl-2'-O-methyluridine (ncm) 5 Um), 5-carboxymethylaminomethyl-2'-O-methyl-uridine (cmnm) 5 Um), 3,2'-O-dimethyluridine (m) 3 Um), 5-(isopentenylaminomethyl)-2'-O-methyl-uridine (inm) 5 Um), 1-thio-uridine, deoxythymidine, 2'-F-ara-uridine, 2'-F-uridine, 2'-OH-ara-uridine, 5-(2-methyl ester vinyl)uridine, 5-[3-(1-E-propenyl amino)uridine, or any other modified uridine known in the art.

[0209] In embodiments, the RNA comprises other modified nucleosides or comprises further modified nucleosides, such as modified cytidines, as described above. For example, in one embodiment, 5-methylcytidine is partially or completely, preferably completely, substituted for cytidine in the RNA. In one embodiment, the RNA comprises 5-methylcytidine and one or more compounds selected from pseudouridine (ψ), N1-methyl-pseudouridine (m1ψ), and 5-methyl-uridine (m5U). In embodiments, the RNA comprises 5-methylcytidine and N1-methyl-pseudouridine (m1ψ). In some embodiments, the RNA comprises 5-methylcytidine in place of each cytidine and N1-methyl-pseudouridine (m1ψ) in place of each uridine.

[0210] cap

[0211] 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 for codon usage.

[0212] "RNA containing a 5'-cap," "RNA provided with a 5'-cap," "RNA modified with a 5'-cap," or "capped RNA" refers to RNA containing a 5'-cap. For example, providing RNA with a 5'-cap can be achieved through in vitro transcription of a DNA template in the presence of the 5'-cap, wherein the 5'-cap is 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 (e.g., a capping enzyme for vaccinia virus). In capped RNA, the 3' position of the first base of the (capped) RNA molecule is linked via a phosphodiester bond to the 5' position of the subsequent base ("the second base") of the RNA molecule.

[0213] In this disclosure, naturally occurring caps are typically selected from unmethylated cap dinucleotides (G(5')ppp(5')N; also known as GpppN) and methylated cap dinucleotides ((m 7 G(5')ppp(5')N; also known as m 7 GpppN). m 7 GpppN (where N is G) is represented by the following formula: .

[0214] The capped RNA disclosed herein can be prepared in vitro and is therefore independent of the capping mechanism in the host cell. Co-transcriptional capping is achieved through all four ribonucleoside triphosphates or their functional analogues and capping reagents such as m 7 G(5')ppp(5')G(also known as m) 7 It functions by in vitro transcription of DNA templates using bacterial or bacteriophage nucleic acid polymerases in the presence of GpppG. 7 The 3'-OH of the guanosine moiety of GpppG initiates transcription by nucleophilicly attacking the α-phosphate of the next templated nucleoside triphosphate (pppN), producing the intermediate m. 7 GpppGpN (where N is the second base in the RNA molecule).

[0215] In a preferred embodiment of this disclosure, the RNA molecule comprises a 5'-cap analog. Cap analogs have been initially described as facilitating the large-scale synthesis of RNA transcripts through in vitro transcription.

[0216] For messenger RNA, several cap analogs (also known as synthetic caps) have been generally described to date, and all of them can be used in the context of this disclosure. Ideally, cap analogs associated with higher translation efficiency and / or increased in vivo and / or increased in vitro degradation resistance are selected.

[0217] Preferably, a cap-like compound that can only be incorporated into the RNA chain in one direction is used. (Pasquinelli) et al. In 1995, RNA J. 1:957-967 demonstrated that during in vitro transcription, bacteriophage RNA polymerase uses a 7-methylguanosine unit to initiate transcription, thereby approximately 40-50% of capped transcripts have an inverted cap dinucleotide (i.e., the initial reaction product is Gpppm). 7 RNA with a reversed cap does not function in the translation of nucleic acid sequences into proteins, compared to RNA with the correct cap. Therefore, it is desirable to incorporate the cap in the correct direction, i.e., to produce RNA with a cap that substantially corresponds to m... 7 RNAs with structures such as GpppGpN, etc., have been shown to have reverse integration of cap dinucleotides inhibited by substitution of the 2'- or 3'-OH group of methylated guanosine units (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 analogues" produces RNA higher than that synthesized in conventional 5'-cap RNA. 7 In the presence of GpppG, in vitro transcribed RNA is translated more efficiently. For this reason, Holtkamp, ​​for example... et al. , 2006, Blood 108:4009-4017 describes a cap analogue in which the 3'OH group of the methylated guanosine unit is replaced by OCH3 (7-methyl(3'-O-methyl)GpppG; anti-reverse cap analogue (ARCA)). ARCA is a suitable cap dinucleotide according to this disclosure: .

[0218] In the implementation, the cap has the effect that RNA with such a cap is substantially insensitive to decapping. This is important because, in general, the amount of protein produced from synthetic mRNA introduced into cultured mammalian cells is limited by the natural degradation of mRNA. One in vivo pathway of mRNA degradation begins with the removal of the mRNA cap. This removal is catalyzed by a heterodimeric pyrophosphatase containing a regulatory subunit (Dcp1) and a catalytic subunit (Dcp2). The catalytic subunit cleaves between the α and β phosphate groups of the triphosphate bridge. In this disclosure, a cap that is insensitive or less sensitive to this type of cleavage can be selected. Suitable cap analogs for this purpose can be selected from cap dinucleotides according to the following formula (I):

[0219] Where R 1Selected from optionally substituted alkyl, optionally substituted alkenyl, optionally substituted ynyl, optionally substituted cycloalkyl, optionally substituted heterocyclic, optionally substituted aryl, and optionally substituted heteroaryl. R 2 and R 3 Independently selected from H, halogen, OH and optionally substituted alkoxy groups, or R 2 and R 3 Together they form OXO, where X is selected from optionally substituted CH2, CH2CH2, CH2CH2CH2, CH2CH(CH3) and C(CH3)2, or R 2 With connection R 2 The hydrogen atoms at the 4' position of the ring combine to form -O-CH2- or -CH2-O-. R 5 Selected from S, Se and BH3, R 4 and R 6 It is independently selected from O, S, Se and BH3.

[0220] n is 1, 2, or 3.

[0221] R was disclosed in WO 2011 / 015347 A1. 1 R 2 R3, R 4 R 5 R 6 The preferred embodiments are those described herein, and may be selected accordingly.

[0222] For example, in embodiments, the RNA molecule of this disclosure includes a thiophosphate-cap-analyte. A thiophosphate-cap-analyte is a specific cap-analyte in which one of the three non-bridging O atoms in the triphosphate chain is replaced by an S atom, i.e., R in formula (I). 4 R 5 or R 6 One of them is S. Thiophosphate-cap-analytes have been developed by Kowalska. et al. ,2008, RNA, 14:1119-1131 describes a solution to an undesirable decapping process, thereby increasing the stability of RNA in vivo. Specifically, replacing the oxygen atom with a sulfur atom at the β-phosphate group of the 5'-cap results in stability against Dcp2. In the preferred embodiment of this disclosure, R in formula (I) 5 It is S; and R 4 and R 6 It is O.

[0223] In another embodiment, the RNA molecule of this disclosure comprises a phosphate thioester-cap-analyte, wherein the phosphate thioester modification of the RNA 5'-cap is combined with an "anti-reverse cap analog" (ARCA) modification. Corresponding ARCA-phosphate thioester-cap-analytes are described in WO 2008 / 157688 A2, and they can all be used in the RNA of this disclosure. In this embodiment, R in formula (I) 2 or R 3 At least one of them is not OH, preferably R. 2 and R 3 One of them is a methoxy group (OCH3), and R 2 and R 3 Another preferred form is OH. In a preferred embodiment, the oxygen atom is replaced by a sulfur atom on the β-phosphate group (so that R in formula (I) is substituted with OH). 5 It is S; and R 4 and R 6 (It is O). It is believed that thiophosphate modification of ARCA ensures that the α, β, and γ thiophosphate groups are precisely located within the active site of the cap-binding protein in both translation and decapping mechanisms. At least some of these analogues are substantially resistant to pyrophosphatases Dcp1 / Dcp2. Thiophosphate-modified ARCA has been described as having a much higher affinity for eIF4E than the corresponding ARCA lacking the thiophosphate group.

[0224] The corresponding cap, particularly preferred in this disclosure, namely, m2 7,2’-O Gpp s pG, referred to as β-S-ARCA (WO 2008 / 157688 A2; Kuhn et al. (Gene Ther. 17:961-971, 2010). Therefore, in one embodiment of this disclosure, the RNA of this disclosure is modified with β-S-ARCA. β-S-ARCA is represented by the following structure: .

[0225] Generally, by replacing oxygen atoms with sulfur atoms at the bridging phosphate, diastereomers of the thiophosphate, known as D1 and D2, are obtained based on their elution patterns in HPLC. In short, the D1 diastereomer of β-S-ARCA, or "β-S-ARCA (D1)", is a diastereomer of β-S-ARCA that elutes first on the HPLC column compared to the D2 diastereomer of β-S-ARCA (β-S-ARCA (D2)) and therefore exhibits a shorter retention time. The determination of stereochemical configuration by HPLC is described in WO 2011 / 015347 A1.

[0226] In a first particularly preferred embodiment of this disclosure, the RNA of this disclosure is modified with the β-S-ARCA (D2) diastereomer. The two diastereomers of β-S-ARCA have different sensitivities to nucleases. It has been shown that RNA carrying the D2 diastereomer of β-S-ARCA is 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 intermediate sensitivity to Dcp2 cleavage (71% cleavage). Further, increased stability to Dcp2 cleavage has been shown to be associated with increased protein expression in mammalian cells. In particular, it has been shown that RNA carrying the β-S-ARCA (D2) cap is translated more efficiently in mammalian cells than RNA carrying the β-S-ARCA (D1) cap. Therefore, in one embodiment of this disclosure, the RNA of this disclosure is modified with a cap analog according to formula (I), characterized by the inclusion of a substituent R in formula (I). 5 The stereochemical configuration at the P atom corresponds to the P atom in the D2 diastereomer of β-S-ARCA. β Stereochemical configuration at the atom. In this embodiment, R in formula (I) 5 It is S; and R 4 and R 6 It is O. Furthermore, R in equation (I) 2 or R 3 At least one of them is preferably not OH, preferably R. 2 and R 3 One of them is a methoxy group (OCH3), and R 2 and R 3 Another preferred component is OH.

[0227] In a second particularly preferred embodiment, the RNA of this disclosure is modified with a β-S-ARCA (D1) diastereomer. This embodiment is particularly suitable for transferring capped RNA into immature antigen-presenting cells, for example, for inoculation purposes. The β-S-ARCA (D1) diastereomer has been shown to be particularly effective in increasing RNA stability, increasing RNA translation efficiency, prolonging RNA translation, increasing total protein expression of RNA, and / or increasing the immune response against antigens or antigenic peptides encoded by said RNA (Kuhn). et al. (GeneTher. 17:961-971, 2010). Therefore, in an alternative embodiment of this disclosure, the RNA of this disclosure is modified with a cap analog according to formula (I), characterized in that formula (I) contains a substituent R. 5The stereochemical configuration at the P atom corresponds to the P atom in the D1 diastereomer of β-S-ARCA. β Stereochemical configuration at the atomic level. WO 2011 / 015347 A1 and Kuhn et al. Each cap analogue and embodiment thereof is described in Gene Ther. 17:961-971, 2010. Any cap analogue described in WO 2011 / 015347A1 may be used in this disclosure, wherein the cap analogue contains the substituent R. 5 The stereochemical configuration at the P atom corresponds to the P atom in the D1 diastereomer of β-S-ARCA. β The stereochemical configuration at the atomic position. Preferably, R in formula (I) 5 It is S; and R 4 and R 6 It is O. Furthermore, R in equation (I) 2 or R 3 At least one of them is preferably not OH, preferably R. 2 and R 3 One of them is a methoxy group (OCH3), and R 2 and R 3 Another preferred component is OH.

[0228] In one embodiment, the RNA of this disclosure is modified with a 5'-cap structure according to formula (I), wherein any phosphate group is replaced by a boron phosphate group or a selenophosphate group. Such a cap exhibits enhanced stability both in vitro and in vivo. Optionally, the corresponding compound has a 2'-O- or 3'-O-alkyl group (wherein the alkyl group is preferably methyl); the corresponding cap analogues are referred to as BH3-ARCA or Se-ARCA. Compounds particularly suitable for capping mRNA include β-BH3-ARCA and β-Se-ARCA, as described in WO2009 / 14925A2. For these compounds, the P corresponding to the D1 diastereomer of β-S-ARCA... β The stereochemical configuration at the atom contains the substituent R in formula (I). 5 The stereochemical configuration at the P atom is preferred.

[0229] In some embodiments, the RNA includes a cap, which may be applicable in the following cases: This disclosure is for cap O (methylation of the first nucleobase, e.g.) m7 GpppN), Hat 1 ( m7 Additional methylation of the ribose of adjacent nucleotides of GpppN), cap 2 ( m7 Additional methylation of the ribose of the second nucleotide downstream of GpppN), cap 3 ( m7Additional methylation of the ribose of the third nucleotide downstream of GpppN), cap 4 ( m7 Additional methylation of the ribose of the fourth nucleotide downstream of GpppN), ARCA (anti-reverse cap analogue), modified ARCA (e.g., phosphate thioester modified ARCA, such as β-S-ARCA), inosine, N1-methyl-guanosine, 2'-fluoro-guanosine, 7-denitro-guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine, and 2-azido-guanosine.

[0230] In some implementations, the RNA includes a cap, which 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, Ramanathan A et al. Figure 1 and Decroly E et al. Figure 1 .

[0231] In some embodiments, the cap 0 contains the guanosine nucleoside methylated at the 7th position of guanine ( m7 G). In some implementations, the cap 0 is linked to the RNA via a 5'-to-5'-triphosphate bond, and is also referred to herein as... m7 Gppp or m7 G(5')ppp(5').

[0232] In some embodiments, cap 1 contains a guanosine nucleoside methylated at the 7th position of guanine ( m7 G or 7m G) and the first nucleotide methylated at 2'O in RNA (G) 2'OMe N1 or N12'OMe or N1 2’OMe In some embodiments, cap 1 is linked to RNA via a 5'-to-5'-triphosphate bond; in some embodiments, cap 1 may 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.

[0233] In some implementation schemes, m7 G(5')ppp(5')(N1 2’OMeCap 1 contains a second nucleotide, and N2 is an A, G, C, or U nucleotide located proximal to the cap at position +2. In some embodiments, such cap 1 is represented as ( m7 G(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.

[0234] In some implementations, cap 1 is or contains m7 G(5')ppp(5')(A1 2’OMe pG2, where A1 is A near the cap at position +1 and G2 is G near the cap at position +2; and has the following structure: .

[0235] In some implementations, cap 1 is or contains m7 G(5')ppp(5')(A1 2’OMe pU2, where A1 is A near the cap at position +1 and U2 is U near the cap at position +2; and has the following structure: .

[0236] In some implementations, cap 1 is or contains m7 G(5')ppp(5')(G1 2’OMe pG2, where G1 is the G near the cap at position +1 and G2 is the G near the cap at position +2; and has the following structure: .

[0237] In some embodiments, cap 1 contains a guanosine nucleoside methylated at the 7th position of guanine ( m7 G) and one or more additional modifications, such as methylation on the ribose and 2'-O-methylation of the first nucleotide in the RNA. In some embodiments, cap 1 comprises guanosine nucleotide methylated at position 7 and 3'-O methylation on the ribose (m7G3'OMe or 7m G 3’OMe ), and the first nucleotide of 2'O methylation in RNA (N1 2’OMe In some implementations, the cap 1 is linked to the RNA via a 5'-to-5'-triphosphate bond, and is also referred to herein as (m7G3'OMe)ppp(2'OMeN1) or ( m7 G 3’OMe (5')ppp(5')( 2’OMeN1). 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.

[0238] In some implementation schemes, ( m7 G 3’OMe (5')ppp(5')(N1 2’OMe Cap 1 contains a second nucleotide, and N2 is a nucleotide proximal to the cap at position 2 and is 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.

[0239] In some implementations, cap 1 is or contains ( m7 G 3’OMe (5')ppp(5')(A1 2’OMe pG2, where A1 is A near the cap at position +1 and G2 is G near the cap at position +2; and has the following structure: .

[0240] In some implementations, cap 1 is or contains ( m7 G 3’OMe (5')ppp(5')(G1 2’OMe pG2, where G1 is the G near the cap at position +1 and G2 is the G near the cap at position +2; and has the following structure: .

[0241] In some embodiments, the second nucleotide in cap 1 may contain one or more modifications, such as methylation. In some embodiments, cap 1 containing a second nucleotide with 2'O methylation is a cap 2 structure.

[0242] In some embodiments, RNA polynucleotides containing cap 1 have increased translation efficiency, increased translation rate, and / or increased expression of the coding payload relative to a suitable reference. In some embodiments, those containing ( m7 G 3’OMe (5')ppp(5')(A1 2’OMe The cap 1 RNA polynucleotide of pG2 (where A1 is the nucleotide at position +1 and G2 is the nucleotide at position +2) relative to the nucleotide containing ( m7 G3’OMe (5')ppp(5')(G1 2’OMe The cap 1 RNA polynucleotide of 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) has increased translation efficiency. In some embodiments, the increased translation efficiency is assessed when the RNA polynucleotide is administered to cells or organisms.

[0243] In some implementations, the cap analogue for RNA polynucleotides is m7 G 3’OMe Gppp(m1 2’-OMe ApG (sometimes also called m2) 7,3’-OMe G(5')ppp(5')m 2’-OMe ApG or ( m7 G 3’OMe (5')ppp(5')(A 2’OMe pG), which has the following structure: .

[0244] The following is an example cap 1 RNA, which contains RNA and m2. 7,3’OMe G(5')ppp(5')m 2’-OMe ApG: .

[0245] Here is another example cap 1 RNA: .

[0246] UTR

[0247] The term “untranslated region” or “UTR” refers to a region in a DNA molecule that is transcribed but not translated into an amino acid sequence, or to a corresponding region in an RNA molecule (such as an mRNA molecule). Untranslated regions (UTRs) can be located at the 5' (upstream) end of an open reading frame (5'-UTR) and / or the 3' (downstream) end of an open reading frame (3'-UTR).

[0248] The 3'-UTR, if present, is located at the 3' end of the gene, downstream of the stop codon in the protein-coding region, but 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.

[0249] The 5'-UTR, if present, is located at the 5' end of the gene, upstream of the start codon in the protein-coding region. The 5'-UTR can also be downstream of the 5'-cap, for example, directly adjacent to it.

[0250] According to this disclosure, 5'- and / or 3'-untranslated regions can be functionally linked to open reading frames, thereby associating these regions with the open reading frames in a manner that increases the stability and / or translation efficiency of the RNA containing the open reading frames.

[0251] In some embodiments, the RNA molecule according to this disclosure contains a 5'-UTR and / or a 3'-UTR.

[0252] UTRs are related to RNA stability and translation efficiency. In addition to structural modifications involving a 5'-cap and / or a 3'-poly(A)-tail as described herein, both can be improved by selecting specific 5' and / or 3' untranslated regions (UTRs). Sequence elements within the UTR are generally understood to influence translation efficiency (primarily the 5'-UTR) and RNA stability (primarily the 3'-UTR). The presence of an active 5'-UTR is preferred to increase RNA translation efficiency and / or stability. Independently or additionally, the presence of an active 3'-UTR is preferred to increase the translation efficiency and / or stability of the RNA molecule.

[0253] Regarding the first nucleic acid sequence (e.g., UTR), the terms "active to increase translation efficiency" and / or "active to increase stability" mean that the first nucleic acid sequence can modify the translation efficiency and / or stability of the second nucleic acid sequence in a co-transcript with the second nucleic acid sequence in such a way that the translation efficiency and / or stability are increased compared to the translation efficiency and / or stability of the second nucleic acid sequence in the absence of the first nucleic acid sequence.

[0254] The 5'-UTR according to this disclosure may contain any combination of more than one nucleic acid sequence, said nucleic acid sequences optionally separated by linkers. The 3'-UTR according to this disclosure may contain any combination of more than one nucleic acid sequence, said nucleic acid sequences optionally separated by linkers.

[0255] According to the terminology of this disclosure, a "linker" refers to a nucleic acid sequence added between two nucleic acid sequences to link the two nucleic acid sequences. There are no particular limitations on the linker sequence.

[0256] 3'-UTRs typically range in length from 200 to 2000 nucleotides, for example, 500 to 1500 nucleotides. The 3'-untranslated regions (UTRs) of immunoglobulin mRNAs are relatively short (less than about 300 nucleotides), while those of other genes are relatively long. For example, the 3'-UTR 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'-UTRs of mammalian mRNAs often contain a homologous region called the AAUAAA hexanucleotide sequence. This sequence may be a poly(A) linker signal, typically located 10 to 30 bases upstream of the poly(A) linker site. The 3'-UTR may contain one or more inverted repeat sequences that can fold to form stem-loop structures, which act as a barrier to exonucleases or interact with proteins known to increase RNA stability, such as RNA-binding proteins.

[0257] Human β-globin 3'-UTR, especially two consecutive identical copies of human β-globin 3'-UTR, contributes to high transcript stability and translation 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 the human β-globin 3'-UTR. Thus, it is located at the 5'... The 3' direction includes: (a) an optional 5'-UTR; (b) an open reading frame; (c) a 3'-UTR; said 3'-UTR comprising two consecutive identical copies of human β-globin 3'-UTR, fragments thereof, or variants of human β-globin 3'-UTR or fragments thereof.

[0258] In embodiments, the RNA molecule according to this disclosure contains a 3'-UTR that is active to increase translation efficiency and / or stability, but is not the human β-globin 3'-UTR, a fragment thereof, or a variant or fragment thereof.

[0259] In the embodiments, the RNA molecule according to this disclosure contains a 5'-UTR, which is active to increase translation efficiency and / or stability.

[0260] poly(A) sequence

[0261] In some embodiments, the RNA molecule according to this disclosure contains a 3'-poly (A) sequence.

[0262] According to this disclosure, in one embodiment, the poly(A) sequence comprises, substantially 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, particularly up to 150 A nucleotides, particularly about 120 A nucleotides. In this context, "substantially consists of" means that the majority of the nucleotides in the poly(A) sequence are typically at least 50%, preferably at least 75%, of the nucleotides in the poly(A) sequence, and are A nucleotides (adenosine), but the remaining nucleotides are permitted to be nucleotides other than A nucleotides, such as U nucleotides (uridine), G nucleotides (guanosine), and C nucleotides (cytidine). In this context, "consist of" means that all the nucleotides in the poly(A) sequence, i.e., 100% of the nucleotides in the poly(A) sequence are A nucleotides. The term "A nucleotide" or "A" refers to adenosine.

[0263] In fact, the 3'-poly (A) sequence of approximately 120 A nucleotides has been shown to have a beneficial effect on RNA levels in transfected eukaryotic cells, as well as on protein levels translated from open reading frames present upstream (5') of the 3'-poly (A) sequence (Holtkamp). et al. , 2006, Blood, vol. 108, pp. 4009-4017).

[0264] This disclosure provides a 3'-poly(A) sequence linked during RNA transcription, i.e., during the preparation of RNA transcribed in vitro, based on a DNA template containing repeating dT nucleotides (deoxythymidines) in a strand complementary to the coding strand. The DNA sequence (coding strand) encoding the poly(A) sequence is referred to as a poly(A) box.

[0265] In some embodiments of this disclosure, the 3'-poly(A) box present in the coding strand of the DNA template molecule is essentially composed of dA nucleotides, but interrupted by a random sequence having an equal distribution of four nucleotides (dA, dC, dG, dT). The length of such a random sequence can be 5 to 50 nucleotides, preferably 10 to 30, more preferably 10 to 20 nucleotides. Such a box is disclosed in WO 2016 / 005004 A1. Any poly(A) box disclosed in WO 2016 / 005004 A1 can be used in this disclosure. The poly(A) box displays plasmid DNA at the DNA level in *E. coli* (…). E. coliConstant proliferation in the poly(A) box, and still associated at the RNA level with beneficial properties supporting RNA stability and translation efficiency, is essentially composed of dA nucleotides but interrupted by random sequences of four equally distributed nucleotides (dA, dC, dG, dT) with lengths of, for example, 5 to 50 nucleotides.

[0266] Therefore, in some embodiments of this disclosure, the 3'-poly (A) sequence contained in the RNA molecule described herein consists essentially of A nucleotides, but is interrupted by a random sequence of four nucleotides (A, C, G, U) having an equal distribution. The length of such random sequence can be from 5 to 50 nucleotides, preferably from 10 to 30 nucleotides, and more preferably from 10 to 20 nucleotides.

[0267] Codon usage

[0268] Generally, the degeneracy of the genetic code allows the substitution of certain codons (base triplets) present in an RNA sequence with other codons (base triplets) while maintaining the same coding capacity (such that the substituted codon encodes the same amino acid as the substituted codon). In some embodiments of this disclosure, at least one codon of an open reading frame contained in the RNA molecule differs from the corresponding codon of the corresponding open reading frame in the species from which the open reading frame originates. In this embodiment, the coding sequence of the open reading frame is referred to as "adapted" or "modified." The coding sequence of the open reading frame contained in the RNA molecule can be adjusted.

[0269] For example, when adjusting the coding sequence of an open reading frame (OPF), frequently used codons can be selected: WO 2009 / 024567 A1 describes adjustments to the coding sequence of nucleic acid molecules, including replacing rare codons with more frequently used ones. Since the frequency of codon use depends on the host cell or host organism, this type of adjustment is suitable for adapting the nucleic acid sequence to expression in a specific host cell or organism. Generally, more frequently used codons are usually translated more efficiently in the host cell or organism, although it is not always necessary to adjust all codons in the OPF.

[0270] For example, when adjusting the coding sequence of an open reading frame, the content of G (guanosine monophosphate) and C (cytidine monophosphate) residues can be altered by selecting codons with the highest GC enrichment for each amino acid. RNA molecules with GC-rich open reading frames have been reported to have the potential to reduce immune activation and improve RNA translation and half-life (Thess et al. ,2015, Mol. Ther. 23:1457-1465).

[0271] Particles

[0272] To overcome obstacles to safe and efficient nucleic acid delivery, nucleic acids can be administered together with one or more delivery mediators that protect the nucleic acids from degradation, maximize delivery to target cells, and minimize exposure to off-target cells. Such nucleic acid delivery mediators can be compounded with or encapsulate nucleic acids and comprise a variety of materials, including polymers and lipids. In some embodiments, such nucleic acid delivery mediators can form particles with nucleic acids, preferably RNA.

[0273] The RNA, particularly mRNA, described herein can be present in particles containing (i) RNA and (ii) at least one cation or cationic ionizable compound, such as a polymer or lipid, complexed with RNA. Electrostatic interactions between positively charged molecules, such as polymers and lipids, and negatively charged RNA are involved in particle formation. This leads to the complexation and spontaneous formation of nucleic acid (particularly RNA) particles.

[0274] In some embodiments, the compositions described herein contain one or more RNA molecules in the particles.

[0275] Different types of nucleic acid-containing particles have been previously described as suitable for delivering RNA in particulate form (see, for example, Kaczmarek, JC et al., 2017, Genome Medicine 9, 60). For non-viral RNA delivery mediators, the encapsulation of nucleic acid nanoparticles physically protects the nucleic acid from degradation and, depending on specific chemistry, can facilitate cellular uptake and endosome escape.

[0276] In the context of this disclosure, the term "particle" refers to a structured entity formed of molecules or molecular complexes, particularly compounds that form particles. In some embodiments, the particle contains a coating (e.g., one or more layers or lamellae) made of one or more types of amphiphilic substances (e.g., amphiphilic lipids). In this context, the expression "amphiphilic substance" means that the substance has both hydrophilic and lipophilic properties. The coating may also contain additional substances that are not necessarily amphiphilic (e.g., additional lipids). Thus, the particle can be a monolayer or multilayer structure, wherein the substances constituting one or more layers or lamellae comprise one or more types of amphiphilic substances (particularly selected from amphiphilic lipids), optionally combined with additional substances that are not necessarily amphiphilic (e.g., additional lipids). In some embodiments, the term "particle" refers to a micrometer or nanometer-sized structure, such as a dense structure at the micrometer or nanometer scale. According to this disclosure, the term "particle" includes nanoparticles.

[0277] "DNA particles," "RNA particles," or "DNA and RNA particles" can be used to deliver DNA and / or RNA to a target site (e.g., cells, tissues, organs, etc.). DNA and / or RNA particles can be formed from lipids containing at least one cation or cationic ionizable lipid. It is not desired to be bound by any theory that cationic or cationic ionizable lipids bind with nucleic acids to form aggregates, and that such aggregation results in colloidally stable particles.

[0278] The RNA particles described in this article include formulations based on lipid nanoparticles (LNP) and lipoplexes (LPX).

[0279] The lipoplex (LPX) described herein can be obtained by mixing two aqueous phases: a phase containing RNA and a phase containing lipid dispersions. In some embodiments, the lipid phase contains liposomes.

[0280] In some embodiments, liposomes are self-enclosed monolayer or multilayer vesicle particles, wherein the lamellae comprise a lipid bilayer and the encapsulated cavity contains an aqueous phase. A prerequisite for using liposomes to form nanoparticles is that the desired lipids in the mixture are capable of forming a lamellae (bilayer) phase in the aqueous environment of the application.

[0281] In some embodiments, liposomes comprise a single or multiple phospholipid bilayer encapsulating an aqueous core (also referred to herein as an aqueous cavity). They can be prepared from materials having polar head (hydrophilic) groups and nonpolar tail (hydrophobic) groups. In some embodiments, the cationic lipids used to formulate liposomes designed for RNA delivery are amphiphilic in nature and consist of a positively charged (cationic) amine head group linked via glycerol to a hydrocarbon chain or cholesterol derivative.

[0282] In some embodiments, the lipoplex is a multilayered liposome-based formulation that forms upon electrostatic interaction between cationic liposomes and nucleic acids. In some embodiments, the formed lipoplex has a different internal arrangement of molecules resulting from the transformation from a liposome structure to a compact RNA-lipoplex.

[0283] In some embodiments, LPX particles comprise amphiphilic lipids as described herein, particularly cationic or cationically ionizable amphiphilic lipids, and RNA (particularly mRNA). In some embodiments, electrostatic interactions between positively charged liposomes (made from one or more amphiphilic lipids, particularly cationic or cationically ionizable amphiphilic lipids) and negatively charged RNA (particularly mRNA) lead to the recombination and spontaneous formation of RNA lipoplex particles. Positively charged liposomes can typically be synthesized using cationic or cationically ionizable amphiphilic lipids (e.g., DOTMA and / or DODMA) and optional additional lipids (e.g., DOPE or DSPC).

[0284] Generally, lipid nanoparticles (LNPs) can be obtained by directly mixing RNA in an aqueous phase with lipids in a phase containing an organic solvent such as ethanol. In this case, lipids or lipid mixtures can be used for particle formation, which does not form a thin-layered (bilayer) phase in water.

[0285] In some embodiments, the LNP comprises, or is composed of, cationic / cationically ionizable lipids and accessory lipids (e.g., phospholipids, cholesterol, and / or polymerically conjugated lipids (e.g., polyethylene glycol (PEG) lipids)). In some embodiments, in the RNA LNP described herein, RNA (particularly mRNA) is bound by cationically ionizable lipids occupying a central core of the LNP. In some embodiments, polymerically conjugated lipids, together with phospholipids, form the surface of the LNP. In some embodiments, charged and uncharged forms of cholesterol and cationically ionizable lipids may be distributed throughout the LNP.

[0286] In some embodiments, the RNA (e.g., mRNA) described herein may bind non-covalently to the particles described herein. In embodiments, the RNA (especially mRNA) may adhere to the outer surface of the particle (surface RNA (especially surface mRNA)) and / or may be contained within the particle (encapsulated RNA (especially encapsulated mRNA)).

[0287] In some embodiments, the size (e.g., diameter) of the particles described herein (e.g., LNP and LPX) ranges from about 10 nm 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 at most about 1900 nm (e.g., at most about 1800 nm, at most about 1700 nm, at most about 1600 nm, at most about 1500 nm, at most about 1400 nm, at most about 1300 nm, at most about 1200 nm, at most about 1100 nm, at most about 1000 nm, at most about 950 nm). nm, up to about 900 nm, up to about 850 nm, up to about 800 nm, up to about 750 nm, up to about 700 nm, up to about 650 nm, up to about 600 nm, up to about 550 nm, or up to about 500 nm), for example, the range of about 20 nm to about 1500 nm, for example, about 30 nm to about 1200 nm, about 40 nm to about 1100 nm, about 50 nm to about 1000 nm, about 60 nm to about 900 nm, about 70 nm to about 800 nm, about 80 nm to about 700 nm, about 90 nm to about 600 nm, or about 50 nm to about 500 nm, or about 100 nm to about 500 nm, for example, the range of 10 nm to 1000 nm, 15 nm to 500 nm, 20 nm to 450 nm, 25 nm to 400 nm, 30 nm to 350 nm, 40 nm to 300 nm, 50 nm to 250 nm, 60 nm to 200 nm, 70 nm to 150 nm, or 80 nm to 150 nm. In some embodiments, the size (e.g., diameter) of the particles described herein (e.g., LNP and LPX) ranges from about 40 nm to about 200 nm, such as 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.

[0288] In some embodiments, the average diameter of the particles described herein (e.g., LNP and LPX) is, in some embodiments, about 50 nm to about 1000 nm, about 50 nm to about 800 nm, about 50 nm to about 700 nm, about 50 nm to about 600 nm, about 50 nm to about 500 nm, about 50 nm to about 450 nm, about 50 nm to about 400 nm, about 50 nm to about 350 nm, about 50 nm to about 300 nm, about 50 nm to about 250 nm, about 50 nm to about 200 nm, about 100 nm to about 1000 nm, about 100 nm to about 800 nm, about 100 nm to about 700 nm, about 100 nm to about 600 nm, about 100 nm to about 500 nm, about 100 nm to about 450 nm, about 100 nm to about 400 nm, about 100 nm to about 350 nm, about 100 nm to about 300 nm, about 100 nm to about 25 ... nm to about 200 nm, about 150 nm to about 1000 nm, about 150 nm to about 800 nm, about 150 nm to about 700 nm, about 150 nm to about 600 nm, about 150 nm to about 500 nm, about 150 nm to about 450 nm, about 150 nm to about 400 nm, about 150 nm to about 350 nm, about 150 nm to about 300 nm, about 150 nm to about 250 nm, about 150 nm to about 200 nm, about 200 nm to about 1000 nm, about 200 nm to about 800 nm, about 200 nm to about 700 nm, about 200 nm to about 600 nm, about 200 nm to about 500 nm, about 200 nm to about 450 nm, about 200 nm to about 400 nm, about 200 nm to about 350 nm, about 200 nm to about 300 nm, about 200 nm to about 250 nm, or about 80 The average diameter of the particles described herein (e.g., LNP and LPX) is from about 40 nm to about 200 nm, for example, from about 50 nm to about 180 nm, from about 60 nm to about 160 nm, from about 80 nm to about 150 nm, or from about 80 nm to about 120 nm.

[0289] The RNA particles (especially mRNA particles) described herein can exhibit a polydispersity index (PDI) of less than about 0.5, less than about 0.4, less than about 0.3, less than about 0.2, less than about 0.1, or less than about 0.05. As an example, RNA particles can exhibit a polydispersity index ranging from about 0.01 to about 0.4 or from about 0.1 to about 0.3.

[0290] The N / P ratio gives the proportion of nitrogen groups in lipids to phosphate groups in nucleic acids. It is related to the charge ratio because nitrogen atoms (depending on pH) are generally positively charged, while phosphate groups are negatively charged. An N / P ratio at which charge balance exists depends on pH. Lipid formulations can form at N / P ratios greater than 4 to 12 because positively charged nanoparticles can facilitate transfection. In this case, RNA is considered to be completely bound to the nanoparticles.

[0291] This disclosure describes compositions comprising RNA (particularly mRNA) and at least one cationic or cationically ionizable lipid, said lipid associating with the RNA to form RNA particles, and formulations comprising such particles. The RNA particles may comprise RNA compounded in various forms through non-covalent interactions with the particles. The particles described herein are not viral particles, particularly infectious viral particles, i.e., they cannot infect cells.

[0292] Suitable cations or cationically ionizable lipids are those that form RNA particles and are included in the terms "particle-forming component" or "particle-forming agent." The terms "particle-forming component" or "particle-forming agent" refer to any component that associates with RNA that forms RNA particles. Such components include any component that can be part of the RNA particle.

[0293] In some implementations, RNA particles (especially mRNA particles) contain more than one type of RNA molecule, wherein the molecular parameters of the RNA molecules may be similar to or different from each other, for example, in terms of molar mass or basic structural elements such as molecular structure, capping, coding regions or other features.

[0294] In particulate formulations, each RNA species can be formulated into a separate particulate formulation. In this case, each separate particulate formulation will contain one RNA species. The separate particulate formulations can exist as separate entities, for example, in separate containers. Such formulations can be obtained by providing each RNA species (usually in the form of an RNA-containing solution) and a particulate forming agent separately, thereby allowing the formation of particles. When particles (separate particulate formulations) are formed, each particle will contain only the specific RNA species provided. In some embodiments, compositions, such as pharmaceutical compositions, contain more than one separate particulate formulation. The corresponding pharmaceutical compositions are referred to as mixed particulate formulations. Mixed particulate formulations according to this disclosure can be obtained by the steps of separately forming separate particulate formulations and then mixing the separate particulate formulations. Through the mixing step, a formulation containing a mixed population of RNA-containing particles is obtained. The separate particle populations can be together in a container containing the mixed population of separate particulate formulations. Alternatively, all RNA species of a pharmaceutical composition can be formulated together as a combined particulate formulation. Such a formulation can be obtained by providing a combined formulation (usually a combined solution) of all RNA species and a particulate forming agent, thereby allowing the formation of particles. Unlike mixed particle formulations, combined particle formulations typically contain particles with more than one type of RNA. In combined particle compositions, different RNA types are usually present together in a single particle.

[0295] polymer

[0296] Given their high chemical flexibility, polymers are commonly used materials for nanoparticle-based delivery. Typically, cationic polymers are used to electrostatically concentrate negatively charged RNA into particles, particularly nanoparticles. These positively charged groups are usually composed of amines with altered protonation states in a pH range of 5.5 to 7.5, which are thought to cause ionic imbalances leading to endosome disruption. Polymers such as poly-L-lysine, polyamide amines, protamine, and polyethyleneimine, as well as naturally occurring polymers such as chitosan, have been used for nucleic acid delivery and are suitable as cationic polymers in this paper. Furthermore, some researchers have synthesized polymers specifically for nucleic acid delivery. In particular, poly(β-amino esters) have been widely used for nucleic acid delivery due to their ease of synthesis and biodegradability. Such synthetic polymers are also suitable as cationic polymers in this paper.

[0297] As used in this article, "polymer" is given its usual meaning, referring to a molecular structure containing one or more repeating units (monomers) linked by covalent bonds. The repeating units may be all identical, or in some cases, more than one type of repeating unit may be present in a polymer. In some cases, polymers are biologically derived, i.e., biopolymers such as proteins. In other cases, additional parts may also be present in polymers, such as targeting moieties.

[0298] A polymer is called a "polymer" if it contains more than one type of repeating unit. It should be understood that the polymer used herein can be a copolymer. The repeating units forming a copolymer can be arranged in any manner. For example, the repeating units can be arranged in a random order, an alternating order, or as a "block" copolymer, i.e., a "block" copolymer comprising one or more regions, each containing a first repeating unit (e.g., a first block), and one or more regions, each containing a second repeating unit (e.g., a second block), etc. Block copolymers can have two (diblock copolymers), three (triblock copolymers), or more different blocks.

[0299] In some embodiments, the polymer is biocompatible. A biocompatible polymer is one that typically does not cause significant cell death at moderate concentrations. In some embodiments, the biocompatible polymer is biodegradable, i.e., the polymer is capable of chemical and / or biodegradation within physiological environments, such as in vivo.

[0300] In some embodiments, the polymer may be protamine or polyalkylene imide.

[0301] The term "protamine" refers to any of a variety of relatively low molecular weight, strongly basic proteins rich in arginine, and found to be associated particularly with DNA, rather than with somatic cell histones in the sperm cells of various animals (such as fish). Specifically, the term "protamine" refers to proteins found in fish sperm that are strongly basic, water-soluble, not thermally coagulated, 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.

[0302] According to this disclosure, as used herein, the term "protamine" means any protamine amino acid sequence obtained or derived from natural or biological sources, including fragments thereof and polymeric forms of said amino acid sequence or fragments thereof, as well as (synthetic) polypeptides that are artificial and specifically designed for a particular purpose and cannot be isolated from natural or biological sources.

[0303] In one embodiment, the polyalkylene imide comprises polyethyleneimine and / or polypropyleneimine, preferably polyethyleneimine. A preferred polyalkylene imide is polyethyleneimine (PEI). The average molecular weight of PEI is preferably 0.75. 102 Da to 107 Da, preferably 1000 Da to 105 Da, more preferably 10000 Da to 40000 Da, more preferably 15000 Da to 30000 Da, and even more preferably 20000 Da to 25000 Da.

[0304] According to this disclosure, linear polyalkylene imides, such as linear polyethyleneimine (PEI), are preferred.

[0305] The cationic polymers (including polycationic polymers) considered for use herein include any cationic polymer capable of electrostatically binding nucleic acids. In one embodiment, the cationic polymers considered for use herein include any cationic polymer capable of associating with nucleic acids, for example, by forming a complex with nucleic acid or forming vesicles that encapsulate or encapsulate nucleic acids.

[0306] The particles described herein may also contain polymers other than cationic polymers, namely non-cationic polymers and / or anionic polymers. Anionic and neutral polymers are collectively referred to herein as non-cationic polymers.

[0307] lipids

[0308] The terms “lipid” and “lipid-like material” are broadly defined herein as molecules comprising one or more hydrophobic moieties or groups and optionally one or more hydrophilic moieties or groups. Molecules comprising both hydrophobic and hydrophilic moieties are also commonly referred to as amphiphiles. Lipids are generally insoluble or sparingly soluble in water, but soluble in many organic solvents. In an aqueous environment, the amphiphilic nature allows molecules to self-assemble into organized structures and different phases. One such phase consists of lipid bilayers, as they exist in vesicles, multilayer / monolayer liposomes, or membranes in an aqueous environment. Hydrophobicity can be imparted by including nonpolar groups, including but not limited to long-chain saturated and unsaturated aliphatic hydrocarbon groups and such groups substituted with one or more aromatic, alicyclic, or heterocyclic groups. Hydrophilic groups can include polar and / or charged groups and include carbohydrates, phosphates, carboxyl groups, sulfates, amino groups, thiol groups, nitro groups, hydroxyl groups, and other similar groups.

[0309] As used herein, the term "hydrophobic" means 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. The monovalent group of a hydrocarbon is referred to herein as a hydrocarbon group. A hydrophobic group may have functional groups (e.g., ethers, esters, halides, etc.) and atoms other than carbon and hydrogen, provided that the group satisfies the condition of being substantially immiscible or insoluble in aqueous solution.

[0310] The term "hydrocarbon" includes acyclic, such as linear (straight-chain) or branched hydrocarbon groups, such as 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, such as halogens, oxygen, or sulfur. Unless otherwise stated, hydrocarbon groups may also include cyclic (alkyl, alkenyl, or alkynyl) or aryl groups, provided that the overall polarity of the hydrocarbon remains relatively nonpolar.

[0311] The term "alkyl" refers to a saturated linear or branched monovalent hydrocarbon moiety that may have one to thirty, typically one to twenty, and often six to eighteen carbon atoms. Exemplary nonpolar alkyl groups include, but are not limited to, methyl, ethyl, propyl, isopropyl, hexyl, decyl, dodecyl, tetradecyl, hexadecyl, octadecyl, etc.

[0312] The term "alkenyl" refers to a linear or branched monovalent hydrocarbon moiety having at least one carbon-carbon double bond, wherein the total number of carbon atoms can be six to thirty, typically six to twenty, and often six to eighteen. 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, and if the number of carbon atoms in the alkenyl group is odd, the result of the division is rounded to the next integer. For example, for an alkenyl group having nine carbon atoms, the maximum number of carbon-carbon double bonds is four. Preferably, the alkenyl group has one to six (e.g., one to four) carbon-carbon double bonds, i.e., one, two, three, four, five, or six carbon-carbon double bonds.

[0313] The term "alkynyl" refers to a linear or branched monovalent hydrocarbon moiety having at least one carbon-carbon triple bond, wherein the total number of carbon atoms can be six to thirty, typically six to twenty, and often six to eighteen. The alkynyl may optionally have one or more carbon-carbon double bonds. Generally, the maximum number of carbon-carbon triple bonds in the alkynyl can be equal to an integer calculated by dividing the number of carbon atoms in the alkynyl by 2, and if the number of carbon atoms in the alkynyl is odd, the result of the division is rounded to the next integer. For example, for an alkynyl having nine carbon atoms, the maximum number of carbon-carbon triple bonds is four. Preferably, the alkynyl has one to six (e.g., one to four), i.e., one, two, three, four, five, or six, more preferably one or two carbon-carbon triple bonds.

[0314] The term "alkylene" refers to a saturated linear or branched divalent hydrocarbon moiety that may have one to thirty, typically two to twenty, and often four to twelve carbon atoms. Exemplary nonpolar alkylenes include, but are not limited to, methylene, ethylene, trimethylene, hexamethylene, decamethylene, dodecylene, tetradecamethylene, hexadecylene, octadecylene, etc.

[0315] The term "alkenyl" refers to a linear or branched divalent hydrocarbon moiety having at least one carbon-carbon double bond, wherein the total number of carbon atoms can be from two to thirty, typically from two to twenty, and often from four to twelve. 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, and if the number of carbon atoms in the alkenyl group is odd, the result of the division is rounded to the next integer. For example, for an alkenyl group having nine carbon atoms, the maximum number of carbon-carbon double bonds is four. Preferably, the alkenyl group has one to six (e.g., one to four), that is, one, two, three, four, five, or six carbon-carbon double bonds.

[0316] The term "cycloalkyl" preferably refers to a cyclic non-aromatic form of "alkyl" and "alkenyl" having 3 to 14 carbon atoms, such as 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 (e.g., 3, 4, 5, 6, 7, 8, 9, or 10 carbon atoms), more preferably 3 to 7 carbon atoms. Exemplary cycloalkyl groups include cyclopropyl, cyclopropenyl, cyclobutyl, cyclobutenyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cycloheptyl, cycloheptenyl, cyclooctyl, cyclooctenyl, cyclononyl, cyclononenyl, cyclodecyl, cyclodecenyl, and adamantyl. A cycloalkyl group can consist of one ring (monocyclic), two rings (bicyclic), or more than two rings (polycyclic).

[0317] The term "aryl" refers to a monovalent group in an aromatic cyclic hydrocarbon. Preferably, the aryl group contains 3 to 14 (e.g., 5, 6, 7, 8, 9, or 10, such as 5, 6, or 10) carbon atoms, which may be arranged in a single ring (e.g., phenyl) or two or more fused rings (e.g., naphthyl). Exemplary aryl groups include cyclopropenylium, cyclopentadienyl, phenyl, indenyl, naphthyl, azulel, fluorenyl, anthracene, 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. Aryl groups do not include fullerenes.

[0318] In the context of hydrocarbons, the term "aromatic" means that the entire molecule must be aromatic. For example, if a monocyclic aryl group is hydrogenated (partially or completely), the resulting hydrogenated cyclic structure is classified as cycloalkyl for the purposes of this disclosure. Similarly, if a di- or polycyclic aryl group (e.g., naphthyl) is hydrogenated, the resulting hydrogenated di- or polycyclic structure (e.g., 1,2-dihydronaphthyl) is classified as cycloalkyl for the purposes of this disclosure (even if one ring, for example in 1,2-dihydronaphthyl, is still aromatic).

[0319] As used herein, the term "amphiphilic" refers to a molecule having both a polar and a nonpolar moiety. Typically, amphiphilic compounds have a polar head attached to a long hydrophobic tail. In some embodiments, the polar moiety is soluble in water, while the nonpolar moiety is insoluble in water. Furthermore, the polar moiety may have either a positive or a negative charge. Optionally, the polar moiety may have both a positive and a negative charge and may be an amphoteric ion or an inner salt. For the purposes of this disclosure, the amphiphilic compound may be, but is not limited to, one or more natural or non-natural lipids and lipid-like compounds.

[0320] The terms "lipid-like material," "lipid-like compound," or "lipid-like molecule" refer to substances that are structurally and / or functionally related to lipids but may not be strictly considered lipids, particularly amphiphilic substances. For example, the term includes compounds capable of forming 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 portions. Generally, the term includes molecules containing hydrophilic and hydrophobic portions with different structural organization, which may be structurally similar to or dissimilar to lipids. Examples of lipid-like compounds capable of spontaneously integrating into cell membranes include functional lipid constructs such as synthetic functional-spacer-lipid constructs (FSL), synthetic functional-spacer-sterol constructs (FSS), and artificial amphiphilic molecules. Lipids containing two long alkyl chains and a polar head group are typically 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 water-insoluble planar bilayers. Conventional surfactant monomers, consisting of only a linear alkyl chain and a hydrophilic head group, are typically conical. The hydrophilic head group tends to occupy more molecular space than the linear alkyl chain. In some embodiments, surfactants tend to aggregate into water-soluble spherical or elliptical micelles. Although lipids also possess the same general structure as surfactants—a polar hydrophilic head group and a nonpolar hydrophobic tail—lipids differ from surfactants in terms of 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 materials, unless otherwise indicated herein or clearly contradicted by the context.

[0321] Lipids are generally classified into eight classes: fatty acids, glycerolipids, glycerophospholipids, sphingolipids, glycolipids, polyketides (derived from the condensation of ketoacyl subunits), sterol lipids, and prenol lipids (derived from the condensation of isoprene subunits). Although the term "lipid" is sometimes used synonymously with fat, fat is a subgroup of lipids called triglycerides. Lipids also include 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, cholesterolanol, cholesterol ketone, coprostinol, cholesterol-2'-hydroxyethyl ether, cholesterol-4'-hydroxybutyl ether, tocopherol and its derivatives, and mixtures thereof.

[0322] Fatty acids, or fatty acid residues, are a group of distinct molecules composed of hydrocarbon chains terminated by carboxylic acid groups; this arrangement gives the molecule a polar hydrophilic end and a nonpolar hydrophobic end that is insoluble in water. The carbon chain, typically 4 to 24 carbon atoms long, can be saturated or unsaturated and can be linked to functional groups containing oxygen, halogens, nitrogen, and sulfur. If a fatty acid contains a double bond, there is a possibility of cis or trans geometric isomerism, which significantly affects the molecular configuration. Cis-double bonds cause the fatty acid chain to bend, a result of complexation with more cis-double bonds in the chain. Other major lipid classes within the fatty acid category are fatty acid esters and fatty acid amides.

[0323] Glycerol lipids consist of mono-, di-, and tri-substituted glycerols, the most well-known being fatty acid triesters of glycerol, called triglycerides. The term "triacylglycerol" is sometimes used synonymously with "triglyceride." In these compounds, the three hydroxyl groups of glycerol are each esterified, usually by different fatty acids. An additional subclass of glycerol lipids is represented by glycosylglycerol, characterized by the presence of one or more sugar residues linked to glycerol via glycosidic bonds.

[0324] Glycerophospholipids are amphiphilic molecules (containing both hydrophobic and hydrophilic regions) with a glycerol core consisting of two fatty acid-derived "tails" linked by ester bonds and a "head" group linked by phosphate ester bonds. Examples of glycerophospholipids, commonly referred to as phospholipids (although sphingomyelins are also classified as phospholipids), include phosphatidylcholine (also known as PC, GPCho, or lecithin), phosphatidylethanolamine (PE or GPEtn), and phosphatidylserine (PS or GPSer).

[0325] Sphingolipids are a complex family of compounds sharing a common structural feature: the sphingoid base skeleton. The major sphingoid base in mammals is generally referred to as sphingosine. Ceramides (N-acyl-sphingoid bases) are the major subclass of sphingoid base derivatives of fatty acids with amide linkages. Fatty acids are typically saturated or monounsaturated with chain lengths of 16 to 26 carbon atoms. The major phosphospholipid in mammals is sphingomyelin (ceramide phosphocholine), while insects primarily contain ceramide phosphoethanolamine, and fungi possess phytoceramide phosphoinositol and a mannose-containing head group. Glycosphingolipids are a different family of molecules composed of one or more sugar residues linked to a sphingoid base via glycosidic bonds. Examples of these are simple and complex glycosphingolipids, such as cerebrosides and gangliosides.

[0326] Sterol lipids, such as cholesterol and its derivatives, or tocopherol and its derivatives, together with glycerophospholipids and sphingomyelin, are important components of membrane lipids.

[0327] Glycolipids are compounds in which fatty acids are directly linked to a glycoskeletal backbone, forming a structure compatible with the membrane bilayer. In glycolipids, monosaccharides replace the glycerol backbone present in glycerol lipids and glycerophospholipids. The most familiar glycolipid is the acylated glucosamine precursor of the lipid A component of lipopolysaccharides in Gram-negative bacteria. A typical lipid A molecule is a disaccharide of glucosamine, derivatized with up to seven fatty acyl chains. The smallest lipopolysaccharide required for growth in *E. coli* is Kdo2-lipid A, a hexaacylated disaccharide of glucosamine glycosylated by two 3-deoxy-D-mannose-octulose acid (Kdo) residues.

[0328] Polyketides are synthesized by polymerizing acetyl and propionyl subunits using classical enzymes and iterative and modular enzymes that share mechanical features with fatty acid synthases. They include a wide range of secondary metabolites and natural products from animal, plant, bacterial, fungal, and marine sources, and exhibit tremendous structural diversity. Many polyketides are cyclic molecules, and their backbones are often further modified by glycosylation, methylation, hydroxylation, oxidation, or other methods.

[0329] According to this disclosure, lipids and lipid-like materials can be cationic, anionic, or neutral. Neutral lipids or lipid-like materials exist in an uncharged or neutral zwitterionic form at a selected pH.

[0330] Cation / Cation-ionizable lipids

[0331] In some embodiments, the RNA compositions, formulations, and nucleic acid particles described herein comprise at least one cationic or cationically ionizable lipid as a particle-forming agent. Cationic or cationically ionizable lipids considered for use herein include any cationic or cationically ionizable lipid (including lipid-like materials) capable of electrostatically binding nucleic acids. In some embodiments, cationic or cationically ionizable lipids considered for use herein are capable of associating with nucleic acids, for example, by forming a complex with nucleic acid or forming vesicles that encapsulate or block nucleic acids.

[0332] As used herein, “cationic lipid” refers to lipids or lipid-like materials that have a net positive charge. Cationic lipids bind to negatively charged nucleic acids through electrostatic interactions. Typically, cationic lipids have a lipophilic moiety, such as a sterol, acyl chain, diacyl, or more acyl chains, and the head group of the lipid usually carries a positive charge.

[0333] In some embodiments, the cationic lipids possess a net positive charge only at certain pH values, particularly acidic pH values, and preferably not at different, preferably higher, pH values, such as physiological pH values, i.e., they are neutral. This ionizable behavior is thought to enhance efficacy by facilitating endosome escape and reducing toxicity compared to particles that remain cationic at physiological pH values.

[0334] As used herein, “cationically ionizable lipid” refers to a lipid or lipid-like material having a net positive charge or being neutral, i.e., it is not a permanent cation. Therefore, a cationically ionizable lipid is either positively charged or neutral depending on the pH of the composition in which it is dissolved. For the purposes of this disclosure, cationically ionizable lipids are covered by the term “cationic lipid” unless it contradicts environmental norms.

[0335] In some embodiments, the cation or cation-ionizable lipid comprises a head group containing at least one nitrogen atom (N) that is positively charged or capable of being protonated (e.g., under physiological conditions).

[0336] Examples of cationic or cationically ionizable lipids include, but are not limited to, N,N-dimethyl-2,3-dioleoyloxypropylamine (DODMA), 1,2-dioleoyl-3-trimethylammonium propane (DOTAP), 1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA), 3-(N-(N',N'-dimethylaminoethane)-carbamoyl)cholesterol (DC-Chol), dimethylbis(octadecylammonium) (DDAB); 1,2-dioleoyl-3-dimethylammonium-propane (DODAP); 1,2-diacyloxy-3-dimethylammonium propane; 1,2-dialkoxy-3-dimethylammonium propane; bis(octadecyldimethylammonium chloride) (DODAC), 1,2-distearate-N... N-Dimethyl-3-aminopropane (DSDMA), 2,3-Di(tetradecoxy)propyl-(2-hydroxyethyl)-dimethylazonium (DMRIE), 1,2-Dimyristoyl-sn-glycerol-3-ethylphosphocholine (DMEPC), 1,2-Dimyristoyl-3-trimethylammonium propane (DMTAP), 1,2-dioleoylpropyl-3-dimethyl-hydroxyethylammonium bromide (DORIE), 2,3-dioleoyloxy-N-[2-(sperminecarbamate)ethyl]-N,N-dimethyl-1-propanetrifluoroacetate (DOSPA), 1,2-dilinoleoyloxy-N,N-dimethylaminopropane (DLinDMA), 1,2-dilinoleoyloxy-N,N-dimethylaminopropane DLenDMA, DOGS, CLinDMA, CpLinDMA, N,N-dimethyl-3,4-dioleoylbenzylamine, DOcarbDAP, 2-[5'-(cholest-5-en-3-β-oxapranoxy)-3-dimethyl-1-(cis,cis-9,12-octadecadienoxy)propane, DMOBA, DOcarbDAP, 2,3-dilinoleoyloxy -N,N-Dimethylpropylamine (DLinDAP), 1,2-N,N'-Dilinoleoylcarbamoyl-3-dimethylaminopropane (DLincarbDAP), 1,2-Dilinoleoylcarbamoyl-3-dimethylaminopropane (DLinCDAP), 2,2-Dilinoleoyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), 2,2-Dilinoleoyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-K-XTC2-DMA), 2,2-Dilinoleenyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA), heptatrien-6,9,28,31-Tetraen-19-yl-4-(dimethylamino)butyrate (DLin-MC3-DMA), N-(2-hydroxyethyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)-1-propanium bromide (DMRIE), (±)-N-(3-)-aminopropyl)-N,N-dimethyl-2,3-bis(cis-9-tetradecyloxy)-1-propanium bromide (GAP-DMORIE), (±)-N-(3-aminopropyl)-N,N-dimethyl-2,3-bis(dodecyloxy)-1-propanium bromide (GAP-DLRI) E), (±)-N-(3-aminopropyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)-1-propaneammonium bromide (GAP-DMRIE), N-(2-aminoethyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)-1-propaneammonium bromide (βAE-DMRIE), N-(4-carboxybenzyl)-N,N-dimethyl-2,3-bis(oleoyloxy)propane-1-ammonium (DOBAQ), 2-({8-[(3β)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,1 2Z)-octadec-9,12-dien-1-oxy]prop-1-amine (octyl-ClinDMA), 1,2-dimyristoyl-3-dimethylammonium-propane (DMDAP), 1,2-dipalmitoyl-3-dimethylammonium-propane (DPDAP), N1-[2-((1S)-1-[(3-aminopropyl)amino]-4-[di(3-aminopropyl)amino]butylformylamino)ethyl]-3,4-di[oleyloxy]-benzamide (MVL5), 1,2-dioleoyl-sn-glycerol-3-ethylphosphocholine (DOEP) C), 2,3-bis(dodecyloxy)-N-(2-hydroxyethyl)-N,N-dimethylprop-1-ammonium bromide (DLRIE), N-(2-aminoethyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)prop-1-ammonium bromide (DMORIE), di((Z)-non-2-en-1-yl)8,8'-(((((2-(dimethylamino)ethyl)thio)carbonyl)azanyl)dioctanoate (ATX)), N,N-dimethyl-2,3-bis(dodecyloxy)prop-1-amine (DLDMA), N,N-dimethyl-2,3-Bis(tetradecoxy)prop-1-amine (DMDMA), di((Z)-non-2-en-1-yl)-9-((4-(dimethylaminobutyryl)oxy)heptadecanoic acid ester (L319), N-dodecyl-3-((2-dodecylcarbamoyl-ethyl)-{2-[(2-dodecylcarbamoyl-ethyl)-2-{(2-dodecylcarbamoyl-ethyl)-[2-(2-dodecylcarbamoyl-ethylamino)-ethyl]-amino}-ethylamino)propionamide (lipid 98N12-5), 1-[2-[bis(2-hydroxydodecyl)amino]ethyl-[2-[4-[2-[bis(2-hydroxydodecyl)amino]ethyl]piperazin-1-yl]ethyl]amino]dodecane-2-ol (lipid C12-200).

[0337] In some embodiments, the cationic or cationically ionizable lipid is DOTMA. In some embodiments, the cationic or cationically ionizable lipid is DODMA.

[0338] DOTMA is a cationic lipid with a quaternary ammonium head group. The structure of DOTMA can be represented as follows: .

[0339] DODMA is an ionizable cationic lipid with a tertiary amine head group. The structure of DODMA can be represented as follows: .

[0340] In some embodiments, the cationic or cationically ionizable lipids may account for about 10 mol% to about 95 mol%, about 20 mol% to about 95 mol%, about 20 mol% to about 90 mol%, about 30 mol% to about 90 mol%, about 40 mol% to about 90 mol%, or about 40 mol% to about 80 mol% of the total lipids present in the particles.

[0341] Additional lipids

[0342] The RNA compositions and formulations described herein, as well as the RNA particles, may also contain lipids (including lipid-like materials) other than cationic or cationic ionizable lipids (collectively referred to herein as cationic lipids), i.e., non-cationic lipids (including non-cationic or non-cationic ionizable lipids or lipid-like materials). Generally, anionic and neutral lipids or lipid-like materials are referred to herein as non-cationic lipids. In addition to cationic or cationic ionizable lipids, optimizing the formulation of RNA particles by adding other hydrophobic components, such as cholesterol and lipids, can enhance particle stability and nucleic acid delivery efficiency.

[0343] One or more additional lipids may or may not affect the total charge of the RNA particle. In some embodiments, the one or more additional lipids are noncationic lipids or lipid-like materials. Noncationic lipids may comprise, for example, one or more anionic lipids and / or neutral lipids. As used herein, “anionic lipid” means any lipid that carries a negative charge at a selected pH. As used herein, “neutral lipid” means any of a variety of lipids that are present at a selected pH in an uncharged or neutral zwitterionic form.

[0344] In some embodiments, the RNA compositions and formulations described herein, as well as the RNA particles, comprise cationic or cationically ionizable lipids and one or more additional lipids.

[0345] Without being bound by theory, the amount of cationic or cationically ionizable lipids, compared to the amount of one or more additional lipids, can affect important RNA particle properties, such as charge, particle size, stability, tissue selectivity, and RNA bioactivity. Therefore, in some embodiments, the molar ratio of cationic 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.

[0346] In some embodiments, one or more additional lipids contained in the RNA compositions and formulations described herein, as well as in the RNA particles, include one or more of the following: neutral lipids, steroids, and combinations thereof.

[0347] In some embodiments, one or more additional lipids include neutral lipids, said neutral lipids being phospholipids. In some embodiments, the phospholipids are selected from phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidic acid, phosphatidylserine, and sphingomyelin. Specific phospholipids that may be used include, but are not limited to, phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidic acid, phosphatidylserine, or sphingomyelin. Such phospholipids particularly include diacylphosphatidylcholine, such as distearylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dimyristoylphosphatidylcholine (DMPC), octadecylphosphatidylcholine, dilauroylphosphatidylcholine, dipalmitoylphosphatidylcholine (DPPC), eicosylphosphatidylcholine (DAPC), bis(docosahexadecylphosphatidylcholine) (DBPC), triacylphosphatidylcholine (DTPC), diacetylphosphatidylcholine (DLPC), palmitoyloleoylphosphatidylcholine (POPC), 1,2-di-O-octadecenyl-sn-glycerol-3-phosphate choline (18:0Diether PC), 1-oleoyl-2-cholestenylhemisuccinoyl-sn-glycerol-3-phosphate choline (OChemsPC), and 1-hexadecyl-sn-glycerol-3-phosphate choline (C16 Lyso PC) and phosphatidylethanolamines, especially diacylphosphatidylethanolamines, such as dioleoylphosphatidylethanolamine (DOPE), distearyl-phosphatidylethanolamine (DSPE), dipalmitoyl-phosphatidylethanolamine (DPPE), dimyristoyl-phosphatidylethanolamine (DMPE), dilauroyl-phosphatidylethanolamine (DLPE), diphyranoyl-phosphatidylethanolamine (DPyPE), 1,2-di-(9Z-octadecenoyl)-sn-glycerol-3-phosphate choline (DOPG), 1,2-dipalmitoyl-sn-glycerol-3-phosphate-(1'-rac-glycerol) (DPPG), 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphate ethanolamine (POPE), N-palmitoyl-D-erythrosine phosphatidylcholine (SM), and other phosphatidylethanolamine lipids with different hydrophobic chains. In some embodiments, the neutral lipid is selected from DSPC, DOPC, DMPC, DPPC, POPC, DOPE, DOPG, DPPG, POPE, DPPE, DMPE, DSPE, and SM. In some embodiments, the neutral lipid is DSPC. In some embodiments, the neutral lipid is DOPE.

[0348] In some embodiments, the additional lipid comprises one of the following: (1) phospholipids, (2) cholesterol or a derivative thereof; or (3) a mixture of phospholipids and cholesterol or a derivative thereof. Examples of cholesterol derivatives include, but are not limited to, cholesterol, cholesterolone, coprostinol, cholesterol-2'-hydroxyethyl ether, cholesterol-4'-hydroxybutyl ether, tocopherol and its derivatives, and mixtures thereof.

[0349] Therefore, in some embodiments, the RNA compositions and formulations described herein, as well as the RNA particles, comprise (1) cationic or cationically ionizable lipids and phospholipids, such as DSPC or DOPE, or (2) cationic or cationically ionizable lipids and phospholipids, such as DSPC or DOPE, and cholesterol.

[0350] In some embodiments, the RNA particles described herein (particularly particles containing mRNA) contain (1) DOTMA and DOPE, (2) DOTMA, DOPE and cholesterol, (3) DODMA and DOPE or (4) DODMA, DOPE and cholesterol.

[0351] DSPC is a neutral phospholipid. The structure of DSPC can be represented as follows: .

[0352] DOPE is a neutral phospholipid. The structure of DOPE can be represented as follows: .

[0353] The structure of cholesterol can be represented as follows: .

[0354] In some embodiments, the RNA compositions and formulations described herein, as well as the RNA particles, do not include polymer-conjugated lipids, such as PEGylated lipids. The term "PEGylated lipid" refers to a molecule comprising a lipid portion and a polyethylene glycol portion. PEGylated lipids are known in the art.

[0355] In some embodiments, additional lipids (e.g., one or more phospholipids and / or cholesterol) may account for 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) account for about 10 mol%, about 15 mol%, or about 20 mol% of the total lipids present in the particles.

[0356] In some embodiments, the additional lipid comprises 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.

[0357] Polymer-conjugated lipids

[0358] In some embodiments, the RNA compositions and formulations described herein, as well as the RNA particles, may comprise at least one polymer-conjugated lipid. Polymer-conjugated lipids are typically molecules comprising a lipid moiety and a polymer moiety conjugated thereto. In some embodiments, the polymer-conjugated lipid is a PEG-conjugated lipid, also referred to herein as a PEGylated lipid or PEG-lipid. The term "polyethylene glycol-conjugated lipid" refers to a molecule comprising a lipid moiety and a polyethylene glycol moiety. Polyethylene glycol-conjugated lipids are known in the art. In some embodiments, the polymer-conjugated lipid is a polysarcosine-conjugated lipid, also referred herein as a sarcosine-conjugated lipid or pSar-lipid. The term "sarcosine-conjugated lipid" refers to a molecule comprising a lipid moiety and a polysarcosine moiety.

[0359] In some embodiments, the polymer-conjugated lipids are designed to spatially stabilize the lipid particles by forming a protective hydrophilic layer that protects the hydrophobic lipid layer. In some embodiments, when such lipid particles are administered in vivo, the polymer-conjugated lipids can reduce their association with serum proteins and / or the resulting uptake by the reticuloendothelial system.

[0360] Polyethylene glycol (PEG) conjugated lipids

[0361] In some embodiments, the RNA compositions / formulations and RNA particles described herein contain PEG-conjugated lipids.

[0362] In some embodiments, the PEG-conjugated lipid (PEGylated lipid) is a lipid having the following general formula:

[0363] Or its pharmaceutically acceptable salts, tautomers or stereoisomers, wherein: Each of R12 and R13 is independently a straight-chain or branched alkyl or alkenyl chain containing 10 to 30 carbon atoms, wherein the alkyl / alkenyl chain is optionally interrupted by one or more ester bonds; and the average value of w ranges from 30 to 60.

[0364] In some embodiments of this formula, each of R12 and R13 is independently a straight-chain alkyl chain containing 10 to 18 carbon atoms, preferably 12 to 16 carbon atoms.

[0365] In some embodiments of this formula, R12 and R13 are the same. In some embodiments, each of R12 and R13 is a straight-chain alkyl chain containing 12 carbon atoms. In some embodiments, each of R12 and R13 is a straight-chain alkyl chain containing 14 carbon atoms. In some embodiments, each of R12 and R13 is a straight-chain alkyl chain containing 16 carbon atoms.

[0366] In some embodiments of this formula, R12 and R13 are different. In some embodiments, one of R12 and R13 is a straight-chain alkyl chain containing 12 carbon atoms, and the other of R12 and R13 is a straight-chain alkyl chain containing 14 carbon atoms.

[0367] In some implementations of this formula, the average value of w ranges from 40 to 50, for example, the average value is 45.

[0368] In some embodiments of this formula, w is in the range of 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, causing the PEG portion of the polyethylene glycol-modified lipid to have an average molecular weight of about 400 to about 6000 g / mol. In some embodiments, each of R12 and R13 is a straight-chain alkyl chain containing 14 carbon atoms, and the average value of w is 45.

[0369] Various PEG-conjugated lipids are known in the art, including but not limited to PEGylated diacylglycerols (PEG-DAG) (e.g., 1-(monomethoxy-polyethylene glycol)-2,3-dimyristoylglycerol (PEG-DMG)), PEGylated phosphatidylethanolamine (PEG-PE), and PEGylated diacylglycerol succinate (PEG-S-DAG) (e.g., 4-O-(2',3'-bis(tetradecanoyloxy)propyl-1-O-( PEG-S-DMG, PEGylated ceramide (PEG-cer), or PEG-dialkoxypropyl carbamate (e.g.) -Methoxy(polyethoxy)ethyl-N-(2,3-di(tetradecyloxy)propyl)carbamate or 2,3-di(tetradecyloxy)propyl-N-( Methoxy (polyethoxy) ethyl (urethane) esters, etc.

[0370] In some embodiments, the PEG-conjugated lipid (polyethylene glycol-modified lipid) is or contains 2-[(polyethylene glycol)-2000]-N,N-bistetradecylacetamide. In some embodiments, the polyethylene glycol-modified lipid has the following structure: .

[0371] In some embodiments, the PEG-conjugated lipid (PEGylated lipid) is, for example, DMG-PEG2000 having the following structure: .

[0372] In some embodiments, the PEG-conjugated lipids (PEGylated lipids) have the following structure:

[0373] The average value of n ranges from 30 to 60, for example, about 50. In one embodiment, the PEG-conjugated lipid (PEGylated lipid) is PEG2000-C-DMA, which preferably refers to 3-N-[(ω-methoxypoly(ethylene glycol)2000)carbamoyl]-1,2-dimyristoxypropylamine (MPEG-(2 kDa)-C-DMA) or methoxy-polyethylene glycol-2,3-bis(tetradecoxy)propylcarbamate (2000).

[0374] In some embodiments, the RNA compositions / formulations described herein may comprise one or more PEG-conjugated lipids or PEGylated lipids, as described in WO 2017 / 075531 and WO 2018 / 081480, the entire contents of which are incorporated herein by reference for the purposes described herein.

[0375] In some embodiments, PEGylated lipids comprise about 1 mol% to about 10 mol% of the total lipids present in the RNA compositions / formulations and RNA particles described herein, preferably about 1 mol% to about 5 mol%, more preferably about 1 mol% to about 2.5 mol%.

[0376] L Implementation plan for ipoplex particles

[0377] In some embodiments of this disclosure, the RNA described herein may be present in RNA lipoplex particles.

[0378] Lipoplex (LPX) is an electrostatic complex that is typically formed by mixing a pre-formed cationic lipid liposome with anionic nucleic acid. The resulting lipoplex has a unique internal molecular arrangement resulting from the transformation of the liposome structure into a compact RNA-lipoplex.

[0379] In some embodiments, the RNA lipoplex particles comprise cationic lipids and additional lipids. In an exemplary embodiment, the cationic lipid is DOTMA, and the additional lipid is DOPE.

[0380] In some embodiments, the molar ratio of at least one cationic lipid to at least one additional lipid is about 10:0 to about 1:9, about 4:1 to about 1:2, or about 3:1 to about 1:1. In specific embodiments, the molar ratio may be about 3:1, about 2.75:1, about 2.5:1, about 2.25:1, about 2:1, about 1.75:1, about 1.5:1, about 1.25:1, or about 1:1. In an exemplary embodiment, the molar ratio of at least one cationic lipid to at least one additional lipid is about 2:1.

[0381] The average diameter of the RNA lipoplex particles described herein is, in some embodiments, about 200 nm to about 1000 nm, about 200 nm to about 800 nm, about 250 nm to about 700 nm, about 400 nm to about 600 nm, about 300 nm to about 500 nm, or about 350 nm to about 400 nm. In specific embodiments, the average diameter of the RNA lipoplex particles is about 200 nm, about 225 nm, about 250 nm, about 275 nm, about 300 nm, about 325 nm, about 350 nm, about 375 nm, about 400 nm, about 425 nm, about 450 nm, about 475 nm, about 500 nm, about 525 nm, about 550 nm, about 575 nm, about 600 nm, about 625 nm, about 650 nm, about 675 nm, about 700 nm, about 725 nm, about 750 nm, about 775 nm, or about 800 nm. The diameters are approximately 825 nm, 850 nm, 875 nm, 900 nm, 925 nm, 950 nm, 975 nm, or 1000 nm. In some embodiments, the average diameter of the RNA lipoplex particles ranges from approximately 250 nm to approximately 700 nm. In some embodiments, the average diameter of the RNA lipoplex particles is from approximately 300 nm to approximately 500 nm. In an exemplary embodiment, the average diameter of the RNA lipoplex particles is approximately 400 nm.

[0382] The RNA lipoplex particles and compositions containing RNA lipoplex particles described herein can be used to deliver RNA to target tissues after parenteral administration, particularly after intravenous administration.

[0383] Spleen-targeting RNA lipoplex particles are described in WO 2013 / 143683 (which is incorporated herein by reference). It has been found that RNA lipoplex particles with a net negative charge can be used to preferentially target spleen tissue or spleen cells, such as antigen-presenting cells, particularly dendritic cells. Therefore, RNA accumulation and / or RNA expression occur in the spleen after administration of the RNA lipoplex particles. Therefore, the RNA lipoplex particles of this disclosure can be used to express RNA in the spleen. In embodiments, no or substantially no RNA accumulation and / or RNA expression occurs in the lungs and / or liver after administration of the RNA lipoplex particles. In some embodiments, RNA accumulation and / or RNA expression occur in specialized antigen-presenting cells, such as 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 lymphatic system, particularly secondary lymphatic organs, more specifically the spleen. If the applied RNA is RNA encoding a vaccine antigen, targeting the lymphatic system, particularly secondary lymphatic organs, is especially preferred, more specifically the spleen. 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.

[0384] The charge of the RNA lipoplex particle disclosed herein is the sum of the charge present in at least one cationic lipid and the charge present in the RNA. The charge ratio is the ratio of the positive charge present in at least one cationic lipid to the negative charge present in the RNA. The charge ratio of the positive charge present in at least one cationic lipid to the negative charge present in the RNA is calculated by the following equation: Charge ratio = [(Cationic lipid concentration (mol))] [(Total positive charge in cationic lipids)] / [(RNA concentration (mol)) (Total negative charge in RNA). The concentration of RNA and the amount of at least one cationic lipid can be determined by those skilled in the art using conventional methods.

[0385] In some embodiments, at physiological pH, the charge ratio of positive to negative charge in RNA lipoplex particles is about 1.6:2 to about 1:2, or about 1.6:2 to about 1.1:2. In specific embodiments, at physiological pH, the charge ratio of positive to negative charge in RNA lipoplex particles is about 1.6:2.0, about 1.5:2.0, about 1.4:2.0, about 1.3:2.0, about 1.2:2.0, about 1.1:2.0, or about 1:2.0.

[0386] Implementation scheme of lipid nanoparticles (LNP)

[0387] In some embodiments, the RNA described herein exists in the form of lipid nanoparticles (LNPs). LNPs typically comprise four components: cationic ionizable lipids, neutral lipids such as phospholipids, steroids such as cholesterol, and polymer-conjugated lipids such as PEG-lipids. LNPs can be prepared by mixing lipids dissolved in ethanol with an aqueous buffer containing RNA.

[0388] In some embodiments, in the RNA LNP described herein, RNA is bound to a cationicly ionizable lipid occupying a central core of the LNP. The polymer-conjugated lipids, together with phospholipids, form the surface of the LNP. In some embodiments, cholesterol and cationicly ionizable lipids may be distributed throughout the LNP.

[0389] In some embodiments, the LNP comprises one or more cationic ionizable lipids and one or more stable lipids. Stable lipids include neutral lipids and polymer-conjugated lipids.

[0390] In some embodiments, the LNP comprises cationic ionizable lipids, neutral lipids, steroids, polymer-conjugated lipids; and RNA encapsulated within or associated with lipid nanoparticles.

[0391] In some embodiments, the LNP comprises 35 mol% to 65 mol%, 40 mol% to 60 mol%, 40 mol% to 55 mol%, 45 mol% to 55 mol%, or 45 mol% to 50 mol% of cationic ionizable lipids.

[0392] In some implementations, the neutral lipids are present at concentrations of 5 mol% to 15 mol%, 7 mol% to 13 mol%, or 9 mol% to 11 mol%.

[0393] In some embodiments, the steroid is present at a concentration of 30 mol% to 50 mol%, 30 mol% to 45 mol%, 35 mol% to 45 mol%, or 35 mol% to 43 mol%.

[0394] In some embodiments, the LNP comprises 1 mol% to 10 mol%, 1 mol% to 5 mol%, or 1 mol% to 2.5 mol% of polymer-conjugated lipids.

[0395] In some embodiments, the LNP comprises 45 mol% to 55 mol% of cationic ionizable lipids; 5 mol% to 15 mol% of neutral lipids; 30 mol% to 45 mol% of steroids; 1 mol% to 5 mol% of polymer-conjugated lipids; and RNA encapsulated within or associated with lipid nanoparticles.

[0396] In some embodiments, the molar percentage is determined based on the total molar number of lipids present in the lipid nanoparticles. In some embodiments, the molar percentage is determined based on the total molar number of cationic ionizable lipids, neutral lipids, steroids, and polymer-conjugated lipids present in the lipid nanoparticles.

[0397] In some embodiments, the neutral lipid is selected from DSPC, DPPC, DMPC, DOPC, POPC, DOPE, DOPG, DPPG, POPE, DPPE, DMPE, DSPE, and SM. In some embodiments, the neutral lipid is DSPC.

[0398] In some implementations, the steroid is cholesterol.

[0399] In some embodiments, the polymer-conjugated lipid is a polyethylene glycol-modified lipid, such as the polyethylene glycol-modified lipid described above.

[0400] In some embodiments, the cationic ionizable lipid component of LNP has the structure of formula (III):

[0401] (III)

[0402] Or a pharmaceutically acceptable salt, tautomer, prodrug, or stereoisomer thereof, wherein: 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 and G2 are each independently unsubstituted C1-C12 alkylene or C1-C12 alkenylene; G3 is C1-C24 alkylene, C1-C24 alkenylene, C3-C8 cycloalkylene, or C3-C8 alkenylene. Ra is H or a C1-C12 alkyl group; R1 and R2 are each independently C6-C24 alkyl or 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 a C1-C6 alkyl group; and x is 0, 1, or 2.

[0403] In some of the foregoing embodiments of formula (III), the lipid has one of the following structures (IIIA) or (IIIB): or

[0404] (IIIA) (IIIB)

[0405] in: A is a 3- to 8-membered cycloalkyl or cyclohexane ring; R6 is independently H, OH or C1-C24 alkyl each time it appears; n is an integer ranging from 1 to 15.

[0406] In some of the aforementioned embodiments of formula (III), the lipid has structure (IIIA), and in other embodiments, the lipid has structure (IIIB).

[0407] In other embodiments of formula (III), the lipid has one of the following structures (IIIC) or (IIID): or

[0408] (IIIC) (IIID)

[0409] Where y and z are each an independent integer from 1 to 12.

[0410] In any of the foregoing embodiments of formula (III), one of L1 or L2 is -O(C=O)-. For example, in some embodiments, each of L1 and L2 is -O(C=O)-. In some different embodiments of any of the foregoing, L1 and L2 are each independently -(C=O)O- or -O(C=O)-. For example, in some embodiments, each of L1 and L2 is -(C=O)O-.

[0411] In some different embodiments of formula (III), the lipid has one of the following structures (IIIE) or (IIIF): or

[0412] (IIIE) (IIIF)

[0413] In some of the aforementioned embodiments of formula (III), the lipid has one of the following structures: (IIIG), (IIIH), (IIII), or (IIIJ): ; ; (IIIG) (IIIH) or .

[0414] (IIII) (IIIJ)

[0415] In some of the foregoing embodiments of equation (III), n is an integer from 2 to 12, such as 2 to 8 or 2 to 4. For example, in some embodiments, n is 3, 4, 5, or 6. In some embodiments, n is 3. In some embodiments, n is 4. In some embodiments, n is 5. In some embodiments, n is 6.

[0416] In some other of the foregoing embodiments of equation (III), y and z are each independently an integer from 2 to 10. For example, in some embodiments, y and z are each independently an integer from 4 to 9 or from 4 to 6.

[0417] In some of the foregoing embodiments of formula (III), R6 is H. In other foregoing embodiments, R6 is a C1-C24 alkyl group. In other embodiments, R6 is OH.

[0418] 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.

[0419] In some other aforementioned embodiments of formula (III), R1 or R2, or both, are C6-C24 alkenyl groups. For example, in some embodiments, R1 and R2 each independently have the following structures: , in: R7a and R7b are independently H or C1-C12 alkyl groups each time they appear; and a is an integer from 2 to 12. R7a, R7b, and a are each chosen such that R1 and R2 each independently contain 6 to 20 carbon atoms. For example, in some embodiments, a is an integer from 5 to 9 or from 8 to 12.

[0420] In some of the foregoing embodiments of formula (III), R7a, which appears at least once, is H. For example, in some embodiments, R7a is H every time it appears. In other different foregoing embodiments, R7b, which appears at least once, is a C1-C8 alkyl group. For example, in some embodiments, the C1-C8 alkyl group is methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-hexyl, or n-octyl.

[0421] In different embodiments of formula (III), R1 or R2 or both have one of the following structures: ; ; ; ; ; ; ; ; ; .

[0422] 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.

[0423] In various different embodiments, the cationic lipid of formula (III) has one of the structures listed in the table below.

[0424] Representative compounds of formula (III).

[0425]

[0426] Other representative cationic ionizable lipids are as follows.

[0427]

[0428] In some embodiments, the RNA described herein is formulated in an LNP composition comprising cationic ionizable lipids (e.g., cationic ionizable lipids as shown above), neutral lipids, steroids, and polymer-conjugated lipids.

[0429] In some embodiments, the RNA described herein is formulated in an LNP composition comprising a cationic ionizable lipid of formula III, a neutral lipid, a steroid, and a polymerically conjugated lipid.

[0430] In some embodiments, the RNA described herein is formulated in an LNP composition comprising cationic ionizable lipids, neutral lipids, steroids, and polymer-conjugated lipids as shown in the table above.

[0431] In some embodiments, the RNA described herein is formulated in an LNP composition comprising 3D-P-DMA, neutral lipids, steroids, and polymer-conjugated lipids.

[0432] In some embodiments, the RNA described herein is formulated in an LNP composition comprising ALC-0366, neutral lipids, steroids, and polymer-conjugated lipids.

[0433] In some embodiments, the RNA described herein is formulated in an LNP composition comprising ALC-0315, neutral lipids, steroids, and polymer-conjugated lipids.

[0434] In some embodiments, the neutral lipid is DSPC. In some embodiments, the steroid is cholesterol. In some embodiments, the polymer-conjugated lipid is a polyethylene glycol-modified lipid, such as DMG-PEG 2000, PEG2000-C-DMA, or ALC-0159.

[0435] In some embodiments, the RNA described herein is formulated in an LNP composition comprising cationic ionizable lipids (e.g., cationic ionizable lipids as shown above), neutral lipids, steroids, and PEGylated lipids.

[0436] In some embodiments, the RNA described herein is formulated in an LNP composition comprising a cationic ionizable lipid of formula III, a neutral lipid, a steroid, and a polyethylene glycol-modified lipid.

[0437] In some embodiments, the RNA described herein is formulated in an LNP composition comprising the cationic ionizable lipids, neutral lipids, steroids, and PEGylated lipids shown in the table above.

[0438] In some embodiments, the RNA described herein is formulated in an LNP composition comprising 3D-P-DMA, neutral lipids, steroids, and PEGylated lipids.

[0439] In some embodiments, the RNA described herein is formulated in an LNP composition comprising ALC-0366, neutral lipids, steroids, and PEGylated lipids.

[0440] In some embodiments, the RNA described herein is formulated in an LNP composition comprising ALC-0315, neutral lipids, steroids, and PEGylated lipids.

[0441] 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.

[0442] In some embodiments, the RNA described herein is formulated in an LNP composition comprising cationic ionizable lipids (e.g., cationic ionizable lipids as shown above), DSPC, cholesterol, and PEGylated lipids.

[0443] In some embodiments, the RNA described herein is formulated in an LNP composition comprising a cationic ionizable lipid of formula III, DSPC, cholesterol, and PEGylated lipids.

[0444] In some embodiments, the RNA described herein is formulated in an LNP composition comprising the cationic ionizable lipids, DSPC, cholesterol, and PEGylated lipids shown in the table above.

[0445] In some embodiments, the RNA described herein is formulated in an LNP composition comprising 3D-P-DMA, DSPC, cholesterol, and PEGylated lipids.

[0446] In some embodiments, the RNA described herein is formulated in an LNP composition comprising ALC-0366, DSPC, cholesterol, and PEGylated lipids.

[0447] In some embodiments, the RNA described herein is formulated in an LNP composition comprising ALC-0315, DSPC, cholesterol, and PEGylated lipids.

[0448] In some implementations, the PEGylated lipid is DMG-PEG 2000, PEG2000-C-DMA, or ALC-0159.

[0449] In some embodiments, the RNA described herein is formulated in an LNP composition comprising cationic ionizable lipids (e.g., cationic ionizable lipids as shown above), DSPC, cholesterol, and DMG-PEG 2000.

[0450] In some embodiments, the RNA described herein is formulated in an LNP composition comprising a cationic ionizable lipid of formula III, DSPC, cholesterol, and DMG-PEG 2000.

[0451] In some embodiments, the RNA described herein is formulated in an LNP composition comprising the cationic ionizable lipids, DSPC, cholesterol, and DMG-PEG 2000 shown in the table above.

[0452] In some embodiments, the RNA described herein is formulated in an LNP composition comprising 3D-P-DMA, DSPC, cholesterol, and DMG-PEG 2000.

[0453] In some embodiments, the RNA described herein is formulated in an LNP composition comprising ALC-0366, DSPC, cholesterol, and DMG-PEG 2000.

[0454] In some embodiments, the RNA described herein is formulated in an LNP composition comprising ALC-0315, DSPC, cholesterol, and DMG-PEG 2000.

[0455] In some embodiments, the RNA described herein is formulated in an LNP composition comprising cationic ionizable lipids (e.g., cationic ionizable lipids as shown above), DSPC, cholesterol, and PEG2000-C-DMA.

[0456] In some embodiments, the RNA described herein is formulated in an LNP composition comprising a cationic ionizable lipid of formula III, DSPC, cholesterol, and PEG2000-C-DMA.

[0457] In some embodiments, the RNA described herein is formulated in an LNP composition comprising the cationic ionizable lipids, DSPC, cholesterol, and PEG2000-C-DMA shown in the table above.

[0458] In some embodiments, the RNA described herein is formulated in an LNP composition comprising 3D-P-DMA, DSPC, cholesterol, and PEG2000-C-DMA.

[0459] In some embodiments, the RNA described herein is formulated in an LNP composition comprising ALC-0366, DSPC, cholesterol, and PEG2000-C-DMA.

[0460] In some embodiments, the RNA described herein is formulated in an LNP composition comprising ALC-0315, DSPC, cholesterol, and PEG2000-C-DMA.

[0461] In some embodiments, the RNA described herein is formulated in an LNP composition comprising cationic ionizable lipids (e.g., cationic ionizable lipids as shown above), DSPC, cholesterol, and ALC-0159.

[0462] In some embodiments, the RNA described herein is formulated in an LNP composition comprising a cationic ionizable lipid of formula III, DSPC, cholesterol, and ALC-0159.

[0463] In some embodiments, the RNA described herein is formulated in an LNP composition comprising the cationic ionizable lipids, DSPC, cholesterol, and ALC-0159 shown in the table above.

[0464] In some embodiments, the RNA described herein is formulated in an LNP composition comprising 3D-P-DMA, DSPC, cholesterol, and ALC-0159.

[0465] In some embodiments, the RNA described herein is formulated in an LNP composition comprising ALC-0366, DSPC, cholesterol, and ALC-0159.

[0466] In some embodiments, the RNA described herein is formulated in an LNP composition comprising ALC-0315, DSPC, cholesterol, and ALC-0159.

[0467] 3D-P-DMA: (6Z,16Z)-12-((Z)-dec-4-en-1-yl)docosa-6,16-dien-11-yl 5-(dimethylamino)valerate

[0468] ALC-0366: ((3-hydroxypropyl)azanidinediyl)bis(nonane-9,1-diyl)bis(2-butyloctanoate)

[0469] ALC-0315: ((4-hydroxybutyl)azanidinediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate) / 6-[N-6-(2-hexyldecanoyloxy)hexyl-N-(4-hydroxybutyl)amino]hexyl-2-hexyldecanoate

[0470] DMG-PEG 2000:

[0471] PEG2000-C-DMA: 3-N-[(ω-methoxypoly(ethylene glycol)2000)carbamoyl]-1,2-dimyristoxypropylamine (MPEG-(2 kDa)-C-DMA or methoxy-polyethylene glycol-2,3-bis(tetradecoxy)propylcarbamate (2000))

[0472] The average value of n ranges from 30 to 60, for example, about 50.

[0473]

[0474] ALC-0159: 2-[(polyethylene glycol)-2000]-N,N-bistetradecylacetamide / 2-[2-(ω-methoxy(polyethylene glycol 2000)ethoxy]-N,N-bistetradecylacetamide

[0475] DSPC: 1,2-Distearyl-sn-glycerol-3-phosphocholine

[0476] cholesterol:

[0477] The N / P value is preferably at least about 4. In some embodiments, the N / P value ranges from 4 to 20, 4 to 12, 4 to 10, 4 to 8, or 5 to 7. In some embodiments, the N / P value is about 6.

[0478] The term "invisible" as used herein describes a host's immune system that is unable to detect the particles described herein, and then isolates and / or degrades them, or has difficulty detecting the particles described herein, and then isolates and / or degrades them, and / or is able to detect the particles described herein, and then subsequently isolates and / or degrades them.

[0479] Macrophages constitute one of the most important components of the immune system and play a major role in eliminating foreign particles, including liposomes and other colloidal particles, from the bloodstream. At the molecular level, particle clearance occurs in two steps: opsonization occurs by depositing serum proteins (or "opsonins") on the particle surface, followed by recognition and capture of opsonized particles by macrophages.

[0480] Modifying the particle surface with hydrophilic and flexible polymer chains, such as poly(ethylene glycol) type polymers, provides them with spatial protection by preventing opsonins from reaching the particle surface.

[0481] In some embodiments, the amphiphilic derivatives of the polymers used herein have hydrophobic groups (e.g., lipids) as specified herein. In some embodiments, the amphiphilic derivatives of the polymers used herein have phospholipids as hydrophobic groups (e.g., lipids), such as biodegradable phospholipids, such as phosphatidylethanolamine. In some embodiments, the phospholipids are selected from DSPE (distearylphosphatidylethanolamine), DPPE (dispalmitoylphosphatidylethanolamine), DOPE (dioleoylphosphatidylethanolamine), and POPE (palmitoyloleoylphosphatidylethanolamine), and mixtures thereof. In some embodiments, DSPE will be used as a phospholipid due to its stability properties in the particles described herein. Furthermore, as hydrophobic groups (e.g., lipids), compounds having at least one alkyl chain providing a hydrophobic anchoring to the particles as described herein can be used.

[0482] In some embodiments, the polymers used herein are selected from 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).

[0483] In some embodiments, the polymer is designed to spatially stabilize the particles by forming a protective hydrophilic layer. In some embodiments, when such particles are administered in vivo, the polymer can reduce particle association with serum proteins and / or the resulting uptake by the reticuloendothelial system.

[0484] In some embodiments, PEG is an optionally substituted linear or branched polymer of 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, hydroxyl, or aryl groups. In some embodiments, the molecular weight of PEG is from about 130 to about 50,000; in another embodiment, the molecular weight of PEG is from about 150 to about 30,000; in another embodiment, the molecular weight of PEG is from about 150 to about 20,000; in another embodiment, the molecular weight of PEG is from about 150 to about 15,000; in another embodiment, the molecular weight of PEG is from about 150 to about 10,000; in another embodiment, the molecular weight of PEG is from about 150 to about 6,000; in another embodiment, the molecular weight of PEG is from about 150 to about 5,000; in another embodiment, the molecular weight of PEG is from about 150 to about 4,000; in another embodiment, the molecular weight of PEG is from about 150 to about 3,000; in another embodiment, the molecular weight of PEG is from about 300 to about 3,000; in another embodiment, the molecular weight of PEG is from about 1,000 to about 3,000; and in yet another embodiment, the molecular weight of PEG is from about 1,500 to about 2,500.

[0485] In some embodiments, the PEG moiety of the amphiphilic derivative of the polymer has a molecular weight of 1000 or higher. In some embodiments, the PEG moiety of the amphiphilic derivative of the polymer contains 10 or more units of the formula (O-CH2-CH2)n. In some embodiments, the PEG contains 20 to 200 ethylene oxide units, for example, about 45 ethylene oxide units.

[0486] In some implementations, PEG contains “PEG2k”, also known as “PEG 2000”, which has an average molecular weight of about 2,000 Daltons.

[0487] In some implementations, DSPE-PEG2000, DSPE-PEG3000 and DSPE-PEG5000 are used as amphiphilic derivatives of the polymer.

[0488] In some implementations, pSar contains 2 to 200 sarcosine units, such as 5 to 100 sarcosine units, 10 to 50 sarcosine units, 15 to 40 sarcosine units, such as about 23 sarcosine units.

[0489] In some implementations, pSar includes the structure of the following general formula:

[0490] Where s is the number of sarcosine units.

[0491] In some embodiments, the POX and / or POZ polymer comprises 2 to 200, 2 to 190, 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 to 150, 5 to 140, 5 to 130, 5 to 120, 5 to 110, 5 to 100, 5 to 90, 5 to 80, 5 to 70, 10 to 200, 10 to 190, 10 to 180, 10 to 170, 10 to 160, 10 to 150, 10 to 140, 10 to 130, 10 to 120, 10 to 110, 10 to 100, 10 to 90, 10 to 80 or 10 to 70 POX and / or POZ repeating units.

[0492] In some implementations, the POX and / or POZ polymers comprise the following general formula: , Where a is an integer from 1 to 2; R11 is an alkyl group, particularly a C1-3 alkyl group, such as methyl, ethyl, isopropyl, or n-propyl, and is chosen independently for each repeating unit; and m refers to the number of POX and / or POZ repeating units.

[0493] In some embodiments, the POX and / or POZ polymers are polymers of POX and contain repeating units of the following general formula: .

[0494] In some embodiments, the POX and / or POZ polymers are polymers of POZ and contain repeating units of the following general formula: .

[0495] In any of the above embodiments, 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 to 150, 5 to 14 ... 0, 5 to 130, 5 to 120, 5 to 110, 5 to 100, 5 to 90, 5 to 80, 5 to 70, 10 to 200, 10 to 190, 10 to 180, 10 to 170, 10 to 160, 10 to 150, 10 to 140, 10 to 130, 10 to 120, 10 to 110, 10 to 100, 10 to 90, 10 to 80, or 10 to 70. In some embodiments, m is 2 to 180, for example 4 to 160, 6 to 140, 8 to 120, or 10 to 100, for example 20 to 80, 30 to 70, or 40 to 50.

[0496] In some embodiments, the POX and / or POZ polymers are copolymers comprising repeating units of the following general formula: , The number of repeating units shown on the left in the copolymer is 1 to 199; the number of repeating units of the formula on the right in the copolymer is 1 to 199; and the sum of the number of repeating units of the formula on the left and the number of repeating units of the formula on the right in the copolymer is 2 to 200.

[0497] In some embodiments of the oxazoline- and / or oxazine-modified hydrophobic moieties (e.g., lipids), the number of repeating units of the left-hand formula in the copolymer is 1 to 179, for example 1 to 159, 1 to 139, 1 to 119, or 1 to 99; the number of repeating units of the right-hand formula in the copolymer is 1 to 179, for example 1 to 159, 1 to 139, 1 to 119, or 1 to 99; and the sum of the number of repeating units of the left-hand formula and the number of repeating units of the right-hand formula in the copolymer is 2 to 180, for example 4 to 160, 6 to 140, 8 to 120, or 10 to 100, for example 20 to 80, 30 to 70, or 40 to 50.

[0498] In some of the above embodiments, R11 may be the same alkyl group each time it appears (i.e., in each repeating unit) (e.g., R11 may be methyl in each repeating unit). In some alternative embodiments, R11 in at least one repeating unit is different from R11 in another repeating unit (e.g., for at least one repeating unit, R11 is a 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.

[0499] In any of the above embodiments, R11 is preferably methyl or ethyl, more preferably methyl. Therefore, in some embodiments, each R11 is methyl or each R11 is ethyl. In some alternative embodiments, for each repeating unit, R11 is independently selected from methyl and ethyl, wherein R11 is methyl in at least one repeating unit and ethyl in at least one repeating unit.

[0500] 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 derivatives thereof.

[0501] In some embodiments, the polymer comprises the following general formula:

[0502] in

[0503] X2 and X1 together are optionally substituted amides, optionally substituted thioamides, or esters; Y is -CH2-, -(CH2)2-, or -(CH2)3-; z is between 2 and 24; and n is between 1 and 100.

[0504] In some implementation schemes, (i) When X1 is -C(O)-, then X2 is -NR1-; (ii) When X1 is -NR1-, then X2 is -C(O)-; (iii) When X1 is -C(S)-, then X2 is -NR1-; (iv) When X1 is -NR1-, then X2 is -C(S)-; (v) When X1 is -C(O)-, then X2 is -O-; or (vi) When X1 is -O-, then X2 is -C(O)-; R1 is hydrogen or a C1-8 alkyl group.

[0505] In some embodiments, X1 is -C(O)- and X2 is -NR1-, wherein R1 is hydrogen or a C1-8 alkyl group. In some embodiments, X1 is -C(O)- and X2 is -NR1-, wherein R1 is hydrogen or a methyl group. In some embodiments, X1 is -C(O)- and X2 is -NR1-, wherein R1 is hydrogen.

[0506] In some implementations, Y is -CH2- or -(CH2)2-. In some implementations, Y is -CH2-.

[0507] In some embodiments, the polymer comprises the following general formula:

[0508] in

[0509] R1 is hydrogen or a C1-8 alkyl group; z is between 2 and 24; and n is between 1 and 100.

[0510] In some embodiments of the above formula, z is 2 to 10. In some embodiments, z is 2 to 7. In some embodiments, z is 2 to 5. In some embodiments, z is 2 or 3. In some embodiments, z is 2.

[0511] In some embodiments, the polymer comprises the following general formula:

[0512] in

[0513] R1 is hydrogen or a C1-8 alkyl group; and

[0514] n is between 1 and 100.

[0515] In some embodiments of the above formula, R1 is hydrogen or methyl. In some embodiments, R1 is hydrogen.

[0516] In some embodiments, the polymer comprises the following general formula:

[0517] in

[0518] n is between 1 and 100.

[0519] In some embodiments of the above formula, n is 5 to 50. In some embodiments, n is 5 to 25. In some embodiments, n is 7 to 14. In some embodiments, n is 10 to 25. In some embodiments, n is 14 to 17. In some embodiments, n is 8 or 14.

[0520] In some embodiments, the molar ratio of the amphiphilic derivative of the polymer integrated into the particles is 0.5 mol% to 20 mol%, preferably 1 mol% to 10 mol%, of the lipid molecules constituting the particles.

[0521] Pharmaceutical Composition

[0522] The particles or compositions described herein may be administered in a pharmaceutical composition or medicament, and may be administered in any suitable pharmaceutical composition form. In some embodiments, the pharmaceutical composition is used for therapeutic or preventative treatment, such as for treating or preventing diseases involving antigens, such as cancer or infectious diseases, particularly HIV infection.

[0523] The term "pharmaceutical composition" refers to a composition comprising a therapeutically effective agent, preferably together with a pharmaceutically acceptable carrier, diluent, and / or excipient. By administering the pharmaceutical composition to a subject, the pharmaceutical composition can be used to treat, prevent, or reduce the severity of a disease.

[0524] The pharmaceutical compositions disclosed herein may contain one or more adjuvants or may be administered in combination with one or more adjuvants. In some embodiments, the pharmaceutical composition does not contain an adjuvant. The term "adjuvant" refers to a compound that prolongs, enhances, or accelerates an immune response. Adjuvants include a heterogeneous group of compounds, such as oil emulsions (e.g., Freund's adjuvant), mineral compounds (e.g., alum), bacterial products (e.g., Bordetella pertussis toxin), or immunostimulatory complexes. Examples of adjuvants include, but are not limited to, 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-α. Other known adjuvants are aluminum hydroxide, Freund's adjuvant, or oils, such as Montanide® ISA51. Other suitable adjuvants for use in this disclosure include lipopeptides, such as Pam3Cys, and lipophilic components, such as saponins, trehalose-6,6-dibenzenesulfonate (TDB), monophosphoryl lipid-A (MPL), monomycoloyl glycerol (MMG), or glucopyranoyl lipid adjuvant (GLA).

[0525] The pharmaceutical compositions disclosed herein may be in a storable form (e.g., frozen or lyophilized / freeze-dried form) or an "applied-to-use" form (i.e., a form that can be immediately administered to a subject, e.g., without any processing such as dilution). Therefore, a storable form of pharmaceutical composition must be processed or converted into an applied-to-use or appliqué form before it can be administered. For example, a frozen pharmaceutical composition must be thawed, or a lyophilized pharmaceutical composition must be reconstituted, for example, by using a suitable solvent (e.g., deionized water, such as water for injection) or a liquid (e.g., an aqueous solution).

[0526] The pharmaceutical compositions according to this disclosure are generally used in a “pharmaceuticalally effective amount” and a “pharmaceuticalally acceptable formulation”.

[0527] The term "pharmaceutical acceptable" refers to the non-toxicity of materials that do not interact with the active component of a pharmaceutical composition.

[0528] The term "pharmaceutical effective amount" refers to the amount, alone or in combination with other doses, that achieves the desired response or desired effect. In some embodiments involving the treatment of a specific disease, the desired response may involve inhibiting the disease progression. 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 delaying the onset of the disease or condition or its symptoms, or preventing the onset of the disease or condition or its symptoms. The effective amount of the pharmaceutical composition described herein will depend on the condition to be treated, the severity of the disease, the individual parameters of the patient, including age, physiological condition, body size and weight, duration of treatment, type of concomitant treatment (if present), specific route of administration, and similar factors. Therefore, the dosage of the pharmaceutical composition described herein may depend on a variety of such parameters. In cases where the patient's response to the initial dose is insufficient, a higher dose may be used (or an effective higher dose achieved through a different, more localized route of administration).

[0529] The pharmaceutical compositions disclosed herein may contain buffers, preservatives, and optionally other therapeutic agents. In some embodiments, the pharmaceutical compositions disclosed herein comprise one or more pharmaceutically acceptable carriers, diluents, and / or excipients.

[0530] Suitable preservatives used in the pharmaceutical compositions disclosed herein include, but are not limited to, benzalkonium chloride, chlorobutanol, para-hydroxybenzoate and thimerosal.

[0531] As used herein, the term "excipient" refers to a substance that may be present in the pharmaceutical compositions of this disclosure but is not an active ingredient. Examples of excipients include, but are not limited to, carriers, binders, diluents, lubricants, thickeners, surfactants, preservatives, stabilizers, emulsifiers, buffers, flavoring agents, or coloring agents.

[0532] The term "diluent" refers to the diluting and / or thinning of reagents. Furthermore, the term "diluent" includes any one or more fluid, liquid, or solid suspensions and / or mixtures. Examples of suitable diluents include ethanol, glycerol, and water.

[0533] The term "carrier" refers to a component, which may be natural, synthetic, organic, or inorganic, in which the active component is combined to facilitate, enhance, or enable the administration of a pharmaceutical composition. As used herein, a carrier may be one or more compatible solid or liquid fillers, diluents, or encapsulating substances suitable for administration to a subject. Suitable carriers include, but are not limited to, sterile water, Ringer's lactate, Ringer's lactate, sterile sodium chloride solution, isotonic saline, polyalkylene glycols, hydrogenated naphthalene, and particularly biocompatible lactide polymers, lactide / glycolic acid copolymers, or polyoxyethylene / polyoxypropylene copolymers. In some embodiments, the pharmaceutical compositions of this disclosure comprise isotonic saline.

[0534] Pharmaceutically acceptable carriers, excipients, or diluents for therapeutic purposes are well known in the pharmaceutical industry and are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (A. RGennaro edit. 1985).

[0535] The drug carrier, excipient, or diluent can be selected based on the intended route of administration and standard pharmaceutical practice.

[0536] Treatment

[0537] The agents, compositions, and methods described herein can be used to treat subjects suffering from diseases (e.g., diseases characterized by HIV infection). The agents, compositions, and methods described herein can be used for therapeutic or preventative treatment of various diseases. In some embodiments, the agents, compositions, and methods described herein can be used for preventative and / or therapeutic treatment of diseases involving antigens.

[0538] This antigen can serve as a target for immune effector cells that express antigen receptors. For example, if the antigen is derived from a virus, the agents, compositions, and methods can be used to treat viral diseases caused by said virus. If the antigen is a tumor antigen, the agents, compositions, and methods can be used to treat cancerous diseases in which cancer cells express said tumor antigen.

[0539] The term "disease" refers to an abnormal condition affecting an individual's body. Disease is generally interpreted as a medical condition associated with specific symptoms and signs. Disease can be caused by factors originating from external sources, such as infectious diseases, or it can be caused by internal dysfunctions, such as autoimmune diseases. In humans, "disease" is often used more broadly to refer to any condition that causes pain, dysfunction, suffering, social problems, or death in the affected individual, or similar problems that come into contact with the individual. In this broader sense, it sometimes includes injuries, disabilities, impairments, syndromes, infections, isolated symptoms, atypical behaviors, and atypical changes in structure and function, which may be considered distinguishable categories in other contexts and for other purposes. Disease often affects an individual not only physically but also emotionally, as contracting and living with many diseases can alter a person's perspective on life and their personality.

[0540] The term "infectious disease" refers to any illness (e.g., the common cold) that can be transmitted from person to person or from organism to organism and is caused by a microbial agent. Infectious diseases are 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 can be, for example, hepatitis, sexually transmitted diseases (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.

[0541] In this context, the terms "treatment," "treating," or "therapeutic intervention" refer to the management and care of a subject for the purpose of combating a condition (e.g., disease or symptom). This term is intended to include a full spectrum of treatment for a given condition suffered by the subject, such as the administration of therapeutically effective compounds to relieve symptoms or complications, delay the progression of the disease, symptom, or condition, alleviate or reduce symptoms and complications, and / or cure or eliminate the disease, symptom, or condition, as well as prevention of the condition, wherein prevention should be understood as the management and care of an individual for the purpose of combating the disease, symptom, or condition, and includes the administration of active compounds to prevent the onset of symptoms or complications.

[0542] The term "therapeutic treatment" refers to any treatment that improves health status and / or prolongs (increases) an individual's lifespan. Such treatment may eliminate disease in an individual, stop or slow the progression of disease in an individual, inhibit or slow the progression of disease in an individual, reduce the frequency or severity of symptoms in an individual, and / or reduce recurrence in an individual currently suffering from or previously suffering from disease.

[0543] The term "prophylactic treatment" or "preventive treatment" refers to any treatment designed to prevent the occurrence of a disease in an individual. The terms "prophylactic treatment" or "preventive treatment" are used interchangeably in this document.

[0544] The terms “individual” and “object” are used interchangeably herein. They refer to a human or another mammal (e.g., a mouse, rat, rabbit, dog, cat, cow, pig, sheep, horse, or primate) that may or may not have a disease or condition (e.g., cancer). In many embodiments, an individual is a human being. Unless otherwise stated, the terms “individual” and “object” do not indicate a specific age and therefore include adults, older adults, children, and newborns. In embodiments of this disclosure, “individual” or “object” is a “patient.”

[0545] The term "patient" refers to an individual or object of treatment, especially an individual or object suffering from a disease.

[0546] The terms "disease involving an antigen," "disease involving cells expressing an antigen," or similar terms refer to any disease involving an antigen, such as a disease characterized by the presence of an antigen. A disease involving an antigen can be an infectious disease, a cancerous disease, or simply cancer. As mentioned above, the antigen can be a disease-associated antigen, such as a tumor-associated antigen, a viral antigen, or a bacterial antigen. In some embodiments, a disease involving an antigen is a disease involving cells that preferably express an antigen on their cell surface.

[0547] The term "infectious disease" refers to any illness that can be transmitted from one individual to another or from one organism to another and is caused by a microbial agent (such as HIV). Infectious diseases are 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 can be, for example, hepatitis, sexually transmitted diseases (such as chlamydia or gonorrhea), tuberculosis, HIV / Acquired Immunodeficiency Syndrome (AIDS), diphtheria, hepatitis B, hepatitis C, cholera, severe acute respiratory syndrome (SARS), avian influenza, and influenza.

[0548] The methods and medications described in this article are particularly useful for treating HIV infection.

[0549] The terms “cell-mediated immunity,” “cellular immunity,” “cellular immune response,” or similar terms are intended to include cellular responses against cells characterized by antigen expression, particularly those characterized by antigen presentation with class I or class II MHC. Cellular responses involve cells called T cells or T lymphocytes, which act as “helper cells” or “killer cells.” Helper T cells (also known as CD4+ T cells) play a crucial role in regulating the immune response and killing diseased cells (also known as cytotoxic T cells, cytolytic T cells, CD8+ T cells, or CTLs) to prevent the proliferation of more diseased cells.

[0550] The term "antigen-presenting cell" (APC) refers to a variety of cells capable of displaying, acquiring, and / or presenting at least one antigen or antigen fragment on their cell surface (or on their cell surface). Antigen-presenting cells can be classified into professional antigen-presenting cells and non-professional antigen-presenting cells.

[0551] The term "professional antigen-presenting cell" refers to antigen-presenting cells that constitutively express major histocompatibility complex II (MHC class II) molecules required for interaction with naïve T cells. If a T cell interacts with the MHC class II molecular complex on the antigen-presenting cell membrane, the antigen-presenting cell produces co-stimulatory molecules that induce T cell activation. Professional antigen-presenting cells include dendritic cells and macrophages.

[0552] The term "non-professional antigen-presenting cells" refers to antigen-presenting cells that do not constitutively express MHC class II molecules, but express them upon stimulation by certain cytokines such as interferon-γ. Exemplary non-professional antigen-presenting cells include fibroblasts, thymic epithelial cells, thyroid epithelial cells, glial cells, pancreatic β cells, or vascular endothelial cells.

[0553] "Antigen processing" refers to the degradation of an antigen into processed products, which are fragments of the antigen (e.g., proteins degraded into peptides), and one or more of these fragments (e.g., via binding) associate with MHC molecules in order to be presented by a cell (e.g., an antigen-presenting cell) to a specific T cell.

[0554] In some embodiments, the compositions, granules, or pharmaceutical compositions described herein may be administered intravenously, intra-arterially, subcutaneously, intradermally, intradermally, intranodally, intramuscularly, intratumorally, or peritumorally. In some embodiments, the compositions, granules, or pharmaceutical compositions described herein may be administered intramuscularly. In some embodiments, the compositions, granules, or pharmaceutical compositions are formulated for local or systemic administration. Systemic administration may include enteral administration, which involves absorption via the gastrointestinal tract, or parenteral administration. As used herein, “parenteral administration” means administration by any means other than the gastrointestinal tract, such as intravenous injection. In some embodiments, the compositions, granules, or pharmaceutical compositions are formulated for systemic administration. In some embodiments, systemic administration is via intravenous administration. In some embodiments, the compositions, granules, or pharmaceutical compositions are formulated for intramuscular administration.

[0555] The present invention has been described and illustrated in detail with reference to the accompanying drawings and embodiments, which are for illustrative purposes only and are not intended to be limiting. Further embodiments, also included in the invention, will be understood by those skilled in the art due to the description and embodiments.

[0556] sequence list Attached Figure Description

[0557] Figure 1 : Figure 1The image shows the string representations of two RNA molecules. The first RNA molecule (string 1) encodes an immunogenic peptide containing 33 segments, and the second RNA molecule (string 2) encodes an immunogenic peptide containing 32 segments. Each pair of segments is separated by up to four enhanced cleavage amino acids. The immunogenic peptide is approximately 550 amino acids in length.

[0558] Figure 2 : Figure 2 The string representations of two RNA molecules are shown. The first RNA molecule (string 1) encodes an immunogenic peptide containing 33 segments, and the second RNA molecule (string 2) encodes an immunogenic peptide containing 32 segments. Each pair of segments is separated by a 10-amino acid non-immunogenic linker, flanked by amino acids that enhance cleavage (up to 4). The immunogenic peptide is approximately 950 amino acids in length.

[0559] Figure 3 Amino acid changes between RNA strings 1 and 2 and RNA strings 1.1 and 2.1.

[0560] Figure 4 In vitro expression of mRNA-encoded peptides for two mRNA strings (string 1 non-GS, string 2 non-GS, and string 2 GS) with or without a flexible glycine-serine (GS) linker. For each string, three distinct nucleotide sequences (nt1, nt2, and nt3) were generated, 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 unmodified (uRNA) or methylpseuuridine (modRNA). For string 2 GS, only nt3 was tested. Each mRNA string, i.e., uRNA / modRNA, nt1-3, string 1-2, and GS / non-GS, was transfected in human cell lines with or without the proteasome inhibitor MG132. The relative abundance of each peptide in the following categories is shown: (A) two trypsin peptides common to each string tested with modRNA; (B) trypsin peptides specific to string 1 non-GS, modRNA, and uRNA; and (C) the relative abundance of each peptide in the 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 the peptides encoded by mRNA strings 1 (non-GS) (D) and 2 (non-GS and GS) (E). String-specific trypsin peptide locations for strings 1 (non-GS), TSTLQEQIGWAR (D), and strings 2 (GS and non-GS), QNYTPGPGVR (E) are also shown.

[0561] Figure 5 Immunopeptidomics was used to evaluate the in vitro processing and presentation of HLA-I alleles for mRNA-encoding peptides from two modRNA strings (string 1 and string 2). Each string was transfected in an A375 cell line encoding a single HLA class I allele, and targeted mass spectrometry analysis was performed to detect HIV-specific epitopes in string 1 (AB) and string 2 (CD). The locations of the detected epitopes are annotated below string 1 (A) and string 2 (C). Detailed information associated with each detected epitope, such as the location of the HLA class I allele, the fragment edge, and the sequence match between the non-HIV linker sequence (edge) and string 1, is shown in plot (B), and the corresponding information for string 2 is shown in plot (D). Legend: Epitope sequence perfectly matches 2021 common B = ConB; Epitope sequence perfectly matches 2021 common C = ConC; Epitope sequence perfectly matches both common B and C = ConB / C; Epitope does not perfectly match either common B or C = Polymorphism.

[0562] Figure 6 TCR engagement of epitopes processed and presented by different mRNA strings in the K562 cell line. Jurkat cell lines expressing a single TCR were co-incubated with K562 cells expressing a single HLA-I allele to measure downstream TCR signaling at a log2-fold signal for luciferase expression. K562 cells were pulsed with peptides encoded by the test string (PP) as a positive control to control TCR activity of the tested specific epitope or transfected with mRNA encoding the peptide (stripe column). 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 for their homologous TCR responses. Processing and presentation of the precise epitopes KF11 and IW9 were studied from different mRNA strings. The polymorphic version of DA9 encoded by string 2, labeled +DA9+, cross-reacted with non-homologous DA9-specific TCRs. Use A 11:01 Testing AK11, using A EL9 was tested at 26:01 using B. 57:01 Test KF11, using B DA9 was tested at 14:01, and the results were obtained via the homologous HLA-I allele B. 57:03 and non-homologous B 57:01 Alleles (marked as +B for both string 1 and string 2) 57:01 +) Presenting IW9. Text description: AK11: ACQGVGGPGHK; DA9: DRFYKTLRA; +DA9+: DRFFKTLRA; KF11: KAFSPEVIPMF; IW9: ISPRTLNAW.

[0563] Figure 7 TCR binding of epitopes processed and presented by different mRNA strings in iDCs generated from two donors. Jurkat cell lines expressing a single TCR were co-incubated with iDCs to measure downstream TCR signaling expressed as a log2-fold signal of luciferase. The iDCs were pulsed with peptides encoded by the test strings. To control TCR activity, the pulsed iDCs were pre-transfected with mRNA encoding the HLA class I allele (PP), or without co-transfection and expressing only endogenous HLA-I from the donor (PP + eHLA) to control potentially endogenous HLA-I-mediated background presentation. The iDCs were co-transfected with mRNA encoding the peptide and the homologous HLA class I allele (stripe column). 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), responses to six (AK11, DA9, +DA9+, KF11, EL9, and IW9) and five (AK11, DA9, +DA9+, EL9, and IW9) unique epitopes with their homologous TCRs were tested, respectively. The processing and presentation of epitope IW9 were investigated from two distinct mRNA strings (string 1 and string 2). The polymorphic version of DA9, labeled +DA9+ and encoded by the string 2 version, cross-reacted with non-homologous DA9-specific TCRs. Using A... 11:01 Testing AK11, using A EL9 was tested at 26:01 using B. 57:01 Test KF11, using B DA9 was tested at 14:01, and the results were obtained via the homologous HLA-1 allele B. 57:03 and non-homologous B 57:01 Alleles (marked as +B for both string 1 and string 2) 57:01+) Presented IW9. Text description: AK11: ACQGVGGPGHK; DA9: DRFYKTLRA; +DA9+: DRFFKTLRA; KF11: KAFSPEVIPMF; IW9: ISPRTLNAW; EL9: EVIPMFSAL.

[0564] Figure 8Responses of CD8+ and CD4+ T cells to two different mRNA strings (string 1 and string 2): Elispot data on IFN-γ production from isolated CD8+ T cells and isolated CD4+ T cells stimulated with a peptide pool. From HLA-A 02:01 T cells were isolated from CB6F1 transgenic mice and stimulated with a peptide pool for 18 hours on Elispot plates coated with anti-IFN-γ antibodies. CD8+ T cells from groups immunized with string 1 (A) or string 2 (B) (n = 5 mice / group) were incubated with an 11mer peptide pool covered with a vaccine fragment containing components from string A. 02:01 (white box - black circle) or at least one known / predicted HIV-1 epitope (proctrk < 1%) of H2b / H2d (striped box - white square) or neither (diamond box - white triangle). CD8+ T cells from groups immunized with string 1 (C) or string 2 (D) are then compared with heterologous minimal A. 02:01 (white box - black circle) or H2b / H2b (striped box - white square) HIV epitope pools (i.e., the smallest HIV epitopes (8-11-mer) containing at least one polymorphism compared to the immune string sequence) were incubated together. Pools I / K tested on the string 1 immune group and pools G / H tested on the string 2 immune group were exact matches of sequences found only in the corresponding strings 2 and 1. Isolated CD4+ T and CD8+ T cells (E) were incubated together with 15-mer peptide pools spanning the entire tetanus toxin. CD8+ and CD4+ T isolated cells from the designated immune groups were tested with an AH-1 unrelated 15-mer peptide pool negative control (F) and a concanavalin A positive control (G). Quantification of IFNg spot-forming units per 100,000 cells per peptide pool was performed. Graph (H) shows an overview of the experimental setup.

[0565] Figure 9 Responses of CD8+ and CD4+ T cells to two different mRNA strings (string 1 and string 2): Elispot data on IFN-γ production from isolated CD8+ T cells and isolated CD4+ T cells stimulated with a peptide pool. From HLA-A 02:01 T cells were isolated from CB6F1 transgenic mice and stimulated with a peptide pool for 18 hours on Elispot plates coated with anti-IFN-γ antibodies. CD8+ T cells were derived from groups immunized with a single mRNA (string 1 (10 µg) or string 2 (10 µg)) or co-immunized with two mRNAs (string 1 + string 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 alone or in combination (A, C) or with string 2 alone or in combination (B, D) were coated with a peptide pool containing anti-IFN-γ antibodies from A. 02:01 (white box - black circle) or H2b / H2d (striped box - white square) 11mer peptide pools (pools A, B, C and D, E, F respectively) containing at least one known / predicted HIV epitope (prctrk < 1%) or neither. 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 (8-11-mer) are incubated together, said epitope pools being encoded only by string 1 (pools G and H) or string 2 (pools I / K). Similarly, CD4+ T cells from groups immunized with string 1 (C) or string 2 (D) are incubated with minimal A 02:01 (white box - black circle) or H2b / H2b (striped box - white square) HIV epitope pools (8-11-mer) were incubated together. Isolated CD4+ T and CD8+ T cells (E) were incubated together with 15-mer peptide pools spanning the entire tetanus toxin. CD8+ and CD4+ T cells isolated from designated immunization groups were tested with an AH-1 unrelated 15-mer peptide pool negative control (F) and a concanavalin A positive control (G). Quantification of IFNg spot-forming units per 100,000 cells per peptide pool was performed. Graph (H) shows an overview of the experimental setup.

[0566] Figure 10TCR binding of epitopes processed and presented by different mRNA strings in the K562 cell line: Jurkat cell lines expressing a single TCR were co-incubated with a single HLA-I allele, K562, to measure downstream TCR signaling expressed as a log2-fold signal of luciferase. K562 was pulsed with a peptide encoded by the test string (PP) as a positive control to control TCR activity of the specific epitope being tested, or transfected with the mRNA sequence encoding the peptide. PHA-L was used as a positive control for TCR activity. Five unique epitopes and their homologous TCR responses were tested. Four different mRNA strings with different amino acid sequences were tested, each with two nucleotide sequence optimizations (nt2 and nt3). String 1 and its derivative, String 1.1 (with four point mutations), were tested in K562 cells transfected with 3 μg (A) or 0.3 μg (B) of methylpseudouridine mRNA. All displayed epitopes were perfectly matched in the sequences of these mRNA strings. String 2 and its derivative String 2.1 (1 point mutation) were tested in K562 cells by transfecting cells with 3 μg (C) or 0.3 μg (D) of methylpseudo-uridine mRNA. Using A 11:01 Testing AK11, using A EL9 was tested at 26:01 using B. 57:01 Test KF11, using B DA9 was tested at 14:01 using the HLA-I allele B. 57:03 Test IW9. Compared with the sequences of these mRNA strings, use a star-shaped ( The epitopes marked with ) contain at least one polymorphism.

[0567] Figure 11TCR binding of epitopes processed and presented by different mRNA strings in the K562 cell line in iDCs generated from two donors: Jurkat cell lines expressing a single TCR effector were co-incubated with iDCs transfected with the target single HLA-I allele K562 or HLA-I from the donor to measure downstream TCR signaling expressed as a log2-fold signal of luciferase. Target cells were pulsed with a peptide encoded by a test string (PP) as a positive control to control TCR activity of the specific epitope being tested, or transfected with an mRNA string encoding the peptide. Pulsed peptides without co-transfection with the HLA allele were also used to control background presentation of endogenous HLA-I from the donor (PP 10µM + eHLA) to test target iDCs. PHA-L was used as a positive control for TCR activity. Three unique epitopes and their homologous TCR responses were tested. Four different mRNA strings with different amino acid sequences were tested, each with two nucleotide sequence optimizations (nt2 and nt3). Strings 1 and 2 were tested by transfecting cells with 3 μg of methylpseudo-uridine mRNA in target K562 cells (A) or target iDCs (B, C) from two donors. The TL9 and RI8 epitopes tested were both encoded by strings 1 and 2, with complete sequence matches. In parallel, the epitope TW10 encoded by string 2 contained a single mutant polymorphism, while also perfectly matching string 1. TL9 was tested using the HLA allele B. 39:10 test, E18 uses HLA allele B 52:01 test, TW10 uses HLA allele B 57:01, B 57:03 and B Test at 58:01.

[0568] Figure 12 Responses of CD8+ and CD4+ T cells to two different mRNA strings (string 1 and string 2) via intramuscular lipid nanoparticle (LNP) nanocarriers: Elispot data on IFN-γ production from CD8+ T cell knockout splenocytes stimulated with peptide pools and CD4+ T cell knockout splenocytes. From HLA-A 02:01 Spleen cells were isolated from CB6F1 transgenic mice and stimulated with a peptide pool for 18 hours on Elispot plates coated with anti-IFN-γ antibody. CD4+ T cell knockout spleen cells and CD8+ T cell knockout spleen cells were generated from animal groups (n=5 mice / group) that were co-immunized with a 1:1 ratio (2.5 μg + 2.5 μg) of a single LNP-prepared mRNA string 1 + string 2 combination or received a saline placebo. The mRNA combination was tested using string 1 + string 2 as unmodified (uRNA) or methyl pseudouridine (modRNA). Animals were immunized with a single intramuscular injection, and spleen cells were harvested 7 days later. CD4+ T cell knockout spleen cells from animals immunized with the string 1 + string 2 modRNA or uRNA combination ( Figure 12 A) or CD8+ T cell knockout spleen cells ( Figure 12 B) Incubate with a pool of 11mer peptides covering the vaccine fragment, said vaccine fragment containing peptides from A 02:01 (white box - black circle) or at least one known / predicted HIV epitope (prctrk < 1%) of H2b / H2d (striped box - white square) or neither (diamond box - white triangle). Spleen cells knocked out by CD4+ T cells and spleen cells knocked out by CD8+ T cells were incubated with a pool of 15-mer peptides spanning the entire tetanus toxin. Figure 12 C). Background IFN-γ signal from CD4+ T cell knockout splenocytes and CD8+ T cell knockout splenocytes incubated with DMSO peptide dilution control was indicated as the median signal measured in all groups tested in plot (A) or (B) – “median DMSO”. IFN-γ spot-forming units per 10e6 cells per peptide pool were quantified. Figure 12 D shows a summary of the experiment overview.

[0569] Example

[0570] Example 1

[0571] HLA-I restrictive epitopes have been selected to improve coverage and evade mutation restrictions. As described below, these groups of CD8+ T cell epitopes have been assembled into multi-epitope cassettes to produce a vaccine.

[0572] A list of highly networked epitopes on CD8+ T cells has been compiled, providing broad coverage based on HLA-I class allele prevalence; this list will be used to design a rational RNA vaccine. We will use Gaiha... et al.Structural topology defines protective CD8+ T cell epitopes in the HIVproteome. A highly networked scoring method described in Science 364, 480-484 (2019) generates an initial list of epitopes of interest based on their potentially high mutation thresholds and publicly available data indicative of human immunogenicity. We evaluated their binding using an internal pMHC prediction tool and performed pMHC binding assays on epitope-MHC pairs that failed internal checks. We identified the sequence of each epitope in the HIV Clades B and C consensus 2021 and evaluated conservation. For each amino acid within epitopes with less than 75% conservation of patient sequences (according to the Los Alamos HIV database), we considered adding epitopes with polymorphic sequences to achieve coverage of most patients. Epitopes were organized and processed into fragments distributed on two different RNAs. Fragments were separated by supplementing residues using two linker methods, resulting in two sets of two RNAs.

[0573] Each epitope has been matched with the HLA class I alleles to which it can bind, and their amino acid sequences are defined in the context shared by HIV clades B and C, and the most frequent polymorphisms are screened within the clade; see Table 1 below.

[0574] 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 restriction were selected. After multiple rounds of selection, 70 epitopes were ranked first, with network scores above the selection threshold and literature-defined proven human immunogenicity and their HLA-I class-I binding couples.

[0575] Table 1: Highly networked epitopes on CD8+ T cells from HIV

[0576] Epitope-allele pairs reported in LADB were collected from numerous studies, including further evaluations. We identified corresponding sequences from clades B and C 2021 of LADB. If the LADB epitope sequence differed from clades B / C, it was discarded, and only the shared sequence was retained. To further validate published information on epitope / HLA interactions, we used a peptide-MHC binding prediction tool. Our analysis showed that approximately 40% of published epitope-allele pairs were predicted as non-binding. Among these pairs, we aimed to examine epitope binding where the prevalence of HLA-I class-I mating alleles was observed to be >2% in Caucasian European or Southern African populations.

[0577] To check binding, we used a pMHC stability assay. Because the MHC monomers of some alleles were unavailable, not all required epitope-allele pairs could be tested. Of the available alleles, 18 epitope / allele pairs were identified as binding pairs and retained, while 37 epitope / allele pairs were non-binding pairs and excluded from vaccine design. Epitope / allele pairs for which binding could not be checked remained in the vaccine but were not used for coverage calculations.

[0578] The selected epitopes were structurally important and should be enriched in conserved regions of the HIV proteome. To verify this and ensure that no relevant polymorphisms were missing, we examined the percentage of patient sequences matching conserved sequences in clades B and C by comparing sequence data from proteins of interest (Gag / Pol / Env / Nef) from LADB. Each amino acid within each epitope with a match less than 75% was added to the most prevalent polymorphic version of the epitope. A total of 17 polymorphic fragments were added to the vaccine.

[0579] Overlapping epitopes were combined and incorporated into peptide fragments to reduce the number of required linkers and decrease the overall RNA cassette size. Fragments were designed to maximally remove predicted HIV-specific class II core epitopes to limit unwanted activation of HIV-specific CD4 responses and minimize coding sequence length.

[0580] We distributed epitopes and fragments between two coding RNA molecules, where epitopes were combined into 26 fragments (out of a total of 52) represented in the clade B and clade C shared sequences, adding 17 polymorphic fragments. Four of the polymorphic fragments matched the same clade B / C shared sequences. To reduce redundancy and limit the final RNA template size, we swapped the shared fragment from clade C with its polymorphic version. The vaccine is bivalent and contains two RNAs: one matching the clade B shared sequence (26 fragments) and the second matching the clade C shared sequence (26 fragments plus 4 polymorphic fragments). We distributed the remaining 13 polymorphic fragments across the two RNA strings, 7 on the clade B RNA string and 6 on the clade C RNA string. In our final design, RNA string 1 (“clade B string”) comprises 33 segments with a cumulative length of 411 amino acids, while string 2 (“clade C string”) comprises 32 segments with a total length of 405 amino acids, as shown below. Figure 1 and 2 As shown.

[0581] String 1 (“clade B string”) (SEQ ID NO: 183 and 187) contains the following 33 fragments, which contain 1, 2, 3, 4, 5, 6, and 7 of the above epitopes 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, FR DYVDRFYKTLRA, AEQASQDVKNWM, RAIEAQQHL, VRMYSPTSILDI, PIQKETWEAWWTEYW, HTQGYFPDWQNYT, EIYKRWIILGLNK, GLNKIVRMY, TSTLQEQI GW, PIQKEIWETWWTDYW, GQMVHQPLSPRTLNAW, YTPGPGTRYPLTFGW, GQMVHQAISPRTLNAWVKVV, TPGIRYQYNVL, ACQGVGGPGHKARVL and GIPPHPAGLK.

[0582] String 2 (“clade C string”) (SEQ ID NO: 185 and 189) contains the following 32 fragments, which contain 1, 2, 3, 4, 5, 6, and 7 of the above epitopes from the pol, gag, env, and nef proteins: IVTDSQYAL,AEQATQEVKNWM,TSTLQEQIAW,GIPHPAGLK,VRMYSPVSILDI,GLNKIVRMY,GHQAAMQMLKETI,AKNPEIVIY,GQMVHQALSPRTLNAWVK VI, TPGIRYQYNVL, AEQATQDVKNWM, SFNCRGEFF, YTPGPGVRFPLTFGWCF, AQNPEIVIYQYMDDL, QNYTPGPGVRYPLTFGWCFKL, FKRKGGIGGY, NTQG YFPDW, HTQGFFPDWQNYT, PIQKETWETWWTDYW, GQMVHQPISPRTLNAW, GERIIDIIA, LIGPTPVNIIGRNML, DCKTILRAL, VKVIEEKAFSPEVIPMFTAL , PIQKEIWETWWTEYW, FRDYVDRFFKTLRA, RAIEAQQHM, TALSEGATPQDLNTMLNT, DIYKRWIILGLNK, KLTPLCVTL, ACQGVGGPSHKARVL and TAFTIPST.

[0583] String 1.1 (“clade B string”) (SEQ ID NO: 205) contains the following 33 fragments, which contain 1, 2, 3, 4, 5, 6, and 7 of the above epitopes 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, FR DYVDRFYKTLRA, AEQASQDVKNWM, RAIEAQQHL, VRMYSPTSILDI, PIQKETWEAWWTEYW, HTQGYFPDWQNYT, EIYKRWIILGLNK, GLNKIVRMY, TSTLQEQI GW, PIQKETWEIWWTDYW, GQMVHQPLSPRTLNAW, YTPGPGTRYPLTFGW, GQMVHQAISPRTLNAWVKVV, TPGIRYQYNVL, ACQGVGGPGHKARVL and GIPPHPAGLK.

[0584] String 2.1 (“clade C string”) (SEQ ID NO: 208) contains the following 32 fragments, which contain 1, 2, 3, 4, 5, 6, and 7 of the above epitopes from the pol, gag, env, and nef proteins: IVTDSQYAL,AEQATQEVKNWM,TSTLQEQIAW,GIPHPAGLK,VRMYSPVSILDI,GLNKIVRMY,GHQAAMQMLKETI,AKNPEIVIY,GQMVHQALSPRTLNAWVK VI, TPGIRYQYNVL, AEQATQDVKNWM, SFNCRGEFF, YTPGPGVRFPLTFGWCF, AQNPEIVIYQYMDDL, QNYTPGPGVRYPLTFGWCFKL, FKRKGGIGGY, NTQG YFPDW, HTQGFFPDWQNYT, PIQKETWETWWTDYW, GQMVHQPISPRTLNAW, GERIIDIIA, LIGPTPVNIIGRNML, DCKTILRAL, VKVIEEKAFSPEVIPMFTAL , PIQKETWETWWTEYW, FRDYVDRFFKTLRA, RAIEAQQHM, TALSEGATPQDLNTMLNT, DIYKRWIILGLNK, KLTPLCVTL, ACQGVGGPSHKARVL and TAFTIPST.

[0585] We supplement each string with additional non-human sequence domains enriched in universal non-HIV class II epitopes to induce CD4+ T cells that can help activate CD8+ T cells.

[0586] To concatenate the segments in each string, two strategies are employed: 1) An internal algorithm determines whether to directly add up to four amino acids to enhance the cut between every two segments. In this strategy, the final string length is approximately 550 amino acids. Figure 1 ) 2) Flexible, non-immunogenic linkers (10 amino acids) flanked by enhanced cleavage amino acids (up to 4) between each pair of segments. In this strategy, the final string length is approximately 950 amino acids. Figure 2 ) like Figure 3 As shown, mRNA strings 1 and 2 differ from mRNA strings 1.1 and 2.1 in that they have very few amino acid changes.

[0587] An algorithm with an additional parameter selects complementary residues to enhance the cleavage: the selected residues must not match natural flanking residues present in the shared B or shared C sequences. This prevents our fragments from extending to non-networked residues and avoids the addition of unwanted non-networked HIV class I epitopes or complementary HIV class II epitopes.

[0588] Fragments were randomized on each RNA, with different orders for string 1 and string 2, to improve downstream RNA analysis and the repetition of linker epitopes.

[0589] Example 2 - Mass Spectrometry Proteomics Analysis

[0590] In short, 5 × 10 mRNAs encoding strings 1 and 2 were transfected with MessengerMax Lipofectamine reagent. 6 HEK293T cells were incubated for 24 hours with or without 1 μM proteasome inhibitor MG132. 24 hours post-transfection, cells were harvested, washed in 1 mL PBS, flash-frozen, and stored until analysis. The frozen cell pellet was thawed and 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 lysate was diluted to reduce the urea content in the lysis buffer to 1 μM. Trypsin / Lys-C was added at a ratio of 1 μg per 50 μg of total protein, and the sample was 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 the incorporated heavy isotope-labeled synthetic peptides. The relative abundance of each peptide was calculated by measuring the area under the curve (AUC) of the top ten most abundant fragment ions. Loading normalization was calculated using a group of peptides derived from housekeeping proteins with constant intracellular abundance. The AUC of each housekeeping peptide was normalized to the mean AUC of the samples, and the median of the mean AUC for each housekeeping protein group was calculated. 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 mean AUC of the samples.

[0591] Figure 4The in vitro expression of mRNA-encoded peptides for two mRNA strings (string 1 non-GS, string 2 non-GS, and string 2 GS) with or without the flexible glycine-serine (GS) linker is shown. For each string, three distinct nucleotide sequences (nt1, nt2, and nt3) were generated, 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 unmodified (uRNA) or methylpseuuridine (modRNA). For string 2 GS, only nt3 was tested. Each mRNA string, i.e., uRNA / modRNA, nt1-3, string 1-2, and GS / non-GS, was transfected in human cell lines with or without the proteasome inhibitor MG132. The relative abundance of each peptide in the following categories is shown: (A) two trypsin peptides common to each string tested with modRNA; (B) trypsin peptides specific to string 1 non-GS, modRNA, and uRNA; and (C) the relative abundance of each peptide in the 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 the peptides encoded by mRNA strings 1 (non-GS) (D) and 2 (non-GS and GS) (E). String-specific trypsin peptide locations for strings 1 (non-GS), TSTLQEQIGWAR (D), and strings 2 (GS and non-GS), QNYTPGPGVR (E) are also shown.

[0592] Overall, the experiments showed that modRNA achieved higher in vitro expression levels compared to uRNA. Detection of the IYSYFPSVISK trypsin peptide revealed that its different strings and codon-optimized versions were translated into the C-terminal region of the encoded polypeptide. Codon optimization affected the translation levels from different strings and appeared to be sequence-dependent. The presence of the proteasome inhibitor MG132 led to an increase in detectable polypeptide, indicating the proteasome degradation pathway required for epitope processing of the translated polypeptide.

[0593] Example 3 - Immunopeptidomics

[0594] A375 cells were engineered to stably express the target allele labeled with BAP, or the target allele was overexpressed intracellularly and used for transfection. Five × 10⁷ engineered cells were transfected with 1 μg mRNA encoding modRNA strings 1 and 2 and Messenger Max Lipofectamine reagent before harvest. Transfected cells were lysed and removed before treatment. For BAP-labeled cell lines, the clarified lysate was biotinylated with biotin, ATP, and BirA before incubation with NeutrAvidin beads to affinity enrich the biotinylated HLA-peptide complex. For overexpression cell lines, agarose beads were loaded with pan-I class antibody and incubated with the clarified lysate to isolate all HLA-peptide complexes. Peptides were washed and eluted from the antibody-bound HLA complexes and molecular weight filtration was performed to separate the peptides. The separated peptides were then labeled with TMTzero, reduced with TCEP, alkylated with iodoacetamide (IAA), and desalted before analysis with nLC-MS / MS. The sample was resuspended in 3% acetonitrile and 5% formic acid, with 150 femtomoles added per injection for each TMT-131C-labeled resynthetic peptide. The peptides were separated using Vanquish Neo uHPLC with an Aurora Ultimate packed emission column and heated at 60°C during separation. The peptides were eluted into an Orbitrap Ascend Tribrid mass spectrometer equipped with a nanospray FlexIon source. Data were obtained using parallel reaction monitoring triggered by an internal standard. A rapid, low-resolution preliminary scan was used to search for m / z values ​​in the inclusion list associated with the TMT-131C-labeled resynthetic peptide internal standard. When an m / z value was observed in the inclusion list, a rapid, low-resolution tandem mass spectrometry (MS / MS) investigation scan was performed, monitoring for 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 shift equal to the difference between the TMT-131C-labeled resynthetic peptide and the TMTzero-labeled target HLA peptide.

[0595] Data analysis was performed using Skyline-daily software. Retention times and peptide fragments were identified by matching them with synthetic peptides doped with heavy isotope-labeled peptides. The relative abundance of each peptide was calculated by measuring the AUC of the top ten most abundant fragment ions.

[0596] Figure 5This paper demonstrates the in vitro processing and presentation of HLA-I alleles encoding peptides from two modRNA strings (string 1 and string 2) using immunopeptidomics. Each string was transfected in an A375 cell line encoding a single HLA class I allele, and targeted mass spectrometry analysis was performed to detect HIV-specific epitopes in string 1 (AB) and string 2 (CD). The locations of the detected epitopes are annotated below string 1 (A) and string 2 (C). Detailed information associated with each detected epitope, such as the HLA class I allele, the location of the fragment edge, and the sequence match between the non-HIV linker sequence (edge) and string 1, is shown in plot (B), and the corresponding information for string 2 is shown in plot (D). Legend: Epitope sequence perfectly matches 2021 common B = ConB; Epitope sequence perfectly matches 2021 common C = ConC; Epitope sequence perfectly matches both common B and C = ConB / C; Epitope does not perfectly match either common B or C = Polymorphism.

[0597] These datasets confirm the processing and presentation of epitopes from mRNA-encoded peptides on human MHC class I alleles. Detection of peptide cleavage between fragments in non-HIV junction regions was confirmed by pulling down epitopes located at fragment edges.

[0598] Example 4 - T cell activation assay using K562

[0599] NFAT-TCR / CD3 effector cells were purchased from Promega as cryopreserved cells. These Jurkat T cells expressed luciferase as a reporter, driven by the NFAT response element (NFAT-RE). Endogenous TCR and β2M- genes had been removed from Jurkat reporter cells via CRISPR-Casp9-mediated knockout. The α and β chains of the CD8-co-receptor were stably inserted into Jurkat reporter cells via transposon. Reporter NFAT-luciferase cells were co-electroplated with two mRNAs encoding the α and β chains of the TCR clone. After transfection, 2 × 10⁴ Jurkat cells were co-cultured with K562 cells at a 2:1 ratio in 384-well plates containing 25 μL of medium (RPMI 1640 + 10% non-thermally inactivated FBS) per 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 specific to the TCRs used, K562 cells transfected with only HLA-I-encoding mRNA pulsed with minimal HIV-epitope target were co-cultured with Jurkat reporter cells transfected with mRNA encoding each TCR. Furthermore, stimulation with 2 μg / ml phytohemagglutinin-L (PHA-L) was used to confirm TCR expression and downstream signaling. Transient expression of transfected HLA-I classes was confirmed by flow cytometry after staining with HLA-A or HLA-B specific antibodies. Sixteen hours later, 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. The luminescence signals measured in different wells corresponded to the TCR-mediated activation levels in Jurkat cells. For each TCR, the log2-fold change in luminescence compared to the "effecton-only control" was calculated, and a cutoff value of two-fold change was used to determine the specific TCR.

[0600] Figure 6This study demonstrates TCR binding to epitopes processed and presented by different mRNA strings in the K562 cell line. Jurkat cell lines expressing a single TCR were co-incubated with K562 cells expressing a single HLA-I allele to measure downstream TCR signaling at a log2-fold signal as luciferase. K562 cells were pulsed with peptides encoded by the tested string (PP) as a positive control to control TCR activity for the tested specific epitope, or transfected with mRNA encoding the peptide (striped column). 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. The %unique epitopes AK11, DA9, +DA9+, KF11, and IW9 were tested for their homologous TCR responses. Processing and presentation of the precise epitopes KF11 and IW9 were investigated from different mRNA strings. The polymorphic version of DA9, labeled +DA9+ and encoded by string 2, cross-reacted with non-homologous DA9-specific TCRs. Similarly, through the homologous HLA-I allele B 57:03 and non-homologous B 57:01 Alleles (marked as +B for both string 1 and string 2) 57:01 +) Presenting IW9. Text description: AK11: ACQGVGGPGHK; DA9: DRFYKTLRA; +DA9+: DRFFKTLRA; KF11: KAFSPEVIPMF; IW9: ISPRTLNAW.

[0601] These datasets confirm that all mRNA-string-encoded peptides can be enzymatically processed to produce epitopes, which are then presented on their homologous HLA class I alleles. Epitopes: The HLA-I complex can participate in downstream TCR signaling in vitro. These data also show the impact of nucleotide sequence optimization on the amplitude of TCR-mediated responses.

[0602] Example 5 - Assay of T cell activation using iDC

[0603] Cells were used and prepared as described in Example 4. Following transfection, 2 × 10⁴ Jurkat cells were co-cultured with immature dendritic cells (iDCs) at a 2:1 ratio in 384-well plates containing 25 μL of medium (RPMI 1640 + 10% non-heat-inactivated FBS) per well. Prior to co-culture, iDCs were generated from donor PBMCs. Briefly, CD14+ monocytes were positively isolated from human PBMCs and cultured at 1 × 10⁶ cells / mL for 5 days in RPMI 1640 7 / 5% pooled human serum (PHS) / 1% sodium pyruvate / 0.5% penicillin-streptomycin (supplemented with 1000 U / mL IL-4 and 1600 U 7mL GM-CSF) to generate iDCs. The iDCs were then transfected with mRNA encoding HIV-derived peptides (strings 1 and 2) and mRNA encoding HLA class alleles before co-incubation with Jurkat reporter cells. As a positive control for TCR specificity, iDCs transfected with only HLA-I-encoding mRNA pulsed with minimal HIV-epitope peptide targets were co-cultured with Jurkat reporter cells transfected with mRNA encoding each TCR. To evaluate the effect of endogenous HLA-I alleles from PBMC donors, a control including iDCs pulsed only with peptides was included. Furthermore, stimulation with 2 μg / ml phytohemagglutinin-L (PHA-L) was used to confirm TCR expression and downstream signaling. 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. The luminescence signals measured in different wells corresponded to the TCR-mediated activation levels in Jurkat cells. For each TCR, the log2-fold change in luminescence compared to the "effecton-only control" was calculated, and a cutoff value of two-fold change was used to determine the specific TCR.

[0604] Figure 7This study demonstrates TCR binding to epitopes processed and presented by different mRNA strings in iDCs generated from two donors. Jurkat cell lines expressing a single TCR were co-incubated with iDCs to measure downstream TCR signaling at a log2-fold signal equivalent to luciferase. The iDCs were pulsed with peptides encoded by the tested strings. To control TCR activity, the pulsed iDCs were pre-transfected with mRNA encoding the HLA class I allele (PP), or without co-transfection and expressing only endogenous HLA-I from the donor (PP + eHLA) to control potentially endogenous HLA-I-mediated background presentation. The iDCs were co-transfected with mRNA encoding the peptide and the homologous HLA class I allele (striped column). 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), responses of six (AK11, DA9, +DA9+, KF11, EL9, and IW9) and five (AK11, DA9, +DA9+, EL9, and IW9) unique epitopes to their homologous TCRs were tested, respectively. The processing and presentation of epitope IW9 were investigated from two different mRNA strings (string 1 and string 2). The polymorphic version of DA9, labeled +DA9+ and encoded by the string 2 version, cross-reacted with non-homologous DA9-specific TCRs. Similarly, responses were also investigated via the homologous HLA-1 allele B. 57:03 and non-homologous B 57:01 Alleles (marked as +B for both string 1 and string 2) 57:01+) Presented IW9. Text description: AK11: ACQGVGGPGHK; DA9: DRFYKTLRA; +DA9+: DRFFKTLRA; KF11: KAFSPEVIPMF; IW9: ISPRTLNAW; EL9: EVIPMFSAL.

[0605] These datasets confirm that all mRNA-string-encoded peptides can be enzymatically processed to produce epitopes, which are then presented on their homologous HLA class I alleles in PBMC-derived human iDCs. Epitopes: The HLA-I complex can participate in downstream TCR signaling in vitro. These data also show the impact of nucleotide sequence optimization on the amplitude of TCR-mediated responses.

[0606] Example 6

[0607] use Figure 1 and Figure 2The RNA molecules shown were used in immunogenicity studies in BALB-C (wild-type) and HLA knock-in mice. For this purpose, lipid-containing compositions for intramuscular (im) or intravenous (iv) delivery were combined with the RNA molecules.

[0608] Vaccination of mice

[0609] Wild-type mice: Wild-type mice were inoculated with the lipid nanoparticle formulation (IM) or the lipoplex formulation (IV). Wild-type mice received the vaccine, either co-formulated with 2–4 RNAs or single-formulated with 2–4 RNAs, at up to 3 doses (low, medium, and high) following different primary / booster regimens (i.e., the time between injections and booster doses). Vaccine-induced CD8+ T cells were quantified from spleen cells and whole blood via the peptide pool ELISPOT after culling. Drained lymph nodes were harvested for immunostaining of relevant markers. Cytokine levels in the blood were measured by multiplex ELISA during the experiment. The magnitude of HIV-specific CD8+ T cells was compared across regimens, doses, formulations (if applicable), and RNA platforms (if applicable) to determine the optimal vaccination schedule for both injection routes.

[0610] HLA knock-in (HLA KI) mice: HLAKI mice were inoculated with a lipid nanoparticle formulation im or a lipoplex formulation iv. HLA groups were engineered to cover relevant HLA-I alleles that were highly prevalent in the population or associated with negative / positive outcomes during HIV infection. HLA KI mice received a vaccine according to the optimal regimen defined for wild-type mice for each RNA platform / lipoplex formulation. Vaccine-induced CD8+ T cells were quantified from spleen cells and whole blood via ELISPOT using a specific peptide of the HLA-I under investigation. Drained lymph nodes were harvested for immunostaining of relevant markers. These experiments confirmed the correct presentation and immunogenicity of the selected epitopes.

[0611] Example 7

[0612] Sample preparation and immunization

[0613] Endotoxin levels were assessed using mRNA generated via in vitro transcription and formulated into lipoplex lipid nanocarriers. Each mRNA was individually prepared 24 h prior to injection at concentrations of 0.1 mg / mL, 0.2 mg / mL, or 0.25 mg / mL and maintained at 4°C. On the day of immunization, the particles were gently resuspended and, when applicable, diluted in PBS 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 via tail vein injection (29G needle) on days 0 (primary), 7 (booster), and 14 (booster). Six hours post-immunization, blood samples were collected from each animal and stored at -80°C. On day 21, one week after the final booster, animals were sacrificed and their spleens were recovered for immediate isolation of CD4+ and CD8+ T cells. Animal health was monitored throughout the experiment until sacrifice.

[0614] T cell isolation and ELISpot

[0615] Freshly recovered spleen cells were treated using negative CD4- or CD8- isolation kits (Miltenyi biotec Cat 130-104-454 and 130-104-075). Briefly, 10e7 cells were incubated with a biotinylate antibody mixture at 4°C for 5 minutes. The antibody / cell mixture was then incubated with anti-biotin microbeads (2 µl / 10e6 cells) at 4°C for 10 minutes. Unlabeled CD4+ or CD8+ T cells were separated from labeled cells via magnetic separation by recovering the flowthrough of the cell suspension. Cells were immediately used for IFNg-ELISpot assays. 5.10e4 BMDCs from CB6F1 were pulsed at 37°C for 2 hours with a 2-6 μg / mL peptide test or control, followed by overnight co-incubation with 1.10e5 isolated T cells on an anti-IFNg coated plate (Mabtech 321-4HPW-2). The spots were detected using a biotinylated anti-mouse IFNg detection antibody, followed by streptavidin-HRP incubation.

[0616] CD8+ and CD4+ T cell responses to two different mRNA strings (string 1 and string 2)

[0617] Elispot data generated from IFN-γ produced by isolated CD8+ T cells and isolated CD4+ T cells stimulated with a peptide pool. From HLA-A 02:01 T cells were isolated from CB6F1 transgenic mice, and the T cells were stimulated with a peptide pool for 18 hours on an Elispot plate coated with anti-IFN-γ antibody. The T cells were then processed using string 1 ( Figure 8 A) or string 2 ( Figure 8 B) CD8+ T cells from the immunized group (n = 5 mice / group) were incubated together with a pool of 11mer peptides covering a vaccine fragment containing components from A 02:01 (white box - black circle) or at least one known / predicted HIV-1 epitope (proctrk < 1%) or H2b / H2d (striped box - white square) or neither (diamond box - white triangle). This will be derived from string 1 ( Figure 8 C) or string 2 ( Figure 8 D) CD8+ T cells in the immune group and the heterologous minimum A 02:01 (white box - black circle) or H2b / H2b (striped box - white square) HIV epitope pools (i.e., the smallest HIV epitopes (8-11-mer) containing at least one polymorphism compared to the immune string sequence) are incubated together. Pool I / K tested on string 1 immune group and pool G / H tested on string 2 immune group are exact matches of sequences found only in the corresponding strings 2 and 1. Isolated CD4+ T and CD8+ T cells ( Figure 8 E) Incubate with a pool of 15-mer peptides spanning the entire tetanus toxin. Use an AH-1-independent 15-mer peptide pool as a negative control. Figure 8 F) and concanavalin A positive control ( Figure 8 G) Test CD8+ and CD4+ T cells isolated from the specified immune group. Quantification of IFNg spot-forming units per 100,000 cells per peptide pool. Figure 8 H shows an overview of the experimental setup.

[0618] Regardless of capping or sequence, all tested strings were immunogenic in the CB6F1 mouse model. The mRNA strings used for immunization elicited responses from mouse MHC-I and human HLA-1 alleles A. 02:01 Restricted HIV-specific CD8+ T cell response ( Figure 8 A and B). Using a heterologous minimal peptide pool for CD8+ T cell stimulation showed that T cells generated by mRNA strings cross-reacted with polymorphic versions of the epitopes they encode. Figure 8 C and D). A specific 15-mer tetanus toxin peptide pool has been used to validate the use of the P2P16 domain in the sequence to support HIV CD8+ T cells by binding to non-HIV-specific CD4+ T cells. Figure 8 E). The tetanus toxin (TT) peptide pool did not appear to elicit a detectable TT-specific CD8+ T cell response, limiting competition between HIV- and TT-specific CD8+ T cells. Figure 8 E).

[0619] Example 8

[0620] Sample preparation and immunization, as well as T cell isolation and ELISpot method, were performed as in Example 7.

[0621] Responses of CD8+ and CD4+ T cells to two different mRNA strings (string 1 and string 2): Elispot data generated by IFN-γ from isolated CD8+ T cells and isolated CD4+ T cells stimulated with a peptide pool. From HLA-A T cells were isolated from CB6F1 transgenic mice at 02:01 and stimulated with a peptide pool for 18 hours on Elispot plates coated with anti-IFN-γ antibodies. CD8+ T cells were derived from groups immunized with a single mRNA (string 1 (10 µg) or string 2 (10 µg)) or co-immunized with two mRNAs (string 1 + string 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 alone or in combination ( Figure 9 A, Figure 9 C) or animals immunized with string 2 alone or in combination ( Figure 9 B. Figure 9 D) and the coverage contains elements from A 02:01 (white box - black circle) or H2b / H2d (striped box - white square) 11mer peptide pools (pools A, B, C and D, E, F respectively) containing at least one known / predicted HIV epitope (prctrk < 1%) or neither of these, and vaccine fragments. From string 1 ( Figure 9 C) or string 2 ( Figure 9 D) The CD8+ T cells of the immune group also showed similar activity to the minimum A. 02:01 (white box - black circle) or H2b / H2b (striped box - white square) HIV epitope pools (8-11-mer) are incubated together, said epitope pools being encoded only by string 1 (pools G and H) or string 2 (pools I / K). Similarly, CD4+ T cells from groups immunized with string 1 (C) or string 2 (D) are incubated with minimal A 02:01 (white box - black circle) or H2b / H2b (striped box - white square) HIV epitope pool (8-11-mer) incubated together. Isolated CD4+ T and CD8+ T cells ( Figure 9 E) Incubate with a pool of 15-mer peptides spanning the entire tetanus toxin. Use an AH-1-independent 15-mer peptide pool as a negative control. Figure 9 F) and concanavalin A positive control ( Figure 9G) Test CD8+ and CD4+ T cells isolated from the specified immune group. Quantification of IFNg spot-forming units per 100,000 cells per peptide pool. Figure 9 H shows an overview of the experimental setup.

[0622] Immunization with a single mRNA string and a bivalent combination vaccine (string 1 + string 2) was immunogenic in the CB6F1 model. The single or combined mRNA strings evoked responses influenced by mouse MHC-I and human HLA-1 alleles A. 02:01 Restricted HIV-specific CD8+ T cell response ( Figure 9 (C and D). Responses to the pool of unsaturated HIV minimal epitope peptides (pools G, H, I, and K) showed a dose-response to the bivalent vaccine. In contrast, responses to A... 02:01 and H2b / d MHC-I allele-restricted low / intermediate levels of HIV-specific CD4+ T cell responses ( Figure 9 C and D) are vaccine-induced, ensuring the inclusion of the P2P6 tetanus toxin helper epitope. A specific 15-mer tetanus toxin peptide pool has been used to validate the use of the P2P16 domain in the sequence to support HIV CD8+ T cells by binding to non-HIV-specific CD4+ T cells. Figure 9 E). The tetanus toxin (TT) peptide pool did not appear to elicit a detectable TT-specific CD8+ T cell response, limiting competition between HIV- and TT-specific CD8+ T cells. Figure 9 E).

[0623] Example 9

[0624] TCR binding of different mRNA string processing and presentation epitopes in the K562 cell line

[0625] Jurkat cell lines expressing a single TCR were co-incubated with a single HLA-I allele, K562, to measure downstream TCR signaling at a log2-fold signal for luciferase expression. K562 was pulsed with a peptide encoded by the test string (PP) as a positive control to control TCR activity at the specific epitope being tested, or transfected with the mRNA sequence encoding the peptide. PHA-L was used as a positive control for TCR activity. Five unique epitopes and their homologous TCR responses were tested. Four distinct mRNA strings with different amino acid sequences were tested, each with two nucleotide sequence optimizations (nt2 and nt3). String 1 and its derivative, string 1.1 (with four point mutations), were tested in K562 cells using 3 μg ( Figure 10 A) or 0.3 μg ( Figure 10B) was tested by transfecting cells with methyl pseudouridine mRNA. All displayed epitopes were perfectly matched in the sequences of these mRNA strings. String 2 and its derivative string 2.1 (1 point mutation) were tested in K562 cells with 3 μg ( Figure 10 C) or 0.3 μg ( Figure 10 The cells were transfected with methyl pseudouridine mRNA (D) for testing. The sequence of these mRNA strings was compared using a star-shaped (D) molecule. The epitopes marked with ) contain at least one polymorphism.

[0626] String 1 and its derivative String 1.1 (including 4 point mutations) encode all five tested epitopes, with complete sequence matches. Epitopes AK11, DA9, and IW9 can be detected by transfecting 3 µg mRNA with each string. Figure 10 A). Sequence optimization of strings 1a and 2a allowed for supplemental TCR binding to detect the KF11 epitope, despite a high untreated background. The EL9 epitope was consistently undetectable in this assay, but this was not due to a lack of TCR reactivity, as its function was observed under both nonspecific PHA-L and specific peptide pulse (PP) conditions. Transfection with suboptimal mRNA at 0.3 µg resulted in the loss of AK11, KF11, and DA9 signals in all or some strings. This observation was expected to be associated with lower overall polyprotein translation of the encoded strings. Therefore, using the strongest response (3 µg) Figure 10 AK11 (A-IW9, DA9) was still detectable at 0.3 µg transfection. String 2 and its derivative String 2.1 (one point mutation) encoded polymorphic versions of three test epitopes. Only the IW9 and KF11 epitopes had complete sequence matches with String 2 and String 2.1. Therefore, AK11 and EL9 were not detected in this assay. Figure 10 (C and D). However, the polymorphic version of DA9 encoded in the mRNA strings of these groups appears to be cross-reactive and efficiently binds to the TCR at 3 µg mRNA. Figure 10 C), but at only 0.3 µg, it is insufficient to be detected. Figure 10 D). Consistent with previous observations in strings 1 and 2, IW9 triggered a strong response at 3 µg. Figure 10 C), and it is still detectable at 0.3 µg. Figure 10 D). However, only some codon-optimized sequences, namely string 2 nt2 and string 2.1 nt3, appear to produce detectable responses at 3 µg, which are identified by the TCR of KF11 detection.

[0627] Text description: AK11: ACQGVGGPGHK; DA9: DRFYKTLRA; KF11: KAFSPEVIPMF; IW9: ISPRTLNAW; EL9: EVIPMFSAL, polymorphism in string 2 and string 2.1 DA9: DRFFKTLRA (in Figure 6 (referred to as +DA9+ in Chinese).

[0628] Example 10

[0629] In iDCs generated from two donors, TCR binding of epitopes for different mRNA string processing and presentation in the K562 cell line: Jurkat cell lines expressing a single TCR effector were co-incubated with iDCs transfected with the target single HLA-I allele K562 or HLA-I from the donor to measure downstream TCR signaling expressed as a log2-fold signal of luciferase. Target cells were pulsed with a peptide encoded by a test string (PP) as a positive control to control TCR activity of the specific epitope being tested, or transfected with an mRNA string encoding the peptide. Pulsed peptides without co-transfection with the HLA allele were also used to control background presentation of endogenous HLA-I from the donor (PP 10µM + eHLA) to test target iDCs. PHA-L was used as a positive control for TCR activity. Three unique epitopes and their homologous TCR responses were tested. Four different mRNA strings with different amino acid sequences were tested, each with two nucleotide sequence optimizations (nt2 and nt3). Strings 1 and 2 were tested in target K562 cells (… Figure 11 A) or target iDC from 2 donors ( Figure 11 In B and C), cells were transfected with 3 μg of methylpseudo-uridine mRNA for testing. The TL9 and RI8 epitopes tested were both encoded by strings 1 and 2, with complete sequence matches. In parallel, the epitope TW10 encoded by string 2 contained a single mutant polymorphism, while perfectly matching string 1. TL9 was tested using the HLA allele B. 39:10 test, E18 uses HLA allele B 52:01 test, TW10 uses HLA allele B 57:01, B 57:03 and B Test at 58:01.

[0630] String 1 encodes all three test epitopes, with complete sequence matches. In contrast, string 2 encodes only the completely matching epitopes TL9 and RI8, but contains a single amino acid mutation in epitope TW10. Strings 1 and 2 were tested with different nucleotide optimizations (nt2 and nt3). The cleavage, processing, and presentation of each test epitope on their homologous HLA derived from peptides translated from the mRNA templates of strings 1 and 2 were evaluated. The function of the TCR clones of all tests was tested using selective T-cell mitogens (PHA-L). Figure 11 (A, B, and C). The specificity of TCR clones for their epitopes: HLA-I complex was verified using monoexpression of the HLA-I allele and peptide pulse (PP). Figure 11 A). It ...

Claims

1. 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, wherein each fragment comprises at least one epitope, wherein the epitope is derived from an amino acid sequence encoded by human immunodeficiency virus (HIV), and wherein the epitope is contained within a sequence selected from any one of SEQ ID NO: 1 to 148 and 198 to 202 or a variant thereof, or contains a sequence selected from any one of SEQ ID NO: 1 to 148 and 198 to 202 or a variant thereof.

2. The composition of 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 of the segments is different from the epitope in at least one other segment or all other segments.

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 said fragments do not contain any epitopes contained in another fragment.

5. The composition according to any one of claims 1 to 4, wherein the sequences of said epitopes overlap in at least one of said segments.

6. The composition according to any one of claims 1 to 5, wherein the sequences of said epitopes do not overlap in at least one of said segments.

7. The composition according to any one of claims 1 to 6, wherein the 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.

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 of those described in SEQ ID NO: 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 to minimize the length of the RNA molecule.

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

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

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

14. The composition according to any one of claims 1 to 13, comprising a common flanking amino acid sequence of HIV amino acid sequences at the 5' and 3' of two or more non-overlapping epitopes within one or two or more fragments, without flanking branches deriving said epitopes.

15. The composition according to any one of claims 1 to 14, wherein the connectors separate at least two segments.

16. The composition according to any one of claims 1 to 15, wherein the variant of the epitope is a polymorph of the epitope, wherein the epitope is a common sequence from at least two different clones of HIV and / or wherein the variant differs from the epitope in 1, 2, 3, 4 or 5 amino acids.

17. The composition according to any one of claims 1 to 16, wherein the composition further comprises a second RNA molecule, wherein the second RNA molecule comprises a second expression cassette encoding a second immunogenic peptide, the peptide comprising at least two fragments, wherein each fragment comprises at least one epitope, wherein the epitope is derived from an amino acid sequence encoded by the human immunodeficiency virus (HIV), and wherein the epitope is contained within a sequence selected from any one of SEQ ID NO: 1 to 148 and 198 to 202 or a variant thereof, or contains a sequence selected from any one of SEQ ID NO: 1 to 148 and 198 to 202 or a variant thereof.

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

19. The composition of claim 17 or 18, wherein the epitope in one fragment of the second immunogenic peptide is different from the epitope 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 fragments of the second immunogenic peptide do not contain any epitopes 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 fragment 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 fragment of the second immunogenic peptide.

23. The composition according to any one of claims 17 to 22, wherein the second 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.

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 of those described in SEQ ID NO: 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 incorporated within the expression cassette of the second immunogenic RNA molecule to minimize the length of the RNA molecule.

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

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

29. The composition according to any one of claims 17 to 28, comprising a common flanking amino acid sequence of HIV amino acid sequences at the 5' and 3' of two or more non-overlapping epitopes within one fragment of the second immunogenic peptide or two or more fragments of the second immunogenic peptide, without flanking branches of said epitopes.

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

31. The composition according to any one of claims 17 to 30, wherein the linker separates at least two segments 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 polymorph of the epitope, wherein the epitope is a common sequence from at least two different clones of HIV and / or wherein the variant differs from the epitope in 1, 2, 3, 4 or 5 amino acids.

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

34. The composition of claim 33, wherein 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 contains the same epitope in both clades.

36. The composition according to any one of claims 33 to 35, wherein each immunogenic peptide comprises a polymorphic epitope from each corresponding 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 a CD8 minimal 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 length of the epitope is 9 or more amino acids.

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

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

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

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

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

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

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

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

49. The composition according to any one of claims 1 to 48, wherein the length of said fragment ranges from 9 to 21 amino acids.

50. The composition according to any one of claims 1 to 49, wherein the peptide comprises the following amino acid sequence: a) SEQ ID NO: 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) SEQ ID NO: 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.

51. The composition according to any one of claims 1 to 49, wherein the peptide comprises the following amino acid sequence: a) SEQ ID NO: 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) SEQ ID NO: 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.

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 composition according to any one of claims 17 to 49, wherein The first peptide comprises the following amino acid sequence: a) SEQ ID NO: 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) SEQ ID NO: 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, The second peptide contains the following amino acid sequence: c) SEQ ID NO: 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) SEQ ID NO: 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.

54. The composition according to any one of claims 17 to 49, wherein a) The first peptide comprises the amino acid sequence of SEQ ID NO: 183 or 205 and the second peptide comprises the amino acid sequence of SEQ ID NO: 185 or 208, or b) The first peptide contains the amino acid sequence of SEQ ID NO: 187 and the second peptide contains the amino acid sequence of SEQ ID NO:

189. Optionally, the first peptide comprises the amino acid sequence of SEQ ID NO:205 and the second peptide comprises the amino acid sequence of SEQ ID NO:

208.

55. The composition according to any one of claims 1 to 54, wherein the RNA molecule comprises the nucleotide sequence of SEQ ID NO: 184, 186, 188, 190, 206, 207, 209, 210 or 211 to 218.

56. The composition according to any one of claims 1 to 55, wherein the epitope is a non-protective epitope capable of causing CD8+ T cell protection collapse.

57. The composition of claim 56, wherein the protective collapse is T cell escape, 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 cell surface.

59. The composition according to claim 58, wherein the cell is an immune cell.

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

61. The composition according to any one of claims 1 to 60, wherein the peptide further comprises a 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 auxiliary epitope.

64. The composition of claim 63, wherein the auxiliary epitope is derived from Clostridium tetani (… Clostridium tetani The P2 and / or P16 amino acid sequences of tetanus toxoid (TT).

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

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

67. The composition of claim 66, wherein the 5' cap is a modified cap, an 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 of 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 of 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 1-70, wherein the expression cassette further comprises a poly A structure.

72. The composition of claim 71, wherein the poly A structure is an interrupted poly A structure.

73. The composition of claim 72, wherein the poly A structure comprises the nucleotide sequence of SEQ 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 SEQ ID NO:

197.

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

75. The composition of claim 74, wherein the reproducible RNA molecule further encodes an RNA-dependent RNA polymerase (replicaase) capable of replicating the reproducible RNA molecule.

76. The composition of claim 74, wherein the reproducible RNA molecule does not encode an RNA-dependent RNA polymerase (replicaase).

77. The composition according to any one of claims 74 to 76, wherein the composition further comprises a non-replicating RNA molecule encoding an RNA-dependent RNA polymerase (replicaase) capable of replicating the replicable 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 removing double-stranded RNA.

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

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

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

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 in the composition together with at least one lipid.

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

86. The composition of claim 85, wherein the particles are lipid nanoparticles (LNP), lipoplexes (LPX), or liposomes.

87. The composition according to claim 85 or 86, wherein the particles are nanoparticles, wherein: (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 lower.

88. The composition according to claim 87, wherein the charge ratio of positive 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 of 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 PEGylated lipid.

93. The composition according to any one of claims 84 to 92, wherein the composition further comprises at least one auxiliary lipid.

94. The composition according to claim 93, wherein the auxiliary 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-trimethylammonium propane (DOTMA), 1,2-dioleoyloxy-3-dimethylaminopropane (DODMA), and / or 1,2-dioleoyl-3-trimethylammonium propane (DOTAP).

96. The composition according to any one of claims 93 or 94, wherein the at least one auxiliary lipid comprises 1,2-bis-(9Z-octadecenoyl)-sn-glycerol-3-phosphate ethanolamine (DOPE), cholesterol (Chol), 1,2-dioleoyl-sn-glycerol-3-phosphate choline (DOPC) and / or 1,2-distearate-sn-glycerol-3-phosphate choline (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 auxiliary 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 comprising DODMA 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, and wherein the charge ratio of the positive charge in DODMA to the negative charge in the 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 comprising DODMA and cholesterol in a molar ratio of 10:0 to 1:9, preferably 8:2 to 3:7, and more preferably 7:3 to 5:5, and wherein the charge ratio of the positive charge in the DODMA to the negative charge in the 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 comprising DODMA and DSPC in a molar ratio of 10:0 to 1:9, preferably 8:2 to 3:7, and more preferably 7:3 to 5:5, and wherein the charge ratio of the positive charge in DODMA to the negative charge in the 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 lipoplexes comprising 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 lipoplexes comprising DOTMA 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, and wherein the charge ratio of the positive charge in DOTMA to the negative charge in the 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 lipoplexes comprising DOTMA and cholesterol in a molar ratio of 10:0 to 1:9, preferably 8:2 to 3:7, and more preferably 7:3 to 5:5, and wherein the charge ratio of the positive charge in the DOTMA to the negative charge in the 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 lipoplexes comprising 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, and wherein the charge ratio of the positive charge in DOTMA to the negative charge in the 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 particle is an LNP that is compounded with and / or encapsulates the RNA molecule.

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

107. The composition according to any one of claims 1 to 84, wherein the RNA molecule is formulated in a composition comprising a polyalkylene imide, preferably, the composition being a polyalkylene imide.

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

0.

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

110. The composition according to claims 107 to 109, wherein the formed particles are a polymeric complex.

111. The composition according to any one of claims 107 to 110, wherein the polyalkylene imide comprises the following general formula (I): , in R is H, an acyl group, or a group containing the following general formula (II): , Where R1 is H or a group containing the following general formula (III): , n, m, and l are independently selected from integers from 2 to 10; and p, q, and r are integers, where the sum of p, q, and r results in an average molecular weight of 1.5 for the polymer. 102 Da to 107 Da, preferably 5000 Da to 105 Da, more preferably 10000 Da to 40000 Da, more preferably 15000 Da to 30000 Da, and even more preferably 20000 Da to 25000 Da.

112. The composition according to any one of claims 107 to 111, wherein the polyalkylene imide 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 polyalkylimide are protonable.

114. The composition according to any one of claims 1 to 113, wherein the composition is a pharmaceutical composition.

115. The composition of claim 114, wherein the composition further comprises a pharmaceutically acceptable carrier or excipient.

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

117. The composition according to any one of claims 1 to 115, wherein the composition is lyophilized.

118. The composition according to any one of claims 1 to 115, wherein the composition is frozen.

119. The composition according to claim 118, wherein the temperature of the composition is -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 buffering substances, sugars, stabilizers, cryoprotectants, lyophilization protectants, and chelating agents.

121. The composition of claim 120, wherein the buffering substance comprises at least one selected from 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid (HEPES), 2-(N-morpholine) ethanesulfonic acid (MES), 3-morpholine-2-hydroxypropanesulfonic acid (MOPSO), acetic acid, acetic acid buffer and analogues, phosphate and phosphate buffer, and citric acid and citrate buffer.

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

123. The composition according to any one of claims 120 to 122, wherein the cryoprotectant comprises at least one selected from glycols and glycerol, wherein the glycols are, for example, ethylene glycol and propylene glycol.

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

125. The composition according to any one of claims 1 to 124, wherein the composition is a vaccine.

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

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

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

129. The kit product according to claim 127 or 128, wherein the RNA molecule is lyophilized.

130. The kit product according to any one of claims 127 to 129, wherein the kit product further comprises instructions for applying the RNA molecule.

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

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

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

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

136. The method according to any one of claims 131 to 135, wherein the method involves only a single application of the composition.

137. The method according to any one of claims 131 to 135, wherein the method comprises applying the composition multiple times.

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

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

140. The method of claim 139, wherein the injection is performed using a needle or using a needleless injection device.

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

142. The composition according to any one of claims 1 to 125, used in a method of preventing or treating HIV infection in a subject, the method comprising applying the composition to the subject.

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

144. The composition according to any one of claims 1 to 125, for use in the preparation of a medicament for the prevention or treatment of HIV infection in a subject.

145. A method for treating or preventing HIV infection, comprising administering a first RNA molecule and a second RNA molecule to a subject. The first RNA molecule contains a nucleotide sequence encoding a first peptide, which contains the following amino acid sequence: a) SEQ ID NO: 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) SEQ ID NO: 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; and The second RNA molecule contains a nucleotide sequence encoding a second peptide, which contains the following amino acid sequence: c) SEQ ID NO: 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) SEQ ID NO: 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.

146. The method of claim 145, wherein... 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.

147. The method of 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 RNA molecule and the second RNA molecule are administered at least 2 weeks apart.

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

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